Effect Of High Glucose And High Insulin On Signaling Molecules Of Dilation And Constriction Of Vascular Smooth Muscle Cells

Background and Objective: Disturbance of glucose metabolism and hypertension are intimately associated. According to lots of clinical researches, patients with diabetes are more likely to be complicated with hypertension. On the other hand, approximately half of the patients who suffered from hypertension encounter with IR (insulin resistance). IR is a clinical state that the sensitivity and reactivity of the target cells to insulin decreases. In other words, the biological effect of insulin declines, which is always followed by compensatory hyperinsulinemia in the early stage. As a result, hyperinsulinemia is generally considered to be a compensatory reaction to IR as well as a symbol for it. In the clinic, hyperglucemia and hyperinsulinemia are more likely to coexist when diabetes patients come with hypertension. Therefore, to identify the modulatory effects of hyperglucemia and hyperinsulinemia on vascular functions, especially on the relaxation and contraction of vascular smooth muscle cells, are vital to mechanism research of developing hypertension or antherosclerosis which follows diabetes or IR.At present, NO (nitric oxide) is considered to be the most effective vasodilatory factor known in humans. In human beings, endogenous NO are derived from the reaction of L-arginine and oxygen to L-ornithine, in which NOS (nitric oxide synthase) is the catalyzer. In this reaction, NOS plays a vital role in the production of NO.The GTP-binding protein RhoA and its downstream target molecules ROCK (Rho- associated kinase) are able to inhibit MLCP (myosin light-chain phosphatase) of VSMC. As a result, the activity of MLCK (myosin light-chain kinase) is promoted, resulting in contraction of VSMC. However, the directly determinate factor of VSMC function is the intracellular free Ca2+ concentration ([Ca2+]i),which is closely associated with NOS,RhoA, as well as ROCK. But, it is still largely unknown about the impact of high glucose or high insulin on these molecules nowadays. The aim of this research is to identify the effect of high glucose and high insulin on these signaling molecules of relaxation and contraction of vascular smooth muscle cells. This study makes us more aware of the related molecular mechanisms between disturbance of glucose metabolism and abnormity of vascular function as well as in the field of controlling hypertension which follows diabetes.Methods:1. Culture and group of vascular smooth muscle cells (VSMC).Vascular smooth muscle cells were obtained from thoracic aorta of a healthy male 10-week-old SD rat. The aorta was dissected, carefully freed from connective tissue and endothelium, placed in M199 medium, and then cultured. Cultures were maintained at 37°C in a humidified atmosphere of 95% air/5% CO2. To verify whether the cultured cells were VSMC or not, detection of immunocytochemical localization of smooth muscle-specific alpha actin was done. After identifying, the 4～10-passage cells were divided into the following groups randomly:1) Control, 2)High glucose (HG), 3)High insulin (HI), 4)Normal insulin (NI), 5)High glucose and high insulin (HG+HI), 6)High glucose and normal insulin (HG+NI). When the cells were 90%-100% confluent, glucose and/or insulin were added into the medium as shown above.2. Detection of protein expression of VSMC24 hours later, cellular proteins were extracted and western blotting was taken out to detect protein expression of iNOS,eNOS,RhoA,ROCK-1;3. Detection of intracellular free Ca2+ concentration.Cells were cultured in 6-well plates. When they were 90%-100% confluent, glucose and/or insulin were added into the plates as described above. 30 mins later, the alternation of cytosolic calcium level of VSMC to angiotensin II (AngII) was measured using PTI ratio fluorescence spectrometers.Results:1. The expression of protein iNOS as well as eNOS decreased, while the expression of protein RhoA,ROCK-1 and the concentration of [Ca2+]i were enhanced in HG group;2. In HI group, the expression of iNOS or eNOS was apparently strengthened. On the other side, the intracellular Ca2+ level declined. And the expression of RhoA or ROCK-1 had no changement;3. In NI group, iNOS or eNOS expression was heightened, while the others kept the same with Control group; 4. In HG+HI group or HG+NI group, insulin had the effect against the impact of glucose on vascular smooth muscle cells.Conclusions:1. High glucose stimulation may result in constriction in VSMC via activation of Rho/ROCK pathway and suppression of NOS.2. Insulin, no matter in physiological or pathological concentration, is against to the impairing effect of high glucose on regulating molecules of relaxant and contractive function of VSMC.