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.

Sodium/bicarbonate co-transporters (NBC) are crucial in the regulation of intracellular pH (pH(i)) and HCO(3)(-) metabolism. Electrogenic NBC1 catalyzes HCO(3)(-) fluxes in mammalian kidney, pancreas, and heart cells. Carbonic anhydrase IV (CAIV), which is also present in these tissues, is glycosylphosphatidyl inositol-anchored to the outer surface of the plasma membrane where it catalyzes the hydration-dehydration of CO(2)/HCO(3)(-). The physical and functional interactions of CAIV and NBC1 were investigated. NBC1 activity was measured by changes of pH(i) in NBC1-transfected HEK293 cells subjected to acid loads. Cotransfection of CAIV with NBC1 increased the rate of pH(i) recovery by 44 +/- 3%, as compared to NBC1-alone. In contrast, CAIV did not increase the functional activity of G767T-NBC1 (mutated on the fourth extracellular loop (EC4) of NBC1), and G767T-NBC1, unlike wild-type NBC1, did not interact with CAIV in glutathione-S-transferase pull-down assays. This indicates that G767 of NBC1 is directly involved in CAIV interaction. NBC1-mediated pH(i) recovery rate after acid load was inhibited by 40 +/- 7% when coexpressed with the inactive human CAII mutant, V143Y. V143Y CAII competes with endogenous CAII for interaction with NBC1 at the inner surface of the plasma membrane, which indicates that NBC1/CAII interaction is needed for full pH(i) recovery activity. We conclude that CAIV binds EC4 of NBC1, and this interaction is essential for full NBC1 activity. The tethering of CAII and CAIV close to the NBC1 HCO(3)(-) transport site maximizes the transmembrane HCO(3)(-) gradient local to NBC1 and thereby activates the transport rate.

Carbonic anhydrases (CA) catalyze the reversible conversion of CO2 to HCO3−. Some bicarbonate transporters bind CA, forming a complex called a transport metabolon, to maximize the coupled catalytic/transport flux. SLC26A6, a plasma membrane Cl−/HCO3− exchanger with a suggested role in pancreatic HCO3− secretion, was found to bind the cytoplasmic enzyme CAII. Mutation of the identified CAII binding (CAB) site greatly reduced SLC26A6 activity, demonstrating the importance of the interaction. Regulation of SLC26A6 bicarbonate transport by protein kinase C (PKC) was investigated. Angiotensin II (AngII), which activates PKC, decreased Cl−/HCO3− exchange in cells coexpressing SLC26A6 and AT1a-AngII receptor. Activation of PKC reduced SLC26A6/CAII association in immunoprecipitates. Similarly, PKC activation displaced CAII from the plasma membrane, as monitored by immunofluorescence. Finally, mutation of a PKC site adjacent to the SLC26A6 CAB site rendered the transporter unresponsive to PKC. PKC therefore reduces CAII/SLC26A6 interaction, reducing bicarbonate transport rate. Taken together, our data support a mechanism for acute regulation of membrane transport: metabolon disruption

Abstract-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 ϩ /H ϩ exchanger (NHE), we studied the relationship between myocardial stretch and intracellular pH (pH i ). 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. pH i was measured by epifluorescence with the acetoxymethyl ester form of the pH-sensitive dye 2Ј,7Ј-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM). Each muscle was progressively stretched to reach maximal developed force (L max ) and maintained in a length that was Ϸ92% L max (L i ). During the "stretch protocol," muscles were quickly stretched to L max for 10 minutes and then released to L i ; pH i significantly increased during stretch and came back to the previous value when the muscle was released to L i . The increase in pH i was eliminated by (1) Key Words: stretch, myocardial Ⅲ pH, intracellular Ⅲ Na ϩ /H ϩ exchange Ⅲ angiotensin Ⅲ endothelin A lthough it is well known that mechanical stimuli cause a variety of effects on the structure and function of the myocardial cells, little is known about how cells sense the mechanical stimuli, transmit the information to messenger systems, and finally regulate function and growth.1,2 Highly regarded experiments demonstrate that the release of angiotensin II (Ang II) contributes to stretch-induced hypertrophy in cultured neonatal cardiac myocytes, [2][3][4][5] and that the effect was suppressed by the AT 1 -receptor antagonist TCV-116. 4 The release of Ang II may involve an autocrine or paracrine mechanism because stretch-conditioned media mimicked the effect of stretch when transferred to nonstretched neonatal cardiomyocytes.2 An increase in PKC activity is an effect detected after stretching cultured neonatal cardiomyocytes 2 and the adult heart. 6 Because Ang II, by mechanisms still unresolved but probably linked to PKC, activates the Na ϩ /H ϩ exchanger (NHE), 7-8 the logical expectation is a rise in intracellular pH (pH i ) after myocardial stretch. Although we are unaware of measurements of myocardial pH i before and during stretch, pressure overload increased NHE-1 mRNA levels in hearts.9 Furthermore, the activity of mitogen-activated protein kinase (MAP kinase) was increased by stretch in cultured cardiomyocytes, and this increase was partially eliminated by NHE inhibition.9 Nevertheless, an unknown is whether stretch alters NHE activity in multicellular preparations from adult hearts. This point is critical, because Ang II released after stretch might increase NHE activity 10 -14 and promote the expression of endothelin (ET) as well as the upregulation of ET rec...

Hypertrophic cardiomyocyte growth contributes substantially to the progression of heart failure. Activation of the plasma membrane Na + -H + exchanger (NHE1) and Cl − -HCO 3 − exchanger (AE3) has emerged as a central point in the hypertrophic cascade. Both NHE1 and AE3 bind carbonic anhydrase (CA), which activates their transport flux, by providing H + and HCO 3 − , their respective transport substrates. We examined the contribution of CA activity to the hypertrophic response of cultured neonatal and adult rodent cardiomyocytes. Phenylephrine (PE) increased cell size by 37 ± 2% and increased expression of the hypertrophic marker, atrial natriuretic factor mRNA, twofold in cultured neonatal rat cardiomyocytes. Cell size was also increased in adult cardiomyocytes subjected to angiotensin II or PE treatment. These effects were associated with increased expression of cytosolic CAII protein and the membrane-anchored isoform, CAIV. The membrane-permeant CA inhibitor, 6-ethoxyzolamide (ETZ), both prevented and reversed PE-induced hypertrophy in a concentration-dependent manner in neonate cardiomyocytes (IC 50 = 18 µM). ETZ and the related CA inhibitor methazolamide prevented hypertrophy in adult cardiomyocytes. In addition, ETZ inhibited transport activity of NHE1 and the AE isoform, AE3, with respective EC 50 values of 1.2 ± 0.3 µM and 2.7 ± 0.3 µM. PE significantly increased neonatal cardiomyocyte Ca 2+ transient frequency from 0.33 ± 0.4 Hz to 0.77 ± 0.04 Hz following 24 h treatment; these Ca 2+ -handling abnormalities were completely prevented by ETZ (0.28 ± 0.07 Hz). Our study demonstrates a novel role for CA in mediating the hypertrophic response of cardiac myocytes to PE and suggests that CA inhibition represents an effective therapeutic approach towards mitigation of the hypertrophic phenotype.

Bicarbonate facilitate more than 50% of pH recovery in the acidotic myocardium, and have roles in cardiac hypertrophy and steady-state pH regulation. To determine which bicarbonate transporters are responsible for this activity, we measured the expression levels of all known HCO3 − -anion exchange proteins in mouse heart, by quantitative real time RT-PCR. Bicarbonate-anion exchangers are members of either the SLC4A or the SLC26A gene families. In neonatal and adult myocardium, AE1 (Slc4a1), AE2 (Slc4a2), AE3 (Slc4a3) (AE3fl and AE3c variants), Slc26a3 and Slc26a6 were expressed. Adult hearts expressed Slc26a3 and Slc4a1-3 mRNAs at similar levels, while Slc26a6 mRNA was about seven-fold higher than AE3, which was more abundant than any other. Immunohistochemistry revealed that Slc26a6 and AE3 are present in the plasma membrane of ventricular myocytes. Slc26a6 expression levels were higher in ventricle than atrium, whereas AE3 was detected only in ventricle. Cl − -HCO

Carbonic anhydrase II (CAII) binds to and regulates transport by the NHE1 isoform of the mammalian Na(+)/H(+) exchanger. We localized and characterized the CAII binding region on the C-terminal tail of the Na(+)/H(+) exchanger. CAII did not bind to acidic sequences in NHE1 that were similar to the CAII binding site of bicarbonate transporters. Instead, by expressing a variety of fusion proteins of the C-terminal region of the Na(+)/H(+) exchanger, we demonstrated that CAII binds to the penultimate group of 13 amino acids of the cytoplasmic tail. Within this region, site-specific mutagenesis demonstrated that amino acids S796 and D797 form part of a novel CAII binding site. Phosphorylation of the C-terminal 26 amino acids by heart cell extracts did not alter CAII binding to this region, but phosphorylation greatly increased CAII binding to a protein containing the C-terminal 182 amino acids of NHE1. This suggested that an upstream region of the cytoplasmic tail acts as an inhibitor of CAII binding to the penultimate group of 13 amino acids. The results demonstrate that a novel phosphorylation-regulated CAII binding site exists in distal amino acids of the NHE1 tail.

BACKGROUND AND PURPOSENa + /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. EXPERIMENTAL APPROACHWe generated two antibodies against putative extracellular loop domains 3 (a-L3) and 4 (a-L4) of NBCe1 which recognized NBCe1 on immunoblots and immunostaining experiments. pHi was monitored using epi-fluorescence measurements in cat ventricular myocytes. Transport activity of total NBC and of NBCe1 in isolation were evaluated after an ammonium ioninduced acidosis (expressed as H + flux, JH, in mmol·L -1 min -1 at pHi 6.8) and during membrane depolarization with high extracellular potassium (potassium pulse, expressed as DpHi) respectively. KEY RESULTSThe potassium pulse produced a pHi increase of 0.18 Ϯ 0.006 (n = 5), which was reduced by the a-L3 antibody (0.016 Ϯ 0.019). The a-L-3 also decreased JH by 50%. Surprisingly, during the potassium pulse, a-L4 induced a higher pHi increase than control,(0.25 Ϯ 0.018) whereas the recovery of pHi from acidosis was faster (JH was almost double the control value). In perforated-patch experiments, a-L3 prolonged and a-L4 shortened action potential duration, consistent with blockade and stimulation of NBCe1-carried anionic current respectively. CONCLUSIONS AND IMPLICATIONSBoth antibodies recognized NBCe1, but they had opposing effects on the function of this transporter, as the a-L3 was inhibitory and the a-L4 was excitatory. These antibodies could be valuable in studies on the pathophysiology of NBCe1 in cardiac tissue, opening a path for their potential clinical use.