bluuebunny/crossref_metadata_2025 huggingface dataset:
Na+/Ca2+exchange in catfish retina horizontal cells: regulation of intracellular Ca2+ store function
Authors: Micci M. A. , Christensen Burgess N.
Published: December 2017

The role of the Na+/Ca2+exchanger in intracellular Ca2+regulation was investigated in freshly dissociated catfish retinal horizontal cells (HC). Ca2+-permeable glutamate receptors and L-type Ca2+ channels as well as inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive intracellular Ca2+ stores regulate intracellular Ca2+ in these cells. We used the Ca2+-sensitive dye fluo 3 to measure changes in intracellular Ca2+ concentration ([Ca2+]i) under conditions in which Na+/Ca2+exchange was altered. In addition, the role of the Na+/Ca2+exchanger in the refilling of the caffeine-sensitive Ca2+ store following caffeine-stimulated Ca2+ release was assessed. Brief applications of caffeine (1–10 s) produced rapid and transient changes in [Ca2+]i. Repeated applications of caffeine produced smaller Ca2+ transients until no further Ca2+ was released. Store refilling occurred within 1–2 min and required extracellular Ca2+. Ouabain-induced increases in intracellular Na+ concentration ([Na+]i) increased both basal free [Ca2+]iand caffeine-stimulated Ca2+release. Reduction of external Na+concentration ([Na+]o) further and reversibly increased [Ca2+]iin ouabain-treated HC. This effect was not abolished by the Ca2+ channel blocker nifedipine, suggesting that increases in [Na+]ipromote net extracellular Ca2+influx through a Na+/Ca2+exchanger. Moreover, when [Na+]owas replaced by Li+, caffeine did not stimulate release of Ca2+ from the caffeine-sensitive store after Ca2+ depletion. The Na+/Ca2+exchanger inhibitor 2′,4′-dimethylbenzamil significantly reduced the caffeine-evoked Ca2+response 1 and 2 min after store depletion.
Inflammatory cytokines (IL-1α, TNF-α) and LPS modulate the Ca2+ signaling pathway in osteoblasts
Authors: Tam Vincent K. , Schotland Sandra , Green Jacob
Published: December 2017

Locally derived growth factors and cytokines in bone play a crucial role in the regulation of bone remodeling, i.e., bone formation and bone resorption processes. We studied the effect of interleukin (IL)-1α, tumor necrosis factor (TNF)-α, and Escherichia coli lipopolysaccharide (LPS) on the hormone-activated Ca2+ message system in the osteoblastic cell line UMR-106 and in osteoblastic cultures derived from neonatal rat calvariae. In both cell preparations, IL-1α, TNF-α, and LPS did not alter basal intracellular Ca2+ concentration ([Ca2+]i) but attenuated Ca2+ transients evoked by parathyroid hormone (PTH) and PGE2 in a dose (1–100 ng/ml)- and time (8–24 h)-dependent fashion. The cytokines modulated hormonally induced Ca2+ influx (estimated by using Mn2+ as a surrogate for Ca2+) as well as Ca2+ mobilization from intracellular stores. The latter was linked to suppressed production of hormonally induced inositol 1,4,5-trisphosphate. The effect of cytokines on [Ca2+]iwas abolished by the tyrosine kinase inhibitor herbimycin A (50 ng/ml). The cytokine’s effect was, however, independent of nitric oxide (NO) production, since NO donors (sodium nitroprusside) as well as permeable cGMP analogs augment, rather than attenuate, hormonally induced Ca2+ transients in osteoblasts. Given the stimulatory role of cytokines on NO production in osteoblasts, the disparate effects of cytokines and NO on the Ca2+ signaling pathway may serve an autocrine/paracrine mechanism for modulating the effect of calciotropic hormones on bone metabolism.
Effect of clenbuterol on sarcoplasmic reticulum function in single skinned mammalian skeletal muscle fibers
Authors: Bakker Anthony J. , Head Stewart I. , Wareham Anthony C. , Stephenson D. George
Published: December 2017

We examined the effect of the β2-agonist clenbuterol (50 μM) on depolarization-induced force responses and sarcoplasmic reticulum (SR) function in muscle fibers of the rat ( Rattus norvegicus; killed by halothane overdose) that had been mechanically skinned, rendering the β2-agonist pathway inoperable. Clenbuterol decreased the peak of depolarization-induced force responses in the extensor digitorum longus (EDL) and soleus fibers to 77.2 ± 9.0 and 55.6 ± 5.4%, respectively, of controls. The soleus fibers did not recover. Clenbuterol significantly and reversibly reduced SR Ca2+loading in EDL and soleus fibers to 81.5 ± 2.8 and 78.7 ± 4.0%, respectively, of controls. Clenbuterol also produced an ∼25% increase in passive leak of Ca2+ from the SR of the EDL and soleus fibers. These results indicate that clenbuterol has direct effects on fast- and slow-twitch skeletal muscle, in the absence of the β2-agonist pathway. The increased Ca2+ leak in the triad region may lead to excitation-contraction coupling damage in the soleus fibers and could also contribute to the anabolic effect of clenbuterol in vivo.
Interaction of reactive oxygen species with ion transport mechanisms
Authors: Kourie Joseph I.
Published: December 2017

The use of electrophysiological and molecular biology techniques has shed light on reactive oxygen species (ROS)-induced impairment of surface and internal membranes that control cellular signaling. These deleterious effects of ROS are due to their interaction with various ion transport proteins underlying the transmembrane signal transduction, namely, 1) ion channels, such as Ca2+ channels (including voltage-sensitive L-type Ca2+currents, dihydropyridine receptor voltage sensors, ryanodine receptor Ca2+-release channels, andd- myo-inositol 1,4,5-trisphosphate receptor Ca2+-release channels), K+ channels (such as Ca2+-activated K+ channels, inward and outward K+ currents, and ATP-sensitive K+ channels), Na+ channels, and Cl− channels; 2) ion pumps, such as sarcoplasmic reticulum and sarcolemmal Ca2+pumps, Na+-K+-ATPase (Na+ pump), and H+-ATPase (H+ pump); 3) ion exchangers such as the Na+/Ca2+exchanger and Na+/H+exchanger; and 4) ion cotransporters such as K+-Cl−, Na+-K+-Cl−, and Pi-Na+cotransporters. The mechanism of ROS-induced modifications in ion transport pathways involves 1) oxidation of sulfhydryl groups located on the ion transport proteins, 2) peroxidation of membrane phospholipids, and 3) inhibition of membrane-bound regulatory enzymes and modification of the oxidative phosphorylation and ATP levels. Alterations in the ion transport mechanisms lead to changes in a second messenger system, primarily Ca2+ homeostasis, which further augment the abnormal electrical activity and distortion of signal transduction, causing cell dysfunction, which underlies pathological conditions.
Partial restoration of cAMP-stimulated CFTR chloride channel activity in ΔF508 cells by deoxyspergualin
Authors: Jiang Canwen , Fang Shaona L. , Xiao Yong-Fu , O’Connor Sean P. , Nadler Steven G. , Lee Des W. , Jefferson Douglas M. , Kaplan Johanne M. , Smith Alan E. , Cheng Seng H.
Published: December 2017

Deletion of the codon encoding phenylalanine 508 (ΔF508) is the most common mutation in cystic fibrosis (CF) and results in a trafficking defect. Mutant ΔF508-CF transmembrane conductance regulator (CFTR) protein retains functional activity, but the nascent protein is recognized as abnormal and, in consequence, is retained in the endoplasmic reticulum (ER) and degraded. It has been proposed that this retention in the ER is mediated, at least in part, by the cellular chaperones heat shock protein (HSP) 70 and calnexin. We have investigated the ability of deoxyspergualin (DSG), a compound known to compete effectively for binding with HSP70 and HSP90, to promote trafficking of ΔF508-CFTR to the cell membrane. We show that DSG treatment of immortalized human CF epithelial cells (ΔF508) and cells expressing recombinant ΔF508-CFTR partially restored cAMP-stimulated CFTR Cl−channel activity at the plasma membrane. Although there are several possible explanations for these results, one simple interpretation is that DSG may have altered the interaction between ΔF508-CFTR and its associated chaperones. If this is correct, agents capable of altering the normal functioning of cellular chaperones may provide yet another means of restoring CFTR Cl− channel activity to CF subjects harboring this class of mutations.
Physiological effects of Na+/Ca2+exchanger knockdown by antisense oligodeoxynucleotides in arterial myocytes
Authors: Slodzinski Martin K. , Blaustein Mordecai P.
Published: December 2017

Antisense oligodeoxynucleotides (AS-oligos) targeted to the Na+/Ca2+exchanger (NCX) inhibit NCX-mediated Ca2+ influx in mesenteric artery (MA) myocytes [ Am. J. Physiol.269 ( Cell Physiol. 38): C1340–C1345, 1995]. Here, we show AS-oligo knockdown of NCX-mediated Ca2+ efflux. In initial experiments, the cytosolic free Ca2+ concentration ([Ca2+]cyt) was raised, and sarcoplasmic reticulum (SR) Ca2+ sequestration was blocked with caffeine and cyclopiazonic acid; the extracellular Na+-dependent (NCX) component of Ca2+ efflux was then selectively inhibited in AS-oligo-treated cells but not in controls (no oligos or nonsense oligos). In contrast, the La3+-sensitive (plasmalemma Ca2+ pump) component of Ca2+ efflux was unaffected in AS-oligo-treated cells. Knockdown of NCX activity was reversed by incubating AS-oligo-treated cells in normal media for 5 days. Transient [Ca2+]cytelevations evoked by serotonin (5-HT) at 15-min intervals in AS-oligo-treated cells were indistinguishable from those in controls. When cells were stimulated every 3 min, however, the peak amplitudes of the second and third responses were larger, and [Ca2+]cytreturned to baseline more slowly, in AS-oligo-treated cells than in controls. Peak 5-HT-evoked responses in the controls, but not AS-oligo-treated cells, were augmented more than twofold in Na+-free media. This implies that NCX is involved in Na+ gradient modulation of SR Ca2+ stores and cell responsiveness. The repetitive stimulation data suggest that the NCX may be important during tonic activation of arterial myocytes.
Reaction of nitric oxide with superoxide inhibits basolateral K+ channels in the rat CCD
Authors: Lu Ming , Wang Wen-Hui
Published: December 2017

We previously demonstrated that nitric oxide (NO) stimulates the basolateral small-conductance K+channel (SK) via a cGMP-dependent pathway [M. Lu and W. H. Wang. Am. J. Physiol. 270 ( Cell Physiol. 39): C1336–C1342, 1996]. Because NO at high concentration has been shown to react with superoxide ([Formula: see text]) to form peroxynitrite (OONO−) [W. A. Pryor and G. L. Squadrito. Am. J. Physiol. 268 ( Lung Cell. Mol. Physiol. 12): L699–L722, 1995 and M. S. Wolin. Microcirculation 3: 1–17, 1996], we extended our study to examine, using patch-clamp technique, the effect of high concentrations of NO on SK in cortical collecting duct (CCD) of rat kidney. Addition of NO donors [100–200 μM S-nitroso- N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP)] reduced channel activity, defined as the product of channel number and open probability, to 15 and 25% of the control value, respectively. The inhibitory effect of NO was completely abolished in the presence of 10 mM Tiron, an intracellular scavenger of [Formula: see text]. NO donors, 10 μM SNAP or SNP, which stimulate channel activity under control conditions, can also inhibit SK in the presence of an[Formula: see text] donor, pyrogallol, or in the presence of an inhibitor of superoxide dismutase, diethyldithiocarbamic acid. The inhibitory effect of NO is still observed in the presence of exogenous cGMP, suggesting that the NO-induced inhibition is not the result of decreased cGMP production. We conclude that the inhibitory effect of NO on channel activity results from an interaction between NO and [Formula: see text].
Vascular smooth muscle cell growth and insulin regulation of mitogen-activated protein kinase in hypertension
Authors: Begum Najma , Song Young , Rienzie Jennifer , Ragolia Louis
Published: December 2017

Hyperinsulinemia (HI) and insulin resistance (IR) are frequently associated with hypertension and atherosclerosis. However, the exact roles of HI and IR in the development of hypertension are unclear. Mitogen-activated protein kinases (MAPK) are well-characterized intracellular mediators of cell proliferation. In this study, we examined the contribution of MAPK pathway in insulin-stimulated mitogenesis using primary vascular smooth muscle cells (VSMCs) isolated from aortas of normotensive Wistar-Kyoto rats (WKY) and spontaneous hypertensive rats (SHR). VSMCs were grown to confluence in culture, serum starved, and examined for DNA synthesis {using [3H]thymidine (TDR), immunoprecipitated MAPK activity, and MAPK phosphatase (MKP-1) induction}. Basal rate of TDR incorporation into DNA was twofold higher in SHR compared with WKY ( P < 0.005). Insulin caused a dose-dependent increase in TDR incorporation (150% over basal levels with 100 nM in 12 h). Stimulation was sustained for 24 h with a decline toward basal in 36 h. Pretreatment with insulin-like growth factor I (IGF-I) receptor antibody did not abolish mitogenesis mediated by 10–100 nM insulin, suggesting that insulin effect is mediated via its own receptors. Insulin had a small mitogenic effect in WKY (33% over basal). Insulin-stimulated mitogenesis was accompanied by a dose-dependent increase in MAPK activity in SHR, with a peak activation (>2-fold over basal) between 5 and 10 min with 100 nM insulin. Insulin had very small effects on MAPK activity in WKY. In contrast, serum-stimulated MAPK activation was comparable in WKY and SHR. Pretreatment with MEK inhibitor, PD-98059, completely blocked insulin’s effect on MAPK activation and mitogenesis. Inhibition of phosphatidylinositol 3-kinase with wortmannin also prevented insulin’s effects on MAPK activation and mitogenesis. In WKY, insulin and IGF-I treatment resulted in a rapid induction of MKP-1, the dual-specificity MAPK phosphatase. In contrast, VSMCs from SHR were resistant to insulin with respect to MPK-1 expression. We conclude that insulin is mitogenic in SHR, and the effect appears to be mediated by sustained MAPK activation due to impaired insulin-mediated MKP-1 mRNA expression, which may act as an inhibitory feedback loop in attenuating MAPK signaling.
Regulation of Na+/Ca2+exchange activity by cytosolic Ca2+ in transfected Chinese hamster ovary cells
Authors: Fang Yu , Condrescu Madalina , Reeves John P.
Published: December 2017

Transfected Chinese hamster ovary cells stably expressing the bovine cardiac Na+/Ca2+exchanger (CK1.4 cells) were used to determine the range of cytosolic Ca2+ concentrations ([Ca2+]i) that activate Na+/Ca2+exchange activity. Ba2+ influx was measured in fura 2-loaded, ionomycin-treated cells under conditions in which the intracellular Na+concentration was clamped with gramicidin at ∼20 mM. [Ca2+]iwas varied by preincubating ionomycin-treated cells with either the acetoxymethyl ester of EGTA or medium containing 0–1 mM added CaCl2. The rate of Ba2+ influx increased in a saturable manner with [Ca2+]i, with the half-maximal activation value of 44 nM and a Hill coefficient of 1.6. When identical experiments were carried out with cells expressing a Ca2+-insensitive mutant of the exchanger, Ba2+influx did not vary with [Ca2+]i. The concentration for activation of exchange activity was similar to that reported for whole cardiac myocytes but approximately an order of magnitude lower than that reported for excised, giant patches. The reason for the difference in Ca2+regulation between whole cells and membrane patches is unknown.
Quantification of myosin heavy chain mRNA in somatic and branchial arch muscles using competitive PCR
Authors: Jung Hak Hyun , Lieber Richard L. , Ryan Allen F.
Published: December 2017

The purpose of this study was to quantify the type and amount of myosin heavy chain (MHC) mRNA within muscles of different developmental origins to determine whether the regulation of gene expression is comparable. Seven MHC isoforms were analyzed in rat adult limb (extensor digitorum longus, tibialis anterior, and soleus) and nonlimb (extraocular, thyroarytenoid, diaphragm, and masseter) muscles using a competitive PCR assay. An exogenous template that included oligonucleotide sequences specific for seven rat sarcomeric MHC isoforms (β-cardiac, 2A, 2X, 2B, extraocular, embryonic, and neonatal) as well as β-actin was constructed and used as the competitor. Only the extraocular muscle contained all seven isoforms. All seven muscles contained type 2A and type 2X MHC transcripts in varying percentages. As expected, the soleus muscle contained primarily β-cardiac MHC (87.8 ± 2.6%). Extraocular MHC was found only in the extraocular and thyroarytenoid muscles and in relatively small proportions (7.4 ± 1.5% and 4.0 ± 0.7%, respectively). Neonatal MHC was identified in extraocular (7.9 ± 0.3%), thyroarytenoid (4.4 ± 0.4%), and masseter (1.0 ± 0.2%) muscles, and embryonic MHC was identified both in extraocular (1.2 ± 0.5%) and, unexpectedly, in soleus (0.6 ± 0.1%) muscles. Absolute MHC mRNA mass was greatest in the masseter (106 pg/0.5 μg RNA) and least for the tibialis anterior (64 pg/0.5 μg RNA). These values suggest that MHC mRNA represents from 4 to 17% of the total mRNA pool in various skeletal muscles. Differences in MHC profile between somatic and branchial arch muscles suggest that the developmental origin of a muscle may, at least in part, be responsible for the MHC expression program that is implemented in the adult. An inverse relationship between the expression of β-cardiac and type 2B MHC transcripts across muscles was noted, suggesting that the expression of these two isoforms may be reciprocally regulated.
A rat parotid gland cell line, Par-C10, exhibits neurotransmitter-regulated transepithelial anion secretion
Authors: Turner John T. , Redman Robert S. , Camden Jean M. , Landon Linda A. , Quissell David O.
Published: December 2017

Because of the lack of salivary gland cell lines suitable for Ussing chamber studies, a recently established rat parotid acinar cell line, Par-C10, was grown on permeable supports and evaluated for development of transcellular resistance, polarization, and changes in short-circuit current ( I sc) in response to relevant receptor agonists. Par-C10 cultures reached confluence in 3–4 days and developed transcellular resistance values of ≥2,000 Ω ⋅ cm2. Morphological examination revealed that Par-C10 cells grew as polarized monolayers exhibiting tripartite junctional complexes and the acinar cell-specific characteristic of secretory canaliculi. Par-C10 I sc was increased in response to muscarinic cholinergic and α- and β-adrenergic agonists on the basolateral aspect of the cultures and to ATP and UTP (through P2Y2 nucleotide receptors) applied apically. Ion replacement and inhibitor studies indicated that anion secretion was the primary factor in agonist-stimulated I sc. RT-PCR, which confirmed the presence of P2Y2 nucleotide receptor mRNA in Par-C10 cells, also revealed the presence of mRNA for the cystic fibrosis transmembrane conductance regulator and ClC-2 Cl− channel proteins. These findings establish Par-C10 cells as the first cell line of salivary gland origin useful in transcellular ion secretion studies in Ussing chambers.
Intracellular ionic strength regulates the volume sensitivity of a swelling-activated anion channel
Authors: Cannon Carolyn L. , Basavappa Srisaila , Strange Kevin
Published: December 2017

Cell swelling activates an outwardly rectifying anion channel termed VSOAC (volume-sensitive organic osmolyte/anion channel). Regulation of VSOAC by intracellular electrolytes was characterized in Chinese hamster ovary cells by whole cell patch clamp. Elevation of intracellular CsCl concentration from 40 to 180 mM resulted in a concentration-dependent decrease in channel activation. Activation of VSOAC was insensitive to the salt gradient across the plasma membrane, the intracellular concentration of specific anions or cations, and the total intracellular concentration of cations, anions, or electrolytes. Comparison of cells dialyzed with either CsCl or Na2SO4solutions demonstrated directly that VSOAC activation is modulated by intracellular ionic strength (μi). The relative cell volume at which VSOAC current activation was triggered, termed the channel volume set point, decreased with decreasing ionic strength. At μi = 0.04, VSOAC activation occurred spontaneously in shrunken cells. The rate of VSOAC activation was nearly 50-fold higher in cells with μi = 0.04 vs. those with μi = 0.18. We propose that μi modulates the volume sensor responsible for channel activation.
Purification and reconstitution of an outwardly rectified Cl− channel from tracheal epithelia
Authors: Jovov Biljana , Shlyonsky Vadim G. , Berdiev Bakhram K. , Ismailov Iskander I. , Benos Dale J.
Published: December 2017

We reported the identification of three outwardly rectified Cl− channel (ORCC) candidate proteins (115, 85, and 52 kDa) from bovine tracheal epithelia. We have raised polyclonal antibodies against these isolated proteins. Incorporation into planar lipid bilayers of material partly purified from bovine tracheal apical membranes with one of these antibodies as a ligand (anti-p115) resulted in the incorporation of an ORCC identical in biophysical characteristics to one we previously described. We developed a new purification procedure to increase the yield and purity of this polypeptide. The purification scheme that gave the best results in terms of overall protein yield and purity was a combination of anion- and cation-exchange chromatography followed by immunopurification. By use of this purification scheme, 7 μg of the 115-kDa protein were purified from 20 mg of tracheal apical membrane proteins. Incorporation of this highly purified material into planar lipid bilayers revealed a DIDS-inhibitable channel with the following properties: linear conductance of 87 ± 9 pS in symmetrical Cl− solutions, halide selectivity sequence of I−> Cl− > Br−, and lack of sensitivity to protein kinase A, Ca2+, or dithiothreitol. Using anti-Gαiantibodies to precipitate Gαiprotein(s) from the partly purified preparations, we demonstrated that the loss of rectification of the ORCC was due to uncoupling of Gαi protein(s) from the ORCC protein and that the 115-kDa polypeptide is an ORCC.
Distinct Ca2+- and cAMP-dependent anion conductances in the apical membrane of polarized T84 cells
Authors: Merlin Didier , Jiang Lianwei , Strohmeier Gregg R. , Nusrat Asma , Alper Seth L. , Lencer Wayne I. , Madara James L.
Published: December 2017

Monolayers of the human colonic epithelial cell line T84 exhibit electrogenic Cl− secretion in response to the Ca2+ agonist thapsigargin and to the cAMP agonist forskolin. To evaluate directly the regulation of apical Cl−conductance by these two agonists, we have utilized amphotericin B to permeabilize selectively the basolateral membranes of T84 cell monolayers. We find that apical anion conductance is stimulated by both forskolin and thapsigargin but that these conductances are differentially sensitive to the anion channel blocker DIDS. DIDS inhibits thapsigargin-stimulated responses completely but forskolin responses only partially. Furthermore, the apical membrane anion conductances elicited by these two agonists differ in anion selectivity (for thapsigargin, I− > Cl−; for forskolin, Cl− > I−). However, the DIDS-sensitive component of the forskolin-induced conductance response exhibits anion selectivity similar to that induced by thapsigargin (I− > Cl−). Thus forskolin-induced apical anion conductance comprises at least two components, one of which has features in common with that elicited by thapsigargin.
Extracellular-regulated protein kinase cascades are activated in response to injury in human skeletal muscle
Authors: Aronson Doron , Wojtaszewski Jørgen F. P. , Thorell Anders , Nygren Jonas , Zangen David , Richter Erik A. , Ljungqvist Olle , Fielding Roger A. , Goodyear Laurie J.
Published: December 2017

The mitogen-activated protein (MAP) kinase signaling pathways are believed to act as critical signal transducers between stress stimuli and transcriptional responses in mammalian cells. However, it is not known whether these signaling cascades also participate in the response to injury in human tissues. To determine whether injury to the vastus lateralis muscle activates MAP kinase signaling in human subjects, two needle biopsies or open muscle biopsies were taken from the same incision site 30–60 min apart. The muscle biopsy procedures resulted in striking increases in dual phosphorylation of the extracellular-regulated kinases (ERK1 and ERK2) and in activity of the downstream substrate, the p90 ribosomal S6 kinase. Raf-1 kinase and MAP kinase kinase, upstream activators of ERK, were also markedly stimulated in all subjects. In addition, c-Jun NH2-terminal kinase and p38 kinase, components of two parallel MAP kinase pathways, were activated following muscle injury. The stimulation of the three MAP kinase cascades was present only in the immediate vicinity of the injury, a finding consistent with a local rather than systemic activation of these signaling cascades in response to injury. These data demonstrate that muscle injury induces the stimulation of the three MAP kinase cascades in human skeletal muscle, suggesting a physiological relevance of these protein kinases in the immediate response to tissue injury and possibly in the initiation of wound healing.
Carbachol-induced desensitization of PLC-β pathway in rat myometrium: downregulation of Gqα/G11α
Authors: Lajat Sandrine , Harbon Simone , Tanfin Zahra
Published: December 2017

In the estrogen-treated rat myometrium, carbachol increased the generation of inositol phosphates by stimulating the muscarinic receptor-Gq/G11-phospholipase C-β3 (PLC-β3) cascade. Exposure to carbachol resulted in a rapid and specific (homologous) attenuation of the subsequent muscarinic responses in terms of inositol phosphate production, PLC-β3 translocation to membrane, and contraction. Refractoriness was accompanied by a reduction of membrane muscarinic binding sites and an uncoupled state of residual receptors. Protein kinase C (PKC) altered the functionality of muscarinic receptors and contributed to the initial period of desensitization. A delayed phase of the muscarinic refractoriness was PKC independent and was associated with a downregulation of Gqα/G11α. Atropine failed to induce desensitization as well as Gqα/G11α downregulation, indicating that both events involve active occupancy of the receptor. Prolonged exposure to A[Formula: see text] reduced subsequent A[Formula: see text] as well as carbachol-mediated inositol phosphate responses and similarly induced downregulation of Gqα/G11α. Data suggest that a decrease in the level of Gqα/G11α is subsequent to its activation and may account for heterologous desensitization.
Age-associated increase in PGE2 synthesis and COX activity in murine macrophages is reversed by vitamin E
Authors: Wu Dayong , Mura Casilda , Beharka Alison A. , Han Sung Nim , Paulson K. Eric , Hwang Daniel , Meydani Simin Nikbin
Published: December 2017

We previously showed that increased macrophage and PGE2 production with age is due to enhanced cyclooxygenase (COX) activity and COX-2 expression. This study determined the effect of vitamin E supplementation on macrophage PGE2 synthesis in young and old mice and its underlying mechanism. Mice were fed 30 or 500 parts per million vitamin E for 30 days. Lipopolysaccharide (LPS)-stimulated macrophages from old mice produced significantly more PGE2 than those from young mice. Vitamin E supplementation reversed the increased PGE2 production in old mice but had no effect on macrophage PGE2production in young mice. In both LPS-stimulated and unstimulated macrophages, COX activity was significantly higher in old than in young mice at all intervals. Vitamin E supplementation completely reversed the increased COX activity in old mice to levels comparable to those of young mice but had no effect on macrophage COX activity of young mice or on COX-1 and COX-2 protein or COX-2 mRNA expression in young or old mice. Thus vitamin E reverses the age-associated increase in macrophage PGE2 production and COX activity. Vitamin E exerts its effect posttranslationally, by inhibiting COX activity.
Regulation of apical membrane Na+/H+exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe
Authors: McSwine Rebecca L. , Musch Mark W. , Bookstein Crescence , Xie Yue , Rao Mrinalini , Chang Eugene B.
Published: December 2017

We examined the regulation of the Na+/H+exchangers (NHEs) NHE2 and NHE3 by expressing them in human intestinal C2/bbe cells, which spontaneously differentiate and have little basal apical NHE activity. Unidirectional apical membrane22Na+influxes were measured in NHE2-transfected (C2N2) and NHE3-transfected (C2N3) cells under basal and stimulated conditions, and their activities were distinguished as the HOE-642-sensitive and -insensitive components of 5-( N, N-dimethyl)amiloride-inhibitable flux. Both C2N2 and C2N3 cells exhibited increased apical membrane NHE activity under non-acid-loaded conditions compared with nontransfected control cells. NHE2 was inhibited by 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate and thapsigargin, was stimulated by serum, and was unaffected by cGMP- and protein kinase C-dependent pathways. In contrast, NHE3 was inhibited by all regulatory pathways examined. Under acid-loaded conditions (which increase apical Na+ influx), NHE2 and NHE3 exhibited similar patterns of regulation, suggesting that the second messenger effects observed were not secondary to effects on cell pH. Thus, in contrast to their expression in nonepithelial cells, NHE2 and NHE3 expressed in an epithelial cell line behave similarly to endogenously expressed intestinal apical membrane NHEs. We conclude that physiological regulation and function of epithelium-specific NHEs are dependent on tissue-specific factors and/or conditional requirements.
Optical measurement of stimulus-evoked membrane dynamics in single pancreatic acinar cells
Authors: Giovannucci David R. , Yule David I. , Stuenkel Edward L.
Published: December 2017

Stimulation of pancreatic acinar cells induces the release of digestive enzymes via the exocytotic fusion of zymogen granules and activates postfusion granule membrane retrieval and receptor cycling. In the present study, changes in membrane surface area of rat single pancreatic acinar cells were monitored by cell membrane capacitance ( C m) measurements and by the membrane fluorescent dye FM1-43. When measured with the C mmethod, agonist treatment evoked a graded, transient increase in acinar cell surface area averaging 3.5%. In contrast, a 13% increase in surface area was estimated using FM1-43, corresponding to the fusion of 48 zymogen granules at a rate of 0.5 s−1. After removal of FM1-43 from the surface-accessible membrane, a residual fluorescence signal was shown by confocal microscopy to be localized in endosome-like structures and confined to the apical regions of acinar cells. The development of an optical method for monitoring the membrane turnover of single acinar cells, in combination with measurements of C m changes, reveals coincidence of exocytotic and endocytotic activity in acinar cells after hormonal stimulation.
Regulation by retinoids of P2Y2 nucleotide receptor mRNA in human uterine cervical cells
Authors: Gorodeski George I. , Burfeind Peter , Gan Shu Uin , Pal Dipika , Abdul-Karim Fadi W.
Published: December 2017

Extracellular ATP stimulates acute changes in paracellular permeability across cultures of human uterine cervical epithelial cells [G. I. Gorodeski, D. E. Peterson, B. J. De Santis, and U. Hopfer. Am. J. Physiol. 270 ( Cell Physiol. 39): C1715–C1725, 1996]. In this paper, we characterize mRNA for a P2Y2 nucleotide receptor in human cervical cells. Using oligonucleotide primers based on the sequence of human airway epithelium P2Y2receptor, a single 632-bp cDNA band was identified in RT-PCR experiments in extracts of human endocervical and ectocervical tissues and in lysates of human cervical CaSki cells, but not in 3T3 fibroblasts. The nucleotide sequence was homologous to the corresponding human airway epithelium P2Y2 receptor. Northern blot analyses revealed hybridization of the P2Y2 receptor probe to a 2.0-kb mRNA fragment, as well as to 2.2-, 3.0-, and 4.6-kb species, indicating that human cervical cells express P2Y2 receptor mRNA. Incubation of CaSki cells in retinoid-free medium abolished the ATP-induced changes in permeability and decreased the expression of the P2Y2 receptor mRNA; treatment with retinoids restored the responses to ATP and upregulated the P2Y2 receptor mRNA, suggesting that the receptor mediates ATP-related changes in permeability. Treatment with actinomycin D decreased the expression of the P2Y2 receptor RNA, but the ratio density of the receptor RNA relative to glyceraldehyde-3-phosphate dehydrogenase RNA remained unchanged, suggesting that retinoids upregulate transcription of the receptor mRNA. We conclude that retinoid-dependent modulation of the P2Y2 receptor expression, and hence of the responses to ATP, may be an important mechanism for the regulation of secretion of cervical mucus in vivo.
PKA induces Ca2+ release and enhances ciliary beat frequency in a Ca2+-dependent and -independent manner
Authors: Braiman Alex , Zagoory Orna , Priel Zvi
Published: December 2017

The intent of this work was to evaluate the role of cAMP in regulation of ciliary activity in frog mucociliary epithelium and to examine the possibility of cross talk between the cAMP- and Ca2+-dependent pathways in that regulation. Forskolin and dibutyryl cAMP induced strong transient intracellular Ca2+ concentration ([Ca2+]i) elevation and strong ciliary beat frequency enhancement with prolonged stabilization at an elevated plateau. The response was not affected by reduction of extracellular Ca2+concentration. The elevation in [Ca2+]iwas canceled by pretreatment with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM, thapsigargin, and a phospholipase C inhibitor, U-73122. Under those experimental conditions, forskolin raised the beat frequency to a moderately elevated plateau, whereas the initial strong rise in frequency was completely abolished. All effects were canceled by H-89, a selective protein kinase A (PKA) inhibitor. The results suggest a dual role for PKA in ciliary regulation. PKA releases Ca2+ from intracellular stores, strongly activating ciliary beating, and, concurrently, produces moderate prolonged enhancement of the beat frequency by a Ca2+-independent mechanism.
The opt1 gene ofDrosophila melanogaster encodes a proton-dependent dipeptide transporter
Authors: Roman Gregg , Meller Victoria , Wu Kwok Hang , Davis Ronald L.
Published: December 2017

We have cloned and characterized the opt1 gene of Drosophila melanogaster. This gene encodes a protein with significant similarity to the PTR family of oligopeptide transporters. The OPT1 protein is localized to the apical epithelial membrane domains of the midgut, rectum, and female reproductive tract. The opt1 message is maternally loaded into developing oocytes, and OPT1 is found in the α-yolk spheres of the developing embryo. It is also found throughout the neuropil of the central nervous system, with elevated expression within the α- and β-lobes of the mushroom bodies. Transport activity was examined in HeLa cells transiently expressing OPT1. This protein is a high-affinity transporter of alanylalanine; the approximate K mconstant is 48.8 μM for this substrate. OPT1 dipeptide transport activity is proton dependent. The ability of selected β-lactams to inhibit alanylalanine transport suggests that OPT1 has a broad specificity in amino acid side chains and has a substrate requirement for an α-amino group. Together these data suggest an important role for OPT1 in regulating amino acid availability.
Control of AMP deaminase 1 binding to myosin heavy chain
Authors: Hisatome Ichiro , Morisaki Takayuki , Kamma Hiroshi , Sugama Takako , Morisaki Hiroko , Ohtahara Akira , Holmes Edward W.
Published: December 2017

AMP deaminase (AMPD) plays a central role in preserving the adenylate energy charge in myocytes following exercise and in producing intermediates for the citric acid cycle in muscle. Prior studies have demonstrated that AMPD1 binds to myosin heavy chain (MHC) in vitro; binding to the myofibril varies with the state of muscle contraction in vivo, and binding of AMPD1 to MHC is required for activation of this enzyme in myocytes. The present study has identified three domains in AMPD1 that influence binding of this enzyme to MHC using a cotransfection model that permits assessment of mutations introduced into the AMPD1 peptide. One domain that encompasses residues 178–333 of this 727-amino acid peptide is essential for binding of AMPD1 to MHC. This region of AMPD1 shares sequence similarity with several regions of titin, another MHC binding protein. Two additional domains regulate binding of this peptide to MHC in response to intracellular and extracellular signals. A nucleotide binding site, which is located at residues 660–674, controls binding of AMPD1 to MHC in response to changes in intracellular ATP concentration. Deletion analyses demonstrate that the amino-terminal 65 residues of AMPD1 play a critical role in modulating the sensitivity to ATP-induced inhibition of MHC binding. Alternative splicing of the AMPD1 gene product, which alters the sequence of residues 8–12, produces two AMPD1 isoforms that exhibit different MHC binding properties in the presence of ATP. These findings are discussed in the context of the various roles proposed for AMPD in energy production in the myocyte.
Estrogen increases the permeability of the cultured human cervical epithelium by modulating cell deformability
Authors: Gorodeski George I.
Published: December 2017

Estrogens increase secretion of cervical mucus in females. The objective of this research was to study the mechanisms of estrogen action. The experimental models were human CaSki (endocervical) and hECE (ectocervical) epithelial cells cultured on filters. Incubation in steroid-free medium increased transepithelial electrical resistance ( R TE) and decreased epithelial permeability to the cell-impermeant acid pyranine. Estrogen treatment reversed the effects, indicating estrogen decreases epithelial paracellular resistance. The estrogen effect was time and dose related (EC50 ∼1 nM) and specific (estradiol = diethylstilbestrol > estrone, estriol; no effect by progesterone, testosterone, or cortisol) and was blocked by progesterone, tamoxifen, and ICI-182780 (an estrogen receptor antagonist). Estrogen treatment did not modulate dilution potential or changes in R TE in response to diC8 or to low extracellular Ca2+ (modulators of tight junctional resistance). In contrast, estrogen augmented decreases in R TE in response to hydrostatic and hypertonic gradients [modulators of resistance of lateral intercellular space ( R LIS)], suggesting estrogen decreases R LIS. Estrogen decreased cervical cell size, shortened response time relative to changes in cell size after hypertonic challenge, and augmented the decrease in cell size in response to hypertonic and hydrostatic gradients. Lowering luminal NaCl had no significant effect on R TE, and the Cl− channel blocker diphenylamine-2-carboxylate attenuated the hypertonicity-induced decrease in cell size to the same degree in control and estrogen-treated cells, suggesting estrogen effects on permeability and cell size are not mediated by modulating Na+ or Cl− transport. In contrast, estrogen increased cellular G-actin levels, suggesting estrogens shift actin steady-state toward G-actin and the cervical cell cytoskeleton toward a more flexible structure. We suggest that the mechanism by which estrogens decrease R LIS and increase permeability is by fragmenting the cytoskeleton and facilitating deformability and decreases in cervical cell size.
Energy-dependent redox state of hemea +a 3 and copper of cytochrome oxidase in perfused rat brain in situ
Authors: Matsunaga Akira , Nomura Yasutomo , Kuroda Satoshi , Tamura Mamoru , Nishihira Jun , Yoshimura Nozomu
Published: December 2017

Using the blood-free perfused rat brain, we examined the redox behavior of cytochrome oxidase of two chromophores, heme a + a 3 and copper. When perfusate inflow was stopped to induce global ischemia, the reduction of heme a + a 3 was triphasic, with a rapid phase, a slow phase, and a second rapid phase. In contrast, the reduction of copper was monophasic after the rapid phase of heme a + a 3. The triphasic reduction of heme a + a 3 was diminished by energy-depleting treatments, such as addition of an uncoupler. The time course of the reduction of copper was not affected by the energy depletion. During global ischemia the decrease in creatine phosphate nearly paralleled the reduction of heme a + a 3, whereas ATP remained at the control level until ∼60% of heme a + a 3 was reduced in the rapid phase. In the slow phase, ATP started to decrease with the reduction of copper. The redox behavior of copper was similar to the slow phase of the reduction of heme a+ a 3 because of the higher oxygen affinity of copper than of heme a + a 3. Therefore, the rapid phase of the reduction of heme a + a 3 can be used as an alarm before a decrease in ATP, whereas the reduction of copper indicates a decrease in ATP under severe hypoxia. Thus the copper signal in noninvasive near-infrared spectroscopy is a useful parameter for the clinical setting.
Modeling cell volume regulation in nonexcitable cells: the roles of the Na+ pump and of cotransport systems
Authors: Hernández Julio A. , Cristina Ernesto
Published: December 2017

The purpose of this study is to contribute to understanding the role of Na+-K+-ATPase and of ionic cotransporters in the regulation of cell volume, by employing a model that describes the rates of change of the intracellular concentrations of Na+, K+, and Cl−, of the cell volume, and of the membrane potential. In most previous models of dynamic cellular phenomena, Na+-K+-ATPase is incorporated via phenomenological formulations; the enzyme is incorporated here via an explicit kinetic scheme. Another feature of the present model is the capability to perform short-term cell volume regulation mediated by cotransporters of KCl and NaCl. The model is employed to perform numerical simulations for a “typical” nonpolarized animal cell. Basically, the results are consistent with the view that the Na+ pump mainly plays a long-term role in the maintenance of the electrochemical gradients of Na+ and K+ and that short-term cell volume regulation is achieved via passive transport, exemplified in this case by the cotransport of KCl and NaCl.
Proton secretion in the male reproductive tract: involvement of Cl−-independent HCO 3 − transport
Authors: Breton Sylvie , Hammar Katherine , Smith Peter J. S. , Brown Dennis
Published: December 2017

The lumen of the epididymis is the site where spermatozoa undergo their final maturation and acquire the capacity to become motile. An acidic luminal fluid is required for the maintenance of sperm quiescence and for the prevention of premature activation of acrosomal enzymes during their storage in the cauda epididymis and vas deferens. We have previously demonstrated that a vacuolar H+-ATPase [proton pump (PP)] is present in the apical pole of apical and narrow cells in the caput epididymis and of clear cells in the corpus and cauda epididymis and that this PP is responsible for the majority of proton secretion in the proximal vas deferens. We now show that PP-rich cells in the vas deferens express a high level of carbonic anhydrase type II (CAII) and that acetazolamide markedly inhibits the rate of proton secretion by 46.2 ± 6.1%. The rate of acidification was independent of Cl− and was strongly inhibited by SITS under both normal and Cl−-free conditions (50.6 ± 5.0 and 57.5 ± 6.0%, respectively). In the presence of Cl−, diphenylamine-2-carboxylate (DPC) had no effect, whereas SITS inhibited proton secretion by 63.7 ± 11.3% when applied together with DPC. In Cl−-free solution, DPC markedly inhibited proton efflux by 45.1 ± 7.6%, SITS produced an additional inhibition of 18.2 ± 6.6%, and bafilomycin had no additive effect. In conclusion, we propose that CAII plays a major role in proton secretion by the proximal vas deferens. Acidification does not require the presence of Cl−, but DPC-sensitive Cl− channels might contribute to basolateral extrusion of[Formula: see text] under Cl−-free conditions. The inhibition by SITS observed under both normal and Cl−-free conditions indicates that a Cl−/[Formula: see text]exchanger is not involved and that an alternative[Formula: see text] transporter participates in proton secretion in the proximal vas deferens.
Collagen gel overlay induces apoptosis of polarized cells in cultures: disoriented cell death
Authors: Tang Ming-Jer , Hu Jeng-Jay , Lin Hsi-Hui , Chiu Wen-Tai , Jiang Si-Tse
Published: December 2017

In this study, we attempted to investigate the response of polarized cells to inappropriate interaction with the extracellular matrix. Cell lines of epithelial [Madin-Darby canine kidney (MDCK) and LLC-PK1], endothelial [bovine aortic endothelial cells (BAEC)], and mesenchymal (ESK-4 and NIH/3T3) origins were employed. With collagen gel overlay, MDCK cells underwent membrane remodeling and gradually developed lumen formation within 24 h. Apoptosis could also be observed following cell remodeling. The ratio of apoptosis was enhanced from 12.1 ± 2.4% within 24 h to 58.4 ± 9.8% at day 3, and finally the monolayer was disintegrated. Collagen gel overlay-induced apoptosis was not a result of physical stress, since agarose gel overlay did not induce any morphological alterations. All epithelial and endothelial cells examined developed apoptosis in response to collagen overlay. In contrast, collagen overlay did not affect growth of fibroblasts at all, although their growth under agarose gel was slightly hindered due to physical stress. Collagen overlay-induced apoptosis seems to be a unique phenomenon for polarized cells and thus is defined as “disoriented cell death.” Furthermore, anti-α2-integrin antibody could abolish collagen overlay-induced morphological changes and apoptosis in MDCK cells, indicating that signals through α2-integrin on the apical membrane are required for disoriented cell death. Finally, Bcl-2 overexpression prolonged survival of MDCK cells in response to collagen overlay, but these cells eventually developed apoptosis due to downregulation of Bcl-2 protein. These findings indicate that inappropriate cell-matrix interaction results in apoptosis, which may account for cell death mechanisms during developmental processes or under pathological conditions.
Electrophysiological characteristics of the proton-coupled peptide transporter PEPT2 cloned from rat brain
Authors: Wang Hong , Fei You-Jun , Ganapathy Vadivel , Leibach Frederick H.
Published: December 2017

We have cloned a peptide transporter from rat brain and found it to be identical to rat kidney PEPT2. In the present study we characterize the transport function of the rat brain PEPT2, with special emphasis on electrophysiological properties and interaction with N-acetyl-l-aspartyl-l-glutamate (NAAG). When heterologously expressed in HeLa cells and in SK-N-SH cells, PEPT2 transports several dipeptides but not free amino acids in the presence of a proton gradient. NAAG competes with other peptides for the PEPT2-mediated transport process. When PEPT2 is expressed in Xenopus laevis oocytes, substrate-induced inward currents are detectable with dipeptides of differing charge in the presence of a proton gradient. Proton activation kinetics are similar for differently charged peptides. NAAG is a transportable substrate for PEPT2, as evidenced by NAAG-induced currents. The Hill coefficient for protons for the activation of the transport of differently charged peptides, including NAAG, is 1. Although the peptide-to-proton stoichiometry for negatively charged peptides is 1, the transport nonetheless is associated with transfer of positive charge into the oocyte, as indicated by peptide-induced inward currents.