Journal of the American College of Cardiology Vol. 52, No. 11, by the American College of Cardiology Foundation ISSN /08/$34.

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1 Journal of the American College of Cardiology Vol. 52, No. 11, by the American College of Cardiology Foundation ISSN /08/$34.00 Published by Elsevier Inc. doi: /j.jacc Evidence for Involvement of Calcitonin Gene-Related Peptide in Nitroglycerin Response and Association With Mitochondrial Aldehyde Dehydrogenase-2 (ALDH2) Glu504Lys Polymorphism Ren Guo, MD, Xiao-Ping Chen, MD, PHD, Xin Guo, MD, Lei Chen, MS, Dai Li, MD, Jun Peng, MD, PHD, Yuan-Jian Li, MD Hunan, China Objectives Background Methods Results Conclusions This study sought to determine whether calcitonin gene-related peptide (CGRP) is involved in glyceryl trinitrate (GTN) response in humans, and its association with mitochondrial aldehyde dehydrogenase-2 (ALDH2) Glu504Lys (ALDH2*2) polymorphism. In animal models, CGRP contributes to the cardiovascular effects of GTN. The enzyme principally responsible for GTN bioactivation is ALDH2. The common ALDH2*2 polymorphism is associated with a lack of GTN efficacy. In 18 ALDH2*2-genotyped Chinese volunteers, we observed the changes in plasma concentrations of CGRP after sublingual GTN administration and its correlation with GTN response, and assessed the expression of CGRP messenger ribonucleic acid (mrna) in cultured peripheral blood mononuclear cells (PBMCs) pre-treated with 10 5 mol/l GTN. In contrast to carriers of the ALDH2*2 allele, ALDH2*1/*1 homozygotes showed a significantly higher extent of absolute changes in both systolic blood pressure ( SBP) and HR ( HR) at several time points after GTN administration. Plasma concentrations of CGRP were increased significantly 12 min after GTN administration, the percentage increase in plasma concentrations of CGRP correlated positively with both SBP and HR, and percentage increase in plasma concentrations of CGRP was significantly higher in ALDH2*1/*1 homozygotes. In addition, PBMCs from ALDH2*1/*1 homozygotes showed a higher-fold increase in both CGRP I and CGRP II mrna after GTN stimulation, and the GTN-induced increase in CGRP mrna expression in PBMCs from ALDH2*1/*1 homozygotes was inhibited by the ALDH2 inhibitor chloral hydrate. We found that CGRP is associated with the cardiovascular effect of GTN through an ALDH2-dependent pathway in humans. (J Am Coll Cardiol 2008;52:953 60) 2008 by the American College of Cardiology Foundation Nitroglycerin (glyceryl trinitrate, GTN) is an old drug that has been widely used in ischemic heart disease and congestive heart failure. It is widely accepted that the direct See page 961 anti-ischemic and vasodilatory properties of GTN are mediated by its final metabolic product nitric oxide (NO) From the Department of Pharmacology, School of Pharmaceutical Science, Central South University, Changsha, Hunan, China. Supported by the Chinese National Science Foundation (No ) and China New Century Excellent Talents of Ministry of Education (NCET ) to Dr. X.P. Chen. Drs. Guo and X.P. Chen contributed equally to this work. Manuscript received February 1, 2008; revised manuscript received April 28, 2008, accepted May 19, through NO/cyclic guanosine monophosphate (cgmp) pathway (1). The neuropeptide calcitonin gene-related peptide (CGRP) is the most potent endogenous vasodilator identified thus far (2). Two isoforms of human CGRP, namely CGRP-1 and CGRP-2, have been identified. The CGRP is originally thought to be synthesized and released by the capsaicin-sensitive sensory nerves fibers (3), and is mainly detected in perivascular nerve terminal (4). Previous animal studies have suggested that GTN can activate sensory nerves fibers to release CGRP (5,6), and the vasodilatory effect of GTN is reduced by both selective CGRP receptor antagonist and depletion of CGRP with capsaicin (7 10). Human studies have also shown that the increase in plasma CGRP correlates positively with the intensity of delayed migraine

2 954 Guo et al. JACC Vol. 52, No. 11, 2008 Calcitonin Gene-Related Peptide Is Involved in GTN Response September 9, 2008: Abbreviations and Acronyms DBP absolute decrease in diastolic blood pressure from baseline HR absolute increase in heart rate from baseline SBP absolute decrease in systolic blood pressure from baseline ALDH2 (mtaldh) mitochondrial aldehyde dehydrogenase-2 CGRP calcitonin generelated peptide CH chloral hydrate GTN glyceryl trinitrate HR heart rate NO nitric oxide PBMC peripheral blood mononuclear cell headache induced by GTN (11), and sumatriptan improves GTNinduced migraine by decreasing peripheral plasma CGRP concentration and CGRP release from perivascular axons (12). Because CGRP can be released in experimental models by NO-mediated mechanisms (13), we hypothesized that the cardiovascular effect of GTN is mediated, at least in part, by CGRP in humans. No studies, however, have ever addressed this question so far. It has been reported recently that the mitochondrial aldehyde dehydrogenase-2 (ALDH2, mtaldh) catalyzes the formation of 1,2-glyceryl dinitrate and nitrite from GTN, and leads to NO-based bioactivity of GTN (14,15). In mtaldh selectively deleted mice, the vascular responsiveness to low GTN was eliminated (16). Study in humans has also observed that the change in forearm blood flow (FBF) is reduced by ALDH2 inhibitor (14). A common Glu504Lys mutation (also called ALDH2*2) of ALDH2, which accounts for decreased ALDH2 activity and the flushing response to alcohol in about 30% to 50% of Asian populations in an autosomal-dominant manner (17), was observed to be associated with decreased GTN efficacy and blunted FBF response (14,18). High prevalence of the ALDH2*2 polymorphism in Asians gives us the advantage to further explore mechanisms of action of GTN in this population. To clarify the role that CGRP plays in the GTN response, we examined the association between changes in blood pressure (BP) and plasma concentrations of CGRP after sublingual GTN administration, and assessed the effect of ALDH2*2 polymorphism on both GTN response and CGRP release in healthy Chinese volunteers in this study. Because CGRP is also expressed in cells such as peripheral blood mononuclear cells (PBMCs) (19), the effects of GTN on CGRP expression were also explored in this study. Methods Subjects. Forty unrelated male volunteers were enrolled and screened for their ALDH2 Glu504Lys genotypes. Nine wild-type homozygotes (ALDH2*1/*1) and 9 subjects with the ALDH2*2 allele (8 ALDH2*1/*2 heterozygotes and 1 ALDH2*2/*2 homozygote), respectively, were randomly selected with a mean age of 28 years (range: 25 to 32 years). All subjects were nonsmokers and healthy as determined by medical history, physical examination, and laboratory tests before participating in the study. No medication or ethanol consumption was allowed for at least 2 weeks before the study. None of the individuals was following a particular type of diet before the study. Written informed consent was obtained from all subjects. The study was performed with the approval of the Ethical Committee of the College of Pharmaceutical Science of Central South University. Both the volunteers and the investigators performing the clinic study were blinded with respect to the ALDH2 genotype of each participant. On the day of nitroglycerin study, baseline venous blood samples of 13 ml were collected at 8:00 AM after a 30-min rest period for determination of CGRP level and isolation of PBMCs. All subjects received a sublingual administration of 0.5 mg GTN after the first blood samples were drawn. The 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. Each measure was repeated 3 times. A second venous blood sample of 3 ml was taken 12 min after GTN administration. Blood samples for CGRP analysis were drawn into ice-cold tubes containing 7.5% ethylenediaminetetracetic acid (EDTA) and aprotinin (400 mol/l). After centrifugation at 1,800 g for 15 min (4 C), the plasma was aspirated and stored at 70 C until analysis. Blood samples for isolation of PBMCs were drawn into heparinized tubes. Determination of ALDH2 Glu504Lys genotypes. Venous blood samples of 5 ml were drawn and genomic DNA samples from peripheral blood leukocytes were extracted according to standard phenol/chloroform protocols. The ALDH2 genotype was carried out by polymerase chain reaction restriction fragment length polymorphism as described previously (17). Determination of the concentration of CGRP in plasma. Concentration of CGRP in plasma was measured by a sensitive radioimmunoassay kit with antisera raised against human CGRP, 125 I-labelled CGRP, and human CGRP standard (Puerweiye Biotech Co. LTD, Beijing, China) according to the manufacturer s instructions. Isolation and culture of human PBMCs. The PBMCs were obtained from buffy coats, after centrifugation at 2,000 g for 30 min over 5 ml Ficoll-Hypaque gradients, washed twice with ice-cold phosphate-buffered saline, and resuspended in Roswell Park Memorial Institute 1640 containing 10% fetal calf serum, 50 U/ml penicillin, and 50 g/ml streptomycin. Cells were then seeded in 12-well plates at a concentration of /ml and cultured at 37 C in a humidified atmosphere enriched with 5% CO 2 for 24 h. The medium was removed, and cells were cultured with Roswell Park Memorial Institute 1640 containing 1% fetal calf serum for another 24 h. Cultures were challenged with or without (control) 10 5 mol/l GTN for 24 h, and then cells were harvested. The PBMCs from all 18 volunteers were used for each experimental condition. The PBMCs from 3 ALDH2*1/*1 homozygotes were also pretreated with 1 mmol/l chloral hydrate (CH) 30 min before the addition of GTN. Real-time polymerase chain reaction analysis. Total ribonucleic acid was extracted from PBMCs using Trizol

3 JACC Vol. 52, No. 11, 2008 September 9, 2008: Guo et al. Calcitonin Gene-Related Peptide Is Involved in GTN Response 955 reagent according to the manufacturer s instructions. Aliquots of 1 g ribonucleic acid from each sample were reverse-transcribed. Quantitative analysis using the SYBR Green method of CGRP messenger ribonucleic acid (mrna) expression was performed by using the ABI 7300 real-time polymerase chain reaction system. Sequences of primers were as follows: CGRP I: 5=-CCCAGAAGAGAGCCTGTGACA-3= (sense) and 5=-CTTCACCACACCCCCTGATC-3= (antisense); CGRP II: 5=-TCTTTCGGAGCCATCCTGTT-3= (sense) and 5=-GATTTCACGTCCCCCTAAGGTT-3= (antisense); GAPDH (endogenous control): 5=-CTGCACCAC- CAACTGCTTAG-3= (sense); 5=-AGGTCCACCACT- GACACGTT-3= (antisense). A 25- l reaction mixture containing 1 l cdna template, 12.5 l SYBR Master mix, and 0.20 l of each primer was amplified using the following thermal parameters: denaturing at 95 C for 10 min and 45 cycles of the amplification step (denaturation at 95 C for 15 s, annealing and extension at 60 C for 1 min). All amplification reactions were performed in triplicate, and the averages of the threshold cycles were used to interpolate curves using 7300 System SDS software (version 1.2.3, Applied Biosystems, Foster City, California). Results were expressed as the ratio of CGRP 1 or CGRP II mrna with GAPDH mrna, respectively. Statistical analysis. Statistical analysis of the data was carried out using the software SSPS (version 11.0, SPSS Inc., Chicago, Illinois). Data were expressed as means SD and, for clarity, as mean SEM in figures. Differences in BP, HR, and CGRP concentrations between time points after GTN administration and baseline were compared with the paired-sample t test. Differences in plasma concentration of CGRP and hemodynamic parameters between ALDH2 genotypes were compared with the Student t test. The Spearman correlation coefficient was calculated to assess the correlation of percentage change in plasma concentration of CGRP with absolute decrease in systolic blood pressure from baseline ( SBP) and absolute increase in heart rate from baseline ( HR). The difference in expression of CGRP mrna among groups was Figure 1 Changes in Blood Pressure and Heart Rate After GTN Administration Variation of SBP (A), DBP (B), HR(C), absolute decrease from baseline in SBP ( SBP) (D) and DBP ( DBP) (E), and absolute increase from baseline in HR ( HR) (F) in response to sublingual GTN administration in ALDH2*1/*1 homozygotes (solid circles; n 9) and carriers of ALDH2*2 allele (open circles; n 9). Values are mean SEM. *p 0.05, #p 0.01, **p compared with corresponding time point of carriers of ALDH2*2 allele. ALDH2 mitochondrial aldehyde dehydrogenase-2; DBP diastolic blood pressure; GTN glyceryl trinitrate; HR heart rate; SBP systolic blood pressure.

4 956 Guo et al. JACC Vol. 52, No. 11, 2008 Calcitonin Gene-Related Peptide Is Involved in GTN Response September 9, 2008: compared by analysis of variance. Significant difference was regarded as p Results Genotype distribution of ALDH2*2 polymorphism and individual selection. Genotype analysis of 40 healthy volunteers identified 25 ALDH2*1/*1 homozygotes and 15 individuals carrying at least 1 ALDH2*2 allele (13 homozygotes, 2 heterozygotes), with an incidence of 37.5% for carriers of the ALDH2*2 allele. Nine ALDH2*1/*1 homozygotes and 9 carriers of the ALDH2*2 allele (8 heterozygotes and 1 homozygote), respectively, were selected randomly for the following study. SBP and HR after GTN administration was associated with ALDH2*2 polymorphism. Both SBP and diastolic blood pressure (DBP) were declined significantly, whereas HR was increased significantly 2 min after GTN administration. Mean maximal decrease in SBP ( SBP max, 12 6 mm Hg) and DBP ( DBP max,8 7 mm Hg), and mean maximal increase in HR ( HR max,16 2beats/min) occurred at 10 min, 8 min, and 5 min, respectively, after GTN administration. The HR recovered gradually since 5 min, whereas SBP and DBP recovered gradually since 15 min after GTN administration. The DBP recovered completely, whereas SBP did not (110 8 mm Hg vs mm Hg, 35 min vs. baseline, p 0.001) 35 min after GTN administration. Both SBP and DBP were comparable between ALDH2 genotypes at baseline. As compared with carriers of the ALDH2*2 allele, ALDH2*1/*1 homozygotes showed significantly lower SBP at 10 min after GTN administration (p Figure 2 Change in Plasma CGRP After GTN Administration Concentration of CGRP in plasma in each individual at baseline and 12 min after sublingual GTN administration was measured. Solid circles (n 9) represent ALDH2*1/*1 homozygotes, open circles (n 9) represent carriers of ALDH2*2 allele. CGRP calcitonin gene-related peptide; other abbreviations as in Figure 1. Increase After Association Sublingual Plasma WithGTN ALDH2 CGRP Administration Glu504Lys Concentration Mutation and 12 Min Table 1 Increase in Plasma CGRP Concentration 12 Min After Sublingual GTN Administration and Association With ALDH2 Glu504Lys Mutation GTN Genotype Baseline (pg/ml) Treatment (pg/ml) % Increase ALDH2*1/* * ALDH2*1/*2 ALDH2*2/* * Overall * *p compared with baseline. p compared with ALDH2*1/*1. ALDH2 mitochondrial aldehyde dehydrogenase-2; CGRP calcitonin gene-related peptide; GTN glyceryl trinitrate. 0.05) (Fig. 1A). The ALDH2*1/*1 homozygotes also showed significantly higher SBP than carriers of the ALDH2*2 allele at several time points after GTN administration (Fig. 1D). As compared with carriers of the ALDH2*2 allele, SBP max was also significantly higher in ALDH2*1/*1 homozygotes (17 3 mm Hg vs. 8 4mm Hg, ALDH2*1/*1 homozygotes vs. carriers of the ALDH2*2 allele, p 0.001). No difference in either DBP or DBP was observed between ALDH2 genotypes in any time point after GTN administration (Figs. 1B and 1E). No difference in HR was observed between ALDH2 genotypes at any time point before and after GTN administration, either. However, ALDH2*1/*1 homozygotes showed significantly higher HR than carriers of the ALDH2*2 allele at several time points after GTN administration (Fig. 1F). The HR recovered to baseline at 20 min in carriers of the ALDH2*2 allele, while not recovered in ALDH2*1/*1 homozygotes even at 35 min (p 0.001) after GTN administration. Percentage increase in plasma concentrations of CGRP after GTN administration was associated with ALDH2*2 polymorphism. Plasma concentrations of CGRP were increased in all 18 individuals 12 min after GTN administration (Fig. 2). The percentage increase in CGRP concentrations was % (varied from 3.6% to 130.3%) in overall subjects (Table 1). The ALDH2*1/*1 homozygotes showed a significantly higher degree of percentage increase in plasma concentrations of CGRP than carriers of the ALDH2*2 allele ( % vs %, p 0.002) (Table 1). Plasma concentration of CGRP after GTN administration trended to be higher in ALDH2*1/*1 homozygotes. However, no significant difference in plasma concentrations of CGRP was observed between ALDH2 genotypes either before or after GTN administration. Percentage increase in plasma concentrations of CGRP correlated positively with both SBP and HR after GTN administration. We further analyzed the correlation between percentage increase in plasma concentrations of CGRP and SBP or HR. As shown in Figure 3, percentage increase in plasma concentrations of CGRP correlated positively with both SBP (Spearman R 0.491, p 0.038) and HR (Spearman R 0.490, p 0.046) 10 min after GTN administration.

5 JACC Vol. 52, No. 11, 2008 September 9, 2008: Guo et al. Calcitonin Gene-Related Peptide Is Involved in GTN Response 957 Figure 3 Correlation Between SBP or HR and Increase in Plasma CGRP A significant correlation was observed between SBP (A) or HR (B) at 10 min after GTN administration and percentage increase in plasma CGRP concentration at 12 min after GTN administration. Abbreviations as in Figures 1 and 2. Upregulation of CGRP mrna expression in PBMCs by GTN was associated with ALDH2*2 polymorphism. The expression of both CGRP 1 and CGRP II mrna in PBMCs were increased significantly after 10 5 mol/l GTN treatment for 24 h (Fig. 4). As compared with PBMCs from carriers of the ALDH2*2 allele, PBMCs from ALDH2*1/*1 homozygotes showed significantly higher fold increase in both CGRP I mrna ( vs , n 9, respectively, p 0.01) (Fig. 4A) and CGRP II mrna ( vs , n 9, respectively, p 0.01) (Fig. 4B) expression. The increase in the expression of both CGRP 1 mrna and CGRP II mrna induced by GTN in PBMCs from ALDH2*1/*1 homozygotes was inhibited significantly by 1 mmol/l CH pretreatment (p 0.05) (Fig. 5). Discussion In this study, we assessed the role of CGRP in GTN responsiveness in a group of ALDH2 genotype-known Chinese volunteers. We found that the plasma concentration of CGRP was increased significantly after sublingual GTN administration, and the percentage increase in plasma CGRP content correlated positively with both SBP and HR when GTN showed the maximal vasodilatory effects. We also observed that the changes in plasma CGRP, SBP, and HR were associated with ALDH2*2 polymorphism. Furthermore, we found that PBMCs from ALDH2*1/*1 homozygotes showed a higher degree of upregulation in CGRP mrna expression after GTN treatment, which could be inhibited by the ALDH2 inhibitor CH. CGRP is involved in GTN immediate response in humans. Our study is the first to examine the role of CGRP in GTN immediate response in humans. Previous studies by Juhasz et al. (11,12) have shown that CGRP is involved in the delayed migraine headache induced by GTN. However, no direct evidence has shown whether CGRP underlies the mechanism of immediate response of GTN in humans. We observed that the percentage increase in plasma concentration of CGRP after GTN administration correlated positively with both SBP and HR. Our findings show strong evidence that the increase in plasma CGRP is associated with the cardiovascular effects of NTG, and CGRP may partly underlie the mechanism of GTN action in vivo in humans. According to the Spearman correlation coefficient, the increase in plasma CGRP accounts for the approximately 24% variation in SBP caused by sublingual GTN. However, because specific CGRP receptor antagonists are still in clinical trials (20,21), it is difficult to evaluate the exact magnitude that CGRP contributes in GTN response. Further studies on the role of CGRP in GTN response are required in patients to confirm our findings. We found that HR max occurred earlier than both SBPmax and DBP max after GTN administration. Because CGRP is a vasodilator and also exerts a positive chronotropic effect on the heart (22), it is presumable that the early increase in HR is mediated by a direct action of CGRP, whereas the later increase in HR resulted from a reflex increase in sympathetic activity caused by vasodilation. Some other nitrates are also widely used in clinics. A previous report shows that NO donors such as nitroprusside and S-nitroso-N-acetylpenicillamine also stimulate the secretion of CGRP from trigeminal neurons (23). Whether CGRP is involved in the actions of other nitrates deserves further study.

6 958 Guo et al. JACC Vol. 52, No. 11, 2008 Calcitonin Gene-Related Peptide Is Involved in GTN Response September 9, 2008: Figure 5 CH Inhibited GTN-Induced CGRP Expression Peripheral blood monuclear cells (PBMCs) from ALDH2*1/*1 homozygotes were seeded in the presence of GTN (10 5 mol/l) for 24 h with or without 1 mmol/l chloral hydrate (CH) pre-treatment (control group; GTN group: 10 5 mol/l GTN; GTN CH group: 10 5 mol/l GTN 1 mmol/l CH; CH group: 1 mmol/l CH). The CGRP I and CGRP II mrna expression in PBMCs was analyzed by realtime polymerase chain reaction and expressed as percentage of control (mean SEM, n 3, respectively). *p 0.05 compared with control. #p 0.05 compared with corresponding GTN group. Abbreviations as in Figures 1, 2, and 4. CGRP (Fig. 6), and the magnitude that CGRP contributes to the GTN response is dependent on ALDH2 activity. As reported previously, CGRP mediates the delayed migraine attack induced by GTN several hours after drug discontinuation (11,12). This suggests that GTN may stimulate the expression of CGRP. In addition, other NO donors, such as S-nitroso-N-acetylpenicillamine, can stim- Figure 4 CGRP mrna Expression After GTN Treatment The expression of CGRP 1 and CGRP II messenger ribonucleic acid (mrna) in cultured peripheral blood monuclear cells (PBMCs) in the presence of 10 5 mol/l GTN or not (control) for 24 h was studied. The CGRP I (A) and CGRP II (B) mrna expression in PBMCs from ALDH2*1/*1 homozygotes (solid bars) and carriers of the ALDH2*2 allele (open bars) was analyzed by real-time polymerase chain reaction and expressed as percentage of control (mean SEM, n 9, respectively). *p compared with carriers of the ALDH2*2 allele. GAPDH glyceraldehyde-3-phosphate dehydrogenase; other abbreviations as in Figures 1 and 2. Contribution of CGRP in GTN response in an ALDH2- dependent manner. We also observed the effect of ALDH2*2 polymorphism on GTN response. In support of previous clinical studies (18,24), we found that individuals with low ALDH2 activity because of ALDH2*2 polymorphism showed a decreased GTN response as well as a lower degree of increase in plasma CGRP. Because NO, the active metabolite of GTN mediated by ALDH2, can stimulate the release of CGRP (11,13), our observation suggests that ALDH2- mediated bioactivation of GTN can lead to the release of Figure 6 Scheme of the Interaction Between GTN/CGRP and ALDH2 sgc soluble guanylate cyclase; other abbreviations as in Figures 1 and 2.

7 JACC Vol. 52, No. 11, 2008 September 9, 2008: Guo et al. Calcitonin Gene-Related Peptide Is Involved in GTN Response 959 ulate CGRP promoter activity (23). In this study, we observed that the therapeutic concentration of GTN induced the mrna expression of CGRP obviously in cultured PBMCs. Furthermore, the increase in CGRP mrna expression showed 3- to 4-fold higher in PBMCs with the wild-type ALDH2. To clarify whether upregulation of CGRP expression by GTN also depends on its bioactivation, an ALDH2 inhibitor CH was also applied. In accord with our initial assumption, the upregulation of CGRP mrna expression in PBMCs from individuals with the wild-type ALDH2 was inhibited completely by CH, which further suggests that ALDH2-mediated bioactivation of GTN is crucial for upregulation of CGRP expression by GTN. Of note, we could not observe an association between the decrease in DBP and ALDH2*2 polymorphism in our study. It is known that SBP and DBP are determined mainly by cardiac output and total peripheral resistance of blood vessels, respectively. Our observation is reasonable because, in usual doses of GTN, the magnitude of systemic venous dilatation (mainly affects cardiac output) is greater than systemic arterial dilatation (mainly affects peripheral resistance). The SBP was also used as a parameter to study the hemodynamic effect of GTN elsewhere (25). Development of nitrate tolerance after exposure to GTN for a long time is a major drawback that limits its clinical use. An increase in the production of reactive oxygen species and reactive oxygen species-induced inactivation of ALDH2 accounts for mechanism-based GTN tolerance in animals and in vitro models (26 29). Evidence has shown that GTN-induced release of endogenous CGRP during GTN desensitization attenuates the degree of tolerance (6), although the exact mechanism remains undetermined. However, the release of CGRP is reduced in response to subsequent GTN stimulation once nitrate tolerance is established (8,9,30). Our finding that CGRP is involved in the pharmacological action of GTN may also lead to a better understand of mechanisms underlying GTN tolerance. We could hypothesize from our study that decrease in CGRP release caused by reactive oxygen species-induced ALDH2 inhibition may also contribute to GTN tolerance in humans. Of course, further studies on these issues are needed. Summary and clinical implications. We confirmed for the first time that the neuropeptide CGRP is associated with GTN response in humans in an ALDH2-dependent pathway. Our findings provide new insights regarding the mechanisms of the cardiovascular effects of GTN. Further studies concerning the role of CGRP in GTN response and tolerance in patients are deserved in the future. 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