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Atrial fibrillation (AF) is the most common arrhythmia seen in general practice. Muscarinic ACh receptors (M2R, M3R) are involved in vagally induced AF. M2R and M3R activate the heterotrimeric G proteins, [G.sub.i] and [G.sub.q], respectively, by promoting GTP binding, and these in turn activate distinct [K.sup.+] channels. Signaling is terminated by GTP hydrolysis, a process accelerated by regulator of G protein signaling (RGS) proteins. RGS2 is selective for [G.sub.q] and thus may regulate atrial M3R signaling. We hypothesized that knockout of RGS2 ([RGS2.sup.-/-] ) would render the atria more susceptible to electrically induced AF. One-month-old male [RGS2.sup.-/-] and C57BL/6 wild-type (WT) mice were instrumented for intracardiac electrophysiology. Atrial effective refractory periods (AERPs) were also determined in the absence and presence of carbachol, atropine, and/or the selective M3R antagonist darifenacin. Susceptibility to electrically induced AF used burst pacing and programmed electrical stimulation with one extrastimulus. Real-time RT-PCR measured atrial and ventricular content of RGS2, RGS4, M2R, M3R, and M4R mRNA. AERP was lower in [RGS2.sup.-/-] compared with WT mice in both the high right atrium (HRA) (30 [+ or -] 1 vs. 34 [+ or -] 1 ms, P < 0.05) and mid right atrium (MRA) (21 [+ or -] 1 vs. 24 [+ or -] 1 ms, P < 0.05). Darifenacin eliminated this difference (HRA: 37 [+ or -] 2 vs. 39 [+ or -] 2 ms, and MRA: 30 [+ or -] 2 vs. 30 [+ or -] 1, P > 0.4). [RGS2.sup.-/-] were more susceptible than WT mice to atrial tachycardia/fibrillation (AT/F) induction (11/22 vs. 1/25, respectively, P < 0.05). Muscarinic receptor expression did not differ between strains, whereas M2R expression was 70-fold higher than M3R (P < 0.01). These results suggest that RGS2 is an important cholinergic regulator in the atrium and that [RGS2.sup.-/-] mice have enhanced susceptibility to AT/F via enhanced M3 muscarinic receptor activity. autonomic; atrial fibrillation; electrophysiology; murine doi: 10.1152/ajpheart.00779.2009

Singh J, Mehendiratta V, Del Galdo F, Jimenez SA, Cohen S, DiMarino AJ, Rattan S. Immunoglobulins from scleroderma patients inhibit the muscarinic receptor activation in internal anal sphincter smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 297: G1206-G1213, 2009. First published September 24, 2009; doi: 10.1152/ajpgi.00286.2009.--Systemic sclerosis (SSc) IgGs affecting the [M.sub.3]-muscarinic receptor ([M.sub.3]-R) have been proposed to be responsible for the gastrointestinal (GI) dysmotility in this disease. However, the effect of SSc IgGs on smooth muscle cell (SMC) function has not been studied. We determined the effect of SSc IgGs on the muscarinic receptor activation by bethanechol (BeCh; methyl derivate of carbachol) in SMC and smooth muscle strips from rat internal anal sphincter. IgGs were purified from GI-symptomatic SSc patients and normal volunteers, with protein G-Sepharose columns. SMC lengths were determined via computerized digital micrometry. The presence of [M.sub.3]-R and IgG-[M.sub.3]-R complex was determined by Western blot. IgGs from SSc patients but not from normal volunteers caused significant and concentration-dependent inhibition of BeCh response (P < 0.05). The maximal shortening of 22.2 [+ or -] 1.2% caused by [10.sup.-4] M BeCh was significantly attenuated to 8.3 [+ or -] 1.2% by 1 mg/ml of SSc IgGs (P < 0.05). Experiments performed in smooth muscle strips revealed a similar effect of SSc IgG that was fully reversible. In contrast to the effect on BeCh, the SSc IgGs caused no significant effect (P > 0.05) on [K.sup.+] depolarization and [[alpha].sub.1]- adrenoceptor activation by phenylephrine. Western blot studies revealed the specific presence of SSc IgG-[M.sub.3]-R complex. SSc IgGs attenuated [M.sub.3]-R activation, which was reversible with antibody removal. These data suggest that SSc GI dysmotility may be caused by autoantibodies that inhibit the muscarinic neurotransmission. Future treatment of SSc patients may be directed at the removal or neutralization of these antibodies. systemic sclerosis; rectoanal function; muscarinic receptor; autoantibodies doi: 10.1152/ajpgi.00286.2009

Information regarding the role of cholinergic nerves in mediating vaginal smooth muscle contraction is sparse, and in vitro studies of the effects of muscarinic agonists on vaginal smooth muscle are discrepant. The goal of this study was to determine the expression of muscarinic receptors in the vaginal wall of the rat. In addition, we sought to determine the effect of the muscarinic receptor agonist carbachol on contractility and inositol phosphate production of the proximal and distal rat vaginal muscularis. RT-PCR analysis indicated that both [M.sub.2] and [M.sub.3] receptor transcripts were expressed within the proximal and distal rat vagina. Carbachol dose-dependently ([10.sup.-7]-[10.sup.-4] M) contracted the rat vaginal muscularis with a greater maximal contractile response in the proximal vagina (P < 0.01) compared with the distal vagina. The contractile responses of the rat vaginal muscularis to carbachol were dose dependently inhibited by the [M.sub.3] antagonist para-fluoro-hexahydrosiladefenidol, and a [pK.sub.B] of 7.78 and 7.95 was calculated for the proximal and distal vagina, respectively. Inositol phosphate production was significantly increased in both regions of the vagina following 20-min exposure to 50 [micro]M carbachol with higher levels detected in the proximal vagina compared with the distal (P < 0.05). Preliminary experiments indicated the presence of [M.sub.2] and [M.sub.3] receptors in the human vaginal muscularis as well as contraction of human vaginal muscularis to carbachol, indicating that our animal studies are relevant to human tissue. Our results provide strong evidence for the functional significance of [M.sub.3] receptor expression in the vaginal muscularis. vagina; smooth muscle; muscarinic receptor; female sexual response doi: 10.1152/ajpregu.90516.2008.

The forebrain cholinergic system promotes higher brain function in part by signaling through the [M.sub.1] muscarinic acetylcholine receptor (mAChR). During Alzheimer's disease (AD), these cholinergic neurons degenerate, therefore selectively activating [M.sub.1] receptors could improve cognitive function in these patients while avoiding unwanted peripheral responses associated with non-selective muscarinic agonists. We describe here benzyl quinolone carboxylic acid (BQCA), a highly selective allosteric potentiator of the [M.sub.1] mAChR. BQCA reduces the concentration of ACh required to activate [M.sub.1] up to 129-fold with an inflection point value of 845 nM. No potentiation, agonism, or antagonism activity on other mAChRs is observed up to 100 [micro]M. Furthermore studies in [M.sub.1.sup.-/-] mice demonstrates that BQCA requires [M.sub.1] to promote inositol phosphate turnover in primary neurons and to increase c-fos and arc RNA expression and ERK phosphorylation in the brain. Radioligand-binding assays, molecular modeling, and sitedirected mutagenesis experiments indicate that BQCA acts at an allosteric site involving residues Y179 and W400. BQCA reverses scopolamine-induced memory deficits in contextual fear conditioning, increases blood flow to the cerebral cortex, and increases wakefulness while reducing delta sleep. In contrast to [M.sub.1] allosteric agonists, which do not improve memory in scopolamine-challenged mice in contextual fear conditioning, BQCA induces [beta]-arrestin recruitment to [M.sub.1], suggesting a role for this signal transduction mechanism in the cholinergic modulation of memory. In summary, BQCA exploits an allosteric potentiation mechanism to provide selectivity for the [M.sub.1] receptor and represents a promising therapeutic strategy for cognitive disorders.

Muscarinic receptors (mAChRs) have been identified in the urothelium, a tissue that may be involved in bladder sensory mechanisms. This study investigates the expression and function of mAChRs using cultured urothelial cells from the rat. RT-PCR established the expression of all five mAChR subtypes. Muscarinic agonists acetylcholine (ACh; 10 [mciro]M), muscarine (Muse; 20 [micro]M), and oxotremorine methiodide (OxoM; 0.001-20 [micro]M) elicited transient repeatable increases in the intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) in ~50% of cells. These effects were blocked by the mAChR antagonist atropine methyl nitrate (10 [micro]M). The sources of [[Ca.sup.2+]].sub.i] changes included influx from external milieu in 63% of cells and influx from external milieu plus release from internal stores in 27% of cells. The use of specific agonists and antagonists (10 [micro]M [M.sub.1] agonist MeN-A-343; 10 [micro]M [M.sub.2], [M.sub.3] antagonists AF-DX 116, 4-DAMP) revealed that [M.sub.1], [M.sub.2], [M.sub.3] subtypes were involved in [[Ca.sup.2+]].sub.i] changes. The PLC inhibitor U-73122 (10 [micro]M) abolished OxoM-elicited [Ca.sup.2+] responses in the presence of the [M.sub.2] antagonist AF-DX 116, suggesting that [M.sub.l], [M.sub.3], or [M.sub.5] mediates [[[Ca.sup.2+]].sub.i] increases via PLC pathway. ACh (0.1 [micro]M), Muse (10 [micro]M), oxotremorine sesquifumarate (20 [micro]M), and MeN-A-343 (1 [micro]M) acting on [M.sub.1], [M.sub.2], and [M.sub.3] mAChR subtypes stimulated ATP release from cultured urothelial cells. In summary, cultured urothelial cells express functional [M.sub.1], [M.sub.2], and [M.sub.3] mAChR subtypes whose activation results in ATP release, possibly through mechanisms involving [[[Ca.sup.2+]].sub.i] changes. bladder; Fura-2 calcium imaging; ATP

Hyperpolarization current ([I.sub.f]) is an important player in controlling heart rate and is stimulated by cAMP and inhibited by members of the pertussis toxin-sensitive G-protein [G.sub.i]/[G.sub.o] family. We have successfully derived cardiocytes from embryonic stem cells lacking [G.sub.o] or [G.sub.i2] and [G.sub.i3]. We have established that both basal and isoproterenolstimulated activities of [I.sub.f] in these cardiocytes have typical nodal-atrial characteristics and are unaffected by targeted gene inactivation of the G proteins [G.sub.o] or [G.sub.i2] and [G.sub.i3]. Under basal conditions, both [[G.sub.o] and [G.sub.i] are required for muscarinic inhibition of [Is.bu.f] activity via a mechanism that involves the generation of nitric oxide, whereas, with prior stimulation by [beta]-agonists, only [[G.sub.o] is required and [G.sub.i] and nitric oxide production are not. Our findings establish an essential role for Go in the antiadrenergic effect of muscarinic agent on [I.sub.f]. G proteins; ion channels; nitric oxide; muscarinic receptors

Suppressed parasympathetic nervous system (PSNS) function has been found in a variety of cardiovascular diseases, such as hypertension, heart failure, and diabetes. However, whether impaired PSNS function plays a significant role in ventricular dysfunction remains to be investigated. Cardiac regulation by the PSNS is primarily mediated by the [M.sub.2] muscarinic acetylcholine receptor ([M.sub.2]-AChR). In this study, we tested the hypothesis that lack of [M.sub.2]-AChR-mediated PSNS function may adversely impact cardiac ventricular function. Using [M.sub.2]-AChR knockout (KO) and wild-type (WT) mice, we found that the basal levels of heart rate and left ventricular function were similar in [M.sub.2]-AChR KO and WT mice. A bolus injection of isoproterenol (Iso) induced a greater increase in heart rate in [M.sub.2]-AChR KO mice than in WT mice. However, the responses of change in pressure over time (dP/dt) to Iso were similar in the two groups. After chronic infusion with Iso for 1 wk, the baseline values of left ventricular function were increased to similar extents in [M.sub.2]-AChR KO and WT mice. However, the [M.sub.2]-AChR KO mice exhibited impaired ventricular function, indicated as attenuated dP/dt and increased end-diastolic pressure, during an increase in cardiac afterload induced by a bolus injection of phenylephrine. Furthermore, chronic Iso infusion significantly increased matrix metalloproteinase (MMP) activity in the heart in [M.sub.2]-AChR KO mice. In primary culture of mixed neonatal rat cardiac fibroblast and cardiomyocytes, cotreatment with muscarinic agonist bethanechol reversed phenylephrine-induced increase in MMP-9 activation. These data suggest that [M.sub.2]-AChR may mediate an inhibitory regulation on MMP function. The overall results from this study suggest that [M.sub.2]-AChR-mediated PSNS function may provide cardiac protection. Lack of this protective mechanism will increase the susceptibility of the heart to cardiac stresses. muscarinic; cardiac; contractility; matrix metalloproteinase; isoproterenol

Contractile responses of airway smooth muscle (ASM) determine airway resistance in health and disease. Caveolae microdomains in the plasma membrane are marked by caveolin proteins and are abundant in contractile smooth muscle in association with nanospaces involved in [Ca.sup.2+] homeostasis. Caveolin-1 can modulate localization and activity of signaling proteins, including trimeric G proteins, via a scaffolding domain. We investigated the role of caveolae in contraction and intracellular [Ca.sup.2+] ([[[Ca.sup.2+]].sub.i]) mobilization of ASM induced by the physiological muscarinic receptor agonist, acetylcholine (ACh). Human and canine ASM tissues and cells predominantly express caveolin-1. Muscarinic [M.sub.3] receptors ([M.sub.3]R) and G[[alpha].sub.q/11] cofractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with [beta]-cyclodextrin in canine tracheal strips reduced sensitivity but not maximum isometric force induced by ACh. In fura-2-1oaded canine and human ASM cells, exposure to methyl-[beta]-cyclodextrin (m[beta]CD) reduced sensitivity but not maximum [[[Ca.sup.2+]].sub.i] induced by ACh. In contrast, both parameters were reduced for the partial muscarinic agonist, pilocarpine. Fluorescence microscopy revealed that m[beta]CD disrupted the colocalization of caveolae-1 and [M.sub.3]R, but [N-methyl-[sup.3]H]scopolamine receptor-binding assay revealed no effect on muscarimc receptor availability or affinity. To dissect the role of caveolin-1 in ACh-induced [[[Ca.sup.2+]].sub.i] flux, we disrupted its binding to signaling proteins using either a cell-permeable caveolin-1 scaffolding domain peptide mimetic or by small interfering RNA knockdown. Similar to the effects of m[beta]CD, direct targeting of caveolin-1 reduced sensitivity to ACh, but maximum [[[Ca.sup.2+]].sub.i] mobilization was unaffected. These results indicate caveolae and caveolin-1 facilitate [[[Ca.sup.2+]].sub.i] mobilization leading to ASM contraction induced by submaximal concentrations of ACh. caveolin; G protein-coupled receptor; asthma; histamine; G[[alpha].sub.q]

The extent to which gut motility and smooth muscle contractility are altered by intestinal inflammation depends on the nature of the underlying immune activation. The muscarinic receptor on smooth muscle plays a critical role in mediating acetylcholine-driven motor function. We examined the ability of cytokines to influence muscarinic receptor characteristics on intestinal longitudinal muscle and related the findings to studies on carbachol-induced contraction. Cells were isolated from longitudinal muscle myenteric plexus (LMMP). Cytokine receptor expression, muscle contractility, and muscarinic agonist receptor characteristics were examined by agonist displacement of [N-methyl-[sup.3]H]scopolamine ([[sup.3]H]NMS) binding. The TGF-[beta]1 receptor (543 bp) and the IFN-[gamma]/receptor 1 (660 bp) were identified on smooth muscle cells. Scatchard analysis revealed dissociation constant and maximum binding values for [[sup.3]H]NMS of 2.6 nM and 2.4 x [10.sup.4] sites/cell, respectively, in control cells. Nematode infection was accompanied by a reduction in inhibitory constant of the high-affinity sites ([K.sub.H]), and this was independent of signal transduction and activator of transcription 6. Preincubation with TGF-[beta]1 enhanced longitudinal muscle contractility and decreased the [K.sub.H] to 2.2 pM (increased muscarinic receptor affinity), whereas preincubation with IFN-[gamma] increased the [K.sub.H] to 0.4 [micro]M (decreased muscarinic receptor affinity) and decreased longitudinal muscle contractility. Preincubation of LMMP with IL-13 decreased the [K.sub.H] to 0.2 nM. Cytokines exert differential effects on the muscarinic receptor on intestinal longitudinal smooth muscle. These findings explain the basis for altered muscle contractility observed in Th1 and Th2 models of inflammation, as well as in the post-nematode-infected state. interleukin-13; transforming growth factor-[beta]; intestinal motility doi:10.1152/ajpgi.00545.2006

Administration of abdominal radiotherapy results in small intestinal motor dysfunction. We have developed a rat radiation enteritis model that, after exposure in vivo, shows high-amplitude, long-duration (HALD) pressure waves in ex vivo ileal segments. These resemble in vivo dysmotility where giant contractions migrate both antegradely and retrogradely. Mediation of these motor patterns is unclear, although enteric neural components are implicated. After the induction of acute radiation enteritis in vivo, ileal segments were isolated and arterially perfused. TTX, hexamethonium, atropine, or the selective muscarinic antagonists pirenzepine ([M.sub.1]), methoctramine ([M.subb.2]), and 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; M3) were added to the perfusate. The baseline mean rate per minute per channel of HALD pressure waves was 0.35 [+ or -] 0.047. This was significantly reduced by TTX (83.3%, P < 0.01), hexamethonium (90.3%, P < 0.03), and atropine (98.4%, P < 0.01). The HALD pressure wave mean rate per minute per channel was significantly reduced by pirenzepine (81.1%, P < 0.03), methoctramine (96.8%, P < 0.001), and 4-DAMP (93.1%, P < 0.03) compared with predrug baseline data. As an indicator of normal motility patterns, the frequency of low-amplitude, short-duration pressure waves was also assessed. The mean rate per minute per channel of 5.15 [+ or -] 0.98 was significantly increased by TTX (19%, P < 0.05) but significantly reduced by pirenzepine (35.1%, P < 0.02) and methoctramine (75%, P < 0.0003). However, the rate of small-amplitude pressure waves was not affected by hexamethonium, atropine, or the [M.sub.3] antagonist 4-DAMP. The data indicate a role for neuronal mechanisms and the specific involvement of cholinergic receptors in generating dysmotility in acute radiation enteritis. The effect of selective [M.sub.3] receptor antagonism suggests that [M.sub.3] receptors may provide specific therapeutic targets in acute radiation enteritis. radiation enteritis; high-amplitude contractions doi:10.1152/ajpgi.00469.2006

G protein-coupled receptors play a pivotal role in regulating cardiac automaticity. Their function is controlled by regulator of G protein signaling (RGS) proteins acting as GTPase-activating proteins for Get subunits to suppress G[[alpha].sub.i] and G[[alpha].sub.q] signaling. Using knock-in mice in which G[[alpha].sub.i2]-RGS binding and negative regulation are disrupted by a genomic Gc[[alpha].sub.i2]G184S (GS) point mutation, we recently (Fu Y, Huang X, Zhong H, Mortensen RM, D'Alecy LG, Neubig RR. Circ Res 98: 659-666, 2006) showed that endogenous RGS proteins suppress muscarinic receptor-mediated bradycardia. To determine whether this was due to direct regulation of cardiac pacemakers or to alterations in the central nervous system or vascular responses, we examined isolated, perfused hearts. Isoproterenol-stimulated beating rates of heterozygote (+/GS) and homozygote (GS/GS) hearts were significantly more sensitive to inhibition by carbachol than were those of wild type (+/+). Even greater effects were seen in the absence of isoproterenol; the potency of muscarinic-mediated bradycardia was enhanced fivefold in GS/GS and twofold in +/GS hearts compared with +/+. Al-adenosine receptor-mediated bradycardia was unaffected. In addition to effects on the sinoatrial node, +/GS and GS/GS hearts show significantly increased carbachol-induced third-degree atrioventricular (AV) block. Atrial pacing studies demonstrated an increased PR interval and AV effective refractory period in GS/GS hearts compared with +/+. Thus loss of the inhibitory action of endogenous RGS proteins on G[[alpha].sub.i2] potentiates muscarinic inhibition of cardiac automaticity and conduction. The severe carbachol-induced sinus bradycardia in G[[alpha].sub.i2]G184S mice suggests a possible role for alterations of G[[alpha].sub.i2] or RGS proteins in sick sinus syndrome and pathological AV block. G protein signaling; heart rate; muscarinic receptors; arrhythmias; adenosine receptors doi:10.1152/ajpheart.01391.2006.

Airway hyperreactivity in antigen-challenged animals is mediated by eosinophil major basic protein (MBP) that blocks inhibitory [M.sub.2] muscarinic receptors on parasympathetic nerves, increasing acetylcholine release onto [M.sub.3] muscarinic receptors on airway smooth muscle. Acutely, anticholinergics block hyperreactivity in antigen-challenged animals and reverse asthma exacerbations in the human, but are less effective in chronic asthma. We tested whether atropine, given before antigen challenge, affected hyperreactivity, [M.sub.2] receptor function, eosinophil accumulation, and activation. Sensitized guinea pigs received atropine (1 mg/kg ip) 1 h before challenge and 6 h later. Twenty-four hours after challenge, animals were anesthetized, vagotomized, paralyzed, and ventilated. Airway reactivity to electrical stimulation of the vagi and to intravenous acetylcholine was not altered by atropine pretreatment in nonsensitized animals, indicating that atropine was no longer blocking postjunctional muscarinic receptors. Antigen challenge induced airway hyperreactivity to vagal stimulation that was significantly potentiated by atropine pretreatment. Bronchoconstriction induced by acetylcholine was not changed by antigen challenge or by atropine pretreatment. [M.sub.2] receptor function was lost in challenged animals but protected by atropine pretreatment. Eosinophils in bronchoalveolar lavage and within airway tissues were significantly increased by challenge but significantly reduced by atropine pretreatment. However, extracellular MBP in challenged airways was significantly increased by atropine pretreatment, which may account for reduced eosinophils. Depleting eosinophils with antibody to IL-5 before challenge prevented hyperreactivity and significantly reduced MBP in airways of atropine-pretreated animals. Thus atropine pretreatment potentiated airway hyperreactivity by increasing eosinophil activation and degranulation. These data suggest that anticholinergics enhance eosinophil interactions with airway nerves. anticholinergic; asthma; muscarinic receptors; parasympathetic nerves doi:10.1152/ajplung.00455.2006

Caveolae are abundant plasma membrane invaginations in airway smooth muscle that may function as preorganized signalosomes by sequestering and regulating proteins that control cell proliferation, including receptor tyrosine kinases (RTKs) and their signaling effectors. We previously demonstrated, however, that p42/p44 MAP kinase, a critical effector for cell proliferation, does not colocalize with RTKs in caveolae of quiescent airway myocytes. Therefore, we investigated the subcellular sites of growth factor-induced MAP kinase activation. In quiescent myocytes, though epidermal growth factor receptor (EGFR) was almost exclusively found in caveolae, p42/p44 MAP kinase, Grb2, and Raf-1 were absent from these membrane domains. EGF induced concomitant phosphorylation of caveolin-1 and p42/p44 MAP kinase; however, EGF did not promote the localization of p42/p44 MAP kinase, Grb2, or Raf-1 to caveolae. Interestingly, stimulation of muscarinic [M.sub.2] and [M.sub.3] receptors that were enriched in caveolae-deficient membranes also induced p42/p44 MAP kinase phosphorylation, but this occurred in the absence of caveolin-1 phosphorylation. This suggests that the localization of receptors to caveolae and interaction with caveolin-1 is not directly required for p42/p44 MAP kinase phosphorylation. Furthermore, we found that EGF exposure induced rapid translocation of EGFR from caveolae to caveolae-free membranes. EGFR trafficking coincided temporally with EGFR and p42/p44 MAP kinase phosphorylation. Collectively, this indicates that although caveolae sequester some receptors associated with p42/p44 MAP kinase activation, the site of its activation is associated with caveolae-free membrane domains. This reveals that directed trafficking of plasma membrane EGFR is an essential element of signal transduction leading to p42/p44 MAP kinase activation. epidermal growth factor receptor; muscarinic receptor; airway smooth muscle; p42/p44 mitogen-activated protein kinase doi:10.1152/ajplung.00471.2006

In the heart, ACh activates the ACh-activated [K.sup.+]current ([I.sub.K,ACh) via the [M.sub.2] muscarinic receptor. The relationship between desensitization of [I.sub.K,ACh] and internalization of the [ M.sub.2] receptor has been studied in rat atrial cells. On application of the stable muscarinic agonist carbachol for 2 h, [I.sub.K,ACh] declined by ~62% with time constants of 1.5 and 26.9 rain, whereas ~83% of the [M.sub.2] receptor was internalized from the cell membrane with time constants of 2.9 and 51.6 min. Transfection of the cells with [beta]-adrenergic receptor kinase 1 (G protein-receptor kinase 2) and [beta]-arrestin 2 significantly increased [I.sub.K,ACh] desensitization and [M.sub.2] receptor internalization during a 3-min application of agonist. Internalized [M.sub.2] receptor in cells exposed to carbachol for 2 h was colocalized with clathrin and not caveolin. It is concluded that a G protein-receptor kinase 2- and [beta]-arrestin 2-dependent internalization of the [M.sub.2] receptor into clathrin-coated vesicles could play a major role in [I.sub.K,ACh] desensitization. acethylcholine-activated potassium current; acetylcholine; Kir3.1; Kir3.4; caveolin

We evolved muscarinic receptors in yeast to generate a family of G protein-coupled receptors (GPCRs) that are activated solely by a pharmacologically inert drug-like and bioavailable compound (clozapine-N-oxide). Subsequent screening in human cell lines facilitated the creation of a family of muscarinic acetylcholine GPCRs suitable for in vitro and in situ studies. We subsequently created lines of telomerase-immortalized human pulmonary artery smooth muscle cells stably expressing all five family members and found that each one faithfully recapitulated the signaling phenotype of the parent receptor. We also expressed a [G.sub.i]-coupled designer receptor in hippocampal neurons (h[M.sub.4]D) and demonstrated its ability to induce membrane hyperpolarization and neuronal silencing. We have thus devised a facile approach for designing families of GPCRs with engineered ligand specificities. Such reverse-engineered GPCRs will prove to be powerful tools for selectively modulating signal-transduction pathways in vitro and in vivo. cell engineering | molecular evolution | receptorome

To effect movement, motoneurons must respond appropriately to motor commands. Their responsiveness to these inputs, or excitability, is regulated by neuromodulators. Possible sources of modulation include the abundant cholinergic 'C boutons' that surround motoneuron somata. In the present study, recordings from motoneurons in spinal cord slices demonstrated that cholinergic activation of [m.sub.2]-type muscarinic receptors increases excitability by reducing the action potential afterhyperpolarization. Analyses of isolated spinal cord preparations in which fictive locomotion was elicited demonstrated that endogenous cholinergic inputs increase motoneuron excitability during locomotion. Anatomical data indicate that C boutons originate from a discrete group of interneurons lateral to the central canal, the medial partition neurons. These results highlight a unique component of spinal motor networks that is critical in ensuring that sufficient output is generated by motoneurons to drive motor behavior. afterhyperpolarization | C bouton | central pattern generator | muscarinic receptors | spinal cord

The involvement of muscarinic receptors in neurogenic responses of the ileum was studied in wild-type and muscarinic-receptor (M-receptor) knockout (KO) mice. Electrical field stimulation to the wild-type mouse ileum induced a biphasic response, a phasic and sustained contraction that was abolished by tetrodotoxin. The sustained contraction was prolonged for an extended period after the termination of electrical field stimulation. The phasic contraction was completely inhibited by atropine. In contrast, the sustained contraction was enhanced by atropine. Ileal strips prepared from [M.sub.2]-receptor KO mice exhibited a phasic contraction similar to that seen in wild-type mice and a sustained contraction that was larger than that in wild-type mice. In [M.sub.3]-receptor KO mice, the phasic contraction was smaller than that observed in wild-type mice. Acetylcholine exogenously administrated induced concentration-dependent contractions in strips isolated from wild-type, [M.sub.2]- and [M.sub.3]-receptor KO mice. However, contractions in [M.sub.3]-receptor KO mice shifted to the right. The sustained contraction was inhibited by capsaicin and neurokinin N[K.sub.2] receptor antagonist, suggesting that it is mediated by substance P (SP). SP-induced contraction of [M.sub.2]-receptor KO mice did not differ from that of wild-type mice. SP immunoreactivity was located in enteric neurons, colocalized with [M.sub.2] receptor immunoreactivity. These results suggest that atropine-sensitive phasic contraction is mainly mediated via the [M.sub.3] receptor, and SP-mediated sustained contraction is negatively regulated by the [M.sub.2] receptor at a presynaptic level. [M.sub.2]- and [M.sub.3]-receptor knockout mouse; neurogenic response; acetylcholine-mediated phasic contraction; substance P-mediated tonic contraction

[Ca.sup.2+] influx through voltagegated R-type ([Ca.sub.v]2.3) [Ca.sup.2+] channels is important for hormone and neurotransmitter secretion and other cellular events. Previous studies have shown that [Ca.sub.v]2.3 is both inhibited and stimulated through signaling mechanisms coupled to muscarinic ACh receptors. We previously demonstrated that muscarinic stimulation of [Ca.sub.v]2.3 is blocked by regulator of G protein signaling (RGS) 2. Here we investigated whether muscarinic inhibition of [Ca.sub.v]2.3 is antagonized by RGS3. RGS3 is particularly interesting because it contains a lengthy (~380 residue) amino-terminal domain of uncertain physiological function. [Ca.sub.v]2.3, [M.sub.2] muscarinic ACh receptors ([M.sub.2]R), and various deletion mutants of RGS3, including its native isoform RGS3T, were expressed in HEK293 cells, and agonist-dependent inhibition of [Ca.sub.v]2.3 was quantified using whole cell patch-clamp recordings. Full-length RGS3, RGS3T, and the core domain of RGS3 were equally effective in antagonizing inhibition of [Ca.sub.v]2.3 through [M.sub.2]R. These results identify RGS3 and RGS3T as potential physiological regulators of R-type [Ca.sup.2+] channels. Furthermore, they suggest that the signaling activity of RGS3 is unaffected by its extended amino-terminal domain. Confocal microscopy was used to examine the intracellular locations of four RGS3-enhanced green fluorescent protein fusion proteins. The RGS3 core domain was uniformly distributed throughout both cytoplasm and nucleus. By contrast, full-length RGS3, RGS3T, and the amino-terminal domain of RGS3 were restricted to the cytoplasm. These observations suggest that the amino terminus of RGS3 may serve to confine it to the cytoplasmic compartment where it can interact with cell surface receptors, heterotrimeric G proteins, and other signaling proteins. calcium channels; regulator of G protein signaling proteins; muscarinic acetylcholine receptors; enhanced green fluorescent protein-fusion proteins; voltage-gated R-type calcium channels

TREK-2 is a member of the two-pore domain [K.sup.+] channel family and provides part of the background [K.sup.+] current in many types of cells. Neurotransmitters that act on receptors coupled to [G.sub.q] strongly inhibit TREK-2 and thus enhance cell excitability. The molecular basis for the inhibition of TREK-2 was studied. In COS-7 cells expressing TREK-2 and [M.sub.3] receptor, acetylcholine (ACh) applied to the bath solution strongly inhibited the whole cell current, and this was markedly reduced in the presence of U-73122, an inhibitor of PLC. The inhibition was also observed in cell-attached patches when ACh was applied to the bath solution. In inside-out patches, direct application of guanosine 5'-O-(3-thiotriphosphate) (10 [micro]M), [Ca.sup.2+] (5 [micro]M), or diacylglycerol (DAG; 10 [micro]M) produced no inhibition of TREK-2 in >75% of patches tested. Phosphatidic acid, a product of DAG kinase, had no effect on TREK-2. Pretreatment of cells with 20 [micro]M wortmannin, an inhibitor of phosphatidylinositol kinases, did not affect the inhibition or the recovery from inhibition of TREK-2, suggesting that phosphatidylinositol 4,5-bisphosphate depletion did not mediate the inhibition. Pretreatment of cells with a protein kinase C inhibitor (bisindolylmaleimide, 10 [micro]M) markedly inhibited ACh-induced inhibition of TREK-2. Mutation of two putative PKC sites (S326A, S359C) abolished inhibition by ACh. Mutation of these amino acids to aspartate to mimic the phosphorylated state resulted in diminished TREK-2 current and no inhibition by ACh. These results suggest that the agonist-induced inhibition of TREK-2 via [M.sub.3] receptor occurs primarily via PKC-mediated phosphorylation. two-pore domain potassium channel; Gq protein; background potassium conductance; protein kinase C; phosphatidylinositol 4,5-bisphosphate

TNF has a critical mediator role in inflammation and is an important therapeutic target. We recently discovered that TNF production is regulated by neural signals through the vagus nerve. Activation of this 'cholinergic antiinflammatory pathway' inhibits the production of TNF and other cytokines and protects animals from the inflammatory damage caused by endotoxemia and severe sepsis. Here, we describe a role for central muscarinic acetylcholine receptors in the activation of the cholinergic antiinflammatory pathway. Central muscarinic cholinergic activation by muscarine, the M1 receptor agonist McN-A-343, and the M2 receptor antagonist methoctramine inhibited serum TNF levels significantly during endotoxemia. Centrally administered methoctramine stimulated vagus-nerve activity measured by changes in instantaneous heart-rate variability. Blockade of peripheral muscarinic receptors did not abolish antiinflammatory signaling through the vagus nerve, indicating that peripheral muscarinic receptors on immune cells are not required for the cytokine-regulating activities of the cholinergic antiinflammatory pathway. The role of central muscarinic receptors in activating the cholinergic antiinflammatory pathway is of interest for the use of centrally acting muscarinic cholinergic enhancers as antiinflammatory agents. muscarinic receptors | systemic inflammation | TNF | vagus nerve

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. [M.sub.3] muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and [M.sub.3] mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular [Ca.sup.2+] signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands. translocation; aquaporin-5

Muscarinic acetylcholine receptors play an important role in the regulation of gastric acid secretion stimulated by acetylcholine nonetheless, the precise role of each receptor subtype ([M.sub.1]-[M.sub.5]) remains unclear. This study examined the involvement of [M.sub.1], [M.sub.3], and [M.sub.5] receptors in cholinergic regulation of acid secretion using muscarinic receptor knockout (KO) mice. Gastric acid secretion was measured in both mice subjected to acute gastric fistula production under urethane anesthesia and conscious mice that had previously undergone pylorus ligation. [M.sub.3] KO mice exhibited impaired gastric acid secretion in response to carbachol. Unexpectedly, [M.sub.1] KO mice exhibited normal intragastric pH, serum gastrin and mucosal histamine levels, and gastric acid secretion stimulatied by carbachol, histamine, and gastrin. Pirenzepine, known as an [M.sub.1]-receptor antagonist, inhibited carbachol-stimulated gastric acid secretion in a dose-dependent manner in [M.sub.1] KO mice as well as in wild-type (WT) mice, suggesting that the inhibitory effect of pirenzepine on gastric acid secretion is independent of [M.sub.1]-receptor antagonism. Notably, [M.sub.5] KO mice exhibited both significantly lower carbachol-stimulated gastric acid secretion and histamine-secretory responses to carbachol compared with WT mice. RT-PCR analysis revealed [M.sub.5]-mRNA expression in the stomach, but not in either the fundic or antral mucosa. Consequently, cholinergic stimulation of gastric acid secretion is clearly mediated by [M.sub.3] (on parietal cells) and [M.sub.5] receptors (conceivably in the submucosal plexus), but not [M.sub.1] receptors. knockout

We investigated cellular mechanisms that mediate or modulate the vascular response to muscarinic receptor activation (ACh) in pulmonary veins (PV). Isometric tension was measured in isolated canine PV rings with endothelium (E+) and without endothelium (E-). Tension and intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) were measured simultaneously in fura-2-loaded E- PV strips. In the absence of preconstriction, ACh (0.01-10 [micro]M) caused dose-dependent contraction in E+ and E- rings. ACh contraction was potentiated by removing the endothelium or by nitric oxide (NO) synthase inhibition (N-nitro-L-arginine methyl ester, P = 0.001). Cyclooxygenase inhibition (indomethacin) reduced ACh contraction in both E+ and E- PV rings (P = 0.013 and P = 0.037, respectively). ACh contraction was attenuated by inhibitors of voltage-operated [Ca.sup.2+] channels (nifedipine, P < 0.001), inositol-1,4,5-trisphosphate (I[P.sub.3])-mediated [Ca.sup.2+] release (2-aminoethoxydiphenyl borate, P = 0.001), PKC (bisindolyl-maleimide I, P = 0.001), Rho-kinase (Y-27632, P = 0.002), and tyrosine kinase (TK; tyrphostin 47, P = 0.015) in E- PV rings. ACh (1 [micro]M) caused a leftward shift in the [[[Ca.sup.2+]].sub.i]-tension relationship (P = 0.015), i.e., ACh increased myofilament [Ca.sup.2+] sensitivity. Inhibition of PKC, Rho-kinase, and TK attenuated the ACh-induced increase in myofilament [Ca.sup.2+] sensitivity (P < 0.001, P < 0.001, and P = 0.024, respectively). These findings indicate that in canine PV, ACh contraction is modulated by NO and partially mediated by metabolites of the cyclooxygenase pathway and involves [Ca.sup.2+] influx through voltage-operated [Ca.sup.2+] channels and I[P.sub.3]-mediated [Ca.sup.2+] release. In addition, ACh induces increased myofilament [Ca.sup.2+] sensitivity, which requires the PKC, Rho-kinase, and TK pathways. endothelium; nitric oxide; myofilament calcium sensitivity; calcium influx and release

Muscarinic acetylcholine receptors (mAChRs) modulate the activity of an extraordinarily large number of physiological functions. Individual members of the mAChR family ([M.sub.1]-[M.sub.5]) are expressed in a complex, overlapping fashion in most tissues and cell types. However, the identification of the precise physiological roles of individual mAChR subtypes remains a challenging task because, with the exception of a few snake toxins, mAChR ligands that can activate or inhibit specific mAChR subtypes with a high degree of selectivity are not yet available. Knowledge of the specific roles of mAChR subtypes is of considerable interest for the development of novel, clinically useful mAChR ligands. In this article, recent studies of mutant mouse strains developed, using gene targeting techniques, to be deficient in one of the three [G.sub.q]-coupled mAChR subtypes ([M.sub.1], [M.sub.3] and [M.sub.5]) are discussed. These investigations have led to many important new insights into the physiological roles of these receptor subtypes.

The inotropic effects of ACh and adenosine on ferret ventricular cells were investigated with the action potential-clamp technique. Under current clamp, both agonists resulted in action potential shortening and a decrease in contraction. Under action potential clamp, both agonists failed to decrease contraction substantially. In the absence of agonist, application of the short action potential waveform (recorded previously in the presence of agonist) also resulted in a decrease in contraction. Under action potential clamp, application of ACh resulted in a [Ba.sup.2+] -sensitive outward current with the characteristics of muscarinic [K.sup.+] current ([I.sub.K,ACh]); the presence of the muscarinic [K.sup.+] channel was confirmed by PCR and immunocytochemistry. In the absence of agonist, on application of the short ACh action potential waveform, the decrease in contraction was accompanied by loss of the inward [Na.sup.+]/[Ca.sup.2+] exchange current ([I.sub.NaCa]). ACh also inhibited the background inward [K.sup.+] current ([I.sub.K,1]). It is concluded that ACh activates [I.sub.K,ACh], inhibits [I.sub.K,1], and indirectly inhibits [I.sub.NaCa]; this results in action potential shortening, decrease in contraction, and, as a result of the inhibition of [I.sub.K,1], minimum decrease in excitability. heart; acetylcholine; muscarinic [K.sup.+] channel

Vitamin A deficiency (VAD) remains an important health problem among children in developing countries. Children living in these areas have a higher mortality from respiratory infections, which likely results in part from suboptimal nutrition, including VAD. Bronchial hyperreactivity can follow viral respiratory infections and may complicate the recovery. To investigate whether VAD promotes bronchial hyperreactivity, we have assessed methacholine-induced bronchoconstriction in VAD and vitamin A-sufficient rats. Bronchial constriction developed at lower concentrations of inhaled methacholine in VAD than in vitamin A-sufficient rats. This did not result from an increase in the bronchial wall thickness or the clearance of a small molecule (with a size similar to methacholine) from the air space. The function and abundance of the muscarinic [M.sub.2] receptors in bronchial tissue were reduced in VAD rats, suggesting that this receptor may contribute to these animals' diminished ability to limit cholinergic-mediated bronchoconstriction. A similar reduction in muscarinic [M.sub.2] receptor function has been observed in asthma. Vitamin A (retinol) and its congeners (retinoids) may be required to regulate bronchial responsiveness in addition to maintaining a normal bronchial epithelium. asthma; retinoids; retinol; cholinergic

The present study characterizes the ionic conductances activated by acetylcholine (ACh) and ATP, two candidate neuromodulators, in isolated spiral ganglion neurons (SGNs). Brief application (1 s) of ACh evoked in a dose-dependent manner (E[C.sub.50] = 4.1 [micro]M) a reversible inward current with a long latency (average 1.3 s), at holding potential ([V.sub.h]) = -50 mV. This current was reversibly blocked by atropine and mimicked by muscarine. Application of ATP also evoked a reversible inward current at [V.sub.h] = -50 mV, but the current showed two components. A fast component with a short latency was largely reduced when N-methyl-D-glucamine (NMDG) replaced extracellular sodium, implying a P2X-like ionotropic conductance. The second component had a longer latency (average 1.1 s) and was presumably activated by metabotropic P2Y-like receptors. The second component of ATP-evoked current shared similar characteristics with the responses evoked by ACh: the current reversed near 0 mV, displayed inward rectification, could be carried by NMDG, and was insensitive to extracellular and intracellular calcium. This ACh-/ATP-evoked conductance was reversibly inhibited by preapplication of ionomycin. These results suggest that muscarinic receptors and purinergic metabotropic receptors activate a similar large nonselective cation conductance via a common intracellular pathway in SGNs, a candidate mechanism to regulate neuronal excitability of SGNs. acetylcholine; adenosine 5'-triphosphate; cochlear neuron

Bead injection spectroscopy (BIS) techniques are introduced for automated measurement of pharmacological antagonism by functional assay. Chinese hamster ovary cells that express the rat type 1 muscarinic receptor are cultured on microbeads and used as a renewable biological target for muscarinic receptor antagonist ligands. A flow injection instrument is used to reproducibly sample and capture the cells in a jet ring chamber. The effect of the antagonist pirenzepine on the carbachol-induced intracellular calcium response of the cells is measured with a fluorescence microscope photometry system. The BIS functional assay is used to quantify both equilibrium and kinetic pharmacological values for pirenzepine. In addition, two muscarinic receptor antagonists (pirenzepine and atropine) are assayed to compare their relative efficacy at diminishing the calcium response. Due to the precision of the automated fluid/bead handling protocols, and reproducibility of the measured calcium response, the quantification of useful pharmacological information from living cells by BIS techniques is demonstrated.

The authors present research examining the effects on muscarinic cholinergic receptor binding characteristics in rats' brains and lymphocytes when exposed to low levels of methylmercury.

There are five known subtypes of muscarinic receptors ([M.sub.1]-[M.sub.5]). We have used knockout mice lacking the [M.sub.1], [M.sub.2], or [M.sub.4] receptors to determine which subtypes mediate modulation of voltage-gated [Ca.sup.2+] channels in mouse sympathetic neurons. Muscarinic agonists modulate N- and L-type [Ca.sup.2+] channels in these neurons through two distinct G-protein-mediated mechanisms. One pathway is fast and membrane-delimited and inhibits N- and P/Q-type channels by shifting their activation to more depolarized potentials. The other is slow and voltage-independent and uses a diffusible cytoplasmic messenger to inhibit both [Ca.sup.2+] channel types. Using patch-clamp methods on acutely dissociated sympathetic neurons, we isolated each pathway by pharmacological and kinetic means and found that each one is nearly absent in a particular knockout mouse. The fast and voltage-dependent pathway is lacking in the [M.sub.2] receptor knockout mice; the slow and voltage-independent pathway is absent from the [M.sub.1] receptor knockout mice; and neither pathway is affected in the [M.sub.4] receptor knockout mice. The knockout effects are clean and are apparently not accompanied by compensatory changes in other muscarinic receptors.

Horses with recurrent airway obstruction (RAO) exhibit bronchospasm and tracheobronchial tree inflammation. Equine bronchochasm is mediated by cholinergic mechanisms that involve the release of acetylcholine (ACh). These mechanisms, however, can be disrupted by inhibitory of neuronal (alpha)2- and (beta)2 adrenoreceptors. The effects of physiological antagonists epinephrine and norepinephrine and the (beta)2 agonists RR and RR/SS formoterol on ACH release are also investigated. Results provide an explanation for neuronal muscarinic-autoreceptor dysfunction in equine RAO.

Hyperreactivity to histamine in antigen-challenged guinea pigs is believed to be due to M2 receptor dysfunction. This hypothesis is tested by examining the mechanism of bronchoconstriction in antigen-challenged animals. Results show that hyperreactivity to histamine is vagally mediated. It is also demonstrated that hyperreactivity is the result of the antagonistic action of eosinophil major basic protein on M2 muscarinic receptors. These results provide valuable insights that help explain the hyperreactivity of agents in patients with asthma.

The effects of saline versus atropine infusion of gastric emptying were investigated using a radiolabeled method that permits simultaneous measurement of the solid and liquid components of a meal in lean and obese male and female subjects. Results showed that obese subjects exhibit altered gastric cholinergic activity compared with lean subjects and that gender differences in gastric emptying may be due to differences in autonomic tone.

Members of the muscarinic acetylcholine receptor family (M1-M5) are known to be involved in a great number of important central and peripheral physiological and pathophysiological processes. Because of the overlapping expression patterns of the M1-M5 muscarinic receptor subtypes and the lack of ligands endowed with sufficient subtype selectivity, the precise physiological functions of the individual receptor subtypes remain to be elucidated. To explore the physiological roles of the M2 muscarinic receptor, we have generated mice lacking functional M2 receptors by using targeted mutagenesis in mouse embryonic stem cells. The resulting mutant mice were analyzed in several behavioral and pharmacologic tests. These studies showed that the M2 muscarinic receptor subtype, besides its well documented involvement in the regulation of heart rate, plays a key role in mediating muscarinic receptor-dependent movement and temperature control as well as antinociceptive responses, three of the most prominent central muscarinic effects. These results offer a rational basis for the development of novel muscarinic drugs.

Research was conducted to examine which heterotrimeric G protein couples muscarinic receptors to cytoskeletal reorganization in human airway smooth muscle (ASM) cells. An antisense approach that is capable of selectively down-regulating individual G protein alpha-subunits was used. Results provide evidence that G-alpha(sub i-2) protein couples muscarinic receptors to cytoskeletal reorganization in human ASM cells.

Tolterodine is a novel muscarinic receptor antagonist intended for the treatment of symptoms of bladder overreactivity and urge incontinence. Studies have shown that Tolterodine is more selective in its action having a more pronounced effect on the bladder. Asymmetrical total synthesis of this drug is accomplished via conjugate addition of nucleophiles to an unsaturated acid derivative serving as the substrate.

A sample of nonpregnant rat myometrium was exposed to carbachol resulting in increased generation of inositol phosphates. Exposure also caused a rapid and specific attenuation of the subsequent muscarinic responses in terms of inositol phosphate production, phospholipase C-beta3 translocation to membrane and contraction. This refractoriness was accompanied by a reduction of membrane muscarinic binding sites and an uncoupled state of residual receptors.

Since the cloning and expression of many of the G protein-coupled receptors during the 1980s, there has been a massive increase in our understanding of many aspects of their function. The use of molecular biology to engineer and express mutant receptors has made it possible to determine key amino acids involved in receptor function. Although advances in molecular biology have contributed greatly to our understanding of the pharmacology and structure of the five subtypes of muscarinic receptor, much remains to be learned about the factors that regulate their expression and function. This review by El-Bdaoui Haddad and Jonathan Rousell describes the current state of awareness and highlights recent advances made in the elucidation of the mechanisms involved in muscarinic receptor regulation. Because most is known about the regulation of expression of the [M.sub.2] receptor subtype, particular attention will be paid to it. Furthermore, this receptor subtype plays an important role in regulating acetylcholine output from airway cholinergic nerves, and there is substantial evidence from studies both in vivo and in vitro in human and animal models that these receptors are dysfunctional in asthma.

Polymerase chain reaction and several muscarinic receptor antagonists were utilized in the identification and characterization of the receptors present in isolated acinar cells of rat pancreas. The response of the cells to the various antagonist was monitored by measuring the rate of amylase secretion and proteolytic zymogen processing. Results reveal that the most potent inhibitor of these responses is the M1 selective antagonist telenzepine.

Site directed mutagenesis was used to determine the relative contribution of various amino acids at particular positions in the third intracellular loop of the m5 muscarinic receptor responsible for G-protein binding. The effects of the substitutions are documented. The influence of the amino acid residues at two positions necessary for G-protein coupling is investigated. The intracellular loop appear to be composed of a hydrophobic core with a positively charged lip.

The muscarinic receptor mediated regulation of bladder contractility was investigated in vitro using muscle strips from rat bladder and a variety of antagonists and receptor blockers. Localization of receptor type was undertaken using reverse transcriptase polymerase chain reaction. Results reveal the presence of three muscarinic receptors controlling the contractility of rat bladder, prejunctional M1 which facilitates contraction, prejunctional M2 which inhibits contraction and postjunctional M3 which modulates contraction.

Muscarinic acetylcholine receptors are members of the G protein-coupled receptor superfamily expressed in neurons, cardiomyocytes, smooth muscle, and a variety of epithelia. Five subtypes of muscarinic acetylcholine receptors have been discovered by molecular cloning, but their pharmacological similarities and frequent colocalization make it difficult to assign functional roles for individual subtypes in specific neuronal responses. We have used gene targeting by homologous recombination in embryonic stem cells to produce mice lacking the m1 receptor. These mice show no obvious behavioral or histological defects, and the m2, m3, and m4 receptors continue to be expressed in brain with no evidence of compensatory induction. However, the robust suppression of the M-current potassium channel activity evoked by muscarinic agonists in sympathetic ganglion neurons is completely lost in m1 mutant mice. In addition, both homozygous and heterozygous mutant mice are highly resistant to the seizures produced by systemic administration of the muscarinic agonist pilocarpine. Thus, the m1 receptor subtype mediates M current modulation in sympathetic neurons and induction of seizure activity in the pilocarpine model of epilepsy.

We have studied the effect of the cholinergic agonist carbachol on the spontaneous release of glutamate in cultured rat hippocampal cells. Spontaneous excitatory postsynaptic currents (sEPSCs) through glutamatergic [Alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type channels were recorded by means of the patch-clamp technique. Carbachol increased the frequency of sEPSCs in a concentration-dependent manner. The kinetic properties of the sEPSCs and the amplitude distribution histograms were not affected by carbachol, arguing for a presynaptic site of action. This was confirmed by measuring the turnover of the synaptic vesicular pool by means of the fluorescent dye FM 1-43. The carbachol-induced increase in sEPSC frequency was not mimicked by nicotine, but could be blocked by atropine or by pirenzepine, a muscarinic cholinergic receptor subtype M1 antagonist. Intracellular [Ca.sup.2+] signals recorded with the fluorescent probe Fluo-3 indicated that carbachol transiently increased intracellular [Ca.sup.2+] concentration. Since, however, carbachol still enhanced the sEPSC frequency in bis (2.aminophenoxy) ethane-N,N,N[prime],N[prime]-tetra-acetate-loaded cells, this effect could not be attributed to the rise in intracellular [Ca.sup.2+] concentration. On the other hand, the protein kinase inhibitor staurosporine as well as a down-regulation of protein kinase C by prolonged treatment of the cells with 4[Beta]-phorbol 12-myristate 13-acetate inhibited the carbachol effect. This argues for an involvement of protein kinase C in presynaptic regulation of spontaneous glutamate release. Adenosine, which inhibits synaptic transmission, suppressed the carbachol-induced stimulation of sEPSCs by a G protein-dependent mechanism activated by presynaptic A1-receptors.

A study was conducted on the influence of four putative protein kinase C inhibitors on acetylcholine-induced Ca2+ sensitization using a Beta-escin permeabilized canine tracheal smooth muscle. The four pharmacological protein kinase C inhibitors employed are chelerythrine chloride, calphostin C, staurosporine and a pseudosubstrate protein kinase C inhibitor. None of the inhibitors were able to reverse or prevent increase in Ca2+ sensitization induced by acetylcholine plus GTP.

The hypothesis that muscarinic receptor subtypes are differentially distributed across the rat pontine reticular formation was investigated using in vitro autoradiographic techniques. The results showed that the pontine reticular formation contains a homogenous distribution of M2 binding sites along the rostral to caudal extent. This indicated that the anatomic specificity within the pontine reticular formation for cholinoceptive evocation of rapid eye movement sleeplike state cannot be accounted for by the differential density of muscarinic receptors.

The m5 muscarinic receptor constitutive activation was investigated to determine the structural implications and mechanisms involved in the phenomenon. The study utilized a series of mutations at the extracellular end of transmembrane 6. Results of the research showed that the primary effect of substitution favors the formation of the active state without the need for agonists. The results also prove the significant role of a two-state model of receptor function in the activation of muscarinic receptors.

The effect of the autonomic blockade on power spectrum of heart rate variability (HRV) during exercise was investigated using an exercise context to selectively block parasympathetic and sympathetic outflow. Results of the research revealed that HRV is a valid technique for noninvasive measurement of parasympathetic tone during exercise. However, its validity as measure of sympathetic tone during exercise is still ambiguous.

Experiment was conducted to characterize a cromakalim-induced current and its regulation by muscarinic receptors in tracheal smooth muscle cells using the patch-clamp technique. Cromakalim activated a steady-state increase of 33-292 pA in whole cell current, consistent with the activation of ATP-sensitive K+ channels [K sub (ATP)]. Results indicate that K sub (ATP) channels may play a role in the regulation of membrane potential and contractility in airway smooth muscle.