Effect of halothane on the guanosine 5' triphosphate binding activity of G-protein alphai subunits.
BACKGROUND
Receptor-mediated increases in the force produced by airway smooth muscle are attenuated by anesthetics such as halothane. Guanosine 5'-triphosphate (GTP) binding protein alpha subunits (Galpha(i)) are known to participate in the regulation of force in airway smooth muscle. The authors hypothesized that halothane would inhibit the ability of Galpha(i) subunits to bind a nonhydrolyzable analog of GTP (GTPgammaS).


METHODS
The effect of halothane on both GTPase-specific activity and [35S]GTPgammaS binding were assayed using purified, recombinant Galpha(i1). In separate experiments, [35S]GTPgammaS binding to Galpha(i) in crude airway smooth muscle membrane preparations was assayed using an immunoprecipitation technique in the presence and absence of halothane.


RESULTS
The steady state GTPase-specific activity of the recombinant Galpha(i1) was 0.033 +/- 0.018 (mean +/- SD) mole P(i) mole Galpha(i1)-1 min-1 under control conditions and 0.035 +/- 0.015 mole P(i) mole Galpha(i1)-1 min-1 in the presence of 1.1 +/- 0.2 mm halothane, a difference that is not significant. The mole fractions of recombinant Galpha(i1) bound to [35S]GTPgammaS were 0.49 +/- 0.02 and 0.60 +/- 0.02 at 10 and 20 min, respectively. The addition of halothane (1.26 +/- 0.07 mm) did not significantly change these values. Halothane did not affect the binding of [35S]GTPgammaS to Galpha(i) subunits in membrane fractions of airway smooth muscle as measured using immunoprecipitation. Validity of the assays was confirmed using suramin, an inhibitor of GTP binding.


CONCLUSIONS
These results suggest that halothane, which inhibits receptor-activated Galpha(i)-coupled pathways in intact airway smooth muscle, must functionally target a component of the G protein-coupled receptor complex other than Galpha(i).
Streiff
John
J
Department of Anesthesiology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
Jones
Kristofer
K
Perkins
William J
WJ
Warner
David O
DO
Jones
Keith A
KA
D000251:Adenosine Triphosphatases / Q000378:metabolism; D018685:Anesthetics, Inhalation / Q000494:pharmacology*; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000187:drug effects / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006221:Halothane / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D009130:Muscle, Smooth / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry
D018685:Anesthetics, Inhalation; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013498:Suramin; D006160:Guanosine Triphosphate; D014364:Tryptophan; D000251:Adenosine Triphosphatases; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D006221:Halothane