Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC350

Poster Communications

Effect of high external potassium on spontaneous calcium oscillations in isolated ICC from the rabbit urethra.

B. J. Drumm1, M. A. Hollywood1, G. P. Sergeant1, K. D. Thornbury1, B. J. Harvey2, N. G. McHale1

1. Smooth Muscle Research Centre, DkIT, Dundalk, Ireland. 2. Molecular Medicine Laboratories, Royal College of Surgeons in Ireland, Dublin, Ireland.

The rabbit urethra contains interstitial cells of Cajal (ICC) which are non-contractile and spontaneously active showing regular propagated intracellular calcium waves1. The frequency of these is modulated by calcium influx across the cell membrane and increased when external potassium concentration is increased. The purpose of the present study is to elucidate the mechanism of this response. Urethra was removed from rabbits killed by lethal injection of pentobarbitone. Strips were dispersed in Ca2+-free solution containing collagenase1. Smooth muscle cells (approx 95%) and ICC (5%) were released. These were loaded with fluo-4/AM and measurements made of intracellular Ca2+ using fast confocal microscopy1. When [K+]o was increased from 5.9 to 60 mM (by replacing NaCl with KCl) frequency of calcium waves in ICC increased from 5.6 ± 0.85 min-1 to 11.65 ± 1.33, mean ± SEM, n=33. It is known, however, that decreasing [Na+]o can of itself increase frequency of spontaneous waves so, to control for this, Na was replaced with equimolar N-methyl D-glucamine (NMDG). Lowering [Na+]o by 60 mM increased wave frequency from 3.35 ± 0.6 to 4.7 ± 0.8 (ns) whereas replacing NMDG with K increased frequency to 10.2 ± 2.6 (p < 0.05, repeated measures ANOVA, n=8). To examine whether this was mediated by activation of voltage-dependent L-type calcium channels, nifedipine 1 µM was added during the K replacement. High [K+]o caused an increase in frequency from 3.9 ± 1.18 to 10.13 ± 2.6, n=6 but this was not significantly reduced by nifedipine (8.2 ± 1.98). When this experiment was repeated with smooth muscle cells the results were quite different. There was a sustained increase in intracellular calcium on raising [K+]o and the cell contracted to 50% of its resting length. Nifedipine (1 µM) reversed the calcium rise and caused the cell to relax. Another possible mediator of the high K response in ICC was the voltage dependent T-type channel and indeed 30 µM NiCl2 (a known blocker of T-channels) reduced the frequency in high [K+]o from 4.96 ± 1.2 to 2.7 ± 0.7, n=4, p < 0.05. However mibefradil 300 nM, a more specific T-channel blocker had no significant effect (11.07 ± 2.59 to 10.2 ± 2.4, n=6). This suggested that nickel might be acting by another mechanism and, since it is known to block NCX (sodium/calcium exchange), the possibility that high [K+]o was acting via this pathway was examined using the reverse mode NCX blocker SEA0400. 1µM SEA decreased frequency in high [K+]o from 16.3 ± 2.98 to 9.0 ± 2.44 n=8, p < 0.05. We conclude that high [K+]o increases firing rate of intracellular calcium waves by increasing calcium influx via reverse mode NCX.

Where applicable, experiments conform with Society ethical requirements