RESUMO
The effects of hormonal status on protein kinase activity was examined in homogenates of rat liver. Protein kinase activity was evaluated from incorporation of 32P from [gamma-32P]ATP into protamine or histone as receptor substrates. Protamine phosphorylation in the presence or absence of cyclic AMP exceeded histone phosphorylation by at least a factor or two. Hypophysectomy markedly increased protamine phosphorylation in the presence or absence of saturating amounts of cyclic AMP. In contrast, hypophysectomy only slightly increased cyclic AMP independent phosphorylation of histone. These results could not be amounted for by differences in ATPase or protein phosphase activities. Cortisone (2 mg/day x 3) decreased total protein kinase activity in livers of hypophysectomized rats when protamine was substrate, but had no effect on the total activity toward histone. Growth hormone (100 mug/day x 3) significantly increased histone, but not protamine phosphorylation in livers of hypophysectomized rats. Administration of 5 mug of triiodothyonine/day to hypophysectomized rats also markedly increased the phosphorylation of histone, but not protamine when saturating amounts of cyclic AMP were present. These results support the hypothesis that liver may contain more than one type of protein kinase activity and that the different protein kinase activities can be separately affected by hormones. Such control distal to cyclic AMP might allow selective modulation of cyclic AMP-dependent processes in cells which carry out more than one such process.
Assuntos
Fígado/enzimologia , Proteínas Quinases/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Cortisona/farmacologia , AMP Cíclico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Hipofisectomia , Fígado/efeitos dos fármacos , Protamina Quinase/metabolismo , Protaminas , Ratos , Tri-Iodotironina/farmacologiaRESUMO
The effects of epinephrine on cyclic AMP content and protein kinase activity were examined in an in situ rat heart preparation. Bolus injection of epinephrine into the superior vena cava caused an increase in the activity ratio (-cyclic AMP/"cyclic AMP) of 12 000 X g supernatant protein kinase. The increase was significant within 5 s and maximal in 10 s. Epinephrine produced a dose-dependent increase in both protein kinase activity ratio and cyclic AMP content. The increases in both parameters exhibited a high degree of correlation. The increase in protein kinase activity ratio observed with low doses of epinephrine (less than or equal to 1 microgram/kg) resulted from an increase in independent protein kinase activity (-cyclic AMP) without a change in total protein kinase activity (+cyclic AMP). However, the increase in the activity ratio observed with higher doses of epinephrine (greater than 1 microgram/kg) was due mainly to a decrease in total protein kinase activity rather than a further increase in independent protein kinase activity. The loss of supernatant total protein kinase activity could be accounted for by an increase in activity associated with particulate fractions obtained from the homogenates. A similar redistribution of protein kinase could be demonstrated by the addition of cyclic AMP to homogenates prepared from hearts not stimulated with epinephrine. These results demonstrate that epinephrine over a wide dose range produces a parallel increase in the content of cyclic AMP and the activation of soluble protein kinase. The findings also suggest that protein kinase translocation to particulate material may depend on the degree of epinephrine-induced enzyme activation.
Assuntos
Epinefrina/farmacologia , Miocárdio/enzimologia , Proteínas Quinases/metabolismo , Animais , AMP Cíclico/metabolismo , Citosol/enzimologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Feminino , Cinética , Masculino , Membranas/enzimologia , Ratos , SolubilidadeRESUMO
Recent studies have reported cellular effects of 1,25-dihydroxyvitamin D3 within 15 minutes, a time period too rapid to be mediated by nuclear activation. The vitamin increases hepatocyte cytosolic calcium levels in the absence of extracellular calcium within 5 minutes. Since metabolites of phosphatidylinositol have been implicated as second messengers in the regulation of cytosolic calcium, we examined the effect of 1,25-dihydroxyvitamin D3 on hepatocyte phosphatidylinositol turnover and compared these effects to those produced by vasopressin. In isolated hepatocytes labeled with [3H]inositol, 1,25-dihydroxyvitamin D3 (4 nM) increased [3H]glycerophosphorylinositol by 16% (p less than 0.01) within 2.5 minutes, by 18% (p less than 0.01) after 5 minutes, and by 11% (p less than 0.05) after 10 minutes. At a concentration of 20 nM, 1,25-dihydroxyvitamin D3 increased [3H]glycerophosphorylinositol by 27% (p less than 0.01) after 5 minutes. Vitamin D did not affect [3H]inositol polyphosphates. Conversely, vasopressin had no effect on [3H]glycerophosphorylinositol but significantly increased [3H]inositol phosphate, [3H]inositol bisphosphate, and [3H]inositol triphosphate. 1,25-Dihydroxyvitamin D3 (4 nM) decreased [3H]phosphatidylinositol by 10% (p less than 0.05) after 5 minutes and by 16% (p less than 0.01) after 10 minutes. At a concentration of 20 nM, 1,25-dihydroxyvitamin D decreased [3H]phosphatidylinositol by 18% (p less than 0.01) after 5 minutes. The vitamin did not affect [3H]phosphatidylinositol bisphosphate or [3H]phosphatidylinositol trisphosphate. 24,25-Dihydroxyvitamin D had no effect on inositol phospholipids. The effects of 1,25-dihydroxyvitamin D3 on inositol phospholipids were blocked by quinacrine. Bromophenacylbromide inhibited the effects of 1,25-dihydroxyvitamin D3 on inositol phospholipids and also blocked the vitamin-induced increments in cytosolic calcium.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Calcitriol/farmacologia , Fígado/metabolismo , Fosfatidilinositóis/metabolismo , Fosfolipases A/metabolismo , Fosfolipases/metabolismo , Animais , Cálcio/análise , Cromatografia por Troca Iônica , Esterificação , Técnicas In Vitro , Ponto Isoelétrico , Fígado/enzimologia , Masculino , Ratos , Ratos Endogâmicos , Solubilidade , Vasopressinas/farmacologiaRESUMO
1 alpha,25-Dihydroxyvitamin D3 rapidly increases cytosolic calcium and alters membrane phospholipid metabolism in hepatocytes. To define the causal relationship between these events, we examined the effects of 1 alpha,25-dihydroxyvitamin D3 on 32P-labeled lysophosphatidylinositol levels and cytosolic calcium as affected by pertussis toxin and 1 beta,25-dihydroxyvitamin D3, the biologically inactive analog. 32P-labeled lysophosphatidylinositol was determined by two-dimensional thin-layer chromatography. Cytosolic calcium was measured in cells loaded with quin-2AM. Within 5 min, 1 alpha,25-dihydroxyvitamin D3 increased hepatocyte cytosolic calcium by 31% (p less than 0.05) and 32P-labeled lysophosphatidylinositol by 38% (p less than 0.05). Pertussis toxin inhibited the hormone-induced rise in cytosolic calcium but not the increase in 32P-labeled lysophosphatidylinositol. Exposure to exogenous lysophosphatidylinositol for 5 min increased cytosolic calcium by 40% (p less than 0.05), an effect that was also inhibited by pertussis toxin. 1 beta,25-Dihydroxyvitamin D3 had no effect on either hepatocyte cytosolic calcium or 32P-labeled lysophosphatidylinositol but prevented the 1 alpha,25-dihydroxyvitamin D3-induced increments. The results suggest that a G protein sensitive to pertussis toxin is required for the transduction of the lysophosphatidylinositol signal but not the generation of the signal. The ability of 1 beta,25-dihydroxyvitamin D3 to inhibit the 1 alpha,25-dihydroxyvitamin D3-induced changes in phospholipids suggests that the epimer may compete with 1 alpha,25-dihydroxyvitamin D3 for an initiating receptor.
Assuntos
Calcitriol/farmacologia , Cálcio/metabolismo , Fígado/efeitos dos fármacos , Lisofosfolipídeos/metabolismo , Animais , Células Cultivadas , Citosol/metabolismo , Fígado/metabolismo , Lipídeos de Membrana/metabolismo , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The mechanism that underlies activation of lipolysis by GH is not yet understood. Although cAMP is thought to be involved, the biochemical linkages between GH and cAMP are unknown, and lipolysis produced by GH differs from that produced by such typical activators of adenylate cyclase as the catecholamines with regard to time course, maximum response, and dependence on other factors such as glucocorticoids or theophylline. The present studies were undertaken to evaluate the possibility that activation of protein kinase C by GH may play an important role in the production of the delayed increase in lipolysis. Phorbol myristate acetate (PMA), a known activator of protein kinase C, increased lipolysis in segments of adipose tissue of hypophysectomized rats, and, as with GH, this effect was potentiated by theophylline. The lipolytic effects of PMA were concentration-dependent, required a shorter lag period than those of human GH, increased as the concentration of PMA was raised from 0.1 to 10 microM, and were additive at all concentrations with lipolysis produced by saturating concentrations of GH. The lipolytic actions of GH, but not PMA, were potentiated by dexamethasone in adipose tissue of normal rats. Sphingosine and staurosporine, which are known to inhibit protein kinase C, blocked the lipolytic effects of PMA and severely reduced lipolysis in response to GH in tissues of both normal and hypophysectomized rats. Although higher concentrations of sphingosine interfered with the specific binding of 125I-labeled human GH to isolated adipocytes, the inhibitory effects of sphingosine cannot be attributed to interference with GH binding, since it decreased lipolysis by at least 50% when used at concentrations that were too low to reduce binding significantly. Staurosporine produced little (approximately 20%) or no decrease in binding. At concentrations that severely reduced lipolysis in response to GH and dexamethasone, sphingosine had little or no effect on lipolysis in response to dibutyryl cAMP or forskolin. Staurosporine and sphingosine, however, severely inhibited lipolysis in response to isoproterenol. We conclude that protein kinase C activity plays an important role in hormone-stimulated lipolysis probably by an action exerted on the transduction pathway proximal to cAMP. The present data are equally consistent with the possibilities that GH and/or isoproterenol activate protein kinase C, or that protein kinase C is constitutively active to some extent. It is likely that protein kinase C activity is permissive for, rather than a mediator of, the lipolytic actions of GH and isoproterenol.
Assuntos
Hormônio do Crescimento/farmacologia , Isoproterenol/farmacologia , Lipólise/efeitos dos fármacos , Proteína Quinase C/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Alcaloides/farmacologia , Animais , Bucladesina/farmacologia , Dexametasona/farmacologia , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Hipofisectomia , Masculino , Ratos , Esfingosina/farmacologia , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologia , Teofilina/farmacologiaRESUMO
GH exerts a number of metabolic effects on adipose tissue. Depending on the circumstances, it may increase or decrease glucose metabolism and lipolysis. These effects appear to be mediated by a single class of receptors, which bind GH with high affinity. Incubation of isolated rat adipocytes with a variety of lipolytic agents, including catecholamines, forskolin, or (Bu)2cAMP, decreased the specific binding of [125I]human (h) GH within 10 min. In the presence of 10 microM forskolin, GH binding declined to less than 20% of the control value within 50 min. Cholera and pertussis toxins, which increase cAMP secondary to ADP ribosylation of guanine nucleotide-binding proteins associated with hormone receptors, also decreased the binding of GH. None of these agents affected the rate of loss of cell-associated 125I when added to cells that had previously equilibrated with [125I]hGH. The inhibitory effects of forskolin and (Bu)2cAMP were at least as great when binding was measured in the presence of the protease inhibitor leupeptin, suggesting that increased rates of internalization and processing of bound hormone could not account for the decline in binding. Scatchard plots of data obtained in the presence of forskolin or (Bu)2cAMP were linear and parallel to control plots, indicating that the decline in binding could be accounted for by a decrease in the number of binding sites, with no change in affinity. To determine whether phosphorylation affected binding to receptors already present in the membrane or modified the turnover of receptors, we studied adipocyte ghosts, whose cellular apparatus for receptor turnover is disrupted. Incubation of adipocyte ghosts with cAMP-dependent protein kinase decreased the binding of [125I]hGH by 25%. The data suggest that cAMP-dependent phosphorylation of the GH receptor or a closely associated membrane protein renders the receptor incapable of binding GH.
Assuntos
Tecido Adiposo/metabolismo , AMP Cíclico/fisiologia , Receptores da Somatotropina/metabolismo , Animais , Bucladesina/farmacologia , Membrana Celular/metabolismo , Toxina da Cólera/farmacologia , Colforsina/farmacologia , Epinefrina/farmacologia , Hormônio do Crescimento/metabolismo , Técnicas In Vitro , Cinética , Lipólise/efeitos dos fármacos , Masculino , Toxina Pertussis , Ratos , Receptores da Somatotropina/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The effects of hypophysectomy and GH on blood pressure and renin secretion were studied. Within 2 weeks after removal of the pituitary gland of rats, mean arterial blood pressure declined 30%, and heart rate fell 50%. No significant effect of hypophysectomy on blood volume or hematocrit was noted. Within 2 h after the iv administration of 10 micrograms ovine GH to hypophysectomized rats, blood pressure, but not heart rate, was restored to normal. Despite the hypotension, the PRA of hypophysectomized rats was not significantly greater than that of intact animals. This relatively low PRA could not be accounted for by a lack of renin per se, since kidneys of hypophysectomized rats contained at least as much renin as kidneys from intact rats. Neither the PRA of hypophysectomized rats nor the kidney renin content was significantly altered 24 h after a single injection of GH (100 micrograms, ip). When perfusions were performed at 100 mm Hg, the rate of renin secretion by isolated kidneys obtained from hypophysectomized rats was markedly lower than that of kidneys from intact rats. Reduction of mean perfusion pressure to 50 mm Hg or adding isoproterenol to the perfusate produced much smaller increases in renin secretion by kidneys of hypophysectomized rats than that observed in kidneys from intact rats. Kidneys obtained from hypophysectomized rats treated with GH 24 h earlier had a renin content similar to that of kidneys from untreated animals, but secreted renin at a much higher rate. Kidneys from hormone-treated hypophysectomized rats also exhibited a greater renin secretory response to low perfusion pressure or isoproterenol. These data indicate that hypophysectomy severely impairs the renin secretory response of the isolated kidney and that a single injection of GH can reverse this impairment. These observations suggest the possibility that the hypotension characteristic of hypophysectomized rats may, in part, reflect the lack of GH and alterations in the renin-angiotensin system.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Renina/metabolismo , Animais , Dexametasona/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Hipofisectomia , Rim/efeitos dos fármacos , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Renina/sangueRESUMO
The effects of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25-(OH)2D3) on Ca2+ levels and phospholipid metabolism were studied in isolated nuclei prepared from rat liver. Nuclear Ca2+ concentration was estimated with the fluorescent indicator Fura 2. In agreement with previous reports, ATP (1 mM) produced a rapid increase in nuclear Ca2+ from 188 +/- 25 to 593 +/- 121 nM. Exposure to 1 alpha,25-(OH)2D3 (20 nM) also produced a rapid increase in nuclear Ca2+ to 402 +/- 71 nM. The 1 beta epimer of 1 alpha,25-(OH)2D3 had no effect. Nuclear phosphatidylinositol was labeled by incubation with [gamma-32P]ATP for 3 h. 1 alpha,25-(OH)2D3 produced a two-fold increase in [32P]lysophosphatidylinositol (LPI) within 5 min from 44 +/- 11 to 87 +/- 19 cpm/2.5 x 10(7) nuclei. 1 beta,25-(OH)2D3 had no effect on [32P]LPI production. Exposure of nuclei to exogenous LPI (15 microM) produced an instantaneous increase in nuclear Ca2+ to 372 +/- 81 nM, comparable to ATP and 1 alpha,25-(OH)2D3. The rapid effects of 1 alpha,25-(OH)2D3 on phospholipid metabolism and Ca2+ in isolated nuclei suggest that the steroid may exert effects distinct from the well-characterized receptor-mediated changes in gene expression.
Assuntos
Calcitriol/farmacologia , Cálcio/metabolismo , Núcleo Celular/metabolismo , Fosfolipídeos/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Técnicas In Vitro , Fígado , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/farmacologia , Masculino , Fosfatidilinositóis/metabolismo , Ratos , Ratos Endogâmicos , Estereoisomerismo , Fatores de TempoRESUMO
Na+/H+ exchange differs kinetically in mesenteric arteries from spontaneously hypertensive rats (SHR). Both the maximal transport rate and the sensitivity to [H+]i are greater in SHR than in Wistar Kyoto (WKY) rats. Kinetic changes are not seen in prehypertensive SHR or in SHR rendered normotensive by captopril therapy. Thus, blood pressure affects the properties of Na+/H+ exchange in SHR. To determine whether elevated pressure would have similar effects on the WKY transporter, WKY rats were rendered hypertensive by treatment with deoxycorticosterone acetate (DOCA). Mesenteric arteries from these animals were loaded with the pH-sensitive dye BCECF and Na+/H+ exchange activity was assessed by measuring the Nao-dependent, dimethyl amiloride-sensitive recovery of intracellular pH after acid loading. Treatment with DOCA/salt (two injections of 30 mg of DOCA/kg per week for 4 weeks + 1% NaCl in the drinking water) produced a significant increase in mean arterial pressure (181 +/- 29 v 108.1 +/- 6.2 in DOCA/salt v untreated controls), but had no effect on Na+/H+ exchange. Maximal transport rates for untreated, DOCA/salt-treated, and salt-treated rats were virtually identical (0.088 +/- 0.001, 0.103 +/- 0.011, and 0.093 +/- 0.006 mmol/L H+/sec, respectively. Moreover, the sensitivity of Na+/H+ exchange to internal H+ (ie, the Hill coefficient) was unchanged (n(app) = 4.3, 3.7, and 4.0, respectively). Because elevation in blood pressure produced no significant alterations in the WKY Na+/H+ exchanger, it seems likely that there are strain differences in the exchanger and/or in processes regulating its activity.
Assuntos
Pressão Sanguínea/fisiologia , Músculo Liso Vascular/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Desoxicorticosterona , Fluoresceínas , Corantes Fluorescentes , Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , Masculino , Artérias Mesentéricas , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Cloreto de SódioRESUMO
The kinetic properties of Na+/H+ exchange are significantly different in segments of mesenteric arteries from spontaneously hypertensive rats (SHR) as compared to normotensive Wistar-Kyoto (WKY) rats. Both the maximal rate of Na+/H+ exchange and the sensitivity of Na+/H+ exchange to intracellular [H+] are greater in vessels from hypertensive animals. Prehypertensive SHR do not exhibit these features, thus the present studies examined the role of blood pressure in mediating the response. Both SHR and WKY rats were treated with captopril for 4 weeks, after which the characteristics of the Na+/H+ exchanger were examined. The Na+/H+ exchange was monitored in tissue segments by measuring Nao-dependent, dimethyl amiloride-sensitive recovery of intracellular pH after acid loading (pHi determined using the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein). In SHR, captopril reduced mean arterial pressure from 153 to 118 mm Hg and altered Na+/H+ exchange. Maximal transport rate decreased from 0.164 to 0.056 mmol/L H+/sec and the sensitivity of the Na+/H+ exchange to internal H+ decreased (ie, the Hill coefficient decreased from 6.6 to 2.2). In WKY rats captopril reduced blood pressure from 108 to 71 mm Hg, but had no effect on the Na+/H+ exchange. Since reduced pressure affected Na+/H+ exchange in SHR, but not in WKY, it is likely that the Na+/H+ exchanger, or the cellular processes regulating its properties, differ in the two rat strains. Supporting this notion was the finding that ouabain treatment reduced the Hill coefficient of SHR vessels but had no effect in WKY vessels.
Assuntos
Proteínas de Transporte/metabolismo , Hipertensão/metabolismo , Artérias Mesentéricas/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Captopril/farmacologia , Concentração de Íons de Hidrogênio , Masculino , Ouabaína/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Trocadores de Sódio-HidrogênioRESUMO
Using 125I-labeled neurotensin (NT), porcine brain membranes were found to contain two types of high-affinity receptors, one class (approximately 1/3 of total) with an apparent Kd of 0.12 nM and another with an apparent Kd of 1.4 nM. Nonhydrolyzable analogs of GTP inhibited NT binding in a dose-dependent manner. In the presence of 60 microM guanosine 5'-(3-thio) 5'-(beta, gamma-imino) triphosphate. NT binding was decreased by 35% with an associated decrease in the number of binding sites and little change in the Kd. Cross-linking of 125I-labeled NT to brain membranes using disuccinimidyl suberate was found to specifically label two substances of approximately 120 kDa and approximately 160 kDa, which could represent different binding proteins or complexes. For a series of NT analogs, there was close agreement between the IC50 in the binding assay and the ED50 in a bioassay based on ability to contract the guinea pig ileum. In addition, metal ions inhibited NT binding and the contractile action of NT with the same order of potency (Hg++ > Zn++ > Cu++ > Mn++ > Mg++ > Li++). There was a linear relationship between the standard reduction potential for these ions and the logarithm of the IC50 in the binding assay. The results suggest that porcine brain contains high-affinity, G-protein-linked receptors for NT, the functioning of which depends upon group(s), perhaps sulfhydryl(s), which can interact strongly with certain heavy metal ions.
Assuntos
Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanilil Imidodifosfato/farmacologia , Metais/farmacologia , Neurotensina/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Técnicas In Vitro , Membranas/metabolismo , Dados de Sequência Molecular , Peso Molecular , Neurotensina/análogos & derivados , Neurotensina/química , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Receptores de Neurotensina , Receptores de Neurotransmissores/química , Receptores de Neurotransmissores/metabolismo , SuínosRESUMO
In the present study changes in the extent of 32P labelling of membrane phospholipids were correlated with the alpha 1-adrenoceptor-induced events of increased 45Ca influx, 45Ca release and contraction in the rabbit aorta. Under basal conditions 32P incorporation into all phospholipids proceeded without saturation through 80 min of labelling. During a 5 min exposure to 10(-5) M norepinephrine (NE) after 25 min of prelabelling the incorporation of 32P into certain phospholipids was substantially increased. Phosphatidic acid (PA) labelling was increased above basal levels by 4.1 fold, phosphatidylinositol (PI) 2.5 fold and phosphatidylcholine (PC) 1.8 fold. Half maximal stimulation of 32P labelling of PA occurred at 2.0 microM, which was similar to the EC50 value for stimulation of 45Ca influx (2.5 microM) and 45Ca release (2.1 microM) but slightly higher than the value for contractile response (0.9 microM). Antagonist sensitivity studies reinforced the alpha 1 receptor subtype character of the rabbit aorta. Prazosin (10(-7) M) reduced agonist-induced events by 63-82% while yohimbine (10(-7) M) was without influence. Phenoxybenzamine (10(-8) M) reduced agonist-induced events by 56-76%. A temporal comparison showed that agonist stimulation of PA labelling was slower than 45Ca release, but similar to the time course of 45Ca influx. Hydrolysis of 32P-labelled phosphatidylinositol diphosphate (PIP2) was more rapid and paralleled 45Ca release. These findings suggest that PIP2 hydrolysis may account for the rapid phase of norepinephrine-induced contraction in rabbit aorta while PA or its immediate precursor diacylglycerol may account for receptor-induced Ca2+ influx.
Assuntos
Cálcio/metabolismo , Canais Iônicos/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Fosfatidilinositóis/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Feminino , Hidrólise , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Norepinefrina/farmacologia , Fenoxibenzamina/farmacologia , Prazosina/farmacologia , Coelhos , Ioimbina/farmacologiaRESUMO
The interaction of cholinergic agonists and antagonists with smooth muscle muscarinic receptors has been investigated by measurement of displacement of the muscarinic antagonist [3H]QNB (quinuclidinyl benzilate) in membranes prepared from toad stomach. The binding of [3H]QNB was saturable, reversible and of high affinity (KD = 423 pM). The muscarinic receptor subtypes present in gastric smooth muscle were classified by determining the relative affinities for the selective antagonists pirenzepine (M1), AF-DX 116 (M2) and 4-DAMP (M3). The results from these studies indicate the presence of a heterogeneous population of muscarinic receptor subtypes, with a majority (88%) exhibiting characteristics of M3 receptors and a much smaller population (12%) exhibiting characteristics of M2 receptors. The binding curve for the displacement of [3H]QNB binding by the agonist oxotremorine was complex and was consistent with presence of two affinity states: 24% of the receptors had a high affinity (KD = 4.7 nM) for oxotremorine and 76% displayed nearly a 1,000-fold lower affinity (KD = 4.4 microM). When oxotremorine displacement of [3H]QNB binding was determined in the presence GTP gamma S, high affinity binding was abolished, indicating that high affinity agonist binding may represent receptors coupled to G proteins. Moreover, pertussis toxin pretreatment of membranes also abolished high affinity agonist binding, indicating that the muscarinic receptors are coupled to pertussis toxin-sensitive G proteins. Reaction of smooth muscle membranes with pertussis toxin in the presence [32P]NAD caused the [32P]-labelling of a 40 dD protein that may represent the alpha subunit(s) of G proteins that are known to by NAD-ribosylated by the toxin.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Músculo Liso/efeitos dos fármacos , Parassimpatolíticos/farmacologia , Parassimpatomiméticos/farmacologia , Receptores Muscarínicos/efeitos dos fármacos , Adamantano/análogos & derivados , Adamantano/farmacologia , Adenosina Difosfato Ribose/metabolismo , Animais , Bufo marinus , Antagonistas do Ácido Fólico/farmacologia , Guanosina Trifosfato/farmacologia , Técnicas In Vitro , Oxotremorina/farmacologia , Toxina Pertussis , Pirenzepina/análogos & derivados , Pirenzepina/farmacologia , Quinuclidinil Benzilato/farmacologia , Estômago/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Adipocytes are physiological targets for GH in both growing and nongrowing individuals. In adipocytes that have been deprived of GH for at least 3 h, GH initially produces a response that is characterized by increased metabolism of glucose and inhibition of the lipolytic effects of catecholamines. This insulin-like effect disappears within 2-3 h despite continued stimulation and cannot be elicited again unless cells are deprived of GH for at least 3 h. Despite refractoriness to the insulin-like action of GH, the lipolytic effect of GH is evident at this time. Although termination of the insulin-like response and induction of both refractoriness and lipolysis all depend upon synthesis of RNA and proteins, these 3 effects of GH appear to be neither temporally nor causally related. Scatchard analysis of ligand binding data suggests that these various effects are produced by interaction of GH with a single class of receptors. However, since modification of either the hormone or the carbohydrate moiety of the receptor can selectively attenuate either the insulin-like or the lipolytic response, more than one hormone receptor interaction is likely. Northern analysis indicates the presence of at least 2 alternately spliced mRNA transcripts for the GH receptor, and at least 3 different complexes are seen after GH is covalently crosslinked to intact adipocytes. Refractoriness does not result from changes in either the number or affinity of GH receptors, but may result from increased cytosolic calcium. Although the protein kinase C activator phorbol myristate acetate mimics both the insulin-like and lipolytic actions of GH, increased activity of protein kinase C probably does not mediate either action of GH. The intracellular mediators of the diverse actions of GH are unknown at this time.
Assuntos
Tecido Adiposo/citologia , Hormônio do Crescimento/farmacologia , Tecido Adiposo/efeitos dos fármacos , Animais , HumanosRESUMO
An increased turnover of phosphatidate and phosphatidyl inositol has been found in many tissues where hormones or neurotransmitters are postulated to raise Ca2+ influx, for example in smooth muscle. However, the relationship between changes in phospholipid metabolism and changes in Ca2+ permeability was unknown. Following recent reports on the interactions of Ca2+ with phosphatidic acid in membranes and artificial systems, we investigated the hypothesis that phosphatidate accumulation mediates the action of cholinergic and other stimuli on Ca2+ influx. We report here that synthesis and accumulation of phosphatidate was accelerated in smooth muscle cells stimulated by carbamylcholine with a similar time course to that of contraction. This alteration in phosphatidate metabolism does not seem to result from an increase in intracellular Ca2+ or depolarisation of the cell membrane. Furthermore, submicromolar concentrations of phosphatidate rapidly produce contractions of isolated smooth muscle cells. These results support the contention that cholinergic-induced changes in membrane Ca2+ permeability in smooth muscle could be mediated by phosphatidate accumulation.
Assuntos
Carbacol/farmacologia , Músculo Liso/metabolismo , Ácidos Fosfatídicos/metabolismo , Receptores Colinérgicos/efeitos dos fármacos , Receptores Muscarínicos/efeitos dos fármacos , Animais , Bufo marinus , Cálcio/metabolismo , Canais Iônicos/metabolismo , Contração MuscularRESUMO
A method for combined light and fluorescent microscopic imaging of nucleolar organizer regions and cellular rRNA is described. Nucleolar organizer regions were detected by silver staining (Ag-NOR), and rRNA was detected by fluorescent in situ hybridization (FISH). MG-63 human fibrosarcoma cells were silver stained prior to in situ hybridization. To quantitate Ag-NOR within individual cells, brightfield images were digitized, and the total Ag-NOR area/nucleus was determined. Fluorescent images were digitized, and the total cellular fluorescence was calculated after correction for nonuniformity of illumination. By using this method, it was shown that the Ag-NOR procedure did not significantly affect the fluorescence intensity related to FISH. Furthermore, the hybridization procedure did not interfere with quantitation of Ag-NOR. With this method, both Ag-NOR and rRNA product can be quantitated within the same cell. Because the relationship of rRNA content to cell proliferation is well established, correlation to quantitative Ag-NOR parameters within individual cells will contribute to the better definition of the relationship of quantitative Ag-NOR indices with cellular proliferation.
Assuntos
Hibridização in Situ Fluorescente/métodos , Região Organizadora do Nucléolo/ultraestrutura , RNA Ribossômico/ultraestrutura , Coloração pela Prata/métodos , Fibrossarcoma , Humanos , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Células Tumorais CultivadasRESUMO
The possibility that activation of cyclic 3':5'-nucleotide phosphodiesterase is a component of muscarinic inhibition of cyclic AMP accumulation was investigated in WI-38 fibroblasts. At 0.2 to 20 microM, 1-isoamyl-3-isobutylxanthine, an inhibitor of fibroblast phosphodiesterase activity, attenuated the fall in WI-38 cyclic AMP content seen in response to 1 microM carbachol. The inhibitory effect of carbachol on WI-38 cyclic AMP metabolism was also suppressed by the inclusion of 0.1 to 10 microM trifluoperazine in cell incubation media. Exposure of WI-38 cultures to 1 microM carbachol was associated with elevated phosphodiesterase activity in the corresponding broken cell preparations. Both 1-isoamyl-3-isobutylxanthine and trifluoperazine interfered with the ability of 10 microM phosphatidate to mimic carbachol-inhibition of WI-38 cyclic AMP accumulation. Fibroblast calmodulin-dependent phosphodiesterase preparations were activated by micromolar dispersions of phosphatidate. This action of the phospholipid did not appear to require calcium and was blocked by trifluoperazine. These data lend support to the notion that increased cyclic nucleotide phosphodiesterase activity is at least partially responsible for the fall in WI-38 cyclic AMP levels seen in response to cholinergic stimulation. The results also suggest that the effects of cholinergic agents on WI-38 cyclic AMP hydrolysis may be related to changes in phospholipid metabolism, notably the accumulation of phosphatidate.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Carbacol/farmacologia , AMP Cíclico/metabolismo , Ácidos Fosfatídicos/farmacologia , 1-Metil-3-Isobutilxantina/análogos & derivados , 1-Metil-3-Isobutilxantina/farmacologia , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1 , Feminino , Fibroblastos/metabolismo , Humanos , Trifluoperazina/farmacologiaRESUMO
To test the hypothesis that phosphatidic acid (PhA) is involved in the carbachol inhibition of hormone stimulated accumulation of cAMP we observed the effects of PhA on PGE1-stimulation of cAMP in WI-38 fibroblasts. PhA inhibited PGE1-stimulated cAMP accumulation of WI-38 fibroblasts; maximum inhibition (approximately 50-80%) occurred at a PhA concentration of 1.0 microM and significant inhibition was observed with a concentration of 0.1 microM. The full effects of PhA were evident within 15 sec after the co-addition of PGE1 and PhA. Addition of PhA to cells which had been pre-stimulated with PGE1 resulted in the rapid decay of cAMP levels to a new steady state level with a t 1/2 of approximately 65 sec. The inhibition produced by PhA did not appear to be simply attributable to a depolarization or increased intracellular Ca2+, since addition of either KCl or the Ca2+ ionophore A23187 did not lower PGE1-stimulated cAMP accumulation. When intact cells were pretreated with PhA then lysed and adenylate cyclase immediately assayed, no detectable changes in broken cell adenylate cyclase activities were observed. Also, PhA added directly to adenylate cyclase assays at concentrations as high as 100 microM produced no detectable inhibition of the membrane fraction adenylate cyclase activities. Nonetheless, our results suggest that adenylate cyclase activity in intact cells may be directly affected by physiological levels of PhA . Further, the similarities of carbachol [Butcher, R. W., Journal of Cyclic Nucleotide Research, 4:411 (1978)] and PhA inhibition support the hypothesis that carbachol (acetylcholine) exerts its effect on adenylate cyclase through alterations of the plasma membrane phospholipid composition.
Assuntos
Carbacol/farmacologia , AMP Cíclico/metabolismo , Ácidos Fosfatídicos/farmacologia , Prostaglandinas E/farmacologia , Adenilil Ciclases/metabolismo , Atropina/farmacologia , Calcimicina/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Fosfolipídeos/farmacologia , Fito-Hemaglutininas/farmacologia , Cloreto de Potássio/farmacologia , Estimulação Química , Fatores de TempoRESUMO
The mechanism of beta-adrenergic relaxation was investigated in isolated smooth muscle cells. Beta-adrenergic agents stimulate cyclic AMP-dependent phosphorylation, enhance Na+/K+ transport and induce relaxation. The stimulation of Na+/K+ transport is obligatory for relaxation, and we suggest that this stimulation induces relaxation through enhanced Na+/Ca2+ exchange.