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Query: UNIPROT:P06889 (Mol)
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1. Nicotinic acetylcholine receptors (nAChR)4 from BC3H1 cells (which express a skeletal muscle-type receptor) and from Torpedo californica electric organ were expressed in Xenopus laevis oocytes and studied with a voltage-clamp technique. 2. We found that bath application of ATP in the micromolar to millimolar range increased the ACh-elicited current in both muscle and electrocyte receptors. The effect of ATP increased with successive applications. This "use-dependent" increase in potentiation was Ca2+ dependent, while the potentiation itself was not. 3. Four other nucleotides were tested on muscle nAChR: ADP, AMP, adenosine, and GTP. Of these, only ADP was a potentiator, but its effect was not use dependent. Neither ATP nor ADP affected the resting potential of the oocyte membrane. 4. ADP potentiated the response to suberyldicholine and nicotine, as well as ACh. 5. Finally, ADP reversed the phencyclidine-induced block of ACh currents in oocytes expressing muscle nAChR.
Cell Mol Neurobiol 1990 Sep
PMID:Regulation of nicotinic acetylcholine receptor function by adenine nucleotides. 225 64

The effect of extracellular and intracellular application of forskolin on the voltage-sensitive calcium current, ICa, was studied in myocytes isolated from frog ventricle. Myocytes were isolated by enzymatic dissociation, and ICa was measured using the whole-cell configuration of the patch clamp technique modified to permit intracellular perfusion of various substances. Intracellular perfusion with forskolin (0.1 to 10 microM) had a negligible effect on ICa: ICa was increased 15 +/- 13% (mean +/- SE; N = 5). In contrast, superfusion of the cell with forskolin increased ICa significantly. The EC50 for the forskolin effect was 0.4 microM. A maximal 4.5-fold increase in ICa occurred with 3 microM forskolin. This is somewhat less than the maximal response to isoprenaline seen in this same series of experiments. The effects of forskolin, isoprenaline, and intracellular cAMP were not additive. In contrast, the effects of isoprenaline or intracellular cAMP and calcium channel agonists, such as Sandoz (+)202-791, were additive. This supports the hypothesis that the positive inotropic effects of forskolin are at least partly mediated by an increase in intracellular cAMP and a stimulation of ICa. The effects of forskolin were antagonized by acetylcholine (1 microM) or intracellular perfusion with cGMP. Acetylcholine on the average decreased forskolin-stimulated ICa 57 +/- 11% (N = 17). The relevance of these results to the suggestion that acetylcholine acts by mechanisms other than inhibition of adenylate cyclase is discussed.
Mol Pharmacol 1987 Nov
PMID:Effect of forskolin and acetylcholine on calcium current in single isolated cardiac myocytes. 244 14

A new GTP-binding protein, which serves as a substrate for pertussis toxin, was prepared from porcine brain. The new G protein was separated from other GTP-binding proteins, Gi and Go, by an anion-exchange column chromatography. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha subunit of the new G protein was between those of alpha subunits of Gi and Go. Evidence that the alpha subunit is not a proteolytic fragment of the alpha subunit is not a proteolytic fragment of the alpha subunit of Gi or Go was provided by experiments involving partial hydrolysis of these G proteins with thermolysin and their interaction with an antibody raised against the amino terminal peptide of the alpha subunit of Gi. In addition, the gamma subunit of the new G protein was indicated to be different from the gamma subunits of Gi and Go, because the latter were found to be phosphorylated by protein kinase C but the former was not. GTP-sensitive high affinity binding of muscarinic receptors with acetylcholine was observed when muscarinic receptors purified from porcine cerebrum were reconstituted in phospholipid vesicles with the new G protein as well as with Gi or Go. The proportion of the high affinity sites increased with the concentrations of the G proteins, the potency of the new G protein being similar to that of Gi but a little lower than that of Go. This GTP-sensitive high affinity binding was not observed when each G protein was pretreated with pertussis toxin and then reconstituted with muscarinic receptors. Acetylcholine accelerated the dissociation of [3H]GDP from the new G protein as well as from Gi and Go, which were reconstituted with muscarinic receptors. These results indicate that muscarinic receptors interact with at least the above three kinds of G proteins, in a pertussis toxin-sensitive manner.
Mol Pharmacol 1989 Mar
PMID:Cerebral muscarinic acetylcholine receptors interact with three kinds of GTP-binding proteins in a reconstitution system of purified components. 249 27

The rules underlying muscarinic acetylcholine receptor (mAChR) regulation in an in vitro cortical slice preparation of adult rats were examined following various alterations of bioelectric activity and following agonist stimulation. Muscarinic ACh antagonists [3H]N-methyl scopolamine ([3H]NMS) or [3H]quinuclidinyl benzylate ([3H]QNB) were used to label cell surface vs total (i.e. surface and internal) receptors, respectively. Depolarization of neural membranes for 4 h at 22-37 degrees C using veratridine or high external potassium (K+) led to a temperature-dependent down-regulation of surface mAChR of 26.2% and 11.3%. Total mAChRs decreased by 37.6% and 8.1%. Addition of picrotoxin and glutamic acid also led to decreases in mAChRs. Increases in inward chloride ion current induced by gamma-aminobutyric acid (GABA) or gold chloride had no significant effect on mAChRs. Blockade of calcium channels and synaptic transmission by magnesium or cobalt and postsynaptic calcium channels with nifedipine showed a significant effect on mAChRs only in the latter case. In contrast, agonist stimulation using carbachol led to a large down-regulation for both [3H]NMS and [3H]QNB (26.1%, 35.9%). ACh decreased [3H]QNB binding by 33.9%, but had little effect on [3H]NMS binding (6.3%). For [3H]QNB binding sites the effects of carbachol appeared to summate with those of veratridine. Down-regulation of [3H]NMS labelled mAChRs by carbachol and veratridine had an estimated half-time of 30 min and 2 h, respectively. Neither the effects of veratridine nor carbachol could be antagonized by tetrodotoxin (TTX), showing that the effects were not due to an increase in sodium ion currents. However, a common thread linking the various agents which induce mAChR down-regulation appears to involve changes in potassium (K+) current. Potassium channel blockers tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP) and apamin had little independent effect on mAChR number, but prevented veratridine-induced down-regulation, presumably through a blockade of K+- and Ca2+-dependent K+-channels. Only TEA and 4-AP diminished carbachol-induced down-regulation suggesting that this effect involves only the non Ca2+-dependent K+-channels. It thus appears that mAChR regulation in the rat cerebral cortex is linked to changes in active K+-channel currents: activation of the K+-channel by depolarization-induced changes in K+ current or by agonist stimulation leading to changes in the selective K+ currents stimulate mAChR down-regulation; blockage of the K+-channels prevents this down-regulation.
Brain Res Mol Brain Res 1989 Jan
PMID:A role for potassium channels in the regulation of cortical muscarinic acetylcholine receptors in an in vitro slice preparation. 253 5

The effects of ischemia, reperfusion and hypoxia on the cardiac acetylcholine, choline, norepinephrine and cyclic AMP contents were investigated in isolated, spontaneously beating rat hearts perfused under constant pressure (100 cm H2O) with Krebs-Henseleit solution gassed with 95% O2-5% CO2. Acetylcholine, choline and norepinephrine were determined by high performance liquid chromatography with electrochemical detection. Cyclic AMP was determined by radioimmunoassay. One min reperfusion following 15 min ischemia (termination of perfusion) caused a significant decrease in both cardiac acetylcholine (P less than 0.05) and norepinephrine (P less than 0.01) contents, but had no significant effect on the cardiac norepinephrine/acetylcholine content ratio, or choline or cyclic AMP content. By contrast, 16 min ischemia did not significantly affect the cardiac acetylcholine, norepinephrine, choline or cyclic AMP content. Also, 16 min hypoxia (perfusion with Krebs Henseleit solution gassed with 95% N2 5% CO2) decreased the cardiac norepinephrine content significantly (P less than 0.01) and norepinephrine/acetylcholine content ratio slightly but not significantly. However, hypoxia had no significant effect on the cardiac acetylcholine, choline or cyclic AMP content. Pre-treatment with 10 microns atropine sulfate prevented the decrease in the cardiac acetylcholine content caused by reperfusion but caused a significant depletion in the cardiac norepinephrine content in the control (P less than 0.01) and ischemia (P less than 0.05) groups and a significant decrease in the norepinephrine/acetylcholine content ratio in all three groups (all, P less than 0.05). Extending the reperfusion period to 5 and 10 min following 15 min ischemia also caused a significant decrease in both cardiac acetylcholine and norepinephrine contents compared with the control groups. However, no significant difference in these contents was found between 1 min reperfusion group and 5 or 10 min reperfusion group. Twenty or 25 min ischemia alone did not significantly affect these contents. These findings suggest that reperfusion disturbs both the sympathetic and parasympathetic nervous systems in the heart and that pre-treatment with atropine adversely affects the balance of the autonomic nervous system.
J Mol Cell Cardiol 1989 Feb
PMID:Effect of reperfusion on the cardiac acetylcholine and norepinephrine contents in rat hearts. 254 84

Responsiveness to catecholamines may be blunted after prolonged exposure to an agonist; this phenomenon, termed desensitization, is often mediated by receptor down-regulation. beta-Adrenergic receptors mediate relaxation of vascular smooth muscle. We have examined the possibility that this response may be desensitized after prolonged exposure to increased concentrations of epinephrine. Rats were treated with epinephrine infusions (300 micrograms/kg/hr from a minipump) for 7 days and had levels of plasma epinephrine 70-fold greater than those of controls. The mesenteric artery rings from the epinephrine-treated rats contracted normally when exposed to serotonin; however, the extent of relaxation promoted by the beta-adrenergic agonist isoproterenol was blunted (86 +/- 4 vs. 43 +/- 9%; p less than 0.05). Acetylcholine and nitroglycerine, which may act through a cyclic GMP mechanism, caused virtually identical relaxation responses in both control and epinephrine-treated groups. To determine the mechanism for the loss in responsiveness to isoproterenol, we measured adrenergic receptors in individual mesenteric arteries using [125I]cyanopindolol. Specific binding of [125I]cyanopindolol was found to have the expected characteristics of interaction with beta receptors. There was no difference in the number of beta-adrenergic receptors between control and epinephrine-treated animals (24 +/- 5 vs. 26 +/- 6 fmol/mg of protein), although there was significantly marked down-regulation of beta-adrenergic receptors in hearts (23 +/- 2 vs. 10 +/- 1 fmol/mg of protein; p less than 0.001) and lungs (172 +/- 29 vs. 76 +/- 7 fmol/mg of protein; p less than 0.01) in the same rats. The ability of isoproterenol to stimulate cyclic AMP production in the mesenteric arteries from the two groups was not significantly different (20.3 +/- 3.5 vs. 23.8 +/- 4.7 pmol of cAMP/mg of protein/2 min). Furthermore, mesenteric artery relaxation was found to be decreased in response to the cyclic AMP analogue dibutyryl cyclic AMP (45 +/- 2.0 vs. 28 +/- 2.0%; p less than 0.001) in the epinephrine-infused rats. These data suggest that the desensitization of beta-adrenergic receptor-mediated smooth muscle relaxation may be caused by a mechanism distal to cyclic AMP production.
Mol Pharmacol 1985 Feb
PMID:Desensitization of beta-adrenergic receptor-mediated vascular smooth muscle relaxation. 257 3

Triphenylmethylphosphonium (TPMP) blocked the nerve-elicited twitch tension of frog sartorius muscles by 50% at a concentration of about 20 microM. This neuromuscular blockade by TPMP, which originated at the level of the nicotinic receptor, was due in part to the ability of the drug to block the quiescent receptor by degrees that depended on the TPMP concentration and on membrane potential. In addition, the blockade was markedly enhanced when the receptor was activated by acetylcholine. Finally, TPMP shortened the lifetimes of ACh-activated ion channels. These findings were interpreted as follows. Under resting conditions, TPMP shifted the equilibrium of the receptor channel complex toward the desensitized state. TPMP united with the activated ion channel to shorten channel lifetime and to deepen the blockade. High concentrations (greater than or equal to 50 microM) of TPMP altered muscle action potentials and often increased, by about 30-fold, the frequency of miniature endplate potentials.
Mol Pharmacol 1985 Feb
PMID:Triphenylmethylphosphonium blocks the nicotinic acetylcholine receptor noncompetitively. 257 4

Experiments are described, showing the presence of putative nicotinic cholinoreceptors in the egg after fertilization. The experiments were carried out on gametes and early embryos of the sea urchin Paracentrotus lividus, by using nicotinic agonists and antagonists. 1 mM Acetylcholine (ACh), 100 microM nicotine, 100 nM alpha-bungarotoxin (alpha-BuTx) and 100 microM curare inhibit sperm motility and fertilization, while they have no effect on unfertilized eggs. The drugs added within 1 min. after the raising of the fertilization layer had stronger effects on cleavage and development; when added more than 15 min. after the raising of the fertilization layer, they had lesser effects on further development up to pluteus stage. In all the experiments, nicotine was the most effective drug. The binding of fluorescein-labelled alpha-BuTx did not point out any affinity sites on unfertilized eggs, while they were localized on the sperms and on the eggs fertilized by sperms, but not on the eggs activated artificially. The binding was prevented by pretreatment of sperms and activated eggs with 10 nM native alpha-BuTx and 10 microM curare. We conclude that, in the fertilized egg, putative nicotinic cholinoreceptors are present, which are able to bind alpha-BuTx and curare. Fertilization by sperms is needed to trigger the formation of alpha-BuTx receptors.
Cell Mol Biol 1989
PMID:Localization of putative nicotinic cholinoreceptors in the early development of Paracentrotus lividus. 273 Nov 92

Several presynaptic cholinergic markers were measured in subcellular fractions of the rat cardiac atrium. A P2 fraction consisting of isolated terminals, free mitochondria, glycogen and other subcellular organelles was prepared first by sucrose density centrifugation. No intact cells were recovered in this fraction, as determined by electron microscopy. Hemicholinium-3 sensitive choline transport coupled to acetylcholine synthesis was concentrated over 10-fold in the P2 fraction compared to minced atria when expressed per mg protein. Six subfractions were recovered after centrifugation of the P2 fraction over a sucrose-metrizamide density gradient. One of these (fraction 4) consistently contained higher levels of choline acetyltransferase activity and hemicholinium-3 sensitive [3H]acetylcholine synthesis than were present in the P2 fraction of the other five subfractions. Electron microscopy of fraction 4 revealed isolated nerve terminals among other subcellular organelles. Kinetic analysis of total [3H]choline uptake in the P2 fraction revealed two apparent uptake processes, with Kts of approximately 11 microM and 219 microM. [3H]Acetylcholine synthesis was partially sodium-dependent in the P2 fraction, and reached a maximal level between choline concentrations of 100 to 200 microM. Some of the newly synthesized [3H]-acetylcholine in the P2 fraction was released by 50 mM K+ depolarization in a calcium-dependent manner, arguing for a neuronal localization. This depolarization-induced release was attenuated by 10 to 100 microM oxotremorine in an atropine-sensitive manner, but was not affected by 1 microM tetrodotoxin.
J Mol Cell Cardiol 1989 Mar
PMID:Cholinergic properties of rat atrial subcellular preparations. 274 56

The influence of cAMP on the cell-to-cell diffusion of Lucifer Yellow CH in heart muscle was further investigated. It was found that isoproterenol (10(-5) M) enhanced the diffusion coefficient (D) from 4 +/- 0.63 X 10(-7) cm2/s (control) to 2.4 +/- 0.66 X 10(-6) cm2/s. Dibutyryl-cAMP (5 X 10(-4) M) plus theophylline (0.4 mM) also increased the cell-to-cell diffusion of Lucifer Yellow CH (D = 3.2 +/- 0.69 X 10(-6) cm2/s). The effect of dBcAMP was increased by theophylline. Since the permeability of the surface cell membrane to the dye is negligible in presence of these drugs and the binding of Lucifer Yellow CH to cytoplasmic proteins was not altered, it is thus concluded that the enhanced longitudinal diffusion of the dye was due to an increase in junctional permeability. As the enhanced cell-to-cell diffusion of the dye caused by isoproterenol was quickly reversed the hypothesis of a greater synthesis of intercellular channels seems unlikely. Acetylcholine (10(-5) M) that increases the intercellular concentration of cGMP had no effect on cell-to-cell diffusion of the dye. The present results support the view that cAMP is a modulator of junctional permeability in heart muscle.
J Mol Cell Cardiol 1987 Aug
PMID:Further studies on the influence of cyclic nucleotides on junctional permeability in heart. 282 96


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