Adenylyl Cyclase Isoforms Research Articles

Effects of Straight Chain Alcohols on Specific Isoforms of Adenylyl Cyclase

Our previous studies showed that the activity of adenylyl cyclase (AC) was enhanced by pharmacologically relevant concentrations of ethanol, that this enhancing effect of ethanol on AC activity was AC isoform specific, and that the alcohol cutoff effect for n-alkanol potentiation of AC activity was also AC isoform specific. Therefore, we hypothesized that within the cyclic AMP-generating system, AC is the target of ethanol's action and that alcohols interact directly with the AC molecules. To characterize the interaction between alcohols and AC proteins, the effects of a series of straight chain alcohols would be very valuable in understanding alcohol action at the molecular level. To our knowledge, straight chain alcohols other than n-alkanols and 1,Omega-diols have not been used extensively to study alcohol effects on the activity of AC or other proteins important in the alcohol research field. The effects of a series of straight chain alcohols on D1A dopamine receptor-stimulated activity of AC isoforms type 6, 7, and 9 (AC6, AC7, and AC9) were examined in transfected Hela cells by a cAMP accumulation assay. In general, all 3 AC isoforms responded to a series of straight chain alcohols in a similar manner. The order of responsiveness is as follows: monoalcohol > diol > triol and tetraol. Within monoalcohols, 1-alcohols had larger effects than 2-alcohols. Two of 3 stereoisomers of 2,3-butanediol, [D-(-)-2,3-butanediol and meso-2,3-butanediol] showed similar enhancing effects on all 3 AC isoforms. However, the third stereoisomer, L-(+)-2,3-butanediol, inhibited AC7 activity, while it stimulated AC6 and AC9. The number and the position of hydroxyl groups in straight chain alcohols play an important role in the magnitude of the enhancement on AC activity. Regardless of AC isoforms, the most effective of the straight chain alcohols seems to be the 1-alcohol (n-alkanol) for a given chain length. We found that one of the stereoisomers of 2,3-butanediol had opposite effects on AC activity depending on the AC isoform. Overall, the results are consistent with the hypotheses and demonstrate that a series of straight chain alcohols can be a valuable tool to study AC-alcohol interactions.

Nerve Growth Factor-Induced Differentiation of PC12 Cells Is Accompanied by Elevated Adenylyl Cyclase Activity

Rat pheochromocytoma (PC12) cells characteristically undergo differentiation when cultured with nerve growth factor (NGF). Here we show that NGF dramatically increased the adenylyl cyclase-activating property of forskolin in PC12 cells. This effect of NGF was well maintained even when NGF was removed after 4 days, even though the morphological features of neuronal differentiation were rapidly lost on removal of NGF. The enhanced cAMP production in response to forskolin could be due to a synergistic interaction between forskolin and endogenously released agonists acting on G_s-coupled receptors. However, responses to forskolin were not attenuated by antagonists of adenosine A2 receptors or pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, suggesting that adenosine and PACAP were not involved. Adenylyl cyclases 3, 6 and 9 were the predominant isoforms expressed in PC12 cells, but we found no evidence for NGF-induced changes in expression levels of any of the 9 adenylyl cyclase isoforms, nor in the expression of Gα_s. These findings highlight that NGF has a subtle influence on adenylyl cyclase activity in PC12 cells which may influence more than the neurite extension process classically associated with neuronal differentiation.

AC3: A Novel Gene Plays a Role in the Regulation of Body Weight

AC3 is one of adenylyl cyclase isoforms involved in cAMP and insulin signaling pathway. A previous study using Goto-Kakizaki rat, a non-obese type 2 diabetes model, indicates that AC3 is over-expressed in pancreatic islets. A recent genetic study has demonstrated that AC3 DNA polymorphisms are associated with body mass index (BMI) in the subjects with obesity and type 2 diabetes. Furthermore, AC3 knock out mice exhibit obese when they age mainly due to low locomoter activity, hyperphagia and leptin insensitivity. These findings suggest that AC3 plays an important role in the regulation of body weight. This review summarizes genetic and biological relevancies of AC3 in the regulation of body weight and also discusses about the potential development of anti-obesity drug using AC3 as a target.

A timely characterization of vasopressin-sensitive adenylyl cyclase isoforms in the mouse inner medullary collecting duct

second messenger signaling in the kidney mediates several of the organ's basic physiological functions including the regulation of blood pressure, control of fluid volume, and maintenance of the body's water homeostasis in times of duress. Tight regulation of the second messenger cAMP is critical

Disparate developmental neurotoxicants converge on the cyclic AMP signaling cascade, revealed by transcriptional profiles in vitro and in vivo

Cell-signaling cascades are convergent targets for developmental neurotoxicity of otherwise unrelated agents. We compared organophosphates (chlorpyrifos, diazinon), an organochlorine (dieldrin) and a metal (Ni 2+) for their effects on neuronotypic PC12 cells, assessing gene transcription involved in the cyclic AMP pathway. Each agent was introduced during neurodifferentiation at a concentration of 30 μM for 24 or 72 h and we assessed 69 genes encoding adenylyl cyclase isoforms and regulators, G-protein α-and β,γ-subunits, protein kinase A subtypes and the phosphodiesterase family. We found strong concordance among the four agents across all the gene families, with the strongest relationships for the G-proteins, followed by adenylyl cyclase, and lesser concordance for protein kinase A and phosphodiesterase. Superimposed on this pattern, chlorpyrifos and diazinon were surprisingly the least alike, whereas there was strong concordance of dieldrin and Ni 2+ with each other and with each individual organophosphate. Further, the effects of chlorpyrifos differed substantially depending on whether cells were undifferentiated or differentiating. To resolve the disparities between chlorpyrifos and diazinon, we performed analyses in rat brain regions after in vivo neonatal exposures; unlike the in vitro results, there was strong concordance. Our results show that unrelated developmental neurotoxicants can nevertheless produce similar outcomes by targeting cell signaling pathways involved in neurodifferentiation during a critical developmental period of vulnerability. Nevertheless, a full evaluation of concordance between different toxicants requires evaluations of in vitro systems that detect direct effects, as well as in vivo systems that allow for more complex interactions that converge on the same pathway.

Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells

Microdomains have been proposed to explain specificity in the myriad of possible cellular targets of cAMP. Local differences in cAMP levels can be generated by phosphodiesterases, which control the diffusion of cAMP. Here, we address the possibility that adenylyl cyclases, the source of cAMP, can be primary architects of such microdomains. Distinctly regulated adenylyl cyclases often contribute to total cAMP levels in endogenous cellular settings, making it virtually impossible to determine the contribution of a specific isoform. To investigate cAMP dynamics with high precision at the single-isoform level, we developed a targeted version of Epac2-camps, a cAMP sensor, in which the sensor was tagged to a catalytically inactive version of the Ca(2+)-stimulable adenylyl cyclase 8 (AC8). This sensor, and less stringently targeted versions of Epac2-camps, revealed opposite regulation of cAMP synthesis in response to Ca(2+) in GH(3)B(6) pituitary cells. Ca(2+) release triggered by thyrotropin-releasing hormone stimulated the minor endogenous AC8 species. cAMP levels were decreased by inhibition of AC5 and AC6, and simultaneous activation of phosphodiesterases, in different compartments of the same cell. These findings demonstrate the existence of distinct adenylyl-cyclase-centered cAMP microdomains in live cells and open the door to their molecular micro-dissection.

Direct demonstration of discrete Ca2+ microdomains associated with different isoforms of adenylyl cyclase

Ca(2+)-sensitive adenylyl cyclases (ACs) orchestrate dynamic interplay between Ca(2+) and cAMP that is a crucial feature of cellular homeostasis. Significantly, these ACs are highly selective for capacitative Ca(2+) entry (CCE) over other modes of Ca(2+) increase. To directly address the possibility that these ACs reside in discrete Ca(2+) microdomains, we tethered a Ca(2+) sensor, GCaMP2, to the N-terminus of Ca(2+)-stimulated AC8. GCaMP2-AC8 measurements were compared with global, plasma membrane (PM)-targeted or Ca(2+)-insensitive AC2-targeted GCaMP2. In intact cells, GCaMP2-AC8 responded rapidly to CCE, but was largely unresponsive to other types of Ca(2+) rise. The global GCaMP2, PM-targeted GCaMP2 and GCaMP2-AC2 sensors reported large Ca(2+) fluxes during Ca(2+) mobilization and non-specific Ca(2+) entry, but were less responsive to CCE than GCaMP2-AC8. Our data reveal that different AC isoforms localize to distinct Ca(2+)-microdomains within the plasma membrane. AC2, which is regulated via protein kinase C, resides in a microdomain that is exposed to a range of widespread Ca(2+) signals seen throughout the cytosol. By contrast, a unique Ca(2+) microdomain surrounds AC8 that promotes selectivity for Ca(2+) signals arising from CCE, and optimizes CCE-mediated cAMP synthesis. This direct demonstration of discrete compartmentalized Ca(2+) signals associated with specific signalling proteins provides a remarkable insight into the functional organization of signalling microdomains.

Characterization of vasopressin-responsive collecting duct adenylyl cyclases in the mouse

Little is known about collecting duct adenylyl cyclase (AC) isoforms or regulation in the mouse. We performed RT-PCR for AC isoforms 1-9 in microdissected cortical (CCD) and outer medullary (OMCD) and acutely isolated inner medullary (IMCD) collecting duct. All collecting duct regions contained AC3, AC4, and AC6 mRNA, while CCD and OMCD, but not IMCD, also contained AC5 mRNA. Acutely isolated IMCD expressed AC3, AC4, and AC6 proteins by Western blot analysis. The mIMCD3 cell line expressed AC2, AC3, AC4, AC5, and AC6 mRNA; M-1 CCD cells expressed AC2, 3, 4, and 6, while mpkCCD cell lines contained AC3, AC4, and AC6 mRNA. AVP stimulated cAMP accumulation in acutely isolated mouse IMCD; this was reduced by chelation of extracellular calcium (EGTA) and almost completely abolished by blockade of calmodulin (W-7). Blockade of calmodulin kinase with KN-93 or endoplasmic reticulum calcium ATPase (thapsigargin) also reduced the AVP response. A similar inhibitory effect of W-7, KN-93, and thapsigargin was seen on forskolin-stimulated cAMP content in acutely isolated mouse IMCD. These three agents had the same pattern of blockade of AVP- or forskolin-stimulated AC activity in acutely isolated rat IMCD. AVP responsiveness in primary cultures of mouse IMCD was also reduced by W-7, KN-93, and thapsigargin. Small interfering RNA (siRNA) designed to knock down AC3 or AC6 in primary cultured mouse IMCD significantly reduced AVP-stimulated cAMP accumulation. Together, these data are consistent with a role of AC3 and AC6 in the activation of mouse collecting duct by AVP.

Inhibition of specific adenylyl cyclase isoforms by lithium and carbamazepine, but not valproate, may be related to their antidepressant effect

Lithium, valproate, and carbamazepine decrease stimulated brain cyclic-AMP (cAMP) levels. Adenylyl cyclase (AC), of which there are nine membrane-bound isoforms (AC1-AC9), catalyzes the formation of cAMP. We have recently demonstrated preferential inhibition of AC5 by lithium. We now sought to determine whether carbamazepine and valproate also preferentially inhibit specific AC isoforms or decrease cAMP levels via different mechanisms. COS7 cells were transfected with one of AC1-AC9, with or without D1-dopamine receptors. Carbamazepine's and valproate's effect on forskolin- or D1 agonist-stimulated ACs was studied. The effect of Mg(2+) on lithium's inhibition was studied in membrane-enriched fraction from COS7 cells co-expressing AC5 and D1 receptors. AC5 knockout mice were tested for a behavioral phenotype similar to that of lithium treatment. Carbamazepine preferentially inhibited forskolin-stimulated AC5 and AC1 and all D1 agonist-stimulated ACs, with AC5 and AC7 being the most sensitive. When compared to 1 or 3 mM Mg(2+), 10 mM Mg(2+) reduced lithium-induced AC5 inhibition by 70%. In silico modeling suggests that among AC isoforms carbamazepine preferentially affects AC1 and AC5 by interacting with the catechol-estrogen site. Valproate did not affect any forskolin- or D1 receptor-stimulated AC. AC5 knockout mice responded similarly to antidepressant- or lithium-treated wild-types in the forced-swim test but not in the amphetamine-induced hyperactivity mania model. Lithium and carbamazepine preferentially inhibit AC5, albeit via different mechanisms. Lithium competes with Mg(2+), which is essential for AC activity; carbamazepine competes for AC's catechol-estrogen site. Antidepressant-like behavior of AC5 knockout mice in the forced-swim test supports the notion that AC5 inhibition is involved in the antidepressant effect of lithium and carbamazepine. The effect of lithium and carbamazepine to lower cAMP formation in AC5-rich dopaminergic brain regions suggests that D1-dopamine receptors in these regions are involved in the antidepressant effect of mood stabilizers.

Dopamine D1, D2 and mu-opioid receptors are co-expressed with adenylyl cyclase 5 and phosphodiesterase 7B mRNAs in striatal rat cells

Intracellular cAMP levels are regulated by cAMP synthesis and degradation rate. Nine isoforms of cAMP-synthesizing enzymes called adenylyl-cyclases (ACs) and eleven phosphodiesterases (PDEs) that degrade cyclic nucleotides have been identified. Both types of enzymes exhibit variations not only in their expression pattern distribution throughout the brain, but also in their regulatory characteristics. Different isoforms of ACs and PDEs may be co-expressed in a single cell, thus a gradient of cAMP intracellular levels is formed, which accounts for the diversity of cell responses. Among these isoforms, AC5 and PDE7B are highly expressed in striatum, where the cAMP pathway is implicated in diverse behavioural functions. Striatal AC5 is involved in drug reinforcing actions and motor activity. Less is known about the role of the PDE7B isoenzyme. We performed a double in situ hybridization analysis of the co-expression patterns of AC5 and PDE7B with μ-opioid-receptor (MOR), D1- and D2-receptor mRNAs to contribute to a better understanding in the regulation of cAMP levels under dopamine or opioidergic pathway activation in striatum. We found co-expression of AC5 and PDE7B mRNAs in caudate-putamen and nucleus accumbens; we also encountered that more than 50% of MOR, D2- and D1-expressing cells contained AC5 and PDE7B mRNAs. The presence of AC5 and PDE7B mRNAs in D1- and D2-containing cells suggests the participation of these enzymes in striatal functions involving dopaminergic pathways. Co-localization of both isoenzyme mRNAs with MOR expressing cells suggests their involvement in opioid reinforcing effects.

Adenylyl Cyclase Type 5 Contributes to Corticostriatal Plasticity and Striatum-Dependent Learning

Dopamine (DA)-dependent corticostriatal plasticity is thought to underlie incremental procedural learning. A primary effector of striatal DA signaling is cAMP, yet its role in corticostriatal plasticity and striatum-dependent learning remains unclear. Here, we show that genetic deletion of a striatum-enriched isoform of adenylyl cyclase, AC5 knock-out (AC5KO), impairs two forms of striatum-dependent learning and corticostriatal synaptic plasticity. AC5KO mice were severely impaired in acquisition of a response strategy in the cross maze, a striatum-dependent task requiring a correct body turn to find a goal arm. In addition, AC5KO mice were impaired in acquisition of a motor skill, as assessed by the accelerated rotarod. Slice electrophysiology revealed a deficit in corticostriatal long-term depression (LTD) after high-frequency stimulation of tissue from AC5KO mice. LTD was rescued by activation of either presynaptic cannabinoid type 1 (CB(1)) receptors or postsynaptic metabotropic glutamate receptors (mGluRs), suggesting a postsynaptic role of AC5-cAMP, upstream of endocannabinoid release. In striatopallidal-projecting medium spiny neurons, DA D(2) receptors are negatively coupled to cAMP production, and activation of these receptors is required for endocannabinoid release and corticostriatal LTD. Recordings from striatopallidal neurons indicated that this is mediated by AC5, because coactivation of D(2) and mGluRs could induce LTD in wild-type but not in AC5KO neurons. To further examine the role of cAMP in corticostriatal plasticity, we elevated cAMP in striatal neurons of wild-type mice via the recording electrode. Under these conditions, corticostriatal LTD was eliminated. Together, these data suggest an AC5-cAMP-endocannabinoid-CB(1) signaling pathway in corticostriatal plasticity and striatum-dependent learning.

Adenylyl cyclase type 5 protein expression during cardiac development and stress

Adenylyl cyclase (AC) types 5 and 6 (AC5 and AC6) are the two major AC isoforms expressed in the mammalian heart that mediate signals from beta-adrenergic receptor stimulation. Because of the unavailability of isoform-specific antibodies, it is difficult to ascertain the expression levels of AC5 protein in the heart. Here we demonstrated the successful generation of an AC5 isoform-specific mouse monoclonal antibody and studied the expression of AC5 protein during cardiac development in different mammalian species. The specificity of the antibody was confirmed using heart and brain tissues from AC5 knockout mice and from transgenic mice overexpressing AC5. In mice, the AC5 protein was highest in the brain but was also detectable in all organs studied, including the heart, brain, lung, liver, stomach, kidney, skeletal muscle, and vascular tissues. Western blot analysis showed that AC5 was most abundant in the neonatal heart and declined to basal levels in the adult heart. AC5 protein increased in the heart with pressure-overload left ventricular hypertrophy. Thus this new AC5 antibody demonstrated that this AC isoform behaves similarly to fetal type genes, such as atrial natriuretic peptide; i.e., it declines with development and increases with pressure-overload hypertrophy.

Adenylyl Cyclase–A-kinase Anchoring Protein Complexes: The Next Dimension in cAMP Signaling

The formation of multiprotein complexes is a repeated theme in biology ranging from the regulation of the extracellular signal-regulated kinase and cAMP signaling pathways to the formation of postsynaptic density complexes or tight junctions. A-kinase anchoring proteins (AKAPs) are well known for their ability to scaffold protein kinase A and components upstream and downstream of cAMP production, including G protein-coupled receptors, cAMP-dependent Rap-exchange factors, and phosphodiesterases. Specific adenylyl cyclase (AC) isoforms have also been identified as components of AKAP complexes, namely AKAP79, Yotiao, and mAKAP. In this review, we summarize recent evidence for AC-AKAP complexes and requirements for compartmentalization of cAMP signaling. The ability of AKAPs to assemble intricate feedback loops to control spatiotemporal aspects of cAMP signaling adds yet another dimension to the classic cAMP pathway.

An Adenylyl Cyclase-mAKAPβ Signaling Complex Regulates cAMP Levels in Cardiac Myocytes

Protein kinase A-anchoring proteins (AKAPs) play important roles in the compartmentation of cAMP signaling, anchoring protein kinase A (PKA) to specific cellular organelles and serving as scaffolds that assemble localized signaling cascades. Although AKAPs have been recently shown to bind adenylyl cyclase (AC), the functional significance of this association has not been studied. In cardiac myocytes, the muscle protein kinase A-anchoring protein beta (mAKAPbeta) coordinates cAMP-dependent, calcium, and MAP kinase pathways and is important for cellular hypertrophy. We now show that mAKAPbeta selectively binds type 5 AC in the heart and that mAKAPbeta-associated AC activity is absent in AC5 knock-out hearts. Consistent with its known inhibition by PKA phosphorylation, AC5 is inhibited by association with mAKAPbeta-PKA complexes. AC5 binds to a unique N-terminal site on mAKAP-(245-340), and expression of this peptide disrupts endogenous mAKAPbeta-AC association. Accordingly, disruption of mAKAPbeta-AC5 complexes in neonatal cardiac myocytes results in increased cAMP and hypertrophy in the absence of agonist stimulation. Taken together, these results show that the association of AC5 with the mAKAPbeta complex is required for the regulation of cAMP second messenger controlling cardiac myocyte hypertrophy.

The role of calcium in the regulation of renin secretion.

Renin is the enzyme which is the rate-limiting step in the formation of the hormone angiotensin II. Therefore, the regulation of renin secretion is critical in understanding the control of the renin-angiotensin-aldosterone system and its many biological and pathological actions. Renin is synthesized, stored in, and released from the juxtaglomerular (JG) cells of the kidney. While renin secretion is positively regulated by the "second messenger" cAMP, unlike most secretory cells, renin secretion from the JG cell is inversely related to the extracellular and intracellular calcium concentrations. This novel relationship is referred to as the "calcium paradox." This review will address observations made over the past 30 years regarding calcium and the regulation of renin secretion, and focus on recent observations which address this scientific conundrum. These include 1) receptor-mediated pathways for changing intracellular calcium; 2) the discovery of a calcium-inhibitable isoform of adenylyl cyclase associated with renin in the JG cells; 3) calcium-sensing receptors in the JG cells; 4) calcium-calmodulin-mediated signals; 5) the role of phosphodiesterases; and 6) connexins, gap junctions, calcium waves, and the cortical extracellular calcium environment. While cAMP is the dominant second messenger for renin secretion, calcium appears to modulate the integrated activities of the enzymes, which balance cAMP synthesis and degradation. Thus this review concludes that calcium modifies the amplitude of cAMP-mediated renin-signaling pathways. While calcium does not directly control renin secretion, increased calcium inhibits and decreased calcium amplifies cAMP-stimulated renin secretion.

Differential Inhibition of Various Adenylyl Cyclase Isoforms and Soluble Guanylyl Cyclase by 2′,3′-O-(2,4,6-Trinitrophenyl)-Substituted Nucleoside 5′-Triphosphates

Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP and play a key role in signal transduction. In a recent study (Mol Pharmacol 70:878-886, 2006), we reported that 2',3'-O-(2,4,6-trinitrophenyl)-substituted nucleoside 5'-triphosphates (TNP-NTPs) are potent inhibitors (K(i) values in the 10 nM range) of the purified catalytic subunits VC1 and IIC2 of membranous AC (mAC). The crystal structure of VC1:IIC2 in complex with TNP-ATP revealed that the nucleotide binds to the catalytic site with the TNP-group projecting into a hydrophobic pocket. The aims of this study were to analyze the interaction of TNP-nucleotides with VC1:IIC2 by fluorescence spectroscopy and to analyze inhibition of mAC isoforms, soluble AC (sAC), soluble guanylyl cyclase (sGC), and G-proteins by TNP-nucleotides. Interaction of VC1:IIC2 with TNP-NDPs and TNP-NTPs resulted in large fluorescence increases that were differentially reduced by a water-soluble forskolin analog. TNP-ATP turned out to be the most potent inhibitor for ACV (K(i), 3.7 nM) and sGC (K(i), 7.3 nM). TNP-UTP was identified as the most potent inhibitor for ACI (K(i), 7.1 nM) and ACII (K(i), 24 nM). TNP-NTPs inhibited sAC and GTP hydrolysis by G(s)- and G(i)-proteins only with low potencies. Molecular modeling revealed that TNP-GTP and TNP-ATP interact very similarly, but not identically, with VC1:IIC2. Collectively, our data show that TNP-nucleotides are useful fluorescent probes to monitor conformational changes in VC1:IIC2 and that TNP-NTPs are a promising starting point to develop isoform-selective AC and sGC inhibitors. TNP-ATP is the most potent sGC inhibitor known so far.

Differential interactions of the catalytic subunits of adenylyl cyclase with forskolin analogs

The diterpene forskolin (FS) binds to, and activates, mammalian membranous adenylyl cyclase (AC) isoforms I–VIII. Diterpenes without C 1–OH group do not activate ACs. The C 1–OH group forms a hydrogen bond with the backbone oxygen of Val506 of the C1 catalytic subunit of AC (isoform V numbering). To better understand the mechanism of AC activation we examined the interactions of FS and eight FS analogs with purified catalytic AC subunits C1 (AC V) and C2 (AC II) by fluorescence spectroscopy, using 2′,3′- O-( N-methylanthraniloyl)-guanosine 5′-triphosphate (MANT-GTP) as fluorescent reporter probe, and by enzymatic activity. FS analogs induced C1/C2 assembly as assessed by fluorescence resonance energy transfer from Trp1020 of C2 to MANT-GTP and by increased direct MANT-GTP fluorescence in the order of efficacy FS ∼ 7-deacetyl-FS ∼ 6-acetyl-7-deacetyl-FS ∼ 9-deoxy-FS > 7-deacetyl-7-(N-methylpiperazino-γ-butyryloxy)-FS > 1-deoxy-FS ∼ 1,9-dideoxy-FS ∼ 7-deacetyl-1-deoxy-FS ∼ 7-deacetyl-1,9-dideoxy-FS. In contrast, FS analogs activated catalysis in the order of efficacy FS > 7-deacety-FS ∼ 6-acetyl-7-deacetyl-FS ∼ 9-deoxy-FS > 7-deacetyl-7-( N-methylpiperazino-γ-butyryloxy)-FS ≫ 1-deoxy-FS, 1,9-dideoxy-FS, 7-deacetyl-1-deoxy-FS and 7-deacetyl-1,9-dideoxy-FS (all ineffective). 1-Deoxy-FS analogs inhibited FS-stimulated catalysis by an apparently non-competitive mechanism. Our data suggest a two-step mechanism of AC activation by diterpenes. In the first step, diterpenes, regardless of their substitution pattern, promote C1/C2 assembly. In the second and yet poorly understood step, diterpenes that form a hydrogen bond between C 1–OH and Val506 promote a conformational switch that results in activation of catalysis. The apparent non-competitive interaction of FS with 1-deoxy-FS analogs is explained by impaired ligand exchange due to strong hydrophobic interactions with C1/C2.

Localization and coupling of adenylyl cyclase isoforms 2, 3 and 6 with G protein‐coupled receptors in mouse bronchial smooth muscle cells

Several GPCR stimulate adenylyl cyclase (AC) to increase cAMP and promote relaxation in smooth muscle. β‐adrenergic receptors only couple to certain AC isoforms due to compartmentation in lipid rafts. Previous data show AC2, AC3, AC4 and AC6 are expressed in primary mouse bronchial smooth muscle cells (mBSMC). We overexpressed AC2, AC3 or AC6 in mBSMC using recombinant adenoviruses and assessed their localization by cell fractionation and confocal microscopy then determined the coupling of various GPCR to each AC isoform by examining the arborization response to receptor‐specific agonists. Lipid rafts were isolated using a detergent‐based fractionation method and fractions were analyzed by immunoblotting. AC3 and AC6 were expressed primarily in caveolin‐rich fractions, whereas the AC2 expression was excluded from these domains. Forskolin‐induced arborization of mBSMC, measured by time‐lapse microscopy, was enhanced by overexpression of all three AC isoforms. Isoproterenol‐ or beraprost‐mediated arborization was only accelerated by AC6 overexpression. In contrast, butaprost‐mediated arborization was only accelerated by AC2 overexpression. AC3 overexpression did not significantly alter the response to any of these agonists. Our results imply that particular GPCR couple to discreet AC isoforms, likely based upon their colocalization. Supported by HL079166.

The Level of Cardiac Specific Overexpression of Adenylyl Cyclase Type 2 Dictates the Response to Chronic Pressure Overload

The major adenylyl cyclase (AC) isoforms in the heart are AC5 and AC6, which when overexpressed, exert different effects on the response to chronic pressure overload (CPO). Accordingly, the goal of this study was to examine the effects of overexpression (OE) of an AC isoform, which does not normally play a major role in the heart, eg, AC2, on the response to CPO. Two lines were generated: one with moderate AC2 OE of the transgene and the other with a higher level. The AC2 protein was 2 times greater in the line with higher OE. At least 6 WT and 6 AC2 OE from each group were subjected to transverse aortic constriction (TAC) or sham (S) operation for 1 week. With moderate AC2 OE, CPO did not affect the LV ejection fraction (EF) measured with echocardiography (75±1%) vs WT banded (72±1%), or with sham operated animals (76±1%). With higher AC2 OE, 1 week of CPO leads to a significant decrease in LVEF compared with WT (47±4 vs 72±1%) and increase in LVEDP (22.1±7.8 vs 3.5±0.5 mmHg). These were associated with more LV hypertrophy [LV weight/body weight (BW) ratio increased 121% vs 31%] and development of heart failure, as reflected by increased lung weight/BW (278%) vs WT (11%), and 3‐fold more fibrosis on histopathological examination. Thus, cardiac expression of AC2 at a moderate level does not affect the response to CPO adversely, but at a high level, results in more LV hypertrophy and reduced cardiac function leading to heart failure.

Capturing adenylyl cyclases as potential drug targets

Cyclic AMP (cAMP) is an important intracellular signalling mediator. It is generated in mammals by nine membrane-bound and one soluble adenylyl cyclases (ACs), each with distinct regulation and expression patterns. Although many drugs inhibit or stimulate AC activity through the respective upstream G-protein coupled receptors (for example, opioid or beta-adrenergic receptors), ACs themselves have not been major drug targets. Over the past decade studies on the physiological functions of the different mammalian AC isoforms as well as advances in the development of isoform-selective AC inhibitors and activators suggest that ACs could be useful drug targets. Here we discuss the therapeutic potential of isoform-selective compounds in various clinical settings, including neuropathic pain, neurodegenerative disorders, congestive heart failure, asthma and male contraception.