bluuebunny/crossref_metadata_2025 huggingface dataset:
ATP depletion alters myosin I beta cellular location in LLC-PK1 cells
Authors: Wagner M. C. , Molitoris B. A.
Published: December 2017

The brush border (BB) of the proximal tubule cell (PTC) requires dynamic membrane events for function. The actin cytoskeleton is necessary for structure and function in this region. ATP depletion disrupts both structure and function. In this report, myosin 1 beta location in LLC-PK1 cells was followed during ATP depletion and repletion using immunofluorescence and Western blot techniques. Myosin I beta colocalized with F-actin in the microvilli and cell periphery, but no colocalization was observed with stress fibers. ATP depletion increased the apical F-actin, and myosin I beta was colocalized there. In addition, after ATP depletion, myosin I beta was extracted less by Triton X-100. These changes were reversed after ATP repletion. Finally, immunofluorescence of kidney sections shows myosin I beta in the BB. These results place this motor in a dynamic region of the PTC where its actin and membrane binding domains can contribute to PTC function.
Temporal expression of PDGF receptors and PDGF regulatory effects on osteoblastic cells in mineralizing cultures
Authors: Yu X. , Hsieh S. C. , Bao W. , Graves D. T.
Published: December 2017

Platelet-derived growth factor (PDGF) is mitogenic and chemotactic for osteoblastic cells in vitro. It is expressed during osseous wound healing and stimulates formation of new bone in vivo. PDGF stimulates cells by binding to specific cell surface receptors. The purpose of this study was to examine the effects of PDGF on osteoblastic proliferation and differentiation in long-term mineralizing cultures. Utilizing Northern blot analysis, we found that continuous PDGF treatment increased histone expression, indicative of enhanced proliferation, but suppressed osteoblast differentiation, demonstrated by inhibition of alkaline phosphatase, type I collagen, and osteocalcin expression. The inhibitory effect of PDGF on the differentiated function of osteoblasts was further established by findings that PDGF significantly inhibited nodule formation. The expression of PDGF receptors varied at different stages of culture. PDGF receptor mRNA expression increased when the cells had achieved a mature phenotype, during the stage of matrix maturation, and then decreased. However, as demonstrated by thymidine incorporation assays, the capacity of PDGF to stimulate DNA synthesis actually decreased during osteoblast maturation, as receptor expression increased. To investigate this apparent contradiction, tyrosyl phosphorylation and immunoblot assays were performed to assess changes in PDGF activation of their cognate receptors. The pattern of PDGF-induced tyrosyl phosphorylation remained relatively constant. This suggests that the diminished mitogenic activity of PDGF that occurs after osteoblast differentiation is regulated at a postreceptor level.
Amino acids Val115-Ile126 of rat gastric H(+)-K(+)-ATPase confer high affinity for Sch-28080 to Na(+)-K(+)-ATPase
Authors: Lyu R. M. , Farley R. A.
Published: December 2017

Na(+)-K(+)-ATPase is inhibited by cardiac glycosides and is insensitive to Sch-28080, an inhibitor of gastric H(+)-K(+)-ATPase. Gastric H(+)-K(+)-ATPase is not inhibited by cardiac glycosides. Both ouabain and, Sch-28080 binding are inhibited by K+, and it has been suggested that the inhibitors bind to corresponding regions on the alpha-subunit of each ion pump. For identification of regions of each pump that interact with the specific inhibitors, chimeric alpha-subunits consisting of selected regions from Na(+)-K(+)-ATPase and gastric H(+)-K(+)-ATPase have been prepared. One chimera (gM1/2) has been constructed from cDNA of the sheep alpha1-subunit of Na(+)-K(+)-ATPase by replacement of the last 12 amino acids of the first predicted transmembrane region (Ile99-Ile110) with corresponding amino acids from rat gastric H(+)-K(+)-ATPase. gM1/2 was expressed in yeast cells together with either the rat Na(+)-K(+)-ATPase beta 1-subunit (NK beta 1) or rat gastric H(+)-K(+)-ATPase beta-subunit (HK beta). Western blots show that the expression level of the chimeric alpha-subunit was comparable to the Na(+)-K(+)-ATPase alpha 1. Ouabain binds with high affinity to gM1/2+NK beta 1 [ouabain binding affinity (Kd) = 9.5 nM] but not to gM1/2+HK beta. The Kd for ouabain binding to Na(+)-K(+)-ATPase was 7.8 nM. Na(+)-K(+)-ATPase activity of gM1/2+NK beta 1 was inhibited both by ouabain and Sch-28080. The 50% inhibition concentration for Sch-28080 was 20-60 nM. Sch-28080 at 10 microM did not inhibit Mg(2+)- and Pi-dependent ouabain binding to gM1/2+NK beta 1. Ouabain (0.75 mM) inhibited palytoxin-induced K+ efflux from yeast cells expressing either gM1/2+NK beta 1 or gM1/2+NK beta, and Sch-28080 increased the palytoxin-induced K+ efflux from yeast cells expressing gM1/2+NK beta 1 or gM1/2+HK beta. These results implicate a small number of amino acids in the first transmembrane part of gastric H(+)-K(+)-ATPase as partial determinants of the sensitivity to Sch-28080. The data also suggest that ouabain and Sch-28080 do not bind to the same site on the chimera.
Ca2+ flux in human placental trophoblasts
Authors: Karl P. I. , Chusid J. , Tagoe C. , Fisher S. E.
Published: December 2017

Intracellular Ca2+ is an important second messenger. In the placenta, regulation of intracellular Ca2+ concentration ([Ca2+]i) by extracellular factors has received relatively little attention. Cultured human placental trophoblasts were treated with a series of potential Ca(2+)-mobilizing ligands. After 3 days in culture, there was an increase in [Ca2+]i in response to angiotensin, endothelin, transforming growth factor-alpha, and ATP in approximately 8, 54, 17, and 100% of the cells, respectively. The response to ATP was dose dependent. At low ATP concentrations (1-10 microM), the response to repeat ATP application remained unchanged, whereas at 100 microM, response to repeat stimulation resulted in lower peak value. The order of potency for the ATP derivatives was ATP = UTP > benzoylbenzoic-ATP > ATP gamma S > ADP beta S > ADP > alpha,beta-MeATP > AMP. This suggests action via the P2u purinergic receptor. Removal of extracellular Ca2+ decreased the ATP-induced Ca2+ response by 45%; this indicates that a substantial portion of the increase in [Ca2+]i was due to influx from extracellular space. Finally, ATP rapidly induced inositol 1,4,5-trisphosphate formation in cultured trophoblasts. Therefore, ATP-induced changes in Ca2+ flux may be due in part to activation of phosphoinositide-specific phospholipase C. In summary, ATP is a potent calciotropic ligand in human placental trophoblasts, acting through the P2u receptor. The effect of ATP on [Ca2+]i may prove to be involved in the modulation of various trophoblast functions, including hormone secretion and active transport of nutrients.
Eicosanoid production from porcine neutrophils and platelets: differential production with various agonists
Authors: Stahl G. L. , Morse D. S. , Martin S. L.
Published: December 2017

Polymorphonuclear neutrophils (PMN) and platelets interact to produce both inflammatory and anti-inflammatory lipid mediators during human disease. Because swine models of human disease are used, it is important to understand the mechanisms involved in the formation of lipid mediators from porcine PMN-platelet interactions. In the present study, we investigated the mechanism of thromboxane (Tx) A2 and lipoxin A4 (LXA4) formation from porcine PMN and platelets, respectively. PMN (10(7)/ml) and platelet (30 x 10(7)/ml) suspensions stimulated with porcine C5a (pC5a), but not recombinant human C5a (rhC5a), significantly enhanced TxB2 formation. After cytochalasin B treatment, pC5a or rhC5a significantly and equally enhanced TxB2 formation from PMN-platelet suspensions. A-23187-induced TxB2 formation from platelets was not significantly augmented by the presence of PMN in these suspensions. A-23187 induced significant LXA4 production from porcine PMN that was not augmented by addition of platelets. Flow cytometric analysis of PMN-platelet suspensions revealed activated platelets adherent to PMN following pC5a stimulation. CV-6209, a platelet-activating factor (PAF) receptor antagonist, dose dependently prevented pC5a-induced platelet adherence to PMN and TxB2 formation. These data demonstrate that 1) porcine PMN alone can biosynthesize LXA4 without the assistance of platelets, which is in sharp contrast to human PMN-platelet interactions, and 2) in the absence of cytochalasin B, pC5a stimulates PAF biosynthesis from porcine PMN, resulting in TxB2 formation from platelets.
IL-6 secreted by astroglial cells regulates Na-K-Cl cotransport in brain microvessel endothelial cells
Authors: Sun D. , Lytle C. , O'Donnell M. E.
Published: December 2017

We showed previously that cerebral microvessel endothelial cells (CMEC) exhibit a prominent Na-K-Cl cotransporter that functions to regulate intracellular volume and may also mediate vectorial ion transport across the blood-brain barrier (BBB). Astrocytes and their conditioned media induce BBB properties of the endothelium. Our previous studies demonstrated that exposure of CMEC to astroglial cells markedly increases cotransport activity, upregulates cotransporter protein expression, and also increases cotransporter protein phosphorylation. In the present study, we evaluated the possibility that astroglial effects on the Na-K-Cl cotransporter are mediated by astroglial cell-secreted interleukin-6 (IL-6). Cotransporter activity was assessed as bumetanide-sensitive K influx, and protein expression was evaluated by Western blot analysis. Exposing CMEC to IL-6 for 48 h caused a dose-dependent stimulation of Na-K-Cl cotransport activity. Both C6 glial cell-conditioned medium (C6CM)-induced and IL-6-induced increases in cotransport activity were neutralized by anti-IL-6 antibodies. A 48-h exposure of cells to IL-6 or C6CM also resulted in increased cotransporter protein expression. Furthermore, using an enzyme-linked immunosorbent assay for IL-6, we found significant amounts of IL-6 in C6CM. These data suggest that astroglia-secreted IL-6 may mediate the observed astroglial cell effects on CMEC Na-K-Cl cotransport and further support the hypothesis that astrocytes participate in maintenance of cerebral ionic homeostasis by regulating Na-K-Cl cotransporter function at the BBB.
Actions of sulfhydryl reagents on single ryanodine receptor Ca(2+)-release channels from sheep myocardium
Authors: Eager K. R. , Roden L. D. , Dulhunty A. F.
Published: December 2017

Effects of the reactive disulfides, 2,2'- and 4,4'-dithiodipyridine, on single cardiac ryanodine receptor (RyR) ion channels incorporated into lipid bilayers are reported. RyRs are activated within minutes of addition of the reactive disulfides (10(-7) to 10(-3) M) with an irreversible loss of channel activity after the activation at concentrations > or = 10(-4) M. This activation, followed by loss of activity, is seen over a wide range of cytoplasmic (cis) Ca2+ concentration between 10(-9) and 2 x 10(-2) M and occurs more rapidly with higher reactive disulfide concentrations or when RyRs are initially active at 10(-5) or 10(-3) M Ca2+. The reactive disulfides increase the channel open probability by introducing long components into the open time distributions, increasing the mean channel open time by up to 50-fold. Closed time distributions are not altered by the sulfhydryl reagents. The effects of the reactive disulfides are prevented by the reducing agents dithiothreitol and glutathione (1–10 mM). The results suggest that cysteine residues on the RyR complex can regulate the ion channel gating mechanisms.
Differential processing of guanylyl cyclase C along villus-crypt axis of rat small intestine
Authors: Scheving L. A. , Chong K. M.
Published: December 2017

Many strains of enterotoxigenic Escherichia coli produce a heat-stable peptide enterotoxin (STa) that binds to the intestinal receptor guanylyl cyclase C (GC-C). STa receptors are structurally heterogeneous, but the molecular events causing this heterogeneity remain obscure. We examined the influence of cell position along the villus-crypt axis on STa receptor heterogeneity by fractionating EDTA-dissociated cells that detached in a villus-to-crypt direction. STa affinity labeling experiments revealed that the initially released villus “tip” fraction had four major STa binding proteins (STBPs), with relative molecular weight (M(r)) of 150,000, 135,000, 125,000, and 95,000, that did not react with a GC-C carboxy-terminal antibody. Yet succeeding villus cell fractions had major immunoreactive STBPs with M(r) of 275,000 and 250,000. Limited proteolysis of these larger GC-C isoforms produced 1) smaller STBPs that had M(r) similar to those in the initial villus fraction, 2) a 65,000 M(r) protein GC-C isoform that did not bind STa, and 3) elevated basal and STa-induced cyclase activity. Our data show that STBP structural heterogeneity in the intact intestine arises largely from multisite proteolytic processing of GC-C.
TCA cycle flux estimates from NMR- and GC-MS-determined [13C]glutamate isotopomers in liver
Authors: Vogt J. A. , Yarmush D. M. , Yu Y. M. , Zupke C. , Fischman A. J. , Tompkins R. G. , Burke J. F.
Published: December 2017

Infusion of 13C-labeled lactate into rabbits and the subsequent measurement of glutamate isotopomers by 13C nuclear magnetic resonance (NMR) spectroscopy enables one to calculate relative flow rates associated with the tricarboxylic acid (TCA) cycle, albeit with a lower precision than one would obtain using a perfused organ. Two factors contribute to the lower precision in the determination of relative flow rates for the in vivo system: 1) a poorly defined pyruvate input and 2) low levels of 13C-enriched oxaloacetate and acetyl-CoA isotopomers, which give rise to weaker glutamate isotopomer NMR signals. To help overcome these limitations, we introduce a procedure to 1) include experimental data from gas chromatography-mass spectrometry (GC-MS) and 2) account for the uncertainty in the labeling of the input to pyruvate by creating the labeling as a measurement that is subject to measurement error. The effects of the uncertainties in the input labeling, NMR data, and MS data are evaluated via a Monte Carlo method. The change in the precision of the relative fluxes for the cases of high/low NMR and high/low MS precision is given. An uncertainty in the lactate measurements of up to 10% does not add significantly to the imprecision of the relative flow rates.
Overexpression of protein kinase C-zeta isoform increases cyclooxygenase-2 and inducible nitric oxide synthase
Authors: Miller B. W. , Baier L. D. , Morrison A. R.
Published: December 2017

Cyclooxygenase (COX) catalyzes the formation of prostaglandins from arachidonic acid. Nitric oxide synthase catalyzes the production of nitric oxide, a short-lived messenger molecule involved in many diverse cellular processes. Both of these enzymes have inducible forms [COX-2 and inducible nitric oxide synthase (iNOS), respectively] that respond to environmental stresses, chemicals, and extracellular ligands such as interleukin-1, epidermal growth factor, and platelet-derived growth factor. The precise cascade of intracellular events that leads to the expression of either COX-2 or iNOS is not known. Protein kinase C (PKC) is a family of 11 serine-threonine kinases conserved throughout eukaryotic species that transduce a wide variety of signals critical for cellular functions. Using a retroviral vector to overexpress the zeta-isoform of PKC in rat mesangial cells, we demonstrate markedly increased COX-2, prostaglandin E2 (PGE2), iNOS, and altered cellular morphology compared with mesangial cells expressing a control retroviral vector and untransfected mesangial cells. NIH/3T3 cells overexpressing PKC-zeta showed no change in morphology, PGE2 production, COX-2 expression, or iNOS expression at basal conditions. This suggests a role for PKC-zeta in the expression of these enzymes in mesangial cells.
cAMP-dependent sulfate secretion by the rabbit distal colon: a comparison with electrogenic chloride secretion
Authors: Freel R. W. , Hatch M. , Vaziri N. D.
Published: December 2017

The ability of a Cl-secreting epithelium to support net secretion of an anion other than a halide was investigated with 35SO4 flux measurements across the isolated, short-circuited rabbit distal colon. In most experiments, 36Cl fluxes were simultaneously measured to validate the secretory capacity of the tissues. Serosal addition of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 0.5 mM) stimulated a sustained net secretion of SO4 (about -3.0 nmol.cm-2.h-1 from a 0.20 mM solution) via an increase in the serosal-to-mucosal unidirectional flux, whereas Ca ionophore A-23187 (1 microM, serosal) produced a more transient stimulation of SO4 and Cl secretion. Net adenosine 3',5'-cyclic monophosphate (cAMP)-dependent SO4 and Cl secretion were strongly voltage sensitive, principally through the potential dependence of the serosal-to-mucosal fluxes, indicating an electrogenic transport process. Symmetrical replacement of either Na, K, or Cl inhibited cAMP-dependent SO4 secretion, whereas HCO3-free buffers had no effect on SO4 secretion. Serosal bumetanide (50 microM) or furosemide (100 microM) reduced DBcAMP-stimulated SO4 and Cl secretion, whereas serosal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (50 microM) blocked DBcAMP-induced SO4 secretion while enhancing net Cl secretion and short-circuit current. Mucosal 5-nitro-2-(3-phenylpropylamino)benzoic acid partially inhibited SO4 secretion and completely inhibited Cl secretion. It is concluded that secretagogue-stimulated SO4 secretion, like Cl secretion, may be an electrogenic process mediated by diffusive efflux through an apical anion conductance. Cellular accumulation of SO4 across the basolateral membrane appears to be achieved by a mechanism that is distinct from that employed by Cl.
Swelling-activated efflux of taurine and other organic osmolytes in endothelial cells
Authors: Manolopoulos V. G. , Voets T. , Declercq P. E. , Droogmans G. , Nilius B.
Published: December 2017

We used a combined biochemical, pharmacological, and electrophysiological approach to study the effects of hyposmotic swelling on organic osmolyte efflux in endothelial cells (EC). In [3H]taurine-loaded monolayers of calf pulmonary artery EC (CPAEC), hyposmolality activated time- and dose-dependent effluxes of [3H]taurine. Swelling-activated [3H]taurine efflux (Jtau swell)in CPAEC was inhibited by the anion channel blockers tamoxifen, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), fenamates, and also quinine (in a pH-dependent manner), ATP, and the phospholipase A2 inhibitor 4-bromophenacyl bromide. In contrast, Jtau swell was partly or totally insensitive to bumetanide, forskolin, phorbol 12-myristate 13-acetate, and staurosporine. Swelling also activated myo-[3H]inositol efflux that was blocked by tamoxifen, NPPB, DIDS, and niflumic acid. Moreover, the cellular content of taurine and other amino acids was significantly reduced in osmotically activated CPAEC. Finally, in whole cell patch-clamp experiments, taurine, glycine, aspartate, and glutamate exhibited significant permeability for swelling-activated anion channels. In conclusion, hyposmotic swelling activates efflux of taurine and other organic osmolytes in EC. In addition, our results suggest that anion channels may provide a pathway for swelling-activated efflux of organic osmolytes in EC.
bFGF induces BCK promoter-driven expression in muscle via increased binding of a nuclear protein
Authors: Kim L. , Steves A. , Collins M. , Fu J. , Ritchie M. E.
Published: December 2017

Changes in gene expression occurring during skeletal muscle differentiation are exemplified by downregulation of brain creatine kinase (BCK) and induction of muscle creatine kinase (MCK). Although both are transcriptionally regulated, there appears to be no transcription factor-element overlap, suggesting that their coordinate expression results from culture medium-related influences. Basic fibroblast growth factor (bFGF) prevents myogenesis and represses MCK expression by inhibiting transcriptional activation. It was hypothesized that bFGF similarly influenced BCK by inducing its expression. Accordingly, BCK promoter constructs were transiently transfected into C2C12 cells and, after a switch to differentiation medium, were treated with bFGF, bFGF plus herbimycin, adenosine 3',5'-cyclic monophosphate (cAMP), or phorbol 12-myristate 13-acetate (PMA). Analyses demonstrated that bFGF responsiveness was contained within a 33-base pair element. Electromobility shift assays showed that bFGF induction increased the abundance of the nuclear factor binding the element. Both effects were prevented by herbimycin. Neither cAMP nor PMA specifically induced the construct containing the bFGF-responsive element. The induced factor required phosphorylation to bind, implying that bFGF-mediated increases in binding may be due to transcription factor phosphorylation.
Possible thiol group involvement in intracellular pH effect on low-conductance Ca(2+)-dependent K+ channels
Authors: Riquelme G. , Diaz M. , Sepulveda F. V.
Published: December 2017

We have studied the effect of intracellular pH (pHi) shifts on the activity of Ca(2+)-dependent, inwardly rectifying K+ channels of HeLa cells. Recordings of macroscopic currents in symmetrical 145 mK K+ and internal pH of 7.4 gave moderate inward rectification of the current. At pH 6.4, inward rectification was more marked, whereas it switched to outward rectification at pH 8.2. In excised inside-out membrane patches, similar changes in pHi did not affect the single-channel conductance of the channels underlying the Ca(2+)-dependent K+ currents. At neutral pH, the open state probability (Po) was independent of voltage in the range from -70 to 70 mV. At alkaline pH, Po became voltage dependent, decreasing at negative potentials and increasing with depolarization compared with pH 7.4. These changes accounted for the pH-dependent changes in rectification of the macroscopic current. The possibility that voltage dependence might arise from the ionization of a thiol group was tested by using thiol-directed reagents. The decrease in Po produced by intracellular alkalinization at negative potential was reverted by treatment with N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic acid), and 2,2'-dithiodipyridine. The effect of intracellular alkalinization is speculated to occur through ionization of a cysteine group(s) within the field of the membrane affecting gating.
Morphological and biochemical evidence of muscle hyperplasia following weight-lifting exercise in rats
Authors: Tamaki T. , Akatsuka A. , Tokunaga M. , Ishige K. , Uchiyama S. , Shiraishi T.
Published: December 2017

We used a rat model of weight lifting to examine the serial biochemical and morphological changes following muscle fiber hyperplasia during 14 days of exercise. [3H]thymidine and [14C]leucine labeling were used to determine the serial changes in cellular mitotic activity and the level of amino acid uptake and myosin synthesis. Morphological changes were assessed with light and transmission electron microscopy, whereas proliferation of cells was evaluated immunohistochemically with 5-bromo-2'-deoxyuridine (BrdU). The intensity of the exercise and degree of muscle damage were monitored by serum creatine kinase (CK) activity. Damaged fibers were sparsely distributed, and a significant CK leakage was observed 30-60 min after exercise. Anti-BrdU-positive cells were observed in damaged fibers and at the periphery of undamaged fibers. Changes typical of muscle regeneration were observed; however, the formation of new fibers in the interstitial space was also evident. The mitotic activity also changed and reflected the appearance of anti-BrdU-positive cells and activated satellite cells. Amino acid uptake increased during the first week of exercise, probably reflecting muscle hypertrophy and synthesis of other noncontractile related proteins. The uptake also increased during the second week, probably due to hyperplasia, a finding also supported by electron microscopy. Our results suggest that one bout of weight-lifting exercise in untrained rats induced muscle hyperplasia following regeneration. The process of muscle hyperplasia was activated by muscle fiber damage in our model.
Mechanisms of electrolyte transport across the endometrium. II. Regulation by GRP and substance P
Authors: Vetter A. E. , O'Grady S. M.
Published: December 2017

The purpose of this study was to investigate the regulation of electrolyte transport across the porcine endometrium by gastrin-releasing peptide (GRP) and substance P (SP). Luminal addition of GRP, neuromedin B (NMB), SP, or neurokinin A(NKA) to mucosal tissues mounted in Ussing chambers produced a multiphasic change in short-circuit current (Isc) characterized by an initial rapid increase and subsequent decrease in current. A similar response was obtained after addition of ionomycin or thapsigargin to the tissues. The Isc response to the peptides or Ca ionophore was inhibited by pretreatment of the tissues with luminal amiloride or benzamil. GRP and SP were more potent [50% effective concentration (EC50) of 3 nM] than NMB or NKA (EC50 values of 46 and 26 nM, respectively) in producing the decrease in Isc. Pretreatment with the GRP receptor antagonist 3-Phe-His-Trp-Ala-Val-D-Ala-His-D-Pro-psi Phe-NH2 blocked the Isc response to GRP and NMB but not to SP or NKA, whereas the NMB receptor antagonist D-Nal-[Cys-Try-D-Trp-Orn-Val-Cys]-Nal-NH2 was ineffective in inhibiting the Isc response to any of the peptides. In contrast, pretreatment of the tissue with the nonpeptide SP receptor antagonist CP-99,994 blocked the Isc response to SP and NKA but not to GRP or NMB. Experiments with amphotericin B-permeabilized tissues showed that GRP, SP, ionomycin, and thapsigargin increased current through an outwardly rectifying K conductance located on the apical membrane of the cells. The K-to-Na selectivity ratio of this conductance was calculated to be 2.5:1. These experiments showed that GRP and SP, acting through different receptors, produced an increase in K efflux through a Ca-dependent K conductance present in the apical membrane of surface endometrial epithelial cells. In addition, immunohistochemistry data showed that GRP-like immunoreactivity was localized to surface and glandular epithelial cells, whereas GRP receptor antibody labeling was observed in both epithelial and stromal cells. These results suggest that GRP functions as both an autocrine and paracrine regulatory peptide in the endometrium.
Role of concentration and size of intracellular macromolecules in cell volume regulation
Authors: Summers J. C. , Trais L. , Lajvardi R. , Hergan D. , Buechler R. , Chang H. , Pena-Rasgado C. , Rasgado-Flores H.
Published: December 2017

To gain insight into the mechanism(s) by which cells sense volume changes, specific predictions of the macromolecular crowding theory (A. P. Minton. In: Cellular and Molecular Physiology of Cell Volume Regulation, edited by K. Strange. Boca Raton, FL: CRC, 1994, p. 181-190. A. P. Minton, C. C. Colclasure, and J. C. Parker. Proc. Natl. Acad. Sci. USA 89: 10504-10506, 1992) were tested on the volume of internally perfused barnacle muscle cells. This preparation was chosen because it allows assessment of the effect on cell volume of changes in the intracellular macromolecular concentration and size while maintaining constant the ionic strength, membrane stretch, and osmolality. The predictions tested were that isotonic replacement of large macromolecules by smaller ones should induce volume decreases proportional to the initial macromolecular concentration and size as well as to the magnitude of the concentration reduction. The experimental results were consistent with these predictions: isotonic replacement of proteins or polymers with sucrose induced volume reductions, but this effect was only observed when the replacement was > or = 25% and the particular macromolecule had an average molecular mass of < or = 20 kDa and a concentration of at least 18 mg/ml. Volume reduction was effected by a mechanism identical with that of hypotonicity-induced regulatory volume decrease, namely, activation of verapamil-sensitive Ca2+ channels.
Two mechanisms by which ATP depletion potentiates induction of the mitochondrial permeability transition
Authors: Simbula G. , Glascott P. A. , Akita S. , Hoek J. B. , Farber J. L.
Published: December 2017

The present and a previous study [J. W. Snyder, J. G. Pastorino, A. M. Attie, and J. L. Farber, Am. J. Physiol. 264 (Cell Physiol. 33): C709-C714, 1993] define two mechanisms whereby ATP depletion promotes liver cell death. ATP depletion and cell death are linked by the mitochondrial permeability transition (MPT). Mitochondrial deenergization promotes the MPT, and ATP maintains a membrane potential by reversal of ATP synthase. With an increased influx of Ca2+ induced by the ionophore A-23187, oligomycin depleted the cells of ATP without loss of the mitochondrial membrane potential and further elevated the intracellular Ca2+ concentration. Cyclosporin A (CyA) prevented the accompanying cell killing. Fructose also preserved the viability of the cells. With the increased cytosolic Ca2+ imposed by A-23187, viability is maintained by ATP-dependent processes. Upon depletion of ATP, Ca2+ homeostasis cannot be maintained, and the MPT is induced. Rotenone also depleted the cells of ATP, and A-23187 accelerated the loss of the mitochondrial membrane potential occurring with rotenone alone. CyA and fructose prevented the cell killing with rotenone and A-23187. Oligomycin did not prevent this action of fructose. We conclude that ATP is needed to maintain Ca2+ homeostasis to prevent the MPT and the resultant liver cell death. ATP is also needed to maintain mitochondrial energization when electron transport is inhibited.
Adaptation to low-K+ media increases H(+)-K(+)-ATPase but not H(+)-ATPase-mediated pHi recovery in OMCD1 cells
Authors: Guntupalli J. , Onuigbo M. , Wall S. , Alpern R. J. , DuBose T. D.
Published: December 2017

Studies in rat and rabbit outer medullary collecting duct of inner stripe origin (OMCDis) suggest that both H(+)-ATPase and H(+)-K(+)-ATPase participate in H+ secretion. However, the relative contributions of these transporters, and, in particular, that of H(+)-K(+)-ATPase to K+ absorption have not been defined precisely. The present study was designed to delineate more clearly the response of these two transporters to hypokalemia and acidosis in a newly developed mouse OMCD1 cell line. In cells grown in normal K+ (5 mM) media, intracellular pH (pHi) recovery was similar either in the presence or absence of K+ in the perfusate (delta pHi/min = 0.014 +/- 0.001 vs. 0.017 +/- 0.003, not significant). The inhibitory effects of Sch-28080 (10 microM) and bafilomycin A1 (10 nM) on pHi recovery were evident only in the presence and absence of K+ in the perfusate, respectively. In cells grown in low-K+ (2.5 mM) media to simulate chronic hypokalemia, pHi recovery was significantly faster than in cells grown in normal K+ media (delta pHi/min = 0.045 +/- 0.01 vs. 0.014 +/- 0.001, P < 0.01) and was inhibited specifically by Sch-28080, not by bafilomycin A1. In contrast, in cells preconditioned to low pH (7.0) to simulate chronic acidosis, the enhanced pHi recovery was abolished by bafilomycin A1 but not by Sch-28080. 86Rb+ uptake, when used as a K+ congener, was inhibited by Sch-28080. The K(m) for 86Rb+ uptake (H(+)-K(+)-ATPase activity) and the 50% inhibitory concentration for Sch-28080 were 270 and 5.0 microM, respectively. These studies provide evidence that, in morphologically homogeneous OMCD1 cells, 1) both H(+)-K(+)-ATPase and H(+)-ATPase participate in pHi regulation, 2) the H(+)-K(+)-ATPase is selectively upregulated by preconditioning in low-K+ media, and 3) conversely, preconditioning in low-pH media stimulates only the H(+)-ATPase. Thus, in OMCDis, the H(+)-K(+)-ATPase and H(+)-ATPase respond selectively and independently to chronic hypokalemia and acidosis, respectively.
Differential activation of NF-kappa B in human aortic endothelial cells conditioned to specific flow environments
Authors: Mohan S. , Mohan N. , Sprague E. A.
Published: December 2017

Endothelial cell-monocyte interaction plays an important role in atherogenesis. The expressions of some endothelial cell adhesion molecules involved in endothelial cell-monocyte interactions are regulated by transcription factor NF-kappa B. Because low shear stress has been known to influence endothelial monocyte adhesion, the differential activation of NF-kappa B under different flow regimens across time (0.5-24 h) was investigated. Nuclear proteins from flow-conditioned human aortic endothelial cells (HAEC) were analyzed by electrophoretic mobility shift assay using [gamma-32P]dATP-labeled NF-kappa B-specific oligonucleotide. Our results demonstrated that NF-kappa B activation was significantly elevated in HAEC exposed to prolonged (> 2 h) steady low shear (2 dyn/cm2) and pulsatile low shear (2 +/- 2 dyn/cm2) compared with HAEC exposed to high shear (16 dyn/cm2). In contrast, at 30 min, high shear-exposed HAEC exhibited an early, transient increase in NF-kappa B activity, relative to low shear-exposed cells, which reversed on continued exposure to high shear. Maximum activity in both low shear- and pulsatile low shear-conditioned HAEC was observed at 16 h compared with HAEC exposed to prolonged high shear. These results indicate that exposure of HAEC to prolonged low shear conditions is associated with significantly increased and prolonged NF-kappa B activity. This observation might provide a mechanism to explain the increased monocyte adhesion in atherosclerosisprone arterial sites exposed to chronic low-shear flow patterns.
Functional properties of conditioned skeletal muscle: implications for muscle-powered cardiac assist
Authors: Trumble D. R. , LaFramboise W. A. , Duan C. , Magovern J. A.
Published: December 2017

Latissimus dorsi (LD) muscles of six canines were studied to assess changes induced by electrical conditioning and to quantify the capacity of these muscles to perform hemodynamic work. Muscles were conditioned using burst stimuli delivered over an 8-wk period. Contralateral LD were used as control. Muscles were tested in situ to simulate anticipated linear-pull cardiac assist conditions. This training process reduced muscle mass and cross-sectional area by 16 and 17%, respectively. Muscle phenotype shifted to a predominantly “slow” form by coordinate reduction of myosin heavy chain (MHC) 2A expression and increased expression of the MHC beta/slow form. Force generation was reduced by 54%, and contractile duration increased 13%. Fatigue resistance was markedly enhanced, and chronic stroke work increased from 0.19 to 0.72 mJ/g. The highest steady-state power output (2.06 mW/g) was obtained from one muscle fully converted to a slow phenotype. These data suggest that single LD trained via conventional techniques can provide energy sufficient for partial cardiac assistance but cannot sustain work levels needed to achieve total circulatory support.
Cholinergic modulation of the Ca2+ response to bradykinin in neuroblastoma cells
Authors: Coggan J. S. , Thompson S. H.
Published: December 2017

Fura 2 imaging was used to measure intracellular Ca2+ signals in N1E-115 mouse neuroblastoma cells during combined activation of bradykinin (BK) and cholinergic receptors. BK and carbachol (CCh) both activate phospholipase C (PLC) and cause Ca2+ release from inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores. The Ca2+ signal in response to CCh is prolonged by the activation of Ca2+ influx, but BK does not appear to activate the influx pathway. When BK and CCh are applied together (BK+CCh), the Ca2+ response is composed of both Ca2+ release and Ca2+ influx. Ca2+ influx is also activated by BK+CCh in a subset of cells that does not respond with a intracellular Ca2+ concentration increase when CCh is presented by itself. This suggests that CCh stimulates a Ca(2+)-silent cholinergic receptor that is not coupled to Ca2+ release but acts synergistically with BK receptors to activate Ca2+ influx. Pertussis toxin reduces influx without affecting release, indicating that the G protein that modulates the influx pathway is different from the G protein responsible for activating PLC. Cholinergic stimulation also causes progressive heterologous desensitization of BK-evoked Ca2+ release. Desensitization has the unique property of continuing to develop after the cholinergic agonist is removed and the cholinergic Ca2+ response has fully recovered. Heterologous desensitization is not the result of Ca2+ store depletion or a long-lasting inhibition of PLC or IP3-dependent Ca2+ release. Instead, it appears to involve an early step in the BK-signaling cascade, possibly at the level of the B2 receptor or associated G proteins.
Suppression of neuropeptide Y1 receptor function in SK-N-MC cells by nitric oxide
Authors: Dotsch J. , Hanze J. , Beste O. , Behrendt J. , Weber W. M. , Dittrich K. , Rascher W.
Published: December 2017

The neuropeptide Y1 receptor (NPY1) predominantly mediates the vasoconstrictor effects of NPY in smooth muscle cells. The present experiments were planned to study the direct influence of the vasodilator nitric oxide (NO) on NPY1-receptor function. SK-N-MC and CHP-234 cells expressing Y1 and Y2 receptor, respectively, were incubated with the NO donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), and S-nitroso-N-acetyl-penicillamine (SNAP). Receptor binding, Y1-receptor mRNA expression by Northern blot, and adenosine 3',5'-cyclic monophosphate (cAMP) and intracellular Ca2+ concentration ([Ca2+]i) responses were studied. SNP, SIN-1, and SNAP decreased normal binding of NPY to the NPY1 receptor in SK-N-MC cells in a concentration-dependent manner. SNP (500 microM), SIN-1 (1,000 microM), and SNAP (500 microM) significantly decreased binding to approximately 50%. The cell viability was not reduced. None of the NO donors affected Y2 receptor binding. Pretreatment with SNP significantly attenuated NPY-induced inhibition of cAMP formation in SK-N-MC cells but had no effect on CHP cells. The NPY-induced [Ca2+]i response was reduced to 50% by SNP pretreatment. NPY1 mRNA expression was reduced to one-third after SNAP treatment of SK-N-MC cells. In vitro, NPY1 receptor function of SK-N-MC cells is inhibited by NO-donor incubation on an mRNA level.
Dual modulation of Na/H antiport by atrial natriuretic factor in rat aortic smooth muscle cells
Authors: Ricci R. , Baldini P. , Bogetto L. , De Vito P. , Luly P. , Zannetti A. , Incerpi S.
Published: December 2017

The aim of the present work was to study the effect of the atrial natriuretic factor (ANF) on the Na/H antiport in rat aorta smooth muscle cells, evaluated as intracellular pH (pHi) recovery after an acid load with ammonium chloride. The Na/H antiport was studied using a fluorescent probe, sensitive to pHi, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Our data indicate that ANF modulates the activity of the Na/H antiport in both a dose- and time-dependent manner. Hormone concentrations of 10(-10) M activate the antiport, increasing both the rate of recovery and the set point by approximately 0.2 pH units. This effect is mediated by diacylglycerol as a result of phospholipid hydrolysis by a phospholipase C, even if an involvement of adenosine 3',5'-cyclic monophosphate (cAMP) cannot be ruled out. ANF (10(-7) M) inhibits the antiport, decreasing both the rate of recovery and the set point by approximately 0.3 pH units, because of guanosine 3',5'-cyclic monophosphate production. Both inhibition and stimulation of pHi by ANF were more pronounced when the hormone was given before the acid load, perhaps because of the longer time exposure. We present new hypotheses on the mechanism of action of this paracrine/autocrine factor.
Effects of P2 purinergic receptor stimulation in brown adipocytes
Authors: Lee S. C. , Pappone P. A.
Published: December 2017

Sympathetic stimulation of brown adipocytes plays a major role in body energy homeostasis by activating energy-wasting pathways. Sympathetic neuronal input initiates a variety of metabolic, developmental, and membrane responses in brown fat cells. Many of these actions are mediated by adrenergic pathways mobilized by released norepinephrine. However, since sympathetic stimulation may also release vesicular ATP, we tested brown fat cells for ATP responses. Micromolar concentrations of extracellular ATP had a number of effects on brown adipocytes. We have shown previously that ATP elicits substantial (average of approximately 30%) increases in cell membrane capacitance (P. A. Pappone and S. C. Lee, J. Gen. Physiol. 108: 393-404, 1996). Here, we show that cytosolic calcium levels were increased by ATP, both through release from intracellular stores and through influx, as assessed by fura 2 imaging. In addition, ATP indirectly activated a nonselective cation conductance that was independent of cytosolic calcium levels in patch voltage-clamped brown fat cells. Similar calcium, conductance, and capacitance responses could be activated by 2-methylthio-ATP and ADP, consistent with mediation by a P2 type purinergic receptor. Calorimetric measurements from cell suspensions showed that ATP increased basal heat production of isolated brown fat cells by approximately 40% but had no effect on the greater than fivefold increase in heat production seen with maximal adrenergic stimulation. These myriad responses to extracellular ATP suggest that P2 receptor-mediated signaling is important in brown adipocyte physiology and that sympathetic stimulation may normally activate purinergic as well as adrenergic pathways in brown fat.
Effects of epinephrine on insulin-stimulated glucose uptake and GLUT-4 phosphorylation in muscle
Authors: Lee A. D. , Hansen P. A. , Schluter J. , Gulve E. A. , Gao J. , Holloszy J. O.
Published: December 2017

beta-Adrenergic stimulation has been reported to inhibit insulin-stimulated glucose transport in adipocytes. This effect has been attributed to a decrease in the intrinsic activity of the GLUT-4 isoform of the glucose transporter that is mediated by phosphorylation of GLUT-4. Early studies showed no inhibition of insulin-stimulated glucose transport by epinephrine in skeletal muscle. The purpose of this study was to determine the effect of epinephrine on GLUT-4 phosphorylation, and reevaluate the effect of beta-adrenergic stimulation on insulin-activated glucose transport, in skeletal muscle. We found that 1 microM epinephrine, which raised adenosine 3',5'-cyclic monophosphate approximately ninefold, resulted in GLUT-4 phosphorylation in rat skeletal muscle but had no inhibitory effect on insulin-stimulated 3-O-methyl-D-glucose (3-MG) transport. In contrast to 3-MG transport, the uptakes of 2-deoxyglucose and glucose were markedly inhibited by epinephrine treatment. This inhibitory effect was presumably mediated by stimulation of glycogenolysis, which resulted in an increase in glucose 6-phosphate concentration to levels known to severely inhibit hexokinase. We conclude that 1) beta-adrenergic stimulation decreases glucose uptake by raising glucose 6-phosphate concentration, thus inhibiting hexokinase, but does not inhibit insulin-stimulated glucose transport and 2) phosphorylation of GLUT-4 has no effect on glucose transport in skeletal muscle.
Mechanisms of renal tubular cell hypertrophy: mitogen-induced suppression of proteolysis
Authors: Franch H. A. , Curtis P. V. , Mitch W. E.
Published: December 2017

The combination of epidermal growth factor (EGF) plus transforming growth factor-beta 1 (TGF-beta 1) causes hypertrophy in renal epithelial cells. One mechanism contributing to hypertrophy is that EGF induces activation of the cell cycle and increases protein synthesis, whereas TGF-beta 1 prevents cell division, thereby converting hyperplasia to hypertrophy. To assess whether suppression of proteolysis is another mechanism causing hypertrophy induced by these growth factors, we measured protein degradation in primary cultures of proximal tubule cells and in cultured NRK-52E kidney cells. A concentration of 10(-8) M EGF alone or EGF plus 10(-10) M TGF-beta 1 decreased proteolysis by approximately 30%. TGF-beta 1 alone did not change protein degradation. Using inhibitors, we examined which proteolytic pathway is suppressed. Neither proteasome nor calpain inhibitors prevented the antiproteolytic response to EGF + TGF-beta 1. Inhibitors of lysosomal proteases eliminated the antiproteolytic response to EGF + TGF-beta 1, suggesting that these growth factors act to suppress lysosomal proteolysis. This antiproteolytic response was not caused by impaired EGF receptor signaling, since lysosomal inhibitors did not block EGF-induced protein synthesis. We conclude that suppression of lysosomal proteolysis contributes to growth factor-mediated hypertrophy of cultured kidney cells.
Cytoplasmic codiffusion of fatty acids is not specific for fatty acid binding protein
Authors: Luxon B. A. , Milliano M. T.
Published: December 2017

The intracellular movement of fatty acids is thought to be facilitated through codiffusion with fatty acid binding protein (FABP). Previous work suggested that FABP decreases fatty acid binding to immobile membranes, causing faster cytoplasmic diffusion. However, the specificity for binding to FABP has not been addressed. The aim of the current study was to determine whether specific FABP binding is required or whether binding to other proteins will produce the same effect. A model cytoplasm consisted of a fatty acid, proteins, and liposomes to simulate intracellular membranes. Laser photobleaching (fluorescence recovery after photobleaching) was used to measure the movement of the fluorescent fatty acid 12-N-methyl-7-nitrobenzo-2-oxa-1,3-diazoaminostearate (NBD-stearate) in model cytoplasm, in normal and permeabilized Hep G2 cells, and after incubation of permeabilized cells with bovine serum albumin (BSA) or FABP. Increasing protein in the model cytoplasm increased the diffusion rate in proportion to the extent of protein binding. Cell permeabilization reduced diffusion of NBD-stearate to < 5% of controls. Incubation of permeabilized cells with FABP or BSA resulted in a concentration-dependent increase in the NBD-stearate diffusion rate. BSA was more effective than FABP in binding NBD-stearate and increasing its diffusion rate after permeabilization. Proteins like FABP promote the diffusion of fatty acids. Removal of these proteins drastically reduces cytoplasmic diffusion. Substitution with BSA reestablishes the diffusive flux, suggesting that specific binding to FABP is not required. These data support a role for intracellular binding proteins in facilitating the cytoplasmic movement of fatty acids.
Xanthine affects [Ca2+]i and contractile responses of ventricular cardiocytes to electrical stimulation
Authors: Rohn T. T. , Sauvadet A. , Pavoine C. , Pecker F.
Published: December 2017

Xanthine, a major purine by-product of ATP, accumulates during myocardial ischemia. In the present study, we show that xanthine (0.5-1 mM) impaired the occurrence of cytosolic Ca2+ concentration ([Ca2+]i) transients, visualized in fura 2-loaded cells, and twitches of contraction in ventricular cardiocytes in response to electrical stimulation. This effect of xanthine was independent of superoxide anion production. That it was a result of decreased membrane excitability was supported by the following: 1) it was reversed by increasing either the amplitude of the stimulus voltage required to stimulate cardiocytes or the extracellular concentration of NaCl; and 2) xanthine reversed the depolarization following electrical stimulation in cardiocytes loaded with the voltage-sensitive dye bis-oxonol. P2 purinergic-agonists, including ATP (10 microM), but not P1 purinergic agonists reproduced the effects seen with xanthine. In addition, a lack of additivity between xanthine and ATP at maximal concentrations was observed. We conclude that xanthine, through activation of a P2 purinoceptor, may contribute to myocardial arrhythmia occurring during ischemia-reperfusion injury.