P2 purinoceptors stimulate inositol phosphate release in the organ of Corti.

We examined the effects of purinoceptor agonists on inositol phosphate (IP) release in the guinea-pig organ of Corti. The P2y receptor agonist ATP-gamma-S (200 microM) increased IPs 4-fold; identical concentrations of alpha, beta-methylene ATP, a P2x agonist, and adenosine, a P1 agonist, did not significantly affect IP release. In calcium-free incubations, simulated IP release decreased by 35% indicating partial dependence of ATP-mediated phosphoinositide hydrolysis on calcium influx. ATP-stimulated IP release was not enhanced by the cholinergic agonist carbachol known to increase IPs via muscarinic receptors in the organ of Corti. This is consistent with the notion that ATP and carbachol have a common target, most likely outer hair cells. P2 purinoceptors coupled to the phosphoinositide cascade suggest ATP as an afferent neuromodulator or efferent neurotransmitter in the cochlea.     

Effect of in vivo exposure to hypoxia on muscarinic cholinergic receptor-coupled phosphoinositide turnover in the rat brain

We examined the effects of a transient exposure of rats to severe (5% O2, 95% N2 for 30 min) or mild (10% O2, 90% N2 for 30 min) hypoxia and a chronic exposure to mild hypoxia (10% O2, 90% N2 up to 48 h) on carbachol-stimulated phosphoinositide (PI) turnover and [3H]quinuclidinyl benzilate (QNB) binding in 4 regions of the brain (cerebral cortex, striatum, hippocampus and cerebellum). When the rats were exposed to hypoxia transiently, significant changes were caused only by severe hypoxia. In all 4 regions examined, basal incorporation of [3H]inositol into inositol phosphates (IPs) in the absence of carbachol was significantly increased following transient severe hypoxia and remained high for 48 h. Carbachol stimulation of PI turnover (% of basal) was significantly enhanced only in the hippocampus. The increase in carbachol stimulation in the hippocampus was observed only after a 6-h exposure to room air following the severe hypoxia and lasted for at least 48 h. In Ca2+-free incubation medium, the increase in basal incorporation of [3H]inositol into IPs elicited by severe hypoxia was not observed but carbachol still had an enhanced stimulatory effect. Binding studies showed that maximum binding capacity (Bmax values) of [3H]QNB binding was significantly increased only in the hippocampus and only after a 6-h exposure to room air following the severe hypoxic exposure and remained increased for 48 h. Kd values showed no significant change. While a transient exposure of rats to mild hypoxia caused no significant change either in carbachol-stimulated PI turnover or in [3H]QNB binding parameters, a chronic exposure to mild hypoxia for more than 6 h caused a significant increase in [3H]QNB binding capacity localized in the hippocampus, which was accompanied by an enhanced stimulatory effect of carbachol on PI turnover. These changes observed with mild hypoxia, however, were transient and tended to return to control levels following a 48-h exposure. These results suggest an up-regulation of muscarinic cholinergic receptor-coupled PI turnover in the rat brain caused either by transient severe hypoxia or by chronic mild hypoxia.     

Developmental changes in the chemosensitivity of rat brain synaptoneurosomes to excitatory amino acids, estimated by inositol phosphate formation

The evolution of excitatory amino acids-(EAA) stimulated inositol phosphates (IPs) turnover during postnatal development was investigated in synaptoneurosomes prepared from rat forebrains. The two main EAA agonists which induce the IPs synthesis were quisqualate (QA) and N-methyl-D-aspartate (NMDA). The QA and NMDA stimulations of IPs formation present a particular developmental pattern, characterized by an active phase during rat synaptogenesis. The QA-evoked IPs accumulation peaked in synaptoneurosomes prepared from 8-day-old rat forebrains while that evoked by NMDA peaked in synaptoneurosomes from 12-day-old rats. These two developmental patterns are specific of the EAA agonists since the other various neuroactive substances tested (carbachol (Carb), noradrenaline, and high concentrations of potassium) induced an IPs accumulation, which increases during development and reaches a maximum in synaptoneurosomes of adult animals. Aging leads to a decrease in the capability of EAAs and muscarinic agonists to stimulate IPs formation in synaptoneurosomes, whereas the stimulation of IPs turnover by noradrenaline remains constant. Taken together, these results suggest that EAAs play a key role during brain development by sequentially activating two receptor subtypes, a new QA receptor, and a NMDA receptor, linked to the phosphoinositide metabolism. They may also indicate that these EAA-induced IPs responses are related to neuronal plastic events, the amplitude of which decreases with aging.     

Muscarinic cholinergic receptor regulation and acetylcholinesterase inhibition in response to insecticide exposure during development

Neonatal rats were exposed to parathion, an acetylcholinesterase inhibiting organophosphorus pesticide, during a rapid phase of cholinergic receptor development. Rats were given subcutaneous injections of 1.5 mg/kg/day from postnatal days 8–20. The immediate effects of subchronic developmental exposure were assessed in 21-day-old animals and more persistent effects assessed in 36-day-old animals. There was a 61% inhibition of acetylcholinesterase and a 27% decrease of muscarinic receptor density in 21-day-old treated rats. The reduction in receptor density was dose-dependent and a significant correlation was found between the level of acetylcholinesterase inhibition produced by parathion and the reduction in receptor density. It was estimated that a minimum of at least 15% prolonged inhibition of forebrain acetylcholinesterase by parathion was necessary to reduce receptor density. Regional analyses of receptor autoradiograms of 21-day-old animals indicated muscarinic receptors in the cortex and hippocampus were preferentially lost. The anterior thalamus was notable in having a high density of cholinergic receptors which were unaffected by parathion treatment. No changes were found in the affinity of [³H]QNB for the receptor or in the binding of the agonist, acetylcholine, in competition binding studies. AChE activity and muscarinic receptor density returned to normal after a 16 day recovery period. Parathion treated animals were growth inhibited but, growth retardation induced by undernutrition did not alter receptor density or affinity of QNB for muscarinic receptors. Thus, the transient decrease in receptor density in parathion exposed animals was similar to the response previously observed in adults and was not secondary to growth retardation or undernutrition. Receptor densities and acetylcholinesterase levels were regulated back to normal values after a 16 day recovery period in spite of the perturbation of cholinergic function during cholinergic synapse and receptor development.     

Activation of muscarinic cholinergic receptors stimulates inositol phosphates synthesis in the developing avian cochlear duct

We previously reported that the inositol phosphates (IPs) synthesis is induced by muscarinic agonists in the rat cochlea and that this stimulation is maximal at postnatal day 12. This peak response is concomitant with the onset of the efferent synaptogenesis at the outer hair cell level. Whether the correlation between this neuronal plasticity and the enhanced IPs formation is unique to the rat or a general feature of the developing vertebrate cochlea is not known. To examine this question, we measured, in the presence of LiCl, the accumulation of (3H)-IPs induced by carbachol, in the developing chick cochlear duct during a period ranging from embryonic day (E) 8 to post-hatching day (P) 20. Carbachol (1 mM) causes a significant increase of IPs formation relative to basal values at all ages. This IPs accumulation is maximal at E8 (1854% of the basal level), then, rapidly decreases until P13 when it reaches a steady-state level of 294% of the basal level. Strikingly, this gradual decline in IPs formation is interrupted between E15 and E19, by a transient increase in IPs synthesis. This rise peaks at E16 with a stimulation value of 757% of the control level. This maximal stimulation is inhibited by atropine in a dose-dependent manner, as is the case at E9, suggesting the involvement of muscarinic receptors. Interestingly, the occurrence of the peak response is concomitant with the plastic events associated with the maturation of the efferent innervation of the cochlear duct. Thus, these results suggest that there may be a correlation between cochlear plasticity and enhanced IPs synthesis, which is not species-specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of muscarinic cholinergic receptors in the submandibular gland of the rat

The specific muscarinic ligand [3H]quinuclidinyl benzilate ([3H]QNB) was used to label acetylcholine receptors in the submandibular gland of the rat. Specific binding of [3H]QNB increased linearly with tissue concentration in the range of 0.02-0.3 mg of protein/ml. Kinetic analysis of [3H]QNB binding revealed the presence of a single population of high affinity binding sites, with a dissociation constant of 87.2 pM and a Hill coefficient of 0.95. The binding was saturable and the receptor density was 214 fmol/mg of protein. The rate constants at 37 degrees C for association and dissociation of the [3H]QNB-receptor complex were 5.98 X 10(-8) M-1 X min-1 and 6.6 X 10(-3) X min-1, respectively. The ratio k-1/k+1 gave a Kd value of 11.1 pM, similar to the Kd value (13.1 pM) determined by kinetic parameters when extrapolated at infinitely low receptor concentration. Muscarinic antagonists displaced [3H]QNB from muscarinic receptors with a Hill coefficient near to 1.0. Displacement curves for muscarinic agonists and for the atypical antagonist pirenzepine had Hill values significantly less than one. In the presence of 0.1 mM GPP(NH)P, the potency of agonists but not antagonists in displacing [3H]QNB binding decreased 2 to 3-fold. The [3H]QNB binding site was sensitive to the inhibitory effect of various sulfhydryl reagents. Repeated treatments of rats with an acetylcholinesterase inhibitor led to a decreased density of muscarinic receptors in the submandibular gland. This alteration was specific for the muscarinic recognition site and was paralleled by a reduced sensitivity to carbachol.     

Effect of chronic lithium on cholinergically mediated responses and [3H]QNB binding in rat brain

Lithium (Li) has been previously reported to increase acetylcholine turnover and release in rat brain and to potentiate the neurotoxicity of cholinergic agents. We studied the effect of chronic Li administration, alone and in combination with the muscarinic antagonist, scopolamine, on two cholinergically-mediated responses and on muscarinic cholinergic receptor (MCR) binding in rat brain. Administered separately, Li and scopolamine enhanced the cataleptic and hypothermic responses to pilocarpine; combined administration resulted in an additive effect on both these measures. [3H]Quinuclidinyl benzilate ([3H]QNB) binding was increased by Li in the corpus striatum but not in the cortex, hippocampus and hypothalamus. Scopolamine increased [3H]QNB binding in the striatum, cortex and hippocampus; Li and scopolamine effects on striatal MCR were not additive. Contrary to a previous report, antagonist-induced MCR supersensitivity was not prevented by concurrent Li administration in any of the brain areas studied. The additive effect of Li and scopolamine on pilocarpine-induced catalepsy and a trend in this direction for pilocarpine-induced hypothermia suggest that the actions of the two agents to enhance cholinergically mediated responses may be achieved by different mechanisms. Supersensitive responses following scopolamine may be attributed to antagonist-induced up-regulation of postsynaptic muscarinic receptors as demonstrated in the binding studies. The effects of Li to enhance cholinergically-mediated catalepsy and hypothermia are interpreted as extending previous reports that Li stimulates brain cholinergic function by a presynaptic increase in acetylcholine turnover and release.     

Human retinal pigment epithelial cells possess muscarinic receptors coupled to calcium mobilization

Human retinal pigment epithelial (RPE) cells in culture demonstrated saturable specific binding of [3H]quinuclidinyl benzilate (QNB). Specific binding represents about 75% of total binding. Scatchard analysis yields a Kd of 0.178 nM and Bmax of 42 fmol/mg protein. Atropine and carbachol show typical displacement curves, and a Hill plot has a slope of 0.96, suggesting a homogeneous population of receptors. Muscarinic agonists have no effect on intracellular cyclic adenosine monophosphate levels in RPE cells measured by radioimmunoassay, nor do they alter the isoproterenol-induced stimulation of adenylate cyclase. However, both acetylcholine and carbachol cause a rapid increase in intracellular calcium concentration measured by the fluorescent indicator quin 2. Atropine reverses the calcium rise when added after agonist and prevents the rise when added prior to agonist. These data suggest that human RPE cells possess muscarinic receptors coupled to calcium mobilization.     

Effect of chronic lithium on cholinergically mediated responses and [H]QNB binding in rat brain

Lithium (Li) has been previously reported to increase acetylcholine turnover and release in rat brain and to potentiate the neurotoxicity of cholinergic agents. We studied the effect of chronic Li administration, alone and in combination with the muscarinic antagonist, scopolamine, on two cholinergically-mediated responses and on muscarinic cholinergic receptor (MCR) binding in rat brain. Administered separately, Li and scopolamine enhanced the cateleptic and hypothermic responses to pilocarpine; combined administration resulted in an additive effect on both these measures. [³H]Quinuclidinyl benzilate ([³H]QNB) binding was increased by Li in the corpus striatum but not in the cortex, hippocampus and hypothalamus. Scopolamine increased [³H]QNB binding in the striatum, cortex and hippocampus; Li and scopolamine effects on striatal MCR were not additive. Contrary to a previous report, antagonist-induced MCR supersensitivity was not prevented by concurrent Li administration in any of the brain areas studied. The additive effect of Li and scopolamine on pilocarpine-induced catalepsy and a trend in this direction for pilocarpine-induced hypothermia suggest that the actions of the two agents to enhance cholinergically mediated responses may be achieved by different mechanisms. Supersensitive responses following scopolamine may be attributed to antagonist-induced up-regulation of postsynaptic muscarinic receptors as demonstrated in the binding studies. The effects of Li to enhance cholinergically-mediated catalepsy and hypothermia are interpreted as extending previous reports that Li stimulates brain cholinergic function by a presynaptic increase in acetylcholine turnover and release.     

Soman- or kainic acid-induced convulsions decrease muscarinic receptors but not benzodiazepine receptors

[3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors decreased in the rat forebrain after convulsions induced by a single dose of either soman, a potent inhibitor of acetylcholinesterase, or kainic acid, an excitotoxin. A Rosenthal plot revealed that the receptors decreased in number rather than affinity. When the soman-induced convulsions were blocked, the decrease in muscarinic receptors at 3 days was less extensive than when convulsions occurred and at 10 days they approached control levels in most of the brain areas. The most prominent decrements in QNB binding were in the piriform cortex where the decline in QNB binding is probably related to the extensive convulsion-associated neuropathology. The decrements in QNB binding after convulsions suggest that the convulsive state leads to a down-regulation of muscarinic receptors in some brain areas. In contrast to the decrease in QNB binding after convulsions, [3H]flunitrazepam binding to benzodiazepine receptors did not change even in the piriform cortex where the loss in muscarinic receptors was most prominent. Thus, it appears that those neuronal processes that bear muscarinic receptors are more vulnerable to convulsion-induced change than those with benzodiazepine receptors.     

Muscarinic receptors modulate intracellular calcium level in chick sensory neurons

In the present work we have studied the variation of intracellular calcium levels induced by muscarinic agonists in chick dorsal root ganglia neurons. Muscarinic agonists such as muscarine and oxotremorine cause an increase of intracellular calcium levels in fura-2AM-loaded DRG neurons of E18 chick embryos. This increase was abolished following treatment with 1 microM atropine but not by 1 microM mecamylamine, indicating that the observed intracellular calcium increase, was dependent on muscarinic receptor activation. Stimulation in absence of external calcium or pre-incubation of the DRG cultures with thapsigargin or Mn(2+) demonstrated that [Ca(2+)](i) increase is mainly due to its release from intracellular stores. The use of selective antagonists of muscarinic receptor subtypes also indicated that M(1) and to a lesser extent M(3) receptor subtypes are responsible for the observed intracellular calcium mobilization. Finally pre-treatment of DRG cultures with pertussis toxin showed that the variation of [Ca(2+)](i) levels was dependent on PTX-insensitive G-protein. Moreover muscarinic agonists induce in DRG also the increase of IPs level, suggesting that the variations of intracellular calcium levels may be due at least in part to the activation of the phosphoinositide transduction pathway. In conclusion the reported observations demonstrate the activity of muscarinic receptors in sensory neurons, suggesting a functional role for acetylcholine in sensory transduction.     

Regional responses of rat brain muscarinic cholinergic receptors to immobilization stress

The effect of immobilization stress (IM-stress) on the muscarinic cholinergic (m-Ch) receptor binding was determined in 8 brain regions using [³H]quinuclidinyl benzilate (QNB). IM-stress produced an increase in specific QNB binding in the septum, striatum, hippocampus and pons + medulla oblongata. Scatchard panalyisis revealed that IM-stresss produced an increase in the affinity of m-Ch receptors in the septum, hippocampus andm pons + medulla oblangata, but did not alter the maximum number of binding sites (B_max). In the striatum, an increase in specific QNB binding was due to both the increase in B_max and reduction of the dissociation constant (K_d). The present study suggest that IM-stress induces supersensitivity of postsynaptic m-Ch receptors probably due to a deacrease in presynaptic cholinergic activities in the septum, striatum, hippocampus and pons + medulla oblongata. As the m-Ch receptors in the striatum and pons + medulla oblongata. Scatchard analysis revealed that IM-stress produced an increase in the affinity of m-Ch receptors stressful situations in this regions as well as in the septum, hippocampus and cerebral cortex.     

Antagonism of muscarinic receptors in the rabbit iris-ciliary body by 8-OH-DPAT and other 5-HT1A receptor agonists

Summary Physiological studies have shown that serotonin and 5-HT_1A agonists can influence muscarinic function in the rabbit iris-ciliary body (ICB). The purpose of this study was to examine whether a direct interaction exists between muscarinic and 5-HT_1A receptors in the ICB. At high concentrations, the 5-HT_1A agonist 8-OH-DPAT attenuated the carbachol-induced stimulation of inositol phosphates (InsPs) production, but this was not blocked by the presence of 5-HT_1A antagonists. In contrast, serotonin failed to influence carbachol-induced InsPs formation. Moreover, 8-OH-DPAT but not serotonin displayed affinity for [³H]QNB binding sites in the ICB. The combined data suggest that activation of 5-HT_1A receptors in the ICB does not cause a modulation of muscarinic receptor-stimulated phosphoinositide turnover. The data instead suggest that, at high concentrations, 8-OH-DPAT acts as an antagonist at muscarinic receptors and in this way influences muscarinic receptor function. The mechanism of 5-HT-induced modulation of muscarinic function in the ICB therefore remains to be elucidated.     

Rat Schwann cells express M1-M4 muscarinic receptor subtypes

The expression of different muscarinic receptor subtypes was analyzed in immature Schwann cells obtained from sciatic nerve of 2-day neonatal rats. By using RT-PCR analysis, we demonstrated the presence of M1, M2, M3, and M4 receptor subtypes in cultured Schwann cells, with M2 displaying the highest expression levels. Muscarinic subtypes were also quantified by immunoprecipitation and [3H]QNB binding. With this approach, we found the levels of receptor expression to be M2 > M3 > M1. M4 is expressed at very low levels, and M5 receptor was not detectable. Moreover, we also demonstrated that stimulation of the receptors by muscarinic agonists activates previously described signal transduction pathways, leading to a decrease of cAMP and an increase of IP3 levels not associated with an efficient intracellular Ca2+ release. The presence and activity of particular muscarinic receptors in immature Schwann cells suggest that ACh may play an important role in Schwann cell development.     

Kindled seizure-induced reduction of muscarinic cholinergic receptors in rat hippocampal formation: evidence for localization to dentate granule cells

The binding of [3H] quinuclidinyl benzilate ( [3H] QNB) to muscarinic cholinergic receptors in dentate gyrus of rat hippocampal formation was analyzed by membrane binding assay and in vitro autoradiography. The destruction of dentate granule cells, either by neonatal irradiation or colchicine injection, resulted in nearly complete elimination of [3H] QNB binding sites in the molecular and granule cell layers. By contrast, neither perforant path transection nor destruction of the septal-hippocampal cholinergic afferents caused a decline of [3H] QNB binding sites. Amygdala kindled seizures resulted in a 30% reduction of [3H] QNB binding sites which was distributed uniformly across the entire molecular and granule cell layers. Thus, most, if not all, of the muscarinic cholinergic receptors present in dentate gyrus appear to reside on the somata and dendritic trees of the dentate granule cells. We propose that this kindled seizure-induced decline of muscarinic receptors represents an endogenous compensatory mechanism designed to stabilize granule cell excitability.     

Calcium store-mediated signaling in sustentacular cells of the mouse olfactory epithelium

Sustentacular cells have structural features that allude to functions of secretion, absorption, phagocytosis, maintenance of extracellular ionic gradients, metabolism of noxious chemicals, and regulation of cell turnover. We present data detailing their dynamic activity. We show, using a mouse olfactory epithelium slice model, that sustentacular cells are capable of generating two types of calcium signals: intercellular calcium waves where elevations in intracellular calcium propagate between neighboring cells, and intracellular calcium oscillations consisting of repetitive elevations in intracellular calcium confined to single cells. Sustentacular cells exhibited rapid, robust increases in intracellular calcium in response to G-protein coupled muscarinic and purinergic receptor stimulation. In a subpopulation of sustentacular cells, oscillatory calcium transients were evoked. We pharmacologically characterized the properties of purinergic-evoked increases in intracellular calcium. Calcium transients were elicited by release from intracellular stores and were not dependent on extracellular calcium. BAPTA-AM, a cytosolic calcium chelator, and cyclopiazonic acid, an endoplasmic reticulum Ca(2+)-ATPase inhibitor irreversibly blocked the purinergic-induced calcium transient. Phospholipase C antagonist U73122 inhibited the purinergic-evoked calcium transient. 2-Aminoethoxydiphenyl borate, an inositol-1,4,5-trisphosphate (IP(3)) receptor antagonist, and the ryanodine receptor (RyR) antagonists tetracaine and ryanodine, inhibited the UTP-induced calcium transients. Collectively, these data suggest that activation of the phospholipase C pathway, IP(3)-mediated calcium release, and subsequent calcium-induced-calcium release is involved in ATP-elicited increases in intracellular calcium. Our findings indicate that sustentacular cells are not static support cells, and, like glia in the central nervous system, have complex calcium signaling.     

The ontogeny of m1-m5 muscarinic receptor subtypes in rat forebrain.

The ontogeny of muscarinic receptor subtypes in rat forebrain has been determined utilizing a panel of antisera recognizing unique epitopes of the m1-m5 receptor proteins. Total receptor density in forebrain, as measured by the non-selective antagonist, [3H]quinuclidinyl benzilate (QNB), was found to increase from 507 fmol/mg in neonates (3.5 days) to 1727 fmol/mg in mature animals (45 days). Adult levels were reached two weeks post-partum. A recently developed precipitation protocol was then used to further characterize receptor ontogeny. Cumulatively, 90% of [3H]QNB labelled muscarinic receptors from adult forebrain were immunoprecipitated with subtype selective antibodies (m1-m5). In 3- to 4-day-old neonates, m1 receptors are expressed at 31% of adult levels, m2 receptors at 32% of adult levels, m3 receptors at 36% of adult levels, and m4 receptors at 20% of adult levels. In mature animals, m1 receptors were equal to 35%, m2 equal to 18%, m3 equal to 11%, and m4 equal to 26%, of total receptors, respectively. Levels of m5 receptors are consistently very low at all ages tested (less than or equal to 1% of total receptor density). Although there were subtle differences in the time courses of development between muscarinic receptor subtypes, in general, they developed with similar ontogenic profiles. Subtypes coupled preferentially to inhibition of adenylate cyclase (m2 and m4) comprised 35% of total receptors expressed in neonates. The combined m2/m4 receptor density increased at a uniform rate during development from 173 to 757 fmol/mg. Receptors preferentially coupled to phosphoinositide turnover (m1, m3, and m5) were found in newborns at approximately 270 fmol/mg.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

An IP3-assisted form of Ca2+-induced Ca2+ release in neocortical neurons

Calcium increases induced by single action potentials in rat visual cortex layer II/III pyramidal neurons were shown to be augmented by muscarinic acetylcholine receptor (mAchR) stimulation. This augmentation was drastically reduced by intracellular injection of heparin but not ruthenium red, therefore involving inositol-1,4,5-trisphosphate (IP3)-sensitive rather than ryanodine-sensitive calcium stores. Only the calcium increase induced by the second or later spike of a spike train, but not that induced by the first spike, was augmented, indicating the requirement of both spike-induced calcium increase and mAchR activation. The calcium store depletor thapsigargin abolished this augmentation use-dependently. These findings suggest a neocortical occurrence of calcium-induced calcium release from IP3-sensitive calcium stores that have been sensitized beforehand by IP3 through mAchR-mediated mechanisms.     

Muscarinic receptor localization and function in rabbit carotid body

Acetylcholine and muscarinic agonists inhibit chemosensory activity in the rabbit carotid sinus nerve (CSN). Because the mechanism of this inhibition is poorly understood, we have investigated the kinetics and distribution of muscarinic receptors in the rabbit carotid body with the specific muscarinic antagonist [3H]quinuclidinylbenzilate ([3H]QNB). Equilibrium binding experiments identified displaceable binding sites (1 microM atropine) with a Kd = 71.46 pM and a Bmax = 9.23 pmol/g tissue. These binding parameters and the pharmacology of the displaceable [3H]QNB binding sites are similar to specific muscarinic receptors identified in numerous other nervous, muscular and glandular tissues. Comparisons of specific binding in normal and chronic CSN-denervated carotid bodies suggest that muscarinic receptors are absent on afferent terminals in the carotid body; however, nearly 50% of the specific [3H]QNB binding is lost following chronic sympathectomy, suggesting the presence of presynaptic muscarinic receptors on the sympathetic innervation supplying the carotid body vasculature. Autoradiographic studies have localized the remainder of [3H]QNB binding sites to lobules of type I and type II parenchymal cells. In separate experiments, the muscarinic agonists, oxotremorine (100 microM) stimulation of the in vitro carotid body. Our data suggest that muscarinic inhibition in the rabbit carotid body is mediated by receptors located on type I cells which are able to modulate the excitatory actions of acetylcholine at nicotinic sites.