Effect of isoflurane on proliferation and Na+,K+-ATPase activity of alveolar type II cells injured by hydrogen peroxide

The influence of isoflurane (Iso) on proliferation and Na+,K+-ATPase activity of alveolar type II cells (ATII cells) injured by hydrogen peroxide (H2O2) was investigated. ATII cells isolated and purified from adult Sprague-Dawley rats were randomly divided into six groups: control group, 0.28 mM Iso group, 2.8 mM Iso group, 75 microM H2O2 group, 75 microM H2O2 + 0.28 mM Iso group, and 75 microM H2O2 + 2.8 mM Iso group. After primary culture for 32 hours, the proliferation of ATII cells was detected by MTT assay, and after culture for 24 hours the activity of Na+,K+-ATPase and lactate dehydrogenase (LDH) in the cells, and malonaldehyde (MDA) content of the culture medium, were measured by colorimetry. It was found that 0.28 mM and 2.8 mM Iso had no effect on the proliferation of ATII cells (p > 0.05), but 75 microM H2O2 inhibited their proliferation (p < 0.05) compared with untreated controls; 0.28 mM and 2.8 mM Iso significantly decreased Na+,K+-ATPase activity of ATII cells compared with untreated control cells (p < 0.05), and 75 microM H2O2 markedly decreased Na+,K+-ATPase activity of ATII cells (p < 0.01) with untreated control cells. 0.28 mM and 2.8 mM Iso aggravated the decrease of Na+,K+-ATPase activity induced by H2O2. Iso had no effect on LDH activity and MDA content of the culture medium of normal ATII cells, but significantly increased LDH activity and MDA content of the culture medium of ATII cells injured by H2O2. These findings suggest that Iso itself may decrease the activity of Na+,K+-ATPase of ATII cells in vitro and further damage the cells' function under peroxidation conditions, but has no effect on the proliferation of ATII cells.     

Bumetanide reduces insulin release by a direct effect on the pancreatic beta-cells

The effect of the loop diuretic bumetanide on glucose-induced insulin release, 45Ca2+ uptake, 36Cl- fluxes and 86Rb+ (K+ analogue) efflux was tested in isolated beta-cell-rich mouse pancreatic islets. Low concentrations of bumetanide (0.1-10 microM) reduced glucose-induced insulin release as well as 45Ca2+ uptake. High concentrations (0.5-1 mM) augmented glucose-induced insulin release and an intermediate concentration (100 microM) had no effect. Bumetanide (0.01-1 mM) reduced the islet accumulation of 36Cl-. The net efflux of 36Cl- in the presence of 20 mM D-glucose was reduced by a concentration (10 microM) that lowered glucose-induced insulin release. Bumetanide (10 microM) did not affect the rate coefficient for 36Cl- efflux, which suggests that chloride permeability is not affected. Bumetanide (10 microM) reduced 86Rb+ efflux from preloaded islets. The data show that bumetanide reduces insulin release by a direct effect on pancreatic beta-cells and suggest that this may be due to reduced chloride accumulation by a Na+, K+, Cl- co-transport system. It is suggested that the reduced chloride level is responsible for the decrease in glucose-induced chloride efflux and insulin release.     

Effect of a non-sulphonylurea hypoglycaemic agent, KAD-1229 on hormone secretion in the isolated perfused pancreas of the rat.

1. We examined the cooperative effect of a newly synthesized oral hypoglycaemic agent, KAD-1229 with glucose on insulin, glucagon and somatostatin secretion in the isolated perfused pancreas of the rat. 2. KAD-1229 stimulated concentration-dependently the first phase of insulin secretion without the second phase in the presence of 2.8 mM glucose, while it stimulated both the first and the second phase of insulin release in the presence of 5.6 mM glucose. It was confirmed that the first phase of insulin release is depolarization-induced release with no other additional signal transduction. 3. KAD-1229 also enhanced insulin release evoked by 16.7 mM glucose, a concentration known to inhibit the ATP-sensitive K+ current completely. 4. A low concentration (2.8 mM) of glucose stimulated somatostatin release transiently, while a higher concentration (16.7 mM) of glucose exerted a sustained stimulation. KAD-1229 stimulated somatostatin secretion in a concentration-dependent manner irrespective of glucose concentrations. 5. When glucagon release was stimulated with 2.8 mM glucose, KAD-1229 inhibited this hypoglycaemia-induced glucagon secretion. 6. When pancreata from rats pretreated with streptozotocin (STZ) 60 mg kg-1 were perfused, the basal secretion of glucagon was markedly elevated, and the glucagon response to the low glucose was abolished. Further, the insulin and somatostatin responses to KAD-1229 were largely attenuated. KAD-1229 showed transient enhancement followed by inhibition of the glucagon release from the STZ-pretreated rat pancreas. 7. We conclude that KAD-1229 stimulates insulin and somatostatin release, while it inhibits glucagon release following transient stimulation.     

Nitric oxide modulates Na+, K+-ATPase activity through cyclic GMP pathway in proximal rat trachea

The present work demonstrated that nitric oxide (NO) modulates Na+, K+-ATPase activity in the proximal rat trachea. Sodium nitroprusside induced concentration-dependent (10-100 microM) stimulation in proximal trachea Na+, K+-ATPase activity. The effect was specific for Na+, K+-ATPase since Mg-ATPase activity was unaffected. This NO-donor changed neither Na+, K+-ATPase nor Mg-ATPase activity in the distal segment. The modulatory action on Na+, K+-ATPase induced by sodium nitroprusside was linked to an increase in nitrates/nitrites and cyclic GMP levels in proximal segments. Modulation of proximal Na+, K+-ATPase activity by sodium nitroprusside was mimicked by S-nitroso-N-acetylpenicillamine (100 microM) and 8-bromo-cyclic GMP (100 microM). Both sodium nitroprusside and 8-bromo-cyclic GMP effects on Na+, K+-ATPase activity of proximal segments of trachea were blocked by 2 microM of KT 5823 (a cyclic GMP-dependent protein kinase inhibitor), but not by 0.5 microM of KT 5720 (a cyclic AMP-dependent protein kinase inhibitor). Both kinase inhibitors decreased proximal Na+, K+-ATPase activity, but did not change Mg-ATPase activity. Okadaic acid (1 microM), a phosphatase-1 inhibitor, increased proximal Na+, K+-ATPase but not Mg-ATPase activity. The effect of okadaic acid was non-additive with that of 8-bromo-cGMP on Na+, K+-ATPase activity. Our results suggest that NO modulates proximal rat trachea Na+, K+-ATPase activity through cyclic GMP and cyclic GMP-dependent protein kinase.     

Inhibition of Na+,K+-ATPase by cisplatin and its recovery by 2-mercaptoethanol in human squamous cell carcinoma cells

Na+,K+-ATPase (EC 3.6.1.37) is assumed to be involved in the transport of cisplatin [cis-diamminedichloroplatinum(II)] into cells and to act as a modulator of 5-fluorouracil (5-FU) in combination therapy of cisplatin and 5-FU. Whereas inhibition of Na+,K+-ATPase activity by cisplatin is expected to have effects on both anti-cancer therapy and nephrotoxicity, the inhibition mechanism remains to be elucidated. We studied the inhibition of Na+,K+-ATPase activity by cisplatin using an enzyme partially purified from Ca9-22 cells derived from a human squamous cell carcinoma of the gingiva. Cisplatin inhibited the Na+,K+-dependent ATP hydrolysis activity, and this inhibition depended on both the concentration of cisplatin and the preincubation time with cisplatin. The time-dependent inhibition was thought to be caused by a slow change of cisplatin from the inactive to the active form. We further tested the effect of cisplatin on the partial reactions of the enzyme, Na+-dependent ATP hydrolysis and K+-dependent pnitrophenylphosphate hydrolysis activities to determine which step in the reaction sequence of Na+,K+-ATPase was inhibited. Cisplatin inhibited both activities depending on its concentration and the preincubation time, whereas the Na+-dependent ATP hydrolysis activity was inhibited even at lower concentrations. Formation of a phosphointermediate of Na+,K+-ATPase was also inhibited by cisplatin depending on the concentration and preincubation time. Cisplatin (500 microM) and 8-fold higher concentration of 2-mercaptoethanol (2-ME; 4 mM) prevented inactivation of the enzyme by cisplatin, and the Na+,K+-ATPase activity inhibited by pretreatment with cisplatin was also recovered almost completely by 2-ME. These results suggest that the active form of cisplatin inhibits the Na+,K+-ATPase activity by inhibiting the formation of a phosphointermediate of the enzyme and that the inhibition by cisplatin is arrested by an addition of thiol group.     

Effects of sulphonylureas and diazoxide on insulin secretion and nucleotide-sensitive channels in an insulin-secreting cell line.

1. The effects of various sulphonylureas and diazoxide on insulin secretion and the activity of various channels have been studied using tissue culture and patch-clamp methods in an insulin-secreting cell line derived from a rat islet cell tumour. 2. Tolbutamide, glibenclamide and HB699 increased the rate of insulin release by 2-5 fold. The concentrations of tolbutamide and glibenclamide giving half-maximum effects on insulin secretion were approximately 40 microM and 0.2 microM, respectively. 3. Diazoxide (0.6-1.0 mM) per se, had either no effect or produced a small increase in insulin secretion, whereas when secretion was maximally stimulated by the combination of glucose (3 mM) and leucine (20 mM), it produced inhibition. Tolbutamide-induced release was also inhibited by diazoxide. 4. Tolbutamide, glibenclamide, HB699 and HB985 reduced the open-state probability of the ATP-K+ channel in a dose-dependent manner. Tolbutamide and glibenclamide were shown to be effective regardless of which side of the membrane they were applied. 5. In whole cell recording, in which the total ATP-sensitive K+ conductance of the cell could be measured, dose-inhibition curves for tolbutamide and glibenclamide were constructed, resulting in Ki values of 17 microM and 27 nM, respectively. The value of Ki for tolbutamide was unchanged when ATP (0.1 mM) was present in the electrode. 6. Diazoxide (0.6 mM) activated the ATP-K+ channels only when they had first been inhibited by intracellular ATP (0.1 mM) or bath applied tolbutamide (3-30 microM). The inhibition produced by glibenclamide could not be reversed by diazoxide. 7. Neither tolbutamide (1.0 mM) nor glibenclamide (10 microM) altered the open-state probability of the Ca2+-activated K+ channel or the Ca2+-activated non-selective cation channel which are present in this cell line. 8. It is concluded that the sulphonylureas and related hypoglycaemic drugs and diazoxide regulate insulin secretion by direct effects on the ATP-K+ channel or a protein closely associated with this channel.     

Facilitation of insulin release by N-p-tosylglycine

N-p-tosylglycine, which inhibits transglutaminase activity in islet homogenates, was found to cause a rapid and sustained facilitation of insulin release evoked by D-glucose, L-leucine or the association of Ba2+ and theophylline in intact islets. Such a facilitating action could not be attributed to any obvious effect upon either nutrient oxidation or 45Ca net uptake and outflow. It failed to be reproduced by glycine, N alpha-p-tosyl-L-arginine methyl ester or N alpha-p-tosyl-L-lysine methyl ester. N-p-tosylglycine (5.0 mM) slightly enhanced insulin release evoked by a high concentration of glucose (16.7 mM) and failed to affect significantly the secretory response to the association of L-leucine and L-glutamine or that of D-glucose and gliclazide. N-p-tosylglycine failed to affect the incorporation of [2,5-3H]histamine in trichloroacetic acid-precipitable material in intact islets. These results suggest that N-p-tosylglycine interferes with a late event in the secretory sequence, possibly at the level of the cell boundary, rather than inhibiting the crosslinking of intracellular proteins.     

Effects of mercuric chloride on [3H]dopamine release from rat brain striatal synaptosomes

Electrophysiological studies employing amphibian neuromuscular preparations have shown that mercuric chloride (HgCl2) in vitro increases both spontaneous and evoked neurotransmitter release. The present study examines the effect of HgCl2 on the release of [3H]dopamine from synaptosomes prepared from mammalian brain tissue. Mercuric chloride (3-10 microM) produces a concentration-dependent increase in spontaneous [3H]dopamine release from "purified" rat striatal synaptosomes, in both the presence and absence of extra-synaptosomal calcium. The effects of HgCl2 on transmitter release from amphibian neuromuscular junction preparations resemble those produced by the Na+, K+-ATPase inhibitor ouabain. Experiments were performed to determine whether the HgCl2 effects on mammalian synaptosomal dopamine release are a consequence of Na+, K+-ATPase inhibition. Na+, K+-ATPase activity in lysed synaptosomal membranes is inhibited by HgCl2 (IC50 = 160 nM). However, mercuric chloride in the presence of 1 mM ouabain still increased [3H]dopamine release. The specific inhibitor of Na+-dependent, high-affinity dopamine transport, RMI81,182 inhibited ouabain-induced [3H]dopamine release whereas it had no effect on HgCl2-induced [3H]dopamine release. These data suggest that augmentation of spontaneous [3H]dopamine release by HgCl2 probably is not mediated by an inhibition of Na+, K+-ATPase and HgCl2 does not act directly on the dopamine transporter.     

Stimulation of insulin secretion by glucose in the absence of diminished potassium (86Rb+) permeability.

Two inhibitors of the nucleotide-sensitive K+ (KATP) channel, tolbutamide and quinine, were utilized in order to assess the role of this channel in glucose-stimulated insulin release from perifused rat islets. In the absence of these drugs, the addition of 15 mM glucose elicited a marked biphasic stimulation of insulin secretion concomitant with a reduction in the rate of 86Rb+ efflux. In the presence of either 500 microM tolbutamide or 100 microM quinine, a reduced rate of efflux of 86Rb+ was observed together with an elevated rate of insulin release. Under such conditions, the addition of 15 mM glucose retained the ability to stimulate insulin secretion though this was associated with a marked increase in 86Rb+ efflux. It is concluded that a net reduction in beta-cell K+ permeability is not an obligatory step in glucose-stimulated insulin release. Thus, glucose is likely to exert depolarizing actions on the beta-cell in addition to the closure of K+ channels.     

Effects of putative activators of K+ channels in mouse pancreatic beta-cells.

1 The vasodilator and antihypertensive properties of pinacidil, cromakalim (BRL 34915), nicorandil and minoxidil sulphate may be due, at least in part, to their ability to open K+ channels in vascular smooth muscles. In this study, mouse pancreatic islets were used to determine whether these drugs affect insulin release by acting on K+ channels of beta-cells. Their effects were compared to those of diazoxide. 2 Diazoxide caused a dose-dependent inhibition of insulin release by islets incubated with 15 mM glucose (93% at 100 microM). Pinacidil inhibited release by 36 and 72% at 100 and 500 microM, respectively. Cromakalim and nicorandil were less effective (35 and 25% inhibition at 500 microM). Minoxidil sulphate increased insulin release at 500 microM. 3 In the presence of 7 mM glucose and in the absence of Ca2+ (to avoid activation of Ca2+-dependent K+ channels), 86Rb efflux from islet cells was increased by 100-500 microM pinacidil and 500 microM nicorandil, which were, however, less potent than diazoxide. Cromakalim was ineffective, whereas 500 microM minoxidil sulphate decreased the efflux rate. In the absence of glucose and presence of Ca2+, 500 microM cromakalim and minoxidil sulphate inhibited 86Rb efflux. 4 Like diazoxide, pinacidil (500 microM) abolished glucose-induced electrical activity in beta-cells and hyperpolarized the membrane. 5 ATP-sensitive K+ currents were studied in single beta-cells by the whole cell patch-clamp technique. Pinacidil increased the current less than did diazoxide. In contrast, cromakalim and minoxidil sulphate decreased K+-currents whilst nicorandil was without effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of potassium concentration on the inhibitory effect of amiodarone on guinea-pig microsomal Na(+)-K(+)-ATPase activity.

The effects of varying incubation K+ concentration on the inhibitory action of amiodarone on the Mg(2+)-dependent ATP hydrolysis by myocardial Na(+)-K(+)-ATPase (EC 3.6.1.3) were studied in guinea pig heart preparations. In the first part of the study, it was established that the activity of the enzyme increased with growing concentrations up to approximately 20 mM K+. The concentration-response relationships for amiodarone were investigated in incubation media containing 2.5, 5.0 and 10 mM K+ respectively. Amiodarone exhibited similar concentration-dependent inhibitory effects in the range of 0.01 nM-80 microM at 2.5 mM, 0.13-150 microM at 5.0 mM and 0.3-700 microM at 10.0 mM K+. The corresponding IC50 values were 10.4 +/- 3.2 microM, 28.3 +/- 7.6 microM at 5.0 mM and 33.3 +/- 9.2 microM at 10.0 mM K+, respectively. Thus, reduction in the K+ concentration from the "standard" 5.0 to 2.5 mM was accompanied by a significant right-to-left shift in the inhibitory potency of amiodarone, the effective concentrations being shifted from microM into nM ranges. Increasing K+ concentration to 10 mM on the other hand attained opposite but less remarkable effects. The results show that the in vitro inhibition of myocardial Na(+)-K(+)-ATPase activity by amiodarone is related to the K+ concentration of the incubation medium. These effects may be pertinent to the mechanism by which this drug interferes with the electrogenic Na+/K+ pump activity of the enzyme, thereby probably contributing to the mechanism(s) responsible for some of its cardiac actions.     

Ouabain binding and the conformational change in Na+, K+ -ATPase

The addition of ouabain to the Na+, K+-ATPase [EC.3.6.1.3] of pig kidney modified with N-[p-(2-benzimidazolyl) phenyl] maleimide gradually increased the fluorescence and the amount of phosphoenzyme from Pi with the same time course in the presence of 0.43 mM Mg2+, 16 mM Na+, 27 microM ADP and 27 microM Pi. The extent of the increment of the fluorescence intensity was dependent on the concentration of ouabain. A Hill plot of the data showed that n (Hill coefficient) and K1/2 (apparent affinity) were equal to 0.27 and 0.84 microM, respectively. Addition of ouabain to give 93 microM increased the intensity to the highest level, similar to that of K+-sensitive phosphoenzyme (E2P), and increased the extent of phosphorylation to half the amount of E2P formed with Mg2, Na+ and ATP. ADP inhibited the phosphorylation from Pi without affecting the binding of ouabain. The extent of the fluorescence intensity induced by ouabain in the presence of 0.43 mM Mg2+, 16 mM Na+ and 27 microM ADP was the same irrespective of the presence of 27 microM Pi. Addition of inorganic phosphate to give 2.6 mM accelerated the rate of fluorescence increase and 27 microM ADP retarded it without affecting the extent of the increment. The addition of ouabain to the Na+-bound enzyme increased the fluorescence with time to a level similar to that of E2P. These results and those of others indicate that ouabain can bind to nonphosphorylated Na+, K+-ATPase, and the relative fluorescence intensity of ouabain bound Na+, K+-ATPase was similar to that of E2P irrespective of the phosphorylation.     

Effects of phenothiazine derivatives on the microclimate-pH in the rat jejunum

The effects of several phenothiazine derivatives (PTDs) and quinidine (QD) on the jejunal microclimate-pH in rats were studied using a microelectrode. Chlorpromazine, thioridazine, chlorpromazine sulfoxide (CPZSO), trifluoperazine, prochlorperazine, and QD at concentrations of 1 mM increased this microclimate-pH by 0.15-0.3 pH units, while 1 mM diethazine and 1 mM promethazine had little effect on it. The increases in the microclimate-pH caused by PTDs and QD were concentration dependent and reversible. We studied the effects of PTDs on the fluidity of intestinal brush border membranes and on the release of proteins from the intestinal tissue to the lumen. The PTD-induced changes in microclimate-pH could not be explained by either of these nonspecific effects on the membranes. Then, the effects of PTDs on Na+,K+-ATPase activity and Mg2+-ATPase activity were studied using the jejunal homogenate. Each PTD inhibited Na+,K+-ATPase and Mg2+-ATPase activity to some extent. The inhibitory effects on Na+,K+-ATPase and Mg2+-ATPase activity were compared with the PTD-induced increases in the microclimate-pH. No good correlation was obtained between the IC50 values of PTDs for Na+,K+-ATPase activity and the concentrations required to increase the microclimate-pH by 0.1 pH unit, while IC50 values of PTDs for Mg2+-ATPase activity showed a relatively good correlation, except for that of CPZSO. These findings suggest that the effects of PTDs on the microclimate-pH were not nonspecific, although the increases in the microclimate-pH caused by PTDs cannot be fully explained by the inhibitory effects of these compounds on either Na+,K+-ATPase activity or Mg2+-ATPase activity alone.     

Influence of lorcainide on microsomal Na+, K(+)-ATPase in guinea-pig isolated heart preparations.

1. The effects of lorcainide on the myocardial Mg2(+)-dependent, Na+ and K(+)-activated adenosine triphosphatase (Na+, K(+)-ATPase) were compared in guinea-pig heart preparations with those of ouabain, a specific inhibitor of the enzyme activity. 2. Both ouabain and lorcainide inhibited the microsomal Na+, K(+)-ATPase activity in a concentration-dependent fashion. Their inhibitory effective ranges were 0.05-100 microM and 0.15-125 microM, respectively, and the concentrations for half maximal inhibition (IC50 values) were 2.1 +/- 0.3 and 33.5 +/- 7.3 microM, respectively. 3. In a second series of experiments, the combined effects of the two drugs on the enzyme activity were studied. In these experiments, lorcainide produced a concentration-dependent potentiation of the inhibitory effects of ouabain on Na+, K(+)-ATPase activity. 4. The present study demonstrates that lorcainide is a potent inhibitor of myocardial Na+, K(+)-ATPase.     

Ligand effects on membrane lipids associated with sodium, potassium-activated adenosine triphosphatase: comparative spin probe studies with rat brain and heart enzyme preparations

Physical properties of membrane lipids associated with rat or dog brain and heart Na+, K+-ATPase preparations were compared using an electron spin resonance probe, 5-doxyl stearate. The degree of acyl chain order of the membrane lipids was greater for brain enzyme than for heart enzyme preparations; membrane lipids in the rat heart enzyme preparations were the most disordered. In the absence of added ligands, membrane lipids did not appear to undergo a detectable temperature-dependent rearrangement or structural transition. An apparent transition was observed in the simultaneous presence of Na+, Mg2+, and ATP. These ligands increased lipid order at temperatures above the structural transition, but not below it. In the presence of the above ligands, K+ caused a marked decrease in the transition temperature in the rat brain enzyme preparations, but only a modest decrease in rat heart enzyme preparations. Arrhenius plots of rat brain and heart Na+, K+-ATPase activity revealed a break point roughly corresponding to respective membrane lipid transition temperatures observed in the presence of Na+, K+, Mg2+, and ATP. A low concentration of ouabain (1 microM) failed to affect either the lipid transition temperature estimated by the spin probe or the value of lipid order of the rat brain enzyme preparations observed in the presence of Na+, Mg2+, and ATP, but markedly reduced the effect of K+ to lower the transition temperature observed in the presence of the above ligands. A high concentration (100 microM) of ouabain which was needed to completely inhibit rat heart enzyme eliminated the thermally induced structural rearrangement observed in the presence of Na+, Mg2+, and ATP, apparently through a nonspecific lipid perturbation. These results indicate that differences in the physical properties of the membrane lipids per se are unlikely to account for the low affinity of rat heart Na+, K+-ATPase for ouabain and also suggest that the use of high concentrations of ouabain required to completely inhibit Na+, K+-ATPase activity may cause nonspecific changes in addition to inhibition of Na+, K+-ATPase or the sodium pump.     

Effects of ethanol administration on rat liver plasma membrane-bound enzymes

Changes in the properties of rat liver plasma membranes were examined in studies designed to differentiate between direct and metabolic effects of acute and chronic ethanol ingestion. One hour after a single dose of ethanol (3 g/kg body weight) there were increases in Na+,K+-ATPase (32%) and 5'-nucleotidase (36%), and hepatic concentrations of ethanol and acetaldehyde were approximately 23 mM and 50 microM, respectively. Na+,K+-ATPase and 5'-nucleotidase activities in liver plasma membranes from control rats were not significantly changed by in vitro addition of 30 microM acetaldehyde or 50 mM ethanol. Increases in Na+,K+-ATPase (approximately 20%) and 5'-nucleotidase (approximately 30%) were also observed in liver plasma membranes isolated from rats 16 hr after feeding ethanol or sucrose supplements for 17 days. The intake of calories from dietary protein and lipid was decreased by about 25% in both the ethanol and sucrose-fed animals. Na+,K+-ATPase activities in liver plasma membranes isolated from control rats were inhibited (approximately 20%) by 100 mM ethanol in vitro, whereas no inhibition was observed using membrane preparations from rats fed ethanol or sucrose supplements. Our results show that changes in liver plasma membrane enzyme activities associated with a single dose of ethanol are not a direct effect correlated with blood, hepatic or plasma membrane concentrations of ethanol or acetaldehyde. Chronic ingestion of ethanol or sucrose supplements had similar effects on liver plasma membrane enzyme characteristics and parallel changes in nutrient intake may be a more feasible explanation of these results than any analogous direct effects of the two compounds.     

Evidence against an involvement of Na+, K+-ATPase in antiarrhythmic mechanism of phenytoin

Phenytoin (Diphenylhydantoin, DPH) did not activate Na+,K+-ATPase activity prepared from both canine cardiac and renal tissues at any ratio of NA+ to K+ in standard assay medium and this drug failed to relieve the inhibitory effect of ouabain on Na+,K+-ATPase. With the Na+,K+-ATPase partially purified from the cardiac tissue the maximum number of ouabain binding sites was 50 pmol ouabain per mg enzyme and the dissociation constant (Kd) was 4 X 10(-8) mol/l. Scatchard analysis of ouabain binding to Na+,K+-ATPase indicates that DPH did not significantly alter these parameters. The release of ouabain from Na+,K+-ATPase and ouabain complex was also not significantly influenced by DPH which indicates that the antiarrhythmic action of DPH against digitalis-induced arrhythmia is not due to a simple displacement of ouabain from Na+,K+-ATPase molecules.     

Effect of vanadium in the +5, +4 and +3 oxidation states on cardiac force of contraction, adenylate cyclase and (Na+ + K+)-ATPase activity

The influence of vanadium in the nominally +5 (NH4VO3; referred to as V5+), +4 (C10H14O5V and VOSO4; V4+) and +3 oxidation states (VCl3; V3+) on cardiac force of contraction, adenylate cyclase and (Na+ + K+)-ATPase activity was investigated in order to determine which form of vanadium mediates the cardiac effects. V5+, V4+ and V3+ (300 microM each) increased the force of contraction of isolated electrically driven cat papillary muscles by about 100%. In the presence of the reducing agent ascorbic acid (5 mM) none of the three compounds led to any distinct increase in force of contraction. On the particulate adenylate cyclase preparation from feline right ventricles only V5+ stimulated the enzyme activity by about 100%, whereas V4+ and V3+ were ineffective. In the presence of 5 mM ascorbic acid all three compounds were ineffective. In contrast, in the presence of the oxidizing agent diamide (azodicarboxylic acid-bis-dimethylamide; 1 mM) all three compounds became stimulatory. On the isolated (Na+ + K+)-ATPase V5+ (500 microM) alone reduced the basal activity by about 95%. In the presence of ascorbic acid the inhibitory effect of V5+ was greatly diminished. Similar results were obtained with V4+, V3+ (100 microM) alone inhibited (Na+ + K+)-ATPase activity only by about 40%. In the presence of ascorbic acid V3+ was ineffective. From the results it is concluded that positive inotropism, stimulation of adenylate cyclase and inhibition of (Na+ + K+)-ATPase by vanadium compounds likewise result from an action of vanadium in the +5 oxidation state.     

牛磺酸对2型糖尿病大鼠胰腺线粒体氧化应激的影响

目的探讨牛磺酸对糖尿病大鼠胰腺线粒体氧化应激的影响。方法将30只Wistar大鼠随机分为正常对照组、糖尿病组(DM组)和牛磺酸治疗组(Tau组,采用20g.L-1牛磺酸生理盐水溶液治疗,200mg·kg-1),前两组注射等体积的生理盐水溶液。8wk后,测3组大鼠血浆葡萄糖、胰岛素、丙二醛(MDA),胰腺线粒体MDA、Ca2+、超氧化物歧化酶(SOD)及Na+,K+-ATP酶(Na+,K+-ATPase)和Ca2+,Mg2+-ATP酶(Ca2+,Mg2+-ATPase)的活性。结果①DM组大鼠血糖、MDA和胰腺线粒体MDA、Ca2+含量明显高于对照组(P<0.01),而血浆胰岛素水平、SOD、Na+,K+-AT-Pase和Ca2+,Mg2+-ATPase活性明显降低(P<0.05)。②Tau组大鼠血糖、MDA及胰腺线粒体Ca2+、MDA含量较DM组明显降低(P<0.05),血浆胰岛素水平、SOD、Na+,K+-ATPase和Ca2+,Mg2+-ATPase活性明显升高(P<0.05)。结论牛磺酸可减轻2型糖尿病大鼠胰腺线粒体氧化应激水平。     

Stimulation of neuronal Na+, K+-ATPase by calcium

The effect of calcium on ATP-phosphohydrolase activity of rat brain homogenates has been investigated. In both the presence and absence of the chelating agent EDTA, free calcium within the concentration range 1.2 x 10(-7) to 5.0 x 10(-4) moles/l consistently affected only the activity of Na+, K+-ATpase; the activities of Mg2+-ATPase and Na+-ATPase were essentially unchanged by Ca2+; Ca2+-ATPase could not be demonstrated. In either the presence or absence of EDTA, concentrations of free-Ca2+ above 3 x 10(-6) moles/l caused an inhibition of Na+,K+-ATPase activity. In the presence of EDTA, concentrations of free-Ca2+ below 3 x 10(-6) moles/l were ineffective at altering Na+, K+-ATPase activity but, in the absence of EDTA, free-Ca2+ in this concentration range caused a marked stimulation of the enzyme. Evidence is presented to show that the stimulation of Na+, K+-ATPase by calcium is modulated by the regulatory protein calmodulin. Since the stimulation occurs over the range of concentrations at which calcium would be expected to be encountered within the cell, it is suggested that this is the major physiological effect of calcium on Na+, K+-ATPase.