bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution of the Na+/Ca2+ exchange protein in developing rabbit myocytes
Authors: Chen F. , Mottino G. , Klitzner T. S. , Philipson K. D. , Frank J. S.
Published: December 2017

The Na+/Ca2+ exchanger is a major pathway for transmembrane flux of Ca2+ in cardiac cells. Immunolabeling in adult rabbit myocytes showed localization of the Na+/Ca2+ exchanger to the peripheral sarcolemma and especially in the T tubules. Previous studies have also demonstrated higher Na+/Ca2+ exchanger activity in fetal and newborn rabbit hearts in which the T tubular system is not completely developed. Indirect immunofluorescent studies were performed to localize the Na+/Ca2+ exchanger in isolated myocytes from immature (5, 11, 17, and 30 days) and adult rabbits. Cells were incubated with a monoclonal antibody to the exchanger followed by fluorescein-labeled goat anti-mouse antibody. It is found that at 5 days of age the immunofluorescent labeling was very intense and confined to the peripheral sarcolemma. After 11 days of age, localization of labeling followed the development of the T tubules. The exchanger appeared in the T tubules as soon as they were formed. The Na+/Ca2+ exchange protein is abundantly localized to the peripheral sarcolemma before and during the development of T tubule system.
Vascular smooth muscle cell migration mediated by thrombin and urokinase receptor
Authors: Noda-Heiny H. , Sobel B. E.
Published: December 2017

To determine whether thrombin directly modifies mobility of vascular smooth muscle cells (SMC), in Transwell systems (modified Boyden chambers), we exposed SMC to alpha-thrombin. In concentrations as low as 1 NIH U/ml, thrombin induced migration as well as proliferation of SMC. Inhibition of protein synthesis by cycloheximide (2 micrograms/ml) obviated thrombin's chemotactic effect. Neither gamma-thrombin nor D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK)-inactivated alpha-thrombin (both used as controls) exerted a chemotactic effect. Concomitant hirudin or antithrombin III plus heparin inhibited chemotaxis by thrombin when added up to 2 h after addition of thrombin. alpha-Thrombin increased SMC synthesis of urokinase receptor (uPAR) and its cell surface expression as shown by metabolic labeling and immunoprecipitation as well as by flow cytometry. Thus alpha-thrombin, in concentrations thought to be present in vivo at sites of vascular injury, can stimulate not only proliferation but also migration of vascular SMC though a mechanism(s) possibly involving synthesis of uPAR, which is known to influence migration in diverse types of cells. Accordingly, both proliferation and migration dependent on thrombin may accelerate atherosclerosis, restenosis, or both after interventions such as angioplasty.
The Na+/H+ antiport in renal mitochondria
Authors: Sastrasinh M. , Young P. , Cragoe E. J. , Sastrasinh S.
Published: December 2017

In isolated renal mitochondria, Na+ and Li+ stimulated H+ efflux from the mitochondrial matrix. In submitochondrial particles (SMP), Na+ flux was also coupled to H+ transport in the opposite direction. The overshoot of Na+ uptake in SMP with an outwardly directed H+ gradient indicated that downhill efflux of H+ through the mitochondrial membrane induced uphill transport of Na+. Similar to the Na+/H+ antiport in other types of mitochondria, the antiport in renal mitochondria was more sensitive to amiloride derivatives than to amiloride itself. Benzamil and ethylisopropylamiloride (EIPA), but not amiloride, inhibited the antiport, with 50% inhibition of 10(-4) M for both benzamil in mitochondria and EIPA in SMP. The Na+/H+ antiport in renal mitochondria had simple saturation kinetics for external Na+ [Michaelis-Menten constant (Km) = 3.27 +/- 0.63 mM; maximal velocity (Vmax) = 0.022 +/- 0.002 pH units/s] and Li+ (Km = 3.62 +/- 0.75 mM; Vmax = 0.022 +/- 0.002 pH units/s). NH4Cl and NH4 acetate stimulated Na+ efflux and inhibited Na+ uptake in SMP. Comparable results with NH4 acetate and chloride suggested that NH4+ modified Na+ transport through its direct interaction with the Na+/H+ antiport, rather than through the alkalinization of intra-SMP space from non-ionic diffusion of NH3. These results suggested that the Na+/H+ antiport may be a factor in the exit of NH4+ from renal mitochondria.
Protein kinase C-dependent stimulation of Na(+)-K(+)-ATP epsilon in rat proximal convoluted tubules
Authors: Feraille E. , Carranza M. L. , Buffin-Meyer B. , Rousselot M. , Doucet A. , Favre H.
Published: December 2017

In rat proximal convoluted tubule (PCT), activation of protein kinase C (PKC) by phorbol 12,13-dibutyrate (PDBu) was previously reported to inhibit Na(+)-K(+)-ATPase, a paradoxical finding in view of the known stimulatory effect of PKC on Na+ reabsorption. Because this inhibition occurs via phospholipase A2 activation, a pathway stimulated by hypoxia, we evaluated the influence of oxygen supply on PKC action on Na(+)-K(+)-ATPase. Results confirmed that PDBu inhibited PCT Na(+)-K(+)-ATPase activity under usual conditions. In contrast, when oxygen supply was increased, PDBu had no effect on Na(+)-K(+)-ATPase hydrolytic activity, but it dose-dependently stimulated ouabain-sensitive 86Rb+ uptake. This latter effect, which was abolished by PKC inhibitors, resulted from an increment of the Na+ sensitivity of Na(+)-K(+)-ATPase. Thus, in oxygenated rat PCTs, activation of PKC primarily stimulated Na(+)-K(+)-ATPase. This likely contributes to increase solute reabsorption. Inhibition of Na(+)-K(+)-ATPase was observed only under hypoxic conditions. It may represent an adaptation to protect PCTs against deleterious effects of hypoxia.
Fractional SR Ca release is regulated by trigger Ca and SR Ca content in cardiac myocytes
Authors: Bassani J. W. , Yuan W. , Bers D. M.
Published: December 2017

The release of sarcoplasmic reticulum (SR) Ca in cardiac muscle during excitation-contraction coupling is known to be graded by the amount of activating Ca outside the SR (i.e., Ca-induced Ca release). However, little is known about how intra-SR Ca affects the release process. In this study we assessed how the fractional SR Ca release as described by Bassani et al. [Am. J. Physiol. 265 (Cell Physiol. 34): C533-C540, 1993] is affected by alteration of trigger Ca and of SR Ca content. Experiments were done with isolated ferret ventricular myocytes using indo 1 to measure Ca concentration, perforated patch to measure Ca current (ICa), caffeine application to release SR Ca, and thapsigargin to completely block SR Ca uptake. For what we consider a Normal SR Ca load and trigger Ca [action potential at 0.5 Hz with 2 mM extracellular Ca concentration ([Ca]o)], 35 +/- 3% of the SR Ca content was released at a twitch. Changing trigger Ca by altering [Ca]o (to 0.5 and 8 mM) at a test twitch changed this fractional SR Ca release to 10 +/- 2 and 59 +/- 6%, with the same SR Ca load (and peak ICa changed in a parallel manner in separate voltage-clamp experiments). Three different levels of SR Ca load were studied (Low, Normal, and High; by action potential stimulation at different frequencies from 0.05 to 0.8 Hz) using the same standard test trigger Ca (2 mM). Surprisingly, the High-load condition only increased SR Ca content by approximately 4% but appeared to be very close to the limiting SR Ca capacity.(ABSTRACT TRUNCATED AT 250 WORDS)
pHi and serum regulate AE2-mediated ClŐ/HCOŐ3, exchange in CHOP cells of defined transient transfection status
Authors: Jiang L. , Stuart-Tilley A. , Parkash J. , Alper S. L.
Published: December 2017

Page C855: L. Jiang, A. Stuart-Tilley, J. Parkash, and S. L. Alper. “pHi and serum regulate AE2-mediated Cl-/HCO-3, exchange in CHOP cells of defined transient transfection status.” The acknowledgment section omitted our thanks to Genetics Institute and Dana Dubois for subsidizing the additional cost of publication in color.
Extracellular matrix hyaluronan is a determinant of the endothelial barrier
Authors: Qiao R. L. , Wang H. S. , Yan W. , Odekon L. E. , Del Vecchio P. J. , Smith T. J. , Malik A. B.
Published: December 2017

We measured the hydraulic conductivity (Lp) of the extracellular matrix (ECM) obtained after detaching bovine pulmonary microvascular endothelial (BPMVEC) and bovine pulmonary arterial endothelial cell (BPAEC) monolayers from the ECM at different days postseeding. From day 1 to day 5 in culture, the total Lp (i.e., of cell monolayer + ECM) decreased from basal values of 17.1 +/- 4.0 to 8.5 +/- 1.6 x 10(-6) cm.s-1.cmH2O-1 in BPAEC (P < 0.05) and 7.6 +/- 1.1 to 3.7 +/- 0.8 in BPMVEC (P < 0.05), respectively, and on day 5 the total Lp values were lower in BPMVEC than in BPAEC (P < 0.05). On the 5th day, ECM Lp was 55.0 +/- 8.3 in BPAEC and 10.7 +/- 0.9 cm.s-1.cmH2O-1 in BPMVEC (P < 0.05), indicating that the contribution of ECM to the total Lp was greater in BPMVEC than in BPAEC. Treatment of [3H]acetate-labeled ECM with Streptomyces hyaluronidase (HAse; 6 U/ml for 10 min) released sixfold greater radioactivity in BPMVEC compared with untreated BPMVEC controls; a similar treatment of BPAEC did not release detectable radioactivity indicative of a higher hyaluronan content in the BPMVEC ECM. HAse treatment reduced the differences in total Lp between BPMVEC and BPAEC at different days postseeding. Moreover, on the 5th day after seeding, the ECM Lp of BPMVEC increased to a greater extent after HAse treatment than the ECM of BPAEC. These data indicate that the hyaluronan component of the ECM is an important determinant of the endothelial liquid-exchange barrier.
Ca(2+)-dependent Cl- current in canine tracheal smooth muscle cells
Authors: Janssen L. J. , Sims S. M.
Published: December 2017

Our goal was to investigate the role of Ca2+ entry in regulating Cl- current (ICl) in smooth muscle cells from canine trachealis. When studies were done using the perforated patch configuration, depolarization elicited a dihydropyridine-sensitive Ca2+ current (ICa), followed in many cells by a sustained current. This sustained current reversed direction close to the Cl- equilibrium potential, consistent with its representing ICl. The ICl was also apparent as slowly deactivating tail currents seen upon repolarization to negative potentials. The Cl- channel blocker niflumic acid abolished both the sustained and tail currents, without affecting ICa. Several observations indicated that the ICl was dependent on Ca2+ entry. ICl was increased in magnitude when Ca2+ influx was augmented [by prolonging the depolarization or using BAY K 8644 or acetylcholine (ACh)] and decreased in magnitude when Ca2+ influx was reduced (using nifedipine). Based on these findings, we conclude that depolarization causes Ca2+ entry, with resultant elevation of cytosolic free Ca2+ concentration leading to activation of ICl (ICl(Ca)). We investigated whether Ca(2+)-induced Ca2+ release from the sarcoplasmic reticulum was involved in activation of ICl(Ca), by depleting intracellular stores of Ca2+ using cyclopiazonic acid to block the sarcoplasmic Ca(2+)-adenosinetriphosphatase and repeated stimulation with ACh. In such Ca(2+)-depleted cells, depolarization-mediated Ca2+ entry continued to activate ICl(Ca), suggesting that Ca(2+)-induced Ca2+ release was not required for its activation. We conclude that Ca2+ entry can activate Cl- channels in tracheal smooth muscle. This represents a positive-feedback system, which would promote excitation and contraction of airway muscle.
K+ self-exchange by the Na+ pump: regulation by P(i) and metabolic perturbations
Authors: Nonaka T. , Warden D. H. , Matsushita K. , Stokes J. B.
Published: December 2017

We have previously demonstrated that the Na(+)-K+ pump on the basolateral membrane of the rabbit cortical collecting duct can function in the K+/K+ exchange mode. Increasing intracellular phosphate in red blood cells inhibits the Na+ pump and increases K+/K+ exchange. We found that maneuvers designed to increase intracellular phosphate in collecting duct cells caused an increase in K+/K+ exchange. Subjecting the cells to a metabolic insult (cyanide) increased K+/K+ exchange by the pump as judged by its ouabain sensitivity and lack of electrogenic or conductive characteristics. The results demonstrate that the rate of K+/K+ exchange by the Na(+)-K+ pump can be altered by changes in intracellular phosphate over a range that is physiologically or pathologically achievable. The results also suggest a mechanism for inhibition of vectorial Na+ transport during metabolic stress.
Tyrosine kinase activation is necessary for inducible nitric oxide synthase expression by interleukin-1 beta
Authors: Tetsuka T. , Morrison A. R.
Published: December 2017

The inflammatory cytokine interleukin-1 (IL-1) induces the inducible form of nitric oxide synthase (iNOS) with an increase in nitric oxide in rat mesangial cells. However, the cellular mechanisms that underlie the induction of iNOS by IL-1 beta in mesangial cells has not been clarified. Because we have shown that tyrosine kinase inhibitors attenuate IL-1 beta-induced cyclooxygenase expression and prostaglandin production, we investigated the effect of tyrosine kinase inhibitors on IL-1 beta-induced nitrite production and iNOS mRNA expression in rat mesangial cells. The tyrosine kinase inhibitors genistein and herbimycin A attenuated IL-1 beta-induced nitrite production in a dose-dependent manner. In addition, both of these inhibitors blocked IL-1 beta-induced iNOS mRNA expression. These data suggest that tyrosine kinase(s) plays a central role in IL-1 beta signaling to induce iNOS in rat mesangial cells.
Receptor activation induces short-term modulation of arterial contractions: memory in vascular smooth muscle
Authors: Ratz P. H.
Published: December 2017

This study examined the hypothesis that arteries retain a memory of receptor activation, resulting in temporary modulation of stimulus-contraction coupling. When pretreated for 30 min with 10(-5) M phenylephrine (PE), histamine, or prostaglandin F2 alpha (PGF2 alpha) and then relaxed fully for 10 min, steady-state increases in stress (S/So) and myosin light-chain phosphorylation (MLC20P/MLC20) produced by KCl in femoral arteries were weaker (0.33-0.57 S/So and 0.29-0.30 MLC20P/MLC20) than control responses (approximately 0.91 S/So and approximately 0.41 MLC20P/MLC20). The inhibitory effect lasted for at least 2 h and was not as strong in tissues pretreated for a 10-fold shorter duration or a 10-fold lower concentration of PE. When pretreated with low concentrations of PE (10(-7) M) and PGF2 alpha (10(-6) M), the early portion of subsequent KCl-induced contractile responses reached levels higher than control responses (0.79-0.86 S/So compared with approximately 0.70 S/So). These data support the hypothesis that receptor activation of arteries not only caused contractions but also stimulated another system, a response modulator that appeared to serve as memory of receptor activation.
Differential expression and effect of 1,25-dihydroxyvitamin D3 on myosin in arterial tree of rats
Authors: Ishibashi K. , Evans A. , Shingu T. , Bian K. , Bukoski R. D.
Published: December 2017

The hypothesis that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3, also known as calcitriol] modulates myosin expression in vascular smooth muscle was tested. Wistar-Kyoto or spontaneously hypertensive rats given intraperitoneal injections of 25 ng 1,25(OH)2D3/100 g body weight for varying periods of time showed a greater than twofold increase in aortic mRNA encoding the myosin regulatory light chain relative to 18S rRNA (P < 0.05). 1,25(OH)2D3 administration to Wistar rats caused a significant increase in the aortic content of total myosin regulatory light chain and total myosin heavy chain. The increase in myosin light chain was the result of a specific increase in expression of its smooth muscle isoform [control = 65.2 +/- 3.4% vs. 1,25(OH)2D3 = 78.7 +/- 3.6%, P = 0.020]. 1,25(OH)2D3 had no effect on total myosin light chain or heavy chain in the superior mesenteric artery. The hormone did, however, increase the proportion of the smooth muscle isoform of the light chain in this vessel [control = 81.4 +/- 2.6% vs. 1,25(OH)2D3 = 88.8 +/- 2.1%, P = 0.048]. In branch II and III mesenteric resistance arteries, 1,25(OH)2D3 significantly increased the active stress response to 10 mumol/l norepinephrine but was without effect on total myosin light chain or heavy chain content or on the relative expression of the myosin light chain isoforms [control = 94.0 +/- 1.4% vs. 1,25(OH)2D3 = 95.8 +/- 1.1%, P = 0.33].(ABSTRACT TRUNCATED AT 250 WORDS)
Redistribution of aquaporin-2 water channels induced by vasopressin in rat kidney inner medullary collecting duct
Authors: Marples D. , Knepper M. A. , Christensen E. I. , Nielsen S.
Published: December 2017

Aquaporin-2 (AQP2) is the predominant vasopressin-regulated water channel of the renal collecting duct. We tested whether vasopressin induces translocation of AQP2 from intracellular vesicles into the apical plasma membrane. AQP2 was quantitated in plasma membrane and intracellular vesicle fractions prepared from the inner medulla of one kidney from each rat before or 20 min after intravenous 1-desamino-8-D-arginine vasopressin (DDAVP) treatment, using immunoblotting and densitometry. Contralateral kidneys were prepared for immunofluorescence and immunoelectron microscopy. Immunoblotting revealed that, compared with untreated controls, DDAVP treatment significantly increased the fraction of AQP2 protein associated with the plasma membrane fraction relative to intracellular vesicles. This increase averaged 2.0-fold in untreated rats and 2.9-fold in rats water loaded for 12 h. Water loading, presumably by suppressing circulating vasopressin levels, decreased the fraction of AQP2 associated with the plasma membrane by 55%, suggesting retrieval of AQP2 from the plasma membrane. In rats sequentially thirsted for 48 h to increase expression and then water loaded for 72 h to minimize plasma membrane labeling, DDAVP caused a 12-fold increase in the plasma membrane to intracellular vesicle labeling ratio. The accentuation of the DDAVP response seen after water loading is consistent with the observed increase in the fraction of AQP2 in the intracellular pool available for insertion. Immunofluorescence confirmed a marked DDAVP-induced redistribution of AQP2 from intracellular to plasma membrane domains. Furthermore, quantitative immunoelectron microscopy demonstrated a 3.4-fold increase in apical plasma membrane to intracellular vesicle labeling ratio. These results provide a direct in vivo demonstration of vasopressin-induced translocation of AQP2 into the apical plasma membrane.
M-creatine kinase gene expression in mechanically overloaded skeletal muscle of transgenic mice
Authors: Tsika R. W. , Hauschka S. D. , Gao L.
Published: December 2017

The molecular pathways and regulatory molecules that underlie changes in gene transcription during mechanical overload of skeletal muscle remain obscure. To better understand this process, we have examined mouse muscle creatine kinase (MCK) gene expression in mechanically overloaded plantaris (OP) muscle of transgenic and nontransgenic mice. Northern blot analysis revealed that endogenous MCK-specific mRNA transcripts were decreased 150% in the OP muscles after 6 wk. To identify the MCK gene regions involved in the response to mechanical overload, three different mouse MCKCAT transgenes were studied by measuring chloramphenicol acetyltransferase (CAT assays) activity in OP and sham-operated (control plantaris) muscles. Mouse lines carrying (+enh206)117MCKCAT and -1256MCKCAT transgenes exhibited 30 and 40% lower CAT levels, whereas two mouse lines carrying -3300MCKCAT transgenes exhibited average decreases of 430%. Nearly identical results, including measurements of exogenous CAT mRNA, were obtained 2 days postoverload. Six weeks or 2 days of mechanical overload led to an average decrease in MM-CK isoprotein of 140%. These data provide evidence that mechanical overload induces changes in MCK gene expression that appear to be regulated by at least two portions of the MCK gene: the 206 base pair 5' enhancer and the -3,300 to -1,257 region.
Myosin phosphorylation augments force-displacement and force-velocity relationships of mouse fast muscle
Authors: Grange R. W. , Cory C. R. , Vandenboom R. , Houston M. E.
Published: December 2017

Two studies were conducted to examine the effect of myosin regulatory light chain (R-LC) phosphorylation on the rate and extent of shortening in submaximally activated mouse extensor digitorum longus muscles in vitro at 25 degrees C. For each study, R-LC phosphate content was increased fivefold by application of a 5-Hz, 20-s conditioning stimulus (CS) to 0.65-0.68 mol phosphate/mol R-LC; this level was sustained between 10 and 40 s after the CS. Maximum isometric twitch force and the maximum rate of force development (+dF/dtmax) were potentiated in the range 13-17% and 9-17% (P < 0.05), respectively, after the CS. In study 1, the maximal rate and extent of shortening were significantly enhanced by 10 and 21% (P < 0.001), respectively, when measured using a twitch zero-load clamp technique. In study 2, the force-velocity and force-displacement relationships were both augmented when determined with the twitch afterload technique. Displacement was enhanced between 20 and 82% for loads that ranged from 3 to 75% of active peak twitch force, whereas velocity was increased 6-8% over the same range (P < 0.05), including the predicted maximum velocity (Vmax; 5.08 vs. 4.69 muscle length/s). In both studies the increase in velocity likely represents a shift along the force-velocity relationship toward true Vmax that reflects a decrease in relative load due to force potentiation. Furthermore, with the decrease in relative load, displacement at a given load was also increased. Potentiated displacement and extent of R-LC phosphorylation also decreased in parallel when studied for 5 min after the CS. The increase in muscle shortening is a novel finding and suggests a function for R-LC phosphorylation with respect to movement because both peak work and power were also enhanced by up to 22%. These effects are consistent with an R-LC phosphorylation-induced increase in fapp, the apparent rate constant that describes the cross-bridge transition from the non-force-generating to the force-generating state.
Voltage-dependent potentiation of neuronal L-type calcium channels due to state-dependent phosphorylation
Authors: Sculptoreanu A. , Figourov A. , De Groat W. C.
Published: December 2017

Modulation of Ca2+ channels during repetitive activity in excitable cells can have an important role in altering cellular function. In mammalian parasympathetic and dorsal root ganglion neurons, L-type Ca2+ channels are potentiated by single depolarizing prepulses or trains of short high-frequency depolarizing pulses. This type of potentiation takes place regardless of whether Ca2+ or Ba2+ is the charge carrier and requires phosphorylation by a adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. The magnitude of facilitation was correlated with frequency of conditioning trains, was enhanced by 8-bromoadenosine 3',5'-cyclic monophosphate or the Sp diastereomer of adenosine 3',5'-cyclic monophosphothioate (cAMPS), and reduced by Rp-cAMPS or a peptide inhibitor of cAMP-dependent protein kinase. The N-type Ca2+ channels exhibited the opposite response to these agents. We propose that the potentiation of L-type Ca2+ channel currents in neurons is due to state-dependent phosphorylation by cAMP-dependent protein kinase (Sculptoreanu, A., T. Scheuer, and W. A. Catterall. Nature Lond. 364: 240-243, 1993; Sculptoreanu, A., E. Rotman, M. Takahashi, T. Scheuer, and W. A. Catterall. Proc. Natl. Acad. Sci. USA 90: 10135-10139, 1993.). Thus state-dependent phosphorylation in neurons may be a mechanism for the regulation of various functions including transmitter release.
Lung epithelial Na channel subunits are differentially regulated during development and by steroids
Authors: Tchepichev S. , Ueda J. , Canessa C. , Rossier B. C. , O'Brodovich H.
Published: December 2017

Because the alpha-subunit of the rat lung epithelial Na channel (rENaC) is not expressed until late fetal gestation, the developmental immaturity of alpha-rENaC may be involved in the premature fetal lung's inability to mount a Na-absorptive response to appropriate agonists. As previous work has shown that the beta- and gamma-rENaC subunits of the Na channel are required for maximal alpha-rENaC activity, we determined their developmental expression in the fetal lung. In addition, because thyroid and corticosteroid therapy can mature the in vivo fetal lamb lung's ability to transport Na, we wished to determine whether such treatment increased the expression of alpha-, beta-, and gamma-rENaC. Lungs were harvested from normal rat fetuses of 17 through 22 days gestation (term = 22 days), normal rat pups during the first week of life, and adult rats. Initial expression of alpha-rENaC was detected at 19 days gestation and progressively increased in utero. beta- and gamma-rENaC mRNA were not detected until 21 and 22 days gestation, and then only at very low levels. During the first week after birth, the levels of alpha-rENaC declined, whereas beta- and gamma-rENaC mRNA levels increased. This pre- and postnatal pattern of alpha-rENaC expression correlates with the endogenous glucocorticosteroid levels in the fetus and the rat pup's early postnatal corticosteroid resistance. Combined or separate treatment of pregnant rats (16 through 22 days gestational age) with thyroid-releasing hormone (TRH) and/or dexamethasone (Dex) for 48 h showed that Dex, but not TRH, could increase fetal lung alpha-rENaC mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)
Flow-mediated NO release from endothelial cells is independent of K+ channel activation or intracellular Ca2+
Authors: O'Neill W. C.
Published: December 2017

The role of K+ channels and intracellular [Ca2+] in flow-induced nitric oxide (NO) production was investigated in bovine aortic endothelial cells in culture. NO release (measured as nitrite production) and K+ channel activity (measured as 86Rb+ efflux) were measured in cells grown on collagen-coated microcarrier beads and perfused in a column. An eightfold increase in flow produced a rapid (within 1 min), sustained, and reversible sixfold increase in NO release. Efflux of 86Rb+ also increased but rapidly returned to baseline and then transiently decreased when flow was decreased. This was probably due to boundary layer washout rather than to K+ channel activation, because an identical pattern was seen for release of [3H]ouabain. Neither tetraethylammonium nor increasing medium [K+] to block K+ currents prevented flow-induced NO release. Removal of medium Ca2+ or chelation of intracellular Ca2+ also did not block flow-mediated NO release. The results demonstrate that flow rapidly increases NO release from endothelial cells but that this increase in NO release is not dependent on activation of K+ channels or changes in intracellular [Ca2+].
Magnetic fields applied to collagen-coated ferric oxide beads induce stretch-activated Ca2+ flux in fibroblasts
Authors: Glogauer M. , Ferrier J. , McCulloch C. A.
Published: December 2017

The ability to apply controlled forces to the cell membrane may enable elucidation of the mechanisms and pathways involved in signal transduction in response to applied physical stimuli. We have developed a magnetic particle-electromagnet model that allows the application of controlled forces to the plasma membrane of substrate-attached fibroblasts. The system allows applied forces to be controlled by the magnitude of the magnetic field and by the surface area of cell membrane covered with collagen-coated ferric beads. Analysis by single-cell ratio fluorimetry of fura 2-loaded cells demonstrated large calcium transients (50-300 nM) in response to the magnetic force applications. Experiments using either the stretch-activated channel blocker gadolinium chloride or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to eliminate external calcium ions, or addition of extracellular manganese ions, indicated that there was a calcium influx through putative stretch-activated channels. The probability of a calcium influx in single cells was increased by higher surface bead loading and the degree of cell spreading. Depolymerization of actin filaments by cytochalasin D increased the amplitude of calcium response twofold. The regulation of calcium flux by filamentous actin content and by cell spreading indicates a possible modulatory role for the cytoskeleton in channel sensitivity. Magnetic force application to beads on single cells provides a controlled model to study mechanisms and heterogeneity in physical force stimulation of cation-permeable channels.
Ca(2+)-permeable large-conductance nonselective cation channels in rat basophilic leukemia cells
Authors: Obukhov A. G. , Jones S. V. , Degtiar V. E. , Luckhoff A. , Schultz G. , Hescheler J.
Published: December 2017

Spreading of Ca2+ signals in rat basophilic leukemia (RBL) cells occurs by release of ATP. Therefore we studied the effect of ATP on membrane currents. ATP (1-10 microM) activated large-conductance channels. Single channel events were resolved in the whole cell mode. Similar channel activity was observed in RBL cells transfected with the muscarinic M1 receptor after stimulation with carbachol as well as after intracellular infusion of aluminum fluoride. Activation was independent of internal Ca2+ (0-10 microM). The channels had a conductance of 250 pS in 135 mM Na+ and 70 pS in 100 mM Ca2+. The permeability (P) ratio was PCa/PNa/PCs/PMg = 16:1:0.6:0.6. These channels may contribute to secretory responses by allowing Ca2+ entry, leading to high Ca2+ concentrations in the vicinity of the channel pore.
Intracellular Ca2+ stores in chick cerebellum Purkinje neurons: ontogenetic and functional studies
Authors: Sacchetto R. , Cliffer K. D. , Podini P. , Villa A. , Christensen B. N. , Volpe P.
Published: December 2017

The molecular composition of intracellular Ca2+ stores in developing chicken cerebellum Purkinje neurons from embryonic day 11 (E11) to posthatching day 2 (P2) was studied by immunocytochemistry using specific antibodies for three molecular constituents, the receptor (R) and/or channel sensitive to inositol 1,4,5-trisphosphate (IP3), Ca(2+)-adenosinetriphosphatase (ATPase), and calsequestrin (CS). CS, IP3R, and Ca(2+)-ATPase were first detected by light-microscopic immunofluorescence in migrating Purkinje cells at E11-E12 and throughout late phases of embryonic development. Ontogenesis of CS, IP3R, and Ca(2+)-ATPase accompanied well-defined stages of cerebellum histogenesis and cytogenesis and was accomplished before hatching. High-resolution immunogold electronmicroscopy revealed that, at E18-P1, CS was still largely distributed to the endoplasmic reticulum (ER) lumen and began to be segregated to ER subcompartments (calciosomes) only by P2, whereas the IP3R was concentrated into ER cisternal stacks as early as E18. Both ionotropic and metabotropic plasma membrane receptors were present in dissociated single chicken Purkinje cells from E16 onward, as indicated by measurements of membrane currents (whole cell recording mode) and of cytoplasmic Ca2+ transients monitored with the cell-trappable fluorescent indicator fura 2-acetoxymethyl ester, respectively. Cytoplasmic Ca2+ transients were detected after either activation of glutamate metabotropic receptors, i.e., evidence of IP3-sensitive Ca2+ channels, or application of caffeine, i.e., evidence of ryanodine-sensitive Ca2+ channels. Intracellular Ca2+ stores appear to be functional during embryonic development.
Leucine activates system A amino acid transport in L6 rat skeletal muscle cells
Authors: McDowell H. E. , Christie G. R. , Stenhouse G. , Hundal H. S.
Published: December 2017

In this study, we present evidence showing that leucine is involved in the upregulation of system A amino acid transport activity in the L6 rat skeletal muscle cell line. At leucine concentrations of > or = 0.05 mM, the uptake of N-methylamino-alpha-isobutyric acid (MeAIB), a paradigm system A substrate, was stimulated by up to 50%. Kinetic analysis revealed that this stimulation was a result of an increase in the maximal transport rate of MeAIB uptake, from 327 +/- 26 to 450 +/- 8 pmol.min-1.mg protein-1 after incubation of cells with leucine. No significant change in the concentration at which MeAIB transport was half maximal was observed. System A activation was biphasic, reaching an initial plateau after 3 h, with a second phase of activation being observed after 5 h. The initial activation of system A transport occurred by a mechanism distinct from that activated by insulin-like growth factor-I (IGF-I) (3 nM), since the effects of leucine and IGF-I were additive. This activation was not due to transstimulation, since 2-amino-2-norbornane-carboxylic acid, a specific system L substrate, did not stimulate system A. Leucine's keto acid, ketoisocaproic acid, prevented the activation of system A transport, whereas aminooxyacetate, a transaminase inhibitor, augmented the increase in system A activity by leucine. Both cycloheximide and actinomycin D inhibited the leucine-induced increase in MeAIB uptake. The present results indicate that leucine, or some cellular component regulated by it, is capable of stimulating system A transport through control of DNA transcription, possibly of a gene encoding either a repressor or enhancer molecule of system A or perhaps of the gene encoding system A itself.
Quantitative confocal imaging along the crypt-to-surface axis of colonic crypts
Authors: Chu S. , Brownell W. E. , Montrose M. H.
Published: December 2017

Quantitative confocal microscopy methods are used to measure events along the crypt-to-surface axis in living mouse colonic mucosa. Experiments visualize carboxyseminaphthorhodofluor-1 (SNARF-1; a pH-sensitive fluorescent dye) in the extracellular fluid to measure extracellular pH within an intact epithelium. Lucifer yellow (LY; a pH-insensitive dye) is used to control for fidelity of the optical path. Light scatter from colonic tissue caused SNARF-1 or LY fluorescence to decrease 3% per micrometer focal distance into tissue at both 640- and 580-nm emission wavelengths. However, dual emission ratios of LY fluorescence were constant as a function of focal distance into tissue or in the presence of short-chain fatty acids (SCFA). SCFA, known to cause changes in extracellular pH, cause maximal changes in crypt luminal pH at 10 microns from the crypt base. Maximal changes of pH in lamina propria occur higher along the crypt-to-surface axis than maximal changes in luminal pH. Lateral intercellular spaces between colonocytes are a separate microdomain in which pH is constant in absence vs. presence of SCFA and along the base-to-apex axis of colonocytes.
Biosynthetic and growth abnormalities are associated with high-level expression of CFTR in heterologous cells
Authors: Schiavi S. C. , Abdelkader N. , Reber S. , Pennington S. , Narayana R. , McPherson J. M. , Smith A. E. , Hoppe H. , Cheng S. H.
Published: December 2017

An inducible gene amplification system was utilized to study the effects of overexpression of cystic fibrosis transmembrane conductance regulator (CFTR) in vitro. BTS, a monkey kidney cell line expressing a temperature-sensitive simian virus 40 (SV-40) large T antigen was stably transfected at the nonpermissive temperature with a plasmid containing an SV-40 origin of replication and the cDNA for either the wild-type CFTR or the mutant G551D-CFTR. Shift of the isolated cell lines to the permissive temperature resulted in induction and accumulation to high levels of the CFTR plasmid, mRNA, and protein. However, high-level expression of CFTR was transient in both BTS-CFTR and BTS-G551D cells due to a decrease in their respective levels of CFTR mRNA. Because G551D-CFTR only exhibits residual Cl channel activity, this suggests that the observed downregulation with BTS-G551D cells may have been induced by either the physical presence of high amounts of CFTR or some low threshold level of Cl- channel activity. Examination of cell growth properties revealed a correlation between high-level expression of wild-type CFTR and growth arrest of the cells at the G2/M phase. However, similar induction of the G551D-CFTR mutant showed only a slight growth inhibition and little enrichment of cells at the G2/M phase. Cytofluorographic analysis further revealed that BTS-CFTR cells were significantly larger than parental BTS or BTS-G551D cells at all stages of the cell cycle. These results indicate that CFTR overexpression is capable of inducing its own downregulation and that high levels of Cl- channel activity can result in increased cell volume and subsequent cell growth abnormalities.
Maximum shortening velocity and myosin isoforms in single muscle fibers from young and old rats
Authors: Li X. , Larsson L.
Published: December 2017

Maximum velocity of unloaded shortening (Vo) and myosin heavy (MHC) and light chain (MLC) isoform compositions were determined in 185 single fibers from the extensor digitorum longus (EDL) and soleus muscles in 3- to 6- and 20- to 24-mo-old rats. In the soleus, fibers expressing the type I MHC isoform dominated in young and old animals. In the EDL, most fibers in the young animals expressed type IIb MHC or a combination of types IIx and IIb (type IIxb), whereas in the old animals type IIxb MHC fibers predominated. Vo was significantly (P < 0.01) lower (0.59 +/- 0.28 ml/s, n = 55) in soleus fibers from old than from young animals (1.12 +/- 0.46 ml/s, n = 48), despite the fact that all fibers expressed the type I MHC and slow MLC isoforms. In the EDL, Vo values in single fibers did not differ between young (2.18 +/- 0.58 ml/s, n = 43) and old animals (2.10 +/- 0.53 ml/s, n = 39). The mechanism underlying age-related slowing in soleus fibers is not known, but it has been suggested that there could be more than one beta/slow MHC isoform and that there is an age-related transition within these isoforms.
Influence of amphotericin B on the sodium pump of porcine lens epithelium
Authors: Delamere N. A. , Dean W. L. , Stidam J. M. , Moseley A. E.
Published: December 2017

Active transport by Na(+)-K(+)-ATPase in the monolayer of lens epithelium is vital for the regulation of sodium and potassium levels within the mass of fiber cells that make up the bulk of the lens. In this study, experiments were conducted using porcine lenses to test whether Na(+)-K(+)-ATPase activity in the epithelium is altered when the permeability of lens cell plasma membranes is increased by the ionophore amphotericin B. After 24 h, sodium was significantly (P < 0.01) elevated in lenses exposed to 5 or 10 microM amphotericin B. Amphotericin B stimulated 86Rb uptake, probably through an increase of cytoplasmic sodium concentration due to increased inward sodium leak; the rate of ouabain-sensitive potassium (86Rb) uptake by intact lenses was significantly increased by amphotericin B at 5 microM (P < 0.05) and 10 microM (P < 0.01). After 24 h, the epithelium from lenses exposed to amphotericin B had an Na(+)-K(+)-ATPase activity that was more than twofold higher (P < 0.01) than the Na(+)-K(+)-ATPase activity in control lenses. By immunoblot, there was an increase in Na(+)-K(+)-ATPase catalytic (alpha) subunit immunoreactive polypeptide in the epithelium of lenses exposed to amphotericin B. The increase stemmed from a marked increase of Na(+)-K(+)-ATPase alpha 2-immunoreactive polypeptide but little change in the amount of alpha 1-immunoreactive protein. As judged by immunoblot experiments, the amount of Na(+)-K(+)-ATPase beta 1-immunoreactive polypeptide also appeared to be higher in the epithelium of amphotericin B-treated lenses compared with control lenses. In summary, these results suggest that in response to a permeability challenge with amphotericin B, the porcine lens epithelium is able to increase the activity of Na(+)-K(+)-ATPase. The same permeability challenge also appears to stimulate the biosynthesis of Na(+)-K(+)-ATPase catalytic subunit as well as glycoprotein subunit polypeptides.
Regulation of ICAM-1 by dexamethasone in a human vascular endothelial cell line EAhy926
Authors: Burke-Gaffney A. , Hellewell P. G.
Published: December 2017

Regulation by dexamethasone of intercellular adhesion molecule-1 (ICAM-1) in cultured monolayers of the human umbilical vein endothelial cell line EAhy926 was investigated. Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in combination or lipopolysaccharide (LPS) alone gave time- and dose-dependent increases in ICAM-1. Sustained expression of ICAM-1 was observed after short exposure (30 min) to TNF-alpha + IFN-gamma, but not to LPS. LPS-induced ICAM-1 expression was not inhibited by interleukin-1 (IL-1) receptor antagonist (0.01-100 micrograms/ml). Dexamethasone (1,000 nM) did not inhibit TNF-alpha + IFN-gamma-induced ICAM-1 expression or mRNA induction. In contrast, dexamethasone dose dependently (0.1-1,000 nM) inhibited LPS-induced ICAM-1 expression; however, its effect on mRNA was not established, because ICAM-1 mRNA induced by LPS was not detected at the time points investigated in this study (3 and 20 h). Adhesion of unstimulated human neutrophils to EAhy926 monolayers activated with TNF-alpha + IFN-gamma or LPS was increased in the presence of dexamethasone at low doses, whereas neutrophil adhesion to LPS- but not cytokine-stimulated endothelial cells was significantly reduced (P < 0.05) in the presence of a high dose of dexamethasone (1,000 nM). In conclusion, dexamethasone was demonstrated to regulate the expression and function of ICAM-1 in a stimulus-dependent manner.
Mitogenic inhibition by phorbol esters is associated with decreased phosphatidylinositol-3 kinase activation
Authors: Weiss R. H. , Yabes A. P.
Published: December 2017

In contrast to their role as potent tumor promoters, phorbol esters can cause inhibition of cell growth. Because the effect of phorbol esters occurs through activation of protein kinase C (PKC) and because activated PKC is translocated to the membrane placing it in a position to act on the intracellular portion of the growth factor receptor, we asked whether this inhibitory effect is mediated through the action of phorbol 12-myristate 13-acetate (PMA) on receptor association with the signal transfer proteins. When added to rat vascular smooth muscle (VSM) cells concurrently with basic fibroblast growth factor (bFGF), PMA at 100 ng/ml completely inhibits bFGF-stimulated DNA synthesis. Under the same growth-inhibitory conditions of PMA addition, aggregation of phosphatidylinositol 3-kinase (PI3K) to the fibroblast growth factor receptor and tyrosine phosphorylation of the 85-kDa regulatory component of the signal transfer protein PI3K are reduced by 94 and 79%, respectively. PI3K catalytic activity, as measured by conversion of phosphatidylinositol to phosphatidylinositol 3-phosphate, is decreased 88% by PMA addition. This effect is not specific to PI3K, since aggregation of phospholipase C-gamma 1 to the activated bFGF receptor is also decreased by PMA treatment. In addition, the PI3K inhibitor wortmannin markedly attenuates bFGF-stimulated VSM cell growth in a dose-dependent manner. These data suggest that the site of growth inhibition by PMA in VSM cells lies upstream of signal transfer particle aggregation and that such growth arrest may be mediated through inhibition of activation of PI3K.
Characteristics of mitochondria isolated from type I and type IIb skeletal muscle
Authors: Jackman M. R. , Willis W. T.
Published: December 2017

Mitochondria isolated from rabbit soleus (98% type I) and gracilis (99% type IIb) skeletal muscle were compared for compositional differences. Whole muscle mitochondrial contents were 14.5 +/- 1.2 mg/g of wet weight in soleus and 5.3 +/- 0.6 mg/g in the gracilis muscle, a 2.7-fold difference. Maximal pyruvate plus malate oxidase activity in gracilis mitochondria was roughly 75% of that in soleus mitochondria. In contrast, glycerol 3-phosphate (G-3-P) oxidation was 10-fold greater in gracilis mitochondria. Both soleus and gracilis mitochondria exhibited additive pyruvate and G-3-P oxidase activities. In general, citric acid cycle enzyme activities were higher in soleus mitochondria. A notable exception was isocitrate dehydrogenase, which was twofold higher in gracilis mitochondria. Substrate cytochrome c reductase activities indicated that the electron transport chain (ETC) of soleus mitochondria possess roughly twice the capacity for both NADH and succinate oxidation. Similarly, the maximal activities of NADH dehydrogenase and succinate dehydrogenase were roughly twofold higher in soleus mitochondria. The findings demonstrate that mitochondria isolated from types I and IIb skeletal muscle differ substantially in composition. Furthermore, the relatively similar pyruvate plus malate oxidase activities in the face of markedly different ETC capacities suggest that the interaction between matrix dehydrogenases and the ETC may differ in mitochondria isolated from types I and IIb skeletal muscle.
Role of weight-bearing function on expression of myosin isoforms during regeneration of rat soleus muscles
Authors: Bigard X. A. , Merino D. , Serrurier B. , Lienhard F. , Guezennec Y. C. , Bockhold K. J. , Whalen R. G.
Published: December 2017

The expression of myosin isoforms was studied in regenerated rat soleus muscle during either normal or altered postural activity. Regeneration was induced following injury by venom from the Notechis scutatus scutatus snake. Immunohistochemical analysis showed that, in regenerating soleus muscle after 3 wk of hindlimb suspension, nearly all fibers reacted positively with the myosin heavy chain (MHC) antibody associated with fast-twitch muscle fibers (fast MHC). When 3 wk of recovery with normal weight-bearing activity followed hindlimb suspension, the regeneration soleus muscle exhibited a nearly homogeneous staining with the MHC antibody associated with the slow-twitch muscle fibers (slow MHC). These findings were in accordance with quantitative analysis of the electrophoretic separation of the native myosin isoforms. Immunohistochemical data showed that removal of weight bearing in the 21-day old regenerated soleus muscles resulted in an increase in fast MHC expression. Together, the results of the present study clearly demonstrate that the postural load is an important component in the induction of slow MHC in regenerating muscle and that the control of the expression of MHC in muscle comprising a homogeneous population of fibers deriving from satellite cells appears more homogeneous and more complete than in a nondegenerated one.
Regulation of colonic ion transport by GRP. II. GRP modulates the epithelial response to PGE2
Authors: Traynor T. R. , O'Grady S. M.
Published: December 2017

The purpose of this study was to examine the potential modulatory effects of gastrin-releasing peptide (GRP) on prostaglandin (PG) E2-stimulated electrolyte transport across the distal colon epithelium. In an earlier study, PGE2 was shown to reduce net Cl absorption without altering the serosal-to-mucosal unidirectional Cl flux in porcine distal colon (19). In the present study, tissues were pretreated with serosal or mucosal GRP and subsequently stimulated with PGE2. The resulting increase in short-circuit current (ISC) was 152% (serosal GRP) and 49% (mucosal GRP) greater than control PGE2 responses alone. Serosal, but not mucosal, GRP also enhanced the ISC response to vasoactive intestinal peptide. On the basis of flux measurements, the combined effects of serosal GRP and PGE2 resulted in the activation of a transcellular pathway for Cl secretion, which was not activated by either mediator alone. The time course of the PGE2 response was also affected by GRP. Serosal GRP shortened the time to maximum ISC by 35%, whereas mucosal peptide lengthened the time to maximum ISC by 68% These results suggest that GRP acts as a modulator of PG action on electrolyte transport in the distal colon.