M2 Muscarinic Receptor Subtypes Research Articles

Forebrain acetylcholine regulates adult hippocampal neurogenesis and learning

Hippocampus-mediated learning enhances neurogenesis in the adult dentate gyrus (DG), and this process has been suggested to be involved in memory formation. The hippocampus receives abundant cholinergic innervation and acetylcholine (ACh) plays an important role in learning and Alzheimer's disease (AD) pathophysiology. Here, we show that a selective neurotoxic lesion of forebrain cholinergic input with 192 IgG-saporin reduces DG neurogenesis with a concurrent impairment in spatial memory. Conversely, systemic administration of the cholinergic agonist physostigmine increases DG neurogenesis. We find that changes of forebrain ACh levels primarily influence the proliferation and/or the short-term survival rather than the long-term survival or differentiation of the new neurons. We further demonstrate that these newly born cells express the muscarinic receptor subtypes M1 and M4. Our data provide evidence that forebrain ACh promotes neurogenesis, and suggest that the impaired cholinergic function in AD may in part contribute to deficits in learning and memory through reductions in the formation of new hippocampal neurons.

Muscarinic stimulation of pancreatic insulin and glucagon release is abolished in m3 muscarinic acetylcholine receptor-deficient mice.

Pancreatic muscarinic acetylcholine receptors play an important role in stimulating insulin and glucagon secretion from islet cells. To study the potential role of the M(3) muscarinic receptor subtype in cholinergic stimulation of insulin release, we initially examined the effect of the muscarinic agonist, oxotremorine-M (Oxo-M), on insulin secretion from isolated pancreatic islets prepared from wild-type (WT) and M(3) receptor-deficient mice (M3(+/-) and M3(-/-) mice). At a stimulatory glucose level (16.7 mmol/l), Oxo-M strongly potentiated insulin output from islets of WT mice. Strikingly, this effect was completely abolished in islets from M3(-/-) mice and significantly reduced in islets from M3(+/-) mice. Additional in vitro studies showed that Oxo-M-mediated glucagon release was also virtually abolished in islets from M3(-/-) mice. Consistent with the in vitro data, in vivo studies showed that M3(-/-) mice displayed reduced serum insulin and plasma glucagon levels and a significantly blunted increase in serum insulin after an oral glucose load. Despite the observed impairments in insulin release, M3(-/-) mice showed significantly reduced blood glucose levels and even improved glucose tolerance, probably due to the reduction in plasma glucagon levels and the fact that M3(-/-) mice are hypophagic and lean. These findings provide important new insights into the metabolic roles of the M(3) muscarinic receptor subtype.

The M2 muscarinic receptor mediates in vitro bladder contractions from patients with neurogenic bladder dysfunction.

Bladder muscle specimens from seven patients with neurogenic bladder dysfunction were analyzed to determine whether the muscarinic receptor subtype mediating contraction shifts from M(3) to the M(2) subtype as found in the denervated, hypertrophied rat bladder. Seven bladder specimens were analyzed from six female and one male patients. Six of the patients had traumatic cervical spinal cord injuries (C(4)-C(7)), and the other patient had an L(1) congenital myelomeningocele. This was compared with results from bladder specimens obtained from eight organ transplant donors. The affinities of three subtype-selective muscarinic receptor antagonists for inhibition of carbachol-induced contractions were determined. The affinity of the M(3) selective antagonists darifenacin or p-fluoro-hexahydrosiladifenadol (p-F-HHSiD) was determined in six of the seven spinal injury patient specimens. The affinity was consistent with M(2)-mediated contractions in four of these six specimens, intermediate between M(2) and M(3) in one specimen, and within the M(3) range in one specimen. The other specimen, tested only with the M(2) selective antagonist methoctramine, showed an M(3) affinity. In the organ donors, the affinity of p-F-HHSiD was within the M(2) range for six of seven specimens, whereas the affinity of darifenacin was within the M(3) range for five of six and intermediate between M(2) and M(3) for the other specimen tested. The affinity of methoctramine in both organ donor specimens tested was within the M(3) range. Whereas normal detrusor contractions are mediated by the M(3) receptor subtype, in patients with neurogenic bladder dysfunction as well as certain organ transplant donors, contractions can be mediated by the M(2) muscarinic receptor subtype.

Histamine H1 Receptor Down-Regulation Mediated by M3 Muscarinic Acetylcholine Receptor Subtype

Heterologous down-regulation of histamine H1 receptor (H1R) mediated by muscarinic acetylcholine receptor subtype was investigated using five kinds of Chinese hamster ovary (CHO) cells stably co-expressing the human H1R and one of the five (M1 –M5) muscarinic acetylcholine receptors, CHO-H1/M1, CHO-H1/M2, CHO-H1/M3, CHO-H1/M4, and CHO-H1/M5 cells. Among the CHO-H1/M1, CHO-H1/M3, and CHO-H1/M5 cells, carbachol treatment of the CHO-H1/M3 cells time-dependently led to remarkable down-regulation of the H1R to 60% of the control level. In contrast, stimulation of CHO-H1/M1 cells by carbachol induced negligible effect on the down-regulation. Stimulation of CHO-H1/M5 cells by carbachol induced significant but only small H1R down-regulation. M2 and M4 muscarinic receptors showed negligible effect on the down-regulation. H1R-mediated accumulation of inositol phosphates in CHO-H1/M3 cells with long-term expose to carbachol was decreased to 60% compared with non-treated cells. Heterologous phosphorylation of H1R was induced by the stimulation of each muscarinic receptor. H1R was phosphorylated by about twofold from the basal level through five subtypes of muscarinic receptor. The M3 muscarinic receptor-mediated phosphorylation of H1R was reversed by the inhibition of protein kinase C. In the present study we demonstrated that the M3 muscarinic acetylcholine receptor mediated remarkable down-regulation of the H1R with decreased receptor signaling.

Mechanisms mediating cholinergic antral circular smooth muscle contraction in rats

To investigate the pathway (s) mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent, bethanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction. Circular smooth muscle strips from the antrum of Sprague-Dawley rats were mounted in muscle baths in Krebs buffer. Isometric tension was recorded. Cumulative concentration-response curves were obtained for (+)-cis-dioxolane (cD), a nonspecific muscarinic agonist, at 10(-8)-10(-4) mol/L, in the presence of tetrodotoxin (TTX, 10(-7) mol/L). Results were normalized to cross sectional area. A repeat concentration-response curve was obtained after incubation of the muscle for 90 min with antagonists for M1 (pirenzepine), M2 (methoctramine) and M3 (darifenacin) muscarinic receptor subtypes. The sensitivity to PTX was tested by the ip injection of 100 mg/kg of PTX 5 d before the experiment. The antral circular smooth muscles were removed from PTX-treated and non-treated rats as strips and dispersed smooth muscle cells to identify whether PTX-linked pathway mediated the contractility to bethanechol. A dose-dependent contractile response observed with bethanechol, was not affected by TTX. The pretreatment of rats with pertussis toxin decreased the contraction induced by bethanechol. Lack of calcium as well as the presence of the L-type calcium channel blocker, nifedipine, also inhibited the cholinergic contraction, with a reduction in response from 2.5+/-0.4 g/mm2 to 1.2+/-0.4 g/mm(2) (P<0.05). The dose-response curves were shifted to the right by muscarinic antagonists in the following order of affinity: darifenacin (M(3))>methocramine (M(2)) >pirenzepine (M(1)). The muscarinic receptors-dependent contraction of rat antral circular smooth muscles was linked to the signal transduction pathway(s) involving pertussis-toxin sensitive GTP-binding proteins and to extracellular calcium via L-type voltage gated calcium channels. The presence of the residual contractile response after the treatment with nifedipine, suggests that an additional pathway could mediate the cholinergic contraction. The involvement of more than one muscarinic receptor (functionally predominant type 3 over type 2) also suggests more than one pathway mediating the cholinergic contraction in rat antrum.

The anti-apoptotic response of the Gq/11-coupled muscarinic receptor family.

It is now clear that G-protein-coupled receptors can regulate programmed cell death (apoptosis) through a variety of mechanisms that are dependent on cell type and receptor subtype. Here we present evidence that the G(q/11)-coupled subtypes of the muscarinic receptor family (namely M(1), M(3) and M(5)-muscarinic receptor subtypes) are able to protect against apoptotic cell death. In particular we demonstrate that the C-terminal tail of the M(3)-muscarinic receptor is an essential structural element for signalling to the anti-apoptotic pathway. Removal of the distal portion of the C-terminal tail results in a receptor that is coupled normally to the G(q/11)/phospholipase C pathway and the mitogen-activated protein kinase pathway, but is unable to couple to the anti-apoptotic pathway. Furthermore, a poly-basic region conserved within the C-terminal tail of the G(q/11)-coupled muscarinic receptor subtypes appears to be the structural determinant of coupling to the anti-apoptotic pathway.

Endothelin-1 synthesis and secretion in human retinal pigment epithelial cells (ARPE-19): differential regulation by cholinergics and TNF-alpha.

Endothelin (ET)-1 can produce nerve damage analogous to that in optic neuropathies such as glaucoma. The precise source of endothelin in the posterior segment of the eye remains unclear. The present study was conducted to determine whether the retinal pigment epithelium (RPE), which helps maintain the outer blood-retinal barrier, is a local source for ET-1 and whether the amount of ET-1 secreted by the RPE is be differentially regulated by cholinergics and the cytokine TNF-alpha. Human retinal pigment epithelial cells (ARPE-19) were cultured either to a mature state (mRPE) for 4 weeks, with well-defined tight junctions, or to a young state (yRPE) for 4 days, with incompletely formed tight junctions. ET-1-like immunoreactivity was determined by immunocytochemistry, and secreted ET-1 was quantified by radioimmunoassay in both cell types. Cells were stimulated with the cholinergic agonist carbachol or with the cytokine TNF-alpha for specific periods. The expression of muscarinic receptor subtypes M1 and M3 and the peripheral membrane protein ZO-1 were analyzed by immunoblot and immunocytochemistry, respectively. Expression of preproendothelin-1 (ppET-1) mRNA after application of different stimuli at specific time points was determined by real-time RT-PCR. Carbachol-mediated elevation in intracellular calcium ([Ca2+]i) was also measured in the presence or absence of a selective muscarinic receptor antagonist. Constitutive synthesis and secretion of ET-1 was greater in mRPE than in yRPE cells. TNF-alpha caused a significant increase in ppET-1 mRNA levels and ET-1 secretion in both phenotypes. The disruption and subsequent breakdown of the tight junction barrier was evident in either phenotype after treatments with TNF-alpha. There was a concentration-dependent increase in [Ca2+]i in both y- and mRPE cells in response to CCh. CCh at 1 microM significantly increased ET-1 secretion, a response observed in yRPE but not in mRPE cells. This effect may be mediated primarily by the M3 receptor subtype and is phospholipase C (PLC) dependent. Regulation of ET-1 release in ARPE-19 cells was differentially regulated by TNF-alpha and CCh and was dependent on the age of the culture. RPE may be a source for ET-1 in the retina, and its increased release may become more important during breakdown of the blood-retinal barrier, as seen after TNF-alpha treatment.

Tiotropium (Spiriva) - a long-acting inhaled anticholinergic for the treatment of chronic obstructive pulmonary disease (COPD)

Anticholinergics are agents of first choice for the symptomatic treatment of patients with COPD. Tiotropium (Ba 679 BR, Spiriva) is a long-acting inhaled anticholinergic designed for once-daily bronchodilator treatment of COPD. Tiotropium is a selective antagonist of pulmonary M1 and M3 muscarinic receptor subtypes, that produces a long-lasting (24 hours), dose-dependent bronchodilation and bronchoprotection against constrictive stimuli, e. g. methacholine, following inhalation of single doses. Clinical trials with tiotropium in COPD patients over a maximum treatment duration of one year have confirmed a persisting bronchodilator effect of tiotropium compared with placebo and ipratropium, as well as meaningful clinical improvements in lung function, hyperinflation, exercise tolerance, symptom control and quality of life. Moreover, recent trials indicate that treatment with tiotropium also reduces the frequency of COPD exacerbations and hospitalizations. Comparative trials further suggest that the bronchodilator potency of tiotropium may be superior to those of available COPD treatments. Besides a higher incidence of dry mouth, the side effect profile was comparable to ipratropium bromide. In conclusion, present clinical data suggest that tiotropium has the potential of a first-line treatment for patients with COPD.

Nuclear factor κb activation by muscarinic receptors in astroglial cells: effect of ethanol

Activation of muscarinic receptors leads to proliferation of astroglial cells and this effect is inhibited by ethanol. Among the intracellular pathways involved in the mitogenic action of muscarinic agonists, activation of the atypical protein kinase C ζ (PKC ζ) appears to be of most importance, and is also affected by low ethanol concentrations. PKC ζ has been reported to activate nuclear factor κB (NF-κB), a transcription factor that has been shown to play an important role in cell proliferation. The aim of this study was, therefore, to determine whether muscarinic receptors would activate NF-κB in astroglial cells, whether such activation would play a role in the mitogenic action of muscarinic agonists, and whether it would represent a possible target for ethanol. Carbachol activated NF-κB in human 1321N1 astrocytoma cells, as evidenced by translocation of the p65 subunit of NF-κB to the nucleus, phosphorylation and degradation of IκBα in the cytosol, and increase NF-κB binding to DNA. Carbachol also induced translocation of p65 to the nucleus in primary rat astrocytes. Carbachol-induced NF-κB activation was mediated by the M3 subtype of muscarinic receptors and appeared to involve Ca2+ mobilization and activation of PKC ϵ and PKC ζ, but not PI3-kinase and mitogen-activated protein kinase. The NF-κB peptide inhibitor SN50, but not the inactive peptide SN50M, strongly inhibited carbachol-induced astrocytoma cells proliferation and p65 translocation to the nucleus. Increased DNA synthesis was also antagonized by the IκBα kinase inhibitor BAY 11–7082. Ethanol (25–100 mM) inhibited the translocation of p65 and the binding of NF-κB to DNA in both 1321N1 astrocytoma cells and primary rat cortical astrocytes. Together, these results suggest that activation of NF-κB by muscarinic receptors in astroglial cells is important for carbachol-induced DNA synthesis and that ethanol-mediated inhibition of cell proliferation may be due in part to inhibition of NF-κB activation.

Muscarinic receptors control glucagon-like peptide 1 secretion by human endocrine L cells.

Glucagon-like peptide 1 (GLP-1) released from distal intestinal endocrine L cells after food intake is a potent glucose-dependent stimulant of insulin secretion. Plasma levels of GLP-1 rise rapidly after nutrient ingestion through an indirect mechanism triggered from the proximal intestine and involving the vagus nerve. Our previous studies showed the involvement of M1 muscarinic receptors expressed by the L cells in the regulation of postprandial GLP-1 secretion in rats. The goal of this study was to explore the involvement of muscarinic receptors in the regulation of GLP-1 secretion by human L cells using a newly described human L cell line (NCI-H716). Phorbol 12-myristate 13-acetate (positive control) stimulated GLP-1 secretion to 252 +/- 38% of the control (P < 0.001). Bethanechol, a nonselective muscarinic agonist, significantly stimulated GLP-1 secretion to 187 +/- 20% of the control (P < 0.01, n = 8). Pirenzepine (M1 antagonist; 10-1000 microM) and gallamine (M2 antagonist; 10-1000 microM) completely inhibited bethanechol-induced GLP-1 secretion, whereas 4-diphenylacetoxy-N-methylpiperidine (M3 antagonist) had no effect on bethanechol-stimulated GLP-1 secretion. McN-A-343 (M1 muscarinic agonist) dose dependently stimulated GLP-1 secretion (to 252 +/- 50% of control at 1000 microM; P < 0.01), whereas oxotremorine (M3 agonist) had no effect. M1, M2, and M3 muscarinic receptors were shown to be expressed in NCI-H716 cells by Western blot, immunohystochemistry, and RT-PCR. Expression of the M1, M2, and M3 muscarinic receptor subtypes was also confirmed in paraffin-embedded human small intestine sections by double immunofluorescent staining. These results demonstrate the role of M1 and M2 muscarinic receptors expressed by human L cells in the control of GLP-1 secretion.

Two distinct calcium-sensitive and -insensitive PKC up- and down-regulate an alpha-bungarotoxin-resistant nAChR1 in insect neurosecretory cells (DUM neurons).

While there is mounting knowledge about the structure and diversity of insect neuronal nicotinic acetylcholine receptors, less attention has been directed towards their intracellular regulation by calcium-mediated activation or inhibition of protein phosphorylation. The main goal of this work was to delineate the chain of molecular events that lead to the up- and down-regulation by two protein kinase Cs of an insect neuronal alpha-bungarotoxin-resistant nicotinic acetylcholine receptor (called nAChR1). The native nicotinic acetylcholine receptor intracellular regulation was studied on dissociated adult dorsal unpaired median neurons isolated from the terminal abdominal ganglion of the cockroach Periplaneta americana using whole-cell patch-clamp technique and calcium imaging. We report that under 0.5 micro malpha-bungarotoxin treatment, the inward current produced by pressure ejection application of nicotine onto the cell body was differentially sensitive to specific protein kinase C activators and inhibitors. The phorbol ester PMA produced a calcium-dependent increase in current amplitude blocked by chelerythrine. By contrast, the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol produced a calcium-independent reduction of the nicotinic response, reversed by rottlerin and chelerythrine. This indicated that two protein kinase C isozymes ('classical' and 'novel' protein kinase C, named PKC1 and PKC2, respectively) up- and down-regulated nicotinic acetylcholine receptor function. PMA and 1,2-dioctanoyl-sn-glycerol effects were mimicked by pirenzepine-sensitive M1 muscarinic receptor subtype coupled to phospholipase C second messenger pathway. Low concentration of muscarine elevated internal calcium levels, which thereby activated PKC1. By contrast, a high concentration of muscarine strongly increased [Ca 2+]i, which induced inhibition of PKC1. This effect was reversed by FK506, suggesting the implication of PP2B which unmasked PKC2 activity mediating down-regulation of nicotinic acetylcholine receptor.

Cooperative enhancement of insulinotropic action of GLP-1 by acetylcholine uncovers paradoxical inhibitory effect of beta cell muscarinic receptor activation on adenylate cyclase activity

The cooperative effect of glucagon-like peptide 1 (GLP-1) and acetylcholine (ACh) was evaluated in a beta cell line model (BRIN BD11). GLP-1 (20 nM) and ACh (100 μM) increased insulin secretion by 24–47%, whereas in combination there was a further 89% enhancement of insulin release. Overnight culture with 100 ng/mL pertussis toxin (PTX) or 10 nM PMA significantly reduced the combined insulinotropic action ( P<0.05 and P<0.001, respectively) and the sole stimulatory effects of GLP-1 (PTX treatment; P<0.01) or ACh (PMA treatment; P<0.05). Under control conditions, ACh (50 nM–1 mM) concentration-dependently inhibited by up to 40% ( P<0.001) the 10-fold ( P<0.001) elevation of cyclic 3′,5′-adenosine monophosphate (cAMP) induced by 20 nM GLP-1. The paradoxical inhibitory action of ACh was abolished by PTX pre-treatment, suggesting involvement of G i and/or G o G protein alpha subunit. Effects of selective muscarinic receptor antagonists on the concentration-dependent insulinotropic actions of ACh (50 nM–1 mM) on 20 nM GLP-1 induced insulin secretion revealed inhibition by ρ-FHHSiD (M3 antagonist, P<0.05), stimulation with pirenzepine (M1 antagonist, P<0.001) and no significant effects of either methoctramine (M2 antagonist) or MT-3 (M4 antagonist). Antagonism of M2, M3 and M4 muscarinic receptor effects with methoctramine (3–100 nM), ρ-FHHSiD (3–30 nM) or MT-3 (10–300 nM) did not significantly affect the inhibitory action of ACh on GLP-1 stimulated cAMP production. In contrast, M1 receptor antagonism with pirenzepine (3–300 nM) resulted in a concentration-dependent decrease in the inhibitory action of ACh on GLP-1 stimulated cAMP production ( P<0.001). These data indicate an important functional cooperation between the cholinergic neurotransmitter ACh and the incretin hormone GLP-1 on insulin secretion mediated through the M3 muscarinic receptor subtype. However, the insulinotropic action of ACh was associated with a paradoxical inhibitory effect on GLP-1 stimulated cAMP production, achieved through a novel PTX- and pirenzepine-sensitive M1 muscarinic receptor activated pathway. An imbalance between these pathways may contribute to dysfunctional insulin secretion.

Effects of muscarinic toxins MT2 and MT7, from green mamba venom, on m1, m3 and m5 muscarinic receptors expressed in Chinese Hamster Ovary cells

Several small proteins called muscarinic toxins (MTs) have been isolated from venom of green mamba ( Dendroaspis angusticeps). They have previously been shown in radioligand binding studies to have high selectivity and affinity for individual muscarinic receptor subtypes, but less is known of their functional effects. This study has examined the actions of two of these MTs, MT2 and MT7, using changes in cytosolic Ca 2+ ([Ca 2+] i ) measured using the fluorescent indicator fura-2 in Chinese Hamster Ovary (CHO) cells stably transfected with individual muscarinic receptor subtypes, m1, m3 and m5. MT2 activated the m1 receptor: at concentrations above 100 nM it caused significant and concentration-dependent increases in [Ca 2+] i . From 25 to 800 nM MT2 also produced increases in [Ca 2+] i by activating m3 receptors, although these increases in [Ca 2+] i were not strictly concentration-dependent with only intermittent responses being recorded (i.e. it was not always possible to obtain a response to the agonist with each application of the compound). MT2 (800–1600 nM) also caused significant increases in [Ca 2+] i in CHO cells expressing the m5 muscarinic receptor subtype. MT7 (1 μM) displayed no agonist activity at any of the muscarinic receptors but was a potent non-competitive antagonist (at 20 nM) at the m1 muscarinic receptor subtype. It had no antagonist activity at the m3 or m5 subtypes. These results indicate that MT7 is a highly specific antagonist at the m1 muscarinic receptor subtype as suggested by results from radioligand binding studies. However, MT2 is less selective for the m1 muscarinic receptor than previously described as it also exhibits agonist activity at the m3 and m5 muscarinic receptors, which was not detected in radioligand binding studies.

The Role of M2 Muscarinic Receptor Subtypes Mediating Contraction of the Circular and Longitudinal Smooth Muscle of the Pig Proximal Urethra

The Role of M2 Muscarinic Receptor Subtypes Mediating Contraction of the Circular and Longitudinal Smooth Muscle of the Pig Proximal Urethra

The Role of M2 Muscarinic Receptor Subtypes Mediating Contraction of the Circular and Longitudinal Smooth Muscle of the Pig Proximal Urethra

We investigated the characterization of muscarinic receptor subtypes in the female pig urethra. The affinities of muscarinic antagonists against carbachol responses were calculated in normal and cyclic adenosine monophosphate (cAMP) elevated tissues (contraction with KCl and relaxation with isoprenaline) using longitudinal and transverse strips of urethra. In displacement experiments with 1-quinuclidinyl [phenyl-4-3H] benzilate inhibitory constant (pKi) values of 4-diphenylacetoxyl-N-methyl-piperidine methiodide (DAMP) M3 selective antagonist) and methoctramine (M2 antagonist) indicated the presence of the M2 receptor. In functional studies contraction responses to carbachol were greater in longitudinal than in circular muscle. After cAMP elevation the contraction response increased in circular muscles to the level close to that of longitudinal muscles but did not change significantly in cAMP elevated longitudinal muscle. On normal circular tissues 4-DAMP had high mean affinity (mean apparent pKB value 9.3) but with a Schild slope of less than unity (0.76). Methoctramine competitively antagonized carbachol responses (mean affinity [pA2] value 6.9). On normal longitudinal tissues 4-DAMP and methoctramine competitively antagonized carbachol responses (mean pA2 9.0 and 6.2, respectively). After cAMP elevation in circular tissues mean pA2 values for 4-DAMP (8.7) were significantly lower (p = 0.0015), and those for methoctramine (7.3) were significantly higher (p = 0.0193) than in normal tissues. In longitudinal tissues the mean pA2 value for methoctramine (6.9) was significantly greater than in normal tissues (p <0.0001) but the value for 4-DAMP (8.8) did not alter. Pig urethra appears to have predominantly M2 muscarinic receptors. Contraction of the normal urethra appears to be predominantly mediated by M2 and M3 receptors in circular muscle but by M3 receptors in longitudinal muscle. After cAMP elevation a contribution to contraction of M2 receptors appeared to be demonstrated in the 2 tissues but the involvement of M2 receptors appeared greater in circular muscle.

Immunohistochemical localization of subtypes of muscarinic receptors in human inferior turbinate mucosa.

The regulation of glandular secretions and vasomotor tone in human nasal mucosa implicates muscarinic receptors. There are 5 recognized classes (m1 through m5) of muscarinic receptor subtypes, and the aim of our study was to localize muscarinic receptor subtypes (m1 through m5) in human inferior turbinate mucosa by an immunohistochemical method. We found m1 and m2 receptors distributed on glands, arteries, veins, and epithelia; m4 receptors were found around arteries; and m5 receptors were identified on glands and arteries. We found m3 receptors to be the most extensively distributed on glands, arteries, and veins of all of the muscarinic receptor subtypes. The m3 receptor is probably important in the physiology of the human inferior turbinate. This study may help identify the best target for more selective muscarinic drugs and guide the treatment of allergic and nonallergic rhinitis.

Septal cholinergic mediation of hippocampal theta in the cat

The effects of intraseptally microinjected muscarinic (atropine sulfate, pirenzepine and gallamine) and nicotinic (hexamethonium) antagonists on spontaneous, sensory and electrically-induced hippocampal (HPC) theta EEG activity were investigated in the freely behaving cat. Administration of hexamethonium failed to elicit a detectable effect on HPC theta. Injections of atropine and pirenzepine abolished, whereas the injection of gallamine only reduced hippocampal theta. Moreover, a gradual recovery of theta amplitude and power was observed, while frequency recovered rapidly. Our data provide further evidence that the septal M1 and M2 muscarinic receptor subtypes mediate the HPC theta in this species. Intraseptal microinjection of cholinergic agonist (carbachol) produced almost a continuous HPC theta with increased amplitude and power. The contribution of the medial septal cholinergic projections to HPC theta frequency and amplitude was also discussed.

Characterization and gene organization of Taiwan banded krait (Bungarus multicinctus) gamma-bungarotoxin.

gamma-Bungarotoxin was isolated from Bungarus multicinctus (Taiwan banded krait) venom using a combination of chromatography on a SP-Sephadex C-25 column and a reverse-phase high-performance liquid chromatography column. Circular dichroism (CD) measurement revealed that its secondary structure was dominant with beta-sheet structure as is that of snake venom alpha-neurotoxins and cardiotoxins. gamma-Bungarotoxin exhibits activity on inhibiting the binding of [3H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine receptor subtype, and competes weakly with radioiodinated alpha-bungarotoxin for binding to the Torpedo nicotinic acetylcholine receptor. Moreover, the toxin inhibits collagen-induced platelet aggregation, with an IC50 of approximately 200 nM. The genomic DNA encoding the gamma-bungarotoxin precursor is amplified by polymerase chain reaction (PCR). The gene is organized with three exons separated by two introns, and shares virtually identical overall organization with those reported for alpha-neurotoxin and cardiotoxin genes, including similar intron insertions. The intron sequences of these genes share sequence identity up to 85%, but the exon sequences are highly variable. These observations suggest that gamma-bungarotoxin, alpha-neurotoxins, and cardiotoxins originate from a common ancestor, and the evolution of these genes shows a tendency to diversify the functions of snake venom proteins.

Diagnosing, managing, and preventing salivary gland disorders.

Salivary function provides host protection, assists in the initiation of food and fluid intake, and enables communication through speech. Without adequate salivary output, oral and pharyngeal health declines along with a person’s quality of life. The complaint of a dry mouth (xerostomia) and the objective finding of salivary dysfunction are common occurrences in older individuals, producing transient and permanent oral and systemic problems. Salivary dysfunction, however, is not a normal consequence of growing older, and is due to systemic diseases, medications, and head and neck radiotherapy. Diagnosis of salivary disorders begins with a careful medical history, head, and neck examination. While complaints of xerostomia may be indicative of a salivary gland disorder, salivary diseases can present without symptoms. Therefore, routine examination of salivary function must be part of any head, neck, and oral examination. Therapies are designed to prevent the development of oral and pharyngeal sequelae of salivary hypofunction. Current xerostomia-based treatments include replacement therapies and gustatory, masticatory, and pharmacological stimulants. Healthcare professionals can play a vital role in identifying patients at risk for developing salivary dysfunction, and should provide appropriate preventative and interventive techniques that will help preserve a person’s health, function, and quality of life.

Muscarinic receptor subtypes are differentially distributed in the rat cochlea

Five different genes encode the muscarinic acetylcholine receptors. The muscarinic receptor subtypes M1, M3, and M5 are typically coupled to activation of the Gα q/11-phosphatidyl inositol pathway, whereas the M2 and M4 subtypes are typically linked to Gα i and adenylyl cyclase inhibition. In order to localize muscarinic receptors in the rat cochlea, we applied polyclonal antibodies for subtypes M1, M2, M3, and M5, and monoclonal antibody for subtype M4 to paraffin sections. In the organ of Corti, outer hair cells exhibited strong immunoreactivity for M3 and weak immunoreactivity for M1. Deiters’ cells were strongly immunoreactive to antibodies for the M1 and M2 subtypes, with weak staining observed for M3, and weaker yet for M5. Inner hair cells showed moderate immunoreactivity for the M1 subtype, weaker staining for the M5 subtype, and slight staining for the M3 subtype. Among the spiral ganglion neurons, weak to moderate immunoreactivity was detected for M3 and M5 subtypes and weak staining was observed for the M1 subtype. The efferent fibers of the intraganglionic spiral bundle were positive for M2 and M5. In the lateral wall, weak to moderate staining was detected for M5 in the stria vascularis corresponding in position to the basolateral extensions of marginal cells. Staining for M3 was observed associated with capillaries. Fibrocytes of the spiral ligament exhibited limited but selective subtype immunoreactivity. No immunoreactivity was detected in the cochlea for the M4 subtype. From the present findings we suggest that M3 is the primary muscarinic receptor subtype in outer hair cells mediating a postsynaptic response to the medial olivocochlear cholinergic efferent input. The muscarinic receptor subtypes M1, M3, and M5 appear to subserve the action of cholinergic lateral olivocochlear efferent stimulation on postsynaptic responses in type I afferents. Whether M1, M3, and M5 protein in inner hair cells indicates constitutive or vestigial expression remaining from development is unknown. M2 and M5 muscarinic receptors expressed presynaptically may modulate the efferent signal. Finally, expression by Deiters’ cells of several muscarinic subtypes raises the possibility that cholinergic efferents couple to these non-sensory cells through muscarinic receptors.