Subcellular distribution of SERCA and calcium-activated ATPase in rabbit and human urinary bladder smooth muscle.

Abstract

Previous studies have demonstrated that calcium storage and release from IP-3-dependent sites in the sarcoplasmic reticulum play an important role in the contractile response of the rabbit urinary bladder to both field stimulation (mediated via neurotransmitter release) and bethanechol (direct muscarinic stimulation). In view of the importance of SERCA in urinary bladder smooth muscle function, we studied the distribution of SERCA by two methods: using Western blotting to quantitate the protein concentration and by enzyme analysis using thapsigargin to specifically inhibit SERCA. Rabbit and human samples of urinary bladder smooth muscle were homogenized and the homogenate separated into three particulate fractions by differential centrifugation: nuclear-cell wall, mitochondrial, and microsomal. The protein concentration of these three particulate fractions was determined and the SERCA protein level quantitated by Western blotting using SERCA-2 antibodies. The calcium-ATPase activity was quantitated using standard enzymatic analysis and the thapsigargin sensitivity determined. The results demonstrated that: (1) the concentration of SERCA was significantly greater in the microsomal 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 ATPase were evenly divided among the three fractions, and (3) the enzymatic activity of both total calcium-activated ATPase and thapsigargin-sensitive calcium ATPase of the rabbit exceeded that of the human. In conclusion, the distribution of SERCA and calcium-ATPase of the rabbit bladder smooth muscle was similar to that in the human bladder smooth muscle, although activities in rabbit were significantly greater than those of human tissue.