QNB Binding Research Articles

Studies of blocking mechanisms of carbachol-induced polyphosphoinositide turnover in rat cortical synaptosomes by neuroactive drugs

1. 1. The incorporation of 32Pi into 4 phospholipids of rat cortical synaptosomes was altered in the presence of carbachol (1 mM), viz. a decrease of phosphatidylinositol-4,5-bisphosphate and phosphatidyl-4-phosphate by 34 and 21%, and an increase of phosphatidylinositol and phosphatidic acid by 52 and 96% of basal controls respectively. 2. 2. The IC 30 values calculated from the dose-response curves for drugs affecting carbachol-induced 32Pi incorporation into these phospholipids, and [ 32H]QNB binding to the cortical synaptosomes were similar for the typical antimuscarinic agents (i.e. atropine, pirenzepine and trihexyphenidyl), and tricyclic antidepressants (i.e. amitryptyline, doxepin and imipramine) studied. 3. 3. The IC 30 values obtained for drugs affecting carbachol-induced 32Pi incorporation into these phospholipids, and high potassium-induced 45Ca 2+-uptake by this preparation were similar for neuroselective calcium channel blockers (i.e. cinnarizine and flunarizine) studied. 4. 4. Our results suggest that the neuroactive drugs studied can either act at, or beyond the receptor level, perhaps on the availability of calcium ion, to block carbachol-induced polyphosphoinositide turnover in rat cortical synaptosomes.

Effects of long-term dietary choline and phosphatidylcholine administration on muscarinic receptors in aged mouse brain.

It has been suggested that choline and/or phosphatidylcholine may be beneficial in improving the memory deficits associated with aging and Alzheimer's disease. The effects of long-term choline or phosphatidylcholine treatment on cholinergic receptors in the brain have been investigated. Mice were maintained on one of four diets from 50 days of age until sacrificed at 20-24 months. [3H]-QNB binding in the cortex, hippocampus and striatum was specific, saturable and of high affinity. Animals treated with a phosphatidylcholine-enriched diet displayed a down-regulation of muscarinic receptors in the cortex and hippocampus, reflected by a decrease in Bmax. There were no significant differences in the binding affinities among the treatment groups. Choline levels were unaffected by the various diets, however phosphatidylcholine treatment resulted in an increase in phosphatidylcholine in the cortex and somewhat in the hippocampus. This study indicates that choline and phosphatidylcholine have different effects after long-term dietary administration. Phosphatidylcholine treatment results in a down-regulation of muscarinic receptors in certain brain areas which appears to be related to an increase in phosphatidylcholine concentration. Any potentially beneficial effects derived from chronic phosphatidylcholine treatment must overcome the apparent down-regulation of muscarinic receptors which may occur.

Changes in affinity states during down-regulation of muscarinic receptors in tracheal smooth muscle of organophosphate-treated swine.

1. Subacute (daily) treatment of male swine with the organophosphate acetylcholinesterase inhibitor diisopropylfluorophosphate (DFP) resulted in tolerance to the effects of DFP within 5-6 days. 2. Subacute administration of DFP resulted in a 98% inhibition of tissue cholinesterase after 7 days and in a decrease of [3H]quinuclidinyl benzilate [( 3H]QNB) binding sites in homogenates of tracheal smooth muscle by 77%. The maximal density of receptors (Bmax) decreased from 1.8 +/- 0.4 to 0.5 +/- 0.1 pmole mg-1 protein. There was no significant change in the dissociation constant (Kd) for [3H]QNB binding. 3. Pirenzepine displacement of [3H]QNB binding was best described by a single binding site model, with a Ki of 230 +/- 40 nM. This value was unchanged following seven days of DFP treatment (250 +/- 30 nM). The low affinity for this M1 antagonist suggests that there is predominantly a single population of [3H]QNB binding sites of the M2 subtype in tracheal smooth muscle. 4. Carbachol displacement of [3H]QNB binding yielded data best fit by a two-binding site model. The dissociation constants were KiL = 210 +/- 60 microM (61 +/- 1%) and KiH = 1.2 +/- 0.4 microM (39 +/- 1%) respectively (n = 7) for the low and high affinity states. Seven-day treatment with DFP reduced the percent of high affinity receptors to 25 +/- 4%. 5. Addition of Mg++ to the incubation medium prevented this shift in the proportion of low and high affinity receptors. Gpp(NH)p and Mg++ together decreased the proportion of the high affinity receptors when added to the incubation medium in control tissue (to 25%), but not tissue from 7-day DFP-treated swine. NEM increased the proportion of muscarinic receptors in the high affinity state both for controls and for the DFP-treated swine, in both cases yielding receptors with identical binding properties. 6. Thus, subacute administration of DFP causes not only a decrease in the number of receptors, but also a change in the affinity of the receptors for agonists which is related to the interaction of the guanine nucleotide binding protein and the muscarinic receptor.

Inhibition of phosphoinositide turnover by selective muscarinic antagonists in the rat striatum: Correlation with receptor occupancy

In the rat corpus striatum, receptor occupancy and the inhibition of phosphoinositide turnover by muscarinic antagonists have been examined under very similar conditions with respect to tissue preparation and buffer composition. The results suggest a good correlation between receptor occupancy and inhibition by muscarinic antagonists, of the carbachol-stimulated turnover of inositol phospholipids, measured by the accumulation of [ 3H]inositol phosphates in the presence of 5 mM LiCl. In the presence of 10 mM carbachol (CCh), the accumulation of labeled inositol phosphates was increased 8-fold above basal levels ( ec 50 = 95 μ M ). Inclusion of antagonists resulted in a dose-dependent inhibition of the 0.1 mM CCh-stimulated inositol phosphate accumulation, with a rank order of potency of atropine > trihexyphenidyl > pirenzepine ⪢ gallamine. Radioligand binding studies with [ 3H]- l-quinucliclinyl benzilate ([ 3H]QNB) in a cell aggregate preparation revealed a single class of saturable, high affinity [ 3H]QNB binding sites exhibiting a K d of 74 pM and a B max, of 2.85 pmol/mg protein. The antagonists examined were able to inhibit the binding of [ 3H]QNB with the same rank order of potency as for the inhibition of carbachol-stimulated phosphoinositide turnover (atropine > trihexyphenidyl > pirenzepine ⪢ gallamine). Although the inhibition of phosphoinositide turnover and [ 3H]QNB binding by the nonselective antagonist atropine was best described by interaction at a single site, inhibition of phosphoinositide turnover and [ 3H]QNB binding by both pirenzepine, which is selective for M1 receptors, and gallamine, which is selective for M2 receptors, is complex. Pirenzepine was much more potent than gallamine for both binding to receptors and inhibiting phosphoinositide turnover. Nonlinear curve-fitting analysis indicated that slope factors for inhibition of phosphoinositide turnover (analogous to Hill coefficient for binding) by only subtype selective antagonists were significantly less than unity. The above-mentioned antagonist interactions together with the apparently multicomponent stimulation of phosphoinositide turnover by carbachol suggest that phosphoinositide turnover may be coupled to more than one muscarinic receptor subtype in the corpus striatum.

Quantitative distribution of muscarinic receptors and choline acetyltransferase in rat medulla: examination of transmitter-receptor mismatch

Recent reports have identified discrepancies between the anatomical distribution of transmitters and their receptors, a phenomenon known as transmitter-receptor mismatch. However, quantitative determinations of transmitter activity and receptor density in individual brain regions have not been conducted in parallel. We therefore sought to determine quantitatively the relationship between muscarinic acetylcholine receptor density and the density of cholinergic innervation as reflected by activity of the biosynthetic enzyme for acetylcholine, choline acetyltransferase (ChAT). To assure sampling of equivalent regions using the two methods, an ‘electronic micropunch’ technique was developed to allow measurement of [ 3H]quinuclidinyl benzilate ([ 3H]QNB) binding within the corresponding cylinders of tissue obtained by the micropunch cannula. Nineteen regions of the rat medulla (1 mm diameter, 1 mm height) were studied. The micropunch region containing the gracile nucleus, the area postrema and the choroid plexus of the fourth ventricle contained the highest ChAT activity, but exhibited little [ 3H]QNB binding to muscarinic receptors. However, among the remaining 18 regions a strong correlation was obtained between uncorrected muscarinic receptor density and ChAT activity within each micropunched region ( r = 0.89, n = 18). Correction for autoradiographic efficiency weakened the overall relationship between receptor density and ChAT activity ( r = 0.58, n = 18). This was due to a relatively high density of receptors associated with fiber tract regions containing low ChAT activity. The presence of receptors within white matter is ordinarily obscured by high tritium quenching. This is consistent with the hypothesis that a portion of muscarinic receptors are located extrasynaptically and may be present within axons. When the 4 areas with the highest white matter content, as indicated by low autoradiographic efficiency, were considered separately, corrected muscarinic receptor density and ChAT activity were highly correlated in this group ( r = 0.86, n = 4) and in the remaining regions ( r = 0.70, n = 14). Thus, cholinergic innervation is a major determinant of muscarinic receptor density in both white matter and grey matter regions. We conclude that muscarinic receptor density is correlated with the extent of cholinergic innervation as indicated by ChAT activity. The transmitter-receptor mismatch which did occur could be accounted for by the existence of receptors undergoing axonal transport. These findings do not support the hypothesis that acetylcholine and other neurotransmitters diffuse across large distances to act on non-innervated receptors.

Contractile responses of tracheal smooth muscle in organophosphate‐treated swine: 2. Effects of antagonists

1. Swine tracheal smooth muscles (TSM) developed spontaneous contractions following the acute administration of DFP in vivo and/or in vitro which could be blocked pharmacologically using atropine (2 x 10(-7) M), pirenzepine (3 x 10(-7) M), or hemicholinium 3 (HC3, 5 x 10(-6) M). 2. Treatment of TSM in vitro with DFP caused them to become responsive to ACh concentrations as low as 10(-10) M. 3. Atropine and pirenzepine (at 2 and 3 x 10(-7) M respectively) increased the EC50 concentrations for ACh approximately 300- and 8-fold respectively. The shifts caused by atropine and pirenzepine in the dose-response curves for ACh were not parallel after in vitro treatment of the muscle with DFP. By contrast, the shifts in the dose-response curves were parallel when muscles from swine injected for 7 days with DFP were used. 4. HC3 had no effect on the control dose-response curve for ACh, but steepened the dose-response curve after in vitro treatment of the muscle with DFP. The ACh dose-response curve obtained in the presence of HC3 and DFP was identical to that obtained using muscles from swine treated for 7 days with DFP. 5. McN-A-343, a partial agonist at muscarinic receptors, also induced contraction although the maximal tension induced was 56% of the maximal tension obtained using ACh. In vitro treatment of the muscle with DFP caused a leftward shift in the dose-response curve for McN-A-343. The muscles from animals treated for 7 days with DFP did not respond to McN-A-343 at doses up to 10(-3) M. 6. McN-A-343 competition for [3H]QNB binding suggested that the loss of the contractile response can be correlated with the loss of a high affinity site for McN-A-343 from the muscle. 7. We conclude that tolerance to DFP with subacute treatment results in part from the reduction in sensitivity of neural elements associated with swine tracheal smooth muscle to ACh. In addition the response to the partial agonist McN-A-343 is lost after subacute DFP treatment.

Muscarinic receptors in preoptic area and hypothalamus: effects of cyclicity, sex and estrogen treatment

Cholinergic muscarinic receptor binding was measured in the preoptic area (POA) and whole hypothalamus (HTH) of adult Sprague-Dawley rats using the tritiated antagonist quinuclidinyl benzilate ([ 3H]QNB) as the ligand. Binding of [ 3H]QNB expressed as fmol/mg protein was 30% higher in POA than in HTH from gonadectomized rats. Cyclic changes were observed in the POA with the highest binding at proestrus and the lowest binding at diestrus. In HTH, no significant changes occurred over the estrous cycle. Estrogen treatment (10 μg of estradiol benzoate (EB)/120 g b. wt./48 and 24 h before sacrifice) increased [ 3H]QNB binding by 42% in the POA and 17% in HTH, relative to the ovariectomized controls. The enhancement of [ 3H]QNB binding in POA as compared with controls was evident with both the filtration and the centrifugation methods, although binding levels were higher when centrifugation assay was used. A lower estrogen dose (2 μg EB/rat/48 and 24 h before sacrifice) which is routinely used to activate lordotic behavior in female rats increased muscarinic binding by 26% in the POA but had no appreciable effect in HTH. A significant sex difference was found in the ability of estrogen to induce [ 3H]QNB binding in the central nervous system (CNS). Estrogen was ineffective in altering [ 3H]QNB binding in either brain region of castrated males, although the level and pattern of cholinergic binding between untreated gonadectomized males and females were similar. These data suggest that physiological changes in estrogen secretion over the estrous cycle are capable of modulating cholinergic muscarinic binding in the POA and these changes may be physiological relevance. The failure of estrogen to enhance binding in either the POA or HTH of male rats is consistent with the hypothesis that cholinergic mechanisms are involved in mediating sensitivity to estrogenic hormones and implicates the sexual differentiation process in its regulation.

Sensory deafferentation fails to modify muscarinic receptor binding in raccoon somatosensory cortex

The characteristics and distribution of muscarinic acetylcholine (mACh) receptor binding in primary somatosensory (SI) cortex and the caudate nucleus of raccoons were studied using [ 3H]-QNB, a muscarinic antagonist. The binding characteristics were similar to reported values in rat and cat. Autoradiographs produced from tissue sections labeled with [ 3H]-QNB showed the distribution of mACh receptors in the forebrain of the raccoon. [ 3H]-QNB binding was highest in cerebral cortex, neostriatum and hippocampus. Within SI cortex, binding was high in layers I–III and VI and relatively low in layers IV and V. Autoradiographs obtained from animals that had undergone peripheral deafferentation of part of the forepaw revealed no changes in [ 3H]-QNB binding in the affected cortical region during the time that physiological reorganization is known to occur.

Effects of 3,3′,4,4′-tetrachlorobiphenyl in the brain of the neonatal mouse

To study the retention of TCB in the brain of the immature mouse and its effects on the muscarinic cholinergic receptors in the cerebral cortex and hippocampus, [ 14C]TCB and TCB were administered to 10-day-old mice as a single peroral dose of 1.5 MBq/kg body wt and 0.41 mg or 41 mg/kg body wt, respectively. The mice were killed 24 h or 7 days after treatment. The amount of radioactivity in the whole brain decreased by 78% between 24 h and 7 days after administration. The density of muscarinic receptors was measured using the muscarinic antagonist [ 3H]QNB. A significant decrease (16%) in specific [ 3H]QNB binding was observed in the hippocampus 7 days after treatment. Despite the relatively rapid elimination of TCB from the immature mouse brain, a single low dose of TCB given during the period of rapid development of the cholinergic system appeared to affect the muscarinic cholinergic receptors in the immature mice.

Binding of Two Specific Bradycardic Agents, Alinidine and AQ-A 39, to Muscarinic Receptors of Guinea Pig Atria and Ventricle

The mechanism of action of the two "specific bradycardic agents" alinidine and AQ-A 39 (falipamil) is still a matter of controversy. Their binding properties to atrial and ventricular myocardium of the guinea pig and rat were, therefore, investigated by the radioligand binding technique. In competition studies against the nonselective antagonists [125I]3-quinuclidinyl 4-iodobenzilate [( 125I]QNB) and 1-N-methyl-[3H]scopolamine methylchloride [( 3H]NMS), both alinidine and AQ-A 39 competitively displaced the radioligands with I50 values (corrected for radioligand concentration) of 1-2 microM (alinidine/[125I]QNB) and 4 microM (alinidine/[3H]NMS), respectively. The I50 values for AQ-A 39 were lower by a factor of two. Slope factors (pseudo Hill coefficients) were 0.7-0.8 (AQ-A 39) and 0.8-0.9 (alinidine), and significantly lower than unity in both atria and ventricle. The guanosine triphosphate (GTP) (100 microM) and 5'-guanylimido-di-phosphate [Gpp(NH)p] (100 microM) slightly creased [3H]QNB binding and produced no or only a small (factor 2-3) rightward shift of alinidine and AQ-A 39 competition curves. At high concentration (1 mM), AQ-A 39 drastically decreased [125I]QNB dissociation rate from both atrial and ventricular receptors (t1/2 control, 19 min; plus AQ-A 39, 75 min) while alinidine (1 mM) decreased dissociation half-life in ventricle with no change in atria. It is concluded that both bradycardic agents possess some but not all characteristics of weak agonists in binding studies, and that they also bind to an allosteric site of the muscarinic receptors. Association with this site could possibly activate a mixed Na+/K+ inward pacemaker current (if) resulting in bradycardia.

Binding of Two Specific Bradycardic Agents, Alinidine and AQ-A 39, to Muscarinic Receptors of Guinea Pig Atria and Ventricle

Summary: The mechanism of action of the two “specific bradycardic agents” alinidine and AQ-A 39 (falipamil) is still a matter of controversy. Their binding properties to atrial and ventricular myocardium of the guinea pig and rat were, therefore, investigated by the radioligand binding technique. In competition studies against the nonselective antagonists [125I]3-quinuclidiny1 4-iodoben-zilate ([125I]QNB) and 1-N-methyl-[3H]scopolamine methylchloride ([3H]NMS), both alinidine and AQ-A 39 competitively displaced the radioligands with I50 values (corrected for radioligand concentration) of 1-2 μM (alinidine/[125I]QNB) and 4 μM (alinidine/[3H]NMS), respectively. The I50 values for AQ-A 39 were lower by a factor of two. Slope factors (pseudo Hill coefficients) were 0.7-0.8 (AQ-A 39) and 0.8-0.9 (alinidine), and significantly lower than unity in both atria and ventricle. The Guanosine triphosphate (GTP) (100 μM) and 5′-guanylimido-di-phosphate [Gpp(NH)p] (100 μM) slightly creased [3H]QNB binding and produced no or only a small (factor 2-3) rightward shift of alinidine and AQ-A 39 competition curves. At high concentration (1 mM), AQ-A 39 drastically decreased [125I]QNB dissociation rate from both atrial and ventricular receptors (t1/2control, 19 min; plus AQ-A 39, 75 min) while alinidine (1 mM) decreased dissociation half-life in ventricle with no change in atria. It is concluded that both bradycardic agents possess some but not all characteristics of weak agonists in binding studies, and that they also bind to an allosteric site of the muscarinic receptors. Association with this site could possibly activate a mixed Na+/K+ inward pacemaker current (if) resulting in bradycardia.

Polyphosphoinositides, generation of second messengers, myosin light chain phosphorylation and contraction in rabbit iris sphincter smooth muscle.

There is general agreement now that elevation of intracellular calcium ion concentration [( Ca2+]i) mediates the physiological actions of a number of hormones, neurotransmitters and other pharmacological agents. In smooth muscle one way by which these agents elevate [Ca2+]i and induce contraction involves formation of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). Our initial observation that the muscarinic-stimulated breakdown of PIP2 into IP3 and DG in the iris smooth muscle is a primary event that could couple activated receptors to contraction is now supported by the following: 1. In the iris sphincter contractions by carbachol (CCh) indicated close correlations, using different concentrations of CCh, atropine- and pirenzepine antagonists, time course, temperature and Ca2+, between the stimulated hydrolysis of PIP2, myosin light chain (MLC) phosphorylation and muscle contraction. Further, studies on the relationships between receptor occupancy of muscarinic acetylcholine receptors in this tissue, measured by [3H]QNB binding, and the CCh-stimulated PIP2 hydrolysis, MLC phosphorylation and contraction revealed that all of these responses are coupled to the M2 receptor subtype. 2. NaF, which activates G proteins by promoting the dissociation of their subunits, produced a concentration-dependent activation of phospholipase C, measured as IP3 accumulation, and contraction in the iris sphincter muscle. 3. In permeabilized smooth muscle fibers, IP3 has been shown to release Ca2+ from the SR and induce contraction. 4. In the iris sphincter, as well as in other smooth muscles, phorbol 12,13-dibutyrate, which mimics the action of DG, induced MLC phosphorylation and contraction in a dose- and time-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Piracetam elevates muscarinic cholinergic receptor density in the frontal cortex of aged but not of young mice.

Chronic treatment (2 weeks) with piracetam (500 mg/kg, once daily PO) elevated m-cholinoceptor density in the frontal cortex of aged (18 months) female mice by about 30-40%, but had no effect on m-cholinoceptor density in the frontal cortex of young (4 weeks) mice. The effect of piracetam on m-cholinoceptor density as determined by the specific binding of tritiated QNB was not affected by concomitant daily treatment with either choline (200 mg/kg) or scopolamine (4 mg/kg). It is concluded that the effect of piracetam on m-cholinoceptor density could explain the positive effects which have been reported for combinations of cholinergic precursor treatment with piracetam on memory and other cognitive functions in aged experimental animals and patients and could also represent part of the possible mechanism of action of piracetam alone.

Muscarinic stimulation of submucosal glands in swine trachea.

The properties of muscarinic acetylcholine receptors (mAChR) on tracheal explants and isolated submucosal gland cells were determined using [3H]quinuclidinyl benzilate ([3H]QNB) and N-[3H]methylscopolamine ([3H]NMS) as ligands. Analysis of competitive displacement of ([3H]NMS binding by pirenzepine demonstrated the presence of M1- (27 +/- 2%) and M2G- (73 +/- 2%) receptors on isolated tracheal submucosal gland cells (TSGC's) in control. Daily administration of diisopropylfluorophosphate (DFP) inhibited cholinesterase activity by greater than 95%. After 7 days of DFP treatment, [3H]QNB binding to intact TSGC's decreased from 14.2 +/- 0.6 to 6.3 +/- 0.8 fmol/10(6) cells; similarly, [3H]NMS binding fell from 8.1 +/- 1.9 to 2.0 +/- 0.8 fmol/10(6) cells. The loss of mAChR's was predominantly of the M2G subtype with the relative proportion dropping to 33%. In addition, 90% of the receptors assumed the high-affinity state for carbachol displacement of [3H]NMS. Mucus secretion was quantitated by measuring the release of 3H-labeled mucus macromolecules from explants of tracheal submucosal glands and isolated cells. Acetylcholine (ACh), 2 X 10(-5) M, stimulated mucus secretion by 2.5 and 2.3 times the basal rate, respectively. Elimination of acetylcholinesterase (AChe) by DFP increased the ACh sensitivity by 18- and 5-fold. Tracheal explants or TSGC's obtained 2 h after an in vivo DFP treatment showed a 6- and 3-fold ACh stimulation. This ACh sensitivity decreased during the continued daily dosing with DFP such that only a 1.3- and 1.1-fold ACh stimulation was apparent after 7 days of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

P-48 - Induction time-dependent changos in desensitization of muscarinic cholinergic receptors in guinea pig taenia caecum: Contraction and [3H]-QNB binding characteristics

P-48 - Induction time-dependent changos in desensitization of muscarinic cholinergic receptors in guinea pig taenia caecum: Contraction and [3H]-QNB binding characteristics

Muscarinic stimulation of inositol phosphate formation in rat retina: Developmental changes

The stimulation of inositol phosphates by various cholinergic agonists and the effect of antagonists was studied in rat retinal slices of different ages. The potency order of muscarinic agonists and antagonists for receptors linked to inositol phosphate production was the same for retinas of all ages. The maximum stimulation of inositol phosphate production varied between retinas of different ages, being greatest in the younger animals and lowest in the adult. However, no difference was found in the EC 50 values. This contrasts with the neurotransmitters, noradrenaline and serotonin, both known to stimulate phosphoinositide metabolism; neither was found to have the same developmental profile of carbachol-induced stimulation of inositol phosphates. In contrast to the muscarinic receptors linked to phosphoinositide hydrolysis, the density of muscarinic binding sites measured by [ 3H]QNB binding increased in retinas during development. The affinity constants ( K d) remained constant, however. The results are interpreted as suggesting that muscarinic receptors linked to inositol phosphate production are more efficiently coupled (to a guanine regulator protein) in retinas of young animals than in the adult.

Muscarinic cholinergic receptor subtypes in normal human brain and Alzheimer's presenile dementia

Brain muscarinic M1 and M2 binding sites, as defined by their affinity for pirenzepine, were studied in 24 regions of 3 normal control brains. For each region, the binding of [ 3H]QNB, a non-subtype selective antagonist, was saturable with a mean K D of 0.117 ± 0.066 nM. Computer-assisted analysis of the pirenzepine competition binding curves yielded the amount of high affinity (M1) and low affinity sites (M2) as well as their respective K i value for this ligand. The neocortex contained a mixed population of 67% M1 and 33% M2 sites, without locoregional heterogeneity. The muscarinic receptors of the caudate nucleus, putamen, pallidum, hippocampus and nucleus amygdalis were predominantly of the M1 type as well. The cerebellum and the brain-stem are examples of regions containing over 90% M2 sites. White matter structures like the centrum ovale appeared to contain low concentrations of [ 3H]QNB binding sites, predominantly of the M1 subtype. The data for the frontal cortex were compared with data obtained in 3 patients who died from Alzheimer's disease with very early onset: the muscarinic receptor concentrations were somewhat lower but their M1 M2 ratio remained unchanged. GTP caused an appreciable rightward shift and steepening of the carbachol competition binding curve in M2 predominant regions such as the pons, whereas only a slight shift was observed in the cortex. In the presence of GTP, the alkylating reagent N-ethylmaleimide caused a 35-fold increase of the affinity for carbachol in M2 predominant regions. In contrast, regions where the receptors are predominantly of the M1 type, N-ethylmaleimide caused only a 5-fold increase in agonist affinity. These findings confirm our previously formulated hypothesis that the ability of N-ethylmaleimide to modulate the agonist affinity is an additional criterion for the characterization of M1 and M2-type receptors.

Impaired performance of rats in the Morris swim-maize test late in abstinence following long-term sodium barbital treatment

Rats were tested for place learning in the Morris swim maze on days 110 –114 of abstinence following 48 weeks of treatment with sodium barbital. A retarded acquisition of the swim-maze task, that could not be ascribed to motor impairments, was found in the barbital-treated rats. There was a significant difference in brain weight, but there were no significant differences between the control and barbital-treated rats in the frontal cortical concentrations of noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), nor in the intra- and extrasynaptosomal activities of cerebral cortical monoamine oxidase towards NA and 5-HT. Postsynaptically, neither the cerebral cortical inositol phospholipid breakdown responses to carbachol and NA (mediated by muscarinic and alpha 1-adrenergic receptors, respectively), nor the striatal and cortical densities of muscarinic receptors labelled by [ 3H]quinuclidinyl benzilate ([ 3H]QNB) were found significantly to be altered in the barbital-treated rats. A strong correlation between the density of striatal and cortical [ 3H]QNB binding sites was seen for the barbital-treated ( r = 0.91) but not for the control ( r = − 0.05) rats. It is suggested that the deficit in performance of the barbitaltreated rats in the Morris maze may be related to a cholinergic dysfunction.

3H]AF-DX 116 labels subsets of muscarinic cholinergic receptors in rat brain and heart

The in vitro binding properties of the novel muscarinic antagonist [ 3H]AF-DX 116 were studied using a rapid filtration technique. Association and dissociation rates of [ 3H]AF-DX 116 binding were rapid at 25°C (2.74 and 2.70 × 10 7 min −1 M −1 for k +1; 0.87 and 0.93 min −1 for k −1) but 20–40 times slower at 0–4°C (0.13 and 0.096 × 10 7 min −1 M −1 for k +1; 0.031 and 0.022 min −1 for k −1 in cerebral cortical and cardiac membranes, respectively). Kinetic dissociation constants (K ds) were estimated to be 31.8 nM and 30.9 nM at 25°C ; 23.1 nM and 22.9 nM at 0–4°C for the cerebral cortex and heart, respectively. In saturation studies, [ 3H]AF-DX 116 labeled 29 percent of the total [ 3H](−)QNB binding sites in the cerebral cortical membranes and 87 percent in the cardiac membranes, with K d values of 28.9 nM and 17.9 nM, respectively. Muscarinic antagonists inhibited [ 3H]AF-DX 116 binding in a rank order of potency of atropine > dexetimide > AF-DX 116 > PZ > levetimide in both tissues. Except for PZ/[ 3H]AF-DX 116 and AF-DX 116/[ 3H]AF-DX 116 in the cerebral cortex, all the antagonist competition curves had Hill coefficients close to one. Carbachol and oxotremorine produced shallow inhibition curves against [ 3H]AF-DX 116 binding in both tissues. Regional distribution studies with [ 3H](−)QNB, [ 3H]PZ and [ 3H]AF-DX 116 showed that most of the muscarinic receptors in the cerebral cortex, hippocampus, nucleus accumbens and corpus striattum are of the M 1 subtype while those in the brainstem, cerebellum and other lower brain regions are of the M 2 subtype. These results indicate that [ 3H]AF-DX 116 is a useful probe for the study of heterogeneity of muscarinic cholinergic receptors.

Effects of calcium antagonists on muscarinic receptor subtypes in the rat brain

The effects of several calcium antagonists, including nicardipine, nifedipine, verapamil, and diltiazem, on muscarinic acetylcholine antagonist binding were studied in the P 2 fraction of the rat cerebral cortex using either [ 3H]quinuclidinyl benzilate ([ 3H]QNB) or [ 3H]pirenzipine as the radioactive ligand. (1) The potency of [ 3H]QNB binding inhibition was in the ordernicardipine>verapamil>diltiazem>nifedipine. The IC 50 values of nicardipine, verapamil, and diltiazem were 2.56 × 10 −6 M, 1.28 × 10 −5 M, and 6.00 × 10 −5 M, respectively. (2) The inhibition of [ 3H]QNB binding by nicardipine was significantly decreased in the presence of Ca ions. (3) In saturation experiments of [ 3H]QNB binding in the presence of nicardipine, the K d value appeared to be significantly affected, but the B max value was unchanged. This indicated that nicardipine probably inhibited [ 3H]QNB binding allosterically. On the other hand, (4) nicardipine inhibited [ 3H]pirenzipine binding completely with an IC 50 value of 7.87 × 10 −7 M. It was concluded that nicardipine had an inhibitory effect on M 1-receptor binding.