bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Na-K-2Cl cotransport inhibition on myocardial Na and Ca during ischemia and reperfusion
Authors: Anderson S. E. , Dickinson C. Z. , Liu H. , Cala P. M.
Published: December 2017

In the context of the "pump-leak" hypothesis (37), changes in myocardial intracellular Na (Nai) during ischemia and reperfusion have historically been interpreted to be the result of changes in Na efflux via the Na-K pump. We investigated the alternative hypothesis that changes in Nai during ischemia are the result of changes in the Na "leak" rather than changes in the pump. More specifically, we hypothesize that the increase in Nai during ischemia is in part the result of increased Na uptake mediated by Na/H exchange. Furthermore, we present data consistent with the interpretation that the Na-K-2Cl cotransporter is active (or, alternatively, displaced from equilibrium) during ischemia and may contribute an additional Na efflux pathway during reperfusion. Thus inhibition of Na efflux via Na-K-2Cl cotransport during ischemia and reperfusion could result in increased Nai and therefore decreased force driving Ca efflux via Na/Ca exchange and ultimately increased intracellular Ca concentration ([Ca]i). Nai (in meq/kg dry wt) and [Ca]i (in nM) were measured in isolated Langendorff-perfused rabbit hearts using nuclear magnetic resonance spectroscopy. Except, during the 65 min of ischemia, hearts were perfused with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Krebs-Henseleit solution equilibrated with 100% O2 at 23 degrees C and pH 7.4 +/- 0.05. During ischemia, Nai rose from 16.6 +/- 0.3 to 62.9 +/- 5.1 (delta Nai approximately 46) meq/kg dry wt and decreased during subsequent reperfusion (mean +/- SE, n = 3 hearts). To measure Na uptake ("leak") in the absence of efflux via the Na-K pump, in all of the protocols described below, the perfusate was nominally K-free solution containing 1 mM ouabain for 10 min before ischemia and during the 30-min reperfusion. After K-free perfusion, Nai rose from 20.2 +/- 0.5 to 79.1 +/- 5.3 (delta Nai approximately 59) meq/kg dry wt (n = 3) during ischemia and decreased during K-free reperfusion. When amiloride (1 mM) was added to the K-free perfusate to inhibit Na/H exchange, Nai rose from 16.3 +/- 0.9 to 44.7 +/- 5.1 (delta Nai approximately 28) meq/kg dry wt (n = 3) during ischemia; i.e., amiloride decreased Na uptake. When bumetanide (20 microM) was added to the nominally K-free perfusate to inhibit Na-K-2Cl contransport, Nai rose from 22.5 +/- 3.9 to 83.8 +/- 13.9 (delta Nai approximately 61 meq/kg dry wt (n = 3) during ischemia and did not decrease during reperfusion; i.e., bumetanide inhibited Na recovery during reperfusion (P < 0.05 compared with bumetanide free). For the same protocol, the presence of bumetanide resulted in increased [Ca]i during ischemia and reperfusion (P < 0.05); these increases in [Ca]i are interpreted to be the result of increased Nai. Thus the results are consistent with the hypotheses.
Insulin and diabetes cause reciprocal changes in the association of eIF-4E and PHAS-I in rat skeletal muscle
Authors: Kimball S. R. , Jefferson L. S. , Fadden P. , Haystead T. A. , Lawrence J. C.
Published: December 2017

We have investigated the roles of eukaryotic initiation factor 4E (eIF-4E), the cap-binding protein, and the translational regulator, PHAS-I, in the effects of insulin and alloxan-induced diabetes on protein synthesis in rat skeletal muscle. Diabetes increased the amount of eIF-4E found in the inactive PHAS-I.eIF-4E complex by threefold, explaining in part the inhibitory effect of insulin deficiency on translation initiation. Insulin treatment of diabetic rats caused dissociation of the complex, consistent with the action of the hormone on reversing the inhibitory effect of diabetes on translation initiation. The effects of both insulin and diabetes on PHAS-I binding to eIF-4E appeared to be due to changes in PHAS-I phosphorylation. Neither insulin nor diabetes changed the phosphorylation state of eIF-4E. The results indicate that the effects of both insulin and diabetes on protein synthesis in skeletal muscle involve modulation of the interaction of PHAS-I and eIF-4E.
Nitric oxide is necessary for a switch from stationary to locomoting phenotype in epithelial cells
Authors: Noiri E. , Peresleni T. , Srivastava N. , Weber P. , Bahou W. F. , Peunova N. , Goligorsky M. S.
Published: December 2017

The restitution of epithelial integrity is accomplished in part by cell migration. Studying this process, we have found that nitric oxide (NO) release migrating epithelial BSC-1 cells displayed a biphasic response to the inflicted wounds; an initial transient release of NO is followed by a delayed sustained elevation. Whereas the constitutive endothelial NO synthase (NOS) did not show any spatial or temporal changes associated with wounding, the inducible NOS became expressed 3 h after wounding and showed higher abundance at the edges of epithelial wounds. L-Arginine (L-Arg) or NO donor, S-nitroso-N-acetyl-DL-penicillamine, exerted motogenic effect in epithelial and endothelial cells. Inhibition of NOS with NG-nitro-L-arginine methyl ester (L-NAME) or a selective knockout of inducible NOS with antisense oligodeoxynucleotides reduced the rate of spontaneous or epidermal growth factor (EGF)-induced BSC-1 cell migration. Migrating cells showed the polarized expression of NOS, suggesting a head-to-rear NO gradient. Several growth factors (EGF, insulin-like growth factor I, hepatocyte growth factor, and fibroblast growth factor) were motogenic for BSC-1 cells, but this effect was abrogated by pretreatment with L-NAME. We conclude that endogenous NO production is a prerequisite for BSC-1 cell migration. A vectorial NO release may be essential for the spatially and temporally coordinated reciprocal phenomena that occur at the leading and trailing edge of locomoting epithelial cells. Although the exact mode of NO action remains uncertain, it is conceivable that the production of NO serves as a cellular switch from the stationary to the locomoting epithelial phenotype.
Role of glucosamine synthesis in the stimulation of TGF-alpha gene transcription by glucose and EGF
Authors: Roos M. D. , Han I. O. , Paterson A. J. , Kudlow J. E.
Published: December 2017

Transforming growth factor-alpha (TGF-alpha) gene transcription is regulated by both epidermal growth factor (EGF) and glucose. Previous studies have suggested that the metabolism of glucose to glucosamine through the enzyme L-glutamine: D-fructose-6-phosphate amidotransferase (GFAT) plays a critical role in the glucose signaling. In this paper, we compared the role of GFAT in the glucose and EGF signals. We found that, although EGF stimulates GFAT mRNA accumulation in MDA-MB-468 cells, this effect of EGF occurred several hours after TGF-alpha transcription increased. MDA-MB-468 cells also exhibited a TGF-alpha transcriptional response to low concentrations of glucose. The TGF-alpha response to glucose but not EGF could be inhibited by a blocker of GFAT activity. Blockade of GFAT was confirmed by using Western blotting with the RL2 antibody, which recognizes an epitope on proteins containing N-acetylglucosamine. Exposure of cells to glucose increased the RL2 signal on several polypeptides, but this change could be blocked by inhibition of GFAT. These results support the notion that glucose stimulation of TGF-alpha expression requires GFAT, but EGF stimulation does not.
Nitric oxide: a modulator for the epidermal growth factor receptor expression in developing ovarian granulosa cells
Authors: Hattori M. , Sakamoto K. , Fujihara N. , Kojima I.
Published: December 2017

the present study was designed to assess the involvement of nitric oxide (NO) in the regulation of the epidermal growth factor (EGF) receptor during development of rat granulosa cells, which were prepared by puncturing ovaries of diethylstilbestrol-treated rats. The immature cells were cultured for 48 h with follicle-stimulating hormone (FSH) to be transformed into mature cells. A marked accumulation of guanosine 3',5' -cyclic monophosphate (cGMP) was observed during development. The accumulation of cGMP, but not of adenosine 3',5'-cyclic monophosphate (cAMP), was suppressed by specific inhibitors of NO synthase, L-NG-monomethyl-L-arginine (L-NMMA) and L-N(G)-nitro-L-arginine, and a selective inhibitor of the inducible NO synthase, aminoguanidine. The L-NMMA-induced suppression was partially reversed by addition of L-arginine to cultures but not D-arginine, indicating that NO formation is inhibited by competing with analogues of L-arginine for NO synthase. Treatment with 2-(4-carboxyphenyl)-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide , an antagonist of NO, caused suppression in the increase of EGF binding sites, whereas exposure of the cells to sodium nitroprusside (SNP), an NO donor, caused elevation in EGF binding sites, with increasing extra- and intracellular cGMP levels. Analysis of the EGF receptor by affinity labeling with 125I-labeled EGF revealed that the intensity of the cross-linked receptor molecular with a molecular mass of 180 kDa was increased by exposure to SNP. The facilitatory effect of SNP on the EGF receptor was observed when the cAMP-dependent pathway was fully activated by FSH. However, the NO effect may be mediated by a cGMP-independent pathway, as 8-bromo-cGMP did not mimic the action of SNP. These results indicate that the L-arginine-NO system may contribute to the regulation of EGF receptor expression in developing granulosa cells stimulated by FSH.
The effect of temperature on charge movement repriming in amphibian skeletal muscle fibers
Authors: Gonzalez A. , Caputo C.
Published: December 2017

Cut twitch muscle fibers, mounted in a triple Vaseline-gap chamber, were used to study the effects of temperature on intramembranous charge movement and, in particular, on the repriming of charge 1 (the intramembranous charge that normally moves in the potential range between -100 and +40 mV). Changing the holding potential from -90 to 0 mV modified the voltage distribution of charge movement but not the maximum movable charge. Temperature changes between 16 and 5 degrees C did not modify the fiber linear capacitance, the maximum nonlinear intramembranous charge, or the voltage distribution of charge 1 and charge 2 (the intramembranous charge moving in the membrane potential range between approximately -4 and -160 mV). We used a pulse protocol designed to study the repriming time course of charge 1, with little contamination from charge 2. The time course of charge movement repriming at 15 degrees C is described by a double exponential with time constants of 4.2 and 25 s. Repriming kinetics were found to be highly temperature dependent, with two rate-limiting steps having Q10 (increase in rate of a process by raising temperature 10 degrees C) values of 1.7 and 7.1 above and below 11.5 degrees C, respectively. This is characteristic of processes with a high energy of activation and could be associated with a conformational change of the voltage sensor or with the interaction between the voltage sensor and the calcium release channel.
AlphaB-crystallin protects glial cells from hypertonic stress
Authors: Kegel K. B. , Iwaki A. , Iwaki T. , Goldman J. E.
Published: December 2017

AlphaB-crystallin and the small stress protein, heat shock protein of 27 kDa (HSP27), share structural similarities and are coordinately induced by classical stress stimuli. We have recently observed that hypertonic stress produced by high NaCl concentrations selectively induces alphaB-crystallin in glial cells. To examine divergence of the functional properties of these two related proteins, we have constructed stable alphaB-crystallin-expressing glial cell lines from the U-251 MG glioma cells, which are normally deficient in alphaB-crystallin expression but constitutively express HSP27. These transfected cells lines are more resistant to acute hypertonic stress than the parental line from which they were derived. Moreover, the parental line acclimates to stepwise increases in hypertonicity and upregulates endogenous alphaB-crystallin in the process but not HSP27. The overexpression of HSP27 and alphaB-crystallin in NIH/3T3 fibroblasts, a cell line that normally expresses little alphaB-crystallin and no HSP27, does not result in increased survival. This suggests that alphaB-crystallin interacts with cell-type specific mechanisms to aid in protection from hypertonic stress.
Rapid kinetics of second messenger production in bitter taste
Authors: Spielman A. I. , Nagai H. , Sunavala G. , Dasso M. , Breer H. , Boekhoff I. , Huque T. , Whitney G. , Brand J. G.
Published: December 2017

The tasting of bitter compounds may have evolved as a protective mechanism against ingestion of potentially harmful substances. We have identified second messengers involved in bitter taste and show here for the first time that they are rapid and transient. Using a quench-flow system, we have studied bitter taste signal transduction in a pair of mouse strains that differ in their ability to taste the bitter stimulus sucrose octaacetate (SOA); however, both strains taste the bitter agent denatonium. In both strains of mice, denatonium (10 mM) induced a transient and rapid increase in levels of the second messenger inositol 1,4,5-trisphosphate (IP3) with a maximal production near 75-100 ms after stimulation. In contrast, SOA (100 microM) brought about a similar increase in IP3 only in SOA-taster mice. The response to SOA was potentiated in the presence of GTP (1 microM). The GTP-enhanced SOA-response supports a G protein-mediated response for this bitter compound. The rapid kinetics, transient nature, and specificity of the bitter taste stimulus-induced IP3 formation are consistent with the role of IP3 as a second messenger in the chemoelectrical transduction of bitter taste.
Effect of Ca2+ influx on intracellular free Ca2+ responses in antigen-stimulated RBL-2H3 cells
Authors: Smith G. D. , Lee R. J. , Oliver J. M. , Keizer J.
Published: December 2017

We undertake a quantitative investigation of changes in intracellular free Ca2+ concentration ([Ca2+]i) in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells, which include contributions of both Ca2+ store release and Ca2+ influx from the medium. Following Keizer and De Young (J. Keizer and G. De Young. Biophys. J. 61: 649-660, 1992), we develop a highly constrained mathematical model for [Ca2+]i oscillations in RBL-2H3 cells, which includes activation of the inositol trisphosphate receptor (IP3R) by inositol 1,4,5-trisphospate, indirect Ca2+ activation of the IP3R via Ca2+ -dependent activity of phospholipase C-gamma, slow inhibition of the IP3R by cytosolic Ca2+, refilling of Ca2+ stores by a Ca2+ -ATPase (SERCA)-type pump, and a simple representation of the dependence of plasma membrane (PM) fluxes on experimental conditions. Using this full (open cell) model, we simulate [Ca2+]i responses for protocols in which antigen concentration and external Ca2+ are manipulated and compare out calculations with experimental data. In protocol A, cells are stimulated in the presence of external Ca2+, in protocols B and C, cells are stimulated in the absence of external Ca2+, with external Ca2+ later reapplied in protocol C. We are able to reproduce quantitatively the important features of all three protocols, including the dose response of protocol B, the [Ca2+]i response to thapsigargin, and lag time results, and we provide qualitative explanations for the responses derived from our calculations. We also develop a simplified (closed cell) version of the model in which PM fluxes are neglected and total free Ca2+ concentration ([Ca2+]T) is a slowly varying parameter. This permits us to explain in a simple graphical fashion how PM fluxes may influence [Ca2+]i responses in RBH-2H3 cells through modulation of [Ca2+]T.
Mechanical regulation of cardiac myocyte protein turnover and myofibrillar structure
Authors: Simpson D. G. , Sharp W. W. , Borg T. K. , Price R. L. , Terracio L. , Samarel A. M.
Published: December 2017

Mechanical forces play an essential role in regulating the synthesis and assembly of contractile proteins into the sarcomeres of cardiac myocytes. To examine if physical forces might also regulate the turnover of contractile proteins at a posttranslational site of control, beating and nonbeating neonatal cardiac myocytes (NCM) were subjected to a 5% static stretch. The L-type calcium channel blocker nifedipine (12 microM) was used to inhibit contraction. Pulse-chase biosynthetic labeling experiments demonstrated that contractile arrest accelerated the loss of isotopic tracer from the total myofibrillar protein fraction, myosin heavy chain (MHC), and actin, but not desmin. Myofibrillar abnormalities developed in parallel with these metabolic changes. A 5% static load appeared to partially stabilize myofibrillar structure in nonbeating NCM and suppressed the loss of isotopic tracer from the total myofibrillar protein fraction, MHC, and actin in beating and nonbeating NCM. Contractile activity and/or a static stretch promoted the accumulation of MHC, actin, and desmin. Applying a static load to myocytes that lacked preexisting myofibrils did not promote the assembly of sarcomeres or alter protein turnover. These data indicate that the turnover of MHC and actin is correlated with the organizational state of the myofibrillar apparatus.
EGF receptor mRNA and protein in rat lacrimal acinar cells: evidence of its EGF-dependent phosphotyrosilation
Authors: Marechal H. , Jammes H. , Rossignol B. , Mauduit P.
Published: December 2017

This study was designed to demonstrate the presence of the epidermal growth factor (EGF) receptor in the rat exorbital lacrimal gland. EGF receptor gene transcription was demonstrated 1) by reverse transcription-polymerase chain reaction analysis of lacrimal gland and acinar cells from RNA with a set of specific primers deduced from the rat EGF receptor sequence and 2) by Northern blot analysis of rat lacrimal gland mRNA. Lacrimal acinar cell preparations contain a low but detectable amount of specific 125I-EGF binding sites and efficiently internalize the ligand on binding at 37 degrees C. A sheep polyclonal antibody, directed against the human EGF receptor, detects a protein of 170 kDa by Western blot analysis of membrane proteins of the whole gland. This protein can be immunoprecipitated by the same antibody from whole gland membrane proteins as well as from solubilized acinar cells. Incubations of acinar cells in the presence of EGF results in an increased content of tyrosine-phosphorylated residues in immunoprecipitated 170-kDa protein. Taken together, these results demonstrate for the first time both EGF receptor gene transcription and protein expression in a lacrimal tissue, i.e., the rat exorbital lacrimal gland. These results also suggest a specific cellular location of the EGF receptor in a cell population contained in acinar cell preparations.
Characterization of cell volume loss in CEM-C7A cells during dexamethasone-induced apoptosis
Authors: Benson R. S. , Heer S. , Dive C. , Watson A. J.
Published: December 2017

A reduction in cell volume is a fundamental feature of apoptosis. We have characterized changes in cell volume, together with nuclear changes, occurring in dexamethasone-induced apoptosis in CEM-C7A lymphoblastoid cells. Cell volume was measured by electronic cell sizing and flow cytometry, and two distinct phases of volume loss were observed. The first phase began 12 h after addition of dexamethasone (5 microM) and progressed until 36 h when chromatin condensation was detected in intact cells. Removal of dexamethasone before 36 h (the precommitment period) resulted in reversal of the volume decrease and prevented the appearance of nuclear changes. Cell shrinkage in the first 24 h of dexamethasone exposure was associated with a net loss of potassium but no change in cellular buoyant density. There were no significant differences in the rates of volume recovery after either hypertonic or hypotonic stimuli. These observations favor a mechanism of cell shrinkage involving loss of the entire cytoplasmic contents, possibly following proteolysis, rather than loss of only osmolytes and water. The second phase of volume loss was coincident with chromatin condensation and was associated with cellular fragmentation and a reduction in cellular density. We conclude that volume loss in this model of apoptosis is mediated by multiple mechanisms that are both dependent and independent of cellular fragmentation.
Wortmannin inhibits contraction without altering electrical activity in canine gastric smooth muscle
Authors: Burke E. P. , Gerthoffer W. T. , Sanders K. M. , Publicover N. G.
Published: December 2017

Wortmannin, an inhibitor of myosin light-chain kinase (10-30 microM), completely and irreversibly abolished (in 75% of tissues from canine gastric antrum) phase contractions caused by slow waves with no significant effects on resting membrane potential or the frequency, amplitude, or duration of spontaneous slow waves. Responses to agents that normally cause hyperpolarization (cromakalim, sodium nitroprusside, and forskolin) were unaffected by wortmannin treatment. It was also possible to study the excitatory effects of agents and conditions that normally result in loss of intracellular impalements: 1) elevated extracellular K+ concentrations altered membrane potential close to values predicted by the Nernst equation, and 2) high concentrations of acetylcholine produced depolarization and rapid oscillations in membrane potential coincident with contractile activity. Cholinergic increases in myosin light-chain phosphorylation and contractions were partially blocked by wortmannin. In canine antrum, wortmannin inhibition of contraction was irreversible, although in other tissue types, partial recovery of contractions was observed when wortmannin was removed. Wortmannin can be a useful agent to investigate the electrophysiology of some smooth muscles when movement might lead to recording artifacts or loss of signal.
Growth hormone regulates cytosolic free calcium in rat fat cells by maintaining L-type calcium channels
Authors: Gaur S. , Yamaguchi H. , Goodman H. M.
Published: December 2017

In freshly isolated individual rat adipocytes, cytosolic free Ca2+ concentration ([Ca2+]i) as measured with fura 2 slowly declined during incubation but was sustained, or even somewhat increased, by brief treatment with growth hormone (GH) at the beginning of a 3-h incubation period. GH-treated adipocytes were more permeable to Ca2+ than GH-deprived cells as indicated, using Mn2- as a surrogate and monitoring influx by the rate of quenching of fura 2 fluorescence. Blockage of Ca2- channels with 100 nM nimodipine lowered [Ca2+]i in GH-treated cells to the level seen in GH-deprived cells. Increases in [Ca2+]i or the rate of Mn2+ entry were twofold greater in GH-treated than in GH-deprived cells when extracellular K+ was increased to 30 mM. Similarly, the Ca2+ channel agonist BAY K 5552 or the diacylglycerol analogue 1,2-dioctanoyl-sn-glycerol increased [Ca2+]i more in GH-treated than in GH-deprived adipocytes. Ca(2+)-ATPase activity was two times higher in plasma membranes isolated from GH-treated than from GH-deprived cells. Continued synthesis of Ca(2+)-ATPase may depend on [Ca2+]i, since the effects of GH on [Ca2+]i and Ca(2+)-ATPase were blocked by a cycloheximide or verapamil. We suggest that voltage-sensitive L-type Ca2+ channels regulate steady-state [Ca2+]i in rat adipocytes and that GH maintains the number or functional integrity of these channels.
Growth hormone increases calcium uptake in rat fat cells by a mechanism dependent on protein kinase C
Authors: Gaur S. , Yamaguchi H. , Goodman H. M.
Published: December 2017

Growth hormone (GH; 500 ng/ml) rapidly doubled cytosolic free Ca2+ concentration ([Ca2+]i) in rat adipocytes as determined with the Ca2+ indicator fura 2. No response was seen in Ca(2+)-free medium, suggesting that the increase in [Ca2+]i was due to Ca2+ influx. GH also doubled the influx of Mn2- as inferred from the rate of fluorescence quenching. Depolarization with 30 mMK+ also increased [Ca2+]i, and the increase in [Ca2+]i due to either GH or 30 mMK+ was blocked by 100 nM nimodipine, suggesting that GH increases [Ca2+]i by activating voltage-sensitive L-type Ca2+ channels. GH increased [Ca2+]i even when K+ channels were blocked, suggesting that activation of Ca2+ uptake was not secondary to closure of K+ channels and consequent depolarization. A diacylglycerol (PAG) analogue, 1,2-dioctanoyl-sn-glycerol (50 microM), duplicated, and the protein kinase C(PKC) inhibitors calphostin C (100 nM), chelerythrine (1 microM), and bis-indolylmaleimide (250 nM) inhibited the effects of GH on [Ca2+]i. Xanthogenate tricyclodecan-9-yl (D609), a specific inhibitor of phospholipase C(PLC), abolished the increase in [Ca2+]i due to GH but not to DAG. The results suggest that GH increases [Ca2+]i by activation of PLC, release of DAG, and activation of a Ca(2+)-independent isoform of PKC. PKC-catalyzed phosphorylation of either the Ca2+ channels or a protein that regulates them may account for the influx of Ca2+ produced by GH.
Delta F508-CFTR channels: kinetics, activation by forskolin, and potentiation by xanthines
Authors: Haws C. M. , Nepomuceno I. B. , Krouse M. E. , Wakelee H. , Law T. , Xia Y. , Nguyen H. , Wine J. J.
Published: December 2017

Trafficking, activation, and kinetics of delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) and CFTR were compared in stably transduced C127I mouse mammary epithelial cells. Western blots detected a small amount of fully glycosylated delta F508-CFTR Efflux of 125I was stimulated by forskolin with the same mean effective concentration (EC50; approximately 0.5 microM) for CFTR and delta F508-CFTR cells, but the maximum response was reduced more than fivefold and its latency increased approximately threefold in delta F508-CFTR cells. In delta F508-CFTR cells, 3-isobutyl-1-methylxanthine (IBMX; EC50 = 1.45 microM) and 8-cyclopentyl-1,3-dipropylxanthine (CPX; EC50 = 58 microM) increased the peak forskolin-stimulated efflux rate approximately 2.5-fold and decreased the time to peak. A sevenfold increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels accompanied potentiation of forskolin-induced 125I efflux by IBMX but not by CPX. Elevation of intracellular cAMP increased linear voltage-independent whole cell currents 30-fold in CFTR and 4-fold in delta F508-CFTR cells; the response rate in delta F508-CFTR cells was much slower. Single-channel currents were detected in 57 of 68 cell-attached patches from forskolin-prestimulated CFTR cells vs. 6 of 35 patches in delta F508-CFTR cells. Mean number of active channels per patch was 4.1 for CFTR [open probability (Po) = 0.34] and 0.2 for delta F508-CFTR (Po = 0.11). The lower Po of delta F508-CFTR resulted from an approximately threefold longer mean interburst interval. We estimate that forskolin-stimulated chloride conductance of delta F508-CFTR C127I cells is < 5% of CFTR cells. CPX is approximately 25-fold more potent than IBMX in potentiating delta F508-CFTR and may operate by a mechanism other than elevation of cAMP.
Cl channel blockers inhibit the volume-activated efflux of Cl and taurine in cultured neurons
Authors: Sanchez-Olea R. , Morales M. , Garcia O. , Pasantes-Morales H.
Published: December 2017

The effects of the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 1,9-dideoxyforskolin (DDF), dipyridamole, and niflumic acid and of the polyunsaturated fatty acids arachidonic, linolenic, and linoleic acids on regulatory volume decrease (RVD) and associated 125I and [3H]taurine fluxes in cultured rat cerebellar granule neurons were examined. Dose-response curves of NPPB, DDF, and dipyridamole showed 20-100% inhibition of RVD and osmolyte fluxes. Niflumic acid was less potent, requiring 150-600 microM to show effects of this magnitude. The polyunsaturated fatty acids (5-20 microM) inhibited 80-90% RVD and osmolyte fluxes, with arachidonic acid exhibiting the most potent effect. The volume-associated taurine efflux was somewhat higher in younger neurons, but the pharmacological sensitivity was essentially the same in immature and mature cells. The effects of all tested drugs on 125I and [3H]taurine fluxes were remarkably similar, indicating a close pharmacological sensitivity of the transport mechanism for the two osmolytes. This is in line with the suggestion of a common pathway for the volume-associated release of Cl and amino acids functioning as osmolytes.
Computational studies of ion-water flux coupling in the airway epithelium. II. Role of specific transport mechanisms
Authors: Novotny J. A. , Jakobsson E.
Published: December 2017

Ion and water balance by the in vivo airway epithelium was investigated utilizing dynamic computer modeling. Parameters of the osmotically significant transport processes were varied to assess the sensitivity of water transport and fluid composition to transport perturbations. Establishment and regulation of water secretion represent a coordinated function of at least seven different ion transport processes: basolateral passive potassium transport, basolateral active sodium-potassium transport, basolateral sodium-potassium-chloride cotransport, apical passive sodium and chloride transport, and diffusion of sodium and chloride across the paracellular path. We found that apical chloride permeability at a level reported for cystic fibrosis is sufficient to cause the airway dehydration characteristic of cystic fibrosis. Given the reduction in apical chloride permeability in cystic fibrosis, a reduction in apical sodium permeability can potentially compensate completely for the airway dehydration associated with the cystic fibrosis genetic defect. Other simulations presented here address the importance of various membrane transport processes in airway epithelium water balance and the sensitivity of epithelium water balance to ion transport perturbations.
Capsaicin and nicotine both activate a subset of rat trigeminal ganglion neurons
Authors: Liu L. , Simon S. A.
Published: December 2017

Nicotine and capsaicin produce many similar physiological responses that include pain, irritation, and vasodilation. To determine whether neuronal nicotine acetylcholine receptors (nAChR) are present on capsaicin-sensitive neurons, whole cell patch-clamp recordings were performed on rat trigeminal ganglion cells. It was found that approximately 20% of the total number of neurons tested was activated by both 100 microM nicotine and 1 nM capsaicin. Other subsets of neurons were activated by only one of these compounds, whereas a fourth subset was not activated by either compound. At -60 mV, the magnitude of the capsaicin-activated currents was about three times larger than the magnitude of the nicotine-activated currents. The current-voltage relationship of the nAChR exhibited marked rectification, such that for voltages > or = 0 mV the current was essentially zero. In contrast, the current-voltage relationship of the capsaicin-activated current was ohmic from +/- 60 mV. These data indicate the existence of subsets of capsaicin-sensitive afferent neurons.
ANG II-induced tyrosine phosphorylation stimulates phospholipase C-gamma 1 and Cl-channels in mesangial cells
Authors: Marrero M. B. , Schieffer B. , Ma H. , Bernstein K. E. , Ling B. N.
Published: December 2017

Angiotensin II (ANG II)-induced, activation of phospholipase C (PLC) and Ca(2+)-dependent Cl-channels is an important signal transduction pathway for mesangial cell contraction and growth. Although ANG II receptors are traditionally though to be G protein coupled, recent evidence suggests that they may also mediate protein tyrosine phosphorylation. In cultured rat mesangial cells, 10(-7) MANG II stimulated the tyrosine phosphorylation of PLC-gamma 1 and elevation of intracellular inositol 1,4,5-trisphosphate (IP3) and Ca2+ levels; peak response occurred within 0.5 min. In cell-attached patches, ANG II stimulated the activity of Ca(2+)-dependent, 3- to 4-pS Cl-channels (number of channels x open probability) from 0.063 +/- 0.022 to 0.77 +/- 0.20. Tyrosine kinase inhibition with genistein or herbimycin A blocked all four ANG II-induced responses. We conclude the following. 1) Stimulation of inositol phosphate hydrolysis by PLC, release of IP3-dependent intracellular Ca2+ stores, and activation of Ca(2+)-dependent C1-channels by ANG II are dependent on the tyrosine phosphorylation of PLC-gamma 1.2) This ANG II-induced signal transduction cascade provides a possible mechanism for both the contractile and growth-stimulating effects of ANG II on glomerular mesangial cells.
Preservation of structural and functional polarity in isolated epithelial cells
Authors: Torres R. J. , Altenberg G. A. , Copello J. A. , Zampighi G. , Reuss L.
Published: December 2017

We describe a method to isolate epithelial cells from gallbladders of Necturus maculosus with preserved structural and functional polarity. Isolation was carried out with a mixture of collagenase and protease, with only a brief exposure to a divalent-cation-free medium. About 40% of the isolated epithelial cells had a “figure-eight” shape and retained metabolic and cell membrane integrity. Figure-eight cells display features consistent with preserved polarity for several hours, including the following: 1) the “apical” and “basolateral” membrane domains were differentially labeled by a hydrophobic fluorescent dye; 2) freeze fracture electron microscopy verified two plasma membrane domains differing in the presence of microvilli and folds and separated by tight junctions; 3) proteins such as ZO-1, NHE3, and Na(+)-K(+)-ATPase remained localized in the junctional, apical, and basolateral regions, respectively; 4) after apical surface exposure to wheat germ agglutinin, the label remained in the apical membrane after cell isolation; and 5) patch-clamp experiments demonstrated polarized expression of K+ channels. Polarity was rapidly lost after removal of extracellular Ca2+, exposure to trypsin, or ATP depletion. Therefore, this preparation allows for structural and functional studies of epithelial transport in single cells retaining the essential features present in the assembled epithelium.
Protein kinase C-dependent phosphorylation of Na(+)-K(+)-ATPase alpha-subunit in rat kidney cortical tubules
Authors: Carranza M. L. , Feraille E. , Favre H.
Published: December 2017

We have previously shown that, in oxygenated rat kidney proximal convoluted tubules (PCT), activation of protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) directly stimulates Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity. PKC modulation of Na(+)-K(+)-ATPase activity by phosphorylation of its alpha-subunit was the postulated mechanism. The present study was therefore designed to investigate the relationship between PKC-mediated phosphorylation of the catalytic alpha-subunit and the cation transport activity of the Na(+)-K(+)-ATPase. In a suspension of rat kidney cortical tubules, activation of PKC by 10(-7) M PDBu increased the level of phosphorylation of the Na(+)-K(+)-ATPase alpha-subunit and stimulated the ouabain-sensitive 86Rb uptake by 47 and 42%, respectively. Time and dose dependence of the PDBu-induced increase in Na(+)-K(+)-ATPase activity and phosphorylation was strongly linearly correlated. The effects of PDBu on phosphorylation and activity of Na(+)-K(+)-ATPase were prevented by GF-109203X, a specific PKC inhibitor, whereas H-89, a specific PKA inhibitor, was ineffective. These results demonstrate that PKC activation induces phosphorylation of the catalytic alpha-subunit of Na(+)-K(+)-ATPase, which may participate in the stimulation of its cation transport activity in the rat PCT.
Angiotensin II type 1 receptor modulation of neuronal K+ and Ca2+ currents: intracellular mechanisms
Authors: Sumners C. , Zhu M. , Gelband C. H. , Posner P.
Published: December 2017

Angiotensin II (ANG II) elicits an ANG II type 1 (AT1) receptor-mediated decrease in voltage-dependent K+ current (Ik) and an incrase in voltage-dependent Ca2+ current (ICa) in neurons cocultured from newborn rat hypothalamus and brain stem. Modulation of these currents by ANG II involves intracellular messengers that result from an AT1 receptor-mediated stimulation of phosphoinositide hydrolysis. For example, the effects of ANG II on IK and ICa were abolished by phospholipase C antagonists. The reduction in IK produced by ANG II was attenuated by either protein kinase C (PKC) antagonists or by chelation of intracellular Ca2+. By contrast, PKC antagonism abolished the stimulatory effect of ANG II on ICa. Superfusion of the PKC activator phorbol 12-myristate 13-acetate produced effects on IK and ICa similar to those observed after ANG II. Furthermore, intracellular application of inositol 1,4,5-trisphosphate (IP3) elicited a significant reduction in IK. This suggests that the AT1 receptor-mediated changes in neuronal K+ and Ca2+ currents involve PKC (both IK and ICa) and IP3 and/or intracellular Ca2+ (IK).
Sarcoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle is regulated by a novel pathway
Authors: Grover A. K. , Xu A. , Samson S. E. , Narayanan N.
Published: December 2017

Coronary artery smooth muscle expresses an alternative splice (SERCA2b) of the sarcoplasmic reticulum (SR) Ca2+ pump gene SERCA2, which is also expressed in cardiac muscle (SERCA2a), but how the activity of this transporter is regulated in the coronary artery is not known. SERCA2a in the cardiac muscle can be regulated via phospholamban or, as recently reported, by a direct phosphorylation of this protein by calmodulin kinase (Xu, A., C. Hawkins, and N. Narayanan. J.Biol. Chem. 268:8394-8397, 1993). Because both SERCA2a and SERCA2b contain this calmodulin kinase phosphorylation site, we examined the effect of endogenous calmodulin kinase phosphorylation of the SR Ca2+ pump in the coronary artery. SR-enriched membranes were isolated from coronary artery smooth muscle and washed in ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to remove bound calmodulin. When these membranes were incubated with MgATP2- in the presence of Ca2+/calmodulin, a 115-kDa protein was phosphorylated. This phosphorylated 115-kDa protein was identified as SERCA2b in Western blots and by immunoprecipitation using a SERCA2-selective antibody. Preincubating the membranes in MgATP2- in the presence of Ca2+/calmodulin stimulated the subsequent Ca2+ uptake in the presence of oxalate plus MgATP2- and azide. The stimulation of Ca2+ uptake was inhibited by including the SR Ca2+ pump inhibitors thapsigargin and cyclopiazonic acid in the Ca2+ uptake medium or by including the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide or the calmodulin kinase II peptide fragment 290-309 in the phosphorylation solution. Thus an endogenous calmodulin-dependent kinase phosphorylated SERCA2b and activated it. Phospholamban could not be detected in these membranes in Western blots. Therefore, the regulation of the SR Ca2+ pump activity in coronary artery smooth muscle may involve a direct phosphorylation of the pump protein by an endogenous calmodulin-dependent kinase.
Transient activation of K+ channels by carbachol in bovine pigmented ciliary body epithelial cells
Authors: Stelling J. W. , Jacob T. J.
Published: December 2017

The action of carbachol (CCh) on isolated pigmented ciliary epithelial cells was examined using whole cell patch-clamp recording. Application of 100 microM CCh caused transient, occasionally oscillatory, increases in the inward and outward currents, followed by a long-term decrease in both currents. Caffeine produced transient responses similar to those of CCh. The responses to CCh were blocked by the muscarinic receptor antagonist atropine and the inositol 1,4,5-trisphosphate receptor blocker heparin (200 micrograms/ml in patch pipette). Manipulation of the internal ionic concentrations indicated that only K+ conductances were affected by CCh. Changing intracellular Ca2+ concentration ([Ca2+]i) with the calcium ionophore ionomycin demonstrated that both the inward rectifier K+ current and the outward current exhibited Ca2+ dependence. There was no Cl- current stimulated either directly by CCh or indirectly by modulators of [Ca2+]i, and any Cl- currents present arose from osmotic effects. In the short term, muscarinic stimulation will activate K+ channels by causing a transient increase in [Ca2+]i. This effect only lasts for 1-5 min, however, and, in the long term, the conductance decreases below its original level. The effect of such a transient increase in [Ca2+]i on secretion would be complex, involving effects on gap junction communication between the pigmented and nonpigmented cell layers and the activation state of Cl- channels in the nonpigmented cells. This complexity probably accounts for the variable reports of the effects of muscarinic stimulation of the ciliary body in vivo.
Human connexin 43 gap junction channel gating: evidence for mode shifts and/or heterogeneity
Authors: Brink P. R. , Ramanan S. V. , Christ G. J.
Published: December 2017

The gating parameters of human connexin 43 (Cx43) gap junction channels were determined using dual whole cell patch clamp and methods designed for analysis of multichannel recordings. Under steady-state conditions, the mean open time (MOT) of Cx43 gap junction channels was computed and it ranged from 0.43 to 5.25 s. The computed mean closed times (MCT) varied from 0.21 to 1.49 s. Analysis showed that, while the MOT declined with increasing transjunctional voltage (Vj), the apparent decline in the MCT with Vj was not statistically significant. The calculated open probabilities ranged from 0.50 to 0.95. Inspection of the data showed that there was a prolonged decay in junctional current, which had a time course of 60-150 s. The analysis excluded the possibility of a homogeneous voltage inactivated/deactivated population of independent and identical Cx43 gap junction channels. The analysis provided evidence for a homogeneous population of Cx43 channels, which can mode shift under the influence of voltage. The latter case cannot be distinguished from a heterogeneous population of Cx43 channels in which one population is voltage inactivated/deactivated and another is unaffected or weakly inactivated/deactivated by voltage.
Effect of ablation of phospholamban on dynamics of cardiac myocyte contraction and intracellular Ca2+
Authors: Wolska B. M. , Stojanovic M. O. , Luo W. , Kranias E. G. , Solaro R. J.
Published: December 2017

We compared mechanical activity and Ca2+ transients of ventricular myocytes isolated from wild-type and phospholamban (PLB)-deficient mouse hearts in control conditions and during beta-adrenergic stimulation. Compared with wild-type controls, cells isolated from PLB-deficient mouse hearts showed 1) a 2-fold increase in extent of cell shortening, 2) a 3-fold increase in maximal shortening velocity, and 3) a 3.4-fold increase in maximal relengthening velocity. PLB-deficient myocytes also demonstrated significant increases in the peak amplitude of the fura 2 fluorescence ratio and the rates of rising and falling phases of the Ca2+ transient. The fura 2 diastolic ratios were similar in both groups, suggesting no change in intracellular Ca2+ during diastole. In PLB-deficient myocytes, 0.05 microM isoproterenol induced an increase in the twitch amplitude by 152 +/- 11% (n = 6) compared with 290 +/- 31% (n = 6) in wild-type cells. Maximal shortening velocity was increased by 183 +/- 10% (n = 6) in PLB-deficient myocytes, compared with 398 +/- 62% (n = 6) in wild-type cells. The isoproterenol-induced increase in maximum relengthening velocity was increased by 168 +/- 8% (n = 6) in PLB-deficient cells compared with 445 +/- 71% (n = 6) in wild-type myocytes. In both groups, these changes in contractile parameters were accompanied by changes in the Ca2+ transient. Our results indicate that phosphorylation of sites other than PLB may play an important role in regulation of contraction-relaxation dynamics of heart cells responding to beta-adrenergic stimulation.
Swelling and cAMP on hyperpolarization-activated Cl- conductance in rat Leydig cells
Authors: Noulin J. F. , Fayolle-Julien E. , Desaphy J. F. , Poindessault J. P. , Joffre M.
Published: December 2017

We have used the whole cell patch-clamp technique to characterize changes in membrane conductance induced by osmotic swelling in mature rat Leydig cells dialyzed with ATP (control cells) or adenosine 3',5'-cyclic monophosphate (cAMP) plus ATP (cAMP cells). A spontaneous current activation occurs in both groups in isosmotic conditions (300/295 mosM in/out). This development is entirely counteracted in control cells and partly inhibited in cAMP cells by exposing them to a hyperosmotic (350 mosM) bath solution, and these currents increase again in a hyposmotic (205 mosM) bath solution. These currents are sensitive to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, a Cl- channel blocker. Taken together, the results indicate that, in the control cells (ATP alone) as well as in the presence of intracellular cAMP, osmotic swelling activates the background hyperpolarization-activated Cl- conductance, osmotic swelling and cAMP appearing to act synergistically.
Potentiation of smooth muscle contractility after rapid changes in isotonic force
Authors: Meiss R. A.
Published: December 2017

The sudden application of step increases in afterload (0.4-3.0 s in duration) arrested the isotonic shortening of electrically stimulated ovarian ligament smooth muscle strips from rabbits. Force steps were chosen to produce, after initial rapid yielding, a quasi-steady state in which muscle length and force remained constant. Removal of the extra afterload allowed renewed shortening that began with a velocity transiently greater than that measured before the force step (at same muscle length). The rate of force redevelopment was also transiently potentiated under isometric conditions after the removal of the extra load. Both types of potentiation depended on force-step duration, and the transients decayed exponentially with a time constant of approximately 0.25 s. The stiffness of the muscle during the force step was initially depressed but then increased along an exponential time course while force and length remained constant. These observations are consistent with an initial detachment of a portion of the cross-bridge array, which then reattached during the course of the force step, with potentiation being due to either a transient increase in cycling rate or a time-dependent reconfiguration of cytoskeletal elements supporting the contractile system.
cAMP promotes gap junctional coupling in T84 cells
Authors: Chanson M. , White M. M. , Garber S. S.
Published: December 2017

The effect of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on dye and electrical coupling was studied in T84 cells, a cell line often used as a model for epithelial cell fluid secretion. Injections of lucifer yellow (LY) into single cells within a cluster of control cells resulted in LY localization to 1.3 +/- 0.2 (mean +/- SE) cells within a cluster. Twenty-six percent of control T84 cell pairs were electrically coupled as assayed by the dual patch-clamp technique. Treatment of cells with agents that either increase intracellular cAMP and/or activate protein kinase A (PKA) increased dye localization to 3.8 +/- 0.6 cells and the proportion of electrically coupled cell pairs to 65%. No electrical coupling was observed in the presence of the Rp diastereomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS), a PKA antagonist. Excess of Rp-cAMPS prevented cell coupling elicited by 20 microM of the Sp diastereomer of adenosine-3',5'-cyclic monophosphothioate. Expression of connexin 32 mRNA, but not of connexins 26, 43, or 45, was detected by reverse transcription-polymerase chain reaction. These results suggest that communication between T84 cells is modulated by PKA, providing a mechanism for regulating multicellular activity, such as fluid secretion.