bluuebunny/crossref_metadata_2025 huggingface dataset:
Control of heat production by the Ca2+-ATPase of rabbit and trout sarcoplasmic reticulum
Authors: De Meis Leopoldo
Published: December 2017

The sarcoplasmic reticulum Ca2+-ATPase of rabbit skeletal muscle can convert the energy derived from a Ca2+ gradient into heat (L. de Meis, M. L. Bianconi, and V. A. Suzano. FEBS Lett. 406: 201–204, 1997). In this report, it is shown that this conversion varies depending on the temperature and on whether rabbit (endotherm) or trout (poikilotherm) sarcoplasmic reticulum vesicles are used. The gradient doubled the yield of heat produced during ATP hydrolysis and the calorimetric enthalpy of ATP hydrolysis (Δ H cal) value found with both rabbit and trout varied between −10 and −12 kcal/mol in leaky vesicles (no gradient) and between −20 and −22 kcal/mol with intact vesicles (gradient). For the rabbit, the difference of Δ H cal measured with and without gradient was detected in the range of 30–35°C and disappeared when the temperature was decreased below 30°C. For the trout, the difference was detected between 20 and 25°C and disappeared below 20°C. The effect of the gradient on the Δ H cal for ATP hydrolysis was modified by DMSO, trifluoperazine, and heparin sodium.
Extracellular ATP and bradykinin increase cGMP in vascular endothelial cells via activation of PKC
Authors: Castro A. F. , Amorena C. , Müller A. , Ottaviano G. , Tellez-Iñon M. T. , Taquini A. C.
Published: December 2017

Vasodilation by agents such as bradykinin and ATP is dependent on nitric oxide, the endothelium-dependent relaxing factor (EDRF). The release of EDRF results in elevation of cGMP in endothelial and smooth muscle cells (9). The signaling pathway that leads to increases in cGMP is not completely understood. The role of protein kinase C (PKC) in the elevation of cGMP induced by ATP and bradykinin was studied in cultured porcine aortic endothelial cells, by measuring PKC phosphorylation of a substrate and by measuring cGMP levels by radioimmunoassay. Extracellular ATP and bradykinin simultaneously elevated cGMP levels and PKC activity. The PKC inhibitors staurosporine, calphostin C, and Cremophor EL (T. Tamaoki and H. Nakano. Bio/Technology 8: 732–735, 1990; F. K. Zhao, L. F. Chuang, M. Israel, and R. Y. Chuang. Biochem. Biophys. Res. Commun. 159: 1359–1367, 1989) prevented the elevation of cGMP elicited by ATP and reduced that produced by bradykinin. Cremophor did not affect the elevation of cGMP by nitroprusside, an agent that directly increases guanylate cyclase activity (9). The PKC activator phorbol 12-myristate 13-acetate, but not a phorbol ester analog inactive on PKC, also elevated cGMP levels. These results suggest that EDRF agonists elevate cGMP in endothelial cells via PKC stimulation.
Effects of divalent cations on single-channel conduction properties of XenopusIP3 receptor
Authors: Mak Don-On Daniel , Foskett J. Kevin
Published: December 2017

The effects of Mg2+ and Ba2+ on single-channel properties of the inositol 1,4,5-trisphosphate receptor (IP3R) were studied by patch clamp of isolated nuclei from Xenopusoocytes. In 140 mM K+ the IP3R channel kinetics and presence of conductance substates were similar over a range (0–9.5 mM) of free Mg2+. In 0 mM Mg2+ the channel current-voltage ( I-V) relation was linear with conductance of ∼320 pS. Conductance varied slowly and continuously over a wide range (SD ≈ 60 pS) and sometimes fluctuated during single openings. The presence of Mg2+ on either or both sides of the channel reduced the current (blocking constant ∼0.6 mM in symmetrical Mg2+), as well as the range of conductances observed, and made the I-V relation nonlinear (slope conductance ∼120 pS near 0 mV and ∼360 pS at ±70 mV in symmetrical 2.5 mM Mg2+). Ba2+ exhibited similar effects on channel conductance. Mg2+ and Ba2+ permeated the channel with a ratio of permeability of Ba2+ to Mg2+ to K+ of 3.5:2.6:1. These results indicate that divalent cations induce nonlinearity in the I-V relation and reduce current by a mechanism involving permeation block of the IP3R due to strong binding to site(s) in the conduction pathway. Furthermore, stabilization of conductance by divalent cations reveals a novel interaction between the cations and the IP3R.
Hypotonicity activates a lanthanide-sensitive pathway for K+ release in A6 epithelia
Authors: De Smet Patrick , Li Jinqing , Van Driessche Willy
Published: December 2017

The nature of the pathway for K+ release activated during regulatory volume decrease (RVD) in A6 epithelia was investigated by measuring cell thickness (Tc) as an index of cell volume and by probing K+ efflux with86Rb as tracer for K+(RRb). Cell swelling was induced by sudden reduction of basolateral osmolality (from 260 to 140 mosmol/kgH2O). Experiments were performed in the absence of Na+transport. Apical RRb was negligible in iso- and hyposmotic conditions. On the other hand, osmotic shock increased basolateral RRb([Formula: see text]) rapidly, reaching a maximum 7 min after the peak in Tc. Quinine (0.5 mM) completely inhibited RVD and [Formula: see text]. Also verapamil (0.2 mM) impeded volume recovery considerably; lidocaine (0.2 mM) did not exert a noticeable effect. The K+ channel blocker Ba2+ (30 mM) delayed RVD but could not prevent complete volume recovery. Cs+ inhibited RVD noticeably at concentrations <40 mM. With large Cs+ concentrations (>40 mM), the initial osmometric swelling was followed by a gradual increase of Tc, suggesting activation of Cs+ influx. Chronic exposure of the basolateral surface to 0.5 mM La3+ or Gd3+ completely abolished RVD and[Formula: see text]. Acute administration of lanthanides at the time of osmolality decrease did not affect the initial phase of RVD and reduced [Formula: see text]only slightly. Apical Gd3+ exerted an inhibitory effect on RVD and [Formula: see text]. The effect of Gd3+ should therefore be localized at an intracellular site. The role of Ca2+ entry could be excluded by failure of extracellular Ca2+removal to inhibit volume recovery. In contrast to lanthanides, chronically and acutely administered Mg2+ (0.5 mM) inhibited RVD and[Formula: see text] by ∼50%. These data suggest that K+ excretion during RVD occurs through a rather poorly selective pathway that does not seem to be directly activated by membrane stretch.
Tyrosine kinase inhibitors and antioxidants modulate NF-κB and NOS-II induction in retinal epithelial cells
Authors: Faure Violaine , Courtois Yves , Goureau Olivier
Published: December 2017

Bovine retinal pigmented epithelial (RPE) cells express an inducible nitric oxide synthase (NOS-II) after activation with interferon-γ (IFN-γ) and lipopolysaccharide (LPS). Experiments were performed to investigate the effects of tyrosine kinase inhibitors (genistein and herbimycin A) and antioxidants [pyrrolidine dithiocarbamate (PDTC) and butyl hydroxyanisol] on NOS-II induction. The LPS-IFN-γ-induced nitrite release was inhibited in a concentration-dependent manner by these compounds. Analysis by Northern blot showed that this inhibitory effect correlated with a decrease in NOS-II mRNA accumulation. Analysis by electrophoretic mobility shift assay of the activation of the transcription factor nuclear factor-κB (NF-κB) involved in NOS-II induction demonstrated that LPS alone or combined with IFN-γ induced NF-κB binding. NF-κB activation was not changed by the presence of tyrosine kinase inhibitors but was totally prevented by PDTC pretreatment. Immunocytochemistry experiments confirmed the reduction of the nuclear translocation of NF-κB only by PDTC. Our results demonstrated the existence in retinal pigmented epithelial cells of different intracellular signaling pathways in NOS-II induction, since tyrosine kinase inhibitors blocked NOS-II mRNA accumulation without inhibiting NF-κB activation. Furthermore, the LPS-IFN-γ-induced NOS-II mRNA accumulation was sensitive to cycloheximide, suggesting that, in addition to NF-κB, transcriptional factors that require new protein synthesis are involved in NOS-II induction.
Ca2+ and protein kinase C activation of mucin granule exocytosis in permeabilized SPOC1 cells
Authors: Scott C. E. , Abdullah Lubna H. , Davis C. William
Published: December 2017

Mucin secretion by airway goblet cells is under the control of apical P2Y2, phospholipase C-coupled purinergic receptors. In SPOC1 cells, the mobilization of intracellular Ca2+ by ionomycin or the activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) stimulates mucin secretion in a fully additive fashion [L. H. Abdullah, J. D. Conway, J. A. Cohn, and C. W. Davis. Am. J. Physiol. 273 ( Lung Cell. Mol. Physiol. 17): L201–L210, 1997]. This apparent independence between PKC and Ca2+ in the stimulation of mucin secretion was tested in streptolysin O-permeabilized SPOC1 cells. These cells were fully competent to secrete mucin when Ca2+ was elevated from 100 nM to 3.1 μM for 2 min following permeabilization; the Ca2+EC50 was 2.29 ± 0.07 μM. Permeabilized SPOC1 cells were exposed to PMA or 4α-phorbol at Ca2+ activities ranging from 10 nM to 10 μM. PMA, but not 4α-phorbol, increased mucin release at all Ca2+ activities tested: at 10 nM Ca2+ mucin release was 2.1-fold greater than control and at 4.7 μM Ca2+ mucin release was maximal (3.6-fold increase). PMA stimulated 27% more mucin release at 4.7 μM than at 10 nM Ca2+. Hence, SPOC1 cells possess Ca2+-insensitive, PKC-dependent, and Ca2+-dependent PKC-potentiated pathways for mucin granule exocytosis.
Effect of in vivo corticosteroids on Na+ transport across airway epithelia
Authors: Grubb B. R. , Boucher R. C.
Published: December 2017

We have investigated the role in vivo of mineralocorticoid and glucocorticoid hormones in regulating the rate of electrogenic amiloride-sensitive Na+ absorption across murine airway tissue studied in vivo (nasal potential difference) and in vitro (Ussing chambers). We found that elevating the plasma aldosterone concentration 10-fold (low-Na+diet) had no significant effect on amiloride-sensitive Na+ absorption across tracheal or nasal epithelia. High doses of dexamethasone for 1 wk likewise did not change the rate of amiloride-sensitive Na+ absorption across airway epithelia. In contrast, both hormonal manipulations elevated the rate of colonic Na+ absorption. Furthermore, adrenalectomy (both normal and cystic fibrosis mice) also failed to alter Na+ absorption across airway epithelia. We conclude that, in vivo, neither the mineralocorticoid nor the glucocorticoid hormones significantly regulate the rates of amiloride-sensitive electrogenic Na+ absorption across airway epithelia in the adult mouse.
An analysis of Ca2+ release by DGEA: mobilization of two functionally distinct internal stores in Saos-2 cells
Authors: McCann T. J. , Terranova G. , Keyte J. W. , Papaioannou S. S. , Mason W. T. , Meikle M. C. , McDonald F.
Published: December 2017

Osteoblasts can be activated by their collagen matrix and in particular the DGEA peptide motif. We have reported that DGEA is able to activate Ca2+ signaling pathways in the human osteoblast-like cell line, Saos-2, by a tyrosine kinase-dependent pathway (T. J. McCann, W. T. Mason, M. C. Meikle, and F. McDonald. Matrix Biol. 16: 271–280, 1997). In the present study, we show that this activity is due to coupling of the signal to intracellular Ca2+ stores, since the DGEA action is not blocked by La3+ but is lost when Ca2+ stores are depleted with 2 μM and blocked by 10 μM ryanodine. The activated stores also differ functionally from those activated by thrombin, as blockade with U-73122 obstructs only thrombin-activated Ca2+ release. We have shown that the DGEA activity was not due to its high-charge density, since the two acidic residues can be substituted with their uncharged homologues (asparagine and glutamine) without significant loss of activity. This was in turn measured by an adhesion assay that also demonstrated this level of specificity. Furthermore, by constructing DGEA bound to FITC, we have shown that DGEA binding was dependent on divalent cations. We have also demonstrated that an intact actin cytoskeleton is not required for Ca2+ activation by inhibiting actin polymerization with the addition of cytochalasin B. These data strengthen the argument that collagen has a significant role in regulating osteoblast function via this peptide motif.
Effects of local anesthetics on Na+ channels containing the equine hyperkalemic periodic paralysis mutation
Authors: Sah Rajan L. , Tsushima Robert G. , Backx Peter H.
Published: December 2017

We examined the ability of local anesthetics to correct altered inactivation properties of rat skeletal muscle Na+channels containing the equine hyperkalemic periodic paralysis (eqHPP) mutation when expressed in Xenopusoocytes. Increased time constants of current decay in eqHPP channels compared with wild-type channels were restored by 1 mM benzocaine but were not altered by lidocaine or mexiletine. Inactivation curves, which were determined by measuring the dependence of the relative peak current amplitude after depolarization to −10 mV on conditioning prepulse voltages, could be shifted in eqHPP channels back toward that observed for wild-type (WT) channels using selected concentrations of benzocaine, lidocaine, and mexiletine. Recovery from inactivation at −80 mV (50-ms conditioning pulse) in eqHPP channels followed a monoexponential time course and was markedly accelerated compared with wild-type channels (τWT= 10.8 ± 0.9 ms; τeqHPP= 2.9 ± 0.4 ms). Benzocaine slowed the time course of recovery (τeqHPP,ben = 9.6 ± 0.4 ms at 1 mM) in a concentration-dependent manner. In contrast, the recovery from inactivation with lidocaine and mexiletine had a fast component (τfast,lid = 3.2 ± 0.2 ms; τfast,mex = 3.1 ± 0.2 ms), which was identical to the recovery in eqHPP channels without drug, and a slow component (τslow,lid = 1,688 ± 180 ms; τslow,mex = 2,323 ± 328 ms). The time constant of the slow component of the recovery from inactivation was independent of the drug concentration, whereas the fraction of current recovering slowly depended on drug concentrations and conditioning pulse durations. Our results show that local anesthetics are generally incapable of fully restoring normal WT behavior in inactivation-deficient eqHPP channels.
Expression of the transforming growth factor-β gene during growth inhibition following polyamine depletion
Authors: Patel Anami R. , Li Ji , Bass Barbara L. , Wang Jian-Ying
Published: December 2017

Polyamine depletion and cytokine transforming growth factor-β (TGF-β) inhibit cell proliferation. The current study tests the hypothesis that polyamine depletion results in growth inhibition by altering expression of the TGF-β gene in intestinal epithelial cells. Studies were conducted in the IEC-6 cell line derived from rat small intestinal crypt cells. Cells were grown in DMEM in the presence or absence of α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine biosynthesis, for 6 and 12 days. Administration of DFMO not only depleted intracellular polyamines but also significantly increased the mRNA levels of TGF-β. Increased TGF-β mRNA in DFMO-treated cells was paralleled by an increase in TGF-β content. Depletion of intracellular polyamines by DFMO had no effect on the rate of TGF-β gene transcription, as measured by nuclear run-on assay. The half-life of mRNA for TGF-β in normal cells was ∼65 min and increased to >16 h in cells treated with DFMO for 6 or 12 days. Exogenous polyamine, when given together with DFMO, prevented the increased half-life of TGF-β mRNA in IEC-6 cells. TGF-β added to the culture medium significantly decreased the rate of DNA synthesis and final cell number in normal and polyamine-deficient cells. Furthermore, growth inhibition caused by polyamine depletion was partially but significantly blocked by addition of immunoneutralizing anti-TGF-β antibody. These results indicate that 1) depletion of intracellular polyamines induces the activation of the TGF-β gene through posttranscriptional regulation and 2) increased expression of the TGF-β gene plays an important role in the process of growth inhibition following polyamine depletion.
Activation of ΔF508 CFTR in an epithelial monolayer
Authors: Bebök Zsuzsa , Venglarik Charles J. , Pánczél Zita , Jilling Tamás , Kirk Kevin L. , Sorscher Eric J.
Published: December 2017

The ΔF508 mutation leads to retention of cystic fibrosis transmembrane conductance regulator (CFTR) in the endoplasmic reticulum and rapid degradation by the proteasome and other proteolytic systems. In stably transfected LLC-PK1(porcine kidney) epithelial cells, ΔF508 CFTR conforms to this paradigm and is not present at the plasma membrane. When LLC-PK1 cells or human nasal polyp cells derived from a ΔF508 homozygous patient are grown on plastic dishes and treated with an epithelial differentiating agent (DMSO, 2% for 4 days) or when LLC-PK1 cells are grown as polarized monolayers on permeable supports, plasma membrane ΔF508 CFTR is significantly increased. Moreover, when confluent LLC-PK1 cells expressing ΔF508 CFTR were treated with DMSO and mounted in an Ussing chamber, a further increase in cAMP-activated short-circuit current (i.e., ∼7 μA/cm2; P < 0.00025 compared with untreated controls) was observed. No plasma membrane CFTR was detected after DMSO treatment in nonepithelial cells (mouse L cells) expressing ΔF508 CFTR. The experiments describe a way to augment ΔF508 CFTR maturation in epithelial cells that appears to act through a novel mechanism and allows insertion of functional ΔF508 CFTR in the plasma membranes of transporting cell monolayers. The results raise the possibility that increased epithelial differentiation might increase the delivery of ΔF508 CFTR from the endoplasmic reticulum to the Golgi, where the ΔF508 protein is shielded from degradative pathways such as the proteasome and allowed to mature.
The mechanism of IL-5 signal transduction
Authors: Adachi Tetsuya , Alam Rafeul
Published: December 2017

Cytokines are important regulators of hematopoiesis. They exert their actions by binding to specific receptors on the cell surface. Interleukin-5 (IL-5) is a critical cytokine that regulates the growth, activation, and survival of eosinophils. Because eosinophils play a seminal role in the pathogenesis of asthma and allergic diseases, an understanding of the signal transduction mechanism of IL-5 is of paramount importance. The IL-5 receptor is a heterodimer of α- and β-subunits. The α-subunit is specific, whereas the β-subunit is common to IL-3, IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF) receptors and is crucial for signal transduction. It has been shown that there are two major signaling pathways of IL-5 in eosinophils. IL-5 activates Lyn, Syk, and JAK2 and propagates signals through the Ras-MAPK and JAK-STAT pathways. Studies suggest that Lyn, Syk, and JAK2 tyrosine kinases and SHP-2 tyrosine phosphatase are important for eosinophil survival. In contrast to their survival-promoting activity, Lyn and JAK2 appear to have no role in eosinophil degranulation or expression of surface adhesion molecules. Raf-1 kinase, on the other hand, is critical for eosinophil degranulation and adhesion molecule expression. Btk is involved in IL-5 stimulation of B cell function. However, it does not appear to be important for eosinophil function. Thus a clear segregation of signaling molecules based on their functional importance is emerging. This review describes the signal transduction mechanism of the IL-3/GM-CSF/IL-5 receptor system and compares and contrasts IL-5 signaling between eosinophils and B cells.
Inducible expression of protein kinase Cα suppresses steroidogenesis in Y-1 adrenocortical cells
Authors: Reyland Mary E. , Williams David L. , White Elizabeth K.
Published: December 2017

We have previously shown that protein kinase C (PKC) suppresses steroidogenesis in Y-1 adrenocortical cells. To ask directly if the PKCα isoform mediates this suppression, we have developed Y-1 cell lines in which PKCα is expressed from a tetracycline-regulated promoter. Induction of PKCα expression in these cell lines results in decreased P450 cholesterol side-chain cleavage enzyme (P450-SCC) activity as judged by the conversion of hydroxycholesterol to pregnenolone. Transcription of a P450-SCC promoter-luciferase construct is also reduced when PKCα expression is increased. However, expression of PKCα has no effect on 8-bromo-cAMP induction of steroidogenesis, indicating that these pathways function independently to regulate steroidogenesis. To determine the relationship between endogenous PKC activity and steroidogenesis, we examined 12 Y-1 subclones that were isolated by limited dilution cloning. In each of these subclones, steroid production correlates inversely with total PKC activity and with the expression of PKCα but not PKCε or PKCζ. These studies define for the first time the role of a specific PKC isoform (PKCα) in regulating steroidogenesis and P450-SCC activity in adrenocortical cells.
Effect of Bcl-2 on oxidant-induced cell death and intracellular Ca2+mobilization
Authors: Ichimiya Masato , Chang Seung H. , Liu Hui , Berezesky Irene K. , Trump Benjamin F. , Amstad Paul A.
Published: December 2017

The mechanism by which Bcl-2 inhibits cell death is unknown. It has been suggested that Bcl-2 functions as an antioxidant. Because Bcl-2 is localized mainly to the membranes of the endoplasmic reticulum (ER) and the mitochondria, which represent the main intracellular storage sites for Ca2+, we hypothesized that Bcl-2 might protect cells against oxidant injury by altering intracellular Ca2+homeostasis. To test this hypothesis, we examined the effect of oxidant treatment on viability in normal rat kidney (NRK) cells and in NRK cells stably transfected with Bcl-2 in the presence or absence of intracellular Ca2+, and we compared the effect of Bcl-2 expression on oxidant-induced intracellular Ca2+ mobilization and on ER and mitochondrial Ca2+pools. NRK cells transfected with Bcl-2 (NRK-Bcl-2) were significantly more resistant to H2O2-induced cytotoxicity than control cells. EGTA-AM, an intracellular Ca2+ chelator, as well as the absence of Ca2+ in the medium, reduced H2O2-induced cytotoxicity in both cell lines. Compared with controls, cells overexpressing Bcl-2 showed a delayed rise in intracellular Ca2+ concentration ([Ca2+]i) after H2O2treatment. After treatment with the Ca2+ ionophore ionomycin, Bcl-2-transfected cells showed a much quicker decrease after the maximal rise than control cells, suggesting stronger intracellular Ca2+ buffering, whereas treatment with thapsigargin, an inhibitor of the ER Ca2+-ATPases, transiently increased [Ca2+]iin control and in Bcl-2-transfected cells. Estimates of mitochondrial Ca2+ stores using an uncoupler of oxidative phosphorylation show that NRK-Bcl-2 cells have a higher capacity for mitochondrial Ca2+storage than control cells. In conclusion, Bcl-2 may prevent oxidant-induced cell death, in part, by increasing the capacity of mitochondria to store Ca2+.
Cytosolic pH regulatesG Cl through control of phosphorylation states of CFTR
Authors: Reddy M. M. , Kopito Ron R. , Quinton P. M.
Published: December 2017

Our objective in this study was to determine the effect of changes in luminal and cytoplasmic pH on cystic fibrosis transmembrane regulator (CFTR) Cl− conductance ( G Cl). We monitored CFTR G Cl in the apical membranes of sweat ducts as reflected by Cl− diffusion potentials ( V Cl) and transepithelial conductance ( G Cl). We found that luminal pH (5.0–8.5) had little effect on the cAMP/ATP-activated CFTR G Cl, showing that CFTR G Cl is maintained over a broad range of extracellular pH in which it functions physiologically. However, we found that phosphorylation activation of CFTR G Cl is sensitive to intracellular pH. That is, in the presence of cAMP and ATP [adenosine 5′- O-(3-thiotriphosphate)], CFTR could be phosphorylated at physiological pH (6.8) but not at low pH (∼5.5). On the other hand, basic pH prevented endogenous phosphatase(s) from dephosphorylating CFTR.After phosphorylation of CFTR with cAMP and ATP, CFTR G Cl is normally deactivated within 1 min after cAMP is removed, even in the presence of 5 mM ATP. This deactivation was due to an increase in endogenous phosphatase activity relative to kinase activity, since it was reversed by the reapplication of ATP and cAMP. However, increasing cytoplasmic pH significantly delayed the deactivation of CFTR G Cl in a dose-dependent manner, indicating inhibition of dephosphorylation. We conclude that CFTR G Cl may be regulated via shifts in cytoplasmic pH that mediate reciprocal control of endogenous kinase and phosphatase activities. Luminal pH probably has little direct effect on these mechanisms. This regulation of CFTR may be important in shifting electrolyte transport in the duct from conductive to nonconductive modes.
Amino acid effects on translational repressor 4E-BP1 are mediated primarily by l-leucine in isolated adipocytes
Authors: Fox Heather L. , Pham Phuong T. , Kimball Scot R. , Jefferson Leonard S. , Lynch Christopher J.
Published: December 2017

Previous studies indicated that amino acids may activate the protein kinase activity of the target of rapamycin (TOR) and thereby augment and/or mimic the effects of insulin on protein synthesis, p70S6k phosphorylation, and multicellular clustering in adipocytes. To identify the individual amino acids responsible for these effects, the present study focused on the TOR substrate and translational repressor 4E-BP1. A complete mixture of amino acids stimulated the phosphorylation of 4E-BP1, decreasing its association with eukaryotic initiation factor eIF-4E. Studies on subsets of amino acids and individual amino acids showed that l-leucine was the amino acid responsible for most of the effects on 4E-BP1 phosphorylation; however, the presence of other amino acids was required to observe a maximal effect. The stimulatory effect of leucine was stereospecific and not mimicked by other branched chain amino acids but was mimicked by the leucine metabolite α-ketoisocaproate (α-KIC). The effect of α-KIC, but not leucine, was attenuated by the transaminase inhibitor (aminooxy)acetate. The latter result indicates that the effects of α-KIC required its conversion to leucine. Half-maximal stimulation of 4E-BP1 phosphorylation occurred at ∼430 μM; therefore, the response was linear within the range of circulating concentrations of leucine found in various nutritional states.
Membrane lipid diffusion and band 3 protein changes in human erythrocytes due to acute hypobaric hypoxia
Authors: Celedón Gloria , González Gustavo , Sotomayor Carlos P. , Behn Claus
Published: December 2017

Because it has been reported that hypoxia in rats may promote lipid peroxidation and other free radical reactions that could modify membrane lipids and proteins, the effect of acute hypobaric hypoxia on human erythrocyte membranes was investigated. 12-(1-Pyrene)dodecanoic acid fluorescent probe was used to assess short-range lateral diffusion status in the membrane bilayer. Membrane protein modification was detected by SDS-PAGE. Healthy young men were exposed for 20 min to the hypobaric hypoxia, simulating an altitude of 4,500 m. Under this condition, erythrocyte membrane lipids reached a state of higher lateral diffusivity with respect to normobaric conditions and membrane band 3 protein was modified, becoming more susceptible to membrane-bound proteinases. These observations suggest that acute hypobaric hypoxia may promote an oxidative stress condition in the erythrocyte membrane.
Endogenous expression of the renal high-affinity H+-peptide cotransporter in LLC-PK1 cells
Authors: Wenzel Uwe , Diehl Daniela , Herget Martina , Daniel Hannelore
Published: December 2017

The reabsorption of filtered di- and tripeptides as well as certain peptide mimetics from the tubular lumen into renal epithelial cells is mediated by an H+-coupled high-affinity transport process. Here we demonstrate for the first time H+-coupled uptake of dipeptides into the renal proximal tubule cell line LLC-PK1. Transport was assessed 1) by uptake studies using the radiolabeled dipeptided-[3H]Phe-l-Ala, 2) by cellular accumulation of the fluorescent dipeptided-Ala-Lys-AMCA, and 3) by measurement of intracellular pH (pHi) changes as a consequence of H+-coupled dipeptide transport. Uptake ofd-Phe-l-Ala increased linearly over 11 days postconfluency and showed all the characteristics of the kidney cortex high-affinity peptide transporter, e.g., a pH optimum for transport ofd-Phe-l-Ala of 6.0, an apparent K m value for influx of 25.8 ± 3.6 μM, and affinities of differently charged dipeptides or the β-lactam antibiotic cefadroxil to the binding site in the range of 20–80 μM. pHi measurements established the peptide transporter to induce pronounced intracellular acidification in LLC-PK1 cells and confirm its postulated role as a cellular acid loader.
Thyroid hormone stimulates the renal Na/H exchanger NHE3 by transcriptional activation
Authors: Cano Adriana , Baum Michel , Moe Orson W.
Published: December 2017

Thyroid hormone stimulates renal proximal tubule NaCl and NaHCO3 absorption in part by activating the apical membrane Na/H exchanger NHE3. We used a renal epithelial cell line, the opossum kidney (OK) cell, to define the mechanism by which 3,5,3′-triiodothyronine (T3) increases NHE3 activity. T3 stimulated NHE3 activity, an effect that was blocked by inhibition of cellular transcription or translation. The increase in activity was associated with increases in steady-state cell surface and total cellular NHE3 protein and NHE3 transcript abundance. T3stimulated transcription of the NHE3 gene and had no effect on NHE3 transcript stability. The transcriptional activity of the 5′-flanking region of the rat NHE3 gene was stimulated by T3 when expressed in OK cells. When heterologously expressed rat NHE3 transcript levels were clamped constant with a constitutive promoter in OK cells, T3 has no effect on rat NHE3 protein abundance, suggesting the absence of regulation of NHE3 protein stability or translation. These studies demonstrate that T3 stimulates NHE3 activity by activating NHE3 gene transcription and increasing NHE3 transcript and protein abundance.
Squalamine, a novel cationic steroid, specifically inhibits the brush-border Na+/H+exchanger isoform NHE3
Authors: Akhter S. , Nath S. K. , Tse C. M. , Williams J. , Zasloff M. , Donowitz M.
Published: December 2017

Squalamine, an endogenous molecule found in the liver and other tissues of Squalus acanthias, has antibiotic properties and causes changes in endothelial cell shape. The latter suggested that its potential targets might include transport proteins that control cell volume or cell shape. The effect of purified squalamine was examined on cloned Na+/H+exchanger isoforms NHE1, NHE2, and NHE3 stably transfected in PS120 fibroblasts. Squalamine (1-h pretreatment) decreased the maximal velocity of rabbit NHE3 in a concentration-dependent manner (13, 47, and 57% inhibition with 3, 5, and 7 μg/ml, respectively) and also increased K′[H+]i. Squalamine did not affect rabbit NHE1 or NHE2 function. The inhibitory effect of squalamine was 1) time dependent, with no effect of immediate addition and maximum effect with 1 h of exposure, and 2) fully reversible. Squalamine pretreatment of the ileum for 60 min inhibited brush-border membrane vesicle Na+/H+activity by 51%. Further investigation into the mechanism of squalamine’s effects showed that squalamine required the COOH-terminal 76 amino acids of NHE3. Squalamine had no cytotoxic effect at the concentrations studied, as indicated by monitoring lactate dehydrogenase release. These results indicate that squalamine 1) is a specific inhibitor of the brush-border NHE isoform NHE3 and not NHE1 or NHE2, 2) acts in a nontoxic and fully reversible manner, and 3) has a delayed effect, indicating that it may influence brush-border Na+/H+exchanger function indirectly, through an intracellular signaling pathway or by acting as an intracellular modulator.
KDR activation is crucial for VEGF165-mediated Ca2+ mobilization in human umbilical vein endothelial cells
Authors: Cunningham Sonia A. , Tran Tuan M. , Arrate M. Pia , Bjercke Robert , Brock Tommy A.
Published: December 2017

We have prepared a polyclonal mouse antibody directed against the first three immunoglobulin-like domains of the kinase insert domain-containing receptor (KDR) tyrosine kinase. It possesses the ability to inhibit binding of the 165-amino acid splice variant of vascular endothelial cell growth factor (VEGF165) to recombinant KDR in vitro as well as to reduce VEGF165 binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the first three immunoglobulin-like domains of KDR are involved in VEGF165 interactions. The anti-KDR antibody is able to completely block VEGF165-mediated intracellular Ca2+ mobilization in HUVEC. Therefore, it appears that binding of VEGF165 to the fms-like tyrosine kinase (Flt-1) in these cells does not translate into a Ca2+ response. This is further exemplified by the lack of response to placental growth factor (PlGF), an Flt-1-specific ligand. Additionally, PlGF is unable to potentiate the effects of submaximal concentrations of VEGF165. Surprisingly, the VEGF-PlGF heterodimer was also very inefficient at eliciting a Ca2+ signaling event in HUVEC. We conclude that KDR activation is crucial for mobilization of intracellular Ca2+ in HUVEC in response to VEGF165.
Activation of ERK by Ca2+store depletion in rat liver epithelial cells
Authors: Maloney Judith A. , Tsygankova Oxana M. , Yang Lijun , Li Qiuyang , Szot Agnieszka , Baysal Kemal , Williamson John R.
Published: December 2017

In rat liver epithelial (WB) cells, Ca2+ pool depletion induced by two independent methods resulted in activation of extracellular signal-regulated protein kinase (ERK). In the first method, Ca2+ pool depletion by thapsigargin increased the activity of ERK, even when rise in cytosolic Ca2+ was blocked with the Ca2+ chelator BAPTA-AM. For the second method, addition of extracellular EGTA at a concentration shown to deplete intracellular Ca2+pools also increased ERK activity. In each instance, ERK activation, as measured by an immunocomplex kinase assay, was greatly reduced by the tyrosine kinase inhibitor genistein, suggesting that Ca2+ store depletion increased ERK activity through a tyrosine kinase pathway. The intracellular Ca2+-releasing agent thapsigargin increased Fyn activity, which was unaffected by BAPTA-AM pretreatment, suggesting that Fyn activity was unaffected by increased cytosolic free Ca2+. Furthermore, depletion of intracellular Ca2+ with EGTA caused inactivation of protein phosphatase 2A and protein tyrosine phosphatases. ANG II-induced activations of Fyn, Raf-1, and ERK were augmented in cells pretreated with BAPTA-AM, but ANG II-induced expression of the dual-specificity phosphatase mitogen-activated protein kinase phosphatase-1 was blocked by BAPTA-AM pretreatment. Together these results indicate that ERK activity is regulated by the balance of phosphorylation vs. dephosphorylation reactions in intact cells and that the amount of Ca2+stored in intracellular pools plays an important role in this regulation.
Detection of local ATP release from activated platelets using cell surface-attached firefly luciferase
Authors: Beigi Reza , Kobatake Eiry , Aizawa Masuo , Dubyak George R.
Published: December 2017

We have developed a method for measuring the local concentration of ATP at the extracellular surface of live cells. This method relies on the specific attachment to the cell surface of a chimeric protein that consists of the IgG-binding domain of Staphylococcus aureus protein A fused in-frame with the complete sequence for firefly luciferase (proA-luc). Expression of proA-luc in Escherichia coli and its one-step affinity purification are straightforward. Attachment to cells is demonstrated to be specific and antibody dependent using several suspended and adherent cell types. Light production by cell surface-attached luciferase is continuous, linearly related to ATP concentration, and sufficient to provide nanomolar sensitivity. The spatial resolution of this method enables the observation of strictly local changes in extracellular ATP during its secretion from activated platelets. Furthermore, the activity of cell-attached luciferase is relatively refractory to the inclusion of nucleotidases in the medium, arguing for its effectiveness in cell systems possessing potent ecto-ATPases.
Regulation of ET-1 biosynthesis in cerebral microvascular endothelial cells by vasoactive agents and PKC
Authors: Yakubu Momoh A. , Leffler Charles W.
Published: December 2017

Endothelin-1 (ET-1) is the most potent vasoconstrictor agent known. ET-1 is elevated in the cerebrospinal fluid following hemorrhage and brain injury and can compromise cerebral microvascular homeostasis. The modulation of ET-1 production by cerebral microvascular endothelial cells and the mechanism by which such changes take place are very important in our understanding of the pathological roles of ET-1. In the present study, we investigated the effects of vasoconstrictor agents that can be released from hemolyzed blood, cAMP-dependent dilators, and the role of protein kinase C (PKC) in the regulation of ET-1 production by piglet cerebral microvascular endothelial cells in culture. ET-1 was measured by RIA. 1) Cerebral microvascular endothelial cells synthesize and release ET-1 into the media; 2) 5-hydroxytryptamine (5-HT), lysophosphatidic acid (LPA), thromboxane analog U-46619, fetal bovine serum (20%), and phorbol 12-myristate 13-acetate significantly increase ET-1 production; 3) basal and vasoconstrictor agent-induced increases in ET-1 production by endothelial cells may be mediated via PKC; 4) cAMP-dependent vasodilators attenuate the basal production of ET-1 by cerebral microvessels; and 5) pretreatment of endothelial cells with a higher concentration of LPA, U-46619, or 5-HT counterbalances the cAMP-dependent dilator agent-induced reduction in basal ET-1 production. Therefore, by-products of hemolyzed blood can stimulate the production of ET-1 by a PKC-mediated mechanism. cAMP-dependent dilators can attenuate the vasoconstrictor agent-induced elevation in ET-1 production. These results suggest that cerebral microvascular homeostasis could be compromised by effects of interactions among vasoactive agents released during conditions injurious to the brain and they may further the understanding of potential contributions of hemolyzed blood clots to subarachnoid hemorrhage-induced vasospasm.
Adenosine and its nucleotides activate wild-type and R117H CFTR through an A2Breceptor-coupled pathway
Authors: Clancy John P. , Ruiz Fadel E. , Sorscher Eric J.
Published: December 2017

ATP and its metabolites stimulate Cl−secretion in human epithelium in vitro and in vivo. The specific purinergic receptor subtypes that govern these effects have been difficult to separate, in part due to multiple parallel pathways for Cl− secretion in respiratory and intestinal epithelia. In a simplified model using COS-7 cells, we demonstrate acquisition of an ATP-, ADP-, AMP-, and adenosine (ADO)-regulated halide permeability specifically following expression of wild-type (wt) cystic fibrosis transmembrane conductance regulator (CFTR). This halide permeability is blocked by the P1 purinergic receptor antagonist 8-phenyl theophylline, sensitive to the protein kinase A inhibitor H-89, and associated with a modest, dose-dependent increase in cellular cAMP concentration. Phorbol esters poorly activate halide permeability compared with ADO, and ADO-stimulated efflux was not affected by treatment with the protein kinase C inhibitor bisindolylmaleimide I. The A2 ADO receptor (AR) agonists 5′- N-ethylcarboxamide adenosine and ADO were strong activators, whereas the A1 AR agonist R-phenylisopropyladenosine failed to activate halide permeability. Metabolic conversion of ADO nucleotides by surface ecto-5′-nucleotidase to more active (less phosphorylated) forms contributes to anion transport activation in these cells. Immunoprecipitation with anti-A2B AR antibody identified a 31-kDa protein in both COS-7 and human bronchial epithelial cells. Together, these findings indicate that ADO and its nucleotides are capable of activating wtCFTR-dependent halide permeability through A2B AR and that this AR subtype is present in human bronchial epithelium. We also present data showing that this pathway can activate clinically significant mutant CFTR molecules such as R117H.
Shrinkage-induced activation of Na+/H+exchange in rat renal mesangial cells
Authors: Bevensee Mark O. , Bashi Esther , Schlue Wolf-Rüdiger , Boyarsky Gregory , Boron Walter F.
Published: December 2017

Using the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we examined the effect of hyperosmolar solutions, which presumably caused cell shrinkage, on intracellular pH (pHi) regulation in mesangial cells (single cells or populations) cultured from the rat kidney. The calibration of BCECF is identical in shrunken and unshrunken mesangial cells if the extracellular K+concentration ([K+]) is adjusted to match the predicted intracellular [K+]. For pHi values between ∼6.7 and ∼7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH2O) is threefold larger than in unshrunken cells (∼300 mosmol/kgH2O). In the nominal absence of CO2/[Formula: see text], exposing cell populations to a HEPES-buffered solution supplemented with ∼300 mM mannitol (600 mosmol/kgH2O) causes steady-state pHi to increase by ∼0.4. The pHi increase is due to activation of Na+/H+exchange because, in single cells, it is blocked in the absence of external Na+ or in the presence of 50 μM ethylisopropylamiloride (EIPA). Preincubating cells in a Cl−-free solution for at least 14 min inhibits the shrinkage-induced pHi increase by 80%. We calculated the pHi dependence of the Na+/H+exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) the pHi dependence of the total acid-extrusion rate and 2) the pHi dependence of the EIPA-insensitive acid-loading rate. Shrinkage alkali shifts the pHi dependence of Na+/H+exchange by ∼0.7 pH units.
Cellular microbiology: can we learn cell physiology from microorganisms?
Authors: Fasano Alessio
Published: December 2017

Cellular microbiology is a new discipline that is emerging at the interface between cell biology and microbiology. The application of molecular techniques to the study of bacterial pathogenesis has made possible discoveries that are changing the way scientists view the bacterium-host interaction. Today, research on the molecular basis of the pathogenesis of infective diarrheal diseases of necessity transcends established boundaries between cell biology, bacteriology, intestinal pathophysiology, and immunology. The use of microbial pathogens to address questions in cell physiology is just now yielding promising applications and striking results.
Desensitization of P2Y2receptor-activated transepithelial anion secretion
Authors: Clarke Lane L. , Harline Matthew C. , Otero Miguel A. , Glover Geraldine G. , Garrad Richard C. , Krugh Brent , Walker Nancy M. , González Fernando A. , Turner John T. , Weisman Gary A.
Published: December 2017

Desensitization of P2Y2 receptor-activated anion secretion may limit the usefulness of extracellular nucleotides in secretagogue therapy of epithelial diseases, e.g., cystic fibrosis (CF). To investigate the desensitization process for endogenous P2Y2 receptors, freshly excised or cultured murine gallbladder epithelia (MGEP) were mounted in Ussing chambers to measure short-circuit current ( I sc), an index of electrogenic anion secretion. Luminal treatment with nucleotide receptor agonists increased the I sc with a potency profile of ATP = UTP > 2-methylthioATP >> α,β-methylene-ATP. RT-PCR revealed the expression of P2Y2 receptor mRNA in the MGEP cells. The desensitization of anion secretion required a 10-min preincubation with the P2Y2receptor agonist UTP and increased in a concentration-dependent manner (IC50 ≈ 10−6 M). Approximately 40% of the anion secretory response was unaffected by maximal desensitizing concentrations of UTP. Recovery from UTP-induced desensitization was rapid (<10 min) at preincubation concentrations less than the EC50 (1.9 × 10−6 M) but required progressively longer time periods at greater concentrations. UTP-induced total inositol phosphate production and intracellular Ca2+ mobilization desensitized with a concentration dependence similar to that of anion secretion. In contrast, maximal anion secretion induced by Ca2+ ionophore ionomycin was unaffected by preincubation with a desensitizing concentration of UTP. It was concluded that 1) desensitization of transepithelial anion secretion stimulated by the P2Y2 receptor agonist UTP is time and concentration dependent; 2) recovery from desensitization is prolonged (>90 min) at UTP concentrations >10−5 M; and 3) UTP-induced desensitization occurs before the operation of the anion secretory mechanism.
Targeted disruption of the murine Nhe1 locus induces ataxia, growth retardation, and seizures
Authors: Bell Sheila M. , Schreiner Claire M. , Schultheis Patrick J. , Miller Marian L. , Evans Richard L. , Vorhees Charles V. , Shull Gary E. , Scott William J.
Published: December 2017

In most cells, the ubiquitously expressed Na+/H+exchanger isoform 1 (NHE1) is thought to be a primary regulator of pH homeostasis, cell volume regulation, and the proliferative response to growth factor stimulation. To study the function of NHE1 during embryogenesis when these cellular processes are very active, we targeted the Nhe1 gene by replacing the sequence encoding transmembrane domains 6 and 7 with the neomycin resistance gene. NHE activity assays on isolated acinar cells indicated that the targeted allele is functionally null. Although the absence of NHE1 is compatible with embryogenesis, Nhe1 homozygous mutants (−/−) exhibit a decreased rate of postnatal growth that is first evident at 2 wk of age. At this time, Nhe1 −/− animals also begin to exhibit ataxia and epileptic-like seizures. Approximately 67% of the −/− mutants die before weaning. Postmortem examinations frequently revealed an accumulation of a waxy particulate material inside the ears, around the eyes and chin, and on the ventral surface of the paws. Histological analysis of adult tissues revealed a thickening of the lamina propria and a slightly atrophic glandular mucosa in the stomach.
Cloning of Trp1β isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein
Authors: Wang Weiching , O’Connell Brian , Dykeman Raymond , Sakai Takayuki , Delporte Christine , Swaim William , Zhu Xi , Birnbaumer Lutz , Ambudkar Indu S.
Published: December 2017

The Trp gene product has been proposed as a candidate protein for the store-operated Ca2+channel, but the Trp protein(s) has not been identified in any nonexcitable cell. We report here the cloning of a rat brain Trp1β cDNA and detection and immunolocalization of the endogenous and expressed Trp1 protein. A 400-bp product, with >95% homology to mouse Trp1, was amplified from rat submandibular gland RNA. Rat-specific primers were used for cloning of a full-length rat brain Trp1β cDNA (rTrp1), encoding a protein of 759 amino acids. Northern blot analysis demonstrated the transcript in several rat and mouse tissues. The peptide (amino acids 523–536) was used to generate a polyclonal antiserum. The affinity-purified antibody 1) immunoprecipitated human Trp1 (hTrp1) from transfected HEK-293 cells, 2) reacted with a protein of ∼92 kDa, but not with hTrp3, in membranes of hTrp3-expressing HEK-293 cells, and 3) reacted with proteins of 92 and 56 kDa in human and rat brain membranes. Confocal microscopy and cell fractionation demonstrated that endogenous and expressed hTrp1 and expressed hTrp3 proteins were localized in the plasma membrane of HEK-293 cells, consistent with their proposed role in Ca2+ influx. The data demonstrate for the first time the presence of Trp1 protein in a nonexcitable cell.