bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyclic GMP accumulation during cholinergic stimulation of eccrine sweat glands
Authors: Sato K. , Sato F.
Published: December 2017

The possibility that guanosine 3'5'-cyclic monophosphate (cGMP) may be an intracellular mediator of cholinergic stimulation [methacholine chloride (MCh)] was explored by comparing the relationship between the time course of cGMP accumulation and sweat secretion by use of isolated monkey palm eccrine sweat glands. Isolated sweat glands were incubated with MCh or other agents, and tissue levels of cGMP were determined by radioimmunoassay. In parallel experiments, sweat secretion was induced from cannulated single sweat glands in vitro. Stimulation with MCh produced a Ca-dependent transient elevation of cGMP level from 10 to 80 fmol/gland, peaking at 1-2 min but returning to the basal level by 5 min. The MCh-induced cGMP level was dose dependent and was inhibited by atropine. Ionophore A23187 (2 X 10(-4) M), however, caused persistent elevation of cGMP level for at least 20 min. Neither 10(-4) M MNNG, which elevated the cGMP level comparably with MCh stimulation, nor 8-bromo-cGMP (2 mM) induced sweat secretion. Thus although a parallelism between the cGMP level and sweating rate appears to hold for the initial stage of MCh-induced sweating, it does not hold for the steady state of sweat secretion. Data could not be interpreted to favor the notion that cGMP may be the intracellular mediator of cholinergic sweat secretion.
Characterization of potassium permeability of cochlear duct by perilymphatic perfusion of barium
Authors: Marcus D. C.
Published: December 2017

The relative transepithelial "permeabilities" of the cochlear duct to K, Na, and Cl were investigated so as to identify the K-selective tissues and to determine the cellular origin of this selectivity. Single-ion substitutions were made for K, Na, and Cl with the impermeant species N-methyl-D-glucamine (NMDG) for K and Na and gluconate or sulfate for Cl in perilymph. Transepithelial potential changes were relatively slow and small for Na and Cl substitutions. However, either K for Na or K for NMDG substitutions demonstrated a pronounced K selectivity (rapid changes of electrical potential) of only the sensory-cell tissue (organ of Corti). The response to the K for Na substitution was most clearly seen after electrogenic K transport was inhibited by ischemia while the sensory cells were metabolically sustained via perilymphatic perfusion. Under this condition, perfusion of a medium containing 154 mM K gluconate reduced the negative potential (typically -25 to -40 mV) to within a few millivolts of zero. In a control medium, perilymphatic barium (0.5-5 mM) produced qualitatively similar effects, suggesting that this K selectivity is localized primarily at the basolateral membrane of the sensory cells rather than at the junctional complexes.
Amiloride-sensitive Na+-H+ exchange in platelets and leukocytes: detection by electronic cell sizing
Authors: Grinstein S. , Goetz J. D. , Furuya W. , Rothstein A. , Gelfand E. W.
Published: December 2017

A new method was developed to detect the activity of the Na+-H+ exchange system as changes in cell volume. The cytoplasmic pH of isolated cells in suspension was lowered by incubation in Na-propionate medium, due to permeation of the protonated acid. This resulted in activation of Na+-H+ countertransport, measurable either as a Na+-dependent alkalinization or as an increase in 22Na+ uptake, both of which are amiloride sensitive. The continued operation of the antiport on prolonged exposure to Na-propionate results in a considerable increase in Na+ (and presumably propionate-) content. This is accompanied by an osmotic water shift and cell swelling, detectable by electronic sizing. This method was used to investigate the presence of the Na+-H+ exchanger in human platelets, neutrophils, lymphocytes, and monocytes as well as in cultured cell lines of B and T lymphoblasts and of macrophages. All these cell types displayed an amiloride-sensitive swelling when suspended in Na-propionate media. The results suggest the ubiquity of the Na+-H+ exchange system in the plasma membrane of nucleated mammalian blood cells.
Muscle metabolism and performance in perfused rat hindquarter during heavy exercise
Authors: Spriet L. L. , Matsos C. G. , Peters S. J. , Heigenhauser G. J. , Jones N. L.
Published: December 2017

An isolated perfused rat hindquarter model was used to examine muscle metabolism and performance during heavy muscular contraction. A one-pass system was used to perfuse the hindquarter for 30 min at rest and 20 min while electrically stimulated at 0.5 Hz with tetanic stimuli (100 Hz). The isometric tension generated by the gastrocnemius-plantaris-soleus muscle group was recorded continuously, and muscle biopsies were taken pre- and postperfusion. Peak tension was 2,648 +/- 55 g, decreasing to 69.3 and 51.6% of peak following 5 and 20 min of stimulation, respectively. Hindquarter O2 uptake increased from 0.56 mumol X min-1 X g perfused muscle-1 to 2.60 mumol X min-1 X g working muscle-1 after 3 min of stimulation and declined slightly thereafter. Lactate release by the hindquarter increased at the onset of stimulation, peaked at 2-4 min (23.4 +/- 1.4 mumol X min-1), and decreased to a constant amount in the final 10 min. Five minutes of stimulation produced large decreases in glycogen, creatine phosphate (CP), and ATP levels and increased lactate concentrations in all muscles sampled except the soleus. An additional 15 min of stimulation further decreased glycogen concentrations while lactate concentrations decreased in all muscles. Calculations based on the measured amounts of O2 and CP consumed and the amount of lactate produced revealed a heavy glycolytic contribution to energy production during the initial 5 min of stimulation (23%) and an aerobic domination during the final 15 min (90%). With this model heavy muscular contraction can be sustained to permit the measurement of uptake and release of substrates and products of the main energy-yielding pathways.
Electrical activity, cAMP concentration, and insulin release in mouse islets of Langerhans
Authors: Eddlestone G. T. , Oldham S. B. , Lipson L. G. , Premdas F. H. , Beigelman P. M.
Published: December 2017

The influence of forskolin and 3-iso-butyl-1-methylxanthine (IBMX) on mouse pancreatic beta-cell electrical activity, whole islet cAMP content, and insulin release were investigated. The two drugs potentiated to a similar extent both glucose-stimulated electrical activity and insulin release. In terms of the electrical response, both drugs potentiated the silent depolarization of the membrane in response to low (substimulatory) glucose concentrations, whereas at higher (stimulatory) glucose concentrations they caused an increase in the plateau fraction, with a response similar to the effect of increasing the glucose concentration. Both phases of insulin release were increased by each of the drugs. Ten micromolar forskolin and 100 microM IBMX caused an increase in intraislet adenosine 3',5'-cyclic monophosphate (cAMP) in the presence of 11.1 mM glucose, the former a 17-fold and the latter a 2-fold increase over the cAMP concentration in the presence of glucose alone. Because the two drugs lead to an increase in islet cAMP content, it is proposed that protein phosphorylation resulting from an activation of beta-cell cAMP-dependent protein kinases is responsible for the potentiation of the glucose-induced insulin release and beta-cell electrical activity. The observed effects on electrical activity are compatible with the hypothesis that cAMP-dependent phosphorylation induces alteration of the kinetics of the calcium-sensitive potassium permeability of the beta-cell plasma membrane. The increase in calcium entry into the beta-cell that would result from these alterations may be responsible for the cAMP-dependent potentiation of insulin release.
Magnesium transport in murine S49 lymphoma cells: pharmacology and divalent cation selectivity
Authors: Grubbs R. D. , Wetherill C. A. , Kutschke K. , Maguire M. E.
Published: December 2017

The murine S49 lymphoma cell transports Mg2+ by a system distinct from systems responsible for Ca2+ influx (J. Physiol. London 337: 351-371, 1983). We have now determined the ability of various cations, anions, and drugs to modulate Mg2+ influx. Neither sulfate, nitrate, phosphate, nor bicarbonate altered Mg2+ influx. Among cations only T1+, Ba2+, Zn2+, Mn2+, Sc3+, and La3+ potently inhibited Mg2+ influx without causing obvious cell toxicity. Seventeen other cations were ineffective at maximal nontoxic concentrations. T1+ inhibition (Ki = 300 micron) is noncompetitive and apparently derives from its ability to dissipate membrane potential. The noncompetitive nature of and the rather poor inhibition constants for Ca2+ (Ki approximately equal to 5 mM) and Mn2+ (Ki = 200 micron) indicate that neither cation is an effective physiological antagonist of Mg2+ influx. Only Ba2+ exhibited competitive inhibition of Mg2+ influx (Ki = 1 mM). Cisplatin and Ca2+ channel antagonists also did not inhibit Mg2+ influx. These data further differentiate Mg2+ transport systems from those for Ca2+. In addition, the selectivity series for group IIa cation inhibition of influx (Mg2+ greater than Ba2+ much greater than Ca2+ greater than or equal to Sr2+) has not been observed previously in biological systems and is indicative of a very high anionic field strength at the Mg2+ recognition site.
Specific binding sites for ovine prolactin in three amphibian cell lines
Authors: Dunand M. , Aubert M. L. , Kraehenbuhl J. P. , Rossier B. C.
Published: December 2017

Established cell lines (TB-6c and TB-M) obtained by continuous culture of epithelial cells from toad Bufo marinus urinary bladder, which, in culture, maintained a high degree of functional differentiation, exhibited a significant number of high-affinity (KA = 1-2 X 10(10) M-1) binding sites detected both with radioiodinated (125I) ovine prolactin (oPRL) and human growth hormone (hGH). Binding capacity was higher in the case of TB-6c cells (7,573 +/- 581 sites/cell) than with the TB-M cells (1,160 +/- 87). Similarly, binding sites for oPRL were characterized on Xenopus laevis kidney-derived cell line A6. With oPRL used both as tracer and standard, significant cross-reaction was observed with hGH, less with human or rat prolactin (PRL), and none with human chorionic somatomammotropin, bovine growth hormone, and rat luteinizing hormone or follicle-stimulating hormones. B. marinus pituitary extracts completely displaced the binding of 125I-oPRL to toad bladder binding sites. This finding of specific sites for PRL on amphibian bladder and kidney cells confirms that PRL exerts specific biological actions for the control of electrolyte and water metabolism in the amphibians.
Influence of pH change on redox state and CCK-induced secretion in isolated perfused rat pancreas
Authors: Asada N. , Kanno T.
Published: December 2017

The isolated perfused rat pancreas was used to examine the influence of the acid-base status of the perfusing solution on the redox states of cytochromes and secretory responses. Lowering the pH in a perfusing solution (pHe) from 7.3 to 6.8 induced simultaneous oxidation of cytochromes aa3, b, and c + c1, and further lowering to 6.0 induced larger oxidation of the cytochromes. Raising the pHe from 7.3 to 8.0 induced reduction of the cytochromes. Continuous stimulation with synthetic C-terminal octapeptide of cholecystokinin (CCK-8) at 100 pM induced the maximum secretory responses (pancreatic juice flow, protein output, and amylase output) during perfusion with the standard solution. The responses were not inhibited at pHe 6.8 but were inhibited significantly at pHe 6.0 or 8.0. The responses induced by stimulation with 20 pM CCK-8 were completely inhibited when pHe was lowered to 6.0 and CaCl2 was removed from the perfusing solution.
Serotonin, histamine, and norepinephrine mediation of endothelial and vascular smooth muscle cell movement
Authors: Bottaro D. , Shepro D. , Peterson S. , Hechtman H. B.
Published: December 2017

The effects of serotonin (5-HT), histamine (H), and norepinephrine (NE) on bovine aortic endothelial cell (BAE) and vascular smooth muscle cell (VSM) movement were quantitated using a phagokinetic tracking assay. BAE movement was significantly reduced by 5-HT (31%), H (48%), and NE (62%) at concentrations ranging from 10(-10) to 10(-4) M (P less than 0.01). VSM motility was significantly enhanced by 5-HT (17%) and H (25%) at concentrations of 10(-8) and 10(-4)M, respectively (P less than 0.01). NE (10(-4)M) reduced VSM motility by 63% (P less than 0.01). Ketanserin (1 microM) reduced the 5-HT-associated inhibition of BAE movement by 75% (P less than 0.01). Diphenhydramine (1 microM) reduced the H-associated inhibition of BAE movement by 66% (P less than 0.01). Propranolol (1 microM) reduced NE-associated inhibition of BAE movement by 50% (P less than 0.01) and 5-HT-associated inhibition by 45% (P less than 0.05), but phenoxybenzamine had no significant effect. The results suggest 1) BAE and VSM are affected qualitatively in opposite ways by 5-HT and H; 2) the H-associated inhibition of BAE movement appears to be at least partially mediated by H1 receptors; 3) the 5-HT-associated inhibition of BAE movement may be mediated directly by a 5-HT binding component but indirectly by beta-adrenergic receptors; and 4) the NE-associated inhibition of BAE movement appears to be mediated by beta-adrenergic receptors.
Role of Ca2+ in serum-stimulated Na+ influx in normal and transformed cells
Authors: Owen N. E. , Villereal M. L.
Published: December 2017

Previous studies in human foreskin fibroblasts suggested that the mechanism by which serum stimulates Na+ influx is via a Ca2+-calmodulin-mediated event. In the present experiments in normal WI-38 cells (human lung fibroblasts), both the intracellular Ca2+ antagonist 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) and the potent calmodulin antagonist trifluoperazine (TFP) blocked serum-stimulated Na+ influx [TMB-8 concentration causing half-maximal inhibition (Ki) = 15 microM and TFP Ki = 10 microM]. Similar results were obtained in Swiss 3T3 cells. In contrast, in transformed WI-38 or Swiss 3T3 cells neither TMB-8 nor TFP had any effect on serum-stimulated Na+ influx (TMB-8 Ki greater than 100 microM and TFP Ki greater than 100 microM). In addition, when 45Ca2+ efflux measurements were made on normal and transformed cells, serum stimulated significant 45Ca2+ efflux (P less than 0.05) from WI-38 and Swiss 3T3 cells, while having no effect on 45Ca2+ efflux from simian virus 40 (SV40)-WI-38 or SV40-Swiss 3T3 cells. However, an elevation of intracellular Ca2+ can stimulate Na+ influx, since it was found that A23187 mimicked the effects of serum in both normal and transformed cells. These results suggest that the Ca2+-calmodulin-mediated event, which is thought to be involved in serum-stimulated Na+ influx in normal cells, may be bypassed or overridden in transformed cells.
Lectin-gold cytochemistry reveals intercalated cell heterogeneity along rat kidney collecting ducts
Authors: Brown D. , Roth J. , Orci L.
Published: December 2017

The lectin-gold technique was used to detect Helix pomatia and Dolichos biflorus lectin binding sites directly on semithin and thin sections of rat kidney collecting ducts. Intercalated cell apical plasma membranes and the membranes of apical cytoplasmic vesicles were heavily labeled in the cortex and outer stripe of the outer medulla but were negative or very weakly labeled in the inner stripe and inner medulla. In contrast, clear cell apical membranes were labeled along the entire length of the collecting duct. Double labeling of semithin cryostat sections with a specific antibody and lectin-gold complexes was used to demonstrate that the intercalated cells in all regions studied contained carbonic anhydrase, even though the lectin binding differed. These results indicate that, in terms of their glycocalyx composition, intercalated cells represent a heterogeneous population in different regions of the collecting duct.
Phorbol ester-induced changes of cytoplasmic pH in neutrophils: role of exocytosis in Na+-H+ exchange
Authors: Grinstein S. , Elder B. , Furuya W.
Published: December 2017

Cytoplasmic pH homeostasis was studied in intact and granule-free porcine neutrophils following activation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). In intact cells, TPA activated at least two separate processes: a Na+-independent and amiloride-insensitive acidification, and a compensatory acid extrusion. The latter is Na+ dependent and blocked by amiloride and is likely to represent Na+-H+ exchange. Enucleated and degranulated neutrophils (cytoplasts) were prepared by sedimentation of cytochalasin B-treated neutrophils through a discontinuous density gradient. Cytoplasts responded to an artificially imposed acid load with activation of an amiloride-sensitive Na+-H+ exchange. TPA also activated both acid production and Na+-dependent acid extrusion in cytoplasts. The magnitude of the responses was comparable in intact neutrophils and in cytoplasts. These data suggest that 1) the nucleus and the secretory granules are not involved in the acidifying response to TPA, and 2) exocytosis of secretory vesicles is not required for activation of Na+-H+ exchange during acid loading or following treatment with the phorbol ester TPA.
Cultured rat vascular smooth muscle cells: extracellular calcium and Na+-K+ regulation
Authors: Kino M. , Tokushige A. , Tamura H. , Hopp L. , Searle B. M. , Khalil F. , Aviv A.
Published: December 2017

This study explores the relationship between extracellular calcium (Cao) and Na+-K+ regulation as it particularly pertains to the activity of the Na+ pump in cultured vascular smooth muscle cells (VSMCs) originating from Sprague-Dawley rats. As compared with cells incubated in media containing 0.5, 2.0, or 4.0 mM calcium, when the Na pump is active, VSMCs incubated in a Ca-deficient medium show a marked increase in intracellular sodium and no significant change in intracellular potassium. Associated with the rise in intracellular sodium there is an augmented activity of the Na pump. When the Na pump is inhibited, VSMCs incubated in either high-Ca medium (Cao = 4.0 mM) or Ca-deficient medium manifest a greater decline in intracellular potassium than cells incubated in media containing 0.5 or 2.0 mM calcium. Furthermore, when the Na pump is inhibited, VSMCs incubated in a Ca-deficient medium exhibit higher intracellular sodium levels in comparison with their counterparts incubated in media containing calcium. Flux experiments indicate that the aforementioned changes reflect increased membrane permeabilities to Na+ and K+. It is concluded that by regulating the permeability of the VSMC membrane, Cao plays an important role in the intracellular Na+-K+ homeostasis and that its effect on the Na pump is mediated via perturbations in the intracellular Na+ and K+ concentrations.
Regulation of glutamine production by skeletal muscle cells in culture
Authors: Durschlag R. P. , Smith R. J.
Published: December 2017

Cells of the L6 rat myogenic line consume glutamine in contrast to skeletal muscle in vivo, which produces glutamine. The basis of glutamine consumption in the cultured cells and the conditions under which net glutamine production occurs were investigated. After treating cultures with cytosine-1-beta-D-arabinofuranoside to eliminate replicating myoblasts remaining after differentiation, the L6 cells continued to consume glutamine from medium containing normal plasma levels (650 microM). Consumption declined when cells were incubated with 325 microM glutamine, and, at 0 microM, net production occurred. In addition to the tendency to consume glutamine, the cells rapidly consumed glutamic acid. When medium glutamic acid was increased from 70 to 150, 500, and 1,000 microM, glutamine production increased. Alterations in levels of NH3, the other substrate for glutamine synthesis, did not have this effect. The L6 cells were found to have glutamine synthetase activity similar to normal rat skeletal muscle but markedly increased glutaminase activity. In spite of the imbalance in synthetic and degradative activities, however, inhibitor studies with L-methionine sulfoximine and 6-diazo-5-oxo-L-norleucine showed that flux through both pathways influenced net glutamine production.
Cation and anion transport pathways in volume regulatory response of human lymphocytes to hyposmotic media
Authors: Sarkadi B. , Cheung R. , Mack E. , Grinstein S. , Gelfand E. W. , Rothstein A.
Published: December 2017

The regulatory volume decrease of osmotically swollen human peripheral blood lymphocytes can be inhibited by agents acting on volume-activated K+- or Cl--transport pathways. Quinine, cetiedil, and 3,3'-dipropylthiadicarbocyanine were found to block the volume-induced K+ transport by interaction with sites on the outside face of the membrane, perhaps by competition with external K+. Drugs known to influence calmodulin action inhibit both volume-induced K+ and Cl- transport to varying degrees. Those inhibitors, particularly of K+ transport, are correlated with their calmodulin-antagonist activity. Penetrating sulfhydryl (SH) reagents (in contrast to nonpenetrating ones) are potent inhibitors of both volume-induced K+ and Cl- movements, indicating the presence of functionally important SH groups located within the membrane or at the cytoplasmic face. A number of agents, such as dipyridamole and oligomycin C, are specific inhibitors of the volume-activated anion pathway. In all respects studied, the inhibition characteristics of the volume-activated K+ pathway of lymphocytes resemble those of the Ca2+-activated K+ channel of red cells. In contrast, the volume-induced anion permeability differs from the primary anion-transport pathway of red cells.
Dihydropyridine Ca2+ antagonists: potent inhibitors of secretion from normal and transformed pituitary cells
Authors: Enyeart J. J. , Aizawa T. , Hinkle P. M.
Published: December 2017

Three dihydropyridine (DHP) Ca2+ antagonists were compared with several other organic Ca2+ antagonists with respect to their ability to inhibit depolarization-dependent hormone secretion from the GH4C1 pituitary cell line and from normal rat pituitary cells. The three DHP, nimodipine, nisoldipine, and nifedipine, potently and specifically inhibited KCl-stimulated prolactin secretion from GH4C1 cells (estimated IC50 values: 1.8, 1.8, and 6.0 nM, respectively). Both basal and thyrotropin-releasing hormone-stimulated secretion from GH4C1 cells were much less sensitive to inhibition by the DHP. The inhibition by the DHP was reversible, and their potency was independent of depolarizing concentrations of KCl between 18.8 and 53.8 mM. Other organic antagonists, including verapamil, cinnarizine, and diltiazem, blocked secretion from GH4C1 cells but at much higher concentrations. The estimated IC50 values for these three were 1,000, 1,100, and 3,500 nM, respectively. Depolarization-stimulated prolactin secretion from normal pituitaries was inhibited by the DHP and verapamil at the same concentrations found effective in GH4C1 cells. KCl-stimulated 45Ca2+ uptake by GH4C1 cells was also blocked by DHP at concentrations that inhibited secretion. Since depolarization-stimulated secretion and 45Ca2+ uptake are probably triggered by Ca2+ entering through voltage-sensitive channels, the above results suggest that DHP antagonists potently block these channels in both normal and transformed pituitary cells. These Ca2+ channels appear to be identical in this respect. These findings further suggest a similarity between the Ca2+ channels of endocrine cells and those of smooth muscle and other excitable cells.
Subcontracture depolarizations increase sarcoplasmic ionized calcium in frog skeletal muscle
Authors: Snowdowne K. W.
Published: December 2017

The hypothesis that the sarcoplasmic concentration of ionized calcium (Cai) of frog skeletal muscle may control its rate of metabolism was examined by monitoring the changes in Cai due to perturbations that are known, from the work of previous investigators, to alter the rate of metabolism. Cai was measured with aequorin, which was microinjected into isolated fibers in sufficient quantity to detect basal Cai. When these fibers were exposed to 5-18 mM KCl, 75 mM RbCl2, or 100 mM CsCl2, there was an increase in the aequorin signal. The potassium-evoked increase in the aequorin signal was diminished by an increase in the extracellular concentration of Ca or by Ca-free media containing 3.6 mM Mg, Mn, Sr, or Zn but was enhanced by subcontracture concentrations of caffeine or media containing nitrate instead of chloride. These results are consistent with the hypothesis that Cai may control the rate of metabolism in frog skeletal muscle fibers.
Intracellular pH in human fibroblasts: effect of mitogens, A23187, and phospholipase activation
Authors: Muldoon L. L. , Dinerstein R. J. , Villereal M. L.
Published: December 2017

Serum-deprived human fibroblasts (HSWP cells) were loaded with either the fluorescent pH indicator 6-carboxy-4',5'-dimethylfluorescein or the calcium indicator quin 2, and the fluorescence of the intracellular probes was continuously monitored with a microspectrofluorometer. Addition of a cocktail of peptide growth factors causes intracellular alkalinization, which is blocked by amiloride or by replacement of extracellular Na+ with choline, confirming that mitogenic stimulation activates a Na+-H+ exchanger in HSWP cells. The exchanger is also activated by A23187, acid loading the cells, and stimulation of phospholipase activity with melittin. Growth factors and melittin activation of the exchanger have been demonstrated to be dependent on the mobilization of Ca2+ from intracellular stores. The intracellular alkalization and increase in Ca2+ activity due to both melittin and growth factors is inhibited by phospholipase inhibitors, indicating that phospholipase activity is necessary for the activation of the Na+-H+ exchanger and the mobilization of intracellular Ca2+.
A Na+-Ca2+ exchange process in isolated sarcolemmal membranes of mesenteric arteries from WKY and SHR rats
Authors: Matlib M. A. , Schwartz A. , Yamori Y.
Published: December 2017

The existence of a Na+-Ca2+ exchange process in cell membrane vesicles isolated from mesenteric arteries of Wistar-Kyoto normotensive (WKY) and spontaneously hypertensive (SHR) rats was investigated. Membranes from cleaned mesenteric arteries were isolated by sucrose density gradient centrifugation, which yielded three distinct membrane fractions. The lighter membrane fraction of both WKY and SHR rats was enriched in 5'-nucleotidase activity, a marker for cell membrane, by about 10-fold, based on the activity in the homogenate, and was higher in membranes of SHR compared with WKY rats. Ouabain-sensitive Na+-K+-ATPase activity, another marker for cell membrane, was also concentrated in the lighter membrane fraction and was lower in the membranes of SHR compared with WKY rats. Higher activities of 5'-nucleotidase and Na+-K+-ATPase of both WKY and SHR rats was taken as evidence that the lighter membrane fraction was enriched in plasma membrane. Electron microscopic examination indicated that the membranes were in vesicular form. When the vesicles were loaded with Na+, a time-dependent uptake of Ca2+ was observed if the assay was carried out in high potassium to create a Na+ concentration gradient across the membrane of the vesicles. Very little Ca2+ uptake was observed when the vesicles were loaded with K+ or when the uptake of Ca2+ was carried out under conditions in which the Na+ gradient across the vesicle membranes was reduced. Ca2+ uptake in Na+-loaded vesicles of SHR rats was only slightly increased compared with WKY rats. The data indicate that a Na+-Ca2+ exchange process exists in the cell membrane of rat mesenteric arteries.
Myosin phosphorylation and contraction of feline esophageal smooth muscle
Authors: Weisbrodt N. W. , Murphy R. A.
Published: December 2017

We tested the hypothesis that phosphorylation of the 20,000-Da light chain of myosin (LC 20) is related to mechanical activation of esophageal smooth muscle. Circular muscle layer strips of cat esophagus were taken from the lower esophageal sphincter (LES) and the distal esophageal body (EB). The LES strips developed tone spontaneously, and the EB strips were tonically contracted with carbachol. Both tissues relaxed in response to electrical-field stimulation. Phosphorylation of the LC 20 was determined in tissues quick-frozen during relaxation and during stress redevelopment after cessation of field stimulation. Stress and phosphorylation levels were low after 30 s of field stimulation, and a rapid contraction followed field stimulation. Phosphorylation in the LES increased from 0.043 +/- 0.029 to 0.328 +/- 0.043 mol Pi/mol LC 20 within 10 s after stimulation of the inhibitory nerves was terminated, while stress was still rising rapidly. Phosphorylation in the LES then declined to a steady-state value of 0.162 +/- 0.034 mol Pi/mol LC 20 after 10 min. Isotonic shortening velocities at a constant afterload following a quick release showed changes with time that were proportional to the level of phosphorylation. This was also true for values of maximal shortening velocity estimated for zero external load and for the rate of stress redevelopment after a step shortening. Comparable measurements were made in the carbachol-contracted EB. These results indicate that visceral smooth muscles, which normally function tonically (LES) or phasically (EB), exhibit an initial rapid mechanical activation associated with myosin phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)
Regulation of polypeptide-hormone biosynthesis at the level of the genome
Authors: Habener J. F.
Published: December 2017

Understanding of gene control mechanisms has greatly accelerated largely due to the application of recombinant DNA techniques. Polypeptide hormone genes encode multiexonic-intronic transcriptional sequences, the exons of which in turn encode polyprotein precursors or prohormones from which the hormones are cleaved during posttranslational processing of the prohormones. Transcriptional processes are regulated by at least two qualitatively different modes of gene regulation. The first mode includes the factors and structural components of a gene that determine whether a gene can or cannot be expressed in a given tissue when the appropriate inducer is present. The second mode is the physiological induction and regulation of the gene that can normally be expressed in a particular tissue. Both cis and trans regulatory mechanisms appear to operate in both tissue-specific expression and physiological regulation. Tissue-specific enhancer sequences consisting of short nucleotide sequences of from 10 to 50 base pairs have been identified in or around genes that are expressed in specific tissues. In many instances trans-acting DNA binding proteins have been found to repress or activate the transcription of the genes. Physiological regulation of hormone genes involves at least two different classes of macromolecules, steroid hormone receptors and phosphoproteins that are formed in response to the binding of ligands to specific surface-located receptors. Although the precise mechanisms by which information encoded in cellular effectors is coupled to cellular responses is incomplete, continued investigations should lead to a more complete understanding of gene control mechanisms and the eventual ability to alter the expression of specific genes.
Single-channel recordings from amiloride-sensitive epithelial sodium channel
Authors: Hamilton K. L. , Eaton D. C.
Published: December 2017

We report here the first evidence in intact epithelial cells of unit conductance events from an amiloride-sensitive Na+ channel. The events were observed when patch-clamp recordings were made from the apical surface of cultured epithelial kidney cells (A6). The channel characteristics are as follows. Single-channel conductance ranged between 7 and 10 pS (mean = 8.4 +/- 1.3), the current-voltage (I-V) relationship displayed little if any nonlinearity over a range of +/- 80 mV (with respect to the patch pipette), and the channel Na+/K+ selectivity was approximately 3-4:1. Amiloride, a cationic blocker of the channel, reduced channel mean open time and increased channel mean closed time as the voltage of the cell interior was made more negative. Amiloride induced channel flickering at increased negative potentials (intracellular potential with respect to the patch) but did not alter the single-channel conductance or the I-V relationship from that observed in control patches.
Passive sodium and potassium movements in sickle erythrocytes
Authors: Berkowitz L. R. , Orringer E. P.
Published: December 2017

Deoxygenation causes an increase in passive Na and K movements across the membrane of the sickle erythrocyte. Some investigators find that these ion movements are accompanied by cell dehydration, while others find no evidence for cell water loss with sickling. Because gelation of hemoglobin S would be enhanced by cell water loss, we reinvestigated Na and K movements in sickle cells to define further the role that ion movements might play in the pathogenesis of sickling. With deoxygenation, we found that sickle cells gained Na and lost K without losing cell water. These net ion movements were not seen in control red blood cells. For sickle cells, deoxygenation also increased passive unidirectional influxes of Na and K, effects not observed when control red blood cells were deoxygenated. The deoxygenation-induced passive influxes of Na and K in sickle cells were not diminished by anion substitution or by the addition of the diuretic furosemide. We also found differences in passive Na and K fluxes between oxygenated sickle cells and normal red blood cells. The addition of furosemide or replacement of Cl with NO3 or SCN, maneuvers that largely reduced passive Na and K movements in oxygenated normal cells, had no effect on Na and K movements in oxygenated sickle cells. These findings militate against the idea that solute and water loss occur as a consequence of deoxygenation but do indicate that there are acquired membrane abnormalities in sickle red blood cells.
Dissociation of effects of insulin and contraction on glucose transport in rat epitrochlearis muscle
Authors: Nesher R. , Karl I. E. , Kipnis D. M.
Published: December 2017

The effects of insulin and contraction on glucose transport and metabolism were investigated in rat epitrochlearis muscles in vitro. Insulin dose-response curves showed a threshold (approximately 50 microunits/ml) and saturation-type (approximately 1 mU/ml) kinetics, whereas isometric contraction activated glucose transport and metabolism in a linear fashion with no evidence of a threshold. Insulin and contraction increased the apparent maximal rate of uptake of the hexose transport system with minimal effect on its apparent Km. The stimulatory effects of insulin and contraction were additive; similar results were obtained with 2-deoxy-D-glucose. Contraction stimulated glucose transport in three different preparations of muscles depleted of insulin: 1) exhaustively washed for 2 h, 2) rats infused with anti-insulin serum, and 3) chronically (streptozotocin-induced) diabetic rats. Prostaglandin E2 augmented the effect of a submaximal concentration of insulin on glucose transport without exerting any effect by itself but had no effect on contraction-augmented glucose transport. It is concluded that insulin and contraction activate glucose transport and metabolism via independent mechanisms.
Passive calcium-buffering capacity of a rabbit ventricular homogenate preparation
Authors: Pierce G. N. , Philipson K. D. , Langer G. A.
Published: December 2017

This study characterizes the passive Ca2+-binding capacity of a crude homogenate fraction prepared from rabbit ventricular tissue. The soluble and particulate fractions of the homogenate were separated by centrifugation, and the Ca2+-binding capacity of each fraction was measured separately with Ca2+-selective electrodes and then summed to obtain the Ca2+-binding capacity of the total tissue homogenate. Qualitatively, the Ca2+ bound to the homogenate exhibited both relatively high- (Km less than 1 microM) and low-affinity (Km greater than 10 microM) sites over the physiological range of free Ca2+ concentrations. The total homogenate bound 72, 128, and 308 mumol Ca2+/kg wet wt tissue at 1, 10, and 50 microM free Ca2+ concentrations, respectively. The Ca2+ bound to the homogenate was mostly La3+ displaceable, which suggests that it represents a largely noncompartmentalized, rapidly exchangeable fraction of Ca2+. Myofibrillar ATPase activity was examined as a function of free Ca2+ concentration in an ionic medium similar to that used in the Ca2+-binding experiments. At free Ca2+ concentrations that stimulated myofibrillar ATPase activity half maximally, the measured rise in Ca2+ bound to the homogenate above resting values was greater than 100 mumol/kg wet wt. These data provide base-line values for the passive Ca2+-buffering capacity of the myocardium, although relevance to the physiological setting must be interpreted with caution.
ATP depletion and loss of cell integrity in anoxic hepatocytes and silica-treated P388D1 macrophages
Authors: Kane A. B. , Petrovich D. R. , Stern R. O. , Farber J. L.
Published: December 2017

The relationship between ATP depletion and the loss of cell integrity was examined in the killing of hepatocytes by anoxia and P388D1 macrophages by silica. ATP depletion is a feature of the reaction to either hazard. Treatment of hepatocytes, however, with antimycin, oligomycin, sodium azide, or N,N'-dicyclohexylcarbodiimide produced a rate and extent of ATP depletion comparable with anoxia without significant loss of viability. Treatment of P388D1 cells with 2-deoxyglucose plus antimycin, oligomycin, or sodium azide reproduced the loss of ATP accompanying silica particle intoxication. Again, there was no loss of viability. These data dissociate the loss of cellular ATP from the genesis of lethal injury in both cell types. ATP depletion was, however, associated with a loss of lysosomal integrity. With the metabolic inhibitors, loss of lysosomal integrity occurred in the absence of irreversible cell injury over the time course that anoxia and silica intoxication significantly damaged the cells. This implies that neither hazard produces lethal damage through mechanisms dependent on intracellular lysosomal enzyme release. While ATP depletion can cause lysosomal rupture in P388D1 macrophages, phagocytosis of silica particles in the absence of extracellular Ca2+ ions is associated with release of lysosomal contents without depletion of ATP or loss of cell integrity. Silica particles are concluded to interact directly with both the plasma and lysosomal membranes. The former leads to Ca2+ influx with resultant cell death and ATP depletion. The latter leads to release of lysosomal contents that is not followed by irreversible cell injury.
Passive K+-Cl- fluxes in low-K+ sheep erythrocytes: modulation by A23187 and bivalent cations
Authors: Lauf P. K.
Published: December 2017

A fraction of the ouabain-resistant (OR) K+ flux of low-K+ (LK) sheep erythrocytes is Cl- dependent (K+-Cl- transport) and is activated reversibly by cell swelling or irreversibly by treatment with N-ethylmaleimide (NEM). The effect of the ionophore A23187 plus bivalent cations (Me2+) or ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) was studied on K+-Cl- transport in control or NEM-treated LK cells. The following observations were made. 1) A23187 (6 microM), at a hematocrit of 10% (vol/vol) and in the presence of 1 mM EGTA, activated severalfold OR K+-Cl- transport in shrunken or swollen cells but failed to stimulate further K+-Cl- flux in NEM-treated cells. 2) In the absence of EGTA, but at very low external Ca2+ concentrations [( Ca2+]o = 10(-7) M), A23187 stimulated OR K+-Cl- flux in controls less than with EGTA and inhibited it slightly in NEM-treated cells. 3) When [Ca2+]o was raised to 10(-3) M, an almost complete inhibition of OR K+-Cl- fluxes occurred in shrunken, swollen, or NEM-treated cells. 4) Other Me2+ inhibited OR K+-Cl- flux in the presence of A23187 in the following order of decreasing potency: Mn2+ much greater than Ca2+ greater than Mg2+ greater than Sr2+ much much greater than Ba2+. 5) Stimulation of OR K+-Cl- flux by A23187 +/- EGTA and inhibition by A23187 + Ca2+ were reversible and did not alter significantly cellular ATP. 6) The stimulatory effect of A23187 plus EGTA, perhaps by Me2+ removal, on K+-Cl- flux and its inhibition by Ca2+ were reversibly abolished in metabolically depleted cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Control of enzyme activities in individual myotubes cultured without nerve
Authors: Nemeth P. M. , Solanki L. , Lawrence J. C.
Published: December 2017

The activities of lactate dehydrogenase, malate dehydrogenase, phosphorylase, and adenylate kinase were measured in single myotubes dissected from primary cultures of rat skeletal muscle. For a given enzyme, activities among the spontaneously contracting cells varied as much as eightfold. When the myotubes were paralyzed with tetrodotoxin, the variability in enzyme levels was markedly decreased. These and other findings suggest that differences in enzyme levels among individual myotubes may arise as a result of differences in their pattern of contractile activity.
Basolateral membrane chloride transport in isolated epithelia of frog skin
Authors: Stoddard J. S. , Jakobsson E. , Helman S. I.
Published: December 2017

Isotopic methodology was used to characterize Cl- transport in isolated epithelia of frog skin (northern Rana pipiens) bathed in Cl--rich Ringer solution and short-circuited. Cl- content of epithelia measured when loaded to 36Cl specific activity equilibrium averaged 139.6 neq/mg dry wt. The kinetics of 36Cl efflux was biexponential and consistent with binding or compartmentalization of approximately 30% of tissue Cl- within the intracellular pool. Because efflux of 36Cl to the apical solution was immeasurable, it was concluded that apical membranes were virtually impermeable to Cl- and that basolateral membranes were highly permeable to Cl- with a mean unidirectional Cl- efflux of 21.7 microA/cm2. Both furosemide (1 mM) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (5 X 10(-4) M) inhibited markedly the basolateral membrane chloride fluxes within seconds, as measured in chamber experiments. As inhibition of Cl- flux occurred in the absence of a change of the electrical parameters of apical and basolateral membranes, the mechanisms of Cl- transport appeared to be electroneutral and, for the most part at least, not coupled to the fluxes of Na+ and K+. Transepithelial Cl- fluxes averaged near 1 microA/cm2, proceeding via transport routes in parallel to the cells of the stratified epithelium. No correlation existed between the "shunt" resistance measured in the presence of 100 microM amiloride (greater than 1,000 omega X cm2) and the partial conductance to Cl-.
A radioimmunoblotting method for measuring myosin light chain phosphorylation levels in smooth muscle
Authors: Hathaway D. R. , Haeberle J. R.
Published: December 2017

A method for measuring the molar stoichiometry of myosin light chain phosphorylation in intact smooth muscle has been developed. Antiserum to the 20,000-Da light chains of bovine aortic smooth muscle was harvested from rabbits and used to label light chains by a radioimmunoblotting procedure. In the initial characterization it was found that the 20,000-Da light chains could be transferred by electroblotting from polyacrylamide gels to nitrocellulose paper with an efficiency of approximately 80% over a protein range of 0.1-5.0 micrograms. At a dilution of 1:500, the unpurified light chain antiserum required approximately 10-12 h at 22 degrees C to reach equilibrium binding to the transferred light chains. Moreover, equilibrium labeling of the light chain-antibody complex with 125I-protein A required 4-6 h of incubation at 22 degrees C. By using these conditions, a radioimmunoassay for the 20,000-Da light chains was developed that was linear over a protein range of 0.1-5.0 micrograms (5-250 pmol). As little as 20 ng of light chains could be measured if a second antibody procedure (goat anti-rabbit immunoglobulin G Fab fragments) was used. Phosphorylated and unphosphorylated myosin light chains were separated by glycerol-urea polyacrylamide gel electrophoresis. This procedure, combined with radioimmunoblot, gave similar estimates of phosphorylation levels when compared with direct assay for phosphate or scanning of Coomassie blue-stained gels. Moreover, when applied to intact uterine smooth muscle, the glycerol-urea gel radioimmunoblot gave values of myosin light chain phosphorylation for relaxed and contracted muscles that were not statistically different from those obtained with a two-dimensional electrophoretic method.(ABSTRACT TRUNCATED AT 250 WORDS)
Phorbol ester stimulation of active anion secretion in intestine
Authors: Chang E. B. , Wang N. S. , Rao M. C.
Published: December 2017

Phorbol 12,13-dibutyrate (PDB) stimulates electrogenic anion secretion in rabbit and chicken distal ileum. The 50% effective dose in each case was 15 nM, and the maximal short-circuit current (Isc) responses seen at 10(-6) M were 20 and 140 microA/cm2, respectively. Isc was also stimulated by the diacylglycerol 1-oleoyl-2-acetyl glycerol (10(-5) M) but not by the inactive phorbol ester analogue 4 alpha-phorbol 12,13-didecanoate. Removal of Ca from the serosal bathing medium and pretreatment of tissues with atropine (10(-6) M) or tetrodotoxin (2 X 10(-7) M) did not affect PDB-stimulated responses. PDB also did not alter basal intracellular free Ca levels in isolated chicken enterocytes loaded with quin 2 or basal adenosine 3',5'-cyclic monophosphate levels in intact tissues from rabbit and chicken. In the presence of PDB, Ca- and phospholipid-dependent phosphorylation of several proteins in particulate and soluble fractions from isolated chicken enterocytes were seen. Pretreatment of chicken and rabbit tissues with indomethacin or mepacrine inhibited the Isc responses to PDB and carbamylcholine, suggesting a role for prostaglandins. These results suggest that electrogenic anion secretion stimulated by phorbol esters and diacylglycerol may be mediated by activation of protein kinase C.