Effect of forskolin, isoprenaline and propranolol on spontaneous contractions of the isolated atrium of guinea pigs

Forskolin (0.375 mumol 1(-1)) produced positive inotropic and chronotropic effects on the isolated, spontaneously beating atria of the guinea pig. The same effects were also observed or even increased in the presence of various concentrations of isoprenaline (0.024 and 0.12 mumol 1(-1)) in the organ bath. The effects of forskolin on the isometric contraction and the atrial rate of the isolated, spontaneously beating atria of the guinea-pig were significantly inhibited by propranolol (9 mumol 1(-1)). These results indicate that the action of forskolin on the spontaneously beating atria of the guinea-pig is mediated by stimulation of the adenylate cyclase system, but in some steps of this action, a direct stimulation of beta-adrenoceptors might also be implicated.     

Forskolin stimulates adenylate cyclase and iodine metabolism in thyroid

Forskolin is a potent activator of the cyclic AMP-generating system in many tissues. In dog thyroid slices, the enhancement of cyclic AMP level was rapid, sustained in the presence of forskolin, but easily reversible after its withdrawal. Contrary to TSH, forskolin induced little apparent desensitization. Forskolin potentiated the effects of TSH, PGE1 and cholera toxin. However, the forskolin-induced cyclic AMP accumulation was still sensitive to inhibitors of dog thyroid adenylate cyclase such as iodide, norepinephrine and adenosine. As fluoride, but contrary to TSH and PGE1, forskolin stimulated adenylate cyclase in a medium where Mg2+ was replaced by Mn2+. This suggests that in thyroid, as in other tissues, forskolin acts beyond the receptor level but, as it potentiates hormone action and does not impair modulation by inhibitors, it may interact with the nucleotide-binding regulatory proteins. Forskolin mimicked the effect of TSH on iodide organification and secretion.     

Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase.

The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase involves a cyclic AMP-dependent lesion that is after the G-protein.     

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.     

Relationship between cyclic AMP production and lipolysis induced by forskolin in rat fat cells.

Forskolin (7 beta-acetoxy-8, 13-epoxy-1 alpha,6 beta,9 alpha-trihydroxy-labd-14-ene-11-one) induced both cyclic AMP production and lipolysis in intact fat cells, but stimulated lipolysis without increasing cyclic AMP at a concentration of 10(-5) M. Homogenization of fat cells elicited lipolysis without elevation of cyclic AMP. Forskolin did not stimulate lipolysis in the homogenate. Forskolin stimulated both cyclic AMP production and lipolysis in a cell-free system consisting of endogenous lipid droplets and a lipoprotein lipase-free lipase fraction prepared from fat cells. However, at a concentration of 10(-6) M, it induced lipolysis without increase in the cyclic AMP content in this cell-free system. In the cell-free system, homogenization of the lipid droplets resulted in marked increase in lipolysis to almost the same level as that with 10(-4) M forskolin without concomitant increase in cyclic AMP. Addition of forskolin to a cell-free system consisting of homogenized lipid droplets and lipase did not stimulate lipolysis further. Phosphodiesterase activities were found to be almost the same both in the presence and absence of forskolin in these reaction mixtures. Although 10(-3) M forskolin produced maximal concentrations of cyclic AMP: 6.7 x 10(-7) M in fat cells and 2.7 x 10(-7) M in the cell-free system, 10(-4) M cyclic AMP did not stimulate lipolysis in the cell-free system. In a cell-free system consisting of lipid droplets and the lipase, pyrophosphate inhibited forskolin-induced cyclic AMP production, but decreased forskolin-mediated lipolysis only slightly. Based on these results, mechanism of lipolytic action of forskolin was discussed.     

Activation of renal adenylate cyclase by forskolin: assessment of enzymatic activity in animal models of the secondary hyperparathyroid state

The effects of forskolin on kidney slice cyclic AMP content and membrane adenylate cyclase activity were studied in order to determine whether or not activation of the enzyme by forskolin was affected in experimental animal models of the secondary hyperparathyroid state. Forskolin was found to be a potent activator of renal adenylate cyclase in rats and chicks, and the diterpene produced a marked potentiation of the cyclic AMP response to parathyroid hormone (PTH). The diterpene had no effect on the binding of PTH to renal receptors. Activity of adenylate cyclase in the presence of forskolin was similar in renal membranes from either vitamin D-deficient rats or chicks compared to control. Forskolin did not restore full responsiveness to PTH in renal slices from chicks raised on diets that were deficient in either vitamin D or calcium although the diterpene was capable of potentiating the cyclic AMP response to PTH in these tissues. Forskolin also augmented the activation of membrane adenylate cyclase by PTH although this effect of the diterpene was much less prominent in membrane preparations than that observed in renal slices. This study provided additional evidence that the downregulation of renal PTH-dependent adenylate cyclase in experimental models of secondary hyperparathyroidism is due to a specific reduction in receptor-mediated regulation of cyclic AMP formation. Adenylate cyclase activity as assessed by forskolin-stimulated enzyme activity was fully maintained in kidney membranes from these animal models. Thus, forskolin appears to be a useful drug for measuring total enzymatic activity in situations where altered responsiveness of adenylate cyclase to hormones has been demonstrated to be mediated by changes in hormone receptors.     

Forskolin stimulation of pepsinogen secretion from frog esophageal mucosa is partly mediated by intrinsic cholinergic neurons

Forskolin was found to stimulate pepsinogen secretion from frog esophageal mucosa. The stimulation was dose-dependent and was accompanied with a great increase in tissue cAMP content. The response to forskolin mimicked the action of bethanechol and was not additive with bethanechol. The stimulatory effect of forskolin was inhibited by 50% in the presence of either atropine or tetrodotoxin. On the other hand, incubation in a calcium-free medium not only reduced the response to forskolin by 45% but also eliminated the influence of atropine and tetrodotoxin. These results indicate that forskolin may stimulate pepsinogen secretion from the frog esophageal mucosa via activating adenylate cyclase, and part of its effect may arise from eliciting acetylcholine release from the intrinsic neurons.     

Photoaffinity Labelling of MSH Receptors on Anolis Melanophores: Effects of Catecholamines,Calcium and Forskolin

Abstract

Photoaffinity labelling of MSH receptors on Anolis melanophores was used as a tool for studying the effects of catecholamines, calcium and forskolin on hormone-receptor interaction and receptor-adenylate cyclase coupling. Covalent attachment of photoreactive α-MSH to its receptor was suppressed in calcium-free buffer but was hardly influenced by catecholamines or forskolin. The longlasting signal generated by the covalent MSH-receptor complex was readily and reversibly abolished by adrenaline, noradrenaline, dopamine or clonidine or by the absence of calcium. The suppression of pigment dispersion by catecholamines was blocked by the simultaneous presence of yohimbine but not prazosin, indicating that the catecholamines antagonize the α-MSH signal by inhibitory action on the adenylate cyclase system through an alpha-2 receptor. Forskolin, which stimulates melanophores by direct action on the catalytic unit of the adenylate cyclase and at about the same speed as α-MSH, produced a slower and weaker response in the presence of noradrenaline. If MSH receptors were covalently labelled and then exposed to noradrenaline, the characteristics of the forskolin-induced response were identical to those of unlabelled cells that had not been exposed to noradrenaline. This may point to a partial restoration of receptor-adenylate cyciase coupling by forskolin. The results show that the longlasting stimulation of Anolis melanophores by photoaffinity labelling proceeds via a permanently stimulated adenylate-cyclase system whose coupling to the receptor depends on calcium and is abolished by alpha-2 receptor agonists. Calcium is also essential for hormone-receptor binding.     

Augmentation of cholinergic-mediated amylase release by forskolin in mouse parotid gland

Cholinergic-mediated amylase release in mouse parotid acini was augmented by forskolin; the potency but not the maximal response to carbachol was altered. Amylase released by carbachol plus forskolin was dependent on extracellular calcium and was mimicked by the calcium ionophore, A23187 plus forskolin. Forskolin was also shown to enhance carbachol-stimulated 45Ca2+ uptake into isolated acini. Hydroxylamine, nitroprusside, and 8-bromo-c-GMP each in combination with forskolin mimicked the effects of carbachol plus forskolin on amylase release. In the presence of carbachol (10(-8)M) forskolin did not augment c-AMP levels. However, in the presence of carbachol (5 X 10(-7) M) or hydroxylamine (50 microM) forskolin did significantly augment c-AMP accumulation. These results suggest that calcium and c-GMP may mediate the augmentation of cholinergic-mediated amylase release by effects on c-AMP metabolism.     

Effects of forskolin on adenylate cyclase,cyclic AMP,protein kinase and intermediary metabolism of the thyroid gland

Forskolin (40 μM) stimulated adenylate cyclase activities of bovine thyroid plasma membranes without pthe addition of guanine nucleotides. GDP had little effect on the forskolin-stimulated adenylate cyclase activity while Gpp[NH]p (0.1–1.0 μM) decreased it. In the presence of TSH (10 mU/0.11), Gpp[NH]p no longer caused inhibition. Forskolin did not affect phosphodiesterase activities of thyroid homogenates. Forskolin (10 μM) rapidly increased cAMP levels in bovine thyroid slices both in the absence and presence of a phosphodiesterase inhibitor. The effect of TSH (50 mU/ml) on cAMP levels was additive or greater than additive to that of forskolin. An initial 2-h incubation of slices with forskolin did not decrease their subsequent cAMP responses to either forskolin and/or TSH while similar treatment of slices with TSH induced desensitization of the cAMP response to TSH, but not to forskolin. Forskolin (10 μM) as well as TSH (50 mU/ml) activated cAMP-dependent protein kinase of slices in the absence of a phosphodiesterase inhibitor. Although forskolin activated the adenylate cyclase cAMP system, it did not stimulate iodide organification or glucose oxidation, effects which have been attributed to cAMP. In fact, forskolin inhibited these parameters and ³²P incorporation into phospholipids as well as their stimulation by TSH. These results indicate that an increase in cAMP levels and cAMP-dependent protein kinase activity in thyroid slices may not necessarily reproduce the effects of TSH on the thyroid.     

Cyclic AMP inhibits secretion from bovine adrenal chromaffin cells evoked by carbamylcholine but not by high K+

The role of cAMP in the control of secretion from bovine adrenal chromaffin cells was examined using the adenylate cyclase activator, forskolin. Treatment of chromaffin cells with forskolin resulted in a rise in cAMP levels. Forskolin inhibited catecholamine release elicited by carbamylcholine or nicotine but had no effect on secretion evoked by 55 mM K+. Inhibition of carbamylcholine-stimulated release by forskolin was half-maximal at 10 microM forskolin. The inhibition by forskolin of secretion evoked by carbamylcholine was at a step distal to the rise in intracellular free calcium concentration ([Ca2+]i), since this rise was not inhibited by forskolin, which itself produced a small rise in [Ca2+]i. The results suggest that secretion evoked by carbamylcholine is due to the activation of an additional second messenger pathway acting with the rise in [Ca2+]i. This additional pathway may be the target for cAMP action.     

Inhibition by forskolin of insulin-stimulated glucose transport in L6 muscle cells.

The cardioactive diterpene forskolin is a known activator of adenylate cyclase, but recently a specific interaction of this compound with the glucose transporter has been identified that results in the inhibition of glucose transport in several human and rat cell types. We have compared the sensitivity of basal and insulin-stimulated hexose transport to inhibition by forskolin in skeletal muscle cells of the L6 line. Forskolin completely inhibited both basal and insulin-stimulated hexose transport when present during the transport assay. The inhibition of basal transport was completely reversible upon removal of the diterpene. In contrast, insulin-stimulated hexose transport did not recover, and basal transport levels were attained instead. This effect of inhibiting (or reversing) the insulin-stimulated fraction of transport is a novel effect of the diterpene. Forskolin treatment also inhibited the stimulated fraction of transport when the stimulus was by 4 beta-phorbol 12,13-dibutyrate, reversing back to basal levels. Half-maximal inhibition of the above-basal insulin-stimulated transport was achieved with 35-50 microM-forskolin, and maximal inhibition with 100 microM. Forskolin did not inhibit 125I-insulin binding under conditions where it caused significant inhibition of insulin-stimulated hexose transport. Forskolin significantly elevated the cyclic AMP levels in the cells; however its inhibitory effect on the above basal, insulin-stimulated fraction of hexose transport was not mediated by cyclic AMP since: (i) 8-bromo cyclic AMP and cholera toxin did not mimic this effect of the diterpene, (ii) significant decreases in cyclic AMP levels caused by 2',3'-dideoxyadenosine in the presence of forskolin did not prevent inhibition of insulin-stimulated hexose transport, (iii) isobutylmethylxanthine did not potentiate forskolin effects on glucose transport but did potentiate the elevation in cyclic AMP, and (iv) 1,9-dideoxyforskolin, which does not activate adenylate cyclase, inhibited hexose transport analogously to forskolin. We conclude that forskolin can selectively inhibit the insulin- and phorbol ester-stimulated fraction of hexose transport under conditions where basal transport is unimpaired. The results are compatible with the suggestions that glucose transporters operating in the stimulated state (insulin or phorbol ester-stimulated) differ in their sensitivity to forskolin from transporters operating in the basal state, or, alternatively, that a forskolin-sensitive signal maintains the stimulated transport rate.     

2-Arachidonoyl glycerol sensitizes the pars distalis and enhances forskolin-stimulated prolactin secretion in Syrian hamsters

2-Arachidonoyl glycerol (2-AG) is a major endocannabinoid and an important regulator of neuroendocrine system. In Syrian hamster and human, we found that 2-AG is synthesized in the hypophysial pars tuberalis (PT), an interface between photoperiodic melatonin signals and neuroendocrine output pathways. The target of 2-AG produced in the PT is likely to be the pars distalis (PD). Here we demonstrate that 2-AG in combination with forskolin stimulated prolactin secretion from PD organ cultures of Syrian hamsters, whereas incubation with 2-AG alone had no effect. Forskolin-induced prolactin secretion was also significantly enhanced when cultured PD tissue was preincubated with 2-AG. The stimulatory effects of 2-AG on prolactin secretion were blocked by AM251, a selective CB1 antagonist, and were still observed in the presence of quinpirole, a D2-class dopamine receptor agonist. 2-AG also enhanced prolactin secretion in the presence of adenosine, while it had little effect when applied together with adenosine diphosphate (ADP) and adenosine triphosphate (ATP). Moreover, the effect of forskolin was mimicked by adenosine in a dose-dependent manner. In conclusion, our data suggest that 2-AG sensitizes the PD tissue to potentiate the stimulating effects of forskolin and adenosine on prolactin secretion and thus provide novel insight into the mode of action of 2-AG in the PD.     

Effects of forskolin and analogues on nicotinic receptor-mediated sodium flux,voltage-dependent calcium flux,and voltage-dependent rubidium efflux in pheochromocytoma PC12 cells

1. Forskolin, a naturally occurring diterpene that activates adenylate cyclase, HL706, a water-soluble derivative of forskolin (6 beta-[(piperidino)acetoxy]-7-desacetylforskolin) that is less potent than forskolin in activating adenylate cyclase, and 1,9-dideoxyforskolin, an analogue that does not activate adenylate cyclase, were examined for effects on the nicotinic receptor-mediated 22Na+ flux, a high potassium-induced 45Ca2+ flux through L-type calcium channels, and a high potassium-induced 86Rb+ efflux through a calcium-dependent potassium channels in PC12 cells. 2. Forskolin and analogues at 30 microM completely blocked carbamylcholine-elicited flux of 22Na+ through the nicotinic receptor-gated channel. 1,9-Dideoxyforskolin had an IC50 value of 1.6 microM with forskolin and HL706 being two- to three fold less potent. 3. Forskolin and its analogues appear to be noncompetitive blockers of the neuronal nicotinic receptor-channel complex in PC12 cells, but unlike many noncompetitive blockers, did not markedly enhance desensitization. Instead, forskolin, but not HL706 or 1,9-dideoxyforskolin, slightly antagonized the desensitization evoked by high concentrations of carbamylcholine. N-Ethylcarboxamidoadenosine, an adenosine analogue that elevates cyclic AMP and 8-bromo-cyclic AMP had no effect on desensitization. 4. Forskolin, HL706, and 1,9-dideoxyforskolin in the presence of carbamylcholine inhibited the binding of a noncompetitive blocker, [3H]perhydrohistrionicotoxin, to the muscle-type nicotinic receptor-channel complex in Torpedo electroplax membranes with IC50 values of 20 microM. Forskolin had no effect on [3H]perhydrohistrionicotoxin binding in the absence of carbamylcholine, while HL706 and 1,9-dideoxyforskolin still inhibited binding in the absence of carbamylcholine. 5. Forskolin, but not HL706 or 1,9-dideoxyforskolin had a slight inhibitory effect on the binding of [125I]alpha-bungarotoxin to acetylcholine recognition sites in Torpedo membranes. 1,9-Dideoxyforskolin at 30 microM, but not forskolin or HL706, markedly inhibited depolarization-evoked 45Ca+ flux and 86Rb+ efflux in PC12 cells, suggesting that 1,9-dideoxyforskolin has nonspecific inhibitory effects on a variety of ion channels.     

Inhibition of 3-O-methylglucose transport in human erythrocytes by forskolin

The effect of forskolin, an activator of adenylate cyclase, was investigated on glucose transport in human erythrocytes. Forskolin was found to be a potent inhibitor of 3-O-methylglucose (3-O-MG) influx in human erythrocytes. The inhibition of 3-O-MG transport was instantaneous and reversible. The inhibitory effect of forskolin was concentration-dependent, having an IC50 value of 7.5 microM. Forskolin caused a decrease in Vmax of carrier-mediated 3-O-MG transport from 35.32 to 1.56 mumol/ml of cell X min in the presence of 50 microM forskolin. Inhibition of influx was not reversed at high concentrations of 3-O-MG. In addition, forskolin inhibited the influx of other carbohydrates including galactose, ribose, and fructose. In contrast, forskolin was without effect on adenosine transport. To unravel the underlying mechanism responsible for the inhibitory action of forskolin, the possible involvement of cyclic AMP in controlling glucose transport was examined. Erythrocytes treated with 50 microM forskolin exhibited an increase in cyclic AMP content from the basal levels of 258 fmol/ml of cell to 334 fmol/ml of cell within 10 s after forskolin exposure. However, erythrocytes in which cyclic AMP was allowed to accumulate in excess of 10,000 times the basal level, by means of preincubation with exogenous cyclic AMP, displayed 3-O-MG transport indistinguishable from that of cyclic AMP-poor control cells. In view of the finding that cyclic AMP plays no discernible role in the erythrocyte 3-O-MG transport, it is suggested that the forskolin inhibition is mediated by a mechanism other than by stimulating adenylate cyclase activity. Moreover, forskolin appears to directly inactivate the 3-O-MG transport system since glucose-sensitive cytochalasin B binding to erythrocyte membranes is virtually abolished by 50 microM forskolin.     

Forskolin Mediates the Survival of Nerve Growth Factor-Dependent Sympathetic Neurons of Chick Embryo by a Cyclic AMP-Independent Mechanism

Forskolin has become an invaluable tool for exploring the involvement of cyclic AMP in a variety of cellular functions. The diterpine directly activates the catalytic subunit of adenylate cyclase, causing a marked increase in cyclic AMP content. Because of this well-characterized action, practically all the observed effects of forskolin are commonly attributed to cyclic AMP-dependent processes. We show here that forskolin exerts a neurotrophic action that is almost identical to that of nerve growth factor (NGF) and phorbol 12,13-dibutyrate (PDB) but independent of cyclic AMP. Sympathetic neurons of the chick embryo supported in culture for 2 days by NGF, forskolin plus 3-isobutyl-1-methylxanthine (IBMX), or PDB had almost identical levels of cyclic AMP (between 9 and 12 pmol/mg protein). Neurons supported in culture for 2 days by NGF or PDB when challenged with forskolin plus IBMX showed almost a 15-fold increase in cyclic AMP, but those supported by forskolin plus IBMX and then exposed to the same combination of drugs did not show an increase in cyclic AMP, exhibiting a marked down-regulation. Exposure of neurons to forskolin for 2 h was ineffective in supporting long-term survival, suggesting that an initial increase in cyclic AMP formation is not sufficient but the continued presence of the drug is essential for survival. Effects of forskolin on the survival of these neurons could be observed even in the presence of dideoxyadenosine, and inhibitor of adenylate cyclase. Neurons supported by PDB for 2 days in culture when exposed to NGF for the first time did not show any increase in cyclic AMP, providing clear evidence that NGF does not affect this second messenger in its target cells. Similarly, neurons supported by NGF for 2 days when exposed to PDB did not show an increase in cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of bile acid active transport related to increased mucosal cyclic AMP content in rat ileum in vitro

The regulation of bile acid transport in rat ileum was studied in vitro using the adenylate cyclase stimulator forskolin, or 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Forskolin 20 microM as well as 100 microM IBMX enhanced mucosal cyclic AMP to 3-fold the control levels. As a physiological response, net fluid absorption in everted ileal sacs was reduced. Taurocholate (10-500 microM) transfer in everted perfused segments of rat ileum was measured using a three compartment dual label method suitable for measuring active transport. Transport asymmetry with absorption exceeding its counterflux by 26-fold, was measured at 500 microM taurocholate. Forskolin increased absorption of taurocholate still further, by 68%, and reduced the serosal to mucosal flux. Enhanced intracellular accumulation of taurocholate indicated a stimulatory action of forskolin on active transport at the mucosal brush-border membrane. In uptake studies, accumulation of taurocholate was enhanced by 100 microM IBMX also. Forskolin-induced uptake stimulation could also be shown for chenodeoxycholate and cholate. In the presence of the neuronal blocker tetrodotoxin, uptake stimulation was still effective. Results indicate that the ileal bile acid transporter is included within the group of sodium-dependent cotransporters of the rat small intestine which are subject to a cyclic AMP-related stimulation at the mucosal cellular level.     

Effect of forskolin on action potential, slow inward current and tension of frog atrial fibers

The new nonhormonal activator of adenylate cyclase forskolin was studied on frog atrial trabeculae by current clamp and voltage clamp methods using a double sucrose gap technique. Forskolin (5 X 10(-6) M to 2 X 10(-5) M) dose-dependently increased action potential duration, the height of the plateau and twitch tension. The time constant for inactivation of the slow inward current and the steady state kinetic variables of calcium channels d infinity and f infinity remained uneffected. Forskolin increased the amplitude of slow inward calcium current isi and of the phasic tension related to it. The maximal conductance gsi increased. These effects were indistinguishable from those obtained earlier on cardiac fibers with hormonal and nonhormonal activators of cyclic AMP-dependent phosphorylation. The beta-adrenoreceptor antagonist propranolol 10(-6)M did not decrease the effect of forskolin. Forskolin had no effect when slow inward current was previously increased by saturating concentrations of the beta-adrenergic agonist isoproterenol (10(-4)M). Our results are in favour of the hypothesis that cyclic AMP-dependent phosphorylation of membrane proteins modulates the Ca-entry in the heart cells through the membrane slow calcium channels.     

Induction of osteoblastic cell differentiation by forskolin. Stimulation of cyclic AMP production and alkaline phosphatase activity

The effects of forskolin on differentiation of osteoblastic cells (clone MC3T3-E1) cultured in alpha-minimum essential medium containing 0.1% bovine serum albumin were investigated by assays of intracellular cyclic AMP level and alkaline phosphatase activity in the cells. Forskolin increased cyclic AMP production in the cells in a dose-related manner, the maximum increase being 250-fold above that of the controls. Alkaline phosphatase activity in the cells was also elevated as early as 24 h and rose to nearly its maximum at 48 h. The elevation was dose-dependent, with a maximum increase at 5 X 10(-6) M forskolin. Forskolin and prostaglandin E2 showed a supraadditive effect on cyclic AMP production in the cells and had an additive effect on alkaline phosphatase activity, whereas forskolin and dibutyryl cyclic AMP had little additive effect on either cyclic AMP production or enzyme activity. These results suggest that cyclic AMP is closely linked to the differentiation of osteoblastic cells in vivo.     

The regulation of adenylate cyclase activity in turkey erythrocyte membranes by forskolin

The mechanisms by which forskolin stimulates adenylate cyclase activity in turkey erythrocyte membranes and is influenced by manganese and Gpp(NH)p were studied. Forskolin-dependent adenylate cyclase activity in particulate turkey erythrocyte membranes is enhanced following preincubation of membranes with isoproterenol and GMP (cleared membranes). In contrast, solubilization of turkey erythrocyte membranes, previously cleared, renders them relatively refractory to forskolin but not to Gpp(NH)p. Whereas adenylate cyclase activity due to the simultaneous presence of forskolin and Mn2+ in particulate turkey erythrocyte membranes is additive, their copresence becomes synergistic after solubilization. The apparent Kact for forskolin activation of adenylate cyclase is not influenced by clearance or by the presence of Mn2+ in particulate turkey erythrocyte membranes. Following solubilization, the Vmax for forskolin-dependent adenylate cyclase activation determined in the presence of Mn2+ is also independent of clearance. Forskolin activation of turkey erythrocyte adenylate cyclase appears to be influenced at sites in addition to the catalytic unit.