bluuebunny/crossref_metadata_2025 huggingface dataset:
Model Organisms: New Insights Into Ion Channel and Transporter Function. Stomatin homologues interact inCaenorhabditis elegans
Authors: Sedensky M. M. , Siefker J. M. , Morgan P. G.
Published: December 2017

In C. elegans the protein UNC-1 is a major determinant of anesthetic sensitivity and is a close homologue of the mammalian protein stomatin. In humans stomatin is missing from erythrocyte membranes in the hemolytic disease overhydrated hereditary stomatocytosis, despite an apparently normal stomatin gene. Overhydrated hereditary stomatocytosis is characterized by alteration of the normal transmembrane gradients of sodium and potassium. Stomatin has been shown to interact genetically with sodium channels. It is also postulated that stomatin is important in the organization of lipid rafts. We demonstrate here that antibodies against UNC-1 stain the major nerve tracts of Caenorhabditis elegans, with very intense staining of the nerve ring. We also found that a gene encoding a stomatin-like protein, UNC-24, affects anesthetic sensitivity and is genetically epistatic to unc-1. In the absence of UNC-24, the staining of the nerve ring by anti-UNC-1 is abolished, despite normal transcriptional levels of the unc-1 mRNA. Western blots indicate that UNC-24 probably affects the stability of the UNC-1 protein. UNC-24 may therefore be necessary for the correct placement of UNC-1 in the cell membrane and organization of lipid rafts.
Inhibitory actions of ceramide upon PKC-ε/ERK interactions
Authors: Bourbon Nicole A. , Yun Jong , Berkey Danielle , Wang Yizheng , Kester Mark
Published: December 2017

We have previously shown that interleukin-1 receptor-generated ceramide induces growth arrest in smooth muscle pericytes by inhibiting an upstream kinase in the extracellular signal-regulated kinase (ERK) cascade. Here, we now report the mechanism by which ceramide inhibits ERK activity. Ceramide renders the human embryonic kidney 293 cells (HEK 293) resistant to the mitogenic actions of growth factors and activators of protein kinase C (PKC). A role for PKC to mediate ceramide inhibition of growth factor-induced ERK activity and mitogenesis is suggested, as exogenous ceramide directly inhibits both immunoprecipitated and recombinant PKC-ε activities. To confirm that PKC-ε is necessary for ceramide-inhibited ERK activity, HEK 293 cells were transfected with a dominant-negative mutant of PKC-ε (ΔPKC-ε). These transfected cells respond to insulin-like growth factor I (IGF-I) with a significantly decreased ERK activity that is not further reduced by ceramide treatment. Coimmunoprecipitation studies reveal that the treatment with IGF-I induces the association of ERK with PKC-ε but not with PKC-ζ. Ceramide treatment significantly inhibits the IGF-I-induced PKC-ε interaction with bioactive phosphorylated ERK. Ceramide also inhibits IGF-I-induced PKC-ε association with Raf-1, an upstream kinase of ERK. Together, these studies demonstrate that ceramide exerts anti-mitogenic actions by limiting the ability of PKC-ε to form a signaling complex with Raf-1 and ERK.
Differential effects of apical and basolateral uridine triphosphate on intestinal epithelial chloride secretion
Authors: Smitham Jane E. , Barrett Kim E.
Published: December 2017

Our goal was to examine the sidedness of effects of the purinergic agonist, uridine 5′-triphosphate (UTP), on Cl− secretion in intestinal epithelial cells. We hypothesized that UTP might exert both stimulatory and inhibitory effects. All studies were conducted with T84 intestinal epithelial cells. UTP induced Cl− secretion in a concentration-dependent fashion. Responses to serosally added UTP were smaller and more transient than those evoked by mucosal addition, but there was no evidence that mucosal responses involved cAMP-dependent mechanisms. Pretreatment with serosal UTP inhibited subsequent Ca2+-dependent Cl− secretion induced by carbachol or thapsigargin, or secretion induced by mucosal UTP, in a manner that was reversed by a tyrosine kinase inhibitor. The inhibitory effect of serosal UTP on Cl− secretion was not additive with that of carbachol, known to exert its inhibitory effects through the tyrosine kinase-dependent generation of inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6) P 4]. Moreover, responses to both serosal and mucosal UTP were reduced by prior treatment of T84 cells with carbachol. Finally, serosal, but not mucosal, UTP evoked an increase in Ins(3,4,5,6) P 4. We conclude that different signaling mechanisms lie downstream of apical and basolateral UTP receptors in epithelial cells, at least in the intestine. These differences may be relevant to the use of UTP as a therapy in cystic fibrosis.
Collagen gel overlay induces two phases of apoptosis in MDCK cells
Authors: Wang Yang-Kao , Lin Hsi-Hui , Tang Ming-Jer
Published: December 2017

We previously demonstrated that collagen gel overlay induced cell remodeling to form lumen and apoptosis in Madin-Darby canine kidney cells. In the present study, we established that collagen gel overlay-induced apoptosis was initiated at areas exclusive of cell remodeling within 24 h (first phase) and extended into areas of cell remodeling within 48 h (second phase). Collagen gel overlay-induced apoptosis was accompanied by selective proteolysis of focal adhesion kinase (FAK), talin, p130cas, and c- src. Upon collagen gel overlay, FAK was initially degraded into a 90-kDa product during the first phase and subsequently into a 80-kDa product during the second phase. Collagen gel overlay-induced apoptosis of focal adhesion complex proteins and apoptosis of the first phase could be blocked only by a protease inhibitor cocktail. In addition, we found that both DEVD-fmk and ZVAD-fmk inhibited secondary proteolysis of FAK, but only ZVAD-fmk blocked collagen gel overlay-induced apoptosis of the second phase. Finally, collagen gel overlay-induced apoptosis and proteolysis of focal adhesion complex proteins were completely inhibited by the combination of protease inhibitor cocktail and ZVAD-fmk. Taken together, collagen gel overlay induces two phases of apoptosis; the first phase is dependent on proteolysis of focal adhesion complex proteins, and the second phase on activation of caspases.
Normal Ca2+ extrusion by the Ca2+ pump of intact red blood cells exposed to high glucose concentrations
Authors: Raftos Julia E. , Edgley Amanda , Bookchin Robert M. , Etzion Zipora , Lew Virgilio L. , Tiffert Teresa
Published: December 2017

The ATPase activity of the plasma membrane Ca2+ pump (PMCA) has been reported to be inhibited by exposure of red blood cell (RBC) PMCA preparations to high glucose concentrations. It has been claimed that this effect could have potential pathophysiological relevance in diabetes. To ascertain whether high glucose levels also affect PMCA transport function in intact RBCs, Ca2+extrusion by the Ca2+-saturated pump [PMCA maximal velocity ( V max)] was measured in human and rat RBCs exposed to high glucose in vivo or in vitro. Preincubation of normal human RBCs in 30–100 mM glucose for up to 6 h had no effect on PMCA V max. The mean V max of RBCs from 15 diabetic subjects of 12.9 ± 0.7 mmol · 340 g Hb−1 · h−1 was not significantly different from that of controls (14.3 ± 0.5 mmol · 340 g Hb−1 · h−1). Similarly, the PMCA V max of RBCs from 11 streptozotocin-diabetic rats was not affected by plasma glucose levels more than three times normal for 6–8 wk. Thus exposure to high glucose concentrations does not affect the ability of intact RBCs to extrude Ca2+.
Model of ionic transport for bovine ciliary epithelium: effects of acetazolamide and HCO 3 −
Authors: To Chi-Ho , Do Chi-Wai , Zamudio Aldo C. , Candia Oscar A.
Published: December 2017

The possible existence of transepithelial bicarbonate transport across the isolated bovine ciliary body was investigated by employing a chamber that allows for the measurement of unidirectional, radiolabeled fluxes of CO2 + HCO[Formula: see text]. No net flux of HCO[Formula: see text] was detected. However, acetazolamide (0.1 mM) reduced the simultaneously measured short-circuit current ( I sc). In other experiments in which36Cl− was used, a net Cl− flux of 1.12 μeq · h−1 · cm−2 (30 μA/cm2) in the blood-to-aqueous direction was detected. Acetazolamide, as well as removal of HCO[Formula: see text] from the aqueous bathing solution, inhibited the net Cl− flux and I sc. Because such removal should increase HCO[Formula: see text] diffusion toward the aqueous compartment and increase the I sc, this paradoxical effect could result from cell acidification and partial closure of Cl−channels. The acetazolamide effect on Cl− fluxes can be explained by a reduction of cellular H+ and HCO[Formula: see text] (generated from metabolic CO2production), which exchange with Na+ and Cl−via Na+/H+ and Cl−/HCO[Formula: see text] exchangers, contributing to the net Cl− transport. The fact that the net Cl−flux is about three times larger than the I sc is explained with a vectorial model in which there is a secretion of Na+ and K+ into the aqueous humor that partially subtracts from the net Cl− flux. These transport characteristics of the bovine ciliary epithelium suggest how acetazolamide reduces intraocular pressure in the absence of HCO[Formula: see text] transport as a driving force for fluid secretion.
Effect of cyclic stretch on α-subunit mRNA expression of Na+-K+-ATPase in aortic smooth muscle cells
Authors: Sevieux Nancy , Alam Jawed , Songu-Mize Emel
Published: December 2017

We previously demonstrated that protein expression of both α1- and α2-catalytic subunits of the Na+-K+-ATPase is elevated after a 2- to 4-day chronic cyclic stretch regimen in cultured aortic smooth muscle cells (ASMC). In the present study, we investigated whether cyclic stretch affects mRNA expression of the α-isoforms of the Na+-K+-ATPase. Using a stretch apparatus, rat ASMC were cyclically stretched 10 or 20% of their length for 1, 3, or 6 h. α-Isoform mRNA levels were measured using Northern analysis. A 3-h 10% stretch had no significant affect on mRNA expression for either isoform, but a 20% stretch increased mRNA of both isoforms approximately twofold. Whereas a 6-h 20% stretch increased α1 mRNA by 3.3-fold, α2 was not affected any further. Actinomycin D blocked the stretch-induced stimulation of mRNA expression of both α-subunits. In conclusion, cyclic stretch stimulates the mRNA expression of both α1- and α2-subunits of Na+-K+-ATPase. The sensitivity of the two genes to the degree and duration of stretch is different. The stretch-induced increase of mRNA may be a result of increased transcription.
Identified neurons in C. elegans coexpress vesicular transporters for acetylcholine and monoamines
Authors: Duerr Janet S. , Gaskin Jennifer , Rand James B.
Published: December 2017

We have identified four neurons (VC4, VC5, HSNL, HSNR) in Caenorhabditis elegans adult hermaphrodites that express both the vesicular acetylcholine transporter and the vesicular monoamine transporter. All four of these cells are motor neurons that innervate the egg-laying muscles of the vulva. In addition, they all express choline acetyltransferase, the synthetic enzyme for acetylcholine. The distributions of the vesicular acetylcholine transporter and the vesicular monoamine transporter are not identical within the individual cells. In mutants deficient for either of these transporters, there is no apparent compensatory change in the expression of the remaining transporter. This is the first report of neurons that express two different vesicular neurotransmitter transporters in vivo.
Preconditioning improves function and recovery of single muscle fibers during severe hypoxia and reoxygenation
Authors: Kohin Suzanne , Stary Creed M. , Howlett Richard A. , Hogan Michael C.
Published: December 2017

Reperfusion following prolonged ischemia induces cellular damage in whole skeletal muscle models. Ischemic preconditioning attenuates the deleterious effects. We tested whether individual skeletal muscle fibers would be similarly affected by severe hypoxia and reoxygenation (H/R) in the absence of extracellular factors and whether cellular damage could be alleviated by hypoxic preconditioning. Force and free cytosolic Ca2+([Ca2+]c) were monitored in Xenopus single muscle fibers ( n = 24) contracting tetanically at 0.2 Hz during 5 min of severe hypoxia and 5 min of reoxygenation. Twelve cells were preconditioned by a shorter bout of H/R 1 h before the experimental trial. In preconditioned cells, force relative to initial maximal values (P/Po) and relative peak [Ca2+]c fell ( P< 0.05) during 5 min of hypoxia and recovered during reoxygenation. In contrast, P/Po and relative peak [Ca2+]c fell more during hypoxia ( P < 0.05) and recovered less during reoxygenation ( P < 0.05) in control cells. The ratio of force to [Ca2+]c was significantly higher in the preconditioned cells during severe hypoxia, suggesting that changes in [Ca2+]c were not solely responsible for the loss in force. We conclude that 1) isolated skeletal muscle fibers contracting in the absence of extracellular factors are susceptible to H/R injury associated with changes in Ca2+handling; and 2) hypoxic preconditioning improves contractility, Ca2+ handling, and cell recovery during subsequent hypoxic insult.
ANG II-mediated inhibition of neuronal delayed rectifier K+ current: role of protein kinase C-α
Authors: Pan Sheng-Jun , Zhu Mingyan , Raizada Mohan K. , Sumners Colin , Gelband Craig H.
Published: December 2017

It was previously determined that ANG II and phorbol esters inhibit Kv current in neurons cultured from newborn rat hypothalamus and brain stem in a protein kinase C (PKC)- and Ca2+-dependent manner. Here, we have further defined this signaling pathway by investigating the roles of “physiological” activators of PKC and different PKC isozymes. The cell-permeable PKC activators, diacylglycerol (DAG) analogs 1,2-dioctanoyl- sn-glycerol (1 μmol/l, n = 7) and 1-oleoyl-2-acetyl- sn-glycerol (1 μmol/l, n = 6), mimicked the effect of ANG II and inhibited Kv current. These effects were abolished by the PKC inhibitor chelerythrine (1 μmol/l, n = 5) or by chelation of internal Ca2+ ( n = 8). PKC antisense (AS) oligodeoxynucleotides (2 μmol/l) against Ca2+-dependent PKC isoforms were applied to the neurons to manipulate the endogenous levels of PKC. PKC-α-AS ( n = 4) treatment abolished the inhibitory effects of ANG II and 1-oleoyl-2-acetyl- sn-glycerol on Kv current, whereas PKC-β-AS ( n = 4) and PKC-γ-AS ( n = 4) did not. These results suggest that the angiotensin type 1 receptor-mediated effects of ANG II on neuronal Kv current involve activation of PKC-α.
TNF receptor I is required for induction of macrophage heat shock protein 70
Authors: Heimbach Julie K. , Reznikov Leonid L. , Calkins Casey M. , Robinson Thomas N. , Dinarello Charles A. , Harken Alden H. , Meng Xianzhong
Published: December 2017

Expression of heat shock proteins (HSP) is an adaptive response to cellular stress. Stress induces tumor necrosis factor (TNF)-α production. In turn, TNF-α induces HSP70 expression. However, osmotic stress or ultraviolet radiation activates TNF-α receptor I (TNFR-I) in the absence of TNF-α. We postulated that TNF-α receptors are involved in the induction of HSP70 by cellular stress. Peritoneal Mφ were isolated from wild-type (WT), TNF-α knockout (KO), and TNFR (I or II) KO mice. Cells were cultured overnight and then heat stressed at 43 ± 0.5°C for 30 min followed by a 4-h recovery at 37°C. Cellular HSP70 expression was induced by heat stress or exposure to endotoxin [lipopolysaccharide (LPS)] as determined by immunoblotting. HSP70 expression induced by either heat or LPS was markedly decreased in TNFR-I KO Mφ, whereas TNFR-II KO Mφ exhibited HSP70 expression comparable to that in WT mice. Expression of HSP70 after heat stress in TNF-α KO Mφ was also similar to that in WT mice, suggesting that induction of HSP70 by TNFR-I occurs independently of TNF-α. In addition, levels of steady-state HSP70 mRNA were similar by RT-PCR in WT and TNFR-I KO Mφ despite differences in protein expression. Furthermore, the effect of TNFR-I appears to be cell specific, since HSP70 expression in splenocytes isolated from TNFR-I KO was similar to that in WT splenocytes. These studies demonstrate that TNFR-I is required for the synthesis of HSP70 in stressed Mφ by a TNF-independent mechanism and support an intracellular role for TNFR-I.
Inhibition of VRAC by c-Src tyrosine kinase targeted to caveolae is mediated by the Src homology domains
Authors: Trouet Dominique , Carton Iris , Hermans Diane , Droogmans Guy , Nilius Bernd , Eggermont Jan
Published: December 2017

We used the whole cell patch-clamp technique in calf pulmonary endothelial (CPAE) cells to investigate the effect of wild-type and mutant c-Src tyrosine kinase on I Cl,swell, the swelling-induced Cl−current through volume-regulated anion channels (VRAC). Transient transfection of wild-type c-Src in CPAE cells did not significantly affect I Cl,swell. However, transfection of c-Src with a Ser3Cys mutation that introduces a dual acylation signal and targets c-Src to lipid rafts and caveolae strongly repressed hypotonicity-induced I Cl,swell in CPAE cells. Kinase activity was dispensable for the inhibition of I Cl,swell, since kinase-deficient c-Src Ser3Cys either with an inactivating point mutation in the kinase domain or with the entire kinase domain deleted still suppressed VRAC activity. Again, the Ser3Cys mutation was required to obtain maximal inhibition by the kinase-deleted c-Src. In contrast, the inhibitory effect was completely lost when the Src homology domains 2 and 3 were deleted in c-Src. We therefore conclude that c-Src-mediated inhibition of VRAC requires compartmentalization of c-Src to caveolae and that the Src homology domains 2 and/or 3 are necessary and sufficient for inhibition.
Characterization of a novel 132-bp exon of the human maxi-K channel
Authors: Korovkina Victoria P. , Fergus Daniel J. , Holdiman Amanda J. , England Sarah K.
Published: December 2017

The large-conductance Ca2+-activated voltage-dependent K+ channel (maxi-K channel) induces a significant repolarizing current that buffers cell excitability. This channel can derive its diversity by alternative splicing of its transcript-producing isoforms that differ in their sensitivity to voltage and intracellular Ca2+. We have identified a novel 132-bp exon of the maxi-K channel from human myometrial cells that encodes 44 amino acids within the first intracellular loop of the channel protein. Distribution analysis reveals that this exon is expressed predominantly in human smooth muscle tissues with the highest abundance in the uterus and aorta and resembles the previously reported distribution of the total maxi-K channel transcript. Single-channel K+ current measurements in fibroblasts transfected with the maxi-K channel containing this novel 132-bp exon demonstrate that the presence of this insert attenuates the sensitivity to voltage and intracellular Ca2+. Alternative splicing to introduce this 132-bp exon into the maxi-K channel may elicit another mode to modulate cell excitability.
Regulation of I Cl,swell in neuroblastoma cells by G protein signaling pathways
Authors: Estevez Ana Y. , Bond Tamara , Strange Kevin
Published: December 2017

Guanosine 5′- O-(3-thiotriphosphate) (GTPγS) activated the I Cl,swell anion channel in N1E115 neuroblastoma cells in a swelling-independent manner. GTPγS-induced current was unaffected by ATP removal and broadly selective tyrosine kinase inhibitors, demonstrating that phosphorylation events do not regulate G protein-dependent channel activation. Pertussis toxin had no effect on GTPγS-induced current. However, cholera toxin inhibited the current ∼70%. Exposure of cells to 8-bromoadenosine 3′,5′-cyclic monophosphate did not mimic the effect of cholera toxin, and its inhibitory action was not prevented by treatment of cells with an inhibitor of adenylyl cyclase. These results demonstrate that GTPγS does not act through Gαi/o GTPases and that Gαs/Gβγ G proteins inhibit the channel and/or channel regulatory mechanisms through cAMP-independent mechanisms. Swelling-induced activation of I Cl,swell was stimulated two- to threefold by GTPγS and inhibited by 10 mM guanosine 5′- O-(2-thiodiphosphate). The Rho GTPase inhibitor Clostridium difficile toxin B inhibited both GTPγS- and swelling-induced activation of I Cl,swell. Taken together, these findings indicate that Rho GTPase signaling pathways regulate the I Cl,swell channel via phosphorylation-independent mechanisms.
Differential contribution of sialic acid to the function of repolarizing K+ currents in ventricular myocytes
Authors: Ufret-Vincenty Carmen A. , Baro Deborah J. , Santana L. F.
Published: December 2017

We investigated the contribution of sialic acid residues to the K+ currents involved in the repolarization of mouse ventricular myocytes. Ventricular K+ currents had a rapidly inactivating component followed by slowly decaying and sustained components. This current was produced by the summation of three distinct currents: I to, which contributed to the transient component; I ss, which contributed to the sustained component; and I K,slow, which contributed to both components. Incubation of ventricular myocytes with the sialidase neuraminidase reduced the amplitude of I to without altering I K,slow and I ss. We found that the reduction in I to amplitude resulted from a depolarizing shift in the voltage of activation and a reduction in the conductance of I to. Expression of Kv4.3 channels, a major contributor to I to in the ventricle, in a sialylation-deficient Chinese hamster ovary cell line (lec2) mimicked the effects of neuraminidase on the ventricular I to. Furthermore, we showed that sialylated glycolipids have little effect on the voltage dependence of I to. Finally, consistent with its actions on I to, neuraminidase produced an increase in the duration of the action potential of ventricular myocytes and the frequency of early afterdepolarizations. We conclude that sialylation of the proteins forming Kv4 channels is important in determining the voltage dependence and conductance of I to and that incomplete glycosylation of these channels could lead to arrhythmias.
Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store
Authors: Mohanty Madhumita Jena , Ye Maian , Li Xingli , Rossi Noreen F.
Published: December 2017

Hypotonic swelling increases the intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle cells (VSMC). The source of this Ca2+ is not clear. To study the source of increase in [Ca2+]i in response to hypotonic swelling, we measured [Ca2+]i in fura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a 40.7-nM increase in [Ca2+]i that was not inhibited by EGTA but was inhibited by 1 μM thapsigargin. Prior depletion of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores with vasopressin did not inhibit the increase in [Ca2+]i in response to hypotonic swelling. Exposure of 45Ca2+-loaded intracellular stores to hypotonic swelling in permeabilized VSMC produced an increase in45Ca2+ efflux, which was inhibited by 1 μM thapsigargin but not by 50 μg/ml heparin, 50 μM ruthenium red, or 25 μM thio-NADP. Thus hypotonic swelling of VSMC causes a release of Ca2+ from the intracellular stores from a novel site distinct from the IP3-, ryanodine-, and nicotinic acid adenine dinucleotide phosphate-sensitive stores.
Contractile regulation of the Na+-K+-2Cl− cotransporter in vascular smooth muscle
Authors: Akar Fatma , Jiang Gengru , Paul Richard J. , O'Neill W. Charles
Published: December 2017

Vasoconstrictors activate the Na+-K+-2Cl− cotransporter NKCC1 in rat aortic smooth muscle, but the mechanism is unknown. Efflux of86Rb+ from rat aorta in response to phenylephrine (PE) was measured in the absence and presence of bumetanide, a specific inhibitor of NKCC1. Removal of extracellular Ca2+ completely abolished the activation of NKCC1 by PE. This was not due to inhibition of Ca2+-dependent K+ channels since blocking these channels with Ba2+ in Ca2+-replete solution did not prevent activation of NKCC1 by PE. Stimulation of NKCC1 by PE was inhibited 70% by 75 μM ML-9, 97% by 2 μM wortmannin, and 70% by 2 mM 2,3-butanedione monoxime, each of which inhibited isometric force generation in aortic rings. Bumetanide-insensitive Rb+efflux, an indication of Ca2+-dependent K+channel activity, was reduced by ML-9 but not by the other inhibitors. Stretching of aortic rings on tubing to increase lumen diameter to 120% of normal almost completely blocked the stimulation of NKCC1 by PE without inhibiting the stimulation by hypertonic shrinkage. We conclude that activation of the Na+-K+-2Cl− cotransporter by PE is the direct result of smooth muscle contraction through Ca2+-dependent activation of myosin light chain kinase. This indicates that the Na+-K+-2Cl− cotransporter is regulated by the contractile state of vascular smooth muscle.
Inhibition of TASK-1 potassium channel by phospholipase C
Authors: Czirják Gábor , Petheő Gábor L. , Spät András , Enyedi Péter
Published: December 2017

The two-pore-domain K+ channel, TASK-1, was recently shown to be a target of receptor-mediated regulation in neurons and in adrenal glomerulosa cells. Here, we demonstrate that TASK-1 expressed in Xenopus laevis oocytes is inhibited by different Ca2+-mobilizing agonists. Lysophosphatidic acid, via its endogenous receptor, and ANG II and carbachol, via their heterologously expressed ANG II type 1a and M1 muscarinic receptors, respectively, inhibit TASK-1. This effect can be mimicked by guanosine 5′- O-(3-thiotriphosphate), indicating the involvement of GTP-binding protein(s). The phospholipase C inhibitor U-73122 reduced the receptor-mediated inhibition of TASK-1. Downstream signals of phospholipase C action (inositol 1,4,5-trisphosphate, cytoplasmic Ca2+ concentration, and diacylglycerol) do not mediate the inhibition. Unlike the Gq-coupled receptors, stimulation of the Gi-activating M2 muscarinic receptor coexpressed with TASK-1 results in an only minimal decrease of the TASK-1 current. However, additional coexpression of phospholipase C-β2 (which is responsive also to Giβγ-subunits) renders M2 receptor activation effective. This indicates the significance of phospholipase C activity in the receptor-mediated inhibition of TASK-1.
Decrease in rat taste receptor cell intracellular pH is the proximate stimulus in sour taste transduction
Authors: Lyall Vijay , Alam Rammy I. , Phan Duy Q. , Ereso Glenn L. , Phan Tam-Hao T. , Malik Shahbaz A. , Montrose Marshall H. , Chu Shaoyou , Heck Gerard L. , Feldman George M. , DeSimone John A.
Published: December 2017

Taste receptor cells (TRCs) respond to acid stimulation, initiating perception of sour taste. Paradoxically, the pH of weak acidic stimuli correlates poorly with the perception of their sourness. A fundamental issue surrounding sour taste reception is the identity of the sour stimulus. We tested the hypothesis that acids induce sour taste perception by penetrating plasma membranes as H+ ions or as undissociated molecules and decreasing the intracellular pH (pHi) of TRCs. Our data suggest that taste nerve responses to weak acids (acetic acid and CO2) are independent of stimulus pH but strongly correlate with the intracellular acidification of polarized TRCs. Taste nerve responses to CO2 were voltage sensitive and were blocked with MK-417, a specific blocker of carbonic anhydrase. Strong acids (HCl) decrease pHi in a subset of TRCs that contain a pathway for H+ entry. Both the apical membrane and the paracellular shunt pathway restrict H+ entry such that a large decrease in apical pH is translated into a relatively small change in TRC pHi within the physiological range. We conclude that a decrease in TRC pHi is the proximate stimulus in rat sour taste transduction.
Regulation of glutamate transport and transport proteins in a placental cell line
Authors: Novak Donald , Quiggle Forrest , Artime Carlos , Beveridge Mark
Published: December 2017

We utilized HRP.1 cells derived from midgestation rat placental labyrinth to determine that the primary pathway for glutamate uptake is via system X[Formula: see text], a Na+-dependent transport system. Kinetic parameters of system X[Formula: see text]activity were similar to those previously determined in rat and human placental membrane vesicle preparations. Amino acid depletion caused a significant upregulation of system X[Formula: see text] activity at 6, 24, and 48 h. This increase was reversed by the addition of glutamate and aspartate but not by the addition of α-(methylamino)isobutyric acid. Immunoblot analysis of the three transport proteins previously associated with system X[Formula: see text] activity indicated a trend toward an increase in GLT1, EAAC1, and GLAST1 immunoreactive protein contents by 48 h; cell surface expression of the same was enhanced by 24 h. Inhibition analysis suggested key roles for EAAC1 and GLAST1 in basal anionic amino acid transfer, with an enhanced role for GLT1 under conditions of amino acid depletion. In summary, amino acid availability as well as intracellular metabolism regulate anionic amino acid uptake into this placental cell line.
Regulation of cytoplasmic calcium levels by two nitric oxide receptors
Authors: Bowman Charles L. , Yohe Lori , Lohr James W.
Published: December 2017

We examined the effects of dissolved nitric oxide (NO) gas on cytoplasmic calcium levels ([Ca2+]i) in C6 glioma cells under anoxic conditions. The maximum elevation (27 ± 3 nM) of [Ca2+]i was reached at 10 μM NO. A second application of NO was ineffective if the first was >0.5 μM. The NO donor diethylamine/NO mimicked the effects of NO. Acute exposure of the cells to low calcium levels was without effect on the NO-evoked response. Thapsigargin (TG) increased [Ca2+]iand was less effective if cells were pretreated with NO. Hemoglobin inhibited the effects of NO at a molar ratio of 10:1. 8-Bromo-cGMP was without effect on the NO-evoked response. If cells were pretreated with TG or exposed chronically to nominal amounts of calcium, NO decreased [Ca2+]i. The results suggest that C6 glioma cells have two receptors for NO. One receptor (NOA) elevates [Ca2+]i and resides on the endoplasmic reticulum (ER). The other receptor (NOB) decreases [Ca2+]i and resides on the plasmalemma or the ER. The latter receptor dominates when the level of calcium within intracellular stores is diminished.
Effect of pH, ionic charge, and osmolality on cytochromec-mediated caspase-3 activity
Authors: Segal Mark S. , Beem Elaine
Published: December 2017

Cytochrome c-mediated activation of caspase-3 is the final common pathway for most signals that induce apoptosis. Before release of cytochrome c from mitochondria, K+ and Cl− efflux and intracellular acidification must occur. We have utilized an in vitro assay to examine the role of pH, cations, anions, and uncharged molecules on the process of cytochrome c-mediated activation of procaspase-3. In this cell-free system, a pH above 7.4 severely suppressed the activation of procaspase-3 but not the activity of caspase-3. KCl, NaCl, and other salts all inhibited caspase activation, but uncharged molecules did not. Comparison of the inhibitory capacity of various salts suggests that the crucial element in causing suppression is the cation. The inhibition of alkaline pH could be overcome by increasing concentrations of cytochrome c, whereas the inhibition of ionic charge could not, suggesting that pH and salts affect the activation of caspase-3 by different mechanisms.
Role of Na+/H+ exchanger during O2 deprivation in mouse CA1 neurons
Authors: Yao Hang , Gu Xiang-Qun , Douglas Robert M. , Haddad Gabriel G.
Published: December 2017

To determine the role of membrane transporters in intracellular pH (pHi) regulation under conditions of low microenvironmental O2, we monitored pHi in isolated single CA1 neurons using the fluorescent indicator carboxyseminaphthorhodafluor-1 and confocal microscopy. After total O2 deprivation or anoxia (Po 2≅ 0 Torr), a large increase in pHi was seen in CA1 neurons in HEPES buffer, but a drop in pHi, albeit small, was observed in the presence of HCO[Formula: see text]. Ionic substitution and pharmacological experiments showed that the large anoxia-induced pHi increase in HEPES buffer was totally Na+ dependent and was blocked by HOE-694, strongly suggesting the activation of the Na+/H+exchanger (NHE). Also, this pHi increase in HEPES buffer was significantly smaller in Na+/H+ exchanger isoform 1 (NHE1) null mutant CA1 neurons than in wild-type neurons, demonstrating that NHE1 is responsible for part of the pHiincrease following anoxia. Both chelerythrine and H-89 partly blocked, and H-7 totally eliminated, this anoxia-induced pHiincrease in the absence of HCO[Formula: see text]. We conclude that 1) O2 deprivation activates Na+/H+ exchange by enhancing protein kinase activity and 2) membrane proteins, such as NHE, actively participate in regulating pHi during low-O2states in neurons.
Selective inhibition of Cl− conductance in toad skin by blockers of Cl− channels and transporters
Authors: Nagel Wolfram , Somieski Petra , Katz Uri
Published: December 2017

We compared the effects exerted by two classes of Cl−transport inhibitors on a Cl−-selective, passive anion transport route across the skin of Bufo viridis, the conductance ( G Cl) of which can be activated by transepithelial voltage perturbation or high cAMP at short circuit. Inhibitors of antiporters (erythrosine, eosin) or cotransporters (furosemide) reduced voltage-activated G Cl with IC50 of 6 ± 1, 54 ± 12, and 607 ± 125 μM, respectively; they had no effect on the cAMP-induced G Cl. The voltage for half-maximal activation of G Cl ( V 50) increased compared with controls, but effects on the maximal G Cl at more positive clamp potentials were small. Cl− channel blockers from the diphenylamino-2-carboxylic acid (DPC) family [dichloro-DPC, niflumic acid, flufenamic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid] reduced the voltage-activated G Cl with IC50 of 8.3 ± 1.2, 10.5 ± 0.6, 16.5 ± 3.4, and 36.5 ± 11.4 μM, respectively, and also inhibited the cAMP-induced G Cl, albeit with slightly larger IC50. V 50 was not significantly changed compared with controls; the maximal G Clwas strongly reduced. We conclude that the pathway for Cl−is composed of the conductive pore proper, which is blocked by the derivatives of DPC, and a separate, voltage-sensitive regulator, which is influenced by blockers of cotransporters or antiporters. This influence is partly overcome by increasing the clamp potential and removed by high concentrations of cAMP, which renders the pathway insensitive to voltage.
Effect of glutamine supplementation on exercise-induced changes in lymphocyte function
Authors: Krzywkowski Karen , Petersen Emil Wolsk , Ostrowski Kenneth , Kristensen Jens Halkjær , Boza Julio , Pedersen Bente Klarlund
Published: December 2017

The purpose of this study was to investigate the possible role of glutamine in exercise-induced impairment of lymphocyte function. Ten male athletes participated in a randomized, placebo-controlled, double-blind crossover study. Each athlete performed bicycle exercise for 2 h at 75% of maximum O2 consumption on 2 separate days. Glutamine or placebo supplements were given orally during and up to 2 h postexercise. The trial induced postexercise neutrocytosis that lasted at least 2 h. The total lymphocyte count increased by the end of exercise due to increase of both CD3+TCRαβ+ and CD3+TCRγδ+ T cells as well as CD3−CD16+CD56+ natural killer (NK) cells. Concentrations of CD8+ and CD4+ T cells lacking CD28 and CD95 on their surface increased more than those of cells expressing these receptors. Within the CD4+ cells, only CD45RA− memory cells, but not CD45RA+ naive cells, increased in response to exercise. Most lymphocyte subpopulations decreased 2 h after exercise. Glutamine supplementation abolished the postexercise decline in plasma glutamine concentration but had no effect on lymphocyte trafficking, NK and lymphokine-activated killer cell activities, T cell proliferation, catecholamines, growth hormone, insulin, or glucose. Neutrocytosis was less pronounced in the glutamine-supplemented group, but it is unlikely that this finding is of any clinical significance. This study does not support the idea that glutamine plays a mechanistic role in exercise-induced immune changes.
Modulation of pulmonary alveolar type II cell phenotype and communication by extracellular matrix and KGF
Authors: Isakson Brant E. , Lubman Richard L. , Seedorf Gregory J. , Boitano Scott
Published: December 2017

The alveolar epithelium consists of two cell types, alveolar type I (AT1) and alveolar type II (AT2) cells. We have recently shown that 7-day-old cultures of AT2 cells grown on a type I collagen/fibronectin matrix develop phenotypic characteristics of AT1 cells, display a distinct connexin profile, and coordinate mechanically induced intercellular Ca2+ changes via gap junctions (25). In this study, we cultured AT2 cells for 7 days on matrix supplemented with laminin-5 and/or in the presence of keratinocyte growth factor. Under these conditions, cultured AT2 cells display AT2 type morphology, express the AT2-specific marker surfactant protein C, and do not express AT1-specific cell marker aquaporin 5, all consistent with maintenance of AT2 phenotype. These AT2-like cells also coordinate mechanically induced intercellular Ca2+ signaling, but, unlike AT1-like cells, do so by using extracellular nucleotide triphosphate release. Additionally, cultured cells that retain AT2 cell-specific markers express connexin profiles different from cultured cells with AT1 characteristics. The parallel changes in intercellular Ca2+ signaling with cell differentiation suggest that cell signaling mechanisms are an intrinsic component of lung alveolar cell phenotype. Because lung epithelial injury is accompanied by extracellular matrix and growth factor changes, followed by extensive cell division, differentiation, and migration of AT2 progenitor cells, we suggest that similar changes may be vital to the lung recovery and repair process in vivo.
Increased expression of utrophin in a slow vs. a fast muscle involves posttranscriptional events
Authors: Gramolini Anthony O. , Bélanger Guy , Thompson Jennifer M. , Chakkalakal Joe V. , Jasmin Bernard J.
Published: December 2017

In addition to showing differences in the levels of contractile proteins and metabolic enzymes, fast and slow muscles also differ in their expression profile of structural and synaptic proteins. Because utrophin is a structural protein expressed at the neuromuscular junction, we hypothesize that its expression may be different between fast and slow muscles. Western blots showed that, compared with fast extensor digitorum longus (EDL) muscles, slow soleus muscles contain significantly more utrophin. Quantitative RT-PCR revealed that this difference is accompanied by a parallel increase in the expression of utrophin transcripts. Interestingly, the higher levels of utrophin and its mRNA appear to occur in extrasynaptic regions of muscle fibers as shown by immunofluorescence and in situ hybridization experiments. Furthermore, nuclear run-on assays showed that the rate of transcription of the utrophin gene was nearly identical between EDL and soleus muscles, indicating that increased mRNA stability accounts for the higher levels of utrophin in slow muscles. Direct plasmid injections of reporter gene constructs showed that cis-acting elements contained within the utrophin 3′-untranslated region (3′-UTR) confer greater stability to chimeric LacZ transcripts in soleus muscles. Finally, we observed a clear difference between EDL and soleus muscles in the abundance of RNA-binding proteins interacting with the utrophin 3′-UTR. Together, these findings highlight the contribution of posttranscriptional events in regulating the expression of utrophin in muscle.
Ouabain and substrate affinities of human Na+-K+-ATPase α1β1, α2β1, and α3β1when expressed separately in yeast cells
Authors: Müller-Ehmsen Jochen , Juvvadi Padmaja , Thompson Curtis B. , Tumyan Lusine , Croyle Michelle , Lingrel Jerry B. , Schwinger Robert H. G. , McDonough Alicia A. , Farley Robert A.
Published: December 2017

Human Na+-K+-ATPase α1β1, α2β1, and α3β1heterodimers were expressed individually in yeast, and ouabain binding and ATP hydrolysis were measured in membrane fractions. The ouabain equilibrium dissociation constant was 13–17 nM for α1β1 and α3β1at 37°C and 32 nM for α2β1, indicating that the human α-subunit isoforms have a similar high affinity for cardiac glycosides. K 0.5 values for antagonism of ouabain binding by K+ were ranked in order as follows: α2 (6.3 ± 2.4 mM) > α3(1.6 ± 0.5 mM) ≈ α1 (0.9 ± 0.6 mM), and K 0.5 values for Na+ antagonism of ouabain binding to all heterodimers were 9.5–13.8 mM. The molecular turnover for ATP hydrolysis by α1β1 (6,652 min−1) was about twice as high as that by α3β1 (3,145 min−1). These properties of the human heterodimers expressed in yeast are in good agreement with properties of the human Na+-K+-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-D Horisberger, L Lelievie, and K Geering. J Biol Chem275: 1976–1986, 2000). In contrast to Na+ pumps expressed in Xenopus oocytes, the α2β1 complex in yeast membranes was significantly less stable than α1β1 or α3β1, resulting in a lower functional expression level. The α2β1 complex was also more easily denatured by SDS than was the α1β1 or the α3β1 complex.
Sequential and opposite regulation of two outward K+ currents by ET-1 in cultured striatal astrocytes
Authors: Bychkov R. , Glowinski J. , Giaume C.
Published: December 2017

In the brain, astrocytes represent a major target for endothelins (ETs), a family of peptides that can be released by several cell types and that have potent and multiple effects on astrocytic functions. Four types of K+ currents ( I K) were detected in various proportions by patch-clamp recordings of cultured striatal astrocytes, including the A-type I K, the inwardly rectifying I K IR, the Ca2+-dependent I K( I K Ca), and the delayed-rectified I K ( I K DR). Variations in the shape of current-voltage relationships were related mainly to differences in the proportion of these currents. ET-1 was found to regulate with opposite effects the two more frequently recorded outward K+ currents in striatal astrocytes. Indeed, this peptide induced an initial activation of I K Ca(composed of SK and BK channels) and a delayed long-lasting inhibition of I K DR. In current-clamp recordings, the activation of I K Ca correlated with a transient hyperpolarization, whereas the inhibition of I K DR correlated with a sustained depolarization. These ET-1-induced sequential changes in membrane potential in astrocytes may be important for the regulation of voltage gradients in astrocytic networks and the maintenance of K+ homeostasis in the brain microenvironment.
Modulation of glucagon receptor expression and response in transfected human embryonic kidney cells
Authors: Ikegami Tadashi , Cypess Aaron M. , Bouscarel Bernard
Published: December 2017

The modulation of glucagon receptor (GR) expression and biological response was investigated in human embryonic kidney cell (HEK-293) clones permanently expressing the GR with different densities. The GR mRNA expression level in these clones was upregulated by cellular cAMP accumulation and presented a good correlation with both the protein expression level and the maximum number of glucagon binding sites. However, the determination of glucagon-induced cAMP accumulation in these cell lines revealed that the enhancement of receptor expression did not lead to a proportional increase in cAMP formation. Under these conditions, the maximum cAMP production induced by NaF and forskolin was not significantly different among selected clones, regardless of the receptor expression level. High receptor-expressing clones showed the greatest susceptibility for agonist-induced desensitization compared with clones with lower GR expression levels. The results of the present study suggest that the GR can recruit non-GR-specific desensitization mechanism(s). Furthermore, the partial inhibition or alteration of the overall cAMP synthesis pathway at the receptor level may be a necessary adaptive step for a cell in response to a massive increase in membrane receptor expression level.
NHLBI workshop report: endothelial cell phenotypes in heart, lung, and blood diseases
Authors: Stevens Troy , Rosenberg Robert , Aird William , Quertermous Thomas , Johnson Frances L. , Garcia Joe G. N. , Hebbel Robert P. , Tuder Rubin M. , Garfinkel Susan
Published: December 2017

Endothelium critically regulates systemic and pulmonary vascular function, playing a central role in hemostasis, inflammation, vasoregulation, angiogenesis, and vascular growth. Indeed, the endothelium integrates signals originating in the circulation with those in the vessel wall to coordinate vascular function. This highly metabolic role differs significantly from the historic view of endothelium, in which it was considered to be merely an inert barrier. New lines of evidence may further change our understanding of endothelium, in regard to both its origin and function. Embryological studies suggest that the endothelium arises from different sites, including angiogenesis of endothelium from macrovascular segments and vasculogenesis of endothelium from microcirculatory segments. These findings suggest an inherent phenotypic distinction between endothelial populations based on their developmental origin. Similarly, diverse environmental cues influence endothelial cell phenotype, critical to not only normal function but also the function of a diseased vessel. Consequently, an improved understanding of site-specific endothelial cell function is essential, particularly with consideration to environmental stimuli present both in the healthy vessel and in development of vasculopathic disease states. The need to examine endothelial cell phenotypes in the context of vascular function served as the basis for a recent workshop sponsored by the National Heart, Lung, and Blood Institute (NHLBI). This report is a synopsis of pertinent topics that were discussed, and future goals and research opportunities identified by the participants of the workshop are presented.
Functional and molecular adaptations in skeletal muscle of myoglobin-mutant mice
Authors: Grange Robert W. , Meeson Annette , Chin Eva , Lau Kim S. , Stull James T. , Shelton John M. , Williams R. Sanders , Garry Daniel J.
Published: December 2017

Myoglobin is a cytoplasmic hemoprotein that is restricted to cardiomyocytes and oxidative skeletal myofibers and facilitates oxygen delivery during periods of high metabolic demand. Myoglobin content in skeletal muscle increases in response to hypoxic conditions. However, we previously reported that myoglobin-null mice are viable and fertile. In the present study, we define important functional, cellular, and molecular compensatory adaptations in the absence of myoglobin. Mice without myoglobin manifest adaptations in skeletal muscle that include a fiber type transition (type I to type II in the soleus muscle), increased expression of the hypoxia-inducible transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2 (endothelial PAS domain protein), stress proteins such as heat shock protein 27, and the angiogenic growth factor vascular endothelial growth factor (soleus muscle), as well as increased nitric oxide metabolism (extensor digitorum longus). The resulting changes in angiogenesis, nitric oxide metabolism, and vasomotor regulation are likely to account for preserved exercise capacity of animals lacking myoglobin. These results demonstrate that mammalian organisms are capable of a broad spectrum of adaptive responses that can compensate for a potentially serious defect in cellular oxygen transport.