HALOTHANE DOES NOT ALTER CA2+ AFFINITY OF TROPONIN-C

Troponin C has been suggested as a possible target for the negative inotropic action of volatile anesthetics. This study has examined the effect of halothane on the structure and response of isolated cardiac troponin C to Ca2+ and the response of skinned soleus and cardiac muscle fibers to Ca2+. The high-affinity Ca2+-binding sites of cardiac troponin C were assessed by measurement of the change in intrinsic tyrosine fluorescence and ultraviolet circular dichroism in response to Ca2+ in the presence and absence of halothane. Halothane (0.9 mM, 1.4%) did not alter the 45% enhancement in intrinsic tyrosine fluorescence that occurs with saturation of the high-affinity sites or change the Ca2+ concentration at which half-maximal enhancement occurred. The molar ellipticity in the far ultraviolet region, a measure of the secondary structure, increased to a similar extent with addition of 10(-6) M Ca2+ in the absence and presence of 1.0 mM (1.6%) halothane. The binding rate of the sulfhydryl reagent, 5,5'-dithiobis (2-nitrobenzoic acid), to troponin C in response to Ca2+ titration was used as a measure of the integrity of the low-affinity Ca2+-binding site in troponin C in the presence and absence of 1.0 mM (1.6%) halothane. The rate of reaction was stimulated twofold, and the half maximal effect was observed at pCa 4.8 +/- 0.2 in both control and halothane-treated samples. Halothane (5 mM; 7.8%) did not change the pCa/tension response of skinned soleus fibers; the data were fit to the Hill equation and yielded dissociation constants of 6.2 x 10(-7) M for control and halothane-treated specimens. Cardiac skinned fibers were not altered by exposure to 1.0 mM (1.6%) halothane; Ca/tension curves were essentially identical and yielded dissociation constants of 5.9 x 10(-7) M and 6.0 x 10(-7) M for control and halothane-treated specimens, respectively. Halothane had no effect on the maximal tension achieved by either skinned soleus or cardiac fibers. The authors conclude from these observations that halothane does not alter the affinity of cardiac troponin C for Ca2+ and that the myofibrils are not an important site for the negative inotropic effect of halothane.