Specific combinations of G-protein subunits discriminate hormonal signalling in rat pituitary (GH(3)) cells in culture.

It was previously shown that hormone receptor coupling to voltage-dependent calcium channels in prolactin and growth hormone-producing GH(3) cells was heavily dependent on the specific heterotrimeric combinations of alpha, beta, and gamma subunits of the guanosine triphosphate (GTP)-binding protein family. Consequently, we assessed whether this was also the case for hormonal modulation of the adenylate cyclase (AC) and phospholipase C (PL-C) effector enzymes in GH(3) cells in culture. By employing polyclonal antibodies directed towards C-terminal decapeptides of various alpha subunits in membrane assays, as well as antisense oligonucleotides towards certain beta- and gamma-subunit genes in whole-cell incubations, it was possible to unravel a tentative profile of heterotrimers preferred by some of the seven-transmembrane-stretch receptors in their modulation of AC and PL-C activities. Vasoactive intestinal peptide (VIP) and thyroliberin (TRH) activate membrane-bound AC through alpha(s)beta(2)gamma(2), while somatostatin (SRIH) and dopamine (DA) inhibited the AC through alpha(i2)beta(1)gamma(3). TRH activated membrane-bound PL-C through alpha(q/11)beta(4)gamma(2), while DA inhibition of the PL-C was accomplished via alpha(o)beta(3)gamma(4). Hence, it seems that not only the specificity of alpha subunits determines the coupling between G protein-associated receptors in GH cells, the receptor binding to G proteins also requires certain combinations of beta and gamma subunits.

Diverse expression of G-proteins in human sarcoma cell lines with different osteogenic potential: evidence for the involvement of Gi2 in cell proliferation.

Previously, it has been shown that the GTP-binding protein Gi2 is implicated in cellular growth [1,2] and differentiation [2,3]. In the present paper we demonstrate that this is also the case for human sarcoma cells. Six human osteosarcoma and three soft tissue sarcoma clonal cell lines were analyzed for levels of G-protein mRNA and polypeptide expression and effector enzyme (i.e., adenylate cyclase and phospholipase C) activation, which were all compared with individual growth rates. Unexpectedly, it appeared that the various strains exhibited large inter-individual variations in G-protein expression and signaling system activation. However, cell doubling time in the exponential phase of growth was inversely correlated (r = 0.71, P < 0.05) to immunodetected levels of intrinsic Gi2 alpha. Furthermore, cells stably transfected with a retroviral (pZipNeo(SV)X) construct containing the activating or inactivating Gi2 alpha-R179E or Gi2 alpha-G204A point mutations consistently reduced or enhanced individual cell strain doubling time, respectively. It appeared that other parameters investigated, including cellular alkaline phosphatase and monoclonal antibody epitope binding, both being markers of the proliferating osteoblast, did not correlate with cell doubling times.

Direct effects of vitamin D3 analogues on G-protein mediated signalling systems in rat osteosarcoma cells and rat pituitary adenoma cells.

In normal rats treated with 1,25(OH)2D3 or 24,25(OH)2D3, serum Ca2+, ALP, PRL and GH are significantly altered. In order to study the primary effect of vitamin D3 analogues on target organ function, rat UMR 106 osteosarcoma and GH3 pituitary adenoma cells in monolayer culture were exposed accordingly. Surprisingly, prolonged exposure of these cell lines to physiological levels of either 1,25(OH)2D3 or 24,25(OH)2D3 did not significantly affect the secretory parameters (ALP, PRL or GH) tested. However, 1,25(OH)2D3 exposure significantly reduced PTH- and Gpp(NH)p-elicited AC as well as Gpp(NH)p-stimulated PLC activities in the UMR 106 cells. These changes were accompanied by an increase and decrease in the membrane contents of the G-protein subunits G36 beta and Gq/11 alpha, respectively. In contrast, 24,25(OH)2D3 remained without significant biological effect on these signalling systems despite concomitantly augmented levels of G36 beta. TRH- and Gpp(NH)p-elicited PLC activities in the GH3 cells were significantly reduced by 1,25(OH)2D3 with a concurrent reduction in cellular amounts of Gq/11 alpha, however, 24,25(OH)2D3 did not significantly alter any signalling systems nor G-proteins analyzed. It is concluded that the osteoblastic and pituitary cell secretion of ALP, PRL and GH remain unaffected by the presence of 1,25(OH)2D3 and 24,25(OH)2D3, despite distinct alterations in components of G-protein mediated signalling pathways. Hence, other factors like ambient Ca2+ may be responsible for the perturbed secretory patterns of ALP and PRL seen in vitamin D3 treated rats.

Correlation of the chemical structure of 4-nitroquinolines inactivating human cytomegalovirus and established in vivo carcinogenicity tests.

Inactivation of the infectivity of human cytomegalovirus (CMV) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) has been observed following exposure to 4-nitroquinoline 1-oxide (NQO) or its metabolite, 4-hydroxyaminoquinoline 1-oxide (HAQO). The present study of the specificity of the chemical structure of 4-nitroquinolines demonstrated that both the 4-nitro and 1-oxide groups were required for inactivation of virus infectivity. Reduction of the 4-nitro group to a 4-hydroxyamino group enhanced activity, while further reduction to an amino group resulted in loss of activity against virus infectivity. The capacity to inactivate virus was also lost by substitution of the pyridine ring for the quinoline nucleus of NQO. The relationship between the chemical structure and the ability to inactivate viruses studied here correlates well with earlier in vivo carcinogenicity studies of the same group of chemicals.