Adenylyl Cyclase Type Research Articles

Adenylyl Cyclase type 3, a marker of primary cilia, is reduced in primary cell culture and in lumbar spinal cord in situ in G93A SOD1 mice

BackgroundThe primary cilium is a solitary organelle important in cellular signaling, that projects from the cell surface of most growth-arrested or post-mitotic cells including neurons in the central nervous system. We hypothesized that primary cilial dysfunction might play a role in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), and as a first step, report on the prevalence of primary cilial markers on cultured motor neurons from the lumbar spinal cord of embryonic wildtype (WT) and transgenic G93A SOD1 mice, and on motor neurons in situ in the lumbar spinal cord.ResultsAt 7 days in culture there is no difference in the proportion of G93A SOD1 and WT motor neurons staining for the cilial marker ACIII. However, at 21 days there is a large relative drop in the proportion of ciliated G93A SOD1 motor neurons. In situ, at 40 days there was a slight relative drop in the proportion of ciliated motor neurons in G93A SOD1 mice. At 98 days of age there was no change in motor neuron ciliation in WT mice, but there was motor neuron loss and a large reduction in the proportion of surviving motor neurons bearing a primary cilium in G93A SOD1 mice.ConclusionsIn primary culture and in situ in G93A SOD1 mice there is a large reduction in the proportion of motor neurons bearing a primary cilium.

Effects of Alcohols on Recombinant Adenylyl Cyclase Type 7 Expressed in Bacteria

Our previous studies showed that ethanol enhanced the activity of adenylyl cyclase (AC) in an isoform-specific manner and that alcohol cutoff point of AC was isoform specific. Recently, we showed that 2,3-butanediol inhibited AC type 7 (AC7) activity in a stereoisomer-specific manner and that this inhibition was also AC isoform specific. These observations strongly suggest that a major target of alcohol action on cAMP signaling is AC. We hypothesized that alcohols exhibit their effect on AC activity by direct interaction with AC proteins. However, experimental systems employed in past studies such as intact cells and membrane preparations are too complex and do not allow us to unequivocally test this hypothesis. In attempt to bypass, these complications of the membrane-bound AC, we decided to study the effect of alcohols on AC recombinant proteins expressed in bacteria. A recombinant AC, designated as AC7sol, consisting of the C(1a) and C(2) domains of the human AC7 was designed and expressed in bacteria. The activity of AC7sol was examined using lysate prepared from bacteria expressing AC7sol. The activity of AC7sol was stimulated by manganese or by the α subunit of G protein that stimulates AC (G(sα) ). Forskolin by itself did not stimulate the activity of AC7sol. However, in the presence of activated G(sα) , forskolin stimulated the activity of AC7sol. A series of n-alkanols including ethanol enhanced the manganese-stimulated activity of AC7sol. The alcohol cutoff point of AC7sol was pentanol. Ethanol and butanol increased V(max) and K(M) values of AC7sol. These results are consistent with our hypothesis and suggest that the enhancing effect of alcohols on AC activity is because of the increase in turnover number of AC. The current study demonstrates for the first time that the effect of alcohols requires only the C(1a) and C(2) domains of AC and no other domains of AC as well as no other mammalian proteins.

Phosphorylation of G Protein-coupled Receptor Kinase 1 (GRK1) Is Regulated by Light but Independent of Phototransduction in Rod Photoreceptors

Phosphorylation of rhodopsin by G protein-coupled receptor kinase 1 (GRK1, or rhodopsin kinase) is critical for the deactivation of the phototransduction cascade in vertebrate photoreceptors. Based on our previous studies in vitro, we predicted that Ser(21) in GRK1 would be phosphorylated by cAMP-dependent protein kinase (PKA) in vivo. Here, we report that dark-adapted, wild-type mice demonstrate significantly elevated levels of phosphorylated GRK1 compared with light-adapted animals. Based on comparatively slow half-times for phosphorylation and dephosphorylation, phosphorylation of GRK1 by PKA is likely to be involved in light and dark adaptation. In mice missing the gene for adenylyl cyclase type 1, levels of phosphorylated GRK1 were low in retinas from both dark- and light-adapted animals. These data are consistent with reports that cAMP levels are high in the dark and low in the light and also indicate that cAMP generated by adenylyl cyclase type 1 is required for phosphorylation of GRK1 on Ser(21). Surprisingly, dephosphorylation was induced by light in mice missing the rod transducin α-subunit. This result indicates that phototransduction does not play a direct role in the light-dependent dephosphorylation of GRK1.

Sex-Specific Role for Adenylyl Cyclase Type 7 in Alcohol Dependence

BackgroundAlcohol has been shown to critically modulate cyclic adenosine-3′,5′ monophosphate (cAMP) signaling. A number of downstream effectors that respond to the cAMP signals (e.g., protein kinase A, cAMP response element binding protein) have, in turn, been examined in relation to alcohol consumption. These studies did not, however, delineate the point at which the actions of alcohol on the cAMP cascade might translate into differences in drinking behavior. To further understand the role of cAMP synthesis in alcohol drinking and dependence, we investigated a specific adenylyl cyclase isoform, adenylyl cyclase (AC) Type 7, whose activity is selectively enhanced by ethanol.MethodsWe measured alcohol consumption and preference in mice in which one copy of the Adcy7 gene was disrupted (Adcy7+/−). To demonstrate relevance of this gene for alcohol dependence in humans, we tested the association of polymorphisms in the ADCY7 gene with alcohol dependence in a sample of 1703 alcohol-dependent individuals and 1347 control subjects.ResultsWe show that Adcy7+/− female mice have higher preference for alcohol than wild-type mice, whereas there is little difference in alcohol consumption or preference between Adcy7+/− male mice and wild-type control subjects. In the human sample, we found that single nucleotide polymorphisms in ADCY7 associate with alcohol dependence in women, and these markers are also associated with ADCY7 expression (messenger RNA) levels.ConclusionsThese findings implicate adenylyl cyclase Type 7 as a critical component of the molecular pathways contributing to alcohol drinking and the development of alcohol dependence.

Inhibition of Adenylyl Cyclase Type 5 Protects Against Obesity and Diabetes

We reported adenylyl cyclase type 5 (AC5) disruption (knock out, KO) increases food intake, but reduces weight, implying a metabolic profile consistent with protection against obesity and diabetes. We compared AC5 KO and wild type littermates (WTL) on a normal diet and examined the effects of a pharmacological inhibitor, specific for AC5, on WT mice on a high fat diet (HFD). The AC5 KO mice, despite eating more, weighed 12% less compared against WTL (P<0.05). At sacrifice, fat pads were isolated and adiposity index calculated. In the KO mice adiposity index was 34% less than WTL. The pharmacolgical inhibitor of AC5 (Vidarabine), was administered to WT mice via a subcutaneous osmotic pump (30 mg/kg/day). The specificity of this inhibitor was demonstrated by the degree of inhibition of forskolin-stimulated AC activity in cardiac myocytes as follows: AC5 overexpression (OE) > WT, AC6 OE > AC5 KO. Mice on HFD treated with the AC5 inhibitor ate similarly but gained 41% less weight (p<0.05), compared to vehicle treated mice and the adiposity index was reduced by 30% (p<0.05), with a 32% reduction in visceral fat cell size (p<0.05). The AC5 inhibitor also normalized the impaired glucose tolerance in mice on the HFD. Thus, inhibiting AC5 reduces obesity and improves glucose tolerance, suggesting a potentially novel therapeutic approach to obesity and diabetes.

Collecting Duct‐Specific Knockout of Adenylyl Cyclase Type VI Causes Urinary Concentration Defect in Mice

Adenylyl cyclase VI (AC6) has been implicated in arginine vasopressin (AVP)‐stimulated renal water reabsorption. To evaluate the AC6 role we generated mice with collecting duct (CD)‐specific KO of AC6 using the Cre/lox system. KO and floxed controls were studied on normal water, chronically water loaded, and water deprived for 18 hours, ± DDAVP clamp 25ng/hr. Food, water intake, body weight, urine volume and osmolality, and inner medullary collecting duct (IMCD) AVP stimulated cAMP levels were measured. Baseline urine osmolality (Uosm) was reduced (1427±91 KO vs. 1938±189 mOsm/L con, p&lt;0.04). After 18 hr water deprivation, Uosm was also reduced (2271±160 KO vs. 2777±108 mOsm/L con, p&lt;0.02). There was no change with chronic water loading. With DDAVP infusion there was no difference in Uosm with normal conditions, or chronic water loading. With water deprivation Uosm decreased (3142±50 KO vs 3332±63 mOsm/L con p=0.032). There was no difference in urine volume or fluid intake. In acutely isolated IMCD AVP stimulated cAMP accumulation was reduced (0.09±0.015 KO vs. 0.16±0.015 pmol/μg con p=0.016). Thus, AC6 plays an important role in AVP stimulated water reabsorption and cAMP accumulation in CD.

Identification of a Specific Assembly of the G Protein Golf as a Critical and Regulated Module of Dopamine and Adenosine-Activated cAMP Pathways in the Striatum

In the principal neurons of striatum (medium spiny neurons, MSNs), cAMP pathway is primarily activated through the stimulation of dopamine D1 and adenosine A2A receptors, these receptors being mainly expressed in striatonigral and striatopallidal MSNs, respectively. Since cAMP signaling pathway could be altered in various physiological and pathological circumstances, including drug addiction and Parkinson’s disease, it is of crucial importance to identify the molecular components involved in the activation of this pathway. In MSNs, cAMP pathway activation is not dependent on the classical Gs GTP-binding protein but requires a specific G protein subunit heterotrimer containing Gαolf/β2/γ7 in particular association with adenylyl cyclase type 5. This assembly forms an authentic functional signaling unit since loss of one of its members leads to defects of cAMP pathway activation in response to D1 or A2A receptor stimulation, inducing dramatic impairments of behavioral responses dependent on these receptors. Interestingly, D1 receptor (D1R)-dependent cAMP signaling is modulated by the neuronal levels of Gαolf, indicating that Gαolf represents the rate-limiting step in this signaling cascade and could constitute a critical element for regulation of D1R responses. In both Parkinsonian patients and several animal models of Parkinson’s disease, the lesion of dopamine neurons produces a prolonged elevation of Gαolf levels. This observation gives an explanation for the cAMP pathway hypersensitivity to D1R stimulation, occurring despite an unaltered D1R density. In conclusion, alterations in the highly specialized assembly of Gαolf/β2/γ7 subunits can happen in pathological conditions, such as Parkinson’s disease, and it could have important functional consequences in relation to changes in D1R signaling in the striatum.

Mice lacking adenylyl cyclase type 5 (AC5) show increased ethanol consumption and reduced ethanol sensitivity

The adenylyl cyclase (AC)/cAMP system is believed to be a key component in regulating alcohol-drinking behavior. It was reported that adenylyl cyclase-5 (AC5) is expressed widely in the brain, with a preferential concentration in the dorsal striatum and nucleus accumbens, brain regions which are important for addiction and emotion. AC5 has been shown to be an essential mediator of morphine addiction and dopamine receptor function; however, it remains unknown whether or not AC5 plays a role in ethanol preference and sensitivity in animals. This work was carried out to determine the role of AC5 in alcohol consumption and the hypnotic response to alcohol using AC5 knockout (KO) mice. In the test for ethanol preference employing a two-bottle free-choice paradigm, AC5 KO mice showed increased ethanol consumption and preference compared with the wild-type mice. Ethanol-induced hypothermia was weakly reduced in AC5 KO mice. AC5 KO mice exhibited sedation/behavioral sleep to high-dose ethanol, but their responses were greatly suppressed compared with the wild-type mice. These results suggest that AC5 is an important signaling molecule regulating alcohol sensitivity and preference in animals. These data provide critical information for AC5 activation as a candidate target for the treatment of alcoholism.

Beneficial Effects of Adenylyl Cyclase Type 6 (AC6) Expression Persist Using a Catalytically Inactive AC6 Mutant

Cardiac-directed expression of AC6 has pronounced favorable effects on cardiac function possibly not linked with cAMP production. To determine rigorously whether cAMP generation is required for the beneficial effects of increased AC6 expression, we generated a catalytically inactive AC6 mutant (AC6mut) that has markedly diminished cAMP generating capacity by replacing aspartic acid with alanine at position 426 in the C1 domain (catalytic region) of AC6. Gene transfer of AC6 or AC6mut (adenovirus-mediated) in adult rat cardiac myocytes resulted in similar expression levels and intracellular distribution, but AC6mut expression was associated with marked reduction in cAMP production. Despite marked reduction in cAMP generation, AC6mut influenced intracellular signaling events similarly to that observed after expression of catalytically intact AC6. For example, both AC6 and AC6mut reduced phenylephrine-induced cardiac myocyte hypertrophy and apoptosis (p < 0.001), expression of cardiac ankyrin repeat protein (p < 0.01), and phospholamban (p < 0.05). AC6mut expression, similar to its catalytically intact cohort, was associated with increased Ca2+ transients in cardiac myocytes after isoproterenol stimulation. Many of the biological effects of AC6 expression are replicated by a catalytically inactive AC6 mutant, indicating that the mechanisms for these effects do not require increased cAMP generation.

Combined pharmacogenetic effect of ADCY9 and ADRB2 gene polymorphisms on the bronchodilator response to inhaled combination therapy

Inhaled combination therapy composing of long-acting β2-agonist and corticosteroid has been widely applied in the management of asthma, but observed treatment responses vary. The aim of this study was to evaluate the pharmacogenetic effect of the adenylyl cyclase type 9 (ADCY9) gene polymorphism on combination therapy. Eighty-six mild to moderate Korean asthmatics were enrolled in this clinical trial. After the 2-week 'run-in' period, patients received budesonide (an inhaled corticosteroid) and formoterol (long-acting β2-agonist) during the following 12-week active treatment period. Forced expiratory volume in 1 s (FEV(1) ) and maximum mid-expiratory flow (MMEF) levels were measured at all visits as primary outcome. ADCY9 (Ile772Met, 150127 C/T, 150130 C/T, 150397 C/T, 150479 C/T, TTTA (5/4) ) and β2-adrenergic receptor (ADRB2, Arg16Gly) gene polymorphisms were genotyped. Significant associations were observed between the ADCY9 single nucleotide polymorphisms and percent changes in FEV(1) (Ile772Met T/C, P = 0·030) and MMEF (150397 C/T, P = 0·016) after 8 weeks of combination therapy. Haplotype associations were also observed with respect to percent changes in FEV(1) after 8 weeks of therapy (Ht3[TTCC], P = 0·017). Additive therapeutic effect was observed in those with the ADCY9 Ile772Met and ADRB2 Arg16Gly gene polymorphisms in terms of percent change in FEV(1) after 8 and 12 weeks of therapy (P = 0·002 and P = 0·027 respectively). Our results suggest that ADCY9 gene polymorphisms may alone, and in combination with ADRB2 gene polymorphisms, contribute to individual response to combination therapy in mild to moderate asthmatics.

L-DOPA-induced dyskinesia in hemiparkinsonian rats is associated with up-regulation of adenylyl cyclase type V/VI and increased GABA release in the substantia nigra reticulata

l-DOPA treatment induces abnormal involuntary movements (AIMs) in Parkinson's patients and experimental animals. We examined the relationship between the development of AIMs (dyskinesia) and changes in [ 3H]-GABA release and cAMP signaling in striatonigral terminals of rats with unilateral 6-OHDA lesions. Analysis of AIMs scores in hemiparkinsonian rats treated with l-DOPA for 20 days was fitted by the sum of two Gaussian distributions showing the presence of two populations: one with mild and the other with severe dyskinesia. cAMP signaling was evaluated in the two populations by determining changes in cAMP formation, Gα olf and adenylyl cyclase type V/VI levels. In animals that were not treated with l-DOPA, all the parameters were significantly increased in the denervated side. In the animals that had mild dyskinesia, l-DOPA treatment normalized these parameters. In contrast, in the animals in which l-DOPA treatment induced severe dyskinesia all the parameters, except for Gα olf levels, were significantly higher in the denervated side. Similarly, D1-stimulated [ 3H]-GABA release was not elevated in l-DOPA-treated animals with mild dyskinesia but was increased in animals with severe dyskinesia. Changes in Gα olf and adenylyl cyclase type V/VI levels in the striatum paralleled the response in the SNr. The linkage between the changes in [ 3H]-GABA release and cAMP activity was further evaluated with the selective adenylyl cyclase V/VI antagonist NKY80. This inhibitor blocked the increases of both [ 3H]-GABA release and cAMP production. These results indicate that increased expression of adenylyl cyclase V/VI is a major determinant of increased GABAergic transmission in the substantia nigra pars reticulata of animals in which l-DOPA induces severe dyskinesia.

Disruption of adenylyl cyclase type V does not rescue the phenotype of cardiac-specific overexpression of Gαqprotein-induced cardiomyopathy

Adenylyl cyclase (AC) is the principal effector molecule in the β-adrenergic receptor pathway. AC(V) and AC(VI) are the two predominant isoforms in mammalian cardiac myocytes. The disparate roles among AC isoforms in cardiac hypertrophy and progression to heart failure have been under intense investigation. Specifically, the salutary effects resulting from the disruption of AC(V) have been established in multiple models of cardiomyopathy. It has been proposed that a continual activation of AC(V) through elevated levels of protein kinase C could play an integral role in mediating a hypertrophic response leading to progressive heart failure. Elevated protein kinase C is a common finding in heart failure and was demonstrated in murine cardiomyopathy from cardiac-specific overexpression of G(αq) protein. Here we assessed whether the disruption of AC(V) expression can improve cardiac function, limit electrophysiological remodeling, or improve survival in the G(αq) mouse model of heart failure. We directly tested the effects of gene-targeted disruption of AC(V) in transgenic mice with cardiac-specific overexpression of G(αq) protein using multiple techniques to assess the survival, cardiac function, as well as structural and electrical remodeling. Surprisingly, in contrast to other models of cardiomyopathy, AC(V) disruption did not improve survival or cardiac function, limit cardiac chamber dilation, halt hypertrophy, or prevent electrical remodeling in G(αq) transgenic mice. In conclusion, unlike other established models of cardiomyopathy, disrupting AC(V) expression in the G(αq) mouse model is insufficient to overcome several parallel pathophysiological processes leading to progressive heart failure.

Modulation of β-adrenergic receptor signaling in heart failure and longevity: targeting adenylyl cyclase type 5

Despite remarkable advances in therapy, heart failure remains a leading cause of morbidity and mortality. Although enhanced beta-adrenergic receptor stimulation is part of normal physiologic adaptation to either the increase in physiologic demand or decrease in cardiac function, chronic beta-adrenergic stimulation has been associated with increased mortality and morbidity in both animal models and humans. For example, overexpression of cardiac Gsalpha or beta-adrenergic receptors in transgenic mice results in enhanced cardiac function in young animals, but with prolonged overstimulation of this pathway, cardiomyopathy develops in these mice as they age. Similarly, chronic sympathomimetic amine therapy increases morbidity and mortality in patients with heart failure. Conversely, the use of beta-blockade has proven to be of benefit and is currently part of the standard of care for heart failure. It is conceivable that interrupting distal mechanisms in the beta-adrenergic receptor-G protein-adenylyl cyclase pathway may also provide targets for future therapeutic modalities for heart failure. Interestingly, there are two major isoforms of adenylyl cyclase (AC) in the heart (type 5 and type 6), which may exert opposite effects on the heart, i.e., cardiac overexpression of AC6 appears to be protective, whereas disruption of type 5 AC prolongs longevity and protects against cardiac stress. The goal of this review is to summarize the paradigm shift in the treatment of heart failure over the past 50 years from administering sympathomimetic amine agonists to administering beta-adrenergic receptor antagonists, and to explore the basis for a novel therapy of inhibiting type 5 AC.

Effects of cardiac overexpression of type 6 adenylyl cyclase affects on the response to chronic pressure overload.

Adenylyl cyclase (AC) type 5 (AC5) and AC type 6 (AC6) are the two major AC isoforms in the heart. Cardiac overexpression of AC6 has been shown to be protective in response to several interventions. In this investigation, we examined the effects of chronic pressure overload in AC6 transgenic (TG) mice. In the absence of any stress, AC6 TG mice exhibited enhanced contractile function compared with their wild-type (WT) littermates, i.e., increased (P < 0.05) left ventricular (LV) ejection fraction (EF) (75 +/- 0.9 vs. 71 +/- 0.5%) and LV dP/dt (7,850 +/- 526 vs. 6,374 +/- 315 mmHg/s). Forskolin (25 microg x kg(-1) x min(-1) for 5 min) increased LVEF more (P < 0.05) in AC6 TG mice (14.8 +/- 1.0%) than in WT mice (7.7 +/- 1.0%). Also, isoproterenol (0.04 microg x kg(-1) x min(-1) for 5 min) increased LVEF more (P < 0.05) in AC6 TG mice (18.0 +/- 1.2%) than in WT mice (11.6 +/- 2.1%). Pressure overload, induced by 4 wk of transverse aortic constriction (TAC), increased the LV weight-to-body weight ratio and myocyte cross-sectional area similarly in both groups, but reduced LVEF more in AC6 TG mice (22%) compared with WT mice (9%), despite the higher starting level of LVEF in AC6 TG mice. LV systolic wall stress increased more in AC6 TG mice than in WT mice, which could be responsible for the reduced LVEF in AC6 TG mice with chronic pressure overload. In addition, LV dP/dt was no longer elevated in AC6 TG mice after TAC compared with WT mice. LV end-diastolic diameter was also greater (P < 0.05) in AC6 TG mice (3.8 +/- 0.07 mm) than in WT mice (3.6 +/- 0.05 mm) after TAC. Thus, in contrast to other interventions previously reported to be salutary with cardiac AC6 overpression, the response to chronic pressure overload was not; actually, AC6 TG mice fared worse than WT mice. The mechanism may be due to the increased LV systolic wall stress in AC6 TG mice with chronic pressure overload.

Impaired water reabsorption in mice deficient in the type VI adenylyl cyclase (AC6)

Adenylyl cyclase (AC) type VI (AC6) is a calcium-inhibitable enzyme which produces cAMP upon stimulation. Herein, we characterized the specific role of AC6 in the kidneys using two AC6-knockout mouse lines. Immunohistochemical staining revealed that AC6 exists in the tubular parts of the nephron and collecting duct. Activities of AC evoked by forskolin or a selective agonist of the V2 vasopressin receptor were lower in the kidneys of AC6-null mice compared to those of wildtype mice. Results of a metabolic cage assay and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) showed for the first time that AC6 plays a critical role in regulating water homeostasis.

Implication of adenylyl cyclase 8 in pathological smooth muscle cell migration occurring in rat and human vascular remodelling

Recently, we discovered on primary cell cultures that adenylyl cyclase type 8 (AC8) was involved in the transition of rat vascular smooth muscle cells (VSMCs) to an inflammatory phenotype. Here we demonstrate, in human vessels displaying early or advanced atherosclerotic lesions, that: (a) only intimal VSMCs strongly express AC8; and (b) very few AC8-positive VSMCs were detected in the medial layer, either in atherosclerotic or healthy arteries. Furthermore, over-expressing AC8 in primary rat VSMC cultures triggered the recolonization of a wounded zone and similar results were obtained in the presence of mitomycin, a potent inhibitor of proliferation. This phenomenon was prevented by silencing AC8. Indeed, in IL-1 beta-treated cells, AC8 silencing halted migration and decreased the matrix-metalloproteinases 2/9 secretion, known to be involved in VSMC migration. In vivo, we showed: (a) a pronounced up-regulation of AC8 expression in highly migrating VSMCs of the injured rat carotid artery; (b) an undetectable AC8 labelling in re-endothelized vessels where neo-intimal thickening had stopped. From our data, we conclude that AC8 expression appears closely linked to the properties developed by VSMCs in atherosclerosis and post-angioplasty neo-intima formation leading to restenosis. In addition, it reinforces the idea that VSMC responses to their cell environment greatly depend on the AC isoforms expressed and attributes a new role for AC8 in these pathological vascular processes.

Mutations in AKAP5 Disrupt Dendritic Signaling Complexes and Lead to Electrophysiological and Behavioral Phenotypes in Mice

AKAP5 (also referred to as AKAP150 in rodents and AKAP79 in humans) is a scaffolding protein that is highly expressed in neurons and targets a variety of signaling molecules to dendritic membranes. AKAP5 interacts with PKA holoenzymes containing RIIα or RIIβ as well as calcineurin (PP2B), PKC, calmodulin, adenylyl cyclase type V/VI, L-type calcium channels, and β-adrenergic receptors. AKAP5 has also been shown to interact with members of the MAGUK family of PSD-scaffolding proteins including PSD95 and SAP97 and target signaling molecules to receptors and ion channels in the postsynaptic density (PSD). We created two lines of AKAP5 mutant mice: a knockout of AKAP5 (KO) and a mutant that lacks the PKA binding domain of AKAP5 (D36). We find that PKA is delocalized in both the hippocampus and striatum of KO and D36 mice indicating that other neural AKAPs cannot compensate for the loss of PKA binding to AKAP5. In AKAP5 mutant mice, a significant fraction of PKA becomes localized to dendritic shafts and this correlates with increased binding to microtubule associated protein-2 (MAP2). Electrophysiological and behavioral analysis demonstrated more severe deficits in both synaptic plasticity and operant learning in the D36 mice compared with the complete KO animals. Our results indicate that the targeting of calcineurin or other binding partners of AKAP5 in the absence of the balancing kinase, PKA, leads to a disruption of synaptic plasticity and results in learning and memory defects.

Heterologous sensitization of human AC5: dependence on heterotrimeric G proteins

We investigated the role of heterotrimeric G proteins in heterologous sensitization of adenylyl cyclase type 5 (AC5). Although acute stimulation of Gαi‐coupled receptors inhibits AC5 activity, persistent receptor activation enhances AC5 activity. This phenomenon is known as heterologous sensitization of AC, and has been implicated in drug dependence.Previous work revealed an absolute requirement for Gαs in heterologous sensitization of AC5. To further elucidate the mechanism, we assessed sensitization in cells devoid of endogenous Gαs, co‐expressing the D2L receptor, AC5, and Gαs mutants. Our data showed that AC5‐Gαs, but not Gβγ‐Gαs interactions or palmitoylation of Gαs, were required for heterologous sensitization of AC5.Next, we explored the role of Gβγ‐AC5 interactions in heterologous sensitization. Sequestering Gβγ with βARK‐ct partially attenuated heterologous sensitization. Deletion of a novel N‐terminal Gβγ interacting domain of AC5 abolishes Gβγ binding, but does not greatly alter other forms of acute regulation. However, the AC5 deletion mutant was selectively impaired in heterologous sensitization. Together, these studies support a complex model of heterologous sensitization of AC5 where Gαs is directly interacting with AC5. In contrast, Gβγ appears to have multiple and indirect roles in heterologous sensitization of AC5.Supported by P.U. and grants VJW‐MH060397 and CWD‐GM060419.

Microarray Analysis Provides a Link between Adenylyl Cyclase Type 5 and Pressure Overload Hypertrophy

Adenylyl cyclase type 5 and 6 (AC5 and AC6) are the major cardiac isoforms, but may affect cardiac pathophysiology differently. To investigate genes regulated by AC5 and AC6 regarding hypertrophy, we performed microarray‐based gene expression analysis of transgenic mice (TG) with cardiac specific overexpression of AC5 or AC6, and compared them with wild type with transverse aortic constriction (WT‐TAC). We detected a positive correlation of global gene expression profiles between AC5 TG and WT‐TAC, but not between AC6 TG and WT‐TAC. Baseline measures of AC5 high‐overexpression resulted in increased LV/BW (3.5±0.2) compared with WT (3.1±0.1) and AC6 TG (3.2±0.0). We found that commonly up‐regulated genes in both AC5 TG and WT‐TAC, but down‐regulated in AC6 TG were significantly, p&lt;0.001, associated with extracellular matrix (ECM) organization and biogenesis. In addition hypertrophy related genes were up‐regulated in both AC5 TG and WT‐TAC, but not in AC6 TG. We confirmed those results by quantitative real‐time PCR. In summary, AC5 TG mice, even at baseline, have a similar gene expression profile to WT mice with TAC, which could explain the predisposition of AC5 TG to pressure overload hypertrophy. The microarray data also suggest that ECM related genes, which are regulated differently in AC5 and AC6 may be responsible for the adverse response of LV remodeling after pressure overload in AC5.

Effect of alcohols on a soluble form of adenylyl cyclase type 7 expressed in bacteria

Cyclic AMP (cAMP) signal transduction has been postulated to play an important role in the development of alcoholism in humans. Our previous studies showed that generation of cAMP by mammalian membrane‐bound adenylyl cyclase (AC) is enhanced in an isoform specific manner and that alcohol‐cutoff phenomena are also isoform‐specific. We hypothesized that alcohols exhibit their effect on AC activity by direct interaction with AC proteins. However, experimental systems employed in the past such as intact cells and membrane preparations are too complex and do not allow us to unequivocally test this hypothesis. We decided to study the effect of alcohols on AC recombinant proteins expressed in bacteria. A type 7 AC (AC7) recombinant protein consisting of the C1a and C2 domains was designed and expressed in E. coli. The AC activity of the soluble form of AC7 recombinant protein was significantly increased by manganese and forskolin. In addition, n‐alkanols (ethanol to hexanol) significantly enhanced manganese‐stimulated activity of the AC7 recombinant protein. The alcohol cutoff for this recombinant protein is pentanol, which is same as that of wild type observed in mammalian system. These results suggest that the AC7 recombinant protein retains alcohol response similar to that of the parent enzyme. The results are consistent with our working hypothesis. Supported by NIH Grant AA13148 (MY).