bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunofluorescent localization of the ubiquitin-activating enzyme, E1, to the nucleus and cytoskeleton
Authors: Trausch J. S. , Grenfell S. J. , Handley-Gearhart P. M. , Ciechanover A. , Schwartz A. L.
Published: December 2017

Ubiquitin, a 76-amino acid protein, is covalently attached to abnormal and short-lived proteins, thus marking them for ATP-dependent proteolysis in eukaryotic cells. Ubiquitin is found within the cytoplasm, nucleus, microvilli, autophagic vacuoles, and lysosomes. The ubiquitin-activating enzyme, E1, catalyzes the first step in ubiquitin conjugation. To date, very little is known about the subcellular distribution of this enzyme. We have utilized immunofluorescence and immunoblotting to examine the cellular distribution of E1 in several eukaryotic cell lines, including HeLa, smooth muscle A7r5, choriocarcinoma BeWo, Pt K1, and Chinese hamster ovary (CHO) E36. E1 was identified in both cytoplasmic and nuclear compartments in all cell lines examined. However, the relative abundance within these compartments differed markedly between the cell lines. Even within a single cell line, nuclear distribution was not uniform, and certain cells demonstrated an absence of nuclear staining. E1 resides predominantly within the nucleus in BeWo. In contrast, its distribution in CHO and Pt K1 cells is mainly cytoplasmic. Within the cytoplasm, three pools of E1 were identified by double-label immunofluorescence. The first of these colocalized with phalloidin, indicating association of E1 with actin filaments. A second cytoplasmic pool colocalized with tubulin and was predominantly perinuclear in its distribution. The third pool associated with intermediate filaments. This suggests that E1 is associated with all three components of the cytoskeleton. The distribution of E1 was unaltered in a mutant line of CHO E36 designated ts20, in which the E1 can be thermally inactivated. The variable distribution of E1 among cell lines, including its apparent cytoskeletal association, suggests pleiotropic functions of this enzyme and the ubiquitin-conjugating system.
Metabolism of extracellular adenine nucleotides by human endothelial cells exposed to reactive oxygen metabolites
Authors: Aalto T. K. , Raivio K. O.
Published: December 2017

Endothelial cells have ectonucleotidases that rapidly catabolize extracellular nucleotides. Our aim was to study whether the metabolism of extracellular nucleotides and adenosine are influenced by exposure of endothelial cells to reactive oxygen metabolites at concentrations relevant to human pathology. Human umbilical vein endothelial cells were incubated with hypoxanthine (100 microM) and xanthine oxidase (80 mU/ml), to generate superoxide, or with hydrogen peroxide (100 microM). The cells were then washed, and the metabolism of radioactive substrates was followed. After exposure to hypoxanthine-xanthine oxidase the half time of disappearance of [14C]ATP (5 microM) was prolonged from 9.9 +/- 5 to 28.3 +/- 15.6 min and that of [14C]AMP from 9.5 +/- 2.5 to 25.0 +/- 9.9 min. The conversion of extra- into intracellular nucleotides via adenosine was also decreased (mean for [14C]ATP 0.25 vs. 0.90 and for [14C]AMP, 0.075 vs. 0.75 nmol/10(6) cells in 30 min compared with parallel controls, respectively). Hydrogen peroxide or trypsin had no significant effect on the metabolism of extracellular adenine nucleotides and neither did a short (up to 15 min) exposure to the superoxide-generating system. The conversion of [14C]adenosine into intracellular nucleotides and hypoxanthine was not influenced by either hypoxanthine-xanthine oxidase or by hydrogen peroxide. We conclude that superoxide radicals inhibit the catabolism of extracellular adenine nucleotides by the ectonucleotidases of endothelial cells and may thus modify the pathophysiology of ischemia-reperfusion injury.
Na(+)-K(+)-ATPase subunit isoform pattern modification by mitogenic insulin concentration in 3T3-L1 preadipocytes
Authors: Russo J. J. , Sweadner K. J.
Published: December 2017

When 3T3 preadipocyte cell lines are induced to differentiate to the adipocyte phenotype, the expression of Na(+)-K(+)-ATPase isoforms changes. Some disparities have been noted by investigators who used different hormonal conditions to stimulate adipocyte conversion, however. In the present report we investigated the effect of high concentrations of insulin on the 3T3-L1 cell line, to determine whether it affected Na(+)-K(+)-ATPase expression separately from its ability to promote phenotypic conversion. The effect of insulin was compared with that of dexamethasone and 3-isobutyl-1-methylxanthine. Changes in Na(+)-K(+)-ATPase subunit expression were seen regardless of the hormonal stimulus. Induction of alpha 2-mRNA and reduction of beta 1-mRNA were always observed. At the protein level, too, induction of alpha 2-protein was noted; alpha 1-protein levels were not markedly affected. The change in alpha-isoform protein and mRNA levels did not correspond quantitatively with the fraction of cells that were morphologically converted, suggesting that it is an early event in differentiation.
Corticosterone induces 11 beta-HSD and mineralocorticoid specificity in an amphibian urinary bladder cell line
Authors: Gaeggeler H. P. , Duperrex H. , Hautier S. , Rossier B. C.
Published: December 2017

We have examined the mineralocorticoid specificity in a TBM 18-23 cell line derived from the toad bladder epithelium. In cells grown on porous substrate, corticosterone was more potent than aldosterone in stimulating a sodium transport response, measured by the short-circuit current method 6 h after hormone addition [mean affinity constant (K0.5) for corticosterone = 1 nM vs. K0.5 for aldosterone = 8 nM]. The time course of effects and saturation kinetics were identical for both agonists, suggesting interaction with identical receptors. Whereas the dose-response relationship for aldosterone did not change with time of incubation (6 vs. 24 h), the dose-response curve for corticosterone became biphasic at 24-h incubation (apparent K0.5 as high as 40 nM), demonstrating that corticosterone became apparently less potent with time. Pretreatment with carbenoxolone, a potent inhibitor of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), restored full sensitivity at 24-h incubation to corticosterone. The 11 beta-HSD activity was low during the first 3 h of incubation in the presence of 3 nM corticosterone, and only a small fraction (approximately 7%) of corticosterone was metabolized. At 24-h incubation, 11 beta-HSD activity increased approximately 2.5-fold (P < 0.001, n = 8). We conclude that 11 beta-HSD activity is induced by its own substrate in TBM cells in parallel with the induction of the carbenoxolone-sensitive sodium transport response.
Effect of membrane potential on K-Cl transport in human erythrocytes
Authors: Kaji D. M.
Published: December 2017

We examined whether swelling-activated K-Cl cotransport is electrogenic in human erythrocytes. Baseline membrane potential, measured by the change in fluorescence of the carbocyanine dye diS-C3-5, was not different in hypotonically swollen (-7.6 mV) or isosmotically swollen cells (-9.5 mV). We used hemisodium, a new highly selective Na ionophore, in varying concentrations, in the presence of a fixed outwardly directed Na gradient (intracellular Na, 75 mM; external Na, 1 mM) to vary membrane potential over a wide range despite identical K and Cl concentrations. The membrane potential varied between -8 and -90 mV. K influx increased slightly with hyperpolarization in swollen and nonswollen cells. However, the difference between the two fluxes, swelling-activated K influx, a measure of K-Cl cotransport, was unaffected by voltage changes, as was swelling-activated K efflux. We conclude that K-Cl cotransport in human erythrocytes is electroneutral and by inference has a 1:1 stoichiometry.
Involvement of arachidonic acid in the chloride secretory response of intestinal epithelial cells
Authors: Barrett K. E. , Bigby T. D.
Published: December 2017

The inflammatory mediator, adenosine, induces chloride secretion from the human colonic epithelial cell line, T84, in a manner apparently independent of increases in adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, or cytoplasmic Ca2+. This prompted a search for other messengers that might account for the secretory response. A possible role for arachidonic acid or a metabolite in the response to adenosine has been demonstrated 1) by showing a relationship between arachidonic acid mobilization and chloride secretion induced by the adenosine agonist 5'-(N-ethylcarboxamido)adenosine (NECA) and 2) by determining that exogenous arachidonic acid affects T84 cell function. Addition of NECA to T84 cells induces chloride secretion and release of radioactivity from cells preloaded with [3H]arachidonic acid with similar dose dependencies. The effect of NECA on chloride secretion is inhibited by the phospholipase A2 inhibitor 4-bromophenacyl bromide or the diglyceride lipase inhibitor RG80267 but is unaffected by inhibitors of lipoxygenase or cyclooxygenase. Arachidonic acid has a small but significant effect on chloride secretion when added alone to T84 cells and synergistically enhances, as does NECA, responses to calcium-dependent secretogogues. Thus receptor-stimulated release of arachidonic acid in T84 cells may provide a second-messenger system promoting chloride secretion, in addition to calcium and cyclic nucleotides.
Cell surface labeling of CFTR in T84 cells
Authors: Prince L. S. , Tousson A. , Marchase R. B.
Published: December 2017

To distinguish cystic fibrosis transmembrane conductance regulator (CFTR) at the surface of epithelial cells from that present in intracellular membranes, intact T84 cells were treated with periodate and biotin-LC-hydrazide to derivatize exposed glycoconjugates. Cell lysates were then passed over a monomeric avidin column, which allows reversible avidin-biotin binding. After washing, biotinylated molecules were eluted with 2 mM biotin. CFTR was then immunoprecipitated with a mouse monoclonal antibody from both the unbound and biotin eluent fractions, radioactively labeled by in vitro phosphorylation, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Nonimmune mouse immunoglobulin G failed to precipitate any CFTR, and CFTR was detectable only in the wash fractions when cells were periodate treated but not labeled with biotin hydrazide. In biotinylated cells, CFTR levels were approximately equal in the unbound fraction and the biotin eluent. The proportion of biotinylated CFTR did not significantly increase when cells were labeled after treatment with 10 microM forskolin. These data demonstrate that in T84 cells CFTR is constitutively expressed on the cell surface and that activation of CFTR does not primarily depend on the cAMP-dependent trafficking of CFTR to the plasma membrane. The large unbiotinylated pool of maturely glycosylated CFTR suggests that CFTR resides in an intracellular compartment as well as being present at the cell surface.
Fab fragments from amyotrophic lateral sclerosis IgG affect calcium channels of skeletal muscle
Authors: Delbono O. , Magnelli V. , Sawada T. , Smith R. G. , Appel S. H. , Stefani E.
Published: December 2017

Amyotrophic lateral sclerosis (ALS) is a human disease involving upper and lower motoneurons. In this paper we studied the action of specific antigen-binding site (Fab) fragments of immunoglobulins from ALS patients on dihydropyridine (DHP)-sensitive Ca2+ channel function in situ. Ca2+ channels in single mammalian skeletal muscle fibers tested by the double Vaseline gap technique and single Ca2+ channels reconstituted into bilayer were tested in these experiments. Although the observed current-voltage relationship was not modified by the addition of Fab fragments (1.5 mg/ml), peak Ca2+ current (ICa) was significantly reduced. The effect of these Fab fragments on the peak ICa reached a stable value after 60 min of incubation. ALS Fab fragments also slowed the ICa rising phase and increased the rate of tail current deactivation. Studies with double pulses demonstrated that ICa inactivation time course, voltage dependence, and recovery were not modified by ALS Fab fragments. Fab fragments from normal subjects and heat-inactivated Fab fragments from ALS patients did not induce any modification on the charge movement and ICa. In single channel studies, ALS Fab fragments reduced channels amplitude. These data support the concept of an immunological interaction between the circulating antibodies from ALS patients and DHP-sensitive Ca2+ channels.
Staurosporine induced Ca2+ increase in DDT1MF-2 smooth muscle cells
Authors: Himpens B. , De Smedt H. , Casteels R.
Published: December 2017

The free calcium concentrations in nucleus ([Ca2+]n) and cytoplasm ([Ca2+]c) of cultured DDT1MF-2 smooth muscle cells were estimated using the fluorescent dye indo-1 and laser confocal microscopy. The alkaloid staurosporine mainly increased [Ca2+]c during the initial minutes of stimulation and the nucleocytoplasmic gradient was maintained. Thereafter [Ca2+]n increased further while [Ca2+]c decreased, resulting after 10 min in a reversion of the nucleocytoplasmic gradient. Staurosporine increased the Ca2+ influx but also released intracellular Ca2+. In Ca(2+)-free solution no inversion of the nucleocytoplasmic gradient was seen in most cells. Ca2+ increased more rapidly in the area of the perinuclear sarcoplasmic reticulum (SR) than in the central cytoplasm. This is suggestive of the release of Ca2+ from this region. Ca2+ concentration remained also enhanced near the plasmalemma during this time period. This might indicate that the efflux mechanism is also affected. It is unlikely that these changes are due to inhibition of protein kinase C, since phorbol esters are ineffective and since downregulation by prolonged incubation with phorbol 12-myristate 13-acetate only affected the time periods required to attain the described [Ca2+]c and [Ca2+]n peaks. The slow time course of the staurosporine effects allowed demonstration of the spatiotemporal Ca2+ gradients between the nucleus and the cytoplasm and also inside the cytosol. The different time course of the increases in [Ca2+]c and [Ca2+]n suggests that two different Ca2+ mobilization events are present. The occurrence of an inversion of the nucleocytoplasmic gradient whereby [Ca2+]n increased while [Ca2+]c values decreased indicates that the increase in [Ca2+]n might result partially from release sites inside the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)
Alpha 2-adrenergic, but not imidazole, agonists activate NaCl cotransport in rabbit tracheal epithelial cells
Authors: Liedtke C. M. , Furin J. , Ernsberger P.
Published: December 2017

The adrenergic agonist clonidine activates NaCl cotransport in rabbit tracheocytes. With the use of the high-affinity analogue p-[125I]iodoclonidine, binding of clonidine to cells was determined to fit a two-site model, with one site of high specificity for alpha 2-adrenergic (alpha 2-AR) and the other with a high affinity for I1-imidazol(in)e (I1) receptors. Total density of binding sites for both receptors was similar at 18 fmol/mg protein. Moxonidine displayed a 166-fold greater specificity for I1 receptors compared with cimetidine. Bumetanide-sensitive Na or Cl transport was stimulated by the alpha 2-AR agonists clonidine or guanabenz but not by the I1 agents cimetidine or moxonidine. I1 agonists-stimulated Na transport was detected only in the presence of bumetanide. Prazosin did not block clonidine-stimulated NaCl uptake or efflux, indicating the presence of an alpha 2A-AR subtype. Addition of clonidine either before or after incubation with l-isoproterenol or forskolin did not attenuate the time- and dose-dependent increase in adenosine 3',5'-cyclic monophosphate (cAMP) levels. Thus clonidine stimulates NaCl cotransport in rabbit tracheocytes through an alpha 2A-AR mechanism that does not require cAMP for signal transduction. In addition, I1-imidazol(in)e receptors stimulate Na transport in rabbit tracheocytes through an unidentified pathway.
cAMP-regulated whole cell chloride currents in pancreatic duct cells
Authors: Gray M. A. , Plant S. , Argent B. E.
Published: December 2017

Using the whole cell configuration of the patch-clamp technique, we have identified an adenosine 3',5'-cyclic monophosphate (cAMP)-regulated chloride conductance in pancreatic duct cells. Basal whole cell currents in single isolated cells were very low (approximately 5 pA/pF) but could be stimulated 17-fold by elevation of intracellular cAMP. The cAMP-activated currents exhibited 1) a high chloride selectivity, 2) a near linear current-voltage relationship, 3) time and voltage independence, 4) block by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and 5) an anion selectivity sequence based on permeability ratios of SCN > NO3 > Br > Cl > I > HCO3 > F > ClO4 > gluconate. Currents in single cells ran down within a few minutes; however, stable chloride currents could be recorded from duct cell clusters in which four or five cells were in electrical communication. We present evidence suggesting that these cAMP-regulated currents are carried by cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. Physiologically, these CFTR channels act in parallel with chloride-bicarbonate exchangers to facilitate bicarbonate secretion across the apical plasma membrane of the duct cell.
Refilling the inositol 1,4,5-trisphosphate-sensitive Ca2+ store in neuroblastoma x glioma hybrid NG108-15 cells
Authors: Lo T. M. , Thayer S. A.
Published: December 2017

Bradykinin-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) were recorded in single NG108-15 cells with indo-1-based dual-emission microfluorimetry (50% effective concentration, 16 nM). A 1-min exposure to 30 nM bradykinin completely depleted the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store; refilling the store required extracellular Ca2+ (half time, 2 min). Refilling the IP3-sensitive store was completely blocked by 1 microM La3+ and 10 microM nitrendipine, but not 10 microM verapamil, 10 microM flunarizine, 1 microM nitrendipine, or 0.1 microM La3+. Thapsigargin irreversibly depleted the Ca2+ store and prevented its refilling (half-maximal inhibitory concentration, 3 nM). Influx of Ca2+ across the plasma membrane did not increase after depletion of the IP3-sensitive store by exposure to bradykinin, although maintained presence of the agonist produced significant Ca2+ influx. Similarly, Mn2+ and Ba2+ influx, as measured by indo-1 quenching and spectral shifts, did not increase following depletion of IP3-sensitive store. In contrast to depletion of the IP3-sensitive Ca2+ store by bradykinin, thapsigargin (10 nM) treatment produced Ca2+ and Ba2+ influx. We conclude that after Ca2+ mobilization, the IP3-sensitive Ca2+ store in NG108-15 cells is refilled with cytoplasmic Ca2+ via a thapsigargin-sensitive Ca(2+)-Mg(2+)-ATPase. Cytoplasmic Ca2+ is replenished by a persistent leak of Ca2+ across the plasma membrane. This leak is not modulated by the status of the intracellular Ca2+ store. In NG108-15 cells, agonist and thapsigargin-evoked Ca2+ entry are mediated by activation of plasmalemmal Ca2+ channels independent of the status of the IP3-sensitive intracellular Ca2+ store.
Elevation of inositol tetrakisphosphate parallels inhibition of Ca(2+)-dependent Cl- secretion in T84 cells
Authors: Kachintorn U. , Vajanaphanich M. , Barrett K. E. , Traynor-Kaplan A. E.
Published: December 2017

Carbachol and histamine both stimulate calcium-dependent chloride secretion in the colonic epithelial cell line, T84. However, pretreatment of cell monolayers with carbachol blocks subsequent chloride secretion induced by thapsigargin but not the calcium elevation stimulated by this agent, whereas histamine pretreatment blocks neither thapsigargin-induced chloride secretion nor calcium elevation. To examine whether inositol phosphate metabolism might account for this difference, we measured levels of radiolabeled inositol phosphates: Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins-(1,3,4,6)P4, Ins(3,4,5,6)P4, InsP5, and InsP6 after cell stimulation. Although both carbachol and histamine increase Ins (1,4,5)P3 at 5 s, there is a greater and more persistent increase in the levels of Ins(1,3,4)P3 and InsP4 at later time points after carbachol than histamine, which corresponded to the suppression of the chloride secretory response.
Effects of exercise training on skeletal muscle glucose uptake and transport
Authors: Etgen G. J. , Brozinick J. T. , Kang H. Y. , Ivy J. L.
Published: December 2017

Exercise training increases the concentration of GLUT-4 protein in skeletal muscle that is associated with an increase in maximal insulin-stimulated glucose transport. The purpose of this study was to determine whether exercise training results in a long-lasting increase in insulin-stimulated glucose transport in rat skeletal muscle. Glucose uptake and skeletal muscle 3-O-methyl-D-glucose (3-MG) transport were determined during hindlimb perfusion in the presence of a maximally stimulating concentration of insulin (10 mU/ml). Hindlimb glucose uptake was approximately 29% above sedentary (Sed) levels in rats examined within 24 h (24H) of their last exercise session. However, when rats were examined 48 h (48H) after their last exercise session, hindlimb glucose uptake was not different from Sed levels. Maximal 3-MG transport was enhanced, above Sed levels, in red (RG; 72% increase) and white (WG; 44% increase) gastrocnemius and plantaris (Plan; 67% increase) muscles, but not soleus (Sol), of 24H rats. GLUT-4 protein content was significantly elevated in those muscles that exhibited enhanced 3-MG transport in 24H rats. GLUT-4 protein content was also elevated in RG, WG, and Plan of 48H rats and was not different from 24H rats. Despite the elevated GLUT-4 protein content, 3-MG transport in 48H rats was only slightly, although statistically not significantly, higher than in Sed rats. These results provide evidence that exercise training does not result in a persistent increase in skeletal muscle glucose uptake or transport, despite an increase in GLUT-4 protein content.
Deoxygenation-induced cation fluxes in sickle cells. III. Cation selectivity and response to pH and membrane potential
Authors: Joiner C. H. , Morris C. L. , Cooper E. S.
Published: December 2017

Deoxygenation of sickle cells increases membrane permeability to Na, K, and Ca and contributes to cellular cation depletion. This study examines the physiological nature of the pathway that mediates deoxygenation-induced movements of monovalent cations. Deoxygenation-induced Rb influx was a linear function of external Rb concentration, with no evidence of saturation. Activation of the deoxygenation-induced pathway was fostered by alkaline pH (7.5), whereas ion movements via the activated pathway were optimal between pH 6.9 and 7.0 in cells incubated in media in which NO3 replaced Cl to eliminate KCl cotransport. The deoxygenation-induced pathway exhibited no selectivity among the alkali metal cations Li, Na, K, Rb, or Cs, but the monovalent organic cations tetramethylammonium, tetraethylammonium, and N-methylglucamine were excluded. Sickle cells incubated in low-Cl media (external Cl, 40 mM) to depolarize the membrane exhibited increased deoxygenation-induced K efflux and reduced Na influx. Cells treated with valinomycin to hyperpolarize the membrane showed increased deoxygenation-induced Na influx. These characteristics of the deoxygenation-induced transport pathway, linear concentration dependence, lack of cation selectivity, and response to membrane potential, argue against a carrier-mediated mechanism in favor of a diffusional process. The exclusion of small organic cations, however, suggests that factors other than ion size influence deoxygenation-induced permeability.
Intracellular pH determination by 13C-NMR spectroscopy
Authors: Chacko V. P. , Weiss R. G.
Published: December 2017

A noninvasive method for the determination of pH by the 13C-nuclear magnetic resonance (NMR) chemical shift of the C-3 carbon of sn-glycerol 3-phosphate is described. Nonlinear least-squares analysis of the chemical shift variation of the C-3 resonance of sn-glycerol 3-phosphate with pH at 37 degrees C in solutions and in perchloric acid extracts of tissue yielded a pKa of 6.2, making it a very sensitive indicator of pH in the approximate range of 5-7. Intracellular pH determined by the present 13C-NMR method correlated well with simultaneous measurements of pH by 31P-NMR spectroscopy over a wide range during normal perfusion and ischemic conditions in intact rat hearts. These findings indicate that this approach is particularly suited for quantification of intracellular pH over the physiological range in intact tissues and that observed in ischemic myocardium.
Activation of protein kinase C increases Ca2+ sensitivity of secretory response of GH3 pituitary cells
Authors: Haymes A. A. , Hinkle P. M.
Published: December 2017

The effect of protein kinase C on the secretory response of GH3 pituitary cells to Ca2+ was investigated. Activation of protein kinase C with 100 nM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 40 min reduced the rise in intracellular free calcium concentration ([Ca2+]i) stimulated by depolarization with high K+ but did not affect the threefold increase in prolactin secretion stimulated by 50 mM K+. Both [Ca2+]i and prolactin release were measured for control and TPA-treated cells over a range of [Ca2+]i values attained by adding the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)-ethane -N,N,N',N'- tetraacetic acid (BAPTA/AM) to reduce [Ca2+]i or high K+ with or without BAY K 8644 to increase [Ca2+]i. Half-maximal prolactin secretion occurred at lower [Ca2+]i concentrations for cells treated with TPA (approximately 160 nM) than for control cells (approximately 270 nM), but the rate of secretion at high [Ca2+]i was the same. GH3 cells also secreted more prolactin in response to thyrotropin-releasing hormone (TRH) after protein kinase C activation, although TRH evoked a smaller Ca2+ transient. Fluorescence ratio imaging revealed that GH3 cells undergo spontaneous [Ca2+]i oscillations (4-12/min) and that TPA nearly abolishes [Ca2+]i oscillations as well as inhibits the increase in [Ca2+]i stimulated by depolarization. These results demonstrate that activation of protein kinase C increases the Ca2+ sensitivity of the secretory response in GH3 cells, causing up to a twofold increase in the rate of secretion at typical intracellular Ca2+ concentrations.
Phorbol ester-stimulated exocytosis in lacrimal gland: PKC might not be the sole effector
Authors: Zoukhri D. , Sergheraert C. , Rossignol B.
Published: December 2017

In this work we show that, although both phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-dibutyrate (PdBu) stimulate the protein discharge in the rat lacrimal gland with the same half-maximal effective concentration (EC50 approximately 2 x 10(-7) M), PdBu is more efficient in eliciting this response compared with PMA. We also show that sphingosine and chelerythrine have no inhibitory effect on the protein discharge stimulated by PMA or PdBu at concentrations up to 2 x 10(-4) and 3 x 10(-5) M, respectively. With staurosporine, a complete inhibition could not be obtained even at 1 microM. However, only with trifluoperazine (TFP) we obtained a complete inhibition of the PMA-induced protein discharge at 10(-4) M TFP. On the other hand, we show that three diacylglycerol-permeant analogues (1-oleoyl-2-acetyl-sn-glycerol, 1,2-dioctanoyl-sn-glycerol, and 1,2-didecanoyl-sn-glycerol) do not stimulate protein discharge. In a previous report from our laboratory (30), we showed that the rat lacrimal gland expresses the alpha-isoform of protein kinase C (PKC). In this study, using specific antibodies directed against the newly identified isoforms of PKC, we show on a diethylaminoethyl-cellulose fraction that, besides PKC-alpha, the rat lacrimal gland expresses PKC-epsilon, as previously suggested by Dartt et al. (11), and PKC-delta. Our results question the direct implication of PKC activity as a sole effector of the phorbol ester-stimulated protein secretion in the rat lacrimal gland.
Unbiased estimates of quantal release parameters and spatial variation in the probability of neurosecretion
Authors: Provan S. D. , Miyamoto M. D.
Published: December 2017

A procedure was developed for dealing with two problems that have impeded the use of quantal parameters in studies of transmitter release. The first, involving temporal and spatial biasing in the estimates for the number of functional release sites (n) and probability of release (p), was addressed by reducing temporal variance experimentally and calculating the bias produced by spatial variance in p (var(s)p). The second, involving inaccuracies in the use of nerve-evoked endplate potentials (EPPs), was circumvented by using only miniature EPPs (MEPPs). Intracellular recordings were made from isolated frog cutaneous pectoris, after decapitation and pithing of the animals, and the concentration of K+ ([K+]) was raised to 10 mM to increase the level of transmitter release. The number of quanta released (m) by the EPP was replaced by the number of MEPPs in a fixed time interval (bin), and 500 sequential bins used for each quantal estimate. With the use of 50-ms bins, estimates for var(s)p were consistently negative. This was due to too large a bin (and introduction of undetected temporal variance) because the use of smaller bins (5 ms) produced positive estimates of var(s)p. Increases in m, n, and p but not var(s)p were found in response to increases in [K+] or [Ca2+]/[Co2+]. La3+ (20 microM) produced increases in m and n, which peaked after 20 min and declined toward zero. There were also large increases in p and var(s)p, which peaked and declined only to initial control values. The increase in var(s)p was presumed to reflect La(3+)-induced release of Ca2+ from intracellular organelles. The results suggest that this approach may be used to obtain unbiased estimates of n and p and that the estimates of var(s)p may be useful for studying Ca2+ release from intraterminal organelles.
Properties of cAMP-dependent and Ca(2+)-dependent whole cell Cl- conductances in rat epididymal cells
Authors: Huang S. J. , Fu W. O. , Chung Y. W. , Zhou T. S. , Wong P. Y.
Published: December 2017

Single rat epididymal cell studied under whole cell patch-clamp condition responded to 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP) (500 microM) and to ionomycin (1 microM) by an increase in whole cell conductance. A major part of the stimulated current was carried by Cl-, although a small part was due to nonselective cation current. After elimination of the cation current component by using impermeant cation, the cells revealed different Cl- conductance properties in response to adenosine 3',5'-cyclic monophosphate (cAMP) and ionomycin. The cAMP-stimulated Cl- conductance was independent of time and voltage and showed a linear current-voltage relationship. The anion permselectivity was NO3- > Br- > Cl- approximately I- >> SO(4)2-. The ionomycin-stimulated Cl- conductance showed marked time and voltage dependency. In contrast to the cAMP-induced anion permselectivity, the ionomycin-induced anion permselectivity was I- > Br- approximately NO3- > Cl- >> SO(4)2-. These results indicate that the epididymal epithelial cells exhibit different Cl- conductances sensitive to cAMP and Ca2+. The cAMP-activated conductance has properties resembling the type associated with the cystic fibrosis transmembrane conductance regulator found in cystic fibrosis-affected epithelia. This finding supports the notion that the epididymis is a cystic fibrosis epithelium.
Lysophosphatidylcholine causes cGMP-dependent verapamil-sensitive Ca2+ influx in vascular smooth muscle cells
Authors: Stoll L. L. , Spector A. A.
Published: December 2017

Lysophosphatidylcholine (lyso-PC) is a vasoactive phospholipid present in oxidized low-density lipoprotein. We used a coculture model of the vascular wall to study its interaction with endothelial cells (EC) and vascular smooth muscle cells (SMC). Lyso-PC was taken up readily by SMC and gradually acylated to phosphatidylcholine. Low concentrations (< or = 1 microM) of lyso-PC present in the interstitial medium of an EC-SMC coculture system were taken up primarily by the SMC. Lyso-PC produced a rapid two- to three-fold increase in SMC guanosine 3',5'-cyclic monophosphate (cGMP) levels, reaching a maximum in 1 min. This increase was associated with decreased SMC proliferation and increased calcium influx. The increase in intracellular calcium was inhibited by verapamil and KT5823, a specific cGMP-dependent kinase inhibitor, while a similar increase was produced by the membrane-permeant cGMP analogue 8-bromoguanosine 3',5'-cyclic monophosphate. These studies suggest that SMC are the primary target for the biological effects of lyso-PC present in the vessel wall and that the responses are mediated by calcium influx, possibly due to opening of a verapamil-sensitive cGMP kinase-dependent channel.
Rapid kinetic measurements of second messenger formation in olfactory cilia from channel catfish
Authors: Restrepo D. , Boekhoff I. , Breer H.
Published: December 2017

The effect of stimulating olfactory cilia from the channel catfish (Ictalurus punctatus) with odorant amino acids on the formation of adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, and inositol 1,4,5-trisphosphate (IP3) was studied in the subsecond time scale using a quenched flow technique. L-Alanine (L-Ala) and L-cysteine (L-Cys) (100 microM) elicited a transient elevation in IP3 levels that peaked at 25 ms. In contrast, even at high concentration a mixture of odorant amino acids (1 mM L-Ala, L-Cys, L-norleucine, L-glutamate, L-proline, and L-arginine) did not elicit a change in cAMP levels in this time scale and caused only relatively slow and minor increases in cGMP. The dose-response relationship for the IP3 response of L-Cys and L-Ala in the range from 10 nM to 1 mM is consistent with previous electrophysiological and ligand binding experiments. Odorant amino acid-stimulated IP3 formation was GTP dependent and was inhibited by guanosine 5'-O-(2-thiodiphosphate), suggesting that the response was G protein mediated. These results are consistent with a mediatory role for IP3 in amino acid olfactory signal transduction in catfish.
Cytosolic pH sensitivity of an expressed human NHE-1 Na(+)-H+ exchanger
Authors: Takaichi K. , Balkovetz D. F. , Van Meir E. , Warnock D. G.
Published: December 2017

These studies examined the effects of protein kinase C activation and calmodulin inhibition on the amiloride-sensitive NHE-1 isoform of the Na(+)-H+ exchanger in defined host cells. Our objective was to define differences in the cellular regulatory responses using a specified isoform of the Na(+)-H+ exchanger. Suspended cells were loaded with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and preacidified to a cytosolic pH of 6.2. Wild-type mouse Ltk- cells, human A-431 cells, and mutant mouse fibroblasts stably transfected with the human NHE-1 isoform (LAP+ cells) were examined to define the maximal rate of transport (Vmax) in response to 140 mM external Na+, the Hill stoichiometric coefficient, and the cytosolic pH at which the NHE-1 isoform was half-maximally stimulated (pH50). The mouse NHE-1 isoform had a greater affinity for cytosolic H+ than the human NHE-1 isoforms. Calmodulin antagonism with N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide reduced the Vmax and shifted the pH50 in the acidic direction, especially in the A-431 cells. Protein kinase C stimulation had a similar effect in A-431 cells and little effect in the wild-type (Ltk-) and transfected (LAP+) mouse cells. While the NHE-1 isoform contains several potential phosphorylation sites, the cellular milieu in which the isoform is expressed has an important effect on the modulation of NHE-1 activity.
Ca(2+)-dependent Cl- channels in undifferentiated human colonic cells (HT-29). I. Single-channel properties
Authors: Morris A. P. , Frizzell R. A.
Published: December 2017

The patch-clamp technique was combined with camera-based intracellular Ca2+ concentration ([Ca2+]i) imaging to identify the single-channel basis of the Ca(2+)-dependent Cl- conductance in human colonic adenocarcinoma cells (HT-29). Cl- channels were activated when membrane patches were excised into solutions containing high (1 microM) Ca2+ concentrations. Their single-channel conductance, measured by amplitude histogram analysis, averaged 13 pS at -90 mV and 16 pS at +90 mV membrane potential (MP). In multiple channel patches, Cl- currents showed properties similar to Ca(2+)-activated whole cell currents: outward rectification and time-dependent activation at depolarizing MP. Channel activity disappeared shortly after patch excision from the cell. In cell-attached patches, Cl- channel opening was infrequent at resting [Ca2+]i values (96 +/- 18 nM), but when [Ca2+]i was increased by the Ca2+ ionophore ionomycin (1 microM), Cl- channels were activated with a time course that paralleled the [Ca2+]i rise. Repetitive ionophore exposure produced equivalent rises in [Ca2+]i, but the corresponding Cl- channel activity became progressively reduced. The Ca(2+)-mediated agonist neurotensin (50 nM) elicited a transient Cl- channel activation that preceded the generalized cellular [Ca2+]i rise. Channel activation with neurotensin occurred in the absence of pipette Ca2+ but was abolished by preloading cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus, in response to the Ca(2+)-mediated agonist neurotensin, Cl- channel activation results from Ca2+ mobilization from intracellular pools localized within the vicinity of the plasma membrane. The Ca2+ dependency, voltage sensitivity, and kinetics of this 15-pS Cl- channel indicate that it is the basis of the whole cell Ca(2+)-activated Cl- current.
Myosin light chain phosphorylation in vertebrate striated muscle: regulation and function
Authors: Sweeney H. L. , Bowman B. F. , Stull J. T.
Published: December 2017

The regulatory light chain of myosin (RLC) is phosphorylated in striated muscles by Ca2+/calmodulin-dependent myosin light chain kinase. Unique biochemical and cellular properties of this phosphorylation system in fast-twitch skeletal muscle maintain RLC in the phosphorylated form for a prolonged period after a brief tetanus or during low-frequency repetitive stimulation. This phosphorylation correlates with potentiation of the rate of development and maximal extent of isometric twitch tension. In skinned fibers, RLC phosphorylation increases force production at low levels of Ca2+ activation, via a leftward shift of the force-pCa relationship, and increases the rate of force development over a wide range of activation levels. In heart and slow-twitch skeletal muscle, the functional consequences of RLC phosphorylation are probably similar, and the primary physiological determinants are phosphorylation and dephosphorylation properties unique to each muscle. The mechanism for these physiological responses probably involves movement of the phosphorylated myosin cross bridges away from the thick-filament backbone. The movement of cross bridges may also contribute to the regulation of myosin interactions with actin in vertebrate smooth and invertebrate striated muscles.
Diaphragmatic fatigue assessed by 31P-magnetic resonance spectroscopy in vivo
Authors: Nichols D. G. , Buck J. R. , Eleff S. M. , Shungu D. C. , Robotham J. L. , Koehler R. C. , Traystman R. J.
Published: December 2017

We tested whether fatigue of the piglet diaphragm is associated with inadequate oxidative metabolism as measured by magnetic resonance spectroscopy (MRS). An MRS measured ratio of inorganic phosphate to phosphocreatine (Pi/PCr) > or = 1 was taken as evidence of inadequate oxidative metabolism. Piglets (n = 10) underwent phrenic nerve pacing for 90 min with stimulation frequency of 30 Hz and duty cycle of 0.33. In a separate group of six piglets PCr, Pi, ATP, and intracellular pH were measured by in vivo MRS, and diaphragmatic blood flow was measured with radioactive microspheres at control, 2, 10, 45, 60, and 90 min of pacing. Transdiaphragmatic pressure fell from 25 +/- 3 to 15 +/- 2 mmHg (61 +/- 5%) at 2 min and remained depressed in a separate group of four piglets (P < 0.05). Conversely, compound action potential amplitude remained constant for the first 10 min of pacing and fell to 68 +/- 5% of control at 45 min (P < 0.05). Pi/PCr rose from a control value of 0.32 +/- 0.06 to 0.92 +/- 0.23 at 2 min and 0.79 +/- 0.03 at 10 min (P < 0.05) before returning toward control at 45-90 min. O2 delivery increased from 4.6 +/- 1.2 to 24.7 +/- 4.8 ml.min-1.100 g-1 at 2 min and 18.4 +/- 2.2 ml.min-1.100 g-1 at 10 min (P < 0.05) but then fell to lower levels at 45-90 min. ATP and intracellular pH remained constant except for a decline in pH to 6.98 +/- 0.09 at 45 min (P < 0.05) from the control value of 7.26 +/- 0.06.(ABSTRACT TRUNCATED AT 250 WORDS)
Ontogeny of alpha 1- and beta 1-isoforms of Na(+)-K(+)-ATPase in fetal distal rat lung epithelium
Authors: O'Brodovich H. , Staub O. , Rossier B. C. , Geering K. , Kraehenbuhl J. P.
Published: December 2017

Because immature, in contrast to mature, fetal lungs have ineffective Na transport, we wished to determine the ontogeny of Na(+)-K(+)-ATPase expression in fetal distal lung epithelium (FDLE). FDLE and fibroblasts (FLF) from 17- to 22-day gestational age fetal rats (term = 22 days) were grown in primary culture. Northern and slot-blot analyses utilizing isoform-specific cDNA probes determined that alpha 1- (3.7 kb) and beta 1- (2.7, 2.3, and 1.9 kb) transcripts were present in FDLE at levels approximately fivefold higher than in FLF. alpha 2-, alpha 3-, or beta 2-isoforms of Na(+)-K(+)-ATPase were not detected. In 17-day gestational age FDLE, only small amounts of alpha 1-mRNA levels were detectable, and there were approximately 10-fold less beta 1-isoform transcripts. By 20 days gestational age, the level of alpha 1-transcripts roughly doubled, whereas beta 1-levels increased approximately sixfold. Thus, during the transition from the canalicular to saccular stages of lung development, FDLE have a differentially regulated surge in mRNA levels of alpha 1- and beta 1-Na(+)-K(+)-ATPase isoforms and do not switch isoforms during lung development. Levels for both isoform transcripts then fell before birth, reaching values less than those seen for 17-day gestational age FDLE. FDLE vesicle Na(+)-K(+)-ATPase activity did not increase until 22 days gestational age.
Lys-430, site of irreversible inhibition of band 3 Cl- flux by eosin-5-maleimide, is not at the transport site
Authors: Liu S. Q. , Knauf P. A.
Published: December 2017

Although eosin-5-maleimide (EM) covalently labels band 3 and has been thought to react at the external-facing anion transport site, EM reversibly inhibits Cl- exchange at 0 degrees C in a noncompetitive fashion, indicating that under these conditions it does not bind to the transport site [Knauf, P.A., N.M. Strong, J. Penikas, R.B. Wheeler, Jr., and S.J. Liu. Am. J. Physiol. 264 (Cell Physiol. 33): C1144-C1154 1993]. To see whether or not the covalent labeling by EM takes place at the same noncompetitive site as the reversible binding, we examined the dependence of reaction rate on EM concentration. The reaction rate saturates with increasing EM concentration, indicating that reversible binding precedes covalent reaction and that EM therefore acts as an affinity label. A more complex model in which reversible binding prevents a bimolecular reaction at a different site cannot, however, be ruled out. Cl- gradients across the membrane affect EM reversible binding in a manner suggesting that EM binds preferentially to the Eo form of band 3, with the transport site unloaded and facing outward. Thus EM binds to and probably reacts covalently with a site that is different from the transport site, but whose conformation is affected by the orientation of the transport site. Lysine-430, the amino acid residue which is covalently labeled by EM (4), may be near the transport site but does not seem to be directly involved in the binding of transported substrates such as chloride. EM binding to one band 3 monomer decreases the reactivity of the adjacent monomer but does not decrease the affinity constant of the reversible binding step that precedes covalent reaction. Although a small fraction (approximately 1%) of band 3 monomers fail to react with EM, EM nearly completely inhibits transport in those monomers with which it reacts.
Role of calcium in organic osmolyte efflux when MDCK cells are shifted from hypertonic to isotonic medium
Authors: Bagnasco S. M. , Montrose M. H. , Handler J. S.
Published: December 2017

Madin-Darby canine kidney (MDCK) cells accumulate the nonperturbing osmolytes myo-inositol and betaine when grown in hypertonic medium. When returned to isotonic conditions, there is a transient basolateral efflux of these osmolytes, contributing to regulatory volume decrease. Using fura-2 fluorescence, we estimated intracellular calcium concentrations after switching MDCK cells from 500 to 300 mosM medium. Cell calcium increased 565 +/- 93 nM within 5 min. Lowering extracellular calcium inhibited the increase in cell calcium and osmolyte efflux when cells were shifted from 500 to 300 mosM medium. The calcium channel blockers lanthanum and nifedipine also inhibited osmolyte efflux after the shift from 500 to 300 mosM. In the absence of change in medium tonicity, increasing cell calcium by exposure to 1 microM ionomycin did not alter osmolyte efflux. As in PAP-HT25 cells, the cytochrome P-450 inhibitors ketoconazole and SKF-525A inhibited the efflux of both osmolytes caused by a reduction in osmolarity. Thus an early rise in cell calcium that is dependent on extra-cellular calcium and a pathway blocked by inhibitors of cytochrome P-450 oxidase are critical in regulation of osmolyte efflux when MDCK cells are shifted from hypertonic to isotonic medium.
Identification of a novel smooth muscle myosin heavy chain cDNA: isoform diversity in the S1 head region
Authors: White S. , Martin A. F. , Periasamy M.
Published: December 2017

Smooth muscle myosin heavy chain (SMHC) isoforms, SM1 and SM2, are the products of alternative splicing from a single gene (P. Babij and M. Periasamy. J. Mol. Biol. 210: 673-679, 1989). We have previously shown that this splicing occurs at the 3'-end of the mRNA, resulting in proteins that differ at the carboxyterminal (R. Nagai, M. Kuro-o, P. Babij, and M. Periasamy. J. Biol. Chem. 264: 9734-9737, 1989). In the present study we demonstrate that additional SMHC isoform diversity occurs in the globular head region by isolating and characterizing two distinct rat SMHC cDNA (SMHC-11 = SM1B and SMHC-5 = SM1A). Sequence comparison of the two clones reveals that they are completely identical in their coding regions, except at the region encoding the 25/50 kDa junction of the myosin head, where the SM1B isoform contains an additional seven amino acids. This divergent region is located adjacent to the Mg(2+)-ATPase site, and differences in this region may be of functional importance. Ribonuclease protection analysis demonstrates that the corresponding SM1B and SM1A mRNA messages are coexpressed in all smooth muscle tissues; however, the proportion of the two mRNA present differs significantly between tissues. The SM1A-type mRNA predominates in most smooth muscle tissues, with the exception of intestine and urinary bladder, which contain greater proportions of the SM1B message. The differential distribution of these two isoforms may provide important clues toward understanding differences in smooth muscle contractile properties.