Effects of cannabinoids in Krox-24 targeted mice.

Krox-24 is an immediate early gene encoding a zinc-finger transcription factor implicated in several adaptive responses, and its induction by cannabinoids has been reported. We used mice targeted in the Krox-24 gene to specifically dissect the role of this protein in the acute and chronic central actions of cannabinoids. We report here on the ability of cannabinoids to activate G-proteins and to inhibit adenylyl cyclase, and to elicit behavioral responses in wild-type and mutant mice. The behavioral parameters and the biochemical correlates of abstinence after delta9-THC withdrawal were evaluated. We show that Krox-24 is not involved in the acute analgesic effects of delta9-THC and in the SR precipitated delta9-THC withdrawal syndrome.

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).

Radioligand binding properties of VV-hemorphin 7, an atypical opioid peptide.

Receptor binding properties of the hemoglobin-derived nonapeptide VV-hemorphin 7 (Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe-OH) were studied using both the unlabelled form and tritium-labelled derivative of the peptide. In binding studies using selective opioid radioligands, VV-hemorphin 7 exhibited a rank order of potency of mu > kappa >> delta. VV-hemorphin 7 was tritiated resulting in a compound with 1.03 TBq/mmol (27.8 Ci/mmol) specific radioactivity. The maximal number of binding sites was found to be 66.5 pmol/mg protein with an affinity of 82.1 nM in rat brain membranes. In competition studies, marked similarity was observed to the binding profile of the naturally occurring opioid heptapeptide Met-enkephalin-Arg-Phe (MERF) and its analogues to their naloxone-insensitive binding site. The common -Arg-Phe sequence at the carboxyl terminal end, which is similar to those of other endogenous peptides (-Arg-Phe-NH(2) in neuropeptide FF and FMRF-NH(2)) brings attention to the C-terminal end of the molecule and points to the possible existence of a common nonopioid binding site in mammals.

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.

Specific activation of the mu opioid receptor (MOR) by endomorphin 1 and endomorphin 2.

The recently discovered endomorphin 1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin 2 (Tyr-Pro-Phe-Phe-NH2) were investigated with respect to their direct receptor-binding properties, and to their ability to activate G proteins and to inhibit adenylyl cyclase in both cellular and animal models. Both tetrapeptides activated G proteins and inhibited adenylyl cyclase activity in membrane preparations from cells stably expressing the mu opioid receptor, an effect reversed by the mu receptor antagonist CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2), but they had no influence on cells stably expressing the delta opioid receptor. To further establish the selectivity of these peptides for the mu opioid receptor, brain preparations of mice lacking the mu opioid receptor gene were used to study their binding and signalling properties. Endomorphin 2, tritiated by a dehalotritiation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), labelled the brain membranes of wild-type mice with a Kd value of 1.77 nM and a Bmax of 63.33 fmol/mg protein. In membranes of mice lacking the mu receptor gene, no binding was observed, and both endomorphins failed to stimulate [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding and to inhibit adenylyl cyclase. These data show that endomorphins are capable of activating G proteins and inhibiting adenylyl cyclase activity, and all these effects are mediated by the mu opioid receptors.

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.

Opioid binding profiles of new hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxymorphinans.

Several hydrazone, oxime, carbazone and semicarbazone derivatives of 14-alkoxycodeinones and 14-alkoxydihydrocodeinones were synthesised [1] and characterised in in vitro radioligand binding assays in rat brain membrane preparations. The tested compounds show the highest affinity for the mu opioid binding sites and most of them have agonist character. Subtype analysis of the binding shows mu2 specificity. However, some of these ligands are able to block partially (40-60%) the high affinity (putative mu1) opioid binding sites while all of them act as reversible ligands at the low affinity (putative mu2) sites.

In vitro binding and signaling profile of the novel mu opioid receptor agonist endomorphin 2 in rat brain membranes.

The recently discovered endogenous mu receptor selective endomorphin 2 was prepared in tritiated form by a catalytic dehalogenation method resulting in a specific radioactivity of 1.98 TBq/mmol (53.4 Ci/mmol), and used for in vitro labelling of rat brain membranes. The binding was saturable, stereospecific and of high affinity (Kd: 0.97 and 1.12 nM based on kinetic and equilibrium binding studies, respectively). The maximal number of binding sites (Bmax) was found to be 114.8 fmol/mg protein. [3H]Endomorphin 2 was displaced by mu-receptor selective specific peptides and heterocyclic compounds with high affinity, whereas kappa and delta receptor specific ligands were much less potent. The Ki values of endomorphin 1 and 2 in inhibiting [3H]naloxone binding increased by 15-fold in the presence of 100 mM NaCl which indicates the agonist property of these peptides. Endomorphins stimulated [35S]GTPgammaS binding and inhibited adenylyl cyclase activity which also provides evidence for the agonist character of endomorphins.

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.

Behavioural and biochemical evidence for signs of abstinence in mice chronically treated with delta-9-tetrahydrocannabinol.

Tolerance and dependence induced by chronic delta-9-tetrahydrocannabinol (THC) administration were investigated in mice. The effects on body weight, analgesia and hypothermia were measured during 6 days of treatment (10 or 20 mg kg(-1) THC twice daily). A rapid tolerance to the acute effects was observed from the second THC administration. The selective CB-1 receptor antagonist SR 141716A (10 mg kg(-1)) was administered at the end of the treatment, and somatic and vegetative manifestations of abstinence were evaluated. SR 141716A administration precipitated several somatic signs that included wet dog shakes, frontpaw tremor, ataxia, hunched posture, tremor, ptosis, piloerection, decreased locomotor activity and mastication, which can be interpreted as being part of a withdrawal syndrome. Brains were removed immediately after the behavioural measures and assayed for adenylyl cyclase activity. An increase in basal, forskolin and calcium/calmodulin stimulated adenylyl cyclase activities was specifically observed in the cerebellum of these mice. The motivational effects of THC administration and withdrawal were evaluated by using the place conditioning paradigm. No conditioned change in preference to withdrawal associated environment was observed. In contrast, a conditioned place aversion was produced by the repeated pairing of THC (20 mg kg(-1)), without observing place preference at any of the doses used. This study constitutes a clear behavioural and biochemical model of physical THC withdrawal with no motivational aversive consequences. This model permits an easy quantification of THC abstinence in mice and can be useful for the elucidation of the molecular mechanisms involved in cannabinoid dependence.