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The role of calcium and actions of calcium antagonists in human colonic smooth muscle

The role of calcium and actions of calcium antagonists in human colonic smooth muscle
The role of calcium and actions of calcium antagonists in human colonic smooth muscle

Macroscopically normal human colon was obtained from patients undergoing colonic resection. In muscle strips suspended under tension, KCl (20-120mM) induced biphasic activity, with peak phasic followed by sustained tonic components. Contracture was abolished in Ca2+-free/EGTA Krebs solution. Nitrendipine, verapamil and diltiazem (10-10-10-6M) caused concentration-dependent inhibition, with no significant discrimination between phasic and tonic KCl responses. Calmodulin antagonists, TMB8 and W7 (10-5-10-3M), also non-selectively suppressed KCl contracture. ACh rapidly stimulated human colonic muscle, inducing recurrent contraction/relaxation spiking over a tonic contraction. Ca2+ antagonists (5x10-6-10-5M) selectively abolished spike activity without reducing sustained tone. BAY K 8644 (10-8-10-6M) caused slow-developing contraction/relaxation spiking, but the effect was abolished at > 5 x 10-6M. Nitrendipine, verapamil and diltiazem (10-7-10-5M) inhibited BAY K 8644 contracture, indicating closely-linked drug receptor sites. [3H]-nitrendipine binding to human colonic membrane fractions was saturable, reversible, and of high affinity. Scatchard analysis of saturation studies gave an average Kd ofg 0.98nM, and Bmax of 161.5fmoles/mg protein, assuming a single dihydropyridine binding site. [3H]-nitrendipine binding, with Ki values of 1.82 x 10-13, 8.59 x 10-13 and 1.40 x 10-8M, respectively. Verapamil showed incomplete displacement and diltiazem caused specific binding enhancement, suggesting allosteric receptor interaction. 45Ca efflux was studied in 20mg muscle pieces, preloaded in 45Ca-Krebs solution (10μCi/ml). Perfusion with non-isotopic buffer (1.0ml/min) resulted in biphasic efflux, with fast extracellular Ca^2+ washout followed by slower removal of intracellular Ca^2+ stores. 80mM KCl stimulated slow ^45Ca efflux, with the response abolished in Ca^2+ free/EGTA or 20mMLa^3+ Krebs solution. Pretreatment with 10^-6M nitrendipine also inhibited KCl-stimulated efflux. Similarly ^45Ca influx, by the lanthanum method, could be stimulated by 60mM KCl and was significantly inhibited by 10^-6M nitrendipine (p < 0.05). Functionally intact, viable smooth muscle cells were successfully isolated from human colonic muscularis by collagenase (1.5mg/ml), hyaluronidase (0.75mg/ml) digestion. Muscle cell yield averaged 1.97 x 10^-5 cells/g wet weight tissue (viability, > 95% purity, 63%). Preliminary measurement of intracellular calcium transients was achieved in Fura-2 loaded, isolated muscle cells, confirming their functional integrity. It is concluded that activator calcium mobilisation, from both extracellular and intracellular sources, is central to the contractile response of human colonic muscle. The contribution of each depends upon the stimulant and phase of mechanical activity studied. Extracellular calcium influx is predominantly via voltage-dependent calcium channels.

University of Southampton
Butler, Deborah Jane
Butler, Deborah Jane

Butler, Deborah Jane (1990) The role of calcium and actions of calcium antagonists in human colonic smooth muscle. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Macroscopically normal human colon was obtained from patients undergoing colonic resection. In muscle strips suspended under tension, KCl (20-120mM) induced biphasic activity, with peak phasic followed by sustained tonic components. Contracture was abolished in Ca2+-free/EGTA Krebs solution. Nitrendipine, verapamil and diltiazem (10-10-10-6M) caused concentration-dependent inhibition, with no significant discrimination between phasic and tonic KCl responses. Calmodulin antagonists, TMB8 and W7 (10-5-10-3M), also non-selectively suppressed KCl contracture. ACh rapidly stimulated human colonic muscle, inducing recurrent contraction/relaxation spiking over a tonic contraction. Ca2+ antagonists (5x10-6-10-5M) selectively abolished spike activity without reducing sustained tone. BAY K 8644 (10-8-10-6M) caused slow-developing contraction/relaxation spiking, but the effect was abolished at > 5 x 10-6M. Nitrendipine, verapamil and diltiazem (10-7-10-5M) inhibited BAY K 8644 contracture, indicating closely-linked drug receptor sites. [3H]-nitrendipine binding to human colonic membrane fractions was saturable, reversible, and of high affinity. Scatchard analysis of saturation studies gave an average Kd ofg 0.98nM, and Bmax of 161.5fmoles/mg protein, assuming a single dihydropyridine binding site. [3H]-nitrendipine binding, with Ki values of 1.82 x 10-13, 8.59 x 10-13 and 1.40 x 10-8M, respectively. Verapamil showed incomplete displacement and diltiazem caused specific binding enhancement, suggesting allosteric receptor interaction. 45Ca efflux was studied in 20mg muscle pieces, preloaded in 45Ca-Krebs solution (10μCi/ml). Perfusion with non-isotopic buffer (1.0ml/min) resulted in biphasic efflux, with fast extracellular Ca^2+ washout followed by slower removal of intracellular Ca^2+ stores. 80mM KCl stimulated slow ^45Ca efflux, with the response abolished in Ca^2+ free/EGTA or 20mMLa^3+ Krebs solution. Pretreatment with 10^-6M nitrendipine also inhibited KCl-stimulated efflux. Similarly ^45Ca influx, by the lanthanum method, could be stimulated by 60mM KCl and was significantly inhibited by 10^-6M nitrendipine (p < 0.05). Functionally intact, viable smooth muscle cells were successfully isolated from human colonic muscularis by collagenase (1.5mg/ml), hyaluronidase (0.75mg/ml) digestion. Muscle cell yield averaged 1.97 x 10^-5 cells/g wet weight tissue (viability, > 95% purity, 63%). Preliminary measurement of intracellular calcium transients was achieved in Fura-2 loaded, isolated muscle cells, confirming their functional integrity. It is concluded that activator calcium mobilisation, from both extracellular and intracellular sources, is central to the contractile response of human colonic muscle. The contribution of each depends upon the stimulant and phase of mechanical activity studied. Extracellular calcium influx is predominantly via voltage-dependent calcium channels.

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Published date: 1990

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Local EPrints ID: 458320
URI: http://eprints.soton.ac.uk/id/eprint/458320
PURE UUID: 6b78a3e4-10f3-4723-837a-51894a5b9135

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Date deposited: 04 Jul 2022 16:46
Last modified: 04 Jul 2022 16:46

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Author: Deborah Jane Butler

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