Muscimol increases acetylcholine release by directly stimulating adult striatal cholinergic interneurons

Because GabaA ligands increase acetylcholine (ACh) release from adult striatal slices, we hypothesized that activation of GabaA receptors on striatal cholinergic interneurons directly stimulates ACh secretion. Fractional [³H]ACh release was recorded during perifusion of acutely dissociated, [³H]choline-labeled, adult male rat striata. The GabaA agonist, muscimol, immediately stimulated release maximally 300% with EC₅₀=1 μM. This action was enhanced by the allosteric GabaA receptor modulators, diazepam and secobarbital, and inhibited by the GabaA antagonist, bicuculline, by ligands for D2 or muscarinic cholinergic receptors or by low calcium buffer, tetrodotoxin or vesamicol. Membrane depolarization inversely regulated muscimol-stimulated secretion. Release of endogenous and newly synthesized ACh was stimulated in parallel by muscimol without changing choline release. Muscimol pretreatment inhibited release evoked by K⁺ depolarization or by receptor-mediated stimulation with glutamate. Thus, GabaA receptors on adult striatal cholinergic interneurons directly stimulate voltage- and calcium-dependent exocytosis of ACh stored in vesamicol-sensitive synaptic vesicles. The action depends on the state of membrane polarization and apparently depolarizes the membrane in turn. This functional assay demonstrates that excitatory GabaA actions are not limited to neonatal tissues. GabaA-stimulated ACh release may be prevented in situ by normal tonic dopaminergic and muscarinic input to cholinergic neurons.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates melatonin synthesis from rat pineal gland

The effect of pituitary adenylate cyclase polypeptide (PACAP) on rat pineal was examined. PACAP stimulated melatonin release from cultured dissociated pinealocytes with a 10⁴ higher potency than isoproterenol (EC₅₀ were 30 pM and 250 nM, respectively). The 10⁻⁹ M PACAP stimulation was not inhibited by 5 × 10⁻⁶ M VIP antagonist whereas that of 10⁻⁹ M VIP was reduced by 54%. Kinetic analysis of melatonin release indicated that PACAP acts postsynaptically via receptor activation.     

Characteristics of dopamine and β-adrenergic sensitive adenylate cyclases in the frontal cerebral cortex of the rat. Comparative effects of neuroleptics on frontal cortex and striatal dopamine sensitive adenylate cyclases

Homogenates of frontal cerebral cortex of the rat were prepared from microdiscs punched out in areas rich in dopaminergic terminals. Under optimal assay conditions, dopamine (10^?4M) stimulated an adenylate cyclase present in these homogenates by 80–100%. This stimulation reached 200% when microdiscs were punched out from the medial part of the frontal cerebral cortex, adjacent to the forceps minor.Dopamine interacted with an homogeneous population of receptor sites which had an apparent affinity (K_D) of 3.8 ± 0.9 × 10^?6M (N= 4). The dopamine receptor was blocked by fluphenazine and phentolamine but had no affinity for pindolol, propranolol orl-isoproterenol.The affinities of several neuroleptics having different chemical structures were simultaneously determined on striatal and on frontal cerebral cortex dopamine sensitive adenylate cyclases. Fluphenazine was more potent in blocking the striatal than the frontal cerebral cortex dopaminergic receptors. In contrast, in all experiments, haloperidol had an higher affinity for the cerebral frontal cortex than for the striatal dopaminergic receptors. Thus, haloperidol was less effective than fluphenazine in blocking the striatal dopaminergic receptors, and equally potent than fluphanazine in inhibiting the frontal cerebral cortex dopamine sensitive adenylate cyclase. Chlorpromazine, thioridazine and clozapine had the same affinity for the two dopaminergic adenylate cyclase systems.l-Isoproterenol interacted with an homogeneous population of β-adrenergic receptor sites (K_D = 3 ± 2 × 10^?7M; N = 4) coupled with adenylate cyclase distinct from the dopamine sensitive adenylate cyclase. This β-receptor had no affinity for dopamine or fluphenazine but was blocked by propranolol or pindolol.l-Norepinephrine was shown to stimulate both the dopamine (K_D = 1.8 ± 1 × 10^?5;N =4 and the β-adrenergic (K_D = 8 ± 3 × 10^?7M; N = 4) senstive adenylate cyclases. Thus, thel-norepinephrine effect was totally blocke in the combined presence of fluphenazine and pindolol.     

Regulation of Melatonin Release and N-Acetyltransferase Activity in Ovine Pineal Cells

A suspension culture of ovine pineal cells was developed to investigate the regulation of melatonin synthesis and release. Dosedependent stimulation of melatonin release by a series of adrenergic agonists yielded a typical β-adrenergic profile. Serotonin N-acetyltransferase (NAT), the rate-limiting enzyme in melatonin biosynthesis, was also stimulated by a β-adrenergic mechanism. However, NAT activity appeared less sensitive than melatonin release to β-adrenergic stimulation. No evidence was obtained for a contribution of α₁-adrenergic receptors to the regulation of NAT activity and melatonin release. Activation of adenylate cyclase by forskolin or addition of a cyclic AMP analogue increased both melatonin release and NAT activity. In contrast, the Ca²⁺ ionophore A₂₃₁₈₇ stimulated melatonin release without a detectable increase in NAT activity. Together, the present data argue for a β-adrenergic regulation of both NAT activity and melatonin release in ovine pinealocytes. The evidence also suggests that two intracellular mechanisms may control melatonin release in ovine pinealocytes: a cyclic AMP-dependent mechanism, associated with an increase in NAT activity and a Ca²⁺-dependent mechanism, independent of NAT activity.     

Peptidergic modulation of G-protein coupled cyclic-AMP accumulation in the rat caudate nucleus.

Somatostatin, substance P, and vasoactive intestinal polypeptide were incubated in an adenylate cyclase assay with a particulate fraction of caudate-putamen tissue of the rat in order to examine the effect of the neuropeptides on G-protein coupled adenylate cyclase in vitro. Somatostatin induced an enhancement of cyclic AMP formation in presence of guanine nucleotides and cholera toxin but inhibited pertussis toxin and forskolin enzyme stimulation. Pertussis toxin and cholera toxin also depressed forskolin-induced stimulation as described previously. Somatostatin was able to antagonize these inhibitory effects of both toxins. On the contrary, substance P reduced GTP and cholera toxin stimulated striatal adenylate cyclase, without affecting forskolin activation. In our preparation, VIP did not influence basal adenylate cyclase activity or the stimulation by guanine nucleotides, cholera toxin, and pertussis toxin. VIP potently inhibited the enhancement of cyclic AMP formation by forskolin and completely antagonized the inhibitory effect of cholera toxin on forskolin activation. These results suggest that neuromodulatory effects of somatostatin, substance P, and VIP are mediated by the inhibitory as well as stimulatory guanine nucleotide proteins G-i and G-s coupled to an adenylate cyclase system.     

D2 dopamine receptor activation inhibits basal and forskolin-evoked acetylcholine release from dissociated striatal cholinergic interneurons

We tested whether D2 ligands inhibit basal and forskolin-stimulated [³H]ACh release from dissociated striata, as opposed to striatal slices. Quinpirole inhibited both basal (40% maximal inhibition; IC₅₀ ≈ 50 nM) and 10 μM forskolin-stimulated release (80% inhibition; IC₅₀≈25nM quinpirole) and both actions were blocked by a D2 antagonist. Vesamicol prevented the quinpirole and forskolin actions. The ability of D2 agonists to inhibit basal and cyclase-stimulated acetylcholine release emanating from vesamicol-sensitive vesicles appears to be tonically suppressed by inhibitory elements within striatal circuitry.     

Characterization of the excitatory amino acid receptor-mediated release of [H]acetylcholine from rat striatal slices

The pharmacological nature of the interaction of excitatory amino acids with striatal cholinergic neurons was investigated in vitro. Agonists of excitatory amino acid receptors evoked the release of [³H]acetylcholine from slices of rat striatum, in the presence of magnesium (1.2 mM). Removal of magnesium from the medium markedly increased the release of [³H]acetylcholine evoked by all excitatory amino acid receptor agonists tested, with the exception of kainate. In the absence but not the presence of magnesium, a clear rank order of potency was found: N-methyl-dl-aspartate = ibotenate >l-glutamate >l-aspartate cysteate > kainate = quisqualate.The excitatory amino acid receptor mediating [³H]acetylcholine release resembles the N-methyl-d-aspartate preferring (N-type) receptor, as previously characterized electrophysiologically, according to 3 criteria: (1) rank order of potency of agonists; (2) magnesium-sensitivity; and (3) antagonism by 2-amino-5-phosphonovalerate.The release of [³H]acetylcholine evoked by N-methyl-dl-aspartate was blocked by tetrodotoxin (0.5 μM). Moreover, N-methyl-dl-aspartate failed to evoke [³H]acetylcholine release from slices of hippocampus, where cholinergic afferents, rather than interneurons, are found. These results suggest that excitatory amino acids act at receptors on the dendrites of striatal cholinergic interneurons, giving rise to action potentials and release of acetylcholine from cholinergic nerve terminals.     

No alterations in the 5-HT1A-mediated inhibition of forskolin-stimulated adenylate cyclase activity in the hippocampal membranes from rats chronically treated with lithium or antidepressants

Summary In order to determine the relevance of 5-HT_1A-related signal transduction in the mode of action of lithium and antidepressants, the effects of long-term treatment with these drugs on the 5-HT_1A-mediated inhibition of forskolin-stimulated adenylate cyclase activity were investigated in the rat hippocampal membranes. Chronic administration of antidepressants altered neither the [³H]8-hydroxy-2-(di-n-propylamino)tetralin ([³H]8-OH-DPAT) binding sites nor the inhibition of forskolin-stimulated adenylate cyclase activity by 5-HT. Long-term treatment with lithium did not affect the inhibitory effect of 5-HT on the forskolin-stimulated adenylate cyclase activity, either. Neither the stimulation by forskolin nor the inhibition by guanyl-5-ylimidodiphosphate (Gpp(NH)p) of adenylate cyclase activity was not influenced by lithium treatment, suggesting that lithium has no effects on the components of adenylate cyclase system distal to the 5-HT_1A receptors.These results indicate that the 5-HT_1A-mediated neural transmission has not such an important relevance in the mechanisms of action of lithium or antidepressants.     

α2-adrenoceptor-GTP binding regulatory protein-adenylate cyclase system in cerebral cortical membranes of adult and senescent rats

The characterization of [³H]clonidine binding and effects of GTP, forskolin, islet-activating protein (IAP) and cholera toxin on adenylate cyclase activity were investigated in cerebral cortical membranes from 70-day-old and 2-year-old rats. Neither K_d nor B_max values in [³H]clonidine binding were changed between day 70 and year 2. The activation of adenylate cyclase by forskolin was significantly higher in senescent than in adult animals. The inhibitory effect of adrenaline, which was completely abolished by the pretreatment with IAP/NAD on forskolin/GTP-stimulated cyclase activity, was low in senescent rats compared to that in adult ones. The stimulatory effect of cholera toxin/NAD was also low at the senescent stage compared to that at the adult stage. It is suggested that ligand binding affinity and the density in α₂-adrenoceptors do not change between day 70 and year 2 but that GTP binding and/or coupling activity of inhibitory as well as stimulatory GTP binding regulatory protein to catylytic unit decrease in synaptic membranes of 2-year-old compared to those of 70-day-old rat brain.     

Involvement of cholinergic neurons in the release of dopamine elicited by stimulation of μ-opioid receptors in striatum

The involvement of striatal cholinergic neurons in the release of dopamine (DA) elicited by the (μ-opioid receptor agonist DAGO [d-Ala², NMePhe⁴-Gly⁵(ol)]enkephalin) was explored. The striatal release of DA was measured by microdialysis in rats anesthetized with chloral hydrate. When infused in the striatum, through the microdialysis probe, DAGO increased the extracellular levels of DA. The previous injection in striatum of AF 64-A, a toxin for cholinergic neurons, or the concomitant infusion of the M₂-muscarinic antagonist methoctramine abolished the effect of DAGO on the DA release. It is concluded that stimulation of μ-opioid receptors, by inhibiting the acetylcholine release which stimulates tonically M₂-muscarinic receptors likely associated with dopaminergic nerve endings, indirectly increases the striatal DA release.     

Differential changes in presynaptic modulation of transmitter release during aging

The purpose of this study was to assess the functional role of presynaptic α2-autoreceptors in noradrenergic transmission in the hippocampus and dopamine-2 heteroreceptors in cholinergic transmission in the striatum in young, adult, and senescent rats. Male and female Wistar rats (4, 12, and 24 months old) were used and the release of radioactivity from striatal and hippocampal slices that had been loaded either with [³H]choline or with [³H]norepinephrine was measured at rest and in response to field stimulation (2 Hz, 360 shocks). The release was challenged by sulpiride, a selective dopamine-2 receptor antagonist, and CH-38083, a selective α2-adrenoceptor antagonist. The dissociation constant and the number of α2-adrenoceptors was also determined by binding studies using [³H]yohimbine as ligand in crude membrane preparations of frontal cortex. There were an age-related changes in α2-adrenoceptor-mediated negative feedback modulation of norepinephrine release and in the density and dissociation constant of α2-adrenoceptors. They were reduced in senescent rats. In contrast the presynaptic modulation of striatal cholinergic transmission by dopamine-2 receptors was not altered during aging, but the storage capacity of and the release of acetylcholine from cholinergic interneurons was significantly lower.     

Adenosine A2 receptor-mediated excitation of a subset of AH/Type 2 neurons and elevation of cAMP levels in myenteric ganglia of guinea-pig ileum

Abstract The aim of the study was to test the hypothesis that excitatory A₂ and inhibitory A₁ receptors coexist on myenteric AHIType 2 neurons, and are positively coupled to adenylate cyclase to stimulate cAMP formation. The A₂ agonists NECA and CGS 21680 increased excitability and depolarized the membrane in 40% of 71 AH/Type 2 neurons. In the remainder, the agonists depressed excitability and hyperpolarized the neurons. In 13% of neurons, A₂ agonists caused a concentration-dependent depolarization at nanomolar concentrations, followed by hyperpolarization at higher concentrations. CGS 21680 (EC₅₀=0.15 nM) was 133-fold more potent than NECA (EC₅₀= 20 nM) in depolarizing AH/Type 2 neurons. The A₁ agonist, CCPA, caused hyperpolarization and depressed excitability in more than 90% of neurons. The potency profile of agonists for depolarization was CGS 21680 ≫ NECA ≫> CCPA. NECA augmented at nanomolar and inhibited at micromolar concentrations, excitatory depolarizing responses to forskolin in AH/Type 2 neurons; whereas, CCPA only inhibited the action of forskolin. In parallel studies on enzymatically dissociated myenteric ganglia, when the ganglia were exposed to priming concentrations of forskolin (5 μM) in the presence of Ro-20 1724, NECA enhanced the stimulatory action of forskolin on CAMP formation. This effect was abolished by the adenosine receptor antagonist DPSPX. The potency of NECA for stimulation of adenylate cyclase equalled that for depolarization of the AH/Type 2 neurons. The results suggest that high affinity excitutory A₂ receptors are coupled to adenylate cyclase in a minority subset of AH/Type 2 myenteric neurons, and that inhibitory A₁ and excitatory A₂ receptors are co-localized on some AH/Type 2 neurons.     

Biogenic amine-sensitive adenylate cyclases in primary culture of neuronal or glial cells from mesencephalon

Primary cultures of virtually pure mesencephalic neurons (5 days) or glials (4 weeks) from 14-day-old mouse embryo were obtained using appropriate medium.Membranes prepared from neuronal cells contained mainly serotonin and β₁-adrenergic-sensitive adenylate cyclases. However, a low but significant classical dopamine-sensitive adenylate cyclase activity (D₁ receptor) was detected. Contrasting with the data obtained from a previous study on striatal neurons¹⁴ no adenosine-sensitive adenylate cyclase was found on mesencephalic neurons. Study on the additive effects of the 3 biogenic amines-sensitive adenylate cyclases indicated that: (1) all neuronal cells having dopamine receptors possess β₁-adrenergic receptors (no additivity); (2) β₁-adrenergic and serotonin receptors on the one hand, and dopamine and serotonin receptors on the other hand, were coupled with independent adenylate cyclase systems localized either on two different domains of the same cell or on different cells (complete additivity).Membranes prepared from primary mesencephalic cultures of glial cells contained a mixture of β₁- and β₂-adrenergic receptor subtypes coupled with an adenylate cyclase (70% and 30%, respectively). No dopamine- or serotonin-sensitive adenylate cyclase was detected on mesencephalic glial cells.     

Effects of GTP and sodium on rat striatal dopamine receptors labeled with lisuride

[³H]Lisuride binding to rat striatal membranes appeared to be stereospecifically displaced by the dopamine antagonist butaclamol.Sodium increased the number of [³H]lisuride binding sites (B_max) without changing the dissciation constant (K_d). GTP did not affect [³H]lisuride binding characteristics, either with or without sodium.These results suggest that dopamine receptor sites labeled by lisuride are at least in part sodium-dependent, possibly the D₂-receptors not involved in adenylate cyclase stimulation.     

Muscarinic and dopaminergic receptor subtypes on striatal cholinergic interneurons

Unilateral stereotaxic injection of small amounts of the cholinotoxin, AF64A, caused minimal nonselective tissue damage and resulted in a significant loss of the presynaptic cholinergic markers [3H]hemicholinium-3 (45% reduction) and choline acetyltransferase (27% reduction). No significant change from control was observed in tyrosine hydroxylase or tryptophan hydroxylase activity; presynaptic neuronal markers for dopamine- and serotonin-containing neurons, respectively. The AF64A lesion resulted in a significant reduction of dopamine D2 receptors as evidenced by a decrease in [3H]sulpiride binding (42% reduction) and decrease of muscarinic non-M1 receptors as shown by a reduction in [3H]QNB binding in the presence of 100 nM pirenzepine (36% reduction). Saturation studies revealed that the change in [3H]sulpiride and [3H]QNB binding was due to a change in Bmax not Kd. Intrastriatal injection of AF64A failed to alter dopamine D1 or muscarinic M1 receptors labeled with [3H]SCH23390 and [3H]pirenzepine, respectively. In addition, no change in [3H]forskolin-labeled adenylate cyclase was observed. These results demonstrate that a subpopulation of muscarinic receptors (non-M1) are presynaptic on cholinergic interneurons (hence, autoreceptors), and a subpopulation of dopamine D2 receptors are postsynaptic on cholinergic interneurons. Furthermore, dopamine D1, muscarinic M1 and [3H]forskolin-labeled adenylate cyclase are not localized to striatal cholinergic interneurons.     

Inhibition of brain adenylate cyclase by A1 adenosine receptors: Pharmacological characteristics and locations

When tested under conditions reducing the endogenous production of adenosine (presence of adenosine deaminase (ADA) 1.6 IU/ml; and deoxyadenosine triphosphate (d-ATP)), and in the presence of both NaCl and GTP, the ADA-resistant analog phenylisopropyladenosine (PIA) inhibited the adenylate cyclase of several tissues. These tissues included:(1) 5 brain areas of adult rats (frontal and parietal cortex, cerebellum cortex, hippocampus and striatum) — hypothalamus and mid-brain adenylate cyclases were not inhibited by PIA; (2) astrocytes in primary cultures prepared from cerebral cortex of newborn mice; and (3) neurons in primary cultures prepared from striata of 15-day-old mouse embryos.The specificity profile of the adenosine receptor involved in the inhibition was determined in astrocytes. It was typical of an A₁ adenosine receptor (high affinity of PIA;K_aapp: 9 ± 5 × 10⁻⁹M(n= 4) compared to the affinity of 5′-N-ethylcar☐amide adenosine (NECA);K_aapp: 1.3 ± 0.6 × 10⁻⁷M (n= 3). There was an excellent correlation between the affinities of several adenosine agonists and antagonists for A₁ receptors coupled with an adenylate cyclase in astrocytes and for the receptors labeled with N⁶-cyclohexyl-[³H]adenosine in brain cortex.In adult rat striatum as well as in astrocytes and striatal neurons in culture the adenylate cyclase was inhibited by low PIA concentrations through A₁ receptors and stimulated by higher concentrations through A₂ receptors. In contrast, A₂ receptors were not detected in adult rat cerebral cortex.In adult rat striatum, A₁ and dopamine receptors coupled with an adenylate cyclase seemed to be located on different cell populations. In contrast, in astrocytes A₁ and β-adrenergic receptors coupled with adenylate cyclase were apparently located on the same cells.     

Reduced striatal acetylcholine efflux in the R6/2 mouse model of Huntington's disease: an examination of the role of altered inhibitory and excitatory mechanisms

Huntington's disease (HD) is a genetic neurodegenerative disorder that is characterized by the progressive onset of cognitive, psychiatric, and motor symptoms. In parallel, the neuropathology of HD is characterized by progressive loss of projection neurons in cortex and striatum; striatal cholinergic interneurons are relatively spared. Nonetheless, there is evidence that striatal acetylcholine (ACh) function is altered in HD. The present study is the first to examine striatal ACh function in awake, behaving animals, using the R6/2 mouse model of HD, which is transgenic for exon 1 of the mutant huntingtin gene. Physiological levels of extracellular striatal ACh were monitored in R6/2 mice and wild type controls using in vivo microdialysis. Results indicate that spontaneous ACh release is reduced in R6/2 mice relative to controls. Intrastriatal application of the GABA_A antagonist bicuculline methiodide (10.0 μM) significantly elevated ACh levels in both R6/2 mice and wild type controls, while overall ACh levels were reduced in the R6/2 mice compared to the wild type group. In contrast, systemic administration of the D₁ dopamine receptor partial agonist, SKF-38393 (10.0 mg/kg, IP), elevated ACh levels in control animals, but not R6/2 mice. Taken together, the present results suggest that GABA-mediated inhibition of striatal ACh release is intact in R6/2 mice, further demonstrating that cholinergic interneurons are capable of increased ACh release, whereas D₁ receptor-dependent activation of excitatory inputs to striatal cholinergic interneurons is dysfunctional in R6/2 mice. Reduced levels of extracellular striatal ACh in HD may reflect abnormalities in the excitatory innervation of cholinergic interneurons, which may have implications ACh-dependent processes that are altered in HD, including corticostriatal plasticity.     

Forskolin attenuates the evoked release of [H]GABA from striatal neurons in primary culture

The actions of the diterpene forskolin, and cyclic AMP analogues, on the evoked release of [³H]GABA (γ-aminobutyric acid) was examined in intact striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to forskolin (100 μM) resulted in a 40–55% attenuation of [³H]GABA release evoked by either KCl (30 mM) or veratrine (2 μg/ml), while baseline levels of release were unaffected. The dose-dependence for forskolin in striatal neurons. Exposure of striatal neurons to membrane-permeable identical to the dose-dependent elevation of cyclic AMP levels by forskolin in striatal neurons. Exposure of striatal neurons to membrane-permeable analogues of cyclic AMP, such as p-chlorophenylthio cyclic AMP (0.5 mM) and dibutyryl cyclic AMP (1 mM), resulted in a 25 and 26% attenuation of [³H]GABA release, respectively; dibutyryl cyclic GMP (1 mM) was without effect. The similarity between the actions of forskolin and the cyclic AMP analogues suggests that, in striatal neurons in primary culture, the elevation of cyclic AMP levels results in the attenuation of the evoked release of [³H]GABA. The greater effectiveness of forskolin, compared to the cyclic AMP analogues, may be related to the recently reported, additional direct actions of forskolin on neuronal membrane ion channels.     

Chronic opioid treatment attenuates carbachol-mediated polyphosphoinositide hydrolysis in chick embryo neuronal cultures

Opiate binding sites on cultured neurons derived from 6-day-old (E6) chick embryo cerebral hemispheres (CH), shown to be cholinergic by choline acetyltransferase immunostaining, were labeledd with [³H]etorphine (μ and δ opiate receptors expression) and [³H]morphine (mostly μ). When examined by light microscope autoradiography, opiate receptors were found to be expressed by most neurons and were distributed predominantly on neuronal perikarya. Muscarnic and opiate receptors in E6CH cultured neurons were found to be functionally coupled when the effects of opiate receptor occupancy on the inositol phosphate-linked muscarinic receptors was studied. Carbachol stimulated the release of [³H]inositol phosphates (InsP) from cultures preincubated with [³H]inositol and LiCl, in a dose-dependent manner, and the functional expression of muscarinic receptors peaked in number at day 7 in culture, declining thereafter. Short-term (<1h) treatment of E6 neuronal cultures with 1 μM opioid peptides such as morphiceptin or d-Ala²-d-Leu⁵-enkephalin (DADLE) did not not inhibit the release of inositol phosphates in response to 1 mM carbachol whereas forskolin, which also activates adenylate cyclase and raises cAMP levels, inhibited InsP release by about 25%. In contrast, long-term (48 h) opioid treatment with either morphiceptin or DADLE (1–10 μM) inhibited the carbachol-stimulated inositol phosphate release by 50%. Prolonged treatment with morphiceptin also inhibited the bradykinin-mediated release of InsP from E6CH cells. In both cases, the inhibition was blocked by the continuous presence of naloxone, suggesting that the inhibition was mediated through opiate receptors. When E6CH cells were depolarized by 30 mM K⁺, allowing [Ca²⁺]i to increase, the inhibition of inositol phosphate formation caused by long-term morphiceptin exposure was partially relieved. This 48 h (chronic) exposure to opioids may involve some depletion of available [Ca²⁺]i in cholinergic neurons. We propose that these interactions between the opiate and muscarinic receptor transducing systems may represent the molecular basis of the neuromodulating activity of opioids upon the cholinergic system early in development.