RESUMO
Local TNF-alpha production in different organs may affect HIV replication and pathogenesis. Alveolar macrophages (AMs) obtained by bronchoalveolar lavage from asymptomatic HIV-seropositive and HIV-seronegative individuals did not spontaneously release TNF-alpha, but LPS stimulation of these cells significantly increased TNF-alpha production. We tested whether NF-kappa B affects TNF-alpha production by AMs using N-tosyl-
Assuntos
Soropositividade para HIV/imunologia , Proteínas I-kappa B , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , NF-kappa B/fisiologia , Fator de Necrose Tumoral alfa/biossíntese , Adulto , Adesão Celular/genética , Adesão Celular/imunologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidor de NF-kappaB alfa , NF-kappa B/genética , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase , Marcação in Situ com Primers , Estudos Prospectivos , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , Inibidores de Serina Proteinase/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Tosilfenilalanil Clorometil Cetona/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fosfolipases Tipo C/antagonistas & inibidores , Tirosina/análogos & derivados , Tirosina/farmacologiaRESUMO
Complementary DNA encoding a rat bone PTH/PTHrP receptor was stably expressed in the murine corticotroph cell line, AtT-20. Several clones, expressing variable numbers of PTH/PTHrP receptors, were developed. In contrast to the relatively low binding affinity (apparent Kd = 15 nM) observed in COS-7 cells transiently expressing the PTH/PTHrP receptor, all AtT-20 stable transfectants bound [Nle8,18,Tyr34]bPTH(1-34)NH2 (NlePTH) with an affinity that was indistinguishable from that observed in ROS 17/2.8 cells expressing native PTH/PTHrP receptors. Additionally, NlePTH dramatically increased cAMP accumulation and ACTH release in AtT-20 cells expressing the PTH/PTHrP receptor with an ED50 of 0.6 +/- 0.3 and 0.3 +/- 0.1 nM, respectively. The high binding affinity and the high efficacy of NlePTH in stimulating cAMP accumulation and ACTH release indicate that the PTH/PTHrP receptor is efficiently coupled to the intracellular signalling system responsible for stimulation of ACTH release in AtT-20 cells. No additivity of cAMP accumulation or of ACTH release was observed when these cells were treated with maximally active concentrations of both NlePTH and CRF. This suggests that the receptors for both of these hormones share the same intracellular effectors, and that intracellular signaling in AtT-20 cells is not compartmentalized. Additionally, the ability of NlePTH to stimulate ACTH release in AtT-20 cells, a function that is normally performed by CRF, demonstrates promiscuity between activated receptors and distal biological functions.
Assuntos
Hormônio Adrenocorticotrópico/metabolismo , AMP Cíclico/metabolismo , Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/farmacologia , Proteínas/metabolismo , Receptores de Superfície Celular/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Linhagem Celular , Hormônio Liberador da Corticotropina/farmacologia , Relação Dose-Resposta a Droga , Biblioteca Gênica , Cinética , Camundongos , Proteína Relacionada ao Hormônio Paratireóideo , Neoplasias Hipofisárias , Ratos , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/genética , Receptores de Hormônios Paratireóideos , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Transfecção , Células Tumorais CultivadasRESUMO
The osteoblast-like cells, UMR 106-01, express PTH receptors that are coupled to adenylate cyclase. Recently, we reported the isolation of a UMR 106-01 subclone, UMR 4-7, that is stably transfected with a Zn(++)-inducible mutant of the regulatory subunit of protein kinase A. Incubation of UMR 4-7 cells with Zn++ renders the cells unresponsive to cAMP agonists. This subclone, therefore, seemed particularly suitable for studies of PTH receptor regulation. In UMR 106-01 cells, PTH receptors are strikingly down-regulated by pretreatment with 8-Br-cAMP or 3-isobutyl-1-methylxanthine for 2 days. In UMR 4-7 cells, this effect is totally prevented by prior and concurrent treatment with Zn++. Zn++ addition to UMR 106 cells does not modify these responses. Treatment with the PTH agonist [Nle8,18,Tyr34]bovine PTH(1-34)NH2 [(NlePTH(1-34)] also markedly down-regulates PTH receptors in UMR 106 cells, but this effect is only partially inhibited in Zn(++)-induced UMR 4-7 cells. At high doses, the PTH antagonist, [Nle8,18,Tyr34]bovine PTH(3-34)NH2 [NlePTH(3-34)] also (partially) reduces PTH receptor availability. Receptor regulation by NlePTH(3-34) is not blocked in the cAMP-resistant cells, however. Coincubation of submaximal doses of NlePTH(1-34) (1 nM) with NlePTH(3-34) (1 microM) reduces receptor availability more than when the cells are exposed to either ligand alone. This decrease is only partially inhibited in Zn(++)-induced UMR 4-7 cells. In contrast to its additive effect on receptor regulation, NlePTH(3-34) efficiently competes for binding to the PTH receptor in UMR 106-01 cells and antagonizes the stimulatory effects of NlePTH(1-34) on both intracellular cAMP accumulation and gene expression driven by a transiently transfected synthetic cAMP-responsive enhancer. In conclusion, homologous down-regulation of PTH receptors is mediated by activation of both cAMP-dependent (via protein kinase A) and cAMP-independent pathways. PTH activates both pathways, whereas the effect of NlePTH(3-34) appears to be exclusively cAMP-independent. These results give new insights into mechanisms of PTH receptor regulation.