Effect of moxibustion on M2 and P2X3 receptors of bladder tissue in rats with neurogenic bladder of detrusor areflexia after lumbar-sacral spinal cord injury / 中国针灸

OBJECTIVE@#To observe the effect of moxibustion at "Guanyuan" (CV 4) and "Shenque" (CV 8) on acetylcholine (Ach), adenosine triphosphate (ATP) and muscarinic-type choline receptor (M2) and purine receptor P2X3 in bladder tissue in the rats with neurogenic bladder (NB) of detrusor areflexia after lumbar-sacral spinal cord injury and explore the underlying mechanism of moxibustion for promoting detrusor contraction.@*METHODS@#Sixty SD rats were randomly divided into a model preparation group (n=45) and a sham-operation group (n=15). In the model preparation group, the modified Hassan Shaker spinal cord transection method was used to prepare the model of NB. In the sham-operation group, the spinal cord transection was not exerted except laminectomy and spinal cord exposure. Among the rats with successfully modeled, 30 rats were selected and divided randomly into a model group and a moxibustion group, with 15 rats in each one. On the 15th day after the operation, moxibustion was applied at "Guanyuan" (CV 4) and "Shenque" (CV 8) in the moxibustion group, 10 min at each acupoint, once a day. The consecutive 7-day treatment was as one course and the intervention for 2 courses was required. Urodynamic test was adopted to evaluate bladder function in rats. Using HE staining, the morphological changes in bladder tissue were observed. The content of Ach and ATP in bladder tissue was measured with biochemical method, and the protein and mRNA expression levels of M2 and P2X3 receptors in bladder tissue were detected with Western blot and real-time fluorescence quantification PCR method.@*RESULTS@#Compared with the sham-operation group, the maximum bladder capacity, leakage point pressure and bladder compliance were increased in the rats of the model group (P<0.05). Compared with the model group, the maximum bladder capacity, the leakage point pressure and bladder compliance were decreased in the rats of the moxibustion group (P<0.05). In the model group, the detrusor fibres were arranged irregularly, bladder epithelial tissues were not tightly connected and cell arrangement was disordered, combined with a large number of vacuolar cells. In the moxibustion group, compared with the model group, the detrusor fibres were arranged regularly, bladder epithelial cells were well distributed and vacuolar cells were reduced. Compared with the sham-operation group, the content of Ach and ATP in bladder tissue was decreased (P<0.05), the protein and mRNA expression levels of M2 and P2X3 receptors were reduced (P<0.05) in the model group. In the moxibustion group, the content of Ach and ATP in bladder tissue was increased (P<0.05) and the protein and mRNA expression levels of M2 and P2X3 receptors were increased (P<0.05) as compared with the model group.@*CONCLUSION@#Moxibustion at "Guanyuan" (CV 4) and "Shenque" (CV 8) may effectively improve bladder function in the rats with NB of detrusor areflexia after lumbar-sacral spinal cord injury and its underlying mechanism is related to promoting the release of Ach and up-regulating the expression of M2 receptor, thereby enhancing the release of ATP and increasing the expression of P2X3 receptor. Eventually, detrusor contraction is improved.

Impacts on urodynamic parameters and the protein expressions of M2 and M3 receptors of detrusor in the rats of detrusor hyperreflexia treated with ginger-salt-isolated moxibustion at "Shenque" (CV 8) / 中国针灸

<p><b>OBJECTIVE</b>To discuss the effects on detrusor hyperreflexia treated with ginger-salt-isolated moxibustion at "Shenque" (CV 8) and its mechanism.</p><p><b>METHODS</b>Thirty female adult SD rats were selected. The model of detrusor hyperreflexia was prepared with complete spinal transection at T, of which, 20 rats were randomized into a model group (10 rats) and a moxibustion group (10 rats). A sham-operation group (10 rats) was set up for sham-spinal transection. In the moxibustion group, when urine incontinence occurred (about in 2 weeks of modeling), the ginger-salt-isolated moxibustion at "Shenque" (CV 8) was given, 3 moxa cones each time, once a day, continuously for 7 days. After treatment, in each group, the urodynamic parameters were determined, after which, the bladder detrusor was collected. Western blot was used to determine the protein expressions of M2 and M3 receptors.</p><p><b>RESULTS</b>Compared with the sham-operation group, the micturition interval was shortened apparently (<0.01); the maximal bladder pressure was increased apparently (<0.01); the protein expression of M2 receptor in the detrusor was increased significantly (<0.05) and that of M3 receptor had no apparent change (>0.05) in the rats of the model group. Compared with the model group, the micturition interval was longer apparently (<0.01), the maximal bladder pressure was reduced apparently (<0.01), the protein expression of M2 receptor in the detrusor was reduced significantly (<0.05) and that of M3 receptor had no apparent change (>0.05) in the rats of the moxibustion group.Compared with the sham-operation group, the results of the above indicators were not different significantly in the moxibustion group (all>0.05).</p><p><b>CONCLUSIONS</b>The ginger-salt-isolated moxibustion at "Shenque" (CV 8) suppresses the overactive bladder in the rat with spinal transection and its effect mechanism is possibly relevant with reducing the protein expression of detrusor M2 and inhibiting the excessive contraction of the detrusor.</p>

Relaxing Effect of Acetylcholine on Phenylephrine-Induced Contraction of Isolated Rabbit Prostate Strips Is Mediated by Neuronal Nitric Oxide Synthase

PURPOSE: The location of acetylcholinesterase-containing nerve fibers suggests a role for acetylcholine in both contractility and secretion in the prostate gland. The colocalization of nitrergic nerves with cholinergic nerves, and the cotransmission of nitric oxide with acetylcholine in cholinergic nerves, has been demonstrated in the prostate glands of various species. Thus, we investigated the effects of acetylcholine on phenylephrine-induced contraction and the correlation between cholinergic transmission and nitric oxide synthase by using isolated prostate strips of rabbits. MATERIALS AND METHODS: Isolated prostate strips were contracted with phenylephrine and then treated with cumulative concentrations of acetylcholine. Changes in acetylcholine-induced relaxation after preincubation with NG-nitroarginine methyl ester, 7-nitroindazole, and aminoguanidine were measured. The effects of selective muscarinic receptor antagonists were also evaluated. RESULTS: In the longitudinal phenylephrine-contracted strip, the cumulative application of acetylcholine (10(-9) to 10(-4) M) elicited a concentration-dependent relaxation effect. Acetylcholine-induced relaxation was inhibited not only by nitric oxide synthase inhibitors (10 microM L-NAME or 10 microM 7-nitroindazole) but also by 10 microM atropine and some selective muscarinic receptor antagonists (10(-6) M 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one and 10(-6) M 4-diphenylacetoxy-N-methyl-piperidine). In contrast, relaxation was significantly increased by pretreatment of the strips with 10 mM L-arginine. CONCLUSIONS: Acetylcholine relaxed phenylephrine-induced contractions of isolated rabbit prostate strips. This relaxation may be mediated via both cholinergic and constitutive nitric oxide synthase with both the M2 and M3 receptors possibly playing key roles.

ZMS regulation of M_2 muscarinic receptor stability mediated by de novo synthesis of protein / 上海交通大学学报（医学版）

Objective To explore the mechanism of ZMS regulation of M_2 muscarinic receptor mRNA expression. Methods In vitro cultured CHOm2 cells were divided into ZMS 1 group (treatment with 1 × 10~(-5) mol/L ZMS for 24 h), ZMS 2 group (treatment with 1 × 10~(-5) mol/L ZMS for 24 h and 1 μg/mL cycloheximide for 12 h) and ZMS 3 group (treatment with 1 μg/mL cycloheximide for 4 h and 1 × 10 ~(-5) mol/L ZMS for 24 h), and their corresponding control groups were also established (substitution of ZMS by DMSO). Actinomycin D was added to cultured CHOm2 cells of each group to inhibit the synthesis of mRNA. CHOm2 cell samples were taken at different time points, the relative expression of M_2 receptor mRNA was detected by Real-time PCR, and half life of M_2 receptor mRNA was calculated. Results Compared with corresponding control groups, the half life of M_2 receptor mRNA of CHOm2 cells in ZMS 1 group and ZMS 2 group was significantly prolonged [(4.75h± 0.54) h vs (2.13 ±0.23) h, P<0.05; (5.43 ±1.13) h vs (2.46 ±0.09) h, P<0.05]. There was no significant difference in half life of M_2 receptor mRNA of CHOm2 cells between ZMS 3 group and its corresponding control [ ( 3.06 ±0.23) h vs (3.00 ± 0.20) h, P > 0.05]. Conclusion De novo protein synthesis is required for the enhancement of M_2 receptor mRNA stability regulated by ZMS.

Regulatory effect of catalpol from Radix Rehmanniae on M_2 receptor density in M_2 receptor transfected CHO cells / 中国药理学通报

Aim To investigate the effect of catalpol from Radix Rehmanniae on M_2 receptor density in CHO cells transfected with gene of M_2 receptors(CHO m2).Methods Cultured CHOm2 cells were randomly divided into 4 groups:three concentrations of catalpol :10-6、10-5、10-4 mol?L-1,and saline control.After addition of catalpol and saline for 72 h,M_2 Receptor density was measured by single point 3H-NMS binding assay,the content of protein was measured with Lowry's method.Competitive binding assay using the binding system of 3H-NMS was performed to address the question about whether catalpol could occupy the M receptor binding site.Addition of catapol to brain homogenate and measuring the enzyme activity with the Ellman's colorimetric method were performed to address the question about whether catalpol could in-hibit acetylcholinesterase activity.Results Catalpol can elevate the M_2 receptor density in CHO m2 cells significantly at the doses of 10-5、10-4 mol?L-1(P

Muscarine M2 Receptor-mediated Presynaptic Inhibition of GABAergic Transmission in Rat Meynert Neurons

Cholinergic modulation of GABAergic spontaneous miniature inhibitory postsynaptic currents (mIPSCs) by the activation of muscarine receptors was investigated in mechanically dissociated rat nucleus basalis of the Meynert neurons using the conventional whole-cell patch recording configuration. Muscarine (10microM) reversibly and concentration-dependently decreased mIPSC frequency without affecting the current amplitude distribution. Muscarine action on GABAergic mIPSCs was completely blocked by 1microM methoctramine, a selective M2 receptor antagonist, but not by 1microM pirenzepine, a selective M1 receptor antagonist. NEM (10microM), a G-protein uncoupler, attenuated the inhibitory action of muscarine on GABAergic mIPSC frequency. Muscarine still could decrease GABAergic mIPSC frequency even in the Ca2+-free external solution. However, the inhibitory action of muscarine on GABAergic mIPSCs was completely occluded in the presence of forskolin. The results suggest that muscarine acts presynaptically and reduces the probability of spontaneous GABA release, and that such muscarine-induced inhibitory action seems to be mediated by G-protein-coupled M2 receptors, via the reduction of cAMP production. Accordingly, M2 receptor-mediated disinhibition of nBM neurons might play one of important roles in the regulation of cholinergic outputs from nBM neurons as well as the excitability of nBM neurons themselves.