Hippocampal sympathetic ingrowth and cholinergic denervation uniquely alter muscarinic receptor subtypes in the hippocampus

Following cholinergic denervation of the hippocampus by medial septal lesions, an unusual neoronal reorganization occurs, in which peripheral sympathetic fibers, originating from the superior cervical ganglia, grow into the hippocampus. Previously, we have found that both hippocampal sympathetic ingrowth (HSI) and cholinergic denervation (CD), alone, altered the total number and affinity of muscarinic cholinergic receptors (mAChR). In this study, we utilized the muscarinic antagonist [³H]Pirenzepine, in combination with membrane radioligand binding techniques, to determine the effects of HSI and CD on hippocampal M₁ and M₁ + M₃ mAChR subtypes, 4 weeks after MS lesions. In both the dorsal and ventral hippocampus, HSI was found to markedly diminish the number of M₁ AChRs, while CD was found to increase the number of M₁ AChRs. Neither treatment affected the affinity of the M₁ AChR. However, when M₁ + M₃ binding was assessed, CD was found to decrease the affinity in both hippocampal regions, without altering the number of receptors. Neither affinity nor number of M₁ + M₃ receptors was altered by HSI. The results of this study suggest that both cholinergic denervation and hippocampal sympathetic ingrowth uniquely affect hippocampal muscarinic receptors.     

Antagonism by cholinomimetic drugs of the turning induced by intrastriatal pirenzepine in mice

In order to investigate the behavioural effect of selective blockade of M1 muscarinic receptors in the forebrain, and to characterize a new model for the evaluation of muscarinic agonistic activity, the effect of intrastriatally injected pirenzepine was studied in mice. The direct injection of pirenzepine (0.01–1 g/mouse) into the right striatum of conscious mice resulted in contralateral turning behaviour. When injected intraperitoneally (IP) 15 min before pirenzepine (1 g), the muscarinic receptor agonists arecoline and pilocarpine (0.3–3 mg/kg), oxotremorine (0.003–0.03 mg/kg) and RS 86 (0.03–1 mg/kg) antagonized pirenzepine-induced turning, as did the choline-esterase inhibitor physostigmine (0.01–0.1 mg/kg) and the nootropic drug aniracetam (10–30 mg/kg). Haloperidol (0.03–0.3 mg/kg IP) weakly, but significantly, decreased the effect of pirenzepine, whereas (±) sulpiride (3–100 mg/kg) failed to affect it. Finally, (+)-amphetamine (0.1–3 mg/kg IP), citalopram (1–30 mg/kg IP) and muscimol (0.03–0.3 mg/kg IP) failed to modify pirenzepine-induced turning when administered prior to intrastriatal pirenzepine.These results suggest an involvement of M1 muscarinic receptors in rotational behaviour, and indicate that pirenzepine-induced turning may represent a new model for studying the central activity of cholinomimetic drugs.     

Dose-response curves of pirenzepine in man in relation to M1- and M2-cholinoceptor occupancy

Summary The aim of the present study was to investigate the M-cholinoceptor subtype selectivity of pirenzepine in man. In parallel with effects on the heart rate and salivary flow, M-cholinoceptor subtype occupancy by antagonist present in plasma samples was detected in radioreceptor assays. Bovine cerebral cortex membranes labelled with ³H-pirenzepine (M₁) and rat salivary gland membranes labelled with ³H-N-methylscopolamine (M₂) were used in these in vitro assays. A half-maximal occupancy of M₁-cholinoceptors in the in vitro assay of plasma samples was detected after 0.25 mg of pirenzepine i.v. The respective half-maximal M₂-cholinoceptor occupancy was observed after 10 mg. Doses < 3 mg decreased the heart rate by maximally 10.7 beats/min with an ED₅₀ of about 0.1 mg. An increase in heart rate (relative to control values) was observed at doses > 10 mg. This bivalent dose-response relationship was also observed after -blockade. Salivary flow tended to increase at doses < 1 mg and was half-maximally inhibited after 10 mg. Combining the in vitro and in vivo results, the typical antimuscarinic effects (tachycardia and inhibition of salivary flow) can be attributed to the blockade of M₂-cholinoceptors, whereas the reduction of heart rate coincides with the blockade of the M₁-subtype. With respect to the typical antimuscarinic effects, pirenzepine was 70-fold less potent than atropine; in contrast, with respect to the reduction of heart rate, pirenzepine was equipotent with atropine. It is concluded that pirenzepine does not discriminate between cardiac and salivary gland M₂-cholinoceptors but shows pronounced selectivity for the M₁-cholinoceptors through which it mediates the decrease in heart rate. The latter effect may be explained by inhibitory M₁-auto-receptors. Send offprint requests to H. F. Pitschner at the above addressParts of the results were reported at the spring meeting 1987 of the Deutsche Gesellschaft für Pharmakologie und Toxikologie (Wellstein et al. 1987)     

Effect of combination therapy with cimetidine and pirenzepine on plasma parathyroid hormone and calcitonin levels in hemodialyzed patients

Summary In this study we evaluated the effects of an oral combination therapy with cimetidine and pirenzepine on plasma parathyroidhormone (PTH) and calcitonin (CT) levels in 24 patients on maintenance hemodialysis (mean age: 50 years; mean duration of dialysis treatment: 23 months). As compared to the pre-treatment plasma levels of PTH and CT, there were no significant changes of their plasma concentrations during a 4-week administration of 800 mg cimetidine or 100 mg pirenzepine daily, and the concentrations also did not change significantly during the following 4 weeks of combination therapy with cimetidine and pirenzepine in the above mentioned dosage. Serum concentrations of calcium and phosphate and the activity of the alkaline phosphatase showed no significant changes either. Therefore, we suggest that this therapeutic approach cannot be considered for the treatment of uremic hyperparathyroidism.     

Selectivity of pirenzepine in the central nervous system. III. Differential effects of multiple pirenzepine and scopolamine administrations on muscarinic receptors as measured autoradiographically

The effects of intrahippocampal injections of scopolamine and pirenzepine on muscarinic receptor binding were examined by quantitative autoradiographic techniques. Brain slices from animals which had received 7 injections of either scopolamine (n = 5) or pirenzepine (n = 5) over a 22-day injection schedule were compared with slices from 5 saline-injected controls for receptor binding to the whole slice and within selected regions of the brain as measured autoradiographically. The total number of receptors was determined from direct binding assays with 1-[3H]quinuclidinyl-benzilate ([3H]-1-QNB), while the binding of the selective ligands pirenzepine, carbamylcholine, and scopolamine was examined through inhibition studies. The data from the whole slices indicated that pirenzepine-treated animals contained more receptors for [3H]-1-QNB than either saline- or scopolamine-injected controls. Slices from the same animals also displayed a lower affinity for pirenzepine. Slices from scopolamine-injected animals revealed neither an increase in receptor number nor a decrease in antagonist affinity, although the binding of the agonist carbamylcholine was increased. Quantitative analysis of the autoradiograms generated from the slices indicated that the increase in receptor number for pirenzepine-injected animals was predominantly within the cerebral and cingulate cortices. The inhibition by pirenzepine was also lower in these areas in the same group of animals. Agonist inhibition was altered in the central layers of the cerebral cortex and in the pretectal area in scopolamine-treated animals. The results suggest separate mechanisms of drug action and adaptation for pirenzepine and scopolamine.     

Differential effects on [35S]GTPgammaS binding using muscarinic agonists and antagonists in the gerbil brain

In this work, we studied the in vitro G-protein activation induced by muscarinic agonists using [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) autoradiographic methods to characterize the M(2) and M(4) muscarinic subtypes response. Thus, we describe a detailed characterization of the increases in [(35)S]GTPgammaS binding elicited by carbachol (Cch) and oxotremorine (OXO) (binding in the presence minus binding in the absence of agonist) throughout the gerbil brain (Meriones unguiculatus). For both agonists, the strongest stimulations were found in the superficial gray layer of the superior colliculus, the anteroventral and anteromedial thalamic nuclei, the anterior paraventricular thalamic nucleus, and the caudate-putamen. The comparative study using OXO and Cch suggested that OXO is able to detect differences in the response of structures enriched in M(4) muscarinic receptors, showing a lower potency to stimulate these brain areas. Furthermore, using increasing concentrations of selective M(2) (AF-DX 116) and M(1)/M(4) (pirenzepine) antagonists to inhibit specific Cch- or OXO-induced [(35)S]GTPgammaS binding, significant differences were observed in M(2)-enriched structures but not in M(4)-enriched ones such as the caudate-putamen. These data indicate that appropriate muscarinic agonist stimulation, together with selective inhibition of this effect using functional autoradiography, can be used as a tool to unravel the M(2)- and M(4)-muscarinic subtype-mediated response.     

Significance of radioautography of soluble compounds in analysis of cholinergic innervation in rat gastric mucosa

To clarify the cholinergic regulation of the gastric mucosa, the localization of the muscarinic receptors in the rat fundic mucosa was studied using the radioautography of soluble compounds of³H-quinuclidinyl benzilate (QNB) and³H-pirenzepine (PZ) in comparison with the routine fixation method. Through the radioautography of soluble compounds, the binding sites of PZ, corresponding to the localization of M₁ receptors, were located on the nerve endings and enterochromaffin-like (ECL) cells; while the binding sites of QNB, representing the muscarinic receptors, were seen on the epithelial cells, endothelial cells and smooth muscle cells as well as on the nerve endings and ECL cells. By contrast, the routine fixation method was of little use in showing the localization of diffusible compounds in the gastric mucosa.     

Distinct kinetic binding propeties of N-[3H]-methylscopolamine afford differential labeling and localization of M1, M2, and M3 muscarinic receptor subtypes in primate brain

Three classes of muscarinic receptors in mammalian brain have been postulated on the basis of equilibrium and kinetic binding data. However, equilibrium binding assays alone have not permitted a clear demonstration of the localization of putative M1, M2, and M3 receptor subtypes in the brain because of the overlaping affinities of virtually all muscarinic antagonists. In the present study, the conditions for selective occupancy of the M1, M2, and M3 receptor subtypes in the brain of the rhesus monkey were based on the distinct kinetic and equilibrium binding properties of N-[³H]-methylscopolamine (NMS) at cloned m1–m4 muscarinic receptor subtypes expressed in A9L transfected cells. Quantitative autoradiography of the M1, M2, and M3 muscarinic receptor subtypes in the primate brain was performed according to the following strategy. The M1 (m1) receptor subtype was labeled directly with a non-saturating concentration of [³H]-pirenzepine. The M2 (m2) subtype was labeled by incubations consisting of short, two minute pulses of [³H]-NMS after a preincubation with 0.3 μM pirenzepine to occlude m1, m3, and m4 sites. Selective occupancy of the M3 (m3) receptor (subtype) was achieved by pre-incubation with 0.5 nM unlabeled NMS to partially occlude the m1, m2, and m4 sites, equilibrium with 0.5 nM [³H]-NMS, followed by a 60 minute tracer dissociation in the presence of 1 μM atropine. In vitro autoradiography demonstrated that the M1 receptor subtype was confined to forebrain structures. M1 receptors were prevalent throughout the cerebral cortical mantle, amygdala, hippocampus, and the striatum. Low to background levels of the M1 receptor subtype were measured over the thalamus, hypothalamus, and brainstem. The M2 subtype was widely distributed with elevated densities of binding sites seen over all primary sensory cortical areas, and within discrete thalamic, hypothalamic, and brainstem nuclei. The distribution of the M3 receptor subtype was largely coincident with the pattern of the M1 sites labeled by non-saturating concentrations of [³H]-pirenzepine with some notable exceptions. Within the cerebral cortical mantle, the M3 receptor exhibited an elevated gradient over the orbitofrontal gyrus and the temporal lobe. Within the striatum, the M3 subtype was elevated over the anterior and dorsal part of the caudate nucleus, while the M1 receptors were most prevalent over the ventromedial sector. Selective labeling of M3 receptors was seen over the medial division of the globus pallidus and within the substantia nigra pars reticulata. In contrast to the pattern of the M1 receptor subtype, M3 receptors were prevalent also over midline nuclei of the hypothalamus. These results demonstrate that the distinct kinetic and equilibrium binding profiles of N-methylscopolamine and pirenzepine for cloned muscarinic receptors provide a viable ligand autoradiographic strategy for mapping the distribution of M1, M2, and M3 receptors in brain. © 1993 Wiley-Liss, Inc.     

哌吡唑酮对消炎痛引起大鼠胃粘膜损伤的作用

比较哌吡唑酮和阿托品、甲氰咪呱三种药物对消炎痛所致胃粘膜损伤的抑制作用。哌吡唑酮大、中、小三种剂量灌胃,结果三组的胃粘膜损伤指数分别由对照组的87.9±69.7降至1.0±2.2、9.4±17.2和6.4±7.6。发病率也由对照组的100%降至30%、63.6%和60%。三组与对照组相比均有显著性差异。哌吡唑酮大中小三种剂量灌胃的作用均强于甲氰咪呱大剂量皮下注射,而弱于阿托品大剂量腹腔注射。初步探讨了其抗粘膜损伤的机制。     

哌仑西平的药理作用

本文报告了哌仑西平和阿托品的药理实验结果。1.哌仑西平和阿托品对大鼠的幽门结扎、冷水应激和利血平诱导的胃溃疡均有预防作用。2.两化合物在大鼠胃肠蠕动实验中有重要差别。哌仑西平的抑制作用弱于阿托品。3.关于毛果芸香碱刺激兔的唾液分泌,哌仑西平的抑制率明显的低于阿托品。4.兔的瞳孔直径测量表明:阿托品的扩瞳效应比哌仑西平强得多,且有效期也比哌仑西平长。