bluuebunny/crossref_metadata_2025 huggingface dataset:
Noninvasive measurement of chloride concentration in rat olfactory receptor cells with use of a fluorescent dye
Authors: Kaneko Hiroshi , Nakamura Tadashi , Lindemann Bernd
Published: December 2017

Inwardly directed Ca2+-dependent chloride currents are thought to prolong and boost the odorant-induced transient receptor currents in olfactory cilia. Cl− inward current, of course, requires a sufficiently high intracellular Cl− concentration ([Cl−]i). In previous measurements using a fluorescent Cl− probe, N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), [Cl−]i of newt olfactory cells was estimated to be only 40 mM. This low value led us to reexamine the [Cl−]i by an improved procedure. When isolated rat olfactory neurons were bathed in Tyrode's solution (150 mM Cl−) at room temperature, the [Cl−] was 81.5 ± 13.5 mM (mean ± SE) in the tip of the dendrite (olfactory knob) and 81.8 ± 10.2 mM (mean ± SE) in the soma. The corresponding Cl− equilibrium potentials were −15.4 and −15.3 mV, respectively. Therefore, at resting potentials in the range of −90 to −50 mV, Cl− currents are predicted to be inward and capable of contributing to the depolarization induced by odorants. Yet, if the cell was depolarized beyond −15 mV, somal Cl− currents would be outward and facilitate repolarization during excitation. The measured [Cl−] in soma and knob are of interest, because in the cilia the chloride content may be expected to equilibrate with that of the knob in the resting state. They provide a starting point for the decrease in ciliary [Cl−] predicted to occur during transduction.
Protease-activated receptor-2-mediated inhibition of ion transport in human bronchial epithelial cells
Authors: Danahay Henry , Withey Louise , Poll Christopher T. , van de Graaf Stan F. J. , Bridges Robert J.
Published: December 2017

A cytoprotective role for protease-activated receptor-2 (PAR2) has been suggested in a number of systems including the airway, and to this end, we have studied the role that PARs play in the regulation of airway ion transport, using cultures of normal human bronchial epithelial cells. PAR2 activators, added to the basolateral membrane, caused a transient, Ca2+-dependent increase in short-circuit current ( I sc), followed by a sustained inhibition of amiloride-sensitive I sc. These phases corresponded with a transient increase in intracellular Ca2+ concentration and then a transient increase, followed by decrease, in basolateral K+ permeability. After PAR2 activation and the addition of amiloride, the forskolin-stimulated increase in I sc was also attenuated. By contrast, PAR2 activators added to the apical surface of the epithelia or PAR1 activators added to both the apical and basolateral surfaces were without effect. PAR2 may, therefore, play a role in the airway, regulating Na+ absorption and anion secretion, processes that are central to the control of airway surface liquid volume and composition.
Twisting integrin receptors increases endothelin-1 gene expression in endothelial cells
Authors: Chen Jianxin , Fabry Ben , Schiffrin Ernesto L. , Wang Ning
Published: December 2017

A magnetic twisting stimulator was developed based on the previously published technique of magnetic twisting cytometry. Using ligand-coated ferromagnetic microbeads, this device can apply mechanical stresses with varying amplitudes, duration, frequencies, and waveforms to specific cell surface receptors. Biochemical and biological responses of the cells to the mechanical stimulation can be assayed. Twisting integrin receptors with RGD (Arg-Gly-Asp)-containing peptide-coated beads increased endothelin-1 (ET-1) gene expression by >100%. In contrast, twisting scavenger receptors with acetylated low-density lipoprotein-coated beads or twisting HLA antigen with anti-HLA antibody-coated beads did not lead to alterations in ET-1 gene expression. In situ hybridization showed that the increase in ET-1 mRNA was localized in the cells that were stressed with the RGD-coated beads. Blocking stretch-activated ion channels with gadolinium, chelating Ca2+ with EGTA, or inhibiting tyrosine phosphorylation with genistein abolished twist-induced ET-1 mRNA elevation. Abolishing cytoskeletal tension with an inhibitor of the myosin ATPase, with an inhibitor of myosin light chain kinase, or with an actin microfilament disrupter blocked twisted-induced increases in ET-1 expression. Our results are consistent with the hypothesis that the molecular structural linkage of integrin-cytoskeleton is an important pathway for stress-induced ET-1 gene expression.
Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells
Authors: Ekhterae Daryoush , Platoshyn Oleksandr , Krick Stefanie , Yu Ying , McDaniel Sharon S. , Yuan Jason X.-J.
Published: December 2017

Cell shrinkage is an incipient hallmark of apoptosis in a variety of cell types. The apoptotic volume decrease has been demonstrated to attribute, in part, to K+ efflux; blockade of plasmalemmal K+ channels inhibits the apoptotic volume decrease and attenuates apoptosis. Using combined approaches of gene transfection, single-cell PCR, patch clamp, and fluorescence microscopy, we examined whether overexpression of Bcl-2, an anti-apoptotic oncoprotein, inhibits apoptosis in pulmonary artery smooth muscle cells (PASMC) by diminishing the activity of voltage-gated K+ (Kv) channels. A human bcl-2gene was infected into primary cultured rat PASMC using an adenoviral vector. Overexpression of Bcl-2 significantly decreased the amplitude and current density of Kv currents ( I Kv). In contrast, the apoptosis inducer staurosporine (ST) enhanced I Kv. In bcl-2-infected cells, however, the ST-induced increase in I Kv was completely abolished, and the ST-induced apoptosis was significantly inhibited compared with cells infected with an empty adenovirus (− bcl-2). Blockade of Kv channels in control cells (− bcl-2) by 4-aminopyridine also inhibited the ST-induced increase in I Kv and apoptosis. Furthermore, overexpression of Bcl-2 accelerated the inactivation of I Kv and downregulated the mRNA expression of the pore-forming Kv channel α-subunits (Kv1.1, Kv1.5, and Kv2.1). These results suggest that inhibition of Kv channel activity may serve as an additional mechanism involved in the Bcl-2-mediated anti-apoptotic effect on vascular smooth muscle cells.
Dynamics of nuclear localization sites for COX-2 in vascular endothelial cells
Authors: Parfenova Helena , Parfenov Vladimir N. , Shlopov Boris V. , Levine Vladimir , Falkos Sheryl , Pourcyrous Massroor , Leffler Charles W.
Published: December 2017

We investigated the relationships among expression, activity, and spatial organization of cyclooxygenase (COX-1 and COX-2) in endothelial cells from porcine and human cerebral microvessels and from human umbilical vein. In quiescent cells, COX-1 was detected in the perinuclear zone and the cytoplasm, while COX-2 was mainly a nuclear resident possibly connected with the nuclear matrix. COX-2 immunogold labeling was situated in the nuclear envelope, at the nuclear pores, and in connection with the perichromatin regions of the nucleus, considered to be the sites of active transcription. In human endothelial cells transcriptionally activated by interleukin (IL)-1β, the nucleus remained a major COX-2 localization site during the first 12 h of stimulation, when COX-2 expression was maximally induced. The continuous rise in prostanoid synthesis at 17–23 h of stimulation was associated with COX-2 relocation from the nucleus to the nuclear envelope and the cytoplasm. IL-1β did not affect COX-1 expression, activity, and localization. COX-2 nuclear localization sites and trafficking between the nucleus and the cytoplasm in endothelial cells may indicate a novel function of COX-2 in regulating gene expression.
Incorporation of caged cysteine and caged tyrosine into a transmembrane segment of the nicotinic ACh receptor
Authors: Philipson Kenneth D. , Gallivan Justin P. , Brandt Gabriel S. , Dougherty Dennis A. , Lester Henry A.
Published: December 2017

The nonsense codon suppression technique was used to incorporate o-nitrobenzyl cysteine or o-nitrobenzyl tyrosine (caged Cys or Tyr) into the 9′ position of the M2 transmembrane segment of the γ-subunit of the muscle nicotinic ACh receptor expressed in Xenopusoocytes. The caged amino acids replaced an endogenous Leu residue that has been implicated in channel gating. ACh-induced current increased substantially after ultraviolet (UV) irradiation to remove the caging group. This represents the first successful incorporation of caged Cys into a protein in vivo and the first incorporation of caged amino acids within a transmembrane segment of a membrane protein. The bulky nitrobenzyl group does not prevent the synthesis, assembly, or trafficking of the ACh receptor. When side chains were decaged using 1-ms UV light flashes, the channels with caged Cys or caged Tyr responded with strikingly different kinetics. The increase in current upon photolysis of caged Cys was too rapid for resolution by the voltage-clamp circuit [time constant (τ) <10 ms], whereas the increase in current upon photolysis of caged Tyr was dominated by a phase with τ ∼500 ms. Apparently, the presence of a bulky o-nitrobenzyl Tyr residue distorts the receptor into an abnormal conformation. Upon release of the caging group, the receptor relaxes, with τ ∼500 ms, into a conformation that allows the channel to open. Tyr at the 9′ position of the γ-subunit greatly increases the ability of ACh to block the channel by binding within the channel pore. This is manifested in two ways. 1) A “rebound,” or increase in current, occurs upon removal of ACh from the bathing medium; and 2) at ACh concentrations >400 μM, inward currents are decreased through the mutated channel. The ability to incorporate caged amino acids into proteins should have widespread utility.
Modeling of transcellular Ca transport in rat duodenum points to coexistence of two mechanisms of apical entry
Authors: Slepchenko Boris M. , Bronner Felix
Published: December 2017

Employing realistic parameters, we have demonstrated that a relatively simple mathematical model can reproduce key features of steady-state Ca2+ transport with the assumption of two mechanisms of Ca2+ entry: a channel-like flux and a carrier-mediated transport. At low luminal [Ca2+] (1–5 mM), facilitated entry dominates and saturates with K m = 0.4 mM. At luminal [Ca2+] of tens of millimolar, apical permeability is dominated by the channel flux that in turn is regulated by cytosolic Ca2+. The model reproduces the linear relationship between maximum Ca2+ transport rate and intestinal calbindin D9K (CaBP) content. At luminal [Ca2+] > 50 mM, local sensitivity analysis shows transcellular transport to be most sensitive to variations in CaBP. At low luminal [Ca2+], transport becomes sensitive to apical entry regulation. The simulations have been run within the Virtual Cell modeling environment, yielding the time course of external Ca2+ and spatiotemporal distributions of both intracellular Ca2+ and CaBP. Coexistence of two apical entry mechanisms accords with the properties of the duodenal Ca2+ transport protein CaT1 and the epithelial Ca2+ channel ECaC.
Polyamine transport system mediates agmatine transport in mammalian cells
Authors: Satriano Joseph , Isome Masato , Casero Robert A. , Thomson Scott C. , Blantz Roland C.
Published: December 2017

Agmatine is a biogenic amine with the capacity to regulate a number of nonreceptor-mediated functions in mammalian cells, including intracellular polyamine content and nitric oxide generation. We observed avid incorporation of agmatine into several mammalian cell lines and herein characterize agmatine transport in mammalian cells. In transformed NIH/3T3 cells, agmatine uptake is energy dependent with a saturable component indicative of carrier-mediated transport. Transport displays an apparent Michaelis-Menten constant of 2.5 μM and a maximal velocity of 280 pmol · min−1 · mg−1 protein and requires a membrane potential across the plasma membrane for uptake. Competition with polyamines, but not cationic molecules that utilize the y+ system transporter, suppresses agmatine uptake. Altering polyamine transporter activity results in parallel changes in polyamine and agmatine uptake. Furthermore, agmatine uptake is abrogated in a polyamine transport-deficient human carcinoma cell line. These lines of evidence demonstrate that agmatine utilizes, and is dependent on, the polyamine transporter for cellular uptake. The fact that this transport system is associated with proliferation could be of consequence to the antiproliferative effects of agmatine.
Intercellular communication in cultured human vascular smooth muscle cells
Authors: Wang Hong-Zhan , Day Nancy , Valcic Mira , Hsieh Ken , Serels Scott , Brink Peter R. , Christ George J.
Published: December 2017

Intercellular communication through gap junction channels plays a fundamental role in regulating vascular myocyte tone. We investigated gap junction channel expression and activity in myocytes from the physiologically distinct vasculature of the human internal mammary artery (IMA, conduit vessel) and saphenous vein (SV, capacitance vessel). Northern and Western blots documented the presence of connexin43 (Cx43) in frozen tissues and cultured cells from both vessels. Northern blots also confirmed the presence of Cx40 mRNA in cultured IMA and SV myocytes. Dual whole cell patch-clamp experiments revealed that macroscopic junctional conductance was voltage dependent and characteristic of that observed for Cx43. In the majority of records, in both vessels, single-channel activity was dominated by a main-state conductance of 120 pS, with subconducting events comprising less than 10% of the amplitude histograms. However, some records showed “atypical” unitary events that had a conductance similar to Cx40 (∼140–160 pS), but gating behavior like that of Cx43. As such, it is conceivable that the presence and coexpression of Cx40 and Cx43 in IMA and SV myocytes may result in heteromeric channel formation. Nonetheless, in terms of gating, Cx43-like behavior clearly dominates.
Phosphatidylinositol 3-kinase-dependent stabilization of α1(I) collagen mRNA in human lung fibroblasts
Authors: Ricupero Dennis A. , Poliks Christine F. , Rishikof David C. , Cuttle Kelly A. , Kuang Ping-Ping , Goldstein Ronald H.
Published: December 2017

We investigated the role of phosphatidylinositol 3-kinase (PI3K) in the expression of α1(I) collagen mRNA. We report that the basal level of α1(I) collagen mRNA was reduced when PI3K activity was inhibited by either LY-294002 or wortmannin. These PI3K inhibitors also blocked increases of α1(I) collagen mRNA levels after the addition of transforming growth factor-β. The effect of PI3K inhibition was abolished by the removal of the inhibitor or by the addition of cycloheximide. Inhibition of PI3K activity decreased the stability of the α1(I) collagen mRNA with no change in the rate of transcription of the α1(I) collagen gene as assessed by Northern blotting with actinomycin D-treated fibroblasts and nuclear run-on assays. Expression of a truncated α1(I) collagen minigene driven by a cytomegalovirus promoter in murine fibroblasts was decreased by LY-294002 treatment. These data indicate that PI3K activation results in increased stabilization of α1(I) collagen mRNA. In vivo, the PI3K activity in fibroblasts may regulate basal levels of α1(I) collagen mRNA expression.
Functionally distinct pools of actin in secretory cells
Authors: Ammar David A. , Nguyen Phuong N. B. , Forte John G.
Published: December 2017

Acid secretion by the gastric parietal cell is controlled through movement of vesicles containing the proton pump, the H+-K+-ATPase (HK). We have used latrunculin B (Lat B), which binds to monomeric actin, to investigate actin turnover in the stimulated parietal cell. In isolated gastric glands, relatively high concentrations of Lat B were required to inhibit acid accumulation (ED50 ∼70 μM). Cultured parietal cells stimulated in the presence of low Lat B (0.1–1 μM) have reduced lamellipodia formation and some aberrant punctate phalloidin-stained structures, but translocation of HK and vacuolar swelling appeared unaffected. High Lat B (10–50 μM) resulted in gross changes in actin organization (punctate phalloidin-stained structures throughout the cell and nucleus) and reduced translocation of HK and vacuolar swelling. Resting parietal cells treated with high Lat B showed minor effects on morphology and F-actin staining. If resting cells treated with high Lat B were washed immediately before stimulation, they exhibited a normal stimulated morphology. These data suggest distinct pools of parietal cell actin: a pool highly susceptible to Lat B primarily involved in motile function of cultured cells; and a Lat B-resistant pool, most likely microvillar filaments, that is essential for secretion. Furthermore, the stimulation process appears to accentuate the effects of Lat B, most likely through Lat B binding to monomer actin liberated by the turnover of the motile actin filament pool.
Focal adhesion kinase signaling is decreased in polyamine-depleted IEC-6 cells
Authors: Ray Ramesh M. , Viar Mary Jane , McCormack Shirley A. , Johnson Leonard R.
Published: December 2017

Polyamines are essential to the migration of epithelial cells in the intestinal mucosa. Cells depleted of polyamines do not attach as rapidly to the extracellular matrix and do not form the actin stress fibers essential for migration. Because both attachment and stress fiber formation depend on integrin signaling and the formation of focal adhesions, we examined these and related processes in polyamine-depleted IEC-6 cells. There was general decreased tyrosine phosphorylation of focal adhesion kinase (FAK), and, specifically, decreased phosphorylation of Tyr-925, the paxillin binding site. In control cells, FAK phosphorylation was rapid after attachment to the extracellular matrix, while attached cells depleted of polyamines had significantly delayed phosphorylation. FAK activity was also significantly inhibited in polyamine-depleted cells as was the phosphorylation of paxillin. Polyamine-depleted cells failed to spread normally after attachment, and immunocytochemistry showed little colocalization of FAK and actin compared with controls. Focal adhesion complex formation was greatly reduced in the absence of polyamines. These data suggest that defective integrin signaling may, at least in part, account for the decreased rates of attachment, actin stress fiber formation, spreading, and migration observed in polyamine-depleted cells.
Epidermal growth factor regulation of rat NHE2 gene expression
Authors: Xu Hua , Collins James F. , Bai Liqun , Kiela Pawel R. , Lynch Ronald M. , Ghishan Fayez K.
Published: December 2017

Epidermal growth factor (EGF) is involved in acute regulation of Na+/H+ exchangers (NHEs), but the effect of chronic EGF administration on NHE gene expression is unknown. The present studies showed that EGF treatment increased NHE2-mediated intestinal brush-border membrane vesicle Na+ absorption and NHE2 mRNA abundance by nearly twofold in 19-day-old rats. However, no changes were observed in renal NHE2 mRNA or intestinal and renal NHE3 mRNA abundance. To understand the mechanism of this regulation, we developed the rat intestinal epithelial (RIE) cell as an in vitro model to study the effect of EGF on NHE2 gene expression. EGF increased functional NHE2 activity and mRNA abundance in cultured RIE cells, and this stimulation could be blocked by actinomycin D (a transcriptional inhibitor). Additionally, NHE2 promoter reporter gene assays in transiently transfected RIE cells showed an almost twofold increase in promoter activity after EGF treatment. We conclude that rat NHE2 activity can be stimulated by chronic EGF treatment and that this response is at least partially mediated by gene transcription.
Negative regulation of ligand-initiated Ca2+uptake by PKC-βII in differentiated HL60 cells
Authors: Korchak Helen M. , Corkey Barbara E. , Yaney Gordon C. , Kilpatrick Laurie E.
Published: December 2017

In phagocytic cells, fMet-Leu-Phe triggers phosphoinositide remodeling, activation of protein kinase C (PKC), release of intracellular Ca2+ and uptake of extracellular Ca2+. Uptake of extracellular Ca2+ can be triggered by store-operated Ca2+channels (SOCC) and via a receptor-operated nonselective cation channel(s). In neutrophilic HL60 cells, the PKC activator phorbol myristate acetate (PMA) activates multiple PKC isotypes, PKC-α, PKC-β, and PKC-δ, and inhibits ligand-initiated mobilization of intracellular Ca2+ and uptake of extracellular Ca2+. Therefore PKC is a negative regulator at several points in Ca2+ mobilization. In contrast, selective depletion of PKC-β in HL60 cells by an antisense strategy enhanced fMet-Leu-Phe-initiated Ca2+ uptake but not mobilization of intracellular Ca2+. Thapsigargin-induced Ca2+ uptake through SOCC was not affected by PKC-βII depletion. Thus PKC-βII is a selective negative regulator of Ca2+ uptake but not release of intracellular Ca2+ stores. PKC-βII inhibits a receptor-operated cation or Ca2+ channel, thus inhibiting ligand-initiated Ca2+ uptake.
A role for MAP kinase in regulating ectodomain shedding of APLP2 in corneal epithelial cells
Authors: Xu Ke-Ping , Zoukhri Driss , Zieske James D. , Dartt Darlene A. , Sergheraert Christian , Loing Estelle , Yu Fu-Shin X.
Published: December 2017

We previously reported an increased secretion of amyloid precursor-like protein 2 (APLP2) in the healing corneal epithelium. The present study sought to investigate signal transduction pathways involved in APLP2 shedding in vitro. APLP2 was constitutively shed and released into culture medium in SV40-immortalized human corneal epithelial cells as assessed by Western blotting, flow cytometry, and indirect immunofluorescence. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) caused significant increases in APLP2 shedding. This was inhibited by staurosporine and a PKC-ε-specific, N-myristoylated peptide inhibitor. Epidermal growth factor (EGF) also induced APLP2 accumulation in culture medium. Basal APLP2 shedding as well as that induced by PMA and EGF was blocked by a mitogen-activated protein kinase (MAPK) kinase inhibitor, U-0126. Our results suggest that MAPK activity accounts for basal as well as PKC- and EGF-induced APLP2 shedding. In addition, PKC-ε may be involved in the induction of APLP2 shedding in corneal epithelial cells.
Alterations in airway ion transport in NKCC1-deficient mice
Authors: Grubb B. R. , Pace A. J. , Lee E. , Koller B. H. , Boucher R. C.
Published: December 2017

Airways of Na+-K+-2Cl−(NKCC1)-deficient mice (−/−) were studied in Ussing chambers to determine the role of the basolateral NKCC1 in transepithelial anion secretion. The basal short-circuit current ( I sc) of tracheae and bronchi from adult mice did not differ between NKCC1−/− and normal mice, whereas NKCC1−/− tracheae from neonatal mice exhibited a significantly reduced basal I sc. In normal mouse tracheae, sensitivity to the NKCC1 inhibitor bumetanide correlated inversely with the age of the mouse. In contrast, tracheae from NKCC1−/− mice at all ages were insensitive to bumetanide. The anion secretory response to forskolin did not differ between normal and NKCC1−/− tissues. However, when larger anion secretory responses were induced with UTP, airways from the NKCC1−/− mice exhibited an attenuated response. Ion substitution and drug treatment protocols suggested that HCO[Formula: see text]secretion compensated for reduced Cl− secretion in NKCC1−/− airway epithelia. The absence of spontaneous airway disease or pathology in airways from the NKCC1−/− mice suggests that the NKCC1 mutant mice are able to compensate adequately for absence of the NKCC1 protein.
Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms
Authors: Song Jaekyung Cecilia , Hanson Celina M. , Tsai Vance , Farokhzad Omid C. , Lotz Margaret , Matthews Jeffrey B.
Published: December 2017

The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibits Cl− secretion (short-circuit current, I sc) and decreases barrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes in this response, we compared PMA with two non-phorbol activators of PKC (bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozyme selectivity profiles. PMA sequentially inhibited cAMP-stimulated I sc and decreased TER, as measured by voltage-current clamp. By subcellular fractionation and Western blot, PMA (100 nM) induced sequential membrane translocation of the novel PKCε followed by the conventional PKCα and activated both isozymes by in vitro kinase assay. PKCδ was activated by PMA but did not translocate. By immunofluorescence, PKCε redistributed to the basolateral domain in response to PMA, whereas PKCα moved apically. Inhibition of I sc by PMA was prevented by the conventional and novel PKC inhibitor Gö-6850 (5 μM) but not the conventional isoform inhibitor Gö-6976 (5 μM) or the PKCδ inhibitor rottlerin (10 μM), implicating PKCε in inhibition of Cl− secretion. In contrast, both Gö-6976 and Gö-6850 prevented the decline of TER, suggesting involvement of PKCα. Bryostatin-1 (100 nM) translocated PKCε and PKCα and inhibited cAMP-elicited I sc. However, unlike PMA, bryostatin-1 downregulated PKCα protein, and the decrease in TER was only transient. Carbachol (100 μM) translocated only PKCε and inhibited I sc with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1 and carbachol inhibition of I sc. We conclude that basolateral translocation of PKCε inhibits Cl−secretion, while apical translocation of PKCα decreases TER. These data suggest that epithelial transport and barrier function can be modulated by distinct PKC isoforms.
PI 3-kinases and Src kinases regulate spreading and migration of cultured VSMCs
Authors: Yamboliev Ilia A. , Chen Jennifer , Gerthoffer William T.
Published: December 2017

Pulmonary artery smooth muscle cell (PASMC) adhesion, spreading, and migration depend on matrix-stimulated reorganization of focal adhesions. Platelet-derived growth factor (PDGF) activates intracellular signal transduction cascades that also regulate adhesion, spreading, and migration, but the signaling molecules involved in these events are poorly defined. We hypothesized that phosphatidylinositol (PI) 3-kinases and Src tyrosine kinases translate matrix and PDGF-initiated signals into cell motility. In experiments with cultured canine PASMCs, inhibition of PI 3-kinases with wortmannin (0.3 μM) and LY-294002 (50 μM) and of Src kinase with PP1 (30 μM) did not decrease spontaneous (nonstimulated) or PDGF-stimulated (10 ng/ml) adhesion onto collagen. PI 3-kinase and Src kinase activities, however, were necessary for cell spreading: PP1 inhibited cell spreading and Src Tyr-418 phosphorylation in a concentration-dependent manner. Inhibition of PI 3-kinase and Src partially reduced cell migration, while at 10 and 30 μM, PP1 eliminated migration, likely due to inhibition of PDGF receptors. In conclusion, both PI 3-kinases and Src tyrosine kinases are components of pathways that mediate spreading and migration of cultured PASMCs on collagen.
Regulation of phospholipase C-γ1 by the actin-regulatory protein villin
Authors: Panebra A. , Ma S.-X. , Zhai L.-W. , Wang X.-T. , Rhee S. G. , Khurana S.
Published: December 2017

The actin-regulatory protein villin is tyrosine phosphorylated and associates with phospholipase C-γ1(PLC-γ1) in the brush border of intestinal epithelial cells. To study the mechanism of villin-associated PLC-γ1 activation, we reconstituted in vitro the tyrosine phosphorylation of villin and its association with PLC-γ1. Recombinant villin was phosphorylated in vitro by the nonreceptor tyrosine kinase c-src or by expression in the TKX1 competent cells that carry an inducible tyrosine kinase gene. Using in vitro binding assays, we demonstrated that tyrosine-phosphorylated villin associates with the COOH-terminal Src homology 2 (SH2) domain of PLC-γ1. The catalytic activity of PLC-γ1was inhibited by villin in a dose-dependent manner with half-maximal inhibition at a concentration of 12.4 μM. Villin inhibited PLC-γ1 activity by sequestering the substrate phosphatidylinositol 4,5-bisphosphate (PIP2), since increasing concentrations of PIP2 reversed the inhibitory effects of villin on PLC activity. The inhibition of PLC-γ1 activity by villin was reversed by the tyrosine phosphorylation of villin. Further, we demonstrated that tyrosine phosphorylation of villin abolished villin's ability to associate with PIP2. In conclusion, tyrosine-phosphorylated villin associates with the COOH-terminal SH2 domain of PLC-γ1and activates PLC-γ1 catalytic activity. Villin regulates PLC-γ1 activity by modifying its own ability to bind PIP2. This study provides biochemical proof of the functional relevance of tyrosine phosphorylation of villin and identifies the molecular mechanisms involved in the activation of PLC-γ1 by villin.
S-adenosyl-l-homocysteine hydrolase is necessary for aldosterone-induced activity of epithelial Na+ channels
Authors: Stockand James D. , Zeltwanger Shawn , Bao Hui-Fang , Becchetti Andrea , Worrell Roger T. , Eaton Douglas C.
Published: December 2017

The A6 cell line was used to study the role of S-adenosyl-l-homocysteine hydrolase (SAHHase) in the aldosterone-induced activation of the epithelial Na+channel (ENaC). Because aldosterone increases methylation of several different molecules, and because this methylation is associated with increased Na+ reabsorption, we tested the hypothesis that aldosterone increases the expression and activity of SAHHase protein. The rationale for this work is that general methylation may be promoted by activation of SAHHase, the only enzyme known to metabolize SAH, a potent end-product inhibitor of methylation. Although aldosterone increased SAHHase activity, steroid did not affect SAHHase expression. Antisense SAHHase oligonucleotide decreased SAHHase expression and activity. Moreover, this oligonucleotide, as well as a pharmacological inhibitor of SAHHase, decreased aldosterone-induced activity of ENaC via a decrease in ENaC open probability. The kinetics of ENaC in cells treated with antisense plus aldosterone were similar to those reported previously for the channel in the absence of steroid. This is the first report showing that active SAHHase, in part, increases ENaC open probability by reducing the transition rate from open states in response to aldosterone. Thus aldosterone-induced SAHHase activity plays a critical role in shifting ENaC from a gating mode with short open and closed times to one with longer open and closed times.
Direct estimate of 1:1 stoichiometry of K+-Cl− cotransport in rabbit erythrocytes
Authors: Jennings Michael L. , Adame Mark F.
Published: December 2017

This work was undertaken to obtain a direct measure of the stoichiometry of Na+-independent K+-Cl− cotransport (KCC), with rabbit red blood cells as a model system. To determine whether 86Rb+ can be used quantitatively as a tracer for KCC, 86Rb+ and K+effluxes were measured in parallel after activation of KCC with N-ethylmaleimide (NEM). The rate constant for NEM-stimulated K+ efflux into isosmotic NaCl was smaller than that for86Rb+ by a factor of 0.68 ± 0.11 (SD, n = 5). This correction factor was used in all other experiments to calculate the K+ efflux from the measured86Rb+ efflux. To minimize interference from the anion exchanger, extracellular Cl− was replaced with SO[Formula: see text], and 4,4′-diisothiocyanothiocyanatodihydrostilbene-2,2′-disulfonic acid was present in the flux media. The membrane potential was clamped near 0 mV with the protonophore 2,4-dinitrophenol. The Cl−efflux at 25°C under these conditions is ∼100,000-fold smaller than the uninhibited Cl−/Cl− exchange flux and is stimulated ∼2-fold by NEM. The NEM-stimulated36Cl− flux is inhibited by okadaic acid and calyculin A, as expected for KCC. The ratio of the NEM-stimulated K+ to Cl− efflux is 1.12 ± 0.26 (SD, n = 5). We conclude that K+-Cl− cotransport in rabbit red blood cells has a stoichiometry of 1:1.
Hypertonicity rescues T cells from suppression by trauma-induced anti-inflammatory mediators
Authors: Loomis William H. , Namiki Sachiko , Hoyt David B. , Junger Wolfgang G.
Published: December 2017

Trauma causes the release of anti-inflammatory factors thought to cause infections by inhibiting T cells. We have found that hypertonic saline (HS) enhances functions of normal T cells. Here we studied if HS can rescue T cells from suppression by costimulating interleukin (IL)-2 production. Human peripheral blood mononuclear cells were treated with the immunosuppressive factors IL-4, IL-10, transforming growth factor (TGF)-β1, and PGE2 and with serum of trauma patients and stimulated with phytohemagglutinin, and IL-2 production was measured. Costimulation with HS tripled IL-2 production of normal cells. IL-4, IL-10, TGF-β1, and PGE2suppressed IL-2 production with IC50 of 500, 1, 36,000, and 0.01 pg/ml, respectively. Costimulation of suppressed cells with HS restored IL-2 production and increased IC50 values >70-fold. Serum from trauma patients could completely suppress normal cells; however, costimulation with HS restored IL-2 production by up to 80% of the control response. These findings show that HS can restore the function of suppressed T cells, suggesting that HS resuscitation of trauma patients could reduce posttraumatic sepsis.
Regulation of ATP-sensitive K+ channels by protein kinase C in murine colonic myocytes
Authors: Jun Jae Yeoul , Kong In Deok , Koh Sang Don , Wang Xuan Yu , Perrino Brian A. , Ward Sean M. , Sanders Kenton M.
Published: December 2017

We investigated the regulation of ATP-sensitive K+ (KATP) currents in murine colonic myocytes with patch-clamp techniques. Pinacidil (10−5 M) activated inward currents in the presence of high external K+ (90 mM) at a holding potential of −80 mV in dialyzed cells. Glibenclamide (10−5 M) suppressed pinacidil-activated current. Phorbol 12,13-dibutyrate (PDBu; 2 × 10−7 M) inhibited pinacidil-activated current. 4-α-Phorbol ester (5 × 10−7 M), an inactive form of PDBu, had no effect on pinacidil-activated current. In cell-attached patches, the open probability of KATP channels was increased by pinacidil, and PDBu suppressed openings of KATP channels. When cells were pretreated with chelerythrine (10−6 M) or calphostin C (10−7M), inhibition of the pinacidil-activated whole cell currents by PDBu was significantly reduced. In cells studied with the perforated patch technique, PDBu also inhibited pinacidil-activated current, and this inhibition was reduced by chelerythrine (10−6 M). Acetylcholine (ACh; 10−5 M) inhibited pinacidil-activated currents, and preincubation of cells with calphostin C (10−7 M) decreased the effect of ACh. Cells dialyzed with protein kinase C ɛ-isoform (PKCɛ) antibody had normal responses to pinacidil, but the effects of PDBu and ACh on KATP were blocked in these cells. Immunofluorescence and Western blots showed expression of PKCɛ in intact muscles and isolated smooth muscle cells of the murine proximal colon. These data suggest that PKC regulates KATP in colonic muscle cells and that the effects of ACh on KATP are largely mediated by PKC. PKCɛ appears to be the major isozyme that regulates KATP in murine colonic myocytes.
Function and structure of heterodimeric amino acid transporters
Authors: Wagner Carsten A. , Lang Florian , Bröer Stefan
Published: December 2017

Heterodimeric amino acid transporters are comprised of two subunits, a polytopic membrane protein (light chain) and an associated type II membrane protein (heavy chain). The heavy chain rbAT (related to b0,+ amino acid transporter) associates with the light chain b0,+AT (b0,+ amino acid transporter) to form the amino acid transport system b0,+, whereas the homologous heavy chain 4F2hc interacts with several light chains to form system L (with LAT1 and LAT2), system y+L (with y+LAT1 and y+LAT2), system x[Formula: see text](with xAT), or system asc (with asc1). The association of light chains with the two heavy chains is not unambiguous. rbAT may interact with LAT2 and y+LAT1 and vice versa; 4F2hc may interact with b0,+AT when overexpressed. 4F2hc is necessary for trafficking of the light chain to the plasma membrane, whereas the light chains are thought to determine the transport characteristics of the respective heterodimer. In contrast to 4F2hc, mutations in rbAT suggest that rbAT itself takes part in the transport besides serving for the trafficking of the light chain to the cell surface. Heavy and light subunits are linked together by a disulfide bridge. The disulfide bridge, however, is not necessary for the trafficking of rbAT or 4F2 heterodimers to the membrane or for the functioning of the transporter. However, there is experimental evidence that the disulfide bridge in the 4F2hc/LAT1 heterodimer plays a role in the regulation of a cation channel. These results highlight complex interactions between the different subunits of heterodimeric amino acid transporters and suggest that despite high grades of homology, the interactions between rbAT and 4F2hc and their respective partners may be different.
Fluid transport by human nonpigmented ciliary epithelial layers in culture: a homeostatic role for aquaporin-1
Authors: Patil Rajkumar V. , Han Zhiqiang , Yiming Maimaiti , Yang Junjie , Iserovich Pavel , Wax Martin B. , Fischbarg Jorge
Published: December 2017

We report for the first time that cultured nonpigmented human ciliary epithelial (NPE) cell layers transport fluid. Cells were grown to confluence on permeable membrane inserts, and fluid transport across the resulting cell layers was determined by volume clamp at 37°C. These cell layers translocated fluid from the apical to the basal side at a steady rate of 3.6 μl · h−1 · cm−2( n = 4) for 8 h. This fluid movement was independent of hydrostatic pressure and was completely inhibited by 1 mM ouabain, suggesting it arose from fluid transport. Mercuric chloride, a nonspecific but potent blocker of Hg2+-sensitive aquaporins, and aquaporin-1 antisense oligonucleotides both partially inhibited fluid transport across the cell layers, which suggests that water channels have a role in NPE cell homeostasis. In addition, these results suggest that of the two ciliary epithelial layers in tandem, the NPE layer by itself can transport fluid. This cultured layer, therefore, constitutes an interesting model that may be useful for physiological and pharmacological characterization of ciliary epithelial fluid secretion.
Participation of cAMP in a signal-transduction pathway relating erythrocyte deformation to ATP release
Authors: Sprague Randy S. , Ellsworth Mary L. , Stephenson Alan H. , Lonigro Andrew J.
Published: December 2017

Previously, we reported that red blood cells (RBCs) of rabbits and humans release ATP in response to mechanical deformation and that this release of ATP requires the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). It was reported that cAMP, acting through a cAMP-dependent protein kinase, PKA, is an activator of CFTR. Here we investigate the hypothesis that cAMP stimulates ATP release from RBCs. Incubation of human and rabbit RBCs with the direct activator of adenylyl cyclase, forskolin (10 or 100 μM), with IBMX (100 μM), resulted in ATP release and increases in intracellular cAMP. In addition, epinephrine (1 μM), a receptor-mediated activator of adenylyl cyclase, stimulated ATP release from rabbit RBCs. Moreover, incubation of human and rabbit RBCs with an active cAMP analog [adenosine 3′5′-cyclic monophosphorothioate Sp-isomer (Sp-cAMP, 100 μM)] resulted in ATP release. In contrast, forskolin and Sp-cAMP were without effect on dog RBCs, cells known not to release ATP in response to deformation. When rabbit RBCs were incubated with the inactive cAMP analog and inhibitor of PKA activity, adenosine 3′,5′-cyclic monophosphorothioate Rp-isomer (100 μM), deformation-induced ATP release was attenuated. These results are consistent with the hypothesis that adenylyl cyclase and cAMP are components of a signal-transduction pathway relating RBC deformation to ATP release from human and rabbit RBCs.
Resistance of macrophages to the suppressive effect of interleukin-10 following thermal injury
Authors: Schwacha Martin G. , Schneider Christian P. , Bland Kirby I. , Chaudry Irshad H.
Published: December 2017

The activation of a macrophage (Mφ)-dependent proinflammatory cascade following thermal injury plays an important role in the development of immunosuppression and increased susceptibility to subsequent sepsis in burn patients. In contrast, although interleukin (IL)-10, an anti-inflammatory cytokine that can downregulate Mφ activity, has also been implicated in postburn immune dysfunction, its role in the regulation of Mφ function postburn remains unclear. To study this, C57BL/6 female mice were subjected to a 25% total body surface area third-degree scald burn, and splenic Mφs were isolated 7 days later. Lipopolysaccharide (LPS)-stimulated IL-10, IL-6, tumor necrosis factor (TNF)-α, and nitric oxide (NO) production were significantly increased in the burn group compared with shams. Blockade of endogenous IL-10 activity enhanced IL-6 and TNF-α release, but not NO release, in both groups. The addition of exogenous IL-10 to the Mφ cultures dose dependently suppressed production of these inflammatory mediators in both groups. The timing of IL-10 addition to the cultures in relation to LPS stimulation, however, was critical. The suppressive effect of exogenous IL-10 was attenuated in both groups when the cells were exposed to IL-10 at 4–6 h after LPS stimulation; however, Mφs from injured mice were significantly better able to maintain inflammatory mediator-productive capacity. The resistance of Mφs from injured mice to IL-10-mediated suppression correlated with decreased IL-10 receptor (IL-10R) expression and increased CD11b expression. These findings suggest that Mφs, following thermal injury, display resistance to suppression by IL-10 due in part to downregulation of IL-10R expression.
Two distinct caveolin-1 domains mediate the functional interaction of caveolin-1 with protein kinase A
Authors: Razani Babak , Lisanti Michael P.
Published: December 2017

Numerous components of the cAMP-based signaling cascade, namely G-proteins and G- protein coupled receptors, adenylyl cyclase, and protein kinase A (PKA) have been localized to caveolae and shown to be regulated by the caveolar marker proteins, the caveolins. In order to gain mechanistic insights into these processes in vivo, we have assessed the functional interaction of caveolin-1 (Cav-1) with PKA using mutational analysis. As two regions of Cav-1 had previously been implicated in PKA signaling in vitro, we constructed Cav-1 molecules with mutations/deletions in one or both of these domains. Examination of these mutants shows that Cav-1 requires the presence of either the scaffolding domain or the COOH-terminal domain (but not both) to functionally interact with and inhibit PKA. Interestingly, in contrast to the wild-type protein, these Cav-1 mutants are not localized to caveolae microdomains. However, upon coexpression with wild-type Cav-1, a substantial amount of the mutants was recruited to the caveolae membrane fraction. Using the Cav-1 double mutant with both disrupted scaffolding and COOH-terminal domains, we show that wild-type Cav-1's inhibition of PKA signaling can be partially abrogated in a dose-responsive manner; i.e., the mutant acts in a dominant-negative fashion. Thus, this dominant-negative caveolin-1 mutant will be extremely valuable for assessing the functional role of endogenous caveolin-1 in regulating a variety of other signaling cascades.
Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues
Authors: Timmer Richard T. , Klein Janet D. , Bagnasco Serena M. , Doran John J. , Verlander Jill W. , Gunn Robert B. , Sands Jeff M.
Published: December 2017

A new polyclonal antibody to the human erythrocyte urea transporter UT-B detects a broad band between 45 and 65 kDa in human erythrocytes and between 37 and 51 kDa in rat erythrocytes. In human erythrocytes, the UT-B protein is the Kidd (Jk) antigen, and Jk(a+b+) erythrocytes express the 45- to 65-kDa band. However, in Jk null erythrocytes [Jk(a−b−)], only a faint band at 55 kDa is detected. In kidney medulla, a broad band between 41 and 54 kDa, as well as a larger band at 98 kDa, is detected. Human and rat kidney show UT-B staining in nonfenestrated endothelial cells in descending vasa recta. UT-B protein and mRNA are detected in rat brain, colon, heart, liver, lung, and testis. When kidney medulla or liver proteins are analyzed with the use of a native gel, only a single protein band is detected. UT-B protein is detected in cultured bovine endothelial cells. We conclude that UT-B protein is expressed in more rat tissues than previously reported, as well as in erythrocytes.
Role of PLCγ and Ca2+ in VEGF- and FGF-induced choroidal endothelial cell proliferation
Authors: McLaughlin A. P. , De Vries G. W.
Published: December 2017

Although both vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) receptors have been shown to be important in the regulation of vascular endothelial cell growth, the roles of phospholipase C (PLC)γ and Ca2+ in their downstream signaling cascades are still not clear. We have examined the effects of VEGF and FGF on PLCγ phosphorylation and on changes in intracellular Ca2+ levels in primary endothelial cells. VEGF stimulation leads to PLCγ activation and increases in intracellular Ca2+, which are correlated with mitogen-activated protein (MAP) kinase (MAPK) activation and cell growth. Inhibition of Ca2+ increases by the Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane- N,N,N′,N′-tetraacetic acid (BAPTA)-AM resulted in marked inhibition of MAPK activation, which was shown to be linked to regulation of cell growth in these cells. In contrast, FGF stimulation did not lead to PLCγ activation or to changes in intracellular Ca2+ levels, although MAPK phosphorylation and stimulation of cell proliferation were observed. Neither BAPTA-AM nor the PLC inhibitor U-73122 had an effect on these FGF-stimulated responses. These data demonstrate a direct role for PLCγ and Ca2+ in VEGF-regulated endothelial cell growth, whereas this signaling pathway is not linked to FGF-mediated effects in primary endothelial cells. Thus endothelial cell-specific factors regulate the ability of VEGF receptors and FGF receptors to couple to this signaling pathway.
Actin depolymerization via the β-adrenoceptor in airway smooth muscle cells: a novel PKA-independent pathway
Authors: Hirshman Carol A. , Zhu Defen , Panettieri Reynold A. , Emala Charles W.
Published: December 2017

Actin is a major functional and structural cytoskeletal protein that mediates such diverse processes as motility, cytokinesis, contraction, and control of cell shape and polarity. While many extracellular signals are known to mediate actin filament polymerization, considerably less is known about signals that mediate depolymerization of the actin cytoskeleton. Human airway smooth muscle cells were briefly exposed to isoproterenol, forskolin, or the cAMP-dependent protein kinase A (PKA) agonist stimulatory diastereoisomer of adenosine 3′,5′-cyclic monophosphate (Sp-cAMPS). Actin polymerization was measured by concomitant staining of filamentous actin with FITC-phalloidin and globular actin with Texas red DNase I. Isoproterenol, forskolin, or Sp-cAMPS induced actin depolymerization, indicated by a decrease in the intensity of filamentous/globular fluorescent staining. The PKA inhibitor Rp diastereomer of adenosine 3′,5′-cyclic monophosphothioate (Rp-cAMPS) completely inhibited forskolin-stimulated depolymerization, whereas it only partially inhibited isoproterenol-induced depolymerization. The protein tyrosine kinase inhibitors genistein or tyrphostin A23 also partially inhibited isoproterenol-induced actin depolymerization. In contrast, the combination of Rp-cAMPS and either tyrosine kinase inhibitor had an additive effect at inhibiting isoproterenol-induced actin depolymerization. These results suggest that both PKA-dependent and -independent pathways mediate actin depolymerization in human airway smooth muscle cells.