bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular localization of markers within injected or cut frog muscle fibers
Authors: Eisenberg B. R. , Mathias R. T. , Gilai A.
Published: December 2017

Many experimental procedures require drastic alterations of muscle fibers, such as cutting the fiber or injecting molecular probes through microelectrodes. We report the ultrastructure of similarly altered muscle fibers and the intracellular distribution of injected horseradish peroxidase (HRP). Cut fibers appear structurally normal at distances greater than 500 microM from the cut end, however, the structure deteriorates nearer to the cut. HRP diffuses longitudinally about 2,000 micrometer from the cut end and the concentration is uniform over the fiber's cross section. If HRP is introduced intracellularly either by pressure injection or through a nick in the sarcolemma, it distributes in a C-shaped annulus extending approximately 2,000 micrometer longitudinally and 1–20 micrometer radially. The ultrastructure of injected or nicked fibers appears normal. The HRP freely entered the junctional gap between T-system and sarcoplasmic reticulum (SR) but was excluded from either structure. Occasionally, a light pillar could be seen between T-system and SR; the space of these pillars suggest they are the central area of the “feet” appearing light against the dark marker.
Energetics of sugar transport by isolated intestinal epithelial cells: effects of cytochalasin B
Authors: Kimmich G. A. , Randles J.
Published: December 2017

The capability of isolated intestinal epithelial cells to establish concentration gradients of 3-O-methylglucose (3-OMG) by a Na+-dependent transport system is limited by concomitant function of a Na+-independent, facilitated diffusion transport system. Monosaccharides accumulated by the active system are continuously lost via the passive system, which acts to lower steady-state sugar gradients maintained by the cell. Cytochalasin B is a potent inhibitor of the passive system and allows the cells to establish a sugar gradient that is much higher than normal. When extracellular [3-;OMG] is 1 mM, cytochalasin induces sugar accumulation ratios of 30-;fold (+/- phlorizin) in contrast to control ratios of approximately 10-;fold. When [3-;OMG] is 0.1 mM, cytochalasin (0.1 mM) induces 40-;fold accumulation ratios. When changes in extracellular sugar concentration are considered, steady-state concentration gradients observed are 70-;fold. For a Na:sugar coupling stoichiometry of 1:1, gradients of this magnitude represent the approximate theoretical maximum for a transport system driven exclusively by the transmembrane electrochemical potential for Na+.
Cyclic variation of K+ conductance in pancreatic beta-cells: Ca2+ and voltage dependence
Authors: Ribalet B. , Beigelman P. M.
Published: December 2017

Pulses of hyperpolarizing current were injected through the microelectrode recording the electrical activity of beta-cells in order to measure input resistance. Increase in resistance during depolarization of the slow oscillation ("burst") indicates inactivation of an outward current, probably K+. Decrease in resistance as the plateau commences suggests that the previous depolarization causes activation of an inward current, probably calcium. The postburst hyperpolarization, caused by a late activation of potassium permeability (PK), would result from the increase of intracellular free calcium. An intracellular buffering system may control this intracellular free calcium level. By restoring the silent phases, in the presence of ouabain or high potassium, injection of hyperpolarizing current shows also a voltage dependency of the PK involved in the postburst hyperpolarization. Glucose, by stimulating intracellular binding of calcium, would cause membrane depolarization at glucose levels below threshold and elongation of the plateau phase at higher concentrations.
Effect of Amytal on metabolism of perfused rat heart: relationship between glycolysis and oxidative phosphorylation
Authors: Nishiki K. , Erecinska M. , Wilson D. F.
Published: December 2017

In perfused rat hearts, infusion of increasing concentration of Amytal caused progressive inhibition of respiration and increase in glycolytic activity. At maximal inhibition of respiration, with glucose as the substrate, glycolysis provided about 60% of the total ATP produced. The myocardial content of ATP remained constant irrespective of the infused Amytal concentration but [CrP]/[Cr] and [ATP]/[ADP]f[Pi] progressively decreased. Changes in the concentrations of glycolytic intermediates were observed, the most pronounced of which were increases in fructose 1,6-diphosphate and lactate contents and a decrease in the pyruvate level. Myocardial levels of oxaloacetate, malate, and alanine were elevated and so was alanine release from the tissue. Substitution of glucose with pyruvate caused a large increase in the concentrations of the tricarboxylic acid cycle intermediates and consequent accumulation of reducing equivalents in the mitochrondria. With the latter substrate, in the presence of Amytal, the rates of mitochondrial ATP production were higher than those with glucose as the substrate. The metabolic picture of the Amytal block resembles biochemical manifestations of human myopathies of mitochondrial origin, and therefore Amytal inhibition is a convenient model system for exploration of intermediary metabolism in these defects.
Role of Ca in isoproterenol-induced increases in cAMP levels in rat uterus
Authors: Meisheri K. D. , McNeill J. H.
Published: December 2017

The influence of alteration in the Ca2+ environment of the tissue on isoproterenol-induced increases in cAMP levels and relaxation was studied in rat uterus. In muscles depolarized with 47.5 mM K+ (with or without Na+), the ability of isoproterenol to increase cAMP levels and to produce relaxation was found to be inversely related to external calcium concentration. The pretreatment of the muscle with D600 or EGTA restored the cAMP response to isoproterenol in the depolarized uterus to a level observed in nondepolarized muscle. The study with Ro 20-1724, a phosphodiesterase (PDE) inhibitor indicated that the failure of isoproterenol to elevate cAMP levels in the depolarized uterus could not be related to the activation of PDE by Ca2+. The exposure of rat uterus to a zero-Ca2+ solution accentuated the increases in cAMP levels produced by isoproterenol. These results have raised the question of a possible regulatory role of Ca2+ in beta-adrenoceptor-induced increases in cAMP levels in uterine smooth muscle.
Effects of two synaptic activators, calcium and ethanol, on MEPP distribution in time
Authors: Velussi C. , Danieli-Betto D. , Boschiero R.
Published: December 2017

Miniature end-plate potentials (MEPPs) were recorded intracellularly from sartorius muscle of Rana esculenta. Tracings were divided into time bins whose duration approximated one-fifth of the mean interval between consecutive potentials. The observed number of bins containing 0, 1, 2, ... MEPPs was compared, by the X2 test, with the number calculated from the Poisson equation. MEPP timing was analyzed in the absence as well in the presence of Ca2+ (1 mM, 2.5 MM, and 15 mM). In half of the experiments, 0.5% ethanol was added to the bathing solution. In the absence of Ca2+, MEPP timing fitted the Poisson predictions. On adding Ca2+, the fit became poor and MEPPs showed the tendency to cluster. At 15 mM Ca2+, no experiment proved to be Poissonian. Though increasing the frequency of MEPPs similarly to Ca2+, ethanol maintained a Poissonian release of transmitter at any concentration of Ca2+. It is suggested that ethanol masks the effects of Ca2+ on MEPP timing by also inducing the discharge of transmitter outside the Ca2+-dependent sites of exocytosis in the presynaptic membrane.
Role of ATP in respiratory control and active transport in tobacco hornworm midgut
Authors: Mandel L. J. , Riddle T. G. , Storey J. M.
Published: December 2017

The intracellular ATP, ADP, AMP, and orthophosphate (Pi) levels were measured in the midgut of Manduca sexta. The nucleotide levels were identical in tissues either “fresh” frozen or equilibrate in regular (32 mM) K or low (8 mM) K solutions. The calculated [ATP]/[ADP][Pi]ratio was approximately 300 M-1, which is low compared to other tissues. Given the ability of this ratio to control the respiratory rate, it is speculated that this low value may cause the maximal uncontrolled respiration normally observed in the midgut. The kinetics to anoxia of active transport (Isc) and the redox level of the mitochondrial cytochromes were measured simultaneously in the midgut. The cytochromes became reduced with a time constant of 0.75 +/- 0.15 min, whereas that for Isc inhibition was 2.1 +/- 0.15 min after a delay of 0.25 min. The difference between these two kinetic rates indicates that an intermediate form of energy exists in this tissue to energize active K transport. Measurements of ATP levels during the transition to anoxia indicate that its decay kinetics are sufficiently slow for ATP to be the immediate energy source for active transport in this tissue.
Contractile properties of transplanted extensor digitorum longus muscles of cats
Authors: Faulkner J. A. , Niemeyer J. H. , Maxwell L. C. , White T. P.
Published: December 2017

Following autologous transplantation of whole extensor digitorum longus (EDL) muscles of cats into the EDL site, we investigated the degree and time course of restoration of contractile properties of autografts toward control values. Isometric and isotonic contractile properties of 66 autografted EDL muscles were measured in situ from 40 to 440 days following transplantation. The control value for maximum tetanus tension (P0) was 27 N. The mean P0 for the autografts increased from 2% of control 40 days after transplantation to 26% 440 days after transplantation. When P0 was normalized per square centimeter of muscle fiber cross-sectional area, no significant difference was observed between the P0 of autografts (28 N/cm2) and controls. Compared to controls, autografts 40-179 days following transplantation had slower time to peak twitch tensions, half-relaxation times, and maximum velocities of shortening and smaller twitch-tetanus tension ratios. Between 180 and 440 days after transplantation, values for autografts approached control values. Autografts fatigued more than twice as rapidly as controls.
Effect of protein cross-linking reagents on membrane currents of squid axon
Authors: Horn R. , Brodwick M. S. , Eaton D. C.
Published: December 2017

The effects of the protein cross-linking reagents glutaraldehyde, tannic acid, and formaldehyde were examined in voltage-clamped and internally perfused squid axons. All three reagents reduced sodium and potassium currents and reduced or abolished sodium inactivation. Glutaraldehyde and tannic acid produced an alteration of potassium current kinetics.
Secretory response of dispersed rat submandibular cells. II. Mucin secretion
Authors: Quissell D. O. , Barzen K. A.
Published: December 2017

The secretory response of dispersed rat submandibular cells as it relates to the secretion of D-[1-14C]glucosamine hydrochloride-labeled mucin following sympathomimetic and parasympathomimetic stimulation was evaluated. The adrenergic agonists (-)-norepinephrine and (-)-epinephrine were found to have equal efficacy and potency with a median effective concentration (EC50) of 7.1 x 10(-7) M. (-)-Isoproterenol was found to be acting as a "partial" agonist and had an EC50 of 3.9 x 10(-7) M. (-)-Phenylephrine addition resulted in a small, but significant, secretion of mucin at higher doses tested (10(-4) M--10(-3) M). Neither cholinergic nor alpha-adrenergic receptor stimulation was able to elicit a net increase in the secretion of mucin. However alpha-adrenergic receptor activation in conjunction with beta-adrenergic receptor activation facilitated the rate of secretion. Extracellular Ca2+ and Mg2+ were not required for the secretion of mucin, but extracellular Ca2+ enhanced the rate of secretion following alpha- and beta-adrenergic receptor activation. However extracellular Ca2+ did not enhance mucin secretion following beta-adrenergic receptor activation. Both cellular Ca2+ and beta-adrenergic receptor activation were required to elicit a secretory response following sympathomimetic stimulation.
Anion selectivity of the chloride-transport process in dog tracheal epithelium
Authors: Widdicombe J. H. , Welsh M. J.
Published: December 2017

We have measured the fluxes of 82Br or 131I across short-circuited dog tracheal epithelium following complete replacement of chloride in the bathing medium by either bromide or iodide. In addition, we have partly replaced the bathing chloride with bromide, iodide, or thiocyanate and studied the effects of these anions on 36Cl fluxes. Our results show that the affinity sequence for the chloride transport process is I > Br congruent to Cl, that Br is transported, but that I is not. Thiocyanate is a potent inhibitor of chloride transport, and its effects are greater than those predicted by a simple competitive interaction with chloride for transport sites.
Temperature acclimation: effects on membrane physiology of an identified snail neuron
Authors: Zecevic D. , Levitan H.
Published: December 2017

The neuronal basis for thermal acclimation was examined by comparing the short- and long-term effects of temperature change on the physiological properties of an identified neuron in the isolated ganglion of Hexis aspersa. Using intracellular electrophysiological techniques, we found that the frequency of spontaneous action potentials and excitability of neurons from warm-acclimated animals was depressed by abruptly cooling from 20 to 5 degrees C. After a 2-wk period of acclimation to 5 degrees C, the levels of spontaneous activity and excitability were comparable to those of warm-acclimated neurons at 20 degrees C. Conversely, abrupt warming of neurons from cold-acclimated animals greatly increased the frequency of spontaneous activity, but after acclimation to 20 degrees C the frequency decreased. Although the duration of the action potential and the cell's electrogenic Na-K pump were temperature sensitive, thermal acclimation had no obvious effects on these parameters. Membrane permeability to Na and PNa/PK decreased with cooling, whereas PRb/PK and PCs/PK increased. Warming had the opposite effect on the relative alkali cation permeability (PX/PK). With acclimation PX/PK underwent compensatory changes.
Penetration-induced hyperpolarization as evidence for Ca2+ activation of K+ conductance in isolated smooth muscle cells
Authors: Walsh J. V. , Singer J. J.
Published: December 2017

Single smooth muscle cells, freshly isolated by enzymatic digestion of the stomach muscularis of the toad Bufo marinus were studied under direct microscopic observation using standard electrophysiological techniques. Following penetration with a microelectrode, a hyperpolarization lasting many seconds occurred before the membrane depolarized to a steady-state level. The following lines of evidence indicate that the penetration-induced hyperpolarization results from an increase in K+ conductance caused by Ca2+ that enters the cell at the time of penetration: 1) The cell contracted at the time of penetration indicating that [Ca2+]i was elevated even though no action potential had occurred; the cell subsequently relaxed. 2) The input resistance was much lower during the hyperpolarization than during the steady-state resting potential. In the steady state all cells displayed outward-going rectification. 3) At constant [Ca2+]0, the amplitude of the hyperpolarization varied with log[K+]0 (1.3-56 mM) to a much greater degree than did the steady-state potential. Tetraethylammonium chloride (TEA) (18.2 mM) reduced the hyperpolarization. 4) At constant [K+]0, the amplitude of the hyperpolarization increased as the [Ca2+]0 was raised (1.8-52.1 mM). 5) With [Ca2+]0 low (less than or equal to 0.16 mM), the hyperpolarization was almost completely abolished in the presence of a high concentration of Ba2+ (80 mM) or Mn2+ (79.2 mM); this was not the case with Sr2+.
Sialic acid: regulation of electrogenesis in cultured heart cells
Authors: Nathan R. D. , Fung S. J. , Stocco D. M. , Barron E. A. , Markwald R. R.
Published: December 2017

Removal of up to 50% of the sialic acid from aggregates of 7-day chick embryo heart cells during incubation in a highly purified preparation of neuraminidase resulted in hyperpolarization of the maximum diastolic potential, reduction in the slope of diastolic depolarization leading to slowing of beating, negative shifts of threshold potential and the voltage at which upstroke velocity was maximal, and an initial increase in the action potential overshoot. These effects were opposite to those induced by phospholipase C, a possible contaminant. The modification of electrical properties by neuraminidase is suggested to result from an enhanced influx of calcium ions that might "screen" or bind specifically to internal negative fixed charges and thereby shift the voltage dependence of conductance and kinetic parameters to more negative potentials. This hypothesis is supported by the results above and by greater uptake of 45Ca following release of sialic acid, augmentation of enzyme-induced changes in elevated Ca2+, and the similarity of effects produced by A23187, and inophore which also increased 45Ca uptake. However, other mechanisms cannot be ruled out at the present time.
An electrogenic component of resting potential in rabbit ventricular muscle?
Authors: Ruiz-Ceretti E. , Nguyen T. A. , Schanne O. F. , Caille J. P.
Published: December 2017

The resting potential and the intracellular Na and K concentrations (Nai, Ki) were determined at several extracellular K concentrations (Ko) between 0.5 and 18 mM and after inhibition of the sodium pump with 0.5 microM ouabain. Exposure to low Ko (0.5 mM) produced a transient hyperpolarization (from -80 to -100 mV) followed by a depolarization that led to a stable potential of -60 mV within 25 min. Similar potential levels were observed in the presence of ouabain regardless of the Ko/Ki ratio. Intracellular sodium increased at Ko < 5 mM, whereas Ki rose at Ko less than or equal to 1.0 mM. Because of the large decrease of Ki at Ko = 0.5 mM, Ko/Ki was the same at 0.5 and 1 mM. However, the resting potentials at the steady state differed by 50 mV at these concentrations. A PNa/PK of 0.032 for the control conditions was obtained with the Mullins-Noda equation using 2.5 as the Na-K coupling ratio. This PNa/PK value yielded a Goldman potential of -69 mV; so we estimated that electrogenic sodium extrusion contributed -10 mV to the resting potential. The size of the electrogenic potential increased as Ko was lowered from 5 to 1 mM. This finding suggests that the control of the Na-K coupling ratio may be independent of the mechanism that controls the pumping rate.
Inhibition of tetanus tension by elevated extracellular calcium concentration
Authors: Howell J. N. , Snowdowne K. W.
Published: December 2017

Extracellular [Ca2+] in the range of 5-20 mM produces a concentration-dependent reversible reduction in tetanus tension in single frog skeletal muscle fibers. Both peak tension and ability to sustain tension during tetanus is reduced. The effect is unrelated to osmotic effects and independent of stimulation frequency in the range 100-200 Hz. The effect occurs both at 8 and 24 degrees C. Tetanus tension is most strongly inhibited by elevated extracellular [Ca2+] at short muscle lengths, but the effect can be seen at all lengths. Microelectrode recordings during tetanus indicate that action potentials remain undiminished in amplitude and duration throughout the tetanus. The evidence suggests that the inhibition results from a failure of action potentials propagation within the transverse tubular system.
Effects of inhibitors of F0-F1 proton-translocating ATPase on urinary acidification
Authors: Fanestil D. D. , Park C. S.
Published: December 2017

A proton pump in diverse biological systems consists of two structural units with separate but integrated functions, the F0-F1-ATPase. We tested by chemical perturbation the possibility that such a proton pump might be involved in urinary acidification conducted by urinary epithelia (UF0-UF1-ATPase). Tyrosine-reactive chemicals and N,N'-dicyclohexylcarbodiimide, known to block the proton channel unit (F0), also inhibited urinary acidification, as measured by the reverse short-circuit current (RSCC) in urinary bladders from toads and turtles. Since these chemicals were equally effective under aerobic and anaerobic conditions, the inhibition appears to occur directly on UF0 rather than on mitochondria. In contrast, an inhibitor of F0-F1-ATPase, oligomycin, only inhibited aerobic RSCC and was ineffective on anaerobic RSCC. Thus, oligomycin appears to inhibit mitochondrial F0-F1 rather than any UF0+UF1. Another inhibitor of the F1-ATPase unit, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline, was without effect on RSCC at a concentration (0.3 mM) that produced over 50% inhibition of short-circuit current. These results support the concept that acidifying urinary epithelia contain a plasma membrane proton channel, UF0, but that UF1, if it exists, is unique in that it is resistant to known inhibitors.
Possible mechanisms of stimulus-induced vacuolation in serous cells of tracheal secretory glands
Authors: Quinton P. M.
Published: December 2017

Several factors affecting the stimulus-induced vacuolation in cat tracheal submucosal glands described in a companion paper [Am. J. Physiol. 241 (Cell Physiol. 10): C18–C24, 1981] were examined. Stimulation with predominantly alpha-adrenergic, beta-adrenergic, and cholinergic agonists at various concentrations and in the presence of appropriate blockers showed that alpha-adrenergic or cholinergic stimulation is more effective in inducing vacuolation than beta-adrenergic stimulation. In addition, Ca- or HCO3- deficient incubation medium or low incubation temperature either blocked or significantly decreased the vacuolation response. Stimulation in the presence of the Na-K-ATPase inhibitor ouabain prevented vacuolation. The known roles of these factors in fluid transport are consistent with the possibility that stimulus-induced vacuolation possibly arises in response to disturbances in cytoplasmic fluid, which are induced in the secretory cell when the fluid component of secretion is intensely stimulated. A model is presented that possibly relates the observed responses to intracellular event and suggests the possibility of ion pumps in the apical membrane.
Locus of N-ethylmaleimide action on sugar transport in nucleated erythrocytes
Authors: Whitfield C. F. , Schworer M. E.
Published: December 2017

Goose red blood cells were studied as a model for metabolic regulation of sugar transport. In contrast to their action in human erythrocytes, sulfhydryl-blocking agents such as N-ethylmaleimide (NEM) stimulated 3-O-methylglucose transport markedly in goose red blood cells. The effect of NEM was further enhanced when adenosine 5'-triphosphate (ATP) was first depleted by 2,4-dinitrophenol treatment or anoxia. Only sulfhydryl-blocking agents that enter the cell were effective transport stimulators, and the effect was not altered by substrates of the transporter. In nucleated red blood cell ghosts, NEM inhibited 3-O-methylglucose transport. Results of these studies with intact cells were consistent with the hypothesis that free sulfhydryl groups are essential for regulation of transporter activity rather than for the transport process itself. The locus of NEM action appears to be either on the cytoplasmic side of the membrane or partially located in the cytoplasm. ATP depletion may expose previously masked sulfhydryl groups, producing an enhanced reaction with sulfhydryl-blocking agents and a highly stimulated rate of sugar transport.
Separate turnover of cytochrome c and myoglobin in the red types of skeletal muscle
Authors: Hickson R. C. , Rosenkoetter M. A.
Published: December 2017

The purpose of this investigation was to determine whether cytochrome c and myoglobin have similar turnover rates in the three types of skeletal muscles. Exercise (endurance training) was used as an inducing stimulus to increase their concentrations. The half-lives (t 1/2) were subsequently estimated from the time course of return to base-line levels after cessation of exercise. When exercise was stopped, cytochrome c concentration returned rapidly to control levels; the lengths of t 1/2 were approximately 8 days in fast-twitch red, 5 days in slow-twitch red, and 9 days in mixed muscles. These findings confirm previous results of cytochrome c turnover. The concentration of myoglobin decreased at a slower rate than that observed for cytochrome c during detraining in fast-red slow-red, and plantaris muscles, and did not return to sedentary control levels throughout the 50-day detraining period. Myoglobin concentration in fast-twitch white muscle did not increase with the training. These results provide evidence that the degradation rate of myoglobin differs from that of cytochrome c in the red types of skeletal muscle. These elevated myoglobin levels may, in part, provide one explanation for the slow rate of decline in aerobic power that has been observed when individuals stop exercising.
An improved liquid ion exchanger for chloride ion-selective microelectrodes
Authors: Baumgarten C. M.
Published: December 2017

A new chloride liquid ion exchanger, Corning 477913, suitable for fabricating ion-selective microelectrodes (ISE) is described. It differs from the standard chloride exchanger. Corning 477315, in that the concentration of organophilic ligand is increased fivefold. The properties of ISE (tip diameter less than 1 micrometer) made from the new material are superior to those of ISE made from the standard material. The yield of acceptable ISE was greater (77 vs. 34%); the ISE resistance was decreased 5.3-fold; the average slope was improved (-52.8 vs. -45.5 mV); and the selectivity over some interfering anions was increased. Measurement of intracellular chloride activity in rabbit ventricle with ISE made from the new exchanger gave results similar to those reported previously with the standard exchanger. This suggests that 477913 is satisfactory for use in biological tissue. The new formulation should be particularly advantageous for measurements requiring ISE with very fine tips and in fabrication of multibarrel ISE.
Mucociliary frequency of frog palate epithelium
Authors: Puchelle E. , Zahm J. M. , Sadoul P.
Published: December 2017

The depleted frog palate epithelium is extensively used as a model for studying the transport velocity of mucus. It is speculated that the epithelium can be depleted of endogenous gel mucus without affecting the ciliary frequency, provided the sol phase where the cilia beat remains present. To control the stability of the ciliary frequency during the measurement of mucous transport velocity, we have used a photoelectric method for measuring mucociliary frequency and have carried out experiments in excised frog palates in different areas and at successive stages of depletion. From the distribution function of the mucociliary frequency obtained by the fast Fourier transform analysis, we have calculated the mean frequency (Fm) and the dispersion of the spectrum defined as spectrum width (Sw). Fm and Sw are similar in different areas throughout the zone selected for mucous transport velocity measurements. A significant decrease in Fm (21%) and parallel increase in Sw (22.8%) are observed at the stage of depletion, but the values return to normal when a drop of frog's mucus is added. These results suggest that the depleted frog palate is a good model for studying the in vitro transport rate of mucus and investigating the effects of mucus on ciliary frequency.
Effects of a thioreactive agent, diamide, on neuromuscular transmission in lobster
Authors: Colton C. A. , Colton J. S.
Published: December 2017

Diamide[diazine-dicarboxylic acid-bis(dimethylamide)], a thiol-oxidizing agent, has both pre- and postsynaptic actions on the glutaminergic neuromuscular junction of the lobster walking leg. Postsynaptically, diamide produced an increase in the response to exogenously applied glutamate, whereas the effect of diamide on presynaptic transmitter release involved two major changes: 1) a decrease in excitatory junction potential amplitude and 2) an increase in miniature junction potential frequency. Short-term facilitation also decreased. Equilibration with 1,4-dithiothreitol (a sulfhydryl-reducing agent) reversed the decline in excitatory junction potential frequency, and the fall in short-term facilitation. The miniature junction potential frequency increase induced by diamide was independent of external Ca2+, as diamide in a Ca2+-free solution produced a similar response to that in a Ca2+-containing solution. We propose that the action of diamide on transmitter release is similar to the action of polyvalent cations, i.e., diamide has two sites of action, a blockade of inward Ca2+ flux and an increased release of Ca2+ inside the terminal.
Intracellular pH mediates action of insulin on glycolysis in frog skeletal muscle
Authors: Fidelman M. L. , Seeholzer S. H. , Walsh K. B. , Moore R. D.
Published: December 2017

In a glucose-free bicarbonate Ringer (5% CO2 in N2), insulin increased intracellular pH (pHi), as determined by [14C]dimethadione, by 0.12 +/- 0.02 and stimulated glycolysis, as monitored by anaerobic lactate production, by 42.9 +/- 3.5% in paired frog sartorius muscles. The effect of insulin on glycolysis was shown to vary approximately linearly with log [Na+]0, being converted in 0.12 mM Na+ Ringer to a 51.5 +/- 8.4% inhibition of glycolysis. As the Na+ free-energy gradient was varied by decreasing [Na+]0 from 104 to 6.8 mM, the changes in glycolytic flux produced by insulin consistently paralleled the changes in pHi produced by the hormone. The relationship between the change in pHi and percent change in glycolytic flux was the same regardless of whether the effects were produced by insulin or by changing CO2. When glycolysis was either stimulated or inhibited, intracellular levels of fructose 6-phosphate varied inversely with glycolytic flux. This indicates that the effect on glycolysis of either insulin or changes in CO2 is due to a change in the activity of phosphofructokinase. The results support the model that the acute effect of insulin on glycolysis is mediated by a change in pHi, consequent to activation by insulin of Na:H exchange at the plasma membrane.
Metabolite changes in individual rat muscle fibers during stimulation
Authors: Hintz C. S. , Chi M. M. , Fell R. D. , Ivy J. L. , Kaiser K. K. , Lowry C. V. , Lowry O. H.
Published: December 2017

Rat plantaris and soleus muscles were stimulated intensely in vivo for 1 and 15 min, freeze-clamped, and freeze-dried, and individual fibers were dissected free. Fibers, assigned to four groups on the basis of lactate dehydrogenase and malate dehydrogenase, were each separately analyzed for ATP, P-creatine, glycogen, glucose, glucose-6-phosphate (glucose-6-P), lactate, citrate, and malate. Some fibers were also analyzed for fructose 1,6-phosphate, total adenylate and total creatine. Although each group as a whole showed significant and often large differences in control composition and response to stimulation, individual fibers varied enough to create an almost continuous spectrum of metabolite levels from one extreme to the other. The data suggest that the slowest twitch fibers were the most active in the control state. Stimulation for 1 min caused a small increase in ATP in all groups with a large decrease in P-creatine in "fast white" fibers and a modest decrease in the rest. After 15-min stimulation, fast white fibers had lost 60% of initial ATP and 97% of initial P-creatine, whereas in other fiber types these compounds underwent little further change. Metabolite changes with stimulation were also greatest in fast white fibers. Glucose-6-P rose 15-fold in 1 min, then fell to below control by 15 min when glycogen had been exhausted; lactate rose two to six times more than in other types. Glucose rose in all groups to levels at 15 min, compatible with equilibrium with blood plasma.
Contractile properties of hindlimb muscles in rat during surgical overload
Authors: Freeman P. L. , Luff A. R.
Published: December 2017

Structural and dynamic properties of the extensor digitorum longus (EDL) and soleus muscles of the rat under pentobarbital sodium anesthesia have been determined 4, 28, and 400 days after extirpation of the tibialis anterior and compared with the properties of muscles from the contralateral limb of the same animal. At 4 days the mass of the EDL increased by about 17%, but the maximum tetanic tension and tension per unit cross-sectional area decreased. The soleus showed a slight decrease in mass of about 6%. At 28 days the mass of the EDL had increased to 126% of the control value. Maximum isometric twitch and tetanic tensions increased, whereas tetanic tension per unit cross-sectional area returned to normal values; the number of sarcomeres in series increased, which led to an increase in the whole muscle speed of shortening. It is concluded that the synergistic EDL undergoes compensatory hypertrophy by the addition of contractile material in series and in parallel but without any change in the intrinsic properties of the muscle. The fibers of the antagonistic soleus shorten slightly but are otherwise unchanged.
Sodium-calcium exchange and sarcolemmal enzymes in ischemic rabbit hearts
Authors: Bersohn M. M. , Philipson K. D. , Fukushima J. Y.
Published: December 2017

We have investigated alterations in sarcolemmal function that occur during myocardial ischemia. Rabbit ventricles were incubated at 37 degrees C for time periods ranging from 5 min to 2 h. The ischemic tissue was homogenized, and activities of the sarcolemmal enzymes Na+-K+-ATPase, K+-p-nitrophenylphosphatase (K+-pNPPase), and adenylate cyclase were measured in the crude homogenate. Na+-K+-ATPase and K+-pNPPase were substantially inhibited after only 10 min of ischemia, and activities for all three enzymes declined progressively up to 1 h of ischemia, when activities were 37–59% of control. Highly purified sarcolemmal membranes prepared from control tissue and myocardium that had been made ischemic for 1 h showed similar purification of sarcolemmal enzymes, passive Ca2+ binding, and passive permeability to Ca2+. However, the velocity of Na+-Ca2+ exchange in ischemic sarcolemmal vesicles was reduced approximately 50% due to a reduction in Vmax. Although the parallel decline in activities of several sarcolemmal functions might suggest a change in membrane structure, phospholipid and cholesterol contents in ischemic sarcolemma were the same as control.
Spontaneous sweat secretion in calcium-free strontium medium
Authors: Sato K. , Sato F.
Published: December 2017

Sr2+ substitutes for Ca2+ in methacholine (MCH)-evoked sweat secretion in isolated simian eccrine sweat glands, but four to eight times greater concentrations of Sr2+ were required to achieve the same effect as did a given Ca2+ concentration. In Ca2+-free Sr2+ medium the anticholinergic effect of atropine was reduced by about four orders of magnitude. In the Sr2+ medium, spontaneous sweat secretion (SSS) was also observed in the absence of any pharmacological stimuli. SSS occurs slowly, reaching a plateau after 30 min incubation. The maximal SSS is a saturation function of Sr2+ concentration reaching a maximum at 8 mM. Sr2+-induced SSS was inhibitable by removal of Sr2+, low bath temperature (10–14 degrees C), ouabain (5 X 10(-6) M), and D 600 (10(-3) M). Ca2+ was found to inhibit Sr2+-induced SSS, but a Schild plot for the Ca2+ and Sr2+ dose relationship failed to show a slope of unity. The data suggest that Sr2+-induced SSS represents actual secretory processes, which are triggered by Sr2+ leaking into the cell through as yet undefined membrane channels.
Glucose-induced proton uptake in secretory granules of beta-cells in monolayer culture
Authors: Pace C. S. , Sachs G.
Published: December 2017

The weak base acridine orange (AO) has been shown to be accumulated by the insulin-containing secretory granules of cultured beta-cells in response to high glucose. Various lines of evidence indicate that this accumulation is due to a pH gradient. Thus ionophores such as monensin and nigericin abolish the glucose-induced accumulation, and a high concentration of the weak base, benzylamine, results in swelling of the granules. In the absence of glucose, ATP addition to digitonin-permeabilized cells also results in dye uptake. These data also suggest that a primary or secondary active accumulation mechanism for hydrogen ions exists across the granule membrane.
A simple method for the accurate determination of free [Ca] in Ca-EGTA solutions
Authors: Bers D. M.
Published: December 2017

A simple method for the accurate determination of free [Ca] in ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)-buffered Ca solutions is described. This method is useful for calibration of Ca macro- and microelectrodes to low free [Ca] and should improve the reliability of calculated free [Ca] in more complex solutions. Briefly, free [Ca] in Ca-EGTA solutions is measured with a Ca electrode, bound Ca is calculated, and Scatchard and double-reciprocal plots are resolved for the total [EGTA] and the apparent Ca-EGTA association constant (K'Ca) in the solutions used. The free [Ca] is then recalculated using the determined parameters, giving a more accurate knowledge of the free [Ca] in these solutions and providing an accurate calibration curve for the Ca electrode. These solutions can then be used to calibrate other Ca electrodes (e.g., Ca microelectrodes) or the calibrated Ca electrode can be used to measure free [Ca] in solutions containing multiple metal ligands. This method allows determination of free [Ca], K'Ca, and total [EGTA] in the actual solutions used regardless of pH, temperature, or ionic strength. It does not require accurate knowledge of K'Ca or EGTA purity and circumvents many potential errors due to assumption of binding parameters. K'Ca was found to be 2.45 +/- 0.04 X 10(6) M-1 in 100 mM KCl, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 1 mM EGTA at pH 7.00 and 23 degrees C. Total [EGTA] varied with supplier but was always less than quoted.
Effect of GABA and isogabaculine on ornithine decarboxylase and putrescine metabolism
Authors: Diekema K. A. , McCann P. P. , Lippert B. J.
Published: December 2017

Isogabaculine (3-amino-1,3-cyclohexadienyl carboxylic acid; RMI 71,932), an irreversible inhibitor of GABA transaminase, when added to mouse neuroblastoma cells in spinner culture at the time of induction of cell proliferation, increased ornithine decarboxylase (ODC) activity threefold above that of normal control cells and twofold above that of GABA (gamma-aminobutyric acid)-treated cells. Isogabaculine did not affect ODC activity of rat glioma (C6) or rat hepatoma (HTC) cells. As determined by half-life measurements of ODC and intracellular GABA concentrations, isogabaculine apparently has a direct stabilizing effect on ODC in neuroblastoma cells that is unrelated to the accumulation of GABA due to GABA transaminase inhibition. Putrescine metabolism to GABA or spermidine was determined in C6, HTC, and neuroblastoma cells in the presence or absence of isogabaculine and/or GABA. Neither GABA nor isogabaculine treatment dramatically altered the metabolism of putrescine to GABA or spermidine in neuroblastoma, C6 glioma, or HTC cells. However, the appreciable amount of labeled GABA formed from putrescine indicated that this metabolic route may be more important than was previously thought.
Effect of cholera toxin on cAMP levels and Na+ influx in isolated intestinal epithelial cells
Authors: Hyun C. S. , Kimmich G. A.
Published: December 2017

Freshly isolated chicken intestinal cells contain approximately 20 pmol adenosine 3',5'-cyclic monophosphate (cAMP)/mg cellular protein. Incubation with 3 micrograms/ml cholera toxin (CT) at 37 degrees C induces an elevation of cellular cAMP beginning 10-15 min after initial exposure. The response is linear with time for 40-50 min and causes a six- to eightfold increase over control levels at steady state. Dibutyryl cAMP and agents that increase cAMP production inhibit Na+ influx into the isolated enterocytes. Chlorpromazine completely abolishes the toxin-induced elevation of cAMP in the isolated cells and also reverses the effect on Na+ entry. The data provide evidence for a cAMP-mediated control of intestinal cell Na+ uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na+ during induction of intestinal secretory activity. Studies on the time-dependent effects of chlorpromazine on both intracellular cAMP concentration and Na+ influx suggest that the reactivation of the Na+ transport system after cAMP-induced inhibition is slow relative to the disappearance of cAMP.
Mechanism of glucocorticoid effect on renal transport of phosphate
Authors: Turner S. T. , Kiebzak G. M. , Dousa T. P.
Published: December 2017

We explored whether glucocorticoid administration, a known stimulus of renal gluconeogenesis (GNG), could decrease avid inorganic phosphate (Pi) reabsorption in rats stabilized on low-phosphorus diet (LPD). Rats adapted to LPD were injected with the glucocorticoid (GCD) triamcinolone acetonide (1.25 or 2.5 mg.100 g body wt-1.day-1 ip) for 2 days; they showed a profound increase in urinary excretion of Pi during the injection period. In clearance studies GCD increased the clearance and fractional excretion of Pi but did not change the filtered load of Pi. Initial "uphill" Na+-gradient (Nao+ greater than Nai+)-dependent uptake of 32Pi by luminal brush-border membrane (BBM) vesicles prepared from renal cortex of rats treated with GCD was markedly (greater than 40%) decreased compared with control rats; Na+-gradient-dependent uptake of D-[3H]glucose was not diminished. At the "equilibrium" time interval, measured at 120 min, BBM vesicles from control and GCD-treated rats did not differ in the uptake of 32Pi or D-[3H]glucose. With kinetic analysis, BBM from GCD-treated rats showed a marked decrease (-40%) in the maximum velocity (Vmax) of initial Na+-dependent 32Pi uptake, but the apparent affinity of the BBM transport system for Pi (apparent Km = 0.078 mM Pi) was not different from that of controls. Alkaline phosphatase specific activity was much lower (-40%) in BBM from GCD-treated rats compared with controls, but the activities of three other BBM enzymes (maltase, leucine aminopeptidase, and gamma-glutamyl transferase) were not different. The addition of triamcinolone to BBM in vitro had no effect on either Na+-dependent uptake of 32Pi or alkaline phosphatase activity. The rate of GNG from alpha-ketoglutarate was significantly increased in cortical slices from GCD-treated rats adapted to LPD. Also, the NAD+-to-NADH ratio was higher in the renal cortex of GCD-treated rats, although the total content of NAD [NAD+ + NADH] was not different from controls. Renal excretory, BBM, and metabolic changes elicited controls. Renal excretory, BBM, and metabolic changes elicited by GCD treatment were similar in intact and thyroparathyroidectomized rats. Phosphaturia elicited in rats fed LPD by GCD administration in vivo appears to be at least in part due to a decreased capacity of luminal BBM of proximal tubules for decreased capacity of luminal BBM of proximal tubules for Na+-dependent uptake of Pi. Although the causal relationship between observed parameters is not established, our results are compatible with the interpretation that an increase in the rate of renal GNG, perhaps via action of NAD+ on BBM (J. Clin. Invest. 67: 1347-1360, 1981), decreases luminal uptake and reabsorption of Pi in proximal tubules.
Nature of anthopleurin-B-induced release of norepinephrine from adrenergic nerves
Authors: Ohizumi Y. , Shibata S.
Published: December 2017

Anthopleurin-B (AP-B), a new polypeptide from sea anemone (Anthopleura xanthogrammica), markedly increased the amount of norepinephrine (NA) released from the guinea pig isolated vas deferens. The AP-B-induced release of NA was inhibited or abolished by pretreatment with reserpine and by guanethidine or procaine but remained almost unaffected by mecamylamine. D 600, nifedipine, diltiazem, Mn2+, and Mg2+ markedly inhibited the NA releasing action of AP-B. The AP-B-induced release of NA increased in a linear fashion with increasing Na+ concentrations (85-150 mM). Also the NA release by AP-B increased with an increase in the concentration of external Ca2+ from 0 to 0.8 mM but decreased with an increase in the Ca2+ concentration from 0.8 to 2.0 mM. These results suggest that AP-B increases the permeability across the nerve cell membrane to both Na+ and Ca2+ and that this plays an important role in the NA release from the adrenergic nerve.
Reduction of hematopoietic stem cells and adaptive increase in cell cycle rate in rickets
Authors: Wientroub S. , Hagan M. P. , Reddi A. H.
Published: December 2017

The close but complex physiological interactions between osteogenesis and hematopoiesis during differentiation, development, and function are unclear. As vitamin D has a major role in bone metabolism and function, we have investigated the effects of vitamin D deficiency on bone marrow cellular kinetics. Using rats in vitamin D-deficient status in utero as well as in postfetal life, we have investigated changes in hematopoiesis. Total cellularity, colony-forming unit (CFU) content, and the cycled rate were determined from the axial compartment of the femoral marrow. A marked reduction in the CFU content and a corresponding increase in the cycle fraction began at 30 days of age and stabilized by 45 days of age at 4.2 +/- 0.4 CFUs per 1 X 10(5) nucleated cells in the vitamin D-deficient animals as compared with 9.4 +/- 1.6 in the control group. During the same period of time the CFU generation time decreased from 61 +/- 5 h in the normal animals to 24 +/- 6 h in the vitamin D-deficient animals. Administration of either 1,25-dihydroxyvitamin D [1,25-(OH)2D3] or 24,25-(OH)2D3 at 45 days of age on a daily basis for 7-14 days failed to correct the defect although the serum calcium and phosphate were corrected. The data indicate that factors influencing the development of bones may affect bone marrow cells as well. Vitamin D metabolites may be needed for normal development and function of marrow-colony-forming cells in utero.
High-affinity pH-dependent passive Ca binding by myometrial plasma membrane vesicles
Authors: Grover A. K. , Kwan C. Y. , Daniel E. E.
Published: December 2017

Rat myometrium plasma membrane-(PM) enriched fraction N1 binds calcium passively in a pH-dependent manner at a Ca2+ concentration of 1 microM. The Ca binding increases with increasing pH from 6.27 to 7.47 with a half maximum near 6.8. The difference between binding at 6.27 and 7.07 (the pH-dependent Ca binding) depends on the pH of the reaction medium rather than the pH of the medium in which the membranes had previously been suspended. The pH-dependent Ca binding is not an artifact due to EGTA, the pH buffer used, or soluble protein trapped inside the membrane vesicles. The pH-dependent Ca binding occurs with a dissociation constant value of 0.28 microM and Hill coefficient of 2.37 for Ca2+. The high affinity pH-dependent Ca uptake and the release of Ca2+ from the membranes is virtually complete in 10 s in the presence of 1 microM A23187 but not in its absence. The distribution of the pH-dependent Ca binding in the various rat myometrium subcellular fractions parallels the activity of 5'-nucleotidase in these fractions and not the activities of NADPH-dependent or succinate-dependent cytochrome c reductases. The high affinity and rapid binding and release of Ca at the pH-dependent Ca binding sites in the PM-enriched fraction suggests that the binding and release from these sites may play a key role in excitation-contraction coupling of the smooth muscle.