RESUMO
Norepinephrine and to a lesser extent epinephrine increased the resting membrane potential of earthworm body wall muscle cells. Ouabain, phentolamine, propranolol, and replacement of Ca(2+) with Mg(2+) in the incubation medium abolished this effect. External 3'5'-cAMP in high concentration, dibutyryl cAMP, and dibutyryl cGMP did not induced hyperpolarization of muscle cell membranes. It was concluded that norepinephrine and epinephrine increased the resting membrane potential in earthworm body wall muscle cells via activation of Na(+), K(+) pump. This process involves Ca(2+) and sarcolemmal adrenergic structures, similar to alpha-adrenoceptors in vertebrate, but not the cyclic nucleotide systems.
Assuntos
Epinefrina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Norepinefrina/farmacologia , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Dibutiril GMP Cíclico/metabolismo , Magnésio/metabolismo , Músculos/metabolismo , Oligoquetos , Receptores Adrenérgicos alfa/metabolismo , Sarcolema/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de Tempo , Vasoconstritores/farmacologiaRESUMO
Nitric oxide (NO) modulates processes of synaptic transmission at pre- and postsynaptic levels. In the present work we studied the mechanisms of action of NO on [gamma-14C]amino-n-butyric acid ([14C]GABA) release in rat cortical synaptosomes. NO donors--S-nitroso-L-cysteine and hydroxylamine (but not sodium nitroprusside)--inhibited the neurotransmitter efflux in a concentration range from 10 microM to 1 mM. Nitrosocysteine completely and selectively suppressed the Ca2+-dependent (vesicular) [14C]GABA release, while not affecting the Ca2+-independent component of the [14C]GABA transport. The influence of NO donors was not related to activation of guanylyl cyclase, since the membrane-permeable cGMP analog dibutyryl-cGMP did not mimic and the guanylyl cyclase inhibitor methylene blue did not change the NO effects. In contrast, the membrane-permeable SH-reagent N-ethylmaleimide (NEM) resembled the effects of NO donors on the Ca2+-dependent [14C]GABA release. The degree of inhibition of the release by nitrosocysteine, hydroxylamine, and NEM correlated with their ability to oxidize intra-synaptosomal SH-groups. These data suggest that synaptosomal sulfhydryl groups are the target for NO action at the presynaptic level. The NO-induced oxidation of thiols may be involved in physiological and, especially, pathological effects of nitric oxide in the central nervous system.
Assuntos
Encéfalo/metabolismo , Cálcio/metabolismo , Isótopos de Carbono/metabolismo , Cisteína/análogos & derivados , Doadores de Óxido Nítrico/farmacologia , S-Nitrosotióis , Sinaptossomos/metabolismo , Ácido gama-Aminobutírico/metabolismo , Domínios de Homologia de src/fisiologia , Animais , GMP Cíclico/metabolismo , Cisteína/farmacologia , Dibutiril GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Guanilato Ciclase/metabolismo , Hidroxilamina/farmacologia , Cinética , Masculino , Azul de Metileno/metabolismo , Nitroprussiato/farmacologia , Compostos Nitrosos/farmacologia , Potássio/metabolismo , Ratos , Ratos Wistar , Reagentes de Sulfidrila/farmacologia , Fatores de TempoRESUMO
Change of osmolality surrounding spawned sperm from isotonic to hypotonic causes the initiation of sperm motility in the common carp. Here we show that membrane-permeable cAMP does not initiate motility of carp sperm that is quiescent in isotonic solution, and that motility of the demembranated sperm can be reactivated without cAMP. Furthermore, the cAMP level does not change during the initiation of sperm motility, and inhibitors of protein kinase do not affect sperm motility, suggesting that no cAMP-dependent system is necessary for the regulation of sperm motility. Sperm motility could not be initiated in Ca(2+)-free hypoosmotic solutions, and significant increase in the intracellular Ca(2+) level was observed by a Ca-sensitive fluorescence dye during hypoosmolality-induced active motion period. The demembranated sperm cells were fully reactivated in the solutions containing 10(-7) to 10(-5) M Ca(2+). Ca(2+) channel blockers such as verapamil and omega-conotoxin reversibly inhibited the initiation of sperm motility, suggesting that Ca(2+) influx is the prerequisite for the initiation of carp sperm motility. Motility of intact sperm was completely blocked; however, that of the demembranated sperm was not inhibited by the calmodulin inhibitor W7, suggesting that the calmodulin bound close to the plasma membrane participated in the initiation of sperm motility. Flow cytometric membrane potential measurements and spectrophotometric measurements by using fluorescence dyes showed transient membrane hyperpolarization on hypoosmolality-induced motility. This article discusses the role of membrane hyperpolarization on removal of inactivation of Ca(2+) channels, leading to Ca(2+) influx at the initiation of carp sperm motility.
Assuntos
Canais de Cálcio/metabolismo , Motilidade dos Espermatozoides/fisiologia , Animais , Bucladesina/metabolismo , Bucladesina/farmacologia , Calcimicina/farmacologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Carpas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico , Dibutiril GMP Cíclico/metabolismo , Dibutiril GMP Cíclico/farmacologia , Ionóforos/farmacologia , Masculino , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases , Motilidade dos Espermatozoides/efeitos dos fármacos , Valinomicina/farmacologiaRESUMO
We studied the effect of lipopolysaccharide (LPS) on the proliferation of culture-stimulated rat stellate cells. DNA synthesis as determined by [3H]-thymidine incorporation was significantly suppressed by up to 52% compared with the control culture in the presence of LPS (> 5 ng/mL). Such an inhibitory effect of LPS was dramatically augmented in the presence of interferon-gamma (IFNgamma). Lipopolysaccharide alone or in combination with IFNgamma activated transcription factors AP-1 and NF-kappaB, and elicited nitric oxide (NO) production by stellate cells by inducing NO synthase. Inhibition of NO production by the addition of L-arginine antagonists to the culture, partially cancelled such an inhibitory effect of LPS and/or IFNgamma on DNA synthesis without affecting the activation of AP-1 and NF-kappaB and the NO synthase level. The cellular level of cyclic guanosine monophosphate (cGMP) increased in response to LPS and IFNgamma, and dibutyryl cGMP or 8-bromo-cGMP inhibited the incorporation of [3H]-thymidine in a dose-dependent manner. These results indicate that LPS is potent in modulating stellate cell proliferation by some NO- and cGMP-dependent mechanism.
Assuntos
Lipopolissacarídeos/farmacologia , Fígado/citologia , Óxido Nítrico/fisiologia , Animais , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , DNA/biossíntese , Dibutiril GMP Cíclico/metabolismo , Interferon gama/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , NF-kappa B/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Ratos , Ratos Wistar , Fator de Transcrição AP-1/metabolismoRESUMO
Sterol regulatory element (SRE) has been recognized to regulate various key genes coding for especially low density lipoprotein (LDL)-receptor, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase known to play a crucial role in the cholesterol feedback mechanism. The deranged cholesterol feedback mechanism has been widely recognised in initiation as well as progression of various types of cancers including chronic myeloid leukaemia (CML). Consequently, the present study was addressed to understand this phenomenon and revealed the existence of a unique 47 kDa protein factor having affinity for this SRE sequence in lymphocytes from normal subjects as well as its absence in lymphocytes from untreated CML patients. However, this factor appeared when the CML patients achieved complete haematological remission (CHR) through alpha-interferon therapy. Further, an inverse relationship was also observed between sterol modulated LDL-receptor gene transcription and the binding affinity of this 47 kDa factor to the SRE sequence. Based upon these results we propose that alpha-interferon through its receptor initiates phosphatidic acid dependent signalling which in turn regulates the affinity of 47 kDa sterol regulatory element binding factor as well as LDL-receptor gene transcription in lymphocytes from CML patients.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Linfócitos/metabolismo , Receptores de LDL/metabolismo , Esteróis/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Bucladesina/metabolismo , Dibutiril GMP Cíclico/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Fosforilação , Receptores de LDL/genética , Zinco/metabolismoRESUMO
A role for cGMP in the control of capacitative Ca2+ influx was identified in rat pituitary GH3 cells. Application of 50 microM - 1 mM of the non-specific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), or the specific cGMP-phosphodiesterase inhibitor, zaprinast, induced a dose-dependent increase in the intracellular free Ca2+ concentration [Ca2+]i of the pituitary cell line, as assessed by video ratio imaging using fura-2. Response onset times were identical and response profiles were similar in all cells analysed. Application of 50 microM dibutyryl cGMP to GH3 cells resulted in heterogeneous Ca2+ responses, consisting of single or multiple transients with varying onset times. In all cases, increases in [Ca2+]i were predominantly due to Ca2+ influx, since no responses were detected in low Ca2+ medium, or following pre-incubation of cells with 1 microM verapamil, or nicardipine. Depleting intracellular Ca2+ stores by prior treatment of cells with 1 microM thapsigargin resulted in a dramatic potentiation in the Ca2+ influx mediated by both phosphodiesterase inhibitors and dibutyryl cGMP, suggesting that cGMP modulates a dihydropyridine-sensitive Ca2+ entry mechanism in GH3 cells which is possibly regulated by the state of filling of Ca2+ stores.
Assuntos
Cálcio/metabolismo , Dibutiril GMP Cíclico/metabolismo , Hipófise/metabolismo , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Células Cultivadas , Di-Hidropiridinas/metabolismo , Di-Hidropiridinas/farmacologia , Nucleotídeos Cíclicos/metabolismo , Nucleotídeos Cíclicos/farmacologia , Inibidores de Fosfodiesterase/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Ratos , Terpenos/metabolismo , Terpenos/farmacologia , TapsigarginaRESUMO
The effects of Trolox C (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a vitamin E analogue, (60-900 microM) and SIN-1 (3-morpholino sydnonimine), a nitric oxide donor, (30-3000 microM) on arachidonic acid metabolism and on cyclic GMP formation in calcium ionophore A23187 (calcimycin)-stimulated human polymorphonuclear leukocytes were investigated. Trolox C elicited a dose dependent decrease in leukotriene B4 levels and increase in prostaglandin E2 levels but did not affect cyclic GMP levels. SIN-1 dose dependently inhibited leukotriene B4 and stimulated prostaglandin E2 and cyclic GMP formation. Dibutyryl cyclic GMP did not affect the formation of leukotriene B4 and prostaglandin E2. Trolox C (180 microM), which itself had no effect on cyclic GMP levels, enhanced the effect of SIN-1 (100 microM) on cyclic GMP levels more than 5-fold. The effects of SIN-1 on arachidonic acid metabolism seem to be independent of cyclic GMP and are probably due to nitric oxide. In this experimental model both Trolox C and SIN-1 have similar actions on the prostaglandin/leukotriene ratio, and Trolox C potentiates the SIN-1-induced increase in cyclic GMP levels.
Assuntos
Antioxidantes/farmacologia , Ácido Araquidônico/metabolismo , Cromanos/farmacologia , GMP Cíclico/metabolismo , Molsidomina/análogos & derivados , Neutrófilos/efeitos dos fármacos , Calcimicina/farmacologia , Centrifugação com Gradiente de Concentração , Dibutiril GMP Cíclico/metabolismo , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Guanilato Ciclase/metabolismo , Humanos , Leucotrieno B4/metabolismo , Molsidomina/farmacologia , Neutrófilos/metabolismo , Radioimunoensaio , Vasodilatadores/farmacologia , Vitamina E/análogos & derivadosRESUMO
The effects of endogenous and exogenous cyclic AMP on the synthesis of pancreatic lipase, colipase, and amylase were studied. Pancreatic lobules were prepared and incubated with forskolin, dibutyryl cyclic AMP (dbcAMP), and dibutyryl cyclic GMP (dbcGMP), respectively, in the presence of 35S-cystine. The individual pancreatic enzymes were isolated by polyacrylamide gel electrophoresis, and the incorporation of radioactive cystine into lipase, colipase, and amylase was determined. Incubation with forskolin (25 microM) rapidly increased lipase synthesis rate within 30 min, followed by an increase in colipase synthesis rate after 60 min of incubation. Amylase synthesis rate did not change during the 1st h of incubation but decreased slightly when incubated for 2 h. Incubation of pancreatic lobules with dbcAMP (1 mM) for 1 h also stimulated the incorporation of cysteine into lipase and colipase by 21% and 25%, respectively, whereas incubation with dbcGMP had no effect on the synthesis rates of lipase and colipase. Neither dbcAMP nor dbcGMP had any effect on synthesis rate of amylase. It is concluded that cyclic AMP might be an important intracellular signal for the synthesis of pancreatic lipase and colipase in the rat.
Assuntos
Colipases/biossíntese , AMP Cíclico/fisiologia , Lipase/biossíntese , Pâncreas/enzimologia , Amilases/biossíntese , Animais , Bucladesina/metabolismo , Colforsina/farmacologia , Dibutiril GMP Cíclico/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Ratos , Ratos Sprague-DawleyRESUMO
Dibutyryl cyclic GMP, but not dibutyryl cyclic AMP, derepresses sporulation and synthesis of mycobacillin and dipicolinic acid under conditions of glucose repression in Bacillus subtilis strain B34. Neither of these compounds appears to affect sporulation and synthesis of mycobacillin and dipicolinic acid in this strain under normal physiological conditions. Mutants insensitive to glucose repression were indifferent to the addition of either of the nucleotides both in the presence and in the absence of glucose. A role for dibutyryl cyclic GMP in annulling the repressing effect of glucose on sporulation and on synthesis of mycobacillin and dipicolinic acid is thus indicated.
Assuntos
Antifúngicos/biossíntese , Bacillus subtilis/fisiologia , GMP Cíclico/metabolismo , Glucose/metabolismo , Micobacilina/biossíntese , Ácidos Picolínicos/biossíntese , Dibutiril GMP Cíclico/metabolismo , Mutação , Esporos BacterianosRESUMO
The abilities of the pancreatic cholecystokinin (CCK) receptor antagonists dibutyryl cyclic GMP, proglumide, benzotript, CBZ-tryptophan, CBZ-cysteine and CCK-27-32-amide to inhibit CCK binding to its receptor in the pancreas and brain of mice and guinea pigs was examined. In both species, the same relative potencies of the antagonists in brain and pancreas was seen except that dibutyryl cyclic GMP was considerably more potent on pancreas than on cerebral cortex CCK receptors. CCK-27-32-amide was the most potent inhibitor for both brain and pancreas but was more potent in the guinea pig than in the mouse. Proglumide, a relatively weak antagonist, was a more potent inhibitor of the guinea pig than of the mouse pancreas receptor. Thus, these data suggest that there are both tissue-specific and species-specific differences in CCK antagonist interactions with the CCK receptor.
Assuntos
Encéfalo/metabolismo , Pâncreas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Benzamidas/metabolismo , Ligação Competitiva , Cisteína/análogos & derivados , Cisteína/metabolismo , Dibutiril GMP Cíclico/metabolismo , Cobaias , Técnicas In Vitro , Masculino , Camundongos , Oligopeptídeos/metabolismo , Proglumida/metabolismo , Receptores da Colecistocinina , Relação Estrutura-Atividade , Triptofano/análogos & derivados , Triptofano/metabolismoRESUMO
The regulation of a putative neuronal nicotinic acetylcholine receptor, the alpha-bungarotoxin (alpha-Btx)-binding protein, was investigated in primary cultures of chick embryo retina. Depolarization of the cells by veratrum alkaloids, or by a high K+ concentration of the culture medium, reduced the accumulation of cell surface alpha-Btx receptors in these cultures. In contrast, the addition of a blocker of voltage-dependent Na+ channels, tetrodotoxin, increased alpha-Btx receptor accumulation in differentiated retina cultures. Derivatives of cyclic adenosine 3':5'-monophosphate also increased the number of alpha-Btx receptor sites, whereas dibutyryl cyclic guanosine 3':5'-monophosphate, but not 8-bromo-cyclic guanosine 3':5'-monophosphate, had the opposite effect. The blocker of voltage-dependent Ca2+ channels, D600, and media containing a reduced Ca2+ concentration also increased alpha-Btx receptor levels. All of these different culture conditions altered the rate of receptor loss after blocking protein synthesis by cycloheximide. These results show that the synthesis of the neuronal alpha-Btx receptor is regulated by membrane depolarization, cyclic nucleotides, and Ca2+ in a fashion analogous to the regulation of muscle acetylcholine receptor.
Assuntos
GMP Cíclico/metabolismo , Junção Neuromuscular/metabolismo , Receptores Colinérgicos/metabolismo , Receptores Nicotínicos , Retina/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Embrião de Galinha , GMP Cíclico/análogos & derivados , Dibutiril GMP Cíclico/metabolismo , Potássio/metabolismo , Tetrodotoxina/metabolismo , Receptor Nicotínico de Acetilcolina alfa7RESUMO
N2,O2'-dibutyryl cyclic guanosine 3':5'-monophosphate has been reported to inhibit the activity of cholecystokinin on several different end organ responses and in several species. Although the mechanism of this inhibition is unclear, competitive antagonism at the level of the receptor has been suggested. We have investigated an alternate possibility, that N2,O2'-dibutyryl cyclic guanosine 3':5'-monophosphate inhibits cholecystokinin action by interacting directly with the peptide while both are in solution. We used antisera with specificities for different regions of cholecystokinin-gastrin peptides and radioiodinated cholecystokinin-33, cholecystokinin-8, and gastrin-17. Our results support the possibility of a soluble interaction between N2,O2'-dibutyryl cyclic guanosine 3':5'-monophosphate and the peptides of cholecystokinin-gastrin family that is specific for the COOH-terminal (receptor binding) region of these peptides, that is dependent on the concentration of N2,O2'-dibutyryl cyclic guanosine 3':5'-monophosphate, and that correlates with concentrations that inhibit biologic activity. The proposed N2,O2'-dibutyryl cyclic guanosine 3':5'-monophosphate-cholecystokinin complex is dispersed by gel filtration chromatography, and is prevented from being formed by certain detergents.
Assuntos
Colecistocinina/antagonistas & inibidores , GMP Cíclico/análogos & derivados , Dibutiril GMP Cíclico/farmacologia , Reações Antígeno-Anticorpo , Ligação Competitiva , Colecistocinina/imunologia , Colecistocinina/metabolismo , Dibutiril GMP Cíclico/imunologia , Dibutiril GMP Cíclico/metabolismo , Peptídeos/imunologia , Radioimunoensaio , SolubilidadeAssuntos
Colecistocinina/farmacologia , GMP Cíclico/análogos & derivados , Dibutiril GMP Cíclico/antagonistas & inibidores , Reações Antígeno-Anticorpo , Ligação Competitiva , Colecistocinina/imunologia , Colecistocinina/metabolismo , Dibutiril GMP Cíclico/imunologia , Dibutiril GMP Cíclico/metabolismoAssuntos
Fatores Inibidores da Migração de Leucócitos/biossíntese , Linfócitos/imunologia , Linfocinas/biossíntese , Fito-Hemaglutininas/farmacologia , Bucladesina/metabolismo , Carbacol/farmacologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Dibutiril GMP Cíclico/metabolismo , Humanos , Imidazóis/farmacologia , Papaverina/farmacologia , Biossíntese de Proteínas , Proteínas/metabolismo , Fatores de TempoRESUMO
We have prepared (125)I-labeled cholecystokinin and have examined the kinetics, stoichiometry, and chemical specificity with which the labeled peptide binds to dispersed acini from guinea pig pancreas. Binding of (125)I-labeled cholecystokinin was reversible, temperature-dependent, saturable, specific, and localized to the plasma membrane. Each acinar cell possessed approximately 9000 binding sites, and binding of the labeled peptide to these sites could be inhibited by cholecystokinin and structurally related peptides (e.g., gastrin and caerulein) as well as by nonpeptide competitive antagonists of the action of cholecystokinin. Binding was not inhibited by other pancreatic secretagogues such as secretin, vasoactive intestinal peptide, glucagon, physalaemin, eledoisin, kassinin, substance P, carbamoylcholine, litorin, or ranatensin or by bovine pancreatic polypeptide, atropine, neurotensin, leucineenkephalin, methionine-enkephalin, or cyclic somatostatin. With agonists as well as antagonists there was a good correlation between occupation of cholecystokinin binding sites and changes in acinar cell function. With each of six different peptide agonists maximal stimulation of enzyme secretion occurred with 40% receptor occupation and occupation of the remaining 60% caused a progressive decrease in stimulated amylase release. Agonists, but not antagonists, accelerated the dissociation of bound (125)I-labeled cholecystokinin, and these findings suggest that, in pancreatic acini, radiolabeled cholecystokinin binds to at least one class of interacting binding sites whose affinities are influenced by the extent to which these sites are occupied by agonists but not the extent to which they are occupied by antagonists.
Assuntos
Colecistocinina/metabolismo , Pâncreas/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Membrana Celular/metabolismo , GMP Cíclico/metabolismo , Dibutiril GMP Cíclico/metabolismo , Cobaias , Cinética , Fragmentos de Peptídeos/metabolismo , Relação Estrutura-Atividade , TemperaturaAssuntos
Cálcio/fisiologia , Nucleotídeos Cíclicos/metabolismo , Potássio/fisiologia , Sistema Nervoso Simpático/fisiologia , Teofilina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Anuros , Bucladesina/metabolismo , Bucladesina/farmacologia , Diazepam/farmacologia , Dibutiril GMP Cíclico/metabolismo , Dibutiril GMP Cíclico/farmacologia , Compostos de Metacolina/farmacologia , Papaverina/farmacologia , Rana catesbeiana , Sistema Nervoso Simpático/metabolismoRESUMO
In female rats aspirin-induced gastrin mucosal damage was increased and glycoprotein synthesis decreased by fasting and by insulin administration. Glucose added to the drinking water during the fasting period reduced mucosal damage and increased glycoprotein synthesis to control levels. Alloxan diabetes did not affect mucosal damage or glycoprotein synthesis. Alloxan diabetes plus insulin restored blood glucose levels to normal, and susceptibility to aspirin damage and glycoprotein synthesis were also normal. Alloxan diabetes plus fasting restored blood glucose levels to normal but increased aspirin-induced mucosal damage and reduced glycoprotein synthesis. In vitro incubation of gastric mucosal homogenates showed that diburyryl cyclic AMP and theophylline inhibited glycoprotein synthesis but dibutyryl cyclic GMP had no significant effects. The importance of an adequate supply of glucose to the gastric mucosa and the effects of cyclic nucleotides on glycoprotein synthesis are discussed.