Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
Mais filtros








Base de dados
Intervalo de ano de publicação
1.
Brain Res ; 920(1-2): 84-96, 2001 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-11716814

RESUMO

Pre-clinical and clinical studies are currently underway to evaluate the potential of phosphodiesterase-4 (PDE4) inhibitors for the treatment of chronic obstructive pulmonary disease and other inflammatory conditions of the airways. The most common side effect associated with this class of compounds is emesis. The squirrel monkey provides a model for evaluating the efficacy of PDE4 inhibitors and their emetic potential. The distribution of three PDE4 isoforms (A, C and D) has been investigated in the squirrel monkey medulla and nodose ganglion to determine which isoform(s) could be responsible for the emetic adverse effects. The distribution of PDE4 isoforms was delineated using immunohistochemistry with antibodies specific for PDE4A, PDE4C and PDE4D and by in situ hybridization with isoform-selective riboprobes. PDE4A was present in the medulla where expression was mostly restricted to glial cells and the vasculature. PDE4C was not detected in either the medulla or nodose ganglion. Finally, the PDE4D isoform was localized to neurons in the nodose ganglion and found through many structures of medulla including the area postrema, neurons of the nucleus tractus solitarius and locus coeruleus. These data are consistent with a role for PDE4D in the emetic response.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Bulbo/enzimologia , Gânglio Nodoso/enzimologia , Animais , Sequência de Bases , Western Blotting , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Feminino , Imuno-Histoquímica , Hibridização In Situ , Isoenzimas/metabolismo , Masculino , Microscopia de Fluorescência , Dados de Sequência Molecular , Sondas RNA , Reflexo/fisiologia , Saimiri , Substância P/metabolismo , Vômito/fisiopatologia
2.
Cell Biochem Biophys ; 29(1-2): 159-78, 1998.
Artigo em Inglês | MEDLINE | ID: mdl-9631244

RESUMO

A CHO-K1 cell line stably expressing a recombinant full-length human PDE-IVa (HSPDE4A4B) enzyme was established under hygromycin B selection. Full-length expression of the protein was determined by Western blot analysis, which revealed the presence of a 125-kDa immunoreactive band using rabbit anti-PDE-IVa antibodies. The potency of inhibitor compounds was examined by their ability to increase cAMP in the whole-cell, and by their ability to inhibit cAMP hydrolysis in a 100,000 g supernatant (soluble enzyme preparation) obtained from the same cell line. Inhibition of the expressed PDE-IVa activity by selective PDE-IV inhibitors--(R) and (S)-rolipram, RS 14203, and CDP 840--at 100 nM substrate demonstrated that RS 14203 and CDP 840 were the most potent with IC50 = 9 nM, followed by (R)-rolipram (IC50 = 110 nM) and (S)-rolipram (IC50 = 420 nM). The rank order of potencies of the inhibitors in elevating cAMP in the whole-cell assay was quite different from that on the soluble enzyme. RS 14203 was still the most potent compound in elevating cAMP. Moreover, the relative rank order of potencies between CDP 840 and (R)-rolipram changed dramatically, such that (R)-rolipram was more potent than CDP 840 = (S)-rolipram. An apparent 30-fold stereoselectivity between (R)- and (S)-rolipram was also noted. The whole-cell rank order of potencies was also maintained when PKA activity ratios were measured in place of cAMP levels. The ability of the compounds to elevate cAMP in the stable CHO-K1 cells appeared to track better with the potency of the compounds against the high-affinity (Sr) conformer of the enzyme rather than the low-affinity catalytic state.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/biossíntese , 3',5'-AMP Cíclico Fosfodiesterases/genética , Células CHO/enzimologia , Proteínas Recombinantes/biossíntese , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Células CHO/química , Catálise/efeitos dos fármacos , Cricetinae , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Eosinófilos/enzimologia , Eosinófilos/metabolismo , Cobaias , Humanos , Hidrólise/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Transfecção
3.
Am J Physiol ; 273(2 Pt 1): L363-73, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9277448

RESUMO

We studied the intracellular free Ca2+ concentration ([Ca2+]i) response to histamine in a cystic fibrosis airway epithelial cell line (CF/T43). Histamine (100 microM; duration approximately 10 min) biphasically increased [Ca2+]i, with a rapid initial peak (30-45 s) followed by a smaller second peak that lasted for several minutes before returning to baseline. Neither peak specifically depended on Ca2+ influx. Exposure to bradykinin (10 microM) elicited a single peak that lasted 3-3.5 min before returning to baseline. Bradykinin increased intracellular inositol 1,4,5-trisphosphate (IP3), which peaked and returned to baseline within 150 s. Histamine also increased IP3 monophasically, but the peak was brief (< 20 s). Both phases of the Ca2+ response to histamine exhibited similar responsiveness to histamine concentration and sensitivity to antagonists. Cimetidine or thioperamide (1 mM) had no effect on the second peak. Pyrilamine blocked the second peak at concentrations similar to those required to block the initial peak. Activation of the second peak was observed at a threshold concentration of 1 microM comparable with the threshold of the initial peak. Neither adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, nor cyclic ADP (cADP)-ribose altered the second phase of the histamine response.


Assuntos
Cálcio/metabolismo , Fibrose Cística/metabolismo , Membranas Intracelulares/metabolismo , Mucosa Nasal/metabolismo , Traqueia/metabolismo , Bradicinina/farmacologia , Linhagem Celular Transformada , Fibrose Cística/patologia , Epitélio/metabolismo , Epitélio/patologia , Histamina/farmacologia , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Mucosa Nasal/patologia , Sistemas do Segundo Mensageiro , Fatores de Tempo , Traqueia/patologia
4.
Exp Lung Res ; 22(5): 509-23, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8886756

RESUMO

This study examined the production of stored mucosubtances in rats after repeated exposure to aerosolized endotoxin, a common contaminant of bioaerosols. Male Fischer 344 rats were exposed to aerosolized saline (sham control) or endotoxin (target concentrations of 0.05, 0.5, and 5.0 micrograms/m3) for 3 h/day, 5 days/week for 4 weeks. Following the final exposure, the left lung of each animal was lavaged and the right lung and nasal cavity were fixed with buffered formalin. Morphometric examination of Alcian blue/Periodic acid Schiffs-stained (AB/PAS) lung sections demonstrated dose-dependent increases in stored intraepithelial mucosubstances in the intrapulmonary airways of endotoxin-exposed rats. Threefold and eightfold increases in stored mucosubstances were observed in generation 5 airways of animals exposed to 0.5 or 5.0 microgram/m3 endotoxin, respectively (p < .05). This mucous cell metaplasia in the intrapulmonary airways was not accompanied by evidence of lung inflammation or increased AB/PAS-staining high molecular weight material in lavage fluid. Furthermore, despite significant deposition of endotoxin aerosols (mass median aerodynamic diameter of 1.9 microns) in the nasal cavity, no significant changes in stored mucosubstances were observed in the nasal septum. In animals repeatedly exposed to 5.0 micrograms/m3 endotoxin and allowed to recover for 1 month, stored mucosubstances in the intrapulmonary airway were still more than fivefold greater than control values. Thus, in rats, repeated exposure to inhaled endotoxin produced a persistent mucous cell metaplasia only in the intrapulmonary airways.


Assuntos
Brônquios/efeitos dos fármacos , Endotoxinas/toxicidade , Muco/efeitos dos fármacos , Administração por Inalação , Animais , Brônquios/metabolismo , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Relação Dose-Resposta a Droga , Endotoxinas/administração & dosagem , Processamento de Imagem Assistida por Computador , Masculino , Muco/metabolismo , Nariz/patologia , Ratos , Ratos Endogâmicos F344 , Sistema Respiratório/patologia
5.
Am J Respir Cell Mol Biol ; 13(2): 133-43, 1995 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-7626283

RESUMO

The ovalbumin-sensitized guinea pig is commonly used as a small animal model of allergic asthma. This animal model exhibits many of the hallmark characteristics observed in patients afflicted with asthma including nonspecific airway hyperreactivity, airway eosinophilia, early and late phase bronchoconstriction, and plasma extravasation into the airways. In addition, mucous hypersecretion in the airways of asthmatic patients is thought to be responsible for the plugging of distal airways and to contribute to the morbidity and mortality associated with the disease process. In this study we examined whether the allergic guinea pig model exhibits an increase in airway high molecular weight glycoconjugate (HMWG) secretion in response to an antigen challenge and whether dexamethasone exerts any modulatory effects upon the response. Ovalbumin (OVA) -sensitized guinea pigs were challenged with OVA 2 wk following the initial exposure. Trachobronchoalveolar lavages (TBAL) were performed, and the samples were assayed for total eosinophil cell number, eosinophil peroxidase activity (EPO), and both acidic and neutral HMWG content. Morphometric analysis of mucous-containing cells was also performed on tissue sections prepared from the trachea, mainstem bronchus, and three lobes of the left lung. Within 24 h of an antigen challenge, TBAL samples obtained from the allergic guinea pigs exhibited increases in eosinophil cell number, measured EPO enzyme activity, and acidic HMWG content compared to TBAL samples prepared from vehicle-exposed animals. These antigen-induced changes were dependent on the concentration of aerosolized OVA administered. Exposing the animals to 0.3% OVA provoked a 6.23-fold increase in airway eosinophils, 15-fold elevation in TBAL EPO enzyme activity, and 175% increase in TBAL acidic HMWG. No significant changes in TBAL neutral HMWG were measured. The changes in measured EPO activity correlated with the levels of acidic HMWG found in the TBAL samples (r = 0.73, P < or = 0.001). The measured increase in TBAL acidic HMWG was time dependent and was found to be maximal at 2 h post-antigen challenge. Morphometric analysis of Alcian blue (pH 2.5) -stained airway sections showed a decline in stored mucosubstances following the antigen exposure, supporting the notion that the allergic guinea pig model exhibits a mucosecretory component. Pretreating the animals with dexamethasone attenuated the antigen-induced release of HMWG and changes in measured EPO activity. In conclusion, these data indicate that the allergic guinea pig may be a useful model for examining the neural and cellular mechanisms underlying mucus hypersecretion in individuals afflicted with bronchial asthma.


Assuntos
Dexametasona/farmacologia , Glicoconjugados/metabolismo , Hipersensibilidade/tratamento farmacológico , Ovalbumina/metabolismo , Animais , Asma/imunologia , Líquido da Lavagem Broncoalveolar/química , Modelos Animais de Doenças , Eosinófilos/efeitos dos fármacos , Eosinófilos/metabolismo , Glicoconjugados/análise , Cobaias , Masculino , Peso Molecular , Muco/metabolismo , Ovalbumina/imunologia
6.
Am J Respir Crit Care Med ; 151(6): 1931-8, 1995 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7767542

RESUMO

Chronic exposure of rats to high concentrations of SO2 gas causes pathologic changes in airway similar to those seen in human chronic bronchitis. The purpose of this study was to examine the pulmonary mechanical correlates of these changes and to quantify the extent of mucous hypersecretion by measuring changes in mucous glycoproteins. Female Sprague-Dawley rats were exposed to 250 ppm SO2 gas, 5 h/d, 5 d/wk, for a period of 4 wk. Control rats were exposed to air only. On the day after the last SO2 exposure, rats were anesthetized, instrumented for the measurement of pulmonary resistance (RL) and dynamic compliance (Cdyn), and ventilated. Chronic SO2 exposure caused a small but significant increase in RL and decrease in Cdyn. Airway responsiveness to inhaled aerosolized methacholine was increased in SO2-exposed rats, as indicated by approximately 6.6- and 4.6-fold decreases respectively, in the doses of inhaled methacholine required to double RL or decrease Cdyn to 50% of baseline. SO2 exposure had no effect on the contractile response of the trachea measured in vitro. Tracheae and lungs from SO2-exposed animals exhibited 140 and 535% increases in measured neutral mucous glycoproteins, respectively, and 33 and 37% increases in acid glycoproteins. Our results indicate that this animal model of chronic bronchitis mimics the mucous hypersecretion, airway obstruction, and increased airway responsiveness observed in human bronchitis and may allow us to begin to probe their mechanistic basis.


Assuntos
Hiper-Reatividade Brônquica/fisiopatologia , Bronquite/fisiopatologia , Animais , Brônquios/metabolismo , Brônquios/fisiopatologia , Testes de Provocação Brônquica , Bronquite/induzido quimicamente , Bronquite/diagnóstico , Feminino , Glicoproteínas/metabolismo , Pulmão/fisiopatologia , Cloreto de Metacolina , Mucinas/metabolismo , Muco/química , Muco/metabolismo , Ratos , Ratos Sprague-Dawley , Dióxido de Enxofre , Traqueia/fisiopatologia
7.
Can J Physiol Pharmacol ; 72(9): 1025-30, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7842384

RESUMO

The ability of aerosolized sodium metabisulfite to induce hypertrophic and hyperplastic changes in rat airway secretory epithelial cells was investigated. A 10% solution of sodium metabisulfite was aerosolized into a Plexiglas exposure chamber, using an ultrasonic humidifier. The level of SO2 gas generated by this apparatus was measured to be 500 ppm. Measured levels of neutral and acidic mucous glycoproteins in extracts from tracheal and lung tissue were used as indices of hypertrophic (increases in mucus content per cell) and hyperplastic (increased numbers of cells containing mucus per gram of tissue) changes occurring in mucus-secreting cells of the airways. Exposing rats to sodium metabisulfite for 3 weeks resulted in profound increases in total neutral mucous glycoproteins found in tracheal and lung tissue (6.2-fold and 10.1-fold, respectively), compared with the H2O-treated counterparts. Total acidic mucous glycoproteins were significantly elevated in lung tissue only (13.5-fold). In addition, neutral and acidic mucous glycoproteins were elevated 20-fold and 9-fold, respectively, in bronchoalveolar lavage samples prepared from sodium metabisulfite exposed animals. These results indicate that aerosolized sodium metabisulfite may be a useful agent for developing small animal models of mucus hypersecretion.


Assuntos
Epitélio/efeitos dos fármacos , Glicoproteínas/análise , Muco/metabolismo , Sulfitos/administração & dosagem , Aerossóis , Animais , Modelos Animais de Doenças , Epitélio/metabolismo , Epitélio/patologia , Hiperplasia/induzido quimicamente , Hipertrofia/induzido quimicamente , Pulmão , Pneumopatias Obstrutivas/induzido quimicamente , Masculino , Ratos , Ratos Sprague-Dawley , Projetos de Pesquisa , Traqueia
8.
Am J Respir Cell Mol Biol ; 10(6): 625-34, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7516172

RESUMO

Determination of hyperplastic and hypertrophic changes of mucus-secreting cells in animal airways has been performed in the past by using histologic, immunologic, and/or molecular biologic approaches. Histologic techniques are tedious and time-consuming. The other approaches require specific antibodies and cDNA probes that have proved difficult to develop. Described here is a method for the rapid estimation of hyperplastic and hypertrophic changes of secretory epithelial cells in rat airways. The assay specifically measures acidic and neutral mucoproteins in a linear fashion from 0.5 microgram to at least 10 micrograms. Male Sprague-Dawley rats were exposed to metabisulfite mist (10% wt/vol) for 5 days/wk for 3 wk. The lungs were removed and homogenized in a phosphate-buffered solution containing reducing agents and protease inhibitors. The particulate matter was removed by centrifugation, and the soluble extract was applied to a column packed with Sepharose CL-6B. The material eluting in the void volume was applied to a PVDF membrane and stained for either acidic or neutral mucosubstances using Alcian blue or periodic acid-Schiff (PAS) staining, and the absorbance was read using a 96-well plate reader. Lungs from sodium metabisulfite-exposed animals showed a 7-fold and 3.5-fold increase in PAS-positive and Alcian blue-positive material, respectively. The increase in both PAS and Alcian blue staining was hyaluronidase and chondroitinase insensitive. The observed changes are consistent with morphometric measurements of mucus-containing cells in histologic sections of the tissues. This assay may be useful in determining which neurohumoral mediators might be involved in mucus cell hypertrophy and hyperplasia in animal models of chronic obstructive pulmonary disease.


Assuntos
Azul Alciano , Histocitoquímica/métodos , Pulmão/patologia , Mucinas/análise , Reação do Ácido Periódico de Schiff , Administração por Inalação , Animais , Cromatografia em Agarose , Epitélio/patologia , Hiperplasia , Hipertrofia , Pulmão/química , Masculino , Mucinas/efeitos dos fármacos , Mucinas/isolamento & purificação , Proteoglicanas/análise , Ratos , Ratos Sprague-Dawley , Coloração e Rotulagem , Sulfitos/administração & dosagem , Sulfitos/farmacologia , Traqueia/química
9.
J Pharmacol Exp Ther ; 269(3): 891-6, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8014876

RESUMO

Agonist-induced changes in intracellular calcium ion concentration ([Ca++]i) were examined in human monocytic leukemia THP-1 cells loaded with fura 2/acetoxymethyl ester (fura 2/AM). Leukotriene (LT)D4 induced a concentration-dependent biphasic response consisting of a transient phase (up to 5-fold peak increase) followed by a sustained phase, showing characteristics of a receptor-operated calcium channel. Homologous desensitization to LTD4 was observed. The responses to LTD4 were reduced by 80 to 90% in calcium-free buffer. The responses to LTD4 in a calcium-free buffer were dependent upon the duration of prior exposure of the cells to a calcium-free environment. The response at 30 or 60 min after exposure to calcium-free buffer was greater than that at earlier time points (time-dependent sensitization). Similar responses were obtained with THP-1 cells exposed to EDTA-containing buffer. It is speculated that such time-dependent sensitization is a result of changes at the receptor level. The responses to LTD4 were blocked by two specific LTD4 antagonists, MK-0571 and ICI-204,219, in a concentration-dependent manner. When given after addition of LTD4, MK-0571 or ICI-204,219 reversed the sustained phase of the LTD4-induced response, suggesting that maintenance of the response requires persistent activation of the LTD4 receptor. ICI-204,219 was 5 to 10 times more potent than MK-0571 (IC50 values of 1.1 and 9.3 nM, respectively), in agreement with results from radioligand binding studies reported separately.


Assuntos
Cálcio/metabolismo , Leucemia Mieloide/metabolismo , Leucotrieno D4/farmacologia , Proteínas de Membrana , Receptores de Leucotrienos/fisiologia , Citosol/metabolismo , Humanos , Indóis , Antagonistas de Leucotrienos , Fenilcarbamatos , Propionatos/farmacologia , Quinolinas/farmacologia , Sulfonamidas , Compostos de Tosil/farmacologia , Células Tumorais Cultivadas
10.
Eur J Pharmacol ; 245(2): 119-27, 1993 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-8387925

RESUMO

We have investigated the cellular signalling pathway by which vasopressin stimulates a Ca2(+)-dependent Cl- conductance and the effects of two known Cl- channel blockers in cultured rat A7r5 aortic smooth muscle cells using anion efflux and fluorescent Ca2+ imaging studies. Addition of vasopressin (100 nM) to A7r5 cells enhanced 125I (Cl- substitute) efflux from the cells through a V1 receptor-mediated pathway. Maximal increases in the rate of efflux were observed 1 min following addition of vasopressin (4-fold above basal levels). Activation of the V1 pathway was demonstrated by an increase in inositol trisphosphate (IP3) formation and lack of cAMP accumulation by the cells following the addition of vasopressin. Fluorescent ratio imaging with fura-2 revealed that addition of vasopressin to the cells results in an increase of [Ca2+]i which peaks within 20 s and does not return to resting levels during the 100 s observation period. The addition of a Ca2+ ionophore mimicked the vasopressin-induced efflux from the cells. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and a chloro-substituted compound (cpd 149) inhibited the vasopressin-stimulated 125I efflux from the cells. The concentrations of NPPB and cpd 149 required to inhibit 125I efflux from the cells were similar to those which also attenuated vasopressin-induced Ca2+ transients in the cells. NPPB and cpd 149 had no effects on the ionomycin stimulated efflux. The mechanism(s) by which cpd 149 exerts its effect on stimulated efflux was examined by measuring its action on vasopressin-induced changes in IP3. Compound 149 inhibited IP3 generation in response to vasopressin.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Cálcio/metabolismo , Homeostase/efeitos dos fármacos , Proteínas de Membrana/antagonistas & inibidores , Músculo Liso Vascular/metabolismo , ortoaminobenzoatos/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Linhagem Celular , Canais de Cloreto , Colforsina/farmacologia , AMP Cíclico/metabolismo , Fosfatos de Inositol/metabolismo , Iodo/metabolismo , Radioisótopos do Iodo , Músculo Liso Vascular/efeitos dos fármacos , Nitrobenzoatos/farmacologia , Ratos , Vasopressinas/farmacologia
11.
J Biol Chem ; 267(35): 25444-50, 1992 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-1281159

RESUMO

The effect of a cAMP-dependent secretogogue (VIP) on the phosphorylation of an endogenous, membrane-bound protein (pp170) was assessed in an intact cell preparation from the avian salt gland. The addition of VIP, in the presence of 100 microM isobutylmethylxanthine, resulted in a concentration-dependent increase in phosphorylation of pp170. This effect was rapid and transient with a 3-5-fold increase in phosphorylation occurring 1 min after the addition of VIP. Under similar incubation conditions, VIP stimulated a 4.6-fold increase in cAMP accumulation that paralleled phosphorylation. Exposure of cells to either forskolin or 8-Br-cAMP resulted in a 5-8-fold increase in the phosphorylation of pp170. The effect of forskolin was dose dependent with an EC50 similar to that for stimulation of secretion (35 nM). These results implicate an involvement for a cAMP-dependent protein kinase in the phosphorylation of pp170. The identity of pp170 was assessed utilizing a monoclonal antibody (Q3) directed against pp170. Q3 recognized a single 170-kDa band on Western blots of salt gland membrane protein. Immunoprecipitation of pp170 from salt gland cells resulted in the selective extraction of a single protein whose phosphorylation state was increased approximately 5-fold in response to carbachol or VIP. The identity of pp170 was established using two criteria. First, Q3 recognized affinity-purified Na:K:Cl cotransporter preparations from shark rectal gland membranes. Second, pp170 was selectively immunoprecipitated by monoclonal antibodies (J3, J4, and J7) that recognize different epitopes of the shark transport protein. These results suggest that pp170 is homologous to the shark rectal gland Na-K-Cl cotransporter, and thus the proteins may be functionally similar.


Assuntos
1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , AMP Cíclico/metabolismo , Proteínas de Membrana/metabolismo , Glândula de Sal/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Western Blotting , Carbacol/farmacologia , Proteínas de Transporte/isolamento & purificação , Membrana Celular/metabolismo , Colforsina/farmacologia , Patos , Cinética , Proteínas de Membrana/isolamento & purificação , Fosfatos/metabolismo , Fosforilação , Simportadores de Cloreto de Sódio-Potássio , Fatores de Tempo
12.
Am J Physiol ; 261(3 Pt 1): C543-9, 1991 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-1887875

RESUMO

The effect of cholinergic stimulation of cellular protein phosphorylation was studied using an intact cell preparation isolated from the avian salt gland. Isolated cells were allowed to incorporate 32Pi into the cellular ATP pool and then challenged with compounds known to induce ion secretion in this tissue. Addition of carbachol resulted in a time- and concentration-dependent (EC50 = 500 nM) increase in 32Pi content of a 170-kDa protein (pp170). The stimulated phosphorylation could be blocked by the inclusion of atropine (100 microM). Subcellular fractionation studies localized pp170 to the plasma membrane fraction of the tissue. The integral nature of this protein was demonstrated by detergent-solubilization experiments with Triton X-100. The possibility that carbachol stimulates phosphorylation of pp170 via activation of protein kinase C (PKC) was investigated. Incubating salt gland cells with 4 beta-phorbol 12-myristate 13-acetate (PMA; 1 microM) or carbachol (100 microM) resulted in a translocation of soluble PKC from the cytosol to a plasma membrane fraction. Addition of either PMA (1 microM) or ionomycin (1 microM) alone did not enhance phosphorylation of pp170. A 4.5-fold increase in the phosphorylation state of pp170 was only observed when PMA and ionomycin were added concurrently. Preincubation of salt gland cells with PKC inhibitors H-7 (50 microM) or staurosporine (10 microM) inhibited the carbachol-stimulated phosphorylation of pp170. These findings suggest that carbachol mediates its secretomimetic effects via activation of PKC and that pp170 may represent a novel integral membrane PKC substrate protein.


Assuntos
Carbacol/farmacologia , Fosfatos/metabolismo , Proteínas/metabolismo , Glândula de Sal/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Alcaloides/farmacologia , Animais , Atropina/farmacologia , Patos , Ionomicina/farmacologia , Isoquinolinas/farmacologia , Cinética , Peso Molecular , Fosfoproteínas/isolamento & purificação , Fosforilação , Piperazinas/farmacologia , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases , Glândula de Sal/efeitos dos fármacos , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologia
13.
Mol Endocrinol ; 5(1): 34-41, 1991 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1850109

RESUMO

Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.


Assuntos
Adenilil Ciclases/metabolismo , Receptores Adrenérgicos beta/fisiologia , Neoplasias do Córtex Suprarrenal , Hormônio Adrenocorticotrópico/farmacologia , Animais , Expressão Gênica , Iodocianopindolol , Isoproterenol/farmacologia , Camundongos , Mutação , Pindolol/análogos & derivados , Pindolol/metabolismo , Plasmídeos , Propanolaminas/metabolismo , Receptores Adrenérgicos beta/genética , Fluoreto de Sódio/farmacologia , Transfecção , Células Tumorais Cultivadas
14.
Endocr Res ; 17(1-2): 19-42, 1991.
Artigo em Inglês | MEDLINE | ID: mdl-1679008

RESUMO

Y1 adrenocortical tumor cells (Y1DS) and Y1 mutants resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were transfected with a gene encoding the mouse beta 2-adrenergic receptor (beta 2-AR). Transfectants expressed beta 2-ARs that were able to stimulate adenylyl cyclase activity and steroid biosynthesis. These transfectants were used to explore the basis for the DR mutation in Y1 cells. We demonstrate that beta-adrenergic agonists desensitize the adenylyl cyclase system in transfected Y1DS cells whereas transfected Y1DR cells are resistant to desensitization by beta-adrenergic agonists. The fate of the beta 2-ARs during desensitization was evaluated by photoaffinity labelling with [125I]iodocyanopindolol diazerine. Desensitization of Y1DS transfectants was accompanied by a modest loss in receptor density that was insufficient to account for the complete loss of responsiveness to beta-adrenergic agonists. The extent of receptor loss induced by beta-adrenergic agonists in Y1DR transfectants exceeded that in the Y1DS transfectants indicating that the mutation which protects Y1DR cells from agonist-induced desensitization is prior to receptor down-regulation in the desensitization pathway. From these results we infer that ACTH and isoproterenol desensitize adenylyl cyclase by a common pathway and that receptor loss is not a major component of the desensitization process in these cells.


Assuntos
Adenilil Ciclases/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Agonistas Adrenérgicos beta/farmacologia , Hormônio Adrenocorticotrópico/farmacologia , Iodocianopindolol/análogos & derivados , Isoproterenol/farmacologia , Neoplasias do Córtex Suprarrenal/enzimologia , Neoplasias do Córtex Suprarrenal/ultraestrutura , Animais , Diazometano/análogos & derivados , Radioisótopos do Iodo , Pindolol/análogos & derivados , Receptores Adrenérgicos beta/efeitos dos fármacos , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/enzimologia , Células Tumorais Cultivadas/patologia
15.
Am J Physiol ; 258(6 Pt 1): C1036-43, 1990 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-2163195

RESUMO

This study characterized the various adenosine 3',5'-cyclic monophosphate (cAMP)-binding proteins in epithelial cells cultured from human sweat glands to identify potential pathways of cAMP action in this tissue. The cAMP-binding proteins were identified by specific labeling with the photoprobe 8-azido-[32P]-cAMP and visualized by autoradiography after electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. Three cAMP-binding proteins, with molecular masses of 48, 52, and 54 kDa, were identified in subcellular fractions of the cultured sweat glands. On the basis of their relative electrophoretic mobilities and reactivity with specific antisera, the 48-kDa protein was identified as the regulatory subunit of the type 1 cAMP-dependent protein isozyme, and the 54-kDa protein was identified as the regulatory subunit of the type 2 cAMP-dependent protein kinase isozyme. The 52-kDa cAMP-binding protein appeared to be a distinct isoform of the regulatory subunit, possibly related to the subclass associated with neural tissue. As determined from the relative distributions of the photolabel, the 48-, 52-, and 54-kDa binding proteins were present in the cell cytosol at a ratio of 1:2:1. The 48- and 52-kDa proteins bound the photoprobe with apparent dissociation constants (Kd) of 20-40 nM, whereas the 54-kDa protein bound the photoprobe with much lower affinity (Kd 230 nM). Only the 48- and 52-kDa proteins were associated with cell membranes. They were present in the membrane fractions in approximately equal amounts and bound the photoprobe with apparent Kd values of greater than 100 nM.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Proteínas de Transporte/metabolismo , Proteína Receptora de AMP Cíclico , Glândulas Écrinas/metabolismo , Glândulas Sudoríparas/metabolismo , Marcadores de Afinidade/metabolismo , Azidas/metabolismo , Proteínas de Transporte/isolamento & purificação , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Eletroforese em Gel de Poliacrilamida , Epitélio/metabolismo , Humanos , Cinética , Substâncias Macromoleculares , Peso Molecular , Proteínas Quinases/metabolismo
16.
Biochem Cell Biol ; 66(7): 683-90, 1988 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2846010

RESUMO

Membrane-bound protein kinase C of rat submandibular gland was characterized and the cytosolic kinase C of the tissue was partially purified. The membrane-bound kinase could be activated by Triton X-100 but not EGTA in the presence of both Ca2+ and phosphatidylserine (PS). The Km values for Ca2+ and PS were 150 microM and 5 micrograms, respectively. Addition of 10(-6) M diacylglycerol resulted in an increased affinity of the kinase for Ca2+ (Km = 10 microM). Phorbol 12,13-dibutyrate activated the kinase in the absence of exogenous Ca2+ and PS, suggesting that adequate amounts of each activator are present in the membrane itself. Polymyxin B inhibited the stimulated kinase C activity in a concentration-dependent manner. This inhibition could be overcome by addition of PS. The cytosolic kinase was partially purified 133-fold by chromatography on columns of DEAE-Sephacel and S-300 Sephacryl. The total kinase activity increased with respect to the kinase activity measured in the starting material with column chromatography, suggesting that an inhibitor is present in the cytosolic fraction of the tissue.


Assuntos
Proteína Quinase C/análise , Glândula Submandibular/enzimologia , Animais , Citosol/enzimologia , Membranas Intracelulares/enzimologia , Masculino , Mitocôndrias/enzimologia , Proteína Quinase C/isolamento & purificação , Ratos , Ratos Endogâmicos , ATPase Trocadora de Sódio-Potássio/metabolismo
17.
Biochim Biophys Acta ; 927(3): 392-401, 1987 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-3028497

RESUMO

This study examined the changes in protein phosphorylation in response to cholinergic (muscarinic) stimulation of salivary secretion in the rat submandibular gland. Carbachol stimulation was associated with phosphorylation in a number of protein bands as detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and autoradiography. The molecular masses (Mr) of two proteins, in which the amount of phosphorylation more than doubled in response to carbachol, were 22,000 and 96,000. The Mr 96,000 protein precipitated at 120,000 X g while most of the Mr 22,000 protein remained in the supernatant at this speed. The effect of carbachol on the phosphorylation of the Mr 22,000 and 96,000 proteins was blocked by atropine, indicating that the cholinergic receptor involved is muscarinic. The time course of phosphorylation of the Mr 22,000 protein consisted of a rapid increase in phosphorylation within the first min of carbachol stimulation. This increased phosphorylation persisted for less than 1 min. The increased phosphorylation of the Mr 96,000 protein also occurred within the first min but it persisted for at least 10 min. However, removal of the muscarinic agonist, carbachol, resulted in the rapid dephosphorylation of this protein. When the plasma membranes were purified, the Mr 96,000 protein was phosphorylated by ATP in the presence of Na+ and Mg2+. It was dephosphorylated by K+. This proves that the Mr 96,000 dalton protein is the alpha-subunit of the (Na+ + K+)-ATPase.


Assuntos
Carbacol/farmacologia , Fosfoproteínas/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Glândula Submandibular/metabolismo , Animais , Técnicas In Vitro , Substâncias Macromoleculares , Masculino , Ouabaína/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Fosforilação , Ratos , Ratos Endogâmicos , Glândula Submandibular/efeitos dos fármacos
18.
Can J Biochem Cell Biol ; 63(7): 774-7, 1985 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-2994863

RESUMO

A low molecular weight substance is present in the soluble fraction of the rat submandibular gland which activates Na+,K+-ATPase. Varying amounts of cytosolic protein from rat submandibular gland were added to a heavy microsomal preparation of the same tissue. A dose-dependent activation of the Na+,K+-ATPase was observed, with a peak activation of approximately 82% occurring when 9.0 micrograms/mL of cytosolic protein was included in the assay. The activating factor is heat stable and soluble following heat treatment. The factor elutes at a molecular mass of 600 daltons as determined by molecular sieve column chromatography. Ultraviolet-visible scanning of the elute material results in an absorbance at 210 nm, which is characteristic of a low molecular weight peptide.


Assuntos
Proteínas/fisiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Glândula Submandibular/enzimologia , Animais , Citosol/metabolismo , Estabilidade de Medicamentos , Ativação Enzimática , Temperatura Alta , Cinética , Masculino , Proteínas/isolamento & purificação , Ratos , Ratos Endogâmicos , Solubilidade
19.
Am J Physiol ; 245(3): G364-8, 1983 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-6412565

RESUMO

Oxygen consumption of slices of rat submandibular gland was monitored with an oxygen electrode method. Carbachol stimulated an immediate increase in tissue respiration that was inhibitable by ouabain. The stimulation required the presence of calcium in the incubation medium and was blocked by atropine. The calcium ionophore A23187 also stimulated ouabain-sensitive oxygen consumption in the tissue slices. The results show that the mechanism using the extra energy during cholinergic stimulation is the sodium pump. Amiloride at a 1, 10, or 100 microM concentration had no effect on stimulation of ouabain-sensitive respiration by carbachol. Since amiloride, which is known to block the sodium reabsorption process in the ductal segment, has no effect on the stimulation, the increased sodium pump activity is probably located in the acinar region and is associated with the primary fluid secretion process.


Assuntos
Carbacol/farmacologia , Ouabaína/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Glândula Submandibular/metabolismo , Amilorida/farmacologia , Animais , Atropina/farmacologia , Calcimicina/farmacologia , Relação Dose-Resposta a Droga , Cinética , Masculino , Ratos , Ratos Endogâmicos , Glândula Submandibular/efeitos dos fármacos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA