APStracts 2:0077H, 1995.

Gtp requirement for isoproterenol activation of calcium channels in vascular myocytes. Shi, Qing-Yao, and Robert H. Cox. Bockus Research Institute, The Graduate Hospital, and Department of Physiology, University of Pennsylvania, Philadelphia, PA.

APStracts 2:0077H, 1995.

The effects of activating the [beta]-adrenoceptor pathway on calcium current (ICa) in rabbit portal vein (PV) were studied in myocytes freshly isolated by collagenase and elastase treatment. ICa was measured at room temperature (20 C) using whole cell, voltage clamp methods from a holding potential of -60 mV in cells dialyzed with a pipette solution containing (mM) 100 CsCl, 20 TEACl, 5 NaCl, 5 MgATP, 20 HEPES and 10 BAPTA. The cells were superfused with a solution containing (mM) 140 NaCl, 5 KCl, 1 MgCl2, 5 CaCl2, 10 HEPES and 10 glucose. Only L-type ICa was present in these myocytes averaging 3.5+/-0.3 pA/pF at +10 mV under control conditions. With 0.1 mM GTP added to the pipette solution, 1 [mu]M isoproterenol (ISO) or forskolin (FOR) uniformly increased ICa: ISO by 45+/-5% and FOR by 88+/-11%. This augmentation of ICa was not associated with significant changes in the voltage-dependence of activation or inactivation but was associated with a small increase in the rate of inactivation of ICa. FOR was also associated with an increased rate of ICa activation. The ISO effect was blocked by pretreatment with 1 [mu]M propranolol and reversed by propranolol after ISO exposure. The ICa response to 10[mu]M ISO or FOR was smaller than the response to 1[mu]M with some cells showing a steady-state reduction in ICa. When the latter occurred, the voltage-dependence of availability was shifted to the left by 5+/-0.4 mV. In the absence of pipette GTP, superfusion with drug-free solution was associated with an 18+/-5% decrease in ICa at +10 mV when measured with the same temporal protocols as used with ISO, suggesting ICa rundown. Without [GTP]i, 1 [mu]M ISO produced no significant average change in ICa but the responseto 1[mu]M forskolin was not effected. With 0.1 mM GTP[gamma]S added to the pipette solution, control ICa was greatly augmented but 1 [mu]M ISO had no further effect on ICa. With 1.0 mM GDP[beta]S added to the pipette solution, the initial ICa was not changed and ICa was not significantly affected by 1 [mu]M ISO (+10+/-6%). These results suggest that [beta]-adrenergic activation of portal vein myocytes augments ICa by a mechanism that involves adenylyl cyclase and G-protein activation, and requires intracellular GTP.

Received 1 August 1994; accepted in final form 9 February 1995.
APS Manuscript Number H677-4.
Article publication pending Am. J. Physiol. (Heart Circ. Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 21 March 1995.