RESUMEN
The neuropeptide CAP2b stimulates fluid transport obligatorily via calcium entry, nitric oxide, and cGMP in Drosophila melanogaster Malpighian (renal) tubules. We have shown by RT-PCR that the Drosophila L-type calcium channel alpha1-subunit genes Dmca1D and Dmca1A (nbA) are both expressed in tubules. CAP2b-stimulated fluid transport and cytosolic calcium concentration ([Ca2+]i) increases are inhibited by the L-type calcium channel blockers verapamil and nifedipine. cGMP-stimulated fluid transport is verapamil and nifedipine sensitive. Furthermore, cGMP induces a slow [Ca2+]i increase in tubule principal cells via verapamil- and nifedipine-sensitive calcium entry; RT-PCR shows that tubules express Drosophila cyclic nucleotide-gated channel (cng). Additionally, thapsigargin-induced [Ca2+]i increase is verapamil sensitive. Phenylalkylamines bind with differing affinities to the basolateral and apical surfaces of principal cells in the main segment; however, dihydropyridine binds apically in the tubule initial segment. Immunocytochemical evidence suggests localization of alpha1-subunits to both basolateral and apical surfaces of principal cells in the tubule main segment. We suggest roles for L-type calcium channels and cGMP-mediated calcium influx in both calcium signaling and fluid transport mechanisms in Drosophila.
Asunto(s)
Canales de Calcio/fisiología , Calcio/metabolismo , Proteínas de Drosophila , Proteínas de Insectos/fisiología , Túbulos Renales/metabolismo , Neuropéptidos/metabolismo , Oligopéptidos/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , GMP Cíclico/farmacología , Drosophila melanogaster , Inhibidores Enzimáticos/farmacología , Proteínas de Insectos/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Túbulos Renales/citología , Neuropéptidos/efectos de los fármacos , Nifedipino/farmacología , Oligopéptidos/efectos de los fármacos , Ácido Pirrolidona Carboxílico/análogos & derivados , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tapsigargina/farmacología , Verapamilo/farmacologíaRESUMEN
Brca1 mRNA was detectable in female mouse mammary gland tissue from adult virgins, during pregnancy and early lactation. It was associated with phases of mammary epithelial cell proliferation and differentiation but the pattern of Brca1 expression was dissociable from that of a true differentiation marker, beta-casein, by virtue of its significant expression in the virgin gland and termination of its expression in early lactation. In a primary cell culture model, association of a laminin-rich extracellular matrix (ECM) with mammary epithelial cells was required for cell survival and cell differentiation and suppressed Brca1 expression in these cells. ECM-association may significantly contribute to the final restriction in Brca1 expression in the lactating gland in vivo. Interestingly, our results suggest that mammary epithelial cells undergo apoptosis both when expressing and when not expressing Brca1, depending on whether the dying cell populations had been terminally differentiated or not.
Asunto(s)
Apoptosis/fisiología , Proteína BRCA1/genética , Células Epiteliales/fisiología , Matriz Extracelular/fisiología , Regulación de la Expresión Génica , Genes BRCA1 , Laminina/fisiología , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/fisiología , Animales , Diferenciación Celular , Supervivencia Celular , Células Epiteliales/citología , Femenino , Lactancia , Ratones , Reacción en Cadena de la Polimerasa , EmbarazoRESUMEN
Mammary epithelial cells in primary cell culture require both growth factors and specific extracellular matrix (ECM)-attachment for survival. Here we demonstrate for the first time that inhibition of the ECM-induced Erk 1/Erk 2 (p42/44 MAPK) pathway, by PD 98059, leads to apoptosis in these cells. Associated with this cell death is a possible compensatory signalling through the p38 MAP kinase pathway the inhibition of which, by SB 203580, leads to a more rapid onset of apoptosis. This provides evidence for a hitherto undescribed Erk 1/Erk 2 to p38 MAP kinase pathway 'cross-talk' that is essential for the survival of these cells. The cell death associated with inhibition of these two MAP kinase pathways however, occurred in the presence of insulin that activates the classical PI-3 kinase-dependent Akt/PKB survival signals and Akt phosphorylation. Cell death induced by inhibition of the MAP kinase pathways did not affect Akt phosphorylation and may, thus, be independent of PI-3 kinase signalling.
Asunto(s)
Apoptosis , Matriz Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Glándulas Mamarias Animales/citología , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular , Células Cultivadas , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Femenino , Flavonoides/farmacología , Imidazoles/farmacología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piridinas/farmacología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The leucokinin (LK) family of neuropeptides has been found widely amongst invertebrates. A member of this family was purified from adults of the fruit fly Drosophila melanogaster. The peptide sequence for Drosophila leucokinin (DLK) was determined as Asn-Ser-Val-Val-Leu-Gly-Lys-Lys-Gln-Arg-Phe-His-Ser-Trp-Gly-amide, making it the longest member of the family characterized to date. Synthetic DLK peptide was shown to act to stimulate fluid secretion in D. melanogaster Malpighian (renal) tubules by approximately threefold, with an EC(50) of approximately 10(-)(10 )mol l(-)(1), and a secondary effect at approximately 10(-)(7 )mol l(-)(1). DLK also acted to elevate intracellular [Ca(2+)] in the Malpighian tubules by approximately threefold, with an EC(50) of 10(-)(10) to 10(-)(9 )mol l(-)(1). Responses were detected in stellate cells and occasionally in principal cells, although at no concentration tested did [Ca(2+)] in the principal cell increase significantly above background. In stellate cells, DLK produced a biphasic rise in intracellular [Ca(2+)] from resting levels of 80-100 nmol l(-)(1), with a transient peak being followed by a slower rise that peaked at 200-300 nmol l(-)(1) after 3 s, then decayed over approximately 10 s. The wide range of concentrations over which DLK acts suggests the involvement of more than one receptor. The genomic sequence encoding the DLK peptide has been identified, and the gene has been named pp. The gene resides at cytological location 70E3-70F4 of chromosome 3L. The localisation of this first Drosophila LK gene in a genetic model permits a genetic analysis of the locus.