Evidence that diphenylhydantoin does not affect adenosine triphosphatases from brain.

The effects of diphenylhydantoin on brain sodium- and potassium-dependent adenosine triphosphatase (NaK ATPase), p-nitrophenylphosphatase and the calcium-dependent adenosine triphosphatase (Ca ATPase) were examined. The results indicated that: (1) diphenylhydantoin did not stimulate the NaK ATPase of beef brain sodium iodide-treated microsomes or rat brain synaptosomes at nonsaturating or saturating levels of sodium and potassium: (2) the apparent stimulation by diphenylhydantoin under high sodium: potassium ratios reported by other workers can be explained by a potassium contamination in the diphenylhydantoin; (3) diphenylhydantoin did not alter the Michaelis constant for ATP for the NaK ATPase; (4) diphenylhydantoin did not slowly interact with or alter the NaK ATPase; (5) diphenylhydantoin did not alter the calcium inhibition of the enzyme and (6) diphenylhydantoin did not alter the p-nitrophenylphosphatase or Ca ATPase. Based on these considerations, diphenylhydantoin must be exerting its pharmacological effect by altering a membrane constituent other than the NaK ATPase.     

Plasma membrane adenosine triphosphatases in rat peritoneal mast cells and macrophages--the relation of the mast cell enzyme to histamine release.

Adenosine triphosphatases activated by calcium or magnesium have been demonstrated on the outer surface of rat peritoneal mast cells and macrophages. The plasma membrane ATPases in the two types of cells have similar but not identical properties. Mg²⁺ is somewhat more effective than Ca²⁺ in stimulating both the enzymes. They are not influenced by sodium and potassium and not inhibited by ouabain and oligomycin. Ethacrynic acid inhibits both, but the mast cell enzyme is more sensitive to it. The enzyme on the macrophage has five to thirty-seven times higher activity (average seventeen times) than that on the mast cell. The apparent K_m of the enzymes in intact cells, incubated with adenosine triphosphate for 5 min, is estimated to be 36 μM for mast cells and 30 μM for macrophages. The optimal pH for the mast cell and the macrophage enzymes is 6.7 and 7.1 respectively. The activities of the two enzymes rise similarly with temperature up to 37° but differ at 47°, the macrophage enzyme being less active at this temperature than at 37°. Phosphatidyl serine, which stimulates anaphylactic and dextran-induced histamine release, causes about 40 per cent stimulation of the plasma membrane ATPase of mast cells in the absence of Ca²⁺ and Mg²⁺ but has no appreciable effect in their presence. No change in the mast cell enzyme could, however, be observed in relation to histamine release induced by dextran, compound $\text{48}\text{80}$ and ATP. But ethacrynic acid, which in 1 mM concentration inhibits 50 per cent of the mast cell enzyme activity, also causes pronounced inhibition of histamine release induced by all the three agents in the same concentration. The inhibition is not influenced by the presence of glucose, suggesting that ethacrynic acid does not inhibit histamine release by blocking energy metabolism. Ethacrynic acid apparently acts at another site. The site of action could very well be plasma membrane ATPase. There is also a correlation between the inhibition of the mast cell enzyme by sodium fluoride and lack of calcium and their inhibitory effect on histamine release. The possible involvement of the plasma membrane ATPase of mast cells in the process of exocytosis leading to histamine release is discussed.     

Estradiol inhibition of catecholamine elicited eating in the famale rat

Two experiments test the hypothesis that estradiol suppresses food intake by inhibiting β dopamine hydroxylase in the anterior diencephalon where norepinephrine sensitive neurons activate feeding behavior. In the first experiment, dopamine or norepinephrine was injected intracerebrally in female rats during estrous and diestrous phases of the estrous cycle. Dopamine injections in the anterior diencephalon of rats elicited eating (p<0.05) only during diestrus when circulating estradiol is low; norepinephrine elicited eating during both diestrous (p<0.02) and estrous (p<0.001) phases of the cycle. In the second experiment the catecholamines were administered to ovariectomized rats with and without estradiol replacement. Again dopamine was effective in eliciting food intake (p<0.05) only in the absence of circulating estradiol while the increases following norepinephrine were independent of estradiol treatment.     

Effects of tetrabenazine and of chlorpromazine on submitochondrial adenosine triphosphatases of rat brain in the presence of the soluble fraction

The effects of tetrabenazine and of chlorpromazine on Na⁺-K⁺-ATPase and Mg²⁺-ATPase in two submitochondrial fractions prepared from rat brain were studied. The soluble fraction inhibits the ATPases in the two particulate fractions. Tetrabenazine, like the catecholamines, counteracts the inhibition by the soluble fraction. Chlorpromazine inhibits differently the ATPases in the two fractions. In the presence of the soluble fraction chlorpromazine can reduce both the inhibition by the soluble fraction and the stimulation by dopamine or tetrabenazine. It is suggested that an endogenous inhibitor and the catecholamines regulate the ATPase activity of certain membrane structures of the rat brain. Drugs like tetrabenazine and chlorpromazine might affect this regulation.     

Inhibition by uranyl nitrate of adenosine triphosphatases derived from animal and human tissues

Inhibition of adenosine triphosphatase (ATPase) by uranyl nitrate (UO₂²⁺ or U⁶⁺) was studied in microsomal fractions and tissue homogenates of several organs and species. U⁶⁺ inhibited ouabain-sensitive (Na⁺ + K⁺-dependent) ATPase and ouabain-insensitive (Mg²⁺-dependent) ATPase with I50 of 2 × 10^?5 to 2 × 10^?4m. Higher concentrations of U⁶⁺ were required to inhibit the enzyme in homogenates than in microsomal fractions. Mg²⁺ ATPase was somewhat more sensitive to U⁶⁺ than was Na⁺ + K⁺ ATPase when data were corrected for protein content of enzyme preparations. The inhibition of Na⁺ + K⁺ ATPase, but not Mg²⁺ ATPase, was markedly antagonized by Na⁺. This suggests that U⁶⁺ may inhibit Na⁺ + K⁺ ATPase at the Na⁺ site on the enzyme, whereas ouabain inhibits at the K⁺ site. ATP decreased and Mg²⁺ increased the inhibition of both enzymes. K⁺ had no effect. The remaining studies were done with Na⁺ + K⁺ ATPase. Increasing pH enhanced inhibition. The enzyme was protected by bovine serum albumin and citric acid. Ascorbic acid increased inhibition possibly by reducing U⁶⁺ to U⁴⁺, thus rendering the new ionic species reactive with sulfhydryl groups in addition to organic anions.     

Pentobarbitone inhibition of catecholamine secretion

1. The perfused isolated cow adrenal gland was used to investigate the effect of barbituric acid, phenobarbitone and pentobarbitone on catecholamine secretion.2. Pentobarbitone reduced catecholamine secretion induced by a number of drugs which cause exocytosis. The concentration of pentobarbitone which caused a 50% inhibition of catecholamine secretion was for acetylcholine 5.6 x 10(-5)M, for carbachol 6.3 x 10(-5)M, for histamine 1.6 x 10(-4)M, for (+)-amphetamine 4.4 x 10(-5)M and for potassium chloride 1.5 x 10(-4)M. The degree of inhibition by pentobarbitone was not dependent on the concentration of the secretagogue.3. Pentobarbitone (up to 10(-3)M) did not inhibit the catecholamine release that was induced by acetyldehyde or by calcium chloride; it inhibited slightly (34%) the catecholamine secretion induced by tyramine.4. Catecholamine release induced by carbachol was also inhibited by phenobarbitone (50% inhibition at 2.8 x 10(-4)M (n=7)) but was unaffected by barbituric acid.5. Pentobarbitone had no effect on spontaneous or on (+)-amphetamine- or tyramine-induced release of catecholamines from isolated chromaffin vesicles of cow adrenal medulla.6. It is concluded that pentobarbitone inhibits catecholamine release by preventing a configurational change in the structure of the membrane of the chromaffin cell which is a necessary link between receptor activation and catecholamine release.     

Ascorbic acid-like effect of the soluble fraction of rat brain on adenosine triphosphatases and its relation to catecholamines and chelating agents

The inhibitory effect of the soluble fraction of rat brain on the Na⁺, K⁺-ATPase, Mg²⁺-ATPase and K⁺-p-nitrophenylphosphatase activities of rat brain subcellular particles and its antagonization by catecholamines have been studied. The soluble fraction could be replaced by l-ascorbic acid in the process, but dehydroascorbic acid or cytoplasmic reducing agents such as l-cysteine and l-glutathione did not show the same effect. Catecholamines could be replaced by chelating agents; EDTA, EGTA, o-phenanthroline and α,α'-dipyridyl prevented the inhibition by the soluble fraction and by l-ascorbic acid. The inhibition of the enzyme activities was not prevented by catecholamines or chelators when they were added after preincubation of the enzyme preparation in the presence of soluble fraction or of l-ascorbic acid. The effect of EGTA was eliminated by an excess of Ca²⁺ and the effect of EDTA by an excess of Ni²⁺. Excesses of Ca²⁺, Mn²⁺ and Co²⁺ did not influence the effect of EDTA. It is supposed that a reoxidizable heavy metal bound to the membrane structures is responsible for the inhibitory effect of the soluble fraction and of l-ascorbic acid. Catecholamines may act by chelating this metal. The possible role of iron in the phenomenon has been investigated and discussed.     

Metabolism and uptake of adenosine in rat isolated lung and its inhibition

The metabolism of adenosine perfused through the pulmonary circulation of isolated lungs from rats was investigated radiochemically. Following a 10 s infusion of radioactive [14C]- or [3H]-adenosine, the recovery of radioactivity in effluent from the lung after 1 min increased from 30% at 0.5 microM to 80% at 1 mM adenosine. Unchanged adenosine comprised the major radioactive species in effluent, being about a third of the total up to 100 microM. Uptake of radioactivity was saturable at high concentrations with an apparent Km of 215 microM. Radioactivity retained in lung comprised over 80% as ATP and about 2% as adenosine at all concentrations. Perfusion of lungs with Krebs solution containing dipyridamole (1-100 microM) or adenine (10 microM) increased the rate of radioactive efflux, decreased uptake of radioactivity by lung and decreased metabolites of adenosine (inosine and hypoxanthine) in the effluent. Dipyridamole (10 microM) was more potent in decreasing uptake in guinea-pig lungs than in rat lungs. From these results we conclude that the pulmonary circulation in rat lung exhibits a significant inactivation process for adenosine. The isolated lung provides a convenient preparation for studying in situ pharmacological or pathological modifications of this vascular inactivation process.     

The mechanism of alpha-adrenergic inhibition of catecholamine release.

1 The effect of alpha-adrenoceptor agonists on membrane adenosine triphosphatase (ATPase) activity was studied in membranes from the bovine adrenal medulla and the rat submaxillary gland. 2 alpha-Adrenoceptor agonists (10(-7) to 10(-5) M) enhanced significantly Na,K-ATPase activity but not Mg-ATPase activity in adrenal medulla. This effect was not observed in membranes from phaeochromyocytoma. Phenylephrine (10(-5) M), naphazoline (10(-5) M) and clonidine (10(-5) M) caused a significant increase of the activity of Na,K-ATPase (but not of Mg-ATPase) in the submaxillary gland. The enhancement became more prominent after ligature of the submaxillary duct but disappeared completely after superior cervical ganglionectomy. Thus, the effect of the alpha-adrenoceptor agonists was due to an action on adrenergic nerve terminals in the submaxillary gland. 3 Phenylephrine and naphazoline did not affect 45Ca uptake but enhanced the rate of 45Ca efflux from adrenal medullary slices in vitro. 4 Phenylephrine enhanced the rate of 45Ca efflux from slices of submaxillary gland (with previous ligation of the duct); this was blocked by phentolamine and sympathetic denervation. Therefore phenylephrine was acting on the adrenergic nerve terminals. 5 It is suggested that the inhibition by alpha-adrenoceptor agonists of the exocytotic release of catecholamines from adrenergic nerve terminals and from chromaffin cells may be due to activation of the sodium pump, which results in enhancement of calcium efflux, causing a reduction of free intracellular Ca2+.     

Uptake of 5-hydroxytryptamine by rat blood platelets and its inhibition by adenosine 5'-diphosphate.

1 The uptake of 5-hydroxytryptamine (5-HT) by rat blood platelets in citrated plasma was linear for at most 10 s and was substantially complete within 3 minutes. 2 Adenosine 5'-diphosphate (ADP) was a potent inhibitor of 5-HT uptake (Ki=0.38 muM) and kinetic analysis revealed that the inhibition was not competitive. 3 Inhibition of 5-HT uptake by ADP was abolished in the presence of prostaglandin E1 and 2-n-amylthio-AMP, which also inhibit the stimulant actions of ADP on blood platelets. 4 It is concluded that ADP could inhibit 5-HT uptake by changing the Na+/K+ distribution across the cell membrane, and the biological significance of this is discussed.     

Adrenocorticotropin binding sites in rat cardiac tissue

Using the ACTH analog [125I-Tyr23,Phe2,Nle4] ACTH(1-24), the existence of specific binding sites for ACTH in atrial membrane preparations was demonstrated. The dissociation constants (Kd), determined by Scatchard analysis were not significantly different for membrane preparations of adrenal gland or atrial tissue (being 6.40 x 10(-12)M and 8.86 x 10(-12)M respectively). No binding was observed to membrane preparations from kidney or lung. While the binding of the ACTH(1-24) analog to atrial membranes was inhibited by ACTH(1-24), it was not affected by norepinephrine or epinephrine. It was proposed that the ACTH(1-24) analog may bind to sites located on the adrenergic nerve endings associated with the cardiac tissue, and that such binding would interfere with the neuronal reuptake of the catecholamines.     

Further studies on the inhibition of adenosine uptake into rat brain synaptosomes by adenosine derivatives and methylxanthines

Various adenosine derivatives, methylxanthines and other compounds were tested for their abilities to inhibit the rapid uptake of adenosine by rat cerebral cortical synaptosomes. Several pharmacologically potent derivatives of adenosine were weak inhibitors of uptake with IC20 values in excess of 10(-5) M. Derivatives in this category were adenosine-5'-N-ethylcarboxamide, adenosine-5'-cyclopropylcarboxamide, N6-cyclohexyladenosine, L-N6-phenylisopropyladenosine, 1-methylisoguanosine, 2-phenylaminoadenosine and 5-iodotubercidin. Several methylxanthines were very weak inhibitors of adenosine uptake. These included pentoxifylline, n-hexyltheophylline, n-butyltheobromine, and isoamyltheobromine. HL 725, a pyrimido-isoquinoline with potent phosphodiesterase inhibitory activity, inhibited adenosine uptake with an IC20 of 2.0 X 10(-6) M. PK 11195, a putative ligand for the peripheral benzodiazepine binding site did not alter uptake at a concentration of 10(-4) M.     

A1 adenosine receptor inhibition of cyclic AMP formation and radioligand binding in the guinea-pig cerebral cortex.

1. A1 adenosine receptors were investigated by radioligand binding and functional studies in slices and particulate preparations from guinea-pig cerebral cortex. 2. Binding of the adenosine receptor antagonist radioligand, 8-cyclopentyl-[3H]-1,3-dipropylxanthine (DPCPX) to guinea-pig cerebral cortical membranes exhibited high density (1410 +/- 241 fmol mg-1 protein) and high affinity (Kd 3.8 +/- 0.3 nM). 3. [3H]-DPCPX binding to guinea-pig cerebral cortical membranes was displaced in a monophasic manner by adenosine receptor antagonists with the rank order of affinity (Ki values, nM): DPCPX (6) < xanthine amine congener (XAC, 153) < PD 115,199 (308). 4. Agonist displacement of [3H]-DPCPX binding was biphasic and exhibited the following rank order at the low affinity site (Ki values): 2-chloro-N6-cyclopentyl-adenosine (CCPA, 513 nM) = N6-R-phenylisopropyladenosine (R-PIA, 526 nM) = N6-cyclopentyladenosine (CPA, 532 nM) < 2-chloroadenosine (2CA, 3.2 microM) = 5'-N-ethylcarboxamidoadenosine (NECA, 4.6 microM) < N6-S-phenylisopropyladenosine (S-PIA, 19.9 microM). 5. In cerebral cortical slices, [3H]-DPCPX binding was displaced by antagonists and agonists in an apparently monophasic manner with the rank order of affinity (Ki values, nM): DPCPX (14) < XAC (45) < R-PIA (266) < PD 115,199 (666) < S-PIA (21000). 6. Cyclic AMP accumulation stimulated by 30 microM forskolin in guinea-pig cerebral cortical slices was inhibited by R-PIA, CCPA and CPA up to 1 microM in a concentration-dependent fashion with IC50 values of 14, 18, and 22 nM, respectively. All three analogues inhibited the forskolin response to a similar extent (82-93% inhibition).(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional characterization of the adenosine receptor mediating inhibition of peristalsis in the rat jejunum.

1. The non-selective adenosine agonist, 5'-N-ethylcarboxamidoadenosine (NECA), is a potent inhibitor of morphine withdrawal diarrhoea in rats. More recently we found that NECA exerts its antidiarrhoeal effect by inhibiting secretion in both the jejunum and ileum and also by inhibiting peristalsis in the ileum. The specific aim of this study was to characterize the receptor in the rat jejunum mediating inhibition of peristalsis via functional studies using a range of metabolically stable adenosine analogues based on the pharmacological criteria of relative agonist and antagonist potencies. 2. Peristalsis in the rat isolated jejunum was achieved by raising the pressure to between 7-11 cmH2O for 3 min followed by a 3 min rest period (pressure at zero). The mean rate of peristalsis during inflation was 7.3 +/- 0.1 peristaltic waves per 3 min and this rate remained consistent for up to 30 min, in 5 separate tissues. The inhibitory effects of the adenosine analogues were quantified by expressing their effects as a % reduction in the mean number of peristaltic contractions derived from the control tissues. 3. The rank order of agonist potency to reduce the rate of peristalsis was: N6-cyclopentyladenosine (CPA) > NECA > R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA) > chloroadenosine (2-CADO) > S-PIA > 2-phenylaminoadenosine (CV-1808). This order complies well with the rank order of agonist potency that represents the activation of the A1 receptor subtype (CPA > R-PIA = CHA = > NECA > 2-CADO > S-PIA > CV-1808).(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic inhibition of sympathetic neurotransmission by adenosine in the rat kidney

The effect of adenosine on responses to sympathetic nerve stimulation was studied in the isolated perfused rat kidney. Adenosine at 1, 3 and 10 micrograms/ml caused significant impairment of renal sympathetic neurotransmission as evidenced in a dose-dependent reduction in the vasoconstrictor responses elicited by periarterial nerve stimulation at 2, 4 and 8 Hz. Vasoconstriction to exogenous norepinephrine was unaffected by adenosine except at 10 micrograms/ml where there was a slight reduction in response to norepinephrine. Theophylline, an adenosine receptor antagonist, inhibited the above action of adenosine at the frequencies of 2 and 4 Hz. At 2 Hz, 10 microM theophylline completely blocked the effect of 1 and 3 micrograms/ml adenosine and reduced by 50% the inhibitory action of 10 micrograms/ml. Higher concentration of theophylline (50 microM) was required to antagonize the action of adenosine at 4 Hz. These results indicate that adenosine can inhibit sympathetic neurotransmission in the rat kidney through a presynaptic purinergic mechanism. The physiological and pharmacological significance of this presynaptic action of adenosine is discussed.     

Sodium load and high affinity ouabain binding in rat and guinea-pig cardiac tissue.

An estimation of the actual Na/K-ATPase transport activity in intact cardiac cells was made by measuring the binding of [3H]-ouabain to rat and guinea-pig ventricular strips. At the low [3H]-ouabain concentration of 1 nM equilibrium binding was hardly obtained after an incubation time of five hours. Different procedures known to alter the sodium load of the cardiac preparations influenced [3H]-ouabain binding: the sodium ionophore monensin enhanced [3H]-ouabain binding, the local anaesthetic dibucaine and a reduction of external sodium ion concentration diminished [3H]-ouabain binding; [3H]-ouabain binding was similarly affected by these procedures in the rat and guinea-pig. Since [3H]-ouabain binding occurred predominantly at the high-affinity binding sites of rat myocardium under the applied experimental conditions, it was concluded that these binding sites represent Na/K-ATPase molecules involved in sodium ion transport.     

Muscarinic receptor binding and behavioral effects of atropine following chronic catecholamine depletion or acetylcholinesterase inhibition in rats

Rats were subjected to one of two experimental treatments: (1) intraventricular infusion of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA), known to permanently reduce brain dopamine and norepinephrine levels, or (2) chronic administration of the irreversible acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP). Both treatments are believed to produce relative overactivity of cholinergic systems and to suppress forward locomotion. The anticholinergic agent atropine sulfate yielded excessive forward walking in otherwise chronically akinetic 6-OHDA-treated rats, whereas atropine slightly decreased locomotion in controls. The hypothesis that such supersensitivity to atropine may be related to a reduction in the density of muscarinic cholinergic receptors was not supported: First, ³H-quinuclidinyl benzilate (QNB) binding to membrane preparations was not decreased in the 6-OHDA-treated rats; secondly, atropine did not induce excessive forward locomotion in the DFP-treated rats in which ³H-QNB binding was decreased. There were other changes in the DFP-treated rats consistent with muscarinic receptor alteration, including tolerance to the locomotor suppressive effects of DFP, cross tolerance to the cholinergic agonist pilocarpine, and exaggerated atropine-induced increases in core temperature and stereotypy. It is concluded that 6-OHDA and DFP produce different long-term changes in cholinergic brain systems and atropine-sensitive behaviors.