Proteinase-activated receptors 1 and 2 in rat olfactory system: layer-specific regulation of multiple signaling pathways in the main olfactory bulb and induction of neurite retraction in olfactory sensory neurons.

Proteinase-activated receptors (PARs) are a family of four G protein-coupled receptors that are widely distributed in the CNS and involved in neural cell proliferation, differentiation and survival. The olfactory system undergoes continuous neurogenesis throughout life and may represent a critical target of PAR cellular actions. In the present study we investigated the functional activity of PAR1 and PAR2 in microdissected tissue preparations of olfactory nerve-glomerular layer (ON-GL), external plexiform layer (EPL) and granule cell layer (GRL) of the rat main olfactory bulb and in primary cultures of olfactory neuroepithelial cells. Activation of either PAR1 or PAR2 regulated multiple signaling pathways, including activation of pertussis-toxin sensitive Gi/o proteins, inhibition of cyclic AMP formation, stimulation of Gq/11-mediated phosphoinositide (PI) hydrolysis, phosphorylation of Ca2+/calmodulin-dependent protein kinase II and activation of the monomeric G protein Rho, predominantly in ON-GL, whereas only activation of Rho was detected in the deeper layers. Olfactory nerve lesion by nasal irrigation with ZnSO4 induced a marked decrease of PAR signaling in ON-GL. In primary cultures of olfactory neurons, double immunofluorescence analysis showed the localization of PAR1 and PAR2 in cells positive for olfactory-marker protein and neuron-specific enolase. Cell exposure to either nanomolar concentrations of thrombin and trypsin or PAR-activating peptides caused rapid neurite retraction. This study provides the first characterization of the laminar distribution of PAR1 and PAR2 signaling in rat olfactory bulb, demonstrates the presence of the receptors in olfactory sensory neurons and suggests a role of PARs in olfactory sensory neuron neuritogenesis.

Purification and characterization of high-affinity cyclic adenosine 5'-monophosphate phosphodiesterases from human acute myelogenous leukemic cells.

Soluble, high-affinity cyclic adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterases extracted from blast cells of patients with acute myelogenous leukemia have been characterized by physical, kinetic, and immunological criteria and fractionated to a high degree of purity. Procedures used in this study were similar to those used to purify the high-affinity enzyme from dog kidney. Two forms of high-affinity enzyme were found in blast cells. Form A was similar to the known type IV phosphodiesterases, including those of normal lymphocytes and monocytes. It showed a molecular weight near 60,000, a rate of hydrolysis of cyclic AMP 7 to 10 times that of cyclic guanosine 3':5'-monophosphate (cyclic GMP), competitive inhibition by cyclic GMP for cyclic AMP hydrolysis, and identical immunoreactivity by Western transfer analysis. This enzyme form was purified to apparent homogeneity by physical criteria but showed a low maximum velocity relative to other phosphodiesterase forms. A second, different form of high-affinity phosphodiesterase (Form B) was also resolved and partially purified. By comparison with Form A, this enzyme eluted from diethylaminoethyl cellulose at slightly lower ionic strength, had a lower molecular weight, appeared specific for cyclic AMP as substrate, showed no inhibition of cyclic AMP hydrolysis by cyclic GMP, and displayed no immunological cross-reactivity to the Mr 60,000 enzyme. Neither enzyme form was activated by calmodulin or proteolysis, whereas both showed comparable inhibition by 6,7-dimethoxy-1-veratrylisoquinoline, 1-methyl-3-isobutylxanthine, and 1,3-dimethylxanthine.

Regulation by a beta-adrenergic receptor of a Ca2+-independent adenosine 3',5'-(cyclic)monophosphate phosphodiesterase in C6 glioma cells.

The hormonal control of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity has been studied by using as a model the isoproterenol stimulation of cyclic AMP phosphodiesterase activity in C6 glioma cells. A 2-fold increase in cyclic AMP phosphodiesterase specific activity was observed in homogenates of isoproterenol-treated cells relative to control. This increase reached a maximum 3 h after addition of isoproterenol, was selective for cyclic AMP hydrolysis, was reproduced by incubation with 8-Br cycl AMP but not with 8-Br cyclic GMP and was limited to the soluble enzyme activity. The presence of 0.1 mM EGTA did not alter the magnitude of the increase in phosphodiesterase activity. Moreover, the calmodulin content in the cell extracts was not changed after isoproterenol. DEAE-Sephacel chromatography of the 100000 X g supernatant resolved two peaks of phosphodiesterase activity. The first peak hydrolyzed both cyclic nucleotides and was activated by Ca2+ an purified calmodulin. The second peak was specific for cyclic AMP but it was Ca2+- and calmodulin-insensitive. Isoproterenol selectively increased the specific activity of the second peak. Kinetic analysis of the cyclic AMP hydrolysis by the induced enzyme revealed a non-linear Hofstee plot with apparent Km values of 2-5 microM. Cyclic GMP was not hydrolyzed by this enzyme in the absence or presence of calmodulin and failed to affect the kinetics of the hydrolysis of cyclic AMP. Gel filtration chromatography of the induced DEAE-Sephacel peak resolved a single peak of enzyme activity with an apparent molecular weight of 54000.

Apomorphine hydrochloride-induced improvement in Huntington's chorea: stimulation of dopamine receptor.

Four patients affected by Huntington's chorea (HC) with a well defined family history of the disease were injected intramuscularly with apomorphine hydrochloride in nonemetic doses, ranging from 1 to 4 mg. Soon after treatment, all patients showed a marked decrease in abnormal involuntary movements. Pretreatment with haloperidol (2 mg intramuscularly) or sulpiride (100 mg intramuscularly) 30 minutes prior to apomorphine treatment, prevented the therapeutic effect of this compound. It is suggested that apomorphine-induced improvement in Huntington's Chorea is mediated by the stimulation of a special kind of dopamine receptor, leading to inhibition of the activity of dopaminergic neurons.

Snake toxins with high selectivity for subtypes of muscarinic acetylcholine receptors.

There are five subtypes of muscarinic acetylcholine receptors (M(1) to M(5)) which control a large number of physiological processes, such as the function of heart and smooth muscles, glandular secretion, release of neurotransmitters, gene expression and cognitive functions as learning and memory. A selective ligand is very useful for studying the function of a subtype in presence of other subtypes, which is the most common situation, since a cell or an organ usually has several subtypes. There are many non-selective muscarinic ligands, but only few selective ones. Mambas, African snakes of genus Dendroaspis have toxins, muscarinic toxins, that are selective for M(1), M(2) and M(4) receptors. They consist of 63-66 amino acids and four disulfides which form four loops. They are members of a large group of snake toxins, three-finger toxins; three loops are extended like the middle fingers of a hand and the disulfides and the shortest loop are in the palm of the hand. Some of the toxins target the allosteric site which is located in a cleft of the receptor molecule close to its extracellular part. A possible explanation to the good selectivity is that the toxins bind to the allosteric site, but because of their size they probably also bind to extracellular parts of the receptors which are rather different in the various subtypes. Some other allosteric ligands also have good selectivity, the alkaloid brucine and derivatives are selective for M(1), M(3) and M(4) receptors. Muscarinic toxins have been used in several types of experiments. For instance radioactively labeled M(1) and M(4) selective toxins were used in autoradiography of hippocampus from Alzheimer patients. One significant change in the receptor content was detected in one region of the hippocampus, dentate gyrus, where M(4) receptors were reduced by 50% in patients as compared to age-matched controls. Hippocampus is essential for memory consolidation. M(4) receptors in dentate gyrus may play a role, since they decreased in Alzheimers disease which destroys the memory. Another indication of the role of M(4) receptors for memory is that injection of the M(4) selective antagonist muscarinic toxin 3 (M(4)-toxin 1) into rat hippocampus produced amnesia.

Mixed agonist-antagonist properties of clozapine at different human cloned muscarinic receptor subtypes expressed in Chinese hamster ovary cells.

We recently reported that clozapine behaves as a partial agonist at the cloned human m4 muscarinic receptor subtype. In the present study, we investigated whether the drug could elicit similar effects at the cloned human m1, m2, and m3 muscarinic receptor subtypes expressed in the Chinese hamster ovary (CHO) cells. Clozapine elicited a concentration-dependent stimulation of [3H]inositol phosphates accumulation in CHO cells expressing either the m1 or the m3 receptor subtype. Moreover, clozapine inhibited forskolin-stimulated cyclic AMP accumulation and enhanced [35S] GTP gamma S binding to membrane G proteins in CHO cells expressing the m2 receptor. These agonist effects of clozapine were antagonized by atropine. The intrinsic activity of clozapine was lower than that of the full cholinergic agonist carbachol, and, when the compounds were combined, clozapine potently reduced the receptor responses to carbachol. These data indicate that clozapine behaves as a partial agonist at different muscarinic receptor subtypes and may provide new hints for understanding the receptor mechanisms underlying the antipsychotic efficacy of the drug.

GABA(B) receptor-mediated stimulation of adenylyl cyclase activity in membranes of rat olfactory bulb.

Previous studies have shown that GABA(B) receptors facilitate cyclic AMP formation in brain slices likely through an indirect mechanism involving intracellular second messengers. In the present study, we have investigated whether a positive coupling of GABA(B) receptors to adenylyl cyclase could be detected in a cell-free preparation of rat olfactory bulb, a brain region where other Gi/Go-coupled neurotransmitter receptors have been found to stimulate the cyclase activity. The GABA(B) receptor agonist (-)-baclofen significantly increased basal adenylyl cyclase activity in membranes of the granule cell and external plexiform layers, but not in the olfactory nerve-glomerular layer. The adenylyl cyclase stimulation was therefore examined in granule cell layer membranes. The (-)-baclofen stimulation (pD2=4.53) was mimicked by 3-aminopropylphosphinic acid (pD2=4.60) and GABA (pD2=3.56), but not by (+)-baclofen, 3-aminopropylphosphonic acid, muscimol and isoguvacine. The stimulatory effect was counteracted by the GABA(B) receptor antagonists CGP 35348 (pA2=4.31), CGP 55845 A (pA2=7.0) and 2-hydroxysaclofen (pKi=4.22). Phaclofen (1 mM) was inactive. The (-)-baclofen stimulation was not affected by quinacrine, indomethacin, nordihydroguaiaretic acid and staurosporine, but was completely prevented by pertussis toxin and significantly reduced by the alpha subunit of transducin, a betagamma scavenger. The betagamma subunits of transducin stimulated the cyclase activity and this effect was not additive with that produced by (-)-baclofen. In the external plexiform and granule cell layers, but not in the olfactory nerve-glomerular layer, (-)-baclofen enhanced the adenylyl cyclase stimulation elicited by the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) 38. Conversely, the adenylyl cyclase activity stimulated by either forskolin or Ca2+/calmodulin-(Ca2+/CaM) was inhibited by (-)-baclofen in all the olfactory bulb layers examined. These data demonstrate that in specific layers of rat olfactory bulb activation of GABA(B) receptors enhances basal and neurotransmitter-stimulated adenylyl cyclase activities by a mechanism involving betagamma subunits of Gi/Go. This positive coupling is associated with a widespread inhibitory effect on forskolin- and Ca2+/CaM-stimulated cyclic AMP formation.

Antagonism of striatal muscarinic receptors inhibiting dopamine D1 receptor-stimulated adenylyl cyclase activity by cholinoceptor antagonist used to treat Parkinson's disease.

A number of cholinoceptor antagonists used in the treatment of Parkinson's disease were examined for their ability to antagonize either the muscarinic receptor-mediated inhibition of dopamine D1 receptor-stimulated adenylyl cyclase or the muscarinic receptor-mediated stimulation of [3H]-inositol phosphates ([3H]-IPs) formation in rat striatal membranes. The drugs were found to block the receptors inhibiting adenylyl cyclase activation with high affinity and more potently than those stimulating [3H]-IPs formation. Moreover, their rank order of potencies for the former effect showed good correlation with their clinical efficacies. These data suggest that the blockade of the muscarinic receptor-mediated inhibition of striatal dopamine D1 receptor activation may be one of the mechanisms by which cholinoceptor blocking drugs exert their antiparkinsonian effect.

Effects of clozapine on rat striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity and on the human cloned m4 receptor.

1. Clozapine has recently been claimed to behave as a selective and full agonist at the cloned m4 muscarinic receptor artificially expressed in Chinese hamster ovary (CHO) cells. In the present study we have investigated whether clozapine could activate the rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity, considered as pharmacologically equivalent to the m4 gene product. In addition, we have examined the effect of the drug on various functional responses following the activation of the cloned m4 receptor expressed in CHO cells. 2. In rat striatum, clozapine (1 nM-10 microM) caused a slight inhibition of forskolin-stimulated adenylyl cyclase activity, which was not counteracted by 10 microM atropine. On the other hand, clozapine antagonized the inhibitory effect of acetylcholine with a pA2 value of 7.51. Moreover, clozapine (1 microM) failed to inhibit dopamine D1 receptor stimulation of adenylyl cyclase activity, but counteracted the inhibitory effect of carbachol (CCh). Clozapine displaced [3H]-N-methylscopolamine ([3H]-NMS) bound to striatal M4 receptors with a monophasic inhibitory curve and a pKi value of 7.69. The clozapine inhibition was not affected by the addition of guanosine-5'-O-(thio)triphosphate (GTPgammaS). 3. In intact CHO cells, clozapine inhibited forskolin-stimulated cyclic AMP accumulation with an EC50 of 31 nM. This effect was antagonized by atropine. CCh produced a biphasic effect on cyclic AMP levels, inhibiting at concentrations up to 1 microM (EC50=50 nM) and stimulating at higher concentrations (EC50 = 7 microM). Clozapine (0.3-5 microM) antagonized the CCh stimulation of cyclic AMP with a pKi value of 7.47. Similar results were obtained when the adenylyl cyclase activity was assayed in CHO cell membranes. 4. In CHO cells pretreated with the receptor alkylating agent 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (10 microM), the maximal inhibitory effect of clozapine on cyclic AMP formation was markedly reduced, whereas the CCh inhibitory curve was shifted to the right with no change in the maximum. 5. As in rat striatum, in CHO cell membranes the displacement of [3H]-NMS binding by clozapine yielded a monophasic curve which was not affected by GTPgammaS. 6. Clozapine (10 nM-10 microM) had a small stimulant effect (approximately 20%) on the binding of [35S]-GTPgammaS to CHO cell membranes, whereas CCh caused a 250% increase of radioligand binding. Moreover, clozapine (50 nM-5 microM) antagonized the CCh-stimulated [35S]-GTPgammaS binding with a pA2 value of 7.48. 7. These results show that at the striatal M4 receptors clozapine is a potent and competitive antagonist, whereas at the cloned m4 receptor it elicits both agonist and antagonist effects. Thus, clozapine behaves as a partial agonist, rather than as a full agonist, at the m4 receptor subtype, with intrinsic activity changing as a function of the coupling efficiency of the receptor to effector molecules.

Antagonism by (R)- and (S)-trihexyphenidyl of muscarinic stimulation of adenylyl cyclase in rat olfactory bulb and inhibition in striatum and heart.

1. Activation of muscarinic receptors in rat olfactory bulb stimulates adenylyl cyclase activity. This response was competitively antagonized by the (R)- and (S)-enantiomers of trihexyphenidyl with pA2 values of 8.84 and 6.09, respectively. 2. Similarly, in rat striatal homogenates, muscarinic inhibition of adenylyl cyclase activity was antagonized by the (R)- and (S)-enantiomers with pA2 values of 8.75 and 6.12, respectively. 3. In contrast, in rat myocardium the muscarinic inhibition of the adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation was more weakly antagonized by trihexyphenidyl, with a particularly marked loss (15 fold) in activity of the (R)-enantiomer. The (R)- and (S)-enantiomers had pA2 values of 7.64 and 5.72, respectively. 4. Each muscarinic response was completely antagonized by increasing concentrations of (R)-trihexyphenidyl with a Hill coefficient not significantly different from unity. 5. The present study shows that the muscarinic receptors coupled to stimulation of adenylyl cyclase in the olfactory bulb display high stereoselectivity for the enantiomers of trihexyphenidyl. The affinities of these receptors for the antagonists are similar to those shown by the striatal receptors. This finding supports the hypothesis that both the muscarinic stimulation of adenylyl cyclase in the olfactory bulb and the muscarinic inhibition of the enzyme in striatum are mediated by activation of a receptor subtype pharmacologically equivalent to the m4 gene product. On the other hand, the weaker affinities and the lower stereoselectivity for the trihexyphenidyl enantiomers exhibited by the muscarinic inhibition of adenylyl cyclase in the heart are consistent with the involvement of M2 receptors in this response.

Rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity: potent block by the selective m4 ligand muscarinic toxin 3 (MT3).

1. In rat striatal membranes, muscarinic toxin 3 (MT3), a selective ligand of the cloned m4 receptor subtype, antagonized the acetylcholine (ACh) inhibition of forskolin- and dopamine D1 receptor-stimulated adenylyl cyclase activities with pA2 values of 8.09 and 8.15, respectively. 2. In radioligand binding experiments, MT3 increased the Kd but did not change the Bmax value of [3H]-N-methylscopolamine (3H]-NMS) binding to rat striatal muscarinic receptors. The toxin displaced the major portion of the [3H]-NMS binding sites with a Ki of 8.0 nM. 3. In rat myocardium, MT3 antagonized the ACh inhibition of adenylyl cyclase with a Ki value of 860 nM. 4. In rat cerebral cortical membranes prelabelled with [3H]-myo-inositol, MT3 counteracted the methacholine stimulation of [3H]-inositol phosphates formation with a Ki value of 113 nM. 5. The present study shows that MT3 is a potent antagonist of the striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity. This finding provides strong evidence for the classification of these receptors as pharmacologically equivalent to the m4 gene product (M4). On the other hand, the weaker potencies of MT3 in antagonizing the muscarinic responses in cerebral cortex and in the heart are consistent with the reported lower affinities of the toxin for the cloned m1 and m2 receptor subtypes, respectively.

Inhibition of acetylcholine muscarinic M(1) receptor function by the M(1)-selective ligand muscarinic toxin 7 (MT-7).

MT-7 (1 - 30 nM), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M(1) receptor, inhibited the acetylcholine (ACh)-stimulated [(35)S]-guanosine-5'-O-(3-thio)triphosphate ([(35)S]-GTPgammaS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M(1) receptor subtype. MT-7 failed to affect the ACh-stimulated [(35)S]-GTPgammaS binding in membranes of CHO cells expressing either the M(2), M(3) or M(4) receptor subtype. In N1E-115 neuroblastoma cells endogenously expressing the M(1) and M(4) receptor subtypes, MT-7 (0.3 - 3.0 nM) inhibited the carbachol (CCh)-stimulated inositol phosphates accumulation, but failed to affect the CCh-induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38-stimulated cyclic AMP accumulation. In both CHO/M(1) and N1E-115 cells the MT-7 inhibition consisted in a decrease of the maximal agonist effect with minimal changes in the agonist EC(50) value. In CHO/M(1) cell membranes, MT-7 (0.05 - 25 nM) reduced the specific binding of 0.05, 1.0 and 15 nM [(3)H]-N-methylscopolamine ([(3)H]-NMS) in a concentration-dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT-7 (3 nM) decreased the dissociation rate of [(3)H]-NMS by about 5 fold. CHO/M(1) cell membranes preincubated with MT-7 (10 nM) and washed by centrifugation and resuspension did not recover control [(3)H]-NMS binding for at least 8 h at 30 degrees C. It is concluded that MT-7 acts as a selective noncompetitive antagonist of the muscarinic M(1) receptors by binding stably to an allosteric site.

Pharmacological and biochemical characterization of dopamine receptors mediating stimulation of a high affinity GTPase in rat striatum.

In synaptic plasma membranes of rat striatum, activation of dopamine receptors stimulates a high affinity GTPase activity. The rank order of potency of various dopamine receptor agonists in increasing GTP hydrolysis is the following: (-)-propylnorapomorphine greater than (-)-apomorphine = (+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene [(+/-)-A-6,7-DTN] greater than dopamine = LY 171555 greater than noradrenaline. The selective D-1 dopamine receptor agonist, SKF 38393, does not produce a significant increase in GTP hydrolysis. Moreover, the dopamine-stimulated GTPase activity is completely reversed by the D-2 receptor antagonists, 1-sulpiride and zetidoline, but not by the selective D-1 antagonist SCH 23390. Na+ modulates the dopamine receptor-regulated GTP hydrolysis by increasing the percentage of stimulation and decreasing the agonist potency. Intrastriatal injection of pertussis toxin, which impairs the function of the inhibitory guanine nucleotide binding regulatory protein (Ni) of adenylate cyclase, significantly reduces the dopamine stimulation of striatal GTPase activity and the dopamine inhibition of adenylate cyclase. In contrast, cholera toxin, which blocks the stimulation of GTPase activity by hormones which increase adenylate cyclase activity, does not modify the dopamine-stimulated GTPase activity. These data indicate that the stimulation of GTPase activity elicited by dopamine results from activation of the D-2 type of dopamine receptors and is expression of the increased turnover of GTP at the level of Ni. The results are consistent with the idea that Ni is involved in the inhibitory coupling of striatal D-2 receptors to adenylate cyclase.

Beta gamma-mediated enhancement of corticotropin-releasing hormone-stimulated adenylyl cyclase activity by activation of gamma-aminobutyric acid(B) receptors in membranes of rat frontal cortex.

A number of studies have shown that activation of gamma-aminobutyric acid(B) (GABA(B)) receptors potentiates neurotransmitter-induced accumulation of cyclic AMP in brain slices, but the mechanisms involved in the facilitatory effect have not been fully elucidated. In the present study, we showed that in membranes of rat frontal cortex the GABA(B) receptor agonist (-)baclofen increased basal adenylyl cyclase activity and potentiated the maximal enzyme stimulation elicited by corticotropin-releasing hormone (CRH). The less active enantiomer (+)baclofen had no effect on cyclic AMP formation, whereas the natural agonist GABA mimicked the stimulatory action of (-)baclofen. In radioligand-binding experiments, the affinity and maximal binding capacity of (125)I-Tyr-CRH was not affected by (-)baclofen. The GABA(B) receptor antagonist CGP 55845A competitively counteracted the (-)baclofen potentiation of CRH-stimulated adenylyl cyclase activity with a pA(2) value of 6.70. Moreover, both (-)baclofen and GABA, but not (+)baclofen, caused a concentration-dependent stimulation of [(35)S]GTP gamma S binding to membrane G-proteins. The intracerebral injection of pertussis toxin significantly reduced the facilitatory effects of (-)baclofen on both basal and CRH-stimulated adenylyl cyclase activities. Moreover, membrane incubation with the GDP-bound form of the alpha subunit of transducin, a scavenger of G protein beta gamma subunits, blocked the stimulatory effects of (-)baclofen. The data indicate that in rat frontal cortex activation of GABA(B) receptors potentiates the CRH stimulation of adenylyl cyclase activity through a mechanism involving the beta gamma subunits of the pertussis toxin-sensitive G protein G(i)/G(o).

Stimulation of phosphoinositide hydrolysis by muscarinic receptor activation in the rat olfactory bulb.

The effect of muscarinic receptor activation on phosphoinositide hydrolysis in the rat olfactory bulb was investigated by determining either the inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) mass or the accumulation of [3H]inositol phosphates ([3H]InsPs). In miniprisms of rat olfactory bulb, carbachol produced an atropine-sensitive increase in Ins(1,4,5)P3 concentration. In a membrane preparation, the formation of Ins(1,4,5)P3 was stimulated by guanosine-5'-(3-O-thio) triphosphate (GTP gamma S), but not by carbachol. However, carbachol potentiated the GTP gamma S stimulation when the two agents were combined. In miniprisms prelabelled with [3H]myo-inositol, carbachol increased the accumulation of [3H]InsPs and this effect was significantly reduced by tissue treatment with either 1 microM phorbol 12-myristate 13-acetate or 1 mM dibutyryl cyclic AMP. Analysis of concentration-response curves indicated that carbachol (EC50 = 96 microM) and oxotremorine-M (EC50 = 8.2 microM) behaved like full agonists, whereas oxotremorine, BM5, arecoline and bethanechol were partial agonists. The carbachol stimulation of [3H]InsPs accumulation was counteracted with high affinity by the M1 antagonist pirenzepine (pA2 = 8.26), and less potently by the M3 antagonist para-fluorohexahydro-sila-difenidol (pA2 = 6.7) and the M2 antagonist AF-DX 116 (pA2 = 6.12). The biochemical and pharmacological properties of the muscarinic stimulation of phosphoinositide hydrolysis were compared with those displayed by the muscarinic stimulation of adenylate cyclase in the rat olfactory bulb.

Mediation by G protein betagamma subunits of the opioid stimulation of adenylyl cyclase activity in rat olfactory bulb.

In the rat olfactory bulb, activation of opioid receptors enhances basal adenylyl cyclase (EC 4.6.1.1) activity and potentiates enzyme stimulation by Gs-coupled neurotransmitter receptors in a pertussis toxin-sensitive manner. In the present study, we investigated the involvement of G protein betagamma subunits by examining the effects of betagamma scavengers and exogenously added betagamma subunits of transducin (betagamma(t)). The QEHA fragment of type II adenylyl cyclase (50 microM), a peptide that binds to and inactivates betagamma, inhibited the maximal stimulation of adenylyl cyclase activity elicited by Leu-enkephalin (Leu-enk) by about 50%. Similarly, the GDP-bound form of the alpha subunit of transducin (5 nM-1.5 microM), another betagamma scavenger, reduced both the opioid stimulation of basal adenylyl cyclase activity and the potentiation of vasoactive intestinal peptide-stimulated enzyme activity. Under the same experimental conditions, these agents failed to affect the stimulation of the enzyme activity elicited by activation of beta-adrenergic receptors with 1-isoproterenol. Moreover, the addition of betagamma(t)(400 nM) stimulated basal adenylyl cyclase by 80%, and this effect was not additive with that produced by Leu-enk. The data indicate that opioids enhance adenylyl cyclase activity in rat olfactory bulb by promoting the release of betagamma subunits from pertussis toxin-sensitive G proteins Gi/Go.

Presynaptic dopamine autoreceptors and second messengers controlling tyrosine hydroxylase activity in rat brain.

In brain areas enriched of dopaminergic nerve terminals presynaptic dopamine (DA) autoreceptors control the state of activation of tyrosine hydroxylase (TH) by regulating the extent of phosphorylation of the enzyme. Evidence is presented indicating that this autoinhibitory control may involve a decrease in the cyclic AMP-dependent activation of TH through an inhibitory coupling of presynaptic DA autoreceptors to adenylate cyclase. As indicated by the insensitivity of the DA inhibition of TH to changes in the extracellular concentrations of Ca++, to the addition of the Ca++ ionophore A 23187 and of different K+ channel blockers, a reduction of Ca++ influx and an increase in the K+ channel activity do not seem to be involved in the presynaptic regulation of TH activity by DA autoreceptors at least under basal conditions.

[Phe1phi(CH2-NH)Gly2]nociceptin-(1-13)-NH2 acts as a partial agonist at ORL1 receptor endogenously expressed in mouse N1E-115 neuroblastoma cells.

The nociceptin derivative [Phe1phi(CH2-NH)Gly2]-nociceptin-(1-13)-NH2 (Phe(phi)noc) has been reported to act either as a simple antagonist or as a full agonist at the opioid receptor-like (ORL1) receptor. In the present study, we identified the expression of the ORL1 receptor in murine N1E-115 neuroblastoma cells and used this neuronal system to investigate the pharmacological activity of Phe(phi)noc. Like nociceptin, Phe(phi)noc stimulated the binding of [35S]GTPgammaS (EC50 = 120 nM) and inhibited forskolin-stimulated [3H]cAMP formation (EC50 = 3.3 nM). However, Phe(phi)noc elicited maximal effects lower than those induced by nociceptin, and when combined with nociceptin potently antagonized the responses to the natural agonist (Ki = 0.9 nM). These data indicate that Phe(phi)noc acts as a partial agonist at the ORL1 receptor endogenously expressed in N1E-115 cells.

G protein-coupled corticotropin-releasing hormone receptors in rat retina.

The presence of corticotropin-releasing hormone (CRH) receptors in rat retinal membranes was investigated by using [125I-Tyro]-ovine CRH ([125I]oCRH) as radioligand. The receptor binding was rapid, reversible, saturable and specific. The [125I]oCRH binding was completely displaced by different CRH-related peptides with a rank order of potency similar to that displayed in stimulating rat retinal adenylyl cyclase activity. Two populations of binding sites were detected: one with high affinity (Kd = 1.7 nM) and the other with low-affinity (Kd = 130 nM). The GTP analogue guanosine 5'-O-(3'-thiotriphosphate) reduced the high-affinity binding and increased the relative proportion of sites with low-affinity. Incubation of rat retinal membranes with the RM/1 antibody, which recognizes the carboxyl-terminus of the alpha subunit of the G protein Gs, prevented the CRH stimulation of adenylyl cyclase. In immunoblots, the RM/1 antibody recognized an immunoreactive protein band of 45 kDa and a protein with a similar electrophoretic mobility was ADP-ribosylated by cholera toxin. These data provide evidence for the presence of specific CRH receptors in rat retina and contribute to define the CRH signalling system in this tissue.

Corticotropin-releasing hormone stimulates adenylyl cyclase activity in the retinas of different animal species.

In the present study we investigated the presence of corticotropin-releasing hormone (CRH)-stimulated adenylyl cyclase activity in the retinas of different animal species. CRH significantly stimulated adenylyl cyclase activity in homogenates of calf, pig, rabbit and guinea pig retinas. The stimulatory effects were concentration-dependent with half-maximal responses occurring at 20-30 nM CRH. The enzyme activities increased by 37-80% at the maximal concentration of CRH (1 microM). On the other hand, adenylyl cyclase activities of chicken and pigeon retinas were poorly stimulated by CRH. In calf, pig and rabbit retinas, the CRH effect was completely antagonized by the CRH receptor antagonist alpha-helical CRH 9-41 and required the presence of GTP. The stimulatory response elicited by CRH was also found to be not additive with that produced by either vasoactive intestinal peptide or dopamine. These results provide evidence for the presence in retinas of different animal species of functional CRH receptors, an important criterion for the classification of CRH as a retinal neurotransmitter.