1er. CONGRESO VIRTUAL DE CARDIOLOGIA
RESUMENES DE TEMAS LIBRES

FARMACOLOGIA CARDIOVASCULAR

Index

2471
Acción de los ácidos grasos de la dieta y efecto de la epinefrina en la frecuencia de aurículas aisladas de ratas.
Alfonso, P.C.; Lamanna, N.V.; Rodríguez, R.M.; Pérez-González, M.
Sección de Investigaciones Cardio-Renales. Universidad Central de Venezuela.
Caracas. Venezuela

Los ácidos grasos de los lípidos de la dieta producen cambios en el perfil de los ácidos grasos en las membranas y pueden actuar como biorreguladores endógenos. Los ácidos grasos libres poli-insaturados incrementan las corrientes de calcio en los miocitos aislados de cobayos. El objetivo de este trabajo es estudiar los efectos de dietas enriquecidas con distintos aceites vegetales, sobre la respuesta cronotrópica a la epinefrina en aurículas aisladas de ratas. Se utilizaron 18 ratas machos Sprague Dawley y se dividieron en tres grupos de 6 ratas. La dieta basal fue Ratarina® con 2% de lípidos ( Grupo R ); La dieta del Grupo M fue enriquecida con 5% de mezcla de aceites de oleína de palma más soya y la dieta del Grupo S fue enriquecida con 5% de aceite de soya. La relación de 18:2,n-6/16:0 y 18:1,n-9/16:0 en las dietas fueron : 2,6 y 1,6 en la ratarina, 1,5 y 1,3 en la mezcla, 3,9 y 1,8 en la soya, respectivamente. Después de 6 semanas, las ratas fueron anestesiadas con pentobarbital sódico, se aislaron las aurículas, se fijaron al transductor en un baño de órgano con solución tyrode, 37º C y burbujeada con 95% 02/ 5% C02 . Se realizó una curva dosis respuesta a Epinefrina. Se encontró que la respuesta cronotrópica máxima a la epinefrina obtenida en el grupo M fue significativamente mayor que la del grupo S ( p<0,05), la frecuencia espontánea de las preparaciones antes de añadir la droga al baño no fue distinta para los tres grupos experimentales. En conclusión, la composición de los ácidos grasos podría regular a nivel del microambiente lipídico la respuesta del agonista y/o la corriente If.

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2462
Acción de un ionoforo de Ca ++ sobre la liberación endotelial coronaria de oxido nítrico.
Catrip Torres J, González Garduño A, Torres Narváez J.C, Del Valle Mondragón L, Pastelín G
Departamento de Farmacología. Instituto Nacional de Cardiología «Ignacio Chávez»
Ciudad de México. México

Introducción: La síntesis y liberación de óxido nítrico (ON) por el endotelio vascular esta determinada por factores de diversa naturaleza como los relacionados con la neurotransmisión, mediadores químicos o estímulos físicos. Se sabe que su síntesis requiere del ión calcio (Ca++) como cofactor. La fricción «shear stress», que ejerce la sangre sobre el endotelio vascular, es un factor de naturaleza física que induce la síntesis de ON en este tejido.
Objetivo: Analizar la influencia de distintos grados de «shear stress» sobre la síntesis de ON por el endotelio vascular coronario en presencia de un ionóforo de Ca++ (Lasalocid).
Metodología: Se aislaron corazones de cobayo según el método de Langendorff. El corazón se conecta por la aorta al sistema de perfusión, se utilizaron flujos (F) de 5, 10 y 20 ml/min. En el líquido efluente se midió la concentración de ON en base al método de Schrader, en condiciones control y en presencia de Lasalocid (L), a concentraciones de 0.125 a 4.000µM.
Resultados: La velocidad de perfusión coronaria induce en relación directa, una mayor síntesis de ON (F: 5 ml = 27.42; 10 ml = 33.66; 20 ml = 48.62 pmoles/ml ON). En presencia de L se observa un incremento en la liberación de ON, cuando el flujo coronario es bajo (5ml/min: L a 0.125 µM = 48.62 pmoles/ml ON, a 4.000 µM = 140.8 pmoles/ml ON). Cuando el flujo coronario es alto (20 ml/min) la síntesis de ON no se incrementa por acción del L.
Discusión: La entrada sarcolemal de Ca++ inducida por L a flujos coronarios bajos incrementa este cofactor de la sintasa de ON constitutiva y da lugar a un incremento en la síntesis de ON.
Conclusión: El efecto de «shear stress» derivado del flujo coronario utiliza la sintasa de ON constitutiva y presenta una limitante desconocida a flujos coronarios altos.

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2511
Acidosis enhances isoproterenol-induced phospholamban phosphorylation in the intact heart by increasing phosphorylation of Thr 17 residue.
Mundiña-Weilenmann Cecilia, Vittone Leticia, Ortale Manuel, Chiappe de Cingolani Gladys, Mattiazzi Alicia R
Centro de Investigaciones Cardiovasculares, La Plata, Argentina

Previous experiments showed that acidosis enhances isoproterenol-induced phospholamban (PHL) phosphorylation (Am J Physiol 270:C107-114, 1996). The availability of phosphorylation site-specific antibodies to PHL prompted us to re-examine the issue in isolated Langendorff perfused rat hearts to gain further insight into the mechanism involved in this effect. At normal pHo (7.40), PHL phosphorylation -measured in 32P labelled hearts- and phosphorylation of Thr17 and Ser16 residues, attained a «plateau» at 30 nM isoproterenol. Acidosis (pHo 6.70) produced a further increase in 32P incorporation into PHL from 132 ± 26 (30 nM Isoproterenol, pHo 7.40) to 243 ± 38 pmol 32P/mg membrane protein (30 nM Isoproterenol, pHo 6.70) (n=5). This increase was exclusively due to an increase in phosphorylation of Thr17 (n=5) and was associated with a significant enhancement of isoproterenol-induced relaxant effect. Since acidosis increases [Ca]i (J Physiol 384:431-449, 1987), we explored the possibility that the enhancement of Thr17 phosphorylation could be due to an activation of Ca-calmodulin-dependent protein kinase (CaMKII). Increasing [Ca]o at pHo 7.40 in the presence of 30 nM isoproterenol produced an increase in Thr17 phosphorylation (n=3), suggesting that CaMKII can be further activated in the presence of isoproterenol. In addition, acidosis produced a significant inhibition of sarcoplasmic reticulum associated phosphatase activity which can contribute to phosphorylation of PHL. The experiments indicate that acidosis enhancement of isoproterenol-induced PHL phosphorylation is exclusively due to Thr17 phosphorylation and may occur by simultaneous activation of CaMKII and inhibition of PHL phosphatase

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2523
Acidosis enhancement of isoproterenol-induced phosphorylation of Thr 17 of Phospholamban (PHL) could be mimicked by either increasing extracellular calcium ([Ca]o) or administration of okadaic acid (OA).
Mundiña-Weilenmann Cecilia, Said Matilde, Vittone Leticia, Mattiazzi Alicia.
Centro de Investigaciones Cardiovasculares.
La Plata, Argentina

Acidosis enhances the phosphorylation of PHL produced by 30 nM isoproterenol, (Iso), due to the phosphorylation of Thr17 residue (Biophys J 72:A168, 1997). To further study this effect, phosphorylation-site specific antibodies were used to assess the effect of acidosis (pHo 6.80) on the phosphorylation of Ser16 and Thr17 residues of PHL at different Iso concentrations, in Langendorff-perfused rat hearts. In the absence of Iso, acidosis induced a non significant increase in Thr17 phosphorylation (measured by optical densitometry and expressed as % of the maximal value obtained in each experimental series at pHo 7.40). At all Iso concentrations (3 to 300 nM) Thr17 phosphorylation was significantly higher at pHo 6.80 than at pHo 7.40 without significant changes in Ser16 phosphorylation. Either increasing [Ca]o to 3.85 mM or addition of 1 µM OA in the presence of Iso were both able to mimick the increase in Iso-induced Thr17 phosphorylation produced by acidosis. The results suggest that acidosis may increase Iso-induced Thr17 phosphorylation by either activation of CaMKII (by increasing [Ca]i (J Physiol 384:431, 1987), inhibition of phosphatases (Am J Physiol 270:C107, 1996) or both.

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2394
Activation of the Na +/H+ exchanger underlies the slow increase in force (SIF) that follows myocardial stretch.
Pérez Néstor G, Ennis Irene L, Alvarez Bernardo V, Camilión de Hurtado María C, Cingolani Horacio E
Centro de Investigaciones Cardiovasculares. La Plata, Argentina

Rat trabeculae were stretched ~10 % (from ~88 to ~98% of their maximal length) and intracellular Na+ (Na+i) was estimated by rationing the SBFI 340/380 nm fluorescence signals. The rapid increase in developed force (DF) was followed by a second progressive increase that developed in ~10 minutes. The SIF accounted for 24 ± 5 % of the total increase in DF and when it was expressed as a percentage of the initial phase, it was 14 ± 3 % (0.473 of 3.667 g/mm2) (P<0.05, n=5). During the SIF, the SBFI ratio increased by 4.6 ± 0.4 % (0.033 of 0.730) (P<0.05, n=5). The increase in Na+i and the SIF were both prevented by blocking the Na+/H+ exchanger (NHE) with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, 1 µmol/L). The activation of the NHE was also prevented by blocking the subtype AT1 of the angiotensin II receptors (1µmol/L losartan). The data indicate that activation of the NHE by an autocrine-paracrine mechanism underlies the increase in Na+i that in turn may cause the increase in Ca2+i through the Na+/Ca2+ exchanger.

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2496
Angiotensin II activates NA +-independent Cl-/HCO3- exchange in ventricular myocardium.
Ennis Irene L, de Hurtado María C, Alvarez Bernardo V, Pérez Néstor Gustavo, Cingolani Horacio E
Centro de Investigaciones Cardiovasculares. La Plata (1900), Argentina.

The effect of angiotensin II (ANG II) on the activity of the cardiac Na+-independent Cl-/HCO3- exchanger (AE) was explored in cat papillary muscles. Intracellular pH (pHi) was measured by epifluorescence with BCECF/AM. ANG II (500 nM) induced a EIPA-sensitive increase in pHi from 7.01±0.05 to 7.11±0.02 (n=4,P<.05) in the absence of external HCO3- (HEPES buffer), consistent with the stimulatory action of the hormone on Na+/H+ exchange (NHE). This alkalinizing effect was not detected in the presence of a CO2/HCO3- buffer (pHi: 7.07±0.02 and 7.08±0.02 before and after ANG II, respectively, n=17), suggesting that the ANG II-induced activation of NHE could have been overcome by a HCO3- dependent acidifying mechanism. This hypothesis was examined by measuring the velocity of myocardial pHi recovery after imposition of an intracellular alkali load (trimethylamine hydrochloride prepulse) in a HCO3- containing solution either with or without ANG II. The rate of myocardial pHi recovery from alkalosis was augmented from a control value of 0.009±0.003 to 0.018±0.004 pH unit/min in the presence of ANG II (n=6, P<.05), indicating a stimulatory effect on AE. A similar enhancement of the activity of this exchanger by ANG II was detected when the AE activity was reversed by extracellular Cl- removal: thus the rate of intracellular alkalinization induced under this conditions was almost doubled (from 0.053±0.016 to 0.108±0.026 pH unit/min,n=6, P<.05) in the presence of ANG II. This effect was cancelled either by the presence of the AT1-receptor antagonist, losartan (1 µM), or by the previous inhibition of protein kinase C (PKC) with chelerythrine (10 µM). The above results show, for the first time, that ANG II activates the AE in ventricular myocardium by a PKC-dependent regulatory pathway linked to the AT1-receptors.

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2493
Angiotensin II activates the Na + independent Cl-/HCO3- exchanger by release/production of endothelin-1
Alvarez Bernardo V, Ennis Irene L, Camilión de Hurtado María C, Cingolani Horacio E
Centro de Investigaciones Cardiovasculares.
La Plata, Argentina

In heart cells, the recovery of intracellular pH (pHi) from alkalosis is mediated by the Na+ independent Cl-/HCO3- exchanger (AE) (1 external Cl- exchanged by 1 internal HCO3-). This AE activity, being dependent on pHi, with alkalization increasing exchange activity. It was previously demonstrated by our lab that angiotensin II (Ang II), through its AT1 receptor, activates the AE by a protein-kinase dependent pathway (M.C. de Hurtado et al., Circ.Res. 83:473-481,1998). Since some of the effects of Ang II thought to be unique to this hormone were recently reported to be mediated by endothelin (ET-1), the possibility that the activation of the AE by Ang II could be the result of an effect mediated by ET-1 was examined. Experiments were performed in cat papillary muscles in which pHi was increased by 40 mM trimethylamine and the activity of the AE was assessed by measuring the velocity of pHi recovery after the alkaline load. pHi after the alkaline load increased from 7.05 ± 0.02 to 7.48 ± 0.02 (n=16) without significant differences among the experimental groups studied.
Velocity of pHi recovery (pHunits.103/min) (*indicate P<0.05 vs control)

Since the ETA blocker BQ123 abolished the changes in pHi without blocking the positive inotropic changes, the possibility of an AT1 blockade induced by BQ123 was ruled out. Our data are consistent with an Ang II-induced activation of the AE as the result of ET-1 release/production. Since we are using a multicellular preparation the origin of ET-1 remains to be determined.

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2509
Angiotensin II–induced positive inotropic effect in feline miocardium: calcium, myofilament responsiveness or both?
Vila Petroff Martín G, Aiello Ernesto, Palomeque Julieta, Cingolani Horacio E, Mattiazzi Alicia R
Centro de Investigaciones Cardiovasculares. La Plata. Argentina

Angiotensin II (ANG II) exerts a potent positive inotropic effect (PIE) in several species. However, the subcellular mechanisms involved in this effect are still a matter of major controversy. The present study examines the role of intracellular calcium (Cai) and pH (pHi) in the ANG II-induced PIE in isolated cat ventricular myocytes. For this purpose, either pHi (via SNARF/AM fluorescence) or Cai (via INDO-1/AM fluorescence) were measured simultaneously with cell shortening. In HEPES buffer ANG II induced a pronounced increase in contraction amplitude that reached steady state approximately 10 minutes after administration of the drug. The increase in contraction amplitude (223±38% above control) was associated with an increase in the Cai transient amplitude (CaiT) (38.5±7% above control) (n=6) and an intracellular alkalinization (0.058±0.01 pH units)(n=5). However, before reaching steady state there was a window of time (up to 5 min after administration of ANG II) where the PIE and CaiT increase induced by ANG II occurred without changes in pHi. With the aim of dilucidating whether an increase in myofilament responsiveness to calcium contributes to ANG II-induced PIE, Cai was measured in a parallel group of experiments where increases in contraction amplitude similar to those evoked by ANG II at either 5 or 10 minutes were produced by increasing extracellular [Ca]o. For a comparable increase in contractility, the ANG II-induced increase in CaiT was significantly lower than that induced by raising [Ca]o. We conclude that in feline cardiac myocytes, both an increase in CaiT and in myofilament responsiveness to calcium are responsible for the ANG II-induced PIE. Furthermore, at least in the initial phase (where no pHi changes were detected) an increase in pHi does not underlie the augmented myofilament responsiveness to calcium.

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2614
Assessment of load-independent inotropic effects of amrinone in closed-chest dogs
Lic. Negroni Jorge A, Lic. Lascano Elena, Dr. Crottogini Alberto, Dr del Valle Héctor
Universidad Favaloro, Instituto de Investigación en Ciencias Básicas Buenos Aires, Argentina

Introduction: It is controversial whether amrinone (Am) has a dominant inotropic or vasodilator effect on cardiac function, since due to load dependence of pump function indexes it is impossible to assess a purely inotropic response.
Methods: To evaluate the load-independent action of Am, the following pump function indexes: end-diastolic pressure (EDP), ejection fraction (EF), stroke work (SW), maximum first derivative of ventricular pressure (dP/dtmax) and stroke volume (SV) were measured in 12 anesthetized, closed-chest dogs at 3-5 preloads (end-diastolic volume, VD; vena cava occlusion), in three afterloading conditions (peripheral resistance, R): basal, increased (aortic occlusion) and decreased (nitroprusside). Each index was fitted to a second order model as a function of VD, R and heart rate (HR), and the load-independent inotropic increase (IE) was calculated from the fitted plane at the same VD, R and HR as control.
Results: Amrinone produced 11.5 ± 8.5 % decrease in VD, 11.6± 10 % decrease in R and 7.3 ± 7.1 % increase in HR (p<0.01). Its effect on the indexes in uncontrolled loading conditions (%DE) and on %IE was:

Mean ± SD; * p<0.05† p<0.01; ‡ p<0.001, t test against no effect.

Conclusions: The present model allowed the assessment of the purely inotropic effect of Am in intact animals and can be employed to evaluate other inotropic drugs whose action on VD, R and HR affect load-dependent indexes.

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2453
Aumento del inotropismo y del margen de seguridad en análogos sintéticos de digitoxigenina con mayor electronegatividad estructural
Del Valle Leonardo; Torres Juan Carlos; Zarco Gabriela; Pastelín Gustavo
Departamento de Farmacología, Instituto Nacional de Cardiología «Ignacio Chávez». Ciudad de México, México.

Pese a su reducido índice terapéutico, los digitálicos siguen siendo utilizados en la insuficiencia cardiaca congestiva y la fibrilación auricular crónica. Es a partir del descubrimiento de su composición química, que se originan estudios de remodelación estructural tratando de disminuir su toxicidad. Tales investigaciones reportan que la potencia de un digitálico, radica en la densidad electrónica negativa sobre los anillos «C» y «D» esteroidales. En el presente trabajo damos cuenta de la obtención por síntesis y caracterización química y biológica de una serie de compuestos, derivados de Digitoxigenina, con grupos que sustituyen sobre el anillo «D» a la lactona e hidroxilo. La obtención de los derivados 14ß,17ß-ciclocetoester-3ß-hidroxi-androstáno (INCICH-D7), 14ß,17ß-ciclohidramida-3 alpha, 3ß-dihidroxi-androstáno, 14ß,17ß-cicloanhídrido- 3ß-hidroxi - androstáno, 12ß,17ß-ciclodicetovinilol-3 alpha, 3ß-dihidroxi-androstáno y el 12ß,14ß-ciclocarbadiol-3ß-hidroxi-androstáno, se ratificó por reactividad química y cristalografía por difracción de rayos-X. La electronegatividad de los mismos, se determinó por el método de Duhamm bajo radiación infrarroja. El estudio farmacológico se realizó en corazón aislado de cobayo, comparando el efecto de los seis derivados con su precursor, mediante curvas dosis-respuesta evaluando la fuerza de contracción. El margen de seguridad del INCICH-D7, se determinó bajo el modelo de Starling en corazón de perro.
Resultados: El análisis químico y cristalográfico confirmaron la estructura de los derivados de Digitoxigenina. La evaluación de electronegatividad estructural, indicó que ésta es mayor en los seis productos sintetizados que en el precursor. El incremento en la fuerza de contracción inducida por estos compuestos presenta una relación directa con el aumento en la electronegatividad sobre los anillos «C» y «D». El margen de seguridad del INCICH-D7 aumenta significativamente con respecto a la Digitoxigenina. Conclusiones: Se confirmó que al incrementar la electronegatividad sobre los anillos «C» y «D» en la Digitoxigenina, sustituyendo la lactona e hidroxilo por grupos de alta electronegatividad, se logra una mayor actividad inotrópica y un margen de seguridad más amplio.

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2321
Autonomic and hemodynamic improvement with the association of beta-blockers with flurocortisone in postural tachycardia syndrome (POTS) patients.
Freitas Joao, Santos Rosa, Azevedo Elsa, Costa Ovidio, Carvalho Mario, Falcão de Freitas Antonio
Centro de Estudos da Função Autonómica – Hospital São João. Porto, Portugal

Introduction: Orthostatic intolerance (POTS) is a frequent misdiagnosis disabling condition.
Objective: To evaluate the efficacy of drugs in the treatment of the autonomic/hemodynamic disturbance that characterizes POTS patients.
Material and Methods: 9 women, with a mean age 31 years (21 to 57 years), with POTS were evaluated: four pts received oral betablocker (B) therapy, 2 pts flurocortisone (F) and 3 pts association of B and F. The patients were study in basal and tilt position, before (b) and after (a) treatment. HRV, SBPV and spontaneous baroreceptor gain were calculated by FFT and alpha index. The hemodynamic data was assessed by modelflow-TNO ® analysis.
Results:

*p<0.05 and **p<0.01 after treatment;+ p<0.05 and ++ p<0.01after tilt before treatment; Units: HR in bpm; SV in ml; HRV in nu; SBPV in mmHg2; BR in ms/mmHg; TPR in dyn.s.cm-5; SBP in mmHg.

Conclusion: 1- Betablockers and/or flurocortisone improve the clinical and autonomic/hemodynamic disturbance observed in POTS. 2- All medical personnel must be alert to the diagnosis of this disabling misdiagnosis syndrome with such an easy treatment and marked clinical improvement.

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2442
Cambios en la actividad mitocondrial de corazón de ratas producidos por la ingesta de aceite de pescado.
Crespo-Armas A., Azavache V., Anchustegui B, Guercio MG, Planchart A
Cátedra de Fisiología. Instituto de Medicina Experimental. Facultad de Medicina. Universidad Central de Venezuela. Caracas. Venezuela

La composición de los ácidos grasos (AG) de las células cardíacas se modifica con la ingesta de diferentes dietas lipídicas. La eficiencia de fosforilación mitocondrial de diferentes tejidos, se altera al cambiar la composición lipídica de las membranas de estas organelas.
En este trabajo se estudió el efecto de la administración de una dieta suplementada con aceite de pescado, en la composición lipídica y la actividad de las mitocondrias de corazón de rata.
Se utilizaron ratas machos alimentadas por 3 semanas con Ratarinaâ y con Ratarinaâ suplementada con AP al 8% (p/p). Los animales se sacrificaron, se aislaron las mitocondrias del corazón. A estas organelas se les midió: a.- la composición porcentual de los AG de los lípidos totales por cromatografía gas-líquido y b.- el consumo de oxígeno utilizando un electrodo tipo Clarck y succinato como sustrato oxidable. Se calculó: a.- la relación ADP/O, b.- las velocidades de respiración en estados 3 (VR3) y 4 (VR4) y c.- el índice de control respiratorio (ICR).
La suplementación de la dieta con AP cambió la composición porcentual de los AG de los lípidos totales de las mitocondrias. El contenido de C18:2 y C20:4 disminuyeron en un 12 % (p< 0.0001) y 8 % (p< 0.0001), respectivamente; y aumentó el contenido de C22:6 en 10 % (p< 0.001). Esta suplementación no modificó la relación ADP/O, aunque sí aumentó las VR3 y VR4 en un 35 y 48 % (p< 0.0001), respectivamente.
Los cambios observados en la actividad mitocondrial pueden ser debidos a la alteración de la composición porcentual de los AG de los lípidos totales de las membranas mitocondriales, que a su vez pudiera modificar las propiedades de fluidez de la membrana y en consecuencia, las actividades enzimáticas, de transporte y permeabilidad de las mismas, ocasionando los resultados en VR3 y VR4.

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2457
Características del efecto inhibidor del INCICH-D7 sobre ATPasa-Na+,K+
Ramírez Margarita, Del Valle Leonardo, Pastelín Gustavo
Departamento de Farmacología. Instituto Nacional de Cardiología «Ignacio Chávez» México D.F., México

Introducción: La interacción de los digitálicos con su receptor la ATPasa-Na+,K+ involucra un sitio de unión para el azúcar y otro para la genina; los estudios clásicos donde se relaciona la estructura de los digitálicos con la actividad farmacológica señalan que los compuestos 14ß-hidroxiesteroides sustituidos en posición 17ß con un anillo lactónico insaturado son los que poseen mayor actividad digitálica. El INCICH-D7 [14ß,17ß-ciclocetoéster-3ß-OH androstano] (D7) compuesto sintetizado a partir de la Digitoxigenina no posee el -OH en posición 14 ni el anillo lactónico en posición 17, entre estas dos posiciones se ha logrado una fusión heterocíclica de tipo cetoéster, éste derivado retiene su actividad inotrópica positiva y posee un margen de seguridad superior al de la Digitoxigenina y al de la ouabaína.
Objetivo: Averiguar si el D7 retiene además la capacidad de inhibir a la ATPasa-Na+,K+ y conocer las características de la interacción del nuevo derivado con esta enzima.
Métodos: Trabajamos con una preparación enzimática purificada obtenida de riñón de perro. Se midió la velocidad de hidrólisis de ATP en presencia de concentraciones crecientes de Ouabaína, Digitoxigenina y D7
Resultados: El D7 inhibe a la ATPasa-Na+,K+ siguiendo un comportamiento no-competitivo, la capacidad de inhibición del D7: IC50=4µM es 5 veces más baja que la de la Ouabaína: IC50=0.8µM y 70 veces más baja que la de Digitoxigenina: IC50=0.06µM.
Conclusiones: D7 retiene la capacidad inhibidora sobre la ATPasa-Na+,K+. La sustitución del anillo lactónico y del grupo OH en posiciones 17 y 14 respectivamente por el heterociclo en la molécula de Digitoxigenina produce una significante disminución del efecto inhibidor sobre la ATPasa-Na+,K+.

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2470
Cambios inducidos por la dieta sobre la respuesta contráctil en la aorta de la rata. Efecto de la hidralazina.
Rodríguez RM, Lamanna NV, Alfonso PC, Pérez-González M.
Facultad de Medicina. Universidad Central de Venezuela. Caracas, Venezuela

La cantidad y tipo de ácidos grasos ingeridos en la dieta, son un factor de riesgo para las enfermedades cardiovasculares. Una dieta rica en ácido linoleico, disminuye los valores de presión sanguínea y es capaz de prevenir la enfermedad arterial. El objetivo del presente estudio es comparar el efecto de varias dietas enriquecidas con diferentes ácidos grasos sobre la respuesta contráctil inducida por concentraciones crecientes de norepinefrina (NE) sobre la aorta de la rata y evaluar el efecto que sobre dicha respuesta tiene la hidralazina. Fueron utilizadas 16 ratas Sprague-Dawley que se dividieron en tres grupos:

Luego de seis semanas, se sacrificaron los animales y se obtuvieron anillos de aorta de 5 mm de longitud que fueron colocados en un baño para órgano aislado (15 ml) con solución de Kreb´s’-Henseleit a 37° C (95% O2, 5% CO2). Se realizó una curva de dosis respuesta a la NE en ausencia y en presencia de hidralazina (5 x 10-4 M). En el grupo 3, hubo un desplazamiento de la curva hacia la derecha en una unidad logarítmica (p < 0,05), y una disminución del efecto máximo en presencia de hidralazina del 24% (p < 0,05), no observándose diferencias significativas entre el grupo control y el grupo 2. Estos resultados nos sugieren que una dieta rica en ácido linoleico, puede alterar la composición lipídica de la membrana, haciéndola menos susceptible a la acción de la NE. En conclusión, la composición de la dieta modifica la respuesta de la NE sobre la aorta de la rata y la hidralazina incrementa su poder relajante en ese grupo de animales.

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2325
Cardiotoxicidad en la intoxicación por cocaína
Ferrer Marrero, Daisy; Sirgo Patiño, Irene; Pérez Alvarez, Halina; Montalvo Vidal, Elisa
Instituto de Medicina Legal Ciudad de la Habana, Cuba

La toxicidad cardiovascular inducida por la cocaína es un hecho evidente. Se ha demostrado la relación entre el consumo de cocaína y los síndromes isquémicos coronarios, miocarditis, arritmias, crisis hipertensivas, cardiomiopatía dilatada y la disección aórtica. En el presente trabajo se confirman los efectos cardiotóxicos de la cocaína en tres fallecidos que ingresaron en el Instituto de Medicina Legal cuyas procedencias fueron hospitalización, domicilio y cuerpo de guardia respectivamente. Se establecieron los diagnósticos clínicos de las complicaciones no cardiovasculares en el primer caso, no así en el segundo y tercer caso dada la procedencia. Durante la necropsia, el hallazgo más relevante y común correspondió a una hemorragia pulmonar severa. El estudio con microscopía óptica reveló daño miocárdico al confirmarse en el primer caso el diagnóstico de miocarditis intersticial y en el segundo y tercer caso la existencia de un infarto agudo del miocardio. La aplicación del estudio químico toxicológico a todos los casos demostró cuali y cuantitativamente la presencia de cocaína y sus metabolitos en sangre y orina. Se discuten los resultados y se establecen comparaciones con lo reportado en la literatura revisada.

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2474
Comparación del efecto vasodilatador del cromakalim y verapamil sobre las arterias pulmonar y aorta de la rata.
Rodríguez RM, Lamanna V, Alfonso PC, Pérez-González M.
Facultad de Medicina Universidad Central. Caracas, Venezuela

El cromakalim y verapamil son agentes vasodilatadores con mecanismo de acción diferente. El presente trabajo tiene como objetivo evaluar su efecto in vitro sobre la respuesta contráctil de las arterias pulmonar (Ap) y aorta (Ao) de la rata. Se tomaron anillos de 3-4 mm de longitud provenientes de ratas macho Sprague-Dawley con un peso de 437,92 ± 5,47 y se colocaron en baños para órgano aislado (15 ml) que contenían solución de Kreb’s-Henseleit a 37º C. La contracción de los vasos se indujo con norepinefrina (NE) (1 x 10-7 M) o bien despolarizando la preparación con KCl = 40 mM. Al estabilizarse la respuesta se añadieron dosis crecientes de cromakalim y verapamil. Los resultados encontrados fueron los siguientes:

DROGA RELAJANTE (* p < 0,05)

Al estimular con NE y KCl (40 mM), la concentración que produjo el efecto máximo en ambas arterias cuando se utilizó CRM fue de 1 x 10-5 M. Al estimular con NE y KCl (40 mM), la concentración que produjo el efecto máximo en ambas arterias cuando se utilizó verapamil fue de 1 x 10-4 M. En conclusión, cuando se utiliza verapamil, la Ap depende más de la entrada de calcio por los canales operados por receptor cuando se estimula con NE, mientras que cuando se utiliza como vasodilatador el CRM, este presenta un mayor efecto en la Ao por un mecanismo que no está relacionado con la hiperpolarización de la membrana.

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2497
Chronic administration of nifedipine induces up regulation of functional calcium channels in rat myocardium.
Aiello Ernesto A, Morgan Patricio E, Chiappe de Cingolani Gladys E, Mattiazzi Alicia R, Cingolani Horacio E
Centro de Investigaciones Cardiovasculares. La Plata, Argentina

The objective of this study was to determine the effect of chronic nifedipine (NIFE) treatment (a single 10-mg oral dose/kg/day for 28 days) on rat myocardial Ca2+ channels. Ca2+ channel density was assessed by specific binding at the dihydropyridine receptors with [methyl-3H]PN 200-110 in rat ventricular membranes. Chronic NIFE treatment produced up-regulation of Ca2+ channels, being the maximal binding capacities 222±19 fmol/mg protein (n=14) and 310±21 fmol/mg protein (n=11) in untreated and treated animals, respectively (p<0.05). The functional consequences of this up-regulation of Ca2+ channels was determined in isolated ventricular myocytes by measuring L type Ca2+ currents (ICa) with the whole-cell configuration of patch-clamp technique and by intracellular Ca2+ (Ca2+i) transients estimated by the Indo-1/AM fluorescence ratio (410/482) simultaneously monitored with cell shortening. Peak ICa density recorded at 0 mV was 39.5 % greater in myocytes isolated from the treated group than in those obtained from the untreated group (-11.51±0.78 pA/pF (n=8) vs -8.24±0.69 pA/pF (n=6); p<0.05). Ca2+i transient amplitude and cell shortening were significantly higher in ventricular myocytes obtained from NIFE-treated rats than in myocytes isolated from untreated animals. Ca2+i transient and shortening were 438±59 nM and 10.3±1.2 % of resting length (Lo) in myocytes from treated rats (n=10) and 212±22 nM and 5.32±0.52 % of L0 in myocytes from control rats (n=6, p<0.05). The results demonstrate an up-regulation of functionally active cardiac Ca2+ channels after chronic NIFE treatment.

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2510
Chronic treatment with enalapril improves insulin resistance in spontaneously hypertensive rats.
Caldiz Claudia I, Chiappe de Cingolani Gladys E
Centro de Investigaciones Cardiovasculares. La Plata . Argentina

It is frequently observed the coexistence of hypertension with insulin resistance (IR). Whether the increase in peripheral resistance is the cause or the result of IR is controversial. We have compared the resistance to insulin-stimulated glucose uptake in adipocytes isolated from spontaneously hypertensive (SHR) and normotensive (WKY) rats with and without 5 weeks-treatment (in mg/Kg/day) with losartan (Los, 40), enalapril (Enal, 20) or Enal + Hoe 140 (0.150). All treatments decreased blood pressure in SHR from 180 ± 5 to 139 ± 1 mm Hg (SHR-Los), 125 ± 6 (SHR_Enal) and 143 ± 0.8 mm Hg (SHR-Enal-Hoe) (P < 0.05). Glucose uptake was measured as glucose incorporated into lipids. The adipocytes (105 cells) were incubated with 3 mM 14C-Glucose (± 0.01 to 5 nM insulin). Maximal effect (Emax) of insulin-stimulated glucose uptake (in µmoles of glucose /105 cells) was lower in SHR (0.39 ± 0.34) than in WKY (1.08 ± 0.19) (P<0.05). The Pretreatment with Los did not improve insulin responsiveness in SHR whereas the Emax in SHR-ENAL (1.02 ± 0.04) was similar to that obtained with untreated WKY. The improvement in IR induced by ENAL was abolished when the SHR were treated with ENAL and the Bradykinin (Bk)-B2 receptor blocker HOE 140. The results suggest 1) IR in SHR is not related to changes in peripheral resistance and 2) Pretreatment with the angiotensin converting enzyme inhibitor Enal improves IR in adipocytes from SHR due to an increase of B.

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2499
Contribution of Ser 16 and Thr17 to the isoproterenol-induced phospholamban phosphorylation and relaxant effect.
Mattiazzi Alicia; Mundiña Cecilia; Vittone Leticia; Said Matilde.
Centro de Investigaciones Cardiovasculares. La Plata. Argentina.

The relative contribution of Ser16 and Thr17 phosphorylation to the Isoproterenol (Iso)-induced phospholamban phosphorylation (PHL-P) and relaxant effect is not presently known. Phosphorylation site-specific antibodies to PHL and assessment of 32P incorporation into PHL were combined with measurements of mechanical parameters in isolated perfused rat hearts. 30nM Iso maximally increased PHL, Ser16 and Thr17 phosphorylation. In the presence of Iso, a decrease in [Ca]o was paralleled by a decrease in PHL-P (32 ± 3 %), exclusively produced by a decrease in Thr17 phosphorylation, and was associated to a 38% decrease of the Iso-induced relaxant effect. In the virtual absence of Ca supply to the cell, the Iso-induced PHL-P decreased by 58 ± 4%, due to the decrease in Thr17 phosphorylation, which reached basal levels. An increase in Thr17 phosphorylation (similar to that evoked by 30nM Iso) without significant changes in Ser16 phosphorylation was obtained by increasing [Ca]o under acidosis and produced 62% of the Iso-induced relaxant effect. The results would indicate that Ser16 and Thr17 phosphorylation contributed to about 50% each to the Iso-induced PHL-P and relaxant effect.

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2506
Does phosphorylation of THR17 residue of phospholamban (PHL) contribute to isoproterenol-induced PHL phosphorylation at submaximal isoproterenol concentrations?
Mattiazzi Alicia, Mundiña Cecilia, Vittone Leticia, Said Matilde, Chiappe Gladys
Centro de Investigaciones Cardiovasculares, Facultad de Medicina. La Plata, Argentina

Phospholamban (PHL) is a regulatory protein of cardiac sarcoplasmic reticulum (SR) which reversibly inhibits the activity of the SR Ca2+-ATPase. Phosphorylation of PHL by either cAMP-dependent protein kinase (PKA) at Ser16 residue or Ca2+-calmodulin-dependent protein kinase (CaMKII) at Thr17 residue, relieves this inhibition. In the intact heart, PHL is phosphorylated by isoproterenol (Iso) at Ser16 and Thr17 sites. However, the relative contribution of each phosphorylation site to the total phosphorylation of PHL induced by Iso is not known whereas the functional role of Thr17 phosphorylation remains controversial. Previous experiments have shown that phosphorylation of Thr17 contributes to approximately 50% in the total PHL phosphorylation produced by 30nM Iso. To further study this issue experiments were performed in isolated rat hearts perfused with different concentrations of Iso. Iso produced a dose-dependent increase in 32P incorporation into PHL from 49±16 (0.3nM Iso) to 225±27 (300nM Iso) pmol32P/mg protein. This increase was associated with a dose-dependent increase in the phosphorylation of Ser16 and Thr17 (Immunodetection technique) and in the Iso relaxant effect. Suppression of Ca2+ supply to the cells decreased the Iso-induced PHL phosphorylation by approximately 50% in the concentration range of Iso between 3 and 300nM. This decrease was produced by a significant decrease in Thr17 phosphorylation. The results indicate that phosphorylation of Thr17 contributes in about 50% to the total Iso-induced PHL phosphorylation and suggest its participation in the Iso-induced relaxant effect.

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2473
Efecto de bajas concentraciones de cadmio sobre la frecuencia espontánea y la fuerza contráctil de aurículas aisladas de cobayos
Alfonso P.C, Lamanna N.V, Rodríguez R.M., Pérez-González, M
Sección de Investigaciones Cardio-renales. Universidad Central de Venezuela. Caracas. Venezuela

El cadmio es más potente que el níquel y el manganeso en bloquear los canales lentos de calcio. En estudios previos demostramos que a bajas concentraciones tanto el níquel (0,2 mM), como el manganeso (0,2 mM), producen un efecto cronotrópico bimodal. Aquí describiremos los efectos de bajas concentraciones de CdCl2, sobre la frecuencia sinusal (FS) y la fuerza contráctil (FC) de aurículas aisladas de cobayos. Utilizamos cuarenta y un cobayos de 574 ± 51 g que fueron anestesiados con pentobarbital por vía intraperitoneal. Las aurículas, se colocaron en un baño de 15ml a 35° C en una solución Tyrode con Ca+2 normal (1,7 mM), bajo Ca+2 (0,85) mM o alto Ca+2 ( 9mM), pH=7,4 con 95% O2 más 5%CO2. Después de 30 min de estabilización se obtuvo la FS o FC control y se agregó el Cd+2 (0,02mM ; 0,05mM ó 0,08mM). Para estudiar la FC de las aurículas izquierdas se estimularon a 3Hz, con pulsos bipolares de 3 mseg. El Cd+2 produce un efecto cronotrópico bimodal a bajas concentraciones. A 0,02 mM indujo aceleración de FS en el primer minuto que alcanzó su máximo de 18,1% a los 5 min. Después de lavar y colocar la misma dosis de Cd+2, se observó una depresión de la FS de 77,5 ± 5,3% ( X ± DS) del control. A 0,08 mM la FS fue 10,75 ± 3,8 % por encima del control (p<0,05). y luego de 15 min la FS disminuye . A las tres concentraciones distintas de calcio se obtuvo una aceleración significativa inicial a los 3 y 5 min después de añadir Cd+2. El Cd+2 a bajas concentraciones produce un efecto inotrópico negativo. En conclusión, el cadmio a bajas concentraciones induce un efecto inotrópico negativo, presumiblemente por bloqueo de canales tipo L, pero también induce un efecto cronotrópico positivo que no se corresponde a ese mecanismo. Este cronotropismo positivo no se obtiene a dosis elevadas (0,1 mM) y quizás se relaciona con un transporte de carga a través de los canales tipo T.

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2475
Efecto de la hidralazina sobre la vasoconstricción pulmonar hipóxica en la rata
Rodríguez RM, Lamanna V., Alfonso C. y Pérez-González M
Facultad de Medicina. Universidad Central de Venezuela. Caracas, Venezuela

La vasoconstricción pulmonar hipóxica (VPH) es una propiedad que se ha desarrollado como un mecanismo de protección pulmonar que puede producir profundas consecuencias hemodinámicas, incluyendo una disminución en el gasto cardíaco, un aumento en la permeabilidad vascular pulmonar e insuficiencia ventricular derecha. La finalidad del presente trabajo es evaluar el efecto que sobre la HPV ejercen dos agentes vasodilatadores con mecanismos de acción diferentes (la hidralazina y el verapamil) y determinar si dicha respuesta requiere o no la presencia de endotelio intacto. Utilizamos ratas Sprague-Dawley (250-350 g). Se sacrificaron los animales y se obtuvieron anillos de arteria pulmonar que fueron colocados en un baño para órgano aislado (15 ml) con solución de Kreb´s –Henseleit a 37º C. En algunos experimentos se procedió a remover el endotelio de la arteria pulmonar sin dañar al músculo liso. Los experimentos mostraron que los segmentos de arteria pulmonar con endotelio expuestos a hipoxia in vitro, presentan en los primeros minutos una disminución en la tensión, seguida por un incremento en la fuerza contráctil (VPH), y luego una disminución en el tono vascular . Esta respuesta no fue observada en ausencia de endotelio. La hidralazina, fue capaz de disminuir de una manera significativa la VPH, mientras que el verapamil no. Posiblemente la hidralazina pueda eliminar o bloquear el efecto de la hipoxia sobre el sarcolema y/o el retículo sarcoplásmico (RS), disminuyendo la liberación de calcio intracelular. El hecho de no encontrar efecto del verapamil sobre la VPH sugiere que esta contracción se debe fundamentalmente a la liberación de calcio del retículo sarcoplásmico u otro depósito intracelular. En conclusión, nuestros resultados apoyan la hipótesis que la VPH está relacionada con el movimiento intracelular de calcio en la arteria pulmonar; para observar dicha respuesta se requiere del endotelio intacto.

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2454
Efecto del bloqueo de canales T y L en la liberación de óxido nítrico inducido por el flujo coronario
González Garduño Amalia Enriqueta 1; Torres Narváez Juan Carlos1; Del Valle Mondragón Leonardo1; Suárez Munguía Jorge2; Pastelín Hernández Gustavo1.
1) Departamento de Farmacología. Instituto Nacional de Cardiología «Ignacio Chávez». Ciudad de México. México. 2) Escuela Superior de Medicina, Sección de Graduados, I.P.N. Ciudad de México. México

Introducción: En el endotelio vascular se liberan una gran cantidad de sustancias bioactivas, entre las que se encuentra el óxido nítrico (ON) que es un factor de relajación importante para la regulación del tono vascular. Se sintetiza en el endotelio por medio de la óxido nítrico sintasa (ONS), la cual, presenta tres isoformas: la inducible, la endotelial y la constitutiva, esta última es dependiente de Ca++. El flujo coronario estimula la liberación de ON por un mecanismo desconocido en donde está involucrado un incremento en la concentración de Ca++.
Objetivo: Explorar la participación de los canales de Ca++ tipo «T» y «L» en la liberación de ON en respuesta al flujo.
Metodología. Se utilizó el modelo de Langendorff para corazón aislado de cobayo a diferentes velocidades de perfusión arterial en condiciones basales y con inhibidores específicos para canales de Ca ++ tipo «L» (Verapamil) y tipo «T» (Mibefradil). La cuantificación de ON se estimó midiendo la reducción de oxihemoglobina por el ON a metahemoglobina.
Resultados: El bloqueo de canales tipo «T» con mibefradil inhibió la liberación de ON. El verapamil (bloqueador de los canales tipo «L») provocó una inhibición menor. La combinación de los dos antagonistas de Ca++ potenció el efecto inhibitorio.
Conclusiones: Los antagonistas de Ca++ estudiados, inhiben la liberación de ON inducido por el flujo coronario indicando que los canales «T» y «L» están relacionados con el efecto del flujo.

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2514
Efectos de la acidosis sobre la fosforilación de fosfolamban (FL) inducida por isoproterenol (Iso): posibles mecanismos involucrados.
Mundiña Cecilia, Vittone Leticia, Said Matilde, Mattiazzi Alicia R
Centro de Investigaciones Cardiovasculares. La Plata, Argentina

La FL es una proteína del retículo sarcoplasmático (RS) cardíaco que al fosforilarse aumenta la retoma activa de Ca+2 por el RS. La FL es fosforilada por las proteínas quinasas dependientes de AMPc (PKA) y de Ca+2-calmodulina (CaMKII) en Ser16 y Thr17 respectivamente. En el miocardio la acidosis aumenta la fosforilación de FL inducida por Iso. La acidosis produce además un aumento del [Ca+2]i, que activaría a la CaMKII, y una inhibición de las fosfatasas. El presente trabajo fue diseñado con la intención de dilucidar cuáles de estos mecanismos están involucrados en el aumento de la fosforilación de FL producido por la acidosis en presencia de Iso. Se estudió el efecto de la acidosis (pHo 6.80) sobre la fosforilación de Ser16 y Thr17 de FL a distintas concentraciones de Iso en corazones aislados y perfundidos de rata. La fosforilación de FL en Ser16 o Thr17 se determinó por el reconocimiento con anticuerpos específicos y se cuantificó por densitometría óptica. La fosforilación de Thr17 fue significativamente mayor a pHo 6.80 que a pHo 7.40, para todas las concentraciones de Iso estudiadas. Dicho aumento fue de 25±6, 52±14, y 70±32 % a 3, 30 y 300 nM Iso respectivamente. En ninguna situación hubo cambios en la fosforilación de Ser16. En presencia de 30 nM Iso, el aumento de [Ca]o y la administración de ácido ocadaico 1 µM (AO), un inhibidor de fosfatasas mimetizaron el efecto de la acidosis sobre la fosforilación de Thr17 inducida por Iso. La fosforilación de Thr17 subió 84±35 % ([Ca]o alto + Iso) y 86±41 % (AO + Iso) con respecto a la fosforilación observada con Iso. El aumento de [Ca]o y el AO produjeron una disminución y un aumento respectivamente de la fosforilación de Ser16. Los resultados indican que la acidosis aumenta la fosforilación de FL inducida por Iso debido exclusivamente a fosforilación del residuo Thr17. Dicho aumento podría deberse tanto a una activación de la CaMKII como a una inhibición de la fosfatasa de FL ya que el aumento de [Ca]o y el AO mimetizan los efectos de la acidosis sobre la fosforilación de la Thr17. El hecho de que la acidosis no haya aumentado la fosforilación de Ser16 producida por Iso, como debería esperarse por una inhibición de fosfatasas, podría explicarse por el efecto opuesto que sobre dicha fosforilación tiene el aumento del [Ca2+]i que también ocurre durante la acidosis.

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2512
Efectos de la fosforilación del residuo Thr 17 de fosfolamban (FL) sobre la relajación miocárdica.
Vittone Leticia, Mundiña Cecilia, Said Matilde, Mattiazzi Alicia R
Centro de Investigaciones Cardiovasculares. La Plata. Argentina

La fosfolamban (FL) es una proteína del retículo sarcoplasmático cardíaco (RS) que inhibe reversiblemente a la bomba de Ca2+ del RS. La fosforilación de FL libera esta inhibición lo que lleva a un aumento del secuestro de Ca2+ por el RS y a una mayor relajación miocárdica. La FL se fosforila en dos residuos consecutivos, Ser16 y Thr17 por la proteína quinasa AMPc dependiente (PKA) y la Ca+2-calmodulina dependiente (CaMKII) respectivamente. El significado funcional de la fosforilación del sitio de Thr17 es poco conocido. Con el propósito de estudiar el tema se perfundieron corazones de rata aislados aumentando el [Ca]o para activar a la CaMKII, en presencia y ausencia de estimulación ß-adrenérgica. Se midió la incorporación de 32P a la FL, la fosforilación de los residuos Ser16 y Thr17 con anticuerpos específicos y la actividad mecánica. El aumento de [Ca]o en presencia de isoproterenol 30nM (Iso) aumentó la incorporación de 32P en FL de 117±18 a 236±31 pmoles32P/mg prot RS, n=7. Este incremento fue debido exclusivamente a un aumento de la fosforilación de Thr17 y se asoció con aumentos de la contractilidad y relajación miocárdicas. El aumento de [Ca]o en ausencia de Iso aumentó la fosforilación de FL sólo en presencia de intervenciones que inhiben la fosfatasa de FL, como acidosis (pHo 6.80) y ácido ocadaico (0.1µM), desde 34.5±2.5 pmoles32P/mg prot RS (control, n=6) a 56.5±8.4 (acidosis, n=5) y 83±15 (ácido ocadaico, n=8). En ambos casos la mayor fosforilación fue debida a incrementos en la fosforilación de Thr17, 38±14 % (acidosis, n=5) y 45±14 % (ácido ocadaico, n=5) y se asoció con un efecto relajante. Se concluye que la fosforilación de Thr17 de FL está asociada a un efecto relajante en el miocardio. En ausencia de estimulación ß-adrenérgica la fosforilación de Thr17 sólo se detecta cuando la activación de CaMKII ocurre simultáneamente con la inhibición de la fosfatasa de FL.

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2617
Effect of loading and heart rate changes induced by inotropic drugs on clinically used cardiac indexes
Dr. del Valle Héctor, Lic. Lascano Elena, Lic. Negroni Jorge, Dr. Crottogini Alberto
Universidad Favaloro, Instituto de Investigación en Ciencias Básicas. Buenos Aires, Argentina

Introduction: Cardiac indexes are generally influenced by loading and heart rate (HR) changes induced by inotropic drugs (ID), limiting their assessment of a purely inotropic effect. Thus, it is necessary to separately calculate the error introduced by loading and HR variations on the index evaluation of the contractile state, in order to select the index less affected for clinical purposes.
Methods: Eleven amrinone(Am)-treated and anesthetized closed-chest dogs were studied at 3-5 preloads (vena cava occlusions) in 3 afterloading conditions; basal, increased (aortic occlusion) and decreased (nitroprusside). The following indexes: ejection fraction (EF), cardiac index (CI), stroke work (SW) and maximal rate of rise of ventricular pressure (P’max) were fitted to a second order model as a function of end-diastolic volume (VD), peripheral resistance (R) and heart rate (HR). To quantify the load and HR dependance a relative error function that takes partial derivatives of each index with respect to control (no Am) VD, R and HR was used.
Results: Asuming 10% of variation for VD, R and HR, the relative errors were

ANOVA, * p < 0.05 ** p < 0.01, t test against EF.

Conclusions : Application of the model to analyze Index response to loading and HR modulation shows that EF is the Index less affected by load and HR changes and can thus be clinically employed to evaluate inotropic state after ID administration.

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2803
Effects of adenosine on postischemic dysfunction in the isolated rabbit heart
M. Donato, C. Morales, A. Bagnarelli, V. D’Annunzio, O. Scapin, R. J. Gelpi
Laboratorio de Fisiopatología Cardiovascular, Departamento de Patología, Facultad de Medicina, Universidad de Buenos Aires y Grupo de Estudios Multicéntricos en Argentina. Buenos Aires, Argentina

It is well known that adenosine lessens the systolic alterations of the «Myocardial Stunning». The aim was to determine the effect of adenosine on systolic and diastolic functions in the «Myocardial Stunning». A second goal was to determine whether adenosine modifies the release of CPK and LDH. Isolated isovolumic hearts were perfused, and subjected to 15 min of global ischemia and 30 min reperfusion (control group, G1). Adenosine was added to the perfusate at the beginning of reperfusion (group 2, G2). The left ventricular developed pressure (LVDP, mmHg), and end-diastolic pressure (LVEDP, mmHg) (diastolic stiffness) were measured, and the time constant of isovolumic relaxation (t , mseg) was calculated. The CPK and LDH were measured in the effluent.

Adenosine administered at the beginning of reperfusion, lessened systolic alteration and diastolic stiffness in the «Myocardial Stunning» without modifying the isovolumic relaxation. The release of CPK and LDH was not modified. *P<0.05 vs stunned group.

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2621
Effects of Carvedilol in patients with idiopathic dilated cardiomyopathy
Musumeci Giuseppe, Biadi Ombretta, Gigli Nicoletta, Rossini Roberta, Dell’Anna Rita, Cecchini Maurizio, Papi Laura, Mariotti Rita, Mariani Mario
Cardiotoracic Department. University of Pisa. Pisa. Italy

Objectives: the aim of this study is to evaluate clinical effects of carvedilol on NYHA class, exercise tolerance and ejection fraction of left ventricular (EF%) in patients (pts) with idiopathic dilated cardiomyopathy (DCM).
Material and Methods: Twenty-seven pts (19 M and 8 F, middle age 51,3± 5,2 years) affected by DCM, with EF < 45% and in NYHA class I-III, underwent clinical examination, ECG, Echocardiography (ECHO), and Cardiopulmonary stress test (CPX). Patients, in therapy with digoxin, diuretics and an ACE inhibitor, were randomly assigned (double blind) to either Placebo or Carvedilol. The dosage of double blind medication was initially 6,25 mg bid and was increased gradually, if tolerate, over six weeks to 25 mg bid. Thirteen pts (9 M and 4 F, middle age 50,8± 6,4 years) were treated with carvedilol (Group A) and 14 pts (10 M and 4F, middle age 52,4± 4,7 years) were treated with placebo (Group B). During the study, patient benefit was assessed periodically and ECHO and CPX were repeated at one year.
Results: Compared with placebo carvedilol therapy was associated with significant improvement in NYHA functional class (from 2,9 to 1,4 p<0,001in Group A vs 3,1 to 2,9 in Group B), in EF% (from 24,5± 3,9 to 29,7± 4,6; p<0,05 in Group A vs 25,3 to 25,8 in Group B), in Peak Oxygen consumption (VO2 peak: from 15,5 7 ± 0,8 to 19,4 ± 2,1 ml/min/Kg; p<0,001 in Group A vs 16,8 to 15,9 ml/min/Kg in Group B) and in anaerobic threshold (AT: from 19,4 2,3 to 19,4 1,9 ml/min/Kg; p<0,001 in Group A vs 12,28 to 12,01 ml/min/Kg in Group B). In Group A one patient died for brain haemorrhage (patient treated with warfarin) in Group B two pts died for sudden death.
Conclusions: This study indicates that the addiction of carvedilol to conventional therapy produces significant hemodynamic and clinical benefits in pts with DCM.

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2805
Effects of sevofluorane on postischemic dysfunction in isolated rabbit hearts
J. C. Marini, M. Donato, M. Rodríguez, R. J. Gelpi
Laboratorio de Fisiopatología Cardiovascular, Departamento de Patología, Facultad de Medicina, Universidad de Buenos Aires e Instituto de Cardiología de Corrientes «Juana F. Cabral».
Buenos Aires, Argentina

The volatile anesthetic agents Halotane, Enfluorane and Isofluorane, protect the myocardium from postischemic dysfunction, (Warltier et al. Anesthesiology, 69:552, 1988; White et al. Br. J. Anaesth. 73:214, 1994; Freedman et al. Anesthesiology 62:29, 1985). The aim of this study was to determine the effects of Sevofluorane (S), one of the newest volatile agents, on stunned myocardium. Isolated isovolumic rabbit hearts were perfused, and subjected to 15 min of global ischemia (I) and 30 min of reperfusion (R) (control group, G1). S was bubbled into the perfusate 10 min before I (group 2, G2) at 2 vol %, and discontinued during reperfusion. The left ventricular developed pressure (LVDP) and end diastolic pressure (LVEDP) (Diastolic Stiffness), were measured and the time required for pressure to fall to 50 % of its peak value was calculated (t1/2, msec) (isovolumic relaxation). Mean values ± SE.

We can conclude that, in our experimental conditions, Sevofluorane does not attenuate systolic and diastolic alterations of myocardial stunning in the isolated and isovolumic rabbit hearts.

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2500
Effects of sodium nitroprusside (NP) and isoproterenol (ISO) on phospholamban (PHL) phosphorylation in cat aorta.
Vittone Leticia, Mundiña Cecilia, Rinaldi Gustavo, Said Matilde, Chiappe de Cingolani Gladys, Mattiazzi Alicia R
Centro de Investigaciones Cardiovasculares. La Plata. Argentina

Recent experiments in PHL-deficient mice have emphasized the role of PHL in the regulation of smooth muscle contractility. However, the effect of PHL on relaxation was less clear (Circ. Res. 80: 506, 1997). In the present experiments, specific antibodies to PHL and to Ser16 and Thr17-phosphorylated PHL peptides were used to determine the effects of ISO and NP on phosphorylation of Ser16 and Thr17 residues and on mechanical relaxation of KCl-induced contractures, measured in cat aortic rings. NP produced a dose-dependent increase in phosphorylation of Ser16, which was correlated with a relaxant effect in the same aorta. 1µM NP produced a 19,4 and a 15,5 7 of the maximal relaxant effect and maximal Ser16 phosphorylation respectively. Thr17 phosphorylation was not modified by NP. Unexpectedly, 1µM Iso did not change either relaxation or Ser16 and Thr17 phosphorylation of PHL. The results indicated that in cat aorta, NP increased PHL phosphorylation by increasing Ser16 phosphorylation exclusively, an effect that was in close association with the relaxant action of NP. Iso did not affect either PHL phosphorylation or relaxation.

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2677
Effects of Sotalol in reperfusion-induced arrhythmias
Bátiz LF, Ponce Zumino A, Baiardi G, Carrión A, Verdaguer MN
Cátedra de Fisiología Normal, Facultad de Ciencias Médicas, U.N.Cuyo, Mendoza, Argentina

Reperfusion of the ischemic myocardium is responsible for the occurrence of ventricular arrhythmias, such as ventricular tachycardia (VT) and ventricular fibrillation(VF). This arrhythmias have been suggested as a possible explanation for the sudden cardiac death, one of the major causes of mortality in the western world. This fact has driven to make several researches in order to understand the electrophysiological mechanisms involved in the initiation and the maintenance of ventricular arrhythmias during ischemia and reperfusion. Such an approach may highlight potential therapies for these life-threatening arrhythmias.
Even though in the last years there have been solid results, the electrophysiological mechanisms and principally the cellular mechanisms are still not clearly identified. Many authors coincide in that this ischemia and reperfusion-induced arrhythmias represent a complex phenomena involving both reentrant and non-reentrant arrhythmias and at cellular level, the principal effects are modifications of potassium (K+) and calcium (Ca++) homeostasis: In relation to the K+, some authors point out that the reduction in intracellular ATP content activate ATP-sensitive potassium channels, with the corresponding exit of this ion toward the extracellular liquid; others sustain that alterations in the delayed outward rectifier current (IK) would also be involved in this phenomenon.
In relation to the Ca++, most of the authors coincide in that the intracellular accumulation of this ion may have an important role in initiating the arrhythmias during reperfusion favouring the delayed after-depolarizations appearance. The increase in the concentration of the citosolic calcium is owed, on one hand, to alterations in the activity of the ionic channels (ICa) and the exchangers (Na+/H+ and Na+/Ca++); on the other hand, it is secondary to catecholamine accumulation, free radical production and/or phospholipid catabolism. Summing up, the increase in citosolic calcium would act as a common final via of several factors involved in the genesis of these arrhythmias.
Most of these alterations at electrophysiological level lead to a reduced conduction velocity, an alteration of the rest potential (depolarization), a decrease in ventricular action potential duration (APD) and refractoriness (ERP) and may also be responsible for the initiation of after-depolarizations.
Because of this multiplicity of events it is that it becomes difficult to get an appropriate therapy, but it suggests that the agent should possess multiple anti-arrythmic actions. Among the drugs that could satisfy that condition, the Sotalol is. This drug, classified as a class III anti-arrythmic, has very particular actions: 1- Beta-blocking activity and 2- Capacity to prolong the repolarization (increasing the APD and the ERP) of the myocardium fibres due to an important reduction of the delayed outward rectifier current (IK) associated with a small decrease in the inward rectifier current (IK1).

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2355
Effects of the slow-releasing oral Lidocaine in suppression of ventricular arrhytmia in patients after myocardial infarction
Ioan Axente Gutiu*, Victor Voicu**, Constantin Mircioiu**, Mariana Jinga**, Laurentiu Ioan Gutiu**
* St. Mary Hospital  ** Army Research Center
Bucuresti, Romania

Background: Lidocaine is an efficient anti-arrhythmic drug used intensively on intravenous way. But our pharmacodynamic/pharmacokinetic studies on the supposed effects of oral slow-releasing Lidocaine proved that this drug can be used as a useful drug in treatment of ventricular arrhythmia, by mouth, in many clinical situations. In this preliminary study we analyse the effects of oral Lidocaine on ventricular arrhythmia in patients after myocardial infarction.
Subjects: We studied 14 patients with myocardial infarction in their recent history (the past 6 months). Mean age 62 years; 11 males, 3 women.
Method: Initially, after a 24 hours Holter monitoring, we gave to patient slow-releasing Lidocaine in tablets of 350 mg, three-times in day. In following day, we repeated the Holter monitoring and then, the tape analysis.
Results: The results in the table show the significant reduction of the ventricular premature beats, of salvos and of ventricular tachycardia episodes.

We did not noted important side effects of this treatment, only moderate dizziness in 2 patients, at 25-30 minutes after the second dose.
Conclusion: This preliminary study show that oral slow-releasing Lidocaine is efficient in treatment of the ventricular arrhythmia in the patients in recent stage after myocardial infarction. However, is necessary a more large clinical study with the serum determination of the drug for establishing of the all therapeutic capacity.

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2520
Enalapril treatment improves insulin sensitivity in spontaneously hypertensive rats
Chiappe de Cingolani Gladys, Caldiz Claudia I, Palacios Rosa A
Centro de Investigaciones Cardiovasculares. La Plata. Argentina

A decrease of insulin sensitivity has been found to be a common feature of essential hypertension. Whether hypertension decreases insulin sensitivity or the former is the consequence of insulin resistance is still unresolved. We have studied the effect of insulin on glucose uptake in adipocytes from spontaneously hypertensive (SHR) and normotensive (WKY) rats untreated and treated with 25 mg/kg/day of enalapril for 5 weeks. Enalapril decreases blood pressure in SHR from 182.9 ± 3.2 to 131.1 ± 4.4 mmHg. Glucose uptake was measured in adipocytes incubated with and without 0.1 to 5 nM insulin in the presence of 0.1 mM 3-O-[methyl-3H]glucose (glucose transport) or 3 mM [14C]glucose (lipogenesis). In untreated animals the effect of insulin either on glucose transport or lipogenesis was lower in SHR than in WKY. At 5 nM insulin, the increase in glucose transport was 632 ± 83 (WKY) and 369 ± 69 (SHR) pmoles/g dry wt (P < 0.05) and the increase in lipogenesis 0.905 ± 0.110 (WKY) and 0.435 ± 0.128 (SHR) mmoles/ g dry wt (P < 0.05). When treated with enalapril, glucose uptake in response to insulin in SHR and WKY was similar to untreated WKY. The increase in lipogenesis at 5 nM insulin was 1.090 ± 0.244 (SHR-enalapril) and 1.183 ± 0.295 (WKY-enalapril). The results show that decreasing blood pressure with enalapril-treatment returns insulin sensitivity to normal values in adipocytes from SHR.

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2472
Estudio del efecto de bajas concentraciones de níquel y bario sobre la frecuencia espontánea y la fuerza contráctil de aurículas aisladas de cobayos
Alfonso PC, Lamanna NV, Rodríguez RM, Pérez-González M
Universidad Central de Venezuela. Facultad de Medicina. Sección de Investigaciones cardio-renales. Caracas. Venezuela

El manganeso, el níquel y el cadmio se han utilizado a concentraciones mayores de 1 mM para bloquear los canales lentos de calcio. Estudiaremos la acción de bajas concentraciones de NiCl2 y BaCl2 sobre la frecuencia sinusal (FS) y la fuerza contráctil (FC) de aurículas aisladas de cobayo. Ambas aurículas se colocaron en un baño de órgano con solución de Tyrode a 35°C y burbujeada con 95%02, 5%C02. Para estudiar FC se montaron aurículas izquierdas y se estimularon a 3 Hz. El Ni+2 0,2 mM aumentó la FS hasta 25% por encima del 25% por encima del control en los primeros minutos, para luego volver a valores control en minutos. Una concentración de 0,8 mM causó aumento de 18,5% por encima del control a los tres minutos y luego disminuyó por debajo del control. El efecto inotrópico del Ni+2 (0,2 mM) se mantuvo en presencia de propranolol (1 x 10-5 M) y de verapamil (3 x 10-7 M). También se mantuvo en presencia de[Ca+2]e= 0,85 mM. En cambio, con [Ca+2]e= 9 mM no hubo aumento de FC. Al añadir 0,2 mM de Ni+2 se incrementó la FC en los 3 minutos pero luego esta cayó por debajo del control. De manera que el Ni+2 a bajas dosis produce inicialmente un efecto cronotrópico e inotrópico positivo, que persiste en presencia de propranolol o verapamil y podría ser debido al paso de un ion a través de los canales lentos, ya que, la [Ca+2]e= 9 mM elimina el efecto cronotrópico positivo del Ni+2. El Ba+2 (0,2 mM) produjo una pequeña pero significativa caída de la FS (13,5%); a 0,8 mM esta caida fue mayor (34%). Sobre la FC hubo un marcado efecto: esta aumentó en un 100 y un 140% por encima del control (0,2 y 0,8 mM respectivamente). El efecto inotrópico ha sido descrito, pero no el efecto cronotrópico negativo, este podría explicarse por la disminución de la If o el intercambiador Na+ / Ca+2.

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2452
Estudio farmacológico de un digitálico sintetizado a partir de la digitoxigenina
Zarco Gabriela, Del Valle Leonardo, Pastelín Gustavo
Departamento de Farmacología, Instituto Nacional de Cardiología «Ignacio Chávez». México, México

Introducción: Los digitálicos aumentan la contractilidad del miocardio (inotropismo positivo) y son de utilidad en el tratamiento de la insuficiencia cardíaca. Estos, poseen un hidroxilo en el carbón 14 del núcleo esteroidal y un anillo de lactona en el carbón 17, los cuales se consideran importantes para la actividad del digitálico. Así, estudiamos la relación que existe entre la estructura química y la actividad biológica de un derivado sintético de la digitoxigenina en donde se hizo reaccionar entre si a la lactona y al hidroxilo, de tal manera que ambas estructuras dieron origen a un cetoéster que cicliza la posición 14 y 17 de la base digitoxigénica, compuesto que posteriormente fue glucosilado en el carbón 3 esteroidal, dando finalmente el producto 14b,17b-cicloester-3b-D(+)-glucopiranosa-androstano, denominado INCICH-D7G. Este compuesto se ensayó en corazón de perro en la preparación cardiopulmonar de Starling. Se indujo insuficiencia cardíaca con una dosis controlada de 100 mg de pentobarbital, para estudio del efecto de reversibilidad de la insuficiencia por el INCICH-D7G. Este compuesto se aplico en infusión continua (0.09 mmoles/Kg de corazón/min). Se midió el gasto cardíaco y se obtuvieron las dosis: mínima terapéutica, de intoxicación y letal.
Resultados: Se ha observado que con el glucósido sintético la regresión de la insuficiencia cardíaca es más rápida que con la digitoxigenina y su margen de seguridad aumenta significativamente en 4 veces.
Conclusiones: Estos resultados nos permiten concluir que la modificación estructural realizada en la digitoxigenina, aunado a la glucosilación en el carbón-3, acrecentan significativamente el índice terapéutico.

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2517
Evidences for an electrogenic Na +/HCO3- cotransport in rat cardiac myocytes
Aiello Ernesto A, Vila Petroff Martín G, Mattiazzi Alicia R, Cingolani Horacio E
Centro de Investigaciones Cardiovasculares. La Plata, Argentina

Perforated whole-cell configuration of patch clamp and the pH fluorescent indicator SNARF were used to determine the electrogenecity of the Na+/HCO3- cotransport in isolated rat ventricular myocytes. Switching from HEPES- to HCO3--buffer at constant extracellular pH (pHo) hyperpolarized resting membrane potential (Em) by 3.03±0.52 mV (n=7, p<0.05). In the presence of HCO3-, the anion blocker, SITS (0.1 mM), depolarized Em by 3.05±0.75 mV (n=3, p<0.05). No HCO3- induced hyperpolarization was observed in the absence of extracellular Na+. The duration of action potential measured at 50% of repolarization time (APD50) was 31.8±6.8% shorter in the presence of HCO3- than in its abscence (n=4, p<0.05). Quasi-steady-state currents were evoked by 8 second duration voltage-clamped ramps ranging from -130 to +30 mV. The development of a novel component of SITS sensitive steady-state outward current was observed in the presence of HCO3-. The Na+/HCO3- cotransport current (INaBic) was studied in isolation in standard whole-cell experiments. INaBic reversed at -96±1.9 mV, consistent with the influx of 2 HCO3- ions per Na+ ion by the Na+/HCO3- cotransporter. Depolarizing Em with 70 mM extracellular K+ caused a significant pHi alkalinization (0.24±0.02 pH units; n=4, p<0.05) in the presence of external HCO3- while no change in pHi was observed in HEPES (0.02±0.01 pH units; n=4, NS). Whereas increments in stimulation frequency from 0.25 to 1.5 Hz induced a decreased in pHi of 0.041±0.006 pH units (n=7, p<0.05) in HEPES, a slight alkalinization was detected in the presence of external HCO3- (0.023±0.003 pH units; n=8, p<0.05). These data are in agreement with an increase in Na+/HCO3- current driving force induced by the repetitive depolarizations. The above results allow us to conclude that the Na+/HCO3- cotransport is electrogenic and has an influence on Em and APD of rat ventricular cells.

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2522
Immunodetection of phosphorylation sites of phospholamban (PHL) in aortic smooth muscle. Effects of sodium nitroprusside (NP)
Vittone Leticia, Mundiña-Weilenmann Cecilia, Rinaldi Gustavo, Said Matilde, Chiappe de Cingolani Gladys, Mattiazzi Alicia R
Centro de Investigaciones Cardiovasculares. La Plata. Argentina

The importance of PHL phosphorylation in the regulation of myocardial contractility and relaxation is well established. Recent experiments have emphasized that PHL modulation of the sarcoplasmic reticulum Ca-ATPase (SERCA) may also play a major role in the regulation of contractility and relaxation of smooth muscle (Am J Physiol 80:506, 1997). Furthermore, several types of evidence in vascular smooth muscle suggested that activation of cGMP-dependent protein kinase has a pivotal role in the decrease of [Ca]i by activation of SERCA via phosphorylation of PHL. In the present experiments, specific antibodies to PHL and to Ser16 and Thr17-phosphorylated phospholamban peptides were used to determine in cat aorta streeps 1) the presence of PHL and 2) the effect of increasing NP concentration (1.5 x 10-10 to 1.5 x 10-4M) on phosphorylation of Ser16 and Thr17 residues of PHL. These biochemical measurements were compared with the mechanical assessment of the effect of NP on the relaxation of KCl-induced contractures. The results demonstrated that PHL is present in cat aorta. In addition NP produced a dose-dependent increase in phosphorylation of Ser16, which was correlated with NP-induced relaxant effect, i.e. 1.5 µM NP evoked a 52.5±3.5 % increase in Ser16 phosphorylation and produced a relaxant effect of 51.5 ± 9.48 %. Thr17 phosphorylation was not affected by NP. Conclusions: In cat aorta, NP increases PHL phosphorylation by increasing Ser16 phosphorylation exclusively. This effect is in close association with the relaxant effect of NP.

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2722
Importance of the electromechanical uncoupling point in the volatile cardioplegy induced by halothane
Gomes OM, Neves HJ, Gomes ES, Carvalho JI
Fundaçâo Cardiovascular Sâo Francisco de Assis ServCor/HSFA. Brazil

Sixteen wistar rats were anesthetized by ether inhalation and their hearts were perfused in a Langendorff system with Krebs-Henseleit solution (36 Cº; 90 cm H2O pressure). After a 15- min period for stabilization the control values for heart rate, force (T) and dT/dt were recorded and a halothane-enriched (8%) solution (same temperature and pressure) was perfused until cardiac standstill with electromechanical uncoupling (Group I) or total cardiac arrest was obtained (Group II). The same Krebs-Henseleit solution was reperfused and the parameters recorded after 2, 5, 10 and 15 min. In the Group I hearts the following values (mean) were obtained regarding the heart rate: control, 274.3+-22.6; uncoupling, 221.7+-43.1 (p<0.001) and 266.7+-24.9, 269.3+-19.4, 270.6+-19.7 and 271.6+-19.7 bpm in the 2,5, 10 and 15-min, respectively (p>0.05). In the Group II hearts, excepting for the total arrest period all others values obtained for the heart rate were not statistically different from the control. The force and the dT/dt parameters were not statistically different in the control and uncoupling standstill periods between both Groups, but in the recovery periods the Group II hearts were unable to recover their normal mechanical performance, five hearts being arrested in the 15- min recovery period. These data demonstrated that the electromechanical uncoupling point was the maximum effect compatible with safe normothermic cardioplegy induced by halothane, suggesting that the blockade of the ryanodine calcium channels system of the sarcoplasmic reticulum can be the main site of action of the halothane when depressing the cardiac contractions.

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2501
Increased L type Ca 2+ current in rat ventricular myocytes after suppression of chronic treatment with nifedipine
Aiello Ernesto A, Cingolani Horacio E
Centro de Investigaciones Cardiovasculares. La Plata, Argentina

Despite the wide use of Ca2+ channel blockers in the treatment of hypertension, the chronic effects of these compounds on cardiac function is controversial. The goal of the present study was to determine the effect of suppression of nifedipine (NIFE) treatment (one 10 mg oral dose/Kg/day for 28 days) on L type Ca2+ current (ICa) of rat ventricular myocytes. Previous experiments from our lab demonstrated an up-regulation of Ca2+ channels assessed by specific binding at the dihydropiridine receptor with [methyl-3H]PN 200-110 in ventricular membranes from NIFE-treated rats (Circulation 98:I-820, 1998). The functional role of this up-regulation of Ca2+ channels was determined by measuring ICa with the whole-cell configuration of patch-clamp technique. Peak ICa density recorded at 0 mV was 30 % greater in myocytes isolated from the treated group than those obtained from the untreated group (-8.75±0.75 pA/pF (n=7) vs -6.11±0.70 pA/pF (n=7); p<0.05). Neither the kinetics of activation or inactivation nor the voltage-dependence of steady-state inactivation of ICa were altered by the NIFE-treatment. The latter results suggest the absence of remaining NIFE in the pore of the Ca2+ channels present in the isolated myocytes after 18 hours of suppression of the chronic NIFE-treatment. The results demonstrate an up-regulation of functionally active cardiac Ca2+ channels after NIFE-treatment and offer a possible explanation for a «withdrawal effect» at myocardial level after suppression of the treatment with this drug.

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2723
Influence of propafenone and procainamide on the contractile activity of the rat’s latissimus dorsi muscle isolated in organ chamber
Gomes OM, Simões R, Machado ELG, Freitas OGA
Fundaçâo Cardiovascular Sâo Francisco de Assis. ServCor/HSFA. Brazil

This study was designed to evaluate the effect of the propafenone and procainamide in the contractile force of the Latissimus dorsi muscle, nowadays used to treat heart failure in the cardiomyoplasty procedure.
Three groups of ten muscles were isolated from Whistar rats and studied in organ chamber, being: Group I - Control; Group II, with procainamide (71,2 ug/ml) added in the bath; Group III, with propafenone (9.8 ug/ml) added.
The following mean results were obtained regarding contraction tensions observed: Group I, 2.78g; Group II, 2.75g; Group III, 2.75g, in the first period (control). Group I 2.75 and 2.68g; Group II 2.43 and 1.98; Group III 2.21 and 1.86 g, in the second and third period of study, respectively.
Based on statistical analysis of the results it is concluded that both procainamide and propafenone have a depressing effect upon the contractile force of the Latissimus dorsi muscle of rats.

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2724
Influence of the myocardium contractile state in the coronary flow deviation phenomenon
Gomes OM, Fiorelli AI, Brum JMG, Gomes ES, Moraes NLTB
Fundaçâo Cardiovascular Sâo Francisco de Assis. ServCor/HSFA. Brazil

Twenty-four mongrel dogs were anesthetized and submitted to normothermic extracorporeal circulation and to selective perfusion of the coronary arteries with flows of 10.0,5.0 and 2.5 ml/kg body weight/min. A catheter was introduced inside the coronary sinus, in a 2.0 to 2.5 cm extension, for the flow measurements. The following conditions where investigated: Group I - with hearts beating spontaneously; Group II - under ventricular fibrilation; Group III - under stimulation by isoproterenol (200 meg); Group IV - under artificial estimulation by pacemaker. Results in Group I hearts demonstrated an inverse relationship between the coronary arterial flow and its proportional drainage in coronary sinus. This phenomenon was not altered (p<=0.05) in Group II (fibrilating) and IV (pacemaker, 80-180 bpm). In Group II hearts, under isoproterenol stimulation an inversion of the above pattern occurred with linear increasing of the coronary sinus proportional drainage with the coronary arterial flow augmentation. It is concluded that the coronary flow distribution pattern is altered by the improvement in myocardial contractility induced by isoproterenol.

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2606
Inhibition of agonist-induced rises in internal calcium in human endothelial cells by Ginkgo biloba extract (EGb 761) and rolipram
Campos-Toimil Manuel, Lugnier Claire, Takeda Kenneth
Pharmacologie et Physico-Chimie des Interactions Moleculaires et Cellulaires CNRS, UMR 653491
Université Louis Pasteur Strasbourg, France

EGb 761 is a standardized extract which has been shown to have complex and multiple cardiovascular effects, including increases in peripheral and cerebral blood flow and microcirculation, inhibition of platelet aggregation and a decrease in capillary permeability. It also relaxes spasmatic blood vessels and contracts those that are abnormally dilated. In the present work, the effects of EGb 761 on five isolated vascular cyclic nucleotide phosphodiesterase (PDE) isoforms were evaluated. EGb 761 preferentially inhibits PDE4 (IC50 = 25±3 mg/L), the isoform which is mainly present in endothelial cells, in a competitive manner (Ki = 12.5 mg/L). Since changes in cyclic nucleotide levels may affect intracellular calcium levels ([Ca2+]i) in endothelial cells, we examined the effects of EGb 761 on both resting [Ca2+]i levels and agonist-induced rises in [Ca2+]i in single human umbilical vein endothelial cells (HUVEC) in culture. The effects of EGb 761 were compared to those of rolipram, a selective PDE4 inhibitor which increases cellular cAMP levels, and the cAMP analogue dibutyryl-cAMP (db-cAMP). EGb 761 (20, 100 mg/L), rolipram (50 µmol/L) and db-cAMP (100 µmol/L) significantly inhibit histamine-, ATP- and thrombin-induced [Ca2+]i increases in HUVEC, without modifying resting [Ca2+]i levels. Similar results were obtained using a Ca2+-free bathing solution. EGb 761 (100 mg/L), but not rolipram (50 µmol/L) or db-cAMP (100 µmol/L), also inhibits the capacitative Ca2+ entry in cells having thapsigargin-depleted internal Ca2+ stores bathed in Ca2+-free external solution. Our results are consistent with an inhibition of PDE activity causing a reduction of agonist-induced increases in [Ca2+]i in HUVEC, mainly by inhibition of Ca2+ mobilization from internal stores. Thus, it may also be that the cardiovascular effects of EGb 761 involve an inhibition of PDE4 activity, and a subsequent modification of Ca2+ signaling in endothelial cells.

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2513
Mechanism of the increase in contractility and calcium transient produced by chronic nifedipine (NIFE) treatment
Patricio E. Morgan, Alicia R. Mattiazzi, Gladys Chiappe de Cingolani, Horacio E. Cingolani
Centro de Investigaciones Cardiovasculares. La Plata, Argentina

Previous reports of our laboratory have demonstrated that chronic treatment with nifedipine (Nife), a dihydropyridine receptor blocker produced an up-regulation of rabbit and rat cardiac L type calcium channels and an enhancement of contractility. The aim of the present study, was to further investigate whether this increase in contractility was due to either an increase in intracellular calcium or in calcium myofilament responsiveness. To this purpose experiments were performed in isolated ventricular myocytes of rats chronically treated with Nife (10 mg/Kg/day for 28 days). Intracellular calcium transients (CaiT) estimated by the Indo-1 fluorescence ratio (410/482 nm), were simultaneously monitored with the cell shortening, (CS) expressed as % of resting length (Lo). Both CaiT and CS were significantly higher in ventricular myocytes obtained from Nife-treated rats (367 ± 55 nM and 10.31 ± 1.2% of Lo respectively) than in myocytes isolated from untreated animals (184 ± 19 nM and 5.32 ± 0.52% of Lo respectively; n=6, p<0.05). Possible changes in calcium myofilaments responsiveness induced by chronic Nife treatment were explored by studying the relationship between CS and CaiT in both groups of myocytes at two different extracellular calcium. The myocytes from treated and non treated rats follow a similar linear relationship. The results indicate that the chronic treatment with Nife enhances myocytes contractility by increasing intracellular CaiT without significant alterations in calcium myofilaments responsiveness.

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2503
Mechanisms involved in the relaxant effect of sodium nitroprusside (NP) on cat aorta smooth muscle
Vittone Leticia, Rinaldi Gustavo, Mundiña Cecilia, Said Matilde, Chiappe Gladys, Mattiazzi Alicia
Centro de Investigaciones Cardiovasculares. Facultad de Medicina. La Plata Argentina

Different mechanisms have been hypothetized to account for guanosine 3’,5’-cyclic monophosphate (cGMP)-mediated relaxation of arterial smooth muscle. The present experiments attempt to dissect among these mechanisms, the relative role of sarcoplasmic (endoplasmic) reticulum (SR) Ca2+-pump (SERCA2) and of phospholamban, a protein that when phosphorylated, enhance the activity of SERCA2, in the relaxant effect of NP in cat aortic strips.. 100 µM NP completely relaxed the norepinephrine (Ne) -induced contractures ( 98 ± 1.8% ) but relaxed by only 40.5 ± 4 % the contractures induced by increasing the external KCl. Similar results to those obtained with high KCl were obtained in the Ne -induced contractures in the presence of tetraethylamonium (TEA) used to block K+ channels. These results indicated that NP-induced K+-channels activation would account for almost 60% of the total NP-induced relaxant effect. 1µM thapsigargin (TH) used to inhibit SERCA2, significantly attenuated the relaxant effect of NP in KCl-induced contractures. At each NP concentration, the difference between the NP induced relaxant effect in the absence and in the presence of TH would represent the participation of the SR in the NP-induced relaxant effect. This difference was 22 % of the total relaxant effect evoked by 100 µM NP. At all NP concentrations, the NP-relaxant effect attributed to the SR was closely associated to an increase in phosphorylation of Ser16 residue of PHL from 35 98 % of basal values at 0.01 µM NP to 144 ± 42% at 100 µM NP.
Conclusions: In cat aorta smooth muscle, phosphorylation of PHL would contribute to approximately 22 % to the total relaxant effect induced by 100 µM NP, whereas . 59% of this effect would correspond to K+ channels activation. The remainder relaxant effect might be due to an increase in Ca2+ efflux or a decrease in Ca 2+ influx or in Ca2+ myofilament responsiveness.

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2746
Mecanismos de la acción vasoselectiva de dos nuevas dihidropiridinas: Elgodipina y Oxodipina
Galán Loipa, Talavera Karel, Vassort Guy, Alvarez Julio
Instituto de Cardiología y Cirugía Cardiovascular La Habana, Cuba U-390 INSERM Montpellier, Francia

Introduction: There is still a need for more vascular selective calcium antagonists due to the potentially life-threatening effects of their cardiac depressant action. Elgodipine and oxodipine are second generation dihydropyridines that are known to strongly depress smooth muscle contraction. However, their mechanisms of action on calcium channels have not been fully characterized.
Objectives : The present study was undertaken to compare the pharmacological properties of elgodipine and oxodipine and to characterize their mechanisms of action on calcium channels in isolated rat ventricular cardiomyocytes.
Material and Methods : Contractions were recorded on KCl-depolarized rat and rabbit abdominal aortic rings and on electrically-stimulated rat right ventricular strips. Patch-clamp recordings of the whole-cell calcium currents were done in rat cultured neonatal ventricular myocytes and in enzymatically rat isolated adult ventricular myocytes.
Results: Elgodipine and oxodipine depressed aortic contraction with stronger potency than cardiac contraction. The vascular selectivity index (IC50 ventricular/IC50 aorta) was greater for elgodipine (680) than for oxodipine (72). Elgodipine and oxodipine decreased the L-type Ca current (ICaL) of neonatal ventricular myocytes with IC50 of 0.33 and 0.24 µM respectively. Oxodipine was mor _e potent on the T-type Ca current (ICaT) than elgodipine (IC50 = 0.41 vs 2.18 µM). Both compounds were less potent in inhibiting ICaL of adult cardiomyocytes. Elgodipine and oxodipine increased their potency of action on ICaT and ICaL by at least one order of magnitude when the cells were partially depolarized. Elgodipine in addition exhibited a frequency-dependent block of both currents.
Discussion and Conclusions: Both dihydropyridines are highly vascular selective and the underlying mechanism of this vascular selectivity is the voltage-dependent block of calcium currents. In the case of elgodipine, which shows a vascular selectivity higher than that of oxodipine, its use-dependent inhibition of calcium currents could also contribute to this action.

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2557
Papel de los canales iónicos que se activan por estiramiento y del calcio en la síntesis de óxido nítrico en corazón aislado
Torres Juan Carlos*, Del Valle Leonardo*, Pastelín Gustavo*, Suárez Jorge**
* Instituto Nacional de Cardiología «Ignacio Chávez» ** Escuela Superior de Medicina Sección de Graduados I.P.N. Ciudad de México. México

El estrés mecánico generado por el flujo sanguíneo, estimula a la célula endotelial para liberar sustancias constrictoras y relajantes, entre las que se encuentra el óxido nítrico (ON). En la circulación coronaria no se conoce el mecanismo por el cual la liberación de este gas sea inducida por los estímulos mecánicos que genera el flujo sobre la célula, ni tampoco es muy claro el mecanismo de transducción de esos estímulos para la regulación del tono vascular. Se sabe que la síntesis de ON es a través de la óxido nítrico sintasa cuya isoforma constitutiva es dependiente de calcio, mientras que la isoforma inducible es independiente de calcio.
El objetivo de este trabajo es explorar una de las vías de entrada de calcio a la célula: los canales iónicos que se activan por estiramiento (CIAE), analizando su participación en la síntesis de óxido nítrico en el corazón aislado así como la participación de los canales de calcio tipo T y L.
Metodología. Utilizamos el método de Langendorff para corazón aislado de cobayo. La cuantificación de óxido nítrico se realizó por conversión de oxihemoglobina a metahemoglobina a diferentes velocidades de perfusión, en condiciones control y con aplicación de gadolinio para bloquear los CIAE y bloqueadores específicos para canales de calcio tipo T y tipo L.
Resultados. Con el bloqueo de CIAE se disminuye significativamente la concentración de ON, mientras que el bloqueo de los canales tipo T y tipo L por separado, generan disminución en la concentración de ON que no es tan potente como con gadolinio.
Conclusión. Los CIAE tienen un papel importante en la entrada de calcio a la célula y por lo tanto en la regulación de la síntesis de ON. Estos fenómenos son en función del estrés mecánico sobre la célula endotelial generado por el flujo.

Tope

2498
pH-independent increase in myofilament responsiveness to calcium mediates angiotensin II-induced positive inotropic effect in feline myocardium
Vila-Petroff Martín G, Aiello Ernesto A, Morgan Patricio E, Cingolani Horacio E, Mattiazzi Alicia R
Centro de Investigaciones Cardiovasculares. La Plata, Argentina

Angiotensin II (ANG II) exerts potent positive inotropic actions in several species. However, the subcellular mechanisms involved are still a matter of major controversy. The present study examines the intracellular calcium (Cai) and pH (pHi) dependence of the ANG II-induced positive inotropic effect in isolated cat ventricular myocytes. For this purpose, pHi (via SNARF/AM fluorescence) and Cai (via INDO-1/AM fluorescence) were measured simultaneously with cell shortening. In HEPES buffer (n=4) there was a temporal dissociation between the positive inotropic effect and the alkalinizing effect of ANG-II (1 µM). While the positive inotropic effect of Ang II reached its maximum approximately 10 minutes after administration of the drug (82±7% above control shortening), no changes in pHi were detected during this time interval. The increase in pHi became significant (0.04±0.01 pH units) only after 20 min of ANG II incubation, when the positive inotropic effect was already fading. In spite of increasing contraction amplitude, ANG II did not increase Cai transient amplitude (CaiT) (n=6). A similar increase in contraction amplitude produced by increasing extracellular [Ca]o increased CaiT by 63,7±7.6 above control (n=4). No effect of ANG II on peak L-type calcium current (ICa) density was observed using the patch clamp technique in the whole cell configuration. The values measured at +10 mV were -5.4±1pA/pF in control and -4.71±1pA/pF after 10 min of ANG II (n=4). We conclude that in feline cardiac myocytes the initial increase in contractile response evoked by ANG II, observed in the absence of changes in CaiT, ICa and pHi, is mainly mediated by a pH-independent increase in myofilament responsiveness to calcium. Whether the increase in myofilament responsiveness to calcium is due to changes in maximal force or to changes in myofilament calcium sensitivity is under current investigation.

Tope

2504
Phosphorylation of Ser 16 is not a pre-requisite for Thr17 phosphorylation of phospholamban in vivo
Mundiña Cecilia, Said Matilde, Vittone Leticia, Mattiazzi Alicia R
Centro de Investigaciones Cardiovasculares. La Plata, Argentina

Phospholamban (PHL), the cardiac regulator of the sarcoplasmic reticulum Ca-ATPase is phosphorylated at Ser16 by the cAMP-dependent protein kinase (PKA) and at Thr17 by the Ca2+-calmodulin dependent protein kinase (CaMKII) during isoproterenol (Iso) stimulation of the heart. It has been shown that phosphorylation of Thr17 is independent of Ser16 phosphorylation in vitro but it is controvertial whether similar results could be obtained in vivo. Experiments using transgenic mice, lacking Ser16 site, suggest that phosphorylation of Thr17 appears to require prior phosphorylation of Ser16 in vivo. To elucidate this issue experiments were performed in which rat hearts were perfused at high [Ca]o (3.85 mM) to activate CaMKII pathway in the absence or the presence of phosphatase inhibition (1 µM okadaic acid or low pH (pH 6.8)). In the absence of phosphatase inhibition, phosphorylation site-specific antibodies to PHL were not able to detect phosphorylation of either Thr17 or Ser16 residues. In the presence of phosphatase inhibition (pH 6.8), phosphorylation of Thr17 increased 73.80 ± 11.60 % (n=4) without significant changes in Ser16 phosphorylation (results are expressed as percentage of maximal site-specific phosphorylation obtained with 30 nM Iso). In order to abolish basal Ser16 phosphorylation, an inhibitor of PKA (1 µM H-89) was included when [Ca]o was increased under acidotic conditions. Again Thr17 was the only site phosphorylated (80.33 ± 14.64 %, n=3). The results suggest that Thr17 phosphorylation could be detected independently of phosphorylation of Ser16 in vivo.

Tope

2521
Phosphorylation of Thr 17 residue of phospholamban (PHL). New insights into the physiological role of CaMKII pathway of PHL phosphorylation
Mattiazzi Alicia, Vittone Leticia, Mundiña-Weilenmann Cecilia, Said Matilde
Centro de Investigaciones Cardiovasculares. La Plata. Argentina

The physiological meaning of Thr17 phosphorylation of PHL remains uncertain. To study this issue experiments were performed in isolated Langendorff perfused rat hearts in which phosphorylation site-specific antibodies to PHL and quantitative assessment of 32P incorporation into PHL were combined with measurements of mechanical parameters. Increasing [Ca]o to activate CaMKII pathway in the presence of 30 nM isoproterenol (Iso), increased 32P incorporation into PHL. This increase was exclusively due to an increase in Thr17 phosphorylation and was associated to a significant positive inotropic and relaxant effect in agreement with our own previous findings (J Biol Chem 271:33561, 1996). Increasing [Ca]o in the absence of Iso increased PHL phosphorylation only at low extracellular pH (6.80) or when okadaic acid (OA, 0.1-1 µM) was present. These two interventions (acidosis and OA) inhibit PHL phosphatase. In both cases, the increase in PHL phosphorylation was exclusively due to an increase in Thr17 phosphorylation which was 42.2±15 % (acidosis, n=5) and 50.4±12.4 % (OA, n=6) of the maximun increase obtained with 30 nM Iso. This increase in Thr17 phosphorylation was associated to a significant relaxant effect. Conclusions: 1) Phosphorylation of Thr17 residue of PHL has a physiological role being responsible of part of the relaxant effect of Iso. 2) Phosphorylation of Thr17 residue is associated to a relaxant effect even in the absence of Iso. This phosphorylation seems to require simultaneous activation of kinases and inhibition of phosphatases.

Tope

2502
Second phase of force increase after stretch is due to the activation of Na+/H+ exchanger in rat myocardium
Pérez Néstor G, Alvarez Bernardo V, Camilión de Hurtado María C, Cingolani Horacio E
Cent