SUBCELLULAR-DISTRIBUTION OF SERCA AND CALCIUM-ACTIVATED ATPASE IN RABBIT AND HUMAN URINARY-BLADDER SMOOTH-MUSCLE

Previous studies have demonstrated that calcium storage and release fr om IP-3-dependent sites in the sarcoplasmic reticulum play an importan t role in the contractile response of the rabbit urinary bladder to bo th field stimulation (mediated via neurotransmitter release) and betha nechol (direct muscarinic stimulation). In view of the importance of S ERCA in urinary bladder smooth muscle function, we studied the distrib ution of SERCA by two methods: using Western blotting to quantitate th e protein concentration and by enzyme analysis using thapsigargin to s pecifically inhibit SERCA. Rabbit and human samples of urinary bladder smooth muscle were homogenized and the homogenate separated into thre e particulate fractions by differential centrifugation: nuclear-cell w all, mitochondrial, and microsomal. The protein concentration of these three particulate fractions was determined and the SERCA protein leve l quantitated by Western blotting using SERCA-2 antibodies. The calciu m-ATPase activity was quantitated using standard enzymatic analysis an d the thapsigargin sensitivity determined. The results demonstrated th at: (1)the concentration of SERCA was significantly greater in the mic rosomal fraction than in either of the other fractions for both rabbit and human bladder smooth muscle; (2) the enzymatic activities of both total calcium-activated ATPase and thapsigargin-sensitive calcium ATP ase were evenly divided among the three fractions? and (3) the enzymat ic activity of both total calcium-activated ATPase and thapsigargin-se nsitive calcium ATPase of the rabbit exceeded that of the human. In co nclusion, the distribution of SERCA and calcium-ATPase of the rabbit b ladder smooth muscle was similar to that in the human bladder smooth m uscle, although activities in rabbit were significantly greater than t hose of human tissue.