Molecular basis for angiotensin II-induced increase of chloride/bicarbonate exchange in the myocardium.

Plasma membrane anion exchangers (AEs) regulate myocardial intracellular pH (pH(i)) by Na(+)-independent Cl(-)/HCO(3)(-) exchange. Angiotensin II (Ang II) activates protein kinase C (PKC) and increases anion exchange activity in the myocardium. Elevated anion exchange activity has been proposed to contribute to the development of cardiac hypertrophy. Our Northern blots showed that adult rat heart expresses AE1, AE2, AE3fl, and AE3c. Activity of each AE isoform was individually measured by following changes of pH(i), associated with bicarbonate transport, in transfected HEK293 cells. Exposure to the PKC activator, PMA (150 nmol/L), increased the transport activity of only the AE3fl isoform by 50+/-11% (P<0.05, n=6), consistent with the increase observed in intact myocardium. Cotransfection of HEK293 cells with AE3fl and AT1(a)-Ang II receptors conferred sensitivity of anion transport to Ang II (500 nmol/L), increasing the transport activity by 39+/-3% (P<0.05, n=4). PKC inhibition by chelerythrine (10 micromol/L) blocked the PMA effect. To identify the PKC-responsive site, 7 consensus PKC phosphorylation sites of AE3fl were individually mutated to alanine. Mutation of serine 67 of AE3 prevented the PMA-induced increase of anion transport activity. Inhibition of MEK1/2 by PD98059 (50 micromol/L) did not affect the response of AE3fl to Ang II, indicating that PKC directly phosphorylates AE3fl. We conclude that following Ang II stimulation of cells, PKCepsilon phosphorylates serine 67 of the AE3 cytoplasmic domain, inducing the Ang II-induced increase in anion transport observed in the hypertrophic myocardium.

Stimulation of myocardial Na(+)-independent Cl(-)-HCO(3)(-) exchanger by angiotensin II is mediated by endogenous endothelin.

Experiments were performed in isolated cat papillary muscles loaded with the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in the esterified form to study the effect of endothelin-1 (ET-1) on the activity of the Na(+)-independent Cl(-)-HCO(3)(-) exchanger. Exposure to ET-1 (10 nmol/L) raised pH(i) by 0.13+/-0.03 U (P<0.05) in papillary muscles superfused with nominally HCO(3)(-)-free solution, whereas no significant change was detected under CO(2)/HCO(3)(-)-buffered medium. However, if ET-1 was applied to muscles pretreated with the anion exchanger inhibitor 4-acetamido-4'-isothiocyanato-stilbene-2, 2'-disulfonic acid, pH(i) increased by 0.09+/-0.02 U (P<0.05) in the presence of CO(2)/HCO(3)(-) buffer. The rate of pH(i) recovery from trimethylamine hydrochloride-induced intracellular alkaline load was enhanced so that net HCO(3) efflux increased about three times in the presence of ET-1 (2.74+/-0.25 versus 9.66+/-1.29 mmol. L(-1). min(-1) at pH(i) 7.55, P<0.05). This effect was canceled by previous exposure to either 50 nmol/L PD 142,893 (nonselective endothelin receptor blocker) or 300 nmol/L BQ 123 (selective blocker of ET(A) receptors). BQ 123 also abolished angiotensin II-induced activation of the Na(+) independent Cl(-)-HCO(3)(-) exchanger. These results show that ET-1 increases the activity of the Na(+)-independent Cl(-)-HCO(3)(-) exchanger in cardiac tissue through the ET(A) receptors. Furthermore, our data suggest that the previously described angiotensin II-induced stimulation of the anion exchanger activity is mediated by endogenous ET-1.

Mechanisms underlying the increase in force and Ca(2+) transient that follow stretch of cardiac muscle: a possible explanation of the Anrep effect.

Myocardial stretch produces an increase in developed force (DF) that occurs in two phases: the first (rapidly occurring) is generally attributed to an increase in myofilament calcium responsiveness and the second (gradually developing) to an increase in [Ca(2+)](i). Rat ventricular trabeculae were stretched from approximately 88% to approximately 98% of L(max), and the second force phase was analyzed. Intracellular pH, [Na(+)](i), and Ca(2+) transients were measured by epifluorescence with BCECF-AM, SBFI-AM, and fura-2, respectively. After stretch, DF increased by 1.94+/-0.2 g/mm(2) (P<0.01, n = 4), with the second phase accounting for 28+/-2% of the total increase (P<0.001, n = 4). During this phase, SBFI(340/380) ratio increased from 0.73+/-0.01 to 0.76+/-0.01 (P<0.05, n = 5) with an estimated [Na(+)](i) rise of approximately 6 mmol/L. [Ca(2+)](i) transient, expressed as fura-2(340/380) ratio, increased by 9.2+/-3.6% (P<0.05, n = 5). The increase in [Na(+)](i) was blocked by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA). The second phase in force and the increases in [Na(+)](i) and [Ca(2+)](i) transient were blunted by AT(1) or ET(A) blockade. Our data indicate that the second force phase and the increase in [Ca(2+)](i) transient after stretch result from activation of the Na(+)/H(+) exchanger (NHE) increasing [Na(+)](i) and leading to a secondary increase in [Ca(2+)](i) transient. This reflects an autocrine-paracrine mechanism whereby stretch triggers the release of angiotensin II, which in turn releases endothelin and activates the NHE through ET(A) receptors.

Stretch-induced alkalinization of feline papillary muscle: an autocrine-paracrine system.

Myocardial stretch is a well-known stimulus that leads to hypertrophy. Little is known, however, about the intracellular pathways involved in the transmission of myocardial stretch to the cytoplasm and nucleus. Studies in neonatal cardiomyocytes demonstrated stretch-induced release of angiotensin II (Ang II). Because intracellular alkalinization is a signal to cell growth and Ang II stimulates the Na+/H+ exchanger (NHE), we studied the relationship between myocardial stretch and intracellular pH (pHi). Experiments were performed in cat papillary muscles fixed by the ventricular end to a force transducer. Muscles were paced at 0.2 Hz and superfused with HEPES-buffered solution. pHi was measured by epifluorescence with the acetoxymethyl ester form of the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM). Each muscle was progressively stretched to reach maximal developed force (Lmax) and maintained in a length that was approximately 92% Lmax (Li). During the "stretch protocol," muscles were quickly stretched to Lmax for 10 minutes and then released to Li; pHi significantly increased during stretch and came back to the previous value when the muscle was released to Li. The increase in pHi was eliminated by (1) specific inhibition of the NHE (EIPA, 5 micromol/L), (2) AT1-receptor blockade (losartan, 10 micromol/L), (3) inhibition of protein kinase C (PKC) (chelerythrine, 5 micromol/L), (4) blockade of endothelin (ET) receptors with a nonselective (PD 142,893, 50 nmol/L) or a selective ETA antagonist (BQ-123, 300 nmol/L). The increase in pHi by exogenous Ang II (500 nmol/L) was also reduced by both ET-receptor antagonists. Our results indicate that after myocardial stretch, pHi increases because of stimulation of NHE activity. This involves an autocrine-paracrine mechanism in which protein kinase C, Ang II, and ET play crucial roles.

Enalapril induces regression of cardiac hypertrophy and normalization of pHi regulatory mechanisms.

Intracellular pH is under strict control in myocardium; H+ are extruded from the cells by sodium-dependent mechanisms, mainly Na+/H+ exchanger and Na+/HCO3- symport, whereas Na+-independent Cl-/HCO3- exchanger extrudes bases on intracellular alkalinization. Hypertrophic myocardium from spontaneously hypertensive rats (SHR) exhibits increased Na+/H+ exchange activity that is accompanied by enhanced extrusion of bases through Na+-independent Cl-/HCO3- exchange. The present experiments were designed to investigate the effect of enalapril-induced regression of cardiac hypertrophy on the activity of these exchangers. Male SHR and normotensive Wistar-Kyoto rats (WKY) received enalapril maleate (20 mg/kg per day) in the drinking water for 5 weeks. Gender- and age-matched SHR and WKY were used as untreated controls. Enalapril treatment significantly reduced systolic blood pressure in SHR and completely regressed cardiac hypertrophy. Na+/H+ activity was estimated in terms of both steady pHi value in HEPES buffer and the rate of pHi recovery from CO2-induced acid load. Na+-independent Cl-/HCO3- activity was assessed by measuring the rate of pHi recovery from intracellular alkalinization produced by trimethylamine exposure. Regression of cardiac hypertrophy was accompanied by normalization of Na+/H+ and Na+-independent Cl-/HCO3- exchange activities. Inhibition of protein kinase C (PKC) activity with chelerythrine (10 mmol/L) or calphostin C (50 nmol/L) returned both exchange activities to normal values. These results show that angiotensin-converting enzyme inhibition normalizes the enhanced activity of both exchangers while regressing cardiac hypertrophy. Because normalization of exchange activities could be also achieved by PKC inhibition, the data would suggest that PKC-dependent mechanisms play a significant role in the increased ion exchange activities of hypertrophic myocardium and in their normalization by angiotensin-converting enzyme inhibition.

Angiotensin II activates Na+-independent Cl--HCO3- exchange in ventricular myocardium.

The effect of angiotensin II (Ang II) on the activity of the cardiac Na+-independent Cl--HCO3- exchanger (anionic exchanger [AE]) was explored in cat papillary muscles. pHi was measured by epifluorescence with BCECF-AM. Ang II (500 nmol/L) induced a 5-(N-ethyl-N-isopropyl)amiloride-sensitive increase in pHi in the absence of external HCO3- (HEPES buffer), consistent with its stimulatory action 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). Moreover, in Na+-free HCO3--buffered medium, in which neither NHE nor Na+-HCO3- cotransport are acting, Ang II decreased pHi, and this effect was canceled by previous treatment with SITS. These findings suggested that the Ang II-induced activation of NHE was masked, in the presence of the physiological buffer, by a HCO3--dependent acidifying mechanism, probably the AE. This hypothesis was confirmed on papillary muscles bathed with HCO3- buffer that were first exposed to 1 micromol/L S20787, a specific inhibitor of AE activity in cardiac tissue, and then to 500 nmol/L Ang II (n=4). Under this condition, Ang II increased pHi from 7.05+/-0.05 to 7.22+/-0.05 (P<.05). The effect of Ang II on AE activity was further explored by measuring the velocity of myocardial pHi recovery after the imposition of an intracellular alkali load in a HCO3--containing solution either with or without Ang II. The rate of myocardial pHi recovery was doubled in the presence of Ang II, suggesting a stimulatory effect on AE. The enhancement of the activity of this exchanger by Ang II was also detected when the AE activity was reversed by the removal of extracellular Cl- in a Na+-free solution. Under this condition, the rate of intracellular alkalinization increased 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 canceled either by the presence of the AT1 receptor antagonist, losartan, or by the previous inhibition of protein kinase C with chelerythrine or calphostin C. The above results allow us to conclude that Ang II, in addition to its stimulatory effect on alkaline loading mechanisms, activates the AE in ventricular myocardium and that the latter effect is mediated by a protein kinase C-dependent regulatory pathway linked to the AT1 receptors.

Role of an electrogenic Na(+)-HCO3- cotransport in determining myocardial pHi after an increase in heart rate.

The contribution of electrogenic Na(+)-HCO3- cotransport to pHi regulation during changes in heart rate was explored in cat papillary muscles loaded with BCECF-AM in bicarbonate-free (HEPES) medium and in CO2/HCO3(-)-buffered medium. Stepwise increments in the frequency of contraction from 15 to 100 bpm induced a reversible increase in the pHi from 7.13 +/- 0.03 to 7.36 +/- 0.03 (P < .05, n = 5) in the presence of CO2/ HCO3- buffer. The same increase in the frequency of stimulation, however, decreased pHi from 7.10 +/- 0.02 to 6.91 +/- 0.06 (P < .05, n = 5), in the absence of bicarbonate. Moreover, in CO2/HCO3(-)-superfused muscles pretreated with SITS (0.1 mmol/L), this effect of increasing the contraction frequency was reversed, and a decrease of pHi from 7.03 +/- 0.04 to 6.88 +/- 0.06 (P < .05, n = 4) was observed when the pacing rate was increased stepwise from 15 to 100 bpm. High [K+]o-induced depolarization of cell membrane alkalinized myocardial cells in the presence of HCO3- ions, whereas acidification was observed as a consequence of hyperpolarization induced by low external [K+]o. Myocardial resting membrane potential became hyperpolarized upon exposure to HCO3(-)-buffered media. This HCO3(-)-induced hyperpolarization was not blocked by the inhibition of Na+,K(+)-ATPase activity by ouabain (0.5 mumol/L) but was prevented by SITS. The results suggested that membrane depolarization during cardiac action potential causes an increase in electrogenic Na(+)-HCO3- cotransport. Such depolarizations occurring as a consequence of increases in heart rate would thus, by means of elevated bicarbonate influxes, substantially increase the myocardial cell's ability to recover from an enhanced proton production.

pHi regulation in myocardium of the spontaneously hypertensive rat. Compensated enhanced activity of the Na(+)-H+ exchanger.

To elucidate the mechanisms controlling pHi in myocardium of the spontaneously hypertensive rat (SHR), experiments were performed in papillary muscles (isometrically contracting at 0.2 Hz) from SHR and age-matched normotensive Wistar-Kyoto (WKY) rats loaded with the pH-sensitive fluorescent probe BCECF-AM. An enhanced activity of the Na(+)-H+ exchanger was detected in the hypertrophic myocardium of SHR. This conclusion was based on the following: (1) The myocardial pHi was more alkaline in SHR (7.23 +/- 0.03) than in WKY rats (7.10 +/- 0.03) (P < .05) in HEPES buffer. (2) SITS (0.1 mmol/L in HEPES buffer) did not alter pHi in the SHR (pHi 7.26 +/- 0.03 and 7.28 +/- 0.03 before and after SITS, respectively). (3) The fall in pHi observed after 20 minutes of Na(+)-H+ exchanger inhibition [5 mumol/L 5-(N-ethyl-N-isopropyl)amiloride (EIPA)] was greater in SHR (-0.16 +/- 0.01) than in WKY rats (-0.09 +/- 0.02, P < 0.05). (4) The velocity of pHi recovery from an intracellular acid load was faster in SHR than in WKY rats (0.068 +/- 0.02 versus 0.014 +/- 0.002 pH units/min at pHi 6.99, P < .05). (5) After EIPA inhibition, the rate of pHi recovery from the same acid load decreased to a similar value in both rat strains (0.0032 +/- 0.002 pH units/min in SHR and 0.0032 +/- 0.002 pH units/min in WKY rats). Under the more physiological HCO3(-)-CO2 buffer, no significant difference in steady state myocardial pHi was detected between rat strains (7.15 +/- 0.03 in SHR and 7.11 +/- 0.05 in WKY rats).(ABSTRACT TRUNCATED AT 250 WORDS)