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Clonidine induces calcitonin gene-related peptide expression via nitric oxide pathway in endothelial cells
Peptides 30 (2009) 1746–1752
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Clonidine induces calcitonin gene-related peptide expression via nitric oxide pathway in endothelial cells Yi-Min Zhang a,b,1, Jun Peng a,c,1, Chang-Ping Hu a, Qiu-Tao Jiang b, Guo-Long Jiang b, Yuan-Jian Li a,* a
Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, 110 Xiang-Ya Road, Changsha, Hunan 410078, China Hunan Yongzhou Food and Drug Administration, Yongzhou, Hunan 425600, China c Institute of Hypertension, Xiang-Ya Hospital of Central South University, Changsha 410008, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 February 2009 Received in revised form 30 May 2009 Accepted 1 June 2009 Available online 9 June 2009
The present study was to determine whether clonidine could induce calcitonin gene-related peptide (CGRP) production and the underlying mechanisms. Human umbilical vein endothelial cells were treated with clonidine and the dose–effect or time–effect relationship of clonidine on CGRP production was examined. Youhimbine (a a2-adrenoceptor blocker) and L-NAME (an antagonist of nitric oxide synthase, NOS) were chosen to explore the role of a2-adrenoceptor and nitric oxide pathway in the effect of clonidine on endothelial cell-derived CGRP production. The level of CGRP mRNA or protein was detected by Real Time-PCR or radioimmunoassay. Nitric oxide content was measured by nitroreduction assay. The study showed that clonidine was able to induce CGRP mRNA (a- and b-isoforms) expression in a dose-dependent manner in endothelial cells. The effect of clonidine on endothelial cell-derived CGRP synthesis and secretion was attenuated in the presence of youhimbine. L-NAME treatment could also inhibit clonidine-induced CGRP synthesis and secretion concomitantly with the decreased NO content in culture medium. These results suggest that clonidine could stimulate CGRP synthesis and secretion in endothelial cells through the activation of a2-adrenoceptor, which is related to the NO pathway. ß 2009 Published by Elsevier Inc.
Keywords: a2-Adrenoceptor Calcitonin gene-related peptide Clonidine Nitric oxide
1. Introduction Calcitonin gene-related peptide (CGRP), one of the major neurotransmitters of capsaicin-sensitive sensory nerves, is widely distributed in the nervous and cardiovascular systems [1]. The major site of CGRP synthesis is the dorsal root ganglion that contains the cell bodies of sensory nerves which terminate peripherally on blood vessels and other tissues. It is the most potent vasodilator discovered to date and participates in regulating the vascular tone and regional organ blood flows both under physiological and pathophysiological conditions [7]. In addition to nervous cells, other type of cells such as lymphocytes and endothelial cells has been also reported to synthesize CGRP [14,21]. Interestingly, it has been shown that local source of CGRP may have special physiological significance. There are reports that lymphocyte was able to produce and secrete CGRP, which were supposed to participate in the modulating lymphocyte function in response to immune stimulation [21]. Endothelial cell is able to synthesize and secrete CGRP, too. Our
* Corresponding author. Tel.: +86 731 2355078; fax: +86 731 2355078. E-mail address: [email protected] (Y.-J. Li). 1 These authors contributed equally to this work. 0196-9781/$ – see front matter ß 2009 Published by Elsevier Inc. doi:10.1016/j.peptides.2009.06.001
recent work has demonstrated that endothelial cell-derived CGRP was involved in heat stress-induced protection of endothelial function [26]. Clonidine is a centrally acting agonist of a2-adrenergic receptor prescribed historically as an anti-hypertensive drug. The mechanism of the anti-hypertensive effect of clonidine is not completely understood but probably reduction of central sympathetic tone must play a very important role. Activation of a2-adrenoceptors located in the central nervous system seems to be the most important mechanism of the sympathetic inhibition [29]. There is evidence that clonidine also stimulates peripheral a2 receptors and induces vasodilatation through the activation of a2 receptors in endothelial cells, an effect which was blocked by L-NAME, the inhibitor of NOS, suggesting that NO pathway may involve in this process [17]. Since CGRP is the most potent vasodilator and endothelial cell is able to produce CGRP as mentioned above, in the present study, we therefore examined whether clonidine could induce CGRP synthesis and secretion in endothelial cell through activating a2adrenoceptor. Since clonidine is able to induce NO production and NO has been demonstrated to participate in regulating CGRP synthesis and release [3,16], we therefore also explored whether clonidine-induced CGRP production is mediated by the NO pathway in endothelial cells.
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2. Materials and methods 2.1. Cell culture Human umbilical vein endothelial cells (HUVECs) were cultured according to standard procedure. Briefly, cells were seeded at a density of 3 105 per 100-mm dish in DMEM, supplemented with 20 mM HEPES and 20% FCS. The cultures were maintained at 37 8C with a gas mixture of 5% CO2/95% air. All media were supplemented with 5 U/ml heparin, 100 IU/ ml penicillin and 100 mg/ml streptomycin. Medium was changed every 2–3 days. Endothelial cells of the forth to sixth passages in the actively growing condition were used for experiments. 2.2. Protocols To explore the effect of clonidine on the synthesis and secretion of CGRP in endothelial cells and its underlying mechanisms, following series of experiments were performed. The first series was designed to evaluate the dose–effect relationship of clonidine on CGRP expression in HUVECs. Cells were incubated with clonidine at concentrations of 108, 107 or 106 M for 24 h before collecting for the analysis of CGRP (a and b) mRNA expression. The second series was designed to evaluate the time–effect relationship of clonidine on CGRP expression in HUVECs, the cells were incubated with clonidine at concentration of 106 M for 12, 24, 36 or 48 h before collecting for the analysis of CGRP mRNA (a and b) expression. The third series was designed to examine whether the effect of clonidine on regulating CGRP mRNA expression in HUVECs is through the activation of a2-adrenergic receptor and whether NO pathway is involved. Cells were divided into six groups: (1) control; (2) clonidine, cells were incubated with clonidine (106 M) for 24 h; (3) youhimbine, cells were incubated with youhimbine (105 M), the antagonist of a2-adrenergic receptor, for 24 h; (4) clonidine plus youhimbine, cells were incubated with clonidine (106 M) and youhimbine (105 M) for 24 h; (5) LNAME, cells were incubated with L-NAME (105 M) for 24 h; (6) clonidine plus L-NAME, cells were incubated with clonidine (106 M) and L-NAME (105 M) for 24 h. At the end of experiments, culture medium and cells were collected for the analysis of CGRP level and CGRP expression (mRNA and protein), respectively.
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2.3.2. Real Time-PCR analysis To verify the results of RT-PCR, Real Time-PCR for CGRP mRNA analysis was performed as previously described [11]. Briefly, a 10-ml reaction mixture containing 2 ml cDNA template, 5 ml SYBR Master mix, 0.20 ml ROX, 2.4 ml H2O, and 0.20 ml of each primer was amplified by the following thermal parameters: denaturing at 95 8C for 10 min and 45 cycles of the amplification step (denaturation at 95 8C for 15 s, annealing and extension at 60 8C for 1 min). Each cDNA sample tested for quantitative a- or b-CGRP mRNA was also subjected to GAPDH analysis. All amplification reactions were performed in triplicate and the averages of the threshold cycles were used to interpolate curves using 7300 System SDS Software. Results were expressed as the ratio of a- or b-CGRP to GAPDH mRNA, and the value of a- or b-CGRP expression level in group of Control was regarded as 1. 2.4. Determination of CGRP protein level To determine the CGRP protein level secreted from endothelial cells, culture medium from each sample (10 106 cells) was collected and purified on a sep-pak C18 column, lyophilized and stored at 80 8C until used. To determine the CGRP protein level in HUVECs, cells (10 106) were collected and first boiled in 1 ml 1 M acetic acid for 10 min, then sonicated and centrifuged at 3000 rpm for 15 min at 4 8C. The supernatants were collected, lyophilized and stored at 80 8C until used. As previously described [12], CGRP-like immunoreactivity (CGRP-LI) was determined by radioimmunoassay kits (Dongya Immunity Technology Institution, China), using an antibody of rabbit anti-rat CGRP, which showed a 93% cross-reactivity toward human CGRP. The assay was performed as recommended by the supplier. There is no crossreactivity with adrenomedullin, adrenomedullin-2 (intermedin), amylin and substance P. 2.5. Determination of nitric oxide level The level of NO in the conditioned medium was determined indirectly as the content of nitrite and nitrate. The level of nitrite/ nitrate in the conditioned medium was measured as described previously. Briefly, nitrate was converted to nitrite with aspergillums nitrite reductase, and the total nitrite was measured with the Griess regent. The absorbance was determined at 540 nm with a spectrophotometer. 2.6. Reagents
2.3. Determination of CGRP mRNA level 2.3.1. RT-PCR analysis Total RNA isolation from the cells and semi-quantitative RT-PCR were performed according to standard techniques. The specific primers pairs and the size of the expected products were as follows (forward and reverse, respectively): a-CGRP, 50 -CTAAGCGGTGCGGTAATC-30 and 50 -CCTCCCATCTGAAGTTTGA-30 (326 bp); b-CGRP, 50 CAGCTCCACCAAACCTTA-30 and 50 -GTTCACAAATGGCACAAT-30 (131 bp); b-actin, 50 -CTGTCCCTGTATGCCTCTG-30 and 50 -ATGTCACGCACGATTTCC-30 (218 bp). The PCR amplification profiles consisted of denaturation at 94 8C for 45 s, annealing at 58 8C for 45 s and elongation at 72 8C for 1 min. The linear exponential phases for CGRP (alpha and beta) and b-actin PCR were 38 and 26 cycles, respectively. Equal amounts of corresponding a-CGRP, b-CGRP and b-actin PCR products were loaded on 1.7% agarose gels. To control for possible difference in loading of the RNA samples, we normalized the RNA levels to those for b-actin. Optical densities of anthodium bromide-stained DNA bands were quantitated and results were expressed as CGRP/b-actin ratios.
Human umbilical vein endothelial cell line was obtained from American Type Culture Collection (ATCC, USA). Clonidine, yohimbine and L-NAME were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Sijiqing Biological Engineering Materials (Hangzhou, China). Dulbecco’s modified Eagle’s medium (DMEM) and Trizol reagent was purchased from GIBCO BRL (USA). Primers for PCR were synthesized in TaKaRa Biotechnology (Dalian, China). The RT-PCR kits were purchased from MBI (Lithuania). Radioimmunoassay kits for measurement of CGRP were purchased from the Immunity Institute of Dongya (Beijing, China). Real Time-PCR kits were purchased from TaKaRa Biotechnology (Dalian, China). 2.7. Statistical analysis Data are expressed as means S.E.M. All values were analyzed by using ANOVA and multiple comparison test (the Student– Newman–Keuls t-test) by using SPSS 10.0. The acceptable value of significance was p < 0.05.
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3. Results 3.1. Effect of clonidine on the expression of endothelial cell-derived CGRP As shown in Fig. 1, there is baseline CGRP (a- and b-isoforms) mRNA expression in HUVECs, consistent with our previous report [26]. Treating the cells with clonidine, an agonist of a2adrenoreceptor, at concentrations of 108, 107 or 106 M for 24 h significantly induced CGRP (a and b) mRNA expression in a dose-dependent manner, indicating that a2-adrenergic signal pathway is involved in regulation of CGRP expression. Next we examined the time–effect relationship of clonidine on CGRP expression in HUVECs. As displayed in Fig. 2, treating the cells with clonidine at concentration of 106 M for 12, 24, 36 or 48 h, the expression level of CGRP (a and b) mRNA increased
gradually and reached the highest point at 24 h, and then went down after that. 3.2. a2-Adrenergic and NO signal pathway involve in clonidineinduced expression of endothelia cell-derived CGRP To verify clonidine induced CGRP synthesis and release in endothelia cells is through activation of a2-adrenergic receptor, yohimbine, the antagonist of a2-adrenoreceptor, has been used. In Fig. 3, it shows that clonidine treatment significantly induced CGRP mRNA expression in endothelia cells, an effect which was inhibited in the presence of yohimbine. In-line with these results, clonidine treatment also significantly increased CGRP content from 13.1 4.2 (control) to 21.7 7.6 pg/107 cells (clonidine) in culture medium or from 26.2 8.5 (control) to 62.0 14.5 pg/107 cells (clonidine) in cells (Fig. 4). These effects were abolished by
Fig. 1. Dose–effect relationship of clonidine on the expression of endothelial cell-derived CGRP. (A) Representative sample of RT-PCR products for a-CGRP, b-CGRP and bactin. M: DNA marker; 1, 2, 3 and 4 stands for Con, Clo (L), Clo (M) and Clo (H), respectively. (B) The results of densitometric scanning from DNA bands of each group, which were expressed as the ratio of a- or b-CGRP/b-actin. (C) The results of Real Time-PCR for each group, which were expressed as the ratio of a- or b-CGRP/GAPDH. Con: control; Clo (L), Clo (M) and Clo (H): clonidine treatment at 108, 107 and 106 M, respectively. All values are means S.E.M., four independent experiments were carried out in each group. *p < 0.05 and **p < 0.01 vs. Con.
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Fig. 2. Time-effect relationship of clonidine on the expression of endothelial cell-derived CGRP. (A) Representative sample of RT-PCR products for a-CGRP, b-CGRP and bactin, respectively. M: DNA Marker; 1, 2, 3, 4 and 5 stands for Con, Clo (12 h), Clo (24 h), Clo (36 h) and Clo (48 h), respectively. (B) The results of densitometric scanning from DNA bands of each group, which were expressed as the ratio of a- or b-CGRP/b-actin. (C) The results of Real Time-PCR for each group, which were expressed as the ratio of aor b-CGRP/GAPDH. Con: control; Clo (12 h), Clo (24 h), Clo (36 h) and Clo (48 h): clonidine treatment for 12, 24, 36 and 48 h, respectively. All values are means S.E.M., four independent experiments were carried out in each group. *p < 0.05 and **p < 0.01 vs. Con.
pretreatment with yohimbine, supporting the role of a2-adrenergic signal pathway in clonidine-induced expression of endothelia cellderived CGRP. Since NO pathway has been shown involved in CGRP production, we therefore chose L-NAME, an inhibitor of nitric oxide synthase, to examine its role in clonidine-induced CGRP synthesis and release. Similar to yohimbine, L-NAME is also able to block the effect of clonidine on CGRP mRNA and protein expression in endothelial cells (Fig. 4), confirming the role of NO pathway in modulating CGRP production induced by clonidine. 3.3. Effect of clonidine on NO production The level of NO in the conditioned medium was determined indirectly by the content of nitrite and nitrate. As shown in Fig. 5, clonidine treatment (106 M) for 24 h significantly increased the
NO level in the culture medium, from 22.0 7.2 (control) to 39.5 11.4 mmol/107 cells (clonidine). These effects were abolished in the presence of yohimbine or L-NAME, consistent with the change of CGRP levels mentioned above. 3.4. Discussion In present study, we have demonstrated that clonidine was able to induce CGRP synthesis and secretion in cultured HUVECs through the activation of a2-adrenergic receptor. Furthermore, we provided evidence that clonidine-induced CGRP production is related to NO pathway because the effect of clonidine on endothelial cells was blocked in the presence of L-NAME. It is well recognized that the blood vessels are widely innervated both by sympathetic and capsaicin-sensitive sensory nerves, which play significant roles in controlling the resistance
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Fig. 3. Role of a2-adrenoceptor and NO pathway in clonidine-induced CGRP mRNA expression in endothelial cells. (A) Representative sample of RT-PCR products for a-CGRP, b-CGRP and b-actin, respectively. M: DNA Marker; 1, 2, 3, 4, 5 and 6 stands for Con, Clo (H), Yoh, Yoh + Clo (H), L-NAME and L-NAME + Clo (H), respectively. (B) The results of densitometric scanning from DNA bands of each group, which were expressed as the ratio of a- or b-CGRP/b-actin. (C) The results of Real Time-PCR for each group, which were expressed as the ratio of a- or b-CGRP/GAPDH. Con, control; Clo (H), clonidine (106 M); Yoh, yohimbine (105 M); Yoh + Clo (H), yohimbine (105 M) + clonidine (106 M); L-NAME + Clo (H), L-NAME (105 M) + clonidine (106 M). All values are means S.E.M., four independent experiments were carried out in each group. *p < 0.05; **p < 0.01 vs. Con; +p < 0.05; ++p < 0.01 vs. Clo (H).
vascular tone through the release of two classes of vasoactive substances, vasoconstrictors and vasodilators [1]. Among the vasodilators, CGRP is the most potent one to date. It is approximately 100–1000 times more potent than other vasodilators such as adenosine, substance P or acetylcholine [4,16]. There is plenty of evidence that CGRP plays an important role in regulating blood pressure [7,11,15]. Early reports from clinical studies showed that plasma levels of CGRP were significantly decreased in patients with essential hypertension [18,19]. Our recent work has demonstrated that plasma levels of CGRP were also decreased spontaneously in hypertensive rats, the decreased plasma levels of CGRP might be one of the major factors involved in
the pathogenesis of essential or spontaneous hypertension [11]. However, in acquired hypertension patients (such as hyperadrenocortism) or in 2 kidneys 1 clip hypertensive rats (2K1C), the plasma levels of CGRP were increased [6], and the increased plasma levels of CGRP in the secondary hypertension could be considered as the beneficial compensation against high blood pressure. Clonidine is a very potent anti-hypertensive agent. Although the effect of clonidine on reducing central sympathetic tone is well established, its role in regulating peripheral vascular tone remains poorly understood. A recent study has demonstrated that endothelial a2-adrenoceptors may be involved in the anti-hypertensive
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Fig. 4. Role of a2-adrenoceptor and NO pathway in clonidine-induced CGRP protein synthesis and secretion in endothelial cells. (A) CGRP protein levels in culture medium. (B) CGRP protein levels within cells. Con, control; Clo (H), clonidine (106 M); Yoh, yohimbine (105 M); Yoh + Clo (H), yohimbine (105 M) + clonidine (106 M); L-NAME + Clo (H), L-NAME (105 M) + clonidine (106 M). Values are presented as mean S.E.M., six independent experiments were carried out in each group. **p < 0.01 vs. Con; ++p < 0.01 vs. Clo (H).
action of clonidine because in isolated rat mesenteric arteries, clonidine was able to induce endothelium-dependent relaxation which was inhibited by pretreatment with a2-adrenoceptor blocker yohimbine [17]. L-NAME or ODQ (a guanylyl cyclase inhibitor) pretreatment could also reduce the clonidine-induced vascular vasodilation in isolated mesenteric arteries, suggesting that clonidine-induced vasodilation is related to activation of NO-cGMP pathway. There is strong evidence that CGRP plays an important role in mediating vascular vasodilation. It has been shown that vasodilator responses to nitroglycerin, a NO donor, in feline cerebral arterioles or in rat aortas were associated with endogenous CGRP [2,23]. Others have reported that L-arginine, a substrate of NO synthase (NOS), enhanced the release of CGRP evoked by endotoxin in the isolated mesenteric arterial bed, an effect which was attenuated by NG-nitro-L-arginine, a selective inhibitor of NOS [22]. The role of NO in regulating CGRP release is consistent with our recent studies. In the isolated rat aorta, preincubation with nitroglycerin induced significant release of CGRP and led to vascular relaxation in a concentration-dependent manner, and the effect was attenuated by pretreatment with CGRP-(8–37), a selective CGRP receptor antagonist, or by capsaicin, which
Fig. 5. Effect of clonidine on nitrite and nitrate production in endothelial cells. Con, control; Clo (H), clonidine (106 M); Yoh, yohimbine (105 M); Yoh + Clo (H), yohimbine (105 M) + clonidine (106 M); L-NAME + Clo (H), L-NAME (105 M) + clonidine (106 M). Values are presented as mean S.E.M., six independent experiments were carried out in each group. **p < 0.01 vs. Con; ++ p < 0.01 vs. Clo (H).
specifically depletes the transmitter content of sensory nerves [27,28]. Based on the studies from the others and ours, we therefore postulate that clonidine-induced vascular relaxation may be mediated by its stimulatory effect on the CGRP release. However, a recent study has shown that clonidine inhibited perivascular sensory outflow (CGRP release) by the activation of a2adrenoceptor [20], indicating that CGRP from sensory nerves may not be the source that is responsible for clonidine-induced vascular relaxation. As mentioned before, besides nervous cells, endothelial cells are also able to produce and secrete CGRP. Therefore, we hypothesize that clonidine-induced vascular relaxation is through stimulating CGRP secretion from endothelial cells. In clinic, the therapeutic doses most commonly employed ranged from 0.2 to 0.6 mg per day given in divided doses. The reported circulating levels of clonidine ranged from 0.65 to 1.34 ng/ml (equal to 2.4 106 to 5.0 106 M) [9,24]. In the present study, we first evaluated the dose–effect relationship of clonidine on CGRP expression at concentrations of 108, 107 and 106 M, and then chose 106 M for the following studies because it is more clinically relevant. The results from this study have clearly shown that clonidine induced CGRP expression and secretion in HUVECs, an effect which was attenuated in the presence of a2-adrenoceptor blocker yohimbine, supports our hypothesis. CGRP has two subtypes namely a- and b-CGRP, which are encoded in different genes, and differ from each other by only one or three amino acids in rats or in humans. There is evidence that the subtype of CGRP differs from different tissues or species. For example, only b-CGRP is presented in alveolar type II epithelial cells and rat lymphocytes while both a- and b-CGRP are expressed in neuron cells and human lymphocytes [10,13,21,25]. Although previous reports showed that there existed CGRP expression in endothelial cells, it could not identify the subtype of CGRP by using methods of immunohistochemistry or in situ hybridization [5,8]. In the present study, by using RT-PCR, we extended previous reports and demonstrated that both a- and b-CGRP were expressed in HUVECs, consistent with our recent report [14]. Interestingly, it seems like clonidine-induced CGRP expression was dominated by a-CGRP and the physiological significance of this phenomenon remains to be elucidated. Because NO has been repeatedly shown to participate in regulating CGRP synthesis and release and clonidine was able to stimulate NO production, we therefore examined the correlation between NO and CGRP in this study. As expected, clonidineinduced CGRP production in HUVECs was blocked by pretreatment with L-NAME concomitantly with the reduced NO level in culture
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medium, supporting that clonidine-induced CGRP expression in endothelial cells is closely associated with NO pathway. In summary, we have demonstrated for the first time that clonidine could stimulate CGRP synthesis and secretion in endothelial cells through the activation of a2-adrenoceptor, which is involved in the NO pathway. It may provide novel insight into the mechanisms of anti-hypertensive effect of clonidine. Acknowledgements This work was supported by grants from the National Basic Research Program of China (973 Program), No. 2007CB512000 and the National Natural Science Foundation of China, No. 30430740. References [1] Bell D, McDermott BJ. Calcitonin gene-related peptide in the cardiovascular system: characterization of receptor populations and their (patho)physiological significance. Pharmacol Rev 1996;48:253–88. [2] Booth BP, Nolan TD, Fung HL. Nitroglycerin-inhibited whole blood aggregation is partially mediated by calcitonin gene-related peptide—a neurogenic mechanism. Br J Pharmacol 1997;122:577–83. [3] Booth BP, Tabrizi-Fard MA, Fung H. Calcitonin gene-related peptide-dependent vascular relaxation of rat aorta. An additional mechanism for nitroglycerin. Biochem Pharmacol 2000;59:1603–9. [4] Brain SD, Williams TJ, Tippins JR, Morris HR, MacIntyre I. Calcitonin generelated peptide is a potent vasodilator. Nature 1985;313:54–6. [5] Cai WQ, Dikranian K, Bodin P, Turmaine M, Burnstock G. Colocalization of vasoactive substances in the endothelial cells of human umbilical vessels. Cell Tissue Res 1993;274:533–8. [6] Deng PY, Ye F, Zhu HQ, Cai WJ, Deng HW, Li YJ. An increase in the synthesis and release of calcitonin gene-related peptide in two-kidney, one-clip hypertensive rats. Regul Pept 2003;114:175–82. [7] Deng PY, Li YJ. Calcitonin gene-related peptide and hypertension. Peptides 2005;26:1676–85. [8] Doi Y, Kudo H, Nishino T, Kayashima K, Kiyonaga H, Nagata T, et al. Synthesis of calcitonin gene-related peptide (CGRP) by rat arterial endothelial cells. Histol Histopathol 2001;16:1073–9. [9] Frisk-Holmberg M, Paalzow L, Wibell L. Relationship between the cardiovascular effects and steady-state kinetics of clonidine in hypertension. Demonstration of a therapeutic window in man. Eur J Clin Pharmacol 1984;26:309–13. [10] Hastings RH, Hua XY. Expression of calcitonin gene-related peptide by cultured rat alveolar type II cells. Am J Respir Cell Mol Biol 1995;13:563–9. [11] Li D, Peng J, Xin HY, Luo D, Zhang YS, Zhou Z, et al. Calcitonin gene-related peptide-mediated antihypertensive and anti-platelet effects by rutaecarpine in spontaneously hypertensive rats. Peptides 2008;29:1781–8. [12] Li D, Chen BM, Peng J, Zhang YS, Li XH, Yuan Q, et al. Role of anandamide transporter in regulating calcitonin gene-related peptide production and blood pressure in hypertension. J Hypertens 2009;27:1224–32.
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Clonidine induces calcitonin gene-related peptide expression via nitric oxide pathway in endothelial cells Yi-Min Zhang a,b,1, Jun Peng a,c,1, Chang-Ping Hu a, Qiu-Tao Jiang b, Guo-Long Jiang b, Yuan-Jian Li a,* a
Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, 110 Xiang-Ya Road, Changsha, Hunan 410078, China Hunan Yongzhou Food and Drug Administration, Yongzhou, Hunan 425600, China c Institute of Hypertension, Xiang-Ya Hospital of Central South University, Changsha 410008, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 February 2009 Received in revised form 30 May 2009 Accepted 1 June 2009 Available online 9 June 2009
The present study was to determine whether clonidine could induce calcitonin gene-related peptide (CGRP) production and the underlying mechanisms. Human umbilical vein endothelial cells were treated with clonidine and the dose–effect or time–effect relationship of clonidine on CGRP production was examined. Youhimbine (a a2-adrenoceptor blocker) and L-NAME (an antagonist of nitric oxide synthase, NOS) were chosen to explore the role of a2-adrenoceptor and nitric oxide pathway in the effect of clonidine on endothelial cell-derived CGRP production. The level of CGRP mRNA or protein was detected by Real Time-PCR or radioimmunoassay. Nitric oxide content was measured by nitroreduction assay. The study showed that clonidine was able to induce CGRP mRNA (a- and b-isoforms) expression in a dose-dependent manner in endothelial cells. The effect of clonidine on endothelial cell-derived CGRP synthesis and secretion was attenuated in the presence of youhimbine. L-NAME treatment could also inhibit clonidine-induced CGRP synthesis and secretion concomitantly with the decreased NO content in culture medium. These results suggest that clonidine could stimulate CGRP synthesis and secretion in endothelial cells through the activation of a2-adrenoceptor, which is related to the NO pathway. ß 2009 Published by Elsevier Inc.
Keywords: a2-Adrenoceptor Calcitonin gene-related peptide Clonidine Nitric oxide
1. Introduction Calcitonin gene-related peptide (CGRP), one of the major neurotransmitters of capsaicin-sensitive sensory nerves, is widely distributed in the nervous and cardiovascular systems [1]. The major site of CGRP synthesis is the dorsal root ganglion that contains the cell bodies of sensory nerves which terminate peripherally on blood vessels and other tissues. It is the most potent vasodilator discovered to date and participates in regulating the vascular tone and regional organ blood flows both under physiological and pathophysiological conditions [7]. In addition to nervous cells, other type of cells such as lymphocytes and endothelial cells has been also reported to synthesize CGRP [14,21]. Interestingly, it has been shown that local source of CGRP may have special physiological significance. There are reports that lymphocyte was able to produce and secrete CGRP, which were supposed to participate in the modulating lymphocyte function in response to immune stimulation [21]. Endothelial cell is able to synthesize and secrete CGRP, too. Our
* Corresponding author. Tel.: +86 731 2355078; fax: +86 731 2355078. E-mail address: [email protected] (Y.-J. Li). 1 These authors contributed equally to this work. 0196-9781/$ – see front matter ß 2009 Published by Elsevier Inc. doi:10.1016/j.peptides.2009.06.001
recent work has demonstrated that endothelial cell-derived CGRP was involved in heat stress-induced protection of endothelial function [26]. Clonidine is a centrally acting agonist of a2-adrenergic receptor prescribed historically as an anti-hypertensive drug. The mechanism of the anti-hypertensive effect of clonidine is not completely understood but probably reduction of central sympathetic tone must play a very important role. Activation of a2-adrenoceptors located in the central nervous system seems to be the most important mechanism of the sympathetic inhibition [29]. There is evidence that clonidine also stimulates peripheral a2 receptors and induces vasodilatation through the activation of a2 receptors in endothelial cells, an effect which was blocked by L-NAME, the inhibitor of NOS, suggesting that NO pathway may involve in this process [17]. Since CGRP is the most potent vasodilator and endothelial cell is able to produce CGRP as mentioned above, in the present study, we therefore examined whether clonidine could induce CGRP synthesis and secretion in endothelial cell through activating a2adrenoceptor. Since clonidine is able to induce NO production and NO has been demonstrated to participate in regulating CGRP synthesis and release [3,16], we therefore also explored whether clonidine-induced CGRP production is mediated by the NO pathway in endothelial cells.
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2. Materials and methods 2.1. Cell culture Human umbilical vein endothelial cells (HUVECs) were cultured according to standard procedure. Briefly, cells were seeded at a density of 3 105 per 100-mm dish in DMEM, supplemented with 20 mM HEPES and 20% FCS. The cultures were maintained at 37 8C with a gas mixture of 5% CO2/95% air. All media were supplemented with 5 U/ml heparin, 100 IU/ ml penicillin and 100 mg/ml streptomycin. Medium was changed every 2–3 days. Endothelial cells of the forth to sixth passages in the actively growing condition were used for experiments. 2.2. Protocols To explore the effect of clonidine on the synthesis and secretion of CGRP in endothelial cells and its underlying mechanisms, following series of experiments were performed. The first series was designed to evaluate the dose–effect relationship of clonidine on CGRP expression in HUVECs. Cells were incubated with clonidine at concentrations of 108, 107 or 106 M for 24 h before collecting for the analysis of CGRP (a and b) mRNA expression. The second series was designed to evaluate the time–effect relationship of clonidine on CGRP expression in HUVECs, the cells were incubated with clonidine at concentration of 106 M for 12, 24, 36 or 48 h before collecting for the analysis of CGRP mRNA (a and b) expression. The third series was designed to examine whether the effect of clonidine on regulating CGRP mRNA expression in HUVECs is through the activation of a2-adrenergic receptor and whether NO pathway is involved. Cells were divided into six groups: (1) control; (2) clonidine, cells were incubated with clonidine (106 M) for 24 h; (3) youhimbine, cells were incubated with youhimbine (105 M), the antagonist of a2-adrenergic receptor, for 24 h; (4) clonidine plus youhimbine, cells were incubated with clonidine (106 M) and youhimbine (105 M) for 24 h; (5) LNAME, cells were incubated with L-NAME (105 M) for 24 h; (6) clonidine plus L-NAME, cells were incubated with clonidine (106 M) and L-NAME (105 M) for 24 h. At the end of experiments, culture medium and cells were collected for the analysis of CGRP level and CGRP expression (mRNA and protein), respectively.
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2.3.2. Real Time-PCR analysis To verify the results of RT-PCR, Real Time-PCR for CGRP mRNA analysis was performed as previously described [11]. Briefly, a 10-ml reaction mixture containing 2 ml cDNA template, 5 ml SYBR Master mix, 0.20 ml ROX, 2.4 ml H2O, and 0.20 ml of each primer was amplified by the following thermal parameters: denaturing at 95 8C for 10 min and 45 cycles of the amplification step (denaturation at 95 8C for 15 s, annealing and extension at 60 8C for 1 min). Each cDNA sample tested for quantitative a- or b-CGRP mRNA was also subjected to GAPDH analysis. All amplification reactions were performed in triplicate and the averages of the threshold cycles were used to interpolate curves using 7300 System SDS Software. Results were expressed as the ratio of a- or b-CGRP to GAPDH mRNA, and the value of a- or b-CGRP expression level in group of Control was regarded as 1. 2.4. Determination of CGRP protein level To determine the CGRP protein level secreted from endothelial cells, culture medium from each sample (10 106 cells) was collected and purified on a sep-pak C18 column, lyophilized and stored at 80 8C until used. To determine the CGRP protein level in HUVECs, cells (10 106) were collected and first boiled in 1 ml 1 M acetic acid for 10 min, then sonicated and centrifuged at 3000 rpm for 15 min at 4 8C. The supernatants were collected, lyophilized and stored at 80 8C until used. As previously described [12], CGRP-like immunoreactivity (CGRP-LI) was determined by radioimmunoassay kits (Dongya Immunity Technology Institution, China), using an antibody of rabbit anti-rat CGRP, which showed a 93% cross-reactivity toward human CGRP. The assay was performed as recommended by the supplier. There is no crossreactivity with adrenomedullin, adrenomedullin-2 (intermedin), amylin and substance P. 2.5. Determination of nitric oxide level The level of NO in the conditioned medium was determined indirectly as the content of nitrite and nitrate. The level of nitrite/ nitrate in the conditioned medium was measured as described previously. Briefly, nitrate was converted to nitrite with aspergillums nitrite reductase, and the total nitrite was measured with the Griess regent. The absorbance was determined at 540 nm with a spectrophotometer. 2.6. Reagents
2.3. Determination of CGRP mRNA level 2.3.1. RT-PCR analysis Total RNA isolation from the cells and semi-quantitative RT-PCR were performed according to standard techniques. The specific primers pairs and the size of the expected products were as follows (forward and reverse, respectively): a-CGRP, 50 -CTAAGCGGTGCGGTAATC-30 and 50 -CCTCCCATCTGAAGTTTGA-30 (326 bp); b-CGRP, 50 CAGCTCCACCAAACCTTA-30 and 50 -GTTCACAAATGGCACAAT-30 (131 bp); b-actin, 50 -CTGTCCCTGTATGCCTCTG-30 and 50 -ATGTCACGCACGATTTCC-30 (218 bp). The PCR amplification profiles consisted of denaturation at 94 8C for 45 s, annealing at 58 8C for 45 s and elongation at 72 8C for 1 min. The linear exponential phases for CGRP (alpha and beta) and b-actin PCR were 38 and 26 cycles, respectively. Equal amounts of corresponding a-CGRP, b-CGRP and b-actin PCR products were loaded on 1.7% agarose gels. To control for possible difference in loading of the RNA samples, we normalized the RNA levels to those for b-actin. Optical densities of anthodium bromide-stained DNA bands were quantitated and results were expressed as CGRP/b-actin ratios.
Human umbilical vein endothelial cell line was obtained from American Type Culture Collection (ATCC, USA). Clonidine, yohimbine and L-NAME were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) was obtained from Sijiqing Biological Engineering Materials (Hangzhou, China). Dulbecco’s modified Eagle’s medium (DMEM) and Trizol reagent was purchased from GIBCO BRL (USA). Primers for PCR were synthesized in TaKaRa Biotechnology (Dalian, China). The RT-PCR kits were purchased from MBI (Lithuania). Radioimmunoassay kits for measurement of CGRP were purchased from the Immunity Institute of Dongya (Beijing, China). Real Time-PCR kits were purchased from TaKaRa Biotechnology (Dalian, China). 2.7. Statistical analysis Data are expressed as means S.E.M. All values were analyzed by using ANOVA and multiple comparison test (the Student– Newman–Keuls t-test) by using SPSS 10.0. The acceptable value of significance was p < 0.05.
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3. Results 3.1. Effect of clonidine on the expression of endothelial cell-derived CGRP As shown in Fig. 1, there is baseline CGRP (a- and b-isoforms) mRNA expression in HUVECs, consistent with our previous report [26]. Treating the cells with clonidine, an agonist of a2adrenoreceptor, at concentrations of 108, 107 or 106 M for 24 h significantly induced CGRP (a and b) mRNA expression in a dose-dependent manner, indicating that a2-adrenergic signal pathway is involved in regulation of CGRP expression. Next we examined the time–effect relationship of clonidine on CGRP expression in HUVECs. As displayed in Fig. 2, treating the cells with clonidine at concentration of 106 M for 12, 24, 36 or 48 h, the expression level of CGRP (a and b) mRNA increased
gradually and reached the highest point at 24 h, and then went down after that. 3.2. a2-Adrenergic and NO signal pathway involve in clonidineinduced expression of endothelia cell-derived CGRP To verify clonidine induced CGRP synthesis and release in endothelia cells is through activation of a2-adrenergic receptor, yohimbine, the antagonist of a2-adrenoreceptor, has been used. In Fig. 3, it shows that clonidine treatment significantly induced CGRP mRNA expression in endothelia cells, an effect which was inhibited in the presence of yohimbine. In-line with these results, clonidine treatment also significantly increased CGRP content from 13.1 4.2 (control) to 21.7 7.6 pg/107 cells (clonidine) in culture medium or from 26.2 8.5 (control) to 62.0 14.5 pg/107 cells (clonidine) in cells (Fig. 4). These effects were abolished by
Fig. 1. Dose–effect relationship of clonidine on the expression of endothelial cell-derived CGRP. (A) Representative sample of RT-PCR products for a-CGRP, b-CGRP and bactin. M: DNA marker; 1, 2, 3 and 4 stands for Con, Clo (L), Clo (M) and Clo (H), respectively. (B) The results of densitometric scanning from DNA bands of each group, which were expressed as the ratio of a- or b-CGRP/b-actin. (C) The results of Real Time-PCR for each group, which were expressed as the ratio of a- or b-CGRP/GAPDH. Con: control; Clo (L), Clo (M) and Clo (H): clonidine treatment at 108, 107 and 106 M, respectively. All values are means S.E.M., four independent experiments were carried out in each group. *p < 0.05 and **p < 0.01 vs. Con.
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Fig. 2. Time-effect relationship of clonidine on the expression of endothelial cell-derived CGRP. (A) Representative sample of RT-PCR products for a-CGRP, b-CGRP and bactin, respectively. M: DNA Marker; 1, 2, 3, 4 and 5 stands for Con, Clo (12 h), Clo (24 h), Clo (36 h) and Clo (48 h), respectively. (B) The results of densitometric scanning from DNA bands of each group, which were expressed as the ratio of a- or b-CGRP/b-actin. (C) The results of Real Time-PCR for each group, which were expressed as the ratio of aor b-CGRP/GAPDH. Con: control; Clo (12 h), Clo (24 h), Clo (36 h) and Clo (48 h): clonidine treatment for 12, 24, 36 and 48 h, respectively. All values are means S.E.M., four independent experiments were carried out in each group. *p < 0.05 and **p < 0.01 vs. Con.
pretreatment with yohimbine, supporting the role of a2-adrenergic signal pathway in clonidine-induced expression of endothelia cellderived CGRP. Since NO pathway has been shown involved in CGRP production, we therefore chose L-NAME, an inhibitor of nitric oxide synthase, to examine its role in clonidine-induced CGRP synthesis and release. Similar to yohimbine, L-NAME is also able to block the effect of clonidine on CGRP mRNA and protein expression in endothelial cells (Fig. 4), confirming the role of NO pathway in modulating CGRP production induced by clonidine. 3.3. Effect of clonidine on NO production The level of NO in the conditioned medium was determined indirectly by the content of nitrite and nitrate. As shown in Fig. 5, clonidine treatment (106 M) for 24 h significantly increased the
NO level in the culture medium, from 22.0 7.2 (control) to 39.5 11.4 mmol/107 cells (clonidine). These effects were abolished in the presence of yohimbine or L-NAME, consistent with the change of CGRP levels mentioned above. 3.4. Discussion In present study, we have demonstrated that clonidine was able to induce CGRP synthesis and secretion in cultured HUVECs through the activation of a2-adrenergic receptor. Furthermore, we provided evidence that clonidine-induced CGRP production is related to NO pathway because the effect of clonidine on endothelial cells was blocked in the presence of L-NAME. It is well recognized that the blood vessels are widely innervated both by sympathetic and capsaicin-sensitive sensory nerves, which play significant roles in controlling the resistance
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Fig. 3. Role of a2-adrenoceptor and NO pathway in clonidine-induced CGRP mRNA expression in endothelial cells. (A) Representative sample of RT-PCR products for a-CGRP, b-CGRP and b-actin, respectively. M: DNA Marker; 1, 2, 3, 4, 5 and 6 stands for Con, Clo (H), Yoh, Yoh + Clo (H), L-NAME and L-NAME + Clo (H), respectively. (B) The results of densitometric scanning from DNA bands of each group, which were expressed as the ratio of a- or b-CGRP/b-actin. (C) The results of Real Time-PCR for each group, which were expressed as the ratio of a- or b-CGRP/GAPDH. Con, control; Clo (H), clonidine (106 M); Yoh, yohimbine (105 M); Yoh + Clo (H), yohimbine (105 M) + clonidine (106 M); L-NAME + Clo (H), L-NAME (105 M) + clonidine (106 M). All values are means S.E.M., four independent experiments were carried out in each group. *p < 0.05; **p < 0.01 vs. Con; +p < 0.05; ++p < 0.01 vs. Clo (H).
vascular tone through the release of two classes of vasoactive substances, vasoconstrictors and vasodilators [1]. Among the vasodilators, CGRP is the most potent one to date. It is approximately 100–1000 times more potent than other vasodilators such as adenosine, substance P or acetylcholine [4,16]. There is plenty of evidence that CGRP plays an important role in regulating blood pressure [7,11,15]. Early reports from clinical studies showed that plasma levels of CGRP were significantly decreased in patients with essential hypertension [18,19]. Our recent work has demonstrated that plasma levels of CGRP were also decreased spontaneously in hypertensive rats, the decreased plasma levels of CGRP might be one of the major factors involved in
the pathogenesis of essential or spontaneous hypertension [11]. However, in acquired hypertension patients (such as hyperadrenocortism) or in 2 kidneys 1 clip hypertensive rats (2K1C), the plasma levels of CGRP were increased [6], and the increased plasma levels of CGRP in the secondary hypertension could be considered as the beneficial compensation against high blood pressure. Clonidine is a very potent anti-hypertensive agent. Although the effect of clonidine on reducing central sympathetic tone is well established, its role in regulating peripheral vascular tone remains poorly understood. A recent study has demonstrated that endothelial a2-adrenoceptors may be involved in the anti-hypertensive
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Fig. 4. Role of a2-adrenoceptor and NO pathway in clonidine-induced CGRP protein synthesis and secretion in endothelial cells. (A) CGRP protein levels in culture medium. (B) CGRP protein levels within cells. Con, control; Clo (H), clonidine (106 M); Yoh, yohimbine (105 M); Yoh + Clo (H), yohimbine (105 M) + clonidine (106 M); L-NAME + Clo (H), L-NAME (105 M) + clonidine (106 M). Values are presented as mean S.E.M., six independent experiments were carried out in each group. **p < 0.01 vs. Con; ++p < 0.01 vs. Clo (H).
action of clonidine because in isolated rat mesenteric arteries, clonidine was able to induce endothelium-dependent relaxation which was inhibited by pretreatment with a2-adrenoceptor blocker yohimbine [17]. L-NAME or ODQ (a guanylyl cyclase inhibitor) pretreatment could also reduce the clonidine-induced vascular vasodilation in isolated mesenteric arteries, suggesting that clonidine-induced vasodilation is related to activation of NO-cGMP pathway. There is strong evidence that CGRP plays an important role in mediating vascular vasodilation. It has been shown that vasodilator responses to nitroglycerin, a NO donor, in feline cerebral arterioles or in rat aortas were associated with endogenous CGRP [2,23]. Others have reported that L-arginine, a substrate of NO synthase (NOS), enhanced the release of CGRP evoked by endotoxin in the isolated mesenteric arterial bed, an effect which was attenuated by NG-nitro-L-arginine, a selective inhibitor of NOS [22]. The role of NO in regulating CGRP release is consistent with our recent studies. In the isolated rat aorta, preincubation with nitroglycerin induced significant release of CGRP and led to vascular relaxation in a concentration-dependent manner, and the effect was attenuated by pretreatment with CGRP-(8–37), a selective CGRP receptor antagonist, or by capsaicin, which
Fig. 5. Effect of clonidine on nitrite and nitrate production in endothelial cells. Con, control; Clo (H), clonidine (106 M); Yoh, yohimbine (105 M); Yoh + Clo (H), yohimbine (105 M) + clonidine (106 M); L-NAME + Clo (H), L-NAME (105 M) + clonidine (106 M). Values are presented as mean S.E.M., six independent experiments were carried out in each group. **p < 0.01 vs. Con; ++ p < 0.01 vs. Clo (H).
specifically depletes the transmitter content of sensory nerves [27,28]. Based on the studies from the others and ours, we therefore postulate that clonidine-induced vascular relaxation may be mediated by its stimulatory effect on the CGRP release. However, a recent study has shown that clonidine inhibited perivascular sensory outflow (CGRP release) by the activation of a2adrenoceptor [20], indicating that CGRP from sensory nerves may not be the source that is responsible for clonidine-induced vascular relaxation. As mentioned before, besides nervous cells, endothelial cells are also able to produce and secrete CGRP. Therefore, we hypothesize that clonidine-induced vascular relaxation is through stimulating CGRP secretion from endothelial cells. In clinic, the therapeutic doses most commonly employed ranged from 0.2 to 0.6 mg per day given in divided doses. The reported circulating levels of clonidine ranged from 0.65 to 1.34 ng/ml (equal to 2.4 106 to 5.0 106 M) [9,24]. In the present study, we first evaluated the dose–effect relationship of clonidine on CGRP expression at concentrations of 108, 107 and 106 M, and then chose 106 M for the following studies because it is more clinically relevant. The results from this study have clearly shown that clonidine induced CGRP expression and secretion in HUVECs, an effect which was attenuated in the presence of a2-adrenoceptor blocker yohimbine, supports our hypothesis. CGRP has two subtypes namely a- and b-CGRP, which are encoded in different genes, and differ from each other by only one or three amino acids in rats or in humans. There is evidence that the subtype of CGRP differs from different tissues or species. For example, only b-CGRP is presented in alveolar type II epithelial cells and rat lymphocytes while both a- and b-CGRP are expressed in neuron cells and human lymphocytes [10,13,21,25]. Although previous reports showed that there existed CGRP expression in endothelial cells, it could not identify the subtype of CGRP by using methods of immunohistochemistry or in situ hybridization [5,8]. In the present study, by using RT-PCR, we extended previous reports and demonstrated that both a- and b-CGRP were expressed in HUVECs, consistent with our recent report [14]. Interestingly, it seems like clonidine-induced CGRP expression was dominated by a-CGRP and the physiological significance of this phenomenon remains to be elucidated. Because NO has been repeatedly shown to participate in regulating CGRP synthesis and release and clonidine was able to stimulate NO production, we therefore examined the correlation between NO and CGRP in this study. As expected, clonidineinduced CGRP production in HUVECs was blocked by pretreatment with L-NAME concomitantly with the reduced NO level in culture
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medium, supporting that clonidine-induced CGRP expression in endothelial cells is closely associated with NO pathway. In summary, we have demonstrated for the first time that clonidine could stimulate CGRP synthesis and secretion in endothelial cells through the activation of a2-adrenoceptor, which is involved in the NO pathway. It may provide novel insight into the mechanisms of anti-hypertensive effect of clonidine. Acknowledgements This work was supported by grants from the National Basic Research Program of China (973 Program), No. 2007CB512000 and the National Natural Science Foundation of China, No. 30430740. References [1] Bell D, McDermott BJ. Calcitonin gene-related peptide in the cardiovascular system: characterization of receptor populations and their (patho)physiological significance. Pharmacol Rev 1996;48:253–88. [2] Booth BP, Nolan TD, Fung HL. Nitroglycerin-inhibited whole blood aggregation is partially mediated by calcitonin gene-related peptide—a neurogenic mechanism. Br J Pharmacol 1997;122:577–83. [3] Booth BP, Tabrizi-Fard MA, Fung H. Calcitonin gene-related peptide-dependent vascular relaxation of rat aorta. An additional mechanism for nitroglycerin. Biochem Pharmacol 2000;59:1603–9. [4] Brain SD, Williams TJ, Tippins JR, Morris HR, MacIntyre I. Calcitonin generelated peptide is a potent vasodilator. Nature 1985;313:54–6. [5] Cai WQ, Dikranian K, Bodin P, Turmaine M, Burnstock G. Colocalization of vasoactive substances in the endothelial cells of human umbilical vessels. Cell Tissue Res 1993;274:533–8. [6] Deng PY, Ye F, Zhu HQ, Cai WJ, Deng HW, Li YJ. An increase in the synthesis and release of calcitonin gene-related peptide in two-kidney, one-clip hypertensive rats. Regul Pept 2003;114:175–82. [7] Deng PY, Li YJ. Calcitonin gene-related peptide and hypertension. Peptides 2005;26:1676–85. [8] Doi Y, Kudo H, Nishino T, Kayashima K, Kiyonaga H, Nagata T, et al. Synthesis of calcitonin gene-related peptide (CGRP) by rat arterial endothelial cells. Histol Histopathol 2001;16:1073–9. [9] Frisk-Holmberg M, Paalzow L, Wibell L. Relationship between the cardiovascular effects and steady-state kinetics of clonidine in hypertension. Demonstration of a therapeutic window in man. Eur J Clin Pharmacol 1984;26:309–13. [10] Hastings RH, Hua XY. Expression of calcitonin gene-related peptide by cultured rat alveolar type II cells. Am J Respir Cell Mol Biol 1995;13:563–9. [11] Li D, Peng J, Xin HY, Luo D, Zhang YS, Zhou Z, et al. Calcitonin gene-related peptide-mediated antihypertensive and anti-platelet effects by rutaecarpine in spontaneously hypertensive rats. Peptides 2008;29:1781–8. [12] Li D, Chen BM, Peng J, Zhang YS, Li XH, Yuan Q, et al. Role of anandamide transporter in regulating calcitonin gene-related peptide production and blood pressure in hypertension. J Hypertens 2009;27:1224–32.
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