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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

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

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.

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

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

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

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.

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.

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.

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

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

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.

Research was conducted to examine the regulatory transposition in the chimera of mammalian guanylyl and adenylyl cylases (GC/AC chimera) by coexpressing guanylyl cyclase and adenylyl cylase halves. Results demonstrate the ability of functional complement at the catalytic sites in both cyclases. Findings obtained from the enzymatic characterization of the chimeric cyclase suggest an important role of the N-terminal adenylyl cyclase half for stimulatory actions.

Correlation of potential changes in the structure and dynamics of the Ca-ATPase with changes in the phosphatidylethanolamine (PE) content of the bilayer was studied using frequency-domain phosphorescence spectroscopy. The Ca-ATPase structure was specifically modified at Lys 464 within the phosphorylation domain with the long-lived phosphorescent probe erythrosin isothiocyanate. Correlations between the maximal ATP hydrolytic activities of Ca-ATPase and the dynamic structure of the phosphorylation domain suggest that PE headgroups enhance the catalytic activity of Ca-ATPase.

An experiment was conducted to test the theory that regional left ventricular function and regional alterations in left ventricular beta-adrenergic receptor signaling would be tightly connected. The confirmation of a close link would suggest that factors within the left ventricle are more important than systemic factors in the pathogenesis of myocardial adrenergic desensitization in heart failure.

Research was conducted to examine the potential role of adenylyl cyclases (ACs) in gene regulation and signaling in the developing mouse skeletal muscle. Using Northern blot analysis, four isoforms of AC, namely, AC2, AC6, AC7 and AC9, were detected in gastrocnemius muscle. AC activity was measured in the presence of forskolin, isoproterenol, epinephrine and amylin and in the absence of stimuli in different stages of development. Results demonstrate that AC mRNAs and AC activity are critical in the different physiopathological states of skeletal muscle development.

The process of development in Dictyostelium is prompted by starvation-induced DdMyb2 activation. This leads to adenylyl activity and produces cAMP, which triggers differentiation-inducing signals. The DdMyb2 gene is a DNA-binding motif of Myb-related transcription factors. Mutants without DdMyb2 can be rescued with ectopic expression of ACA.

Research was conducted to study the influence of galanin or galanin-derived peptides on cAMP production, MAPK activity and the accumulation of inositol phosphate in the Chinese hamster ovary cell lines expressing the rat galanin receptor subtypes. Cells were split into 12-well plates and incubated for one to two days while DNA and protein sequence studies were carried out using the DNA* software. The physiological functions showed by galanin indicated the involvement of multiple galanin receptors activating distinct intracellular signaling pathways.

The marine diatoms Thalassiosira rotula, Odontella sinensis and Lauderia annulata were isolated from plankton samples of the North Sea and exposed to ultraviolet (UV) radiation of various wavebands to study the effects of the radiation on pigmentation, amino acid pools, adenylate contents and (sup 15)N-ammonium uptake of the diatoms. L. annulata and O. sinensis were exposed to Philips lamps with cut-off filters for UV-B and UV-A. T. rotula was exposed to special Philips lamps. Results showed a reduction in the pigment content of all three diatoms upon exposure to UV-B and UV-B plus UV-A radiation.

Escherichia coli samples are studied in an experiment to determine if the protein IIA(super Glc) of the phosphoenolpyruvate:sugar phosphotransferase system can affect the basal activity of adenylyl cyclase. The samples were mutated via site-directed mutagenesis to glutamate and glutamine. Results show that the tested protein did not affect basal activity. However, partial and full activators of adenylyl cyclase were identified.

Research was conducted to test whether the phosphoinositol/Ca2+ pathway associated with the adenylate cyclase/adenosine 3',5'-cyclic monophosphate (cAMP) pathway in the cardiac Kappa-receptor. A collagenase perfusion method was utilized to take isolate ventricular myocytes from the heart of male Sprague-Dawley rats. Results showed that the activation of the pathway suppressed the AC/cAMP pathway via an intracellular Ca2+ concentration.

Research was conducted to investigate the effects of glucocorticoids on G protein-regulated phospholipase C (PLC) activity in osteoblastic cells. Dexamethasone increased basal and parathyroid hormone (PTH) activated phospholipase C correlated by a differential increase in the expression of Galpha1-11 and Galphas in these cells. Dexamethasone treatment also increased the response to PTH over the response in control cells. Results indicated that the synthetic glucocorticoid Dex is a potent regulator of hormone-stimulated PLC in the UMR cells.

The effect of arginine vassopressin (AVP) on agonist-stimulated adenosine 3', 5'-cyclic monophosphate (cAMP) accumulation was examined in vascular smooth muscles cultured (VSMC) from rat thoracic aorta. Results of the experiment indicated that AVP activates V1 formation by a Ca2+-calmodulin-dependent mechanism. Furthermore, type III adenylyl cyclase provides a focal point in the VSMC for processes between constrictor and dilator pathways.

The role of protein kinase C (PKC) in beta2 adrenoreceptor function in cultured rat proximal tubule epithelial cells was analyzed using proximal epithelial cells in culture. Results of the experiment showed that beta2-adrenogenic receptor (AR)-dependent increases in Na-K-ATPase activity and sodium flux are tranduced by PKC. The activation of PKC by phorbal esters inhibits beta2-AR-dependent increases in sodium transport and Na-K-ATPase activity .

Research was conducted to examine the effect of triiodothyronine (T3) administration to euthyroid rats on Beta3-adrenoceptor (Beta3-AR) expression and on the different components of the adenylyl cyclase system in brown adipose tissue. In rats treated with T3, the Beta3-AR density (assessed by the binding of [3H]CGP-12177) showed a decrease of 50%, as did their mRNA, as analyzed by reverse transcriptase-polymerase chain reaction. Results indicate that downregulation of the Beta3-AR by T3 was counterbalanced by changes in other components of the AC cascade.

cAMP assay and measurement of expression of cAMP-regulated genes were conducted to determine the relationship between external glucose concentration and cAMP levels inside Escherichi coli. Results reveal that E. coli controls its cAMP levels through two or more transitions influenced by external glucose concentration and does not respond to decreasing saturation of glucose transport. Furthermore, results present implications for models of phosphotransferase-mediated regulation of adenylate cyclase.

The antagonism of adenylyl cyclase activity is unnecessary for the carbachol-induced inhibition of positive inotropic responses of ventricular muscle to adenosine 3',5'-cyclic monophosphate (cAMP) -generating agents. The elevation of cAMP levels in the particulate fraction is related to the positive inotropic response to cAMP-elevating agents. This elevation is independent of selective coupling of muscarinic receptors to the pool of adenylyl cyclase.

Membrane potential modulates adenylylcyclase (AC) in sea urchin sperms. Hyperpolarization of Lytechinus pictus sperms with valinomycin in artificial sea water (ASW) increases c-AMP in the absence of K+. The increase also occurs on dilution of ASW containing Strogylocentrotus purpuratus sperms without Na+, and in the absence of extracellular Ca2+. A phosphodiesterase inhibitor, IBMX, enhances the c-AMP increase. Increase in AC levels which were attributed to increases in intracellular Ca2+ and pH are improbable in the absence of Na+ and Ca2+.

Adenyl cyclase is involved in the the accumulation and deposition of brown adipose tissue (BAT) soon after birth. The activation of adenyl cyclase in BAT was investigated in newborn rats. These rats exhibited a high rate of expression of an adenyl cyclase subtype, AC-III, which accounted for all of the adenyl cyclase activity at this developmental stage. The appearance of AC-III signaled the start of AC activity under stimulation by isoproterenol.

Previous research has shown low cAMP levels in Escherichia coli grown on glucose 6-phosphate (Glc6P). To explain this phenomenon, the mechanism leading to the low cAMP level when Glc6P is the carbon source is investigated. Results indicate that Flc6P is not directly responsible for the low cAMP level in E. coli but rather that the level is determined by a flux in the Glc6P through the UhpT.

Catalyzed by cholera toxin, ADP ribosylation of the stimulatory G protein-alpha (Gs-alpha), associated with adrenergic stimulation of adenylyl cyclase, increases with age in the rat liver. These age related changes in Gs are probably responsible for the increase in the beta-adrenergic-responsiveness of adenylyl cyclase. ADP ribosylation factors appear to be involved in the changes in Gs. However, immunoreactive alpha- and beta-subunits of Gs and the inhibitory G protein are unaffected in rats between 6 to 24 months of age. Toxin labeling and Gs activity increase from 6-18 months, but decrease at 24 months.

The nonconserved domains C(sub 1b) and C(sub 2b) of adenylyl cyclase are not essential for regulating the enzymatic activity by G(sub s-alpha) and forskolin. This is observed for truncation mutants of adenylyl cyclase from the recombinant baculovirus. When some of the most conserved residues in the two domains are replaced by alanine, the enzyme shows very low G(sub s-alpha) binding activity, even if expressed fully. Its low P-site inhibition and high K(sub m, ATP) suggests that ATP and P-site inhibitors probably bind to the same site with different conformations.