1. The rate of heat production (heat rate) and isometric twitch tension of ventricular trabeculae isolated from guinea-pig heart were measured at 37°C in order to determine the relative contributions of actomyosin-ATPase, Ca2+-ATPase and Na+,K+-ATPase to myocardial energy metabolism. 2. The increase in heat rate recorded during isometric contractions at optimal length (contraction-related heat production) was 19-1 + 1P2 mW cm-3 at a stimulation rate of 2 Hz. The tension-time integral of individual contractions measured under the same conditions was 147 + 15 mN s cm2. 3. The heat production of the actomyosin-ATPase was determined by inhibiting the contractile proteins with 2,3-butanedione monoxime (BDM). Contraction-related heat production was reduced by 0-219 + 0 010 and the isometric tension-time integral was reduced by 0-288 + 0-016 in the presence of 1 mm BDM. From these data an estimate of 0-76 for the relative contribution of the actomyosin-ATPase to contraction-related heat production was derived.4. The heat production related to actomyosin-ATPase plus Ca2+-ATPase was studied by blocking Ca2+ influx into the myocardial cells with a solution containing 100 /M Ca2+ and 400 #M Ni2+. In this solution contraction-related heat production was reduced by 0'907 + 0-012. Comparison of this value with the component attributable to the actomyosin-ATPase yields an estimate of 0-15 for the relative contribution of the Ca2+-ATPase to contraction-related heat production. 5. The heat production related to the Na+,K+-ATPase in resting preparations was studied by blocking the sodium pump with 400 uM dihydro-ouabain (DHO). DHO produced a transient decrease in heat rate lasting 1-2 min, which was followed by a secondary increase. From the heat transient produced by DHO the heat rate related to the Na+,K+-ATPase in the steady state was extrapolated. The relative contribution of the sodium pump to resting heat production was estimated to be 0-17.6. The heat production related to the Na+,K+-ATPase in contracting preparations was studied by first blocking Ca2+ influx with 100 uM Ca2+ and 400,uM Ni2+, and then inhibiting the sodium pump with 400 uM dihydro-ouabain (DHO). The relative contribution of the sodium pump to contraction-related heat production extrapolated from these data was 0 10, which agreed well with the fraction of contraction-related heat production persisting after blockage of actomyosin-ATPase and Ca2+-ATPase (0 09).7. It is concluded that contraction-related heat production can be completely accounted for by the three major ATPases, and that under our experimental conditions actomyosinATPase, Ca2+-ATPase and Na+,K+-ATPase contribute about 76, 15 and 9%, respectively, to myocardial energy expenditure. 8. In cardiac muscle contracting under aerobic conditions the heat rate measured in the steady state is proportional to the rate of ATP hydrolysis, 1 1uW corresponding to about 11 pmol ATP s-5. From our heat and tension measurements the mechanical impulse per ATP molecule was calculated to be 0-027 pN s.

1. Inwardly rectifying K+ ('K(IR)) currents of freshly dissociated coronary endothelial cells from guinea-pig heart were investigated with the perforated-patch technique. 2. The whole-cell current-voltage relationship of endothelial cells showed strong inward rectification. Increasing the extracellular K+ resulted in an increase of inward currents. The slope conductance of the cells in the potential range negative to the calculated potassium equilibrium potential (EK) with 5, 60 and 150 mm external potassium was 0-18 + 0'14, 0.55 + 0 50 and 0-63 + 0-29 nS (mean + S.D.), respectively. 3. To quantify the steepness of inward rectification, the voltage dependence of the chord conductance of the cells was fitted with a Boltzmann function. The slope factor k describing the steepness of the relationship was 6-8 + 1'5 mV. 4. Extracellular barium induced a potential-and time-dependent block of inward currents through endothelial KIR channels. Half-maximum inhibition of IK(IR) currents was achieved with < 1 AM barium at a membrane potential of -70 mV in a solution containing 60 mM K+.5. Whole-cell inward currents revealed the opening and closing of single KIR channels. The single-channel conductance was 26 + 3 pS with 60 mm external K+ and 33 + 6 pS with 150 mm external K+. 6. Our results suggest that the electrical properties of freshly dissociated endothelial cells are to a large extent determined by five to sixty active strong inwardly rectifying K+ (KIR) channels.

Glibenclamide closes and cromakalim opens potassium channels in smooth muscle cells of coronary arterioles. The voltage dependence of the steady state current changes suggests that the current activated by cromakalim is not carried by the same channels as the current inhibited by glibenclamide. The glibenclamide sensitive channels make a significant contribution to the membrane potential of isolated arterioles.

This study demonstrates possible ways to estimate the rate constants of reaction kinetic models for ion transport from steady-state current-voltage data as measured at various substrate concentrations. This issue is treated theoretically by algebraic reduction and extension of a reaction kinetic four-state model for uniport. Furthermore, an example for application is given; current-voltage data from an open K+ selective channel (Schroeder, J.I., R. Hedrich, and J.M. Fernandez, 1984, Nature (Lond.), 312:361-362) supplemented by some new data have been evaluated. The analysis yields absolute numerical estimates of the 14 rate constants of a six-state model, which is discussed in a wider context.

Summary, The contribution of the major K + channel to the slow electrodynamic properties of the Acetabularia membrane has been investigated by patch-clamp techniques in the cell-attached mode using physiologically intact protoplasmic drops. This study comprises recording and statistical analysis of opening and closing events over long periods of time at various membrane voltages V~, as well as measurement and evaluation of individual or averaged current responses of single channels upon large voltage steps. Although detailed observations reveal a variety of different states (such as bursts or various levels of conductance or noise), a serial three-state reaction kinetic model was adequate for the description of the channel properties which are relevant to the macroscopic electrical behavior. This model consists of an open state (o) and two closed states (1 and 2) with the approximate rate constants ko, = 1 sec t, k~o = I sec -~, k~2 = 20 -exp(-V,,e/2kT) sec -I and kzl = 4 . exp(V,~e/2kT) sec -1. The current-voltage relationship of the open channel and the equilibrium ko~/kjo are relatively constant, whereas the (voltage-dependent) equilibrium k~z/kzi can spontaneously change within an order of magnitude at constant voltage.Key Words Acetabularia 9 patch clamp 9 potassium channel 9 relaxation analysis 9 channel kinetics 9 channel statistics 9 reaction kinetic model