Forskolin Activation Of Adenylyl Cyclase Research Articles

Insulin, dibutyryl-cAMP, and glucose modulate expression of patatin-like domain containing protein 7 in cultured human myotubes.

Expression of patatin-like phospholipase domain containing protein 7 (PNPLA7), also known as neuropathy target esterase-related esterase (NRE), a lysophospholipase, increases with fasting and decreases with feeding in mouse skeletal muscle, indicating it is regulated by insulin, counterregulatory hormones, such as glucocorticoids and catecholamines, and/or nutrients. In cultured mouse adipocytes insulin reduces Pnpla7 expression, underscoring the possibility that insulin regulates PNPLA7 in skeletal muscle. The first aim of this study was to establish whether PNPLA7 is functionally expressed in cultured human skeletal muscle cells. The second aim was to determine whether PNPLA7 is regulated by insulin, glucocorticoids, cAMP/protein kinase A pathway, and/or glucose. Cultured human skeletal muscle cells expressed PNPLA7 mRNA and protein. Gene silencing of PNPLA7 in myoblasts reduced the phosphorylation of 70 kDa ribosomal protein S6 kinase and ribosomal protein S6 as well as the abundance of α1-subunit of Na+,K+-ATPase and acetyl-CoA carboxylase, indirectly suggesting that PNPLA7 is functionally important. In myotubes, insulin suppressed PNPLA7 mRNA at 1 g/L glucose, but not at low (0.5 g/L) or high (4.5 g/L) concentrations. Treatment with synthetic glucocorticoid dexamethasone and activator of adenylyl cyclase forskolin had no effect on PNPLA7 regardless of glucose concentration, while dibutyryl-cAMP, a cell-permeable cAMP analogue, suppressed PNPLA7 mRNA at 4.5 g/L glucose. The abundance of PNPLA7 protein correlated inversely with the glucose concentrations. Collectively, our results highlight that PNPLA7 in human myotubes is regulated by metabolic signals, implicating a role for PNPLA7 in skeletal muscle energy metabolism.

Forskolin Regulates L-Type Calcium Channel through Interaction between Actinin 4 and β3 Subunit in Osteoblasts.

Voltage-dependent L-type calcium channels that permit cellular calcium influx are essential in calcium-mediated modulation of cellular signaling. Although the regulation of voltage-dependent L-type calcium channels is linked to many factors including cAMP-dependent protein kinase A (PKA) activity and actin cytoskeleton, little is known about the detailed mechanisms underlying the regulation in osteoblasts. Our present study investigated the modulation of L-type calcium channel activities through the effects of forskolin on actin reorganization and on its functional interaction with actin binding protein actinin 4. The results showed that forskolin did not significantly affect the trafficking of pore forming α1c subunit and its interaction with actin binding protein actinin 4, whereas it significantly increased the expression of β3 subunit and its interaction with actinin 4 in osteoblast cells as assessed by co-immunoprecipitation, pull-down assay, and immunostaining. Further mapping showed that the ABD and EF domains of actinin 4 were interaction sites. This interaction is independent of PKA phosphorylation. Knockdown of actinin 4 significantly decreased the activities of L-type calcium channels. Our study revealed a new aspect of the mechanisms by which the forskolin activation of adenylyl cyclase - cAMP cascade regulates the L-type calcium channel in osteoblast cells, besides the PKA mediated phosphorylation of the channel subunits. These data provide insight into the important role of interconnection among adenylyl cyclase, cAMP, PKA, the actin cytoskeleton, and the channel proteins in the regulation of voltage-dependent L-type calcium channels in osteoblast cells.

Chuanxiongzine relaxes isolated corpus cavernosum strips and raises intracavernous pressure in rabbits

It has been shown that there are many Chinese traditional herbals that can enhance sexual activity. Chuanxiongzine is a vasoactive ingredient that has been isolated and purified from Ligusticum chuanxiong Hort. In previous studies, it has been found that chuanxiongzine was effective in relaxing rabbit corpus cavernosum smooth muscle. We determined the effects of chuanxiongzine on relaxation of isolated corpus cavernosum strips in vitro and on increase of intracavernous pressure (ICP) in vivo in rabbits. Chuanxiongzine caused a concentration-dependent relaxation of phenylephrine precontracted isolated corpus cavernosum strips (EC50 1.58 × 10−4 mol l−1), which were endothelium independent and NO independent. However, the guanylyl cyclase inhibitor 1-H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one significantly shifted the chuanxiongzine concentration–response relationship to the right. Although there was no significant difference in the level of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) in isolated corpus cavernosum strips treated with chuanxiongzine or vehicle, chuanxiongzine caused a significant rise in the level of cGMP and cAMP in isolated corpus cavernosum strips pretreated with the activator of adenylyl cyclase forskolin and the source of NO sodium nitroprusside. In an in vivo study, chuanxiongzine dose-dependently raised ICP after the intracavernous injection of its cumulative doses (0.5, 1, 2 and 5 mg kg−1). The ICP increased from baseline to 19.1±3.7, 24.8±2.1, 30.2±4.8 and 39.7±6.1 mm Hg, respectively, and the duration of tumescence ranged from 8.5±2.8 to 22.9±7.3 min. Our results show that chuanxiongzine can relax isolated corpus cavernosum strips of rabbits in vitro and increase ICP of rabbits in vivo, which is neither endothelium dependent nor NO dependent, but may be partly mediated by the inhibition of cAMP phosphodiesterase or cGMP phosphodiesterase.

Depolarisation and suppression of burst firing activity in the mouse subthalamic nucleus by dopamine D1/D5 receptor activation of a cyclic-nucleotide gated non-specific cation conductance

Neuronal burst firing in the subthalamic nucleus (STN) is one of the hallmarks of dopamine depletion in Parkinson's disease. Here, we have determined the postsynaptic effects of dopamine in the STN and the functional consequences of dopamine receptor modulation on burst firing in vitro. STN cells displayed regular spiking activity at a rate of 7.9 ± 0.5 Hz. Application of dopamine (30 μM) induced membrane depolarisations accompanied by an increase in firing rate of mean 12.0 ± 0.6 Hz in all 69 cells. The dopamine effect was mimicked by the dopamine D1/D5 receptor agonist SKF38393 (10 μM, 17 cells) and the dopamine D2-like receptor agonist quinpirole (10 μM, 35 cells), partly reduced by D1/D5 antagonist SCH23390 (2 μM, seven cells), but unaffected by the D2 antagonists sulpiride (10 μM, seven cells) or eticlopride (10 μM, six cells). Using voltage ramps, dopamine induced an inward current of 69 ± 9.4 pA at a holding potential of −60 mV ( n = 17). This current was accompanied by an increase in input conductance of 1.55 ± 0.35 nS which reversed at −30.6 ± 2.3 mV, an effect mimicked by SKF38393 (10 μM, nine cells). Similar responses were observed when measuring instantaneous current evoked by voltage steps and in the presence of the I h blocker, ZD7288, indicating effects independent of I h. The increase in conductance was blocked by SCH23390 (2 μM, n = 4), mimicked by the activator of adenylyl cyclase forskolin (10 μM, n = 7) and blocked by H-89, an inhibitor of cyclic AMP dependent protein kinase A (10 μM, n = 6). These results indicate that the dopamine depolarisation is in part mediated by D1/D5 receptor mediated activation of a cyclic-nucleotide gated (CNG) non-specific cation conductance. This conductance contributes to the membrane depolarisation that changes STN neuronal bursting to more regular activity by significantly increasing burst duration and number of spikes per burst.

Agonist‐independent, high constitutive activity of the human mela‐nocortin 1 receptor

The melanocortin hormones act on epidermal melanocytes to increase eumelanogenesis, melanocyte dendricity and likely melanosome transfer to keratinocytes. These actions are mediated by the melanocortin 1 receptor (MC1R), positively coupled to adenylyl cyclase. Gain‐of‐function Mc1r alleles are associated with dark, eumelanic skin. Conversely, loss‐of‐function variants or overexpression of agouti, the natural antagonist, yield yellow, pheomelanic furs. In humans, loss‐of‐function MC1R variants are associated with fair skin, poor tanning and increased skin cancer risk. Therefore, MC1R is a key regulator of mammalian pigmentation. An induction of constitutive pigmentation in amelanotic mouse melanoma cells following the expression of MC1R has been reported, suggesting that this receptor might display agonist‐independent activity, although this aspect has not yet been comparatively studied for MC1R and Mc1r. We show that the expression of MC1R in heterologous systems leads to high agonist‐independent increases in intracellular cAMP. This basal signalling is a function of the quantity of receptor expressed, is considerably higher for MC1R than Mc1r and is also observed in human melanoma cells overexpressing MC1R. Moreover, MC1R basal signalling is abolished or reduced by point mutations impairing the response to agonists. Lastly, the expression of wild‐type MC1R, but not of loss‐of‐function mutants potently stimulates forskolin activation of adenylyl cyclase, a feature characteristic of constitutively active G‐coupled receptors. Therefore, we conclude that MC1R displays a strong agonist‐independent constitutive activity.

Agonist-independent, high constitutive activity of the human melanocortin 1 receptor.

The melanocortins (alpha-melanocyte-stimulating hormone and adrenocorticotropin) act on epidermal melanocytes to increase melanogenesis, the eumelanin/pheomelanin ratio and dendricity. These actions are mediated by the heptahelical melanocortin 1 receptor (MC1R), positively coupled to adenylyl cyclase. Gain-of-function mouse Mc1r alleles are associated with a dark, eumelanic coat. Conversely, loss-of-function variants, or overexpression of agouti, a natural melanocortin antagonist, yield yellow, pheomelanic furs. In humans, loss-of-function MC1R variants are associated with fair skin, poor tanning, propensity to freckle and increased skin cancer risk. Therefore, MC1R is a key regulator of mammalian pigmentation. Several observations such as induction of constitutive pigmentation in amelanotic mouse melanoma cells following expression of MC1R indicate that the receptor might display agonist-independent activity. We report a systematic and comparative study of MC1R and Mc1r constitutive activity. We show that expression of MC1R in heterologous systems leads to an agonist-independent increase in cyclic adenosine monophophate (cAMP). Basal signalling is a function of receptor expression and is two to fourfold higher for MC1R than for Mc1r. Moreover, it is observed in human melanoma cells over-expressing the MC1R. Constitutive signalling is abolished or reduced by point mutations of MC1R impairing the response to agonists, and is only doubled by the Lys94Glu mutation, mimicking the constitutively active mouse E(so-3J) allele. Stable or transient expression of wild-type MC1R, but not of loss-of-function mutants, potently stimulates forskolin activation of adenylyl cyclase, a common feature of constitutively active Gs-coupled receptors. Therefore, human MC1R displays a strong agonist-independent constitutive activity.

Cyclic AMP-mediated upregulation of the expression of neuronal NO synthase in human A673 neuroepithelioma cells results in a decrease in the level of bioactive NO production: analysis of the signaling mechanisms that are involved.

The expression level of neuronal nitric oxide synthase (nNOS) can vary depending on the (patho)physiological conditions. Here we document a marked induction of nNOS mRNA, protein, and total NO production in response to dibutyryl cyclic AMP (db-cAMP) in human A673 neuroepithelial cells. However, the upregulation of nNOS was associated with a decreased level of production of bioactive NO and by an increase in the level of generation of reactive oxygen species (ROS). ROS production could be prevented by the NOS inhibitor L-NAME, suggesting nNOS itself is involved in ROS generation. Sepiapterin supplementation of db-cAMP-treated A673 cells could restore full bioactive NO production, most likely by preventing the uncoupling of nNOS. nNOS was upregulated by other stable analogues of cAMP, by the activator of adenylyl cyclase forskolin, by isoproterenol or by dopamine through activation of D1 receptors, and by inhibitors of phosphodiesterase. cAMP did not change the half-life of the nNOS mRNA. Inhibitors of protein kinase A (PKA), H-89 and R(p)-cAMPS, produced a partial inhibition of basal and cAMP-induced nNOS expression. cAMP response element binding and modulator transcription factors (CREB and CREM), typical target proteins of PKA, were expressed in A673 cells, as was the coactivator CREB binding protein (CBP). cAMP-stimulated induction of nNOS was significantly enhanced in A673 cells stably transfected with wild-type CREB and almost abolished in cells transfected with KCREB (containing a mutation of the DNA binding domain). In A673 cells transfected with CREB(133) (containing a mutation of the phosphorylatable serine 133), the overall level of nNOS expression was reduced, but the expressional stimulation by cAMP remained. This suggests that CREB bypasses, in part, the classical requirement for phosphorylation and association with CBP. Three members of the recently described four-and-a-half-LIM-domain proteins (FHL1-FHL3) were found to be expressed in A673 cells; FHL-1 and FHL-3 were upregulated by cAMP. These proteins can provide direct activation function to both CREB and CREM, and may be responsible for the PKA-independent component of CREB and CREM activity.

Dual Effects of DAMGO [d-Ala2,N-Me-Phe4,Gly5-ol]-Enkephalin and CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) on Adenylyl Cyclase Activity: Implications for μ-Opioid Receptor GsCoupling

The mu-opioid receptor (MOR) couples to multiple G proteins, of which coupling to Gs has long been debated. As expected, in opioid naive Chinese hamster ovary cells expressing recombinant MOR, the predominant action of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) is inhibitory. However, inactivation of Gi/Go proteins via pertussis toxin (PTX) unmasks its ability to facilitate forskolin activation of adenylyl cyclase (AC) activity. Tolerance develops to this effect of DAMGO, which can also be attenuated by cholera toxin (CTX). The latter suggests G mediation. D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), previously considered to be a neutral MOR antagonist, also produces a facilitation of forskolin (FSK) activation of AC that is augmented by chronic morphine. Facilitative effects of CTAP in naive as well as its augmentation in tolerant membranes are both substantially reduced by CTX. This suggests that not only Gs mediation but also G(salpha)-linked signaling is critical to the chronic morphine-induced enhanced facilitative action of CTAP. Interestingly, the (augmented) CTAP facilitation of FSK-stimulated AC activity that is observed in opioid tolerant (but not in naive) membranes is also sensitive to PTX. This can best be explained by postulating the involvement of Gi-derived G(betagamma), which would stimulate type 2 ACs, conditional on the presence of activated G(salpha). The emergence of a G(betagamma) dimension of AC stimulation by CTAP after chronic morphine could explain its ability to augment the stimulatory action of CTAP on AC. These results support putative MOR coupling to Gs and underscore the multifaceted nature and plasticity of MOR G protein coupling.

Interaction between the mu opioid receptor and filamin A is involved in receptor regulation and trafficking.

The carboxyl tail of the human mu opioid receptor was shown to bind the carboxyl terminal region of human filamin A, a protein known to couple membrane proteins to actin. Results from yeast two-hybrid screening were confirmed by direct protein-protein binding and by coimmunoprecipitation of filamin and mu opioid receptor from cell lysates. To investigate the role of filamin A in opioid receptor function and regulation, we used the melanoma cell line M2, which does not express filamin A, and its subclone A7, transfected with human filamin A cDNA. Both cell lines were stably transfected with cDNA encoding myc-tagged human mu opioid receptor. Fluorescent studies, using confocal microscopy, provided evidence that filamin and mu opioid receptors were extensively colocalized on the membranes of filamin-expressing melanoma cells. The immunostaining of mu opioid receptors indicated that the lack of filamin had no detectable effect on membrane localization of the receptors. Moreover, mu opioid receptors function normally in the absence of filamin A, as evidenced by studies of opioid binding and DAMGO inhibition of forskolin-stimulated adenylyl cyclase. However, agonist-induced receptor down-regulation and functional desensitization were virtually abolished in cells lacking filamin A. The level of internalized mu-opioid receptors, after 30-min exposure to agonist, was greatly reduced, suggesting a role for filamin in mu opioid receptor trafficking. During these studies, we observed that forskolin activation of adenylyl cyclase was greatly reduced in filamin-lacking cells. An even more unexpected finding was the ability of long-term treatment with [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin of M2 cells, containing mu opioid receptors, to restore normal forskolin activation. The mechanism of this effect is currently unknown. It is postulated that the observed effects on mu opioid receptor regulation by filamin A and, by implication, of the actin cytoskeleton may be the result of its role in mu opioid receptor trafficking.

Metabotropic glutamate receptor agonists protect from oxygen–glucose deprivation- and colchicine-induced apoptosis in primary cultures of cerebellar granule cells

The effects of the metabotropic glutamate receptor agonists against apoptosis induced by oxygen–glucose deprivation or colchicine were studied in the primary cultures of mature cerebellar granule cells. Oxygen–glucose deprivation (90 min) or addition of colchicine (1 μM) resulted in neuronal damage as revealed by Trypan Blue assay 12 h later. Further analysis demonstrated that the cells exposed to oxygen–glucose deprivation or colchicine exhibit typical features of apoptosis: internucleosomal DNA fragmentation, condensation and fragmentation of chromatin and typical DNA ladder on agarose gel electrophoresis. Metabotropic glutamate receptor agonist, (1S,3R)-1-aminocycloheptane- trans-1,3-dicarboxylic acid, acting at group I and II receptors, and selective agonist, (2S,1′R,2R′,3R′)-2(2,3-dicarboxycyclopropyl)glycine, acting at group II receptors, added to cells recovering from oxygen-glucose deprivation exerted neuroprotective action against oxygen–glucose deprivation-induced apoptosis. Similar neuroprotective effects of metabotropic glutamate receptor agonists were observed against colchicine-induced apoptosis. The results thereby provide evidence that metabotropic glutamate receptor agonists have therapeutic potential in the treatment of pathologies associated with increased neuronal apoptosis. The selective protein kinase C inhibitor bisindolylmaleimide (100 nM) abolished the neuroprotective action of (1S,3R)-1-aminocycloheptane- trans-1,3-dicarboxylic acid, whereas the activator of adenylyl cyclase forskolin (10 μM) abolished the neuroprotective action of (2S,1′R,2R′,3R′)-2(2,3-dicarboxycyclopropyl)glycine (30 μM) against colchicine-induced apoptosis. It is concluded that both phosphoinositide hydrolysis with consequent activation of protein kinase C and inhibition of adenylyl cyclase seem to contribute to the neuroprotective action of metabotropic glutamate receptor agonists against neuronal apoptosis in the primary culture of cerebellar granule cells.

Depolarization-dependent effect of flavonoids in rat uterine smooth muscle contraction elicited by CaCl 2

1. 1. The effects of the flavonoids genistein (3–60 μM), kaempferol (3–60 μM) and quercetin (1–100 μM) on KCI (60 μM)-induced tonic contraction in rat uterus and their modifications with the inhibitor of cAMP-dependent protein kinases (TPCK, 3 μM), the inhibitor of ornithine decarboxylase [α-difluoromethyl ornithine (DFMO), 10 mM] and the polyamine spermine (1 mM) have been assayed. The effects of the three flavonoids were also studied on the contraction elicited by CaCl 2 (30 μM to 10 mM) on rat uterus incubated in medium lacking calcium and supplemented with 33, 60 or 90 μM of KCI. For comparison, the effects of the calcium channel blockers nifedipine and verapamil and the activator of adenylyl cyclase forskolin were assayed on contractions induced by KCl and CaCl 2. 2. 2. Genistein (IC 50: 20.2± 1.0 μM, n=11), kaempferol (IC 50:10.1±0.8 μM, n=8) and quercetin (IC 50:13.2±0.5 μM, n=8) relaxed the tonic contraction induced by KCI (60 mM) in a concentration-dependent way. Verapamil (IC 50:70.1±5.8 μM, n=7), nifedipine (IC 50:8.4±0.7 nM, n=6) and forskolin (IC 50: 0.62±0.08 μM, n=14) also relaxed the KCl-induced contraction. TPCK (3 μM) significantly antagonized the effect of quercetin, kaempferol and forskolin ( P<0.01) but did not modify the effect of genistein. 3. 3. Spermine (1 mM) increased the effects of genistein and verapamil and antagonized the effect of quercetin but did not modify those of kaempferol and forskolin. DFMO (10 mM) did not modify the effect of quercetin but increased that of genistein and antagonized those of kaempferol and forskolin. The addition of spermine (1 mM) plus DFMO (10 mM) antagonized the effect of quercetin. Spermine counteracted the effect of DFMO on forskolin but not on genistein. 4. 4. KCI (33, 60 or 90 mM) did not produce contraction in calcium-free solution, but CaCl 2 (30 μM to 10 mM) induced concentration-dependent contraction after depolarizing with KC1. The EC 50 values for CaCl 2 were: 0.74±0.08 ( n=12), 0.34±0.03 ( n=14) and 0.48±0.02 ( n=12) mM in a medium with 33, 60 or 90 mM of KCI, respectively. 5. 5. Genistein (20 μM), kaempferol (10 μM), quercetin (15 μM), verapamil (70 nM), nifedipine (10 nM) and forskolin (0.5 μM) inhibited the concentration-response curve to CaCl 2 in medium supplemented with 33, 60 or 90 mM of KCI. The effect of kaempferol was independent of the concentration of KCl in the incubation medium. However, the inhibitory effect of genistein on CaCl 2-induced contraction was inversely related to the concentration of KCI in the medium. On the contrary, the effect of quercetin was directly related to the concentration of KCl in the medium. 6. 6. The antagonism of verapamil, nifedipine and forskolin on CaC1 2-induced contraction seems to be related to the degree of depolarization because increasing the KCl in the medium counteracted their effects. 7. 7. Our results suggest that (1) cAMP contributes to the relaxant effects of quercetin and kaempferol on KCI (60 mM)-induced tonic contraction; (2) polyamines are involved in the relaxant effects of forskolin and kaempferol on KCl-induced tonic contraction but not on CaCl 2-induced contraction in the depolarized uterus, and (3) the flavonoids assayed also possess a calcium antagonist action but show a different behavior toward the calcium channel blockers and the cAMP enhancer forskolin.

Atypical pattern of adenylyl cyclase activity in the adrenal medulla with age.

Concurrent reduced physiological responsiveness often occurs in those tissues in which signal transduction coupled to adenylyl cyclase is diminished with age. In the adrenal medulla, the major physiological functions are catecholamine synthesis and release, and these functions are increased with age. To examine whether the sensitivity or efficacy for adenylyl cyclase stimulation is altered with age, we determined the vasoactive intestinal peptide (VIP) and forskolin activation of adenylyl cyclase in adrenal medullary membranes from young (4 month) and senescent (24 month) male F-344 rats. Stimulation by VIP (10 microns) and GTP (10 microns) were unchanged with age. In contrast, the maximal stimulation by forskolin (100 microns) and the sensitivity for forskolin stimulation were increased with age. These data indicate that the pattern of increased adenylyl cyclase stimulation with age is comparable to that in liver tissue but atypical with respect to all other tissues in the rat.

A method for isolating and culturing vascular smooth muscle cells from porcine cerebral arteries

Cerebral blood vessels have unique properties when compared to most peripheral vascular beds. One such property is that large cerebral vessels are involved in the regulation of cerebral vascular resistance. Studying smooth muscle cells isolated from these vessels will determine how phenotypic properties of these cells contribute to unique cerebrovascular function. Therefore we developed a method of culturing smooth muscle cells from explants of cerebral arteries of porcine brains obtained, gratis, from a local slaughter house. Cells isolated and cultured by the methods described herein were of smooth muscle origin as indicated by histochemical staining for smooth muscle α-actin. Further, we examined the response of the cultured cells to agonists which activate the cGMP dependent vasodilator system by stimulating soluble guanylyl cyclase (nitroglycerin and sodium nitroprusside) or particulate guanylyl cyclase (C-type natriuretic peptide). Also, forskolin activation of adenylyl cyclase was examined. These agents stimulated an increase in intracellular cGMP and cAMP in a manner that was reproducible in every cell isolation (20 brains) and which remained unchanged through nine passages. Additionally, the cells could be frozen, thawed and replated without loss of responsiveness to these agents. The protocol reported here provides a method for culturing cerebral artery smooth muscle cells that is inexpensive, relatively simple, and which yields cells that can be utilized through multiple passages.

Effects of guanine nucleotides and divalent cations on forskolin activation of rabbit luteal adenylyl cyclase: evidence for the existence of an inhibitory guanine nucleotide-binding regulatory component.

The effects of guanine nucleotides and divalent cations on the activation of rabbit luteal adenylyl cyclase by the diterpene forskolin were investigated. Saturating concentrations of forskolin elicited 10- to 15-fold stimulation of adenylyl cyclase activity in the absence of added guanine nucleotide. No lag was observed in the time course of forskolin-induced activation. Addition of 10 microM guanosine triphosphate (GTP) and guanyl-5'-yl imidodiphosphate [GMP-P(NH)P] inhibited forskolin activation by 10-15% and 30-40%, respectively, in the presence of 3.0 mM MgCl2. GMP-P(NH)P was more potent than GTP in inhibiting forskolin activation of adenylyl cyclase having an IC50 of 36 nM compared to 610 nM for GTP. In contrast, the Kact for stimulating adenylyl cyclase activity by both GMP-P(NH)P and GTP were similar, 1.00 and 0.86 microM, respectively. GMP-P(NH)P-induced inhibition of the forskolin-activated enzyme was not due to the hysteretic nature of GMP-P(NH)P activation of luteal adenylyl cyclase, as addition of GMP-P(NH)P to an enzyme that had been treated 5 min earlier with forskolin resulted in the immediate inhibition of enzymatic activity. Addition of GMP-P(NH)P to a concentration-effect curve for forskolin increased the Kact value for forskolin form 7.18 to 26.8 microM. There was a different MgCl2 concentration requirement for maximal stimulation of luteal cyclase by GMP-P(NH)P (8 mM MgCl2) and maximal inhibition of forskolin-stimulated activity by GMP-P(NH)P (0.5-0.6 mM MgCl2). Further, MnCl2 concentrations above 1.0 mM completely abolished the inhibitory action of GMP-P(NH)P on forskolin activation of luteal cyclase. In fact, at 2.0 mM MnCl2, adenylyl cyclase activity in the presence of GMP-P(NH)P plus forskolin was greater than that of forskolin alone. Thus taken together, these findings suggest that the rabbit corpus luteum contains an inhibitory guanine nucleotide-binding regulatory component in addition to a stimulatory regulatory component. Further, these components demonstrate differing requirements for both guanine nucleotides and divalent cations in order to interact with the catalytic moiety of adenylyl cyclase. The existence of such an inhibitory component suggests the presence of an inhibitory receptor in the corpus luteum which could negatively regulate adenylyl cyclase resulting in the inhibition of cAMP production and reduced progesterone output from the corpus luteum under normal physiological conditions.