Amiloride-sensitive 22Na Uptake Research Articles

Impairment of cation transport in A549 cells and rat alveolar epithelial cells by hypoxia.

A reduced cation reabsorption across the alveolar epithelium decreases water reabsorption from the alveoli and could diminish clearing accumulated fluid. To test whether hypoxia restricts cation transport in alveolar epithelial cells, cation uptake was measured in rat lung alveolar type II pneumocytes (AII cells) in primary culture and in A549 cells exposed to normoxia and hypoxia. In AII and A549 cells, hypoxia caused a PO2-dependent inhibition of the Na-K pump, of Na-K-2Cl cotransport, and of total and amiloride-sensitive 22Na uptake. Nifedipine failed to prevent hypoxia-induced transport inhibition in both cell types. In A549 cells, the inhibition of the Na-K pump and Na-K-2Cl cotransport occurred within approximately 30 min of hypoxia, was stable >20 h, and was reversed by 2 h of reoxygenation. There was also a reduction in cell membrane-associated Na-K-ATPase and a decrease in Na-K-2Cl cotransport flux after full activation with calyculin A, indicating a decreased transport capacity. [14C]serine incorporation into cell proteins was reduced in hypoxic A549 cells, but inhibition of protein synthesis with cycloheximide did not reduce ion transport. In AII and A549 cells, ATP levels decreased slightly, and ADP and the ATP-to-ADP ratio were unchanged after 4 h of hypoxia. In A549 cells, lactate, intracellular Na, and intracellular K were unchanged. These results indicate that hypoxia inhibits apical Na entry pathways and the basolateral Na-K pump in A549 cells and rat AII pneumocytes in culture, indicating a hypoxia-induced reduction of transepithelial Na transport and water reabsorption by alveolar epithelium. If similar changes occur in vivo, the impaired cation transport across alveolar epithelial cells might contribute to the formation of hypoxic pulmonary edema.

Na+/H+ exchange and osmotic shrinkage in isolated trout hepatocytes

The ability of rainbow trout liver cells to regulate their intracellular pH (pHi) was studied using two methods on hepatocytes isolated by collagenase digestion: (i) by monitoring pHi with the fluorescent dye BCECF-AM, and (ii) by measuring the amiloride-sensitive uptake of 22Na, which represents Na+/H+ exchange. In low-Na+ medium (¾16mmoll-1), Na+ uptake was reduced by approximately 70% in the presence of amiloride derivatives (DMA or MPA, 10(-4)moll-1). Changing separately either the extracellular pH (pHe) or the intracellular pH (pHi, clamped by treating the cells with nigericin in the presence of 140mmoll-1 K+) between 6 and 8 induced an increase in the rate of Na+ uptake when pHe was raised or when pHi was reduced. When transferred to hypertonic medium, hepatocytes shrank to nearly 72% of their initial volume, and thereafter a slow and partial regulatory volume increase phase was observed, with an increase in the amiloride-sensitive rate of Na+ uptake and an increase in intracellular pH. As DIDS-sensitive Cl- uptake was concomitantly enhanced, it is suggested that hypertonic stress activates Na+/H+ and Cl-/HCO3- exchange.

Platelet sodium/hydrogen ion exchange in normal pregnancy and non-proteinuric pre-eclampsia.

In non-pregnant individuals, abnormalities in cation transport in vascular tissues have been linked to essential hypertension. In the present study, we consider whether Na+/H+ exchange (NHE) is affected in non-proteinuric pre-eclampsia (NPP). Platelet NHE characteristics and plasma cholesterol were measured in a cross-sectional study of normal primigravidae at 14 +/- 0.5 (n = 9), 29 +/- 0.7 (n = 7), 39 +/- 0.4 (n = 8) weeks gestation, in women with NPP (n = 15) and in non-pregnant women (n = 8). Amiloride-sensitive 22Na uptake was measured in platelets which had been acid loaded, to stimulate NHE, by suspension in isotonic potassium propionate buffer (pH 6.7). Intraplatelet radioactivity was used to calculate the affinity (Km) and the capacity (Vmax) of Na+ uptake. In normotensive women, Vmax (mean +/- s.e.) at 14, 29, 39 weeks gestation and 6 weeks postpartum were 452 +/- 46, 469 +/- 33, 713 +/- 101 and 562 +/- 77 pmolNa+/10(6) cells/min respectively; the third trimester values were higher (P < 0.05) than those in the first and second trimester and were also higher than those of non-pregnant women (415 +/- 20). Vmax of patients with NPP in the third trimester (712 +/- 44) was not different from gestational age-matched controls. Km values were not affected by gestational age or NPP. Plasma cholesterol concentration was positively correlated with Vmax values during normotensive pregnancy (r = 0.493, P < 0.05). In conclusion, the capacity for amiloride-sensitive Na+ uptake by platelets correlates positively with gestational age during normal pregnancy. However, neither the capacity nor affinity for Na+ was altered in NPP platelets suggesting that NHE is not implicated in the pathophysiology of this condition.

Role of glucocorticoids in the maturation of renal cortical Na+/H+ exchanger activity during fetal life in sheep

We have studied the role of glucocorticoids in inducing the maturation in activity of the proximal tubule Na+/H+ exchanger that follows birth. Renal cortical microvillus membrane vesicles were prepared from 132-day gestation sheep fetuses (n = 8) that had received intraperitoneal cortisol (13 micrograms.kg-1.h-1) for the previous 48 h. Membrane vesicles were also obtained from sham-operated twin controls (n = 8). Amiloride-sensitive uptake of 22Na+ by these vesicles was measured, and Woolf-Augustinsson-Hofstee plots were used to determine the Michaelis constant (Km) and maximal velocity (Vmax). There was no significant difference in Km; however, the Vmax was 61% higher in cortisol-treated fetuses. Posttreatment circulating cortisol levels were significantly higher in the treated fetuses. Total RNA was collected from renal cortex of the eight pairs of twins when killed. Renal cortex Na+/H+ exchanger 3 (NHE3) mRNA levels were approximately fourfold higher in cortisol-treated than in control fetuses. Although proximal tubule Na+/H+ exchanger activity and renal cortex NHE3 mRNA levels increased significantly in cortisol-treated fetuses, cortisol infusion did not stimulate renal sodium reabsorption in the fetus but rather produced a natriuresis. These results demonstrate that glucocorticoids can induce an increase in both Na+/H+ exchanger activity and NHE3 mRNA levels during the last trimester of gestation in sheep. However, these changes are not associated with an increased ability of the fetal kidney to reabsorb sodium.

Maturation of proximal tubule Na+/H+ antiporter activity in sheep during transition from fetus to newborn

We have studied maturational changes in the kinetics of the proximal tubule Na+/H+ antiporter. Microvillus membrane vesicles were prepared from renal cortex of fetal and newborn lambs. Amiloride-sensitive uptake of 22Na+ by these vesicles was measured and Woolf-Augustinsson-Hofstee plots were used to determine the Michaelis constant (Km) and rate of maximal uptake (Vmax). Initial studies of fetal lambs at 130-132 days gestation (n = 5; term is 145 days) and 3- to 4-day-old lambs (n = 5) revealed no maturational change in Km (7.27 +/- 1.25 for fetuses and 9.01 +/- 1.03 mM for lambs); however, there was a 242% increase in Vmax (from 1.28 +/- 0.33 in the fetuses to 4.37 +/- 0.85 nmol.s-1.mg protein-1 in the lambs, P = 0.005). Further definition of the developmental change in Na+/H+ antiporter Vmax was obtained when 144-day-gestation fetuses (n = 5) were compared with 24-h-old sibling lambs (n = 5) that had been delivered by cesarean section at 144 days gestation. Again, no significant difference was seen in Na+/H+ antiporter Km (14.9 +/- 6.5 for fetuses and 12.5 +/- 3.4 mM for lambs); however, a significant increase in Na+/H+ antiporter Vmax occurred (from 1.41 +/- 0.51 in the fetuses to 3.32 +/- 0.37 nmol.s-1.mg protein-1 in the lambs, P < 0.01). This study shows that there is a maturational increase in renal cortical Na+/H+ antiporter Vmax during the transition from fetal to newborn life. This increase parallels the increase in renal tubular Na+ reabsorption that occurs at this time.

Endogenous renal dopamine production regulates phosphate excretion

We examined the effect of endogenous dopamine production on Pi and citrate excretion by Wistar rats. Carbidopa (20-40 mumol/kg ip) decreased dopamine, Pi, and citrate excretion within 20 min (86%, 47%, and 38%, respectively); Pi reabsorption increased 11 +/- 4% (P = 0.03). The decreases were sustained for at least 18 h. 3-Hydroxybenzylhydrazine (45 mumol/kg ip) reduced Pi excretion 24%. Benserazide (40 mumol/kg ip and 0.1 mumol/min iv) reduced dopamine excretion (94%) and blocked the effect of carbidopa on Pi and citrate excretion. In isolated perfused kidneys benserazide, carbidopa, and 3-hydroxybenzylhydrazine all decreased Pi excretion. Dopamine (1 mumol/l) added to cortical minceates reduced brush-border membrane vesicle (BBMV) 32P uptake by 8% (P < 0.02) and amiloride-inhibitable 22Na uptake by 19%. Carbidopa added to minceates increased 32P uptake by 12%. Carbidopa pretreatment increased (75%) amiloride-sensitive 22Na uptake into BBMV of rats fed a high-salt diet. Uptake was not increased into BBMV from rats fed a low-salt diet. Carbidopa increased (17%) basolateral membrane Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) gradually over 4 h. Na(+)-K(+)-ATPase did not increase in rats fed a low-phosphorous diet, but did increase when dopa was added to the diet. Thus endogenous dopamine appears to directly control Na(+)-Pi and Na+/H+ transport and secondarily alter basolateral membrane Na(+)-K(+)-ATPase.

Cell cycle-dependent and kinase-specific regulation of the apical Na/H exchanger and the Na,K-ATPase in the kidney cell line LLC-PK1 by calcitonin.

Calcitonin (CT), which regulates serum calcium through its actions in bone and the kidney tubule, also has a potent natriuretic effect in vivo. Na reabsorption in the proximal kidney tubule is mostly dependent on the activity of the Na,K-ATPase and the apical Na/H exchanger. We have previously shown that CT regulates the activity of the Na,K-ATPase in the proximal kidney tubule cell line LLC-PK1 in a cell cycle-dependent manner. We report here that, in the same cells, CT also regulates the Na/H exchanger through a cell cycle-specific activation of the Ca/calmodulin-dependent protein kinase II. In G2 phase, no changes in ethylisopropyl amiloride-sensitive 22Na uptake is observed, despite an increase in cAMP. In contrast, the hormone inhibits the apical exchanger when the cells are in S phase, resulting in an 80% inhibition of 22Na uptake. These results demonstrate that CT affects the activity of the two major proximal tubule Na transport systems and may help clarify the mechanisms by which CT regulates Na+ reabsorption.

Dual effect of cyclic GMP on renal brush border Na-H antiporter.

The Na-H antiporter of renal-brush border membranes is inhibited by cyclic AMP and stimulated by protein kinase C. The proximal tubule contains guanylate cyclase and is capable of cyclic GMP production. The effect of cGMP on renal Na-H antiporter activity was analyzed in phosphorylated brush border membranes by 22Na uptake in the presence or absence of 1 mM amiloride. 8-Bromo cyclic GMP (1 microM) increased the amiloride-sensitive 22Na uptake in control from 1.26 +/- 0.13 to 1.54 +/- 0.12 nmol/mg/protein/10 sec, P less than 0.01, without altering the amiloride-insensitive component. In the absence of exogenous ATP, cGMP also stimulated the amiloride-sensitive 22Na uptake, which can be explained by the presence of endogenous ATP in concentrations of up to 50 microM in the membranes. In ATP-depleted membrane vesicles, however, cGMP inhibited the amiloride-sensitive 22Na uptake. These data indicate that cGMP acts on the Na-H antiporter by at least two different mechanisms, one of which is ATP dependent. It is likely that cGMP-dependent protein kinase mediates the stimulatory effects seen in the presence of ATP, and the inhibition seen in ATP-depleted membranes results from cGMP direct action on the Na-H antiporter.

Cl − permeability of the basolateral membrane of the Rana esculenta epithelium: activation of Cl −/HCO 3− exchange by alkaline intracellular pH

We have investigated Cl − transport mechanism(s) located in the basolateral membranes of the frog skin epithelium and in particular activation of Cl −/HCO 3 − exchange following an alkaline load. We found that 87% of the total 36Cl uptake by the epithelial cells occurs across the basolateral membranes ( J Cl − b) and submitting the epithelium to an alkaline load (HCO 3 −-Ringer solution, pH 8.1) increased J Cl − b. Intracellular Cl − activity ( a Cl − i), measured with ion-sensitive microelectrodes, increased when the Ringer solution bathing the basolateral membranes was changed from a Ringer solution equilibrated in air (pH 7.4) to one containing CO 2/HCO 3 − (pH 7.4). pH i recovery following an alkaline load was dependent on Cl − since it did not occur in serosal Cl −-free media, indicating the presence of a Cl −-dependent regulatory mechanism. Acid loading of the epithelial cells (5% CO 2, HCO 3 −-free Ringer) produced no change in J Cl − b but stimulated an amiloride-sensitive 22Na uptake across the basolateral membranes of the epithelium, compatible with an activation of a Na +/H + exchanger, previously described in this tissue. J Cl − b was partially blocked by SITS (5 · 10 −4 mmol/l), niflumic acid (5 · 10 −5 mmol/l), furosemide or bumetanide. Simultaneous addition of furosemide and niflumic acid produced an inhibition of J Cl − b which was not different with furosemide alone. Substitution of Na + by choline had no effect on J Cl − b and furosemide did not block the 22Na + uptake, suggesting that J Cl − b is not a Na +-dependent process (cotransport). We conclude that a significant Cl − permeability at the basolateral membranes of the epithelial cells is due to the presence of a Cl −/HCO 3 − exchanger which is essential for the recovery of pH i following an alkaline load.

High-affinity binding sites for VIP in renal cortical membranes: Possible role of VIP in renal transport

We studied binding and degradation of vasoactive intestinal peptide (VIP) by highly purified brush border and basolateral membranes from rabbit kidney cortex. Brush border and basolateral membranes were capable of 73 and 49% degradation of VIP after 20 minutes, and the degradation was totally prevented by bacitracin. There was 66 and 87% specific binding of 125I-VIP to brush border and basolateral membranes, respectively. 125I-VIP binding to renal membrane was displaced in a dose dependent fashion by unlabeled VIP with half maximal displacement at 2 x 10(-7) M. Other related peptides failed to displace VIP. Scatchard analysis showed one single class of receptors for VIP in both membranes with similar Kd (0.5 x 10(-7) M), but higher number of binding sites (Bmax) in the basolateral membranes than in the brush border membranes (22.0 vs. 4.4 pmol/mg protein), respectively. Forty-eight percent of VIP binding to brush border membranes could be explained by cross contamination of these membranes with basolateral membranes. We examined the effect of VIP on Na-H antiporter, Na-dependent glucose uptake and Na-dependent phosphate uptake by isolated proximal tubule suspension. In acid loaded proximal tubules VIP (10(-6) M) inhibited total and amiloride-sensitive 22Na uptake by 35 and 75%, respectively, as compared to control. On the other hand VIP failed to inhibit Na-dependent methyl alpha-14C-glucopyranoside and Na-dependent 32phosphate uptake. VIP failed to stimulate cyclic AMP generation by proximal tubule suspension while PTH showed the expected stimulation. Our results demonstrate the presence of specific binding for VIP in highly purified cortical membranes and suggest an effect of VIP to inhibit the Na-H antiporter by a mechanism independent of cyclic AMP.

Ion specificity of cardiac sarcolemmal Na+/H+ antiporter.

In bovine cardiac sarcolemmal vesicles, an outward H+ gradient stimulated the initial rate of amiloride-sensitive uptake of 22Na+, 42K+, or 86Rb+. Release of H+ from the vesicles was stimulated by extravesicular Na+, K+, Rb+, or Li+ but not by choline or N-methylglucamine. Uptakes of Na+ and Rb+ were half-saturated at 3 mM Na+ and 3 mM Rb+, but the maximal velocity of Na+ uptake was 1.5 times that of Rb+ uptake. Na+ uptake was inhibited by extravesicular K+, Rb+, or Li+, and Rb+ uptake was inhibited by extravesicular Na+ or Li+. Amiloride-sensitive uptake of Na+ or Rb+ increased with increase in extravesicular pH and decrease in intravesicular pH. In the absence of pH gradient, there were stimulations of Na+ uptake by intravesicular Na+ and K+ and of Rb+ uptake by intravesicular Rb+ and Na+. Similarly, there were trans stimulations of Na+ and Rb+ efflux by extravesicular alkali cations. The data suggest the existence of a nonselective antiporter catalyzing either alkali cation/H+ exchange or alkali cation/alkali cation exchange. Since increasing Na+ caused complete inhibition of Rb+/H+ exchange, but saturating K+ caused partial inhibitions of Na+/H+ exchange and Na+/Na+ exchange, the presence of a Na(+)-selective antiporter is also indicated. Although both antiporters may be involved in pH homeostasis, a role of the nonselective antiporter may be in the control of Na+/K+ exchange across the cardiac sarcolemma.

Agonist-induced activation of Na+/H+ exchange in rat parotid acinar cells

The present studies were designed to test our previous suggestion that Na+/H+ exchange was activated by muscarinic stimulation of rat parotid acinar cells. Consistent with this hypothesis, we demonstrate here that intact rat parotid acini stimulated with the muscarinic agonist carbachol in HCO3- -free medium show an enhanced recovery from an acute acid load as compared to similarly challenged untreated preparations. Amiloride-sensitive 22Na uptake, due to Na+/H+ exchange, was also studied in plasma membrane vesicles prepared from rat parotid acini pretreated with carbachol. This uptake was stimulated two-fold relative to that observed in vesicles from control (untreated) acini. This stimulation was time dependent, requiring approximately 15 min of acinar incubation with carbachol to reach completion, and was blocked by the presence of the muscarinic antagonist atropine (2 x 10(-5) M) in the pretreatment medium. The effect of carbachol was dose dependent with K0.5 approximately 3 x 10(-6) M. Stimulation of the exchanger was also seen in vesicles prepared from acini pretreated with the alpha-adrenergic agonist epinephrine, but not with the beta-adrenergic agonist isoproterenol, or with substance P. Kinetic analysis indicated that the stimulation induced by carbachol was due to an alkaline shift in the pH responsiveness of the exchanger in addition to an increased apparent transport capacity. Taken together with previous results from this and other laboratories, these results strongly suggest that the Na+/H+ exchanger and its regulation are intimately involved in the fluid-secretory response of the rat parotid.

Functional expression of the amiloride-sensitive sodium channel in Xenopus oocytes.

Expression of the amiloride-sensitive sodium channel was examined in Xenopus oocytes that were microinjected with A6 cell mRNA. Amiloride-inhibitable 22Na flux could be measured in intact oocytes 2-3 days after injection with 25 ng of poly(A)+ RNA isolated from aldosterone-treated A6 cells. The rate of 22Na uptake was approximately 15-fold greater in oocytes microinjected with 25 ng of poly(A)+ RNA than in water-injected control oocytes. An increase in 22Na uptake by mRNA-injected oocytes occurred whether the mRNA was isolated from A6 cells grown on a porous or nonporous support. In the presence of 4 mM NaCl, amiloride caused dose-dependent inhibition of 22Na uptake in mRNA-injected oocytes, which was half-maximal at 6 x 10(-8) M. Both 1 microM amiloride and 0.1 microM benzamil inhibited 22Na uptake in mRNA-injected oocytes by greater than 95%, whereas less than 50% inhibition occurred with 1 microM 5-(N-ethyl-N-isopropyl)amiloride. When A6 cell mRNA was size fractionated by sucrose density-gradient centrifugation, amiloride-sensitive 22Na uptake was expressed predominantly by oocytes injected with mRNA from two contiguous fractions.

Distinct activation of Na+-H+ exchange by gastrin and CCK peptide in acini from guinea pig

The effect of cholecystokinin (CCK)-gastrin family peptides (caerulein, unsulfated gastrin-17, and pentagastrin) and secretin in activating amiloride-sensitive 22Na uptake were investigated in guinea pig pancreatic acini. Secretin had no effect, but CCK-gastrin peptides stimulated the amiloride-sensitive 22Na uptake. The effect of caerulein was inhibited by dibutyryl guanosine 3',5'-cyclic monophosphate (cGMP) and asperlicin, indicating that activation of the Na+-H+ antiport caused by caerulein is mediated by CCK receptors. The effect of gastrin was dibutyryl cGMP and asperlicin insensitive, whereas the effect of pentagastrin was inhibited by the CCK antagonists but with a low affinity, indicating that the effect of gastrin and that of pentagastrin was CCK receptor independent. The calcium ionophore A23187 caused an increase in amiloride-sensitive 22Na uptake. However, the effect of caerulein, which increased internal calcium concentration, was not modified after depletion of intracellular calcium, and that of CCK-gastrin family peptides was not dependent on external calcium concentration. Activation of amiloride-sensitive 22Na uptake was also induced by 12-O-tetradecanoylphorbol 13-acetate and 1-oleoyl-2-acetyl-glycerol. Activation of protein kinase c may be involved in the mechanism of caerulein or gastrin in activating the Na+-H+ exchange.

CAMP-associated inhibition of Na+-H+ exchanger in rabbit kidney brush-border membranes.

Adenosine 3',5'-cyclic monophosphate (cAMP) inhibits the rate of bicarbonate reabsorption and the rate of Na+-H+ exchange transport in the apical membrane of the proximal convoluted tubule. To study the relation between cAMP, cAMP-dependent protein kinase, and Na+-H+ exchange transport, brush-border membrane vesicles from the rabbit kidney were phosphorylated in vitro. The rate of proton gradient-stimulated amiloride-inhibitable 22Na+ uptake was measured as an index of Na+-H+ exchange transport activity. The inclusion of cAMP (10(-6) M) in a phosphorylating solution containing ATP decreased the 10-s uptake of amiloride-sensitive sodium from 2.25 +/- 0.21 nmol/mg protein in controls to 1.94 +/- 0.19 (P less than 0.001). Incubation of vesicles in the presence of purified catalytic subunit of cAMP-dependent protein kinase inhibited the amiloride-sensitive uptake of 22Na+ at 10 s from 2.35 +/- 0.49 nmol/mg protein to 2.05 +/- 0.44 (P less than 0.005). The inhibitory effect of both cAMP and catalytic subunit of cAMP-dependent protein kinase was blocked by the specific thermostable protein inhibitor of the kinase. These studies demonstrate that activation of endogenous membrane-bound cAMP-dependent protein kinase or exposure to exogenous catalytic subunit of cAMP-dependent protein kinase inhibits the rate of Na+-H+ exchange transport in the brush-border membrane of the rabbit kidney.

Characterization of the activation of Na+/H+ exchange in lymphocytes by phorbol esters: change in cytoplasmic pH dependence of the antiport.

Phorbol 12-myristate 13-acetate and other phorbol esters induce an intracellular alkalinization in rat thymic lymphocytes. An extracellular acidification can be recorded concomitantly. This transmembrane H+ (equivalent) flux is dependent on external Na+ and is amiloride sensitive. Phorbol esters also stimulate an amiloride-sensitive uptake of 22Na+, suggesting activation of Na+o/H+i exchange. Only those phorbol derivatives that are tumor promoters and activators of protein kinase C stimulate the antiport. Activation of the Na+/H+ exchange is brought about by a change in the cytoplasmic pH sensitivity of the antiport. Activation of the Na+/H+ exchanger by phorbol esters results in membrane hyperpolarization, due to indirect stimulation of the electrogenic Na+/K+ pump by the increased intracellular Na+ concentration. Increased Na+/H+ exchange also produces cell swelling, which may be one of the earliest manifestations of the growth-promoting properties of the phorbol esters.