Sulphhydryl-modifying Reagents Alter Ototoxin Block of Muscarinic Receptor-linked Phosphoinositide Turnover in the Cochlea

In the 12-day-old rat cochlea, the synthesis of inositol phosphates (IPs) can be activated via M3 cholinoceptors. This stimulation is blocked by ototoxins (mercury, ethacrynate, cisplatin, neomycin), drugs with side effects that lead to damage of hair cells and strial cells. As these toxic effects can be reversed in vivo by thiol molecules, we investigated whether modifications of thiol compounds could be involved in ototoxin-induced inhibition of the IP turnover in the cochlea. For this purpose, we assessed whether the sulphhydryl-modifying reagents N -ethylmaleimide and cadmium modify the carbachol-stimulated formation of IPs in the 12-day-old rat cochlea. Both molecules inhibit the carbachol effect on a dose-dependent way without altering the basal metabolism of IPs. As cadmium may block some calcium channels, the effect of verapamil, another calcium channel antagonist, was tested. Verapamil (1 –50 μM) does not alter carbachol-evoked IP formation, suggesting that the inhibitory effect of cadmium is not due to a calcium influx block. Binding experiments with the muscarinic ligand quinuclidinyl benzylate (QNB) showed that the sulphhydryl-modifying reagents do not displace QNB from binding sites. Combining ototoxins and reagents shows that N -ethylmaleimide acts synergistically with all ototoxins but ethacrynate while cadmium does so only with mercury. Both N -ethylmaleimide and cadmium have additive effects with ethacrynate. As a supplement, disulphide bond-modifying agents do not alter the carbachol-enhanced metabolism of IPs. These results suggest that molecules having thiol-modifying properties inhibit the carbachol-induced turnover of IPs without acting at the muscarinic sites. Since thiol modifiers and ethacrynate share similar features in both QNB binding and IP response it is hypothesized that they strike common targets, possibly G proteins.     

Effects of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin on the dopaminergic and cholinergic receptors as evaluated by positron emission tomography in the Rhesus monkey

Summary The effects of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (R-THBP) on the central cholinergic and dopaminergic systems in the Rhesus monkey brain were investigated by positron emission tomography (PET) with the muscarinic cholinergic receptor ligands (N-[¹¹C]methyl-benztropine) and dopaminergic receptor ligands selective for D₁ D₂, and D₃ subtypes ([¹¹C]SCH23390, N-[¹¹C]methyl-spiperone, and (+)[¹¹C]UH232, respectively). None of the doses (3, 10, and 30 mg/kg i.v.) of R-THBP used significantly affected the regional cerebral blood flow (rCBF as determined by Raichle's H₂ ¹⁵O method), and 10 mg/kg of R-THBP had little effect on the regional cerebral metabolic rate of glucose (rCMRglc) in the Rhesus monkey brain, as assessed by the graphical [¹⁸F]fluoro-deoxyglucose method. The effect of R-THBP on the muscarinic cholinergic system was dose dependent; while 3 mg/kg of R-THBP did not significantly alter the uptake ratio of N-[¹¹C]methyl-benztropine in several brain regions to that in the cerebellum, 10 and 30 mg/kg of R-THBP significantly reduced the uptake ratio in the thalamus, as well as in the frontal and temporal cortices. None of the doses (3, 10, and 30 mg/kg i.v.) of R-THBP tested affected [¹¹C]SCH23390 (dopamine D₁ receptor) binding. However, the k3 value for N-[¹¹C]methyl-spiperone (dopamine D₂ receptor) binding, which represents the association rate × B_max value, was significantly decreased in the striatum. The uptake ratio of (+)[¹¹C]UH232 (dopamine D₃ receptor) in the striatum to that in the cerebellum was also decreased by administration of R-THBP (3 and 30 mg/kg i.v.). These findings suggest that R-THBP acts on dopamine D₂ and D₃ receptors selectively without markedly affecting dopamine D₁ receptor binding. Furthermore, the changes in cholinergic and dopamine D₂ and D₃ receptors in vivo can not be attributed to a change in rCBF but may depend on the action of R-THBP.Abbreviations R-THBP 6R-L-erythro-5,6,7,8-tetrahydrobiopterin - PET positron emission tomography - rCBF regional cerebral blood flow - rCMRglc regional cerebral metabolic rate of glucose     

Effect of Central Muscarine Receptor Blockade with DKJ-21 on the Blood Pressure and Heart Rate in Stress-Induced Hypertensive Rats

Da-Nian Zhu, Long-Mei Xue, Peng Li. Effect of central muscarine receptor blockade. with DKJ-21 on the blood pressure and heart rote in stress-induced hypertensive rats.

The experiments were performed on Wistar or Sprague-Dawley rats of both sexes divided at random into stress and control groups. The rats in the stress groups were put into cages and subjected to electric foot-shocks and noises for 9-15 days, which caused an increase in blood pressure (BP) and heart rate (HR). In hypertensive rats DKJ-21 (4mg/lml) was injected intravenously (i.v.), and 0.5-1.0h after administration the BP and HR dropped from the high level to normotensive level. In normotensive rats, however, administration of DKJ-21 had no effect on BP or HR. In separate groups of normotensive rats, pretreatment of DKJ-21 (4 mg/l ml, i.v.) blocked the pressor and tachycardiac effect induced by microinjection of physostigmine (0.4μg/0.1 /μl/site), corticosterone (40μg/0.1μl/site) or aldosterone (40 μg/0.1 μl/site) into the rostral ventrolateral medulla (rVLM). Furthermore, DKJ-21 also attenuated the enhancement of the pressor response to stimulation of the defense area in the midbrain, which was induced by microinjection of drugs (mentioned above) into the rVLM. These results indicate that i.v. DKJ-21 can selectively block the muscarinic receptors in the rVLM in stress-induced hypertensive rats, which suggests that abnormal enhancement of cholinergic mechanism in the rVLM may be related to hypertensive effects of corticoids in this area.     

Effects of phorbol ester on carbachol-induced contraction in bovine ciliary muscle: possible involvement of protein kinase C

The aim of the research was to characterize muscarinic receptors of bovine ciliary muscle and to investigate the desensitization process. The role of protein kinase C was analyzed. The results show that muscarinic receptors of bovine ciliary muscle have the pharmacological characteristics of the M₃ subtype. Acute exposure to phorbol esters (1 μM phorbol 12,13-dibutyrate, PDB, or 0.1 μM phorbol 12-myristate 13-acetate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensitization and loss of phorbol ester-induced desensitization. Staurosporine (3 μM) and H₇ [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 μM), protein kinase C inhibitors, produced a significant potentiation of the contractile effect of carbachol, reduced the desensitization produced by repeated addition of carbachol and suppressed that induced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [³H]quinuclidinyl benzilate. In vitro incubation with PDB and PMA produced, as expected, a significant translocation of protein kinase C from the cytosol to the membrane. The incubation of the ciliary muscle with carbachol, using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ciliary muscle is correlated, at least partially, to the translocation of protein kinase C from the cytosol to the membrane. The desensitization by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the density and affinity of muscarinic receptors is not involved.     

Galanin-like innervation of rat submandibular and sublingual salivary glands: origin and effect on acinar cell membranes.

The distribution and source of a galanin-like innervation of rat salivary glands has been examined. Additionally, submandibular and sublingual acinar cell membrane responses to galanin or a cholinergic agonist were studied. Galanin-immunoreactive fibers were observed throughout the submandibular and sublingual glands in association with ducts and acini. A subset of submandibular ganglion cells expresses galanin immunoreactivity. Parasympathectomy resulted in a marked decrease in galanin immunoreactivity in the glands. Sympathectomy resulted in marked reduction of dopamine beta-hydroxylase immunoreactivity with no appreciable change in galanin immunoreactivity. Retrograde labeling experiments demonstrated that galanin-immunoreactive sensory neurons in the trigeminal ganglion do not innervate the submandibular or sublingual gland. These results indicate that the galanin-like innervation of rat salivary glands is derived from parasympathetic nerves to the glands. Since rat sublingual glands contain largely mucous acini while rat submandibular gland acini are seromucous, electrophysiological responses to galanin and the muscarinic agonist, bethanechol, were compared. Agonist-induced voltage shifts varied between the two glands. The galanin-induced response at the level of the resting membrane potential in submandibular acinar cells was a hyperpolarization, while that in sublingual acinar cells was a depolarization. There was also a greater voltage dependence to the galanin-induced submandibular response than to the sublingual response. Differences were also noted in the acinar cell response to cholinergic stimulation between these glands. These results demonstrate the existence of a galanin-like innervation to salivary glands that may be functionally relevant. Moreover, the results challenge the idea that agonist-induced membrane responses are similar among acinar cells of different glands.     

Cholinergic therapy in dementia

After reviewing the evidence for cholinergic pathology in Alzheimer's disease and related disorders, this paper reviews strategies for treating dementia using cholinomimetic drugs. Special attention is paid to cholinesterase inhibitors, particularly tacrine, the drug recently approved by the FDA. New studies suggesting that muscarinic and nicotinic cholinergic receptor active drugs may be more effective will be reviewed. Brief mention will be made of strategies to slow the progression of Alzheimer's disease.     

老年大鼠脑M_1亚型受体的变化及黄芪的调节作用

目的：观察老年大鼠不同脑区胆碱能Ｍ１亚型受体的变化和黄芪对其的调节作用。方法：采用放射自显影技术显示大鼠脑Ｍ１受体，并用图像分析仪进行灰度分析，以反映Ｍ１受体在不同脑区的相对定量分布。结果：所得脑切片自显影灰度层次清晰，主要分布在大脑皮质、海马、纹状体部位，非特异结合灰度很低，老年大鼠皮质、海马、纹状体的灰度显著低于青年鼠，分别降低１５７８％，８６９％，１２３６％（Ｐ＜００５），老年服黄芪组三个部位的灰度均明显高于老年对照组，分别升高１６６３％，９８１％，１０３２％，（Ｐ＜００５）。结论：黄芪对老年大鼠降低的脑胆碱能Ｍ１亚型受体具有上调作用。     

Inhibition of ligand binding to g protein-coupled receptors by arachidonic acid

Arachidonic acid (AA), released in response to muscarinic acetylcholine receptor (mAChR) stimulation, previously has been reported to function as a reversible feedback inhibitor of the mAChR. To determine if the effects of AA on binding to the mAChR are subtype specific and whether AA inhibits ligand binding to other G protein-coupled receptors (GPCRs), the effects of AA on ligand binding to the mAChR subtypes (M₁, M₂, M₃, M₄, and M₅) and to the μ-opioid receptor, β₂-adrenergic receptor (β₂-AR), 5-hydroxytryptamine receptor (5-HTR), and nicotinic receptors were examined. AA was found to inhibit ligand binding to all mAChR subtypes, to the β₂-AR, the 5-HTR, and to the μ-opioid receptor. However, AA does not inhibit ligand binding to the nicotinic receptor, even at high concentrations of AA. Thus, AA inhibits several types of GPCRs, with 50% inhibition occurring at 3–25 μM, whereas the nicotinic receptor, a non-GPCR, remains unaffected. Further research is needed to determine the mechanism by which AA inhibits GPCR function.     

β-Adrenergic and muscarinic receptor mRNA accumulation in the sinoatrial node area of adult and senescent rat hearts

The sinoatrial (SA) node is the cardiac pacemaker and changes in its adrenergic-muscarinic phenotype have been postulated as a determinant of age-associated modifications in heart rate variability. To address this question, right atria were microdissected, the SA node area was identified by acetylcholinesterase staining, and, using a RT-PCR method, the accumulation of mRNA molecules encoding β₁- and β₂-adrenergic (β₁- and β₂-AR) and muscarinic (M₂-R) receptor was quantified to define the proportion between β-AR and M₂-R mRNAs within the sinoatrial area of adult (3 months) and senescent (24 months) individual rat hearts. In adult hearts, the highest M₂-R/β-AR mRNA ratio was observed within the sinoatrial area compared with adjacent atrial myocardium, while in the senescent hearts, no difference was observed between sinoatrial and adjacent areas. This change was specific of the sinoatrial area since adult and senescent whole atrial or ventricular myocardium did not differ in their M₂-R/β-AR mRNA ratio, and was associated with a fragmentation of acetylcholinesterase staining of the senescent SA node. Quantitative changes in the expression of genes encoding proteins involved in heart rate regulation specifically affect the sinoatrial area of the senescent heart.     

Muscarinic supersensitivity in the rat urinary bladder after capsaicin pretreatment

The effects of capsaicin on urinary bladder function have been investigated in adult rats. Ten days after capsaicin treatment immunocytochemical investigations showed a nearly complete disappearance of substance P (SP) and calcitonin gene-related peptide (CGRP) in all parts of the bladder. Recordings of micturition patterns and cystometrical investigations in conscious animals revealed no functional effects of capsaicin treatment. In-vitro experiments showed that the contractile response to substance P was similar before and after capsaicin treatment and CGRP exerted no contractile effects on the urinary bladder in either group of rats. The concentration–response curve to carbachol as well as the frequency-response curve to electrical stimulation were significantly shifted to the left in bladder muscle after capsaicin treatment. However, the maximal responses were similar in control and capsaicin-treated bladders. In the presence of scopolamine the maximal response to electrical stimulation was clearly lower in bladders subjected to capsaicin treatment than in controls. In conclusion, depletion of substance P and CGRP in the rat urinary bladder by capsaicin induced no supersensitivity to these peptides. However, the increased sensitivity to carbachol and to electrical stimulation seen after capsaicin treatment indicates the development of a supersensitivity to muscarinic receptor stimulation. Despite this supersensitivity in vitro no functional effects of capsaicin treatment were found in vivo.