Regulation of the receptor-mediated cyclic AMP response of kidney to parathyroid hormone in the vitamin D-deficient rat

Rats fed a diet deficient in vitamin D were found to exhibit a refractory cyclic AMP response of kidney slices to parathyroid hormone and a marked decrease in membrane parathyroid hormone-dependent adenylate cyclase activity. Both the characteristic calcium deficiency (hypocalcemia) and secondary elevation of circulating parathyroid hormone appeared before the first noticeable decrease in hormone-dependent enzyme activity. After repletion of D-deficient rats with vitamin D₂, we found that serum calcium and parathyroid hormone were both restored to normal levels before the depressed enzyme response to the hormone was reversed. Moreover, infusion of parathyroid hormone into vitamin D-replete rats led to a marked reduction in parathyroid hormone-dependent adenylate cyclase activity, which was partly restored to control level 3 hours after discontinuing the hormone infusion. Taken as a whole, this study suggests that the elevated endogenous parathyroid hormone in the vitamin D-deficient rat is involved in the “down-regulation” of renal cyclic AMP responsiveness to the hormone. However, these experiments do not rule out the possibility that calcium deficiency and/or vitamin D per se participate in the regulation of the renal cyclic AMP response to parathyroid hormone.     

Activation of Cyclic AMP-Generating Systems in Brain Membranes and Slices by the Diterpene Forskolin: Augmentation of Receptor-Mediated Responses

The diterpene forskolin markedly activates adenylate cyclase in membranes from various rat brain regions and elicits marked accumulations of radioactive cyclic AMP in adenine-labeled slices from cerebral cortex, cerebellum, hippocampus, striatum, superior colliculi, hypothalamus, thalamus, and medulla-pons. In cerebral cortical slices, forskolin has half-maximal effects at 20-30 microM on cyclic AMP levels, both alone and in the presence of the phosphodiesterase inhibitor ZK 62771. The presence of a very low dose of forskolin (1 microM) can augment the response of brain cyclic AMP-generating systems to norepinephrine, isoproterenol, histamine, serotonin, dopamine, adenosine, prostaglandin E2, and vasoactive intestinal peptide. Forskolin does not augment responses to combinations of histamine-norepinephrine adenosine-norepinephrine, or histamine-adenosine. For norepinephrine and isoproterenol in rat cerebral cortical slices and for histamine in guinea pig cerebral cortical slices, the presence of 1 microM-forskolin augments the apparent efficacy of the amine, whereas for adenosine, prostaglandin E2, and vasoactive intestinal peptide, the major effect of 1 microM-forskolin is to increase the apparent potency of the stimulatory agent. In rat striatal slices, forskolin reveals a significant response of cyclic AMP systems to dopamine and augments the dopamine-elicited activation of adenylate cyclase in rat striatal membranes. The activation of cyclic AMP systems by forskolin is rapid and reversible, and appears to involve both direct activation of adenylate cyclase and facilitation and/or enhancement of receptor-mediated activation of the enzyme.     

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.     

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.     

Forskolin stimulates adenylate cyclase and iodine metabolism in thyroid

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

Evidence that forskolin activates turkey erythrocyte adenylate cyclase through a noncatalytic site

The diterpene forskolin has been reported to activate adenylate cyclase in a manner consistent with an interaction at the catalytic unit. However, some of its actions are more consistent with an interaction at the coupling unit that links the hormone receptor to the adenylate cyclase activity. This report adds support to the latter possibility. Under conditions that lead to stimulation of adenylate cyclase in turkey erythrocyte membranes by GTP, forskolin also becomes more active. Additional evidence to support an influence of forskolin upon adenylate cyclase via the GTP-coupling protein N includes the following: (i) forskolin, at submaximal concentrations, leads to enhanced sensitivity and responsiveness of isoproterenol-dependent adenylate cyclase activity in turkey erythrocyte membranes; (ii) under specified conditions, the nucleotide GDP, an inhibitor of the stimulating nucleotide GTP and its analog, guanyl imidodiphosphate (Gpp(NH)p), also markedly inhibits the action of forskolin; (iii) both Gpp(NH)p and forskolin are associated with a decrease in agonist affinity for the beta-adrenergic receptor. However, actions of forskolin in the turkey erythrocyte are not identical to those of GTP: (i) forskolin is never as potent as Gpp(NH)p in activating adenylate cyclase; (ii) the magnitude of synergism between isoproterenol and forskolin is not equal to that observed with isoproterenol and Gpp(NH)p; (iii) at high concentrations, forskolin inhibits antagonist binding to the beta-receptor. Forskolin appears to have several sites of action in the turkey erythrocyte membrane, including an influence upon the adenylate cyclase regulatory protein N.     

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.     

Increase of adenylate cyclase catalytic-unit activity by dexamethasone in rat osteoblast-like cells.

Glucocorticoids are known to increase the cyclic AMP response to parathyroid hormone (PTH) in cultured bone organs or bone cells. Using the osteoblast-like cell line ROS 17/2.8, which possesses receptors for both PTH and glucocorticoids, we investigated which component of the complex hormone receptor-guanine nucleotide regulatory unit--adenylate cyclase was affected by dexamethasone treatment. In response to PTH, isoproterenol or forskolin, a compound that is supposed to act directly on the catalytic unit, cyclic AMP production by intact cells and adenylate cyclase activity in purified plasma membrane were markedly increased by dexamethasone. Whereas NaF, guanosine 5'-[beta gamma-imido]triphosphate and Mn/ stimulated adenylate cyclase activity were similarly enhanced in membranes isolated from glucocorticoid-treated cells, the activity of the stimulatory guanine nucleotide regulatory unit, as assessed by reconstitution into membranes from the CYC- clone, which is genetically devoid of this component, was not altered. Thus in osteoblast-like cells dexamethasone appears to increase cyclic AMP synthesis by influencing the catalytic unit. Moreover, since it has been reported that glucocorticoids may produce changes in cell calcium metabolism, we evaluated cytoplasmic free Ca2+ concentration ([Ca2+]i) and intracellular Ca2+ stores mobilizable by the bivalent-cationophore ionomycin, by using the intracellular fluorescent indicator Quin-2. The results indicated that dexamethasone treatment did not influence [Ca2+]i but markedly decreased ionomycin-releasable Ca2+ stores.     

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.     

Steroidogenic effect of ent-kaur-16-en-15 beta-ol (kaurenol) on isolated rat adrenal cells

In an in vitro bioassay system for adrenocorticotropic hormone using isolated rat adrenal cells, kaurenol, a diterpene alcohol, stimulated corticosterone production and augmented the steroidogenic effect of adrenocorticotropin or forskolin, dose-dependently. Kaurenol had no effect on cyclic AMP production by the cells. The diterpene also had no stimulatory effect on the adrenal adenylate cyclase activity in a cell free system. The results suggest that this particular diterpene exerts a steroidogenic effect through a mechanism independent of cyclic AMP generation.     

Studies in vivo on the effects of parathyroid hormone upon kidney cyclic adenosine 3',5'-monophosphate content using rapid tissue fixation by microwave irradiation.

Microwave irradiation is shown to be a useful method for simultaneously killing chicks and fixing tissues. Renal adenylate cyclase and phosphodiesterase activities were rapidly abolished by microwaving. The increase in chick kidney cyclic adenosine 3',5'-monophosphate (cyclic AMP) content produced by intravenous bovine parathyroid hormone (PTH) injection was much greater in microwaved birds than in those killed by cervical dislocation with subsequent tissue fixation in liquid nitrogen. After PTH injection there was a prolonged elevation of renal cyclic AMP content. At the time of maximum response (2 minutes), log. dose-response curves were linear in the dose range 0.1-10 U. The responses to three different bovine PTH preparations were indistinguishable. Arginine vasopressin, arginine vasotocin, salmon calcitonin and prostaglandin E1 did not affect kidney cyclic AMP content within 2 minutes. Because of its specificity and precision, the method is of use for the in vivo bioassay of PTH. Injection of CaCl2 (20 mumoles) 1 minute before, or conjointly with, bovine PTH inhibited the subsequent increase in kidney cyclic AMP content. The synthetic bovine PTH peptide fragments BPTH (1-34) and BPTH (2-34) both increased chick kidney cyclic AMP content. The use of such fragments allows investigation of the structural requirements of PTH for interaction with the systems regulating cyclic AMP metabolism in the kidney in vivo.     

Effects of parathyroid hormone and calcitonin on adenylate cyclase in murine mononuclear phagocytes

Peritoneal mononuclear phagocytes elicited by thioglycollate demonstrate responsiveness to parathyroid hormone (PTH) and calcitonin (CT) which differs from that seen in the normal resident population. PTH causes a twofold stimulation of adenylate cyclase activity in elicited cells but inhibits this activity in resident cells. CT causes a greater stimulation of adenylate cyclase in elicited than in resident cells. Both CT and PTH cause an increase in cyclic AMP accumulation in cultures of elicited mononuclear phagocytes. These results indicate that cells of the mononuclear phagocyte lineage have functional receptors for both PTH and CT. This is the first biochemical evidence to support the hypothesis that mononuclear phagocytes are precursors of the bone resorbing osteoclast.     

Bioactivity of amino terminal fragments of parathyroid hormone and parathyroid hormone related peptide in rat kidney slices: no effect of dietary phosphate deprivation

Humoral hypercalcemia of malignancy has been associated with the production of a recently cloned peptide human parathyroid hormone related protein (hPTHRP). One of the markers of this disease is an increased urinary excretion of cyclic AMP. The postreceptor mechanism of action and physiological role of hPTHRP remain obscure. To study the activity of hPTHRP 1-34 compared to rat and human parathyroid hormone (PTH) 1-34 we incubated these peptides with rat kidney slices and measured the cyclic AMP generated in the supernatant. hPTHRP 1-34 was equipotent with human PTH 1-34 but both were 5 times less active than rat PTH 1-34. Previous studies have suggested that a low dietary phosphate intake results in renal resistance to the phosphaturic action of PTH perhaps mediated by reduced adenylate cyclase activation by PTH. To determine whether, during dietary phosphate restriction, hPTHRP 1-34 has actions different from hPTH 1-34 we studied their effects following dietary phosphate deprivation. Dietary phosphate restriction had no significant effect on the cyclic AMP generating activity of any of the peptides. We conclude that hPTHRP 1-34 may be operating through similar mechanisms as human PTH 1-34 and that the previously observed effects of dietary phosphate deprivation on PTH mediated cyclic AMP generation in a broken cell preparation do not occur in intact cell preparations.     

Alpha-2 adrenergic activation inhibits forskolin-stimulated adenylate cyclase activity and lipolysis in human adipocytes

Forskolin at 10 muM caused a 100-fold increase in the intracellular concentration of cyclic AMP and a 6-fold increase in glycerol release in the human adipocyte. These responses are comparable to those prompted by 10 muM isoproterenol. The effects of forskolin on cyclic AMP and lipolysis were dose-dependent. Alpha-2 adrenergic activation, achieved with 10 muM epinephrine and 30 muM propranolol, significantly inhibited forskolin-stimulated cyclic AMP accumulation and glycerol release, shifting the dose-response curves to the right. Forskolin at 10 muM caused a 4.5-fold increase in the adenylate cyclase activity of human adipocyte membranes. When either isoproterenol or epinephrine (0.1 mM) was combined with forskolin, the magnitude of response was substantially greater than the sum of responses achieved by each agent incubated alone.     

Forskolin potentiates adrenocorticotropin-induced cyclic AMP production and steroidogenesis in isolated rat adrenal cells

Forskolin, a unique diterpene which directly activates the adenylate cyclase, stimulated production of both cyclic AMP and corticosterone in isolated rat adrenal cells, in vitro. This agent also potentiated the action of adrenocorticotropin and/or cholera toxin on cyclic AMP production and steroidogenesis at lower concentrations. It augmented both an early (cyclic AMP production) and a late (steroidogenesis) action of the hormone in the adrenal gland.     

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

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

Electroconvulsive Shock and Cyclic AMP Signal Transduction: Effects Distal to the Receptor

Chronic electroconvulsive shock (ECS) induced a significant decrease in noradrenaline- and forskolin-stimulated cyclic AMP production in rat cortical slices, whereas a single ECS had a much smaller effect. In a cortical membrane preparation, adenylate cyclase activity in response to stimulation by forskolin, guanosine-5'-(beta,gamma-imido) triphosphate, and Mn2+ ions was significantly increased in membranes derived from rats that had received chronic ECS, but was either unchanged or reduced in membranes from rats that received a single treatment only. The results are interpreted in terms of changes occurring at components of the adenylate cyclase enzyme distal to the receptor.     

Dissociation by cooling of hormone and cholera toxin activation of adenylate cyclase in intact cells.

Cholera toxin, through adenylate cyclase activation reproduced cyclic AMP-mediated effects of thyroid-stimulating hormone (TSH) in dog thyroid slices, i.e. protein iodination, [1-14C]glucose-oxidation and hormone secretion. Iodide and carbamylcholine decreased the cyclic AMP accumulation induced by cholera toxin as well as by TSH, which supports the hypothesis of an action of these agents beyond the steps of hormone-receptor and receptor-adenylate cyclase interaction. Cooling to 20 degrees C did not impair the TSH induced cyclic AMP accumulation in thyroid slices, but completely suppressed the cholera toxin effect. This observation has been extended to other hormones and target tissues, such as the parathyroid hormone (PTH) (kidney cortex), adrenocorticotropic hormone (ACTH) (adrenal cortex) and luteinizing hormone (LH) (ovary systems). As in thyroid, cooling dissociated the cholera toxin and hormonal effects on cyclic AMP accumulation. In homogenate, cooling decreased cyclic AMP generation in the presence of cholera toxin but at 20 degrees C and 16 degrees C a cholera toxin stimulation was still observed. These results bear strongly against the hypothesis that the glycoprotein hormones TSH and LH acetivate adenylate cyclase by a mechanism identical to cholera toxin.     

Effect of prostaglandin E on the adenyl cyclase-cyclic AMP system and gluconeogenesis in rat renal cortical slices.

Prostaglandin E was found to increase the formation of cyclic acdenosine 3',5'-monophosphate (cyclic AMP) by renal cortical slices. This increased release of cyclic AMP was not influenced by the absence of Ca2+ in the incubating media. The enhanced production of cyclic AMP was probably mediated by stimulation of membrane-bound adenylate cyclase activity. An increase in adenyl cyclase activity was observed with increasing concentrations of prostaglandin E. Furthermore, prostaglandin E augmented glucose production from alpha-ketoglutarate. This effect on gluconeogenesis was abolished by the removal of Ca2+ from the incubating medium. These effects are similar to those described for parathyroid hormone and suggest that the renal cortex is a prostaglandin-dependent system. Prostaglandin E decreased cyclic AMP production and glucose production (from alpha-ketoglutarate) in response to submaximal doses of parathyroid hormone, suggesting that prostaglandin may be important in modulating the intracelluar action of parathyroid hormone in the kidney cortex.     

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.