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 IP3-dependent sites in the sarcoplasmic reticulum play an important role in the contractile response of the rabbit urinary bladder to bot h field stimulation (mediated via neurotransmitter release) and bethan echol (direct muscarinic stimulation). In view of the importance of SA RCO (see text) in urinary bladder smooth muscle function, we studied t he distribution of SERCA by two methods; using Western blotting to qua ntitate the protein concentration and by enzyme analysis using thapsig argin to specifically inhibit SERCA. Rabbit and human samples of urina ry bladder smooth muscle were homogenized and the homogenate separated into three particulate fractions by differential centrifugation: the cell wall-nuclear, mitochondrial, and microsomal. The protein concentr ation of these three particulate fractions was determined and the SERC A protein level quantitated by Western blotting using SERCA-2 antibodi es. The calcium ATPase activity was quantitated using standard enzymat ic analysis and the thapsigargin sensitivity determined. The results d emonstrated that (1) the concentration of SERCA was significantly grea ter in the microsomal fraction than in either of the other fractions f or both rabbit and human bladder smooth muscle; (2) the enzymatic acti vities of both total calcium-activated ATPase and thapsigargin-sensiti ve calcium ATPase were evenly divided among the three fractions, and ( 3) the enzymatic activity of both total calcium-activated ATPase and t hapsigargin-sensitive calcium ATPase of the rabbit exceeded that of th e human. In conclusion, the distribution of SERCA and calcium ATPase o f the rabbit bladder smooth muscle was similar to that in the human bl adder smooth muscle, although activities in rabbit were significantly greater than those of human tissue.