GTP REQUIREMENT FOR ISOPROTERENOL ACTIVATION OF CALCIUM CHANNELS IN VASCULAR MYOCYTES

The effects of activating the beta-adrenoceptor pathway on calcium cur rent (I-Ca) in rabbit portal vein (PV) were studied in myocytes freshl y isolated by collagenase and elastase treatment. I-CA was measured at room temperature (20 degrees C) using whole cell, voltage-clamp metho ds 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 I-Ca was present in these myocytes, averaging 3.5 +/- 0.3 pA/pF a t +10 mV under control conditions. With 0.1 mM guanosine 5'-triphospha te (GTP) added to the pipette solution, 1 mu M isoproterenol (Iso) or forskolin (Fsk) uniformly increased I-Ca: Iso by 45 +/- 5% and Fsk by 88 +/- 11%. This augmentation of I-Ca was not associated with signific ant changes in the voltage dependence of activation or inactivation bu t was associated with a small increase in the rate of inactivation of I-Ca. Fsk was also associated with an increased rate of I-Ca activatio n. The Iso effect was blocked by pretreatment with 1 mu M propranolol and reversed by propranolol after Iso exposure. The I-Ca response to 1 0 mu M Iso or Fsk was smaller than the response to 1 mu M, with some c ells showing a steady-state reduction in I-Ca. When the latter occurre d, 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 I-Ca at +10 mV when measured with the same temporal protocols as used with Iso, sugge sting I-Ca rundown. Without intracellular GTP, 1 mu M Iso produced no significant average change in I-Ca, but the response to 1 mu M Fsk was not affected. With 0.1 mM guanosine 5'-O-(3-thiotriphosphate) added t o the pipette solution, control I-Ca was greatly augmented, but 1 mu M Iso had no further effect on I-Ca. With 1.0 mM guanosine 5'-O-(2-thio diphosphate) added to the pipette solution, the initial I-Ca was not c hanged, and I-Ca was not significantly affected by 1 mu M Iso (+10 +/- 6%). These results suggest that beta-adrenergic activation of PV myoc ytes augments I-Ca by a mechanism that involves adenylyl cyclase and G protein activation and requires intracellular GTP.