Effects Of Cholinergic Agonists Research Articles

In vitro effect of cholinergic agonists and antagonists, activators and blockers of voltage-sensitive Ca++-channels and Na+-channels on arylalkylamine N-acetyltransferase 2 mRNA levels in pineal organ of the catfish, Clarias gariepinus

The fish pineal organ is innervated by cholinergic nerve fibres. But there is no information on cholinergic regulation of aanat2 gene in the fish pineal organ. Therefore, we investigated in vitro effect of specific agonists and antagonists of cholinergic receptors on aanat2 mRNA levels in the pineal organ of catfish Clarias gariepinus. Acclimatized mature male fish was decapitated and the pineal organ was rapidly removed and placed in culture medium and pre-incubated for 1h. After pre-incubation, the culture medium was replaced with the culture medium containing desired concentrations of specific ago-nists and antagonists of cholinergic receptors, blockers and activators of volt-age-sensitive Ca++ channels and Na+ channels for 1h, after which the pineal organs were collected and processed for measuring relative expression of aanat2 gene by measuring AANAT2 mRNA with the help of Real Time RT-PCR. Acetylcholine and nicotine produced dose-dependent inhibitory and stimulatory effect on aanat2 transcript levels, while carbachol increased aa-nat2 transcript levels in a dose-dependent manner. In vitro treatment of the pineal organ with atropine, hexamethonium, the blockers of voltage-sensitive Ca++ channels and Na+ channels significantly blocked the stimulato-ry effect of carbachol and nicotine on aanat2 transcript levels, respectively. Based on these findings, we suggest that cholinergic mechanism plays an important and direct role in regulation of aanat2 gene expression in the fish pineal organ, and stimulates the gene transcription via both muscarinic and nicotinic receptors. Further, muscarinic and nicotinic receptors seem to in-crease aanat2 transcript levels by affecting voltage-sensitive Ca++ channels and Na+ channels, respectively.

Acute Detubulation of Ventricular Myocytes Amplifies the Inhibitory Effect of Cholinergic Agonist on Intracellular Ca2+ Transients.

Muscarinic receptors expressed in cardiac myocytes play a critical role in the regulation of heart function by the parasympathetic nervous system. How the structural organization of cardiac myocytes affects the regulation of Ca2+ handling by muscarinic receptors is not well-defined. Using confocal Ca2+ imaging, patch-clamp techniques, and immunocytochemistry, the relationship between t-tubule density and cholinergic regulation of intracellular Ca2+ in normal murine ventricular myocytes and myocytes with acute disruption of the t-tubule system caused by formamide treatment was studied. The inhibitory effect of muscarinic receptor agonist carbachol (CCh, 10 μM) on the amplitude of Ca2+ transients, evoked by field-stimulation in the presence of 100 nM isoproterenol (Iso), a β-adrenergic agonist, was directly proportional to the level of myocyte detubulation. The timing of the maximal rate of fluorescence increase of fluo-4, a Ca2+-sensitive dye, was used to classify image pixels into the regions functionally coupled or uncoupled to the sarcolemmal Ca2+ influx (ICa). CCh decreased the fraction of coupled regions and suppressed Ca2+ propagation from sarcolemma inside the cell. Formamide treatment reduced ICa density and decreased sarcoplasmic reticulum (SR) Ca2+ content. CCh did not change SR Ca2+ content in Iso-stimulated control and formamide-treated myocytes. CCh inhibited peak ICa recorded in the presence of Iso by ∼20% in both the control and detubulated myocytes. Reducing ICa amplitude up to 40% by changing the voltage step levels from 0 to –25 mV decreased Ca2+ transients in formamide-treated but not in control myocytes in the presence of Iso. CCh inhibited CaMKII activity, whereas CaMKII inhibition with KN93 mimicked the effect of CCh on Ca2+ transients in formamide-treated myocytes. It was concluded that the downregulation of t-tubules coupled with the diminished efficiency of excitation–contraction coupling, increases the sensitivity of Ca2+ release and propagation to muscarinic receptor-mediated inhibition of both ICa and CaMKII activity.

Have you herb about this; the effect of cholinergic agonists on motor control

Have you herb about this; the effect of cholinergic agonists on motor control

The Role of Muscarinic Receptors in the Pathophysiology of Mood Disorders: A Potential Novel Treatment?

The central cholinergic system has been implicated in the pathophysiology of mood disorders. An imbalance in central cholinergic neurotransmitter activity has been proposed to contribute to the manic and depressive episodes typical of these disorders. Neuropharmacological studies into the effects of cholinergic agonists and antagonists on mood state have provided considerable support for this hypothesis. Furthermore, recent clinical studies have shown that the pan-CHRM antagonist, scopolamine, produces rapid-acting antidepressant effects in individuals with either major depressive disorder (MDD) or bipolar disorder (BPD), such as bipolar depression, contrasting the delayed therapeutic response of conventional mood stabilisers and antidepressants. This review presents recent data from neuroimaging, post-mortem and genetic studies supporting the involvement of muscarinic cholinergic receptors (CHRMs), particularly CHRM2, in the pathophysiology of MDD and BPD. Thus, novel drugs that selectively target CHRMs with negligible effects in the peripheral nervous system might produce more rapid and robust clinical improvement in patients with BPD and MDD.

Abstract 035: Cholinergic Agonists Protect from the Development of Hypertension during Chronic Inflammatory Disease

Systemic lupus erythematosus (SLE) is an autoimmune disorder with prevalent hypertension. Previous studies using a genetic mouse model of SLE (NZBWF1) suggest chronic inflammation is an important contributor to SLE hypertension. A novel neuroimmune pathway involving the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR) suppresses splenic cytokine release and reduces systemic inflammation upon stimulation. To test whether activation of this ‘cholinergic anti-inflammatory pathway’ at the level of the α7nAChR attenuates the development of hypertension during SLE, female SLE and control (NZW) mice were infused with nicotine hydrogen tartrate salt (2 mg/kg/day, SC) or saline for 7 days. Nicotine-treated SLE mice had lower splenic protein expression of TNF-α and IL-6 (normalized to β-actin) relative to saline-treated SLE mice (1.09±0.06 vs. 1.37±0.06 and 0.36±0.04 vs. 0.55±0.10; all p<0.05), suggesting efficacy of the therapy. Mean arterial pressure (MAP; mmHg) was increased in SLE mice compared to controls (140±4 vs. 114±2; p<0.001). Nicotine prevented the rise in MAP in SLE mice (129±4; p=0.022), but not controls (121±3). This protection from hypertension coincided with a 46±5% lower renal cortical TNF-α in nicotine-treated SLE mice compared to saline-treated SLE mice (0.39±0.04 vs. 0.73±0.18), which is important because it has been previously shown that renal TNF-α plays a mechanistic role in the development of hypertension during SLE. Because nicotine acts on both ganglionic and peripheral cholinergic receptors, in a subsequent study mice were administered the selective α7nAChR agonist, PNU-282987 (0.38 mg/kg/day, IP), or vehicle for 28 days. PNU-282987-treated SLE mice had lower splenic protein expression of TNF-α and IL-6 relative to saline-treated SLE mice (0.33±0.01 vs. 0.54±0.03 and 0.40±0.08 vs. 0.86±0.05; all p<0.05). MAP was increased in SLE mice compared to controls (138±2 vs. 122±5). PNU-282987 prevented the rise in MAP in SLE mice (128±4), but not controls (125±5). These data suggest the anti-inflammatory effects of cholinergic agonists may protect from SLE hypertension and that the cholinergic anti-inflammatory pathway may be an important target in hypertensive patients with chronic inflammatory diseases.

Effects of Cholinergic Agonists and Antagonists under Conditions of Spleen Denervation in Rats with Endotoxic Shock

Model experiments on rats with endotoxic shock induced by intraperitoneal injection of LPS Salmonella Typhi strain ty-4441 (20 mg/kg) showed that crossing of the vagus nerve innervating the spleen increased HR, stimulated production of antibodies, and moderated serotonergic activity of splenocytes. Pharmacological correction of the shock with muscarinic receptor antagonist atropine and its combinations with anticholinesterase agent galantamine or muscarinic and nicotinic cholinoreceptor agonist choline alfoscerate 30 min before shock modeling moderated HR and normalized B cell functions and serotonin level in the spleen.

Cholinergic submandibular effects and muscarinic receptor expression in blood vessels of the rat

In order to functionally characterise the muscarinic vasodilator responses, effects of cholinergic agonists were studied on isolated preparations of the rat submandibular artery and vein and carotid and jugular vessels. Tentatively, a cholinergic regulatory mechanism having different effects on the arterial and venous vessels would enhance vascular fluid recruitment for the secretory response. In vitro functional findings were correlated to the expression and cellular location of the different receptors that were assessed by immunohistochemistry. In order to find in vivo correlates to the in vitro findings, the influence of muscarinic receptors on permeability was studied on the vasculature of the submandibular gland in anaesthetised rats. Staining for muscarinic M1 receptors occurred in the endothelium, and muscarinic M5 receptors, and possibly M3 also, were detected in the arterial smooth muscle. In venous endothelium, muscarinic M1 and M4 receptors occurred. In the jugular smooth muscle layer, staining for M1, and possibly also for M3, appeared. Muscarinic agonists caused arteries to relax and veins to contract. The nitric oxide synthase inhibitor Nω-nitro- l-arginine ( l-NNA; 10 −4 M) markedly reduced the cholinergic-evoked relaxation of pre-contracted carotid arterial preparations. In the presence of 4-DAMP (10 −7 M), the relaxation to cholinergic agonists was inhibited. Pirenzepine (10 −5 M) did not only inhibit the relaxatory effects, but even reversed the effects, while it in the jugular vein abolished the cholinergic effects. The arterial nitric oxide-dependent response to muscarinic receptor stimulation consisted of two parts—one sensitive to pirenzepine and 4-DAMP and the other to 4-DAMP only. Inhibition of the former part only, resulted in cholinergic arterial contraction. Also, the submandibular artery and vein responses to muscarinic receptor stimulation show a resemblance with those of the carotid and jugular vessels, i.e. a pronounced arterial relaxation and a contractile component in the venous response. In vivo examination of submandibular glandular vasculature by studying glandular permeability to Evans blue, confirmed the in vitro observations indicating muscarinic M1 receptors preserving perfusion pressure during the secretory process.

Alpha 9 acetylcholine receptors are essential for epidermal differentiation

Extraneuronally, acetylcholine (Ach) is synthesized, stored and secreted in the tegumental cells covering the inner and outer surfaces of the body. It acts via nicotinic (nAchR) and muscarinic (mAchR) receptors in a paracrine and autocrine fashion influencing various keratinocyte functions. Using in vitro studies, we present evidence that effects of cholinergic agonists and antagonists on cutaneous biology are dependent upon specific Ach‐R, localised in the different compartments of the skin. Using immunohistochemistry on frozen section of normal skin we could previously demonstrate a differential distribution of alpha 3*, alpha 7, alpha 9 and beta 1 nAchR and m1, m3–5 mAchR in human epidermis. Here we show the functional significance of AchR blockage using organotypical keratinocytes cultures. Cholinergic agonists and antagonists were added on the day of the lift to the air‐liquid interface. After 5 days, blockage of all AchR using mecamylamine and atropine resulted in complete inhibition of terminal differentiation. Mecamylamine and atropine alone had only a retarding effect suggesting an additive mechanism of nAchR and mAchR blockage. Specific inhibition of α9 homooligomers with strychnine produced the strongest effects leaving behind only a keratinocytes monolayer. Stimulation of AchR with either nicotine or muscarine in short term organotypic cultures did not produce dramatic changes of epidermal morphology. We conclude that alpha 9 AchR that are located in the basal and lower suprabasal layers of the epidermis, are crucially involved in terminal differentiation of keratinocytes. The functional significance of other nAchR expressed in the epidermis remains unclear, due to the lack of specific agonists and antagonists.

Quantitative changes in gastric mucosal glycoproteins: effect of cholinergic agonist and vagal nerve stimulation in the rat

The role of the vagus nerve and cholinergic mechanisms in the control of the rat gastric mucin and protein (PROT) release in vivo was investigated. Under urethane anaesthesia (1.25 g kg(-1)), the rats had their gastric lumen perfused with saline. Mucus secretion was measured as a function of adherent mucus on the mucosa surface and the luminal content of sialic acids (SIA), galactose (GAL), pyruvate and PROT. Electrical stimulation of the vagi significantly increased the levels of mucus (3.23 +/- 025 microg g(-1) tissue, P < 0.05), free sialic acid (FS) (0.18 +/- 0.04 mg mL(-1), P < 0.05) and PROT (0.25 +/- 0.003 mg mL(-1), P < 0.05) when compared with control animals. Bilateral cervical vagotomy had no significant effect on adherent mucus or basal levels of PROT, SIA and GAL (P > 0.05) with respect to the control. In both vagotomized and vagal intact animals, the cholinergic agonist (carbachol, 200 mg kg(-1)) significantly increased PROT, adherent mucus and FS (P < 0.05) and decreased bound sialic acid (P > 0.05). There were no visible haemorrhagic streaks on the gastric mucosa of vagotomized, vagal intact and carbachol-treated animals. The results suggest that vagus nerve does not exert a tonic control on gastric glycoprotein secretion in vivo and that cholinergic effect on the mucus secreting cells may be implemented via the intrinsic nerves of the enteric nervous system.

Prolonged treatment with glycerophosphocholine, an acetylcholine precursor, does not disclose the potentiating effect of cholinesterase inhibitors on GHRH-induced somatotroph secretion in anorexia nervosa.

Unlike normal subjects, in patients with anorexia nervosa (AN) the GH response to GHRH is refractory to the increasing and inhibitory effect of cholinergic agonists and antagonists, respectively. This cholinergic impairment could reflect malnutrition-induced exhaustion of acetylcholine (Ach) precursors. We studied whether treatment with glycerophosphocholine (GLY), an Ach precursor, could disclose the potentiating effect of pyridostigmine (PD) on the GH response to GHRH in AN. In 6 young women with AN (AW) we studied the GH response to iv GHRH (1.0 microg/kg) alone and combined with oral PD (120 mg) before and after 1 month of oral treatment with GLY (400 mg thrice daily). Eight age-matched normal women (NW) were studied as controls. Before GLY, basal GH levels in AW were higher (p < 0.05) than in NW. The GH response to GHRH in AW was higher (p < 0.05) than in NW. PD failed to modify the GHRH-induced GH rise in AW, while it enhanced it in NW (p < 0.05). One month treatment with GLY in AW did not modify the GH response to GHRH either alone or combined with PD. This study shows the existence of a derangement in the cholinergic control of somatotroph function in AN and indicates that treatment with Ach precursors does not exert any effect on this impairment. This could reflect primary alterations of cholinergic neurons, though the effectiveness of more prolonged treatment and/or higher doses of cholinergic precursors needs to be verified.

Effect of cholinergic agonists on resting membrane potential of earthworm body wall muscle cells.

Carbachol in a concentration of 5x10(-8) mol/liter does not hyperpolarize, and in a concentration of 5x10(-6) mol/liter depolarizes the membrane of somatic muscle cell in earthworm. d-tubocurarine, alpha-bungarotoxin, atropine, and hexamethonium added to the incubation medium did not abolish the carbachol-induced decrease in resting membrane potential. Each of these drugs alone had no effect on resting membrane potential in muscle cells. Presumably, the acetylcholine-sensitive receptor-channel membrane complex in earthworm muscle cell differs from acetylcholine receptor in skeletal muscle fibers and peripheral neurons of vertebrates.

Cirri of the stalked crinoid Metacrinus rotundus: neural elements and the effect of cholinergic agonists on mechanical properties.

Sea lilies are enigmatic animals due to their scarcity and their biology is comparatively neglected. Cirri, arranged in whorls of five along the sea lily stalk, anchor and support the animal. They consist of ossicles interconnected by collagenous ligaments and by a central canal. Cirri have a well-developed nervous system but lack muscular cells. A light and electron microscopic study was performed to clarify the morphology of the nervous system of the cirri. Two cellular networks were found, one of neuron-like cells and one of cells filled with bullet-shaped organelles. Both networks ramify throughout the cirral ossicles up to the interossicle ligaments. Mechanical tests were performed to analyse the influence of cholinergic agonists on the mechanical properties of these ligaments. In the tests, the cirral ligaments softened after the application of acetylcholine, muscarinic agonists and nicotinic agonists. The reaction time to muscarinic agonists was much slower than to acetylcholine and nicotinic agonists. At low concentrations, muscarinic agonists caused active development of force. No reaction to stimuli was observed in anaesthetized cirri. The data clearly establish the existence of catch connective tissue which can change its mechanical properties under nervous control mediated via nerves with cholinergic receptors. The possible sources of the observed force production are discussed and it is concluded that active contraction of collagenous ligaments causes movement of cirri.

Modulation of inhibitory synaptic potentials in the piriform cortex.

Modulation of inhibitory synaptic potentials in the piriform cortex. Intracellular recordings from pyramidal neurons in brain slice preparations of the piriform cortex were used to test results from a computational model about the effects of cholinergic agonists on inhibitory synaptic potentials induced by stimulation of afferent fibers in layer Ia and association/intrinsic fibers in layer Ib. A simple model of piriform cortex as an associative memory was used to analyze how suppression of inhibitory synaptic transmission influenced performance of the network. Levels of suppression of excitatory synaptic transmission were set at levels determined in previous experimental work. Levels of suppression of inhibitory synaptic transmission were then systematically varied within the model. This modeling work demonstrated that suppression of inhibitory synaptic transmission in layer Ib should be stronger than suppression of inhibitory synaptic transmission in layer Ia to keep activity levels high enough for effective storage. Experimental data showed that perfusion of the cholinergic agonist carbachol caused a significant suppression of inhibitory postsynaptic potentials (IPSPs) in the pyramidal neurons that were induced by stimulation of layer Ib, with a weaker effect on IPSPs induced by stimulation of layer Ia. As previously described, carbachol also selectively suppressed excitatory postsynaptic potentials (EPSPs) elicited by intrinsic but not afferent fiber stimulation. The decrease in amplitude of IPSPs induced by layer Ib stimulation did not appear to be directly related to the decrease in EPSP amplitude induced by layer Ib stimulation. The stimulation necessary to induce neuronal firing with layer Ia stimulation was reduced in the presence of carbachol, whereas that necessary to induce neuronal firing with layer Ib stimulation was increased, despite the depolarization of resting membrane potential. Thus physiological data on cholinergic modulation of inhibitory synaptic potentials in the piriform cortex is compatible with the functional requirements determined from computational models of piriform cortex associative memory function.

Nicotine-evoked nitric oxide release in the rat hippocampal slice

The effects of cholinergic agonists on nitric oxide (NO) release in hippocampal slices from male Sprague–Dawley rats were investigated using electrochemical recording procedures using Nafion and o-phenylenediamine-treated carbon fiber microelectrodes. These microelectrodes are highly selective for NO versus other interferents. Acetylcholine (Ach) with neostigmine, or nicotine was delivered by pressure ejection from pipettes placed within 300 μm of the NO sensors. Both Ach and nicotine produced NO signals ranging from 0.04 to 2.14 μM in the CA1, CA3, and dentate gyrus of the rat hippocampus that lasted for 2–5 min. The Ach responses were not antagonized by the muscarinic antagonist atropine. However, nicotine-evoked responses were partially antagonized by α-bungarotoxin, a finding consistent with α7-nicotinic cholinergic receptors being involved with the effects of nicotine. These data support the hypothesis that nicotine is capable of evoking long lasting NO release in the hippocampus.

Sex Differences in Anti-allodynic Effects of Cholinergic Agonists in a Model of Neuropathic Pain

Sex Differences in Anti-allodynic Effects of Cholinergic Agonists in a Model of Neuropathic Pain

Electrophysiological properties and cholinergic responses of rat ventral oral pontine reticular neurons in vitro

In order to characterize the electrophysiological properties of morphologically identified neurons of the ventral part of the oral pontine reticular (vRPO) nucleus and the effects of cholinergic agonists on them, intracellular recordings were obtained from 45 cells in a rat brain-slice preparation. Intracellular staining was performed with 2% biocytin in potassium acetate (1 M)-filled micropipettes. Results demonstrated the presence of two types of vRPO neurons. Type I cells ( n=12, 24%) were characterized by a break with a decrease of the depolarizing slope following hyperpolarizing pulses which delayed the return to the resting V m and subsequent spike-firing. The delay was antagonized by 4-AP (200–500 μM) which specifically blocks the transient outward K +-mediated current I A. Type II neurons ( n=38, 76%) displayed a typical depolarizing sag during hyperpolarizing current pulses which was blocked by Cs +. This behavior is characteristic of the hyperpolarization-activated current I Q. These two neuronal types displayed different morphological features. Most type I and II cells (100 and 73.7%, respectively) were depolarized by acetylcholine (1–15 μM), carbachol (0.5–1 μM) and muscarine (1–10 μM) through the activation of post-synaptic muscarinic receptors. The remaining type II cells (26.3%) were hyperpolarized (1–10 min, 3–15 mV) through the activation of post-synaptic muscarinic receptors. Results are consistent with the hypothesis that the vRPO could be a neuronal target of Cch in eliciting paradoxical sleep because most of its neurons are activated by muscarinic agonists. © 1979 Elsevier Science B.V. All rights reserved.

Spin-Label Studies on Synaptic Membrane Lipids: Effect of Cholinergic Agonists, Antagonists, and Cholinesterase Inhibitors

The effects on membrane fluidity of inhibitors of acetylcholinesterase (A ChE) as well as those of certain nicotinic agonists have been examined in synaptosomes prepared from the electroplax of Torpedo californica. Electron paramagnetic resonance (EPR) spectrometry was used to monitor changes in the order parameters of three spin-labeled fatty acid probes, doxyl stearic acid derivatives, intercalated in the lipid bilayer of synaptosomal membranes. Neither inhibition of A ChE nor the application of nicotinic ligands significantly influenced the viscosity of the membrane bilayer as judged by changes in the order parameters of the probes. A small, narrow line component of the EPR spectra disappeared when high concentrations of acetylcholine (ACh) were added to the synaptosomes. This effect was not seen with other cholinergic agonists, e.g., car-bachol. The results of this investigation suggest that if anti-A ChEs produce damaging effects on the lipid components of nerve membranes, the effects are indirect, resulting from accumulation of high ACh levels and not from a direct action of the agent on the membrane lipid.

Effect of cholinergic agonists on gastrin release from primary cultures of human antral G cells

BACKGROUND & AIMS: There is conflicting evidence concerning whether muscarinic regulation of gastrin release in humans is a direct or indirect effect on antral G cells. The present experiments were designed to resolve this question using an isolated human G-cell preparation.METHODS: The ability of muscarinic agonists to stimulate or inhibit gastrin release was assessed with or without an immunoneutralizing somatostatin antibody or an m3 receptor antagonist. The effect of secretogogues on G and D cells was monitored by intracellular calcium imaging.RESULTS: Muscarinic agonists failed to stimulate gastrin release, even after the removal of somatostatin- induced inhibition. Our group has previously shown that muscarinic agonists stimulated somatostatin release from antral D cells. Methacholine (100 mumol/L) increased intracellular calcium levels in cocultured D cells; this increase was inhibited by the m3 receptor antagonist 4-diphenylacetoxy-n-methylpiperidine methiodide. Gastrin cells in the same field of view lacked a response to methacholine but showed a clear response to 10 nmol/L bombesin.CONCLUSIONS: The experiments indicate that vagal control of gastrin release in humans is indirect; stimulation would be achieved by the activation of intrinsic gastrin-releasing peptide neurons, and inhibition would be via the paracrine action of somatostatin released from adjacent D cells.(Gastroenterology 1997 Feb;112(2):357-63)

Muscarinic and nicotinic receptors raise intracellular Ca2+ levels in rat carotid body type I cells.

1. The effects of cholinergic agonists upon intracellular free Ca2+ levels ([Ca2+]i) have been studied in enzymically isolated rat carotid body single type I cells, using indo-1. 2. Acetylcholine (ACh) dose-dependently increased [Ca2+]i in 55% of cells studied (EC50 = 13 microM). These [Ca2+]i rises were partially inhibited by atropine or mecamylamine. 3. Specific nicotinic and muscarinic agonists also elevated [Ca2+]i in a dose-dependent manner (nicotine, EC50 = 15 microM; methacholine, EC50 = 20 microM). 4. While the majority of the ACh-sensitive cells responded to both classes of cholinergic agonist, 29% responded exclusively to nicotinic stimulation and 9% responded exclusively to muscarinic stimulation. 5. In the presence of nicotinic agonists, Ca2+i responses were transient. In the presence of muscarinic agonists, Ca2+i responses consisted of an initial rise, which then declined to a lower plateau level. 6. Nicotinic responses were rapidly abolished in Ca(2+)-free medium, suggesting that they are dependent on Ca2+ influx. 7. The plateau component of the muscarinic-activated response was also abolished in Ca(2+)-free conditions. The rapid initial [Ca2+]i rise, however, could still be evoked after several minutes in Ca(2+)-free medium. Muscarine also increased Mn2+ quenching of intracellular fura-2 fluorescence. These data suggest that the full muscarinic response depends on both Ca2+ release from intracellular stores and Ca2+o influx. 8. The results indicate that, in rat carotid body type I cells, both nicotinic and muscarinic acetylcholine receptors increase [Ca2+]i, but achieve this via different mechanisms. ACh may therefore play a role in carotid body function by modulating Ca2+i in the chemosensory type I cells.

Effect of cholinergic agonists on bulbospinal C1 neurons in rats.

Cholinergic inputs to the rostral ventrolateral medulla (RVLM) may contribute to sympathetic tone generation. The present study analyzes the response of RVLM neurons to cholinergic agonists. In chloralose-anesthetized rats iontophoresis of carbachol excited RVLM sympathoexcitatory neurons (+69% from resting level of 11.9 +/- 2 spikes/s; n = 28). This effect was reduced 85% by iontophoresis of methylatropine and abolished by intravenous scopolamine. Iontophoresis of nicotine or hexamethonium was ineffective. In contrast, most RVLM respiratory units were inhibited by carbachol. Whole cell recordings of bulbospinal RVLM neurons were made in neonatal rat brain slices (54 cells, 24 C1 adrenergic neurons). In current-clamp recordings (without tetrodotoxin) carbachol produced depolarization, increased postsynaptic potential frequency, and decreased input resistance. In voltage-clamp recording (-50 to -60 mV; 1 microM tetrodotoxin) carbachol produced inward current [50% effective concentration (EC50): 10 +/- 1 microM; 12.6 +/- 2 pA at 30 microM; n = 16] that persisted in low Ca2+/high Mg2+ (n = 6). Muscarine (30 microM) caused smaller inward currents (2.6 +/- 0.6 pA; n = 16). The carbachol-induced current was reduced 46% by 5 microM methylatropine (n = 15) and 84% by 200 microM hexamethonium (n = 9). The current was linear as a function of the holding potential (extrapolated reversal potential: -22 +/- 2 mV). In conclusion, carbachol exerts both pre- and postsynaptic effects on C1 and other putative sympathoexcitatory RVLM neurons. In vitro the postsynaptic effect of carbachol has a mixed nicotinic and muscarinic pharmacology. In vivo, iontophoretically applied carbachol produces muscarinic excitation of barosensitive RVLM neurons.