The Effects Of The Activity Changes Of Large Conductance Ca²⁺-activeted K⁺ Channels In Vascular Smooth Muscle Cells On The Expression Of P16 And P21

Objective: To investigate the effects of the activity changes of large conductance Ca2+-activeted K+ channels in vascular smooth muscle cells on the expression of p16 and p21 so as to explore the mechanism of large conductance Ca2+-activeted K+ channels during vascular aging. Methods: (1) Experimental animals: second order healthy male Sprague-Dawley(SD) rats,①young group: n= 12②elderly group: n= 12. The rat VSMC was primary cultured by enzyme digestion method and was detected SM-α-actinin for identification by immunocytochemical staining. The 2nd passage VSMCs were applied for experiments and were divided into 3 group, NS1619,IBTX and control groups. (2) To observe the effects of the activity changes of BKCa in vascular smooth muscle cells from different ages on vascular aging by beta galactosidase staining method. (3) To analyze the expression levels of p16 and p21 of young and elder VSMC primary cultured with DMEM/F12 containing with BKCa activator and blocker by RT-PCR. Results: (1) Under contrast view microscope, cells are shaped like long shuttle, growing like typical"peak and vallige". Immunocytochemical staining shows yellow in cytoplasam and VSMCs accout for above 98%. (2)Results of the expression of beta galactosidase staining: Compared with those in control group, whatever the rat's age is, the number of senile VSMC containing with BKCa activator decreased, while the cell activity containing with BKCa blocker increased(P<0.01). Whatever the activities of BKCa channels are, the percentage of beta galactosidase staining positive VSMC from elder rats higher than those from young rats(P<0.01). At the same time, there exists interaction between the factors of age and BKCa activity on the effect of senile VSMC (P<0.01), which make the effect of the activity changes of BKCa in VSMC on the elder rats are more significant than young rats. (3) There was a weak mRNA expression of p16 in VSMC primary cultured with BKCa activator NS1619. However,an obvious expression of p16 Was seen in the control senescent cells. On the contrary, there was a more obvious expression of p16 in IBTX group than the control group(P<0.05). Otherwise, there was a more obvious expression of p21 in elder group than young group(P<0.05). Additionally,no difference of p21 level between the four group cultured VSMC cells was seen(P>0.05). Conclusion: (1)NS1619 significantly decrease the number of the senile VSMC, on the contrary, IBTX increase the number of the senile VSMC。The activity of BKCa decreased with vascular aging, and low activity of BKCa make vascular aging more serious. (2)There was no effect of activity changes of BKCa on the expression of p21. But NS1619 could strongly inhibit p16 expression of VSMC,IBTX could increase p16 expression of VSMC ,which may contribute to the pathogenesis of cardiovascular diseases during aging. (3) whatever the activities of BKCa channels are, the percentage of beta galactosidase staining positive VSMC from elder rats higher than those from young rats, the levels of both p16 and p21 mRNA expression in elder group higher than those in young group. It indicates that aging itself is independent risk factor in the process of vascular aging. Besides, there exists interaction between the factors of age and BKCa activity on the effect of senile VSMC, which make the effect of the activity changes of BKCa in VSMC on the elder rats are more significant than that of young rats, it could be concerned with elder cell which provide a tendency toward senescence. It indicates that change of BKCa during aging probably is the pathogenesis of VSMC senescence.