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PubMed1994

Localization of the forskolin labeling sites to both halves of P-glycoprotein: similarity of the sites labeled by forskolin and prazosin.

An iodinated derivative of forskolin, 6-O-[[2-[3-(4-azido-3-[125I] iodophenyl)propionamido]ethyl]carbamyl]forskolin ([125I]6-AIPP-Fsk), photolabels the multidrug efflux pump P-glycoprotein in membranes prepared from the multidrug-resistant cell lines KB-V1 and KB-C1. The labeling site for [125I]6-AIPP-Fsk was localized by immunoprecipitation of tryptic fragments of P-glycoprotein labeled in KB-C1 membranes. A 6-kDa, photolabeled, tryptic fragment was immunoprecipitated by antiserum raised against residues 348-419 of P-glycoprotein, PEPG9, but not by antisera raised against flanking regions PEPG7 and PEPG11. A peptide that corresponds to residues 343-359 of P-glycoprotein inhibited immunoprecipitation of the 6-kDa fragment by antiserum against PEPG9 but had no effect on the immunoprecipitation of photolabeled fragments by antiserum against PEPG7. A second peptide, corresponding to residues 360-376, had no effect on the immunoprecipitation by antiserum against PEPG9. [125I]6-AIPP-Fsk labels the carboxyl-terminal half of P-glycoprotein, because low molecular mass tryptic fragments were immunoprecipitated by three carboxyl-terminal antisera. Therefore, [125I]6-AIPP-Fsk labels both halves of P-glycoprotein, and labeling in the amino-terminal half can be localized to residues 291-359, which span proposed transmembrane regions 5 and 6. KB-V1 membranes photolabeled with [125I]6-AIPP-Fsk and [125I]iodoarylazidoprazosin were digested with either Staphylococcus aureus V8 protease or chymotrypsin and had similar digestion patterns, suggesting that the two drugs label the same sites on P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Positive inotropic effect of the novel forskolin derivative 6-(3-dimethylaminopropionyl)forskolin hydrochloride.

The effect of NKH477 (6-(3-dimethylaminopropionyl)forskolin hydrochloride, CAS 138605-00-2), a novel water-soluble forskolin derivative, on the energy metabolism of the heart was examined and compared with that of dobutamine. The isolated rat heart was perfused with Krebs-Henseleit bicarbonate buffer with a gas mixture containing 95% O2 + 5% CO2 according to the Langendorff's technique. NKH477 (1 or 10 mumol/l) increased maximum rate of rise of left ventricular pressure (LVdP/dtmax) (positive inotropic action), heart rate (positive chronotropic action), and coronary flow. On the other hand, dobutamine (50 or 500 nmol/l) exhibited the positive inotropic action only. Both NKH477 (10 mumol/l) and dobutamine (500 nmol/l) increased the tissue adenosine monophosphate level, and decreased the tissue creatine phosphate level (a decrease in energy store) in the heart. Nevertheless, neither NKH477 (1 or 10 mumol/l) nor dobutamine (50 or 500 nmol/l) modified the tissue adenosine triphosphate level and the value of energy charge potential, an index of myocardial energy state. These results suggest that NKH477 produces both positive inotropic and positive chronotropic actions, while it does not disturb myocardial energy state, although it decreases the energy store.

Adrenergic beta-1 Receptor Agonists↗
PubMed1985

Forskolin potentiation of cholera toxin-stimulated cyclic AMP accumulation in intact C6-2B cells. Evidence for enhanced Gs-C coupling.

Forskolin directly stimulates adenylate cyclase activity and acts synergistically with receptor-mediated agonists which stimulate cyclic AMP production. We have previously observed that a 3-hr incubation of C6-2B rat astrocytoma cells with 6 nM cholera toxin in the presence of 1 microM forskolin results in cyclic AMP accumulation 9-fold greater than in the absence of forskolin. Since the action of cholera toxin is mediated by the stimulatory guanine nucleotide-binding regulatory component (GS) of the adenylate cyclase complex, we proposed that the mechanism by which forskolin augments hormone responses involves an enhanced coupling of GS with the adenylate cyclase catalytic component (C). In the present communication, we report the detailed characterization of the synergistic interaction between forskolin and cholera toxin as effectors of cyclic AMP accumulation in intact C6-2B cells. After a 3-hr incubation, maximal cholera toxin-stimulated cyclic AMP accumulation was 990 +/- 34 pmol/mg of protein. In the presence of 1 microM forskolin, the response to cholera toxin increased to 13,137 +/- 1,595 pmol of cyclic AMP/mg of protein. The half-maximally effective cholera toxin concentrations estimated by nonlinear least squares regression analysis determined in the absence or presence of 0.1 mM forskolin were 56.6 and 57.5 pM, respectively. The highly reproducible lag in forskolin-stimulated cyclic AMP accumulation in C6-2B cells was abolished by cholera toxin pretreatment, indicating a possible role for GS-associated GTPase in the mechanism of forskolin action. Cholera toxin treatment markedly augmented forskolin-stimulated cyclic AMP accumulation and shifted the forskolin concentration-response curve to the left approximately 1.5 log units. When C6-2B cells were treated for 1 min with 10 nM cholera toxin, the response to forskolin was significantly potentiated by 10 min. No significant increase in cellular cyclic AMP content in the absence of a forskolin challenge was apparent for up to 45 min. It appears that prior promotion of GS-C coupling by cholera toxin treatment enhances the ability of forskolin to stimulate cyclic AMP accumulation. Whether or not forskolin interacts (i.e., binds) exclusively to C remains to be proven. However, the actions of forskolin to stimulate cyclic AMP formation and potentiate agonist-stimulated cyclic AMP formation are modulated by the activity state of GS, and at least part of the response to forskolin is mediated by GS.

Adenosine Diphosphate Ribose↗
PubMed1987

Effects of forskolin on bone resorption in the absence and presence of parathyroid hormone and calcitonin.

Release of previously incorporated 45Ca from fetal rat long bones was determined with the diterpene forskolin, both in the absence and presence of parathyroid hormone (PTH) and calcitonin (CT). In the absence of hormone, increased bone resorption was observed with 10(-7)M forskolin, but biphasic responses, consisting of initial decreases in 45Ca release that were followed by increased calcium mobilization, were produced with 10(-6)M and 10(-5)M forskolin. Inhibition of 45Ca release was pronounced and delayed more with 10(-5)M forskolin while the greatest stimulation of bone resorption was elicited by 10(-6)M forskolin, a response that was inhibited by 100 mU/ml CT. In the presence of 250 ng/ml PTH, a synergistic enhancement of 45Ca release occurred with 10(-7)M forskolin treatment while, in contrast, calcium mobilization was inhibited by 10(-6)M and 10(-5)M forskolin. Inhibition by 10(-6)M forskolin was characterized by "escape" while that of 10(-5)M forskolin was continuous over a 5 day interval. Inhibition throughout the experimental period also was noted when 10(-5)M forskolin was combined with 2.5 ng/ml PTH, but no effect on calcium mobilization was observed upon addition of 10(-7)M forskolin and, rather than inhibition, an enhancement of 45Ca release occurred when 10(-6)M forskolin was combined with 2.5 ng/ml PTH. Inhibition of 250 ng/ml PTH, but lack of inhibition of 2.5 ng/ml PTH by 10(-6)M forskolin suggests a 10(-6)M forskolin-sensitive portion of PTH-mediated calcium efflux. Absence of "escape" when 10(-5) M forskolin is combined with 250 ng/ml PTH suggests that heterologous desensitization may not play a major role in the "escape" which occurs with 10(-6) M forskolin.

Animals↗
PubMed1991

Forskolin inhibits and reverses the effects of brefeldin A on Golgi morphology by a cAMP-independent mechanism.

Brefeldin A (BFA) causes rapid redistribution of Golgi proteins into the ER, leaving no definable Golgi apparatus, and blocks transport of proteins into post-Golgi compartments in the cell. In this study we follow the disassembly of the Golgi apparatus in BFA-treated, living cells labeled with NBD-ceramide and demonstrate that forskolin can both inhibit and reverse this process. Long, tubular processes labeled with NBD-ceramide were observed emerging from Golgi elements and extending out to the cell periphery in cells treated with BFA for 5 min. With longer incubations in BFA, the NBD label was dispersed in a fine reticular pattern characteristic of the ER. Treatment with forskolin inhibited these effects of BFA as well as BFA's earliest morphologic effect on the Golgi apparatus: the redistribution to the cytosol of a 110-kD Golgi peripheral membrane protein. In addition, forskolin could reverse BFA's block in protein secretion. Forskolin inhibition of BFA's effects was dose dependent and reversible. High concentrations of BFA could overcome forskolin's inhibitory effect, suggesting forskolin and BFA interact in a competitive fashion. Remarkably, in cells already exposed to BFA, forskolin could reverse BFA's effects causing the 110-kD Golgi peripheral membrane protein to reassociate with Golgi membrane and juxtanuclear Golgi complexes to reassemble. Neither membrane permeant cAMP analogues nor cAMP phosphodiesterase inhibitors could replicate or enhance forskolin's inhibition of BFA. 1,9-Dideoxyforskolin, which does not activate adenylyl cyclase, was equally as effective as forskolin in antagonizing BFA. A derivative of forskolin, 7-HPP-forskolin, that is less potent than forskolin at binding to adenylyl cyclase, was also equally effective as forskolin in antagonizing BFA. In contrast a similar derivative, 6-HPP-forskolin, that is equipotent with forskolin at binding to adenylyl cyclase, did not inhibit BFA's effects. These results suggest that forskolin acts as a competitive antagonist to BFA, using a cAMP-independent mechanism to prevent and reverse the morphologic effects induced by BFA.

4-Chloro-7-nitrobenzofurazan↗
PubMed1987

Stimulation of adenylate cyclase by water-soluble analogues of forskolin.

Analogues of forskolin that are more soluble in water than forskolin have been synthesized and tested for their ability to interact with adenylate cyclase. These analogues are esterified with various heterocyclic amino acids at the 6 beta-hydroxyl position of forskolin or at the 6 beta-hydroxyl or 7 beta-hydroxyl position of 7-desacetyl forskolin. Analogues were tested for their ability to activate rat brain adenylate cyclase, activate detergent-solubilized rat brain adenylate cyclase, increase cyclic AMP in intact S49 wild-type cells, and inhibit the binding of 3H-forskolin to rat brain membranes. Forskolin activated rat brain adenylate cyclase with an EC50 of 4 microM and increased cyclic AMP in intact S49 cells with an EC50 of 5 microM. Analogues esterified at the 7 beta-hydroxyl position had EC50 values that ranged from 4 microM to 15 microM for activating adenylate cyclase in membranes and solubilized preparations, and for increasing cyclic AMP in S49 cells. Analogues esterified at the 6 beta-hydroxyl position with no acyl group at the 7 beta-hydroxyl position were generally less potent than the corresponding 7-acyl analogues with EC50 values that ranged from 30 microM to 100 microM. Interestingly, the diacyl analogues of forskolin containing an acetate group at the 7 beta-hydroxyl position and esterified with heterocyclic amino acids at the 6 beta-hydroxyl position were very potent at stimulating adenylate cyclase, with EC50 values that ranged from 1 microM to 25 microM. The 7-acyl analogues and the 6,7-diacyl analogues inhibited the binding of 3H-forskolin to rat brain membranes with IC50 values that ranged from 20 microM to 70 microM, while the 6-acyl analogues had much higher IC50 values that ranged from 100 nM to 375 nM. Aqueous solutions of forskolin were also produced by dissolving forskolin in solutions of hydroxypropyl-gamma-cyclodextrin. These aqueous solutions of forskolin were equipotent with alcoholic solutions of forskolin in stimulating adenylate cyclase. In conclusion, water-soluble derivatives of forskolin may be useful for increasing cyclic AMP in broken cell preparations or in intact cell preparations where the presence of organic solvents, which are necessary to solubilize forskolin, are detrimental. Alternatively, aqueous solutions of forskolin can be produced by dissolving forskolin in solutions of hydroxypropyl-gamma-cyclodextrin.

Adenylyl Cyclases↗
PubMed1986

Conditional inhibition of forskolin-activated adenylate cyclase by guanosine diphosphate and its analog.

Forskolin-activated adenylate cyclases (AC) in intact membranes, solubilized with Lubrol or eluted following adsorption on a forskolin-Sepharose column, were examined for inhibition by GDP and GDP beta S. AC in intact membranes of rat or rabbit adipocytes was activated by 100 microM forskolin and further potentiated by 10 microM Gpp(NH)p in combination with either 230 microM epinephrine or 50 mU X ml-1 ACTH. GDP (0-1 mM) or GDP beta S (0-500 microM) inhibited activation in a dose-dependent manner to a level similar to or slightly below that produced by 100 microM forskolin alone. Forskolin at 100 microM stimulated solubilized AC of rabbit adipocytes and rat liver membranes for 10 +/- 4 to 160 +/- 10 and from 26 +/- 2 to 274 +/- 21 pmol(mg X min)-1, respectively, in the absence of GDP beta S; forskolin-activated activity decreased from 160 +/- 10 to 157 +/- 6 and from 274 +/- 21 to 238 +/- 14 pmol(mg X min)-1 in the presence of 500 microM GDP beta S. Forskolin-activated solubilized enzyme was further potentiated by 10 microM Gpp(NH)p from 160 +/- 10 to 289 +/- 52 and from 274 +/- 21 to 702 +/- 50 pmol(mg X min)-1. GDP beta S at 500 microM inhibited 93 and 103% of the Gpp(NH)p-potentiated activity. AC of rat adipocytes eluted from forskolin-Sepharose affinity column with 500 mM NaCl and 100 microM forskolin was not significantly activated by Gpp(NH)p nor inhibited by GDP beta S. However, it was activated by forskolin. The lack of inhibition of unmodified forskolin-activated activity by GDP or GDP beta S in contrast to the inhibition of Gpp(NH)p-activated enzyme or Gpp(NH)p-potentiated forskolin-activated enzyme may be a general phenomenon descriptive of the action of forskolin on AC. Furthermore, inhibition of forskolin-activated AC by GDP and its analog may be a useful index in analyzing the degree of guanine nucleotide potentiation of this enzyme.

Adenylyl Cyclases↗
PubMed1986

Forms of adenylate cyclase, activation and/or potentiation by forskolin.

Activation of different forms of adenylate cyclases (AC) by forskolin and displacement of [14,15-3H]dihydroforskolin binding from membranes by forskolin in the absence or presence of specific stimulatory hormone and beta, gamma-imidoguanosine 5'-triphosphate (Gpp(NH)p) have been studied. These conditions have been used to generate forskolin dose-response curves of AC activation. A plot of enzyme activation versus apparent forskolin-binding showed a linear and a nonlinear relationship, respectively, in the absence or presence of the other two stimulators. The latter relationship can be fitted by two linear regression lines with a defined intercept, the slopes of which represent two distinct binding-activation (B-A) effects. The B-A effects of forskolin for rat adipocyte and liver membranes in the absence of stimulatory hormone and Gpp(NH)p were 10 and 8 (pmol X min-1) X (pmol)-1, respectively. The B-A effects for the same membranes in the presence of the other two stimulators were 69 (high) and 13 (low) (pmol X min-1) X (pmol)-1 for adipocyte membrane, and 83 (high) and 9 (low) (pmol X min-1) X (pmol)-1 for liver membrane. The ratio of potentiation of forskolin-activated enzyme activity to the unmodified forskolin-stimulated activity (P-A ratio) was determined without the binding data. At 3 microM forskolin, with and without 230 epinephrine and 10 microM Gpp(NH)p, the P-A ratio was 3.7, decreasing to 1.1 with the addition 100 microM forskolin. The line representing a high B-A effect and a resulting high P-A ratio appears to describe the interactions between forskolin and the AC stimulated by epinephrine and Gpp(NH)p. The line of low B-A effect may represent the interaction between forskolin and the basal AC. Two peaks of AC activity were eluted from forskolin-Sepharose column. They have apparent differences in sensitivity to Gpp(NH)p and affinity for forskolin. Based on the results available thus far, with consideration for known limitations of the methodology, a working model has been proposed for forskolin activation of AC.

Adenylyl Cyclases↗
PubMed1985

Deacetylation of forskolin catalyzed by bovine brain membranes.

Radiolabeled forskolin, 7-(3H-acetyl)-forskolin, was synthesized to explore interactions between forskolin and bovine brain membrane preparations. The radiolabeled derivative was chemically characterized, and found to be indistinquishable from unlabeled forskolin in its ability to stimulate bovine brain adenylate cyclase. Preliminary binding data demonstrated that binding of 7-(3H-acetyl)-forskolin to membranes was concentration dependent. However, competition binding studies using a constant concentration of 7-(3H-acetyl)-forskolin with increasing levels of unlabeled forskolin showed enhanced binding of the labeled derivative. This suggested that 7-(3H-acetyl)-forskolin was degraded by membranes and protected by native forskolin. Incubation of forskolin with membranes and analysis of the products by thin layer chromatography and mass spectroscopy showed the formation of 7-desacetylforskolin. The deacetylation of forskolin was monitored by quantitating the release of [3H]acetate from 7-(3H-acetyl)-forskolin. The reaction was linear with time and protein concentration. These data illustrate that forskolin can be degraded by membranes and indicate that ligand binding studies using labeled forskolin and membrane preparations should be cautiously interpreted.

Acetylation↗
PubMed1995

1-Acetyl-7-deacetylforskolin: a potential non-specific inactive analog of forskolin for estimation of its specific high-affinity binding and adenylyl cyclase stimulation in vitro.

Labeled and unlabeled 1-acetyl-7-deacetylforskolin and forskolin were synthesized by acetylation of 7-deacetylforskolin with labeled and unlabeled acetyl chloride. The binding of 1-acetyl[1-acetyl-11C]-7-deacetylforskolin ([11C]1-acetyl-7-deacetylforskolin) and [7-acetyl-11C]forskolin ([11C]forskolin) to rat brain membranes was studied using filtration assay. The [11C]forskolin binding was decreased with an increasing load of unlabeled forskolin, whereas [11C]1-acetyl-7-deacetyl-forskolin binding was always very low, the level of which agreed with that of the non-specific binding in forskolin. However, binding of [7-acetyl-11C]1,9-dideoxyforskolin, which has been used as a non-specific inactive analog of forskolin, had a higher binding ratio than that of the non-specific binding of forskolin. The binding of [11C]forskolin was not affected by an increased load of cold 1-acetyl-7-deacetylforskolin. Forskolin activated adenylyl cyclase (AC) in cultured human endothelial cells, whereas 1-acetyl-7-deacetylforskolin did not. These data show that the 1-acetyl-7-deacetylforskolin lacks specific binding affinity and the ability to stimulate AC, while it has similar physical properties with forskolin. The compound 1-acetyl-7-deacetylforskolin would be a suitable "non-specific inactive analog" of forskolin with which to estimate its specific high-affinity binding capacity and to validate forskolin-specific AC stimulation in vitro.

Adenylyl Cyclases↗
PubMed1986

Use of forskolin to study the relationship between cyclic AMP formation and bone resorption in vitro.

The effect of the adenylate cyclase activator forskolin on bone resorption and cyclic AMP accumulation was studied in an organ-culture system by using calvarial bones from 6-7-day-old mice. Forskolin caused a rapid and fully reversible increase of cyclic AMP, which was maximal after 20-30 min. The phosphodiesterase inhibitor rolipram (30 mumol/l), enhanced the cyclic AMP response to forskolin (50 mumol/l) from a net cyclic AMP response of 1234 +/- 154 pmol/bone to 2854 +/- 193 pmol/bone (mean +/- S.E.M., n = 4). The cyclic AMP level in bones treated with forskolin (30 mumol/l) was significantly increased after 24 h of culture. Forskolin, at and above 0.3 mumol/l, in the absence and the presence of rolipram (30 mumol/l), caused a dose-dependent cyclic AMP accumulation with an calculated EC50 (concentration producing half-maximal stimulation) value at 8.3 mumol/l. In 24 h cultures forskolin inhibited spontaneous and PTH (parathyroid hormone)-stimulated 45Ca release with calculated IC50 (concentration producing half-maximal inhibition) values at 1.6 and 0.6 mumol/l respectively. Forskolin significantly inhibited the release of 3H from [3H]proline-labelled bones stimulated by PTH (10 nmol/l). The inhibitory effect by forskolin on PTH-stimulated 45Ca release was significant already after 3 h of culture. In 24 h cultures forskolin (3 mumol/l) significantly inhibited 45Ca release also from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxycholecalciferol (0.1 mumol/l). The inhibitory effect of forskolin on spontaneous and PTH-stimulated 45Ca release was transient. A dose-dependent stimulation of basal 45Ca release was seen in 120 h cultures, at and above 3 nmol of forskolin/l, with a calculated EC50 value at 16 nmol/l. The stimulatory effect of forskolin (1 mumol/l) could be inhibited by calcitonin (0.1 unit/ml), but was insensitive to indomethacin (1 mumol/l). Forskolin increased the release of 3H from [3H]proline-labelled bones cultured for 120 h and decreased the amount of hydroxyproline in bones after culture. Forskolin inhibited PTH-stimulated release of Ca2+, Pi, beta-glucuronidase and beta-N-acetylglucosaminidase in 24 h cultures. In 120 h cultures forskolin stimulated the basal release of minerals and lysosomal enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Kinetics and thermodynamics of the binding of forskolin to the galactose-H+ transport protein, GalP, of Escherichia coli.

The binding of the transport inhibitor, forskolin, to the galactose-H+ symporter, GalP, of Escherichia coli was evaluated by equilibrium and time-resolved fluorescence measurements. A quench in protein fluorescence of 8-12% was observed upon the binding of forskolin. The overall dissociation constant (Kd) for forskolin determined by fluorescence titration ranged between 1.2 and 2.2 microM, which is similar to that reported from equilibrium dialysis measurements of the binding of [3H]forskolin (Kd = 0.9-1.4 microM). The kinetics of forskolin binding were measured by stopped-flow fluorescence methods. The protein fluorescence was quenched in a biphasic manner; the faster of these two rates was dependent on the concentration of forskolin and was interpreted as the initial binding step from which both the association (kon) and dissociation (koff) rate constants were determined. The association and dissociation rate constants were 5.4-6.2 microM-1.s-1 and 5.1-11.5 s-1 respectively, and the Kd was calculated to be 1.5 microM. The binding of forskolin was inhibited by D-galactose, but not by L-galactose, and displacement by sugar provided an additional method to calculate the dissociation rate constant for forskolin (koff = 12.4-13.0 s-1). The rate of the slow change in protein fluorescence (3-5 s-1) was independent of the forskolin concentration, indicating an isomerization of the transporter between different conformations, possibly outward- and inward-facing forms. These kinetic parameters were determined at a series of temperatures, so that the thermodynamics of forskolin binding and transporter re-orientation could be analysed. The binding process was entropically driven (delta S = 83.7 J.K-1.mol-1; delta H = 8.25 kJ.mol-1), similar to that for cytochalasin B, which is also an inhibitor of GalP. Measurements of the binding of [3H]forskolin by equilibrium dialysis revealed competitive displacement of bound forskolin by cytochalasin B, possibly suggesting that the sugar, forskolin and cytochalasin B binding sites are overlapping; the Kds for forskolin and cytochalasin B were calculated to be 0.85 microM and 4.77 microM respectively, and the concentration of binding sites was 10.2 nmol.mg-1.

Bacterial Proteins↗
PubMed1986

3',5'-cyclic adenosine monophosphate as an intracellular second messenger of luteinizing hormone: application of the forskolin criteria.

The role of adenosine 3',5'-cyclic monophosphate (cAMP) as an intracellular second messenger of luteinizing hormone (LH) was reinvestigated in vitro with diterpene forskolin, a highly specific activator of adenylate cyclase. Treatment of cultured testicular cells from adult hypophysectomized rats with increasing concentrations (10(7)-10(-4) M) of forskolin produced dose-dependent increments in cAMP and testosterone accumulation. Concomitant blockade of cAMP-phosphodiesterase activity with 3-isobutyl-1-methyl-xanthine (10(-4) M) resulted in significant (P less than 0.05) enhancement of the forskolin effect for all but the 10(-4) M forskolin dose. Potency evaluation as judged by half-maximal stimulation of testosterone accumulation revealed median effective doses (mean +/- SE) of 1.25 +/- 0.2 x 10(-5), 1.7 +/- 0.5 x 10(-5), and 2.5 +/- 0.4 x 10(-10) M for forskolin, N6, O2'-dibutyryl cAMP (Bt2cAMP), and human chorionic gonadotropin (hCG), respectively. Examination of the time requirements of forskolin disclosed time-dependent increments in the accumulation of extracellular cAMP and testosterone, the earliest significant (P less than 0.05) increases being noted by 6 hr of treatment. In comparison, a minimal time requirement of less than or equal to 12 hr was noted for hCG- and choleragen-stimulated androgen biosynthesis, whereas the apparent onset of action of Bt2cAMP was delayed to the 24-hr time point. Although 10(-7) M of forskolin by itself did not alter the accumulation of testosterone, its addition resulted in substantial amplification of the hCG effect, producing a 4.6-fold reduction in the median effective dose (ED50) of hCG. Moreover, concurrent treatment with this functionally inert dose of forskolin rendered steroidogenically inert doses of hCG (eg, 10(-11) or 3 x 10(-11) M) steroidogenically potent. However, combined treatment with maximally stimulatory doses of Bt2cAMP (10(-4) M) and one of several testicular cell agonists [forskolin (10(-4) M), choleragen (10(-9) M) or hCG (10(-9) M)] did not prove additive. Taken together, our findings indicate that forskolin, like LH, is capable of stimulating testicular cAMP generation as well as androgen biosynthesis and that a functionally inert low dose of forskolin can significantly amplify LH hormonal action. Inasmuch as forskolin-stimulated and forskolin-amplified hormonal action are acceptable as novel criteria of cAMP dependence, our observations provide new evidence in keeping with the notion that cAMP may be in intracellular second messenger of LH.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Forskolin inhibits platelet-activating factor binding to platelet receptors independently of adenylyl cyclase activation.

The effect of forskolin on platelet-activating factor (PAF) receptor was investigated. Rabbit platelets treated with forskolin showed approximately a 9-fold increase in cAMP levels over the control. After treatment of platelets with forskolin prior to PAF binding, a 30-40% (P < 0.005) decrease in PAF binding was observed. The decrease in PAF binding caused by forskolin was concomitant with a decrease in the physiological responses of platelets induced by PAF. However, this forskolin-induced decrease in PAF binding was not a consequence of cAMP formation as the addition of a cAMP analog could not mimic the action of forskolin. Additionally, the inactive analog of forskolin, dideoxyforskolin, which does not activate adenylyl cyclase, also reduced PAF binding to its receptor. Reduction of PAF binding by forskolin and dideoxyforskolin was also observed with isolated platelet membranes. To understand the mechanism of forskolin induced changes in PAF binding, the involvement of a G-protein in this process was investigated. Cells treated with GTP gamma S showed approximately a 25% reduction in PAF binding. Addition of forskolin to the GTP gamma S treated cells resulted in a further reduction in PAF binding, suggesting the action of forskolin was independent of G-protein activation. The data suggests that the action of forskolin was independent of adenylyl cyclase or G-protein involvement. It is speculated that the action of forskolin on PAF binding was due to a direct effect of this molecule and its analog on the PAF receptor itself or to components of the post-receptor signalling for PAF.

Adenylyl Cyclases↗
PubMed1987

Forskolin refractoriness. Exposure to the diterpene alters guanine nucleotide-dependent adenylate cyclase and calcium-uptake activity of cells cultured from the rat aorta.

Cells with the morphological properties of endothelial cells were cultured from the rat aorta. The cultured cells accumulated 45Ca2+ from the medium in a manner which was stimulated by forskolin and by 8-bromo-cyclic AMP. Pretreating the cultures for 20 h with forskolin diminished forskolin-dependent Ca2+-uptake activity. Adenylate cyclase activity of cultured cell homogenates was stimulated by guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) and forskolin, and by isoprenaline in the presence, but not in the absence, of guanine nucleotide. p[NH]ppG increased forskolin sensitivity and caused a leftward shift in the forskolin dose-response curve. Pretreating the cultured cells with forskolin for 20 h, conditions that decreased forskolin-dependent Ca2+ uptake, increased basal and guanine nucleotide-dependent adenylate cyclase activity, but not forskolin-dependent activity determined in the absence of p[NH]ppG. Forskolin pretreatment diminished p[NH]ppG's capacity to increase forskolin sensitivity, but did not have a significant effect on either the sensitivity of adenylate cyclase to p[NH]ppG or its responsiveness to isoprenaline. These results suggest that the Ca2+-uptake mechanism is cyclic AMP-dependent and that guanine nucleotides mediated forskolin-dependent cyclic AMP production by the intact cells. In addition, there may be different guanine nucleotide requirements for hormone-receptor coupling and forskolin activation.

Adenylyl Cyclases↗
PubMed1988

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.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Effects of guanine nucleotides and divalent cations on forskolin activation of rabbit luteal adenylyl cyclase: evidence for the existence of an inhibitory guanine nucleotide-binding regulatory component.

The effects of guanine nucleotides and divalent cations on the activation of rabbit luteal adenylyl cyclase by the diterpene forskolin were investigated. Saturating concentrations of forskolin elicited 10- to 15-fold stimulation of adenylyl cyclase activity in the absence of added guanine nucleotide. No lag was observed in the time course of forskolin-induced activation. Addition of 10 microM guanosine triphosphate (GTP) and guanyl-5'-yl imidodiphosphate [GMP-P(NH)P] inhibited forskolin activation by 10-15% and 30-40%, respectively, in the presence of 3.0 mM MgCl2. GMP-P(NH)P was more potent than GTP in inhibiting forskolin activation of adenylyl cyclase having an IC50 of 36 nM compared to 610 nM for GTP. In contrast, the Kact for stimulating adenylyl cyclase activity by both GMP-P(NH)P and GTP were similar, 1.00 and 0.86 microM, respectively. GMP-P(NH)P-induced inhibition of the forskolin-activated enzyme was not due to the hysteretic nature of GMP-P(NH)P activation of luteal adenylyl cyclase, as addition of GMP-P(NH)P to an enzyme that had been treated 5 min earlier with forskolin resulted in the immediate inhibition of enzymatic activity. Addition of GMP-P(NH)P to a concentration-effect curve for forskolin increased the Kact value for forskolin form 7.18 to 26.8 microM. There was a different MgCl2 concentration requirement for maximal stimulation of luteal cyclase by GMP-P(NH)P (8 mM MgCl2) and maximal inhibition of forskolin-stimulated activity by GMP-P(NH)P (0.5-0.6 mM MgCl2). Further, MnCl2 concentrations above 1.0 mM completely abolished the inhibitory action of GMP-P(NH)P on forskolin activation of luteal cyclase. In fact, at 2.0 mM MnCl2, adenylyl cyclase activity in the presence of GMP-P(NH)P plus forskolin was greater than that of forskolin alone. Thus taken together, these findings suggest that the rabbit corpus luteum contains an inhibitory guanine nucleotide-binding regulatory component in addition to a stimulatory regulatory component. Further, these components demonstrate differing requirements for both guanine nucleotides and divalent cations in order to interact with the catalytic moiety of adenylyl cyclase. The existence of such an inhibitory component suggests the presence of an inhibitory receptor in the corpus luteum which could negatively regulate adenylyl cyclase resulting in the inhibition of cAMP production and reduced progesterone output from the corpus luteum under normal physiological conditions.

Adenylyl Cyclases↗