Analysis of adenosine vascular effect in isolated rat aorta: possible role of Na+/K+-ATPase

The present experiments were undertaken in order to examine the effect of adenosine in isolated rat aorta, to investigate the possible role of intact endothelium and endothelial relaxing factors in this action and to determine which population of adenosine receptors is involved in rat aorta response to adenosine. Adenosine (0.1-300 microM) produced concentration-dependent (intact rings: pD2=4.39+/-0.09) and endothelium-independent (denuded rings: pD2=4.52+/-0.12) relaxation of isolated rat aorta. In the presence of high concentration of K+ (100 mM) adenosine-evoked relaxation was significantly reduced (maximal relaxation in denuded rings: control - 92.1+/-9.8 versus K+- 54.4+/-5.0). Similar results were obtained after incubation of ouabain (100 microM) or glibenclamide (1 microM). In K+-free solution, K+ (1-10 mM)-induced rat aorta relaxant response was significantly inhibited by ouabain (100 microM). Application of indomethacin (10 microM), NG-nitro-L-arginine (10 microM) or tetraethylammonium (500 microM) did not alter the adenosine-elicited effect in rat aorta. 8-(3-Chlorostyril)-caffeine (0.3-3 microM), a selective A2A-receptor antagonist, significantly reduced adenosine-induced relaxation of rat aorta in a concentration-dependent manner (pKB=6.57). Conversely, 1,3-dipropyl-8-cyclopentylxanthine (10 nM), an A1-receptor antagonist, did not affect adenosine-evoked dilatation. These results indicate that in isolated rat aorta, adenosine produces endothelium-independent relaxation, which is most probably dependent upon activation of smooth muscle Na+/K+-ATPase, and opening of ATP-sensitive K+ channels, to a smaller extent. According to receptor analysis, vasorelaxant action of adenosine in rat aorta is partly induced by activation of smooth muscle adenosine A2A receptors.     

Species difference in temperature dependence of cardiac (Na+ + K+)-ATPase activity

Cardiac (Na+ + K+)-ATPase from several species of animals were comparatively studied with respect to their molecular form and temperature dependence. The molecular weight of the catalytic subunit varied a little among species, but the difference did not correlate with the sensitivity of the enzyme to ouabain inhibition. Analysis of Arrhenius plots of the activities of the enzymes showed that enzymes showing break points of 24-25 degrees were relatively insensitive to ouabain inhibition whereas those enzymes with break points of 29-31 degrees were much more sensitive to the glycoside. This suggests that there is a difference in the interaction of cardiac (Na+ + K+)-ATPase with lipids between the ouabain-sensitive and -insensitive animals.     

肾上腺素能受体对心肌钠钾泵的亚基特异性调节

钠钾泵是镶嵌在哺乳动物细胞膜上的一种蛋白质,在维持细胞内离子平衡、能量代谢和信号传递中都发挥着重要作用。其功能的紊乱和调节异常会引起严重的病理生理变化。肾上腺素能受体是钠钾泵的一个重要调节因子。该文即对肾上腺素能受体对心肌钠钾泵的亚基特异性调节及研究进展进行的综述。     

Inhibition of synaptosomal (Na+ + K+)-ATPase activity by lysophosphatidic acid: its possible role in membrane depolarization

Lysophosphatidic acid (LPA), a metabolite of phosphatidic acid (PA) by phospholipases of the A2 type, markedly inhibited the synaptic membrane (Na+ + K+)-ATPase activity from rat cerebral cortices in incubation media containing free Ca2+ concentrations below 4.7 x 10(-6) M. This effect of LPA was dose-dependent, and the minimum effective concentration was 10(-6) g/ml. The inhibitory action of LPA was more potent than that of PA. Lysophosphatidylinositol (LPI) and lysophosphatidylethanolamine (LPE) at 10(-5) g/ml had little or no effect on this enzyme activity. These results suggest that LPA may partly play a role in the depolarization and/or increase in intracellular calcium concentrations in the brain.     

Involvement of tropomyosin in the sensitivity of Na+ + K+ ATPase to ouabain

The Na⁺/K⁺ ATPase sensitivity to ouabain was shown to be increased by 300 to 1000-fold after treatment of the plasma membrane by EDTA. Addition of proteins detached from the plasma membrane with Ca²⁺ ions to EDTA treated membranes reconstituted the original Na⁺/K⁺ ATPase resistance to ouabain inhibition. Tropomyosin with Ca²⁺ ions (not with Mg²⁺ ions) induced the same effect. When suboptimal doses of tropomyosin were used for such a reconstitution, the dose-response curve indicated a full reconstitution of a given percentage of enzyme molecules. This observation led us to assume a direct or indirect effect of tropomyosin on Na⁺/K⁺ ATPase functions.     

Inhibition of rat brain microsomal Na+/K(+)-ATPase and ouabain binding by mercuric chloride

This study concerned the effects of mercuric chloride on Na+/K(+)-ATPase and [3H]ouabain binding in rat brain microsomes in vitro. The data showed that HgCl2 inhibited Na+/K(+)-ATPase effectively at micromolar concentrations. The degree of inhibition was decreased with increases in enzyme concentration and incubation time. Variations in the ionic strength of Na+ and K+ did not alter the percent inhibition of Na+/K(+)-ATPase activity by HgCl2. Repeated washings partially restored enzyme activity. The binding of [3H]ouabain to microsomal membranes was inhibited by HgCl2 in a concentration-dependent manner. Cumulative inhibition studies with HgCl2 and ouabain indicated that these inhibitors did not act concurrently and independently on Na+/K(+)-ATPase.     

Digitalis-induced signaling by Na+/K+-ATPase in human breast cancer cells

Because beneficial effects of digitalis treatment in breast cancer patients have been suggested by epidemiological studies, we explored the mechanism of the growth inhibitory effects of these drugs on the estrogen receptor-negative human breast cancer cell line MDA-MB-435 s. Ouabain concentrations (100 nM or lower) that caused less than 25% inhibition of the pumping function of Na+/K+-ATPase had no effect on cell viability but inhibited proliferation. At the same concentrations, ouabain 1) activated Src kinase and stimulated the interaction of Src and Na+/K+-ATPase with epidermal growth factor receptor (EGFR); 2) caused a transient and then a sustained activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2); 3) increased the expression of p21Cip1 but decreased that of p53; and 4) activated c-Jun NH2-terminal kinase (JNK) but not p38 kinase. These data, in conjunction with our previous findings on the signaling role of Na+/K+-ATPase in other cells, suggest that ouabain-induced activation/transactivation of Src/EGFR by Na+/K+-ATPase leads to activation of ERK1/2, the resulting increase in the level of cell cycle inhibitor p21Cip1, and growth arrest. Cooperation of JNK with ERK1/2 in this process is also suggested. Digoxin and digitoxin concentrations close to or at the therapeutic plasma levels had effects on proliferation and ERK1/2 similar to those of ouabain, supporting the proposed potential value of digitalis drugs for the treatment of breast cancer.     

Reversible inhibition of (Na+ + K+)-ATPase with a cardiac glycoside.

The effect of a semisynthetic cardiac glycoside, Actinogen (Ay22241), on Na+ + K+ - ATPase was studied. Ay22241 was found to be as an effective inhibitor of the enzyme as ouabain, Ay22241 inhibition was a time dependent process and was completely reversible. While ouabain inhibition was also time dependent, it was only partially reversible. This reversibility with Ay22241 should make it a useful tool in studying the mode of action of cardiac glycosides.     

Differences in the role of Na+/K+-ATPase during alpha 1-adrenergic events in rat and rabbit aorta

Transport activity of the Na+/K+-ATPase was studied in rat and rabbit aorta under basal and agonist-stimulated conditions. Basal ouabain-sensitive 86Rb+ uptake was 2.9-fold higher in rat tissues as compared to rabbit tissues. This higher uptake in the rat was associated with a greater sensitivity to the Na+/H+ exchange inhibitor amiloride. Stimulation of alpha 1-adrenergic receptors by norepinephrine (NE) or phenylephrine (PE) resulted in an increase in ouabain-sensitive 86Rb+ uptake, whose temporal pattern differed between arteries of the two species. In rat aorta the increase was maximal during the first 2 min of agonist exposure reaching approximately a 50% higher rate of uptake than controls while rabbit aorta exhibited a steady rise in 86Rb+ uptake. Removal of extracellular Ca2+ by EGTA (1 mM) for 10 min resulted in an activation of Na+/K+-ATPase-related 86Rb+ uptake after which PE was still capable of causing a further increase, suggesting that Ca2+ influx is not responsible for the receptor-induced stimulation. Removal of extracellular Na+ reduced the PE-induced stimulation, while amiloride did not block the agonist effect. To characterize the role of the Na+/K+-ATPase during contractile events, receptor-induced 45Ca2+ uptake, 45Ca2+ release and contraction were compared in rat and rabbit aorta following Na+/K+-ATPase inhibition. Rat responses to PE were readily inhibited by ouabain or K+-free conditions, while rabbit responses were relatively resistant. 45Ca2+ extrusion and relaxation following alpha 1-adrenergic receptor stimulation were both highly dependent on activity of the Na+/K+-ATPase in rat aorta. These species' differences in Na+/K+-ATPase transport activity and its role in the regulation of contractility illustrate an example of heterogeneity in the ionic control of arterial tone.     

Evidence against a regulation of Na+/K+-ATPase by Gi proteins. Failure to detect an influence of G proteins on Na+/Ca2+-exchange in cardiac sarcolemmal membranes

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (G_i proteins) mediate negative chronotropic and negative inotropic effects by activation of K⁺ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of G_i proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na⁺/Ca²⁺ exchange and a carbachol-induced inhibition of the Na⁺/K⁺-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A₁ (Maximum number of receptors, B _max 0.033 pmol/mg) and muscarinic M₂ (B _max 2.9 pmol/mg) receptors and contained G_i2 and G_i3, two G_i protein isoforms, and Go, another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A₁-selective agonist, (–)-N ⁶-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase activity as well as those of the ouabain-sensitive, K⁺-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na⁺/K⁺-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (–)-N ⁶-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na⁺/Ca²⁺ exchange and Na⁺/K⁺-ATPase are not regulated by Gi proteins in canine cardiac sarcolemmal membranes.     

Isolated rat inferior mesenteric artery response to adenosine: possible participation of Na+/K+-ATPase and potassium channels

Adenosine (10(-7)-3 x 10(-4) M) produced concentration-dependent and endothelium-independent relaxation of isolated rat inferior mesenteric artery. Application of indomethacin (10(-5) M) or N(G)-nitro-L-arginine (10(-5) M) did did not alter adenosine-elicited relaxation. Conversely, in the presence of high concentration of K+ (100 mM), ouabain (10(-4)) or combination of tetraethylammonium (5 x 10(-4) M) and glibenclamide (10(-6) M), adenosine-evoked relaxant effect was significantly reduced. In K+-free solution, 1-3 mM potassium induced relaxation, which was partially reversed by ouabain (10(-4) M). 1,3-Dipropyl-8-cyclopentylxanthine (10(-9) M), an A1-receptor antagonist, did not affect adenosine-evoked relaxation. Oppositely, 8-(3-chlorostyryl)-caffeine (3 x 10(-7)-10(-6) M), a selective A2A receptor antagonist, significantly inhibited adenosine-induced relaxation in a concentration-dependent manner (pA2 = 6.74). These results indicate that in the isolated rat inferior mesenteric artery, adenosine produces endothelium-independent relaxation, which is partly induced by activation of smooth muscle adenosine A2A receptors, and further mediated by the activation of smooth muscle Na+/K+-ATPase and opening of mixed population of K+ channels.     

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, Mg²⁺-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 V_max with no change in K_m. Similarly, activation of K⁺ PNPPase by PNPP in the presence of MIC showed a decrease in V_max with no change in K_m. 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. Received: 3 November 1994/Accepted: 23 February 1995     

Active ingredients in Chinese medicines promoting blood circulation as Na+/K+ -ATPase inhibitors

The positive inotropic effect of cardiac glycosides lies in their reversible inhibition on the membrane-bound Na(+)/K(+)-ATPase in human myocardium. Steroid-like compounds containing a core structure similar to cardiac glycosides are found in many Chinese medicines conventionally used for promoting blood circulation. Some of them are demonstrated to be Na(+)/K(+)-ATPase inhibitors and thus putatively responsible for their therapeutic effects via the same molecular mechanism as cardiac glycosides. On the other hand, magnesium lithospermate B of danshen is also proposed to exert its cardiac therapeutic effect by effectively inhibiting Na(+)/K(+)-ATPase. Theoretical modeling suggests that the number of hydrogen bonds and the strength of hydrophobic interaction between the effective ingredients of various medicines and residues around the binding pocket of Na(+)/K(+)-ATPase are crucial for the inhibitory potency of these active ingredients. Ginsenosides, the active ingredients in ginseng and sanqi, substantially inhibit Na(+)/K(+)-ATPase when sugar moieties are attached only to the C-3 position of their steroid-like structure, equivalent to the sugar position in cardiac glycosides. Their inhibitory potency is abolished, however, when sugar moieties are linked to C-6 or C-20 position of the steroid nucleus; presumably, these sugar attachments lead to steric hindrance for the entrance of ginsenosides into the binding pocket of Na(+)/K(+)-ATPase. Neuroprotective effects of cardiac glycosides, several steroid-like compounds, and magnesium lithospermate B against ischemic stroke have been accordingly observed in a cortical brain slice-based assay model, and cumulative data support that effective inhibitors of Na(+)/K(+)-ATPase in the brain could be potential drugs for the treatment of ischemic stroke.     

Inotropic effects and Na+,K+-ATPase inhibition of ouabain in isolated guinea-pig atria and diaphragm

Effects of ouabain on force of contraction were compared in electrically driven isolated tissue preparations of guinea-pig left atria and diaphragm. A distinct and steady positive inotropic effect of ouabain was observed in atrial preparations, whereas in diaphragm preparations, ouabain produced only a slight and transient positive inotropic effect, followed by the negative inotropic phase. The transient positive inotropic effect of ouabain was observed even in the absence of extracellular calcium, but was markedly dependent on the extracellular sodium concentration. In vitro [3H]ouabain binding studies revealed that the affinity of Na+,K+-ATPase for ouabain was about eight times higher and tissue concentration of the enzyme was significantly lower in diaphragm than in cardiac tissue. The Ki value for ouabain inhibition of the cardiac Na+,K+-ATPase was also approximately ten times higher than for the diaphragm enzyme. Ouabain-sensitive 86Rb uptake, an estimate of sodium pump activity, was inhibited by ouabain at a time when it produced its transient positive inotropic effect in diaphragm preparations. These results indicate that the lack of a distinct and steady positive inotropic effect of ouabain in diaphragm was due neither to the difference in the ouabain-Na+,K+-ATPase interaction between diaphragm and cardiac tissues nor the failure of sodium pump inhibition by ouabain in diaphragm.     

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.