Adenylyl Cyclase Type Research Articles

An Opiate Cocktail that Reduces Morphine Tolerance and Dependence

Morphine is an exceptionally effective analgesic whose utility is compromised by the development of tolerance and dependence to the drug. Morphine analgesia and dependence are mediated by its activity at the mu opioid peptide (MOP) receptor [1]. The MOP receptor is activated not only by morphine, but also by other opiate drugs such as methadone and endogenous opioids such as endorphins. Morphine, however, is a unique opioid agonist ligand because it fails to induce endocytic trafficking of the MOP receptor [2], whereas the endogenous ligands and methadone do facilitate endocytosis [3]. Using the unique pharmacology of the MOP receptor and its proposed existence as an oligomeric structure [4], we designed a pharmacological cocktail that facilitates endocytosis of the MOP receptor in response to morphine. This cocktail consists of morphine and a small dose of methadone. Importantly, this cocktail, while retaining full analgesic potency, does not promote morphine dependence. We further demonstrate that dependence is reduced, at least in part, because endocytosis of the MOP receptor in response to morphine prevents the upregulation of N-methyl-D-aspartate (NMDA) receptors.

Adenylyl cyclase type-VIII activity is regulated by G βγ subunits

The Ca 2+-activated adenylyl cyclase type VIII (AC-VIII) has been implicated in several forms of neural plasticity, including drug addiction and learning and memory. It has not been clear whether G i/o proteins and G-protein coupled receptors regulate the activity of AC-VIII. Here we show in intact mammalian cell system that AC-VIII is inhibited by μ-opioid receptor activation and that this inhibition is pertussis toxin sensitive. Moreover, we show that G βγ subunits inhibit AC-VIII activity, while constitutively active α i/o subunits do not. Different G β isoforms varied in their efficacies, with G β1γ2 or G β2γ2 being more efficient than G β3γ2 and G β4γ2, while G β5 (transfected with γ 2) had no effect. As for the G γ subunits, G β1 inhibited AC-VIII activity in the presence of all γ subunits tested except for γ 5 that had only a marginal activity. Moreover, cotransfection with proteins known to serve as scavengers of G βγ dimers, or to reduce G βγ plasma membrane anchorage, markedly attenuated the μ-opioid receptor-induced inhibition of AC-VIII. These results demonstrate that G βγ (originating from agonist activation of these receptors) and probably not G αi/o subunits are involved in the agonist inhibition of AC-VIII.

Dexras1 blocks receptor-mediated heterologous sensitization of adenylyl cyclase 1

Dexras1/AGS1/RasD1 is a member of the Ras superfamily of monomeric G proteins and has been suggested to disrupt receptor-G protein signaling. We examined the ability of Dexras1 to modulate dopamine D 2L receptor regulation of adenylyl cyclase (AC) type 1 in HEK293 cells. Acute D 2L receptor-mediated inhibition of A23187-stimulated AC1 activity (IC 50, 4.0 ± 1.4 nM; 50 ± 3% inhibition) was not altered in the presence of Dexras1 (IC 50, 2.4 ± 1.3 nM, 50 ± 1% inhibition); however, Dexras1 blocked acute D 2L receptor-mediated activation of ERK 1/2 by approximately 50%. Heterologous sensitization of AC1 induced by persistent activation of D 2L receptors was completely blocked by Dexras1 under basal and A23187-stimulated conditions. The block of sensitization was concentration-dependent and was not observed with a nucleotide binding-deficient Dexras1G31V mutant. Sensitization of AC1 was Gβγ-dependent as demonstrated using the C-terminus of β-adrenergic receptor kinase (βARK-ct). These data suggest that Dexras1 selectively regulates receptor-mediated Gβγ signaling pathways.

Molecular properties and pharmacogenetics of a polymorphism of adenylyl cyclase type 9 in asthma: interaction between β-agonist and corticosteroid pathways

In asthma, the response to beta-agonists acting at beta2-adrenergic receptors (beta2AR) displays extensive interindividual variation. One effector for airway beta2AR, adenylyl cyclase type 9 (AC9), was considered a candidate locus for predicting beta-agonist efficacy in the absence and presence of corticosteroid treatment. One non-synonymous AC9 polymorphism has been identified, which results in substitution of Met for Ile at amino acid 772. Under standard culture conditions in stably transfected cells, we found decreased catalytic activity of Met772. However, cells cultured in the presence of glucocorticoid expressing Met772 had a significantly increased albuterol-stimulated adenylyl cyclase response (approximately 80%) when compared with those expressing Ile772 (approximately 20%, P=0.02). An equivalent increase in beta2AR expression was observed in both lines due to glucocorticoid, but AC9 expression was unaffected. The hypothesis that Met772-AC9 is associated with an improved albuterol bronchodilator response in asthmatics was investigated in 436 asthmatic children who were followed for 4 years and randomized to receive placebo or the inhaled corticosteroid budesonide. Met772 carriers on budesonide showed a significant improvement in forced expiratory volume in 1 s (P=0.005). Moreover, a highly significant interaction (P=0.002) was found for budesonide treatment and the AC9 polymorphism. These in vitro and human association studies are consistent with this AC9 polymorphism altering albuterol responsiveness in the context of concomitant inhaled corticosteroid administration, which is a common asthma regimen. The Met772-AC9 polymorphism represents one of most likely several multi-gene polymorphisms along the receptor-relaxation axis, which together may provide for a composite pharmacogenetic index for asthma therapy.

Calmodulin Is Required for Vasopressin-stimulated Increase in Cyclic AMP Production in Inner Medullary Collecting Duct

Calmodulin plays a critical role in regulation of renal collecting duct water permeability by vasopressin. However, specific targets for calmodulin action have not been thoroughly addressed. In the present study, we investigated whether Ca2+/calmodulin regulates adenylyl cyclase activity in the renal inner medullary collecting duct. Rat inner medullary collecting duct suspensions were incubated in the presence or absence of 0.1 nM vasopressin and the calmodulin inhibitors, monodansylcadaverine, W-7, and trifluoperazine, followed by measurement of cAMP. Vasopressin-stimulated cAMP elevation was significantly attenuated in the presence of calmodulin inhibitors. Analysis of transglutaminase 2 knock-out mice confirmed that these compounds were not acting through inhibition of transglutaminase 2 activity. Calmodulin inhibitors also blocked both cholera toxin- and forskolin-stimulated cAMP accumulation. In isolated perfused tubules, W-7 reversibly blocked vasopressin-stimulated urea permeability, a process that requires a rise in intracellular cAMP but does not appear to involve protein trafficking to the apical plasma membrane. These results suggest that calmodulin is required for vasopressin-stimulated adenylyl cyclase activity in the intact inner medullary collecting duct. Reverse transcription-PCR, immunoblotting, and immunohistochemistry revealed the presence of the calmodulin-sensitive adenylyl cyclase type 3 in the rat collecting duct, an isoform previously not known to be expressed in the collecting duct. Long-term treatment of Brattleboro rats with a vasopressin analog markedly decreased adenylyl cyclase type 3 protein abundance, providing an explanation for long-term down-regulation of vasopressin response in the collecting duct. These studies demonstrate the importance of calmodulin in the regulation of collecting duct adenylyl cyclase activity and transport function.

Conditional ablation of mature olfactory sensory neurons mediated by diphtheria toxin receptor

The vertebrate olfactory epithelium provides an excellent model system to study the regulatory mechanisms of neurogenesis and neuronal differentiation due to its unique ability to generate new sensory neurons throughout life. The replacement of olfactory sensory neurons is stimulated when damage occurs in the olfactory epithelium. In this study, transgenic mice, with a transgene containing human diphtheria toxin receptor under the control of the olfactory marker protein promoter (OMP-DTR), were generated in which the mature olfactory sensory neurons could be specifically ablated when exposed to diphtheria toxin. Following diphtheria toxin induced neuronal ablation, we observed increased numbers of newly generated growth associated protein 43 (GAP43)-positive immature olfactory sensory neurons. OMP-positive neurons were continuously produced from the newly generated GAP43-positive cells. The expression of the signal transduction components adenylyl cyclase type III and the G-protein alpha subunit G(alpha olf) was sensitive to diphtheria toxin exposure and their levels decreased dramatically preceding the disappearance of the OMP-positive sensory neurons. These data validate the hypothesis that OMP-DTR mice can be used as a tool to ablate the mature olfactory sensory neurons in a controlled fashion and to study the regulatory mechanisms of the neuronal replacement.

Regulation of multiple dopamine signal transduction molecules by retinoids in the developing striatum

Increasing evidence based on pharmacological and genetic studies suggests that retinoid signaling plays an important role in developmental control of striatal neurons. In the present report, we screened for genes that might be regulated by retinoids in the developing striatum. We cultured tissue explants from the lateral ganglionic eminence (striatal primordium), and for regional comparison, its adjacent structures of the cerebral cortex and the medial ganglionic eminence in embryonic day 15 rat telencephalon. Using the ribonuclease protection assay, we found that both all- trans retinoic acid and 9- cis retinoic acid significantly up-regulated dopamine D1 receptor, heterotrimeric G protein olfactory, adenylyl cyclase type V and dopamine- and cyclic adenosine 3′:5′-monophosphate-regulated phosphoprotein mRNAs in the lateral ganglionic eminence culture. By contrast, neither all- trans retinoic acid nor 9- cis retinoic acid significantly altered D1 receptor, heterotrimeric G protein olfactory, adenylyl cyclase type V and dopamine- and cyclic adenosine 3′:5′-monophosphate-regulated phosphoprotein mRNAs in the cortical and the medial ganglionic eminence cultures except that D1 receptor mRNA was dramatically induced in the medial ganglionic eminence by retinoic acid treatments. To test whether the induction of multiple dopamine signaling molecules in the lateral ganglionic eminence was due to a general enhancement of neuronal differentiation by retinoic acid, we assayed the effects of retinoic acid on other differentiation markers, including glutamate decarboxylase 65, NR1 subunit of glutamate NMDA receptor and microtubule-associated protein-2. None of these genes were significantly altered by retinoic acid treatments in the lateral ganglionic eminence culture, indicating the specificity of gene regulation by retinoic acid signaling. As D1 receptor, heterotrimeric G protein olfactory, adenylyl cyclase type V and dopamine- and cyclic adenosine 3′:5′-monophosphate-regulated phosphoprotein are important molecules involved in propagation of striatal dopamine neurotransmission, our study raises the hypothesis that retinoid signaling may coordinately activate the transcriptional program that is associated with the dopamine signaling pathway in developing striatal neurons. Such coordinate regulation by retinoids may be part of the mechanisms by which the complex yet highly organized neurochemical constituents of the striatum are established during development.

Ultra-low-dose naloxone suppresses opioid tolerance, dependence and associated changes in mu opioid receptor–G protein coupling and Gβγ signaling

Opiates produce analgesia by activating μ opioid receptor-linked inhibitory G protein signaling cascades and related ion channel interactions that suppress cellular activities by hyperpolarization. After chronic opiate exposure, an excitatory effect emerges contributing to analgesic tolerance and opioid-induced hyperalgesia. Ultra-low-dose opioid antagonist co-treatment blocks the excitatory effects of opiates in vitro, as well as opioid analgesic tolerance and dependence, as was demonstrated here with ultra-low-dose naloxone combined with morphine. While the molecular mechanism for the excitatory effects of opiates is unclear, a switch in the G protein coupling profile of the μ opioid receptor and adenylyl cyclase activation by Gβγ have both been suggested. Using CNS regions from rats chronically treated with vehicle, morphine, morphine+ultra-low-dose naloxone or ultra-low-dose naloxone alone, we examined whether altered μ opioid receptor coupling to G proteins or adenylyl cyclase activation by Gβγ occurs after chronic opioid treatment. In morphine-naïve rats, μ opioid receptors coupled to Go in striatum and to both Gi and Go in periaqueductal gray and spinal cord. Although chronic morphine decreased Gi/o coupling by μ opioid receptors, a pronounced coupling to Gs emerged coincident with a Gβγ interaction with adenylyl cyclase types II and IV. Co-treatment with ultra-low-dose naloxone attenuated both the chronic morphine-induced Gs coupling and the Gβγ signaling to adenylyl cyclase, while increasing Gi/o coupling toward or beyond vehicle control levels. These findings provide a molecular mechanism underpinning opioid tolerance and dependence and their attenuation by ultra-low-dose opioid antagonists.

Inhibition and Superactivation of the Calcium-Stimulated Isoforms of Adenylyl Cyclase: Role of G<SUB>βγ</SUB> Dimers

It was shown previously that chronic exposure to opiate agonists increases adenylyl cyclase (AC) activity, a phenomenon termed AC superactivation (or supersensitization). More recently, we showed that acute Gi/o- coupled receptor activation inhibits the activity of several AC isozymes, including Ca2+/calmodulin-stimulated AC-I and -VIII, whereas chronic receptor activation induces their superactivation. Here, we report that both acute Mu-opioid receptor-induced inhibition and chronic induced superactivation of AC-I and -VIII are pertussis toxin sensitive. In addition, we show that proteins that interfere with the activity of Gbetagamma subunits (Gbetagamma scavengers) strongly attenuate the acute inhibition of AC-I and -VIII and the superactivation of AC-I, and abolish the superactivation of AC-VIII. Based on these results, we suggest that Gbetagamma is involved in the acute inhibition and chronic agonist-induced superactivation of AC types I and VIII.

Protein kinase C and epidermal growth factor stimulation of Raf1 potentiates adenylyl cyclase type 6 activation in intact cells.

Adenylyl cyclase type 6 (AC6) activity is inhibited by protein kinase C (PKC) in vitro; however, in intact cells, PKC activation does not inhibit the activity of transiently expressed AC6. To investigate the effects of PKC activation on AC6 activity in intact cells, we constructed human embryonic kidney (HEK) 293 cells that stably express wild-type AC6 (AC6-WT) or an AC6 mutant lacking a PKC and cyclic AMP-dependent protein kinase (PKA) phosphorylation site, Ser674 (AC6-S674A). In contrast to in vitro observations, we observed a PKC-mediated enhancement of forskolin- and isoproterenol-stimulated cyclic AMP accumulation in HEK-AC6 cells. Phorbol 12-myristate 13-acetate also potentiated cyclic AMP accumulation in cells expressing endogenous AC6, including Chinese hamster ovary cells and differentiated Cath.a differentiated cells. In HEK-AC6-S674A cells, the potentiation of AC6 stimulation was significantly greater than in cells expressing AC6-WT. The positive effect of PKC activation on AC6 activity seemed to involve Raf1 kinase because the Raf1 inhibitor 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) inhibited the PKC potentiation of AC6 activity. Furthermore, the forskolin-stimulated activity of a recombinant AC6 in which the putative Raf1 regulatory sites have been eliminated was not potentiated by activation of PKC. The ability of Raf1 to regulate AC6 may involve a direct interaction because AC6 and a constitutively active Raf1 construct were coimmunoprecipitated. In addition, we report that epidermal growth factor receptor activation also enhances AC6 signaling in a Raf1-dependent manner. These data suggest that Raf1 potentiates drug-stimulated cyclic AMP accumulation in cells expressing AC6 after activation of multiple signaling pathways.

Nitroprusside Increases Gene Transfer Associated with Intracoronary Delivery of Adenovirus

Efficient gene transfer by vectors that can be easily delivered to target organs is desirable in clinical gene therapy. We tested the hypothesis that intracoronary infusion of the nitric oxide donor nitroprusside would increase the efficiency of adenovirus vector-mediated gene transfer to the heart. Intracoronary delivery of an adenovirus encoding murine adenylyl cyclase type VI (Ad.AC(VI)) was performed in adult pigs with and without simultaneous intracoronary infusion of nitroprusside. Animals were killed 12-14 days after Ad.AC(VI) delivery and myocardial adenylyl cyclase activity was measured. Addition of nitroprusside during intracoronary infusion of Ad.AC(VI) was associated with a 4-fold increase in cAMP-generating capacity in the left ventricle. Transgene expression was confirmed by immunoblotting. Intracoronary nitroprusside produced mild dose-dependent changes in blood pressure and heart rate during infusion. Intracoronary nitroprusside infusion is a safe and effective means to increase the extent of cardiac gene transfer with intracoronary delivery of adenovirus vectors.

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.

Differential expression of adenylyl cyclase subtypes in human cardiovascular system

In human myocardium, β 1-adrenoceptor stimulation achieves maximal inotropic response but less than 50% of maximal adenylyl cyclase activation, whereas the reverse is true of the β 2-adrenoceptor. Four types of adenylyl cyclase, type IV-VII, have been described in mammalian heart, but their expression and relative distribution in human heart and blood vessels is not known. We found that type IV, V, VI and VII adenylyl cyclases were all expressed in cardiomyocytes. Whereas types IV and VII RNA were more abundant in extra-cardiac than cardiac tissues, both absolute and relative expression of type VI was greatest in heart, and lower in tissues lacking a β 1-adrenoceptor. Type V expression was virtually confined to atrium. In situ mRNA hybridisation showed that the β 1-adrenoceptor co-localised with type VI adenylyl cyclase but not other subtypes in juxtoglomerular cells of human kidney. The tissue specific expression of these adenylyl cyclase subtypes may favour its coupling to corresponding receptors expressed in the given tissue type.

Adenylyl cyclase type VI corrects cardiac sarcoplasmic reticulum calcium uptake defects in cardiomyopathy.

Calcium malfunction plays a central role in heart failure. Here, we provide evidence that adenylyl cyclase type VI restores sarco(endo)plasmic reticulum 2a (SERCA2a) affinity for calcium and maximum velocity of cardiac calcium uptake by sarcoplasmic reticulum in murine dilated cardiomyopathy. Restoration of normal SERCA2a affinity for calcium is associated not only with decreased phospholamban protein expression but also with increased phospholamban phosphorylation by PKA activation. The ratio of phosphorylated ryanodine receptor 2 (RyR2) to RyR2 protein was increased, but the amount of phosphorylated RyR2 was unaffected. These data provide a possible mechanism by which adenylyl cyclase type VI (in contrast to other signaling elements associated with increased cAMP generation) has a salutary effect in the failing heart.

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 …

Intracoronary adenovirus encoding adenylyl cyclase VI increases left ventricular function in heart failure.

We tested the hypothesis that intracoronary delivery of an adenovirus encoding adenylyl cyclase type VI (Ad.AC(VI)) would be associated with increased left ventricular (LV) function in pigs with congestive heart failure. Pigs (52+/-6 kg; n=16) underwent placement of pacemakers, LV pressure transducers, and left atrial and aortic catheters. Physiological and echocardiographic studies were obtained from conscious animals 13 days later, and pacing was initiated (220 bpm). Seven days later, measures of LV function were reduced, documenting severe LV dysfunction and dilation. Pigs then received intracoronary Ad.AC(VI) (1.4x10(12) vp; n=7) or saline (PBS; n=9) (randomized, blinded), with concomitant infusion of nitroprusside (50 microg/min, 6.4 minutes) to increase gene transfer. Pacing was continued for 14 days, and final studies were obtained. The a priori key end point was change in LV dP/dt during isoproterenol infusion (pre-Ad.AC(VI) value minus value after 21 days of pacing). Pigs receiving Ad.AC(VI) showed a smaller decrease in both LV +dP/dt (P=0.0014) and LV -dP/dt (P=0.0008). Serial echocardiography showed that Ad.AC(VI) treatment was associated with increased LV function and reduced LV dilation and that end-systolic wall stress was reduced. AC-stimulated cAMP production was increased 1.7-fold in LV samples from Ad.AC(VI)-treated pigs (P=0.006), and B-type natriuretic peptide was reduced (0.035). Gene transfer was confirmed by polymerase chain reaction. AC(VI) gene transfer increases LV function and attenuates deleterious LV remodeling in congestive heart failure.

Nitric Oxide Inhibition of Adenylyl Cyclase Type 6 Activity Is Dependent upon Lipid Rafts and Caveolin Signaling Complexes

Several cell types, including cardiac myocytes and vascular endothelial cells, produce nitric oxide (NO) via both constitutive and inducible isoforms of NO synthase. NO attenuates cardiac contractility and contributes to contractile dysfunction in heart failure, although the precise molecular mechanisms for these effects are poorly defined. Adenylyl cyclase (AC) isoforms type 5 and 6, which are preferentially expressed in cardiac myocytes, may be inhibited via a direct nitrosylation by NO. Because endothelial NO synthase (eNOS and NOS3), beta-adrenergic (betaAR) receptors, and AC6 all can localize in lipid raft/caveolin-rich microdomains, we sought to understand the role of lipid rafts in organizing components of betaAR-G(s)-AC signal transduction together with eNOS. Using neonatal rat cardiac myocytes, we found that disruption of lipid rafts with beta-cyclodextrin inhibited forskolin-stimulated AC activity and cAMP production, eliminated caveolin-3-eNOS interaction, and increased NO production. betaAR- and G(s)-mediated activation of AC activity were inhibited by beta-cyclodextrin treatment, but prostanoid receptor-stimulated AC activity, which appears to occur outside caveolin-rich microdomains, was unaffected unless eNOS was overexpressed and lipid rafts were disrupted. An NO donor, SNAP, inhibited basal and forskolin-stimulated cAMP production in both native cardiac myocytes and cardiac myocytes and pulmonary artery endothelial cells engineered to overexpress AC6. These effects of SNAP were independent of guanylyl cyclase activity and were mimicked by overexpression of eNOS. The juxtaposition of eNOS with betaAR and AC types 5 and 6 results in selective regulation of betaAR by eNOS activity in lipid raft domains over other G(s)-coupled receptors localized in nonraft domains. Thus co-localization of multiple signaling components in lipid rafts provides key spatial regulation of AC activity.

Indirect intracoronary delivery of adenovirus encoding adenylyl cyclase increases left ventricular contractile function in mice.

We performed indirect intracoronary delivery of adenovirus vectors in mice and explored techniques including hypothermia and pharmacological means to increase cardiac gene transfer. Mice were maintained in a normothermic state or cooled to 25 degrees C. The aorta or both the pulmonary artery and aorta were clamped while a needle was advanced into the left ventricular cavity to deliver adenovirus vectors encoding enhanced green fluorescent protein (EGFP) or murine adenylyl cyclase type VI (AC(VI)) with saline, sodium nitroprusside, acetylcholine, or serotonin. Clamping was maintained for 30 s (normothermia) or 2 min (25 degrees C) after adenovirus administration. Mice were killed 7 or 21 days later, and hearts were examined for EGFP expression. Compared with clamping the aorta alone and with no cooling, gene transfer was increased as follows: 1) 1.3-fold with hypothermia to extend dwell time; 2) 4.5-fold by clamping the aorta and the pulmonary artery; 3) 11.4-fold with nitroprusside administration; 4) 11.8-fold with serotonin addition, and 5) 14.3-fold with acetylcholine delivery. Gene expression remained substantial at 21 days, and no significant inflammatory response was seen. Efficacy of the method was tested by performing gene transfer of adenovirus encoding AC(VI). Fourteen days after gene transfer, hearts isolated from mice that received adenovirus encoding AC(VI) showed increased contractile function. Indirect intracoronary delivery of adenovirus vectors in mice is associated with efficient cardiac gene transfer and increased left ventricular function after AC(VI) gene transfer.

A common polymorphism in the adenylyl cyclase type 9 (AC9) gene does not alter vascular responses to isoproterenol

Adenylyl cyclase (AC) is one of the downstream effectors of β2-adrenergic receptor (BAR2) stimulation. A common functionally significant polymorphism in the AC9 gene, an A to G substitution at nucleotide 2312, resulting in an Ile to Met at codon 772, has been shown to decrease basal and BAR2-mediated AC activity and blunt catalytic function in vitro (Small et al., 2003). We hypothesized that the Met772 variant would impair sensitivity to an agonist acting through AC. Venodilation in response to increasing doses of isoproterenol (Iso) (1 - 480 ng/min) infused into a phenylephrine-preconstricted dorsal hand vein was measured using a linear variable differential transformer. A 66-fold individual variation was observed in the ED50 for Iso (2.8 – 199.2 ng/min) in 26 healthy women (6 whites and 20 Hispanics, aged 18–42 years). The log ED50 for Iso was not significantly different between the subjects with Ile/Ile (n=17) and Ile/Met (n=8) or Met/Met (n=1) [1.26 ± 0.52 (SD) versus 1.49 ± 0.56; P = 0.31]. No differences in the Emax for Iso were observed (101.3% ± 11.8 versus 102.6% ± 13.7; P = 0.80). These findings suggest that the Met772 variant of AC9 is not a major determinant of variability in vasodilatory response to a BAR2 agonist. Clinical Pharmacology & Therapeutics (2004) 75, P70–P70; doi: 10.1016/j.clpt.2003.11.263

CAMP-elevating agents and adenylyl cyclase overexpression promote an antifibrotic phenotype in pulmonary fibroblasts

Pulmonary fibroblasts are recruited to sites of lung injury, where they are activated to produce extracellular matrix proteins and to facilitate repair. However, these cells become dysregulated in pulmonary fibrosis, producing excess collagen at sites of injury and forming fibrotic loci that impair lung function. In this study, we used WI-38 human lung fibroblasts and evaluated the ability of G protein-coupled receptor agonists to increase cAMP production and regulate cell proliferation and collagen synthesis. WI-38 cells increase cAMP in response to the beta-adrenergic agonist isoproterenol (Iso), prostaglandin E(2) (PGE(2)), certain prostanoid receptor-selective agonists (beraprost, butaprost), an adenosine receptor agonist, and the direct adenylyl cyclase (AC) activator forskolin (Fsk). Responses to Iso, PGE(2), and Fsk were studied in more detail. Each induced a dose-dependent inhibition of serum-stimulated cell proliferation (as measured by [(3)H]thymidine incorporation) and collagen synthesis (as measured by [(3)H]proline incorporation, collagenase-sensitive [(3)H]proline incorporation, or levels of procollagen type 1 C-peptide). Quantitative RT-PCR analyses indicated that elevation in cellular cAMP levels decreases expression of collagen types 1alpha(II) and 5alpha(I) and increases expression and activity of matrix metalloproteinase 2 (MMP-2). Overexpression of AC type 6 or inhibition of cyclic nucleotide phosphodiesterases also increased cellular cAMP levels and decreased cell proliferation and collagen synthesis. Thus multiple approaches that increase cAMP signaling reduce proliferation and differentiated function in human pulmonary fibroblasts. These results suggest that therapies that raise cAMP levels may prove useful in the treatment of pulmonary fibrosis.