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cAMP and [Ca.sup.2+] are antagonistic intracellular messengers for the regulation of vascular smooth muscle tone; rising levels of [Ca.sup.2+] lead to vasoconstriction, whereas an increase of cAMP induces vasodilatation. Here we investigated whether [Ca.sup.2+] interferes with cAMP signaling by regulation of phophodiesterases (PDEs) or adenylyl cyclases (ACs). We studied regulation of cAMP concentrations by [Ca.sup.2+] signals evoked by endogenous purinergic receptors in vascular smooth muscle cells (VSMCs). The fluorescence resonance energy transfer (FRET)-based cAMP sensor Epac 1-camps allowed the measurement of cAMP levels in single-living VSMCs with subsecond temporal resolution. Moreover, in vitro calibration of Epacl-camps enabled us to estimate the absolute cytosolic cAMP concentrations. Stimulation of purinergic receptors decreased cAMP levels in the presence of the [beta]-adrenergic agonist isoproterenol. Simultaneous imaging of cAMP with Epaclcamps and of [Ca.sup.2+] with Fura 2 revealed a rise of intracellular [Ca.sup.2+] in response to purinergic stimulation followed by a decline of cAMP. Chelation of intracellular [Ca.sup.2+] and overexpression of CaZ+-independent AC4 antagonized this decline of cAMP, whereas pharmacological inhibition of [Ca.sup.2+]-activated PDE1 had no effect. AC assays with VSMC membranes revealed a significant attenuation of isoproterenol-stimulated cAMP production by the presence of 2 [micro]M [Ca.sup.2+]. Furthermore, small interfering RNA (siRNA) knockdown of AC5 and AC6 (the two ACs known to be inhibited by [Ca.sup.2+]), significantly reduced the decrease of cAMP upon purinergic stimulation of isoproterenol-prestimulated VSMCs. Taken together, these results implicate a [Ca.sup.2+]-mediated inhibition of AC5 and 6 as an important mechanism of purinergic receptor-induced decline of cAMP and show a direct cross talk of these signaling pathways in VSMCs. cAMP; adenylyl cyclases; fluorescence resonance energy transfer; vascular smooth muscle cells; purinergic receptors doi: 10.1152/ajpcell.00197.2009

pH homeostasis is essential for life, yet it remains unclear how animals sense their systemic acid/base (A/B) status. Soluble adenylyl cyclase (sAC) is an evolutionary conserved signaling enzyme that produces the second messenger cAMP in response to bicarbonate ions (HC[O.sub.3.sup.-]). We cloned the sAC ortholog from the dogfish, a shark that regulates blood A/B by absorbing and secreting protons ([H.sup.+]) and HC[O.sub.3.sup.-] at its gills. Similar to mammalian sAC, dogfish soluble adenylyl cyclase (dfsAC) is activated by HC[O.sub.3.sup.-] and can be inhibited by two structurally and mechanistically distinct small molecule inhibitors, dfsAC is expressed in the gill epithelium, where the subset of base-secreting cells resides. Injection of inhibitors into animals under alkaline stress confirmed that dfsAC is essential for maintaining systemic pH and HC[O.sub.3.sup.-] levels in the whole organism. One of the downstream effects of dfsAC is to promote the insertion of vacuolar proton pumps into the basolateral membrane to absorb [H.sup.+] into the blood, sAC orthologs are present throughout metazoans, and mammalian sAC is expressed in A/B regulatory organs, suggesting that systemic A/B sensing via sAC is widespread in the animal kingdom. cAMP | pH | proton pump | dogfish | gill www.pnas.org/cgi/doi/10.1073/pnas.0911790107

Activation of soluble adenylyl cyclase (sAC) by bicarbonate causes local cAMP generation, indicating that sAC might act as a pH and/or bicarbonate sensor in kidney cells involved in acid-base homeostasis. Therefore, we examined the expression of sAC in renal acid-base transporting intercalated cells (IC) and compared its distribution to that of the vacuolar proton pumping ATPase (V-ATPase) under different conditions. In all IC, sAC and V-ATPase showed considerable overlap under basal conditions, but sAC staining was also found in other cellular locations in the absence of V-ATPase. In type A-IC, both sAC and V-ATPase were apically and subapically located, whereas in type B-IC, significant basolateral colocalization of sAC and the V-ATPase was seen. When apical membrane insertion of the V-ATPase was stimulated by treatment of rats with acetazolamide, sAC was also concentrated in the apical membrane of A-IC. In mice that lack a functional B1 subunit of the V-ATPase, sAC was colocalized apically in A-IC along with V-ATPase containing the alternative B2 subunit isoform. The close association between these two enzymes was confirmed by coimmuno-precipitation of sAC from kidney homogenates using anti-V-ATPase antibodies. Our data show that sAC and the V-ATPase colocalize in IC, that they are concentrated in the IC plasma membrane under conditions that 'activate' these proton secretory cells, and that they are both present in an immunoprecipitated complex. This suggests that these enzymes have a close association and could be part of a protein complex that is involved in regulating renal distal proton secretion. proton pump; kidney; intercalated cells; acid-base homeostasis

BACKGROUND: Organophosphate developmental neurotoxicity involves multiple mechanisms converging on neural cell replication and differentiation. OBJECTIVES: We evaluated mechanisms contributing to the adverse effects of chlorpyrifos (CPF) on DNA synthesis, cell number and size, and cell signaling mediated by adenylyl cyclase (AC) in PC12 cells, a neuronotypic cell line that recapitulates the essential features of developing mammalian neurons. RESULTS: In undifferentiated cells, cholinergic receptor antagonists had little or no protective effect against the antimitotic actions of CPF; however, when nerve growth factor was used to evoke differentiation, the antagonists showed partial protection against deficits in cell loss and alteration in cell size elicited by CPF, but were ineffective in preventing the deterioration of AC signaling. Nicotine, which stimulates nicotinic acetylcholine receptors but also possesses a mixture of prooxidant/antioxidant activity, had adverse effects by itself but also protected undifferentiated cells from the actions of CPF and had mixed additive/protective effects on cell number in differentiating cells. The antioxidant vitamin E also protected both undifferentiated and differentiating cells from many of the adverse effects of CPF but worsened the impact on AC signaling. Theophylline, which prevents the breakdown of cyclic AMP, was the only agent that restored AC signaling to normal or supranormal levels but did so at further cost to cell replication. CONCLUSIONS: Our results show definitive contributions of cholinergic hyperstimulation, oxidative stress, and interference with AC signaling in the developmental neurotoxicity of CPF and point to the potential use of this information to design treatments to ameliorate these adverse effects. KEY WORDS: adenylyl cyclase, brain development, chlorpyrifos, cholinergic antagonists, neurotoxicity, nicotine, organophosphate insecticides, oxidative stress, theophylline, vitamin E. Environ Health Perspect 115:1306-1313 (2007). doi:10.1289/ehp.10194 available via http://dx.doi.org/ [Online 14 June 2007], The nearly ubiquitous exposure of the human population to organophosphate pesticides has raised increasing concern about their propensity to elicit developmental neurotoxicity at exposures that go undetected because of the [...]

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 Gi 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.sup.338] on raf-1 kinase, indicative of activation. IL-1[beta] 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. GW5074; mitogen-activated protein kinase; phosphorylation; immunoblot; cell culture doi:10.1152/ajplung.00123.2006.

We previously identified a sperm-specific [Na.sup.+]/[H.sup.+] exchanger (sNHE) principally localized to the flagellum. Disruption of the sNHE gene in mice resulted in absolute male infertility associated with a complete loss of sperm motility. Here, we show that the sNHE-null spermatozoa fail to develop the cAMP-dependent protein tyrosine phosphorylation that coincides with the functional maturation occurring upon incubation in capacitating conditions in vitro. Both the sperm motility defect and the lack of induced protein tyrosine phosphorylation are rescued by the addition of cell-permeable cAMP analogs, suggesting that cAMP metabolism is impaired in spermatozoa lacking sNHE. Our analyses of the bicarbonate-dependent soluble adenylyl cyclase (sAC) signaling pathway in sNHE-null sperm cells reveal that sNHE is required for the expression of full-length sAC, and that it is important for the bicarbonate stimulation of sAC activity in spermatozoa. Furthermore, both codependent expression and coimmunoprecipitation experiments indicate that sNHE and sAC associate with each other. Thus, these two proteins appear to be components of a signaling complex at the sperm flagellar plasma membrane. We propose that the formation of this complex efficiently modulates intracellular pH and bicarbonate levels through the rapid and effective control of sAC and sNHE activities to facilitate sperm motility regulation. cAMP | sperm motility

The pituitary adenylate cyclase-activating polypeptide (PACAP) receptor is a class II G protein-coupled receptor that contributes to many different cellular functions including neurotransmission, neuronal survival, and synaptic plasticity. The solution structure of the potent antagonist PACAP (residues 6'-38') complexed to the N-terminal extracellular (EC) domain of the human splice variant hPAC1-R-short (hPAC1-[R.sub.s]) was determined by NMR. The PACAP peptide adopts a helical conformation when bound to hPAC1-[R.sub.s] with a bend at residue A18' and makes extensive hydrophobic and electrostatic interactions along the exposed [beta]-sheet and interconnecting loops of the N-terminal EC domain. Mutagenesis data on both the peptide and the receptor delineate the critical interactions between the C terminus of the peptide and the C terminus of the EC domain that define the high affinity and specificity of hormone binding to hPAC1-[R.sub.s]. These results present a structural basis for hPAC1-[R.sub.s] selectivity for PACAP versus the vasoactive intestinal peptide and also differentiate PACAP residues involved in binding to the N-terminal extracellular domain versus other parts of the full-length hPAC1-[R.sub.s] receptor. The structural, mutational, and binding data are consistent with a model for peptide binding in which the C terminus of the peptide hormone interacts almost exclusively with the N-terminal EC domain, whereas the central region makes contacts to both the N-terminal and other extracellular parts of the receptor, ultimately positioning the N terminus of the peptide to contact the transmembrane region and result in receptor activation. NMR | vasoactive intestinal peptide | G protein-coupled receptor

Although the [Na.sup.+]/[H.sup.+] exchanger (NHE) is considered to be involved in regulation of intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) through the [Na.sup.+]/[Ca.sup.2+] exchanger, the exact mechanisms of its participation in [Ca.sup.2+] handling by cardiomyocytes are not fully understood. Isolated rat cardiomyocytes were treated with or without agents that are known to modify [Ca.sup.2+] movements in cardiomyocytes and exposed to an NHE inhibitor, 5-(N-methyl-Nisobutyl)amiloride (MIA). [[[Ca.sup.2+]].sub.i] in cardiomyocytes was measured spectrofluorometrically with fura 2-AM in the absence or presence of KCl, a depolarizing agent. MIA increased basal [[[Ca.sup.2+]].sub.1] and augmented the KCl-induced increase in [[[Ca.sup.2+]]sub.i] in a concentrationdependent manner. The MIA-induced increase in basal [[[Ca.sup.2+]].sub.i] was unaffected by extracellular [Ca.sup.2+], antagonists of the sarcolemmal (SL) L-type [Ca.sup.2+] channel, and inhibitors of the SL [Na.sup.+]/[Ca.sup.2+] exchanger, SL [Ca.sup.2+] pump ATPase and mitochondrial [Ca.sup.2+] uptake. However, the MIA-induced increase in basal [[[Ca.sup.2+]].sub.i] was attenuated by inhibitors of SL [Na.sup.+]-[K.sup.+]-ATPase and sarcoplasmic reticulum (SR) [Ca.sup.2+] transport. On the other hand, the MIA-mediated augmentation of the KCl response was dependent on extracellular [Ca.sup.2+] concentration and attenuated by agents that inhibit SL L-type [Ca.sup.2+] channels, the SL [Na.sup.+]/[Ca.sup.2+] exchanger, SL [Na.sup.+]-[K.sup.+]-ATPase, and SR [Ca.sup.2+] release channels and the SR [Ca.sup.2+] pump. However, the effect of MIA on the KCl-induced increase in [[[Ca.sup.2+]].sub.i] remained unaffected by treatment with inhibitors of SL [Ca.sup.2+] pump ATPase and mitochondrial [Ca.sup.2+] uptake. MIA and a decrease in extracellular pH lowered intracellular pH and increased basal [[[Ca.sup.2+]].sub.i], whereas a decrease in extracellular pH, in contrast to MIA, depressed the KCl-induced increase in [[[Ca.sup.2+]].sub.i] in cardiomyocytes. These results suggest that NHE may be involved in regulation of [[[Ca.sup.2+]].sub.i] and that MIA-induced increases in basal [[[Ca.sup.2+]].sub.i], as well as augmentation of the KCl-induced increase in [[[Ca.sup.2+]].sub.i], in cardiomyocytes are regulated differentially. sodium-potassium adenosinetriphosphatase; calcium-handling proteins; sarcolemmal calcium transport; sarcoplasmic reticulum calcium transport; 5-(N-methyl-N-isobutyl) amiloride

Three receptors for VIP and pituitary adenylate cyclase-activating peptide (PACAP) have been cloned and characterized: [PAC.sub.1], with high affinity for PACAP, and [VPAC.sub.1] and [VPAC.sub.2] with equally high affinity for VIP and PACAP. The existence of a VIP-specific receptor ([VIP.sub.s]) in guinea pig (GP) teniae coli smooth muscle was previously surmised on the basis of functional studies, and its existence was confirmed by cloning of a partial N[H.sub.2]-terminal sequence. Here we report the cloning of the full-length cDNAs of two receptors, a [VPAC.sub.2] receptor from GP gastric smooth muscle and [VIP.sub.s] from GP teniae coli smooth muscle. The cDNA sequence of the [VIP.sub.s] encodes a 437-amino acid protein ([M.sub.r] 49,560) that possesses 87% similarity to [VPAC.sub.2] receptors in rat and mouse and differs from the [VPAC.sub.2] receptor in GP gastric smooth muscle by only two amino-acid residues, [F.sup.40][F.sup.41] in lieu of [L.sup.40][L.sup.41]. In COS-1 cells transfected with the GP teniae coli smooth muscle receptor, only VIP bound with high affinity ([IC.sub.50] 1.4 nM) and stimulated cAMP formation with high potency ([EC.sub.50] 1 nM). In contrast, in COS-1 cells transfected with the GP gastric smooth muscle receptor, both VIP and PACAP bound with equally high affinity ([IC.sub.50] 2.3 nM) and stimulated cAMP with equally high potency ([EC.sub.50] 1.5 nM). We conclude that the receptor cloned from GP teniae coli smooth muscle is a [VIP.sub.s] distinct from [VPAC.sub.1] and [VPAC.sub.2] receptors. The ligand specificity in this species is determined by a pair of adjacent phenylalanine residues ([L.sup.40][L.sup.41]) in the [NH.sup.2]-terminal ligand-binding domain.

Chimeric G proteins made by replacing the COOH-terminal heptapeptide of [G.sub.[alpha]]q with the COOH-terminal heptapeptide of [G.sub.[alpha]]s or [G.sub.[alpha]]i were used to assess the relative coupling of [[beta].sub.3]-adrenergic receptor ([[beta].sub.3]-AR) splice variants ([[beta].sub.3A] and [[beta].sub.3B]) to [G.sub.[alpha]]s and [G.sub.[alpha]]i. The [G.sub.[alpha]]q/s and [G.sub.[alpha]]q/i chimeras transformed the response to receptor activation from regulation of adenylyl cyclase to mobilization of intracellular calcium ([Ca.sup.2+]i). Complementary high-throughput and single-cell approaches were used to evaluate agonist-induced coupling of the receptor to the G protein chimeras. In cells stably transformed with rat [[beta].sub.3]-AR, transfected with the G protein chimeras, and evaluated using a scanning fluorometer, [[beta].sub.3]-AR-induced coupling to [G.sub.[alpha]]q/s produced a rapid eightfold increase in [Ca.sup.2+]i followed by a slow decay to levels 25% above baseline. [G.sub.[alpha]]q/i also linked rat [[beta].sub.3]-AR to mobilization of [Ca.sup.2+]i in a similar time- and agonist-dependent manner, but the net 2.5-fold increase in [Ca.sup.2+]i was only 30% of the response obtained with [G.sub.[alpha]]q/s. Activation of the rat [[beta].sub.3]-AR also increased GTP binding to endogenous [G.sub.[alpha]]i threefold in membranes from CHO cells stably transformed with the receptor. A complementary single-cell imaging approach was used to assess the relative coupling of mouse [[beta].sub.3A]- and [[beta].sub.3B]-AR to [G.sub.[alpha]]i under conditions established to produce equivalent agonist-dependent coupling of the receptor splice variants to [G.sub.[alpha]]q/s and to increases in intracellular cAMP through endogenous [G.sub.[alpha]]s. The [[beta].sub.3A]- and [[beta].sub.3B]-AR coupled equivalently to [G.sub.[alpha]]q/i, but the temporal patterns of [Ca.sup.2+]i mobilization indicated that coupling was significantly less efficient than coupling to [G.sub.[alpha]]q/s. Collectively, these findings indicate less efficient but equivalent coupling of [[beta].sub.3A]- and [[beta].sub.3B]-AR to [G.sub.[alpha]]i vs. [G.sub.[alpha]]s and suggest that differential expression of the splice variants would not produce local differences in signaling networks linked to [[beta].sub.3]-AR activation. [beta]-adrenergic receptor; G proteins; cyclic adenosine monophosphate; signaling plasticity

Human sperm development and the role of adenylyl cyclase in this process is examined.

There is increasing awareness of the compartmentalization of cAMP signalling--the means by which cAMP levels change in discrete domains of the cell with discrete local consequences. Current developments in understanding the organization of adenylyl cyclases in the plasma membrane are illuminating how the earliest part of cAMP compartmentalization could occur. This review focuses on recent findings regarding three levels of adenylyl cyclase organization--oligomerization, positioning to lipid rafts and participation in multiprotein signalling complexes. This organization, coupled with the role of scaffolding proteins in arranging the downstream effectors of cAMP, helps to identify complexes that greatly facilitate the translation of enzyme activation into local consequences.

We previously showed that rat taste buds express several adenylyl cyclases (ACs) of which only AC8 is known to be stimulated by [Ca.sup.2+]. Here we demonstrate by direct measurements of cAMP levels that AC activity in taste buds is stimulated by treatments that elevate intracellular [Ca.sup.2+]. Specifically, 5 [micro]M thapsigargin or 3 [micro]M A-23187 (calcium ionophore), both of which increase intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]), lead to a significant elevation of cAMP levels. This calcium stimulation of AC activity requires extracellular [Ca.sup.2+], suggesting that it is dependent on [Ca.sup.2+] entry rather than release from stores. With immunofluorescence microscopy, we show that the calcium-stimulated AC8 is principally expressed in taste cells that also express phospholipase C[[beta].sub.2] (i.e., cells that elevate [[[Ca.sup.2+]].sub.i] in response to sweet, bitter, or umami stimuli). Taste transduction for sucrose is known to result in an elevation of both cAMP and calcium in taste buds. Thus we tested whether the cAMP increase in response to sucrose is a downstream consequence of calcium elevation. Even under conditions of depletion of stored and extracellular calcium, the cAMP response to sucrose stimulation persists in taste cells. The cAMP signal in response to monosodium glutamate stimulation is similarly unperturbed by calcium depletion. Our results suggest that tastant-evoked cAMP signals are not simply a secondary consequence of calcium modulation. Instead, cAMP and released [Ca.sup.2+] may represent independent second messenger signals downstream of taste receptors. calcium-sensitive adenylyl cyclase; capacitative entry; cross talk; taste transduction doi:10.1152/ajpcell.00303.2005

Heterogeneity of heart failure (HF) phenotypes indicates contributions from underlying common polymorphisms. We considered polymorphisms in the [[beta].sub.1]-adrenergic receptor ([[beta].sub.1]AR), a [beta]-blocker target, as candidate pharmacogenomic loci. Transfected cells, genotyped human nonfailing and failing ventricles, and a clinical trial were used to ascertain phenotype and mechanism. In nonfailing and failing isolated ventricles, [[beta].sub.1]-Arg-389 had respective 2.8 [+ or -] 0.3- and 4.3 [+ or -] 2.1-fold greater agonist-promoted contractility vs. [[beta].sub.1]-Gly-389, defining enhanced physiologic coupling under relevant conditions of endogenous expression and HF. The [beta]-blocker bucindolol was an inverse agonist in failing Arg, but not Gly, ventricles, without partial agonist activity at either receptor; carvedilol was a genotype-independent neutral antagonist. In transfected cells, bucindolol antagonized agonist-stimulated cAMP, with a greater absolute decrease observed for Arg-389 (435 [+ or -] 80 vs. 115 [+ or -] 23 fmol per well). Potential pathophysiologic correlates were assessed in a placebo-controlled trial of bucindolol in 1,040 HF patients. No outcome was associated with genotype in the placebo group, indicating little impact on the natural course of HF. However, the Arg-389 homozygotes treated with bucindolol had an age-, sex-, and race-adjusted 38% reduction in mortality (P = 0.03) and 34% reduction in mortality or hospitalization (P = 0.004) vs. placebo. In contrast, Gly-389 carriers had no clinical response to bucindolol compared with placebo. Those with Arg-389 and high baseline norepinephrine levels trended toward improved survival, but no advantage with this allele and exaggerated sympatholysis was identified. We conclude that [[beta].sub.1]AR-389 variation alters signaling in multiple models and affects the [beta]-blocker therapeutic response in HF and, thus, might be used to individualize treatment of the syndrome. adenylyl cyclase | G protein-coupled receptor | genetics | myocardium | signaling

Abstract: We have previously shown the augmented levels of Giα-2 and Giα-3 proteins (isoforms of inhibitory guanine nucleotide regulatory protein (G-protein)), and not of Gsα, in the hearts and aortas [...]

Schizosaccharomyces pombe senses environmental glucose through a cAMP-signaling pathway, activating cAMP-dependent protein kinase A (PKA). This requires nine git (glucose insensitive transcription) genes that encode adenylate cyclase, the PKA catalytic subunit, and seven 'upstream' proteins required for glucose-triggered adenylate cyclase activation, including three heterotrimeric G-protein subunits and its associated receptor. We describe here the cloning and characterization of the git[1.sup.+] gene. Girl is distantly related to a small group of uncharacterized fungal proteins, including a second S. pombe protein that is not functionally redundant with Git1, as well as to members of the UNC-13/Munc13 protein family. Mutations in git[1.sup.+] demonstrate functional roles for the two most highly conserved regions of the protein, the C2 domain and the MHD2 Munc homology domain. Cells lacking Git1 are viable, but display phenotypes associated with cAMP-signaling defects, even in strains expressing a mutationally activated G[alpha]-subunit, which activates adenylate cyclase. These cells possess reduced basal cAMP levels and fail to mount a cAMP response to glucose. In addition, Girl and adenylate cyclase physically interact and partially colocalize in the cell. Thus, Git1 is a critical component of the S. pombe glucose/cAMP pathway.

Association studies suggest [[beta].sub.1]adrenergic receptor ([[beta].sub.1]-AR) polymorphisms are disease modifiers in heart failure. The Arg389 variant has increased coupling to [G.sub.s] in transfected cells and evokes enhanced ventricular function in transgenic mice. Here, we assessed the differential effects of the human Gly389 and Arg389 [[beta].sub.1]-AR polymorphisms on myocardial recovery after ischemic injury. Function was studied in transgenic mice with cardiac-specific expression of either human Gly389 or Arg389 [[beta].sub.1]AR at baseline and after 20 min of ex vivo ischemia and reperfusion (I/R). In 3-mo-old mice of either genotype, there was poor recovery after I/R (~38% vs. ~68% for nontransgenic). Paradoxically, at 6 mo of age, functional recovery remained severely depressed in Gly389 hearts (~32%) but was similar to nontransgenic for Arg389 hearts (~60%). In Arg389 hearts, agonist-promoted adenylyl cyclase activities were depressed by ~35% at 6 mo of age, and G protein-coupled receptor kinase (GRK) activity was increased by approximately twofold compared with Gly389. Furthermore, I/R evoked an approximately threefold increase in ERK2 phosphorylation in Arg389 but an approximately twofold decrease in Gly389 hearts. Individually, these changes have been shown to mitigate I/R injury: thus the Arg389-[[beta].sub.1]-AR uniquely evokes specialized pathways that act to protect against I/R injury. The improved recovery of function after I/R in Arg389 hearts relative to Gly389 appears to be due to an adaptive multimechanism program with allele-specific alterations in receptor signaling, GRK activity, and ERK2. Thus genetic variation of the human [[beta].sub.1]-AR may play a role in cardiac functional recovery after ischemic injury. genetics; adenylyl cyclase; mitogen-activated protein kinase; G protein-coupled receptor kinase

A capillary electrophoresis laser-induced fluorescence (CE-LIF) assay was developed for detection of adenylyl cyclase (AC) activity using BODIPY FL ATP (BATP) as substrate. In the assay, BATP was incubated with AC and the resulting mixture of BATP and enzyme product (BODIPY cyclic AMP, BcAMP) separated in 5 min by CELIF. Substrate depletion and product accumulation were simultaneously monitored during the course of the reaction. The rate of product formation depended upon the presence of AC activators forskolin or G[[alpha].sub.s]-GTP[gamma]S as evidenced by a more rapid BATP turnover to BcAMP compared to basal levels. The CE-LIF assay detected E[C.sub.50] values for forskolin and G[[alpha].sub.s]-GTP[gamma]S of 27 [+ or -] 6 [micro]M and 317 [+ or -] 56 nM, respectively. These E[C.sub.50] values compared well to those previously reported using [[alpha]-[sup.32]p]ATP as substrate. When AC was concurrently activated with 2.5 [micro]M forskolin and 25 nM G[[alpha].sub.s]-GTP[gamma]S, the amount of BcAMP formed was 3.4 times higher than the additive amounts of each activator alone indicating a positively cooperative activation by these compounds in agreement with previous assays using radiolabeled substrate. Inhibition of AC activity was also demonstrated using the AC inhibitor 2'-(or-3')-O-(N-methylanthraniloyl) guanosine 5'-triphosphate with an I[C.sub.50] of 9 [+ or -] 6 nM. The use of a fluorescent substrate combined with CE separation has enabled development of a rapid and robust method for detection of AC activity that is an attractive alternative to the AC assay using radioactive nucleotide and column chromatography. In addition, the assay has potential for high-throughput screening of drugs that act at AC.

Opioid drugs produce their pharmacological effects by activating inhibitory guanine nucleotide-binding regulatory protein-linked [mu], [delta], and [kappa] opioid receptors. One major effector for these receptors is adenylyl cyclase, which is inhibited upon receptor activation. However, little is known about which of the ten known forms of adenylyl cyclase are involved in mediating opioid actions. Here we show that all of the major behavioral effects of morphine, including locomotor activation, analgesia, tolerance, reward, and physical dependence and withdrawal symptoms, are attenuated in mice lacking adenylyl cyclase type 5 (AC5), a form of adenylyl cyclase that is highly enriched in striatum. Furthermore, the behavioral effects of selective [mu] or [delta] opioid receptor agonists are lost in AC[5.sup.-/-] mice, whereas the behavioral effects of selective [kappa] opioid receptor agonists are unaffected. These behavioral data are consistent with the observation that the ability of a [mu] or [delta] opioid receptor agonist to suppress adenylyl cyclase activity was absent in striatum of AC[5.sup.-/-] mice. Together, these results establish AC5 as an important component of [mu] and [delta] opioid receptor signal transduction mechanisms in vivo and provide further support for the importance of the cAMP pathway as a critical mediator of opioid action. striatum | opioid receptors | analgesia | addiction | cAMP

Mutations affecting the Schizosaccharomyces pombe cAMP phosphodiesterase (PDE) gene cgs[2.sup.+] were identified in a screen for suppressors of mutant alleles of the adenylate cyclase gene (git[2.sup.+]/cyr[1.sup.+]), which encode catalytically active forms of the enzyme that cannot be stimulated by extracellular glucose signaling. These mutations suppress both the git[2.sup.-] mutant alleles used in the suppressor selection and mutations in git[1.sup.+], git[3.sup.+], git[5.sup.+], git[7.sup.+], git[10.sup.+], and git[11.sup.+], which are all required for adenylate cyclase activation. Notably, these cgs2 mutant alleles fail to suppress mutations in gpa[2.sup.+], which encodes the G[alpha] subunit of a heterotrimeric G protein required for adenylate cyclase activation, although the previously identified cgs2-2 allele does suppress loss of gpa[2.sup.+]. Further analysis of the cgs2-s1 allele reveals a synthetic interaction with the gpa[2.sup.R176H]-activated allele, with respect to derepression of fbp1-lacZ transcription in glucose-starved cells. In addition, direct measurements of cAMP levels show that cgs2-s1 cells maintain normal basal cAMP levels, but are severely defective in feedback regulation upon glucose detection. These results suggest that PDE activity in S. pombe may be coordinately regulated with adenylate cyclase activity as part of the feedback regulation mechanism to limit the cAMP response to glucose detection.

Inactivation of the gene encoding the periplasmic protease DegP confers a high-temperature-sensitive phenotype in Escherichia coli. We have previously demonstrated that a degP mutant of E. coli strain CBM (W3110 pldA1) is not temperature sensitive and showed that this was most likely due to constitutive activation of the sigma E and Cpx extracytoplasmic stress regulons in the parent strain. In this study, further characterization of this strain revealed a previously unknown cryptic mutation that rescued the degP temperature-sensitive phenotype by inducing the extracytoplasmic stress regulons. We identified the cryptic mutation as an 11-bp deletion of nucleotides 1884 to 1894 of the adenylate cyclase-encoding cyaA gene (cyaA[DELTA]11). The mechanism in which cyaA[DELTA]11 induces the sigma E and Cpx regulons involves decreased activity of the mutant adenylate cyclase. Addition of exogenous cyclic AMP (cAMP) to the growth medium of a cyaA[DELTA]11 mutant strain that contains a Cpx- and sigma E-inducible degP-lacZ reporter fusion decreased [beta]-galactosidase expression to levels observed in a cya[A.sup.+] strain. We also found that a cyaA null mutant displayed even higher levels of extracytoplasmic stress regulon activation compared to a cyaA[DELTA]11 mutant. Thus, we conclude that the lowered concentration of cAMP in cyaA mutants induces both sigma E and Cpx extracytoplasmic stress regulons and thereby rescues the degP temperature-sensitive phenotype.

Bicarbonate stimulates the activities of several class III adenylyl cyclases studied to date. However, we show here that bicarbonate decreased [V.sub.max] and substrate affinity in Cya1, a major adenylyl cyclase in the cyanobacterium Synechocystis sp. strain PCC 6803. This indicates that manifestation of the bicarbonate responsiveness is specifically modulated in Cya1.

We identified the human ortholog of soluble adenylyl cyclase (hsAC) in a locus linked to familial absorptive hypercalciuria and cloned it from a human cDNA library. hsAC transcripts were expressed in multiple tissues using RT-PCR and RNA blotting. RNA blot analysis revealed a predominant 5.1-kb band in a multiple human tissue blot, but three splice transcript variants were detected using RT-PCR and confirmed by performing sequence analysis. Immunoblot analysis showed 190- and 80-kDa bands in multiple human cell lines from gut, renal, and bone origins in both cytosol and membrane fractions, including Caco-2 colorectal adenocarcinomas, HEK-293 cells, HOS cells, and primary human osteoblasts, as well as in vitro induced osteoclast-like cells. The specificity of the antiserum was verified by peptide blocking and reduction using sequence-specific small interfering RNA. Confocal immunofluorescence cytochemistry localized hsAC primarily in cytoplasm, but some labeling was observed in the nucleus and the plasma membrane. Cytoplasmic hsAC colocalized with microtubules but not with microfilaments. To test the function of hsAC, tour constructs containing catalytic domains I and II (aa 1-802), catalytic domain II (aa 231-802), noncatalytic domain (aa 648-1,610), and full-length protein (aa 1-1,610) were expressed in Sf9 insect cells. Only catalytic domains I and II or full-length proteins showed adenylyl cyclase activity. [Mg.sup.2+], [Mn.sup.2+], and [Ca.sup.+] all increased adenylyl cyclase activity in a dose-dependent manner. While hsAC had a minimal response to HC[O.sup.-.sub.3] in the absence of divalent cations, HC[O.sup.-.sub.3] robustly stimulated [Mg.sup.2+]-bound hsAC but inhibited [Mn.sup.24]-bound hsAC in a dose-dependent manner. In summary, hsAC is a divalent cation and HC[O.sup.-.sub.3] sensor, and its HC[O.sup.-.sub.3] sensitivity is modulated by divalent cations. bicarbonate sensor; calcium homeostasis; hypercalciuria

Class III adenylyl cyclases contain catalytic and regulatory domains, yet structural insight into their interactions is missing. We show that the mycobacterial adenylyl cyclase Rv1264 is rendered a pH sensor by its N-terminal domain. In the structure of the inhibited state, catalytic and regulatory domains share a large interface involving catalytic residues. In the structure of the active state, the two catalytic domains rotate by 55° to form two catalytic sites at their interface. Two α helices serve as molecular switches. Mutagenesis is consistent with a regulatory role of the structural transition, and we suggest that the transition is regulated by pH., Adenylyl cyclases (ACs) synthesize the universal second messenger 3',5'-cyclic adenosine monophosphate (cAMP) (1). Most ACs belong to class III, such as all mammalian and many bacterial enzymes (2), and are [...]

We have previously reported that a receptor-type adenylyl cyclase (CyaA) of Myxococcus xanthus undergoes an osmosensor mainly during spore germination (Y. Kimura et al., J. Bacteriol. 184:3578-3585, 2002). In the present study, we cloned another receptor-type adenylyl cyclase gene (cyaB) and characterized the function of the cyaB-encoded protein. Disruption of cyaB generates a mutant that showed growth retardation at high ionic (NaCI) or high nonionic (sucrose) osmolarity. When vegetative cells were stimulated with 0.15 M NaCl, the increases in intracellular cyclic AMP levels of cyaB mutant cells were lower than those of wild-type cells. Under nonionic osmostress, the cyaB mutant exhibited reduced spore germination; however, the germination rate of the cyaB mutant was significantly higher than that of the cyaA mutant.

Adenylyl cyclase (AC) is a prototypical cell-signaling molecule expressed in virtually all organisms form bacteria to man. 7Clb-S meets basic criteria to serve as a model protein for the Clb region and may be used as a prototype to develop other isoform Clb soluble model proteins.

Previous research has indicated that volatile odorants are detected through the main olfactory epithefium (MOE), whereas pheromones are detected via the vomeronasal organ (VNO). Gene disruption studies have established that olfactory signaling through the MOE is mediated through receptor stimulation of type 3 adenylyl cyclase (AC3). Mice lacking AC3 cannot detect odorants through the MOE. Recently, it was discovered using olfactory-based behavioral assays that AC3 mutant mice can detect some volatile odorants. An analysis of these mutant mice led to the surprising discovery that some odorants are detected through the VNO. Key words: adenylyl cyclase, olfaction, vomeronasal organ.

Adenylyl cyclase type VI corrects cardiac sarcoplasmic reticulum calcium uptake defects in cardiomyopathy. Am J Physiol Heart Circ Physiol 287: H1906-H1912, 2004. First published July 8, 2004; doi:10.1152/ajpheart.00356.2004.--Calcium malfunction plays a central role in heart failure. Here, we provide evidence that adenylyl cyclase type VI restores sarco(endo)plasmic reticulum 2a (SERCA2a) affinity for calcium and maximum velocity of cardiac calcium uptake by sarcoplasmic reticulum in murine dilated cardiomyopathy. Restoration of normal SERCA2a affinity for calcium is associated not only with decreased phospholamban protein expression but also with increased phospholamban phosphorylation by PKA activation. The ratio of phosphorylated ryanodine receptor 2 (RyR2) to RyR2 protein was increased, but the amount of phosphorylated RyR2 was unaffected. These data provide a possible mechanism by which adenylyl cyclase type VI (in contrast to other signaling elements associated with increased cAMP generation) has a salutary effect in the failing heart. cAMP; [beta]-adrenergic receptor; myocardium; heart failure

Time-resolved FRET and 2-aminopurine (2AP) fluorescence are used to examine the structure and dynamics of the polyadenylation inhibition element (PIE) RNA. Distance distributions of the RNA structures show that the intramolecular distance between the arms of the PIE RNA varies on the time scale of the fluorescence measurements.

I. Introduction II. Native GAB[A.sub.B] Receptors A. Coupling to G proteins B. Coupling to [Ca.sup.2+] channels C. Coupling to [K.sup.+] channels D. Coupling to adenylyl cyclase E. Pharmacology of native [...]

Studies done to explore the possible catalytic roles of Gln212, Asn270 and Arg301 aminoacids through site-directed mutagenesis are reported. Results of study reveals that Gln212, Asn270, and Arg301 are indispensable to catalysis by adenylosuccinate lyase and probably interact noncovalently with the carboxylate anions of the substrates 5-aminoimidazole-4(N-succinylocarboxamide)ribonucleotide and adenylosuccinate, optimizing their bound orientations.

To acquire the ability to fertilize, spermatozoa undergo complex, but at present poorly understood, activation processes. The intracellular rise of cAMP produced by the bicarbonate-dependent soluble adenylyl cyclase (sAC) has been suggested to play a central role in initiating the cascade of the events that culminates in spermatozoa maturation. Here, we show that targeted disruption of the sAC gene does not affect spermatogenesis but dramatically impairs sperm motility, leading to male sterility, sAC mutant spermatozoa are characterized by a total loss of forward motility and are unable to fertilize oocytes in vitro. Interestingly, motility in sAC mutant spermatozoa can be restored on cAMP loading, indicating that the motility defect observed is not caused by a structural defect. We, therefore, conclude that sAC plays an essential and nonredundant role in the activation of the signaling cascade controlling motility and, therefore, in fertility. The crucial role of sAC in fertility and the absence of any other obvious pathological abnormalities in sAC-deficient mice may provide a rationale for developing inhibitors that can be applied as a human male contraceptive.

Bicarbonate-responsive 'soluble' adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation.

In mammals, [Ca.sup.2+] and HC[O.sup.-.sub.3] ions play a critical role in the regulation of sperm function, most likely by regulation of cAMP levels. Mammalian germ cells contain a soluble adenylyl cyclase (sAC) with properties distinct from the well characterized membrane-bound enzymes Here we investigated whether the cyclase expressed in mature spermatozoa has the properties of sAC and whether it is regulated by [Ca.sup.2+]. In addition to an HC[O.sup.-.sub.3]-dependent activation, the cyclase endogenous to human spermatozoa is stimulated 2- to 3-fold by [Ca.sup.2+]. In a concentration-dependent manner (E[C.sub.50] [approximately equal to] 400 nM). In a similar fashion, [Ca.sup.2+] activates the recombinant rat and human full-length sAC with similar E[C.sub.50] values. The [Ca.sup.2+] stimulation was also observed when sAC was activated with HC[O.sup.-.sub.3], was independent of calmodulin, and was associated with an increase in [V.sub.max] without changes in [K.sub.m] for ATP-[Mg.sup.2+]. An increase in intracellular [Ca.sup.2+] by ionophore or by a muscarinic cholinergic receptor agonist increases cAMP in cells transfected with FL-hsAC, but not in mock-transfected cells. Similarly, both [Ca.sup.2+] and HC[O.sup.-.sub.3] stimulate cAMP accumulation in human spermatozoa. These findings provide evidence that human spermatozoa express a cyclase with the properties of sAC and that [Ca.sup.2+] can substitute for HC[O.sup.-.sub.3] in the stimulation of this enzyme, underscoring an important role for sAC in the control of sperm functions.

The sympathetic nervous system is designed to respond to stress. Adenylyl cyclase (AC) is the keystone of sympathetic transmission, yet its role in response to acute overload in the heart or in the pathogenesis of heart failure is controversial. We examined the effects of pressure overload, induced by thoracic aortic banding, in mice in which type 5 AC, a major cardiac AC isoform, was disrupted (AC[5.sup.-/-]). Left ventricular weight/tibial length ratio (LVW/TL) was not different between the WT and AC[5.sup.-/-] at baseline and increased progressively and similarly in both groups at 1 and 3 wk after aortic banding. However, LV ejection fraction (LVEF) fell in WT at 3 wk after banding (from 70 [+ or -] 2.8 to 57 [+ or -] 3.9%, P < 0.05), and this decrease was associated with LV dilatation, indicating incipient cardiac failure. In contrast, AC[5.sup.-/-] mice did not exhibit a fall in LVEF from 74 [+ or -] 2.2%. The number of apoptotic myocytes was similar at baseline, but it increased roughly 4-fold in WT at both 1 and 3 wk after banding, and significantly less, P < 0.05, in AC[5.sup.-/-]. Importantly, the increase in apoptosis occurred before the decline in LVEF in WT. The protective mechanism seems to involve Bcl-2, which was up-regulated significantly more in AC[5.sup.-/-] mice with pressure overload. Our findings suggest that limiting type 5 AC plays a protective role in response to pressure overload and the development of heart failure, potentially through limiting the incidence of myocardial apoptosis.

The intracellular levels of cAMP play a critical role in the meiotic arrest of mammalian oocytes. However, it is debated whether this second messenger is produced endogenously by the oocytes or is maintained at levels inhibitory to meiotic resumption via diffusion from somatic cells. Here, we demonstrate that adenylyl cyclase genes and corresponding proteins are expressed in rodent oocytes. The mRNA coding for the AC3 isoform of adenylyl cyclase was detected in rat and mouse oocytes by RT-PCR and by in situ hybridization. The expression of AC3 protein was confirmed by immunocytochemistry and immunofluorescence analysis in oocytes in situ. Cyclic AMP accumulation in denuded oocytes was increased by incubation with forskolin, and this stimulation was abolished by increasing intraoocyte [Ca.sub.2+] with the ionophore A23187. The [Ca.sub.2+] effects were reversed by an inhibitor of [Ca.sub.2+], calmodulin-dependent kinase II. These regulations of cAMP levels indicate that the major cyclase that produces cAMP in the rat oocyte has properties identical to those of recombinant or endogenous AC3 expressed in somatic cells. Furthermore, mouse oocytes deficient in AC3 show signs of a defect in meiotic arrest in vivo and accelerated spontaneous maturation in vitro. Collectively, these data provide evidence that an adenylyl cyclase is functional in rodent oocytes and that its activity is involved in the control of oocyte meiotic arrest. Keywords: cAMP; Adenylyl cyclase; Oocyte; Meiosis; Cell cycle

Byline: Kentaro Takei (1), Yasumasa Dekishima (2), Tadashi Eguchi (2), Tomoyuki Yamaya (1), Hitoshi Sakakibara (1) Keywords: Adenylate isopentenyltransferase; Cytokinin; Isopentenyladenine; Purine-nucleoside phosphorylase; Radioisotope labeling; trans-Zeatin Abstract: We describe a new enzymatic reaction method for the preparation of the radioisotope-labeled cytokinins isopentenyladenine (iP), trans-zeatin (tZ), and their ribosides. The method is based on the three enzyme activities of an adenylate isopentenyltransferase (IPT EC 2.5.1.27) from Arabidopsis thaliana, an alkaline phosphatase (EC 3.1.3.1) from calf intestine, and a purine-nucleoside phosphorylase (EC 2.4.2.1) from Escherichia coli. The A. thaliana IPT, AtIPT7, utilized both dimethylallyldiphosphate and 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate as isoprenoid donors. The dual specificity of the substrates enabled us to produce iP-type and tZ-type cytokinins separately in the same system simply by switching the substrates. Our method affords a much higher yield of the labeled products than the chemical reaction methods previously used. These labeled compounds will be useful tools for cytokinin research, such as receptor--ligand assays and cell metabolism studies. Author Affiliation: (1) Plant Science Center, RIKEN (The Institute of Physical and Chemical Research), 1-7-22 Suehiro, Tsurumi, Yokohama 230-0045, Japan (2) Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro, Tokyo 152-8551, Japan Article History: Received Date: 29/12/2002 Accepted Date: 06/03/2003 Online Date: 01/05/2003

Byline: Shamsher S. Saini (1), Deborah L. Gessell-Lee (1), Johnny W. Peterson (1) Keywords: COX-2; Celecoxib; adenylyl cyclase; enzyme kinetics; inflammation; cholera toxin Abstract: Nonsteroidal anti-inflammatory drugs (NSAIDs) are well-known causes of acute renal insufficiency and gastropathy in patients with chronic inflammatory diseases. This action is presumed to result from nonselective inhibition of both constitutive and inducible forms of prostaglandin H synthases, also known as the cyclooxygenase enzymes (i.e., COX-1 amd COX-2). Celecoxib (Celebrex.sup.(r)) is a COX-2 enzyme inhibitor and has emerged as a preferred therapeutic agent for the treatment of rheumatoid arthritis as compared to other NSAIDs. Celecoxib has recently been the subject of criticism for its side effects, mainly arterial thrombosis and renal hemorrhage, although it is considered a superior drug in protecting the gastrointestinal tract. In the present study, we report that celecoxib not only inhibited COX-2, but also exhibited the property of inhibiting adenylyl cyclase, an important enzyme forming the intracellular second messenger 3 ,5 -adenosine monophosphate (cAMP) from adenosine triphosphate (ATP). Celecoxib also inhibited cholera toxin-stimulated cAMP formation, which indicated its ability to permeate cell membranes in order to reach intracellular adenylyl cyclase. It inhibited in vitro adenylyl cyclase activity in both human colonic epithelial cells and purified adenylyl cyclase from Bordetella pertussis. The IC.sub.50 of celecoxib for B. pertussis adenylyl cyclase was calculated to be 0.375 mM. Lineweaver--Burk analysis showed that the type of enzyme inhibition was competitive. The apparent K .sub.m and V .sub.max of adenylyl cyclase was calculated as 25.0 nM and 7.14 nmol/min/mg, respectively. Celecoxib changed the K .sub.m value to 66.6 nM without affecting the V .sub.max. The current study suggests that apart from inflammation, celecoxib therapy could be further extended to diseases involving cAMP upregulation either by endogenous reactions or exogenous agents. These new data showing inhibition of adenylyl cyclase should be considered in light of the drug's pathological effects or in patients specifically excluded from treatment (e.g., asthmatics). Author Affiliation: (1) Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas Article History: Registration Date: 20/10/2004

Byline: Y. Zhao (), D. Xu (), J.M. Quaegebeur (), C.W. Emala (), L.S. Sun () Abstract: We have previously demonstrated that both basal and isoproterenol-stimulated activities of myocardial adenylyl cyclase were greater in cyanotic patients with tetralogy of Fallet (TOF) than those in acyanotic patients. However, it was not determined whether increased enzyme activity was related to a similar increase in adenylyl cyclase protein and mRNA expression. In the current study, we examined the mRNA and protein expression of cardiac adenylyl cyclase, types V and VI, in cyanotic and acyanotic patients with TOF. Ribonuclease protection assays and immunoblotting were performed on myocardial specimens obtained from cyanotic patients with TOF and acyanotic patients with TOF or ventricular septal defect. We demonstrated that in both cyanotic and acyanotic patients, there was more type V adenylyl cyclase mRNA than type VI. Types V and VI cardiac adenylyl cyclase mRNA were significantly increased in myocardium of the cyanotic group compared to the acyanotic group. Protein expression of both V and VI adenylyl cyclases was correspondingly upregulated in cyanotic patients compared to acyanotic patients. Our results indicate that gene and protein expression of cardiac adenylyl cyclases, types V and VI, is increased in the cyanotic myocardium. These results suggest that chronic hypoxemia may regulate the expression of adenylyl cyclase enzymes. Author Affiliation: () Department of Anesthesiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA, US () Department of Surgery, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA, US () Departments of Anesthesiology and Pediatrics, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA, US

Byline: Tomoko Tada (1), Hiroyuki Sekimoto (1), Masayuki Ohmori (1) Keywords: Keywords: Adenylate cyclase, Cyanobacterium, Forskolin, GAF domain, PAS domain Abstract: gene encodes a novel type of adenylate cyclase. The catalytic domain is located in the carboxyl-terminal half, while the GAF and PAS domains are conserved in the amino-terminal half. Recombinant CyaB1 and a truncated CyaB1 lacking the amino-terminal domain (IN--CyaB1) were purified and characterized. The purified CyaB1 is activated by divalent cations, such as Mg.sup.2+ and Mn.sup.2+, like other types of adenylate cyclase. The activity of CyaB1 was slightly elevated by forskolin, but was not affected by cGMP, irrespective of the presence of the cGMP binding motif in the GAF domain. The specific activity of IN--CyaB1 is one-eighteenth that of CyaB1, whereas the Km values of both proteins are almost the same. The results suggest that the amino-terminal half has a positive regulatory effect on the catalytic activity. Author Affiliation: (1) Department of Life Sciences, Graduate School of Arts and Sciences, the University of Tokyo, 3--8--1 Komaba, Meguro, Tokyo, 153--8902 Japan, JP Article note: Received 27 April 2001/ Accepted in revised form 6 July 2001

Type V adenylyl cyclase has been overexpressed in the transgenic mice hearts. Results demonstrate that adenylyl cyclase levels influence basal activities and cardiac functions.

The two large cytoplasmic domains (C1 and C2) of adenylyl cyclases (AC), when expressed separately and mixed together, reconstitute enzyme activity that can be regulated by various modulators. Therefore, we have used the C1 or its C1a subdomain and C2 regions from type I AC (ACI) and type V AC (ACV) to identify the region on ACI that interacts with [Beta][Gamma] subunits of heterotrimeric G proteins. In addition, we also used a chimeric C1 domain (VC1aIC1b) in which the C1a region was derived from ACV and the C1b region was from ACI. By mixing the C1 or C1a or VC1aIC1b domains with C2 regions of ACI or ACV, we have shown that the C1a region (amino acids 236-471) of ACI is sufficient to observe [Beta][Gamma]-mediated inhibition of enzyme activity, which is stimulated by either constitutively active [G.sub.s[Alpha]] ([[G.sub.s[Alpha].sup.*]]) or [Ca.sup.2+]/calmodulin (CAM). Although the C1b region and C2 domain of ACI were by themselves not sufficient for inhibition of activity by [Beta][Gamma]subunits, the presence of both of these regions formed another [Beta][Gamma] interaction site that was sufficient to observe [[G.sub.s[Alpha].sup.*]]- or [Ca.sup.2+]/CaM-stimulated activity. Inhibition of AC activity attributable to interaction of [Beta][Gamma] subunits at either of the two sites was blocked by a peptide (QEHA) that has previously been shown to inhibit the effects of [Beta][Gamma] on various effectors. Moreover, the C1 region of ACI was sufficient to observe [G.sub.i[Alpha]1]-elicited inhibition of [Ca.sup.2+]/CaM-stimulated activity. Although the C1a region of ACV was sufficient for inhibition of activity by [G.sub.i[Alpha]1], the presence of C1b region from either ACI or ACV increased sensitivity to inhibition by the inhibitory G protein. Thus, the inhibitory influences of [G.sub.i[Alpha]1] are mediated on the C1 regions of both ACI and ACV. The effects of [Beta][Gamma] on ACI can be mediated by interactions with the C1a region and a [Beta][Gamma] interacting site formed by the C1b and C2 domains of this enzyme.

Activation of cAMP-dependent protein kinase (PKA) triggers terminal differentiation in Dictyostelium, without an obvious requirement for the G-protein-coupled adenylyl cyclase, ACA, or the osmosensory adenylyl cyclase, ACG. A third adenylyl cyclase, ACB, was recently detected in rapidly developing mutants. The specific characteristics of ACA, ACG, and ACB were used to determine their respective activities during development of wild-type cells. ACA was highly active during aggregation, with negligible activity in the slug stage. ACG activity was not present at significant levels until mature spores had formed. ACB activity increased strongly after slugs had formed with optimal activity at early fruiting body formation. The same high activity was observed in slugs of ACG null mutants and ACA null mutants that overexpress PKA (acaA/PKA), indicating that it was not due to either ACA or ACG. The detection of high adenylyl cyclase activity in acaA/PKA null mutants contradicts earlier conclusions (B. Wang and A. Kuspa, Science 277, 251-254, 1997) that these mutants can develop into fruiting bodies in the complete absence of cAMP. In contrast to slugs of null mutants for the intracellular cAMP-phosphodiesterase REGA, where both intact cells and lysates show ACB activity, wild-type slugs only show activity in lysates. This indicates that cAMP accumulation by ACB in living cells is controlled by REGA. Both REGA inhibition and PKA overexpression cause precocious terminal differentiation. The developmental regulation of ACB and its relationship to REGA suggest that ACB activates PKA and induces terminal differentiation. Key Words: adenylyl cyclase B; protein kinase A; rapidly developing mutants; terminal differentiation; Dictyostelium discoideum.

The hypothesis that differences in enzyme regulation of cyclic adenylic acid (cAMP) synthesis and degradation uniquely establish an elevated content in pulmonary microvascular endothelial cells (PMVECs) was tested. This was because the mechanism that enables PMVECs to form a more restrictive barrier to macromolecular flux than pulmonary arterial endothelial cells was unknown. The results showed that suppressed calcium entry and low adenosine triphosphate-to-cAMP conversion intrinsincally influenced calcium sensitivity.

Research was conducted to examine the mechanism of action of cholera toxin (CTX) on the opossum internal anal sphincter (IAS) smooth muscle. The objective was to investigate the effects of CTX on the signal transduction related to the adenylate cyclase (AC) pathway on the basal tone of the IAS that was not affected by the neurotoxins TTX and omega-conotoxinor the nitric oxide synthase inhibitor N(super G)-nitro-L-arginine. Results demonstrate that a major part of the inhibitory action of CTX in the IAS is through the direct response of the smooth muscle cell that is linked with G(sub S) protein to the AC pathway.

A study was conducted to determine whether endothelin-1 (ET-1)-induced Na+/H+ antiporter activation is related with Na+/H+ exchanger isoform 3 (NHE3) activation and phosphorylation. Results suggest that ET-1 activates NHE3. Binding of ET-1 to ET (sub B) receptors results in a two- to three-fold elevation in NHE3 phosphorylation with a dose and time dependence that parallels that of the change in activity. Serine and threonine residues are involved in phosphorylation at multiple sites.