bluuebunny/crossref_metadata_2025 huggingface dataset:
Abnormal brown adipose tissue in hamsters with muscular dystrophy
Authors: Himms-Hagen J. , Gwilliam C.
Published: December 2017

The size (wet weight, total protein, total cytochrome oxidase) of interscapular brown adipose tissue is reduced to about one-half of normal in the cardiomyopathic hamster (BIO 14.6). The mitochondria are normal in binding of purine nucleotides [guanosine 5'-diphosphate (GDP)] and in proportion of polypeptides in the region of 32,000, both indices of the thermogenic proton conductance pathway, and in specific activity of cytochrome oxidase. Brown adipose tissue of the cardiomyopathic hamster can grow during acclimation to 4 degrees C, but its size remains smaller than in cold-acclimated normal hamsters. Mitochondrial polypeptide composition is not altered by acclimation to cold, but a large increase in mitochondrial GDP binding occurs in both normal and cardiomyopathic hamsters. The reduced calorigenic response of cardiomyopathic hamsters to catecholamines (Horwitz, B.A., and G.E. Hanes, Proc. Soc. Exp. Biol. Med. 147: 393-395, 1974) may, at least in part, be explained by a reduction in the amount of brown adipose tissue, the major site of this response. A defect in control of growth of this tissue in the cardiomyopathic hamster is suggested.
Effects of deuterium oxide on mechanics and energetics of skeletal muscle contraction
Authors: Rall J. A.
Published: December 2017

Mechanics and energetics of contraction were studied in isolated frog semitendinosus muscles (n = 15) in deuterium oxide (D2O) at 5 degrees C. Twitch force and energy liberation (heat + work) in 99.8% D2O-Ringer at lo were 4 +/- 0.8 and 22 +/- 1%, respectively, of control values. Tetanus force was slowed in development, and developed tetanus force was reduced to 89 +/- 7% of control values. With repeated tetanic contractions, the rate of force development increased, and peak force increased above control values in some muscles. The twitch was potentiated 10-fold (10 +/- 2) after repeated tetanic contractions. Energy liberation increased but not in proportion to force. Ca2+ cycling energy liberation or activation heat, estimated from stretched muscles, was 60 +/- 3% of control values. With maximum posttetanic twitch potentiation, the activation heat increased by 25 +/- 3%. Peak twitch force and energy liberation occurred at longer muscle lengths than in normal Ringer. After correcting for small differences in force development at lo and 1.38 +/- 0.01 lo, it was possible to estimate the effect of length per se on energy liberation. Under these conditions, there was an 8 +/- 2% decrease in energy liberation at 1.38 lo.
Uptake of sulfobromophthalein-glutathione conjugate by isolated hepatocytes
Authors: Schwarz L. R. , Gotz R. , Klaassen C. D.
Published: December 2017

Uptake of sulfobromophthalein-glutathione conjugate (BSP-GS) was studied using isolated hepatocytes and was compared with previous results with the parent BSP (Schwenk et al., Eur. J. Biochem. 64: 189-197, 1976). Adsorption of BSP-GS to the liver cell membrane has two sites with different affinities and binding capacities. Uptake of the conjugate is decreased by two metabolic inhibitors and appears to be saturable up to 12.5 microM with an apparent Km of 4 microM (7 for BSP) and a Vmax of 0.16 nmol x mg protein-1 x min-1 (2.6 for BSP). Similar to the parent BSP at higher concentrations a second mechanism of uptake is observed indicated by the nonlinear Hanes plot. SP-GS transport is competitively inhibited by BSP with a Ki of about 1 microM, and, similar to BSP, replacement of sodium ions in the medium had little effect on the rates of uptake. These results suggest a common transport mechanism for BSP and its glutathione conjugate. Furthermore, the effect of organic anions, bases, and steroids on uptake of BSP-GS has been tested.
Passive properties of the membrane of single freshly isolated smooth muscle cells
Authors: Singer J. J. , Walsh J. V.
Published: December 2017

Single, smooth muscle cells were isolated from the stomach muscularis of the toad Bufo marinus and studied on the same day as isolation using standard electrophysiological techniques and direct microscopic observation at high magnification. Following penetration a period of hyperpolarization occurred that appeared to be caused by an increase in K+ conductance activated by Ca2+ entering the cell upon penetration. Ion substitution studies showed that the stable steady-state resting potential was dependent on both [Na+]0 and [K+]0. At [Ca2+]0 = 1.8 mM, active responses could be elicited which, at the higher [Ca2+]0 (< 8mM) generally employed, became action potentials with overshoots. Calculations employing the equations for a short cable and the observed change of membrane potential as a single exponential in response to a small hyperpolarizing current step both indicated that the length constant (lambda) was sufficiently greater than the cell length so that the cell behaved as an isopotential surface during subthreshold perturbations. From photomicrographic measurements of each cell studied and the input resistance, values of specific membrane resistance (Rm) were obtained that ranged as high as 152 k omega x cm2 depending on the ionic environment, most notably on [Ca2+]0. The membrane capacity (Cm) referred to the surface area measured with light microscopy was 1.3 +/- 0.3 microF/cm2 (mean +/- SD). When the best estimate of caveolar membrane area was included, Cm referred to total membrane area (caveolar plus noncaveolar) was approximately 0.8 microF/cm2.
Calcium action potentials in single freshly isolated smooth muscle cells
Authors: Walsh J. V. , Singer J. J.
Published: December 2017

The ionic basis of the action potential was investigated using intracellular microelectrodes in single smooth muscle cells freshly isolated from the stomach of the toad Bufo marinus. When [Ca2+]0 was elevated (> 8mM), action potentials were readily elicited, which had similar characteristics to those found in many tissue preparations of visceral smooth muscle. There was a decrease in membrane resistance at the peak of the action potential and during the undershoot. The following evidence indicated that the inward current is carried by Ca2+: 1) Raising [Ca2+]0 from 15 to 49.6 mM in the presence of 18.2 mM tetraethylammonium chloride (TEA) increased the maximum rate of rise and the overshoot amplitude, the latter by 15 mV, i.e., 29.5 mV/10-fold change in [Ca2+]0. Changing [Na2+]0 from 11.8 to 81.8 mM had no significant effect on the maximum rate of rise or the overshoot. 2) The action potentials were blocked by 8 mM Mn2+ ([Ca2+]0 = 14.6 mM) but not by 14.3 microM tetrodotoxin (TTX) ([Na2+]0 = 100 mM). 3) Action potentials could be elicited when [Ba2+]0 or [Sr2+]0 were present in high concentrations ([Ca2+]0 less than or equal to 31 microM,[Na2+]0 = 11.8 mM). Both the maximum rate of rise and overshoot amplitude of the action potential increased as the membrane potential became more negative, suggesting increased activation of the inward current. Both TEA and Ba2+ prolonged the action potential, suggesting that a K+ current is responsible for repolarization. Action potentials could also be elicited on anode break at elevated [K+]0 (91 mM).
Rectifying properties of the membrane of single freshly isolated smooth muscle cells
Authors: Singer J. J. , Walsh J. V.
Published: December 2017

Single smooth muscle cells freshly isolated from the stomach muscularis of the toad Bufo marinus were studied under direct microscopic observation using intracellular microelectrodes. The deviation of the membrane potential from rest was recorded when steps of current were injected into the cell. Outward-going rectification was consistently observed both in the presence of 1.8 mM and higher external concentrations of Ca2+. There was no indication of inward-going rectification even under conditions favoring its demonstration, i.e., when the external concentration of K+ was high (108 mM) and Cl-, low (39.6 mM). In the presence of tetraethylammonium chloride (TEA), there was a marked decrease in the rectification normally observed with depolarizing currents, suggesting that a K+ conductance contributes to the outward-going rectification. This K+ conductance increased by almost two orders of magnitude over the range from -20 to 0 mV, and displayed an e-fold increase with a depolarization as small as 4-7 mV. In response to hyperpolarizing currents, the membrane potential did not always reach a plateau but at times continued to become more negative. The feasibility of the depletion of ions from the caveolae as an explanation for this observation is discussed.
Binding of aldosterone to cytoplasmic and nuclear receptors of the rabbit kidney
Authors: Farman N. , Vandewalle A. , Bonvalet J. P.
Published: December 2017

The binding characteristics of [3H]aldosterone ([3H]A) in the rabbit kidney were compared with those described in other species, and experimental conditions suitable for both the isolation of renal tubules and the retention of aldosterone receptors were examined. Specific binding of [3H]A to cytoplasmic (C) and nuclear (N) receptors was determined on kidney slices incubated at +30 degrees C in rabbits fed a low K+-high Na+ diet. Results indicate the presence of at least two binding sites in N fractions, one of high affinity (Kd (I)) 1.8 X 10(-8) M) and low capacity (Nmax (I) 4.6 X 10(-14) mol/mg DNA), called type I sites, and the other of lower affinity (Kd (II) 8.9 X 10(-8) M) and more numerous sites (Nmax (II) 13.1 X 10(-14) mol/mg DNA). Competition studies showed that type I sites have higher affinity for [3H]A than for other steroids. Incubation at +37 degrees C resulted in a loss of type I sites. In adrenalectomized rabbits the C binding was improved but not the N binding (Kd (I) 1.2 X 10(-8) M; Kd (II) 5.6 X 10(-8) M). In rabbits treated with aminoglutethimide, an inhibitor of steroid synthesis, the N binding was lower than in other series. It was concluded that 1) [3H]A binding in the rabbit kidney does not differ from that described in other species; 2) a low K+-high Na+ diet is a satisfactory condition for study of [3H]A binding; and 3) incubation at +30 degrees C, a temperature necessary for microdissection procedures, is suitable for aldosterone binding studies.
Calcium affects insulin release and membrane potential in islet beta-cells
Authors: Frankel B. J. , Atwater I. , Grodsky G. M.
Published: December 2017

Insulin release from perfused rat pancreas was compared with membrane potentials of single beta-cells from perifused mouse islets during glucose stimulation (11.1 mM) in the presence of varying Ca and Mg concentrations. Depolarization was associated with insulin release and hyperpolarization with its suppression, irrespective of Ca concentration. After sudden reduction of Ca and Mg (to 0.05 and 0.01 mM, respectively), glucose-stimulated insulin release was maintained while the cell membrane depolarized, leading to a reversed pattern of burst activity. Readdition of Ca and Mg caused suppression of insulin release that paralleled hyperpolarization of the cell membrane. This suppression was transient, lasting < 5 min, and was due mainly to readdition of Ca. Patterns of insulin release during reduction of Ca and Mg in the presence of valinomycin (1 microM), diphenylhydantoin (25 microgram/ml), and ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (0.037 and 1.0 mM) were also studied. In conclusion, the relative concentrations of Ca and Mg and the membrane potential per se are important in the release of insulin. Also, under certain experimental conditions, Ca can block glucose-stimulated insulin release, possibly by increasing K+ permeability.
Tetraethylammonium modifies gap junctions between pancreatic beta-cells
Authors: Sheppard M. S. , Meda P.
Published: December 2017

Gap junctions between pancreatic beta-cells were quantitatively assessed in freeze-fracture replicas of isolated rat islets of Langerhans incubated for 90 min with or without the potassium conductance blocker tetraethylammonium (TEA). The results show that TEA increases the median number of particles per beta-cell gap junction but not the frequency of gap junctions at both nonstimulating and threshold-stimulating concentrations of glucose. TEA increased the relative gap junctional area at both concentrations of glucose. TEA had no effect on insulin release at a basal concentration of glucose but potentiated that release at the threshold glucose level. Thus TEA modifies beta-cell gap junctions independently of its effect on insulin release. However, the junctional changes observed were greater when insulin release was also elevated.
Glucose-induced electrical activity in the pancreatic beta-cell: effect of veratridine
Authors: Tarvin J. T. , Pace C. S.
Published: December 2017

Veratridine was used in the presence of glucose to assess the role of the Na pump in the regulation of glucose-induced burst activity. In the presence of 8.4 mM glucose, veratridine elicited a silent hyperpolarization, followed by burst activity. The magnitude of depolarization to plateau potential and the duration of the silent phase were increased. The addition of tetrodotoxin (TTX) restored the pattern of electrical activity to that observed in the absence of veratridine. Similar results were observed when veratridine was used in the presence of 16.7 mM glucose and tetraethylammonium (blocks voltage-dependent potassium permeability). TTX or ouabain blocked the effects of veratridine, and produced depolarization and continuous spike activity. Quinine (blocks Ca-dependent potassium permeability) elicited continuous spike activity in the presence of 16.7 mM glucose. The addition of veratridine induced only a transient return to burst activity, followed by a return to continuous spike activity. These results suggest that an electrogenic Na pump is an important factor in maintaining the transmembrane potential at an optimum level for operation of a voltage- and Ca-sensitive potassium permeability: changes in potassium permeability operating on a background of electrogenic current may be responsible for the voltage transitions associated with burst activity.
Introduction
Authors: Wright Ernest M.
Published: December 2017

Pig kidney cell line LLC-PK1 cultured on a collagen-coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by circumferential occluding junctions. In the presence of 5.5 mM D-glucose, a potential difference (PD) of 2.8 mV, apical bath negative, short-circuit current Isc of 13.2 microA . cm-2, and transepithelial resistance of 211 omega . cm2 were recorded. Isc and PD were reduced by phlorizin added to the apical bath but were unaffected when phlorizin was placed in the basolateral bath. Ouabain or the replacement of Na by tris-(hydroxymethyl)aminomethane or choline abolished the Isc. The sugar concentrations required to produce the half-maximal Isc were 0.13 mM beta-methyl-D-glucoside, 0.28 mM D-glucose, 0.65 mM alpha-methyl-D-glucoside, 0.77 mM 6-deoxy-D-glucose, 4.8 mM D-galactose, and 29 mM 3-O-methylglucose. When [Na] was reduced, the D-glucose required for half-maximal SCC increased. Isotopically 3H- and 14C-labeled D-glucose were used to determine simultaneous bidirectional fluxes; a resultant net apical-to-basolateral flux was present and could be abolished by phlorizin. The transported isotope cochromatographed with labeled D-glucose, indicating negligible metabolism. The cell culture model provides advantages for investigation of mechanisms of transepithelial glucose transport.
Occluding junctions in cultured epithelial monolayers
Authors: Cereijido M. , Meza I. , Martinez-Palomo A.
Published: December 2017

When MDCK cells are cultured in monolayers, they synthesize, assemble, and seal occluding junctions that limit the paracellular route. These processes may be impaired by inhibitors of the protein synthesis but not by inhibitors of the synthesis of RNA. Once established, the occluding junctions confer to the monolayer an overall electrical resistance of 80–600 omega . cm2. At the microscopical level, the resistance of individual junctions have large variations along the perimeter of a given cell. This agrees with the images of freeze-fracture electron microscopy where the network of the junction varies abruptly from 1 to 10 strands. The junctions are impermeable to macromolecular tracers, have a 9 to 1 Na+/Cl- discrimination, and a cation selectivity following the order: K+ greater than Na+ greater than Rb+ greater than Cs+ greater than Li+. Sealing requires extracellular Ca2+, but the junctions open when the concentration of Ca2+ in the cytoplasm increases. The structural components of the cytoskeleton (microtubules and microfilaments) seem to be involved in the junctional events as revealed by staining with immunofluorescent specific antibodies. If the cells are treated with cytochalasin B, actin microfilaments disorganize, the junctions open, and the electrical resistance across the monolayers falls. The resealing of the tight junction is inhibited by this drug.
Effects of ovarian steroids and pregnancy on adrenergic nerves of uterus and oviduct
Authors: Marshall J. M.
Published: December 2017

This review concerns the influence of ovarian steroids and of pregnancy on norepinephrine (NE) metabolism in the adrenergic neurons of the female reproductive tract and speculates on the physiological consequences of this influence. Estrogen and progesterone affect not only the NE content of these nerves but also the turnover of NE, the activity of its synthetic enzyme, and releases of NE from nerve terminals. During pregnancy additional factors including stretch-induced hypertrophy come into play and cause degeneration of the nerves in the uterine corpus. This degeneration makes the muscle supersensitive to NE and may also induce morphological changes in the muscle cell membrane. As a result there may be a withdrawal of neural inhibitory influences on the corpus, allowing spontaneous myogenic contractions to intensify. Although the physiological significance of the steroid-transmitter interactions are still unclear, these nerves per se are of interest because they represent a model system for the study of neuroendocrine regulation in the peripheral nervous system.
Na--Ca exchange in rat myometrium membrane vesicles highly enriched in plasma membranes
Authors: Grover A. K. , Kwan C. Y. , Daniel E. E.
Published: December 2017

When isolated rat myometrium vesicles highly enriched in plasma membranes were preincubated with 100 mM NaCl and then diluted 21-fold in Na-free media, an ATP-independent Ca uptake value of 4.10 +/- 0.23 mumol/g protein occurred, compared to a value of 2.87 +/- 0.16 for a similar uptake by vesicles preincubated in Na-free media. Brief (less than 10 s) exposure of the membrane vesicles to 5 mM ethyleneglycol-bis(beta-aminoethyl)-N,N'-tetraacetic acid (EGTA) after the Ca uptake showed that the NaCl preincubated vesicles retained more Ca than the sucrose or KCl preincubated vesicles. A NaCl concentration in the membrane fractions identical to its concentration in the Ca uptake medium did not enhance the Ca uptake by the vesicles did not show an increased Ca uptake. NaCl added to plasma membrane vesicles actively loaded with Ca caused retention of less Ca than the control. NaCl added to actively loaded vesicles along with EGTA also enhanced calcium efflux compared to EGTA alone. Sucrose, K+, Rb+, or Cs+ could not replace Na+ for the Na+-dependent Ca uptake or release, while Li+ was a poor substitute in both the instances. Na+-dependent Ca-uptake distribution in the various fractions correlated very well with their 5'-nucleotidase activity but not with their NADPH- or succinate-dependent cytochrome c reductase activities. The results have been discussed using a Na--Ca exchange model as well as by a model in which Na+ competes for calcium binding to the membranes.
Effects of monensin on amylase release from mouse parotid acini
Authors: Watson E. L. , Farnham C. J. , Friedman J. , Farnham W.
Published: December 2017

Isolated mouse parotid acinar cells (acini) were prepared by enzyme digestion, divalent cation depletion, and mechanical shearing. Acini were found to be morphologically intact, i.e., 95% viable as judged by trypan blue exclusion. Amylase release by the cholinergic agonist carbachol, by the beta-adrenergic agonist isoproterenol, and by monensin was similar to responses obtained in mouse parotid fragments. Monensin-stimulated amylase release was associated with enhanced 22Na+ uptake and 45Ca2+ efflux; monensin did not affect 45Ca2+ uptake. In the absence of extracellular Na+, the response to monensin (50 microM) was reduced from 162 +/- 33.5 to 12.4 +/- 0.5%; monensin also failed to stimulate 45Ca2+ efflux. Similar results were obtained with isoproterenol (10(-6) M). The results suggest that Na+ ions may play a role in amylase release possibly by releasing Ca2+ from internal stores.
Mobilization of a common source of smooth muscle Ca2+ by norepinephrine and methylxanthines
Authors: Deth R. C. , Lynch C. J.
Published: December 2017

The ability of norepinephrine (NE), caffeine (CAF), theophylline (THEO), dinitrophenol (DNP), and potassium (high K) to mobilize cellular Ca2+ in rabbit aorta was examined using 45Ca-efflux techniques. After 10 min of Ca2+ deprivation using either Ca-free EGTA or Ca-free lanthanum (La3+) buffers, NE, CAF, and DNP still caused an increase in 45Ca-efflux rate, indicating a cellular source of 45Ca, while high K did not. Contractile behavior after Ca removal paralleled 45Ca-efflux events. CAF (10 mM) inhibited NE contractile responses, and this inhibition was associated with the depletion of the NE-releasable Ca2+ store. Previous exposure to CAF during 45Ca efflux reduced subsequent stimulation of 45Ca efflux was not additive. THEO caused a stimulation of 45Ca efflux similar to CAF. CAF, THEO, and 3-isobutyl-l-methylxanthine caused a two- to threefold increase in cAMP levels in association with their stimulation of 45Ca efflux. These results suggest that NE and methylxanthines mobilize a common cellular Ca2+ source that is associated with contraction in the case of NE but relaxation in the case of methylxanthines.
Adaptive responses of rats to prolonged treatment with epinephrine
Authors: Fell R. D. , Terblanche S. E. , Winder W. W. , Holloszy J. O.
Published: December 2017

Rats were given a daily injection of L-epinephrine, 100 micrograms/100 g body wt, for 6 wk. The hearts of the epinephrine-treated animals were heavier (11.5%), and blood glucose and plasma insulin concentrations were lower than those of control rats. Acute responses to epinephrine were compared in the two groups. An increase in blood glucose and decreases in plasma insulin, liver glycogen, and muscle glycogen occurred in both groups. The magnitude of these responses were similar in the two groups except for the decrease in muscle glycogen, which was smaller in the chronic epinephrine-treatment group. There were no changes in respiratory capacity, citrate synthase or succinate dehydrogenase activities, or in cytochrome c concentration in skeletal muscle in response to 6 wk of epinephrine treatment. These results are compatible with the suggestion that catecholamines may play a role in some of the metabolic and cardiac adaptations to exercise training. However, they argue strongly against the hypothesis that catecholamines are responsible for inducing the increase in muscle mitochondria that occurs in response to exercise training.
Effects of divalent cations on beta-cell electrical activity
Authors: Ribalet B. , Beigelman P. M.
Published: December 2017

The effects of various divalent cations, added to the external medium, upon beta-cell action potential were studied using intracellular microelectrodes. Changes of spike peak potential, as a function of external cation concentration, indicate that Sr2+ or Ba2+ may substitute for Ca2+ as a charge carrier. Complete blockage by Mn2+ of electrical activity elicited by Sr2+, Ba2+, or Ca2+ suggests that these cations penetrate the membrane though the same Ca2+ channel. The increase of maximum rate of depolarization, dV(d)/dtmax, and decrease of maximum rate of repolarization, dV(r)/dtmax, when Sr2+ is substituted for Ca2+ suggest that Sr2+ penetrates more readily the Ca2+ channel but is less effective than Ca2+ in activating K permeability. Reversal of these effects by addition of equimolar Ca2+ to Sr2+ indicates that Ca2+ has a greater affinity than Sr2+ for the receptor site. The blockage of electrical activity by Ba2+ at a depolarized membrane level suggests that Ba2+ markedly reduces all K+ permeabilities. Analysis of dV(d)/dtmax at various Ca2+ concentrations, in the presence of nonpermeant divalent cations (Co2+, Mn2+, and Mg2+), shown that these cations bind competitively at the same receptor site with differing dissociation constants, For all of these divalent cations, the order of binding would be Co2+ greater than Mn2+ greater than Ca2+ greater than Sr2+, Mg2+.
A mass spectrometer to measure transepithelial unidirectional labeled water fluxes
Authors: Jarrell J. A. , Mills J. W. , King J. G.
Published: December 2017

A sealed quartz micropipette forms a liquid-vacuum interface that together with a uranium reduction element and a mass spectrometer functions as an in vitro real-time labeled water-selective microelectrode. Its performance is demonstrated by monitoring unidirectional fluxes across toad urinary bladder. This instrument has direct applicability to studies of water flux across epithelial membranes and could also be useful in monitoring water fluxes across such preparations as isolated tubules or capillaries.
Inward rectification in mouse macrophages: evidence for a negative resistance region
Authors: Gallin E. K. , Livengood D. R.
Published: December 2017

The electrical properties of cultured mouse thioglycollate-induced peritoneal macrophages were investigated using intracellular recording techniques. Thirty-five percent of the cells studied had membrane potentials ranging from -65 to -95 mV and exhibited S-shaped, steady-state current-voltage (I-V) relationships containing a transitional region. Analysis of currents in the transitional region from the rate of rise and fall of the voltage responses to current pulses indicated the presence of a negative resistance region in this area. Tetrodotoxin (3 × 10(-5) M), cobalt chloride (3 mM), 4-aminopyridine (4 mM), and tetraethylammonium chloride (8 mM) did not eliminate the transitional region of the I-V curves, whereas addition of barium chloride (4 mM) and rubidium chloride (3 mM) did. Increasing the external concentration of potassium shifted the I-V relationship horizontally along the current axis but did not eliminate the transitional region. These data indicate that the inward rectification and the negative resistance region probably result from a voltage-dependent potassium conductance.
Fatigue and metabolism of frog muscle fibers during stimulation and in response to caffeine
Authors: Nassar-Gentina V. , Passonneau J. V. , Rapoport S. I.
Published: December 2017

Tension and metabolite concentrations were measured in single frog muscle fibers at 15 degrees C in vitro, in response to electrical stimulation or to immersion in caffeine- or potassium chloride-Ringer. Sarcomere length equaled 2.3 micrometers. Interrupted stimulation for 150 s at 20 Hz or stimulation for 7.5 min at 1 Hz was followed by at least 20 min of fatigue, evidenced by a reduced 200-ms test contraction. Fatigued fibers contracted maximally in potassium chloride- or caffeine-Ringer. They had high lactate and glucose 6-phosphate concentrations and a reduced phosphocreatine (PCr) concentration. Adenosine 5'-triphosphate (ATP) concentration was approximately normal but was markedly reduced by a caffeine contracture. A plot of PCr consumption against the tension-time integral at different stimulation frequencies (25, 35, or 50 Hz) and durations had an intercept of 25.5 nmol PCr/mg protein at time zero and a corrected slope of 0.65 nmol approximately P/mg protein per kg . s . cm-2. Prolonged fatigue is not due to energy exhaustion or to the inability of muscle fibers to consume residual ATP but probably arises from long-lasting interference in excitation-contraction coupling, which can be reversed by KCl- or caffeine-induced release of Ca2+ from intracellular stores.
Release of sarcoplasmic enzymes from frog skeletal muscle
Authors: Suarez-Kurtz G. , Eastwood A. B.
Published: December 2017

Isolated, intact frog muscles bathed in control saline release creatine kinase (CK) and lactate dehydrogenase (LDH) at constant rates for several hours. The basal rates of release from “toe” muscles (CK 0.087%/min; LDH 0.105%/min) were one order of magnitude greater than those from semitendinosus muscles. This is attributed to differences in muscle mass and geometry, and to the smaller diameter of toe muscle fibers. Enzyme release rates were not affected by Na-free or Cl-free solutions, whereas LDH release rate doubled during exposure to Ca-free (EGTA-containing) saline or in the presence of isosmotic solutions containing 120 mM KCl or potassium propionate. Following mechanical injury or detergent treatment (Brij 58), the enzyme release rates into Ca-free medium reached peak values 4 and 16 times (toe muscle), and 16 and 20–30 times (semitendinosus), respectively, the control rates. The greater effect of detergent treatment is ascribed to a larger area of sarcolemmal damage plus possible changes in the state of the enzymes in the sarcoplasm.
Turnover and regulation of Na-K-ATPase in HeLa cells
Authors: Pollack L. R. , Tate E. H. , Cook J. S.
Published: December 2017

HeLa cells in log growth have 10(6) surface Na-K-ATPase molecules as estimated by the specific binding of [3H]-ouabain. Studies utilizing ouabain as a label show that the ligand is internalized at a rate corresponding to the turnover of three sets of Na-K-ATPase enzymes per generation. The label is taken up exclusively into a particulate cell compartment where it is codistributed with beta-hexosaminidase, identifying the internal compartment as lysosomal. Turnover is an important parameter in the recovery of the cells from glycoside intoxication. The unmetabolized glycoside is subsequently released by exocytosis. 13C-density-labeled Na-K-ATPase has been identified by specific phosphorylation of its catalytic subunit with [32P]ATP or [33P]ATP, and the rate of turnover of the density label is shown to be the same as the internalization of the ouabain-labeled site. There is a transit time of about 4 h from the onset of synthesis of the catalytic subunit to its insertion in the surface membrane; 2,800 catalytic subunits are synthesized per minute per cell, and 2,100 are turned over K+-starved cells respond to the stress in 24-30 h with modulation of the surface density of Na-K-ATPase the synthetic rate remains constant; the number of functional enzymes per cell is controlled by change in the rate constant for turnover.
Isoproterenol-induced current changes in glands of frog skin
Authors: Thompson I. G. , Mills J. W.
Published: December 2017

In this study we report on isoproterenol-induced changes in short-circuit current (SCC) that are located in the glands of the frog skin. Following complete inhibition of the SCC with amiloride (10(-4) M), the addition of isoproterenol (9.0 X 10(-7) M) to the serosal side of the skin elicits a significant increase in SCC from 0.6 +/- 0.1 to 3.9 +/- 0.5 microA/cm2 (p less than 0.001). The conductance is doubled, and both effects are blocked completely with the beta-blocking agent propranolol. In the split-skin preparation, in which no glands are present, there is no current response to isoproterenol following amiloride. This indicates that the amiloride-insensitive isoproterenol-stimulated SCC resides in the glands. In the intact skin, removal of sodium (choline replacement) from the serosal (but not the mucosal) medium abolishes this current change. The postisoproterenol current change in amiloride-inhibited skins is also dependent on the presence of chloride in the serosal bath. Preincubation of the amiloride-inhibited skin with ouabain (10(-4) M) for 10 min prior to the addition of isoproterenol reduces the isoproterenol response by 50%. These results indicate that there is a sodium- and chloride-dependent ouabain-sensitive secretory mechanism in the skin glands of the frog that is responsible for the development of the isoproterenol-stimulated SCC observed in the presence of amiloride.
Glucose-induced electrical activity in pancreatic beta-cell: modulation by pH
Authors: Tarvin J. T. , Sachs G. , Pace C. S.
Published: December 2017

Studies have been undertaken to examine the influence of changes in pH on the electrical activity in mouse islet beta-cells. Extracellular acidification or the presence of the permeable weak acid glycodiazine was found to alter the cyclic nature of glucose-induced electrical activity leading to depolarization and constant spike activity. Conversely, the presence of the permeable weak base imidazole was found to transiently lead to hyperpolarization with concomitant inhibition of electrical activity. Monensin, an electroneutral Na:H antiporter, was found to inhibit glucose-induced electrical activity, even in the presence of glycodiazine. These results suggest that protons may play an important role in the regulation and/or generation of the oscillatory pattern of electrical activity in the beta-cell.
Trophic effect of a sciatic nerve extract on fast and slow myosin heavy chain synthesis
Authors: Thibault M. C. , Havaranis A. S. , Heywood S. M.
Published: December 2017

Myosin heavy chain (MHC) synthesis in cultures from chick pectoralis muscle cells was determined by [35S]methionine incorporation. Two types of MHC, migrating as 200,000-dalton components on sodium dodecyl sulfate polyacrylamide gels, were distinguished with antibodies against adult fast and slow MHC. Their synthesis was revealed by autoradiography. The effect of a sciatic nerve extract on the synthesis of the two types of MHC was also determined. Control experiments show that fast MHC is primarily synthesized in 48-h cultures. At a later stage of development (5- to 7-day cultures), slow MHC is also produced. The nerve extract promotes muscle cell differentiation and stimulates the synthesis of the slow type of MHC at an earlier stage of development (i.e., at 48 h as compared with 5-7 day in controlled cultures). It is concluded therefore that presumptive fast muscle cells in culture synthesize initially fast MHC and later both types of MHC (slow and fast). These results also suggest that the sciatic nerve extract is capable either of activating the transcription of the structural gene for slow MHC or of activating the translation of preexisting messenger RNA coding for this protein.
Tonic force maintenance with reduced shortening velocity in arterial smooth muscle
Authors: Dillon P. F. , Murphy R. A.
Published: December 2017

The isotonic shortening velocity of swine carotid media tissues contracting in response to high K+, histamine, norepinephrine, or AC electrical stimulation rapidly increased to a maximum value and then declined to a steady-state level while force was still increasing or steady. The maximum shortening velocity calculated for no external load on the tissue (Vo) also decreased during the course of contractions when active stress remained constant. The fall in velocity with time was not Ca2+ dependent, because reductions in the [Ca2+] in high K+ solutions that significantly reduced the maximum stress (Fo) had no effect on Vo in the steady state. On washout of high K+ solutions, the ability of the tissue to shorten on isotonic quick release fell rapidly to low levels before isometric stress exhibited significant declines. The data indicate that cross-bridge cycling rates are modulated in this tissue. We suggest that this reflects the formation of attached, noncycling cross bridges (termed latch bridges), which constitute an internal load on the contractile system during tonic contractions.
Binding of peanut lectin to specific epithelial cell types in kidney
Authors: LeHir M. , Kaissling B. , Koeppen B. M. , Wade J. B.
Published: December 2017

The binding of peanut agglutinin (PNA) to epithelial membranes of the rabbit kidney was evaluated at the light- and electron-microscope level using PNA conjugated to horseradish peroxidase. In the renal cortex and outer stripe of the medulla PNA appears to bind exclusively to the luminal membrane of intercalated cells in connecting tubules and collecting ducts. PNA also binds to the thin descending limb of the loop of Henle in the inner stripe and inner zone of the medulla. This very specific affinity of PNA should be useful in the isolation and characterization of specific cell types in cytologically heterogeneous epithelia.
Depolarization-induced contractile activity of smooth muscle in calcium-free solution
Authors: Mangel A. W. , Nelson D. O. , Rabovsky J. L. , Prosser C. L. , Connor J. A.
Published: December 2017

In calcium-free solution, strips of cat intestinal muscle developed slow, rhythmic electrical potential changes that triggered contractions. Some strips failed to develop spontaneous electrical activity in calcium-free solution but responded with contractions to depolarization by direct electrical stimulation or by treatment with barium chloride, potassium chloride, or acetylcholine. Similar results were obtained with segments of cat stomach, colon, esophagus, bladder, uterus, and vena cava, as well as with rabbit vena cava. In calcium-free saline, rat small intestinal muscle showed fast electrical activity with accompanying development of a tetanuslike contraction. After 60 min in calcium-free solution, cat small intestinal muscle retained 17.7% of its original concentration of calcium. It is concluded that in some smooth muscles, depolarization-triggered release of intracellular calcium does not require an associated influx of calcium.
Development of Na+-dependent hexose transport in a cultured line of porcine kidney cells
Authors: Amsler K. , Cook J. S.
Published: December 2017

LLC-PK1 cells in culture do not concentrate alpha-methylglucoside (alpha-meG) during their early growth phase but develop the capacity to concentrate this hexose as the growth rate decreases in confluent cultures. The concentrating ability is dependent on the Na+ electrochemical gradient and is inhibited by phlorizin with KI,0.5 approximately 0.2 microM. The development of the concentrative capacity can be accelerated by the Friend cell inducer hexamethylene bisacetamide (HMBA) and by the phosphodiesterase inhibitors dibutyryl cAMP, theophylline, and 1-methyl-3-isobutylxanthine (MIX). In cultures treated with any of these differentiation-accelerating chemicals, the development of alpha-meG concentrating capacity is severely inhibited by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) but not by inactive (in tumor promotion) analogs of TPA. In all cases, an early event in the development of alpha-meG accumulating capacity is an elevated intracellular cAMP concentration; however the results suggest that this increase in cAMP may be necessary but not sufficient to induce the differentiated hexose-accumulating capacity.
Impairment of trophic response of brown fat to cold in guanethidine-treated rats
Authors: Mory G. , Ricquier D. , Nechad M. , Hemon P.
Published: December 2017

The aim of the present study was to investigate the role of the sympathetic nervous system in the trophic response of brown adipose tissue (BAT) to chronic cold exposure. The hyperplasia and the development of the mitochondria characterizing this response are usually considered as mainly controlled by the sympathetic activity in BAT, but this has never been clearly demonstrated. In the present work rats were sympathectomized by chronic administration of guanethidine and then exposed to cold during two weeks. The treatment induced a strong reduction of the noradrenaline content of BAT. The trophic response of the tissue to cold was largely impaired: no increase of the tissue weight, weak increase in the DNA, protein, and phospholipid content of the tissue. The development of the mitochondria was almost abolished. The increase in the proportion of the 32,000-dalton protein, a protein which regulates heat production by BAT, was suppressed and the GDP-binding to mitochondria, which is an index of BAT thermogenic capacity, was not increased as in normal cold-adapted rats. The ultrastructure of the tissue remained the same as in warm-adapted animals. It is concluded that the sympathetic nervous system plays an essential role in the control of the trophic response of BAT to the cold.
Reconstitution of D-glucose transporter from human placental microvillous plasma membranes
Authors: Bissonnette J. M. , Black J. A. , Thornburg K. L. , Acott K. M. , Koch P. L.
Published: December 2017

Proteins from microvillous plasma membrane vesicles of the maternal surface of human trophoblast were solubilized with octyl beta-D-glucoside. After incorporation of the soluble protein into phospholipid liposomes D-glucose uptake exceeded that of L-glucose. The reconstituted system showed that D-glucose uptake was not sodium dependent and was inhibited by cytochalasin B. Efflux of D-glucose from the proteoliposomes was retarded by cytochalasin B. D-Glucose uptake, but not L-glucose, was proportional to the amount of protein used in the reconstitution procedure. Membrane protein was also solubilized with octylglucoside from vesicles that had been extracted previously by dimethylmaleic anhydride. Proteoliposomes prepared from these latter proteins showed D-glucose uptake threefold greater than that from octylglucoside solubilization alone, but sodium dodecyl sulfate polyacrylamide gel electrophoresis of the extracted protein showed no clear difference between the double extraction procedure and the pattern obtained with the single detergent.
Effects of ATP on lysosomes: inhibition of the loss of latency caused by cooling
Authors: Ruth R. C. , Weglicki W. B.
Published: December 2017

Triton-filled lysosomes from rat liver and a crude lysosomal fraction from livers of rats not injected with Triton have been used to study the ability of ATP to protect lysosomes against the effects of incubation in vitro at 37 degrees C. When incubation was carried out in a sucrose-NH4Cl medium at pH 8.0 or in buffer containing 0.15 M KCl at pH 7.4, ATP protected against the loss of lysosomal enzyme latency that occurred at 37 degrees C and also against the additional loss of latency that occurred when the incubated lysosomes were cooled to 0 degrees C. The inhibition of the effects of cooling was found to result from a decrease in the loss of latency caused specifically by cooling the lysosomes through the temperature range of the membrane phase transition (15-0 degrees C); ATP did not affect the basic rate of loss of latency at 0 degrees C. Protection was minimal when incubation was carried out in 1 mM ethylenediaminetetraacetic acid without added divalent metal ion and was enhanced by the addition of either Mg2+ or Ca2+. Kinetic studies were carried out on the ATP-protective action against the effects of cooling by adding ATP to lysosomes that had been incubated at 37 degrees C and that were then cooled to 0 degrees C at various times after addition of the ATP. Susceptibility to the effects of cooling continued to decrease until the ATP had been present at 37 degrees C for approximately 3 min before cooling. The concentration of ATP required for maximum initial rates of protection was found to be 10(-3) M or possibly lower. The rate of decrease in susceptibility to the effects of cooling was much slower when ATP was added at 22 than at 37 degrees C, suggesting that enzyme action may be involved in the protective mechanism. Possible mechanisms for the ATP-protective action are discussed.
Influence of ATP on sarcoplasmic reticulum function of vascular smooth muscle
Authors: Ford G. D. , Hess M. L.
Published: December 2017

A vesicular fraction isolated from bovine aorta and enriched in fragmented sarcoplasmic reticulum (FSR) exhibited active calcium transport and ATPase activity. By use of a hypotonic NaHCO3 extraction solution, an active preparation was isolated that retained activity for up to 4 days. A small but significant (P less than 0.05) Ca2+-stimulated, Mg2+-dependent ATPase associated with calcium transport was demonstrated with a specific activity of 0.33 mumol inorganic phosphate (Pi).mg-1.min-1. The basal Mg2+ ATPase demonstrated Michaelis-Menten kinetics [Km(Mg2+-ATP) = 0.44 +/- 0.01 X 10(-3) M; Vmax = 2.22 +/- 0.01 mumolPi.mg-1.min-1]. The Ca2+-stimulated, Mg2+-ATPase demonstrated apparent substrate inhibition (Ks approximately 10 mM) with no evidence for end-product (ADP) or excess added Ca2+ contributing to this inhibition. Oxalate-supported active calcium uptake velocities also exhibited quantitatively similar substrate inhibition. These results suggest that FSR from vascular smooth muscle contains either two enzymes or one enzyme with two isomeric forms, one of which is associated with the calcium uptake activity of this structure and the other of unknown function.
Optical measurements of oxygen delivery and consumption in gerbil cerebral cortex
Authors: Bashford C. L. , Barlow C. H. , Chance B. , Haselgrove J. , Sorge J.
Published: December 2017

Oxygen metabolism of the cerebral cortex of anesthetized gerbils was monitored by surface fluorescence and reflectance spectrophotometry both in vivo and in specimens freeze-trapped by the surface-freezing technique. Fiber optic light guides were used for optical coupling for determining mitochondrial flavoprotein and pyridine nucleotide fluorescence and for recording dual-wavelength reflectance spectra that indicated the levels of ferrocytochromes aa3 and c + c1 in the cortex and the oxygen saturation of cortical hemoglobin. During anoxic episodes increases in ferrocytochromes aa3 and c + c1 and in reduced pyridine nucleotides and flavoproteins were observed only when the cortical hemoglobin was more than 85% disoxygenated. Indeed the steady-state levels of oxidation-reduction of mitochondrial respiratory-chain components remained constant over a wide range of oxygen delivery, increased reduction being observed only when the fraction of inspired oxygen fell below 6%; these properties of mitochondria in vivo resemble those found in vitro. However, in normoxic animals the absorbance at 605 nm that may arise from ferri- or ferrocytochrome aa3 is larger than would be expected of purified mitochondria and may represent a pool of mitochondria that remains reduced at all levels of tissue oxygenation or a pigment not involved in oxygen metabolism.
Ca2+ dependence of calcium uptake by rat myometrium plasma membrane-enriched fraction
Authors: Grover A. K. , Kwan C. Y. , Daniel E. E.
Published: December 2017

ATP-dependent Ca uptake by rat myometrium plasma membrane fraction occurs only in the presence of magnesium. Studies of the membrane protein-concentration dependence of the ATP-dependent Ca uptake showed that at 33 micrograms protein/ml the uptake increased linearly with time up to 2 min and deviated from linearity at longer times. Studies of the Ca2+ concentration dependence of initial Ca-uptake velocity (reaction time 2 min) of the uptake reaction showed that 1) ATP-dependent Ca uptake increased with increasing Ca2+ concentration from 0.1 to 1 microM, and a further increase in Ca2+ concentration above 1 microM did not enhance the uptake further and 2) the Ca-uptake dependence on Ca2+ concentration was nonhyperbolic with a Hill coefficient of 1.3–1.7 and half saturation occurring at 0.4–0.5 microM Ca2+. These data suggest that the ATP-dependent Ca movement by rat myometrium plasma membranes may play a very important role in smooth muscle relaxation. Physiological implications of the nonhyperbolic kinetics are also discussed.