The Effects Of TRPP2-STIM1 Complex Regulating Vascular Tension Via SOCE On High-salt Induced Hypertensive Rats

High-salt diet is a risky factor to induce hypertension. In the early stage of high-salt diet-induced hypertension, rat aortic contraction began to enhance, indicating that abnormal vascular smooth muscle cell(VSMC) contraction was appeared in the early stage of the high-salt diet-induced hypertension. Cytosolic Ca2+ is a key factor to induce VSMC contraction. Therefore, Ca2+ channels play a crucial role in the formation and development of hypertension.Depletion of intracellular Ca2+ stores will activate Ca2+ channels on the cell membrane mediating extracellular Ca2+ influx, which process is so called store-operated Ca2+ entry(SOCE). SOCE is a key pathway to mediate Ca2+ influx in the live cells. In recent years, numerous studies have demonstrated that STIM1 proteins are located in the endoplasmic reticulum(ER) membrane functioning as Ca2+ concentration sensor of Ca2+ store. Calcium store depletion will activate STIM1 and cause STIM1 aggregation(STIM1 puncta) and translocation. After then, STIM1 puncta moves to the plasma membrane(PM) and ER connecting region. These aggregated STIM1 s interact with and activate Orai1 channels evoking SOCE. But the regulatory mechanism and physiological significance of SOCE are not fully understood. To date, the study in this filed is still a hot issue. TRPP2 is a member of transient receptor potential(TRP) channels and can permeate Ca2+. TRPP2 proteins are expressed in the ER membrane and PM. Even both TRPP2 and STIM1 are located in the ER membrane, no evidence is showing whether there is the physical interaction between TRPP2 and STIM1. Moreover, no study was performed to identify the functional role of TRPP2 in the development of the high-salt diet-induced hypertension in the literature.In the present study, we firstly investigated the physical interaction between TRPP2 and STIM1. Secondly, we identified the functional role of TRPP2 in the development of the high-salt diet-induced hypertension:(1) TRPP2 interacts with STIM1 to regulate SOCE and participates in agonist-induced VSMC contraction;(2) change of TRPP2-STIM1 complex expression in VSMCs and its effect on the blood pressure in the high-salt diet-induced hypertension rat. Through this study, we can provide a new target for the treatment of the high-salt diet-induced hypertension and a theoretic evidence for the clinical treatment. 1. TRPP2 interacts with STIM1 to regulate SOCE and participates in agonist-induced VSMC contractionIn the study, we found that(1) in HEK293 cells, co-localization, co-immunoprecipitation and fluorescence resonance energy transfer(FRET) data indicate that TRPP2 and STIM1 physically associate each other in the ER membrane in VSMCs;(2) Suppressing TRPP2 or STIM1 expression via specific siRNA decreased adenosine-triphosphate(ATP)- and thapsigargin(TG)-induced SOCE in HEK293 cells and VSMCs, and STIM1 puncta formation in VSMCs; 3) Suppressing TRPP2 or STIM1 expression via specific siRNA reduced mouse aorta contraction evoked by phenylephrine(Phe) and Endothelin 1(ET-1); 4) Inositol 1,4,5-trisphosphate(IP3) receptor(IP3R) antagonist heparin eliminates TRPP2 or STIM1 siRNA effect on vascular tone. Taken together, these results compulsively suggested that TRPP2-STIM1 complex is crucially responsible for agonist-induced SOCE of VSMCs and blood vessel contraction. In addition, IP3 R has an important role in this process. 2. Change of TRPP2-STIM1 complex expression in VSMCs and its effect on the blood pressure in the high-salt diet-induced hypertension ratHigh-salt diet hypertensive rats were prepared and used to study the pathogenesis of salt-sensitive hypertension. The rats were fed diets containing 4%(w/w) NaCl in the high-salt group and 0.4% NaCl in the regular-salt group for 4 weeks. The blood pressures were monitored by the tail cuff method weekly. After 4 weeks, compared with the control rats, systolic blood pressures of high-salt diet rats were increased significantly. As we all known that increased resistant arterial tension is a key factor affecting blood pressure. Therefore, blood vessel tones of mesenteric artery were examined. In the mesenteric artery, the third branch was used for our study. The results indicate that high-salt intake caused enhanced agonist-induced contraction in the mesenteric artery and thoracic aorta compared with the control rats. TRPP2 and STIM1 expression levels in high-salt diet rat mesenteric artery and aorta were detected by Western blot and immunofluorescence. It was found that TRPP2 and STIM1 expression levels in the mesenteric artery and thoracic aorta were significantly increased in the high-salt intake rats. In order to further investigate the function of TRPP2, we used TRPP2 shRNA lentivirus to infect normal rats. The systolic blood pressure and the agonist-induced contraction in the mesenteric artery and thoracic aorta were significantly decreased in TRPP2 shRNA lentivirus-infected rats. The study also suggests that the expression levels of TRPP2 in the mesenteric artery were significantly higher than those in the thoracic aorta, and the inhibitory effect of TRPP2 shRNA on the agonist-induced contraction in the mesenteric artery was stronger than that in thoracic aorta. Furthermore, the response of agonist-induced contraction was much stronger in the mesenteric artery than in thoracic aorta. Therefore, we conclude that upregulated TRPP2 has a critical role in increased VSMC contraction and blood pressure in high-salt diet-induced hypertension.