From vasopressin receptor to water channel: intracellular traffic, constraint and by-pass.
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Adenylyl Cyclases↗
Biomedical subjects
J Hanoune
Publications and source records attributed to J Hanoune.
Different expression of adenylyl cyclase isoforms after retinoic acid induction of P19 teratocarcinoma cells.
We have investigated the adenylyl cyclase (AC) activity and gene expression in retinoic acid (RA)-primed murine P19 teratocarcinoma cells, which recapitulate in vitro the first stages of neuroectodermal formation. Here we show that the P19 stem cells possess a basal Ca2+/CaM-stimulated AC activity, which increases about 10-fold after RA induction. The rise of AC activity is associated with a stage-specific up-regulation of AC2, AC5 and AC8 mRNAs and a down-regulation of AC3 mRNA. P19 cells provide a powerful model to investigate the role and specific regulation of AC isoforms during neuronal differentiation.
Adenylyl Cyclases↗
Adenylyl cyclases: structure, regulation and function in an enzyme superfamily.
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Adenylyl Cyclases↗
Effects of repeated methamphetamine administration on dopamine D1 receptor, D2 receptor and adenylate cyclase type V mRNA levels in the rat striatum.
Dopamine D1 receptor (D1R), D2 receptor (D2R) and adenylate cyclase type V (AC5) mRNAs in the rat dorsal striatum (CPu) and ventral striatum (Acb) were measured by in situ hybridization histochemistry (ISHH) after repeated methamphetamine (MAP) administration. MAP (4 mg/kg) or an equivalent volume of saline was administered intraperitoneally once daily for 10 days to male Wistar rats. After 7 days of abstinence, behavioral experiment with MAP-challenge revealed that a significant behavioral sensitization was achieved in MAP-treated animals. The animals were sacrificed 7 days after the last injection and brain sections were analyzed by ISHH using 35S-labeled antisense oligonucleotide probes. We found that D1R mRNA levels in the CPu were significantly increased (124% of control, P < 0.01). In contrast, repeated MAP-treatment did not significantly affect the expression of D1R mRNA in Acb or mRNA for D2R or AC5. It was concluded that MAP-induced behavioral sensitization is accompanied by increased D1R mRNA levels in CPu.
Adenylyl Cyclases↗
Differential expression of type I, II, and V adenylyl cyclase gene in the postnatal developing rat brain.
The developmental changes in the expression of mRNA encoding three major brain adenylyl cyclase (AC; EC 4.6.1.1) subtypes, type I (AC1), II (AC2), and V (AC5), were examined by in situ hybridization in rat brain from neonate to adult. During the early postnatal stage, levels of AC1 transcripts were very high in the cerebral cortex, striatum, thalamus, brainstem, and inferior colliculus. Then, AC1 mRNA levels rapidly decreased to the levels observed in the adult brain. In contrast, AC1 transcripts were very low at the early postnatal stage in the cerebellum and hippocampus and markedly increased during the second postnatal week. AC2 mRNA was widely distributed in rat brain throughout the development, and levels did not vary with different ages of the animal. AC5 mRNA was expressed to a limited extent in the neonatal brain, but levels dramatically increased during the second postnatal week in restricted regions, including the striatum, nucleus accumbens, and olfactory tubercle. The developing profiles of three AC gene transcripts were confirmed by northern blot analyses with mRNA isolated from different brain regions at different postnatal stages. In addition, the basal and forskolin-, GTP gamma S-, or Ca2+/calmodulin-stimulated AC activity in plasma membrane preparations obtained from different brain regions at different ages were correlated with the age-dependent changes in the region-specific AC mRNA levels. These results demonstrate that different AC subtypes are expressed in the developing rat brain in a region- and age-specific manner, suggesting specific roles not only in the synaptic transmission but also in the differentiation and maturation of neuronal cells in the developing brain.
Adenylyl Cyclases↗
Changes in the expression of adenylyl cyclases in the rat uterus during the course of pregnancy.
The changes in the expression of the various adenylyl cyclases (ACs) in the rat uterus during the course of pregnancy and after delivery were examined by Northern blot analysis and AC assay. Northern blot analysis revealed that five isoforms of ACs (AC2, AC4, AC6, AC7, and AC9) were expressed in the rat uterus, AC6 being the most abundant. The level of expression of these ACs increased 1.7- to 3.4-fold during the course of pregnancy and diminished near term and after delivery. The highest level of expression in each type of AC was consistently seen on Day 17 of pregnancy, and the relative increase of expression, as compared to that in nonpregnant rats, was as follows: AC2 > AC4 > AC7 > AC9 > AC6. In agreement with these findings, both basal and forskolin-stimulated AC activities exhibited a 2- to 3-fold increase during the course of pregnancy, followed by a decrease near term. Our data indicate that post-receptor events, namely marked changes in the level of AC mRNA (and presumably proteins) occur during pregnancy and after delivery and that they contribute to the essential role of cAMP in maintaining uterus quiescence.
Adenylyl Cyclases↗
Expression of adenylyl cyclase mRNAs in the adult, in developing, and in the Brattleboro rat kidney.
The activity and expression of adenylyl cyclases (AC) were examined in the adult rat renal cortex and medulla. Northern blot analysis and in situ hybridization demonstrated that AC-6 was the predominant isoform in the adult rat kidney, whereas AC-4, -5, and -9 had a lower expression. AC-4 expression was higher in the cortex, and AC-5 and AC-6 were higher in the medulla. AC-9 expression was at the same level in both regions. AC activity was high in the fetus and declined in the adult. At all stages, AC activity was sensitive to parathyroid hormone, whereas no stimulation by vasopressin and isoproterenol was found in the fetus and the neonate. AC-5 and AC-6 mRNAs increased at day 1 and then markedly decreased, paralleling the decline in AC activity. The mRNA of AC-4 did not change and that of AC-9 increased markedly until adult. In the homozygous Brattleboro rat kidney, the expression of all these isoforms was decreased.
Adenylyl Cyclases↗
Localization and differential expression of adenylyl cyclase messenger ribonucleic acids in rat adrenal gland determined by in situ hybridization.
The expression of adenylyl cyclases (ACs) in the adult rat adrenal gland was examined. In situ hybridization revealed specific patterns of AC messenger RNA (mRNA) distribution. AC1 was limited exclusively to the adrenal medulla. AC5 and AC6 were mainly expressed in the adrenal medulla, with a weak expression in the zona glomerulosa. AC9 was found in all the three regions of the adrenal cortex but not in the adrenal medulla. All these ACs were detected on postnatal day 1 (PN1), and their pattern of expression was unchanged on PN7, PN21, and PN90 (adult). We analyzed the response of these ACs to various physiological conditions known to affect the synthesis of aldosterone and corticosterone in the adrenal cortex. Our study demonstrates a specific increase of AC6 but not AC5 mRNA in the zona glomerulosa of rats given a low sodium diet. AC9 mRNA was increased in all the three cortical zones of rats treated with ACTH. We suggest that AC6 and AC9 play important roles in different pathways associated with the regulation of aldosterone and corticosteroid production.
Adenylyl Cyclases↗
Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene.
Despite tremendous efforts in the search for safe, efficacious and non-addictive opioids for pain treatment, morphine remains the most valuable painkiller in contemporary medicine. Opioids exert their pharmacological actions through three opioid-receptor classes, mu, delta and kappa, whose genes have been cloned. Genetic approaches are now available to delineate the contribution of each receptor in opioid function in vivo. Here we disrupt the mu-opioid-receptor gene in mice by homologous recombination and find that there are no overt behavioural abnormalities or major compensatory changes within the opioid system in these animals. Investigation of the behavioural effects of morphine reveals that a lack of mu receptors abolishes the analgesic effect of morphine, as well as place-preference activity and physical dependence. We observed no behavioural responses related to delta- or kappa-receptor activation with morphine, although these receptors are present and bind opioid ligands. We conclude that the mu-opioid-receptor gene product is the molecular target of morphine in vivo and that it is a mandatory component of the opioid system for morphine action.
Analgesics↗
Diurnal variation of the adenylyl cyclase type 1 in the rat pineal gland.
Nocturnal melatonin production in the pineal gland is under the control of norepinephrine released from superior cervical ganglia afferents in a rhythmic manner, and of cyclic AMP. Cyclic AMP increases the expression of serotonin N-acetyltransferase and of inducible cAMP early repressor that undergo circadian oscillations crucial for the maintenance and regulation of the biological clock. In the present study, we demonstrate a circadian pattern of expression of the calcium/calmodulin activated adenylyl cyclase type 1 (AC1) mRNA in the rat pineal gland. In situ hybridization revealed that maximal AC1 mRNA expression occurred at midday (12:00-15:00), with a very low signal at night (0:00-3:00). We established that this rhythmic pattern was controlled by the noradrenergic innervation of the pineal gland and by the environmental light conditions. Finally, we observed a circadian responsiveness of the pineal AC activity to calcium/calmodulin, with a lag due to the processing of the protein. At midday, AC activity was inhibited by calcium (40%) either in the presence or absence of calmodulin, while at night the enzyme was markedly (3-fold) activated by the calcium-calmodulin complex. These findings suggest (i) the involvement of AC1 acting as the center of a gating mechanism, between cyclic AMP and calcium signals, important for the fine tuning of the pineal circadian rhythm; and (ii) a possible regulation of cyclic AMP on the expression of AC1 in the rat pineal gland.
Adenylyl Cyclases↗
Reduction of morphine abstinence in mice with a mutation in the gene encoding CREB.
Chronic morphine administration induces an up-regulation of several components of the cyclic adenosine 5'-monophosphate (cAMP) signal transduction cascade. The behavioral and biochemical consequences of opiate withdrawal were investigated in mice with a genetic disruption of the alpha and Delta isoforms of the cAMP-responsive element-binding protein (CREB). In CREBalphadelta mutant mice the main symptoms of morphine withdrawal were strongly attenuated. No change in opioid binding sites or in morphine-induced analgesia was observed in these mutant mice, and the increase of adenylyl cyclase activity and immediate early gene expression after morphine withdrawal was normal. Thus, CREB-dependent gene transcription is a factor in the onset of behavioral manifestations of opiate dependence.
Adenylyl Cyclases↗
Identification and characterization of a widely expressed form of adenylyl cyclase.
A novel mammalian adenylyl cyclase was identified by reverse transcription-polymerase chain reaction amplification using degenerate primers based on a conserved region of previously described adenylyl cyclases (Premont, R. T. (1994) Methods Enzymol. 238, 116-127). The full-length cDNA sequence obtained from mouse brain predicts a 1353-amino acid protein possessing a 12-membrane span topology, and containing two regions of high similarity with the catalytic domains of adenylyl cyclases. Comparison of this novel adenylyl cyclase with the eight previously described mammalian enzymes indicates that this type 9 adenylyl cyclase sequence is the most divergent, defining a sixth distinct subclass of mammalian adenylyl cyclases. The AC9 gene has been localized to human chromosome band 16p13.3-13.2. The 8.5-kb mRNA encoding the type 9 adenylyl cyclase is widely distributed, being readily detected in all tissues tested, and is found at very high levels in skeletal muscle and brain. AC9 mRNA is found throughout rat brain but is particularly abundant in hippocampus, cerebellum, and neocortex. An antiserum directed against the carboxyl terminus of the type 9 adenylyl cyclase detects native and expressed recombinant AC9 protein in tissue and cell membranes. Levels of the AC9 protein are highest in mouse brain membranes. Characterization of expressed recombinant AC9 reveals that the protein is a functional adenylyl cyclase that is stimulated by Mg2+, forskolin, and mutationally activated Gsalpha. AC9 activity is not affected by Ca2+/calmodulin or by G protein betagamma-subunits. Thus AC9 represents a functional G protein-regulated adenylyl cyclase found in brain and in most somatic tissues.
Adenylyl Cyclases↗
Protein kinases in the rat nucleus accumbens are involved in the aversive component of opiate withdrawal.
The specific participation of protein kinases in the expression of the somatic signs of morphine withdrawal has been previously demonstrated, suggesting that changes in intracellular signalling systems are involved in opioid addiction. In the present study, the involvement of protein kinases in the aversive/dysphoric effects of morphine abstinence has been investigated in the nucleus accumbens, because of the critical role played by the mesolimbic system in the rewarding effects of opioids. Rats were chronically treated with morphine, twice a day for 5 days, with doses progressively increased from 5 to 30 mg/kg (i.p.). In addition, microinjections into the nucleus accumbens of the serine-threonine kinase inhibitors H7 or H8 (1 or 10 nmol per side) or saline once daily were also given, both in control and in morphine-treated animals. After these chronic treatments, withdrawal syndrome was induced by naloxone administration (0.1 mg/kg, s.c.), and the motivational component of morphine abstinence was studied using the place aversion paradigm. When administered at the highest dose (10 nmol), H7 and H8 strongly reduced the place aversion induced by naloxone in morphine dependent animals. Protein kinase inhibitors did not induce significant behavioural responses in non-dependent animals. Chronic morphine treatment induced a selective up-regulation of adenylate cyclase activity in the amygdala, without affecting other brain regions. The morphine-increased adenylate cyclase activity in amygdala was reversed by the chronic intra-accumbens microinjections of H7 and H8. These results suggest that serine-threonine kinases in the nucleus accumbens play an important role in the emotional/dysphoric properties which characterize opiate withdrawal.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
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.
Adenylyl Cyclases↗
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.
Adenylyl Cyclases↗
Chronic morphine administration causes region-specific increase of brain type VIII adenylyl cyclase mRNA.
The up-regulation of the cyclic AMP system and enhanced phosphorylation of protein substrates after either sudden interruption of chronic opioid treatment or antagonist administration has been proposed to account for the various behavioral responses observed during withdrawal. Using in situ hybridization histochemistry, we show here for the first time that type VIII adenylyl cyclase mRNA is selectively increased, as early as 12 h after morphine treatment in the locus coeruleus and the amygdala, two brain regions suggested to be important in morphine dependence expression. Moreover, the time course of morphine-induced changes in type VIII adenylyl cyclase mRNA in locus coeruleus is related to the incidence of jumping, the most important sign of morphine withdrawal in mice. In addition, the overexpression of type VIII adenylyl cyclase mRNA in thalamic nuclei could add to morphine tolerance. These findings offer a strong support, at the molecular level, for an altered regulation of the cyclic AMP system in opiate tolerance and dependence. The present measure relates only to type VIII adenylyl cyclase mRNA and similar tests with other cyclases are needed to explore fully this relationship.
Adenylyl Cyclases↗
Testis-specific transcription start site in the aspartate aminotransferase housekeeping gene promoter.
We have studied the expression and regulation of the rat testis cytosolic aspartate aminotransferase gene. The cytosolic aspartate aminotransferase activity was 5-fold lower in the testis than in the liver and kidney. A 1.9-kilobase mRNA form was detected in the rat testis in contrast to the 2.1- and 1.8-kilobase forms present in other organs. Using Northern blot and S1 mapping analyses, we found that the proximal polyadenylation site was almost exclusively used in the testis as opposed to other organs where the distal site was preferentially used. RNase protection and primer extension analysis showed that transcription was initiated at multiple sites in all organs, but the pattern of those start sites was different in the testis; in particular, a novel transcription start site was specifically detected in this organ (at position -115 from the translation start site). This site was first observed in 29-day-old rats and was maximally utilized in the adult testis. DNase I footprinting using testis nuclear extracts revealed the presence of three sites of DNA-protein interaction in the 250-base pair proximal promoter, a pattern similar to the one found using liver nuclear extracts. However, the proteins bound had different properties as shown by gel retardation experiments. We conclude that the pattern of transcription initiation and the polyadenylation site selection of a housekeeping gene can be tissue-specific.
Animals↗
Phorbol esters inhibit the glucocorticoid-mediated stimulation of cytosolic aspartate aminotransferase gene transcription.
The regulation of cytosolic aspartate aminotransferase (cAspAT) gene expression by phorbol esters was investigated in the highly differentiated hepatoma cell line Fao. Phorbol 12,13-dibutyrate (PdBu) had no effect on basal activity but partially inhibited the induction of cAspAT by dexamethasone. The extent of inhibition (40%) was similar to that obtained with insulin or vanadate. The inhibitory effects of PdBu and vanadate were additive. In the case of PdBu, the inhibitory effects could be eliminated by first incubating the cells with PdBu, which down-regulates protein kinase C. In contrast, inhibition by insulin was not modified by this treatment. The molecular mechanism of PdBu action was investigated. Northern blot analysis showed that the steady-state mRNA levels of cAspAT were decreased by PdBu in the presence of dexamethasone. In addition, the transcription rate, as measured by run-on experiments, was also decreased under the same conditions. Finally, a 2.4 kb promoter fragment driving the chloramphenicol acetyltransferase gene was stably transfected into the Fao cells. The regulation of the activity of this promoter fragment by dexamethasone and PdBu was similar to the regulation of the endogenous cAspAT activity. We conclude that PdBu acts by regulating the promoter activity of the cAsPAT gene.
Animals↗