Tumor necrosis factor alpha and glutathione interplay in chronic heart failure.

The pro-inflammatory cytokine, tumor necrosis factor alpha (TNF alpha), in concert with neurohormones, contributes to chronic heart failure (CHF) progression. This implies that TNF a antagonism may constitute an important target for CHF therapy. However, clinical trials in CHF patients using compounds that trap TNF alpha, comprising infliximab, an antibody directed to TNF alpha, and etanercept, a soluble recombinant receptor of TNF alpha, gave disappointing results bringing back to light the dual, short-term beneficial and long-term harmful effect of TNF alpha. This review focuses on the dual, concentration- and time-related effects of TNF alpha, the yin and yang action of TNF alpha in cardiac ischemia/reperfusion and contraction. Importantly, the harmful effects of TNF a are related to glutathione deficiency, a common hallmark to several other chronic inflammatory diseases. Recently, in rat models of CHF, oral administration of the glutathione precursor, N-acetylcysteine (NAC), was shown to hinder pathways of TNF alpha harmful signalling and to rescue cardiac structure and function. These results suggest that glutathione deficiency in association with TNF alpha activation may play a role in the pathophysiology of CHF and that NAC may represent a potential therapy in CHF.

Regulation and role of adenylyl cyclase isoforms.

At least nine closely related isoforms of adenylyl cyclases (ACs), the enzymes responsible for the synthesis of cyclic AMP (cAMP) from ATP, have been cloned and characterized in mammals. Depending on the properties and the relative levels of the isoforms expressed in a tissue or a cell type at a specific time, extracellular signals received through the G-protein-coupled receptors can be differentially integrated. The present review deals with various aspects of such regulations, emphasizing the role of calcium/calmodulin in activating AC1 and AC8 in the central nervous system, the potential inhibitory effect of calcium on AC5 and AC6, and the changes in the expression pattern of the isoforms during development. A particular emphasis is given to the role of cAMP during drug and ethanol dependency and to some experimental limitations (pitfalls in the interpretation of cellular transfection, scarcity of the invalidation models, existence of complex macromolecular structures, etc).

Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase.

The present review focuses on the potential physiological regulations involving different isoforms of adenylyl cyclase (AC), the enzymatic activity responsible for the synthesis of cAMP from ATP. Depending on the properties and the relative level of the isoforms expressed in a tissue or a cell type at a specific time, extracellular signals received by the G protein-coupled receptors can be differently integrated. We report here on various aspects of such regulations, emphasizing the role of Ca(2+)/calmodulin in activating AC1 and AC8 in the central nervous system, the potential inhibitory effect of Ca(2+) on AC5 and AC6, and the changes in the expression pattern of the isoforms during development. A particular emphasis is given to the role of cAMP during drug dependence. Present experimental limitations are also underlined (pitfalls in the interpretation of cellular transfection, scarcity of the invalidation models, and so on).

Enhanced cardiac function in transgenic mice expressing a Ca(2+)-stimulated adenylyl cyclase.

The predominant functional adenylyl cyclases normally expressed in cardiac tissue and coupled to beta-adrenergic receptors are inhibited by micromolar Ca(2+) concentration. To modify the overall balance of activities, we have generated transgenic mice expressing the Ca(2+)-stimulatable adenylyl cyclase type 8 (AC8) specifically in the heart. AC activity is increased by at least 7-fold in heart membranes from transgenic animals and is stimulated by Ca(2+) in the same range of concentration that inhibits the endogenous activity. Moreover, the in vivo basal protein kinase A activity was augmented 4-fold. Overexpression of AC8 in the heart has no detrimental consequences on global cardiac function. Basal heart rate and contractile function, measured by noninvasive echocardiography, were unchanged. In contrast, on release of parasympathetic tone, the intrinsic contractility is heightened and unresponsive to further beta-adrenergic receptor stimulation. AC8 transgenic mice thus represent an original model to investigate the relative influence of Ca(2+) and cAMP on cardiac function within a phenotype of enhanced cardiac contractility and relaxation.

Cannabinoid withdrawal is dependent upon PKA activation in the cerebellum.

Region-specific up-regulation of the cyclic AMP pathway is considered an important molecular mechanism in the origin of the somatic manifestations of the withdrawal syndrome to known drugs of abuse. Nevertheless, the existence of a withdrawal syndrome after prolonged cannabinoid administration has long been a controversial issue. Recent studies, in different species, have shown that withdrawal to prolonged cannabinoid exposure precipitated by the cannabinoid antagonist SR141716A is characterized by physical signs underlying impairment of motor coordination. Interestingly, cannabinoid withdrawal is accompanied by an increase of adenylyl cyclase activity in the cerebellum. Here, we investigate the functional role of the cyclic AMP pathway in the cerebellum in the establishment of cannabinoid withdrawal. We show that after SR141716A precipitation of cannabinoid withdrawal, basal and calcium-calmodulin-stimulated adenylyl cyclase activities as well as active PKA in the cerebellum increase in a transient manner with a temporal profile which matches that of the somatic expression of abstinence. Selectively blocking the up-regulation of the cyclic AMP pathway in the cerebellum, by microinfusing the cyclic AMP blocker Rp-8Br-cAMPS in this region, markedly reduced both PKA activation and the somatic expression of cannabinoid withdrawal. Our results (i) directly link the behavioural manifestations of cannabinoid withdrawal with the up-regulation of the cyclic AMP pathway in the cerebellum, pointing towards common molecular adaptive mechanisms for dependence and withdrawal to most drugs of abuse; (ii) suggest a particular role for the cerebellum as a major neurobiological substrate for cannabinoid withdrawal.

Expression of multiple adenylyl cyclase isoforms in human and dog thyroid.

Although the TSH receptor and Galpha(s), which activate the cAMP cascade in the thyroid gland have been much studied, nothing is known about the adenylyl cyclase (AC) isoforms which are actually involved in this pathway. To characterize the cAMP generation in the dog and human thyroid gland, resulting from the presence of distinct adenylyl cyclase families, the responses to various agents (Ca2+, calmodulin (CaM), phorbol esters (TPA) and thapsigargin (Tg)) were studied. These experiments suggest a role of at least two families of cyclases: cyclases negatively modulated by Ca2+ (ACV or ACVI) and cyclases positively modulated by PKC (ACII, ACIII or ACVII). To further analyze by other experimental procedures the expression pattern of the cyclase isoforms in the thyroid gland, Northern blotting, Western blotting and RT-PCR experiments were performed. The results clearly suggest that in both species, three different adenylyl cyclases ACIII, ACVI and ACIX are mainly expressed in thyrocytes.

Molecular identification of adenylyl cyclase 3 in bovine corpus luteum and its regulation by prostaglandin F2alpha-induced signaling pathways.

The involvement of cAMP in various aspects of ovarian steroidogenic cells functions has been extensively studied. However, the adenylyl cyclase (AC) types expressed in ovarian cells, of any species, are not yet determined. The present study was undertaken to identify AC types present in bovine luteal cells and their regulation by various stimuli. AC isoforms 2, 3, 5, 6, 7, 8, and 9 were detected in the bovine brain by Northern blotting analysis, whereas the bovine corpus luteum (CL) only expressed AC3 and 6 mRNAs, with AC3 being more abundant than AC6. The use of AC3-specific primers in RT-PCR reaction verified the presence of AC3 mRNA in both bovine and rat CL tissue as well as in bovine steroidogenic luteal cells. Because these two AC isoforms, AC3 and 6, exhibit distinct regulatory patterns we have next examined the effects of various signaling pathways on AC activity in luteal cells. These studies have shown that: 1) prostaglandin (PG) F2alpha and phorbol 12-myristate 13-acetate markedly elevated agonist-stimulated cAMP synthesis (these effects were inhibited by addition of highly specific PKC inhibitor, bisindolylmaleimide); 2) depletion of Ca2+ from the incubation medium inhibited AC activity; 3) physiological concentrations of Ca2+ ions (up to 5 mM) significantly stimulated cAMP production in luteal cells; and 4) the effects of Ca2+ on cAMP synthesis were evident only in the presence of forskolin. These regulatory characteristics of AC activity are consistent with the molecular identification of ACs indicating the presence of AC3 in luteal cells. The reported data may delineate the cross-talk between physiological activators of AC in the CL (such as LH, PGE2, and PGI2) and other ligands (such as PGF2alpha and endothelin-1), which indirectly modulate AC activity. Therefore, the identification of AC isoforms present in luteal cells is an important step toward understanding the mode of action of a wide array of hormones regulating ovarian cells.

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.

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.

Adenylyl cyclase activity and gene expression during mesodermal differentiation of the P19 embryonal carcinoma cells.

DMSO-primed P19 pluripotent cells, which recapitulate the first stages of mammalian cardiogenesis and endodermal formation, were used as an in vitro model to analyze the variations in activity and expression of the different adenylyl cyclase (AC) isoforms during the early events of embryonic cell differentiation. Here, we show that the total AC activity, which increases up to 10-fold after differentiation of P19 cells, is mainly associated with increases in AC2, AC5, and AC6 mRNA levels. Particularly, the marked increase in AC5 mRNA correlates with the appearance of beating cardiomyocytes and with the transcription of the atrial myosin light chain (MLC1A) gene which encodes a protein specifically involved in the cardiac muscle cell contractile phenotype. Together, the results strongly suggest that 1) a rise in cyclic AMP (cAMP) may be associated with cardiomyocyte and endodermal cell differentiation during mammalian embryogenesis; and 2) AC5 gene expression starts very early during normal mouse cardiogenesis and correlates with the differentiation of cardiomyocytes.

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.

Stability of the vasopressin V2 receptor-adenylyl cyclase system in rat kidney.

In the Brattleboro rat with diabetes insipidus vasopressin V2 receptor mRNA and the mRNA of various adenylyl cyclase (AC) isoforms are moderately reduced compared with those of normal rats. In the present study renal vasopressin V2 receptor mRNA was modestly higher (by 34%), as was expression of AC 5, 6 and 9 mRNAs (up to 22% greater), in BDI rats treated with the vasopressin V2 receptor agonist desamino-[Arg8] vasopressin than in untreated controls. AC 4 mRNA was decreased by 17% following desamino-[Arg8s] vasopressin treatment. While the stimulatory Gsalpha mRNA was little affected by the desamino-[Arg8] vasopressin treatment, two of the inhibitory G proteins were raised (Galphai-2 by 54% and Galphai-3 by 57%). Treatment of Sprague-Dawley rats with a specific vasopressin V2 receptor antagonist (SR 121463A) was not associated with any marked changes in mRNA expression. These results indicate that the vasopressin V2 receptor adenylyl cyclase system mediating the antidiuretic response to vasopressin is relatively stable. The Gi proteins may be involved in the stabilizing mechanism.

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.

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.

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.

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.

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.

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.

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.