Adenylyl Cyclase Isoforms Research Articles

Antidepressant treatment increases cAMP signaling by translocating Gas from lipid rafts and increasing association with type 6 adenylyl cyclase (AC6) independent of action at monoamine transporters.

Antidepressants of different chemical classes promote sustained increases in cAMP, due, at least in part to the redistribution of Gαs from lipid rafts into non‐raft membrane fractions resulting in increased Gαs functional coupling with adenylyl cyclase (AC). This has been demonstrated in both rats and cultured neural and glial cells by a number of techniques, including cell fractionation, functional assays, and imaging studies such as Fluorescence Recovery After Photobleaching (FRAP). Unlike animal models, C6 glioma lack monoamine reuptake transporters, suggesting that mechanisms of antidepressant response entail more than inhibition of neurotransmitter reuptake inhibition. This is noteworthy, since inhibition of 5HT or NE uptake is quite rapid even though clinical effects of antidepressants require several weeks (and several days in the cellular models). Furthermore, while these neural and glial cells showed an “antidepressant response”, Gαs localization and cAMP production in kidney epithelial cells like COS7 and HEK293 were unchanged by antidepressant treatment. Similarly, membranes from liver and kidney of rats treated chronically with antidepressant did not show the same response as brain from those same animals. In this study we sought to determine whether cellular antidepressant response, with respect to increased cAMP signaling and Gαs localization, is dependent on the type of AC isoform expression. Cell lines “insensitive” to antidepressant treatment, such as HEK293, become responsive after transfection with AC6, showing translocation of Gαs from lipid rafts, both by cell fractionation and by FRAP. Likewise, knockdown of AC6 but not other adenylyl cyclase isoforms abolishes the antidepressant response in responsive cell lines such as C6 glioma. Thus, it is suggested that AC6 performs a anchoring function for Gαs outside of rafts, affixing the translocated Gαs into the non‐raft domain and facilitating an antidepressant‐induced increase in adenylyl cyclase activity.

Adenylyl Cyclase Isoform Specific Effects in Lipid Raft and Non‐Lipid Raft Membrane Domains Regulate cAMP Compartmentation in Human Airway Smooth Muscle Cells

The formation of distinct macromolecular signaling complexes allows different G‐protein coupled receptors to produce diverse functional responses, even while sharing a common second messenger such as cAMP. In human airway smooth muscle (HASM) cells, segregation of specific receptors into different membrane microdomains is thought to critically aid in generating compartmentalized cAMP responses. Whereas, E type prostaglandin receptors (EPRs) have been shown to be expressed in non‐lipid raft domains of the plasma membrane, β‐Adrenergic receptors (βARs) are predominantly expressed in caveolar lipid rafts. In the present study, we tested the hypothesis that adenylyl cyclase type 2 (AC2) preferentially couples to EPRs in a non‐lipid raft domain, while adenylyl cyclase type 6 (AC6) selectively couples to βARs in lipid rafts. To do this, we examined the effect of overexpressing AC2 and AC6 on cAMP responses detected using genetically‐encoded FRET‐based biosensors targeted to lipid raft and non‐lipid raft domains of the plasma membrane, as well as the bulk cytosolic compartment in HASM cells. This approach has the advantage of measuring the kinetics of cAMP production in living cells without the use of PDE inhibitors. Overexpression of AC2 enhanced the cAMP response to EPR activation associated with non‐lipid raft domains, without significantly affecting responses detected elsewhere. AC2 overexpression also had no effect on cAMP responses to βAR activation detected in any subcellular location. These data confirm the hypothesis that AC2 couples exclusively with EPRs in a non‐lipid raft membrane compartment. Overexpression of AC6, on the other hand, actually decreased the response to βAR stimulation associated with lipid rafts, without significantly affecting responses elsewhere. AC6 overexpression also had no effect on the responses to EPR activation detected anywhere in the cell. The ability of AC6 overexpression to inhibit βAR production of cAMP in lipid raft domains was reversed by inhibition of PDE4 activity with rolipram. It was also reversed by overexpression of Ht31 peptide, which disrupts the interaction of protein kinase A with A‐kinase anchoring proteins (AKAPs). These results suggests that AC6 overexpression upregulates and/or recruits PKA and PDE4 activity, which then reduces βAR production of cAMP associated specifically with lipid raft domains.Support or Funding InformationSupport by: NIH R01 GM107094

Importance of adenylyl cyclase 6 in developing lithium‐induced nephrogenic diabetes insipidus

Nephrogenic diabetes insipidus (NDI) develops in approximately 50% of psychiatric patients being treated with lithium (Li+). The mechanism behind Li+‐induced NDI (Li‐NDI) is only poorly understood, but it occurs at least partly due to reduced aquaporin‐2 (AQP2) expression, leading to lower water reabsorption in collecting ducts (CD) of the kidney. A suggested mechanism inducing these effects is that Li+ inhibits adenylyl cyclase (AC) activity and thereby decreases cAMP levels and subsequently reduces AQP2 abundance and membrane targeting. In the present study, we tested the hypothesis that Li‐NDI would not develop in mice lacking AC6, the AC isoform responsible for the majority of vasopressin‐induced inner medullary (IM) cAMP formation. Total‐body AC6 knockout (AC6−/−) mice and newly developed connecting tubule/principal cell‐specific AC6 knockout mice (AC6loxloxCre) were studied. Similar to AC6−/− mice, the novel AC6loxloxCre mice had lower urine osmolality [mean ± SE (n): control 2363 ± 114 mmol/kg (17), AC6loxloxCre 1219 ± 49 mmol/kg (9); P < 0.05] and higher water intake [control 0.19 ± 0.01 g/g BW/24h (7), AC6loxloxCre 0.28 ± 0.02 g/g BW/24h (6); P < 0.05]. In both controls, AC6−/−, and in AC6loxloxCre mice, dietary Li+ administration increased the water intake (P < 0.05) and reduced the urine osmolality (P < 0.05). Similar to AC6−/− mice, the AC6loxloxCre mice showed under standard conditions lower medullary levels of AQP2 (42% reduction, P < 0.05) and pS256‐AQP2 and (51% reduction, P < 0.05) and the levels were further reduced by respectively 54% and 57% after Li+ administration (P < 0.05). Furthermore, Li+ induced an increase in the numbers of proliferating cell nuclear antigen‐positive cells in the IMCD in both AC6loxloxCre (~2200% increase, P < 0.05) and control mice (~2600% increase, P < 0.05). However, compared to control mice, a greater % of H+‐ATPase B1 subunit‐positive cells were found in the AC6loxloxCre mice under both standard condition (32%, P < 0.05) and after Li+‐administration (33%, P < 0.05). Collectively, AC6 plays a minor role in the development of Li‐NDI, but could have an important function in determining the intercalated‐to‐principal cell ratio, which is altered during Li‐NDI.Support or Funding InformationFunding to R.A.F. is provided by the Novo Nordisk Foundation, the Lundbeck Foundation and Danish Medical Research Council. T.R. is supported by NIDDK 1R01DK110621‐01, the O'Brien Center for Acute Kidney Injury Research Grant P30DK079337, the Diabetes Endocrinology Research Center P30DK063491, the AHA 15BGIA22410018 and Satellite Healthcare (a not‐for‐profit renal care provider).

Type 2 inositol trisphosphate receptor gene expression in hepatocytes is regulated by cyclic AMP

The type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) is the principal intracellular Ca2+ release channel in hepatocytes, and so is important for bile secretion and other functions. IP3R2 activity is regulated in part by post-translational modifications but little is known about transcriptional regulation of its expression. We found that both IP3R2 mRNA and protein levels in liver were increased during fasting. Treatment of hepatocytes with forskolin or 8-CPT-cAMP also increased IP3R2, and this was reduced by actinomycin D. Analysis of the IP3R2 promoter revealed five CREs, and CREB potently increased promoter activity. Mutation of CRE4 or CRE5 decreased induction by CREB, and ChIP assay showed recruitment of CREB to these sites. Adenylyl cyclase (AC) 6 and 9 were the principal AC isoforms detected in rat hepatocytes, and silencing either one decreased organic anion secretion, which depends on IP3R2. Secretion furthermore was increased by overnight but not acute treatment with forskolin or 8-CPT-cAMP. These findings provide evidence that IP3R2 expression is transcriptionally regulated by cAMP via CREB binding to CRE elements in its promoter. The findings furthermore suggest that this mechanism is relevant for hormonal regulation of bile secretion.

International Union of Basic and Clinical Pharmacology. CI. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases.

Adenylyl cyclases (ACs) generate the second messenger cAMP from ATP. Mammalian cells express nine transmembrane AC (mAC) isoforms (AC1-9) and a soluble AC (sAC, also referred to as AC10). This review will largely focus on mACs. mACs are activated by the G-protein Gαs and regulated by multiple mechanisms. mACs are differentially expressed in tissues and regulate numerous and diverse cell functions. mACs localize in distinct membrane compartments and form signaling complexes. sAC is activated by bicarbonate with physiologic roles first described in testis. Crystal structures of the catalytic core of a hybrid mAC and sAC are available. These structures provide detailed insights into the catalytic mechanism and constitute the basis for the development of isoform-selective activators and inhibitors. Although potent competitive and noncompetitive mAC inhibitors are available, it is challenging to obtain compounds with high isoform selectivity due to the conservation of the catalytic core. Accordingly, caution must be exerted with the interpretation of intact-cell studies. The development of isoform-selective activators, the plant diterpene forskolin being the starting compound, has been equally challenging. There is no known endogenous ligand for the forskolin binding site. Recently, development of selective sAC inhibitors was reported. An emerging field is the association of AC gene polymorphisms with human diseases. For example, mutations in the AC5 gene (ADCY5) cause hyperkinetic extrapyramidal motor disorders. Overall, in contrast to the guanylyl cyclase field, our understanding of the (patho)physiology of AC isoforms and the development of clinically useful drugs targeting ACs is still in its infancy.

G-Protein-coupled receptors: Decoding mixed signals.

A new mechanism of functional crosstalk between two distinct G-protein-coupled receptors (GPCRs)—the parathyroid hormone receptor (PTHR) and β2-adrenergic receptor (β2 Ar)—that occurs at the level of G protein βγ subunits and a specific adenylyl cyclase isoform is identified. This crosstalk augments cAMP signaling by the PTHR from endosomes, and thus promotes the actions of PTH ligands in bone target cells.

Molecular Modeling Evaluation of the Enantiomers of a Novel Adenylyl Cyclase 2 Inhibitor.

Adenylyl cyclase 2 (AC2) is one of nine membrane-bound isoforms of adenylyl cyclase that converts ATP into cyclic AMP (cAMP), an important second messenger molecule. Upregulation of AC2 is linked to cancers like pancreatic and small intestinal neuroendocrine tumors (NETs). The structures of the various isoforms of adenylyl cyclases are highly homologous, posing a significant challenge to drug discovery efforts for an effective, isoform-selective modulator of AC2. In a previous study, a screen identified a potential isoform-selective and noncompetitive inhibitor of AC2, SKF83566. In the present study, molecular modeling is used to explore the mode of inhibition of AC2 by SKF83566 and to investigate the active enantiomer of SKF83566. Homology models of hAC2 were built based on canine AC5-C1a and rat AC2-C2a templates. With these models, a combination of flexible docking, molecular dynamics simulations, and free energy calculations using the MM/GBSA methodology suggested an allosteric mechanism in which (S)-SKF83566 binds to an allosteric site near ATP and alters the protein conformation of the ATP binding site, potentially preventing the adenosine moiety of ATP from forming an archlike shape to form cAMP. The predicted binding preference for the (S)-SKF83566 enantiomer and the predicted free energy are consistent with the experimental data.

Adenylyl cyclase 3/adenylyl cyclase-associated protein 1 (CAP1) complex mediates the anti-migratory effect of forskolin in pancreatic cancer cells.

Pancreatic cancer is one of the most lethal human malignancies. A better understanding of the intracellular mechanism of migration and invasion is urgently needed to develop treatment that will suppress metastases and improve overall survival. Cyclic adenosine monophosphate (cyclic AMP) is a second messenger that has shown to regulate migration and invasion of pancreatic cancer cells. The rise of cyclic AMP suppressed migration and invasion of pancreatic ductal adenocarcinoma cells. Cyclic AMP is formed from cytosolic ATP by the enzyme adenylyl cyclase (AC). There are ten isoforms of ACs; nine are anchored in the plasma membrane and one is soluble. What remains unknown is the extent to which the expression of transmembrane AC isoforms is both modified in pancreatic cancer and mediates the inhibitory effect of forskolin on cell motility. Using real-time PCR analysis, ADCY3 was found to be highly expressed in pancreatic tumor tissues, resulting in a constitutive increase in cyclic AMP levels. On the other hand, ADCY2 was down-regulated. Migration, invasion, and filopodia formation in two different pancreatic adenocarcinoma cell lines, HPAC and PANC-1 deficient in AC1 or AC3, were studied. We found that AC3, upon stimulation with forskolin, enhanced cyclic AMP levels and inhibited cell migration and invasion. Unlikely to be due to a cytotoxic effect, the inhibitory effects of forskolin involved the quick formation of AC3/adenylyl cyclase-associated protein 1 (CAP1)/G-actin complex, which inhibited filopodia formation and cell motility. Using Western blotting analysis, forskolin, through AC3 activation, caused phosphorylation of CREB, but not ERK. The effect of CREB phosphorylation is likely to be associated with long-term signaling changes. © 2016 Wiley Periodicals, Inc.

Glucagon-Like Peptide 1 Receptor: A Novel Pharmacological Target for Treating Human Bronchial Hyperresponsiveness.

Asthma is associated with several comorbidities, such as type 2 diabetes mellitus, which may lead to bronchial hyperresponsiveness (BHR). Because glucagon-like peptide (GLP) 1 regulates glucose homeostasis, we pharmacologically investigated the influence of the GLP1 receptor (GLP1-R) agonist, exendin-4, on BHR induced in human isolated airways. The effect of exendin-4 was assessed in human isolated airways undergoing overnight passive sensitization and high-glucose stimulation, two conditions mimicking ex vivo the BHR typical of patients with asthma and diabetes, respectively. GLP1-R activation modulated the bronchial contractile tone induced by transmural stimulation (maximum effect -56.7 ± 3.6%; onset of action, 28.2 ± 4.4 min). Exendin-4 prevented BHR induced by both high-glucose stimulation and passive sensitization (-37.8 ± 7.5% and -74.9 ± 3.9%, P < 0.05 versus control, respectively) through selective activation of GLP1-R and in an epithelium-independent manner. The cAMP-dependent protein kinase A inhibitor, KT5720, reduced the protective role of exendin-4 (P > 0.05 versus passively sensitized tissues). The GLP1-R stimulation by overnight incubation with exendin-4 induced the overexpression of adenylyl cyclase isoform V (+48.4 ± 1.3%, P < 0.05 versus passively sensitized tissues) and restored the cAMP levels depleted by this procedure (+330.8 ± 63.3%, P < 0.05 versus passively sensitized tissues). In conclusion, GLP1-R may represent a novel target for treating BHR by activating the cAMP-dependent protein kinase A pathway in human airways, and GLP1-R agonists could be used as a "new" class to treat patients with asthma and patients with type 2 diabetes mellitus with BHR.

Comparative electrostatic analysis of adenylyl cyclase for isoform dependent regulation properties

The enzyme adenylyl cyclase (AC) plays a pivotal role in a variety of signal transduction pathways inside the cell, where it catalyzes the cyclization of adenosine triphosphate (ATP) into the second-messenger cyclic adenosine monophosphate (cAMP). Among other roles, AC regulates processes involved in neural plasticity, innervation of smooth muscles of the heart and the endocrine system of the pancreas. The functional diversity of AC is manifested in its different isoforms, each having a specific regulation pattern. There is an increasing amount of data available concerning the regulatory properties of AC isoforms, however little is known about the interactions on a structural level. Here, we conducted a comparative electrostatic analysis of the catalytic domains of all nine transmembrane AC isoforms with the aim of detecting, verifying and predicting the binding sites of molecular regulators on AC. The results provide support for the positioning of the binding site of the inhibitory protein Gi α at a pseudo-symmetric position to the stimulatory Gs α binding site. They also provide a structural interpretation of the Gβγ interaction with ACs 2, 4, and 7 and suggest a new binding site for RGS2. Comparison of the small molecule binding sites on AC shows that overall they have high electrostatic similarity, but regions of electrostatic differences are identified. These could provide a basis for the development of novel compounds with isoform-specific modulatory effects on AC. Proteins 2016; 84:1844-1858. © 2016 Wiley Periodicals, Inc.

Oscillation in expression of Adenylyl Cyclase isoforms: new insight to regulation of molecular clock

Background: Mammalian cells contain a cell-autonomous circadian clock that couples endogenous rhythms with changes of cellular environment. Some recent reports have been shown that circadian clock regulates glucose homeostasis; but, their molecular basis is little understood. Adenylyl Cyclase (AC) is a key enzyme in glucose metabolism. We aimed to investigate circadian mRNA expression of two circadian clock genes Cry1, Cry2, and nine Adenylyl Cyclase isoforms.Methods: MCF7 cell culture shocked using horse serum. Every four hours (over a 48-h period), total RNA was extracted and cDNA was synthesized. Expression level of AC isoforms and Cry genes was evaluated by quantitative RT-PCR.Results: Two AC isoforms; AC3, AC6 showed a circadian expression pattern in which they had a significant correlation with Cry1 and Cry2 transcripts. AC3 isoform was also up-regulated after serum shock. Time lags results indicated that AC3 expression was coincided with Cry2 and leads Cry1 expression.Conclusions: Adenylyl Cyclase isoforms, especially AC3, may play an important role in molecular clock regulation and response to external factors.

Role of an adenylyl cyclase isoform in ethanol's effect on cAMP regulated gene expression in NIH 3T3 cells

Previous research has indicated that the cyclic AMP (cAMP) signal transduction system plays an important role in the predisposition to and development of ethanol abuse in humans. Our laboratory has demonstrated that ethanol is capable of enhancing adenylyl cyclase (AC) activity. This effect is AC isoform-specific; type 7 AC (AC7) is most enhanced by ethanol. Therefore, we hypothesized that the expression of a specific AC isoform will play a role on the effect of ethanol on cAMP regulated gene expression. We employed NIH 3T3 cells transfected with AC7 or AC3 as a model system. To evaluate ethanol's effects on cAMP regulated gene expression, a luciferase reporter gene driven by a cAMP inducing artificial promoter was utilized. Stimulation of AC activity leads to an increase in the reporter gene activity. This increase was enhanced in the presence of ethanol in cells expressing AC7, while cells expressing AC3 did not respond to ethanol. cAMP reporter gene expression was increased in the presence of 8-bromo-cAMP; this expression was not enhanced by ethanol. These observations are consistent with our hypothesis. The basal level of CREB phosphorylation was high and did not change by cAMP stimulation or in the presence of ethanol. However, there were significant changes in the TORC3 amount in nuclei depending on stimulation conditions. The results suggest that nuclear translocation of TORC3 plays a more important role than CREB phosphorylation in the observed changes in the cAMP driven reporter gene activity.

Expression and Immunohistochemical Localisation of the Gβγ-activated and Calcineurin-Inhibited Adenylyl Cyclase Isoforms in Rat Articular Chondrocytes.

To determine the expression and localisation of the Gβγ-activated adenylyl cyclase (AC) isoforms 2, 4, and 7 and calcineurin-inhibited AC isoform 9 in rat articular chondrocytes. Experimental study. Jumma Research Laboratory and Histology Laboratory, The Aga Khan University, Karachi, from 2009 to 2011. Fresh slices of articular cartilage were taken from various synovial joints of rats of different age groups. The expression of AC isoforms was determined by RT-PCR and immunohistochemistry was performed to localise these isoforms in articular chondrocytes. Tissue sections were processed for immunostaining with respective antibodies. The color was developed by diaminobenzidine. All the studied AC isoforms were found to be differentially expressed in different zones of the rat articular cartilage. Generally, expression of all AC isoforms studied increased with age. The expression of the AC isoforms through PCR was almost consistent with the localisation of these isoforms by immunohistochemistry. These data add to the information about signalling cascades possibly involved in articular chondrocytes. Variable expression of AC isoforms 2, 4, 7, and 9 suggest a role for the signalling cascades regulated by the AC isoforms in articular chondrocytes.

Pancreatic Beta Cell G-Protein Coupled Receptors and Second Messenger Interactions: A Systems Biology Computational Analysis.

Insulin secretory in pancreatic beta-cells responses to nutrient stimuli and hormonal modulators include multiple messengers and signaling pathways with complex interdependencies. Here we present a computational model that incorporates recent data on glucose metabolism, plasma membrane potential, G-protein-coupled-receptors (GPCR), cytoplasmic and endoplasmic reticulum calcium dynamics, cAMP and phospholipase C pathways that regulate interactions between second messengers in pancreatic beta-cells. The values of key model parameters were inferred from published experimental data. The model gives a reasonable fit to important aspects of experimentally measured metabolic and second messenger concentrations and provides a framework for analyzing the role of metabolic, hormones and neurotransmitters changes on insulin secretion. Our analysis of the dynamic data provides support for the hypothesis that activation of Ca2+-dependent adenylyl cyclases play a critical role in modulating the effects of glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and catecholamines. The regulatory properties of adenylyl cyclase isoforms determine fluctuations in cytoplasmic cAMP concentration and reveal a synergistic action of glucose, GLP-1 and GIP on insulin secretion. On the other hand, the regulatory properties of phospholipase C isoforms determine the interaction of glucose, acetylcholine and free fatty acids (FFA) (that act through the FFA receptors) on insulin secretion. We found that a combination of GPCR agonists activating different messenger pathways can stimulate insulin secretion more effectively than a combination of GPCR agonists for a single pathway. This analysis also suggests that the activators of GLP-1, GIP and FFA receptors may have a relatively low risk of hypoglycemia in fasting conditions whereas an activator of muscarinic receptors can increase this risk. This computational analysis demonstrates that study of second messenger pathway interactions will improve understanding of critical regulatory sites, how different GPCRs interact and pharmacological targets for modulating insulin secretion in type 2 diabetes.

CB1 cannabinoid receptor-mediated increases in cyclic AMP accumulation are correlated with reduced Gi/o function.

CB1 cannabinoid receptors (CB1Rs) stimulate Gi/o-dependent signaling pathways. CB1R-mediated cAMP increases were proposed to result from Gs activation, but CB1R-stimulated GTPγS binding to Gs has not heretofore been investigated. Three models of CB1R-stimulated cAMP production were tested: pertussis toxin disruption of Gi/o in N18TG2 cells; L341A/A342L-CB1R expressed in Chinese hamster ovary (CHO) cells; and CB1 and D2 dopamine receptors endogenously co-expressed in MN9D cells. cAMP was assayed by [3H]cAMP binding competition. G protein activation was assayed by the antibody-targeted scintillation proximity assay. In L341A/A342L-CB1-CHO cells, cannabinoid agonists significantly stimulated cAMP accumulation over vehicle; (-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxyl propyl] cyclohexan-1-ol (CP55940)-stimulated [35S]GTPγS binding to Gi1/2/3 was reversed, whereas binding to Gs was not different from CB1R. In MN9D cells, CB1 agonist HU210 or D2 agonist quinpirole alone inhibited forskolin-activated cAMP accumulation, whereas HU210 plus quinpirole increased cAMP accumulation above basal. HU210 alone stimulated [35S]GTPγS binding to Gi1/2/3, whereas co-stimulation with quinpirole reversed HU210-stimulated [35S]GTPγS binding to Gi1/2/3. CB1R couples to Gs but with low efficacy compared to Gi/o. The L341A/A342L mutation in CB1R reversed CP55940 activation of Gi to an inhibition, but had no effect on Gs. Combined CB1 plus D2 agonists in MN9D cells converted the CB1 agonist-mediated activation of Gi to inhibition of Gi. In these models, the CB1 agonist response was converted to an inverse agonist response at Gi activation. Cannabinoid agonist-stimulated cAMP accumulation can be best explained as reduced activation of Gi, thereby attenuating the tonic inhibitory influence of Gi on the major isoforms of adenylyl cyclase.

The IBMX‐insensitive PDE8A is expressed in human airway smooth muscle cells and selectively regulates signaling through AC6

The second messenger cAMP is produced by several different adenylyl cyclase (AC) isoforms in human airway smooth muscle (HASM). We previously defined two distinct cAMP signaling compartments in these cells, one containing AC6 and another containing AC2 (Naunyn‐Schmiedebergs Arch Pharmacol, 387(4):329–39, 2014). We hypothesized that phosphodiesterase (PDE) isozymes may also be compartmentized in HASM. We detected mRNA and protein expression of the IBMX‐insensitive PDE8A in cultured HASM cells. Using lentiviral delivery of shRNA we knocked down PDE8A expression and measured cAMP levels stimulated by forskolin. The broad‐spectrum PDE inhibitor, IBMX, was included in these assays to eliminate contributions by most other PDE isoforms. 1 μM forskolin stimulated 2‐fold higher cAMP in HASM with PDE8A knockdown than in cells treated with control lentivirus. Overexpression of AC2 did not alter this response, but overexpression of AC6 increased this response an additional 80%. We used TaqMan PCR to quantify the expression of AREG (a gene whose expression is regulated by cAMP from both AC2 and AC6) and NR4A2 (a gene regulated only by cAMP from AC6). PDE8A knockdown increased forskolin‐stimulated AREG and NR4A2 expression in AC6 overexpressing HASM but had no effect on either of these genes when AC2 was overexpressed. Quantitative phosphoproteomic analysis of whole cell lysates show that forskolin treatment causes PDE8A phosphorylation in HASM but that only AC6 overexpression increased this phosphorylation. To determine receptor‐mediated signaling regulated by PDE8A, we examined cAMP production in response to isoproterenol (β2AR, which couple primarily to AC6) and butaprost (EP2R, which couple to only AC2). PDE8A knockdown did not alter butaprost‐stimulated cAMP in HASM overexpressing AC2, consistent with no role of this PDE isoform in AC2‐mediated cAMP signaling. Isoproterenol‐stimulated cAMP was reduced by 70% in HASM overexpressing AC6, as effect opposite of what was predicted based on forskolin‐mediated responses. We conclude that PDE8A selectively hydrolyzes cAMP produced by AC6 with no effect on cAMP emanating from AC2. However, knockdown of PDE8A expression causes an uncoupling of β2AR to cAMP production through an undetermined mechanism.Support or Funding InformationSupported by NIH (GM107094) and AHA (14GRNT20380762)

Isoforskolin from Native Plant &amp;lt;i&amp;gt;Coleus forskohlii&amp;lt;/i&amp;gt; of Yunnan, China Plays Multiple Biological Roles

Isoforskolin (ISOF) was isolated from Coleus forskohlii native to Yunnan in China. It is identified as one analog of diterpene forskolin (FSK) which comes from the Indian plant Coleus forskohlii. Researches found that the Yunnan native plant Coleus forskohlii contained rich ISOF but not FSK. ISOF was reported to activate adenylyl cyclase (AC) isoforms. The ophthalmologic and cardiovascular effects of ISOF were firstly reported in the 1990s. The researchers in Kunming, China, found ISOF could lower blood pressure and intraocular pressure. Recent researches found the respiratory tract effects of ISOF, and showed that ISOF significantly elevated cyclic adenosine monophosphate (cAMP) levels in rat lung homogenate, and relaxed the histamine-induced contraction of isolated guinea pig trachea and lung smooth muscle, isoforskolin pretreatment attenuates acute lung injury induced by lipopolysaccharide (LPS) in several models, lowered LPS-induced secretion of the inflammatory cytokine in human monocytes.

The Spatiotemporal Regulation of cAMP Signaling in Blood Platelets-Old Friends and New Players.

Atherothrombosis, the pathology underlying numerous cardiovascular diseases, is a major cause of death globally. Hyperactive blood platelets play a key role in the atherothrombotic process through the release of inflammatory mediators and formation of thrombi. In healthy blood vessels, excessive platelet activation is restricted by endothelial-derived prostacyclin (PGI2) through cyclic adenosine-5′-monophosphate (cAMP) and protein kinase A (PKA)-dependent mechanisms. Elevation in intracellular cAMP is associated with the control of a number of distinct platelet functions including actin polymerisation, granule secretion, calcium mobilization and integrin activation. Unfortunately, in atherosclerotic disease the protective effects of cAMP are compromised, which may contribute to pathological thrombosis. The cAMP signaling network in platelets is highly complex with the presence of multiple isoforms of adenylyl cyclase (AC), PKA, and phosphodiesterases (PDEs). However, a precise understanding of the relationship between specific AC, PKA, and PDE isoforms, and how individual signaling substrates are targeted to control distinct platelet functions is still lacking. In other cells types, compartmentalisation of cAMP signaling has emerged as a key mechanism to allow precise control of specific cell functions. A-kinase anchoring proteins (AKAPs) play an important role in this spatiotemporal regulation of cAMP signaling networks. Evidence of AKAP-mediated compartmentalisation of cAMP signaling in blood platelets has begun to emerge and is providing new insights into the regulation of platelet function. Dissecting the mechanisms that allow cAMP to control excessive platelet activity without preventing effective haemostasis may unleash the possibility of therapeutic targeting of the pathway to control unwanted platelet activity.

Lipopolysaccharide-induced pulmonary endothelial barrier disruption and lung edema: critical role for bicarbonate stimulation of AC10

Bacteria-induced sepsis is a common cause of pulmonary endothelial barrier dysfunction and can progress toward acute respiratory distress syndrome. Elevations in intracellular cAMP tightly regulate pulmonary endothelial barrier integrity; however, cAMP signals are highly compartmentalized: whether cAMP is barrier-protective or -disruptive depends on the compartment (plasma membrane or cytosol, respectively) in which the signal is generated. The mammalian soluble adenylyl cyclase isoform 10 (AC10) is uniquely stimulated by bicarbonate and is expressed in pulmonary microvascular endothelial cells (PMVECs). Elevated extracellular bicarbonate increases cAMP in PMVECs to disrupt the endothelial barrier and increase the filtration coefficient (Kf) in the isolated lung. We tested the hypothesis that sepsis-induced endothelial barrier disruption and increased permeability are dependent on extracellular bicarbonate and activation of AC10. Our findings reveal that LPS-induced endothelial barrier disruption is dependent on extracellular bicarbonate: LPS-induced barrier failure and increased permeability are exacerbated in elevated bicarbonate compared with low extracellular bicarbonate. The AC10 inhibitor KH7 attenuated the bicarbonate-dependent LPS-induced barrier disruption. In the isolated lung, LPS failed to increase Kf in the presence of minimal perfusate bicarbonate. An increase in perfusate bicarbonate to the physiological range (24 mM) revealed the LPS-induced increase in Kf, which was attenuated by KH7. Furthermore, in PMVECs treated with LPS for 6 h, there was a dose-dependent increase in AC10 expression. Thus these findings reveal that LPS-induced pulmonary endothelial barrier failure requires bicarbonate activation of AC10.

Deficits in behavioral sensitization and dopaminergic responses to methamphetamine in adenylyl cyclase 1/8-deficient mice.

The cAMP/protein kinase A pathway regulates methamphetamine (METH)‐induced neuroplasticity underlying behavioral sensitization. We hypothesize that adenylyl cyclases (AC) 1/8 mediate these neuroplastic events and associated striatal dopamine regulation. Locomotor responses to METH (1 and 5 mg/kg) and striatal dopamine function were evaluated in mice lacking AC 1/8 (DKO) and wild‐type (WT) mice. Only 5 mg/kg METH induced an acute locomotor response in DKO mice, which was significantly attenuated versus WT controls. DKO mice showed a marked attenuation in the development and expression of METH‐induced behavioral sensitization across doses relative to WT controls. While basal and acute METH (5 mg/kg)‐evoked accumbal dialysate dopamine levels were similar between genotypes, saline‐treated DKO mice showed elevated tissue content of dopamine and homovanillic acid in the dorsal striatum (DS), reflecting dysregulated dopamine homeostasis and/or metabolism. Significant reductions in DS dopamine levels were observed in METH‐sensitized DKO mice compared to saline‐treated controls, an effect not observed in WT mice. Notably, saline‐treated DKO mice had significantly increased phosphorylated Dopamine‐ and cAMP‐regulated phosphoprotein levels, which were not further augmented following METH sensitization, as observed in WT mice. These data indicate that AC 1/8 are critical to mechanisms subserving drug‐induced behavioral sensitization and mediate nigrostriatal pathway METH sensitivity. Calcium/calmodulin‐stimulated adenylyl cyclase (AC) isoforms 1 and 8 were studied for their involvement in the adaptive neurobehavioral responses to methamphetamine. AC 1/8 double knockout (DKO) mice showed heightened basal locomotor activity and dorsal striatal dopamine responsivity. Conversely, methamphetamine‐induced locomotor activity was attenuated in DKO mice, accompanied by reductions in dopamine and HVA content and impaired DARPP‐32 activation. These findings indicate AC 1/8 signaling regulates the sensitivity of the nigrostriatal pathway subserving stimulant and neuroadaptive sensitizing effects of methamphetamine. 3‐MT, 3‐methoxytyramine; Ca2+, calcium; CaM, calmodulin; cdk5; cyclin‐dependent kinase 5; DA, dopamine; DARPP‐32, dopamine‐ and cAMP‐regulated phosphoprotein; D1R, dopamine D1 receptor; HVA, homovanillic acid; PKA, protein kinase A.