This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 90/4/374    most recent hh0402.105373v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Aiello, E. A. Articles by Cingolani, H. E. Search for Related Content PubMed PubMed Citation Articles by Aiello, E. A. Articles by Cingolani, H. E. Related Collections Arrythmias-basic studies Ion channels/membrane transport

(Circulation Research. 2002;90:374.)
© 2002 American Heart Association, Inc.

Reports

Autocrine Stimulation of Cardiac Na⁺-Ca²⁺ Exchanger Currents by Endogenous Endothelin Released by Angiotensin II

Ernesto A. Aiello, María C. Villa-Abrille, Horacio E. Cingolani

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

Correspondence to Dr Ernesto A. Aiello or Dr Horacio E. Cingolani, Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Médicas, 60 y 120, La Plata 1900, Argentina. E-mail cicme{at}atlas.med.unlp.edu.ar

Abstract

The goal of the present study was to evaluate the effects of Ang II on the current produced by the Na⁺-Ca²⁺ exchanger (I_NCX) working in the reverse mode and the possible autocrine role played by the release of endothelin (ET) in these actions. I_NCX was studied in isolation in cat cardiac myocytes. Angiotensin II (Ang II) (100 nmol/L) increased I_NCX at potentials higher than 0 mV (at +60 mV: 2.07±0.22 pA/pF in control versus 2.73±0.22 pA/pF in Ang II, n=9; P<0.05). The increase in I_NCX induced by Ang II was prevented by the treatment of the cells with the unspecific blocker of the ET receptors, TAK 044 (1 µmol/L) (at +60 mV: 2.15±0.27 pA/pF in control versus 2.01±0.26 pA/pF in Ang II, n=5, NS). These results show, for the first time, that the effect of Ang II on I_NCX is the result of the autocrine actions of ET released by the octapeptide.

Key Words: sodium-calcium exchanger • cardiac myocytes • angiotensin II • endothelin

Previous experimental evidence indicates that some cardiovascular effects initially thought to be mediated by angiotensin II (Ang II) were, in fact, due to the release of endogenous endothelin (ET). In cultured neonatal rat cardiomyocytes, Ito et al¹ reported inhibition of Ang II–induced hypertrophy with a blocker of ET_A receptors (BQ123) or antisense oligonucleotides directed against mRNA of preproET. In rats, the elevation of blood pressure induced by infusion of Ang II was reversed by the ET_A blocker PD 155080.² The goal of the present study was to evaluate, in isolated myocytes, the effects of Ang II on the current produced by the NCX (I_NCX) working in the reverse mode and the possible autocrine role played by the release of ET in these actions. We present novel evidence that demonstrates, for the first time, that the reverse mode of the cardiac NCX is stimulated by endogenous ET released by the myocytes on Ang II regulation.

Materials and Methods

Cat cardiac myocytes were isolated according to the technique previously described.³ Animals were provided by a local supplier (San Cayetano, Monte Grande, Argentina) and procedures were performed according to the Guide for the Care and Use of Laboratory Animals (US Department of Health and Human Services). The determination of the NCX current (I_NCX) was performed under the conditions previously described by Hobai and O’Rourke⁴ with some modifications. In some experiments, L-type Ca²⁺ current (I_Ca) was recorded. The composition of solutions and other details are described in an expanded Materials and Methods section that can be found in the online data supplement, available at http://www.circresaha.org.

Results and Discussion

Whereas the contribution of the reverse mode of the NCX to basal cardiac contraction is controversial,⁵ increases in contractility due to stimulation of this mode of the NCX have been reported to be present after the effects of agonists such as Ang II⁶ or ET.⁷ Therefore, since the initial goal of the present study was the evaluation of the effects of Ang II on the reverse mode of the NCX, we measured I_NCX under recording conditions that favor this mode over the forward mode. Figure 1 shows the inhibitory effects of the NCX blocker KB-R7943 (KBR, 10 µmol/L) on whole-cell currents recorded under conditions of I_NCX isolation. Note that KBR blocked both forward and reverse modes of NCX, as it was previously reported for this concentration of the agent.⁸ Lower concentrations of KBR were proven to block selectively the reverse mode of NCX.⁸ Consistently, no effects of this drug on the forward mode were observed when 5 µmol/L KBR was used (shown in the online data supplement). Ni²⁺ (10 mmol/L), another NCX antagonist, blocked the current registered at +60 mV by 85±5% (n=11). On average, the apparent reversal potentials (E_rev) of I_NCX were -44.4±2.5 mV (n=6) and -39.5±4.1 mV (n=11) estimated as the zero current potential of the KBR- and Ni²⁺-sensitive currents, respectively. These values are similar to the I_NCX E_rev values reported by other authors working in similar conditions to those of the present study.⁴ The above experiments allowed us to conclude that the whole-cell currents registered were mainly underlain by the NCX (I_NCX).

View larger version (24K): [in this window] [in a new window]   Figure 1. Characterization of INCX. A, Families of representative whole-cell currents evoked by depolarizing pulses between -80 to +100 mV from a holding potential of -40 mV, recorded in a myocyte in the absence and presence of KBR (10 µmol/L). Pulses (260 ms in duration) were delivered at 0.1 Hz. B, Voltage dependence of the average current density measured in 6 myocytes exposed to KBR (10 µmol/L). C, Average current-voltage relations of the KBR-sensitive currents, representing INCX (n=6).

Ballard and Schaffer⁹ reported that Ang II or ET increased the activity of the NCX in canine cardiac sarcolemmal vesicles. Thus, we next examined whether Ang II was able to affect I_NCX in isolated cat cardiac myocytes. Figure 2A depicts the time course of the effect of Ang II (100 nmol/L) on the current recorded at +60 mV in a single myocyte. Ang II induced a gradual increase in the current that reaches steady-state values after 5 minutes of administration of the peptide. Average data of the current recorded at +60 mV in the absence and presence of Ang II are shown in Figure 2C. This current increased by 24.7±4.9% (n=26) in the presence of Ang II. The Ang II type 1 receptor (AT₁) antagonist losartan (1 µmol/L) reversed the effects of Ang II (data not shown).

View larger version (22K): [in this window] [in a new window]   Figure 2. Ang II effects on INCX. A, Time course of effects of Ang II on the current registered at +60 mV from a holding potential of -40 mV, recorded in a single myocyte. Pulses were delivered at 0.1 Hz. B, Representative traces of whole-cell currents corresponding to the points indicated in panel A. C, Average data of current density recorded at +60 mV before and after the addition of Ang II (100 nmol/L) (n=26). D, Ang II (100 nmol/L) and KBR (10 µmol/L) effects on average current-voltage relations evoked by the voltage protocol of Figure 1 (n=9). E, Ang II effects on average current-voltage relations for the KBR-sensitive current, corresponding to INCX (n=9). *Ang II statistically different from control.

The outward current recorded at +60 mV is mostly but not totally I_NCX because a small remnant current is present after the effects of the NCX blockers Ni²⁺ or KBR. Thus, the possibility that the Ni²⁺-insensitive current could be affected by Ang II was evaluated. No increase in outward current was induced by Ang II (100 nmol/L) in the presence of Ni²⁺ (10 mmol/L) (0.75±0.31 pA/pF in Ni²⁺ and 0.78±0.36 pA/pF in Ni²⁺ plus Ang II, n=5; NS), indicating that the current affected by Ang II is most likely I_NCX. Moreover, to confirm that Ang II affects I_NCX, we studied the effects of this peptide on the KBR-sensitive currents. Figure 2D shows average current density-voltage relations obtained in 9 myocytes exposed to 10 µmol/L KBR after having obtained the increase in the whole-cell current by Ang II. Ang II induced a significant increase in outward currents at potentials positive to 0 mV. KBR blocked the currents recorded after Ang II to the same extent of those recorded in the absence of the peptide (Figure 1). Figure 1E shows the effects of Ang II on the average current-voltage relation for the KBR-sensitive currents, corresponding to I_NCX, obtained by subtracting the currents in KBR to those recorded in both control and in Ang II. Ang II significantly increased I_NCX at potentials positive to 0 mV. At +60 mV, I_NCX increased by 38.7±14.6% in the presence of Ang II (n=9).

We next examined whether the Ang II–induced increase in I_NCX observed in the present study was produced by release of endogenous ET. For this purpose, we pretreated the cells with the nonselective blocker of ET receptors, TAK 044 (1 µmol/L). This blocker did not affect basal current (at +60 mV: 2.59±0.15 pA/pF in control versus 2.45±0.12 pA/pF in TAK 044-treated myocytes, n=13; NS). Figure 3A shows the time course of effects of Ang II in the presence of TAK 044 on the current registered at +60 mV. In the presence of the ET receptor blocker, Ang II failed to induce enhancement of this current. On average, no effects of Ang II were observed in the presence of TAK 044 on the current recorded at +60 mV (Figure 3C) or at any of the tested voltages (Figure 3D). In addition, no effects of Ang II in the presence of TAK 044 were observed on the Ni²⁺-sensitive currents, representing I_NCX (at +60 mV: 2.15±0.27 pA/pF in TAK 044 versus 2.01±0.27 pA/pF in Ang II plus TAK 044, n=5; NS).

View larger version (16K): [in this window] [in a new window]   Figure 3. Inhibition of Ang II effects on INCX by the ET receptor antagonist TAK 044. A, Time course of effects of Ang II in the presence of TAK 044 on the current registered at +60 mV from a holding potential of -40 mV, recorded in a single myocyte. Pulses were delivered at 0.1 Hz. B, Representative traces of whole-cell currents corresponding to the points indicated in panel A. C, Average data of current density recorded at +60 mV before and after the addition of Ang II (100 nmol/L) to the solution containing TAK 044 (1 µmol/L) (n=13). D, Ang II effects in the presence of TAK 044 on average current-voltage relations evoked by the same voltage protocol of Figure 1 (n=7).

To rule out the possibility that the ET receptor blocker TAK 044 is acting on the Ang II receptors, we performed experiments in which the L-type calcium current (I_Ca) was measured in the absence and presence of Ang II (100 nmol/L) with or without TAK 044 (1 µmol/L) in the extracellular solution. We have recently reported that Ang II enhances I_Ca via activation of AT₁ receptors in cat cardiac myocytes.³ Under the recording conditions of the present study, the peak I_Ca recorded at 0 mV increased by 23±8% (n=5) after 10 minutes in the presence of Ang II. A similar increment in I_Ca after 10 minutes of Ang II was observed in the presence of TAK 044 (21±7%, n=5). These results suggest that this ET receptor blocker did not unspecifically bind to AT₁ receptors and that the endogenous ET released by Ang II did not affect I_Ca. The previously reported failure of ET-1 to affect I_Ca when standard whole-cell configuration is used can account for the last result.¹⁰

Taken together, the above results allow us to suggest that in cardiac myocytes the stimulation of I_NCX induced by Ang II is, in fact, mediated by endogenous ET released by Ang II. The major finding of this study is the demonstration at the cardiac single-cell level of the existence of an autocrine mechanism in the heart involving the myocyte as the source and target of ET. Additional observations and extended discussion of the results are presented in the online data supplement.

Acknowledgments

This study was supported by a grant "Carrillo-Oñativia" (to H.E.C.) from the Ministerio de Salud de La Nación, Argentina. M.C.V.A. is a recipient of a fellowship of the Comisión de Investigaciones Científicas (CIC, Buenos Aires) and sponsored by a grant "Carrillo-Oñativia" (Ministerio de Salud, Argentina). E.A.A. and H.E.C are Established Investigators of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.

Received November 5, 2001; revision received January 10, 2002; accepted January 10, 2002.

References

1. Ito H, Hirata Y, Adachi S, Tanaka M, Tsujino M, Koike A, Nogami A, Marumo F, Hiroe M. Endothelin-1 is an autocrine/paracrine factor in the mechanism of angiotensin II-induced hypertrophy in cultured rat cardiomyocytes. J Clin Invest. 1993; 92: 398–403.[Medline] [Order article via Infotrieve]

2. Rajagopalan S, Laursen JB, Borthayre A, Kurz S, Keiser J, Haleen S, Giaid A, Harrison DG. Role for endothelin-1 in angiotensin II–mediated hypertension. Hypertension. 1997; 30: 29–34.[Abstract/Free Full Text]

3. Aiello EA, Cingolani HE. Angiotensin II stimulates cardiac L-type Ca²⁺ current by a Ca²⁺- and protein kinase C-dependent mechanism. Am J Physiol. 2001; 280: H1528–H1536.

4. Hobai IA, O’Rourke B. Enhanced Ca²⁺-activated Na⁺-Ca²⁺ exchange activity in canine pacing-induced heart failure. Circ Res. 2000; 87: 690–698.[Abstract/Free Full Text]

5. Blaustein MP, Lederer WJ. Sodium/calcium exchange: its physiological implications. Physiol Rev. 1999; 79: 763–854.[Abstract/Free Full Text]

6. Fujita S, Endoh M. Influence of a Na⁺/H⁺ exchange inhibitor ethylisopropylamiloride, Na⁺/Ca²⁺ exchange inhibitor KB-R7943 and their combination on the increases in contractility and Ca²⁺ transient induced by angiotensin II in isolated adult rabbit ventricular myocytes. Naunyn Schmiedebergs Arch Pharmacol. 1999; 360: 575–584.[CrossRef][Medline] [Order article via Infotrieve]

7. Yang HT, Sakurai K, Sugawara H, Watanabe T, Norota I, Endoh M. Role of Na⁺/Ca²⁺ exchange in endothelin-1-induced increases in Ca²⁺ transient and contractility in rabbit ventricular myocytes: pharmacological analysis with KB-R7943. Br J Pharmacol. 1999; 126: 1785–1795.[CrossRef][Medline] [Order article via Infotrieve]

8. Watano T, Kimura J, Morita T, Nakanishi H. A novel antagonist, No. 7943, of the Na⁺/Ca²⁺ exchange current in guinea-pig ventricular cells. Br J Pharmacol. 1996; 119: 555–563.[Medline] [Order article via Infotrieve]

9. Ballard C, Schaffer S. Stimulation of the Na⁺/Ca²⁺ exchanger by phenylephrine, angiotensin II and endothelin 1. J Mol Cell Cardiol. 1996; 28: 11–17.[CrossRef][Medline] [Order article via Infotrieve]

10. Kelso E, Spiers P, McDermott B, Schofield N, Silke B. Dual effects of endothelin-1 on the L-type Ca²⁺ current in ventricular cardiomyocytes. Eur J Pharmacol. 1996; 308: 351–355.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				L. Ferron, V. Capuano, Y. Ruchon, E. Deroubaix, A. Coulombe, and J.-F. Renaud Angiotensin II Signaling Pathways Mediate Expression of Cardiac T-Type Calcium Channels Circ. Res., December 12, 2003; 93(12): 1241 - 1248. [Abstract] [Full Text] [PDF]

				N. G Perez, M. C Villa-Abrille, E. A Aiello, R. A Dulce, H. E Cingolani, and M. C Camilion de Hurtado A low dose of angiotensin II increases inotropism through activation of reverse Na+/Ca2+ exchange by endothelin release Cardiovasc Res, December 1, 2003; 60(3): 589 - 597. [Abstract] [Full Text] [PDF]

				M. G Vila Petroff, J. Palomeque, and A. R Mattiazzi Na+-Ca2+ Exchange Function Underlying Contraction Frequency Inotropy in the Cat Myocardium J. Physiol., August 1, 2003; 550(3): 801 - 817. [Abstract] [Full Text] [PDF]

				Y. Shimoni and X.-F. Liu Role of PKC in autocrine regulation of rat ventricular K+ currents by angiotensin and endothelin Am J Physiol Heart Circ Physiol, April 1, 2003; 284 (4): H1168 - H1181. [Abstract] [Full Text] [PDF]

				H. E Cingolani, N. G Perez, B. Pieske, D. von Lewinski, and M. C Camilion de Hurtado Stretch-elicited Na+/H+ exchanger activation: the autocrine/paracrine loop and its mechanical counterpart Cardiovasc Res, March 15, 2003; 57(4): 953 - 960. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 90/4/374    most recent hh0402.105373v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Aiello, E. A. Articles by Cingolani, H. E. Search for Related Content PubMed PubMed Citation Articles by Aiello, E. A. Articles by Cingolani, H. E. Related Collections Arrythmias-basic studies Ion channels/membrane transport