Forskolin对放射性转化细胞TGF基因及部分癌基因表达的影响

3~H-TdR放射性转化细胞(TC3H/10)经1×10~(-5)mol/L Foskolin处理24 h后,TGFα、c-myc、c-K-ras基因的mRNA表达下降,TGFβ、c-fos基因表达无明显变化。非转化细胞(NC3H/10)经相同条件处理,TGFα、TGFβ、c-myc及c-K-ras基因表达无显著改变。提示:Forskolin介导的转化细胞的生长抑制作用与TGFa、cmyc、c-K-ras基因的转录表达下降有关。     

Effect of Forskolin on Progesterone and Plasminogen Activator Secretion by Perfused Rat Corpora Lutea during Pseudopregnancy

Effects of forskolin on progesterone and plasminogen activator production in pseudopregnant ratcorpora lutea was investigated using isolated in vitro perfused ovaries.Progesterone andplasminogen activator production were measured on day 1,8 and 18 of hCG-inducedpseudopregnancy.The results indicated:different concentrations of forskolin(100,200,400 and800 μg)administered to ovaries on the 8th day of pseudopregnancy caused elevation of progesteronesecretion in a dose-dependent manner.After 8 hours of perfusion,PA contents increasedsignificantly in ovaries treated with forskolin.With exogenous PA-urokinase(800 U)added to theperfusion solution,progesterone secretion increased significantly as compared to control group andremained on high level throughout the perfusion period.Though exerting no apparent effects in lowdosage(5 mM),AMCHA,a PA inhibitor,administered in higher dosage(10 and 15 mM)led tomarked reduction in PA activity and progesterone secretion as compared to control group.Thusforskolin causes significant elevation of level of progesterone secretion and PA activity inpseudopregnant rat ovary perfused in vitro.And PA seems to regulate progesterone secretion in theperfused rat corpora luteum.     

Adrenocorticotropin/α-Melanocyte-Stimulating Hormone (ACTH/MSH)-Like Peptides Modulate Adenylate Cyclase Activity in Rat Brain Slices: Evidence for an ACTH/MSH Receptor-Coupled Mechanism

Abstract: The regulation of adenylate cyclase activity by adrenocorticotropin/α-melanocyte–stimulating hormone (ACTH/MSH)-like peptides was investigated in rat brain slices using a superfusion method. Adenylate cyclase activity was concentration-dependently increased by ACTH-(1–24), α-MSH (EC₅₀ values 16 and 6 nM, respectively), and [Nle⁴,D-Phe⁷]α-MSH (EC₅₀ value 1.6 nM), in the presence of forskolin (1 μM, optimal concentration). 1-9-Dideoxy-forskolin did not augment the response of adenylate cyclase to ACTH-(1–24). Various peptide fragments were tested for their ability to enhance [³H]cyclic AMP production. [Nle⁴,D-Phe⁷]α-MSH increased [³H]cyclic AMP formation with a maximal effect of 30% and was more potent than ACTH-(1–24), ACTH-(1–16)-NH₂, α-MSH, ACTH-(1–13)-NH₂, [MetO⁴]α-MSH, [MetO₂⁴,D-Lys⁸,Phe⁹]ACTH-(4–9), ACTH-(7–16)-NH₂, ACTH-(1–10), and ACTH-(11–24), in order of potency. This structure–activity relationship resembles that found for the previously described peptide-induced display of excessive grooming. ACTH-(1–24) stimulated adenylate cyclase activity in both striatal (maximal effect, ?20%) and septal slices (maximal effect, ?40%), but not in hippocampal or cortical slices. Lesioning of the dopaminergic projections to the striatum did not result in a diminished effect of [Nle⁴,D-Phe⁷]α-MSH on [³H]cyclic AMP accumulation, which indicates that the ACTH/MSH receptor–stimulated adenylate cyclase is not located on striatal dopaminergic terminals. ACTH-(1–24) did not affect the dopamine D₁ or D₂ receptor–mediated modulation of adenylate cyclase activity. Based on the present data, we suggest that the binding of endogenous ACTH or α-MSH to a putative ACTH/MSH receptor in certain brain regions leads to the activation of a signal transduction pathway using cyclic AMP as a second messenger.     

Heterogeneity of murine adherent interleukin-2-activated killer cells differential effect of prostaglandin E2 and forskolin

We have studied the relationship between cytotoxic activity, size and granularity of murine interleukin-2-activated adherent killer cells issued from spleen cells cultured with high levels of IL-2. The effects of prostaglandin E₂ (PGE₂) and forskolin upon these cells were assessed. All adherent spleen cells obtained after 5 days of culture were large granular lymphocytes but presented a heterogeneity in size and granularity. After fractionation on a discontinuous-density Percoll gradient, four cellular subpopulations were isolated. Fluorescence-activated cell sorting analysis showed that cells of the lightest fraction (F1) were the largest, while the cells found in the heaviest fraction (F4) were much more granular than the cells collected in the two intermediate fractions (F2 and F3). The serine esterases level was higher in F4 than in unfractionated cells and diminished to about 40% in cells of fractions F2 and F3, which expressed a cytotoxic activity against YAC-1 cells higher than that in unfractionated cells or in F1 or F4, which presented the lowest cytotoxic activity. When AK cells were cultured for 48 h in the presence of either PGE₂ or forskolin, which induce an intracellular increase of cAMP, we observed that PGE₂ (1M) inhibited the cytotoxic activity, but surprisingly forskolin (2M) exerted a stimulating effect on the induction of cytotoxic activity. After fractionation on a discontinuous Percoll gradient we observed the same cellular distribution among PGE₂ or forskolin-treated or-untreated cells, but PGE₂ induced an increase of size and granularity. This effect of PGE₂ was more potent on the cells collected in F4. However this variation of granularity was not associated with any variation in the serine esterase level. The cytotoxic activity of PGE₂- or forskolin-treated cells did not present any significant variation relative to the control for cells collected in F2 and F3: on the other hand, forskolin-treated cells collected in F4 showed a significantly higher cytotoxicity than did the corresponding untreated or PGE₂-treated cells.     

Bidirectional Regulation of GABAA Receptor α1 and α6 Subunit Expression by a Cyclic AMP-Mediated Signalling Mechanism in Cerebellar Granule Cells in Primary Culture

Abstract: Forskolin treatment of cerebellar granule cells in culture resulted in bidirectional regulation of the expression of GABA_A receptor α1 and α6 subunits. Thus, forskolin applied at 2 days in vitro (DIV) increased expression of the α1 subunit but decreased the expression of the α6 subunit. Values with respect to control cultures, both assayed at 9 DIV by immunoblotting, were 310 ± 48% for α1 and 25 ± 16% for the α6 subunit. Similar effects were evoked following chronic treatment with both dibutyryl cyclic AMP and 3-isobutyl-1-methylxanthine. Dideoxyforskolin had no effect on the level of expression of either the α1 or the α6 GABA_A receptor subunits. The changes in subunit expression were accompanied by a 1.7-fold increase in number of total specific [³H]Ro 15-4513 binding sites expressed by intact cerebellar granule cells. This increase in total binding sites was accommodated by a 2.7-fold increase in number of diazepam-sensitive Ro 15-4513 binding sites in accordance with the observed increase in α1 subunit expression. The number of diazepam-insensitive subtype of binding sites were not significantly changed. These results suggest that GABA_A receptor subtype expression can be differentially regulated by intracellular cyclic AMP concentration.     

Lysophosphatidic Acid-Induced Neurite Retraction in PC12 Cells: Neurite-Protective Effects of Cyclic AMP Signaling

Abstract: Effects of the cyclic AMP second messenger system were studied on the retraction of neurites elicited by the phospholipid mediator lysophosphatidic acid (LPA) in PC12 cells. LPA stimulation inhibited adenylyl cyclase, indicating that the LPA receptor couples to the heterotrimeric G_i proteins. However, pertussis toxin or expression of dominant negative Ras did not prevent neurite retraction. In contrast, cholera toxin, forskolin, and application of dibutyryl-cyclic AMP prevented neurite retraction. The neurite-protective effect of forskolin was blocked by Rp -adenosine 3',5'-phosphorothioate. Forskolin and dibutyryl-cyclic AMP both failed to protect neurites in A126-1B2 and 123.7 cells, which lack cyclic AMP-activated protein kinase. Data indicate that elevation of cyclic AMP levels triggers a cyclic AMP-activated protein kinase-dependent mechanism that opposes the functioning of the morphoregulatory signaling activated by LPA. ADP-ribosylation of Rho by the Clostridium botulinum C-3 toxin in 123.7 cells caused neuronal differentiation, indicated by neurite extension, and blocked LPA-induced neurite retraction. LPA activates G_q- and G_i-linked signaling in parallel; therefore, a morphoregulatory signaling network hypothesis is proposed versus the simplistic approach of a signaling pathway. The signaling network integrates the receptor-activated individual, sequential, and parallel signaling events into an interactive network whose individual components may fulfill required and permissive functions encoding the cellular response.     

A Cyclic AMP Mechanism Mediates the Serotonin-Induced Increase in Glutamic Acid Decarboxylase Activity in the Preoptic Area and Hypothalamus

Previous studies have shown that the injection of 5-hydroxytryptamine (5-HT) into the third ventricle of rats on the afternoon of proestrus increases glutamic acid decarboxylase (GAD) activity in the preoptic area and the hypothalamus. In the present report we examine the adenylate cyclase-cyclic AMP (cAMP) system as mediator of that effect. The increase in GAD activity induced by intraventricular injection of 5-HT was completely blocked by injecting an antiserum against cAMP into the third ventricle 30 min earlier, whereas an injection of serum from normal rabbits was ineffective. On the contrary, activation of adenylate cyclase activity by intraventricular injection of forskolin increased GAD activity, an effect that was also blocked by anti-cAMP serum. Anti-cAMP serum also lowered GAD activity in the preoptic area and hypothalamus when injected on the morning of proestrus but not when injected in the afternoon, when the values of GAD activity were already low. The results suggest that a cAMP mechanism may be involved in the changes in preoptic-area and hypothalamic GAD activity such as the rise in enzyme activity induced by intraventricular injection of 5-HT.     

Relationship Among Calmodulin-, Forskolin-, and Guanine Nucleotide-Dependent Adenylate Cyclase Activities in Cerebellar Membranes: Studies by Limited Proteolysis

Adenylate cyclase activity in bovine cerebellar membranes is regulated by calmodulin, forskolin, and both stimulatory (Ns) and inhibitory (Ni) guanine nucleotide-binding components. The susceptibility of the enzyme to chymotrypsin proteolysis was used as a probe of structure-function relationships for these different regulatory pathways. Pretreatment of membranes with low concentrations of chymotrypsin (1-2 micrograms/ml) caused a three- to fourfold increase in basal adenylate cyclase activity and abolished the Ca2+-dependent activation of the enzyme by calmodulin. In contrast, the stimulation of the enzyme by GTP plus isoproterenol was strongly potentiated after protease treatment, an effect that mimics the synergistic activation of adenylate cyclase by Ns and calmodulin in unproteolyzed membranes. Limited proteolysis revealed low- and high-affinity components in the activation of adenylate cyclase by forskolin. The low-affinity component was readily lost on proteolysis, together with calmodulin stimulation of the enzyme. The activation via the high-affinity component was resistant to proteolysis and nonadditive with the Ns-mediated activation of the enzyme, suggesting that both effectors utilize a common pathway. The inhibitory effect of low concentrations (10(-7) M) of guanyl-5'-yl imidodiphosphate [Gpp(NH)p] on forskolin-activated adenylate cyclase was retained after limited proteolysis of the membranes, indicating that the proteolytic activation does not result from an impairment of the Ni subunit. Moreover, in the rat cerebellum, proteolysis as well as calmodulin was found to enhance strongly the inhibitory effect of Gpp(NH)p on basal adenylate cyclase activity. Our results suggest that calmodulin and Ns/Ni interact with two structurally distinct but allosterically linked domains of the enzyme. Both domains appear to be involved in the mode of action of forskolin.     

Metabolic effects and cyclic AMP levels produced by glucagon, (1-Nα-trinitrophenylhistidine,12-homoarginine)glucagon and forskolin in isolated rat hepatocytes

[1-N^α-Trinitrophenylhistidine,12-homoarginine]glucagon (THG) is a potent antagonist of the effects of glucagon on liver membrane adenylate cyclase. In isolated hepatocytes, this glucagon analogue was an extremely weak partial agonist for cAMP accumulation, and it blocked the stimulation of cAMP accumulation produced by glucagon. However, THG was a full agonist for the stimulation of glycogenolysis, gluconeogenesis and urea synthesis in rat hepatocytes, and did not antagonize the metabolic effects of glucagon under most of the conditions examined. Forskolin potentiated the stimulation of cAMP accumulation produced by glucagon or THG, but did not potentiate their metabolic actions. A much larger increase in cAMP levels seemed to be required for the stimulation of hepatocyte metabolism by forskolin than by glucagon or THG. This may suggest the existence of a functional compartmentation of cAMP in rat hepatocytes. The possible existence of compartments in cAMP-mediated hormone actions and the involvement of factors, besides cAMP, in mediating the effects of THG and glucagon is suggested.     

Effects of Cell Division, Cell Density, and Cyclic Nucleotides on Choline Acetyltransferase Activity in a Cholinergic Neuroblastoma Cell Line (S-20Y)

We investigated the effects of a number of experimental perturbations on choline acetyltransferase (ChAT) in a cholinergic mouse neuroblastoma cell line (S-20Y). ChAT specific activity increased by 4.5-fold during growth, suggesting that enzyme activity is dependent on increased cell density. This was confirmed by assessing enzyme activity at differential initial seeding densities. ChAT activity was also markedly enhanced by 1 mM dibutyryl cyclic-3',5'-AMP (dBcAMP), an effect that was blocked by cycloheximide. Confirmation of the dBcAMP effect was achieved with forskolin, a compound known to enhance intracellular cyclic AMP; forskolin (100 microM) caused a significant increase in ChAT activity. After a 20-h latent interval ChAT activity was also enhanced significantly by cytosine arabinoside. The common element in these diverse effects on ChAT activity may be cessation of cell division, although cell-cell interactions at the level of the cell membrane may also be important in the control of ChAT in S-20Y.