RESUMEN

The cloning, sequencing and functional expression of Sgbeta1, a novel locust (Schistocerca gregaria) non-alpha nicotinic acetylcholine receptor (nAChR) subunit is described. This subunit shows 80% identity with the Drosophila melanogaster Dbeta1 and 92% identity with the Locusta migratoria beta1, non-alpha subunits but only 38% identity to Sgalpha1 (also referred to as alphaL1), a previously cloned S. gregaria nAChR alpha-subunit. When expressed in Xenopus laevis oocytes, Sgbeta1 does not respond to nicotine. Responses to nicotine are observed, however, in oocytes co-expressing Sgalpha1 and Sgbeta1, but the pharmacology is indistinguishable from that of currents produced by expressing Sgalpha1 alone. We conclude that either Sgbeta1 does not co-assemble with Sgalpha1, or that it is unable to contribute to the functional properties of the receptor, in the Xenopus oocyte expression system.

Asunto(s)

Drosophila melanogaster/fisiología , Saltamontes/fisiología , Proteínas de Insectos/genética , Receptores Nicotínicos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico

RESUMEN

Somatostatin (somatotropin release inhibitory factor; SRIF) initiates its biological activity by interacting with a family of highly homologous integral membrane receptors (sst(1) -sst(5)). SRIF neuronal actions regulate protein phosphorylation levels, control second messenger production and modulate neuronal membrane potential. Recently, our understanding of SRIF neurobiology has been driven by new pharmacological and molecular biological tools. SRIF receptor subtype specific antibodies have identified a distinctive, yet overlapping, expression pattern for this receptor family, with multiple subtypes co-localizing in the central and peripheral nervous system. This complex expression profile has confounded efforts to establish each receptor's role in the nervous system in part by the possible homo- and heteroligomerization of the receptor proteins. However, the recent discovery of SRIF receptor subtype selective ligands, supplemented by in vitro and in vivo models with inactivated SRIF receptor genes, now provides opportunities to clearly delineate each receptor's neuronal role. The convergence of these pharmacologic, immunologic and molecular biologic approaches extend our understanding of SRIF neurobiology while promising new therapeutic avenues for SRIF research.

Asunto(s)

Enfermedades del Sistema Nervioso Central/fisiopatología , Neuronas/metabolismo , Transducción de Señal/fisiología , Somatostatina/fisiología , Enfermedad de Alzheimer/fisiopatología , Animales , Sistema Nervioso Central/citología , Sistema Nervioso Central/metabolismo , Epilepsia del Lóbulo Temporal/fisiopatología , Humanos , Enfermedad de Huntington/fisiopatología , Enfermedad de Parkinson/fisiopatología , Adenohipófisis/citología , Adenohipófisis/metabolismo , Receptores de Somatostatina/clasificación , Receptores de Somatostatina/efectos de los fármacos , Receptores de Somatostatina/metabolismo

RESUMEN

Somatostatin (SRIF) initiates its biological activities by interacting with five homologous G-protein-coupled receptor subtypes (sst(1--5)). In the mammalian nervous system, sst(1--5) receptor mRNA expression patterns have been localized by in situ hybridization studies, or at the protein level with receptor-specific antibodies. Cortical responses to SRIF have been demonstrated, although a functional relationship between an SRIF effect and an individual receptor subtype is lacking. The recent development of novel, subtype-selective SRIF receptor ligands now provides a means to correlate receptor subtype expression patterns with the corresponding biological function. In cultured monolayers of E17-18 rat embryonic cortical neurons, 10(-7) M SRIF-28 inhibited 10(-6) M forskolin-stimulated cAMP accumulation by 37%, a level of inhibition that was mimicked by L-797,591, a potent sst(1)-selective agonist. SRIF-14 or L-797,591 inhibited forskolin-stimulated cAMP accumulation in a concentration-dependent fashion, with EC(50)s (effective concentration for 50% maximal response) of 8.0 x 10(-10) M and 7.0 x 10(-10) M, respectively. No similar concentration-dependent effect on forskolin-stimulated cAMP levels was observed with sst(2)-, sst(3)- or sst(4)-selective agonists. Furthermore, both SRIF-14 and L-797,591 inhibited 10(-7) M CRH-induced cAMP in the embryonic neurons. These results are the first evidence demonstrating that sst(1) regulates intracellular cAMP levels in embryonic neurons and may inhibit CRH-mediated effects in the embryonic cortex.

Asunto(s)

Corteza Cerebral/metabolismo , AMP Cíclico/metabolismo , Indoles , Neuronas/metabolismo , Receptores de Somatostatina/fisiología , Amidas/farmacología , Animales , Corteza Cerebral/citología , Corteza Cerebral/embriología , Colforsina/farmacología , Hormona Liberadora de Corticotropina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Neuronas/efectos de los fármacos , Embarazo , Ratas , Ratas Sprague-Dawley , Receptores de Somatostatina/agonistas , Somatostatina/farmacología , Somatostatina-28

RESUMEN

The alpha chemokine receptor CXCR4 and its only characterized chemokine ligand, stromal cell-derived factor-1 (SDF-1), are postulated to be important in the development of the B-cell arm of the immune system. In addition, CXCR4 is a critical coreceptor in support of viral entry by T-cell line tropic strains (X4) of the Human Immunodeficiency Virus Type 1 (HIV-1), viral variants which predominate in some infected individuals in end stage disease. SDF-1 can block X4-tropic HIV-1 infection of CD4+ target cells in vitro, and allelic variants of the human gene encoding SDF-1 in vivo correlate with delayed disease progression. Therefore, CXCR4 may be an appropriate target for therapeutic intervention in acquired immunodeficiency syndrome (AIDS), and knowledge of the pharmacology of SDF-1 binding to its cognate receptor will be important in the interpretation of these experiments. We report here a Kd derived using a competition binding assay of 4.5 nM for CXCR4 endogenously expressed on peripheral blood monocytes and T-cells. This affinity is similar to that which SDF-1 exhibits when binding to endogenous CXCR4 on an established immortal Jurkat T-cell line as well as recombinant CXCR4 transfected into Chinese Hamster Ovary (CHO) cells. We also demonstrate that the determined affinity of SDF-1 for CXCR4 is reflective of its ability to induce a CXCR4-mediated signal transduction in these different cell types. Furthermore, using Bordetella pertussis toxin, we observe that high affinity binding of SDF-1 to CXCR4 is independent of the G-protein coupled state of the receptor, as uncoupling of G-protein did not lead to the appearance of measurable low affinity SDF-1 binding sites. Moreover, binding affinity and receptor number were unaffected by uncoupling for both recombinant and endogenously expressed CXCR4. Thus, SDF-1 is novel among agonist ligands of G protein-coupled receptors in that it appears to have equal affinity for both the G protein-coupled and uncoupled states of CXCR4.

Asunto(s)

Quimiocinas CXC/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Receptores CXCR4/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Células CHO , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Colforsina/farmacología , Cricetinae , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Expresión Génica , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Células Jurkat , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Toxina del Pertussis , Receptores CXCR4/agonistas , Receptores CXCR4/genética , Transducción de Señal/efectos de los fármacos , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factores de Virulencia de Bordetella/farmacología

RESUMEN

Somatostatin (SST) potently inhibits insulin and glucagon release from pancreatic islets. Five distinct membrane receptors (SSTR1-5) for SST are known, and at least two (SSTR2 and SSTR5) have been proposed to regulate pancreatic endocrine function. Our current understanding of SST physiology is limited by the receptor subtype selectivity of peptidyl SST analogs, making it difficult to assign a physiological function to an identified SST receptor subtype. To better understand the physiology of SSTRs we studied the in vitro effects of potent subtype-selective nonpeptidyl SST analogs on the regulation of pancreatic glucagon and insulin secretion in wild-type (WT) and in somatostatin receptor 2 knockout (SSTR2KO) mice. There was no difference in basal glucagon and insulin secretion between islets isolated from SSTR2KO and WT mice; however, potassium/arginine-stimulated glucagon secretion was approximately 2-fold higher in islets isolated from SSTR2KO mice. Neither SST nor any SSTR-selective agonist inhibited basal glucagon or insulin release. SST-14 potently inhibited stimulated glucagon secretion in islets from WT mice and much less effectively in islets from SSTR2KO mice. The SSTR2 selective analog L-779,976 inhibited glucagon secretion in islets from WT, but was inactive in islets from SSTR2KO mice. L-817,818, an SSTR5 selective analog, slightly reduced glucagon release in both animal groups, whereas SSTR1, -3, and -4 selective analogs were inactive. SST and L-817,818 inhibited glucose stimulated insulin release in islets from WT and SSTR2KO mice. L-779,976 much less potently reduced insulin secretion from WT islets. In conclusion, our data demonstrate that SST inhibition of glucagon release in mouse islets is primarily mediated via SSTR2, whereas insulin secretion is regulated primarily via SSTR5.

Asunto(s)

Glucagón/metabolismo , Antagonistas de Hormonas/farmacología , Indoles , Antagonistas de Insulina/farmacología , Islotes Pancreáticos/metabolismo , Naftalenos , Receptores de Somatostatina/genética , Somatostatina/farmacología , Amidas/farmacología , Animales , Glucosa/farmacología , Técnicas In Vitro , Islotes Pancreáticos/citología , Islotes Pancreáticos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Somatostatina/agonistas , Somatostatina/análogos & derivados , Somatostatina-28 , Estimulación Química

RESUMEN

Somatostatin (SST) regulates growth hormone (GH) secretion from pituitary somatotrophs by interacting with members of the SST family of G-protein-coupled receptors (sst1-5). We have used potent, nonpeptidyl SST agonists with sst2 and sst5 selectivity to determine whether these receptor subtypes are involved in regulating growth hormone releasing hormone (GHRH) stimulated secretion. GHRH stimulated GH release from pituitary cells in a dose-dependent manner, and this secretion was inhibited by Tyr(11)-SST-14, a nonselective SST analog. A sst2 selective agonist, L-779,976, potently inhibited GHRH-stimulated GH release. In addition, L-817, 818, a potent sst5 receptor selective agonist, also inhibited GH secretion, but was approximately 10-fold less potent (P < 0.01, ANOVA) in inhibiting GH release than either Tyr(11)-SST-14 or L-779, 976. These results show that both sst2 and sst5 receptor subtypes regulate GHRH-stimulated GH release from rat pituitary cells.

Asunto(s)

Hormona Liberadora de Hormona del Crecimiento/farmacología , Hormona del Crecimiento/metabolismo , Indoles , Adenohipófisis/efectos de los fármacos , Receptores de Somatostatina/agonistas , Somatostatina/agonistas , Amidas/farmacología , Animales , Relación Dosis-Respuesta a Droga , Masculino , Adenohipófisis/citología , Ratas , Ratas Wistar , Somatostatina/análogos & derivados , Somatostatina/farmacología

RESUMEN

Fentanyl, and its structural analogs lofentanil and sufentanil, are potent analgesics used clinically in the management of pain. However, the high analgesic potency of these compounds is limited by the development of tolerance after chronic use. To investigate whether their tolerance development may be related to mu receptor desensitization, the cloned mouse mu receptor as well as mutant forms of the receptor were stably expressed in HEK 293 cells and tested for their response to continuous opioid treatment. Fentanyl and its analogs potently bound to the mu receptor and effectively inhibited cAMP accumulation. Three-hour pretreatment of mu receptors with fentanyl and its analogs desensitized the mu receptor by uncoupling it from adenylyl cyclase. The fentanyl analogs caused a slight internalization of the mu receptor as accessed by antibody binding to the epitope-tagged mu receptor. Truncation of the mu receptor by removal of its carboxyl terminus at Glu341 did not affect the ability of the fentanyl analogs to bind to and activate the mu receptor nor did it prevent the fentanyl analogs from desensitizing the receptor. In a previous study we showed that morphine did not desensitize the cloned mu receptor even though it is a potent and effective agonist at the mu receptor. Mutagenesis studies revealed that morphine interacts differently with the mu receptor to activate it than do the fentanyl analogs which may explain its lack of desensitization of the mu receptor. These results indicate that desensitization of the mu receptor may be a molecular basis for the development of tolerance to fentanyl and its analogs.

Asunto(s)

Analgésicos Opioides/farmacología , AMP Cíclico/metabolismo , Fentanilo/farmacología , Receptores Opioides mu/efectos de los fármacos , Animales , Células Cultivadas , Fentanilo/análogos & derivados , Ratones , Mutagénesis , Receptores Opioides mu/genética , Sufentanilo/farmacología , Transfección

RESUMEN

To investigate the role of Asp114 in the cloned rat mu-opioid receptor for ligand binding, the charged amino acid was mutated to an asparagine to generate the mutant mu receptor D114N. The wild-type mu receptor and the D114N mutant were then stably expressed in human embryonic kidney 293 cells, and the binding affinities of a series of opioids were investigated. The mu-selective agonists [D-Ala2,MePhe4,Gly-ol5]enkephalin and morphine and the endogenous peptides Met-enkephalin and beta-endorphin exhibited greatly reduced affinities for the D114N mutant compared with the wild-type mu receptor, as did the potent synthetic agonist etorphine. In contrast to the full agonists, the partial agonists buprenorphine and nalorphine and the antagonists diprenorphine and naloxone bound with similar affinities to the wild-type and D114N mutant mu receptors. The reduced affinities of the full agonists for the D114N mutant did not involve an uncoupling of the receptor from G proteins because methadone and etorphine stimulated the D114N mu receptors to inhibit adenylyl cyclase. Although the Asp114 to Asn114 mutation reduced full-agonist binding, mutation of His297 to Asn297 in the mu receptor did not but, in contrast, did reduce binding affinity of the partial agonist buprenorphine and the antagonist diprenorphine. These results indicate that some partial agonists and antagonists may have different determinants for binding to the mu receptor than do the prototypical full agonists.

Asunto(s)

Mutagénesis , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Buprenorfina/metabolismo , Línea Celular , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Mutagénesis/fisiología , Antagonistas de Narcóticos/metabolismo , Narcóticos/metabolismo , Ratas , Receptores Opioides mu/agonistas

RESUMEN

An aspartic acid at residue 95 (Asp95) in the delta receptor has previously been shown to be critical for the binding affinity of selective delta agonists. To gain a better understanding of the functional consequence of agonist action at the delta receptor, the Asp95 residue was mutated to an asparagine (D95N) and opioids were tested for binding and functional activation of the wild-type and mutant delta receptors. Selective agonists such as [D-Ser2,D-Leu5]enkephalin-Thr6 (DSLET) and [D-Ala2,D-Leu5]enkephalin (DADLE) had greatly reduced affinity for the D95N mutant receptor but still inhibited cAMP accumulation, which indicated that the mutant receptor was still functionally coupled to adenylyl cyclase. Antagonist binding was not affected by the Asp95 mutation. Similarly, the partial agonist buprenorphine bound with equally high affinity to the D95N mutant and the wild-type delta receptor, which indicated that Asp95 is not essential for the binding affinity of this opioid. Buprenorphine did not affect cAMP accumulation in HEK 293 cells expressing the D95N mutant, and it blocked the ability of DSLET and bremazocine to inhibit cAMP accumulation via the D95N mutant, which indicated that buprenorphine acts as an antagonist at the D95N mutant. These findings confirm the essential role of Asp95 in the activation of the delta receptor by agonists and reveal a molecular basis of the unique property of buprenorphine.

Asunto(s)

Analgésicos Opioides/farmacología , Buprenorfina/farmacología , Antagonistas de Narcóticos/farmacología , Receptores Opioides delta/efectos de los fármacos , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Encefalina Leucina/análogos & derivados , Encefalina Leucina/farmacología , Leucina Encefalina-2-Alanina/farmacología , Ratones , Mutagénesis , Naltrexona/análogos & derivados , Naltrexona/metabolismo , Receptores Opioides delta/química

RESUMEN

Opioid analgesics are used extensively in the management of pain. Although the clinically effective opioids bind with high affinity to the mu-opioid receptor, studies have suggested that the delta-opioid agonists might represent more ideal analgesic agents, with fewer side effects. A limitation to opiate effectiveness is the development of tolerance, an event that has been linked to opioid receptor desensitization. To gain a better understanding of delta-receptor agonist regulation, the cloned mouse delta receptor was stably expressed in human embryonic kidney 293 cells, and the functional effects of agonist pretreatment were examined. With a 3-hr pretreatment protocol, the delta-selective agonists [D-Pen2,D-Pen5]enkephalin, [D-Ala2,D-Leu5]enkephalin, and [D-Ser2,Leu5]enkephalin-Thr and the nonselective opioids levorphanol, etorphine, and ethylketocyclazocine were found to desensitize delta receptors. [D-Pen2,D-Pen5]enkephalin, [D-Ser2,Leu5]enkephalin-Thr, [D-Ala2,D-Leu5]enkephalin, and etorphine treatments also caused a pronounced internalization of the epitope-tagged delta receptor, suggesting that the desensitization and internalization may be related. In contrast, levorphanol pretreatment did not internalize the receptor but still resulted in a 400-fold reduction in potency, suggesting that prolonged treatment with levorphanol only uncoupled the delta receptor from adenylyl cyclase. In contrast to the desensitization induced by peptide-selective delta agonists, pretreatment with the delta-selective nonpeptide agonist 7-spiroindanyloxymorphone and morphine sensitized the opioid inhibition of forskolin-stimulated cAMP accumulation. This differential regulation of the delta receptor may be due to variations in the ability of agonists to bind to the receptor. This hypothesis was supported by the finding that a point mutation that converted Asp128 to Asn128 (D128N) diminished the ability of delta-selective agonists to inhibit cAMP accumulation while increasing the potency of morphine to reduce cAMP accumulation. In particular, a lack of desensitization of the delta receptor by morphine may contribute to our understanding of the molecular basis of development of morphine-induced tolerance and dependence.

Asunto(s)

Leucina Encefalina-2-Alanina/farmacología , Encefalina Leucina/análogos & derivados , Encefalinas/efectos de los fármacos , Etorfina/farmacología , Receptores Opioides delta/metabolismo , Adenilil Ciclasas/metabolismo , Animales , Anticuerpos Monoclonales , Línea Celular , Colforsina/farmacología , AMP Cíclico/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Encefalina D-Penicilamina (2,5) , Encefalina Leucina/farmacología , Proteínas de Unión al GTP/fisiología , Humanos , Metadona/farmacología , Ratones , Morfina/farmacología , Mutación Puntual , Ensayo de Unión Radioligante , Receptores Opioides delta/agonistas , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad

RESUMEN

Opiates are potent analgesics used clinically in the treatment of pain. A significant drawback to the chronic use and clinical effectiveness of opiates is the development of tolerance. To investigate the cellular mechanisms of tolerance, the cloned human kappa-opioid receptor was stably expressed in human embryonic kidney (HEK 293) cells, and the effects of opioid agonist treatment were examined. The receptor-expressing cells showed specific high-affinity membrane binding for a kappa-selective opioid, 3H-labeled (+)-(5alpha,7alpha,8beta)-N-methyl-N-[7-(1-pyrrolidiny l)-1-oxaspiro [4,5] dec-8-yl] benzeneacetamide ([3H]U69,593), and a nonselective opioid antagonist, [3H]diprenorphine. Pretreatment with pertussis toxin or guanosine 5'-O-(3-thiotriphosphate) reduced [3H]69,593 binding, indicating that the human K receptor coupled to G proteins of the Gi or Go families in HEK 293 cells. The receptor-mediated inhibition of adenylyl cyclase was abolished by pertussis toxin pretreatment and was blocked by a kappa-selective antagonist, norbinaltorphimine. A 3-h pretreatment with a kappa-selective agonist, (+/-)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide (U50,488), caused receptor down-regulation, whereas no receptor down-regulation was found after levorphanol pretreatment. U50,488 or dynorphin A(1-17) pretreatments (3 h) desensitized the ability of U50,488 or dynorphin A(1-17) to inhibit cyclic AMP accumulation, as evidenced by a decrease in functional potency. Also, U50,488 pretreatment desensitized the ability of levorphanol to inhibit forskolin-stimulated cyclic AMP accumulation. In contrast, pretreatment of cells with either levorphanol or a potent nonselective opioid, etorphine, resulted in no apparent receptor desensitization. Taken together, these results demonstrate that the human kappa receptor is differentially regulated by selective and nonselective opioid agonists, with selective agonists able to desensitize the receptor.

Asunto(s)

Bencenoacetamidas , Narcóticos/agonistas , Receptores Opioides kappa/efectos de los fármacos , Receptores Opioides kappa/metabolismo , 3,4-Dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclohexil)-bencenacetamida, (trans)-Isómero , Toxina de Adenilato Ciclasa , Línea Celular , AMP Cíclico/antagonistas & inhibidores , AMP Cíclico/metabolismo , Diprenorfina/metabolismo , Dinorfinas/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Levorfanol/farmacología , Antagonistas de Narcóticos/metabolismo , Narcóticos/farmacología , Toxina del Pertussis , Pirrolidinas/metabolismo , Pirrolidinas/farmacología , Receptores Opioides kappa/agonistas , Factores de Virulencia de Bordetella/farmacología

RESUMEN

Mu opioid receptors mediate the analgesia induced by morphine. Prolonged use of morphine causes tolerance development and dependence. To investigate the molecular basis of tolerance and dependence, the cloned mouse mu opioid receptor with an amino-terminal epitope tag was stably expressed in human embryonic kidney (HEK) 293 cells, and the effects of prolonged opioid agonist treatment on receptor regulation were examined. In HEK 293 cells the expressed mu receptor showed high affinity, specific, saturable binding of radioligands and a pertussis toxin-sensitive inhibition of adenylyl cyclase. Pretreatment (1 h, 3 h, or overnight) of cells with 1 microM morphine or [D-Ala2MePhe4,Gly(ol)5]enkephalin (DAMGO) resulted in no apparent receptor desensitization, as assessed by opioid inhibition of forskolin-stimulated cAMP levels. In contrast, the morphine and DAMGO pretreatments (3 h) resulted in a 3-4-fold compensatory increase in forskolin-stimulated cAMP accumulation. The opioid agonists methadone and buprenorphine are used in the treatment of addiction because of a markedly lower abuse potential. Pretreatment of mu receptor-expressing HEK 293 cells with methadone or buprenorphine abolished the ability of opioids to inhibit adenylyl cyclase. No compensatory increase in forskolin-stimulated cAMP accumulation was found with methadone or buprenorphine; these opioids blocked the compensatory effects observed with morphine and DAMGO. Taken together, these results indicate that methadone and buprenorphine interact differently with the mouse mu receptor than either morphine or DAMGO. The ability of methadone and buprenorphine to desensitize the mu receptor and block the compensatory rise in forskolin-stimulated cAMP accumulation may be an underlying mechanism by which these agents are effective in the treatment of morphine addiction.

Asunto(s)

Receptores Opioides mu/metabolismo , Toxina de Adenilato Ciclasa , Analgésicos/metabolismo , Animales , Buprenorfina/farmacología , Colforsina/farmacología , AMP Cíclico/metabolismo , Diprenorfina/metabolismo , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/metabolismo , Etorfina/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Levorfanol/farmacología , Metadona/farmacología , Ratones , Morfina/farmacología , Toxina del Pertussis , Factores de Virulencia de Bordetella/farmacología

RESUMEN

Opioid actions are initiated at membrane receptors which couple to cellular effectors through G protein-mediated pathways. In the central nervous system opioids reduce neuronal activity through the inhibition of voltage-dependent Ca2+ channels, the activation of K+ channels and the inhibition of adenylyl cyclase. A significant clinical limitation to opioid therapy is the development of tolerance, a biological event that has been linked to agonist effects at the receptor level. Molecular studies on the consequences of opioid receptor regulation will provide a better understanding of the cellular mechanisms involved in the agonist-mediated events in tolerance development.

Asunto(s)

Receptores Opioides/metabolismo , Animales , Humanos

RESUMEN

Opiate receptors mediate the physiological actions of opioid peptides and the clinical effects of the synthetic opioid agonists and antagonists. Site-directed mutagenesis studies have revealed regions of opiate receptors that are essential for ligand recognition, and this could aid the design of more selective opioid ligands.

Asunto(s)

Péptidos/metabolismo , Receptores Opioides/metabolismo , Relación Estructura-Actividad , Sitios de Unión , Clonación Molecular , Modelos Moleculares , Estructura Molecular , Antagonistas de Narcóticos , Receptores Opioides/agonistas

RESUMEN

Localization in the nervous system of Drosophila melanogaster of a cloned Drosophila muscarinic acetylcholine receptor (mAChR) was investigated using a polyclonal antiserum raised against a peptide corresponding to the predicted receptor carboxyl terminal domain. Immunocytochemical studies on fly sections indicated that the product of the Dm1 mAChR gene was localized in the antennal lobes and in other regions of the brain and thoracic nervous system. Intense staining in the glomeruli of the antennal lobes, the region of the nervous system containing terminals of antennal olfactory sensory neurones and mechanosensory neurones, indicates possible roles for this mAChR gene product in the processing of olfactory and mechanosensory signals in the fly. The staining of a discrete group of neurosecretory cells in the pars intercerebralis of the brain indicates a possible new role for this mAChR in the regulation of neurosecretion. Very little staining is detected in the thoracic nervous system.

Asunto(s)

Drosophila melanogaster , Sistema Nervioso/química , Receptores Muscarínicos/análisis , Animales , Química Encefálica , Clonación Molecular , Extremidades/inervación , Ganglios de Invertebrados/química , Inmunohistoquímica , Masculino , Mecanorreceptores/química , Neuronas/química , Vías Olfatorias/química , Receptores Muscarínicos/genética , Tórax

RESUMEN

The pharmacological properties of a cloned Drosophila muscarinic acetylcholine receptor (mAChR) were investigated using two independent transient expression systems. The binding characteristics of the expressed receptor were determined using transfected COS-7 cells, whereas the mAChR functional properties were analyzed using nuclearly injected Xenopus oocytes. Competition displacement studies with transfected COS-7 cell membranes showed that N-[3H]methylscopolamine binding was displaced most effectively by atropine, followed by 4-diphenylacetoxy-N-methylpiperidine methiodide, pirenzepine, and AFDX-116. This same order of effectiveness (4-diphenylacetoxy-N-methylpiperidine methiodide > pirenzepine > AFDX-116) was observed in oocytes expressing Dm1 when carbamylcholine-induced currents were inhibited by the same antagonists. Thus, the expressed Drosophila mAChR (Dm1) exhibits a pharmacology that broadly resembles that of the vertebrate M1 and M3 mAChR subtypes. To determine the anatomical localization of the Drosophila mAChR, polyclonal antiserum was raised against a peptide corresponding to the predicted carboxyl-terminal domain of the receptor. Immunocytochemistry on fly sections demonstrated that the mAChR gene product was found in the nervous system and was not seen in skeletal muscle. The most intense staining was localized to the glomeruli of the antennal lobes, an area of the insect brain where first-order synaptic processing of olfactory information occurs.

Asunto(s)

Drosophila/anatomía & histología , Fenómenos Fisiológicos del Sistema Nervioso , Sistema Nervioso/ultraestructura , Receptores Muscarínicos/fisiología , Animales , Unión Competitiva , Células Cultivadas , Clonación Molecular , Electrofisiología , Femenino , Inmunohistoquímica , Fosfatos de Inositol/metabolismo , Membranas/efectos de los fármacos , Membranas/ultraestructura , Antagonistas Muscarínicos , N-Metilescopolamina , Sistema Nervioso/química , Oocitos/efectos de los fármacos , Oocitos/fisiología , Quinuclidinil Bencilato/metabolismo , Quinuclidinil Bencilato/farmacología , Receptores Muscarínicos/genética , Derivados de Escopolamina/metabolismo , Derivados de Escopolamina/farmacología , Estimulación Química , Transfección , Tritio , Xenopus laevis

RESUMEN

The hexapeptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6) and GH-releasing factor (GHRH) produced a rapid release of GH upon perifusion of dispersed rat pituitary cells. In contrast to the native hormone GHRH, GHRP-6 elicited a response of short duration. When perifusion of each secretagogue was continued until the cells no longer released GH, a challenge by the alternative secretagogue immediately resulted in a secondary release of GH. These results are consistent with each secretagogue causing desensitization of discrete receptor-linked second messenger pathways. Cells which were perifused for 1 min with GHRP-6 required continued perifusion with culture medium alone for 60 min before they completely regained responsiveness to a subsequent challenge with GHRP-6. Somatostatin (SRIF) was able to inhibit the action of either secretagogue completely. However, when both GHRH and GHRP-6 were perifused together, SRIF attenuated but did not block GH secretion. These perifusion data add support to conclusions derived from static cell culture studies, that GHRH and GHRP-6 act through different receptor sites and that through discrete signalling pathways their individual effects on GH release are amplified.

Asunto(s)

Hormona Liberadora de Hormona del Crecimiento/farmacología , Hormona del Crecimiento/metabolismo , Hormonas/farmacología , Oligopéptidos/farmacología , Hipófisis/efectos de los fármacos , Animales , Células Cultivadas , Masculino , Perfusión , Hipófisis/citología , Hipófisis/metabolismo , Ratas , Ratas Endogámicas , Sistemas de Mensajero Secundario/fisiología , Somatostatina/farmacología , Estimulación Química

RESUMEN

BACKGROUND: Cancer treatments without proved efficacy have achieved new levels of popularity, particularly among well-educated patients. The value of these therapies is vigorously debated. METHODS: We compared the length of survival and quality of life in patients who received treatment at a prominent unorthodox cancer clinic in addition to conventional treatment and in matched control patients from an academic cancer center who received only conventional treatment. All the patients had documented extensive malignant disease associated with a predicted median survival time of less than one year. The study sample consisted of 78 pairs of patients matched according to sex, race, age, diagnosis, and time from the diagnosis of metastatic or recurrent disease, who were enrolled over a period of 3 1/2 years. Periodic follow-up (approximately every two months) continued until death. RESULTS: There was no difference between the two patient groups in length of survival. Median survival for both groups was 15 months (P = 0.22; relative risk, 1.23; 95 percent confidence interval, 0.88 to 1.72). Quality-of-life scores were consistently better among conventionally treated patients from enrollment on. CONCLUSIONS: For this sample of patients with extensive disease and for this particular unorthodox treatment regimen, conventional and unorthodox treatments produced similar results.

Asunto(s)

Terapias Complementarias , Neoplasias/terapia , Calidad de Vida , Adulto , Anciano , Demografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/mortalidad , Tasa de Supervivencia , Estados Unidos/epidemiología

RESUMEN

The deletion of residues 239-272 from the hamster beta-adrenergic receptor resulted in a loss of the ability of the receptor, expressed in mouse L cells, to stimulate adenylate cyclase (Dixon, R. A. F., Sigal, I. S., Rands, E., Register, R. B., Candelore, M. R., Blake, A. D., and Strader, C. D. (1987) Nature 326, 73-77). This mutant receptor (D(239-272)beta AR) bound the agonist isoproterenol with a single class of binding sites, in contrast to the wild-type beta-adrenergic receptor, which exhibited two classes of agonist affinity sites. We now report that the affinity of D(239-272)beta AR for isoproterenol is relatively insensitive to detergent solubilization or to treatment with either GTP or NaF, indicating the absence of a receptor-Gs interaction. Whereas deletions within the region of amino acids 229-258 did not reduce the ability of the receptor to couple to Gs or to stimulate adenylate cyclase, the deletion of either of the regions 222-229 or 258-270 resulted in receptors which were unable to couple to Gs. The affinities of D(222-229)beta AR, D(239-272)beta AR, and D(258-270)beta AR toward isoproterenol were greater than that observed for the low affinity, uncoupled form of the wild-type receptor. These results suggest a role for the regions of the beta-adrenergic receptor encompassing amino acids 222-229 and 258-270, which are predicted to form amphiphilic helices, in the agonist-promoted activation of Gs.

Asunto(s)

Agonistas Adrenérgicos beta/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores Adrenérgicos beta/genética , Adenilil Ciclasas/metabolismo , Animales , Unión Competitiva , Deleción Cromosómica , Cricetinae , Yodocianopindolol , Isoproterenol/metabolismo , Ratones , Pindolol/análogos & derivados , Pindolol/metabolismo , Receptores Adrenérgicos beta/metabolismo , Fluoruro de Sodio/farmacología , Relación Estructura-Actividad

RESUMEN

Desensitization of the beta-adrenergic receptor has been correlated in some cell systems with receptor phosphorylation. Various kinases have been implicated in these phosphorylation processes, including both cAMP-dependent protein kinase and protein kinase C. In the present study, we have utilized the protein sequence information obtained from the cloning of the mammalian beta-adrenergic receptor to prepare synthetic peptides corresponding to regions of the receptor which would be predicted to act as possible substrates for these kinases in vivo. Two of these receptor-derived peptides were found to serve as substrates for these protein kinases. A peptide corresponding to amino acids 257-264 of the beta-receptor is the preferred substrate for the cAMP-dependent protein kinase, while protein kinase C showed a marked preference for phosphorylation of a peptide corresponding to residues 341-351 of the beta-adrenergic receptor.

Asunto(s)

Oligopéptidos/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Quinasas/metabolismo , Receptores Adrenérgicos beta/metabolismo , Secuencia de Aminoácidos , Animales , Cricetinae , Cinética , Oligopéptidos/síntesis química , Fosforilación , Serina/metabolismo , Relación Estructura-Actividad