Mechanism Of Dietary Caffeine And Menthol Regulating Blood Pressure And Vascular Function

Background and Purpose: Hypertension is a global chronic disease. It has been the main casue of stroke, coronary heart disease, heart failure and kidney failure. The incidence of hypertension is still rising year by year. Environmental factors contribute about 60% in the development of hypertension. Epidemiological studies has proved that more than 50% hypertension is salt sensitive hypertension, and cold is also an important environmental factor. Many natural components in food, such as caffeine and menthol, have an effect on blood pressure regulation. Therefore, to explore the effect and mechanism that dietary factors on blood pressure regulation is of great importance for hypertension prevention and treatment.Caffeine(1, 3, 7-trimethylxanthine) is the most commonly used psychoactive agent, which has diuretic and natriuretic effect. However, due to the effect of activating sympathetic nervous system, caffeine has been found to acutely increase blood pressure. Recent studies have found chronic caffeine intake improves metabolic syndrome and lower blood pressure, however, the mechanism of caffeine lower blood pressure is still unclear. High salt intake is an important environmental factor in the occurrence and development of hypertension. Epithelial sodium channel(ENa C) plays a key role in sodium reabsorption and blood pressure regulation. It has been reported that caffeine can influence kinase s regulating ENa C in several kinds of cells, however, it is still unclear whether caffeine influences those kinases in cortical collecting duct cells and subsequently regulating ENa C and sodium reabsorption. It has been rarely reported the effect of chronic caffeine intake on renal sodium reabsorption and blood pressure.Menthol is the main component of mint and peppermint essential oil. Recent studies have found menthol activates the transient receptor potential melastatin 8(TRPM8), which is non-selective cation channel mediates influx of Ca2+, Na+ and K+. It is report that TRPM8 expressed in nerve endings, and also expressed in vascular smooth muscle. Menthol can activate TRPM8 and increase the calcium signal in the vascular smooth muscle cells. However, the molecular mechanism of menthol regulating vascular contraction and relaxation are still unclear. It has been reported that TRPM8 regulating both Ca2+ influx and SR Ca2+ release. Therefore, we hypothesized that activation of TRPM8 by menthol might regulate Ca2+ signaling and related kinases, which inhibits vascular contraction and lower blood pressure.In the present study, we focus on dietary factors, caffeine and menthol regulating blood pressure by two sections: The first section evaluates caffeine on blood pressure regulation. The aim of this section is to observe the effect of chronic caffeine intake on blood pressure in Dahl’s salt sensitive rats fed with high salt diet, to explore the mechanism of chronic caffeine regulating blood pressure in the cellular and molecular levels, and to observe the effect of caffeine on healthy volunteer. The second section observes menthol on blood pressure regulation. The aim of this section is to explore the underlying mechanism of TRPM8 agonist menthol on vasoconstriction and vasodilation,.Materials and methods:The present study includes in vivo and in vitro experiments. In vivo models include Dahl’s salt sensitive rats fed high salt diet with or without caffeine, or wild-type mice and TRPM8 knockout mice fed with or without methol. In vitro models were cortial collecting duct cells(M1CCD) in the caffeine experiment, or vascular smooth muscle cells and mesenteric arteries from WT and TRPM8-/- mice in the menthol experiment.1. Dahl’s salt sensitive rats were randomly divided into two groups:(control group) were given 8% high salt diet and(caffeine group) were given 8% high salt diet + 0.1% caffeine in water intervention. We continuously monitored the blood pressure and locomotor activity of rats by telemetry. And the daily water intake, food intake and body weight were also recorded. After fifteen days of high salt diet with or without caffeine intervention, we measured rats’ fecal and urinary electrolytes. Rats were intraperitoneal injected ENa C specific inhibitors amiloride, thiazide-sensitive Na+/Cl- cotransporter(NCC) inhibitors hydrochlorothiazide, then sodium reabsorption were calculated in both control group and caffeine group.2. Little animals ultrasonic were used to measure the cardiac structure and function in Dahl’s salt sensitive rats after fifteen days of high salt diet with or without caffeine intervention. Then rats were killed and the endothelium-dependent and independent vasodilation and electric stimulation(Electrical field stimulation, EFS) mediated vasoconstriction of mesenteric artery were measured. Ussing Chamber was used to evaluate the effect of caffeine on intestinal electrolytes transport.3. Western blot were used to measure expression of ENa C and ENa C regulating kinases in renal cortical collecting ducts from Dahl’s salt sensitive rats. We observed the effect of caffeine on ENa C activity by in vitro experiments.4. We measured the effect of long-term coffee drinking on 24 h ambulatory blood pressure and 24 hours urinary electrolytes of healthy volunteers.5. We detected activation of TRPM8 by menthol on Rho A/ROCK signaling pathway in vascular smooth muscle cells by western blot. We evaluated the effect of menthol on phasic and tonic components of vasoconstriction in vitro experiments. We observed the effect of removing endothelium on menthol-induced vasodilation.Results:1. Caffeine group has a significant lower systolic blood pressure and mean arterial pressure, but no effects on locomotor activity, heart rate, cardiac structure, plasma catecholamines and vascular function in Dahl-S rat.2. Caffeine group had a significant higher urinary sodium concentration and total 24 h urinary sodium excretion compared with control group in Dahl-S rat after 15 days of intervention.3. After injection of the ENa C specific inhibitors amiloride, the increased urinary sodium excretion in caffeine group significantly lower compared with control group. While after injection of thiazide-sensitive Na+/Cl- cotransporter(NCC) inhibitors hydrochlorothiazide, there is no significant differences in increased urinary sodium excretion between two groups.4. Long-term caffeine intake decreased αENa C expression in cortical collecting ducts of rats, but expression of βENa C, γENa C and NCC had no significant changes.5. Long-term caffeine intake decreased the intestinal sodium hydrogen cotransporter(NHE) mediated sodium water absorption. However, caffeine has no effect on fecal electrolytes.6. Caffeine significantly increased the short circuit current(Isc) of intestinal mucosal epithelium. Inhibiting c AMP abolished caffeine induced Isc increase. Moreover, blocking SR ryanodine receptor and Ca2+ activated K+ channel decreased caffeine induced Isc.7. Caffeine intervention decreased expression of αENa C in cortical collecting duct cells(M1CCD) and its open probability by activation of AMPK.8. Chronic coffee drinking incresed 24 hours urinary sodium and urine output when compared with their baseline levels in healthy volunteers. The systolic and diastolic blood pressure sightly decreased after chronic caffeine intake.9. Menthol activation of TRPM8 inhibited U46619 induced phasic and tonic components of mesenteric vascular contraction. Administration of thapsigargin to deplete internal Ca2+ stores abolished U46619-induced tonic constriction, but had no effect on phasic contraction. The effect of menthol on tonic constriction mimicked the actions of thapsigargin. ROCK inhibitor Y27632 significantly reduced the tonic constriction in mesenteric arteries.10. When the vessel is under relaxation, menthol caused concentration dependent vasoconstriction, though the amplitude of vasoconstriction was small. Menthol induced vasodilation is endothelium independent.Conclusion:1. Chronic caffeine intake reduced high salt-induced increased in systolic blood pressure in Dahl-S rat. Chronic caffeine intake increased 24 h urinary sodium excretion in Dahl-S rat.2. Caffeine activation of AMPK reduced αENa C expression, thus reducing the ENa C mediated sodium reabsorption in renal cortical collecting duct cells.3. Chronic caffeine intake increased 24 h urinary sodium excretion, and sightly decreased blood pressure in healthy individual.4. Menthol inhibited tonic component of vasoconstriction by inhibition of Rho A/ROCK-related calcium signaling. Menthol induced vasodilatation was endothelium independent.