Enhancement of Stimulation-Evoked Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells by Forskolin

Acetylcholine (ACh) increased cyclic AMP levels in cultured bovine chromaffin cells with a peak effect at 1 min after the addition. Pretreatment with forskolin (0.3 microM) enhanced the ACh-evoked cyclic AMP increase. The catecholamine (CA) release induced by ACh was enhanced by forskolin, but forskolin alone did not enhance the CA release. The effect of forskolin increased dose-dependently up to 1 microM, but decreased at higher concentrations. Dibutyryl cyclic AMP (DBcAMP) also enhanced ACh-evoked CA release, but the effect was less potent than that of forskolin. Forskolin enhanced both [3H]norepinephrine ([3H]NE) and endogenous CA release evoked by 30 mM K+ from cells that were preloaded with [3H]NE. The effects of forskolin were substantial when CA release was evoked with low concentrations of ACh or excess K+, but decreased with higher concentrations of the stimulants. Forskolin also enhanced the CA release induced by ionomycin and veratrine, or by caffeine in Ca2+-free medium. The potentiation by forskolin of the ACh-evoked CA release was manifest in low Ca2+ concentrations in the medium, but decreased when Ca2+ concentration was increased. These results suggest that cyclic AMP may play a role in the modulation of CA release from chromaffin cells.     

Characteristics of Tyrosine Hydroxylase Activation by K+-Induced Depolarization and/or Forskolin in Rat Striatal Slices

The mechanisms of tyrosine hydroxylase (TH) activation by depolarization or exposure of dopaminergic terminals to cyclic AMP have been compared using rat striatal slices. Tissues were incubated with veratridine or 60 mM K+ (depolarizing conditions), on the one hand, and forskolin or dibutyryl cyclic AMP, on the other. K+-(or veratridine-)induced depolarization triggered an activation of TH (+75%) that persisted in soluble extracts of incubated tissues. This effect disappeared when drugs (EGTA, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, Gallopamil) preventing Ca2+- and calmodulin-dependent processes were included in the incubating medium. In contrast, prior in vivo reserpine treatment or in vitro addition of benztropine did not affect the depolarization-induced activation of TH. In vitro studies of soluble TH extracted from depolarized tissues indicated that activation was associated with a marked increase in the enzyme Vmax but with no change in its apparent affinity for the pteridin cofactor 6-methyl-5,6,7,8-tetrahydropterin (6-MPH4) or tyrosine. Furthermore, the activated enzyme from depolarized tissues exhibited the same optimal pH (5.8) as native TH extracted from control striatal slices. In contrast, TH activation resulting from tissue incubation in the presence of forskolin or dibutyryl cyclic AMP was associated with a selective increase in the apparent affinity for 6-MPH4 and a shift in the optimal pH from 5.8 to 7.0-7.2. Clear distinction between the two activating processes was further confirmed by the facts that heparin- and cyclic AMP-dependent phosphorylation stimulated TH activity from K+-exposed (and control) tissues but not that from striatal slices incubated with forskolin (or dibutyryl cyclic AMP). In contrast, the latter enzyme but not that from depolarized tissues could be activated by Ca2+-dependent phosphorylation. These data strongly support the concept that Ca2+- but not cyclic AMP-dependent phosphorylation is responsible for TH activation in depolarized dopaminergic terminals.     

Adenosine deaminase normalizes cyclic AMP responses of hypothyroid rat fat cells to forskolin, but not beta-adrenergic agonists

Lipolysis and cyclic AMP accumulation in response to beta-adrenergic agonists or forskolin are severely impaired in fat cells from the hypothyroid rat. Incubating hypothyroid rat fat cells with adenosine deaminase normalizes the cyclic AMP response to forskolin, but not to beta-adrenergic agonists. Increased sensitivity to adenosine action in the hypothyroid state appears to be the basis for the impaired cyclic AMP response to forskolin, but does not appear to be the underlying defect responsible for the impaired response to beta-adrenergic agonists.     

Inhibition of forskolin-stimulated cardiac adenylate cyclase activity by short-chain alcohols

The diterpene forskolin stimulated rat cardiac adenylate cyclase activity at least 20-fold and potentiated the effect of NaF. The stimulatory effect of forskolin was reduced in the presence of Gpp(NH)p. Ethanol markedly reduced the stimulation of adenylate cyclase by forskolin while potentiating NaF and Gpp(NH)p stimulation. The inhibitory effect of ethanol on forskolin stimulation appeared to be of a mixed type with both a competitive and a non-competitive component. Three other short-chain linear alcohols (methanol, propanol, butanol) also inhibited forskolin-stimulation, this effect being proportional to the number of carbon atoms.     

Muscarinic Receptor-Stimulated Phosphoinositide Turnover in Human SK-N-SH Neuroblastoma Cells: Differential Inhibition by Agents that Elevate Cyclic AMP

The possibility that an increased intracellular concentration of cyclic AMP (cAMP) can regulate the extent of muscarinic receptor-stimulated phosphoinositide (PPI) turnover in the human neuroblastoma cell line SK-N-SH was examined. Addition of either forskolin (or its water-soluble analog, L-85,8051), theophylline, isobutylmethylxanthine, or cholera toxin, agents that interact with either the catalytic unit of adenylate cyclase, cAMP phosphodiesterase, or the guanine nucleotide binding protein linked to adenylate cyclase activation, resulted in a 45-181% increase in cAMP concentration and a 27-70% inhibition of carbachol-stimulated inositol phosphate release. Through the use of digitonin-permeabilized cells, the site of inhibition was localized to a step at, or distal to, the guanine nucleotide binding protein that regulates phospholipase C activity. In contrast, when intact SK-N-SH cells were exposed to prostaglandin E1, the ensuing increases in cAMP were not accompanied by an inhibition of stimulated PPI turnover. These differential effects of increased cAMP concentrations on stimulated PPI turnover may reflect the compartmentation of cAMP within SK-N-SH cells.     

Serotonin Levels in the Rabbit Retina Are Elevated Following Intraocular Injection of Forskolin

Analysis of the mammalian retina for serotonin immunoreactivity suggests an absence of the amine. However, following an intraocular injection of forskolin (1 microM) into a rabbit eye 1 h before analysis of the retina, serotonin immunoreactivity is associated with a subpopulation of amacrine cells. These cells correspond in size and position to the "indoleamine-accumulating cells" of the retina. Biochemical experiments show that forskolin treatment produces an increase in levels of endogenous serotonin and 5-hydroxytryptophan but has no effect on the uptake of serotonin or tryptophan or the metabolism of 5-hydroxytryptophan. These results suggest that the "indoleamine-accumulating cells" in the retina are "serotonergic cells" and that the level of amine is elevated sufficiently for localisation following forskolin treatment. It would appear that forskolin either directly or indirectly activates tryptophan hydroxylase.     

Swelling and Ca2+-activated Anion Conductances in C127 Epithelial Cells Expressing WT and ΔF508-CFTR

CFTR is a chloride channel that is required for fluid secretion and salt absorption in many exocrine epithelia. Mutations in CFTR cause cystic fibrosis. CFTR expression influences some ion channels, but the range of channels influenced, the mechanism of the interaction and the significance for cystic fibrosis are not known. Possible interactions between CFTR and other ion channels were studied in C127 mouse mammary epithelial cell lines stably transfected with CFTR, ΔF508-CFTR, or vector. Cell lines were compared quantitatively using an ¹²⁵I efflux assay and qualitatively using whole-cell patch-clamp recording. As expected, ¹²⁵I efflux was significantly increased by forskolin only in the CFTR line, and forskolin-stimulated whole-cell currents were time- and voltage independent. All three lines responded to hypotonic challenge with large ¹²⁵I efflux responses of equivalent magnitude, and whole-cell currents were outwardly rectified and inactivated at positive voltages. Unexpectedly, basal ¹²⁵I efflux was significantly smaller in the ΔF508-CFTR cell line than in either the CFTR or control cell lines (P < 0.0001), and the magnitude of the efflux response to ionomycin was largest in the vector cell line and smallest in the cell line expressing ΔF508-CFTR (P < 0.01). Whole-cell responses to ionomycin had a linear instantaneous I-V relation and activated at depolarizing voltages. Forskolin responses showed simple summation with responses to ionomycin or hypotonic challenge. Thus, we found no evidence for interactions between CFTR and the channels responsible for swelling-mediated responses. Differences were found in basal and ionomycin-stimulated efflux, but these may arise from variations in the clonally selected cell lines that are unrelated to CFTR expression. Received: 15 November 1995/Revised: 16 February 1996     

毛喉萜对人胃癌细胞BGC－823中蛋白激酶C及其亚类的影响

以人胃癌细胞ＢＧＣ－８２３为模型,研究了毛喉萜（ｆｏｒｓｋｏｌｉｎ）对胃癌细胞中蛋白激酶Ｃ活性及其亚类基因表达的作用,同时也观察了毛喉萜对癌基因ｃ－ｊｕｎ及抑癌基因ｐ５３表达的影响．结果表明,２×１０~（－５）ｍｏｌ／Ｌ毛喉萜处理ＢＧＣ－８２３细胞７２ｈ,细胞质、膜和细胞核ＰＫＣ活性下降,ＰＫＣ亚类β,γ基因表达被抑制,癌基因ｃ－ｊｕｎ的表达也明显降低,而抑癌基因ｐ５３表达升高,上述变化可能是毛喉萜抑制胃癌细胞增殖等生理效应的重要分子事件。