bluuebunny/crossref_metadata_2025 huggingface dataset:
Method for recovering ATP content and mitochondrial function after chemical anoxia in renal cell cultures
Authors: Doctor R. B. , Bacallao R. , Mandel L. J.
Published: December 2017

Cultured renal cells provide a highly reproducible and malleable model to study cellular responses to metabolic perturbations. Nevertheless, there is currently no good method to achieve metabolic inhibition and complete recovery in cultured cells. This study describes a specific method for reversibly inhibiting both glycolytic and oxidative metabolism. Glycolysis was inhibited by removing all glycolytic substrates, and mitochondrial respiration was inhibited with rotenone, a site I inhibitor of the electron transport chain. Within 30 min, ATP values were decreased by 98%. Glycolysis was restored through the reintroduction of glucose. Oxidative metabolism was restored by the addition of heptanoate, a short odd-chain fatty acid, which supplies reducing equivalents to site II of the electron transport chain. Employing Madin-Darby canine kidney and LLC-PK1 cell lines, this protocol caused the immediate and complete recovery of mitochondrial respiration and, by 60 min, the complete recovery of cellular ATP levels. Application of this protocol should allow the investigation of the cellular effects and alterations that occur within cells recovering from sublethal energy depletion.
Soleus muscle nascent polypeptide chain elongation slows protein synthesis rate during non-weight-bearing activity
Authors: Ku Z. , Thomason D. B.
Published: December 2017

Protein synthesis rate of the soleus muscle decreases rapidly during non-weight-bearing activity. We isolated polysomes from 18-h non-weight-bearing soleus muscle to investigate the mechanism of this phenomenon. The distribution of polysomal alpha-actin mRNA and 18S rRNA on sucrose density gradients shows that polysomes shift to larger sizes (more ribosomes per mRNA) during non-weight-bearing activity. Furthermore, RNA is mobilized into the polysome pool of the non-weight-bearing soleus muscle; these data indicate that initiation of protein synthesis is not rate limiting. We explain these results as the slowing of nascent polypeptide chain elongation, such that there is a “traffic jam” of ribosomes on the mRNAs, increasing the number of ribosomes per mRNA while, at the same time, decreasing protein synthesis rate. In support of this hypothesis, myoblasts treated with a low dose of cycloheximide (a specific elongation inhibitor) show a similar shift in polysome size. A numerical model of protein synthesis further shows that elongation is more effective than initiation and termination in affecting protein synthesis and polysome size. We conclude that the non-weight-bearing-induced decrease in postural muscle protein synthesis rate is initially caused by slowing of nascent polypeptide chain elongation.
TGF-beta-induced macrophage colony-stimulating factor gene expression in various mesenchymal cell lines
Authors: Takaishi T. , Matsui T. , Tsukamoto T. , Ito M. , Taniguchi T. , Fukase M. , Chihara K.
Published: December 2017

We report here that transforming growth factor-beta (TGF-beta) can increase the expression level of macrophage colony-stimulating factor (M-CSF) mRNA in a variety of mesenchymal cell lines derived from osteoblasts, bone marrow stromal cells, fibroblasts, and myoblasts. The M-CSF activity in the conditioned medium of mouse osteoblast-like MC3T3-E1 cells was increased by TGF-beta as well as interleukin-1 (IL-1) treatment. The increase of M-CSF mRNA expression was observed as early as 2 h after TGF-beta or IL-1 addition and was superinduced by cycloheximide treatment. Nuclear run-off assays revealed that the increase in M-CSF mRNA by TGF-beta as well as IL-1 occurred, at least in part, at the transcriptional level. Platelet-derived growth factor (PDGF) also enhanced the M-CSF production in MC3T3-E1 cells. Furthermore, TGF-beta and IL-1 distinctly induced both PDGF-A and PDGF-B chain mRNA in MC3T3-E1 with different time courses. Our present studies suggest that PDGF autocrine loop-dependent and loop-independent pathways could modulate the M-CSF production stimulated by TGF-beta or IL-1 and account for the complexity of the cytokine network involving M-CSF in vivo under various physiological and pathological conditions.
Skeletal muscle junctional membrane protein content in pigs with different ryanodine receptor genotypes
Authors: Mickelson J. R. , Ervasti J. M. , Litterer L. A. , Campbell K. P. , Louis C. F.
Published: December 2017

The content of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, transverse tubule dihydropyridine receptor (DHPR), and SR ryanodine receptor (RyR) was determined in muscle of pigs homozygous for the normal RyR allele and homozygous or heterozygous for the malignant hyperthermia-susceptible (MHS) RyR allele. Total muscle membranes isolated from 1-day-old pigs of the three different genotypes did not differ in the content of any of these proteins. However, at 28 days of age, crude membranes and total muscle homogenates from homozygous MHS pigs exhibited only 61-81% of the [3H]PN 200-110 or [3H]ryanodine binding of identical preparations isolated from normal pigs; these MHS membranes also contained only 50% of the normal content of each of the DHPR subunits. The crude membranes and muscle homogenates from heterozygous pigs were intermediate to both types of homozygotes in terms of [3H]PN 200-110 binding, [3H]ryanodine binding, and the content of the DHPR subunits. However, membrane preparations enriched in triadic junctional proteins isolated from 3- to 4-mo-old pigs of the three different genotypes did not differ in their [3H]PN 200-110 binding, [3H]ryanodine binding, or Ca(2+)-ATPase activities. We conclude that, although the stoichiometry of the RyR to DHPR is not altered, the presence of the MHS RyR allele during muscle development results in a decreased relative content of these two proteins. This is probably due to a lower junctional membrane content and may be an important ultrastructural consequence of the altered sarcoplasmic Ca2+ regulation in MHS muscle.
Anomalies in ion transport in CF mouse tracheal epithelium
Authors: Grubb B. R. , Paradiso A. M. , Boucher R. C.
Published: December 2017

The cystic fibrosis (CF) mouse trachea has become a model for gene transfer. To characterize ion transport properties of tracheal epithelium from normal and CF mice, tracheas were excised, mounted in Ussing chambers, and basal properties and responses to pharmacological agents and/or ion substitution protocols measured. No difference in basal short-circuit (Isc) was observed between normal (29.1 +/- 3.8 muA/cm2, n = 21) and CF (34.7 +/- 4.5 muA/cm2, n = 16) tracheas. The relative contribution of Na+ transport to basal Isc was small (30-40%). Ionomycin stimulated large increases in Isc in both normal and CF murine tracheas [change in Isc (delta Isc) with ionomycin: 30.5 +/- 8.8 muA/cm2, n = 11, normal; 27.3 +/- 6.7 muA/cm2, n = 6, CF]. Unexpectedly, forskolin increased Isc in both CF and normal amiloride-pretreated tracheas (delta Isc: 10.5 +/- 2.1 muA/cm2, n = 21, normal; 13 +/- 2.3 muA/cm2, n = 16, CF). Forskolin was observed to increase intracellular Ca2+ in both normal and CF tracheal cells, suggesting this as a mechanism to induce Cl- secretion. These similarities in ion transport, in part reflecting the dominance of Ca(2+)-regulated Cl- conductance, suggest that the murine trachea is not an ideal target for assessment of CF correction by gene transfer.
Characterization of Na(+)-H+ antiporter activity associated with human cheek epithelial cells
Authors: McMurchie E. J. , Burnard S. L. , Patten G. S. , Lee E. J. , King R. A. , Head R. J.
Published: December 2017

Na+ transport activity was characterized in human cheek epithelial cells obtained from normotensive adult subjects. The cells were isolated using a mouth-wash procedure and assayed for Na+ uptake using a radioactive (22Na+) rapid filtration assay. Cheek cells displayed proton-dependent Na+ uptake activity that was dependent on the magnitude of the externally directed proton gradient measured using the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to determine intracellular pH. Amiloride, ethylisopropylamiloride (EIPA), 5-(N,N-dimethyl)-amiloride, 5-(N-methyl-N-isobutyl)-amiloride (MIA), and 5-(N,N-hexamethylene)-amiloride (NNHA) all inhibited proton-dependent Na+ uptake, with MIA, EIPA, and NNHA being the most potent. The Michaelis constant (Km) for extracellular Na+ was 5.7 mM, while the maximum velocity for Na(+)-H+ antiporter activity was 4.3 nmol Na+.mg protein-1.30s-1. The Km for intracellular H+ was 0.17 microM, with a Hill coefficient of 0.7. Stimulation by ouabain and inhibition by bumetanide of cheek cell proton-dependent Na+ uptake indicated only relatively low activities of Na(+)-K(+)-ATPase and Na(+)-K(+)-2Cl- cotransport, respectively. These results are consistent with the presence of Na(+)-H+ antiporter activity in cheek cells. Cheek cells therefore provide a convenient, relatively noninvasive source of tissue for examining Na(+)-H+ antiporter activity in human subjects.
Interferon-gamma induces a cell surface phenotype switch on T84 intestinal epithelial cells
Authors: Colgan S. P. , Parkos C. A. , Matthews J. B. , D'Andrea L. , Awtrey C. S. , Lichtman A. H. , Delp-Archer C. , Madara J. L.
Published: December 2017

Intestinal epithelia are in intimate contact with submucosal and intraepithelial lymphocytes. The concentration of intraepithelial lymphocytes increases during inflammatory processes, and, when stimulated, these cells generate cytokines such as interferon-gamma (IFN-gamma). In this study, we examined the effect of recombinant human IFN-gamma on ion transport events in T84 cells, a crypt epithelial cell line widely used to study electrogenic Cl- secretion, the transport event responsible for mucosal hydration. Epithelial exposure to IFN-gamma brought about a marked attenuation in stimulated Cl- secretion, as measured by generation of short-circuit current (ISC). This IFN-gamma-elicited decrease in the Cl- secretory response was present for a variety of specific agonists, appeared largely due to IFN-gamma interactions with the basolateral surface, and did not result from a defect in second messenger generation. Efflux and uptake studies were utilized to functionally define the individual cell surface transport proteins that participate in Cl- secretion and revealed that, in response to epithelial exposure to IFN-gamma, apical Cl- channels and basolateral Na(+)-K(+)-2Cl- cotransporters, K+ channels, and Na-K-adenosinetriphosphatase were all functionally downregulated. [3H]bumetanide binding assays suggested that surface expression of the cotransporter was diminished by > 70% after IFN-gamma preexposure. Concurrently, surface immunofluorescence studies revealed that epithelial exposure to IFN-gamma brought about the induction of major histocompatibility complex (MHC) class II molecule expression on T84 epithelial monolayers and markedly increased MHC class I surface expression. Finally, neutrophil-epithelial adhesion studies revealed that preexposure of epithelial monolayers to IFN-gamma elicited a beta 2-integrin-dependent induction of neutrophil adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)
Inhibition of acrosin by protein C inhibitor and localization of protein C inhibitor to spermatozoa
Authors: Zheng X. , Geiger M. , Ecke S. , Bielek E. , Donner P. , Eberspacher U. , Schleuning W. D. , Binder B. R.
Published: December 2017

Protein C inhibitor (PCI) is synthesized by cells throughout the male reproductive tract and is present in high concentrations (220 micrograms/ml) in seminal plasma. Seminal plasma as well as the acrosome of spermatozoa are rich in possible target proteases for PCI. We analyzed the interaction of PCI with acrosin, a serine protease stored in its zymogen form in the acrosome of spermatozoa. Purified human PCI inhibited the amidolytic activity of purified boar acrosin with an apparent second-order rate constant of 3.7 x 10(4) M-1.s-1. Inhibition was paralleled by the degradation of PCI from its 57- to its 54-kDa form. Human PCI also inhibited the amidolytic activity of activated human sperm extracts and formed complexes with acrosin as determined by an enzyme-linked immunosorbent assay. Immunocytochemistry revealed that morphologically abnormal spermatozoa stained for PCI antigen, whereas morphologically normal spermatozoa were negative. In immunoelectron microscopy, PCI was exclusively localized in the immediate vicinity of disrupted acrosomal membranes of sperm heads. These data suggest that PCI might function as a scavenger of prematurely activated acrosin, thereby protecting intact surrounding cells and seminal plasma proteins from possible proteolytic damage.
Selection for MDR1/P-glycoprotein enhances swelling-activated K+ and Cl- currents in NIH/3T3 cells
Authors: Luckie D. B. , Krouse M. E. , Harper K. L. , Law T. C. , Wine J. J.
Published: December 2017

The relationship between multidrug resistance (MDR) P-glycoprotein expression and swelling-activated Cl- and K+ conductance was investigated in mouse NIH/3T3 fibroblasts and their colchicine-selected counterparts (COL1000, high P-glycoprotein). Whole cell patch-clamp and isotopic flux experiments confirmed that swelling-activated Cl- currents were induced by 20-30% bath dilution only in the MDR-expressing cell line. However, at bath dilutions > 30%, both cell lines developed Cl- currents that reached similar large magnitudes at higher dilution levels. Thus the apparent absolute difference in cell lines at lower dilutions is due to a shift in the response curve relating hypotonicity to Cl- conductance. At all dilutions and in both cell lines, the swelling-activated Cl- currents were outwardly rectifying, active at negative cell voltages, and inactivated at positive voltages. Verapamil (100 microM) and 1,9-dideoxyforskolin (100 microM), which inhibit P-glycoprotein drug transport, did not significantly inhibit the swelling-activated Cl- conductance efflux in the COL1000 cells also showed a leftward shift in the response curve to hypotonicity. These results indicate that response curve to hypotonicity. These results indicate that colchicine-selection for increased P-glycoprotein expression did not lead to the expression of swelling-activated Cl- channels, but instead enhanced a step in the pathway from bath dilution to regulatory volume decrease that is common to both K+ and Cl- channels.
Activity and density of the Na+/H+ antiporter in normal and transformed human lymphocytes and fibroblasts
Authors: Siczkowski M. , Davies J. E. , Ng L. L.
Published: December 2017

The turnover number for the sodium-hydrogen exchanger isoform 1 (NHE-1) has been determined in human lymphocytes and MRC5 fibroblasts and in their virally transformed counterparts. Using fluorometric methods, we have determined the intracellular pH and Na+/H+ antiport activity of these cells. Intracellular pH was elevated in both lines of transformed cells. In contrast, Na+/H+ antiport activity was apparently unchanged in simian virus 40-transformed MRC5 fibroblasts (MRC5 SV1 TV1 28.9 +/- 5.2 mM/min compared with MRC5 fibroblasts 26.5 +/- 5.3 mM/min) but slightly increased in Epstein-Barr virus-transformed lymphoblasts (16.7 +/- 1.0 mM/min compared with lymphocytes 13.5 +/- 2.3 mM/min, P < 0.05). With the use of specific antisera to NHE-1, viral transformation was associated with a decreased number of NHE-1 molecules per cell in fibroblasts (from 441,504 +/- 53,428 to 64,745 +/- 7,151 sites/cell, P < 0.001) but an increased number in lymphocytes (from 14,066 +/- 3,100 to 22,474 +/- 4,050 sites/cell, P < 0.01). The NHE-1 density per cell yielded very similar turnover numbers for NHE-1 in the untransformed cells (lymphocytes, 3,161 +/- 833 cycles/s; MRC5 fibroblasts, 3,026 +/- 441 cycles/s), which were significantly elevated in the transformed cells (lymphoblasts, 8,471 +/- 1,177 cycles/s; MRC5 SV1 TV1, 10,521 +/- 2,299 cycles/s, P < 0.001 compared with untransformed cells). We conclude that viral transformation has different effects on Na+/H+ antiport activity and NHE-1 density per cell in different cell types, but the turnover number of NHE-1 is significantly increased after viral transformation, which correlates with the increased proliferation rate of these transformed cells.
Effect of isosmotic removal of extracellular Na+ on cell volume and membrane potential in muscle cells
Authors: Pena-Rasgado C. , Summers J. C. , McGruder K. D. , DeSantiago J. , Rasgado-Flores H.
Published: December 2017

Isosmotic removal of extracellular Na+ (Nao) is a frequently performed manipulation. With the use of isolated voltage-clamped barnacle muscle cells, the effect of this manipulation on isosmotic cell volume was studied. Replacement of Nao by tris(hydroxymethyl)aminomethane produced membrane depolarization (approximately 20 mV) and cell volume loss (approximately 14%). The membrane depolarization was verapamil insensitive but depended on extracellular Ca2+ (Cao) and was probably due to activation of intracellular Ca2+ (Cai)-dependent nonselective cation channels. The cell volume loss did not require membrane depolarization but depended on Cao. This was probably due to an increase in Cai, mediated by activation of Ca2+ influx via Na+/Ca2+ exchange. Nao replacement by Li+ also promoted membrane depolarization (approximately 20 mV) and cell volume loss (20%). Both effects were reduced (approximately 73%) but were not abolished by Cao removal. Under this condition, the remaining membrane depolarization was probably due to a higher membrane permeability of Li+ over Na+. The remaining cell volume loss was due to membrane depolarization, which probably induced Ca2+ release from intracellular stores.
cAMP and inositol 1,4,5-trisphosphate increase Ca2+ in HT-29 cells by activating different Ca2+ influx pathways
Authors: Denning G. M. , Clark R. A. , Welsh M. J.
Published: December 2017

Ca2+ plays a central role in regulating transepithelial fluid and electrolyte transport in intestinal epithelial cells. To investigate the mechanisms regulating the cytosolic free Ca2+ concentration ([Ca2+]c), we examined the effect of secretory agonists on [Ca2+]c in the intestinal epithelial cell line HT-29 clone 19A cells. We found that [Ca2+]c increased after addition of either adenosine 3',5'-cyclic monophosphate (cAMP)-dependent agonists or a D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]dependent agonist carbachol. Several lines of evidence suggest that cAMP- and Ins(1,4,5)P3-dependent agonists act through separate pathways. First, isoproterenol and forskolin increased cellular levels of cAMP but not Ins(1,4,5)P3, whereas carbachol increased cellular levels of Ins(1,4,5)P3 and stimulated inositol phosphate turnover without increasing cAMP. Second, carbachol increased [Ca2+]c by stimulating the release of Ca2+ from intracellular stores and influx of extracellular Ca2+. In contrast, cAMP agonists increased [Ca2+]c by stimulating Ca2+ influx alone. Third, the responses to maximal concentrations of cAMP agonists and carbachol were approximately additive. Finally, Ins(1,4,5)P3- but not cAMP agonist-dependent Ca2+ influx was inhibited by inorganic Ca2+ channel blockers. Thus, in intestinal epithelial cells, [Ca2+]c is regulated by at least two different second-messenger pathways, involving Ins(1,4,5)P3 or cAMP. In addition, cAMP stimulates influx of extracellular Ca2+ through a pathway distinct from that mediated by Ins(1,4,5)P3.
Labeling of the Amphiuma erythrocyte K+/H+ exchanger with H2DIDS
Authors: Maldonado H. M. , Cala P. M.
Published: December 2017

Subsequent to swelling, the Amphiuma red blood cells lose K+, Cl-, and water until normal cell volume is restored. Net solute loss is the result of K+/H+ and Cl-/HCO3- exchangers functionally coupled through changes in pH and therefore HCO3-. Whereas the Cl-/HCO3- exchanger is constitutively active, K+/H+ actively is induced by cell swelling. The constitutive Cl-/HCO3- exchanger is inhibited by low concentrations (< 1 microM) of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or H2DIDS, yet the concentration of H2DIDS > 25 microM irreversibly modifies the K+/H+ exchanger in swollen cells. We exploited the volume-dependent irreversible low-affinity reaction between H2DIDS and the K+/H+ to identify the protein(s) associated with K+/H+ exchange activity. Labeling of the membrane proteins of intact cells with 3H2DIDS results in high-affinity labeling of a broad 100-kDa band, thought to be the anion exchanger. Additional swelling-dependent low-affinity labeling at 110 kDa suggests the possibility of a volume-induced population of anion exchangers. Finally, the correlation between volume-sensitive K+/H+ modification and low-affinity labeling suggests that transport activity is associated with a protein of approximately 85 kDa. Although a 55-kDa protein is also labeled, it is a less likely candidate, since label incorporation and transport modification are less well correlated than that of the 85- and 110-kDa proteins.
Modulation of ATP-sensitive K+ channels by internal acidification in insulin-secreting cells
Authors: Fan Z. , Tokuyama Y. , Makielski J. C.
Published: December 2017

The effect of intracellular acidification (low pHi) on open probability of the ATP-sensitive K+ (KATP) channel was examined in insulin-secretion cells using an inside-out configuration of the patch-clamp technique. In an insulin-secreting cell line beta-TC3, KATP single-channel currents (IKATP) were readily recorded in the absence of internal ATP. ATP (50 microM and 0.5 mM) dramatically decreased the channel activity. A step decrease of intracellular pH (pHi) from 7.4 to 6.7 or 6.3 in the presence of ATP gradually increased the channel activity. In addition, low pHi in the presence of ATP could partially restore channel activity lost in a process called "rundown." Kinetic analysis revealed a change in channel gating at low pHi with ATP. The bursting durations of IKATP at pHi 6.3 in the presence of ATP were significantly longer than those at pHi 7.4 in the absence of ATP. These results suggest that the increased channel activity at low pHi might have resulted from a mechanism involving an alteration of channel conformation. We also observed an inhibitory effect of low pHi on channel activity. However, the inhibitory effect was much more apparent at pHi 5.7 and was only partially reversible. The activation effect of low pHi on IKATP in the presence of ATP was also observed in acutely isolated rat islet cells and in another insulin-secretion cell line RINm5F, although the effect was weaker and was variable among experiments. We conclude that, as in frog skeletal muscle and cardiac muscle, an increase in channel activity at low pHi is one of the mechanisms underlying proton modulation of IKATP in insulin-secreting cells.
Endotoxin tolerance is associated with decreased prostaglandin H synthases-1 and -2
Authors: Geisel J. , Cook J. A. , Ashton S. H. , Wise W. C. , Halushka P. V.
Published: December 2017

Lipopolysaccharide (LPS) induces macrophage protein and eicosanoid synthesis. Previous studies have shown that LPS induction of eicosanoid metabolism is transcription dependent and that prostaglandin (PG) H synthase is the committed step in the conversion of arachidonic acid (AA) to thromboxane (Tx) B2. LPS tolerance induced by sublethal in vivo injections of LPS renders rats resistant to LPS in vivo and macrophages refractory to LPS-stimulated eicosanoid synthesis in vitro. This study examined potential activity and content changes in constitutive and mitogen inducible PGH synthase in LPS-stimulated control and tolerant macrophages. TxB2 levels were measured to evaluate basal PGH synthase activity and stimulation by Salmonella enteritidis LPS (50 micrograms/ml), calcium ionophore A-23187, and AA. All tolerant macrophage groups demonstrated decreased TxB2 synthesis. TxB2 synthesis stimulated by AA in tolerant cells was decreased by 70% (P < 0.05) compared with control macrophages. In subsequent studies changes in PGH synthase content were examined in rat peritoneal macrophages from tolerant and control rats incubated with and without LPS. Immunoblot analysis of PGH synthase-1 (constitutive) demonstrated no increase in cells stimulated with LPS compared with basal but was diminished by 62 +/- 9% (n = 4, P < 0.05) in tolerant macrophages compared with control cells. Immunoblot analysis of PGH synthase-2 (mitogen inducible) demonstrated induction in response to LPS that was maximal between 12 and 24 h. PGH synthase-2 was also induced in tolerant macrophages in response to LPS but was less than in control cells. The results demonstrate that endotoxin tolerance is associated with reduced activity and content of PGH synthase-1 and a decreased LPS induction of PGH synthase-2.(ABSTRACT TRUNCATED AT 250 WORDS)
ATP-activated chloride channel inhibited by an antibody to P glycoprotein
Authors: Zhang J. J. , Jacob T. J.
Published: December 2017

In this report, we present the characteristics of a Cl- channel found in lens fiber cells. The single channel has a conductance of 17 pS, a linear current-voltage curve, is activated by ATP or strong depolarization and is blocked by verapamil, quinidine, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 5-nitro-2-(3- phenylpropylamino)benzoate, dideoxyforskolin, and tamoxifen. These properties are similar to those reported for a volume-activated Cl- channel associated with the multidrug resistance (MDR) gene product, P glycoprotein (24). Confirming this connection, we demonstrate that our lens Cl- channel is inhibited by an antibody to P glycoprotein. The data we present here may, therefore, be the first characterization of the single channel activity of the Cl- channel associated with P glycoprotein.
Endothelins stimulate mitogen-activated protein kinase cascade through either ETA or ETB
Authors: Wang Y. , Rose P. M. , Webb M. L. , Dunn M. J.
Published: December 2017

Endothelin (ET) has been shown to activate mitogen-activated protein kinase (MAPK). However, it has been unclear which of the ET receptors is coupled to MAPK activation. In the present study, we conducted experiments to determine which ET receptor is linked to MAPK activation. We found that both human ETA and ETB were coupled to the MAPK cascade in ETA or ETB cDNA-transfected Chinese hamster ovary cells. ET-1 was more potent than ET-3 in the activation of p42 MAPK, induction of MAPK kinase (MAPKK) gel retardation and uptake of [3H]thymidine in ETA-transfected cells, whereas sarafotoxin (S6c) showed no stimulatory effect on the kinases and [3H]thymidine uptake. ET-1, ET-3, and S6c had approximately the same potency to activate p42 MAPK, MAPKK gel retardation, and [3H]thymidine uptake in ETB-transfected cells. These data suggest that 1) ET isopeptides, through either ETA or ETB receptors, induce the MAPK cascade as well as cell proliferation; and 2) the different potencies of ET isopeptides for activation of the MAPK cascade and induction of cell growth are mainly due to their different affinities toward ETA and ETB.
Endothelin receptors in human smooth muscle cells: antagonist potency differs on agonist-evoked responses
Authors: Wu-Wong J. R. , Chiou W. J. , Huang Z. J. , Vidal M. J. , Opgenorth T. J.
Published: December 2017

The present study characterized endothelin (ET) receptors in human pericardium smooth muscle cells (HPSMC) and examined the potency of antagonists on ET-evoked signal transduction and DNA synthesis. HPSMC contain both ETA and ETB receptors. ET-1 binding was completely inhibited by a nonselective antagonist (Ro-46-2005) or a combination of ETA-selective and ETB-selective ligands (BQ-123 and ET-3). The molecular masses for ETA and ETB receptors were 69 and 42 kDa, respectively. ET-1, but not ET-3, stimulated phosphatidylinositol hydrolysis and arachidonic acid release in a dose- and time-dependent manner, reaching a plateau within 20-40 min. These immediate effects of ET-1 on signal transduction were completely inhibited by 1 microM, BQ-123, ET-1, but not ET-3, stimulated DNA synthesis in a dose-dependent manner, and the effect became prominent after 24 h. BQ-123 (1 microM) or Ro-46-2005 (10 microM) did not completely inhibit this mitogenic effect of ET-1. The reduced potency of BQ-123 and Ro-46-2005 on the delayed effect of ET-1 was not the result of ligand degradation or a difference in receptor internalization; rather, the decrease in potency was due to the fact that antagonist binding was more reversible than ET-1 binding.
Angiotensin II-dependent proximal tubule sodium transport is mediated by cAMP modulation of phospholipase C
Authors: Schelling J. R. , Singh H. , Marzec R. , Linas S. L.
Published: December 2017

Angiotensin II (ANG II) stimulates proximal tubule sodium transport by decreasing adenylyl cyclase activity. The role of ANG II-dependent phospholipase C is less certain. To determine the contribution of phospholipase C and adenylyl cyclase to apical (AP) ANG II-dependent sodium transport, unidirectional (AP to basolateral) 22Na flux was measured in rat proximal tubule cells cultured on permeable supports. AP ANG II (100 nM)-dependent sodium flux was prevented by preincubation with concentrations of the phospholipase C inhibitor U-73122 (1 microM) that blocked ANG II-dependent inositol phosphate formation. AP ANG II-dependent sodium flux was also abolished by preincubation with the intracellular calcium mobilization inhibitor 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), further suggesting that ANG II-dependent sodium transport was mediated by inositol phosphates. Neither U-73122 nor TMB-8 prevented ANG II-dependent adenosine 3',5'-cyclic monophosphate (cAMP) decreases. Incubation with dibutyryl cAMP (10 microM) or forskolin (10 microM) prevented ANG II-dependent sodium flux as well as ANG II-dependent inositol phosphate formation. In conclusion, ANG II-dependent proximal tubule sodium transport in cultured cells was transduced by phospholipase C and adenylyl cyclase. The adenylyl cyclase effect on ANG II-dependent sodium transport was mediated by phospholipase C.
Agonist-induced redistribution of calponin in contractile vascular smooth muscle cells
Authors: Parker C. A. , Takahashi K. , Tao T. , Morgan K. G.
Published: December 2017

Calponin is a thin filament-associated protein that has been implicated in playing an auxiliary regulatory role in smooth muscle contraction. We have used immunofluorescence and digital imaging microscopy to determine the cellular distribution of calponin in single cells freshly isolated from the ferret portal vein. In resting cells calponin is distributed throughout the cytosol, associated with filamentous structures, and is excluded from the nuclear area of the cell. The ratio of surface cortex-associated calponin to cytosol-associated calponin (R) was found to be 0.639 +/- 0.021. Upon depolarization of the cell with physiological saline solution containing 96 mM K+, the distribution of calponin did not change from that of a resting cell (R = 0.678 +/- 0.025, P = 0.369). Upon stimulation with an agonist (10 microM phenylephrine) that is known to activate protein kinase C (PKC) in these cells, the cellular distribution of calponin changed from primarily cytosolic to primarily surface cortex associated (R = 1.24 +/- 0.085, P < 0.001). This agonist-induced redistribution of calponin was partially inhibited by the PKC inhibitor calphostin, overlapped in time with PKC translocation, and preceded contraction of these cells. These results suggest that the physiological function of calponin may be to mediate agonist-activated contraction via a PKC-dependent pathway.
Agonist-induced changes of [Ca2+]i and membrane currents in single bovine aortic endothelial cells
Authors: Himmel H. M. , Rasmusson R. L. , Strauss H. C.
Published: December 2017

Cultured bovine aortic endothelial cells (BAECs) possess an inward rectifier K+ current (IK1), a Ca(2+)-activated K+ current, a nonselective cation current (INS), and a Ca(2+)-activated Cl- current; however, their relative roles remain to be established. In single BAECs, cytosolic free Ca2+ concentration ([Ca2+]i) [K5-fura 2 (50 microM), ratio 340/380 nm] was measured simultaneously with whole cell currents at 22 degrees C. Bradykinin (BK, 2 microM), ATP (10 microM), ionomycin (100 nM), or 2,5-di-(tert-butyl)-1,4-benzohydroquinone (10 microM) were used as agonists. In physiological salt solution (PSS), agonist exposure caused a rapid [Ca2+]i increase, followed by an increase in outward current (greater than -50 mV) and a smaller increase in inward current (greater than -80 mV). Chelation of [Ca2+]i with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid attenuated agonist-induced [Ca2+]i and current responses. Inactivation of the cyclooxygenase pathway by acetylsalicyclic acid and ibuprofen (50 microM each) did not affect the BK-induced [Ca2+]i transient but abolished the current response. In K(+)-free solution, agonist-stimulated outward currents (at +50 mV) were 10 times smaller than in PSS and were consistent with the activation of both INS and a Cl- current. In Cl(-)-free solution, the outward current response following agonist exposure was virtually abolished; at the same time, a linear inward current component with a reversal potential near the equilibrium potential for Na+ was activated. The maximal amplitude of the agonist-induced outward current decreased with decreasing symmetrical Cl- concentrations. Our results suggest that 1) IKI is the dominant current in resting BAECs; 2) K+, Cl-, and nonselective cation conductances contribute to the agonist-induced current response; and 3) most of the agonist-induced activation of currents depends on increased [Ca2+]i and is sensitive to cyclooxygenase inhibitors.
Loss of red blood cell glutathione during Mg deficiency: prevention by vitamin E, D-propranolol, and chloroquine
Authors: Mak I. T. , Stafford R. , Weglicki W. B.
Published: December 2017

Mg deficiency results in loss of red blood cell glutathione and was thought to be due to decreased Mg-dependent synthesis. The effects of vitamin E, D-propranolol, and chloroquine on red blood cell glutathione levels in Mg-deficient rats were examined. Feeding the rats a Mg-deficient diet for 3 wk resulted in an approximately 80% decrease in serum Mg and a 55% loss of red blood cell glutathione; concomitantly, plasma thiobarbituric acid reactive (TBAR) materials rose 240%. All three drug treatments had no effect on the plasma Mg levels but significantly inhibited the rise in TBAR content and attenuated (60-80% effective) the loss of glutathione. Red blood cell ghost membranes from the Mg-deficient rats also exhibited 2.3-fold higher TBAR content, which was attenuated by vitamin E treatment. With isolated red blood cells from Mg-sufficient rats, loss of glutathione could be induced by a chemical oxyradical system. Direct protective effects were afforded by alpha-tocopherol and D-propranolol but not by chloroquine. The data suggest that 1) the loss of glutathione during Mg deficiency was due to increased oxidative degradation, 2) both vitamin E and D-propranolol protected by a membrane antiperoxidative action, and 3) chloroquine probably protected by diminishing prooxidant activity secondary to its inhibition of cytokine induction during Mg deficiency.
Multiple modes of regulation of airway epithelial chloride secretion by extracellular ATP
Authors: Stutts M. J. , Fitz J. G. , Paradiso A. M. , Boucher R. C.
Published: December 2017

Cultured normal and cystic fibrosis (CF) airway epithelia were exposed to 5'-(N-ethylcarboxamido)-adenosine (NECA), ATP, or ionomycin. NECA activated a sustained, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-insensitive Cl- secretory response in normal but not CF, consistent with stimulation of the CF transmembrane conductance regulator (CFTR). In normal and CF, ionomycin or ATP induced Cl- secretion with an initial peak that was inhibited > 50% by DIDS, but in normals there was a prolonged current that was not inhibited by DIDS. The ATP and ionomycin responses in CF were of greater magnitude, and the prolonged phase was inhibited by DIDS. Although we expected ATP to regulate Cl- conductance through intracellular Ca2+ activity, ATP further stimulated Cl- secretion in tissues pretreated to maximally elevate intracellular Ca2+ activity. ATP also activated whole cell Cl- currents in cells dialyzed with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Thus ATP and ionomycin regulate a Cl- conductance that is distinct from CFTR, but the regulation by ATP is not tightly coupled to intracellular Ca2+ activity. Alternatively, ATP regulates separate Ca(2+)-sensitive and Ca(2+)-insensitive Cl- conductances. Furthermore, extracellular ATP activates DIDS-resistant Cl- secretion in normal but not CF cultured epithelia, consistent with activation of CFTR by extracellular ATP.
Kinetics of prostacyclin synthesis in PGHS-1-overexpressed endothelial cells
Authors: Sanduja S. K. , Tsai A. L. , Matijevic-Aleksic N. , Wu K. K.
Published: December 2017

The availability of a human endothelial cell overexpressed with prostaglandin H synthase-1 (PGHS-1) by retrovirus-mediated gene transfer made it possible to quantify the kinetics of prostacyclin [prostaglandin (PG)I2] synthesis and PGHS-1 turnover. Prostacyclin synthesis in response to arachidonate (AA) and ionophore A-23187 fit a single exponential kinetics. The rate constants for AA- and ionophore-treated cells were 0.064 min-1 [half-life (t1/2) of 11 min] and 0.032 min-1 (t1/2 = 22 min), respectively. The rate constant of PGI2 synthesis from PGH2 was 0.13 min-1. Using kinetic analysis coupled with computer modeling, the PGHS-1 half-life was determined to be 10.8 min. PGI2 production under successive treatments with AA or ionophore was reduced by only approximately 30% after each treatment. The decline of PGI2 synthesis corresponded to the reduction of PGHS-1 mass. The half-life of PGI2 synthesis from this analysis was at least an order of magnitude higher than that estimated from the single-dose experiment. These findings indicate that approximately 30% of PGHS-1 was degraded during each catalysis-induced autoinactivation and that the extent and duration of PGI2 synthesis are governed by the level of PGHS-1 mass.
Swelling-activated K+ fluxes in vascular endothelial cells: role of intracellular Ca2+
Authors: Perry P. B. , O'Neill W. C.
Published: December 2017

Swelling of bovine aortic endothelial cells activates Ca(2+)-dependent K+ channels. To determine the role of Ca2+ in this response, we examined the effect of cell swelling on intracellular Ca2+ concentration ([Ca2+]i), and the role of [Ca2+]i in swelling-activated K+ efflux. Basal [Ca2+]i, measured by fura 2 fluorescence, was 62 nM and increased by 36 nM in hypotonic medium (220 mosmol/l) compared with a 277 nM increase in response to extracellular ATP. In cells loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA), the increases induced by swelling and by ATP were reduced to 13 and 20 nM, respectively. Exposure to hypotonic medium (220 mosmol/kg) or to the Ca2+ ionophore A-23187 stimulated a furosemide-insensitive 86Rb efflux consistent with activation of K+ channels. The swelling-activated efflux was inhibited 16% by 5 mM tetraethylammonium and 24% by 23 mM tetrabutylammonium, but not by 100 microM quinidine, a pattern similar to that previously observed for swelling-activated K+ channels in cell-attached patches. The effects of A-23187 and hypotonic swelling on 86Rb efflux were completely additive, suggesting Ca(2+)-independent activation by cell swelling. Removal of Ca2+ from the external medium or loading of cells with BAPTA to buffer intracellular Ca2+ blocked the activation of 86Rb efflux by A-23187, but not by hypotonic swelling. Hypertonic medium (440 mosmol/kg by the addition of sucrose) attenuated the increased 86Rb efflux in response to A-23187. We conclude that the activation of K+ efflux in swollen endothelial cells occurs independently of changes in [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)
Cloning, functional expression, and characterization of a PKA-activated gastric Cl- channel
Authors: Malinowska D. H. , Kupert E. Y. , Bahinski A. , Sherry A. M. , Cuppoletti J.
Published: December 2017

cDNA encoding a Cl- channel was isolated from a rabbit gastric library, sequenced, and expressed in Xenopus oocytes. The predicted protein (898 amino acids, relative molecular mass 98,433 Da) was overall 93% similar to the rat brain ClC-2 Cl- channel. However, a 151-amino acid stretch toward the COOH-terminus was 74% similar to ClC-2 with six amino acids deleted. Two new potential protein kinase A (PKA) phosphorylation sites (also protein kinase C phosphorylation sites) were introduced. cRNA-injected Xenopus oocytes expressed a Cl- channel that was active at pHtrans 3 and had a linear current-voltage (I-V) curve and a slope conductance of 29 +/- 1 pS at 800 mM CsCl. A fivefold Cl- gradient caused a rightward shift in the I-V curve with a reversal potential of +30 +/- 3 mV, indicating anion selectivity. The selectivity was I- > Cl- > NO3-. The native and recombinant Cl- channel were both activated in vitro by PKA catalytic subunit and ATP. The electrophysiological and regulatory properties of the cloned and the native channel were similar. The cloned protein may be the Cl- channel involved in gastric HCl secretion.
pHi and serum regulate AE2-mediated Cl-/HCO3-, exchange in CHOP cells of defined transient transfection status
Authors: Jiang L. , Stuart-Tilley A. , Parkash J. , Alper S. L.
Published: December 2017

Page C855: L. Jiang, A. Stuart-Tilley, J. Parkash, and S. L. Alper. “pHi and serum regulate AE2-mediated Cl-/HCO3-, exchange in CHOP cells of defined transient transfection status.” The acknowledgment section omitted our thanks to Genetics Institute and Dana Dubois for subsidizing the additional cost of publication in color.
cGMP antagonizes angiotensin-mediated phosphatidylcholine hydrolysis and C kinase activation in mesangial cells
Authors: Barnett R. L. , Ruffini L. , Ramsammy L. , Pasmantier R. , Friedlaender M. M. , Nord E. P.
Published: December 2017

Previous studies from this laboratory have shown that in cultured rat mesangial cells (MC), angiotensin II (ANG II) mediates its effects via activation of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PC-PLD). In addition, guanosine 3',5'-cyclic monophosphate (cGMP)-elevating maneuvers that stimulate particulate and soluble guanylate cyclase [atrial natriuretic factor (ANF) and sodium nitroprusside (SNP), respectively] antagonize ANG II-mediated PI-PLC activation. The current study explored whether cGMP impairs ANG II-mediated PC-PLC and PLD activity. The ANG II-stimulated release of the water-soluble metabolites of PC breakdown (phosphorylcholine and choline) was blocked by ANF and SNP. ANG II-stimulated phosphatidic acid and phosphatidylethanol formation were significantly reduced by ANF and SNP, confirming that cGMP blunted PLD activity. The inhibitory effect of cGMP on PLD could be reversed by N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide, a blocker of cGMP-dependent protein kinase. In parallel experiments, ANF and SNP abrogated sustained diacylglycerol (DAG) accumulation derived from ANG II stimulation of PC hydrolysis, confirming that cGMP diminished PC-PLC activity. Inhibition of PC-derived DAG accumulation by cGMP was associated with a concomitant decrement in ANG II-mediated translocation of protein kinase C (PKC) activity from the cytosol to the membrane. In summary, in MC, cGMP antagonizes ANG II-mediated PC hydrolysis, DAG formation, and PKC activation. We propose that cGMP-mediated inhibition of phospholipid metabolism and PKC translocation plays an important role in MC vasorelaxation.
Modulation of a potassium conductance in developing skeletal muscle
Authors: Wieland S. J. , Gong Q. H.
Published: December 2017

K+ conductances dominate and potentially modulate the resting potential of skeletal muscle cells. The expression and modulation of a major K+ conductance were examined during in vitro differentiation of the mouse myoblast cell line C2C12. The inwardly rectifying K+ conductance (IKi) increased from unmeasurable levels in undifferentiated myoblasts to approximately 1.56 +/- 0.51 nA (n = 17) in myoballs derived from myotubes at 5 days after induction of differentiation. The inward rectifier was subject to modulation by intracellular signals. Exposure of cytoplasm to guanosine 5'-O-(3-thiotriphosphate) during whole cell recording produced a concentration (5-100 microM)- and time (1-20 min)-dependent inhibition of the mean conductance. Elevation of intracellular free Ca2+ (> 200 nM) also inhibited IKi. These findings demonstrate a potential mechanism for modulation of the resting potential of muscle fibers via the control of skeletal muscle IKi.
Perichondrial localization of ETA receptor in rat tracheal and xiphoid cartilage and in fetal rat epiphysis
Authors: Lodhi K. M. , Sakaguchi H. , Hirose S. , Shibabe S. , Hagiwara H.
Published: December 2017

Autoradiographic studies using 125I-labeled endothelin-1 (ET-1) on sections of rat cartilage tissues, including the trachea, xiphisternum, and fetal rat epiphysis, revealed dense localization of endothelin receptors in the perichondrium. In contrast, the binding of ET-1 was not detected in the chondrocytes, cartilage matrix, and other connective tissues of the cartilage tissues tested. The perichondrial binding of 125I-ET-1 was completely abolished with BQ-123 [an endothelin receptor subtype A (ETA) antagonist] but not with BQ-3020 (an ETB agonist), and we demonstrated the perichondrial localization of ETA receptors. [3H]thymidine incorporation in vitro was significantly increased in rat xiphoid cartilage tissues exposed to ET-1. These findings suggest that the ET-1/ETA receptor system plays an important role in regulating cartilage metabolism and endochondral bone formation.
Further evidence of incomplete neural control of muscle properties in cat tibialis anterior motor units
Authors: Unguez G. A. , Roy R. R. , Pierotti D. J. , Bodine-Fowler S. , Edgerton V. R.
Published: December 2017

To examine the influence of a motoneuron in maintaining the phenotype of the muscle fibers it innervates, myosin heavy chain (MHC) expression, succinate dehydrogenase (SDH) activity, and cross-sectional area (CSA) of a sample of fibers belonging to a motor unit were studied in the cat tibialis anterior 6 mo after the nerve branches innervating the anterior compartment were cut and sutured near the point of entry into the muscle. The mean, range, and coefficient of variation for the SDH activity and the CSA for both motor unit and non-motor unit fibers for each MHC profile and from each control and each self-reinnervated muscle studied was obtained. Eight motor units were isolated from self-reinnervated muscles using standard ventral root filament testing techniques, tested physiologically, and compared with four motor units from control muscles. Motor units from self-reinnervated muscles could be classified into the same physiological types as those found in control tibialis anterior muscles. The muscle fibers belonging to a unit were depleted of glycogen via repetitive stimulation and identified in periodic acid-Schiff-stained frozen sections. Whereas muscle fibers in control units expressed similar MHCs, each motor unit from self-reinnervated muscles contained a mixture of fiber types. In each motor unit, however, there was a predominance of fibers with the same MHC profile. The relative differences in the mean SDH activities found among fibers of different MHC profiles within a unit after self-reinnervation and those found among fibers in control muscles were similar, i.e., fast-2 < fast-1 < or = slow MHC fibers.(ABSTRACT TRUNCATED AT 250 WORDS)