Mechanism of Galpha i-mediated inhibition of type V adenylyl cyclase

The topology of mammalian adenylyl cyclase reveals an integral membrane protein composed of an alternating series of membrane and cytoplasmic domains (C1 and C2). The stimulatory G protein, Galpha(s), binds within a cleft in the C2 domain of adenylyl cyclase while Galpha(i) binds within the opposite cleft in the C1 domain. The mechanism of these two regulators also appears to be in opposition. Activation of adenylyl cyclase by Galpha(s) or forskolin results in a 100-fold increase in the apparent affinity of the two domains for one another. We show herein that Galpha(i) reduces C1/C2 domain interaction and thus formation of the adenylyl cyclase catalytic site. Mutants that increase the affinity of C1 for C2 decrease the ability of Galpha(i) to inhibit the enzyme. In addition, Galpha(i) can influence binding of molecules to the catalytic site, which resides at the C1/C2 interface. Adenylyl cyclase can bind substrate analogs in the presence of Galpha(i) but cannot simultaneously bind Galpha(i) and transition state analogs such as 2'd3'-AMP. Galpha(i) also cannot inhibit the membrane-bound enzyme in the presence of manganese, which increases the affinity of adenylyl cyclase for ATP and substrate analogs. Thus homologous G protein alpha-subunits promote bidirectional regulation at the domain interface of the pseudosymmetrical adenylyl cyclase enzyme.     

Mechanism of cicaprost-induced desensitization in rat pulmonary artery smooth muscle cells involves a PKA-mediated inhibition of adenylyl cyclase

Long-term infusion of prostacyclin, or its analogs, is an effective treatment for severe pulmonary arterial hypertension. However, dose escalation is often required to maintain efficacy. The aim of this study was to investigate the mechanisms of prostacyclin receptor desensitization using the prostacyclin analog cicaprost in rat pulmonary artery smooth muscle cells (PASMCs). Desensitization of the cAMP response occurred in 63 nM cicaprost after a 6-h preincubation with agonist. This desensitization was reversed 12 h after agonist removal, and resensitization was inhibited by 10 microg/ml of cycloheximide. Desensitization was heterologous since desensitization to other G(s)alpha-adenylyl cyclase (AC)-coupled agonists, isoproterenol (1 microM), adrenomedullin (100 nM), or bradykinin (1 microM), was also reduced by preincubation with cicaprost. The reduced cAMP response to prolonged cicaprost exposure appeared to be due to inhibition of AC activity since the responses to the directly acting AC agonist forskolin (3 microM) and the selective AC5 activator NKH-477 were similarly reduced. Expression of AC2 and AC5/6 protein levels transiently decreased after 1 h of cicaprost exposure. The PKA inhibitor H-89 (1 microM) added 1 h before cicaprost preincubation (6 h, 63 nM) completely reversed cicaprost-induced desensitization, whereas the PKC inhibitor bisindolylmaleimide (100 nM) was only partly effective. Desensitization was not prevented by the G(i) inhibitor pertussis toxin. In conclusion, chronic treatment of PASMCs with cicaprost induced heterologous, reversible desensitization by inhibition of AC activity. Our data suggest that heterologous G(s)alpha desensitization by cicaprost is mediated predominantly by a PKA-inhibitable isoform of AC, most likely AC5/6.     

Human airway smooth muscle expresses 7 isoforms of adenylyl cyclase: a dominant role for isoform V

Adenylyl cyclases are a nine-member family of differentially regulated enzymes responsible for the synthesis of cAMP. cAMP is an important second messenger that contributes to the regulation of airway smooth muscle tone. However, little is known regarding the expression and regulation of adenylyl cyclase isoforms in airway smooth muscle cells. Nondegenerate specific primers were designed for all nine known isoforms of human adenylyl cyclase. RT-PCR experiments were performed using total RNA extracted from whole human brain (positive control), whole rat brain (negative control), whole human trachea, human airway smooth muscle, and primary cultures of human airway smooth muscle cells. Seven of the nine known isoforms of adenylyl cyclase (isoforms I, III-VII, and IX) were expressed at the mRNA level in both human airway smooth muscle and primary cultures of human airway smooth muscle cells. Immunoblot and adenylyl cyclase functional assay indicated that isoform V is likely among the functionally predominant isoforms of adenylyl cyclase in human airway smooth muscle. These results suggest that multiple isoforms of adenylyl cyclase enzymes are coexpressed in human airway smooth muscle cells and that isoform V is among the functionally important isoforms.     

Decreased adenylyl cyclase protein and function in airway smooth muscle by chronic carbachol pretreatment

Cellular levels of cAMP arean important determinant of airway smooth muscle tone. We havepreviously shown that chronic (18 h) but not acute (30 min or 2 h)pretreatment with the muscarinic receptor agonist carbachol resulted indecreased adenylyl cyclase activity in response to GTP, isoproterenol,or forskolin via a pathway blocked by the protein kinase C inhibitorstaurosporine. The present study was designed to determine ifcarbachol-induced decreases in adenylyl cyclase activity were due toregulatory events at the level of either G_s or adenylylcyclase. Detergent-solubilized G_s from control orcarbachol-pretreated bovine airway smooth muscle had similar adenylylcyclase activity in response to either NaF or guanosine5'-O-(3-thiotriphosphate) (GTPS) when reconstituted intoS49 cyc membranes that lack endogenous G_s(carbachol pretreated: GTPS, 93 ± 13% of control;NaF/AlCl₃, 99 ± 8.6% of control; n = 4). Exogenous G_s solubilized from red blood cells failedto restore normal adenylyl cyclase activity when reconstituted intocarbachol-pretreated bovine airway smooth muscle (carbachol pretreated:GTP, 36 ± 10% of control; NaF/AlCl₃, 54 ± 11%of control; n = 4). [³H]forskolinradioligand saturation binding assays revealed a decreased quantity oftotal adenylyl cyclase protein after carbachol pretreatment (maximalbinding: 152 ± 40 and 107 ± 31 fmol/mg protein in control and carbachol-pretreated airway smooth muscle, respectively). Theseresults suggest that chronic activation of muscarinic receptors downregulates the expression of adenylyl cyclase protein in bovine airway smooth muscle.

     

Nitric oxide modulates Gi-protein expression and adenylyl cyclase signaling in vascular smooth muscle cells

We have previously shown that treatment of rats with the nitric oxide (NO) synthase inhibitor N6-nitro-L-arginine methyl ester for 4 weeks resulted in the augmentation of blood pressure and enhanced levels of Gialpha proteins. The present studies were undertaken to investigate if NO can modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMC) and primary cultured cells from aorta of Sprague-Dawley rats were used for these studies. The cells were treated with S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) for 24 h and the expression of Gialpha proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [32P]cAMP formation for [alpha-32P]ATP. Treatment of cells with SNAP (100 microM) or SNP (0.5 mM) decreased the expression of Gialpha-2 and Gialpha-3 by about 25-40% without affecting the levels of Gsalpha proteins. The decreased expression of Gialpha proteins was reflected in decreased Gi functions (receptor-independent and -dependent) as demonstrated by decreased or attenuated forskolin-stimulated adenylyl cyclase activity by GTPgammaS and inhibition of adenylyl cyclase activity by angiotensin II and C-ANP4-23, a ring-deleted analog of atrial natriuretic peptide (ANP) that specifically interacts with natriuretic peptide receptor-C (NPR-C) in SNAP-treated cells. The SNAP-induced decreased expression of Gialpha-2 and Gialpha-3 proteins was not blocked by 1H[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylyl cyclase, or KT5823, an inhibitor of protein kinase G, but was restored toward control levels by uric acid, a scavenger of peroxynitrite and Mn(111)tetralis (benzoic acid porphyrin) MnTBAP, a peroxynitrite scavenger and a superoxide dismutase mimetic agent that inhibits the production of peroxynitrite, suggesting that NO-mediated decreased expression of Gialpha protein was cGMP-independent and may be attributed to increased levels of peroxynitrite. In addition, Gsalpha-mediated stimulation of adenylyl cyclase by GTPgammaS, isoproterenol, and forskolin was significantly augmented in SNAP-treated cells. These results indicate that NO decreased the expression of Gialpha protein and associated functions in VSMC by cGMP-independent mechanisms. From these studies, it can be suggested that NO-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which NO regulates blood pressure.     

Chromosomal mapping of human adenylyl cyclase genes type III,type V and type VI

Adenylyl cyclase activity plays a central role in the regulation of most cellular processes. At least eight different adenylyl cyclases have been identified, which are endowed with various and sometimes opposing regulatory properties. Recently we have localized the human genes encoding two of these adenylyl cyclases: the gene for type 11 adenylyl cyclase is located on chromosome 2 (sub-band 2p15.3), the gene for type VIII is located on chromosome 8 (sub-band 8824.2). More recently the type I gene has been located on chromosome 7 (sub-band 7pl2–7p13). Using in situ hybridization, we have now localized the genes for three other adenylyl cyclases: the type III gene has been localized on chromosome 2 in the sub-band 2p22–2p24, the type V gene on chromosome 3 at position 3q13.2–3q21, and the type VI gene on chromosome 12 at position 12q12–12q13. It therefore appears that all adenylyl cyclase genes, known at present are located on different chromosomes and thus are likely to be independently regulated.     

Type-specific regulation of adenylyl cyclase. Selective pharmacological stimulation and inhibition of adenylyl cyclase isoforms

Crystallographic studies have elucidated the binding mechanism of forskolin and P-site inhibitors to adenylyl cyclase. Accordingly, computer-assisted drug design has enabled us to identify isoform-selective regulators of adenylyl cyclase. After examining more than 200 newly synthesized derivatives of forskolin, we found that the modification at the positions of C6 and C7, in general, enhances isoform selectivity. The 6-(3-dimethylaminopropionyl) modification led to an enhanced selectivity for type V, whereas 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] and 6-(4-acrylbutyryl) modification led to an enhanced selectivity for type II. In contrast, 2'-deoxyadenosine 3'-monophosphate, a classical and 3'-phosphate-substituted P-site inhibitor, demonstrated a 27-fold selectivity for inhibiting type V relative to type II, whereas 9-(tetrahydro-2-furyl) adenine, a ribose-substituted P-site ligand, showed a markedly increased, 130-fold selectivity for inhibiting type V. Consequently, on the basis of the pharmacophore analysis of 9-(tetrahydro-2-furyl) adenine and adenylyl cyclase, a novel non-nucleoside inhibitor, 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), was identified after virtual screening of more than 850,000 compounds. NKY80 demonstrated a 210-fold selectivity for inhibiting type V relative to type II. More importantly, the combination of a type III-selective forskolin derivative and 9-(tetrahydro-2-furyl) adenine or NKY80 demonstrated a further enhanced selectivity for type III stimulation over other isoforms. Our data suggest the feasibility of adenylyl cyclase isoform-targeted regulation of cyclic AMP signaling by pharmacological reagents, either alone or in combination.     

Suppression of soluble adenylyl cyclase protects smooth muscle cells against oxidative stress-induced apoptosis

Apoptosis of vascular smooth muscle cells (VSMC) significantly contributes to the instability of advanced atherosclerotic plaques. Oxygen radicals are an important cause for VSMC death. However, the precise mechanism of oxidative stress-induced VSMC apoptosis is still poorly understood. Here, we aimed to analyse the role of soluble adenylyl cylclase (sAC). VSMC derived from rat aorta were treated with either H₂O₂ (300 µmol/L) or DMNQ (30 µmol/L) for 6 h. Oxidative stress-induced apoptosis was prevented either by treatment with 30 µmol/L KH7 (a specific inhibitor of sAC) or by stable sAC-knockdown (shRNA-transfection). A similar effect was found after inhibition of protein kinase A (PKA). Suppression of the sAC/PKA-axis led to a significant increase in phosphorylation of the p38 mitogen-activated protein kinase under oxidative stress accompanied by a p38-dependent phosphorylation/inactivation of the pro-apoptotic Bcl-2-family protein Bad. Pharmacological inhibition of p38 reversed these effects of sAC knockdown on apoptosis and Bad phosphorylation, suggesting p38 as a link between sAC and apoptosis. Analysis of the protein phosphatases 1 and 2A activities revealed an activation of phosphatase 1, but not phosphatase 2A, under oxidative stress in a sAC/PKA-dependent manner and its role in controlling the p38 phosphorylation. Inhibition of protein phosphatase 1, but not 2A, prevented the pro-apoptotic effect of oxidative stress. In conclusion, sAC/PKA-signaling plays a key role in the oxidative stress-induced apoptosis of VSMC. The cellular mechanism consists of the sAC-promoted and protein phosphatase 1-mediated suppression of p38 phosphorylation resulting to activation of the mitochondrial pathway of apoptosis.     

A kinetic analysis of activation of smooth muscle adenylate cyclase by forskolin

Forskolin action was studied using uterine smooth muscle adenylate cyclase, an enzyme form that is slowly and irreversibly activated by treatment with nonhydrolyzable GTP analogs. Activation of the particulate smooth muscle enzyme by prolonged treatment with Gpp[NH]p (guanyl-5′-yl imidodiphosphate) at 24 °C followed simple Michaelis-Menten kinetics with respect to the guanine nucleotide. Under these treatment conditions, forskolin increased both the V_max and the K_m for Gpp[NH]p, suggesting diterpene action affected the guanine nucleotide-binding coupling factor. Sensitivity of a detergent-solubilized form of the enzyme to stimulation by both Gpp[NH]p and forskolin was much more labile at 4 °C than was the Mn⁺² sensitivity of the catalytic subunit. In the particulate form, the catalytic subunit was more resistant to the denaturing effects of N-ethylmaleimide than was its sensitivity to stimulation by Gpp[NH]p or forskolin. Forskolin stimulation of the particulate form of the enzyme followed simple Michaelis-Menten kinetics with respect to the concentration of the diterpene. Denaturation of the enzyme by treatment with N-ethylmaleimide lowered the V_max and increased the K_m for forskolin, further suggesting that forskolin had an indirect effect on the activity of the catalytic subunit. These results could be accounted for if the diterpene, like Gpp[NH]p, was bound by the coupling factor.     

The PI3K-mediated activation of CRAC independently regulates adenylyl cyclase activation and chemotaxis

The ability of a cell to detect an external chemical signal and initiate a program of directed migration along a gradient comprises the fundamental process called chemotaxis. Investigations in Dictyostelium discoideum and neutrophils have established that pleckstrin homology (PH) domain-containing proteins that bind to the PI3K products PI(3,4)P2 and PI(3,4,5)P3, such as CRAC (cytosolic regulator of adenylyl cyclase) and Akt/PKB, translocate specifically to the leading edge of chemotaxing cells. CRAC is essential for the chemoattractant-mediated activation of the adenylyl cyclase ACA, which converts ATP into cAMP, the primary chemoattractant for D. discoideum. The mechanisms by which CRAC activates ACA remain to be determined. We now show that in addition to its essential role in the activation of ACA, CRAC is involved in regulating chemotaxis. Through mutagenesis, we show that these two functions are independently regulated downstream of PI3K. A CRAC mutant that has lost the capacity to bind PI3K products does not support chemotaxis and shows minimal ACA activation. Finally, overexpression of CRAC and various CRAC mutants show strong effects on ACA activation with little effect on chemotaxis. These findings establish that chemoattractant-mediated activation of PI3K is important for the CRAC-dependent regulation of both chemotaxis and adenylyl cyclase activation.     

Raf-1 kinase mediates adenylyl cyclase sensitization by TNF-alpha in human airway smooth muscle cells

Tumor necrosis factor (TNF)-alpha is a potent inflammatory cytokine implicated in the exacerbation of asthma. Chronic exposure to TNF-alpha has been reported to induce G protein-coupled receptor desensitization, but adenylyl cyclase sensitization, in airway smooth muscle cells by an unknown mechanism. Cyclic AMP, which is synthesized by adenylyl cyclases in response to G protein-coupled receptor signals, is an important second messenger involved in the regulation of the airway muscle proliferation, migration, and tone. In other cell types, TNF-alpha receptors transactivate the EGF receptor, which activates raf-1 kinase. Further studies in transfected cells show that raf-1 kinase can phosphorylate and activate some isoforms of adenylyl cyclase. Cultured human airway smooth muscle cells were treated with TNF-alpha in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, or G(i) proteins. TNF-alpha caused a significant dose- (1-10 ng/ml) and time-dependent (24 and 48 h) increase in forskolin-stimulated adenylyl cyclase activity, which was abrogated by pretreatment with GW5074 (a raf-1 kinase inhibitor), was partially inhibited by an EGF receptor inhibitor, but was unaffected by pertussis toxin. TNF-alpha also increased phosphorylation of Ser(338) on raf-1 kinase, indicative of activation. IL-1beta and EGF sensitization of adenylyl cyclase activity was also sensitive to raf-1 kinase inhibition by GW5074. Taken together, these studies link two signaling pathways not previously characterized in human airway smooth muscle cells: TNF-alpha transactivation of the EGF receptor, with subsequent raf-1 kinase-mediated activation of adenylyl cyclase.     

An important functional role of the N terminus domain of type VI adenylyl cyclase in Galphai-mediated inhibition

We show herein that removal of the first 86 amino acids (aa) of the N terminus (designated N) of type VI adenylyl cyclase (ACVI) caused the resultant ACVI mutant (ACVI-DeltaA87) to be more greatly inhibited by a Galpha(i)-coupled receptor or activated Galpha(i) protein. Moreover, in vitro binding of the full-length N and C1a domain (designated C1a), which interacts with Galpha(i), was detected. A truncated N terminus (aa 1-86) also interacted with C1a, suggesting that the C1a-interacting region is located within aa 1-86. Mutation analyses further revealed that N might interact with C1a in the region (aa 434-505) where Galpha(i) is bound. Mutations of two residues (Leu-472 and Val-476) located in this N-binding region of C1a suppressed the interaction between recombinant N and C1a and markedly reduced Galpha(i)-mediated inhibition of ACVI-DeltaA87. Further biochemical analyses of the effect of internal mutations of Leu-472/Val-476 on Galpha(i)-mediated inhibition of wild-type ACVI and ACVI-DeltaA87 suggested that N modulates the Galpha(i)-mediated inhibition of ACVI via binding to C1a when the level of Galpha(i) is low (i.e. around the IC(50) value) and that a more complicated interfering mode results when the level of Galpha(i) is high (i.e. approximately 10- to 20-fold of the IC(50) value). Collectively, data presented herein suggest a novel function of the N terminus of ACVI in Galpha(i)-mediated regulation.     

Cyclic GMP modulates the expression of Gi protein and adenylyl cyclase signaling in vascular smooth muscle cells

We have recently shown that the nitric oxide (NO) donor, SNAP, decreased the expression of Giα proteins and associated functions in vascular smooth muscle cells. Because NO stimulates soluble guanylyl cyclase and increases the levels of guanosine 3′,5′-cyclic monophosphate (cGMP), the present studies were undertaken to investigate whether cGMP can also modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMCs) and primary cultured cells from aorta of Sprague Dawley rats were used for these studies. The cells were treated with 8-bromoguanosine 3′,5′-cyclic monophosphate (8Br-cGMP) for 24 h and the expression of Giα proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [³²P]cAMP formation for [α-³²P]ATP. Treatment of cells with 8-Br-cGMP (0.5 mM) decreased the expression of Giα-2 and Giα-3 by about 30–45%, which was restored towards control levels by KT5823, an inhibitor of protein kinase G. On the other and hand, the levels of Gsα protein were not altered by this treatment. The decreased expression of Giα proteins by 8Br-cGMP treatment was reflected in decreased Gi functions. For example, the inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity by low concentrations of GTPγS (receptor-independent Gi functions) was significantly decreased by 8Br-cGMP treatment. In addition, exposure of the cells to 8Br-cGMP also resulted in the attenuation of angiotensin (Ang) II- and C-ANP_4–23 (a ring-deleted analog of atrial natriuretic peptide [ANP]-mediated inhibition of adenylyl cyclase activity (receptor-dependent functions of Gi). On the other hand, Gsα-mediated stimulations of adenylyl cyclase by GTPγS, isoproterenol and FSK were significantly augmented in 8Br-cGMP-treated cells. These results indicated the 8Br-cGMP decreased the expression of Giα proteins and associated functions in VSMCs. From these studies, it can be suggested that 8Br-cGMP-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which cGMP regulates vascular tone and thereby blood pressure.     

Ca2+-dependent inhibition of smooth muscle adenylate cyclase activity

We have examined the inhibitory regulation by Ca2+ of the adenylate cyclase activity associated with microsomes isolated from bovine aorta smooth muscle. In the presence of 2 mM MgCl2, Ca2+ (0.8-100 microM) inhibited in a noncompetitive manner activation of the enzyme by GTP, Gpp[NH]p, or forskolin. In all instances the value for half-maximal inhibition was between 2 and 3 microM. In contrast, Ca2+ inhibited the activation by MgCl2 (2-50 mM), alone or in the presence of GTP, in a competitive manner. The inhibition of adenylate cyclase by 10 microM Ca2+ was reversed in the presence of either 5 or 25 microM calmodulin or troponin C. These data show that (i) Ca2+, at concentrations similar to those which activate smooth muscle contraction, inhibits the stimulation of adenylate cyclase by several activators; (ii) Ca2+ and Mg2+ compete for a common site on the smooth muscle adenylate cyclase complex; and (iii) the reversal of Ca2+-dependent inhibition by Ca2+-binding proteins may be produced by chelation of the metal by these proteins.     

Intramolecular signaling in tandem-GAF domains from PDE5 and PDE10 studied with a cyanobacterial adenylyl cyclase reporter

The dimeric mammalian phosphodiesterases (PDEs) are regulated by N-terminal domains. In PDE5, the GAF-A subdomain of a GAF-tandem (GAF-A and -B) binds the activator cGMP and in PDE10 GAF-B binds cAMP. GAF-tandem chimeras of PDE5 and 10 in which the 36 aa linker helix between GAF-A and -B was swapped lost allosteric regulation of a reporter adenylyl cyclase. In 16 consecutive constructs we substituted the PDE10 linker with that from PDE5. An initial stretch of 10 amino acids coded for isoform specificity. A C240Y substitution uncoupled cyclase activity from regulation, whereas C240F, L or G did not. The C240Y substitution increased basal activity to stimulated levels. Notably, over the next 12 substitutions basal cyclase activity decreased linearly.Further targeted substitutions were based on homology modeling using the PDE2 structure. No combination of substitutions within the initial 10 linker residues caused loss of regulation. The full 10 aa stretch was required. Modeling indicated a potential interaction of the linker with a loop from GAF-A. To interrupt H-bonding a glycine substitution of the loop segment was generated. Despite reduction of basal activity, loss of regulation was maintained. Possibly, the orientation of the linker helix is determined by formation of the dimer at the initial linker segment. Downstream deflections of the linker helix may have caused loss of regulation.     

Phosphoinositide 3-kinase activity controls the chemoattractant-mediated activation and adaptation of adenylyl cyclase

The binding of chemoattractants to cognate G protein-coupled receptors activates a variety of signaling cascades that provide spatial and temporal cues required for chemotaxis. When subjected to uniform stimulation, these responses are transient, showing an initial peak of activation followed by a period of adaptation, in which activity subsides even in the presence of stimulus. A tightly regulated balance between receptor-mediated stimulatory and inhibitory pathways controls the kinetics of activation and subsequent adaptation. In Dictyostelium, the adenylyl cyclase expressed during aggregation (ACA), which synthesizes the chemoattractant cAMP, is essential to relay the signal to neighboring cells. Here, we report that cells lacking phosphoinositide 3-kinase (PI3K) activity are deficient in signal relay. In LY294002-treated cells, this defect is because of a loss of ACA activation. In contrast, in cells lacking PI3K1 and PI3K2, the signal relay defect is because of a loss of ACA adaptation. We propose that the residual low level of 3-phosphoinositides in pi3k(1-/2-) cells is sufficient to generate the initial peak of ACA activity, yet is insufficient to sustain the inhibitory phase required for its adaptation. Thus, PI3K activity is poised to regulate both ACA activation and adaptation, thereby providing a link to ensure the proper balance of counteracting signals required to maintain optimal chemoresponsiveness.     

A MAP kinase necessary for receptor-mediated activation of adenylyl cyclase in Dictyostelium

Analysis of a developmental mutant in Dictyostelium discoideum which is unable to initiate morphogenesis has shown that a protein kinase of the MAP kinase/ERK family affects relay of the cAMP chemotactic signal and cell differentiation. Strains in which the locus encoding ERK2 is disrupted respond to a pulse of cAMP by synthesizing cGMP normally but show little synthesis of cAMP. Since mutant cells lacking ERK2 contain normal levels of both the cytosolic regulator of adenylyl cyclase (CRAC) and manganese-activatable adenylyl cyclase, it appears that this kinase is important for receptor-mediated activation of adenylyl cyclase.     

The dopamine D(2) receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle

Dopamine receptors are G protein-coupled receptors that are divided into two subgroups, "D(1)-like" receptors (D(1) and D(5)) that couple to the G(s) protein and "D(2)-like" receptors (D(2), D(3), and D(4)) that couple to G(i). Although inhaled dopamine has been reported to induce bronchodilation in patients with asthma, functional expression of dopamine receptor subtypes has never been described on airway smooth muscle (ASM) cells. Acute activation of G(i)-coupled receptors inhibits adenylyl cyclase activity and cAMP synthesis, which classically impairs ASM relaxation. In contrast, chronic activation of G(i)-coupled receptors produces a paradoxical enhancement of adenylyl cyclase activity referred to as heterologous sensitization. We questioned whether the dopamine D(2)-like receptor is expressed on ASM, whether it exhibits classical G(i)-coupling, and whether it modulates ASM function. We detected the mRNA encoding the dopamine D(2) receptor in total RNA isolated from native human ASM and from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the dopamine D(2) receptor protein in both native human and guinea pig ASM and cultured HASM cells. The dopamine D(2) receptor protein was immunohistochemically localized to both human and guinea pig ASM. Acute activation of the dopamine D(2) receptor by quinpirole inhibited forskolin-stimulated adenylyl cyclase activity in HASM cells, which was blocked by the dopamine D(2) receptor antagonist L-741626. In contrast, the chronic pretreatment (1 h) with quinpirole potentiated forskolin-stimulated adenylyl cyclase activity, which was inhibited by L-741626, the phospholipase C inhibitor U73122, or the protein kinase C inhibitor GF109203X. Quinpirole also stimulated inositol phosphate synthesis, which was inhibited by L-741626 or U73122. Chronic pretreatment (1 h) of the guinea pig tracheal rings with quinpirole significantly potentiated forskolin-induced airway relaxation, which was inhibited by L-741626. These results demonstrate that functional dopamine D(2) receptors are expressed on ASM and could be a novel therapeutic target for the relaxation of ASM.     

Cloning and expression of human adenylyl cyclase type VI in normal thyroid tissues

The adenylyl cyclase type VI gene expressed in human normal thyroid tissue was cloned and sequenced. The cDNA sequence (6463 nt) is susceptible to code for a 1168 aa protein. Northern blots using specific probes showed that the expression of adenylyl cyclase type VI gene was significantly higher in one hyperfunctioning thyroid tumor than in normal thyroid tissue, in one follicular cold adenoma or in one papillary carcinoma.