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At least 19 recordsLinked to original sources

PubMed1990

Irreversible loss of [3H]forskolin binding sites in human platelets by alpha-haloacetyl analogs of forskolin.

The 7-bromoacetyl-7-desacetyl (BrAcFsk) and 7-chloroacetyl-7-desacetyl (CIAcFsk) analogs of forskolin were synthesized as alkylating agents to study the high affinity binding sites for forskolin. BrAcFsk and CIAcFsk activated adenylate cyclase in human platelet membranes with EC50 values of about 20 and 12 microM, respectively. Both analogs increased cyclic AMP in human platelets; however, they were less potent that forskolin. Forskolin inhibited [3H]forskolin binding to human platelet membranes with an IC50 of 20 nM, whereas BrAcFsk and CIAcFsk inhibited [3H] forskolin binding with IC50 values of 0.1 microM. Pretreatment of intact platelets with 10 microM BrAcFsk caused a 90% irreversible loss in [3H]forskolin binding sites, whereas pretreatment with 10 microM CIAcFsk led to a loss of 55% of the binding sites. The loss of binding sites occurred within 5 min for BrAcFsk and within 30 min for CIAcFsk. The time required for the loss of binding sites produced by either alkylating agent was increased by the inclusion of 200 microM forskolin in the pretreatment buffer. The inactive bromoacetyl analog of forskolin 7-bromoacetyl-7-desacetyl-1.9-dideoxyforskolin (1,9-dideoxy-BrAcFsk) did not activate adenylate cyclase, inhibit [3H]forskolin binding, or cause an irreversible loss of [3H]forskolin binding sites. Adenylate cyclase was assayed in membranes from platelets treated with either 10 microM BrAcFsk or 10 microM 1,9-dideoxy-BrAcFsk. The stimulation of adenylate cyclase by prostaglandin E1, guanosine-5'-O-(3-thio)triphosphate, and AIF4 was inhibited by about 50% in membranes from platelets treated with BrAcFsk. However, the stimulation of adenylate cyclase by forskolin was unaffected by preincubation with BrAcFsk. Pretreatment of human platelets with 1,9-dideoxy-BrAcFsk had no effect on the stimulation of adenylate cyclase by prostaglandin E1, AIF4, or forskolin.

Adenylyl Cyclases↗

PubMed1992

Interaction of aminoalkylcarbamates of forskolin with adenylyl cyclase: synthesis of an iodinated derivative of forskolin with high affinity for adenylyl cyclase.

7-(2-Aminoethyl)aminocarbonyl-7-desacetylforskolin (7-AEC-Fsk) and 6-(2-aminoethyl)aminocarbonylforskolin (6-AEC-Fsk) were synthesized and tested for their ability to activate adenylyl cyclase and inhibit the high affinity binding of [3H]forskolin to bovine brain membranes. Forskolin and 7-AEC-Fsk were equipotent in activating adenylyl cyclase, with EC50 values of about 4 microM, whereas 6-AEC-Fsk had an EC50 of about 2 microM. 6-AEC-Fsk and 7-AEC-Fsk stimulated adenylyl cyclase about 7-fold over basal levels at 100 microM, whereas forskolin produced a 5-fold stimulation. Forskolin and 6-AEC-Fsk inhibited the binding of [3H]forskolin to bovine brain membranes with Kd values of 41 nM and 28 nM, respectively, whereas 7-AEC-Fsk had a Kd of 83 nM. The 3-(3-iodo-4-hydroxyphenyl)propionamide derivative of 6-AEC-Fsk (6-I-HPP-Fsk) was more potent than forskolin in inhibiting [3H]forskolin binding to bovine brain membranes, with a Kd of 14 nM. 6-AEC-Fsk was reacted with 125I-labeled Bolton-Hunter reagent to produce 6-125I-HPP-Fsk with a specific activity of 2175 Ci/mmol. 6-125I-HPP-Fsk bound to bovine brain membranes with a Kd of 13 nM and a Bmax of 3.8 pmol/mg of protein. Forskolin inhibited the binding of 6-125I-HPP-Fsk to bovine brain membranes with a Kd of 31 nM, whereas 1,9-dideoxyforskolin only slightly inhibited the binding at 10 microM. The binding of 6-125I-HPP-Fsk was not inhibited by agents that inhibit forskolin binding to the glucose transporter, such as D-glucose or cytochalasin B. There was no displaceable binding of 6-125I-HPP-Fsk to red blood cell membranes, which contain a large concentration of the glucose transporter. Pretreatment of bovine brain membranes with an alkylating derivative of forskolin, 7-bromoacetyl-7-desacetylforskolin (BrAcFsk), led to an irreversible decrease in the binding of [3H]forskolin and 6-125I-HPP-Fsk. The time dependence and concentration dependence for the BrAcFsk-induced decrease in [3H]forskolin binding sites were identical to those observed for the decrease in 6-125I-HPP-Fsk binding sites. 6-125I-HPP-Fsk binding was determined in human platelet membranes in the presence of Mg2+ alone and in combination with guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or AIF4-. The presence of GTP gamma S or AIF4- increased the binding of 6-125I-HPP-Fsk by 4.5-fold and 4-fold, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclases↗

PubMed1991

(Aminoalkyl)carbamates of forskolin: intermediates for the synthesis of functionalized derivatives of forskolin with different specificities for adenylyl cyclase and the glucose transporter.

(Aminoalkyl)carbamates of forskolin were synthesized at the 6- and 7-hydroxyl positions of forskolin with the length of the alkyl chain varying from ethyl to heptyl. Two of these derivatives, 7-[[(2-aminoethyl)amino]carbonyl]-7-desacetylforskolin (2) and 6-[[(2-aminoethyl)amino]carbonyl]forskolin (3), were used to synthesize iodinated derivatives of forskolin that bind with high affinity to adenylyl cyclase in bovine brain membranes and the glucose transporter in human erythrocyte membranes, respectively. Hydroxyphenyl derivatives of forskolin were prepared from the (aminoalkyl)carbamates and tested for their ability to bind to adenylyl cyclase in bovine brain membranes and the glucose transporter in human erythrocyte membranes. The 6-derivative (18) of forskolin had a Kd of 9 nM at adenylyl cyclase and was more potent than either the 7-derivatives or the 6-derivatives of 7-desacetylforskolin. The 7-derivatives were more potent at binding to the glucose transporter than forskolin. In contrast, the 6-derivatives had Kd's greater than 100 microM at the glucose transporter. Isothiocyanates and N-bromoacetyl derivatives were synthesized from 2 and 3 as potential alkylating agents for forskolin binding sites. The alkylating agents produced an irreversible loss of forskolin binding to adenylyl cyclase. In contrast, the alkylating agents bound reversibly to the glucose transporter.

Adenylyl Cyclases↗

PubMed1990

Effects of forskolin and 1,9-dideoxy-forskolin on cochlear potentials.

Endocochlear potential (EP) and cochlear microphonics (CM) were recorded during the perilymphatic perfusion with forskolin known as an adenylate cyclase stimulant. Forskolin produced a reversible EP elevation in a dose-dependent manner. Perfusion with 1,9-dideoxy-forskolin, an analogue of forskolin that does not stimulate adenylate cyclase, had no effect on EP, whereas perfusions with other agents that raise the cAMP-level (IBMX, a phosphodiesterase inhibitor, and dbcAMP) duplicated the effect of forskolin. The vigorous CM during the EP elevation and the large negative EP induced by anoxia superimposed on the elevated EP indicate that the K+ diffusion potential through the hair cell membrane cannot be altered by forskolin. The results suggest that the adenylate cyclase system in the stria vascularis and/or Reissner's membrane may modulate the generation of EP.

1-Methyl-3-isobutylxanthine↗

PubMed1990

Effects of alpha 2-adrenergic agonist preincubation on subsequent forskolin-stimulated adenylate cyclase activity and [3H]forskolin binding in membranes from HT29 cells.

alpha 2-Adrenergic agonist preincubation resulted in a leftward shift in the subsequent concentration-response curve to forskolin-stimulated adenylate cyclase activity in membranes from HT29 cells, a human colon adenocarcinoma cell line. This effect was much less pronounced than the effect seen in the intact cell cyclic AMP production assays. Removal of GTP from the assay caused a further slight leftward shift in the concentration-response curve. In [3H]forskolin binding experiments, alpha 2-adrenergic agonist preincubation caused a doubling of the maximal number of binding sites (80 vs 31 fmol/mg protein) compared to control. The addition of MgCl2 and NaF to the assay buffer increased control binding 5-fold. With agonist preincubation, there was a further increase in binding in the presence of MgCl2 and NaF which was not significantly different from the appropriate control. Pertussis toxin pretreatment blocked both the leftward shift in the forskolin concentration-response curve and the increase in maximal number of binding sites, indicating that a pertussis toxin sensitive protein is involved in these changes. Activation of cyclic AMP production in the intact cell by cholera toxin followed by norepinephrine preincubation and then stimulation by forskolin resulted in a degree of sensitization similar to that seen in the membrane adenylate cyclase and binding assays. Pertussis toxin also blocked this sensitization. It appears that if the cyclase system is highly activated, then the degree of sensitization is similar in the membrane and intact cell assay.

Adenylate Cyclase Toxin↗

PubMed1989

Effects of either forskolin, the 1,9-dideoxy derivative of forskolin, or 8-bromocyclic AMP on cyclic AMP and melatonin production in the Syrian hamster pineal gland in organ culture.

The possible involvement of cyclic AMP in the regulation of melatonin production was investigated in cultured Syrian hamster pineal glands. Addition of forskolin, an adenylate cyclase activator, to the incubation medium caused marked increases in both cyclic AMP and melatonin levels in glands collected in the second half of the dark period. 8-Bromocyclic AMP, an analogue of cyclic AMP, also increased melatonin production. The 1,9-dideoxy derivative of forskolin, which is unable to activate the cyclase, was ineffective in stimulating either cyclic AMP levels or melatonin production. These results support a primary role for cyclic AMP in the nocturnal increase of melatonin production in the Syrian hamster pineal gland.

8-Bromo Cyclic Adenosine Monophosphate↗

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↗

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↗

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↗

PubMed1990

High affinity forskolin inhibition of L-type Ca2+ current in cardiac cells.

The diterpene forskolin is widely known for its ability to directly activate adenylyl cyclase and consequently increase intracellular cAMP. In cardiac cells, one result is a cAMP-mediated increase in the L-type Ca2(+)-channel current (ICa). However, forskolin was also shown recently to affect a number of ionic channels in noncardiac cells by mechanisms that do not involve activation of adenylyl cyclase. The present study reveals such an effect of forskolin on cardiac Ca2+ channels. Indeed, under appropriate conditions, forskolin was found to cause an inhibition of ICa. Although the stimulation of adenylyl cyclase and ICa requires micromolar concentrations of forskolin, the inhibitory effect of forskolin was observed in the nanomolar range of concentrations, i.e., 2-3 orders of magnitude lower. This high affinity forskolin inhibition of ICa was observed when ICa was previously enhanced via a cAMP-dependent pathway, but not when ICa was at its basal level or when the current was elevated by the dihydropyridine Bay K 8644. The inhibitory effect occurred at a site of action remote from adenylyl cyclase, because forskolin similarly inhibited ICa that had been previously elevated by isoprenaline (a beta-adrenergic agonist) or directly by intracellular perfusion with cAMP. Under these conditions, forskolin was inhibitory when applied to either side of the cell membrane, but only in its lipid-soluble form. The inhibitory effect of forskolin appeared to be independent of membrane potential and was not accompanied by a change in the time constants of ICa activation and inactivation. This may indicate that forskolin mainly reduces the number of functional Ca2+ channels without changing the gating of individual channels. However, the reduction in ICa amplitude was not equally distributed among the different exponential components that constitute ICa, which suggests that forskolin also modifies the resting state of the channels. This novel high affinity forskolin inhibition of ICa may take place at some step in the pathway between cAMP and Ca2+ channel phosphorylation and/or at Ca2+ channels only after they have been phosphorylated.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1989

Forskolin inhibits the Gs-stimulated adenylate cyclase in rat ascites hepatoma AH66F cells.

Forskolin increased intracellular cyclic AMP and augmented cyclic AMP formation by prostaglandin E1 (PGE1) in normal rat hepatocytes and ascites hepatoma AH66 cells. However, in AH66F cells which were derived from the AH66 cell line, the diterpene only slightly increased the cyclic AMP level, and dose-dependently inhibited the accumulation caused by PGE1. Forskolin dose-dependently activated adenylate cyclase in these membranes, and the magnitude of activation by forskolin was largest in the following order: hepatocytes, AH66 cells, and AH66F cells. This difference may be based on the number of forskolin-binding sites. The binding affinity of forskolin for each cell membrane was similar. The number and affinity of forskolin-binding sites in these cells were not influenced by 5'-guanylylimidodiphosphate [Gpp(NH)p]. In hepatocytes and AH66 cells, forskolin and other adenylate cyclase activators such as PGE1, GTP, Gpp(NH)p, F-, and Mn2+ synergistically increased the enzyme activity. In AH66F cells, the forskolin-stimulated activity was hardly influenced by the GTP analog, and forskolin diminished the activities induced by the GTP analog in a manner similar to that of diterpene alone. Forskolin (10 microM) also significantly inhibited the activities induced by PGE1, GTP, and F-. The effect of forskolin with Mn2+ was additive in AH66F cells. The data suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide-binding protein and the catalytic unit in the membrane of normal hepatocytes and AH66 cells, but it interferes with the coupling in AH66F cells.

Adenylyl Cyclases↗

PubMed1990

Reduction of voltage-activated K+ currents by forskolin is not mediated via cAMP in pleural sensory neurons of Aplysia.

1. Forskolin is often used to activate adenylate cyclase in studies relating adenosine 3',5'-cyclic monophosphate (cAMP) to the modulation of membrane current. There is growing concern, however, that some actions of forskolin are independent of cAMP. With the use of two-electrode voltage-clamp techniques, we compared the effects of analogues of cAMP to the effects of forskolin on K+ currents in somata of sensory neurons that were isolated from pleural ganglia of Aplysia californica. 2. Analogues of cAMP did not reduce the peak amplitude of either the transient K+ current (IA) or the voltage-dependent K+ current (IK.V). Analogues of cAMP did reduce the previously described cAMP-sensitive S K+ current (IK.S). In contrast, forskolin reduced the peak amplitude of both IA and IK.V. Furthermore, both IA and IK.V were reduced by 1,9-dideoxy-forskolin, a derivative of forskolin that does not activate adenylate cyclase. These results indicate that the effects of forskolin and 1,9-dideoxy-forskolin on IA and IK.V were not mediated via cAMP. 3. Bath application of a modified form of forskolin (7-deacetyl-6-[N-acetylglycyl]-forskolin), which has enhanced water solubility and activates adenylate cyclase, reduced IK.S, but did not alter either IA or IK.V. Thus it appears that certain derivatives of forskolin can be used to activate adenylate cyclase and avoid some of the nonspecific actions on membrane current that are associated with forskolin.

Adenylyl Cyclase Inhibitors↗

PubMed1989

Forskolin activates voltage-dependent Ca2+ channels in bovine but not in rat fasciculata cells.

The action of forskolin on bovine and rat fasciculata cells was examined in freshly prepared cells. Bovine cells show a close parallelism between production of steroids and production of cAMP as a function of the concentration of ACTH up to 10(-8) M. By contrast, forskolin (10(-7)-10(-5) M) causes a similar increase in steroid synthesis but relatively little effect on the production of cAMP. cAMP-dependent protein kinase shows a similar response to ACTH but no response to forskolin in the same range of concentrations. ACTH and forskolin, at submaximal concentrations, cause greater steroid production when added together than when added separately, but the two agents at high concentrations produce the same response whether added together or separately. The inhibitors of voltage-dependent Ca2+ channels inhibit the steroidogenic response to forskolin (IC50 for nifedipine is 0.1 microM and for Py108-068 is 0.4 microM). A Ca2+ channel agonist (BAY K8644) increases the steroidogenic response of bovine adrenal cells to forskolin, but not that of ACTH. Finally, forskolin causes a concentration-dependent uptake of Ca2+ by these cells; in the concentration range of 0.1-10 microM, forskolin caused an increase in [Ca2+] from 185 nM to 345 nM. By contrast, forskolin caused some stimulation of the production of cAMP, but not that of steroids in rat fasciculata cells. It is concluded that in bovine fasciculata cells forskolin activates voltage-dependent Ca2+ channels with a consequent increase in steroid synthesis. This effect is independent of the well known action of forskolin on adenylate cyclase. Rat fasciculata cells, on the other hand, do not possess such Ca2+ channels and do not show a steroidogenic response to forskolin.

Adenylyl Cyclase Inhibitors↗

PubMed1992

Inhibition of 25-hydroxyvitamin D3-24-hydroxylase by forskolin: evidence for a 3',5'-cyclic adenosine monophosphate-independent mechanism.

Forskolin has long been used to demonstrate the involvement of cAMP in the regulation of cellular function, by virtue of its ability to stimulate adenylate cyclase directly. Recently, however, forskolin has been shown to affect plasma membrane transporter and channel function in a manner unrelated to cAMP. The present study examines whether forskolin-mediated inhibition of a mitochondrial membrane-associated enzyme, 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), also occurs by a cAMP-independent mechanism. Both forskolin and PTH stimulated cAMP accumulation and inhibited 24-hydroxylase activity in a dose-dependent manner in fresh mouse renal tubules. However, the level of inhibition of 24-hydroxylase achieved with forskolin was consistently greater than that obtained with PTH, at comparable levels of cAMP. 1',9'-Dideoxyforskolin, a cyclase-inactive analog of forskolin, also inhibited 24-hydroxylase activity, without stimulating cAMP production. Moreover, both forskolin and 1',9'-dideoxyforskolin directly inhibited 24-hydroxylase in isolated renal mitochondria. Kinetic analysis revealed a competitive mode of inhibition for both agents; however, 1',9'-dideoxyforskolin proved to be a more potent inhibitor of 24-hydroxylase than forskolin (inhibitory constant, 0.25 vs. 22 microM, respectively). Finally, both forskolin and 1',9'-dideoxyforskolin also inhibited inducible 24-hydroxylase in renal tubules prepared from 1,25-(OH)2D3-treated mice. However, inducible 24-hydroxylase activity was less susceptible to inhibition by the diterpenes than the basal enzyme activity. The present study provides evidence for cAMP-independent inhibition of 24-hydroxylase by forskolin and represents the first demonstration of a cAMP-independent effect of forskolin on a protein that is not a plasma membrane-associated transporter or channel. Our data advocate caution in the interpretation of studies using forskolin to assess the role of cAMP in cellular processes.

Animals↗

PubMed1990

Is adenosine involved in inhibition of forskolin-stimulated cyclic AMP accumulation by caffeine in rat brain?

Caffeine potently inhibited forskolin-stimulated cyclic AMP accumulation in slices of rat cerebral cortex, with an IC50 of 21 +/- 3 microM. Because caffeine competitively blocks adenosine receptors, we examined whether the action of forskolin involved endogenous adenosine or whether caffeine was acting through some novel mechanism. Inhibition by caffeine was observed at all forskolin concentrations examined, although the degree of inhibition decreased at higher concentrations of forskolin. The effect of caffeine was not blocked by the presence of a phosphodiesterase inhibitor but was mimicked by several other methylxanthines. The most potent of these was 8-(p-sulfophenyl)-theophylline, which does not readily cross cell membranes, arguing for an extracellular site of action. Addition of either adenosine or the adenosine uptake blocker dipyridamole potentiated the forskolin response, suggesting that forskolin and adenosine act synergistically in increasing cyclic AMP accumulation. The nonxanthine adenosine receptor antagonist CGS 15943 potently blocked cyclic AMP responses to forskolin, adenosine, and combinations. 3-Isobutyl-1-methylxanthine potently blocked the response to adenosine but caused little or no inhibition of the response to forskolin. Adenosine deaminase (ADA) was added to eliminate contributions of endogenous adenosine. ADA inhibited the response to both adenosine and forskolin; however, 200 times as much enzyme was necessary to inhibit the forskolin response. Inhibition of added ADA with 2'deoxycoformycin dramatically increased the concentration of ADA required to inhibit the adenosine response, without altering the concentration required to inhibit the forskolin response. These results suggest that forskolin-stimulated cyclic AMP accumulation may be partially dependent on endogenous adenosine but that the inhibition observed with caffeine is not solely due to blockade of adenosine receptors.

Adenosine↗

PubMed1990

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.

Adrenal Gland Neoplasms↗

PubMedDate not supplied

Vasodilation produced by forskolin compared with that produced by adenosine in rabbit coronary artery.

We compared the relaxing action of forskolin with that of adenosine in rabbit coronary artery and analyzed the pharmacological properties of this effect of forskolin. In preparations of this artery precontracted by 10-16 mM KCl, the addition of forskolin (10(-9)-10(-5) M) and adenosine (10(-9)-10(-4) M) to the organ bath produced dose-dependent relaxations. The mean EC50 for the relaxing action of forskolin was 4.5 X 10(-8) M and that of adenosine was 3.6 X 10(-7) M, forskolin being 10 times more potent than adenosine. Relaxation produced by forskolin was not affected by treatment with propranolol (10(-5) M), atropine (10(-6) M), or 8-phenyltheophylline (10(-6) M), but was competitively inhibited by ouabain (10(-6) M). The relaxation produced by adenosine was inhibited by 8-phenyltheophylline (10(-6) M) competitively and by ouabain (10(-6) M) noncompetitively. In preparations precontracted by a higher concentration of KCl, 40 mM, forskolin produced full relaxation with a shift of the dose-response curve to the right; adenosine did not produce full relaxation. Both forskolin and adenosine attenuated the maximum contraction produced by Ca2+. These findings indicate that the vasorelaxing effect of forskolin was not due to activation of beta-adrenoceptors, muscarinic receptors, or adenosine receptors, whereas that of adenosine was due to activation of adenosine receptors. Increased availability of intracellular Ca2+ competitively inhibited the relaxation induced by forskolin and noncompetitively inhibited the relaxation induced by adenosine. Both forskolin and adenosine noncompetitively inhibited contraction induced by Ca2+.

Adenosine↗

PubMed1990

Forskolin stimulates prostaglandin synthesis in rabbit heart by a mechanism that requires calcium and is independent of cyclic AMP.

Infusion of forskolin, an adenylate cyclase activator, in concentrations (2 microM) that do not alter basal prostaglandin (PG) synthesis inhibit synthesis of PG elicited by isoproterenol in rabbit heart. This inhibitory action of forskolin appears to be dependent on cyclic AMP (cAMP). Bolus injection of forskolin (75 nmol), however, was found to stimulate PG synthesis in rabbit heart. The purpose of this study was to elucidate the mechanism of the stimulatory action of forskolin on PG synthesis (prostaglandin I2 measured as 6-ketoprostaglandin F1 alpha [6-keto-PGF1 alpha]) in isolated perfused rabbit heart. Forskolin enhanced PG production in a dose-dependent manner. 1,9-Dideoxyforskolin, a forskolin analogue devoid of adenylate cyclase-stimulating activity, also enhanced PG synthesis. The cAMP analogue chlorophenylthio-cAMP failed to stimulate output of 6-keto-PGF1 alpha, although this agent produced dose-related changes in mechanical function in rabbit heart. Furthermore, the adenylate cyclase inhibitor (-)-N6-(R-phenylisopropyl)adenosine potentiated, whereas the phosphodiesterase inhibitor cilostamide attenuated, forskolin-stimulated PG production. (-)-N6-(R-Phenylisopropyl)adenosine and cilostamide had no effect on the mechanical actions of chlorophenylthio-cAMP, suggesting selectivity of these agents for adenylate cyclase and phosphodiesterase, respectively. 6-Keto-PGF1 alpha output elicited by forskolin was abolished by reduction of calcium in the perfusion fluid as well as by the calcium channel blocker diltiazem. The intracellular calcium antagonists TMB-8 and ryanodine also abolished forskolin-stimulated PG synthesis in rabbit heart. PG synthesis stimulated by 1,9-dideoxyforskolin was also prevented by reduced extracellular calcium, diltiazem, and ryanodine. The calmodulin antagonists trifluoperazine, W-7, and calmidazolium failed to significantly alter PG production in response to forskolin. These results indicate that forskolin-stimulated PG synthesis in rabbit heart is independent of cAMP and requires calcium from both extracellular and intracellular sources.

6-Ketoprostaglandin F1 alpha↗