bluuebunny/crossref_metadata_2025 huggingface dataset:
Bastadins relate ryanodine-sensitive and -insensitive Ca2+ efflux pathways in skeletal SR and BC3H1 cells
Authors: Pessah I. N. , Molinski T. F. , Meloy T. D. , Wong P. , Buck E. D. , Allen P. D. , Mohr F. C. , Mack M. M.
Published: December 2017

Bastadins potently interact with the FK-506-binding protein of 12 kDa (FKBP12)-ryanodine receptor (Ry1R) complex in skeletal muscle to enhance a high-affinity ryanodine binding conformation (M. M. Mack, T. F. Molinski, E. D. Buck, and I. N. Pessah. J. Biol. Chem. 269: 23236-23249, 1994). Bastadins are used to examine the relationship between ryanodine-sensitive and ryanodine-insensitive Ca2+ efflux pathways that coexist in junctional sarcoplasmic reticulum (SR) vesicles from rabbit skeletal muscle and differentiated BC3H1 cells. Complete block of caffeine-sensitive Ca2+ channels with micromolar ryanodine or ruthenium red does not alter the steady-state loading capacity of SR. Inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps with thapsigargin unmasks a ryanodine- and ruthenium red-insensitive Ca2+ efflux pathway. Bastadin 5 alone does not inhibit Ca2+ efflux unmasked by inhibition of SERCA pumps, but, in combination with blocking concentrations of ryanodine or ruthenium red, it eliminates the ryanodine-insensitive Ca2+ "leak" and enhances steady-state loading capacity of SR vesicles approximately 2.5-fold. These actions of bastadins occur in the same concentration range that enhances the number of high-affinity binding sites for [3H]ryanodine (50% effective concentration of approximately 2 microM). Similar effects on SR Ca2+ transport are found with FK-506 and ryanodine in combination. Block of Ry1R in intact BC3H1 cells with ryanodine does not eliminate the prominent Ca2+ leak unmasked by thapsigargin. A membrane-permeant mixture of bastadins in combination with ryanodine nearly eliminates the Ca2+ leak unmasked by thapsigargin, even though the Ca2+ stores are replete. The requirement of both a known Ry1R blocker and bastadins in combination provides a pharmacological link between ryanodine-sensitive Ca2+ channels and ryanodine-insensitive leak pathways in isolated junctional SR and BC3H1 cells. Together, these results strongly suggest that bastadins, through their modulatory actions on the FKBP12-Ry1R complex, convert ryanodine-insensitive leak states into ryanodine-sensitive channels that recognize [3H]ryanodine with high affinity.
Activation of diacylglycerol in cultured endothelial cells exposed to cyclic strain
Authors: Evans L. , Frenkel L. , Brophy C. M. , Rosales O. , Sudhaker C. B. , Li G. , Du W. , Sumpio B. E.
Published: December 2017

Confluent bovine aortic endothelial cells (EC) were grown on flexible membranes and subjected to 10% average strain at 60 cycles/min for up to 500 s. A biphasic increase in diacylglycerol (DAG) occurred, with an initial transient peak at 10 s followed by sustained elevation to 500 s. The early peak corresponded to the transient formation of inositol 1,4,5-trisphosphate, demonstrating hydrolysis of L-alpha-phosphatidylinositol (PI) by PI-specific phospholipase C. To determine the origin of the sustained DAG phase, we incubated confluent bovine aortic EC with 1 microCi/ml [14C]myristate overnight and subjected them to cyclic strain. There was a decrease in phosphatidylcholine (PC) and a corresponding increase in DAG at 10 s and 250 s, suggesting PC hydrolysis with the generation of DAG at both an early (10 s) and a late (250 s) phase. [14C]phosphatidylethanol, a specific product of phospholipase D (PLD) in the presence of 1% ethanol, was measured in EC preincubated with [14C]myristate. Cyclic strain led to an immediate and sustained activation of PLD. Increased ethanol concentration led to a consistent decrease in DAG. Furthermore, when EC were pretreated with 1% ethanol, the strain-induced proliferative response was attenuated.
Hypertension alters rapid cooling contractures in single rat cardiocytes
Authors: Stauffer B. L. , Palmer B. M. , Hazel A. , Cheung J. Y. , Moore R. L.
Published: December 2017

Previous work has demonstrated that, in single, paced left ventricular (LV) myocytes isolated from rats with hypertension, the extent of myocyte shortening and the amplitude of the cytosolic Ca2+ concentration transient are decreased relative to normal myocytes. These findings suggest that reduced sarcoplasmic reticular (SR) Ca2+ release could be responsible for hypertension-induced attenuation of the myocyte contractile response. Hypertension-induced reductions in SR Ca2+ release could be due to 1) a decrease in releasable SR Ca2+ content relative to the sarcoplasmic volume into which it is released or 2) alterations in the SR Ca2+ release mechanism such that the fractional release of SR Ca2+ is reduced. Using rapid cooling contractures (RCCs) to provide an index of SR Ca2+ content, we conducted a series of experiments designed to test the former hypothesis. Single LV myocytes were isolated from normotensive control rats and from rats with hypertension, which was induced by abdominal aortic banding (for approximately 4 mo). The extent of myocyte shortening during an RCC is taken to be directly proportional to SR Ca2+ content. As expected, the amplitudes of both twitches and RCCs decreased as pacing frequency increased from 0.2 to 1.0 Hz across both control and hypertensive groups, although the effect was greatest in control myocytes. A significant finding of this study was that, at both pacing frequencies, RCC magnitude was attenuated in hypertensive relative to control myocytes. These results suggest that in hypertension cellular Ca2+ homeostasis is altered and there is a mismatch between releasable SR Ca2+ content and the sarcoplasmic volume into which it is released.
Sphingosine 1-phosphate stimulates Na+/H+ exchange in thyroid FRTL-5 cells
Authors: Tornquist K.
Published: December 2017

Sphingosine derivatives are potent mitogens in several cell types. Many mitogens activate the Na+/H+ exchange, although the interrelationships between Na+/H+ exchange and mitogenesis are unclear. The present investigation in thyroid FRTL-5 cells shows that sphingosine 1-phosphate (SPP) activates Na+/H+ exchange in a dose-dependent manner in acid-loaded cells. The effect of SPP was abolished in a Na+-free buffer and by pretreatment of the cells with ethylisopropylamiloride. SPP did not affect basal intracellular pH (pHi). SPP stimulated the release of sequestered Ca2+ and a substantial entry of Ca2+. The effect of SPP on pH(i) was abolished in cells incubated in a Ca2+-free buffer, and in cells loaded with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Furthermore, the effect of SPP was abolished in pertussis toxin (PTX)-treated cells. PTX decreased Ca2+ entry only, without affecting the release from intracellular stores. Phosphatidic acid (PA) did not activate Na+/H+ exchange, suggesting that the effect of SPP was not mediated via activation of phospholipase D and the production of PA. Thus one mechanism of action of SPP in FRTL-5 cells appears to be to activate Na+/H+ exchange. This action is mediated via a G protein-dependent mechanism and requires an increase in intracellular free Ca2+.
Dual role for AlF4(-)-sensitive G proteins in the function of T84 epithelial cells: transport and barrier effects
Authors: Ries J. , Stein J. , Traynor-Kaplan A. E. , Barrett K. E.
Published: December 2017

T84 monolayers were studied to determine the effect of AlF4, an activator of heterotrimeric G proteins, on Cl-secretion and intestinal barrier function. Basolateral (but not apical) addition of AlF4- increased short-circuit current (I(sc)) and decreased transepithelial resistance. Preincubation with the heavy metal chelator deferoxamine showed that both effects were dependent on Al3+. The effect on I(sc) was abolished by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid or in Cl(-)-free solutions, whereas the decrease in resistance was unaffected. AlF4- also increased intracellular Ca2+, as assessed via fura 2 fluorometry. AlF4- had no effect on adenosine 3',5'-cyclic monophosphate (cAMP) or guanosine 3',5'-cyclic monophosphate (cGMP) levels in T84 cells. The effect of AlF4- on transepithelial resistance was accompanied by a decrease in cellular F-actin as well as increased transepithelial fluxes of the paracellular markers mannitol and inulin. The results indicate that AlF4(-)-sensitive G proteins regulate both epithelial secretory and barrier functions, but via different pathways. AlF4- increases Cl- secretion via a Ca2+-dependent and cAMP- and cGMP-independent mechanism in T84 cells, whereas the decrease in resistance is independent of Ca2+.
Mechanism of anion permeation in the basolateral membrane of isolated rabbit renal proximal tubule S3 segment
Authors: Seki G. , Yamada H. , Taniguchi S. , Uwatoko S. , Suzuki K. , Kurokawa K.
Published: December 2017

Conventional and double-barreled microelectrodes were used to examine the anion selectivity of Cl- conductance in the basolateral membrane of rabbit renal proximal tubule S3 segment. The permeability sequence determined by anion replacements in the presence of K+ channel blocker quinine was SCN- > I- > Br- > Cl- > gluconate in both nonperfused and luminally perfused tubules. The anion-selective microelectrodes with higher sensitivity to I- enabled us to measure intracellular I- activities. With these electrodes, we could compare the conductive movements of Cl- and I- in response to the increase in bath K+ concentrations and confirmed that the conductance sequence was also I- > Cl-. Although the basolateral potential changes generated by Cl- and Br- currents were stimulated by adenosine 3',5'-cyclic monophosphate (cAMP), the potential changes by SCN- and I- currents were somewhat inhibited by cAMP. In addition, the conductive uptake of I- was, in contrast to that of Cl-, inhibited by cAMP These results are consistent with the existence of at least two distinct anion conductances in this membrane, which are differently regulated by cAMP.
Intracellular acidosis activates c-Src
Authors: Yamaji Y. , Tsuganezawa H. , Moe O. W. , Alpern R. J.
Published: December 2017

The purpose of the present studies was to determine whether acidosis activates protein tyrosine kinase pathways. Incubation of MCT cells, a renal proximal tubule cell line, in acid media caused increased phosphotyrosine content of 60- to 70- and 120-kDa cytosolic proteins. Media acidification induced a twofold increase in c-Src activity that occurred within 30 s. Significant activation occurred with media pH changes as small as 0.07 pH unit accompanied by cell acidification of 0.06 pH unit. Sodium propionate addition, NH4Cl prepulse, and nigericin addition, maneuvers that decrease intracellular pH in the absence of changes in extracellular pH, activated c-Src. Significant activation by sodium propionate was seen with cell pH changes as small as 0.07 pH unit. Sodium orthovanadate, a protein tyrosine phosphatase inhibitor, prevented c-Src activation by media acidification but did not prevent protein tyrosine phosphorylation. In summary, decreased intracellular pH activates c-Src. Acid activation of c-Src represents a novel mechanism of c-Src activation that may be relevant to many cellular responses to acidosis.
Regulation of intracellular calcium oscillations in porcine tracheal smooth muscle cells
Authors: Prakash Y. S. , Kannan M. S. , Sieck G. C.
Published: December 2017

Using real-time confocal microscopy, we examined the dynamic intracellular Ca2+ concentration ([Ca2+]i) response of porcine tracheal smooth muscle (TSM) cells to acetylcholine (ACh). Exposure to ACh caused regenerative, propagating [Ca2+]i oscillations. The amplitude and fall time of the [Ca2+]i oscillations were inversely correlated to basal [Ca2+]i, whereas the frequency and rise time were directly correlated to basal [Ca2+]i. ACh-induced [Ca2+]i oscillations were initiated in the absence of extracellular Ca2+ and after membrane depolarization with KCl, suggesting that 1) [Ca2+]i oscillations primarily arise by release from internal stores such as the sarcoplasmic reticulum (SR), and 2) Ca2+ influx is necessary for maintenance of oscillations. Exposure to both caffeine and ryanodine inhibited ongoing ACh-induced [Ca2+]i oscillations, suggesting a role for caffeine-sensitive ryanodine receptor (RyR) SR Ca2+ channels. Inhibition of SR Ca2+ reuptake by thapsigargin increased basal [Ca2+]i and decreased [Ca2+]i oscillation amplitude, suggesting that Ca2+ reuptake is also essential. The present results suggest that [Ca2+]i oscillations in porcine TSM cells involve repetitive Ca2+ release and reuptake from RyR channels, perhaps through a Ca2+ -induced Ca2+ release mechanism.
Psoralens: novel modulators of Cl- secretion
Authors: Devor D. C. , Singh A. K. , Bridges R. J. , Frizzell R. A.
Published: December 2017

We evaluated effects of psoralens on Cl- secretion (short-circuit current, I(sc)) across T84 monolayers. Methoxsalen failed to increase I(sc). Several observations suggest that psoralens open cystic fibrosis transmembrane conductance regulator Cl- channels. 1) After activation of the Ca2+-dependent basolateral membrane K+ channel (K(Ca)) by 1-ethyl-2-benzimidazolinone or thapsigargin, methoxsalen (10 microM) further increased I(sc). 2) When added before carbachol (CCh), methoxsalen potentiated the I(sc) response to CCh, as predicted, if it increased apical Cl- conductance. 3) After establishment of a mucosal-to-serosal Cl- gradient and permeabilization of basolateral membrane with nystatin, psoralens increased Cl- current, which was inhibited by glibenclamide. In contrast, neither TS-TM calix[4]arene nor Cd2+, inhibitors of outwardly rectifying Cl- channels and the ClC-2 Cl-channel, respectively, inhibited psoralen-induced Cl- current. In contrast to their effects on Cl- conductance, psoralens failed to significantly affect basolateral membrane K+ conductance; subsequent addition of 1-ethyl-2-benzimidazolinone induced a large increase in K+ conductance. Also, in excised patches, methoxsalen failed to activate K(Ca). In addition to potentiating the peak response to CCh, psoralens induced a secondary, sustained response. Indeed, when added up to 60 min after return of CCh-induced I(sc) to baseline, psoralens induced a sustained I(sc). This sustained response was inhibited by atropine, demonstrating the requirement for continuous muscarinic receptor activation by CCh. This sustained response was inhibited also by verapamil, removal of bath Ca2+, and charybdotoxin. These results suggest that return of I(sc) to baseline after CCh stimulation is not due to downregulation of Ca2+ influx or K(Ca). Finally, we obtained similar results with psoralens in rat colon and primary cultures of murine tracheal epithelium. On the basis of these observations, we conclude that psoralens represent a novel class of Cl- channel openers that can be used to probe mechanisms underlying Ca2+-mediated Cl- secretion.
Defining the volume dependence of multiple K flux pathways of trout red blood cells
Authors: Berenbrink M. , Weaver Y. R. , Cossins A. R.
Published: December 2017

The volume sensitivity of different K flux pathways has been determined in trout red blood cells subjected to volume perturbation. Gentle hyposmotic swelling induced a K influx in a Cl-containing saline but not in NO3- or methanesulfonate (MeSF)-containing salines, consistent with the activation of a Cl-dependent flux. Extreme hyposmotic swelling led to larger K fluxes in all salines but with reduced anion discrimination of the Cl-dependent flux. In contrast to these graded responses, isosmotic swelling using ammonium chloride or beta-adrenergic stimulation activated only Cl-dependent fluxes in an all-or-none fashion. The relationship between the hyposmotically and isosmotically induced pathways was studied by coactivation using either ammonium chloride or isoproterenol with anisosmotic treatment. Cells in ammonium chloride-containing hyposmotic salines showed no additive K flux over that induced by hyposmotic treatment alone, indicating that the isosmotically induced Cl-dependent flux was identical to the hyposmotically induced Cl-dependent flux. However, cells coactivated by hyposmotic and beta-adrenergic treatment showed a small Cl-dependent flux in addition to that induced by hyposmotic treatment alone. This small third component was unaffected by anisosmotic treatment. We conclude that the major Cl-dependent and Cl-independent K flux pathways are distinct and separate and that the former has an anion dependence that varies with cell volume and a volume sensitivity that varies with ionic strength.
Cholera and pertussis toxins increase acidification of endocytic vesicles without altering ion conductances
Authors: Van Dyke R. W.
Published: December 2017

Acidification of endocytic vesicles, driven by the vacuolar H+ pump, is affected by parallel ion transporters. Because adenosine 3',5'-cyclic monophosphate (cAMP) and heterotrimeric G proteins may alter ion transporters, I tested whether cholera and pertussis toxins affected acidification of rat liver endosomes. Fluorescein-labeled dextran-loaded "10-min" endosomes from cholera toxin-treated rats exhibited ATP-dependent rates of acidification in the presence and absence of Cl- or K+ that were approximately 60-120% (P < 0.05) faster than rates from control endosomes. This increase was greater for "older" "20-min" endosomes and less for 'early" "2-min" endosomes. Ion transport functions of 10-min and 20-min toxin-exposed endosomes were similar to those of 2-min control endosomes. Cholera toxin also increased ATP-dependent steady-state intravesicular H+ concentration by 38-218% (P < 0.05). Pertussis toxin increased endosome acidification rates by 20-54% (P < 0.05). Both toxins increased liver cAMP content, and endosomes prepared from perfused livers exposed to 0.75 mM dibutyryl cAMP exhibited similar increases in acidification rates. These studies indicate that both cholera and pertussis toxins markedly alter the function of rat liver endosomes. The mechanism is unlikely to reflect major changes in vesicle ion transporters but rather may indicate either an increase in the number of H+ pumps per endosome and/or changes in fusion, remodeling, and maturation of early endocytic vesicles in response to cAMP.
Myosin isoform expression and force generation in cultured resistance arteries
Authors: Ishibashi K. , Bukoski R. D.
Published: December 2017

Organ culture of mesenteric resistance arteries results in a loss of force-generating ability, which is prevented by 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3]. We have tested the hypothesis that the culture-induced decrease in active stress is associated with altered myosin isoform expression. Rat mesenteric resistance arteries were studied immediately (fresh) or after incubation at 37 degrees C for 48 h in culture medium (control), with 300 pg/ml 1,25(OH)2D3, or with 5 microg/ml insulin. Isometric force was measured by myography; myosin heavy chain (MHC) and regulatory myosin light chain isoform (MLC) contents were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal active stress to 100 mM K+ (mN/mm2) was greater for fresh (147.8 +/- 4.9) than control (109.2 +/- 4.6, P = 0.001) or insulin (79.6 +/- 8.6, P < 0.001) but not 1,25(OH)2D3 (137.4 +/- 9.5, P = 0.197). Organ culture did not alter MLC or MHC smooth muscle (SM)-1 isoform content. MHC SM-2 content (nmol/mg protein) was greater in fresh (0.038 +/- 0.003) than control (0.026 +/- 0.003, P = 0.012) and insulin (0.027 +/- 0.002, P = 0.018) but not 1,25(OH)2D3 (0.036 +/- 0.003, P = 0.693); nonmuscle MHC (NMM) was observed in insulin. The maximal active stress response to K+ significantly correlated with SM-2 MHC isoform content (r2 = 0.483, P < 0.001). We conclude that 1) arterial organ culture alters MHC isoform content, 2) SM-2 MHC isoform content positively correlates with active stress generation, 3) 1,25(OH)2D3 maintains force-generating capacity by preventing the shift of MHC isoform expression, and 4) insulin impairs force-generating ability by lowering MHC SM-2 content and stimulating NMM expression.
Fatty acid modulation of K+ channels in taste receptor cells: gustatory cues for dietary fat
Authors: Gilbertson T. A. , Fontenot D. T. , Liu L. , Zhang H. , Monroe W. T.
Published: December 2017

In an attempt to determine the chemosensory cues, if any, provided by fats in the oral cavity, we have performed patch-clamp recordings on isolated rat taste receptor cells during application of free fatty acids. Cis-polyunsaturated fatty acids, when applied extracellularly, inhibit delayed-rectifying K+ channels. In a subset of cells, these fatty acids also enhance inwardly rectifying K+ currents. Saturated, monounsaturated, and trans-polyunsaturated fatty acids have no significant effect on K+ currents. These effects do not involve activation of G protein-mediated pathways, including protein kinase C and protein kinase A, lipoxygenase pathways, cyclooxygenase pathways, or cytochrome P-450 pathways, consistent with direct effects on these ion channels or closely associated proteins. The net effect of fatty acids is to prolong stimulus-induced depolarizations of taste receptor cells, and we propose the effects on K+ channels represent the mechanism by which fats are detected by receptor cells in the oral cavity.
NH4Cl activates AE2 anion exchanger in Xenopus oocytes at acidic pHi
Authors: Humphreys B. D. , Chernova M. N. , Jiang L. , Zhang Y. , Alper S. L.
Published: December 2017

In the course of experiments to define regulation by intracellular pH (pHi) of the AE2 anion exchanger expressed in Xenopus oocytes, we discovered an unexpected regulation of AE2 by NH4+. Intracellular acidification produced by extracellular acidification or produced by equimolar substitution of NaCl with sodium acetate each inhibited AE2 activity. In contrast, intracellular acidification by equimolar substitution of NaCl with NH4Cl activated AE2-associated, trans-anion-dependent, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 36Cl- influx and efflux. Regulation by NH4+ was isoform specific, since neither erythroid nor kidney AE1 was activated. AE2 activation was maximal at <5 mM NH4Cl; was not mimicked by extracellular KCl, chloroquine, or polyamines; and was insensitive to amiloride, bumetanide, barium, and gadolinium. Whether NH4Cl acts directly on AE2 or on another target remains to be determined. Activation of AE2 by NH4+ may serve to sustain Cl-/HCO3- exchange activity in the presence of acidic pH in renal medulla, colon, abscesses, and other AE2-expressing acidic locales exposed to elevated NH4+ concentration.
Effect of chronic hypoxia on K+ channels: regulation in human pulmonary vascular smooth muscle cells
Authors: Peng W. , Hoidal J. R. , Karwande S. V. , Farrukh I. S.
Published: December 2017

We investigated the effects of chronic hypoxia on the major outward K+ currents in early cultured human main pulmonary arterial smooth muscle cells (HPSMC). Unitary currents were measured from inside-out, outside-out, and cell-attached patches of HPSMC. Chronic hypoxia depolarized resting membrane potential (Em) and reduced the activity of a charybdotoxin (CTX)- and iberiotoxin-sensitive, Ca2+-dependent K+ channel (KCa). The 4-aminopyridine-sensitive and CTX-insensitive channel or the delayed rectifier K+ channel was unaffected by chronic hypoxia. Chronic hypoxia caused a +33- to +53-mV right shift in voltage-dependent activation of K(Ca) and a decrease in K(Ca) activity at all cytosolic Ca2+ concentrations ([Ca2+]i) in the range of 0.1-10 microM. Thus the hypoxia-induced decrease in K(Ca) activity was most likely due to a decrease in K(Ca) sensitivity to Em and [Ca2+]i. Chronic hypoxia reduced the ability of nitric oxide (NO.) and guanosine 3',5'-cyclic monophosphate (cGMP) to activate K(Ca). The cGMP-dependent protein kinase-induced activation of K(Ca) was also significantly inhibited by chronic hypoxia. In addition, inhibiting channel dephosphorylation with calyculin A caused significantly less increase in K(Ca) activity in membrane patches excised from chronically hypoxic HPSMC compared with normoxic controls. This suggests that the mechanism by which hypoxia modulates NO.-induced K(Ca) activation is by decreasing the NO./cGMP-mediated phosphorylation of the channel.
Phospholipase D activity facilitates Ca2+-induced aggregation and fusion of complex liposomes
Authors: Blackwood R. A. , Smolen J. E. , Transue A. , Hessler R. J. , Harsh D. M. , Brower R. C. , French S.
Published: December 2017

Phospholipase D (PLD) activation in stimulated neutrophils results in the conversion of membrane phosphatidylcholine (PC) to phosphatidic acid (PA). This change in membrane phospholipid composition has two potentially positive effects on degranulation. It 1) replaces a nonfusogenic phospholipid with a fusogenic one and 2) increases the potential for interactions between membranes and the annexins. Modeling neutrophil degranulation, we examined the effect of PLD (Streptomyces chromofuscus) hydrolysis on the aggregation and fusion of liposomes in the presence and absence of annexin I. We found that PLD-mediated conversion of PC to PA lowered the [Ca2+] required for fusion. Annexin I increased the rate of fusion in the presence of PA, although it did not lower threshold [Ca2+], which remained above the physiological range. However, after hydrolysis by PLD, annexin I lowered the [Ca2+] required for aggregation by almost three orders of magnitude, to near physiological concentrations. These studies indicate that the activation of PLD and the production of PA may play a role in annexin-mediated membrane-membrane apposition.
Cytokine control of PMN phagocytosis: regulatory effects of hypoxemia and hypoxemia-reoxygenation
Authors: Knowles R. , Keeping H. , Graeber T. , Nguyen K. , Garner C. , D'Amico R. , Simms H. H.
Published: December 2017

We investigated the effects of hypoxemia and hypoxemia-reoxygenation (H/R) on interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), or IL-1beta stimulation of whole blood polymorphonuclear leukocyte (PMN) phagocytosis and bactericidal activity. Whole blood PMN were rendered hypoxemic (venous PO2 < 15 mmHg), normoxic (venous PO2 60-80 mmHg), or reoxygenated after hypoxemia (H/ R = venous PO2 150-200 mmHg) and were incubated with IL-8, TNF-alpha, or IL-1beta before sequential addition of serum-opsonized fluorescent microspheres and fluorescein isothiocyanate-conjugated mouse anti-human CD64, CD32w, CD16, CD35, or CD11b/CD18. Concomitant two-color flow cytometric analyses were then performed measuring mean channel fluorescence and the percentage of PMN positive for phagocytosis, with simultaneous subset receptor analysis on populations of PMN that exceeded control levels of phagocytosis. During hypoxemia, whole blood PMN phagocytosis in the presence of IL-8, TNF-alpha, or IL-1beta was increased compared with normoxia. Northern blot analyses revealed an increase in steady-state mRNA levels for CD32w during hypoxemia + IL-8 and CD64 during hypoxemia + IL-1beta. During reoxygenation, both whole blood PMN phagocytosis and bactericidal activity were reduced in the presence of IL-8, TNF-alpha, or IL-1beta, and in subsets of PMN with reduced phagocytosis H/R reduced CD64, CD32w, CD16, CD35, and CD11b/CD18 expression in the presence of each cytokine. Northern blot analyses revealed that H/R reduced mRNA levels for opsonic receptors primarily for IL-1beta-stimulated PMN. These results demonstrate a direct regulatory effect of hypoxemia and H/R on whole blood PMN phagocytosis, receptor expression, and steady-state mRNA levels of both Fc(gamma) and complement receptors.
Modes of operation of an electroneutral Na+/Li+ countertransport in human skin fibroblasts
Authors: Zerbini G. , Mangili R. , Gabellini D. , Pozza G.
Published: December 2017

An elevated activity of erythrocyte Na+/Li+ countertransport (SLC) is an intermediate phenotype of human essential hypertension, but cells other than erythrocytes have not been studied. Therefore, we have examined several transport modes of Na+/Li+ exchange in human skin fibroblasts. External Na+-stimulated Li+ efflux was 152 +/- 31 (SE) nmol x mg protein(-1) x min(-1) (n = 8). At intracellular pH 7.3, intracellular Na+-stimulated Li+ influx, intracellular Li+-stimulated Na+ influx, and external Li+-stimulated Na+ efflux were very similar, indicating the presence of a tightly coupled 1:1 SLC. This pathway was not affected by 5-(N,N-dimethyl)-amiloride and changes in the membrane potential, but phloretin and intracellular acidification (intracellular pH 6.8) were markedly inhibitory. Kinetic analyses of the external Na+ site also compared with SLC, although the internal site appeared to show a low affinity for Li+. We conclude that an SLC pathway similar to that in human erythrocytes is expressed in human skin fibroblasts.
Telokin expression in A10 smooth muscle cells requires serum response factor
Authors: Herring B. P. , Smith A. F.
Published: December 2017

Telokin transcription is initiated from a smooth muscle-specific promoter located in an intron of the smooth muscle myosin light chain kinase gene. We have previously identified a 310-base pair fragment of the promoter that mediates A10 smooth muscle cell-specific expression of telokin. In the current study, telokin-luciferase reporter gene assays in A10 cells and REF52 nonmuscle cells revealed that the promoter region between -81 and +80 contains the regulatory elements required to mediate the in vitro cell specificity of the promoter. Several positive-acting elements, including an E box, myocyte enhancer factor 2 (MEF2)-TATA box, and CArG-serum response element, were identified within this region. Telokin transcription in A10 smooth muscle cells requires all three transcription initiation sites and an AT-rich sequence between -71 and -62 that includes a TATA box. MEF2 interacts with the AT-rich region with low affinity; however, MEF2 binding is not required for transcriptional activity in A10 cells. Binding of serum response factor (SRF) to a CArG element proximal to the TATA sequence is also critical for high levels of transcription in A10 cells. Together these data suggest that an AT-rich motif, acting in concert with SRF and an unusual transcription initiation mechanism, is required for the cell-specific expression of the telokin promoter in A10 smooth muscle cells.
Measurement of changes in cell volume based on fluorescence quenching
Authors: Srinivas S. P. , Bonanno J. A.
Published: December 2017

The intracellular fluorescence of 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ), a Cl(-) -sensitive fluorescent dye, is quenched by intracellular organic anions and proteins of unknown identity. The concentration of these intracellular quenchers (ICQs), however, is dependent on cell volume. In the absence of Cl-, changes in the observed SPQ fluorescence may therefore reflect changes in cell volume. This concept has been applied to determine relative changes in cell volume of cultured corneal endothelium in response to anisosmotic shocks, using NO3- as the Cl- substituent. SPQ fluorescence increased with decreasing osmolarity and vice versa. A 20 mosM hypertonic shock was needed to detect a change in SPQ fluorescence with a signal-to-noise ratio of >25. Assuming dynamic quenching by ICQs, we applied an extension of the Stern-Volmer equation to develop a simple relationship between the measured SPQ fluorescence and relative changes in cell volume. For large hyposmotic shocks, regulatory volume decrease (RVD) was observed. The rate of RVD could be enhanced by exposure to 0.5 microM gramicidin in low-Na+ Ringer solution (i.e., K+-NO3- efflux), indicating that K+ conductance is rate limiting for RVD. These results demonstrate the principle of using fluorescence quenching to measure changes in cell volume in real time. Because SPQ is sensitive to Cl-, its usefulness as a quenching probe is limited. However, a structure-activity study can be expected to yield useful Cl(-)-insensitive analogs.
Gap junctional units are functionally expressed before first cleavage in the early ascidian embryo
Authors: Tosti E.
Published: December 2017

Manually apposed ascidian zygotes established electrical communication within 50 min of fertilization and before cytokinesis. Junctional conductance between zygotes was 14.5 +/- 2.9 nS (n = 7), similar to that previously reported for ascidian two-cell-stage blastomeres, suggesting that zygotes and blastomeres express an equivalent number of gap junctional half-channels. Because puromycin at 400 microM does not inhibit the functional expression of these half-channels, they appear to be of maternal origin and their activation does not require protein synthesis. Loading zygotes with 500 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid or exposing zygotes to 10 microM of the calcium ionophore A-23187 shows that these half-channels are regulated by intracellular calcium, consistent with the behavior of these channels in adult tissues. The results show that gap junctional units are expressed in the ascidian at the zygote stage.
Noncoordinate regulation of epithelial Na channel and Na pump subunit mRNAs in kidney and colon by aldosterone
Authors: Escoubet B. , Coureau C. , Bonvalet J. P. , Farman N.
Published: December 2017

Distal colon and renal cortical collecting ducts are major effectors of aldosterone-dependent Na homeostasis. Na is absorbed by entry through an apical amiloride-sensitive Na channel and extruded by Na-K-ATPase at the basolateral membrane. Using a ribonuclease protection assay, we studied, in vivo, aldosterone regulation of alpha-, beta-, gamma-subunits of the rat epithelial Na channel (rENaC) and alpha 1- and beta 1-subunits of Na-K-ATPase. In the kidney, Na-K-ATPase mRNAs were also assayed over discrete tubular segments by in situ hybridization. In rat colon, all three rENaC mRNAs were decreased by adrenalectomy, with a major effect on beta- and gamma-subunits, and were restored with 7 days, but not 2 days, of aldosterone treatment; in the kidney, however, only alpha-transcripts varied. Na-K-ATPase alpha 1- and beta 1-subunit mRNAs in both organs were not (in the case of the beta 1-subunit) or were mildly (in the case of the alpha 1-subunit) affected after adrenalectomy. Our conclusions are as follows: 1) Transcripts of rENaC and Na-K-ATPase subunits are not coordinately regulated by aldosterone in vivo; i.e., modulation involves mainly the Na channel, not Na-K-ATPase; the effect is not of comparable magnitude on each subunit mRNA and differs between tissues. 2) The delay of the aldosterone effect on transcripts is much longer than that required to restore normal Na transport in adrenalectomized rats, indicating that rENaC and Na-K-ATPase subunit transcript levels may depend on unidentified early aldosterone-induced proteins.
Submandibular enzymatic vasoconstrictor increases DNA and phosphoinositol synthesis by mesenchymal cells
Authors: Carbini L. A. , Scicli A. G.
Published: December 2017

Submandibular enzymatic vasoconstrictor (SEV, rK9) induces vascular contraction and platelet aggregation by a mechanism requiring intact enzymatic activity. On the basis of a published report demonstrating growth-promoting enzymatic activity in an extract of the rat submandibular gland, we hypothesized that SEV would affect DNA synthesis. Recombinant SEV (rSEV), expressed in a baculovirus system, increased DNA synthesis 3- to 25-fold in Chinese hamster lung (CCL39) fibroblasts and in rabbit and rat vascular smooth muscle cells in a dose-dependent manner dose eliciting 50% of maximal response: 0.1-1 nM); this effect was inhibited by pertussis toxin (PTX). Inactive rSEV failed to enhance DNA synthesis. In CCL39 fibroblasts, rSEV increased total phosphoinositol (PI) formation (6- to 10-fold at 10 nM), which was inhibited 49% by PTX; it was also partially inhibited by the tyrosine kinase inhibitor genistein (33%) but was not affected by the protein kinase C inhibitor bisindolylmaleimide. These results show that rSEV increases DNA synthesis and PI formation in mesenchymal cells in a dose- and enzymatic activity-dependent manner through a pathway partially mediated by a PTX-sensitive G protein. Thus SEV can induce growth-associated responses, perhaps through a protease-activated receptor mechanism.
Extracellular ATP modulates [Ca2+]i in retinoic acid-treated embryonic chondrocytes
Authors: Hung C. T. , Allen F. D. , Mansfield K. D. , Shapiro I. M.
Published: December 2017

When treated with low doses of retinoic acid (RA), cephalic chondrocytes of the chick embryonic sternum mature and express phenotypic characteristics of postmitotic hypertrophic cells. In concert with these maturation-dependent changes, cells release adenine nucleotides into the culture medium. To ascertain if these compounds modulate chondrocyte function, we challenged chondrocytes with nucleotides and measured one determinant of the signal transduction pathway, intracellular Ca2+ concentration ([Ca2+]i). In the presence of micromolar concentrations of ATP, there was a dose-dependent elevation in chondrocyte [Ca2+]i; ADP caused a small but significant rise in the peak [Ca2+]i response. We found that the change in the [Ca2+]i response is linked to retinoid-dependent maturation of chondrocytes. Thus the [Ca2+]i rise was dependent on the RA concentration and treatment time. Immature caudal chondrocytes, cells that were not affected by RA, were used as control cells for this study. When treated with ATP, these cells did not exhibit a [Ca2+]i response. Although the purinergic subtype receptor was not characterized, the observation that cells responded to ATP and ADP but were refractory to AMP and adenosine suggested that P2 purinoceptors were expressed by chondrocytes. Because, during the same culture period, chondrocytes exhibited many of the unique characteristics of the terminally differentiated cell, the acquisition of purinergic receptors represents a new feature associated with expression of the mature phenotype. Finally, to ascertain if the ATP-dependent response was due to release of Ca2+ from intracellular stores, cells were treated with thapsigargin. Since this compound significantly reduced the [Ca2+]i signal, we concluded that the ATP response is mediated by release of cation, from the endoplasmic reticulum.
Quantification of ion transport in perfused rat heart: 133Cs+ as an NMR active K+ analog
Authors: Schornack P. A. , Song S. K. , Ling C. S. , Hotchkiss R. , Ackerman J. J.
Published: December 2017

Proper ion balance between intra- and extracellular compartments is necessary for normal physiological function. Conversely, alterations in membrane ion transport occur in numerous pathological states. As a noninvasive, nondestructive spectroscopic technique, nuclear magnetic resonance (NMR) offers a powerful approach to the study of ion balance in intact biological systems. Unfortunately, rare NMR active nuclides that are isotopes of the 100% naturally abundant 23Na+ and 39K+ are not available for tracer kinetic studies of Na1 and K+ transport. However, Cs is a biologically active analog of K+, and the 100% naturally abundant NMR active 133Cs+ nuclide can be employed to examine K+ transport (Davis, D. G., E. Murphy, and R. E. London. Biochemistry 27: 3547-3551, 1988). The distinguishing feature of 133Cs+ is that it naturally gives two separate well-resolved NMR resonances for intra- and extra-cellular 133Cs+, permitting study of the time course changes of either of these compartments independent of the other. In this report, the experimental procedures and compartmental modeling formalism are developed that allow quantitative analysis of Cs+ membrane transport in the perfused rat heart. Intracellular 133Cs+ is shown to be 100% visible by solution-state NMR methods and its influx transport to be markedly inhibited by ouabain, a confirmation of findings previously reported by others. Intracellular 133Cs+ spin-lattice and spin-spin relaxation times at 7 T were determined to be 2.1 +/- 0.3 (SD)s (n = 8) and 0.065 +/- 0.007 (SD) s (n = 8), respectively, for T1 and T2. The rate constant for Na(+)-K(+)-ATPase pump dominated intracellular influx was measured to be 0.25 +/- 0.07 (SD) min-1 (n = 27) and that for efflux 0.005 +/- 0.001 (SD) min-1 (n = 14). The rate constant for 133Cs+ equilibration in the extracellular space at supraphysiological perfusate flow rate (20 ml/min) was found to be 4.6 +/- 0.9 (SD) min-1 (n = 20). Thus extracellular diffusion limitations do not dominate the 133Cs+ transport measurements.
Inhibition of ion transport in septic rat heart: 133Cs+ as an NMR active K+ analog
Authors: Schornack P. A. , Song S. K. , Hotchkiss R. , Ackerman J. J.
Published: December 2017

Sepsis, the systemic response to severe infection, and the resulting multiorgan failure it induces are major contributors to intensive care unit morbidity and mortality. A number of abnormalities in ion transport processes and intracellular free Na+ ([Na+]i) and K+ ([K+]i) concentrations have been reported to occur during sepsis/endotoxemia. An effect of sepsis on the NA(+)-K(+)-ATPase may be an important contribution to changes in intracellular ion balance and the resultant pathophysiology of the disorder. The purpose of this study was to examine the effect of sepsis on the Na(+)-K(+)-ATPase in the isolated perfused rat heart using 133Cs+ nuclear magnetic resonance (NMR). Cs+ is a K+ analog, and 133Cs-NMR offers the opportunity to examine Na(+)-K(+)-ATPase activity in the intact organ via tracer kinetics. Sepsis was induced in halothane-anesthetized male Sprague-Dawley rats using the cecal ligation and perforation (CLP) model. Twenty-four to thirty-six hours after surgery, hearts from CLP or sham-operated rats were perfused with Krebs-Henseleit buffer containing 1.25 mM Cs+. The influx rate constant for Cs+ was decreased by 24% in septic rat hearts, i.e., 0.25 +/- 0.08 (SD) min 1 for controls and 0.19 +/- 0.04 (SD) min-1 for septic animals (P = 0.003). There was no difference for Cs+ efflux [0.005 +/- 0.001 (SD) min-1 for controls and 0.005 +/- 0.002 (SD) min-1 for septic animals; P = 0.8]. These results are consistent with an inhibition of the Na(+)-K(+)-ATPase pump during sepsis/endotoxemia. A decrease in the activity of the Na(+)-K(+)-ATPase pump may be responsible for or contribute to the changes in [Na+]i and [K+]i during the disorder.
Altered pHi regulation in 3T3/CFTR clones and their chemotherapeutic drug-selected derivatives
Authors: Wei L. Y. , Hoffman M. M. , Roepe P. D.
Published: December 2017

Recently (L. Y. Wei, M. J. Stutts, M. M. Hoffman, and P. D. Roepe. Biophys. J. 69: 883-895, 1996), 3T3 cells overexpressing the cystic fibrosis transmembrane conductance regulator (CFTR) were found to exhibit chemotherapeutic drug resistance and other traits of multidrug resistant (MDR) cells. In the present work, NIH 3T3/CFTR clones were selected with either doxorubicin or vincristine in incremental fashion to generate series of stable MDR cell lines that exhibit increasing levels of drug resistance. Thus C3D6 (grown in the presence of 600 nM doxorubicin) was selected from C3D4 (grown in the presence of 400 nM doxorubicin), which was selected from C3D1 (grown in the presence of 100 nM doxorubicin), which was in turn selected from the original 3T3/CFTR clone C3 (M. J. Stutts, S. E. Gabriel, J. C. Olsen, J. T. Gatzy, T. L. O'Connell, E. M. Price, and R. C. Boucher. J. Biol. Chem. 268: 20653-20658, 1993), which was not grown in the presence of chemotherapeutic drug. A similar series was generated via selection with vincristine. In both series, as well as series derived from a different CFTR clone, initial low-level drug selection increases CFTR expression without promoting MDR 1 or multidrug resistance-associated protein expression. On continued selection at higher drug concentrations, CFTR mRNA levels decrease while MDR 1 mRNA levels concomitantly increase. At each incremental step of selection, intracellular pH (pHi) increases (e.g., pHi of C3D6 > C3D4 > C3D1 > C3). Cl-/HCO3- exchange activity is significantly reduced in the drug-selected derivatives overexpressing MDR 1 but not the parental CFTR clones. The apparent set point of Na+/H+ exchange activity is significantly lower for the non-drug-selected 3T3/CFTR clones, relative to controls, but it increases on initial selection with chemotherapeutic drug. Overexpression of MDR 1 in the higher-level selectants does not appear to further perturb apparent Na+/H+ exchange. These data further describe how CFTR and MDR proteins may affect pHi regulation.
Cytoskeletal mechanics in confluent epithelial cells probed through integrins and E-cadherins
Authors: Potard U. S. , Butler J. P. , Wang N.
Published: December 2017

Mechanical forces associated with the cytoskeleton (CSK) and transmitted to adjacent cells or to the extracellular matrix (ECM) influence cellular functions. We investigated the force transfer across cell-to-ECM and cell-to-cell connections using magnetic twisting cytometry. We probed the CSK through integrins and E-cadherins in confluent epithelial cell lines (MCF7). At high applied stress (> 10 dyn/cm2), stiffness (stress/strain) of the CSK coupled through integrins was greater than stiffness coupled through E-cadherins. The stiffness reduction after microfilament or microtubule disruption with cytochalasin D or colchicine was greater for integrins. At low applied stress, disruption of microfilaments had very little effect on stiffness probed through either receptor type, indicating a correspondingly small contribution of microfilaments to the CSK mechanics in these confluent cells. This differs from results in nonconfluent MCF7 cells and from predictions that are based on prestressed models in which tensile stresses presumably associated with the microfilaments are the origin of prestress and, in consequence, cell stiffness. In addition, there was substantial cell spreading on collagen I-coated dishes, in contrast to little spreading on dishes coated with E-cadherin antibody. This result, together with observations of a relatively high cell stiffness probed through integrins compared with the small stiffness probed through E-cadherins, suggests that mechanical force transmission might also be important in regulating cell spreading. We conclude that the degree of confluency may be associated with different mechanics and functions of the CSK network.
A synthetic peptide derived from glycine-gated Cl- channel induces transepithelial Cl- and fluid secretion
Authors: Wallace D. P. , Tomich J. M. , Iwamoto T. , Henderson K. , Grantham J. J. , Sullivan L. P.
Published: December 2017

M2GlyR is a synthetic 23-amino acid peptide that mimics the second membrane-spanning region of the alpha-subunit of the postsynaptic glycine receptor. This peptide has been shown to form an anion-selective channel in phospholipid bilayers. We have investigated the possibility that the peptide may incorporate into the apical membrane of secretory epithelia and induce the secretion of Cl- and water. We improved the solubility of this peptide by adding four lysine residues to the carboxy terminus, C-K4-M2GlyR, and assayed its channel-forming activity using a subculture of Madin-Darby canine kidney (MDCK) cells. The addition of 100 microM C-K4-M2GlyR to the apical surface of MDCK monolayers significantly increased short-circuit current (Ise), hyperpolarized transepithelial potential difference, and induced fluid secretion. The increase in Ise was inhibited by 100 microM bumetanide and by Cl- channel inhibitors. The effectiveness of the channel blockers followed the sequence niflumic acid > or = 5-nitro-2-(3-phenylpropylamino)benzoate > diphenylamine-2-carboxylate (DPC) > glibenclamide. The effect of the peptide was not inhibited by 4.4'-diisothiocyanostilbene-2-2'-disulfonic acid. Removing Cl from the bathing solutions also inhibited the effect of the peptide. The Cl- efflux pathway induced by C-K4-M2GlyR differs from the native pathway activated by the adenosine 3',5'-cyclic monophosphate (cAMP) agonist, forskolin. First, intracellular cAMP levels were unaffected. Second, the concentration of DPC required to inhibit the effect of the peptide was much lower than that needed to block the forskolin response (100 microM vs. 3 mM). These results support the hypothesis that the synthetic peptide C-K4-M2GlyR can from Cl -selective channels in the apical membrane of secretory epithelial cells and can induce sustained transepithelial secretion of Cl- and fluid.