bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetic analysis of the downregulation of epidermal growth factor receptors in rats in vivo
Authors: Yanai S. , Sugiyama Y. , Iga T. , Fuwa T. , Hanano M.
Published: December 2017

We previously clarified the specific binding sites for epidermal growth factor (EGF) in several organs in rats based on in vivo kinetic analysis (D. C. Kim, Y. Sugiyama, H. Sato, T. Fuwa, T. Iga, and M. Hanano. J. Pharm. Sci. 77: 200-207, 1988). In the present study, we have determined the extent of the receptor downregulation and the recovery rate of the available receptors for EGF in several organs in vivo. At the specified times (30 min-24 h) after intravenous administration of excess unlabeled EGF (300 micrograms/kg), the early-phase (less than 3 min) uptake clearances (k1) of the tracer amount of 125I-EGF, which are proportional to the cell-surface available receptor densities, were determined in the liver, kidney, duodenum, jejunum, ileum, stomach, and spleen. As the result, the k1 value in each organ at 30 min after intravenous administration of unlabeled EGF was lowered close to the receptor-independent clearance value, indicating that the cell-surface receptors were almost completely downregulated, and thereafter, the k1 value showed gradual recovery to the control level. Furthermore, the recovery half-lives showed interorgan differences, namely the half-life (20 min) in the liver was much shorter than those (2-4.5 h) in other organs. These results were considered to reflect the processes of the recycling of internalized EGF receptors to the cell-surface or recruitment of new receptors. It was concluded that the recovery rate of the downregulated receptors in the liver, which is most responsible for the plasma clearance of EGF, is much faster than those in other organs.
Kinetic mechanism of ATP action in Na(+)-K(+)-Cl- cotransport of HeLa cells determined by Rb+ influx studies
Authors: Ikehara T. , Yamaguchi H. , Hosokawa K. , Miyamoto H.
Published: December 2017

The kinetics of Na(+)-K(+)-Cl- cotransport were studied by measuring ouabain-insensitive furosemide-sensitive Rb+ influx (JRb) into HeLa cells while varying the cellular ATP and the extracellular Rb+ and Na+ concentrations. Results reveal that ATP stimulates JRb by increasing the affinity of the cotransporter for Rb+ (K+), and the apparent Michaelis constant (Km) for ATP was 0.95 +/- 0.03 mmol/l cell water. Two ATP molecules may relate to the uptake of one Rb+ by the cotransport pathway, as examined by the nonlinear least-squares method for goodness-of-fit and a Hill plot, JRb was strengthened by an increase in the inward chemical gradient associated with cell swelling on preincubation in a low-Na+ high-K+ medium, accompanying an increase in the affinity of the transporter for ATP. JRb was apparently activated by extracellular Na+, and the activation was enhanced by an increase in the cellular ATP concentration. Lactate production stimulated by 2 microM carbonylcyanide m-chlorophenyl hydrazone (CCCP) was reduced by 10 microM ouabain but not altered by further addition of 0.1 mM furosemide. Increases in cellular adenosine 3',5'-cyclic monophosphate (cAMP) caused by treatment with 0.1 mM isoproterenol plus 0.5 mM 3-isobutyl-1-methylxanthine or with 0.1 mM dibutyryl cAMP did not influence JRb. From this and previous studies, we propose a general and a specific model of Na(+)-K(+)-Cl- cotransport, which elucidate the order of binding of extracellular ions and reaction of cellular ATP.
Calcium entry in rat parotid acini: activation by carbachol and aluminum fluoride
Authors: Mertz L. M. , Horn V. J. , Baum B. J. , Ambudkar I. S.
Published: December 2017

Entry of extracellular Ca2+ into the cytosol of exocrine cells plays an important role in the process of fluid transport, especially during periods of prolonged secretion. However, in parotid acinar cells, the process of Ca2+ entry and the identity of factors which regulate it remain obscure. In this report, we demonstrate that AlF-4, like carbachol, activates Ca2+ entry into dispersed rat parotid acini. In physiological Ca2(+)-containing (1.28 mM) medium, both agents elicit three phases of cytosolic Ca2+ change, an initial transient increase (intracellular Ca2+ dependent) followed sequentially by a decrease (intra- and extracellular Ca2+ dependent) and a small sustained increase (extracellular Ca2+ dependent). Cytosolic Ca2+ concentration ([Ca2+]i) during the last two phases is influenced by variations in extracellular [Ca2+]. Elevation of extracellular [Ca2+], at any time after the initial transient increase, results in a rise of cytosolic [Ca2+], thus demonstrating the existence of a Ca2+ entry pathway during the two later phases. These data suggest the likelihood that in parotid acini, G protein activation is involved in stimulating this Ca2+ entry pathway. Because in AlF-4-treated acini entry into the cytosol is detectable only after the initial intracellular Ca2+ release phase, we suggest that this Ca2+ entry process does not accompany initial intracellular Ca2+ mobilization. Furthermore, the sustained cytosolic [Ca2+] elevation which can be observed 15-30 min after initial stimulation of acini is likely determined by this Ca2+ entry process which, in physiological conditions, could support sustained fluid secretion.
Voltage-dependent gating of gap junction channels in embryonic chick ventricular cell pairs
Authors: Veenstra R. D.
Published: December 2017

The dependence of macroscopic gap junctional conductance (Gj) on transjunctional voltage (Vj) was studied in paired myocytes after enzymatic dissociation of 7-day-old embryonic chick ventricles. The membrane voltage of both cells was independently controlled by separate patch-clamp circuits in the whole cell configuration. Two distinctive unitary junctional conductances were identified in recordings from seven different cell pairs. The larger channel had a mean conductance of 166 +/- 37 pS (n = 6 pairs), whereas a second channel averaged 58 +/- 10 pS (n = 3). Instantaneous Gj remained linear over a Vj range of -100 to +100 mV, whereas the steady-state Gj declined when voltages exceeded +/- 30 mV. Both decay and recovery phases of Gj follow exponential time courses, with the recovery time constant being four times slower than inactivation, requiring 1.1 s at 80 mV. The normalized steady-state Gj-Vj curve could be defined by a two-state Boltzmann distribution, assuming an effective gating charge of 1.72, a half-inactivation voltage of 45 mV, and a residual voltage-insensitive Gj of 27% of maximum. Single-channel recordings revealed closure of 160-pS channels on a Vj step to 80 mV, and the ensemble average of five such records produced an exponentially decaying junctional current with a time constant of 184 ms. The single-channel current-voltage relationship remains linear with a slope of 145 pS over the entire Vj range. The results support the hypothesis that a population of 160-pS gap junction channels is gated by transjunctional potentials.
Effect of norepinephrine on Na(+)-K+ pump and Na+ influx in sheep cardiac Purkinje fibers
Authors: Chae S. W. , Wang D. Y. , Gong Q. Y. , Lee C. O.
Published: December 2017

Effects of norepinephrine and Ca+ on Na(+)-K+ pump and pacemaker current were investigated by simultaneous measurement of intracellular Na+ activity (aiNa) and membrane potential in driven (1 Hz) and quiescent sheep cardiac Purkinje fibers. Concurrently, twitch force was measured in driven fibers, in which norepinephrine (NE) produced a decrease in aiNa, a prolongation in action potential duration, and a hyperpolarization in diastolic membrane potential, Vdm. In contrast, in quiescent fibers, NE produced an increase in aiNa and a depolarization in resting membrane potential, Vm. The decrease in aiNa, prolongation in action potential duration, and hyperpolarization in Vdm produced by NE were blocked by 5 x 10(-6) M strophanthidin, presumably through inhibition of the Na(+)-K+ pump. The increase in aiNa and membrane depolarization caused by NE were abolished by high [K+]o or Cs+, presumably through inhibition of the pacemaker current, if. These results indicate that in driven fibers NE stimulates predominantly the Na(+)-K+ pump, producing a decrease in aiNa and that in quiescent fibers it increases predominantly if, producing an increase in aiNa. The effect of NE on driven and quiescent fibers differs because of the voltage dependence of if and perhaps the Na(+)-K+ pump. Consequently, the relative magnitude of the two opposing effects of NE on aiNa appears to be dependent on membrane potential. In quiescent fibers, Cs+ monotonically decreased aiNa to a steady-state value, while Cs+ hyperpolarized membrane potential and then slowly depolarized to a steady-state level, producing a transient hyperpolarization. In driven fibers, Cs+ decreased aiNa, shortened action potential duration, and depolarized Vdm. Cs+ decreased aiNa more in quiescent fibers than in driven fibers. The decrease in aiNa and hyperpolarization in membrane potential produced by Cs+ in quiescent fibers were abolished by depolarization induced by high K+ extracellular concentration (25.4 mM) but were not abolished or reduced by 5 x 10(-6) M strophanthidin. These results suggest that the decrease in aiNa and hyperpolarization in membrane potential by Cs+ are caused by blockage of if but not by stimulation of the Na(+)-K+ pump and that if is an important source of Na+ loading into cells.
Regulation of 5'-aminolevulinate synthase activity in overloaded skeletal muscle
Authors: Essig D. A. , Kennedy J. M. , McNabney L. A.
Published: December 2017

The regulation of the mitochondrial enzyme 5'-aminolevulinate synthase (ALV synthase) activity during chronic weight-bearing activity (overload) in chicken skeletal muscle was investigated. Maximal enzyme activity was increased 2.5- and 4.0-fold after 3 and 7 days of overload. The content of ALV synthase mRNA (ng/mg total RNA) was not changed after 3 days but increased (20%; P less than 0.05) after 7 days of overload. Normalizing the content of ALV synthase mRNA relative to the increase in total RNA indicated that ALV synthase mRNA increased by 1.6- and 2.0-fold at 3 and 7 days, respectively. On this basis, the increase in enzyme activity per gram protein exceeded the increase in mRNA content per gram protein by 60-70%. During overload, the activity of cytochrome oxidase was unchanged after 3 days but increased by 1.5-fold (P less than 0.05) after 7 days of overload. The data indicate that 1) the initial rise in ALV synthase mRNA and activity due to overload occurs in the absence of a prior change in the level of cytochrome oxidase, an enzyme that requires heme for its assembly, and 2) induction of ALV synthase activity is regulated largely by processes at the translational or posttranslational steps.
Selective turnover of sarcolemmal phospholipids with lethal cardiac myocyte injury
Authors: Miyazaki Y. , Gross R. W. , Sobel B. E. , Saffitz J. E.
Published: December 2017

To delineate the biochemical mechanisms responsible for the transition from reversible to irreversible ischemic injury, we used quantitative electron microscopic autoradiography. Specific alterations of phospholipid catabolism in individual subcellular organelles of cardiac myocytes associated with simulated ischemic injury were identified. Neonatal rat cardiac myocytes were incubated with 5 nM [3H]arachidonic acid to label loci of phospholipid turnover and were exposed to 30 microM iodoacetate to produce reversible and irreversible injury. Although only minute amounts of arachidonic acid were incorporated into sarcolemmal phospholipids under control conditions, 20- and 96-fold increases were observed under conditions leading to reversible and irreversible cell injury, respectively. Increases of 5- and 28-fold in the specific radioactivity of sarcolemmal phospholipids in reversibly and irreversibly injured cells occurred in the absence of significant alterations in the specific radioactivity of other subcellular compartments, demonstrating that accelerated phospholipid catabolism was confined essentially to the sarcolemma. Selective catabolism of sarcolemmal phospholipids, known to be highly enriched in arachidonic acid, is likely to augment local accumulation of arachidonic acid, identified recently as a second messenger regulating myocardial K+ channels. Because the biochemical integrity of the sarcolemma is critical to both electrophysiological function and viability of myocytes, the observed selective alterations of sarcolemmal phospholipid metabolism appear to be pivotal determinants of lethal myocardial injury.
Nifedipine inhibits calcium-activated K transport in human erythrocytes
Authors: Kaji D. M.
Published: December 2017

The effect of nifedipine on K transport across human erythrocytes was investigated. Nifedipine had no effect on K influx mediated by the Na-K pump, Na-K-2Cl cotransport, or the passive residual K flux. However, it inhibited the K and water loss from ATP-depleted cells in the presence of external Ca (Cao). Similar inhibition of Ca-activated K [K(Ca)] efflux was observed in fresh cells exposed to Cao and A23187 or ionomycin. The inhibition was observed even when nifedipine was added after initiation of the K(Ca) efflux and was not readily reversed by washing cells with drug-free media. When K(Ca) efflux was plotted as a function of external free Ca, nifedipine reduced the maximum K(Ca) efflux but had no effect on the Ca concentration required for half-maximum K(Ca) efflux. The inhibition of K(Ca) efflux by nifedipine was not consequent to its effect on conductive Cl permeability, because valinomycin-induced K efflux in Cl media was enhanced rather than reduced by nifedipine and because the inhibition was also seen with SCN, a nonlimiting anion. Nifedipine inhibited the K(Ca) efflux with a dissociation constant (Kd) of 4 microM. The inhibitory capacity of nifedipine was reduced by increasing external K. Nifedipine reduced not only the basic conductance but also the zero-current K conductance with a Kd of 23 microM. Other Ca-channel blockers, such as verapamil and diltiazem, did not inhibit K(Ca) efflux, but other dihydropyridines, including BAY K 8644, a Ca-channel agonist, were effective in inhibiting K(Ca) efflux.(ABSTRACT TRUNCATED AT 250 WORDS)
Development and biological applications of chloride-sensitive fluorescent indicators
Authors: Verkman A. S.
Published: December 2017

Chloride movement across cell plasma and internal membranes, is of central importance for regulation of cell volume and pH, vectorial salt movement in epithelia, and, probably, intracellular traffic. Quinolinium-based chloride-sensitive fluorescent indicators provide a new approach to study chloride transport mechanisms and regulation that is complementary to 36Cl tracer methods, intracellular microelectrodes, and patch clamp. Indicator fluorescence is quenched by chloride by a collisional mechanism with Stern-Volmer constants of up to 220 M-1. Fluorescence is quenched selectively by chloride in physiological systems and responds to changes in chloride concentration in under 1 ms. The indicators are nontoxic and can be loaded into living cells for continuous measurement of intracellular chloride concentration by single-cell fluorescence microscopy. In this review, the structure-activity relationships for chloride-sensitive fluorescent indicators are described. Methodology for measurement of chloride transport in isolated vesicle and liposome systems and in intact cells is evaluated critically by use of examples from epithelial cell physiology. Future directions for synthesis of tailored chloride-sensitive indicators and new applications of indicators for studies of transport regulation and intracellular ion gradients are proposed.
Effects of ouabain on background and voltage-dependent currents in canine colonic myocytes
Authors: Burke E. P. , Sanders K. M.
Published: December 2017

Previous studies have suggested that the membrane potential gradient across the circular muscle layer of the canine proximal colon is due to a gradient in the contribution of the Na(+)-K(+)-ATPase. Cells at the submucosal border generate approximately 35 mV of pump potential, whereas at the myenteric border the pump contributes very little to resting potential. Results from experiments in intact muscles in which the pump is blocked are somewhat difficult to interpret because of possible effects of pump inhibitors on membrane conductances. Therefore, we studied isolated colonic myocytes to test the effects of ouabain on passive membrane properties and voltage-dependent currents. Ouabain (10(-5) M) depolarized cells and decreased input resistance from 0.487 +/- 0.060 to 0.292 +/- 0.040 G omega. The decrease in resistance was attributed to an increase in K+ conductance. Studies were also performed to measure the ouabain-dependent current. At 37 degrees C, in cells dialyzed with 19 mM intracellular Na+ concentration [( Na+]i), ouabain caused an inward current averaging 71.06 +/- 7.49 pA, which was attributed to blockade of pump current. At 24 degrees C or in cells dialyzed with low [Na+]i (11 mM), ouabain caused little change in holding current. With the input resistance of colonic cells, pump current appears capable of generating at least 35 mV. Thus an electrogenic Na+ pump could contribute significantly to membrane potential.
Fluorescent stilbene (BADS) binding proteins in anion-transporting epithelia
Authors: Pearce S. F. , Zadunaisky J. A.
Published: December 2017

Chloride transport occurs at the interface between the internal and external environments of a cell where chloride uptake or efflux is regulated through a variety of mechanisms that involve cotransport of cations, exchange mechanism with anions, or movement through channels. One of these mechanisms, a chloride-bicarbonate exchange found in the human red blood cell, is well characterized and is mediated by a protein commonly known as band 3. To ascertain the presence of this or other mechanisms in epithelia, the sensitivity of epithelial membranes toward stilbenes was examined. Structure function activities of stilbene derivatives with red cell ghosts show that stilbene molecules block anion transport sites. One of these stilbenes, 4-benzamido-4'-aminostilbene-2-2'-disulfonic acid (BADS), chosen for its property of enhanced fluorescence on binding to hydrophobic sites, was used as a probe to examine the presence or absence of similar sites on epithelial membranes. With the use of nonlinear curve fitting, a single class of sites was found for BADS in the rat kidney cortex (1.6 microM), rat kidney medulla (2.1 microM), rat small intestine (2.2 microM), rat pancreatic islets (5.8 microM), frog cornea (4.3 microM), and shark rectal gland (1.5 microM). In the presence of chloride, the affinity for BADS decreased in all tissues except the frog corneal epithelium where it remained unchanged. The binding of BADS could be displaced by loop diuretics (furosemide, bumetanide, and piretanide) and thiocyanate anion in the kidney, intestine, and shark rectal gland; 50% displacement occurred at approximately 40 microM concentrations for furosemide with an order of magnitude less for bumetanide. The near-millimolar concentrations required for the displacement of BADS by loop diuretics indicate that this effect is nonspecific. However, the effect of chloride, thiocyanate, and loop diuretics on the binding of BADS indicates that BADS possibly interacts with an anion site.
Relationship between proliferation and glucose metabolism in primary cultures of rabbit proximal tubules
Authors: Tang M. J. , Tannen R. L.
Published: December 2017

Primary cultures of rabbit proximal tubules, which revert to a glycolytic profile as reflected by increased activity of pyruvate kinase (PK) paralleled by increased glucose consumption and lactate formation, were utilized to explore the relationship between glycolytic metabolism and proliferation. Tubules placed in serum-free, hormonally defined Dulbecco's modified Eagle's medium with 5 mM glucose exhibited logarithmic growth beginning on day 3 in culture. The increase in PK activity lagged approximately 1 day behind, suggesting that the reversion to glycolysis is a consequence of rather than a prerequisite for cellular proliferation. Tubules cultured in 0.5 mM as contrasted with 25 mM glucose exhibited heightened proliferation reflected by an increase in protein content and cell number on day 5 in culture. The heightened proliferation was accompanied by increased PK activity. On day 9, after confluency had been achieved, no differences in protein content or PK activity were detected between tubules cultured in different glucose concentrations. These findings indicate that a low glucose concentration is mitogenic for renal proximal tubules and that the proliferative process in some fashion up-regulates the activity of the glycolytic enzyme PK. Furthermore, because accelerated growth proceeds in the presence of glucose restriction, the energy from glycolysis is not required for the proliferative process.
Alterations in the functional properties of skinned fibers from denervated rabbit skeletal muscle
Authors: Trachez M. M. , Sudo R. T. , Suarez-Kurtz G.
Published: December 2017

Isometric tension was recorded in vitro from chemically skinned fibers obtained from normal and 14-day-denervated extensor digitorum longus muscles of the rabbit. Denervation potentiated the tensions elicited by pCa 6.0 but did not modify the pCa value (5.6) required for maximum tension. Ca2+ transport across the membranes of the sarcoplasmic reticulum (SR) was markedly affected by denervation. Thus the rate of ATP-dependent net Ca2+ uptake increased significantly, and the spontaneous release ("leakage") of the Ca2+ stored in the SR was significantly reduced in denervated fibers. These effects lead to increased accumulation of Ca2+ in the lumen of the SR. The dose-response curve for the halothane-induced contractures of Ca2(+)-loaded skinned fibers was displaced to the left after denervation. Thus 0.7 mM halothane, a concentration that elicited no tension in 10 control fibers, induced contractures in the 10 denervated fibers tested. The potentiation of the halothane-induced tensions is attributed mainly to the larger stores of Ca2+ in the SR of denervated fibers. The possibility that denervation may also affect the interaction of halothane with the SR membranes is discussed.
Extracellular magnesium-dependent sodium efflux in squid giant axons
Authors: Gonzalez-Serratos H. , Rasgado-Flores H.
Published: December 2017

Experiments were designed to determine whether the putative Na(+)-Mg2+ exchanger previously demonstrated to mediate Mg2+ efflux (R. DiPolo and L. Beague. Biochim. Biophys. Acta 946: 424-428, 1988) could also mediate the efflux of Na+ (presumably a Na+ efflux-Mg2+ influx exchange) in squid giant axons. The effects of external Mg2+ (Mg(o)) on 22Na efflux were measured in internally dialyzed, ATP-fueled axons in which the contribution to Na+ efflux by other pathways was inhibited. To facilitate measurement of Mg(o)-dependent Na+ efflux, the intracellular concentration of Na+ was increased. To prevent Na(+)-Na+ exchange, external Na+ was replaced by tris(hydroxymethyl)aminomethane. To assess the effect of Mg(o) on Na+ efflux without altering the total divalent cation concentrations, Mg(o) was replaced mole-for-mole by external Ba2+ (Ba(o)). This manipulation produced reversible reductions in Na+ efflux. These reductions were neither due to membrane hyperpolarization nor to a direct effect of Bao but were due instead to the reduction in Mg(o). The Mg(o)-dependent Na+ efflux was inhibited by external amiloride but was spared by bumetanide. In the absence of external Na+, the Mgo-dependent Na+ efflux increased as a function of external Mg2+ with Michaelis-Menten kinetics. These results indicate that the Na(+)-Mg2+ exchange can mediate the efflux of Na+ (operate in Na+ efflux-Mg2+ influx mode of exchange).
Loss of bradykinin receptors and TPA-stimulated Na+ influx in SV40-transformed WI-38 cells
Authors: Etscheid B. G. , Jamieson G. A. , Toscas K. , Villereal M. L.
Published: December 2017

Mitogenic stimulation of Na(+)-H+ exchange activity, as defined by the level of 5-(N,N-hexamethylene)amiloride (HMA)-sensitive Na+ influx, was compared in WI-38 and SV40 virus-transformed WI-38 fibroblasts. Serum or bradykinin dramatically stimulated HMA-sensitive Na+ influx in WI-38 cells, whereas in SV40-transformed WI-38 cells, serum, but not bradykinin, produced a large increase in HMA-sensitive Na+ influx. This lack of a bradykinin response was traced to a dramatic reduction in the number of bradykinin receptors, from 470 fmol/mg protein in WI-38 cells to 29 fmol/mg protein in the SV40-transformed WI-38 cells. Transformation of WI-38 cells with SV40 virus also altered the mechanism by which HMA-sensitive Na+ influx is stimulated. In WI-38 cells, 12-O-tetradecanoylphorbol 13-acetate (TPA) dramatically stimulated HMA-sensitive Na+ influx. In SV40-transformed WI-38 cells, TPA alone had no effect on HMA-sensitive influx and inhibited serum-stimulated HMA-sensitive Na+ influx. Down-regulation of protein kinase C activity decreased serum- and TPA-stimulated HMA-sensitive Na+ influx in the WI-38 cells and relieved the TPA inhibition of serum-stimulated HMA-sensitive Na+ influx in the SV40-transformed WI-38 cells.
Time-varying magnetic fields increase cytosolic free Ca2+ in HL-60 cells
Authors: Carson J. J. , Prato F. S. , Drost D. J. , Diesbourg L. D. , Dixon S. J.
Published: December 2017

Electromagnetic fields have been reported to cause a variety of biological effects. It has been hypothesized that many of these phenomena are mediated by a primary effect on the concentration of cytosolic free calcium ([Ca2+]i). We investigated the effects of exposure to electromagnetic fields on [Ca2+]i in HL-60 cells using the Ca2(+)-sensitive fluorescent indicator indo-1. Indo-1-loaded cell samples were exposed to a radiofrequency electromagnetic field, a static magnetic field, and a time-varying magnetic field, which were generated by a magnetic resonance imaging (MRI) unit. We found that a 23-min exposure to all three fields, in combination, induced a significant increase in [Ca2+]i of 31 +/- 8 (SE) nM (P less than 0.01, n = 13) from a basal level of 121 +/- 8 nM. Also, cells exposed to only the time-varying magnetic field had a mean [Ca2+]i that was 34 +/- 10 nM (P less than 0.01, n = 11) higher than parallel control samples. Separate exposure to the radio-frequency (6.25 MHz) or static field (0.15 T) had no detectable effects. These results demonstrate that time-varying magnetic fields alter [Ca2+]i and suggest that at least some of the reported biological effects of time-varying magnetic fields may arise from elevation of [Ca2+]i.
Effect of oxygen tension on catecholamine-induced formation of cAMP and on swelling of carp red blood cells
Authors: Salama A. , Nikinmaa M.
Published: December 2017

We studied the effects of beta-adrenergic stimulation on the formation of adenosine 3',5'-cyclic monophosphate (cAMP) and on the cell volume in carp red blood cells in normoxia (PO2 = 150 mmHg) and hypoxia (PO2 = 8 mmHg). Accumulation of cAMP was the prerequisite for adrenergic cell swelling. Cell swelling was induced by beta-agonists, forskolin, and 8-bromo-cAMP. The amount of cAMP required for adrenergic swelling was minimal; swelling was observed at cAMP concentrations greater than 100 nM. Maximal increase in cell volume was observed at 200 nM cAMP. These values were independent of both the oxygen tension and the beta-agonist used. Norepinephrine caused the largest accumulation of cAMP, followed by isoproterenol and epinephrine. At hypoxic conditions, the cAMP concentrations obtained after stimulation with the natural catecholamines norepinephrine and epinephrine were greater than at normoxic conditions. The catecholamines caused appreciable cell swelling at lower concentrations in hypoxia than in normoxia. Thus the number of beta-adrenergic receptors, and their inherent ligand affinities, may be higher in hypoxic than in normoxic carp red blood cells. Oxygen tension had a pronounced effect on the magnitude of the adrenergic swelling. The maximal increase in cell volume was 5-7% in hypoxia, whereas in normoxia it was only approximately 2%. This was not due to differences in cAMP formation, but possibly to a greater activity of the Na(+)-H+ exchanger in hypoxic than in normoxic conditions.
Alteration in the myosin phosphorylation pattern of smooth muscle by phorbol ester
Authors: Seto M. , Sasaki Y. , Sasaki Y.
Published: December 2017

Pretreatment with a high concentration of phorbol 12-myristate 13-acetate (PMA, 100 nM) increased the degree of tension and extent of myosin light chain (MLC20) phosphorylation in the K(+)-stimulated rabbit aortic artery. Pretreatment with 100 nM PMA did not alter the relationship between MLC20 phosphorylation and the tension seen with K+ stimulation in the initial phase and steady state of contraction. However, a low concentration of PMA (10 nM) potentiated only the MLC20 phosphorylation during the steady state of contraction with no effect on the tension. In contrast, the prostaglandin (PG) F2 alpha-induced tension development and the MLC20 phosphorylation were not affected by PMA pretreatment at both low and high concentrations. The inhibitory action of nifedipine on the K(+)-induced contraction was not affected by pretreatment with 100 nM PMA; the concentration producing half-maximal inhibition of nifedipine for the K(+)-induced contraction (33 nM) was the same as that of the K+ plus 100 nM PMA-induced contraction (32 nM). Our results suggest that PMA may increase the level of myosin light chain kinase-dependent MLC20 phosphorylation and the tension in the K(+)-stimulated artery, an effect which differs from that seen with increases in K+ concentrations. The regulatory mechanism for the contraction involving PGF2 alpha stimulation may differ from that seen in the case of K+ stimulation.
Ca sensitivity and acetylcholine receptor currents of twitch and tonic snake muscle fibers
Authors: Ruff R. L. , Spiegel P.
Published: December 2017

Myofibrillar Ca sensitivity and single-channel acetylcholine receptor (AChR) currents were studied in garter snake (Thamnophis sirtalis sirtalis) costocutaneous muscle fibers. Nomarski optics were used to identify tonic and fast- and slow-twitch fibers. Measurements of tension generation were made using chemically skinned fibers. The maximum tensions of the three types of fibers were similar, and the fast- and slow-twitch fibers had similar Ca sensitivities. Compared with twitch fibers, tonic fibers had lower threshold Ca concentrations for tension generation and a larger range of Ca concentrations between threshold and maximum tension. The AChR channels were studied by enzymatically removing the nerve terminals and performing patch-clamp recordings on the exposed postsynaptic membrane. Twitch fibers had only one class of end-plate channel with a conductance of approximately 51 pS. Tonic fibers had two types of synaptic channels. One AChR channel in the tonic fibers resembled the type seen in twitch fibers. The other channel in tonic fibers had a smaller conductance of approximately 33 pS and resembled extrajunctional AChRs on denervated twitch fibers.
Regulation of cytosolic pH of cultured mesangial cells by prostaglandin F2 alpha and thromboxane A2
Authors: Mene P. , Dubyak G. R. , Scarpa A. , Dunn M. J.
Published: December 2017

Prostaglandins (PG) and thromboxane A2 (TxA2) have marked vasoactive effects on the renal glomerular microcirculation. Exposure of cultured mesangial cells to PGF2 alpha and TxA2 mimetics results in a rapid elevation of free cytosolic Ca2+ ([Ca2+]i) followed by contraction and cell proliferation. We studied whether other ionic changes mediate these effects of eicosanoids on cells of rat and human origin. Cytoplasmic pH (pHi) was monitored in cells loaded with the fluorescent, intracellularly trapped pH-sensitive probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. PGF2 alpha in rat cells and the TxA2 mimetic U-46619 in human cells induced rapid, dose-dependent cytosolic acidification followed by recovery and net alkalinization mediated by enhanced Na(+)-H+ exchange. The early acidification was also stimulated by ionomycin and Ca2(+)-mobilizing peptides, implicating a Ca2(+)-dependent mechanism. Alkalinization was abolished by removal of extracellular Na+ and by amiloride. Both components of the responses were inhibited by phorbol myristate acetate, which could mimic alkalinization, suggesting a regulatory role of protein kinase C in activation of the Na(+)-H+ exchanger by eicosanoids. Vasoconstrictor arachidonate metabolites may control glomerular cell function by a signaling mechanism centered on concurrent changes of pHi and [Ca2+]i.
Regulation of calcium-activated potassium efflux by neurotensin and other agents in HT-29 cells
Authors: Wu H. , Franklin C. C. , Kim H. D. , Turner J. T.
Published: December 2017

Neurotensin receptors have been shown previously to be coupled to polyphosphoinositide turnover and intracellular Ca2+ ([Ca2+]i) mobilization in HT-29 colonic epithelial cells (Bozou et al. Biochem. J. 264: 871, 1989; Turner et al. J. Pharmacol. Exp. Ther. 253: 1049, 1990). In this study, neurotensin was found to enhance dramatically the Ba2(+)- and tetraethylammonium chloride-sensitive K(+)-efflux rate (measured with 86Rb+) in the presence of ouabain and bumetanide, with basal efflux increasing 4.5 +/- 0.5-fold with 10 nM neurotensin. The K(+)-efflux rate, which was partially dependent on the extracellular Ca2+ concentration, was also increased by carbachol and ATP, two other [Ca2+]i-mobilizing agonists in HT-29 cells, as well as by the Ca2+ ionophores ionomycin and A23187, suggesting that the efflux was through Ca2(+)-activated K+ channels. Pretreatment of cells with neurotensin, carbachol, or ATP desensitized subsequent neurotensin-stimulated efflux by 82, 57, and 63%, respectively, confirming our previous results which indicated homologous and heterologous desensitization of the neurotensin receptor-signal transduction pathway. Pretreatment of cells with the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and mezerein did not affect [Ca2+]i mobilization or K+ efflux directly but desensitized neurotensin-stimulated efflux by greater than 80%. Pretreatment (2 h) with PMA also decreased K+ efflux in response to ionomycin by 59%, although ionomycin-induced [Ca2+]i mobilization was not inhibited. Downregulation of protein kinase C by overnight pretreatment with PMA resulted in recovery of ionomycin-stimulated efflux. These results suggest that agonist-stimulated Ca2(+)-activated K+ channels in HT-29 cells are regulated at multiple steps in the signal transduction pathway.
Ryanodine receptor in different malignant hyperthermia-susceptible porcine muscles
Authors: Ervasti J. M. , Strand M. A. , Hanson T. P. , Mickelson J. R. , Louis C. F.
Published: December 2017

The sarcoplasmic reticulum (SR) ryanodine receptor was studied in SR vesicles isolated from the vastus intermedius skeletal muscle and cardiac muscle of malignant hyperthermia-susceptible (MHS) and normal pigs. MHS and normal heavy SR preparations isolated from the vastus intermedius muscle had similar yields, polyacrylamide gel electrophoretic patterns, Ca2(+)-ATPase activities, mitochondrial enzyme activities, calsequestrin contents, and maximal [3H]ryanodine-binding activities. However, while half-maximal calcium concentrations (Ca0.5) for stimulation of MHS and normal vastus intermedius SR [3H]ryanodine binding were not significantly different, the Ca0.5 for inhibition of [3H]ryanodine binding to MHS vastus intermedius SR (76 +/- 17 microM) was significantly greater than to normal SR (16 +/- 9 microM). MHS vastus intermedius SR also exhibited a significantly lower Kd value (62 +/- 15 nM) for [3H]ryanodine binding compared with normal SR (Kd = 284 +/- 102 nM). These values for MHS and normal vastus intermedius SR are similar to those reported using SR isolated from a muscle composed of predominantly fast-twitch fibers, indicating the similarity of the ryanodine receptor in fast- and slow-twitch skeletal muscles. In contrast, there were no differences in the properties of the ryanodine receptor of porcine cardiac SR isolated from MHS and normal pigs. We therefore conclude that there is a defect in the SR ryanodine receptor of both slow- and fast-twitch skeletal muscle fiber types but not in cardiac muscle of MHS individuals.
Prolonged incubation of skeletal muscle increases system A amino acid transport
Authors: Gulve E. A. , Cartee G. D. , Youn J. H. , Holloszy J. O.
Published: December 2017

During the course of experiments involving prolonged incubation of skeletal muscle, we observed large increases in system A amino acid transport activity. System A activity was monitored with the nonmetabolizable amino acid analogue alpha-(methylamino)isobutyrate (MeAIB). When rat epitrochlearis muscles are incubated in Krebs-Henseleit buffer supplemented with 0.1% bovine serum albumin and 8 mM glucose, basal MeAIB transport doubles after 5 h and is elevated approximately sevenfold after 9 h compared with rates measured in muscles incubated for 1 h. Insulin-stimulated transport also doubles after 5 h and increases by fourfold after 9 h. The increases in basal and insulin-stimulated system A transport over time can be prevented by incubating muscles in the presence of cycloheximide. Addition of minimum essential medium essential amino acids (EAA) to the incubation medium blocks the increase in basal and insulin-stimulated MeAIB transport measured after 9 h by 85-90 and 60%, respectively. A single amino acid, glutamine, can account for half of the inhibitory effect of EAA on the time-dependent increase in basal system A transport. Amino acid metabolism is not necessary for inhibition of the rise in basal MeAIB transport. At concentrations normally present in minimum essential medium, nonessential amino acids are less effective (51% inhibition) in preventing the rise in basal transport occurring over 9 h. At three times normal concentrations, however, the ability of nonessential amino acids to prevent the time-dependent increases in basal and insulin-stimulated MeAIB transport is comparable to that of EAA. These changes in MeAIB transport with prolonged incubation are not due to muscle deterioration.(ABSTRACT TRUNCATED AT 250 WORDS)
Functional biology of intestinal goblet cells
Authors: Specian R. D. , Oliver M. G.
Published: December 2017

Goblet cells reside throughout the length of the small and large intestine and are responsible for the production and maintenance of the protective mucus blanket by synthesizing and secreting high-molecular-weight glycoproteins known as mucins. To elucidate the role of goblet cells in the biology of the intestinal tract, an overview of the physiological implications of the mucus gel is presented, including a concise review of the products secreted by the cell. Because of the unique nature of this highly polarized exocrine cell, the maturational reorganization of the cytoarchitecture and the cellular mechanisms by which goblet cells secrete their products are discussed. This includes elucidation of the baseline secretory pathway, which is dependent on the cytoskeleton for granule movement, and the accelerated secretory pathway, which is independent of the cytoskeleton but requires an extracellular signal to occur. Finally, the involvement of goblet cell mucins in the pathophysiology of intestinal neoplasia and ulcerative colitis are presented.
Simultaneous measurement of intracellular pH and Ca2+ using the fluorescence of SNARF-1 and fura-2
Authors: Martinez-Zaguilan R. , Martinez G. M. , Lattanzio F. , Gillies R. J.
Published: December 2017

Upon cell stimulation with hormones and other mitogens, a variety of biochemical and physiological responses occur within the first few minutes. Changes in both intracellular pH (pHin) and intracellular Ca2+ concentration ([Ca2+]in) are prominent and play a major role in the signal transduction mechanism leading to the physiological response, i.e., secretion, neurotransmission, proliferation, or differentiation. However, it is not clear whether these ions work independently in the activation pathway leading to a particular physiological response. The fluorescence characteristics of most Ca2+ indicators are pH sensitive, and quantitative estimates of [Ca2+]in cannot be made without knowledge of pHin. Thus it is desirable to have a technique to simultaneously monitor these two ions with relatively high time resolution. Here we have developed experimental conditions that allow us to use optimum emission conditions for a pH fluorescent indicator SNARF-1 and optimum excitation conditions for the Ca2+ indicator fura-2. The fluorescence spectra of these compounds are sufficiently different to allow simultaneous measurement of pH and Ca2+ both in vitro and in situ. We have observed simultaneous changes in both pHin and [Ca2+]in in BALB/c 3T3 cells on treatment with the nonfluorescent Ca2+ ionophore 4-bromo-A23187. This temporal relationship between pHin and Ca2+ gives further credence to the interrelationship between these two second messengers in the expression of physiological responses.
Effect of cromakalim and lemakalim on slow waves and membrane currents in colonic smooth muscle
Authors: Post J. M. , Stevens R. J. , Sanders K. M. , Hume J. R.
Published: December 2017

The effects of cromakalim (BRL 34915) and its optical isomer lemakalim (BRL 38227) were investigated in intact tissue and freshly dispersed circular muscle cells from canine proximal colon. Cromakalim and lemakalim hyperpolarized resting membrane potential, shortened the duration of slow waves by abolishing the plateau phase, and decreased the frequency of slow waves. Glyburide, a K channel blocker, prevented the effect of cromakalim on slow-wave activity. The mechanisms of these alterations in slow-wave activity were studied in isolated myocytes under voltage-clamp conditions. Cromakalim and lemakalim increased the magnitude of a time-independent outward K current, but cromakalim also reduced the peak outward K current. Glyburide inhibited lemakalim stimulation of the time-independent background current. Nisoldipine also reduced the peak outward current, and in the presence of nisoldipine, cromakalim did not affect the peak outward component of current. This suggested that cromakalim may block a Ca-dependent component of the outward current. Lemakalim did not affect the peak outward current. We tested whether the effects of cromakalim on outward current might be indirect due to an effect on inward Ca current. Cromakalim, but not lemakalim, was found to inhibit L-type Ca channels; however, glyburide did not alter cromakalim inhibition of inward Ca current. We conclude that the effects of cromakalim and lemakalim on membrane potential and slow waves in colonic smooth muscle appear to result primarily from stimulation of a time-independent background K conductance. The effects of these compounds on channel activity may explain the inhibitory effect of these compounds on contractile activity.
Length-dependent activation by Ba2+ and Sr2+ of skinned cardiac and skeletal muscle of the rabbit
Authors: Grundeman R. L. , de Beer E. L. , van den Berg C. , van Buuren K. J. , Schiereck P.
Published: December 2017

Over a wide range of sarcomere lengths, force activation by Ca2+, Ba2+, and Sr2+ was studied in papillary muscle and in fast skeletal fibers of the gracilis muscle of the rabbit, both skinned by means of freeze drying. The length-tension relations of Ba2+ activation differ significantly from those of Sr2+ and Ca2+ activation with respect to both the value and the position of the maximum. At (almost) full activation, force induced in gracilis muscle by Ba2+ was 50% of the developed force induced by Ca2+. The position of the Sr2+ sensitivity curve for papillary muscle preparations is independent of sarcomere length, in contrast to the position of the Ca2+ sensitivity curves. The binding of Sr2+ to the papillary preparation proves to be very stable as observed from the long-lasting relaxation after activation. Immersion of the papillary preparation in the relaxation fluid after activation with Ba2+ results in a tension transient: a rise in tension followed by a decrease was observed. The maximal value of the tension transient was up to twice the steady tension, dependent on Ba2+ concentration. The steady-state tension was approximately 50% of the Ca2(+)-induced tension. Ba2+ sensitivity curves are not sigmoidal but show a maximum. Above [Ba2+] greater than 10(-5) to 10(-4) M (dependent on sarcomere length) tension decreased. These observations suggest that two counteracting processes govern Ba2+ contraction in papillary muscle preparations, namely activation and inhibition.
Disruption of microtubules alters polarity of basement membrane proteoglycan secretion in epithelial cells
Authors: de Almeida J. Bruno , Stow Jennifer L.
Published: December 2017

Basement membrane proteins such as the heparan sulfate proteoglycan (HSPG) are secreted in a polarized fashion from the basolateral membrane of epithelial cells. We have used the microtubule-disrupting drug colchicine to study the role of the microtubule network in directing constitutive secretion to the basolateral membrane of LLC-PK1 renal epithelial cells. Microtubule depolymerization induced by colchicine resulted in fragmentation and redistribution of fluorescently labeled trans-Golgi membranes. Increased immunofluorescent staining of HSPG was associated with these dispersed Golgi cisternae. The biosynthetic processing of HSPG was not significantly altered by the loss of microtubules or by the dispersal of the Golgi elements. The most striking effect of microtubule disruption was the loss of polarity of HSPG secretion. Immunoprecipitation studies showed that HSPG was secreted from both apical and basolateral surfaces of LLC-PK1 cells treated with colchicine, and a similar result was found for the delivery of laminin, another basement membrane protein. In contrast, there was no change in the distribution of an integral basolateral membrane protein, Na+-K+-ATPase, following colchicine treatment. Our results provide the first demonstration that microtubules are involved in the constitutive trafficking of basolateral secretory proteins. These data also suggest that there may be an inherent difference in the targeting or delivery of membrane and secretory proteins to the basolateral cell surface.

polarized secretion; heparan sulfate proteoglycans; sorting; Golgi processing

Submitted on July 10, 1990

Accepted on November 12, 1990
Brain natriuretic peptide stimulates K and Cl secretion across porcine distal colon epithelium
Authors: Traynor T. R. , O'Grady S. M.
Published: December 2017

Porcine distal colon epithelium was mounted in Ussing chambers and bathed with porcine Ringer solution. The serosal addition of brain natriuretic peptide (BNP; 50 nM) or atriopeptin III (AP-III; 500 nM) produced significant increases (50-75 microA/cm2) in short-circuit current (Isc). These increases in Isc were not inhibited by pretreatment with tetrodotoxin (TTX) or 5,8,11,14-eicosatetraynoic acid (ETYA). Analysis of concentration-response relationships revealed that BNP was 5.8-fold more potent than AP-III in stimulating the Isc. BNP and AP-III significantly increased the serosal-to-mucosal (S----M) Cl flux and reduced net Cl absorption by 38 and 41%, respectively. The BNP-stimulated S----M Cl flux was abolished when HCO3 was removed. In contrast, the vasoactive intestinal peptide (VIP)-stimulated S----M Cl flux was not affected by HCO3 replacement. In addition to their effects on Cl transport, BNP and AP-III increased net Rb secretion by 79 and 58%, respectively. BNP-stimulated Rb secretion was reduced by 76% after HCO3 replacement. These results indicate that natriuretic peptides stimulate K- and HCO3-dependent Cl secretion which is not present under basal conditions or after VIP stimulation. The difference in potency between BNP and AP-III suggests that ANP-B receptors may mediate their effects on ion transport in the porcine colon.