Activation Of Thermo-TRPs Are Involved In Regulation Of Vascular Function And Artery Blood Pressure

Background and Purpose:Transient receptor potential (TRP) channel is a non-selective cation channel in the plasma membrane. Ten subtypes are recognized as thermosensitive TRPs (thermo-TRPs), in the sense that they exhibit temperature-sensitive properties and are activated at specific temperatures in the range from noxious heat to painful cold, including TRPA1, C5, M2, M4, M5, M8, V1, V2, V3and V4. Thermo-TRPs are classified into three groups by different temperature thresholds for activation, noxious heat receptor, warm receptor and cold receptor. TRPV1and TRPV2belong to noxious heat receptors and cold receptor comprises TRPA1and TRPM8. Thermo-TRPs expressed in primary sensory neurons, are activated by ambient thermal change, and temperature information is ransmitted to the central nervous system in the form of electrophysiological signals, subsequently elevating sympathetic tone, inducing peripheral vascular contraction or relaxation and facilitating heat production or dissipation. Abnormal expression and function of these channels would lead to hypersensitivity or desensitization of thermosensation. In addition, thermo-TRPs are expressed in vascular tissue and activated by the change of osmotic pressure or stretch stress, the natural or synthetic chemicals and endogenous compounds, which are involved in the regulation of blood vessels physiological functions. Vascular dysfunction promotes the pathogenetic progression of hypertension, coronary heart disease and stroke, including abnormal vascular tone, endothelium-dependent vasodilation disorder, high permeability of endothelial, excessive proliferation and migration of vascular smooth muscle cells(VSMCs), as well as the hyperreactivity for inflammatory mediators, oxidative stress and vasoactive substances. Currently cardiovascular disease remains the primary threat to human health. it is the inevitable passing to improve blood vessel function and effectively control the disease at early stage. Normal calcium metabolism is critically important for virtually every aspect of cell function in the cardiovascular system, and most of thermo-TRPs channels are permeable to calcium ions, which have opened up our understanding of the vascular disease control and prevention.Menthol, transient receptor potential melastatin subtype8(TRPM8) agonists, dilates forearm cutaneous vessels and decreased mean arterial pressure slightly (not statistically significant) in humans upon topical application of menthol to the skin, indicating activation of TRPM8has a vasodilatory effect. A previous study reported that menthol induces Ca2+influx in rat VSMCs required for vasoconstriction, but menthol is shown to inhibited rat tail artery vasoconstriction-induced by agonist. This effect appeared as a small and transient contraction followed by sustained relaxation, and the mechanism is still confused. Another study showed that menthol induces relaxation and inhibits contraction in rat arteries through inhibiting Ca2+influx via nifedipine-sensitive Ca2+channels in VSMCs, but this is inconsistent with the argument that store-operated channels (SOCs) and small Ca2+influx is required for myocyte tonic contraction, as well as calcium sensitization involved Rho/Rho kinase (ROCK) pathway. Cooling-induced contraction of the rat fundus is mediated by activation of TRPM8via a mechanism involving activation of ROCK, indicating that there is a essential link between TRPM8and ROCK pathway. However, peppermint used clinically to relieve intestinal smooth muscle spasm. Thus, we assume that TRPM8activation directly affects the function of SOCs and calcium sensitization involved RhoA/ROCK pathway, which improve vascular function and decrease blood pressure.Noxious heat receptor TRPV1is expressed in ECs, VSMCs and perivascular sensory neure, and involved in regulation of vasomotor function. long-term activation of TRPV1can attenuate the arterial pressure of spontaneous hypertensive rats(SHR). However, it is unclear whether activation of TRPV1is still a protective effect under pathological conditions of diabetes. Diabetes is identified as an independent risk for cardiovascular disease, and hyperglycemia impaired endothelial cells(ECs) function through increasing oxidative stress. Increased reactive oxygen species (ROS) mainly resulted from mitochondria, reduced biological activity of endothelial nitric oxide synthase (eNOS), and reduced NO production. Uncoupling protein2(UCP2) expressed in mitochondrial is a regulatory factor ROS generation and is regulated by c AMP/protein kinase A (PKA). TRPV1activation by dietary capsaicin can activate Ca2+-sensitive PKA, regulate eNOS activity and increase UCP2expression in liver and adipose tissue. Accordingly, we propose that TRPV1 activation by dietary capsaicin might attenuate vascular endothelial injury in diabetic mouse mediated by PKA/UCP2pathway.This present study was divided into two parts. Part one:To discuss the effect of TRPM8activation on vascular function and the mechanism involved in this process. Section1: Protein and mRNA expression of TRPM8in aortic and mesenteric arteries from mice. Section2:Vascular tone and arterial blood pressure in murine after long-term activation of TRPM8by dietary menthol. Section3:Protein expression of RhoA/ROCK pathway after TRPM8activation. The vascular tone of mice mesenteric arteries after administration with vascular activator. Calcium image in vascular endothelial cells with menthol, thapsigargin and U46619(thromboxane A2receptor agonist). Section4:Blood pressure and vascular function in prehypertensive subjects before and after oral menthol capsule intervention. Part two:To discuss the effect of TRPV1activation on vascular function in diabetic mouse and the mechanism involved in this process. After long-term activation of TRPV1by dietary capsaicin, the investigation were performed in diabetic mouse that included arterial blood pressure, vascular reactivity, protein expression of phospho-PKA/UCP2/p22phox/eNOS in ECs of mesenteric arteries.Materials and Methods:This study includes in vivo, in vitro experiments and clinical trial. In vitro experiments were operated on cultured VSMCs from mice mesenteric arteries. In vivo experiments, TRPM8deficient miceCTRPM-/-), wild type (C57/BL6J) littermates and SHRs were assigned into normal forage and forage plus menthol group, intervening28weeks; wild type (C57BL/KsJ) and db/db mice were assigned into normal forage and forage plus capsaicin group, intervening14weeks. Clinical trial is an8-week, randomized, double-blind, placebo-controlled intervention trial. Eligible subjects with prehypertensive subjects were assigned into placebo and menthol groups.1. Expression of TRPM8protein and mRNA in VSMCs from arota in mice were detected by immunoblotting and RT-PCR respectively. Distribution of TRPM8in VSMCs and mesenteric arteries was shown by immunofluorescence.2. Vasorelaxant responses of mesenteric artery from murine after TRPM8activation with menthol was evaluated using isotonic myograph, as well as arota and mesenteric artery from db/db mice.. 3. Ambulatory mean arterial pressure were monitored by implantable radio-telemetry in mice or rat after dietary intervention. Arterial blood pressure was performed using tail-cuff plethysmography.4. Expression of RhoA/ROCK pathway protein and TRPV1/phospho-PKA/UCP2/p22phox/eNOS were detected by immunoblotting.5. Calcium image in VSMCs was detected by fluorescence, with menthol, thapsigargin or U46619(thromboxane A2receptor agonist) intervention.6. Superoxide anion levels and NO levels in mesenteric resistance arteries from db/db mice were assessed by dihydroethidium (DHE) and DAF-2DA fluorescence respectively.7. Office blood pressure was measured by conventional sphygmomanometric methods every two weeks. Ambulatory blood pressure monitoring was recorded using Spacelabs90217monitor before and after intervention.8. Vasodilation of prehypertensive subjects was determined using high-frequency ultrasound probe before and after intervention.Major Research Results:1. TRPM8mRNA and protein expression were detected in both cultured VSMCs and freshly isolated aortas from WT mice.TRPM8expression was also detected clearly by immunofluorescence both in the cultured VSMCs and in the mesenteric arterial medial layers from WT.2. Chronic dietary menthol treatment significantly reduced U46619-induced vasoconstriction and lowered24-hour ambulatory arterial pressure in WT mice and SHR, but not in TRPM8-/-mice.3. Acute or chronic activation by menthol also inhibited Rho A, ROCK-2, and p-MYPT-1expression in mesenteric arteries from WT mice and SHR, but not in TRPM8-/-mice.4. TRPM8activation by menthol attenuated both tonic and phasic contractile induced by U46619in concentration-dependent manner, and these effects were inhibited by AMTB (TRPM8antagonist). After calcium store depleted, the menthol completely inhibited U46619-induced calcium influx in VSMCs from WT, but not in TRPM8-/-mice.5. Menthol capsules lower blood pressure and ambulatory diastolic blood pressure in prehypertensive participants. In addition, menthol capsules improve flow-mediated vasodilation and nitroglycerin-mediated vasodilation.6. TRPV1activation by dietary capsaicin promotes endothelial PKA phosphorylation and increases UCP2levels, attenuates endothelial oxidative stress and increases NO levels in diabetic mice.7.Chow plus capsaicin diet ameliorates endothelium-dependent relaxation in db/db mice.Conclusion:1. These results suggest that dietary menthol attenuates vasoconstriction and lowers high blood pressure in murine by inhibiting the RhoA/ROCK pathway in a TRPM8-dependent manner. The inhibitory effect of menthol on mesenteric artery constriction was associated with menthol reducing U46619-induced Ca2+influx in TRPM8-dependent manner.2.Menthol capsules administration favorably lowered blood pressure and improved vasodilation in human prehypertension.3. Prolonged administration of dietary capsaicin promotes endothelium-dependent relaxation in diabetic mice. The mechanism is that TRPV1activation by dietary capsaicin ameliorates vascular oxidative stress and increases NO levels through promoting endothelial PKA phosphorylation and increasing the expression level of UCP2.