Differential regulation of vacuolar H+-ATPase and Na+/H+ exchanger 3 in rat cholangiocytes after bile duct ligation

The cholangiocytes lining the intrahepatic bile ducts modify the primary secretion from the hepatocytes. The cholangiocytes secrete HCO₃⁻ into bile when stimulated with secretin in many species, including man. However, in rats, secretin stimulation neither affects biliary HCO₃⁻ concentration nor bile flow, whereas following bile duct ligation (BDL) it induces hypercholeresis with significant increase of NaHCO₃ concentration. We hypothesized that BDL might affect the expression of cholangiocyte H⁺ transporters and thereby choleresis, and determined the expression and localization of the 31 kDa vacuolar type H⁺-ATPase (V-ATPase) subunit and of Na⁺/H⁺ exchanger NHE3 in the livers of control and BDL rats by real-time PCR, in situ hybridization, immunoblotting, and immunohistochemistry. In controls, secretin had no effect on bile flow, whereas following BDL, secretin increased bile flow ∼threefold. V-ATPase and NHE3 were expressed in control cholangiocytes showing intracellular and apical distribution, respectively. BDL significantly up-regulated V-ATPase mRNA and protein expression and was associated with redistribution to the apical pole in ∼60% of the cholangiocytes lining the small bile ductules. In contrast, NHE3 expression was significantly down-regulated by BDL at the mRNA and protein level. The data demonstrate expression of V-ATPase in rat cholangiocytes. BDL-induced down-regulation of NHE3 may contribute to a reduction of Na⁺ and HCO₃⁻ reabsorption and thus to their net secretion into bile. Apical localization of V-ATPase in cholangiocytes may indicate its involvement in pH regulation and/or HCO₃⁻ salvage to compensate for NHE3 down-regulation in BDL.     

Adenylyl cyclase isoforms in rat testis and spermatozoa from the cauda epididymidis

Expression of adenylyl cyclase genes in rat testis and spermatozoa from the cauda epididymidis was investigated using RT-PCR analysis. Genes encoding the transmembrane adenylyl cyclases (tmAC) II, III, IV, V, VI, VII, and VIII were expressed in the testis, whereas only the gene for tmAC III was expressed in caudal spermatozoa. Immunocytochemistry was used to investigate which tmAC were translated into putative, functional proteins in spermatozoa. Indirect immunofluorescence localized the tmAC II enzyme to a region on the head occupied by the acrosome. The tmAC III enzyme was localized to the posterior margin of the head and to the flagellum, whereas tmAC V and/or VI was localized to the region where the ventral surface of the acrosomal equatorial segment is located. The tmAC VII and VIII enzymes were localized to the convex margin of the head, covering the dorsal region of the acrosomal crescent. To our knowledge, this is the first demonstration that five apparently different tmAC enzymes are localized to discrete subcellular regions of mammalian spermatozoa. These findings provide a fundamental basis for future studies, to determine the physiological roles of tmAC in testis and mature spermatozoa.This work was supported by a grant from the Australian Research Council/Department of Education, Training and Youth Affairs (ARC A40001141)     

Tissue distribution of Na+/H+ exchanger isoforms NHE2 and NHE4 in rat intestine and kidney

We present evidence that tissue distribution of two highlyconservedNa⁺/H⁺exchanger isoforms, NHE2 and NHE4, differs significantly from previously published reports. Riboprobes unique to each of these antiporters, from 5' (noncoding and coding) and 3' codingregions, were used to analyze mRNA from adult rat kidney and intestine by ribonuclease protection assay and in situ hybridization. In contrastto earlier work that concluded that both NHE2 and NHE4 were expressedthroughout the intestine and in the kidney, our data show that there isno NHE2 message in the kidney and NHE4 is not expressed in small orlarge intestine. Analyses of intestinal epithelial and kidney membraneproteins by an NHE2-specific antibody identified a doublet at <90 kDain intestine but not in kidney. NHE2 is highly expressed in theNa⁺-absorptive epithelium ofjejunum, ileum, and ascending and descending colon. NHE4 mRNA messageis found in the inner medulla of the kidney as previously reported (C. Bookstein, M. W. Musch, A. DePaoli, Y. Xie, M. Villereal, M. C. Rao,and E. B. Chang. J. Biol. Chem. 269:29704-29709, 1994) and not in the intestine. From these data, wespeculate that neither NHE2 nor NHE4 has a role in renalNa⁺ absorption. NHE2 is likelyinvolved in gut Na⁺ absorption,whereas NHE4 may have a specialized role in cell volume rectificationof inner medullary collecting duct cells. Knowledge of the correcttissue and cell-specific distribution of these two antiporters shouldhelp significantly in understanding their physiological roles.

     

Regulation of the Na+/H+ exchanger in fibroblasts overexpressing the Na+/Ca2+ exchanger

To assess the role of Ca²⁺in regulation of theNa⁺/H⁺exchanger (NHE1), we used CCL-39 fibroblasts overexpressing theNa⁺/Ca²⁺exchanger (NCX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca²⁺ rise andlong-lasting cytoplasmic alkalinization (60-80% inhibition) induced by -thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant withthe calmodulin (CaM)-binding domain deleted (amino acids 637-656),suggesting that the effect of NCX1 transfection involves Ca²⁺-CaM binding. Expression ofNCX1 only slightly inhibited platelet-derived growth factor BB-inducedalkalinization and did not affect hyperosmolarity- or phorbol12-myristate 13-acetate-induced alkalinization. Downregulation ofprotein kinase C (PKC) inhibited thrombin-induced alkalinization partially in control cells and abolished it completely inNCX1-transfected cells, suggesting that the thrombin effect is mediatedexclusively via Ca²⁺ and PKC. Onthe other hand, deletion mutant study revealed that PKC-dependentregulation occurs through a small cytoplasmic segment (amino aids566-595). These data suggest that a mechanism involving directCa²⁺-CaM binding lasts for arelatively long period after agonist stimulation, despite apparentshort-lived Ca²⁺ mobilization, andfurther support our previous conclusion that Ca²⁺- and PKC-dependent mechanismsare mediated through distinct segments of the NHE1 cytoplasmic domain.

     

Expression and characterization of the Na+/H+ exchanger in the mammalian myocardium

We examined two expression systems for studying the Na⁺/H⁺ exchanger in the mammalian myocardium. Mammalian NHE1 with a hemagglutinin (HA) tag and was cloned behind the alpha myosin heavy chain promoter. Transgenic mice were made with wild type NHE1 protein or with a hyperactive NHE1 protein mutated at the calmodulin-binding domain. Three lines of transgenic mice were made of each cDNA with expression levels of each type varying from high to low. Higher levels and activity of the Na⁺/H⁺ exchanger were associated with decreased long-term survival of mice, and with dilated or hypertrophic cardiomyopathy. The exogenous NHE1 protein was present in freshly made cardiomyocytes from transgenic mice, however, expression from the alpha myosin heavy chain promoter declined rapidly and little exogenous NHE1 was apparent on the fourth day after cardiomyocyte isolation. To express NHE1 protein in isolated cardiomyocytes, we transferred a mutated form of the protein into an adenoviral expression system. Infection of neonatal rat cardiomyocytes resulted in robust expression of the exogenous NHE1 protein. The mutant form of the NHE1 protein could be distinguished from the endogenous Na⁺/H⁺ exchanger by its resistance to inhibition by amiloride analogs. Our results suggest that for in vivo studies on intact hearts and animals, expression in transgenic mice is an appropriate system, however for long-term studies on cardiomyocytes, this model is inappropriate due to waning expression from the alpha myosin heavy chain promoter. Therefore, infection by adenovirus is a superior system for long-term studies on cardiomyocytes in culture.     

Regulation of mesangial cell Na+/Ca2+ exchanger isoforms

An isoform of the Na(+)/Ca(2+) exchanger (SDNCX1.10) was cloned from mesangial cells of Sprague-Dawley rat. Regulation of this isoform was compared to two other clones that were derived from the Dahl/Rapp salt sensitive (SNCX) and salt resistant rat (RNCX). All isoforms differ at the alternative splice site and at amino acid 218 for SNCX. PKC activates RNCX but not SNCX while SDNCX1.10 was also activated by PKC. Regulation of exchanger activities by intracellular calcium ([Ca(2+)](i)), pH, and kinases was assessed using Na-dependent (45)Ca(2+) uptake assays in OK-PTH cells expressing the vector, RNCX, SNCX, or SDNCX1.10. [Ca(2+)](i) was elevated from 50 to 125 nM (n = 4) with thapsigargin (40 nM) and reduced from 50 to 29 nM (n = 4) and 18 nM (n = 4) with 10 or 20 microM BAPTA, respectively. RNCX was active at all three [Ca(2+)](i) while SNCX and SDNCX1.10 were only active at lower [Ca(2+)](i). Varying extracellular pH (pH(e), without nigericin) or pH(e) and intracellular pH (pH(i), with 10 microM nigericin) from pH 7.4 to 6.2, 6.8, or 8.0 showed that SNCX activity was attenuated at both low and high pHs. SDNCX1.10 activity was attenuated only at pH 6.2 and 6.8 (with or without nigericin) while RNCX activity was attenuated at pH 6.2 (with or without nigericin) and pH 6.8 (with nigericin). Finally, only SDNCX1.10 activity was stimulated by 250 microM CPT-cAMP or 250 microM DB-cGMP treatment. Thus the differential regulation of [Ca(2+)](i) by these exchangers is dependent upon the pattern of cellular Na(+)/Ca(2+) exchanger isoform expression.     

Prenatal programming of rat proximal tubule Na+/H+ exchanger by dexamethasone

Prenatal administration of dexamethasone causes hypertension in rats when they are studied as adults. Although an increase in tubular sodium reabsorption has been postulated to be a factor programming hypertension, this has never been directly demonstrated. The purpose of this study was to examine whether prenatal programming by dexamethasone affected postnatal proximal tubular transport. Pregnant Sprague-Dawley rats were injected with intraperitoneal dexamethasone (0.2 mg/kg) daily for 4 days between the 15th and 18th days of gestation. Prenatal dexamethasone resulted in an elevation in systolic blood pressure when the rats were studied at 7-8 wk of age compared with vehicle-treated controls: 131 +/- 3 vs. 115 +/- 3 mmHg (P < 0.001). The rate of proximal convoluted tubule volume absorption, measured using in vitro microperfusion, was 0.61 + 0.07 nl.mm(-1).min(-1) in control rats and 0.93+ 0.07 nl.mm(-1).min(-1) in rats that received prenatal dexamethasone (P < 0.05). Na(+)/H(+) exchanger activity measured in perfused tubules in vitro using the pH-sensitive dye BCECF showed a similar 50% increase in activity in proximal convoluted tubules from rats treated with prenatal dexamethasone. Although there was no change in abundance of NHE3 mRNA, the predominant luminal proximal tubule Na(+)/H(+) exchanger, there was an increase in NHE3 protein abundance on brush-border membrane vesicles in 7- to 8-wk-old rats receiving prenatal dexamethasone. In conclusion, prenatal administration of dexamethasone in rats increases proximal tubule transport when rats are studied at 7-8 wk old, in part by stimulating Na(+)/H(+) exchanger activity. The increase in proximal tubule transport may be a factor mediating the hypertension by prenatal programming with dexamethasone.     

Cytoplasmic pH in isolated rat enterocytes. Role of Na+/H+ exchanger

The cytoplasmic pH (pHi) was determined in isolated rat intestinal cells with four methods. The pHi of cells in physiological saline buffered with Hepes (pH 7.3) at 37 degrees C was close to 7.0. The most reliable method, using the fluorescent pH indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), furnished a mean value of 7.03 +/- 0.05 (n = 42). The buffering capacity of intestinal cells determined with this fluorescent indicator was 62 +/- 5 mmol.l-1.pH-1. The mechanism governing the control of cytoplasmic pH was also investigated with BCECF, varying the Na+ concentration inside and outside the cells. When intestinal cells were suspended in a sodium-free medium in the presence or absence of ouabain, they became acidified. The process was reversed when Na+ was added to the incubation medium. An identical phenomenon occurred when the cells were artificially acidified with NH4Cl. Additional experiments led to the conclusion that isolated rat intestinal cells have an Na+/H+ exchanger independent of Cl- and inhibited by amiloride. This exchanger plays an important but not exclusive role in the control of pHi. The presence of other exchangers and the high buffering power of the cells explains the high stability of pHi noted in this study.     

The co-presence of Na+/Ca2+-K+ exchanger and Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

We have previously shown that there is high Na(+)/Ca(2+) exchange (NCX) activity in bovine adrenal chromaffin cells. In this study, by monitoring the [Ca(2+)](i) change in single cells and in a population of chromaffin cells, when the reverse mode of exchanger activity has been initiated, we have shown that the NCX activity is enhanced by K(+). The K(+)-enhanced activity accounted for a significant proportion of the Na(+)-dependent Ca(2+) uptake activity in the chromaffin cells. The results support the hypothesis that both NCX and Na(+)/Ca(2+)-K(+) exchanger (NCKX) are co-present in chromaffin cells. The expression of NCKX in chromaffin cells was further confirmed using PCR and northern blotting. In addition to the plasma membrane, the exchanger activity, measured by Na(+)-dependent (45)Ca(2+) uptake, was also present in membrane isolated from the chromaffin granules enriched fraction and the mitochondria enriched fraction. The results support that both NCX and NCKX are present in bovine chromaffin cells and that the regulation of [Ca(2+)](i) is probably more efficient with the participation of NCKX.     

Combined blockade of the Na+ channel and the Na+/H+ exchanger virtually prevents ischemic Na+ overload in rat hearts

Blocking either the Na⁺ channel or the Na⁺/H⁺ exchanger (NHE) has been shown to reduce Na⁺ and Ca²⁺ overload during myocardial ischemia and reperfusion, respectively, and to improve post-ischemic contractile recovery. The effect of combined blockade of both Na⁺ influx routes on ionic homeostasis is unknown and was tested in this study. [Na⁺]_i, pH_i and energy-related phosphates were measured using simultaneous ²³Na- and ³¹P-NMR spectroscopy in isolated rat hearts. Eniporide (3 μM) and/or lidocaine (200 μM) were administered during 5 min prior to 40 min of global ischemia and 40 min of drug free reperfusion to block the NHE and the Na⁺ channel, respectively. Lidocaine reduced the rise in [Na⁺]_i during the first 10 min of ischemia, followed by a rise with a rate similar to the one found in untreated hearts. Eniporide reduced the ischemic Na⁺ influx during the entire ischemic period. Administration of both drugs resulted in a summation of the effects found in the lidocaine and eniporide groups. Contractile recovery and infarct size were significantly improved in hearts treated with both drugs, although not significantly different from hearts treated with either one of them.     

Epoxyeicosatrienoic acids activate Na+/H+ exchange and are mitogenic in cultured rat glomerular mesangial cells

The present study examined responses of cultured rat glomerular mesangial cells to exogenous exposure of epoxyeicosatrienoic acids (EET's), products of cytochrome P450 epoxygenase. One day after administration of 8,9- or 14,15-EET, cultured rat mesangial cells demonstrated significant increases in [3H]thymidine incorporation (10(-7) M 14,15-EET: 120 +/- 7% of control; n = 6; P less than 0.025; 10(-6) M 14,15-EET: 145 +/- 10%; n = 20; P less than 0.0005; 10(-6) M 8,9-EET: 167 +/- 31%; n = 9; P less than 0.05), which was not affected by addition of the cyclooxygenase inhibitor indomethacin. In addition to stimulation of [3H]thymidine incorporation, the epoxides stimulated mesangial cell proliferation. 14,15-EET administration induced intracellular alkalinization of 0.2-0.3 pH units, which was prevented by extracellular Na+ removal and blunted by amiloride (0.5 mM). Following intracellular acidification with NH4Cl addition and removal, greater than 85% of 3 mM 22Na uptake into mesangial cells was inhibited by 1 mM amiloride, indicating Na+/H+ exchange. Under these conditions, 14,15-EET stimulated Na+/H+ exchange by 42% and 8,9-EET stimulated Na+/H+ exchange by 59%. Neither protein kinase C depletion nor addition of the protein kinase C inhibitor, staurosporine, affected this stimulation. In [3H]myo-inositol loaded mesangial cells, no significant stimulation of phosphoinositide hydrolysis was detected in response to administration of 14,15-EET. Twenty-four hours after addition of [14C]14,15-EET, greater than 90% was preferentially esterified to cellular lipids, with predominant incorporation into phosphatidylinositol, phosphatidylethanolamine, and diacylglycerol. Thus, these results demonstrate epoxyeicosatrienoic acids stimulate Na+/H+ exchange and mitogenesis in mesangial cells. These effects do not appear to be mediated via phospholipase C activation. In addition, 14,15-EET was selectively incorporated into cellular lipids known to mediate signal transduction. These observations extend the potential biologic roles of c-P450 arachidonate metabolites to include stimulation of cell proliferation and suggest a role for these compounds in vascular and renal injury.