GTP requirement for isoproterenol activation of calcium channels in vascular myocytes

Abstract

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 degrees 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 tetraethylammonium chloride, 5 NaCl, 5 MgATP, 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (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 guanosine 5'-triphosphate (GTP) added to the pipette solution, 1 microM isoproterenol (Iso) or forskolin (Fsk) uniformly increased ICa: Iso by 45 +/- 5% and Fsk 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. Fsk was also associated with an increased rate of ICa activation. The Iso effect was blocked by pretreatment with 1 microM propranolol and reversed by propranolol after Iso exposure. The ICa response to 10 microM Iso or Fsk was smaller than the response to 1 microM, 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.(ABSTRACT TRUNCATED AT 250 WORDS)