Kinetics of Na(+)-dependent conformational changes of rabbit kidney Na+,K(+)-ATPase

The kinetics of Na(+)-dependent partial reactions of the Na+,K(+)-ATPase from rabbit kidney were investigated via the stopped-flow technique, using the fluorescent labels N-(4-sulfobutyl)-4-(4-(p-(dipentylamino)phenyl)butadienyl)py ridinium inner salt (RH421) and 5-iodoacetamidofluorescein (5-IAF). When covalently labeled 5-IAF enzyme is mixed with ATP, the two labels give almost identical kinetic responses. Under the chosen experimental conditions two exponential time functions are necessary to fit the data. The dominant fast phase, 1/tau 1 approximately 155 s-1 for 5-IAF-labeled enzyme and 1/tau 1 approximately 200 s-1 for native enzyme (saturating [ATP] and [Na+], pH 7.4 and 24 degrees C), is attributed to phosphorylation of the enzyme and a subsequent conformational change (E1ATP(Na+)3-->E2P(Na+)3 + ADP). The smaller amplitude slow phase, 1/tau 2 = 30-45 s-1, is attributed to the relaxation of the dephosphorylation/rephosphorylation equilibrium in the absence of K+ ions (E2P<==>E2). The Na+ concentration dependence of 1/tau 1 showed half-saturation at a Na+ concentration of 6-8 mM, with positive cooperatively involved in the occupation of the Na+ binding sites. The apparent dissociation constant of the high-affinity ATP-binding site determined from the ATP concentration dependence of 1/tau 1 was 8.0 (+/- 0.7) microM. It was found that P3-1-(2-nitrophenyl)ethyl ATP, tripropylammonium salt (NPE-caged ATP), at concentrations in the hundreds of micromolar range, significantly decreases the value of 1/tau 1, observed. This, as well as the biexponential nature of the kinetic traces, can account for previously reported discrepancies in the rates of the reactions investigated.     

Modulation of mouse cerebral Na+,K(+)-ATPase activity by oxygen free radicals

There is increasing evidence that oxygen free radicals (OFR) are involved in cerebral ischaemia-reperfusion injury, possibly via a modulation of Na+,K(+)-ATPase activity, one of the major membrane pumps responsible for ionic homeostasis. We measured OFR-mediated modulation of this enzymatic activity and examined the roles of lipid and/or protein alterations. Using mouse brain microsomes exposed to UV-C irradiation, our results show a good correlation between activity inhibition and lipoperoxidation estimated by PUFA loss as well as malondialdehyde production. The protective effect of thiourea (OH scavenger) and the lack of effect noted with DTT (thiol protector) suggest that the functionality of the Na+,K(+)-ATPase is altered by perturbation of membrane integrity rather than by a structural alteration of the protein itself.     

Coexisting NPY and NE synergistically regulate renal tubular Na+, K(+)-ATPase activity

The sympathetic renal nerves are of central importance for the regulation of sodium balance. Sodium excretion decreases following renal nerve activation and increases following denervation. These effects have been attributed to norepinephrine (NE) acting on alpha-adrenergic receptors. In the present study, using isolated permeabilized rat renal proximal convoluted tubule (PCT) cells, neuropeptide Y (NPY) was shown to stimulate Na+, K(+)-ATPase activity. This 36-amino acid peptide is a messenger molecule in the sympathetic nervous system which is co-stored with NE and dopamine-beta-hydroxylase (DBH), the NE synthesizing enzyme in the renal nerves. The effect is likely to be mediated via the NPY Y2 receptor, a pertussis toxin (PTX)-sensitive G-protein, and calcium. It is partially antagonized by alpha-adrenergic antagonists, and enhanced by the subthreshold doses of alpha-adrenergic agonists. Our results suggest an important role for this peptide in the regulation of the sodium balance in the kidney.     

A putative fourth Na+,K(+)-ATPase alpha-subunit gene is expressed in testis

The Na+,K(+)-ATPase alpha subunit has three known isoforms, alpha 1, alpha 2 and alpha 3, each encoded by a separate gene. This study was undertaken to determine the functional status of a fourth human alpha-like gene, ATP1AL2. Partial genomic sequence analysis revealed regions exhibiting sequence similarity with exons 3-6 of the Na+,K(+)-ATPase alpha isoform genes. ATP1AL2 cDNAs spanning the coding sequence of a novel P-type ATPase alpha subunit were isolated from a rat testis library. The predicted polypeptide is 1028 amino acids long and exhibits 76-78% identity with the rat Na+,K(+)-ATPase alpha 1, alpha 2 and alpha 3 isoforms, indicating that ATP1AL2 may encode a fourth Na+,K(+)-ATPase alpha isoform. A 3.9-kb mRNA is expressed abundantly in human and rat testis.     

Separation and characterization of Na+,K(+)-ATPase containing vesicles

Na+,K(+)-ATPase was reconstituted in vesicles prepared by a dialysis method. Ion-exchange chromatography was used to obtain well characterized fractions from the inhomogeneous vesicle preparation. Lipid and protein content was determined by optical methods during the elution process. It was possible to separate fractions with distinct enzymatic and transport activities. A protocol was set up, which allowed to calculate the average number of 5-IAF labeled ion pumps per vesicle in the different fractions. The dependence of the number of protein molecules per vesicle was studied as function of the initial protein concentration added to the lipid solution before dialysis. The transport activity disappears completely at very low protein concentrations (3.3 micrograms protein per mg lipid). This observation is in favor of the proposal discussed in the literature, that the heterodimer (alpha beta)2 is the transport-active form of the Na+,K(+)-ATPase. The presented method can be applied to all reconstituted vesicle preparations in which the proteins can be labeled quantitatively with a fluorescence dye.     

Electrogenic property of Na+, K(+)-ATPase through computer simulation

A computer simulation of the electrogenic nature of the membrane-bound Na+, K(+)-ATPase is presented. The model involves coupling two simulation systems for passive and active transports, using a minimum of empirical parameters, and studies the contribution of the pump to the membrane potential. The simulation results indicate that electrogenic active transport accelerates the restoration of the resting electrochemical gradients and contributes approximately 0.44-1.1 mV to the resting potential of the membrane, depending on the Na:K coupling ratio. The effect of membrane potential and the physical positioning of the enzyme from the passive transporting channel on the enzyme function is also presented. The validity of the model is checked by comparing our results with reported literature values.     

Effect of methyl isocyanate on rabbit cardiac Na+, K(+)-ATPase

The present study describes the effect of methyl isocyanate (MIC) on rabbit cardiac microsomal Na+, K(+)-ATPase. Addition of MIC in vitro resulted in dose-dependent inhibition of Na+, K(+)-ATPase, Mg(2+)-ATPase and K(+)-activated p-nitrophenyl phosphatase (K(+)-PNPPase). Activation of Na+, K(+)-ATPase by ATP in the presence of MIC showed a decrease in Vmax with no change in Km. Similarly, activation of K+ PNPPase by PNPP in the presence of MIC showed a decrease in Vmax with no change in Km. The circular dichroism spectral studies revealed that MIC interaction with Na+, K(+)-ATPase led to a conformation of the protein wherein the substrates Na+ and K+ were no longer able to bind at the Na(+)- and K(+)-activation sites. The data suggest that the inhibition of Na+, K(+)-ATPase was non-competitive and occurred by interference with the dephosphorylation of the enzyme-phosphoryl complex.     

L-phenylalanine effect on rat brain acetylcholinesterase and Na+,K(+)-ATPase

The effect of different L-phenylalanine (Phe) concentrations (0.1-12.1 mM), on acetylcholinesterase (AChE) and Na+,K(+)-ATPase activities of brain homogenate and pure enzymes, was investigated at 37 degrees C. AChE and Na+,K(+)-ATPase activities were determined according to Ellman G. L., Courtney D., Andres V. and Featherstone R. M. (1961), Biochem. Pharmacol. 7, 88-95 and Bowler K. and Tirri R. (1974), J. Neurochem. 23, 611-613) respectively, after preincubation with Phe. AChE activity in brain homogenate or in pure eel E.electricus enzyme showed a decrease, which reached up to 18% with concentrations of 0.9-12.1 mM. Brain homogenate Na+,K(+)-ATPase activity showed an increase 16-65% with 0.24-0.9 mM of Phe, while an activity increase of 60-65% appeared with 0.9-12.1 mM of Phe. Pure enzyme activity (from porcine cerebral cortex) was not affected by high Phe concentrations, while it was increased by low concentrations. The above results suggest: a) A direct effect of Phe on AChE, b) A direct effect of low Phe concentrations and an indirect effect of high ones on Na+,K(+)-ATPase.     

Na+,K(+)-ATPase of human placenta during gestational hypertension: a biochemical-biophysical study

1. Na+,K(+)-ATPase is the membrane enzyme catalysing the active transport of Na+ and K+ across the plasma membrane of animal cells. A reduced activity of Na+,K(+)-ATPase has been described in gestational hypertension in a variety of cell types, in agreement with the hypothesis that gestational hypertension can induce membrane transport modifications similar to those reported for essential hypertension. The causes of the reduced Na+,K(+)-ATPase activity are still debated. 2. The aim of the present work was to investigate the molecular mechanism of the reduced enzymic activity in gestational hypertension using as a model Na+,K(+)-ATPase purified from human placenta. Na+,K(+)-ATPase obtained from term placentas of eight healthy pregnant women and eight age-matched women with gestational hypertension was purified as previously described. 3. We observed in gestational hypertension: (i) a significant increase in the activation energies above transition temperature; (ii) a significant decrease in the fluorescence polarization of 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (i.e. increased fluidity) and an increase in the mean lifetime (modified hydrophobicity); (iii) a lower Kq, suggesting an enzymic structural modification; and (iv) an increased mean lifetime and rotational relaxation time of pyrene isothiocyanate, indicating a modified ATP binding site.     

Regulation of Na+,K(+)-ATPase activity by dopamine in cultured rat aortic smooth muscle cells

We investigated the effect of dopamine on Na+,K(+)-ATPase activity in cultured aortic smooth muscle cells. Na+,K(+)- ATPase activity was measured by a coupled enzyme assay. Our results demonstrate that dopamine and dopamine receptor agonists, SKF-38393 (a D1 receptor agonist) and quinpirole (a D2 receptor agonist) produced 62%, 50% and 49% inhibition of Na+,K(+)-ATPase activity in aortic smooth muscle cells, respectively. The combination of the two agonists produced inhibition similar to that of dopamine. Dopamine- and the agonist-induced Na+,K(+)-ATPase inhibition was blocked by selective receptor antagonists. The Na+,K(+)-ATPase inhibition by SKF-38393 but not by quinpirole was abolished by pertussis toxin. Na+,K(+)-ATPase inhibition was also achieved by guanosine triphosphate analog GTP-gamma-S. SKF-38393 but not quinpirole stimulated phosphoinositide hydrolysis rate in rat aortic slices. SKF-38393-induced phosphoinositide hydrolysis stimulation was reversed by SCH-23390, a dopamine D1 receptor antagonist, and attenuated by pertussis toxin. In conclusion, our observations indicate that dopamine and dopamine receptor agonists inhibit Na+,K(+)-ATPase activity through specific vascular receptors. Dopamine D1 receptors are linked to pertussis toxin sensitive-mechanism(s) and a GTP-binding protein appears to be coupled to the enzyme inhibition. Finally, the inhibition of Na+,K(+)-ATPase activity in response to dopamine D1 receptor activation may be mediated by the phospholipase C signaling pathway.     

Captopril ameliorates the decreased Na+,K(+)-ATPase activity in the retina of streptozotocin-induced diabetic rats

PURPOSE: To examine the effect of captopril, an angiotensin-converting enzyme (ACE) inhibitor, on the activity of retinal sodium-potassium ATPase (Na,K-ATPase) and the activity of ACE in the serum and retina of streptozotocin (STZ)-induced diabetic rats. METHODS: Experimental diabetes was induced in male Long-Evans rats by a single intraperitoneal injection of STZ (55 mg/kg body weight). Some groups of normal and diabetic animals were treated with captopril (10 mg/kg per day) added to the drinking water for either a week or a month. After 2 and 4 months of diabetes, the specific activity of retinal total Na,K-ATPase was determined. The components of the activity of Na,K-ATPase caused by the alpha 1 and alpha 3 isoforms were pharmacologically separated by their different sensitivity to ouabain. The activity of ACE in the serum and retina was measured by radioassay using benzoyl-gly-gly-gly as substrate (10(5) cpm, 5 mM). RESULTS: The total Na,K-ATPase activity was decreased significantly after 2 (16%, P < 0.02) and 4 months (15%, P < 0.02) of diabetes. At both time points examined, the activities of the alpha 1-low-ouabain-affinity isoform and the alpha 3-high-ouabain-affinity isoform of retinal Na,K-ATPase were significantly reduced compared to those of age-matched controls (alpha 1, 9% to 14%, P < 0.05; alpha 3, 14% to 19%, P < 0.05 and P < 0.02 respectively). After 1 month of captopril administration, the activities of both Na,K-ATPase isoforms were at control level in 2-month diabetic rats, whereas they were restored only partially in 4-month diabetic rats. In age-matched normal animals, 1 month of captopril treatment did not alter the specific activities of either Na,K-ATPase isoform. One week or 1 month of captopril administration to diabetic rats did not change the activities of retinal Na,K-ATPase isoforms. Serum ACE activity was elevated significantly in both groups of untreated STZ rats (55% and 40%, respectively). One month of captopril administration further increased the ACE levels in 2- and 4-month diabetic rats (101% and 94%, respectively) and also enhanced significantly the serum ACE activity in normal animals (131%) versus the basal values. In contrast, retinal ACE activity was decreased significantly in both groups of untreated STZ rats (approximately 37%). Captopril exerted a significant inhibitory effect on the retinal ACE activity in 2- and 4-month diabetic rats (37% and 31%, respectively) compared to untreated diabetic animals as well as in normal rats (29%). CONCLUSIONS: These data suggest that stimulation of retinal Na,K-ATPase activity in diabetes is most likely one of the mechanisms through which captopril can improve retinal complications. The effect of captopril seems to be related to local effects in the retina. Whether the inhibition of retinal ACE is part of the mechanism of action of captopril requires further study.     

Na+,K(+)-ATPase activity is selectively increased in thalamus in thiamine deficiency prior to the appearance of neurological symptoms

OBJECTIVES: We analyzed the relationship between menstrual and reproductive history and risk of hip fractures in post-menopausal women using data from an Italian case-control study. METHODS: Cases were 206 post-menopausal women admitted for fractures of the hip proximal femur to a network of teaching and general hospitals in Milan, Italy. The comparison group consisted of 590 post-menopausal women admitted to the same network of hospitals for acute, non-neoplastic, non-hormone-related conditions, other than traumatic or orthopedic disorders. Odds ratios (OR) of hip fracture were derived from unconditional multiple logistic regression. RESULTS: No relation emerged between risk of hip fractures and age at menarche, lifelong menstrual cycle pattern and age at menopause. In comparison with women with age at menopause > or = 53 years, the multivariate OR of hip fractures were 1.2, 1.1, 1.2 and 0.5 in women with menopause at 50-52, 45-49, 40-44 and before 40 years (X2(1) trend 0.21). In comparison with nulliparae, the estimated age-adjusted OR was 0.6 (95% confidence interval, CI, 0.4-0.9) for parous women, but the multivariate estimate was not significant (OR 0.8, 95% CI 0.6-1.3) and the multivariate trend in risk with number of births was not significant either. No relation emerged between hip fractures and age at first and last birth, and history of abortions. CONCLUSIONS: This study found no relevant influence of menstrual and reproductive factors on the risk of hip fractures in post-menopausal women. However, this is not in contrast with the observation of a short-term effect of menopause and more in general. female hormone levels on osteoporosis and hence on hip fractures.     

Inhibition of Na+,K(+)-ATPase by platelet-activating factor in dog submandibular glands

Physiological stimulation of dog submandibular gland has been shown to generate platelet-activating factor (PAF). However, PAF is not released from cells in the tissue. To assess its intracellular activity, the effect of PAF on Na+,K(+)-ATPase was examined in dog submandibular gland cells. PAF inhibited Na+,K(+)-ATPase in membrane preparations, and the inhibitory effect was dependent on the protein concentration in the enzyme preparation. The inhibitory effect of a low concentration of PAF was antagonized by a PAF-receptor antagonist, BN 50,739, but at high concentrations, PAF was not antagonized. Kinetic analysis of PAF inhibition of Na+,K(+)-ATPase suggests that the inhibition of Na+,K(+)-ATPase by PAF is not due to competition by PAF at K(+)- or Na(+)-binding sites on the enzyme, but by complex inhibitory mechanisms. These results suggest that PAF may interact with specific and nonspecific site of action resulting in the inhibition of Na+,K(+)-ATPase. Ouabain increased mucin release from dog submandibular gland cells. Because Na+,K(+)-ATPase and ion exchange pathways are important in the secretory responses of acinar cells, PAF may regulate intracellularly the secretory function of acinar cells by modulating Na+,K(+)-ATPase and ionic homeostasis.     

Inhibitory effect of several nitric oxide-generating compounds on purified Na+,K(+)-ATPase activity from porcine cerebral cortex

Nitric oxide (NO)-generating compounds (NO donors) such as sodium nitroprusside, S-nitroso-N-acetylpenicillamine, S-nitroso-L-glutathione, 3-morpholino-sydnonimine (SIN-1), (DL)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-5-3-hexenamide, and 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene inhibited the Na+,K(+)-ATPase activity purified from porcine cerebral cortex. NO-reducing or -scavenging agents, such as superoxide dismutase or N-(dithiocarbamate)-N-methyl-D-glucamine sodium salt, L-ascorbic acid; and sulfhydryl (SH) compounds, such as dithiothreitol or the reduced form of glutathione, but not alpha-tocopherol, prevented the inhibition of the enzyme activity by all NO donors except sodium nitroprusside. Enzyme inhibition could also be reversed by these SH compounds, but not by superoxide dismutase, L-ascorbic acid, and alpha-tocopherol. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide (PTIO), which is able to scavenge NO radicals and generate nitrogen dioxide radicals (.NO2), potentiated the inhibition of this enzyme activity induced by all NO donors (except SIN-1). PTIO did not potentiate, but rather attenuated, the SIN-1-induced inhibition. SIN-1 has been reported to release both NO and superoxide and thereby to rapidly form peroxynitrite (ONOO-). These potentiated and attenuated inhibitions of the enzyme activity induced by PTIO plus all of the NO donors except sodium nitroprusside were prevented by SH compounds, but not by superoxide dismutase, L-ascorbic acid, and alpha-tocopherol. These results suggest that NO donors may release NO or NO-derived products, presumably .NO2 and ONOO-, and may inhibit the Na+,K(+)-ATPase activity by interacting with a SH group at the active site of the enzyme.     

Na+,K+-ATPase activity in cultured bovine retinal pigment epithelium

Ibogaine (Endabuse) is a psychoactive indole alkaloid found in the West African shrub, Tabernanthe iboga. This drug interrupts cocaine and amphetamine abuse and has been proposed for treatment of addiction to these stimulants. However, the mechanism of action that explains its pharmacological properties is unclear. Since previous studies demonstrated differential effects of psychotomimetic drugs (cocaine and methamphetamine) on neuropeptides such as neurotensin (NT), the present study was designed to determine: (1) the effects of ibogaine on striatal, nigral, cortical, and accumbens neurotensin-like immunoreactivity (NTLI); (2) the effects of selective dopamine antagonists on ibogaine-induced changes in NT concentrations in these brain areas; and (3) the effects of ibogaine pretreatment on cocaine-induced changes in striatal, nigral, cortical and accumbens NTLI content. Ibogaine treatments profoundly affected NT systems by increasing striatal, nigral, and accumbens NTLI content 12 h after the last drug administration. In contrast, NTLI concentrations were not significantly increased in the frontal cortex after ibogaine treatment. The ibogaine-induced increases in NTLI in striatum, nucleus accumbens and substantia nigra were blocked by coadministration of the selective D1 receptor antagonist, SCH 23390. The D2 receptor antagonist, eticlopride, blocked the ibogaine-induced increase in nigral NTLI, but not in striatum and nucleus accumbens. Ibogaine pretreatment significantly blocked the striatal and nigral increases of NTLI resulting from a single cocaine administration. Whereas many of the responses by NT systems to ibogaine resembled those which occur after cocaine, there were also some important differences. These data suggest that NT may contribute to an interaction between ibogaine and the DA system and may participate in the pharmacological actions of this drug.     

Vasoinhibitory effect of leminoprazole, a H+,K(+)-ATPase inhibitor, on rat aortic rings

The effects of the marine ascidian compound bistratene A on in vitro cultures of Plasmodium falciparum were assessed. Concentrations from 0 to 1.5 micrograms ml(-1) of the compound were tested. The parasitaemia in asynchronous cultures treated with bistratene A increased normally over the first 40 h, then decreased leaving only gametocytes. When synchronized cultures were treated with a constant dose of 50 ng ml(-1), gametocytes developed more rapidly than they did in control cultures. In addition, gametocytes developed in drug-treated cultures of P. falciparum that normally do not produce gametocytes. Bistratene A appears to inhibit merozoite invasion as well as to induce gametocytogenesis.     

E31-K352, the minimal cation binding moiety of Na+,K(+)-ATPase

While examining an imported Indonesian mangrove monitor, Varanus indicus (Reptilia: Sauria: Varanidae), for helminths, a new species of Hastospiculum was collected and is described as Hastospiculum spiralis n. sp. This species differs from all other members of the genus in caudal papillae number and arrangement, a pair of large cephalic papillae, and a spirally twisted left spicule in males. Additionally, H. spiralis n. sp. differs from certain Hastospiculum species by the right and left spicule lengths, egg shape, and the final host.     

Overexpression of the Na+,K+-ATPase alpha1 subunit increases Na+,K+-ATPase function in A549 cells

Ron (the receptor for Macrophage Stimulating Protein) has never been implicated before in human malignancies or in cell transformation. In this report we show that Ron can acquire oncogenic potential by means of two amino acid substitutions-D1232V and M1254T-affecting highly conserved residues in the tyrosine kinase domain. The same mutations in Kit and Ret have been found associated with two human malignancies, mastocytosis and Multiple Endocrine Neoplasia type 2B (MEN2B), respectively. Both mutations caused Ron-mediated transformation of 3T3 fibroblasts and tumour formation in nude mice. Moreover, cells transformed by the oncogenic Ron mutants displayed high metastatic potential. The Ron mutant receptors were constitutively active and the catalytic efficiency of the mutated kinase was higher than that of wild-type Ron. Oncogenic Ron mutants enhanced activation of the Ras/MAPK cascade with respect to wild type Ron, without affecting the JNK/SAPK pathway. Expression of Ron mutants in 3T3 fibroblasts led to different patterns of tyrosine-phos-phorylated proteins. These data show that point mutations altering catalytic properties and possibly substrate specificity of the Ron kinase may force cells toward tumorigenesis and metastasis.     

Functional activity of Na+,K(+)-pump in normal and pathological tissues

Na+,K(+)-ATPase, supporting the ionic homeostasis of the cell, is under control of Na+, K+, Mg2+, and ATP. The regulating effect of Mg2+ is rather unclear, whereas the Na+/K+ ratio in the cytoplasm is a potent regulatory factor, especially for osmotic balance in excitable cells. We have demonstrated two possibilities for regulation of ion pumping activity: First, via the number of Na+,K(+)-ATPase molecules under operation, and second, via changes in the turnover rate of the active molecules. In the presence of low ATP concentration, which is typical for cells with membrane damage (ischemic cardiac myocytes, tumor cells, fatigued muscles) Na+,K(+)-ATPase is transformed to a regime of the decreased efficiency. Radiation inactivation study demonstrates the weakening of the interprotein interactions in the enzyme complexes during ATP deficiency. Thus, measurements of ATPase activity of the purified enzyme under optimal conditions in vitro may be useless for the discrimination of pathological from normal tissues. In such a case, the estimation of lipid composition and microviscosity of the membranes under study could be important. This review briefly discusses several basic mechanisms of the regulation of Na+,K(+)-ATPase--an integral protein of the outer cell membranes.     

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.