| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1992: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1991: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
Isolated parotid acinar cells were perifused in small columns by embedding them in Bio-Gel P-2 beads as an inert supporting matrix, and the effect of carbamylcholine (CCh), substance P, and isoproterenol on the rate of amylase release was examined by measuring amylase activity in the effluent. Amylase release by continuous stimulation with CCh and substance P in the presence of calcium was biphasic. They produced a rapid and large peak (about 10-15 times above resting level) in the rate of amylase release that reached maximum 30 to 60s after the stimulation, followed by a rapid decline to a lower sustained level (about 15-20% of the peak for CCh and 5% for substance P) that was maintained as long as the agonists were present. The effect of CCh was dose-dependent, with the EC_<50> for the peak and plateau being about 8 and 6 muM, respectively. The CCh-induced plateau level was increased by increasing extracellular calcium from 1 to 10 mM, but the magnitude of the initial peak was not. I
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n the absence of calcium, CCh evoked only the initial peak, the magnitude of which decreased if the cells were preincubated in calcium-free medium.
Switching to a calcium-free solution during the CCh-induced plateau rapidly decreased amylase secretion. When calcium was added again to the perifusion solution, a second sharp peak in amylaee secretion was observed with its magnitude being higher than the CCh-induced sustained plateau. The second peak was observed when nickel, cobalt or high potassium was used in place of calcium elimination. CCh evoked biphasic changes in cytosolic free calcium concentration ([Ca^<2+>]_i) in cell suspensions in a stirred cuvette with its plateau being about 40% of the peak height. Removal of calcium during the CCh-induced plateau rapidly decreased [Ca^<2+>]_i to nearly the resting level; restoration of medium calcium restored the sustained plateau, but did not produce the second sharp peak. These results show that CCh evokes biphasic changes in both [Ca^<2+>]_i and amylase release, but amylase release induced by CCh dose not always change in parallel with [Ca^<2+>]_i. The results also show that the second sharp peak in amylase, which was observed in response to restoring calcium to the perifusion medium after continuous stimulation with CCh in the absence of calcium, cannot be explained by the changes in [Ca^<2+>]_i.
Amylase release by continuous stimulation with isoproterenol, on the other hand, developed more slowly with the peak rate being attained at about 6 min after the onset of stimulation. Thus refractoriness was not observed in the effect of isoproterenol. The maximum effect in the rate of amylse release attained by CCh or substance P was higher than that by isoproterenol. The apparent small effect of CCh and substance P on amylase release reported earlier by using batch systems is suggested to be probably due to the rapid development of refractoriness to these secretagogues, but not to isoproterenol. Less
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