Fig. 2. Malate-dependent respiration at increasing halothane concentrations. ( A ) The effect of halothane on state 3 respiration via complex I. The dashed line denotes the respiratory rate of N2 mitochondria at the EC50for immobility of the whole animal. This value, approximately 80 nA O · min−1· mg protein−1, is higher than the respiratory rate for gas-1(fc21) mitochondria in the absence of anesthetic. ( B ) The effect of halothane on state 4 respiration via complex I. State 4 respiration was not significantly increased by halothane, even at high doses, ruling out that halothane acts as an uncoupler. ( C ) 2,4-dinitrophenol uncoupled respiration of malate in the presence of halothane. Note that the inhibition of complex I–dependent function is not removed when the mitochondria are uncoupled, indicating that halothane does not inhibit respiration by blocking adenosine triphosphate synthesis. Bars and error bars represent means and SDs, respectively. n =number of repeats for each experiment (n =3 for N2; n =4 for gas-1 ).