Expression of glutathione transferase isoenzymes in the human H295R adrenal cell line and the effect of forskolin

In previous studies in our laboratory (L. Mankowitz, L. Staffas, M. Bakke, and J. Lund, Biochem J, 1995, 305, 111-118; L. Staffas, L. Mankowitz, M. S?derstr?m, A. Blanck, I. Porsch-H?llstr?m, C. Sundberg, B. Mannervik, B. Olin, J. Rydstr?m, and J.W. DePierre, Biochem J, 1992, 286, 65-72) isoenzymes of GST, primarily of the mu class, have been shown to be downregulated by adrenocorticotropic hormone (ACTH) in rat and mouse adrenal cells. In the present investigation the human adrenal H295R cell line (W.E. Rainey, I.M. Bird, and J.I. Mason, Mol Cell Endocrinol, 1994, 100, 45-50) was examined in a similar manner. Analysis by reverse-phase HPLC revealed that these cells express four isoenzymes of GST, i.e., A1, A2, P1, and M4, as well as another unidentified protein that was retained by our affinity column (elution time of 32 min) and, thus, presumably binds glutathione. Among these forms, A1 was present at the highest level. Upon addition of forskolin (an activator of adenylate cyclase which has been shown previously to mimic the effect of ACTH on adrenal cells) to the culture medium, the level of A1 decreased approximately 70% by forskolin, whereas the levels of the other isoenzymes were slightly increased, and that of the unknown form doubled. Thus, the influence of ACTH on expression of GST isoenzymes in this human adrenal cell line differs from that in rat and mouse adrenal cells.     

Regulation of Dopamine Release from PC12 Pheochromocytoma Cell Cultures During Stimulation with Elevated Potassium or Carbachol

To examine the role of cyclic AMP in the process of catecholamine release experiments have been performed with cultures of PC12 pheochromocytoma cells. Elevated potassium (56 mM) and carbamylcholine (carbachol, 10(-4) M) cause rapid increases in cyclic AMP levels in the cultures that show a time course similar to that of evoked dopamine release. These secretogogue-induced increases in cyclic AMP levels are well correlated with release in terms of relative magnitude and calcium dependence. Forskolin (a direct activator of adenylate cyclase) causes dose-related increases in cyclic AMP levels in PC12 cell cultures that are synergistic with those caused by either elevated potassium or carbachol. At low concentrations forskolin significantly increases evoked release, whereas at higher concentrations it increases both spontaneous and evoked release. These results suggest that cyclic AMP may be involved in the process of dopamine release from PC12 cells in culture.     

Stimulation-Evoked Ca2+ Fluxes in Cultured Bovine Adrenal Chromaffin Cells Are Enhanced by Forskolin

Forskolin, 1 microM, increased acetylcholine (ACh)-stimulated 45Ca uptake by chromaffin cells. The stimulatory effects of forskolin decreased with increasing concentration of ACh. The attenuation of the effect of forskolin on 45Ca uptake as a function of ACh concentration correlated well with changes in the forskolin effect on ACh-evoked catecholamine (CA) release. Forskolin increased excess KCl- and veratrine-evoked CA release and 45Ca uptake. Forskolin by itself stimulated 45Ca efflux and enhanced ACh-, excess KCl-, and veratrine-stimulated 45Ca efflux. High doses of forskolin inhibited both ACh-evoked 45Ca uptake and CA release. The inhibitory action of forskolin was specific to receptor-mediated response because excess KCl- and veratrine-stimulated 45Ca uptake and CA release were not inhibited. Forskolin, 0.3-30 microM, dose-dependently increased caffeine-stimulated CA release and 45Ca efflux in the absence of Ca2+ in the medium, and the effects were mimicked by dibutyryl cyclic AMP. These results suggest that cyclic AMP increases stimulation-induced CA release by enhancing calcium uptake across the plasma membrane and/or altering calcium flux in an intracellular calcium store.     

Activation of Tyrosine Hydroxylase in PC12 Cells by the Cyclic GMP and Cyclic AMP Second Messenger Systems

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is subject to regulation by a variety of agents. Previous workers have found that cyclic AMP-dependent protein kinase and calcium-stimulated protein kinases activate tyrosine hydroxylase. We wanted to determine whether cyclic GMP might also be involved in the regulation of tyrosine hydroxylase activity. We found that treatment of rat PC12 cells with sodium nitroprusside (an activator of guanylate cyclase), 8-bromocyclic GMP, forskolin (an activator of adenylate cyclase), and 8-bromocyclic AMP all produced an increase in tyrosine hydroxylase activity measured in vitro or an increased conversion of [14C]tyrosine to labeled catecholamine in situ. Sodium nitroprusside also increased the relative synthesis of cyclic GMP in these cells. In the presence of MgATP, both cyclic GMP and cyclic AMP increased tyrosine hydroxylase activity in PC12 cell extracts. The heat-stable cyclic AMP-dependent protein kinase inhibitor failed to attenuate the activation produced in the presence of cyclic GMP. It eliminated the activation produced in the presence of cyclic AMP. Sodium nitroprusside also increased tyrosine hydroxylase activity in vitro in rat corpus striatal synaptosomes and bovine adrenal chromaffin cells. In all cases, the cyclic AMP-dependent activation of tyrosine hydroxylase was greater than that of the cyclic GMP-dependent second messenger system. These results indicate that both cyclic GMP and cyclic AMP and their cognate protein kinases activate tyrosine hydroxylase activity in PC12 cells.     

Is Dopamine-Induced Inhibition of Adenylate Cyclase Involved in the Autoreceptor-Mediated Negative Control of Tyrosine Hydroxylase in Striatal Dopaminergic Terminals?

Abstract The mechanism of the negative control of tyrosine hydroxylase (TH) activity induced by the stimulation of presynaptic 3,4-dihydroxyphenylethylamine (dopamine, DA) autoreceptors was investigated using rat striatal slices and synaptosomes incubated under control ([K⁺] = 4.8 mM) or depolarizing ([K⁺] = 60 mM) conditions. The stimulation of DA autoreceptors by 7-hydroxy-2-(di-n-propylamino) tetralin (1 μM 7-OH-DPAT) produced a significant decrease in TH activity extracted from striatal slices maintained under control conditions. This effect was associated with the complete conversion of TH into an enzyme form with a low affinity for its pterin cofactor (K_m～0.80 mM). Furthermore, compared to TH extracted from control tissues, that from 7-OH-DPAT-exposed striatal slices was more sensitive to the stimulatdry effects of exogenous heparin and cyclic AMP-dependent phosphorylation. Such changes were opposite to those induced by incubating striatal slices with the adenylate cyclase activator forskolin. Indeed, forskolin treatment completely converted TH into an enzyme form with a high affinity for its pterin cofactor (K_m～0.16 mM). Such conversion was associated with a shift in the optimal pH for TH activity from 5.8 (control) to 7.2 (forskolin). Under depolarizing conditions, the blockade by (—)-sulpiride of the stimulation of DA autoreceptors by endogenous DA was associated with a marked activation of TH. Modifications of enzymatic characteristics triggered by (—)-sulpiride were then similar to those induced by forskolin treatment. These data suggest that presynaptic DA autoreceptors modulate the activity of TH by controlling the degree of cyclic AMP-dependent phosphorylation of the enzyme. The blockade by Pertussis toxin of the 7-OH-DPAT-induced inhibition of TH activity is coherent with a possible negative coupling of presynaptic DA autoreceptors (closely related to the D₂ type) with adenylate cyclase. Such negative coupling would account for the reduction of TH activity when presynaptic DA autoreceptors are stimulated.     

Phosphorylation of DARPP-32 Is Regulated by GABA in Rat Striatum and Substantia Nigra

Abstract: In the medium-sized spiny neurons of the striatonigral pathway, a cascade of events involving the activation of dopamine D₁ receptors, an increase in cyclic AMP, and activation of cyclic AMP-dependent protein kinase causes the phosphorylation of DARPP-32 on Thr³⁴, converting DARPP-32 into a powerful inhibitor of protein phosphatase-1. In the present study, the incubation of striatal or substantia nigra slices with GABA also increased the phosphorylation of DARPP-32 on Thr³⁴. GABA did not significantly increase cyclic AMP levels in slices. The phosphorylation of DARPP-32 by GABA was blocked in both brain regions by pretreatment of slices with the GABA_A receptor antagonist, bicuculline, but not with the GABA_B receptor antagonist, phaclofen. Moreover, the threonine phosphorylation of DARPP-32 produced by maximally effective doses of either forskolin (in striatum) or l -3,4-dihydroxyphenylalanine (in substantia nigra) was increased further by GABA. The data are consistent with a model in which GABA increases the phosphorylation state of DARPP-32 by inhibiting dephosphorylation of the protein by the calcium/calmodulin-dependent protein phosphatase, calcineurin.     

毛喉萜对小鼠骨髓细胞和腹腔巨噬细胞表面胰岛素受体和GM－CSF受体的下调作用

放射性标记受体分析表明毛喉萜可以降低小鼠骨央细胞和腹腔巨噬细胞表面的胰岛素和粒细胞－巨噬细胞集落刺激因子受体数目,而且对ＧＭ－ＣＳＦ的作用有剂量和时间依赖性,经ＦＳＫ处理的巨噬细胞酸性磷酸酶活性增加,微丝更加舒展。     

小牛睾丸中G蛋白对FSH受体亲和性和腺苷酸环化酶活性的调节

本文用NEM(N-ethylmaleimide)为探针研究了G蛋白(鸟嘌呤核苷酸调节蛋白,Gp)对小牛睾丸中FSH受体的亲和性及腺苷酸环化酶活性的调节作用。证据表明,①在小牛睾丸细胞膜G蛋白上存在两种类型鸟嘌呤核苷酸结合位点(下简称GTP结合位点),高亲和性低容量结合位点及低亲和性高容量结合位点;②高亲和性结合位点(对NEM敏感)调节腺苷酸环化酶活性,而对NEM相对不敏感的低亲和性位点则不直接参与该酶活性的调节;③G蛋白对受体亲和性的调节则不仅要高亲和性位点的参与,而且主要受低亲和性位点的调节。     

Regulation of Cyclic AMP by the μ-Opioid Receptor in Human Neuroblastoma SH-SY5Y Cells

The human neuroblastoma clonal cell line SH-SY5Y expresses both mu- and delta-opioid receptors (ratio approximately 4.5:1). Differentiation with retinoic acid (RA) was previously shown to enhance the inhibition of adenylyl cyclase (AC) by mu-opioid agonists. We tested here the inhibition of cyclic AMP (cAMP) accumulation by morphine under a variety of conditions: after stimulation with prostaglandin E1 (PGE1), forskolin, and vasoactive intestinal peptide (VIP), both in the presence and in the absence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Morphine inhibition of the forskolin cAMP response (approximately 65%) was largely unaffected by the presence of IBMX. In contrast, deletion of IBMX enhanced morphine's inhibition of the PGE1 and VIP cAMP response from approximately 50 to approximately 80%. The use of highly mu- and delta-selective agents confirmed previous results that inhibition of cAMP accumulation by opioids is mostly mu, and not delta, receptor mediated in SH-SY5Y cells, regardless of the presence or absence of IBMX. Because of the large morphine inhibition and the high cAMP levels even in the absence of IBMX, PGE1-stimulated, RA-differentiated SH-SY5Y cells were subsequently used to study narcotic analgesic tolerance and dependence in vitro. Upon pretreatment with morphine over greater than or equal to 12 h, a fourfold shift of the PGE1-morphine dose-response curve was observed, whether or not IBMX was added. However, mu-opioid receptor number and affinity to the mu-selective [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin were largely unaffected, and Na(+)- and guanyl nucleotide-induced shifts of morphine-[3H]naloxone competition curves were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Phorbol Esters and Forskolin on Basal and Histamine-induced Accumulation of Inositol Phosphates in Cultured Bovine Adrenal Chromaffin Cells

The effect of phorbol esters and forskolin pretreatment on basal and histamine-induced accumulation of inositol phosphates and catecholamine release was examined in cultures of bovine adrenal chromaffin cells. Histamine caused a dose-dependent, Ca2+-dependent accumulation of total inositol phosphates with an EC50 at approximately 1 microM and an eight- to 10-fold increase at 100 microM within 30 min of incubation. Histamine (10 microM) also caused the release of cellular catecholamines amounting to some 2.8% of cellular stores released over a 20-min period. Both the inositol phosphate and catecholamine responses were completely blocked by the H1-antagonist mepyramine and were insensitive to the H2-antagonist cimetidine. Examination of the time course of accumulation of the individual inositol phosphates stimulated by histamine revealed an early and sustained rise in inositol 1,4-bisphosphate content but not inositol 1,4,5-trisphosphate content at 1 min and the overall largest accumulation of inositol monophosphate after 30 min of stimulation. Pretreatment with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a dose-dependent, time-dependent inhibition of histamine-induced inositol phosphate formation and catecholamine secretion. In this inhibitory action, PMA exhibited high potency (IC50 of approximately 0.5 nM), an effect not shared by the inactive phorbol ester 4-alpha-phorbol 12,13-didecanoate. Pretreatment with forskolin, on the other hand, only marginally inhibited the histamine-induced inositol phospholipid metabolism and catecholamine secretion. These data suggest that protein kinase C activation in chromaffin cells may mediate a negative feedback control on inositol phospholipid metabolism.