Involvement of ion channels in the induction of proenkephalin A gene expression by nicotine and cAMP in bovine chromaffin cells

The involvement of Na+ and Ca2+ channels in the stimulatory effect of nicotine and cAMP upon proenkephalin A mRNA (mRNA ENK) levels in primary cultures of bovine adrenal chromaffin cells was analyzed. Nicotine (10 microM) caused about a 2-3-fold increase in mRNA ENK which was abolished by the nicotinic receptor antagonist tubocurarine (4 X 10(-7) M), inhibited by the Ca2+ channel antagonist nifedipine (100 nM) abolished by the Ca2+ channel blocker D600 (10 microM), and augmented by the Ca2+ channel agonist BayK 8644 (100 nM). In contrast, blockade of the Na+ channel by tetrodotoxin (1 microM) did not modulate the nicotine-induced increase in mRNA ENK. Incubation of the cells with forskolin (25 microM) and 8-bromo-cAMP (1 mM) also resulted in an increase in mRNA ENK levels that was inhibited by the Ca2+ channel blocker verapamil (50 microM) and nifedipine (100 nM), whereas it was enhanced by BayK 8644 (100 nM). In addition, the effect of forskolin and 8-bromo-cAMP was decreased by the Na+ channel blocker tetrodotoxin (1 microM). These results suggest that the induction of proenkephalin A gene expression by cAMP and nicotine involves the modulation of ion channels. It appears that changes in Ca2+ flux are involved in mediating this induction. The dihydropyridines nifedipine and BayK 8644 and the Ca2+ channel blockers verapamil and D600 all modulate 45Ca uptake. In addition, we show that incubation of the cells with A23187 (10(-7) M), a Ca2+ ionophore, resulted in an increase in mRNA ENK, indicating that changes in intracellular Ca2+ levels may indeed modulate proenkephalin A gene expression. Although it appears that an elevation of mRNA ENK upon nicotinic receptor activation occurs rapidly (an increase could be detected after 2 h incubation), the findings that the rise in mRNA ENK could be abolished by the Ca2+ channel blocker D600 but not affected by tetrodotoxin (1 microM), and that agents such as KCl (20 mM) and veratridine (5 microM) that increase mRNA ENK by activation of voltage-dependent Ca2+ channels do not result in an increase in intracellular cAMP, provide no evidence for a major role of the adenylate cyclase system in the inducing effect of nicotine upon proenkephalin A gene expression.     

Differentiation to a neuronal phenotype in bovine chromaffin cells is repressed by protein kinase C and is not dependent on c-fos oncoproteins

We investigated the intracellular signals underlying the neurotrophic response of adult bovine chromaffin cells to histamine and basic fibroblast growth factor (bFGF). Histamine produced significant neurite outgrowth within 48 hr, whereas the response to bFGF developed after 1 week. H7, a protein kinase C (PKC) inhibitor potentiated both the histamine and the bFGF responses, while another PKC antagonist, staurosporine, induced a rapid and efficient differentiation response when applied alone. These observations suggest that basal PKC activity is required for stabilization of the endocrine phenotype in these cells. They contrast with findings on NGF induction of neurite outgrowth in PC12 cells where PKC promotes differentiation, apparently by activating the fos/jun complex. Thus, we examined the role of c-fos in our model. Both histamine and bFGF induced c-fos gene expression transiently. To determine whether increased levels of c-fos oncoprotein were essential to the differentiation process, we used a hybrid arrest approach employing an innovative transfection technique applicable to primary culture systems. Transfection with plasmid pSVsof, producing antisense c-fos mRNA, reduced c-fos oncoprotein levels but did not diminish histamine-induced neurite outgrowth. We infer that histamine-induced differentiation in bovine chromaffin cells is independent of increased levels of c-fos oncoprotein.     

Up-regulation of fibroblast growth factor (FGF) receptor mRNA levels by basic FGF or testosterone in androgen-sensitive mouse mammary tumor cells.

Since we had previously shown that both basic fibroblast growth factor (bFGF) and testosterone stimulate the growth of mouse mammary carcinoma cells (SC-3) in serum-free culture, we tested the effect of bFGF or testosterone on FGF receptor mRNA levels. Northern blot analyses revealed that stimulation with bFGF resulted in a 5-fold increase in FGF receptor mRNA levels at 6-8 h followed by a decline to the unstimulated levels at 24 h. Simultaneous addition of cycloheximide blocked bFGF-induced accumulation of FGF receptor mRNA, although exposure of SC-3 cells to cycloheximide alone caused marginal increase in its basal level. Neither phorbol ester nor forskolin stimulated FGF receptor mRNA expression, but testosterone could raise FGF receptor mRNA levels. To obtain the maximum stimulation, however, testosterone required the longer stimulation period (12 h) than bFGF, suggesting that testosterone-induced FGF receptor mRNA accumulation is mediated through an induction of FGF-like growth factor.     

Importance of histamine in the cytokine network in the lung through H2 and H3 receptors: stimulation of IL-10 production

Histamine, a well-known inflammatory mediator, has been implicated in various immunoregulatory effects that are poorly understood. Thus, we tested the hypothesis that histamine inhibits the release of a proinflammatory cytokine, namely TNF, by stimulating the release of an anti-inflammatory cytokine, IL-10. Alveolar macrophages (AMs) from humans, Sprague Dawley rats, and the AM cell line, NR8383, were treated with different concentrations of histamine (10-5-10-7 M) for 2 h prior to their stimulation with suboptimal concentration of LPS (1 ng/ml) for 4 h. Histamine inhibited TNF release in a dose-dependent manner. This inhibition was mimicked by H2 and H3 receptor agonists, but not by H1 receptor agonist. Furthermore, we demonstrated the expression of H3 receptor mRNA in human AMs. Interestingly, treatment of AMs with anti-IL-10, anti-PGE2, or a NO synthase inhibitor (Nomega-nitro-l -arginine methyl ester) before the addition of histamine abrogated the inhibitory effect of the latter on TNF release. Histamine treatment (10-5 M) increased the release of IL-10 from unstimulated (2.2-fold) and LPS-stimulated (1. 7-fold) AMs. Unstimulated AMs, NR8383, express few copies of IL-10 mRNA, as tested by quantitative PCR, but expression of IL-10 was increased by 1.5-fold with histamine treatment. Moreover, the stimulation of IL-10 release by histamine was abrogated by pretreatment with anti-PGE2 or the NO synthase inhibitor, Nomega-nitro-l -arginine methyl ester. Thus, histamine increases the synthesis and release of IL-10 from AMs through PGE2 and NO production. These results suggest that histamine may play an important role in the modulation of the cytokine network.