The Regulatory Mechanisms And The Pathophy-siological Significance For The Synthesis And Release Of Endothelium-derived Calcitonin Gene-related Peptide

Chapter 1Upregulation of endothelium-derived CGRP production in phenol-induced hypertension via activation ofα2-adrenergic receptorBACKGROUNDCalcitonin gene-related peptide (CGRP), a very potent vasodilator, is the predominant neurotransmitter in capsaicin-sensitive sensory nerves and widely distributes in the nerve and cardiovascular systems. In peripheral nerve system, the dorsal root ganglia (DRG) are the major site for CGRP synthesis. Besides nerve cells, other type of cells such as human lymphocytes, human typeⅡalveolar epithelial cells, Langerhans cells and endothelial cells has been also reported to synthesize CGRP. There is evidence that local source of CGRP may have special physiological functions. Our recent work has demonstrated that endothelial cell-derived CGRP was involved in heat stress-induced protection of endothelial function. Although the nerve cell-derived CGRP production is decreased in SHR or in phenol-induced hypertensive rats, little is known about the change of endothelia cell-derived CGRP and the underlying mechanisms. In most of cases, the activity of sympathetic nervous system is increased in hypertension. It has been shown that the plasma level of norepinephrine (NE) was elevated in phenol-induced hypertensive rats. Recently, we have found that activation of a2-adrenoceptor can induce CGRP production in endothelial cells. As a compensatory vasodilator mechanism to counteract the increased blood pressue, we postulate that the endothelium-derived CGRP production is likely upregulated in phenol-induced hypertension through the activation ofα2-adrenegic receptor.In the present study, by using the established phenol-induced hypertensive rat model and the cultured endothelial cells, we explored the changes in endothelium-derived CGRP production and the involved signal transduction pathway.METHODSHypertensive rats were induced by injecting 50μl of 10% phenol in the lower pole of the left kidney. Fifteen days after phenol treatment, the thoracic aorta and mesenteric artery were excised to determine CGRP peptide and mRNA by immunohistochemistry and in situ hybridization, respectively. In order to test the efect of prazosin on the expression of CGRP, the rats was treated with prazosin (3 mg/kg/d, i.g) for 14 days.In the cultured HUVEC-12, cells were treated with NE at concentration of 10-9,10-8,10-7,10-6 or 10-5 M for 24 h to evaluate the dose-effect relationship on CGRP expression or incubated with NE at concentration of 10"6M for 6,12,24,36 or 48 h to evaluate the time-effect relationship. Next, the cells were incubated with NE (10-5M) for 6 h after the pretreatment with yohimbine, prazosin or L-NAME for 30 min to determine the likely involved subtype of adrenergic signaling pathway in CGRP mRNA expression and to determine whether NO pathway was also involved. The level of CGRP mRNA was detected by RT-PCR. The level of nitrite/nitrate in the conditioned medium was measured with the Griess regent.RESULTSThe immunostaining signal of CGRP was significantly increased in the intima layer of aorta or mesenteric artery in the hypertension group, which was attenuated by prazosin treatment. Consistent with the immunostaining results of CGRP protein, the results of in situ hybridization showed that CGRP mRNA expression was significantly increased in the intima layer of aorta or mesenteric artery in the hypertension group, which was attenuated by prazosin treatment.NE significantly induced CGRP mRNA expression in a dose-dependent manner and increased gradually and reached the highest point at 6 h, and then went down after that, the effect of which was inhibited by yohimbine and L-NAME but not by prazosin, indicating that it isα2-but notα1-adrenoreceptor. NE treatment (10-5M) for 6 h significantly increased the NO level in the culture medium. These effects were attenuated in the presence of yohimbine or L-NAME but not prazosin, consistent with the change of CGRP mRNA expression mentioned above.CONCLUSIONThe present results suggest that the endothelium-derived CGRP production was increased in phenol-induced hypertension, which may represent an endogenous compensatory mechanism to counteract the elevated blood pressure. The up-regulation of endothelium-derived CGRP production is through activation ofα2-adrenergic receptor, which was associated with the NO pathway. Chapter 2The effect of clonidine on calcitonin gene-related peptide expression in cultured endothelial cellsBACKGROUNDIn addition to nerve cells, other type of cells such as lymphocytes and endothelial cells has been also reported to synthesize CGRP. It has been shown that local source of CGRP may have special physiological significance. For example, lymphocyte was able to produce and secret CGRP, which were supposed to participate in the modulating lymphocyte function in response to immune stimulation. Endothelial cell is able to synthesis and secret CGRP, too. Our recent work has demonstrated that endothelial cell-derived CGRP was involved in heat stress-induced protection of endothelial function.Clonidine is a centrally acting agonist ofα2 adrenergic receptor prescribed historically as an antihypertensive 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. There is evidence that clonidine is able to stimulate the peripheralα2 receptors and induce vasodilatation through activation ofα2 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. In the present study, we examined whether clonidine could induce CGRP synthesis and secretion in endothelial cell through activating a2-adrenoceptor and whether clonidine-induced CGRP production is mediated by the NO pathway.METHODSEndothelial cells were treated with clonidine at concentration of 10-8, 10-7 or 10-6 M for 24 h to evaluate the dose-effect relationship on CGRP expression or incubated with clonidine at concentration of 10-6M for 12, 24,36 or 48 h to evaluate the time-effect relationship. Next, the cells were incubated with clonidine (10-6M) for 24 h after the pretreatment with yohimbine or L-NAME for 30 min 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. The level of CGRP mRNA was detected by RT-PCR and Real time-PCR, while protein level was measured by radioimmunoassay (RIA). The level of nitrite/nitrate in the conditioned medium measured with the Griess regent.RESULTSTreating the cells with clonidine significantly induced CGRP mRNA expression in a dose-dependent manner. The expression of CGRP mRNA increased gradually and reached the highest point at 24 h, and then went down after that, the effect of which was inhibited by yohimbine or L-NAME. In line with these results, clonidine treatment also significantly increased CGRP content. These effects were abolished by pretreatment with yohimbine or L-NAME. Clonidine treatment (10-5M) for 24 h significantly increased the NO level in the culture medium. These effects were attenuated in the presence of yohimbine or L-NAME consistent with the change of CGRP mRNA expression mentioned above.CONCLUSIONThe present results suggest that clonidine could stimulate CGRP synthesis and secretion in endothelial cells through activation of a2-adrenoceptor, which is involved in the NO pathway. Chapter 3The role of endothelial cell-derived calcitonin gene-related peptide in angiotensinⅡ-induced endothelial cell apoptosisBACKGROUNDThe increase in RAS activity is one of the important mechanisms in the development of hypertension. It has been shown that the synthesis and release of CGRP was increased with the reduced blood pressure after taking captopril or losartan in renal hypertension rat and SHR, suggesting that RAS participated in regulating the synthesis and release of CGRP. It is well known that RAS plays a very important role in vascular remodeling and the decelopment of hypertension. There were reports that RAS participated in the regulation of nerve-derived CGRP. However, little is known about the role of RAS in regulating the endothelium-derived CGRP.As a major member of RAS, AngⅡpossesses multiplebiological functions. It involves in many pathophysiological process such as vascular remodeling, cellular apoptosis, oxidative stress, etc. Endothelial cell apoptosis has been thought closely related to the development of many cardiovascular diseases such as atherosclerosis and myocardial infarction. There is evidence that CGRP has the property of anti-apoptosis. It is not known whether AngⅡ-induced endothelial cell apoptosis is related to the inhibition of endothelium-derived CGRP expression.Our recent works has demonstrated that vascular endothelial cell can situ synthesis CGRP and express VR1, and capsaicin can stimulate the expression of endothelial cell-derived CGRP, which is abolished by antagonist of VR1 receptor caspazepine. In the present study, therefore, we explored the role of endothelial cell-derived CGRP in the AngⅡ-induced endothelial cell apoptosis.METHODSEndothelial cells were treated with AnglⅡat concentration of 10-8, 10-7 or 10-6 M for 24 h to evaluate the dose-effect relationship on CGRP expression or incubated with AngⅡat concentration of 10"6M for 6,12, 24,36 or 48 h to evaluate the time-effect relationship. Next, the cells were incubated with AngⅡ(10-5M) for 24 h after the pretreatment with losartan for 30 min to determine whether AT1 receptor was involved. The level of CGRP mRNA was detected by Real time-PCR and theprotein level was measured by radioimmunoassay.To explore the effect of CGRP on the AngⅡ-induced apoptosis, endothelial cells were pretreated with CGRP or capsaicin before AngⅡtreatment. Cell apoptosis was examined by hoechst 33258 staining and FITC-AnnexinⅤflow cytometry. Activity of Caspase 3 was measured by colorimetry. The level of CGRP, Bcl-2 and Bax mRNA were detected by RT-PCR. RESULTSAngⅡsignificantly decreasedα-andβ-CGRP mRNA expression and the content of CGRP in cultured endothelial cell in a dose-dependent manner, which was inhibited by pretreatment with losartan. Treating the cells with AngⅡat concentration of 10-6M for 6,12,24,36 or 48 h, the expression level of CGRP(α-andβ-) mRNA decreased gradually from the time point of 6 h.Pretreatment the cells with exogenous CGRP decreased AngⅡ-induced endothelial cell apoptosis accompanied by the decreased caspase-3 activity, which was attenuated in the presence of CGRP8-37, the antagonist of CGRP receptorTreating the cells with capsaicin at concentration of 10-8,10-7 or 10-6 M for 24 h, the expression level of CGRP(αandβ) mRNA increased in a dose-dependent manner, which was inhibited by capsazepine, the antagonist of VR1 receptorPretreatment the cells with capsaicin decreased AngⅡ-induced endothelial cell apoptosis accompanied by the increased Bcl-2 mRNA expression, the decreased Bax mRNA expression and caspase-3activity which was attenuated in the presence of capsazepine or CGRP8-37.CONCLUSIONAngⅡis able to inhibit the synthesis and release of CGRP in the cultured HUVECs, which may contribute to AngⅡ-induced endothelial cell apoptosis. Exogenous administration of CGRP or stimulating the synthesis of endogenous CGRP may provide a novel approach to prevent the AngⅡ-induced endothelial cell apoptosis.