This review describes recent progress concerning the molecular aspects of the Na+/H+ exchanger. The Na+/H+ exchanger is an important regulator for intracellular pH, cell volume, and transepithelial Na+ transport. It exists in virtually all cells with cell type-dependent pattern of isoform expression, and it is regulated in response to a variety of extracellular stimuli, among them not only agonists such as growth factors and hormones but also mechanical stimuli such as osmotic stress and cell spreading. Thus this transporter is also an excellent model to study the signal transduction. Since the first molecular cloning of the Na+/H+ exchanger, detailed studies revealed many interesting features of this transporter. At present, at least five different isoforms of the Na+/H+ exchanger are known. These isoforms differ in tissue localization, sensitivity of inhibitors, and mode of transcriptional and posttranscriptional regulation, allowing them to participate in different physiological processes. We have only started to understand an intriguing mechanism underlying these functional differences among the exchanger isoforms. Because the Na+/H+ exchanger is relatively simple in terms of its kinetic features, e.g., a simple 1:1 stoichiometry of Na+ and H+ and no input of metabolic energy such as ATP hydrolysis, the study of its structural and mechanistic aspects would also serve as a good model to understand the general mechanism of various ion transporters.

No.7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), a selective inhibitor of the Na+/Ca2+ exchanger (NCX1), has been newly synthesized. It dose-dependently inhibited Na+i-dependent 45Ca2+ uptake and Na+i-dependent [Ca2+]i increase in cardiomyocytes, smooth muscle cells, and NCX1-transfected fibroblasts (IC50 = 1.2-2.4 microM). Inhibition was observed without prior incubation with the agent and was completely reversed by washing cells with buffer for 1 min. Interestingly, No.7943 was much less potent in inhibiting Na+o-dependent 45Ca2+ efflux and Na+o-induced [Ca2+]i decline (IC50 = >30 microM), indicating that it selectively blocks the reverse mode of Na+/Ca2+ exchange in intact cells. In cardiac sarcolemmal preparations consisting mostly of inside-out vesicles, the agent inhibited Na+i-dependent 45Ca2+ uptake and Na+o-dependent 45Ca2+ efflux with similar, but slightly lower, potencies (IC50 = 5.4-13 microM). Inhibition was noncompetitive with respect to Ca2+ and Na+ in both cells and sarcolemmal vesicles. These results suggest that No.7943 primarily acts on external exchanger site(s) other than the transport sites in intact cells, although it is able to inhibit the exchanger from both sides of the plasma membrane. No.7943 at up to 10 microM does not affect many other ion transporters nor several cardiac action potential parameters. This agent at these concentrations also did not influence either diastolic [Ca2+]i or spontaneous beating in cardiomyocytes. Furthermore, No.7943 markedly inhibited Ca2+ overloading into cardiomyocytes under the Ca2+ paradox conditions. Thus, No.7943 is not only useful as a tool with which to study the transport mechanism and physiological role of the Na+/Ca2+ exchanger but also has therapeutic potential as a selective blocker of excessive Ca2+ influx mediated via the Na+/Ca2+ exchanger under pathological conditions.

Abstract-Changes in membrane tension resulting from membrane stretch represent one of the key elements in blood flow regulation in vascular smooth muscle. However, the molecular mechanisms involved in the regulation of membrane stretch remain unclear. In this study, we provide evidence that a vanilloid receptor (TRPV) homologue, TRPV2 is expressed in vascular smooth muscle cells, and demonstrate that it can be activated by membrane stretch. Cell swelling caused by hypotonic solutions activated a nonselective cation channel current (NSCC) and elevated intracellular Ca 2ϩ ([Ca 2ϩ ] i ) in freshly isolated cells from mouse aorta. Both of these signals were blocked by ruthenium red, an effective blocker of TRPVs. The absence of external Ca 2ϩ abolished this increase in [Ca 2ϩ ] i caused by the hypotonic stimulation and reduced the activation of NSCC. Significant immunoreactivity to mouse TRPV2 protein was detected in single mouse aortic myocytes. Moreover, the expression of TRPV2 was found in mesenteric and basilar arterial myocytes. Treatment of mouse aorta with TRPV2 antisense oligonucleotides resulted in suppression of hypotonic stimulationinduced activation of NSCC and elevation of [Ca 2ϩ ] i as well as marked inhibition of TRPV2 protein expression. In Chinese hamster ovary K1 (CHO) cells transfected with TRPV2 cDNA (TRPV2-CHO), application of membrane stretch through the recording pipette and hypotonic stimulation consistently activated single NSCC. Moreover, stretch of TRPV2-CHO cells cultured on an elastic silicon membrane significantly elevated [Ca 2ϩ ] i . These results provide a strong basis for our purpose that endogenous TRPV2 in mouse vascular myocytes functions as a novel and important stretch sensor in vascular smooth muscles. Key Words: TRPV2 Ⅲ vanilloid receptor Ⅲ mouse aorta Ⅲ membrane stretch Ⅲ vascular smooth muscle D etection of mechanical stimuli is essential for diverse biological functions including audition, touch, and maintenance of vascular myogenic tone. In the latter, elevation of intravascular pressure depolarizes vascular smooth muscle cells via membrane stretch. 1,2 This depolarization activates voltage-dependent L-type Ca 2ϩ channels (VDCC) and increases [Ca 2ϩ ] i , resulting in vasoconstriction and/or myogenic tone. 3 A large component of the elevation of [Ca 2ϩ ] i by myogenic tone can be inhibited by blockers of VDCC. However, some components are resistant to these agents, and instead can be accounted for by a separate nonselective cation channel that is permeable to Ca 2ϩ and also is activated by the intravascular pressure. 4,5 Because stretchactivated channels play obligatory roles in regulation of the myogenic tone, extensive studies have been performed to identify the molecular entity of these channels. Originally, a yeast MID1 gene product (MID1) was shown to be a eukaryotic stretch-activated channel, because CHO cells expressing MID1 responded to membrane stretch. 6 A member of the transient receptor potential channels (TRP), specifically TRPC6, is sensitive ...

The membrane topology of the human Na ؉ /H ؉ exchanger isoform 1 (NHE1) was assessed by substituted cysteine accessibility analysis. Eighty-three cysteine residues were individually introduced into a functional cysteineless NHE1, and these mutants were expressed in the exchanger-deficient PS120 cells. The topological disposition of introduced cysteines was determined by labeling with a biotinylated maleimide in the presence or absence of preincubation with the membrane-impermeable sulfhydryl reagent, 2-trimethylammoniumethylmethanethiosulfonate in streptolysin O-permeabilized or nonpermeabilized cells. We proposed a new model for the topology of NHE1 that is significantly different from the model derived from hydropathy analysis. In this model, NHE1 is composed of 12 transmembrane segments (TMs) with the N and C termini located in the cytosol. The large, last extracellular loop in the membrane domain of the original model was suggested to comprise an intracellular loop, a new transmembrane segment (TM11), and an extracellular loop in the new model. Interestingly, cysteines at 183 and 184 and at 324 and 325 mapped to intracellular loops connecting TMs 4 and 5 (IL2) and TMs 8 and 9 (IL4), respectively, were accessible to sulfhydryl reagents from the outside. Furthermore, exchange activities of two mutants, R180C and Q181C, within IL2 were markedly inhibited by external MTSET. These data suggest that part of IL2 or IL4 may be located in a pore-lining region that is accessible from either side of the membrane and involved in ion transport.

Despite unfavorable conditions, a single species of fish, Osorezan dace, lives in an extremely acidic lake (pH 3.5) in Osorezan, Aomori, Japan. Physiological studies have established that this fish is able to prevent acidification of its plasma and loss of Na(+). Here we show that these abilities are mainly attributable to the chloride cells of the gill, which are arranged in a follicular structure and contain high concentrations of Na(+)-K(+)-ATPase, carbonic anhydrase II, type 3 Na(+)/H(+) exchanger (NHE3), type 1 Na(+)-HCO(3)(-) cotransporter, and aquaporin-3, all of which are upregulated on acidification. Immunohistochemistry established their chloride cell localization, with NHE3 at the apical surface and the others localized to the basolateral membrane. These results suggest a mechanism by which Osorezan dace adapts to its acidic environment. Most likely, NHE3 on the apical side excretes H(+) in exchange for Na(+), whereas the electrogenic type 1 Na(+)-HCO(3)(-) cotransporter in the basolateral membrane provides HCO(3)(-) for neutralization of plasma using the driving force generated by Na(+)-K(+)-ATPase and carbonic anhydrase II. Increased expression of glutamate dehydrogenase was also observed in various tissues of acid-adapted dace, suggesting a significant role of ammonia and bicarbonate generated by glutamine catabolism.