The Change Of Store-operated Ca²⁺ Entry In Aged Rat Vascular Smooth Muscle Cells And Its Mechanism Study

ObjectiveTaking thoracic aorta and carotid artery as the research objectives, we explored thechange of store-operated calcium entry (SOCE) and its effect on vessel contraction inaged rat. Moveover, we investigated expression level changes of SOCE components forexample Orai1, STIM1and TRPC1in aged rat. Therefore, we can determine thechange of the store-operated Ca2+entry in aged rat vascular smooth muscle cells and itsmechanism.Method1. Animal grouping: SD male rat, clean level,3-4months: young rat,16-18months:aged rat.2. Preparation of thoracic aortic and carotid arterial rings: I took thoracic aortaimmediately after using CO2to kill rat, then cut it off using surgical scissors and placedit into krebs solution which has already saturated with95%O2and5%CO2. In thesame way, I separated the carotid artery in both sides of the neck.3. Vessel contraction of thoracic aorta and carotid artery: To Use verapamil whichblocks L-type calcium channel specially in plasma membrane to prevent extracellularcalcium influx through L-type channel and add thapsigargin which blocks calciumbump specially in endoplasmic reticulum to deplete the calcium store and to explore thechange of SOCE induced vessel contraction in the thoracic aorta and carotid artery fromyoung and aged rats.4. Immunofluorescence in carotid artery: To use antibodies including anti-Orai1and anti-STIM1respectively to detect the change of Orai1and STIM1protein invascular smooth muscle cells of carotid arteries from young and aged rats.5. Calcium image in thoracic aorta: To use fluorescent dye fluo-8to detect thechange of SOCE induced calcium increase via laser scanning confocal microscope. Toexplore the relationship between contractive function and SOCE channel.6. Western blot in thoracic aorta: To use the antibodies including anti-Orai1,anti-STIM1and anti-TRPC1, respectively to detect the expression of Orai1, STIM1and TRPC1protein in vascular smooth muscle cells of aged and young rats.7. Statistical analysis: All statistical analysis was performed by sigmaplot software.The data among different groups was compared by using the independent t test andvalues are expressed as Mean±SEM.Results1. Vessel tension of carotid artery and thoracic aorta: The results from theexperiments of vessel tension show that60mmol/L high K~+-induced vessel contractionsignificantly increased in aged rat carotid artery compared to that in young ra（tP<0.05).However, the percentage of SOCE induced vessel contraction in high K+-inducedcontractionsignificantly decreased（P<0.05）. The results from the experiments of vesseltension in thoracic aorta show that it did not significantly increased in60mmol/L highK+-induced vessel contraction in aged rat carotid artery compared to that in young rat（P>0.05. The percentage of SOCE-induced vessel contraction in high K+solution-induced contraction significantly decreasedP<0.05）.2. Immunofluorescence in carotid artery: The results from immunofluorescence showthat the expression of Orai1, STIM1proteins is decreased in aged rat vascular smoothmuscle cells compared with that in young rat.3. Calcium imaging: Through the calcium image via laser scanning confocalmicroscope, and the fluorescent dye fluo-8, we detected the change of SOCE induced calcium increase. The results show that SOCE induced calcium increase significantlydecreased in aged rat compared with that in young rat.4. Western blot result in thoracic aorta: The results from western blot show that theexpression of Orai1and TRPC1proteins decreased in VSMC from aged rat comparedwith that from young rat. However, we did not find a significant change of STIM1protein expression level between aged and young rats.Conclusion1. The calcium increase induced by SOCE significantly decreased in VSMC from agedrat carotid artery compared with that from young rat. This is because of decreasedexpression of Orai1and STIM1proteins.2. The calcium increase induced by SOCE significantly decreased in VSMC from agedrat thoracic aorta compared with that from young rat. This is because of decreasedexpression of Orai1and TRPC1proteins.