Involvement Of CGRP In Pharmacological Actions Of Nitrates And Association With Related Drug Metabolism Enzymes

Organic nitrates nitrates are widely used for myocardial ischemia and chronic congestive heart failure.It is widely accepted that nitrates are a group of parent drugs and act through release of NO,the latter can activate the soluble guanylate cyclase(sGC),increase the the concentration of cyclic guanosine monophosphate(cGMP),and finally lead to vasodilatation.No has been regarded as the only effector molecular of nitrates.Calcitonin gene-related peptide(CGRP),the major transmitter in sensory nerves,is the most potent endogenous vasodilator identified so far.Previous studies in our group performed in animals have shown that CGRP is involved in the cardiovascular effects of nitroglycerin,and the decrease in synthesis and release of CGRP contribute to the development of nigroglycerin tolerance.Several drug metabolizing enzymes such as the mitochrondrial aldehyde dehydrogenase-2(ALDH2),glutathione-S-transferases(GSTs), xanthine oxidase and cytochrome P450 are involved in the biotransformation of nitrates.Therefore,the present study was designed to explore the involvement of CGRP in cardiovascular response to nitrates and association with enzymes involved in nitrates biotransformation in healthy Chinese male volunteers. Chapter 1 Evidence for involvement of calcitonin gene-related peptide in nitroglycerin response and association with mitochondrial aldehyde dehydrogenase-2(ALDH2) Glu504Lys polymorphismBACKGROUND:Nitroglycerin(GTN) has been one of most widely used anti-ischemic drugs for more than a century.Organic nitrates are excellent agents for the treatment of stable-effort angina,unstable angina,in patients with myocardial infarction and in patients with chronic congestive heart failure, the therapeutic effect of organic nitrates is due to venous and arterial dilation,decreased oxygen consumption and relaxation of coronary artery.Calcitonin gene-related peptide(CGRP),the major transmitter in sensory nerves,is widely distributed in the cardiovascular system.CGRP is the most potent endogenous vasodilator identified thus far.It has been confirmed in animal experiment that the CGRP plays an important role in the GTN response.It has recently been reported that the mitochondrial aldehyde dehydrogenase-2(ALDH2,mtALDH) catalyzes the formation of 1,2-glyceryl dinitrate and nitrite from GTN,inhibition of ALDH2 or deleted the cellular mitochondria leads to the impaired biotransformation of GTN.Naitrate tolerance is also partly due to the decreased bioactivity of ALDH2.A common Glu504Lys polymorphism(also called ALDH2^*2) of ALDH2,which accounts for decreased ALDH2 activity.In the present study,we explored the invlvement of CGRP in healthy Chinese volunteers and association with ALDH2 polymorphism.METHODS:1.Effect of ALDH2^*2 on CGRP mediated GTN responseEighty unrelated male volunteers were enrolled and screened for their ALDH2 Glu504Lys genotypes.Nine wild-type homozygotes (ALDH2^*1/^*1) and nine subjects with the ALDH2^*2 allele respectively, were randomly selected.All subjects received a sublingual administration of 0.5 mg nitroglycerin after the first blood samples were drawn.Blood pressure(BP) and heart rate(HR) were measured at 0,2,5,8,10,15,20, 25,30 and 35 min,respectively,after GTN administration in a sitting position.A second venous blood sample of 3 ml was taken 12 min after GTN administration for CGRP analysis.2 Effect of GTN on the mRNA expression of CGRPâ… å’ŒCGRPâ…¡in cultured PBMCs10 ml of venous blood sample was drawn to separate and cultivate PBMCs.Experiments were divided into 4 groups:(1) ALDH2^*1/^*1 homozygotes control;(2) ALDH2^*1/^*1 homozygotes+GTN(10^-5M);(3) carriers of the ALDH2^*2 allele control;(4) carriers of the ALDH2^*2 allele+GTN(10^-5M)3 Effect of ALDH2 inhibitor on stimulation of CGRP mRNA expression by GTNThe PBMCs from 3 ALDH2^*1/^*1 homozygotes were obtained from venous blood,experiments were divided into 4 groups:(1) control;(2) GTN(10^-5M);(3) GTN+ 1mM chloral hydrate(CH);(4) chloral hydrate (CH).RESULTS:(1) In contrast with carriers of the ALDH2^*2 allele,ALDH2^*1/^*1 homozygotes showed significantly higher extent of absolute changes in both systolic blood pressure(ΔSBP) and HR(ΔHR) at several time points after GTN administration.(2) Plasma concentrations of CGRP were increased significantly 12 min after GTN administration,and the percentage increase in plasma concentrations of CGRP correlated positively with bothΔSBP andΔHR, and percentage increase in plasma concentrations of CGRP was significantly higher in ALDH2^*1/^*1 homozygotes. (3) PBMCs from ALDH2^*1/^*1 homozygotes showed higher fold increase in both CGRPâ… and CGRPâ…¡mRNA after GTN stimulation.(4) The GTN-induced increase in CGRP mRNA expression in PBMCs from ALDH2^*1/^*1 homozygotes was inhibited by ALDH2 inhibitor chloral hydrate.CONCLUSION:CGRP is involved in the cardiovascular effect of GTN through an ALDH2-dependent pathway in human. Chapter 2 Evidence for involvement of calcitonin gene-related peptide in 5-ISMN response and association with GSTM1 polymorphismBackground:Organic nitrates nitrates such as nitroglycerin,isosorbide dinitrate, isosorbide-5-mononitrate(5-ISMN) can release NO to activate the guanylate cyclase,increase the the concentration of cyclic guanosine monophosphate,then activate the cGMP-dependent kinaseâ… ,decrease the concentration of Ca²⁺ in cells,finally induce vasodilatation.Calcitonine gene-related peptide(CGRP),the major neural transmitter in capsaicin sensitive nerves,comprised of 37 amino-acid residues,and is a potent vasodilator.Two isoforms of human CGRP,namely CGRP-1 and CGRP-2,have been identified,the two isoforms encoded by different gene.It has been reported that the synthesis and release of CGRP is regulated by multiple endogenous substances,incuding NO.There are many metabolic enzymes participate the biotransformation of nitrate drugs,such as mitochondrial aldehyde dehydrogenase-2 (ALDH2),glutathione- S-transferase(GSTs),xanthine oxidase, cytochrome P450.Glutathione-S-transferase is an importantâ…¡phase metabolic enzyme,encoded by a supergene family,it has been reported that Glutathione-S-transferase is involved in the metabolism of nitrates in vivo,there is a polymorphism in GSTM1,leads to decreased activity of the enzyme.The redox reaction mediated by xanthine oxidase is a source of reactive oxygen species in the cardiovascular system.XOR catalyzed organic nitrates,nitrates,and nitrites to NO though a NADH-dependent way.In this study,we explored the invlvement of CGRP in healthy Chinese volunteers and association with GSTM1 polymorphism.METHODS:2.Effect of GSTM1 polymorphism and on CGRP mediated 5-ISMN responseEighty unrelated male volunteers were enrolled and screened for their GSTM1 and ALDH2 Glu504Lys genotypes.Twelve GSTM1 wildtype genetype,twelve GSTM1 null genetype respectively,were randomly selected,included twelve wild-type homozygotes(ALDH2^*1/^*1) and twelve subjects with the ALDH2^*2 allele.All subjects received a oral administration of 20 mg 5-ISMN after the first blood samples were drawn. Blood pressure(BP) and heart rate(HR) were measured at several time points after 5-ISMN administration in a sitting position.Each 3 ml venous blood sample was taken at several time points after 5-ISMN administration for CGRP and blood drug level analysis.2.Effect of 5-ISMN on the mRNA expression of CGRPâ… å’ŒCGRPâ…¡in cultured PBMCs15 ml of venous blood sample was drawn to separate and cultivate PBMCs.PBMC were treated with 5-ISMN(50μM) 24 hour to detcted the mRNA expression of CGRPâ… å’ŒCGRPâ…¡.Cells were pretreated with ethacrvnic acid(1μM) or ALLopurinol(20μM) 30 min before 5-ISMN treatment to explore the effects of these enzymes inhibitor on up-regulated CGRP expression induced by 5-ISMN.RESULTS:(1) As compared with GSTM1 wildtype genetype,GSTM1 null genetype showed significant lower SBP and DBP at several time points after 5-ISMN administration.There is a significant deviation of changes in HR between GSTM1 wildtype genetype,GSTM1 null genetype.(2) As compared with GSTM1 wildtype genetype,GSTM1 null genetype showed significant lower 5-ISMN C_max and AUC_{0→∝}.(3) Plsma concentration of CGRP were increased in all volunteers,as compared with GSTM1 wildtype genetype,plsma concentration of CGRP in GSTM1 null genetype increased earlier and last for a longer time,percentage increase in plasma concentrations of CGRP was significantly higher in GSTM1 null genetype.(4) The XOR mRNA expression in volunteers before 5-ISMN administration correlated positively withΔSBP at 10,11,12 hour after 5-ISMN administration.(5) Treatment with 5-ISMN for 7 hour significantly up-regulated CGRPâ… mRNA expression in PBMC from GSTM1 null genetype,there is no significant deviation in PBMC from GSTM1 wildtype genetype.(6) Treatment with 5-ISMN significantly up-regulated the expression of CGRP 1å’ŒCGRPâ…¡mRNA in PBMC,which was abolished by xanthine oxidase inhibitor ALLopurinol(20μM),but not affected by GSTs inhibitor ethacrvnic acid(1μM). CONCLUSION:CGRP is involved in the cardiovascular effect of 5-ISMN.GSTM null genetype can accelerate the metabolism of 5-ISMN and improve its cardiovascular effect.