Enhancement Of Adenylyl Cyclase Activity Research Articles

Phosphodiesterase 1A modulates cystogenesis in zebrafish.

Substantial evidence indicates the importance of elevated cAMP in polycystic kidney disease (PKD). Accumulation of cAMP in cystic tissues may be, in part, caused by enhanced adenylyl cyclase activity, but inhibition of cAMP degradation by phosphodiesterases (PDE) likely has an important role, because cAMP is inactivated much faster than it is synthesized. PDE1 is the only PDE family activated by Ca(2+), which is reduced in PKD cells. To assess the contribution of the PDE1A subfamily to renal cyst formation, we examined the expression and function of PDE1A in zebrafish. We identified two splice isoforms with alternative starts corresponding to human PDE1A1 and PDE1A4. Expression of the two isoforms varied in embryos and adult tissues, and both isoforms hydrolyzed cAMP with Ca(2+)/calmodulin dependence. Depletion of PDE1A in zebrafish embryos using splice- and translation-blocking morpholinos (MOs) caused pronephric cysts, hydrocephalus, and body curvature. Human PDE1A RNA and the PKA inhibitors, H89 and Rp-cAMPS, partially rescued phenotypes of pde1a morphants. Additionally, MO depletion of PDE1A aggravated phenotypes in pkd2 morphants, causing more severe body curvature, and human PDE1A RNA partially rescued pkd2 morphant phenotypes, pronephric cysts, hydrocephalus, and body curvature. Together, these data indicate the integral role of PDE1A and cAMP signaling in renal development and cystogenesis, imply that PDE1A activity is altered downstream of polycystin-2, and suggest that PDE1A is a viable drug target for PKD.

Development of sensitization to methamphetamine in offspring prenatally exposed to morphine, methadone and buprenorphine

Heroin use among young women of reproductive age has drawn much attention around the world. However, there is lack of information on the long-term effects of prenatal exposure to opioids on their offspring. Our previous study demonstrated that prenatally buprenorphine-exposed offspring showed a marked change in the cross-tolerance to morphine compared with other groups. In the current study, this animal model was used to study effects of methamphetamine (METH)-induced behavioral sensitization in the offspring at their adulthood. The results showed no differences in either basal or acute METH-induced locomotor activity in any of the groups of animals tested. When male offspring received METH injections of 2 mg/kg, i.p., once a day for 5 days, behavioral sensitization was induced, as determined by motor activity. Furthermore, the distance and rate of development (slope) of locomotor activity and conditioned place preference induced by METH were significantly increased in the prenatally buprenorphine-exposed animals compared with those in other groups. The dopamine D1 R in the nucleus accumbens of the prenatally buprenorphine-exposed offspring had lower mRNA expression; but no significant changes in the μ-, κ-opioid, nociceptin, D2 R and D3 R receptors were noted. Furthermore, significant alterations were observed in the basal level of cAMP and the D1 R agonist enhanced adenylyl cyclase activity in the prenatally buprenorphine-exposed group. Overall, the study demonstrates that D1 R and its downregulated cAMP signals are involved in enhancing METH-induced behavioral sensitization in prenatally buprenorphine-exposed offspring. The study reveals that prenatal exposure to buprenorphine caused long-term effects on offspring and affected the dopaminergic system-related reward mechanism.

The dopamine D2receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle

Dopamine receptors are G protein-coupled receptors that are divided into two subgroups, "D(1)-like" receptors (D(1) and D(5)) that couple to the G(s) protein and "D(2)-like" receptors (D(2), D(3), and D(4)) that couple to G(i). Although inhaled dopamine has been reported to induce bronchodilation in patients with asthma, functional expression of dopamine receptor subtypes has never been described on airway smooth muscle (ASM) cells. Acute activation of G(i)-coupled receptors inhibits adenylyl cyclase activity and cAMP synthesis, which classically impairs ASM relaxation. In contrast, chronic activation of G(i)-coupled receptors produces a paradoxical enhancement of adenylyl cyclase activity referred to as heterologous sensitization. We questioned whether the dopamine D(2)-like receptor is expressed on ASM, whether it exhibits classical G(i)-coupling, and whether it modulates ASM function. We detected the mRNA encoding the dopamine D(2) receptor in total RNA isolated from native human ASM and from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the dopamine D(2) receptor protein in both native human and guinea pig ASM and cultured HASM cells. The dopamine D(2) receptor protein was immunohistochemically localized to both human and guinea pig ASM. Acute activation of the dopamine D(2) receptor by quinpirole inhibited forskolin-stimulated adenylyl cyclase activity in HASM cells, which was blocked by the dopamine D(2) receptor antagonist L-741626. In contrast, the chronic pretreatment (1 h) with quinpirole potentiated forskolin-stimulated adenylyl cyclase activity, which was inhibited by L-741626, the phospholipase C inhibitor U73122, or the protein kinase C inhibitor GF109203X. Quinpirole also stimulated inositol phosphate synthesis, which was inhibited by L-741626 or U73122. Chronic pretreatment (1 h) of the guinea pig tracheal rings with quinpirole significantly potentiated forskolin-induced airway relaxation, which was inhibited by L-741626. These results demonstrate that functional dopamine D(2) receptors are expressed on ASM and could be a novel therapeutic target for the relaxation of ASM.

Isoform‐Specific Enhancement of Adenylyl Cyclase Activity by n‐Alkanols

Numerous studies suggest that cAMP signaling pathways play important roles in the development of and predisposition to alcoholism. Our previous study showed that cAMP generation by various isoforms of adenylyl cyclase (AC) exhibits a broad spectrum of responses to ethanol in the human embryonic kidney (HEK) 293 cell system overexpressing individual AC isoforms. These findings suggest that the target of ethanol's action in the cAMP-generating system is AC. However, it is unknown if the action of ethanol is direct or indirect. The effect of a series of n-alkanols (ethanol to decanol) on dopamine (DA)-stimulated activity of AC isoforms type 6, 7, and 9 (AC6, AC7, and AC9) were examined in transfected HEK293 cells by cAMP accumulation assay. n-Alkanols increased DA-stimulated cAMP production in an AC isoform-specific manner, and displayed the alcohol cutoff phenomenon (defined as the carbon chain length beyond which there is no further increase in the potency of an ascending series of n-alkanols). The n-alkanol cutoffs for AC6, AC7, and AC9 are butanol (C4), pentanol (C5), and equal to or greater than decanol (C10), respectively. The results clearly indicate that, in the HEK293 expression system, the alcohol cutoff effect for n-alkanol potentiation of DA-stimulated AC activity is AC isoform specific. These results strongly suggest that alcohols interact directly with AC molecules.

The Cytosolic Phospholipase A2 Pathway, a Safeguard of β2-Adrenergic Cardiac Effects in Rat

We have recently demonstrated that in human heart, beta2-adrenergic receptors (beta2-ARs) are biochemically coupled not only to the classical adenylyl cyclase (AC) pathway but also to the cytosolic phospholipase A2 (cPLA2) pathway (Pavoine, C., Behforouz, N., Gauthier, C., Le Gouvello, S., Roudot-Thoraval, F., Martin, C. R., Pawlak, A., Feral, C., Defer, N., Houel, R., Magne, S., Amadou, A., Loisance, D., Duvaldestin, P., and Pecker, F. (2003) Mol. Pharmacol. 64, 1117-1125). In this study, using Fura-2-loaded cardiomyocytes isolated from adult rats, we showed that stimulation of beta2-ARs triggered an increase in the amplitude of electrically stimulated [Ca2+]i transients and contractions. This effect was abolished with the PKA inhibitor, H89, but greatly enhanced upon addition of the selective cPLA2 inhibitor, AACOCF3. The beta2-AR/cPLA2 inhibitory pathway involved G(i) and MSK1. Potentiation of beta2-AR/AC/PKA-induced Ca2+ responses by AACOCF3 did not rely on the enhancement of AC activity but was associated with eNOS phosphorylation (Ser1177) and L-NAME-sensitive NO production. This was correlated with PKA-dependent phosphorylation of PLB (Ser16). The constraint exerted by the beta2-AR/cPLA2 pathway on the beta2-AR/AC/PKA-induced Ca2+ responses required integrity of caveolar structures and was impaired by Filipin III treatment. Immunoblot analyses demonstrated zinterol-induced translocation of cPLA and its cosedimentation with MSK1, eNOS, PLB, and sarcoplasmic reticulum Ca2+ pump (SERCA) 2a in a low density caveolin-3-enriched membrane fraction. This inferred the gathering of beta2-AR signaling effectors around caveolae/sarcoplasmic reticulum (SR) functional platforms. Taken together, these data highlight cPLA as a cardiac beta2-AR signaling pathway that limits beta2-AR/AC/PKA-induced Ca2+ responses in adult rat cardiomyocytes through the impairment of eNOS activation and PLB phosphorylation.

Phosphoinositide 3-kinase activity is required for biphasic stimulation of cyclic adenosine 3',5'-monophosphate by relaxin.

The G protein-coupled receptors LGR7 and LGR8 have recently been identified as the primary receptors for the polypeptide hormone relaxin and relaxin-like factors. RT-PCR confirmed the existence of mRNA for both LGR7 and LRG8 in THP-1 cells. Whole cell treatment of THP-1 cells with relaxin produced a biphasic time course in cAMP accumulation, where the first peak appeared as early as 1-2 min with a second peak at 10-20 min. Selective inhibitors for phosphoinositide 3-kinase (PI3K), such as wortmannin and LY294002, showed a dose-dependent inhibition of relaxin-mediated increases in cAMP, specific for the second peak of the relaxin time course. Adenylyl cyclase activation by relaxin in purified plasma membranes from THP-1 cells was not inhibited by LY294002, consistent with a mechanism involving direct stimulation by a Galphas-coupled relaxin receptor. However, reconstitution of membranes with cytosol from THP-1 cells enhanced adenylyl cyclase activity and restored LY294002 sensitivity. In addition, relaxin increased PI3K activity in THP-1 cells. Neither the effects of relaxin nor the inhibition of relaxin by LY294002 was mediated by the activity of phosphodiesterases. Taken together, we show that PI3K is required for the biphasic stimulation of cAMP by relaxin in THP-1 cells and present a novel signal transduction pathway for the activation of adenylyl cyclase by a G protein-coupled receptor.

Chronic ethanol treatment reduces adenylyl cyclase activity in human erythroleukemia cells

Characteristic changes of platelet membrane adenylyl cyclase activity have been described in men with alcoholism. We studied the occurrence of these changes in human erythroleukemia (HEL) cells after chronic ethanol treatment. Chronic treatment of the HEL cell with ethanol (50 or 100 mM) for 48 h resulted in significant reduction of prostaglandin E 1-stimulated adenylyl cyclase activity. The acute ethanol (200 mM, 5 min) enhancement of adenylyl cyclase activity was significantly reduced after chronic ethanol treatment. We also observed a reduction in phorbol-12,13-dibutyrate (PDB) enhancement of prostaglandin E 1-stimulation after chronic ethanol treatment. Chronic ethanol treatment (50 or 100 mM) reduced the activity of adenylyl cyclase in response to stimulation by acute ethanol to a greater extent than that of after acute PDB. The increase in cAMP formation by ethanol and PDB was only evident when prostaglandin E 1 was present and under basal conditions (when no stimulatory agent was present) ethanol up to 200 mM, and PDB up to 1 M, had no significant effect on adenylyl cyclase activity. The reduced capacity of ethanol and/or PDB to stimulate adenylyl cyclase activity after chronic ethanol treatment suggests the involvement of a common denominator in the action of ethanol and PDB.

Modification of Adipocyte Membrane Adenylyl Cyclase Activity by NAD: Evidence against NAD-Induced Endogenous ADP-Ribosylation of Gsα Protein

Treatment of saponin-permeabilized adipocytes with NAD enhanced adenylyl cyclase activity stimulated by GTγS, [Al/F4]−, isoproterenol, and forskolin in membrane fractions and potentiated isoproterenol-induced cAMP accumulation in whole cells. In parallel, when permeabilized adipocytes were incubated with [32P]NAD, there was significant incorporation of [32P]ADP-ribose in a 44-kDa acceptor membrane protein. This reaction was inhibited by l-arginine and was enhanced by the addition of GTPγS. Surprisingly, this 44-kDa protein could not be identified as Gs protein: (1) It was not recognized by Gsα specific antibody; (2) it did not comigrate with the major cholera toxin substrates in either 10% SDS-PAGE or two-dimensional electrophoresis; (3) a pretreatment of adipocytes with NAD did not decrease cholera toxin-mediated ADP-ribosylation of Gsα proteins on membrane fractions. Our results indicate that NAD did not induce endogenous ADP-ribosylation of Gsα in permeabilized rat adipocytes but nonetheless modified the adenylyl cyclase response.

Vanadate stimulation of adenylyl cyclase: an index of tyrosine kinase vascular effects.

Beyond their mitogenic effects, hormones such as insulin, which activate receptor tyrosine kinases, regulate vascular tone. Further, we have demonstrated that receptor tyrosine kinase activation enhances adenylyl cyclase activation, a prominent mechanism that mediates vasodilation. However, whether tyrosine kinase-mediated human vascular responses parallel tyrosine kinase-mediated cellular effects on adenylyl cyclase activity is unknown. To assess tyrosine kinase-mediated vascular responses, vascular sensitivity to insulin was assessed with the dorsal hand vein linear variable differential transformer technique. Insulin infusion resulted in a dose-dependent relaxation in all subjects. Cellular responses were assessed by means of the insulinomimetic vanadate-mediated sensitization of vascular adenylyl cyclase activity. Vanadate stimulated a tyrosine kinase-dependent enhancement of adenylyl cyclase function in human and rat aortic vascular smooth muscle cells, human lymphocytes, and human aortic endothelial cells. Further, maximal insulin-mediated vasodilation was significantly positively correlated with maximal vanadate-mediated enhancement of human lymphocyte adenylyl cyclase activity. Insulin-mediated vasodilation is positively correlated with vanadate-mediated enhancement of adenylyl cyclase activity. Vanadate-mediated enhancement of adenylyl cyclase activity in lymphocytes may represent an index of tyrosine kinase-mediated vascular effects.

Chronic activation of ORL1 receptor induces supersensitization of adenylyl cyclase.

The opioid receptor-like (ORL1) receptor regulates a variety of effectors shared by its close relatives, the opioid receptors. Supersensitization of adenylyl cyclase (AC) is a hallmark of cellular tolerance induced by chronic activation of opioid receptors. To examine if chronic activation of the ORL1 receptor leads to a similar adaptation, a HEK293 cell line stably expressing the human ORL1 receptor (293/ORL1) was established. Chronic treatment of 293/ORL1 cells with nociceptin/OFQ resulted in enhanced AC activity in response to forskolin stimulation. The AC supersensitivity was blocked by pertussis toxin, indicating the involvement of Gi/Go proteins. Likewise, chronic activation of endogenous ORL1 receptors in the neuroblastoma SK-N-SH cells led to Gi/Go-mediated AC supersensitization.

Martin Rodbell (1925-1998).

Martin Rodbell (1925-1998).

Nonenzymatic palmitoylation at Cys 3 causes extra-activation of the alpha-subunit of the stimulatory GTP-binding protein Gs.

Treatment of crude stimulatory GTP-binding protein of adenylyl cyclase (Gs) from turkey erythrocyte membranes with hydroxylamine results in twofold enhancement of adenylyl cyclase activity following reconstitution with adenylyl cyclase type V expressed in Spodoptera frugiperda cells (Sf9) cells. Enhancement by hydroxylamine of immunoaffinity purified Gs was still 1.5-fold, while that of Gs purified according to the multiple-step procedure by Northup, J. K., Sternweis, P. C., Smigel, M. D., Schleifer, L. S., Ross, E. M. & Gilman, A. G. (Proc. Natl Acad. Sci. USA 78, 6516-6520, 1980) was close to unity. The alpha-subunit of the stimulatory GTP-binding protein expressed in Escherichia coli (r(alpha)s), likewise, failed to show an effect by hydroxylamine. Surprisingly, guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-liganded r(alpha)s, treated with palmitoyl-CoA for 14 h at 4 degrees C resulted in sixfold enhancement of reconstitutive activity. In contrast, that of the GDP-liganded r(alpha)s(beta)gamma heterotrimer was not improved by palmitoylation and consecutive activation with GTP[S], although incorporation of [3H]palmitate into momomer and heterotrimer was identical. While adenylyl cyclase type-V activity reconstituted by r(alpha)s x GTP[S] was not influenced by betagamma-subunits, that activated by palmitoylated r(alpha)s x GTP[S] was considerably inhibited, suggesting a higher affinity of palmitoylated r(alpha)s for betagamma-subunits. On treatment of either form with the proteinase Lys-C, less than 25% of the label was found in a stable M(r) 38000 fragment with intact C terminus, but lacking the N-terminal portion. Absence of the latter did not affect activation by r(alpha)s, but caused a >90% loss of extra-activation by palmitoylated r(alpha)s. The results also indicate that nonenzymatic, much in the same way as physiological enzymatic, palmitoylation of alpha(s) occurs predominantly on Cys 3.

A Single Dose of Antenatal Betamethasone Enhances Isoprenaline and Prostaglandin E2-Induced Relaxation of Preterm Ovine Pulmonary Arteries

β-Adrenergic agonists and prostaglandin E₂ (PGE₂) play an important role in perinatal pulmonary circulation. We have determined the effect of antenatal glucocorticoid treatment on isoprenaline- and PGE₂-mediated relaxation of pulmonary arteries of newborn preterm lambs. Ovine fetuses (121 days of gestation; term = 150 days) received a single intramuscular dose of betamethasone (0.5 mg/kg) or saline. Fifteen hours after the injection, the lambs were delivered, ventilated for 3 h, and sacrificed. The fourth-generation pulmonary arteries were dissected and cut into rings for study. In endothelin-1-preconstricted vessels, isoprenaline, PGE₂, and forskolin (an activator of adenylyl cyclase) induced greater relaxations of pulmonary arteries of betamethasone-treated lambs than those of controls. 8-Bromo-cyclic adenosine monophosphate, a cell membrane permeable analogue of cyclic adenosine monophosphate, caused similar relaxation of all vessels. When stimulated with isoprenaline and PGE₂, the adenylyl cyclase activity of crude membrane preparations of pulmonary arteries treated with betamethasone was greater than that of controls. These results show that single-dose antenatal betamethasone treatment enhances relaxation of pulmonary arteries of preterm lambs induced by isoprenaline and PGE₂ and that an enhanced adenylyl cyclase activity contributes to the effect of betamethasone on pulmonary arteries of preterm lambs.

Dimeric tubulin-stimulated adenylyl cyclase activity is augmented after long-term amitriptyline treatment.

We have investigated altered association of tubulin dimers interacting with G proteins and modulating adenylyl cyclase (AC) as a result of long-term amitriptyline (AMT) treatment. Gpp(NH)p-stimulated, but not basal or manganese-stimulated, AC activity was significantly augmented in cortex membranes prepared from rats chronically treated with AMT. The enhancement of AC activity by Gpp(NH)p-liganded tubulin (tubulin-Gpp(NH)p) was significantly higher in chronically AMT-treated rats than in control rats. Moreover, in cortex membranes from controls, tubulin-Gpp(NH)p prepared from chronically AMT-treated rats was more effective to activate AC activity than tubulin-Gpp(NH)p from controls. Immunoblotting and photoaffinity guanine nucleotide binding procedures showed no significant differences in the amount and the function of G proteins between controls and AMT-treated groups. It is suggested that long-term AMT treatment causes alteration in the functional interaction between tubulin and G protein, and this modification may participate in enhanced coupling of Gs to the catalytic subunit of AC induced by the chronic antidepressant treatment.

Oxidant Stress Enhances Adenylyl Cyclase Activation

The generation of oxygen-derived free radicals has been implicated in the disordered vascular regulation of inflammation and reperfusion. In the vasculature, oxygen-derived free radicals are vasodilatory. The mechanisms underlying this effect remain unclear. To examine the cellular processes involved, we studied the effects of hydrogen peroxide (H2O2) on adenylyl cyclase activity in A10 cells, a murine vascular smooth muscle cell line. Pretreatment with H2O2 caused a dose-dependent enhancement of forskolin-stimulated adenylyl cyclase activity (ED50, 44 mumol/L to a maximum of 166% of control activity; n = 4). This enhancement was attenuated by iron chelation with deferoxamine and by the intracellular hydroxyl scavenger dimethylthiourea and mimicked by preincubation with purine/xanthine oxidase either alone or in the presence of superoxide dismutase. The effects of H2O2 were completely blocked by the tyrosine kinase inhibitors genistein and tyrphostin A9 but not by its inactive analogue tyrphostin A1 (H2O2 alone, 149 +/- 13%; H2O2 + tyrphostin A9, 100 +/- 9%; H2O2 + tyrphostin A1, 171 +/- 21%; n = 4). H2O2 comparably enhanced adenylyl cyclase activity stimulated by isoproterenol (166 +/- 17% of control, n = 5) and sodium fluoride (177 +/- 18% of control, n = 5). Thus oxygen-derived free radicals enhance adenylyl cyclase activation, probably via tyrosine kinase-mediated effects on the catalytic subunit of adenylyl cyclase. Sensitization of adenylyl cyclase activation may be an important mechanism by which free radicals modulate hormone-mediated vasodilation.

Influence of ischaemia and reperfusion on cardiac signal transduction. G protein content, adenylyl cyclase activity, cyclic AMP content, and forskolin and dibutyryl cyclic AMP-induced inotropy in the rat Langendorff heart.

We investigated whether post-receptor alterations contribute to the diminished beta-adrenergic inotropic effects in the rat Langendorff heart following ischaemia (I) and reperfusion (R). We quantitated immunodetectable Gs and Gi protein alpha-subunit content, basal and stimulated adenylyl cyclase activity and cyclic AMP (cAMP) content in normoxic, ischaemic (30 min) and ischaemic reperfused (30 min) hearts. In addition, we measured the inotropic response of normoxic and reperfused Langendorff hearts to forskolin and dibutyryl cAMP (db-cAMP). Immunodetectable Gs and Gi alpha-subunits were unaltered by I or R. Basal adenylyl cyclase activity was decreased during I, but recovered during R. In membranes from normoxic hearts, isoprenaline, GTP, Gpp(NH)p, NaF, forskolin or Mn2+ enhanced adenylyl cyclase activity. This increase in activity was diminished in ischaemic hearts, but could be restored by R. cAMP content decreased time-dependently during I and did not recover by R, indicating ATP depletion. Forskolin and db-cAMP induced an inotropic response in normoxic hearts, which was virtually abolished after I and R. We conclude that adenylyl cyclase responsiveness is impaired during I. Since adenylyl cyclase responsiveness recovers during R, whereas inotropic responses to forskolin and db-cAMP are virtually absent in reperfused hearts, an additional mechanism downstream of cAMP formation appears to be defective during R, which prevents recovery of inotropic responses to hormonal stimulation.

Adaptive changes in the number of G s- and G i-proteins underlie adenylyl cyclase sensitization in morphine-treated rat striatal neurons

In the present study we investigated the possible role of changes in the number of membrane-bound G-proteins in the sensitization of dopamine D 1 receptor-stimulated adenylyl cyclase, observed in primary cultures of rat striatal neurons chronically exposed to morphine. Whereas exposure of these neurons to 10 μM morphine for 7 days caused a profound increase in cyclic AMP production, induced by the dopamine D 1 receptor agonist SKF 38393 (1 μM), Scatchard analysis of [ 125I]SCH 23982 binding to membrane preparations revealed that neither the B max nor the K d values of dopamine D 1 receptor binding sites were affected. Interestingly, immunoblotting experiments revealed an increase (of more than 50%) in the number of stimulatory G-proteins (G αs) in neurons displaying an enhanced adenylyl cyclase activity. In morphine-treated neurons, the number of inhibitory G-proteins (G αi) appeared to be slightly reduced (by about 16%). Moreover, the observation that cholera toxin (0.1 nM)-stimulated cyclic AMP production, unlike that induced by forskolin (1 μM), was enhanced in morphine-treated neurons, indicates a causal relationship between the reciprocal changes in G-protein number and the increase of dopamine D 1 receptor-stimulated adenylyl cyclase activity. The possible role of these changes in G-protein number in the development of morphine tolerance and dependence is discussed.

Stress alters adenylyl cyclase activity in the pituitary and frontal cortex of the rat

We hypothesized that sustained biosynthesis of proopiomelanocortropin (POMC) from the anterior pituitary during chronic stress might result in enhanced membrane adenylyl cyclase (AC) activity, facilitating amplification of the CRH signal despite falling numbers of CRH receptors. Therefore, we investigated the effects of stress on AC activity in anterior pituitaries from Sprague-Dawley rats exposed to stress. Following 12 h of intermittent, cold, swim stress, stressed rats had plasma corticosterone levels that were 10 fold higher than in nonstressed animals and showed a 40% reduction in the specific binding of 125I-CRH to anterior pituitary membranes. Moreover, stressed rats showed a 3 fold increase in anterior pituitary POMC mRNA levels. To test the hypothesis that factors released during stress enhanced the AC signal transduction system, thereby leading to increased POMC gene expression, we measured anterior pituitary cAMP and assayed AC activity from membranes prepared from anterior pituitary of control and stressed rats. Levels of cAMP were 2 fold higher in pituitaries from stressed rats compared to controls. The significant increase in cAMP was accompanied by a significant increase of AC activity. To test what component(s) of the AC complex are altered by stress, type I and II AC mRNA as well as Gsα, Gi(1–3)α and Gβ protein levels were determined. Type II AC mRNA was significantly increased 1.7 fold in stressed rats compared with controls, whereas no consistent alteration in G-protein levels were detected. Enhanced AC activity following cold swim stress was not limited to the pituitary, to one line of rat, nor one type of stress. In Fisher rats, both cold swim and restraint stress enhanced AC activity in the pituitary and in the frontal cortex. In summary, stress enhances AC activity in the anterior pituitary. The increase in AC activity is associated with increased steady state levels of type II AC mRNA. Factor(s) released during stress may enhance AC signal transduction and allow for persistent elevation in POMC gene expression despite the inhibitory influences of glucocorticoids.

Control of Plasminogen Activator Activity in the Thecal Layer of the Largest Preovulatory Follicle in the Hen Ovary*

Although the existence of plasminogen activator (PA) activity and the factors that regulate it in ovarian granulosa cells of both mammalian and avian species have been extensively documented, very little information has been generated concerning the control of PA activity in the adjacent thecal layer. This study was conducted to evaluate the effects of several physiological and pharmacological agents on PA activity in dispersed cells from the thecal layer of the largest preovulatory follicle in the hen ovary 17-16 h before ovulation. LH (50 and 100 ng) in the presence of 3-isobutyl-1-methylxanthine (0.01 mM) stimulated an approximate 25% increase in cell-associated PA activity, possibly via elevated levels of cAMP. Prostaglandin E1 and E2 (PGE1 and PGE2; 0.1 and 1 microM), but not PGI2 or PGF2 alpha (1 microM), enhanced PA activity and cAMP formation, effects that were potentiated by 0.01 mM 3-isobutyl-1-methylxanthine. Activation of Gs with cholera toxin (0.01-10 ng/tube) or adenylyl cyclase with forskolin (0.01-10 microM) stimulated cAMP formation and PA activity in a dose-dependent manner. Exposure of cells to the cAMP analog 8-bromo-cAMP (0.1-5 mM) caused similar increases in thecal cell PA activity. Incubation of cells with phorbol 12-myristate 13-acetate (PMA; 3.2-162 nM), an agonist known to activate protein kinase-C, resulted in a dose-dependent increase in PA activity. However, an equimolar concentration of phorbol 13-monoacetate (162 nM), an inactive analog of PMA that does not activate protein kinase-C, was without effect. Coincubation of cells with forskolin (1 microM) and PMA (32 nM) resulted in a synergistic stimulation of secreted PA activity, apparently via an enhancement of adenylyl cyclase activity. Treatment of cells with the calcium ionophore A23187 (0.01-1 microM) suppressed basal PA activity. However, PA activity stimulated by PMA (32 nM) was synergistically increased after coincubation with a 0.05-microM concentration of A23187, but was inhibited at doses of 0.5 and 1 microM. Taken collectively, the data indicate that PA activity is present in the thecal layer of the largest preovulatory follicle in the ovary of the domestic hen. Furthermore, several endocrine factors (i.e. LH and PGs) were found to stimulate PA activity, possibly via both the adenylyl cyclase-cAMP-protein kinase-A and phosphoinositide-protein kinase-C pathways. In light of these findings, we propose that the preovulatory increase in PGs and LH activates PA in the thecal layer of the largest preovulatory follicle, resulting in proteolytic degradation of the follicular connective tissue and, ultimately, ovulation.

Dexamethasone increases adenylyl cyclase activity and expression of the alpha-subunit of Gs in GH3 cells.

Glucocorticoids regulate responsiveness of many different cells and tissues to hormones that act by stimulating adenylyl cyclase. In a rat pituitary cell line, GH3, exposure to dexamethasone (150 mM for 72 h) doubled the specific activity of adenylyl cyclase in membrane extracts, whether the enzyme was stimulated by guanine nucleotides, vasoactive intestinal peptide, or forskolin. Dexamethasone treatment of GH3 cells also caused a 5-fold increase in RNA encoding the alpha-chain of Gs (alpha s) as well as a 2-fold increase in membrane-bound alpha s-polypeptide, measured by either of two assays: 1) Biochemical complementation of the alpha s deficiency of S49 lymphoma cyc- membranes in vitro; 2) Cholera toxin-catalyzed ADP ribosylation of 45- and 52-kilodalton alpha s-polypeptides. These observations suggest that glucocorticoid-induced enhancement of adenylyl cyclase activity in GH3 cells is mediated, at least in part, by increased expression of alpha s.