RESUMO
Pancreatic beta-cells and gamma-aminobutyric acid (GABA)-secreting neurons both express the enzyme glutamic acid decarboxylase (GAD) which is a major target of autoantibodies associated with beta-cell destruction and impairment of GABA-ergic neurotransmitter pathways. The predominant form of GAD in pancreatic beta-cells, GAD65, is synthesized as a soluble hydrophilic molecule, which is modified to become firmly membrane anchored. Here we show by immunogold electron microscopy that GAD65 is localized to the membrane of small vesicles which are identical in size to small synaptic-like microvesicles in pancreatic beta-cells. The NH2-terminal domain of GAD65 is the site of a two-step modification, the last of which results in a firm membrane anchoring that involves posttranslational hydroxylamine sensitive palmitoylation. GAD65 can be released from the membrane by an apparent enzyme activity in islets, suggesting that the membrane anchoring step is reversible and potentially regulated. The hydrophobic modifications and consequent membrane anchoring of GAD65 to microvesicles that store its product GABA may be of functional importance and, moreover, significant for its selective role as an autoantigen.
Assuntos
Autoantígenos/metabolismo , Glutamato Descarboxilase/metabolismo , Membranas Intracelulares/metabolismo , Ilhotas Pancreáticas/metabolismo , Isoenzimas/metabolismo , Organelas/metabolismo , Ácidos Palmíticos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Autoantígenos/genética , Baculoviridae/genética , Linhagem Celular , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/imunologia , Hidroxilamina , Hidroxilaminas/farmacologia , Insetos , Membranas Intracelulares/ultraestrutura , Isoenzimas/genética , Isoenzimas/imunologia , Microscopia Imunoeletrônica , Organelas/ultraestrutura , Ácido Palmítico , Fosfatidilinositol Diacilglicerol-Liase , Ligação Proteica , Ratos , TransfecçãoRESUMO
Growth hormone secretagogues (GHSs) are synthetic compounds that induce GH release in several species, including man. The aim of the current study was to identify hypothalamic GHS receptor (GHS-R) agonists. This led to the discovery of adenosine as a GHS-R agonist. We demonstrate that adenosine as well as the A1 adenosine receptor agonist N6-R-phenylisopropyladenosine (R-PIA) induce calcium responses, with EC50 values of 50 nM and 0.5 nM, respectively, in cells which express recombinant human GHS-R. However, neither compound induces a calcium response in nontransfected cells. Binding experiments show that adenosine and the GHS compound MK-0677 bind to membranes from GHS-R expressing cells with nearly identical Bmax values (2.6 +/- 0.1 x 10(-10) mol/mg protein for adenosine and 2.0 +/- 0.3 x 10(-10) mol/mg protein for MK-0677). However, no binding to membranes from nontransfected cells could be detected. Furthermore, we show that the IC50 values for inhibition of the adenosine, R-PIA, and GHS induced calcium responses by the GHS-R antagonist [D-Arg1, D-Phe5, D-Trp7,9, D-Leu11]-substance P are similar. These findings strongly suggest that adenosine and R-PIA are agonists of the GHS-R. Interestingly, neither adenosine nor R-PIA were able to induce GH release from rat pituitary cells in vitro. The implications of the latter finding is discussed.
Assuntos
Adenosina/farmacologia , Hormônio do Crescimento/metabolismo , Receptores de Droga/agonistas , Animais , Cálcio/metabolismo , Cromatografia Líquida de Alta Pressão , Cricetinae , Feminino , Corantes Fluorescentes , Fura-2 , Humanos , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Indóis/farmacologia , Hipófise/citologia , Hipófise/metabolismo , Ratos , Ratos Wistar , Receptores de Droga/genética , Espectrofotometria Ultravioleta , Compostos de Espiro/farmacologia , TransfecçãoRESUMO
The highly potent vanilloid receptor (VR) agonist resiniferatoxin has been radiolabeled with 125I, and the pharmacology to the cloned rodent VR, VR1, and the endogenous VR in rat spinal cord membranes has been characterized. [125I]RTX binding to human embryonic kidney 293 cells expressing VR1 was reversible and with high affinity (Kd = 4.3 nM) in an apparent monophasic manner. In rat spinal cord membranes, [125I]RTX bound with a similar high affinity (Kd = 4.2 nM) to a limited number of binding sites (Bmax = 51 +/- 8 fmol/mg of protein). The pharmacology of recombinant rodent VR1 and the endogenous rat VR1 was indistinguishable when measuring displacement of [125I]RTX binding (i.e., the following rank order of affinity was observed: RTX > I-RTX > olvanil > capsaicin > capsazepine). Capsaicin and RTX induced large nondesensitizing currents in Xenopus laevis oocytes expressing VR1 (EC50 values were 1300 nM and 0.2 nM, respectively), whereas I-RTX induced no current per se at concentrations up to 10 microM. However, I-RTX completely blocked capsaicin-induced currents (IC50 = 3.9 nM). In vivo, I-RTX effectively blocked the pain responses elicited by capsaicin (ED50 = 16 ng/mouse, intrathecally). The present study showed that I-RTX is at least 40-fold more potent than the previously known VR antagonist, capsazepine. Thus, I-RTX as well as its radiolabeled form, should be highly useful for further exploring the physiological roles of VRs in the brain and periphery.
Assuntos
Diterpenos/farmacologia , Receptores de Droga/antagonistas & inibidores , Analgésicos/farmacologia , Animais , Ligação Competitiva , Capsaicina/farmacologia , Células Cultivadas , Diterpenos/uso terapêutico , Interações Medicamentosas , Humanos , Radioisótopos do Iodo , Camundongos , Oócitos , Dor/tratamento farmacológico , Medição da Dor/efeitos dos fármacos , Ensaio Radioligante , Receptores de Droga/metabolismo , Canais de Cátion TRPV , Transfecção , Xenopus laevisRESUMO
In order to isolate mutants with impaired uptake of branched-chain amino acids, mutants were induced which on complex medium were sensitive to an inhibitor of branched-chain amino acid biosynthesis. Eighteen mutants of independent origin were found. Ten of them were assayed for branched-chain amino acid uptake. Three of these were impaired in the uptake of L-valine, L-isoleucine and L-leucine, while the rest were unaffected in uptake of any of the three amino acids. Kinetics of the uptake by one selected mutant and the parental strain S288C were compared to models for one or two systems obeying Michaelis-Menten kinetics. This analysis suggested that a high-affinity system for all three amino acids is absent in the mutant, whereas low-affinity uptake of L-isoleucine and L-leucine by one or more systems remains unaffected. Moreover, medium-affinity uptake components for L-valine and L-leucine, not originally seen in the wild type, were identified in the mutant. In the wild type, 10 mM of any of the amino acids L-alanine, L-cysteine, L-isoleucine, L-leucine, L-tryptophan and L-valine reduce uptake of any of the three branched-chain amino acids. We propose that a permease responsible for high-affinity uptake of the branched-chain amino acids in strain S288C is partially or completely inactive in the mutant. Tetrad analysis shows that the phenotype can be ascribed to a single Mendelian gene. The wild-type allele is denoted BAP1 for branched-chain amino acid permease. The BAP1-dependent system is different from the general amino acid permease.
Assuntos
Aminoácidos de Cadeia Ramificada/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Meios de Cultura , Isoleucina/metabolismo , Cinética , Leucina/metabolismo , Mutação , Saccharomyces cerevisiae/genética , Valina/metabolismoRESUMO
The alpha isoform of protein kinase C (PKCalpha) is a ubiquitous protein kinase, which, upon activation, translocates rapidly from the cytoplasm to the plasma membrane. To follow this translocation, PKCalpha was tagged with a highly fluorescent derivative of green fluorescent protein and stably expressed in baby hamster kidney cells overexpressing the muscarinic type 1 receptor. Addition of the agonist carbamylcholine triggered the onset of translocation within 1 s. Half-maximal and maximal translocation occurred after about 3 and 15 s respectively. Plasma membrane association of the fusion protein was transient and the protein returned to the cytoplasm within about 45 s. A high-resolution study showed an almost homogeneous cytoplasmic distribution of tagged PKCalpha in unstimulated cells and virtually complete translocation to the plasma membrane in response to the phorbol ester, PMA. Simultaneous visualization of intracellular calcium concentration ([Ca2+]i) and PKCalpha translocation in single cells showed a good correlation between these parameters at intermediate and high concentrations of agonist. At low agonist concentration, a small increase in [Ca2+]i was observed, without detectable translocation of PKCalpha. In contrast, PMA induced translocation of PKCalpha without any increase in [Ca2+]i. Neither cytochalasin D nor colcemid influenced the distribution or calcium-dependent translocation of tagged PKCalpha, indicating that PKCalpha translocation may be independent of both actin filaments and microtubules. The time course of PKCalpha translocation is compatible with diffusion of the protein from its cytoplasmic localization to the plasma membrane.
Assuntos
Cálcio/metabolismo , Isoenzimas/metabolismo , Proteínas Luminescentes/metabolismo , Proteína Quinase C/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Transporte Biológico , Carbacol/farmacologia , Citoesqueleto/fisiologia , Proteínas de Fluorescência Verde , Cobaias , Processamento de Imagem Assistida por Computador/métodos , Isoenzimas/genética , Proteínas Luminescentes/genética , Camundongos , Microscopia de Fluorescência/métodos , Proteína Quinase C/genética , Proteína Quinase C-alfa , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
In an attempt to elucidate the origin of the T cell lymphopenia and/or the beta-cell-specific autoimmunity observed in diabetes-prone Bio-Breeding (DP-BB) rats, a thymic cDNA library was subjected to differential screening with thymic cDNA probes of DP-BB rats and nonlymphopenic nondiabetic controls. This approach resulted in the identification of a prominent lack of thymic B cells in DP-BB rats. This deficiency is distinct from a less pronounced peripheral B cell deficiency of different timing. The thymic B cell defect is linked to the lymphopenia trait on chromosome 4 and thereby with susceptibility to diabetes in crosses involving the DP-BB rat. In conclusion, our data suggest that the contribution of thymic B cells to the (negative) selection of thymocytes is inadequate in DP-BB rats, thus providing a plausible explanation for at least some of the spontaneous autoimmune phenomena in this animal model.