Species variation in the ouabain sensitivity of cardiac Na+/K+-ATPase. A possible role for membrane lipids

The role of membrane lipid composition on the modulation of ouabain sensitivity of cardiac Na+/K+-ATPase has been studied in vitro using several animal species. The animals can be grouped as ouabain-sensitive and ouabain-insensitive species. Ouabain-sensitive species (I50; 0.5-2.2 microM) include sheep, marmoset, pig and the guinea pig, whilst rat and mouse form the ouabain-insensitive group (I50; 100-105 microM). Although no species variation in the distribution of major phospholipid classes was observed, significant differences were apparent in the proportions of certain saturated and unsaturated phospholipid fatty acids. Thus, there was a marked increase in the relative proportion of docosahexaenoic (22:6, omega-3) acid in the Na+/K+-ATPase preparations from the rat and mouse compared to ouabain-sensitive species. Despite these differences, all animals had similar proportions of total saturated (sigma SAT) and total unsaturated (sigma Unsat) fatty acids. On the other hand, a good correlation between the unsaturation index of membrane lipids and I50 value for ouabain was observed. It is proposed that acyl chain characteristics (unsaturation and/or chain length) rather than the head group of the phospholipid molecule play a major role in the modulation of Na+/K+-ATPase to inhibition by ouabain.     

Neurogenic stimulation of gastric acid secretion by the Na+,K+-ATPase inhibitor ouabain in mouse isolated stomach

• 1.1. We have previously found the stimulatory effect of ouabain on gastric acid secretion. In the present study, we further studied the ouabain-induced acid secretion in mouse isolated stomach.
• 2.2. In the resting stomach preparation, ouabain produced a transient increase in basal acid secretion, which was followed by a fall to the level lower than the initial basal level.
• 3.3. The ouabain-induced acid secretion was abolished by tetrodotoxin or atropine, and was partially inhibited by hexamethonium or famotidine. High K⁺ solution potentiated the ouabain stimulation.
• 4.4. Endogenous ouabain has been recently identified in plasma of humans, but the physiological roles of this compound have not yet been defined. The present study in mouse isolated stomach showed that ouabain induced a transient increase in gastric acid secretion through the release of endogenous acetyl-choline, and subsequently inhibited acid secretion. It therefore seems likely that endogenous ouabain modifies gastric acid secretion in physiological and pathological conditions.

     

The effects of dietary treatment with essential fatty acids on sciatic nerve conduction and activity of the Na+/K+ pump in streptozotocin-diabetic rats.

1. This study examined the effects of dietary essential fatty acid supplementation (5% (w/w) evening primrose oil) upon sciatic motor nerve conduction velocity and 86Rb+ pumping in sciatic nerve endoneurial preparations in rats with 4 to 5 weeks of streptozotocin-induced diabetes. 2. Control diabetic rats (dietary supplementation with 5% (w/w) hydrogenated coconut oil) exhibited a reduction in motor nerve conduction velocity (16%; P less than 0.05) compared to similarly-fed non-diabetic controls, but there was no significant alteration in ouabain-sensitive 86Rb+ pumping, a parameter reflecting activity of the Na+/K+ pump. 3. Treatment of diabetic rats with evening primrose oil prevented completely the development of the motor nerve conduction velocity deficit without affecting the severity of diabetes. Evening primrose oil treatment did not significantly affect motor nerve conduction velocity of non-diabetic animals. 4. Evening primrose oil treatment caused a significant reduction in activity of the Na+/K+ pump in sciatic nerves of diabetic animals (45%; P less than 0.05). 5. These results suggest that the acute conduction velocity defect arising in streptozotocin-diabetic rats, and the actions of evening primrose oil upon this, are independent of any effect on activity of the Na+/K+ pump. Other putative mechanisms are discussed.     

Assessing the permeability of the rat sciatic nerve epineural sheath against compounds with local anesthetic activity: an ex vivo electrophysiological study

Abstract

Studies have shown that the sciatic nerve epineural sheath acts as a barrier and has a delaying effect on the diffusion of local anesthetics into the nerve fibers and endoneurium. The purpose of this work is to assess and to quantify the permeability of the epineural sheath. For this purpose, we isolated the rat sciatic nerve in a three-chamber recording bath that allowed us to monitor the constant in amplitude evoked nerve compound action potential (nCAP) for over 24?h. For nerves exposed to the compounds under investigation, we estimated the IT₅₀ the time required to inhibit the nCAP to 50% of its initial value. For desheathed nerves, the half-vitality time was denoted as IT₅₀(?) and for the ensheath normal nerves as IT₅₀(+). There was no significant difference between the IT₅₀ of desheathed and ensheathed nerves exposed to normal saline. The IT₅₀(?) for nerves exposed to 40?mM lidocaine was 12.1?±?0.95?s (n?=?14) and the IT₅₀(+) was 341.4?±?2.49?s (n?=?6). The permeability (P) coefficient of the epineural sheath was defined as the ratio IT₅₀(+)/IT₅₀(?). The P coefficient for 40?mM lidocaine and linalool was 28.2 and 3.48, correspondingly, and for 30?mM 2-heptanone was 4.87. This is an indication that the epineural sheath provided a stronger barrier against lidocaine, compared to natural local anesthetics, linalool and 2-heptanone. The methodology presented here is a useful tool for studying epineural sheath permeability to compounds with local anesthetic properties.     

Relationship between the positive inotropic effect of ouabain and its inhibitory effects on Na+, K+ -ATPase and active transport of Rb+ in the dog heart

Two previous reports have indicated that in the dog if sustained positive inotropy without arrhythmias is induced by ouabain infusion, inhibition of myocardial Na+, K+-activated adenosine triphosphatase is not observed. Another previous study has shown that in similar experiments induction of sustained inotropy is accompanied by inhibition of the active uptake of Rb+. To resolve the apparent inconsistencies between these findings, we repeated the experiments and measured the inhibition of the enzyme and the inhibition of Rb+ uptake after the induction of sustained inotropy with ouabain. Utilizing three different ouabain dosage regimens in open-chested dogs, we obtained three different quasi-steady state plasma levels of ouabain. At the highest level, ouabain toxicity accompanied by enzyme inhibition and inhibition to RB+ uptake was observed. With the intermediate levels, positive inotropy without significant toxicity was also accompanied by enzyme inhibition and the inhibition of Rb+ uptake. At the lowest ouabain plasma levels, sustained positive inotropy without significant inhibition of either enzyme activity or +b+ uptake was obtained. The data indicate that in this preparation either a small degree of enzyme inhibition, which is difficult to detect is sufficient to produce pronounced positive inotropy, or that the inhibition of the enzyme is not required for the induction of positive inotropy.     

Influence of highly unsaturated phosphatidylcholine on the effects of ouabain and some cardioactive drugs on cardiac contractile force and Na+, K+-ATPase activity.

Isolated left guinea pig auricles and cardiac Na,K-ATPase from calf heart were incubated with highly unsaturated phosphatidylcholine (PC) for 2 hr. Thereafter the actions of ouabain on Na, K-ATPase, and of ouabain, digoxin, digitoxin, isoproterenol, acetylcholine, pentobarbital and different extracellular Ca²⁺ concentrations on contractile force of the auricles were investigated. PC itself altered neither the ATPase activity nor the intracellular ionic homeostasis, nor contractile force of the auricles. Similarly, the staircase phenomenon, the contractile response to different extracellular Ca²⁺ concentrations, to pentobarbital and ouabain, and the extent of ouabain-induced inhibition of the ATPase were not influenced. In addition the toxicity of ouabain was not altered. However, the rate of development of the ouabain-induced ATPase inhibition, as well as of the positive inotropism and toxic effects of ouabain, digoxin and digitoxin was markedly reduced. The maximum inotropic effect of digoxin and digitoxin, and the toxicity of digitoxin were significantly enhanced. Remarkably, the binding of [³H]digitoxin was significantly diminished in PC-pre-incubated auricles, binding of [³H]ouabain, however, remained unchanged. The dose-response curves to isoproterenol and acetylcholine, the latter also in the presence of physostigmine, were markedly parallel-shifted to the right. The modifying effect of highly unsaturated PC on the action of the drugs studied is suggested to be due to an altered physico-chemical state (fluidity) of the outer surface of the cellular membrane.     

17Beta-oestradiol regulates the expression of Na+/K+-ATPase beta1-subunit, sarcoplasmic reticulum Ca2+-ATPase and carbonic anhydrase iv in H9C2 cells

1. It is necessary to improve our understanding of the effect of 17beta-oestradiol (E2) on the heart at a molecular and cellular level. In the present study, the effects of E2 on Na(+)/K(+)-ATPase, sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and carbonic anhydrase IV (CAIV) in H9C2 cells were investigated. To identify the mechanism of action of E2 on these proteins, the oestrogen receptor (ER) antagonist tamoxifen was used. 2. The results indicated that 1 and 100 nmol/L E2 can enhance the activity of Na(+)/K(+)-ATPase and SERCA and upregulate the expression of the Na(+)/K(+)-ATPase beta1-subunit, SERCA2a and CAIV at both the mRNA and protein level compared with 0 and 0.01 nmol/L E2. 17beta-Oestradiol had the greatest effect at 100 nmol/L; 1 micromol/L E2 did not further protein expression compared with 100 nmol/L E2. 3. Tamoxifen (10 nmol/L) significantly decreased the activity of SERCA, as well as the expression of the Na(+)/K(+)-ATPase beta1-subunit and SERCA at the mRNA and protein level, in H9C2 cells cultured with 1 nmol/L E2. Tamoxifen alone had no significant effect on these proteins in H9C2 cells. 4. It may be hypothesized that a suitable E2 concentration has a protective effect on the heart and that the actual dose of E2 used in hormone-replacement therapy is important in menopausal women.     

In vivo effects of nitrogen dioxide on the blood nitrate level and the Na+,K+-ATPase activity of red blood cells of rats

Male Wistar rats were exposed to 0.4, 1.2 and 4.0 ppm NO2 for 13 weeks to examine the effects of NO2 on the blood nitrate concentration and the Na+,K+-ATPase activity of red blood cells. Exposures to 1.2 and 4.0 ppm NO2 caused an elevation of the blood nitrate level at the first, third and eighth week. The maximum concentration attained was 148% (P less than 0.001, at the third week) and 201% (P less than 0.001, at the eighth week) of the controls at 1.2 and 4.0 ppm NO2, respectively. On the other hand, the nitrate concentration was decreased to the control level at the second, fourth and thirteenth weeks. 0.4 ppm NO2 caused a progressive but slight increase in the blood nitrate concentration from the third week and reached the maximum (122% of the control, P less than 0.001) at the eighth week. The Na+,K+-ATPase activity decreased slightly from the first week upon exposure to 4.0 ppm NO2 and reached the minimum (72% of the control, P less than 0.05) at the third week. Subsequently, the activity was increased to 159% (P less than 0.001) of the control at the eighth week. Exposure to 0.4 and 1.2 ppm NO2 caused fundamentally similar but less significant alterations of the Na+,K+-ATPase activity.     

In vivo and in vitro effects of cadmium on adult rat brain total antioxidant status, acetylcholinesterase, (Na+, K+)-ATPase and Mg2+-ATPase activities: protection by L-cysteine

This study was undertaken in order to investigate a) the short- and long-term in vivo effects of cadmium (Cd) on brain acetylcholinesterase (AChE), (Na+, K+)-ATPase activities in adult rats, b) the concentration-dependent in vitro and in vivo (acute experiment) effects of Cd on the activity of those enzymes, c) the in vivo and in vitro effects of the antioxidant L-cysteine (Cys) on the above enzyme activities, and d) the evaluation of brain total antioxidant status after in vivo Cd, L-cysteine, or L-cysteine+Cd administration in rats. In vitro, CdSO4 inhibited pure and brain AChE in concentrations higher than 0.1 mM, while it activated by approximately 70% (P<0.001) brain Na+, K+ -ATPase in concentrations up to 0.1 mM and inhibited its activity in higher concentrations. Mg2+ -ATPase was not influenced up to 0.1 mM concentration, while it was inactivated (40%, P<0.001) in higher CdSO4 concentrations. A dose-response study of brain CdSO4 (1,2 and 5 mg/kg once 8 hr before decapitation) revealed a dose-dependent decrease (-14 to -30%, P<0.001) of brain AChE activity, an increase of Na+, K+ -ATPase activity (+47 to +200%, P<0.001) and an increase of Mg2+ -ATPase only after the highest dose (5mg/kg) in the short-term treatment of rats. Long-term Cd administration (1 mg/kg rat daily for 4 months) activated brain AChE and Na+, K+ -ATPase about 50-65% (P<0.001) but not Mg2+ -ATPase. Brain total antioxidant status was decreased by Cd (30%, P<0.01), while it was increased by L-cysteine or L-cysteine+Cd (50%, P<0.001) in the short-term in vivo treatment. L-cysteine reversed the enzymatic activity changes observed with Cd alone in the high-dose short-term in vivo treatment of rats, as well as the brain AChE inhibition induced by Cd in the in vitro experiments. These results indicate that: a) Cd can influence in a different way the examined enzyme activities after short- and long-term administration, b) Cd may modulate brain cholinergic mechanism(s), neural excitability and metabolic energy production, and c) L-cysteine can have a protective antioxidant effect on the oxidative stress of the brain induced by Cd.     

Effects of prolonged copper exposure in the marine gulf toadfish (Opsanus beta) II: copper accumulation, drinking rate and Na+/K+ -ATPase activity in osmoregulatory tissues

Gulf toadfish were exposed to sublethal levels of copper (12.8 or 55.2 microM) for 30 days. Drinking in control fish averaged 1 ml kg(-1)h(-1) but exposure to 55.2 microM copper resulted in a complex biophasic pattern with initial (3 h and 1 day) inhibition of drinking rate, followed by an elevation of drinking rate from day 3 onwards. Drinking led to copper accumulation in the intestinal fluids at levels three to five times higher than the ambient copper concentrations, which in turn resulted in intestinal copper accumulation. The gill exhibited more rapid accumulation of copper than the intestine and contributed to early copper uptake leading to accumulation in internal organs. Muscle, spleen and plasma exhibited little if any disturbance of copper homeostasis while renal copper accumulation was evident at both ambient copper concentrations. The liver exhibited the highest copper concentrations and the greatest copper accumulation of all examined internal organs during exposure to 55.2 microM. Elevated biliary copper excretion was evident from measurements of gall bladder bile copper concentrations and appeared to protect partially against internal accumulation in fish exposed to 12.8 microM copper. No inhibition of Na+/K+ -ATPase activity in either gills or intestine was seen despite copper accumulation in these organs. Calculations of inorganic copper speciation suggest that Cu(CO3)(2)2- complexes which dominate in seawater and intestinal fluids are of limited availability for uptake while the low levels of ionic Cu2+, CuOH+ and CuCO3 may be the forms taken up by the gill and the intestinal epithelium.     

Phenytoin dephosphorylates the alpha(-) catalytic subunit of (Na+, K+)-ATPase. A study in mouse, cat and human brain

Phenytoin, a potent antiepileptic drug, has been thought to stimulate Na+, K+ transport across cell membranes, but its influence on (Na+, K+)-ATPase activity remains highly controversial. We have investigated the effects of the drug on the phosphorylation level of (Na+, K+)-ATPase partially purified from mouse, cat and human brain. (Na+, K+)-ATPase catalytic subunits [alpha(+) and alpha(-)] were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis. Previous experiments had shown that phenytoin dephosphorylates the (Na+, K+)-ATPase catalytic subunit by +/- 50% in C57/BL mice. In the present study, we showed that phenytoin (10(-4) M) decreases the phosphorylation level of (Na+, K+)-ATPase catalytic subunit by the same value in cat and human cortex. Moreover, that effect is predominant on the alpha(-) subunit, thought to be the predominant enzymatic form in non-neuronal or glial cells. The results are thus favoring the hypothesis that phenytoin stimulates the brain (Na+, K+)-ATPase. They further suggest that phenytoin mainly activates the glial enzymatic form, providing central nervous system with an enhanced ability to regulate extracellular K+.     

Triggered activity induced by K(+)-free, Na(+)-deficient solution in guinea pig ventricular muscle: the effects of ouabain, lidocaine, and Ca2+ channel blockers.

Triggered activity induced by delayed afterdepolarizations has been suggested as a cellular mechanism for some arrhythmias. The development of triggered activity should be favored by conditions that increase myoplasmic Ca2+ and increase membrane resistance. A solution which could create these conditions was devised. After perfusion with normal Tyrode's solution, 24 trabeculae were exposed to the experimental solution. All preparations developed triggered activity after exposure to the experimental solution, 83% developed delayed afterdepolarizations before the onset of triggered activity, and triggered activity stopped in all trabeculae after reperfusion with normal Tyrode's solution. The interaction of ouabain, lidocaine, and three Ca2+ channel blockers with triggered activity induced by the experimental solution is described. Verapamil inhibited triggered activity but not underlying voltage oscillations, whereas nisoldipine and Mn2+ inhibited both triggered activity and voltage oscillations. Lidocaine did not inhibit afterdepolarizations or triggered activity. Exposure to ouabain for 10 min caused delayed afterdepolarizations but not triggered activity. Our results show that the experimental solution induced triggered activity, which was highly reliable and readily reversible. The high reproducibility of this activity enables the study of interactions of pharmacological agents with this triggered activity. This may contribute to the further understanding of the mechanism underlying some arrhythmias.