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Differential effects of lysyl bradykinin and thapsigargin on epithelial chloride secretion
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Differentia] effects of lysyl bradykinin and thapsigargin on epitheli.,d chloride secretion Cuthbert, A.W. and Pickles, R.J.
Department of Pharmacology, University of Cambridge, TenniA Court Road, Cambridge CB2 1QJ, I~K Chloride secretion in epithelia requires that the chloride ion is accumulated within the cell, by carrier mediated transport through the basolateral membrane, followed by exit through chloride channels in the apical membrane. Processes at both membr&nes can be affected by the intracellular mediators cyclic ANP and c',dcium. Two agents causing epithelial chloride secretion, namely lysylbradykinin (LBk) and thapsigargin (Tg) have been investigated. The former acts directly on membrane receptors (Cuthbert and Spayne, 1982) while the latter bypasses the membrane to release intracellular calcium stores (Brayden et al., 1989). Four separate, human colonic epithelia all derived from the same adenocarcinoma have been utilised. They are designated HCA-7, Coiony 1, Colony 3 and Colony 29 (Cuthbert et al., 1987). Chloride secretion was measured as short circuit current (SCC) in cultured monolayers, while intracellular calcium ion concentration was monitored by ~ura-2 fluorescence in cell suspensions. The fable shows the increase in SCC (/tA/cm 2) and Cai (nM) following LBk (. 1 pM) and Tg (0.17/tM). All values are the means of between 6 and 30 measurements. LBK HCA-7 Colony I Colony 3 Colony 29
SSC 46.4 10.0 0.0 64.2
Tg Ca i 95 0 0 345
SCC 17.6 2.4 3.6 10.1
Ca i 214 200 120 207
Chloride secretion was accompanied by a measureable rise in Cai, except in Colony I cells in response to LBk, but there was no simple relationship between secretion and the increase in Ca i. While forskolin, which increases cAMP, produced a marked potentiation of the responses to LBk it had no effect on the calcium signal generated by the peptide. The rate of the rise of Ca i (as the time taken to reach 50% of the maximal) was faster with LBk than Tg (15 sec. and 30 sec. for LBk and Tg respectively in HCA-7). The discrepancy was even greater when the rate of increase of chloride secretion was measured. For example, in Colony 29 cells, the SCC reached 50% of its maximal value 33 sec. after LBk while the corresponding time for Tg was 285 sec. Tg seems to be a useful agent for elucidating the roie of calcium in secretion.
References Brayden, D.J., Hanley, M.R., Thastrup, O. and Cuthbert, A.W. (1989) Br. J. Pharmacoi. 98, 809. Cuthbert, A.W. and $payne, J.A. (1982) Br. J. Phaxmacol. 76, 33. Cuthbert, A.W., Egleme, C., Greenwood, H., Hickman, M.E., Kit'Hand, S.C. and MacVinish, L.J. (1987) Br. J. Pharmacol. 91, 503.
Differentia] effects of lysyl bradykinin and thapsigargin on epitheli.,d chloride secretion Cuthbert, A.W. and Pickles, R.J.
Department of Pharmacology, University of Cambridge, TenniA Court Road, Cambridge CB2 1QJ, I~K Chloride secretion in epithelia requires that the chloride ion is accumulated within the cell, by carrier mediated transport through the basolateral membrane, followed by exit through chloride channels in the apical membrane. Processes at both membr&nes can be affected by the intracellular mediators cyclic ANP and c',dcium. Two agents causing epithelial chloride secretion, namely lysylbradykinin (LBk) and thapsigargin (Tg) have been investigated. The former acts directly on membrane receptors (Cuthbert and Spayne, 1982) while the latter bypasses the membrane to release intracellular calcium stores (Brayden et al., 1989). Four separate, human colonic epithelia all derived from the same adenocarcinoma have been utilised. They are designated HCA-7, Coiony 1, Colony 3 and Colony 29 (Cuthbert et al., 1987). Chloride secretion was measured as short circuit current (SCC) in cultured monolayers, while intracellular calcium ion concentration was monitored by ~ura-2 fluorescence in cell suspensions. The fable shows the increase in SCC (/tA/cm 2) and Cai (nM) following LBk (. 1 pM) and Tg (0.17/tM). All values are the means of between 6 and 30 measurements. LBK HCA-7 Colony I Colony 3 Colony 29
SSC 46.4 10.0 0.0 64.2
Tg Ca i 95 0 0 345
SCC 17.6 2.4 3.6 10.1
Ca i 214 200 120 207
Chloride secretion was accompanied by a measureable rise in Cai, except in Colony I cells in response to LBk, but there was no simple relationship between secretion and the increase in Ca i. While forskolin, which increases cAMP, produced a marked potentiation of the responses to LBk it had no effect on the calcium signal generated by the peptide. The rate of the rise of Ca i (as the time taken to reach 50% of the maximal) was faster with LBk than Tg (15 sec. and 30 sec. for LBk and Tg respectively in HCA-7). The discrepancy was even greater when the rate of increase of chloride secretion was measured. For example, in Colony 29 cells, the SCC reached 50% of its maximal value 33 sec. after LBk while the corresponding time for Tg was 285 sec. Tg seems to be a useful agent for elucidating the roie of calcium in secretion.
References Brayden, D.J., Hanley, M.R., Thastrup, O. and Cuthbert, A.W. (1989) Br. J. Pharmacoi. 98, 809. Cuthbert, A.W. and $payne, J.A. (1982) Br. J. Phaxmacol. 76, 33. Cuthbert, A.W., Egleme, C., Greenwood, H., Hickman, M.E., Kit'Hand, S.C. and MacVinish, L.J. (1987) Br. J. Pharmacol. 91, 503.