bluuebunny/crossref_metadata_2025 huggingface dataset:
ICAM-1-CD18 interaction mediates neutrophil cytotoxicity through protease release
Authors: Barnett Carlton C. , Moore Ernest E. , Mierau Gary W. , Partrick David A. , Biffl Walter L. , Elzi David J. , Silliman Christopher C.
Published: December 2017

Interaction of the β2-integrin complex on the polymorphonuclear neutrophil (PMN) with intercellular adhesion molecule-1 (ICAM-1) has been implicated in PMN-mediated cytotoxicity. This study examined interaction of the CD11a, CD11b, and CD18 subunits of the β2-integrin with ICAM-1, transfected into Chinese hamster ovarian (CHO) cells to avoid effects of other adhesion molecules. Incubation of quiescent PMNs with wild-type and ICAM-1-transfected CHO cells produced nominal cell lysis. Similarly, when phorbol myristate acetate (PMA)-activated PMNs were incubated with wild-type CHO cells, minimal cytotoxicity was produced. However, when ICAM-1-transfected CHO cells were incubated with PMA-activated PMNs, 40% cell lysis occurred. Blockade with a monoclonal antibody (MAb) to ICAM-1 or MAbs to CD11a, CD11b, or CD18 reduced PMN-mediated cytotoxicity to baseline. To examine the role of adhesion in cytotoxicity, we studied β2-integrin-mediated PMN adhesion to ICAM-1-transfected CHO cells and found that MAbs for CD11a, CD11b, and CD18 all abrogated PMN cytotoxicity despite disparate effects on adhesion. To assess the role of CD18, β2-integrin subunits were cross-linked, and CD18 alone mediated protease release. Moreover, ICAM-1 was immunoprecipitated from transfected CHO cells and incubated with PMNs. This soluble ICAM-1 provoked elastase release, similar to PMA, which could be inhibited by MAbs to CD18 but not MAbs to other β2-integrin subunits. In addition, coincubation with protease inhibitors eglin C and AAPVCK reduced PMN-mediated cytotoxicity to control levels. Finally, ICAM-1-transfected CHO cells were exposed to activated PMNs from a patient with chronic granulomatous disease that caused significant cell lysis, equivalent to that of PMNs from normal donors. Collectively, these data suggest that ICAM-1 provokes PMN-mediated cytotoxicity via CD18-mediated protease release.
Androgen receptors in human preadipocytes and adipocytes: regional specificities and regulation by sex steroids
Authors: Dieudonné M. N. , Pecquery R. , Boumediene A. , Leneveu M. C. , Giudicelli Y.
Published: December 2017

Various clinical and epidemiological evidence strongly suggests a major role for sex steroid hormones in the determination of anatomical specificities of fat distribution in human. To date, no studies have examined the possible presence of androgen receptors (AR) in human adipocytes and preadipocytes. We have studied AR in preadipocytes from various anatomical locations (intra-abdominal and subcutaneous) in middle-aged men and women during the proliferation and differentiation processes (adipogenesis). Androgen binding sites quantified by [3H]R-1881-specific binding in whole cell extracts were twofold higher in intra-abdominal than in subcutaneous preadipocytes but identical for the same fat depots in men and women. Western blot analysis revealed 1) the presence of AR in the nuclear and cytosolic fractions of human preadipocytes, 2) a decrease of AR expression during adipogenesis, and 3) an upregulation of AR by androgens in vitro. RT-PCR experiments showed the presence of AR mRNA in human preadipocytes and adipocytes and also the regional specificity of AR distribution. However, AR mRNA expression was found to increase during adipogenesis. The same results were observed in rat preadipocytes. In conclusion, this study clearly demonstrates the presence of AR in human preadipocytes and adipocytes and suggests that androgens may contribute, through regulation of their own receptors, to the control of adipose tissue development.
Rab11a redistributes to apical secretory canaliculus during stimulation of gastric parietal cells
Authors: Calhoun Benjamin C. , Lapierre Lynne A. , Chew Catherine S. , Goldenring James R.
Published: December 2017

Previous investigations in several systems have demonstrated that Rab3 family members redistribute to soluble fractions on fusion of secretory granules with target plasma membranes. Rab proteins are then recycled back onto mature secretory vesicles after reinternalization of the membrane. Although this cycle is well established for Rab3, far less is known about redistribution of other Rab proteins during vesicle fusion and recycling. In the gastric parietal cell, Rab11a is associated with H-K-ATPase-containing tubulovesicles, which fuse with the apical plasma membrane (secretory canaliculus) in response to agonists such as histamine. We have analyzed distribution of Rab11a and other tubulovesicle proteins in resting and histamine-stimulated rabbit parietal cells. Stimulation of isolated gastric glands in the presence of 100 μM histamine and 100 μM 3-isobutyl-1-methylxanthine did not cause a significant increase in soluble Rab11a. H-K-ATPase, Rab11a, Rab25, syntaxin 3, and SCAMPs increased immunoreactivity in stimulus-associated vesicles prepared from rabbits treated with histamine compared with those from ranitidine-treated animals. The large GTPase dynamin was found in both vesicle preparations, but there was no change in amount of immunoreactivity. Immunofluorescence staining of resting and histamine-stimulated primary cultures of parietal cells demonstrated redistribution of H-K-ATPase and Rab11a to F-actin-rich canalicular membranes. Dynamin was present on canalicular membranes in resting and stimulated cells. These results indicate that Rab11a does not cycle off the membrane during the process of tubulovesicle fusion with the secretory canaliculus. Thus Rab11a may remain associated with recycling apical membrane vesicle populations.
Ca2+-sensitizing effects of the mutations at Ile-79 and Arg-92 of troponin T in hypertrophic cardiomyopathy
Authors: Morimoto Sachio , Yanaga Fumi , Minakami Reiko , Ohtsuki Iwao
Published: December 2017

Several mutations in human cardiac troponin T (TnT) gene have been reported to cause hypertrophic cardiomyopathy (HCM). To explore the effects of the mutations on cardiac muscle contractile function under physiological conditions, human cardiac TnT mutants, Ile79Asn and Arg92Gln, as well as wild type, were expressed in Escherichia coli and exchanged into permeabilized rabbit cardiac muscle fibers, and Ca2+-activated force was determined. The free Ca2+ concentrations required for tension generation were found to be significantly lower in the mutant TnT-exchanged fibers than in the wild-type TnT-exchanged fibers, whereas no significant differences were found in tension-generating capability under maximal activating conditions and in cooperativity. These results suggest that a heightened Ca2+ sensitivity of cardiac muscle contraction is one of the factors to cause HCM associated with these TnT mutations.
Swelling-activated cation-selective channels in A6 epithelia are permeable to large cations
Authors: Li Jinqing , De Smet Patrick , Jans Danny , Simaels Jeannine , Van Driessche Willy
Published: December 2017

Effects of basolateral monovalent cation replacements (Na+ by Li+, K+, Cs+, methylammonium, and guanidinium) on permeability to86Rb of volume-sensitive cation channels (VSCC) in the basolateral membrane and on regulatory volume decrease (RVD), elicited by a hyposmotic shock, were studied in A6 epithelia in the absence of apical Na+ uptake. A complete and quick RVD occurred only when the cells were perfused with Na+ or Li+ saline. With both cations, hypotonicity increased basolateral86Rb release ([Formula: see text]), which reached a maximum after 15 min and declined back to control level. When the major cation was K+, Cs+, methylammonium, or guanidinium, the RVD was abolished. Methylammonium induced a biphasic time course of cell thickness (Tc), with an initial decline of Tc followed by a gradual increase. With K+, Cs+, or guanidinium, Tc increased monotonously after the rapid initial rise evoked by the hypotonic challenge. In the presence of K+, Cs+, or methylammonium,[Formula: see text] remained high during most of the hypotonic period, whereas with guanidinium blockage of[Formula: see text] was initiated after 6 min of hypotonicity, suggesting an intracellular location of the site of action. With all cations, 0.5 mM basolateral Gd3+ completely blocked RVD and fully abolished the [Formula: see text] increase induced by the hypotonic shock. The lanthanide also blocked the additional volume increase induced by Cs+, K+, guanidinium, or methylammonium. When pH was lowered from 7.4 to 6.0, RVD and[Formula: see text] were markedly inhibited. This study demonstrates that the VSCCs in the basolateral membrane of A6 cells are permeable to K+, Rb+, Cs+, methylammonium, and guanidinium, whereas a marked inhibitory effect is exerted by Gd3+, protons, and possibly intracellular guanidinium.
Cloning and characterization of fiber type-specific ryanodine receptor isoforms in skeletal muscles of fish
Authors: Franck Jens P. C. , Morrissette Jeffery , Keen John E. , Londraville Richard L. , Beamsley Mark , Block Barbara A.
Published: December 2017

We have cloned a group of cDNAs that encodes the skeletal ryanodine receptor isoform (RyR1) of fish from a blue marlin extraocular muscle library. The cDNAs encode a protein of 5,081 amino acids with a calculated molecular mass of 576,302 Da. The deduced amino acid sequence shows strong sequence identity to previously characterized RyR1 isoforms. An RNA probe derived from a clone of the full-length marlin RyR1 isoform hybridizes to RNA preparations from extraocular muscle and slow-twitch skeletal muscle but not to RNA preparations from fast-twitch skeletal or cardiac muscle. We have also isolated a partial RyR clone from marlin and toadfish fast-twitch muscles that shares 80% sequence identity with the corresponding region of the full-length RyR1 isoform, and a RNA probe derived from this clone hybridizes to RNA preparations from fast-twitch muscle but not to slow-twitch muscle preparations. Western blot analysis of slow-twitch muscles in fish indicates the presence of only a single high-molecular-mass RyR protein corresponding to RyR1. [3H]ryanodine binding assays revealed the fish slow-twitch muscle RyR1 had a greater sensitivity for Ca2+ than the fast-twitch muscle RyR1. The results indicate that, in fish muscle, fiber type-specific RyR1 isoforms are expressed and the two proteins are physiologically distinct.
Na+/Ca2+exchange in neonatal rat heart cells: antisense inhibition and protein half-life
Authors: Slodzinski Martin K. , Blaustein Mordecai P.
Published: December 2017

Cardiac Na+/Ca2+exchanger (NCX) protein half-life ( t ½) and antisense knockdown were studied in primary cultured neonatal rat cardiomyocytes. Protein t ½ was determined using [35S]methionine with a pulse-chase protocol. The 35S signal in NCX was identified by immunoprecipitation and Western blotting. The t ½ of NCX protein was 33 h. Low concentrations (0.5 μM) of chimeric, phosphorothioated antisense oligodeoxynucleotides (AS-oligos) targeted to the region around the start codon of NCX1 transcript were used to knock down NCX protein and activity. Control myocytes (no oligos or scrambled oligos for at least 4 days) exhibited spontaneous Ca2+ transients (measured with fura 2). The sustained (“diastolic”) Ca2+ concentration in the cytosol ([Ca2+]cyt) of control cells was unaffected by cyclopiazonic acid (CPA) plus caffeine (Caf), which promote depletion of sarcoplasmic reticular Ca2+ stores, but [Ca2+]cytrose in control cells when external Na+ was removed. In contrast, ∼60% of cells treated with AS-oligos for at least 4 days did not exhibit spontaneous Ca2+transients or respond to Na+-free medium; however, CPA + Caf did induce a prolonged elevation in [Ca2+]cytin these cells. In all cells, 50 mM K+ increased [Ca2+]cyt. NCX protein was reduced by ∼50% in cells treated with AS-oligos for 7 days but was not reduced after only 2 days. These biochemical data are consistent with the physiological evidence of NCX knockdown in ∼60% of cells.
Mitochondrial uncoupling protein may participate in futile cycling of pyruvate and other monocarboxylates
Authors: Jezek Petr , Borecky Jirí
Published: December 2017

The physiological role of monocarboxylate transport in brown adipose tissue mitochondria has been reevaluated. We studied pyruvate, α-ketoisovalerate, α-ketoisocaproate, and phenylpyruvate uniport via the uncoupling protein (UCP1) as a GDP-sensitive swelling in K+ salts induced by valinomycin or by monensin and carbonyl cyanide- p-(trifluoromethoxy)phenylhydrazone in Na+ salts. We have demonstrated that this uniport is inhibited by fatty acids. GDP inhibition in K+ salts was not abolished by an uncoupler, indicating a negligible monocarboxylic acid penetration via the lipid bilayer. In contrast, the electroneutral pyruvate uptake (swelling in ammonium pyruvate or potassium pyruvate induced by change in pH) mediated by the pyruvate carrier was inhibited by its specific inhibitor α-cyano-4-hydroxycinnamate but not by fatty acids. Moreover, α-cyano-4-hydroxycinnamate enhanced the energization of brown adipose tissue mitochondria, which was monitored fluorometrically by 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide and safranin O. Consequently, we suggest that UCP1 might participate in futile cycling of unipolar ketocarboxylates under certain physiological conditions while expelling these anions from the matrix. The cycle is completed on their return via the pyruvate carrier in an H+ symport mode.
Growth hormone regulates the distribution of L-type calcium channels in rat adipocyte membranes
Authors: Gaur Shikha , Morton Mary E. , Frick G. Peter , Goodman H. Maurice
Published: December 2017

Earlier studies demonstrated that deprivation of growth hormone (GH) for ≥3 h decreased basal and maximally stimulated cytosolic Ca2+ in rat adipocytes and suggested that membrane Ca2+channels might be decreased. Measurement of L-type Ca2+ channels in purified plasma membranes by immunoassay or dihydropyridine binding indicated a two- to fourfold decrease after 3 h of incubation without GH. No such decrease was seen in unfractionated adipocyte membrane preparations. The decrease in plasma membrane channel content was largely accounted for by redistribution of channels to a light microsomal membrane fraction. Immunoassay of α1-, α2/δ-, and β-channel subunits in membrane fractions indicated that the channels redistributed as intact complexes. Addition of GH during the 1st h of incubation prevented channel redistribution, and addition of GH after 3 h restored channel distribution to the GH-replete state of freshly isolated adipocytes. The studies suggest that GH may regulate the abundance of Ca2+ channels in the adipocyte plasma membrane and thereby modulate sensitivity to signals, the expression of which is Ca2+dependent.
Voltage-dependent block of endothelial volume-regulated anion channels by calix[4]arenes
Authors: Droogmans Guy , Prenen Jean , Eggermont Jan , Voets Thomas , Nilius Bernd
Published: December 2017

We have studied the effects of calix[4]arenes on the volume-regulated anion channel (VRAC) currents in cultured calf pulmonary artery endothelial cells. TS- and TS-TM-calix[4]arenes induced a fast inhibition at positive potentials but were ineffective at negative potentials. Maximal block occurred at potentials between 30 and 50 mV. Lowering extracellular pH enhanced the block and shifted the maximum inhibition to more negative potentials. Current inhibition was also accompanied by an increased current noise. From the analysis of the calix[4]arene-induced noise, we obtained a single-channel conductance of 9.3 ± 2.1 pS ( n = 9) at +30 mV. The voltage- and time-dependent block were described using a model in which calix[4]arenes bind to a site at an electrical distance of 0.25 inside the channel with an affinity of 220 μM at 0 mV. Binding occludes VRAC at moderately positive potentials, but calix[4]arenes permeate the channel at more positive potentials. In conclusion, our data suggest an open-channel block of VRAC by calix[4]arenes that also depends on the protonation of the binding site within the pore.
Prostaglandin F2αstimulates CFTR activity by PKA- and PKC-dependent phosphorylation
Authors: Yurko-Mauro Karin A. , Reenstra William W.
Published: December 2017

The cystic fibrosis transmembrane conductance regulator (CFTR) can be activated by protein kinase A (PKA)- or protein kinase C (PKC)-dependent phosphorylation. To understand how activation of both kinases affects CFTR activity, transfected NIH/3T3 cells were stimulated with forskolin (FSK), phorbol myristate acetate (PMA), or prostaglandin F2α (PGF). PGF stimulates inositol trisphosphate and cAMP production in NIH/3T3 cells. As measured by I− efflux, maximal CFTR activity with PGF and FSK was equivalent and fivefold greater than that with PMA. Both PGF and PMA had additive effects on FSK-dependent CFTR activity. PMA did not increase cellular cAMP, and maximal PGF-dependent CFTR activity occurred with ∼20% of the cellular cAMP observed with FSK-dependent activation. Staurosporine, but not H-89, inhibited CFTR activation and in vivo phosphorylation at low PGF concentrations. In contrast, at high PGF concentrations, CFTR activation and in vivo phosphorylation were inhibited by H-89. As judged by protease digestion, the sites of in vivo CFTR phosphorylation with FSK and PMA differed. For PGF, the data were most consistent with in vivo CFTR phosphorylation by PKA and PKC. Our data suggest that activation of PKC can enhance PKA-dependent CFTR activation.
α-Adrenergic receptors regulate human lymphocyte amiloride-sensitive sodium channels
Authors: Bubien James K. , Cornwell Trudy , Bradford Anne Lynn , Fuller Catherine M. , DuVall Michael D. , Benos Dale J.
Published: December 2017

Two independent signal transduction pathways regulate lymphocyte amiloride-sensitive sodium channels (ASSCs), one utilizing cAMP as a second messenger and the other utilizing a GTP-binding protein. This implies that two plasma membrane receptors play a role in the regulation of lymphocyte ASSCs. In this study, we tested the hypothesis that α1- and α2-adrenergic receptors independently regulate lymphocyte ASSCs via the two previously identified second messengers. Direct measurements indicated that norepinephrine increased lymphocyte cAMP and activated ASSCs. The α2-specific inhibitor, yohimbine, blocked this activation, thereby linking α2-adrenergic receptors to ASSC regulation via cAMP. The α1-specific ligand, terazosin, acted as an agonist and activated lymphocyte ASSCs but inhibited ASSC current that had been preactivated by norepinephrine or 8-(4-chlorophenylthio) (CPT)-cAMP. Terazosin had no effect on the lymphocyte whole cell ASSC currents preactivated by treatment with pertussis toxin. This finding indirectly links α1-adrenergic receptors to lymphocyte ASSC regulation via GTP-binding proteins. Terazosin had no direct inhibitory or stimulatory effects on α,β,γ-endothelial sodium channels reconstituted into planar lipid bilayers and expressed in Xenopus oocytes, ruling out a direct interaction between terazosin and the channels. These findings support the hypothesis that both α1- and α2-adrenergic receptors independently regulate lymphocyte ASSCs via GTP-binding proteins and cAMP, respectively.
Mechanical loading regulates expression of talin and its mRNA, which are concentrated at myotendinous junctions
Authors: Frenette Jérôme , Tidball James G.
Published: December 2017

The hypothesis that mechanical loading regulates talin expression in developing and adult muscle was tested using in vitro and in vivo models. Talin was selected for study because it is a key structural link between the cytoskeleton and cell membrane. In the in vitro model, C2C12myotubes were subjected to cyclic strains for 48 h. In the in vivo model, rat hindlimb muscles were unloaded for 10 days, then reloaded for 2 days. Cyclic loading of myotubes resulted in significant increases in the quantity of talin (68%) and its 190-kDa proteolytic fragment (70%), as well as talin mRNA (180%), relative to unloaded myotube cultures. Similarly, talin concentration and its mRNA increased by 68 and 136%, respectively, in soleus muscles reloaded for 2 days relative to ambulatory controls. Immunohistochemistry and in situ RT-PCR showed that talin and its mRNA are concentrated and colocalized at myotendinous junctions. Thus these findings indicate that increased mechanical loading promotes talin synthesis, which occurs principally at myotendinous junctions, according to talin mRNA distribution.
Minor role of a Ca2+-depleted sarcoplasmic reticulum in heterologous desensitization of smooth muscle to K+
Authors: Wardle Robert L. , Murphy Richard A.
Published: December 2017

Exposure of porcine carotid artery smooth muscle (PCASM) to histamine was followed by a large reduction in the rate of force generation in response to 40 mM KCl. This was shown to be a manifestation of slow attainment of a steady-state myoplasmic Ca2+concentration ([Ca2+]i). We hypothesized that if net transsarcolemmal Ca2+ flux into the depolarized PCASM cells is the same before and after a desensitizing histamine treatment, then the transient attenuation of the increase in [Ca2+]imay be due to accelerated uptake of Ca2+ by a partially depleted sarcoplasmic reticulum (SR) acting as a Ca2+ sink or superficial buffer barrier. We tested this hypothesis by eliciting responses of “desensitized PCASM” to 40 mM KCl in the presence of cyclopiazonic acid (CPA), an SR Ca2+-ATPase inhibitor. Contractions of CPA-treated tissues were attenuated less than those of tissues not treated with CPA, but they were not abolished. CPA-insensitive mechanism(s) dominated the desensitization. We conclude that histamine pretreatment reduced net transsarcolemmal Ca2+ flux into PCASM in response to 40 mM KCl.
Increased NHE2 expression in rat intestinal epithelium during ontogeny is transcriptionally mediated
Authors: Collins James F. , Kiela Pawel R. , Xu Hua , Zeng Jiamin , Ghishan Fayez K.
Published: December 2017

We have previously described changes in intestinal brush-border membrane vesicle (BBMV) Na+/H+exchange activity and characterized Na+/H+exchanger (NHE3) expression during rat ontogeny. The current studies were designed to investigate developmental changes in NHE2 expression in rat intestine. In previous studies, pH-dependent uptake of Na+ in jejunal BBMV utilizing HOE-694 inhibition demonstrated that NHE2 functional protein levels were lowest in 2-wk-old rats, higher in 3-wk-old and adult rats, and highest in 6-wk-old rats [Collins et al. Am. J. Physiol. 273 ( Cell Physiol. 42): C1937–C1946, 1997]. In the current investigation, Northern blot analyses showed that NHE2 mRNA levels in the jejunum were similar in 6-wk-old, adult, and 3-wk-old rats and three- to fivefold lower in 2-wk-old rats. In situ hybridization of 2- and 6-wk-old rat intestine with NHE2-specific probes confirmed Northern blot observations. Polyclonal antibodies were developed against an NHE2-specific peptide from amino acids 652–661. Western blots with NHE2 antiserum showed that the intensity of a specific 90-kDa band was lowest in 2-wk-old animals and four- to sixfold higher in 3- and 6-wk-old and adult animals. Immunohistochemical analysis showed specific staining of NHE2 antiserum to only the apical intestinal membrane. Furthermore, nuclear run-on analyses showed a 1.7-fold higher NHE2 transcription rate in 6-wk-old rats than in 2-wk-old rats. Overall, the current data suggest that increases in NHE2 expression upon weaning are mediated by increased gene transcription.
Loading pyranine via purinergic receptors or hypotonic stress for measurement of cytosolic pH by imaging
Authors: Gan Bing Siang , Krump Eric , Shrode Lamara D. , Grinstein Sergio
Published: December 2017

Although used extensively for the measurement of intracellular pH, derivatives of fluorescein such as 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) have suboptimal sensitivity and can generate toxic photoproducts. These limitations can be overcome using the pH-sensitive fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (pyranine), which has improved spectroscopic properties. However, the use of pyranine has been limited by the difficulties encountered in delivering this highly hydrophilic dye to the cell interior. We describe a strategy for intracellular delivery of pyranine based on the reversible activation of purinergic P2x7 receptors, which allow permeation of the dye into otherwise intact cells. When loaded into J774 or RAW cells by this method, pyranine is not only more sensitive than BCECF (the dynamic range is ∼7-fold greater), but is retained better and is less toxic. Pyranine was distributed throughout the cytosol but was not detectable in endomembrane compartments. Repeated illumination resulted in blebbing and loss of functional responsiveness of cells loaded with BCECF, whereas comparably irradiated cells loaded with pyranine remained healthy and responsive. Pyranine can also be loaded into cells not expressing P2x7 receptors by brief exposure to a hypotonic solution. The properties of cells labeled by this method are similar to those loaded via purinergic receptors and compare favorably with those of BCECF-loaded cells. Pyranine thus provides a useful alternative to fluorescein derivatives for the measurement of intracellular pH, particularly when using the high excitation intensities required for microscopic digital imaging.
Effects of cGMP and sodium nitroprusside on odor responses in turtle olfactory sensory neurons
Authors: Inamura Kouhei , Kashiwayanagi Makoto , Kurihara Kenzo
Published: December 2017

The effects of cGMP and sodium nitroprusside (SNP) on odor responses in isolated turtle olfactory neurons were examined. The inward current induced by dialysis of a mixture of 1 mM cAMP and 1 mM cGMP was similar to that induced by dialysis of 1 mM cAMP or 1 mM cGMP alone. After the neurons were desensitized by the application of 1 mM cGMP, 3 mM 8-(4-chlorophenylthio)-cAMP, a membrane-permeable cAMP analog, did not elicit any current, indicating that both cAMP and cGMP activated the same channel. Extracellular application of SNP, a nitric oxide (NO) donor, evoked inward currents in a dose-dependent manner. However, application of SNP did not induce any currents after desensitization of the cGMP-induced currents, suggesting that SNP-induced currents are mediated via the cGMP-dependent pathway. Application of the cAMP-producing odorants to the neurons induced a large inward current even after neurons were desensitized to a high concentration of cGMP or SNP. These results suggest that the transduction pathway independent of cAMP, cGMP, and NO also contributes to the generation of odor responses in addition to the cAMP-dependent pathway.
Human calcitonin receptor is directly targeted to and retained in the basolateral surface of MDCK cells
Authors: Nussenzveig Daniel R. , Matos Maria De Fatima C. , Thaw Colette N.
Published: December 2017

The human calcitonin receptor (hCTR) is expressed in polarized cells of the kidney, bone, and nervous system. In the kidney, hCTRs are found in cells of the distal nephron to which blood-borne calcitonin has access only at the basolateral surface. We expressed hCTR subtypes 1 and 2 in Madin-Darby canine kidney (MDCK) cells to establish a cell model useful for delineating the molecular mechanisms underlying hCTR polarity. Selective cell surface incubation demonstrated functional polarity of hCTRs by equilibrium binding or cross-linking of radioiodinated salmon calcitonin (125I-sCT) and cAMP accumulation stimulated by sCT. We estimated that at the steady state there are 40-fold more hCTRs on the basolateral than on the apical side. Domain-selective cell surface biotinylation followed by immunoblotting of streptavidin-agarose-fractionated biotinylated glycoproteins independently confirmed the polarized distribution of FLAG epitope-tagged hCTR-2 in the basolateral domain. Confocal microscopy of immunostained receptors revealed that hCTRs are concentrated on a lateral subdomain of the basolateral membrane. Cell surface arrival assay of newly formed receptors demonstrated that direct delivery to the basolateral domain is the mechanism by which hCTRs become polarized. Measurement of receptor turnover on the basolateral surface showed that retention contributes to hCTR distribution at the steady state.
ADP-ribose gates the fertilization channel in ascidian oocytes
Authors: Wilding Martin , Russo Gian Luigi , Galione Anthony , Marino Marcella , Dale Brian
Published: December 2017

We report an ion channel in the plasma membrane of unfertilized oocytes of the ascidian Ciona intestinalis that is directly gated by the second messenger ADP-ribose. The ion channel is permeable to Ca2+ and Na+ and is characterized by a reversal potential between 0 and +20 mV and a unitary conductance of 140 pS. Preinjection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA) or antagonists of intracellular Ca2+ release channels into oocytes did not inhibit the ADP-ribose current, demonstrating that the channel is activated in a Ca2+-independent manner. Both the fertilization current and the current induced by the injection of nicotinamide nucleotides are blocked by nicotinamide, suggesting that the ADP-ribose channel is activated at fertilization in a nicotinamide-sensitive manner. These data suggest that ascidian sperm trigger the hydrolysis of nicotinamide nucleotides in the oocyte to ADP-ribose and that this mechanism is responsible for the production of the fertilization current.
An immunologically distinct β-adaptin on tubulovesicles of gastric oxyntic cells
Authors: Okamoto Curtis T. , Jeng Young Y.
Published: December 2017

Clathrin and the γ-adaptin subunit of the AP-1 clathrin adaptor have been previously identified on H-K-ATPase-rich tubulovesicles from gastric acid secretory (oxyntic) cells [C. T. Okamoto, S. M. Karam, Y. Y. Jeng, J. G. Forte, and J. Goldenring. Am. J. Physiol. 274 ( Cell Physiol. 43): C1017–C1029]. We further characterized this AP-1 adaptor from rabbit and hog tubulovesicles biochemically and immunologically. Clathrin coat proteins were stripped from purified tubulovesicular membranes and fractionated by hydroxyapatite chromatography. The AP-1 adaptor appears to elute at 200 mM sodium phosphate, based on the presence of proteins in this fraction that are immunoreactive with antibodies against three of the four subunits of this heterotetrameric complex: the γ-, μ1-, and ς1-adaptin subunits. Although the putative β-adaptin subunit in this fraction is not immunoreactive with the anti-β-adaptin monoclonal antibody (MAb), this β-adaptin is immunoreactive with polyclonal antibodies (PAbs) directed against the peptide sequence Gly625-Asp-Leu-Leu-Gly-Asp-Leu-Leu-Asn-Leu-Asp-Leu-Gly-Pro-Pro-Val640, a region conserved between β1- and β2-adaptins that is thought to be involved in the binding of clathrin heavy chain. Immunoprecipitation of the AP-1 adaptor complex from this fraction with anti-γ-adaptin MAb 100/3 resulted in the coimmunoprecipitation of the β-adaptin that did not react with the anti-β-adaptin MAb but did react with the anti-β-adaptin PAbs. In contrast, immunoprecipitation of the AP-1 adaptor complex from crude clathrin-coated vesicles from brain resulted in the coimmunoprecipitation of a β-adaptin that was recognized by both the anti-β-adaptin MAb and PAbs. These results suggest that the tubulovesicular AP-1 adaptor complex may be distinct from that found in the trans-Golgi network and may contain an immunologically distinct β-adaptin. This immunologically distinct β-adaptin may be diagnostic of apical tubulovesicular endosomes of epithelial cells.
Thyroid hormone modifies mitochondrial phenotype by increasing protein import without altering degradation
Authors: Craig Elaine E. , Chesley Alan , Hood David A.
Published: December 2017

The mitochondrial phenotype within cardiac muscle cells is dramatically altered by thyroid hormone. We report here that this can be accounted for, in part, by modifications in the rate of mitochondrial protein import. The import of matrix-localized precursor proteins malate dehydrogenase (MDH) and ornithine carbamoyltransferase was augmented, whereas the insertion of the outer membrane protein Bcl-2 was unaffected by thyroid hormone treatment. Coincident with increases in the import of these matrix-localized precursors were thyroid hormone-induced elevations in the outer membrane receptor Tom20 and the matrix heat-shock protein mthsp70. The phospholipid cardiolipin was not involved in mediating the thyroid hormone-induced increase in import, as judged from adriamycin inhibition studies. When the import reaction was supplemented with rat heart cytosol, we found that 1) MDH import was stimulated, but Bcl-2 import was inhibited and 2) thyroid hormone did not influence the effect of the cytosol on import rates. Thus distinct requirements exist for the mitochondrial import of precursor proteins, destined for different organellar compartments. Although import of these matrix-localized proteins was augmented by thyroid hormone treatment, the proteolysis of matrix proteins was unaffected as indicated by the degradation of cytob2(167)RIC-dihydrofolate reductase, a chimeric protein missorted to the matrix. Thus our data indicate that at least some thyroid hormone-induced modifications of the mitochondrial phenotype occur due to the compartment-specific upregulation of precursor protein import rates, likely mediated via changes in the expression of protein import machinery components.
Human monocyte stimulus-coupled IL-1β posttranslational processing: modulation via monovalent cations
Authors: Perregaux David G. , Gabel Christopher A.
Published: December 2017

Lipopolysaccharide-activated human monocytes produce prointerleukin (pro-IL)-1β but release little of this inflammatory cytokine as the biologically active species. Efficient externalization of mature 17-kDa cytokine requires that the activated monocytes encounter a secondary stimulus such as ATP. To identify cation requirements of the ATP-induced process, lipopolysaccharide-activated monocytes were treated with ATP in media containing different Cl− salts or sucrose. Media devoid of Na+ did not support IL-1β processing. Titration of NaCl into choline chloride- or sucrose-based media restored 17-kDa IL-1β production. Na+ replacement, however, was not sufficient to support ATP-induced production of 17-kDa IL-1β in the presence of ≥37 mM extracellular K+ or Li+. Inhibition by K+ suggests that efflux of this cation is a necessary component of the stimulus-coupled response. The inhibitory effect achieved by Na+depletion is not due to inactivation of the ATP receptor and is distinct from a caspase-1 inhibitor. Stimulus-coupled IL-1β posttranslational processing, therefore, requires extracellular Na+ for a step downstream of the initiating stimulus but preceding caspase-1 activation.
Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions
Authors: Pavalko Fredrick M. , Chen Neal X. , Turner Charles H. , Burr David B. , Atkinson Simon , Hsieh Yeou-Fang , Qiu Jinya , Duncan Randall L.
Published: December 2017

Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of β1-integrins and α-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of α-actinin that inhibits α-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the α-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.
Mechanical stress-induced Ca2+ entry and Cl− current in cultured human aortic endothelial cells
Authors: Nakao Miki , Ono Kyoichi , Fujisawa Susumu , Iijima Toshihiko
Published: December 2017

A fluid stream through a microtube was applied to cultured human aortic endothelial cells to investigate the endothelial responses of both the ionic currents and intracellular Ca2+concentration ([Ca2+]i) to mechanical stimulation. The fluid stream induced an increase in [Ca2+]ithat was dependent on both the flow rate and the extracellular Ca2+ concentration. Gd3+ and niflumic acid inhibited the fluid stream-induced increase in [Ca2+]i, whereas Ba2+ and tetraethylammonium ion exhibited no effect. The fluid stream-induced [Ca2+]iincrease was accompanied by the activation of an inward current at −52.8 mV. The reversal potential of the fluid stream-induced current shifted to positive potentials when the external Cl− concentration was reduced but was not affected by variation of the external Na+ concentration. During the exposure to the fluid stream, [Ca2+]iwas voltage dependent, i.e., depolarization decreased [Ca2+]i. We therefore conclude that the fluid stream-induced current is largely carried by Cl− and that the Cl− current may thus play a role in modulating the Ca2+ influx by altering the membrane potential of endothelial cells.
Mechanosensitive tyrosine phosphorylation of paxillin and focal adhesion kinase in tracheal smooth muscle
Authors: Tang Dachun , Mehta Dolly , Gunst Susan J.
Published: December 2017

We investigated the role of the integrin-associated proteins focal adhesion kinase (FAK) and paxillin as mediators of mechanosensitive signal transduction in tracheal smooth muscle. In muscle strips contracted isometrically with ACh, we observed higher levels of tyrosine phosphorylation of FAK and paxillin at the optimal muscle length ( L o) than at shorter muscle lengths of 0.5 or 0.75 L o. Paxillin phosphorylation was also length sensitive in muscles activated by K+ depolarization and adjusted rapidly to changes in muscle length imposed after contractile activation by either ACh or K+depolarization. Ca2+ depletion did not affect the length sensitivity of paxillin and FAK phosphorylation in muscles activated with ACh, indicating that the mechanotransduction process can be mediated by a Ca2+-independent pathway. Since Ca2+-depleted muscles do not generate significant active tension, this suggests that the mechanotransduction mechanism is sensitive to muscle length rather than tension. We conclude that FAK and paxillin participate in an integrin-mediated mechanotransduction process in tracheal smooth muscle. We propose that this pathway may initiate alterations in smooth muscle cell structure and contractility via the remodeling of actin filaments and/or via the mechanosensitive regulation of signaling molecules involved in contractile protein activation.
Inducible expression of erythrocyte band 3 protein
Authors: Timmer Richard T. , Gunn Robert B.
Published: December 2017

A permanent cell line with inducible expression of the human anion exchanger protein 1 (hAE1) was constructed in a derivative of human embryonic kidney cells (HEK-293). In the absence of the inducer, muristerone A, the new cell line had no detectable hAE1 protein by Western analysis or additional36Cl flux. Increasing dose and incubation time with muristerone A increased the amount of protein (both unglycosylated and glycosylated). The 4,4′-dinitrostilbene-2,2′-disulfonate (DNDS)-inhibitable rapid Cl exchange flux was increased up to 40-fold in induced cells compared with noninduced cells. There was no DNDS-inhibitable rapid flux component in noninduced cells. This result demonstrates inducible expression of a new rapid Cl transport pathway that is DNDS sensitive. The additional transport of36Cl and35SO4had the characteristics of hAE1-mediated transport in erythrocytes: 1) inhibition by 250 μM DNDS, 2) activation of36Cl efflux by external Cl with a concentration producing half-maximal effect of 4.8 mM, 3) activation of36Cl efflux by external anions that was selective in the order NO3 = Cl > Br > I, and 4) activation of35SO4influx by external protons. Under the assumption that the turnover numbers of hAE1 were the same as in erythrocytes, there was good agreement (±3-fold) between the number of copies of glycosylated hAE1 and the induced tracer fluxes. This is the first expression of hAE1 in a mammalian system to track the kinetic characteristics of the native protein.
Reduced osmotic water permeability of the peritoneal barrier in aquaporin-1 knockout mice
Authors: Yang Baoxue , Folkesson Hans G. , Yang Jian , Matthay Michael A. , Ma Tonghui , Verkman A. S.
Published: December 2017

Aquaporin-1 (AQP1) water channels are expressed widely in epithelia and capillary endothelia involved in fluid transport. To test whether AQP1 facilitates water movement from capillaries into the peritoneal cavity, osmotically induced water transport rates were compared in AQP1 knockout [(−/−)], heterozygous [(+/−)], and wild-type [(+/+)] mice. In (+/+) mice, RT-PCR showed detectable transcripts for AQP1, AQP3, AQP4, AQP7, and AQP8. Immunofluorescence showed AQP1 protein in capillary endothelia and mesangium near the peritoneal surface and AQP4 in adherent muscle plasmalemma. For measurement of water transport, 2 ml of saline containing 300 mM sucrose (600 mosM) were infused rapidly into the peritoneal cavity via a catheter. Serial fluid samples (50 μl) were withdrawn over 60 min, with albumin as a volume marker. The albumin dilution data showed significantly decreased initial volume influx in AQP1 (−/−) mice: 101 ± 8, 107 ± 5, and 42 ± 4 (SE) μl/min in (+/+), (+/−), and (−/−) mice, respectively [ n = 6–10, P < 0.001, (−/−) vs. others]. Volume influx for AQP4 knockout mice was 100 ± 8 μl/min. In the absence of an osmotic gradient,3H2O uptake [half time = 2.3 and 2.2 min in (+/+) and (−/−) mice, respectively], [14C]urea uptake [half time = 7.9 and 7.7 min in (+/+) and (−/−) mice, respectively], and spontaneous isosmolar fluid absorption from the peritoneal cavity [0.47 ± 0.05 and 0.46 ± 0.04 ml/h in (+/+) and (−/−) mice, respectively] were not affected by AQP1 deletion. Therefore, AQP1 provides a major route for osmotically driven water transport across the peritoneal barrier in peritoneal dialysis.
Oxygen modulates Na+absorption in middle ear epithelium
Authors: Portier F. , van den Abbeele T. , Lecain E. , Sauvaget E. , Escoubet B. , Huy P. Tran Ba , Herman P.
Published: December 2017

The physiology of the middle ear is primarily concerned with keeping the cavities air filled and fluid free to allow transmission of the sound vibrations from the eardrum to the inner ear. Middle ear epithelial cells are thought to play a key role in this process, since they actively transport Na+ and water. The [Formula: see text] of the middle ear cavities varies from 44 to 54 mmHg in healthy human ears but may be lower in the course of secretory otitis media. The effect of chronic hypoxia on ion transport was investigated on a middle ear cell line using the short-circuit current technique. Chronic hypoxia reversibly decreased the rate of Na+absorption across the MESV cell line. Although a decrease in cellular ATP content was observed, the decrease of Na+ absorption seemed related to a primary modulation of apical Na+entry. As revealed by RNase protection assay, the decrease in the rate of apical Na+ entry strictly paralleled the decrease in the expression of transcripts encoding the α-subunit of the epithelial Na+channel. This effect of oxygen on Na+ absorption might account for 1) the presence of fluid in the middle ear in the course of secretory otitis media and 2) the beneficial effect of the ventilation tube in treating otitis media that allows the[Formula: see text] to rise and restores the fluid clearance.
Activation of mechanosensitive currents in traumatized membrane
Authors: Wan Xiaodong , Juranka Peter , Morris Catherine E.
Published: December 2017

Mechanosensitive (MS) channels, ones whose open probability varies with membrane tension in patch recordings, are diverse and ubiquitous, yet many are remarkably insensitive to mechanical stimuli in situ. Failure to elicit mechanocurrents from cells with abundant MS channels suggests that, in situ, the channels are protected from mechanical stimuli. To establish what conditions affect MS channel gating, we monitored Lymnaea neuron stretch-activated K (SAK) channels in cell-attached patches after diverse treatments. Mechanosensitivity was gauged by rapidity of onset and extent of channel activation during a step pressure applied to a “naive” patch. The following treatments enhanced mechanosensitivity: actin depolymerization (cytochalasin B), N-ethylmaleimide, an inhibitor of ATPases including myosin, elevated Ca (using A-23187), and osmotic swelling (acutely and after 24 h). Osmotic shrinking decreased mechanosensitivity. A unifying interpretation is that traumatized cortical cytoskeleton cannot prevent transmission of mechanical stimuli to plasma membrane channels. Mechanoprotection and capricious mechanosensitivity are impediments to cloning efforts with MS channels. We demonstrate a potpourri of endogenous MS currents from L-M(TK−) fibroblasts; others had reported these cells to be MS current null and hence to be suitable for expressing putative MS channels.
Resistance to TNF-α cytotoxicity can be achieved through different signaling pathways in rat mesangial cells
Authors: Guo Yan-Lin , Kang Baobin , Williamson John R.
Published: December 2017

We reported previously that Ro-318220 blocked expression of mitogen-activated protein kinase phosphatase-1 (MKP-1) induced by tumor necrosis factor-α (TNF-α) and subsequently caused apopotosis in mesangial cells (Y.-L. Guo, B. Kang, and J. R. Williamson. J. Biol. Chem. 273: 10362–10366, 1998). These data support our hypothesis that a TNF-α-inducible phosphatase may be responsible for preventing sustained activation of c-Jun NH2-terminal protein kinase (JNK) and consequent cell death in these cells (Y.-L. Guo, K. Baysal, B. Kang, L.-J. Yang, and J. R. Williamson. J. Biol. Chem. 273: 4027–4034, 1998). In this study, we investigated the involvement of protein kinase C (PKC) in regulation of MKP-1 expression in mesangial cells together with effects on viability. Although originally characterized as a PKC inhibitor, Ro-318220 inhibited TNF-α-induced MKP-1 expression through a mechanism other than blocking the PKC pathway. Furthermore, inhibition of the PKC pathway neither significantly affected TNF-α-induced MKP-1 expression nor made cells susceptible to toxic effect of TNF-α. Thus PKC activation is not essential for cells to achieve the resistance to TNF-α cytotoxicity displayed by normal mesangial cells. However, activation of PKC by phorbol 12-myristate 13-acetate (PMA) dramatically increased cellular resistance to the apoptotic effect of TNF-α. Coincidentally, PMA stimulated MKP-1 expression and suppressed JNK activation. Therefore, PMA-induced MKP-1 expression may contribute to the protective effect of PMA. These results provide a mechanistic explanation for previous documentation that PKC activation can rescue some cells from apopotosis.