Association in normal human fibroblasts of elevated levels of adenosine 3':5'-monophosphate with a selective decrease in collagen production.

To evaluate the hypothesis that extracellular mediators may affect collagen production by mesenchymal cells via a cyclic AMP coordinated mechanism, normal human fibroblasts were exposed to a variety of agents (prostaglandin E1, isoproterenol, cholera toxin) which independently elevated intracellular cyclic AMP during a 6-h incubation. Concomitantly, each agent caused an average 47% reduction in the percentage of total protein synthesis represented by collagen, yet little change in other major extracellular proteins. Since no active collagenase was found in these cultures, these findings suggest cyclic AMP levels may modulate the differentiated state of normal fibroblasts with respect to collagen production.     

Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells.

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E1 also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E1 is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 muM isoproterenol is increased and approaches that produced by5.6 muM prostaglandin E1. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 muM prostaglandin E1 is reduced by as much as 50%, the concentration of prostaglandin E1 required to induce a maximal increase in cyclic AMP is 1/10 that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E1 can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparation from control or choleragen-treated cells. In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblast choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

Effect of cyclic AMP on the intracellular degradation of newly synthesized collagen

Prostaglandin E1 and cholera toxin increased the intracellular levels of cyclic AMP of human lung fibroblasts. With prostaglandin E1, the increase in cyclic AMP occurred within 10 min followed by a decline to less than one-half of peak values in 6 h. With cholera toxin, the increase occurred within 60 min but the level of cyclic AMP remained increased for 6 h. Both agents caused a decrease in collagen production as expressed as the proportion of newly synthesized protein represented by collagen. The increase in cyclic AMP levels was accompanied by a marked increase in the proportion of newly synthesized collagen which was degraded intracellularly prior to secretion. Analysis of the degraded collagen showed it to be predominantly less than 1000 daltons in molecular mass, but still in peptide linkage. The data are consistent with the hypothesis that cyclic AMP levels in diploid fibroblasts regulate the amount of collagen produced by fibroblasts, at least in part, by modulating the level of intracellular collagen degradation.     

Modulation of adenylate cyclase/cyclic AMP response by thyrotropin and prostaglandin E2 in cultured thyroid cells. 1. Negative regulation.

Isolated porcine thyroid cells, cultured in the presence of thyrotropin (greater than or equal to 0.25 mU/ml) or prostaglandin E2 (greater than or equal to 0.1 micron), showed decreased adenosine 3':5'-monophosphate (cyclic AMP) response to further thyrotropin or prostaglandin E2 stimulation, respectively. Kinetics of the refractory process to thyrotropin and prostaglandin E2 are different: (a) maximal refractoriness to prostaglandin E2 was attained after 2--6 h exposure to prostaglandin E2 while refractoriness to thyrotropin was maximal only after 12--24 h; (b) the degree of refractoriness to prostaglandin E2 was much greater than that to thyrotropin. Refractoriness to thyrotropin or prostaglandin E2 is characterized: by specificity for each thyroid stimulator; by dependence upon the dose of thyrotropin or prostaglandin E2 in culture, e.g. induction of high degree of refractoriness with 0.5 mU/ml thyrotropin (or 1 micron prostaglandin E2), which elicits only a small cyclic AMP increase; by time requirement for induction; by partial effect; by changes of maximum activation of cyclic AMP response; by reversibility. This refractoriness of the cyclic AMP response was not induced by dibutyryl adenosine 3':5'-monophosphate. It was not attributed to increased cyclic AMP-phosphodiesterase activity, but to alterations in the receptor-adenylate cyclase system. Prevention of refractoriness to thyrotropin or prostaglandin E2 by incubation of cells in the presence of actinomycin D, puromycin and cycloheximide suggests that new RNA and protein syntheses are required for the development of the refractory state.     

Progesterone treatment in vitro enhances prostaglandin E and forskolin-promoted cyclic AMP production in human endometrial stromal cells

Confluent human endometrial stromal cell cultures were exposed to steroids for up to 72 h and then stimulated with agonists of adenylate cyclase for 60 min. Neither steroid alone or in combination had significant effect on cyclic AMP production. However, when stromal cell adenylate cyclase was stimulated with a receptor-dependent agonist (prostaglandin E), or with forskolin (which acts at a post-receptor site), progesterone in oestradiol-primed cells markedly enhanced (P less than 0.05) the effect of both agonists. The presence of phenol red, a weak oestrogenic compound, in the standard culture medium was sufficient to allow the progesterone effect to be manifest. Moreover, while oestradiol alone had no significant effect on prostaglandin E or forskolin-stimulated cyclic AMP production, the simultaneous exposure of cells to oestradiol and progesterone was the most effective treatment. Short-term incubation (up to 120 min) with progesterone had no effect on agonist-induced cyclic AMP accumulation, indicating that progesterone elicits its effect by the classic nuclear mechanism of action. It is suggested that the potentiation by progesterone of prostaglandin E-promoted production of cyclic AMP represents an important aspect of the functional role progesterone plays in the preparation of the endometrium for implantation.     

Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E₁ also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E₁ is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 μM isoproterenol is increased and approaches that produced by 5.6 μM prostaglandin E₁. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells, lower concentrations of isoproterenol are more effective in the choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 μM prostaglandin E₁ is reduced by as much as 50%, the concentration of prostaglandin E₁ required to induce a maximal increase in cyclic AMP is $\text{1}\text{10}$ that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E₁ can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparations from control or choleragen-treated cells.In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblasts, choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

Stimulation of prostaglandin E production by concanavalin A in isolated graafian follicles

Addition of Concanavalin A (con A) to isolated rat Graafian follicles induced prostaglandin E (PGE) production after 2 h of incubation. PGE synthesis continued throughout 24 h culture period. Cyclic AMP accumulation was noted after 6 h of incubation with Con A. Aspirin, indomethacin and flufenamate prevented both the stimulation of PGE production and of cyclic AMP accumulation by Con A; antibodies to PGE prevented the cyclic AMP production. These studies indicate that the interaction of Con A with the follicle results in PGE production. It seems that besides the known pathway for the induction of PGE synthesis in the ovarian follicle, via elevation of intracellular cyclic AMP, an additional pathway, via an external signal which is independent of cyclic AMP exists.     

Detection of regional ischemia in perfused beating hearts by phosphorus nuclear magnetic resonance.

Rat Graafian follicles isolated intact responded to 8-Br-cyclic GMP (0.3 and 1.0 mM) with increased prostaglandin E (PGE) production (4-fold and 8-fold, respectively) during a 6 h incubation. The effect of 8-Br-cyclic GMP was noted after a lag period of 2–4 h. 8-Br-cyclic AMP (1.0 mM) also stimulated PGE production (4-fold increase), while 8-Br-cyclic IMP, 8-Br-5′GMP and 8-Br-5′AMP were inactive in this respect. Actinomycin D (10 μg/ml) and cycloheximide (10 μg/ml) given simultaneously with 8-Br-cyclic GMP prevented the stimulatory effect of the cyclic nucleotide. The results suggest that cyclic GMP induces de novo synthesis of a macromolecular component of the ovarian prostaglandin synthetase system, and that this cyclic nucleotide, along with cyclic AMP, may play a role in the known stimulatory action of luteinizing hormone on follicular prostaglandin production.     

Stimulation of prostaglandin E production by concanavalin A in isolated Graafian follicles

Addition of Concanavalin A (Con A) to isolated rat Graafian follicles induced prostaglandin E (PGE) production after 2 h of incubation. PGE synthesis continued throughout 24 h culture period. Cyclic AMP accumulation was noted after 6 h of incubation with Con A. .Aspirin, indomethacin and flufenamate prevented both the stimulation of PGE production and of cyclic AMP accumulation by Con A; antibodies to PGE prevented the cyclic AMP production. These studies indicate that the interaction of Con A with the follicle results in PGE production. It seems that besides the known pathway for the induction of PGE synthesis in the ovarian follicle, via elevation of intracellular cyclic AMP, an additional pathway, via an external signal which is independent of cyclic AMP exists.     

Adenosine 3',5'cyclic monophosphate in adipose tissue of diabetic rats.

Normal male rats were made chronically diabetic by injection of alloxan or acutely diabetic by injection of anti-insulin serum. The concentration of cyclic AMP in epididymal adipose tissue was increased approximately 2 1/2-fold 24 h after alloxan administration and up to 7-fold 72 h post-alloxan. Treatment of alloxan-diabetic rats with insulin for 4 h completely suppressed lipolysis but only partially suppressed cyclic AMP levels; 6 h following insulin treatment cyclic AMP levels were normal. When segments of the epididymal fat bodies were incubated in vitro the high cyclic AMP levels were not maintained but instead decreased spontaneously. Addition of insulin to the incubation media decreased lipolysis in tissues of diabetic rats to levels measured in tissues of normal rats and accelerated the decline in cyclic AMP levels but did not return cyclic AMP levels to normal. Rats rendered acutely insulin deficient by injection of anti-insulin serum showed increased plasma glucose and free fatty acid levels and increased adipose tissue free fatty acid, and cyclic AMP levels 30 min following injection of the antiserum. Plasma glucagon levels increased but not until 2 h following anti-insulin serum, thereby excluding the possibility that an increment in plasma glucagon is the primary stimulus for the acceleration of lipolysis in diabetes. These data are consistent with the view that control of adipose tissue cyclic AMP levels in situ is an important physiologic action of insulin.     

Insulin as an activator of cyclic AMP accumulation in rat fat cells.

In rat fat cells incubated with lipolytic agents and insulin for 30 or 60 minutes the increase in cyclic AMP accumulation due to norepinephrine and theophylline or adenosine deaminase added during the last 2-5 minutes of the incubation period was much greater as compared to cells incubated in the absence of insulin. Protaglandin E1 or nicotinic acid were just as anti-lipolytic as insulin but prior incubation with these agents markedly decreased the subsequent rise in cyclic AMP accumulation due to late catecholamine addition. The ability of insulin to increase cyclic AMP accumulation appeared to be secondary to inhibition of lipolysis. These results indicate that prostaglandin E1 and nicotinic acid are inhibitors of cyclic AMP accumulation while insulin acts by another mechanism to reduce lipolysis.     

Cycloheximide potentiation of prostaglandin E1- and cholera toxin-stimulated cyclic AMP accumulation in NG108-CC15 neuroblastoma-glioma hybrid cells

Previous studies have demonstrated that catecholamine responsiveness in a variety of cells can be altered by inhibitors of RNA and protein synthesis. The neuroblastoma-glioma hybrid, NG108-CC15, which lacks catecholamine-stimulated accumulation of cyclic AMP, was investigated to determine if the responsiveness to prostaglandin E1 (PGE1) could be modified by inhibitors of protein synthesis. Cycloheximide in a time-dependent manner potentiated the ability of prostaglandin E1 to stimulate accumulation of intracellular cyclic AMP. However, the alpha-adrenergic inhibition of the prostaglandin response was not affected by cycloheximide. Withdrawal of norepinephrine following a long-term incubation resulted in a potentiation of subsequent PGE1-stimulated cyclic AMP accumulation. Cycloheximide enhanced this norepinephrine withdrawal effect. Our previous studies have shown that cholera toxin induces refractoriness to beta-adrenergic agonists in C6-2B rat astrocytoma cells and that cycloheximide blocked this action of cholera toxin. In an analogous manner cholera toxin caused refractoriness to subsequent prostaglandin-stimulated cyclic AMP production in NG108-CC15 cells, and cycloheximide reduced cholera toxin-induced prostaglandin refractoriness. Thus cycloheximide potentiates the prostaglandin stimulatory effect, has no effect on the ability of alpha-agonists to inhibit the prostaglandin response, increases the stimulatory effect of PGE1 after norepinephrine withdrawal, and reduces cholera toxin-induced PGE1 refractoriness. these observations suggest that PGE1-stimulated cyclic AMP accumulation in NG108-CC15 cells contains components which are regulated by de novo protein synthesis.     

Adrenocorticotropin-induced unresponsiveness in cultured adrenal tumor cells.

Our results demonstrate that adrenocorticotropin (ACTH)-induced refractoriness occurs in cultured adrenal tumor cells. Cells became 85% refractory to ACTH-induced cyclic AMP formation in 20 min and the effect persisted if the hormone remained in the incubation medium. Refractory cells gradually regained hormone-specific responsiveness within 24 h if cultures were incubated in fresh media containing serum. The observed effect is hormone specific since cyclic AMP could not induce unresponsiveness to ACTH. The addition of ACTH plus inhibitors of protein synthesis partially reversed hormone-specific refractoriness. However, preincubation with cycloheximide or diphtheria toxin led to superinduction of ACTH-induced cyclic AMP formation. These experiments suggest that unresponsiveness, following hormonal activation of adrenal cells, may be related to a decrease in hormone-specific binding sites or to synthesis of an adenylate cyclase inhibitor.     

Correlation of the biosynthesis of prostaglandin and cyclic AMP in monolayer cultures of rabbit articular chondrocytes

We have utilized ionophores to test whether stimulation of chondrocyte prostaglandin biosynthesis is accompanied by an increase in cyclic nucleotide levels in these cells. Radioimmunoassay of prostaglandin E2, 6-oxo-prostaglandin F1 alpha (the stable metabolite of prostaglandin I2) and prostaglandin F2 alpha showed that synthesis of each was stimulated by the divalent-cation ionophore, A23187 after short-term incubation (1-7 min) in serum-free medium. No stimulation of thromboxane B2 was detected. Two monovalent ionophores, lasalocid and monensin failed to stimulate prostaglandin biosynthesis after short-term incubation. Ionophore A23187-stimulated prostaglandin biosynthesis was variably and partially inhibited by sodium meclofenamate, indomethacin and aspirin, but not by sodium salicylate. Ionophore A23187-stimulated prostaglandin biosynthesis was accompanied by a 7.5-fold increase in cyclic AMP levels after 15 min. Sodium meclofenamate, indomethacin and aspirin which inhibited prostaglandin E2 biosynthesis also reduced cyclic AMP levels. Exogenous prostaglandin E2 (1 microgram/ml) stimulated cyclic AMP biosynthesis, which was not inhibited by aspirin. These results indicated that prostaglandins can be considered as one of the local effectors controlling cyclic AMP production in articular cartilage.     

Adenosine 3′,5′cyclic monophosphate in adipose tissue of diabetic rats

Normal male rats were made chronically diabetic by injection of alloxan or acutely diabetic by injection of anti-insulin serum. The concentration of cyclic AMP in epididymal adipose tissue was increased approximately 24 h after alloxan administration and up to 7-fold 72 h post-alloxan. Treatment of alloxan-diabetic rats with insulin for 4 h completely suppressed lipolysis but only partially suppressed cyclic AMP levels; 6 h following insulin treatment cyclic AMP levels were normal. When segments of the epididymal fat bodies were incubated in vitro the high cyclic AMP levels were not maintained but instead decreased spontaneously. Addition of insulin to the incubation media decreased lipolysis in tissues of diabetic rats to levels measured in tissues of normal rats and accelerated the decline in cyclic AMP levels but did not return cyclic AMP levels to normal. Rats rendered acutely insulin deficient by injection of anti-insulin serum showed increased plasma glucose and free fatty acid levels and increased adipose tissue free fatty acid, and cyclic AMP levels 30 min following injection of the antiserum. Plasma glucagon levels increased but not until 2 h following anti-insulin serum, thereby excluding the possibility that an increment in plasma glucagon is the primary stimulus for the acceleration of lipolysis in diabetes. These data are consistent with the view that control of adipose tissue cyclic AMP levels in situ is an important physiologic action of insulin.     

Enhancement of hormonal stimulation in intact cells. Potentiation of GTP-dependent activation of adenylate cyclase.

The ability of prostaglandin E1 (PGE1) and cholera toxin to increase cyclic AMP levels is potentiated 6-fold when normal rat kidney (NRK) cells are treated with picolinic acid or histidinol, or grown in isoleucine-deficient medium. The response to (-)-isoproterenol is increased 2-fold in NRK cells treated with picolinic acid but not in cells subjected to isoleucine deprivation. The increase in agonist responsiveness is time-dependent, reaches its maximum at 40 h, and is quickly reversed following removal of picolinic acid or addition of medium with normal amounts of isoleucine. The cholera toxin response is also increased about 7-fold in simian virus 40-transformed NRK cells and Moloney sarcoma virus-transformed NRK cells treated with picolinic acid. GTP-stimulated, but not fluoride-stimulated, adenylate cyclase activities are increased in membranes from NRK cells treated with picolinic acid or starved for isoleucine, indicating that the increased response is due, at least in part, to a specific potentiation of GTP-dependent functions of the adenylate cyclase system. The results demonstrate that GTP-dependent events in hormonal stimulation of adenylate cyclase can be altered in intact cells to modulate hormonal enhancement of cyclic AMP production.     

Stimulation of prolactin synthesis and of adenosine 3'':5''-cyclic phosphate formation by prostaglandins and thyroliberin in cultured rat pituitary cells.

1. The effects of prostaglandins E2 and F2alpha on prolactin synthesis were examined in a clonal strain of rat pituitary tumour cells, and compared with those of thyroliberin. 2. The prostaglandins and thyroliberin gave a dose-related and time-dependent stimulation of prolactin synthesis. The maximal effects (about twofold increases) were observed after 54h of treatment with 25nM-prostaglandin E2 and 2.5nM-prostaglandin F2alpha. A similar stimulation of prolactin synthesis was observed after 250nM-thyroliberin. The combined treatment with prostaglandins and thyroliberin did not increase prolactin synthesis over and above that obtained with each compound alone. 3. After removal of prostaglandins E2 and F2alpha there was a complete reversal of prolactin synthesis to pre-stimulation values 18h later (t1/2less than or equal to 9h). The rapid reversible effect of prostaglandins was in contrast with that of thyroliberin, where prolactin synthesis returned to control values with a t1/2 of about 42 h. 4. Prostaglandin E2 (5mum) and thyroliberin (5mum) increased cellular concentrations of cyclic AMP eight- and four-fold respectively. Maximal effects were observed after 2-5min of incubation. The increases in cyclic AMP were biphasic; normal values were obtained 60 min after the start of incubation with prostaglandin E2 or thyroliberin. 5. The dose/response curve showed that prostaglandin E2 caused maximal increase of cyclic AMP at 50nM. Concentrations of prostagland in E2 that caused half-maximal stimulation of cyclic AMP accumulation and of prolactin synthesis were 4 and 5nM respectively. 6. Combined treatment with prostaglandin E2 and thyroliberin in concentrations that separately caused maximal cyclic AMP increases did not result in a further increase in this cyclic nucleotide. 7. These results are consistent with a role of cyclic AMP in mediating the effects or prostaglandins and thyroliberin on prolactin synthesis. However, if cyclic AMP is involved as a common intracellular mediator of prolactin synthesis, it cannot alone explain all the effects of prostaglandins and thyroliberin in this cell system.     

Action of luteinizing hormone-releasing hormone and synthesis of prostaglandin in the pituitary gland.

The possibility that prostaglandin E2 (PGE2) may play a role in luteinizing hormone (LH) release was examined using an in vitro model. Addition of luteinizing hormone-releasing hormone (LH-RH) to the culture medium stimulated cyclic AMP accumulation and LH-release by incubated hemipituitaries, but did not affect the level of PGE2 or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE2 or prostaglandin synthetase activity in the gland. Aspirin and indomethacin reduced both prostaglandin synthetase activity and PGE2 content in the pituitary, but did not impair the stimulatory action of LH-RH on either cyclic AMP accumulation or LH-release. Flufenamic acid on its own caused LH-release, but the drug abolished the effect of LH-RH on cyclic AMP accumulation. The mechanism of this action of flufenamic acid is not understood. It is concluded that the stimulatory action of LH-RH on pituitary cyclic AMP production and LH release is not mediated by prostaglandins.     

Effects of delta 1-tetrahydrocannabinol on cyclic AMP in cultured human diploid fibroblasts.

(-)-trans-delta 1-Tetrahydrocannabinol (delta 1-THC) antagonized the cyclic AMP responses of WI-38 fibroblasts to both prostaglandin E1 (PGE1) and catecholamines. Both cellular cyclic AMP accumulation and cyclic AMP escape to the incubation medium were reduced, but the reduction of escape was much more dramatic at all concentrations of the drug. Conversely, long term incubations of cells with delta 1-THC alone resulted in substantial accumulations of cyclic AMP in the incubation medium. This effect was potentiated by the phosphodiesterase inhibitor 1-methyl, 3-isobutylxanthine and appeared to result from weak agonist activity of the cannabinoid as determined by a) stimulation of radioactivity incorporated into cyclic AMP using 3H-adenine prelabelled cells, and b) a rapid and pronounced increase in the activity ratio of cellular protein kinase. The antagonistic effect of delta 1-THC on the cellular response to PGE1 was greater in preconfluent cells than in confluent monolayers. Further, the increased sensitivity of preconfluent cultures to delta 1-THC was associated with the appearance of cytoplasmic vacuoles in the perinuclear region of the cells. Cannabidiol acted similar to delta 1-Thc in affecting cyclic AMP metabolis whereas cannabinol and cannabicyclol showed mixed effects on the various parameters studied.     

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.