bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulated calcium channel in apical membranes renal proximal tubule cells
Authors: Zhang M. I. , O'Neil R. G.
Published: December 2017

Using the single-channel patch-clamp technique, we identified a Ca2+ channel in the apical membranes rabbit cultured proximal tubule cells. The channel is permeable to both Ca2+ and Ba2+ but not to monovalent cations. In on-cell patches, the channel opened infrequently and had a conductance of 4.6 +/- 0.9 pS (n = 5) with 105 mM CaCl2 in the pipette. Although addition of forskolin (12.5 microM) Cl2 is without effect, addition of phorbol 12-myristate 13-acetate (PMA, 1 microM) activated the channel. At 0 mV pipette voltage (resting state) in the on-cell patches, PMA increased open probability (P0) from 0 to 6.9 +/- 2.3% (n = 5) within 1-3 min of PMA application. Likewise, stretching the membrane patch (-10 to -30 mmHg) activated this channel (P0 increased to 5.3 +/- 2.1%, n = 3, at 0 mV applied pipette potential), with results consistent with a mechanosensitive channel. The channel displayed only modest voltage sensitivity, with mild activation on membrane hyperpolarization but with inactivation on strong depolarization. The addition of the L-type Ca2+ channel blocker (antagonist), nifedipine (10 microM), completely blocked this channel in both on-cell and inside-out patches, whereas the agonist, BAY K 8644 (5 microM) was without effect. It is concluded that this channel is a nifedipine-sensitive, protein kinase C-regulated Ca2+ channel and that it may play a role in Ca2+ signaling in the proximal tubule cells, particularly during periods of mechanical stress.
Sodium-glucose cotransporters display sodium- and phlorizin-dependent water permeability
Authors: Loike J. D. , Hickman S. , Kuang K. , Xu M. , Cao L. , Vera J. C. , Silverstein S. C. , Fischbarg J.
Published: December 2017

Expression of Na(+)-glucose cotransporters of the SGLT-1 type by Xenopus laevis oocytes increased the osmotic water permeability (Pf) of oocytes by a factor of 1.9-2.8, in the presence and in the absence of 5 mM extracellular glucose. The Pf increase was correlated with the amount of SGLT-1 cRNA injected. In oocytes expressing SGLT-1, either addition of phlorizin to the medium or the replacement of Na+ by choline inhibited the uptake of methyl-alpha-D-glucopyranoside, a specific substrate for SGLT-1, and returned oocyte Pf to its level in uninjected oocytes. Phlorizin inhibited the SGLT-1-attributable increase in Pf with an inhibition constant (Ki) of 6.1 microM, a value analogous to the Ki for phlorizin inhibition of sugar uptake. However, neither the presence of phlorizin nor the absence of extracellular Na+ significantly affected the increase in Pf elicited in oocytes expressing GLUT-1, a facilitative glucose transporter. These findings suggest that SGLT-1 forms a pore that allows the transmembrane passage of water and that water and glucose traverse the protein through this pore. The finding that removal of extracellular Na+ abolishes the increase in Pf attributable to SGLT-1 suggests that extracellular Na+ is required to maintain patency of this transporter's water-permeable transmembrane pore.
Ibuprofen protects human endothelial cell prostaglandin H synthase from hydrogen peroxide
Authors: Wessels D. A. , Hempel S. L.
Published: December 2017

Human endothelial cells exposed to H2O2 demonstrate decreased prostacyclin (PGI2) synthesis due to decreased prostaglandin H synthase (PGH synthase) activity. We tested the hypothesis that PGH synthase activity could be protected from H2O2 by a reversible nonsteroidal anti-inflammatory drug. Experiments demonstrate that ibuprofen if present during H2O2 exposure, protects endothelial cell PGH synthase against the decrease in prostaglandin formation caused by H2O2. Additional studies demonstrated that decreasing arachidonic acid release from cell phospholipids during H2O2 exposure did not protect PGI2 synthesis following H2O2 exposure. In other experiments, ibuprofen did not chelate Fe2+ in a conformation that inhibited the reactivity of Fe2+. In addition, ibuprofen did not scavenge HO. However, we demonstrate that ibuprofen significantly protects purified PGH synthase cyclooxygenase activity from the effects of H2O2. The results confirm the hypothesis. These findings suggest that ibuprofen displaces oxidant species from the cyclooxygenase site of PGH synthase, thereby preventing oxidation of the functional groups important for PGH synthase activity.
Mutations in the pore region of ROMK enhance Ba2+ block
Authors: Zhou H. , Chepilko S. , Schutt W. , Choe H. , Palmer L. G. , Sackin H.
Published: December 2017

The sequence of the hydrophobic “P” (pore) region of a K(+)-selective channel from the kidney (ROMK2) was altered to match that of the closely related inward rectifier (IRK1) channel by changing two amino acids, leucine (L) 117 and valine (V) 121, to isoleucine (I) and threonine (T), respectively. The mutant channel expressed in Xenopus laevis oocytes had an apparent inhibition constant at zero voltage [Ki(0)] in the presence of Ba2+ of 0.07 +/- 0.01 mM, which was more than 50 times lower than the Ki(0) of the wild-type channel (4.7 +/- 1.0 mM). The increased sensitivity to Ba2+ was accounted for by the point mutation V121T. Single-channel measurements indicated that the increased affinity involved an increase in the on-rate for Ba2+ block and a decrease in the off-rate. Block by Ca+ was also enhanced. The single-channel conductance of the L1171/ V121T mutant was increased by 50%, whereas the degree of inward rectification, ion selectivity, and apparent affinity for K+ were essentially unchanged. When the neutral asparagine residue within the second putative membrane-spanning domain of the ROMK channel was substituted with aspartic acid, the corresponding amino acid in IRK1, the degree of inward rectification was enhanced but Ba2+ block and single-channel inward conductance were unaffected. Thus the site of Ba2+ binding appears to be distinct from the locus of internal Mg2+ block and from at least one of the sites that determines K+ conjuctivity.
Effects of taxol on isolated rat hepatocyte metabolism
Authors: Manzano A. , Roig T. , Bermudez J. , Bartrons R.
Published: December 2017

Taxol is a natural product, isolated from Taxus brevifolia, with increasing clinical applications because of its potent antitumor activity. Although it is mainly metabolized in the liver, its effect on hepatocyte metabolism has been scarcely investigated. In this study, the response of isolated rat hepatocytes to taxol was evaluated by correlating the changes observed in global indexes such as viability and heat dissipation with those produced in oxygen consumption and intracellular metabolites (ATP, fructose 2,6-bisphosphate, and lactate). The results indicate that taxol reduces aerobic metabolism, which induces an insufficient increase of the ATP via anaerobic glycolysis. Moreover, incubations of isolated mitochondria with taxol indicate that the respiratory chain is directly affected by this drug.
Vascular endothelial growth factor affects permeability of brain microvessel endothelial cells in vitro
Authors: Wang W. , Merrill M. J. , Borchardt R. T.
Published: December 2017

Vascular endothelial growth factor (VEGF), which stimulates endothelial cell growth and induces hyperpermeability of the microvasculature, plays an important role in normal and tumor-vasculature development and tumor edema generation. In this study, we investigated the effect of VEGF on the permeability of cultured bovine brain microvessel endothelial cells (BMECs), an in vitro blood-brain barrier (BBB) model. We found that addition of purified VEGF to both the apical and basolateral sides of the BMEC monolayers increased the permeability of the monolayer to [14C]sucrose (approximately 3-fold). A more significant increase in permeability was observed when VEGF was applied to the basolateral side of the monolayer (3-fold) than to the apical side (1.5-fold). The permeability-increasing activity of VEGF on the BMEC monolayers is both dose and time dependent. The VEGF-induced permeability increase in BMECs requires a long incubation time with VEGF, and the effect is durable. These results suggest that this cell culture system may be useful for exploring the role of VEGF in regulating the permeability of the BBB, for studying the mechanism of the permeability-increasing effect of VEGF on the endothelial cells, and for evaluating the strategies to regulate the activity of VEGF.
Ultrastructural and cytochemical characterization of cultured dogfish shark rectal gland cells
Authors: Valentich J. D. , Karnaky K. J. , Ecay T. W.
Published: December 2017

The dogfish shark rectal gland (SRG) is histologically complex, containing connective, nerve, and smooth muscle tissue and at least three types of epithelial cells: secretory tubule, central duct, and endothelial. This cellular heterogeneity precludes studies of the intact SRG from distinguishing direct tubular effects of mediators that modulate chloride secretion from their indirect effects on nonepithelial cells such as neurons. Primary SRG cultures express high levels of secretagogue-stimulated chloride secretion, suggesting that SRG cells retain a significant level of cytodifferentiation in vitro. However, because nontubular cells could contaminate these cultures, the question of whether secretagogues activate chloride secretion through direct or indirect effects on tubular epithelial cells remains unresolved. To address this issue, detailed ultrastructural and cytochemical analyses of SRG cultures were carried out to assess the level of cellular heterogeneity and the degree of cytodifferentiation expressed by SRG cells in vitro. The results demonstrate that, after 15 days, primary SRG monolayer cultures are composed exclusively of tubular epithelial cells with no detectable contamination by central duct cells, fibroblasts, smooth muscle cells, endothelial cells, or neurons. Tubular epithelial cells express most of the structural features of native SRG cells, including numerous mitochondria, massive basolateral surface amplification, complex tight junctions, and an extensive tubulovesicular system in the apical cytoplasm. Cultured SRG cells also display a striking level of polarization of cytoplasmic organelles and plasma membrane secretagogue receptors. These results account for the exceptionally high rates of electrogenic chloride secretion by SRG tubular epithelial cells in vitro and confirm that the effects of secretagogues on transport activity reflect their direct interaction with tubular epithelial cells.
Insulin-mimetic agents vanadate and pervanadate stimulate glucose but inhibit amino acid uptake
Authors: Tsiani E. , Abdullah N. , Fantus I. G.
Published: December 2017

The protein tyrosine phosphatase (PTP) inhibitors vanadate and pervanadate (pV) exert insulin-like biologic effects. In cultured differentiated rat L6 skeletal muscle cells, vanadate and pV stimulated 2-deoxy-D-[3H]glucose uptake in a dose- and time-dependent manner. There was no increase in maximum stimulation by additional insulin. In contrast, whereas insulin stimulated [14C]methylaminoisobutyric acid (MeAIB) uptake, basal uptake was inhibited by vanadate and pV. Insulin-stimulated MeAIB uptake was also inhibited in a dose-dependent manner and completely abolished by 5 mM vanadate or 0.1 mM pV. The inhibitory effect on basal MeAIB uptake was associated with a decrease in transporter affinity and a small decrease in maximum transport capacity, whereas the insulin-stimulated increase in maximum transport capacity was completely inhibited. Inhibition of MeAIB uptake by vanadate and pV was not blocked by cycloheximide, and oubain did not inhibit uptake. Vanadate also inhibited amino acid deprivation-stimulated MeAIB uptake. Insulin-stimulated MeAIB uptake was also inhibited in rat hepatoma cells. Thus vanadate and pV mimic insulin to stimulate glucose uptake but inhibit system A amino acid uptake. The relative inhibitory concentrations of vanadate and pV suggest that the mechanism may involve PTP inhibition.
Glucocorticosteroids increase sodium transport in middle ear epithelium
Authors: Herman P. , Tan C. T. , van den Abbeele T. , Escoubet B. , Friedlander G. , Huy P. T.
Published: December 2017

The effect of glucocorticosteroids on ion transport was investigated on a middle ear cell line with the short-circuit current (Isc) technique. Dexamethasone (DXM) produced a dose- and time-dependent increase in Isc. Concentration of half-maximal stimulation was 2.68 x 10(-8) M. This effect was blunted by the glucocorticoid antagonist RU-38486 and was related to Na+ transport, as evidenced by the inhibition induced by 1) apical addition of the Na+ channel inhibitor benzamil (10(-6) M) or 2) substitution of Na+ with N-methylglucamine in the incubation medium. The increase in Na+ transport resulted from a primary modulation of apical Na+ entry, since 1) the Na(+)-K(+)-ATPase activity of cellular homogenates was not modified by corticosteroids and 2) the DXM-induced increase in the ouabain-sensitive uptake of 86Rb was blunted by benzamil. Ribonuclease protection assay revealed 1) a constitutive expression of the mRNA encoding the alpha-subunit of the epithelial Na+ channel and 2) that DXM increased the expression of this transcript. This increase was dose dependent and paralleled changes in transepithelial Na+ transport. This study suggests that a component of the beneficial effect of steroid therapy for the treatment of otitis media might be related to increased fluid clearance.
Secondary regulatory volume increase conferred on Xenopus oocytes by expression of AE2 anion exchanger
Authors: Jiang L. , Chernova M. N. , Alper S. L.
Published: December 2017

Xenopus oocytes lack volume regulation and Cl/anion-exchange (AE) activity but express endogenous Na+/H+ exchange (NHE). We postulated that expression in oocytes of heterologous anion exchangers might allow regulatory volume increase (RVI) via functional coupling with endogenous NHE. Expression of neither erythroid nor kidney isoforms of AE1 conferred any form of RVI. In contrast, although AE2 expression did not confer primary RVI, it did confer on oocytes secondary RVI, with a requirement for hypotonic swelling before hypertonic shrinkage. This secondary RVI required extracellular Cl- and Na+, was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and amiloride, was bumetanide insensitive, and was blocked by prevention of intracellular alkalinization, all properties consistent with functional coupling of AE2-mediated Cl-/HCO3- exchange and endogenous NHE. RVI was unaffected by CO2-HCO3- or by partial oocyte Cl- depletion and was unrelated to the rate of oocyte shrinkage. Prior hypotonic swelling did not significantly alter subsequent hypertonic stimulation of AE2-mediated 36Cl influx or efflux. We conclude that heterologous AE2 expression suffices to confer volume regulation on Xenopus oocytes that lack intrinsic volume-regulatory mechanisms.
Reduced Mg2+ inhibition of Ca2+ release in muscle fibers of pigs susceptible to malignant hyperthermia
Authors: Owen V. J. , Taske N. L. , Lamb G. D.
Published: December 2017

The inhibitory effect of myoplasmic Mg2+ on Ca2+ release from the sarcoplasmic reticulum (SR) was examined in mechanically skinned skeletal muscle fibers from pigs of different ryanodine-receptor (RyR) genotypes. In fibers from pigs homozygous for the normal RyR allele, the free Mg2+ concentration ([Mg2+]) had to be lowered from the normal resting level of 1 to approximately 0.1 mM to induce Ca2+ release and a force response. Fibers from pigs heterozygous or homozygous for the RyR allele associated with malignant hyperthermia (MH) needed only a smaller reduction in free [Mg2+] to induce Ca2+ release (reduction to 0.1-0.2 and > or = 0.2 mM, respectively). Dantrolene (20 microM) counteracted the effect of this reduced Mg2+ inhibition in MH muscle. The response of muscle fiber bundles to the caffeine-halothane contracture test in the three genotypes correlated well with the responsiveness of single fibers to reduced [Mg2+]. Thus the abnormal responsiveness of MH muscle to various stimuli may largely result from the reduced ability of myoplasmic Mg2+ to inhibit Ca2+ release from the SR.
Volume-sensitive chloride current in pigmented ciliary epithelial cells: role of phospholipases
Authors: Mitchell C. H. , Zhang J. J. , Wang L. , Jacob T. J.
Published: December 2017

The whole cell recording technique was used to examine an outwardly rectifying chloride current activated by hypotonic shock in bovine pigmented ciliary epithelial (PCE) cells. Removal of internal and external Ca2+ did not affect the activation of these currents, but they were abolished by the phospholipase C inhibitor neomycin. The current was blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, and 4,4'-disothiocyanostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner, but tamoxifen, dideoxyforskolin, and quinidine did not affect it. This blocking profile differs from that of the volume-sensitive chloride channel in neighboring nonpigmented ciliary epithelial cells (Wu, J., J. J. Zhang, H. Koppel, and T. J. C. Jacob, J. Physiol, Lond. 491: 743-755, 1996), and this difference implies that the volume responses of the two cell types are mediated by different chloride channels (Jacob, T. J. C., and J. J. Zhang. J. Physiol. Lond. In press). Intracellular administration of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to PCE cells induced a transient, time-independent, outwardly rectifying chloride current that closely resembled the current activated by hypotonic shock. DIDS produced a voltage-dependent block of the GTP gamma S-activated current similar to the block of the hypotonically activated current. Intracellular neomycin completely prevented activation of this current as did incubation of the cells in calphostin C. and inhibitor of protein kinase C (PKC). Removal of Ca2+ did not affect activation of the current by GTP gamma S but extended the duration of the response. Inhibition of phospholipase A2 (PLA2) with p-bromophenacyl bromide prevented the activation of the hypotonically induced current and also inhibited the current once activated by hypotonic solution. The findings imply that the hypotonic response in PCE cells is mediated by both phospholipase C (PLC) and PLA2. Both phospholipases generate arachidonic acid, and, in addition, the PLC pathway regulates the PLA2 pathway via a PKC-dependent phosphorylation of PLA2.
Association of Tyr phosphorylation of GTPase-activating protein with mitogenic action of serotonin
Authors: Lee S. L. , Wang W. W. , Fanburg B. L.
Published: December 2017

We have previously shown that serotonin (5-HT) induces both hyperplasia and hypertrophy of pulmonary artery smooth muscle cells (SMC) but not of endothelial cells (EC) through its high-affinity uptake. The present studies demonstrate rapid enhancement by 5-HT of Tyr phosphorylation of proteins, including p120, which also occurs in SMC but not in EC. The p120 protein was identified as GTPase-activating protein (GAP) by immunoprecipitation. Its phosphorylation occurred within minutes and preceded other events associated with 5-HT-induced mitogenesis. Tyr kinase (TK) and 5-HT uptake inhibitors and 8-bromoadenosine 3',5'-cyclic monophosphate blocked both the 5-HT-induced DNA synthesis and Tyr phosphorylation of GAP. Vanadate elevated DNA synthesis and Tyr phosphorylation of GAP of both control and 5-HT-treated cells. 5-HT failed to alter Tyr phosphorylation of GAP in cellular homogenates, as opposed to intact cells. In the presence of 3-isobutyl-1-methylxanthine, 5-HT inhibited cellular growth, presumably through its action on 5-HT1A or 5-HT4 receptors and elevation of adenosine 3',5'-cyclic monophosphate, but this was not associated with an alteration of Tyr phosphorylation of GAP. Similarly, a 5-HT1 or 5-HT2 receptor agonist failed to stimulate Tyr phosphorylation or DNA synthesis of SMC. Stimulation of cellular proliferation and enlargement produced by 1 microM 5-HT were totally abolished by TK inhibitors that did not affect 5-HT uptake. These data indicate that Tyr phosphorylation of GAP may act as an intermediate signal in 5-HT-induced mitogenesis of SMC which requires cellular internalization of 5-HT rather than its action on a membrane receptor.
Functional properties of cultured endothelial cells derived from large microvessels of human brain
Authors: Spatz M. , Kawai N. , Merkel N. , Bembry J. , McCarron R. M.
Published: December 2017

This report describes the fractional separation of microvessels from human brain for establishment of segmentally derived endothelial cell (EC) cultures. The investigation comprised evaluation of media constituents and purity of the cell culture and focused on functional biochemical characterization of endothelium derived from large microvessels (EC) Cells contained endothelial marker factor VIII (von Willebrand antigen), secreted endothelin-1 (ET-1) and prostaglandins, and took up 86Rb+ as a measure of K+. Exogenous ET-1 stimulated phosphatidylinositol hydrolysis and K+ uptake; BQ-123 (selective ETA receptor antagonist) but not IRL-1038 or BQ-788 (selective ETB receptor antagonists) inhibited both. Ouabain (inhibitor of Na(+)-K(+)-ATPase) and bumetanide (inhibitor of Na(+)-K(+)-Cl- cotransport) reduced (74-80 and 20-40%, respectively) the ET-1-stimulated K+ uptake. Staurosporine [protein kinase C (PKC) inhibitor] selectively reduced Na(+)-K(+)-Cl- cotransport, whereas verapamil but not nifedipine (L-type voltage-dependent Ca2+ channel blockers) decreased Na(+)-K(+)-ATPase activity induced by ET-1. Phorbol 12-myristate 13-acetate (PMA; activator of PKC) stimulated K+ uptake, which was only decreased with bumetanide. N-ethylisopropylamiloride (inhibitor of Na+/H+ exchange) reduced the ET-1-stimulated but not the PMA-induced K+ uptake. Results indicate that phosphatidylinositol hydrolysis and ion transport systems in large microvascular EC are stimulated by ET-1 through activation of ETA receptors. The findings also suggest that the ET-1-stimulated Na(+)-K(+)-ATPase activity, in contrast to Na(+)-K(+)-Cl- cotransport, is not mediated by PKC. In addition, the data suggest a linkage between Na(+)-K(+)-ATPase activity and Na+/H+ exchange.
Protection from apoptosis in human neutrophils is determined by the surface of adhesion
Authors: Ginis I. , Faller D. V.
Published: December 2017

Recent work suggests that various neutrophil agonists affect the rate of apoptosis in these cells. On the basis of these observations, we hypothesized that signals triggered in neutrophils via their adhesion receptors might also modify their life span. This hypothesis has been tested using human neutrophils adherent to tissue culture plastic, either untreated or coated with extracellular matrix (ECM) proteins or with monolayers of human umbilical vein endothelial cells. To detect and quantitate apoptotic changes in adherent cells, we developed a microtiter plate assay using a cell-permeable DNA-binding fluorescent dye, Hoechst 33342. Use of this assay demonstrated that 1) the number of apoptotic cells among neutrophils adherent to plastic after 6-20 h of incubation was significantly lower than that among neutrophils adherent to the ECM proteins fibronectin or laminin; 2) adhesion to interleukin-1-activated endothelial cells delayed apoptosis, whereas adhesion to nonactivated endothelium accelerated neutrophil death; and 3) monoclonal antibodies directed against intercellular adhesion molecule 1 or against the common beta 2-chain of the leukocyte integrins abolished the protective effect of interleukin-1-activated endothelial cells on apoptosis of adherent neutrophils. These results suggest that the life span of adherent neutrophils. depends on the activating signals triggered by the surface of adhesion.
Effect of the organic Ca2+ channel blocker D-600 on sarcoplasmic reticulum Ca2+ uptake in skeletal muscle
Authors: Ortega A. , Gonzalez-Serratos H. , Lepock J. R.
Published: December 2017

Experiments were undertaken to study the possibility that the calcium channel blocker D-600 (gallopamil), which penetrates into muscle cells (20), facilitates excitation-contraction coupling in skeletal muscle (7) by a direct effect on the sarcoplasmic reticulum (SR). The effects of D-600 were studied on single phasic muscle fibers, either intact or split open. D-600 potentiated twitches in intact fibers at concentrations lower than those reported in whole muscles. In split fibers, the force produced by caffeine-induced Ca2+ release from the SR was reversibly inhibited by 5 microM D-600, when added to the Ca2+ loading solution. This inhibitory effect was inversely related to temperature, and it was dose dependent. When D-600 was added after Ca2+ loading and before caffeine exposure, or during the caffeine exposure itself, it did not inhibit Ca2+ release, but rather increased the development of force. We conclude that, apart from the blocking effect that D-600 may have on the voltage sensor, the drug penetrates into the myoplasm and affects excitation-contraction coupling by inhibiting the SR Ca2+ pump. This may be the consequence of a conformational change in the transmembrane Ca2+ binding domain of the ATPase.
BAY K 8644 and ClO4- potentiate caffeine contracture without Ca2+ release channel activation
Authors: Oba T. , Koshita M. , Aoki T. , Yamaguchi M.
Published: December 2017

Effects of perchlorate (ClO4-) and BAY K 8644 on caffeine contracture and Ca2+ release channel current were studied in frog skeletal muscle. Single fibers produced a small transient contracture on addition of 2.2 mM caffeine. ClO4 at 10 mM enhanced caffeine contracture 3.7-fold. This effect was inhibited by 10 microM nifedipine pretreatment. An increase in caffeine contracture was also obtained after exposure to 0.1 microM BAY K 8644 for 1 h. At 20 mM, external K+ potentiated caffeine contracture 2.2-fold. ClO4- (< 10 mM) and BAY K 8644 (0.1-1 microM) did not affect open probability (Po), unitary conductance, and open and closed time constants of the Ca2+ release channel current. BAY K 8644 at 0.1 microM did not further enhance the channel that had been activated by 2 mM caffeine. However, 20-30 mM ClO4 increased Po significantly and led the channel to a long open state by increasing the slow open time constant and decreasing the fast closed time constant. These results suggest that binding of ClO4 and BAY K 8644 to dihydropyridine receptors elicits a further increase in Ca2+ release from the sarcoplasmic reticulum.
Induction of heme oxygenase-1 (HSP32) mRNA in skeletal muscle following contractions
Authors: Essig D. A. , Borger D. R. , Jackson D. A.
Published: December 2017

The capacity of preexisting antioxidant pathways to handle oxidative stress during exercise may be complemented by the synthesis of inducible heat stress proteins (HSP). Our purpose was to determine if the amount of mRNA for HSP32, a major oxidative stress protein, was increased in muscle after repetitive contractions. Reverse transcriptase-polymerase chain reaction analysis showed that HSP32 mRNA (normalized to alpha-actin mRNA) was increased about seven- and about fourfold (P < 0.35) immediately after 1 h of exhaustive running and after 3 h of muscle contractions (10 Hz nerve stimulation), respectively. Northern blot analysis revealed that HSP70 mRNAs were 3.5- to 15.5-fold above control value (P < 0.05), whereas the content of another oxidative stress protein mRNA (macrophage stress protein 23) was unchanged 0 h after contractions. The relative increase in HSP32 mRNA was found to be dependent on active tension generation; passive tension did not increase the HSP32-to-actin mRNA ratio. Increases in HSP32 mRNA may underlie an inducible antioxidant pathway in muscle responsive to metabolic stresses associated with repeated muscle contractions.
Protein kinase A activity modulates natriuretic peptide-dependent cGMP accumulation in renal cells
Authors: Ledoux S. , Dussaule J. C. , Chatziantoniou C. , Ardaillou N. , Vandermeersch S. , Ardaillou R.
Published: December 2017

The purpose of this work was to examine whether the level of cAMP accumulation and protein kinase A (PKA) activity influence atrial natriuretic factor (ANF)-dependent guanosine 3',5'-cyclic monophosphate (cGMP) production in two renal cell types: rabbit cortical vascular smooth muscle cells (RCSMC) and SV-40-transformed human glomerular visceral epithelial cells (HGVEC-SV1). N-[2-(p-bromocinnamylamino)ethyl]- 5-isoquinolinesulfonamide (H-89), a PKA inhibitor, decreased ANF-stimulated cGMP production in RCSMC in a time- and concentration-dependent manner. ANF-stimulated cGMP production was markedly inhibited after prolonged 9- and 18-h incubations with 25 microM H-89 (52 and 65%, respectively) but was not altered after exposure of cells to this agent for 1 h. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine and N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide, protein kinase inhibitors not selective for PKA, did not reproduce the effect of H-89, even at higher concentrations (50 and 100 microM). Cycloheximide (10 microM), a protein synthesis inhibitor, limited the inhibitory effect of H-89, although alone it did not modify the ANF-stimulated cGMP production. H-89 did not affect cGMP production when it was stimulated by SIN-1, a nitric oxide donor. Prolonged incubation (18 h) with 8-bromo cAMP or cholera toxin, an activator of Gs protein resulting in adenylate cyclase stimulation, enhanced ANF-dependent cGMP production by 225 and 176%, respectively. This stimulatory effect was blocked by 25 microM H-89. 125I-ANF binding to RCSMC at 4 degrees C was not affected by preincubation of the cells with H-89. There was a 44% decrease in the expression of ANF C receptors measured as the ANF-(4-23)-displaceable 125I-ANF binding at 37 degrees C, which could not, however, explain the inhibitory effect of H-89 on cGMP production. Modulation of ANF- and C-type natriuretic peptide-dependent cGMP production by H-89 and cholera toxin was also found in HGVEC-SV1 with the same characteristics as in RCSMC. Taken together, these results suggest that PKA activity controls the function of natriuretic peptide guanylate cyclase-coupled receptors in the two cell types studied. PKA-dependent inhibition of a negatively regulatory protein distinct from the receptor itself seems necessary for a full cGMP response.
L antigens of sheep red blood cell membranes and modulation of ion transport
Authors: Dunham P. B. , Blostein R.
Published: December 2017

Sheep are polymorphic with respect to the intracellular Na+ and K+ concentrations of their erythrocytes. Erythrocytes of sheep of the high-K+ (HK) phenotype have high K+ and low Na+ concentrations; erythrocytes from sheep of the allelic low-K+ (LK) phenotype have abnormally low K+ and high Na+ concentrations. The difference is due to differences in rates of cation transport: higher Na+-K+ pump flux in HK cells and higher K+-Cl- cotransport in LK cells. The HK/LK polymorphism is associated with a polymorphism of red blood cell antigens: the L antigen is only on LK cells, and HK cells have only the M antigen. There are two classes of L antigen that assort together: Lp, which is associated with Na+-K+ pumps, and Ll, which is associated with K+-Cl- cotransporters. There are functional consequences of these associations: anti-Lp antibody stimulates the pump and anti-Ll antibody inhibits cotransport. The use of these antibodies has permitted delineation of the roles of the antigens in modulating the function of the transporters. In this review, we summarize the evidence that these antigens are entities distinct from the pump. The Lp antigen reacts reversibly with the Na+-K+ pump; the antigen inhibits the pump, mainly by promoting nonspecific inhibition by intracellular K+. The antigen also modulates pump differentiation in immature cells. In contrast, the Ll antigen stimulates K+-Cl- cotransport. The evidence suggests that the two polymorphisms are controlled by a single genetic locus and that all of the distinct properties of ion transporters in LK cells are attributable to interactions with L antigens.
Interaction of apo E-containing lipoproteins with the LDL receptor-related protein LRP
Authors: Kuchenhoff A. , Harrach-Ruprecht B. , Robenek H.
Published: December 2017

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional cell surface receptor that interacts with apolipoprotein E (apo E)-rich lipoproteins, and alpha2-macroglobulin (alpha2-M) in the activated state (alpha2-M*). Whether LRP is a physiologically relevant lipoprotein receptor for naturally occurring apo E-rich lipoproteins, however, is still under discussion. To address this question, we isolated beta-migrating very low density lipoprotein (beta-VLDL) from rabbits by using gel filtration chromatography. Biochemical analysis of beta-VLDL subfractions demonstrated that we isolated apo E- and cholesterol-rich triglycerides with differences in composition and size. Binding and uptake characteristics of beta-VLDL subfractions and alpha2-M* on mouse peritoneal macrophages (MPM) and Hep G2 cells were examined by electron microscopy. One of the beta-VLDL subfractions, beta-VLDL(II), bound specifically to LRP on MPM and Hep G2. beta-VLDL(II) competed with the binding of alpha2-M* without addition of exogenous apo E. Furthermore, binding and uptake of beta-VLDL(II) and alpha2-M* were not affected by either lactoferrin or Ca2+-free medium. The results indicate that naturally occurring apo E-rich lipoproteins do exist and that they very likely interact with LRP via the same binding site as alpha2-M*.
T cell lymphokines modulate bFGF-induced smooth muscle cell fibrinolysis and migration
Authors: Wang W. , Chen H. J. , Schwartz A. , Cannon P. J. , Rabbani L. E.
Published: December 2017

Smooth muscle cell (SMC) fibrinolysis is necessary for SMC migration. To determine whether the T cell lymphokines interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) modulate SMC fibrinolysis and migration induced by basic fibroblast growth factor (bFGF), we examined the effects of IL-4 and IFN-gamma on human SMC tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (UPA), and plasminogen activator inhibitor 1 (PAI-1) antigen production, determined by enzyme-linked immunosorbent assays. Although IL-4 had no effects on SMC tPA, UPA, and PAI-1 production, it potentiated bFGF-induced tPA, UPA, and PAI-1 antigens. IL-4 plus bFGF resulted in a net increase in SMC fibrinolytic activity. IFN-gamma did not significantly affect bFGF induction of SMC tPA and PAI-1 antigens. However, IFN-gamma significantly decreased bFGF-mediated induction of SMC UPA antigen. IFN-gamma decreased the IL-4 plus bFGF induction of both tPA and UPA antigens. IL-4 increased and IFN-gamma abrogated bFGF induction of in vitro SMC migration through a modified micro-Boyden chamber. Therefore, IL-4 and IFN-gamma modulate bFGF-mediated induction of in vitro vascular SMC fibrinolysis and migration.
Cyclic ADP-ribose signaling in sea urchin gametes: metabolism in spermatozoa
Authors: Chini E. N. , Thompson M. A. , Chini C. C. , Dousa T. P.
Published: December 2017

The molecular mechanism that initiates Ca2+ signaling in sea urchin egg fertilization has not yet been clarified. To determine whether sea urchin sperm may generate and possibly supply cyclic ADP-ribose (cADPR) as a Ca2+-releasing factor in the course of sea urchin egg fertilization, we determined cADPR content and the capacity for cADPR synthesis in sea urchin sperm. cADPR content was determined using the sea urchin egg homogenate Ca2+-release bioassay combined with high-performance liquid chromatography (HPLC). We found that sperm homogenates synthesized cADPR from beta-NAD but did not synthesize cADPR when alpha-NAD was the substrate. The identity of cADPR generated by sperm homogenates was verified by HPLC analysis, use of specific Ca2+-release antagonists, and homologous desensitization of the sea urchin egg homogenate Ca2+-release bioassay. The ambient content of cADPR was approximately 0.3 nmol cADPR/g wet wt sea urchin sperm. Our results show that sperm can synthesize cADPR and that they contain cADPR levels comparable to other tissues.
Loss of plasma membrane structural support in ATP-depleted renal epithelia
Authors: Doctor R. B. , Zhelev D. V. , Mandel L. J.
Published: December 2017

Renal ischemia induces cytoskeletal alterations, membrane perturbations, including bleb formation, and ultimately membrane lysis. The mechanisms that underlie these alterations are largely unknown. Through the use of isolated rat renal proximal tubule fragments and calibrated micropipette techniques, two potential mechanisms for membrane bleb formation during ATP depletion were examined: 1) decreased cytoskeletal retention of the plasma membrane and 2) increased intracellular pressure. Under control conditions, the pressure required to pull the membrane from the underlying cellular matrix was 73 +/- 10 kdyn/cm2. After 30 min of ATP depletion, this pressure was diminished by >95% and blebs began to emerge from the basal membrane. The intracellular pressure within these blebbed cells was only 0.08 +/- 0.02 kdyn/cm2. These observations indicate that, during ATP depletion, the strength of membrane retention diminished until the relatively low intracellular pressure was capable of driving membrane bleb formation. Cytochalasin D, which disrupts the actin cytoskeleton, decreased the strength of membrane retention by 65 +/- 7%. This suggests that, during ATP depletion, alterations of the actin cytoskeleton may mediate the loss of membrane retention.
Regulation of ATP synthase subunit e gene expression by hypoxia: cell differentiation stage-specific control
Authors: Levy F. H. , Kelly D. P.
Published: December 2017

Using the technique of differential display, we identified genes that are expressed differentially under normoxic and hypoxic conditions. One regulated gene encoded subunit e of mitochondrial F1F0-ATP synthase (subunit e). The hypoxia-mediated regulation of subunit e expression in C2C12 cells was influenced by the stage of cellular differentiation. Under normoxic conditions, subunit e expression was markedly upregulated during the transition from myoblast to myotube. After exposure to hypoxia for 24 h, subunit e mRNA expression markedly decreased (>70%) in C2C12 myotubes. In contrast, subunit e mRNA levels increased slightly in response to hypoxia in C2C12 myoblasts. Studies performed with primary rat cardiocytes demonstrated that expression of subunit e mRNA and a cardiac-enriched related transcript was downregulated after a hypoxic exposure. We conclude that expression of subunit e is regulated, at the pretranslational level, by oxygen availability via cell differentiation stage-specific mechanisms consistent with the proposed regulatory role of this protein in cellular ATP production.
cAMP activates an ATP-conductive pathway in cultured shark rectal gland cells
Authors: Cantiello H. F. , Jackson G. R. , Prat A. G. , Gazley J. L. , Forrest J. N. , Ausiello D. A.
Published: December 2017

The molecular mechanisms associated with ATP transport and release into the extracellular milieu are largely unknown. To assess the presence of endogenous ATP-conductive pathway(s) in shark rectal gland (SRG) cells, patch-clamp techniques were applied to primary cultures of SRG cells. Whole cell currents were obtained with either intracellular tris(hydroxymethyl)aminomethane (Tris) or Mg2+ salts of ATP (200 mM nominal ATP) and 280 mM NaCl bathing solution. Basal currents showed a sizable ATP permeability for outward movement of MgATP. Adenosine 3',5'-cyclic monophosphate (cAMP) stimulation significantly increased the whole cell conductance (with either intracellular Tris-ATP or MgATP). Symmetrical whole cell ATP currents were also observed after cAMP activation, thus consistent with ATP as the main charge carrier. The cAMP-inducible ATP currents were insensitive to the Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, diphenylamine-2-carboxylate, and anthracene-9-carboxylic acid but were readily blocked by nifedipine (400 microM) and glibenclamide (400 microM). The nature of the electrodiffusional ATP movement was further assessed by single-channel analysis of either MgATP or Tris-ATP currents in excised inside-out patches, both spontaneous and after activation with protein kinase A. Single-channel ATP currents were inhibited by either nifedipine or glibenclamide. Thus SRG cells express endogenous ATP-permeable pathways both before and after cAMP stimulation. Electrodiffusional ATP movement by SRG cells may play a significant role in the transport and delivery of cellular ATP to the extracellular milieu, which may help coordinate the dynamics of the epithelial secretory response in this cell model.
Acidosis has no effect on the ATP cost of contraction in cat fast- and slow-twitch skeletal muscles
Authors: Harkema S. J. , Adams G. R. , Meyer R. A.
Published: December 2017

Studies of skinned fibers suggest that the rate of ATP turnover in skeletal muscle is depressed by acidosis. To examine whether this occurs in intact muscles, the ATP cost of isometric contractions was measured in ex vivo, arterially perfused cat biceps (predominantly fast-twitch) and soleus (slow-twitch) muscles under normocapnic (5% CO2) and hypercapnic (70% CO2) conditions. Hypercapnia decreased extracellular pH from 7.4 to 6.7 and intracellular pH from 7.1 to 6.5 (soleus) or 6.6 (biceps) but had no significant effect on the phosphocreatine (PCr)-to-ATP ratio in muscles at rest. The ATP cost of contraction was estimated from PCr changes, measured by gating the acquisition of 31P-nuclear magnetic resonance spectra to times before and after brief tetani (1 s at 100 Hz and 2 s at 25 Hz for biceps and soleus, respectively) or 10-s trains of twitches (2 and 1 Hz, respectively). Peak isometric force and the ATP cost of tetanic contraction (PCr/force x time integral) were not significantly different under hypercapnic compared with normocapnic conditions in either muscle (mean: 7.97 and 2.44 micromol x kg(-1) x s(-1) for biceps and soleus, respectively). Twitch force and the ATP cost per twitch decreased by nearly 50% during hypercapnic perfusion in both muscle types. The results indicate that hypercapnic acidosis has no significant effect on the ATPase rate per active myosin head in intact mammalian skeletal muscle.
Noninvasive measurement of phosphocreatine recovery kinetics in single human muscles
Authors: Walter G. , Vandenborne K. , McCully K. K. , Leigh J. S.
Published: December 2017

The rate at which phosphocreatine (PCr) is resynthesized after exercise is related to muscle oxidative capacity (Vmax). With the use of a one-dimensional image-guided, localized nuclear magnetic resonance spectroscopy technique, PCr kinetics were monitored in the medial gastrocnemius of eight healthy subjects after voluntary, short duration, maximal rate exercise. Localized spectra were obtained every 6 s with <5% contamination from nonselected regions. Maximal rate exercise elicited near-maximal to maximal muscle activation, as indicated by the high-PCr hydrolysis rate (2.26 +/- 0.07 mM/s) and extensive PCr depletion. At the end of 9 s of maximal rate exercise, PCr was depleted by 61.4 +/- 2.4% and intracellular pH was 7.04 +/- 0.03. After 9 s of maximal rate exercise, PCr recovered with a rate constant (kPCr) of 1.87 +/- 0.15 min(-1) and a Vmax of 67.2 +/- 6.0 mM/min. Independent of prior activity, aerobic ATP synthesis rates reached 48.6 +/- 4.9 mM/min within 9 s. Extending maximal rate exercise to 30 s resulted in 92.0 +/- 1.2% PCr depletion and an intracellular pH of 6.45 +/- 0.07. The intracellular acidosis separated the direct relationship between kPCr and muscle Vmax but did not affect the initial PCr resynthesis rate.
Endothelial cell-conditioned medium downregulates smooth muscle contractile protein expression
Authors: Vernon S. M. , Campos M. J. , Haystead T. , Thompson M. M. , DiCorleto P. E. , Owens G. K.
Published: December 2017

Smooth muscle cells (SMC) within atherosclerotic lesions proliferate and exhibit phenotypic modulation, but the contribution of vascular endothelium to this process is poorly understood. Our aim was to examine the effects of endothelial cell-conditioned medium (ECCM) on vascular SMC growth and differentiation. Rat aortic ECCM stimulated a ninefold increase in [3H]thymidine incorporation and downregulated smooth muscle-specific myosin heavy chain and alpha-actin synthesis in rat aortic SMC. These effects were not inhibited by antibodies to platelet-derived growth factor (PDGF)-BB or PDGF-AB or with a PDGF beta-receptor subunit. Treatment with PDGF-BB (at a concentration found in ECCM), PDGF-AA, basic fibroblast growth factor, endothelin-1, or transforming growth factor-beta did not reproduce these effects. The ECCM activities were sensitive to heat and trypsinization, were >30 kDa in molecular mass, and bound weakly to heparin-Sepharose. Our data indicate that cultured endothelial cells produce a factor(s) that downregulates contractile protein expression in SMC, which may contribute to SMC dedifferentiation and proliferation.
Mechanism of modulation of the voltage-gated skeletal and cardiac muscle sodium channels by fatty acids
Authors: Bendahhou S. , Cummins T. R. , Agnew W. S.
Published: December 2017

Voltage-gated rat skeletal muscle and cardiac Na+ channels are modulated by exogenous unsaturated fatty acids. Application of 1-10 microM arachidonic or oleic acids reversibly depressed Na+ channel conductance and shifted the inactivation curve to hyperpolarizing potentials. These effects were not prevented by inhibitors of lipoxygenase, cyclooxygenase, cytochrome P-450 epoxygenase, or protein kinase C. Neither palmitic acid nor methyl ester oleate had an effect on the inward Na+ current, suggesting that trivial variations in membrane fluidity are not responsible for the Na+ current depression or kinetic changes. Arachidonic acid altered fast Na+ inactivation without changing the slow inactivation kinetics. Moreover, skeletal muscle Na+ channel gating currents were markedly decreased by 2 microM arachidonic acid. Finally, nonstationary noise analysis indicated that both the number of channels and the open probability were slightly decreased without change in the single-channel conductance. These data suggest that unsaturated fatty acids such as arachidonic and oleic acids 1) specifically regulate voltage-gated Na+ channels and 2) interact directly with Na+ channels, perhaps at a fatty acid binding domain, by decreasing the total gating charge and altering fast-inactivation kinetics.