bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcium is permeable through a maitotoxin-activated nonselective cation channel in mouse L cells
Authors: Estacion M. , Nguyen H. B. , Gargus J. J.
Published: December 2017

The shellfish poison maitotoxin causes the irreversible opening of nonselective cation channels in mouse L cell fibroblasts, consistent with the action of this toxin in other cell types and the previously demonstrated existence of 28-pS voltage-insensitive nonselected cation channels that are activated by platelet-derived growth factor in these cells. Toxin-induced opening of these nonselective cation channels led to increases of intracellular calcium and secondary activation of calcium-activated potassium channel. These effects were completely dependent on influx of extracellular calcium, supporting the conclusion that the maitotoxin-activated nonselective cation channels are permeable to calcium as well as to sodium and potassium. The implication of this finding is that calcium signaling through this channel underlies its links into the growth factor response.
MK-801 reduces uptake and stimulates efflux of excitatory amino acids via membrane depolarization
Authors: Longuemare M. C. , Keung E. C. , Chun S. , Sharp F. R. , Chan P. H. , Swanson R. A.
Published: December 2017

MK-801 and related compounds reduce excitotoxic neuronal injury by blocking N-methyl-D-aspartate (NMDA) receptorgated ion channels. These agents also cause neuronal vacuolization and block glutamate-induced astrocyte swelling, effects that may be unrelated to actions at the NMDA receptor. In the present study, high concentrations of MK-801 (100-1,000 microM) caused uncompetitive inhibition of glutamate uptake in astrocyte and neuronal cultures and stimulated D-aspartate efflux from astrocytes. MK-801 (500 microM) reduced the maximal velocity for glutamate uptake in astrocytes from 31 to 17 nmol.mg protein-1.min-1, whereas competitive NMDA receptor antagonists did not affect glutamate uptake. MK-801 also inhibited uptake of gamma-aminobutyric acid (GABA). Because both GABA uptake and glutamate uptake are electrogenic, one mechanism by which MK-801 could inhibit uptake is by membrane depolarization. Whole cell patch-clamp recording confirmed that MK-801 in the range of 100-1,000 microM caused dose-dependent and reversible depolarization. These concentrations are far higher than necessary to block NMDA receptors, and the findings suggest that actions at sites other than NMDA receptors could contribute to the effects of high doses of MK-801 in some experimental and clinical settings.
Insulin-induced translocation of Na(+)-K(+)-ATPase subunits to the plasma membrane is muscle fiber type specific
Authors: Lavoie L. , Roy D. , Ramlal T. , Dombrowski L. , Martn-Vasallo P. , Marette A. , Carpentier J. L. , Klip A.
Published: December 2017

We have previously shown that an acute insulin treatment induces redistribution of the alpha 2- and beta 1- isoforms of the Na(+)-K(+)-ATPase from intracellular membranes to plasma membranes detected on subcellular fractionation of mixed muscles and immunoblotting with isoform-specific antibodies (H. S. Hundal et al. J. Biol. Chem. 267: 5040-5043, 1992). In the present study we give both biochemical and morphological evidence that this insulin effect is operative in muscles composed mostly of oxidative (red) fibers but not in muscles composed mostly of glycolytic (white) fibers. The redistribution of the Na(+)-K(+)-ATPase alpha 2- and beta 1-isoforms after insulin injection was detected in membranes isolated from and muscles (soleus, red gastrocnemius, red rectus femoris, and red vastus lateralis) but not in membranes from white muscles (white gastrocnemius, tensor fasciae latae, white rectus femoris, and white vastus lateralis). After insulin injection, the potassium-dependent 3-O-methylfluorescein phosphatase activity of the enzyme was higher by 22% in the plasma membrane-enriched fraction and lower by 15% in the internal membrane fraction isolated from red but not from white muscles. Quantitative immunoelectron microscopy of ultrathin muscle cryosections showed that in vivo insulin stimulation augmented the density of Na(+)-K(+)-ATPase alpha 2- and beta 1- isoforms at the plasma membrane of soleus muscle by 80 and 124%, respectively, with no change in white gastrocnemius muscle. The effect of insulin to increase the content of Na(+)-K(+)-ATPase alpha 2- and beta 1-subunits in isolated plasma membranes was still observed when glycemia was prevented from dropping by using hyperinsulinemic-euglycemic clamps. We conclude that the insulin-induced redistribution of the alpha 2- and beta 1-isoforms of the Na(+)-K(+)-ATPase from an intracellular pool to the plasma membrane in restricted to oxidative fiber-type skeletal muscles. This may be related to the selective expression of beta 1-subunits in these fibers and implies that the beta 2-subunit, typical of glycolytic muscles, does not sustain translocation of alpha 2 beta 2-complexes.
Comparative effects of insulin and isoproterenol on lipoprotein lipase in rat adipose cells
Authors: Chiappe de Cingalani G. E. , Goers J. W. , Giannotti M. , Caldiz C. I.
Published: December 2017

The effects of insulin and isoproterenol on lipoprotein lipase mass and enzyme activity were investigated in rat adipocytes. Cells were pulse labeled for 1 h with [35S]methionine to measure immunoprecipitable lipoprotein lipase. The results showed that 80% of the newly synthesized enzyme was membrane associated and 20% was secreted into the cell incubation medium. Enzyme activity was mainly associated with lipoprotein lipase secreted into the medium. A 10-min incubation with 10(-7) M insulin stimulated the secretion of lipoprotein lipase activity and the activity associated with adipocyte membranes. Conversely, 10(-6) M isoproterenol decreased the activity in all fractions. In addition, insulin increased lipoprotein lipase mass associated with cell membranes and decreased that in the incubation medium, whereas isoproterenol induced a decrease in both cell membranes and medium. Insulin and isoproterenol stimulated phosphorylation of lipoprotein lipase. These findings suggest that insulin stimulates the secretion of active lipoprotein lipase and a reuptake of inactive secreted enzyme, and isoproterenol decreases the activity by enzyme degradation. Moreover, because both agents stimulate phosphorylation of lipoprotein lipase, phosphorylation may play a role in the effect of insulin increasing enzyme activity, in secretion or reuptake, and in the effect of isoproterenol inducing degradation of lipoprotein lipase.
Plasticity in epithelial polarity of renal intercalated cells: targeting of the H(+)-ATPase and band 3
Authors: Al-Awqati Q.
Published: December 2017

The intercalated cell is an epithelial cell of the renal collecting tubule that is specialized for H+ and HCO3- transport. These cells exist as two types, alpha and beta. The alpha-cell secretes H+ into the lumen by an apical H(+)-ATPase and a basolateral Cl-/HCO3- exchanger that is a form of band 3 protein (AE1). The beta-cell secretes HCO3- into the lumen by an apical Cl-/HCO3- exchanger and a basolateral H(+)-ATPase. In a previous study, it was suggested that a reversal in epithelial polarity of these cells occurs during the response of the kidney to an acid load (G.J. Schwartz, J. Barasch, and Q. Al-Awqati. Nature Lond. 318: 368-371,1985). Recent studies, however have shown that there are many other subtypes where the distribution of these two proteins does not fit into this neat bipolar classification. This group of investigators recently generated an immortalized cell line of the beta-intercalated cell and found that the apical Cl-/HCO3- exchanger is also AE1. Furthermore, when these cells were seeded at high densities, the polarized targeting of the apical band 3 was reversed to the basolateral membrane. This was produced by the secretion of extracellular matrix protein that by themselves were capable of reversing the polarity of band 3 (J. S. van Adelsberg, J. C. Edwards, J. Takito, B. Kiss, and Q. Al-Awqati. Cell 76: 1053-1061, 1995). A large new extracellular matrix protein, hensin, was identified and found to be present exclusively in the collecting tubule. The extensive recent literature on the biology of alpha- and beta-intercalated cells is reviewed here and found to be compatible with the idea of the reversal of polarity as a mechanism for the regulation of H+ secretion by the tubule
A novel preparation of dissociated renal proximal tubule cells that maintain epithelial polarity in suspension
Authors: Segal A. S. , Boulpaep E. L. , Maunsbach A. B.
Published: December 2017

The functional properties of an epithelium are inextricably linked to its polarized structure. It has been difficult to study polarity at the level of the single cell, since most epithelial cells lose their polarity within minutes after dissociation. We have developed a preparation of native, dissociated, Ambystoma proximal tubule cells that maintain structural and functional polarity for a minimum of 7 days in suspension. We have used these cells to explore cell surface polarity in a single cell. Electron microscope cytochemical and immunocytochemical studies show that alkaline phosphatase is localized exclusively to the apical brush border, whereas the Na(+)-K(+)-ATPase is restricted to the basolateral membrane. Just as in the proximal tubule in situ, a sharp structural transition between the apical and basolateral membrane domains is retained. The ZO-1 protein found at the tight junction in situ is not present on the membrane of the dissociated cells, but rather it is distributed in the cytoplasm. The actin cytoskeleton also remains polarized in the single cells, and its distribution and organization appear to help maintain cell polarity. Electrophysiological measurements show that these cells remain viable at least as long as they remain structurally polarized. Patch-clamp recordings from both the apical and basolateral membranes show that the distribution of several ion channel proteins maintains functional polarity. We hypothesize that, despite loss of the intercellular “gate” and membrane-associated ZO-1, the socalled “fence” function of the tight junctional complex is retained in these dissociated proximal tubule cells. This preparation may serve as a useful single cell model with which to study epithelial polarity and membrane trafficking pathways.
H2O2 modulates twitch tension and increases Po of Ca2+ release channel in frog skeletal muscle
Authors: Oba T. , Koshita M. , Yamaguchi M.
Published: December 2017

The effect of H2O2 was examined to elucidate the basis of muscle injury after exercise. Exposure of single fibers to 1.5-6 mM H2O2 led to twitch potentiation followed by a marked decrease. Then, fibers contracted spontaneously. BAY K 8644 augmented twitch potentiation and slowed the decay of twitches. In 5 mM dithiothreitol (DTT), twitch potentiation and spontaneous contraction were not observed on H2O2 addition. Cytoplasmic application of 1.5-3 mM H2O2 to heavy sarcoplasmic reticulum (SR) vesicles incorporated into planar lipid bilayers increased the open probability of Ca2+ release channels, an effect reversed by DTT. We investigated oxidation of sulfhydryl groups on proteins in SR membrane by H2O2 with N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. Pretreatment of light and heavy SR membranes with 1.5 mM H2O2 exponentially increased fluorescence intensity. The time constant of the intensity increase was increased markedly only in heavy SR in solution containing 50 microM cytoplasmic Ca2+, so Ca2+ release was associated with protein oxidation by H2O2. Thus extracellular H2O2 probably acts by oxidizing sulfhydryls of proteins at two distinct sites: the dihydropyridine receptors, oxidation of which elicits potentiation and subsequent inhibition of twitches, and Ca2+ release channels, whose oxidation elicits spontaneous contraction, resulting in muscle dysfunction.
Role of reactive oxygen metabolites in murine peritoneal macrophage phagocytosis and phagocytic killing
Authors: Takao S. , Smith E. H. , Wang D. , Chan C. K. , Bulkley G. B. , Klein A. S.
Published: December 2017

This study was designed to quantify the role of reactive oxygen metabolites (ROMs) in two distinct components of murine peritoneal macrophage activity, phagocytosis and killing, and to discriminate quantitatively the degree to which each component is dependent on NADPH oxidase and/or xanthine oxidase. A fluorochromatic vital staining technique was modified to simultaneously quantify phagocytosis and microbicidal activity of macrophages incubated with Candida parapsilosis targets. To determine the role of ROMs, macrophages were preincubated with free radical scavengers [superoxide dismutase (SOD) and/or catalase] or with selective inhibitors of xanthine oxidase (XO, e.g., allopurinol) or NADPH oxidase [diphenyleneiodonium (DPI)]. Phagocytosis was not affected by treatment of macrophages with SOD, catalase, allopurinol, or DPI. Candidacidal activity, however, was inhibited by SOD, allopurinol, or DPI. The inhibitory effects of DPI and allopurinol were additive. Histochemical and biochemical assays demonstrated substantial quantities of XO in murine peritoneal macrophages. The findings suggest that the generation of ROMs by XO- and NADPH oxidase-dependent pathways are each important for phagocytic killing by murine peritoneal macrophages.
Electrical properties of diaphragm and EDL muscles during the life of dystrophic mice
Authors: De Luca A. , Pierno S. , Camerino D. C.
Published: December 2017

The membrane electrical properties of diaphragm and extensor digitorum longus (EDL) muscle fibers of dystrophic mdx and control mice from 4 wk to 14-19 mo of age were recorded with the intracellular microelectrode technique. Up to 8 wk of age, the diaphragm and EDL muscles did not differ between the two strains. From 8 up to 20 wk, the mdx diaphragm fibers showed a higher membrane resistance (Rm), which was due to significantly lower values of resting chloride conductance (GCl) and an overexcitability with respect to age-matched controls. Oppositely, the mdx EDL muscle fibers had significantly lower Rm and higher GCl values than age-related controls at 8, 10, and 13 wk, along with a decreased membrane excitability. These differences were no longer detectable at 20 wk. The diaphragm and EDL muscles from 14- to 19-mo-old controls showed a decrease of GCl and an increase of potassium conductance with respect to adult animals. In aged mdx animals, these changes were very dramatic in diaphragm fibers, whereas no differences, with respect to adults, were found in the EDL muscle. Thus GCl is an index of the dystrophic condition of mdx muscles. In the degenerating diaphragm, the impairment of GCl can account for some of the pathological features of the muscle. In the EDL muscle, the changes of GCl can follow the high regenerative potential of the hindlimb muscles of the mdx phenotype.
Bowditch lecture. Integrated regulation of intestinal epithelial transport: intercellular and intracellular pathways
Authors: Barrett K. E.
Published: December 2017

The intestinal epithelium is an important site of active solute transport processes. Such processes include the secretion of electrolytes into the lumen, predominantly chloride and bicarbonate. These secretory mechanisms subserve a variety of functions, both physiological and pathophysiological, including maintenance of the fluidity of intestinal contents and mucosal defense. Both chloride and bicarbonate secretion are the subject of integrated regulatory mechanisms at both the intercellular and intracellular levels. The goal of this article is to discuss data that exemplify these two levels of regulation, which have been the subject of research in my laboratory. It is likely that some of these principles are also broadly applicable to secretory epithelial cells outside of the intestinal tract, such as those in the airway. I also discuss the ways in which we believe these regulatory mechanisms are involved not only in intestinal physiology but also perhaps in the pathogenesis of specific disease states.
Electrogenic Li+/Li+ exchange mediated by the Na+-K+ pump in rabbit cardiac myocytes
Authors: Hemsworth P. D. , Whalley D. W. , Rasmussen H. H.
Published: December 2017

The membrane Na+-K+ pump can be activated at extracellular sites by several different monovalent cations, including Li+, while being highly selective for the physiological activator Na+ at intracellular sites. We examined whether Li+ can replace Na+ as an activator of the Na+-K+ pump at intracellular sites. Single cardiac myocytes were voltage clamped at 0 mV with wide-tipped patch pipettes filled with a K+- and Na+-free solution containing 160 mM Li+. Ouabain induced an inward shift of membrane current when the myocytes were superfused with Na+-Tyrode solution containing 5.6 mM K+. The shift was dependent on the presence of intracellular Li+ (LiCl in pipette filling solution replaced with tetramethylammonium chloride) and extracellular K+. When we replaced Na+ and K+ in the superfusate with Li+ and voltage clamped myocytes using Li+-containing filling solutions in patch pipettes, ouabain induced an inward shift in membrane current similar to that recorded when myocytes were superfused with K+-containing Na+-Tyrode solution. These findings indicate that the Na+-K+ pump can mediate electrogenic exchange of intracellular Li+ for extracellular K+ or Li+.
Roles of cytoplasmic Ca2+ and intracellular Ca2+ stores in induction and suppression of apoptosis in S49 cells
Authors: Bian X. , Hughes F. M. , Huang Y. , Cidlowski J. A. , Putney J. W.
Published: December 2017

The Ca2+-ATPase inhibitors, thapsigargin and cyclopiazonic acid, depleted intracellular Ca2+ stores, induced large increases in intracellular Ca2+ concentration, and caused apoptosis in S49 cells. Removal of extracellular Ca2+ augmented apoptosis due to thapsigargin, indicating that depletion of Ca2+ from intracellular stores is responsible for apoptosis with this agent. Overexpression of the apoptosis suppressor, Bcl-2, inhibited apoptosis due to thapsigargin but did not affect thapsigargin-induced Ca2+ signaling. Dexamethasone induced apoptosis, diminished the size of the endoplasmic reticulum Ca2+ pool, and caused a small elevation of intracellular Ca2+. However, this elevation was not due to Ca2+ influx because the increase was similar in the presence or absence of Ca2+ in the medium. Furthermore, in contrast to the results with thapsigargin, apoptosis due to dexamethasone was unchanged in a Ca2+-free medium. These results indicate that depletion of Ca2+ stores initiates a pathway leading to apoptosis. Elevations in cytoplasmic Ca2+ appears to play a lesser role than previously thought in the actions of Bcl-2 and glucocorticoids.
Confocal imaging analysis of ATP-induced Ca2+ response in individual endothelial cells of the artery in situ
Authors: Ohata H. , Ujike Y. , Momose K.
Published: December 2017

The mechanisms for mobilization of intracellular free Ca2+ have been studied in various types of isolated and cultured cells, but little is known about Ca2+ mobilization in individual cells in situ. We tried to establish imaging analysis of intracellular free Ca2+ concentration ([Ca2+]i) in individual cells loaded with the acetoxymethyl ester of fluo 3 in situ, using laser scanning confocal microscopy. The method permitted us to distinguish signals from endothelial and smooth muscle cells of guinea pig artery. Addition of ATP to the artery caused a transient increase in endothelial [Ca2+]i. It was concluded that the response was induced via P2Y purinoceptors, because adenosine 5'-O-(2-thiodiphosphate), but not UTP, caused a similar response independent of extracellular Ca2+. The percentage of cells that responded to ATP (1-10 microM) and the peak amplitude of the transient increase in [Ca2+]i were dose dependently increased. Using rapid xy-scanning and line-scanning modes, we confirmed that 10 microM ATP induced Ca2+ waves, at a rate of 10-30 microns/s, after a lag time of approximately 3 s. These results show that [Ca2+]i waves within endothelial cells are physiologically induced by ATP via P2Y purinoceptor, but not P2U purinoceptor, in aortic strips in situ. The method should be of use in the study of vascular physiology and pathophysiology.
p70s6k is activated by CCK in rat pancreatic acini
Authors: Bragado M. J. , Groblewski G. E. , Williams J. A.
Published: December 2017

The expression and activity of p70s6k-p85s6k in isolated rat pancreatic acini were revealed by Western blotting, immunoprecipitation, and kinase assay. Cholecystokinin (CCK) stimulation of p70s6k activity was biphasic, with an early phase maximum at 5 min and a late phase maximum at 60 min. The threshold concentration of CCK to increase p70s6k activity was 3 pM, and the maximal effect was seen at 1 nM CCK. Carbachol and bombesin, but not vasoactive intestinal peptide, also activated p70s6k. The protein kinase C (PKC) activator (12-O-tetradecanoylphorbol 13-acetate), the calcium ionophore (ionomycin), and a derivative of adenosine 3',5'-cyclic monophosphate induced only a slight increase in p70s6k activity. Rapamycin potently blocked both the basal and the CCK-stimulated p70s6k activity, and this inhibition was reversed by an excess of FK-506. The phosphatidylinositol 3-kinase inhibitor, wortmannin, potently inhibited p70s6k activation by CCK, whereas the tyrosine kinase inhibitor genistein had only a partial effect. Neither rapamycin nor wortmannin inhibited amylase release at concentrations that inhibited p70s6k activity. Thus the activation pathway of p70s6k by CCK is not mediated by PKC or mobilization of intracellular calcium but seems to be mediated by phosphatidylinositol 3-kinase. The effect of rapamycin to inhibit p70s6k activity is mediated by binding to the immunophyllin FK-506-binding protein of 12 kDa. The p70s6k is not involved in the secretion of digestive enzymes induced by CCK.
Effects of fluid flow on intracellular calcium in bovine articular chondrocytes
Authors: Yellowley C. E. , Jacobs C. R. , Li Z. , Zhou Z. , Donahue H. J.
Published: December 2017

Fluid flow-induced shear stress results in a variety of morphological and metabolic changes in cultured bovine articular chondrocytes (BAC). However, the mechanism by which the flow signal is transduced into a biological response is unknown. Therefore, we investigated the effects of fluid flow on intracellular Ca2+ concentration ([Ca2+]i) in BAC. Cells loaded with fura 2 were exposed to steady and pulsatile (0.5 Hz) flow at 9, 18, and 34 ml/min in a parallel-plate flow chamber. In response to flow, there was a significant and flow rate-dependent increase in the percentage of cells showing a rise in [Ca2+]i, but no effect on the [Ca2+]i response amplitude. There was no significant difference between the [Ca2+]i responses to steady and pulsatile flow. Mean intracellular Ca2+ response values ranged between 26.2 +/- 1.6 (9 ml/min) and 38.0 +/- 6.8 nM (34 ml/min) above basal [Ca2+]i (81.3 +/- 24.1 nM, n = 90). Removal of extracellular Ca2+ or addition of Gd3+ significantly reduced the percentage of cells responding, suggesting that influx of Ca2+, possibly through mechanosensitive channels, contributes to the rise in intracellular Ca2+. Our data suggest fluid flow-induced mobilization of intracellular Ca2+ may contribute to the mechanism by which mechanical loads are transduced by chondrocytes.
Muscarinic signaling pathway for calcium release and calcium-activated chloride current in smooth muscle
Authors: Wang Y. X. , Kotlikoff M. I.
Published: December 2017

We investigated the muscarinic activation of Ca(2+)-activated Cl- currents [ICl(Ca)] in voltage-clamped equine tracheal myocytes. The threshold of cytosolic free Ca2+ concentration ([Ca2+]i) required for activation of ICl(Ca) was 202 +/- 22 nM, and full activation of the current occurred at 771 +/- 31 nM. Hexahydro-sila-difenidol (M3 antagonist) inhibited the methacholine-induced phasic [Ca2+]i increase and ICl(Ca) in a concentration-dependent manner, whereas methoctramine (M2 antagonist) only slightly attenuated the [Ca2+]i increase and ICl(Ca) (14.8 and 21.4%, respectively), consistent with incomplete selectivity. Dialysis of heparin (10 mg/ml) blocked methacholine-induced [Ca2+]i and ICl(Ca) but had no effect on the caffeine-induced Ca2+ release or ICl(Ca); inositol 1,4,5-trisphosphate (100 microM) induced ICl(Ca) and blocked the methacholine current. Conversely, ruthenium red (50 microM) prevented the caffeine-induced [Ca2+]i release and ICl(Ca) but had no effect on methacholine-induced [Ca2+]i or current. Intracellular dialysis of the calmodulin antagonist N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7, 500 microM) or the Ca2+/calmodulin-dependent protein kinase inhibitor KN93 (5 microM) had no effect on the [Ca2+]i increase or ICl(Ca). Pertussis toxin (0.5 mg/ml) did not affect the increase in [Ca2+]i or ICl(Ca). Dialysis with antibodies directed against the alpha-subunit of Gq/G11 (Gq alpha/ G alpha 11) blocked the methacholine-induced ICl(Ca) in a concentration-dependent manner, whereas anti-G alpha i-1/G alpha 1-2 antibodies (1:35) and anti-G alpha i-3/G(o) alpha antibodies (1:35) were without effect. The results indicate that stimulation of phospholipase C via M3/Gq proteins is the predominant signaling pathway for the activation of ICl(Ca); at high agonist concentrations, Ca(2+)-induced Ca2+ release does not appear to play a prominent role in muscarinic signaling.
Ammonia effect on calcium-activated chloride secretion in T84 intestinal epithelial monolayers
Authors: Mayol J. M. , Hrnjez B. J. , Akbarali H. I. , Song J. C. , Smith J. A. , Matthews J. B.
Published: December 2017

We recently showed that ammonia profoundly inhibits cyclic nucleotide-regulated Cl- secretion in model human T84 intestinal epithelia but does not impair the secretory response to the Ca2+ agonist carbachol. Using transepithelial transport, fura 2 fluorescence, and radioisotopic efflux techniques, we further explored this dichotomy and arrived at a preliminary explanation for the inhibitory action of ammonia. The secretory response to the Ca(2+)-adenosinetriphosphatase inhibitor thapsigargin is unaffected by ammonia, which suggests that an increase in intracellular Ca2+ stimulates secretory pathways that are insensitive to ammonia. Surprisingly, Cl- secretion elicited by the Ca2+ ionophores ionomycin and A23187 is markedly blunted in monolayers pretreated with ammonia. However, ammonia posttreatment does not inhibit the secretory response to ionophore, which suggests that ammonia may interfere with the ability of these ionophores to increase intracellular [Ca2+]. This hypothesis is directly supported by fura 2 experiments. The inhibitory action of ammonia parallels the behavior of the K+ channel blocker Ba2+, and ammonia reduces the basolateral 86Rb+ efflux rate constant in forskolin- but not in carbachol-treated monolayers. Ammonia, which is present in high concentrations in the normal gastro-intestinal tract, may serve as a novel endogenous regulator of epithelial electrolyte transport by interfering with a Ba(2+)-sensitive basolateral K+ conductance distinct from the Ca(2+)-activated basolateral K+ conductance.
Volume expansion-sensing outward-rectifier Cl- channel: fresh start to the molecular identity and volume sensor
Authors: Okada Y.
Published: December 2017

The maintenance of a constant volume in the face of extracellular and intracellular osmotic perturbation is essential for the normal function and survival of animal cells. Osmotically swollen cells restore their volume, exhibiting a regulatory volume decrease by releasing intracellular K+, Cl-, organic solutes, and obligated water. In many cell types, the volume regulatory effluxes of Cl- and some organic osmolytes are known to be induced by swelling-induced activation of anion channels that are characterized by their moderate outward rectification, cytosolic ATP dependency, and intermediate unitary conductance (10-100 pS). Recently, simultaneous measurements of cell size by light microscopy and whole cell Cl- current have shown that the Cl- current density is proportionally increased with an increase in the outer surface area, which is mainly achieved through unfolding of membrane invaginations by volume expansion. Thus this anion channel can somehow sense volume expansion and can be called the volume expansion-sensing outwardly rectifying (VSOR) anion channel. Its molecular identity and activation mechanism are yet to be elucidated. Three cloned proteins, ClC-2, P-glycoprotein, and pIcln, have been proposed as candidates for the VSOR anion channel. The unitary conductance, voltage dependency, anion selectivity, pH dependency, and pharmacology of the VSOR anion channel are distinct from the ClC-2 Cl- channel, which is also known to be sensitive to volume changes. Recent patch-clamp studies in combination with molecular biological techniques have shown that P-glycoprotein is not itself the channel protein but is a regulator of its volume sensitivity. Although there is still debate about another candidate protein, pIcln, the most recent study has suggested that this is likely to be a regulator of some other distinct Cl- channel. Identification of the VSOR anion channel protein per se, its volume-sensing mechanism, and its accessory/regulatory proteins at the molecular level is currently a subject of utmost physiological importance.
Polarized distribution of L-type calcium channels in early sea urchin embryos
Authors: Dale B. , Yazaki I. , Tosti E.
Published: December 2017

Using the whole cell clamp technique, we have measured calcium-dependent currents and steady-state conductance in early sea urchin blastomeres. The calcium currents in M phase decreased from 8.5 microA/cm2 at the four-cell stage to 5.4 microA/cm2 at the eight-cell stage. In 16-cell stage embryos, calcium currents were 7.4 microA/cm2 in the mesomeres, 2.3 microA/cm2 in the macromeres, and were not detected in the micromeres. In contrast, the micromeres had a two- to threefold higher steady-state conductance than the mesomeres or macromeres, which may be due to potassium ion conductivity. Nifedipine, an L-type channel antagonist, delays cleavage division at a concentration of 0.05-0.1 mM and causes developmental defects, such as poor skeletal differentiation in later sea urchin embryos.
Receptor-mediated inhibition of renal Na+-K+-ATPase is associated with endocytosis of its α- and β-subunits
Authors: Chibalin Alexander V. , Katz Adrian I. , Berggren Per-Olof , Bertorello Alejandro M.
Published: December 2017

The mechanisms involved in receptor-mediated inhibition of Na+-K+-ATPase remain poorly understood. In this study, we evaluate whether inhibition of proximal tubule Na+-K+-ATPase activity by dopamine is linked to its removal from the plasma membrane and internalization into defined intracellular compartments. Clathrin-coated vesicles were isolated by sucrose gradient centrifugation and negative lectin selection, and early and late endosomes were separated on a flotation gradient. Inhibition of Na+-K+-ATPase activity by dopamine, in contrast to its inhibition by ouabain, was accompanied by a sequential increase in the abundance of the α-subunit in clathrin-coated vesicles (1 min), early endosomes (2.5 min), and late endosomes (5 min), suggesting its stepwise translocation between these organelles. A similar pattern was found for the β-subunit. The increased incorporation of both subunits in all compartments was blocked by calphostin C. The results demonstrate that the dopamine-induced decrease in Na+-K+-ATPase activity in proximal tubules is associated with internalization of its α- and β-subunits into early and late endosomes via a clathrin-dependent pathway and that this process is protein kinase C dependent. The presence of Na+-K+-ATPase subunits in endosomes suggests that these compartments may constitute normal traffic reservoirs during pump degradation and/or synthesis.
Regulation of red blood cell filterability by Ca2+ influx and cAMP-mediated signaling pathways
Authors: Oonishi Tadahiro , Sakashita Kanako , Uyesaka Nobuhiro
Published: December 2017

To investigate the mechanism of the regulation of human red blood cell deformability, we examined the deformability under mechanical stress. Washed human red blood cells were rapidly injected through a fine needle, and their filterability was measured using a nickel mesh filter. The decrease in filterability showed a V-shaped curve depending on the extracellular Ca2+ concentration; the maximum decrease was achieved at ∼50 μM. The decreased filterability was accompanied by no change in cell morphology and cell volume, indicating that the decrease in filterability can be ascribed to alterations of the membrane properties. Ca2+entry blockers (nifedipine and felodipine) inhibited the impairment of filterability under mechanical stress. Prostaglandins E1 and E2, epinephrine, and pentoxifylline, which are thought to modulate the intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) level of red blood cells, improved or worsened the impaired filterability according to their expected actions on the cAMP level of the cells. These results strongly suggest that the membrane properties regulating red blood cell deformability are affected by the signal transduction system, including Ca2+-dependent and cAMP-mediated signaling pathways.
Signal transduction pathways involved in the regulation of protein synthesis by insulin in L6 myoblasts
Authors: Kimball Scot R. , Horetsky Rick L. , Jefferson Leonard S.
Published: December 2017

The phosphorylation states of three proteins implicated in the action of insulin on translation were investigated, i.e., 70-kDa ribosomal protein S6 kinase (p70 S6k ), eukaryotic initiation factor (eIF) 4E, and the eIF-4E binding protein 4E-BP1. Addition of insulin caused a stimulation of protein synthesis in L6 myoblasts in culture, an effect that was blocked by inhibitors of phosphatidylinositide-3-OH kinase (wortmannin), p70 S6k (rapamycin), and mitogen-activated protein kinase (MAP kinase) kinase (PD-98059). The stimulation of protein synthesis was accompanied by increased phosphorylation of p70 S6k , an effect that was blocked by rapamycin and wortmannin but not PD-98059. Insulin caused dephosphorylation of eIF-4E, an effect that appeared to be mediated by the p70 S6k pathway. Insulin also stimulated phosphorylation of 4E-BP1 as well as dissociation of the 4E-BP1 ⋅ eIF-4E complex. Both rapamycin and wortmannin completely blocked the insulin-induced changes in 4E-BP1 phosphorylation and association of 4E-BP1 and eIF-4E; PD-98059 had no effect on either parameter. Finally, insulin stimulated formation of the active eIF-4G ⋅ eIF-4E complex, an effect that was not prevented by any of the inhibitors. Overall, the results suggest that insulin stimulates protein synthesis in L6 myoblasts in part through utilization of both the p70 S6k and MAP kinase signal transduction pathways.
pH-independent retrograde targeting of glycolipids to the Golgi complex
Authors: Schapiro Florencia B. , Lingwood Clifford , Furuya Wendy , Grinstein Sergio
Published: December 2017

A small fraction of the molecules internalized by endocytosis reaches the Golgi complex through a retrograde pathway that is poorly understood. In the present work, we used bacterial toxins to study the retrograde pathway in Vero cells. The recombinant B subunit of verotoxin 1B (VT1B) was labeled with fluorescein to monitor its progress within the cell by confocal microscopy. This toxin, which binds specifically to the glycolipid globotriaosyl ceramide, entered endosomes by both clathrin-dependent and -independent pathways, reaching the Golgi complex. Once internalized, the toxin-receptor complex did not recycle back to the plasma membrane. The kinetics of internalization and the subcellular distribution of VT1B were virtually identical to those of another glycolipid-binding toxin, the B subunit of cholera toxin (CTB). Retrograde transport of VT1B and CTB was unaffected by addition of weak bases in combination with concanamycin, a vacuolar-type ATPase inhibitor. Ratio imaging confirmed that these agents neutralized the luminal pH of the compartments where the toxin was located. Therefore, the retrograde transport of glycolipids differs from that of proteins like furin and TGN38, which require an acidic luminal pH. Additional experiments indicated that the glycolipid receptors of VT1B and CTB are internalized independently and not as part of lipid “rafts” and that internalization is cytochalasin insensitive. We conclude that glycolipids utilize a unique, pH-independent retrograde pathway to reach compartments of the secretory system and that assembly of F-actin is not required for this process.
Cyclic ADP-ribose activates caffeine-sensitive calcium channels from sea urchin egg microsomes
Authors: Pérez Claudio F. , Marengo Juan José , Bull Ricardo , Hidalgo Cecilia
Published: December 2017

Adenosine 5′-cyclic diphosphoribose [cyclic ADP-ribose (cADPR)], a metabolite of NAD+ that promotes Ca2+ release from sea urchin egg homogenates and microsomal fractions, has been proposed to act as an endogenous agonist of Ca2+ release in sea urchin eggs. We describe experiments showing that a microsomal fraction isolated from Tetrapigus nyger sea urchin eggs displayed Ca2+-selective single channels with conductances of 155.0 ± 8.0 pS in asymmetric Cs+ solutions and 47.5 ± 1.1 pS in asymmetric Ca2+ solutions. These channels were sensitive to stimulation by Ca2+, ATP, and caffeine, but not inositol 1,4,5-trisphosphate, and were inhibited by ruthenium red. The channels were also activated by cADP-ribose in a Ca2+-dependent fashion. Calmodulin and Mg2+, but not heparin, modulated channel activity in the presence of cADP-ribose. We propose that these Ca2+ channels constitute the intracellular Ca2+-induced Ca2+ release pathway that is activated by cADP-ribose in sea urchin eggs.
Ets-1 is an early response gene activated by ET-1 and PDGF-BB in vascular smooth muscle cells
Authors: Naito Shinji , Shimizu Shunichi , Maeda Shigeto , Wang Jianwei , Paul Richard , Fagin James A.
Published: December 2017

Ets-1 is a transcription factor that activates expression of matrix-degrading proteinases such as collagenase and stromelysin. To study the control of ets-1 gene expression in rat vascular smooth muscle cells (VSMC), cells were exposed to factors known to regulate VSMC migration and proliferation. Platelet-derived growth factor-BB (PDGF-BB), endothelin-1 (ET-1), and phorbol 12-myristate 13-acetate (PMA) induced a dose-dependent expression of ets-1 mRNA. These effects were abrogated by inhibition of protein kinase C (PKC) by H-7 or chronic PMA treatment. Ets-1 mRNA was superinduced by PDGF-BB and ET-1 in the presence of cycloheximide. The chelation of intracellular Ca2+ by 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-acetoxymethyl ester and the depletion of endoplasmic reticulum intracellular Ca2+concentration ([Ca2+]i) by thapsigargin inhibited PDGF-BB- and ET-1-induced ets-1 mRNA, whereas ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid had no effect. However, [Ca2+]irelease alone was not sufficient to increase ets-1 mRNA. Forskolin blocked ET-1-, PDGF-BB-, and PMA-induced ets-1 mRNA, as well as inositol phosphate formation, consistent with an effect through impairment of PKC activation. Inhibitors of ets-1 gene expression, such as H-7 and herbimycin A, inhibited the ET-1 induction of collagenase I mRNA. We propose that ets-1 may be an important element in the orchestration of matrix proteinase expression and of vascular remodeling after arterial injury.
Charybdotoxin block of Ca2+-activated K+ channels in colonic muscle depends on membrane potential dynamics
Authors: Frey Bret W. , Carl Andreas , Publicover Nelson G.
Published: December 2017

Charybdotoxin (ChTX) is a specific blocker of Ca2+-activated K+ channels. The voltage- and time-dependent dynamics of ChTX block were investigated using canine colonic myocytes and the whole cell patch-clamp technique with step and ramp depolarization protocols. During prolonged step depolarizations, K+ current slowly increased in the continued presence of ChTX (100 nM). The rate of increase depended on membrane potential with an e-fold change for every 60 mV. During ramp depolarizations, the effectiveness of ChTX block depended significantly on the rate of the ramp (50% at 0.01 V/s to 80% at 0.5 V/s). Results are consistent with a mechanism in which ChTX slowly “unbinds” in a voltage-dependent manner. A simple kinetic model was developed in which ChTX binds to both open and closed states. Slow unbinding is consistent with ChTX having little effect on electrical slow waves recorded from circular muscle while causing depolarization and contraction of longitudinal muscle, which displays more rapid “spikes.” Resting membrane potential and membrane potential dynamics are important determinants of ChTX action.
Sequential increases in capillarization and mitochondrial enzymes in low-frequency-stimulated rabbit muscle
Authors: Škorjanc Dejan , Jaschinski Frank , Heine Georg , Pette Dirk
Published: December 2017

To investigate temporal changes in capillarization and increases in mitochondrial enzyme activity, rabbit tibialis anterior muscles underwent chronic low-frequency stimulation for up to 50 days. Capillary density (CD), capillary-to-fiber ratio (C/F), intercapillary distance (ICD), and mean capillary area (MCA), as well as several other parameters of capillarization, were examined. In addition, tissue levels of mRNA specific to vascular endothelial growth factor (VEGF) were assessed by reverse transcriptase-polymerase chain reaction. Citrate synthase (CS) activity, a marker of aerobic-oxidative metabolic potential, was measured in the same muscles. Significant increases in CD and C/F, respectively, and decreases in ICD and MCA were observed after 2 days. These changes reached stable maxima by 14 days. The increases in capillarization occurred in a fiber-type-specific manner, affecting type IId fibers before types IIda and IIa. VEGF mRNA levels increased in a bimodal time pattern with a first elevation (2.5-fold) after 1 day and a second (9-fold) after 6–8 days. Increases in CS were first noted after 8 days. Obviously, increases in capillarization as induced by enhanced contractile activity precede increases in the aerobic-oxidative potential of energy metabolism.
Sulfhydryls associated with H2O2-induced channel activation are on luminal side of ryanodine receptors
Authors: Oba Toshiharu , Ishikawa Tatsuya , Yamaguchi Mamoru
Published: December 2017

The mechanism underlying H2O2-induced activation of frog skeletal muscle ryanodine receptors was studied using skinned fibers and by measuring single Ca2+-release channel current. Exposure of skinned fibers to 3–10 mM H2O2 elicited spontaneous contractures. H2O2 at 1 mM potentiated caffeine contracture. When the Ca2+-release channels were incorporated into lipid bilayers, open probability ( P o) and open time constants were increased on intraluminal addition of H2O2 in the presence of cis catalase, but unitary conductance and reversal potential were not affected. Exposure to cis H2O2 at 1.5 mM failed to activate the channel in the presence of trans catalase. Application of 1.5 mM H2O2 to the transside of a channel that had been oxidized by cis p-chloromercuriphenylsulfonic acid (pCMPS; 50 μM) still led to an increase in P o, comparable to that elicited by trans 1.5 mM H2O2 without pCMPS. Addition of cis pCMPS to channels that had been treated with or without trans H2O2 rapidly resulted in high P o followed by closure of the channel. These results suggest that oxidation of luminal sulfhydryls in the Ca2+-release channel may contribute to H2O2-induced channel activation and muscle contracture.
Distinct domains of the voltage-gated K+ channel Kvβ1.3 β-subunit affect voltage-dependent gating
Authors: Uebele Victor N. , England Sarah K. , Gallagher Daniel J. , Snyders Dirk J. , Bennett Paul B. , Tamkun Michael M.
Published: December 2017

The Kvβ1.3 subunit confers a voltage-dependent, partial inactivation (time constant = 5.76 ± 0.14 ms at +50 mV), an enhanced slow inactivation, a hyperpolarizing shift in the activation midpoint, and an increase in the deactivation time constant of the Kv1.5 delayed rectifier. Removal of the first 10 amino acids from Kvβ1.3 eliminated the effects on fast and slow inactivation but not the voltage shift in activation. Addition of the first 87 amino acids of Kvβ1.3 to the amino terminus of Kv1.5 reconstituted fast and slow inactivation without altering the midpoint of activation. Although an internal pore mutation that alters quinidine block (V512A) did not affect Kvβ1.3-mediated inactivation, a mutation of the external mouth of the pore (R485Y) increased the extent of fast inactivation while preventing the enhancement of slow inactivation. These data suggest that 1) Kvβ1.3-mediated effects involve at least two distinct domains of this β-subunit, 2) inactivation involves open channel block that is allosterically linked to the external pore, and 3) the Kvβ1.3-induced shift in the activation midpoint is functionally distinct from inactivation.
SNAP-25 is essential for cortical granule exocytosis in mouse eggs
Authors: Ikebuchi Yoshihide , Masumoto Nobuyuki , Matsuoka Tetsu , Yokoi Takeshi , Tahara Masahiro , Tasaka Keiichi , Miyake Akira , Murata Yuji
Published: December 2017

Synaptosome-associated protein of 25 kDa (SNAP-25) has been shown to play an important role in Ca2+-dependent exocytosis in neurons and endocrine cells. During fertilization, sperm-egg fusion induces cytosolic Ca2+mobilization and subsequently Ca2+-dependent cortical granule (CG) exocytosis in eggs. However, it is not yet clear whether SNAP-25 is involved in this process. In this study, we determined the expression and function of SNAP-25 in mouse eggs. mRNA and SNAP-25 were detected in metaphase II (MII) mouse eggs by RT-PCR and immunoblot analysis, respectively. Next, to determine the function of SNAP-25, we evaluated the change in CG exocytosis with a membrane dye, tetramethylammonium-1,6-diphenyl-1,3,5-hexatriene, after microinjection of a botulinum neurotoxin A (BoNT/A), which selectively cleaves SNAP-25 in MII eggs. Sperm-induced CG exocytosis was significantly inhibited in the BoNT/A-treated eggs. The inhibition was attenuated by coinjection of SNAP-25. These results suggest that SNAP-25 may be involved in Ca2+-dependent CG exocytosis during fertilization in mouse eggs.
Selective hydrolysis of plasmalogen phospholipids by Ca2+-independent PLA2 in hypoxic ventricular myocytes
Authors: McHowat Jane , Liu Shi , Creer Michael H.
Published: December 2017

Accelerated phospholipid catabolism occurs early after the onset of myocardial ischemia and is likely to be mediated by the activation of one or more phospholipases in ischemic tissue. We hypothesized that hypoxia increases phospholipase A2(PLA2) activity in isolated ventricular myocytes, resulting in increased lysophospholipid and arachidonic acid production, contributing to arrhythmogenesis in ischemic heart disease. The majority of ventricular myocyte arachidonic acid was found in plasmalogen phospholipids. Hypoxia increased membrane-associated, Ca2+-independent, plasmalogen-selective PLA2activity, resulting in increased arachidonic acid release and lysoplasmenylcholine production. Pretreatment with the specific Ca2+-independent PLA2 inhibitor bromoenol lactone blocked hypoxia-induced increases in PLA2 activity, arachidonic acid release, and lysoplasmenylcholine production. Lysoplasmenylcholine produced action potential derangements, including shortening of action potential duration, and induced early and delayed afterdepolarizations in normoxic myocytes. The electrophysiological alterations induced by lysoplasmenylcholine would likely contribute to the initiation of arrhythmogenesis in the ischemic heart.