Ernesto A. Aiello

The Positive Inotropic Effect of Angiotensin II: Role of Endothelin-1 and Reactive Oxygen Species

Many effects believed to be because of angiotensin II (Ang II) are attributable to the action of endothelin (ET)-1, which is released/produced by Ang II. We investigated whether Ang II elicits its positive inotropic effect (PIE) by the action of endogenous ET-1, in addition to the role played by reactive oxygen species (ROS) in this mechanism. Cat cardiomyocytes were used for: (1) sarcomere shortening measurements; (2) ROS measurements by epifluorescence; (3) immunohistochemical staining for preproET-1, BigET-1, and ET-1; and (4) measurement of preproET-1 mRNA by RT-PCR. Cells were exposed to 1 nmol/L Ang II for 15 minutes. This low concentration of Ang II increases sarcomere shortening by 29.2±3.7% (P<0.05). This PIE was abrogated by Na+/H+ exchanger or Na+/Ca2+ exchanger reverse mode inhibition. The production of ROS increased in response to Ang II treatment (&Dgr;ROS respect to control: 68±15 fluorescence units; P<0.05). The Ang II–induced PIE and ROS production were blocked by the Ang II type 1 receptor blocker losartan, the nonselective ET-1 receptor blocker TAK044, the selective ETA receptor blocker BQ-123, or the ROS scavenger N-(2-mercapto-propionyl)glycine. Exogenous ET-1 (0.4 nmol/L) induced a similar PIE and increase in ROS production to those caused by Ang II. Immunostaining for preproET-1, BigET-1, and ET-1 was positive in cardiomyocytes. The preproET-1 mRNA abundance increased from 100±4.6% in control to 241.9±39.9% in Ang II–treated cells (P<0.05). We conclude that the PIE after exposure to 1 nmol/L Ang II is due to endogenous ET-1 acting through the ETA receptor and triggering ROS production, Na+/H+ exchanger stimulation, and Na+/Ca2+ exchanger reverse mode activation.

The positive inotropic effect of endothelin-1 is mediated by mitochondrial reactive oxygen species.

We have previously demonstrated the participation of reactive oxygen species (ROS) in the positive inotropic effect of a physiological concentration of Angiotensin II (Ang II, 1 nM). The objective of the present work was to evaluate the role and source of ROS generation in the positive inotropic effect produced by an equipotent concentration of endothelin-1 (ET-1, 0.4 nM). Isolated cat ventricular myocytes were used to measure sarcomere shortening with a video-camera, superoxide anion (()O(2)(-)) with chemiluminescence, and ROS production and intracellular pH (pH(i)) with epifluorescence. The ET-1-induced positive inotropic effect (40.4+/-3.1%, n=10, p<0.05) was associated to an increase in ROS production (105+/-29 fluorescence units above control, n=6, p<0.05). ET-1 also induced an increase in ()O(2)(-) production that was inhibited by the NADPH oxidase blocker, apocynin, and by the blockers of mitochondrial ATP-sensitive K(+) channels (mK(ATP)), glibenclamide and 5 hydroxydecanoic acid. The ET-1-induced positive inotropic effect was inhibited by apocynin (0.3 mM; 6.3+/-6.6%, n=13), glibenclamide (50 microM; 8.8+/-3.5%, n=6), 5 hydroxydecanoic acid (500 microM; 14.1+/-8.1, n=9), and by scavenging ROS with MPG (2 mM; 0.92+/-5.6%, n=8). ET-1 enhanced proton efflux (J(H)) carried by the Na(+)/H(+) exchanger (NHE) after an acid load, effect that was blocked by MPG. Consistently, the ET-induced positive inotropic effect was also inhibited by the NHE selective blocker HOE642 (5 microM; 9.37+/-6.07%, n=7). The data show that the effect of a concentration of ET-1 that induces an increase in contractility of about 40% is totally mediated by an intracellular pathway triggered by mitochondrial ROS formation and stimulation of the NHE.

Evidence for an electrogenic Na+-HCO3− symport in rat cardiac myocytes

1 The perforated whole‐cell configuration of patch clamp and the pH fluorescent indicator SNARF were used to determine the electrogenicity of the Na+‐HCO3− cotransport in isolated rat ventricular myocytes. 2 Switching from Hepes buffer to HCO3− buffer at constant extracellular pH (pHo) hyperpolarized the resting membrane potential (RMP) by 2.9 ± 0.4 mV (n= 9, P < 0.05). In the presence of HCO3−, the anion blocker SITS depolarized RMP by 2.6 ± 0.5 mV (n= 5, P < 0.05). No HCO3−‐induced hyperpolarization was observed in the absence of extracellular Na+. The duration of the action potential measured at 50 % of repolarization time (APD50) was 29.2 ± 6.1 % shorter in the presence of HCO3− than in its absence (n= 6, P < 0.05). 3 Quasi‐steady‐state currents were evoked by voltage‐clamped ramps ranging from −130 to +30 mV, during 8 s. The development of a novel component of Na+‐dependent and Cl−‐independent steady‐state outward current was observed in the presence of HCO3−. The reversal potential (Erev) of the Na+‐HCO3− cotransport current (INa,Bic) was measured at four different levels of extracellular Na+. A HCO3−:Na+ ratio compatible with a stoichiometry of 2:1 was detected. INa,Bic was also studied in isolation in standard whole‐cell experiments. Under these conditions, INa,Bic reversed at −96.4 ± 1.9 mV (n= 5), being consistent with the influx of 2 HCO3− ions per Na+ ion through the Na+‐HCO3− cotransporter. 4 In the presence of external HCO3−, after 10 min of depolarizing the membrane potential (Em) with 45 mm extracellular K+, a significant intracellular alkalinization was detected (0.09 ± 0.03 pH units; n= 5, P < 0.05). No changes in pHi were observed when the myocytes were pre‐treated with the anion blocker DIDS (0.001 ± 0.024 pH units; n= 5, n.s.), or when exposed to Na+‐free solutions (0.003 ± 0.037 pH units; n= 6, n.s.). 5 The above results allow us to conclude that the cardiac Na+‐HCO3− cotransport is electrogenic and has an influence on RMP and APD of rat ventricular cells.

Aldosterone Stimulates the Cardiac Na+/H+ Exchanger via Transactivation of the Epidermal Growth Factor Receptor

The use of antagonists of the mineralocorticoid receptor in the treatment of myocardial hypertrophy and heart failure has gained increasing importance in the last years. The cardiac Na+/H+ exchanger (NHE-1) upregulation induced by aldosterone could account for the genesis of these pathologies. We tested whether aldosterone-induced NHE-1 stimulation involves the transactivation of the epidermal growth factor receptor (EGFR). Rat ventricular myocytes were used to measure intracellular pH with epifluorescence. Aldosterone enhanced the NHE-1 activity. This effect was canceled by spironolactone or eplerenone (mineralocorticoid receptor antagonists), but not by mifepristone (glucocorticoid receptor antagonist) or cycloheximide (protein synthesis inhibitor), indicating that the mechanism is mediated by the mineralocorticoid receptor triggering nongenomic pathways. Aldosterone-induced NHE-1 stimulation was abolished by the EGFR kinase inhibitor AG1478, suggesting that is mediated by transactivation of EGFR. The increase in the phosphorylation level of the kinase p90RSK and NHE-1 serine703 induced by aldosterone was also blocked by AG1478. Exogenous epidermal growth factor mimicked the effects of aldosterone on NHE-1 activity. Epidermal growth factor was also able to increase reactive oxygen species production, and the epidermal growth factor–induced activation of the NHE-1 was abrogated by the reactive oxygen species scavenger N-2-mercaptopropionyl glycine, indicating that reactive oxygen species are participating as signaling molecules in this mechanism. Aldosterone enhances the NHE-1 activity via transactivation of the EGFR, formation of reactive oxygen species, and phosphorylation of the exchanger. These results call attention to the consideration of the EGFR as a new potential therapeutic target of the cardiovascular pathologies involving the participation of aldosterone.

Subcellular mechanisms of the positive inotropic effect of angiotensin II in cat myocardium

1 Cat ventricular myocytes loaded with [Ca2+]i‐ and pHi‐sensitive probes were used to examine the subcellular mechanism(s) of the Ang II‐induced positive inotropic effect. Ang II (1 μM) produced parallel increases in contraction and Ca2+ transient amplitudes and a slowly developing intracellular alkalisation. Maximal increases in contraction amplitude and Ca2+ transient amplitude were 163 ± 22 and 43 ± 8 %, respectively, and occurred between 5 and 7 min after Ang II administration, whereas pHi increase (0·06 ± 0·03 pH units) became significant only 15 min after the addition of Ang II. Furthermore, the inotropic effect of Ang II was preserved in the presence of Na+‐H+ exchanger blockade. These results indicate that the positive inotropic effect of Ang II is independent of changes in pHi. 2 Similar increases in contractility produced by either elevating extracellular [Ca2+] or by Ang II application produced similar increases in peak systolic Ca2+ indicating that an increase in myofilament responsiveness to Ca2+ does not participate in the Ang II‐induced positive inotropic effect. 3 Ang II significantly increased the L‐type Ca2+ current, as assessed by using the perforated patch‐clamp technique (peak current recorded at 0 mV: ‐1·88 ± 0·16 pA pF−1 in control vs. ‐3·03 ± 0·20 pA pF−1 after 6‐8 min of administration of Ang II to the bath solution). 4 The positive inotropic effect of Ang II was not modified in the presence of either KB‐R7943, a specific blocker of the Na+‐Ca2+ exchanger, or ryanodine plus thapsigargin, used to block the sarcoplasmic reticulum function. 5 The above results allow us to conclude that in the cat ventricle the Ang II‐induced positive inotropic effect is due to an increase in the intracellular Ca2+ transient, an enhancement of the L‐type Ca2+ current being the dominant mechanism underlying this increase.

Endothelin-1 Stimulates the Na+/Ca2+ Exchanger Reverse Mode Through Intracellular Na+ (Na+i)-Dependent and Na+i-Independent Pathways

This study aimed to explore the signaling pathways involved in the positive inotropic effect (PIE) of low doses of endothelin-1 (ET-1). Cat papillary muscles were used for force and intracellular Na+ concentration (Na+i) measurements, and isolated cat ventricular myocytes for patch-clamp experiments. ET-1 (5 nmol/L) induced a PIE and an associated increase in Na+i that were abolished by Na+/H+ exchanger (NHE) inhibition with HOE642. Reverse-mode Na+/Ca2+ exchanger (NCX) blockade with KB-R7943 reversed the ET-1–induced PIE. These results suggest that the ET-1–induced PIE is totally attributable to the NHE-mediated Na+i increase. However, an additional direct stimulating effect of ET-1 on NCX after the necessary increase in Na+i could occur. Thus, the ET-1–induced increase in Na+i and contractility was compared with that induced by partial inhibition of the Na+/K+ ATPase by lowering extracellular K+ (K+o). For a given Na+i, ET-1 induced a greater PIE than low K+o. In the presence of HOE642 and after increasing contractility and Na+i by low K+o, ET-1 induced an additional PIE that was reversed by KB-R7943 or the protein kinase C (PKC) inhibitor chelerythrine. ET-1 increased the NCX current and negatively shifted the NCX reversal potential (ENCX). HOE642 attenuated the increase in NCX outward current and abolished the ENCX shift. These results indicate that whereas the NHE-mediated ET-1–induced increase in Na+i seems to be mandatory to drive NCX in reverse and enhance contractility, Na+i-independent and PKC-dependent NCX stimulation appears to additionally contribute to the PIE. However, it is important to stress that the latter can only occur after the primary participation of the former.

Intracellular signaling following myocardial stretch : an autocrine/paracrine loop

The stretch of adult papillary muscle elicits a chain of autocrine/paracrine events in which the Na(+)/H(+) exchanger (NHE-1) activation is the central step. This activation is induced by a sequential angiotensin II-endothelin (Ang II-ET) release and results in an increase in intracellular Na(+) (Na(+)(i)) without significant changes in intracellular pH. The increase in Na(+)(i) negatively shifts the reverse potential of the Na(+)/Ca(2+) exchanger (NCX) thus inducing cell Ca(2+) influx that augments myocardial contractility. This increase in force represents the mechanical counterpart of the autocrine/paracrine mechanism triggered by stretch and has been called the slow force response (SFR) to stretch.

Binding of carbonic anhydrase IX to extracellular loop 4 of the NBCe1 Na+/HCO3− cotransporter enhances NBCe1-mediated HCO3− influx in the rat heart

Na(+)/HCO(3)(-) cotransporter (NBC)e1 catalyze the electrogenic movement of 1 Na(+):2 HCO(3)(-) into cardiomyocytes cytosol. NBC proteins associate with carbonic anhydrases (CA), CAII, and CAIV, forming a HCO(3)(-) transport metabolon. Herein, we examined the physical/functional interaction of NBCe1 and transmembrane CAIX in cardiac muscle. NBCe1 and CAIX physical association was examined by coimmunoprecipitation, using rat ventricular lysates. NBCe1 coimmunoprecipitated with anti-CAIX antibody, indicating NBCe1 and CAIX interaction in the myocardium. Glutathione-S-transferase (GST) pull-down assays with predicted extracellular loops (EC) of NBCe1 revealed that NBCe1-EC4 mediated interaction with CAIX. Functional NBCe1/CAIX interaction was examined using fluorescence measurements of BCECF in rat cardiomyocytes to monitor cytosolic pH. NBCe1 transport activity was evaluated after membrane depolarization with high extracellular K(+) in the presence or absence of the CA inhibitors, benzolamide (BZ; 100 μM) or 6-ethoxyzolamide (ETZ; 100 μM) (*P < 0.05). This depolarization protocol produced an intracellular pH (pH(i)) increase of 0.17 ± 0.01 (n = 11), which was inhibited by BZ (0.11 ± 0.02; n = 7) or ETZ (0.06 ± 0.01; n = 6). NBCe1 activity was also measured by changes of pH(i) in NBCe1-transfected human embryonic kidney 293 cells subjected to acid loads. Cotransfection of CAIX with NBCe1 increased the rate of pH(i) recovery (in mM/min) by about fourfold (12.1 ± 0.8; n = 9) compared with cells expressing NBCe1 alone (3.1 ± 0.5; n = 7), which was inhibited by BZ (7.5 ± 0.3; n = 9). We demonstrated that CAIX forms a complex with EC4 of NBCe1, which activates NBCe1-mediated HCO(3)(-) influx in the myocardium. CAIX and NBCe1 have been linked to tumorigenesis and cardiac cell growth, respectively. Thus inhibition of CA activity might be useful to prevent activation of NBCe1 under these pathological conditions.

Antibodies against the cardiac sodium/bicarbonate co-transporter (NBCe1) as pharmacological tools

BACKGROUND AND PURPOSE Na+/HCO3‐ co‐transport (NBC) regulates intracellular pH (pHi) in the heart. We have studied the electrogenic NBC isoform NBCe1 by examining the effect of functional antibodies to this protein.

The electrogenic Na+/HCO3− cotransport modulates resting membrane potential and action potential duration in cat ventricular myocytes

Perforated whole‐cell configuration of patch clamp was used to determine the contribution of the electrogenic Na+/HCO3− cotransport (NBC) on the shape of the action potential in cat ventricular myocytes. Switching from Hepes to HCO3− buffer at constant extracellular pH (pHo) hyperpolarized resting membrane potential (RMP) by 2.67 ± 0.42 mV (n= 9, P < 0.05). The duration of action potential measured at 50% of repolarization time (APD50) was 35.8 ± 6.8% shorter in the presence of HCO3− than in its absence (n= 9, P < 0.05). The anion blocker SITS prevented and reversed the HCO3−‐induced hyperpolarization and shortening of APD. In addition, no HCO3−‐induced hyperpolarization and APD shortening was observed in the absence of extracellular Na+. Quasi‐steady‐state currents were evoked by 8 s duration voltage‐clamped ramps ranging from −130 to +30 mV. A novel component of SITS‐sensitive current was observed in the presence of HCO3−. The HCO3−‐sensitive current reversed at −87 ± 5 mV (n= 7), a value close to the expected reversal potential of an electrogenic Na+/HCO3− cotransport with a HCO3−:Na+ stoichiometry ratio of 2: 1. The above results allow us to conclude that the cardiac electrogenic Na+/HCO3− cotransport has a relevant influence on RMP and APD of cat ventricular cells.