bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphagens and intracellular pH in intact rabbit smooth muscle studied by 31P-NMR
Authors: Hellstrand P. , Vogel H. J.
Published: December 2017

Phosphorus-31 nuclear magnetic resonance (NMR) spectra (103.2 MHz) were obtained from rabbit portal vein, urinary bladder, and taenia coli smooth muscle. The muscles were mounted isometrically as strips in a horizontal probe and superfused with oxygenated medium at 23 degrees C. Under these conditions the preparations could remain in a stable metabolic state for at least 24 h. The resonances observed represented phosphocreatine (PC), adenosine triphosphate (ATP), and phosphomonoester. The PC-ATP ratio in relaxed preparations was approximately 1.9 (bladder) and 1.4 (taenia coli), approximately consistent with chemical analysis of tissue extracts by isotachophoresis. However, the levels of adenosine diphosphate (ADP) and inorganic phosphate (Pi) were considerably lower as estimated by NMR, suggesting intracellular compartmentalization. Contraction for 30 min in high-K+ medium caused a reversible 10–30% decline of PC but no change in ATP. Intracellular pH was 7.0 +/- 0.1 and was unchanged by contraction or inhibition of tissue respiration by cyanide. Treatment with 2-deoxyglucose resulted in accumulation of 2-deoxyglucose 6-phosphate, verifying the pH assignment. However, the phosphomonoester resonance of normal spectra is not glucose 6-phosphate. Treatment with Mg2+-free, high-Mg2+ (10 mM), or hyperosmotic media did not alter the Mg2+ saturation of ATP. The results obtained by the nontissue-destructive 31P-NMR measurements are consistent with the view that metabolic steady-state conditions are maintained during contraction in the smooth muscle.
Epithelial properties of human colonic carcinoma cell line Caco-2: effect of secretagogues
Authors: Grasset E. , Bernabeu J. , Pinto M.
Published: December 2017

Human colonic carcinoma Caco-2 cells grown in vitro form epithelial layers of highly polarized cells. Unlike colonic adsorptive cells they possess a mucosal membrane with very limited ionic conductance, even after exposure to aldosterone. When grown on filters, Caco-2 cells were sensitive to various secretagogues; these included 10(-5) M dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and 10(-10) M vasoactive intestinal peptide, both of which, added serosally, enhanced the short-circuit current. The same applied to mucosal forskolin. Caco-2 cell sensitivity to serosal epinephrine was lower. Ion substitutions and 22Na-36Cl flux measurements indicated the possibility of secretagogue-dependent chloride secretion. Measurements on cells grown on Petri dishes and exposed to 1 mM DBcAMP for 1 h enabled detection of more profound modifications. Sustained 20-mV cell depolarization and a large reduction in the relative electrical resistance of the mucosal membrane were concomitant with a sizable decrease in 36Cl accumulation. These results suggest that Caco-2 cells, which to some extent resemble colonic crypt cells, possess the cAMP-dependent mucosal chloride conductance characteristic of secretory cells.
Calcium pump, high-affinity Ca2+-ATPase, and other ATPases in dog antrum smooth muscle plasma membrane
Authors: Grover A. K. , Kwan C. Y. , Oakes P. J.
Published: December 2017

The plasma membrane-enriched fraction from dog antrum smooth muscle is enriched in ATP-dependent azide-insensitive Ca2+ uptake (0.3-0.4 microM Ca2+ required for half-maximal activity), a high-affinity Ca2+-ATPase (Km of 0.3-0.8 microM for Ca2+), a low-affinity Ca2+-ATPase (Km for 250-400 microM for Ca2+), and a Mg2+-ATPase. Studies using membranes washed with EDTA and assay media treated with Chelex 100 showed that the high-affinity Ca2+-ATPase did not depend on contaminating Mg2+. Thus, whereas the ATP-dependent Ca2+ uptake had an absolute requirement for Mg2+, the Ca2+-ATPases did not. Studies using gamma-irradiation showed that the protein responsible for the ATP-dependent Ca2+ uptake was inactivated at significantly lower doses of radiation than the three ATPases. The Ca2+ uptake and the high-affinity Ca2+-ATPase also differed in their inhibition by calmodulin antagonists and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Thus it is unlikely that the high-affinity Ca2+-ATPase by itself is responsible for the ATP-dependent Ca2+ uptake.
Effects of tumor promoters on sodium ion transport across frog skin
Authors: Civan M. M. , Rubenstein D. , Mauro T. , O'Brien T. G.
Published: December 2017

Phorbol esters are tumor promoters and mitogens whose effects may be mediated by changes in ion transport across membranes. Clarification of the transport effects of these agents should be facilitated by using a well-characterized model epithelial system whose intracellular and transmural parameters are readily measurable. The current results constitute a preliminary study of the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibutyrate (PDBU), and phorbol on the short-circuit current (Isc) across frog skin. TPA produced two effects: a stimulation of Isc of variable magnitude and a far more constant inhibition of the natriferic action of vasopressin. These effects appear related to the action of TPA as a tumor promoter insofar as PDBU (an active ester) also inhibited the natriferic response to vasopressin, whereas phorbol (inactive as a tumor promoter) had no significant effect. TPA is largely active from the mucosal medium, inhibits the natriferic response to adenosine 3',5'-cyclic monophosphate (cAMP) as well as that to vasopressin, and does not stimulate Isc in the presence of 10(-4) M mucosal amiloride. Inhibition of prostaglandin E1 production by indomethacin had no effect on the actions of TPA. The results indicate that frog skin is a promising model for studying the transport effects of the phorbol esters. The data further suggest that TPA acts on frog skin by activating the physiological amiloride- and cAMP-sensitive channels gating apical Na+ entry from the mucosal medium into the epithelial cells.
Examination of irreversible platelet-fibrinogen interactions
Authors: Peerschke E. I. , Wainer J. A.
Published: December 2017

The progressive stabilization of fibrinogen binding to ADP-treated platelets has been well described, but the nature of this interaction remains obscure. In the present study, irreversibly bound fibrinogen was defined as that fraction of bound iodinated fibrinogen that failed to dissociate from stimulated human gel-filtered platelets within 10 min of adding 10 mM ethylenediaminetetraacetic acid. It represented 16 +/- 11% (mean +/- SD, n = 10) of fibrinogen bound to ADP-treated platelets after 1 min and 52 +/- 11% of fibrinogen bound to these platelets after 60 min. Similar results were obtained if platelets were stimulated with purified human thrombin (0.1 U/ml) or epinephrine (10 microM). Irreversible fibrinogen binding was significantly reduced at 4 degrees C (27 +/- 9%, mean +/- SD, n = 6) if platelets were preincubated (30 min, 25 degrees C) with 30 micrograms/ml cytochalasin B or D (18 +/- 8%) or stimulated with chymotrypsin (0.5 mg/2-3 X 10(8) platelets) (31 +/- 8%). Formation of irreversible platelet-fibrinogen interactions correlated with the incorporation of actin and actin-binding protein into the Triton X-100-insoluble platelet cytoskeleton and the ability of platelets to retract fibrin clots. Irreversibly bound fibrinogen was available on platelets for digestion by 0.2 U/ml plasmin. The enzyme removed 96 +/- 6% (mean +/- SD, n = 6) of all bound fibrinogen from platelets after 30 min at 25 degrees C. This was not accompanied by significant release of [14C]serotonin or lactate dehydrogenase. Furthermore, platelets incubated with plasmin could bind fibrinogen normally after the enzyme had been neutralized with aprotinin.(ABSTRACT TRUNCATED AT 250 WORDS)
Action of a phorbol ester on B-cells: potentiation of stimulant-induced electrical activity
Authors: Pace C. S. , Goldsmith K. T.
Published: December 2017

The possible role of protein kinase c in regulating the electrical events in the B-cell plasma membrane was examined by using the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), a known activator of this enzyme. TPA has been found to enhance glucose- and sulfonylurea-induced insulin secretion with little or no effect on the fluxes of 86Rb+ or 45Ca2+ across the plasma membrane. TPA, 0.2 microM, did not influence the membrane potential from 0 to 5.6 mM glucose but increased by two- to threefold the fraction of the plateau phase of the oscillatory electrical activity induced by 7.0-11.1 mM glucose. This effect of TPA was completely blocked by 0.5 mM spermidine, an inhibitor of protein kinase c. However, spermidine had no influence on the electrical activity elicited by glucose alone. Glyburide, 10 nM, initiated slow depolarization and constant spike activity after about 18 and 25 min, respectively. TPA or 2.8 mM glucose reduced the lag period for glyburide to elicit an electrical response by about 75%. The duration of the spikes was increased two- to threefold by the presence of glucose or TPA with glyburide. There were also characteristic differences in the shape of the spikes under each experimental condition. Spermidine inhibited the influence of glucose, but not TPA, on the glyburide-induced electrical response. These results indicate that TPA may influence stimulant-induced electrical events via protein kinase c or by directly altering the ionic permeability of the plasma membrane.
Characterization of a coronary vasoconstrictor produced by cultured endothelial cells
Authors: Hickey K. A. , Rubanyi G. , Paul R. J. , Highsmith R. F.
Published: December 2017

The vasoactive effects of media obtained from bovine aortic endothelial cells (EC) in culture were directly tested on isolated rings of the porcine left anterior descending coronary artery. Increasing concentrations of EC-conditioned culture media resulted in progressive dose-dependent increments in isometric tension in porcine, bovine, and canine coronary arteries; the response did not require an intact endothelium. Control (nonconditioned) media and that conditioned by fibroblasts or vascular smooth muscle cells in culture had negligible effects on vessel tone. The vasoconstriction required extracellular Ca2+ and was unaffected by inhibitors of cyclooxygenase and lipoxygenase or by antagonists to the alpha- or beta-adrenergic, serotonergic, histaminergic, or cholinergic receptor systems. Calibrated gel filtration of the media indicated a molecular weight of 8,500 for the vasoactive factor; treatment of the EC-conditioned media with either sodium dodecyl sulfate, trypsin, alkali, or with acid hydrolysis completely abolished the vasoconstrictive effect. These findings and others now provide evidence for the existence of an EC-derived polypeptide vasoconstrictor that may be important in the regulation of vascular smooth muscle contractility.
Regulation of glyceraldehyde-3-phosphate dehydrogenase by a cytosolic protein
Authors: Aithal H. N. , Walsh-Reitz M. M. , Toback F. G.
Published: December 2017

Stimulation of glyceraldehyde-3-phosphate dehydrogenase (G3PD) activity and accelerated growth occur in cultures of monkey kidney epithelial cells (BSC-1 line) that are exposed to medium with a reduced K concentration (3.2 mM). We recently found that this activation of G3PD was mediated by the appearance of a new cytosolic protein with an apparent molecular weight of 62,000. G3PD and this modifier protein were isolated from BSC-1 cells, and the interaction between them was characterized to define the mechanism(s) of enzyme activation. The enzyme protein was purified from cells grown in control medium (5.4 mM K). The enzyme, in the presence of modifier, exhibited an increase in maximal rate of enzyme reaction and a decrease in the apparent Km for NAD+. Analysis using Dixon plots revealed that the presence of modifier increased the Ki for NADH by two- to threefold. Inhibition by NADH was competitive with respect to NAD+, glyceraldehyde-3-phosphate, and inorganic phosphate. ATP also inhibited enzyme activity in a competitive manner with respect to NAD; however, the Ki for ATP was similar both in the presence and absence of modifier. These results suggest that one mechanism by which the cytosolic modifier protein stimulates G3PD activity is to decrease product inhibition by NADH.
Greater ATP dependence than sodium dependence of radiocalcium efflux in bullfrog ventricle
Authors: Brommundt G. , Kavaler F.
Published: December 2017

45Ca efflux was studied in intact bullfrog ventricles following a 2-h period of loading with radiocalcium-containing Ringer solution. The cannulated ventricle was placed in a closed air-filled container to which were applied rhythmic, electronically timed, positive- and negative-pressure pulsations, which induced ventricular volume excursions. The mechanical arrangement and timing circuitry made it possible for each period to be as short in duration as 15 s. By use of this technique, penetration of the extracellular space by [14C]inulin was found to be complete within 30 s, and recovery of the inulin proceeded with a time constant of 17-24 s, indicating a completeness of recovery of 98% within 90 s. Washout of added 45Ca was quantitatively quite close to that of inulin, and in addition the estimated rate of sequestration of the isotope was slow enough to introduce only a small error into the experimental results. 45Ca efflux was only slightly (15%) sensitive to replacement of extracellular sodium but was profoundly sensitive to the inhibitors of ATP synthesis, cyanide and 2-deoxy-glucose.
Effects of metabolic acidosis on viability of cells exposed to anoxia
Authors: Bonventre J. V. , Cheung J. Y.
Published: December 2017

The effects of metabolic acidosis were examined in isolated rat hepatocytes under substrate-free oxygenated or anoxic conditions. Lowering extracellular pH to 6.6 under aerobic conditions had no deleterious effects on the cells as determined by trypan blue exclusion, lactate dehydrogenase (LDH) release, cellular K+ and Ca2+ content, and ability to increase ATP levels after nutrients and adenosine were added to media. Cytosolic pH was measured in aerobic cells at varying extracellular pH using 6-carboxyfluorescein. By using values for cytosolic pH obtained in this manner together with 5,5-dimethyl[2-14C]oxazolidine-2,4-dione (DMO) distribution data, a method was derived for determining intramitochondrial pH. The pH gradient across the mitochondrial membrane was found not to change with a decrease in extracellular pH from 7.4 to 6.9. At pH 6.9 hepatocytes were protected against anoxic injury as compared with cells incubated at pH 7.5 or 6.6. This protection was manifested by a decrease in vital dye uptake and LDH release, maintenance of higher cellular K+ content, less stimulation of respiration with succinate, improved recovery of ATP levels after return to an oxygenated nutrient environment, and maintenance of normal cellular Ca2+ content after reoxygenation. Recovery of cellular ATP content was independent of ATP levels, total adenine nucleotide pool, and energy charge ratio at the end of the anoxic period. Measurement of cytoplasmic pH in anaerobic cells by [14C]DMO distribution showed progressive cellular acidification with lowering of extracellular pH. The protective effects observed at pH 6.9 are not unique to hepatocytes since isolated renal cortical tubules exposed to anoxia have improved ATP levels on reoxygenation at this pH when compared with tubules incubated at pH 7.5.
In vivo membrane potentials of smooth muscle cells in the caudal artery of the rat
Authors: Bryant H. J. , Harder D. R. , Pamnani M. B. , Haddy F. J.
Published: December 2017

Membrane potentials measured in vivo may differ significantly from those measured in vitro in part due to humoral factors, innervation, and wall tension. These studies were initiated to determine whether it is feasible to record membrane potentials from vascular smooth muscle cells in vivo in the caudal artery of the pentobarbital-anesthetized male Wistar rat. Membrane potentials were measured using glass microelectrodes and correlated with systolic, diastolic, and mean blood pressures. For systolic blood pressures between 100 and 140 mmHg the average resting membrane potential was -38.4 +/- 0.48 mV. There was good correlation of systolic, diastolic, and mean blood pressures with membrane potential between 100 and 140 mmHg (r = 0.89, 0.75, and 0.89, respectively). Below 80 mmHg the arterial muscle cells became more depolarized than would be expected if the membrane potential were determined solely by transmural pressure. The depolarized membrane potential at low arterial pressures may be due to enhanced neural input. Spontaneous electrical activity was observed in some of the in vivo cells. When action potentials were present, they were generated at rates between 1-2/s and 6-7/min. These studies indicate that it is feasible to measure membrane potentials from arterial smooth muscle cells in vivo in the caudal artery of the rat.
The platelet strip. I. A low-fibrin contractile model of thrombin-activated platelets
Authors: Salganicoff L. , Loughnane M. H. , Sevy R. W. , Russo M.
Published: December 2017

The ultrastructure and contractile behavior of a new preparation of thrombin-activated human platelets is described. The preparation is referred to as the "platelet strip" because of its similarities to classical vascular smooth muscle strips. The platelet strip consists of a giant platelet aggregate 10 mm long, 4 mm wide, and 200 micron thick. To facilitate handling, the aggregate has a special high-compliance nylon mesh embedded in its mass. Each strip contains 7.3 X 10(8) platelets. Fibrin contamination is 150-fold lower than in platelet-rich plasma clots. Active isometric forces of up to 100 g/cm2 and 6-10 h viability are easily and reproducibly obtained. Platelet strips remain contracted after thrombin activation. The contraction is tonic and partial. Further small increases in force can be produced by depolarizing solutions or pharmacological agents, e.g., ADP, epinephrine, and endoperoxide analogues. These small increases are reversible on washout of the agents. Full relaxation is induced by agents such as prostaglandin E1 or papaverine, which increase adenosine 3',5'-cyclic monophosphate. However, after washout of these agents, recovery of tension is variable depending on the concentration of the drug and the degree of prestretching of the preparation.
Myosin phosphorylation in permeabilized rabbit psoas fibers
Authors: Sweeney H. L. , Kushmerick M. J.
Published: December 2017

The 18,000-Da myosin light chains in segments of rabbit psoas fibers were stably phosphorylated to assess the mechanical effects of this modification. Before and after phosphorylation of the same fiber, the maximal shortening velocity (Vmax) was measured at 12 degrees C by a quick-release slack test and by extrapolation to Vmax of hyperbolic force-velocity curves from isotonic releases. The experiments were performed at saturating concentrations of Ca2+, as determined from isometric force-pCa curves, under conditions in which the pH and ATP-ADP ratio were buffered. No effect of phosphorylation on isometric force, Vmax, or the shape of the force-velocity curve was detected under conditions of maximal calcium activation. Thus we find no mechanical evidence for a modulation by light chain phosphorylation of actomyosin interaction in these fiber segments.
Effect of renal ischemia on cortical microsomal calcium accumulation
Authors: Schieppati A. , Wilson P. D. , Burke T. J. , Schrier R. W.
Published: December 2017

Mitochondrial respiration, Ca2+ content, and Ca2+ kinetics have been found to be profoundly altered in ischemic acute renal failure (ARF). The effect of clamping the bilateral renal artery for 50 and 90 min on microsomal Ca2+ uptake was therefore examined in the rat. The 50-min clamping produced a reversible model of nonoliguric ARF, and the 90-min clamping produced a model of nonreversible oliguric ARF. In the 50-min nonoliguric model, ATP-dependent Ca2+ uptake by microsomes from renal cortex (nmol X mg protein-1 X 30 min-1) was significantly impaired immediately before release of the clamp and before return of renal blood flow (reflow) (191 +/- 11 vs. 83 +/- 11, P less than 0.005). However, in this nonoliguric model of ischemic ARF, microsomal uptake returned completely to normal after 1 h of reflow (sham 189 +/- 11 vs. 167 +/- 14 at 1 h, NS) and persisted at this normal level at 24 h (sham 166 +/- 14 vs. 150 +/- 13 at 24 h, NS). In the oliguric model of ARF the microsomal Ca2+ uptake also was impaired immediately after the clamp release (sham 191 +/- 11 vs. 93 +/- 11, P less than 0.001) as well as after 1 h of reflow (sham 189 +/- 11 vs. 129 +/- 12, P less than 0.005) but not at 24 h (sham 166 +/- 14 vs. 173 +/- 13, NS). The results indicate that impaired microsomal Ca2+ uptake occurs early in both oliguric and nonoliguric ARF and persists after 1 h of reflow in the oliguric model.(ABSTRACT TRUNCATED AT 250 WORDS)
Submicron tip breakage and silanization control improve ion-selective microelectrodes
Authors: Tripathi S. , Morgunov N. , Boulpaep E. L.
Published: December 2017

Probable causes of failure of otherwise well-constructed liquid ion-exchanger (LIE) micro-electrodes of average tip size less than 0.15 micron were examined. The problem could be attributed to two major variables, both localized at the tip: partial tip occlusion during fabrication prevents the generation of an electromotive force (small or absent slope and/or selectivity, high resistance); or poor hydrophobicity of the tip permits water to displace the resin from the tip (small or absent slope and/or selectivity and low electrode resistance). Controlled dry tip breakage on paper coated with glassine to final tip sizes well below 0.5 micron (confirmed by scanning electron microscopy) improves the yield of usable electrodes severalfold. Adequate silanization of the tip and consequent retention of resin at the tip can be predicted from the contact angles observed at the glass-LIE-backfilling solution interface. Satisfactory silanization can be achieved despite high ambient humidity. No evidence of shunting of Na+-LIE microelectrodes by the glass wall was seen. In the isolated perfused proximal tubule of Ambystoma tigrinum, the mean intracellular Na+ activity recorded by broken-tip electrodes (13.7 +/- 1.9 meq, n = 4) was similar to that recorded by intact electrodes (15.5 +/- 1.1 meq, n = 31).
Effect of length history on contractile behavior of canine tracheal smooth muscle
Authors: Gunst S. J.
Published: December 2017

The effects of shortening on force redevelopment were studied in canine tracheal smooth muscles that were tonically contracted with electrical stimulation or acetylcholine. Muscle strips were suspended in vitro so that muscle length, force, and rate of change of force could be continuously monitored. The strips were rapidly shortened from different initial lengths to the same final length, at which the effects of shortening distance on force redevelopment were evaluated. Both the rate and magnitude of force redevelopment declined as the shortening distance was increased. Decreasing the activation of the muscle or increasing the prerelease contraction time enhanced the depressive effect of shortening. The time course of the latter corresponded to a decline in the velocity of unloaded shortening. However, force redevelopment was also substantially decreased by releases performed before the onset of force development. The shortening process per se had a depressive effect on both the rate and magnitude of force redevelopment in contracted canine tracheal smooth muscle, and this effect was reduced as the activation of the muscle was increased.
Phosphorylase kinase isozymes in normal and electrically stimulated skeletal muscles
Authors: Lawrence J. C. , Krsek J. A. , Salsgiver W. J. , Hiken J. F. , Salmons S. , Smith R. L.
Published: December 2017

Phosphorylase kinase was quantitatively immunoprecipitated from extracts of different rabbit skeletal muscles, subjected to electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate, and stained with silver. Amounts of the two isozymes, enzymes r and w. were determined by the staining intensities of the alpha'- and alpha -subunits, respectively. The kinase in muscles composed primarily of slow oxidative fibers was almost entirely enzyme r, but several times more of this isozyme was found in muscles with a high proportion of fast oxidative-glycolytic fibers. Enzyme w predominated in muscles composed mostly of fast glycolytic fibers. To investigate the possible role of muscle activity in controlling isozyme levels, tibialis anterior muscles were activated by chronic electrical stimulation of the peroneal nerves. After 10 wk of continuous stimulation, the amount of phosphorylase kinase was decreased by approximately 80%, and the ratio of enzyme r to enzyme w was doubled. These results demonstrate that increased muscle activity can decrease levels of phosphorylase kinase and suggest that activity may regulate the expression of the isozymes in different muscle fiber types.
Substrate metabolism of isolated jejunal epithelium: conservation of three-carbon units
Authors: Mallet R. T. , Kelleher J. K. , Jackson M. J.
Published: December 2017

This study characterizes the substrate metabolism of isolated jejunal epithelial cells. Utilization of substrates was assessed by spectrophotometric assay. Significant quantities of glucose, glutamine, and ketone bodies were consumed in a 1-h period; lactate and ammonia were produced. [U-14C]glucose was metabolized in this medium to approximately three moles of lactate per mole of CO2. The pattern of tricarboxylic acid (TCA) cycle metabolism was analyzed utilizing media containing different concentrations of potential metabolic substrates and trace quantities of [14C]- succinate. O2 consumption rates indicated that glutamine can serve as an energy source in the absence of other substrates. Relative 14CO2 production from [1,4-14C]succinate versus [2,3-14C]succinate, which estimates flux of TCA cycle intermediates to products other than CO2, was increased more than twofold when glutamine was the only major substrate available. Alanine was produced from TCA cycle intermediates. Analysis of the citrate labeling pattern in the presence of [2,3-14C] succinate suggested that carbon from the TCA cycle does not form a significant fraction of acetyl-CoA used for citrate synthesis and that glutamine carbon was not completely oxidized to CO2. These findings suggest that glucose and glutamine are converted to three-carbon compounds by the jejunal epithelium.
Analysis of intracellular oxygenation of isolated adult cardiac myocytes
Authors: Jones D. P. , Kennedy F. G.
Published: December 2017

The influence of cellular shape, cellular O2 consumption rate, and intracellular diffusion coefficient for O2 on the magnitude of intracellular O2 gradients during hypoxia was analyzed with the model of Boag (Curr. Top. Radiat. Res. 5: 141-195, 1969) to determine whether these parameters could account for the experimentally measured O2 dependence curves for myoglobin (Mb) oxygenation and cytochrome a + a3 oxidation in heart cells. The analysis shows that the intracellular diffusion coefficient for O2 must be below 4 X 10(-6) cm2/s for a substantial intracellular gradient to occur. The intracellular diffusion coefficient was calculated from the difference in half-maximal oxidation (P50) values for isolated Mb and intracellular Mb and was found to be 1.76 X 10(-6) cm2/s. Use of this value and appropriate geometric models satisfactorily described the O2 dependence of Mb oxygenation and cytochrome a + a3 oxidation in cells over an eightfold range of O2 consumption rates. However, the analysis does not account for the correspondence of intracellular P50 values of Mb oxygenation and cytochrome a + a3 oxidation. This implies that there exists an intracellular heterogeneity of either Mb distribution, mitochondrial distribution, or mitochondrial respiratory characteristics. Such heterogeneity would further contribute to diffusion limitation of O2 supply during hypoxia and could be a major factor underlying the cardiac myocyte structure-function relationship.
Calcium and proton buffering and diffusion in isolated cytoplasm from Myxicola axons
Authors: Abercrombie R. F. , Hart C. E.
Published: December 2017

Ion-selective electrodes recorded the pH (7.49 +/- 0.05, n = 8) and pCa (6.72 +/- 0.03, n = 40) in samples (approximately 1 microliter) of isolated Myxicola axoplasm mounted within 760-micron diameter plastic tubes. We determined the interactions between Ca2+ and H+ on axoplasmic buffers by microinjecting CaCl2 or HCl into the axoplasmic samples at a distance 75-125 micron from the tips of the electrodes (distance = r). When axoplasmic pH was lowered 0.97 +/- 0.095 from its resting value (measured at r = 125 micron) by injecting 4 nmol HCl, pCa dropped 0.30 +/- 0.05 (n = 6). When expressed in units of concentration, these data show that a HCl injection of approximately 4 mmol/l axoplasm increased H+ and Ca2+ activity by approximately 0.3 microM. Lowering axoplasmic pCa 2.20 +/- 0.43 (r = 75 micron) (n = 3) by injecting 40 pmol CaCl2 had only a small effect on pH. In other experiments, two Ca2+ electrodes measured the Ca2+ activity 125 and 375 micron from the site of CaCl2 injection. Evidence of Ca2+ buffering was obtained when the Ca2+ activity at these two locations was below that expected for simple Ca2+ diffusion away from the injection site. Centrifuged axoplasm (100,000 g) taken from the bottom of the centrifuge tube had a somewhat greater Ca2+ buffering capacity than that taken from the top of the tube. Electron microscopic studies of the centrifuged axoplasm showed a greater concentration of mitochondria and other axoplasmic vesicles in the bottom of the centrifuge tube. Ruthenium red (20-40 micrograms/ml) greatly reduced Ca2+ buffering. The mitochondrial inhibitors CN (2 mM) and oligomycin (a mixture of oligomycin A, B, and C, 5 micrograms/ml) also reduced Ca2+ buffering but were not as effective as ruthenium red. Axoplasm in which ATP and mitochondrial substrates were removed by dialysis was unable to lower free Ca2+ when the concentration of this ion was elevated to approximately 10 microM. In the presence of oligomycin to block mitochondrial ATPase, and with Mg2+ -ATP as the only source of energy, axoplasm lowered Ca2+ activity slowly; with succinate as the only metabolic substrate, axoplasm rapidly lowered the Ca2+ activity from approximately 10 microM to below 1 microM.
Architecture, composition, and contractile properties of rat soleus muscle grafts
Authors: Segal S. S. , White T. P. , Faulkner J. A.
Published: December 2017

Skeletal muscle grafts have a deficit in tension development compared with control muscles, even after accounting for reduced mass and total muscle cross-sectional area. Our purpose was to determine relationships among the architecture, tissue composition, and contractile properties of rat soleus muscle grafts. Data were compared with control soleus muscles obtained from littermates. Female Wistar rats were anesthetized with pentobarbital sodium for grafting of soleus muscles with nerve implant and for dissection of muscles 56 days after grafting. Compared with control values, the maximum specific tension (N/cm2) of grafts was 76%, the interstitial (inulin) space was 135%, and the connective tissue protein concentration was 177%. For grafts, total muscle length and fiber length were 91 and 123% of control values, respectively. The extrapolated shortening velocity at zero load (fiber lengths/s) for grafts was not different from the control value. The deficit in specific tension of grafts is explained by a greater concentration of noncontractile tissue components. Changes in muscle architecture and composition following grafting had little affect on contraction dynamics.
Characterization of monoclonal antibodies to rabbit renal cortical cells
Authors: Ronco P. , Geniteau M. , Poujeol P. , Melcion C. , Verroust P. , Vandewalle A.
Published: December 2017

The immunological heterogeneity of the rabbit nephron was investigated using monoclonal antibodies. Seventeen antibodies have been produced by fusion of NS1 myeloma cells with spleen cells from BALB/c mice immunized with unfractionated rabbit renal cortical cell preparations. Sixteen antibodies reacted with proximal tubular cells: 11 with the brush border and 5 with basolateral membrane or intracytoplasmic components. Only one of the latter was specific for constituents of the proximal tubule. One antibody reacted with the cortical collecting tubule. Eight of the anti-brush-border antibodies were further characterized by immunoprecipitation of detergent-solubilized radiolabeled brush-border membrane vesicles. Seven proteins with subunits ranging in molecular weight from 90,000 to greater than 340,000 were identified. Systematic survey showed that one of these proteins with a subunit molecular weight of 115,000 exhibited leucine aminopeptidase activity. Selected monoclonal antibodies bound to Sepharose 4B immunoadsorbents were used to deplete solubilized brush-border membrane vesicles of a given antigen and to identify leucine aminopeptidase. Furthermore, the obtention of specific antibodies directed against the proximal tubule allowed us to set up a simple method for renal cell separation: isolated renal cortical cells could be depleted by 80% in proximal cells by passage over columns of Sepharose 6MB covalently linked with three different monoclonal anti-brush-border antibodies, thus leading to cell suspensions considerably enriched in tubule cells originating from the more distal segments of the nephron.
Alterations in active Na-K transport during mineralocorticoid-salt hypertension in the rat
Authors: Magliola L. , McMahon E. G. , Jones A. W.
Published: December 2017

Studies have been conducted to determine which, if any, of the parameters governing active Na-K transport (K and/or ouabain sensitive) are altered in vascular smooth muscle during mineralocorticoid-salt hypertension. Rats with one kidney removed were treated with either aldosterone or deoxycorticosterone acetate plus saline for 3-4 wk. Ion transport was measured in arteries incubated in a physiological salt solution for periods of 4-6 h. Increased active Na efflux was observed in femoral arteries and aortas from the hypertensive group. This alteration resulted primarily from an elevation in the saturation or maximal capacity of the active transport mechanism, which operated with no significant change in cell Na concentration. The transport parameters related to ion selectivity, cooperativity, and temperature dependence were not significantly altered. Measures of active Na efflux and K influx in the same aortic strips indicated that both fluxes were elevated in the hypertensive group. The ratio of Na to K for active transport was significantly greater than one but was unchanged in the hypertensive rats. These studies provide evidence for the electrogenic operation of the Na-K pump in arterial smooth muscle and for the operation of the pump at a higher level during mineralocorticoid-salt hypertension. This alteration may result from increased turnover of individual sites or possibly the incorporation of more sites into the membrane as a result of mineralocorticoid-salt treatment.
Na+-Ca2+ exchange contributes to increase of cytosolic Ca2+ concentration during depolarization in heart muscle
Authors: Sheu S. S. , Sharma V. K. , Uglesity A.
Published: December 2017

The possible role of Na+-Ca2+ exchange in contributing to depolarization-induced increase in cytosolic Ca2+ concentration ([Ca2+]i) of isolated rat ventricular myocytes was investigated. Measured with the Ca2+-sensitive indicator quin 2, [Ca2+]i increased from 177 +/- 12 (mean +/- SE, n = 11) to 468 +/- 41 nM when cells were depolarized with solutions containing 50 mM KCl [high extracellular K+ concentration ([K+]o)]. Approximately 73% of this high-[K+]o-induced increase in [Ca2+]i was abolished by the Ca2+ channel blocker verapamil (5 microM). For cells pretreated with 10 mM caffeine to deplete the Ca2+ stored in sarcoplasmic reticulum, 50 mM KCl still produced an increase in [Ca2+]i, even in the presence of 5 microM verapamil. However, if extracellular Na+ was replaced by Li+ or tris(hydroxymethyl)aminomethane, this increase was completely abolished. The results suggest that, in addition to voltage-sensitive Ca2+ channels, voltage-sensitive Na+-Ca2+ exchange can also contribute to the increase in [Ca2+]i on depolarization. Therefore both Ca2+ transport systems may play important roles in regulating cardiac excitation and contraction.
Influence of surface pH on intracellular pH regulation in cardiac and skeletal muscle
Authors: Vanheel B. , de Hemptinne A. , Leusen I.
Published: December 2017

The influence of the surface pH (pHs) on the intracellular pH (pHi) and the recovery of pHi after an imposed intracellular acid load was investigated in isolated sheep cardiac Purkinje fiber, rabbit papillary muscle, and mouse and rat soleus muscle. pHs and pHi, respectively, were continuously measured by use of single- and double-barreled pH-sensitive glass microelectrodes. Surface acidosis, usually obtained by superfusion with solutions of acid pH, was also produced with low buffered (5 mM N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) solutions at control pH. The pHs decrease (delta pHs) induced by low buffering was smallest (-0.08 pH unit) in Purkinje fiber and largest (-0.31 pH unit) in rat soleus muscle, which already had a more acid surface in control conditions. delta pHs was somewhat dependent on the superfusion rate. Higher superfusion rates decreased but did not abolish delta pHs. Surface acidosis was associated with a small intracellular acidification. Intracellular acid loads were produced by adding and subsequently withdrawing 20 meq/l NH4+ from the superfusate. In all preparations, the rate of recovery of pHi after NH4+ withdrawal was notably decreased at acidified pHs. This effect was amiloride sensitive. It is concluded that, in superfused multi-cellular preparations, pHs and therefore the buffer concentration of a superfusate can considerably influence steady-state pHi and pHi recovery from an imposed intracellular acid load.
Biphasic concentration dependency of stimulation of myoblast differentiation by somatomedins
Authors: Florini J. R. , Ewton D. Z. , Falen S. L. , Van Wyk J. J.
Published: December 2017

It is widely believed that mitogens inhibit in vitro differentiation of myoblasts to form postmitotic myotubes, but we and others have shown that the mitogenic hormones insulin and the insulin-like growth factors (IGFs) stimulate myoblast differentiation. We now report the results of concentration-dependency studies that resolve this disagreement. We found that the IGFs give a biphasic dose-response curve; at low concentrations, there is progressive stimulation of L6 myoblast differentiation; at higher concentrations, there is a progressive decrease. Similar results were obtained with IGF-II and insulin. When differentiation was maximally stimulated (by 1,280 ng/ml insulin), adding rat IGF-II gave decreases in differentiation similar to those reported for other mitogens. Two trivial explanations have been eliminated: stimulation of differentiation (at low concentrations) is not due to enhanced survival or growth of the cells, and inhibition (at higher concentrations) is not a toxic effect. In L6 cells, epidermal growth factor and fibroblast growth factor had no effect on proliferation or differentiation. We conclude that the effects of medium components on myoblast differentiation cannot be generalized to indicate inhibition by all mitogens; depending on the cell lines and concentrations used, certain mitogens may either stimulate or inhibit differentiation.
Photoinactivation of sodium-potassium-chloride cotransport in LLC-PK1/Cl 4 cells by bumetanide
Authors: Amsler K. , Kinne R.
Published: December 2017

Rb+ uptake into LLC-PK1/Cl 4 cells can be subdivided into three components: 1) ouabain-sensitive uptake, 2) bumetanide-sensitive uptake, and 3) ouabain- and bumetanide-insensitive uptake. Exposure of cells to near-UV light in the presence of low concentrations of bumetanide produces a specific, irreversible inhibition of the bumetanide-sensitive uptake component, while not affecting the other two uptake components. Irreversible inhibition of bumetanide-sensitive transport is observed when measuring either cellular uptake or efflux and also when measuring 86Rb+ uptake into membrane vesicles. The irreversible inhibition is both concentration and time dependent and is blocked under conditions where the interaction of bumetanide with the Na+-K+-Cl- cotransporter is disturbed. We conclude that bumetanide, at low concentrations, can specifically and irreversibly inhibit the Na+-K+-Cl- cotransporter of LLC-PK1/Cl 4 cells. We suggest that this irreversible inhibition is the result of the photoactivation of an ether linkage in the bumetanide molecule, leading to a covalent binding of bumetanide to the Na+-K+-Cl- cotransporter.
Inositol trisphosphate enhances calcium release in skinned cardiac and skeletal muscle
Authors: Nosek T. M. , Williams M. F. , Zeigler S. T. , Godt R. E.
Published: December 2017

Experiments from other laboratories suggest that inositol trisphosphate (InsP3) may be involved in the excitation-contraction coupling (ECC) process of cardiac and skeletal muscle. Our results support this hypothesis. Studying fiber bundles (less than 200-microns diameter) from guinea pig papillary muscles skinned with saponin and mechanically skinned single fibers from frog semitendinosus muscle, we find that calcium-induced force oscillations (observed in solutions containing low ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid and pCa 7.0) are enhanced in magnitude and frequency by InsP3 at concentrations as low as 1 microM. InsP3 at 10 microM can often induce such oscillations in mechanically skinned frog skeletal muscle. In skinned cardiac fibers, InsP3 increases the magnitude of caffeine contractures at submaximal caffeine concentrations to a greater extent than at near-maximal caffeine concentrations. InsP3 (30 microM) has no effect on either the calcium sensitivity or maximal force generated by the contractile apparatus of skinned cardiac muscle. We conclude that InsP3 has no direct effect on the contractile machinery but that it can modulate ECC by enhancing the calcium-induced release of calcium from the sarcoplasmic reticulum, possibly from the same pool and through the same mechanism as caffeine.
Two different heavy chains are found in smooth muscle myosin
Authors: Rovner A. S. , Thompson M. M. , Murphy R. A.
Published: December 2017

Two putative myosin heavy chains designated SM1 and SM2 were detected on a 3.5% polyacrylamide-sodium dodecyl sulfate gel electrophoresis system loaded with homogenates of several mammalian smooth muscles. The two polypeptides were present in nearly equal amounts in all smooth muscle tissues tested and in myosin purified from swine carotid media and stomach. Both proteins were equally stained by smooth muscle-specific myosin antibodies. The smaller of the polypeptides had a mobility nearly identical to that of the single heavy chain observed in purified fast-twitch skeletal myosin. Electrophoresis of pyrophosphate extracts from swine carotid media, swine stomach, rabbit thoracic aorta, and guinea pig taenia coli on nondenaturing pyrophosphate gels revealed a single protein band. When subsequently electrophoresed on a sodium dodecyl sulfate gel, the native bands from swine tissue extracts revealed the two putative heavy chains in nearly equal amounts, as well as a large amount of a higher molecular weight peptide whose properties reflect those of filamen. Sodium dodecyl sulfate gel analysis of the myosin band from pyrophosphate gels of purified swine stomach myosin showed exclusively the two heavy chains in a nearly 1:1 ratio. Smooth muscle myosin migrates homogeneously on pyrophosphate gels, and the virtual equality of the two heavy chains may reflect the presence of large amounts of a myosin isoenzyme, which is a heavy-chain heterodimer.
Proliferation and differentiation of brown adipocytes from interstitial cells during cold acclimation
Authors: Bukowiecki L. J. , Geloen A. , Collet A. J.
Published: December 2017

The mechanisms of brown adipocyte proliferation and differentiation during cold acclimation (and/or adaptation to hyperphagia) have been studied by quantitative photonic radioautography. [3H]thymidine was injected to warm-acclimated (25 degrees C) rats and to animals exposed to 5 degrees C for 2 days. Samples of interscapular brown adipose tissue were collected for quantitative analysis of mitotic frequencies at various periods of time (4 h-15 days) after the injection of [3H]thymidine, the rats being maintained at the temperatures to which they were initially exposed. Confirming our previous results [Bukowiecki et al., Am. J. Physiol. 242 (Endocrinol. Metab. 5): E353-E359, 1982], it was found that cold exposure for 2 days markedly enhanced mitotic activity in endothelial cells, interstitial cells, and brown preadipocytes rather than in fully differentiated brown adipocytes. The total tissue labeling index (percent of labeled nuclei) increased approximately 70 times over control values. We now report that cellular labeling progressively increased in mature brown adipocytes during cold acclimation, whereas it correspondingly decreased in interstitial cells and brown preadipocytes. This indicates that the sequence of events for cellular differentiation is interstitial cells----brown preadipocytes----mature brown adipocytes. Remarkable, labeling frequency did not change in endothelial cells during cold acclimation demonstrating that these cells cannot be considered as progenitors of brown adipocytes. It is suggested that brown adipocyte proliferation and differentiation from interstitial cells represent the fundamental phenomena explaining the enhanced capacity of cold-acclimated and/or hyperphagic rats to respond calorigenically to catecholamines.
Hypertonic cell volume regulation in mouse thick limbs. II. Na+-H+ and Cl(-)-HCO3- exchange in basolateral membranes
Authors: Hebert S. C.
Published: December 2017

Differential interference contrast microscopy and standard electrophysiological techniques were used to evaluate the transport processes involved in antidiuretic hormone (ADH)-dependent hypertonic cell volume regulation in the in vitro perfused mouse medullary thick ascending limb of Henle. Hypertonic cell volume regulation appeared to involve NaCl uptake into cells, since the cell volume increase after osmotic shrinkage in hypertonic media could be abolished either by symmetrical removal of Na+ from external solutions or by bath Cl- omission. The volume-regulatory process also required CO2/HCO3- in external media and could be abolished by the lipophilic carbonic anhydrase inhibitor, ethoxzolamide, in the presence of CO2/HCO3-. In addition, ADH-dependent hypertonic cell volume regulation was reduced or abolished by 10(-4) M amiloride, 10(-3) M ouabain, or 10(-4) M 4-acetamido-4'-isothiocyanostilbene-2,2-disulfonic acid in peritubular media or by cooling to 15 degrees C. In contrast, lumen Cl- omission or 10(-4) M amiloride addition to the perfusate had no effect on cell volume regulation in hypertonic peritubular media. These data suggest that ADH-dependent, hypertonic cell volume regulation in the mouse medullary thick limb depends on cell NaCl uptake via a secondary active transport process involving parallel Na+-H+ and Cl(-)-HCO3- exchangers in basolateral cell membranes. Finally, luminal furosemide (10(-4) M) abolished bath ouabain-mediated, rapid cell swelling in isotonic media containing ADH. Thus these exchangers do not appear to be active in the resting, isotonic state. The specific role of ADH in this NaCl transport process remains to be defined.