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

Anion exchanger 1 (AE1; SLC4A1), the plasma membrane Cl−/HCO3−exchanger of erythrocytes, is also expressed in heart. The aim of this study was to assess the role of AE1 in heart function through study of AE1-null (AE1−/−) mice, which manifest severe hemolytic anemia resulting from erythrocyte fragility. Heart weight-to-body weight ratios were significantly higher in the AE1−/−mice than in wild-type (AE1+/+) littermates at both 1–3 days postnatal (3.01 ± 0.38 vs. 1.45 ± 0.04) and at 7 days postnatal (9.45 ± 0.53 vs. 4.13 ± 0.41), indicating that loss of AE1 led to cardiac hypertrophy. Heterozygous (AE1+/−) mice had no signs of cardiac hypertrophy. Morphology of the adult AE1−/−mutant heart revealed an increased left ventricular mass, accompanied by increased collagen deposition and fibrosis. M-mode echocardiography revealed dysfunction of the AE1−/−hearts, including dilated left ventricle end diastole and systole and expanded left ventricular mass compared with AE1+/+hearts. Expression of intracellular pH-regulatory mechanisms in the hypertrophic myocardium of neonate AE1−/−mutant mice was indistinguishable from AE1+/−and AE1+/+mice, as assessed by quantitative real-time RT-PCR. Confocal immunofluorescence revealed that, in normal mouse myocardium, AE1 is sarcolemmal, whereas AE3 and slc26a6 are found both at the sarcolemma and in internal membranes (T tubules and sarcoplasmic reticulum). These results indicate that AE1−/−mice, which suffer from severe hemolytic anemia and spherocytosis, display cardiac hypertrophy and impaired cardiac function, reminiscent of findings in patients with hereditary abnormalities of red blood cells. No essential role for AE1 in heart function was found.

In the course of studying the hypertonicity-activated ion transporters in Xenopus oocytes, we found that activation of endogenous oocyte Na(+)/H(+) exchange activity (xoNHE) by hypertonic shrinkage required Cl(-), with an EC(50) for bath [Cl(-)] of approximately 3mM. This requirement for chloride was not supported by several nonhalide anions and was not shared by xoNHE activated by acid loading. Hypertonicity-activated xoNHE exhibited an unusual rank order of inhibitory potency among amiloride derivatives and was blocked by Cl(-) transport inhibitors. Chelation of intracellular Ca(2+) by injection of EGTA blocked hypertonic activation of xoNHE, although many inhibitors of Ca(2+)-related signaling pathways were without inhibitory effect. Hypertonicity activated oocyte extracellular signal-regulated kinase 1/2 (ERK1/2), but inhibitors of neither ERK1/2 nor p38 prevented hypertonic activation of xoNHE. However, hypertonicity also stimulated a Cl(-)-dependent increase in c-Jun NH(2)-terminal kinase (JNK) activity. Inhibition of JNK activity prevented hypertonic activation of xoNHE but not activation by acid loading. We conclude that hypertonic activation of Na(+)/H(+) exchange in Xenopus oocytes requires Cl(-) and is mediated by activation of JNK.

beta-L-(-)-2',3'-Dideoxy-3'-thiacytidine (3TC) is a cytosine nucleoside analog that potently inhibits the replication of human and duck hepatitis B viruses and human immunodeficiency virus through the activity of its 5'-triphosphate ester metabolite. The present study examined the intracellular decay of 3TC 5'-phosphates and tested strategies for modulating the cellular content of those nucleotides in primary cultures of duck hepatocytes and in human hepatoma 2.2.15 cells and CCRF-CEM T lymphoblasts. Inhibition by deoxycytidine of the 5'-phosphorylation of 3TC in duck hepatocytes confirmed that, as in mammalian cells, deoxycytidine kinase catalyzed 3TC activation. The 5'-mono, 5'-di-, and 5'-triphosphates of 3TC underwent monoexponential elimination from duck hepatocytes and 2.2.15 cells (half-lives, 3.6 to 8.0 h). Thymidine and fludarabine, which are agents that enhance the activity of deoxycytidine kinase, were tested in strategies for increasing the cellular content of 3TC 5'-phosphates. Coordinate treatment of cells with 3TC and thymidine (50 microM) increased the content of 3TC 5'-monophosphate in duck hepatocytes and the content of 3TC 5'-di- and 5'-triphosphates in 2.2.15 cells, but enhancement of 3TC 5'-phosphate levels in CCRF-CEM cells required a higher thymidine concentration (100 microM). Fludarabine (5 microM) did not affect the contents of 3TC 5'-di- and 5'-triphosphates in duck hepatocytes, but modestly increased the contents of those nucleotides in 2.2.15 cells and CCRF-CEM cells. Nitrobenzylthioinosine (NBMPR), an inhibitor of the es facilitated diffusion nucleoside transporter, reduced the level of entry of 3TC into 2.2.15 cells and abolished inward fluxes of thymidine, adenosine, and deoxycytidine. In 2.2.15 cells and CCRF-CEM cells, NBMPR reduced the formation of 3TC 5'-di- and 5'-triphosphates and reversed the thymidine- and fludarabine-induced increases in the formation of those nucleotides. NBMPR protected against the cytotoxicity of 3TC in CCRF-CEM cells, whereas thymidine potentiated that toxicity, apparently by enhancing the formation of 3TC 5'-triphosphate. Taken together, these results indicate that deoxycytidine kinase and the es nucleoside transporter are targets for manipulation of the metabolism and activity of 3TC.