Tyrosine kinase-dependent modulation of calcium entry in rabbit colonic muscularis mucosae

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 a holding potential of -60 mV produced inward currents that were abolished by 1 microM 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 at concentrations of up to 100 microM. Conversely, the tyrosine phosphatase inhibitor, orthovanadate, enhanced peak Ca2+ currents by 30%. Spontaneous transient outward 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 extracellular Ca2+ concentration, 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 Ca(2+)-free media followed by reperfusing with a Ca(2+)-containing solution for 3-5 min. 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 anti-phosphotyrosine antibody, showed a tyrosine phosphorylated protein of 60 kDa in control conditions. This was markedly increased after treatment with epidermal growth factor and carbachol. These results suggest that 1) tyrosine kinase modulates the entry of Ca2+ through L-type channels and through nifedipine-resistant pathways 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.