Tyrosine kinase dependent modulation of calcium entry in rabbit
colonic muscularis mucosae.
Hatakeyama, N., D. Mukhopadhyay, R. K. Goyal, and H. I. Akbarali.
Department of Gastroenterology and Nephrology, Beth Israel Hospital
and Harvard Medical School, 330 Brookline Ave, Boston, MA 02215
APStracts 2:0453C, 1995.
We studied the role of tyrosine kinase in the regulation of Ca2+ entry
in single smooth muscle cells of the rabbit colonic muscularis
mucosae using the whole-cell patch clamp technique. Step
depolarization to +10 mV from holding potential of -60 mV , produced
inward currents that were abolished by 1[mu]M nifedipine, consistent
with the activation of L-type Ca2+ channels. The tyrosine kinase
inhibitors, genistein and tyrphostin B42 dose-dependently inhibited
these Ca2+ currents. The inactive analogue of tyrphostins, tyrphostin
A1, did not affect the currents up to concentrations of 100 [mu]M.
Conversely, the tyrosine phosphatase inhibitor, orthovanadate,
enhanced peak Ca2+ currents by 30%. Spontaneous transient outward K+
currents (STOCS) (50 - 600 pA) were elicited with high K+ in the
pipette and at 0 mV holding potential. STOCS were activated due to
release of Ca2+ from intracellular stores, required the presence of
[Ca2+]o and were insensitive to nifedipine. Genistein abolished
STOCS, however, in its presence outward currents activated by
caffeine or carbachol were not affected. The refilling of the Ca2+
stores was studied by first depleting intracellular Ca2+ with
carbachol in Ca2+-free media followed by reperfusion with a Ca2+
containing solution for 3-5 mins. Under these conditions, a second
application of carbachol evoked an outward current due to Ca2+
release. However, this effect was abolished when the refilling of the
stores was carried out in the presence of genistein. Carbachol-evoked
currents were not attenuated when the refilling was examined in the
presence of orthovanadate. Epidermal Growth Factor (200 ng/ml)
enhanced Ca2+ currents by 60% and markedly increased STOCS by over
200%. Western blot analysis using an antiphosphotyrosine antibody
showed a tyrosine phosphorylated protein of 60 kDa in control
conditions. This was markedly increased on treatment with EGF and
carbachol. These results suggest that 1) tyrosine kinase modulates
the entry of Ca2+ through L-type channels and through nifedipine
-resistant pathway involved in refilling of intracellular stores and
2) stimulation of the kinase by agonists enhances Ca2+ entry in the
smooth muscle cells of the rabbit colonic muscularis mucosae
Received 8 September 1995; accepted in final form 12 December
1995.
APS Manuscript Number C546-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 23 December 95