bluuebunny/crossref_metadata_2025 huggingface dataset:
Energy transport from mitochondria to myofibril by a creatine phosphate shuttle in cardiac cells
Authors: McClellan G. , Weisberg A. , Winegrad S.
Published: December 2017

In hyperpermeable cardiac cells, in which the surface membrane has been made highly permeable to small molecules and ions, resting tension increases when the concentration of ATP falls below 200 microM. Peak resting tension occurs in 10 microM ATP and equals 60% of maximum Ca-activated force in 5 mM ATP. The mitochondria in hyperpermeable cells can maintain an ATP concentration above 200 microM if supplied with O2, substrate, ADP, and inorganic phosphate (Pi). Removal of ATP from the bathing solution does not increase resting tension as long as creatine phosphate is present. However O2, substrate, and Pi cannot lower resting tension in the absence of ATP and creatine phosphate. These results are interpreted as evidence for adenine nucleotide tightly bound to the myofibrils and a creatine phosphate shunt of energy from the mitochondria to the myofibrils.
Differing effects of cGMP and cAMP on ion transport across flounder intestine
Authors: Rao M. C. , Nash N. T. , Field M.
Published: December 2017

The intestinal epithelium of the winter flounder is highly cation selective and actively absorbs NaCl via a bumetanide-sensitive (Na + K + 2Cl) cotransport system; it also actively secretes K+. Combined addition of adenosine 3',5'-cyclic monophosphate (cAMP) and theophylline was previously shown to partially inhibit NaCl absorption and to increase passive Cl- permeability. Because theophylline increases cyclic GMP (cGMP) and cAMP concentrations, we compared the effects of the 8-Bromo analogues of these two nucleotides on ion transport. cGMP inhibits Cl- absorption, K+ secretion, and Cl- and K+ influx across the brush border as effectively as do furosemide and bumetanide. Even at maximal doses, cAMP is less effective than cGMP in inhibiting ion transport; however, unlike cGMP, it abolishes the cation selectivity of the epithelium by greatly increasing Cl- permeability. The effects of the two nucleotides are not additive with each other or with those of bumetanide, although cGMP or bumetanide can further inhibit transport in cAMP-treated tissues.
Na+-K+ transport and volume of rat erythrocytes under dietary K+ deficiency
Authors: Duhm J. , Gobel B. O.
Published: December 2017

Red cell Na+ and K+ content and transport were studied in Sprague-Dawley rats in the course of a dietary K+ depletion ranging 1-6 wk. Plasma K+ fell to below 2 mM, and red cell K+ decreased. Cellular Na+ rose due to an increase of the Na+ leak. Inward Rb+ and outward Na+ transport by the Na+-K+ pump (determined at 2 mM external Rb+) were accelerated by the rise in cell Na+ concentration. K+ depletion caused a cation deficit of up to 30% of total red cell Na+ plus K+ and a consecutive cell shrinkage with an increase in mean cellular hemoglobin content (MCHC). The cell shrinkage, in turn, was paralleled by up to a 10-fold increase in the maximum capacity of the furosemide-sensitive, chloride-dependent Na+-K+ cotransport system. This system participated with up to 50% of the total K+ movements across the red cell membrane in severe K+ deficiency. In normal cells shrunken by osmotic means, Na+-K+ cotransport was similarly accelerated severalfold, indicating that the cell shrinkage occurring during K+ depletion is a major factor inducing the changes in Na+-K+ cotransport. However, a second unknown factor is also involved. It is concluded that in the rat, not only genetic but also environmental parameters contribute in determining the actual activity of the red cell Na+-K+ cotransport system. The cell volume and MCHC must be considered when judging Na+ and K+ transport changes observed in rat erythrocytes under various pathophysiological conditions.
Transport of water and urea in red blood cells
Authors: Macey R. I.
Published: December 2017

Evidence for water channels in red blood cells is reviewed. In an entropically driven reaction, organic mercurials decrease water permeability, elevate the activation energy, and reduce the ratio of osmotic to diffusional water permeabilities to unity so that water transport properties of red blood cells are hardly distinguishable from lipid bilayers. It is concluded that mercurials close the water channels. A variety of kinetic, pharmacological, and comparative evidence converges on the conclusion that urea and other solutes are excluded from water channels. Urea apparently permeates the red cell membrane via a facilitated diffusion system, which plays an important role when red blood cells traverse the renal medulla; rapid urea transport helps preserve the osmotic stability and deformability of the cell, and it helps prevent dissipation of extracellular osmotic gradients. Water apparently traverses the channel via a single-file mechanism; the very low channel permeability of H+ is explained if the channel contains fixed charge, or alternatively, if the mobile water molecules within the channel do not form a continuum. An alternative unitary pore hypothesis for simultaneous transport of water, ions, and small solutes is also discussedl.
Mechanics of resting isolated single vascular smooth muscle cells from bovine coronary artery
Authors: VanDijk A. M. , Wieringa P. A. , van der Meer M. , Laird J. D.
Published: December 2017

The viscoelastic behavior of single resting vascular smooth muscle cells from bovine coronary artery was studied. No maintained passive force could be recorded, even when the cells were stretched to two to four times their initial length; this finding suggests that the smooth muscle cells do not contribute to the parallel elastic component in arterial smooth muscle tissue. The force during stretch of resting arterial cells was proportional to the rate of stretch (which varied between 20 and 60% of the initial length per second). This linear viscous resistance was also found for toad stomach cells when similar stretches were applied. The stress-relaxation curves of the arterial cells could be fitted to the sum of two exponential components (with half-lives of 13.1 and 0.5 s, respectively). As a result of the above findings, a model consisting of two viscoelastic elements in parallel was proposed for a single resting arterial smooth muscle cell. The viscous resistance to stretch of resting cells in a Ca2+-containing solution was not significantly (P greater than 0.01) different from that in a Ca2+-free solution. The same result was obtained for bovine coronary arterial rings. It is concluded that an adequate model for resting arterial smooth muscle should include an intracellular viscous element.
Cellular ultrastructure of Amphiuma distal nephron: effects of exposure to potassium
Authors: Stanton B. , Biemesderfer D. , Stetson D. , Kashgarian M. , Giebisch G.
Published: December 2017

The cellular ultrastructure of the renal distal nephron of the salamander, Amphiuma means, was examined by electron-microscopic and stereological techniques before and after exposure to potassium in the ambient environment. The distal nephron of Amphiuma is composed of three ultrastructurally distinct segments: early distal (or diluting segment), late distal, and collecting tubule. The early distal tubule structurally resembles the mammalian thick ascending limb of Henle's loop. Large renin-like granules are present in the smooth muscle cells of the afferent arteriole in the vicinity of the early distal tubule, suggesting the presence of a rudimentary juxtaglomerular apparatus. Late distal tubules are composed of one large cell type characterized by extensive basal membrane invaginations, often extending to the luminal membrane. Collecting tubules contain principal and intercalated cells that are ultrastructurally similar to cells of the mammalian cortical collecting tubule. Exposure to potassium had no effect on the ultrastructure of early distal cells but led to a sharp increase in the basolateral membrane surface density of principal cells in the collecting tubule (1.17 +/- 0.08-1.63 +/- 0.13 micron2/micron3). Potassium adaptation leads to a similar structural response in the mammalian collecting tubule. Since Amphiuma collecting tubules can be isolated and perfused in vitro and impaled with ion- and voltage-sensitive microelectrodes, the observed structural adaptation suggests that the collecting tubule may be a useful preparation to study the cellular mechanisms of potassium adaptation.
Phosphorus nuclear magnetic resonance of fast- and slow-twitch muscle
Authors: Meyer R. A. , Brown T. R. , Kushmerick M. J.
Published: December 2017

Phosphorus nuclear magnetic resonance (NMR) spectra were obtained at 109.3 MHz from isolated, arterially perfused cat biceps brachi (greater than 75% fast-twitch, glycolytic fibers) and soleus (greater than 92% slow-twitch, oxidative) muscles at 30 degrees C. The perfused muscles were stable with respect to O2 consumption, twitch characteristics, and ATP and phosphocreatine (PC) levels for up to 10 h. NMR spectra showed a higher PC/Pi ratio in the biceps (11) than in the soleus (1.7). Relatively higher Pi levels were observed in extracts of clamp-frozen muscles than in the intact muscles. This difference could be accounted for by artifactual hydrolysis of PC during muscle freezing. Based on the NMR and chemical data, the free cytosolic ADP level, calculated from the creatine kinase equilibrium, was 14 microM in the soleus and less than 1 microM in the biceps. Intracellular Pi concentration was 10 mM in the soleus and 3 mM in the biceps. Intracellular pH, estimated from the chemical shift of phosphate or 2-deoxyglucose 6-phosphate, was 7.0 in both muscles (perfusate pH 7.2). Both extracellular space and pH measurements were obtained from NMR spectra of muscles perfused with 10 mM sodium phenylphosphonate added to the perfusate. These results document larger differences in the phosphate metabolites in the two types of mammalian muscles than previously reported.
Agonist-mediated regulation of alpha 1- and beta 2-adrenergic receptors in cloned MDCK cells
Authors: Meier K. E. , Sperling D. M. , Insel P. A.
Published: December 2017

Chronic exposure of cells to neurotransmitters and hormones can result in a decrease in receptor number (down-regulation). We asked whether two different types of receptors on the same cell that can both respond to a particular hormone are identically regulated. For this study we used MDCK-D-1, a cloned renal epithelial cell line that coexpresses alpha 1- and beta 2-adrenergic receptors, as a model system in which to examine the effects of an adrenergic agonist on the expression and function of these receptors. MDCK-D-1 cells retain differentiated features of renal tubular epithelium with respect to morphology and hormonal responsiveness. When MDCK-D-1 cells were incubated for 21 h with 100 microM epinephrine, alpha 1- and beta 2-receptor numbers decreased by 81 and 90%, respectively. The down-regulation of beta 2-receptors proceeded more rapidly than that of alpha 1-receptors. Down-regulated cells showed a greater than 80% loss of both alpha 1- and beta 2-adrenergic responses (alpha 1: stimulation of prostaglandin E2 release; beta 2: elevation of adenosine 3',5'-cyclic monophosphate levels). We conclude that in renal epithelial cells chronic exposure to a catecholamine agonist can result in a profound decrease in the number of alpha 1- and beta 2-receptors, this down-regulation is accompanied by a loss of adrenergic response, and the kinetics of receptor loss are different for alpha 1- and beta 2-receptors.
Activation of glucose transport in diabetic muscle: responses to contraction and insulin
Authors: Wallberg-Henriksson H. , Holloszy J. O.
Published: December 2017

The purpose of this study was to evaluate the effect of insulin deficiency on the ability of muscle contraction to activate glucose transport. Rats were made severely diabetic by injection of streptozotocin (SZ; 125 mg/kg body wt). Only those diabetic rats in which plasma insulin was too low to measure 3 days after SZ injection were included in this study. Glucose transport activity was evaluated by measuring 3-O-methylglucose uptake in epitrochlearis muscles in vitro. Contractile activity induced a 2.8-fold increase in 3-O-methylglucose uptake rate in epitrochlearis muscles of diabetic rats (P less than 0.01) even after the muscles had been thoroughly washed to remove any traces of insulin remaining in the extracellular fluid. However, this increase in sugar uptake was less than 50% as great as that seen in normal controls. This reduced ability to activate glucose transport was not limited to the effect of contractile activity, as the increase in sugar uptake rate in response to a maximal concentration of insulin was similarly reduced in the diabetic muscles. Thus it appears that contractile activity can accelerate glucose transport in insulin-deficient muscle; however, the maximal capacity to activate glucose transport is decreased in skeletal muscle after sustained insulin deficiency.
Some electrical and pharmacological properties of gap junctions between adult ventricular myocytes
Authors: White R. L. , Spray D. C. , Campos de Carvalho A. C. , Wittenberg B. A. , Bennett M. V.
Published: December 2017

Ventricular myocytes were isolated from adult rat hearts using the technique of Wittenberg and Robinson (Cell Tissue Res. 216: 231-251, 1981). These cells exhibited morphology, input resistance, time constant, and excitability expected for cells in intact cardiac tissue. Pairs of these cells were electronically coupled, and junctional conductance was unaffected by transjunctional potential or hyperpolarization of both cells. Brief exposure of cell pairs to medium equilibrated with 100% CO2 or containing 0.1 mM octanol quickly and reversibly decreased junctional conductance. We conclude that gap junctions between pairs of ventricular myocytes possess physiological properties like those of junctions in many other tissues. This preparation will be useful in evaluating drug action on junctional communication in heart.
Stress protein systems of mammalian cells
Authors: Subjeck J. R. , Shyy T. T.
Published: December 2017

Living organisms are known to react to a heat stress by the selective induction in the synthesis of several polypeptides. In this review we list the major stress proteins of mammalian cells that are induced by heat shock and other environments and categorize these proteins into specific subgroups: the major heat shock proteins, the glucose-regulated proteins, and the low-molecular-weight heat shock proteins. Characteristics of the localization and expression of proteins in each of these subgroups are presented. Specifically, the nuclear/nucleolar locale of certain of the major heat shock proteins is considered with respect to their association with RNA and the recovery of cells after a heat exposure. The induction of these major heat shock proteins and the repression of the glucose-regulated proteins as a result of reoxygenation of anoxic cells or by the addition of glucose to glucose-deprived cultures is described. Changes in the expression of these protein systems during embryogenesis and differentiation in mammalian and nonmammalian systems is summarized, and the protective role that some of these proteins appear to play in protecting the animal against the lethal effects of a severe heat treatment and against teratogenesis is critically examined.
Separation of hexose-transporting from nontransporting LLC-PK1 cells on density gradients
Authors: Weiss E. R. , Cook J. S.
Published: December 2017

Over a period of 2-3 wk after plating, cultured LLC-PK1 (pig kidney) cells develop a high capacity for Na+-dependent accumulation of alpha-methyl-D-glucoside. To further the analysis of this developmental process, we have developed a method for separating transporting from nontransporting cells on the basis of density changes accompanying hexose accumulation and the corresponding uptake of water. Volume regulation was prevented by suspending the cells in a K+-free, Cl(-)-free Na-gluconate medium. Na+-dependent transport was maintained at nearly control levels by addition of low concentrations of (NH4)2SO4, since NH+4 stimulates Na+-K+-ATPase at the K+ site and allows for the extrusion of accumulated Na+; NH+4-stimulated hexose uptake is ouabain sensitive. With volume regulation blocked but with transport near normal, transporting cells exhibited a phlorizin-sensitive density shift in methylglucoside-containing medium and could be separated from nontransporting cells on Percoll gradients.
Cold adaptation in guinea pig at level of isolated brown adipocyte
Authors: Rafael J. , Fesser W. , Nicholls D. G.
Published: December 2017

Isolated brown adipocytes were prepared from guinea pigs acclimated to 28 degrees C or exposed to 4-8 degrees C for periods of up to 3 wk. Cells from warm-adapted animals retained respiratory control when stimulated with norepinephrine. Cells from guinea pigs exposed to cold for 4-21 days showed a much greater respiratory response to norepinephrine due to enhanced uncoupling rather than enhanced substrate supply. After 7 days of cold acclimation, norepinephrine-stimulated respiration became uncontrolled and was limited only by the maximal respiratory capacity of the mitochondria. Three weeks of cold acclimation were accompanied by a doubling of total cell number, a doubling of the mitochondrial protein per adipocyte, and a sixfold increase in the norepinephrine-stimulated respiration per in situ mitochondrion with no change in respiratory chain capacity. The induction of norepinephrine-stimulated respiration correlated with the appearance of high-affinity purine nucleotide binding sites on the mitochondria, diagnostic of the uncoupling protein. If the results are extrapolated to the whole animal, they indicate that brown adipose tissue makes little contribution to thermogenesis in the warm-adapted guinea pig but may account for most or all the increment seen on cold adaptation.
Relations between chord and slope conductances and equivalent electromotive forces
Authors: Thompson S. M.
Published: December 2017

Nonlinear current-voltage relations for ion movement across biological membranes have been observed and significantly complicate the interpretation of electrical measurements on these transport processes. To enable analysis of the electrical measurements two formalisms have evolved, chord and slope, by which equivalent conductances and electromotive forces (emfs) can be obtained. Because, in the presence of nonlinear relations between current and voltage, the chord conductances and emfs are generally not equal to their slope counterparts, it is imperative that they not be intermixed (8). However, when the functional relationship between the current and voltage is known, such as the Goldman-Hodgkin-Katz (GHK) flux equation, it becomes possible to compare the voltage dependencies of these parameters and examine interrelationships between them. In this communication analytical expressions are derived for the chord and slope conductances and emfs for transport of a single ionic species that obeys the GHK flux equation. Using these expressions, it is possible to convert electrical equivalent circuit parameters derived for one formalism to electrical equivalent parameters of the other formalism. Therefore data obtained using either formalism can be used to obtain values for intracellular activity and membrane permeability to the transported ion. Parallel analyses can be applied to other models of ion transport.
The neural cell spectrin skeleton: a review
Authors: Goodman S. R. , Zagon I. S.
Published: December 2017

The discovery of nonerythroid spectrin (Goodman et al., Proc. Natl. Acad. Sci. USA 78: 7570-7574, 1981) has generated interest equivalent to that occurring upon the identification of nonmuscle actin and myosin. Brain spectrin has become the best-studied member of the nonerythroid spectrin family of molecules. In this review, we discuss the structure and functional interactions of brain spectrin, as a prelude to attempting to resolve what are some of the more controversial questions in the field. We finish with a discussion of what may be the most profitable directions for future research.
Analysis of proteins in rabbit pulmonary surfactant using monoclonal antibodies
Authors: King R. J. , Martin H. M. , Baseman J. B. , Morrison-Plummer J.
Published: December 2017

We have used monoclonal antibodies developed against the apolipoproteins associated with pulmonary surfactant purified from rabbit lavage fluid to study the expression of epitopes common to these proteins. The pulmonary surfactant contained nearly 20 proteins, of which at least 10 were not derived from serum. Electrophoresis, with sulfhydryl reduction of these proteins indicated apparent molecular weights of approximately 155, 135, 125, and 115 X 10(3) (high-molecular-weight group); 80, 70, and 60 X 10(3) (intermediate group); and 18 through 10 X 10(3) (low-molecular-weight group). Two-dimensional polyacrylamide gel electrophoresis, in which the proteins were electrophoresed without reduction in the first dimension, but with sulfhydryl reduction in the second dimension, revealed that the 80, 70, and 60 X 10(3) proteins dissociated into proteins of nominal molecular weights of 40, 35, and 30 X 10(3), respectively. In contrast, the 125 and 115 X 10(3) proteins of the high-molecular-weight group contained a protein which could only be reduced to a minimum molecular weight of 55 to 60 X 10(3). Monoclonal antibodies generally were of three types: those that reacted strongly with the high-molecular-weight group and weakly with the intermediate group; those that reacted conversely; and those that reacted only with the low-molecular-weight group. Our results indicate that at least two different surfactant apolipoproteins, with differing minimum molecular weights in SDS-polyacrylamide gel electrophoresis, have common epitopes. Although these results cannot certify a physiological relationship between these proteins, they suggest that the intracellular synthesis or extracellular processing of surfactant apolipoproteins may be more complicated than predicted by the findings of previous experiments, perhaps involving the posttranslational assembly of one surfactant protein into oligomers which resist dissociation under the conditions used for the analyses.
Small vessel membrane potential, sympathetic input, and electrogenic pump rate in SHR
Authors: Stekiel W. J. , Contney S. J. , Lombard J. H.
Published: December 2017

Comparative measurements of transmembrane potential (Em) were made in situ in vascular smooth muscle cells (VSM) of mesenteric small principal arteries and veins with innervation and circulation intact. Vessels were in an externalized, topically suffused jejunal loop in 4- to 5-wk-old (initial hypertension) and 12- to 15-wk-old (established hypertension) anesthetized, spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) normotensive control rats. Comparable in vitro measurements of Em were also made in VSM of isolated intact small mesenteric vessel segments (from the 12- to 15-wk-old animals) maintained at their in situ lengths and suffused with physiological salt solution (PSS). During suffusion in situ with control PSS, VSM of both small veins and arteries in older (but not younger)SHR were less polarized than in WKY. Local chemical sympathetic denervation in situ (with 6-hydroxydopamine) hyperpolarized VSM of both vessel types in older (but not younger) SHR to the same Em levels measured in situ in respective WKY vessels. After local denervation, VSM of small arteries (but not veins) of both SHR and WKY remained less polarized in situ than in vitro, suggesting the presence of one or more circulating factors with a specific depolarizing action on the arterial side in both animal types. In vitro, VSM of both small arteries and veins from WKY but not SHR were depolarized immediately by 10(-3) M ouabain. In contrast, reduction of the PSS suffusate temperature to 16 degrees C caused a significantly greater depolarization in VSM of SHR vessels.(ABSTRACT TRUNCATED AT 250 WORDS)
Phosphorylation of myosin in permeabilized mammalian cardiac and skeletal muscle cells
Authors: Sweeney H. L. , Stull J. T.
Published: December 2017

The effect of myosin phosphorylation on tension production at less than 50% maximal activation by Ca2+ was examined in rabbit psoas and ventricular muscle. For psoas fibers, tension was determined at pCa 6.0, 5.8, 5.6, 5.5, and 5.4. Myosin light chain kinase (0.15 microM) and calmodulin (2 microM) were added, and the fibers were incubated at pCa 5.4, which resulted in an increase in light chain phosphorylation (P-light chain) from 5-10 to 60-75%. After 5 min, the sequence of pCa activations was repeated. An identical protocol was followed for cardiac muscle, except the activation solutions were pCa 6.2, 6.0, 5.9, 5.8, and 5.6. Phosphorylation of P-light chain increased tension in both permeabilized cardiac and skeletal muscle fibers. The effect manifested itself as a leftward shift in the pCa-tension relationship at levels below 50% maximal activation, with a decrease in the slope of the pCa-tension relationship. These results indicate that P-light chain phosphorylation affects actin-myosin interactions in cardiac and skeletal muscles at submaximal levels of Ca2+ activation
Serotonin uptake by bovine pulmonary artery endothelial cells in culture. II. Stimulation by hypoxia
Authors: Lee S. L. , Fanburg B. L.
Published: December 2017

Exposure of bovine pulmonary artery endothelial cells to 3% O2 resulted in approximately twofold stimulation of serotonin (5-HT) uptake after 24-48 h when compared with cells exposed to 20% O2. The enhanced uptake was reversed after 48 h when cells were again placed in 20% O2. The stimulation was not observed after 0.5 or 2 h of exposure to hypoxia. The stimulation was present when iproniazid blocked conversion of 5-HT to 5-hydroxyindole-3-acetic acid, indicating that enhanced uptake did not occur through augmentation of monoamine oxidase activity. Stimulation of uptake by hypoxia occurred at low concentrations of 5-HT (up to 10(-6) M) but not at high 5-HT concentrations (greater than 10(-5) M) and was blocked by imipramine or absence of sodium from the medium, indicating that high-affinity transport and not diffusion of 5-HT was stimulated. Furthermore, exposure of cells to hypoxia did not produce morphological evidence of injury or change in protein content or trypan blue exclusion. The cell number of 3% O2-exposed cells was slightly reduced when compared with controls after 48 h. There was no change in cellular ATP or increase in lactate dehydrogenase in medium of cells exposed to 3% O2. Thus exposure of endothelial cells in culture to hypoxia stimulates the membrane activity of 5-HT accumulation with no evidence of injury to the cell.
Multiple neuropeptides exert a direct effect on the same isolated single smooth muscle cell
Authors: Lassignal N. L. , Singer J. J. , Walsh J. V.
Published: December 2017

The contractile effect of various neuropeptides was examined by pressure ejecting these agents from a pipette onto single smooth muscle cells freshly dissociated from the stomach of Bufo marinus. Substance P, cholecystokinin-octapeptide, and bombesin caused contraction, whereas vasoactive intestinal peptide, secretin, and dopamine inhibited acetylcholine-induced contractions. Acetylcholine and the three peptides which produced contraction were found in some instances to act on the same cell, suggesting that receptors for these agents exist on one and the same cell.
Structural studies of innervation on nonpregnant rat uterus
Authors: Garfield R. E.
Published: December 2017

Whole-mount preparations of the uterus and mesentery from nonpregnant rats were examined after staining with glyoxylic acid or acetylcholinesterase to demonstrate the innervation. Some uterine tissues were also evaluated by electron microscopy. Glyoxylic acid fluorescent nerves were present in the mesentery, mesometrium, and uterine wall exclusively around blood vessels. Acetylcholinesterase positive nerve fibers from Frankenhauser's plexus (cervical ganglia) were associated with blood vessels and muscle in the uterine wall but not in the mesentery. Electron microscopy revealed nerve varicosities with granular vesicles associated with blood vessels and varicosities with agranular vesicles located near blood vessels and muscle cells. Treatment of animals for 3 days with 5- and 6-hydroxydopamine, respectively, increased granular size and damaged the varicosities with granular vesicles but did not change nerves with agranular vesicles or induce the presence of gap junctions. The results of this study suggest that blood vessels in the uterus are highly innervated by both adrenergic and other types of nerves that probably control blood flow. Nonadrenergic but not adrenergic nerves may also directly control myometrial contractility.
Cytoplasmic pH regulation in phorbol ester-activated human neutrophils
Authors: Grinstein S. , Furuya W.
Published: December 2017

Activation of neutrophils by 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by an initial cytoplasmic acidification, followed by an alkalinizing phase due to Na+-H+ countertransport. The source of the acidification, which is fully expressed by activation with TPA in Na+-free or amiloride-containing media, was investigated. The acidification phase was detected also in degranulated and enucleated cytoplasts, ruling out a major contribution by the nucleus or secretory vesicles. Cytoplasmic acidification was found to be associated with an extracellular acidification, suggesting metabolic generation of H+. Two principal metabolic pathways are stimulated in activated neutrophils: the reduction of O2 by NADPH-oxidase and the hexose monophosphate shunt. A good correlation was found between the activity of these pathways and the changes in cytoplasmic pH. Inhibition of superoxide synthesis prevented the TPA-induced cytoplasmic acidification. Moreover, activation of the hexose monophosphate shunt with permeable NADPH-oxidizing agents (in the absence of TPA) also produced a cytoplasmic acidification. Cytoplasmic acidification was also elicited by exogenous diacylglycerol and by other beta-phorbol diesters, which are activators of the kinase, but not by unesterified phorbol or by alpha-phorbol diesters, which are biologically inactive. The results suggest that the cytoplasmic acidification induced by phorbol esters in neutrophils reflects accumulation of H+ liberated during the metabolic burst that follows activation.
Single potassium channels blocked by lidocaine and quinidine in isolated turtle colon epithelial cells
Authors: Richards N. W. , Dawson D. C.
Published: December 2017

The patch-clamp technique for recording single-channel currents across cell membranes was applied to single turtle colon epithelial cells isolated with hyaluronidase. With electrodes fabricated from Corning #7052 glass, high-resistance seals were consistently formed to these cells. In on-cell patches with low K (2.5 mM) in the pipette and high K (114.5 mM) in the bath, outward K currents were recorded that had a slope conductance of 17 pS and a reversal potential greater than -70 mV. Currents through this K channel were blocked by lidocaine, quinidine, and barium. These agents also block a cell swelling-induced K conductance identified by macroscopic current measurements in the basolateral membranes of the intact colonic epithelium, suggesting that the 17 pS K channel identified by single-channel recording in isolated turtle colon cells may be responsible for this macroscopically defined K conductance.
Calcium and initial surface binding phase of pinocytosis in Amoeba proteus
Authors: Prusch R. D.
Published: December 2017

The uptake of membrane-bound solute and external medium by bulk-phase pinocytosis in Amoeba proteus is influenced by the level of Ca2+ in the external medium. Increasing external Ca2+ to approximately 10(-4) M increases pinocytotic intensity, while increases in Ca2+ above this level decrease the intensity of pinocytosis. The initial interaction of pinocytotic inducers and Ca2+ at the surface of Amoeba proteus was therefore examined. Alcian blue and Na+, both inducers of pinocytosis, differ in the manner with which they associate with the amoeba surface, suggesting the possibility of different pinocytosis-inducing sites on the amoeba surface. Low levels of external Ca2+ in the range of 3 X 10(-5) to 1.5 X 10(-4) M increase the amount of cationic inducer associated with the cell surface while, at the same time, decreasing anion association with the cell surface. It is suggested that Ca2+ influences ion association with the cell surface by controlling the availability of negative surface sites, which in turn influences pinocytotic intensity.
Estimates of activation in arterial smooth muscle
Authors: Singer H. A. , Kamm K. E. , Murphy R. A.
Published: December 2017

We have previously described the onset of a "latch" state in the swine carotid media after K+ depolarization. This state was characterized by maintained stress after a decrease in shortening velocities and in the level of cross-bridge phosphorylation. The present experiments were designed to determine whether there were changes in other mechanical properties in swine carotid media associated with the onset of the latch state. Medial strips (less than 500 microM thick), incubated in physiological salt solution (PSS) at 37 degrees C at their optimal length (Lo), were subjected to ramp stretches (5.86 mm/s) of 5% Lo. The active stress (Sa) response to stretch was computed by subtraction of the passive element contribution (as determined from identical stretches after 30 min incubation in Ca2+-free PSS) from the total response in the activated muscle. Transitions in the total and active stress responses to stretch were observed in strips stimulated with 109 mM K+ for 1 min or longer and were interpreted as yielding of the contractile apparatus. Active dynamic stiffness (dS/dLo) calculated from the initial 1% Lo portion of the stretch response, correlated linearly with active stress over a wide range. Maximal stress and dynamic stiffness were reached by 1 min and were maintained for at least 30 min in K+-depolarized preparations. However, yield stress increased significantly between 1 and 10 min, and there was a large increase in the length at which yield was observed (1.09 +/- 0.06 to 1.86 +/- 0.10% Lo; n = 9). These increases were maintained between 10 and 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)
Chronic stimulation of mammalian muscle: changes in enzymes of six metabolic pathways
Authors: Henriksson J. , Chi M. M. , Hintz C. S. , Young D. A. , Kaiser K. K. , Salmons S. , Lowry O. H.
Published: December 2017

Twenty-one enzymes of different metabolic systems were measured in the rabbit fast-twitch tibialis anterior (TA) muscle after electrical stimulation (10 Hz, 24 h/day) for 1 day to 10 wk. Nine analytical methods are either new, (3-oxoacid CoA-transferase, branched-chain-amino-acid aminotransferase, carnitine acetyltransferase, thiolase), improved (glutamate dehydrogenase, glycogen synthase, adenylic acid deaminase), or specially adapted (hexokinase, phosphoglucomutase). The activities (based on protein) of 12 mitochondrial or partly mitochondrial enzymes were lower in control TA than in control (slow) soleus (30-84% of soleus level). After 2 wk, 11 of these had surpassed the control soleus level. Maximal increases (3- to 14-fold) occurred after 2-5 wk, and thereafter six of the enzymes declined, whereas the other five maintained or increased their levels. Five glycolytic and two high-energy phosphate transfer enzymes, originally much higher in control TA than in control soleus, decreased gradually to levels at 8-10 wk only 27-123% higher than in soleus. Noncollagen protein concentration dropped 46%, explained largely by a sixfold increase in extracellular (chloride) space and a modest increase in collagen. The data constitute strong evidence for coordinate regulation of (mainly cytosolic) enzymes of glycolysis, glycogenolysis, gluconeogenesis, and high-energy phosphate transfer. Changes in the (mainly mitochondrial) enzymes of oxidative metabolism were more divergent, partly because of a hitherto undescribed secondary phase in the metabolic response. This phase may reflect a lower energy consumption in muscles adapted to continuous activity.
Missing Cl conductance in cystic fibrosis
Authors: Quinton P. M.
Published: December 2017

The specific conductance of the epithelium of the reabsorptive duct of the human eccrine sweat gland was determined by cable analysis of microperfused segments of the duct taken from normal (N) and cystic fibrosis (CF) subjects. Conditions were designed to compare the total conductance and relative contributions of Cl, Na, and residual ion conductance components in the two populations. The total conductance (Gt) of the N ducts was 125 +/- 14 mS/cm2, which was sixfold higher than the Gt of CF ducts. Removal of Cl from the media decreased Gt of the N ducts to the same value as that of the CF ducts (15 +/- 2 mS/cm2). Addition of amiloride to the Cl-free perfusate resulted in a further decline in Gt of approximately 5 mS/cm2 in both tissues. However, the effect of amiloride on Gt of the N duct was more than an order of magnitude greater in Cl-containing than in Cl-free media, but its effect on Gt of the CF duct (with or without Cl) was the same as on that of the N duct in Cl-free media. We conclude that the Cl impermeability that is characteristic of the CF tissue is due to the almost complete absence of an electrodiffusive shunt for Cl across this epithelium, while the remaining conductance components are probably not altered.
Proteolysis in cultured cells during prolonged serum deprivation and replacement
Authors: Berger J. J. , Dice J. F.
Published: December 2017

Cells in culture show a series of changes in intracellular protein degradation in response to serum deprivation and replacement that are similar to alterations in degradation in tissues of starved and refed animals. Rates of intracellular protein degradation are increased in confluent cultures of IMR-90 human diploid fibroblasts when deprived of serum, but this enhanced proteolysis is transient. By 24-48 h, rates of protein degradation decline to values comparable to or below those for cells incubated in the presence of serum. Longer serum deprivation leads to further reductions in proteolysis. The reduced proteolysis after long-term deprivation cannot be explained by experimental artifacts or by gradual depletion of glucocorticoids or thyroid hormones from cells. Readdition of serum to deprived cells that are still in the enhanced phase of proteolysis restores degradation rates to values comparable to those in nondeprived cells. However, in cells deprived of serum for 24-48 h or longer, readdition of serum to the medium results in a marked reduction in proteolysis to rates below those observed in nondeprived cells. These responses of cultured cells to long-term serum deprivation and readdition may be of considerable physiological importance in that the proteolytic responses of tissues in starved and refed animals may be at least partially due to mechanisms operating at the cellular level.
Isolation, growth, and characterization of a gluconeogenic strain of renal cells
Authors: Gstraunthaler G. , Handler J. S.
Published: December 2017

LLC-PK1 cells, derived from pig kidney, retain several properties of the proximal tubule, but are incapable of gluconeogenesis, due to the lack of fructose-1,6-bisphosphatase (FBPase) [Am. J. Physiol. 248 (Cell Physiol. 17): C181-185, 1985]. Cells incapable of gluconeogenesis require a hexose, pentose, or nucleoside to provide ribose-5-phosphate for RNA biosynthesis. To induce or select cells that express FBPase activity, we cultured LLC-PK1 cells in glucose-free medium. We obtained cells (designated LLC-PK1-FBPase+) that express FBPase activity and are capable of growing in the complete absence of sugars or nucleosides. The cells have apical membrane enzyme activities that differ from those of wildtype cells. Tests of metabolic flow through the gluconeogenic pathway, using 3-mercaptopicolinic acid, a specific inhibitor of phosphoenolpyruvate carboxykinase, confirmed that the cells are gluconeogenic. LLC-PK1-FBPase+ cells grown in medium containing 5 mM glucose for five weekly passages continued to express FBPase activity and apical membrane enzyme activities characteristic of the FBPase+ strain. When switched back to glucose-free medium, they proliferated well. The strain appears to be stable. It should provide a model for studying the relationship between gluconeogenesis and other proximal tubule functions. An incidental finding is that in both strains, the activity of lactate dehydrogenase varied directly with the concentration of glucose in the growth medium, indicating that the expression of lactate dehydrogenase may be regulated by glucose or a metabolite of glucose.
Precursors of ribose 5-phosphate suppress expression of glucose-regulated proteins in LLC-PK1 cells
Authors: Gstraunthaler G. , Harris H. W. , Handler J. S.
Published: December 2017

Withdrawal of glucose from the medium bathing mammalian cells in culture results in cessation of growth and induces the synthesis of two stress proteins (Mr approximately 94-100 kDa and 78-80 kDa) that have been termed glucose-regulated proteins (GRPs). In LLC-PK1 cells, proteins of the same molecular weights, assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography, are fully induced by 24 h in glucose-free medium. The GRPs from LLC-PK1 cells cross-react with antibodies to GRPs of nonpolar cells, confirming their identification. Since glucose is not essential for energy production in cultured cells, but is essential for ribose 5-phosphate and nucleotide biosynthesis (Wice et al. J. Biol. Chem. 256: 7812-7819, 1981), we tested the effect of several precursors of ribose 5-phosphate on the induction of GRPs. The addition of 25 mM fructose or galactose to glucose-free medium suppressed the induction of GRPs. The pyrimidine ribonucleosides uridine and cytidine also suppressed GRP synthesis, ribose and the purine ribonucleosides guanosine and adenosine suppressed partially. The results, coupled with indirect evidence in the literature, lead to the suggestion that cell ribose 5-phosphate or a related metabolite regulates the expression of GRPs.
Endotoxin increases superoxide dismutase in cultured bovine pulmonary endothelial cells
Authors: Shiki Y. , Meyrick B. O. , Brigham K. L. , Burr I. M.
Published: December 2017

Manganous (Mn) and copper zinc (CuZn) superoxide dismutase (SOD) concentrations and glutathione peroxidase (GSH-Px) and catalase (CAT) activities were measured in cultured bovine pulmonary endothelial cells with and without exposure to Escherichia coli endotoxin (10(-1) micrograms/ml) over intervals of 0.5-24 h. The activities of two mitochondrial marker enzymes, fumarase and cytochrome-c oxidase, were also measured. Endotoxin exposure caused a marked increase (9-fold) in endothelial cell Mn SOD content without significant effects on GSH-Px, CAT, fumarase, or cytochrome-c oxidase activities. Endotoxin induced a slight decrease in CuZn SOD content over 24 h. This is the first report of a selective effect of endotoxin on Mn SOD in pulmonary endothelial cells. The response appears to be independent of an increase in mitochondrial activity (no change was observed in cytochrome-c oxidase or fumarase activities). These findings support the notion that endotoxin increases generation of toxic oxygen metabolites within pulmonary endothelial cells. An endotoxin-induced increase in Mn SOD could contribute to the reported protective effect of endotoxin against oxygen toxicity in these cells.
Na/Ca exchange in barnacle muscle cells has a stoichiometry of 3 Na+/1 Ca2+
Authors: Rasgado-Flores H. , Blaustein M. P.
Published: December 2017

The portions of the 45Ca influx and 22Na efflux that were activated by physiological concentrations of intracellular free Ca2+, [Ca2+]i, were studied in internally perfused single giant barnacle muscle cells. Since both fluxes were activated by intracellular Ca2+ (Cai) and the Ca influx was dependent on internal Na+ (Nai), the fluxes appear to be coupled (Na/Ca exchange). Tracer Ca/Ca and Na/Na exchanges were eliminated by employing tris(hydroxymethyl)aminomethane (Tris) as the predominant external cation. Under these circumstances, the ratio of the external Ca2+ (Cao)-dependent, Cai-activated Na+ efflux to the Nai-dependent, Cai-activated Ca influx was 3.1-3.2 Na+/1 Ca2+, when the intracellular Na+ concentration, [Na+]i was either 30 or 46 mM. This is the first direct measurement of the Na/Ca exchange stoichiometry. In many types of cells, the Na/Ca exchange system appears to operate in parallel with a plasma membrane ATP-driven Ca pump that has a lower capacity (maximum velocity), but higher affinity for Ca2+ than the Na/Ca exchanger. The data on the stoichiometry and activation by internal Ca2+ imply that the turnover of the Na/Ca exchanger is modulated during periods of cell activity. When the cells are depolarized, the Na/Ca exchange system is activated by the rising [Ca2+]i, and Ca2+ entry via the exchanger is promoted. Then, at repolarization, Ca2+ exits rapidly, primarily via the exchanger. However, in resting cells, with a low [Ca2+]i, much (but not all) of the Ca2+ efflux is probably mediated by the ATP-driven Ca pump.
How large is the drop in PO2 between cytosol and mitochondrion?
Authors: Clark A. , Clark P. A. , Connett R. J. , Gayeski T. E. , Honig C. R.
Published: December 2017

The subject of this brief review is the size of the local drops in PO2 around consuming mitochondria. We show that large drops (several Torr or more) are in conflict with the predictions of basic diffusion theory, when one uses accepted values for relevant parameters. In particular, oxygen diffusion coefficients must be reduced by at least a factor of 10 below measured values to reconcile Fick's law with large PO2 drops. Experimental evidence offered for large drops is often ambiguous because of system heterogeneities. In those cases where tractable models of heterogeneous systems can be developed, the experimental data are consistent with drops in PO2 on the order of a few hundredths of a Torr between cytosol and mitochondrion.
Human platelet diffusional water permeability measured by nuclear magnetic resonance
Authors: Wong K. R. , Verkman A. S.
Published: December 2017

To examine whether water transport in human platelets is mediated by an aqueous pore or channel, the ratio of osmotic to diffusional water permeability coefficients (Pf/Pd) was measured. Pd was measured from protein spin-lattice relaxation times in a dense platelet suspension (approximately 20% intracellular exchangeable water) using 20 mM solution Mn as a paramagnetic quencher. The decay of magnetization was biexponential with time constants of 1.3 and 6.9 ms (10 MHz, 37 degrees C), corresponding to a Pd of (2.9 +/- 0.2) X 10(-3) cm/s (SE; n = 8). Pd did not depend on concentrations of Mn (6-20 mM) or of platelets (2-4 X 10(10) platelets/ml), but increased to 4.5 X 10(-3) cm/s with addition of gramicidin (6 micrograms/10(10) platelets). 54Mn uptake studies showed less than 1% of equilibrium uptake of Mn into platelets in 30 min at 37 degrees C. The activation energies (Ea) for Pd were 4.5 kcal/mol (less than 28 degrees C) and 16.3 kcal/mol (greater than 28 degrees C). Pf was measured by a stopped-flow light scattering technique as reported previously [M. M. Meyer and A. S. Verkman, Human platelet osmotic water and nonelectrolyte transport, Am. J. Physiol. 251 (Cell Physiol. 20): C549-C557, 1986], in which the time course of platelet volume was measured in response to a 100 mM inwardly directed sucrose gradient. At 37 degrees C, Pf was (2.7 +/- 0.2) X 10(-3) cm/s and independent of [Mn]. The measured platelet Pf/Pd of 0.93 +/- 0.1 suggests that unlike water transport in erythrocytes, platelet water transport is not associated with an aqueous channel.