Developmental changes in beta-adrenoceptors, muscarinic cholinoceptors and Ca2+ channels in rat ventricular muscles.

1. In an attempt to explain the previous electrophysiological data on the ontogeny of beta-adrenergic and muscarinic cholinergic interactions on cardiac Ca2+ current, biochemical studies were performed on the ontogeny of beta-adrenoceptors, muscarinic cholinoceptors and Ca2+ channels in cardiac muscle of developing rats: 16-20 days old foetuses, 0-20 days old neonates, and 2-3 months old adults. 2. Developmental changes in cardiac beta-adrenoceptors, muscarinic cholinoceptors, and Ca2+ channels were determined with the use of specific radioligands, [3H]-dihydroalprenolol (DNA), [3H]-quinuclidinyl benzilate (QNB), and [3H]-nitrendipine (NTD), respectively. 3. The Bmax value (fmol mg-1 tissue) for [3H]-DNA binding started to increase on post-gestation day 20, reached almost its maximum level on neonatal day 6, kept almost the same level until neonatal day 20, and then decreased slightly to its adult level. 4. The Bmax value (fmol mg-1 tissue) for [3H]-QNB binding started to increase on post-gestation day 16, reached almost its maximum level on neonatal day 0, remained almost constant until neonatal day 15, and then decreased to its adult level. 5. The Bmax value (fmol mg-1 tissue) for [3H]-NTD binding increased with age between post-gestation day 18 and neonatal day 15, stayed almost constant until neonatal day 20, and then decreased to its adult level. 6. The Kd values for [3H]-DHA, [3H]-QNB, and [3H]-NTD bindings remained almost constant during the developmental period examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha 1- and beta-adrenergic and muscarinic cholinergic receptors in guinea-pig nasal mucosa

The alpha 1- and beta-adrenergic and muscarinic cholinergic receptors in guinea-pig nasal mucosa were measured for the first time by direct binding techniques using [3H]prazosin, [3H]dihydroalprenolol ([3H]DHA) and [3H]quinuclidinyl benzilate ([3H]QNB). The maximum binding capacities of [3H]prazosin, [3H]DHA and [3H]QNB in guinea-pig nasal mucosa were 20.1, 42.3, 159 fmol/mg protein, respectively. The dissociation constants of [3H]prazosin, [3H]DHA and [3H]QNB in guinea-pig nasal mucosa were 0.37, 0.77 and 1.5 nM, respectively. After the removal of the superior cervical ganglion in the guinea-pig, the number of alpha 1-adrenergic receptors was increased in the nasal mucosa while the number of beta-adrenergic and muscarinic receptors remained unchanged. Thus, alpha 1-adrenergic receptors are probably postsynaptic receptors in the target cells of sympathetic nerves, while beta receptors relate to the circulating catecholamines.     

The binding of [3H]prazosin and [3H]clonidine to rat jejunal epithelial cell membranes

The binding of [3H]prazosin and [3H]clonidine to rat jejunal epithelial cell membranes has been studied. The membrane preparation was enriched in baso-lateral components as determined by Na+, K+ ATPase and alkaline phosphatase activities. The membranes possessed two saturable specific binding sites for [3H]prazosin, a high affinity (Kd 0.17 nM) low capacity (Bmax 27.3 fmole bound per mg protein) and a low affinity (Kd 5.0 nM) high capacity (Bmax 276 fmole bound per mg protein) site. The specificity of both sites was similar and was related to alpha 1-adrenoceptors. [3H]Clonidine bound to the membranes in a saturable fashion (Kd 7.3 nM). The specificity of this site was related to alpha 2-adrenoceptors. The [3H]clonidine binding site was present in the membranes in much lower density (Bmax 22.8 fmole bound per mg protein) suggesting that alpha 1-adrenoceptors predominate in this tissue.     

Muscarinic receptor binding characteristics in rat tissues after oral administration of oxybutynin and propiverine

Ex vivo muscarinic receptor binding of oxybutynin and propiverine, the most commonly used anticholinergic agents for the treatment in patients with urinary incontinence, was investigated in rat tissues. The oral administration of oxybutynin (50.8 and 127 micromol/kg) caused a significant increase in the apparent dissociation constant (Kd) for specific (-)-[3H]QNB binding in the rat bladder, prostate, submaxillary gland, heart and cerebral cortex, compared with each of the control values. Also, in the submaxillary gland of these rats, there was a reduction in the maximal number of binding sites (Bmax) for (-)-[3H]QNB binding. Similarly, oral administration of propiverine at doses of 74.3-297 micromol/kg brought about a significant increase in the Kd values for (-)-[3H]QNB binding in rat tissues including the bladder, and greater increase in Kd values was seen in the rat prostate, heart and submaxillary gland. On the other hand, oral administration of propiverine, unlike oxybutynin, resulted in very little reduction in the Bmax valules for (-)-[3H]QNB binding in the submaxillary gland. In conclusion, the present study has shown that oxybutynin and propiverine, after oral administration, bind significantly to muscarinic receptors in tissues such as the bladder, which is the target organ for the treatment of urinary incontinence, and that oxybutynin appears to exhibit long-term binding to muscarinic receptors in the salivary gland.     

Alpha 2-adrenoceptor subtypes and imidazoline-like binding sites in the rat brain.

1. The binding of [3H]-yohimbine and [3H]-idazoxan to rat cortex and hippocampus is rapid, reversible and of high affinity. Saturation data indicate that a single population of binding sites exist for [3H]-yohimbine in the cortex (Bmax 121 +/- 10 fmol mg-1, protein; Kd 5.2 +/- 0.9 nM) and hippocampus (Bmax 72 +/- 6 fmol mg-1 protein; Kd 5.8 +/- 0.7 nM). [3H]-idazoxan labels one site in the cortex (Bmax 87 +/- 8 fmol mg-1 protein; Kd 4.1 +/- 0.9 nM) and hippocampus (Bmax 30 +/- 6 fmol mg-1 protein; Kd 3.5 +/- 0.5 nM), when 3 microM phentolamine is used to define non-specific binding. A second distinct [3H]-idazoxan binding site (Bmax 110 +/- 21 fmol mg-1 protein; Kd 3.6 +/- 0.07 nM) is identified in rat cortex if 0.3 microM cirazoline is used to define non-specific binding and 3 microM yohimbine is included to prevent binding to alpha 2-adrenoceptors. 2. Displacement studies indicate that the alpha 1-adrenoceptor antagonist prazosin and the 5-HT1 ligands 8-OH-DPAT, RU 24969 and methysergide differentiate [3H]-yohimbine binding into two components; a high and low affinity site. In contrast the displacement of [3H]-idazoxan by each ligand was monophasic. 3. The affinities of 8-OH-DPAT, RU 24969 and methysergide determined against [3H]-idazoxan binding to the cortex and hippocampus correlate significantly with the binding site displaying low affinity for prazosin and previously designated alpha 2A. In contrast, a poor correlation exists for the high affinity site for prazosin designated alpha 2B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased density of alpha 2-adrenoceptors in medulla oblongata of spontaneously hypertensive rats

To study the role of medullary alpha-adrenoceptors in hypertension, we compared specific binding of [3H]prazosin and [3H]clonidine in different brain regions of spontaneously hypertensive rats (SHR), stroke-prone SHR (SHRSP), and normotensive Wistar-Kyoto rats (WKY). As compared with age-matched WKY, Bmax values for specific [3H]clonidine binding in the medulla oblongata were significantly lower in SHR and SHRSP at 16-24 weeks of age. In the SHRSP medulla oblongata, the decrease was more prominent in dorsomedial and ventrolateral regions than in the ventromedial region. Density of alpha 2-adrenoceptor binding sites was also decreased in the medulla oblongata of young (4-5-week-old) SHRSP. In contrast, there was no difference in Kd and Bmax values for medullary [3H]prazosin binding between WKY and SHRSP. The dorsomedial and ventrolateral regions of the SHRSP medulla oblongata showed significantly lower levels of norepinephrine (NE). Thus, the present study demonstrates that there is a specific loss of alpha 2-adrenoceptors in the medulla oblongata of SHR and SHRSP that may be partly involved in the pathogenesis of spontaneous hypertension.     

新抗胆碱药[3H]三环哌酯对人脑M受体的作用

用放射配体受体结合试验法,研究了新化合物三环哌酯与人大脑皮质M受体的结合特性,并与QNB作了比较。饱和实验结果显示,[³H]三环哌酯的结合参数与[³H]QNB相近,两种配体的作用均符合单位点模型。竞争性抑制实验结果表明二者作用强度相当。[³H]三环哌酯的结合和解离速率常数均较[³H]QNB大,且其与皮质M受体的解离受季铵酚的变构调节,结果提示,两种配体与M受体有一些不同的结合特性,在M受体研究中,[³H]三环哌酯可以作为[³H]QNB的补充工具。     

Alpha 2-adrenoceptor and catecholamine-insensitive binding sites for [3H]rilmenidine in membranes from rat cerebral cortex.

The kinetic and pharmacological characteristics of the binding of the oxazoline antihypertensive drug, [3H]rilmenidine, to membranes of rat cerebral cortex have been determined. Computerised resolution of curvi-linear, equilibrium binding isotherms was consistent with the existence of two distinct binding sites for [3H]rilmenidine: Kd 17.3 +/- 7.41 nM, Bmax 0.197 +/- 0.06 pmol/mg protein and Kd 254 +/- 48 nM, Bmax 1.59 +/- 0.08 pmol/mg protein. Moreover, the resolution of two association and dissociation rates also suggested the existence of two binding site populations. Drug inhibition studies revealed that specific binding of [3H]rilmenidine (2 nM) was only inhibited by a maximum of 50% by the catecholamines, adrenaline and noradrenaline, but was completely inhibited by some oxazolines, by guanabenz (a guanidino drug) and by several imidazoline compounds including naphazoline, oxymetazoline and clonidine. Binding isotherms for these drugs were also best fit by a two-site model. The relative Ki values at the high affinity site for [3H]rilmenidine and the number of these high affinity sites are consistent with this site being an alpha 2-adrenoceptor. The high affinity of oxymetazoline and low affinity of prazosin for high affinity [3H]rilmenidine binding sites together with the rank order of potency of oxymetazoline greater than phentolamine greater than SKF 104078 greater than ARC-239 greater than prazosin suggest that [3H]rilmenidine binds to the alpha 2A sub-type of adrenoceptor. Computer-resolved Ki values for drugs at the larger number of lower affinity binding sites were very similar to Ki values determined in the presence of 10 microM adrenaline (used to block alpha 2-adrenoceptor binding).(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of benzodiazepine receptor, adrenoceptor and muscarinic receptor by diazepam in rat parotid gland

This study investigated the influence of diazepam on the binding characteristics of adrenoceptor, muscarinic and benzodiazepine receptors in rat parotid gland membrane using a radioligand binding assay. At a concentration of >10(-6)M, diazepam competed with [(3)H]dihydroalprenolol for β-adrenoceptor, but not [(3)H]prazosin for α-adrenoceptor or [(3)H]quinuclidinyl benzilate for muscarinic receptor. Continuous administration of diazepam at doses of 0.4mg/kg/day, i.p. for 7days in rat significantly decreased pilocarpine (4.0mg/kg, i.p.)-induced parotid salivary flow. Diazepam also produced a significant increase in the dissociation constant (Kd) value for [(3)H]dihydroalprenolol binding, but no change in the maximal binding capacity (Bmax) value, and a decrease in the Kd value for [(3)H]diazepam binding to benzodiazepine receptors, but no change in the Kd or Bmax values for [(3)H]prazosin or [(3)H]quinuclidinyl benzilate binding. These results suggest that continuous administration of diazepam modifies affinity for β-adrenoceptor and benzodiazepine receptor binding sites in parotid gland membrane and that changes in these binding sites may be closely related to diazepam-induced suppression of salivary secretion.     

Effect of acute and chronic morphine administration on brain cholinergic muscarinic receptors

The binding of [3H]quinuclidinyl benzilate (QNB) to various brain regions was determined in rats after acute or chronic treatment with morphine. Morphine and naloxone, in vitro, inhibited the binding of [3H]QNB to striatal membranes only at high concentrations. Thirty minutes after a single injection, morphine (5 or 40 mg/kg s.c.) did not alter the Bmax or Kd values for [3H]QNB binding to striatal receptors. The binding of [3H]QNB to membranes of different brain regions was not changed in morphine tolerant-dependent rats or rats undergoing abrupt or naloxone precipitated withdrawal. The results suggest that central cholinergic muscarinic receptors are unaffected by acute or chronic treatment with morphine, or during abstinence.     

Interaction of phenoxybenzamine with muscarinic receptors and calcium channels

Phenoxybenzamine (POB, 10(-6) - 10(-4) M) inhibited the responses of guinea pig ileal longitudinal smooth muscle to both muscarinic agonists and K+-depolarization but was more effective against the agonist-induced responses. POB inhibited binding of both the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) and the Ca2+ channel antagonist [3H]nitrendipine and was, paralleling its effects on mechanical responses, more effective against [3H]QNB binding. POB reduced specific [3H]QNB binding by a reduction in Bmax with no change in KD, but inhibited [3H]nitrendipine binding by reducing KD with no effect on Bmax. It is suggested that the activity of POB against Ca2+ channels may underlie the ability of POB, and other 2-halogenoethylamines, to inhibit a wide variety of apparently discrete pharmacological events.     

Autoradiographic analysis of receptors on vascular endothelium

Receptor autoradiography was used to examine the distribution of muscarinic cholinoceptors ([3H]QNB), alpha 2-adrenoceptors ([3H]rauwolscine), beta-adrenoceptors ([125I]CYP) and substance P receptors ([125I]BHSP) in rabbit aorta, pulmonary artery, rat aorta, dog aorta, splenic, renal and coronary arteries, bovine aorta and coronary arteries. Muscarinic cholinoceptors and alpha 2-adrenoceptors were not associated with endothelium in any of the blood vessels examined. Substance P receptors were found on endothelium in dog renal but not bovine coronary arteries, and beta-adrenoceptors were found on endothelium in dog coronary arteries but not bovine aorta. The results suggest that endothelium-dependent relaxation can result either from activation of receptors located directly on the endothelial cells or, as is the case for ACh, by an indirect mechanism via activation of receptors located on the vascular smooth muscle.     

Non-adrenergic sites for imidazolines are not directly involved in the alpha 2-adrenergic antilipolytic effect of UK 14304 in rat adipocytes

The binding of the alpha 2-agonist [3H]UK 14304 on Wistar rat adipocyte membranes was separated in two distinct components: one was displaceable by adrenaline or other alpha 2-adrenergic agents and possessed the characteristics of alpha 2-adrenoceptors while the other, non-adrenergic in nature, was only recognized by some imidazoline derivatives [3H]idazoxan binding shared the same characteristics. The non-adrenergic sites labeled by both radioligands are similar to those described for [3H]idazoxan on other tissues such as brain cortex, smooth muscle and kidney. Even though they were about 10-fold more numerous than the true alpha 2-adrenoceptors, the non-adrenergic binding sites were not directly involved in the antilipolytic action of UK 14304 since alpha 2-antagonists devoid of interaction with these sites (yohimbine, phentolamine) totally blocked the UK 14304 effect. However, the existence of such a type of site impairs direct quantification of alpha 2-adrenoceptors in rat adipocytes. The use of [3H]RX 821002 (2-(2-methoxy-1,4-benzodioxan-2yl)imidazoline) allowed an accurate quantification of rat adipocyte alpha 2-adrenoceptors (Bmax = 35 +/- 2 fmol/mg protein, Kd = 2.6 +/- 0.6 nM) since it did not interact with non-adrenergic binding sites and exhibited the highest alpha 2-blocking properties among the various alpha 2-antagonists tested. [3H]RX 821002 binding analysis revealed that alpha 2-adrenoceptors are, on rat adipocytes; (i) less numerous than in other species well known for their alpha 2-adrenergic inhibitory regulation of lipolysis (human, hamster, rabbit); (ii) slightly different in nature from the receptors of these species since they had weaker affinity for clonidine and yohimbine; and however (iii) not of the typical alpha 2-B subtype since the affinity of prazosin was lower than that of oxymetazoline in displacing [3H]RX 821002 or [3H]yohimbine binding.     

Dopamine-induced up-regulation of alpha 2-adrenoceptors in crude synaptic membranes of rat brain

After cerebral cortical membranes were incubated with 0.1-100 microM of dopamine (DA) in 50 mM Tris-HCl buffer (pH 7.7) at 37 degrees C for 30 min, [3H] clonidine binding to alpha 2-receptors was increased in a concentration-dependent manner without changing [3H] WB4101 and [3H] DHA binding to alpha 1- and beta-receptors, respectively. Scatchard analysis of [3H] clonidine binding to cortical membranes showed that DA increased the Bmax in both high- and low-affinity components. The increasing effect of DA on [3H] clonidine was dependent on incubation time and temperature, and it was antagonized by pimozide and cis-flupenthixol. The addition of GTP produced a reduction in DA-induced elevation in [3H] clonidine binding, while that of cyclic AMP did not affect the effect of DA. DA and Mn2+, though both of them increased [3H] clonidine binding, appeared to act at a different site in the membrane. Furthermore, the DA-induced increase in [3H] clonidine was found uniformly in membranes prepared from 7 other regions of the rat brain. These results suggested that DA regulates specifically alpha 2-receptor density by stimulating D1-receptors and/or via other mechanism(s).     

Characterization of alpha 2-adrenergic receptors of calf retina membranes by [3H]-rauwolscine and [3H]-RX 781094 binding

Alpha 2-adrenergic receptors were identified in calf retina membranes by binding of the radiolabelled antagonists [3H]-RX 781094 and [3H]-rauwolscine. When 10 microM phentolamine was used to determine the non-specific binding, both radioligands labelled a single class of non-cooperative sites: Bmax = 1051 +/- 252 fmol/mg protein, Kd = 5.1 +/- 1.5 nM for [3H]-RX 78104 and Bmax = 1167 +/- 449 fmol/mg protein, Kd = 21.0 +/- 4.1 nM for [3H]-rauwolscine. Competition binding experiments showed the typical pharmacological potency order of alpha 2-adrenergic receptors, i.e. phentolamine greater than yohimbine greater than prazosin. Agonist competition binding curves revealed the presence of two receptor populations, having respectively high affinity (70% of the total receptor population) and low affinity for agonists, but with the same affinity for the antagonists. The high affinity sites could be converted into low affinity sites by guanine nucleotides. The non-specific binding of [3H]-RX 781094 was the same if 0.1 mM (-)-epinephrine was used instead of phentolamine. In contrast, the non-specific binding of [3H]-rauwolscine was markedly lower with (-)-epinephrine than with phentolamine. Under this condition, the Scatchard plot of [3H]-rauwolscine saturation binding was curvilinear, indicating the presence of low affinity sites for the radioligand in addition to alpha 2-adrenergic receptors. Competition binding experiments revealed that these low affinity sites were distinct from adrenergic receptors: the catecholamine agonists (-)- and (-)-epinephrine, (-)-norepinephrine, (-)-isoproterenol and dopamine competed with similar Ki values (microM range) whereas clonidine did not interact. Furthermore, these sites bound reserpine and the alpha 2-adrenergic antagonists yohimbine and rauwolscine but not phentolamine.     

Identification of drug receptors in porcine dental pulp by the radioligand binding assay

1. The acetylcholine (ACh), histamine and serotonin (5-HT) receptors in porcine dental pulp were characterized by the radioligand binding assay. 2. For [3H]nicotine binding site, Kd was 8.06 +/- 1.65 nM and Bmax was 270.83 +/- 32.68 fmol/mg protein. 3. For [3H]QNB binding site, Kd was 1.04 +/- 0.14 nM and Bmax was 24.83 +/- 3.09 fmol/mg protein. 4. For [3H]histamine binding site, Kd was 1.22 +/- 0.1 nM and Bmax was 283.15 +/- 33.1 fmol/mg protein. 5. For [3H]5-HT binding site, Kd was 1.41 +/- 0.1 nM and Bmax was 53.1 +/- 3.4 fmol/mg protein. 6. These findings indicate that the specific receptors for ACh, histamine and 5-HT are present in the porcine dental pulp, and that the ACh receptor is predominantly nicotinic.     

Modulation of alpha 1-and alpha 2-adrenoceptor binding sites in the brain of audiogenic seizure susceptible mice (DBA/2J)

The binding of [3H]dihydroalprenolol ([3H]DHA), [3H]prazosin and [3H]clonidine was assayed in whole brain and various brain regions of audiogenic seizure (AS) susceptible DBA/2J (D2) mice aged 10, 24 and 50 days (i.e. before, during and after their period of AS susceptibility, respectively) and in age-matched C57BL/6J (B6) controls. In whole brain, at 24 days, [3H]DHA binding was similar in the two strains, while the binding of [3H]prazosin and [3H]clonidine was significantly lowered in D2 mice. No difference could be detected in 10 and 50 day old mice with any of the ligands. Regional studies indicated an involvement of the cerebral cortex, the olfactory bulbs and the brain-stem. alpha- (but not beta-)adrenoceptor changes were concomitant with the AS susceptibility period. These changes were unevenly distributed in the brain of D2 mice; they suggest that alpha 1- and alpha 2-adrenoceptor subtypes might play different roles in the AS of the D2 mouse strain.     

Influence of long-term treatment with the Ca2(+)-antagonists nifedipine, verapamil, flunarizine and with the calmodulin antagonist trifluoperazine on beta-adrenoceptors in rat cerebral cortex

1. The binding of [3H] dihydroalprenolol ((3H]DHA) to beta-adrenoceptors in cerebral cortex membranes (fraction P2) was studied after 13-day oral treatment of male Wistar rats with the Ca2(+)-antagonists nifedipine (20 mg/kg), verapamil (50 mg/kg), flunarizine (10 mg/kg) and with the calmodulin (CaM)-antagonist trifluoperazine (TFP) (3 mg/kg). 2. A significant reduction in the number (Bmax) of binding sites for [3H]DHA was established after the 3 Ca2(+)-antagonists as well as after TFP. 3. No changes in the affinity values (Kd) [3H]DHA binding were found in all treated groups. 4. The fluidity characteristics of the cerebral cortex membranes in nifedipine- and flunarizine-treated rats did not differ from those in controls. 5. Based on these results, we concluded that long-term treatment with Ca2(+)- and calmodulin-antagonists might induce changes in the central adrenergic mechanisms.     

Functional supersensitivity to the alpha 1-adrenoceptor agonist after repeated treatment with antidepressant drugs is not conditioned by beta-down-regulation

The effect of repeated administration of desipramine, and the (+)- and (-)-enantiomers of oxaprotiline (10 mg/kg i.p., twice daily for 14 days) on the binding of beta- and alpha 1-adrenoceptors in the cortex of the rat brain were studied. The functional consequences of such treatment were measured in a behavioural model, involving the exploratory activity of rats in response to administration of the alpha 1-agonist phenylephrine. Desipramine and (+)-oxaprotiline decreased the binding of [3H]dihydroalprenolol ([3H]DHA) to beta-adrenoceptors in the cortex, did not change the binding of [3H]prazosin to alpha 1-adrenoceptors, but enhanced behavioural responses to phenylephrine. A behavioural facilitation was also observed after administration of (-)-oxaprotiline, a substance which does not change the binding of [3H]DHA. These results indicate that a functional supersensitivity to the alpha 1-adrenoceptor agonist, after repeated treatment with antidepressants is not conditioned by beta-down-regulation.     

Differential distribution of muscarinic receptor subtypes and their regulation by G-protein in rat brain

1. [3H]quinuclidinyl benzilate ([3H]QNB) binding in rat cerebral and cerebellar synaptosomes had different Bmax values, but similar Kd values. 2. These bindings could be displaced by classic muscarinic agents: pilocarpine (partial agonist), and atropine (antagonist), which both had similar binding affinities in rat cerebral and cerebellar synaptosomes. 3. The new muscarinic M1 selective agents: McN-A-343 (agonist), pirenzepine and trihexyphenidyl (antagonists) and higher affinities for receptor sites in the cerebrum than in the cerebellum. 4. The muscarinic M2 selective agents: carbachol, oxotremorine (agonists), and AF-DX-116 (antagonist) had higher affinities for receptor sites in the cerebellum than in the cerebrum. 5. GPP(NH)p (40 microM) decreased the binding affinities of carbachol and oxotremorine in the cerebellum, but not in the cerebrum. However, it did not decrease the binding affinities of all the antagonists studied in both brain regions. 6. These results reveal that more muscarinic M1 sites are present in the cerebrum than in the cerebellum, while the opposite is true for M2 sites. Furthermore, the regulatory role of G-protein on these muscarinic receptor subtypes in the brain is different.