Expression of adenylyl cyclase mRNAs in the denervated and in the developing mouse skeletal muscle.

Changes in the activity and in the expression of adenylyl cyclase (AC) were examined in mouse skeletal muscle after denervation and during development. Four isoforms of AC (AC2, AC6, AC7, and AC9) were detected by Northern blot analysis in gastrocnemius muscle, AC9 being the most abundant. After denervation, the levels of AC2 and AC9 mRNA decreased, whereas those of AC6 and AC7 increased. AC activity in response to several neurotransmitters was increased after denervation. During development, AC activity was high in fetus and neonate and declined in the adult; the sensitivity of AC activity to various neurotransmitters was the highest on the third postnatal day. The levels of AC6 and AC7 mRNAs were high on the third postnatal day and then decreased in adult, paralleling the decline in AC activity. All the characteristics of AC expression and activity in fetus and neonate resembled those observed in denervated adult muscle. These results indicate that changes in AC activity and AC mRNAs play an important role in the various physiopathological states of skeletal muscle, especially during muscle atrophy.

The olfactory adenylyl cyclase type 3 is expressed in male germ cells.

Elements of the olfactory pathway, such as receptors, receptor-desensitization machinery, and cyclic nucleotide-gated channels, are expressed in male germ cells. Here we report the expression, in rat testis, of both adenylyl cyclase type 3 (AC3) and the olfactory G protein subunit, G(alpha)olf. Both are expressed in the same sub-population of germ cells, pachytene spermatocytes to spermatids, and in residual bodies. Neither AC3 nor G(alpha)olf was found in Sertoli or in peritubular cells, as shown by Western blotting and immunocytochemical analyses. It thus appears that male germ cells contain all the elements of the signaling cascade present in olfactory cells.

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.

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.

Protein kinase C isoform expression in normal and failing rabbit hearts.

Protein kinase C (PKC) is activated by alpha-adrenergic stimulation. Molecular analysis showed that PKC consists of a family of at least 12 isozymes. Studies of their distribution in the heart showed conflicting results. The first goal of our study was thus to characterize cardiac PKC in normal rabbits. PKC plays an important role in gene expression, cell growth, and differentiation and is involved in the hypertrophy phase of cardiac overload, but since its expression has never been evaluated in heart failure, the second goal of our study was to evaluate PKC activity and isoform expression in rabbits with heart failure induced by a double hemodynamic overload (aortic insufficiency followed by an aortic stenosis). In the first part of the study, PKC isoform expression analyzed in normal rabbits by immunoblotting showed that isoforms alpha, beta, epsilon, and zeta were expressed along with PKC gamma, which had never been detected in the heart. PKC gamma expression was also identified by polymerase chain reaction, and immunofluorescence techniques showed a localization on intercalated disks associated with the membrane localization observed with the other isoforms. In the second part of the study, PKC activity, content, and isoform expression showed a decrease of 37% in the failing group. PKC immunodetection with a monoclonal antibody (Mab 1.9) recognizing the catalytic domain of all PKC isoforms revealed a 20% decrease in the failing ventricles compared with normal left ventricles. Expressed PKC isoforms quantified by Western blot showed, in the failing heart group compared with the control group, a decrease of 27%, 32%, 16%, and 9% of PKC alpha, PKC beta 1, PKC gamma, and PKC epsilon, respectively, whereas PKC zeta was not significantly modified. These results show that, in heart failure, PKC activity and expression of Ca(2+)-dependent PKC isoforms are decreased. This may lead to alterations of PKC-induced phosphorylations.

Characterization of a large population of mRNAs from human testis.

We present the results of single-pass sequencing of 779 expressed sequence tags from normal human testis cDNA clones. Of the sequences generated, 319 (41%) appeared to be completely unknown and are likely to represent new genes, and 289 (37%) were identified based on exact or nonexact matches to sequences in public databases. In analyses of hybridization of four tissues, testis, brain, liver, and kidney, 6 of 12 cDNAs clones revealed testis-specific expression. This argues for the value of the combination of random sequencing and analysis of cellular expression for large-scale characterization of gene expression in the testis.

Expression of mouse brain soluble guanylyl cyclase and NO synthase during ontogeny.

The spatial and temporal distribution of soluble guanylyl cyclase and nitric oxide synthase mRNA was determined during embryonic and postnatal development of the mouse brain. This was achieved by in situ hybridization of specific probes for soluble beta 1 guanylyl cyclase subunit and nitric oxide synthase mRNA on mouse brain sections at late fetal development (19-day embryo) and different stages of postnatal development (3, 7, 15 days, and adult). In the embryo, soluble guanylyl cyclase transcripts are weakly expressed in the central nervous system. Following birth their expression increases in the striatum and neocortex, and they are widely distributed in the adult brain (layer II and V-VI of the cortex, olfactory bulb, striatum, Purkinje cell layer of the cerebellum). In contrast, nitric oxide synthase mRNA was expressed in several embryonic structures of the brain (different layers of the cortical neuroepithelium, colliculi neuroepithelium, pons), and markedly reduced at early postnatal stage, except in the accessory olfactory bulb and pediculopontine nuclei. Nitric oxide synthase transcripts progressively appear, within two weeks following birth, in the striatum and the cerebral cortex but they were specifically confined to isolated cells. During this period, this mRNA also increased in hippocampus, in discrete nuclei (hypothalamus, pontine) and in the molecular layer of the cerebellum. The situation in the adult was similar to the one observed at 15 days. These results show a general lack of regional colocalization of soluble guanylyl cyclase and NOS mRNA during ontogeny, thus suggesting an independent regulation of the related genes.

Cell-specific regulation of cytosolic aspartate aminotransferase by glucocorticoids in the rat kidney.

The basal expression and hormonal regulation of cytosolic aspartate aminotransferase (cAspAT) were investigated in the rat kidney. In adrenalectomized animals, the basal activity was highest in the renal cortex and in the inner stripe of the outer medulla (0.1-0.15 U/mg protein). The glucocorticoid analogue dexamethasone increased cAspAT activity about twofold in the cortex and in the inner stripe of the outer medulla but not in the papilla. A half-maximal increase in the activity was achieved at doses of approximately 5 micrograms/100 g body wt. The mineralocorticoid aldosterone did not modify the cAspAT activity. The cell specificity of the hormonal regulation was analyzed by in situ hybridization. In untreated adrenalectomized rats, a cAspAT cRNA probe labeled mainly the inner stripe of the outer medulla. After dexamethasone or hydrocortisone treatment, labeling was uniformly increased in this part of the medulla and was heterogeneously increased in the renal cortex. The specific increase in labeling within the cortex was shown to be confined to the distal convoluted tubule and the thick ascending limb. We conclude that, in addition to widespread basal expression, cAspAT is regulated by glucocorticoids in a highly cell-specific manner in the renal cortex. The enzyme may thus participate in the increased energy metabolism elicited by these hormones in these cells.

Localization of adenylyl and guanylyl cyclase in rat brain by in situ hybridization: comparison with calmodulin mRNA distribution.

Cyclic nucleotides are major intracellular mediators in the signal transduction events in synaptic neurotransmission of the CNS. Intracellular Ca2+ is known to regulate adenylyl cyclase (AC) in a calmodulin (CaM)-dependent manner, and guanylyl cyclase (GC), in an indirect manner through CaM-sensitive nitric oxide synthase. To ascertain the physiological significance of cyclic nucleotide second messenger systems, we have localized the mRNAs encoding AC, GC, and CaM in the rat brain by in situ hybridization using 35S-labeled RNA probes. The AC mRNA is widely distributed throughout the brain; strong hybridization signal was observed in the granular layers of the cerebellum, in the pyramidal and granule cells of the hippocampus, and in the olfactory system. These AC mRNA localizations are compatible with the distribution of Ca2+/CaM-sensitive AC activities. In contrast to AC mRNA distribution, GC mRNA has a more limited distribution. Significant signals were observed in the striatum, in the pyramidal and granule cells of the hippocampus, in the olfactory system, in the inferior and superior colliculus, in the Purkinje cells of the cerebellum, in the locus coeruleus, and in many pyramidal cells in the layers II-III and V of the cerebral cortex, and mainly, in the occipital cortex. In some discrete brain regions, a close correlation was found between enzyme activity and mRNA hybridization signal of GC. The distinct distribution of AC and GC mRNAs suggests that different cyclic nucleotide second messenger systems have specialized functions. On the other hand, CaM mRNA was colocalized with the AC and GC mRNA, but its distribution was more abundant and specific for neuronal cells, since there was little hybridization signal with CaM probe in neuronal fiber regions such as the corpus callosum and the anterior commissure. The high expression of CaM mRNA in neuronal cells is in agreement with its biochemical role in the regulation of various enzymes. Results of the present study should help in analyzing the role of cyclic nucleotides and CaM in physiological and pathological situations in the CNS.

Sequence of a human brain adenylyl cyclase partial cDNA: evidence for a consensus cyclase specific domain.

A cDNA coding for a human brain adenylyl cyclase was isolated and sequenced. The deduced partial 675 amino-acid sequence was compared with those of other known adenylyl and guanylyl cyclases. Comparison of this predicted amino-acid sequence with that of bovine brain (type I) and rat olfactory (type III) adenylyl cyclase indicated a significant homology with the carboxyl-terminal halves of both enzymes. The homology between the human adenylyl cyclase and the other two mammalian adenylyl cyclase also appears at the topographic level. Indeed, the human enzyme includes a extremely hydrophobic region containing six potential membrane-spanning segments followed by a large hydrophilic domain. At the beginning of the hydrophilic domain, there is a 250 amino-acid region which shows not only a striking homology with the bovine and rat adenylyl cyclase (86% of similarity and 57% of identity), but also a significant homology with non-mammalian adenylyl cyclase and guanylyl cyclases. We found that this 250 amino-acid domain contains a sequence of about 165 amino-acids which is highly conserved in most of the known nucleotide cyclases suggesting that it includes residues that are critical for the function of the enzymes.

Identification of two isoforms of the catalytic subunit of Na,K-ATPase in myocytes from adult rat heart.

The present study demonstrates that two forms of the alpha catalytic subunit of the Na,K-ATPase are present in rat heart and originate from cardiomyocytes. They were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction and alkylation of the sulfhydryl groups. The two forms were identified on immunoblots using two specific antisera against either the alpha subunit from Bufo marinus kidney and the alpha and beta subunits from lamb kidney. Comparison of the two forms to the alkylated Na,K-ATPase from rat kidney (containing one catalytic subunit) and from rat brain (containing alpha and alpha + subunits) suggested that, in rat cardiac myocytes, the form with a fast migration rate (alpha F) corresponds to the alpha subunit of low ouabain affinity and the one with a slow migration rate (alpha S), to a subunit of high ouabain affinity. Thus, the existence of two isoforms of the catalytic subunit in cardiac myocytes accounts well for the biphasic ouabain inhibition of the Na,K-ATPase activity and for the biphasic inotropic responsiveness to cardiac glycosides of the rat heart.