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Angiotensin II Stimulates L-Type Calcium Ca2+
Currents (ICa) of Cat Cardiac Myocytes
Via a pHi-Independent and PKC
and Ca2+i-Dependent Mechanism.
A Perforated-Patch vs Whole-Cell Study

E. A. Aiello, H. E. Cingolani

Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas,
Universidad Nacional de La Plata, La Plata, Argentina

RESUMEN

SUMMARY
Angiotensin II (Ang II) evokes positive inotropic responses in various species. However, the effects of this peptide on ICa and Ca2+ transients are still controversial. We report in this study that the effects of Ang II on ICa differs depending on the mode of patch-clamp technique used, standard whole-cell (WC) or perforated-patch (PP). Whole-cell currents were evoked by pulses to 0 mV (250 ms) delivered at 0.2 Hz from a holding potential of -80 mV followed by a 500 ms prepulse to -40 mV. No significant effects of Ang II were observed when WC was used (-5.5±0.6 pA/pF in control vs -4.4±0.5 pA/pF in Ang II, n=24, NS). However, when the intracellular milieu was preserved using PP, Ang II (0.5 µM) induced a significant 77±6 % increase on ICa that was reversed by the AT1 receptor antagonist, Losartan (2 µM) (-2.2±0.3 pA/pF in control, -3.9±0.6 pA/pF in Ang II and -2.7±0.4 pA/pF after the addition of Losartan, n=8, p<0.05). Since the increase in ICa after Ang II as a result of activation of the Na+/H+ exchanger (NHE) and the subsequent intracellular alkalization was previously reported, we performed experiments where the cell were pretreated with the NHE inhibitor, HOE 642 (1 µM). The presence of this blocker did not prevent the increase on ICa induced by Ang II (71±7 %). Since Ca2+i was strongly chelated when WC was used, we then dialized the cells with low Ca2+ buffer capacity (EGTA 0.1 mM). Under these conditions, Ang II was able to induce increase on ICa (-3.2±0.3 pA/pF in control vs -4.3±0.3 pA/pF in Ang II, n=9, p<0.05). This increase was prevented when the cells were also dialized with the PKC inhibitors Chelerythrine (50 µM) (-4.6±0.5 pA/pF in control and -4.6±0.6 pA/pF in Ang II, n=7, NS) or Calphostin C (1 µM) (-4.1±0.6 pA/pF in control and -4.2±0.8 pA/pF in Ang II, n=6, NS). Similarly to the phenomenon recently described for a1-agonists, the above results allow us to conclude that preservation of the intracellular milieu is essential to observe the physiological effects of Ang II on ICa. In addition, we propose that these effects are likely mediated by the activation of a Ca2+-sensitive PKC isoform.

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   Most of the results of the present work have been recently published in the following paper: Aiello EA, Cingolani HE. Angiotensin II stimulates cardiac L-type Ca2+ current (ICa) by a Ca2+ and protein kinase C-dependent mechanism. Am. J. Physiol. (Heart and Circ. Physiol.). 280: H1528-H1536 (2001).

INTRODUCTION
   Angiotensin II (Ang II) evokes positive inotropic responses in various species. However, the effects of this peptide on cardiac L-type calcium current (ICa) are still controversial. Early studies using multicellular preparations described increase in ICa after Ang II treatment (5,15). However, more recent observations using isolated myocytes reported contradictory results since increase (3,4,13), no effect (1,9), and even decrease (6) in ICa induced by Ang II was reported. Kaibara and co-authors (13) reported that in rabbit ventricular myocytes Ang II induced an increase in ICa only after stimulation of Na+/H+ exchanger (NHE) and subsequent intracellular alkalization. On the other hand, Ikenouchi and coworkers (9), using the same cell type and species, detected a significant 0.2 pH units increase in pHi after Ang II application without changes in ICa or intracellular Ca2+ transients.

   Ang II type 1 receptors (AT1), together with a1-adrenoceptors and ET1 receptors, belong to a family of Gq-coupled receptors linked to phospholipase C (PLC) activation and consequent production of inositol triphosphate (IP3) and diacylglicerol (DAG), which in turn activates Ca2+-dependent (classic) and Ca2+-independent (novel) isoforms of PKC. The cellular responses that involve these pathways might need the preservation of the intracellular milieu, as intact as possible, in order to observe the physiological effects of these hormones on ICa.

   Similarly to the phenomenon recently described for a1-agonists (21,33) and endothelin (ET) (16), we report in this study that the effects of Ang II on ICa differ depending on the mode of patch-clamp technique used, standard whole-cell (WC) or perforated-patch (PP). In addition, we present novel evidence that indicates that in cardiac myocytes the increase in ICa induced by Ang II is a pHi-independent and Ca2+i- and PKC-dependent mechanism.

MATERIALS AND METHODS
   Cat myocytes were isolated according to the technique previously described (2,24). Isolated cat ventricular myocytes were placed in a perfusion chamber and superfused with bath solution at a flow rate of 1.5 ml/min. The perforated-patch and standard whole-cell configurations of the patch clamp technique (2,7,17) were used for voltage-clamp recordings with a patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, Calif.). The currents (filtered at 1 kHz) were digitally recorded directly to hard disk via an analog-to-digital convertor (Digidata 1200, Axon Instruments) interfaced with an IBM clone computer running pClamp and Axotape software (Axon Instruments). Data analysis was performed with pClamp (Clampfit).

   Voltage-clamp depolarizing pulses (250 ms) were delivered at 0.2 Hz. A holding potential of -80 mV was used in all protocols to prevent slow inactivation and to minimize current rundown (23). A 500 ms prepulse to -40 mV, used to inactivate sodium channels and potential T-type calcium channels, preceded the depolarizing test pulses to different potentials. Nystatin produced good intracellular access after 15-20 minutes of seal formation. The calcium current amplitude was measured as peak inward current with reference to the current measured at the end of the test pulse. For each cell, capacitative current was recorded to determine the membrane capacitance and the currents were normalized for cell capacitance. The average cell capacitance was 148.7 ± 7.7 pF (n=14).

RESULTS
   Figure 1A, shows lack of effect of Ang II in representative traces of WC L-type Ca2+ currents evoked by pulses to 0 mV (250 ms) delivered at 0.2 Hz from a holding potential of -80 mV followed by a 500 ms prepulse to -40 mV. Out of a total of 24 cells, Ang II (0.5 µM) produced no effect in ICa in 12 cells, slight increase in 4 cells and decrease in 8 cells. On average, no statistically significant effects of Ang II were observed when WC was used in these experiments (Figure 1B).

Figure 1: Ang II effects on ICa registered under WC configuration. Panel A: Representative traces of ICa compensated for cell capacitance recorded in a myocyte before and after application of Ang II to the bath solution. The WC currents were evoked by a voltage-clamp depolarizing step to 0 mV (250 ms) from a prepulse potential of -40 mV. Dashed lines represent 0 current level. Panel B: Average data of peak ICa density of 24 cells recorded at 0 mV before (open bar) and after Ang II (solid bar). No statistically significant effects of Ang II on ICa were detected.

   In contrast to the results obtained using WC, when the intracellular milieu was preserved using PP (Figure 2), Ang II (0.5 µM) induced a significant and consistent 77±6 % (n=8) increase in ICa that was reversed by the AT1 receptor antagonist, Losartan (Los) (2 µM). Figure 2A, shows the time course of the effect of Ang II on the peak ICa evoked at 0 mV. The representative traces corresponding to the points indicated in the figure are shown below. Application of Ang II to the bath induced an increase in ICa that started after 1.5 min and reached a maximum plateau value after 6 min. The addition of Los (2 µM) in the continuous presence of Ang II induced a slow decrease in ICa that reached a new steady-state value (20 % above control) after 9 min of exposure of the myocyte to the AT1 receptor antagonist. Figure 2B depicts the average current density-voltage relations at control, after 7-8 minutes of Ang II treatment and after 10 min of the addition of Los in the continuous presence of Ang II. A significant increase in current density was observed in the range of voltage between -20 and +40 mV after application of Ang II to the bath solution, which was almost completely reversed by the addition of Los. These results suggest that preservation of the intracellular milieu with the PP is necessary to observe a consistent ICa enhancement induced by Ang II.

Figure 2: Effects of Ang II on ICa recorded under PP configuration. Panel A: Time course of the effects of Ang II and Ang II plus Losartan on peak ICa density evoked by a step to 0 mV in a single myocyte. Representative traces of ICa corresponding to the points indicated in the upper panel are shown below. Panel B: Current density-voltage relations for average data of peak current density collected from 8 myocytes in control, in the presence of Ang II and Losartan in the continuous presence of Ang II. * represents Ang II statistically different than control. ** represents Ang II statistically different than Losartan plus Ang II. Repeated Measures ANOVA for paired values followed by Bonferroni post-hoc test was employed. A significant increase in ICa was observed after application of Ang II to the bath solution.

   ICa enhancement induced by Ang II due to prior stimulation of the Na+/H+ exchanger (NHE) and the subsequent intracellular alkalization was previously reported (13). One possible explanation for the lack of effect of Ang II in the whole-cell experiments could be that intracellular dialysis with pipette solution containing the pH buffer HEPES (10 mM) might be preventing the increase in pHi induced by the peptide. However, in PP experiments, pretreatment of the cells with the specific NHE-1 inhibitor, HOE 642 (1 mM), did not prevent the increase in ICa induced by Ang II (at 0 mV, 71±7 %; n=5). Figure 3 shows an example of these experiments. Under the continuous presence of the NHE inhibitor, Ang II produced a Los-sensitive enhancement of ICa of a similar magnitude to the one observed in the absence of the blocker.

Figure 3: Effects of Ang II on ICa in the presence of the NHE-1 blocker, HOE 642, recorded under PP configuration. Time course of the effects of HOE 642, Ang II and Ang II plus Losartan on peak ICa density evoked by a step to 0 mV in a single myocyte. Representative traces of ICa corresponding to the points indicated in the upper panel are shown below. Despite the continuous presence of the NHE-1 blocker, Ang II induced a 75% increase in ICa.

   Since Ca2+i is a common second messenger for different receptor-operated intracellular pathways, the absence of consistent effect of Ang II when WC was used could be due to the chelation of Ca2+i by the EGTA (5 mM) present in the pipette solution. In order to test this hypothesis, two sets of experiments using WC were performed: 1) We measured ICa in myocytes dialyzed with the more rapid and efficient Ca2+ chelator BAPTA (10 mM). Under these conditions, Ang II failed to induced ICa enhancement in all the cells studied (at 0 mV, -6±1 pA/pF in control and -4.4±0.5 pA/pF in the presence of Ang II, n=6, NS). Indeed, 2 out of 6 myocytes dialyzed with BAPTA showed a sustained decrease in ICa after Ang II. 2) We dialyzed the cells with low Ca2+ buffer capacity (EGTA 0.1 mM). The myocytes were also dialyzed with a higher concentration of HEPES (30 mM) with the objective of efficiently clamp pHi at a constant value. Under these conditions, Ang II was able to induce increase in ICa, as shown in the representative traces of Figure 4A. Figure 4B depicts the average current density-voltage relation for ICa before and after addition of Ang II to the bath solution. This peptide induced a significant increase in the current in the voltage range between -25 and +40 mV. ICa recorded at 0 mV was 38±4 % higher in the presence of Ang II than in its absence. This value was lower than the one obtained using PP and is in the order of previously reported data in which WC was used (3,4,13). The above results allow us to conclude that the increase in ICa induced by Ang II is a mechanism dependent on Ca2+i and independent of changes in pHi.

Figure 4: Ang II effects on ICa registered under WC configuration in cells dialyzed with 0.1 mM EGTA and 30 mM HEPES. Panel A: Representative traces of ICa compensated for cell capacitance recorded in a myocyte before and after application of Ang II to the bath solution. The WC currents were evoked by a voltage-clamp depolarizing step to 0 mV (250 ms) from a prepulse potential of -40 mV. Dashed lines represent 0 current level. Panel B: Current density-voltage relations for average data of peak current density collected from 13 myocytes, before and after the addition of Ang II. * represents Ang II statistically different than control. A significant increase in ICa was observed after application of Ang II to the bath solution.

   Activation of cardiac AT1 receptors by Ang II leads to the stimulation of several PKC isoforms, including Ca2+-dependent (classic) (28) and Ca2+-independent (novel) (14,28) types. Although Ang II induced enhancement of ICa due to stimulation of PKC was previously suggested (3,4), no convincing evidences were provided linking the hormone, the receptor and the kinase. Figure 5A shows representative traces of ICa before and after bath application of Ang II recorded under WC in myocytes dialyzed with the selective PKC inhibitors Calphostin C (1 mM) or Chelerythrine (50 mM). The increase in ICa was prevented when the cells were dialyzed with these PKC inhibitors. The overall results of these experiments are shown in Figure 5B. These results provide direct evidence that confirm the involvement of PKC in the ICa enhancement induced by Ang II.

Figure 5: Ang II effects on ICa registered under WC configuration in cells dialyzed with 0.1 mM EGTA, 30 mM HEPES and the PKC inhibitors Calphostin C (1 mM) or Chelerythrine (50 mM). Panel A: Representative traces of ICa compensated for cell capacitance recorded before and after application of Ang II to the bath solution in myocytes dialyzed with Calphostin C (upper panel) or Chelerythrine (lower panel). The WC currents were evoked by a voltage-clamp depolarizing step to 0 mV (250 ms) from a prepulse potential of -40 mV. Dashed lines represent 0 current level. Panel B: Average data of peak ICa density of 6 cells dialyzed with Calphostin C (left pair of bars) and 7 cells dialyzed with Chelerythrine (right pair of bars), recorded at 0 mV before (open bar) and after Ang II (solid bar). No statistically significant effects of Ang II on ICa were detected in the presence of the PKC inhibitors.

DISCUSSION
   The present study demonstrates that Ang II activation of AT1 receptors increases cardiac ICa by stimulation of PKC and via a Ca2+-dependent and pHi-independent mechanism. Controversial results were obtained when the effects of Ang II on ICa were studied in isolated myocytes. This peptide was reported to cause increase (3,4,13), no effect (1,9) and even decrease (6) in basal ICa. One possible explanation for these discrepant results could be associated to differences in the effect of the peptide in different types of cardiac cells or the species involved. An alternative explanation could be related to the fact that most of these studies attempted to measure the effect of Ang II on ICa using the WC mode of the patch-clamp technique. As in these previous studies, in the present work Ang II induced variable results when the cells were dialyzed with a conventional pipette solution containing milimolar concentration of the widely used Ca2+ chelator, EGTA. Under these conditions, only few cells exhibited increase in ICa upon Ang II exposure. Since EGTA is a slow Ca2+ buffer, we can speculate that in these cells incoming and/or sub-sarcolemmal Ca2+ was not being properly chelated (32). Moreover, when the more rapid Ca2+ buffer, BAPTA (32), was dialyzed into the cells, none of the myocytes showed positive response after Ang II.

   Kaibara and co-authors (13) described an attractive mechanism to explain the Ang II induced cardiac ICa enhancement. Since ICa is sensitive to pHi changes (12), they proposed that ICa increases after Ang II as a result of activation of NHE and the subsequent intracellular alkalization. The Ang II induced ICa enhancement observed in the present study does not support Kaibara and coworkers hypothesis (13), since this effect was still present in the presence of NHE-1 blockade in the PP experiments and in the presence of high pH buffer capacity in the pipette solution in the WC experiments. The specific NHE-1 blocker, HOE 640, was used in our study while Kaibara and coworkers (13) used amiloride derivatives in their work. Whether these different experimental conditions could explain the discrepant results observed is not apparent to us. We should not disregard that amiloride derivatives are not specific blockers of the NHE and, among other currents, inhibition of ICa by these drugs was previously reported (25). This independence of pHi changes of the ICa enhancement induced by Ang II can be also supported by the experiments of Ikenouchi and coworkers (9) in which, in spite of a significant 0.2 pH units increase in pHi after Ang II, no changes in ICa were detected.

   Direct activation of PKC by phorbol esters leading to increased ICa was previously reported (18,20,27). However, PKC involvement in the Ang II induced ICa enhancement has not been studied carefully. Allen et al. (3) have shown that Ang II increased phosphoinositide hydrolysis and related these effects with a possible role of PKC in the ICa enhancement induced by the peptide. De Mello (4) has recently demonstrated that PKC inhibition prevented the increased in ICa produced by Ang II administered intracellularly, a pathway insensitive to Los, and probably involving receptors different than AT1. Thus, our data assessing PKC inhibition represent a new piece of information for the cardiac ICa enhancement through the pathway involving extracellular Ang II, AT1 receptors and PKC activation.
In the present study we have shown that either Ca2+i chelation or PKC inhibition were needed to prevent the Ang II induced ICa enhancement. An attractive hypothesis would be that Ang II induces increase in ICa through the activation of a Ca2+-dependent PKC isoform. Further studies using selective PKC isoforms blockers are needed to bring new insights in this issue. Nevertheless, our present data do not aim to characterize the PKC isoform involved in the Ang II effect on ICa. In addition, although we could speculate that classic PKC isoforms are the enzymes involved in this mechanism we cannot discard the possibility that a physiological level of Ca2+i would be required to activate PKC via the phosphatidylinositol cycle through the activation of G proteins linked to Ca2+-dependent phospholipase C (26,29,31).

   In summary, results of this study suggest that: 1) Particular attention should be taken when the physiological effects of stimulation of certain receptors are studied using the patch-clamp technique, being in the present case the PP the best configuration to delineate the cellular mechanisms by which Ang II stimulation modulates cardiac ICa. 2) Changes in pHi do not seem to contribute to the Ang II induced cardiac ICa enhancement. 3) A physiological level of Ca2+i is required to observe the increase in cardiac ICa induced by Ang II and 4) PKC activation is needed to produce stimulation of cardiac ICa after extracellular application of Ang II.

REFERENCES

1. Ai, T., M. Horie, K. Obayashi, and S. Sasayama. Pflugers Arch. 436: 168-174, 1998.

2. Aiello, E. A., M. G. Vila Petroff, A. Mattiazzi, and H. E. Cingolani. J. Physiol. 512: 137-148, 1998.

3. Allen, I. S., N. M. Cohen, R. S. Dhallan, S. T. Gaa, W. J. Lederer, and T. B. Rogers. Circ. Res. 62: 524-534, 1988.

4. De Mello, W. C. Hypertension 32: 976-982, 1998.

5. Freer, R. J., A. J. Pappano, M. J. Peach, K. T. Bing, M. J. Mc Lean, S. Vogel, and N. Sperelakis. Circ. Res. 39: 178-183, 1976.

6. Habuchi, Y., L. L. Lu, J. Morikawa, and M. Yoshimura. Am. J. Physiol. 268 (Heart and Circ. Physiol. 37): H1053-H1060, 1995.

7. Hamill, O. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth. Pflugers Archiv. 391: 85-100, 1981.

8. Heath, B. M., and D. A. Terrar. J. Physiol. 522.3: 391-402, 2000.

9. Ikenouchi, I., W. H. Barry, J. H. B. Bridge, E. O. Weiberg, C. S. Apstein, and H. Lorell. J. Physiol. 480: 203-215, 1994.

10. Ishihata, A., and M. Endoh. Br. J. Pharmacol. 108: 999-1005, 1993.

11. Josephson, I. R., and N. Speralakis. Biophys. J. 60: 491-497, 1991.

12. Kaibara, M., and M. Kameyama. J. Physiol. 403: 621-640, 1988.

13. Kaibara, M,. S. Matiarai, K. Yano, and M. Kameyama. Circ. Res. 75: 1121-1125, 1994.

14. Kang, P. M., A. Nakouzi, T. Simpson, J. Scheuer, and P. M. Buttrick. Am. J. Physiol. 270 (Heart and Circ. Physiol. 39): H2177-H2183, 1996.

15. Kass, R. S., and M. L. Blair. J. Mol. Cell. Cardiol. 13: 797-809, 1981.

16. Kelso, E, P. Spiers, B. Mc Dermott, N. Schofield, and B. Silke. Eur. J. Pharmacol. 308: 351-355, 1996.

17. Korn, S. J., A. Marty, J. A. Connor, and R. Horn. Methods in Neurosciences. 4: 364-373, 1991.

18. Lacerda, A. E., D. Rampe, and A. M. Brown. Nature. 335: 249-251, 1988.

19. Le Grand, B., S. Hatem, E. Deroubaix, J. P. Couetil, and E. Coraboeuf. Circ. Res 69: 292-299, 1991.

20. Liu, Q. Y., E. Karpinski, and P. K. T. Pang. Biochem. Biophys. Res. Commun. 191: 796-801, 1993.

21. Liu, S. J., and R. H. Kennedy. Am. J. Physiol. 274 (Heart and Circ. Physiol. 43): H2203-H2207, 1998.

22. Mattiazzi, A., N. G. Pérez, M. G. Vila Petroff, B. V. Alvarez, M. C. Camilión de Hurtado, and H. E. Cingolani. Am. J. Physiol. 272 (Heart and Circ. Physiol. 41): H1131-H1136, 1997.

23. Mc Donald, T. F., S. Pelzer, W. Trautwein, and D. J. Pelzer. Physiological Reviews 74: 365-507, 1994.

24. Morgan, P. E., E. A. Aiello, G. E. Chiappe de Cingolani, A. R. Mattiazzi, and H. E. Cingolani. J. Mol. Cell. Cardiol. 31: 1873-1883, 1999.

25. Pierce, G. N., W. C. Cole, K. Liu, H. Massaeli, T. G. Maddaford, Y. J. Chen, C. D. Mc Pherson, S. Jain, and D. Sontag. J. Pharmacol. Exp. Ther. 265: 1280-1291, 1993.

26. Renard, D., and J. Poggioli. J. Mol. Cell. Cardiol. 22: 13-22, 1990.

27. Singer-Lahat, D., E. Gershon, I. Lotan, R. Hullin, M. Biel, V. Flockerzi, F. Hoffmann, and Dascal N. FEBS Lett. 306: 113-118, 1992.

28. Takeishi, Y., T. Jalili, N. A. Ball, and R. A. Walsh. Circ. Res. 85: 264-271, 1999.

29. Taylor, S. J., H. Z. Chae, S. G. Rhee, and J. H. Exton. Nature. 350: 516-518, 1991.

30. Tiaho, F., J. Nargeot, and S. Richard. Pflugers Arch. 419: 596-602, 1991.

31. Van Heughten, H. A. A., H. W. De Jonge, K. Bezstarosti, and J. M. J. Lamers. J. Mol. Cell. Cardiol. 26: 1081-1093, 1994.

32. You, Y., D. J. Pelzer, and S. Pelzer. Biophys. J. 72: 175-187, 1997.

33. Zhang, S., M. Hiraoka, and Y. Hirano. J. Mol. Cell. Cardiol. 30: 1955-1965, 1998.

 

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Angiotensina II (Ang II) Incrementa la Corriente de Ca2+ tipo-L (ICa) de miocitos ventriculares cardíacos a través de la estimulación de una isoforma Ca2+-dependiente de la Proteína Quinasa C (PKC)

RESUMEN
En el presente estudio se determinaron, en miocitos aislados de ventrículo de gato, los efectos de la Ang II sobre ICa registrada bajo los modos standard Whole-Cell (W-C) y Patch-Perforado (P-P) de la técnica de Patch-Clamp. Cuando se utilizó W-C y se estableció el Ca2+ intracelular (Ca2+i) en un nivel subnanomolar mediante la diálisis con solución de pipeta conteniendo 5 mM de EGTA, 500 nM de Ang II no afectó ICa (a 0 mV, -5.5±0.6 pA/pF en control vs -4.4±0.5 pA/pF en presencia de Ang II, n=24, NS). Cuando se minimizó la perturbación del medio intracelular mediante la utilización del P-P, Ang II indujo un importante incremento de ICa (a 0 mV, -1.7±0.2 pA/pF en control y -2.9±0.3 pA/pF en presencia de Ang II, n=6, p<0.05). Cuando se utilizó W-C y las células fueron dializadas con 0.1 mM de EGTA para permitir que el Ca2+i se eleve a 100 nM, la Ang II aumentó ICa de modo similar a lo observado utilizando P-P (a 0 mV, -3.2±0.3 pA/pF en control vs -4.3±0.3 pA/pF en presencia de Ang II, n=9, p<0.05) indicando la necesaria participación del Ca2+i en la respuesta a Ang II. Bajo estas últimas condiciones, la inhibición de la PKC por Calphostina C o Celeritrina previno el incremento de ICa mediado por Ang II. Estos resultados sugieren que en miocitos ventriculares cardíacos, la Ang II incrementa ICa a través de una isoforma Ca2+-dependiente de PKC.

 

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2nd Virtual Congress of Cardiology

Dr. Florencio Garófalo
Steering Committee
President
Dr. Raúl Bretal
Scientific Committee
President
Dr. Armando Pacher
Technical Committee - CETIFAC
President
fgaro@fac.org.ar
fgaro@satlink.com
rbretal@fac.org.ar
rbretal@netverk.com.ar
apacher@fac.org.ar
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Copyright© 1999-2001 Argentine Federation of Cardiology
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