The Role Of Calcitonin Gene-related Peptide In The Development Of Hypertension And Therapeutic Effect Of Rutaecarpine

BACKGROUNDCalcitonin gene-related peptide (CGRP), the principal neurotransmitter of capsaicin-sensitive sensory nerves, is predominately synthesized in dorsal root ganglia (DRG) in peripheral nervous system. CGRP is widely distributed in cardiovascular system and it is thought as the most powerful vasodilator so far. It is well established that CGRP plays an important role in regulation of blood pressure. There is growing reports to show that, in addition to DRG, many of other type of cells such as leukomonocyte and endothelial cells are also able to synthesize CGRP, which exerts multiple functions through autocrine or paracrine secretion. It has been shown that the synthesis and release of CGRP are regulated by Vanilloid receptor subtype 1(VR1) or capsaicin receptor, which exists in all above mentioned cells.There is increasing evidence to suggest that CGRP plays an important role in the development of hypertensive disease. The decreased level of plasma CGRP is thought as one of the main factors involved in the pathogenesis of essential or spontaneous hypertension. Whereas in acquired hypertension patients (such as hyperadrenocortism) or 2 kidneys 1 clip hypertensive rats (2K1C), the level of plasma CGRP was increased, which was considered as beneficial compensation against high blood pressure. It is unclear, however, the mechanisms underlying the difference in the level of plasma CGRP in different type of hypertensions.Anandamide (AEA), an endogenous ligand of VR1, could be synthesized by multiple cells, such as endothelial cell, macrophage and monocyte. It has been shown that VR1 was located in superficies interna of membrane, so its specific ligands (such as AEA) need to across membrane before binding to it. The AEA uptake of cells is mediated by the so-called AEA transporter which is across the membrane and is the major channel for AEA to enter the cells. Therefore, the plasma level of AEA and the activity of AEA transporter are the two key factors to determine the efficiency of AEA uptake, and in turn to regulate the synthesis and release of CGRP via VR1. It was reported that the activity of AEA transporter was affected by nitric oxide (NO) or exogenous NO synthase inhibitor (L-NAME).To elucidate the mechanisms underlying the possible role of CGRP in the development of essential or spontaneous hypertension, in the present study, we first examined plasma levels of AEA and CGRP in essential hypertension patients, SHR and 2K1C, and then collected the leukomonocytes from hypertensive rats for analyzing the activity of AEA transporter to determine its role in regulating the synthesis and release of CGRP.METHODSClinical studyBlood pressure, plasma CGRP (RIA) and AEA levels (HPLC) were determined in essential hypertension patients (before taking medicine for therapy) and healthy volunteers.Animal experimentBlood pressure (tail-cuff method), plasma levels of CGRP, AEA, endogenous NOS inhibitor ADMA and NO (nitroreduction method) were measured in SHR and age-matched WKY, or 2K1C and control (Sham operation group). The expression ofα-CGRP andβ-CGRP mRNA in DRG were detected (Real-Time PCR). AEA transporter activity was also measured (isotope labeling method).Cell experimentThe peripheral blood lymphocytes were collected from Wistar rats, and the effects of AEA on the expression of CGRP mRNA were determined by Real-Time PCR. AM404, the AEA transporter inhibitor, was selected for confirming the effect of AEA. The effect of ADMA on AEA transporter activity was evaluated by the isotope labeling method. RESULTSIn essential hypertension patients, the plasma levels of CGRP were decreased, whereas the AEA levels were decreased. Similar results were obtained in SHR, plus the reduced expression ofα-CGRP andβ-CGRP mRNA; In 2K1C, both AEA and CGRP levels were elevated, accompanying by the increased expression ofα-CGRP andβ-CGRP mRNA. The activity of AEA transporter in lymphocytes from SHR was decreased; and the ADMA levels were elevated concomitantly with an increased NO level in SHR. There is no significant differences in AEA activity, ADMA and NO levels in 2K1C. AEA was able to stimulate the expression ofα-CGRP andβ-CGRP mRNA in lymphocytes, which was blocked by pretreatment with AEA transporter inhibitor. Treatment with exogenous ADMA inhibited AEA transporter activityCONCLUSIONSThe decrease in plasma levels of CGRP whereas the increase in AEA levels in essential hypertension patients and SHR is likely due to the reduced activity of AEA transporter. The increased level of ADMA may account for, at least in part, the reduced AEA transporter activity. BACKGROUNDHypertension is a common global disease that severely threatens the health of human being. Neuroendocrine dysfunction is one of the key factors that contribute to the pathological process of hypertension. It is well known that central and peripheral vascular system is under control by both sympathetic vasoconstrictor and sensory vasodilator, which plays an important role in regulating the cardiovascular function and maintaining the stability of blood pressure. Calcitonin gene-related peptide (CGRP), the principal transmitter of capsaicin-sensitive nerves, is thought as the most powerful vasodilator to date. It has been reported the plasma level of CGRP was decreased in essential hypertension patients as well as in spontaneous hypertensive rat (SHR). It is possible that dysregulation of CGRP synthesis and release might be a key factor contributed to the development of hypertension.Since high blood pressure is prone to activating the platelet and resulting in thrombus formation, so the occurrence of thrombus diseases is more severe and frequent in essential hypertension patients. The activation of platelet is also the important motivation for acute cardiovascular accidents. The activation and aggregation of platelet are closely associated with abnormal concentration or/and activity of endogenous substance in the essential hypertension. It has been indicated that CGRP, an endogenous vasodilator, also exerts the effects on inhibiting platelet aggregation.It is well documented that activation of Vanilloid receptor subtype 1 (VR1) will lead to the synthesis and release of CGRP. Rutaecarpine is a major component isolated from Evodia Fruit, which is a traditional Chinese medicine with the property of anti-hypertension. Recent studies have shown that rutaecarpine is able to produce multiple cardiovascular effects by stimulating CGRP release via activation of VR1. There are reports to show that rutaecarpine has inhibitory effect on platelet aggregation, but the underlying mechanisms have not been fully elucidated.In the present study, we tested the role of CGRP in the effect of anti-hypertension and anti-platelet exerted by rutaecarpine. Since tissue factor (TF) is the most important initiator of intravascular coagulation, the effect of rutaecarpine or CGRP on TF activity was also explored. METHODSIn vivo experimentsSHR were treated with Rut (10> 20 or 40 mg/kg solid dispersant) by intragastric administration and the blood pressure was monitored (tail-cuff method) timely for 18 consecutive days. After experiment, the blood was collected for the measurement of CGRP concentration (radioimmunity), TF concentration (ELISA), TF activity and platelet aggregation (ADP-induced method); and the dorsal root ganglia (DRG) were saved for CGRP mRNA analysis (Real-Time PCR).In vitro experimentsPlatelet and thoracic aorta isolated from healthy Wistar rats were co-incubated in Krebs-Henseleit solution (95%O₂, 5%CO₂, 37℃) to assess the effect of rutaecarpine on platelet aggregation; The isolated platelet was also used to determine the effects of exogenous CGRP on platelet aggregation and the release of TF.RESULTSRutacarpine decreased blood pressure in a dose-dependent manner, concomitantly with the increased levels of plasma CGRP and CGRP mRNA in DRGRutacarpine inhibited ADP-induced platelet aggregation, concomitantly with the decreased TF concentration and activity in plasma. In vitro experiment, rutacarpine was also able to inhibit platelet aggregation which was blocked by VR1 antagonist capsazepine. Exogenous CGRP inhibited platelet aggregation and platelet-derived TF release, which were blocked by CGRP receptor antagonist CGRP (8-37).CONCLUSIONRutacarpine exerts effects on both anti-hypertension and anti-platelet in SHR, which is related to the synthesis and release of CGRP, and the anti-platelet effect of rutacarpine is mediated by CGRP through inhibition of platelet-derived TF release. BACKGROUNDThe results from experimental and clinical studies have shown that the heart suffers severe damage when blood supply returns to the tissue after a long time ischemia. This reperfusion injury could be exacerbated by certain diseases such as hypertension and diabetes. The pathological process of myocardium ischemia is related to alteration of some endogenous substances level and activity. Therefore, to protect the organs or tissues against reperfusion injury, one important avenue is to seek novel drugs which could increase the beneficial substances but decrease the harmful substances to the body.Calcitonin gene-related peptide (CGRP), the predominate neurotransmitter of capsaicin-sensitive sensory nerves, is widely distributed in the cardiovascular system. Besides being as a systemic depressor, CGRP is also considered as a local functional regulator through axon reflex-controlled release. It has been shown that CGRP is an endogenous substance which is able to exert protective effects on the heart. Rutaecarpine (Rut), a major component of Chinese traditional herb Evodia Fruit, has wide and complicate cardiovascular effects. The pharmacological activity of Rut is mediated by endogenous CGRP via activation of VR1..Our previous works has been demonstrated that Rut exerted cardioprotective effect on ischemic myocardium induced by multiple detrimental factors. In the present study, therefore, we explored the correlation between CGRP and exacerbated reperfusion injury by hypertension, and in turn to explore whether Rut is able to exert the protective effects on reperfusion injury by promoting the release of CGRP.METHODSSHR were treated with rutacarpine (20 or 40 mg/kg solid dispersant) by intragastric administration and blood pressure was monitored (tail-cuff method) for 18 consecutive days. At the end of experiment, the blood was collected for measurement of CGRP concentration (radioimmunity). Hearts were isolated and subjected to 20- min global ischemia and 30-min reperfusion; cardiac functions were recorded; coronary flow rate and creatine kinase (CK) activity were measured. Thoracic aortas were separated for determining endothelium-depended relaxation. In this study, WKY and losartan-treated SHR were used as normal and positive drug control, respectively. Thoracic aortas were isolated from healthy Wistar rats and Rut-induced CGRP release was examined after incubation in K-H solution.RESULTSRut decreased blood pressure in dose-dependent manner, concomitantly with the increased plasma CGRP in SHR. The ischemia reperfusion caused a dramatic decrease in myocardial function and coronary flow, and a significant increase in the release of CK. The degree of reperfusion injury in SHR was more severe than that in WKY. Pretreatment with Rut dramatically improved the recovery of cardiac function and coronary flow, and reduced the release of CK. Similar results were observed in Losartan-treated animals.The endothelium-dependent vasodilatation was impaired in SHR, which could be reversed by Rut. Similar results were observed in Losartan group. In vitro, Rut stimulated CGRP release after incubation with thoracic aorta, which was blocked by capsazepine, the VR1 antagonist.CONCLUSIONHypertension exacerbated the reperfusion injury. The protective effect exerted by Rut on reperfusion injury is related to the increased release of CGRP and the improved the vascular endothelium function.