Synthesis and accumulation of an extremely stable high-energy phosphate compound by muscle, heart, and brain of animals fed the creatine analog, 1-carboxyethyl-2-iminoimidazolidine (homocyclocreatine)

A new creatine analog, 1-carboxyethyl-2-iminoimidazolidine (homocyclocreatine), has been synthesized and compared with other synthetic analogs of creatine as a substrate for creatine kinase under both in vitro and in vivo conditions. Reactivity with rabbit muscle creatine kinase at 2 mM and pH 7.0 occurred in the order: creatine greater than cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) greater than N-ethylguanidinoacetate greater than N-propylguanidinoacetate greater than guanidinoacetate greater than N-methyl-3-guanidinopropionate greater than 3-guanidinopropionate greater than homocyclocreatine. Homocyclocreatine was 10,000-fold less active than creatine. In the reverse direction at 0.2 mM and pH 7.0: creatine-P greater than N-ethylguanidinoacetate-P greater than cyclocreatine-P much greater than homocyclocreatine-P. Homocyclocreatine-P was 200,000-fold less active than creatine-P. The phosphoryl group transfer potential of homocyclocreatine-P was estimated to be 2 kcal/mol lower than that of creatine-P. Chicks fed 5% homocyclocreatine for 16 days synthesized and accumulated homocyclocreatine-P in breast muscle (32 mumol/g wet wt), leg muscle (24 mumol/g), heart (7 mumol/g), intestine (8.5 mumol/g), and brain (2.4 mumol/g). During ischemia homocyclocreatine-P was utilized by muscle much more slowly for the regeneration of ATP than was creatine-P or cyclocreatine-P. Our results suggest that in tissues of homocyclocreatine-fed animals subjected to a sudden large increase in work load or to ischemia, the residual creatine-P system would rapidly equilibrate with the adenylate system at the new lower cytosolic phosphorylation potential, whereas in the same cytosol the (homocyclocreatine-P)/(homocyclocreatine) ratio would exhibit a hysteresis or memory effect and reflect for a considerable period of time the earlier higher (ATP)/(free ADP) ratio rather than the actual lower (ATP)/(free ADP) ratio.     

Effects of environmental factors on survival, growth, and production of American shad larvae

Episodic increases in temperature of 5°C above 20° C, over 48 h or declines in pH of 1·0 unit from pH 7·0 reduced survival of yolk-sac and feeding-stage larvae of American shad Alosa sapidissima . Over 16 days all measures of survival, growth, and production were more favourable at each higher temperature in the 15–25° C range. More favourable responses were also obtained at the higher prey level (500 v . 50 Artemia nauplii l^-1) and at the higher pH (7·5 v . 6·5). Combinations of high temperature and high prey levels, at pH 7·5, led to highest larval production. Little growth or production occurred at 15° C, regardless of pH or prey level. The effect of pH was strong with respect to survival, but weak with respect to growth. In attempts to restore American shad populations by larval stocking, release times and sites can be critical to optimize survival and eventual returns. Releases of larvae potentially will be most effective when made at temperatures >20° C, pH>7·0, and prey levels >50 1^-1. These conditions are most likely to occur in Maryland tributaries of Chesapeake Bay between mid-May and early June.     

The effect of temperature, pH and medium in a surface adhesion immunofluorescent technique for detection of Listeria monocytogenes

A rapid surface adhesion-based immunofluorescence technique was used to detect Listeria monocytogenes from inoculated culture systems. The effect of culture type (pure, mixed and meat), pH (7·00, 6·40, 4·76 and 3·13), acids (citric and HCl) and temperature (25°, 30° and 37°C) on the adhesion of Listeria to the polycarbonate membrane used in this technique was determined. It was found that pH had a significant effect ( P < 0·05) with higher numbers of Listeria adhering at low pH values (4·76). Culture type was also important with significantly higher numbers of Listeria ( P < 0·05) adhering to membranes immersed in meat cultures than in pure or mixed cultures. This effect was seen at 30°C but not at 25° or 37°C. The total viable count (TVC) on the membrane was unaffected by pH but temperature had an influence with optimum adhesion occurring at 25°C. The reasons for observed differences and their implications for the surface adhesion immunofluorescent rapid method are discussed.     

Adenine nucleotide degradation in the thoroughbred horse with increasing exercise duration

Adenine nucleotide (AN) degradation has been shown to occur during intense exercise in the horse and in man, at or close to the point of fatigue. The aim of the study was to compare the concentrations of muscle inosine 5'-monophosphate (IMP) and plasma ammonia (NH3) during intense exercise with the concentrations of muscle and blood lactate. Seven trained thoroughbred horses were used in the study. Each exercised on a treadmill for periods of between 30 s and 150 s, at 11 and/or 12 m.s-1. Blood and muscle samples were taken and analysed for lactate and NH3 and adenosine 5'-triphosphate (ATP), phosphorylcreatine (PCr), IMP, creatine, lactate and glycerol-3-phosphate respectively. Horses showed varying degrees of AN degradation as indicated by plasma [NH3] and muscle [ATP] and [IMP]. Comparisons of [IMP] with muscle [lactate], and plasma [NH3] with that of blood [lactate] indicated a threshold to the start of AN degradation. This threshold corresponded to a lactate content of around 80 mmol.kg-1 dry muscle and 15 mmol.l-1 in blood. We discuss the mechanisms which have been proposed to account for AN degradation and suggest that IMP formation occurs as a result of a sudden rise in the concentration of adenosine 5'-diphosphate (ADP) and consequently the concentration of adenosine 5'-monophosphate. The data suggest a critical pH below which there may be a substantial reduction in the kinetics of ADP rephosphorylation provided by PCr resulting in an increase in [ADP], which is the stimulus to AN degradation during intense exercise.     

Thermally induced phenotypic plasticity of swimming performance in European sea bass Dicentrarchus labrax juveniles

The vulnerability of embryonic and larval stages of European sea bass Dicentrarchus labrax to environmental temperature and the longer-term consequences for the early juveniles was demonstrated. This phenotypic plasticity was highlighted by subjecting D. labrax at 15·2 ± 0·3 or 20·0 ± 0·4° C (mean ± s . d .) up to metamorphosis and then at the same temperature (18·5 ± 0·7° C). After 4–6 weeks at the same temperature, the measurement of critical swimming speed at four exercise temperatures (15, 20, 25 and 28° C) showed a significantly higher swimming capacity in the fish initially reared at 15° C than for fish initially reared at 20° C. This performance was correlated with significant differences in the phenotype of red muscle. Thermally induced phenotypic plasticity was clearly demonstrated as an important mechanism controlling swimming performance in early juveniles of D. labrax .     

The critical thermal limits for juvenile Arctic charr Salvelinus alpinus

The chief objective was to determine the critical thermal limits for alevins, fry and parr of Arctic charr, Salvelinus alpinus , (L.) from four races living in Windermere (northwest England). The experimental fish were reared in a hatchery but were the progeny of wild parents. As comparisons between tethal temperatures at four acclimation temperatures (5, 10, 15, 20° C) revealed few significant racial differences, the data were pooled to estimate the lethal values for survival over 7 days (incipient lethal temperature) and over only 10 min (ultimate lethal temperature) for each life stage. Upper lethal values increased with acclimation temperatures for alevins but this effect was negligible for fry and parr, Alevins were generally less tolerant than fry and parr at lower, but not higher, acclimation temperatures; e.g. after acclimation at 5° C, mean upper ultimate values were 23·3, 25·1 and 25·7° C and mean upper incipient values were 18·7, 21·5 and 21·5° C for alevins, fry and parr respectively; after acclimation at 20° C, mean upper ultimate and incipient values were 26·2, 26·1 and 26·6° C and 20·8, 20·8 and 21·6° C for alevins, fry and parr respectively. The area of the temperature tolerance polygon (expressed as ° C²) for juvenile Arctic charr is amongst the lowest recorded for salmonids; being 409, 439 and 461° C² for alevins, fry and parr respectively. These low values are due to lower upper tolerance limits, not high lower tolerance limits; the latter being close to 0° C (<1°C for parr and fry, <0·3° C for alevins) at all acclimation temperatures. Arctic charr are therefore amongst the least resistant of salmonids to high temperatures but probably the most resistant to low temperatures.     

Effect of temperature on nucleotide pools and monoclonal antibody production in a mouse hybridoma

The specific monoclonal antibody productivity (q(Mab)) of a murine hybridoma (CC9C10) increased with incubation temperature in the range 33 degrees C to 39 degrees C. q(Mab) was constant at each temperature and was independent of the phase of culture. The q(Mab) increased 97% at 39 degrees C and decreased by 21% at 33 degrees C compared with controls at 37 degrees C. Specific rates of substrate (glucose and glutamine) utilization and byproduct (lactate and ammonia) formation also increased with temperature but the yield coefficient, Y(Lac/Llc') was constant for 33 degrees C to 39 degrees C and Y(Amm/Gin) was constant for 37 degrees C to 39 degrees C. Y(Amm/Gin) at 33 degrees C was lower than the control. Changes in specific nucleotide concentrations and ratios were monitored by analysis of intracellular nucleotide pools. The NTP ratio, (ATP + GTP)/(UTP + CTP), increased and the U-ratio (UTP/UDP-GNac) decreased during the course of each culture, whereas the adenylate energy charge, (ATP + 0.5ADP)/(ATP + ADP + AMP), remained relatively constant at a value 0.8. The relative content of UDP-/N acetyl galactosamine, UDP-N acetyl glucosamine, and NAD increased with incubation temperature, whereas the relative ATP content, SA(ATP + ADP + AMP)/SU (UTP + UDP-sugars) ratio, purine/pyrimidine, ATP/GTP, and U-ratio decreased at higher incubation temperatures. It is possible that these nucleotide parameters may have a regulatory role in the changes of q(Mab) observed at the higher temperatures. (c) 1994 John Wiley & Sons, Inc.     

Elevated red myotomal muscle temperatures in the most basal tuna species, Allothunnus fallai

The present study tested the hypothesis that Allothunnus fallai can elevate its slow-oxidative red myotomal muscle (RM) temperature. Measurements on 30 A. fallai (750–850 mm fork length) captured by hook and line off the coast of southern New Zealand revealed that RM temperatures are elevated by mean ± s . d . 8·1 ± 1·3° C (range 6·7–10·0° C) above the mean ± s . d . ambient sea surface temperature 15·3 ± 0·8° C (range 14·3 to 16·4° C). These data provide evidence that the vascular modifications to the central circulation of A. fallai act as a counter-current heat exchanger and that RM heat conservation is a character state present in all extant tuna species.     

Metabolic recovery in herring larvae following strenuous activity

Larvae of spring spawning Clyde herring Clupea harengus L. were reared at 5 and 12° C. Metabolism following burst swimming was studied in 7-day-old larvae at their respective rearing temperatures. Escape responses were repeatedly elicited using tactile stimulation for a period of 3 min. Larval herring were hard to fatigue and still responded to tactile stimuli after 3 min. Whole larvae were freeze-quenched in liquid nitrogen, either immediately after exercise, or after periods of recovery of up to 24 h. Samples were freeze-dried and analysed for whole body creatine (Cr), phosphocreatine (PCr), ATP, ADP, AMP, lactate, glucose, and glycogen using high performance liquid chromatography and enzymatic methods. The exercise regime resulted in a marked decrease in PCr, ATP and glycogen concentrations and an increase in creatine, glucose and lactate concentrations whereas there was no significant change in either AMP or ADP concentrations. The extent of phosphagen hydrolysis (approx. 110 to 15μmol PCr g ⁻¹ dry body mass) and lactate accumulation (approx. 7 to 40 μmol lactate g⁻¹ dry body mass) over the exercise period was similar at the two temperatures, consistent with a relatively constant degree of effort. The rates of recovery of PCr and ATP were essentially the same at 5 and 12° C; returning to resting levels after approximately 30 min. Lactate and glycogen concentrations were restored 60 min after exercise at both temperatures. Maximum lactate clearance rates (1.2 μmol min ⁻¹ g ⁻¹ wet muscle mass) were an order of magnitude faster than reported for adult fish in the literature.     

Impacts of treatment parameters on the inactivation of Campylobacter jejuni by high pressure: a statistical study of main effects and interactions

Aim:  The influence of environmental (temperature and pH) and biological (strain) parameters on the inactivation of Campylobacter jejuni by high hydrostatic pressure (HHP) was investigated.
Methods and Results:  Two clinical strains harvested in stationary phase were pressurized at 20°C and 37°C within a range of 50–400 MPa, in a phosphate (pH 7·0) or a citrate phosphate buffer (pH 5·6), for 10 min. Treatment efficiencies were determined by logarithmic comparisons of culturable cells on blood agar before and after treatment. Results were statistically compared using an anova of culturable cells after treatment to evaluate the effect of all factors. At least a 7-log reduction in cell numbers was observed for both strains. The pH and the strains had no effect on HHP treatment at 20°C while at 37°C, both pH and strain influenced significantly the HHP treatment on C. jejuni .
Conclusions:  The pressure efficacy on C. jejuni eradication was affected by both environmental and biological factors.
Significance and Impact of the Study:  Depending on the treatment conditions, C. jejuni sensitivity to HHP can significantly vary. The determination of the inactivation treatment by HPP has to be normalized considering the interaction of environmental and biological factors.     

Thermal tolerance of a northern population of striped bass Morone saxatilis

Thermal tolerance of age 0+ year Shubenacadie River (Nova Scotia, Canada) striped bass Morone saxatilis juveniles (mean ± s . e . fork length, L _F, 19·2 ± 0·2 cm) acclimated in fresh water to six temperatures from 5 to 30° C was measured by both the incipient lethal technique (72 h assay), and the critical thermal method ( C _m). The lower incipient lethal temperature ranged from 2·4 to 11·3° C, and the upper incipient lethal temperature ( I _U) from 24·4 to 33·9° C. The area of thermal tolerance was 618° C². In a separate experiment, the I _U of large age 2+ year fish (34·4 ± 0·5 cm L _F) was 1·2 and 0·6° C lower ( P < 0·01) than smaller age 1+ year fish (21·8 ± 0·5 cm L _F) at acclimation temperatures of 16 and 23° C. Using the C _m, loss of equilibrium occurred at 27·4–37·7° C, loss of righting response at 28·1–38·4° C and onset of spasms at 28·5–38·8° C, depending on acclimation temperature. The linear regression slopes for these three responses were statistically similar (0·41; P > 0·05), but the intercepts differed (25·3, 26·0 and 26·5° C; P < 0·01). The thermal tolerance of this northern population appears to be broader than southern populations.     

Significance of temperature and preincubation temperature on survival of Listeriamonocytogenes at pH 4·8

Listeria monocytogenes is a food-borne pathogenic bacterium that can be found in softcheese. At the beginning of cheese ripening, the pH is about 4·85–4·90. The aimof this work was to study the influence of temperature, preincubation temperature (temperature atwhich the inoculum was cultivated) and initial bacterial concentration on the survival of L.monocytogenes (strain Scott A) at pH 4·8. It was demonstrated in an earlier study thatthese factors did influence growth kinetics. Survival studies of L. monocytogenes weredone in a laboratory broth simulating cheese composition. Four test temperatures (2, 6, 10 and14°C) and two preincubation temperatures were studied (30°C or the test temperature). Listeria monocytogenes (strain Scott A) was unable to grow at pH 4·8 under allconditions tested. The time for 10% survival was about 11 and 2 d, at 2°C with preincubationat 2°C and 30°C, respectively; 9 d at 6°C with preincubation at 6°C; 4 d at 6°Cwith preincubation at 30°C; and 1 d at 14°C with preincubation at 14°C or at 30°C.The results show that survival of L. monocytogenes (strain Scott A) at pH 4·8 is notdependent on initial bacterial concentration but on both the test and preincubation temperatures.     

Purification and some properties of the citrate synthase from a marine Pseudomonas.

Citrate synthase (citrate-oxaloacetate lyase (CoA acetylating), EC 4.1.3.7) has been purified to electrophoretic homogeneity from a marine Pseudomonas. The enzyme was made up of identical subunits, with a molecular wieght of about 53 000, as determined by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. The native enzyme (citrate synthase II, CS II) could be dissociated by dialysis against 20 mM phosphate (Pi), pH 7; the enzyme thus obtained (citrate synthase I, CS I) was still active, but presented different molecular weight and kinetic and regulatory properties. CS II was activated by adenosine monophosphate (AMP), Pi, and KCl, and inhibited by reduced nicotinamide adenine dinucleotide (NADH), being apparently insensitive to adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The inhibition by NADH was completely counteracted by 0.1 mM AMP, but not by 50 mM Pi or 0.1 M KCl. The activation by KCl and Pi, or by KCl and AMP was nearly additive, whereas that by AMP and Pi was not. The activators acted essentially by increasing Vmax, although they also caused a decrease in the Km values. CS I was inhibited by ATP, ADP, AMP, and KCl, and was insensitive to NADH. CS I could be reassociated after elimination of Pi by dialysis, regaining the higher molecular weight and the activation by AMP characteristic of CS II.     

Reaction mechanism of the magnesium ion-dependent adenosine triphosphatase of frog muscle myosin and subfragment 1.

The Mg2+-dependent ATPase (adenosine 5'-triphosphatase) mechanism of myosin and subfragment 1 prepared from frog leg muscle was investigated by transient kinetic technique. The results show that in general terms the mechanism is similar to that of the rabbit skeletal-muscle myosin ATPase. During subfragment-1 ATPase activity at 0-5 degrees C pH 7.0 and I0.15, the predominant component of the steady-state intermediate is a subfragment-1-products complex (E.ADP.Pi). Binary subfragment-1-ATP (E.ATP) and subfragment-1-ADP (E.ADP) complexes are the other main components of the steady-state intermediate, the relative concentrations of the three components E.ATP, E.ADP.Pi and E.ADP being 5.5:92.5:2.0 respectively. The frog myosin ATPase mechanism is distinguished from that of the rabbit at 0-5 degrees C by the low steady-state concentrations of E.ATP and E.ADP relative to that of E.ADP.Pi and can be described by: E + ATP k' + 1 in equilibrium k' - 1 E.ATP k' + 2 in equilibrium k' - 2 E.ADP.Pi k' + 3 in equilibrium k' - 3 E.ADP + Pi k' + 4 in equilibrium k' - 4 E + ADP. In the above conditions successive forward rate constants have values: k' + 1, 1.1 X 10(5)M-1.S-1; k' + 2 greater than 5s-1; k' + 3, 0.011 s-1; k' + 4, 0.5 s-1; k'-1 is probably less than 0.006s-1. The observed second-order rate constants of the association of actin to subfragment 1 and of ATP-induced dissociation of the actin-subfragment-1 complex are 5.5 X 10(4) M-1.S-1 and 7.4 X 10(5) M-1.S-1 respectively at 2-5 degrees C and pH 7.0. The physiological implications of these results are discussed.     

Effect of pH and inorganic phosphate on creatine kinase inactivation: an in vitro 31P NMR saturation-transfer study.

The pseudo-first-order rate constant of rabbit muscle creatine kinase (CK), in the direction of ATP synthesis (kf), was determined by saturation-transfer 31P NMR. When pH was varied between 6.0 and 7.4, kf increased linearly at both 20 degrees C and 37 degrees c. The corresponding flux is very small between pH 6.0 and 6.5, in contrast to previous studies. Up to 50 h exposure of the CK enzyme to high concentrations of inorganic phosphate (Pi), a known inhibitor in certain situations, had negligible effect on enzymatic flux in the physiological pH range. Thus under in vivo conditions, such as in stroke, where pH falls as low as 6.2 and Pi rises to high levels, the rate of the CK reaction may be severely reduced due to pH but not due to high Pi concentrations.     

Behavioural thermoregulation by subyearling fall (autumn) Chinook salmon Oncorhynchus tshawytscha in a reservoir

This study investigated behavioural thermoregulation by subyearling fall (autumn) Chinook salmon Oncorhynchus tshawytscha in a reservoir on the Snake River, Washington, U.S.A. During the summer, temperatures in the reservoir varied from 23° C on the surface to 11° C at 14 m depth. Subyearlings implanted with temperature-sensing radio transmitters were released at the surface at temperatures >20° C during three blocks of time in summer 2004. Vertical profiles were taken to measure temperature and depth use as the fish moved downstream over an average of 5·6–7·2 h and 6·0–13·8 km. The majority of the subyearlings maintained average body temperatures that differed from average vertical profile temperatures during most of the time they were tracked. The mean proportion of the time subyearlings tracked within the 16–20° C temperature range was larger than the proportion of time this range was available, which confirmed temperature selection opposed to random use. The subyearlings selected a depth and temperature combination that allowed them to increase their exposure to temperatures of 16–20° C when temperatures <16 and >20° C were available at lower and higher positions in the water column. A portion of the subyearlings that selected a temperature c. 17·0° C during the day, moved into warmer water at night coincident with an increase in downstream movement rate. Though subyearlings used temperatures outside of the 16–20° C range part of the time, behavioural thermoregulation probably reduced the effects of intermittent exposure to suboptimal temperatures. By doing so, it might enhance growth opportunity and life-history diversity in the population of subyearlings studied.     

Synthesis of ATP and free radicals in an aqueous solution, containing NADH, riboflavin, ADP and inorganic phosphate

pH-dependence of initial (admixture) adenosine triphosphate (ATP) changes (ATP synthesis and hydrolysis) was studied for aerated and deaerated aqueous solutions during the incubation of adenosine diphosphate (ADP) and inorganic phosphate (Pi) in the presence of reduced nicotinamide adenine dinucleotide (NADH) and riboflavin (Rf). The preferential pH regions were indicated both for ATP synthesis and ATP hydrolysis (pH less than pH 5.4, 5.9 divided by pH 6.5 and pH greater than 6.6, pH 5.5 divided by pH 5.8 respectively). 'Free radical content measurements were paralleled by ESR technique. On the basis of the obtained results it was assumed that a part of ESP signal attributed to ADP radicals was increased during ATP synthesis.     

Temperature magnified postcapture mortality in adult sablefish after simulated trawling

For sablefish Anoplopoma fimbria that had been transferred abruptly from ambient (5·7° C) to temperatures ranging from 15 to 20° C for 30 min followed by 15 min in air (19·5) C), mortality increased with temperature. Mortality occurred at lower temperatures for sablefish that were net-towed for 4 h at ambient temperature before exposure to a rapid increase in temperature. A clear relationship was apparent between serum lactate and temperature with lactate increasing as temperature increased. For treatments in which mortality did not occur, lactate decreased sharply within 24 h, suggesting recovery. It would appear that the critical postcapture temperature for sablefish that reside and are captured at 4-6° C, would be between 12 and 15° C. The results of this study suggest that fishery management strategies designed to increase postcapture survival of sablefish bycatch should include a consideration of the impact of exposure to seasonal thermoclines and seasonally elevated air temperatures.     

Histochemical demonstration of ATP: creatine phosphotransferase in rat skeletal muscle

Summary An attempt was made to locate the ATP: creatine phosphotransferase (creatine kinase, CKase) in rat skeletal muscle by a lead precipitation method. The muscle is not stained at all with creatine phosphate (CP), and only weakly with adenosine diphosphate (ADP) as substrate, while it hydrolyzes adenosine triphosphate (ATP) actively. Taking advantage of this fact, it is possible to demonstrate the CKase activity using both ADP and CP as substrate. The CKase activity thus obtained was located in various profiles of sarcoplasmic reticulum as well as in A bands, the staining being comparable to that obtained with ATP as substrate.A weak activity was found only in cisternal dilatations of sarcoplasmic reticulum when sections were incubated with ADP as substrate.     

Cytosolic adenylates and adenosine release in perfused working heart. Comparison of whole tissue with cytosolic non-aqueous fractionation analyses

Free cytosolic adenylates were examined in relation to adenosine plus inosine released from perfused working guinea-pig hearts. Whole-tissue adenylate data from freeze-clamped hearts were quantitatively compared with corresponding values obtained by subcellular fractionation of homogenized myocardium in non-aqueous media. Adenosine and inosine in venous cardiac effluents were measured by high-performance liquid chromatography. Hearts, perfused at their natural flows, were subjected to various workloads, substrates and catecholamines to alter myocardial energy metabolism and respiration over a wide physiological range. Non-aqueous cytosolic ATP and creatine phosphate (CrP) accounted for more than 80% of the respective total myocardium content. The cytosolic CrP/Pi ratio was in near-quantitative agreement with the overall tissue CrP/Pi ratio when the latter parameter was corrected for extracellular Pi. This was conclusive evidence that ATP, CrP and Pi were predominantly located in the cytosol of the well-oxygenated cardiomyocyte. Measured myocardial oxygen uptake (MVO2) was reciprocally related to the phosphorylation state of CrP [( CrP]/[Cr] X [Pi]) and hence that of ATP [( ATP]/[ADP] X [Pi]) assuming the creatine kinase at near-equilibrium at a near-constant pH of 7.2. On the other hand, calculated mean free cytosolic ADP concentrations increased essentially linearly up to threefold with increasing MVO2 in the presence of virtually unchanged or only slightly decreased ATP levels; this was found both according to the whole tissue and the special subcellular fractionation data. Employing the myokinase mass-action ratio and substituting total cardiac ADP by the mean free cytosolic ADP concentrations, the mean free cytosolic AMP concentrations proved to be in the nanomolar range, i.e. up to three orders of magnitude lower than the overall tissue AMP content. We propose, therefore, that in the normoxic heart, AMP is located predominantly in the mitochondrial compartment. Nevertheless, both free cytosolic AMP concentration and release of adenosine plus inosine were apparently square or even higher-power functions of the rate of cardiac respiration. On the other hand, the mean purine nucleoside release seemed linearly correlated (r = 0.920) with the calculated free cytosolic AMP concentration. Our observations seem to suggest that the concentrations of free ADP and AMP in the cytosol are major determinants of the production of inosine and coronary vasodilator adenosine.(ABSTRACT TRUNCATED AT 400 WORDS)