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PubMed1967

Some factors influencing sodium extrusion by internally dialyzed squid axons.

Squid giant axons were internally dialyzed by a technique previously described. In an axon exposed to cyanide seawater for 1 hr and dialyzed with an ATP-free medium, the Na efflux had a mean value of 1.3 pmole/cm(2)sec when [Na](i) was 88 mM, in quantitative agreement with flux ratio calculations for a purely passive Na movement. When ATP at a concentration of 5-10 mM was supplied to the axoplasm by dialysis, Na efflux rose almost 30-fold, while if phosphoarginine, 10 mM, was supplied instead of ATP, the Na efflux rose only about 15-fold. The substitution of Li for Na in the seawater outside did not affect the Na efflux from an axon supplied with ATP, while a change to K-free Na seawater reduced the Na efflux to about one-half. When special means were used to free an axon of virtually all ADP, the response of the Na efflux to dialysis with phosphoarginine (PA) at 10 mM was very small (an increment of ca. 3 pmole/cm(2)sec) and it can be concluded that more than 96% of the Na efflux from an axon is fueled by ATP rather than PA. Measurements of [ATP] in the fluid flowing out of the dialysis tube when the [ATP] supplied was 5 mM made it possible to have a continuous measurement of ATP consumption by the axon. This averaged 43 pmole/cm(2)sec. The ATP content of axons was also measured and averaged 4.4 mM. Estimates were made of the activities of the following enzymes in axoplasm: ATPase, adenylate kinase, and arginine phosphokinase. Values are scaled to 13 degrees C.

Adenosine Triphosphatases↗
PubMed2006

The lack of mitochondrial complex I in a CMSII mutant of Nicotiana sylvestris increases photorespiration through an increased internal resistance to CO2 diffusion.

The cytoplasmic male sterile II (CMSII) mutant lacking complex I of the mitochondrial electron transport chain has a lower photosynthetic activity but exhibits higher rates of excess electron transport than the wild type (WT) when grown at high light intensity. In order to examine the cause of the lower photosynthetic activity and to determine whether excess electrons are consumed by photorespiration, light, and intercellular CO(2), molar fraction (c(i)) response curves of carbon assimilation were measured at varying oxygen molar fractions. While oxygen is the major acceptor for excess electrons in CMSII and WT leaves, electron flux to photorespiration is favoured in the mutant as compared with the WT leaves. Isotopic mass spectrometry measurements showed that leaf internal conductance to CO(2) diffusion (g(m)) in mutant leaves was half that of WT leaves, thus decreasing the c(c) and favouring photorespiration in the mutant. The specificity factor of Rubisco did not differ significantly between both types of leaves. Furthermore, carbon assimilation as a function of electrons used for carboxylation processes/electrons used for oxygenation processes (J(C)/J(O)) and as a function of the calculated chloroplastic CO(2) molar fraction (c(c)) values was similar in WT and mutant leaves. Enhanced rates of photorespiration also explain the consumption of excess electrons in CMSII plants and agreed with potential ATP consumption. Furthermore, the lower initial Rubisco activity in CMSII as compared with WT leaves resulted from the lower c(c) in ambient air, since initial Rubisco activity on the basis of equal c(c) values was similar in WT and mutant leaves. The retarded growth and the lower photosynthetic activity of the mutant were largely overcome when plants were grown in high CO(2) concentrations, showing that limiting CO(2) supply for photosynthesis was a major cause of the lower growth rate and photosynthetic activity in CMSII.

Carbon Dioxide↗
PubMed1982

Effects of inorganic phosphate on ion exchange, energy state, and contraction in mammalian heart.

Effects of inorganic phosphate (Pi) on contractile function, ionic exchange, and cellular energetic state were investigated in isolated, arterially perfused, interventricular rabbit septa. The addition of 10 or 20 mM Pi to a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffered perfusate showed 1) a decrease in developed tension due to a decrease in free Ca concentration; 2) an increase in diastolic tension; 3) a decrease in 42K uptake at the same time that 42K efflux was depressed; 4) an increase of 24Na activity; 5) an increase in tissue 47Ca activity; and 6) a decrease in tissue adenosine 5'-triphosphate (ATP) levels. If a 10-mM Pi intervention was applied without changing the free Ca concentration of the perfusate, the mechanical record showed a transient positive inotropic effect without changes in K efflux. These results are consistent with Pi-induced stimulated mitochondrial Ca uptake and Na-K pump inhibition that combine to increase cellular Ca uptake. The increase in cellular Ca and the decrease in cellular ATP would both tend to increase diastolic tension. In the presence of antimycin A, respiration-dependent Ca uptake is inhibited, and mitochondrial Ca uptake is then maintained at the expense of cellular ATP. This process of mitochondrial Ca uptake could account for the deficiency of ATP and the rigor type contracture that occurs with antimycin A. This energy-deficient state is modified if mitochondrial ATP consumption is blocked by oligomycin, and such treatment prevents the ATP-deficient rigor. Under these conditions, although mitochondrial Ca uptake is blocked by the presence of antimycin A and oligomycin, Ca uptake remains augmented secondary to Na-K pump inhibition by Pi.

Adenosine Triphosphate↗
PubMed1990

Intracellular calcium and high-energy phosphates in isolated cardiac myocytes.

The relationship between intracellular calcium (Cai) and high-energy phosphates was studied in adult cardiac myocytes. Cai and high-energy phosphates were measured in the same population of cells. Cai, reported by the fluorescence of fura-2, was maintained at normal levels in the presence of increased transsarcolemmal calcium gradients, up to 5 mM extracellular calcium concentration. Cai was experimentally elevated by increasing calcium influx from the extracellular medium and/or by diminishing calcium efflux by Na-Ca exchange. Under these conditions, cells contracted and relengthened repetitively. The regulation of high-energy phosphates was challenged by increasing ATP utilization and by inhibiting ATP synthesis. Cai regulation was not affected by inhibition of glycolysis or NADH oxidation, so long as ATP concentration remained unchanged. High-energy phosphates were not depleted in beating cells with intact NADH oxidation, but inhibition of NADH oxidation caused a significant drop in phosphocreatine, demonstrating the increased rate of ATP consumption during beating. In beating cells, as in the working heart, ATP supply is increased to meet ATP demand, and steady-state ATP and phosphocreatine concentrations remain unchanged.

Adenosine Triphosphate↗
PubMed1994

Effects of lowering external Na+ concentration on cytoplasmic pH and Ca2+ concentration in mouse pancreatic beta-cells: mechanism of periodicity of spike-bursts.

The periodic spike-burst response of pancreatic beta-cells varies in duration with an increase of external glucose within the range 5-20 mM. To elucidate the mechanism determining the length of spike-burst, we studied the low-Na+ induced change in electrical response to glucose which is similar to the change induced by high glucose. Cytoplasmic pH (pHi) and Ca2+ concentration ([Ca2+]i) were measured by the microfluorometric method under normal and low-Na+ conditions in mouse pancreatic islets. Lowering external Na+ concentration from 135 to 25 mM by replacing Na+ with Tris+ induced progressive alkalinization in islet cells in the presence of 11.1 mM glucose. In contrast, reduction of external Na+ by replacement with Li+ caused intracellular acidification. Both manipulations described above caused a marked increase in [Ca2+]i, suggesting the presence of Na+/Ca(2+)-antiport activity. Although the change in pHi induced by decreasing external Na+ varied in direction depending on the species of cations used for replacing Na+, the pattern of electrical activity consistently changed from the spike-burst type to the continuous spike-generation type without regard for the difference in species of cations replacing Na+. These findings lead to the following hypothesis: A decrease in Na+ influx could cause a decrease in ATP-consumption by Na+/K(+)-pumps that prevents the fall of intracellular ATP concentration. The resultant continuation of high concentrations of intracellular ATP may be responsible for the abolishment of the silent phase.

Adenosine Triphosphate↗
PubMed1980

Cardioplegic arrest in pigs. Effects of glucose-containing solutions.

A clinically used cardioplegic solution was evaluated in the laboratory in an attempt to elucidate the value of glucose in maintaining the glycogen and energy stores of the myocardium during aortic cross-clamping. In one group of animals the cardioplegic solution contained glucose; in the other group it did not. Energy stores were determined in full-thickness biopsies of left ventricular myocardium taken prior to bypass, at 60 minutes of cardioplegic arrest, and after 20 minutes of reperfusion. At the end of cardioplegic arrest, glycogen levels were slightly higher than control values, with no differences between the groups. Creatine phosphate (CP) and adenosine triphosphate (ATP) fell by 69% to 73% and 43% to 55%, respectively, from the control values, but there were no statistical differences between these groups. At the end of reperfusion, glycogen and CP stores had returned to control but ATP concentration remained below control values in both groups. These results indicate that glucose is of no value either to maintain myocardial glycogen or to influence the changes in CP and ATP during cardioplegic arrest. The dissociation between the complete and partial restoration of CP and ATP stores to control levels during reperfusion suggests either that ATP consumption is greater than the energy transfer between CP and ADP:ATP systems or that the energy transfer is defective under these experimental conditions.

Adenosine Triphosphate↗
PubMed1991

Quantification of ATP-producing and consuming processes in quiescent pig spleen lymphocytes.

ATP production in quiescent pig spleen lymphocytes was estimated on the basis of their coupled respiration. ATP-consuming processes were assessed from the effects of inhibitors of protein synthesis, proteolysis, RNA synthesis, Na+ K(+)-ATPase and Ca(2+)-ATPase on respiration. About 95% of the total ATP consumption could be assigned to specific processes. More than 50% of the ATP produced appear to be consumed by the cation transport ATPases.

Adenosine Triphosphate↗
PubMed1990

Oxygen consumption for K+ uptake during post-stimulatory activation of Na+, K(+)-ATPase in perfused rat mandibular gland.

1. In order to determine the stoichiometry of K+ uptake and ATP consumption by Na+, K(+)-ATPase in the isolated, perfused mandibular gland of the rat, oxygen consumption and net K+ uptake from the vascular side were measured during the recovery period following K+ depletion by stimulation with acetylcholine in combination with ouabain. 2. Acetylcholine (10(-6) M) induced fluid secretion and an initial, transient release of K+ from the gland. Addition of ouabain suppressed salivary fluid secretion and caused an additional, dose-dependent, transient release of K+. 3. With acetylcholine (10(-6) M), the oxygen consumption of the gland increased to 62.4 +/- 4.2 microliters/(g min) from a resting value of 12.9 +/- 1.2 microliters/(g min). The increased oxygen consumption was suppressed by ouabain, in a dose-dependent fashion. 4. Withdrawal of acetylcholine and ouabain induced a net uptake of K+ and, simultaneously, an increase in oxygen consumption. The cumulative K+ uptake and the increment of oxygen consumption during the recovery period were dependent on the concentration of used ouabain. 5. The rates of K+ uptake and ATP hydrolysis were compared during recovery, assuming that six moles of ATP were hydrolysed for each mole of oxygen consumed. The data obtained during the later phase of the recovery lay on a single straight line with a slope of 1.8 for each of the various concentrations of ouabain, suggesting that the relationship between K+ uptake and energy consumption was linear. 6. Assuming the K+:ATP stoichiometry of the Na+, K(+)-ATPase to be 2:1, K+ would appear to be transported with ca 90% efficiency by Na+, K(+)-ATPase in the rat mandibular gland. Using 1.8-2.0 as a K+/ATP stoichiometry, the rate of primary active K+ uptake and the corresponding passive K+ efflux during secretion were estimated to be 20-22 mumol/(g min) from the oxygen consumption and the net K+ flux. 7. The passive K+ efflux was estimated, from the initial rate of K+ release caused by addition of 10(-3) M-ouabain, to be 30 mumol/g min). The discrepancy between the estimated active K+ uptake and passive K+ release (8-10 mumol/(g min] could be attributed to a secondary active K+ uptake process such as Na(+)-K(+)-2Cl-symport.

Acetylcholine↗
PubMed1989

Dihydropyridine calcium antagonists reduce the consumption of high-energy phosphates in the rat brain. A study using combined 31P/1H magnetic resonance spectroscopy and 31P saturation transfer.

The disappearance of high-energy phosphates (HEPs) in the rat brain during global ischemia induced by cardiac arrest is slowed down significantly by dihydropyridine Ca antagonists (DCAs) compared to controls. Two mechanisms might account for this effect: increased HEP production via anaerobic glycolysis or decreased HEP consumption. In order to obtain more insight into the underlying mechanisms of ATP preservation we have studied in the rat the effect of the DCAs isradipine, darodipine and nimodipine on the cerebral steady-state levels of HEPs and lactate as well as the intracellular pH value during global ischemia using combined 31P/1H magnetic resonance spectroscopy. We have found that the ATP preservation in DCA-treated animals is not associated with significantly higher postischemic lactate levels (lactate/N-acetylaspartate 0.97 +/- 0.08 for isradipine at a dose of 2.5 mg/kg i.p.) or lower pH values (6.40 +/- 0.03) as compared to control rats (lactate/N-acetylaspartate 0.94 +/- 0.13, pH = 6.49 +/- 0.03). This is in contrast to hyperglycemic rats, in which similar preservation of ATP levels during ischemia was observed; however, at the expense of a larger drop in brain pH (6.22 +/- 0.09) and a concomitant increase in cerebral lactate (lactate/N-acetylaspartate = 1.40 +/- 0.09). These results strongly favor reduced ATP consumption to be the cause for the protective effect of DCAs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1998

Ouabain suppresses ATP elevation in response to fuel secretagogues in pancreatic islets.

The alterations of the ATP concentration in response to fuel secretagogues such as glucose, glyceraldehyde, and ketoisocaproate (KIC) were investigated in a single islet. The intraislet ATP concentration was transiently elevated and then decreased to a level slightly higher than basal. To asses the ATP content under conditions of reduced ATP consumption, the Na-K pump blocker ouabain was used. The elevation of ATP concentration was found unexpectedly to be suppressed under ouabain in the islet, even when incubated with any of the secretagogues. High glucose did not elevate the intracellular creatine phosphate during incubation with ouabain. Since the suppression rate for the intraislet ATP elevation was considerably smaller with KIC than with glyceraldehyde and glucose, we conclude that ouabain inhibits ATP production, at least in part, in the glycolytic pathway through a feedback mechanism.

Adenosine Triphosphate↗
PubMed1986

Intracellular ATP in a glucosephosphate isomerase mutant of Saccharomyces cerevisiae.

The degree of ATP depletion caused by glucose in a glucosephosphate isomerase-deficient strain of Saccharomyces cerevisiae was determined. Even in the presence of a sugar normally fermentable by the mutant, the addition of glucose can decrease the intracellular ATP, depending on the competition of the sugars for transport and subsequent phosphorylation. For both parent and mutant cells, a correlation exists between the calculated velocity of ATP formation or ATP consumption during the utilization of different concentrations of sugars and the experimental intracellular ATP level. For initially resting yeast cells, a rate increase of 35 mumol per min per g ATP was calculated to increase the intracellular level of this nucleotide by 1 mumol per g cell mass.

Adenosine Triphosphate↗
PubMed1998

Penicillin biosynthesis: energy requirement for tripeptide precursor formation by delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Acremonium chrysogenum.

In nonribosomal peptide formation by multifunctional enzymes, peptide synthetases catalyze the activation and directed condensation of amino acids. The peptide synthetase involved in penicillin biosynthesis (ACV synthetase) forms the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine from the respective L-amino acids and ATP. So far, the energy requirements for the nonribosomal process have not been clearly established. For ACV synthetase we show that ATP consumption depends on the reaction conditions employed. By simultaneously estimating peptide and AMP production by employing fluorescence detection and UV spectroscopy, respectively, we have determined the energy consumption with high accuracy. Under unfavorable reaction conditions more than 20 mol of ATP are consumed/mol of tripeptide formed, while optimal conditions permit the expected energy requirement of one ATP for each carboxyl group activation, corresponding to three ATP for tripeptide formation. The third ATP is required for the activation of L-valine to maintain the valyl-thioester stage for epimerization and peptide bond formation, and this high-energy bond is sacrificed by hydrolytic removal of the product. No extra energy is required for the directed transport in peptide elongation. Additional energy consumed has been traced to hydrolytic loss of activated intermediates, as has been shown by the analysis of incomplete reaction mixtures.

Acremonium↗
PubMed1999

The responses of rat hepatocytes to glucagon and adrenaline. Application of quantified elasticity analysis.

The internal control of hepatocyte metabolism has been previously analysed using metabolic control analysis. The aim of this paper is to extend this analysis to include the responses of the cells to hormonal stimulus. Hepatocyte metabolism was divided into nine reaction blocks: glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, proton leak, mitochondrial phosphorylation and ATP consumption, linked by five intermediates: mitochondrial membrane potential, cytoplasmic NADH/NAD and total cellular ATP, glucose 6-phosphate and pyruvate. The kinetic responses of the reaction blocks to the intermediates were determined previously in the absence of added hormones. In this study, the changes in flux and intermediate levels that occurred upon addition of either glucagon or adrenaline were measured. From comparison of the fractional changes in fluxes and intermediate levels with the known kinetics of the system, it was possible to determine the primary sites of action of the hormones. The results show that the majority of processes in the cell are responsive to the hormones. The notable exception to this is the failure of adrenaline to have a direct effect on glycolysis. The activity change of each metabolic block observed in the presence of either hormone was quantified and compared to the indirect effects on each block caused by changes in metabolite levels. The second stage of the analysis was to use the calculated activity changes and the known control pattern of the system to give a semiquantitative analysis of the regulatory pathways employed by the hormones to achieve the changes in fluxes and metabolite levels. This was instructive in analysing, for example, how glucagon caused a decrease in flux through glycolysis and an increase in oxidative phosphorylation without large changes in metabolite levels (homeostasis). Conversely, it could be seen that the failure of adrenaline to maintain a constant glucose 6-phosphate concentration was due to the stimulation of glycogen breakdown and inhibition of glucose release.

Adenosine Triphosphate↗
PubMed1996

Isoflurane preserves adenosine triphosphate levels in anoxic isolated rat hepatocytes by stimulating glycolytic adenosine triphosphate formation.

The hypothesis that general anesthetics protect energy reserves by decreasing energy demand is widely accepted but poorly substantiated. Isoflurane at clinical doses preserved adenosine triphosphate (ATP) levels in anoxic isolated hepatocytes. Specific inhibitors were used to block mitochondrial and/or glycolytic ATP formation to ascertain whether pathways of energy supply or demand, or both, were involved in ATP preservation by isoflurane. Hepatocytes were isolated from fed adult male rats after perfusing livers with Krebs buffer containing collagenase. Cells were incubated in Krebs buffer for 0-30 min at 25 degrees C under N2/CO2 (95%/5%) +/- isoflurane 0.63 mM in liquid phase. Oligomycin, iodoacetate, or fasting were used to block mitochondrial and glycolytic ATP formation. Under anoxia alone, ATP levels declined more slowly in the presence than in the absence of isoflurane, confirming the ATP-protective effect of isoflurane reported previously. With oligomycin plus iodoacetate blocking all ATP formation, ATP decline (representing pure ATP consumption) was not slowed by isoflurane. Isoflurane's protective effect recurred when glycolytic ATP supply was restored by incubating with oligomycin only. The protective effect was accompanied by increased lactate accumulation, and both effects-ATP preservation and lactate formation-were similarly dependent on isoflurane concentration. We conclude that the protective effect of isoflurane on energy status in anoxic isolated hepatocytes was not associated with reduced ATP demand but with enhanced ATP supply via stimulation of glycolysis.

Adenosine Triphosphate↗
PubMed1996

The intrinsic rhythmicity of spike-burst generation in pancreatic beta-cells and intercellular interaction within an islet.

The pancreatic beta-cell has four types of Ca2+ channel (L-type, T-type, low-threshold slowly inactivating, and low-threshold non-inactivating Ca2+), although the low-threshold non-inactivating Ca2+ channel has not yet been confirmed experimentally. Beside these, there are at least three types of K+ channels (K(ATP), K(Ca,V), and K(V)), and transporters (GLUT-2, Na+/Ca(2+)-countertransporter, and Na+/K(+)-pump) as schematically shown in Fig.4. Opinions on the mechanism of spike-burst are converging to the following view: At intermediate glucose concentrations, the intracellular ATP/ADP ratio oscillates in the following way. A gradual rise in the ATP/ADP ratio causes gradual progression of depolarization to the threshold for the low-threshold Ca2+ channels, of which the opening causes regenerative depolarization to the plateau potential on which spikes (the L-type Ca2+ channel contributes to spike firing) are superimposed. During the active phase, a fall in the ATP/ADP ratio follows a gradual rise in ATP consumption. Slight repolarization due to the opening of a small fraction of K(ATP) channels triggers regenerative repolarization. With the progress of repolarization, a residual fraction of voltage-gated Ca2+ channels (low-threshold non-inactivating) are deactivated. During the silent phase, a gradual rise in the ATP/ ADP ratio leads to gradual depolarization back to the threshold for the next spike-burst. There are still a diversity of views regarding the mechanism of the initial spike-train. On the basis of observations made in various laboratories including ours, we propose the following working model: At low concentrations of glucose, alpha-cells secret glucagon which induces a rise in cAMP in beta-cells lodged in the same islet. A rise in cAMP itself does not activate the enzymes relevant to glycogenolysis, but merely prepares to activate the enzymes. When extracellular glucose increases, Ca2+ spikes are elicited. Influxed Ca2+ ions, together with cAMP, work to activate the enzymes, resulting in an additional supply of fuel for ATP synthesis. After sometime, the cAMP level falls back to a low level and the additional glucose supply from stored glycogen stops. This reaction sequence may be the mechanism behind the initial spike-train. To substantiate this working model, it may be important to elucidate the dependence of the phosphorylasekinase and glycogenphosphorylase activities on the Ca2+ in beta-cells.

Acetylcholine↗
PubMedDate not supplied

Molecular mechanism of acute ammonia toxicity: role of NMDA receptors.

Acute administration of large doses of ammonia leads to the rapid death of animals. This article reviews the role of excessive activation of N-methyl-D-aspartate (NMDA) receptors in the mediation of ammonia-induced mortality. The studies reviewed here show that acute intoxication with large doses of ammonia leads to the activation of NMDA receptors in brain in vivo. Moreover, excessive activation of NMDA receptors is responsible for ammonia-induced death of animals, which is prevented by different antagonists of NMDA receptors. This article also reviews the studies showing that activation of NMDA receptors is also responsible for the following effects of acute ammonia intoxication: (1) depletion of brain ATP, which, in turn, leads to release of glutamate; (2) activation of calcineurin and dephosphorylation and activation of Na+/K+-ATPase in brain, thus increasing ATP consumption; (3) impairment of mitochondrial function and calcium homeostasis at different levels, thus decreasing ATP synthesis; (4) activation of calpain that degrades the microtubule-associated protein MAP-2, thus altering the microtubular network; (5) increased formation of nitric oxide (NO) formation, which, in turn, reduces the activity of glutamine synthetase, thus reducing the elimination of ammonia in brain.

Acute Disease↗
PubMed2000

Creatine kinase in human retinal pigment epithelium.

Retinal pigment epithelial ion transport activity, and consequent ATP consumption vary significantly as a function of photoreceptor activity. In a variety of cell types, ATP levels are maintained during high-energy usage by phosphocreatine hydrolysis, catalysed by the enzyme creatine kinase. The present work was designed to assess the importance of creatine kinase in retinal pigment epithelial cell metabolism. To this end, activity measurements, non-denaturing gel electrophoresis, Western blot analysis and immunohistochemistry were used to characterize creatine kinase in retinal pigment epithelium. Total creatine kinase activity in the retinal pigment epithelium is approximately 0.05 micromol ATP mg protein(-1) min(-1). The bulk of this activity was mediated by the B-CK isoform. However, by immunoblotting, non-denaturing gel electrophoresis and immunohistochemistry, the presence of the M-CK isoform of creatine kinase was also detected. The M-CK isoform was plasma membrane associated and predominately localized to the apical surface. Creatine kinase in the retinal pigment epithelium could function in a spatial energy shuttle that helps to sustain apical plasma membrane ion transport activity.

Blotting, Western↗
PubMed1990

The regulation of ATP-sensitive K+ channel activity in intact and permeabilized rat ventricular myocytes.

1. In isolated rat heart ventricular myocytes exposed to 2 mM-cyanide in the presence of 10 mM-2-deoxyglucose (complete metabolic blockade), there is a time-dependent increase in ATP-sensitive potassium (KATP) channel activity. The increase in KATP channel activity accompanies the decline of twitch amplitude. Channel activation and decline of the twitch amplitude precede the development of a 'rigor' contracture. 2. We measured KATP channel activity in permeabilized cells using the open-cell attached (O-C-A) patch configuration (by establishing a cell-attached patch and then permeabilizing the cell by exposure to saponin). The apparent ATP dependence of KATP channel activity could be described by a sigmoid curve with ki, ATP (ATP concentration required for half-maximum inhibition of channel activity) = 122 microM and H (Hill coefficient) = 1.225. 3. In the O-C-A patch configuration, 10 mM-creatine phosphate (CrP) decreased the apparent ki, ATP from 122 microM to about 10 microM, and the maximal activity (in zero ATP) was decreased to about 30% of the maximal activity in the absence of CrP. 4. In isolated inside-out (I-O) patches, ATP inhibited KATP channel activity at much lower [ATP] than in the O-C-A patch configuration (ki, ATP = 25 microM, H = 2). CrP was without effect on I-O patches. 5. These results are consistent with the hypothesis that the difference in the ATP dependence of KATP channel activity in the O-C-A and I-O patch configurations arises because of ATP consumption in the O-C-A patch configuration. The results suggest that hydrolysis of ATP to ADP by endogenous ATPases leads to the development of gradients of [ATP] and [ADP] between the bath and the 'inside' of the open cell. By re-phosphorylating ADP, CrP is able to dissipate these gradients, revealing the 'true' ATP dependence of channel activity, which is the same as that in the I-O patch configuration. 6. In order to estimate the contribution of KATP channel activity to the rat cardiac action potential at different [ATP] we have made the following measurements. Using electrodes of resistance 2-8 M omega the density of KATP channels was 10.3 +/- 0.1 channels per patch (n = 162).(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗