Increased ATP and creatine phosphate turnover in phagocytosing mouse peritoneal macrophages.

Resident and thioglycollate-elicited macrophages maintained in culture for 24 h contain approximately 5 x 10(-16) and 12 x 10(-16) mol of ATP per cell, respectively. During particle ingestion, the levels of ATP in these cells did not change. However, the specific activity of ATP extracted from macrophages labeled with [32P]Pi during phagocytosis was 40% lower than ATP extracted from control cells. These results suggested that macrophages contain a high energy phosphate reservoir, in addition to the ATP pool(s). A search for such a reservoir led to the identification of creatine phosphate in both resident and thioglycollate-elicited macrophages at concentrations that are in 3- to 5-fold-molar excess over ATP. Creatine phosphate levels in phagocytosing resident macrophages decreased by 45%, while creatine phosphate levels in phagocytosing thioglycollate-elicited macrophages did not change. Creatine phosphate turnover was measured in macrophages prelabeled with [14C]creatine. Over 90% of the intracellular label was in the form of creatine phosphate. During phagocytosis, there was a 40% decrease in intracellular [14C]creatine phosphate in both resident and thioglycollate-elicited macrophages. These results indicate that creatine phosphate turns over more rapidly during phagocytosis and replenishes the ATP consumed.     

Energy Metabolism and Quantal Acetylcholine Release: Effects of Botulinum Toxin, l-Fluoro-2,4-Dinitrobenzene, and Diamide in the Torpedo Electric Organ

In the Torpedo electric organ, a modified nerve-muscle system, type A botulinum toxin blocked the release of acetylcholine (ACh) quanta, both neurally evoked and spontaneous. At the same time, the toxin increased the release of a class of small miniature potentials (the subminiature potentials), reduced the ATP and more the creatine phosphate content of the tissue, and impaired the activity of creatine kinase (CK). Thus, we compared this pattern of changes with those provoked by 1-fluoro-2,4-dinitrobenzene (FDNB), an efficient inhibitor of CK. As expected, FDNB rapidly inactivated CK, which resulted in a profound depletion of ATP whereas the stores of creatine phosphate were preserved. In addition, FDNB caused conspicuous morphological alterations of nerve endings and ACh depletion. This agent also suppressed evoked and spontaneous quantal release whereas the occurrence of subminature potentials was markedly increased. Diamide, a penetrating thiol oxidizing substance, provoked first a transient rise in quantal ACh release and then blockade of transmission with, again, production of a large number of subminiature potentials. Creatine phosphate was depleted in the tissue by diamide, the ATP content reduced, and CK activity partly inhibited. The morphology of nerve terminals did not show obvious changes with either diamide or botulinum toxin at the stage of transmission failure. Although the three poisons acted by different mechanisms, this resulted in a rather similar pattern of physiological changes: failure of quantal release and enhancement of subquantal release. These results and experiments on synaptosomes indicated that CK inhibition was probably a crucial mechanism for FDNB but not for diamide or botulinum intoxication.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorus nuclear magnetic resonance studies of phosphorus metabolites in frog muscle.

31P-nuclear magnetic resonance was applied to living muscles of bullfrogs, and the time courses of metabolic changes of ATP, creatine phosphate, inorganic phosphate, and sugar phosphates were studied under anaerobic and aerobic conditions. A decrease in creatine phosphate was observed in the resting muscle under anaerobic conditions with a concomitant decrease in the intracellular pH, while the ATP level remained constant. With the use of 2,4-dinitro-1-fluorobenzene and iodoacetic acid, ATP disappeared quickly. When the resting muscle was perfused with oxygen-saturated glucose-Ringer's solution, the amount of creatine phosphate increased gradually. These findings indicate that anaerobic glycolysis is insufficient for even the resting energy consumption whereas oxidative phosphorylation is sufficient. The effects of tetanic stimulation on living muscles were also studied. When glycolysis and oxidative phosphorylation were suppressed, the intracellular energy store was depleted by the tetanic contraction. Anaerobic glycolysis produced rapid recovery of the energy store level, although it was insufficient to reach the initial level. Aerobic oxidative phosphorylation produced sufficient energy to reach the initial level, and this level was never exceeded. This finding suggests the existence of a regulatory mechanism for the energy store level.     

Block of acid secretion by amytal and its partial reversal by menadione with ascorbate in the gastric mucosa of the guinea-pig

The effect of amytal on energy metabolism and acid secretion in an isolated gastric mucosa of the guinea-pig were studied. Determination of adenine nucleotides, creatine phosphate, pyruvate and lactate in the gastric mucosa showed that amytal depressed the levels of ATP, creatine phosphate and energy charge with elevation of the AMP and pyruvate levels. This treatment inhibited concomitantly acid secretion and active chloride transport detected by short circuit current. The addition of menadione with ascorbate to the medium in the presence of amytal partially restored ATP and energy charge levels and also induced a partial recovery of acid secretion and active chloride transport. These results suggest that ATP is a direct energy donor for acid secretion in the gastric mucosa of the guinea-pig.     

BREAKDOWN OF CREATINE PHOSPHATE AND ATP IN NERVE TERMINALS AND ELECTROPLAQUES OF THE TORPEDO ELECTRIC ORGAN: COMPARISON WITH THE ELECTRICAL ENERGY DISSIPATED

Abstract— The electrical work performed by the electric organ of Torpedo was compared with the energy provided by the net breakdown of ATP and creatine phosphate (CrP). The electrical work was calculated for single impulses and for repetitive stimulations. The content in CrP and ATP was measured at different times in the course of stimulation and during the period of recovery. The chemical expenditure due to activity of the nerve terminals was distinguished from the total expenditure by the use of curare which interrupts synaptic transmission but does not interfere to any great extent with the release of acetylcholine. In the presence of curare the breakdown of phosphagen started only after more than 1 min of stimulation; it represented the loss of about 20-25% of the initial store. In untreated tissue the breakdown of CrP and ATP occurred in two phases and continued within the first minute after the end of the stimulation; as much as 77% of the phosphagen content was utilized under these conditions. The recovery of ATP and CrP was completed only 3-5 h after stimulation, a long time after the restoration of the physical capabilities of the tissue. The electrical energy dissipated during activity was smaller than the chemical energy provided by the net breakdown of phosphagens. This suggests that only a fraction of the chemical energy is utilized directly to compensate for the physical work accomplished, i.e. for the restoration of the ionic electromotive force. The electric organ also requires chemical energy for other purposes, particularly in the nerve endings where the presynaptic machinery seems to utilize an important fraction of the high energy phosphates stored in the tissue.     

Bioenergetic protection of failing atrial and ventricular myocardium by vasopeptidase inhibitor omapatrilat

Deficient bioenergetic signaling contributes to myocardial dysfunction and electrical instability in both atrial and ventricular cardiac chambers. Yet, approaches capable to prevent metabolic distress are only partially established. Here, in a canine model of tachycardia-induced congestive heart failure, we compared atrial and ventricular bioenergetics and tested the efficacy of metabolic rescue with the vasopeptidase inhibitor omapatrilat. Despite intrinsic differences in energy metabolism, failing atria and ventricles demonstrated profound bioenergetic deficiency with reduced ATP and creatine phosphate levels and compromised adenylate kinase and creatine kinase catalysis. Depressed phosphotransfer enzyme activities correlated with reduced tissue ATP levels, whereas creatine phosphate inversely related with atrial and ventricular load. Chronic treatment with omapatrilat maintained myocardial ATP, the high-energy currency, and protected adenylate and creatine kinase phosphotransfer capacity. Omapatrilat-induced bioenergetic protection was associated with maintained atrial and ventricular structural integrity, albeit without full recovery of the creatine phosphate pool. Thus therapy with omapatrilat demonstrates the benefit in protecting phosphotransfer enzyme activities and in preventing impairment of atrial and ventricular bioenergetics in heart failure.     

Thiamine and Cholinergic Transmission in the Electric Organ of Torpedo

The electric organ of Torpedo marmorata was found to contain as much as 120 +/- 24 nmol of thiamine per g of fresh tissue. The vitamin was distributed as nonesterified thiamine (32%), thiamine monophosphate (22%), thiamine diphosphate (8%), and an important proportion of thiamine triphosphate (38%). A high level of thiamine triphosphate was found in synaptosomes isolated from the electric organ. In contrast, the synaptic vesicles did not show any enrichment in thiamine, whereas they contained a marked peak of acetylcholine (ACh) and ATP. Thus thiamine seems to be very abundant in cholinergic nerve terminals; its localization is apparently extravesicular, either in the axoplasm or in association with plasma membrane. When calcium was reduced and magnesium increased in the external medium, the efficiency of transmission was diminished, owing to inhibition of ACh release; in a parallel manner the degree of thiamine phosphorylation was found to increase--this condition is known to modify the repartition of ACh between vesicular and extravesicular compartments. Electrical stimulation, which causes periodic variations of the level of ACh and ATP, also caused significant changes in thiamine esters. In addition, related changes of the vitamin and the transmitter were observed under other conditions, suggesting a functional link between the metabolism of thiamine and that of ACh in cholinergic nerve terminals.     

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.     

A Kinetic Study of Stimulus-Induced Vesicle Recycling in Electromotor Nerve Terminals Using Labile and Stable Vesicle Markers

The kinetics of recovery, by recycling electromotor synaptic vesicles, of the biophysical parameters of the reserve population has been studied in perfused blocks of electric organ of Torpedo marmorata prestimulated in vivo, followed by density gradient separation of the extracted vesicles in a zonal rotor using labile (acetylcholine and ATP) and stable (proteoglycan) vesicle markers. Stimulation in vivo at 0.15 Hz for 3.3 h depleted tissue acetylcholine much less than stimulation at 1 Hz for 1 h but nevertheless generated a much larger pool of recycled vesicles that recovered more slowly. At the lower rate of stimulation, recovery of the biophysical characteristics of the reserve population by the recycled vesicles, identified by their content of newly synthesized transmitter, was essentially complete by 8 h. The stable proteoglycan marker was immunochemically assayed and was bimodally distributed in the vesicle-containing portion of the density gradient even in experiments with unstimulated or recovered tissue. The second peak corresponded with that of newly synthesized transmitter and was thus identified as containing the recycled vesicles. Its normalized acetylcholine/proteoglycan ratio was lower than that of the first peak, which is consistent with earlier findings that recycled vesicles, before recovery, are only partially loaded with transmitter. However, as expected, the proportion of total vesicular proteoglycan and acetylcholine associated with the recycled vesicle fraction was very much lower in preparations derived from unstimulated or recovered tissue than in those from recently stimulated tissue.     

Relating structure to mechanism in creatine kinase

Found in all vertebrates, creatine kinase catalyzes the reversible reaction of creatine and ATP forming phosphocreatine and ADP. Phosphocreatine may be viewed as a reservoir of "high-energy phosphate" which is able to supply ATP, the primary energy source in bioenergetics, on demand. Consequently, creatine kinase plays a significant role in energy homeostasis of cells with intermittently high energy requirements. The enzyme is of clinical importance and its levels are routinely used as an indicator of myocardial and skeletal muscle disorders and for the diagnosis of acute myocardial infarction. First identified in 1928, the enzyme has undergone intensive investigation for over 75 years. There are four major isozymes, two cytosolic and two mitochondrial, which form dimers and octamers, respectively. Depending on the pH, the enzyme operates by a random or an ordered bimolecular mechanism, with the equilibrium lying towards phosphocreatine production. Evidence suggests that conversion of creatine to phosphocreatine occurs via the in-line transfer of a phosphoryl group from ATP. A recent X-ray structure of creatine kinase bound to a transition state analog complex confirmed many of the predictions based on kinetic, spectroscopic, and mutagenesis studies. This review summarizes and correlates the more significant mechanistic and structural studies on creatine kinase.     

Spinal mechanisms of electric organ discharge synchronization in Gymnotus carapo

Summary The duration of the electric organ discharge (EOD) in Gymnotus carapo is brief and independent of fish size. Spinal mechanisms involved in electrocyte synchronization were explored by recording spontaneous action potentials of single fibers from the electromotor bulbospinal tract (EBST). Using the field potential of the medullary electromotor nucleus (MEN) as a temporal reference we calculated the orthodromic conduction velocity (CV) of these fibers (range: 10.7–91 m/s).The CVs (in m/s) of fibers recorded at the same level of the spinal cord were significantly different in small and large fish; this difference disappeared when CV were expressed as percentage of body length/ms. Plotting these values against conduction distance (also in %) showed that low CV fibers predominate in the rostral cord while only fast fibers are found at distal levels. Moreover, antidromic stimulation of the distal cord was only effective on high CV fibers. The orthodromic CVs in the distal portion of the recorded fibers were calculated by collision experiments; no significant differences were found between proximal and distal portions.The spatial distribution of CV values within the EBST is proposed to play the main role in synchronizing the electromotoneurons' activity along the spinal cord.Abbreviations EOD electric organ discharge - EO electric organ - EBST electromotor bulbospinal tract - MEN medullary electromotor nucleus - CV conduction velocity - EMN electromotoneuron     

PRE- AND POSTSYNAPTIC UTILIZATION OF ATP AND CREATINE PHOSPHATE AT THE NERVE-ELECTROPLAQUE JUNCTION

Abstract— When the electric organ of the Torpedo fish was stimulated in vivo and in vitro , there was a large rapid breakdown of creatine phosphate (CrP), and a slower decrease in the level of ATP. The acetylcholine content showed a parallel fall to that of ATP. Curare was used to interrupt synaptic transmission, allowing the presynaptic nerve endings to remain sensitive to stimulation, but simultaneously keeping electroplaques inactive. In stimulated curarized tissue, the level of ATP was not changed, while the breakdown of CrP was still 25% of that observed in non-curarized controls. These observations suggest that presynaptic nerve endings, despite their small size, utilize a very high amount of energy.     

The creatine phosphate energy shuttle--the molecular asymmetry of a "pool"

The creatine phosphate shuttle energy transfer mechanism was postulated on the basis of the hexokinase acceptor theory of insulin action. It proposes that the movement of chemical energy from the mitochondrion to the myofibril is in the form of creatine phosphate. This occurs because there are isozymes of creatine phosphokinase bound to the inner membrane of the sarcosome and to the A band of the myofibril. These isozymes have been shown to act as transducers of energy from ATP to creatine phosphate at the translocase site and from creatine phosphate back to ATP at the myofibrillar compartment. Calculations show that there is no significant amount of transformation of creatine phosphate to ATP in the intervening space between the mitochondrion and the myofibril so that, essentially, transport between the oxidative sites and the contractile apparatus is through the creatine phosphate shuttle. There is also evidence that another terminus for this shuttle is the microsome so that muscle activity tends to increase energy supply for protein synthesis.     

Creatine kinase-dependent energy transport in sea urchin spermatozoa. Flagellar wave attenuation and theoretical analysis of high energy phosphate diffusion.

The significance of a phosphocreatine (PCr) shuttle in the energy transport of motile spermatozoa (Tombes, R. M., and B. M. Shapiro, 1985, Cell, 41:325-334) has been tested by a quantitative analysis of motility. Computer-assisted analysis of stroboscopic photomicrographs of live sea urchin spermatozoa whose creatine kinase has been specifically inhibited by fluorodinitrobenzene reveals that motility is impaired due to a progressive damping of bending waves as they propagate along the flagellum. This lesion, which has been defined as attenuation and can be quantified, is repaired when these spermatozoa are demembranated and reactivated to swim with ATP. The implication that attenuation is due to the inhibition of energy transport via a PCr shuttle resulting in the decrease of ATP and accumulation of inhibitory levels of ADP distally has been supported by calculating sperm PCr and ATP levels resulting from diffusion along the flagellum. The specific alterations of motility seen with creatine kinase inhibition and their reversal with ATP are as expected from the model and provide strong support for the PCr shuttle in high energy phosphate transport.     

Investigations on the function of creatine kinase in Ehrlich ascites tumor cells

1. Growth and viability of in vitro cultured Ehrlich ascites tumor cells are not significantly impaired by exogenous creatine up to 40mM. Retardation of cell growth by higher concentrations depends on cell density. 2. Ehrlich cells grown in the presence of high concentrations of creatine accumulate creatine phosphate to high levels (up to 23 nmol/10(6) cells in the presence of 40mM creatine). 3. A nearly complete interruption of glycolytic ATP production or inhibition of the oxidative ATP synthesis reduces the maximal creatine to about 40-50% of controls. 4. Studies on the intracellular distribution of creatine kinase have shown, that the enzyme is only associated with the mitochondrial fraction. Titration of isolated mitochondria with digitonin revealed that the activity is located in the inter-membrane space and partly bound to the outer site of the inner membrane. 5. By growth of Ehrlich cells in creatine-free medium it is possible to obtain "creatine phosphate-depleted" cells (creatine phosphate less than 10% of controls). The growth of creatine phosphate-depleted cells as compared to controls is significantly reduced under energetic stress situations. The protein synthesis of these cells after an energetic stress (lack of glucose and oxygen) is significantly reduced as compared to creatine phosphate containing cells. 6. It is concluded that in these cells creatine kinase/creatine phosphate is a thermodynamic buffer system and not part of an energy shuttle as is postulated for muscle cells.     

The effect of hypoxia on carbohydrate metabolism in flounder (Platichthys flesus L.)—II. High energy phosphate compounds and the role of glycolytic and gluconeogenetic enzymes

• 1.1. ATP, ADP, AMP, energy charge potential and total adenylates in heart, kidney and muscle are relatively unaffected by environmental hypoxia. In the liver, hypoxia causes a 90% drop in ATP, a rise in ADP and AMP, and a drop in energy charge potential and total adenylates. In the muscle tissue ATP concentration is stabilized by a large creatine phosphate pool.
• 2.2. Hexokinase activity in the heart is 20 times higher than in the swimming muscle, and thus the heart has a high potential for utilizing exogenous glucose as an anaerobic substrate.
• 3.3. The role of creatine phosphate in regulating muscle glycolysis is discussed on background of the strong inhibition of muscle phosphofructokinase by physiological concentrations of creatine phosphate.
• 4.4. Flounder heart has a dominating M-type lactate dehydrogenase which is identical to the muscle enzyme by electrophoretic and kinetic criteria. This improves the anaerobic capabilities of the flounder heart compared to other fish hearts.
• 5.5. Both liver and kidney have high activities of the gluconeogenetic enzymes glucose-6-phosphatase, fructose-1,6-diphosphatase, and phosphoenolpyruvate carboxykinase and both are capable of synthesizing glucose from [¹⁴C]lactate. Because of more favorable energy conditions in the kidney this organ may substitute the liver as a gluconeogenetic organ during hypoxia.

     

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.     

Utilization of Cyclocreatine Phosphate, an Analogue of Creatine Phosphate, by Mouse Brain During Ischemia and Its Sparing Action on Brain Energy Reserves

Abstract: Brains of mice fed the creatine analogue cyclocreatine accumulated 10 γmol/g fresh wt. of cyclocreatine, of which 93% occurred as the synthetic phosphagen, cyclocreatine-P (l-carboxymethyl-2-imino-3-phosphonoimidazolidine). In brains containing cyclocreatine-P^2-, creatine-P (phosphocreatine) levels were lowered 40%; levels of ATP, P₁, and glucose were not altered: glutamate levels were lowered 17%: and aspartate levels were lowered 56%, relative to controls. When cyclocreatine was removed from the diet, brain cyclocreatine levels decreased with a half-life of 17 to 28 days. Ischemia was initiated in brains by decapitation of mice previously injected with the centrally acting muscle relaxant mephenesin. The initial creatine-P pool of 2-3 γmol/g was completely depleted within 1 min in ischemic brains of both control and cyclocreatine-fed mice. In brains of cyclocreatine-fed mice, the much larger cyclocreatine-P pool of 9.3 γmol/g decreased to 6 γmol/g after 2 min and to 2.2 γrnol/g after 4 min of ischemia, with a correspondingly increased accumulation of P₁. Levels of total cellular ATP were sustained slightly longer during ischemia in brains containing cyclocreatine-P. Available energy reserves of control brains were almost completely depleted after 2 min of ischemia, whereas generation and utilization of high-energy phosphate continued for more than 3 min after initiation of ischemia in brains of cyclocreatine-fed mice. These data suggest that during ischemic episodes cyclocreatine-P can function as a supplemental reservoir of high-energy phosphate and prolong the time required to exhaust the available energy stores of ischemic brain.     

Selective utilization of L-isomer of lactate in the smooth muscle of the guinea pig taenia caeci

Utilization of D- and L-lactate in the isolated intestinal smooth muscle of the guinea pig taenia caeci was examined by measuring contractile tension, oxygen consumption, and adenosine triphosphate (ATP) and creatine phosphate (PCr) concentrations. In the absence of glucose in the medium, muscle contraction induced by a high concentration of K+ was inhibited and the rate of oxygen consumption and the concentrations of ATP and PCr were decreased. Addition of glucose, L-lactate, and D,L-lactate, but not D-lactate, led to recovery of muscle contraction, rate of oxygen consumption, and ATP and PCr concentrations when the tissue had been incubated in the high K+, glucose-free solution. These results suggest that the isolated guinea pig taenia caeci selectively utilizes the L-isomer of lactate as a substrate for energy metabolism.     

Ontogeny of the electric organ discharge and the electric organ in the weakly electric pulse fish Brachyhypopomus pinnicaudatus (Hypopomidae, Gymnotiformes)

I recorded the electric organ discharges (EODs) of 331 immature Brachyhypopomus pinnicaudatus 6–88 mm long. Larvae produced head-positive pulses 1.3 ms long at 7 mm (6 days) and added a second, small head-negative phase at 12 mm. Both phases shortened duration and increased amplitude during growth. Relative to the whole EOD, the negative phase increased duration until 22 mm and amplitude until 37 mm. Fish above 37 mm produced a “symmetric” EOD like that of adult females. I stained cleared fish with Sudan black, or fluorescently labeled serial sections with anti-desmin (electric organ) or anti-myosin (muscle). From day 6 onward, a single electric organ was found at the ventral margin of the hypaxial muscle. Electrocytes were initially cylindrical, overlapping, and stalk-less, but later shortened along the rostrocaudal axis, separated into rows, and formed caudal stalks. This differentiation started in the posterior electric organ in 12-mm fish and was complete in the anterior region of fish with “symmetric” EODs. The lack of a distinct “larval” electric organ in this pulse-type species weakens the hypothesis that all gymnotiforms develop both a temporary (larval) and a permanent (adult) electric organ. Accepted: 1 March 1997