bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of human endothelial cell apoptosis requires both heat shock and oxidative stress responses
Authors: Wang J. H. , Redmond H. P. , Watson R. W. , Bouchier-Hayes D.
Published: December 2017

Endothelial cell (EC) death may play an important role in the development of increased vascular permeability and capillary leak syndrome during systemic inflammatory response syndrome. However, the mode of EC death and the mechanisms involved remain unclear. In this study we employed the proinflammatory mediators lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha), the chemical reagent sodium arsenite, and heat shock to trigger the stress gene responses. Human ECs were used as surrogates of the microvasculature to test the hypothesis that the induction of the heat shock response and the oxidative stress response might combine to induce apoptosis rather than necrosis in human ECs. Sodium arsenite at 80-320 microM, which induced heat shock protein 72 (HSP72) expression and reactive oxygen intermediate (ROI) generation in ECs, resulted in EC apoptosis. TNF-alpha alone (5-75 ng/ml) increased EC ROI generation but did not induce EC apoptosis. Heat shock alone (42 degrees C, 45 min) or sodium arsenite (40 microM) alone, each of which induced HSP72 expression, did not result in EC apoptosis. However, the combination of TNF-alpha with heat shock or 40 microM sodium arsenite led to EC apoptosis as HSP72 expression and ROI were induced. Furthermore, sodium arsenite (80 microM) in the presence of antioxidants failed to induce EC apoptosis. Apoptotic ECs also exhibited functional disturbances as represented by the depression of intercellular adhesion molecule-1 expression as well as the disruption of EC monolayer integrity. These results indicate that the simultaneous induction of a heat shock response and an oxidative stress response is responsible for human EC apoptosis.
Role of calcium and cross bridges in determining rate of force development in frog muscle fibers
Authors: Wahr P. A. , Rall J. A.
Published: December 2017

The influence of Ca2+ and force-generating cross bridges on the kinetics of force development was examined in skinned frog muscle fibers activated by photolytic release of Ca2+ from caged Ca2+ at 10 degrees C. Isometric force development was fit by a double exponential equation with rates of 44.4 s-1 (kc1) and 6.1 s-1 (kc2); kc1 was not significantly different from the rate of force development observed in intact fibers. Maximum activation by caged Ca2+ from preexisting submaximal force produced rates of contraction similar to those observed with maximum activation from zero force. Decreasing the Ca2+ level to an extent that resulted in 50% of maximum force development produced an approximately sevenfold decrease in kc1 and no change in kc2. Partial extraction of troponin C reduced kc1 only slightly (by 16%), whereas decreasing the number of force-generating cross bridges by vanadate did not decrease kc1. Neither treatment altered kc2. Thus the rate of force development increases dramatically with increases in Ca2+ level.
Distinct effect of hypoxia on endothelial cell proliferation and cycling
Authors: Tucci M. , Hammerman S. I. , Furfaro S. , Saukonnen J. J. , Conca T. J. , Farber H. W.
Published: December 2017

Endothelial cells (EC) occupy a strategic location in the vasculature as a barrier between the intravascular compartment and underlying tissues; as such, they are often exposed to stresses, such as decreases in ambient oxygen, diminished metabolic substrate, or changes in temperature, that could affect their ability to divide and proliferate. The present study characterizes cell counts, cell cycle distribution, and bromodeoxyuridine incorporation in pulmonary artery and aortic EC exposed to acute and/or chronic hypoxia and other cellular stresses. During hypoxia, EC division slows but does not arrest; progression through the G1-to-S transition point and/or progression from S to G2/M is altered with an increased percent of EC in S phase. These changes in EC cell cycle distribution with hypoxia are dependent on the origin of the EC as well as the ambient oxygen concentration; moreover, they are distinct from changes observed with elevated temperature or glucose deprivation. and differ from the quiescent pattern induced by serum deprivation or high-density confluence. These findings demonstrate that hypoxia exerts a distinct effect on the cell cycle distribution and proliferation of EC.
Delivery of newly synthesized Na(+)-K(+)-ATPase to the plasma membrane of A6 epithelia
Authors: Coupaye-Gerard B. , Zuckerman J. B. , Duncan P. , Bortnik A. , Avery D. I. , Ernst S. A. , Kleyman T. R.
Published: December 2017

Na(+)-K(+)-ATPase is localized to the basolateral cell surface of most epithelial cells. Conflicting results regarding the intracellular trafficking of Na(+)-K(+)-ATPase in Madin-Darby canine kidney cells have been reported, with delivery to both apical and basolateral membranes or exclusively to the basolateral cell surface. We examined the delivery and steady-state distribution of Na(+)-K(+)-ATPase in the amphibian epithelial cell line A6 using an antibody raised against Na(+)-K(+)-ATPase alpha-subunit and sulfo-N-hydroxysuccinimidobiotin to tag cell surface proteins. The steady-state distribution of the Na(+)-K(+)-ATPase was basolateral, as confirmed by immunocytochemistry. Delivery of newly synthesized Na(+)-K(+)-ATPase to the cell surface was examined using [35S]methionine and [35S]cysteine in a pulse-chase protocol. After a 20-min pulse, the alpha-subunit and core glycosylated beta-subunit were present at both apical and basolateral cell surfaces. The alpha-subunit and core glycosylated beta-subunit delivered to the apical cell surface were degraded within 2 h. Mature alpha/beta-heterodimer was found almost exclusively at the basolateral surface after a 1- to 24-h chase. These data suggest that immature Na(+)-K(+)-ATPase alpha-subunit and core glycosylated beta-subunits are not retained in the endoplasmic reticulum of A6 cells and apparently lack sorting signals. Mature Na(+)-K(+)-ATPase is targeted to the basolateral surface, suggesting that basolateral targeting of the protein is conformation dependent.
Volume regulation in NIH/3T3 cells not expressing P-glycoprotein. I. Regulatory volume decrease
Authors: Pasantes-Morales H. , Sanchez Olea R. , Miranda D. , Moran J.
Published: December 2017

Exposure of NIH/3T3 fibroblasts not expressing P-glycoprotein to 50, 30, 20, and 10% hyposmotic solutions led to cell volume increases of 70, 32, 21, and 12%, respectively. After swelling, NIH/3T3 cells exhibited regulatory volume decrease (RVD), attaining complete volume recovery after 30 min except in 50% hyposmotic solution, in which volume recovery was 76%. RVD was accelerated by gramicidin and inhibited by the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid and by the K channel, blocker quinidine. RVD was reduced 15% by removal of extracellular Ca. The pathway opened by hypotonicity was highly permeable to K and Rb and only partly permeable to other cations. Most anions were able to permeate, with a permeability ranking of nitrate > benzoate = iodide > thiocyanate > chloride > > gluconate. The pathway was permeable to neutral amino acids, with a permeability ranking of glycine > alanine > glutamate > taurine > gamma-aminobutyric acid > glutamine. The pathway was not permeable to basic amino acids. These results show that, despite the absence of P-glycoprotein, NIH/3T3 cells exhibit RVD with properties similar to those expressed in most cell types.
Heterologous desensitization of smooth muscle to K+ depolarization: retarded stimulus-[Ca2+]i coupling
Authors: Wardle R. L. , Strauss J. D. , Rembold C. M. , Murphy R. A.
Published: December 2017

TTo understand the phenomenon of postreceptor heterologous desensitization, we exposed porcine carotid media to 40 mM KCl physiological saline solution both before and after intervening treatment with histamine. Increasing histamine concentration or duration of exposure or decreasing the interval between histamine exposure and KCl progressively slowed the contractile responses to K+ depolarization. A delay in initiation and a slower rate of rise of KCl-induced stress in histamine-pretreated muscle were preceded by a slower rate of rise of aequorin-estimated myoplasmic Ca2+ concentration ([Ca2+]i), myosin regulatory light chain (MRLC) phosphorylation, and tissue stiffness, with no detectable change in the Ca2+ sensitivity of MRLC phosphorylation. This heterologous desensitization was not a diminished steady-state force but instead a profound slowing of contraction rates. This slowing was a manifestation of retardation of the rate at which [Ca2+]i rises to the level appropriate for the stimulus. The lack of rapid initial [Ca2+]i and cross-bridge phosphorylation transients as a consequence of histamine pretreatment resulted in very slow cross-bridge cycling rates and rates of force development (latch).
Immunolocalization of the ubiquitin-protein ligase Nedd4 in tissues expressing the epithelial Na+ channel (ENaC)
Authors: Staub O. , Yeger H. , Plant P. J. , Kim H. , Ernst S. A. , Rotin D.
Published: December 2017

The epithelial Na+ channel (ENaC) was previously shown to be expressed in several Na(+)- and fluid-absorbing epithelia, particularly those of the kidney, colon, and lung. We have recently identified the ubiquitin-protein ligase Nedd4 as an interacting protein with ENaC and demonstrated that Nedd4 binds by its WW domains to the proline-rich PY motifs of ENaC. These PY motifs were recently shown to be deleted/mutated in patients afflicted with Liddle's syndrome, a hereditary form of systemic renal hypertension. Such mutations cause elevated channel activity by an increase in channel number/stability at the plasma membrane and by increased channel opening. We then proposed that Nedd4, by regulating channel stability/ degradation, may be a suppressor of ENaC. To test whether Nedd4 is localized to those tissues/regions that express ENaC, we performed immunocytochemical analysis of rat Nedd4 (rNedd4) distribution in rat kidney, colon, and lung tissues. Our results show that, in the kidney, rNedd4 is primarily localized to the cortical collecting tubules and outer and inner medullary collecting ducts. These tubular segments were previously shown to express ENaC. The epithelium lining medullary calyxes was also intensely stained, and microvillar borders of proximal convoluted tubules expressed variable amounts of rNedd4. In the lung, rNedd4 was mainly expressed in the epithelia lining the airways, in the submucosal glands and ducts, and in the distal respiratory epithelium. These sites resemble the pattern of ENaC expression. In contrast, in the distal colon, rNedd4 was strongly expressed in the epithelia lining the crypts but not in the ENaC-expressing surface epithelium. Low-salt diet (to elevate serum aldosterone levels) had no effect on rNedd4 distribution in the kidney or colon. Thus Nedd4 is coexpressed and likely colocalizes with ENaC in specific regions within the kidney and lung but not in the colon. We speculate this difference in colocalization may reflect differences in the regulation of channel stability in those tissues.
Blocking ATP-sensitive K+ channel during metabolic inhibition impairs muscle contractility
Authors: Gramolini A. , Renaud J. M.
Published: December 2017

The objectives of this study were to determine the metabolic conditions in which ATP-sensitive K+ channels (K+(ATP) channels) contribute to a decrease in force. Sartorius muscles of the frog Rana pipiens were subjected to a 60-min metabolic inhibition by exposing them to cyanide (2 mM) and iodoacetate (1 mM). Muscles were exposed to glibenclamide (100 microM) to block K+ATP channels either 60 min before or 8 or 18 min into metabolic inhibition. Resting potentials, action potentials, and membrane conductance were measured using intracellular microelectrodes. Tetanic and resting tension were measured with a force transducer. ATP, ADP, and phosphocreatine (PCr) were measured by high-pressure liquid chromatography. Glibenclamide completely blocked the shortening of action potential but only partially blocked the increase in membrane conductance. When glibenclamide was added 60 min before metabolic inhibition, the decrease in tetanic force was faster than in control muscle (no glibenclamide). This faster decrease in tetanic force was associated with significant membrane depolarizations, greater increases in resting tension, greater depletions of ATP and PCr contents, and greater increases in ADP content. Addition of glibenclamide 8 min into metabolic inhibition caused an increase in tetanic force followed by a faster decrease compared with control. Addition of glibenclamide 18 min into metabolic inhibition had no effect on the tetanic force compared with control muscles. The data indicate that K+ATP channels 1) were activated during metabolic inhibition and 2) contributed to the decrease in tetanic force but also 3) had a myoprotective effect protecting skeletal muscle against muscle function impairment.
Regulation of branched-chain ketoacid dehydrogenase flux by extracellular pH and glucocorticoids
Authors: Wang X. , Jurkovitz C. , Price S. R.
Published: December 2017

In muscles of rats with metabolic acidosis, branched-chain alpha-ketoacid dehydrogenase (BCKAD) activity is increased. Potential stimulatory signals include acidemia and/or glucocorticoids. It is unclear whether the signal(s) increases BCKAD activity by changing the activation state of the enzyme or by increasing the amount of enzyme. To separate the influences of extracellular pH and glucocorticoids on leucine catabolism, maximal BCKAD flux and the activation state (the ratio of basal to total flux) were measured in two cell types: 1) cells that do not express glucocorticoid receptors and 2) cells stably transfected to express glucocorticoid receptors. Acidification (pH 6.95) increased 1) the activation state from 67.2% at pH 7.4 to 82.8% at pH 6.95, 2) maximal BCKAD flux by 50%, and 3) the BCKAD subunit contents in both cell types (57, 410, and 270% for E2, E1 alpha, and E1 beta, respectively). Dexamethasone increased the BCKAD activation state from 67.2 to 82.3% in cells expressing glucocorticoid receptors, whereas dexamethasone plus acidification increased the activation state to 98%. The time course of stimulation by dexamethasone was slower than that by acidification. These results demonstrate that BCKAD is differentially regulated by extracellular pH and glucocorticoids.
Cyanide suppression of inwardly rectifying K+ channels in guinea pig chromaffin cells involves dephosphorylation
Authors: Inoue M. , Sakamoto Y. , Yano A. , Imanaga I.
Published: December 2017

Treatment of chromaffin cells with cyanide induced a gradual decrease in an inwardly rectifying K+ current (IIR), and washout of the mitochondrial inhibitor resulted in a rapid recovery of IIR. This diminution of IIR was reversed in a time-dependent manner by infusion of ATP or UTP, but not by that of GTP, ITP, or CTP. The restoration by ATP was not altered by addition to the pipette solution of 50 microM fluorescein 5-isothiocyanate, an inhibitor of various ATPases. A similar recovery of IIR occurred with injection of adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), but not of 5'-adenylylimidodiphosphate or alpha,beta-methyleneadenosine 5'-triphosphate. The ATP gamma S effect was biphasic, resulting in first a run-up of the current in ATP-depleted cells followed by a rundown of the current. This rundown was almost abolished by addition of guanosine 5'-O-(2-thiodiphosphate) to the ATP gamma S solution, suggesting the involvement of a G protein. Bath application of the protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine at 100 microM, but not N-(2-[methylamino]-ethyl)-5-isoquinolinesulfonamide, induced a reversible inhibition of IIR in the presence of pipette ATP, and the inhibition was diminished by 1 microM calyculin A, a phosphatase inhibitor. Bath application of 1 microM phorbol 12,13-dibutyrate did not affect IIR. It is concluded that cyanide suppresses inward rectifier K+ channel activity via dephosphorylation and that protein kinase C, adenosine 3',5'-cyclic monophosphate-dependent kinase, or guanosine 3',5'-cyclic monophosphate-dependent kinase is not involved in modulation of the channel.
Early functional and biochemical adaptations to low-frequency stimulation of rabbit fast-twitch muscle
Authors: Hicks A. , Ohlendieck K. , Gopel S. O. , Pette D.
Published: December 2017

To examine mechanisms underlying force reduction after the onset of chronic low-frequency (10 Hz) stimulation (CLFS), we exposed rabbit tibialis anterior muscles to various durations of CLFS. To follow changes in isometric contractile properties and electromyographic (EMG) activity, we studied stimulated and contralateral muscles during a terminal test at 10 Hz for 10 min. In addition, activities and protein amounts of the sarcoplasmic reticulum Ca(2+)-ATPase, content of Na(+)-K(+)-ATPase, and expression patterns of triad junction components were examined. Force output and EMG amplitude declined abruptly soon after the onset of stimulation, suggesting refractoriness of a large fiber population. Although twitch force and to a lesser extent EMG activity gradually recovered after stimulation for 6 days and longer, the muscles exhibited profoundly altered properties, i.e., enhanced fatigue resistance, absence of twitch potentiation, and prolonged contraction and relaxation times. These changes were associated with significant increases in Na(+)-K(+)-ATPase concentration and significant decreases in Ca(2+)-ATPase, ryanodine receptor, dihydropyridine receptor, and triadin concentrations over the course of the 20 days of stimulation. Alterations in excitability, Ca2+ handling, and excitation-contraction coupling prior to changes in myofibrillar protein isoforms may thus be responsible for early functional alterations.
Inhibition of polyamine synthesis influences contractility of intestinal smooth muscle in culture
Authors: Sward K. , Nilsson B. O. , Hellstrand P.
Published: December 2017

Smooth muscle strips from guinea pig ileum were cultured for 5 days and then tested for contractile properties to investigate whether endogenous polyamines influence excitation-contraction coupling. Inhibition of spermidine and spermine synthesis by culture in the presence of the adenosylmethionine decarboxylase (EC4.1.1.50) inhibitor CGP-48664 (1-10 microM) decreased spermidine and spermine levels by 50% and increased putrescine by 20-fold. After culture with 10 microM, but not 1 microM, CGP-48664, the relationship between extracellular Ca2+ concentration and force in high K(+)-depolarized strips was shifted to the right, and phasic contractile activity as well as sensitivity to muscarinic stimulation was enhanced. When spermidine and spermine (each 50 microM) were available for cellular uptake during culture in the presence of 10 microM CGP-48664, spermidine and spermine concentrations were increased, and the effect on Ca2+ sensitivity was reversed. In strips cultured with 0 or 1 microM CGP-48664 in the presence of 50 microM spermidine and 50 microM spermine, no effect on Ca2+ sensitivity was observed. Force development relative to intracellular Ca2+ concentration was decreased in CGP-48664 (10 microM)-treated strips. The results suggest that endogenous polyamines influence excitation-contraction coupling in smooth muscle, although overall tissue concentrations may not reflect the polyamine pools responsible for this effect.
Characterization of the human pH- and PKA-activated ClC-2G(2 alpha) Cl- channel
Authors: Sherry A. M. , Stroffekova K. , Knapp L. M. , Kupert E. Y. , Cuppoletti J. , Malinowska D. H.
Published: December 2017

A ClC-2G(2 alpha) Cl- channel was identified to be present in human lung and stomach, and a partial cDNA for this Cl- channel was cloned from a human fetal lung library. A full-length expressible human ClC-2G(2 alpha) cDNA was constructed by ligation of mutagenized expressible rabbit ClC-2G(2 alpha) cDNA with the human lung ClC-2G(2 alpha) cDNA, expressed in oocytes, and characterized at the single-channel level. Adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) treatment increased the probability of opening of the channel (Po). After PKA activation, the channel exhibited a linear (r = 0.99) current-voltage curve with a slope conductance of 22.1 +/- 0.8 pS in symmetric 800 mM tetraethylammonium chloride (TEACl; pH 7.4). Under fivefold gradient conditions of TEACl, a reversal potential of +21.5 +/- 2.8 mV was measured demonstrating anion-to-cation discrimination. As previously demonstrated for the rabbit ClC-2G(2 alpha) Cl- channel, the human analog, hClC-2G(2 alpha), was active at pH 7.4 as well as when the pH of the extracellular face of the channel (trans side of the bilayer; pHtrans) was asymmetrically reduced to pH 3.0. The extent of PKA activation was dependent on pHtrans. With PKA treatment, Po increased fourfold with a pHtrans of 7.4 and eightfold with a pHtrans of 3.0. Effects of sequential PKA addition followed by pHtrans reduction on the same channel suggested that the PKA- and pH-dependent increases in channel Po were separable and cumulative. Northern analysis showed ClC-2G(2 alpha) mRNA to be present in human adult and fetal lung and adult stomach, and quantitative reverse transcriptase-polymerase chain reaction showed this channel to be present in the adult human lung and stomach at about one-half the level found in fetal lung. The findings of the present study suggest that the ClC-2G(2 alpha) Cl- channel may play an important role in Cl- transport in the fetal and adult human lung.
Myosin heavy chain gene expression in neonatal rat heart cells: effects of [Ca2+]i and contractile activity
Authors: Qi M. , Puglisi J. L. , Byron K. L. , Ojamaa K. , Klein I. , Bers D. M. , Samarel A. M.
Published: December 2017

To determine if mechanical signals or alterations in intracellular Ca2+ concentration ([Ca2+]i) affect myosin heavy chain (MHC) gene expression in spontaneously beating, neonatal rat ventricular myocytes, contractile activity was inhibited with verapamil, KCl, or 2,3-butanedione monoxime (BDM), and their acute and chronic effects on myocyte shortening, [Ca2+]i, and MHC gene expression were examined. Despite their differing effects on [Ca2+]i, verapamil, KCl, and BDM all inhibited contractile activity and markedly downregulated beta-MHC mRNA levels to 24 +/- 5, 21 +/- 7, and 6 +/- 2% of contracting cells, respectively. In contrast, these inhibitors of contraction upregulated alpha-MHC mRNA levels to 163 +/- 19, 156 +/- 7, and 198 +/- 20% of contracting cells, respectively. Transient transfection with a rat beta-MHC promoter-luciferase expression plasmid demonstrated that all inhibitors of contraction significantly decreased beta-MHC promoter activity. Paradoxically, contractile arrest also inhibited alpha-MHC promoter activity, suggesting that increased alpha-MHC mRNA levels resulted from posttranscriptional mechanisms. Actinomycin D mRNA stability assays indicated that alpha-MHC mRNA half-life was prolonged in noncontracting cells (33 h) compared with contracting myocytes (14 h). Contraction-dependent alterations in MHC gene expression were not dependent on release of angiotensin II or other growth factors into the culture medium. Thus intrinsic mechanical signals rather than alterations in [Ca2+]i regulate alpha-MHC and beta-MHC gene expression by both transcriptional and posttranscriptional mechanisms.
Cystic fibrosis affects specific cell type in sweat gland secretory coil
Authors: Reddy M. M. , Bell C. L. , Quinton P. M.
Published: December 2017

The sweat gland has three distinct cell types: a myoepithelial (ME) cell, a beta-adrenergic-insensitive (beta-I) cell, and a beta-adrenergic-sensitive (beta-S) cell. Using intracellular microelectrodes, we sought to functionally identify the specific cell type(s) affected in cystic fibrosis (CF). We found that in CF secretory coils 1) the ME calls are unaffected, as indicated by normal cell membrane potentials and spontaneous and cholinergically induced depolarizing potentials, 2) the beta-I cells showed normal physiological properties, including a relatively smaller cell membrane potential (approx -25 mV) and a Ba(2+)-inhibitable cholinergic response, and, in contrast, 3) the beta-S cell is abnormal, as shown by the lack of a beta-adrenergically activated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- conductance (GCl). Lack of CFTR GCl in this cell type does not affect either the magnitude of cell membrane potential (approx -56 mV) or the relative cell membrane GCl or the cholinergic response, as compared with that of normal beta-S cells. We conclude that, of the three cell types in secretory coil, only the beta-S cell is specifically affected in the CF secretory tissue of the human sweat gland.
Substrate-dependent expression of Na+ transport and shunt conductance in A6 epithelia
Authors: Helman S. I. , Liu X.
Published: December 2017

A6 epithelia grown in tissue culture vary enormously in their baseline rates of Na+ transport due to differences in growth media, serum, and other unknown factors. To evaluate the effect(s) of substrates on expression of Na+ transport, we determined short-circuit currents, open-circuit voltages, and electrical resistances of mature confluent A6 epithelia grown on a variety of commercially available permeable supports. Because the cells, growth conditions, and all other factors were the same, differences in transport could be attributed alone to the substrate on which the cells were grown. Tissues were grown on both large- and small-diameter inserts of the same type with differing ratios of edge length to area so that the contribution of the edge and tight junction conductances to the combined shunt conductance of the inserts could be evaluated. Shunt and cellular conductances and the cellular Thevenin electromotive force were determined after aldosterone stimulation and amiloride inhibition of Na+ transport. Marked and extreme differences were observed not only for expression of Na+ transport (controls, 0.09-3.94 microA/cm2; aldosterone, 1.53-28.2 microA/cm2) due to changes of apical membrane conductance but also for the development of junctional conductances (3,250 to < infinity omega.cm2) and edge conductances (13,175 to < infinity omega.cm) among substrates.
Regulation of native Kv1.3 channels by cAMP-dependent protein phosphorylation
Authors: Chung I. , Schlichter L. C.
Published: December 2017

We present evidence that activity of native Kv1.3 channels in human T lymphocytes can be increased by inhibiting phosphatases [using okadaic acid (OA)] or by activating protein kinase A (PKA). OA increased the maximal conductance (Gmax) by 40% and shifted the voltage dependence of activation and inactivation, resulting in a significant increase in window current around the normal membrane potential. PKA inhibition [using the PKA inhibitor peptide PKI-(5-24)] decreased Gmax by 43%, whereas PKA activation [by the Sp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Sp-cAMPS)] increased Gmax by 60% and shifted the inactivation curve, producing an increase in the window current. These results are consistent with our previously published work using cell-attached patches but differ from some studies of Kv1.3. Because we previously reported a similar upregulation by protein kinase C (PKC) activation in these cells, we tested whether the PKA and PKC effects were additive. Our results suggest that PKC-dependent phosphorylation acts as a master switch, inasmuch as calphostin C greatly inhibited the current even after Sp-cAMPS, OA, or PKC activation was used to increase protein phosphorylation. Inasmuch as phosphorylation by both kinases (phorbol ester followed by Sp-cAMPS) abrogated the effects of either kinase alone, our results support the view that Kv1.3 is regulated in a complex manner by serine/threonine phosphorylation.
Serum transcriptionally regulates Na(+)-K(+)-ATPase gene expression in vascular smooth muscle cells
Authors: Nemoto J. , Muto S. , Ohtaka A. , Kawakami K. , Asano Y.
Published: December 2017

The present study was designed to examine the effects of serum on Na(+)-K(+)-ATPase alpha 1- and beta 1-subunit gene expression in cultured vascular smooth muscle cells (VSMC) from rat thoracic aortas. Addition of 10% serum to VSMC for 24 h increased Na(+)-K(+)-ATPase activity 1.5-fold and alpha 1- and beta 1-subunit protein levels 1.9-fold. Serum (10%) caused a 3.5-fold increase in alpha 1-mRNA levels and a 6.7-fold increase in beta 1-mRNA levels, with peak elevations at 12 h. The protein synthesis inhibitor cycloheximide abolished serum-mediated beta 1-mRNA induction but did not affect serum-mediated alpha 1-mRNA induction. Protein kinase C (PKC) inhibitors (staurosporine A or calphostin C) or tyrosine kinase (TK) inhibitors (genistein or herbimycin A) significantly reduced serum-mediated beta 1-mRNA induction but had no effect on serum-mediated alpha 1-mRNA induction. Transfection experiments with the 5'-flanking sequences of the alpha 1- or beta 1-subunit genes linked to the luciferase reporter gene revealed that 10% serum caused 2.8- and 6.5-fold increases in luciferase activity, respectively. Among growth factors, only basic fibroblast growth factor (FGF) enhanced luciferase activities for the alpha 1- and beta 1-subunit genes. We conclude that 1) serum stimulates alpha 1- and beta 1-mRNA expression, alpha 1- and beta 1-subunit protein accumulation, and Na(+)-K(+)-ATPase activity; 2) serum-mediated beta 1-mRNA induction partly requires de novo synthesis of intermediate regulatory proteins and activation of PKC and TK, whereas serum-mediated alpha 1-mRNA induction occurs through PKC- and TK-independent mechanisms; 3) the 5'-flanking regions of the alpha 1- and beta 1-subunit genes are serum responsive; and 4) FGF mimics stimulatory effects of serum on promoter activities for the alpha 1- and beta 1-subunit genes.
Upregulation of P2Y2 nucleotide receptors in rat salivary gland cells during short-term culture
Authors: Turner J. T. , Weisman G. A. , Camden J. M.
Published: December 2017

In contrast to the widespread expression of G protein-coupled P2Y2 receptors for extracellular nucleotides in permanent cell lines of salivary gland origin, there is less evidence for robust P2Y2 receptor activity in normal rat salivary gland cells assayed immediately after isolation. We examined the effect of short-term culture (3 h to 6 days) of normal rat submandibular gland (SMG) cells on P2Y2 receptor activity and mRNA expression. Results indicate that increases in the intracellular free Ca2+ concentration in SMG cells in response to the P2Y2 receptor agonist UTP (100 microM) were detectable after 3 h in culture and that after 3 days in culture the magnitude of the response to UTP was similar to that obtained with maximal muscarinic cholinoceptor activation. The Ca2+ mobilization response exhibited the pharmacological profile (UTP = ATP > 2-methylthioadenosine 5'-triphosphate) typical of the P2Y2 receptor subtype and was accompanied by enhanced production of inositol phosphates, reflecting the activation of phospholipase C ubiquitously associated with P2Y2 receptors. The time-dependent increase in P2Y2 receptor activity was accompanied by an increase in the steady-state level of P2Y2 receptor mRNA, as assessed by reverse transcription-polymerase chain reaction. Other studies revealed that the increased P2Y2 receptor activity was independent of cell proliferation, was similar in serum-containing and defined culture media, and was blocked by inhibitors of transcription and translation. Upregulation of the P2Y2 receptor was observed in both acinar cell- and ductal cell-enriched cultures of the SMG and in cells isolated from rat parotid and sublingual glands but not in cells isolated from the pancreas. These in vitro results were complemented by in vivo studies in which P2Y2 receptor activity and mRNA levels were increased in SMG after ligation of the main excretory duct but were not increased in the contralateral, nonligated gland. These findings suggest that changes in the expression and activity of the P2Y2 receptor in salivary gland cells may be related to pathological challenges to the gland in vivo.
Purinergic agonists, but not cAMP, stimulate coupled granule fusion and Cl- conductance in HT29-Cl.16E
Authors: Guo X. W. , Merlin D. , Laboisse C. , Hopfer U.
Published: December 2017

Cl- conductance and capacitance were simultaneously measured in the mucin-secreting cell line, HT29-Cl.16E (Cl.16E), and its sister cell line, HT29-Cl.19A (Cl.19A), which lacks mucin granules. Purinergic stimulation by extracellular ATP transiently increased Cl- conductance in both cell lines with similar peak increases of 0.92 and 1.00 nS/pF in Cl.16E and Cl.19A cells, respectively (baseline of 0.08 nS/pF). Cell capacitance increased only in Cl.16E cells (17% above baseline of 22 pF in Cl.16E and 1% above baseline of 18 pF in Cl.19A cells). Wortmannin inhibited the purinergically activated Cl- conductance and capacitance increases in Cl.16E by 50 and 80%, respectively, but had no effects in Cl.19A cells. In Cl.16E cells, adenosine 3',5'-cyclic monophosphate (cAMP) signaling increased Cl- conductance from 0.08 to 0.52 nS/pF without changing capacitance. Cl- secretion in Cl.16E monolayers was additive in response to supramaximal stimulation of purinergic receptors and adenylyl cyclase, even though granule fusion is nine times greater with purinergic than adenylyl cyclase stimulation. In conclusion, 1) wortmannin does not directly inhibit activation of Cl- conductance, 2) at least 50% of purinergically activated Cl- conductance in Cl.16E is associated with granule fusion, and 3) cAMP-activated Cl- conductance is not associated with granules.
Muscarinic receptor stimulation modulates the effect of halothane on Mn2+ influx in airway smooth muscle
Authors: Warner D. O. , Jones K. A. , Lorenz R. R. , Pabelick C. M.
Published: December 2017

Prior studies suggest that the mechanism of action by which halothane relaxes airway smooth muscle depends on the contractile state of the cell. We hypothesized that halothane would inhibit the influx of Ca2+ into canine airway smooth muscle cells during submaximal, but not maximal, muscarinic stimulation. This hypothesis was tested by using the rate of quenching of fura 2 fluorescence by Mn2+ in strips of canine tracheal smooth muscle as an index of Ca2+ influx. Acetylcholine (ACh) produced a dose-dependent increase in Mn2+ influx. Halothane (0.64 +/- 0.05 microM) significantly decreased Mn2+ influx and intracellular Ca2+ concentration when added to strips stimulated with a submaximal concentration of ACh (0.3 microM) but had no effect on Mn2+ influx or intracellular Ca2+ concentration during maximal stimulation with ACh (100 microM). Similar results were observed when the strips were treated with verapamil. These results demonstrate that anesthetic effects on Ca2+ homeostasis in intact canine tracheal smooth muscle cells may be critically modulated by receptor-linked mechanisms.
Regulation of apolipoprotein A-I gene expression in Hep G2 cells depleted of Cu by cupruretic tetramine
Authors: Wu John Y. J. , Zhang Jin J. , Wang Yenshen , Reaves Scott K. , Wang Yi Ran , Lei Polin P. , Lei Kai Y.
Published: December 2017

Studies were designed to examine the regulation of apolipoprotein (apo) A-I gene expression in Cu-depleted Hep G2 cells. The cupruretic chelator N, N′-bis(2-aminoethyl)-1,3-propanediamine ⋅ 4 HCl (2,3,2-tetramine or TETA) was used to maintain a 77% reduction in cellular Cu in Hep G2 cells. After two passages of TETA treatment, the relative abundance of apoA-I mRNA was elevated 52%. In TETA-treated cells, the rate of apoA-I mRNA decay measured by an actinomycin D chase study was accelerated 108%, and the synthesis of apoA-I mRNA determined by a nuclear runoff assay was enhanced 2.5-fold in TETA-treated cells. All of those changes could be reverted toward the control values with Cu supplementation for only 2 days. In transient transfection assays, a 26.7% increase in chloramphenicol O-acetyltransferase (CAT) activity for the reporter construct −256AI-CAT was observed in the treated cells. However, the ability of apoA-I regulatory protein 1 (ARP-1) to repress the CAT activity was not affected by the depressed Cu status. In addition, gel retardation experiments demonstrated that Cu depletion enhanced the binding of hepatocyte nuclear factor 4 (HNF-4) and other undefined nuclear factors to oligonucleotides containing site A, one of three regulatory sites of the apoA-I gene promoter. Moreover, the relative abundance of HNF-4 mRNA was increased 58% in the Cu-depleted cells. Thus the observed increase in apoA-I gene transcription may be mediated mostly by an elevated level of the regulatory factor, HNF-4. In summary, the present findings established the mechanism by which a depressed cellular Cu status can enhance apoA-I mRNA production and subsequently increase apoA-I synthesis.
Gene expression of natriuretic peptide receptors in rats with DOCA-salt hypertension
Authors: Nuglozeh Edem , Mbikay Majambu , Stewart Duncan J. , Legault Louis
Published: December 2017

In our previous studies, we found that the atrial natriuretic peptide (ANP) binding and guanylyl cyclase activity of A-type natriuretic peptide receptors (NPR-A) were upregulated in renal papillae but downregulated in vascular tissues and glomeruli of rats with deoxycorticosterone acetate (DOCA)-salt hypertension [E. Nuglozeh, G. Gauquelin, R. Garcia, J. Tremblay, and E. L. Schiffrin. Am. J. Physiol. 259 ( Renal Fluid Electrolyte Physiol. 28): F130–F137, 1990]. To further understand the molecular significance of these regulations, we measured the relative abundance of the transcripts of NPR-A and NPR-B by Northern blot in the aorta, mesenteric arteries, adrenal cortex, renal papillae, and lungs in DOCA-salt hypertensive and control rats. In renal papillae we also examined the translation and transcription of NPR-A by ribosome loading and run-on assay. Compared with controls, the steady-state levels of mRNA for NPR-A were increased in the aorta and mesenteric arteries but were decreased in the adrenal cortex and renal papillae in DOCA-salt-treated rats. NPR-B mRNA was decreased in the aorta, mesenteric arteries, and adrenal cortex in hypertensive rats. In lungs the mRNA for both receptors was unchanged. Translation of NPR-A mRNA, as assessed by ribosome loading, was reduced in renal papillae. Transcriptional activity of its gene was not detectable in these tissues. Guanosine 3′,5′-cyclic monophosphate levels generated by NPR-A in renal papillae and by NPR-A and NPR-B in the adrenal cortex, aorta, and mesenteric arteries of DOCA-salt-treated rats remained increased in hypertension. The higher NPR-A activity in the presence of a lower level of its mRNA in renal papillae and the higher NPR-B activity in the presence of a lower level of its mRNA in the vasculature, adrenal cortex, and lungs can alternatively be explained by receptor stabilization or increased receptor recycling.
Prostaglandin H synthase: protein synthesis-independent regulation in bovine aortic endothelial cells
Authors: Rosenstock Moti , Danon Abraham , Rimon Gilad
Published: December 2017

The objective of the present study was to examine whether prostaglandin H synthase (PGHS) can be regulated by pathways independent of de novo synthesis of PGHS. Incubation of bovine aortic endothelial cells (BAEC) for as short as 5 min with NaF (40 mM) resulted in a 60% increase in PGHS activity. PGHS activity induced by NaF was unaffected by either 10 μM cycloheximide or 1 μM actinomycin D. Aspirin (25 μM) completely inhibited resting PGHS activity, and NaF did not induce further stimulation. NS-398 (500 nM), a specific PGHS-2 inhibitor, was ineffective. Basic fibroblast growth factor (bFGF) induced a significant increase in PGHS activity within 30 min and was insensitive to cycloheximide. The levels of PGHS-1 and PGHS-2 proteins, as measured by Western blots, were not affected by NaF or bFGF. The tyrosine kinase inhibitor genistein attenuated PGHS activity that was induced by NaF and bFGF, whereas the tyrosine phosphatase inhibitor, sodium orthovanadate, augmented these responses. The G protein activators 5′-guanylyl imidodiphosphate and guanosine 5′- O-(3-thiotriphosphate) inhibited both resting and NaF-induced PGHS activities. These results suggest that, in BAEC, PGHS-1 activity can be regulated by tyrosine kinase and/or G proteins, independently of de novo protein synthesis.
K+ transport and capacitance of the basolateral membrane of the larval frog skin
Authors: Hillyard Stanley D. , Cantiello Horacio F. , Van Driessche Willy
Published: December 2017

Skin from larval bullfrogs was mounted in an Ussing-type chamber in which the apical surface was bathed with a Ringer solution containing 115 mM K+ and the basolateral surface was bathed with a Ringer solution containing 115 mM Na+. Ion transport was measured as the short-circuit current ( I sc) with a low-noise voltage clamp, and skin resistance ( R m) was measured by applying a direct current voltage pulse. Membrane impedance was calculated by applying a voltage signal consisting of 53 sine waves to the command stage of the voltage clamp. From the ratio of the Fourier-transformed voltage and current signals, it was possible to calculate the resistance and capacitance of the apical and basolateral membranes of the epithelium ( R a and R b, C a and C b, respectively). With [Formula: see text] as the anion, R m decreased rapidly within 5 min following the addition of 150 U/ml nystatin to the apical solution, whereas I sc increased from 0.66 to 52.03 μA/cm2 over a 60-min period. These results indicate that nystatin becomes rapidly incorporated into the apical membrane and that the increase in basolateral K+ permeability requires a more prolonged time course. Intermediate levels of I sc were obtained by adding 50, 100, and 150 U/ml nystatin to the apical solution. This produced a progressive decrease in R a and R b while C a and C b remained constant. With Cl− as the anion, I sc values increased from 2.03 to 89.57 μA/cm2 following treatment with 150 U/ml nystatin, whereas with gluconate as the anion I sc was only increased from 0.63 to 11.64 μA/cm2. This suggests that the increase in basolateral K+permeability produced by nystatin treatment, in the presence of more permeable anions, is due to swelling of the epithelial cells of the tissue rather than the gradient for apical K+ entry. Finally, C b was not different among skins exposed to Cl−,[Formula: see text], or gluconate, despite the large differences in I sc, nor did inhibition of I scby treatment with hyperosmotic dextrose cause significant changes in C b. These results support the hypothesis that increases in cell volume activate K+ channels that are already present in the basolateral membrane of epithelial cells.
FLAG epitope positioned in an external loop preserves normal biophysical properties of CFTR
Authors: Schultz Bruce D. , Takahashi Akira , Liu Chongguang , Frizzell Raymond A. , Howard Marybeth
Published: December 2017

We asked whether inclusion of the FLAG epitope in the fourth extracellular loop of the cystic fibrosis transmembrane conductance regulator (M2–901/CFTR), which permits detection of cell surface expression, affected CFTR’s biophysical properties or channel regulation by kinases, phosphatases, and nucleotides. Channel activity of M2–901/CFTR was evaluated in numerous cell types and expression systems to characterize its gating and regulation. Our results show that M2–901/CFTR required adenosine 3′,5′-cyclic monophosphate-dependent protein kinase phosphorylation to initiate channel activity. Subsequently, ATP alone was sufficient to support channel gating, and ADP inhibited channel opening. Current fluctuation analysis indicated that the nucleotide-dependent gating rates were indistinguishable from those of wild-type (wt) cystic fibrosis transmembrane conductance regulator (CFTR). Channel conductance in symmetric Cl− (11.2 pS), anion permeability ratio (1.66), and block by gluconate indicate that the anion conduction pathway is indistinguishable from wtCFTR. Sulfonylureas (glibenclamide and LY-295501) inhibited M2–901/CFTR channel activity by an identical mechanism to that described for wtCFTR. Finally, CFTR-dependent insertion and retrieval of cell membrane was unaffected by the presence of the FLAG epitope. These results indicate that this structural alteration does not affect the control mechanisms for channel gating and suggest that the fourth extracellular loop of CFTR does not contribute to the ion pore. Detection of M2–901/CFTR by a commercially available monoclonal antibody (M2), together with presentation of normal functional properties, makes M2–901/CFTR a valuable tool to evaluate CFTR protein expression and cellular location.
Modulation of Ca2+-gated cardiac muscle Ca2+-release channel (ryanodine receptor) by mono- and divalent ions
Authors: Liu Wei , Pasek Daniel A. , Meissner Gerhard
Published: December 2017

The effects of mono- and divalent ions on Ca2+-gated cardiac muscle Ca2+-release channel (ryanodine receptor) activity were examined in [3H]ryanodine-binding measurements. Ca2+ bound with the highest apparent affinity to Ca2+activation sites in choline chloride medium, followed by KCl, CsCl, NaCl, and LiCl media. The apparent Ca2+ binding affinities of Ca2+ inactivation sites were lower in choline chloride and CsCl media than in LiCl, NaCl, and KCl media. Sr2+ activated the ryanodine receptor with a lower efficacy than Ca2+. Competition studies indicated that Li+, K+, Mg2+, and Ba2+ compete with Ca2+ for Ca2+ activation sites. In 0.125 M KCl medium, the Ca2+ dependence of [3H]ryanodine binding was modified by 5 mM Mg2+ and 5 mM β,γ-methyleneadenosine 5′-triphosphate (a nonhydrolyzable ATP analog). The addition of 5 mM glutathione was without appreciable effect. Substitution of Cl−by 2-( N-morpholino)ethanesulfonic acid ion caused an increase in the apparent Ca2+affinity of the Ca2+ inactivation sites, whereas an increase in KCl concentration had the opposite effect. These results suggest that cardiac muscle ryanodine receptor activity may be regulated by 1) competitive binding of mono- and divalent cations to Ca2+ activation sites, 2) binding of monovalent cations to Ca2+ inactivation sites, and 3) binding of anions to anion regulatory sites.