bluuebunny/crossref_metadata_2025 huggingface dataset:
Bursting behavior during fixed-delay stimulation of spontaneously beating chick heart cell aggregates
Authors: Kunysz A. M. , Shrier A. , Glass L.
Published: December 2017

Spontaneously beating embryonic chick atrial heart cell aggregates were stimulated with depolarizing current pulses delivered at a fixed delay after each action potential. This preparation is an experimental model of a reentrant tachycardia. During fixed-delay stimulation, bursting behavior was typically observed for a wide range of delays. Episodes of bursting at a rate faster (slower) than control were followed by overdrive suppression (underdrive acceleration). We use a simple nonlinear model, based on the interaction between excitability and overdrive suppression, to describe these dynamics. A modified version of the Shrier-Clay ionic model of electrical activity of the embryonic chick heart cell aggregates that includes a simplified Na+ pump term is also presented. We show that the complex patterns during fixed-delay stimulation arise as a result of delicate interactions between overdrive suppression and phase resetting, which can be described in terms of the underlying ionic mechanisms. This study may provide a basis for understanding incessant tachycardias in the intact heart, as well as an alternative mechanism for the emergence of bursting activity in other biologic tissue.
Force decline during muscle relaxation promotes calcium release to the cytosol
Authors: Baker A. J. , Weiner M. W.
Published: December 2017

During relaxation of skeletal muscle, an initial rapid decline of intracellular Ca2+ concentration ([Ca2+]i) (phase 1), is followed by a brief phase in which the decline of [Ca2+]i is markedly slowed or even reversed (phase 2). Phase 2 appears as a prominent "bump" on records of the time course of declining [Ca2+]i during relaxation. The goal of this study was to test the hypothesis that phase 2 represents a release of Ca2+ to the cytosol that occurs with net cross-bridge detachment during relaxation. The experimental approach was to measure [Ca2+]i with indo 1 in stimulated bullfrog semitendinosus muscles and to determine if phase 2 was diminished during relaxation of contractions in which cross-bridge interactions had been reduced by two different methods: 1) stretching muscles to reduce the overlap between actin and myosin filaments or 2) decreasing stimulus duration. The results showed that, when either method was used to reduce cross-bridge interactions during contraction, then the size of phase 2 during relaxation was also decreased. Phase 2 was eliminated during relaxation of contractions in which cross-bridge interactions had been reduced to a lower contraction force approximately 30% of maximum. These findings are consistent with the hypothesis that the phase 2 of [Ca2+]i decline represents a release of Ca2+ to the cytosol that occurs with net cross-bridge detachment during relaxation. This conclusion is consistent with previous studies that suggest that cross-bridge detachment lowers the affinity of troponin for Ca2+.
Role of extracellular signal-regulated kinase and PKC, in cytosolic PLA2 activation by bradykinin in MDCK-D1 cells
Authors: Xing M. , Tao L. , Insel P. A.
Published: December 2017

Pages C1360–C1387: M. Xing, L. Tao, and P. A. Insel. “Role of extracellular signal-regulated kinase and PKC, in cytosolic PLA2 activation by bradykinin in MDCK-D1 cells.” Page C1380, line 27 from the top of the Abstract, there is a typographical error: the middle initial of P. A. Insel in the cited reference (C. Godson et al.) should be “A” not “S” and should read “C. Godson, K. S. Bell, and P. A. Insel. J. Biol. Chem. 268: 11946–11950, 1993.”
Minimal model of beta-cell mitochondrial Ca2+ handling
Authors: Magnus G. , Keizer J.
Published: December 2017

We develop a simplified, but useful, mathematical model to describe Ca2+ handling by mitochondria in the pancreatic beta-cell. The model includes the following six transport mechanisms in the inner mitochondrial membrane: proton pumping via respiration and proton uptake by way of the F1Fzero-ATPase (adapted from D. Pietrobon and S. Caplan. Biochemistry 24: 5764-5778, 1985), a proton leak, adenine nucleotide exchange, the Ca2+ uniporter, and Na+/Ca2+ exchange. Each mechanism is developed separately into a kinetic model for the rate of transport, with parameters taken from experiments on isolated mitochondrial preparations. These mechanisms are combined in a modular fashion first to describe state 4 (nonphosphorylating) and state 3 (phosphorylating) mitochondria with mitochondrial NADH and Ca2+ concentrations as fixed parameters and then to describe Ca2+ handling with variable mitochondrial Ca2+ concentration. Simulations are compared to experimental measurements and agree well with the threshold for Ca2+ uptake, measured mitochondrial Ca2+ levels, and the influence of Ca2+ on oxygen uptake. In the absence of Ca2+ activation of mitochondrial dehydrogenases, the simulations predict a significant reduction in the rate of production of ATP that involves a “short circuit” via Ca2+ uptake through the uniporter. This effect suggests a potential role for mitochondrial Ca2+ handling in determining the ATP-ADP ratio in the pancreatic beta-cell.
Oxygen dependence of mitochondrial function measured by high-resolution respirometry in long-term hypoxic rats
Authors: Costa L. E. , Mendez G. , Boveris A.
Published: December 2017

Respiration and oxidative phosphorylation were investigated in tightly coupled mitochondria isolated from liver and heart of rats submitted to a simulated altitude of 4,400 m for 14-15 mo and their corresponding controls at sea level. High-resolution respirometry was utilized to determine the apparent Michaelis-Menten constant for ADP and O2 (K(m)-ADP and K(m)-O2, respectively), the latter under active and resting states of mitochondrial respiration. The K(m)-O2 in mitochondria isolated from normoxic rats was higher for active (state 3) than for resting (state 4) respiration; the values decreased from 1.5 and 1.7 to 0.25 and 0.30 microM in heart and liver mitochondria, respectively. The K(m)-O2 values found in the active state suggest a role for the normally occurring intracellular PO2 range reported in the literature in the regulation of cellular respiration. No changes were found in the ADP or O2 dependence of respiration in the mitochondria isolated from long-term acclimatized rats compared with their controls, indicating that the intrinsic properties and the efficiency of mitochondria do not change as a consequence of adaptation to hypoxia.
Chloride channels in primary cultures of seawater fish (Dicentrarchus labrax) gill
Authors: Duranton C. , Tauc M. , Avella M. , Poujeol P.
Published: December 2017

Patch-clamp experiments were undertaken on primary cultures of respiratory cells originating from sea bass (Dicentrarchus labrax) gills. A small-conductance Cl- channel of 8 pS was characterized in cell-attached configuration with 140 mM N-methyl-D-glucamine-Cl in the pipette and bath solutions. No activity was recorded below a membrane holding potential of +20 mV (-Vp, referenced to the pipette solution), and the channel showed an inward rectification. In the inside-out configuration the Cl- channel was active at all membrane holding potentials. Its open probability strongly increased with membrane depolarization. The channel activity could be increased by the application of protein kinase A+ATP. This channel was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoic acid, diphenylamino-2-carboxylic acid, and I- and was insensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The presence of Cl- channels in the apical membrane of respiratory cells provides additional evidence for an important role of this cell type in the control of ion homeostasis of seawater fish.
PKA-mediated phosphorylation and inhibition of Na(+)-K(+)-ATPase in response to beta-adrenergic hormone
Authors: Cheng X. J. , Fisone G. , Aizman O. , Aizman R. , Levenson R. , Greengard P. , Aperia A.
Published: December 2017

The activity of Na(+)-K(+)-ATPase can be regulated by hormones that activate adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA). Here, using a site-directed phosphorylation state-specific antibody, we show that hormonal regulation of Na(+)-K(+)-ATPase can occur via phosphorylation of Ser-943 on its alpha-subunit. cDNAs coding for wild-type rat Na(+)-K(+)-ATPase and Na(+)-K(+)-ATPase in which the PKA phosphorylation site Ser-943 was mutated to Ala were stably and transiently transfected into COS cells. In COS cells expressing wild-type Na(+)-K(+)-ATPase the beta-adrenergic agonist isoproterenol (1 microM) significantly increased the level of phosphorylation of the alpha-subunit. Phosphorylation was accompanied by a significant inhibition of the enzyme activity, as reflected by a decrease in ATP hydrolysis and 86Rb+ transport. The effect of isoproterenol was reproduced by the PKA activator forskolin used in combination with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine and was abolished by the specific PKA inhibitor H-89. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A, enhanced phosphorylation and inhibition of Na(+)-K(+)-ATPase induced by isoproterenol. The changes in activity of Na(+)-K(+)-ATPase linearly correlated with the extent of the alpha-subunit of Na(+)-K(+)-ATPase being phosphorylated. When Ser-943 was replaced by alanine, stimulation of the phosphorylation and inhibition of the activity of Na(+)-K(+)-ATPase induced by isoproterenol, alone or in combination with okadaic acid, were not observed. These results indicate that, in intact cells, modulation of the activity of Na(+)-K(+)-ATPase can be achieved by regulation of the state of phosphorylation of Ser-943. Moreover, they provide a biochemical mechanism by which beta-adrenergic agonists can regulate Na(+)-K(+)-ATPase activity.
Hypertonic induction of the cell adhesion molecule beta 1-integrin in MDCK cells
Authors: Sheikh-Hamad D. , Suki W. N. , Zhao W.
Published: December 2017

Cells of many organisms adapt to osmotic stress by accumulating compatible organic osmolytes. In Madin-Darby canine kidney (MDCK) cells, a renal epithelial cell line widely used as a culture model for the study of osmotic regulation in mammals, extracellular hypertonicity induces genes responsible for the accumulation of organic osmolytes. We have recently cloned from these cells a partial cDNA corresponding to a member of the transmembrane 4 superfamily (tetraspan) family, CD9 antigen, and demonstrated its induction by hypertonicity [D. Sheikh-Hamad, J. D. Ferraris, J. Dragolovich, H. G. Preuss, M. B. Burg, and A. Garcia-Perez. Am. J. Physiol. 270 (Cell Physiol. 39): C253-C258, 1996]. An association between CD9 and potential regulatory proteins, such as the heparin-binding epidermal growth factor-like protein and the cell adhesion molecule beta 1-integrin, has been reported. Here, using beta 1-integrin-specific monoclonal antibodies and an antisense oligonucleotide probe, we demonstrate expression and hypertonic induction of beta 1-integrin in MDCK cells. Induction of the mRNA and protein occurs in 2 h and is maximal at 6 h, consistent with a regulatory role in the adaptation to osmotic stress. In addition, we show that accumulation of organic osmolytes markedly attenuates the hypertonic induction of the mRNA, a feature shared with genes involved in hyperosmotic stress response. Finally, we demonstrate that CD9 and beta 1-integrin are expressed in association at the cell membrane. Our findings suggest the existence of a cluster of integral membrane proteins that includes but may not be limited to CD9 and the adhesion molecule beta 1-integrin, which may play a role in the adaptation of kidney cells to osmotic stress, possibly at the regulatory level.
Differential activation of PKC-delta and -zeta by alpha 1-adrenergic stimulation in human airway epithelial cells
Authors: Liedtke C. M. , Cole T. , Ikebe M.
Published: December 2017

Phorbol ester and alpha 1-adrenergic stimulation of Na-Cl-K cotransport in human tracheal epithelial cells was investigated by determining the expression of protein kinase C (PKC) isotypes and their activation by phorbol 12-myristate 13-acetate (PMA) and methoxamine, an alpha 1-adrenergic agonist. PKC-alpha, -beta II, -delta, -epsilon, and -zeta were expressed in confluent cell cultures. PKC-beta I, -gamma, and -eta were not detected. PKC-alpha and -zeta were localized to the cytosol, and PKC-beta II and -delta were distributed approximately evenly between cytosolic and particulate fractions. Treatment with PMA for 30 min increased PKC activity in subcellular fractions and induced a redistribution of PKC-beta II and -delta to a particulate fraction. PMA treatment for 18 h reduced PKC activity to levels found in untreated cells and reduced, but did not deplete, PKC isotype mass. Methoxamine transiently increased PKC activity, with maximal levels at 40 s, and caused a shift in PKC-delta and -zeta mass to a particulate fraction. Methoxamine selectively induced a sustained increase in PKC-zeta activity but only a transient increase in PKC-delta. These results suggest that PKC-delta and -zeta mediate hormonal activation of Na-Cl-K cotransport.
Expression of dopamine D2 receptor in PC-12 cells and regulation of membrane conductances by dopamine
Authors: Zhu Wylie H. , Conforti Laura , Millhorn David E.
Published: December 2017

PC-12 cells depolarize during hypoxia and release dopamine. The hypoxia-induced depolarization is due to inhibition of an O2-sensitive K+ current. The role of dopamine released during hypoxia is uncertain, but it could act as an autocrine to modulate membrane conductance during hypoxia. The current study was undertaken to investigate this possibility. Reverse transcription-polymerase chain reaction and sequence analysis revealed that the D2 isoform of the dopamine receptor is expressed in rat PC-12 cells. Exogenously applied dopamine and the D2agonist quinpirole elicited inhibition of a voltage-dependent K+ current ( I K) that was prevented by sulpiride, a D2 receptor antagonist. Dopamine and quinpirole applied during hypoxia potentiated the inhibitory effect of hypoxia on I K. We also found that quinpirole caused reversible inhibition of a voltage-dependent Ca2+ current ( I Ca) and attenuation of the increase in intracellular free Ca2+ during hypoxia. Our results indicate that dopamine released from PC-12 cells during hypoxia acts via a D2 receptor to “autoregulate” I K and I Ca.
Hyaluronic acid-specific regulation of cytokines by human uterine fibroblasts
Authors: Kobayashi Hiroshi , Terao Toshihiko
Published: December 2017

The physiological inflammatory response can provide an effective mechanism for delivering the baby at the time of parturition. We characterized the mechanisms by which hyaluronic acid (HA) regulates interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-8 (IL-8) production in human uterine fibroblasts. A dose-dependent increase in cytokine release was observed over an HA concentration range of 10 μg/ml to 1 mg/ml. The action of HA on the cytokine production is mediated by CD44. Under serum-free conditions, HA-induced cytokine generation was significantly less compared with production in the presence of serum, suggesting involvement of serum proteins. Addition of inter-α-trypsin inhibitor (ITI) under serum-free conditions enhanced the HA-induced synthesis of TNF-α, which stimulated the temporary release of IL-8. In addition, HA and IL-1β stimulated the release of hyaluronidase by the fibroblasts. These results indicate that cytokine production in human uterine fibroblasts is regulated in a CD44-HA-ITI-specific fashion. HA may be involved in the regulation of delivery in part through the selective release of cytokines that contribute to uterine cervical ripening.
Partial cloning and characterization of Slc12a2: the gene encoding the secretory Na+-K+-2Cl−cotransporter
Authors: Randall Jeffrey , Thorne Tina , Delpire Eric
Published: December 2017

The Slc12a2 gene encodes a widely expressed bumetanide-sensitive Na+-K+-2Cl−cotransporter that participates in various functions such as Cl− secretion and cell volume regulation. We isolated and characterized 75 kilobases of the murine gene encoding the cotransporter. The cotransport protein is encoded by 27 exons. Ribonuclease protection assay and primer extension demonstrated tissue-specific transcription initiation sites located within 270 base pairs upstream of the start codon. Nucleotide sequence analysis of the proximal 5′-flanking region revealed the presence of a weak TATA box, multiple Sp1/GC consensus sites, and the consensus sequence of a putative transcriptional initiator. Transfection of luciferase reporter gene constructs in mouse inner medullary collecting duct (mIMCD-3) cells confirmed the location of the minimal promoter within a 120-base pair fragment upstream of the cDNA. We also report the identification of an alternatively spliced variant of the cotransporter, expressed primarily in brain. This new spliced variant lacks exon 21, which encodes a 16-amino acid peptide located in the COOH-terminal tail of the protein. The absence of this exon causes the loss of the single protein kinase A consensus site of the cotransport protein.
The Ca2+-sensing receptor: a target for polyamines
Authors: Quinn Stephen J. , Ye Chian-Ping , Diaz Rubin , Kifor Olga , Bai Mei , Vassilev Peter , Brown Edward
Published: December 2017

The Ca2+-sensing receptor (CaR) is activated at physiological levels of external Ca2+(Cao) but is expressed in a number of tissues that do not have well-established roles in the control of Cao, including several regions of the brain and the intestine. Polyamines are endogenous polyvalent cations that can act as agonists for the CaR, as shown by our current studies of human embryonic kidney (HEK-293) cells transfected with the human CaR. Cellular parameters altered by polyamines included cytosolic free Ca2+(Cai), inositol phosphate production, and the activity of a nonselective cation channel. Spermine stimulated Cai transients in CaR-transfected HEK cells, with a concentration producing a half-maximal response (EC50) of ∼500 μM in the presence of 0.5 mM Ca2+, whereas sustained increases in Cai had an EC50 of ∼200 μM. The order of potency was spermine > spermidine >> putrescine. Elevation of Cao shifted the EC50 for spermine sharply to the left, with substantial stimulation below 100 μM. Addition of subthreshold concentrations of spermine increased the sensitivity of CaR-expressing HEK cells to Cao. Parathyroid hormone secretion from bovine parathyroid cells was inhibited by 50% in the presence of 200 μM spermine, a response similar to that elicited by 2.0 mM Cao. These data suggest that polyamines could be effective agonists for the CaR, and several tissues, including the brain, may use the CaR as a target for the actions of spermine and other endogenous polycationic agonists.
Cholecystokinin and EGF activate a MAPK cascade by different mechanisms in rat pancreatic acinar cells
Authors: Dabrowski Andrzej , Groblewski Guy E. , Schäfer Claus , Guan Kun-Liang , Williams John A.
Published: December 2017

The effects of activating the Gqprotein-coupled cholecystokinin (CCK) receptor on different proteins/signaling molecules in the mitogen-activated protein kinase (MAPK) cascade in pancreatic acinar cells were analyzed and compared with the effects of activating the tyrosine kinase-coupled epidermal growth factor (EGF) receptor. Both EGF and CCK octapeptide rapidly increased the activity of the MAPKs [extracellular signal-regulated kinase (ERK) 1 and ERK2], reaching a maximum within 2.5 min when 3.9- and 8.5-fold increases, respectively, were observed. The EGF-induced increase of MAPK activity was transient, with only a slight elevation after 30 min, whereas CCK-stimulated MAPK remained at a high level of activation to 60 min. The protein kinase C inhibitor GF-109203X abolished the activation by phorbol ester and inhibited the effect of CCK by 78% but had no effect on EGF-activated MAPK activity. EGF and CCK activated both forms of MAPK kinase (MEK), with CCK having a much larger effect, activating MEK1 by 6-fold and MEK2 by 10-fold, whereas EGF activated both MEKs by only 2-fold. Immunoblotting revealed three different forms of Raf in pancreatic acinar cells. Of the total basal Raf kinase activity, 3.7% was Raf-A, 89.0% was Raf-B, and 7.3% was c-Raf-1. All three forms of Raf were stimulated to a greater extent by CCK than by EGF, which was especially evident for Raf-A and c-Raf-1. The effect of CCK in activating Rafs was at least partially mimicked by stimulation with the phorbol ester 12- O-tetradecanoylphorbol-13-acetate. EGF significantly increased GTP-bound Ras by 183 and 164% at 2.5 and 10 min, respectively; CCK and TPA had no measurable effect. Our study suggests that CCK and EGF activate the MAPK cascade by distinct mechanisms in pancreatic acinar cells.
P2 purinoceptor of the globular substance in the otoconial membrane of the guinea pig inner ear
Authors: Suzuki Hideaki , Ikeda Katsuhisa , Furukawa Masayuki , Takasaka Tomonori
Published: December 2017

The biological characteristics of the globular substance, a precursor of otoconia, are unclear. In the present study, the ATP-induced internal free Ca2+ concentration ([Ca2+]i) changes of the globular substance and the ATP distribution in the vestibular organ were investigated using a Ca2+ indicator, fluo 3, and an adenine nucleotide-specific fluorochrome, quinacrine, by means of confocal laser scanning microscopy. [Ca2+]i showed a rapid and dose-dependent increase in response to ATP with a 50% effective concentration (EC50) of 16.7 μM. This reaction was independent of external Ca2+, indicating the presence of an internal Ca2+ reservoir. Neither adenosine, α,β-methylene-ATP, 3′- O-(4-benzoylbenzoyl)-ATP, ADP, nor UTP evoked this reaction, whereas 2-methylthio-ATP induced an increase of [Ca2+]i with an EC50 of 14.4 μM. Moreover, P2 antagonists, reactive blue 2 and suramin, and a phospholipase C inhibitor, U-73122, inhibited the ATP-induced [Ca2+]i increase. These findings indicate the presence of a P2Y purinoceptor on the globular substance. In addition, granular fluorescence was observed in the quinacrine-stained macular sensory epithelium, indicating the presence of ATP-containing granules in this tissue. These results suggest that a paracrine mechanism involving ATP may exist in the macula and that this mechanism regulates the biological behavior of the globular substance.
Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat
Authors: Nielsen Søren , King Landon S. , Christensen Birgitte Mønster , Agre Peter
Published: December 2017

The molecular pathways for fluid transport in pulmonary, oral, and nasal tissues are still unresolved. Here we use immunocytochemistry and immunoelectron microscopy to define the sites of expression of four aquaporins in the respiratory tract and glandular epithelia, where they reside in distinct, nonoverlapping sites. Aquaporin-1 (AQP1) is present in apical and basolateral membranes of bronchial, tracheal, and nasopharyngeal vascular endothelium and fibroblasts. AQP5 is localized to the apical plasma membrane of type I pneumocytes and the apical plasma membranes of secretory epithelium in upper airway and salivary glands. In contrast, AQP3 is present in basal cells of tracheal and nasopharyngeal epithelium and is abundant in basolateral membranes of surface epithelial cells of nasal conchus. AQP4 resides in basolateral membranes of columnar cells of bronchial, tracheal, and nasopharyngeal epithelium; in nasal conchus AQP4 is restricted to basolateral membranes of a subset of intra- and subepithelial glands. These sites of expression suggest that transalveolar water movement, modulation of airway surface liquid, air humidification, and generation of nasopharyngeal secretions involve a coordinated network of aquaporin water channels.
Intermediate endocrine-acinar pancreatic cells in duct ligation conditions
Authors: Bertelli Eugenio , Bendayan Moïse
Published: December 2017

When tissues were subjected to 24 h of duct ligation, intermediate pancreatic cells simultaneously displaying endocrine and exocrine phenotypes appeared. Immunocytochemistry by laser scanning confocal microscopy revealed the appearance of a large number of these cells coexpressing insulin and amylase. These cells were located within the islets of Langerhans as well as in the acinar parenchyma. They were also detected in a culture system of isolated pancreatic cells. With the use of immunoelectron microscopy, two types of secretory granules were identified in these cells. One was insulin immunoreactive, whereas the other, resembling zymogen granules, contained amylase. Occasionally, some small granules displayed a double labeling for both secretory proteins. Numerous crinophagic bodies and autophagosomes containing insulin and/or amylase were also present. In situ hybridization, applied with the specific probes, confirmed the presence of both insulin and amylase mRNAs in these cells. Because duct ligation is known to induce insulin cell proliferation, the present results confirm that endocrine-acinar cells do appear in such condition and may represent intermediate steps in a transdifferentiating process.
Regulation of endothelin-1 gene expression by cell shape and the microfilament network in vascular endothelium
Authors: Malek Adel Moussa , Lee Ike W. , Alper Seth L. , Izumo Seigo
Published: December 2017

Endothelial synthesis and release of endothelin-1 (ET-1) are exquisitely regulated by external shear and strain. We tested the hypothesis that manipulation of endothelial cell shape can regulate ET-1 gene expression. Treatment of bovine aortic endothelial cell (BAEC) monolayers with cytochalasin D disrupted F-actin and induced cell retraction and rounding, in parallel with time- and dose-dependent specific decreases in ET-1 mRNA levels. Treatments with forskolin, phorbol 12-myristate 13-acetate, staurosporine, and genistein also induced cell shape change and decreased F-actin staining and ET-1 mRNA levels. BAEC plated onto nonadhesive petri dishes coated with decreasing concentrations of synthetic RGD polymer showed RGD dose-dependent decreases in cell spreading and in F-actin microfilament elaboration. These changes were specifically accompanied by decreases in ET-1 peptide secretion (60%) and, via posttranscriptional mechanisms, ET-1 mRNA (94%) and were not due to decreased cell-cell contact. We conclude that the shape and microfilament network of endothelial cells are potent posttranscriptional regulators of ET-1 gene expression.
Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes
Authors: Qian Ting , Nieminen Anna-Liisa , Herman Brian , Lemasters John J.
Published: December 2017

To simulate ischemia and reperfusion, cultured rat hepatocytes were incubated in anoxic buffer at pH 6.2 for 4 h and reoxygenated at pH 7.4. During anoxia, intracellular pH (pHi) decreased to 6.3, mitochondria depolarized, and ATP decreased to <1% of basal values, but the mitochondrial permeability transition (MPT) did not occur as assessed by confocal microscopy from the redistribution of cytosolic calcein into mitochondria. Moreover, cell viability remained >90%. After reperfusion at pH 7.4, pHi returned to pH 7.2, the MPT occurred, and most hepatocytes lost viability. In contrast, after reperfusion at pH 6.2 or with Na+-free buffer at pH 7.4, pHi did not rise and cell viability remained >80%. After acidotic reperfusion, the MPT did not occur. When hepatocytes were reperfused with cyclosporin A (0.5–1 μM) at pH 7.4, the MPT was prevented and cell viability remained >80%, although pHi increased to 7.2. Reperfusion with glycine (5 mM) also prevented cell killing but did not block recovery of pHi or the MPT. Retention of cell viability was associated with recovery of 30–40% of ATP. In conclusion, preventing the rise of pHi after reperfusion blocked the MPT, improved ATP recovery, and prevented cell death. Cyclosporin A also prevented cell killing by blocking the MPT without blocking recovery of pHi. Glycine prevented cell killing but did not inhibit recovery of pHi or the MPT.
P2Z purinoceptor-associated pores induced by extracellular ATP in macrophages and J774 cells
Authors: Coutinho-Silva Robson , Persechini Pedro Muanis
Published: December 2017

Millimolar concentrations of extracellular ATP (ATPo) can induce the permeabilization of plasma membranes of macrophages and other bone marrow-derived cells to low-molecular-weight solutes, a phenomenon that is the hallmark of P2Z purinoceptors. However, patch-clamp and whole cell electrophysiological experiments have so far failed to demonstrate the existence of any ATPo-induced P2Z-associated pores underlying this permeabilization phenomenon. Here, we describe ATPo-induced pores of 409 ± 33 pS recorded using cell-attached patch-clamp experiments performed in macrophages and J774 cells. These pores are voltage dependent and display several properties of the P2Z-associated permeabilization phenomenon: they are permeable to both large cations and anions, such as tris(hydroxymethyl)aminomethane, N-methyl-d-glucamine, and glutamate; their opening is favored at temperatures higher than 30°C; they are blocked by oxidized ATP and Mg2+; and they can be triggered by 3′- O-(4-benzoylbenzoyl)-ATP but not by UTP or ADP. We conclude that the pores described in this report are associated with the P2Z permeabilization phenomenon.
Synchronized Ca2+ signaling by intercellular propagation of Ca2+ action potentials in NRK fibroblasts
Authors: De Roos Albert D. G. , Willems Peter H. G. M. , Van Zoelen Everardus J. J. , Theuvenet Alexander P. R.
Published: December 2017

The intercellular propagation of Ca2+waves by diffusion of inositol trisphosphate has been shown to be a general mechanism by which nonexcitable cells communicate. Here, we show that monolayers of normal rat kidney (NRK) fibroblasts behave like a typical excitable tissue. In confluent monolayers of these cells, Ca2+ action potentials can be generated by local depolarization of the monolayer on treatment with either bradykinin or an elevation of the extracellular K+ concentration. These electrotonically propagating action potentials travel intercellularly over long distances in an all-or-none fashion at a speed of ∼6.1 mm/s and can be blocked by L-type Ca2+ channel blockers. The action potentials are generated by depolarizations beyond the threshold value for L-type Ca2+ channels of about −15 mV. The result of these locally induced, propagating Ca2+ action potentials is an almost synchronous, transient increase in the intracellular Ca2+ concentration in large numbers of cells. These data show that electrically coupled fibroblasts can form an excitable syncytium, and they elucidate a novel mechanism of intercellular Ca2+ signaling in these cells that may coordinate synchronized multicellular responses to local stimuli.
Amidative peptide processing and vascular function
Authors: Oldham Charlie D. , Li Cuizhen , Feng Jun , Scott Robert O. , Wang Wen Z. , Moore Allison B. , Girard Peggy R. , Huang Jianzhong , Caldwell Ruth B. , Caldwell R. William , May Sheldon W.
Published: December 2017

Substance P (SP), an amidated peptide present in many sensory nerves, is known to affect cardiovascular function, and exogenously supplied SP has been shown to activate nitric oxide synthase (NOS) in endothelial cells. We now report that SP-Gly, the glycine-extended biosynthetic precursor of SP (which is enzymatically processed to the mature amidated SP), causes relaxation of rat aortic strips with an efficacy and potency comparable to that of SP itself. Pretreatment of the aortic strips with 4-phenyl-3-butenoic acid (PBA), an irreversible amidating enzyme inactivator, results in marked inhibition of the vasodilation activity induced by SP-Gly but not of that induced by SP itself. Isolated endothelial cell basal NOS activity is also decreased by pretreatment with PBA, with no evidence of cell death or direct action of PBA on NOS activity. Both bifunctional and monofunctional forms of amidating enzymes are present in endothelial cells, as evidenced by affinity chromatography and Western blot analysis. These results provide evidence for a link between amidative peptide processing, NOS activation in endothelial cells, and vasodilation and suggest that a product of amidative processing provides intrinsic basal activation of NOS in endothelial cells.
Differences in regulation of Ca2+-activated Cl− channels in colonic and parotid secretory cells
Authors: Arreola Jorge , Melvin James E. , Begenisich Ted
Published: December 2017

We investigated the regulation of Ca2+-activated Cl− channels in cells from the human colonic cell line T84 and acinar cells from rat parotid glands. The participation of multifunctional Ca2+- and calmodulin-dependent protein kinase (CaM kinase) II in the activation of these channels was studied using selective inhibitors of calmodulin and CaM kinase II. Ca2+-dependent Cl− currents were recorded using the whole cell patch-clamp technique. Direct inhibition of CaM kinase II by 40 μM peptide 281–302 or by 10 μM KN-62, another CaM kinase inhibitor, did not block the Cl− current in parotid acinar cells, whereas in T84 cells KN-62 markedly inhibited the Ca2+-dependent Cl− current. We also used the calmodulin-binding domain peptide 290–309 (0.5 μM), which competitively inhibits the activation of CaM kinase II. This peptide reduced the Cl− current in T84 cells by ∼70% but was without effect on the channels in parotid acinar cells. We conclude that the Ca2+-dependent Cl− channels in T84 cells are activated by CaM kinase II but that the channels in parotid acinar cells must be regulated by a fundamentally different Ca2+-dependent mechanism that does not utilize CaM kinase II or any calmodulin-dependent process.
MDR1 in taste buds of rat vallate papilla: functional, immunohistochemical, and biochemical evidence
Authors: Jakob Ingrid , Hauser Ingeborg A. , Thévenod Frank , Lindemann Bernd
Published: December 2017

Multidrug resistance P-glycoprotein (MDR1) is a membrane protein of 150–170 kDa that catalyzes the ATP-driven efflux of hydrophobic xenobiotics, including fluorescent dyes, from cells. Expressed in many epithelial tissues and in the endothelia of the blood-brain barrier, the MDR1 protein provides major routes of detoxification. We found that taste cells of the rat vallate papilla (VP; posterior tongue) had only a slow increase in fluorescence due to uptake of the hydrophobic dye calcein acetoxymethyl ester. However, the development of fluorescence was accelerated two- to threefold by substrates and/or inhibitors of MDR1, such as verapamil, tamoxifen, and cyclosporin A, and by addition of the transport-blocking antibody to MDR1, UIC2. Western blots of vallate tissue rich in taste buds with the MDR1-specific monoclonal antibodies C219 and C494 revealed an immunoreactive protein at ∼170 kDa. In contrast, the lingual epithelium surrounding the VP showed a much weaker band with these antibodies. Furthermore, using the antibodies C494 and UIC2 with tissue sections, MDR1-like immunoreactivity was found in taste cells. These results show that MDR1 is present and functional in vallate taste cells of the rat. MDR1-related transport may achieve active elimination of xenobiotics from the sensory cells and thereby protect the peripheral taste organs from potentially harmful molecules contained in an animal’s food.
Angiotensin II stimulates the production of NO and peroxynitrite in endothelial cells
Authors: Pueyo Maria E. , Arnal Jean-François , Rami Jacques , Michel Jean-Baptiste
Published: December 2017

Angiotensin II (ANG II) produces vasoconstriction by a direct action on smooth muscle cells via AT1 receptors. These receptors are also present in the endothelium, but their function is poorly understood. This study was therefore undertaken to determine whether ANG II elicits the release of nitric oxide (NO) from cultured rat aortic endothelial cells. NO production, measured by the accumulation of nitrite and nitrate, was enhanced by 10−7 M ANG II. The biological activity of the NO released by ANG II action was evaluated by measuring its guanylate cyclase-stimulating activity in smooth muscle cells. The guanosine 3′,5′-cyclic monophosphate (cGMP) content of smooth muscle cells was significantly increased by exposure of supernatant from ANG II-stimulated endothelial cells. These effects resulted from the activation of NO synthase, as they were inhibited by the l-arginine analogs. These ANG II actions were mediated by the AT1 receptor, as shown by their inhibition by the AT1 antagonist losartan. The cGMP production by reporter cells was inhibited by the calmodulin antagonist W-7, suggesting that ANG II activates endothelial calmodulin-dependent NO synthase. This hypothesis is also supported by the increase of intracellular free calcium induced by ANG II in endothelial cells. ANG II also stimulated luminol-enhanced chemiluminescence in endothelial cells. This effect was inhibited by N ω-monomethyl-l-arginine and superoxide dismutase, suggesting that this luminol-enhanced chemiluminescence reflected an increase in peroxynitrite production. Thus ANG II stimulates NO release from macrovascular endothelium, which may modulate the direct vasoconstrictor effect of ANG II on smooth muscle cells. However, this beneficial effect may be counteracted by the simultaneous production of peroxynitrite, which could contribute to several pathological processes in the vascular wall.
Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopusoocytes
Authors: Kumar Chandira K. , Nguyen Toai T. , Gonzales Francis B. , Said Hamid M.
Published: December 2017

We recently identified a cDNA clone from mouse small intestine, which appears to be involved in folate transport when expressed in Xenopus oocytes. The open reading frame of this clone is identical to that of the reduced folate carrier (RFC) (K. H. Dixon, B. C. Lanpher, J. Chiu, K. Kelley, and K. H. Cowan. J. Biol. Chem. 269: 17–20, 1994). The characteristics of this cDNA clone [previously referred to as intestinal folate carrier 1 (IFC-1)] expressed in Xenopus oocytes, however, were found to be different from the characteristics of folate transport in native small intestinal epithelial cells. To further study these differences, we determined the characteristics of RFC when expressed in an intestinal epithelial cell line, IEC-6, and compared the findings to its characteristics when expressed in Xenopus oocytes. RFC was stably transfected into IEC-6 cells by electroporation; its cRNA was microinjected into Xenopus oocytes. Northern blot analysis of poly(A)+RNA from IEC-6 cells stably transfected with RFC cDNA (IEC-6/RFC) showed a twofold increase in RFC mRNA levels over controls. Similarly, uptake of folic acid and 5-methyltetrahydrofolate (5-MTHF) by IEC-6/RFC was found to be fourfold higher than uptake in control sublines. This increase in folic acid and 5-MTHF uptake was inhibited by treating IEC-6/RFC cells with cholesterol-modified antisense DNA oligonucleotides. The increase in uptake was found to be mainly mediated through an increase in the maximal velocity ( V max) of the uptake process [the apparent Michaelis-Menten constant ( K m) also changed (range was 0.31 to 1.56 μM), but no specific trend was seen]. In both IEC-6/RFC and control sublines, the uptake of both folic acid and 5-MTHF displayed 1) pH dependency, with a higher uptake at acidic pH 5.5 compared with pH 7.5, and 2) inhibition to the same extent by both reduced and oxidized folate derivatives. These characteristics are very similar to those seen in native intestinal epithelial cells. In contrast, RFC expressed in Xenopus oocytes showed 1) higher uptake at neutral and alkaline pH 7.5 compared with acidic pH 5.5 and 2) higher sensitivity to reduced compared with oxidized folate derivatives. Results of these studies demonstrate that the characteristics of RFC vary depending on the cell system in which it is expressed. Furthermore, the results may suggest the involvement of cell- or tissue-specific posttranslational modification(s) and/or the existence of an auxiliary protein that may account for the differences in the characteristics of the intestinal RFC when expressed in Xenopus oocytes compared with when expressed in intestinal epithelial cells.
Continuous binding of the PAF molecule to its receptor is necessary for the long-term aggregation of platelets
Authors: Suzuki Masahiro , Sugatani Junko , Ino Mitsuhiro , Shimura Masahiko , Akiyama Masaki , Yamazaki Ryuta , Suzuki Yasuo , Miwa Masao
Published: December 2017

Human and rabbit platelets fully aggregated by platelet-activating factor (PAF) underwent slow disaggregation but were rapidly disaggregated by the PAF receptor antagonists WEB-2086, Y-24180, SM-12502, and CV-3988. Whereas the 1-alkyl-2-[3H]acetyl- sn-glycero-3-phosphocholine ([3H]acetyl-PAF) specifically bound to platelet receptors underwent slow and spontaneous dissociation, it dissociated promptly from its receptor when WEB-2086 was added, in parallel with platelet disaggregation and disappearance of P-selectin on the cell surface. Extracellular [3H]acetyl-PAF was rapidly deacetylated by normal rabbit platelets; some of the [3H]acetyl-PAF was bound to the cells and a very small amount of [3H]acetate was detected in the cells. In contrast, when 1-[3H]alkyl-2-acetyl- sn-glycero-3-phosphocholine was added to the platelets, the radioactivity was rapidly incorporated into the 1-alkyl-2-acyl- sn-glycero-3-phosphocholine fraction. These results indicate that 1) continuous binding of PAF to its receptor is necessary for prolonged platelet aggregation, which may be mediated through an unknown signaling system for a long-term cell response rather than a transient signaling system, and 2) most of the [3H]acetyl-PAF bound to platelets is metabolized extracellularly by ecto-type PAF acetylhydrolase, with the lyso-PAF generated being incorporated rapidly into the cells and converted to 1-alkyl-2-acyl- sn-glycero-3-phosphocholine.
Hypoxic regulation of endothelial glyceraldehyde-3-phosphate dehydrogenase
Authors: Graven Krista K. , McDonald Robert J. , Farber Harrison W.
Published: December 2017

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced by hypoxia in endothelial cells (EC). To define the mechanisms by which GAPDH is regulated by hypoxia, EC were exposed to cobalt, other transition metals, carbon monoxide (CO), deferoxamine, or cycloheximide in the presence or absence of hypoxia for 24 h, and GAPDH protein and mRNA levels were measured. GAPDH was induced in cells by the transition metals cobalt, nickel, and manganese and by deferoxamine, and GAPDH mRNA induction by hypoxia was blocked by cycloheximide. GAPDH induction by hypoxia, unlike that of other hypoxia-regulated genes, was not inhibited by CO or by 4,6-dioxoheptanoic acid, an inhibitor of heme synthesis. GAPDH induction was not altered by mediators of protein phosphorylation, a calcium channel blocker, a calcium ionophore, or alterations in redox state. GAPDH induction by hypoxia or transitional metals was partially blocked by sodium nitroprusside but was not altered by the inhibitor of nitric oxide synthase N ω-nitro-l-arginine. These findings suggest that GAPDH induction by hypoxia in EC occurs via mechanisms other than those involved in other hypoxia-responsive systems.
Changes in myosin heavy chain mRNA and protein isoforms of rat muscle during forced contractile activity
Authors: Jaschinski Frank , Schuler Michael , Peuker Heidemarie , Pette Dirk
Published: December 2017

A quantitative reverse transcriptase-polymerase chain reaction was established to determine absolute amounts of mRNAs specific to four myosin heavy chain isoforms [MHCIIb, MHCIId(x), MHCIIa, and MHCIβ] in rat extensor digitorum longus muscle during forced contractile activity by chronic (10 h/day) low-frequency stimulation (CLFS). The induced changes in absolute and relative mRNA amounts were similar. MHCIIb mRNA decreased rapidly after 1 day, and MHCIIa mRNA increased after 3 days. MHCIId(x) started to decrease at day 7. After 42 days, the MHCIIb, MHCIId(x), MHCIIa, and MHCIβ mRNAs amounted to 2, 6, 90, and 2% of total MHC mRNAs, respectively. Changes at the protein level were studied in a second experimental series increasing CLFS (24 h/day, up to 100 days). Also under these conditions, MHCIβ reached only a fraction of 12% (2-fold elevation). The changes at the protein level remained restricted to the MHCIIb to MHCIIa transition, which agrees with the notion that the induced changes in MHC isoform expression primarily resulted from altered pretranslational activities. Rat fast-twitch muscle thus exhibits a restricted capacity for fast-to-slow conversion.
Differences in transglutaminase mRNA after polyamine depletion in two cell lines
Authors: Wang Jian-Ying , Viar Mary Jane , Li Ji , Shi Hui-Jun , Patel Anami R. , Johnson Leonard R.
Published: December 2017

Polyamines serve as natural substrates for the transglutaminase that catalyzes covalent cross-linking of proteins and is involved in cellular adhesion and proliferation. This study tests the hypothesis that intracellular polyamines play a role in the regulation of transglutaminase expression in rat small intestinal crypt cells (IEC-6 cell line) and human colon carcinoma cells (Caco-2 cell line). Treatment with α-difluoromethylornithine (DFMO; a specific inhibitor of polyamine synthesis) significantly depleted the cellular polyamines putrescine, spermidine, and spermine in both cell lines. In IEC-6 cells, polyamine depletion was associated with a decrease in the levels of transglutaminase mRNA. In Caco-2 cells, however, polyamine depletion significantly increased the levels of transglutaminase mRNA and enzyme activity. In both cell lines, ornithine decarboxylase mRNA levels increased and protooncogene c- myc mRNA decreased in the presence of DFMO. Addition of polyamines to cells treated with DFMO reversed the effect of DFMO on the levels of mRNA for these genes in both lines. There was no significant change in the stability of transglutaminase mRNA between control and DFMO-treated IEC-6 cells. In contrast, the half-life of mRNA for transglutaminase in Caco-2 cells was dramatically increased after polyamine depletion. Spermidine, when given together with DFMO, completely prevented increased half-life of transglutaminase mRNA in Caco-2 cells. These results indicate that 1) expression of transglutaminase requires polyamines in IEC-6 cells but is inhibited by these agents in Caco-2 cells, 2) polyamines modulate transglutaminase expression at the level of mRNA through different pathways in these two cell lines, and 3) posttranscriptional regulation plays a major role in the induction of transglutaminase mRNA in polyamine-deficient Caco-2 cells.