bluuebunny/crossref_metadata_2025 huggingface dataset:
ATP depletion induces an increase in the assembly of a labile pool of polymerized actin in endothelial cells
Authors: Hinshaw D. B. , Burger J. M. , Miller M. T. , Adams J. A. , Beals T. F. , Omann G. M.
Published: December 2017

Depletion of cellular ATP is associated with profound effects on the cytoskeleton, particularly disruption of microfilaments. We examined this process in bovine pulmonary artery endothelial cells by inducing differential reductions of cellular ATP using mitochondrial inhibition and variable amounts of glucose. Reduction of cellular ATP to levels < 40% of control produced discrete stages in the visible disruption of microfilaments. Using the deoxyribonuclease I assay, a reversible 11% decrease in monomeric (G) actin occurred in conjunction with microfilament disruption. Polyacrylamide gel electrophoretic (PAGE) analysis of the detergent-insoluble cytoskeleton did not reveal any differences in actin content between normal or ATP-depleted cells. Image analysis of adherent endothelial cells that had been fixed and stained with N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phallacidin revealed that an increase of F-actin of approximately 20% occurred in cells depleted of ATP. If the cells were lysed with detergent before fixation, the increase in F-actin was lost. PAGE analysis and electron microscopy of detergent-soluble material from the cells obtained by ultracentrifugation directly demonstrated the presence of a labile pool of F-actin within the cells, which increased with ATP loss. These observations suggest that ATP may play an important role in the organization and remodeling of microfilaments within cells.
Papilloma virus immortalized tracheal epithelial cells retain a well-differentiated phenotype
Authors: Yankaskas J. R. , Haizlip J. E. , Conrad M. , Koval D. , Lazarowski E. , Paradiso A. M. , Rinehart C. A. , Sarkadi B. , Schlegel R. , Boucher R. C.
Published: December 2017

Human airway epithelial cell lines that retain phenotypic properties representative of the native tissue will be useful physiological models. Human papilloma viral (HPV) genes can immortalize human genital keratinocytes and breast and bronchial epithelia. We transfected cystic fibrosis (CF) and normal tracheobronchial epithelial cell cultures with DNA encoding the HPV-18 E6 and E7 genes and characterized phenotypic properties of resultant cell lines. Of the 11 CF clones isolated, 6 developed a polarized phenotype with vectorial ion transport and membrane-specific expression of histamine and purinergic receptors. The ion transport properties of these lines differed from the normal lines and approximated those of primary CF airway epithelial cell cultures more closely than do those of cell lines transformed with the simian virus 40 large T gene. When transplanted into denuded tracheal grafts, these cells can differentiate into ciliated and secretory phenotypes. We conclude that HPV-18 E6 and E7 genes are sufficient to transform human airway epithelial cells and that the resultant cell lines express differentiated phenotypic properties that approximate those of the native epithelium.
Inhibition of Na-K-2Cl cotransport and bumetanide binding by ethacrynic acid, its analogues, and adducts
Authors: Palfrey H. C. , Leung S.
Published: December 2017

The inhibitory effect of ethacrynic acid (EA) and a variety of its derivatives on Na-K-2Cl cotransport in avian erythrocytes was investigated. The most potent compound tested was the adduct of EA with L-cysteine, with an IC50 of 7.2 x 10(-7) M. EA itself, dihydro-EA, EA-D-cysteine, and adducts of EA with other sulfhydryl (-SH) compounds were much less potent. The mechanism of action of EA and EA-L-cysteine differed in several respects: 1) EA-L-cysteine acted more rapidly than EA (half times of < 1 and 4 min, respectively, at 37 degrees C); 2) the action of EA-L-cysteine was reversible by washing, whereas that of EA was not; and 3) the degree of inhibition by EA-L-cysteine varied with medium [K], whereas that of EA did not. The inhibitory effects of both EA-L-cysteine and EA were affected by medium [Na] and [Cl]. We conclude that EA-L-cysteine does not "deliver" EA to transport-related -SH residues or act as an alkylating agent but has some stereospecific effect on cotransport that is a property of the entire molecule. EA does appear to inhibit cotransport by alkylating -SH residues, as closely related compounds lacking the ability to covalently react with such groups were reversible, and other -SH reagents (e.g., N-ethylmaleimide) also inhibited cotransport. EA, EA-L-cysteine, and EA-D-cysteine all inhibited [3H]bumetanide binding to membranes from activated avian erythrocytes at concentrations similar to those that inhibited cotransport. It is possible that the EA and bumetanide types of diuretics interact with closely apposed sites on the Na-K-2Cl cotransporter.
Isolation of plasma membrane fractions from the intestinal epithelial model T84
Authors: Kaoutzani P. , Parkos C. A. , Delp-Archer C. , Madara J. L.
Published: December 2017

The human intestinal epithelial cell line T84 is widely used as a model for studies of Cl- secretion and crypt cell biology. We report a fractionation approach that permits separation of purified apical and basolateral T84 plasma membrane domains. T84 cellular membranes were isolated by nitrogen cavitation and differential centrifugation from monolayers grown on permeable supports. Membranes were then fractionated by isopycnic sucrose density gradient sedimentation, and fractions were assessed, using enzymatic and Western blot techniques, for apical (alkaline phosphatase) and basolateral (Na(+)-K(+)-ATPase) plasma membrane markers and for cytosolic, lysosomal, Golgi, and mitochondrial markers. Buffer conditions were defined that permitted separation of enriched apical and basolateral markers. The validity of the selected markers for the apical and basolateral domains was verified by selective apical and basolateral surface labeling studies using trace iodinated wheat germ agglutinin or biotinylation. This approach allows for separation of apical and basolateral plasma membranes of T84 cells for biochemical analyses and should thus be of broad utility in studies of this model polarized and transporting epithelium.
ATP stimulates Ca2+ release from a rapidly exchanging pool in cultured rat epididymal cells
Authors: Leung A. Y. , Tai H. L. , Wong P. Y.
Published: December 2017

A study was carried out to investigate an ATP-sensitive Ca2+ pool in rat epididymal cells and its role in transepithelial Cl- secretion. In normal buffered solution containing 2.5 mM free Ca2+, ATP triggered single calcium spikes in a dose-dependent fashion. In nominally Ca(2+)-free solution, the peaks of successive Ca2+ spikes diminished after repeated ATP stimulations. Addition of Sr2+ (2.5 mM) to Ca(2+)-free solution after ATP stimulation did not cause changes in fluorescence signals. However, in the presence of Sr2+, ATP gave rise to apparent repetitive Ca2+ spikes of similar magnitudes after repeated stimulations. Increasing the time of exposure in Ca(2+)-free solution containing 50 microM ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid rapidly decreased the intracellular Ca2+ concentration ([Ca2+]i) response to subsequent ATP stimulation. On the other hand, increasing the time of exposure in Sr(2+)-containing solution in Ca(2+)-depleted cells rapidly increased the apparent [Ca2+]i response to subsequent ATP stimulation. These observations suggested the existence of a Ca2+ pool that was rapidly exchanging with the extracellular compartment. Apical application of ATP elicited a transient rise in short-circuit current across the epididymal epithelium in a dose-dependent fashion, and the response was reduced by prior stimulation with thapsigargin. Ca2+ released from a rapidly exchanging ATP-sensitive store might stimulate Cl- secretion in the epididymis, thereby maintaining the electrolyte contents and fluidity of the epididymal microenvironment.
Kinetics of calcium transport across the lymphocyte plasma membrane
Authors: Balasubramanyam M. , Kimura M. , Aviv A. , Gardner J. P.
Published: December 2017

We have investigated plasma membrane Ca2+ transport by monitoring the fluorescence of human peripheral T-lymphocytes loaded with fura 2. Thapsigargin (TG) was utilized the block the Ca(2+)-ATPase of the endoplasmic reticulum and elevate the cytosolic Ca2+ (Ca2+i). Ca2+ influx was inhibited by chelating extracellular Ca2+ with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The rate of decline in the Ca2+i signal of TG-treated lymphocytes after exposure to EGTA was used to assess Ca2+ extrusion across the plasma membrane. Initial rates of Ca2+i decline were examined in cells suspended in Na(+)-containing and Na(+)-free solutions; initial rates were linearly related to the [Ca2+]i at the onset of the Ca2+i decline and were unaffected by varying the extracellular Ca2+. Extracellular Na+ increased the rate of Ca2+ extrusion and decreased the threshold [Ca2+]i for extrusion, indicating a substantial role for the Na(+)-Ca2+ exchange in Ca2+i homeostasis. Both decreased temperature and calmodulin inhibition significantly slowed the Ca2+i decline in Na(+)-free HEPES-buffered solution, suggesting Ca2+ extrusion under these conditions was mediated by the Ca2+ pump. Protein kinase C (PKC) activation or inhibition did not affect the Ca2+i decline parameters. However, Ca2+ accumulation and Mn2+ (a Ca2+ surrogate) uptake were significantly and Mn2+ (a Ca2+ surrogate) uptake were significantly inhibited by activators of PKC. Cyclic nucleotides altered neither the parameters of the Ca2+i decline nor Mn2+ uptake. Thus human T-lymphocytes exhibit Na(+)- and Ca(2+)-dependent transporters characterized as the Na(+)-Ca2+ exchanger and Ca2+ pump. The main effect of PKC in these cells is the modulation of Ca2+ entry across the lymphocyte plasma membrane.
Urea alters set point volume for K-Cl cotransport, Na-H exchange, and Ca-Na exchange in dog red blood cells
Authors: Parker J. C.
Published: December 2017

Urea equilibrates rapidly across the red blood cells of mammals. It was speculated that urea might affect the cell volume sensor by virtue of its properties as a protein perturbant. At concentrations of 0.1-0.6 M, urea caused a decrease in the set points for shrinkage-induced Na-H exchange, swelling-induced K-Cl cotransport, and swelling-induced Ca-Na exchange of dog red blood cells. Okadaic acid opposed the action of urea on all three pathways. The effects were reversible and not due to cyanate. Formamide and acetamide had actions similar to urea but not as potent. Equimolar concentrations of methanol had no effect. The coordinated influence of urea on three separate volume-activated transporters suggests that it acts on a mutual regulatory system that senses and transduces volume stimuli.
Glycosylation status of endogenous CFTR does not affect cAMP-stimulated Cl- secretion in epithelial cells
Authors: Morris A. P. , Cunningham S. A. , Benos D. J. , Frizzell R. A.
Published: December 2017

Cystic fibrosis (CF) impairs Cl- secretion across epithelial tissues and is caused by mutations in an N-linked glycoprotein, the cystic fibrosis transmembrane conductance regulator (CFTR). We modified the glycosylation pattern of CFTR using inhibitors of oligosaccharide processing and determined their effects on both agonist-induced Cl- secretion and CFTR location in human colon (HT-29) cell lines. In both polarized and unpolarized HT-29 cells, immunoprecipitation of cell extracts using a monoclonal antibody against CFTR gave a single band at 170 kDa. Inhibitors of N-linked glycosylation reduced the molecular mass of this band: swainsonine by 10 kDa, deoxymannojirimycin by 30 kDa, and deoxynojirimycin by 10-20 kDa. However, the transepithelial Cl- current and conductance stimulated by adenosine 3',5'-cyclic monophosphate (cAMP)- or Ca(2+)-dependent secretagogues was not affected. In the polarized cells, CFTR was localized in the apical membrane domain. Treatment of the monolayers with glycoprocessing inhibitors did not affect CFTR's location. Thus, in human colonocytes that endogenously express CFTR, the extent of CFTR glycosylation does not influence the targeting of CFTR to the apical membrane domain or its function as an agonist-stimulated Cl- channel.
Membrane potential oscillation from a novel combination of ion channels
Authors: Stelling J. W. , Jacob T. J.
Published: December 2017

Single pigmented epithelial cells from the ciliary body of the eye were studied using the whole cell voltage and current clamp, permeabilized patch recording, and patch-clamp recording. These cells can produce two types of oscillation. Both are slow, with a period in the range of 1-2 min; one has a low amplitude and oscillates between -60 and -80 mV, and the second is larger, with biphasic hyperpolarizing and depolarizing phases. The latter was seen when the membrane potential was driven negative by a constant current and results from the interplay between the inward rectifier K+ channel and a hyperpolarizing-activated cation channel. The hyperpolarization is caused by the constant current acting on a decreasing conductance as the inward rectifier inactivates, and the depolarization drive results from the activation of cation channels. It is suggested that the constant current would be provided by the Na+ pump in vivo, and such an interplay of channels and pumps could drive the uptake of cations in absorbing epithelia or provide an increased driving force for chloride exit in secretory epithelia.
Vasopressin depolymerizes apical F-actin in rat inner medullary collecting duct
Authors: Simon H. , Gao Y. , Franki N. , Hays R. M.
Published: December 2017

In amphibian bladder, arginine vasopressin (AVP) depolymerizes F-actin in the apical region of the granular cell, promoting fusion of water channel-carrying vesicles with the apical membrane. We now report the effect of AVP on F-actin in the mid- and terminal segments of rat inner medullary collecting duct (IMCD2 and IMCD3). In IMCD3, 5 min of stimulation by 2.5-250 nM AVP significantly depolymerized F-actin by 13-24% in whole cell assays employing the rhodamine-phalloidin binding technique. The IMCD2 was more sensitive, responding to subnanomolar (0.25 nM) AVP with 6 +/- 2% depolymerization. Depolymerization occurred as early as 2 min after 2.5 and 25 nM but not 250 nM AVP. 8-Bromoadenosine 3',5'-cyclic monophosphate depolymerized F-actin in IMCD3 at both 2 and 5 min. Immunogold labeling of the apical actin pool in IMCD3 principal cells was reduced by 26 +/- 5% (P < 0.05) by 2.5 nM AVP; the lateral and basal pools showed no significant changes. Capillary endothelial, thin limb of Henle, and intercalated cells showed no changes in immunogold labeling after AVP. Thus reorganization of the apical actin network by AVP is a consistent finding in both mammalian and amphibian target cells.
Effects of repeated eccentric contractions on structure and mechanical properties of toad sartorius muscle
Authors: Wood S. A. , Morgan D. L. , Proske U.
Published: December 2017

It has been proposed that lengthening of active muscle at long lengths is nonuniformly distributed between sarcomeres, with a few being stretched beyond overlap and most hardly being stretched at all. A small fraction of the overstretched sarcomeres may fail to reinterdigitate on subsequent relaxation, leading to progressive changes in the muscle's mechanical properties. Sartorius muscles of the toad Bufo marinus were subjected to repeated lengthening (eccentric) contractions at long lengths, while controls were passively stretched and then contracted isometrically or stretched at short lengths. The muscles undergoing eccentric contractions showed a progressive shift to the right of the length-tension curve, a fall in the yield point during stretch, an increase in slope of the tension response during stretch, and a fall in isometric tension. In control muscles, changes, if any, were significantly less. In electron micrographs, muscle fibers that had been subjected to a series of eccentric contractions showed sarcomeres with A bands displaced toward one half-sarcomere, leaving no overlap in the other half. Adjacent regions often looked normal. These results are all in agreement with the predictions of the nonuniform stretch of sarcomeres hypothesis.
Sodium-permeable channels in the apical membrane of human nasal epithelial cells
Authors: Chinet T. C. , Fullton J. M. , Yankaskas J. R. , Boucher R. C. , Stutts M. J.
Published: December 2017

We used patch-clamp techniques to study the channels that underlie the Na+ conductance of the apical membrane of human normal nasal epithelial cells. Cells were cultured on permeable supports and studied after confluence. In 172 of 334 (52%) excised membrane patches, we observed 20-pS Na(+)-permeable channels that do not discriminate between Na+ and K+ (pNa/pK = 1.33). These nonselective cation channels contained subpopulations that differed by dependence of open probability on voltage and bath Ca2+ activity, suggesting two or more channel types with similar electrical properties. In the presence of 10(-4) M amiloride in the pipette, the proportion of excised patches with nonselective cation channels decreased to 52 of 139 patches (37%), but the decrease was spread across all subpopulations of nonselective cation channels in excised patches. Thus no distinctive Na(+)-selective amiloride-sensitive channels were identified in excised patches. In cell-attached patches, Na(+)-permeable channels were recorded in 56 of 262 patches (21%). Their conductance was 21.4 +/- 1.5 pS (n = 25), and most were selective for Na+ over K+ (pNa/pK > 6). In the presence of amiloride (10(-4) M) in the pipette, the frequency of lambda Na(+)-permeable channels in cell-attached patches decreased to 8 of 134 patches (6%), revealing a population of Na(+)-selective channels recorded in cell-attached patches that was inhibited by amiloride. We conclude that, in excised patches, Na(+)-permeable channels are nonselective for Na+ over K+ and < 30% appear to be amiloride sensitive. In contrast, in cell-attached patches, most channels that conduct sodium are 1) selective for Na+ over K+ and 2) amiloride sensitive. Although we have not discovered the explanation for the discrepancy between cell-attached and excised patch data, we speculate that the channels recognized on cell account for the amiloride-sensitive Na+ conductance of the apical membrane, whereas the excision process alters the properties of the Na(+)-permeable channels and/or activate new channels.
Effects of adenosine antagonists on hexose uptake and preconditioning in perfused rat heart
Authors: Murphy E. , Fralix T. A. , London R. E. , Steenbergen C.
Published: December 2017

Preconditioning with brief intermittent periods of ischemia has been shown to lessen the detrimental effects of a subsequent sustained (30-60 min) period of ischemia. Because adenosine has been suggested to be the mediator of preconditioning, we were interested in investigating whether adenosine antagonists would block the effect of preconditioning on ionic changes during ischemia. We found that 10 microM of the adenosine antagonist BW-A1433U did not reverse the effect of preconditioning on intracellular pH (pHi). Hearts preconditioned with BW-A1433U had virtually no decrease in pHi during the 30-min sustained period of ischemia; after 30 min of ischemia, the pH in untreated hearts was 5.97 +/- 0.16 compared with 6.52 +/- 0.10 in preconditioned hearts and 6.90 +/- 0.08 in hearts preconditioned plus BW-A1433U. Because anaerobic glycolysis is largely responsible for the fall in pHi during ischemia, we examined the effect of BW-A1433U [and other adenosine antagonists, such as PD-115,199 and 8-cyclopentyl-1,3-dipropylxanthine (CPDPX)] on glucose uptake and phosphorylation during aerobic perfusion using 31P-nuclear magnetic resonance to monitor uptake and phosphorylation of 2-deoxyglucose (2-DG) to 2-deoxyglucose 6-phosphate (2-DG-6-P) when one-half of the glucose in the perfusate was replaced with 2-DG. Uptake of 2-DG-6-P after 15 min was reduced by 66% in the presence of BW-A1433U and 82% in the presence of PD-115,199 as compared with untreated hearts, but was not reduced in the presence of CPDPX. Thus CPDPX was the only adenosine antagonist tested that did not block accumulation of 2-DG-6-P. We also found that CPDPX did not block the beneficial effect of preconditioning on ionic alterations during a sustained 30-min period of ischemia or the improved recovery of function on reflow.
G protein-mediated inhibition of inwardly rectifying K+ channels in guinea pig chromaffin cells
Authors: Inoue M. , Imanaga I.
Published: December 2017

Properties of inwardly directed rectification and its G protein-mediated inhibition in guinea pig chromaffin cells were studied using the whole cell version of the patch-clamp technique. The current-voltage (I-V) relationship for plateau currents in response to a 50-ms pulse showed an inwardly directed rectification between -80 and -140 mV and a negative slope at more negative potentials in normal solution. Replacement of Na+ with N-methyl-D-glucamine (NMDG) in the perfusate did not alter the plateau I-V relationship between -110 and -130 mV but did abolish the negative slope below -140 mV. The zero current or resting membrane potential in the NMDG solution was in fair agreement with the equilibrium potential for K+. The chord conductance-voltage relationship showed a good fit with the Boltzmann equation and shifted along the voltage axis by an approximate change in driving force on K+ when K+ concentration was increased. External Cs+ and Ba2+ produced a voltage-dependent inhibition of the inwardly directed rectification. These results indicate that inwardly rectifying (IR) K+ channels are mediating an inwardly directed rectification. Intracellular dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) produced a complete suppression of this IR K+ channel, irrespective of treatment with pertussis toxin. Adding GTP or guanosine 5'-O-(2-thiodiphosphate) to the patch solution resulted in a decrease in GTP gamma S inhibition of the K+ current. Internal application of vanadate was without effect. Time course of the inhibition of the IR K+ current coincided in part with that of inactivation of a nonselective cation current. In conclusion, IR K+ channels in the chromaffin cell are subject to G protein-mediated inhibition.
Oleic acid differentially affects gap junction-mediated communication in heart and vascular smooth muscle cells
Authors: Hirschi K. K. , Minnich B. N. , Moore L. K. , Burt J. M.
Published: December 2017

The effects of oleic acid (OA) on gap junction-mediated intercellular communication between A7r5 cells and neonatal rat cardiac myocytes were determined. In A7r5 cells the extent of dye coupling was influenced in a biphasic manner by increasing concentrations of OA. Low concentrations of OA (0.1-1 microM) reduced the incidence of dye coupling from 90% (in control cells) to approximately 50%. Further increases in OA concentration, up to 100 microM, had no further effect on extent of dye coupling. In contrast, dye coupling between cardiac myocytes was reduced to near zero levels in a linear fashion by 1-25 microM OA. Whereas high OA concentrations reduce junctional conductance (gj) between heart cells to zero [J. M. Burt, K. D. Massey, and B. N. Minnich. Am. J. Physiol. 260 (Cell Physiol. 29): C439-C448, 1991], gj between A7r5 cells was decreased by a maximum of 45% by OA. These differences in OA sensitivity between the two cell types were not explained by differences in the rate or magnitude of OA uptake by the cells or by differences in the fraction of incorporated OA accessible to albumin washout, i.e., the plasma membrane fraction. Instead, the activity of the individual channel types exhibited different sensitivities to OA. In the presence of increasing concentrations of OA, the activities of first the 70-pS channel population [composed of connexin40 (Cx40)] and then the 108-pS channel population (composed of Cx43) were diminished, leaving predominantly the 140-pS channels (composed of Cx43) at high OA concentrations. The uncoupling effects of OA in both cell types could be reversed by washout with albumin-containing solution; however, higher concentrations of albumin and more vigorous wash conditions were required for full recovery in the A7r5 cells. In addition, albumin also reversed the effects of OA on channel activity. These data suggest that OA binds with greater affinity to the 70- vs. 108- or 140-pS channels and associated with binding is reduced channel activity.
Induction of multidrug resistance downregulates the expression of CFTR in colon epithelial cells
Authors: Breuer W. , Slotki I. N. , Ausiello D. A. , Cabantchik I. Z.
Published: December 2017

The epithelial cell line HT-29, which constitutively expresses the cystic fibrosis transmembrane conductance regulator (CFTR), was induced to become drug resistant by cultivation in the presence of colchicine. The gradual acquisition of drug resistance was associated with a corresponding increase in the expression of the multidrug resistance P-glycoprotein (P-gp) and a marked (> 80%) decrease in the constitutive levels of CFTR protein, as determined by immunoblotting. The reduction in CFTR content occurred at the onset of acquisition of drug resistance when P-gp expression was still relatively low. Reversal of drug resistance by removal of colchicine from the culture medium led to a 70% decrease in P-gp levels and a concomitant 40% increase in CFTR. The levels of other membrane proteins such as Na(+)-K(+)-ATPase and alkaline phosphatase remained relatively constant (< 26% variation). We propose that a selective downregulation of CFTR is elicited by acquisition of the multidrug resistance (MDR) phenotype and that induction of P-gp expression leads to a reversible repression of CFTR biosynthesis. These findings provide an experimental foundation for the complementary patterns of expression of the CFTR and MDR1 genes observed in vivo.
Heterologous expression of delta F508 CFTR results in decreased sialylation of membrane glycoconjugates
Authors: Dosanjh A. , Lencer W. , Brown D. , Ausiello D. A. , Stow J. L.
Published: December 2017

The cystic fibrosis transmembrane conductance regulator (CFTR) is commonly mutated in cystic fibrosis to the delta F508 CFTR. CFTR has been shown to function as a adenosine 3',5'-cyclic monophosphate-dependent Cl- channel at the cell surface, and there is evidence to suggest that CFTR may also have a role in transmembrane Cl- conductance in intracellular membrane compartments. Studies using cells from cystic fibrosis patients or heterologous expression systems have demonstrated that defective Cl- conductance at the cell surface and defective acidification of the Golgi compartment are associated with the presence of mutant forms of CFTR. It is possible that mutation of CFTR could also result in altered Golgi function, consistent with reports of changes in the glycosylation of cell surface and secreted glycoproteins in cystic fibrosis. Glycosylation of cell surface glycoproteins, particularly levels of sialylation, may also be related to the increased binding of Pseudomonas to cystic fibrosis cells. The current study was undertaken to compare the sialylation of cell surface glycoconjugates in heterologous cells overexpressing normal CFTR or delta F508 CFTR. The presence of sialylated residues on cells was assessed by the surface binding of the specific lectins, wheat germ agglutinin and elderberry bark lectin. A fluorescent cholera toxin B subunit probe was used to measure surface binding to sialylated gangliosides in transfected cells. Our studies show that cells lacking CFTR and cells expressing normal CFTR have unaltered levels of sialylation. In contrast, cells expressing the delta F508 CFTR have significantly decreased amounts of sialylated glycoproteins and gangliosides on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)
Soluble alpha v beta 3-integrin ligands raise [Ca2+]i in rat osteoclasts and mouse-derived osteoclast-like cells
Authors: Zimolo Z. , Wesolowski G. , Tanaka H. , Hyman J. L. , Hoyer J. R. , Rodan G. A.
Published: December 2017

We evaluated the possible involvement of intracellular Ca2+ concentration ([Ca2+]i) changes in the action of alpha v beta 3-ligands, known to regulate osteoclast function. Rat osteoclasts or mouse osteoclast-like cells, as examined by microfluorimetry and fura 2, showed a transient [Ca2+]i increase when perfused with (all 0.1 microM) vitronectin, osteopontin, polypeptide echistatin, fibronectin, and Arg-Gly-Asp-Asp and Arg-Gly-Asp-Ser peptides (10(-4) M) but not with laminin, collagen I, collagen IV, or [Ala24]echistatin, in which Ala was substituted for Arg in the Arg-Gly-Asp complex. The threshold for echistatin was 10 pM, the 50% effective concentration was 1 nM, and the median [Ca2+]i increase was 420 nM above the resting level (217 +/- 22 nM) at saturating concentration of 0.1 microM. Echistatin did not cause Mn2+ influx, and 10 microM nifedipine, 10 nM omega-conotoxin, 5 mM Ni2+, or Cd2+ did not prevent [Ca2+]i change. However, extracellular Ca2+ was needed for the [Ca2+]i increase, probably enabling ligand-integrin interaction. Polyclonal and monoclonal (LM609) antibody as well as depletion of [Ca2+]i stores with 5 microM thapsigargin and Ca(2+)-free medium abolished the [Ca2+]i increase, after restoring extracellular Ca2+. Furthermore, the LM609 antibody induced a Ca2+ signal in the presence or absence of extracellular Ca2+, suggesting that the alpha v beta 3-ligand interaction is mediated at least partially by Ca2+ mobilized from intracellular stores.
Simultaneous measurement of intracellular Na+ and Ca2+ during K(+)-free perfusion in isolated myocytes
Authors: Hayashi H. , Satoh H. , Noda N. , Terada H. , Hirano M. , Yamashita Y. , Kobayashi A. , Yamazaki N.
Published: December 2017

To study the relationship between intracellular Na+ concentration ([Na+]i) and intracellular Ca2+ concentration ([Ca2+]i), guinea pig ventricular myocytes were loaded with both the Na(+)-sensitive probe, sodium-binding benzofuran isophthalate (SBFI), and the Ca(2+)-sensitive probe, fluo 3. [Na+]i was measured from the ratio image at 510 nm when excited at 340/380 nm. [Ca2+]i, expressed as the percent change of corrected fluo 3 fluorescence, was measured at 540 nm when excited at 500 nm. The fluorescent spectra of these probes were sufficiently different to allow for simultaneous measurement. After 30 min perfusion of K(+)-free solution, [Na+]i of rod-shaped cells increased from 6.4 +/- 0.5 to 20.6 +/- 2.6 mM, and [Ca2+]i increased to 256 +/- 36% of the control. [Ca2+]i was higher in spontaneously contracting cells and shortened cells than in rod-shaped cells at similar levels of [Na+]i. When Ca(2+)-free solution or Ni2+ (5 mM) was applied, [Ca2+]i was lower than in cells perfused with K(+)-free solution alone. It was suggested that extracellular Ca2+ and the Na(+)-Ca2+ exchange were involved in the increase in [Ca2+]i. In conclusion, we have developed a new method for the simultaneous measurement of [Na+]i and [Ca2+]i in isolated myocytes, which should be useful to study the relation between [Na+]i and [Ca2+]i.
BDM affects nucleotide binding and force generation steps of the cross-bridge cycle in rabbit psoas muscle fibers
Authors: Zhao Y. , Kawai M.
Published: December 2017

The effect of 2,3-butanedione monoxime (BDM) on elementary steps of the cross-bridge cycle was studied with the sinusoidal analysis technique in skinned rabbit psoas muscle fibers. Our results showed that isometric tension and stiffness decreased progressively with an increase in the BDM concentration. The MgATP and MgADP binding constants increased 27 and 6 times, respectively, when BDM was increased from 0 to 18 mM, whereas the phosphate binding constant did not change significantly. The equilibrium constants of the ATP isomerization and detachment step were not sensitive to BDM, whereas the equilibrium constant of the attachment (power stroke) step decreased with BDM. Thus, in the presence of BDM, the number of attached cross bridges decreases; more cross bridges accumulate in the detached state, causing isometric tension and stiffness to decline. However, our detailed analysis shows that the decrease in the number of attached cross bridges is approximately 40%, which is not adequate to account for the 84% decrease in the isometric tension when 18 mM BDM was present. Therefore we suggest that a thin-filament activation mechanism is also affected by BDM.
Modulation of intracellular calcium transients and dopamine release by neuropeptide Y in PC-12 cells
Authors: Chen X. , Westfall T. C.
Published: December 2017

In PC-12 cells differentiated with nerve growth factor, neuropeptide Y (NPY) potentiated the K(+)-evoked increase in intracellular calcium, but this potentiation was not mediated by classical Y1 or Y2 NPY receptors. The potentiation by NPY appeared to occur through the mobilization of calcium from intracellular stores because thapsigargin successfully blocked the potentiation. In contrast, the Y2 agonist, NPY-(13-36), attenuated the K(+)-evoked increase in intracellular calcium by decreasing the influx of extracellular calcium. The effect of NPY-(13-36) on dopamine release from PC-12 cells was next studied. NPY-(13-36) significantly attenuated the K(+)-evoked dopamine release in a concentration-dependent manner. Nifedipine and omega-conotoxin also attenuated the evoked dopamine release. In the presence of nifedipine or omega-conotoxin, NPY-(13-36) produced further inhibition of the evoked dopamine release. Furthermore, NPY-(13-36)-induced inhibition of dopamine release was abolished by pertussis toxin pretreatment. We conclude that the regulatory effects of NPY and analogues on intracellular calcium are mediated by multiple NPY receptor subtypes. Y2 receptor-mediated pertussis toxin-sensitive inhibition of the evoked dopamine release does not seem to be due to interactions with L- or N-type Ca2+ channels.
Protein turnover during metabolic arrest in turtle hepatocytes: role and energy dependence of proteolysis
Authors: Land S. C. , Hochachka P. W.
Published: December 2017

Hepatocytes from the western painted turtle (Chrysemys picta bellii) are capable of a coordinated metabolic suppression of 88% during 10 h of anoxia at 25 degrees C. The energy dependence and role of proteolysis in this suppression were assessed in labile ([3H]Phe-labeled) and stable ([14C]Phe-labeled) protein pools. During anoxia, labile protein half-lives increased from 24.7 +/- 3.3 to 34.4 +/- 3.7 h, with stable protein half-lives increasing from 55.6 +/- 3.4 to 109.6 +/- 7.4 h. The total anoxic mean proteolytic suppression for both pools was 36%. On the basis of inhibition of O2 consumption and lactate production rates by cycloheximide and emetine, normoxic ATP-dependent proteolysis required 11.1 +/- 1.7 mumol ATP.g-1.h-1 accounting for 21.8 +/- 1.4% of total cellular metabolism. Under anoxia this was suppressed by 93% to 0.73 +/- 0.43 mumol ATP.g-1.h-1. Summation of this with protein synthesis ATP turnover rates indicated that under anoxia 45% of total ATP turnover rate was directed toward protein turnover. Studies with inhibitors of energy metabolism indicated that the majority of energy dependence was found in the stable protein pool, with no significant inhibition occurring among the more labile proteins. We conclude that proteolysis is largely energy dependent under normoxia, whereas under anoxia there is a shift to a slower overall proteolytic rate that is largely energy independent and represents loss mostly from the labile protein pool.
Macromolecular crowding and confinement in cells exposed to hypertonicity
Authors: Garner M. M. , Burg M. B.
Published: December 2017

The nonideal properties of solutions containing high concentrations of macromolecules can result in enormous increases in the activity of the individual macromolecules. It has been proposed that molecular crowding and confinement occur in cells and are major determinants of the activity of the proteins and other intracellular macromolecules. This concept has important implications for cell volume regulation because, under crowded conditions, relatively small changes in concentration, consequent to alterations of water content, lead to large changes in macromolecular activity. This review considers several aspects of macromolecular crowding and confinement, including: 1) the physical chemical principles involved; 2) in vitro demonstrations of the effects; 3) relation to water activity; 4) estimates of the actual intracellular activity of water and macromolecules; 5) relation to osmotic regulation in various types of cells, including bacteria, red blood cells, and complex nucleated cells; and 6) the relation to inorganic ions and organic osmolytes in cells stressed by hypertonicity. We conclude that, while there is compelling evidence for important effects of molecular crowding in vitro and in red blood cells, the role of macromolecular crowding and confinement in osmotic regulation of more complex cells is an open question that deserves the extensive attention it is currently receiving.
Na-K-Cl cotransport in nystatin-treated tracheal cells: regulation by isoproterenol, apical UTP, and [Cl]i
Authors: Haas M. , McBrayer D. G.
Published: December 2017

Chloride secretion in mammalian airway epithelia is stimulated by beta-adrenergic agonists via an adenosine 3',5'-cyclic monophosphate (cAMP)-dependent mechanism and by apical triphosphate nucleotides (ATP, UTP) via a cAMP-independent mechanism. Both types of secretagogues are known to stimulate apical Cl channels in airway cells; however, to maintain a stimulated rate of secretion, basolateral Cl influx via Na-K-Cl cotransport must be upregulated in parallel with apical Cl efflux. To examine the regulation of basolateral cotransport activity and its relationship to apical Cl efflux, we examined Cl transport in confluent primary cultures of dog tracheal epithelial cells treated with nystatin, an antibiotic that increases the permeability of plasma membranes to small monovalent ions, including Cl. By applying nystatin to the apical membrane of these cultures, apical Cl permeability could be increased to the point where transepithelial Cl transport is limited by transport across the basolateral membrane, which reflects primarily the activity of the cotransporter. In cultures of tracheal cells not treated with nystatin, transepithelial (basolateral-to-apical) 36Cl flux was increased two- to threefold by exposure to isoproterenol (5 microM, basolateral) or apical UTP (10 microM). Apical application of nystatin (400 units/ml) increased the basal level of transepithelial 36Cl flux approximately 1.5-fold and eliminated UTP stimulation of this flux, although an approximately twofold stimulation by isoproterenol persisted. Nystatin treatment also abolished UTP stimulation of saturable, basolateral [3H]bumetanide binding, a measure of functioning Na-K-Cl cotransporters in these cells; isoproterenol stimulation of binding was only mildly inhibited by nystatin treatment. Lowering intracellular Cl concentration ([Cl]i) by incubating cultures with apical media containing nystatin and reduced [Cl] (NO3 replacement) increased both basolateral-to-apical 36Cl flux and [3H]bumetanide binding in the absence of secretagogues or cell shrinkage. The results support our previous suggestion, based entirely on [3H]bumetanide binding [M. Haas, D. G. McBrayer, and J. R. Yankaskas. Am. J. Physiol. 264 (Cell. Physiol. 32): C189-C200, 1993], that UTP stimulation of basolateral Na-K-Cl cotransport in airway epithelial cells is entirely secondary to, and requires, an increase in apical Cl efflux, and further suggest that a decrease in [Cl]i may be a signal for cotransport activation in response to UTP. In addition, a cAMP-dependent cascade initiated by isoproterenol appears to directly stimulate the cotransporter.
Structure and function of ryanodine receptors
Authors: Coronado R. , Morrissette J. , Sukhareva M. , Vaughan D. M.
Published: December 2017

Membrane depolarization, neurotransmitters, and hormones evoke a release of Ca2+ from intracellular Ca(2+)-storing organelles like the endoplasmic reticulum and, in muscle, the sarcoplasmic reticulum (SR). In turn, the released Ca2+ serves to trigger a variety of cellular responses. The presence of Ca2+ pumps to replenish intracellular stores was described more than 20 years ago. The presence of Ca2+ channels, like the ryanodine receptor, which suddenly release the organelle-stored Ca2+, is a more recent finding. This review describes the progress made in the last five years on the structure, function, and regulation of the ryanodine receptor. Numerous reports have described the response of ryanodine receptors to cellular ions and metabolites, kinases and other proteins, and pharmacological agents. In many cases, comparative measurements have been made using Ca2+ fluxes in SR vesicles, single-channel recordings in planar bilayers, and radioligand binding assays using [3H]ryanodine. These techniques have helped to relate the activity of single ryanodine receptors to global changes in the SR Ca2+ permeability. Molecular information on functional domains within the primary structure of the ryanodine receptor is also available. There are at least three ryanodine receptor isoforms in various tissues. Some cells, such as amphibian muscle cells, express more than a single isoform. The diversity of ligands known to modulate gating and the diversity of tissues known to express the protein suggest that the ryanodine receptor has the potential to participate in many types of cell stimulus-Ca(2+)-release coupling mechanisms.
Integrin-mediated adhesive properties of uroepithelial cells are inhibited by treatment with bacterial toxins
Authors: Elgavish A. , Pattanaik A. , Lloyd K. , Reed R.
Published: December 2017

Gram-negative bacteria are a dominant cause of urinary tract infection, and their ability to produce toxins is an important virulence attribute. Cellular mechanisms triggered by the production of toxins in the lower urinary tract have not been completely defined. Ureteral epithelial cells (UT; A. Elgavish, Infect. Immun. 61: 3304-3312, 1993) have served as an in vitro model to explore the possibility that bacterial toxins act on UT by affecting integrin-mediated adhesive properties. The effect of treatment with lipopolysaccharides (LPS) from three strains of the gram-negative Escherichia coli [055:B5 (LPS-1), 0111:B4 (LPS-4), and 0127:B8 (LPS-5)] and lipoteichoic acids from two gram-positive bacteria, Streptococcus faecalis (LT-2) and Bacillus subtilis (LT-3), were examined. LPS-5 inhibited markedly UT attachment to collagen and fibronectin. LPS-4 had no effect, whereas LPS-1 inhibited UT attachment to collagen but not to fibronectin. The fact that LPS-5 and LT-2 inhibited an Arg-Gly-Asp sequence-sensitive component of UT attachment to fibronectin is consistent with the possibility that these toxins acted via a mechanism involving typical fibronectin receptors. UT spreading was inhibited markedly by LPS-1, LT-2, and LT-3, whereas LPS-4 and LPS-5 had no effect. Because clustering of integrins is a crucial step in integrin-mediated signal transduction, the possibility that toxins inhibited spreading by affecting clustering was tested. Treatment with LT-2, which inhibited spreading dramatically, abolished completely a UT cell population containing more than five to eight beta 1- or beta 4-subunit-containing integrin clusters.(ABSTRACT TRUNCATED AT 250 WORDS)
Lysophosphatidic acids induce proliferation of cultured vascular smooth muscle cells from rat aorta
Authors: Tokumura A. , Iimori M. , Nishioka Y. , Kitahara M. , Sakashita M. , Tanaka S.
Published: December 2017

Lysophosphatidic acids (LPA) with a C18 fatty acyl group accelerated thymidine incorporation into cultured rat aortic vascular smooth muscle cells and stimulated their cell division. LPA acted synergistically with epidermal growth factor and fibroblast growth factor but additively with platelet-derived growth factor. The stimulatory actions of LPA were suggested to be rather specific from the following findings: 1) their stimulation of DNA synthesis increased with an increase in their acyl moiety; 2) lysophosphatidylcholine, a neutral lysophospholipid, had no mitogenic action but was cytotoxic at high concentrations; and 3) LPA induced a rapid external Ca(2+)-independent increase in intracellular Ca2+ concentration ([Ca2+]i) in single fura 2-loaded cells that resembled the receptor-mediated increases in [Ca2+]i triggered by different agonists, whereas lysophosphatidylcholine provoked a slow sustained increase in [Ca2+]i in an external Ca(2+)-dependent manner. These results are discussed in relation to the possible pathophysiological role of LPA.
Coupled secretion of chloride and mucus in skin of Xenopus laevis: possible role for CFTR
Authors: Engelhardt J. F. , Smith S. S. , Allen E. , Yankaskas J. R. , Dawson D. C. , Wilson J. M.
Published: December 2017

We used the isolated skin of Xenopus laevis to investigate the relationship between the secretion of salt, water, and mucus by submucosal glands expressing the cystic fibrosis transmembrane conductance regulator (CFTR). In situ hybridization and immunofluorescence provided evidence for specific expression of CFTR in the mucus-secreting cells of the subepidermal glands. Stimulation of isolated sheets of skin with 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate produced active Cl secretion and a marked increase in tissue conductance that was correlated with mucous cell degranulation and the distention of the glandular ducts. This coordinated increase in active secretion of salt and mucus was abolished by pretreatment of skins with bumetanide or by removing Cl from the bathing solutions. These results provide evidence for an intimate coupling between electrolyte transport and mucus secretion that may illuminate the pathophysiology of mucus-producing glands in cystic fibrosis lung disease.
Participation of Na+ channels in the response of carotid body chemoreceptor cells to hypoxia
Authors: Rocher A. , Obeso A. , Cachero M. T. , Herreros B. , Gonzalez C.
Published: December 2017

The role played by Na+ channels of carotid body (CB) chemoreceptor cells was investigated by studying the effects of tetrodotoxin (TTX) on the release of 3H-labeled catecholamines ([3H]CA) by adult rabbit CBs previously incubated with the precursor [3H]tyrosine. TTX inhibited partially the release of [3H]CA elicited by mild hypoxia (10 or 7% O2) or by depolarizing incubation medium containing 20 or 30 mM KCl, but the response to more intense hypoxia (5 or 2% O2) or to higher KCl concentration (40 or 50 mM) was not significantly affected. The release of [3H]CA elicited by acidic stimuli, either 20% CO2 (pH 6.6) or the protonophore dinitrophenol (100 microM), although comparable in magnitude to that elicited by mild hypoxia, was not modified by TTX. These results provide evidence for the first time that Na+ channels of chemoreceptor cells participate in the transduction of hypoxic stimuli into the neurotransmitter release response of these cells and suggest that Na+ current operates as an amplifying device that enhances the initial cell depolarization mediated by the closure of the O2-sensitive K+ channels. Sympathetic denervation of CBs was followed by a marked reduction in the release of [3H]CA elicited by veratridine or by 20 mM KCl, suggesting that the number of Na+ channels in chemoreceptor cells decreases after denervation.