Characteristics Of Muscarinic Receptors Research Articles

The importance of stereoisomerism in muscarinic activity.

Abstract The muscarinic activities and rates of hydrolysis by acetylcholinesterase of the optical isomers of the acetyl-α- and acetyl-β-methylcholines of known configuration have been determined. The results have been correlated and the possible characteristics of muscarinic receptors outlined.

Cytokine modulation of muscarinic receptors in the murine intestine

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-(3)H]scopolamine ([(3)H]NMS) binding. The TGF-beta1 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 [(3)H]NMS of 2.6 nM and 2.4 x 10(4) sites/cell, respectively, in control cells. Nematode infection was accompanied by a reduction in inhibitory constant of the high-affinity sites (K(H)), and this was independent of signal transduction and activator of transcription 6. Preincubation with TGF-beta1 enhanced longitudinal muscle contractility and decreased the K(H) to 2.2 pM (increased muscarinic receptor affinity), whereas preincubation with IFN-gamma increased the K(H) to 0.4 muM (decreased muscarinic receptor affinity) and decreased longitudinal muscle contractility. Preincubation of LMMP with IL-13 decreased the K(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.

Muscarinic receptor subtypes in the human colon: lack of evidence for atypical subtypes

Characteristics of muscarinic receptors were investigated in circular muscle from normal human colon. In saturation studies ( n=18), binding of [ 3H]quinuclidinyl benzylate (QNB) was of high affinity ( K d 87.3 pM) and capacity ( B max 362±27 fmol/mg protein), with no differences between ascending and sigmoid colon. Kinetic studies gave a K d of 55 pM. Methoctramine and darifenacin displayed biphasic binding profiles, the high affinity components being compatible with a population of approximately 80±5% M 2 and 13±2% M 3 muscarinic receptors, respectively. Pirenzepine, mamba toxin 1 and mamba toxin 3 were very weak competitors, indicating negligible expression of muscarinic M 1 and M 4 receptors. Six other subtype-preferring antagonists exhibited K i values typical of those reported at cloned human muscarinic M 2 receptors. In the presence of methoctramine, pre-treatment with alkylating agent 4-diphenylacetoxy- N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP mustard) inhibited [ 3H]quinuclidinyl benzylate binding to 26% of sites. Following alkylation of muscarinic M 3 receptors, darifenacin bound to a single low affinity site, indicating binding to muscarinic M 2 receptors.

Effect of radiation on cAMP, cGMP and Ca(2+)(i) pathways and their interactions in rat distal colon.

The secretory response implicated in the intestinal response to luminal attack is altered by radiation. The cAMP, cGMP and Ca(2+)(i) pathways leading to secretion as well as the interactions between the cAMP pathway and the cGMP or Ca(2+)(i) pathway were studied in the rat distal colon 4 days after a 9-Gy abdominal X irradiation, when modifications mainly occurred. The secretory response in Ussing chambers and cAMP and cGMP accumulation in single isolated crypts were measured. The muscarinic receptor characteristics were determined in mucosal membrane preparations. The secretory response by the cAMP pathway (stimulated by vasoactive intestinal peptide or forskolin) and the cAMP accumulation in crypts were decreased (P < 0.05) after irradiation. The weak secretory response induced by the cGMP pathway (stimulated by nitric oxide or guanylin) was unaltered by radiation, and the small amount of cGMP determined in isolated crypts from the control group became undetectable in the irradiated group. Inducible NOS was not involved in the hyporesponsiveness to VIP after irradiation (there was no effect of an iNOS inhibitor). The secretory response by the Ca(2+)(i) pathway (stimulated by carbachol) was unaffected despite a decreased number and increased affinity of muscarinic receptors. The non-additivity of VIP and carbachol co-stimulated responses was unmodified. In contrast, VIP and SNP co-stimulation showed that NO enhanced the radiation-induced hyporesponsiveness to VIP through a reduced accumulation of cAMP in crypts. This study provides further understanding of the effect of ionizing radiation on the intracellular signaling pathways.

Pharmacological characterization of muscarinic receptors in dog isolated ciliary and urinary bladder smooth muscle.

1. The pharmacological characteristics of muscarinic receptors mediating contraction of dog isolated ciliary muscle were determined and compared to those mediating contraction of dog urinary bladder smooth muscle. 2. (+)-Cis-dioxolane induced concentration-dependent contractions of ciliary muscle (pEC50=7.18+/-0.07, Emax=453+/-64 mg, n=19) and urinary bladder isolated smooth muscle (pEC50=6.55+/-0.07, Emax=11+/-1 g, n=19). These responses were antagonized by several muscarinic receptor antagonists (pKb values for the ciliary muscle and the bladder smooth muscle, respectively): atropine (8.25+/-0.14 and 9.21+/-0.09), pirenzepine (6.31+/-0.13 and 6.70+/-0.25), tolterodine (7.97+/-0.14 and 8.68+/-0.12), oxybutynin (7.40+/-0.08 and 7.88+/-0.12), zamifenacin (6.46+/-0.19 and 7.69+/-0.11), S-secoverine (6.66+/-0.14 and 8.13+/-0.07), AQ-RA 741 (6.16+/-0.15 and 7.08+/-0.23), p-F-HHSiD (7.10+/-0.27 and 7.35+/-0.07) and responses were not antagonized by PD 102807 (up to 100 nM). 3. In urinary bladder smooth muscle, the profile of antagonist pKB values correlated significantly with pK(i) values at human recombinant m3 muscarinic receptors, suggesting that M3 muscarinic receptors mediated the response. In the ciliary muscle, a significant (P<0.01) correlation was obtained with human recombinant m3 and m5 receptors. 4. Darifenacin displayed insurmountable antagonism at receptors in the bladder. At receptors in the ciliary muscle, it exhibited two phases of antagonism, comprising an initial low affinity (pKB<6) component and a high affinity phase (pKB>8). 5. The role of pigmentation in the atypical behaviour of darifenacin was examined. In blue coloured eyes, darifenacin produced apparent surmountable, competitive antagonism of the responses to (+)-cis-dioxolane (pKB=8.76+/-0.07). The antagonist profile obtained in this tissue suggested the involvement of a site which has the pharmacological attributes of the M5 receptor. 6. We suggest that the dog urinary bladder contracts in response to M3 muscarinic receptor activation. Contraction of the brown-eyed dog ciliary muscle is more complex and may include involvement of at least two receptors, possibly the M5 and M3 receptor, whereas blue-eyed dog ciliary muscle may involve a single population of M5 muscarinic receptors.

In vitro contractile responses of detrusor to carbachol and neurokinin A, in children with recurrent urinary tract infection or day wetting.

To investigate whether a history of recurrent urinary tract infection (UTI) and/or the presence of day-wetting/urge symptoms might influence the contractile responses to the cholinergic agonist carbachol or to the sensory neuropeptide neurokinin A (NKA) in the urinary bladder in children. Small detrusor strips were taken from the margin of the cystotomy incision of the bladder dome during surgery to correct vesico-ureteric reflux (VUR) in 62 children (aged 4 months to 12 years) or for unrelated bladder conditions in five controls (aged 3 months to 13 years). Concentration-response curves to carbachol and NKA were constructed using organ-bath techniques, and results compared for age, sex, weight of the detrusor strip, UTI history or day-wetting syndrome. The contractile responses to NKA were no different for any of the features investigated. The contractile response to carbachol and NKA in detrusor from control and VUR patients was not significantly different. The children with a history of UTI were significantly older than those without. The contractility in response to carbachol was greater in older girls (aged 4-12 years) than younger girls (< 4 years) and than in all boys (< 4 years and 4-12 years; ANOVA P = 0.013). The mean (SEM) maximum contractile response to carbachol in the group of 20 young children (4-30 months) with previous UTI was 3.0 (0.3) g, whereas the maximum response in the age-matched group of 11 without UTI was 1.8 (0.3) g (P = 0.046). There were no significant differences in maximum responses between those with day-wetting and those without (aged > 4 years), although there was a significant difference in pD2 value, at 6.19 (0.13) and 5.58 (0.14), respectively (P = 0.018). Carbachol produced a larger contractile response in detrusor from children with a history of UTI than from those without, indicating possible alterations in muscarinic receptor characteristics. An increased sensitivity to muscarinic stimulation in day-wetting children was also suggested, whereas NKA is unlikely to be involved in any of these pathophysiological conditions.

Characterization of the muscarinic receptor in isolated uterus of sham operated and ovariectomized rats.

1. The pharmacological characteristics of muscarinic receptors in rat isolated uterus were studied in ovariectomized (ov.) and sham operated (sh.) animals. 2. Competition radioligand binding studies, using uterine membranes and [3H]-NMS, were undertaken with several muscarinic receptor antagonists. Most of the antagonists indicated a one-site fit with apparent affinity estimates (pKi) unchanged by ovariectomy. The selective M2 antagonist, tripitramine revealed high (representing 33+/-8 and 38+/-2%) and low (67+/-8 and 62+/-2%) affinity binding sites in both sh. and ov. rat uterus, respectively. These sites likely represented muscarinic M2 and M3 receptors and the proportions were not significantly different in the two conditions. 3. Carbachol induced concentration-dependent contractions which were surmountably antagonized by several muscarinic receptor antagonists (pKB, sh.; ov.): zamifenacin (9.19; 9.18), p-F-HHSiD (8. 50; 9.06), tripitramine (7.23; 7.54), himbacine (7.21; 7.41), methoctramine (6.79; 7.49), pirenzepine (6.48; 7.21), AF DX 116 (6. 26; 6.61), MTx 3 (<7.00; <7.00) and PD 102807 (<7.00; <7.00). 4. The apparent affinity values obtained in functional studies using the uteri from both sh. and ov. animals correlated most closely with values reported at human recombinant muscarinic M3 receptors. This suggests that the muscarinic M3 receptor mediates contraction under both conditions. 5. Radioligand binding experiments indicate the presence of M2 receptors, in addition to M3 receptors, which probably explains the discrepancies between functional and binding affinities. These data further suggest that the pharmacological profile and proportions of the two populations of muscarinic receptors are unaffected by ovariectomy.

Ionizing radiation stimulates muscarinic regulation of rat intestinal mucosal function.

The aim of this study was to determine whether ionizing radiation modifies muscarinic regulation of intestinal mucosal function. Rats exposed to total body 8-Gy gamma-irradiation or sham irradiated were studied up to 21 days after irradiation. Basal and carbachol-stimulated short-circuit current (Isc) and transepithelial conductance (Gt) of stripped ileum were determined in Ussing chambers. Muscarinic receptor characteristics using the muscarinic antagonist [3H]quinuclidinyl benzilate and three unlabeled antagonists were measured in small intestinal plasma membranes together with two marker enzyme activities (sucrase, Na+-K+-ATPase). Enzyme activities were decreased 4 days after irradiation (day 4). Basal electrical parameters were unchanged. Maximal carbachol-induced changes in Isc and Gt were increased at day 4 (maximal DeltaIsc = 195.8 +/- 14.7 microA/cm2, n = 19, vs. 115.4 +/- 8.2 microA/cm2, n = 63, for control rats) and unchanged at day 7. Dissociation constant was decreased at day 4 (0.73 +/- 0.29 nM, n = 10, vs. 2.14 +/- 0.39 nM, n = 13, for control rats) but unchanged at day 7, without change in binding site number. Thus total body irradiation induces a temporary stimulation of cholinergic regulation of mucosal intestinal function that may result in radiation-induced diarrhea.

Cardiac muscarinic receptor function in rats with cirrhotic cardiomyopathy.

The pathogenesis of cirrhotic cardiomyopathy remains unclear. Because ventricular contractility is dependent on the interplay of stimulatory beta-adrenergic and inhibitory muscarinic receptors, we aimed to examine a possible role of muscarinic M2 receptor overactivity in a rat model of cirrhotic cardiomyopathy. Cirrhosis was induced by bile duct ligation (BDL), while controls underwent sham operations. Contractile responses to the muscarinic agonist carbachol were measured in situ in the autonomic-denervated pithed rat and in vitro in isolated ventricular papillary muscles. Ventricular sarcolemmal plasma membranes were isolated by sucrose density gradients, and muscarinic receptor characteristics were studied using 1-[N-methyl-3H]scopolamine (NMS). Membrane adenylyl cyclase activity was tested by a protein binding assay. Maximum first time derivative of peak ventricular systolic pressure (+dP/dt) for sham-operated and cirrhotic rats at baseline was 3,599 +/- 296 versus 1,226 +/- 63 mm Hg/sec (P < .01). Maximum first time derivative of ventricular diastolic relaxation (-dP/dt) for sham and cirrhotic rats at basal levels was -3,040 +/- 235 versus -864 +/- 59 (P < .01). The +dP/dt(max), and -dP/dt(max) responses to carbachol were blunted in the cirrhotic rats. The cirrhotic papillary muscles showed significantly less inhibition to incremental doses of carbachol than control rat muscles. Likewise, isoproterenol-stimulated membrane adenylyl cyclase activity was significantly less inhibited by carbachol doses in the cirrhotic rats. Membrane M2 receptor density and binding affinity in cirrhotic rat hearts were similar to controls. We conclude that muscarinic responsiveness was blunted in cirrhotic hearts, but this was not caused by receptor down-regulation, suggesting changes in postreceptor factors. These changes in muscarinic function are likely compensatory, and M2 receptor overactivity is not involved in the genesis of cirrhotic cardiomyopathy.

Characterization of muscarinic receptors mediating relaxation and contraction in the rat iris dilator muscle.

1. The characteristics of muscarinic receptors mediating relaxation and/or contraction in the rat iris dilator muscle were examined. 2. Relaxation was induced in a dilator muscle by application of acetylcholine (ACh) at low doses (3 microM or less) and contraction was induced by high doses. Methacholine and carbachol also showed biphasic effects similar to those of ACh; in contrast, bethanechol, arecoline, pilocarpine and McN-A-343 induced mainly relaxation but no substantial contraction. 3. After parasympathetic denervation by ciliary ganglionectomy, the relaxant response to muscarinic agonists disappeared upon nerve stimulation. Application of McN-A-343 and pilocarpine induced only small contractions in denervated dilator muscles, indicating that these are partial agonists for contraction. 4. pA2 values of pirenzepine, methoctramine, AF-DX 116, himbacine, and 4-DAMP for antagonism to pilocarpine-induced relaxation in normal dilator muscles and those for antagonism to ACh-induced contraction in denervated dilator muscles were determined. The pA2 values for antagonism to relaxation of all these antagonists were most similar to those for M3-type muscarinic receptors. 5. Although pA2 values for contraction of these antagonists, except for methoctramine, were very close to those for relaxation, contraction was not significantly antagonized by methoctramine. Contraction might be mediated by M3-like receptors which have a very low affinity for methoctramine. 6. In conclusion, ACh-induced biphasic responses in rat iris dilator muscles were clearly distinguished from each other by specific muscarinic agonists and parasympathetic denervation, whereas muscarinic receptors could not be subclassified according to the pA2 values of 5 specific antagonists only.

Methyl mercury during late gestation affects temporarily the development of cortical muscarinic receptors in rat offspring.

Pregnant Sprague-Dawley rats were treated by gavage with a single dose of 8 mg/kg of methyl mercury on gestational day 15. Offspring of control and treated rats were killed at 14, 21 and 60 days of age. The binding characteristics of muscarinic receptors labelled in cortical membrane preparation by 3H-L-quinuclidinyl benzilate were studied together with the assessment of mercury level in the same brain area. Furthermore, the performance in passive avoidance tasks was evaluated in 8 weeks old rats. Perinatal exposure to methyl mercury significantly reduced the maximum number of muscarinic receptors (Bmax) in the brain of 14 (53%) and 21 day old rats (21%), while this change was no more present in 60 day old rats. This phenomenon seems to be strictly related to the presence of mercury in the cortex since it disappeared with the normalization of mercury levels in the brain. Despite the recovery of muscarinic receptor densities in methyl mercury exposed rats at 8 weeks of age, the avoidance latency was reduced in passive avoidance test as an indication of learning and memory deficits in these animals. Results from this study indicate that prenatal methyl mercury exposure induces latent cognitive dysfunction which does not seem to be related to transient muscarinic receptor alteration found in the early period of postnatal life.

Vitamin D status modulates rat colonic M 3 muscarinic receptor characteristics and coupling to guanylate cyclase

The present studies were conducted to determine whether [ 3H]quinuclidinyl benzilate binding in rat colonic membranes and/or carbachol-mediated stimulation of particulate guanylate cyclase were altered by changes in vitamin D status. EC 50 values for the stimulation of colonic guanylate cyclase by carbachol were found to be significantly greater in vitamin D-deficient rats compared to their D-sufficient counterparts. Concomitantly, the density of receptors ( B max) were significantly lower, and dissociation constants ( K d) were significantly higher in D-deficient colonic membranes. In vitamin D-repleted animals, moreover, all of these aforementioned alterations were at least partially corrected.

Muscarinic Receptor Characteristics and Regulation in Rat Cerebral Cortex: Changes during Development, Aging and the Oestrous Cycle

The effects of postnatal development, aging and the oestrous cycle on muscarinic acetylcholine receptor (mAChR) properties were examined in in vitro living slices of rat neocortex. Using the hydrophilic antagonist ([3H]NMS) to label cell surface mAChRs, an increase in both Bmax and Kd was found during the first postnatal weeks. These values peaked at between 20-40 days postnatally and then declined to adult levels. After 3 months of age, a steady decline in receptor number started: it was 10.1% lower at 10 months and 38.7% lower at 17 months of age. In contrast, Kd values increased, being 31.7 and 20% higher respectively at these ages. Carbachol-induced (4 h at 37 degrees C) down-regulation of receptor number was approximately 22.2% in newborn and 26.1% in adult (3-month-old) rats, but only 16.3% at 20-40 days of age. The degree of carbachol-induced down-regulation of mAChR was not affected in the older animals. Veratridine, which increases neural activity, also induced a significant reduction in [3H]NMS binding sites of 11.4% in rats aged 0-20 days and 22.4% in 3-month-old rats, but at 20-40 and 40-60 days of age no significant down-regulation of receptor number was observed. Furthermore, down-regulation was absent in the 10-month-old rats as well. Since a great variation in Bmax and Kd values was seen in 3-month-old females but not in male rats, we investigated mAChR characteristics during the oestrous cycle of female rats. In pro-oestrus, mACh receptor number was increased and affinity decreased in comparison with di-oestrus.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel regulation of muscarinic receptors and their coupling with G proteins in smooth muscle: transient resensitization during desensitizing process.

1. Muscarinic stimulation of the smooth muscle of guinea-pig taenia caeci was produced with 10(-4) M carbachol for 15 s, 30 s, 1 min, 2 min and 30 min, and the time course of developing desensitization was studied by measuring the muscle contractility and the binding characteristics of muscarinic receptors. 2. The contractile response to carbachol was analyzed using dose-response curves. The response to 10(-7) M carbachol was reduced by treatment for 15 s with 10(-4) M carbachol (fast desensitization), but recovered partially after 30 s treatment and completely after 1 min treatment (resensitization). Contractility was reduced again after 2 min and 30 min treatment (re-desensitization). 3. The affinity of carbachol for muscarinic receptors was changed by the carbachol treatment in a manner similar to the contractility. Thus, the affinity was reduced at 15 s, restored slightly at 30 s and completely at 1 and 2 min, and was reduced again at 30 min. 4. 5'-Guanylylimidodiphosphate (GppNHp), a non-hydrolysable analogue of guanosine triphosphate (GTP) reduced the affinity of muscarinic receptors for carbachol via guanine nucleotide-binding regulatory proteins (G proteins). A similar effect was observed in tissues desensitized by 15 s carbachol treatment. This effect disappeared after 30 s, recovered completely after 1 and 2 min, and disappeared again after 30 min carbachol treatment. 5. Neither the dissociation constant (Kd value) nor the maximal binding (Bmax) of [3H]-quinuclidinyl benzilate ([3H]-QNB) to muscarinic receptors were changed by the carbachol treatment. 6. These results indicate that the whole process of desensitization, resensitization and re-desensitization are related to changes in the binding ability of muscarinic receptors, in their coupling with G proteins and in the post-receptor steps of the signal transduction. We emphasize that the desensitizing process involves an early transient phase of resensitization that could be caused by restoration of both the affinity of carbachol for muscarinic receptors and their coupling with G proteins. This novel resensitization mechanism may have some physiological significance for cellular homoeostasis by modulating cellular responsiveness transiently or even in an oscillatory manner during the process of desensitization.

Imaging muscarinic cholinergic receptors in human brain in vivo with Spect, [123I]4-iododexetimide, and [123I]4-iodolevetimide.

A method to image muscarinic acetylcholine receptors (muscarinic receptors) noninvasively in human brain in vivo was developed using [123I]4-iododexetimide ([123I]IDex), [123I]4-iodolevetimide ([123I]ILev), and single photon emission computed tomography (SPECT). [123I]IDex is a high-affinity muscarinic receptor antagonist. [123I]ILev is its pharmacologically inactive enantiomer and measures nonspecific binding of [123I]IDex in vitro. Regional brain activity after tracer injection was measured in four young normal volunteers for 24 h. Regional [123I]IDex and [123I]ILev activities were correlated early after injection, but not after 1.5 h. [123I]IDex activity increased over 7-12 h in neocortex, neostriatum, and thalamus, but decreased immediately after the injection peak in cerebellum. [123I]IDex activity was highest in neostriatum, followed in rank order by neocortex, thalamus, and cerebellum. [123I]IDex activity correlated with muscarinic receptor concentrations in matching brain regions. In contrast, [123I]ILev activity decreased immediately after the injection peak in all brain regions and did not correspond to muscarinic receptor concentrations. [123I]IDex activity in neocortex and neostriatum during equilibrium was six to seven times higher than [123I]ILev activity. The data demonstrate that [123I]IDex binds specifically to muscarinic receptors in vivo, whereas [123I]ILev represents the nonspecific part of [123I]IDex binding. Subtraction of [123I]ILev from [123I]IDex images on a pixel-by-pixel basis therefore reflects specific [123I]IDex binding to muscarinic receptors. Owing to its high specific binding, [123I]IDex has the potential to measure small changes in muscarinic receptor characteristics in vivo with SPECT. The use of stereoisomerism directly to measure nonspecific binding of [123I]IDex in vivo may reduce complexity in modeling approaches to muscarinic acetylcholine receptors in human brain.

Effect of the combination of the benzodiazepine tranquilizer medazepam and the nootropic agent meclofenoxate on the activity of rat brain muscarinic receptors

1. 1. The effect of 7-day treatment with the benzodiazepine tranquilizer medazepam (5 mg/kg), the nootropic agent meclofenoxate (100 mg/kg) and their combination in the same doses on the binding activity of muscarinic receptors in four rat brain structures (cerebral cortex, striatum, hippocampus and hypothalamus) were studied using the antagonist [ 3H]-1-quinuclidinyl benzylate ([ 3H]-QNB) as radio-ligand. 2. 2. Medazepam treatment caused significant decrease of muscarinic receptor binding affinity ( K d) and of the receptor binding capacity ( B max) in the brain structures studied. The number of muscarinic binding sites was unsignificantly decreased only in the hippocampus. 3. 3. Meclofenoxate treatment caused an increase of muscarinic receptor affinity and a decrease of the binding capacity in the cerebral cortex and hypothalamus and an increase of the binding affinity in the striatum and hippocampus. 4. 4. The combination of medazepam and meclofenoxate caused no significant changes of both muscarinic receptor characteristics in the hippocampus and of the receptor affinity in the striatum and hypothalamus in comparison with control rats. The B max values were decreased in the cerebral cortex, striatum and hypothalamus when compared with control animals. The differences observed were slighter than those determined after the comparison of medazepam treated rats with control rats. 5. 5. The results obtained afford an oportunity to suggest that the nootropic agent meclofenoxate acts to moderate the effect of the benzodiazepine tranquilizer medazepam on the activity of rat brain muscarinic receptors.

Effects of the Ca 2+-antagonists nifedipine, verapamil, flunarizine and of the calmodulin antagonist trifluoperazine on muscarinic cholinergic receptors in rat cerebral cortex

1. 1. Studies were made of [ 3H]QNB binding to muscarinic receptors in a membrane fraction from the cerebral cortex of rats treated orally for 13 days with the Ca 2+-antagonists nifedipine (20 mg/kg), verapamil (50 mg/kg), flunarizine (10 mg/kg) and with the calmodulin antagonist trifluoperazine (3 mg/kg). 2. 2. The [ 3H]QNB binding capacity ( B max) was decreased by the three Ca 2+-antagonists: nifedipine, verapamil and flunarizine, the decrease was most pronounced with nifedipine. 3. 3. The decrease in the number of muscarinic receptors after nifedipine and flunarizine was accompanied by an increase in their affinity; verapamil decreased both the number and the affinity of muscarinic receptors. 4. 4. The calmodulin antagonist trifluoperazine changed neither the number nor the affinity of muscarinic receptors. 5. 5. It is suggested that continuous treatment with different Ca 2+ or calmodulin antagonists leads to difference in character and degree of alterations in the basic characteristics of muscarinic receptors in rat cerebral cortex.

Altered smooth muscle contraction and sodium pump activity in the inflamed rat intestine

We examined changes in membrane function underlying the increased contractility of jejunal longitudinal muscle to carbachol in rats infected 6 days previously with Trichinella spiralis. Muscarinic receptor characteristics were examined in particulate fractions using [N-methyl-3H]scopolamine (NMS). There was a significant reduction in the total number of binding sites on muscle from infected rats, but the affinity for NMS was unchanged. Similarly, in competition studies, the binding of carbachol to high or low affinity sites was not significantly different in tissue from control or infected rats. However, we observed an 89% suppression of the activity of K+ -stimulated ouabain-sensitive p-nitrophenylphosphatase (pNPPase), an enzyme marker for the Na+ -K+ pump, in plasma membranes from infected compared with control rats. Similar results were obtained in 86Rb uptake studies. In contractility studies, evidence for the electrogenicity of the Na+ -K+ pump was obtained by demonstrating that pump activation by K+, Rb+, or Cs+ was associated with tissue relaxation with a rank order of potency that was identical to that for stimulation of pNPPase activity by these ions. Conversely, pump inhibition by vanadate increased tone and abolished phasic contractions in muscle from control or infected rats. This was accompanied by an increased response to carbachol in muscle from control but not infected rats. In addition, pump inhibition by removing extracellular K increased tone in control tissue but decreased tone in muscle from T. spiralis-infected rats, presumably because of preexisting pump suppression. These results are consistent with the hypothesis that suppression of electrogenic Na-pump activity contributes to the increased contractility of jejunal muscle in rats infected with T. spiralis.

Autoradiographic studies on the effect of ethanol on muscarinic receptors in rat brain

The effect of ethanol vapour inhalation on the binding characteristics of muscarinic receptors in rat brain has been studied using quantitative autoradiography. After 14 days in ethanol vapour there was increased binding of [ 3H]QNB to the cortex of 4- and 6-week-old animals, but not 11-week-old animals, and to the hippocampus of the 6-week-old animals. Displacement studies indicated that the effect of ethanol was due to an increase in the density of low-affinity carbachol binding sites. There was no effect on the binding of [ 3H]QNB in the presence of pirenzipine. The data strongly suggest that the increase in binding after ethanol exposure is due to an increase in M 1 sites in the younger animals possibly linked to age-related plasticity of the nervous system.

Muscarinic receptor binding in central airway musculature in chronic airflow obstruction

The hypothesis of an increased muscarinic receptor sensitivity in airway musculature of patients with chronic airflow obstruction (CAO) has been investigated by in vitro radioligand binding studies. The receptor binding profiles were determined in membrane homogenates of tracheal and main bronchial smooth muscle, as well as in segmental bronchial tissue preparations. The number of receptors was measured by binding of the radioactive muscarinic antagonist [3H]-(-)-quinuclidinyl benzilate ([3H]-(-)QNB). The affinity for agonists was investigated by studying the inhibition of [3H]-(-)-QNB binding by the full muscarinic agonist methylfurtrethonium. Binding characteristics were determined (1) in tracheal smooth muscle from 7 patients with chronic airflow obstruction (2) in main bronchial smooth muscle of 5 patients with CAO and (3) in segmental bronchial tissue of 10 patients with CAO. The receptor binding properties were compared with values obtained on airway tissue from 31 subjects without CAO. Muscarinic receptors in smooth muscle preparations of trachea and main bronchus were present in normal density, and showed normal [ 3H]-(-)-QNB binding affinity and methylfurtrethonium binding properties. In segmental bronchial tissue preparations which contain smooth muscle and glandular tissue, also normal receptor numbers and [ 3H]-(-)-QNB affinity values were found. Morphometric examination of these preparations revealed normal amounts of smooth muscle and submucosal glands. This suggests that the excessive mucus production of CAO is not accompanied by an increased muscarinic receptor density in submucosal glands. These observations suggest unaltered muscarinic receptor characteristics in central airway smooth muscle of patients with chronic airflow obstruction.