Adenylyl Cyclase Type Research Articles

Adenylyl cyclase VI is necessary for AVP stimulation of ENaC

Vasopressin is an important physiological modulator of renal sodium excretion, which is fine‐tuned in the collecting duct. This action of vasopressin is mediated in part by adenylyl cyclase‐stimulated cAMP regulation of the epithelial Na+ channel (ENaC). Multiple adenylyl cyclase isoforms are expressed in the collecting duct. The adenylyl cyclase isoform that is most important to vasopressin‐regulation of ENaC in the collecting duct is obscure. To assess this, the basal activity of ENaC and the response of this channel to desmopressin and water‐deprivation were compared in wild type mice and mice having collecting duct principal cell‐specific knockout of adenylyl cyclase type VI. Patch clamp analysis of split‐open cortical collecting ducts revealed no difference in basal ENaC activity; however, the stimulatory effects of water‐deprivation and desmopressin on ENaC were absent in knockout mice. These results demonstrate that adenylyl cyclase type VI is necessary for vasopressin stimulation of ENaC in the collecting duct but that this isoform is not necessary for basal channel activity under normal conditions.

Differential Effects of AGS3 Expression on D2L Dopamine Receptor-Mediated Adenylyl Cyclase Signaling

Activator of G protein signaling 3 (AGS3) binds Gα(i) subunits in the GDP-bound state, implicating AGS3 as an important regulator of Gα(i)-linked receptor (e.g., D2 dopamine and μ-opioid) signaling. We examined the ability of AGS3 to modulate recombinant adenylyl cyclase (AC) type 1 and 2 signaling in HEK293 cells following both acute and persistent activation of the D(2L) dopamine receptor (D(2L)DR). AGS3 expression modestly enhanced the potency of acute quinpirole-induced D(2L)DR modulation of AC1 or AC2 activity. AGS3 also promoted desensitization of D(2L)DR-mediated inhibition of AC1, whereas desensitization of D(2L)DR-mediated AC2 activation was significantly attenuated. Additionally, AGS3 reduced D(2L)DR-mediated sensitization of AC1 and AC2. These data suggest that AGS3 is involved in altering G protein signaling in a complex fashion that is effector-specific and dependent on the duration of receptor activation.

Adenosine and Dopamine Receptors Coregulate Photoreceptor Coupling via Gap Junction Phosphorylation in Mouse Retina

Gap junctions in retinal photoreceptors suppress voltage noise and facilitate input of rod signals into the cone pathway during mesopic vision. These synapses are highly plastic and regulated by light and circadian clocks. Recent studies have revealed an important role for connexin36 (Cx36) phosphorylation by protein kinase A (PKA) in regulating cell-cell coupling. Dopamine is a light-adaptive signal in the retina, causing uncoupling of photoreceptors via D4 receptors (D4R), which inhibit adenylyl cyclase (AC) and reduce PKA activity. We hypothesized that adenosine, with its extracellular levels increasing in darkness, may serve as a dark signal to coregulate photoreceptor coupling through modulation of gap junction phosphorylation. Both D4R and A2a receptor (A2aR) mRNAs were present in photoreceptors, inner nuclear layer neurons, and ganglion cells in C57BL/6 mouse retina, and showed cyclic expression with partially overlapping rhythms. Pharmacologically activating A2aR or inhibiting D4R in light-adapted daytime retina increased photoreceptor coupling. Cx36 among photoreceptor terminals, representing predominantly rod-cone gap junctions but possibly including some rod-rod and cone-cone gap junctions, was phosphorylated in a PKA-dependent manner by the same treatments. Conversely, inhibiting A2aR or activating D4R in daytime dark-adapted retina decreased Cx36 phosphorylation with similar PKA dependence. A2a-deficient mouse retina showed defective regulation of photoreceptor gap junction phosphorylation, fairly regular dopamine release, and moderately downregulated expression of D4R and AC type 1 mRNA. We conclude that adenosine and dopamine coregulate photoreceptor coupling through opposite action on the PKA pathway and Cx36 phosphorylation. In addition, loss of the A2aR hampered D4R gene expression and function.

Adenylyl cyclase VI mediates vasopressin-stimulated ENaC activity.

Vasopressin modulates sodium reabsorption in the collecting duct through adenylyl cyclase-stimulated cyclic AMP, which exists as multiple isoforms; the specific isoform involved in vasopressin-stimulated sodium transport is unknown. To assess this, we studied mice deficient in adenylyl cyclase type VI specifically in the principal cells of the collecting duct. Knockout mice had increased urine volume and reduced urine sodium concentration, but regardless of the level of sodium intake, they did not exhibit significant alterations in urinary sodium excretion, arterial pressure, or pulse rate. Plasma renin concentration was elevated in knockout mice, however, suggesting a compensatory response. Valsartan significantly reduced arterial pressure in knockout mice but not in controls. Knockout mice had decreased renal cortical mRNA content of all three epithelial sodium channel (ENaC) isoforms, and total cell sodium channel isoforms α and γ were reduced in these animals. Patch-clamp analysis of split-open cortical collecting ducts revealed no difference in baseline activity of sodium channels, but knockout mice had abolished vasopressin-stimulated ENaC open probability and apical membrane channel number. In summary, these data suggest that adenylyl cyclase VI mediates vasopressin-stimulated ENaC activity in the kidney.

Zymosan activates protein kinase A via adenylyl cyclase VII to modulate innate immune responses during inflammation

Pathogens use a variety of strategies to evade host immune defenses. A powerful way to suppress immune function is to increase intracellular concentrations of cAMP in host immune cells, which dampens inflammatory responses and prevents microbial killing. We found that the yeast cell wall extract, zymosan, is capable of increasing intracellular cAMP and activates the protein kinase A pathway in bone marrow derived macrophage (BMDM) cells from mice. This response is dependent on adenylyl cyclase type VII (AC7) and heterotrimeric G proteins, primarily G12/13. Consequently, zymosan induced production of the inflammatory cytokine, TNFα, was much stronger in BMDMs from AC7 deficient mice compared to the response in wild type cells. In a model of zymosan induced peritonitis, mice deficient of AC7 in the myeloid lineage displayed prolonged inflammation. We propose that zymosan induced increases in cAMP and activation of PKA serve as a mechanism to dampen inflammatory responses in host cells, which consequently favors the survival of microbes. This would also help explain a well documented phenomenon, that the ability of zymosan to stimulate inflammatory cytokine responses via TLR2 appears to be weaker than other stimuli of TLR2.

Abstract 19323: Disruption of Adenylyl Cyclase Type 5, a Novel Target for Obesity, Diabetes and Diabetic Cardiomyopathy

We have previously reported that an adenylyl cyclase type 5 (AC5) deficient mouse model (AC5 knock out, KO) lives one-third longer than wild type (WT) mice. Despite greater daily food intake the AC5KO mice weigh less than WT mice and are protected against cardiac stress. In addition, the AC5KO mice have the same molecular signature of caloric restriction, the most common effective intervention to increase healthy longevity.. Since caloric restriction improves metabolic profiles and protects against insulin resistance, obesity and diabetes, we placed 6-8 week old male AC5KO mice and their corresponding wild type littermates (WTL) controls on a 35.5% high fat diet (HFD) for 100 days. At sacrifice, fat deposits were reduced 36% in AC5KO and cholesterol levels were reduced from 131±9.9 mg/dl to 105±3.1 mg/dl, p<0.05. Indirect calorimetry demonstrated that the AC5KO mice had a 16.7% increase in oxygen consumption during the light cycle (p<0.01) and 29.2% increase during the dark cycle (p<0.01) compared to WTL on HFD, suggesting enhanced metabolic rate as the mechanism for decreased adiposity and body weight. AC5KO mice also displayed improved glucose tolerance with a 32% reduction in area under the curve concomitant with a 111% increase in whole body insulin sensitivity (insulin tolerance test). In parallel, conscious non-stressed euglycemic-hyperinsulinemic clamp studies confirmed the marked enhancement of glucose homeostasis and insulin sensitivity in the AC5KO mice. Moreover, WTL developed reduced cardiac function consistent with diabetic cardiomyopathy following high fat diet feeding (e.g., left ventricular (LV) ejection fraction fell from 71±2% to 53±2%) whereas AC5 KO mice had better preserved LV ejection fraction (61±3%). In addition, AC5 KO mice showed 33% decrease in cardiac fibrosis and 40% decrease in apoptosis compared to WTL on HFD. Thus, reduction of AC5 protects against obesity, diabetes and diabetic cardiomyopathy, suggesting that AC5 is a potential novel therapeutic target for the treatment of obesity, diabetes and associated cardiomyopathy.

Abstract 19060: An Anti-Viral Drug with Properties of Selective Inhibition of Adenylyl Cyclase Type 5 Reduces Infarct Size More when Delivered after than Before Reperfusion

There are at least two reasons why very few of the discoveries from genetically engineered mice have been translated to the clinics: 1.Most therapy requires delivery of a drug, since it is difficult to change a gene in patients; 2. Most cardioprotective agents require pretreatment before coronary artery (CA) reperfusion (R), but patients with myocardial infarction are rarely seen before CAR. We studied the drug 9-beta-D-arabinofuranosyladenine (Ara-Ade), previously used and FDA approved as an antiviral drug, because it has a structure resembling adenosine, but demonstrates selective adenylyl cyclase isoform type 5 (AC5) inhibition properties, i.e., it inhibits responses to beta-adrenergic receptor stimulation much greater in AC5 transgenic (Tg) mice than wild type, but not more in AC6 Tg and not at all in AC5 knockout, both in vitro and in vivo. We examined its ability to reduce infarct size (measured with TTC) after 30 min CA occlusion (CAO) and 24 hr CAR in mice. Surprisingly, infarct size, expressed as a fraction of the area at risk was lower, p<0.05, when the drug was delivered at CAR (16%), than when given before either CAO or CAR (21%) compared to vehicle (36%). p Ara-Ade also reduced infarct size in chronically instrumented conscious pigs, when delivered after CAR. In this model instantaneous measurements of coronary blood flow demonstrated that post reperfusion coronary hyperemia and reactive hyperemia, following a 15 sec CAO, were attenuated, consistent with isolated heart studies showing reduced coronary flow recovery after ischemia. Thus, a novel drug that inhibits AC5 induces even more cardioprotection when delivered after CAR than before, making it potentially more useful the clinical setting, where patients with myocardial infarction require CAR immediately after arriving at the hospital. It is conceivable that the mechanism, in part, involves protection of CAR damage, since the magnitude of coronary hyperemia after CAR has been related to CAR reperfusion damage.

Superresolution STED microscopy reveals differential localization in primary cilia

The primary cilium is an organelle that serves as a signaling center of the cell and is involved in the cAMP, Wnt, and hedgehog signaling pathways. Adenylyl cyclase type III (ACIII) is enriched in primary cilia and acts as a marker that is involved in cAMP signaling, while also playing an important role in regulating ciliogenesis and sensory functions. Ciliary function relies on the transportation of molecules between the primary cilium and the cell, which is facilitated by intraflagellar transport (IFT). The detailed localization and interactions of these important proteins remain unclear due to the limited resolution of conventional microscopy. We conducted superresolution imaging of immunostained ACIII and IFT88 in human fibroblasts using stimulated emission depletion (STED) microscopy. Instead of a homogeneous distribution along a primary cilium, our STED images revealed that ACIII formed a periodic punctate pattern with a roughly equal spacing between groups of puncta. Superresolution imaging of IFT88, an important protein of the IFT complexes, demonstrated two novel distinct distribution patterns at the basal end: a triangle of three puncta with similar fluorescence intensities, and a Y-shaped configuration of a bright punctum connected to two branches. We also performed STED imaging of IFT88 in mouse inner medullary collecting duct cells and mouse embryonic fibroblasts. The similar three-puncta and Y-shape patterns were observed in these cells, suggesting that these distribution patterns are common among primary cilia of different cell types. Our results demonstrate the ability of superresolution STED microscopy to reveal novel structural characteristics in primary cilia.

Superresolution STED imaging reveals a periodic punctate pattern of adenylyl cyclase type III on primary cilia

Adenylyl cyclases type III (ACIII) is a primary cilia marker involved in cAMP signaling, playing important roles in regulating ciliogenesis and sensory function. Despite its importance, detailed ACIII localization and their interactions with other proteins remain unclear due to the limited resolution of conventional microscopy. To determine the morphological characteristics of ACIII in primary cilia, we conducted superresolution imaging of immunostained ACIII in fibroblasts and neurons using stimulated emission depletion (STED) microscopy, which allows us to resolve the localization of ACIII achieving a resolution of 50 nm. In contrast to the previous understanding that ACIII distributes uniformly along a primary cilium, our STED images revealed that ACIII formed a periodic punctate pattern with a roughly equal spacing between groups of puncta. These puncta occupied less than 50% of the area, with the size of 137±20 nm in the axial direction along the primary cilia. The spacing between puncta was 250±67 nm. Some primary cilia even showed two rows of periodic puncta along the axial direction, with a tilted angle of about 12° to 35° between the two rows. The spacing between the two rows was 195±19 nm. In some cells, ACIII was only localized in the basal body, where the periodic punctate pattern was absent. In summary, based on our superresolution studies, we found that ACIII can be transported into a primary cilium, but would only occupy regions approximately equally spaced along the cilium.

Common mechanisms for calorie restriction and adenylyl cyclase type 5 knockout models of longevity

Adenylyl cyclase type 5 knockout mice (AC5 KO) live longer and are stress resistant, similar to calorie restriction (CR). AC5 KO mice eat more, but actually weigh less and accumulate less fat compared with WT mice. CR applied to AC5 KO results in rapid decrease in body weight, metabolic deterioration, and death. These data suggest that despite restricted food intake in CR, but augmented food intake in AC5 KO, the two models affect longevity and metabolism similarly. To determine shared molecular mechanisms, mRNA expression was examined genome-wide for brain, heart, skeletal muscle, and liver. Significantly more genes were regulated commonly rather than oppositely in all the tissues in both models, indicating commonality between AC5 KO and CR. Gene ontology analysis identified many significantly regulated, tissue-specific pathways shared by the two models, including sensory perception in heart and brain, muscle function in skeletal muscle, and lipid metabolism in liver. Moreover, when comparing gene expression changes in the heart under stress, the glutathione regulatory pathway was consistently upregulated in the longevity models but downregulated with stress. In addition, AC5 and CR shared changes in genes and proteins involved in the regulation of longevity and stress resistance, including Sirt1, ApoD, and olfactory receptors in both young- and intermediate-age mice. Thus, the similarly regulated genes and pathways in AC5 KO and CR mice, particularly related to the metabolic phenotype, suggest a unified theory for longevity and stress resistance.

Heteromerization of Ciliary G Protein-Coupled Receptors in the Mouse Brain

Nearly every cell type in the mammalian body projects from its cell surface a primary cilium that provides important sensory and signaling functions. Defects in the formation or function of primary cilia have been implicated in the pathogenesis of many human developmental disorders and diseases, collectively termed ciliopathies. Most neurons in the brain possess cilia that are enriched for signaling proteins such as G protein-coupled receptors and adenylyl cyclase type 3, suggesting neuronal cilia sense neuromodulators in the brain and contribute to non-synaptic signaling. Indeed, disruption of neuronal cilia or loss of neuronal ciliary signaling proteins is associated with obesity and learning and memory deficits. As the functions of primary cilia are defined by the signaling proteins that localize to the ciliary compartment, identifying the complement of signaling proteins in cilia can provide important insights into their physiological roles. Here we report for the first time that different GPCRs can colocalize within the same cilium. Specifically, we found the ciliary GPCRs, melanin-concentrating hormone receptor 1 (Mchr1) and somatostatin receptor 3 (Sstr3) colocalizing within cilia in multiple mouse brain regions. In addition, we have evidence suggesting Mchr1 and Sstr3 form heteromers. As GPCR heteromerization can affect ligand binding properties as well as downstream signaling, our findings add an additional layer of complexity to neuronal ciliary signaling.

The A-kinase Anchoring Protein Yotiao Facilitates Complex Formation between Adenylyl Cyclase Type 9 and the IKs Potassium Channel in Heart

The scaffolding protein Yotiao is a member of a large family of protein A-kinase anchoring proteins with important roles in the organization of spatial and temporal signaling. In heart, Yotiao directly associates with the slow outward potassium ion current (I(Ks)) and recruits both PKA and PP1 to regulate I(Ks) phosphorylation and gating. Human mutations that disrupt I(Ks)-Yotiao interaction result in reduced PKA-dependent phosphorylation of the I(Ks) subunit KCNQ1 and inhibition of sympathetic stimulation of I(Ks), which can give rise to long-QT syndrome. We have previously identified a subset of adenylyl cyclase (AC) isoforms that interact with Yotiao, including AC1-3 and AC9, but surprisingly, this group did not include the major cardiac isoforms AC5 and AC6. We now show that either AC2 or AC9 can associate with KCNQ1 in a complex mediated by Yotiao. In transgenic mouse heart expressing KCNQ1-KCNE1, AC activity was specifically associated with the I(Ks)-Yotiao complex and could be disrupted by addition of the AC9 N terminus. A survey of all AC isoforms by RT-PCR indicated expression of AC4-6 and AC9 in adult mouse cardiac myocytes. Of these, the only Yotiao-interacting isoform was AC9. Furthermore, the endogenous I(Ks)-Yotiao complex from guinea pig also contained AC9. Finally, AC9 association with the KCNQ1-Yotiao complex sensitized PKA phosphorylation of KCNQ1 to β-adrenergic stimulation. Thus, in heart, Yotiao brings together PKA, PP1, PDE4D3, AC9, and the I(Ks) channel to achieve localized temporal regulation of β-adrenergic stimulation.

Reduction of Adenylyl Cyclase Type 5 Protects Obesity Induced Cardiomyopathy

Since mice with adenylyl cyclase type 5 (AC5) gene disruption eat more than wild type (WT), but weigh less, the goal of this study was to determine if these mice are protected against obesity and the cardiomyopathy induced by a high fat diet (HFD) for 100 days. AC5KO mice gained less weight/kcal consumed than wild type (WT) on HFD, p<0.05, and insulin resistance and glucose tolerance were improved compared to WT on HFD. The HFD induced cardiomyopathy was characterized by reduced, p<0.05, left ventricular (LV) ejection fraction (EF) (53±2.4%) compared with normal diet (ND) (69±2.9%), whereas the reduction in LVEF was less, p<0.05, in AC5KO on HFD (61±3.0%). The cardiomyopathy was also characterized by increased myocardial fibrosis, assessed by picric Sirius red staining, and increased apoptosis, expressed as TUNEL positive cells per mm2. The AC5KO exhibited reduced fibrosis (1.2±0.1%) compared with WT (1.7±0.1%) on HFD, p<0.05, as well as reduced apoptosis (0.5±0.04) compared with WT (0.9±0.1) on HFD, p<0.05. We also examined the effects of pharmacologic inhibition of AC5 with AraAde, an antiviral compound that has AC5 inhibitory effects; weight gain, glucose tolerance, insulin resistance, LV function and fibrosis were all improved compared to vehicle treated mice on HFD. Thus inhibition of AC5 protects against obesity and the cardiomyopathy it induces.

Enhanced Exercise Capacity in Adenylyl Cyclase Type 5 Knockout Mimics Chronic Exercise Training

Exercise training improves skeletal muscle strength, affects muscle fiber types and increases exercise endurance, leading to prolonged longevity. Adenylyl cyclase type 5 knockout (AC5 KO) mice exhibit prolonged longevity and demonstrate increased exercise capacity. AC5 KO mice were able to run significantly longer than WT in both distance (817 ± 41m vs. 569±26m) and time (37.4±1.0min vs. 30.8±0.8min). The goal of this investigation was to determine whether AC5 KO mice share similar features with chronic exercise training. AC5 KO mice, like exercise training in WT, demonstrated more oxidative slow‐twitch fibers. Using RNA deep sequencing analysis, we found similar gene expression patterns in skeletal muscle between AC5 KO mice and the exercise‐trained WT mice. There were 72 genes (24%) commonly up‐regulated and 103 genes (27%) commonly down‐regulated between AC5 KO and WT exercise‐trained mice; they shared similar gene profiles in 27 different biological processes, including muscle contraction and metabolic related processes. Thus, AC5 KO mice, like exercise training, not only exhibit increased longevity but also share mechanisms resulting in improved skeletal muscle function and exercise endurance.

Altered store operated calcium entry increases cyclic 3',5'-adenosine monophosphate production and extracellular signal-regulated kinases 1 and 2 phosphorylation in polycystin-2-defective cholangiocytes.

Mutations in polycystins (PC1 or PC2/TRPP2) cause progressive polycystic liver disease (PLD). In PC2-defective mice, cyclic 3',5'-adenosine monophosphate/ protein kinase A (cAMP/PKA)-dependent activation of extracellular signal-regulated kinase/ mammalian target of rapamycin (ERK-mTOR) signaling stimulates cyst growth. We investigated the mechanisms connecting PC2 dysfunction to altered Ca(2+) and cAMP production and inappropriate ERK signaling in PC2-defective cholangiocytes. Cystic cholangiocytes were isolated from PC2 conditional-KO (knockout) mice (Pkd2(flox/-) :pCxCreER™; hence, called Pkd2KO) and compared to cholangiocytes from wild-type mice (WT). Our results showed that, compared to WT cells, in PC2-defective cholangiocytes (Pkd2KO), cytoplasmic and ER-Ca(2+) (measured with Fura-2 and Mag-Fluo4) levels are decreased and store-operated Ca(2+) entry (SOCE) is inhibited, whereas the expression of Ca(2+) -sensor stromal interaction molecule 1 (STIM1) and store-operated Ca(2+) channels (e.g., the Orai1 channel) are unchanged. In Pkd2KO cells, ER-Ca(2+) depletion increases cAMP and PKA-dependent ERK1/2 activation and both are inhibited by STIM1 inhibitors or by silencing of adenylyl cyclase type 6 (AC6). These data suggest that PC2 plays a key role in SOCE activation and inhibits the STIM-dependent activation of AC6 by ER Ca(2+) depletion. In PC2-defective cells, the interaction of STIM-1 with Orai channels is uncoupled, whereas coupling to AC6 is maximized. The resulting overproduction of cAMP, in turn, potently activates the PKA/ERK pathway. PLD, because of PC2 deficiency, represents the first example of human disease linked to the inappropriate activation of store-operated cAMP production.

Superresolution STED Imaging Reveals a Periodic Punctate Pattern of Adenylyl Cyclase Type III on Primary Cilia

Primary cilia are organelles serving essential sensory and signaling functions in nearly all mammalian cell types. They serve as a center of complex signaling, involved in cAMP, Wnt and Shh signaling pathways. Adenylyl cyclases type III (ACIII) is a primary cilia marker involved in cAMP signaling, playing important roles in regulating ciliogenesis and sensory function. Despite its importance, detailed ACIII localization and their interactions with other proteins remain unclear due to the limited resolution of conventional microscopy. To determine the morphological characteristics of ACIII in primary cilia, we conducted superresolution imaging of immunostained ACIII in fibroblasts and neurons using stimulated emission depletion (STED) microscopy, which allows us to resolve the localization of ACIII achieving a resolution of 50 nm. In contrast to the previous understanding that ACIII distributes uniformly along a primary cilium, our STED images revealed that ACIII formed a periodic punctate pattern with a roughly equal spacing between groups of puncta. These puncta occupied less than 50% of the area, with the size of 137±20 nm in the axial direction along the primary cilia. The spacing between puncta was 250±67 nm. Some primary cilia even showed two rows of periodic puncta along the axial direction, with a tilted angle of about 12° to 35° between the two rows. The spacing between the two rows was 195±19 nm. In some cells, ACIII was only localized in the basal body, where the periodic punctate pattern was absent. Based on our superresolution studies, we concluded that ACIII can be transported into a primary cilium, but would only occupy regions approximately equally spaced along the cilium.

Abstract 18237: Inhibition of Adenylyl Cyclase Type 5 Protects Against Obesity and Diabetes

Adenylyl cyclase type 5 (AC5) is one of 9 major isoforms of AC. Mice with AC5 gene disruption (knockout, KO) live one third longer than wild type (WT) littermates. Like caloric restriction, the most commonly studied model of longevity, the AC5KO mice weigh 12% less than WT (p<0.05) and have less body fat, i.e., adiposity index, calculated by dividing the sum weights of the three major fat pads (inguinal, retroperitoneal, gonadal) by the body weight, was 50% lower than WT (p<0.05). The first goal of this study was to determine if AC5KO mice are protected, not only against obesity, but also diabetes. Insulin sensitivity, assessed by blood glucose measurement following insulin injection, was improved by 24% compared to WT. Glucose tolerance was also improved, p<0.05. A major limitation of studies in genetically altered mouse models, is that it is difficult to translate these results to the bedside, since it is not possible to knock out a gene in patients. Accordingly, we identified a pharmacological inhibitor of AC5, vidaribine, and examined the extent to which it ameliorated obesity and diabetes. First we demonstrated its selectivity for AC5; the inhibitor reduced inotropic responses to isoproterenol more than WT only in AC5 cardiac transgenic mice, but not in AC6 cardiac transgenic mice. To induce obesity, WT mice were fed a high fat diet feeding for 2 months, increasing body weight by 51% over mice fed a standard diet. WT mice with high fat diet and AC5 inhibitor on board for an additional 3 months, compared with WT mice on vehicle and high fat diet, increased body weight and adiposity index less, p<0.05, by 20% and 28%, respectively, while insulin resistance was reduced by 76% and glucose tolerance was improved by 66% (p<0.05). In AC5KO mice on the high fat diet insulin resistance was also reduced and glucose tolerance was also improved compared to WT on the high fat diet, similar to that observed with the AC5 inhibitor on the high fat diet. Thus, inhibition of AC5 provides a novel target for obesity and diabetes; the effective pharmacological inhibitor of AC5 enhances its clinical relevance.

Collecting duct-specific knockout of adenylyl cyclase type VI causes a urinary concentration defect in mice

Collecting duct (CD) adenylyl cyclase VI (AC6) has been implicated in arginine vasopressin (AVP)-stimulated renal water reabsorption. To evaluate the role of CD-derived AC6 in regulating water homeostasis, mice were generated with CD-specific knockout (KO) of AC6 using the Cre/loxP system. CD AC6 KO and controls were studied under normal water intake, chronically water loaded, or water deprived; all of these conditions were repeated in the presence of continuous administration of 1-desamino-8-d-arginine vasopressin (DDAVP). During normal water intake or after water deprivation, urine osmolality (U(osm)) was reduced in CD AC6 KO animals vs. controls. Similarly, U(osm) was decreased in CD AC6 KO mice vs. controls after water deprivation+DDAVP administration. Pair-fed (with controls) CD AC6 KO mice also had lower urine osmolality vs. controls. There were no detectable differences between KO and control animals in fluid intake or urine volume under any conditions. CD AC6 KO mice did not have altered plasma AVP levels vs. controls. AVP-stimulated cAMP accumulation was reduced in acutely isolated inner medullary CD (IMCD) from CD A6 KO vs. controls. Medullary aquaporin-2 (AQP2) protein expression was lower in CD AC6 KO mice vs. controls. There were no differences in urinary urea excretion or IMCD UT-A1 expression; however, IMCD UT-A3 expression was reduced in CD AC6 KO mice vs. controls. In summary, AC6 in the CD regulates renal water excretion, most likely through control of AVP-stimulated cAMP accumulation and AQP2.

Palmitoylation Targets AKAP79 Protein to Lipid Rafts and Promotes Its Regulation of Calcium-sensitive Adenylyl Cyclase Type 8

PKA anchoring proteins (AKAPs) optimize the efficiency of cAMP signaling by clustering interacting partners. Recently, AKAP79 has been reported to directly bind to adenylyl cyclase type 8 (AC8) and to regulate its responsiveness to store-operated Ca2+ entry (SOCE). Although AKAP79 is well targeted to the plasma membrane via phospholipid associations with three N-terminal polybasic regions, recent studies suggest that AKAP79 also has the potential to be palmitoylated, which may specifically allow it to target the lipid rafts where AC8 resides and is regulated by SOCE. In this study, we have addressed the role of palmitoylation of AKAP79 using a combination of pharmacological, mutagenesis, and cell biological approaches. We reveal that AKAP79 is palmitoylated via two cysteines in its N-terminal region. This palmitoylation plays a key role in targeting the AKAP to lipid rafts in HEK-293 cells. Mutation of the two critical cysteines results in exclusion of AKAP79 from lipid rafts and alterations in its membrane diffusion behavior. This is accompanied by a loss of the ability of AKAP79 to regulate SOCE-dependent AC8 activity in intact cells and decreased PKA-dependent phosphorylation of raft proteins, including AC8. We conclude that palmitoylation plays a key role in the targeting and action of AKAP79. This novel property of AKAP79 adds an unexpected regulatory and targeting option for AKAPs, which may be exploited in the cellular context.

S12 * YOUNG RESEARCHER SYMPOSIUM I: ALCOHOL DEPENDENCE--NEW FINDINGS FROM THE HPA AND THE HPG AXES * S12.1 * ROLE OF ADENYLYL CYCLASE TYPE 7 IN HPA AXIS ACTIVATION DURING ETHANOL INTOXICATION, ALCOHOL DEPENDENCE AND DEPRESSION

Alcohol use disorders and major depressive disorder (MDD) often co-occur with each other. Patients with such comorbidity will have greater functional impairment and disability and require more frequent and longer hospitalization. Several haplotypes in adenylyl cyclase type 7 (AC7) were shown to be associated with MDD and alcohol dependence (AD). Study aimed to assess the association between TG7AT haplotype in AC7 gene previously shown to be associated with familial MDD and AD indicated that the TG7AT haplotype had a significant predisposing effect on familial MDD OR = 2.5. When males and females were divided into two groups, harmful drinking and non-harmful drinking, in females drinking above harmful levels, there was a statistically significant additive association of the TG7AT haplotype and familial MDD (OR = 3.1). One of the unifying hypotheses for the common etiology of MDD and AD is based on differential responses to life stress and an over reactive stress responsiveness. Previously, we showed that ethanol can increase cAMP signaling by increases in AC7 responsiveness to Gsα. It was also suggested that, in anterior pituitary corticotrophs, CRF receptor type 1 is coupled to AC9 as well as to AC7. In order to understand the interplay between AC7 gene, alcohol and HPA axis responsiveness, we subjected our AC7 genetically modified animals to acute ethanol exposure. It was shown that after acute ethanol exposure, male and female transgenic (Adcy7huTG) mice displayed higher plasma adrenocorticotropin and corticosterone levels than WT and/or knock-down (Adcy7+/−) mice, with female Adcy7huTG displaying the highest corticosterone levels, therefore making them more vulnerable to glucocorticoid-induced changes in brain structure and function. We thus hypothesize that increased AC7 signaling and HPA axis activation may contribute to a greater risk for familial depression in females consuming alcohol at high harmful levels. (This work was supported by NIAAA and Banbury Foundation.)