bluuebunny/crossref_metadata_2025 huggingface dataset:
Ion transport asymmetry and functional coupling in bovine pigmented and nonpigmented ciliary epithelial cells
Authors: Edelman J. L. , Sachs G. , Adorante J. S.
Published: December 2017

The solute and water transport properties of the bovine ciliary epithelium were studied using isolated pigmented (PE) and nonpigmented (NPE) cells. It was shown that these cells were functionally coupled by demonstrating dye diffusion between paired PE and NPE cells after microinjection of lucifer yellow. Electronic cell sizing was used to measure cell volume changes of isolated PE and NPE cells in suspension after anisosmotic perturbations and after transport inhibition under isosmotic conditions. The PE cells showed the presence of a regulatory volume increase when subjected to osmotic shrinkage with NaCl, whereas the NPE cells did not demonstrate a regulatory volume increase under these conditions. In contrast, the NPE cells exhibited a regulatory volume decrease when subjected to osmotic swelling, whereas the PE cells did not recover from swelling. The regulatory volume decrease in NPE cells was inhibited by increased bath K or pretreatment with quinine (1 mM). The presence of a bumetanide-sensitive mechanism capable of moving measurable amounts of solute and water, probably Na-K-2Cl cotransport, was demonstrated in the PE cells but absent in the NPE cells. Bumetanide produced a dose-dependent shrinkage of PE cells at concentrations as low as 1 microM. Isosmotically reducing bath Cl, Na, or K concentration caused a rapid shrinkage of PE cells that was bumetanide inhibitable. The asymmetry of transport properties in PE and NPE cells supports a functional syncytium model of aqueous humor formation (39) across the two layers of the ciliary epithelium wherein ion uptake from the blood is carried out by the PE cells and ion extrusion by the NPE cells. Gap-junction coupling between the cells allows the ions taken up by the PE cells to move into the NPE cells. Extrusion of Na by the Na-K pump across the aqueous facing (basolateral) membranes of the NPE cells, most likely accompanied by Cl, determines the formation of the aqueous humor.
Aldosterone induces early activation and late accumulation of Na-K-ATPase at surface of A6 cells
Authors: Beron J. , Verrey F.
Published: December 2017

In A6 cells cultured on filters, aldosterone (10(-6) M) induces an early increase in the initial rate of ouabain binding to intact monolayers (in K-free buffer) that parallels an early increase in Na transport. This effect is independent of apical Na influx and precedes an increase in the synthesis of Na-K-adenosinetriphosphatase (ATPase) subunits. In the present study we observed in addition a late aldosterone-induced increase in the rate of ouabain binding (2.5 times from 3 to 120 h aldosterone). The role of Na-K-ATPase accumulation and/or translocation to the cell surface was tested by Western blotting, saturation ouabain binding, and cell-surface labeling using sulfosuccinimidobiotin or enzyme-mediated radioiodination. Only cell-surface beta 1-subunit was detected by blotting with streptavidin or autoradiography, because the alpha 1-subunit was not efficiently labeled. Three hours after hormone addition, none of the three parameters had increased significantly, whereas after 20 and 120 h similar increases (approximately 1.6 and 2.3 times, respectively) were detected by all three methods. In addition, it was shown that increasing intracellular Na with amphotericin B or a K-free preincubation also stimulated the rate of ouabain binding without increasing the surface labeling of beta 1-subunits. Taken together, these results suggest that a short aldosterone treatment or an increase in intracellular Na leads to an increase in the rate of ouabain binding that is due to an in situ activation of cell-surface Na-K-ATPase molecules. In contrast, the late increase in the rate of ouabain binding parallels an increase in the number of pumps.
Developmental cell-specific regulation of Na(+)-K(+)-ATPase alpha 1-, alpha 2-, and alpha 3-isoform gene expression
Authors: Herrera V. L. , Cova T. , Sassoon D. , Ruiz-Opazo N.
Published: December 2017

Na(+)-K(+)-activated adenosine triphosphatase (Na(+)-K(+)-ATPase) is the integral membrane protein that maintains the Na(+)-K(+) electrochemical gradient across the plasma membrane. Because of the importance of the Na(+)-K(+) electrochemical gradient to fundamental and specialized cell functions, we investigated the cell-specific modulation of Na(+)-K(+)-ATPase alpha-subunit isoform (alpha 1, alpha 2, and alpha 3) gene expression in different stages of postimplantation mouse embryos and neonatal rat tissues by in situ hybridization with use of isoform-specific rat-derived antisense RNA probes. At early organogenesis (9.5-10.5 days postcoitus), we demonstrated generalized coexpression of alpha 1- and alpha 2-isoforms throughout the mouse embryo with greater levels in the developing but already functional heart, in contrast to the distinct spatially restricted alpha 3-isoform gene expression in the early developing neural tube. At midorganogenesis (15.5-16.5 days postcoitus), differential spatial variation in alpha 1-, alpha 2-, and alpha 3-isoform gene expression was already evident in all organs. Interestingly, region-specific expression patterns within single cell types were noted throughout development and were exemplified by 1) alpha 3-isoform gene expression in marginal cells of the 10.5-day-postcoitus developing neural tube; 2) alpha 1-, alpha 2-, and alpha 3-isoform gene expression in cerebellar granular cells of the 4-day-old rat brain; and 3) alpha 1- and alpha 3-isoform gene expression in 4-day-old rat ventricular cardiomyocytes. These isoform-specific changes in cellular and regional Na(+)-K(+)-ATPase alpha-isoform gene expression may play an active role in development and specialized cell functions.
Detection and quantitation of EP3 prostaglandin E2 receptor mRNA along mouse nephron segments by RT-PCR
Authors: Taniguchi S. , Watanabe T. , Nakao A. , Seki G. , Uwatoko S. , Kurokawa K.
Published: December 2017

mRNA of the EP3 prostaglandin E2 (PGE2) receptor was detected and quantitated in microdissected mouse nephron segments by a modified protocol of reverse transcription-polymerase chain reaction (RT-PCR). At the step of RT, a point mutation was introduced in cDNA, which made a new restriction site for the Mbo I enzyme. PCR was performed using a set of primers on the same exon, and genomic DNA was coamplified with cDNA by these primers. PCR products were treated with Mbo I, and signals from genomic DNA and mRNA were separately detected on gel electrophoresis. The relative amount of mRNA per cell was expressed as a ratio of amount of product from mRNA to that from genomic DNA. EP3 PGE2 receptor mRNA expression was abundant in thick ascending limb of Henle, present to a lesser extent in distal convoluted tubules and collecting ducts, and undetectable in glomeruli, proximal tubules, and descending thin limb. The results support the notion that PGE2 modulates water and solute transport through the EP3 receptor in specific structures of the kidney.
Cytokines affect ion transport in primary cultured thick ascending limb of Henle's loop cells
Authors: Escalante B. A. , Ferreri N. R. , Dunn C. E. , McGiff J. C.
Published: December 2017

Tumor necrosis factor-alpha (TNF) and interleukin-1 (IL-1) affect epithelial cell ion transport. However, the site of action along the nephron has not been elucidated fully for these cytokines. Thus, the effect of TNF and IL-1 on the ion transport function of primary cultured medullary thick ascending limb of Henle's loop (mTALH) cells was determined by measuring rubidium (86Rb) uptake. TNF, IL-1, and lipopolysaccharide (LPS), a known activator of cytokine production, inhibited 86Rb uptake by cultured mTALH cells after a 24-h incubation period but had no effect when incubated with the cells for 1 or 4 h. Furthermore, mTALH cells produced biologically active TNF after stimulation with LPS for 24 h, and the LPS-induced inhibition of 86Rb uptake was abolished in the presence of an anti-TNF antibody, suggesting that TNF produced by the mTALH cells acted in an autocrine manner to inhibit 86Rb uptake. The effects of LPS on 86Rb uptake also were inhibited by the cyclooxygenase inhibitor, indomethacin. As TNF increased prostaglandin E2 synthesis by cultured mTALH cells and as prostaglandin E2 also inhibited 86Rb uptake, LPS presumably inhibited 86Rb uptake by inducing a TNF-mediated increase in prostaglandin synthesis. These data demonstrate that a prostanoid produced by mTALH cells mediates the inhibitory effect of LPS and TNF on 86Rb uptake and imply that endogenous TNF inhibits ion fluxes in the mTALH via a prostaglandin-dependent mechanism.
Transepithelial phosphate transport in rabbit proximal tubular cells adapted to phosphate deprivation
Authors: Scheinman S. J. , Reid R. , Coulson R. , Jones D. B. , Ford S. M.
Published: December 2017

Both renal and nonrenal cells in culture adapt to deprivation of Pi by increasing Na-dependent Pi uptake. We studied whether this change in uptake is reflected in an increased renal transepithelial Pi transport. We grew primary cultures of rabbit renal cortical cells in plastic flasks and subcultured them onto Millicell-HA filters. This produced cell monolayers, which structurally and functionally resembled proximal tubule. These cells performed Na-dependent net transepithelial transport of 32Pi in the apical-to-basolateral direction that was inhibited by phosphonoformic acid in the apical fluid or by ouabain in the basolateral fluid or by preincubation with parathyroid hormone. Overnight incubation at low Pi concentrations led to a progressive increase in 5-min Na-dependent Pi uptake into cell monolayers. Na-dependent Pi uptake was threefold higher following overnight incubation at 25 microM Pi, compared with 3 mM Pi, and the increase was one-half maximal with incubation at an extracellular Pi concentration ([Pi]) of 300 microM. This was associated with a decrease in Na-dependent transepithelial Pi flux to the basolateral fluid by the same cells, which fell dramatically following incubation at < or = 300 microM Pi. There was no change in Na-dependent uptake or transepithelial transport of L-glutamine. This adaptation to Pi deprivation in vitro appears to serve to restore depleted cell stores of Pi rather than to regulate transepithelial Pi transport.
K+ modulation of microglial superoxide production: involvement of voltage-gated Ca2+ channels
Authors: Colton C. A. , Jia M. , Li M. X. , Gilbert D. L.
Published: December 2017

A variety of cytoactive factors produced during injury and inflammation are known to activate the central nervous system (CNS) macrophage, the microglia. Since extracellular potassium levels are known to rise rapidly at sites of injury in the CNS, we examined the possibility that changes in extracellular potassium could mediate changes in microglial function. The effect of an increase in potassium concentration on microglial superoxide anion production was studied in cultured neonatal rat microglia. Rather than directly inducing superoxide anion production, exposure to media containing 25 and 55 mM potassium enhanced the production of superoxide induced by phorbol 12-myristate 13-acetate. This potentiation was blocked by nifedipine, a voltage-gated calcium channel blocker. Treatment of the microglia with BAY K 8644, an agonist for voltage-gated calcium channels, produced an enhancement of superoxide levels similar to that of potassium. Because these data indicated the presence of a voltage-gated calcium channel, we also examined whole cell current in cultured microglia. A small, voltage-dependent inward calcium current was seen that was increased by exposure of the microglia to BAY K 8644. The presence of a small but finite calcium influx via these channels may be an important factor in the regulation of intracellular microglial events such as activation of the NADPH oxidase and the consequent production of superoxide anion.
HSP-72 synthesis is promoted by increase in [Ca2+]i or activation of G proteins but not pHi or cAMP
Authors: Kiang J. G. , Carr F. E. , Burns M. R. , McClain D. E.
Published: December 2017

The family of 70-kDa heat-shock proteins (HSP-70) is evolutionarily highly conserved and has been shown to enhance cell survival from thermal injury. This study characterized HSP-72 induction in human epidermoid A-431 cells exposed to 45 degrees C for 10 min and determined the relationship between HSP-72, intracellular pH (pHi), adenosine 3',5'-cyclic monophosphate (cAMP), G proteins, and intracellular cytosolic free Ca2+ concentration ([Ca2+]i). Heat shock induced HSP-72 production, which was dependent on both temperature and the duration of heating. This HSP-72 induction was confirmed by Western blot analysis. HSP-72 levels in cells that had been heated then returned to 37 degrees C were elevated at 2 h (1.5 +/- 0.1 x control), reached a maximum at 8 h (2.7 +/- 0.1 x control), and remained above baseline for up to 4 days. Levels of HSP-72 mRNA, determined by dot-blot analysis, reached a maximum at 2 h and returned to baseline within 8 h. Both actinomycin D and cycloheximide blocked HSP-72 induction. Because heating causes intracellular acidification and increases in cAMP and [Ca2+]i, we studied the effect of pHi, cellular cAMP, and [Ca2+]i on HSP-72 induction. The reduction of pHi to 6.9 by acid loading did not affect the basal level of HSP-72 in unheated cells. Treatment with pertussis toxin, cholera toxin, or forskolin, but not 8-bromo-cAMP, 3-isobutyl-1-methylxanthine, or N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide potentiated heat-induced HSP-72 production. Inhibition of the heat-induced increase in [Ca2+]i attenuated, but failed to completely block, heat-induced HSP-72 production, mRNA synthesis, and the heat-shock transcriptional factor-heat-shock element binding complex formation, which suggests there are Ca(2+)-dependent and -independent processes involved in HSP-72 synthesis. Our results show that an increase in [Ca2+]i or activation of G proteins, but not pHi and cAMP, enhances HSP-72 induction.
Calcium release by cholecystokinin analogue OPE is IP3 dependent in single rat pancreatic acinar cells
Authors: Gaisano H. Y. , Wong D. , Sheu L. , Foskett J. K.
Published: December 2017

Cholecystokinin (CCK) and carbachol raise intracellular Ca2+ concentration ([Ca2+]i) in pancreatic acinar cells by elevating inositol 1,4,5-trisphosphate (IP3). CCK analogues JMV-180 and OPE stimulate fully efficacious enzyme secretion and [Ca2+]i oscillations but release Ca2+ from intracellular stores by apparently IP3-independent mechanisms in permeabilized acinar cells. In the present study, we investigated whether OPE mobilizes Ca2+ from IP3-sensitive Ca2+ stores and whether IP3 mediates such responses in single intact cells. OPE and JMV-180 similarly elevated IP3 to low levels compared with those elicited by 10 nM CCK. Depletion of IP3-sensitive stores by elevation of intracellular IP3 using carbachol, microinjection of a nonmetabolizable IP3 analogue, or exposure to thapsigargin, in the absence of extracellular Ca2+, depleted the same Ca2+ stores that were sensitive to OPE. In converse experiments, OPE depleted carbachol- or thapsigargin-sensitive stores, indicating that carbachol-, thapsigargin-, IP3-, and OPE-sensitive Ca2+ stores overlap completely and that stores mobilized by OPE are IP3 sensitive. To determine whether IP3 mediates responses to OPE, cells were microinjected with low-molecular-weight heparin, a competitive inhibited the rise of [Ca2+]i in response to carbachol, OPE, or JMV-180, whereas de-N-sulfated heparin, an inactive heparin, was without effect. These results indicate that CCK analogues release Ca2+ from IP3-sensitive Ca2+ stores by mechanisms involving the IP3 receptor.
Osmotic and other properties of isolated human cheek epithelial cells
Authors: Lee E. J. , Patten G. S. , Burnard S. L. , McMurchie E. J.
Published: December 2017

This study describes some biological properties of human cheek (buccal epithelial) cells, isolated by mouth wash. Yields ranged from 6.5 to 20.6 x 10(6) cells, with a mean (+/- SE) of 12.2 +/- 4.2 x 10(6) cells, which gave 0.55 +/- 0.01 x 10(6) cells/mg protein. Vital stain exclusion was similar in cells isolated in either water (89 +/- 2%) or 250 mM sucrose (87 +/- 3%). From our measurements of cell volume and electrolyte content, we estimated intracellular Na+ and K+ concentrations to be between 0.3-0.5 and 7.4-13.0 mM, respectively. In 22 adult subjects, basal, prickle, intermediate, and superficial cells represented 0.3 +/- 1.4, 51 +/- 2.4, 26 +/- 0.9, and 22.7 +/- 1.8%, respectively, of the total sample. Cheek cells exhibited a low endogenous rate of oxygen consumption, which was stimulated by glucose or succinate and inhibited by KCN or NaF. Cheek cells were osmotically stable in a wide range of media, including water. However, they exhibited shrinkage and collapse in hypertonic media, particularly polyethylene glycol.
Adrenergic modulation of intracellular pH in isolated brown fat cells from hamster and rat
Authors: Lee S. C. , Hamilton J. S. , Trammell T. , Horwitz B. A. , Pappone P. A.
Published: December 2017

The activity of the uncoupling protein in brown fat mitochondria is enhanced at alkaline pH, leading to the hypothesis that changes in intracellular pH (pHi) may modulate the thermogenic response to sympathetic stimulation. We employed ratio imaging of the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to measure pHi in acutely isolated single brown fat cells from hamster and neonatal rat and in cultured rat cells. Basal pHi averaged approximately 7.2 in HCO3- media and 0.1-0.15 pH units lower in nominally HCO3(-)-free media in all cell types. In both HCO3- and HCO3(-)-free media, stimulation with norepinephrine (NE) typically caused an alkalinization of approximately 0.05-0.1 pH units, which was followed by a smaller net acidification occurring primarily after NE was removed. Alkalinization seemed to be mediated predominantly by alpha-adrenergic stimulation, while acidification most often followed beta-adrenergic activation. Similar pHi changes were elicited by NE in rat and hamster cells, but responses were more frequent in hamster cells. Assays of recovery from ammonium prepulse-induced acid loads indicated that rat and hamster cells have both Na(+)-H+ and Na(+)- and HCO3(-)-dependent regulatory systems, while hamster cells have, in addition, a Na(+)-independent recovery mechanism activated at acid pHi. We conclude that alpha-adrenergic alkalinization of brown fat may contribute to the control of thermogenesis.
Determinants of apical membrane permeabilities of barrier epithelia
Authors: Lande M. B. , Priver N. A. , Zeidel M. L.
Published: December 2017

Renal collecting duct and thick ascending limb, as well as stomach, exhibit strikingly low permeabilities to water and solutes. However, the apical membrane characteristics responsible for these unique permeabilities remain unknown. While the lipid composition of artificial membranes governs membrane permeability, exoplasmic and cytoplasmic leaflets of biological apical membranes exhibit striking asymmetries in lipid composition. This asymmetry, as well as the presence of membrane proteins, may be critical to barrier function. To determine the role of bulk lipid composition in apical membrane barrier function, we compared permeabilities to water (Pf), protons, ammonia, and several small nonelectrolytes of gastric apical membrane vesicles [native gastric vesicles (NGV)] and liposomes prepared from lipids quantitatively extracted from these vesicles [gastric lipid large unilamellar vesicles (LUV)]. Permeabilities were measured on a stopped-flow fluorimeter by monitoring self- or pH-sensitive quenching of entrapped carboxyfluorescein. NGV exhibited low Pf (2.8 +/- 0.3 x 10(-4) cm/s) while gastric lipid LUV Pf averaged 1.2 +/- 0.1 x 10(-3) cm/s, a fourfold increase compared with the value in NGV. Gastric lipid LUV also demonstrated higher permeabilities to protons, ammonia, propylene glycol, butyramide, ethanolamine, and acetamide compared with values in NGV. In contrast, gastric lipid LUV exhibited the same or lower permeabilities to urea, glycerol, and ammonia compared with values in NGV. We conclude that lipid composition alone can reconstitute membrane permeabilities to some, but not all, molecules. These results indicate that bilayer asymmetry may be required for the unique permeability of "water-tight" apical membranes and reveal different barrier mechanisms for water and protons, as opposed to ammonia, urea, and glycerol.
Phorbol myristate acetate and ATP-sensitive potassium channels in insulin-secreting cells
Authors: Dunne M. J.
Published: December 2017

The actions of 4 beta-phorbol 12-myristate 13-acetate (PMA; 10-100 nM) on the ATP-sensitive K+ channel have been studied in the RINm5F insulin-secreting cell line. These experiments were carried out using the inside-out patch and open-cell recording configurations of the patch-clamp technique. In the presence of intracellular ATP and ADP, PMA was found to have complex effects. Over short periods of time, i.e., less than 5 min, PMA promoted channel inhibition. However, in the sustained presence of the phorbol ester, this inhibition was only transient and was followed by a period of recovery and then stimulation of the channels. There were no effects of PMA on ATP-sensitive K+ (K+ATP) channels in the absence of intracellular ATP/ADP and in patches of membrane excised from cells that had been pretreated overnight with 1 microM PMA to downregulate endogenous C-kinase. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate (10 nM-1 microM) was also found to have no actions on ATP-sensitive K+ channels. Overall, these data may suggest that through the modulation of protein kinase(s) associated with K+ATP channels, PMA is capable of causing both activation and inhibition of K+ channels in RINm5F cells.
Modulation of growth factor and cytokine-induced increases in T84 cell monolayer permeability by media components
Authors: McRoberts J. A. , Riley N. E.
Published: December 2017

Previous studies have shown that insulin, insulin-like growth factors, and interferon-gamma cause an increase the paracellular permeability across T84 human colonic epithelial cell monolayers. The striking similarity in the time course and the structural changes in the actin cytoskeleton prompted us to test whether these factors influenced the paracellular permeability by a common mechanism. T84 cell monolayers were grown in variously modified media, and the effect of all three factors was tested by measuring the conductance of monolayers mounted in Ussing chambers. Media additions or deletions that modify cellular carbohydrate metabolism markedly influenced the response to all these peptides. In particular, the increase in conductance produced by each of the three peptides was 1) inhibited by addition of short-chain fatty acids, principally n-butyrate and propionate; 2) attenuated by the replacement of media glucose with slowly metabolized and nonmetabolized sugars; 3) potentiated by decreasing the media iron concentration; and 4) inhibited by increasing the media iron concentration. The effects of short-chain fatty acids, glucose, and iron were all shown to be dose dependent. In addition, the ability of high media iron to prevent the increase in permeability caused by all three factors was shown to require ambient oxygen. Together, these results strongly suggest that insulin, insulin-like growth factors, and interferon-gamma increase the permeability across T84 cell monolayers through a common mechanism that is modulated by glucose-derived metabolites and oxidative metabolism.
Sarcoplasmic reticulum Ca2+ pump expression in denervated skeletal muscle
Authors: Schulte L. , Peters D. , Taylor J. , Navarro J. , Kandarian S.
Published: December 2017

This study was undertaken as one approach to better understand how contractile activity regulates excitation-contraction coupling phenotype in skeletal muscle. The effects of denervation on the expression of the sarcoplasmic reticulum (SR) Ca(2+)-adenosinetriphosphatase (ATPase), a key protein of the contraction-relaxation cycle, was analyzed in fast-twitch (FT) and slow-twitch (ST) skeletal muscle. Muscle mass, mRNA, and protein expression of specific isoforms of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and contractile parameters related to muscle relaxation were measured in rat soleus and extensor digitorum longus (EDL) muscles at 1, 4, 7, 14, and 28 days after sciatic nerve transection. Wet muscle mass decreased to 35% of control by 28 days of denervation in both soleus and EDL muscles (P < 0.05). Northern and Western analyses showed decreases in mRNA and protein expression of the slow Ca2+ pump isoform (SERCA2a) in the denervated soleus muscle and in the fast Ca2+ pump isoform (SERCA1) in the denervated EDL muscle, particularly at later time points. There was no change in the expression of the alternate isoform in either muscle type. Prolongation of twitch contraction times, slowed rates of tension development, and leftward shifts in frequency-tension curves were consistent with the reduced Ca2+ pump density and suggested slowed Ca2+ handling in SR or denervated ST and FT muscles. The results are in marked contrast with those previously reported in non-weight-bearing muscle induced by biomechanical unloading.
Responses to extracellular ATP of lymphoblastoid cell lines from Duchenne muscular dystrophy patients
Authors: Ferrari D. , Munerati M. , Melchiorri L. , Hanau S. , di Virgilio F. , Baricordi O. R.
Published: December 2017

We have observed a striking difference in the response to extracellular ATP in lymphoblastoid cell lines established from Duchenne muscular dystrophy patients and normal subjects. Duchenne muscular dystrophy cells stimulated by extracellular ATP underwent a large increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) and plasma membrane depolarization, while normal cell lines were little or not at all responsive. These changes in intracellular ion homeostasis were due to activation of an ATP-gated membrane channel permeable to Na+ and Ca2+, with little or no contribution of Ca2+ release from intracellular stores. The channel was selectively activated by ATP, since other purine/pyrimidine nucleotides were ineffective, and it was inhibited by pretreatment with oxidized ATP, a compound previously reported to irreversibly inhibit P2 purinergic receptors. In the presence of extracellular ATP, lymphoblastoid cells established from Duchenne muscular dystrophy patients, but not from healthy controls, underwent rounding and swelling and eventually lysed. The results of this study suggest that lymphoblastoid cells isolated from Duchenne muscular dystrophy patients are eminently sensitive to stimulation by extracellular ATP.
Heparin induces endothelial extracellular matrix alterations and barrier dysfunction
Authors: Guretzki H. J. , Schleicher E. , Gerbitz K. D. , Olgemoller B.
Published: December 2017

We investigated the influence of heparin on the composition of the subendothelial matrix and on endothelial permeability to elucidate the structure-function relationship of matrix composition and permeability. Albumin flux across the confluent endothelial monolayers was used to assess the macromolecular permeability. Lowest values were obtained when 100% serum was used as medium for permeability studies. The endothelial matrix components, fibronectin and basement membrane-associated heparan sulfate proteoglycan (HSPG), were measured by enzyme immunoassay. Treatment of proliferating endothelial cells with heparin (0-900 micrograms/ml) induced a dose-dependent decrease in endothelial HSPG content, whereas the fibronectin content was unaltered. This structural change was accompanied by an increase in albumin permeability. Both heparin effects exhibited similar dose-response curves with half-maximal effects at approximately 5 micrograms/ml heparin. Acute addition of 300 micrograms/ml heparin had no effect on permeability or HSPG content. When endothelial cells were preincubated with an HSPG antiserum, the endothelial permeability increased nearly threefold. Our results indicate that heparin-induced loss of HSPG may cause the increase in endothelial permeability. The data underline the importance of HSPG for the integrity of the endothelial barrier.
Expression and function of P-glycoprotein in human mesangial cells
Authors: Bello-Reuss E. , Ernest S.
Published: December 2017

P-glycoprotein (PGP), responsible for multidrug resistance (MDR) in cancer cells, is normally expressed in kidney proximal tubules and mesangium. PGP expression and function were studied in human mesangial cell cultures. MDR1 gene expression was demonstrated by reverse transcription-polymerase chain reaction. PGP expression was determined using MRK16 monoclonal antibody and its function was assessed by the efflux of rhodamine-123 (R123). R123 efflux had a half time of 25 +/- 5 s. Efflux was inhibited by cyclosporin A (10 microM), verapamil (10 microM), and vinblastine (100 microM) with half times of 380, 535, and 312 s, respectively. Incubation with MDR1-antisense oligonucleotide decreased R123 efflux (half time = 304 s). Verapamil, cyclosporin A, and PSC-833 augmented the cytotoxicity of Adriamycin by reducing the 50% maximal growth-inhibitory dose from 730 nM to 130, 110, and 90 nM, respectively. We conclude that human mesangial cells express MDR1 and demonstrate xenobiotic transport inhibitable by several known PGP substrates. Concomitant exposure of mesangial cells to PGP-transported drugs causes intracellular accumulation of toxic PGP substrates and ultimately damages the mesangial cells.
Response to osmotic stimuli in mesangial cells: role of system A transporter
Authors: Yamauchi A. , Miyai A. , Yokoyama K. , Itoh T. , Kamada T. , Ueda N. , Fujiwara Y.
Published: December 2017

It has been suggested that mesangial cells have an osmoregulatory mechanism like that of renal medullary cells, such as intracellular accumulation of polyols in response to hypertonicity. We examined osmoregulatory role of neutral amino acids transported by system A in cultured mesangial cells. The contents of almost all amino acids increased under hypertonic conditions to more than twice the value in isotonic cells. In hypertonic cells, the system A transport activity, measured by Na(+)-dependent 2-(methylamino)isobutyric acid (MeAIB) uptake, was 3.8-fold the uptake in isotonic cells, reaching a maximum 16 h after the switch to hypertonic medium. The response to hypertonicity was the result of an increase in maximal velocity without change in Michaelis constant and was dependent on RNA and protein synthesis. When medium osmolality decreased from hypertonic to isotonic, MeAIB uptake reverted to the isotonic level within 16 h and a large transient efflux of L-proline occurred within 10 min. These results suggest that mesangial cells respond to extracellular hypertonicity by increasing system A transport activity and neutral amino acids can function as compatible osmolytes in mesangial cells.
G alpha q and G alpha 13 regulate NHE-1 and intracellular calcium in epithelial cells
Authors: Kitamura K. , Singer W. D. , Cano A. , Miller R. T.
Published: December 2017

To understand the mechanisms by which G protein-coupled signaling systems regulate NHE-1 in epithelial cells, we expressed G alpha q and G alpha 13 in a renal epithelial cell line. We studied two signaling systems that have been implicated in NHE-1 regulation [intracellular Ca (Cai) and phospholipase C activity] and measured NHE-1 activity, mRNA, and antigen. Expression of alpha qWT and alpha qQ209L (a GTPase-deficient mutant) increased basal Cai and altered the kinetics of the bradykinin-induced Cai signal. The initial bradykinin-induced spike in Cai was prolonged and the plateau was higher in cells expressing alpha qWT and alpha qQ209L than in control cells. Cells expressing alpha 13WT also had a higher basal Cai and plateau after stimulation by bradykinin, but Ca release from intracellular stores was similar to that in control cells. Expression of all three alpha-chains increased NHE-1 activity, antigen, and mRNA. The alpha qQ209L had the greatest effect increasing activity by approximately twofold. The alpha 13WT increased NHE-1 activity by approximately 1.5-fold, and alpha qWT increased activity 1.2-fold. These studies demonstrate that alpha q and alpha 13 alter regulation of Cai but by different mechanisms. The Ca signal or another signal generated by alpha q and alpha 13 regulate(s) NHE-1 at the levels of activity, antigen, and mRNA.
Kinetics of Na+/H+ exchange in vascular smooth muscle cells from WKY and SHR: effects of phorbol ester
Authors: Hoffmann G. , Ko Y. , Sachinidis A. , Gobel B. O. , Vetter H. , Rosskopf D. , Siffert W. , Dusing R.
Published: December 2017

The kinetic properties of Na+/H+ exchange were investigated in vascular smooth muscle cells (VSMC) in culture from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Antiport activity was measured in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-loaded cells after nigericin-induced cytosolic acidification. Studies were performed without (control) and with pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA; 200 nM). Na+/H+ exchange markedly differed between the two strains with lower Hill coefficients [1.56 +/- 0.17 (SE) vs. 2.62 +/- 0.36] and higher maximal activity (Vmax) values (55.85 +/- 5.24 vs. 31.11 +/- 2.38 mmol H+.l-1.min-1) in SHR compared with WKY cell lines. PMA markedly altered the antiport kinetics in WKY VSMC with a decrease in the Hill coefficient (1.75 +/- 0.14) without affecting Vmax (31.88 +/- 1.55 mmol H+.l-1.min-1). In VSMC from SHR, PMA had no effect on the kinetic variables investigated. Thus two kinetic abnormalities are present with respect to Na+/H+ antiport activity in VSMC from SHR compared with WKY, i.e., increased Vmax and decreased Hill coefficient. The observation that PMA does not affect the kinetics of the Na+/H+ antiport in VSMC from SHR suggests a marked degree of antiporter prestimulation in this animal model of genetic hypertension.
Properties of gap junction channels formed of connexin 45 endogenously expressed in human hepatoma (SKHep1) cells
Authors: Moreno A. P. , Laing J. G. , Beyer E. C. , Spray D. C.
Published: December 2017

We have evaluated the voltage dependence and unitary conductance of gap junctional channels that were recorded in a clone isolated from the hepatoma cell line SKHep1. In this clonal population (designated SKHep1A), Northern blots, immunoprecipitation, and immunohistochemical staining demonstrated the expression of connexin (Cx) 45; no other gap junction protein was identified by these techniques, although weak hybridization with Cx40 was detected. Macroscopic junctional conductance (gj) in these cells was low, averaging 1.3 nS, and was steeply voltage dependent. Parameters of voltage sensitivity were as follows: voltage at which voltage-sensitive conductance is reduced by 50%, 13.4 mV; steepness of relation, 0.115 (corresponding to 2.7 gating charges), and voltage-insensitive fraction of residual to total conductance approximately 0.06. Unitary conductance (gamma j) of these junctional channels averaged 32 +/- 8 pS; although gamma j was independent of transjunctional voltage (Vj), at high Vj values (> 50 mV), smaller conductance values were also detected. Open probabilities of the 30-pS channels at various Vj values closely matched the predicted voltage-dependent component of macroscopic gj, the residual conductance at high Vj might be attributable to the smaller conductance events. The voltage dependence of human Cx45 gap junction channels is as steep as that seen for channels formed by Xenopus Cx38 and is much steeper than that previously reported for channels formed of the highly homologous chick Cx45 and for other mammalian connexins expressed either endogenously or exogenously.
Leukoregulin is a potent inducer of hyaluronan synthesis in cultured human orbital fibroblasts
Authors: Smith T. J. , Wang H. S. , Evans C. H.
Published: December 2017

Leukoregulin, a 50-kDa glycoprotein lymphokine, can regulate the extracellular matrix in dermal fibroblasts. Here we investigate the effects of leukoregulin on the synthesis of glycosaminoglycans in human orbital fibroblasts. We demonstrate that leukoregulin enhances the incorporation of [3H]glucosamine into glycosaminoglycans. The effect is dose dependent in the concentration range tested (0.1-2 U/ml), is maximal at 1 U/ml, and is time dependent. [3H]glycosaminoglycan accumulation is enhanced 7.67 +/- 1.23-fold (SE, n = 7) in orbital fibroblast strains. Pulse-chase studies indicate that this enhanced accumulation is not a result of a decreased rate of macromolecular degradation. Radiolabeled material induced by leukoregulin is sensitive to Streptomyces hyaluronidase digestion. Dexamethasone (10(-8) M) and cycloheximide (10 micrograms/ml) can block the cytokine's stimulation of hyaluronan synthesis. [35S]sulfate incorporation into glycosaminoglycan is unaffected by leukoregulin. In dermal fibroblasts, leukoregulin increased hyaluronan synthesis 3.66 +/- 0.37-fold (n = 5 strains, P < 0.02 compared with orbit). The increase in hyaluronan synthesis in orbital fibroblasts is substantially greater than that observed previously with other cytokines, making leukoregulin a candidate molecular trigger in Graves' ophthalmopathy.
Intestinal epithelia (T84) possess basolateral ligands for CD11b/CD18-mediated neutrophil adherence
Authors: Parkos C. A. , Colgan S. P. , Bacarra A. E. , Nusrat A. , Delp-Archer C. , Carlson S. , Su D. H. , Madara J. L.
Published: December 2017

The process of active intestinal inflammation can be modeled by the migration of neutrophils [polymorphonuclear leukocytes (PMN)] across monolayers of the human intestinal cell line T84. We now evaluate the polarity of early adhesive events occurring between human PMN and ligands on intact T84 monolayers. Transient Ca2+ depletion (tr-Ca-depl) of confluent monolayers grown on permeable supports (2 mM EDTA, 12 min, 37 degrees C) resulted in disruption of tight junctions separating apical and basolateral domains (resistance fell from 973 +/- 35 to 57.4 +/- 3.8 omega.cm2 after 12 min). The columnar T84 phenotype was not grossly altered by tr-Ca-depl. Access to basolateral membrane proteins from the apical surface after tr-Ca-depl was confirmed by enzyme-linked immunosorbant assay and immunofluorescence, using antibodies to the basolateral proteins E-Cadherin and beta 1-integrin. The effects of tr-Ca-depl on early events in PMN adhesion were next assessed. Neutrophils (2 x 10(6)) were applied to the apical surface of monolayers followed by stimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (100 nM, 5 min, 37 degrees C) and quantitation. In comparison with control monolayers, tr-Ca-depl increased subsequent PMN adhesion (1.7 +/- 0.24 vs. 3.1 +/- 0.28 x 10(4) adherent PMN/monolayer; P < 0.005), which was inhibited 80% by anti-CD11b/CD18 antibodies. These experiments suggest that PMN adhere to subjunctional ligands on T84 cells in a CD11b/CD18-dependent fashion.
Phosphorylation of dense-plaque proteins talin and paxillin during tracheal smooth muscle contraction
Authors: Pavalko F. M. , Adam L. P. , Wu M. F. , Walker T. L. , Gunst S. J.
Published: December 2017

Reorganization of cytoskeletal-membrane interactions during contractile stimulation may contribute to the regulation of airway smooth muscle contraction. We investigated the effect of contractile stimulation on the phosphorylation of the actin-membrane attachment proteins talin, vinculin, and paxillin. Stimulation of 32P-labeled canine tracheal smooth muscle strips with acetylcholine (ACh; 10(-3) M) resulted in a rapid 2.6-fold increase in phosphorylation of serine and/or threonine residues, compared with resting levels of 0.22 mol PO4(3-)/mol talin. After stimulation with ACh, phosphorylation of tyrosine residues on paxillin increased approximately threefold. Two-dimensional phosphopeptide mapping of in vivo labeled talin and paxillin indicated phosphorylation on a limited number of sites. Vinculin phosphorylation was undetectable in either resting or ACh-stimulated muscle. We conclude that phosphorylation of talin and paxillin occurs during ACh-stimulated contraction of tracheal smooth muscle and that distinct signaling pathways activate a serine/threonine kinase that phosphorylates talin and a tyrosine kinase that phosphorylates paxillin. The pharmacological activation of airway smooth muscle cells might involve the anchoring of contractile filaments to the membrane.
Dual activation of the cardiac Ca2+ channel alpha 1C-subunit and its modulation by the beta-subunit
Authors: Neely A. , Olcese R. , Baldelli P. , Wei X. , Birnbaumer L. , Stefani E.
Published: December 2017

Ca2+ channels are heteromultimeric proteins in which the alpha 1-subunit forms the voltage-dependent Ca(2+)-selective ionic channel. We reported recently that coexpression of the beta-subunit with the cardiac alpha 1-subunit (alpha 1C) facilitates channel opening without affecting either the amplitude or the time course of the gating currents (13). Here we present evidence for the existence of two modes of channel opening. Xenopus oocytes expressing the alpha 1C-subunit alone display two modes of activation as indicated by the double-exponential time course of macroscopic ionic currents and the two open-time distributions of single channels. Coexpression of the beta-subunit potentiates Ca2+ currents by a relative increase of the fast-activating component, an acceleration of the slow component, and a larger proportion of long openings. We propose that multiple modes of gating are encoded in the alpha 1-subunit and that the beta-subunit increases Ca2+ channel opening by favoring a willing mode of gating in which the final transitions leading to channel opening are facilitated. In addition, we show that the carboxy terminus of alpha 1C also modulates the channel-gating behavior.
Regulation of mouse beta 3-adrenergic receptor gene expression and mRNA splice variants in adipocytes
Authors: Granneman J. G. , Lahners K. N.
Published: December 2017

This study examined the regulation of murine beta 3-receptor mRNA and determined whether the recently described mRNA splice variants are differentially regulated by agents that alter total beta 3-receptor mRNA levels. In vivo treatment of mice with the beta 3-receptor agonist BRL-26830 reduced total beta 3-transcripts by 64% in white adipose tissue but did not alter the mRNA splicing pattern. Further analysis in cultured 3T3-F442A adipocytes showed that isoproterenol, dexamethasone, or phorbol 12-myristate 13-acetate also greatly reduced beta 3-receptor mRNA levels without selectively altering poly-U-containing transcripts. Blockade of transcription with actinomycin D produced a rapid loss of beta 3-receptor mRNA, which was prevented by blockade of mRNA translation with cycloheximide. However, neither actinomycin D nor cycloheximide altered the splicing pattern of beta 3-receptor mRNA. Analysis of transcription rate by nuclear run-off assay indicated that 8-bromoadenosine 3',5'-cyclic monophosphate and phorbol 12-myristate 13-acetate reduce beta 3-receptor gene transcription and that suppression of transcription is sufficient to account for the reduction in beta 3-receptor mRNA levels by these agents.
Improved culture conditions stimulate gluconeogenesis in primary cultures of renal proximal tubule cells
Authors: Nowak G. , Schnellmann R. G.
Published: December 2017

Unlike renal proximal tubule cells (RPTC) in vivo, RPTC cultured in standard conditions are hypoxic, glycolytic, and not gluconeogenic. This study investigated the effects of glucose and lactate on glycolysis and gluconeogenesis in rabbit RPTC cultured in conditions of increased oxygen supply (Shake). Confluent Shake cultures grown in the presence of glucose exhibited increased oxygen consumption and decreased glycolysis compared with stationary (Still) cultures. Addition of 5 mM lactate to a 5 mM glucose medium decreased net glucose consumption and glucose oxidation in Shake cultures by 34 and 50%, respectively, and resulted in net lactate consumption. Addition of 5 mM lactate to a glucose-free medium resulted in a threefold increase in net glucose production (0.024 +/- 0.003 vs. 0.074 +/- 0.013 mumol.mg protein-1.day-1) in Shake cultures. Net glucose production further increased to 0.430 +/- 0.020 and 1.640 +/- 0.040 mumol.mg protein-1.day-1 when glucose reuptake was inhibited by 1 mM phloridzin or 1 mM phloridzin + 1 mM phloretin, respectively. These results show that, under conditions of improved oxygenation and in the presence of lactate and physiological levels of glucose and insulin, RPTC aerobic metabolism increases and glucose metabolism changes from glycolysis and net lactate production to gluconeogenesis and net lactate consumption.
Energy-rich phosphates in slow and fast human skeletal muscle
Authors: Vandenborne K. , Walter G. , Ploutz-Snyder L. , Staron R. , Fry A. , De Meirleir K. , Dudley G. A. , Leigh J. S.
Published: December 2017

We investigated the relationship between energy-rich phosphate content and muscle fiber-type composition in human skeletal muscle using a combination of 31P-nuclear magnetic resonance spectroscopy (NMR), histochemical, and biochemical analyses of muscle biopsies. Localized 31P spectra were collected simultaneously from the predominantly slow-twitch soleus muscle and the mixed (fast-twitch and slow-twitch) medial and lateral gastrocnemius muscles, using B1-insensitive Hadamard Spectroscopic Imaging. Biopsy samples were taken from the soleus and lateral gastrocnemius muscles before NMR investigation and analyzed for fiber type composition and succinic dehydrogenase (SDH) activity. Fiber-type composition was determined based both on myofibrillar actomyosin ATPase activity combined with cross-sectional area and on myosin heavy-chain composition. Localized spectroscopy demonstrated a significantly (P < 0.001) higher P(i)/phosphocreatine ratio in the soleus muscle (0.15 +/- 0.01) compared with the medial (0.12 +/- 0.01) and lateral (0.10 +/- 0.0) gastrocnemius. However, in vitro analysis of muscle biopsies showed only a moderate relationship between the basal phosphate content and myofibrillar actomyosin ATPase-based fiber-type composition and SDH activity, respectively.
Histone-induced damage of a mammalian epithelium: the conductive effect
Authors: Kleine T. J. , Gladfelter A. , Lewis P. N. , Lewis S. A.
Published: December 2017

Human semen has been reported to be cytotoxic to rat descending colon by a mechanism involving polyamines (cationic molecules) and collagenase. In this study, we report that histones, cationic proteins found in human semen, can contribute to semen's cytotoxicity. Histones H1, H4, and H5, when added to the mucosal side of rabbit urinary bladder epithelium, were found to alter the transepithelial conductance (Gt) in a voltage-sensitive manner. When the cell interior was negative, the conductance rapidly increased and plateaued. When the cell interior was positive, the induced conductance decreased to control values. Histone increased the Gt by increasing the apical membrane conductance rather than the tight junction conductance. The magnitude of the Gt increase was dose dependent, and the histone-induced conductance was nonselective for Na+, K+, and Cl-. The induced conductance could be reversed by either increasing mucosal Ca2+ concentration or by removal of histone from the mucosal solution. Prolonged exposure of the epithelium to histone was toxic as determined by the irreversible loss of transepithelial resistance. These results indicate that histone increases membrane ionic permeability, is cytotoxic, and thus may contribute to human semen's toxic effect on colonic epithelium.