bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron paramagnetic resonance reveals age-related myosin structural changes in rat skeletal muscle fibers
Authors: Lowe Dawn A. , Surek Jack T. , Thomas David D. , Thompson LaDora V.
Published: December 2017

We tested the hypothesis that low specific tension (force/cross-sectional area) in skeletal muscle from aged animals results from structural changes in myosin that occur with aging. Permeabilized semimembranosus fibers from young adult and aged rats were spin labeled site specifically at myosin SH1 (Cys-707). Electron paramagnetic resonance (EPR) was then used to resolve and quantify the structural states of the myosin head to determine the fraction of myosin heads in the strong-binding (force generating) structural state during maximal isometric contraction. Fibers from aged rats generated 27 ± 0.8% less specific tension than fibers from younger rats ( P < 0.001). EPR spectral analyses showed that, during contraction, 31.6 ± 2.1% of myosin heads were in the strong-binding structural state in fibers from young adult animals but only 22.1 ± 1.3% of myosin heads in fibers from aged animals were in that state ( P = 0.004). Biochemical assays indicated that the age-related change in myosin structure could be due to protein oxidation, as indicated by a decrease in the number of free cysteine residues. We conclude that myosin structural changes can provide a molecular explanation for age-related decline in skeletal muscle force generation.
Molecular characterization of the 5′ regulatory region of rat sodium-dependent multivitamin transporter gene
Authors: Chatterjee Nabendu S. , Rubin Stanley A. , Said Hamid M.
Published: December 2017

Previous studies have demonstrated the involvement of a specialized, Na+-dependent carrier-mediated system for biotin uptake in mammalian intestine. The molecular identity of the carrier protein, the Na+-dependent multivitamin transporter (SMVT), has recently been identified. Upon characterization of transcript expression in the rat intestine, four distinct transcript variants (I-IV) due to heterogeneity at the 5′-untranslated region were found (Chatterjee NS, Kumar CK, Ortiz A, Rubin SA, and Said HM. Am J Physiol Cell Physiol 277: C605–C613, 1999). This finding raised the possibility that multiple promoters may be involved in driving the transcription of the SMVT gene. To test this possibility, we cloned the 5′ regulatory region of the SMVT gene by genome walking. A 6.5-kb genomic DNA fragment was identified and sequenced. Three putative promoters (P1, P2, and P3) that were separated by exons of the four previously identified transcript variants were, indeed, found. P1 was found to contain multiple putative regulatory regions like GATA-1, AP-1, AP-2, and C/EBP, including several repeats of purine-rich regions and two TATA-like elements. P2 and P3 were GC rich and also revealed the presence of many putative regulatory elements including several SP-1 consensus sequences. The functional identity of each promoter and the minimal regions required for its function were established by the luciferase assay following transfection of rat-derived cultured intestinal epithelial IEC-6 cells. The highest functional activity of the cloned promoters was found to be in the order of P1 > P2 > P3. These findings represent the first characterization of the 5′ regulatory region of any mammalian SMVT gene and should assist in the understanding of transcriptional regulation of this important gene.
Serotonin-elicited inhibition of Cl− secretion in the rabbit conjunctival epithelium
Authors: Alvarez Lawrence J. , Turner Helen C. , Zamudio Aldo C. , Candia Oscar A.
Published: December 2017

The effects of serotonin [5-hydroxytryptamine (5-HT)] on the transepithelial electrical properties of the short-circuited rabbit conjunctiva were examined. With this epithelium, the short-circuit current ( I sc) measures Cl− secretion plus an amiloride-resistant Na+ absorptive process. Apical addition of 5-HT (10 μM) elicited a prompt I screduction from 14.2 ± 1.2 to 10.9 ± 1.2 μA/cm2 and increased transepithelial resistance from 0.89 ± 0.05 to 1.03 ± 0.06 kΩ · cm2(means ± SE, n = 21, P < 0.05). Similar changes were obtained with conjunctivae bathed without Na+ in the apical bath, as well as with conjunctivae preexposed to bumetanide with the Cl−-dependent I sc sustained by the parallel activities of basolateral Na+/H+ and Cl−/HCO[Formula: see text] exchangers. In contrast, the 5-HT-evoked effects were attenuated by the absence of Cl−(Δ I sc = −0.5 ± 0.2, n = 5), suggesting that reduced Cl−conductance(s) is an effect of 5-HT exposure. In amphotericin B-treated conjunctiva and in the presence of a transepithelial K+gradient, 5-HT addition reduced K+ diffusion across the preparation by 13% and increased transepithelial resistance by 4% ( n = 6, P < 0.05), indicating that an inhibition in K+ conductance(s) was also detectable. Significant electrical responses also occurred under physiological conditions when 5-HT was introduced to epithelia pretreated with adrenergic agonists or protein kinase C, phospholipase C, phosphodiesterase, or adenylyl cyclase inhibitors or after perturbation of Ca2+ homeostasis. Briefly, the conjunctiva harbors the only known Cl−-secreting epithelium in which 5-HT evokes Cl− transport inhibition; receptor subtype and signal transduction mechanism were not determined.
Potent NK1 antagonism by SR-140333 reduces rat colonic secretory response to immunocyte activation
Authors: Moriarty Derek , Selve Norma , Baird Alan W. , Goldhill Jon
Published: December 2017

The potent neurokinin receptor 1 (NK1) antagonist SR-140333 has previously been shown to reduce castor oil-induced secretion in animal models. The importance of tachykinins in neuroimmune control of secretion and the effect of SR-140333 on key points in this pathway were elucidated in the present study to determine the type of intestinal dysfunction best targeted by this antagonist. Rat colonic secretion and substance P (SP) release were determined in vitro with the use of Ussing chamber and enzyme immunoassay techniques. NK1 receptors played a secretory role as receptor agonists stimulated secretion and SR-140333 antagonized the response to SP response (pKb = 9.2). Sensory fiber stimulation released SP and evoked a large secretion that was reduced by 69% in the presence of SR-140333 (10 nM). Likewise, mastocytes also released SP. The subsequent secretory response was reduced by 43% in the presence of SR-140333 (50 nM). SP was also released from granulocytes; however, this did not cause secretion. Functional NK3receptors were present in the colon as senktide stimulated secretion, an effect that was increased during stress. We conclude that NK3 receptors may play a role in stress-related disorders, whereas NK1 receptors are more important in mast cell/afferent-mediated secretion.
KCNQ4 channels expressed in mammalian cells: functional characteristics and pharmacology
Authors: Søgaard Rikke , Ljungstrøm Trine , Pedersen Kamilla Angelo , Olesen Søren-Peter , Jensen Bo Skaaning
Published: December 2017

Human cloned KCNQ4 channels were stably expressed in HEK-293 cells and characterized with respect to function and pharmacology. Patch-clamp measurements showed that the KCNQ4 channels conducted slowly activating currents at potentials more positive than −60 mV. From the Boltzmann function fitted to the activation curve, a half-activation potential of −32 mV and an equivalent gating charge of 1.4 elementary charges was determined. The instantaneous current-voltage relationship revealed strong inward rectification. The KCNQ4 channels were blocked in a voltage-independent manner by the memory-enhancing M current blockers XE-991 and linopirdine with IC50 values of 5.5 and 14 μM, respectively. The antiarrhythmic KCNQ1 channel blocker bepridil inhibited KCNQ4 with an IC50 value of 9.4 μM, whereas clofilium was without significant effect at 100 μM. The KCNQ4-expressing cells exhibited average resting membrane potentials of −56 mV in contrast to −12 mV recorded in the nontransfected cells. In conclusion, the activation and pharmacology of KCNQ4 channels resemble those of M currents, and it is likely that the function of the KCNQ4 channel is to regulate the subthreshold electrical activity of excitable cells.
Characterization of nucleoside transport systems in cultured rat epididymal epithelium
Authors: Leung George P. H. , Ward Jeffrey L. , Wong Patrick Y. D. , Tse Chung-Ming
Published: December 2017

The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was Na+ dependent, while 30% was Na+ independent. The Na+-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The Na+-dependent [3H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since Na+-dependent [3H]guanosine uptake was inhibited by thymidine by 20% and Na+-dependent [3H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of Na+-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma.
Cloning of a novel EGFR-related peptide: a putative negative regulator of EGFR
Authors: Yu Yingjie , Rishi Arun K. , Turner Jerrold R. , Liu Dayou , Black Eric D. , Moshier Jeffrey A. , Majumdar Adhip P. N.
Published: December 2017

Although epidermal growth factor receptor (EGFR) plays a key role in regulating cell proliferation, differentiation, and transformation in many tissues, little is known about the factor(s) that may modulate its function. We have isolated a cDNA clone from the rat gastroduodenal mucosa whose full length revealed 1,958 bp that contained 227 bp of 5′-untranslated region (UTR) and an open-reading frame encoding 479 amino acids, followed by 290 bp of 3′-UTR. It showed ∼85% nucleotide homology to the external domain of the rat EGFR. We refer to the product of the newly isolated cDNA as EGFR-related protein (ERRP). In Northern blot analysis with poly(A)+ RNA from different rat tissues, ERRP cDNA hybridized to several mRNA transcripts with the strongest reaction noted with a transcript of ∼2 kb. Maximal expression of the 2-kb mRNA transcript was observed in the small intestine, followed by colon, liver, gastric mucosa, and other tissues. Transfection of ERRP cDNA into a colon cancer cell line, HCT116, resulted in a marked reduction in proliferation in monolayer and colony formation in soft agar compared with the vector-transfected controls. In another colon cancer cell line, Caco-2, with a tetracycline-regulated promoter system, induction of ERRP expression in the absence of doxycycline was associated with a marked reduction in EGFR activation and proliferation. We conclude that the ERRP cDNA may represent a new member of the EGFR gene family and that ERRP plays a role in regulating cell proliferation by modulating the function of EGFR.
Acidification and glucocorticoids independently regulate branched-chain α-ketoacid dehydrogenase subunit genes
Authors: Wang X. , Chinsky J. M. , Costeas P. A. , Price S. Russ
Published: December 2017

Acidification or glucocorticoids increase the maximal activity and subunit mRNA levels of branched chain α-ketoacid dehydrogenase (BCKAD) in various cell types. We examined whether these stimuli increase transcription of BCKAD subunit genes by transfecting BCKAD subunit promoter-luciferase plasmids containing the mouse E2 or human E1α-subunit promoter into LLC-PK1 cells, which do not express glucocorticoid receptors, or LLC-PK1-GR101 cells, which we have engineered to constitutively express the glucocorticoid receptor gene. Dexamethasone or acidification increased luciferase activity in LLC-PK1-GR101 cells transfected with the E2 or E1α-minigenes; acidification augmented luciferase activity in LLC-PK1 cells transfected with these minigenes but dexamethasone did not. A pH-responsive element in the E2 subunit promoter was mapped to a region >4.0 kb upstream of the transcription start site. Dexamethasone concurrently stimulated E2 subunit promoter activity and reduced the binding of nuclear factor-κB (NF-κB) to a site in the E2 promoter. Thus acidification and glucocorticoids independently enhance BCKAD subunit gene expression, and the glucocorticoid response in the E2 subunit involves interference with NF-κB, which may act as a transrepressor.
Calpain 3 mRNA expression in mice after denervation and during muscle regeneration
Authors: Stockholm Daniel , Herasse Muriel , Marchand Sylvie , Praud Christophe , Roudaut Carinne , Richard Isabelle , Sebille Alain , Beckmann Jacques S.
Published: December 2017

Lack of functional calpain 3 in humans is a cause of limb girdle muscular dystrophy, but the function(s) of calpain 3 remain(s) unknown. Special muscle conditions in which calpain 3 is downregulated could yield valuable clues to the understanding of its function(s). We monitored calpain 3 mRNA amounts by quantitative RT-PCR and compared them with those of α-skeletal actin mRNA in mouse leg muscles for different types of denervation and muscle injury. Intact muscle denervation reduced calpain 3 mRNA expression by a factor of 5 to 10, while α-skeletal actin mRNA was reduced in a slower and less extensive manner. Muscle injury (denervation-devascularization), which leads to muscle degeneration and regeneration, induced a 20-fold decrease in the mRNA level of both calpain 3 and α-skeletal actin. Furthermore, whereas in normal muscle and intact denervated muscle, the full-length transcript is the major calpain 3 mRNA, in injured muscle, isoforms lacking exon 6 are predominant during the early regeneration process. These data suggest that muscle condition determines the specific calpain 3 isoform pattern of expression and that calpain 3 expression is downregulated by denervation.
Regulation of ClC-2 chloride channels in T84 cells by TGF-α
Authors: Bali Moëz , Lipecka Joanna , Edelman Aleksander , Fritsch Janine
Published: December 2017

The almost ubiquitously expressed ClC-2 chloride channel is activated by hyperpolarization and osmotic cell swelling. Osmotic swelling also activates a different class of outwardly rectifying chloride channels, and several reports point to a link between protein tyrosine phosphorylation and activation of these channels. This study examines the possibility that transforming growth factor-α (TGF-α) modulates ClC-2 activity in human colonic epithelial (T84) cells. TGF-α (0.17 nM) irreversibly inhibited ClC-2 current in nystatin-perforated whole cell patch-clamp experiments, whereas a superimposed reversible activation of the current was observed at 8.3 nM TGF-α. Both effects required activation of the intrinsic epidermal growth factor receptor (EGFR) tyrosine kinase activity, of phosphoinositide 3-kinase, and of protein kinase C. With microspectrofluorimetry of the pH-sensitive fluorescent dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, TGF-α was shown to reversibly alkalinize T84 cells at 8.3 nM but not at 0.17 nM, suggesting that 8.3 nM TGF-α-induced alkalinization activates ClC-2 current. This study indicates that ClC-2 channels are targets for EGFR signaling in epithelial cells.
Localization of ClC-2 Cl− channels in rabbit gastric mucosa
Authors: Sherry Ann M. , Malinowska Danuta H. , Morris Randal E. , Ciraolo Georgianne M. , Cuppoletti John
Published: December 2017

HCl secretion across the parietal cell apical secretory membrane involves the H+-K+-ATPase, the ClC-2 Cl− channel, and a K+ channel. In the present study, the cellular and subcellular distribution of ClC-2 mRNA and protein was determined in the rabbit gastric mucosa and in isolated gastric glands. ClC-2 mRNA was localized to parietal cells by in situ hybridization and by direct in situ RT-PCR. By immunoperoxidase microscopy, ClC-2 protein was concentrated in parietal cells. Immunofluorescent confocal microscopy suggested that the ClC-2 was localized to the secretory canalicular membrane of stimulated parietal cells and to intracellular structures of resting parietal cells. Immunogold electron microscopy confirmed that ClC-2 is in the secretory canalicular membrane of stimulated cells and in tubulovesicles of resting parietal cells. These findings, together with previous functional characterization of the native and recombinant channel, strongly indicate that ClC-2 is the Cl− channel, which together with the H+-K+-ATPase and a K+ channel, results in HCl secretion across the parietal cell secretory membrane.
Inorganic pyrophosphate generation and disposition in pathophysiology
Authors: Terkeltaub Robert A.
Published: December 2017

Inorganic pyrophosphate (PPi) regulates certain intracellular functions and extracellular crystal deposition. PPi is produced, degraded, and transported by specialized mechanisms. Moreover, dysregulated cellular PPiproduction, degradation, and transport all have been associated with disease, and PPi appears to directly mediate specific disease manifestations. In addition, natural and synthetic analogs of PPi are in use or currently under evaluation as prophylactic agents or therapies for disease. This review summarizes recent developments in the understanding of how PPi is made and disposed of by cells and assesses the body of evidence for potentially significant physiological functions of intracellular PPi in higher organisms. Major topics addressed are recent lines of molecular evidence that directly link decreased and increased extracellular PPi levels with diseases in which connective tissue matrix calcification is disordered. To illustrate in depth the effects of disordered PPi metabolism, this review weighs the roles in matrix calcification of the transmembrane protein ANK, which regulates intracellular to extracellular movement of PPi, and the PPi-generating phosphodiesterase nucleotide pyrophosphatase family isoenzyme plasma cell membrane glycoprotein-1 (PC-1).
Modulation of Na+/H+exchange activity by Cl−
Authors: Aharonovitz Orit , Kapus András , Szászi Katalin , Coady-Osberg Natasha , Jancelewicz Tim , Orlowski John , Grinstein Sergio
Published: December 2017

Na+/H+ exchanger (NHE) activity is exquisitely dependent on the intra- and extracellular concentrations of Na+ and H+. In addition, Cl− ions have been suggested to modulate NHE activity, but little is known about the underlying mechanism, and the Cl− sensitivity of the individual isoforms has not been established. To explore their Cl− sensitivity, types 1, 2, and 3 Na+/H+ exchangers (NHE1, NHE2, and NHE3) were heterologously expressed in antiport-deficient cells. Bilateral replacement of Cl− with nitrate or thiocyanate inhibited the activity of all isoforms. Cl− depletion did not affect cell volume or the cellular ATP content, which could have indirectly altered NHE activity. The number of plasmalemmal exchangers was unaffected by Cl− removal, implying that inhibition was due to a decrease in the intrinsic activity of individual exchangers. Analysis of truncated mutants of NHE1 revealed that the anion sensitivity resides, at least in part, in the COOH-terminal domain of the exchanger. Moreover, readdition of Cl− into the extracellular medium failed to restore normal transport, suggesting that intracellular Cl− is critical for activity. Thus interaction of intracellular Cl− with the COOH terminus of NHE1 or with an associated protein is essential for optimal activity.
Smooth muscle length-dependent PI(4,5)P2 synthesis and paxillin tyrosine phosphorylation
Authors: Sul Donggeun , Baron Carl B. , Broome Raymond , Coburn Ronald F.
Published: December 2017

We studied effects of increasing the length of porcine trachealis muscle on 5.5 μM carbachol (CCh)-evoked phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] synthesis and other parameters of phosphatidylinositol (PI) turnover. PI(4,5)P2 resynthesis rates in muscle held at 1.0 optimal length ( L o), measured over the first 6 min of CCh stimulation, were 140 ± 12 and 227 ± 14% of values found in muscle held at 0.5 L o and in free-floating muscle, respectively. Time-dependent changes in cellular masses of PI(4,5)P2, PI, and phosphatidic acid, and PI resynthesis rates, were also altered by the muscle length at which contraction occurred. In free-floating muscle, CCh did not evoke increases in tyrosine-phosphorylated paxillin (PTyr-paxillin), an index of β1-integrin signaling; however, there were progressive increases in PTyr-paxillin in muscle held at 0.5 and 1.0 L o during contraction, which correlated with increases in PI(4,5)P2 synthesis rates. These data indicate that PI(4,5)P2 synthesis rates and other parameters of CCh-stimulated inositol phospholipid turnover are muscle length-dependent and provide evidence that supports the hypothesis that length-dependent β1-integrin signals may exert control on CCh-activated PI(4,5)P2synthesis.
Mechanisms of reduced SR Ca2+ release induced by inorganic phosphate in rat skeletal muscle fibers
Authors: Duke Adrian M. , Steele Derek S.
Published: December 2017

The effects of inorganic phosphate (Pi) on Ca2+ release from the sarcoplasmic reticulum (SR) were studied in mechanically skinned rat skeletal muscle fibers. Application of caffeine or T-tubule depolarization was used to induce Ca2+ release from the SR, which was detected using fura 2 fluorescence. Addition of Pi (1–40 mM) caused a reversible and concentration-dependent reduction in the caffeine-induced Ca2+ transient. This effect was apparent at low Pi concentration (<5 mM), which did not result in detectable precipitation of calcium phosphate within the SR. The inhibitory effect of Pi exhibited a marked dependence on free Mg2+ concentration. At 0.5 mM free Mg2+, 5 mM Pi reduced the caffeine-induced transient by 25.1 ± 4.1% ( n = 13). However, at 1.5 mM free Mg2+, 5 mM Pi reduced the amplitude of caffeine-induced Ca2+ transients by 68.9 ± 3.1% ( n = 10). Depolarization-induced SR Ca2+release was similarly affected. These effects of Pi may be important in skeletal muscle fatigue, if an inhibitory action of Pi on SR Ca2+ release is augmented by the rise in cytosolic Mg2+ concentration, which accompanies ATP breakdown.
Key role for constitutive cyclooxygenase-2 of MDCK cells in basal signaling and response to released ATP
Authors: Ostrom Rennolds S. , Gregorian Caroline , Drenan Ryan M. , Gabot Kathryn , Rana Brinda K. , Insel Paul A.
Published: December 2017

Madin-Darby canine kidney (MDCK) cells release ATP upon mechanical or biochemical activation, initiating P2Y receptor signaling that regulates basal levels of multiple second messengers, including cAMP ( J Biol Chem 275: 11735–11739, 2000). Data shown here document inhibition of cAMP formation by Gd3+ and niflumic acid, channel inhibitors that block ATP release. cAMP production is stimulated via Ca2+-dependent activation of cytosolic phospholipase A2, release of arachidonic acid (AA), and cyclooxygenase (COX)-dependent production of prostaglandins, which activate prostanoid receptors coupled to Gs and adenylyl cyclase. In the current investigation, we assessed the expression and functional role of the two known isoforms of COX, COX-1 and COX-2. Treatment of cells with either a COX-1-selective inhibitor, SC-560, or COX-2-selective inhibitors, SC-58125 or NS-398, inhibited basal and UTP-stimulated cAMP levels. COX inhibitors also decreased forskolin-stimulated cAMP formation, implying this response is in part attributable to an action of AA metabolites. These findings imply an important role for the inducible form of COX, COX-2, under basal conditions. Indeed, COX-2 expression was readily detectable by immunoblot, and treatments that induce or reduce COX-2 expression in other cells (interleukin-1β, tumor necrosis factor-α, phorbol ester, or dexamethasone) had minimal or no effect on the levels of COX-2 immunoreactivity. RT-PCR using isoform-specific primers detected COX-2 mRNA. We conclude that COX-2 is constitutively expressed in MDCK-D1 cells and participates in basal and P2Y2-mediated signaling, implying a key role for COX-2 in regulation of epithelial cell function.
Signaling by eNOS through a superoxide-dependent p42/44 mitogen-activated protein kinase pathway
Authors: Wang Weihan , Wang Shuibang , Nishanian Ervant V. , Del Pilar Cintron Ana , Wesley Robert A. , Danner Robert L.
Published: December 2017

Expression of endothelial nitric oxide synthase (eNOS) in transfected U-937 cells upregulates phorbol 12-myristate 13-acetate (PMA)-induced tumor necrosis factor-α (TNF-α) production through a superoxide (O[Formula: see text])-dependent mechanism. Because mitogen-activated protein kinases (MAPK) have been shown to participate in both reactive oxygen species signaling and TNF-α regulation, their possible role in eNOS-derived O[Formula: see text] signal transduction was examined. A redox-cycling agent, phenazine methosulfate, was found to both upregulate TNF-α (5.8 ± 1.0 fold; P = 0.01) and increase the phosphorylation state of p42/44 MAPK (3.1 ± 0.2 fold; P = 0.01) in PMA-differentiated U-937 cells. Although S-nitroso- N-acetylpenicillamine, a nitric oxide (NO) donor, also increased TNF-α production, NO exposure led to phosphorylation of p38 MAPK, not p42/44 MAPK. Upregulation of TNF-α production by eNOS transfection was associated with increases in activated p42/44 MAPK ( P = 0.001), whereas levels of phosphorylated p38 MAPK were unaffected. Furthermore, cotransfection with Cu/Zn superoxide dismutase, which blocks TNF-α upregulation by eNOS, also abolished the effects on p42/44 MAPK. Expression of Gln361eNOS, a mutant that produces O[Formula: see text] but not NO, still resulted in p42/44 MAPK phosphorylation. In contrast, two NADPH binding site deletion mutants of eNOS that lack oxidase activity had no effect on p42/44 MAPK. Finally, PD-98059, a p42/44 MAPK pathway inhibitor, blocked TNF-α upregulation by eNOS ( P = 0.02). Thus O[Formula: see text] produced by eNOS increases TNF-α production via a mechanism that involves p42/44 MAPK activation.
MAPK p38 antagonism as a novel method of inhibiting lymphoid immune suppression in polymicrobial sepsis
Authors: Song Grace Y. , Chung Chun-Shiang , Chaudry Irshad H. , Ayala Alfred
Published: December 2017

Although studies indicate that a shift from a Th1 to a Th2 response contributes to a marked suppression of cell-mediated immunity during sepsis, the mechanism by which this occurs remains unknown. Given that the mitogen-activated protein kinase (MAPK) p38 plays a critical role in the activation and function of immune cells, the aim of this study was to determine the contribution of MAPK p38 activation to the immune dysfunction seen in polymicrobial sepsis. To study this, polymicrobial sepsis was induced in C3H/HeN male mice by cecal ligation and puncture (CLP). Splenic lymphocytes and purified T cells were harvested 24 h post-CLP, pretreated with the specific MAPK p38 inhibitor SB-203580, and then stimulated with a monoclonal antibody against the T cell marker CD3. The results indicate that interleukin (IL)-2 release is markedly depressed while the release of the immunosuppressive mediator, IL-10, as well as mRNA levels of IL-10 and IL-4, are augmented after CLP. Inhibition of MAPK p38 suppressed in vitro IL-10 levels as well as IL-10 and IL-4 gene expression while restoring the release of IL-2. To determine whether these in vitro findings could be translated to an in vivo setting, mice were given 100 mg of SB-203580/kg body wt or saline vehicle (intraperitoneal) at 12 h post-CLP. Examination of ex vivo lymphocyte responsiveness indicated that, as with the in vitro finding, septic mouse Th1 responsiveness was restored. In light of our recent finding that delayed in vivo SB-203580 treatment also improved survival after CLP, we believe that these results not only illustrate the role of MAPK p38 in the induction of immunosuppressive agents in sepsis but demonstrate that SB-203580 administration after the initial proinflammatory state of sepsis significantly prevents the morbidity from sepsis.
Plasma interleukin-6 during strenuous exercise: role of epinephrine
Authors: Steensberg Adam , Toft Anders Dyhr , Schjerling Peter , Halkjær-Kristensen Jens , Pedersen Bente Klarlund
Published: December 2017

Exercise induces increased levels of plasma interleukin-6 (IL-6) as well as changes in the concentration of lymphocytes and neutrophils. The aim of this study was to investigate a possible role for epinephrine. Seven healthy men participated in an exercise experiment. One month later they received an epinephrine infusion. The exercise consisted of treadmill running at 75% of maximal O2 consumption for 2.5 h. The infusion trial consisted of 2.5 h of epinephrine infusion calculated to reach the same plasma epinephrine levels seen during the exercise experiment. The plasma concentration of IL-6 increased 29-fold during exercise, with peak levels at the end of exercise. The increase in plasma IL-6 during epinephrine infusion was only sixfold, with the peak value at 1 h after infusion. The lymphocyte concentration increased to the same levels during exercise and epinephrine infusion. The lymphocyte count decreased more in the postexercise period than after epinephrine infusion. The neutrophil concentration was elevated threefold in response to exercise, whereas no change was found in response to epinephrine infusion. In conclusion, the exercise-induced increase in plasma IL-6 could not be mimicked by epinephrine infusion. However, epinephrine induced a small increase in IL-6 and may, therefore, partly influence the plasma levels of IL-6 during exercise. In addition, the results support the idea that epinephrine plays a role in exercise-induced changes in lymphocyte number, whereas epinephrine does not mediate exercise-induced neutrocytosis.
Mechanism of thiamine uptake by human jejunal brush-border membrane vesicles
Authors: Dudeja Pradeep K. , Tyagi Sangeeta , Kavilaveettil Reena J. , Gill Ravinder , Said Hamid M.
Published: December 2017

Thiamine, a water-soluble vitamin, is essential for normal cellular functions, growth and development. Thiamine deficiency leads to significant clinical problems and occurs under a variety of conditions. To date, however, little is known about the mechanism of thiamine absorption in the native human small intestine. The objective of this study was, therefore, to characterize the mechanism of thiamine transport across the brush-border membrane (BBM) of human small intestine. With the use of purified BBM vesicles (BBMV) isolated from the jejunum of organ donors, thiamine uptake was found to be 1) independent of Na+ but markedly stimulated by an outwardly directed H+ gradient (pH 5.5in/pH 7.5out); 2) competitively inhibited by the cation transport inhibitor amiloride (inhibitor constant of 0.12 mM); 3) sensitive to temperature and osmolarity of the incubation medium; 4) significantly inhibited by thiamine structural analogs (amprolium, oxythiamine, and pyrithiamine), but not by unrelated organic cations (tetraethylammonium, N-methylnicotinamide, or choline); 5) not affected by the addition of ATP to the inside and outside of the BBMV; 6) potential insensitive; and 7) saturable as a function of thiamine concentration with an apparent Michaelis-Menten constant of 0.61 ± 0.08 μM and a maximal velocity of 1.00 ± 0.47 pmol · mg protein−1 · 10 s−1. Carrier-mediated thiamine uptake was also found in BBMV of human ileum. These data demonstrate the existence of a Na+-independent, pH-dependent, amiloride-sensitive, electroneutral carrier-mediated mechanism for thiamine absorption in native human small intestinal BBMV.
An inwardly rectifying K+ channel, Kir4.1, expressed in astrocytes surrounds synapses and blood vessels in brain
Authors: Higashi Kayoko , Fujita Akikazu , Inanobe Atsushi , Tanemoto Masayuki , Doi Katsumi , Kubo Takeshi , Kurachi Yoshihisa
Published: December 2017

Glial cells express inwardly rectifying K+ (Kir) channels, which play a critical role in the buffering of extracellular K+. Kir4.1 is the only Kir channel so far shown to be expressed in brain glial cells. We examined the distribution of Kir4.1 in rat brain with a specific antibody. The Kir4.1 immunostaining distributed broadly but not diffusely in the brain. It was strong in some regions such as the glomerular layer of the olfactory bulb, the Bergmann glia in the cerebellum, the ependyma, and pia mater, while little activity was detected in white matter of the corpus callosum or cerebellar peduncle. In the olfactory bulb, Kir4.1 immunoreactivity was detected in a scattered manner in about one-half of the glial fibrillary acidic protein-positive astrocytes. Immunoelectron microscopic examination revealed that Kir4.1 channels were enriched on the processes of astrocytes wrapping synapses and blood vessels. These data suggest that Kir4.1 is expressed in a limited population of brain astrocytes and may play a specific role in the glial K+-buffering action.
Na+-dependent neutral amino acid transporter ATB0 is a rabbit epithelial cell brush-border protein
Authors: Avissar Nelly E. , Ryan Charlotte K. , Ganapathy Vadivel , Sax Harry C.
Published: December 2017

System B0 activity accounts for the majority of intestinal and kidney luminal neutral amino acid absorption. An amino acid transport system, called ATB0 (also known as ASCT2), with functional characteristics similar to those of system B0, has been recently cloned. We generated polyclonal antibodies to human and rabbit ATB0 COOH-terminal peptides and used Western blot analysis to detect ATB0 protein in rabbit tissues, rabbit ileal brush-border membrane vesicles (BBMV), and HeLa cells transfected with plasmids containing ATB0 cDNAs. Immunohistochemistry was used to localize ATB0 in rabbit kidney and intestine. In Western blots of rabbit tissues, ATB0 was a broad smear of 78- to 85-kDa proteins. In transfected HeLa cells, ATB0 appeared as a smear consisting of 57- to 65-kDa proteins. The highest expression was found in the kidney. ATB0 was enriched in rabbit ileal BBMV and in HeLa cells transfected with ATB0 cDNAs. In the kidney and in the intestine, ATB0 was confined to the brush-border membrane (BBM) of the proximal tubular cell and of the enterocyte, respectively. Tissue and intracellular distribution of ATB0 protein parallels that of system B0 activity. ATB0protein could be the transporter responsible for system B0in the BBM of epithelial cells.
Nongenomic regulation of ENaC by aldosterone
Authors: Zhou Zhen-Hong , Bubien James K.
Published: December 2017

Aldosterone is involved in salt and water homeostasis. The main effect is thought to involve genomic mechanisms. However, the existence of plasma membrane steroid receptors has been postulated. We used whole cell patch clamp to test the hypothesis that epithelial sodium channels (ENaC) expressed by renal collecting duct principal cells can be regulated nongenomically by aldosterone. In freshly isolated principal cells from rabbit, aldosterone (100 nM) rapidly (<2 min) increased ENaC sodium current specifically. The aldosterone-activated current was completely inhibited by amiloride. Aldosterone also activated ENaC in cells treated with the mineralocorticoid receptor blocker spiranolactone. Nongenomic activation was inhibited by inclusion of S-adenosyl-l-homocysteine in the pipette solution, which inhibits methylation reactions. Also, the nongenomic activation required 2 mM ATP supplementation in the pipette solution. Aldosterone did not activate any ENaC current in whole cell clamped rat collecting duct principal cells. These functional studies are consistent with aldosterone membrane binding studies, suggesting the presence of a plasma membrane steroid receptor that affects cellular processes by mechanisms unrelated to altered gene expression.
Essential role for extracellular Ca2+ in JNK activation by mechanical stretch in bladder smooth muscle cells
Authors: Kushida Nobuhiro , Kabuyama Yukihito , Yamaguchi Osamu , Homma Yoshimi
Published: December 2017

Mechanical stretch has been implicated in phenotypic changes as an adaptive response to stretch stress physically loaded in bladder smooth muscle cells (BSMCs). To investigate stretch-induced signaling, we examined the mitogen-activated protein kinase (MAPK) family using rat primary BSMCs. When BSMCs were subjected to sustained mechanical stretch using collagen-coated silicon membranes, activation of c-Jun NH2-terminal kinase (JNK) was most relevant among three subsets of MAPK family members: the activity was elevated from 5 min after stretch and peaked at 10 min with an 11-fold increase. Activation of p38 was weak compared with that of JNK, and ERK was not activated at all. JNK activation by mechanical stretch was totally dependent on extracellular Ca2+ and inhibited by Gd3+, a blocker of stretch-activated (SA) ion channels. Nifedipine and verapamil, inhibitors for voltage-dependent Ca2+ channels, had no effect on this JNK activation. Moreover, none of the inhibitors pertussis toxin, genistein, wortmannin, or calphostin C affected stretch-induced JNK activation, indicating that G protein-coupled and tyrosine kinase receptors are unlikely to be involved in this JNK activation. On the other hand, W-7, a calmodulin inhibitor, and cyclosporin A, a calcineurin inhibitor, prevented JNK activation by stretch. These results suggest a novel pathway for stretch-induced activation of JNK in BSMCs: mechanical stretch evokes Ca2+ influx via Gd3+-sensitive SA Ca2+ channels, resulting in JNK activation under regulation in part by calmodulin and calcineurin.
Effect of adrenergic blockade on lymphocyte cytokine production at rest and during exercise
Authors: Starkie R. L. , Rolland J. , Febbraio M. A.
Published: December 2017

To examine the effect of exercise and adrenergic blockade on lymphocyte cytokine production, six men ingested either a placebo (control) or an α- (prazosin hydrochloride) and β-adrenoceptor antagonist (timolol malate) capsule (blockade, or BLK) 2 h before performing 19 ± 1 min of supine bicycle exercise at 78 ± 3% peak pulmonary uptake. Blood was collected before and after exercise, stimulated with phorbol 12-myristate 13-acetate and ionomycin, and surface stained for T (CD3+) and natural killer [NK (CD3−CD56+)] lymphocyte surface antigens. Cells were permeabilized, stained for the intracellular cytokines interleukin (IL)-2 and interferon (IFN)-γ, and analyzed using flow cytometry. BLK had no effect on the resting concentration of stimulated cytokine-positive T and NK lymphocytes or the amount of cytokine they were producing. Exercise resulted in an increase ( P< 0.05) in the concentration of stimulated T and NK lymphocytes producing cytokines in the circulation, but these cells produced less ( P < 0.05) cytokine post- compared with preexercise. BLK attenuated ( P < 0.05) the elevation in the concentration of lymphocytes producing cytokines during exercise; however, BLK did not affect the amount of IL-2 and IFN-γ produced. These results suggest that adrenergic stimulation contributes to the exercise-induced increase in the concentration of lymphocytes in the circulation; however, it does not appear to be responsible for the exercise-induced suppression in cytokine production.
PGE2, Ca2+, and cAMP mediate ATP activation of Cl− channels in pigmented ciliary epithelial cells
Authors: Fleischhauer Johannes C. , Mitchell Claire H. , Peterson-Yantorno Kim , Coca-Prados Miguel , Civan Mortimer M.
Published: December 2017

Purines regulate intraocular pressure. Adenosine activates Cl− channels of nonpigmented ciliary epithelial cells facing the aqueous humor, enhancing secretion. Tamoxifen and ATP synergistically activate Cl− channels of pigmented ciliary epithelial (PE) cells facing the stroma, potentially reducing net secretion. The actions of nucleotides alone on Cl− channel activity of bovine PE cells were studied by electronic cell sorting, patch clamping, and luciferin/luciferase ATP assay. Cl−channels were activated by ATP > UTP, ADP, and UDP, but not by 2-methylthio-ATP, all at 100 μM. UTP triggered ATP release. The second messengers Ca2+, prostaglandin (PG)E2, and cAMP activated Cl− channels without enhancing effects of 100 μM ATP. Buffering intracellular Ca2+activity with 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′- tetraacetic acid or blocking PGE2 formation with indomethacin inhibited ATP-triggered channel activation. The Rp stereoisomer of 8-bromoadenosine 3′,5′-cyclic monophosphothioate inhibited protein kinase A activity but mimicked 8-bromoadenosine 3′,5′-cyclic monophosphate. We conclude that nucleotides can act at >1 P2Y receptor to trigger a sequential cascade involving Ca2+, PGE2, and cAMP. cAMP acts directly on Cl−channels of PE cells, increasing stromal release and potentially reducing net aqueous humor formation and intraocular pressure.
Phenotypic analysis of conditionally immortalized cells isolated from the BPK model of ARPKD
Authors: Sweeney William E. , Kusner Linda , Carlin Cathleen R. , Chang Sharon , Futey Lidia , Cotton Calvin U. , Dell Katherine MacRae , Avner Ellis D.
Published: December 2017

To study the pathophysiology of autosomal recessive polycystic kidney disease (ARPKD), we sought to develop conditionally immortalized control and cystic murine collecting tubule (CT) cell lines. CT cells were isolated from intercross breedings between BPK mice ( bpk +/−), a murine model of ARPKD, and the Immorto mice (H-2Kb- ts-A58 +/+ ). Second-generation outbred offspring (BPK × Immorto) homozygous for the BPK mutation ( bpk −/−; Im +/±; cystic BPK/H-2Kb- ts-A58), were phenotypically indistinguishable from inbred cystic BPK animals ( bpk −/−). Cystic BPK/H-2Kb- ts-A58 mice developed biliary ductal ectasia and massively enlarged kidneys, leading to renal failure and death by postnatal day 24. Principal cells (PC) were isolated from outbred cystic and noncystic BPK/H-2Kb- ts-A58 littermates at specific developmental stages. Epithelial monolayers were under nonpermissive conditions for markers of epithelial cell polarity and PC function. Cystic and noncystic cells displayed several properties characteristic of PCs in vivo, including amiloride-sensitive sodium transport and aquaporin 2 expression. Cystic cells exhibited apical epidermal growth factor receptor (EGFR) mislocalization but normal expression of ZO-1 and E-cadherin. Hence, these cell lines retain the requisite characteristics of PCs, and cystic BPK/H-2Kb- ts-A58 PCs retained the abnormal EGFR membrane expression characteristic of ARPKD. These cell lines represent important new reagents for studying the pathogenesis of ARPKD.
Developmental changes in ryanodine- and IP3-sensitive Ca2+ pools in ovine basilar artery
Authors: Nauli S. M. , Williams J. M. , Akopov S. E. , Zhang L. , Pearce W. J.
Published: December 2017

To explore the hypothesis that cerebrovascular maturation alters ryanodine- and inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pool sizes, we measured total intracellular Ca2+ with 45Ca and the fractions of intracellular Ca2+ released by IP3 and/or caffeine in furaptra-loaded permeabilized basilar arteries from nonpregnant adult and term fetal (139–141 days) sheep. Ca2+ mass (nmol/mg dry weight) was similar in adult (1.60 ± 0.18) and fetal (1.71 ± 0.16) arteries in the pool sensitive to IP3 alone but was significantly lower for adult (0.11 ± 0.01) than for fetal (1.22 ± 0.11) arteries in the pool sensitive to ryanodine alone. The pool sensitive to both ryanodine and IP3 was also smaller in adult (0.14 ± 0.01) than in fetal (0.85 ± 0.08) arteries. Because the Ca2+ fraction in the ryanodine-IP3 pool was small in both adult (5 ± 1%) and fetal (7 ± 4%) arteries, the IP3 and ryanodine pools appear to be separate in these arteries. However, the pool sensitive to neither IP3 nor ryanodine was 10-fold smaller in adult (0.87 ± 0.10) than in fetal (8.78 ± 0.81) arteries, where it accounted for 72% of total intracellular membrane-bound Ca2+. Thus, during basilar artery maturation, intracellular Ca2+ mass plummets in noncontractile pools, decreases modestly in ryanodine-sensitive pools, and remains constant in IP3-sensitive pools. In addition, age-related increases in IP3 efficacy must involve factors other than IP3 pool size alone.
Localization and topology of a urate transporter/channel, a galectin, in epithelium-derived cells
Authors: Rappoport Joshua Z. , Lipkowitz Michael S. , Abramson Ruth G.
Published: December 2017

Recombinant protein produced from a cDNA cloned in our laboratory (UAT) functions in lipid bilayers as a urate transporter/channel. Because UAT is a galectin, a family of proteins presumed to be soluble, the localization and topology of UAT were assessed in living cells. UAT was targeted to plasma membrane in multiple epithelium-derived cell lines and, in polarized cells, was targeted to both apical and basolateral membranes. The amino and carboxy termini of UAT were both detected on the cytoplasmic side of plasma membranes, whereas cell surface biotinylation studies demonstrated that UAT is not merely a cytosolic membrane-associated protein but contains at least one extracellular domain. Madin-Darby canine kidney cells were shown both functionally and immunologically to contain an apparent homolog of UAT; however, transfection with UAT did not modify urate uptake. Because coimmunoprecipitation studies revealed that UAT is capable of forming both homo- and heteromultimers, it is proposed that monomers of endogenous channels are in part replaced by monomers of the protein expressed subsequent to transfection, thereby maintaining constancy of urate uptake at basal levels.
Physiological importance of system A-mediated amino acid transport to rat fetal development
Authors: Cramer Stuart , Beveridge Mark , Kilberg Michael , Novak Donald
Published: December 2017

Fetal growth and development are dependent on the delivery of amino acids from maternal amino acid pools to the fetal blood. This is accomplished via transfer across the apical and basal plasma membrane of the placental syncytiotrophoblast. The aim of this study was to determine whether inhibition of system A (amino acid transporter) was associated with a decrease in fetal weight in the rat. System A is a ubiquitous Na+-dependent amino acid transporter that actively transports small zwitterionic amino acids. In brief, system A was inhibited by infusing a nonmetabolizable synthetic amino acid analog, 2-(methylamino)isobutyric acid from days 7–20 of gestation. On day 20, the rats were killed and tissues (maternal liver, fetuses, and placentas) were collected for analysis. The degree of system A inhibition was determined, as was the impact of said inhibition on fetal and maternal weights, system A-mediated placental transport, and placental system A-mediated transporter expression. Our results suggest that when system A is inhibited, fetal weight is diminished [control group: −3.55 ± 0.04 g ( n = 113), experimental group: −3.29 ± 0.04 g ( n = 128)], implying an integral role for system A transport in fetal growth and development in the rat.