Role of the endocannabinoid system in ethanol-induced inhibition of salivary secretion.

AIM: The aim of the present study was to determine whether the endocannabinoid system could be involved in the ethanol-induced inhibition of salivation in adult male Wistar rats. METHODS: Salivary secretion induced by different concentrations of methacholine, a cholinergic agonist, and the endocannabinoid arachidonoyl ethanolamide (anandamide, AEA) production in the submandibular gland (SMG) were determined in rats after ethanol (3 g/kg) administration by gastric gavage. To study the participation of cannabinod receptors in ethanol action, we evaluated methacholine-induced salivary secretion after ethanol administration when CB1 or CB2 receptors were blocked by intra-SMG injections of their selective antagonists AM251 and AM630, respectively. Additionally, we evaluated the in vitro effect of ethanol (0.1 M) on SMG production of cAMP, alone or combined with AM251 or AM630. RESULTS: Acute ethanol administration increased AEA production in SMG and also inhibited the methacholine-induced saliva secretion that was partially restored by intraglandular injection of AM251 or AM630. In addition, ethanol significantly reduced the forskolin-induced increase in cAMP content in SMG in vitro while treatment with AM251 blocked this response. CONCLUSION: We conclude that the inhibitory effect produced by ethanol on submandibular gland salivary secretion is mediated, at least in part, by the endocannabinoid system.

Adenylate cyclase of bovine adrenal cortex plasma membranes. Divergence between corticotropin and fluoride combined effects with forskolin.

The diterpene forskolin maximally stimulated bovine adrenal cortex adenylate cyclase activity 9-fold with a concentration producing half-maximum effect (ED50) of about 4 microM. The effects of forskolin and the fully active corticotropin fragment ACTH (I 24) were additive over nearly the whole range of concentration of both effectors, indicating separate and independent mechanisms of action. By contrast, 10 mM NaF blocked forskolin action in the nanomolar range of the diterpene concentration, while it allowed a partial stimulation by forskolin in the micromolar range. NaF thus reveals a heterogeneity of forskolin action in the adrenal cortex plasma membranes. Moreover, our data suggest that ACTH and NaF activation effects, both mediated by the stimulatory regulatory protein Gs, proceed through different mechanisms.

Platelet adenylate cyclase activity in Israeli victims of Iraqi Scud missile attacks with post-traumatic stress disorder.

Platelet adenylate cyclase activity was measured in 16 control subjects and 16 patients who developed post-traumatic stress disorder (PTSD) as a result of damage inflicted on their homes during the Iraqi Scud missile attacks on Israel which occurred during the 1991 Gulf War. There were no differences in basal, NaF-stimulated, PGE1-stimulated or forskolin-stimulated activity between controls and PTSD subjects. Epinephrine inhibition of forskolin-stimulated activity, an effect mediated by alpha 2 adrenergic receptors, was slightly but not significantly increased in the PTSD patients compared to the controls, while 5-HT induced inhibition, an effect mediated by putative 5-HT1a-like receptors, was unchanged. The relationship of these activities to measures of anxiety and depression in these patients is discussed.

Platelet adenylate cyclase activity in depression and after clomipramine and lithium treatment: relation to serotonergic function.

Adenylate cyclase activity was measured in platelet membranes from 10 healthy controls, 12 depressed patients, and the same patients after treatment with clomipramine (CMI) followed by lithium carbonate (Li) supplementation, in an attempt to determine whether any evidence for an effect on the serotonergic system could be obtained in peripheral cells. There were no differences in basal, NaF-, PGE1-, or forskolin-stimulated activity either between the control subjects and depressed patients or between activities in the patients measured before treatment, after CMI, and after CMI+Li. The degree of inhibition of forskolin-stimulated adenylate cyclase by 5-HT, an effect putatively mediated by a 5-HT1A-like receptor, was not different in the depressed patients compared to controls or affected by CMI treatment, but was significantly reduced after Li supplementation.

Effects of glucocorticoid on signalling by prostaglandin E2 in osteoblast-like cells.

It is well-known that osteoporosis is a common complication of patients with glucocorticoid excess. We previously reported that dexamethasone inhibits Ca2+ influx induced by prostaglandin E2 (PGE2), a potent bone resorbing agent, in osteoblast-like MC3T3-E1 cells (O. Kozawa, H. Tokuda, J. Kotoyori, A. Suzuki, Y. Ito and Y. Oiso, Prostagland Leuk Essent Fatty Acids, in press). In the present study, we examined the effects of dexamethasone on cyclic adenosine monophosphate (cAMP) accumulation and phosphoinositide hydrolysis by PGE2 in MC3T3-E1 cells. The pretreatment with dexamethasone significantly inhibited cAMP accumulation stimulated by PGE2 in a dose-dependent manner in the range between 10 pM and 1 nM in MC3T3-E1 cells. This effect of dexamethasone was dependent on the time of pretreatment up to 8 h. Dexamethasone also inhibited the cAMP accumulation induced by NaF, an activator of guanosine triphosphate (GTP)-binding protein, or forskolin which directly activates adenylate cyclase. In contrast, dexamethasone had little effect on the formation of inositol phosphates stimulated by PGE2 in MC3T3-E1 cells. These results strongly suggest that glucocorticoid modulates the signal transduction by PGE2 in osteoblast-like cells and that it inhibits PGE2-induced cAMP production without affecting PGE2-induced phosphoinositide hydrolysis.

Sodium fluoride inhibits the antisecretory effect of peptide YY and its analog in rabbit jejunum.

The antisecretory peptide YY (PYY) inhibits jejunal secretion through and inhibitory protein (Gi), whereas sodium fluoride (NaF) is a potent activator of G-proteins. This work was conducted to characterize the role of NaF in the antisecretory effect of PYY. For this purpose, electrogenic chloride secretion was assessed by measuring the in vitro variations in short-circuit current (delta Isc) due to alterations in ionic transport, using Ussing chambers Results: 1) NaF induced a transient increase in Isc at concentrations exceeding 5 mM. 2) 2 mM NaF inhibited the antisecretory effect of 0.1 microM PYY and of its analog P915. 3) stimulation of secretion by forskolin and dbcAMP was halved in the presence of 2 mM NaF. 4) Inhibition of protein kinase C by 0.1 mM bisindolylmaleimide caused a sustained increase in Isc in the presence of 5 mM NaF. In conclusion, these results confirm that PYY inhibits electrogenic chloride secretion and show that NaF stimulates it, and suggest that NaF reduces PYY-induced inhibition via a G-dependent and a G-independent functional pathway.

Enhanced expression of Gialpha protein and adenylyl cyclase signaling in aortas from 1 kidney 1 clip hypertensive rats.

We have previously shown the augmented levels of Gialpha-2 and Gialpha-3 proteins (isoforms of inhibitory guanine nucleotide regulatory protein (G-protein)), and not of Gsalpha, in the hearts and aortas of spontaneously and experimentally induced hypertensive rats. The increased expression of Gialpha and blood pressure was restored toward WKY levels by captopril treatment, suggesting a role for angiotensin (Ang) II in the enhanced expression of Gialpha protein and blood pressure. This study was undertaken to investigate whether 1 kidney 1 clip (1K-1C) hypertensive rats that exhibit enhanced levels of Ang II also express enhanced levels of Gialpha proteins. Aortas from 1K-1C hypertensive rats were used. The expression of G-proteins was determined at protein levels with immunoblotting techniques, using specific antibodies for different isoforms of G-proteins. The levels of Gialpha-2 and Gialpha-3 proteins were significantly higher in aortas from 1K-1C hypertensive rats than in control rats; Gsalpha levels were unchanged. The inhibitory effect of low concentrations of guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) on forskolin (FSK)-stimulated adenylyl cyclase (AC) activity was significantly enhanced in aortas from 1K-1C hypertensive rats; the inhibitory effect of C-ANP(4-23), which specifically interacts with the atrial natriuretic peptide (ANP)-C receptor, and Ang II on AC was attenuated. GTPgammaS, isoproterenol, glucagon, NaF, and FSK stimulated the AC activity in aortas from control and hypertensive rats to varying degrees; however, the stimulations were significantly lower in hypertensive rats than in control rats. These data suggest that aortas from 1K-1C hypertensive rats exhibit enhanced expression of Gialpha proteins and associated functions.

Synthesis of 2',3'-O-(2,4,6-trinitrocyclohexadienylidine)guanosine 5'-triphosphate and a study of its inhibitory properties with adenylate cyclase.

A fluorescent GTP analog 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) guanosine 5'-triphosphate (TNP-GTP) has been prepared and some of its physical properties characterized. TNP-GTP was found to be a potent inhibitor of chick embryo heart adenylate cyclase as activated by guanyl 5'-(beta,gamma-imido)triphosphate (GppNHp), F-, and forskolin with Ki values in the 8-15 microM range. It also appeared to inhibit substantially basal adenylate cyclase in this system. TNP-GTP demonstrated an effective competition with [3H]GppNHp, binding to membranes equivalently to GppNHp and about three times better than GTP. 8-Azidoguanosine 5'-triphosphate (8N3GTP) mimics GTP activation of chick embryo heart adenylate cyclase and [gamma-32P]8N3GTP is effectively photoincorporated into a 42,000- to 44,000-Mr doublet when proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. TNP-GTP effectively prevents this photoincorporation, as does GTP, at concentrations that agree with their respective apparent inhibition and activation binding constants. The data suggest that TNP-GTP could prove to be a valuable tool for studying the mechanisms of GTP regulation of adenylate cyclase and other GTP-regulated systems.

Corticotropin-releasing factor-induced adrenocorticotropin hormone release and synthesis is blocked by incorporation of the inhibitor of cyclic AMP-dependent protein kinase into anterior pituitary tumor cells by liposomes.

Corticotropin-releasing factor (CRF) is the most potent and effective natural stimulant of corticotropin (ACTH) secretion. In a tumor cell line of the mouse anterior pituitary (AtT-20/D16-16) consisting of a homogeneous population of corticotrophs, CRF is known to increase adenylate cyclase and cAMP-dependent protein kinase activities as well as to release ACTH. To determine whether activation of cAMP-dependent protein kinase is essential for CRF to evoke the secretion of ACTH, an inhibitor (PKI) of this kinase was inserted into AtT-20 cells. This was accomplished by first encapsulating PKI into liposomes and then covalently coupling them to protein A for binding to antibodies directed against an AtT-20 cell surface antigen, N-CAM (neural cell adhesion molecule). The binding of the liposomes to the anti-N-CAM antibodies led to the internalization of the PKI into the tumor cells. The PKI treatment greatly attenuated CRF-stimulated ACTH release as well as the secretory response to beta-adrenergic agonists. However, ACTH release in response to caerulein, an agonist of cholecystokinin 8 receptors, was not altered by the PKI treatment. CRF treatment also increased the levels of mRNA for proopiomelanocortin (POMC), the precursor for ACTH in AtT-20 cells. Application of liposomes containing PKI to AtT-20 cells blocked the ability of CRF and 8-bromo-cAMP, but not phorbol ester, to increase POMC mRNA levels. The results revealed an essential role for cAMP in mediating the effect of CRF on ACTH release and POMC gene expression.