Adenylyl Cyclase Catalytic Activity Research Articles

Hypoxia inhibits adenylyl cyclase catalytic activity in a porcine model of persistent pulmonary hypertension of the newborn.

Persistent pulmonary hypertension of the newborn (PPHN) features hypoxemia, pulmonary vasoconstriction, and impaired cardiac inotropy. We previously reported low basal and stimulated cAMP in hypoxic pulmonary artery smooth muscle cells (PASMCs). We now examine pulmonary arterial adenylyl cyclase (AC) activity and regulation in hypoxic PPHN. PPHN was induced in newborn swine by normobaric hypoxia (fraction of inspired oxygen 0.10) for 72 h and compared with age-matched normoxic controls. We studied relaxation of pulmonary arterial (PA) rings to AC activator forskolin and cGMP activator sodium nitroprusside (SNP) by isometric myography, ATP content, phosphodiesterase activity, AC content, isoform expression, and catalytic activity in presence or absence of Gαs-coupled receptor agonists, forskolin, or transnitrosylating agents in human and neonatal porcine PASMCs and HEK293T stably expressing AC isoform 6, after 72 h hypoxia (10% O2) or normoxia (21% O2). Relaxation to forskolin and SNP were equally impaired in PPHN PA. AC-specific activity decreased in hypoxia. PASMC from PPHN swine had reduced AC activity despite exposure to normoxia in culture; transient hypoxia in vitro further decreased AC activity. Prostacyclin receptor ligand affinity decreased, but its association with Gαs increased in hypoxia. Total AC content was unchanged by hypoxia, but AC6 increased in hypoxic cells and PPHN pulmonary arteries. Impairment of AC6 activity in hypoxia was associated with nitrosylation. PPHN PA relaxation is impaired because of loss of AC activity. Hypoxic AC is inhibited because of S-nitrosylation; inhibition persists after removal from hypoxia. Downregulation of AC-mediated relaxation in hypoxic PA has implications for utility of Gαs-coupled receptor agonists in PPHN treatment.

Antidepressant-like effects of curcumin on serotonergic receptor-coupled AC-cAMP pathway in chronic unpredictable mild stress of rats

Serotonergic receptors take their physiologic effects by affecting adenylyl cyclase (AC) catalytic activity and cyclic adenosine monophosphate (cAMP) concentration. AC-cAMP second messenger pathway has been recently suggested to play an important role in depression. Therefore, the compound that regulates the signal pathway may have potential as antidepressant. Curcumin is the main component of Curcuma longa L, a well-known indigenous herb with comprehensive bioactivities. In the present study, we investigated the effects of chronic unpredictable mild stress (CUMS) and curcumin on behaviours and serotonergic receptor-coupled AC-cAMP signal pathway in rats. Curcumin produced beneficial effects on the stressed rats by effectively improving CUMS-induced low sucrose consumption and reducing serum corticosterone levels in rats. Moreover, curcumin enhanced AC activity and cAMP levels in platelet and various brain regions, and up-regulated mRNA expressions of AC subtypes AC 2, AC 8 and cAMP response element binding protein (CREB) in the hippocampus, cortex and hypothalamus of the CUMS rats. Curcumin also attenuated CUMS-induced reductions of 5-hydroxytryptamine (5-HT) levels and high expressions of central 5-HT 1A/1B/7 receptors in rats. These results suggested that the potent antidepressant property of curcumin might be attributed to its improvement of AC-cAMP pathway as well as CREB via suppressing central 5-HT 1A/1B/7 receptors in the CUMS rats. Our findings provided a basis for examining the interaction of serotonergic receptors and AC-cAMP pathway in depression and curcumin treatment.

Ca2+-stimulated Basal Adenylyl Cyclase Activity Localization in Membrane Lipid Microdomains of Cardiac Sinoatrial Nodal Pacemaker Cells

Spontaneous, rhythmic subsarcolemmal local Ca(2+) releases driven by cAMP-mediated, protein kinase A (PKA)-dependent phosphorylation are crucial for normal pacemaker function of sinoatrial nodal cells (SANC). Because local Ca(2+) releases occur beneath the cell surface membrane, near to where adenylyl cyclases (ACs) reside, we hypothesized that the dual Ca(2+) and cAMP/PKA regulatory components of automaticity are coupled via Ca(2+) activation of AC activity within membrane microdomains. Here we show by quantitative reverse transcriptase PCR that SANC express Ca(2+)-activated AC isoforms 1 and 8, in addition to AC type 2, 5, and 6 transcripts. Immunolabeling of cell fractions, isolated by sucrose gradient ultracentrifugation, confirmed that ACs localize to membrane lipid microdomains. AC activity within these lipid microdomains is activated by Ca(2+) over the entire physiological Ca(2+) range. In intact SANC, the high basal AC activity produces a high level of cAMP that is further elevated by phosphodiesterase inhibition. cAMP and cAMP-mediated PKA-dependent activation of ion channels and Ca(2+) cycling proteins drive sarcoplasmic reticulum Ca(2+) releases, which, in turn, activate ACs. This feed forward "fail safe" system, kept in check by a high basal phosphodiesterase activity, is central to the generation of normal rhythmic, spontaneous action potentials by pacemaker cells.

Disruption of Type 5 Adenylyl Cyclase Enhances Desensitization of Cyclic Adenosine Monophosphate Signal and Increases Akt Signal With Chronic Catecholamine Stress

Desensitization of the cyclic adenosine monophosphate signal protects cardiac myocytes against catecholamine stress, thus preventing the development of apoptosis. Molecular mechanisms of desensitization have been well studied at the level of adrenergic receptors but less so at the level of the effector enzyme, adenylyl cyclase (AC). When the effects of long-term (1 to 2 weeks) isoproterenol infusion were compared between type 5 AC-null mice (AC5KO) and wild-type controls, we found that the subsequent responses of left ventricular ejection fraction to sudden intravenous isoproterenol challenge were reduced in AC5KO compared with wild-type mice (ie, physiological desensitization was more effective in AC5KO), consistent with enhanced downregulation of AC catalytic activity in AC5KO. One mechanism for the less effective desensitization in wild-type mice was paradoxical upregulation of type 5 AC protein expression. The number of apoptotic myocytes was similar at baseline but was significantly less in AC5KO after infusion. This was accompanied by a 4-fold greater increase in Bcl-2 and a 3-fold greater increase in phospho-Akt in AC5KO. The latter is most likely mediated by increased membrane localization of phosphoinositide-dependent protein kinase 1, which is known to be inhibited by the cyclic adenosine monophosphate signal. The absence of type 5 AC results in more effective desensitization after long-term catecholamine stress and protects against the development of myocyte apoptosis and deterioration of cardiac function, potentially elucidating a novel approach to the therapy of heart failure.

Anomalous regulation of β-adrenoceptor signaling in brain regions of the newborn rat

Desensitization, an essential homeostatic response to excessive or continued β-adrenoceptor (βAR) stimulation, is deficient in immature cells. To determine the mechanisms underlying anomalous βAR responses in newborn rats, we administered terbutaline, a β 2AR agonist, on postnatal day 2 and evaluated signaling through adenylyl cyclase (AC) in cell membrane preparations 4 h later. Although a small decrement in isoproterenol-stimulated AC was obtained in the forebrain, robust sensitization was seen in the brainstem and cerebellum, in association with heterologous increases in AC catalytic activity: increased basal, dopamine-stimulated and forskolin-stimulated AC. Superimposed on this global increase, there was a small degree of βAR and dopamine receptor desensitization, characterized by decreases in the isoproterenol/forskolin and dopamine/forskolin AC activity ratios. Our results indicate that, in some immature brain regions, βAR desensitization is masked by more substantial increases in the activity of signaling elements downstream from the receptors, resulting in sustained responses in the face of continued receptor stimulation. These effects are likely responsible for the maintenance of βAR activity associated with neurotrophic input during synaptogenesis but may also contribute to adverse effects of βAR agonists used in preterm labor.

Ischemic preconditioning prevents ischemia-induced beta-adrenergic receptor sequestration

Preconditioning enables endogenous protection to repeated myocardial ischemia. However, the effect of preconditioning on beta1 adrenergic receptor (AR) signal remains controversial. We have recently developed receptor assay system using whole cells, in which overexpressed cell surface beta ARs can be readily quantitated without disrupting the cell. Using this technique, we examined the effects of chemical/metabolic ischemia on the beta1 AR sequestration and adenylyl cyclase activity. Isoproterenol treatment, but not forskolin treatment, of HEK293T cells overexpressing beta1 ARs led to a rapid decrease (within 2 hours) in the number of the cell surface receptor, which was negated in the presence of concanavalin A. Similarly, treatment of cells with potassium cyanide and 2-deoxy-D-glucose (chemical/metabolic ischemia) induced similar receptor sequestration. When isoproterenol was superimposed on chemical/metabolic ischemia, the degree of sequestration became greater. However, when cells were pre-exposed to potassium cyanide on the preceding day (chemical preconditioning), the sequestration induced by either isoproterenol or chemical/metabolic ischemia was attenuated. Adenylyl cyclase catalytic activity as assessed by stimulation with forskolin was decreased by chemical/metabolic ischemia but fully recovered after 24 hours, suggesting that chemical/metabolic ischemia treatment did not alter cell viability. Putting together, chemical/metabolic ischemia induced beta1 AR sequestration in a similar manner to isoproterenol. In addition, preconditioning prevented the beta1 AR sequestration induced by both isoproterenol and chemical/metabolic ischemia. Pre-conditioning may play a role in preserving the cell surface beta ARs by inhibiting the sequestration that is usually induced by an ischemic event or beta adrenergic stimulation.

Crystallization of cyclase-associated protein from Dictyostelium discoideum.

Cyclase-associated protein (CAP) is a conserved two-domain protein that helps to activate the catalytic activity of adenylyl cyclase in the cyclase-bound state through interaction with Ras, which binds to the cyclase in a different region. With its other domain, CAP can bind monomeric actin and therefore also carries a cytoskeletal function. The protein is thus involved in Ras/cAMP-dependent signal transduction and most likely serves as an adapter protein translocating the adenylyl cyclase complex to the actin cytoskeleton. Crystals belonging to the orthorhombic space group C222, with unit-cell parameters a = 71.2, b = 75.1, c = 162.9 A, have been obtained from Dictyostelium discoideum CAP carrying a C-terminal His tag. A complete native data set extending to 2.2 A resolution was collected from a single crystal using an in-house X-ray system. The asymmetric unit contains one molecule of CAP.

Differences in type II, IV, V and VI adenylyl cyclase isoform expression between rat preadipocytes and adipocytes

Adenylyl cyclase catalytic activity is low in preadipocyte membranes when compared to adipocytes. Under conditions promoting inhibition of adipocyte adenylyl cyclase activity by Gpp(NH)p, a stable GTP analog, a paradoxical increase in preadipocyte adenylyl cyclase activity was obtained. In order to explain this contradiction, expression of types II, IV, V and VI adenylyl cyclase isoforms was compared in adipocytes and undifferentiated preadipocytes both by western blots and by a semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) assay. Type II, IV, V and VI mRNAs and proteins were present in both adipocytes and preadipocytes. However, in undifferentiated preadipocytes, expression of type II mRNA and protein were significantly higher whereas expression of type IV, V and VI adenylyl cyclase mRNAs and proteins were significantly weaker than in adipocytes. In late differentiated preadipocytes, the adenylyl cyclase subtype mRNA expression pattern was intermediary between the undifferentiated and the full differentiation states except for type IV which remained weakly expressed. Moreover, one of the representative r egulators of G -protein s ignaling (RGS protein), RGS4, was less expressed in undifferentiated preadipocyte membranes and cytosol extracts, which contrasts with adipocytes where RGS4 is clearly expressed. Thus, the preferential expression of type II adenylyl cyclase (G βγ subunit-stimulated) in preadipocytes might explain why Gpp(NH)p elicits stimulation of adenylyl cyclase under conditions designed to promote inhibition. Conversely, the preferential expression of type V and VI adenylyl cyclases and the slightly higher expression of type IV adenylyl cyclase in adipocytes could contribute to explain the elevated total catalytic activity observed in mature fat cells compared to their precursor cells.

Regulation of fetal cardiac and hepatic beta-adrenoceptors and adenylyl cyclase signaling: terbutaline effects.

Terbutaline (Ter), a beta(2)-adrenergic agonist used in preterm labor, stimulates fetal beta-adrenoceptors (beta-ARs). We administered Ter to pregnant rats on gestational days 17-20 and examined beta-ARs and adenylyl cyclase (AC) signaling in heart and liver. Ter produced less downregulation of cardiac beta-ARs than in adults, despite a higher proportion of the beta(2)-subtype, and failed to elicit desensitization of the receptor-mediated AC response. AC stimulants acting at different points indicated an offsetting of homologous desensitization at the level of the beta-AR by heterologous sensitization at the level of AC: induction of total AC catalytic activity and a shift in the catalytic profile or AC isoform. In fetal liver, Ter produced downregulation of beta-ARs, in keeping with the predominance of the beta(2)-subtype; hepatic receptor downregulation was equivalent in fetus and adult. Nevertheless, there was still no desensitization of beta-AR-mediated AC responses and again AC was induced. Our results indicate that, unlike in the adult, fetal beta-AR signaling is not desensitized by beta-agonists and, in fact, displays heterologous sensitization, thus sustaining responses during parturition. At the same time, the inability to desensitize beta-AR AC responses may lead to disruption of cardiac, hepatic, or neural cell development as a consequence of tocolytic therapy with beta-agonists.

Gentamicin Decreases the Abundance of Aquaporin Water Channels in Rat Kidney

The present study was performed to examine whether the gentamicin-induced urinary concentration defect is related to an altered regulation of aquaporin (AQP) water channels in the kidney. Male Sprague-Dawley rats were subcutaneously injected with gentamicin (20, 50 or 100 mg /kg per day) for 6 days. The protein expression of AQP1-3 channels and the catalytic activity of adenylyl cyclase were determined in the kidney. Gentamicin treatment resulted in renal failure associated with decreased tubular free water reabsorption and increased urinary flow rate. The expression of AQP2 proteins was significantly decreased in the kidney, in which the cortex was most susceptible, followed by the outer medulla and inner medulla in order. Gentamicin treatment also decreased the shuttling of AQP2, as evidenced by a decrease of its expression in the membrane fraction in proportion to that in the cytoplasmic fraction. The protein expression of AQP1 as well as that of AQP3 was also decreased in the cortex by treatment with the highest dose of gentamicin. The cAMP generation in response to arginine vasopressin or sodium fluoride was decreased by gentamicin, while that to forskolin was not significantly altered. These findings suggest that the primary impairment in the pathway leading to the generation of cAMP lies at the level of G proteins, resulting in a decreased expression of cAMP-mediated AQP channels. The gentamicin-induced urinary concentration defect may in part be accounted for by a reduced abundance of AQP water channels in the kidney.

Neonatal polyamine depletion by α-difluoromethylornithine: effects on adenylyl cyclase cell signaling are separable from effects on brain region growth

Ornithine decarboxylase (ODC) and the polyamines play an essential role in brain cell replication and differentiation. We administered α-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, to neonatal rats on postnatal days 5–12, during the mitotic peak of the cerebellum, a treatment regimen that leads to selective growth inhibition and dysmorphology. In adulthood, cell signaling responses mediated through the adenylyl cyclase pathway were evaluated in order to determine if synaptic dysfunction extends to regions that appear to be otherwise unaffected by DFMO. Total adenylyl cyclase catalytic activity, evaluated with the direct enzymatic stimulant, Mn 2+, was significantly elevated in male rats both in the cerebellum and in brain regions showing no growth retardation (cerebral cortex, brainstem); there were no significant effects in females. In contrast, signaling mediated through the G proteins that couple neurotransmitter receptors to adenylyl cyclase showed a deficit in the DFMO group, as evaluated with the response to fluoride; in males, there was no corresponding increase in activity as would have been expected solely from the enhancement of adenylyl cyclase, and in females, there was actually a significant decrease in the response to fluoride. Again, the deficits were not restricted to the cerebellum. Stimulation of adenylyl cyclase by isoproterenol, a β-adrenergic receptor agonist that acts through G s, likewise displayed deficits in both males and females, and without distinction by brain region. These results indicate that the ODC/polyamine pathway plays a role in the development of cell signaling, and hence in neurotransmission, above and beyond its role in cell replication and differentiation. Given the fact that numerous drugs and environmental contaminants have been shown to alter ODC and the polyamines in the developing brain, our findings suggest that changes in brain region growth or structure are inadequate to predict the targeting of specific neurotransmitter or signaling pathways, and that gender-selective functional defects may be present despite the absence of morphological differences.

Regulation of the Ca2+-inhibitable Adenylyl Cyclase Type VI by Capacitative Ca2+ Entry Requires Localization in Cholesterol-rich Domains

The endogenous Ca(2+)-inhibitable adenylyl cyclase type VI of C6-2B glioma cells is regulated only by capacitative Ca(2+) entry and not by a substantial elevation of [Ca(2+)](i) from either intracellular stores or via ionophore-mediated Ca(2+) entry (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149-1155; Fagan, K. A., Mons, N., and Cooper, D. M. F. (1998) J. Biol. Chem. 273, 9297-9305). The present studies explored the role of cholesterol-rich domains in maintaining this functional association. The cholesterol-binding agent, filipin, profoundly inhibited adenylyl cyclase activity. Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin did not affect forskolin-stimulated adenylyl cyclase activity and did not affect capacitative Ca(2+) entry. However, cholesterol depletion completely ablated the regulation of adenylyl cyclase by capacitative Ca(2+) entry. Repletion of cholesterol restored the sensitivity of adenylyl cyclase to capacitative Ca(2+) entry. Adenylyl cyclase catalytic activity and immunoreactivity were extracted into buoyant caveolar fractions with Triton X-100. The presence of adenylyl cyclase in such structures was eliminated by depletion of plasma membrane cholesterol. Altogether, these data lead us to conclude that adenylyl cyclase must occur in cholesterol-rich domains to be susceptible to regulation by capacitative Ca(2+) entry. These findings are the first indication of regulatory significance for the localization of adenylyl cyclase in caveolae.

Neonatal chlorpyrifos exposure targets multiple proteins governing the hepatic adenylyl cyclase signaling cascade: implications for neurotoxicity

Chlorpyrifos has been hypothesized to interact with receptors and transduction proteins involved in the production of cyclic AMP, contributing to adverse effects on cell replication and differentiation. We studied the effects of neonatal chlorpyrifos exposure on hepatic adenylyl cyclase (AC) activity, as the liver accumulates the highest concentrations of chlorpyrifos and is the site for generation of its active metabolite, chlorpyrifos oxon. Newborn rats were given 1 mg/kg of chlorpyrifos s.c. on PN1-4. On PN5, 24 h after the last dose, AC catalytic activity was induced as assessed by the response to the direct AC stimulant, Mn 2+. In contrast, AC activation dependent upon interaction of the enzyme with G-proteins (forskolin) did not show any enhancement, suggesting impairment of G-protein function. This conclusion was confirmed by impaired responsiveness to fluoride, which directly activates G-proteins. In addition, the response of AC to hormonal signals was altered in a receptor-selective manner, with an enhanced response to glucagon but not to the β-adrenoceptor agonist, isoproterenol. The effects of chlorpyrifos on AC signaling displayed a critical developmental period of vulnerability, as treatment of older rats (PN11-14) failed to cause substantial induction of AC or interference with G-protein signaling, although it did still enhance the glucagon response. In all cases, the effects of chlorpyrifos disappeared within a few days of discontinuing treatment. These results stand in contrast to the delayed deterioration of AC signaling seen in the brain after the same chlorpyrifos treatment. The temporal and organ selectivity of chlorpyrifos’ effects on the AC cascade suggest that disruption of membrane signaling occurs consequent to selective effects on cell development, rather than representing a direct interaction between chlorpyrifos and signaling proteins.

Ontogeny of G-protein expression: control by β-adrenoceptors

Cardiac cell homeostasis is maintained in the face of excessive β-adrenoceptor stimulation through the process of desensitization. Desensitization is not an inherent property of these cells but rather is acquired during development; neonates given β-agonists actually show heterologous sensitization, involving changes in the expression and catalytic activity of adenylyl cyclase (AC) as well as an increased receptor/G-protein coupling. The current study examines the role of specific G-protein components, G sα and G iα, in the ontogeny of β-adrenoceptor responses and in the transition from agonist-induced sensitization to desensitization. Between postnatal days (PN) 6 and 15 there was a significant decrease in the 52 kDa isoform of G sα with no accompanying change of the 45 kDa form; over the same period, G iα3 also declined substantially. In contrast, the 45 kDa isoform of G sα and G iα1,2 remained fairly constant over the same period and fluoride-stimulated AC activity increased. Treatment with isoproterenol on PN2–5 did not result in any significant changes in G sα expression but robustly decreased G iα1,2. These changes were accompanied by heterologous sensitization of AC activity at the level of AC itself, evidenced by equivalent increases in the enzymatic response to fluoride and forskolin–Mn 2+. Isoproterenol given to older animals (PN11–14) also caused specific loss of G i protein, in this case targeting G iα3, whereas G sα again was unchanged; in contrast to the younger group, the older animals displayed heterologous desensitization of AC at the level of G-protein function (specific loss of the fluoride response). These results indicate that the normal ontogenetic increase of cardiac β-adrenoceptor coupling to AC is not dependent on the absolute amount of G-proteins, nor on the relative balance of stimulatory (G s) and inhibitory (G i) subunits. However, the ability of receptor stimulation to downregulate G iα1,2, an event which is specific to immature cardiac cells, is likely to be an important component of the resistance of the fetal/neonatal heart to agonist-induced desensitization and hypertrophy. The maintenance of cardiac β-adrenoceptor signaling in the face of intense stimulation is likely to play an important role in the physiologic adaptations necessary to the perinatal transition.

Changes in adenylyl cyclase isoforms as a mechanism for thyroid hormone modulation of cardiac β-adrenergic receptor responsiveness

Although thyroid hormones are known to modulate cardiac beta-adrenergic receptor expression, the physiologic implications of these changes in the cardiac manifestations of altered thyroid hormone metabolism have been disputed. This study examined whether thyroid hormone modulates signaling via the cyclic adenosine monophosphate (cAMP) pathway by regulating cardiac adenylyl cyclase (AC) isoform expression. Northern blot analyses and AC enzyme assays were performed on preparations from hypothyroid, euthyroid, and hyperthyroid rat ventricles. Steady-state levels of cardiac AC mRNA types V and VI in hypothyroid ventricles were 173% +/- 8% and 149% +/- 12%, respectively, of the values in euthyroid ventricles (P < .01). This increase in AC mRNA isoforms was accompanied by a 1.5-fold increase (P < .05) in the activation of catalytic AC by forskolin and Mn. In contrast, the relative abundance of transcripts for types V and VI AC was similar in hyperthyroid and euthyroid ventricles, but catalytic AC activation by forskolin and Mn was significantly reduced by 35% in membranes obtained from hyperthyroid ventricles. AC activation through beta-adrenergic receptor stimulation by isoproterenol was not altered by thyroid hormone status. Thus, the effect of thyroid hormone to repress AC catalytic activity would be anticipated to offset the increase in beta-adrenergic receptor expression in hyperthyroidism. These studies identify cardiac AC enzymes as important targets for thyroid hormone-dependent regulation of signaling via the cAMP pathway, and support the finding that cardiac adrenergic responsiveness is unaltered in thyroid disease states.

Acute depression of myocardial beta-adrenergic receptor signaling during cardiopulmonary bypass: impairment of the adenylyl cyclase moiety. Duke Heart Center Perioperative Desensitization Group.

Previously the authors showed that myocardial beta-adrenergic (betaAR) function is reduced after cardiopulmonary bypass (CPB) in a canine model Whether CPB results in similar effects on betaAR function in adult humans is not known. Therefore the current study tested two hypotheses: (1) That myocardial betaAR signaling is reduced in adult humans after CPB, and (2) that administration of long-term preoperative betaAR antagonists prevents this process. After they gave informed consent, 52 patients undergoing aortocoronary surgery were enrolled. Atrial biopsies were obtained before CPB and immediately before discontinuation of CPB. Plasma catecholamine concentrations, myocardial betaAR density, and functional responsiveness (basal, isoproterenol, zinterol, sodium fluoride, and manganese-stimulated adenylyl cyclase activity) were assessed. Catecholamine levels increased significantly during CPB (P < 0.005). Myocardial betaAR adenylyl cyclase coupling decreased during CPB, as evidenced by a 21% decrease in isoproterenol-stimulated adenylyl cyclase activity (750 [430] pmol cyclic adenosine monophosphate per milligram total protein 15 min before CPB compared with 540 [390] at the end of CPB, P = 0.0062, medians [interquartile range]) despite constant betaAR density. Differential activation along the betaAR signal transduction cascade localized the defect to the adenylyl cyclase moiety. Administration of long-term preoperative betaAR antagonists did not prevent acute CPB-induced myocardial betaAR dysfunction. These data indicate that the myocardial adenylyl cyclase response to betaAR agonists decreases acutely in adults during aortocoronary surgery requiring CPB, regardless of whether long-term preoperative betaAR antagonists are administered. The mechanism underlying acute betaAR dysfunction appears to be direct impairment of the adenylyl cyclase moiety. Similar increases in manganese-stimulated activity before and at the end of CPB show preserved adenylyl cyclase catalytic activity, suggesting that other mechanisms (such as decreased protein levels or altered isoform expression or function) may be responsible for decreased adenylyl cyclase function.

Inhibition of Adenylyl Cyclase by Caveolin Peptides*

Caveolae and their principal component caveolin have been implicated in playing a major role in G protein-mediated transmembrane signaling. We examined whether caveolin interacts with adenylyl cyclase, an effector of G protein signaling, using a 20-mer peptide derived from the N-terminus scaffolding domain of caveolin-1. When tissue adenylyl cyclases were examined, cardiac adenylyl cyclase was inhibited more potently than other tissue adenylyl cyclases. The caveolin-1 peptide inhibited type V, as well as type III adenylyl cyclase, overexpressed in insect cells, whereas the same peptide had no effect on type II. The caveolin-3 scaffolding domain peptide similarly inhibited type V adenylyl cyclase. In contrast, peptides derived from the caveolin-2 scaffolding domain and a caveolin-1 nonscaffolding domain had no effect. Kinetic studies showed that the caveolin-1 peptide decreased the maximal rate (Vmax) value of type V without changing the Michaelis constant (Km) value for the substrate ATP. Studies with various truncations and point mutations of this peptide revealed that a minimum of 16 amino acid residues and intact aromatic residues are important for the inhibitory effect. The potency of inhibition was greater when adenylyl cyclase was in stimulated condition vs. basal condition. Thus, caveolin may be another cellular component that regulates adenylyl cyclase catalytic activity. Our results also suggest that the caveolin peptide may be used as an isoform-selective inhibitor of adenylyl cyclase.

Isoform-specific Regulation of Adenylyl Cyclase by Oxidized Catecholamines

Both epinephrine and manganese are known to stimulate cAMP production in cardiac homogenates. When added together, however, they inhibited adenylyl cyclase catalytic activity. Type V adenylyl cyclase, the major isoform in the heart, was also inhibited when an increasing concentration of epinephrine was added in the presence of manganese. Inhibition was not dependent on the condition of stimulation or preparation of the enzyme. However, this inhibition was abolished in the presence of anti-oxidant. Other catecholamines, including dopamine and isoproterenol, as well as adrenochrome, an oxidized product of epinephrine, similarly inhibited the activity of this enzyme. Kinetic analyses revealed that the Kmfor the substrate ATP was unchanged, but the Vmax was significantly decreased. In contrast, type II adenylyl cyclase, a non-cardiac isoform, was resistant to such inhibition by adrenochrome and was somewhat stimulated by it. Thus, catecholamines, when oxidized, directly interacted with adenylyl cyclase in an isoform-specific manner in the absence of G proteins. Our findings suggest that adenylyl cyclase isoforms have different sensitivity to various stresses, including oxidative stress.

Conformation-dependent activation of type II adenylyl cyclase by protein kinase C.

Phorbol ester treatment enhanced the catalytic activity of type II adenylyl cyclase overexpressed in insect cells. In cells coexpressing type II adenylyl cyclase and protein kinase C-alpha, type II adenylyl cyclase catalytic activity was higher even in the absence of phorbol ester treatment; phorbol ester treatment further and markedly enhanced type II adenylyl cyclase catalytic activity. However, this enhancement, either by phorbol ester treatment or by coexpression of protein kinase C-alpha, was lost following membrane solubilization with detergents. This attenuation was unaffected by phosphatase inhibitor or salts. In contrast, membrane solubilization did not affect forskolin-stimulated type II adenylyl cyclase catalytic activity. Purified type II adenylyl cyclase was stimulated by forskolin and Gs alpha, but not by protein kinase C-alpha. Therefore, a specific mammalian protein kinase C isoenzyme can activate type II adenylyl cyclase, but the mechanism clearly differs from that underlying either Gs alpha- or forskolin-mediated stimulation.

Differential changes in left and right ventricular adenylyl cyclase activities in congestive heart failure.

The status of beta-adrenergic receptors and adenylyl cyclase in crude membranes from both left and right ventricles was examined when the left coronary artery in rats was occluded for 4, 8, and 16 wk. The adenylyl cyclase activity in the presence of isoproterenol was decreased in the uninfarcted (viable) left ventricle and increased in the right ventricle subsequent to myocardial infarction. The density of beta1-adrenergic receptors, unlike beta2-receptors, was reduced in the left ventricle, whereas no change in the characteristics of beta1- and beta2-adrenergic receptors was seen in the right ventricle. The catalytic activity of adenylyl cyclase was depressed in the viable left ventricle but was unchanged in the right ventricle. In comparison to sham controls, the basal, as well as NaF-, forskolin-, and 5'-guanylyl imidodiphosphate [Gpp(NH)p]-stimulated adenylyl cyclase activities were decreased in the left ventricle and increased in the right ventricle of the experimental animals. Opposite alterations in the adenylyl cyclase activities in left and right ventricles from infarcted animals were also seen when two types of purified sarcolemmal preparations were employed. These changes in adenylyl cyclase activities in the left and right ventricles were dependent on the degree of heart failure. Furthermore, adenosine 3',5'-cyclic monophosphate contents were higher in the right ventricle and lower in the left ventricle from infarcted animals injected with saline, isoproterenol, or forskolin in comparison to the controls. The results suggest differential changes in the viable left and right ventricles with respect to adenylyl cyclase activities during the development of congestive heart failure due to myocardial infarction.