Activation Of TRPV1 By Dietary Capsaicin Improves Endothelium-Dependent Vasorelaxation And Prevents Hypertension

Background and Objectives: Hypertension is currently one of the most common diseases in the world. Its main complications include stroke, myocardial infarction and renal failure, which are associated with substantial morbidity and mortality. The pathogenesis of primary hypertension is due to both the genetic susceptibility and environmental factors. However, the detail underlying mechanism has not been clarified. Epidemiologic studies demonstrated that arterial blood pressure can be regulated by a number of dietary factors. Over the past decade, reduced salt diet, increased potassium intake and consumption of fruits and vegetables based on the"DASH diet"have emerged as effective strategies for lowering blood pressure. Capsaicin is a major pungent ingredient in red pepper and is used as a food additive. Capsaicin is a highly selective agonist for the transient receptor potential vanilloid receptor 1 (TRPV1). TRPV1 as a nonselective cation channel is expressed in sensory neurons and brain but is although present in various non-neuronal tissues including the vasculature. The effect of capsaicin on vascular tone and blood pressure is somewhat of a paradox. Acute, short-term administration of capsaicin either increases or lowers blood pressure transiently in human and rodents. However, the long-term effect of TRPV1 activation on blood pressure and vascular reactivity were unknown. Our previous study demonstrated that activation of TRPV1 with capsaicin can reduce the adipogenesis of 3T3-L1 preadipocytes and prevent high fat diet induced obesity in mouse through increasing calcium entry. Considering endothelial nitric oxide synthase and PKA implicated in the regulation of blood pressure is dependant on calcium signal, we hypothesized that long-term activation of TRPV1 would improve vascular reactivity and prevent hypertension through PKA/eNOS/NO pathway.To test the above hypothesis, the present study was divided into three parts. Part one: Protein and mRNA expression and the distribution of TRPV1 in vascular endothelial cells and mesenteric arteries from mice and spontaneous hypertensive rats were evaluated by immunoblotting, RT-PCR and immunofluorescence, respectively. Part two, section 1: The vasodilation response of mice mesenteric arteries to acute administration with capsaicin was detected. Part two, section 2: Arterial blood pressure and vascular reactivity in normotensive mice (TRPV1 null mice and wild type littermates) after long-term activation of TRPV1 by oral administration with capsaicin were evaluated. Part two, section 3: Arterial blood pressure and vascular reactivity in spontaneous hypertensive rats after long-term activation of TRPV1 by oral administration with capsaicin were identified. Part two, section 4: The effect of long-term TRPV1 activation by oral administration with capsaicin on the endocannabinoid system. Part three: Calcium image in vascular endothelial cells with capsaicin intervention was detected to confirm the functional expression of TRPV1 channel. Nitric oxide bioavailability, total and phospho-PKA/eNOS were evaluated to identify the underlying molecular mechanisms involved in the effects of TRPV1 on the regulation of arterial blood pressure and vascular reactivity.Material and methods: The whole research includes in vitro and in vivo experiments. In vitro tests were conducted on cultured vascular endothelial cells from mice thoracic aorta and mesenteric arteries. When investigating in vivo, TRPV1 transgenic mice, TRPV1 deficient mice and wild type littermates as well as spontaneous hypertensive rats were assigned into normal chow diet and chow plus capsaicin diet group.1. TRPV1 protein and mRNA expression in vascular endothelial cells and mesenteric arteries were detected using RT-PCR and immunoblotting, respectively. While the distributions of TRPV1 in vascular endothelial cells and mesenteric arteries were evaluated by immunofluorescence.2. Vascular reactivity of mesenteric artery from mice and rats after acute or long-term activation with capsaicin was identified using isotonic myograph.3. Arterial blood pressure was obtained using tail-cuff plethysmography every month; Invasive mean blood pressure from carotid artery response to intravenous acetylcholine and nitroglycerine was evaluated; Arterial blood pressure of rats after oral administration was detected by telemetry.4. Calcium image in vascular endothelial cells from TRPV1 deficient mice and wild type littermates with capsaicin intervention in the presence and absence of TRPV1 specific blocker capsazepine was detected by fluorescence.5. Protein expressions of PKA, phospho-PKA, eNOS and phospho-eNOS in vascular endothelial cells and mesenteric arteries were detected by immunoblotting.6. NO bioavailability in vascular endothelial cells and mesenteric arteries from TRPV1 deficient mice, TRPV1 transgenic mice and wild type littermates with capsaicin intervention in the presence or absence of TRPV1 specific blocker, eNOS inhibitor or PKA inhibitor was detected by fluorescence.Results:1. Protein and mRNA expressions of TRPV1 were detected in vascular endothelial cells and mesenteric arteries from mice and spontaneous hypertensive rats.2. Acute activation of TRPV1 caused concentration-dependent vasodilation in PE-induced precontraction of mice mesenteric arterial rings. TRPV1 gene knockout, endothelium denudation, eNOS inhibitor or PKA inhibitor significantly attenuated the vasodilation response induced by TRPV1 activation. Long-term activation of TRPV1 significantly improved endothelium-dependent vasodilation in mesenteric resistance artery, and markedly blunted angiotensin II-induced vasocontraction in mouse aorta.3. Long-term activation of TRPV1 or TRPV1 overexpression markedly enhanced acetylcholine-induced hypotensive response; moreover, long-term activation of TRPV1 can reduce the arterial blood pressure of spontaneous hypertensive rats.4. Capsaicin dose-dependently increased intracellular free calcium concentration in vascular endothelial cells, while this effect can be inhibited in TRPV1-deficient mice or in the presence of capsazepine.5. After intervention with capsaicin, the protein expression of phospho-PKA and phospho-eNOS were significantly upregulated in endothelial cells from wild type mice, but not from TRPV1 null mice. After long-term activation of TRPV1, the protein expression of phospho-PKA and phospho-eNOS were markedly upregulated in mesenteric arteries from wild type mice and spontaneous hypertensive rats, but not from TRPV1 mutant mice.6. Intervention with capsaicin significantly increased the NO bioavailability, while TRPV1 blocker, eNOS inhibitor or PKA inhibitor markedly attenuated these effects. Similarly, capsaicin markedly increased the NO production in a dose-dependent manner, while TRPV1 blocker or eNOS inhibitor significantly blunted these effects. Capsaicin induced highest amount of NO in mesenteric artery from TRPV1 transgenic mice, wild type mice took the second place, while TRPV1 null mice was the lowest one.Conclusion:1. TRPV1 was expressed in mice vascular endothelial cells and mesenteric arteries from mice and spontaneous hypertensive rats.2. Acute or long-term activation of TRPV1 would improve endothelium-dependent vasodilation in resistance arteries, and reduce arterial blood pressure of spontaneous hypertensive rats.3. Activation of TRPV1 can increase intracellular calcium concentration in vascular endothelial cells, upregulate the protein expression of phospho-PKA and phospho-eNOS, and increase the bioavailability of nitric oxide in vascular endothelial cells and mesenteric arteries. The present study suggest that long-term activation of TRPV1 induced nitric oxide increasing mediated by cAMP/PKA-eNOS signal pathway would be a underlying mechanism for improving vascular function and preventing hypertension.