Chymotrypsin selectively decreases forskolin stimulation of adenylate cyclase

We studied the effects of chymotrypsin on turkey erythrocyte membrane adenylate cyclase activity. Proteolysis with chymotrypsin led to a concentration- and time-dependent increase in activation of adenylate cyclase by isoproterenol + guanine nucleotides, and fluoride, and to a decrease in activation by forskolin. Maximal effects (up to 10-fold increases in fluoride- and isoproterenol + guanine nucleotide-stimulated activity, and up to 100% inhibition of forskolin-stimulated activity) occurred under similar conditions (10-20 micrograms/ml chymotrypsin for 10-15 min at 30 degrees C). Augmentation of isoproterenol + guanosine-3'-O-thiotriphosphate (GTP-gamma-S)-stimulated activity by chymotrypsin occurred only if proteolysis preceded stimulation with isoproterenol + GTP-gamma-S. Addition of isoproterenol + GTP-gamma-S to membranes before proteolysis, however, did not prevent chymotrypsin from augmenting subsequent stimulation by these agents. In contrast, addition of forskolin during proteolysis with chymotrypsin prevented the time- and concentration-dependent decline in forskolin stimulation observed with chymotrypsin. Proteolysis decreased the magnitude of stimulation at any concentration of forskolin, but did not alter the concentration dependence of forskolin stimulation (apparent half-maximum = 3 microM). The data are consistent with the existence of a chymotrypsin-sensitive site essential for forskolin stimulation of adenylate cyclase. In view of the simultaneous effect of chymotrypsin to augment fluoride- and isoproterenol + guanine nucleotide-stimulated activities, it is highly unlikely that the site is on the stimulatory guanine nucleotide binding protein. Since forskolin is thought to act directly on the catalytic unit of adenylate cyclase, and since forskolin can protect against the effect of proteolysis with chymotrypsin, the site involved may be on the catalytic unit itself.     

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.     

Stimulation and inhibition of rat basophilic leukemia cell adenylate cyclase by forskolin

The influence of the diterpene, forskolin, was studied on adenylate cyclase activity in membranes of rat basophilic leukemia cells. Forskolin increased basal adenylate cyclase activity maximally 2-fold at 100 microM. However, adenylate cyclase activity stimulated via the stimulatory guanine nucleotide-binding protein, Ns, by fluoride and the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate), was inhibited by forskolin. Half-maximal and maximal inhibition occurred at about 1 and 10 microM forskolin, respectively. The inhibition occurred without an apparent lag phase, whereas the enzyme stimulation by forskolin was preceded by a considerable lag period. The inhibition was not affected by treating intact cells or membranes with pertussis toxin and proteolytic enzymes, respectively, which have been shown in other cell types to prevent adenylate cyclase inhibition mediated by the guanine nucleotide-binding regulatory component, Ni. The forskolin inhibition of the stable GTP analog-activated adenylate cyclase was impaired by increasing the Mg2+ concentration and was reversed into a stimulation by Mn2+. Under optimal inhibitory conditions, forskolin even decreased basal adenylate cyclase activity. Finally, forskolin largely reduced the apparent affinity of the rat basophilic leukemia cell adenylate cyclase for its substrate, MgATP, which reduction resulted in an apparent inhibition at low MgATP concentrations and a loss of the inhibition at higher MgATP concentrations. The data indicate that forskolin can cause both stimulation and inhibition of adenylate cyclase and, furthermore, they suggest that the inhibition may not be mediated by the Ni protein, but may be caused by a direct action of forskolin at the adenylate cyclase catalytic moiety.     

Forskolin activation of adenylate cyclase in mouse parotid membranes

In mouse parotid membranes forskolin activated adenylate cyclase four-fold; maximal activation of the enzyme occurred with 10 microM forskolin. Activation was not dependent on the guanyl nucleotide GTP nor on the inhibitory guanine nucleotide 5'-0-(2-Thiodiphosphate), GDP beta S. In contrast, stimulation of adenylate cyclase by isoproterenol required GTP and was antagonized by GDP beta S in a dose-dependent manner. These results indicate that the guanyl-binding protein of mouse parotid adenylate cyclase is not a requisite for forskolin activation and lends support for direct interaction of forskolin at the catalytic subunit.     

Activation of adenylate cyclase by the diterpene forskolin does not require the guanine nucleotide regulatory protein

Forskolin, a novel diterpene activator of adenylate cyclase in membranes and intact cells, activates the enzyme in membranes from mutant cyc-S49 murine lymphoma cells and the soluble enzyme from rat testes. Each of these enzymes consists only of the catalytic subunit and does not have a functional guanine nucleotide-binding protein. In both cases forskolin converts the manganese-dependent enzymes to a form which does not require manganese for activity. Forskolin can also stimulate a detergent-solubilized preparation of adenylate cyclase from rat cerebral cortex. Activation of adenylate cyclase by forskolin is therefore not dependent on a perturbation of membrane structure nor does it require a functional guanine nucleotide-binding subunit.     

Regulation by forskolin of octopamine-stimulated adenylate cyclase from brain of the dipterous Ceratitis capitata

Forskolin, a diterpene that exerts several pharmacological effects, activates adenylate cyclase in brain and in some other mammalian tissues. Properties of forskolin activation of adenylate cyclase from central nervous system of the dipterous Ceratitis capitata are described. The interaction of forskolin with the insect adenylate cyclase system was studied by evaluating its effect on metal-ATP kinetics, protection against thermal inactivation, membrane fluidity and enzyme modulation by fluoride, guanine nucleotides, octopamine, and ADP-ribosylation by cholera toxin. The diterpene stimulated basal enzyme activity both in membranes and Triton X-100-solubilized preparations, apparently devoid of functional regulatory unit, this effect being rapidly reversed by washing the membranes. An increase of Vmax accounts for the activation of soluble and membrane adenylate cyclase preparations by forskolin, whereas the affinity of the enzyme for the substrate was not affected. Forskolin apparently protects the membrane enzyme from thermal inactivation, and at concentrations that promote the enzyme activity the diterpene does not alter membrane microviscosity. Forskolin does not appear to alter the sensitivity of insect adenylate cyclase to sodium fluoride, guanine nucleotide, or regulatory subunit ADP ribosylated by cholera toxin, the combined effect of these factors with the diterpene resulting in a nearly additive enzymatic activation. However, forskolin blocks the octopamine stimulatory input. Results obtained with the insect adenylate cyclase system are discussed and compared to what is known about mammalian systems to propose a mechanism of enzyme activation by forskolin.     

The effects of forskolin on adenylate cyclase in S49 wild type and cyc- cells

The effect of forskolin on adenylate cyclase in S49 wild type and cyc- cells was tested. Forskolin stimulated adenylate cyclase activity in cyc- membranes, particularly with Mn++ as cofactor. Forskolin stimulation of adenylate cyclase in wild type membranes was greater than in cyc- membranes, and the ability of forskolin to stimulate cyc- membranes was enhanced by Lubrol PX extracts of human erythrocyte membranes. Compared to its potent effect on intact wild type cells, forskolin was a poor stimulator of cAMP accumulation in cyc- cells. Cyc- cells proliferated in medium containing forskolin, while the growth of wild type cells in such medium was inhibited and the wild type cells ultimately died. Clones selected from a suspension of wild type cells on the basis of forskolin resistance showed the characteristics of cyc- cells. Thus, forskolin does not substantially activate adenylate cyclase activity in intact cyc- cells. Our data indicate that the guanine nucleotide regulatory protein (G/F) enhances forskolin activation of adenylate cyclase.     

Forskolin-activated adenylate cyclase. Inhibition by guanyl-5'-yl imidodiphosphate

Forskolin activated adenylate cyclase of purified rat adipocyte membranes in the absence of exogenous guanine nucleotides. Guanyl-5'-yl imidodiphosphate (Gpp(NH)p) inhibited the forskolin-activated cyclase immediately upon addition of the nucleotide at concentrations too low to activate adenylate cyclase (10(-9) to 10(-7) M). Inhibition seen with a very high concentration of Gpp(NH)p (10(-4) M) lasted for 3-4 min and was followed by an increase in the synthetic rate which remained constant for at least 15 min. The length of the transient inhibition did not vary with forskolin concentrations above 0.05 microM but low Gpp(NH)p (10(-8) M) exhibited a lengthened (6-7 min) inhibitory phase. The transient inhibitory effects of Gpp(NH)p were eliminated by 10(-7) M isoproterenol, high (40 mM) Mg2+, or preincubation with Gpp(NH)p in the absence of forskolin. While forskolin stimulated fat cell cyclase in the presence of Mn2+, this ion blocked the inhibitory effects of Gpp(NH)p. The well documented inhibitory effects of GTP on the fat cell adenylate cyclase system were also observed in the presence of forskolin. However, the inhibition by GTP is not transitory. These findings indicate that Gpp(NH)p regulation of forskolin-stimulated cyclase has at least two components: 1) an inhibitory component which acts through an undetermined mechanism and which acts immediately to decrease cyclase activity; and 2) an activating component which modulates the inhibited cyclase activity through the guanine nucleotide regulatory protein.     

The role of a guanine nucleotide-binding protein in the activation of rat liver plasma-membrane adenylate cyclase by forskolin.

The effects of the photoreactive GTP analogue GTP-gamma-azidoanilide on rat liver plasma-membrane adenylate cyclase are described. U.v. irradiation in the presence of the analogue abolished activation by any effector or combination of effectors that function via the activatory G protein. Partial protection against this inhibition was given by F- and guanosine 5'-[gamma-thio]triphosphate. It is concluded that GTP-gamma-azidoanilide acts by a light-induced covalent reaction with the G protein. In the dark the effects of the analogue were similar to those of GTP. Irradiation in the presence of GTP-gamma-azidoanilide was found to reduce but not to abolish activation of rat liver plasma membrane adenylate cyclase by forskolin. The activation by forskolin and GTP together were greater than the sum of the individual activations. Forskolin doubled adenylate cyclase activity in the presence of glucagon and guanosine 5'-[beta, gamma-imido]triphosphate, which might be expected to activate to the maximum possible extent via the G protein. It is concluded that there are two components to the forskolin activation, a guanine nucleotide-dependent and a guanine nucleotide-independent component.     

Activation of lipolysis and cyclic AMP accumulation in rabbit adipocytes by isoproterenol in the presence of forskolin or pertussis toxin

Adipocytes from rabbits are relatively insensitive to catecholamines or forskolin. However, the combination of catecholamines plus forskolin increased cyclic AMP accumulation and lipolysis much more than either agent alone. Pertussis toxin treatment also restored sensitivity to catecholamines. No defect in activation by catecholamines of adenylate cyclase was seen in isolated membranes incubated in the presence of GTP. Rabbit adipocytes appear to have an excess of the inhibitory guanine nucleotide binding protein (Ni). However, in plasma membranes this protein appeared to be relatively inactive as there was an activation of adenylate cyclase activity by catecholamines in the presence of GTP. These data suggest that in intact rabbit adipocytes catecholamines and forskolin are ineffective as stimulators of adenylate cyclase due to an excess of inhibitory guanine nucleotide binding proteins.     

Regulation of [3H]forskolin binding to human platelet membranes by GppNHp, NaF, and prostaglandin E1

Displaceable binding of [3H]forskolin to human platelet membranes can be detected in the presence of magnesium. There is an increase in the number of [3H]forskolin binding sites when membranes are incubated with GppNHp or NaF in the presence of magnesium. Prostaglandin E1, which stimulates human platelet adenylate cyclase, does not affect the binding of [3H]forskolin in the absence of GppNHp. However, the dose-response curve for the GppNHp-dependent increase in [3H]forskolin binding sites is shifted to lower concentrations in the presence of prostaglandin E1. Prostaglandin E1 potentiates the effect of GppNHp on [3H]forskolin binding most likely by facilitating the binding of the guanine nucleotide at the stimulatory quanine nucleotide regulatory protein of adenylate cyclase.     

Studies on the mechanism of the decreased guanine nucleotide stimulatory effects on adenylate cyclase after adrenalectomy in rat adipocytes

In adipocyte membranes from adrenalectomized rats: (i) the defect in the stimulatory effects of guanine nucleotides on adenylate cyclase is greater with GTP than with GppNHp and appears to concern the GS-C complex and not the HR-GS-C ternary complex; (ii) the GTP-ase activity is enhanced; (iii) maximal alpha S-C interaction (tested by the responses to forskolin, cholera toxin or NaF) is unaltered and (iv) the alpha S affinity for guanine nucleotides and the GDP/GppNHp exchange reaction are both unimpaired. These data suggest that the enhanced GTP-ase activity together with a decrease in the catalytic activity of the alpha S-GTP-C complex are the likely mechanisms whereby adrenalectomy causes a defect in the fat cell adenylate cyclase response to guanine nucleotides.     

Photolytic studies on the carbon monoxide complex of sulphaemoglobin.

Salivary-gland homogenates contain 5-hydroxytryptamine-stimulated adenylate cyclase. Half-maximal stimulation was obtained with 0.1 microM-5-hydroxytryptamine in the presence of added guanine nucleotides. Gramine antagonized the stimulation of cyclase caused by 5-hydroxytryptamine. In the presence of hormone, guanosine 5'-[gamma-thio]triphosphate produced a marked activation of adenylate cyclase activity. Stimulation of adenylate cyclase by forskolin or fluoride did not require the addition of guanine nucleotides or hormone. In the presence of EGTA, Ca2+ produced a biphasic activation of cyclase activity. Ca2+ at 1-100 microM increased activity, whereas 2000 microM-Ca2+ inhibited cyclase activity. The neuroleptic drugs trifluoperazine and chlorpromazine non-specifically inhibited adenylate cyclase activity even in the absence of Ca2+. The cyclic AMP phosphodiesterase activity in homogenates was not affected by Ca2+ or exogenous calmodulin. This enzyme was also inhibited by trifluoperazine in the absence of Ca2+. These results indicate that Ca2+ elevates adenylate cyclase activity, but had no effect on cyclic AMP phosphodiesterase of salivary-gland homogenates.     

Binding of [3H]forskolin to solubilized preparations of adenylate cyclase

The binding of [3H]forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating [3H]forskolin bound to protein from free [3H]forskolin by rapid filtration. The Kd for [3H]forskolin binding to solubilized proteins was 14 nM which was similar to that for [3H]forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for [3H]forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. [3H]forskolin bound to proteins solubilized from membranes with a Bmax of 38 fmol/mg protein which increased to 94 fmol/mg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on [3H]forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmol/mg/min which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmol/mg/min which was not stimulated by GppNHp or forskolin. Thus, the number of high affinity binding sites for [3H]forskolin in solubilized preparations correlated with the activation of adenylate cyclase by GppNHp via the guanine nucleotide binding protein (GS).     

Antibodies directed against transducin beta subunits interfere with the regulation of adenylate cyclase activity in brain membranes

In an attempt to study the mechanisms of action of membrane-bound adenylate cyclase, we have applied to rat brain synaptosomal membranes antibodies raised against purified bovine transducin (T) beta gamma subunits. The antibodies recognized one 36-kDa protein in Western blots of the membranes. Adenylate cyclase activation by GTP non-hydrolyzable analogues was greatly decreased in immune, as compared to preimmune, antibody-treated membranes, whereas the enzyme basal activity was unaffected by both types of antibodies. The inhibition of forskolin-stimulated adenylate cyclase by guanine 5'-(beta, gamma-imino)triphosphate (Gpp-(NH)p) was decreased in membranes preincubated with immune, but not preimmune, antibodies. Anti-T beta antibodies moderately decreased the extent of subsequent adenylate cyclase activation by forskolin, while not affecting activation by Al3+/F-. The enzyme activation by Gpp(NH)p in untreated membranes remained the same upon further incubation in the presence of either type of antibodies. Such results were consistent with the decreased exchange of guanine nucleotides which occurred in membrane treated with immune, but not preimmune antibodies, upon addition of GTP. The blockade of the regulation of adenylate cyclase by Gpp(NH)p observed in membranes pretreated by anti-T beta antibodies thus appears to be caused by the impairment of the guanine nucleotide exchange occurring on Gs alpha subunits. The G beta subunits in the adenylate cyclase complex seem to be instrumental in the guanine nucleotide exchange on G alpha subunits, just as T beta subunits are in the transducin complex.     

Microtubule-associated adenylate cyclase

Twice-cycled bovine brain or rat brain microtubule protein contains an adenylate cyclase activity that passes 0.2 micron filters, is activated 2-7-fold by 30 microM forskolin, shows modest stimulation by fluoride (especially in the presence of added AI3+), but is virtually insensitive to added guanine nucleotides. The activity is insensitive to various hormones or Ca2+/calmodulin. The adenylate cyclase is active with both Mg2+ and Mn2+ but activity is less in the presence of Mg2+ than with Mn2+. The cyclase is inhibited by agonists of the adenosine P site. It is proposed that the catalytic unit of adenylate cyclase and probably small quantities of the guanine nucleotide regulatory protein, Ns, are cycled along with microtubules.     

Differential effect of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on [3H]SCH23390 and [3H]forskolin binding in rat striatum

The binding of [3H]forskolin to a homogeneous population of binding sites in rat striatum was enhanced by NaF, guanine nucleotides and MgCl2. These effects of NaF and guanylylimidodiphosphate (Gpp(NH)p) were synergistic with MgCl2, but NaF and Gpp(NH)p together elicited no greater enhancement of [3H]forskolin binding. These data suggest that [3H]forskolin may label a site which is modulated by the guanine nucleotide regulatory subunit which mediates the stimulation of adenylate cyclase (NS). The D1 dopamine receptor is known to stimulate adenylate cyclase via NS. In rat striatum, the Bmax of [3H]forskolin binding sites in the presence of MgCl2 and NaF was approximately two fold greater than the Bmax of [3H]SCH23390-labeled D1 dopamine receptors. Incubation of striatal homogenates with the protein modifying reagent EEDQ elicited a concentration-dependent decrease in the binding of both [3H]SCH23390 and [3H]forskolin, although EEDQ was approximately 14 fold more potent at inactivating the D1 dopamine receptor. Following in vivo administration of EEDQ there was no significant effect on [3H]forskolin binding sites using a dose of EEDQ that irreversibly inactivated greater than 90% of D1 dopamine receptors. These data suggest that EEDQ is a suitable tool for investigating changes in the stoichiometry of receptors and their second messenger systems.     

Inhibitory action of forskolin on adenylate cyclase activity and cyclic AMP generation

In testicular Leydig cells, forskolin causes the expected stimulation of cAMP and testosterone production and potentiates gonadotropin-induced responses, when present in concentrations of 1-10 microM. In addition, when added at lower doses that did not affect cAMP generation and testosterone responses (100 nM), forskolin caused an increase in sensitivity to hormonal stimulation for all cAMP pools (extracellular, intracellular, and receptor-bound) and a 70% reduction in the ED50 for human chorionic gonadotropin (hCG) stimulation of testosterone production. Forskolin-induced increases in receptor-bound cAMP were less effective than those elicited by hCG in stimulating steroidogenesis. In contrast to the well-known stimulatory actions of forskolin, low doses of the diterpene (in the picomolar to nanomolar range) markedly inhibited the production of cAMP and testosterone. Such inhibitory actions of low-dose forskolin were prevented by preincubation of Leydig cells with pertussis toxin before addition of forskolin and/or hCG. Low concentrations of forskolin also inhibited adenylate cyclase activation by GTP and luteinizing hormone, and this effect was prevented by pretreatment of cell membranes with pertussis toxin. These studies have defined the stimulatory effects of forskolin on Leydig-cell cAMP pools, including potentiation of the hormonal increase in receptor-bound cyclic AMP by forskolin, and have provided additional evidence for the functional importance of cAMP compartmentalization during hormonal stimulation of steroidogenesis. We have also demonstrated a novel, high-affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation, an effect that appears to be mediated by the Ni guanine nucleotide regulatory subunit of adenylate cyclase.     

Protein kinase C interferes with Ni-mediated inhibition of human platelet adenylate cyclase

Addition of phorbol ester-activated, partially purified protein kinase C to membranes of human platelets had no effect on forskolin stimulation of the adenylate cyclase and increased stimulation by prostaglandin E1 only at high GTP concentrations by preventing inhibition by GTP. Hormonal inhibition of the platelet adenylate cyclase by epinephrine was eliminated or largely impaired. At low GTP concentrations, epinephrine even caused a small increase in cyclase activity. The data suggest that activated protein kinase C interferes with GTP- and hormone-induced adenylate cyclase inhibition probably by phosphorylating the inhibitory guanine nucleotide-binding regulatory component Ni.     

Binding of [3H]forskolin to human platelet membranes. Regulation by guanyl-5'-yl imidodiphosphate, NaF, and prostaglandins E1 and D2

[3H]Forskolin binds to human platelet membranes in the presence of 5 mM MgCl2 with a Bmax of 125 fmol/mg of protein and a Kd of 20 nM. The Bmax for [3H]forskolin binding is increased to 455 and 425 fmol/mg of protein in the presence of 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and 10 mM NaF, respectively. The increase in the Bmax for [3H]forskolin in the presence of Gpp(NH)p or NaF is not observed in the absence of MgCl2. The EC50 values for the increase in the number of binding sites for [3H]forskolin by Gpp(NH)p and NaF are 600 nM and 4 mM, respectively. The EC50 value for Gpp(NH)p to increase the number of [3H]forskolin binding sites is reduced to 35 mM and 150 nM in the presence of 50 microM PGE1 or PGD2, respectively. The increase in the number of [3H]forskolin binding sites observed in the presence of NaF is unaffected by prostaglandins. The binding of [3H]forskolin to membranes that are preincubated with Gpp(NH)p for 120 min or assayed in the presence of PGE1 reaches equilibrium within 15 min. In contrast, a slow linear increase in [3H]forskolin binding is observed over a period of 60 min when Gpp(NH)p and [3H]forskolin are added simultaneously to membranes. A slow linear increase in adenylate cyclase activity is also observed as a result of preincubating membranes with Gpp(NH)p. In human platelet membranes, agents that activate adenylate cyclase via the guanine nucleotide stimulatory protein (Ns) increase the number of binding sites for [3H]forskolin in a magnesium-dependent manner. This is consistent with the high affinity binding sites for [3H]forskolin being associated with the formation of an activated complex of the Ns protein and adenylate cyclase. This state of the adenylate cyclase may be representative of that formed by a synergistic combination of hormones and forskolin.