Evidence for two different Na+-dependent [3H]-ouabain binding sites of a Na+-K+-ATPase of guinea-pig hearts.

1. The influence of various Na+ concentrations on [3H]-ouabain binding was studied in experiments on a microsomal Na+-K+-adenosine triphosphatase (ATPase) from guinea-pig hearts. 2. The ATP-independent cardiac glycoside binding was not influenced by increasing Na+ concentrations. However, a good correlation was found between the ATP-dependent [3H]-ouabain binding and Na+ concentration. 3. A more detailed analysis of these results according to Hofstee (1952) revealed two distinct processes involved in this interaction: one ouabain binding process was activated at rather low Na+ concentrations, (K0.5 = 4.5 mM); this type of [3H]-ouabain binding was strongly correlated to the Na+ concentration necessary for half maximum phosphorylation (K0.5 = 1 mM). The other ouabain binding process was predominant at high Na+ concentrations (K0.5 = 69 mM). 4. On the basis of the commonly accepted ATPase reaction cycle a model for the interaction of cardiac glycosides with the Na+-K+-ATPase is proposed, assuming two different binding sites for cardiac glycosides (E2-P and E1-P) and involving a translocation of these drugs from an outer to an inner compartment of the cell membrane.     

Control of [3H]ouabain binding to cerebromicrovascular (Na+ + K+)-ATPase by metal ions and proteins

The (Na+ + K+)-ATPase is localized to the cerebral endothelium, i.e. the blood-brain barrier, and is important for the maintenance of the brain electrolyte environment. Data from the present study indicate that Pb2+ inhibits the binding of [3H]ouabain to the cerebral microvascular (Na+ + K+)-ATPase in a time- and dose-dependent manner. Pb2(+)-induced inhibition developed slowly with a maximum obtained after 40 min. Inhibition of [3H]ouabain binding to the enzyme was 48% at 10 microM Pb2+ and appeared maximal (89%) at 100 microM Pb2+ when compared to [3H]ouabain binding in untreated microvessels at 40 min. In contrast, 100 microM Al3+ caused a 55% increase in [3H]ouabain binding to the (Na+ + K+)-ATPase, relative to untreated microvessels at 40 min. Insulin or bovine serum albumin stimulated [3H]ouabain binding to the enzyme when added at similar concentrations. However, the addition of both insulin and bovine serum albumin did not result in an additive effect. These results show that insulin exerts a nonspecific effect on [3H]ouabain binding to the (Na+ + K+)-ATPase similar to that evoked by bovine serum albumin. However, the metal ions Pb2+ and Al3+ provoke selective alterations in the cerebromicrovascular (Na+ + K+)-ATPase with Pb2+ inhibiting and Al3+ stimulating [3H]ouabain binding.     

Altered cardiac Na+,K+-ATPase activity in prehypertensive spontaneously hypertensive rat

Na⁺,K⁺-ATPase activity, Na⁺-dependent phosphorylation, and [³H]ouabain binding in sarcolemma prepared from 4 week old spontaneously hypertensive rat(SHR) ventricles were compared to the same parameters in sarcolemma from age matched normotensive Wistar-Kyoto (WKY) rat ventricles to examine whether the reduced myocardial Na⁺-pump activity in SHR is an inherited enzymatic defect or a second phenomenon due to sustained hypertension. The total body weights, ventricular weights, and blood pressures were the same for SHR and WKY. No significant differences in sarcolemmal protein content and protein recovery were noted between the two groups. Sarcolemma isolated from SHR ventricles showed significantly less Na⁺, K⁺-ATPase activity and number of phosphorylation sites when, compared to sarcolemma from the WKY ventricles. Equilibrium binding of [³H]ouabain and the tumover number of myocardial Na⁺,K⁺-ATPase, however, were the same for both groups. These results indicate that the low affinity (α, or α₁) isoform for ouabain is reduced in SHR compared to WKY but that the high affinity (α+, or α₂) α isoform is the same in ventricles of SHR and WKY. The reduced amount of isoform of the Na⁺,K⁺-ATPase in, prehypertensive SHR ventricles may play some role in the development of hypertension.     

Comparison of [3H]ouabain binding sites in intact cells and cell homogenates: apparent lack of glycoside receptors unrelated to sarcolemmal Na+, K+-ATPase in guinea-pig heart

In intact heart muscle cells incubated in a physiological solution, i.e. under the condition in which the cardiac glycosides produce pharmacological and toxicological effects, receptors for these actions of the glycosides should be available to ouabain. In cell homogenates, [3H]ouabain binding observed in the presence of Mg2+ and inorganic phosphate represents binding of the glycoside to Na+, K+-ATPase. Therefore, numbers of these two types of [3H]ouabain binding sites were compared using viable myocyte preparations obtained from ventricular muscle of guinea-pig heart. The number of ouabain binding sites observed in viable myocytes in the absence of Ca2+ and K+ was not different from the number of ouabain binding sites on Na+, K+-ATPase observed with sodium dodecylsulfate-treated homogenates prepared from isolated myocytes. These results do not support the hypothesis that there are receptors for the pharmacological or toxic actions of ouabain other than those that are associated with sarcolemmal Na+, K+-ATPase.     

Effect of chronic ethanol treatment on specific [3H]ouabain binding to (Na+ + K+)-ATPase in different areas of cat brain.

Chronic ethanol administration to cats increased specific [3H]ouabain binding by 63% in cerebral cortex, 47% in cerebellum, 84% in amygdala, and 100% in hippocampus when the binding assays were performed in the presence of 160 nM [3H]ouabain. There was no significant change in specific [3H]ouabain binding in hypothalamus, thalamus, corpus striatum, and brain stem following chronic ethanol ingestion. Scatchard analysis revealed that enhancement of specific [3H]ouabain binding following chronic ethanol treatment in some areas of cat brain is primarily due to changes in densities of ouabain binding sites. Since ouabain is a specific inhibitor of (Na+ + K+)-ATPase the present observations suggest that the molecular mechanism for the enhancement of (Na+ + K+)-ATPase activity after chronic ethanol ingestion may be due to increased net rate of synthesis of (Na+ + K+)-ATPase molecules or exposure of non-functional enzyme system following conformational change of plasma membrane.     

Alteration of binding sites for [3H]P1075 and [3H]glibenclamide in renovascular hypertensive rat aorta

Aim: The alterations of the binding sites for ATP-sensitive K channel (KATP) openers and blockers in aortic strips were investigated in hypertensive rats. Methods: Radioligand binding techniques were used to compare the specific binding properties of [3H]P1075 and [3H]glibenclamide (Gli) in normotensive (NWR) and reno-vascular hypertensive rat (RVHR) aortic strips. Results: The KD values of [^3H]P1075 binding were increased by 1.5-fold, while the Bmax values were unchanged in RVHR. The IC50 values of P1075 and pinacidil (Pin) for displacing the [^3H]P1075 binding in RVHR were increased by 1.8-and 1.7-fold, respectively. The kinetic processes of association and dissociation of [^3H]P1075 binding were slower in RVHR. Glibenclamide pretreatment slowed down the kinetic processes of the association and dissociation of [3H]P1075 binding in NWR, but failed to alter the kinetic processes of [^3H]P1075 binding in RVHR. The IC50 values of Gli for displacing the [^3H]Gli binding at high-affinity sites were increased by 3-fold, while those at low-affinity sites remained to be unchanged in RVHR. The kinetic processes of association of [^3H]Gli binding were decreased and those of the dissociation were accelerated in RVHR. The treatment with Pin slowed down the association kinetic processes but accelerated the process of the dissociation of [^3H]Gli binding in NWR, but did not alter the kinetics of [3H]Gli binding in RVHR. Conclusion: The affinity of binding sites for [3H]p 1075 and of high-affinity binding sites for [^3H]Gli are decreased, and the negative allosteric interactions between the two binding sites are impaired in RVHR aorta.     

Heterogeneity of ouabain specific binding sites and (Na+ + K+)-ATPase inhibition in microsomes from rat heart

Cardiac glycoside binding to microsomes prepared from rat heart ventricles and enriched in (Na+ + K+)-ATPase was measured by a rapid filtration technique. The relation between ouabain binding to microsomes and (Na+ + K+)-ATPase activity has also been examined. Data were statistically analysed by means of two different non linear regression methods. The experimental results were fitted the most closely by a model describing that ouabain specific binding occurred at two classes of independent sites. High affinity sites were characterized by a dissociation constant of 0.21 +/- 0.01 microM and a low capacity (9.4 +/- 1.4 pmoles/enzymatic unit). Low affinity sites were characterized by a dissociation constant equal to 13 +/- 3 microM and a capacity equal to 87 +/- 15 pmoles/enzymatic unit. Similar results were obtained with the more lipophilic glycoside digoxin. It was also observed that dihydroouabain, a ouabain derivative with a saturated lactone ring, competes with 3H-ouabain for the binding to the two classes of sites. Binding to these two classes of sites appeared to be associated with a corresponding inhibition of (Na+ + K+)-ATPase activity.     

(Na+ -K+)ATPase activity in erythrocyte membranes of spontaneously, one kidney-one wrapped, and deoxycorticosterone acetate--NaCl hypertensive rats

(Na+ -K+)ATPase activity in erythrocyte membranes of spontaneously (SHR), one kidney-one wrapped, and deoxycorticosterone acetate (DOCA)-NaCl hypertensive rats was studied. (Na+ -K+) ATPase activity decreased in both prehypertensive (6 weeks old) and hypertensive (14 weeks old) stages of SHR, suggesting that the alteration of this enzymic activity may be due to a pre-existing defect in the membrane rather than being a consequence of hypertension. By contrast, (Na+ -K+)ATPase activity remained unchanged in the one kidney-one wrapped hypertensive rats, whereas that of one kidney-one wrapped normotensive rats as well as that of DOCA-NaCl hypertensive rats was increased significantly (P less than 0.05). These changes were specific for (Na+ -K+) ATPase, since Mg2+-ATPase activity was not altered. The susceptibility of (Na+ -K+)ATPase to the inhibitory action of ouabain was not changed significantly. These findings indicate that (Na+ -K+)ATPase activities of erythrocyte membranes isolated from the different types of hypertensive rats were subject to different changes. Whether this phenomenon applies to the clinical distinctions among the various types of hypertension remains a subject for further investigation.     

Erythrosin B inhibits high affinity ouabain binding in guinea-pig heart Na+-K+-ATPase without influence on cardiac glycoside induced contractility.

Binding of [3H]-ouabain to guinea-pig heart membranes enriched in Na+-K+-ATPase revealed two different cardiac glycoside binding sites. High affinity binding was obtained at a KD = 2.2 X 10(-7) mol 1(-1) (Bmax = 16.8 pmol ouabain mg-1 protein) whereas low affinity ouabain binding occurred at a KD much greater than 10(-6) mol 1(-1). To discover whether the two ouabain binding sites are functional in guinea-pig heart muscle, erythrosin B, an inhibitor of the high affinity ouabain binding in rat brain tissue, was tested in guinea-pig isolated heart muscle preparations. Erythrosin B proved to be a potent inhibitor of the Mg2+ (Na+)-dependent-, as well as Na+-K+-activated ATPase (ID50 = 9 X 10(-6) mol 1(-1). Contractility of guinea-pig isolated papillary muscles, however, was not influenced by erythrosin B in concentrations up to 1 X 10(-5) mol 1(-1). Only very high concentrations (4 X 10(-4) mol 1(-1) resulted in a slightly negative inotropic effect (about 20%). Erythrosin B dose-dependently inhibited [3H]-ouabain binding to the Na+-K+-ATPase (KD = - 3.6 X 10(-6) mol 1(-1). In a concentration of 1 X 10(-5) mol 1(-1) the dye abolish high affinity [3H]-ouabain binding without affecting the low affinity binding sites. In contrast, in guinea-pig isolated atria, no functional antagonism between erythrosin B (5 X 10(-5) mol 1(-1) and ouabain was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]-imipramine binding sites in fawn-hooded rats

The existence of high-affinity [3H]-imipramine recognition sites was demonstrated in membranes prepared from the cerebral cortex, hypothalamus and platelets obtained from fawn-hooded rats. The Bmax and Kd values for [3H]-imipramine binding to cerebral cortical membranes were virtually identical to those obtained with cortical membrane preparations of Sprague-Dawley rats. An NBR strain of rats, genetically related to fawn-hooded rats, was found to have significantly higher levels of [3H]-imipramine binding sites in cerebral cortical membranes when compared to fawn-hooded and Sprague-Dawley rats. All four strains of rats examined possessed extremely high densities of [3H]-imipramine binding sites in a purified platelet membrane fraction. These results do not support the finding of others that the cerebral cortex and platelets of fawn-hooded rats are virtually devoid of [3H]-imipramine binding sites.     

Presynaptic imidazoline receptors and non-adrenoceptor[3H]-idazoxan binding sites in human cardiovascular tissues

1. In segments of human right atrial appendages and pulmonary arteries preincubated with [³H]-noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone, the involvement of imidazoline receptors in the modulation of [³H]-noradrenaline release was investigated.
2. In human atrial appendages, the guanidines aganodine and DTG (1,3-di(2-tolyl)guanidine) which activate presynaptic imidazoline receptors, inhibited electrically-evoked [³H]-noradrenaline release. The inhibition was not affected by blockade of α₂-adrenoceptors with 1 μM rauwolscine, but antagonized by extremely high concentrations of this drug (10 and/or 30 μM; apparent pA₂ against aganodine and DTG: 5.55 and 5.21, respectively).
3. In the presence of 1 μM rauwolscine, [³H]-noradrenaline release in human atrial appendages was also inhibited by the imidazolines idazoxan and cirazoline, but not by agmatine and noradrenaline. The inhibitory effects of 100 μM idazoxan and 30 μM cirazoline were abolished by 30 μM rauwolscine.
4. In the atrial appendages, the rank order of potency of all guanidines and imidazolines for their inhibitory effect on electrically-evoked [³H]-noradrenaline release in the presence of 1 μM rauwolscine was: aganodine⩾BDF 6143 [4-chloro-2-(2-imidazolin-2-yl-amino)-isoindoline]>DTG⩾clonidine>cirazoline>idazoxan (BDF 6143 and clonidine were previously studied under identical conditions). This potency order corresponded to that previously determined at the presynaptic imidazoline receptors in the rabbit aorta.
5. When, in the experiments in the human pulmonary artery, rauwolscine was absent from the superfusion fluid, the concentration-response curve for BDF 6143 (a mixed α₂-adrenoceptor antagonist/imidazoline receptor agonist) for its facilitatory effect on electrically-evoked [³H]-noradrenaline release was bell-shaped. In the presence of 1 μM rauwolscine, BDF 6143 and cirazoline concentration-dependently inhibited the evoked [³H]-noradrenaline release.
6. In human atrial appendages, non-adrenoceptor [³H]-idazoxan binding sites were identified and characterized. The binding of [³H]-idazoxan was specific, reversible, saturable and of high affinity (K_D: 25.5 nM). The specific binding of [³H]-idazoxan (defined by cirazoline 0.1 mM) to membranes of human atrial appendages was concentration-dependently inhibited by several imidazolines and guanidines, but not by rauwolscine and agmatine. In most cases, the competition curves were best fitted to a two-site model.
7. The rank order of affinity for the high affinity site (in a few cases for the only detectable site; cirazoline=idazoxan>BDF 6143>DTG⩾clonidine) is compatible with the pharmacological properties of I₂-imidazoline binding sites, but is clearly different from the rank order of potency for inhibiting evoked noradrenaline release from sympathetic nerves in the same tissue.
8. It is concluded that noradrenaline release in the human atrium and, less well established, in the pulmonary artery is inhibited via presynaptic imidazoline receptors. These presynaptic imidazoline receptors appear to be related to those previously characterized in rabbit aorta and pulmonary artery, but differ clearly from I₁ and I₂ imidazoline binding sites.

     

Propentofylline and postischemic brain edema: Relation to Na+-K+-ATPase activity

Following the bilateral occlusion of common carotid arteries in gerbil, an increase in water content and sodium/potassium ratio as well as the inhibition of Na⁺-K⁺-ATPase was found. The xanthine derivative propentofylline (HWA 285) [3-methyl-1-(5-oxohexyl)-7-propylxanthine] given either before or after cerebral ischemia attenuated the development or postischemic brain swelling and the increase in sodium/potassium ratio and prevented the postischemic reduction of Na⁺-K⁺-ATPase activity. It is concluded that the action of propentofylline on brain edema during ischemia is mediated aside from other possible mechanism(s), by the influence of the drug on Na⁺-K⁺-ATPase activity.     

Effect of cyclo(leucyl-glycine) on [3H]spiroperidol binding in the corpus striatum and hypothalamus of spontaneously hypertensive rats

These experiments were performed to detect changes in renal function produced by acute infusions of small amounts of ethanol into the isolated kidney of the rat. Ethanol was infused for 10 min beginning at 40 min to reach a final concentration of approximately 80 mg/100 ml in the recirculating perfusate. Control kidneys were perfused for 90 min without the addition of ethanol. Control and ethanol infused kidneys were compared with respect to the following measurements: glomerular filtration rate, urine volume, urine protein concentration, pressure and fractional excretion of sodium, chloride, potassium, calcium and magnesium. Ethanol concentration in the perfusate was measured by gas chromatography. The only parameter affected by these concentrations of ethanol was pressure. During the ten min ethanol infusion, the pressure in the system rose significantly (P less than 0.01) from 110 +/- 0.3 to 120 +/- 2.8 mmHg. After the ethanol infusion, the pressure decreased towards pre-ethanol levels at a faster rate than the decrease in ethanol concentration in the perfusate.     

Quantitative analysis of the high-affinity binding sites for [3H]ouabain in the rat vas deferens and their immunological identification as the alpha 2 isoform of Na+/K(+)-ATPase

Binding assays were performed with [3H]ouabain to investigate the presence of, and to characterize, a Na+/K(+)-ATPase isoform with high affinity for cardiac glycosides in the rat vas deferens. Nonlinear regression analysis of equilibrium experiments carried out with crude preparations in a Mg-Pi medium indicated the presence of high-affinity sites characterized with good precision (individual coefficients of variation = 11-35%) by their density (Bmax = 0.42 to 0.72 pmol/mg protein) and dissociation constant (Kd = 0.069 to 0.136 microM) values. The values of the dissociation rate constant (kappa-1) and the association rate constant (kappa+1) for these sites were 0.151 to 0.267 min-1 and 2.87 to 3.60 microM-1.min-1, respectively. A higher number of low-affinity sites (Kd around 15 microM), supposed to correspond to the alpha 1 isoform, was also identified, but their Kd and Bmax values were not quantified precisely in this crude preparation. Western blot assays indicated hybridization with specific anti-alpha 1 and anti-alpha 2 isoform antibodies but not with anti-alpha 3 isoform antibody. Taken together, the present results indicate the existence of a low proportion of the alpha 2 isoform of Na+/K(+)-ATPase in the rat vas deferens that can be quantified precisely by [3H]ouabain binding even in a crude membrane preparation that is suitable for studies under conditions of plasticity.     

Axoplasmic transport of [3H]ouabain binding sites and catecholamine secretion from an adrenergic nerve trunk.

The presence of a functional Na+/Ca2+ exchange system was explored in the ligated cat hypogastric nerve, a preparation that has been proposed as a model of giant noradrenergic nerve terminal free of effector cells. The rationale for this study was to monitor noradrenaline secretion from the ligated cat hypogastric nerve promoted by the increase in intracellular Ca2+ levels after ouabain blockade of Na+,K(+)-ATPase molecules present in the plasma membrane of the ligated cat hypogastric nerve. Such an increase in intracellular Ca2+ levels is achieved by activation, in "reverse mode," of the Na+/Ca2+ exchange system. In the present study, [3H]ouabain binding sites were identified on crude preparations of hypogastric nerve membranes. A single, high affinity (Kd around 10 nM), binding site was observed in both ligated and nonligated nerves. The number of binding sites increased with the time of ligation, reaching a peak of about 1 pmol/mg of protein 48 hr after ligation. Blockade of these binding sites by ouabain induced a dose-dependent, Ca(2+)-dependent release of noradrenaline, with an ED50 around 50 microM. The maximum release amounted to 9% of the total noradrenaline content in the cells. As would be expected for ouabain-induced noradrenaline secretion mediated by a Na+/Ca2+ exchange system working in reverse mode, the effect of ouabain was dependent upon the presence of Na+ in the incubation medium, reaching a plateau at an extracellular Na+ concentration of 100 mM. Calcium uptake after Ca2+ reintroduction in ouabain-treated nerves increased with time of ligation, suggesting the incorporation of Na+/Ca2+ exchange carrier molecules into the axolemma of hypogastric nerves. The similarity between ouabain-induced noradrenaline secretion from the ligated cat hypogastric nerve and from other adrenergic systems strongly supports the idea that the ligated cat hypogastric nerve is equipped with a functional Na+/Ca2+ exchange system that would contribute to the regulation of intracellular Ca2+ levels. Furthermore, these data, together with previously published reports, fully characterize, from a biochemical point of view, the ligated hypogastric nerve as a model of giant noradrenergic nerve terminal free of effector cells.     

Intestinal Na+-K+-ATPase activity and molecular events downstream of interferon-gamma receptor stimulation

This study evaluated the effects of human interferon-gamma (IFN-gamma) on Na(+)-K(+)-ATPase activity and the intracellular signaling pathways involved in human intestinal epithelial Caco-2 cells. Na(+)-K(+)-ATPase activity was determined as the difference between total and ouabain-sensitive ATPase. p38 MAP kinase activity was analyzed by Western blotting using the p38 MAP kinase assay kit. Total and phosphorylated STAT1 protein levels were detected using the PhosphoPlus Stat1. IFN-gamma decreased Na(+)-K(+)-ATPase activity in a time- and concentration-dependent manner. The IFN-gamma-induced decrease in Na(+)-K(+)-ATPase activity was accompanied by no changes in the abundance of alpha(1) subunit Na(+)-K(+)-ATPase. Downregulation of protein kinase C (PKC) with phorbol-12,13-dibutyrate (PDBu) prevented the inhibitory effect of IFN-gamma on Na(+)-K(+)-ATPase activity. Inhibition of Raf-1, mitogen-activated protein kinase kinase (MAPKK/MEK), p38 MAPK and STAT1 with, respectively, GW 5074, PD 98059, SB 203580 and epigallocatechin gallate prevented inhibition of Na(+)-K(+)-ATPase activity by IFN-gamma. Treatment with IFN-gamma markedly increased the expression of total and phospho-STAT1, this being accompanied by activation of p38 MAPK. Activation of phospho-STAT1 by IFN-gamma was almost abolished by epigallocatechin gallate and markedly reduced by SB 203580, but insensitive to downregulation of PKC. The increase in short circuit current (I(sc)) by 1.0 and 2.5 micrograms ml(-1) amphotericin B was markedly attenuated in IFN-gamma-treated cells. However, the inhibitory effect of PDBu on the amphotericin B-induced increase in I(sc) was of similar magnitude in vehicle- and IFN-gamma-treated cells. It is concluded that IFN-gamma markedly attenuates Na(+)-K(+)-ATPase activity. The transduction mechanisms set into motion by IFN-gamma involve the activation of PKC downstream STAT1 phosphorylation and Raf-1, MEK, ERK2 and p38 MAPK pathways, in a complex sequence of events.     

Role of Na+/H+ exchanger in acetylcholine-mediated pulmonary artery contraction of spontaneously hypertensive rats

Compared to sympathetic nervous system, the role of parasympathetic innervation on tone development, especially under diseased conditions, of the pulmonary artery is relatively unknown. In this study, the contractile effect of acetylcholine and the type(s) of muscarinic (M) receptor involved in the pulmonary artery (1st intralobar branch; endothelium-denuded, under resting tension) of the normotensive Wistar-Kyoto (WKY) and age-matched (male, 22-26 weeks old) Spontaneously hypertensive rats (SHR) were investigated. Cumulative administration of acetylcholine (> or =0.1 microM) caused a concentration-dependent increase in tension (antagonised by p-fluoro-hexahydro-sila-difenidol and 4-diphenylacetoxy-N-methylpiperidine, both are selective muscarinic M(3) receptor antagonists) and the magnitude of maximum contraction (expressed as % of 50 mM [K(+)](o)-induced contraction) was markedly enhanced in the presence of neostigmine (10 microM, an anti-cholinesterase) (acetylcholine 30 microM, SHR: 72% vs. 35%; WKY: 32% vs. 20%). In SHR only, acetylcholine-elicited contraction was suppressed by 1-[beta-[3-(4-Methoxyphenyl)-propoxyl]-4-methoxyphenethyl]-1H-imidazole (SK&F 96365, 1 microM), amiloride (500 microM), ethyl-isopropyl-amiloride (EIPA, 10 microM), 2-[2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R 7943, 5 microM), 2,4-dichlorobenzamil (10 microM), and an equal molar substitution of [Na(+)](o) (< or =30 mM) with choline or N-methyl-D-glucamine. In nominally [Ca(2+)](o)-free, EGTA (0.5 mM)-containing Krebs' solution, acetylcholine (> or =3 microM) only elicited a small contraction. In conclusion, muscarinic M(3) receptor activation is responsible for the pulmonary artery contraction induced by acetylcholine, with a greater magnitude observed in SHR. The exaggerated contraction in SHR is probably due to an influx of [Na(+)](o) through the Na(+)/H(+) exchanger and the store-operated channels (SOC) into smooth muscle cells. Elevation of cytosolic [Na(+)](i) subsequently leads to an influx of [Ca(2+)](o) through the reverse mode of the Na(+)/Ca(2+) exchanger seems to play a permissive role in mediating the exaggerated contractile response of acetylcholine recorded in the SHR.