Impact Of Chronic Fetal Exposure To Caffeine On Resistance Vessel And Cellular Functions Via RyRs-Bk_Ca Down-Regulation In Rat Offspring

Background: Increasing evidence indicates that prenatal insults may increase the susceptibility to cardiovascular disease in adulthood. Caffeine, a xanthine alkaloid, widely used in form of coffee or tea, is consumed by 68-74% of pregnant women at an average intake of 125-190 mg/day. Caffeine can directly affect the contraction of myocytes and affect cardiovascular function. More and more attention have been paid to the effect of maternal caffeine exposure on cardiovascular functions in the adult offspring. However, there is limited information underlying the vascular mechanism. Few studies have addressed the "programming" in offspring after birth to adulthood during cardiovascular development, especially research involving ion channel mechanisms have not been reported. Multiple approaches are to be used in our research including patch-clamp, fluorescence microscope, synchronous dynamic monitoring of micro-arterial diameter and intravascular calcium change,etc. This project proposes that the intracellular calcium signaling and its coupling to BKCa channel underlies the imbalanced regulation of membrane potential, which therefore affect vascular tone and blood pressure. Part 1 The effect of prenatal caffeine exposure on fetal development and adult offspring bodyweight Objective: To determine the effect of prenatal caffeine exposure on fetal development and adult offspring bodyweight. Methods: Saline(control group:16 mothers) or caffeine(caffeine group: 16 mothers; 20 mg/kg/per dose; twice a day) was subcutaneously injected daily to pregnant mothers from GD 3.5 to 19.5. On GD20.5, after cesarean section delivery, fetal body weight, stem length and tail length was measured. All fetal rats/each group were calculated for in utero growth restriction(IUGR) rate using the reported criteria(IUGR was diagnosed when the body weight of each individual animal from the treated group was two standard deviations less than the mean body weight of the control group).Natural delivery was allowed for other pregnant rats. The male offspring(35~40 form 7~8 mothers/each group) were studied at 5-mont-old. Results: Prenatal caffeine(20mg/kg, twice/day) significantly decreased fetal body weight to 86.73% compared to the control, with 25.1% of intrauterine growth restriction(IUGR). However, there were no significant differences in body weight in the adult offspring at 5-month-old. Conclusion: Prenatal caffeine exposure may lead to intrauterine growth restriction. There is no significant differences in adult offspring body weight(male) between the two groups.Part 2 The effect of prenatal caffeine exposure on Phenylephrine-increased pressor responses, vessel contractions, and SMC depolarization in offspring Objective: To determine the effect of prenatal caffeine exposure on Phenylephrine-increased pressor responses, vessel contractions, and SMC depolarization in the offspring. Method: Polyethylene catheters were respectively placed into the femoral vein and artery of offspring,blood pressure, including systolic pressure(SP),diatolic pressure(DP)and mean arterial pressure(MAP),as well as heart rates were monitored by a Power-Lab Physiological Recorder in free moving rats.Third-order mesenteric arterial branches were isolated microscopically from offspring rats.Vascular tone was measured. Single mesenteric arterial SMCs were isolated, Membrane potential was measured using patch clamp technique. Result: 1) Prenatal caffeine caused significantly higher MAPstimulated by phenylephrine in the offspring; 2) PE-induced MA constriction was significantly enhanced with or without L-NNA in caffeine offspring; 3) PE was more effective in depolarizing the myocytes in the caffeine offspring. Conclusion: Membrane depolarization might be involved in underlying mechanisms for over re-activity of arterioles in the offspring exposed to prenatal caffeine.Part 3 The effect of prenatal caffeine exposure on Ca2+ signaling that contributes to regulation of membrane potential in offspring Objective:To determine the effect of prenatal caffeine exposure on Ca2+ signalingthat contributes to regulation of membrane potential. Methods: Ca2+ current was evaluated using Ba2+ as the charge carrier by eliminating K+current from both intracellular and extracellular solutions, and recorded by conventional whole-cell voltage-clamp configuration. Synchronous dynamic monitoring of micro-arterial diameter and intravascular calcium change was used for testing the activity of ryanodine sensitive Ca2+ release in SR. In addition,protein abundance of LTCCs channels α1c subunits were examined in MAs.The expression level of m RNA and protein of Ry R1, Ry R2 and Ry R3 were investigated. Caffeine was used for detecting the ryanodine sensitive Ca2+ store in the two groups. Results: 1) Reduced LTCCs currents and α1c protein expression in the MA myocytes in the caffeine offspring; 2) Decreased functional and molecular Ry Rs in the MA myocytes. Conclusion: Prenatal caffeine exposure decreased the protein expression of α1c subunit in LTCCs; There were functional and molecular downregulation of ryanodine sensitive Ca2+ release channels.Part 4 The effect of prenatal caffeine exposure on activity of MA BKCa channel in offspring Objective: To determine the effect of prenatal caffeine exposure on activity of MA BKCa channel in offspring. Method: Using patch clamp technique to investigated the biophysical properties, whole-cell currents and spontaneous outward currents(STOCs) of BKCa channels. Compared the protein expression of α-subunit and β1-subunit by western blotting. Results: 1) The expression of BKCa β1 subunits was reduced by 29.30% in the caffeine group, while α subunits were unchanged; 2) The calcium/voltage sensitivity of BKCa channel was reduced by prenatal exposure to caffeine as well as the stability of the channel kenetics. Conclusion: The degraded BKCa biophysical properties accompanied with down-regulated BKCa β1 subunits were accounted for the downregulation of STOCs.