A 31P-nuclear magnetic resonance study of skeletal muscle metabolism in rats depleted of creatine with the analogue β-guanidinopropionic acid

Rats were fed a diet containing 1% β-guanidinopropionic acid (GPA) for 6–10 weeks to deplete their skeletal muscle of creatine. ³¹P-NMR was used to monitor metabolic changes in the gastrocnemius muscle at rest, during stimulated steady-state isometric contraction at 4 Hz and during recovery from stimulation. In resting muscles, the [creatine phosphate] was reduced to 10% (2.8 μmol·g^?1) and the [ATP] to 50% (3.3 μmol·g^?1) of those found in rats fed a control diet. The concentration of the phosphorylated form of the analogue (PGPA) was 23 μmol·g^?1. There was no significant difference in muscle performance or in the relative changes in the [ATP] during stimulation. Intracellular pH decreased rapidly on stimulation and recovered during the stimulation period to near resting values in both groups. In control rats, the initial decrease in pH was greater and the time to recovery was longer than in GPA-fed rats. The rate at which PGPA supplied energy to the contracting muscle (0.027 mM·s^?1) was insignificant relative to the minimum estimated rate of ATP turnover (1 mM·s^?1). The rate of PGPA resynthesis during recovery (0.018 mM·s^?1) is enzyme-limited and provides an independent estimate of creatine kinase flux during this period (18.9 mM·s^?1). The creatine kinase flux (creatine phosphate → ATP) in the resting muscle of GPA-fed rats was 12-fold less than in control animals, 1.3 vs. 15.7 mM·s^?1. These results demonstrate that neither the [creatine phosphate] nor the activity of creatine kinase is critical for aerobic metabolism. Skeletal muscle appears to adapt to a diminished creatine pool by enhancing its aerobic capacity.     

Regulation of creatine kinase during steady-state isometric twitch contraction in rat skeletal muscle

In vivo 31P-NMR saturation transfer measurements of the creatine kinase exchange flux in the direction creatine phosphate----ATP were made in the gastrocnemius muscle of rats at rest and during steady-state isometric twitch contraction at frequencies from 0.25 to 2 Hz. There was no correlation between creatine kinase exchange flux and either free [ADP] or oxygen consumption, both of which increase with stimulation frequency. The flux was found to be nearly constant over all conditions at about 16 mM X s-1, 10-times greater than the highest estimated ATP turnover in this study. The kinetic properties of skeletal muscle creatine kinase in vivo are similar to, but not completely predictable from, the equilibrium exchange fluxes measured on the isolated enzyme. These results are not consistent with strong functional coupling between ATP synthesis and mitochondrial creatine kinase.     

The activity of creatine kinase in frog skeletal muscle studied by saturation-transfer nuclear magnetic resonance.

1. The activity of creatine kinase in intact anaerobic frog muscle at 4 degrees C at rest and during contraction was investigated by using saturation-transfer 31P n.m.r. 2. At rest, the measured forward (phosphocreatine to ATP) reaction flux was 1.7 X 10(-3) M . s-1 and the backward flux was 1.2 X 10(-3) M . s-1. The large magnitude of both fluxes shows that creatine kinase is active in resting muscle, so the observed constancy of [phosphocreatine] demonstrates that the enzyme and its substrates are at equilibrium. 3. The apparent discrepancy between the fluxes must arise largely from an underestimation of the backward flux resulting from interaction of ATP with other systems, e.g. via adenylate kinase. For purposes of further calculation we have therefore adopted 1.6 X 10(-3) M . s-1 as an estimate of both fluxes. 4. During contraction, when the creatine kinase reaction is no longer at equilibrium, the net rate of phosphocreatine breakdown, estimated directly from the change in area of the inorganic phosphate peak, was 0.75 X 10(-3) M . s-1. Saturation transfer indicates that the forward reaction flux remains at approx. 1.6 X 10(-3) M . s-1 and the backward flux decreases to about 0.85 X 10(-3) M . s-1. 5. The activity of creatine kinase during contraction is large enough to account for the well-established observation that, during contraction, the concentration of ATP falls by less than 2-3%. The reaction catalysed by creatine kinase is driven forward during contraction by the large relative increase in the concentration of free ADP, which is more than doubled. 6. The observation that the forward flux does not increase during contraction and that the backward flux decreases can most simply be explained on the basis of competition of reactants for a limited amount of enzyme.     

Biochemical adaptation in the skeletal muscle of rats depleted of creatine with the substrate analogue beta-guanidinopropionic acid.

Rats were fed on a diet containing 1% beta-guanidinopropionic acid (GPA), a creatine substrate analogue, for 6-10 weeks to deplete their muscle of creatine. This manipulation was previously shown to give a 90% decrease in [phosphocreatine] in skeletal and cardiac muscle and a 50% decrease in [ATP] in skeletal muscle only. Maximal activities of creatine kinase and of representative enzymes of aerobic and anaerobic energy metabolism were measured in the superficial white, medial and deep red portions of the gastrocnemius muscle, in the soleus and plantaris muscle and in the heart. Fast-twitch muscles were smaller in GPA-fed animals than in controls, but the size of the soleus muscle was unchanged. The activities of aerobic enzymes increased by 30-40% in all fast-twitch muscle regions except the superficial gastrocnemius, but were unchanged in the soleus muscle. The activities of creatine kinase and phosphofructokinase decreased by 20-50% in all skeletal-muscle regions except the deep gastrocnemius, and the activity of glycogen phosphorylase generally paralleled these changes. There were no significant changes in the activities of any of the enzymes measured in the heart. The glycogen content of the gastrocnemius-plantaris complex was increased by 185% in GPA-fed rats. The proportion of Type I fibres in the soleus muscle increased from 81% in control rats to 100% in GPA-fed rats, consistent with a previous report of altered isometric twitch characteristics and a decrease in the maximum velocity of shortening in this muscle [Petrofsky & Fitch (1980) Pflugers Arch. 384, 123-129]. We conclude that fast-twitch muscles adapt by a combination of decreasing diffusion distances, increasing aerobic capacity and decreasing glycolytic potential. Slow-twitch muscles decrease glycolytic potential and become slower, thus decreasing energy demand. These results suggest that persistent changes in the [phosphocreatine] and [ATP] are alone sufficient to alter the expression of enzyme proteins and proteins of the contractile apparatus, and that fibre-type-specific thresholds exist for the transformation response.     

Energy balance studies in frog skeletal muscles shortening at one-half maximal velocity

High-energy phosphate metabolism and energy liberated as heat and work were measured in 3-s tetani of frog sartorius muscle at 0 degree C. Two contraction periods were studied: (a) a 0.35-s period of shortening near half-maximum velocity beginning after 2 s of isometric stimulation, and (b) a 0.65-s isometric period immediately following the shortening. There were no significant changes in levels of ATP, ADP, or AMP in the two contraction periods. The observed changes in inorganic phosphate and creatine levels indicated that the only significant reaction occurring was phosphocreatine splitting. The mean rate of high-energy phosphate splitting during the shortening, 1.60 +/- 0.23 mumol X g-1 X s-1 (n = 24), was about fivefold higher than that in the 1-s period in the isometric tetanus, 0.32 +/- 0.11 mumol X g-1 X s- 1 (n = 17), observed in our previous study. The mean rate in the post- shortening period, 0.46 +/- 0.13 mumol X g-1 X s-1 (n = 17), was not significantly different from that in the 1-s period in the isometric tetanus. A large amount of heat plus work was produced during the shortening period, and this could be accounted for by simultaneous chemical changes. In the post-shortening period, the observed enthalpy was also accounted for by simultaneous chemical reactions. Thus, the present result is in sharp contrast to that obtained from a similar study performed at a shortening at Vmax, where an enthalpy excess was produced during shortening and an enthalpy deficit was produced during the period following the shortening.     

31P-saturation-transfer nuclear-magnetic-resonance measurements of phosphocreatine turnover in guinea-pig brain slices.

The technique of 31P saturation-transfer n.m.r. was used to determine the forward and the reverse rate constants of creatine phosphotransferase in superfused guinea-pig cerebral tissues in vitro. The calculated forward rate constant of 0.22 +/- 0.03s-1 compared well with a previously reported value for rat brain in vivo [Shoubridge, Briggs & Radda (1982) FEBS Lett. 140, 288-292]. The reverse rate constant was found to be 0.55 +/- 0.10s-1. 3. By using concentrations of ATP and phosphocreatine estimated previously for this superfused preparation [Cox, Morris, Feeney & Bachelard (1983) Biochem. J. 212, 365-370], forward and reverse flux rates were calculated to be 0.68 and 0.72 mumol X s-1 X g-1 respectively. The concordance of forward and reverse fluxes contrasts with the situation observed in vitro in other tissues, and suggests that the creatine phosphotransferase reaction is at equilibrium under the conditions used here. 4. Lowering the concentration of glucose in the superfusing medium from 10mM to 0.5mM had no significant effect on phosphocreatine concentration or on the forward (ATP-generating) flux through creatine phosphotransferase. The results indicate that a normal phosphocreatine content in the presence of lowered glucose availability is reflected by an unchanged turnover rate.     

Combined glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase in catecholamine-stimulated guinea-pig cardiac muscle. Comparison with mass-action ratio of creatine kinase.

The steady-state reactant levels of triose-phosphate isomerase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea-pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3-phosphate were directly measured and compared with those of creatine kinase. Non-working Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near-equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP].[Pi])) was derived from creatine kinase and the free [NAD+]/([NADH].[H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near-zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose-phosphate isomerase was near-equilibrium only in control or potassium-arrested Langendorff hearts as well as in postischemic 'stunned' hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure-volume work the enzyme was displaced from equilibrium. The alternative phosphorylation ratio [ATP]'/([ADP]).[Pi]) was derived from the magnesium-dependent glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1-2.0 mM). As predicted, [ATP]/([ADP].[Pi]) and [ATP]'/([ADP]'.[Pi]') were in excellent agreement when glycolysis was virtually halted by hyperkalemic arrest (flux approximately 0.2 mumol C3.min-1.g dry mass-1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux approximately 5-14 mumol C3.min-1.g dry mass-1). In contrast, when flux increased due to transition from no-work to physiological pressure-volume work (rate increase from approximately 3 to 11 mumol C3.min-1.g dry mass-1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial 'stunning', not due to hydraulic work load per se, glyceraldehyde-3-phosphate levels increased from about 4 microM to greater than or equal to 16 microM. Thus the guinea-pig cardiac glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near-equilibrium catalysis at significant flux provided glyceraldehyde-3-phosphate levels rise, e.g., due to 'stunning' or adrenergic hormones.     

Activity of creatine kinase in a contracting mammalian muscle of uniform fiber type.

We investigated whether the creatine kinase-catalyzed phosphate exchange between PCr and gamma ATP in vivo equilibrated with cellular substrates and products as predicted by in vitro kinetic properties of the enzyme, or was a function of ATPase activity as predicted by obligatory "creatine phosphate shuttle" concepts. A transient NMR spin-transfer method was developed, tested, and applied to resting and stimulated ex vivo muscle, the soleus, which is a cellularly homogeneous slow-twitch mammalian muscle, to measure creatine kinase kinetics. The forward and reverse unidirectional CK fluxes were equal, being 1.6 mM.s-1 in unstimulated muscle at 22 degrees C, and 2.7 mM.s-1 at 30 degrees C. The CK fluxes did not differ during steady-state stimulation conditions giving a 10-fold range of ATPase rates in which the ATP/PCr ratio increased from approximately 0.3 to 1.6. The observed kinetic behavior of CK activity in the muscle was that expected from the enzyme in vitro in a homogeneous solution only if account was taken of inhibition by an anion-stabilized quaternary dead-end enzyme complex: E.Cr.MgADP.anion. The CK fluxes in soleus were not a function of ATPase activity as predicted by obligatory phosphocreatine shuttle models for cellular energetics.     

Gluconeogenesis predominates in periportal regions of the liver lobule

Rates of gluconeogenesis from lactate were calculated in periportal and pericentral regions of the liver lobule in perfused rat livers from increases in O2 uptake due to lactate. When lactate (0.1-2.0 mM) was infused into livers from fasted rats perfused in either anterograde or the retrograde direction, a good correlation (r = 0.97) between rates of glucose production and extra O2 uptake by the liver was observed as expected. Rates of oxygen uptake were determined subsequently in periportal and pericentral regions of the liver lobule by placing miniature oxygen electrodes on the liver surface and measuring the local change in oxygen concentration when the flow was stopped. Basal rates of oxygen uptake of 142 +/- 11 and 60 +/- 4 mumol X g-1 X h-1 were calculated for periportal and pericentral regions, respectively. Infusion of 2 mM lactate increased oxygen uptake by 71 mumol X g-1 X h-1 in periportal regions and by 29 mumol X g-1 X h-1 in pericentral areas of the liver lobule. Since the stoichiometry between glucose production and extra oxygen uptake is well-established, rates of glucose production in periportal and pericentral regions of the liver lobule were calculated from local changes in rates of oxygen uptake for the first time. Maximal rates of glucose production from lactate (2 mM) were 60 +/- 7 and 25 +/- 4 mumol X g-1 X h-1 in periportal and pericentral zones of the liver lobule, respectively. The lactate concentrations required for half-maximal glucose synthesis were similar (0.4-0.5 mM) in both regions of the liver lobule in the presence or absence of epinephrine (0.1 microM). In the presence of epinephrine, maximal rates of glucose production from lactate were 79 +/- 5 and 59 +/- 3 mumol X g-1 X h-1 in periportal and pericentral regions, respectively. Thus, gluconeogenesis from lactate predominates in periportal areas of the liver lobule during perfusion in the anterograde direction; however, the stimulation by added epinephrine was greatest in pericentral areas. Differences in local rates of glucose synthesis may be due to ATP availability, as a good correlation between basal rates of O2 uptake and rates of gluconeogenesis were observed in both regions of the liver lobule in the presence and absence of epinephrine. In marked contrast, when livers were perfused in the retrograde direction, glucose production was 28 +/- 5 mumol X g-1 X h-1 in periportal areas and 74 +/- 6 mumol X g-1 X h-1 in pericentral regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Substrate regulation of the sarcoplasmic reticulum ATPase. Transient kinetic studies.

The rate of phosphorylation of the Ca2+-dependent ATPase of sarcoplasmic reticulum vesicles by ITP and ATP was studied using a millisecond mixing and quenching device. The rate of phosphorylation was slower when the vesicles were preincubated in a Ca2+-free medium than when preincubated with Ca2+, regardless of the substrate used and of the pH of the medium. When the vesicles were preincubated with Ca2+ at pH 7.4 an overshoot of phosphorylation was observed in the presence of ITP. The overshoot was abolished when the pH of the medium was decreased to 6.0 or when the vesicles were preincubated in a Ca2+-free medium. Using vesicles preincubated with Ca2+ the apparent Km for ITP found was 2.5 mM at pH 6.0 and 1.0 mM at pH 7.4. The Vmax observed (77 mumol g-1 s-1) did not change with the pH of the medium. Both at pH 6.0 and 7.4 the apparent Km for ATP was 3 microM when preincubated in a Ca2+-free medium. At pH 6.0 the Vmax for ATP varied from 96 to 33 mumol g-1 s-1 depending on whether the vesicles were preincubated in the presence or absence of Ca2+. At pH 7.4 the Vmax for ATP was 90 mumol g-1 s-1 in both conditions. The rate of phosphorylation of the vesicles was dependent on the relative Ca2+ and Mg2+ concentrations of the reaction medium regardless of the substrate used.     

Facilitated Transport of Glucose from Blood into Peripheral Nerve

D-Glucose is the major substrate for energy metabolism in peripheral nerve. The mechanism of transfer of glucose across the blood-nerve barrier is unclarified. In this study an in situ perfusion technique was utilized, in anesthetized rats, to examine monosaccharide transport from blood into peripheral nerve. Unidirectional influxes of D-[14C]glucose, L-[14C]glucose, and [14C]3-O-methyl-D-glucose across capillaries of the tibial nerve were measured at different perfusate concentrations of unlabelled D-glucose. The permeability-surface area product (PA) for D-[14C]glucose and [14C]3-O-methyl-D-glucose decreased, whereas the PA for L-[14C]glucose remained constant, as the perfusate concentration of D-glucose was increased. In the presence of no added unlabelled D-glucose in the perfusate, the PA for L-[14C]glucose equaled one-fifth the PA for D-[14C]glucose. These results demonstrate self-saturation, competitive inhibition, and stereospecificity of glucose transfer, and for the first time show a unidirectional facilitated transport mechanism for D-monosaccharides at capillaries of mammalian peripheral nerve. The data were fit to a model for facilitated transport and passive diffusion. The half-saturation constant and maximal rate of transport for the saturable component of D-glucose influx equaled 23 +/- 11 mumol X ml-1 and 6.6 +/- 3.2 X 10(-3) mumol X s-1 X g-1, respectively. The constant of nonsaturable glucose influx equaled 0.5 +/- 0.1 X 10(-4) s-1. At normal plasma glucose concentrations, the saturable component comprises about 80% of total D-glucose influx into nerve.(ABSTRACT TRUNCATED AT 250 WORDS)     

The polysome as a terminal for the creatine phosphate energy shuttle

The role of the creatine phosphate shuttle in the energetics of muscle protein synthesis in isolated polysomes, from rat hindlimb muscle, was studied. Triton X-100-treated polysomes, following their centrifugation through a 1 M sucrose gradient, contained 38 mU/mg RNA of bound creatine kinase. In the presence of pH 5 enzyme (obtained from rat liver), 0.5 mM ATP, and 1 microM GTP, amino acid (leucine) incorporation by polysomes in the presence of 8 mM creatine phosphate was twice that in the presence of an exogenous ATP regenerating system of 10 mM phospho(enol)pyruvate and 10 U/ml pyruvate kinase. Since added creatine kinase had no effect on incorporation supported by creatine phosphate it is clear that endogenous creatine kinase allows sufficient regeneration of ATP. These data also suggest that nucleoside diphosphokinase must have been associated with the polysome for phosphate was transferred to GTP from [33P]creatine phosphate, and the specific activities of ATP and GTP increased at equal rates, reaching the specific activity of creatine phosphate at 8 min. We conclude that skeletal muscle polysomes have bound creatine kinase activity and they act as terminals for the creatine phosphate energy shuttle. Creatine phosphate regenerates GTP, probably through an intermediate reaction catalyzed by nucleoside diphosphokinase. This provided an added support for the hypothesis of compartmentation of enzymes and substrates and that the transport form of energy between the mitochondria and energy utilizing sites in muscle is creatine phosphate rather than ATP, which extends the general role of the creatine phosphate energy shuttle.     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart. 31P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by 31P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in phosphocreatine/creatine ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg2+ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 mumol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in [phosphocreatine]/[creatine] ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.     

Inhibition of hepatic proteolysis by insulin. Role of hormone-induced alterations of the cellular K+ balance

1. Proteolysis was measured as [3H]leucine release from isolated perfused livers from rats, which had been labeled in vivo by an intraperitoneal injection of [3H]leucine about 16 h prior to the perfusion experiment. In livers from fed rats, insulin (35 nM) inhibited [3H]leucine release by 24.5 +/- 1.3% (n = 15) and led to an amiloride-sensitive, bumetanide-sensitive and furosemide-sensitive net K+ uptake of 5.53 +/- 0.31 mumol.g-1 (n = 15). Both the insulin effects on net K+ uptake and on [3H]leucine release were diminished by about 65% or 55% in presence of furosemide (0.1 mM) or bumetanide (5 microM), respectively. The insulin-induced net K+ uptake was virtually abolished in the presence of amiloride (1 mM) plus furosemide (0.1 mM). 2. In perfused livers from 24-h-starved rats, both the insulin-stimulated net K+ uptake and the insulin-induced inhibition of [3H]leucine release were about 80% lower than observed in experiments with livers from fed rats. The insulin effects on K+ balance and [3H]leucine release were not significantly influenced in the presence of glycine (2 mM), although glycine itself inhibited [3H]leucine release by 30.3 +/- 0.3% (n = 4) and 13.8 +/- 1.2% (n = 5) in livers from starved and fed rats, respectively. When livers from fed rats were preswollen by hypoosmotic perfusion (225 mOsmol.l-1), both the insulin-induced net K+ uptake and the inhibition of [3H]leucine release were diminished by 50-60%. 3. During inhibition of [3H]leucine release by insulin, further addition of glucagon (100 nM) led to a marked net K+ release from the liver (3.82 +/- 0.24 mumol.g-1), which was accompanied by stimulation of [3H]leucine release by 16.4 +/- 4.6% (n = 4). 4. Ba2+ (1 mM) infusion led to a net K+ uptake by the liver of 3.2 +/- 0.2 mumol.g-1 (n = 4) and simultaneously inhibited [3H]leucine release by 12.4 +/- 1.7% (n = 4). 5. There was a close relationship between the Ba2+ or insulin-induced net K+ uptake and the degree of inhibition of [3H]leucine release, even when the K+ response to insulin was modulated by bumetanide, furosemide, glucagon, hypotonic or glycine-induced cell swelling or the nutritional state. 6. The data suggest that the insulin-induced net K+ uptake involves activation of both NaCl/KCl cotransport and Na+/H+ exchange.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium influx in internally dialyzed squid giant axons

A method has been developed to measure Ca influx in internally dialyzed squid axons. This was achieved by controlling the dialyzed segment of the axon exposed to the external radioactive medium. The capacity of EGTA to buffer all the Ca entering the fiber was explored by changing the free EGTA at constant [Ca++]i. At a free [EGTA]i greater than 200 microM, the measured resting Ca influx and the expected increment in Ca entry during electrical stimulation were independent of the axoplasmic free [EGTA]. To avoid Ca uptake by the mitochondrial system, cyanide, oligomycin, and FCCP were included in the perfusate. Axons dialyzed with a standard medium containing: [ATP] = 2 mM, [Ca++]i = 0.06 microM, [Ca++]o = 10 mM, [Na+]i = 70 mM, and [Na+]o = 465 mM, gave a mean Ca influx of 0.14 +/- 0.012 pmol.cm-2.s-1 (n = 12. Removal of ATP drops the Ca influx to 0.085 +/- 0.007 pmol.cm-2.s-1 (n = 12). Ca influx increased to 0.35 pmol.cm-2,s-1 when Nao was removed. The increment was completely abolished by removing Nai+ and (or) ATP from the dialysis medium. At nominal zero [Ca++]i, no Nai-dependent Ca influx was observed. In the presence of ATP and Nai [Ca++]i activates the Ca influx along a sigmoid curve without saturation up to 1 microM [Ca++]i. Removal of Nai+ always reduced the Ca influx to a value similar to that observed in the absence of [Ca++]i (0.087 +/- 0.008 pmol.cm-2.s-1; n = 11). Under the above standard conditions, 50-60% of the total Ca influx was found to be insensitive to Nai+, Cai++, and ATP, sensitive to membrane potential, and partially inhibited by external Co++.     

Coupled in vivo activity of creatine phosphokinase and the membrane-bound (Na+,K+)-ATPase in the resting and stimulated electric organ of the electric fish Narcine brasiliensis.

Physiological control of the plasma membrane sodium pump, (Na+,K+)-ATPase, is essential for proper function of eukaryotic cells. In the electric organ of the elasmobranch Narcine brasiliensis, the normal demands placed upon the pump during the process of generation of electrical currents call for large and rapid changes in activity of this enzyme, making this a good model for the study of its cellular regulation. 31P NMR spectroscopic techniques were used to study metabolic regulation of membrane pump function in resting and stimulated electric organ and in skeletal muscle of the live, intact N. brasiliensis. Because the ATP synthetic abilities of the electric organ by glycolysis or oxidative phosphorylation are extremely limited, depletion of phosphocreatinine (PCr) could be used to determine the activity of the (Na+,K+)-ATPase after the electric organ was stimulated to discharge, and to measure the net flux from PCr to ATP through the creatine phosphokinase (CPK) reaction in the electric organ. Saturation transfer, an NMR technique which measures exchange rates, was applied to determine the unidirectional flux in the forward direction through the same reaction in the electric organ and in skeletal muscle as a control. The pseudo first-order rate constant kf for the CPK reaction at 24 degrees C in resting electric organ was 0.000 +/- 0.002 s-1 (n = 10) and in skeletal muscle was 0.08 +/- 0.03 s-1 (n = 3). The results demonstrate that in resting electric organ, which is well supplied with CPK, there was no measurable flux through this reaction, although CPK when extracted is highly active. Measured and calculated levels of all substrates for the creatine kinase reaction in the electric organ are similar to those in unstimulated skeletal muscle, where the creatine phosphokinase reaction rates are high in vivo. In contrast to the resting electric organ, during stimulation of the electric organ the measured net rate constant was greater than 0.08 s-1. In addition, as shown by lack of PCr depletion, there was virtually no net turnover of ATP in the resting organ compared to the stimulated organ. The marked difference in the (Na+,K+)-ATPase activity in the resting and activated electric organ confirmed earlier results (Blum, H., Nioka, S., and Johnson, R. G., Jr. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1247-1251). Together, these results suggest that there is a novel method of coordinate regulation of cellular enzymes of great sensitivity and rapidity.     

Role of phosphocreatine in energy transport in skeletal muscle of bullfrog studied by 31P-NMR

To evaluate the energy-shuttle hypothesis of the phosphocreatine/creatine kinase system, diffusion rates for ATP, phosphocreatine and flux through the creatine kinase reaction were determined by 31P-NMR in resting bullfrog biceps muscle. The diffusion coefficient of phosphocreatine measured by 31P-pulsed gradient NMR was 1.4-times larger than ATP in the muscle, indicating the advantage of phosphocreatine molecules for the intracellular energy transport. The flux of the creatine kinase reaction measured by 31P-saturation transfer NMR was 3.6 mmol/kg wet wt. per s in the resting muscle. The flux is equal to the turnover rate of ATP, ADP, phosphocreatine and creatine molecules, therefore, the life-times of these substrates and the average distance traversed after the life-times by the diffusing molecules were calculated using the diffusion coefficients obtained by 31P-NMR. The mean square length of one-dimensional diffusion was 22 microns in ATP molecules and the minimum diffusion length was 1.8 microns in ADP molecules. The latter was calculated using free ADP concentration, 30 mumol/kg wet wt., obtained from the equilibrium constant of the creatine kinase reaction and the diffusion coefficient assumed to be the same of ATP in muscle. Similar diffusion lengths of ADP were calculated using the reported values for the flux of the creatine kinase reaction in heart and smooth-muscle. The diffusion lengths of all substrates involved in the creatine kinase reaction were larger than the radii of myofibrils. Therefore, in the muscles with an alternating arrangement of mitochondria and myofibrils, such as heart and certain skeletal muscles, ATP and ADP molecules can move freely between myofibrils and mitochondria without the aid of the creatine kinase reaction; thus, we conclude that the energy-shuttle hypothesis is not obligatory for energy transport between the mitochondria and the myofibrils.     

Dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space of rat-heart mitochondria

To investigate whether or not the mitochondrial intermembrane space together with the extramitochondrial space form a homogeneous pool for adenine nucleotides, rat-heart mitochondria were studied in reconstituted systems with pyruvate kinase and ADP-producing enzymes with varied localization. In the hexokinase system, ADP is produced extramitochondrially by added yeast hexokinase, whereas in the creatine kinase system mitochondrial creatine kinase is responsible for ADP regeneration in the intermembrane space. The dependence of mitochondrial respiration on the extramitochondrial [ATP]/[ADP] ratio in both systems was investigated experimentally and by means of computer simulation. Near the resting state, higher [ATP]/[ADP] ratios were found in the creatine kinase system than in the hexokinase system at the same rate of respiration. This and the maintaining of a substantial creatine kinase-stimulated respiration in the presence of pyruvate kinase in excess is explained by a two-compartment model considering diffusion limitations of adenine nucleotides. A diffusion rate constant of (8.7 +/- 4.7) 10(4) microliters X mg-1 X min-1 for ADP and ATP was estimated, resulting in rate-dependent concentration differences up to 13.7 microM AdN between the extramitochondrial space and the AdN-translocator at the maximum rate of oxidative phosphorylation of rat-heart mitochondria. The results support the assumption that ADP diffusion towards the AdN-translocator is limited if its extramitochondrial concentration is low, resulting in a dynamic compartmentation of adenine nucleotides in the mitochondrial intermembrane space.     

Cell volume regulatory responses of isolated perfused rat liver. The effect of amino acids

1) Addition of glutamine, glycine, alanine, serine, phenylalanine, proline at a concentration of 3mM, each, or of an amino-acid mixture resembling the physiological amino-acid composition of portal venous blood, to influent perfusate of isolated perfused rat liver led to a 4-6% increase of liver mass without increase of the [3H]inulin space, and biphasic K+ movements across the plasma membrane. These K+ movements consisted of an initial net K+ uptake (0.4-0.9 mumol X g-1 liver) for about 2 min, being followed by a net K+ release (1.0-2.8 mumol X g-1 liver) during the next 10 min. Withdrawal of the amino acids from influent perfusate caused a slow net K+ reuptake by the liver and restored the initial liver mass. No effects on liver mass and K+ fluxes were observed following addition of glutamate or glucose at a concentration of 3mM, each. 2) Aminooxyacetate did not affect the alanine (3 mM) induced increase in liver mass. However, in presence of aminooxyacetate the alanine-induced net K+ release from the liver (i.e. K+ release from 2-10 min minus initial K+ uptake) increased from 0.1 to 2.2 mumol X g-1 liver, whereby simultaneously the alanine tissue level rose from 6.8 to 13.3 mumol X g-1 (corresponding to an increase of the intracellular alanine concentration from about 12 to 25 mM) in presence of aminooxyacetate. 3) When livers were perfused with different glutamine concentrations, a maximal increase in liver mass of 5-6% was observed at glutamine concentrations above 1.5-2mM. A halfmaximal increase in liver mass was observed at 0.6-1.0mM glutamine in influent, i.e. at the physiological portal glutamine concentration.(ABSTRACT TRUNCATED AT 250 WORDS)