bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Ca(2+)-activated K+ channels in electrical activity of longitudinal and circular muscle layers of canine colon
Authors: Carl A. , Bayguinov O. , Shuttleworth C. W. , Ward S. M. , Sanders K. M.
Published: December 2017

The role of Ca(2+)-activated K+ channels (BK channels) in the canine colon was evaluated by testing the effects of charybdotoxin (ChTX) and tetraethylammonium on K+ currents of isolated myocytes and on electrical and mechanical activity of tissue strips. ChTX blocked Ca(2+)-activated outward current [IK(Ca)] in a dose- and voltage-dependent manner. No significant differences in IK(Ca) density, ChTX block, or Ca2+ sensitivity of BK channels were observed between circular and longitudinal myocytes. ChTX (100 nM) blocked 60% of current at +80 mV. Delayed rectifier current was not inhibited by 100 nM ChTX. In the absence of agonists, ChTX did not affect electrical or mechanical activity of circular muscle strips. In the presence of 10(-6) M BAY K 8644 or 10(-6) M acetylcholine, ChTX increased slow-wave duration and amplitude, induced membrane potential oscillations, and potentiated contraction. In unstimulated longitudinal muscle strips, ChTX depolarized the tissue, increased burst duration and spiking frequency, and resulted in an increase in contractions. These results indicate that BK channels are important regulators of colonic motility. In the longitudinal layer, BK channels are involved in setting membrane potential and determine excitability. In the circular layer, ChTX-sensitive channels do not participate in the in vitro basal electrical activity but limit the responses to excitatory agonists. w
Protein kinase inhibitor H-89 reverses forskolin stimulation of cardiac L-type calcium current
Authors: Yuan W. , Bers D. M.
Published: December 2017

Calcium currents (ICa) and barium currents (IBa) were measured in freshly isolated single ferret ventricular myocytes, using the whole cell patch-clamp and perforated patch-clamp techniques with Na and K currents blocked by tetraethylammonium and Cs. The membrane potential (Em) dependence of activation and steady-state inactivation curves were determined using a Boltzmann relation, where E0.5 is the Em at half-maximal conductance. Forskolin (1 microM) increased the rate of ICa inactivation, especially in perforated patch, but slowed IBa inactivation. The acceleration is likely to be due to greater Ca-dependent inactivation of ICa, where the slowing of IBa inactivation may be due to protein kinase A-dependent slowing of Em-dependent inactivation. Forskolin (1-10 microM) also increased ICa amplitude by two- to threefold and shifted the E0.5 for both activation and inactivation to more negative potentials by 7-8 mV. The effect of forskolin on the amplitude of ICa could be reversed by an inhibitor of adenosine 3',5'-cyclic monophosphate-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89; 1-10 microM). However, H-89 did not reverse the shift of E0.5 induced by forskolin. H-89 application by itself does not decrease basal ICa but does shift the E0.5 of both activation and inactivation to more negative values of Em. It is possible that H-89 reverses the shift induced by regulatory phosphorylation (due to forskolin) but induces a coincidental negative shift itself.
Amiloride-sensitive and HCO3(-)-dependent ion transport activated by aldosterone and vasotocin in A6 cells
Authors: Doi Y. , Marunaka Y.
Published: December 2017

We studied the effects of aldosterone (Aldo) and arginine vasotocin (AVT) on ion transport of renal epithelial cell line (A6) by measuring short-circuit current (Isc). AVT induced a rapid, transient increase in Isc, followed by a decrease toward the baseline in cells untreated with Aldo. In cells treated with Aldo, Isc showed a biphasic response to AVT, i.e., both transient and sustained increases over 40 min after addition of AVT. The transient increase was composed only of amiloride-insensitive Isc regardless of Aldo treatment, whereas the sustained increase contained both amiloride-sensitive and amiloride-insensitive components. The main part of the amiloride-insensitive, sustained Isc depended on HCO3(-). In cells treated with Aldo for 1 day, removal of HCO3(-) in the bathing solution enhanced the amiloride-sensitive component and decreased the amiloride-insensitive one. These data suggest that 1) Aldo treatment is necessary for an AVT-induced sustained increase of Isc and 2) a HCO3(-)-dependent Isc mainly contributes to the sustained increase in amiloride-insensitive Isc.
Electrophysiology of adult cat cardiac ventricular myocytes: changes during primary culture
Authors: Schackow T. E. , Decker R. S. , Ten Eick R. E.
Published: December 2017

To investigate the nature of electrophysiological changes in adult cat cardiac ventricular myocytes that may occur when cells are maintained in primary culture for 1-2 wk, the electrophysiology of cells freshly isolated from collagenase-perfused hearts (day 0 controls) was compared with that of cells maintained in primary culture for up to 14 days 1) on a two-dimensional (2D) surface (laminin-coated coverslips), which allowed for changes in cellular morphology, or 2) in a three-dimensional (3D) alginate matrix, which minimized changes in cell shape. Action potentials and whole cell ionic currents were recorded using a conventional whole cell patch technique. Whereas cellular resting potential and the depth of the "notch" terminating phase 1 were diminished relative to controls in 2D- and 3D-cultured cells, the action potential duration and the incidence of early afterdepolarizations (EADs) were increased relative to controls in 2D- but not in 3D-cultured cells. Corresponding alterations in whole cell ionic currents included a 40% reduction in inwardly rectifying K current (IK1) conductance (GK1) and a 90% reduction in transient outward K current (Ito) conductance (Gto) in 2D- and 3D-cultured cells relative to day 0 controls and a 50% increase in L-type Ca current (ICa-L) conductance (GCa-L) in 2D-cultured cells relative to 3D-cultured cells and day 0 controls. The reduction in Gto in long-term culture was half-maximal by days 7 and 8 and could not be attributed to reduced Ito availability, involvement of a noninactivating Ito, the cell culture procedure itself, or the presence of serum in the culture media. Gto was larger in day 0 cells from a heart with right ventricular hypertrophy than in day 0 normal control cells and was reduced subsequent to placement of cells in 3D culture for 19 days. The results suggest that long-term culture and change in cellular morphology can affect the electrophysiology of cardiac ventricular myocytes.
Signaling and growth responses of LLC-PK1/Cl4 cells transfected with the rabbit AT1 ANG II receptor
Authors: Burns K. D. , Harris R. C.
Published: December 2017

Angiotensin II (ANG II) receptors of the AT1 subtype are present on the apical and basolateral membranes of renal proximal tubule cells. Cells of the proximal tubulelike cell line, LLC-PK1/Cl4, were transfected with an expression plasmid containing cDNA encoding the rabbit AT1 ANG II receptor. In transfected cells, specific binding of 125I-ANG II was detected on both apical and basolateral membranes; wild-type LLC-PK1/Cl4 cells did not express ANG II receptors. In transfected cells, apical or basolateral ANG II increased both S6 kinase activity and incorporation of [3H]leucine. In cells pretreated with pertussis toxin, the stimulatory effect of apical or basolateral ANG II on [3H]leucine incorporation was abolished. In contrast, ANG II did not affect mitogenesis, determined by [3H]thymidine incorporation. Apical or basolateral ANG II (10(-6) M) stimulated phosphoinositide turnover by 13.4 +/- 4.4% (n = 8) and 16.3 +/- 4.2% (n = 9), respectively. The activity of protein kinase C, determined by phosphorylation of a specific protein kinase C peptide substrate, was also stimulated by ANG II in transfected cells. Apical or basolateral ANG II had no significant effect on cellular adenosine 3',5'-cyclic monophosphate levels. In permeabilized transfected cells, apical ANG II (10(-6) M) inhibited the phosphorylation of a specific peptide substrate of protein kinase A; lower apical concentrations or basolateral ANG II were without significant effect. These results indicate that AT1 ANG II receptors sort to both apical and basolateral membranes in renal epithelial cells and are coupled to activation of phospholipase C. ANG II stimulates protein synthesis by binding to either apical or basolateral receptors; this effect requires coupling to G proteins and may be mediated by activation of S6 kinase. Because high concentrations of ANG II exist in proximal tubule, binding to apical and basolateral receptors may regulate proximal tubule cell growth under physiological conditions.
Alteration of the sodium current in cat cardiac ventricular myocytes during primary culture
Authors: Schackow T. E. , Sheets M. F. , Decker R. S. , Ten Eick R. E.
Published: December 2017

To determine the response of cardiac Na current (INa) in adult cardiac ventricular myocytes to culture, single isolated ventricular myocytes from collagenase-perfused adult cat hearts were placed in primary culture for up to 2 wk on a two-dimensional (2D) surface (laminin-coated coverslips), which allowed the morphology of the myocytes to change markedly, or in a three-dimensional matrix (3D) of alginate, in which cell shape changed only minimally. Action potentials and INa were recorded from groups of 1) freshly isolated myocytes serving as the control (day 0),2) cells maintained in 2D culture for 9-14 days (2D, day 9-14), and 3) cells cultured in alginate for 9-14 days (3D, day 9-14) with use of a conventional whole cell patch technique. Maximal upstroke velocity (Vmax) of the action potential was reduced by approximately 50% in 2D- and 3D-cultured cells relative to controls. INa in 2D- and 3D-cultured cells was strikingly different from that in control myocytes. Half-maximal voltage (V 1/2) for the chord conductance-voltage relationship was shifted approximately 15 mV negatively to that for controls in 2D- and 3D-cultured cells. INa steady-state availability curve also shifted negatively relative to controls in 2D- and 3D-cultured myocytes, but the magnitude of this shift (approximately 16-20 mV) was greater than that for the chord conductance-voltage curve.(ABSTRACT TRUNCATED AT 250 WORDS)
Thrombin-induced mitogenesis in cultured aortic smooth muscle cells requires prolonged thrombin exposure
Authors: Bachhuber B. G. , Sarembock I. J. , Gimple L. W. , McNamara C. A. , Owens G. K.
Published: December 2017

Thrombin has been implicated in vascular smooth muscle cell (VSMC) proliferation after vessel injury. Its proliferative effects, which are mediated via proteolytic activation of a receptor similar or identical to the cloned thrombin receptor (TR), have markedly delayed kinetics. The present study demonstrates that, despite rapid thrombin receptor activation and similar time to S phase entry compared with classic polypeptide growth factors, prolonged thrombin exposure is required to promote maximal VSMC mitogenesis. Flow cytometric analysis of thrombin-stimulated cells revealed that thrombin induced a progressive increase in the growth fraction over 3 days in culture, an effect that was blocked by hirudin even late after thrombin addition. Northern blot hybridization after thrombin stimulation demonstrated that thrombin upregulates TR mRNA expression within 6 h. These findings indicate that VSMC proliferate in response to prolonged thrombin exposure and suggest that the mitogenic delay may involve not only the thrombin-dependent synthesis and activation of newly made TR but also the progressive thrombin-dependent recruitment of cells into the growth fraction.
Altered expression and function of hepatocyte gap junctions after common bile duct ligation in the rat
Authors: Fallon M. B. , Nathanson M. H. , Mennone A. , Saez J. C. , Burgstahler A. D. , Anderson J. M.
Published: December 2017

Gap junction channels allow intercellular exchange of ions and small molecules between adjacent cells. Such communication coordinates cellular and organ function in tissues, although it is unclear if altered gap junction expression and communication contribute to organ dysfunction in pathological states. We examined the immunofluorescent (IF) localization and mRNA and protein levels of the two hepatocyte gap junction proteins connexin 32 and connexin 26, after hepatic injury induced by common bile duct ligation (CBDL) in the rat. Intercellular communication was measured by comparing gap junction-mediated coordination of hormone-induced Ca2+ signals in isolated rat hepatocyte couplets from control and CBDL animals. Connexin 32 plasma membrane IF, protein, and mRNA levels decreased markedly early after CBDL and remained low at 14 days. Connexin 26 plasma membrane IF and protein levels also decreased markedly after CBDL, but mRNA levels rose, and a partial return in membrane IF and protein levels was noted at 9 and 14 days. Coordination of vasopressin-induced Ca2+ signals between cells in isolated rat hepatocyte couplets 1 day after CBDL was significantly impaired compared with controls. These results demonstrate that hepatocyte gap junction communication is impaired early after CBDL because of decreased connexin protein levels. Disruption of gap junctions after CBDL may contribute to loss of hepatic functions that depend on gap junction communication.
Presence of MRP8 and MRP14 in pancreatic cell lines: differential expression and localization in CFPAC-1 cells
Authors: Fanjul M. , Renaud W. , Merten M. , Guy-Crotte O. , Hollande E. , Figarella C.
Published: December 2017

A complex of two calcium binding proteins, MRP8 [also called cystic fibrosis (CF) antigen] and MRP14, proteins known to be expressed in cells of myeloid origin, has been shown to be present in higher amounts in the serum of CF patients and heterozygotes compared with normal subjects. We demonstrated here for the first time, by dot-blot analysis and immunocytochemistry, the expression and the presence of these S100 calcium binding proteins in the pancreatic cell lines CAPAN-1 and CFPAC-1, the latter provided from a patient with CF. Moreover, using immunocytochemical methods, we showed that the localization of MRP8 and MRP14 on the plasma membrane seems to be restricted to the cells expressing a cystic fibrosis transmembrane conductance regulator (CFTR) wild-type protein such as CAPAN-1 cells and CFPAC-1 cells transfected with a plasmid containing the nonmutated CFTR gene (CFPAC-PLJ-CFTR-6 cells). In CFPAC-1 cells, immunoreactivity remains in the cytoplasm throughout the stationary phase. We also showed an increased level of the mRNAs of the two proteins in the CFPAC-1 cells compared with those transfected with the nonmutated CFTR. The demonstration of a difference in the cellular localization of these two proteins and in their mRNA levels in the cell line of CF origin leads us to assume the existence of a possible correlation in the expression of the MRPs with that of the CFTR protein.
Characterization and inhibition of 15-lipoxygenase in human monocytes: comparison with soybean 15-lipoxygenase
Authors: Gleason M. M. , Rojas C. J. , Learn K. S. , Perrone M. H. , Bilder G. E.
Published: December 2017

These studies characterized a method to measure 15-lipoxygenase (15-LO) activity in human monocytes (HMC) exposed to interleukin-4 (IL-4) and compare the activity with that of soybean 15-LO. 15-LO activity was quantitated by measuring 15-[14C]hydroxyeicosa-5Z,8Z,11Z,13E-tetraenoic acid (15-HETE) production from the substrate [14C]arachidonic acid (AA) after high-performance liquid chromatographic or thin-layer chromatographic separation. 15-HETE production by HMC was significantly elevated after continuous exposure to a single dose (10 ng/ml) of IL-4 for 4 days, was maximal at 5 days, and remained elevated at 6 days. At 6 days 15-LO activity in IL-4-treated HMC was increased significantly (2.81 +/- 0.70 fmol 15-HETE/cell) compared with background levels of 15-HETE in untreated HMC (0.098 +/- 0.31 fmol 15-HETE/cell). 15-HETE production was linear in the range of 5 x 10(4) to 7 x 10(5) HMC/assay, from 2 to 160 microM AA, and during 5-10 min of incubation with AA. Ethyl 2-cyano-3-(3',4'-dihydroxyphenyl)propenoate (a caffeic acid analogue), N-methyl-4-benzyloxy-phenyl acetohydroxamate (RG-6866), esculetin, and four novel lipoxygenase inhibitors, a phenylcyanomethylene analogue (RP-27493) and three benzoxadiazine analogues (RP-64835, RP-65047, and RP-65208), inhibited HMC 15-LO, with concentrations eliciting 50% of maximal inhibition (microM) of 0.21, 0.8, 6.3, 10, 22, 20, 6.3, 3, and 20 and 5.8, 5, 12, 1, 30, 0.8, 0.15, 0.05, and 2.8 for inhibition of soybean 15-LO, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Extracellular glutamate flux regulates intracellular glutaminase activity in LLC-PK1-F+ cells
Authors: Welbourne T. C. , Mu X.
Published: December 2017

The role of extracellular glutamate flux in regulating intracellular glutaminase activity was assessed in confluent monolayers of proximal tubule-like LLC-PK1-F+ cells grown on porous supports. Glutamate is a well-known inhibitor of phosphate-dependent glutaminase (PDG). We hypothesized that, by restricting the flux of glutamate from the extracellular media, cellular level would fall, effecting deinhibition of the cellular glutaminase activity. To test this, cellular glutamate uptake and extracellular production were inhibited for 18 h by the addition of D-aspartate (10 mM) or acivicin (0.7 mM) to both apical and basal media. Inhibiting glutamate flux depressed cellular glutamate content 43 and 41%, respectively. Intracellular relative glutaminase activity, monitored as the breakdown of 14C-radiolabeled glutamine to glutamate, measured over 60 s in the presence of D-aspartate or acivicin showed a 2- to 2.5-fold increase with the fall in cellular glutamate. Interestingly, enhanced glutamine uptake after PDG deinhibition was predominantly expressed on the basal surface. Indeed, measuring glutamine utilization after gamma-glutamyltranspeptidase inhibition over the entire 18-h time course revealed inhibition at the apical surface but relative enhancement of uptake at the basal surface. The increased intracellular glutaminase pathway was also reflected in increased alanine production measured over the 18-h time course, despite the reduction in overall glutamine utilization. These results point to a major role for extracellular glutamate fluxes in regulating cellular glutamine metabolism and suggest that the intracellular pathway may be suppressed under these conditions.
An outwardly rectifying K+ current active near resting potential in human retinal pigment epithelial cells
Authors: Hughes B. A. , Takahira M. , Segawa Y.
Published: December 2017

Currents in freshly dissociated adult human retinal pigment epithelial (RPE) cells were studied using the perforated patch-clamp technique. The zero-current potential (V0) averaged -48.9 +/- 7.7 mV (n = 50). Depolarizing voltage pulses from -70 mV evoked an outward current that activated with first-order kinetics and that did not inactivate during prolonged depolarizations. Repolarizing the membrane potential produced tail currents that reversed near the K+ equilibrium potential, indicating that the sustained outward current was carried mainly by K+. The outwardly rectifying K+ conductance (gK) had an activation threshold voltage near -60 mV and was half-maximal at -37 mV. Approximately 25% of gK was active at the average V0. The K+ current was nearly completely blocked by 2 mM Ba2+ but was relatively insensitive to 20 mM tetraethylammonium. The kinetics, voltage dependence, and blocker sensitivity of this current clearly distinguish it from delayed rectifier K+ currents previously identified in RPE cells. We conclude that the sustained outward K+ current may help establish the resting potential of the apical and/or basolateral membranes and may also participate in K+ transport across the RPE.
MCP-1-stimulated monocytes preferentially utilize beta 2-integrins to migrate on laminin and fibronectin
Authors: Penberthy T. W. , Jiang Y. , Luscinskas F. W. , Graves D. T.
Published: December 2017

Recruitment of monocytes to inflammatory sites involves a series of sequential attachments and detachments to extracellular matrix proteins in response to a chemoattractant gradient. In this study we compared the migration of human peripheral blood monocytes on different extracellular matrix proteins in response to monocyte chemoattractant protein-1 (MCP-1) and N-formylmethionyl-leucyl-phenylalanine. Monocytes migrated more effectively on laminin compared with other extracellular matrix proteins. In contrast, this preference was not observed with neutrophils, suggesting that the monocytes and neutrophils may have differences in their migration on extracellular matrix proteins. To study this further, function-blocking monoclonal antibodies were used to examine mechanistically whether beta 1- and beta 2-integrins were involved in monocyte migration on fibronectin or laminin in response to MCP-1. Monocyte migration on both laminin and fibronectin was blocked 100% (P < 0.05) by intact monoclonal antibody, F(ab') fragments, and F(ab')2 fragments to beta 2-integrins. We also determined that antibodies to beta 2-integrins block monocyte migration that has already been initiated. In contrast, antibody to the beta 1-integrins inhibited monocyte migration by approximately 40% (P < 0.05). Thus monocytes that express both beta 1- and beta 2-integrins require utilization of beta 2-integrins in migration on extracellular matrix proteins. The results also suggest that beta 1-integrins facilitate monocyte migration but that monocyte migration is not absolutely dependent on the interaction of beta 1-integrins with extracellular matrix proteins. In contrast, neutrophil migration is beta 2-integrin dependent and is not facilitated by beta 1-integrins.
NIP- and NAP-taurine bind to external modifier site of AE1 (band 3), at which iodide inhibits anion exchange
Authors: Knauf P. A. , Spinelli L. J.
Published: December 2017

External iodide (I-o) inhibits AE1 (band 3)-mediated anion exchange in human red blood cells by binding to a noncompetitive inhibitory site, the external halide modifier site. External N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate (NAP-taurine) and N-(4-isothiocyano-2-nitrophenyl)-2-aminoethyl sulfonate (NIP-taurine) also inhibit Cl- exchange noncompetitively. Increasing I-o decreases the inhibitory potency of NIP-taurine in a competitive fashion; this effect is not due to I- binding to the transport site, which has little effect on the NIP-taurine affinity. Bis(sulfosuccinimidyl)-suberate (BSSS) abolishes the noncompetitive inhibitory effect of I-o and greatly reduces the inhibitory effect of NAP-taurine. Together with previous work, these data suggest that external halides, such as I-, Br-, and probably also Cl-, bind to the same noncompetitive inhibitory site as do NAP- and NIP-taurine and that these reagents can be used to label the halide modifier site. Lys-539, a probable reaction site of BSSS, lies within the same segment of AE1 that is labeled by NAP-taurine and thus may be part of the modifier site.
Phorbol esters downregulate expression of the sodium/calcium exchanger in renal epithelial cells
Authors: Smith L. , Porzig H. , Lee H. W. , Smith J. B.
Published: December 2017

The Na+/Ca2+ exchanger (NCE) contributes to Ca2+ reabsorption by connecting tubules of the nephron. A line of renal epithelial cells from monkey kidney (LLC-MK2) was used to investigate the regulation of NCE expression. After the activation of protein kinase C (PKC) by phorbol myristate acetate (PMA), NCE activity decreased exponentially by 75% in 48 h (half time approximately 19 h). PMA decreased NCE mRNA by 85% in 24 h. The decrease in NCE transcript preceded the downregulation of NCE activity. NCE protein was quantified with a monoclonal antibody to cardiac NCE. PMA decreased the binding of 3H-labeled antibody to cell sonicates by 40% in 24 h. Immunoblots show that PMA produced a marked and extended increase in membrane-associated PKC-alpha, although PMA depleted total PKC-alpha by 65% in 24 h. In vivo 32P labeling of myristolated alanine-rich C kinase substrate, a specific PKC substrate, confirmed that PMA produced a rapid and extended activation of PKC. 4 alpha-PMA, a stereoisomer of PMA that neither binds nor activates PKC, had no effect on NCE activity or transcript. These findings indicate that activation of PKC with phorbol esters downregulates NCE mRNA, protein, and activity in renal epithelial cells.
Actions of palytoxin on Na+ and Ca2+ homeostasis in human osteoblast-like Saos-2 cells
Authors: Monroe J. J. , Tashjian A. H.
Published: December 2017

Palytoxin (PTx) is a potent membrane-active agent produced by marine coelenterates that acts to stimulate bone resorption in organ culture at nanomolar concentrations. We report here the actions of PTx on Na+ and Ca2+ homeostasis in human osteoblast-like Saos-2 cells. PTx induced a rise in the cytosolic free Na+ concentration ([Na]i) by causing entry of extracellular Na+ (Na(e)+). PTx also caused a concentration-dependent biphasic rise in the cytosolic free Ca2+ concentration ([Ca2+]i) by enhancing entry of extracellular Ca2+ (Ca(e)2+). Entry of Na+ was dependent on the presence of Ca(e)2+ and was prevented by the Na+/Ca2+ exchange antagonist 3,4-dichlorobenzamil (DCB). Entry of Ca2+ was dependent on the presence of Na(e)+ but was not prevented by DCB. The actions of PTx on [Na+]i and [Ca2+]i were completely inhibited by pretreatment of the cells with ouabain. Ouabain alone had no acute effect on [Na+]i or [Ca2+]i in Saos-2 cells. We propose that interaction of PTx with the Na+ pump created a channel that allowed influx of Na(e)+ and Ca(e)2+. The rise in [Ca2+]i then stimulated the activity of the plasma membrane Na+/Ca2+ exchanger, which further enhanced Na(e)+ entry.
Effects of magnesium on nitric oxide synthase activity in endothelial cells
Authors: Howard A. B. , Alexander R. W. , Taylor W. R.
Published: December 2017

Magnesium modulates endothelium-dependent vasodilation in intact blood vessels. Therefore, the effects of magnesium on nitric oxide (NO) release by isolated endothelial cells and nitric oxide synthase (NOS) activity in endothelial cell homogenates were studied. Unstimulated and bradykinin-stimulated NO release by porcine aortic endothelial cell (PAEC) monolayers were unaffected by 30 min of exposure to magnesium concentrations varying from 0.010 to 10.0 mM. In contrast, when A-23187-stimulated cells were exposed to 0.01, 3.16, and 10.0 mM MgCl2, NO release was decreased by 11.3 +/- 1.8, 11.7 +/- 3.0, and 20.3 +/- 7.2%, respectively, compared with cells exposed to 1.0 mM MgCl2 (P < 0.01). These data suggested that a change in the intracellular magnesium concentration had an effect on NO release, in contrast to a change in the extracellular concentration, which did not have an effect. To further assess this possibility, crude NOS extracts were prepared from PAEC and exposed to MgCl2. NOS activity was measured via the conversion of L-[3H]arginine to L-[3H]citrulline. Increasing the concentration of MgCl2 by 1.0, 3.16, and 10.0 mM caused a 16.0 +/- 6.8, 17.1 +/- 1.7, and 38.6 +/- 5.3% decrease in citrulline formation, respectively (P < 0.05), suggesting a direct inhibition of NOS by MgCl2. No significant difference in the degree of inhibition of NOS activity was found between MgSO4 and MgCl2, thus ruling out a nonspecific chloride effect. In addition, increasing the concentration of NaCl to 15 mM had no effect on NOS activity, ruling out a nonspecific osmotic effect [101.6 +/- 10.5% of control activity, P = not significant (NS)].(ABSTRACT TRUNCATED AT 250 WORDS)
Measurement of SR free Ca2+ and Mg2+ in permeabilized smooth muscle cells with use of furaptra
Authors: Sugiyama T. , Goldman W. F.
Published: December 2017

The concentrations of intrasarcoplasmic reticulum (SR) free Ca2+ ([Ca2+]SR) and Mg2+ ([Mg2+]SR) were measured in furaptra-loaded saponin-permeabilized cultured aortic smooth muscle (A7r5) cells. Ca(2+)-independent fluorescence emitted by furaptra trapped within organelles, excited at 346 nm (isosbestic point), decreased with a half time of 30 min. All Ca2+ measurements appeared to be from SR, because the apparent Ca2+ distribution within permeabilized cells was uniform and therefore inconsistent with furaptra loading into mitochondria. Moreover, thapsigargin-induced SR Ca(2+)-adenosinetriphosphatase inhibition caused near-total depletion of Ca2+, and the metabolic poisons oligomycin and rotenone had no effect. Calibration curves relating 370 nm-to-346 nm ratios to [Ca2+] and to [Mg2+] were calculated in situ; dissociation constants for Ca2+ and Mg2+ binding were 49 microM and 6.8 mM, respectively. Resting [Ca2+]SR was 75-130 microM, with a mean of 97.2 +/- 2.2 microM (n = 376), whereas [Mg2+]SR, estimated in the absence of Ca2+, was 1.0 mM. Stimulation with inositol 1,4,5-trisphosphate resulted in time-dependent declines in [Ca2+]SR, and pretreatment with guanosine 5'-triphosphate caused a large increase in the rate of inositol 1,4,5-trisphosphate-evoked SR Ca2+ release, although guanosine 5'-triphosphate had no effect by itself. These observations indicate that furaptra will be a valuable tool with which to directly study [Ca2+]SR and SR function.
Sulfhydryl oxidation and activation of red cell K(+)-Cl- cotransport in the transgenic SAD mouse
Authors: De Franceschi L. , Beuzard Y. , Brugnara C.
Published: December 2017

The SAD mouse is characterized by the expression of human SAD hemoglobin (Hb), a super S Hb with a higher tendency to polymerize than HbS due to the presence of two additional mutations, Antilles beta 23Ile and D Punjab beta 121Glu. Monovalent cation transport was studied in erythrocytes from SAD-1 (Hb SAD = 19%) and beta-thal/SAD-1 (Hb SAD = 26%) mice. Erythrocytes containing Hb SAD exhibited dehydration, increased maximal rate of Na(+)-K+ pump, unchanged Rb+ flux via the Gardos channel, and increased K(+)-Cl- cotransport. K(+)-Cl- cotransport was defined as Cl(-)-dependent (substitution with sulfamate or methanesulfonate) okadaic acid-sensitive K+ efflux. Volume regulatory decrease via K(+)-Cl- cotransport was also increased in swollen SAD erythrocytes compared with controls. K(+)-Cl- cotransport was stimulated by staurosporine in all mouse strains, but the extent of stimulation was reduced in beta-thal/SAD-1 mice. Treatment with dithiothreitol reduced K(+)-Cl- cotransport activity in SAD-1 and beta-thal/SAD-1 mice to levels similar to that of control strains, indicating that reversible sulfhydryl oxidation contributes to the activated state of K(+)-Cl- cotransport in mouse erythrocytes that express transgenic human Hb SAD.
Thiophosphorylation independently activates each head of smooth muscle myosin in vitro
Authors: Harris D. E. , Stromski C. J. , Hayes E. , Warshaw D. M.
Published: December 2017

To determine whether thiophosphorylation of the 20-kDa myosin light chain activates each head of smooth muscle myosin independently of the head with which it is paired, chicken gizzard smooth muscle myosin was randomly thiophosphorylated, producing a mixture of unphosphorylated and singly and doubly thiophosphorylated myosin. Thiophosphorylation levels were measured by glycerol-urea gels, and the activity of this myosin was determined by actin-activated adenosinetriphosphatase measurements and in an in vitro motility assay, where the velocity of actin filaments moving over a myosin-coated surface is measured. Activity at each thiophosphorylation level was similar to that previously observed for mixtures of unphosphorylated and doubly thiophosphorylated myosin (D. E. Harris, S. S. Work, R. K. Wright, N. R. Alpert, and D. M. Warshaw. J. Muscle Res. Cell Motil. 15: 11-19, 1994). All doubly thiophosphorylated myosin was then formed into filaments and removed from randomly thiophosphorylated myosin by centrifugation. The remaining myosin (mixture of unphosphorylated and singly phosphorylated myosin), which could not polymerize because of their conformation, retained approximately 70% activity compared with mixtures of unphosphorylated and doubly thiophosphorylated myosin. Thus a thiophosphorylated smooth muscle myosin head can produce substantial biochemical and mechanical activity, even when it is paired with an unphosphorylated partner.
Stimulation of GLUT-1 glucose transporter expression in response to exposure to calcium ionophore A-23187
Authors: Mitani Y. , Behrooz A. , Dubyak G. R. , Ismail-Beigi F.
Published: December 2017

We tested the hypothesis that an increase in cytosolic calcium concentration stimulates glucose transporter isoform (GLUT-1) gene expression. Exposure of a rat liver cell line (Clone 9) to 3 microM A-23187 for 12 h resulted in 3-, 5-, and 10-fold increases in cytochalasin B-inhibitable 3-O-methyl-D-glucose transport, GLUT-1 protein, and GLUT-1 mRNA content, respectively. The induction of GLUT-1 mRNA in response to A-23187 is not preceded by a significant decrease in cell ATP content. This induction is prevented by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in conjunction with ethylene glycol-bis(beta-aminoethyl ether)-N,N, N',N'-tetraacetic acid. To investigate the mechanism of GLUT-1 mRNA induction, we found that exposure to A-23187 stabilized GLUT-1 mRNA: with the employment of actinomycin D, GLUT-1 mRNA had a half-life of 1.5 and 5.5 h in control and A-23187-treated cells, respectively. In nuclear run-on assays, the rate of GLUT-1 gene transcription was stimulated 1.5- to 1.7-fold in nuclei isolated from cells exposed to A-23187 for either 30 min or 2 h. These results demonstrate that exposure to A-23187 stimulates GLUT-1 gene expression and that the increase in GLUT-1 mRNA content is mediated in part by enhanced GLUT-1 gene transcription as well as decreased GLUT-1 mRNA degradation. The increase in GLUT-1 mRNA content, in turn, is associated with increased cell GLUT-1 content and enhanced glucose transport.
Memory of arterial receptor activation involves reduced [Ca2+]i and desensitization of cross bridges to [Ca2+]i
Authors: Ratz P. H. , Lattanzio F. A. , Salomonsky P. M.
Published: December 2017

Rabbit femoral arteries retain a memory of previous maximum receptor activation for up to 3-4 h after complete cessation of the stimulus, as reflected by a reduction in the steady-state contraction produced by a subsequent exposure to KCl. The present study examined the hypothesis that this modulatory effect involves alterations in postreceptor signal transduction. To quantify the degree of cellular downregulation induced by an episode of alpha 1-adrenoceptor stimulation, tissues were pretreated for 30 min with 10(-5) M phenylephrine (PE), washed for 10 min to cause complete relaxation, and activated with increasing concentrations of KCl. Pretreatment of tissues with PE resulted in a large reduction compared with control tissues in the ability of 20-60 mM KCl to increase stress and myosin light-chain phosphorylation. However, only at low (20 and 26 mM), not high (> 26 mM), KCl concentrations did PE pretreatment reduce the ability of KCl to increase intracellular free Ca2+ concentration ([Ca2+]i). These data support the hypothesis that memory of receptor activation involves reductions in both Ca2+ mobilization and the sensitivity of contractile proteins to [Ca2+]i.
Stimulation of Cl- secretion by extracellular ATP does not depend on increased cytosolic Ca2+ in HT-29.cl16E
Authors: Guo X. , Merlin D. , Harvey R. D. , Laboisse C. , Hopfer U.
Published: December 2017

Extracellular ATP and elevated cytosolic Ca2+ concentration ([Ca2+]i) are major secretagogues for Cl- in the goblet cell-like clone cl.16E derived from colonic HT-29 cells. The involvement of [Ca2+]i as a messenger for the purinergically stimulated Cl- secretion was investigated using whole cell patch-clamp and Ussing chamber techniques, as well as [Ca2+]i measurements using fura 2-loaded cells. Under voltage-clamp conditions, the whole cell current at +50 mV was 3 +/- 1 pA/pF under unstimulated conditions. Stimulation of purinergic receptors with 200 microM extracellular ATP increased the current at +50 mV to 41 +/- 10 pA/pF, with a half-maximal effective dose (ED50) of approximately 3 microM. The current was transient, usually lasting 1-2 min, and the current-voltage relationship was approximately linear between -70 and +50 mV. Evidence that the ATP-stimulated current was carried by Cl- included 1) the reversal potential of the current closely followed the Cl- equilibrium potential, and 2) the stimulated current was absent when Cl- was removed from both bath and pipette solutions. Exposure to ATP also increased [Ca2+]i, with an ED50 of approximately 1 microM and maximal changes (at 200 microM) from baseline (71 +/- 3 nM) to 459 +/- 50 nM. The ATP-dependent Cl- conductance increase was not diminished when [Ca2+]i was clamped at 100 nM using a Ca(2+)-1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering system. However, the ATP effect did require some basal level of Ca2+ because clamping [Ca2+]i at < 10 nM abolished activation of the Cl- conductance. The presence of the protein kinase A inhibitor H-89 or the protein kinase C inhibitor staurosprine did not change the ATP-activated Cl-conductance. These data demonstrate that the ATP-stimulated increase in Cl- current does not require an increase in [Ca2+]i, suggesting the involvement of either another signaling pathway or direct activation of Cl- channels by purinergic receptors.
Arachidonic acid stimulates protein tyrosine phosphorylation in vascular cells
Authors: Buckley B. J. , Whorton A. R.
Published: December 2017

Arachidonic acid and its metabolites are important cellular mediators. In this study, we report a novel role for arachidonic acid in vascular cell signaling. We tested the effects of exogenous arachidonic acid on protein tyrosine phosphorylation in cultured vascular endothelial and smooth muscle cells. Arachidonic acid stimulated the phosphorylation of tyrosine-containing proteins of approximately 58, 93, and 120 kDa in the three cell types studied. This response was dose dependent, with a maximum effect observed with 40 microM arachidonic acid. Phosphorylation was rapid and transient, reaching a peak 0.5 min after the addition of arachidonic acid and returning to baseline by 8 min. A common set of protein substrates was phosphorylated in smooth muscle cells treated with the Ca(2+)-mobilizing agonist endothelin, concomitant with an increase in endogenous unesterified arachidonic acid. To determine whether the protein tyrosine phosphorylation was due to arachidonic acid or to a metabolite, we used inhibitors of cyclooxygenase, lipoxygenase, and epoxygenase pathways. Ibuprofen, nordihydroguaiaretic acid, eicosatriynoic and eicosatetraynoic acids, and 8-methoxypsoralen failed to inhibit the arachidonic acid-mediated response. We also found increased protein tyrosine phosphorylation after treatment with oleic, linolenic and gamma-linoleic acid. These results suggest a mechanism of protein tyrosine phosphorylation that is directly stimulated by unmetabolized unsaturated fatty acids.
Epitope tagging permits cell surface detection of functional CFTR
Authors: Howard M. , DuVall M. D. , Devor D. C. , Dong J. Y. , Henze K. , Frizzell R. A.
Published: December 2017

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated Cl channel responsible for adenosine 3',5'-cyclic monophosphate (cAMP)-induced Cl secretion across the apical membranes of epithelial cells. To optimize its detection for membrane localization studies, we tagged CFTR with epitope sequences at the carboxy terminus or in the fourth external loop. When epitopes were added to the fourth external loop, the N-linked glycosylation sites in that loop were either preserved or they were mutated to produce a deglycosylated CFTR (dgCFTR). Tagged CFTRs were expressed in HeLa cells, and their cAMP-sensitive Cl permeability was assayed using the halide-sensitive fluorophore SPQ. CFTRs containing the M2 epitope showed halide permeability responses to cAMP, whereas cells expressing CFTR with the hemagglutinin (HA) tag showed little or no cAMP response. Xenopus oocytes expressing dgCFTR, with or without the M2 epitope, showed Cl conductance responses that were 20% of the wild-type response, whereas M2-tagged constructs retaining the glycosylation sites responded like wild-type CFTR. External M2-tagged CFTR was detected in the surface membrane of nonpermeabilized cells. The surface expression of the mutant M2-tagged CFTRs correlated with processing of these mutants (Gregory et al. Mol. Cell. Biol. 11:3886-3893, 1991). M2-901/CFTR is a useful reporter for the trafficking of wild-type and mutant CFTRs to the cell surface.
Adrenergic, dopaminergic, and muscarinic receptor stimulation leads to PKA phosphorylation of Na-K-ATPase
Authors: Beguin P. , Beggah A. , Cotecchia S. , Geering K.
Published: December 2017

Na-K-adenosinetriphosphatase (Na-K-ATPase) is a potential target for phosphorylation by protein kinase A (PKA) and C (PKC). We have investigated whether the Na-K-ATPase alpha-subunit becomes phosphorylated at its PKA or PKC phosphorylation sites upon stimulation of G protein-coupled receptors primarily linked either to the PKA or the PKC pathway. COS-7 cells, transiently or stably expressing Bufo marinus Na-K-ATPase wild-type alpha- or mutant alpha-subunits affected in its PKA or PKC phosphorylation site, were transfected with recombinant DNA encoding beta 2- or alpha 1-adrenergic (AR), dopaminergic (D1A-R), or muscarinic cholinergic (M1-AChR) receptor subspecies. Agonist stimulation of beta 2-AR or D1A-R led to phosphorylation of the wild-type alpha-subunit, as well as the PKC mutant, but not of the PKA mutant, indicating that these receptors can phosphorylate the Na-K-ATPase via PKA activation. Surprisingly, stimulation of the alpha 1B-AR, alpha 1C-AR, and M1-AChR also increased the phosphorylation of the wild-type alpha-subunit and its PKC mutant but not of its PKA mutant. Thus the phosphorylation induced by these primarily phospholipase C-linked receptors seems mainly mediated by PKA activation. These data indicate that the Na-K-ATPase alpha-subunit can act as an ultimate target for PKA phosphorylation in a cascade starting with agonist-receptor interaction and leading finally to a phosphorylation-mediated regulation of the enzyme.
Monosodium urate crystals promote neutrophil adhesion via a CD18-independent and selectin-independent mechanism
Authors: Reinhardt P. H. , Naccache P. H. , Poubelle P. E. , de Medicis R. , Kehrli M. E. , Kubes P.
Published: December 2017

The primary objective of this study was to determine whether monosodium urate (MSU) crystals induced neutrophil adhesion to cellular substrata and, if so, then to elucidate the molecular mechanisms involved. Human umbilical vein endothelial cells (HUVEC), as well as various other cellular substrata, were treated with various sized MSU crystals, washed, and then coincubated in the presence of neutrophils for 60 min. HUVEC exposed to MSU crystals but not to silica crystals or uric acid promoted neutrophil adhesion in a dose- and size-dependent manner, an event also observed with monolayers of rabbit synovial cells and rat intestinal epithelial cells. The increased neutrophil adhesion could not be attenuated by anti-CD18, anti-intracellular adhesion molecule-1, or various anti-selectin antibodies, despite the fact that scanning electron microscopy revealed that neutrophils were adhering primarily to the endothelial cells rather than to exposed crystals. CD18-deficient neutrophils adhered to MSU crystal-treated HUVEC as effectively as their CD18-positive counterparts. The neutrophil adhesion was temperature dependent but did not require protein synthesis. Additionally, HUVEC phagocytosis of crystals was necessary for subsequent neutrophil-endothelial cell interactions to transpire. Pretreatment of endothelial cells and neutrophils with colchicine significantly reduced the adhesive interaction. Our data demonstrate that exposure of endothelial and other cells to MSU crystals promotes neutrophil adhesion that occurs by a firm CD18-independent and selectin-independent adhesive mechanism.
Induction of a fast-oxidative phenotype by chronic muscle stimulation: histochemical and metabolic studies
Authors: Mayne C. N. , Sutherland H. , Jarvis J. C. , Gilroy S. J. , Craven A. J. , Salmons S.
Published: December 2017

Chronic electrical stimulation of skeletal muscle at 10 Hz induces fast-to-slow fiber type transformation. Does a lower aggregate amount of activity lead to a less complete transformation, or does it produce the same transformation over a longer time course? We examined this question by subjecting adult rabbit tibialis anterior and extensor digitorum longus muscles to continuous stimulation at 2.5 Hz for 2-12 wk. Most of the fibers acquired the histochemical and immunocytochemical characteristics of type 2A, not type 1, fibers. There was a corresponding rise in oxidative activity, but this was accompanied by a marked decline in anaerobic glycolysis. The activities of hexokinase and 3-oxoacid CoA-transferase stopped increasing after 2 wk, glutamate oxaloacetate transaminase after 4 wk, and beta-hydroxyacyl-CoA dehydrogenase after 6 wk of stimulation. Succinate dehydrogenase, citrate synthase, lactate dehydrogenase, and creatine phosphokinase continued to change up to 12 wk of stimulation. Changes in enzyme activity were not as rapid or as marked as those observed for stimulation at 10 Hz, and none showed the typical two-phase response of oxidative enzyme activities to stimulation at 10 Hz. The latter may therefore be dependent on induction of type 1 myosin isoforms.
Na-K-ATPase alpha-isoform expression in heart and vascular endothelia: cellular and developmental regulation
Authors: Zahler R. , Sun W. , Ardito T. , Kashgarian M.
Published: December 2017

The Na pump (Na-K-ATPase) is important for regulation of membrane potential and transport in smooth muscle and heart. The alpha (catalytic)-subunit of this pump has three isoforms: alpha 1 is ubiquitous, but alpha 2 and alpha 3 are mainly localized to excitable tissue. Physiological differences between isoforms are not completely understood, but alpha 3 pumps appear to have a lower affinity for intracellular Na and a higher ouabain affinity than alpha 1 pumps. The alpha 2-and alpha 3-isoform mRNAs are expressed at high levels in the normal adult rat cardiac conduction system. Although alpha 1 and alpha 3 are both globally expressed in neonatal rat myocardia, there is a switch in the myocardial isoform pattern from alpha 3 to alpha 2 after birth. There are also important species differences in cardiac isoform patterns. Furthermore, changes in Na-K-ATPase isoforms in heart and vascular tissue have been reported in association with hypertension, but little is known about isoform expression in normal endothelia. We therefore studied the cellular distribution of Na pump protein isoforms in neonatal and adult myocardia and endothelia. Immunohistochemical analysis of rat tissues showed that the alpha 1-isoform was expressed throughout atrial and ventricular myocardium, with alpha 1 the only isoform detectable in the adult t tubule system. Although alpha 2 was also present in ventricular myocytes, the signal was markedly stronger in conduction tissue and papillary muscle. In hearts from neonatal rats, the alpha 3-isoform predominated in the cardiac conduction system, whereas alpha 2 was not detectable in any structure except vascular endothelium. In tissues and in cell lines representing a variety of species and vessel sizes, endothelia of large vessels expressed primarily alpha 1, whereas alpha 2 could be detected in endothelia of small vessels in rat heart. No evidence of alpha 3 expression in endothelium was found. Thus the complex spatial and developmental regulation of Na pump isoform expression in cardiovascular tissues may provide additional correlates to distinct physiological roles of these transporters.