Increased cAMP Formation Research Articles

Exclusive expression of the Gs-linked prostaglandin E 2 receptor subtype 4 mRNA in human mononuclear Jurkat and KM-3 cells and coexpression of subtype 4 and 2 mRNA in U-937 cells

Prostaglandin E 2 (PGE 2) is regarded as a potent regulator of the immune system. It can regulate apoptosis in monunuclear cells and modulate the cytokine secretion pattern from T-helper cell subpopulations via an increase in cyclic AMP (cAMP). Of the 4 PGE 2 receptor subtypes (EP1–EP4) that are defined pharmacologically by their affinity to subtype-specific ligands and their coupling to G proteins, EP2 and EP4 receptors couple to Gs. It is as yet unknown which of these two receptor subtypes mediates the immunomodulatory effects. By quantitative RT-PCR, the mRNA for EP4 receptors was demonstrated and quantified in the human mononuclear cell lines Jurkat, KM-3 and U-937. However, EP2 receptor mRNA was only present in U-937 cells and was 100-fold less abundant than EP 4 receptor mRNA. PGE 2 increased cAMP formation with an ED 50 of 50–100 nM in all cell lines. cAMP formation was inhibited by the EP4R-specific antagonist AH23848. Since AH23848 inhibited PGE 2-induced cAMP formation in U-937 cells to a similar extent as in Jurkat and KM-3, EP2 receptors seem to play, if any, only a secondary role for the PGE 2-mediated cAMP formation in U-937 cells.

Expression of glucagon-like peptide 1 receptor in a murine C cell line Regulation of calcitonin gene by glucagon-like peptide 1

We have characterized, by RT-PCR amplification using specific primers, the presence of glucagon-like peptide-1 (GLP-1) receptor mRNA in CA-77 cells, a C cell line derived from a rat medullary thyroid carcinoma. Down-regulation of the GLP-1 receptor mRNA was observed after exposure of CA-77 C cells with GLP-1 (7–37). Increased secretion of both calcitonin gene-related peptide (CGRP) and calcitonin (CT) occurred after treatment with GLP-1 (7–37) associated with elevated steady-state levels of CGRP and CT mRNA. GLP-1 (7–37) increased cAMP formation in CA-77 cells in a dose-dependent manner; exendin (9–39), a GLP-1 receptor antagonist, inhibited cAMP production. The GLP-1 peptide which is produced by intestinal cells could be involved in the control of CT secretion through an entero-thyroidal axis implying GLP-1 receptor and increased CT gene expression.

D 2 and D 1 dopaminergic activity of 7-OH-DPAT

The D 1 and D 2 dopamine receptor agonist properties of 7-hydroxy-2-( N,N-di- n-propylamino) tetraline (7-OH-DPAT) was determined by investigating the effect of this compound on rat striatal acetylcholine (ACh) concentration and increase in cAMP formation in primary cerebellar granule cell cultures. 7-OH-DPAT at low doses (0.01 to 0.1 μmol/kg) had no significant effect, and at high doses (0.3 to 30 μmol/kg) significantly ( P < 0.01) increased striatal ACh levels. Likewise, quinpirole was found to significantly elevate ACh content. Pretreatment with haloperidol, a non-selective antagonist of the D2 family of receptors, significantly ( P < 0.01) blocked 7-OH-DPAT- and quinpirole-induced increases in ACh. U-99194A, a D 3 selective dopamine antagonist, had no significant effect on 7-OH-DPAT-induced increases in striatal ACh. However, raclopride, a D 2 selective dopamine antagonist, completely blocked 7-OH-DPAT-induced elevations in ACh. 7-OH-DPAT in the μmolar range increased cAMP formation in granule cell cultures, and this effect was antagonized by SCH 23390, a D 1 selective dopamine antagonist. The neurochemical study indicates that, at high doses, 7-OH-DPAT has both D 1 and D 2 agonist activities.

Inverse agonism at dopamine D2 receptors haloperidol-induced prolactin release from GH4C1 cells transfected with the human D2 receptor is antagonized by R(-)-n-propylnorapomorphine, raclopride, and phenoxybenzamine

Our earlier observation that the antipsychotic drug haloperidol in the absence of dopamine increases cAMP formation and prolactin release in two prolactin-producing cell lines expressing rat dopamine D2 receptors (GH3, GH4ZR7), but not in similar cells devoid of D2 receptors (GH4C1), prompted us to suggest that haloperidol may act as an inverse (or negative) agonist, rather than as a neutral antagonist, at the D2 receptor (Nilsson and Eriksson 1993). In the present study it is shown that haloperidol elicits a dose-dependent increase in prolactin release also in prolactin-producing GH4C1 cells transfected with the human dopamine D2 receptor (short isoform) (GH4C1-hD2s); in addition, it is shown that another antipsychotic drug, flupenthixol, also causes prolactin release per se in this cell line. The effect of haloperidol on prolactin release in GH4C1-hD2s is calcium dependent and counteracted by pretreatment either with the D2 receptor agonist R(-)-n-propylnorapomorphine or with a D2 receptor antagonist that does not affect prolactin release per se (raclopride). In addition, pretreatment with the alkylating compound phenoxybenzamine at a concentration causing a marked reduction of D2 receptor density in GH4C1-hD2s cells significantly counteracted haloperidol-induced prolactin release.

(1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid-induced increases in cyclic AMP formation in the neonatal rat hippocampus are mediated by a synergistic interaction between phosphoinositide- and inhibitory cyclic AMP-coupled mGluRs.

A pharmacological approach was used to investigate the cellular mechanism and metabotropic glutamate receptor (mGluR) subtypes that mediate stimulation of basal cyclic AMP (cAMP) formation in slices of the neonatal rat hippocampus. (1S,3 R)-1-Aminocyclopentane-1, 3-dicarboxylic acid (1S,3R-ACPD), which is an agonist for phosphoinositide-coupled and inhibitory-coupled cAMP-linked mGluRs in cloned and in situ preparations, produced prominent stimulations of basal cAMP levels (five- to 10-fold). However, the agonists 3,5-dihydroxyphenylglycine (DHPG) and (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), which selectively act on phosphoinositide-coupled and inhibitory cAMP-coupled mGluRs, respectively, only weakly increased cAMP levels. When these two mGluR subtype-selective agonists were added in combination, robust increases in cAMP levels, similar to those observed for 1S,3R-ACPD, were found. Stimulations of cAMP content evoked by 1S,3R-ACPD and combined additions of DHPG plus 2R,4R-APDC occurred at concentrations of these agents that directly couple to other mGluR second messenger responses. However, these stimulatory cAMP responses were prevented by the presence of adenosine deaminase and 8-p-sulfophenyltheophylline (an adenosine receptor antagonist), as well as (+)-alpha-methyl-4-carboxyphenylglycine (an mGluR receptor antagonist). Thus, 1S,3R-ACPD-induced increases in cAMP formation in the neonatal rat hippocampus are mediated by a synergistic interaction between mGluRs coupled to phosphoinositide (group 1) and inhibitory cAMP (group 2), which are indirectly expressed by potentiation of cAMP responses to other agonists (in this case, endogenous adenosine).

Tissue‐specific and Developmental Expression of Pituitary Adenylate Cyclase‐activating Polypeptide (PACAP) Receptors in Rat Brain

The two forms of pituitary adenylate cyclase-activating polypeptide, PACAP27,and PACAP38, are novel members of the vasoactive intestinal peptide (VIP)/secretin/glucagon family of peptides. PACAP receptors that are positively coupled to adenylate cyclase and phospholipase C have been recently identified. We examined the expression of PACAP receptors in the rat cortex, hippocampus, cerebellum and hypothalamus during postnatal development. Functional studies revealed PACAP stimulation of cAMP formation in all the brain areas examined and [3H]inositol monophosphate ([3H]InsP) accumulation only in the cerebellum and hypothalamus. Throughout development, the efficacy or PACAP in stimulating cAMP formation slightly increased in the cortex and hypothalamus and decreased in the hippocampus and cerebellum; PACAP stimulation of [3H]InsP formation decreased in the cerebellum and remained steady in the hypothalamus. The effects of PACAP27 and PACAP38 on cAMP levels and inositol phospholipid hydrolysis were dose-dependent between 1 and 100 nM. In the same brain areas, treatment with VIP increased cAMP formation at doses greater than 100nM and failed to affect [3H]InsP content, thus suggesting the existence of type-1 PACAP receptors. The reverse transcription polymerase chain reaction (RT-PCR) was used to analyse the mRNA expression of type-1 PACAP receptor splice variants. PACAP receptor gene expression in the central nervous system was regulated in a developmental- and tissue-specific manner. The PACAP-R transcript was detected in all the brain areas examined whereas PACAP-R-hop mRNA ocurred only in the cerebellum and hypothalamus. The different expression profiles and functional properties of PACAP receptors in the developing rat brain suggest an involvement of PACAP in histogenesis, maturation and neurotransmission.

Platelet-derived Growth Factor Stimulates Protein Kinase A through a Mitogen-activated Protein Kinase-dependent Pathway in Human Arterial Smooth Muscle Cells

The abilities of platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF-I) to regulate cAMP metabolism and mitogen-activated protein kinase (MAP kinase) activity were compared in human arterial smooth muscle cells (hSMC). PDGF-BB stimulated cAMP accumulation up to 150-fold in a concentration-dependent manner (EC50 approximately 0.7 nM). The peak of cAMP formation and cAMP-dependent protein kinase (PKA) activity occurred approximately 5 min after the addition of PDGF and rapidly declined thereafter. Incubating cells with PDGF and 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) enhanced the accumulation of cAMP and PKA activity by an additional 2.5-3-fold, whereas IBMX alone was essentially without effect. The PDGF-stimulated increase in cAMP was prevented by addition of the cyclooxygenase inhibitor indomethacin, consistent with release of prostaglandins stimulating cAMP. PDGF, but not IGF-I, stimulated MAPK activity, cytosolic phospholipase A2 (cPLA2) phosphorylation, and cAMP synthesis which indicated a key role for MAP kinase in the activation of cPLA2. Further, PDGF stimulated the rapid release of arachidonic acid and synthesis of prostaglandin E2 (PGE2) which could be inhibited by a cPLA2 inhibitor (AACOCF3). Calcium mobilization was required for PDGF-induced arachidonic acid release and PGE2 synthesis but not for MAPK activation, whereas PKC was required for PGE2-mediated activation of PKA. In summary, these results demonstrated that PDGF increases cAMP formation and PKA activity through a MAP kinase-mediated activation of cPLA2, arachidonic acid release, and PGE2 synthesis in human arterial smooth muscle cells.

Activation of FSH-responsive adenylate cyclase by staurosporine: role for protein phosphorylation in gonadotropin receptor desensitization

Prolonged stimulation of gonadotropin receptors in granulosa cells leads to desensitization of the cellular response to gonadotropic hormones which is evident by decrease in cAMP formation. In order to explore the mechanism of desensitization and to examine whether protein phosphorylation may play a role in this phenomenon, we have studied the effect of various stimulators and inhibitors of protein phosphorylation on FSH-induced cAMP formation in the FSH-responsive cell line, GFSHR-17, recently established in our laboratory. Both ovine and human FSH activated the hormone sensitive adenylate cyclase in a dose-dependent manner with an ED 50 of 0.5 nM. This stimulation was followed by a sharp decrease in cAMP formation after 30 min incubation of the cell with the hormone. When cells were preincubated for 60 min with staurosporine, cAMP accumulation during 20 min of FSH stimulation was elevated about 500%, compared to cells stimulated by FSH alone. Staurosporine alone showed a negligible effect on cAMP accumulation in these cells. In cells stimulated with forskolin, a non-specific activator of adenylate cyclase, or with cholera toxin (CT), an inhibitor of GTPase activity associated with Gs of adenylate cyclase, preincubation with staurosporine increased cAMP formation in these cells by only 50–70 or 80–120%, respectively. Preincubation of cells with the protein kinase C (PKC) inhibitors chelerythrine and GF109203X increased FSH-stimulated accumulation of cAMP by 50 and 30%, respectively. These drugs exhibit a similar effect on forskolin-stimulated cells. Preincubation of cells for 60 min with a PKC stimulator, TPA, suppressed FSH-mediated cAMP response in these cells by 40%. Tyrosine kinase inhibitors such as AG18, AG33 and genistein exhibit a modest inhibitory effect of up to 20% on FSH-stimulated cAMP accumulation. All the above results were obtained both in the presence and absence of IBMX, a potent inhibitor of the cellular phosphodiesterases. Upon prolonged incubation with FSH (3 h) cells pretreated with staurosporine exhibited a much slower rate of decline in intracellular cAMP levels. Moreover, in desensitized cells, following 1 or 2 h of continuous stimulation with FSH, staurosporine could markedly enhance cAMP formation in the presence of FSH. Our data suggest that staurosporine-sensitive phosphorylation of serine or threonine in the FSH receptor cyclase system may be responsible for desensitization of the FSH coupled activation of cAMP formation, while reactivation of the system can be achieved by protein dephosphorylation at these specific sites. Because specific inhibition of PKC could not mimic the staurosporine effect on FSH-stimulated cAMP formation, nor could activation of kinase C antagonize it, it is suggested that a specific staurosporine-sensitive receptor kinase may be responsible for modulation of the coupling between the gonadotropin receptor and the adenylate cyclase system.

Parathyroid hormone stimulation of calcium transport is mediated by dual signaling mechanisms involving protein kinase A and protein kinase C.

PTH stimulates calcium absorption by renal distal convoluted tubules. The PTH receptor is capable of coupling to adenylyl cyclase and phospholipase C. However, it is not known whether the actions of PTH require activation of both pathways. Three approaches were taken to identify the signaling pathways responsible for stimulating calcium entry in distal convoluted tubule cells: second messengers formed in response to PTH were identified, the effects on calcium uptake of inhibiting protein kinase A (PKA) or protein kinase C (PKC) with chemical or peptide blockers were determined, and calcium transport was reconstituted by the addition of exogenous second messengers. PTH increased cAMP formation in primary cultures of mouse distal and proximal tubule cells. However, PTH stimulated inositol trisphosphate formation only in proximal tubule cells. Blocking PKA with Rp-cAMPS or the cAMP-dependent protein kinase inhibitor inhibited PTH-stimulated Ca uptake. Likewise, the PKC inhibitors, calphostin C and PKC pseudosubstrate, inhibited PTH-induced calcium uptake. Addition of forskolin (30 nM) or phorbol 12-myristate 13-acetate (10 nM) alone had no effect on Ca uptake. However, when added in combination, Ca uptake was stimulated to nearly the same extent as with concentrations of PTH that maximally stimulate calcium transport. We conclude that stimulation of calcium uptake by distal convoluted tubule cells requires activation of both PKA and PKC.

Effects of exogenous linoleic acid on fatty acid composition, receptor-mediated cAMP formation, and transport functions in rat astrocytes in primary culture.

We have examined the effects of culturing neonatal rat-brain astrocytes in medium containing delipidated serum, with or without added linoleic acid (LA, 18:2 omega 6), on membrane fatty-acid composition and functions. After 18-21 days in culture, polyunsaturated fatty acids (PUFA) constituted approximately equal to 24 mol% of the total fatty acids in the astrocytes grown in delipidated media ("controls'); these proportions were increased by 35-40% to approximately equal to 33 mol% when the cells were supplemented with 35 microM LA. Notable differences in the PUFA profiles of the cells cultured with or without added LA included: (a) higher proportions of omega 6 PUFA in the LA-supplemented astrocytes (approximately equal to 25%, relative to approximately equal to 10% in controls) that were accompanied by an increase in the ratio of omega 6/omega 3 PUFA (from < 2 in controls to approximately equal to 5), and (b) higher proportions of 20:3 omega 9 and 22:3 omega 9 in the control astrocytes (> 5%) relative to the LA-supplemented cells (approximately equal to 1%). The major metabolites in the omega 6 PUFA-enriched cells were arachidonic (20:4 omega 6), adrenic (22:4 omega 6) and docosapentaenoic (22:5 omega 6) acids (15, 5 & 3 mol%, respectively). Enrichment of the astrocytes in omega 6 PUFA did not alter basal levels of cAMP, nor did it affect the amounts of cAMP formed in response to forskolin, isoproterenol, adenosine or histamine. However, dopamine-dependent increases in cAMP formation in the presence of the phosphodiesterase inhibitor, Ro 20-1724, were reduced by approximately equal to 25% relative to those in controls. LA supplementation modified uptake of [3H]adenosine into the astrocytes; values for Kt for a high affinity transport were increased relative to controls, and maximum capacity of a lower affinity process was reduced. Uptake of [3H]glutamate was not altered in the omega 6 PUFA-enriched astrocytes. This study demonstrated that cultured astrocytes take up exogenous linoleic acid and incorporate its metabolites into phospholipid, and that the resulting changes in membrane PUFA composition modify only specific cell functional properties.

Effects of long-term treatment with angiotensin-converting enzyme inhibitor on angiotensin II and prostacyclin release from mesenteric arteries in spontaneously hypertensive rats

To evaluate the long-term effects of an angiotensin-converting enzyme inhibitor (ACEI) on vascular angiotensin II (AII), eicosanoid production and vascular reactivities, we treated spontaneously hypertensive rats (SHR) with alacepril for 6 weeks and perfused the isolated mesenteric arterial bed which contains resistance vessels. Alacepril significantly lowered the arterial blood pressure. Changes in perfusion pressure in response to norepinephrine (NE) were attenuated, and isoproterenol-stimulated AII release from the perfused mesenteric arterial beds was inhibited in the alacepril-treated SHR. Vasodilation induced by acetylcholine (Ach) and prostacyclin (PGI 2) release was significantly increased in the vasculature of the alacepril-treated SHR. Alacepril exerted no effect on cyclic GMP (cGMP) formation, but increased cAMP formation in the vasculature. These findings suggest that ACEI inhibits AII formation and facilitates PGI 2 production in the resistance vessels, which leads to blunting of the pressor response to NE and improvement of endothelial function in SHR. These humoral and mechanical changes in the vasculature may contribute to the depressor and organ protective effects of ACEI.

Regulation of Cumulus Cell Steroidogenesis by the Porcine Oocyte and Preliminary Characterization of Oocyte-Produced Factor(s)

This study was designed to investigate whether porcine oocytes produce a factor(s) that influences cumulus and mural granulosa cell steroid production and to characterize the biochemical nature and mode of action of a such factor(s). Porcine cumulus-oocyte complexes (COC) were collected from 2-5 mm follicles and cultured either intact or after oocytectomy for 48 h. Steroid levels were then measured in the culture media. Conditioned media, obtained by culturing denuded oocytes for 48 h, were subjected to heat treatment of charcoal extraction and utilized to culture intact and oocytectomized COC. FSH-stimulated progesterone, 20 alpha-OH-progesterone, and estradiol were significantly higher in oocytectomized vs. intact COC cultures. Denuded oocytes cultured with granulosa cells significantly inhibited progesterone production compared to control. Also, media conditioned with different numbers of denuded oocytes (0 to 300) significantly inhibited progesterone production by oocytectomized COC in a manner dependent on oocyte number. Charcoal extraction, but not heat treatment, significantly removed the inhibitory effect of the conditioned media on progesterone production by oocytectomized COC. Increased progesterone production by oocytectomized COC was not accompanied by a similar increase in cAMP formation. Heptanol, a gap junction blocker, did not alter progesterone production by intact COC. In conclusion, porcine oocytes secrete a factor(s) that inhibits cumulus and mural granulosa cell steroidogenesis. This factor(s) is heat stable but extractable by charcoal. The factor(s) appears not to be transferred to somatic cells via gap junctions, and its effect is downstream of cAMP formation.

Mechanism of Action of Pituitary Adenylate Cyclase‐Activating Polypeptide (PACAP) in Human Nonfunctioning Pituitary Tumors

Several evidence suggest that pituitary adenylate cyclase activating polypeptides (PACAP-38 and -27) could function as hypophysiotropic factors. Both peptides interact with either the type I receptor, which preferentially binds the two PACAPs and has a much lower affinity for vasoactive intestinal polypeptide (VIP) or the type II receptor, which binds the two PACAPs and VIP with a nearly equal affinity. In addition to the stimulation of adenylyl cyclase (AC) activity, in different cell types PACAP causes an increase of cytosolic calcium levels ([Ca2+]i), consequent to phospholipase-C activation. In the present study, we investigated the effect of PACAP on cAMP formation and [Ca2+]i levels in 16 human nonfunctioning pituitary adenomas (NFPA). PACAP-38 increased cAMP formation in all tumors; the peptide stimulated either AC activity in membrane preparations from 26 +/- 10 to 214 +/- 179 pmol/mg prot/min (P < 0.01) or cAMP efflux from 12 +/- 5.4 to 73.2 +/- 32 pmol/well (P < 0.01) in cultured cells. The effect, detectable at concentrations higher than 1-10 pM, was maximal at 0.1-10 nM. While PACAP-38 and PACAP-27 were nearly equally effective and potent, 100-fold higher concentrations of VIP were required to obtain similar AC activation. GHRH and CRH were ineffective in any NFPA. The PACAP effect was not antagonized by a VIP antagonist, while PACAP fragment 6-27 amide partially reduced the stimulatory effects of both PACAP-27 and VIP in 2 out of 3 tumors tested. PACAP-38 caused a [Ca2+]i rise in cells obtained from 7 NFPA (from 110 +/- 34 to 151 +/- 40 nM [Ca2+]i, P < 0.05) while in the remaining 7 the peptide was ineffective at any concentrations tested (from 1 nM to 10 microM). In the responsive tumors, PACAP-38 effect was not consequence of phospholipase-C activation since removal of extracellular Ca2+ as well as blockade of L-type Ca2+ channels by dihydropyridine antagonists abolished [Ca2+]i increase triggered by the peptide. These data indicate that PACAP is by far the most potent activator of cAMP formation in NFPA and suggest a possible modulatory action of this peptide on cell growth.

Involvement of cAMP in the Regulation of High Affinity Choline Uptake by Rat Brain Synaptosomes

The role of cAMP in the regulation of the high affinity choline uptake (HACU) was investigated in resting and KCl - stimulated rat brain synaptosomes. The data indicate that the permeable cAMP analogue, monobutyryl-8-bromo cAMP, increased dose-dependently the HACU in resting synaptosomes. Treatments of resting synaptosomes by oxotremorine, quinacrine, and promethazine resulted in a reduced cAMP formation with a concomitant decrease of HACU. The reduction of HACU could be completely counteracted by the monobutyryl-8-bromo cAMP following oxotremorine treatment and was only partially inhibited in quinacrine and promethazine treated resting synaptosomes. KCl stimulation resulted in a significant increase in cAMP formation and HACU by the synaptosomes. The different profile of data obtained following the previous pharmacological treatments in KCl - stimulated synaptosomes suggests that both cAMP and phospholipase A 2 pathways may act synergistically to coordinate the neuronal choline incorporation.

Reversal of radiation-induced cisplatin resistance in murine fibrosarcoma cells by selective modulation of the cyclic GMP-dependent transduction pathway.

Cisplatin resistance, induced in murine fibrosarcoma cells (SSK) in vitro or in vivo by low-dose irradiation, can be overcome by activation of the cyclic GMP(cGMP)-dependent transduction pathway. This is mediated either by stimulating cGMP formation with sodium nitroprusside or by replacing cGMP with a selective activator of the cGMP-dependent protein kinase, 8-bromo-cGMP. The cyclic AMP-dependent transduction pathway is not involved in cisplatin resistance. Instead, activation of cAMP sensitises both parental and resistant SSK cells equally to the action of cisplatin. There is a 1.8 to 2.5-fold increase in drug toxicity, depending on the activating agent. Enhancement of cisplatin sensitivity is induced by specific inhibition of cAMP hydrolysis, increase in cAMP formation or by increasing the activation potential to cAMP-dependent protein kinase by specific cAMP analogues. Cells that have lost cisplatin resistance respond to cGMP- or cAMP-elevating agents in the same way as the parental SSK cells. The radiation sensitivity is unchanged in all cell lines, even after activation of cAMP or cGMP. These results suggest that specific DNA repair pathways are altered by radiation but affected only in cisplatin damage repair, which is regulated by cGMP. Although there is ample cooperativity and interaction between the cAMP- and the cGMP-dependent transduction pathways, specific substrate binding by cGMP appears to play an important role in radiation-induced cisplatin resistance.

G protein expression and second messenger formation in human granulosa cells.

The expression of heterotrimeric (alpha beta gamma subunits) GTP-binding regulatory proteins (G proteins) and the activation of G protein-linked receptors in human granulosa cells were investigated. The cells were obtained from stimulated follicles in women undergoing in vitro fertilization and were cultured in serum-supplemented medium. Immunoblotting with specific antibodies showed that granulosa cell membranes express alpha s, alpha i3 alpha i1,2, alpha q,11 and beta subunits. Three antibodies against alpha o failed to detect this protein. The cells responded to hCG and to prostaglandin E2 with a dose-dependent increase in cAMP formation, confirming the functional activation of G alpha s. The alpha 2 adrenoceptor agonist, clonidine, inhibited hCG-stimulated cAMP formation and this effect was blocked with pertussis toxin, thus involving a Gi-type protein, most likely G alpha i2. Oxytocin provoked an increase in formation of inositol phosphates and intracellular calcium concentration, which was partly pertussis toxin resistant, providing evidence of G alpha q,11 activation. However, a significant component of the response to oxytocin could be blocked by pertussis toxin, indicating Gi-mediated phospholipase C activation (by either alpha i or beta gamma subunits). These data demonstrate the presence of G proteins in granulosa cells and suggest a complex regulation of hormonal signalling. The concentration of cAMP in these cells depended on the balance of G alpha s:G alpha i activation, whereas activation of the inositol phospholipid pathway and rises in intracellular calcium involved both Gq,11 and Gi pathways.

Adenylyl cyclase in SH-SY5Y human neuroblastoma cells is regulated by intra- and extracellular calcium

Adenylyl cyclase exists as a family of closely related subtypes which differ in their tissue distribution and regulatory properties. Submicromolar rises in [Ca 2+] i produced via activation of phospholipase C (PLC) or Ca 2+ channel opening, provide a mechanism by which Ca 2+/calmodulin (CaM) or protein kinase C (PKC)-sensitive isoforms of adenylyl cyclase can be regulated. In this study we have examined, in detail, the muscarinic (M 3) regulation of adenylyl cyclase in SH-SY5Y cells and report a role for both [Ca 2+] e and [Ca 2+] i. Carbachol (1 mM) and potassium (100 mM) caused a time ( T 1 2 = 3 and 4 min , respectively) and dose (EC 50 = 6.95 μM and 34.7 mM respectively) related increase in cAMP formation. This amounted to an approximate two-fold increase over basal levels. Carbachol and potassium also caused a biphasic increase in [Ca 2+] i with basal, peak and plateau values of 118.4 nM, 697.6 nM, 253.0 nM and 104.0 nM, 351.6 nM, 181.5 nM, respectively. Calcium channel blockade with nickel (2.5 mM) abolished potassium-stimulated cAMP formation and rises in [Ca 2+] i. However, carbachol-stimulated cAMP formation was significantly decreased only at the later time points, where rises in [Ca 2+] i were also essentially abolished. Further evidence for a role for [Ca 2+] e and [Ca 2+] i is provided by the stimulation of cAMP formation by carbachol in the absence of added Ca 2+, followed by a further increase on its re-addition. Carbachol- and potassium-stimulated cAMP formation were inhibited by the CaM antagonist trifluoperazine (100 μM). The μ-opiate agonists, morphine and fentanyl also inhibited carbachol-stimulated cAMP formation. In addition, cAMP formation in SH-SY5Y cell membranes was significantly increased in the presence of Ca 2+ (1.46 μM), CaM (200 nM) and forskolin (1 μM). PKC inhibition with Ro 31 8220 did not affect carbachol-stimulated cAMP formation. Taken collectively, these data suggest that SH-SY5Y cells express type 1, and possibly type 8 isoforms of adenylyl cyclase, which can be regulated by intra- and extracellular Ca 2+.

Morphine Tolerance and Physical Dependence: Reversal of Opioid Inhibition to Enhancement of Cyclic AMP Formation

This laboratory has previously demonstrated that the mu-selective opiate receptor agonist sufentanil can produce a naloxone-reversible increase or decrease in the stimulated formation of cyclic AMP (cAMP) in the myenteric plexus, depending on the concentration of opioid used. On the basis of these results, it was suggested that mu-opiate receptors are positively as well as negatively coupled to adenylyl cyclase. In the present study, the effect of chronic morphine exposure, in vivo, on the magnitude of electrically stimulated formation of cAMP and its modulation by sufentanil was investigated. In chronic morphine-treated preparations, the magnitude of electrically stimulated cAMP formation, while in the presence of an inhibitory (10(-6) M) concentration of sufentanil, is indistinguishable from the formation that occurs in opiate-naive preparations (in the absence of exogenous opioid). This indicates that the negative modulation of stimulated enteric cAMP formation by sufentanil manifests tolerance. Paradoxically, however, in "addicted tissue" the magnitude of the increase in cAMP formation produced by electrical stimulation in the presence of a previously inhibitory concentration of sufentanil is significantly larger than in its absence. Thus, the equivalence between the magnitude of stimulation-induced increase in cAMP formation observed in naive versus tolerant/dependent tissue, while in the presence of sufentanil, is due to the ability of an originally inhibitory concentration of opioid to enhance or facilitate stimulated formation of cAMP. It is suggested that tolerance/dependence to the opioid inhibition of stimulated cAMP formation results not only from the loss of inhibitory potency but also from its reversal to enhancement.

Bursal antisteroidogenic peptide alters the activity of steroidogenic enzymes in chicken granulosa cells

We have previously reported that a peptide from chicken bursa of Fabricius, bursal antisteroidogenic peptide (BASP), inhibits luteinizing hormone-stimulated progesterone biosynthesis by chicken ovarian granulosa cells. The objective of this study was to determine the site(s) of BASP inhibition within the steroidogenic pathway of chicken granulosa cells. The effects of BASP on key steroidogenic enzymes, including adenylyl cyclase (AC), phosphodiesterase, the cholesterol side-chain cleavage enzyme complex and 3ß-hydroxysteroid dehydrogenase were determined. Luteinizing hormone (10 ng/tube) stimulated a fivefold increase in granulosa cell progesterone production that was inhibited by BASP (0.06, 0.12 or 0.25 bursal equivalents) in a dose-dependent manner. Luteinizing hormone stimulated a sixfold increase in cyclic 3′,5′-adenosine monophosphate (cAMP) formation, and this increase was potentiated by BASP in a dose-dependent manner. In addition, BASP stimulated cAMP formation in the absence of luteinizing hormone without affecting progesterone production. The AC activator forskolin (0.1 mM) stimulated a 4.5-fold increase in progesterone synthesis, which was inhibited by BASP. In the presence of forskolin. BASP increased cAMP formation in a dose-dependent manner. A fivefold increase in progesterone synthesis induced by the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (1.0 mM) was inhibited by BASP. In the presence of 3-isobutyl-l-methylxanthine, BASP increased cAMP formation in a dose-dependent manner. Finally, 22(R)-hydroxycholesterol (250, 500, 1,000, or 2,500 ng/tube) or pregnenolone (50, 100, 200, or 500 ng/tube) resulted in up to 15- or 10-fold increases in progesterone production, respectively. Increasing concentrations of BASP caused a dose-dependent suppression of the conversion of 22(R)-hydroxycholesterol, but not pregnenolone, to progesterone. The inhibition of steroidogenesis by BASP is not associated with reduced cAMP levels, and BASP appears to strongly stimulate AC activity. In addition, these findings suggest that BASP may limit the availability of progesterone precursors by inhibiting the activity of the cholesterol side-chain cleavage enzyme complex.

Role of prostaglandin-mediated cyclic AMP formation in protein kinase C-dependent secretion of atrial natriuretic peptide in rat cardiomyocytes

The role of endogenous prostaglandin production in phorbol diester-induced myocardial atrial natriuretic peptide (ANP) secretion was investigated in cultured spontaneously beating ventricular rat cardiomyocytes. Incubation of cells with 4 beta-phorbol 12-myristate 13-acetate (PMA; 0.1 microM) led to a rapid response in ANP release, a response accompanied by increases in cellular prostacyclin (PGI2) production, cyclic AMP (cAMP) formation and spontaneous contraction frequency. Although PMA-induced ANP secretion exhibited the pharmacological profile of a protein kinase C (PKC)-mediated event, the response was abolished in the presence of the cyclo-oxygenase inhibitors indomethacin (10 microM) and diclofenac (1 microM), indicating that endogenous prostaglandin production is responsible for PMA-induced ANP secretion in this system. Confirming this, PMA-induced ANP secretion was strongly correlated with endogenous formation of 6-oxo-prostaglandin F1 alpha (r = 0.93, P < 0.0005, n = 11), and exogenously applied PGI2, prostaglandin E2 (PGE2) or prostaglandin F2 alpha (PGF2 alpha) elicited simultaneous increases in cAMP formation, contraction frequency and ANP secretion in these cells. Furthermore, PMA-induced cAMP formation was abolished in the presence of either diclofenac or indomethacin, whereas the cAMP-elevating agent forskolin (0.1 microM) mimicked the secretory and chronotropic effect of PMA in these cells. A role for cAMP in PMA-induced ANP secretion was also apparent insofar as PMA-induced ANP release was substantially decreased in the presence of the Rp-diastereomer of 3',5'-cyclic adenosine monophosphorothioate (Rp-cAMPS; 10 microM), whereas the cAMP-mimetic agent dibutyryl cAMP (10 microM) provoked a rapid increase in ANP secretion in this system. Finally, the Ca(2+)-channel antagonist nifedipine (0.1 microM) severely decreased PGI2-, PGE2- and PMA-induced ANP secretion without affecting PGF2 alpha-induced peptide release, suggesting that PGI2 and/or PGE2, but not PGF2 alpha, are the prostanoids involved in PMA-induced ANP release. Taken together, these results suggest that PKC activation induces ANP secretion in spontaneously beating rat ventricular cardiomyocytes via an autocrine pathway involving increased PGI2 and/or PGE2 formation, a response leading to the activation of a myocardial adenylate cyclase and, subsequently, to that of a nifedipine-sensitive Ca2+ channel.