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Previous work demonstrated that the slow force response (SFR) to stretch is due to the increase in calcium transients (Ca2+T) produced by an autocrine-paracrine mechanism of locally produced angiotensin II/endothelin activating Na+-H+ exchange. Although a rise in pHi is presumed to follow stretch, it was observed only in the absence of extracellular bicarbonate, suggesting pHi compensation through the Na+-independent Cl--HCO3- exchange (AE) mechanism. Because available AE inhibitors do not distinguish between different bicarbonate-dependent mechanisms or even between AE isoforms, we developed a functional inhibitory antibody against both the AE3c and AE3fl isoforms (anti-AE3Loop III) that was used to explore if pHi would rise in stretched cat papillary muscles superfused with bicarbonate after AE3 inhibition. In addition, the influence of this potential increase in pHi on the SFR was analyzed. In this study, we present evidence that cancellation of AE3 isoforms activity (either by superfusion with bicarbonate-free buffer or with anti-AE3Loop III) results in pHi increase after stretch and the magnitude of the SFR was larger than when AE was operative, despite of similar increases in [Na+]i and Ca2+T under both conditions. Inhibition of reverse mode Na+-Ca2+ exchange reduced the SFR to the half when the AE was inactive and totally suppressed it when AE3 was active. The difference in the SFR magnitude and response to inhibition of reverse mode Na+-Ca2+ exchange can be ascribed to a pHi-induced increase in myofilament Ca2+ responsiveness.

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.

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.

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.

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.