H+ disposal by rabbit gastric mucosal surface cells

Exposure of isolated gastric mucosal surface cells to NH4+ results in acidification of cells as determined by a fluorescent dye technique using acridine orange. The resulting intracellular pH gradient is maintained when cells are suspended in either buffered HCO3- -free Ringer's or choline chloride solution. Cells suspended in a Na+-containing but K+-free solution exhibit dissipation of the proton gradient. When Na+ is added to cells suspended in Na+, K+-free solution, the gradient rapidly dissipates with a half-maximal response occurring at 56 mM Na+. The effect of Na+ is amiloride sensitive with half-maximal inhibition occurring at 38 microM at a Na+ concentration of 50 mM. The K+ does not cause dissipation of the gradient and neither ouabain nor valinomycin have an effect. Yet, K+ has a modulating influence on Na+/H+ exchange by the isolated surface cells. The addition of K+ to acid-loaded cells resuspended in Na+-free solution decreases the ability of subsequent Na+ addition to evoke gradient dissipation. The data suggest that Na+/H+ exchange appears to be at least one mechanism whereby gastric mucosal surface cells could protect themselves against diffusing acid. This ion exchange mechanism is amiloride sensitive and appears to be unrelated to Na+, K+ adenosine triphosphatase activity, but is affected by the external K+ concentration.     

Transient state kinetic evidence for an oligomer in the mechanism of Na+-H+ exchange.

Pre-steady-state kinetic measurements of 22Na+ uptake by the amiloride-sensitive Na+-H+ exchanger in renal brush border membrane vesicles (BBMV) were performed at 0 degrees C to characterize the intermediate reactions of the exchange cycle. At 1 mM Na+, the initial time course of Na+ uptake was resolved into three separate components: (i) a lag phase, (ii) an exponential or "burst" phase, and (iii) a constant velocity or steady-state phase. Pulse-chase experiments using partially loaded BBMV showed no evidence for 22Na+ back-flux, suggesting that the decline in the rate of Na+ uptake rate following the burst represents completion of the first turnover of the exchanger. Gramicidin completely abolished Na+ uptake, indicating that the burst phase results from the translocation of Na+ rather than from residual Na+ binding to external sites. Raising the [Na+] from 1 to 10 mM at constant pH (internal pH 5.7; external pH 7.7) produced a sigmoidal increase in the amplitude of the burst phase without affecting the lag duration or the apparent burst rate. In contrast, Na+ uptake in the steady state obeyed Michaelis-Menten kinetics. These results suggest that a minimum of two Na+ transport sites must be occupied to activate Na+ uptake in the pre-steady state. The transition to Michaelis-Menten kinetics in the steady state can be explained by a "flip-flop" or alternating site mechanism in which the functional transport unit is an oligomer and only one promoter per cycle is allowed to form a translocation complex with Na+ after the first turnover.     

Serum stimulates the Na+,K+ pump in quiescent fibroblasts by increasing Na+ entry.

Two ionophores (monensin and gramicidin) that carry Na+ into 3T3 cells markedly enhance the rate of 86Rb+ uptake. Ouabain prevents both ionophores from increasing 86Rb+ uptake, indicating that the ionophores activate the Na+,K+ pump. Measurements of 86Rb+ uptake and cell Na+ and K+ over a range of monensin concentrations show that the activity of the Na+,K+ pump in 3T3 cells is limited by the supply of internal Na+ and is extremely sensitive to small changes in internal Na+. Serum rapidly enhances the rate of 22Na+ uptake and net Na+ entry when Na+ exit is inhibited by ouabain. At 0.3 microgram/ml, monensin increases the rate of net Na+ entry and activates the Na+,K+ pump by the same degree as serum. The stimulation of 86Rb+ uptake by serum or the ionophores has an absolute requirement for external Na+. Thus, serum appears to stimulate the Na+,K+ pump in quiescent 3T3 cells by increasing its supply of Na+.     

Developmental changes of sarcolemmal Na+-H+ exchange

We previously demonstrated that the effect of respiratory acidosis on cardiac contractility in the newborn was less than in the adult rabbit, and these data suggested a higher [Na+]i and [Na+]i-[Ca2+]o exchange in the newborn as compared to the adult. In this study, we investigated developmental changes of Na+-H+ exchange in isolated sarcolemmal vesicles. Sarcolemmal purification for Na+-K ATPase was 61.9 and 67.1 fold in the newborn and the adult rabbit heart, respectively. In the presence of an outwardly directed proton gradient across the vesicular membrane, sarcolemmal 22Na uptake rate in the newborn (0.22 +/- 0.01 nmol Na+/mg prot/s) was significantly higher than than in the adult (0.16 +/- 0.01 nmol Na+/mg prot/s). 1.0 mM amiloride inhibited 22Na uptake by 75% and 80% in the newborn and the adult, respectively. In the absence of a pH gradient, vesicular 22Na uptake in the newborn and the adult were not significantly different. In conclusion, the higher Na+-H+ exchange in the newborn may lead to a higher [Na+]i and subsequent calcium influx via Na+-Ca2+ exchange as compared with the adult during acidosis. This may explain the greater recovery of mechanical function in the newborn heart as compared to the adult heart during acidosis.     

Possible evidence for transmembrane K(+)-H+ exchange system in guinea pig myocardium.

The aim of this study was to obtain evidence for a transmembrane K(+)-H+ exchange system in Langendorff-perfused whole hearts and isolated ventricular myocytes of guinea pig. Effluent relation between K+ and pH in the whole hearts perfused with HEPES-buffered Tyrode's solution indicated a significant (p < 0.05) functional coupling of K+ uptake and H+ extrusion that was energy-dependent and omeprazole (OPZ)-sensitive. Administration of OPZ (0.3 mM) or dimethylamiloride (0.1 mM), an inhibitor of Na(+)-H+ antiport, to whole hearts subjected to the repetitive NH4Cl applications implied that both Na(+)-H+ and putative K(+)-H+ countertransports contribute to the regulation of intracellular pH. In isolated myocytes, voltage-dependent L-type Ca current (ICa) was inhibited by OPZ (0.3 mM) under K(-)- and Na(+)-free condition by 11 to 14%, and was inhibited to a greater extent (i.e., by 36 to 40%) by this agent in the presence of K+. OPZ-induced inhibition of the putative K(+)-H+ exchanger likely resulted in subsarcolemmal acidification which was responsible for the rate-independent suppression of ICa. In conclusion, these data provide functional evidence for a myocardial transmembrane K(+)-H+ exchanger.     

Isoproterenol stimulates rapid extrusion of sodium from isolated smooth muscle cells.

beta-Agonists cause an inhibition of contractility and a transient stimulation of Na+/K+ pumping in smooth muscle cells of the stomach from the toad Bufo marinus. To determine if the stimulation of Na+/K+ pumping causes changes in intracellular [Na+] ([Na+]i) that might link Na+ pump stimulation to decrease Ca2+ availability for contraction, [Na+]i was measured in these cells with SBFI, a Na(+)-sensitive fluorescent indicator. Basal [Na+]i was 12.8 +/- 4.2 mM (n = 32) and was uniform throughout the cell. In response to isoproterenol, [Na+]i decreased an average of 7.1 +/- 1.1 mM in 3 sec. Since this decrease in [Na+]i could be completely blocked by inhibition of the Na+ pump, or by blockade of the beta-receptor, [Na+]i reduction is the result of occupation of the beta-receptor by isoproterenol and subsequent stimulation of the Na+ pump. 8-Bromoadenosine 3',5'-cyclic monophosphate and forskolin mimicked the effect of isoproterenol, indicating that the sequence of events linking beta-receptor occupation to Na+ pump stimulation most likely includes activation of adenylate cyclase, production of cAMP, and stimulation of cAMP-dependent protein kinase. The decrease in [Na+]i is sufficiently large and fast that it is expected to stimulate turnover of the Na+/Ca2+ exchanger in the Ca2+ extrusion mode, thereby accounting for the observed linkage between stimulation of the Na+/K+ pump and inhibition of contractility in response to beta-adrenergic agonists.     

Sodium-lithium exchange and sodium-proton exchange are mediated by the same transport system in sarcolemmal vesicles from bovine superior mesenteric artery

Several laboratories have reported that Na+-Li+ countertransport activities are increased in red blood cells from patients with essential hypertension. It has been proposed that the activity of this red blood cell transport system might reflect the activity of a similar system in vascular smooth muscle. We previously demonstrated Na+-Li+ exchange in sarcolemmal vesicles from canine artery and proposed that this transport function might be mediated by the Na+-H+ exchanger. In the present studies, however, we were unable to demonstrate Na+-Li+ countertransport in canine red blood cells. Since bovine red blood cells have a vigorous Na+-Li+ exchanger and we previously demonstrated Na+-H+ exchange in sarcolemmal vesicles from bovine artery, we wished to determine whether bovine sarcolemmal vesicles mediate Na+-Li+ exchange and whether this transport function is mediated via the Na+-H+ exchanger. We found that an outwardly directed proton or Li+ gradient stimulated 22Na+ uptake in sarcolemmal vesicles from bovine superior mesenteric artery. Li+ gradient-stimulated Na+ uptake was not due to electrical coupling between the two ions, was not affected by a change in membrane potential, and could not be explained by the parallel operation of Li+-H+ and Na+-H+ exchange. External Li+ inhibited proton gradient-stimulated Na+ uptake, and external protons inhibited Li+ gradient-stimulated Na+ uptake. Na+ efflux from vesicles was stimulated by inwardly directed gradients for Li+ or protons, and these effects were not additive. Proton efflux from vesicles was stimulated by inwardly directed gradients for Na+ or Li+, and these effects were not additive. Finally, Na+-H+ exchange and Na+-Li+ exchange in sarcolemmal vesicles were inhibited by 5-(N-ethyl-N-isopropyl)amiloride in an identical dose-dependent manner. In conclusion, Na+-Li+ countertransport could not be demonstrated in canine red blood cells, but as is the case with bovine red blood cells, sarcolemmal vesicles from bovine artery mediate Na+-Li+ countertransport. This transport function and sarcolemmal Na+-H+ exchange are mediated via a single 5-(N-ethyl-N-isopropyl)amiloride-sensitive cation exchanger with affinity for Na+, Li+, and protons. The cow, as opposed to the dog, may be a good animal model to test whether the activity of red blood cell Na+-Li+ countertransport is predictive of the activity of Na+-Li+ (and Na+-H+) exchange in vascular smooth muscle.     

Na(+)-Ca2+ exchange, myoplasmic Ca2+ concentration, and contraction of arterial smooth muscle.

Na(+)-Ca2+ exchange is proposed to be an important regulator of myoplasmic intracellular Ca2+ concentration ([Ca2+]i) and contraction in vascular smooth muscle. We investigated the role of Na(+)-Ca2+ exchange in regulating [Ca2+]i in swine carotid arterial tissues that were loaded with aequorin to allow simultaneous measurement of [Ca2+]i and force. Reversal of Na(+)-Ca2+ exchange, by reduction of extracellular Na+ concentration ([Na+]o) to 1.2 mM, induced a large increase in aequorin-estimated [Ca2+]i and a low [Ca2+]i sensitivity. The contraction induced by 1.2 mM [Na+]o was partially caused by depolarization and opening of L-type Ca2+ channels because 10 microM diltiazem partially attenuated the 1.2 mM [Na+]o-induced increases in [Ca2+]i. High dose ouabain (10 microM), a putative endogenous Na+,K(+)-ATPase inhibitor, increased both [Ca2+]i and force. However, the increases in [Ca2+]i and force were mostly blocked by 10 microM phentolamine, suggesting the predominant effect of ouabain was to increase norepinephrine release from nerve terminals. In the presence of 10 microM phentolamine, 10 microM ouabain slightly accentuated 1 microM histamine-induced increases in [Ca2+]i and force. The ouabain dose necessary to induce contraction in the absence of phentolamine was significantly less than the ouabain dose necessary to accentuate histamine-induced contractions in the presence of phentolamine. These results suggest that Na(+)-Ca2+ exchange exists in swine arterial smooth muscle. These data also suggest that ouabain (which should increase [Na+]i and inhibit Na(+)-Ca2+ exchange) primarily enhances contractile function in the swine carotid artery by releasing catecholamines from nerve terminals; direct action of Na+,K(+)-ATPase inhibitors on smooth muscle appears to occur only with very high doses.     

Inhibition of Na+,K+ pump affects nucleic acid synthesis and smooth muscle cell proliferation via elevation of the [Na+]i/[K+]i ratio: possible implication in vascular remodelling

OBJECTIVES : Na+,K+ pump inhibition is known to delay the development of apoptosis in vascular smooth muscle cells (VSMC). This study examines Na+,K+ pump involvement in the regulation of VSMC macromolecular synthesis and proliferation. METHODS : DNA, RNA and protein synthesis in VSMC from the rat aorta was studied by the incorporation of [3H]-labelled thymidine, uridine and leucine. Cell cycle progression was estimated by flow cytometry. Intracellular Na+ and K+ content and Na+,K+ pump activity were quantified as the steady-state distribution of 22Na and 86Rb and the rate of ouabain-sensitive 86Rb uptake in Na+-loaded cells, respectively. RESULTS : Ouabain inhibited the Na+,K+ pump with a Ki of 0.1 mmol/l. At concentrations less than 0.1 mmol/l, neither [Na+]i nor [K+]i was affected by ouabain; elevation of ouabain concentration sharply increased the [Na+]i/[K+]i ratio with a K0.5 of approximately 0.3 mmol/l. At concentrations higher than 0.1 mmol/l, ouabain time- and dose-dependently activated RNA and DNA syntheses in serum-deprived VSMC and inhibited cell cycle progression triggered by serum. In quiescent VSMC, ouabain did not affect protein synthesis, total cell number, but slightly increased the percentage of cells in the S-phase (4.25 versus 1.46%) and attenuated cell death assessed by staining with trypan blue and lactate dehydrogenase release. CONCLUSIONS : Elevation of the [Na+]i/[K+]i ratio caused by Na+,K+ pump inhibition markedly enhances nucleic acid synthesis in quiescent VSMC and blocks cell cycle progression in serum-supplied VSMC. The relative contribution of this phenomenon as well as the anti-apoptotic action of increased [Na+]i/[K+]i ratio to vascular remodelling under augmented content of endogenous Na+,K+ pump inhibitors, seen in volume-expanded hypertension, should be investigated by in-vivo studies.     

Noninactivating, tetrodotoxin-sensitive Na+ conductance in rat optic nerve axons.

The ionic current underlying the upstroke of axonal action potentials is carried by rapidly activating, voltage-dependent Na+ channels. Termination of the action potential is mediated in part by the rapid inactivation of these Na+ channels. We previously demonstrated that an influx of Na+ plays a critical role in the cascade leading to irreversible anoxic injury in central nervous system white matter. We speculated that a noninactivating Na+ conductance mediates this pathological Na+ influx and persists at depolarized membrane potentials as seen in anoxic axons. In the present study we measured the resting compound membrane potential of rat optic nerves using a modified "grease-gap" technique. Application of tetrodotoxin (2 microM) to resting nerves ([K+]o = 3 mM) or to nerves depolarized by 15 or 40 mM K+ resulted in hyperpolarizing shifts of membrane potential. We interpret these shifts as evidence for a persistent, noninactivating Na+ conductance. This conductance is present at rest and persists in nerves depolarized sufficiently to abolish classical transient Na+ currents. PK/PNa ratios were estimated at 35.5, 23.2, and 88 in 3 mM, 15 mM, and 40 mM K+, respectively. We suggest that this noninactivating Na+ conductance may provide an inward pathway for Na+ ions, necessary for the operation of Na+, K(+)-ATPase. Under pathological conditions, such as anoxia, this conductance is the likely route of Na+ influx, which causes damaging Ca2+ entry through reverse operation of the Na(+)-Ca2+ exchanger. The presence of this conductance in white matter axons may provide a therapeutic opportunity for diseases such as stroke and spinal cord injury.     

The sodium/hydrogen exchange system in cardiac cells: its biochemical and pharmacological properties and its role in regulating internal concentrations of sodium and internal pH

This paper describes the properties of the amiloride-sensitive Na+/H+ antiporter in chick cardiac cells, compares them with those known in other cellular systems and analyzes the role of the Na+/H+ exchanger in the regulation of internal Na+ concentrations and internal pH. Among the different properties which have been studied one can mention: (i) The external Na+ concentration [( Na+]o) dependence: the activity increases when [Na+]o increases (KNa+ = 20 mM); (ii) The external pH (pHo) dependence: the activity of the exchanger increases when pHo increases (pHmo = 7.05 and Hill coefficient = 1); (iii) The internal pH (pHi) dependence; the activity of the exchanger increases in a cooperative way when internal pH (pHi) decreases (pHmi = 7.35 and Hill coefficient = 3); (iv) There are derivatives of amiloride which are 200 times more potent than amiloride itself (Kethylisopropylamiloride = 30 nM) and which are selective on the Na+/H+ exchange system v. other Na+ transporting system including the Na+/Ca2+ exchange system. Under physiological conditions, the Na+/H+ exchange system contributes little to the regulation of the internal pH of chick cardiac cells. The antiporter then serves as an uptake system for Na+ using the H+ gradient created by other pHi regulatory mechanisms. Treatment of cardiac cells with ouabain inhibits Na+ efflux and produced an increase in intracellular Na+ activity. Ethylisopropylamiloride was used to show that the Na+/H+ exchange system is the main pathway for Na+ entry and accumulation in digitalis action. As expected amiloride derivatives which block Na+ entry via the Na+/H+ antiporter were found to antagonize ouabain action on cardiac cells. When the internal pH of cardiac cells is lowered, the Na+/H+ exchanger becomes the major pHi regulating system. It is the essential system by which cardiac cells recover from cellular acidosis. The situation is due both to an increased activity of the exchanger at acidic pHi and to a decreased activity of other pHi regulatory systems. We propose in this paper that the Na+/H+ exchange system plays a key role in Na+ accumulation followed by Ca2+ accumulation which is observed when ischemic hearts are reperfused.     

Na+-Ca2+ exchange in cultured vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) contract as intracellular free calcium ([Ca2+]i) rises. While Na+-Ca2+ exchange has been proposed to contribute to transmembrane Ca2+ flux, its role in cultured VSMC is unknown. Accordingly, we have investigated the role of Na+-Ca2+ exchange in unidirectional and net transmembrane Ca2+ fluxes in cultured rat aortic VSMC under basal conditions and following agonist-mediated stimulation. Transmembrane Ca2+ uptake was significantly increased in response to a low external Na+ concentration ([Na+]o) compared with 140 mM [Na+]o. Na+-dependent Ca2+ uptake in response to low [Na+]o was further increased by intracellular Na+ loading by preincubation of the VSMC with 1 mM ouabain. Under steady-state conditions, Ca2+ content varied inversely with [Na+]o, increasing from 1.0 nmol Ca2+/mg protein at 140 mM [Na+]o to 4.0 nmol Ca2+/mg protein at 20 mM [Na+]o. Increasing [K+]o to 55 mM also enhanced Na+-dependent Ca2+ influx. Augmentation of Ca2+ uptake with K+ depolarization was not significantly inhibited by the calcium channel antagonist verapamil. Transmembrane Ca2+ efflux was increased in response to 130 mM [Na+]o compared with zero [Na+]o (iso-osmotic substitution with choline+), and was further stimulated by the vasoconstrictor angiotensin II, which is known to elevate [Ca2+]i. These changes in [Ca2+]i were studied directly using fura-2 fluorescence measurements. Elevated [Ca2+]i levels returned to baseline more rapidly in the presence of normal (130 mM) [Na+]o compared with zero [Na+]o (iso-osmotic substitution with choline+). These findings suggest that a bidirectional Na+-Ca2+ exchange mechanism is present in cultured rat aortic VSMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Na+,K+-ATPase by interferon gamma down-regulates intestinal epithelial transport and barrier function

BACKGROUND & AIMS: To determine how interferon (IFN)-gamma inhibits epithelial barrier and ion transport functions, intestinal T84 cells were studied. METHODS: Acute and chronic effects of IFN-gamma on T84 barrier function, Na+,K+-adenosine triphosphatase (ATPase) activity, and certain ion transport and tight junctional proteins were determined. To assess the role of Na+,K+-ATPase and intracellular Na+, similar studies with the Na+,K+-ATPase inhibitor ouabain and Na+ ionophore monensin were performed. To determine the role of nitric oxide (NO), the NO donor SPER-NO was used. RESULTS: IFN-gamma acutely (<6 hour) decreased cellular Na+,K+-ATPase activity, followed later (>24 hours) by decreases in expression of Na/K/2Cl, the alpha subunit of Na+,K+-ATPase, occludin, and ZO-1. In contrast, cystic fibrosis transmembrane conductance regulator or the Na+ pump beta subunit were unchanged. Ouabain and monensin caused nearly identical changes to IFN-gamma. Incubation in low Na+ media significantly blunted the chronic effects of IFN-gamma. Hypotonic-induced cell swelling, in contrast, had effects similar to IFN-gamma but did not alter the expression of the Na+ pump alpha subunit. The NO donor SPER-NO rapidly inhibited Na+,K+-ATPase and also down-regulated transport and barrier proteins. CONCLUSIONS: IFN-gamma inhibition of Na+,K+-ATPase activity acutely causes increases in intracellular Na(i) concentration and cell volume, which are distinct signaling events that ultimately result in a leaky and dysfunctional epithelium associated with chronic inflammation.     

Vasopressin acts on platelets to generate procoagulant activity

The aim of our study was to examine whether arginine vasopressin (AVP) is able to evoke in human platelets a procoagulant response due to activation of an Na+/H+ exchanger. It was found that treatment of platelets with AVP (20-100 nmol/l) results in generation of a weak calcium signal, activation of Na+/H+ exchanger, aggregation, and development of a procoagulant response. The AVP-evoked procoagulant response was dose and time dependent, weaker than that produced by collagen or monensin (mimics Na+/H+ exchanger), and less pronounced following the inhibition of Na+/H+ exchanger by 5-(N-ethyl-N-isopropyl) amiloride or genistein. Flow cytometry studies reveal that in-vitro platelet treatment with AVP results in an unimodal left shift in the forward and side scatter of the entire platelet population, indicating morphological changes on the plasma membrane. The shift was dose related, weaker than that evoked by collagen, similar to that produced by monensin and strongly reduced in the presence of 5-(N-ethyl-N-isopropyl) amiloride or genistein. Using flow cytometry, we demonstrated enhanced expression of phosphatidylserine on the AVP-treated platelets. AVP-evoked phosphatidylserine exposure was dose dependent, inhibited by 5-(N-ethyl-N-isopropyl) amiloride or genistein and weaker than that produced by collagen. AVP in a dose-dependent manner produced a rise in platelet volume. The swelling was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, and its kinetics was similar to that observed in the presence of monensin. We conclude that prolonged treatment of platelets with AVP results in a procoagulant response, which may occur as a consequence of Na+ influx mediated by Na+/H+ exchanger.     

Functional characteristics of a cloned epithelial Na+/H+ exchanger (NHE3): resistance to amiloride and inhibition by protein kinase C.

We previously cloned an isoform Na+/H+ exchanger (NHE3), which was expressed only in intestine, kidney, and stomach. We show here the functional characteristics of NHE3 as a Na+/H+ exchanger by stably transfecting NHE3 cDNA into PS120 cells, a fibroblast cell line that lacks endogenous Na+/H+ exchangers. NHE3 was 39- and 160-fold more resistant to inhibition by amiloride and ethylisopropyl amiloride, respectively, than NHE1, the housekeeping Na+/H+ exchanger isoform. Although both exchangers were stimulated by serum, NHE3 was inhibited by phorbol 12-myristate 13-acetate (PMA), which stimulated NHE1. Mechanistically, serum and PMA stimulated NHE1 by an increase in the apparent affinity of the exchanger for intracellular H+. In contrast, serum stimulated and PMA inhibited NHE3 by a Vmax change. When NHE3 was stably expressed in Caco-2 cells, an intestinal epithelial cell line, NHE3 was functionally expressed in the apical membrane. Thus, NHE3 is a good candidate to be an epithelial brush border Na+/H+ exchanger. Furthermore, Na+/H+ exchangers can be rapidly regulated by mechanisms that change either the Vmax or the affinity for intracellular H+, depending on the Na+/H+ exchanger subtype.     

Activation of the Na(+)-H+ exchanger modulates angiotensin II-stimulated Na(+)-dependent Mg2+ transport in vascular smooth muscle cells in genetic hypertension.

This study investigated the role of the Na(+)-H+ exchanger (NHE) on angiotensin II (Ang II)-induced activation of Na(+)-dependent Mg2+ transport in vascular smooth muscle cells (VSMCs) from Wistar-Kyoto rats (WKY; n=20) and spontaneously hypertensive rats (SHR; n=20). Intracellular free concentrations of Mg2+ ([Mg2+]i) and Na+ ([Na+]i) and intracellular pH (pHi) were measured with the specific fluorescent probes mag-fura 2-AM, SBFI-AM, and BCECF-AM, respectively. Na+ dependency of Mg2+ transport was assessed in Na(+)-free buffer, and the role of the NHE was determined with the highly selective NHE blocker 5-(N-methyl-N-isobutyl) amiloride (MIA). Basal [Mg2+]i was lower in SHR than WKY (0.59+/-0.01 versus 0.71+/-0.01 mmol/L, P<0.05). Basal pHi and [Na+]i were not different between the 2 groups. Ang II dose dependently increased [Na+]i and pHi and decreased [Mg2+]i. Responses were significantly greater (P<0.05) in SHR versus WKY ([Na+]i E(max)=37.5+/-1.1 versus 33.7+/-1.9 mmol/L; pHi E(max)=7.35+/-0.04 versus 7.20+/-0.01; [Mg2+]i E(min)=0. 28+/-0.09 versus 0.53+/-0.02 mmol/L, SHR versus WKY). In Na(+)-free buffer, Ang II-elicited [Mg2+]i responses were inhibited. MIA (1 micromol/L) inhibited Ang II-stimulated responses in WKY and normalized responses in SHR ([Mg2+]i E(min)=0.49+/-0.02). Ang II-stimulated activation of NHE was significantly increased (P<0.05) in SHR (0.07+/-0.002 DeltapH(i)/s) compared with WKY (0.05+/-0.004 DeltapH(i)/s). These data demonstrate that in VSMCs [Mg2+]i regulation is Na+ dependent, that activation of NHE modulates Na(+)-Mg2+ transport, and that increased activity of NHE may play a role in altered Na(+)-dependent regulation of [Mg2+]i in SHR.     

Disturbances in Na+ Transport Systems Induced by Ethanol in Human Red Blood Cells

The effects of ethanol on fluxes catalyzed by four Na+ transport systems (ouabain-sensitive Na+, K+ pump, bumetanide-sensitive Na+, K+ cotransport system, Na+:Li+- countertransport and anion carrier) and on Na+ and K+ leaks were investigated in human red blood cells. Ethanol concentrations higher than 32 mM were required in order to significantly modify erythrocyte Na+ transport function. The observed changes can be summarized as follows: (a) stimulation of Na+ efflux through the Na+, K+ pump (by 21-32% at 160-400 mM) and Na+:Li+ countertransport (by 34-59% at 160-400 mM); (b) inhibition of outward Na+, K+ cotransport (by 23-34% at 160-400 mM) and LiCO3- influx through the anion carrier (by 17-21% at 64-400 mM); and (c) increase in Na+ and K+ leaks (by 13-16% at 64-400 mM). The effects of ethanol on the Na+,K+ pump and Na+,K+ cotransport system resulted from changes in maximal rates of Na+ efflux (increased and decreased, respectively) without any significant effect on the apparent affinities for internal Na+. Erythrocytes preincubated for 1 hr with 160 mM ethanol, washed and further incubated in flux media, recovered a normal Na+ transport function. In conclusion, high concentrations of ethanol induced reversible perturbations of fluxes catalyzed by erythrocyte Na+ transport systems. The observed effects may reflect disturbances in Na+ transport function associated with severe intoxication.     

Mechanism of monensin-induced hyperpolarization of neuroblastoma-glioma hybrid NG108-15.

Addition of the ionophore monensin to mouse neuroblastoma-rat glioma hybrid NG108-15 cells leads to a 20 to 30-mV increase in the electrical potential across the plasma membrane as shown by direct intracellular recording techniques and by distribution studies with the lipophilic cation [3H]-tetraphenylphosphonium+ (TPP+) [Lichtshtein, D., Kaback, H.R. & Blume, A.J. (1979) Proc. Natl. Acad. Sci. USA 76, 650-654]. The effect is not observed with cells suspended in high K+ medium, is dependent upon the presence of Na+ externally, and the concentration of monensin that induces half-maximal stimulation of TPP+ accumulation is approximately 1 microM. The ionophore also causes rapid influx of Na+, a transient increase in intracellular pH, and a decrease in extracellular pH, all of which are consistent with the known ability of monensin to catalyze the transmembrane exchange of H+ for Na+. Although ouabain has no immediate effect on the membrane potential, the cardiac glycoside completely blocks the increase in TPP+ accumulation observed in the presence of monensin. Thus, the hyperpolarizing effect of monensin is mediated apparently by an increase in intracellular Na+ that acts to stimulate the electrogenic activity of the Na+,K+-ATPase. Because monensin stimulates TPP+ accumulation in a number of other cultured cell lines in addition to NG108-15, the techniques described may be of general use for studying the Na+,K+ pump and its regulation in situ.     

Role of cell volume in K+-induced Ca2+ signaling by rat adrenal glomerulosa cells

The involvement of cell volume in the K+-evoked Ca2+ signaling was studied in cultured rat glomerulosa cells. Previously we reported that hyposmosis (250 mOsm) increased the amplitude of T-type Ca2+ current and, accordingly, enhanced the Ca2+ response of cultured rat glomerulosa cells to K+. In the present study we found that this enhancement is not influenced by the cytoskeleton-disrupting drugs cytochalasin-D (20 microM) and colchicine (100 microM). Elevation of extracellular potassium concentration ([K+]e) from 3.6 to 4.6-8.6 mM induced cell swelling, which had slower kinetics than the Ca2+ signal. Cytoplasmic Ca2+ signal measured in single glomerulosa cells in response to stimulation with 5 mm K+ for 2 min showed two phases: after a rapid rise reaching a plateau within 20-30 sec, [Ca2+]c increased further slowly by approximately one third. When 5 mM K+ was coapplied with elevation of extracellular osmolarity from 290 to 320 mOsm, the second phase was prevented. These results indicate that cell swelling evoked by physiological elevation of [K+]e may contribute to the generation of sustained Ca2+ signals by enhancing voltage-activated Ca2+ influx.     

Participation of the Na+/H+ exchanger in the phospholipase-A2 activation of luteinizing hormone-releasing hormone release in rat hypothalamic fragments.

The role of the Na+/H+ exchanger in the phospholipase-A2 (PLA2) stimulation of LHRH release was investigated using in vitro incubations of rat hypothalamic fragments. It was found that monensin, the Na+/H+ ionophore, increased LHRH release in a dose-related manner. That effect diminished in the absence of calcium as well as after the addition of 2,4'-dibromoacetophenone, a blocker of PLA2 action. Amiloride, a blocker of the Na+/H+ exchanger, did not alter the effect of monensin. However, amiloride significantly diminished the effect of melittin, an activator of PLA2 action. LHRH release under PLA2 did not change when amiloride was added to the incubation medium. Lysophosphatidylcholine also increased LHRH release. These results were interpreted as evidence of the participation of Na+/H+ exchange in PLA2 activation in the release of LHRH in rat hypothalamic fragments. A role of lysophospholipids in this process is also suggested.