3'-5' cyclic-guanosine monophosphate increase in rat brain hippocampus after gamma-hydroxybutyrate administration. Prevention by valproate and naloxone

An increase (123%) of cyclic GMP (cGMP) was observed in the hippocampus of the rat killed by microwave irradiation 45 min after administration of 500 mg/kg gamma-hydroxybutyrate (GHB) IP. This increase is time and dose dependent. No modification in cyclic nucleotide content was observed in striatum and in cerebellum. As the role of GHB has been implicated in neurotransmission, the fact that this compound increases cyclic GMP accumulation in hippocampus in vivo may represent a mechanism by which the actions of GHB are mediated at the cellular level. Valproate (400 mg/kg) or naloxone (10 mg/kg) pretreatment completely abolish the cGMP increase due to GHB. A GABAergic and/or opiate phenomenon may be involved in the mechanism of GHB induced increase of cGMP.     

Mechanism of cyclic GMP inhibition of inositol phosphate formation in rat aorta segments and cultured bovine aortic smooth muscle cells

In order to clarify the mechanism(s) by which cyclic GMP inhibits the generation of inositol phosphates in rat aorta segments and cultured bovine aortic smooth muscle cells, we studied phosphoinositide hydrolysis and GTPase activity in homogenates and membrane preparations of cultured bovine aortic smooth muscle cells. Pretreatment of homogenate preparations with cyclic GMP plus ATP did not inhibit [8-arginine, 3H] vasopressin (AVP) binding, but resulted in a total suppression of the AVP-induced GTPase activation. The pretreatment with cyclic GMP and ATP also inhibited the formation of inositol phosphates induced by AVP in the presence of low concentrations of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S), or by high concentrations of GTP gamma S alone. However, the formation of inositol phosphates by high concentrations of Ca2+ alone was not blocked. These results suggest that the ability of cyclic GMP to inhibit phosphoinositide hydrolysis results from an inhibition of a guanine nucleotide regulatory protein activation, and the interaction between guanine nucleotide regulatory protein and phospholipase C. While the precise site of this inhibition is not presently known, the inhibition by cyclic GMP is dependent upon the addition of ATP and probably entails a phosphorylation event since adenylylimidodiphosphate can not substitute for the ATP requirement.     

Activation of Guanylate Cyclase by Bradykinin in Rat Sensory Neurones Is Mediated by Calcium Influx: Possible Role of the Increase in Cyclic GMP

Bradykinin, which activates polymodal nociceptors, increased cyclic GMP (cGMP) in a capsaicin-sensitive population of cultured sensory neurones from rat dorsal root ganglia (DRG) by stimulating guanylate cyclase, but had no effect on cyclic AMP (cAMP). In nonneuronal cells from DRG, bradykinin increased cAMP, but not cGMP. The bradykinin-induced increase in cGMP in the neurones was completely blocked by removal of extracellular Ca2+, or by incubation of the cells with the calcium channel blockers nifedipine and verapamil. Pretreatment of the neurones with either dibutyryl cGMP or sodium nitroprusside (which elevates cGMP) inhibited bradykinin-induced formation of inositol phosphates. It is possible that cGMP could be involved in the regulation of polyphosphoinositide turnover in DRG neurones.     

Atrial natriuretic peptide induces breakdown of phosphatidylinositol phosphates in cultured vascular smooth-muscle cells

Discrepancies exist between extent of guanylate cyclase activation by atrial natriuretic peptide (ANP) in cell-free systems and ANP-stimulated levels of cyclic GMP in whole cells, and also between receptor affinity and dose effectiveness of ANP. Therefore, we have investigated whether, in addition to receptor-coupled guanylate cyclase activation, other second-messenger cascade systems may be involved in mediating both an increase in cyclic GMP and the physiological response to ANP. Equilibrium 125I-ANP binding studies on cultured thoracic aorta smooth muscle cells revealed the existence of low-affinity (approximately 10(-8) M, 84.5 fmol/10(5) cells) and high-affinity (approximately 10(-10) M, 12.5 fmol/10(5) cells) binding sites. We confirm that ANP elevates intracellular cyclic GMP (EC50 approximately 10(-8) M) and inhibits agonist-(isoproterenol and forskolin)-induced increases in intracellular cyclic AMP (IC50 approximately 10(-9) M). ANP also stimulated breakdown of phosphatidylinositol phosphates and generation of inositol phosphates with a half-maximally effective concentration of approximately 10(-10) M. The extent of phosphatidylinositol polyphosphate hydrolysis was small (120%) in comparison to that of phosphatidylinositol (Ptd-Ins) (200%). Ptd-Ins hydrolysis was paralleled by the appearance of glycerophosphoinositol, and there was also a close temporal relationship between these processes and the accumulation of intracellular cyclic GMP. Smooth muscle cells released [3H]arachidonic acid label in response to ANP (EC50 approximately 10(-10) M). Taken together, the data suggest that the vasorelaxant hormone ANP has stimulatory effects on phosphoinositol lipid metabolism via both phospholipase C (generation of inositol phosphates) and phospholipase A2 (generation of releasable [3H]arachidonic acid and indirectly glycerophosphoinositol). In contrast, stimulation of phosphatidylinositol phosphate breakdown by the vasoconstrictive hormone angiotensin II is not associated with glycerophosphoinositol formation, and neither cyclic GMP nor cyclic AMP levels were influenced by this hormone.     

Muscarinic cholinergic stimulation increases cyclic GMP levels in rat hippocampus.

Abstract— Muscarinic cholinergic agonists increase cyclic GMP levels in a number of neural tissues. Since the rat hippocampus receives a cholinergic innervation from the septum, we decided to test whether cyclic GMP levels of the rat hippocampus are increased by bethanechol, a muscarinic cholinergic agonist. Incubation of rat hippocampi with varying concentrations of bethanechol showed that the increase in cyclic GMP levels is concentration-dependent, 500 pwbethanechol producing a maximum increase of 490% over control values. The bethanechol-evoked increases were blocked by the muscarinic antagonist atropine, and were calcium-dependent. It is concluded that at least some of the cells projecting to the rat hippocampus form muscarinic cholinergic synapses which act via a cyclic GMP-dependent mechanism.     

Plasma from uninephrectomized rats stimulates production of inositol trisphosphates and inositol tetrakisphosphate in renal cortical slices.

Metabolism of inositol phosphates in renal cortical slices was investigated in vitro after addition of plasma from uninephrectomized or sham-operated rats. Plasma from uninephrectomized rats stimulated production of InsP3 (inositol trisphosphate) when obtained within the first 3 h after uninephrectomy. With different amounts of added plasma a graded response of InsP3 production was obtained. This effect could be prevented by 0.1 microM-TPA (12-O-tetradecanoylphorbol 13-acetate). When analysis of inositol phosphates was performed by h.p.l.c., plasma from uninephrectomized rats stimulated a rapid increase in Ins(1,4,5)P3 radioactivity, whereas the increase in inositol 1,3,4,5-tetrakisphosphate and Ins(1,3,4)P3 radioactivity was slower. Plasma from uninephrectomized rats decreased cyclic AMP concentration in renal cortical slices. Similar effect was obtained when slices were incubated with TPA (0.05 microM). Plasma from uninephrectomized rats increased cyclic GMP concentration in renal cortical slices, but this effect was abolished when extracellular Ca2+ had been chelated with 4 mM-EGTA. Results indicate that plasma from uninephrectomized rats stimulates phospholipase C, increases cyclic GMP concentration and decreases cyclic AMP concentration in renal cortical slices. Increases in cyclic GMP depend on extracellular Ca2+, whereas the decrease in cyclic AMP concentration is mediated by protein kinase C.     

Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells. Evidence for phospholipase C generated second messengers in the action of luteinizing hormone.

The following studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases cellular levels of cyclic AMP, also provokes increases in 'second messengers' derived from inositol lipid metabolism (i.e. inositol phosphates and diacylglycerol). Rat granulosa cells isolated from mature Graafian follicles were prelabelled for 3 h with myo-[2-3H]inositol. LH provoked rapid (5 min) and sustained (up to 60 min) increases in the levels of inositol mono-, bis, and trisphosphates (IP, IP2 and IP3, respectively). Time course studies revealed that IP3 was formed more rapidly than IP2 and IP following LH treatment. The response to LH was concentration-dependent with maximal increases at LH concentrations of 1 microgram/ml. LiCl (2-40 mM) enhanced the LH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. The effectiveness of LH, however, was dependent on the concentration of lithium employed; maximal increases in IP were observed at 10 mM-LiCl, whereas maximal increases in IP2 and IP3 were observed at 20 mM- and 40 mM-LiCl, respectively. The stimulatory effects of LH on inositol phosphate and progesterone accumulation were also compared with changes in cyclic nucleotide levels. LH rapidly increased levels of inositol phosphates, progesterone and cyclic AMP, but transiently reduced levels of cyclic GMP. These results demonstrate that LH increases both cyclic AMP and inositol trisphosphate (and presumably diacylglycerol) in rat granulosa cells. Our findings suggest that two messenger systems exist to mediate the action of LH in granulosa cells.     

A Method to Measure Simultaneously Cyclic AMP and Inositol Phosphate Accumulation in Rat Brain Slices

The simultaneous measurement of the accumulation of cyclic AMP and inositol phosphates in rat cerebral cortical slices is described. After stimulation, the separation of cyclic AMP and inositol phosphates was achieved using ion-exchange chromatography and their concentrations were determined by means of a double-labeling technique, the substrates adenine and inositol being labeled with 14C and 3H, respectively. The recoveries were 70-80% for inositol phosphates and 40-50% for cyclic AMP. To test the applicability of the method, norepinephrine was chosen as an agonist, because it is known to stimulate the production of these two second messengers by interacting with alpha- and beta-adrenergic receptors. This procedure is an improvement over existing methods, because we obtained the simultaneous formation of 3H-inositol phosphates and [14C]cyclic AMP in a concentration-dependent process. EC50 values were similar for the two, 8.5 +/- 3.9 microM for 3H-inositol phosphates and 20.2 +/- 6.3 microM for [14C]cyclic AMP, and close to the values obtained when each process was studied alone. The action of adrenergic antagonists was also tested. Propranolol blocked the norepinephrine stimulation of [14C]cyclic AMP, but did not inhibit the norepinephrine stimulation of 3H-inositol phosphates. The opposite results were observed with prazosin. Our results suggest that this method could be a useful tool to examine the interaction between these two receptor-coupled effectors.     

Modulation of Second Messengers in the Nervous System of Larval Manduca sexta by Muscarinic Receptors

Abstract: Measurements were made of the effects of muscarinic agents on endogenous levels of cyclic AMP and cyclic GMP, and the turnover of radiolabeled inositol phosphates in the abdominal nervous system of larval Manduca sexta . Cyclic AMP levels were increased by treatment with 3-isobutyl-1-methylxanthine or tetrodotoxin, but the muscarinic agonist oxotremorine-M and the muscarinic antagonist scopolamine had no consistent effects. In contrast, cyclic GMP levels were significantly increased by oxotremorine-M and by oxotremorine-M in the presence of 3-isobutyl-1-methylxanthine and tetrodotoxin but not in the presence of scopolamine. Using lithium to inhibit the recycling of inositol phospholipid metabolites in isolated nerve cords, we detected a small but consistent increase in inositol phosphate production by oxotremorine-M. The primary inositol metabolite generated during a 5-min exposure to oxotremorine-M co-eluted from ion-exchange columns with inositol-1-monophosphate, although other more polar metabolites were also detected. This agonist-evoked increase in inositol phosphate production was unaffected by tetrodotoxin but inhibited by scopolamine, suggesting that it is directly mediated by muscarinic receptors. Further evidence for coupling between muscarinic receptors and inositol metabolism was obtained using a cell-free preparation of nerve cord membranes labeled with [³H]inositol. Incubation with oxotremorine-M evoked a significant increase in labeled inositol bisphosphate, consistent with muscarinic receptors coupling to phosphatidylinositol metabolism. The accumulation of inositol bisphosphate in cell-free preparations suggests that the normal breakdown to inositol monophosphate requires cytosolic components. Together, these results indicate that muscarinic acetylcholine receptors in Manduca couple predominantly to the inositol phospholipid signaling system, although some receptors may modulate cyclic GMP.     

Two Possibly Distinct Prostaglandin E1 Receptors in N1E-115 Clone: One Mediating Inositol Trisphosphate Formation, Cyclic GMP Formation, and Intracellular Calcium Mobilization and the Other Mediating Cyclic AMP Formation

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.     

Neurotensin Stimulates Inositol Phospholipid Metabolism and Calcium Mobilization in Murine Neuroblastoma Clone N1E-115

Murine neuroblastoma cells (clone N1E-115) possess neurotensin receptors that mediate cyclic GMP synthesis. Because of the hypothesized relationship between phospholipid metabolism, intracellular Ca2+, and cyclic GMP synthesis, we determined with these cells the effects of neurotensin on 32P labeling of phospholipids, release of inositol phosphates, and intracellular Ca2+ (as determined with the use of Quin-2, a fluorescent probe sensitive to free Ca2+ levels). Neurotensin stimulated incorporation of 32P into phospholipids, especially phosphatidylinositol and phosphatidate. Neurotensin also stimulated the release of [3H]inositol phosphates with an EC50 of about 1 nM. Mean basal Ca2+ concentration in these cells was 134 nM and this level was increased in a rapid and dose-dependent manner by neurotensin, with an EC50 of 4 nM. Since the EC50 for neurotensin in stimulating cyclic GMP synthesis is 1.5 nM and the KD for binding of [3H]neurotensin at 0 degrees C is 11 nM, all these different effects appear to be shared proximal consequences of neurotensin receptor activation.     

Serotonin Stimulates Both Cytosolic and Membrane-Bound Guanylate Cyclase in NG108–15 Cells

The cyclic GMP (cGMP) content was rapidly (greater than 30 s) increased by serotonin [5-hydroxytryptamine (5-HT)] (EC50 = 10 microM), and the increase lasted for greater than 10 min in NG108-15 cells. The 5-HT-induced elevation of cGMP level (EC50 = 10 microM) at 20 s ("fast" elevation) was inhibited by ICS 205-930 or MDL 72,222 and by Ca2+ deficiency in the reaction medium but not by organic Ca2+ antagonists. The 5-HT effect at 10 min ("slow" elevation) was not inhibited by several antagonists for 5-HT receptors of the 1A, 1B, 1C, 1D, 2, and 3 subtypes and was independent from external Ca2+ concentration. The fast and slow effects of 5-HT were similar to the effects of bradykinin and atrial natriuretic peptide (ANP), respectively, in aspects of both Ca2+ dependency and time course of the effects. Bradykinin transiently stimulated formation of inositol phosphates as well as accumulation of cGMP, a finding suggesting that intracellular Ca2+ is involved in bradykinin-induced cGMP accumulation as shown in the fast response to 5-HT. ANP, an activator of membrane-associated guanylate cyclase (mGC), slowly (approximately 60 s) increased the cGMP content (EC50 = 10 nM), a result lasting for greater than 10 min, and the effects were independent from external Ca2+, as shown in the slow response to 5-HT. 5-HT and ANP did not induce formation of inositol phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of inositol phosphates to arrestin.

Arrestin binds to phosphorylated rhodopsin in its light-activated form (metarhodopsin II), blocking thereby its interaction with the G-protein, transducin. In this study, we show that highly phosphorylated forms of inositol compete against the arrestin-rhodopsin interaction. Competition curves and direct binding assays with free arrestin consistently yield affinities in the micromolar range; for example, inositol 1,3,4,5-tetrakisphosphate (InP4) and inositol hexakisphosphate (InP6 bind to arrestin with dissociation constants of 12 microM and 5 microM, respectively. Only a small control amount of inositol phosphates is bound, when arrestin interacts with phosphorylated rhodopsin. This argues for a release of bound inositol phosphates by interaction with rhodopsin. Transducin, rhodopsin kinase, or cyclic GMP phosphodiesterase are not affected by inositol phosphates. These observations open a new way to purify arrestin and to inhibit its interaction with rhodopsin. Their physiological significance deserves further investigation.     

Isolation and characterization of the inositol cyclic phosphate products of phosphoinositide cleavage by phospholipase C. Metabolism in cell-free extracts

The phosphoinositides are metabolized by phospholipase C in response to hormone or agonist stimulation in many cell types to produce diglyceride and water-soluble inositol phosphates. We have recently shown that the phospholipase C reaction products include cyclic phosphate esters of inositol. One of these, inositol 1, 2-cyclic 4,5-trisphosphate, is active in promoting Ca2+ mobilization in platelets and in inducing changes in conductance in Limulus photoreceptors similar to those produced by light (Wilson, D. B., Connolly, T. M., Bross, T. E., Majerus, P. W., Sherman, W. R., Tyler, A., Rubin, L. J., and Brown, J. E. (1985) J. Biol. Chem. 260, 13496-13501. In the current study, we have examined the metabolism of the inositol phosphates. We find that both cyclic and non-cyclic inositol trisphosphates are metabolized by inositol 1,4,5-trisphosphate 5-phosphomonoesterase, to inositol 1,2-cyclic bisphosphate and inositol 1,4-bisphosphate, respectively. However, the apparent Km of the enzyme for the cyclic substrate is approximately 10-fold higher than for the non-cyclic substrate. These inositol bisphosphates are more slowly degraded to inositol 1,2-cyclic phosphate and inositol 1-phosphate, respectively. Inositol 1,2-cyclic phosphate is then hydrolyzed to inositol 1-phosphate, which in turn is degraded to inositol and inorganic phosphate by inositol 1-phosphate phosphatase. The human platelet inositol 1,2-cyclic phosphate hydrolase enzyme and a similar rat kidney hydrolase do not utilize the cyclic polyphosphate esters of inositol as substrates. These results suggest that the inositol cyclic phosphates and the non-cyclic inositol phosphates are metabolized separately by phosphatases to cyclic and non-cyclic inositol monophosphates. The cyclic monophosphate is then converted to inositol 1-phosphate by a cyclic hydrolase. We suggest that the enzymes that metabolize the inositol phosphates may serve to regulate cellular responses to these compounds.     

Activation of Muscarinic and of α1-Adrenergic Receptors on Astrocytes Results in the Accumulation of Inositol Phosphates

Astrocyte-enriched cultures prepared from the newborn rat cortex incorporated [3H]myo-inositol into intracellular free inositol and inositol lipid pools. Noradrenaline and carbachol stimulated the turnover of these pools resulting in an increased accumulation of intracellular [3H]inositol phosphates. The effects of noradrenaline and carbachol were dose-dependent and blocked by specific alpha 1-adrenergic and muscarinic cholinergic receptor antagonists, respectively. The increase in [3H]inositol phosphate accumulation caused by these receptor antagonists was virtually unchanged when cultures were incubated in Ca2+-free medium, but was abolished when EGTA was also present in the Ca2+-free medium. Cultures of meningeal fibroblasts, the major cell type contaminating the astrocyte cultures, also accumulated [3H]myo-inositol, but no increased accumulation of [3H]inositol phosphates was found in response to either noradrenaline or carbachol.     

Modulation of Neuronal Signal Transduction Systems by Extracellular ATP

The secretion of ATP by stimulated nerves is well documented. Following repetitive stimulation, extracellular ATP at the synapse can accumulate to levels estimated to be well over 100 microM. The present study examined the effects of extracellular ATP in the concentration range of 0.1-1.0 mM on second-messenger-generating systems in cultured neural cells of the clones NG108-15 and N1E-115. Cells in a medium mimicking the physiological extracellular environment were used to measure 45Ca2+ uptake, changes in free intracellular Ca2+ levels by the probes aequorin and Quin-2, de novo generation of cyclic GMP and cyclic AMP from intracellular GTP and ATP pools prelabeled with [3H]guanosine and [3H]adenine, respectively, and phosphoinositide metabolism in cells preloaded with [3H]inositol and assayed in the presence of LiCl. Extracellular ATP induced a concentration-dependent increase of 45Ca2+ uptake by intact cells, which was additive with the uptake induced by K+ depolarization. The increased uptake involved elevation of intracellular free Ca2+ ions, evidenced by measuring aequorin and Quin-2 signals. At the same concentration range (0.1-1.0 mM), extracellular ATP induced an increase in [3H]cyclic GMP formation, and a decrease in prostaglandin E1-stimulated [3H]cyclic AMP generation. In addition, extracellular ATP (1 mM) caused a large (15-fold) increase in [3H]inositol phosphates accumulation, and this effect was blocked by including La3+ ions in the assay medium. In parallel experiments, we found in NG108-15 cells surface protein phosphorylation activity that had an apparent Km for extracellular ATP at the same concentration required to produce half-maximal effects on Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of second messengers in response to activation of ion channels in excitable cells

1. Depolarization of excitable cells of the central nervous system results in the formation of the second messengers cyclic AMP, cyclic GMP, inositol phosphates, and diacylglycerides. 2. Depolarization-evoked accumulation of cyclic AMP in brain preparations can be accounted for mainly by the release of adenosine, which subsequently interacts with stimulatory adenosine receptor linked to adenylate cyclase. 3. Depolarization-evoked formation of cyclic GMP in brain preparations is linked to activation of voltage-dependent calcium channels, presumably leading to activation of guanylate cyclase by calcium ions. 4. In brain slices depolarization-evoked stimulation of phosphoinositide breakdown and subsequent formation of inositol phosphates and diacylglycerides are linked to activation of voltage-dependent calcium channels, which are sensitive to dihydropyridines, presumably leading to activation of phospholipase(s) C by calcium ions. 5. In the synaptoneurosome preparation depolarization-evoked stimulation of phosphoinositide breakdown does not involve activation of dihydropyridine-sensitive calcium channels and, instead, appears to be regulated primarily by the intracellular concentration of sodium ions. Thus, agents that induce increases in intracellular sodium--such as toxins that open or delay inactivation of voltage-dependent sodium channels; ouabain, an inhibitor of Na+/K+ ATPase that transports sodium outward and a sodium ionophore--all stimulate phosphoinositide breakdown. Mechanistically, increases in intracellular sodium either might directly affect phospholipase(s) C or might lead to influx of calcium ions through Na+/Ca2+ transporters. 6. Depolarization-evoked stimulation of cyclic AMP formation and phosphoinositide breakdown can exhibit potentiative interactions with responses to receptor agonists, thereby providing mechanisms for modulation of receptor responses by neuronal activity. 7. Since all these second messengers can induce phosphorylation of ion channels through the activation of specific kinases, it is proposed that depolarization-evoked formation of second messengers represents a putative feedback mechanism to regulate ion fluxes in excitable cells.     

Evidence for a G protein-coupled gamma-hydroxybutyric acid receptor

gamma-Hydroxybutyric acid (GHB) is a naturally occurring metabolite of GABA that has been postulated to exert ubiquitous neuropharmacological effects through GABA(B) receptor (GABA(B)R)-mediated mechanisms. The alternative hypothesis that GHB acts via a GHB-specific, G protein-coupled presynaptic receptor that is different from the GABA(B)R was tested. The effect of GHB on regional and subcellular brain adenylyl cyclase in adult and developing rats was determined and compared with that of the GABA(B)R agonist (-)-baclofen. Also, using guanosine 5'-O:-(3-[(35)S]thiotriphosphate) ([(35)S]GTPgammaS) binding and low-K:(m) GTPase activity as markers the effects of GHB and (-)-baclofen on G protein activity in the brain were determined. Neither GHB nor baclofen had an effect on basal cyclic AMP (cAMP) levels. GHB significantly decreased forskolin-stimulated cAMP levels by 40-50% in cortex and hippocampus but not thalamus or cerebellum, whereas (-)-baclofen had an effect throughout the brain. The effect of GHB on adenylyl cyclase was observed in presynaptic and not postsynaptic subcellular tissue preparations, but the effect of baclofen was observed in both subcellular preparations. The GHB-induced alteration in forskolin-induced cAMP formation was blocked by a specific GHB antagonist but not a specific GABA(B)R antagonist. The (-)-baclofen-induced alteration in forskolin-induced cAMP formation was blocked by a specific GABA(B)R antagonist but not a specific GHB antagonist. The negative coupling of GHB to adenylyl cyclase appeared at postnatal day 21, a developmental time point that is concordant with the developmental appearance of [(3)H]GHB binding in cerebral cortex, but the effects of (-)-baclofen were present by postnatal day 14. GHB and baclofen both stimulated [(35)S]GTPgammaS binding and low-K:(m) GTPase activity by 40-50%. The GHB-induced effect was blocked by GHB antagonists but not by GABA(B)R antagonists and was seen only in cortex and hippocampus. The (-)-baclofen-induced effect was blocked by GABA(B)R antagonists but not by GHB antagonists and was observed throughout the brain. These data support the hypothesis that GHB induces a G protein-mediated decrease in adenylyl cyclase via a GHB-specific G protein-coupled presynaptic receptor that is different from the GABA(B)R.     

Cyclic GMP Formation and Inositol Phosphate Accumulation Do Not Share Common Origins in Rat Brain Slices

Cyclic GMP formation and inositol phospholipid hydrolysis were studied in rat brain slices to determine if the two processes have common origins. Muscarinic cholinergic stimulation enhanced [3H]inositol phosphate ([ 3H]IP) accumulation from slices prelabelled with [3H]inositol but did not affect cyclic GMP formation in the cortex, striatum, or cerebellum. An elevated level of extracellular K+ stimulated accumulation of both cyclic GMP and [3H]IP in cortex slices. The former, but not the latter, was reduced by lipoxygenase and phospholipase A2 inhibition. Calcium channel activation enhanced and blockade reduced K+-stimulated [3H]IP formation without affecting the cyclic GMP level, and there were differences in the Ca2+ requirements for the two responses. Thus, there is no support for the concept that guanylate cyclase activation inevitably accompanies inositol phospholipid breakdown, and the evidence presented demonstrates that K+ stimulation promotes cyclic GMP and [3H]IP accumulation by different transducing pathways.     

Cyclic GMP regulates cromakalim-induced relaxation in the rat aortic smooth muscle: role of cyclic GMP in K(ATP)-channels.

Recent studies have shown that nitric oxide (NO) modulates K+-channel activity which play an important role in controlling vascular tone. The formation of cyclic guanosine 3',5'-monophosphate (cyclic GMP) has also been recognized to be associated with the vasodilatory effect of NO. Both cyclic GMP and NO increase whole-cell K+-current by activating Ca2+-activated K+-channels (K(Ca)-channels). Here, we show evidence that activators of soluble guanylyl cyclase sodium nitroprusside or 3-morpholino-sydnonimine (SIN-1), and an analogue of cyclic GMP 8-bromo-cyclic GMP enhance the relaxation induced by cromakalim which is blocked by glibenclamide (a specific inhibitor of ATP-sensitive K+-channels [K(ATP)-channels]), and partially attenuated by methylene blue (an inhibitor of cyclic GMP formation). However, this is not due to the increase of cyclic GMP level by cromakalim itself because the relaxation induced by cromakalim is not associated with the changes of cyclic GMP level formed in the aortic smooth muscle. Thus, it is most likely that cyclic GMP also modulates activity of K(ATP)-channels, in addition to K(Ca)-channels, in the rat aorta.