Expression Of Adenylyl Cyclase Type Research Articles

Expression pattern of olfactory receptor genes in human cumulus cells as an indicator for competent oocyte selection.

Odorant or olfactory receptors are mainly localized in the olfactory epithelium for the perception of different odors. Interestingly, many ectopic olfactory receptors with low expression levels have recently been found in nonolfactory tissues to involve in local functions. Therefore, we investigated the probable role of the olfactory signaling pathway in the surrounding microenvironment of oocyte. This study included 22 women in intracytoplasmic sperm injection cycle. The expression of olfactory target molecules in cumulus cells surrounding the growing and mature oocytes was evaluated by Western blotting and real-time polymerase chain reaction. Additionally, integrated bioinformatics analyses were carried out and 6 ectopic olfactory receptors were selected for further evaluation. The initiation of olfactory transduction cascade in cumulus cells of competent oocytes was confirmed by analyzing the expression of adenylyl cyclase type 3 and olfactory market protein. Moreover, the expression pattern of the selected olfactory receptors was evaluated and OR10H2 was selected due to a high level of expression in mature fertile oocytes. We suggested that OR10H2 could be considered as a reliable biomarker for oocyte selection in assisted reproduction technique programs. However, further studies are required to elucidate the role of olfactory transduction cascade in embryo quality and implantation.

Cardiac-directed expression of a catalytically inactive adenylyl cyclase 6 protects the heart from sustained β-adrenergic stimulation.

ObjectivesIncreased expression of adenylyl cyclase type 6 (AC6) has beneficial effects on the heart through cyclic adenosine monophosphate (cAMP)-dependent and cAMP-independent pathways. We previously generated a catalytically inactive mutant of AC6 (AC6mut) that has an attenuated response to β-adrenergic receptor stimulation, and, consequently, exhibits reduced myocardial cAMP generation. In the current study we test the hypothesis that cardiac-directed expression of AC6mut would protect the heart from sustained β-adrenergic receptor stimulation, a condition frequently encountered in patients with heart failure.Methods and resultsAC6mut mice and transgene negative siblings received osmotic mini-pumps to provide continuous isoproterenol infusion for seven days. Isoproterenol infusion caused deleterious effects that were attenuated by cardiac-directed AC6mut expression. Both groups showed reduced left ventricular (LV) ejection fraction, but the reduction was less in AC6mut mice (p = 0.047). In addition, AC6mut mice showed superior left ventricular function, manifested by higher values for LV peak +dP/dt (p = 0.03), LV peak -dP/dt (p = 0.008), end-systolic pressure-volume relationship (p = 0.003) and cardiac output (p<0.03). LV samples of AC6mut mice had more sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) protein (p<0.01), which likely contributed to better LV function. AC6mut mice had lower rates of cardiac myocyte apoptosis (p = 0.016), reduced caspase 3/7 activity (p = 0.012) and increased B-cell lymphoma 2 (Bcl2) expression (p = 0.0001).ConclusionMice with cardiac-directed AC6mut expression weathered the deleterious effects of continuous isoproterenol infusion better than control mice, indicating cardiac protection.

TRPV1 modulates morphine-induced conditioned place preference via p38 MAPK in the nucleus accumbens

Emerging evidence suggests that the transient receptor potential vanilloid type 1 channel (TRPV1) is a novel target for the treatment of drug addiction, such as cocaine and morphine. Previously we reported that TRPV1 inhibition reduced morphine reward in the dorsal striatum (DSt) of mice and morphine self-administration through a decrease in accumbal activity in rats. However, the role of TRPV1 on morphine-conditioned reward in addiction-related brain regions, such as the nucleus accumbens (NAc), has not been previously established. Here, we investigated the effects of TRPV1 on morphine conditioned place preference (CPP) and intracellular mechanisms of TRPV1 using Western blot analysis and immunohistochemistry (IHC) in morphine-administered mice. TRPV1 knockout mice did not exhibit morphine reward responses, and both i.p. and intra-NAc injections of SB366791, a selective TRPV1 antagonist, reduced morphine-induced CPP in wild-type mice. Furthermore, i.p. injection of SB203580, a selective p38 MAPK inhibitor, also dampened morphine-induced CPP. To determine the molecular mechanisms of the TRPV1/p38 MAPK pathway in morphine CPP, we investigated the expression of adenylyl cyclase type 1 (AC1) and phospho-p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) in the NAc. Either SB366791 or SB203580 decreased the protein expression levels of phospho-p38 MAPK, phosphor-NF-κB, and AC1 in the NAc of morphine CPP mice. Taken together, our findings suggest that TRPV1 may modulate morphine-induced conditioned reward effects via the p38 MAPK signaling pathway in the NAc. Therefore, blockade of TRPV1 may provide a novel therapeutic approach for the prevention and treatment of opioid addiction.

Adenylyl cyclase type 6 regulates Akt activity via PHLPP2

Adenylyl cyclase (AC) is the enzyme that catalyzes ATP to generate cyclic AMP (cAMP). Cardiac‐directed expression of adenylyl cyclase type 6 (AC6) has pronounced favorable effects on heart function in normal and failing hearts. The mechanisms explaining the favorable effects of AC6 are not entirely known, but may reflect effects on cellular events that are not replicated by cAMP production. Because of the central role of Akt in intracellular signaling, and its importance to cardiac myocyte survival and function, we tested the hypothesis that increased AC6 expression influences Akt signaling. Studies were performed in cultured neonatal cardiac myocytes; AC6 gene transfer was performed using recombinant adenovirus encoding AC6 (Ad.AC6). In the basal (unstimulated) state, AC6 gene transfer increased Akt phosphorylation and activity 5‐fold. However, when these cardiac myocytes were stimulated with a ß‐adrenergic receptor agonist (isoproterenol) or with an adenylyl cyclase activator (forskolin), AC6‐associated Akt phosphorylation rapidly was reduced (&lt;2 min). This reduction in Akt phosphorylation required an Akt‐specific phosphatase, PH domain leucine‐rich phosphatase (PHLPP2), but was cAMP‐independent and reversible. The Mg2+ binding domain in the C1 region of AC6 is required for agonist‐induced activation of PHLPP2. This novel bidirectional regulation of Akt activity may contribute to the unexpected favorable effects of AC6 on the failing heart.

Increased Cardiac Adenylyl Cyclase Expression Is Associated With Increased Survival After Myocardial Infarction

Cardiac-directed expression of adenylyl cyclase type VI (AC(VI)) in mice results in structurally normal hearts with normal basal heart rate and function but increased responses to catecholamine stimulation. We tested the hypothesis that increased left ventricular (LV) AC(VI) content would increase mortality after acute myocardial infarction (MI). Transgenic mice with cardiac-directed AC(VI) expression and their transgene-negative littermates (control) underwent coronary ligation, and survival, infarct size, and LV size and function were assessed 1 to 7 days after MI. Mice with increased AC(VI) expression had increased survival (control 41%, AC(VI) 74%; P = 0.004). Infarct size and myocardial apoptotic rates were similar in AC(VI) and control mice; however, AC(VI) mice had less LV dilation (P < 0.001) and increased ejection fractions (P < 0.03). Three days after MI, studies in isolated perfused hearts showed that basal LV +dP/dt was similar, but graded dobutamine infusion was associated with a more robust LV contractile response in AC(VI) mice (P < 0.05). Increased LV function was associated with increases in cAMP generation (P = 0.0002), phospholamban phosphorylation (P < 0.04), sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) affinity for calcium (P < 0.015), and reduced AV block (P = 0.04). In acute MI, increased cardiac AC(VI) content attenuates adverse LV remodeling, preserves LV contractile function, and reduces mortality.

Rhythmic Expression of Adenylyl Cyclase VI Contributes to the Differential Regulation of Serotonin N-Acetyltransferase by Bradykinin in Rat Pineal Glands

The rhythmic nocturnal production of melatonin in pineal glands is controlled by the periodic release of norepinephrine from the superior cervical ganglion. Norepinephrine binds to the beta-adrenergic receptor and stimulates an increase in intracellular cAMP levels, leading to the transcriptional activation of serotonin N-acetyltransferase, which in turn promotes melatonin production. In the present study, we report that bradykinin inhibits basal- and forskolin-stimulated adenylyl cyclase activity, norepinephrine-induced cAMP generation, and N-acetyltransferase expression in a calcium-dependent manner. These effects were blocked by pretreatment with U73122 (a selective phospholipase C inhibitor), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (a Ca(2+) chelator), but not pertussis toxin. The calcium ionophore, ionomycin, inhibited isoproterenol-mediated cAMP generation, similar to bradykinin. Interestingly, the inhibitory effect of bradykinin was evident only during the daytime. At midday, bradykinin inhibited the cAMP level by approximately 50% but markedly stimulated cAMP production (by approximately 50%) at night. Northern blotting and immunoblotting data disclosed circadian expression of calcium-inhibitable adenylyl cyclase type 6. Expression of adenylyl cyclase type 6 was maximal at Zeitgeber Time (ZT) 01 and very low at ZT 13. Our results suggest that bradykinin-induced calcium inhibits melatonin synthesis through the mediation of adenylyl cyclase type 6 expression.

Adenylyl Cyclase Type VI Gene Transfer Reduces Phospholamban Expression in Cardiac Myocytes via Activating Transcription Factor 3

Cardiac-directed expression of adenylyl cyclase type VI (AC(VI)) increases stimulated cAMP production, improves heart function, and increases survival in cardiomyopathy. In contrast, pharmacological agents that increase intracellular levels of cAMP have detrimental effects on cardiac function and survival. We wondered whether effects that are independent of cAMP might be responsible for these salutary outcomes associated with AC(VI) expression. We therefore conducted a series of experiments focused on how gene transcription is influenced by AC(VI) in cultured neonatal rat cardiac myocytes, with a particular focus on genes that might influence cardiac function. We found that overexpression of AC(VI) down-regulated mRNA and protein expression of phospholamban, an inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase. We determined that the cAMP-responsive-like element in the phospholamban (PLB) promoter was critical for down-regulation by AC(VI). Overexpression of AC(VI) did not alter the expression of CREB, CREM, ATF1, ATF2, or ATF4 proteins. In contrast, overexpression of AC(VI) increased expression of ATF3 protein, a suppressor of transcription. Following AC(VI) gene transfer, when cardiac myocytes were stimulated with isoproterenol or NKH477, a water-soluble forskolin analog that directly stimulates AC, expression of ATF3 protein was increased even more, which correlated with reduced expression of PLB. We then showed that AC(VI)-induced ATF3 protein binds to the cAMP-responsive-like element on the PLB promoter and that overexpression of ATF3 in cardiac myocytes inhibits PLB promoter activity. These findings indicate that AC(VI) has effects on gene transcription that are not directly dependent on cAMP generation.

Gene Therapy in Congestive Heart Failure

Although research and development of gene therapy have encountered several difficulties in recent years, they have also begun to show potential for the treatment of acquired and inherited human diseases. Gene therapy may have wide applicability for the treatment of numerous cardiovascular diseases, including congestive heart failure (CHF), ischemic heart disease, and cardiomyopathy. CHF remains a leading cause of death and morbidity in industrialized nations, and the number of patients with CHF continues to increase as the population ages. Pharmacological therapies control symptoms, reduce left ventricular (LV) dilation, improve function, and reduce mortality. These therapies control the disease but do not cure it. Currently, however, the growing knowledge of molecular mechanisms involved in myocardial dysfunction has allowed the identification of potential therapeutic targets designed with the aim of curing CHF. Several studies in animal models have provided hope that gene therapy may be one of the future strategies for the treatment of clinical CHF. In these studies, myocardial gene transfer has been used to target at least 3 different biological pathways that play a crucial role in the pathophysiology of CHF: Intracellular calcium signaling, β1-adrenergic receptor (β1-AR) signaling, and antiapoptotic signaling. The activity of sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) is decreased in failing myocardium, and this results in abnormal Ca2+ handling.1 In an animal model of heart failure, Miyamoto et al2 showed that in vivo overexpression of SERCA2a, achieved by catheter injection of an adenovirus carrying the SERCA2a gene into the aortic root, restored systolic and diastolic function to normal levels. In another study, Muller et al3 analyzed the chronic overexpression of SERCA2a in both normal and pressure-overloaded hearts (induced by abdominal aortic banding of transgenic rats). Overexpression of SERCA2a resulted in a …

Regulation of Ca2+-sensitive adenylyl cyclase in gonadotropin-releasing hormone neurons.

In immortalized GnRH neurons, cAMP production is elevated by increased extracellular Ca2+ and the Ca2+ channel agonist, BK-8644, and is diminished by low extracellular Ca2+ and treatment with nifedipine, consistent with the expression of adenylyl cyclase type I (AC I). Potassium-induced depolarization of GT1-7 neurons causes a dose-dependent monotonic increase in [Ca2+]i and elicits a bell-shaped cAMP response. The inhibitory phase of the cAMP response is prevented by pertussis toxin (PTX), consistent with the activation of G(i)-related proteins during depolarization. Agonist activation of the endogenous GnRH receptor in GT1-7 neurons also elicits a bell-shaped change in cAMP production. The inhibitory action of high GnRH concentrations is prevented by PTX, indicating coupling of the GnRH receptors to G(i)-related proteins. The stimulation of cAMP production by activation of endogenous LH receptors is enhanced by low (nanomolar) concentrations of GnRH but is abolished by micromolar concentrations of GnRH, again in a PTX-sensitive manner. These findings indicate that GnRH neuronal cAMP production is maintained by Ca2+ entry through voltage-sensitive calcium channels, leading to activation of Ca2+-stimulated AC I. Furthermore, the Ca2+ influx-dependent activation of AC I acts in conjunction with AC-regulatory G proteins to determine basal and agonist-stimulated levels of cAMP production.

Expression of adenylyl cyclase type IX and calcineurin in synapses of the central nervous system.

Distribution of type IX adenylyl cyclase and protein phosphatase calcineurin in the brain and in cultured hippocampal neurons from albino rat was immunohistochemically studied. Both enzymes were detected simultaneously in all synaptic structures of most cerebral neurons except for presynaptic sites, where calcium-inhibited type IX adenylyl cyclase was absent.

Cloning and expression of human adenylyl cyclase type VI in normal thyroid tissues

The adenylyl cyclase type VI gene expressed in human normal thyroid tissue was cloned and sequenced. The cDNA sequence (6463 nt) is susceptible to code for a 1168 aa protein. Northern blots using specific probes showed that the expression of adenylyl cyclase type VI gene was significantly higher in one hyperfunctioning thyroid tumor than in normal thyroid tissue, in one follicular cold adenoma or in one papillary carcinoma.

Expression of adenylyl cyclase type I in cochlear inner hair cells.

Expression of calcium/calmodulin-activated adenylyl cyclase type I (ACI) mRNA has been determined in the cochlea and in an organ-of-Corti subdissected tissue fraction by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Amplification products of predicted size were obtained from the mouse cochlea and rat organ of Corti with nucleotide sequences corresponding to respective ACI brain transcripts. In addition, ACI template was detected in a rat inner hair cell cDNA library by PCR. Immunoreactivity to ACI has been localized within the organ of Corti to the inner hair cell, with diaminobenzidine staining found in both the cell body and in the stereocilia. Evidence, thus, has been obtained that both ACI transcript and protein are expressed in the inner hair cell, the primary mechanosensory receptor cell of the cochlea. We hypothesize that ACI is activated by calcium influx through a calcium/calmodulin interaction and that this adenytyl cyclase isoform may have a role in modulation of receptoneural afferent transmission and/or mechanosensory transduction in the cochlea.

Cardiac beta-adrenergic signaling pathway alteration in isoproterenol-induced cardiac hypertrophy in male Sprague-Dawley rats.

Isoproterenol was continuously administered to rats at a rate of 2.5 microg/kg/min for 7 days via subcutaneously implanted osmotic minipumps. This treatment induced cardiac hypertrophy with increases in heart rate and systolic blood pressure. Beta-adrenoceptor (beta-AR) density decreased in hypertrophied hearts, but the affinity for 125I-cyanopindolol did not change. The beta-AR density decrease did not accompany the change in expression of beta1-AR mRNA. In hypertrophied hearts, adenylyl cyclase activities in the steady state, and stimulated with isoproterenol, forskolin, and manganese, decreased but there was no change in the expression of adenylyl cyclase type V mRNA. The results suggest that beta-AR downregulation and adenylyl cyclase desensitization in isoproterenol-induced cardiac hypertrophy are caused by post-transcriptional changes.