RESUMEN
Arachidonic acid increased intracellular calcium, in cells expressing green fluorescent protein-tagged human FFA4 receptors, with an EC50 of ~40µM. This action was not blocked by cyclooxygenase or lipoxigenase inhibitors but it was inhibited by AH7614, a FFA4 antagonist. Arachidonic acid induced ERK activation accompanied by EGF receptor transactivation. However, EGF transactivation was not the major mechanism through which the fatty acid induced ERK phosphorylation, as evidenced by the inability of AG1478 to block it. Arachidonic acid increased FFA4 receptor phosphorylation that reached its maximum within 15min with an EC50 of ~30µM; inhibitors of protein kinase C partially diminish this effect and AH7614 blocked it. Arachidonic acid induced rapid and sustained Akt/PKB phosphorylation and FFA4 - ß-arrestin interaction. Confocal microscopy evidenced that FFA4 receptor activation and phosphorylation were associated to internalization. In conclusion, arachidonic acid is a bona fide FFA4 receptor agonist.
Asunto(s)
Ácido Araquidónico/farmacología , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Calcio/metabolismo , Línea Celular , Células HEK293 , Humanos , Fosforilación , Quinazolinas/farmacología , Transducción de Señal/efectos de los fármacos , Tirfostinos/farmacología , beta-Arrestinas/metabolismoRESUMEN
1 The role of the protein tyrosine kinase, c-Src, on the function and phosphorylation of alpha1B-adrenoceptors (alpha1B-AR) and their association with G-protein-coupled receptor kinase (GRK) isozymes was studied. 2 Inhibitors of this kinase (PP2 and Src Inhibitor II) decreased ( approximately 50-75%) noradrenaline- (NA) and phorbol myristate acetate-mediated receptor phosphorylation. Expression of a dominant-negative mutant of c-Src similarly reduced receptor phosphorylation induced by the natural agonists, active phorbol esters and endothelin-1 (ET-1). 3 c-Src, GRK2, GRK3 and GRK5 coimmunoprecipitate with alpha1B-ARs in the basal state. In cells treated with NA or phorbol myristate acetate the amount of coimmunoprecipitated GRK2 and GRK3 increased ( approximately 2- to 3-fold), while treatment with ET-1 only augmented the amount of coimmunoprecipitated GRK2 ( approximately 2-fold). The Src inhibitor, PP2, markedly attenuated all these increases. 4 Cell pretreatment with PP2 amplified the increase in intracellular-free calcium observed with NA, in the basal state and after the stimulation (desensitization) induced by ET-1. 5 The data suggest a role of c-Src in alpha1B-AR desensitization/phosphorylation and in the interaction of these ARs with GRKs.
Asunto(s)
Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Quinasas de Receptores Adrenérgicos beta/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1 , Animales , Células COS , Calcio/metabolismo , Chlorocebus aethiops , Cricetinae , Relación Dosis-Respuesta a Droga , Endotelina-1/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Quinasa 3 del Receptor Acoplado a Proteína-G/metabolismo , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Norepinefrina/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Pirimidinas/farmacología , Ratas , Receptores Adrenérgicos alfa 1/genética , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , TransfecciónRESUMEN
In the present paper, the cloning and expression of the guinea pig alpha(1A)-adrenoceptor is presented. The nucleotide sequence had an open reading frame of 1401 bp that encoded a 466 amino-acid protein with an estimated molecular mass of approximately 51.5 kDa. When the clone was expressed in Cos-1 cells, specific high-affinity binding of [(3)H]prazosin and [(3)H]tamsulosin was observed. Chloroethylclonidine treatment of membranes slightly decreased the total binding with both radioligands. Binding competition experiments using [(3)H]tamsulosin showed the following potency order: (a) for agonists: oxymetazoline >>epinephrine>norepinephrine>methoxamine, and (b) for antagonists: prazosin> or 5-methyl-urapidil=benoxathian>phentolamine>>BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione). Photoaffinity labeling using [(125)I-aryl]azido-prazosin revealed a major broad band with a molecular mass between 70 and 80 kDa. The receptor was functional, as evidenced by an epinephrine-increased production of [(3)H]inositol phosphates that was blocked by prazosin.
Asunto(s)
Receptores Adrenérgicos alfa 1/genética , Agonistas Adrenérgicos/farmacología , Antagonistas Adrenérgicos/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Células COS , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Epinefrina/farmacología , Expresión Génica , Cobayas , Metoxamina/farmacología , Datos de Secuencia Molecular , Norepinefrina/farmacología , Oxatiinas/farmacología , Oximetazolina/farmacología , Fentolamina/farmacología , Piperazinas/farmacología , Prazosina/metabolismo , Prazosina/farmacología , Receptores Adrenérgicos alfa 1/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Sulfonamidas/metabolismo , Tamsulosina , TritioRESUMEN
alpha(1b)-Adrenoceptors immunoprecipitated with protein kinase C alpha, delta, and epsilon isoforms under basal conditions and such coimmunoprecipitations were increased in cells treated with phorbol myristate acetate. The increased coimmunoprecipitations induced by phorbol myristate acetate were concentration-dependent and reached their maxima 1 to 2 min after the addition of the tumor promoter. No coimmunoprecipitation of protein kinase C zeta and alpha(1b)-adrenoceptors was detected. Norepinephrine, endothelin-1, lysophosphatidic acid and epidermal growth factor were also able to increase the coimmunoprecipitation of protein kinase C isoenzymes and alpha(1b)-adrenoceptors. These data support the idea that protein kinase-receptor complexes might form and could be relevant in receptor desensitization.
Asunto(s)
Hormonas/farmacología , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Agonistas alfa-Adrenérgicos/farmacología , Animales , Línea Celular , Endotelina-1/farmacología , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos , Immunoblotting , Lisofosfolípidos/farmacología , Norepinefrina/farmacología , Pruebas de Precipitina , Proteína Quinasa C-alfa , RatasRESUMEN
In rat-1 fibroblasts stably expressing rat alpha(1d)-adrenoceptors, noradrenaline and PMA markedly decreased alpha(1d)-adrenoceptor function (noradrenaline-elicited increases in calcium in whole cells and [(35)S]guanosine 5'-[gamma-thio]triphosphate binding in membranes), suggesting homologous and heterologous desensitizations. Photoaffinity labelling, Western blotting and immunoprecipitation identified alpha(1d)-adrenoceptors as a broad band of 70-80 kDa. alpha(1d)-Adrenoceptors were phosphorylated in the basal state and noradrenaline and PMA increased it. The effect of noradrenaline was concentration-dependent (EC(50) 75 nM), rapid (maximum at 1 min) and transient. Phorbol ester-induced phosphorylation was concentration-dependent (EC(50) 25 nM), slightly slower (maximum at 5 min) and stable for at least 60 min. Inhibitors of protein kinase C decreased the effect of phorbol esters but not that of noradrenaline. Evidence of cross-talk of alpha(1d)-adrenoceptors with receptors endogenously expressed in rat-1 fibroblasts was given by the ability of endothelin, lysophosphatidic acid and bradykinin to induce alpha(1d)-adrenoceptor phosphorylation. In summary, it is shown for the first time here that alpha(1d)-adrenoceptors are phosphoproteins and that receptor phosphorylation is increased by the natural ligand, noradrenaline, by direct activation of protein kinase C and via cross-talk with other receptors endogenously expressed in rat-1 fibroblasts. Receptor phosphorylation has functional repercussions.
Asunto(s)
Norepinefrina/farmacología , Receptores Adrenérgicos alfa 1/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Animales , Secuencia de Bases , Calcio/metabolismo , Línea Celular , Cartilla de ADN , Fosforilación , Pruebas de Precipitina , RatasRESUMEN
Desensitization and phosphorylation of the endogenous angiotensin II AT(1) receptor were studied in clone 9 liver cells. Agonist activation of AT(1) receptors blunted the response to subsequent addition of angiotensin II. Partial inhibition of the angiotensin II-induced calcium response was observed when cells were pretreated with dibutyryl cyclic AMP, tetradecanoyl phorbol acetate (TPA), vasopressin, or lysophosphatidic acid. All of these desensitization processes were associated with receptor phosphorylation. Angiotensin II-induced AT(1) receptor phosphorylation was partially blocked by the protein kinase C inhibitor bisindolylmaleimide I and by phosphoinositide 3-kinase inhibitors (wortmannin and LY294002); the actions of these inhibitors were not additive. Pertussis toxin pretreatment of cells also partially inhibited angiotensin II-induced AT(1) receptor phosphorylation. TPA-induced AT(1) receptor phosphorylation was completely blocked by bisindolylmaleimide I. AT(1) receptor phosphorylation was also induced by vasopressin and lysophosphatidic acid, and these effects were partially inhibited by bisindolylmaleimide I. Angiotensin II increased Akt/PKB (protein kinase B) phosphorylation and protein kinase C membrane association. The effect on Akt/PKB phosphorylation was blocked by phosphoinositide 3-kinase inhibitors. These findings indicate that clone 9 cells exhibit both homologous and heterologous desensitization in association with AT(1) receptor phosphorylation. In these hepatic cells, angiotensin II-induced receptor phosphorylation involves pertussis toxin-sensitive and -insensitive G proteins, and is mediated in part through protein kinase C and phosphoinositide 3-kinase.
Asunto(s)
Hepatocitos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Angiotensina/metabolismo , Angiotensina II/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Toxina del Pertussis , Fosforilación , Ratas , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The effect of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) on the phosphorylation and function of alpha(1b)-adrenoceptors transfected into Rat-1 fibroblasts was studied. EGF and PDGF increased the phosphorylation of these adrenoceptors. The effect of EGF was blocked by tyrphostin AG1478 and that of PDGF was blocked by tyrphostin AG1296, inhibitors of the intrinsic tyrosine kinase activities of the receptors for these growth factors. Wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the alpha(1b)-adrenoceptor phosphorylation induced by EGF but not that induced by PDGF. Inhibition of protein kinase C blocked the adrenoceptor phosphorylation induced by EGF and PDGF. The ability of noradrenaline to increase [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTP[S]) binding in membrane preparations was used as an index of the functional coupling of the alpha(1b)-adrenoceptors and G-proteins. Noradrenaline-stimulated [(35)S]GTP[S] binding was markedly decreased in membranes from cells pretreated with EGF or PDGF. Our data indicate that: (i) activation of EGF and PDGF receptors induces phosphorylation of alpha(1b)-adrenoceptors, (ii) phosphatidylinositol 3-kinase is involved in the EGF response, but does not seem to play a major role in the action of PDGF, (iii) protein kinase C mediates this action of both growth factors and (iv) the phosphorylation of alpha(1b)-adrenoceptors induced by EGF and PDGF is associated with adrenoceptor desensitization.
Asunto(s)
Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas alfa-Adrenérgicos/farmacología , Androstadienos/farmacología , Animales , Línea Celular , Membrana Celular/metabolismo , Cricetinae , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Fibroblastos/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Indoles/farmacología , Norepinefrina/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/farmacología , Unión Proteica , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinazolinas , Ratas , Estaurosporina/farmacología , Factores de Tiempo , Transfección , Tirfostinos/farmacología , WortmaninaRESUMEN
The effect of lysophosphatidic acid on the phosphorylation and function of alpha(1b)-adrenoceptors transfected into rat-1 fibroblasts was studied. This phospholipid mitogen increased in a concentration-dependent fashion (EC(50) approximately 50 nM) the phosphorylation of these adrenoceptors. Lysophosphatidic acid-induced alpha(1b)-adrenoceptor phosphorylation was relatively rapid (t(1/2) approximately 1 min), intense (2.5-fold), and sustained for at least 60 min. The effect of lysophosphatidic acid was blocked by pretreatment with pertussis toxin. The alpha(1b)-adrenoceptor phosphorylation induced by lysophosphatidic acid was not blocked by genistein, a tyrosine kinase inhibitor, but it was inhibited by inhibitors of protein kinase C (bisindolylmaleimide I, staurosporine, and Ro 31-8220) and phosphoinositide 3-kinase (wortmannin and LY 294002). The ability of norepinephrine to increase cytosol calcium concentration was markedly decreased in cells previously challenged with lysophosphatidic acid. Norepinephrine-induced [(35)S]GTPgammaS binding in membrane preparations was used as an index of the functional coupling of the alpha(1b)-adrenoceptors and G proteins. Norepinephrine-stimulated [(35)S]GTPgammaS binding was markedly decreased in membranes from cells pretreated with lysophosphatidic acid. This effect of lysophosphatidic acid was blocked by pretreatment with wortmannin or staurosporine. Our data indicate that: 1) activation of lysophosphatidic acid receptors induce phosphorylation of alpha(1b)-adrenoceptors; 2) this effect is mediated through pertussis toxin-sensitive G proteins, phosphatidylinositol 3-kinase, and protein kinase C; and 3) the phosphorylation of alpha(1b)-adrenoceptors induced by the lipid mitogen is associated to adrenoceptor desensitization.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Lisofosfolípidos/farmacología , Fosfatidilinositol 3-Quinasas/fisiología , Receptores Adrenérgicos alfa 1/metabolismo , Animales , Células Cultivadas , Cricetinae , Fosforilación , Proteína Quinasa C/fisiología , Ratas , Receptores Adrenérgicos alfa 1/fisiologíaRESUMEN
This review focuses on alpha(1)-adrenoceptor phosphorylation and function. Most of what is currently known is based on studies on the hamster alpha(1B)-adrenoceptor. It is known that agonist stimulation leads to homologous desensitization of these receptors and current evidence indicates that such decrease in receptor activity is associated with receptor phosphorylation. Such receptor phosphorylation seems to involve G protein-receptor kinases and the receptor phosphorylation sites have been located in the carboxyl tail (Ser(404), Ser(408), and Ser(410)). There is also evidence showing that in addition to desensitization, receptor phosphorylation is associated with internalization and roles of beta-arrestins have been observed. Direct activation of protein kinase C leads to receptor desensitization/internalization associated with phosphorylation; the protein-kinase-C-catalyzed receptor phosphorylation sites have been also located in the carboxyl tail (Ser(394) and Ser(400)). Activation of G(q)-coupled receptors, such as the endothelin ET(A) receptor induces alpha(1B)-adrenoceptor phosphorylation and desensitization. Such effect involves protein kinase C and a yet unidentified tyrosine kinase. Activation of G(i)-coupled receptors, such as the lysophosphatidic acid receptor, also induces alpha(1B)-adrenoceptor phosphorylation and desensitization. These effects involve protein kinase C and phosphatidyl inositol 3-kinase. Interestingly, activation of epidermal growth factor receptors also induces alpha(1B)-adrenoceptor phosphorylation and desensitization involving protein kinase C and phosphatidyl inositol 3-kinase. A pivotal role of these kinases in heterologous desensitization is evidenced.
Asunto(s)
Receptores Adrenérgicos alfa 1/química , Receptores Adrenérgicos alfa 1/fisiología , Animales , Humanos , Fosforilación , Receptores Adrenérgicos alfa 1/metabolismoRESUMEN
In the present work we studied the effect of protein phosphatase inhibitors on the phosphorylation state and function of alpha(1b)-adrenoceptors. Okadaic acid increased receptor phosphorylation in a time- and concentration-dependent fashion (maximum at 30 min, EC(50) of 30 nM). Other inhibitors of protein phosphatases (calyculin A, tautomycin and cypermethrin) mimicked this effect. Staurosporine and Ro 31-8220, inhibitors of protein kinase C, blocked the effect of okadaic acid on receptor phosphorylation. Neither genistein nor wortmannin altered the effect of okadaic acid. The intense adrenoceptor phosphorylation induced by okadaic acid altered the adrenoceptor-G protein coupling, as evidenced by a small decreased noradrenaline-stimulated [(35)S]GTPgammaS binding. Okadaic acid did not alter the noradrenaline-stimulated increases in intracellular calcium or the production of inositol trisphosphate. Our data indicate that inhibition of protein phosphatases increases the phosphorylation state of alpha(1b)-adrenoceptors; this effect seems to involve protein kinase C. In spite of inducing an intense receptor phosphorylation, okadaic acid alters alpha(1b)-adrenergic actions to a much lesser extent than the direct activation of protein kinase C by phorbol myristate acetate.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ácido Ocadaico/farmacología , Fosfoproteínas Fosfatasas/fisiología , Proteína Quinasa C/fisiología , Piranos , Receptores Adrenérgicos alfa 1/metabolismo , Compuestos de Espiro , Animales , Antifúngicos/farmacología , Relación Dosis-Respuesta a Droga , Indoles/farmacología , Cinética , Toxinas Marinas , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Piretrinas/farmacología , Ratas , Receptores Adrenérgicos alfa 1/fisiología , Estaurosporina/farmacología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Maximal adrenergic responses in Rat-1 fibroblasts expressing alpha(1a)-adrenergic receptors are not blocked by activation of protein kinase C. In contrast, activation of protein kinase C induces the phosphorylation of alpha(1b)-adrenoreceptors and blocks their actions. The effect of norepinephrine and phorbol esters on alpha(1a)-adrenoreceptor phosphorylation and coupling to G proteins were studied. Both stimuli lead to dose-dependent receptor phosphorylation. Interestingly, protein kinase C activation affected to a much lesser extent the actions of alpha(1a)-adrenergic receptors than those of the alpha(1b) subtype (norepinephrine elicited increases in calcium in whole cells and [(35)S]GTPgammaS binding to membranes). Basal phosphorylation of alpha(1a)-adrenergic receptors was much less than that observed with the alpha(1b) subtype. The carboxyl terminus seems to be the main domain for receptor phosphorylation. Therefore, chimeric receptors, where the carboxyl-terminal tails of alpha(1a) and alpha(1b) adrenergic receptors were exchanged, were constructed and expressed. alpha(1a)-Adrenoreceptors wearing the carboxyl tail of the alpha(1b) subtype had a high basal phosphorylation and displayed a strong phosphorylation in response to norepinephrine and phorbol esters. Our results demonstrate that stimulation of alpha(1a)-adrenergic receptor, or activation of protein kinase C, leads to alpha(1a)-adrenergic receptor phosphorylation. alpha(1a)-Adrenoreceptors are affected to a much lesser extent than alpha(1b)-adrenoreceptors by protein kinase C activation.
Asunto(s)
Norepinefrina/farmacología , Receptores Adrenérgicos alfa 1/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cinética , Fosforilación , Etiquetas de Fotoafinidad , Pruebas de Precipitina , Proteína Quinasa C/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
BACKGROUND: Desensitization of G protein-coupled receptors is associated with receptor phosphorylation. Two groups of kinases seem to participate in such receptor phosphorylation, i.e., second messenger-activated protein kinases and G protein-coupled receptor kinases. Calcium seems to play a role in the phosphorylation of some G protein-coupled receptors. The role of calcium in alpha 1b-adrenoceptor phosphorylation has not been critically assessed. METHODS: Rat-1 fibroblasts stably expressing the hamster alpha 1b-adrenergic receptor were used. To study receptor phosphorylation cells metabolically labeled with [32P]Pi were lysed and the receptor immunoprecipitated using a polyclonal antibody generated against the receptor carboxyl terminal decapeptide. Intracellular calcium was determined by using Fura-2 fluorescence. RESULTS: Norepinephrine, endothelin-1, and lysophosphatidic acid increased intracellular calcium concentration. All these agents and phorbol myristate acetate (PMA) induce alpha 1b-adrenoceptor phosphorylation. The intracellular chelator, BAPTA, abolished the increase in intracellular calcium induced by the previously mentioned agents but did not affect the receptor phosphorylation induced by norepinephrine, PMA, or lysophosphatidic acid. Under these conditions, receptor phosphorylation induced by endothelin was slightly but consistently decreased. Thapsigargin increased intracellular calcium concentration but was unable to induce alpha 1b-adrenoceptor phosphorylation and decreased PMA-induced receptor phosphorylation. No increase in receptor phosphorylation was observed when calcium ionophores were used. CONCLUSIONS: Our data indicate that an increase in [Ca2+]i is not sufficient to induce alpha 1b-adrenoceptor phosphorylation and that buffering of [Ca2+]i does not alter the receptor phosphorylation induced by norepinephrine, lysophosphatidic acid, and PMA. A marginal role of calcium in the alpha 1b-adrenoceptor phosphorylation induced by endothelin-1 cannot be discarded.
Asunto(s)
Calcio/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Animales , Cricetinae , Fosforilación , RatasRESUMEN
It is now well documented that changes in gene expression take place during cell isolation and culture. Here, we report the change in the expression of the mRNAs for alpha(1)-adrenoceptor subtypes, during dissociation of guinea pig liver cells with collagenase. Using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assays, it was observed that during the isolation procedure, the mRNA for the alpha(1A)-adrenoceptor, normally expressed in whole liver, was degraded and the mRNA for alpha(1D) subtype, barely expressed in whole liver, increased in an actinomycin D-sensitive manner. When the isolation procedure was performed in the presence of cycloheximide, the mRNA for the alpha(1A)-adrenoceptor did not diminish and the induction of the alpha(1D)-adrenoceptor mRNA was even more evident. Our data indicate that cell isolation alters alpha(1)-adrenoceptor mRNA expression.
Asunto(s)
Hígado/metabolismo , ARN Mensajero/metabolismo , Receptores Adrenérgicos alfa 1/genética , Animales , Cicloheximida/farmacología , Dactinomicina/farmacología , Regulación de la Expresión Génica , Cobayas , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Inhibidores de la Síntesis de la Proteína/farmacología , ARN/efectos de los fármacos , ARN/genética , ARN/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Human alpha(1b)-adrenoceptors stably expressed (B(max) approximately 800 fmol/mg membrane protein) in mouse fibroblasts were able to increase intracellular Ca(2+) and inositol phosphate production in response to noradrenaline. Activation of protein kinase C desensitized the alpha(1b)-adrenergic-mediated actions but did not block the ability of the cells to respond to lysophosphatidic acid. Inhibition or downregulation of protein kinase C also blocked the action of the tumor promoter on the adrenergic effects. Photolabeling experiments indicated that the receptor has an apparent molecular weight of approximately 80 kDa. The receptors were phosphorylated in the basal state and such phosphorylation was increased when the cells were incubated with phorbol myristate acetate or noradrenaline. Incubation of the cells with phorbol myristate acetate or noradrenaline blocked noradrenaline-promoted [35S]GTP-gamma-S binding to membranes, suggesting receptor-G protein uncoupling. The results indicate that activation of protein kinase C blocked/desensitized human alpha(1b)-adrenoceptors and that such effect was associated to receptor phosphorylation.
Asunto(s)
Proteína Quinasa C/fisiología , Receptores Adrenérgicos alfa 1/metabolismo , Animales , Unión Competitiva , Calcio/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Indoles/farmacología , Fosfatos de Inositol/metabolismo , Membranas/efectos de los fármacos , Membranas/metabolismo , Norepinefrina/farmacología , Fentolamina/metabolismo , Fosforilación/efectos de los fármacos , Piperazinas/metabolismo , Prazosina/metabolismo , Pruebas de Precipitina , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ensayo de Unión Radioligante , Receptores Adrenérgicos alfa 1/genética , Radioisótopos de Azufre , Acetato de Tetradecanoilforbol/farmacología , TritioRESUMEN
In C9 rat liver cells bradykinin and kallidin increased (approximately 2-fold) the intracellular concentration of calcium, but the B1 agonist, des-Arg9-bradykinin did not. The effect of bradykinin was inhibited by the B2 antagonists, Hoe 140 and N-alpha-adamantaneacetyl-D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin, but not by the B1 antagonist, des-Arg9-[Leu8]-bradykinin. The action of bradykinin was diminished, but not abolished, in medium without calcium. The peptide was able to increase intracellular calcium concentration in cells treated with thapsigargin. Bradykinin action was not observed in cells previously stimulated with this local mediator: however, under the same conditions, angiotensin II induced a clear increase in intracellular calcium concentration. Our data indicate that activation of bradykinin B2 receptors increase intracellular calcium concentrations by inducing both gating of the cation and intracellular mobilization in C9 liver cells. In addition, homologous desensitization was observed.
Asunto(s)
Calcio/metabolismo , Hígado/metabolismo , Receptores de Bradiquinina/metabolismo , Angiotensina II/farmacología , Animales , Bradiquinina/análogos & derivados , Bradiquinina/farmacología , Línea Celular , Activación Enzimática/efectos de los fármacos , Calidina/farmacología , Hígado/efectos de los fármacos , Ratas , Receptor de Bradiquinina B2 , Receptores de Bradiquinina/agonistas , Tapsigargina/farmacologíaRESUMEN
In rat-1 fibroblasts stably expressing alpha1d-adrenoceptors BMY 7378, phentolamine, chloroethylclonidine and 5-methyl urapidil decreased basal [Ca2+]i. WB 4101 induced a very small effect on this parameter but when added before the other antagonists it blocked their effect. All these agents inhibited the action of norepinephrine. Phorbol myristate acetate also blocked the effect of norepinephrine and decreased basal [Ca2+]i. Staurosporine inhibited these effects of the phorbol ester. Our results suggest that: (1) alpha1d-adrenoceptors exhibit spontaneous ligand-independent activity, (2) BMY 7378, phentolamine, chloroethylclonidine and 5-methyl urapidil act as inverse agonists and (3) protein kinase C activation blocks spontaneous and agonist-stimulated alpha1d-adrenoceptor activity.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Calcio/metabolismo , Receptores Adrenérgicos alfa 1/fisiología , Acetato de Tetradecanoilforbol/farmacología , Agonistas de Receptores Adrenérgicos alfa 1 , Antagonistas de Receptores Adrenérgicos alfa 1 , Animales , Línea Celular , Clonidina/análogos & derivados , Clonidina/farmacología , Dioxanos/farmacología , Relación Dosis-Respuesta a Droga , Endotelina-1/farmacología , Fibroblastos , Norepinefrina/farmacología , Fentolamina/farmacología , Piperazinas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Ratas , Estaurosporina/farmacología , TransfecciónRESUMEN
Alpha 1-adrenoceptors mediate some of the main actions of the natural catecholamines, adrenaline, and noradrenaline. They participate in many essential physiological processes, such as sympathetic neurotransmission, modulation of hepatic metabolism, control of vascular tone, cardiac contraction, and the regulation of smooth muscle activity in the genitourinary system. It is now clear that alpha 1-adrenoceptors mediate, in addition to immediate effects, longer term actions of catecholamines such as cell growth and proliferation. In fact, adrenoceptor genes can be considered as protooncogenes. Over the past years, considerable progress has been achieved in the molecular characterization of different alpha 1-adrenoceptor subtypes. Three main subtypes have been characterized pharmacologically and in molecular terms. Splice variants, truncated isoforms, and polymorphisms have also been detected. Similarly, it is now clear that these receptors are coupled to several classes of G proteins that, therefore, are capable of modulating different signaling pathways. In the present article, some of these aspects are reviewed, together with the distribution of the subtypes in different tissues and some of the known roles of these receptors in health and disease.
Asunto(s)
Receptores Adrenérgicos alfa 1/clasificación , Receptores Adrenérgicos alfa 1/fisiología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Cardiomegalia/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Hiperplasia Prostática/metabolismoRESUMEN
Noradrenaline increased the mRNA levels of c-fos and c-jun in rat-1 fibroblast lines stably expressing the cloned alpha1-adrenoceptor subtypes. The efficacy to induce the expression of c-fos mRNA was similar for the three cell lines (alpha1d = alpha1b = alpha1a) but different for c-jun (alpha1a > or = alpha1b > alpha1d). The EC50 values were also different: approximately 5 nM (c-fos) and approximately 300 nM (c-jun) for cells transfected with the alpha1a subtype, approximately 30 nM (c-fos) and approximately 300 nM (c-jun) for cells transfected with the alpha1b subtype and approximately 300 nM (c-fos and c-jun) for those transfected with the alpha1d subtype. Staurosporine and protein kinase C down-regulation blocked such effects, indicating a role of this protein kinase. Endothelin-1 (10 nM) also increased the levels of c-fos and c-jun mRNAs. These actions of endothelin-1 were unaffected by staurosporine and protein kinase C down-regulation. It is concluded that activation of any of the three cloned subtypes can increase the levels of c-fos and c-jun mRNAs and that protein kinase C plays a major role in mediating such effects.
Asunto(s)
Proteína Quinasa C/biosíntesis , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-jun/biosíntesis , ARN Mensajero/biosíntesis , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas alfa-Adrenérgicos/farmacología , Animales , Autorradiografía , Western Blotting , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Endotelina-1/biosíntesis , Inducción Enzimática/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Norepinefrina/farmacología , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/genética , Ratas , Estaurosporina/farmacologíaRESUMEN
Noradrenaline increased phosphorylase a activity through activation of alpha1B-adrenoceptors in rat hepatocytes. Such effect was inhibited by chloroquine (Ki approximately 55 nM) and only slightly reduced by high concentrations of primaquine. Chloroquine did not inhibit the activation of phosphorylase a induced by vasopressin or angiotensin II. Binding competition experiments using [3H]prazosin showed that both chloroquine and primaquine interact with alpha1B-adrenoceptors, but only at very high concentrations. This indicates that the ability of chloroquine to block the alpha1B-adrenergic action was not due to antagonism at the receptor level. Noradrenaline increased phosphatidylinositol resynthesis and inositol trisphosphate production; these effects were inhibited by chloroquine and phorbol 12-myristate 13-acetate. Staurosporine and Ro 31-8220 (3-[1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3 -yl)maleimide), reduced the inhibitions induced by the active phorbol ester and the antimalarial drug on adrenergic-stimulated phosphatidylinositol resynthesis. Similarly, staurosporine blocked the inhibitory actions of chloroquine and phorbol 12-myristate 13-acetate on noradrenaline-stimulated inositol trisphosphate production. These data suggest the possibility that protein kinases, such as protein kinase C, could be involved in the actions of chloroquine.
Asunto(s)
Antagonistas de Receptores Adrenérgicos alfa 1 , Antagonistas Adrenérgicos alfa/farmacología , Cloroquina/farmacología , Hígado/metabolismo , Agonistas alfa-Adrenérgicos/farmacología , Antagonistas Adrenérgicos beta/farmacología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Norepinefrina/farmacología , Fosfatidilinositoles/metabolismo , Fosforilasa a/metabolismo , Propranolol/farmacología , Proteína Quinasa C/metabolismo , Proteínas Quinasas/metabolismo , Ratas , Ratas WistarRESUMEN
The action of bradykinin was studied in rat-1 fibroblasts stably expressing alpha1b-adrenoceptors. It was observed that bradykinin and kallidin markedly increase cytosol calcium concentration, but that the B1 agonist, des-Arg9-bradykinin, only mimicked this effect to a minimal extent. Antagonists, selective for the B2 subtype, such as Hoe 140, blocked this effect of bradykinin and kallidin. Similarly, bradykinin and kallidin stimulated the production of inositol phosphates and B2 antagonists blocked their actions. The possibility that bradykinin could modulate alpha1b-adrenoceptors was studied. It was observed that bradykinin and kallidin increased alpha1b-adrenoceptor phosphorylation and that such effect was also blocked by Hoe 140. Interestingly, the ability of norepinephrine to increase intracellular calcium concentration was not altered by pretreatment of the cells with bradykinin, i.e. bradykinin induced alpha1b-adrenoceptor phosphorylation but this did not lead to receptor desensitization.