cAMP and human neutrophil chemotaxis. Elevation of cAMP differentially affects chemotactic responsiveness.

Neutrophils (PMN) treated with cAMP elevating agents were evaluated for their chemotactic responsiveness to FMLP and leukotriene B4 (LTB4). PGE1 and isoproterenol, increased PMN cyclic AMP production and inhibited chemotaxis to both FMLP and LTB4. In contrast, forskolin, which activates adenylate cyclase directly, inhibited chemotaxis to FMLP but not to LTB4. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), was required for inhibition of PMN chemotaxis to FMLP by forskolin, PGE1, and isoproterenol. Isoproterenol and PGE1 inhibited PMN chemotaxis to LTB4 in the absence of IBMX and chemotaxis was further inhibited in the presence of IBMX. PMN cAMP levels were stimulated 2- to 3-fold with isoproterenol, 6- to 10-fold with PGE1, and 5- to 7-fold with forskolin over basal levels in the presence of IBMX. These observations demonstrate that total cellular cAMP concentration is not correlated with inhibition of PMN chemotaxis to all stimuli; forskolin, which increased cyclic AMP 5- to 7-fold over basal levels, did not inhibit chemotaxis to LTB4, whereas isoproterenol, which increased cyclic AMP only 2- to 3-fold over basal levels, inhibited chemotaxis to LTB4. PMN cAMP extrusion was determined under basal conditions and in the presence of PGE1, isoproterenol, or forskolin. PMN extruded cAMP under all conditions examined.     

Opioid agonists binding and responses in SH-SY5Y cells.

SH-SY5Y (human neuroblastoma) cultured cells, known to have mu-opioid receptors, have been used to assess and compare the ability of eight representative mu-selective compounds from diverse opioid families to recognize and activate these receptors. A wide range of receptor affinities spanning a factor of 10,000 was found between the highest affinity fentanyl analogs (Ki = 0.1nM) and the lowest affinity analog, meperidine (Ki = 1 microM). A similar range was found for inhibition of PGE1-stimulated cAMP accumulation with a rank order of activities that closely paralleled binding affinities. Maximum inhibition of cAMP accumulation by each compound was about 80%. Maximum stimulation of GTPase activity (approximately 50%) was also similar for all compounds except the lowest affinity meperidine. Both effects were naloxone reversible. These results provide further evidence that mu-receptors are coupled to inhibition of adenylate cyclase and that the SH-SY5Y cell line is a good system for assessment of mu-agonists functional responses.     

Retinoic acid enhances VIP receptor expression and responsiveness in human neuroblastoma cell, SH-SY5Y

Retinoic acid (RA) induces partial differentiation of neuroblastoma (NB) cells in vitro. In the human NB line, SH-SY5Y (a neuroblastic subclone of SK-N-SH), RA was previously shown to enhance the stimulatory (PGE1) and inhibitory (opioid) regulation of adenylyl cyclase. Since these cells are also sensitive to cAMP stimulation by vasoactive intestinal peptide (VIP), we have tested the effects of RA on VIP receptor expression and function. Pretreatment of SH-SY5Y cells with 10 microM RA over 6 days dramatically increased VIP receptor number from approximately 3,000 to approximately 70,000 sites per cell and enhanced threefold the cAMP accumulation after external VIP addition, while VIP immunoreactive content in the cells increased 2-3-fold. In the light of the recently proposed autocrine function of VIP in this cell lineage, the strong enhancement of the VIP system may contribute to the differentiation effects of RA.     

Regulation of human basophil and lung mast cell function by cyclic adenosine monophosphate

Immunologic activation of purified human lung mast cells (HLMC) and basophils with anti-IgE induced histamine release but failed to elicit any changes in cAMP levels. In contrast, histamine release and monophasic rises in cAMP were observed in both rat peritoneal mast cells (RPMC) challenged with concanavalin A (73% enhancement over basal cAMP 20 sec after activation) and a cultured mouse bone marrow-derived mast cell (PT18 cell line) passively sensitized with dinitrophenol-specific IgE and stimulated with antigen (39% increase above basal at 15 sec). The adenylate cyclase activators isoprenaline, prostaglandin E2 (PGE2), and forskolin and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) all induced elevations in cAMP levels in both basophils and HLMC. In basophils, PGE2 and isoprenaline produced approximately twofold increases in cAMP that were maximal at 1 min and decayed thereafter. Forskolin and IBMX produced threefold increases in cAMP that peaked 10 min after activation and persisted for up to 20 min. In HLMC, isoprenaline provoked a rapid monophasic fourfold increase in cAMP that was maximal at 1 min after addition. Levels of cAMP subsequently declined but remained significantly elevated over resting levels for up to 30 min. PGE2, forskolin, and IBMX all produced approximately threefold rises in HLMC cAMP that peaked around 5 min and persisted for 30 min. In both the basophil and HLMC, agonist-induced elevations in cAMP correlated well with the inhibition of mediator release. In basophils, the order IBMX greater than forskolin greater than PGE2 greater than isoprenaline held for both the inhibition of histamine and leukotriene C4 release and the augmentation of cAMP levels. In HLMC, individual agonists elevated cAMP levels to similar degrees and inhibited the release of histamine, leukotriene C4, and PGD2 to comparable extents, although the release of the arachidonate metabolites was generally more sensitive to the inhibitory actions of these agonists. These results suggest that elevations in cAMP, in both the basophil and HLMC, are associated with the inhibition of mediator release but not the initiation of the secretory process.     

Intracellular lithium and cyclic AMP levels are mutually regulated in neuronal cells

In this work, we studied the effect of intracellular 3',5'-cyclic adenosine monophosphate (cAMP) on Li+ transport in SH-SY5Y cells. The cells were stimulated with forskolin, an adenylate cyclase activator, or with the cAMP analogue, dibutyryl-cAMP. It was observed that under forskolin stimulation both the Li+ influx rate constant and the Li+ accumulation in these cells were increased. Dibutyryl-cAMP also increased Li+ uptake and identical results were obtained with cortical and hippocampal neurons. The inhibitor of the Na+/Ca2+ exchanger, KB-R7943, reduced the influx of Li+ under resting conditions, and completely inhibited the effect of forskolin on the accumulation of the cation. Intracellular Ca2+ chelation, or inhibition of N-type voltage-sensitive Ca2+ channels, or inhibition of cAMP-dependent protein kinase (PKA) also abolished the effect of forskolin on Li+ uptake. The involvement of Ca2+ on forskolin-induced Li+ uptake was confirmed by intracellular free Ca2+ measurements using fluorescence spectroscopy. Exposure of SH-SY5Y cells to 1 mm Li+ for 24 h increased basal cAMP levels, but preincubation with Li+, at the same concentration, decreased cAMP production in response to forskolin. To summarize, these results demonstrate that intracellular cAMP levels regulate the uptake of Li+ in a Ca(2+)-dependent manner, and indicate that Li+ plays an important role in the homeostasis of this second messenger in neuronal cells.     

Propranolol inhibits cyclic AMP accumulation and amylase secretion in parotid acinar cells stimulated by isobutylmethylxanthine and forskolin.

Propranolol inhibited cyclic AMP (cAMP) accumulation stimulated by 3-isobutyl-1-methylxanthine (IBMX) or forskolin in rat parotid acinar cells. The inhibition by propranolol was highly potent; 10(-7) M propranolol was sufficient for the maximum inhibition (approx. 50% at 5 min). The inhibitory effect was observed in both intact and saponin-permeabilized parotid cells, but the effect was more prominent in permeabilized cells than in intact cells. Other beta-blockers, like alprenolol and atenolol, were as effective as propranolol, but butoxamine (beta 2-selective) was slightly less effective. The inhibition by propranolol was similarly detected in the cells prepared from pertussis-toxin-pretreated rats, suggesting that inhibitory guanine nucleotide regulatory protein (Gi) is not involved in the inhibitory mechanism. Propranolol also inhibited the exocytosis of amylase stimulated by IBMX or forskolin. In the presence of propranolol and IBMX, the responsiveness of saponin-permeabilized cells to exogenous cAMP was markedly increased, indicating that propranolol neither promotes the degradation of cAMP nor prevents the inhibitory effect of IBMX on cAMP phosphodiesterase.     

Cyclic adenosine 3',5'-monophosphate-induced ovulation in the perfused rat ovary and its mediation by prostaglandins

The role and mechanism of action of cyclic adenosine 3',5'-monophosphate (cAMP) in the ovulatory process was investigated by using the in vitro-perfused rat ovary model. Ovaries of pregnant mare's serum gonadotropin (PMSG, 20 IU)-primed rats were perfused for 21 h beginning in the morning of induced proestrus. In vitro stimulation with luteinizing hormone (LH; 0.1 micrograms/ml) resulted in 2.4 +/- 0.7 ovulations per treated ovary. Ovulations could also be induced by the addition of forskolin (30 microM) or dibutyryl cAMP (dbcAMP, 1 mM) with isobutylmethylxanthine (IBMX, 0.2 mM), with 11.8 +/- 1.9 and 18.6 +/- 4.4 ovulations per treated ovary, respectively. Indomethacin (5 micrograms/ml) significantly decreased the number of ovulations in the forskolin and dbcAMP + IBMX groups. The addition of prostaglandin E2 (PGE2; 1 micrograms/ml three times during the perfusion) to the forskolin + indomethacin group reversed the inhibition of ovulation (21.6 +/- 5.4 ovulations per treated ovary). Ovarian PGE tissue levels were significantly higher 10 h after stimulation with either LH, forskolin, or dbcAMP + IBMX compared to the unstimulated control group. Ovulated oocytes in the LH and forskolin groups resumed meiosis but oocytes in the dbcAMP + IBMX groups remained immature. This study shows that an increase in ovarian cAMP, even if not induced by LH, is sufficient to cause ovulation of preovulatory rat follicles, supporting the involvement of cAMP in the normal ovulatory process of the PMSG-treated rat. Furthermore, prostaglandin involvement in cAMP-induced ovulations is demonstrated.     

Role of Adenylate Cyclase in Presynaptic α2-Adrenoceptor- and μ-Opioid Receptor-Mediated Inhibition of [3H]Noradrenaline Release from Rat Brain Cortex Slices

Rat brain cortex slices, prelabelled with [3H]noradrenaline, were superfused and exposed to electrical biphasic block pulses (1 Hz; 12 mA, 4 ms) or to the Ca2+ ionophore A 23187 (10 microM) in the presence of 1.2 mM Ca2+. Forskolin (10 microM), 8-bromo-cyclic AMP (300 microM), and dibutyryl-cyclic AMP (300 microM) facilitated both the electrically evoked and A 23187-induced [3H]noradrenaline release, whereas the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX, 300 microM) and 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone (ZK 62771, 30 microM) enhanced the electrically evoked release only. The inhibitory effects of clonidine (1 nM-1 microM) and the facilitatory effect of phentolamine (0.01-10 microM) on the electrically evoked [3H]noradrenaline release were strongly reduced in the presence of 8-bromo-cyclic AMP. Clonidine (1 microM) reduced and phentolamine (3 microM) enhanced A 23187-induced [3H]noradrenaline release, provided that the slices were simultaneously exposed to forskolin. The inhibitory effects of morphine (1 microM) and [D-Ala2-D-Leu5]enkephalin (DADLE, 0.3 microM), like that of the Ca2+ antagonist Cd2+ (15 microM), on the electrically evoked release of [3H]noradrenaline were not affected by 8-bromo-cyclic AMP. Moreover, morphine and DADLE did not inhibit A 23187-induced release in the absence or presence of forskolin. These data strongly suggest that in contrast to presynaptic mu-opioid receptors, alpha 2-adrenoceptors on noradrenergic nerve terminals are negatively coupled to adenylate cyclase and may thus reduce neurotransmitter release by inhibiting the feed-forward action of cyclic AMP on the secretion process.     

Protein kinase C activation reduces microglial cyclic AMP response to prostaglandin E2 by interfering with EP2 receptors

We studied the modulation by protein kinase C (PKC) of the cyclic AMP (cAMP) accumulation induced by prostaglandin (PG) E2 in rat neonatal microglial cultures. Short pretreatment of microglia with phorbol 12-myristate 13-acetate (PMA) or 4beta-phorbol 12,13-didecanoate, which activate PKC, but not with the inactive 4alpha-phorbol 12,13-didecanoate, substantially reduced cAMP accumulation induced by 1 microM PGE2. The action of PMA was dose and time dependent, and the maximal inhibition (approximately 85%) was obtained after 10-min preincubation with 100 nM PMA. The inhibitory effect of PMA was mimicked by diacylglycerol and was prevented by the PKC inhibitor calphostin C. As PMA did not affect isoproterenol- or forskolin-stimulated cAMP accumulation, we investigated whether activation of PKC decreased cAMP production by acting directly at PGE2 EP receptors. Neither sulprostone (10(-9)-10(-5) M), a potent agonist at EP3 receptors (coupled to adenylyl cyclase inhibition), nor 17-phenyl-PGE2 (10(-6)-10(-5) M), an agonist of EP1 receptors, modified cAMP accumulation induced by forskolin. On the contrary, 11-deoxy-16,16-dimethyl PGE2, which does not discriminate between EP2 and EP4 receptors, both coupled to the activation of adenylyl cyclase, and butaprost, a selective EP2 agonist, induced a dose-dependent elevation of cAMP that was largely reduced by PMA pretreatment, as in the case of PGE2. These results indicated EP2 receptors as a possible target of PKC and suggest that PKC-activating agents present in the pathological brain may prevent the cAMP-mediated microglia-deactivating function of PGE2.     

Vasoactive intestinal peptide and forskolin regulate proliferation of the HT29 human colon adenocarcinoma cell line.

Although several lines of evidence implicate cAMP in the regulation of intestinal cell proliferation, the precise role of this second messenger in the control of the human colon cancer cell cycle is still unclear. In order to investigate the role of cAMP in HT29 cell proliferation, we have tested the effect of vasoactive intestinal peptide (VIP) and forskolin on DNA synthesis and cell number, focusing on the time-dependent efficacy of the treatment. The cells were arrested in G0/G1 phase by incubation for 24 h in serum-free medium and proliferation was re-initiated by addition of either 85 nM insulin or 0.5% fetal calf serum. In the presence of fetal calf serum, G1/S transition was found to occur earlier than with insulin. Exposure of the HT29 cells to 10(-5) M forskolin in the early stages of growth induction (within 12 h from FCS addition or within 14 h from insulin treatment) resulted in a significant inhibition of DNA synthesis and a delayed entry in the S phase. By contrast, VIP (10(-7) M) was inhibitory only when added within a narrow window (10 to 12 h or 12 to 14 h following FCS or insulin addition, respectively). The difference in efficiency of forskolin and VIP to inhibit cell proliferation may be correlated with their own potency to promote long-lasting cAMP accumulation. The combination of VIP plus forskolin had synergistic effects on both cAMP accumulation and cell-growth inhibition. Taken together, our data indicate that cAMP may act at a step in the late G1 or G1/S transition.     

PACAP-38 induces neuronal differentiation of human SH-SY5Y neuroblastoma cells via cAMP-mediated activation of ERK and p38 MAP kinases

The intracellular signaling pathways mediating the neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP) were investigated in human neuroblastoma SH-SY5Y cells. Previously, we showed that SH-SY5Y cells express the PAC(1) and VIP/PACAP receptor type 2 (VPAC(2)) receptors, and that the robust cAMP production in response to PACAP and vasoactive intestinal peptide (VIP) was mediated by PAC(1) receptors (Lutz et al. 2006). Here, we investigated the ability of PACAP-38 to differentiate SH-SY5Y cells by measuring morphological changes and the expression of neuronal markers. PACAP-38 caused a concentration-dependent increase in the number of neurite-bearing cells and an up-regulation in the expression of the neuronal proteins Bcl-2, growth-associated protein-43 (GAP-43) and choline acetyltransferase: VIP was less effective than PACAP-38 and the VPAC(2) receptor-specific agonist, Ro 25-1553, had no effect. The effects of PACAP-38 and VIP were blocked by the PAC(1) receptor antagonist, PACAP6-38. As observed with PACAP-38, the adenylyl cyclase activator, forskolin, also induced an increase in the number of neurite-bearing cells and an up-regulation in the expression of Bcl-2 and GAP-43. PACAP-induced differentiation was prevented by the adenylyl cyclase inhibitor, 2',5'-dideoxyadenosine (DDA), but not the protein kinase A (PKA) inhibitor, H89, or by siRNA-mediated knock-down of the PKA catalytic subunit. PACAP-38 and forskolin stimulated the activation of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAP; p38 MAP kinase) and c-Jun N-terminal kinase (JNK). PACAP-induced neuritogenesis was blocked by the MEK1 inhibitor PD98059 and partially by the p38 MAP kinase inhibitor SB203580. Activation of exchange protein directly activated by cAMP (Epac) partially mimicked the effects of PACAP-38, and led to the phosphorylation of ERK but not p38 MAP kinase. These results provide evidence that the neurotrophic effects of PACAP-38 on human SH-SY5Y neuroblastoma cells are mediated by the PAC(1) receptor through a cAMP-dependent but PKA-independent mechanism, and furthermore suggest that this involves Epac-dependent activation of ERK as well as activation of the p38 MAP kinase signaling pathway.     

VIP elevates platelet cyclic AMP (cAMP) levels and inhibits in vitro platelet activation induced by platelet-activating factor (PAF)

Platelet-activating factor (PAF), a potent endogenous phospholipid released by a variety of mammalian cells, induces platelet activation in vivo and in vitro. Little is known, however, about the physiological modulation of its actions. We have examined the ability of two naturally occurring compounds which stimulate cAMP production, vasoactive intestinal peptide (VIP) and prostacyclin (PGI2), to inhibit PAF-induced platelet aggregation and secretion in vitro. Washed, [3H]serotonin-labeled, rabbit platelets were incubated 60 sec in the presence of VIP, PGI2 or 3-isobutyl-1-methylxanthine (IBMX) and subsequently stimulated with PAF. In separate studies, cAMP levels were determined in similar aliquots of platelets incubated for 30 sec with VIP, PGI2 or IBMX. VIP, PGI2 and IBMX inhibited platelet aggregation and secretion in a dose-dependent manner. Fifty percent inhibition was achieved at final concentrations of 1.7 X 10(6) M VIP, 3.6 X 10(6) M PGI2 and 6.5 X 10(5) M IBMX. IBMX potentiated the inhibitory effects of VIP and PGI2 on PAF-induced platelet activation. VIP and PGI2 elevated platelet cAMP levels four-fold and 50-fold, respectively, in the presence of 10(3) M IBMX. These findings demonstrate that VIP inhibits PAF-induced platelet activation, with a potency comparable to that of PGI2.     

Cyclic AMP-mediated modulation of immunoglobulin production in B cells by prostaglandin E1.

We had previously demonstrated in a transformed human B cell line, LA350, the existence of an inverse relationship between cyclic adenosine monophosphate (cAMP) content and immunoglobulin secretion using the cAMP-elevating agents such as cholera toxin and forskolin. In this paper we report that cAMP acting as a second messenger for prostaglandin exerts a similar effect on the antibody response of B lymphocytes. Incubation of the cells with PGE1 in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) produced a concentration- and time-dependent elevation of intracellular cAMP. Significant increases of cAMP production were observed at physiologically relevant levels of PGE1 (10(-7) and 10(-8) M). Immunoglobulin production, whether measured as the total number of immunoglobulin-secreting cells by a reverse hemolytic plaque assay or as specific immunoglobulin production (IgM) by an enzyme-linked immunoadsorbent assay, was suppressed in a dose-dependent fashion by the presence of IBMX. This suppression of immunoglobulin production was significantly enhanced by the presence of PGE1. Phorbol myristate acetate-induced IgM production was also inhibited by the presence of PGE1. These results imply that prostaglandins regulate B cell activation and immunoglobulin production by signal transduction mechanisms involving cyclic nucleotides.     

Muscarinic receptor-linked elevation of cAMP in SH-SY5Y neuroblastoma cells is mediated by Ca2+ and protein kinase C.

The mechanisms of muscarinic receptor-linked increase in cAMP accumulation in SH-SY5Y human neuroblastoma cells has been investigated. The dose-response relations of carbachol-induced cAMP synthesis and carbachol-induced rise in intracellular free Ca2+ were similar. The stimulated cAMP synthesis was inhibited by about 50% when cells were entrapped with the Ca2+ chelator BAPTA or in the presence of the protein kinase C (PKC) inhibitor staurosporine. Production of cAMP could be induced also by the Ca2+ ionophore, ionomycin and by TPA, an activator of PKC. When added together TPA and ionomycin had a synergistic effect. When cAMP synthesis was activated with cholera toxin, PGE1 or PGE1 + pertussis toxin carbachol stimulated cAMP production to the same extent as in control cells. Ca2+ and protein kinase C thus seem to be the mediators of muscarinic-receptor linked cAMP synthesis by a direct action on adenylate cyclase.     

Bradykinin inhibition of cyclic AMP accumulation in D384 astrocytoma cells. Evidence against a role of cyclic GMP.

The present studies were performed in order to examine the possible role of cyclic GMP-stimulated phosphodiesterase (cGMP-PDE) activity in the inhibitory action of the inflammatory peptide bradykinin on cyclic AMP (cAMP) accumulation in D384 cells. Bradykinin decreased the forskolin-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor rolipram, and caused a transient 50% rise in cellular cGMP in the presence of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Both basal and bradykinin-stimulated cGMP accumulation were about 8 times higher in the presence of IBMX than in the presence of rolipram. Sodium nitroprusside, which caused a 20-70-fold increase in cGMP levels reduced forskolin stimulated cAMP accumulation, whereas hydroxylamine, which maximally caused a 16-fold increase in cGMP, did not. 8-bromo-cGMP or dibutyryl cGMP had no effect on cAMP accumulation induced by forskolin. The inhibitory effect of nitroprusside was totally reversed by blocking the soluble guanylate cyclase activity by methylene blue treatment; however, the inhibitory action of bradykinin on cAMP accumulation was not changed by this treatment. Additionally, inhibition of nitric oxide synthesis, which is known to be regulated by Ca2+ and in turn stimulates cGMP production, by N omega-nitro-L-arginine (L-NAME) treatment did not alter the inhibitory effect of bradykinin on forskolin-induced cAMP accumulation. These results indicate that large increases in cGMP may regulate cAMP via cGMP-PDE whereas the small increase induced by bradykinin is insufficient and that cGMP is not involved in the inhibitory action of bradykinin on cAMP levels in D384 cells.     

Protein kinase A and/or C inhibitors potentiate isoproterenol-induced cyclic AMP accumulation in intact human lymphocytes

The objective of the present study was to investigate the roles of protein kinase A and/or C in agonist-induced beta adrenoceptor activation in intact human lymphocytes. LYmphocytes from healthy subjects were incubated with isoproterenol and phosphodiesterase inhibitor (IBMX, 1.0 mM) after 20 minutes of preincubation with (or without) various compounds possessing protein kinase A and/or C inhibitory activities. These compounds included the relatively selective protein kinase C (PK-C) inhibitors (W-7, calmidazolium, polymyxin B, neomycin, tamoxifen and clomiphene), purified protein inhibitors of protein kinase A (PK-A) (obtained synthetically, or purified from bovine hearts and porcine hearts) and the two compounds (H-7, H-9), which have been found to inhibit both PK-A and PK-C. The results showed that all PK-C inhibitors alone decreased cellular basal cAMP levels while inhibitors of PK-A as well as both H-7 and H-9 increased basal cAMP levels in a dose dependent manner at certain concentrations. All inhibitors studied potentiated isoproterenol-induced cAMP accumulation. The protein kinase A and C inhibitor, H-7, also potentiated PGE1 (but not forskolin)-induced cAMP accumulation. In contrast, the protein kinase C activator, PMA, inhibited isoproterenol- and PGE1- (but not forskolin) induced cAMP accumulation. These data suggest that the potentiating effects of PK-A and/or C inhibitors may be related to the inhibition of PK-A and/or PK-C, both of which have been shown to be involved in beta 2 adrenoceptor desensitization and phosphorylation.     

Role of cAMP in modulating intrafollicular progesterone levels and oocyte maturation in amphibians (Rana pipiens)

The role of cAMP in regulating follicular progesterone levels and oocyte maturation was investigated following in vitro culture of amphibian (Rana pipiens) ovarian follicles. Intrafollicular levels of cAMP were manipulated with the use of a stimulator of cAMP synthesis (forskolin) or by exogenous addition of cAMP alone or either of these in combination with an inhibitor of cAMP catabolism (3-isobutyl-1-methyl xanthine, IBMX). Follicular progesterone content was determined by RIA and oocyte maturation was assessed cytologically. In the presence of increasing doses of forskolin (0-3 microM), cAMP (0-3 mM), or dibutyryl cAMP (dbcAMP, 0-2.5 mM) increasing but low levels of progesterone were detected. Increasing doses of IBMX (0-0.09 mM) alone had no significant effect on follicular steroid content. Exogenous cAMP, dbcAMP, or IBMX (0.09 mM) suppressed hormone-induced oocyte maturation. Simultaneous exposure of follicles to increasing doses of both forskolin (0-3 microM) and IBMX (0-0.09 mM) markedly increased intrafollicular progesterone levels to those produced by frog pituitary homogenate (FPH). A marked increase in progesterone levels also occurred when follicles were exposed to exogenous cAMP (3 mM) and IBMX (0.09 mM). These results indicate that exogenous cAMP is incorporated by follicle cells and that forskolin effects are mediated through cAMP. Changes in follicular progesterone levels (increase and decrease) over time following FPH or cAMP manipulation (cAMP + IBMX or forskolin + IBMX) were essentially identical. In contrast to cAMP, cGMP was inactive in inhibiting hormone induced GVBD or stimulating follicular progesterone accumulation. Elevation of follicular and medium levels of progesterone resulting from FPH or cAMP stimulation required the presence of the somatic follicular cells. The decrease in follicular progesterone levels with prolonged culture was not associated with a corresponding increase in progesterone levels in the medium. The decrease in follicular progesterone levels appears to reflect steroid catabolism rather than loss of steroid to the culture medium. The results suggest that the level of intracellular cAMP in the follicle cells is modulated by the relative activity of the adenylate cyclase system and phosphodiesterase and that FPH can affect both components. Thus, intracellular levels of cAMP play a key role in regulating follicular progesterone levels and FPH action on the follicle cells. The steroidogenic capacity of follicle cells can be manipulated independently of FPH stimulation.