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1.
Factors affecting the binding of [3H]adenosine 3'':5''-cyclic monophosphate to protein kinase from bovine adrenal cortex.
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Inorganic salts, several proteins and traces of protein precipitants were tested to find out by what mechanisms they modulate the binding of cyclic [3H]AMP to protein kinase (ATP-protein phosphotransferase; EC 2.7.1.37). The separation of free and bound cyclic AMP by (NH4)2SO4 precipitation was unaffected by the above agents and was more reliable than the Millipore filtration technique. Several binding sites for cyclic AMP were revealed in adrenal-cortex extract. When this extract was used as binding reagent in an assay for cyclic AMP, the standard curve was distorted in the presence of KCl because the salt affected the different binding sites to a varying extent. At high ionic strenth the protein kinase isoenzyme I dissociated and showed an extraordinarily high affinity for cyclic AMP. Trichloroacetate and perchlorate at very low concentrations were able to dissociate the protein kinase and modulate its binding characteristics as well. A progressive decrease in the cyclic AMP-binding capacity occurred on prolonged incubations. The binding protein was protected against inactivation by 2-mercaptoethanol, EDTA and several proteins. It was more resistant to denaturation when complexed to cyclic AMP. The enhancement of cyclic AMP binding by bovine serum albumin was investigated in some detail and appeared to be a pure stabilizing effect. It is proposed that the competitive-binding assays for cyclic AMP based on protein kinase be conducted at high ionic strength and in the presence of stabilizers (protein, EDTA, 2-mercaptoethanol). The interference from agents that may dissociate the protein kinase or influence its stability will thus be decreased. 相似文献
2.
Leonard R. Forte Wan-Tsih H. Cho Ronald J. Walkenbach Keith H. Byington 《生物化学与生物物理学报:生物膜》1975,389(1):84-96
Porcine kidney cortex was utilized for the preparation of plasma-membrane-enriched and soluble cytoplasmic (cytosol) fractions for the purpose of examining the relative properties of cyclic [3H]AMP receptor and cyclic AMP-dependent protein kinase activities of these preparations. The affinity, specificity and reversibility of cyclic [3H]AMP interaction with renal membrane and cytosol binding sites were indicative of physiological receptors.Binding sites of cytosol and deoxycholate-solubilized membranes were half-saturated at approx. 50nM and 100 nM cyclic [3H]AMP. Native plasma membranes exhibited multiple binding sites which were not saturated up to 1 mM cyclic [3H]AMP. Modification of the cyclic phosphate configuration or 2′-hydroxyl of the ribose moiety of cyclic AMP produced a marked reduction in the effectiveness of the cyclic AMP analogue as a competitor with cyclic [3H]AMP for renal receptors. The cyclic [3H]AMP interaction with membrane and cytosol fractions was reversible and the rate and extent of dissociation of bound cyclic [3H]AMP was temperature dependent. With the plasma-membrane preparation, dissociation of cyclic [3H]AMP was enhanced by ATP or AMP.Assay of both kidney subcellular fractions for protein kinase activity revealed that cyclic AMP enhanced the phosphorylation of protamine, lysine-rich and arginine-rich histones but not casein. The potency and efficacy of activation of renal membrane and cytosol protein kinase by cyclic AMP analogues such as N6-butyryl-adenosine cyclic 3′,5′-monophosphate or N6,O2-dibutyryl-adenosine cyclic 3′,5′-monophosphate supported the observations on the effectiveness of cyclic AMP analogues as competitors with cyclic [3H]AMP in competitive binding assays.This study suggested that the membrane cyclic [3H]AMP receptors may be closely associated with the membrane-bound catalytic moiety of the cyclic AMP-dependent protein kinase system of porcine kidney. 相似文献
3.
The binding of [3H]cyclic AMP to cell surface receptors of differentiated D. discoideum cells at 25° is an oscillatory process with a periodicity of 2 min. This alternating change in the cells' binding capacity for cyclic AMP may be the basis for the refractory period to cyclic AMP stimulation, an essential feature of the chemotactic system. The incorporation of [32P] by whole cells from [γ32P]ATP is also oscillatory with a periodicity identical to that of [3H]cyclic AMP binding. However, the two processes are inversely related in time such that periods of maximal cyclic AMP binding correspond to periods of minimal cellular phosphorylation. These results suggest a receptor kinase/phosphatase mediated desensitization of the cyclic AMP receptor. 相似文献
4.
A new method for assay of cyclic AMP phosphodiesterase (EC 3.1.4.17) has been developed based on the observation that a mixture of cyclic AMP and AMP can be resolved on a column of florisil (activated magnesium silicate) at pH 7.0. The cyclic nucleotide is retained by the silicate and the AMP which is not adsorbed is virtually quantitatively recovered. The adsorption of cyclic AMP by florisil is greatly influenced by the pH of the buffer but independent of its ionic strength. In the actual assay cyclic[3H]AMP is incubated with the enzyme source in the presence of Mg2+ and the reaction is stopped by the addition of CCl3COOH (0.3 m). The mixture is then neutralized by dilution with 10 vol of 0.5 m sodium phosphate buffer, pH 7.0, and applied on a small (0.4 × 4.0-cm) florisil column equilibrated with the same buffer. The column is eluted with 3 vol of the buffer and the radioactivity of the eluate which contains only [3H]AMP is measured. The use of cyclic[3H]AMP of high specific activity in the assay allows a high degree of sensitivity while the addition of CCl3COOH instantaneously terminates the reaction allowing for increased precision. The assay compares favorably in simplicity and speed with those currently employed for cyclic AMP phosphodiesterase. 相似文献
5.
Adenosine 3':5'-monophosphate (cyclic AMP), a mediator of hormone action in a variety of tissues, has been measured in its free and bound forms in intact cardiac tissue. We have used a rapid high dilution technique which involves tissue homogenization, subcellular fractionation, and separation of bound from free cyclic AMP by Millopore filtration. The precision of this method is dependent upon minimization of binding and dissociation of cyclic AMP that occur during the preparation and handling of tissue homogenates. In each experiment, a tracer of cyclic [3H]AMP prebound to isolated cardiac binding protein was freed of unbound cyclic [3H]AMP by Sephadex gel filtration and added to the tissue just prior to homogenization in cold EDTA buffer. This tracer was therefore treated identically to the sample through all subsequent dilution, fractionation, and filtration procedures, and provided an acurate internal monitor for total cyclic AMP dissociation during the course of the free-bound determination. Each tissue sample was then individually corrected for dissociation. Rapid dilution to produce a 1:1000 homogenate was found to lower endogenous cyclic AMP levels sufficiently to make binding (or rebinding) during the procedure negligible (less than 5%). Spontaneously beating rat right atria (controls) contained 5.96 +/- 0.28 pmol of cyclic AMP/mg of protein (n = 19) of which 41 and 14% were bound to soluble and particulate proteins, respectively. The remaining cyclic AMP was free. Pretreatment of the tissue with 1 muM isoproterenol (30 s at 30 degrees) increased both the bound and free forms of cyclic AMP (n = 8). While free cyclic AMP increased 420% with the catecholamine, the bound forms increased 240% (soluble) and 60% (particulate). Similar results were obtained when atria (n = 6) were treated with the phosphodiesterase inhibitor, methylisobutylxanthine (0.5 mM, 10 min at 30 degrees). When both agents were used together, cyclic AMP bound to soluble proteins was elevated 4-fold over control while free cyclic AMP increased 27-fold (n = 7), indicating saturation of the soluble sites. It could be calculated that less than one-third of these sites are occupied in the unstimulated cell. These sites may represent the R subunit of cyclic AMP-dependent protein kinase. The data suggest that half-maximal binding in vivo occurs at an intracellular free cyclic AMP concentration of about 1 muM. 相似文献
6.
M Ueda 《Comptes rendus des séances de la Société de biologie et de ses filiales》1981,175(3):372-376
The effects of calcium and magnesium ions on the corticosterone binding to rat brain cytosol receptor protein(s) were investigated. The increasing amounts of CaCl2 or MgCl2 up to 5.0 mM were added, the specific [3H] corticosterone binding increased 1.3-fold and 1.5 respectively. The addition of MnCl2 and KCl did not affect this binding. The binding of corticosterone with rat brain cytosol receptor(s) were decreased by increasing amounts of EDTA and complete inhibition was observed at concentration equal to and greater than 2.5 mM. Inhibition of this binding by EDTA was less than by EGTA. Either theophylline or dibutyryl cyclic AMP had no effect on this binding. 相似文献
7.
Changing pattern of cyclic AMP-binding proteins during germination of Mucor racemosus sporangiospores. 总被引:4,自引:0,他引:4
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M Orlowski 《The Biochemical journal》1979,182(2):547-554
Interation of cyclic AMP with a profoundly changing pattern of specific binding proteins was shown during aerobic germination of sporangiospores from the fungus Mucor racemosus. 32P-labeled 8-azido-cycli AMP, an analogue of cyclic AMP that forms a covalent linkage with the binding proteins under u.v. light, was used as the ligand. Binding proteins carrying this photoaffinity label were separated by polyacrylamide-gel electrophoresis and identified by radioautography. Equibiltrium dissociation constants (Kd) and binding-response curves in the presence of competing nucleotides were identical for both 8-azido-cyclic [32P]AMP and cyclic [3H]AMP. A quantitative binding assay with both 8-azido-cyclic [32P]AMP and cyclic [3H]AMP over the time course of sporangiospore germination indicated a parallel relationship between cyclic AMP-binding capacity and the intracellular concentrations of cyclic AMP reported in a previous study [Paznokas & Sypherd (1975) J. Bacteriol. 124, 134--139]. Both of these parameters attained transient high values at a time of development when addition of exogenous cyclic AMP prevents hyphal-germ-tube emergence. The measured Kd values did not change during sport germination. 相似文献
8.
Action of the cardiac alpha 1-adrenergic receptor. Activation of cyclic AMP degradation 总被引:3,自引:0,他引:3
Using purified rat ventricular myocytes and membranes prepared from them, we have previously found that alpha 1-adrenergic stimulation causes decreased cyclic AMP accumulation and decreased activation of cyclic AMP-dependent protein kinase. We have now analyzed the mechanism by which alpha 1 stimulation is linked to cyclic AMP metabolism. In an adenylate cyclase assay in which carbachol inhibits the stimulatory effect of norepinephrine, the addition of prazosin (alpha 1-antagonist) has no effect on the response to norepinephrine. In membranes prepared from myocytes treated with pertussis toxin, norepinephrine competes for alpha 1-receptors (assessed by [3H]prazosin binding) with two components, binding to the high affinity component being sensitive to exogenous GTP, exactly as in membranes prepared from control myocytes. In intact cells labeled with [3H]adenine in which carbachol antagonizes the norepinephrine response, prazosin enhances accumulation of [3H]cyclic AMP due to norepinephrine. Treatment of cells with pertussis toxin eliminates inhibition by carbachol but does not alter prazosin's capacity to enhance the norepinephrine response. Addition of phosphodiesterase inhibitors eliminates this effect of alpha 1 blockade. In [3H]adenine-labeled cells loaded with [3H]cyclic AMP by prior treatment with isoproterenol, alpha 1-adrenergic stimulation enhances disappearance of [3H]cyclic AMP. Measurements of cellular cyclic AMP give results similar to those obtained with the adenine labeling technic. We conclude that occupation of the myocyte alpha 1-receptor results in stimulation of cyclic AMP phosphodiesterase activity. 相似文献
9.
Rats were given intravenous injections of cyclic [3H]AMP and the disappearance of radioactivity from plasma and its appearance in bile were followed. Livers were removed and the cyclic [3H]AMP content was measured. The binding of radioactivity to soluble proteins was measured after preparations of a cytoplasmic fraction. Experiments in vitro to determine the ability of hepatic cytoplasmic proteins to bind cyclic [3H]AMP were also carried out. A role for cytoplasmic proteins in the clearance of cyclic AMP from plasma is discounted. 相似文献
10.
Renal cortical plasma membranes were solubilized with sodium deoxycholate. The membrane-bound cyclic AMP receptors retained biologic activity in the detergent-dispersed state exhibiting the properties of high affinity for cyclic AMP, saturability and specificity. Half-maximal binding of cycle [3H]-AMP to these receptors was found to occur at 0.06 muM and 1.5 pmol of cyclic [3H]AMP was bound per mg membrane protein at saturation (0.5 muM cyclic [3H]AMP). Sodium deoxycholate-solubilized membrane proteins were chromatographed on Biogel A-5m. Cyclic [3H]AMP receptors eluted in the internal volume at positions equivalent to molecular sizes of 50 000 and 20 000 daltons and in the void volume at molecular size greater than 450 000. After photoaffinity labeling the renal membrane receptors with cyclic [3H]AMP, we found peaks of tritium radioactivity which eluted at similar molecular size positions on this Bogel A-5m column. Further treatment of photoaffinity labeled membranes with sodium dodecyl sulfate, mercaptoethanol and urea, followed by polyacrylamide gel electrophoresis, showed bands of tritium-labeled receptor protein with relative mobilities corresponding to molecular sizes of 26 000 and 21 000 daltons. This study shows that porcine renal cortical membranes contain at least two molecular species of cyclic AMP receptors which may be associated with regulation of the membrane-bound cyclic AMP-dependent protein kinase. 相似文献
11.
Low- and high-affinity binding sites for cyclic GMP were found to be associated with the cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from human tonsillar lymphocytes, but neither of them was identical with the cyclic AMP binding site. The enzyme activated by cyclic GMP phosphorylated the same site of calf thymus H2b histone as the cyclic AMP activated enzyme; however, more complex kinetics of activation were found with cyclic GMP. Two classes of cyclic GMP binding site were demonstrated by kinetic analysis of cyclic [3H]GMP binding in the enzyme preparations eluted by 0.1 M potassium phosphate (pH 7.0) from DEAE cellulose. The high-affinity cyclic GMP binding site (Kd about 4 . 10(-8) M) belonged to some complex form of the protein kinase, as evidenced by the mutual inhibition of cyclic AMP binding and high affinity cyclic GMP binding. However, the high-affinity cyclic GMP binding site disappeared on Sephadex G-100 gel chromatography of the enzyme preparation, whereas the cyclic AMP binding activity was recovered quantitively as separate fractions. The low-affinity cyclic GMP binding site (Kd 2--5 . 10(-6) M) was demonstrated by the inhibitory effect of 10(-5) M cyclic GMP on cyclic AMP binding in each cyclic AMP binding fraction obtained by gel chromatography. However, cyclic AMP did not inhibit the binding of cyclic GMP to the low-affinity binding site. 相似文献
12.
M. K. McMillian X. P. He J. S. Hong K. R. Pennypacker 《Journal of neurochemistry》1992,58(4):1308-1312
The effect of dopamine (DA) on the binding of [3H]phorbol 12,13-dibutyrate ([3H]PdBu) in cultured rat striatal cells was examined. DA maximally increased specific [3H]PdBu binding by 70 +/- 10%, an increase comparable to that observed with norepinephrine (NE). This finding suggests that DA activates protein kinase C in cultured striatal cells, because increases in [3H]PdBu binding reflect translocation of protein kinase C. Half-maximal stimulation was observed with 10(-6) M DA. The peak response was observed at 2-3 min after addition of 10(-4) M DA, but [3H]PdBu binding was still increased above basal at 30 min. DA was not acting via an adrenergic receptor. Prazosin (10(-6) M) blocked the response to NE, suggesting mediation by an alpha 1-adrenergic receptor, but had little effect on the response to DA. Conversely, the D1 receptor antagonist SCH-23390 (10(-6) M) blocked the response to DA, but only partially inhibited the response to NE. Morphine (10(-6) M) inhibited the response to DA by 46 +/- 14%, but did not affect significantly the response to NE. The DA effect on [3H]PdBu binding is apparently independent of the increase in cyclic AMP seen on D1 receptor activation. Forskolin, apomorphine, and the D1 agonist SKF-38393 all increased cyclic AMP in striatal cells, but were less effective than DA in stimulating [3H]PdBu binding. The D2 agonist quinpirole was ineffective in stimulating either cyclic AMP or [3H]PdBu binding. 相似文献
13.
Preparation and properties of a cell-free, hormonally responsive adenylate cyclase from guinea pig brain 总被引:7,自引:5,他引:2
—The accumulation of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) was studied in cell-free homogenates of guinea pig brain. Homogenates, prepared in Krebs-Ringer buffer, responded markedly to the addition of neurohormones with an increased rate of cyclic AMP synthesis; preparations from cerebellum, cerebral cortex, and hippocampus responded to a degree approximating that achieved with slices of these areas of guinea pig brain. Adenylatc cyclase activity was seen only when cyclic AMP was measured by a [3H]adenine prelabelling technique or when total cyclic AMP was measured by radioimmunoassay; [32P]ATP did not serve as a substrate for this preparation of the enzyme. The adenylate cyclase was paniculate and required a Krebs Ringer buffer; use of tris, or tris with Mg2+ and Ca2+, resulted in a preparation totally devoid of hormonal stimulation. Digestion by purified beef heart cyclic nucleotide phosphodiesterase, Dowex chromatography, solubility in Ba(OH)2-ZnSO4 mixtures, and two thin layer chromatographic systems demonstrated that the product of the hormonally stimulated adenylate cyclase preparation was cyclic AMP. The selectivity of hormonal stimulation and the adrenergic character of the hormonal receptors from different brain areas were maintained in the cell-free preparation. However, simultaneous stimulation with two different neurohormones resulted in additive responses, rather than in the potentiation observed in preparations of slices of brain. 相似文献
14.
- 1.
- 1. Accumulation of intracellular radioactivity was measured during incubation of isolated bovine thyroid cells with cyclic [32P]AMP, cyclic [8-3H]AMP and dibutyryl cyclic [8-3H]AMP. With cyclic [32P]AMP, 32P cell/medium ratios ranged from 0 to to 0.04 compared to a maximum 3H cell/medium ratio of 0.29 with cyclic [3H]AMP and 0.16 with dibutyryl cyclic [3H]AMP. The excess of intracellular cyclic [3H] over cyclic [32P]AMP radioactivity was due to extracellular formation of more penetrable dephosphorylated cyclic AMP metabolites which probably served as precursor of intra-cellular cyclic AMP. 相似文献
15.
Norepinephrine and serotonin augment by about 2-fold the accumulation of cyclic [3H]AMP elicited by 2-chloroadenosine in [3H]adenine-labeled guinea-pig cerebral cortical slices. Histamine causes a 3-fold augmentation. The first two agents have no effect on cyclic AMP alone, while histamine has only a small effect alone. The augmentation of the 2-chloroadenosine response appears to be mediated by alpha 1-adrenergic, 5HT2-serotonergic and H2-histaminergic receptors. VIP-elicited accumulations of cyclic AMP are also augmented through stimulation of alpha 1-adrenergic, 5HT2-serotonergic and H1-histaminergic receptors. Activation of these amine receptors also increases the turnover of phosphatidylinositols in [3H]inositol-labeled guinea pig cerebral cortical slices. Norepinephrine causes a 5-fold, serotonin a 1.2-fold, and histamine a 2.5-fold increase in accumulations of [3H]inositol phosphates. 2-Chloroadenosine, vasoactive intestinal peptide, baclofen, and somatostatin have no effect on phosphatidylinositol turnover, nor do the last two agents augment accumulations of cyclic AMP elicited by 2-chloroadenosine. The data suggest a possible relationship between turnover of phosphatidylinositol and the augmentations of the cyclic AMP accumulations elicited by biogenic amines in brain slices. 相似文献
16.
J R Kelsoe J C Gillin D S Janowsky J H Brown S C Risch B Lumkin 《Life sciences》1986,38(15):1399-1408
Cultured adult skin fibroblasts were studied for binding and functional evidence of muscarinic receptors in order to assess their utility as a model of cholinergic function in affective illness. Saturable, specific, high affinity binding could be demonstrated in intact cells from some cell lines with [3H]-NMS, but not [3H]-QNB, presumably because of intracellular trapping of unbound [3H]-QNB. [3H]-NMS specific binding indicated a single site with a KD of approximately 210 pM. [3H]-NMS was displaced by cholinergic agonists and antagonists with relative affinities similar to muscarinic receptors in brain. Many cell lines, however, showed no specific binding. No functional response to carbachol could be demonstrated with respect to inhibition of isoproterenol-stimulated cyclic AMP formation, stimulation of cyclic GMP formation or stimulation of phosphoinositide hydrolysis in any cell line regardless of either high or no specific [3H]-NMS binding. 相似文献
17.
The effects of bovine thyrotropin (TSH) on DNA synthesis and cyclic AMP production were studied in porcine thyroid follicles using suspension culture. During the early 72 hours incubation, the time-dependent uptake of [3H]thymidine by the follicles was observed. In the presence of 10 mU/ml TSH, the uptake of [3H] thymidine was significantly depressed at 72 hours incubation. TSH inhibition of [3H] thymidine incorporation was related to its concentration and the 50% inhibition was observed by using 1.0 mU/ml TSH. Under the same conditions, cyclic AMP production was stimulated by TSH and the stimulation was observed to be related to TSH concentration. In these experiments, the incubation time was 30 min. Dibutyryl cyclic AMP, an analogue of cyclic AMP, inhibited the [3H] thymidine uptake at 72 hours incubation. From these results, it is suggested that TSH inhibits DNA synthesis, and that the inhibition may be mediated by cyclic AMP that is produced by TSH stimulation. 相似文献
18.
Differential Regulation by Butyrate and Dibutyryl Cyclic AMP of δ-Opioid, α2 -Adrenergic, and Muscarinic Cholinergic Receptors in NCB-20 Cells 总被引:2,自引:1,他引:1
Long-term treatment of NCB-20 cells with sodium butyrate resulted in a marked increase in the specific binding of [3H]D-Ala2,D-Leu5 enkephalin. This increase was concentration and time dependent, with an EC50 of about 480 microM and a maximal effect detected after 3-day treatment. At saturating concentration of butyrate (1 mM) the increase was three- to fourfold of the untreated control. Scatchard analysis revealed that the butyrate effect was due to an increase in the density of the opioid receptor binding sites. Butyrate also induced a smaller (about twofold) increase in the density of muscarinic cholinergic receptor binding assessed by using [3H]quinuclidinyl benzilate, whereas alpha 2-adrenergic receptor binding assessed by using [3H]clonidine was not significantly affected. The butyrate-induced opioid receptor binding could be totally abolished by the presence of cycloheximide, suggesting that the butyrate effect involves synthesis of the receptor protein. Butyrate treatment did not affect basal and prostaglandin E1-stimulated cyclic AMP levels but caused a three- to fourfold decrease in the IC50 of D-Ala2,D-Leu5 enkephalin for attenuating these cyclic AMP levels and approximately 25% increase in the maximal extent of attenuation. In contrast to the butyrate effect, long-term treatment of NCB-20 cells with 1 mM dibutyryl cyclic AMP induced an 80% decrease in the opioid and alpha 2-adrenergic receptor bindings and a 57% loss of muscarinic cholinergic receptor binding. This down-regulation of muscarinic cholinergic receptor binding sites was associated with a 35% decrease of carbachol-induced phosphoinositide breakdown, whereas the receptor up-regulation induced by butyrate was found to increase the carbachol response by about threefold. The differential regulation by butyrate and dibutyryl cyclic AMP suggests that the butyrate effect is mediated by a mechanism independent of intracellular cyclic AMP. The induction by butyrate of opioid-receptors and muscarinic cholinergic receptors in NCB-20 cells may provide a useful system for studying the regulation of gene expression of these receptor proteins. 相似文献
19.
The effects of various sugars on the simultaneous release of insulin and accumulation of cyclic AMP were studied in collagenase isolated rat pancreatic islets. D-Glucose stimulated the formation of cyclic AMP at 3 and 60 min of incubation, whether measured by a label incorporation technique, or by the protein kinase binding assay of Gilman. Only D-glucose and D-mannose were able to stimulate insulin release and cyclic [3H]AMP accumulation in the absence of other substrate. D-fructose had a stimulatory effect in the presence of 3.3 mM D-glucose only at a high concentration (33.8 mM), and enhanced the effects of 8.3 mM glucose when added at the concentration of 8.3 mM. D-Galactose was effective only together with 8.3 mM D-glucose. The order of potency of these hexoses, both regarding insulin secretion and cyclic [3H]AMP accumulation, was glucose-mannose-fructose-galactose. L-Glucose and 3-O-methylglucose had no effects at 60 min when incubated together with 8.3 mM D-glucose, whereas at 3 min, 3-O-methylglucose induced a small stimulation of the cyclic [3H]AMP response. D-mannoheptulose and D-glucosamine inhibited the insulin and cyclic [3H]AMP responses to 27.7 mM glucose. Mannoheptulose suppressed completely the glucose effect on cyclic nucleotide accumulation within 90 s. Although under all incubation conditions, the threshold stimulatory or inhibitory concentration of a given agent was identical for insulin release and cyclic [3H]AMP accumulation, these two variables showed quantitative differences in incubations of 60 min, the magnitude of the changes in insulin secretion being larger than that for the cyclic nucleotide. It is suggested that modulation of islet cyclic AMP level is an important step in the transmission of the effect of various sugars on insulin release; however, glucose and possibly other sugars may also enhance insulin release by additional mechanisms not involving the adenylate cyclase-cyclic AMP system of the beta-cell. 相似文献
20.
Histamine Increases Phospholipid Methylation and H2 -Receptor-Adenylate Cyclase Coupling in Rat Brain 总被引:2,自引:2,他引:0
Histamine stimulated the enzymatic synthesis of phosphatidylcholine from phosphatidylethanolamine in crude synaptic membranes of rat brain containing the methyl donor S-adenosyl-L-methionine (SAM). In the presence of, but not in the absence of SAM, histamine increased cyclic AMP accumulation at the concentrations that stimulate phospholipid methylation. S-Adenosyl-L-homocysteine, an inhibitor of phospholipid methyltransferases, inhibited histamine-stimulated phospholipid methylation and histamine-induced cyclic AMP accumulation in the presence of SAM in a concentration-dependent manner. Histamine-induced [3H]methyl incorporation into phospholipids exhibited a marked regional heterogeneity in rat brain in the order of cortex greater than medulla oblongata greater than hippocampus greater than striatum greater than midbrain greater than hypothalamus. The regional distribution of histamine-induced cyclic AMP accumulation exactly paralleled histamine-stimulated [3H]methyl incorporation in rat brain. Histamine-induced cyclic AMP accumulation was inhibited by the addition of cimetidine or famotidine, but not by mepyramine or diphenhydramine. The accumulation of cyclic AMP in the presence of SAM was observed by the addition of impromidine or dimaprit, but not by 2-pyridylethylamine. These results indicate that phospholipid methylation is induced by histamine and may participate in H2-receptor-mediated stimulation of adenylate cyclase in rat brain. 相似文献