A glibenclamide sensitive potassium conductance in terminal arterioles isolated from guinea pig heart.

OBJECTIVE: The aim was to isolate terminal arterioles from guinea pig heart, to describe their basic electrical properties, and to obtain evidence for the presence of KATP channels in microvascular coronary smooth muscle cells. METHODS: Terminal arterioles of 20-50 microns diameter were obtained by enzymatic digestion of isolated perfused hearts. The isolated arterioles were viable for up to 8 h and constricted upon application of high potassium solution. Whole cell clamp experiments on smooth muscle cells of these arterioles were performed at room temperature. RESULTS: The resting potential of coronary smooth muscle cells in terminal arterioles showed a bimodal distribution with one peak at -23(SD 8) mV and the other peak at -61(4) mV. Application of glibenclamide (50 microM) to the latter group of arterioles produced a depolarisation to -37(8) mV; application of cromakalim (1 microM) produced a hyperpolarisation to -71(1) mV. The current changes associated with voltage steps and slow voltage ramps in the range -120 to +40 mV indicated that the smooth muscle cells in the arterioles were coupled electrically. The steady state current-voltage relation was sigmoid with a flat region in the range -50 to -30 mV. In the presence of 2-50 microM glibenclamide the slope resistance at potentials negative to -50 mV and positive to -30 mV was markedly increased. In the presence of 1 microM cromakalim the slope resistance was decreased and the current-voltage relation at negative potentials became nearly linear. The crossover point of the current-voltage relations measured under control conditions and in the presence of glibenclamide was near the calculated potassium equilibrium potential. CONCLUSIONS: 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.

KATP channels and basal coronary vascular tone.

After reviewing recent experimental work from various laboratories we have come to the following conclusions. (1) An increase in transmural pressure causes depolarisation of coronary arterioles, which increases smooth muscle tone. Under these conditions the opening of KATP channels can induce a much larger change in membrane potential than in relaxed arteries. Furthermore, the rate of ATP hydrolysis by contractile proteins, and thus the submembrane nucleotide concentrations, might also be changed in the presence of myogenic tone. Therefore care should be taken when extrapolating patch clamp results from isolated coronary smooth muscle cells to the function of KATP channels in vivo. (2) The opening of KATP channels is increased in situations related to energy imbalance, such as hypoxia, adenosine release, intracellular acidification, and lactate accumulation. However, there is increasing evidence that KATP channels also contribute to the setting of the membrane potentials of coronary smooth muscle cells under normoxic conditions. Thus the modulation of KATP channels by intracellular metabolites and by vasoactive autacoids may play an important role in the regulation of coronary blood flow even in the presence of normal intracellular ATP concentrations. (3) The smooth muscle cells of coronary terminal arterioles form an electrical syncytium. The opening of a new KATP channels in smooth muscle cells of a terminal arterioles might induce a spatially homogeneous hyperpolarisation of the entire arteriole. The resulting homogeneous decrease in the tone of the coronary smooth muscle cells of the arteriole may induce a considerable change in vascular resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Enzyme kinetics of the prime K+ channel in the tonoplast of Chara: selectivity and inhibition.

The prime potassium channel from the tonoplast of Chara corallina has been analyzed in terms of an enzymatic kinetic model (Gradmann, Klieber & Hansen 1987, Biophys. J. 53:287) with respect to its selectivity for K+ over Rb+ and to its blockage by Cs+ and by Ca2+. The channel was investigated by patch-clamp techniques over a range of membrane voltages (Vm, referred to an extracytoplasmic electrical potential of zero) from -200 mV to +200 mV under various ionic conditions (0 to 300 mM K+, Rb+, Cs+, Ca2+, and Cl-) on the two sides of isolated patches. The experimental data are apparent steady-state current-voltage relationships under all experimental conditions used and amplitude histograms of the seemingly noisy open-channel currents in the presence of Cs+. The used model for K+ uniport comprises a reaction cycle of one binding site through four states, i.e., (1) K(+)-loaded and charged, facing the cytoplasm, (2) K(+)-loaded and charged facing the vacuole, (3) empty, facing the vacuole, and (4) empty, facing the cytoplasm. Vm enters the system in the form of a symmetric Eyring barrier between state 1 and 2. The numerical results for the individual rate constants are (in 10(6)s-1 for zero voltage and 1 M substrate concentration): k12: 1,410, k21: 3,370, k23: 105,000, k32: 10,600, k34: 194, k43: 270, k41: 5,290, k14: 15,800. For the additional presence of an alternate transportee (here Rb+), the model can be extended in an analog way by another two states ((5) Rb(+)-loaded and charged, facing cytoplasm, and (6) Rb(+)-loaded and charged, facing vacuole) and six more rate constants (k45: 300, k54: 240, k56: 498, k65: 4,510, k63: 4,070, k36: 403). This six-state model with its unique set of fourteen parameters satisfies the complete set of experimental data. If the competing substrate can be bound but not translocated (here Cs+ and Ca2+). k56 and k65 of the model are zero, and the stability constants Kcyt (= k36/k63) and Kvac (= k45/k54) turn out to be Kcyt(Ca2+): 250 M-1 x exp(Vm/(64 mV)), kvac(Ca2+): 10 M-1 x exp(-Vm/(66 mV)), Kcyt(Cs+): 0, and Kvac(Cs+): 46 M-2 x exp(-Vm/(12.25 mV)).(ABSTRACT TRUNCATED AT 400 WORDS)

The energy expenditure of actomyosin-ATPase, Ca(2+)-ATPase and Na+,K(+)-ATPase in guinea-pig cardiac ventricular muscle.

1. The rate of heat production (heat rate) and isometric twitch tension of ventricular trabeculae isolated from guinea-pig heart were measured at 37 degrees C in order to determine the relative contributions of actomyosin-ATPase, Ca(2+)-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 +/- 1.2 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 mM s cm-2. 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 Ca(2+)-ATPase was studied by blocking Ca2+ influx into the myocardial cells with a solution containing 100 microM Ca2+ and 400 microM 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 Ca(2+)-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 microM 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 microM Ca2+ and 400 microM Ni2+, and then inhibiting the sodium pump with 400 microM 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 Ca(2+)-ATPase (0.09).(ABSTRACT TRUNCATED AT 400 WORDS)

Reaction kinetic parameters for ion transport from steady-state current-voltage curves.

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.

Vanadate-sensitive ATPase in the plasmalemma of Acetabularia: biochemical and kinetic characterization.

The electrogenic Cl(-)-pump in the plasmalemma of the marine alga Acetabularia acetabulum (L.) Silva has been suggested to be an unusual type of ATPase (Gradmann, 1989, Methods Enzymol. 174, 490). For a biochemical treatment of this issue, a plasmalemma-rich membrane fraction from Acetabularia has been prepared by phase-partitioning. About 80% of the ATPase activity in this material is inhibited by vanadate (K I50=1-2 µM). The phosphohydrolytic properties of the corresponding enzyme were further investigated. Its primary substrate MgATP(2-) (K m about 270 µM). Compared with other plasmalemma ATPases, it has an extremely alkaline pH optimum (pH 8-8.5), a weak sensitivity to diethyl-stilbestrol and to N,N'-dicyclohexylcarbodiimide, a strong selectivity for Mg(2+) over alternative divalent cations, and a weak selectivity for ATP over other phosphohydrolytic substrates. It is insensitive to KCl at concentrations up to 200 mM. Both ATP(4-) and Mg2ATP inhibit the ATPase, satisfying a relationship for competitive inhibition by 2ATP(4-) (K IATP=1.56 mM) and noncompetitive inhibition by Mg2ATP (K IMg2ATP=1.35 mM). Since no transport experiments are reported in this study, the ion species (H(+) or Cl(-)) that is transferred by this ATPase is not identified.

Inwardly rectifying K+ channels in freshly dissociated coronary endothelial cells from guinea-pig heart.

1. Inwardly rectifying K+ (IK(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 < or = 1 microM 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.