bluuebunny/crossref_metadata_2025 huggingface dataset:
Capillary electrophoretic measurement of tissue metabolites
Authors: Dillon Patrick F. , Sears Patrick R.
Published: December 2017

A method for the measurement of tissue metabolites from rabbit urinary bladder using capillary electrophoresis (CE) has been developed. The method generates a reproducible electropherogram containing >20 peaks, including NAD, NADH, lactate, UDP-glucose, phosphocreatine, creatine, ATP, ADP, GTP, and UTP, from <20 nl of extract solution generated from 1.1 nl (or ∼1.2 μg) of tissue in <40 min. Multiple samples from the same bladder produce SE comparable with enzymatic or nuclear magnetic resonance (NMR) measurements of metabolites: phosphorus-NMR measurement requires 106 more tissue than CE; individual enzymatic measurements using 100 μl/sample require 2,000 μl, a 105 greater volume than required by CE for the same number of metabolites. CE detects about three times more peaks than phosphorus-NMR on a similar time scale. Comparable measurements using enzymatic analysis would require ∼10 times longer. The combination of minimal tissue volume requirements, rapid measurement, and reproducibility makes CE a valuable tool in the investigation of simultaneous changes in multiple metabolites from minute tissue samples.
Inhibition of cell differentiation by Gαq in the renal epithelial cell line LLC-PK1
Authors: Sun Lihyun , Weaver Debora J. , Amsler Kurt , Weiss Ellen R.
Published: December 2017

LLC-PK1, an epithelial cell line derived from the kidney proximal tubule, was used to study the ability of the G protein α-subunit, Gαq, to regulate cell differentiation. A constitutively active mutant protein, αqQ209L, was expressed using the LacSwitch-inducible mammalian expression system. Induction of αqQ209L expression with isopropyl-β-d-thiogalactopyranoside (IPTG) enhanced phospholipase C activity maximally by 6- to 7.5-fold. Increasing concentrations of IPTG progressively inhibited the activity of two differentiation markers, Na+-dependent hexose transport and alkaline phosphatase activity. Induction of αqQ209L expression also caused a change from an epithelial to a spindle-shaped morphology. The effects of αqQ209L expression on cell differentiation were similar to those observed with 12- O-tetradecanoylphorbol 13-acetate (TPA) treatment. However, protein kinase C (PKC) levels were downregulated in TPA-treated cells but not in αqQ209L-expressing cells, suggesting that the regulation of PKC by Gαq may be different from regulation by TPA. Interestingly, the PKC inhibitor GF-109203X did not inhibit the effect of IPTG on the development of Na+-dependent hexose transport in αqQ209L-expressing cells. These data implicate PKCδ and PKCε in the pathway used by Gαq to block the development of Na+-dependent hexose transport in IPTG-treated cells.
Taurine synthesis and cysteine metabolism in cultured rat astrocytes: effects of hyperosmotic exposure
Authors: Beetsch Joel W. , Olson James E.
Published: December 2017

We investigated mechanisms controlling taurine synthesis in cultured rat cerebral astrocytes. The mean ± SE rate of taurine synthesis from extracellular cysteine was 21.2 ± 2.0 pmol ⋅ mg protein−1 ⋅ min−1, whereas taurine degradation was <1.3% of this rate. Eliminating cellular glutathione and inhibiting glutathione biosynthesis increased taurine synthesis from extracellular cysteine by 39%. In cell homogenates, cysteine dioxygenase (CDO) and cysteine-sulfinate decarboxylase activities were 2.4 ± 0.2 and 8.3 ± 2.8 nmol ⋅ mg protein−1 ⋅ min−1, respectively. CDO activity was strongly dependent on cysteine concentration over physiological and pathophysiological ranges of intracellular cysteine concentration. Growth in hyperosmotic medium caused a greater increase in culture medium taurine content than that measured from cells in isosmotic growth medium. Hyperosmotic treatment transiently increased the rate of cysteine accumulation and cellular cysteine and glutathione contents but had no effect on the synthesis rate of taurine from extracellular cysteine. Thus cysteine is accumulated and then metabolized to taurine through CDO, whose activity depends on the intracellular cysteine concentration and appears to be rate limiting for taurine synthesis. Hyperosmotic exposure increases net taurine production yet has no effect on taurine synthesis from exogenously applied cysteine. Availability of substrate from intracellular pools must contribute to maintenance of high intracellular taurine during hyperosmotic exposure.
Differential translocation of protein kinase C isozymes by phorbol esters, EGF, and ANG II in rat liver WB cells
Authors: Maloney Judith A. , Tsygankova Oxana , Szot Agnieszka , Yang Lijun , Li Quiyang , Williamson John R.
Published: December 2017

The protein kinase C (PKC) family represents an important group of enzymes whose activation is associated with their translocation from the cytosol to different cellular membranes. In this study, the spatial distribution of PKC-α, -δ and -ε in rat liver epithelial (WB) cells has been examined by Western blot analysis after subcellular fractionation. Cytosolic, membrane, nuclear, and cytoskeletal fractions were obtained from cells stimulated with phorbol 12-myristate 13-acetate (PMA), angiotensin II (ANG II), or epidermal growth factor (EGF). PMA caused most of the PKC-α, -δ and -ε initially present in the cytosol to be transported to the membrane and nuclear fractions. In contrast, both ANG II and EGF induced only a minor translocation of PKC-α to the membrane fraction but caused a statistically significant membrane-directed movement of PKC-δ and -ε. Translocation of PKC-δ and -ε to the nucleus induced by ANG II and EGF was transient and quantitatively smaller than that induced by PMA. PKC-δ and -ε were present in the cytoskeleton of resting cells, but although PMA, ANG II, and EGF caused some changes in their content, these were variable, suggesting that the cytoskeleton fraction was heterogeneous. PKC depletion inhibited ANG II-induced mitogenesis and the sustained activation of Raf-1 and extracellular regulated protein kinase (ERK). However, although PKC depletion inhibited EGF-induced mitogenesis, the maximum EGF-induced activation of the ERK pathway was only slightly retarded. We hypothesize that PKC-δ and -ε are involved in mitogenesis via both ERK-dependent and ERK-independent mechanisms. These results support the notion that specific PKC isozymes exert spatially defined effects by virtue of their directed translocation to distinct intracellular sites.
Increased prostacyclin and PGE2 stimulated cAMP production by macrophages from endotoxin-tolerant rats
Authors: Makhlouf Michel A. , Fernando Lawrence P. , Gettys Thomas W. , Halushka Perry V. , Cook James A.
Published: December 2017

Sublethal administration of lipopolysaccharide (LPS) renders rats tolerant to multiple lethal stimuli. Tolerant macrophages exhibit differential alterations in LPS-stimulated cytokine and inflammatory mediator release. Increased cAMP levels stimulated by PGE2 or prostacyclin (PGI2) result in differential effects on LPS-induced cytokine release and protect against the pathophysiological changes of endotoxemia. In the present studies, we sought to determine whether PGE2- and PGI2-stimulated cAMP levels are altered in tolerant macrophages. Incubation of macrophages with cicaprost or 11-deoxy-PGE1 in the presence of phosphodiesterase inhibitors resulted in significantly higher (2.5- to 6.5-fold) cAMP concentrations in tolerant macrophages compared with control. In contrast, isoproterenol-stimulated cAMP levels were not significantly different between control and tolerant cells. Also, incubation of tolerant macrophages with LPS did not result in significantly elevated cAMP levels. Prostacyclin (IP) receptor mRNA levels were significantly increased in tolerant cells compared with controls, whereas [3H]PGE2binding and PGE2 EP4 receptor mRNA levels were not significantly changed. These studies suggest that LPS tolerance induces selective alterations in eicosanoid regulation of cAMP formation.
Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+exchange
Authors: Wormmeester Louktje , De Medina Fermin Sanchez , Kokke Freddy , Tse Chung-Ming , Khurana Seema , Bowser Joellyn , Cohen Michael E. , Donowitz Mark
Published: December 2017

Intestinal neutral NaCl absorption, which is made up of brush-border (BB) Na+/H+exchange linked to BB Cl−/[Formula: see text]exchange, is up- and downregulated as part of digestion and diarrheal diseases. Glucocorticoids stimulate ileal NaCl absorption and BB Na+/H+exchange. Intestinal BB contains two Na+/H+exchanger isoforms, NHE2 and NHE3, but their relative roles in rabbit ileal BB Na+/H+exchange has not been determined. A technique to separate the contribution of NHE2 and NHE3 to ileal BB Na+/H+exchange activity was standardized by using an amiloride-related compound, HOE-694. Under basal conditions, both NHE2 and NHE3 contribute ∼50% to ileal Na+/H+exchange. Glucocorticoids (methylprednisolone) increase BB Na+/H+exchange (2.5 times) but increase only ileal NHE3 activity (4.1 times), without an effect on NHE2 activity. Thus ileal BB Na+/H+exchange in animals treated with glucocorticoids is 69% via NHE3. A quantitative Western analysis for NHE3 was developed, using as an internal standard a fusion protein of the COOH-terminal 85 amino acids of NHE3 and maltose binding protein. Glucocorticoid treatment increased the amount of BB NHE3. The quantitative Western analysis showed that NHE3 makes up 0.018% of ileal BB protein in control rabbits and 0.042% (2.3 times as much) in methylprednisolone-treated rabbits. Methylprednisolone treatment did not alter the amount of ileal BB NHE2 protein. NHE3 turnover number was estimated to be 458 cycles/s under basal conditions and 708 cycles/s in glucocorticoid-treated ileum. Thus methylprednisolone stimulates ileal BB Na+/H+exchange activity only by an effect on NHE3 and not on NHE2; it does so primarily by increasing the amount of BB NHE3, although it also increases the NHE3 turnover number.
Basis for late rise in fura 2 R signal reporting [Ca2+]i during relaxation in intact rat ventricular trabeculae
Authors: Jiang Yandong , Patterson Michael F. , Morgan David L. , Julian Fred J.
Published: December 2017

Intact rat ventricular trabeculae were injected with the salt form of fura 2, and the fura 2 ratio signal (R) was used to report intracellular Ca2+ concentration ([Ca2+]i). The fixed end relaxation phase of a twitch is associated with a slowing of the decay of the R signal, or even a reversal, to form a distinct bump, indicating a transient rise in [Ca2+]i. The bump is most prominent at 30°C, and motion artifact is not its cause. Increasing doses of 2,3-butanedione monoxime caused progressive attenuation of the twitch and bump. Increasing the bathing Ca2+ concentration potentiated the twitch and enhanced the bump. Imposed muscle shortening during relaxation caused a much quicker force decline, and this led to the appearance of a much more prominent associated bump. The amplitude of the bump depends on the amplitude of twitch force and the rate of relaxation. These findings can be explained, as in skeletal muscle, by making cross-bridge attachment and Ca2+ binding to troponin C strongly cooperative; therefore, the bump during fast relaxation is produced by a reversal of this cooperativity, leading to rapid dissociation of Ca2+ from troponin C into the myoplasm.
Regulation of the Na+/H+exchanger in fibroblasts overexpressing the Na+/Ca2+exchanger
Authors: Ikeda Toshitaro , Iwamoto Takahiro , Wakabayashi Shigeo , Shigekawa Munekazu
Published: December 2017

To assess the role of Ca2+in regulation of the Na+/H+exchanger (NHE1), we used CCL-39 fibroblasts overexpressing the Na+/Ca2+exchanger (NCX1). Expression of NCX1 markedly inhibited the transient cytoplasmic Ca2+ rise and long-lasting cytoplasmic alkalinization (60–80% inhibition) induced by α-thrombin. In contrast, coexpression of NCX1 did not inhibit this alkalinization in cells expressing the NHE1 mutant with the calmodulin (CaM)-binding domain deleted (amino acids 637–656), suggesting that the effect of NCX1 transfection involves Ca2+-CaM binding. Expression of NCX1 only slightly inhibited platelet-derived growth factor BB-induced alkalinization and did not affect hyperosmolarity- or phorbol 12-myristate 13-acetate-induced alkalinization. Downregulation of protein kinase C (PKC) inhibited thrombin-induced alkalinization partially in control cells and abolished it completely in NCX1-transfected cells, suggesting that the thrombin effect is mediated exclusively via Ca2+ and PKC. On the other hand, deletion mutant study revealed that PKC-dependent regulation occurs through a small cytoplasmic segment (amino aids 566–595). These data suggest that a mechanism involving direct Ca2+-CaM binding lasts for a relatively long period after agonist stimulation, despite apparent short-lived Ca2+ mobilization, and further support our previous conclusion that Ca2+- and PKC-dependent mechanisms are mediated through distinct segments of the NHE1 cytoplasmic domain.
Endothelin-1 activates phospholipases and channels at similar concentrations in porcine coronary arteries
Authors: Jones Allan W. , Magliola Lawrence , Waters Carrie B. , Rubin Leona J.
Published: December 2017

Sensitivity of endothelin-1 (ET-1)-ion channel interactions has been proposed to exceed that of ET-1-phospholipase activation in vascular smooth muscle. We wanted to determine whether short-circuiting ion channels with staphylococcal α-toxin pores would shift the ET-1-force relation to the right as predicted from the above proposal. Medium size porcine coronary arteries (outer diameter 0.7–1.5 mm) were mounted on isometric force transducers. ET-1 concentration response curves were compared between intact rings and those subjected to α-toxin treatment with Ca buffered at 0.1 μM. The EC50 for treated rings (1.5 ± 1.0 nM, n = 5 pigs) was similar to that for intact rings (1.9 ± 0.4 nM). The Ca sensitivity of the α-toxin-treated rings (EC50 = 0.43 ± 0.08 μM) was similar to that reported by other laboratories for intact and α-toxin-treated arteries and was shifted eightfold to the left by a high concentration of ET-1 (10 nM). Measurements of [32P]phosphatidic acid ([32P]PA) levels were used to evaluate phospholipase activity in intact arteries. The time courses for [32P]PA production and contraction were similar in response to high (100 nM) and to low (1 nM) ET-1. Significant increases in both steady-state contraction and [32P]PA occurred over a wide range of ET-1 concentrations tested (0.3–100 nM). Our findings support the concept that ET-1-phospholipase coupling is operative over the whole concentration range that induces contractile responses. It is suggested that both Ca entry and Ca sensitization processes are activated by ET-1 at low concentrations (<EC50) and that both processes contribute significantly to the integrated response.
Regulation of rENaC mRNA by dietary NaCl and steroids: organ, tissue, and steroid heterogeneity
Authors: Stokes John B. , Sigmund Rita D.
Published: December 2017

Rats on a low-NaCl diet have a high Na+ channel activity in colon and kidney. To address the mechanism of this increased activity, we measured mRNA levels of three Na+channel subunits in epithelial tissue (rENaC) from rats having been fed either a low (0.13%)- or high (8%)-NaCl diet for 2–3 wk. The size of the mRNA for each of the rENaC subunits as determined by Northern blot was unaffected by diet. RNase protection assay showed heterogeneity of response by organs and subunit. In lung, there was no effect of diet on any of the three subunits. In descending colon, the low-NaCl diet increased β- and γ-rENaC mRNA, with no effect on α-rENaC mRNA. In the kidney, the response to dietary NaCl was dependent on the region. In cortex and outer medulla, diet had no effect on any of the subunits. Rats fed the low-NaCl diet had greater α-rENaC in inner medulla but not β- or γ-rENaC mRNA. We next asked whether acute administration of pure glucocorticoid (GC) or mineralocorticoid (MC) hormones to adrenalectomized rats reproduced the effects of a low-NaCl diet. Six hours after administration of GC or MC, a somewhat different heterogeneity occurred. In lung, α-rENaC mRNA was increased but only in response to GC. In colon, either GC or MC increased β- or γ-rENaC, and there was no effect on α-rENaC. In kidney, either GC or MC increased α-rENaC, without an effect on β- or γ-rENaC. In contrast to the response to a low-NaCl diet, all three regions were similarly affected by acute steroids. These results demonstrate a striking heterogeneity in response to physiological stimuli that regulate ENaC function. The mRNA levels of each of the rENaC subunits can be determined by the type of steroid and by factors unique to the organ and even to the specific region of the kidney.
Differential effects of phorbol ester (PMA) on blocker-sensitive ENaCs of frog skin and A6 epithelia
Authors: Els Willem J. , Liu Xuehong , Helman Sandy I.
Published: December 2017

Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA) caused complex transient perturbations of amiloride-sensitive short-circuit Na+ currents ( I Na) in A6 epithelia and frog skins that were tissue and concentration dependent. A noninvasive channel blocker pulse method of noise analysis (18) was used to investigate how PMA caused time-dependent changes of apical membrane epithelial Na+ channel (ENaC) single-channel currents, channel open probabilities ( P o), and channel densities ( N T). In A6 epithelia, 5 and 50 nM PMA caused within 7 min concentration-dependent sustained decreases of P o (∼55% below control, 50 nM) and rapid compensatory transient increases of N T within 7 min (∼220% above control, 50 nM), resulting in either small transient increases of I Naat 5 nM PMA or small biphasic decreases of I Na at 50 nM PMA. In contrast to A6 epithelia, 50 and 500 nM PMA in frog skin caused after a delay of at least 10 min transient increases of N T to ∼60–70% above control at 30–60 min. Unlike A6 epithelia, P o was increased ∼15% above control within 7 min and remained within ±10–15% of control for the duration of the 2-h experiments. Despite differences in the time courses of secondary inhibition of transport in A6 epithelia and frog skin, the delayed downregulation of transport was due to time-dependent decreases of N T from their preelevated levels in both tissues. Whereas P o is decreased within minutes in A6 epithelia as measured by noise analysis or by patch clamp (8), the discrepancy in regulation of N T in A6 epithelia as measured by noise analysis and patch clamp is most likely explained by the inability of on-cell patches formed before treatment of tissues with PMA to respond to regulation of their channel densities.
Regulation of ribosomal DNA transcription by insulin
Authors: Hannan Katherine M. , Rothblum Lawrence I. , Jefferson Leonard S.
Published: December 2017

The experiments reported here used 3T6-Swiss albino mouse fibroblasts and H4-II-E-C3 rat hepatoma cells as model systems to examine the mechanism(s) through which insulin regulates rDNA transcription. Serum starvation of 3T6 cells for 72 h resulted in a marked reduction in rDNA transcription. Treatment of serum-deprived cells with insulin was sufficient to restore rDNA transcription to control values. In addition, treatment of exponentially growing H4-II-E-C3 with insulin stimulated rDNA transcription. However, for both cell types, the stimulation of rDNA transcription in response to insulin was not associated with a change in the cellular content of RNA polymerase I. Thus we conclude that insulin must cause alterations in formation of the active RNA polymerase I initiation complex and/or the activities of auxiliary rDNA transcription factors. In support of this conclusion, insulin treatment of both cell types was found to increase the nuclear content of upstream binding factor (UBF) and RNA polymerase I-associated factor 53. Both of these factors are thought to be involved in recruitment of RNA polymerase I to the rDNA promoter. Nuclear run-on experiments demonstrated that the increase in cellular content of UBF was due to elevated transcription of the UBF gene. In addition, overexpression of UBF was sufficient to directly stimulate rDNA transcription from a reporter construct. The results demonstrate that insulin is capable of stimulating rDNA transcription in both 3T6 and H4-II-E-C3 cells, at least in part by increasing the cellular content of components required for assembly of RNA polymerase I into an active complex.
Molecular mechanisms of impaired stimulation of DNA synthesis in cultured hepatocytes of aged rats
Authors: Kitano Shoichi , Fawcett Timothy W. , Yo Yoshikage , Roth George S.
Published: December 2017

We examined epidermal growth factor (EGF)- and epinephrine-stimulated mitogen-activated protein kinase kinase (MEK) 1 and MEK2 activities, DNA polymerase α activity, and EGF-stimulated E2F DNA binding activity in primary cultured hepatocytes from 6- and 24-mo-old rats. MEK stimulation by either EGF or epinephrine was not altered with aging. However, stimulation of DNA polymerase α activity by these agents was 70% and 50% lower, respectively, in cells of aged compared with cells of young rats, consistent with a lesser increase in [3H]thymidine incorporation. EGF-stimulated E2F (a transcription factor that regulates expression of the DNA polymerase α gene) binding to DNA was reduced with age. PD-098059, a specific inhibitor of MEK, inhibited EGF-stimulated MEK1 and MEK2 activities in hepatocytes from 6- and 24-mo-old rats. Although PD-098059 inhibited EGF-stimulated DNA synthesis in hepatocytes from 6-mo-old rats, it had no effect in 24-mo-old rats. Thus the age-related impairment appears to occur before E2F activation, and signal transduction sequences other than the mitogen-activated protein kinase pathway may be involved in stimulated DNA synthesis in hepatocytes from old rats.
PKC regulation of cardiac CFTR Cl− channel function in guinea pig ventricular myocytes
Authors: Middleton Lisa M. , Harvey Robert D.
Published: December 2017

The role of protein kinase C (PKC) in regulating the protein kinase A (PKA)-activated Cl− current conducted by the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (cCFTR) was studied in guinea pig ventricular myocytes using the whole cell patch-clamp technique. Although stimulation of endogenous PKC with phorbol 12,13-dibutyrate (PDBu) alone did not activate this Cl− current, even when intracellular dialysis was limited with the perforated patch-clamp technique, activation of PKC did elicit a significant response in the presence of PKA-dependent activation of the current by the β-adrenergic receptor agonist isoproterenol. PDBu increased the magnitude of the Cl− conductance activated by a supramaximally stimulating concentration of isoproterenol by 21 ± 3.3% ( n = 9) when added after isoproterenol and by 36 ± 16% ( n= 14) when introduced before isoproterenol. 4α-Phorbol 12,13-didecanoate, a phorbol ester that does not activate PKC, did not mimic these effects. Preexposure to chelerythrine or bisindolylmaleimide, two highly selective inhibitors of PKC, significantly reduced the magnitude of the isoproterenol-activated Cl− current by 79 ± 7.7% ( n = 11) and 52 ± 10% ( n = 8), respectively. Our results suggest that although acute activation of endogenous PKC alone does not significantly regulate cCFTR Cl− channel activity in native myocytes, it does potentiate PKA-dependent responses, perhaps most dramatically demonstrated by basal PKC activity, which may play a pivotal role in modulating the function of these channels.
Lasp-1 is a regulated phosphoprotein within the cAMP signaling pathway in the gastric parietal cell
Authors: Chew C. S. , Parente J. A. , Zhou C.-J. , Baranco E. , Chen X.
Published: December 2017

Activation of the cAMP signaling pathway is correlated with increased secretory-related events in a wide variety of cell types including the gastric parietal cell. Within this pathway, as well as in other intracellular signaling pathways, protein phosphorylation serves as a major downstream regulatory mechanism. However, although agonist and cAMP-dependent activation of cAMP-dependent protein kinase (PKA) has been demonstrated, little is currently known about the downstream in vivo phosphoprotein substrates of this enzyme. Here we report the isolation, microsequencing, and cloning of a LIM and SH3 domain-containing, cAMP-responsive, 40-kDa phosphoprotein (pp40) from rabbit gastric parietal cells. The deduced amino acid sequence for pp40 is 93.5%, homologous with the putative protein product of the human gene lasp-1, which was recently identified based on its overexpression in some breast carcinomas. In addition to LIM and SH3 domains, the rabbit homolog contains two highly conserved PKA consensus sequences as well as two conserved SH2 binding motifs and several other putative protein kinase phosphorylation sites, including two for tyrosine kinase(s). Combined Northern and Western blot analyses indicate that pp40/lasp-1 is widely expressed (through a single 3.3-kb message) not only in epithelial tissues but also in muscle and brain. Furthermore, stimulation of isolated parietal cells, distal colonic crypts, and pancreatic cells with the adenylyl cyclase activator forskolin leads to the appearance of a higher molecular weight form of pp40/lasp-1, a finding which is consistent with an increase in protein phosphorylation. Thus pp40/lasp-1 appears to be regulated within the cAMP signaling pathway in a wide range of epithelial cell types. Because the cAMP-dependent increase in pp40 phosphorylation is correlated with secretory responses in the parietal cell and because pp40 appears to be widely distributed among various secretory tissues, this newly defined signaling protein may play an important role in modulating ionic transport or other secretory-related activities in many different cell types.
cGMP-independent mechanism of airway smooth muscle relaxation induced byS-nitrosoglutathione
Authors: Perkins William J. , Pabelick Christina , Warner David O. , Jones Keith A.
Published: December 2017

This study tested the hypothesis that the NO donor S-nitrosoglutathione (GSNO) relaxes canine tracheal smooth muscle (CTSM) in part by a cGMP-independent process that involves reversible oxidation of intracellular thiols. GSNO caused a concentration-dependent relaxation in ACh-contracted strips (EC50 ∼1.2 μM) accompanied by a concentration-dependent increase in cytosolic cGMP concentration ([cGMP]i). The soluble guanylate cyclase inhibitor methylene blue prevented the increase in [cGMP]iinduced by 1 and 10 μM GSNO, but isometric force decreased by 10 ± 4 and 55 ± 3%, respectively. After recovery of [cGMP]i to baseline, GSNO-induced relaxation persisted during continuous ACh stimulation. Dithiothreitol caused a rapid recovery of isometric force to values similar to those obtained with ACh alone in these strips. We conclude that GSNO relaxes CTSM contracted by ACh in part by oxidation of intracellular protein thiols.
Effect of intracellular pH on acetylcholine-induced Ca2+ waves in mouse pancreatic acinar cells
Authors: González Antonio , Pfeiffer Fatima , Schmid Andreas , Schulz Irene
Published: December 2017

We have used fluo 3-loaded mouse pancreatic acinar cells to investigate the relationship between Ca2+ mobilization and intracellular pH (pHi). The Ca2+-mobilizing agonist ACh (500 nM) induced a Ca2+ release in the luminal cell pole followed by spreading of the Ca2+ signal toward the basolateral side with a mean speed of 16.1 ± 0.3 μm/s. In the presence of an acidic pHi, achieved by blockade of the Na+/H+exchanger or by incubation of the cells in a Na+-free buffer, a slower spreading of ACh-evoked Ca2+ waves was observed (7.2 ± 0.6 μm/s and 7.5 ± 0.3 μm/s, respectively). The effects of cytosolic acidification on the propagation rate of ACh-evoked Ca2+ waves were largely reversible and were not dependent on the presence of extracellular Ca2+. A reduction in the spreading speed of Ca2+ waves could also be observed by inhibition of the vacuolar H+-ATPase with bafilomycin A1 (11.1 ± 0.6 μm/s), which did not lead to cytosolic acidification. In contrast, inhibition of the endoplasmic reticulum Ca2+-ATPase by 2,5-di- tert-butylhydroquinone led to faster spreading of the ACh-evoked Ca2+ signals (25.6 ± 1.8 μm/s), which was also reduced by cytosolic acidification or treatment of the cells with bafilomycin A1. Cytosolic alkalinization had no effect on the spreading speed of the Ca2+ signals. The data suggest that the propagation rate of ACh-induced Ca2+ waves is decreased by inhibition of Ca2+ release from intracellular stores due to cytosolic acidification or to Ca2+ pool alkalinization and/or to a decrease in the proton gradient directed from the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool to the cytosol.
Cl− transport in an immortalized human epithelial cell line (NCM460) derived from the normal transverse colon
Authors: Sahi Jasminder , Nataraja Selvaraj G. , Layden Thomas J. , Goldstein Jay L. , Moyer M. P. , Rao Mrinalini C.
Published: December 2017

Cells of a newly described, immortalized, epithelial, human transverse colonic cell line, NCM460, reach ∼90% confluence on plastic and develop transepithelial resistances of 120–250 Ω ⋅ cm2 on porous substrates. Its utility as a model for the transverse human colon was validated by comparing second messenger-mediated Cl− transport, using the fluorescent probe 6-methoxy-quinolyl acetoethyl ester, in NCM460 cells and colonocytes isolated from human transverse crypts. Basal Cl− influx was increased ( P < 0.01) by PGE1 (1 μM), forskolin (1 μM), 8-bromoadenosine 3′5′-cyclic monophosphate (100 μM), heat-stable Escherichia colienterotoxin (STa; 1 μM), 8-bromoguanosine 3′5′-cyclic monophosphate (100 μM), histamine (1 μM), and phorbol 12,13-dibutyrate (1 μM) in both cell types. The Cl− channel blocker diphenylamine 2-carboxylic acid (50 μM) and the Na+-K+-2Cl−cotransport inhibitor furosemide (1 μM), but not the K+ channel blocker Ba2+ (3 mM), inhibited these Cl− permeabilities. These cells possess transcripts for cystic fibrosis transmembrane conductance regulator, Na+-K+-2Cl−cotransporter, STa receptor, and intestine-specific cGMP-dependent protein kinase II. Thus cAMP-, cGMP-, and Ca2+-dependent secretagogues act on NCM460 and primary colonocytes to stimulate Cl− transport. This validates the utility of NCM460 as a model for transverse colonic crypts and is the first demonstration of a colonic cell line whose origin is known.
Luminal l-alanine stimulates exocytosis at the K+-conductive apical membrane ofAplysia enterocytes
Authors: Denton Jerod , Boahene Derek , Moran William M.
Published: December 2017

In Aplysia intestine, stimulation of Na+ absorption with luminal alanine increases apical membrane K+ conductance ( G K,a), which presumably regulates enterocyte volume during stimulated Na+ absorption. However, the mechanism responsible for the sustained increase in plasma membrane K+ conductance is not known for any nutrient-absorbing epithelium. In the present study, we have begun to test the hypothesis that the alanine-induced increase in G K,a in Aplysia enterocytes results from exocytic insertion of K+ channels into the apical membrane. We used the fluid-phase marker horseradish peroxidase to assess the effect of alanine on apical membrane exocytosis and conventional microelectrode techniques to assess the effect of alanine on fractional capacitance of the apical membrane (f C a). Luminal alanine significantly increased apical membrane exocytosis from 1.04 ± 0.30 to 1.39 ± 0.38 ng ⋅ min−1 ⋅ cm−2. To measure f C a, we modeled the Aplysia enterocyte as a double resistance-capacitance (RC) electric circuit arranged in series. Several criteria were tested to confirm application of the model to the enterocytes, and all satisfied the model. When added to the luminal surface, alanine significantly increased f C a from 0.27 ± 0.02 to 0.33 ± 0.04 ( n = 10) after 4 min. There are two possible explanations for our findings: 1) the increase in exocytosis, which adds membrane to the apical plasma membrane, prevents plasma membrane fracture, and 2) the increase in exocytosis delivers K+ channels to the apical membrane by exocytic insertion. After the alanine-induced depolarization of apical membrane potential ( V a), there is a strong correlation ( r = 0.96) between repolarization of V a, which reflects the increase in G K,a, and increase in f C a. This correlation supports the exocytic insertion hypothesis for activation of G K,a.
Functional characterization of alternatively spliced human SERCA3 transcripts
Authors: Poch Esteban , Leach Stephen , Snape Susan , Cacic Tasha , MacLennan David H. , Lytton Jonathan
Published: December 2017

The sarcoplasmic (or endoplasmic) reticulum Ca2+-ATPase (SERCA)-3 has been implicated in the possible dysregulation of Ca2+ homeostasis that accompanies the pathology of hypertension and diabetes. We report the molecular cloning of two alternatively spliced transcripts from the human SERCA3 gene, ATP2A3, that encode proteins that differ at their carboxy termini by 36 amino acids. SERCA3 transcripts were most abundantly expressed in lymphoid tissues, intestine, pancreas, and prostate. The two human SERCA3 proteins encoded by alternatively spliced transcripts were recognized by the monoclonal antibody PL/IM430 and demonstrated Ca2+ uptake and ATPase activity with an apparent Ca2+ affinity 0.5 pCa unit lower than that of other SERCA gene products. The subcellular distribution of SERCA3 protein was indistinguishable from that of SERCA2b, with expression in the nuclear envelope and in the endoplasmic reticulum throughout the cell. Two variant SERCA3 constructs, huS3-I and huS3-II, were isolated that encode proteins with three amino acid differences: Ala-673 (in huS3-I) substituted for Thr (in huS3-II), Ile-817 substituted for Met, and an insertion of Glu-994. huS3-I displayed a 10-fold lower capacity to transport Ca2+ than huS3-II.
Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current
Authors: Gomora Juan Carlos , Enyeart John J.
Published: December 2017

Bovine adrenal zona fasciculata cells (AZF) express a noninactivating K+ current ( I AC) whose inhibition by adrenocorticotropic hormone and ANG II may be coupled to membrane depolarization and Ca2 +-dependent cortisol secretion. We studied I ACinhibition by Ca2 + and the Ca2 +ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette) Ca2 +concentration ([Ca2+]i) buffered to 0.02 μM, I AC reached maximum current density of 25.0 ± 5.1 pA/pF ( n = 16); raising [Ca2+]ito 2.0 μM reduced it 76%. In inside-out patches, elevated [Ca2+]idramatically reduced I AC channel activity. Ionomycin inhibited I AC by 88 ± 4% ( n = 14) without altering rapidly inactivating A-type K+ current. Inhibition of I ACby ionomycin was unaltered by adding calmodulin inhibitory peptide to the pipette or replacing ATP with its nonhydrolyzable analog 5′-adenylylimidodiphosphate. I AC inhibition by ionomycin was associated with membrane depolarization. When [Ca2+]iwas buffered to 0.02 μM with 2 and 11 mM 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid (BAPTA), ionomycin inhibited I AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively ( n = 4). ANG II inhibited I AC significantly more effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising [Ca2+]iinhibits I ACthrough a mechanism not requiring calmodulin or protein kinases, suggesting direct interaction with I AC channels. ANG II may inhibit I AC and depolarize AZF by activating parallel signaling pathways, one of which uses Ca2 + as a mediator.
Contraction-induced injury to single muscle fibers: velocity of stretch does not influence the force deficit
Authors: Lynch Gordon S. , Faulkner John A.
Published: December 2017

We tested the null hypothesis that the severity of injury to single muscle fibers following a single pliometric (lengthening) contraction is not dependent on the velocity of stretch. Each single permeabilized fiber obtained from extensor digitorum longus muscles of rats was maximally activated and then exposed to a single stretch of either 5, 10, or 20% strain [% of fiber length ( L f)] at a velocity of 0.5, 1.0, or 2.0 L f /s. The force deficit, the difference between maximum tetanic isometric force (Po) before and after the stretch expressed as a percentage of the control value for Po before the stretch, provided an estimate of the magnitude of muscle injury. Despite a fourfold range from the lowest to the highest velocities, force deficits were not different among stretches of the same strain. At stretches of 20% strain, even an eightfold range of velocities produced no difference in the force deficit, although 40% of the fibers were torn apart at a velocity of 4 L f /s. We conclude that, within the range of velocities tolerated by single permeabilized fibers, the severity of contraction-induced injury is not related to the velocity of stretch.
Expression of TWIK-1, a novel weakly inward rectifying potassium channel in rat kidney
Authors: Cluzeaud F. , Reyes R. , Escoubet B. , Fay M. , Lazdunski M. , Bonvalet J. P. , Lesage F. , Farman N.
Published: December 2017

Several K+ conductances have been identified in the kidney, with specific properties and localization in distinct cell types and membrane domains. On the other hand, several K+ channels have been characterized at the molecular level. By immunolocalization, we show that a new inward rectifying K+channel, TWIK-1, is specifically expressed in distinct tubular segments and cell types of the rat kidney. In the proximal tubule, TWIK-1 prevails in the initial portions (convoluted part), where it is restricted to the apical (brush-border) membrane. In the collecting duct, immunofluorescence was intracellular or confined to the apical membrane and restricted to intercalated cells, i.e., in cells lacking aquaporin-2, as shown by double immunofluorescence. TWIK was also expressed in medullary and cortical parts of the thick limb of the loop of Henle, identified with an anti-Tamm-Horsfall protein antibody (double immunofluorescence). The intensity of TWIK-1 immunolabeling was unchanged in rats fed a low-Na+ or a low-K+ diet. Because TWIK-1 shares common properties with the low-conductance apical K+ channel of the collecting duct, we propose that it could play a role in K+ secretion, complementary to ROMK, another recently characterized K+ channel located in principal cells of the cortical collecting duct and in the loop of Henle.
Adrenergic stimulation of Na+transport across alveolar epithelial cells involves activation of apical Cl−channels
Authors: Jiang Xinpo , Ingbar David H. , O’Grady Scott M.
Published: December 2017

Alveolar epithelial cells were isolated from adult Sprague-Dawley rats and grown to confluence on membrane filters. Most of the basal short-circuit current ( I sc; 60%) was inhibited by amiloride (IC50 0.96 μM) or benzamil (IC50 0.5 μM). Basolateral addition of terbutaline (2 μM) produced a rapid decrease in I sc, followed by a slow recovery back to its initial amplitude. When Cl− was replaced with methanesulfonic acid, the basal I sc was reduced and the response to terbutaline was inhibited. In permeabilized monolayer experiments, both terbutaline and amiloride produced sustained decreases in current. The current-voltage relationship of the terbutaline-sensitive current had a reversal potential of −28 mV. Increasing Cl− concentration in the basolateral solution shifted the reversal potential to more depolarized voltages. These results were consistent with the existence of a terbutaline-activated Cl− conductance in the apical membrane. Terbutaline did not increase the amiloride-sensitive Na+ conductance. We conclude that β-adrenergic stimulation of adult alveolar epithelial cells results in an increase in apical Cl− permeability and that amiloride-sensitive Na+ channels are not directly affected by this stimulation.
Bumetanide blocks CFTRG Cl in the native sweat duct
Authors: Reddy M. M. , Quinton P. M.
Published: December 2017

Bumetanide is well known for its ability to inhibit the nonconductive Na+-K+-2Cl−cotransporter. We were surprised in preliminary studies to find that bumetanide in the contraluminal bath also inhibited NaCl absorption in the human sweat duct, which is apparently poor in cotransporter activity. Inhibition was accompanied by a marked decrease in the transepithelial electrical conductance. Because the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is richly expressed in the sweat duct, we asked whether bumetanide acts by blocking this anion channel. We found that bumetanide 1) significantly increased whole cell input impedance, 2) hyperpolarized transepithelial and basolateral membrane potentials, 3) depolarized apical membrane potential, 4) increased the ratio of apical-to-basolateral membrane resistance, and 5) decreased transepithelial Cl− conductance ( G Cl). These results indicate that bumetanide inhibits CFTR G Clin both cell membranes of this epithelium. We excluded bumetanide interference with the protein kinase A phosphorylation activation process by “irreversibly” phosphorylating CFTR [by using adenosine 5′- O-(3-thiotriphosphate) in the presence of a phosphatase inhibition cocktail] before bumetanide application. We then activated CFTR G Clby adding 5 mM ATP. Bumetanide in the cytoplasmic bath (10−3 M) inhibited ∼71% of this ATP-activated CFTR G Cl, indicating possible direct inhibition of CFTR G Cl. We conclude that bumetanide inhibits CFTR G Clin apical and basolateral membranes independent of phosphorylation. The results also suggest that >10−5 M bumetanide cannot be used to specifically block the Na+-K+-2Cl−cotransporter.
Intracellular H+ regulates the α-subunit of ENaC, the epithelial Na+ channel
Authors: Chalfant Michael L. , Denton Jerod S. , Berdiev Bakhram K. , Ismailov Iskander I. , Benos Dale J. , Stanton Bruce A.
Published: December 2017

Protons regulate electrogenic sodium absorption in a variety of epithelia, including the cortical collecting duct, frog skin, and urinary bladder. Recently, three subunits (α, β, γ) coding for the epithelial sodium channel (ENaC) were cloned. However, it is not known whether pH regulates Na+ channels directly by interacting with one of the three ENaC subunits or indirectly by interacting with a regulatory protein. As a first step to identifying the molecular mechanisms of proton-mediated regulation of apical membrane Na+ permeability in epithelia, we examined the effect of pH on the biophysical properties of ENaC. To this end, we expressed various combinations of α-, β-, and γ-subunits of ENaC in Xenopusoocytes and studied ENaC currents by the two-electrode voltage-clamp and patch-clamp techniques. In addition, the effect of pH on the α-ENaC subunit was examined in planar lipid bilayers. We report that α,β,γ-ENaC currents were regulated by changes in intracellular pH (pHi) but not by changes in extracellular pH (pHo). Acidification reduced and alkalization increased channel activity by a voltage-independent mechanism. Moreover, a reduction of pHi reduced single-channel open probability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited α,β-ENaC, α,γ-ENaC, and α-ENaC currents. We conclude that pHi but not pHo regulates ENaC and that the α-ENaC subunit is regulated directly by pHi.
Eosinophil peroxidase increases membrane permeability in mammalian urinary bladder epithelium
Authors: Kleine Teri J. , Gleich Gerald J. , Lewis Simon A.
Published: December 2017

Eosinophil peroxidase (EPO), a cationic protein found in eosinophils, has been reported to be cytotoxic independent of its peroxidase activity. This study investigated with electrophysiological methods whether EPO is toxic to mammalian urinary bladder epithelium. Results indicate that EPO, when added to the mucosal solution, increases apical membrane conductance of urinary bladder epithelium only when the apical membrane potential is cell interior negative. The EPO-induced conductance was concentration dependent, with a maximum conductance of 411 μS/cm2 and a Michaelis-Menten constant of 113 nM. The EPO-induced conductance was nonselective for K+ and Cl−. The conductance was partially reversed using voltage but not by removal of EPO from the bulk solution. Mucosal Ca2+reversed the EPO-induced conductance by a mechanism involving reversible block of the conductance. Prolonged exposure (up to 1 h) to EPO was toxic to the urinary bladder epithelium, as indicated by an irreversible increase in transepithelial conductance. These results suggest that EPO is indeed toxic to urinary bladder epithelium via a mechanism that involves an increase in membrane permeability.
Effect of acidification on the location of H+-ATPase in cultured inner medullary collecting duct cells
Authors: Alexander Edward A. , Brown Dennis , Shih Theodora , McKee Mary , Schwartz John H.
Published: December 2017

In previous studies, our laboratory has utilized a cell line derived from the rat inner medullary collecting duct (IMCD) as a model system for mammalian renal epithelial cell acid secretion. We have provided evidence, from a physiological perspective, that acute cellular acidification stimulates apical exocytosis and elicits a rapid increase in proton secretion that is mediated by an H+-ATPase. The purpose of these experiments was to examine the effect of acute cellular acidification on the distribution of the vacuolar H+-ATPase in IMCD cells in vitro. We utilized the 31-kDa subunit of the H+-ATPase as a marker of the complete enzyme. The distribution of this subunit of the H+-ATPase was evaluated by immunohistochemical techniques (confocal and electron microscopy), and we found that there is a redistribution of these pumps from vesicles to the apical membrane. Immunoblot evaluation of isolated apical membrane revealed a 237 ± 34% ( P < 0.05, n = 9) increase in the 31-kDa subunit present in the membrane fraction 20 min after the induction of cellular acidification. Thus our results demonstrate the presence of this pump subunit in the IMCD cell line in vitro and that cell acidification regulates the shuttling of cytosolic vesicles containing the 31-kDa subunit into the apical membrane.
P2Z/P2X7receptor-dependent apoptosis of dendritic cells
Authors: Coutinho-Silva Robson , Persechini Pedro M. , Bisaggio Rodrigo Da Cunha , Perfettini Jean-Luc , Neto Ana Cristina Torres De Sa , Kanellopoulos Jean M. , Motta-Ly Iris , Dautry-Varsat Alice , Ojcius David M.
Published: December 2017

Macrophages and thymocytes express P2Z/P2X7nucleotide receptors that bind extracellular ATP. These receptors play a role in immune development and control of microbial infections, but their presence on dendritic cells has not been reported. We investigated whether extracellular ATP could trigger P2Z/P2X7receptor-dependent apoptosis of dendritic cells. Apoptosis could be selectively triggered by tetrabasic ATP, since other purine/pyrimidine nucleotides were ineffective, and it was mimicked by the P2Z receptor agonist, benzoylbenzoyl ATP, and blocked by magnesium and the irreversible antagonist, oxidized ATP. RT-PCR analysis confirmed the mRNA expression of the P2Z/P2X7receptor and the absence of P2X1. Caspase inhibitors and cycloheximide had only a partial effect on the apoptosis, suggesting that a caspase-independent mechanism may also be operative. Brief treatment with ATP led to an increase in the intracellular calcium concentration and permeabilization of the plasma membrane to Lucifer yellow, which diffused throughout the dendritic cell cytosol. Other small extracellular molecules may thus attain a similar intracellular distribution, perhaps activating endogenous proteases that contribute to initiation of apoptosis.