Soluble adenylyl cyclase as an evolutionarily conserved bicarbonate sensor.

Spermatozoa undergo a poorly understood activation process induced by bicarbonate and mediated by cyclic adenosine 3',5'-monophosphate (cAMP). It has been assumed that bicarbonate mediates its effects through changes in intracellular pH or membrane potential; however, we demonstrate here that bicarbonate directly stimulates mammalian soluble adenylyl cyclase (sAC) activity in vivo and in vitro in a pH-independent manner. sAC is most similar to adenylyl cyclases from cyanobacteria, and bicarbonate regulation of cyclase activity is conserved in these early forms of life. sAC is also expressed in other bicarbonate-responsive tissues, which suggests that bicarbonate regulation of cAMP signaling plays a fundamental role in many biological systems.

A calcium-inhibited Drosophila adenylyl cyclase.

Mammals possess a family of transmembrane, G-protein-responsive adenylyl cyclase isoforms (tmACs) encoded by distinct genes differing in their patterns of expression and modes of biochemical regulation. Our previous work confirmed that Drosophila melanogaster also possesses a family of tmAC isoforms defining the fly as a suitable genetic model for discerning mammalian tmAC function. We now describe a Drosophila tmAC, DAC39E, which employs a novel means for regulating its expression; differential exon utilization results in a developmental switch in DAC39E protein. DAC39E protein sequence is most closely related to mammalian type III AC, and it is predominantly expressed in the central nervous system (CNS) and olfactory organs, suggesting a role in processing sensory signaling inputs. DAC39E catalytic activity is inhibited by micromolar concentrations of calcium; therefore, DAC39E is oppositely regulated by calcium compared to the only other tmAC shown to be expressed in the Drosophila CNS, Rutabaga AC. The presence of both positively and negatively regulated tmACs suggests a complex mode of cross-talk between cAMP and calcium signal transduction pathways in the fly CNS.

Decreased type VI adenylyl cyclase mRNA concentration and Mg(2+)-dependent adenylyl cyclase activities and unchanged type V adenylyl cyclase mRNA concentration and Mn(2+)-dependent adenylyl cyclase activities in the left ventricle of rats with myocardial infarction and longstanding heart failure.

OBJECTIVE: To address the effect of longstanding left ventricular (LV) hypertrophy and failure on LV adenylyl cyclase (AC) gene expression, mRNA concentrations of the main cardiac AC isoforms were measured in the non-infarcted area of LV from rats with myocardial infarction (MI), without (H) or with (F) LV failure, and in control (C) rats. Basal, GTP- and forskolin-stimulated Mg(2+)- and Mn(2+)-dependent AC activities were also measured in F and C rats. METHODS: Two- and six months after MI, steady-state AC mRNA concentrations were assessed by Northern blot analysis and RNase protection assay with isoform-specific cDNA and cRNA probes, respectively. AC activities were assessed on LV microsomal fractions using standard procedures. RESULTS: Types V and VI, and types IV and VII were the major and minor AC mRNA isoforms in both the LVs of F and C rats. Two months after MI, no difference in LV type V or VI mRNA to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratios was observed in rats with H or F compared to C. Six months after MI, no difference in LV type V mRNA concentration was observed between the three rat groups, whether this level was normalized to GAPDH, poly-(A+) or 18S RNAs. In contrast, a 35% decrease in the type VI mRNA to poly-(A+) RNA ratio and a 29% decrease in the type VI mRNA to 18S RNA ratio was observed only in rats with F compared to C (p < 0.05 vs. C for the two comparisons). Two- and six months after MI, basal and forskolin-stimulated Mg(2+)-dependent AC activities were decreased by 30-35% in F rats compared to C (p < 0.05), whereas Mn(2+)-dependent activities were unchanged. CONCLUSION: Longstanding LV hypertrophy and failure resulting from MI in rats is not associated with altered expression of the most abundant, type V, AC mRNA isoform, whereas that of type VI is decreased. The lack of change in Mn(2+)-dependent AC activities in the LV of F rats suggests that this decrease has no functional consequence on overall AC activity and that decreased Mg(2+)-dependent activities are related to alterations occurring upstream.

Cloning and characterization of a Drosophila adenylyl cyclase homologous to mammalian type IX.

A novel Drosophila adenylyl cyclase (AC) was identified by PCR using degenerate primers specific for the known metazoan ACs. The full-length cDNA predicts a protein displaying significant sequence homology with mammalian Type IX AC (AC9). The abundance and size of the message for the Drosophila AC9 homolog (DAC9) changes through development. Biochemical analysis of DAC9 confirms it encodes a functional enzyme which can be activated by forskolin or G protein. Together with the Drosophila Type I AC homolog encoded by the learning and memory gene, rutabaga, the molecular identification of DAC9 demonstrates there is a family of Drosophila AC isoforms reflecting at least part of the diversity of mammalian AC isoforms.

Growth inhibitory properties of endothelin-1 in activated human hepatic stellate cells: a cyclic adenosine monophosphate-mediated pathway. Inhibition of both extracellular signal-regulated kinase and c-Jun kinase and upregulation of endothelin B receptors.

During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype, proliferate, and synthetize fibrosis components. We have shown that endothelin-1 (ET-1) inhibits the proliferation of activated human HSC via endothelin B (ETB) receptors. We now investigate the transduction pathway involved in the growth inhibitory effect of ET-1 in activated HSC. Endothelin-1 and the ETB receptor agonist, sarafotoxin-S6C, increased synthesis of PGI2 and PGE2, leading to elevation of cAMP. The cyclooxygenase inhibitor ibuprofen and the adenylyl cyclase inhibitor SQ22536 both blunted the growth inhibitory effect of ET-1. Analysis of early steps associated with growth inhibition indicated that: (a) similar to ET-1, forskolin decreased c-jun mRNA induction without affecting c-fos and krox 24 mRNA expression; (b) ET-1, sarafotoxin-S6C, as well as forskolin, reduced activation of both c-Jun kinase and extracellular signal-regulated kinase. Finally, forskolin, PGI2, and PGE2 raised by fivefold the number of ET binding sites after 6 h, and increased the proportion of ETB receptors from 50% in control cells to 80% in treated cells. In conclusion, ET-1 inhibits proliferation of activated HSC via ETB receptors, through a prostaglandin/cAMP pathway that leads to inhibition of both extracellular signal-regulated kinase and c-Jun kinase activities. Upregulation of ETB receptors by prostaglandin/cAMP raises the possibility of a positive feedback loop that would amplify the growth inhibitory response. These results suggest that ET-1 and agents that increase cAMP might be of interest to limit proliferation of activated HSC during chronic liver diseases.

[Beta adrenergic signal transduction and heart adenyl cyclase]. / Transduction du signal beta-adrénergique et adénylate cyclases cardiaques.

Transduction of the beta-adrenergic signal plays an important role in the regulation of cardiac contractility. It is mediated by three sarcolemmal proteins: the beta-adrenergic receptor, G proteins and adenylyl cyclase which is the catalytic unit of the system which generates cAMP, the second messenger of the system. Each protein comprises a number of isoforms which yields a wide range of potential regulations, many of which are not yet elucidated. Among the three proteins, the adenylyl cyclase is the one which has been less studied. However, the recent cloning of many of its isoforms allows now investigations of their expression in many tissues and cell types. We have shown in rats that among the five isoforms detected in the myocardium, type V and VI adenylyl cyclase mRNAs are the most abundant ones. Type V and VI adenylyl cyclase mRNA abundance is similar in late fetal hearts. Type V mRNA accumulates in the heart during postnatal development whereas type VI mRNA concentration remains unchanged. Consequently, type V mRNA becomes highly predominant compared to type VI mRNA in the adult rat ventricle (type V/type VI adenylyl cyclase mRNAs approximately 10). Whatever the developmental stage, cardiac adenylyl cyclase activity is inhibited by submicromolar calcium concentrations. In adult ventricles, adenylyl cyclase activity in the presence of 1 mM ATP is at least three times higher than that observed in fetal and new born rat hearts. Since this increase parallels the accumulation of type V adenylyl cyclase mRNA, one can hypothesize that the former is due to the latter. In contrast, our preliminary results seem to indicate that during heart failure in rats, decreased adenylyl cyclase activity is not associated with decreased cardiac concentrations of type V and VI adenylyl cyclase mRNAs. Isoform specific antibodies are now required to understand the reasons for such discrepancy.

Type V, but not type VI, adenylyl cyclase mRNA accumulates in the rat heart during ontogenic development. Correlation with increased global adenylyl cyclase activity.

Type V and VI adenylyl cyclase mRNAs are the two main cyclase isoforms expressed in the mammalian heart. A recent report has shown that their expression is differentially regulated during ontogenic development, but the accumulation of the two mRNA species and their concentration ratio have not been determined. We thus determined the accumulation and the relative amounts of type V and VI adenylyl cyclase mRNA in fetal, neonatal and adult rat hearts, using a sensitive ribonuclease protection assay. In 18-day-old fetuses, the two adenylyl cyclase mRNA isoforms were weakly expressed in approximately equal amounts (type V mRNA/type VI mRNA = 0.93 +/- 0.09). Further development was characterized by a sharp increase in type V adenylyl cyclase mRNA (x 1.9 in neonates v fetuses, P < 0.01; x 2.4 and x 4.5 in adults v neonates and fetuses, respectively, P < 0.01 for both comparisons) and a slight, non-significant fall in type VI mRNA (P = 0.16). As a result, the type V mRNA/type VI mRNA ratio was 2.86 +/- 0.57 and 9.09 +/- 1.21 in neonatal hearts and adult ventricles, respectively (P < 0.01 v ratio in fetal hearts for both comparisons; P < 0.01 for ratio in adult ventricles v ratio in neonatal hearts), and the overall amount of the two mRNA isoforms was 2.3 times greater in adult than in fetal hearts (P < 0.01). This increase was paralleled by an increase in basal and isoproterenol- and forskolin-stimulated adenylyl cyclase activities in adult hearts compared to fetal and neonatal hearts (P < 0.01 for the three comparisons). Our results demonstrate that type V adenylyl cyclase mRNA accumulates in the rat heart after birth to become the highly predominant isoform in the adult heart. They further suggest that the increase in cardiac adenylyl cyclase activity observed during rat development is due to this accumulation.

Molecular cloning of the human type VIII adenylyl cyclase.

A cDNA coding for a human type VIII adenylyl cyclase has been isolated from human newborn brain-stem tissue. This cDNA is 6,005 bp long and encodes for a protein of 1251 amino acids, exhibiting the two sets of six transmembrane spanning regions and the hydrophobicity profile typical of other mammalian adenylyl cyclases. Comparison with the rat form shows that they share 97% identity in amino acids. Type VIII adenylyl cyclase is unique in that it has both a long carboxy terminal and a long amino terminal tail. This is the first report on a complete cDNA clone coding for a human adenylyl cyclase. The distribution and regulation of this particular adenylyl cyclase suggest that it may be involved in learning, in memory and in drug dependence.