Biomedical subjects

W B Im

Publications and source records attributed to W B Im.

At least 73 records · Page 4Linked to original sources

PubMed1992

Block of sodium channel current by anticonvulsant U-54494A in mouse neuroblastoma cells.

(+/- ) -cis-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]- benzamide (U-54494A), structurally related to a kappa opioid agonist U-50448H, is a potent anticonvulsant without analgesic or sedative effects of the opioid agonist in intact animal studies (VonVoigtlander et al., 1987). To explore the mechanism of its anticonvulsant action, we investigated the interaction of U-54494A with the voltage-gated sodium channel using the whole cell patch clamp technique in mouse neuroblastoma cells (NIE-115). The drug reversibly and dose-dependently reduced the tetrodotoxin-sensitive inward Na current without affecting its activation or inactivation kinetics or the reversal potential. Nearly half of this resting block by 50 microM U-54494A at a holding potential of -80 mV was reversed upon further hyperpolarization to -120 mV. We also observed a hyperpolarization shift (9.3 mV) of the steady-state slow inactivation curve in the presence of 50 microM drug with no shift in the steady-state activation or the fast inactivation curves. These results indicate that the drug interacts with the resting and the slowly inactivated channels. The drug appears not to interact with the open state, judging from the absence of a time-dependent block in chloramine-T-treated cells. The recovery rate of the inactivated channel was markedly delayed by the drug, and apparently is responsible for its use-dependent block upon repetitive depolarizations. Our results suggest that voltage- and use-dependent block of the Na channel by U-54494A may be an important pharmacological basis for its anticonvulsant action.

Animals↗

PubMed1992

Characterization of functional interactions of imidazoquinoxaline derivatives with benzodiazepine-gamma-aminobutyric acidA receptors.

U-78875 [imidazo[1,5-a]quinoxalin-4(5H)-one, 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)] belongs to a series of imidazoquinoxaline derivatives, recently discovered ligands with high affinity for benzodiazepine receptors. In this study, we have examined the drug and its analogs for their modes of interaction with the receptors, with a particular emphasis on finding molecular determinants for their functional properties. Changes in the substituents on N5 and C6 of the heterocyclic ring produced no major effects on binding characteristics but yielded drugs of widely varying efficacy (antagonist to full agonist), measured as gamma-aminobutyric acid (GABA)-mediated 36Cl- uptake and t-butylbicyclophosphoro[35S]thionate binding in rat cerebrocortical membranes. The relative binding affinity and efficacy of the analogs measured in brain membranes were similar to those in cloned GABAA receptors of the alpha 1 beta 2 gamma 2 (type I) and alpha 3 beta 2 gamma 2 (type II) subtypes. The imidazoquinoxalines showed no marked subtype selectivity. Their Ki value against [3H]flunitrazepam binding for type I was only 2-3 times lower than that for type II, and their rank order for agonistic activity was the same in the two subtypes, measured as GABA-mediated Cl- currents in human kidney cells (A293) expressing the subtypes of GABAA receptors. According to computational modeling of the drugs using both molecular and quantum mechanics, the agonistic activity of the imidazoquinoxaline derivatives depends on the presence of a bulky alkyl substituent at N5 and the deformation of the substituted portion of the otherwise planar ring system induced by a bulky moiety at N5 or C6. With a fixed N5 substituent (isopropyl), the relative efficacy in the brain membranes, as well as in the cloned receptors, appeared to be dependent on the degree of the ring deformation. This out-of-plane portion of the imidazoquinoxalines can be assigned to the general region occupied by the 5-phenyl group of diazepam and other agonistic functional groups of several nonbenzodiazepine ligands. It seems that this region, apparently common to various agonistic ligands, interacts with an agonistic pocket in type I and type II subtypes of the benzodiazepine receptors in the brain. Our results also provide direct support for the view that the agonists and nonagonists share largely overlapping binding regions in the benzodiazepine receptor, which has been proposed earlier from in vivo efficacy measurements of other series of ligands.

Animals↗

PubMed1992

Block of voltage-gated potassium currents by anticonvulsant U-54494A in mouse neuroblastoma cells.

U-54494A [(+-)-cis-3,4-dichloro-N-methyl-N-[2-(1- pyrrolidinyl)-cyclohexyl]-benzamide], an anticonvulsant and Na channel blocker, was examined for its interaction with delayed rectifier K channels in mouse neuroblastoma cells (NIE-115) using whole-cell and inside-out configurations of the patch clamp techniques. U-54494A at anticonvulsant doses reversibly blocked the tetraethylammonium-sensitive outward K current in a time-dependent manner without affecting the current-voltage relationship. Analysis of the time-dependent block suggests interaction of U-54494A with the open state, but not with the resting state of the K channel. Furthermore, the drug showed no interaction with inactivated states of the K channel because it produced no noticeable effect on the steady-state inactivation curve or the recovery of the channel activity from inactivated states. The drug shifted the steady-state activation curve to a hyperpolarizing direction. This shift seems to result from underestimations of the maximal conductance of the K channel in the presence of U-54494A, because of its progressively increasing potency to block the K currents as the membrane potential became more depolarized. The potency of the drug was enhanced not only by depolarized membrane potentials, but also by raising the external pH. These results suggest that U-54494A interacts with a channel binding site which is located within the membrane electric field and approachable from the cytoplasmic side. The proposal was supported further by single channel recordings with inside-out patches in which U-54494A induced flickery blocks of single channel openings of the delayed rectifier.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Tetraalkylammonium ion block of potassium currents in mouse neuroblastoma cells.

Experiments were performed to compare the mechanism of block of the delayed rectifier K+ channels in cultured mouse neuroblastoma cells by various derivatives of tetraethylammonium (TEA) which have symmetric alkyl chains of one to six carbons. Current from the whole cell was studied using the patch clamp technique. TEA blocked the whole cell K+ current with a Ki of 0.6 mM when applied to the external solution. The Ki for block by other derivatives were (mM): tetrapropylammonium, 9.2; tetrabutylammonium (TBA), 1.9; tetrapentylammonium (TPeA), 0.088; and tetrahexylammonium, (THA), 0.006. Block of the whole cell current by TEA or tetrapropylammonium did not increase with time after a step depolarization. However, block by TBA, TPeA or THA was time dependent. TEA did not compete with TPeA for the same receptor. Block by externally applied TEA was not appreciably voltage dependent, and the receptor for TPeA had an apparent electrical distance of 0.3. These observations suggest that TEA and TPeA block at separate receptors. THA could block the open channel in cell-attached patches when the compound was applied to the bath. This observation and the observation that externally applied TPeA and TEA do not occupy the same receptor suggest that derivatives having long alkyl chain lengths can reach the internal receptor from the external solution.

Animals↗

PubMed1991

Aging: stimulation rate on cardiac intracellular Na+ activity and developed tension.

Previous reports suggested that Na,K-ATPase activity and Na(+)-pump capacity decrease with senescence in left atrial myocardium of F344 rats. Current experiments were designed to determine if this reduction in the Na(+)-pump affects free intracellular Na+ levels. Mean intracellular Na+ ion activity (aiNa) was measured with Na-selective microelectrodes in left atrial muscle isolated from hearts of 4-, 14- and 25-month-old F344 rats. Preparations were stimulated randomly at frequencies between 0 and 12 h. There were no age-associated differences in aiNa measured at any frequency or in the decay of Na+ activity following discontinuation of electrical stimulation. These data indicate that the aging-related decline in Na,K-ATPase does not result in elevated aiNa even at extremely high stimulation frequencies, thus suggesting that other routes of Na+ influx and efflux are also altered in atrial muscle.

Aging↗

PubMed1991

Studies on (H(+)-K+)-ATPase inhibitors of gastric acid secretion. Prodrugs of 2-[(2-pyridinylmethyl)sulfinyl]benzimidazole proton-pump inhibitors.

The synthesis of N-substituted benzimidazole (H(+)-K+)-ATPase or proton-pump inhibitors is described. These compounds were prepared to function as prodrugs of the parent N-H compound and evaluated for their ability to inhibit gastric (H(+)-K+)-ATPase and gastric acid secretion. The prodrugs reported rely on either in vivo esterase hydrolysis for liberation of the parent compound (type I and type II) or require an acid environment for release of the active drug (type III and type IV). The N-(acyloxy)alkyl-substituted benzimidazoles 9, 11, and 24 showed improved chemical stability in the solid state and in aqueous solutions when compared to their parent N-H compounds. When given orally, 24 was found to be twice as potent as omeprazole in both the Shay rat and inactivation of gastric (H(+)-K+)-ATPase in the rat. The N-ethoxy-1-ethyl-substituted benzimidazoles 48-50 were found equally as effective as the N-H compound for inhibition of rat (H(+)-K+)-ATPase activity. In the Shay rat 48 at 10 mg/kg was approximately twice as active as parent timoprazole.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1991

Correlation between gamma-aminobutyric acidA receptor ligand-induced changes in t-butylbicyclophosphoro[35S]thionate binding and 36Cl- uptake in rat cerebrocortical membranes.

We have explored the functional significance of various drug-induced changes in t-[35S]butylbycyclophosporothionate (TBPS) binding to gamma-aminobutyric acidA (GABAA) receptors by comparing them with the actions of the drugs on GABA-induced 36Cl- uptake in rat cerebrocortical membrane preparations. In the presence of micromolar concentrations of GABA, various benzodiazepine receptor agonists, 3 alpha-21-dihydroxy-5 alpha-pregnan-20-one, and pentobarbital inhibited [35S]TBPS binding, whereas ethyl-beta-carboline-3carboxylate (beta-CCE), an inverse agonist, stimulated it, in general agreement with earlier reports [Mol. Pharmacol. 23:326-336 (1983); Mol. Pharmacol. 30:218-225 (1986)]. The drug-induced changes in [35S]TBPS binding, after normalization with respect to the corresponding action of diazepam, were closely related to the relative ability of the drugs to affect 36Cl- uptake, with a correlation coefficient of 0.98 and a slope of 0.85. Upon abolishment of GABA action by the use of bicuculline, however, all the tested drugs stimulated [35S]TBPS binding to various degrees, and their relative changes displayed a lower correlation coefficient of 0.69, with a slope of 2. In particular, the effects of the anesthetic steroid and pentobarbital on [35S]TBPS binding were markedly altered by GABA, which at 2 microM increased not only their maximal effects, but also their half-maximal concentrations severalfold. On the other hand, GABA did not significantly affect these parameters for diazepam under our experimental conditions. Also, the GABA-independent changes in [35S]TBPS binding produced by various benzodiazepine receptor agonists matched reasonably well the actions of the drugs on 36Cl- uptake, with a correlation coefficient of 0.85 and a slope of 1.0. These data suggest more pronounced functional coupling of the GABA sites with those for the steroid and the barbiturate, as compared with the benzodiazepine site. It appears that the degree of [35S]TBPS binding in the presence of GABA closely reflects the functional state of GABAA receptors and may be useful for characterization of allosteric interactions between various sites on the receptors.

Animals↗

PubMed1990

Na(+)-dependent GABA transport system scavenges endogenous external GABA and prevents desensitization of GABAA receptors in rat cerebrocortical synaptoneurosomes.

Muscimol-induced 36Cl- uptake in rat cerebrocortical synaptoneurosomes was reduced upon exposure of the membrane sacs to low Na+ media. This Na+ requirement led us to examine the role of the Na(+)-dependent gamma-aminobutyric acid (GABA) transport system in 36Cl- uptake. Incubation of the synaptoneurosomes with nipecotic acid, a specific inhibitor of the GABA transport system, for 10 min increased the level of endogenous external GABA from less than 10 to 150 microM and induced the same signs of desensitization as observed with high muscimol-treated synaptoneurosomes; a marked reduction of muscimol-induced 36Cl- uptake and an appearance of a slow bicuculline-sensitive 36Cl- uptake, probably due to a continuous recovery of a population of GABAA receptors from desensitization. Similar results were obtained upon dissipation of Na+ electrochemical gradient across the membranes by inhibition of Na+, K(+)-ATPase with ouabain or by blocking energy metabolism with azide or N-ethylmaleimide. We propose that the Na(+)-dependent GABA transport system, its operation being dependent on inwardly directed Na+ electrochemical gradient, is responsible for scavenging endogenous GABA released from the synaptoneurosomes, and thus prevents desensitization of GABAA receptors.

Animals↗

PubMed1990

Studies on the mechanism of interactions between anesthetic steroids and gamma-aminobutyric acidA receptors.

Functional interactions between steroidal anesthetics and gamma-aminobutyric acidA (GABAA) receptors have been examined with 36Cl- uptake measurements in rat cerebrocortical synaptoneurosomes. The primary effect of the steroids was to enhance the affinity of GABA for its receptors without much effect on the maximal uptake rate; the ED50 for GABA decreased from 66.4 +/- 5.7 to 8.9 +/- 1.2 microM in the presence of 20 microM 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one. Stimulation of 36Cl- uptake by high concentrations of the anesthetic steroid in the absence of exogenous GABA was not due to direct stimulation of GABAA receptors, as currently proposed, but is due to enhanced action of endogenous GABA, inasmuch as the steroid markedly increases GABA affinity for the receptors. Typically, endogenous GABA was maintained at near 1 microM by a Na(+)-dependent GABA transport system in the synaptoneurosomes. Elevation of its level with nipecotic acid, a specific inhibitor of the GABA transport system, or reduction with GABase, a GABA-scavenging system, increased or decreased, respectively, the steroid-induced bicuculline-sensitive 36Cl- uptake. At low concentrations of GABA (less than 2 microM), the stimulatory effect of 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one was markedly potentiated by pentobarbital but antagonized by 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one, a partial agonist of higher affinity. These observations, along with the structure-activity relationships of steroid analogs, strongly suggest the existence of a specific binding site for the steroids in GABAA receptors and led us to propose a minimal model in which two key common functional groups of anesthetic steroids, 3 alpha-OH- and 17 beta-polar substituents, interact with GABAA receptors (probably through hydrogen bondings) while their hydrophobic backbone remains in contact with the fatty acyl chains of membrane phospholipids.

Anesthetics↗

PubMed1989

Immobilized GABAA receptors and their ligand binding characteristics.

ConA-sepharose and polylysine-agarose beads effectively bound detergent-solubilized GABAA receptors from rat cerebrocortical membranes. The immobilized receptors showed a single class of high affinity binding sites specific for flunitrazepam or muscimol and displayed GABA-stimulated flunitrazepam binding. Maximal binding capacities of the ConA-immobilized receptor for the ligands were about three times greater than those of the polylysine-immobilized receptors. The relative affinities for each of the ligands were not affected by the method of receptor immobilization. The dissociation constants for muscimol of these immobilized receptors were somewhat dependent on the solubilizing agents used, but were considerably lower than those measured using extensively dialyzed rat cerebrocortical membranes.

Animals↗

PubMed1989

Evidence for the presence of a carbohydrate moiety in fluorescein isothiocyanate labeled fragments of rat gastric (H+-K+)-ATPase.

Limited tryptic digestion of fluorescein isothiocyanate (FITC)-labeled (H+-K+)-ATPase from rat resting light gastric membranes produced a soluble 27-kDa polypeptide which retained the fluorescence of the parent enzyme. Its production was markedly enhanced in the presence of an amphiphilic detergent, Zwittergent 3-14, which potently inhibits the ATPase activity. This increase is probably due to protection of certain tryptic cleavage sites through conformational changes of the membrane enzyme by the detergent. The NH2-terminal sequence of the 27-kDa polypeptide corresponded exactly to that beginning at Asn-369 of the cDNA-deduced primary structure of the rat ATPase. The presence of the phosphorylation site, Asp-385, and FITC-labeled Lys-517, which is known to be a part of the ATP-binding site, indicates that the 27-kDa polypeptide contains a major cytoplasmic portion of (H+-K+)-ATPase. Interestingly, the polypeptide was stained with periodate-Schiff's base, indicating its glycoprotein nature. The carbohydrate group attached to the polypeptide seems to include at least an N-linked high-mannose moiety, since the polypeptide showed Con A binding activity as detected with a Con A-biotin/avidin-peroxidase assay on nitrocellulose transblots. Also, its Con A binding activity was inhibited by excess methyl alpha-D-mannopyranoside and disappeared upon treatment of the polypeptide with endoglycosidase H and N-glycanase. Further tryptic action converted the 27-kDa polypeptide to 2 smaller FITC-labeled polypeptides of 25 and 15 kDa, which lost 18 and 96 amino acid residues, respectively, from the NH2 terminus of the parent polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗

PubMed1987

Effect of lysophosphatidylcholine on K+ transport in rat heavy gastric membranes enriched with (H+-K+)-ATPase.

K+- and ATP-dependent H+-accumulation in rat heavy gastric membrane vesicles enriched with (H+-K+)-ATPase was markedly stimulated by amphiphiles like lysophosphatidylcholine and Zwittergent 3-14 at concentrations of 10(-5) M. Their stimulatory effect was dependent on K+-concentration in the medium and was abolished by SCH 28,080, a specific inhibitor of (H+-K+)-ATPase. Lysophosphatidylcholine at the optimal dose (3 X 10(-5) M) showed dual effects on K+-dependent membrane functions; it stimulated the rate of K+-uptake by nearly 60%, but partially inhibited SCH 28,080-sensitive and K+-dependent ATP-hydrolysis (about 20% reduction). These data indicate that H+-pumping through (H+-K+)-ATPase in the inside-out gastric membrane vesicles was facilitated by the stimulatory effect of lysophosphatidylcholine on membrane K+-transport in spite of its partial inhibition of ATP-hydrolysis. It appears that the rate limiting step for operation of the ATPase is the availability of K+ ions in the luminal side of the pump. We propose that ionic amphiphiles may modulate K+-transport in rat heavy gastric membranes through specific interactions with the putative K+-transporter.

Animals↗

PubMed1987

A protein phosphatase associated with rat heavy gastric membranes enriched with (H+-K+)-ATPase influences membrane K+ transport activity.

Rat stimulated heavy gastric membranes enriched with (H+-K+)-ATPase, a marker for the apical membrane of the parietal cell, displayed a 32P-histone-dephosphorylating activity which appeared to be physically copurified with, but functionally independent of, the ATPase. The protein phosphatase activity was optimal at pH 7.5 and was inhibited by fluoride (50 mM), inorganic phosphate (50 mM), and p-chloromercuribenzoate (0.1 mM), but was insensitive to vanadate (1 mM). The 32P-phosphoproteins in the heavy gastric membranes were also dephosphorylated, apparently by their own membrane-bound phosphatase in the presence of Mg2+ at millimolar concentrations, which is likely to enhance membrane-membrane interaction. Heavy gastric membrane vesicles incubated with Mg2+ (2 mM) exhibited no alterations in K+-dependent ATP-hydrolyzing activity, Cl permeability, and protein and lipid compositions, but irreversibly lost the ATP, K+-dependent H+-pumping activity. Since valinomycin, a K+-specific ionophore, restored the intravesicular acidifying activity and an inhibitor of the protein phosphatase, inorganic phosphate, largely blocked the Mg2+-induced change in the membrane transport function, it is reasonable to propose that the phosphatase action on certain membrane proteins, possibly the putative K+ transporter or regulatory proteins, selectively decreases K+-conductance in the apical membranes of gastric parietal cells.

Adenosine Triphosphatases↗

PubMed1987

Gastric antisecretory activity of cycloheximide due to inhibition of protein synthesis.

Treatment of rats with cycloheximide 1 h before carbachol dose-dependently reduced the secretagogue-stimulated gastric acid secretion in pylorus ligated rats, and partially blocked carbachol- or histamine-induced activation of rat gastric (H+ + K+)-ATPase which includes translocation of reserve intracellular (H+ + K+)-ATPase into the apical membrane of the parietal cells and induction of a KCl pathway. Time-course studies showed that the drug was effective only when administered at least 30 min before the secretagogues. Puromycin showed the same effect as cycloheximide. Pulse labelling studies with [35S]methionine led to identification of two most actively synthesized polypeptides in rat gastric mucosa; the proteins of 38,000 and 14,000 molecular weight. The larger polypeptide was identified as rat pepsinogen. The identity of the smaller protein is not known yet. We suggest that synthesis of nascent polypeptide(s) is required for certain steps of the acid secretory process leading to the activation of the acid pump.

Adenosine Triphosphatases↗

PubMed1987

Intracellular sodium ion activity: reliable measurement and stimulation-induced change in cardiac Purkinje fibers.

Recently Na+-selective microelectrodes (NaSM) have been used to measure quantitatively small changes in intracellular sodium ion activity (aiNa) and to determine a precise time course of comparatively rapid change in aiNa. In such studies, accurate measurement of aiNa requires the following criteria: (i) NaSM should have a fast response time and (ii) an NaSM and a conventional voltage microelectrode should measure the same membrane potential. These criteria were evaluated by measuring aiNa when membrane potential of cardiac Purkinje fibers was suddenly hyperpolarized and depolarized by changing stimulation rate. The NaSM coated with a conductive silver paint had fast response times so that rapid changes in aiNa could be reliably measured. The cardiac Purkinje fibers stimulated at a constant rate generated uniform membrane voltage and the NaSM and conventional microelectrode measured virtually the same membrane potential. This result is somewhat different from that reported under voltage-clamp condition by other investigators. The aiNa of the fibers increased as the stimulation rate was increased over the range of 0.5-3 Hz. In fibers stimulated at 1 Hz, cessation of stimulation was immediately followed by an exponential decline of aiNa with an average time constant of 53 +/- 9 s (SD, n = 8), or rate constant of 0.020 +/- 0.004/s. Restimulation of the fibers produced an exponential rise of aiNa with an average time constant of 65 +/- 12 s (n = 8). Similar results were obtained in fibers stimulated at 2 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Finding of a KCl-independent, electrogenic, and ATP-driven H+-pumping activity in rat light gastric membranes and its effect on the membrane K+ transport activity.

Resting rat light gastric membranes prepared through 2H2O and Percoll gradient centrifugations were enriched not only with (H+-K+)-ATPase and K+ transport activity (Im, W. B., Blakeman, D. P., and Davis, J. P. (1985) J. Biol. Chem. 260, 9452-9460), but also with a K+-independent, ATP-dependent H+-pumping activity. This intravesicular acidification has been ascribed to an oligomycin-insensitive H+-ATPase which differed from (H+-K+)-ATPase in several respects. The H+-ATPase is electrogenic, apparently of lower capacity, required a lower optimal ATP concentration (4 microM for the H+-ATPase and 500 microM for (H+-K+)-ATPase), of lower sensitivity to vanadate and sulfhydryl agents such as p-chloromercuribenzoate and N-ethylmaleimide, and insensitive to SCH 28,080, a known competitive inhibitor of (H+-K+)-ATPase with respect to K+. Operation of the H+-ATPase, however, appeared to interfere with the K+ transport activity in the light gastric membranes, probably through development of intravesicular positive membrane potential; for example, micromolar levels of Mg2+-ATP fully inhibited K+ uptake and stimulated K+ efflux as measured with 86Rb+. Involvement of (H+-K+)-ATPase in the K+ transport is not likely, since the inhibitory effect of Mg2+-ATP continued even after removal of the nucleotide with an ATP-scavenging system. Moreover, nigericin, an electroneutral H+/K+ exchanger, could bypass the inhibitory effect of Mg2+-ATP and equilibrate the membrane vesicles with 86Rb+ while valinomycin, an electrogenic K+ ionophore, could not. Finally, the H+-ATPase could possibly be involved in the acid secretory process, since its H+-pumping activity was removed from the light gastric membrane fraction upon carbachol treatment, along with the K+ transport and (H+-K+)-ATPase activities. We have speculated that the H+-ATPase is responsible for maintaining the K+-permeable intracellular membrane vesicles acidic and K+ free during the resting state of acid secretion and may contribute to basal acid secretion.

Adenosine Triphosphatases↗

PubMed1986

Effect of norepinephrine and cyclic AMP on intracellular sodium ion activity and contractile force in canine cardiac Purkinje fibers.

The effect of norepinephrine on the Na+-K+ pump was investigated by simultaneously measuring intracellular sodium ion activity (aiNa) and contractile force of canine cardiac Purkinje fibers driven at 1.0 Hz in K+-free solution, high K+ solution, and in the presence of tetrodotoxin. In Tyrode solution containing 5.4mM [K+]o, 10(-6) M norepinephrine decreased aiNa, whereas in K+-free solution 10(-6) M norepinephrine did not lower aiNa. 16.2 mM [K+]o decreased aiNa from 8.8 +/- 0.9 mM to 6.5 +/- 0.5 mM (mean +/- SD, n = 5). Exposure to 10(-6) M norepinephrine in the presence of high [K+]o further decreased aiNa by 0.7 +/- 0.4 mM. This further decrease was prevented by exposure to 2.5 X 10(-6) M strophanthidin (n = 4). Blockade of the fast sodium channel with 5 X 10(-6) M tetrodotoxin lowered aiNa from 8.5 +/- 1.3 mM to 7.4 +/- 1.1 mM (n = 4). Exposure to 10(-6) M norepinephrine in the presence of tetrodotoxin further lowered aiNa by 0.9 +/- 0.2 mM. We also studied the effects of the analogues of adenosine 3':5'-cyclic monophosphate, N6, 2'-0-dibutyryladenosine 3':5'-cyclic monophosphate, and 8-(4-chlorophenylthiol)-adenosine 3':5'-cyclic monophosphate on aiNa and twitch tension. Both analogues lowered to aiNa and increased twitch tension mimicking the effects of norepinephrine. Our results support the hypothesis that norepinephrine lowers aiNa by stimulating the Na+-K+ pump in this tissue. This stimulation appears to be mediated by adenosine 3':5'-cyclic monophosphate and does not appear to be due to intercellular K+ accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Preparation of rat gastric heavy and light microsomal membranes enriched in (H+-K+)-ATPase using 2H2O and Percoll gradients.

Gastric heavy microsomal membranes highly enriched in (H+-K+)-ATPase were obtained from cimetidine- or carbachol-treated rats through 2H2O and Percoll gradient centrifugations. Both the resting (cimetidine-treated) and the stimulated (carbachol-treated) heavy membranes which presumably represent the apical membrane of gastric parietal cells were enriched with the polypeptides of 81,000 and 45,000 besides that of 93,000 representing (H+-K+)-ATPase. No apparent differences could be detected between the resting and the stimulated heavy membranes in their polypeptide profiles or their specific activity of (H+-K+)-ATPase. Nevertheless, the level of 86RbCl uptake was greater in the stimulated than the resting heavy microsomal membrane vesicles. The light gastric microsomes which abound in intracellular tubulovesicles containing reserve (H+-K+)-ATPase as isolated from cimetidine-treated rats were similarly purified with respect to (H+-K+)-ATPase. The purified light gastric membranes were largely devoid of the polypeptides of 81,000 and 45,000 found in the heavy gastric membranes. These observations further support the current hypothesis that secretagogues bring about changes in the environment of (H+-K+)-ATPase and induce KCl permeability in the apical membrane of the parietal cells, although at present we have been unable to identify the polypeptide(s) responsible for the KCl pathway.

Actins↗