Hormonal modulation of hepatic plasma membrane lactate transport in cultured rat hepatocytes

Hormonal modulation of hepatic plasma membrane lactate transport was studied in primary cultures of isolated hepatocytes from fed rats to examine the mechanism for the known enhancement of lactate transport in starvation and diabetes. Total cellular lactate entry was increased by 14% in the presence of dexamethasone; this was accounted for by an approximately 40% increase in the carrier-mediated component of entry with no effect on diffusion. A trend of similar magnitude was evident with glucagon. The effects of dexamethasone and glucagon on lactate transport constitute an additional potential mechanism for enhancement of gluconeogenesis by these hormones.     

Ionic properties of the acetylcholine receptor in cultured rat myotubes

The acetylcholine reversal potential (Er) of cultured rat myotubes is -3mV. When activated, the receptor is permeable to K+ and Na+, but not to Cl- ions. Measurement of Er in Tris+-substituted, Na-free medium also indicated a permeability to Tris+ ions. Unlike adult frog muscle the magnitude of Er was insensitive to change in external Ca++ (up to 30 mM) or to changes in external pH (between 6.4 and 8.9). The equivalent circuit equation describing the electrical circuit composed of two parallel ionic batteries (EK and ENa) and their respective conductances (gK and gNa), which has been generally useful in describing the Er of adult rat and frog muscle, could also be applied to rat myotubes when Er was measured over a wide range of external Na+ concentrations. The equivalent circuit equation could not be applied to myotubes bathed in media of different external K+ concentrations. In this case, the Er was more closely described by the Goldman constant field equation. Under certain circumstances, it is known that the receptor in adult rat and frog muscle can be induced to reversibly shift from behavior described by the equivalent circuit equation to that described by the Goldman equation. Attempts to similarly manipulate the responses of cultured rat myotubes were unsussessful. These trials included a reduction in temperature (15 degress C), partial alpha-bungarotoxin blodkade, and activation of responses with the cholinergic agonist, decamethonium.     

Rotational diffusion of acetylcholine receptors on cultured rat myotubes

The rotational mobility of acetylcholine receptors (AChR) in the plasma membrane of living rat myotubes in culture is measured in this study by polarized fluorescence recovery after photobleaching (PFRAP). These AChR are known to exist in two distinct classes, evident by labeling with rhodamine alpha-bungarotoxin; clustered AChR that are aggregated in a pattern of highly concentrated speckles and streaks, with each cluster occupying an area of approximately 1,000 microns 2; and nonclustered AChR that appear as diffuse labeling. PFRAP results reported here show that: (a) most clustered AChR (approximately 86%) are rotationally immobile within a time scale of at least several seconds; and (b) most nonclustered AChR (approximately 76%) are rotationally mobile with characteristic times ranging from less than 50 ms to 0.1 s. External cross-linking with the tetravalent lectin concanavalin A immobilizes many nonclustered AChR. PFRAP experiments in the presence of carbachol or cytochalasin D show that the restraints to rotational motion in clusters are remarkably immune to treatments that disperse clusters or disrupt cytoplasmic actin. The experiments also demonstrate the feasibility of using PFRAP to measure rotational diffusion on selected microscopic areas of living nondeoxygenated cells labeled with standard fluorescence probes over a very wide range of time scales, and they also indicate what technical improvements would make PFRAP even more practicable.     

Activity-dependent accumulation of basal lamina by cultured rat myotubes

Myoblasts from 20-day rat embryos fuse and differentiate in culture to form spontaneously active myotubes. The myotubes acquire an extracellular matrix that includes a patchy basal lamina (BL) and a layer of fibrils that runs among and above the cells. Several antibodies that bind to muscle fiber basement membrane in vivo were used to study the organization of the extracellular matrix and the effect of muscle activity on the accumulation of its components. Light and electron microscopic immunohistochemical methods showed that the composition and organization of myotube BL in vitro resemble those seen in vivo. Antibodies that bind to both synaptic and extrasynaptic muscle fiber BL, in vivo stain the entire myotube BL in vitro, while antisera that bind preferentially to synaptic BL in vivo stain small patches of myotube BL, which are usually associated with regions rich in acetylcholine receptors. The effects of activity on accumulation of BL were studied by comparing control myotubes to myotubes paralyzed with tetrodotoxin or lidocaine. Immunohistochemical and 125I-antibody binding experiments with three antisera that stain the entire BL showed that paralyzed myotubes accumulate less BL than active myotubes. The effects of activity and inactivity are reversible: new BL forms if toxin is removed from cultures and BL is lost if active myotubes are paralyzed. Thus, accumulation of BL by myotubes is dependent, at least in part, on activity. In contrast, the number of patches stained by synapse-specific BL antibodies is increased in inactive cultures. Thus, immunologically distinguishable components of BL are differentially affected by activity.     

Partly ordered synthesis and degradation of glycogen in cultured rat myotubes.

The following questions concerning glycogen synthesis and degradation were examined in cultured rat myotubes. 1) Is synthesis and degradation of the individual glycogen molecule a strictly ordered process, with the last glucosyl unit incorporated into the molecule being the first to be released (the last-in-first-out principle), or is it a random process? 2) Are all glycogen molecules in skeletal muscle synthesized and degraded in phase (simultaneous order) or out of phase (sequential order)? Basal glycogen stores were minimized by fasting and were subsequently replenished in two intervals, the first (0-0.5 h) with tritium-labeled and the second (0.5-3 h) with carbon-labeled glucose as precursor. Glycogen degradation was initiated by addition of forskolin. The kinetics of glycogen accumulation as well as degradation could be approximated by monoexponential equations with rate constants of 0.81 and 1.39 h(-1), respectively. The degradation of glycogen largely followed the last-in-first-out principle, particularly in the initial period. Analysis of the size of the glycogen molecules and the beta-dextrin limit during glycogen accumulation and degradation showed that both synthesis and degradation of glycogen molecules are largely sequential and the small deviation from this order is most pronounced at the beginning of the accumulation and at the end of the degradation period. This pattern may reflect the number of synthase and phosphorylase molecules and fits well with the role of glycogen in skeletal muscle as a readily available energy store and with the known structure of the glycogen molecule. It is emphasized that the observed nonlinear relation between the change in glycogen concentration and release of label during glycogen degradation may have important practical consequences for interpretation of experimental data.     

Effect of fibrillation on acetylcholinesterase mRNA in cultured embryonic rat myotubes

Acetylcholinesterase (AChE) and AChE mRNA were evaluated in spontaneously fibrillating myotubes derived from 20-day-old rat fetuses and in matched cultures in which fibrillation was prevented by adding tetrodotoxin on the fourth day of culture. On the eighth day of culture, the AChE activity of fibrillating and nonfibrillating cultures was 5332 and 1861 pmol ACh hydrolyzed min-1 dish-1, respectively (P less than 0.005). Total mRNA was essentially the same in fibrillating and nonfibrillating cultures (27.4 and 25.4 micrograms/dish, respectively). AChE mRNA was assessed by assaying the AChE produced by Xenopus oocytes microinjected with purified mRNA. The AChE produced by mRNA from fibrillating and nonfibrillating cultures was 0.46 and 0.10 pmol ACh hydrolyzed min-1 oocyte-1, respectively (P less than 0.005).     

Phosphorylation of the cardiac isoform of calsequestrin in cultured rat myotubes and rat skeletal muscle.

Calsequestrin is a high-capacity Ca(2+)-binding protein and a major constituent of the sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle. Two isoforms of calsequestrin, cardiac and skeletal muscle forms, have been described which are products of separate genes. Purified forms of the two prototypical calsequestrin isoforms, dog cardiac and rabbit fast-twitch skeletal muscle calsequestrins, serve as excellent substrates for casein kinase II and are phosphorylated on distinct sites (Cala, S.E. and Jones, L.R. (1991) J. Biol. Chem 266, 391-398). Dog cardiac calsequestrin is phosphorylated at a 50 to 100-fold greater rate than is rabbit skeletal muscle calsequestrin, and only the dog cardiac isoform contains endogenous Pi on casein kinase II phosphorylation sites. In this study, we identified and examined both calsequestrin isoforms in rat muscle cultures and homogenates to demonstrate that the cardiac isoform of calsequestrin in rat skeletal muscle was phosphorylated in vivo on sites which are phosphorylated by casein kinase II in vitro. Phosphorylation of rat skeletal muscle calsequestrin was not detected. In tissue homogenates, cardiac and skeletal muscle calsequestrin isoforms were both found to be prominent substrates for endogenous casein kinase II activity with cardiac calsequestrin the preferred substrate. In addition, these studies revealed that the cardiac isoform of calsequestrin was the predominant form expressed in skeletal muscle of fetal rats and cultured myotubes.     

Organization of acetylcholine receptor clusters in cultured rat myotubes is calcium dependent

The effect of extracellular Ca2+ concentration and myasthenic globulin on the distribution and appearance of acetylcholine receptor (AChR) clusters on rat myotubes was studied with tetramethyl-rhodamine-labeled alpha BTX. Low Ca2+ medium (2.5 X 10(-5) M) caused a time-dependent loss of AChR clusters, and a concomitant increase in small punctate areas of fluorescence. High Ca2+ concentrations (1.5 X 10(-2) M) increased the size of AChR clusters without altering AChR synthesis. These changes were not observed with other divalent ions. In the presence of myasthenic globulin, the rate of AChR turnover increases, and AChR clusters are rapidly dispersed. High Ca2+ concentration partially protects the AChR clusters from dispersal and decreases the rate of receptor turnover.     

Regulation of gluconeogenesis from pyruvate and lactate in the isolated perfused rat liver

The effects of glucagon and the alpha-adrenergic agonist, phenylephrine, on the rate of 14CO2 production and gluconeogenesis from [1-14C]lactate and [1-14C]pyruvate were investigated in isolated perfused livers of 24-h-fasted rats. Both glucagon and phenylephrine stimulated the rate of 14CO2 production from [1-14C]lactate but not from [1-14C]pyruvate. Neither glucagon nor phenylephrine affected the activation state of the pyruvate dehydrogenase complex in perfused livers derived from 24-h-fasted rats. 3-Mercaptopicolinate, an inhibitor of the phosphoenolpyruvate carboxykinase reaction, inhibited the rates of 14CO2 production and glucose production from [1-14C]lactate by 50% and 100%, respectively. Furthermore, 3-mercaptopicolinate blocked the glucagon- and phenylephrine-stimulated 14CO2 production from [1-14C]lactate. Additionally, measurements of the specific radioactivity of glucose synthesized from [1-14C]lactate, [1-14C]pyruvate and [2-14C]pyruvate indicated that the 14C-labeled carboxyl groups of oxaloacetate synthesized from 1-14C-labeled precursors were completely randomized and pyruvate----oxaloacetate----pyruvate substrate cycle activity was minimal. The present study also demonstrates that glucagon and phenylephrine stimulation of the rate of 14CO2 production from [1-14C]lactate is a result of increased metabolic flux through the phosphoenolpyruvate carboxykinase reaction, and phenylephrine-stimulated gluconeogenesis from pyruvate is regulated at step(s) between phosphoenolpyruvate and glucose.     

Mediated transport and metabolism of lactate in rat aorta.

1. Under appropriate conditions L- and D-lactate enter the cells of rat aorta and are metabolized. Oxidation of lactate to CO2 occurs under aerobic conditions. 2. L- and D-lactate are taken up into the cells when oxygen, glucose, or both oxygen and glucose are present in the incubation medium. Both L- and D-lactate are excluded from the cells when neither oxygen nor glucose is present. 3. D,L-Glyceraldehyde prevents the uptake of L-lactate. The effect is apparently not due to the inhibition of glucose metabolism by L-glyceraldehyde. 4. L-lactate (20 mM) markedly inhibits the uptake of 5 mM D-lactate, but 20 mM D-lactate fails to inhibit the uptake of 5 mM L-lactate. 5. Raising the pH of the incubation medium markedly depresses the uptake of L-lactate. 6. The results provide evidence that L- and D-lactate enter the cells of rat aorta by a mediated transport system.     

Kinetics of Myo-inositol transport in rat ocular lens

Myo-inositol (MI) influx as a function of concentration in rat lens consisted of a saturable component, fit by a rectangular hyperbola, and a linear component which was more distinct at high myo-inositol concentrations suggesting passive diffusion. The hyperbolic component was half-maximally saturated (K_t) at 61.3 μM and had a maximal transport rate (J_max) of 44.6 μMol/kg wet wt/h. The linear component had an apparent permeability coefficient of 1.44 × 10^?6 s^?1. Sorbitol, which distributed rapidly in the extracellular space (6.83 ml/100 g wet wt), also appeared to enter the intracellular space with a permeability coefficient of 1.37 × 10^?6 s^?1, similar to that of myo-inositol. The influx of myo-inositol was critically dependent on the concentration of extracellular sodium consistent with a sodium-myo-inositol contransport. The kinetics of influx activation by sodium suggested an apparent 2:1 coupling ratio for sodium and myo-inositol. When potassium was used as sodium substitute, a significantly stronger influx inhibition was observed than with nondepolarizing sodium substitutes, indicating that myoinositol was driven by the electrochemicl gradient of sodium rather than the chemical gradient only. Reducing the extracellular Na concentration increased the MI concentration at which transport was half-maximally activated, suggesting an ordered binding sequence of Na followed by MI. Myo-inositol influx was competitively inhibited by phlorizin with an inhibitory coefficient (K_i) of 35 μM. Phloretin also was capable of inhibition but with a much lesser efficacy. Myoinositol desaturates from the lens at a rate of 0.00862 h^?1. Approximately 19% of the efflux can be inhibited with phlorizin, suggesting that it represents carrier-mediated flux. The phlorizin insensitive flux has a rate of 0.00695 h^?1 or 1.93 × 10^?6 s^?1, similar to the Na-independent passive influx. MI influx is due to a Na-dependent, phlorizin-sensitive active transport while the efflux consists largely of a phlorizin-independent passive leakage. © 1995 Wiley-Liss, Inc.     

Studies on the effects of lactate transport inhibition, pyruvate, glucose and glutamine on amino acid, lactate and glucose release from the ischemic rat cerebral cortex

A rat four vessel occlusion model was utilized to examine the effects of ischemia/reperfusion on cortical window superfusate levels of amino acids, glucose, and lactate. Superfusate aspartate, glutamate, phosphoethanolamine, taurine, and GABA were significantly elevated by cerebral ischemia, then declined during reperfusion. Other amino acids were affected to a lesser degree. Superfusate lactate rose slightly during the initial ischemic period, declined during continued cerebral ischemia and then was greatly elevated during reperfusion. Superfusate glucose levels declined to near zero levels during ischemia and then rebounded beyond basal levels during the reperfusion period. Inhibition of neuronal lactate uptake with alpha-cyano-4-hydroxycinnamate dramatically elevated superfusate lactate levels, enhanced the ischemia/reperfusion evoked release of aspartate but reduced glutamine levels. Topical application of an alternative metabolic fuel, glutamine, had a dose dependent effect. Glutamine (1 mM) elevated basal superfusate glucose levels, diminished the decline in glucose during ischemia, and accelerated its recovery during reperfusion. Lactate levels were elevated during ischemia and reperfusion. These effects were not evident at 5 mM glutamine. At both concentrations, glutamine significantly elevated the superfusate levels of glutamate. Topical application of sodium pyruvate (20 mM) significantly attenuated the decline in superfusate glucose during ischemia and enhanced the levels of both glucose and lactate during reperfusion. However, it had little effect on the ischemia-evoked accumulation of amino acids. Topical application of glucose (450 mg/dL) significantly elevated basal superfusate levels of lactate, which continued to be elevated during both ischemia and reperfusion. The ischemia-evoked accumulations of aspartate, glutamate, taurine and GABA were all significantly depressed by glucose, while phosphoethanolamine levels were elevated. These results support the role of lactate in neuronal metabolism during ischemia/reperfusion. Both glucose and glutamine were also used as energy substrates. In contrast, sodium pyruvate does not appear to be as effectively utilized by the ischemic/reperfused rat brain since it did not reduce ischemia-evoked amino acid efflux.