bluuebunny/crossref_metadata_2025 huggingface dataset:
Amino acids are compatible osmolytes for volume recovery after hypertonic shrinkage in vascular endothelial cells
Authors: Dall’Asta Valeria , Bussolati Ovidio , Sala Roberto , Parolari Alessandro , Alamanni Francesco , Biglioli Paolo , Gazzola Gian C.
Published: December 2017

The response to chronic hypertonic stress has been studied in human endothelial cells derived from saphenous veins. In complete growth medium the full recovery of cell volume requires several hours and is neither associated with an increase in cell K+ nor hindered by bumetanide but depends on an increased intracellular pool of amino acids. The highest increase is exhibited by neutral amino acid substrates of transport system A, such as glutamine and proline, and by the anionic amino acid glutamate. Transport system A is markedly stimulated on hypertonic stress, with an increase in activity roughly proportional to the extent and the duration of the osmotic shrinkage. Cycloheximide prevents the increase in transport activity of system A and the recovery of cell volume. It is concluded that human endothelial cells counteract hypertonic stress through the stimulation of transport system A and the consequent expansion of the intracellular amino acid pool.
Switched single-electrode voltage-clamp amplifiers allow precise measurement of gap junction conductance
Authors: Müller Andreas , Lauven Melani , Berkels Reinhard , Dhein Stefan , Polder Hans-Reiner , Klaus Wolfgang
Published: December 2017

Measurement of gap junction conductance ( g j) with patch-clamp amplifiers can, due to series resistance problems, be subject to considerable errors when large currents are measured. Formulas developed to correct for these errors unfortunately depend on exact estimates of series resistance, which are not always easy to obtain. Discontinuous single-electrode voltage-clamp amplifiers (DSEVCs) were shown to overcome series resistance problems in single whole cell recording. With the use of two synchronized DSEVCs, the simulated g j in a model circuit can be measured with a maximum error of <5% in all recording situations investigated (series resistance, 5–47 MΩ; membrane resistance, 20–1,000 MΩ; g j, 1–100 nS). At a very low g j of 100 pS, the error sometimes exceeded 5% (maximum of 15%), but the error was always <5% when membrane resistance was >100 MΩ. The precision of the measurements is independent of series resistance, membrane resistance, and g j. Consequently, it is possible to calculate g j directly from Ohm’s law, i.e., without using correction formulas. Our results suggest that DSEVCs should be used to measure g j if large currents must be recorded, i.e., if cells are well coupled or if membrane resistance is low.
LXA4, aspirin-triggered 15-epi-LXA4, and their analogs selectively downregulate PMN azurophilic degranulation
Authors: Gewirtz Andrew T. , Fokin Valery V. , Petasis Nicos A. , Serhan Charles N. , Madara James L.
Published: December 2017

The eicosanoid lipoxin A4(LXA4) is biosynthesized in vivo by cells present at inflammatory sites and appears to be an endogenous anti-inflammatory mediator. Further, in the presence of aspirin, the 15-epimer of LXA4(15-epi-LXA4) is biosynthesized and may mediate some of aspirin’s desirable bioactions. LXA4, 15-epi-LXA4, and their stable analogs inhibit inflammation in established animal models, indicating that these compounds may be useful for treating inflammatory disease states. To investigate the cellular mechanisms by which these lipid mediators downregulate inflammation, we investigated whether these eicosanoids could influence receptor-mediated degranulation of human neutrophils, an event thought to play a major causative role in several inflammatory disease states. LXA4, 15-epi-LXA4, and their stable analogs potently (IC50 < 1 nM) and selectively downregulated neutrophil release of azurophilic granule contents but did not affect other neutrophil secretory functions. Thus the cellular basis of action of these natural off-switches to inflammation appears to involve downregulation of neutrophil azurophilic granule release.
Role for IL-4 in macromolecular transport across human intestinal epithelium
Authors: Berin M. Cecilia , Yang Ping-Chang , Ciok Leeann , Waserman Susan , Perdue Mary H.
Published: December 2017

Increased epithelial permeability is associated with intestinal inflammation, but there is little information on factors that regulate barrier function in the absence of or before inflammation. We examined if interleukin (IL)-4, or serum from atopic individuals, could alter the barrier function of human colonic epithelial (T84) monolayers to antigenic-sized macromolecules. IL-4 and atopic serum significantly decreased T84 monolayer resistance and increased transepithelial horseradish peroxidase (HRP) transport. Bidirectional transport studies demonstrated that IL-4 selectively enhanced apical-to-basal movement of HRP. HRP transport induced by IL-4 was inhibited by cold (4°C) and the tyrosine kinase inhibitor genistein, but not the protein kinase C inhibitor staurosporine. Electron microscopic analysis demonstrated that both transcellular and paracellular pathways were affected. Anti-IL-4 antibodies abolished the increase in HRP transport in response to both IL-4 and serum. We speculate that enhanced production of IL-4 in allergic conditions may be a predisposing factor to inflammation by allowing uptake of luminal antigens that gain access to the mucosal immune system.
Metabolic adaptation of endothelial cells to substrate deprivation
Authors: Culic Ognjen , Decking Ulrich K. M. , Schrader Jürgen
Published: December 2017

Endothelial cells are known to be metabolically rather robust. To study the mechanisms involved, porcine aortic endothelial cells (PAEC), cultured on microcarrier beads, were perfused with glucose (10 mM) or with substrate-free medium. Substrate-free perfusion for 2 h induced an almost complete loss of nucleoside triphosphates (31P-NMR) and decreased heat flux, a measure of total energy turnover, by >90% in parallel microcalorimetric measurements. Heat flux and nucleoside triphosphates recovered after addition of glucose. Because protein synthesis is a major energy consumer in PAEC, the rate of protein synthesis was measured ([14C]leucine incorporation). Reduction or blockade of energy supply resulted in a pronounced reduction in the rate of protein synthesis (up to 80% reduction). Intracellular triglyceride stores were decreased by ∼60% after 2 h of substrate-free perfusion. Under basal perfusion conditions, PAEC released ∼30 pmol purine ⋅ mg protein−1 ⋅ min−1, i.e., 16% of the cellular ATP per hour, while ATP remained constant. Substrate deprivation increased the release of various purines and pyrimidines about threefold and also induced a twofold rise in purine de novo synthesis ([14C]formate). These results demonstrate that PAEC are capable of recovering from extended periods of substrate deprivation. They can do so by a massive downregulation of their energy expenditure, particularly protein synthesis, while at the same time using endogenous triglycerides as substrates and upregulating purine de novo synthesis to compensate for the loss of purines.
[3H]taurine andd-[3H]aspartate release from astrocyte cultures are differently regulated by tyrosine kinases
Authors: Mongin Alexander A. , Reddi Jyoti M. , Charniga Carol , Kimelberg Harold K.
Published: December 2017

Volume-dependent anion channels permeable for Cl− and amino acids are thought to play an important role in the homeostasis of cell volume. Astrocytes are the main cell type in the mammalian brain showing volume perturbations under physiological and pathophysiological conditions. We investigated the involvement of tyrosine phosphorylation in hyposmotic medium-induced [3H]taurine andd-[3H]aspartate release from primary astrocyte cultures. The tyrosine kinase inhibitors tyrphostin 23 and tyrphostin A51 partially suppressed the volume-dependent release of [3H]taurine in a dose-dependent manner with half-maximal effects at ∼40 and 1 μM, respectively. In contrast, the release ofd-[3H]aspartate was not significantly affected by these agents in the same concentration range. The inactive analog tyrphostin 1 had no significant effect on the release of both amino acids. The data obtained suggest the existence of at least two volume-dependent anion channels permeable to amino acids in astrocyte cultures. One of these channels is permeable to taurine and is under the control of tyrosine kinase(s). The other is permeable to both taurine and aspartate, but its volume-dependent regulation does not require tyrosine phosphorylation.
Vasoconstrictors and nitrovasodilators reciprocally regulate the Na+-K+-2Cl−cotransporter in rat aorta
Authors: Akar Fatma , Skinner Elizabeth , Klein Janet D. , Jena Madhumita , Paul Richard J. , O’Neill W. Charles
Published: December 2017

Little is known about the function and regulation of the Na+-K+-2Cl−cotransporter NKCC1 in vascular smooth muscle. The activity of NKCC1 was measured as the bumetanide-sensitive efflux of86Rb+from intact smooth muscle of the rat aorta. Hypertonic shrinkage (440 mosmol/kgH2O) rapidly doubled cotransporter activity, consistent with its volume-regulatory function. NKCC1 was also acutely activated by the vasoconstrictors ANG II (52%), phenylephrine (50%), endothelin (53%), and 30 mM KCl (54%). Both nitric oxide and nitroprusside inhibited basal NKCC1 activity (39 and 34%, respectively), and nitroprusside completely reversed the stimulation by phenylephrine. The phosphorylation of NKCC1 was increased by hypertonic shrinkage, phenylephrine, and KCl and was reduced by nitroprusside. The inhibition of NKCC1 significantly reduced the contraction of rat aorta induced by phenylephrine (63% at 10 nM, 26% at 30 nM) but not by KCl. We conclude that the Na+-K+-2Cl−cotransporter in vascular smooth muscle is reciprocally regulated by vasoconstrictors and nitrovasodilators and contributes to smooth muscle contraction, indicating that alterations in NKCC1 could influence vascular smooth muscle tone in vivo.
Inhibition of P-glycoprotein-mediated transport by a hydrophobic contaminant in commercial gluconate salts
Authors: Vanoye Carlos G. , Altenberg Guillermo A. , Reuss Luis
Published: December 2017

The substitution of gluconate for Cl− is commonly used to characterize Cl− transport or Cl−-dependent transport mechanisms. We evaluated the effects of substituting gluconate for Cl− on the transport of the P-glycoprotein substrate rhodamine 123 (R123). The replacement of Ringer solution containing Cl−(Cl−-Ringer) with gluconate-Ringer inhibited R123 efflux, whereas the replacement of Cl− by other anions (sulfate or cyclamate) had no effect. The inhibition of R123 efflux by gluconate-Ringer was absent after chloroform extraction of the sodium gluconate salt. The readdition of the sodium gluconate-chloroform extract to the extracted gluconate-Ringer or to cyclamate-Ringer inhibited R123 efflux, whereas its addition to Cl−-Ringer had no effect. These observations indicate that the inhibition of P-glycoprotein-mediated R123 transport by gluconate is due to one or more chloroform-soluble contaminants and that the inhibition is absent in the presence of Cl−. The results are consistent with the fact that P-glycoprotein substrates are hydrophobic. Care should be taken when replacing ions to evaluate membrane transport mechanisms because highly pure commercial preparations may still contain potent contaminants that affect transport.
Syntaxin 1A inhibits regulated CFTR trafficking inXenopus oocytes
Authors: Peters Kathryn W. , Qi Juanjuan , Watkins Simon C. , Frizzell Raymond A.
Published: December 2017

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial cell Cl channel, whose gating activity and membrane trafficking are controlled by cAMP/protein kinase A (PKA)-mediated phosphorylation. CFTR Cl currents are regulated also by syntaxin 1A (A. P. Naren, D. J. Nelson, W. W. Xie, B. Jovov, J. Pevsner, M. K. Bennett, D. J. Benos, M. W. Quick, and K. L. Kirk. Nature 390: 302–305, 1997), a protein best known for its role in membrane trafficking and neurosecretion. To examine the mechanism of syntaxin 1A inhibition, we expressed these proteins in Xenopusoocytes and monitored agonist-induced changes in plasma membrane capacitance and cell surface fluorescence of CFTR that contains an external epitope tag. cAMP stimulation elicited large increases in membrane capacitance and in cell surface labeling of flag-tagged CFTR. Coexpression of CFTR with syntaxin 1A, but not syntaxin 3, inhibited cAMP-induced increases in membrane capacitance and plasma membrane CFTR content. Injection of botulinum toxin/C1 rapidly reversed syntaxin’s effects on current and capacitance, indicating that they cannot be explained by an effect on CFTR synthesis. Functional expression of other integral membrane proteins, including Na-coupled glucose transporter hSGLT1, inwardly rectified K channel hIK1, P2Y2 nucleotide receptor, and viral hemagglutinin protein, was not affected by syntaxin 1A coexpression. These findings indicate that acute regulation of the number of CFTR Cl channels in plasma membrane is one mechanism by which cAMP/PKA regulates Cl currents. Inhibition of plasma membrane CFTR content by syntaxin 1A is consistent with the concept that syntaxin and other components of the SNARE machinery are involved in regulated trafficking of CFTR.
MAP kinase cascade is required for p27 downregulation and S phase entry in fibroblasts and epithelial cells
Authors: Rivard Nathalie , Boucher Marie-Josée , Asselin Claude , L’Allemain Gilles
Published: December 2017

The present report delineates the critical pathway in the G1 phase involved in downregulation of p27Kip1, a cyclin-dependent kinase inhibitor, which plays a pivotal role in controlling entry into the S phase of the cell cycle. In resting CCL39 fibroblasts and IEC-6 intestinal epithelial cells, protein levels of p27Kip1 were elevated but dramatically decreased on serum stimulation, along with hyperphosphorylation of pRb and increased CDK2 activity. In both cell types, expression of ras resulted in an increase of basal and serum-stimulated E2F-dependent transcriptional activity and a reduction in p27Kip1 protein levels as well. The role of the mitogen-activated protein (MAP) kinase cascade in p27Kip1 reduction and S phase reentry was reinforced by the blockades of serum-induced E2F-dependent transcriptional activity and p27Kip1 downregulation with the MKK-1/2 inhibitor PD-98059. In both cell lines, downregulation of p27Kip1 was associated with a repression of its synthesis, an event mediated by the p42/p44 MAP kinase pathway. Using an antisense approach, we demonstrated that p27Kip1 may control cell cycle exit in both cell types. These data indicate that activation of the MAP kinase cascade is required for S phase entry and p27Kip1 downregulation in fibroblasts and epithelial cells.
K+/Na+antagonism at cytoplasmic sites of Na+-K+-ATPase: a tissue-specific mechanism of sodium pump regulation
Authors: Therien Alex G. , Blostein Rhoda
Published: December 2017

Tissue-distinct interactions of the Na+-K+-ATPase with Na+ and K+, independent of isoform-specific properties, were reported previously (A. G. Therien, N. B. Nestor, W. J. Ball, and R. Blostein. J. Biol. Chem. 271: 7104–7112, 1996). In this paper, we describe a detailed analysis of tissue-specific kinetics particularly relevant to regulation of pump activity by intracellular K+, namely K+ inhibition at cytoplasmic Na+ sites. Our results show that the order of susceptibilities of α1 pumps of various rat tissues to K+/Na+antagonism, represented by the ratio of the apparent affinity for Na+ binding at cytoplasmic activation sites in the absence of K+ to the affinity constant for K+ as a competitive inhibitor of Na+ binding at cytoplasmic sites, is red blood cell < axolemma ≈ rat α1-transfected HeLa cells < small intestine < kidney < heart. In addition, we have carried out an extensive analysis of the kinetics of K+ binding and occlusion to the cytoplasmic cation binding site and find that, for most tissues, there is a relationship between the rate of K+ binding/occlusion and the apparent affinity for K+ as a competitive inhibitor of Na+activation, the order for both parameters being heart ≥ kidney > small intestine ≈ rat α1-transfected HeLa cells. The notion that modulations in cytoplasmic K+/Na+antagonism are a potential mode of pump regulation is underscored by evidence of its reversibility. Thus the relatively high K+/Na+antagonism characteristic of kidney pumps was reduced when rat kidney microsomal membranes were fused into the dog red blood cell.
Effects of temperature on slow and fast inactivation of rat skeletal muscle Na+channels
Authors: Ruff Robert L.
Published: December 2017

Patch-clamp studies of mammalian skeletal muscle Na+ channels are commonly done at subphysiological temperatures, usually room temperature. However, at subphysiological temperatures, most Na+ channels are inactivated at the cell resting potential. This study examined the effects of temperature on fast and slow inactivation of Na+ channels to determine if temperature changed the fraction of Na+ channels that were excitable at resting potential. The loose patch voltage clamp recorded Na+ currents ( I Na) in vitro at 19, 25, 31, and 37°C from the sarcolemma of rat type IIb fast-twitch omohyoid skeletal muscle fibers. Temperature affected the fraction of Na+ channels that were excitable at the resting potential. At 19°C, only 30% of channels were excitable at the resting potential. In contrast, at 37°C, 93% of Na+ channels were excitable at the resting potential. Temperature did not alter the resting potential or the voltage dependencies of activation or fast inactivation. I Na available at the resting potential increased with temperature because the steady-state voltage dependence of slow inactivation shifted in a depolarizing direction with increasing temperature. The membrane potential at which half of the Na+channels were in the slow inactivated state was shifted by +16 mV at 37°C compared with 19°C. Consequently, the low availability of excitable Na+ channels at subphysiological temperatures resulted from channels being in the slow, inactivated state at the resting potential.
Time-dependent changes in myosin heavy chain mRNA and protein isoforms in unloaded soleus muscle of rat
Authors: Stevens Laurence , Sultan Karim R. , Peuker Heidemarie , Gohlsch Bärbel , Mounier Yvonne , Pette Dirk
Published: December 2017

Time-dependent changes in myosin heavy chain (MHC) isoform expression were investigated in rat soleus muscle unloaded by hindlimb suspension. Changes at the mRNA level were measured by RT-PCR and correlated with changes in the pattern of MHC protein isoforms. Protein analyses of whole muscle revealed that MHCI decreased after 7 days, when MHCIIa had increased, reaching a transient maximum by 15 days. Longer periods led to inductions and progressive increases of MHCIId(x) and MHCIIb. mRNA analyses of whole muscle showed that MHCIId(x) displayed the steepest increase after 4 days and continued to rise until 28 days, the longest time period investigated. MHCIIb mRNA followed a similar time course, although at lower levels. MHCIα mRNA, present at extremely low levels in control soleus, peaked after 4 days, stayed elevated until 15 days, and then decayed. Immunohistochemistry of 15-day unloaded muscles revealed that MHCIα was present in muscle spindles but at low amounts also in extrafusal fibers. The slow-to-fast transitions thus seem to proceed in the order MHCIβ → MHCIIa → MHCIId(x) → MHCIIb. Our findings indicate that MHCIα is transiently upregulated in some fibers as an intermediate step during the transition from MHCIβ to MHCIIa.
Odor suppression of voltage-gated currents contributes to the odor-induced response in olfactory neurons
Authors: Sanhueza Magdalena , Bacigalupo Juan
Published: December 2017

Olfactory chemotransduction involves a signaling cascade. In addition to triggering transduction, odors suppress ion conductances. By stimulating with brief odorant pulses, we observed a current associated with odor-induced suppression of voltage-gated conductances and studied its time dependence. We characterized this suppression current in isolated Caudiverbera caudiverberaolfactory neurons. All four voltage-gated currents are suppressed by odor pulses in almost every neuron, and suppression is caused by odors inducing excitation and by those inducing inhibition, indicating a nonselective phenomenon, in contrast to transduction. Suppression has a 10-fold shorter latency than transduction. Suppression was more pronounced when odors were applied to the soma than to the cilia, opposite to transduction. Suppression was also present in rat olfactory neurons. Furthermore, we could induce it in Drosophila photoreceptor cells, demonstrating its independence from the chemotransduction cascade. We show that odor concentrations causing suppression are similar to those triggering chemotransduction and that both suppression and transduction contribute to the odor response in isolated olfactory neurons. Furthermore, suppression affects spiking, implying a possible physiological role in olfaction.
Actin filament organization is required for proper cAMP-dependent activation of CFTR
Authors: Prat Adriana G. , Cunningham C. Casey , Jackson G. Robert , Borkan Steven C. , Wang Yihan , Ausiello Dennis A. , Cantiello Horacio F.
Published: December 2017

Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(−)]. cAMP stimulation of ABP(−) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl− currents. In ABP(−) cells expressing CFTR, cAMP was also without effect on Cl− conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl− currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(−)/CFTR(+) cells. At the single-channel level, protein kinase A plus ATP activated single Cl−channels only in excised patches from ABP(+)/CFTR(+) cells. Furthermore, filamin alone also induced Cl− channel activity in excised patches of ABP(−)/CFTR(+) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.
Characterization of a phosphorylation event resulting in upregulation of the salivary Na+-K+-2Cl−cotransporter
Authors: Kurihara Kinji , Moore-Hoon Marilyn L. , Saitoh Masato , Turner R. James
Published: December 2017

Previous studies from our laboratory have shown a close correlation between increased Na+-K+-2Cl−cotransporter activity and increased cotransporter phosphorylation after β-adrenergic stimulation of rat parotid acinar cells. We demonstrate here that these effects are paralleled by an increase in the number of high-affinity binding sites for the cotransporter inhibitor bumetanide in membranes prepared from stimulated acini. We also show that the sensitivity of cotransporter fluxes to inhibition by bumetanide is the same in both resting and isoproterenol-stimulated cells, consistent with the hypothesis that β-adrenergic stimulation and the accompanying phosphorylation result in the activation of previously quiescent transporters rather than in a change in the properties of already active proteins. In addition, we demonstrate that the increased phosphorylation on the cotransporter resulting from β-adrenergic stimulation is localized to a 30-kDa phosphopeptide obtained by cyanogen bromide digestion. Immunoprecipitation and Western blotting experiments demonstrate that this peptide is derived from the NH2-terminal cytosolic tail of the cotransporter, which surprisingly does not contain the sole protein kinase A consensus site on the molecule.
Colonic H-K-ATPase α- and β-subunits express ouabain-insensitive H-K-ATPase
Authors: Sangan Pitchai , Thevananther Sundararajah , Sangan Sheela , Rajendran Vazhaikkurichi M. , Binder Henry J.
Published: December 2017

Active K absorption in the rat distal colon is energized by an apical H-K-ATPase, a member of the gene family of P-type ATPases. The H-K-ATPase α-subunit (HKcα) has been cloned and characterized (together with the β-subunit of either Na-K-ATPase or gastric H-K-ATPase) in Xenopus oocytes as ouabain-sensitive86Rb uptake. In contrast, HKcα, when expressed in Sf9 cells without a β-subunit, yielded evidence of ouabain-insensitive H-K-ATPase. Because a β-subunit (HKcβ) has recently been cloned from rat colon, this present study was initiated to determine whether H-K-ATPase and its sensitivity to ouabain are expressed when these two subunits (HKcα and HKcβ) are transfected into a mammalian cell expression system. Transfection of HEK-293 cells with HKcα and HKcβ cDNAs resulted in the expression of HKcα and HKcβ proteins and their delivery to plasma membranes. H-K-ATPase activity was identified in crude plasma membranes prepared from transfected cells and was 1) saturable as a function of increasing K concentration with a K m for K of 0.63 mM; 2) inhibited by orthovanadate; and 3) insensitive to both ouabain and Sch-28080. In parallel transfection studies with HKcα and Na-K-ATPase β1 cDNAs and with HKcα cDNA alone, there was expression of ouabain-insensitive H-K-ATPase activity that was 60% and 21% of that in HKcα/HKcβ cDNA transfected cells, respectively. Ouabain-insensitive 86Rb uptake was also identified in cells transfected with HKcα and HKcβ cDNAs. These studies establish that HKcα cDNA with HKcβ cDNA express ouabain-insensitive H-K-ATPase similar to that identified in rat distal colon.
High glucose and insulin inhibit VSMC MKP-1 expression by blocking iNOS via p38 MAPK activation
Authors: Begum Najma , Ragolia Louis
Published: December 2017

Our laboratory has recently demonstrated a role for the phosphatidylinositol 3-kinase-mediated inducible NO synthase (iNOS) signaling pathway in acute regulation of insulin-induced mitogen-activated protein phosphatase-1 (MKP-1) expression in primary cultures of rat aortic vascular smooth muscle cells (VSMCs) (N. Begum, L. Ragolia, M. McCarthy, and N. Duddy. J. Biol. Chem. 273: 25164–25170, 1998). We now show that prolonged treatment of VSMCs with 100 nM insulin and high glucose (25 mM) for 12–24 h, to mimic hyperinsulinemia and hyperglycemia, completely blocked MKP-1 mRNA and protein expression in response to subsequent acute insulin treatment. To understand the mechanism of insulin resistance induced by high glucose and insulin, we studied the regulation of iNOS protein induction in these cells. Both high glucose and chronic insulin treatment caused a marked impairment of iNOS induction in response to acute insulin. Blocking of signaling via the p38 mitogen-activated protein kinase (MAPK) pathway by prior treatment for 1 h with SB-203580, a synthetic p38 MAPK inhibitor, completely prevented the inhibition of iNOS induced by high glucose and insulin and restored MKP-1 induction to levels observed with acute insulin treatment. In contrast, PD-98059, a MEK inhibitor, had no effect. Furthermore, high glucose and chronic insulin treatment caused sustained p38 MAPK activation. We conclude 1) that chronic insulin and high glucose-induced insulin resistance is accompanied by marked reductions in both iNOS and MKP-1 inductions due to p38 MAPK activation that leads to excessive cell growth and 2) that p38 MAPK/extracellular signal-regulated kinase pathways regulate iNOS induction, thereby controlling MKP-1 expression, which in turn inactivates MAPKs as a feedback mechanism and inhibits cell growth.
Reversal of sexual dimorphism in splenic T lymphocyte responses after trauma-hemorrhage with aging
Authors: Kahlke Volker , Angele Martin K. , Schwacha Martin G. , Ayala Alfred , Cioffi William G. , Bland Kirby I. , Chaudry Irshad H.
Published: December 2017

Previous studies have demonstrated that hemorrhagic shock produces immunodepression in young male mice, whereas the immunoresponsivness in young proestrus female mice is enhanced under such conditions. This sexually dimorphic immune response to hemorrhage appears to be related to high estrogen and testosterone levels in females and males, respectively. Nonetheless, it is unknown what impact the age-related decline in the sex steroid levels has on the immune response after hemorrhage. To study this, young (2–3 mo) and aged (18–19 mo) male and female CBA/J NIA mice were subjected to laparotomy (i.e., soft tissue trauma) and hemorrhage (35 ± 5 mmHg for 90 min and fluid resuscitation) or sham operation. Twenty-four hours later, splenocyte responses were assessed in vitro. Splenic T lymphocyte responses [i.e., proliferation, interleukin-2 (IL-2) and interferon-γ (IFN-γ) release] were depressed in young males and enhanced in young females after trauma-hemorrhage. In contrast, in the aged male and female groups these parameters of splenocyte function were reversed after trauma-hemorrhage (i.e., increased proliferation and IL-2 release in aged males compared with suppressed proliferation and IFN-γ release in aged females). Furthermore, the release of the immunosuppressive cytokine IL-10 inversely correlated with the age- and gender-related changes in splenocyte responses after trauma-hemorrhage. Thus the sexually dimorphic immune response in young males and females to trauma-hemorrhage appears to reverse as sex hormone levels decline with age.
A myosin phosphatase targeting subunit isoform transition defines a smooth muscle developmental phenotypic switch
Authors: Dirksen Wessel P. , Vladic Franjo , Fisher Steven A.
Published: December 2017

Smooth muscle myosin phosphatase dephosphorylates the regulatory myosin light chain and thus mediates smooth muscle relaxation. The activity of this myosin phosphatase is dependent upon its myosin-targeting subunit (MYPT1). Isoforms of MYPT1 have been identified, but how they are generated and their relationship to smooth muscle phenotypes is not clear. Cloning of the middle section of chicken and rat MYPT1 genes revealed that each gene gave rise to isoforms by cassette-type alternative splicing of exons. In chicken, a 123-nucleotide exon was included or excluded from the mature mRNA, whereas in rat two exons immediately downstream were alternative. MYPT1 isoforms lacking the alternative exon were only detected in mature chicken smooth muscle tissues that display phasic contractile properties, but the isoform ratios were variable. The patterns of expression of rat MYPT1 mRNA isoforms were more complex, with three major and two minor isoforms present in all smooth muscle tissues at varying stoichiometries. Isoform switching was identified in the developing chicken gizzard, in which the exon-skipped isoform replaced the exon-included isoform around the time of hatching. This isoform switch occurred after transitions in myosin heavy chain and myosin light chain (MLC17) isoforms and correlated with a severalfold increase in the rate of relaxation. The developmental switch of MYPT1 isoforms is a good model for determining the mechanisms and significance of alternative splicing in smooth muscle.
Mechanisms of Pi regulation of the skeletal muscle SR Ca2+ release channel
Authors: Balog Edward M. , Fruen Bradley R. , Kane Patricia K. , Louis Charles F.
Published: December 2017

Inorganic phosphate (Pi) accumulates in the fibers of actively working muscle where it acts at various sites to modulate contraction. To characterize the role of Pi as a regulator of the sarcoplasmic reticulum (SR) calcium (Ca2+) release channel, we examined the action of Pi on purified SR Ca2+ release channels, isolated SR vesicles, and skinned skeletal muscle fibers. In single channel studies, addition of Pi to the cis chamber increased single channel open probability ( P o; 0.079 ± 0.020 in 0 Pi, 0.157 ± 0.034 in 20 mM Pi) by decreasing mean channel closed time; mean channel open times were unaffected. In contrast, the ATP analog, β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP), enhanced P o by increasing single channel open time and decreasing channel closed time. Pi stimulation of [3H]ryanodine binding by SR vesicles was similar at all concentrations of AMP-PCP, suggesting Pi and adenine nucleotides act via independent sites. In skinned muscle fibers, 40 mM Pi enhanced Ca2+-induced Ca2+ release, suggesting an in situ stimulation of the release channel by high concentrations of Pi. Our results support the hypothesis that Pi may be an important endogenous modulator of the skeletal muscle SR Ca2+ release channel under fatiguing conditions in vivo, acting via a mechanism distinct from adenine nucleotides.
MHC polymorphism in rodent plantaris muscle: effects of mechanical overload and hypothyroidism
Authors: Caiozzo Vincent J. , Haddad Fadia , Baker Mike , McCue Sam , Baldwin Kenneth M.
Published: December 2017

In a previous study, it was shown that a combined treatment of hyperthyroidism and hindlimb suspension effectively converted the slow-twitch soleus muscle to a fast-twitch muscle. The objective of this study was to test the hypothesis that hypothyroidism [absence of triiodothyronine (−T3)] and mechanical overload (OV) would convert the plantaris (Plan) muscle from a fast- to a slow-twitch muscle. Single-fiber analyses demonstrated that the normal rodent Plan muscle was composed of ∼13 different fiber types as defined by myosin heavy chain (MHC) isoform content. The largest proportion of fibers (∼35%) coexpressed the fast type IIX and IIB MHC isoforms (i.e., type IIX/IIB fibers). In this context, the combined intervention of −T3 and OV produced a significant reduction in the relative proportion of the fast type IIB MHC isoform and a concomitant increase in the slow type I MHC isoform. These transitions were manifested by a large decrease in the proportion of type IIX/IIB fibers and a large increase in fibers coexpressing all four MHC protein isoforms. The mechanical consequences of these transitions, however, were modest, producing a 15% decrease in maximal shortening velocity. The findings of this study demonstrate that −T3+ OV does produce a partial shift toward a slower phenotype; however, the high degree of polymorphism found in the Plan muscle represents a unique design that appears to minimize the functional consequences of these significant MHC transitions.
Sorting of metabolic pathway flux by the plasma membrane in cerebrovascular smooth muscle cells
Authors: Lloyd Pamela G. , Hardin Christopher D.
Published: December 2017

We used β-escin-permeabilized pig cerebral microvessels (PCMV) to study the organization of carbohydrate metabolism in the cytoplasm of vascular smooth muscle (VSM) cells. We have previously demonstrated (Lloyd PG and Hardin CD. Am J Physiol Cell Physiol 277: C1250–C1262, 1999) that intact PCMV metabolize the glycolytic intermediate [1-13C]fructose 1,6-bisphosphate (FBP) to [1-13C]glucose with negligible production of [3-13C]lactate, while simultaneously metabolizing [2-13C]glucose to [2-13C]lactate. Thus gluconeogenic and glycolytic intermediates do not mix freely in intact VSM cells (compartmentation). Permeabilized PCMV retained the ability to metabolize [2-13C]glucose to [2-13C]lactate and to metabolize [1-13C]FBP to [1-13C]glucose. The continued existence of glycolytic and gluconeogenic activity in permeabilized cells suggests that the intermediates of these pathways are channeled (directly transferred) between enzymes. Both glycolytic and gluconeogenic flux in permeabilized PCMV were sensitive to the presence of exogenous ATP and NAD. It was most interesting that a major product of [1-13C]FBP metabolism in permeabilized PCMV was [3-13C]lactate, in direct contrast to our previous findings in intact PCMV. Thus disruption of the plasma membrane altered the distribution of substrates between the glycolytic and gluconeogenic pathways. These data suggest that organization of the plasma membrane into distinct microdomains plays an important role in sorting intermediates between the glycolytic and gluconeogenic pathways in intact cells.
Impermeability of the GIRK2 weaver channel to divalent cations
Authors: Hou Ping , Di Anke , Huang Ping , Hansen Charlotte B. , Nelson Deborah J.
Published: December 2017

A single amino acid mutation (G156S) in the putative pore-forming region of the G protein-sensitive, inwardly rectifying K+ channel subunit, GIRK2, renders the conductance constitutively active and nonselective for monovalent cations. The mutant channel subunit (GIRK2 wv) causes the pleiotropic weaver disease in mice, which is characterized by the selective vulnerability of cerebellar granule cells and Purkinje cells, as well as dopaminergic neurons in the mesencephalon, to cell death. It has been proposed that divalent cation permeability through constitutively active GIRK2 wv channels contributes to a rise in internal calcium in the GIRK2 wv-expressing neurons, eventually leading to cell death. We carried out comparative studies of recombinant GIRK2 wv channels expressed in Xenopus oocytes and COS-7 cells to determine the magnitude and relative permeability of the GIRK2 wv conductance to Ca2+. Data from these studies demonstrate that the properties of the expressed current differ in the two systems and that when recombinant GIRK2 wv is expressed in mammalian cells it is impermeable to Ca2+.
Mechanical strain memory in airway smooth muscle
Authors: Chan Wah-Lun , Silberstein Jeanette , Hai Chi-Ming
Published: December 2017

We investigated the effect of a single rapid stretch on poststretch force and myosin phosphorylation in bovine tracheal smooth muscle. When unstimulated muscle strips were stretched from suboptimal length to optimal length ( L o), poststretch steady-state force was not significantly different from that of unstretched control at L o. However, when carbachol-activated muscle strips were stretched from suboptimal length to L o, poststretch force and myosin phosphorylation were lower than control and significantly correlated with initial length. When poststretch muscle strips were allowed to relax for 1 h and then activated by K+ depolarization, the developed force remained significantly correlated with initial length. When the same strain was applied in 23 increments to minimize peak stress, poststretch force and myosin phosphorylation increased significantly, approaching the levels expected at L o. Furthermore, poststretch force development increased after each cycle of contraction and relaxation, approaching the control level after four cycles. These results suggest that activated airway smooth muscle cells can retain relatively precise memory of past strain when they are stretched rapidly with high stress.
KChAP as a chaperone for specific K+channels
Authors: Kuryshev Yuri A. , Gudz Tatyana I. , Brown Arthur M. , Wible Barbara A.
Published: December 2017

The concept of chaperones for K+ channels is new. Recently, we discovered a novel molecular chaperone, KChAP, which increased total Kv2.1 protein and functional channels in Xenopus oocytes through a transient interaction with the Kv2.1 amino terminus. Here we report that KChAP is a chaperone for Kv1.3 and Kv4.3. KChAP increased the amplitude of Kv1.3 and Kv4.3 currents without affecting kinetics or voltage dependence, but had no such effect on Kv1.1, 1.2, 1.4, 1.5, 1.6, and 3.1 or Kir2.2, HERG, or KvLQT1. Although KChAP belongs to a family of proteins that interact with transcription factors, upregulation of channel currents was not blocked by the transcription inhibitor actinomycin D. A 98-amino acid fragment of KChAP binds to the channel and is indistinguishable from KChAP in its enhancement of Kv4.3 current and protein levels. Using a KChAP antibody, we have coimmunoprecipitated KChAP with Kv2.1 and Kv4.3 from heart. We propose that KChAP is a chaperone for specific Kv channels and may have this function in cardiomyocytes where Kv4.3 produces the transient outward current, I to.
Separate entry pathways for phosphate and oxalate in rat brain microsomes
Authors: Meng X.-J. , Timmer R. T. , Gunn R. B. , Abercrombie R. F.
Published: December 2017

ATP-dependent 45Ca uptake in rat brain microsomes was measured in intracellular-like media containing different concentrations of PO4 and oxalate. In the absence of divalent anions, there was a transient 45Ca accumulation, lasting only a few minutes. Addition of PO4did not change the initial accumulation but added a second stage that increased with PO4 concentration. Accumulation during the second stage was inhibited by the following anion transport inhibitors: niflumic acid (50 μM), 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS; 250 μM), and DIDS (3–5 μM); accumulation during the initial stage was unaffected. Higher concentrations of DIDS (100 μM), however, inhibited the initial stage as well. Uptake was unaffected by 20 mM Na, an activator, or 1 mM arsenate, an inhibitor of Na-PO4cotransport. An oxalate-supported 45Ca uptake was larger, less sensitive to DIDS, and enhanced by the catalytic subunit of protein kinase A (40 U/ml). Combinations of PO4 and oxalate had activating and inhibitory effects that could be explained by PO4 inhibition of an oxalate-dependent pathway, but not vice versa. These results support the existence of separate transport pathways for oxalate and PO4 in brain endoplasmic reticulum.
cAMP activates an ATP-permeable pathway in neonatal rat cardiac myocytes
Authors: Lader Alan S. , Xiao Yong-Fu , O'Riordan Catherine R. , Prat Adriana G. , Jackson G. Robert , Cantiello Horacio F.
Published: December 2017

The molecular mechanisms associated with intracellular ATP release by the heart are largely unknown. In this study the luciferin-luciferase assay and patch-clamp techniques were used to characterize the pathways responsible for ATP release in neonatal rat cardiac myocytes (NRCM). Spontaneous ATP release by NRCM was significantly increased after cAMP stimulation under physiological conditions. cAMP stimulation also induced an anion-selective electrodiffusional pathway that elicited linear, diphenylamine-2-carboxylate (DPC)-inhibitable Cl− currents in either symmetrical MgCl2 or NaCl. ATP, adenosine 5′- O-(3-thiotriphosphate), and the ATP derivatives ADP and AMP, permeated this pathway; however, GTP did not. The cAMP-induced ATP currents were inhibited by DPC and glibenclamide and by a monoclonal antibody raised against the R domain of the cystic fibrosis transmembrane conductance regulator (CFTR). The channel-like nature of the cAMP-induced ATP-permeable pathway was also determined by assessing protein kinase A-activated single channel Cl− and ATP currents in excised inside-out patches of NRCM. Single channel currents were inhibited by DPC and the anti-CFTR R domain antibody. Thus the data in this report demonstrate the presence of a cAMP-inducible electrodiffusional ATP transport mechanism in NRCM. Based on the pharmacology, patch-clamping data, and luminometry studies, the data are most consistent with the role of a functional CFTR as the anion channel implicated in cAMP-activated ATP transport in NRCM.
Cellular energetics analysis by a mathematical model of energy balance: estimation of parameters in human skeletal muscle
Authors: Vicini Paolo , Kushmerick Martin J.
Published: December 2017

Cellular energy balance requires that the physiological demands by ATP-utilizing functions be matched by ATP synthesis to sustain muscle activity. We devised a new method of analysis of these processes in data from single individuals. Our approach is based on the logic of current information on the major mechanisms involved in this energy balance and can quantify not directly measurable parameters that govern those mechanisms. We use a mathematical model that simulates by ordinary, nonlinear differential equations three components of cellular bioenergetics (cellular ATP flux, mitochondrial oxidative phosphorylation, and creatine kinase kinetics). We incorporate data under resting conditions, during the transition toward a steady state of stimulation and during the transition during recovery back to the original resting state. Making use of prior information about the kinetic parameters, we fitted the model to previously published dynamic phosphocreatine (PCr) and inorganic phosphate (Pi) data obtained in normal subjects with an activity-recovery protocol using31P nuclear magnetic resonance spectroscopy. The experiment consisted of a baseline phase, an ischemic phase (during which muscle stimulation and PCr utilization occurred), and an aerobic recovery phase. The model described satisfactorily the kinetics of the changes in PCr and Pi and allowed estimation of the maximal velocity of oxidative phosphorylation and of the net ATP flux in individuals both at rest and during stimulation. This work lays the foundation for a quantitative, model-based approach to the study of in vivo muscle energy balance in intact muscle systems, including human muscle.
Acid incubation causes exocytic insertion of NHE3 in OKP cells
Authors: Yang Xiaojing , Amemiya Morimasa , Peng Yan , Moe Orson W. , Preisig Patricia A. , Alpern Robert J.
Published: December 2017

Incubation of opossum kidney clone P (OKP) cells in acid media (pH 6.8) causes activation of Na+/H+ exchanger 3 (NHE3) at 6, 12, and 24 h. OKP cell NHE3 protein abundance was increased by 45% at 24 h of acid incubation but was unaffected at 3–12 h. By contrast, apical membrane NHE3, measured by surface biotinylation, increased approximately twofold at 6, 12, and 24 h, mirroring the increase in activity. Acid incubation caused a 76% increase in exocytic insertion of NHE3 into the apical membrane but had no effect on endocytic internalization at 6 h. Latrunculin B, an inhibitor of microfilament organization, inhibited the acid-induced increases in apical membrane NHE3, exocytic insertion of NHE3, and NHE3 activity at 6 h. These studies demonstrate two mechanisms for acid-induced increases in NHE3 activity. Beginning at 6 h, there is an increase in apical membrane NHE3 that is due to stimulated exocytic insertion and is required for increased NHE3 activity. At 24 h, there is an additional increase in total cellular NHE3.