bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of [Arg8]vasopressin on electrogenic chloride secretion in cultured rat epididymal epithelia
Authors: Lai K. B. , Fu W. O. , Ko W. H. , Chan H. C. , Wong P. Y.
Published: December 2017

Primary cultured rat efferent ductal epithelia and cauda epididymal epithelial were mounted in Ussing chambers to study the effect of arginine vasopressin (AVP) on chloride secretion in the male excurrent duct. The regional differences in the signal transduction pathways involved were also investigated. In both the efferent duct and the cauda epididymidis, basolateral addition of AVP resulted in a dose-dependent increase in the short-circuit current (Isc), which was mediated via V1 receptor. Replacement of ambient Cl- with gluconate or pretreatment of a Cl- channel blocker, diphenylamine-2-carboxylate (apical, 1 mM), completely abolished the response, whereas addition of amiloride had no effect on the Isc. Pretreating the epithelia of the efferent duct with indomethacin (apical, 5 microM) or forskolin (basolateral, 1 microM), but not thapsigargin (apical, 1 microM) or trifluoperazine (apical, 20 microM), significantly inhibited the AVP response (P < 0.001). By comparison, pretreating the epithelia of the cauda epididymidis with any of the four agents significantly reduced the AVP-evoked response. These results suggested that the stimulation of chloride secretion by AVP in the efferent duct and the cauda epididymidis is mediated by prostaglandin synthesis and involves adenosine 3',5'-cyclic monophosphate (cAMP) as a second messenger. In the cauda epididymidis, calcium, in addition to cAMP, may play a role in mediating the AVP-induced response.
Differential expression of Isk mRNAs in mouse tissue during development and pregnancy
Authors: Felipe A. , Knittle T. J. , Doyle K. L. , Snyders D. J. , Tamkun M. M.
Published: December 2017

The molecular isoform of the cDNA clone Isk present in the AT-1 atrial tumor cell line was characterized by molecular cloning of Isk cDNA. Since Isk mRNA was found in mouse heart, kidney, and uterus, a complete study of its expression during development in the heart and kidney was performed, in addition to its expression in the uterus during pregnancy. In the heart, Isk showed a 4-fold upregulation during the perinatal period followed by a 20-fold decrease between birth and the adult state. Furthermore, the two 0.9- and 3.4-kb transcripts were differentially regulated after birth. In the kidney, Isk progressively increased 10-fold, reaching steady-state adult values at 21 days. Isk mRNA levels in the uterus increased threefold at late pregnancy and decreased sixfold rapidly after birth. The Isk gene is differentially expressed during development in kidney and cardiac tissue, and both Isk transcripts appeared to be differentially regulated. Furthermore, the drastic changes in transcript levels before delivery and after birth suggest that Isk plays a significant role in myometrium during late pregnancy and delivery.
Polyamine deficiency causes reorganization of F-actin and tropomyosin in IEC-6 cells
Authors: McCormack S. A. , Wang J. Y. , Johnson L. R.
Published: December 2017

In earlier work we have shown that polyamine-deficient IEC-6 cells lose most of their ability to migrate. In this report we describe the effect of polyamine deficiency on the cytoskeleton of migrating IEC-6 cells. Cells were grown on cover slips for 4 days. One-third of the monolayer was removed, and the remainder was incubated for 6 h. The monolayers were fixed and stained with rhodamine phalloidin for actin filaments and by immunocytochemistry for tropomyosin. In control cells, actin filaments were found as stress fibers traversing the cell, in a thin actin cortex often visible on only one edge of the cell, and in fine fibers extending into the lamellipodia. Tropomyosin was found in the same distribution. A Western blot showed that tropomyosin was present as 35- and 37-kDa isoforms. In polyamine-deficient cells, actin stress fibers were less dense, whereas the actin cortex was greatly increased in density and lamellipodia were less extensive. Tropomyosin distribution was similar and included a 30-kDa isoform not seen previously. In spite of the obvious changes in the distribution of these cytoskeletal proteins, the concentrations of filamentous actin, beta-actin mRNA, and the higher molecular weight tropomyosin isoforms did not change. In all cases the addition of putrescine to polyamine-deficient cells prevented the changes described. We conclude that polyamines are essential for migration in this system because of their effects on the organization of cytoskeletal actin, tropomyosin, and perhaps other proteins as well.
Identification of A1 adenosine receptors in rat cochlea coupled to inhibition of adenylyl cyclase
Authors: Ramkumar V. , Ravi R. , Wilson M. C. , Gettys T. W. , Whitworth C. , Rybak L. P.
Published: December 2017

A1 adenosine receptors (A1ARs) are found in a number of tissues in the body where their physiological roles have been identified. In the cochlea, neither the existence of these receptors nor a physiological role of adenosine has been described previously. Membranes prepared from rat cochlea demonstrated high affinity and saturable binding to N6-2-(4-amino-3-[125I]iodophenyl)ethyladenosine ([125I]APNEA), an A1AR agonist, with maximum binding capacity and dissociation constant values being 40.5 +/- 0.5 fmol/mg protein and 1.28 +/- 0.03 nM, respectively. Adenosine analogues competed for [125I]APNEA binding sites with a rank order of potency characteristic of these sites being the A1AR. The [125I]APNEA binding was significantly reduced by pertussis toxin, indicating coupling of these receptors with the Gi and/or Go proteins in cochlear membranes. Photoaffinity labeling of the receptor protein with the A1AR agonist N6-2-(4-azido-3[125I]iodophenyl)ethyladenosine showed specific labeling of a 36-kDa receptor protein. Activation of the A1AR with R-phenylisopropyladenosine (R-PIA) led to inhibition of forskolin-stimulated adenylyl cyclase activity. Amplification of reverse-transcribed RNA derived from cochlear tissue by polymerase chain reaction (using primers for the bovine A1AR) yielded a 770-bp product that hybridized to an A1AR cDNA probe on Southern blots. These data indicate the presence of an inhibitory receptor in the peripheral auditory system, which may play an important role in modulating auditory functions.
Cytoskeletal dissociation of ezrin during renal anoxia: role in microvillar injury
Authors: Chen J. , Doctor R. B. , Mandel L. J.
Published: December 2017

The association/dissociation of ezrin, a microvillar membrane-cytoskeleton linker, was studied to search for the initial step leading to anoxia-induced brush-border breakdown in a rabbit proximal tubule suspension. Electron microscopy studies display time-dependent damage to the microvilli during anoxia; immunoblots demonstrate the dissociation of ezrin from the cytoskeleton, reflected by the significant decrease in Triton X-100-insoluble ezrin from control (91%) to 39% after 30 min. Simultaneously, Triton X-100-soluble and extracellular ezrin increased with no change in total ezrin, Triton X-100 solubility of actin, or total intracellular protein. Parallel immunocytochemistry studies show diffusion of ezrin from the brush border, where ezrin is highly colocalized with F-actin during normoxia into the cytoplasm. Thirty minutes of reoxygenation following 30 min of anoxia causes recovery of the microvillar structure and reassociation of ezrin to the cytoskeleton and the brush border. Application of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (4 mM) or inhibition of intracellular calpain or calcineurin do not prevent the dissociation of ezrin during anoxia. We conclude that ezrin-cytoskeletal dissociation may initiate microvillar breakdown during anoxia via calcium-independent mechanisms.
Glucocorticoids regulate Na+/H+ exchange expression and activity in region- and tissue-specific manner
Authors: Cho J. H. , Musch M. W. , DePaoli A. M. , Bookstein C. M. , Xie Y. , Burant C. F. , Rao M. C. , Chang E. B.
Published: December 2017

Na+/H+ exchangers (NHEs) are integral membrane proteins that exchange Na+ for H+ across membranes. Four isoforms have been cloned (NHE-1-4). NHE-3 localizes to the apical domain, and its expression is increased in dexamethasone-treated rats by Northern analysis. This stimulatory effect on expression is region and tissue specific, being present in ileum and proximal colon, but not in jejunum, distal colon, or kidney. The increase in transcript expression in ileum correlates with an increase in protein expression by immunoblotting. Changes in apical Na+/H+ exchange activity, as measured by 22Na uptake into brush-border membrane vesicles, correlate with expression differences, with significant increases observed in ileum and proximal colon. In situ hybridization showed NHE-3 mRNA only in villus and absorptive cells of control rats, the pattern not being altered by dexamethasone treatment. This suggests that dexamethasone does not increase expression by inducing crypt cells to express NHE-3 prematurely. We conclude that glucocorticoids selectively increase intestinal NHE-3 activity in a region-specific manner and that this effect also appears to be tissue specific.
Ontogeny of cationic amino acid transport systems in rat placenta
Authors: Malandro M. S. , Beveridge M. J. , Kilberg M. S. , Novak D. A.
Published: December 2017

Gestational regulation of the placental transfer of amino acids from maternal to fetal circulations is essential for the proper development of the fetus. The cationic amino acid transport systems of the microvillous (maternal facing) and basal (fetal facing) membranes of the rat placental syncytiotrophoblast were examined. Inhibition analysis documented the presence of three kinetically distinct cationic amino acid transport mechanisms: a single Na(+)-dependent mechanism in the microvillous membrane, which increased in activity from 14 to 20 days gestation but was absent from the basal membrane throughout the entire gestational period (system Bo,+), and two Na(+)-independent transport systems in both membrane domains, one that is completely inhibited by leucine, which increased in activity in both the microvillous and basal membrane domains, and the other that is leucine insensitive, which remained fairly constant in the basal membrane and increased throughout gestation in the microvillous membrane (system y1+). Northern analysis with the system y1+ cDNA revealed a specific band of approximately 7.4-7.9 kb, which increased with increasing gestational age.
Decreased protein phosphorylation induced by anoxia in proximal renal tubules
Authors: Kobryn C. E. , Mandel L. J.
Published: December 2017

Anoxia-induced depletion of cellular ATP may affect the degree of protein phosphorylation due to kinase inhibition. In this study, protein phosphorylation was measured in rabbit kidney proximal tubules under normoxic or anoxic conditions in a medium containing 32P. During the first 20 min of normoxia, phosphate incorporation was linear, averaging 17 +/- 5 pmol.mg protein-1.min-1 and was 70% inhibited by the protein kinase C inhibitor chelerythrine chloride. Phosphorylation measurements initiated simultaneously with anoxic conditions (95% N2-5% CO2) significantly reduced the initial rate to 58% of control, saturating after 15 min, and reaching 28 +/- 5% of the normoxic value after 60 min of incubation. The phosphatase inhibitor calyculin A did not affect the initial rate of phosphate incorporation by anoxic tubules but increased phosphate incorporation at 60 min to 43 +/- 4% of normoxia. Addition of 32P after 15 min of anoxia abolished phosphate incorporation, demonstrating that kinase activity was completely inhibited. Cellular phosphate uptake was measured and found not to be rate limiting for phosphorylation. Chelerythrine chloride increased lactate dehydrogenase (LDH) release during normoxia, and calyculin A decreased anoxia-induced LDH release, suggesting that protein phosphorylation events may control plasma membrane permeability.
G protein effects on Ca2+ release and excitation-contraction coupling in skeletal muscle fibers
Authors: Carney-Anderson L. , Donaldson S. K.
Published: December 2017

Skeletal muscle excitation-contraction (EC) coupling may involve secondary mechanisms, such as those involving G proteins. The aim of this study was to identify possible G protein effects on sarcoplasmic reticulum (SR) Ca2+ release, in general, and on voltage-dependent EC coupling, in particular. Effects of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) were studied using a single peeled rabbit skeletal muscle fiber preparation that is capable of releasing SR Ca2+ in response to transverse tubule (TT) depolarization. Because of possible nonspecific and residual effects of 200 microM GTP gamma S, a lower concentration of 50 microM GTP gamma S was used to stimulate G proteins in the peeled fiber system. Under conditions for steady-state (resting) polarization of TT, GTP gamma S rarely elicited SR Ca2+ release. When the TTs are in steady-state (resting) depolarization, 50 microM GTP gamma S or GTP elicited SR Ca2+ release and associated tension transients in only 69% of fibers tested. In contrast GTP gamma S always augmented Ca2+ release during TT depolarization-induced EC coupling. These results indicate the presence of at least two excitatory G proteins for SR Ca2+ release, only one of which is a modulator, albeit nonessential, of peeled fiber EC coupling.
Method for manipulation of cytosolic pH in cells clamped in the whole cell or perforated-patch configurations
Authors: Grinstein S. , Romanek R. , Rotstein O. D.
Published: December 2017

A number of methods have been developed to manipulate the intracellular pH (pHi) of intact cells. However, such methods are not applicable when cells are studied using the patch-clamp technique, due to the continuity of the cell interior with the recording pipette. The perfused-pipette method can be used to modify pHi in the whole cell configuration, but this approach is slow, technically demanding, and not useful in the case of the perforated-patch configuration. In this report, we introduce a simple procedure that enables the investigator to predictably and reversibly alter pHi in cells clamped in either the whole cell or perforated-patch modes. The method is based on the provision of a virtually unlimited reservoir of an intracellular H+ (equivalent) donor/acceptor system, by inclusion of large concentrations of permeable weak electrolytes in the pipette solution. This system not only provides a means for the imposition and maintenance of a chosen pHi but, by changing the external concentration of the weak electrolyte, enables the investigator to rapidly and reversibly change pHi or the transmembrane delta pH during the course of an experiment. The effectiveness of the procedure was validated in peritoneal macrophages by two methods: 1) direct measurement of pHi in single cells by fluorescence ratio determinations and 2) estimation of the reversal potential of H(+)-selective currents. The pHi clamping procedure is shown to be effective using either organic or inorganic weak bases in the whole cell configuration. In addition, because NH+4/NH3 can readily permeate the pores formed by nystatin or amphotericin, the method is also shown to apply to the perforated-patch configuration.
Female sex hormones inhibit volume regulation in rat brain astrocyte culture
Authors: Fraser C. L. , Swanson R. A.
Published: December 2017

To determine whether sex steroids play any role in the increased morbidity associated with acute symptomatic hyponatremia in menstruant females, we studied the actions of estradiol, progesterone, and testosterone on regulatory volume decrease (RVD) of brain astrocytes in culture. To determine intracellular space with the use of 3-O-[methyl-D-3H] glucose, cells were cultured in media containing either estradiol or progesterone. Those treated with ouabain were unable to regulate volume normally, whereas testosterone-treated cells displayed normal RVD. After 15 s of hypotonic exposure, control cell volume and 100 nM testosterone-treated cell volume increased by 26 and 31%, respectively. Cell volume in control cells changed from 1.74 +/- 0.24 to 2.41 +/- 0.28 microliters/mg protein. At the same time, cells treated with either 10 nM estradiol or 10 nM progesterone significantly (P < 0.01) increased their volume by 129 and 90%, respectively. Both the antiestrogen agent tamoxifen and the antiprogesterone agent mifepristone (RU-486) blocked the effects of estradiol and progesterone. The Na-K-ATPase pump, which plays an important role in cell RVD, was significantly (P < 0.03) inhibited by 32 and 21% in estradiol- and progesterone-treated cells, but significantly (P < 0.001) stimulated (49%) by testosterone treatment. Taken together, these results provide a possible explanation for the increased morbidity associated with acute symptomatic hyponatremia in menstruant females.
NMR-visible intracellular P(i) and phosphoesters during regulation of Na(+)-P(i) cotransport in opossum kidney cells
Authors: Barac-Nieto M. , Spitzer A.
Published: December 2017

There is an inverse relationship between intracellular concentration of P(i) ([P(i)]i) in the kidney and maximum velocity (Vmax) of Na(+)-P(i) cotransport in brush-border membrane vesicles both in P(i)-deprived and growing animals. However, at any given [P(i)]i, the Vmax is substantially higher in growing than in P(i)-deprived animals. This suggests that growth and P(i) depletion act on P(i) transport via different mechanisms. We tested this hypothesis by measuring the nuclear magnetic resonance-visible phosphate and the Vmax of Na(+)-P(i) cotransport in proximal tubule-like cells [opossum kidney (OK) cells] cultured in vitro. OK cells incubated in 1 mM extracellular P(i) had a [P(i)]i of 1.1 +/- 0.2 mM and a P(i) uptake of 1.47 +/- 0.06 nmol/mg in 5 min. Exposure of OK cells to P(i)-free medium decreased [P(i)]i by 80 +/- 7% (P < 0.01) and stimulated P(i) transport by 34 +/- 7% (P < 0.05). Exposure of OK cells to 10(-8) M insulin-like growth factor I (IGF-I) increased P(i) transport by 25 +/- 8% (P < 0.05) but did not affect [P(i)]i. The stimulation of Vmax produced by IGF-I was additive to that due to P(i) restriction. In addition, P(i) deprivation decreased the phosphomonoesters by 0.66 +/- 0.04-fold (P < 0.05) and increased the phosphodiesters by 2.5 +/- 0.5-fold (P < 0.01). Treatment with IGF-I increased both the phosphomonoesters (1.2 +/- 0.1-fold) and the phosphodiesters (4.1 +/- 0.6-fold). These results support the assumption that low P(i) supply and IGF-I stimulate Na(+)-P(i) cotransport by independent mechanisms.
In vivo upregulation of adipocyte alpha 2-adrenoceptors by androgens is consequence of direct action on fat cells
Authors: Bouloumie A. , Valet P. , Dauzats M. , Lafontan M. , Saulnier-Blache J. S.
Published: December 2017

Adipose precursor cells from male hamsters were exposed to testosterone in primary culture. The effect of the sex steroid on alpha 2-adrenoceptor (AR) expression was studied. Testosterone enhanced alpha 2-AR expression during adipose conversion to an identical extent to that determined in vivo without modification of the differentiation state as estimated by glycerol-3-phosphate dehydrogenase activity. The expression of preadipocyte alha 2-ARs from female hamster was also submitted to the testosterone control. The upregulating effect was androgen specific, since only testosterone and dihydrotestosterone were efficient in inducing an increase of alpha 2-AR density, whereas estradiol, dexamethasone, progesterone, and androstenedione were without any action. To conclude, androgens act directly on fat cells by upregulating alpha 2-AR expression. The effects are not mediated by aromatization into estrogens and probably involve androgen receptor interactions.
Renal cells transformed with SV40 contain a high-conductance calcium-insensitive potassium channel
Authors: Teulon J. , Ronco P. M. , Vandewalle A.
Published: December 2017

The inside-out variant of the patch-clamp technique was used to investigate the properties of a K+ channel occurring in 20% of patches from two renal cell lines transformed with the wild-type simian virus 40 (SV40) (Vandewalle et al. J. Cell. Physiol. 141: 203-221, 1989). This channel was practically absent from the primary cultures of renal cortical cells from which the cell lines were originally derived. With identical K(+)-rich solutions on both sides of the membrane patch, the channel showed an inwardly rectifying current-voltage relationship with unit conductances of 151.8 +/- 4.8 pS at negative and 86.4 +/- 5.9 pS at positive voltages (n = 18). When K+ in the bath was replaced by Na+, a mean reversal potential of 57.0 +/- 5.2 mV (n = 6) was observed from which a K(+)-to-Na+ permeability ratio of 13 was calculated. The channel was insensitive to internal Ca2+ and was blocked by internal Ba2+. No clear dependence on voltage was apparent. This channel bears no resemblance to any epithelial K+ channel and may be a novel type of K+ channel. Its occurrence in two transformed cell lines with quite distinct phenotypes, one of proximal cells (RC.SV1) and the other of thick ascending limb cells (RC.SV2), suggests that transformation by SV40 might be responsible for its appearance.
Salt stress increases abundance and glycosylation of CFTR localized at apical surfaces of salt gland secretory cells
Authors: Ernst S. A. , Crawford K. M. , Post M. A. , Cohn J. A.
Published: December 2017

Osmotic stress elicits hypertonic NaCl secretion and promotes structural and biochemical differentiation in avian salt glands. In addition to cholinergic control, Cl- secretion is stimulated by vasoactive intestinal peptide (VIP), suggesting that the cystic fibrosis transmembrane conductance regulator (CFTR) may be present and that its expression may be regulated by chronic salt stress. Anion efflux, assayed by 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence changes in single cells, was stimulated by VIP or 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. Immunoblots with a COOH-terminal peptide antibody to human CFTR revealed approximately 170- and approximately 180-kDa bands in lysates from control and salt-stressed glands, respectively. Both variants reduced to approximately 140 kDa after N-glycanase digestion and gave identical tryptic phosphopeptide maps after immunoprecipitation and phosphorylation by protein kinase A. CFTR was localized to apical membranes by immunofluorescence and, additionally, to subapical vesicles by immunoelectron microscopy. Salt stress induced an approximately twofold increase in CFTR abundance/cell protein (approximately 5-fold/cell) and intensified apical membrane immunofluorescence. For comparison, Na+ pump expression increased approximately fourfold per cell protein with little change in actin. Thus differentiation induced by salt stress is accompanied by alteration in CFTR abundance and glycosylation. Upregulation of CFTR likely contributes to increased efficiency of Cl- secretion.
Muscarinic stimulation of gallbladder epithelium. III. Antagonism of cAMP-mediated effects
Authors: Altenberg G. A. , Subramanyam M. , Reuss L.
Published: December 2017

Elevation of adenosine 3',5'-cyclic monophosphate (cAMP) levels in Necturus gallbladder (NGB) epithelium activates an apical membrane Cl- conductance and decreases transepithelial fluid transport (Jv). Acetylcholine (ACh), which had no effects on Jv by itself, antagonized the electrophysiological effects of forskolin (FSK) and theophylline and the decrease in Jv produced by FSK. By itself, ACh had no effects on basal cAMP levels but antagonized the increases in cAMP induced by FSK and theophylline. ACh had no effect on phosphodiesterase activity and prevented both the electrophysiological response and the elevation in cAMP by theophylline. In conclusion, the effect of ACh is mediated by inhibition of adenylate cyclase. A pertussis toxin (PTX)-sensitive G protein may mediate inhibition of adenylate cyclase because pretreatment with PTX prevented the reversal of the electrophysiological effects of FSK by ACh, and PTX catalyzed the ribosylation of cell membranes from NGB epithelium. ACh could have a physiological role in modulating the effects of secretagogues that act via elevation of cAMP levels.
The volume-sensitive organic osmolyte-anion channel VSOAC is regulated by nonhydrolytic ATP binding
Authors: Jackson P. S. , Morrison R. , Strange K.
Published: December 2017

Efflux of intracellular organic osmolytes to the external medium is a ubiquitous response to cell swelling. Accumulating evidence indicates that volume regulatory loss of structurally unrelated organic osmolytes from cells is mediated by a relatively nonselective volume-sensitive anion channel. In C6 cells, we have termed this channel VSOAC for volume-sensitive organic osmolyte-anion channel. Swelling-induced activation of VSOAC required the presence of ATP or nonhydrolyzable ATP analogues [adenosine 5'-O-(3-thiotriphosphate), adenylylmethyl-enediphosphonate (AMP-PCP), or 5'-adenylylimidodiphosphate] in the patch pipette. Sustained activation of VSOAC also required ATP. Channel rundown was observed when cellular ATP levels were lowered by intracellular dialysis with the patch pipette solution. Rundown was prevented by the ATP analogue AMP-PCP. Passive swelling-induced myo-[3H]inositol and [3H]taurine efflux was blocked by metabolic inhibitors that decreased cellular ATP levels. Titration of cellular ATP levels with azide demonstrated that the apparent dissociation constant (Kd) for ATP of both myo-inositol and taurine efflux was approximately 1.7 mM. The high Kd for ATP indicates that cellular metabolic state plays an important role in modulating organic osmolyte loss. Regulation of VSOAC activity by ATP prevents depletion of metabolically expensive organic osmolytes when cellular energy production is reduced. In addition, ATP-dependent regulation provides essential feedback to minimize the loss of energy-producing carbon sources such as pyruvate, short-chain fatty acids, ketone bodies, and amino acids, which readily permeate this channel.
Spatial and temporal variations in local spike propagation in the myometrium of the 17-day pregnant rat
Authors: Lammers W. J. , Arafat K. , el-Kays A. , el-Sharkawy T. Y.
Published: December 2017

Detailed spatial analysis of propagation of individual action potential was performed during spontaneous bursts of activity in the isolated 17-day pregnant rat myometrium. Use was made of high-resolution mapping with simultaneous recordings from 240 extracellular electrodes. Positioning of the electrode assembly by itself did not have any adverse effects, and no differences were found in the period or duration of spontaneous bursts recorded with and without the electrode assembly touching the tissue. The spread of propagation of individual action potentials was reconstructed at several moments during myometrial spike bursts. Both the direction and the sequence of activation of the myometrium were found to be highly variable and depended on 1) the level and spatial dispersion of excitability and 2) whether conduction occurred predominantly in the longitudinal or the circumferential direction. Furthermore, conduction was frequently complicated by the spontaneous occurrence of 1) lines of conduction block, 2) focal sites of pacemaking, or 3) merging of two or more wavelets into a single wave. In contrast, when the myometrium was divided into small segments, activity became much more regular, and both the location of the pacemaker and the direction of propagation were much more stable than in the whole myometrium. In conclusion, spontaneous spatial variations in local spike propagation at the preterm stage could provide for the necessary asynchrony in activation and play a role in the prevention of forceful contractions and premature labor.
Protein kinase C activity does not mediate the inhibitory effect of carbachol on chloride secretion by T84 cells
Authors: Traynor-Kaplan A. E. , Buranawuti T. , Vajanaphanich M. , Barrett K. E.
Published: December 2017

Carbachol induces calcium-dependent chloride secretion and activates protein kinase C in T84 cells. However, prolonged stimulation with carbachol or direct activation of protein kinase C inhibits subsequent calcium-dependent chloride secretion. Furthermore, the ability of carbachol to elevate inositol tetrakisphosphate levels may be linked to inhibition of chloride secretion. Here we demonstrate that protein kinase C activation increases levels of inositol tetrakisphosphates (1,3,4,6- and 3,4,5,6-isomers) in T84 colonic epithelia. Furthermore, this corresponds to an inhibition of chloride secretion. However, protein kinase C is unlikely to mediate the analogous effects of carbachol. Neither the ability of carbachol to inhibit calcium-dependent chloride secretion nor its effects on inositol 3,4,5,6-tetrakisphosphate levels were reversed by staurosporine. Carbachol also has quantitatively and qualitatively different effects on inositol tetrakisphosphate isomers than protein kinase C activators. Thus protein kinase C activity can increase levels of various inositol tetrakisphosphate isomers within T84 cells but does not mediate carbachol-induced increases in these putative messengers. These data further support the hypothesis that inositol 3,4,5,6-tetrakisphosphate is a negative second messenger, uncoupling epithelial chloride secretion from changes in intracellular calcium.
Caffeine stimulation of malignant hyperthermia-susceptible sarcoplasmic reticulum Ca2+ release channel
Authors: Shomer N. H. , Mickelson J. R. , Louis C. F.
Published: December 2017

The altered caffeine sensitivity of malignant hyperthermia-susceptible (MHS) muscle contracture is one basis of the diagnostic test for this syndrome. To determine whether the Arg615-to-Cys615 mutation of the porcine sarcoplasmic reticulum (SR) Ca2+ release channel is directly responsible for this altered caffeine sensitivity, the single-channel kinetics of purified MHS and normal pig Ca2+ release channels were examined. Initial studies demonstrated that decreasing the pH of the medium in either the cis- or trans-chamber decreased the Ca2+ release channel percent open time (Po). The half-inhibitory pH of MHS channels (6.86 +/- 0.04, n = 17) was significantly different from that of normal channels (7.08 +/- 0.07, n = 14). At pH 7.4, in either 7 or 0.12 microM Ca2+, MHS channel Po was not significantly different from that of normal channels over the range 0-10 mM caffeine. Although at pH 6.8 in 7 microM Ca2+ MHS channel Po was greater than that of normal channels over the range 0-20 mM caffeine, the difference could be eliminated by dividing each mean MHS Po by a scaling factor of 3.2. Thus the MHS Ca2+ release channel mutation does not appear to be directly responsible for the altered caffeine sensitivity of MHS pig muscle contracture. Rather, this altered caffeine sensitivity may result from an altered resting myoplasmic Ca2+ concentration or the altered pH and Ca2+ sensitivity of Ca2+ release channel Po of MHS muscle.
Opposite roles of cAMP and cGMP on volume loss in muscle cells
Authors: Pena-Rasgado C. , Kimler V. A. , McGruder K. D. , Tie J. , Rasgado-Flores H.
Published: December 2017

It is controversial whether changes in adenosine 3',5'-cyclic monophosphate (cAMP) and in the cAMP-to-guanosine 3',5'-cyclic monophosphate (cGMP) ratio are involved with cell swelling and in the activation of volume-regulatory mechanisms. We examined whether these nucleotides are involved in cell volume regulation in skeletal muscle. Isolated (intact and internally perfused) barnacle muscle cells were used because these cells, when exposed to a hyposmotic environment, undergo an extracellular Ca2+ (Cao)-dependent regulatory volume decrease (RVD). Using intact cells we found that dibutyryl cAMP and forskolin significantly promoted RVD in cells exposed to Cao-free solutions and that dibutyryl cGMP significantly inhibited RVD in cells exposed to Cao-containing solutions. In perfused cells in which the intracellular free Ca2+ concentration ([Ca2+]i) was heavily buffered [with 8 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)], cAMP induced a volume loss that was inhibited by presence of cGMP. Furthermore, if perfused cells were exposed to hyposmotic conditions, they swelled and underwent RVD provided that [Ca2+]i buffering was low (with 2 mM EGTA). This effect was inhibited by presence of the cAMP antagonist, [R]-p-adenosine 3',5'-cyclic monophosphorothioate.
Endothelin receptor in osteoblastic cells is coupled to multiple messenger signals
Authors: Green J. , Foellmer O. , Kleeman C. R. , Basic M. M.
Published: December 2017

We analyzed the functional characteristics of endothelin (ET) peptides in the osteoblastic UMR-106 cells by studying receptor binding as well as dose-response curves for ET-1 and ET-3 on two biological responses: 1) induction of Ca2+ transients and 2) activation of the Na(+)-H+ exchanger. ET specifically binds to a single class of receptor with a rank order of affinity ET-1 >> ET-3. ET-1 and ET-3 dose dependently stimulated a rise in intracellular Ca2+ ([Ca2+]i), with ET-1 being two orders of magnitude more potent than ET-3 [50% effective concentration (EC50) = 8 x 10(-10) and 9 x 10(-8) M for ET-1 and ET-3, respectively; P < 0.01]. The effect of ET-1 on [Ca2+]i was 90% inhibitable by the ETA antagonist BQ-123. The activity of Na(+)-H+ exchange was studied by using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein as well as by 22Na+ fluxes. ET-1 and ET-3 activated the exchange in a concentration-dependent manner and with similar potencies (EC50 approximately 10(-10) M). The action of ETs on Na(+)-H+ exchange was mimicked neither by phorbol esters nor by Ca2+ ionophores. It was, however, blocked by BQ-123 as well as by the protein tyrosine kinase inhibitor genistein. We conclude that in UMR-106 cells, a single ET receptor subtype is coupled to multiple effectors, a Ca2+ message system and a tyrosine-kinase system which, in turn, activates the Na(+)-H+ exchanger.
Interactions between inhibitory and excitatory modulatory signals in single submucosal neurons
Authors: Barajas-Lopez C.
Published: December 2017

Intracellular recordings were made in submucosal neurons from the guinea pig ileum to study the actions of norepinephrine and somatostatin on slow depolarizations induced by 2-chloroadenosine (CADO) and substance P. Local application (by pressure) of CADO and substance P induced a slow depolarization that occurred concomitantly with an increase in input membrane resistance. Norepinephrine, UK-14304 (alpha 2-adrenoceptor agonist), and somatostatin blocked the excitatory responses induced by CADO in a concentration-dependent manner. The alpha 2-adrenoceptor antagonists idazoxan and yohimbine antagonized these inhibitory effects of UK-14304 and norepinephrine. UK-14304 also decreased depolarizations induced by forskolin, but not those induced by the adenosine 3',5'-cyclic monophosphate analogue 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. Slow depolarizations induced by substance P were blocked neither by UK-14304 nor by somatostatin. It was previously shown that staurosporine (an inhibitor of various protein kinases) and KT-5720 (an inhibitor of protein kinase A) inhibited slow depolarizations induced by CADO. Here, substance P depolarizations were inhibited by staurosporine and calphostin C (a blocker of protein kinase C) but not by KT-5720. In conclusion, activation of alpha 2-adrenoceptors and somatostatin receptors selectively blocks excitatory responses induced by CADO, most likely by inhibition of adenylyl cyclase and via pertussis toxin-sensitive G proteins. Slow depolarizations induced by substance P are independent of adenylyl cyclase activation and involve activation of protein kinase C.
Selective block of gap junction channel expression with connexin-specific antisense oligodeoxynucleotides
Authors: Moore L. K. , Burt J. M.
Published: December 2017

Gap junctions in vascular smooth muscle provide a cell-to-cell conduction pathway that may contribute to regulation and coordination of changes in vascular tone. A7r5 cells, a cell line derived from embryonic rat aorta, express connexin 43 (Cx43) and connexin 40 (Cx40). Gap junction channels with three distinct unitary conductances (70, 108, and 141 pS) were observed in these cells. Events of each channel type were equally common, with an approximate frequency of 30-35%; however, the frequency at which each channel type was observed in individual cell pairs ranged between 10 and 65%. Treatment of the cells for 24-72 h with an antisense oligodeoxynucleotide (ODN) to Cx43 reduced the relative frequency of the 108- and 141-pS channel events, whereas comparable treatment with antisense Cx40 ODN reduced the frequency at which 70-pS channel events were observed. The simplest explanation of these findings is that Cx43 forms the 108- and 141-pS channels, whereas Cx40 forms the 70-pS channels in A7r5 cells.
Interferon-gamma downregulates CFTR gene expression in epithelial cells
Authors: Besancon F. , Przewlocki G. , Baro I. , Hongre A. S. , Escande D. , Edelman A.
Published: December 2017

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective transepithelial Cl- transport. The regulation of CF gene expression is not fully understood. We report that interferon-gamma (IFN-gamma), but not IFN-alpha or -beta, downregulates CFTR mRNA levels in two colon-derived epithelial cell lines, HT-29 and T84, in a time- and concentration (from 0.1 IU/ml)-dependent manner. IFN-gamma has no effect on the transcription rate of the CFTR gene but reduces CFTR mRNA half-life, indicating that it exerts a posttranscriptional regulation of CFTR expression, at least partly, through destabilization of the transcripts. Cells treated with IFN-gamma contain subnormal amounts of 165-kDa CFTR protein. Assays of adenosine 3',5'-cyclic monophosphate-stimulated 36Cl- efflux and whole cell currents show that CFTR function is diminished in IFN-gamma-treated cells. IFN-gamma and tumor necrosis factor-alpha synergistically reduce CFTR gene expression. Our results suggest that production of these cytokines in response to bacterial infections and inflammatory disorders may alter transmembrane Cl- transport.
Effects of EGTA on calcium signaling in airway epithelial cells
Authors: Harris R. A. , Hanrahan J. W.
Published: December 2017

We have studied the effect of the extracellular calcium buffers ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) as well as varying extracellular calcium concentration on the intracellular free calcium response to histamine in a cystic fibrosis airway epithelial cell line (CF/T43). Histamine stimulates a rapid transient increase in cell calcium followed by a slower second peak lasting several minutes. Bathing cells in nominally calcium-free medium (no added calcium) did not abolish the second peak in the biphasic response to histamine. We examined the effect of including either 1 mM EGTA or BAPTA in the perfusate to investigate whether influx might have been supported by trace amounts of calcium in the nominally calcium-free medium. The second histamine-stimulated peak had a shorter duration but similar amplitude in the presence of BAPTA. In contrast, the second peak was completely abolished by EGTA. To examine if the different histamine responses were due to EGTA's lower dissociation constant or some pharmacological effect of unbound EGTA, extracellular calcium was removed by pretreating the saline with BAPTA covalently bound to polystyrene beads, and effluent from the imaging chamber was collected and analyzed for calcium contamination. Histamine stimulation still produced a biphasic calcium response when extracellular free calcium was approximately 7 nM, a concentration that should reverse the electrochemical gradient for calcium at the plasma membrane. These data suggest that the unbound form of EGTA can interfere with calcium signaling in cells through inhibition of its release from intracellular stores.
Ketamine inhibition of large conductance Ca(2+)-activated K+ channels is modulated by intracellular Ca2+
Authors: Denson D. D. , Eaton D. C.
Published: December 2017

The present investigation was conducted to study the relationship between intracellular Ca2+ and inhibition of large conductance Ca(2+)-activated K+ (BK) currents by ketamine using excised patches from GH3 cells. Five ketamine concentrations were studied in the presence of six Ca2+ concentrations. The half-maximal inhibition for BK channel block by ketamine was increased from 4.1 +/- 0.7 microM at 0.1 microM intracellular Ca2+ to 230 +/- 74 microM at 100 microM intracellular Ca2+. Open probability (Po), Ca2+ concentration, and ketamine concentration data were best described by a competitive inhibition model. The inhibition constant for ketamine was 20.5 +/- 5.2 microM, and the Michaelis-Menten constant (Km) value for Ca2+ was 3.58 +/- 0.49 microM, which was not different from Km for Ca2+ in the absence of ketamine (3.33 +/- 0.37 microM). Taken alone, these data would suggest that Ca2+ and ketamine were competing for the same site on the channel protein. However, examination of open and closed interval data from patches containing only one channel show that ketamine primarily produces a decrease in the frequency of long-lived open events, suggesting that the effect of ketamine on BK channels may not be by a direct effect on channel proteins.
Carrier-mediated transport of lipoxin A4 in human neutrophils
Authors: Simchowitz L. , Fiore S. , Serhan C. N.
Published: December 2017

Lipoxins and other eicosanoids display potent and selective biological effects on leukocytes. In this study, we utilized radiolabeled lipoxin A4 ([3H]LXA4) to investigate whether carrier-mediated transport of LXA4 might occur in human neutrophils. At a concentration of 5 nM, uptake of [3H]LXA4, above that due to specific binding to receptors, amounted to approximately 0.6 fmol.10(6) cells-1.min-1. This influx was sensitive to a number of anionic inhibitors, including 3,5-diiodosalicylic acid (K0.5 12 microM), pentachlorophenol (K0.5 25 microM), alpha-cyano-beta-(1-phenylindol-3-yl) acrylic acid, and the organomercurial agents mersalyl (K0.5 110 microM) and p-hydroxy-mercuribenzoate. Influx, which was Na+ and membrane voltage independent, exhibited a striking dependence on pH (negative log of dissociation 5.9), results compatible with an H+ + LXA4 anion cotransport system. The LXA4 carrier did not appear to interact with arachidonic acid, prostaglandin E2, 15(S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid, or the leukotrienes B4, C4, and D4. Moreover, transport activity was not observed in human erythrocytes, lymphocytes, or platelets, but it was inducible in HL-60 cells on differentiation by exposure to retinoic acid. These findings represent the identification and initial characterization of a novel carrier-mediated pathway in human neutrophils that facilitates transport of LXA4 into cells.
Role of external Na+ and cytosolic pH in agonist-evoked cytosolic Ca2+ response in human platelets
Authors: Kimura M. , Nakamura K. , Fenton J. W. , Andersen T. T. , Reeves J. P. , Aviv A.
Published: December 2017

The role of external Na+ in agonist-evoked platelet Ca2+ response is poorly understood. This was explored in this study. Removal of external Na+ decreased both cytosolic Ca2+ mobilization and external Ca2+ entry, induced by thrombin but not by ADP or vasopressin. That external Na+ regulates thrombin activities was demonstrated by 1) Na+ dependency of the amidolytic activity of thrombin, 2) inhibition of thrombin binding to the high-affinity binding sites in Na(+)-free medium, and 3) attenuation of thrombin-induced inositol 1,4,5-trisphosphate production in Na(+)-free medium. Moreover, Ca2+ response to the thrombin receptor 6-amino acid peptide was independent of external Na+. The role of external Na+ in modifying agonist-evoked Ca2+ response through activation of Na+/H+ antiport and cytosolic alkalinization was then explored. Cytosolic alkalinization by monensin or NH4Cl enhanced thrombin, ADP, and thimerosal-induced external Ca2+ entry. Thimerosal-induced acceleration of external Ca2+ entry was diminished by the inhibition of Na+/H+ antiport. Thus external Na+ enhances thrombin activities, and cytosolic pH mediates store-regulated external Ca2+ entry. However, Na+/H+ antiport activation is not essential for agonist-evoked Ca2+ mobilization and external Ca2+ entry.
Role of redox systems on Fe3+ uptake by transformed human intestinal epithelial (Caco-2) cells
Authors: Nunez M. T. , Alvarez X. , Smith M. , Tapia V. , Glass J.
Published: December 2017

Caco-2 cells were used as a model of human intestinal epithelium to investigate the role of redox systems in transepithelial transport of 59Fe3+. The cells reduced Fe3+ present in the apical medium; the reduction was 50% inhibited by adriamycin and p-chloromercuribenzoate. Addition of [14C]ascorbate to the basolateral medium resulted in accumulation of 14C radioactivity in both cells and apical medium; apical radioactivity increased with time and was probably caused by paracellular flux. The cells provided Fe3+ reduction capacity to the apical incubation medium. Addition of ascorbate to the basolateral medium increased this reduction capacity 2-fold and the cellular uptake of 59Fe3+ 1.8-fold. Adriamycin significantly inhibited both cellular 59Fe uptake and Fe transport into the basolateral side. The results indicate that Caco-2 cells reduce apical Fe3+ by two parallel mechanisms: by a plasma membrane ferrireductase and by the secretion of reductants of either cellular or basolateral origin. The data support a model for Fe3+ intestinal absorption in which cell-mediated Fe3+ reduction occurs before cellular Fe uptake.
Sources of calcium utilized in cholinergic responses in canine colonic smooth muscle
Authors: Sato K. , Sanders K. M. , Gerthoffer W. T. , Publicover N. G.
Published: December 2017

Ratiometric fura 2 fluorescence techniques were used to investigate the sources of Ca2+ that lead to an increase in cytosolic Ca2+ concentration ([Ca2+]i) and the generation of force during cholinergic stimulation of canine colonic circular smooth muscle tissues. Acetylcholine (ACh; 1 microM) caused a biphasic increase in [Ca2+]i and force. The initial upstroke phase was characterized by an increase in [Ca2+]i and a pronounced increase in force. The sustained phase was characterized by concurrent oscillations in [Ca2+]i and force (2-3/min) that persisted as long as ACh was present. The increase in [Ca2+]i in response to ACh was reduced to approximately 30% in the presence of nicardipine (1 microM), suggesting that L-type Ca2+ channels contribute to the rise in [Ca2+]i but that other sources also contribute. Preincubation in caffeine (10 mM) and ryanodine (10 microM) reduced the upstroke phase of the increase in [Ca2+]i and contractile responses to ACh, indicating that release of Ca2+ from intracellular stores contributes only to the initial cholinergic response. Responses to ACh persisted when nicardipine (1 microM) was included after emptying of caffeine-ryanodine-sensitive stores, suggesting the presence of additional sources of Ca2+. Data suggest that cholinergic regulation of [Ca2+]i in colonic smooth muscle occurs by a number of parallel pathways. Influx through voltage-dependent Ca2+ channels, release of Ca2+ from intracellular stores, and possibly Ca2+ entry through additional conductances activated by ACh all contribute to the regulation of [Ca2+]i.