Abstract
The ability of halothane to stimulate phospholipase C (PLC) was examined in turkey erythrocyte membranes prepared from [3H]inositol-labeled turkey erythrocytes by measuring [3H]inositol phosphate formation ([3H]InsP) in the presence and absence of G-protein activation. In the presence of guanosine 5'-3-O-(thio)triphosphate) (GTP gamma S), halothane (0.5-10 mM) caused a dose-dependent activation of PLC. The EC50 value for halothane-induced PLC activation was 2.8 +/- 0.3 mM. Halothane (0.1-30 mM) had no effect on PLC activity in the absence of G-protein activation and did not affect Ca(2+)-dependent PLC activity. The activation of PLC by GTP gamma S occurred after an initial lag period of 60 s which was followed by a linear increase in [3H]InsP. Halothane dose-dependently decreased the lag period for GTP gamma S-induced PLC activation (minimal value 15 s) and increased the rate of [3H]InsP formation at all time points following this lag. As a result, halothane shifted the EC50 value for GTP gamma S-induced PLC activation to the left (4-fold) and increased its maximal response. Halothane also caused a dose-dependent activation of PLC in the presence of AlF4-. Half-maximal stimulation of AlF4(-)-activated PLC occurred with an EC50 value of 2.9 +/- 0.4 mM halothane, which is similar to the halothane dose giving half-maximal stimulation of PLC in the presence of GTP gamma S. At low doses (0.1-0.3 mM) halothane inhibited both isoproterenol- and adenosine 5'-O-(2-thiodiphosphate) (ADP beta S)-induced [3H]InsP formation, whereas at higher concentrations it stimulated PLC independent of the presence of these agonists. At concentrations chosen to reflect their different membrane/buffer partition coefficients, both hexanol (5 mM) and benzyl alcohol (20 mM) fluidized turkey erythrocyte membranes to the same degree as halothane (5 mM). However, these agents had no effect on GTP gamma S- or AlF(4-)-induced PLC activity, indicating that halothane-induced PLC activation was not secondary to changes in bulk lipid fluidity properties. Halothane also stimulated [3H]inositol bisphosphate and [3H]inositol trisphosphate formation in intact erythrocytes. These data demonstrate that the anesthetic halothane can stimulate G-protein-dependent PLC activity and modify the responsiveness of this signaling system to activation by receptor-linked agonists.
Highlights
Halothane (0.1-30mM) had no effect on PLC activity in tahbesence of G-protein activation and did not affect Ca2+-dependent PLC activity.The activation of PLC by GTP-yS occurred after an initial lag period of 60 s which was followed by a linear increase in ['HJInsP
Demonstrate that the anesthetic halothane can stimu- Inthe present study we have examined the effects of late G-protein-dependent PLC activity andmodify the halothane on PLC activity in turkey erythrocyte membranes
Total inositol phosphates were measured by counting 600 pI of stimulate PLC in turkey erythrocyte membranes was determined by examining its effects on [3H]InsP formation in the presence and absence of G-protein activation
Summary
Labeling of Turkey Erythrocytes with pH]Inositol-Turkey eryth- 2 S.E. of data obtained from five to eight separate experiments. Rocytes were prepared essentially according to themethod of Harden et al [20],as described previously [24, 29].Briefly, fresh turkey erythrocytes in Alsever’s solution (72 mM NaCl, 0.8 M glucose, 27 mM trisodium citrate, 10% citric acid, pH 6.1)were centrifuged at 1,100 X g for 5 min andthe resultant supernatant removed by aspiration before resuspending the packed cells in 4-6volumes of icecold HEPES buffer (1.5mM HEPES, 150 mM NaCI, pH 7.2).This centrifugation and washing procedure was repeated afurther two times before washing the cells in inositol-free Dulbecco’s modified Eagle’s medium. All incubations were performed in the absence of ATP oranATP-regenerating system to eliminate the
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