Dissertation
Dissertation > Medicine, health > Pharmacy > Pharmacology > Experimental Pharmacology

Effect and it’s mechanism of DHPs calcium antagonist on the 5-HT receptor of rat stomach fundus smooth muscle

Author LiuLiHong
Tutor ZhaoDeHua;MeiQiBing
School Fourth Military Medical University
Course Pharmacology
Keywords DHPs calcium antagonist 5-HT receptor rat stomach fundus smooth muscle
CLC R965
Type PhD thesis
Year 2001
Downloads 113
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We examined the effects and mechanisms of methyl pentyl 1,4-dihydro-2,6-dimethyl-4-(3-notrophenyl)-3,5-pyridinedicarboxylate (Mn9202), a novel DHPs calcium antagonist and lacidipine on the 5-HT2B receptor in cultured rat stomach fundus smooth muscle cells. 1. The transduction pathway of rat stomach fundal 5-HT receptor.Fluo-3-based Laser Confocal Scanning Microscope was used for measuring changes in intracellular [Ca2+] of cultured rat stomach fundus smooth cells(SFSMCs). 5-HT produced a slow increase in SFSMCs, and the increase persisted in Ca2+-free buffer, but it was abolished by incubating with ryanodine in Ca2+-free buffer. The response was blocked by the selective 5-HT antagonist miaserin and L-type calcium antagonist lacidipine, indicating that the increase in cytosolic [Ca2+] evoked by 5-HT is through CICR(calcium inducedcalcium release) mechnism, and this effect was affected by G-protein, PLC and PKC.2.The antagonistic effect of DHPs calcium antagonist Mn9202 on S-HT in rat stomach fundus.We investigated the effects of Ca2+, miaserin and Mn9202 on the contractile responses to 5-HT in rat isolated perfused stomach fundal strip preparation. The results suggested that in a dose-dependent manner, Mn9202 shifted the cumulative dose response curves of 5-HT to right and reduced the efficacy of 5-HT to 75% and 55% respectively. Remove the extrocellular Ca2+, Mn9202(l u mol/L) shifted the curve to right and reduced the efficacy to 18%. When pre-incubated with 1 u mol/L Mn9202, miaserin could not shift the curve to right, indicating that in the rat stomach fundus, Mn9202 can antagonist 5-HT nonspecifically.Immunocytochemistry and image analysis were used to study the effects of DHPs calcium antagonist Mn9202 and lacidipine on 5-HT >TH and 5-HTR of cultured rat stomach fundus smooth muscle cells, We found that 5-HT > TH and 5-HTR immunoreactive substances were existed in SFSMCs, and the image analysis indicated that the contents of 5-HT > TH and 5-HTR were significantly lower when using Mn9202 , lacidipine and cyprohetadine(antagonist of 5-HT). The results suggested that the rat stomach fundus smooth muscle cells possess 5-HT autocrine function, and DHPs calcium antagonist reduced the number of 5-HT receptor in the rat stomach fundus smooth muscle cells through thereduction of 5-HT synthesis, this results conform with cyprohetadine.By hybridization in situ we found a decrease in mRNA for the 5-HT2B receptor in rat stomach fundus smooth muscle cells when incubated with 1 u mol/L Mn9202 and lacidipine; Western blot results suggest that DHPs calcium antagonist reduced the 5-HT receptor expression. Taken together these studies demonstrate significant changes in 5-HT receptor when treated with DHPs calcium antagonist both at the level of the receptor and mRNA, and may provide one reason why Mn9202 were proved to be a noncompetitive inhibiter of 5-HT-induced contraction of fundal strips.3. The effect of DHPs calcium antagonist on [Ca2+]i and Ca2+ sparks in rat stomach fundus smooth muscle cells induced by 5-HTA Biorad MRC 1024 confocal scanning microscope was used to image the cells with use of lasersharp time-course software. We observed that Mn9202(0.1, 1, 10 u mol/L) decreased [Ca2+]i induced by 5-HT(10 n mol/L) in dose-dependent manner, and 10 u mol/L Mn9202 abolished the effect of 5-HT completely.With the using of line-scan mode of the confocal microscope in combination with Fluo-3/AM, we found that frequency and amplitude of Ca2+ sparks were reduced about 56% and 60% respectively when pre-treated with 10 n mol/L Mn9202 before stimulated by 5-HT; In the same way 10 u mol/L lacidipine reducedthe frequency and amplitude of Ca2+ sparks about 50%. Our results show that the release of intracellular calcium and calcium influx by stimulation of 5-HT receptor in rat stomach fundus are sensitive to blockade by Mn9202 and lacidipine.4.The regulation and its mechanism of DHPs calcium antagonist on rat stomach fundus smooth muscle cell proliferation

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