bluuebunny/crossref_metadata_2025 huggingface dataset:
The human histamine H2 receptor regulates c-jun and c-fos in a differential manner
Authors: Wang L. D. , Wang M. , Todisco A. , Grand E. , del Valle J.
Published: December 2017

Previously, we demonstrated that activation of the human H2 receptor (hH2R) leads to an increase in c- fos transcription and cell proliferation. The purpose of these studies was to examine whether hH2R regulates c- jun expression and, if so, explore the mechanisms by which it does so. Histamine induced an increase in c- jun mRNA in human embryonic kidney cells stably transfected with the hH2R (maximal effect: 554.6 ± 86.8% of control). The protein kinase C (PKC) inhibitors staurosporine (10−6 M) and GF-109203X (10−6 M) significantly inhibited histamine-stimulated c- fos mRNA while not altering c- jun expression. The protein kinase A (PKA) pathway inhibitors Rp-cAMP and protein kinase inhibitor did not affect the action of histamine on c- jun or c- fos mRNA. Histamine (10−4M) stimulated extracellularly regulated kinase 2 tyrosine phosphorylation. The specific inhibitor of the mitogen-activated protein (MAP) kinase pathway, PD-98059 (5 × 10−5 M), significantly inhibited histamine-induced c- fos and c- jun mRNA. Of interest, the p70 S6 kinase inhibitor rapamycin (10−6 M) but not wortmannin decreased histamine-stimulated c- jun mRNA by 58.5 ± 12% (mean ± SE, n = 4) while not significantly altering c- fos message. Histamine (10−4 M) also led to an ∼4.5-fold increase in Jun NH2-terminal kinase activity in a PKC-, PKA-, and MAP kinase-independent but rapamycin-sensitive manner. Our findings suggest that histamine stimulates both c- fos and c- jun mRNA in a differential manner. PKC is involved in histamine-mediated c-fos activation, whereas p70 S6 kinase is important for linkage of this receptor to c- jun.
NO regulates PDGF-induced activation of PKB but not ERK in A7r5 cells: implications for vascular growth arrest
Authors: Sandirasegarane Lakshman , Charles Roger , Bourbon Nicole , Kester Mark
Published: December 2017

In addition to the well-documented role of nitric oxide (NO) as a vasodilator, NO has also been implicated in vascular smooth muscle cell (VSMC) growth arrest. Signaling mechanisms responsible for growth factor receptor-mediated VSMC proliferation include the extracellular signal-regulated kinase (ERK) and possibly the protein kinase B (PKB) cascade. Thus the present study was designed to test the hypothesis that, in A7r5 vascular smooth muscle-derived cells, platelet-derived growth factor (PDGF)-induced activation of either ERK or PKB is regulated by NO, which then modulates cellular proliferation and/or apoptosis. PKB-α was the predominant isoform of PKB expressed in A7r5 cells and was also expressed in rabbit carotid arteries and aortae. Phosphorylation of PKB-α and ERK induced by PDGF-BB was maximal within 5–15 min in A7r5 cells. Preincubation of A7r5 cells with the NO donor S-nitroso- N-acetylpenicillamine (SNAP) resulted in a biphasic regulation of PDGF-stimulated PKB-α phosphorylation and bioactivity. Acute exposure to SNAP significantly augmented PDGF-induced activation of PKB-α, whereas prolonged incubation led to a marked diminution in PDGF-induced activation of PKB-α. In contrast, SNAP did not affect PDGF-induced activation of ERK at any time point. The cGMP-independent effects of SNAP on PDGF-induced activation of PKB-α were established with the use of an inhibitor of soluble guanylyl cyclase, ODQ, as well as a cell-permeable analog of cGMP, 8-bromo-cGMP. Prolonged treatment of A7r5 cells with SNAP led to a significant decrease in DNA synthesis without an appreciable increase in apoptosis. These data suggest that, after prolonged exposure to SNAP, NO selectively attenuates PDGF-induced increase in PKB-α activation, which in turn may contribute to diminished VSMC proliferation by mechanisms involving growth arrest but not apoptosis.
LY-294002-inhibitable PI 3-kinase and regulation of baseline rates of Na+ transport in A6 epithelia
Authors: Păunescu Teodor G. , Blazer-Yost Bonnie L. , Vlahos Chris J. , Helman Sandy I.
Published: December 2017

Blocker-induced noise analysis of epithelial Na+ channels (ENaCs) was used to investigate how inhibition of an LY-294002-sensitive phosphatidylinositol 3-kinase (PI 3-kinase) alters Na+transport in unstimulated and aldosterone-prestimulated A6 epithelia. From baseline Na+ transport rates ( I Na) of 4.0 ± 0.1 (unstimulated) and 9.1 ± 0.9 μA/cm2 (aldosterone), 10 μM LY-294002 caused, following a relatively small initial increase of transport, a completely reversible inhibition of transport within 90 min to 33 ± 6% and 38 ± 2% of respective baseline values. Initial increases of transport could be attributed to increases of channel open probability ( P o) within 5 min to 143 ± 17% (unstimulated) and 142 ± 10% of control (aldosterone) from baseline P o averaging near 0.5. Inhibition of transport was due to much slower decreases of functional channel densities ( N T) to 28 ± 4% (unstimulated) and 35 ± 3% (aldosterone) of control at 90 min. LY-294002 (50 μM) caused larger but completely reversible increases of P o (215 ± 38% of control at 5 min) and more rapid but only slightly larger decreases of N T. Basolateral exposure to LY-294002 induced no detectable effect on transport, P o or N T. We conclude that an LY-294002-sensitive PI 3-kinase plays an important role in regulation of transport by modulating N T and P o of ENaCs, but only when presented to apical surfaces of the cells.
VACM-1, a cullin gene family member, regulates cellular signaling
Authors: Burnatowska-Hledin Maria , Zhao P. , Capps B. , Poel A. , Parmelee K. , Mungall C. , Sharangpani A. , Listenberger L.
Published: December 2017

Vasopressin-activated Ca2+-mobilizing (VACM-1) receptor binds arginine vasopressin (AVP) but does not have amino acid sequence homology with the traditional AVP receptors. VACM-1, however, is homologous with a newly discovered cullin family of proteins that has been implicated in the regulation of cell cycle through the ubiquitin-mediated degradation of cyclin-dependent kinase inhibitors. Because cell cycle processes can be regulated by the transmembrane signal transduction systems, the effects of VACM-1 expression on the Ca2+ and cAMP-dependent signaling pathway were examined in a stable cell line expressing VACM-1 in VACM-1 transfected COS-1 cells and in cells cotransfected with VACM-1 and the adenylyl cyclase-linked V2 AVP receptor cDNAs. Expression of the VACM-1 gene reduced basal as well as forskolin- and AVP-stimulated cAMP production. In cells cotransfected with VACM-1 and the V2 receptor, the AVP- and forskolin-induced increases in adenylyl cyclase activity and cAMP production were inhibited. The inhibitory effect of VACM-1 on cAMP production could be reversed by pretreating cells with staurosporin, a protein kinase A (PKA) inhibitor, or by mutating S730A, the PKA-dependent phosphorylation site in the VACM-1 sequence. The protein kinase C specific inhibitor Gö-6983 further enhanced the inhibitory effect of VACM-1 on AVP-stimulated cAMP production. Finally, AVP stimulatedd- myo-inositol 1,4,5-trisphosphate production both in the transiently transfected COS-1 cells and in the stable cell line expressing VACM-1, but not in the control COS-1 and Chinese hamster ovary cells. Our data demonstrate that VACM-1, the first mammalian cullin protein to be characterized, is involved in the regulation of signaling.
Force relaxes before the fall of cytosolic calcium in the photomechanical response of rat sphincter pupillae
Authors: Krivoshik Andrew P. , Barr Lloyd
Published: December 2017

In the rat sphincter pupillae, as in other smooth muscles, the primary signal transduction cascade for agonist activation is receptor → G protein → phospholipase C → inositol trisphosphate → intracellular Ca2+ concentration ([Ca2+]i) → calmodulin → myosin light chain kinase → phosphorylated myosin → force development. Light stimulation of isolated sphincters pupillae can be very precisely controlled, and precise reproducible photomechanical responses (PMRs) result. This precision makes the PMR ideal for testing models of regulation of smooth muscle myosin phosphorylation. We measured force and [Ca2+]i concurrently in sphincter pupillae following stimulation by light flashes of varying duration and intensity. We sampled at unusually short (0.01–0.02 s) intervals to adequately test a PMR model based on the myosin phosphorylation cascade. We found, surprisingly, contrary to the behavior of intestinal muscle and predictions of the phosphorylation model, that during PMRs force begins to decay while [Ca2+]i is still rising. We conclude that control of contraction in the sphincter pupillae probably involves an inhibitory process as well as activation by [Ca2+]i.
Intracellular Ca2+ stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception
Authors: Vicario I. , Obeso A. , Rocher A. , López-Lopez J. R. , González C.
Published: December 2017

The notion that intracellular Ca2+ (Cai 2+) stores play a significant role in the chemoreception process in chemoreceptor cells of the carotid body (CB) appears in the literature in a recurrent manner. However, the structural identity of the Ca2+ stores and their real significance in the function of chemoreceptor cells are unknown. To assess the functional significance of Cai 2+ stores in chemoreceptor cells, we have monitored 1) the release of catecholamines (CA) from the cells using an in vitro preparation of intact rabbit CB and 2) the intracellular Ca2+ concentration ([Ca2+]i) using isolated chemoreceptor cells; both parameters were measured in the absence or the presence of agents interfering with the storage of Ca2+. We found that threshold [Ca2+]i for high extracellular K+ (Ke +) to elicit a release response is ≈250 nM. Caffeine (10–40 mM), ryanodine (0.5 μM), thapsigargin (0.05–1 μM), and cyclopiazonic acid (10 μM) did not alter the basal or the stimulus (hypoxia, high Ke +)-induced release of CA. The same agents produced Cai 2+transients of amplitude below secretory threshold; ryanodine (0.5 μM), thapsigargin (1 μM), and cyclopiazonic acid (10 μM) did not alter the magnitude or time course of the Cai 2+responses elicited by high Ke +. Several potential activators of the phospholipase C system (bethanechol, ATP, and bradykinin), and thereby of inositol 1,4,5-trisphosphate receptors, produced minimal or no changes in [Ca2+]i and did not affect the basal release of CA. It is concluded that, in the rabbit CB chemoreceptor cells, Cai 2+ stores do not play a significant role in the instant-to-instant chemoreception process.
The effect of rapamycin on single ENaC channel activity and phosphorylation in A6 cells
Authors: Yue Gang , Edinger Robert S. , Bao Hui-Fang , Johnson John P. , Eaton Douglas C.
Published: December 2017

Rapamycin and FK-506 are immunosuppressive drugs that bind a ubiquitous immunophilin, FKBP12, but immunosuppressive mechanisms and side effects appear to be different. Rapamycin binds renal FKBP12 to change renal transport. We used cell-attached patch clamp to examine rapamycin's effect on Na+ channels in A6 cells. Channel NP o was 0.5 ± 0.08 ( n = 6) during the first 5 min but fell close to zero after 20 min. Application of 1 μM rapamycin reactivated Na+ channels ( NP o = 0.47 ± 0.1; n=6), but 1 μM FK-506 did not. Also, GF-109203X, a protein kinase C (PKC) inhibitor, mimicked the rapamycin-induced reactivation in a nonadditive manner. However, rapamycin did not reactivate Na+ channels if cells were exposed to 1 μM FK-506 before rapamycin. In PKC assays, rapamycin was as effective as the PKC inhibitor; however, epithelial Na+ channel (ENaC) phosphorylation was low under baseline conditions and was not altered by PKC inhibitors or activators. These results suggest that rapamycin activates Na+ channels by binding FKBP12 and inhibiting PKC, and, in renal cells, despite binding the same immunophilin, rapamycin and FK-506 activate different intracellular signaling pathways.
Sodium/calcium exchange in amphibian skeletal muscle fibers and isolated transverse tubules
Authors: Cifuentes Fredy , Vergara Julio , Hidalgo Cecilia
Published: December 2017

The Na+/Ca2+ exchanger participates in Ca2+ homeostasis in a variety of cells and has a key role in cardiac muscle physiology. We studied in this work the exchanger of amphibian skeletal muscle, using both isolated inside-out transverse tubule vesicles and single muscle fibers. In vesicles, increasing extravesicular (intracellular) Na+ concentration cooperatively stimulated Ca2+ efflux (reverse mode), with the Hill number equal to 2.8. In contrast to the stimulation of the cardiac exchanger, increasing extravesicular (cytoplasmic) Ca2+ concentration ([Ca2+]) inhibited this reverse activity with an IC50 of 91 nM. Exchanger-mediated currents were measured at 15°C in single fibers voltage clamped at −90 mV. Photolysis of a cytoplasmic caged Ca2+ compound activated an inward current (forward mode) of 23 ± 10 nA ( n = 3), with an average current density of 0.6 μA/μF. External Na+ withdrawal generated an outward current (reverse mode) with an average current density of 0.36 ± 0.17 μA/μF ( n = 6) but produced a minimal increase in cytosolic [Ca2+]. These results suggest that, in skeletal muscle, the main function of the exchanger is to remove Ca2+ from the cells after stimulation.
Physiological functions of the regulatory domains of the cardiac Na+/Ca2+exchanger NCX1
Authors: Pan Yan , Iwamoto Takahiro , Uehara Akira , Nakamura Tomoe Y. , Imanaga Issei , Shigekawa Munekazu
Published: December 2017

Physiological functions of the intracellular regulatory domains of the Na+/Ca2+ exchanger NCX1 were studied by examining Ca2+ handling in CCL39 cells expressing a low-affinity Ca2+ regulatory site mutant (D447V/D498I), an exchanger inhibitory peptide (XIP) region mutant displaying no Na+ inactivation (XIP-4YW), or a mutant lacking most of the central cytoplasmic loop (Δ246–672). We found that D447V/D498I was unable to efficiently extrude Ca2+ from the cytoplasm, particularly during a small rise in intracellular Ca2+concentration induced by the physiological agonist α-thrombin or thapsigargin. The same mutant took up Ca2+ much less efficiently than the wild-type NCX1 in Na+-free medium when transfectants were not loaded with Na+, although it appeared to take up Ca2+ normally in transfectants preloaded with Na+. XIP-4YW and, to a lesser extent, Δ246–672, but not NCX1 and D447V/D498I, markedly accelerated the loss of viability of Na+-loaded transfectants. Furthermore, XIP-4YW was not activated by phorbol ester, whereas XIP-4YW and D447V/D498I were resistant to inhibition by ATP depletion. The results suggest that these regulatory domains play important roles in the physiological and pathological Ca2+ handling by NCX1, as well as in the regulation of NCX1 by protein kinase C or ATP depletion.
Molecular markers expressed in cultured and freshly isolated interstitial cells of Cajal
Authors: Epperson Anne , Hatton William J. , Callaghan Brid , Doherty Philip , Walker Rebecca L. , Sanders Kenton M. , Ward Sean M. , Horowitz Burton
Published: December 2017

Located within the tunica muscularis of the gastrointestinal (GI) tract are networks of cells known as interstitial cells of Cajal (ICC). ICC are critical for important basic functions of GI motility such as generation and propagation of slow-wave pacemaker activity and reception of regulatory inputs from the enteric nervous system. We have developed a novel procedure to identify and isolate individual ICC from freshly dispersed cell preparations of the murine small intestine and gastric fundus and to determine differential transcriptional expression We have compared the expression profiles of pacemaker ICC isolated from the murine small intestine (IC-MY) and ICC involved in neurotransmission from the gastric fundus (IC-IM). We have also compared expression profiles between ICC and smooth muscle cells (SMC) and between freshly isolated ICC and cultured ICC. Cultured ICC express smooth muscle myosin, whereas freshly dispersed ICC do not. All cell types express muscarinic receptor types M2 and M3, neurokinin receptors NK1 and NK3, and inhibitory receptor VIP-1, whereas only cultured ICC and SMC express VIP-2. Both cultured and freshly dispersed IC-IM and IC-MY express the soluble form of stem cell factor, whereas SMC from the gastric fundus express only the membrane-bound form.
Potassium channels in basolateral membrane vesicles fromNecturus enterocytes: stretch and ATP sensitivity
Authors: Dubinsky William P. , Mayorga-Wark Otilia , Schultz Stanley G.
Published: December 2017

We have previously reported that ATP-inhibitable K+channels, in vesicles derived from the basolateral membrane of Necturus maculosus small intestinal cells, exhibit volume regulatory responses that resemble those found in the intact tissue after exposure to anisotonic solutions. We now report that increases in K+ channel activity can also be elicited by exposure of these vesicles to isotonic solutions containing glucose or alanine that equilibrate across these membranes. We also demonstrate that swelling after exposure to a hypotonic solution or an isotonic solution containing alanine or glucose reduces inhibition of channel activity by ATP and that this finding cannot be simply attributed to dilution of intravesicular ATP. We conclude that ATP-sensitive, stretch-activated K+ channels may be responsible for the well-established increase in basolateral membrane K+ conductance of Necturus small intestinal cells after the addition of sugars or amino acids to the solution perfusing the mucosal surface, and we propose that increases in cell volume, resulting in membrane stretch, decreases the sensitivity of these channels to ATP.
Functional cross talk after activation of P2 and P1 receptors in oviductal ciliated cells
Authors: Morales Bernardo , Barrera Nelson , Uribe Pablo , Mora Claudio , Villalón Manuel
Published: December 2017

The presence of ATP and adenosine receptors and their role in controlling ciliary activity in oviductal ciliated cells was studied by measuring the ciliary beat frequency (CBF) in oviductal tissue cultures. ATP, adenosine, and related compounds increased the CBF in a dose-dependent manner. We established that P2 receptors of subtype 2Y2 and P1 receptors of subtype A2a mediated the responses to ATP and adenosine, respectively. We found evidence to suggest that stimulation of ciliary activity by ATP requires d- myo-inositol 1,4,5-trisphosphate [Ins(1,4,5) P 3] metabolism, intracellular Ca2+ mobilization, and protein kinase C activation. On the other hand, the adenosine effect is mediated by activation of a Gs protein-dependent pathway that enhances cAMP intracellular levels. To study the interaction between P2 and P1 receptors, cells were stimulated simultaneously with both agonists. We observed a synergistic increase of the CBF even at agonist concentrations (100 nM) that did not produce a significant response when added separately to the culture. Furthermore, a blocker of the cAMP pathway produced a reduction of the ATP response, whereas a blocker of the Ins(1,4,5) P 3 pathway also produced an inhibition of the adenosine response. Our evidence demonstrates that both ATP and adenosine receptors are present in a single ciliated cell and that a mechanism of cross talk could operate in the transduction pathways to control ciliary activity.
Differences in regulation of pHi in large (≥10 nuclei) and small (≤5 nuclei) osteoclasts
Authors: Lees Rita L. , Heersche Johan N. M.
Published: December 2017

Osteoclasts are multinucleated cells that resorb bone by extrusion of protons and proteolytic enzymes. They display marked heterogeneity in cell size, shape, and resorptive activity. Because high resorptive activity in vivo is associated with an increase in the average size of osteoclasts in areas of greater resorption and because of the importance of proton extrusion in resorption, we investigated whether the activity of the bafilomycin A1-sensitive vacuolar-type H+-ATPase (V-ATPase) and amiloride-sensitive Na+/H+ exchanger differed between large and small osteoclasts. Osteoclasts were obtained from newborn rabbit bones, cultured on glass coverslips, and loaded with the pH-sensitive indicator 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Intracellular pH (pHi) was recorded in single osteoclasts by monitoring fluorescence. Large (≥10 nuclei) and small (≤5 nuclei) osteoclasts differed in that large osteoclasts had a higher basal pHi, their pHi was decreased by bafilomycin A1 addition or removal of extracellular Na+, and the realkalinization upon readdition of Na+ was bafilomycin A1 sensitive. After acid loading, a subpopulation of large osteoclasts (40%) recovered by V-ATPase activity alone, whereas all small osteoclasts recovered by Na+/H+ exchanger activity. Interestingly, in 60% of the large osteoclasts, pHi recovery was mediated by both the Na+/H+ exchanger and V-ATPase activity. Our results show a striking difference between pHi regulatory mechanisms of large and small osteoclasts that we hypothesize may be associated with differences in the potential resorptive activity of these cells.
Modification of biophysical properties of lung epithelial Na+ channels by dexamethasone
Authors: Lazrak A. , Samanta A. , Venetsanou K. , Barbry P. , Matalon S.
Published: December 2017

There is considerable interest in identifying the basic mechanisms by which dexamethasone alters ion transport across the adult alveolar epithelium. Herein, we incubated synchronized A549 cells, a human alveolar epithelial cell line, with dexamethasone (1 μM) for 24–48 h. When normalized to HPRT (a housekeeping gene), A549 β- and γ-subunit mRNA levels for the human amiloride-sensitive epithelial sodium channel (hENaC), assessed by RT-PCR, increased by 1.6- and 17-fold respectively, compared with control values ( P < 0.05). These changes were abolished by actinomycin D, indicating transcriptional regulation. Western blotting studies revealed that dexamethasone also increased expression of β- and γ-hENaC protein levels. In contrast, α-hENaC mRNA increased by onefold ( P > 0.05) and α-hENaC protein level was unchanged. Incubation of A549 cells with dexamethasone increased their whole cell amiloride-sensitive sodium currents twofold and decreased the K 0.5 for amiloride from 833 ± 69 to 22 ± 5.4 nM (mean ± SE; P < 0.01). Single channel recordings in the cell-attached mode showed that dexamethasone treatment increased single channel open time and open probability threefold and decreased channel conductance from 8.63 ± 0.036 to 4.4 ± 0.027 pS (mean ± SE; P < 0.01). We concluded that dexamethasone modulates the amiloride-sensitive Na+ channels by differentially regulating the expression of β- and γ-subunits at the mRNA and protein levels in the human A549 cell line, with little effect on α-hENaC subunit.
Cloning and functional expression of human retinal Kir2.4, a pH-sensitive inwardly rectifying K+ channel
Authors: Hughes Bret A. , Kumar Gyanendra , Yuan Yukun , Swaminathan Anuradha , Yan Denise , Sharma Ashish , Plumley Lisa , Yang-Feng Teresa L. , Swaroop Anand
Published: December 2017

To identify novel potassium channel genes expressed in the retina, we screened a human retina cDNA library with an EST sequence showing partial homology to inwardly rectifying potassium (Kir) channel genes. The isolated cDNA yielded a 2,961-base pair sequence with the predicted open reading frame showing strong homology to the rat Kir2.4 (rKir2.4). Northern analysis of mRNA from human and bovine tissues showed preferential expression of Kir2.4 in the neural retina. In situ hybridization to sections of monkey retina detected Kir2.4 transcript in most retinal neurons. Somatic hybridization analysis and dual-color in situ hybridization to metaphase chromosomes mapped Kir2.4 to human chromosome 19 q13.1–q13.3. Expression of human Kir2.4 cRNA in Xenopus oocytes generated strong, inwardly rectifying K+ currents that were enhanced by extracellular alkalinization. We conclude that human Kir2.4 encodes an inwardly rectifying K+ channel that is preferentially expressed in the neural retina and that is sensitive to physiological changes in extracellular pH.
Effects of microtubules and microfilaments on [Ca2+]i and contractility in a reconstituted fibroblast fiber
Authors: Obara Kazuo , Nobe Koji , Nobe Hiromi , Kolodney Michael S. , De Lanerolle Primal , Paul Richard J.
Published: December 2017

We used a reconstituted fiber formed when 3T3 fibroblasts are grown in collagen to characterize nonmuscle contractility and Ca2+ signaling. Calf serum (CS) and thrombin elicited reversible contractures repeatable for >8 h. CS elicited dose-dependent increases in isometric force; 30% produced the largest forces of 106 ± 12 μN ( n = 30), which is estimated to be 0.5 mN/mm2 cell cross-sectional area. Half times for contraction and relaxation were 4.7 ± 0.3 and 3.1 ± 0.3 min at 37°C. With imposition of constant shortening velocities, force declined with time, yielding time-dependent force-velocity relations. Forces at 5 s fit the hyperbolic Hill equation; maximum velocity ( V max) was 0.035 ± 0.002 Lo/s. Compliance averaged 0.0076 ± 0.0006 Lo/Fo. Disruption of microtubules with nocodazole in a CS-contracted fiber had no net effects on force, V max, or stiffness; force increased in 8, but decreased in 13, fibers. Nocodazole did not affect baseline intracellular Ca2+ concentration ([Ca2+]i) but reduced (∼30%) the [Ca2+]i response to CS. The force after nocodazole treatment was the primary determinant of stiffness and V max, suggesting that microtubules were not a major component of fiber internal mechanical resistance. Cytochalasin D had major inhibitory effects on all contractile parameters measured but little effect on [Ca2+]i.
Conformational modulation of slow skeletal muscle troponin T by an NH2-terminal metal-binding extension
Authors: Jin Jian-Ping , Chen Aihua , Ogut Ozgur , Huang Qi-Quan
Published: December 2017

Troponin T (TnT) is an essential element in the thin filament Ca2+-regulatory system controlling striated muscle contraction. Alternative RNA splicing generates developmental and muscle type-specific TnT isoforms differing in the hypervariable NH2-terminal region. Using avian fast skeletal muscle TnT containing a metal-binding segment, we have demonstrated a role of the NH2-terminal domain in modulating the conformation of TnT (Wang J and Jin JP. Biochemistry 37: 14519–14528, 1998). To further investigate the structure-function relationship of TnT, the present study constructed and characterized a recombinant protein in which the metal-binding peptide present in avian fast skeletal muscle TnT was fused to the NH2 terminus of mouse slow skeletal muscle TnT. Metal ion or monoclonal antibody binding to the NH2-terminal extension induced conformational changes in other domains of the model TnT molecule. This was shown by the altered affinity to a monoclonal antibody against the COOH-terminal region and a polyclonal antiserum recognizing multiple epitopes. Protein binding assays showed that metal binding to the NH2-terminal extension had effects on the interaction of TnT with troponin I, troponin C, and most significantly, tropomyosin. The data indicate that the NH2-terminal Tx [4–7 repeats of a sequence motif His-(Glu/Ala)-Glu-Ala-His] extension confers a specific conformational modulation in the slow skeletal muscle TnT.
Excitation wavelengths for fura 2 provide a linear relationship between [Ca2+] and fluorescence ratio
Authors: Palmer Bradley M. , Moore Russell L.
Published: December 2017

We proposed and tested the use of nontraditional excitation wavelengths (λ1 and λ2) and an emission wavelength (λem) to define conditions under which free calcium concentration and a fluorescence ratio are linearly related. Fluorescence spectra were determined for aqueous solutions that contained 25 μM fura 2, 125 mM K+, and either 0 mM or 0.1 mM Ca2+. Effectively linear relationships between [Ca2+] and a fluorescence ratio, i.e., <5% bias when [Ca2+] ≤ 5 × dissociation constant, were apparent when λ1 ≥ 400 nm, λ2 ≤ 370 nm, and λem ≥ 510 nm. Combinations with longer λ1and λem and/or with shorter λ2 reduced this bias further. Although the method described does not obviate the complications that surround the correction for fluorescence background, choosing a nontraditional combination of excitation and emission wavelengths offers several practical advantages over more traditional fura 2 fluorescence methodologies in a variety of experimental settings.
Influence of lipoxin A4 and other lipoxygenase-derived eicosanoids on tissue factor expression
Authors: Maderna Paola , Godson Catherine , Hannify Gary , Murphy Madeline , Brady Hugh R.
Published: December 2017

Lipoxins (LX) are eicosanoids generated via transcellular biosynthetic routes during inflammation, hypersensitivity reaction, and after angioplasty. LXs are modulators of leukocyte trafficking and vascular tone. Their influence on the coagulation cascade has not been determined. In this study, we evaluated the influence of LXs on the expression of tissue factor (TF), a key regulator of coagulation. TF activity was measured in lysates of monocytes, human umbilical vein endothelial cells, and ECV304 cells using a one-stage clotting assay. LXA4 stimulated TF activity in each cell type. The influence of LXA4 on TF activity by ECV304 cells was studied further to explore the mechanism of induction of TF expression. LXA4-induced TF activity was dose dependent, cycloheximide sensitive, and associated with increased TF mRNA levels. Induction of TF activity was specific for LXA4 and was not observed with LXB4, the other major lipoxin generated by mammalian cells. Furthermore, ECV304 cell TF expression was not influenced by 15( R/S)-methyl-LXA4 or 16-phenoxy-LXA4, synthetic analogs of LXA4 that activate the myeloid LXA4 receptor, and was not modulated by SKF-104353, which blocks LXA4 bioactivities transduced through the putative shared LXA4/LTD4 receptor. LXA4-stimulated TF expression was blunted by pertussis toxin and by GF-109203X, an inhibitor of protein kinase C, and was not associated with degradation of IκBα. Our results establish that LXA4 induces TF activity via cell signaling pathways with different structural and receptor requirements from those described for inhibition of leukocyte-endothelial cell interactions. They suggest a role for LXA4 as a modulator of TF-related vascular events during inflammation and thrombosis.
Nitric oxide inhibits chondrocyte response to IGF-I: inhibition of IGF-IRβ tyrosine phosphorylation
Authors: Studer R. K. , Levicoff E. , Georgescu H. , Miller L. , Jaffurs D. , Evans C. H.
Published: December 2017

Chondrocytes in arthritic cartilage respond poorly to insulin-like growth factor I (IGF-I). Studies with inducible nitric oxide synthase (iNOS) knockout mice suggest that NO is responsible for part of the cartilage insensitivity to IGF-I. These studies characterize the relationship between NO and chondrocyte responses to IGF-I in vitro, and define a mechanism by which NO decreases IGF-I stimulation of chondrocyte proteoglycan synthesis. Lapine cartilage slices, chondrocytes, and cartilage from osteoarthritic (OA) human knees were exposed to NO from the donors S-nitroso- N-acetylpenicillamine (SNAP) or ( Z)-1-[2-(2-aminoethyl)- N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate] (DETA NONOate), by transduction with adenoviral transfer of iNOS (Ad-iNOS), or by activation with interleukin-1 (IL-1). NO synthesis was estimated from medium nitrite, and proteoglycan synthesis was measured as incorporation of 35SO4. IGF-I receptor phosphorylation was evaluated with Western analysis. SNAP, DETA NONOate, endogenously synthesized NO in Ad-iNOS-transduced cells, or IL-1 activation decreased IGF-I-stimulated proteoglycan synthesis in cartilage and monolayer cultures of chondrocytes. OA cartilage responded poorly to IGF-I; however, the response to IGF-I was restored by culture with N G-monomethyl-l-arginine(l-NMA). IGF-I receptor phosphotyrosine was diminished in chondrocytes exposed to NO. These studies show that NO is responsible for part of arthritic cartilage/chondrocyte insensitivity to anabolic actions of IGF-I; inhibition of receptor autophosphorylation is potentially responsible for this effect.
A KATP channel deficiency affects resting tension, not contractile force, during fatigue in skeletal muscle
Authors: Gong B. , Miki T. , Seino S. , Renaud J. M.
Published: December 2017

The objective of this study was to determine how an ATP-sensitive K+ (KATP) channel deficiency affects the contractile and fatigue characteristics of extensor digitorum longus (EDL) and soleus muscle of 2- to 3-mo-old and 1-yr-old mice. KATP channel-deficient mice were obtained by disrupting the Kir6.2 gene that encodes for the protein forming the pore of the channel. At 2–3 mo of age, the force-frequency curve, the twitch, and the tetanic force of EDL and soleus muscle of KATPchannel-deficient mice were not significantly different from those in wild-type mice. However, the tetanic force and maximum rate of force development decreased with aging to a greater extent in EDL and soleus muscle of KATP channel-deficient mice (24–40%) than in muscle of wild-type mice (7–17%). During fatigue, the KATP channel deficiency had no effect on the decrease in tetanic force in EDL and soleus muscle, whereas it caused a significantly greater increase in resting tension when compared with muscle of wild-type mice. The recovery of tetanic force after fatigue was not affected by the deficiency in 2- to 3-mo-old mice, whereas in 1-yr-old mice, force recovery was significantly less in muscle of KATP channel-deficient than wild-type mice. It is suggested that the major function of the KATP channel during fatigue is to reduce the development of a resting tension and not to contribute to the decrease in force. It is also suggested that the KATP channel plays an important role in protecting muscle function in older mice.
Effect of HCO3 − on TPA- and IBMX-induced anion conductances in Necturus gallbladder epithelial cells
Authors: Lyall Pamela , McD. Armstrong William , Lyall Vijay
Published: December 2017

Effects of HCO3 − on protein kinase C (PKC)- and protein kinase A (PKA)-induced anion conductances were investigated in Necturus gallbladder epithelial cells. In HCO3 −-free media, activation of PKC via 12- O-tetradecanoylphorbol 13-acetate (TPA) depolarized apical membrane potential ( V a) and decreased fractional apical voltage ratio (FR). These effects were blocked by mucosal 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a Cl− channel blocker. In HCO3 −media, TPA induced significantly greater changes in V a and FR. These effects were blocked only when NPPB was present in both mucosal and basolateral compartments. The data suggest that TPA activates NPPB-sensitive apical Cl− conductance ( g Cl a) in the absence of HCO3 −; in its presence, TPA stimulated both NPPB-sensitive g Cl a and basolateral Cl− conductance ( g Cl b). Activation of PKA via 3-isobutyl-1-methylxanthine (IBMX) also decreased V a and FR; however, these changes were not affected by external HCO3 −. We conclude that HCO3 − modulates the effects of PKC on g Cl b. In HCO3 − medium, TPA and IBMX also induced an initial transient hyperpolarization and increase in intracellular pH. Because these changes were independent of mucosal Na+ and Cl−, it is suggested that TPA and IBMX induce a transient increase in apical HCO3 −conductance.
Molecular characterization of a novel UT-A urea transporter isoform (UT-A5) in testis
Authors: Fenton R. A. , Howorth A. , Cooper G. J. , Meccariello R. , Morris I. D. , Smith C. P.
Published: December 2017

Urea movement across plasma membranes is modulated by specialized transporter proteins that are products of two genes, termed UT-A and UT-B. These proteins play key roles in the urinary concentrating mechanism and fluid homeostasis. We have isolated and characterized a 1.4-kb cDNA from testes encoding a new isoform (UT-A5) belonging to the UT-A transporter family. For comparison, we also isolated a 2.0-kb cDNA from mouse kidney inner medulla encoding the mouse UT-A3 homologue. The UT-A5 cDNA has a putative open reading frame encoding a 323-amino acid protein, making UT-A5 the smallest UT-A family member in terms of molecular size. Its putative topology is of particular interest, because it calls into question earlier models of UT-A transporter structure. Expression of UT-A5 cRNA in Xenopus oocytes mediates phloretin-inhibitable urea uptake and does not translocate water. The distribution of UT-A5 mRNA is restricted to the peritubular myoid cells forming the outermost layer of the seminiferous tubules within the testes and is not detected in kidney. UT-A5 mRNA levels are coordinated with the stage of testes development and increase 15 days postpartum, commensurate with the start of seminiferous tubule fluid movement.
Agonist-induced isometric contraction of smooth muscle cell-populated collagen gel fiber
Authors: Oishi Kazuhiko , Itoh Yuko , Isshiki Yasuhiro , Kai Chikatoshi , Takeda Yasushi , Yamaura Kazuhiro , Takano-Ohmuro Hiromi , Uchida Masaatsu K.
Published: December 2017

String-shaped reconstituted smooth muscle (SM) fibers were prepared in rectangular wells by thermal gelation of a mixed solution of collagen and cultured SM cells derived from guinea pig stomach. The cells in the fiber exhibited an elongated spindle shape and were aligned along the long axis. The fiber contracted in response to KCl (140 mM), norepinephrine (NE; 10−7 M), epinephrine (10−7 M), phenylephrine (10−6 M), serotonin (10−6 M), and histamine (10−5 M), but not acetylcholine (10−5 M). Phentolamine (10−7 M) produced a parallel rightward shift of the NE dose-response curve. Moreover, NE-induced contraction was partially inhibited by nifedipine and completely abolished by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid acetoxymethyl ester, the myosin light chain kinase inhibitor ML-9, the Rho kinase inhibitor Y-27632, and papaverine. A [3H]quinuclidinyl benzilate binding study revealed that the loss of response to acetylcholine was due to the loss of muscarinic receptor expression during culture. The expression of contractile proteins in the fibers was similar to that in cultured SM cells. These results suggest that, although the fiber is not a model for fully differentiated SM, contractile mechanisms are maintained.
Phenylarsine oxide inhibits heat shock protein 70 induction in cultured guinea pig gastric mucosal cells
Authors: Rokutan Kazuhito , Miyoshi Mami , Teshima Shigetada , Kawai Tomoko , Kawahara Tsukasa , Kishi Kyoichi
Published: December 2017

Phenylarsine oxide (PAO) forms a stable ring complex with vicinal dithiols that can be reversed with 2,3-dimercaptopropanol (DMP) but not by dithiothreitol (DTT) or 2-mercaptoethanol (2-ME). PAO at 2 μM or higher inhibited heat shock protein 70 (HSP70) induction within minutes in cultured guinea pig gastric mucosal cells exposed to heat (43°C) for 30 min. PAO did not affect the nuclear translocation and phosphorylation of heat shock factor 1 (HSF1) induced by heat stress, but it completely blocked the binding activity of HSF1 to the heat shock element (HSE), leading to the block of expression of HSP70 mRNA and accumulation of HSP70 in the cells. These inhibitions were completely reversed with 2 μM DMP but not with 0.1 mM DTT or 1 mM 2-ME, suggesting specific interactions between PAO and vicinal dithiol-containing molecules. Thioredoxin (Trx) reversed the inhibition of the binding activity of HSF1 in whole cell extracts prepared from PAO-treated, heat-stressed cells. Our results suggest that PAO may react with vicinal-containing molecules including Trx and specifically block the interaction between HSF1 and HSE.
Ca2+-ATPase protein expression in mammary tissue
Authors: Reinhardt Timothy A. , Filoteo Adelaida G. , Penniston John T. , Horst Ronald L.
Published: December 2017

Protein expression of plasma membrane Ca2+-ATPases (PMCAs) and the putative Golgi secretory pathway Ca2+-ATPase (SPCA) was examined in rat mammary tissue. As lactation started, PMCA protein expression increased dramatically, and this increased expression paralleled milk production. Mammary PMCA was primarily PMCA2b but was ∼4,000 daltons larger than expected. RT-PCR showed that the primary mammary PMCA2b transcript was alternatively spliced, at splice site A, to include an additional 135 bp, resulting in the insertion of 45 amino acids. This splice form is designated 2bw. PMCA2bw is secreted into milk, associated with the milk fat globule membrane. Therefore, PMCA2bw is located on the apical membrane of the secretory cell. Smaller amounts of PMCA1b and 4b protein were found in mammary tissue. PMCA4b was the major PMCA expressed in developing tissue, and its level declined as lactation started. PMCA1b expression increased moderately during lactation. SPCA protein expression increased 1 wk before parturition and increased further as lactation proceeded. The abundance and cell location of PMCA2b suggest that it is important for macro-Ca2+ homeostasis in lactating tissue. The pattern of expression and abundance of SPCA suggest that it is a candidate for the Golgi Ca2+-ATPase.
Epinephrine correction of impaired platelet thromboxane receptor signaling
Authors: Dunlop Patricia C. , Leis Linda A. , Johnson Gerhard J.
Published: December 2017

This study evaluated the mechanism of epinephrine potentiation of platelet secretion induced by thromboxane A2 (TXA2). Dog platelets that do not secrete in response to TXA2 alone (TXA2−) were compared with dog platelets that do secrete (TXA2+) and with human platelets. TXA2− platelets had impaired TXA2receptor (TP receptor)-G protein coupling, indicated by 1) impaired stimulated GTPase activity, 2) elevated basal guanosine 5′- O-(3-thiotriphosphate) binding, and 3) elevated Gαq palmitate turnover that was corrected by preexposure to epinephrine. Kinetic agonist binding studies revealed biphasic dog and human platelet TP receptor association and dissociation. TXA2− and TP receptor-desensitized TXA2+ dog and human platelets had altered ligand binding parameters compared with untreated TXA2+ or human platelets. These parameters were reversed, along with impaired secretion, by epinephrine. Basal phosphorylation of TXA2− platelet TP receptors was elevated 60% and was normalized by epinephrine. Epinephrine potentiates platelet secretion stimulated by TXA2 by reducing basal TP receptor phosphorylation and facilitating TP receptor-G protein coupling in TXA2− platelets and, probably, in normal platelets as well.
Expression of the chloride channel ClC-2 in the murine small intestine epithelium
Authors: Gyömörey Katalin , Yeger Herman , Ackerley Cameron , Garami Elizabeth , Bear Christine E.
Published: December 2017

The chloride channel ClC-2 has been implicated in neonatal airway chloride secretion. To assess its role in secretion by the small intestine, we assessed its subcellular expression in ileal segments obtained from mice and studied the chloride transport properties of this tissue. Chloride secretion across the mucosa of murine ileal segments was assessed in Ussing chambers as negative short-circuit current ( I sc). If ClC-2 contributed to chloride secretion, we predicted on the basis of previous studies that negative I sc would be stimulated by dilution of the mucosal bath and that this response would depend on chloride ion and would be blocked by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid but not by DIDS. In fact, mucosal hypotonicity did stimulate a chloride-dependent change in I sc that exhibited pharmacological properties consistent with those of ClC-2. This secretory response is unlikely to be mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) channel because it was also observed in CFTR knockout animals. Assessment of the native expression pattern of ClC-2 protein in the murine intestinal epithelium by confocal and electron microscopy showed that ClC-2 exhibits a novel distribution, a distribution pattern somewhat unexpected for a channel involved in chloride secretion. Immunolabeled ClC-2 was detected predominantly at the tight junction complex between adjacent intestinal epithelial cells.
Differential renal distribution of NHERF isoforms and their colocalization with NHE3, ezrin, and ROMK
Authors: Wade James B. , Welling Paul A. , Donowitz Mark , Shenolikar Shirish , Weinman Edward J.
Published: December 2017

Na+/H+ exchanger regulatory factor (NHERF) and NHERF2 are PDZ motif proteins that mediate the inhibitory effect of cAMP on Na+/H+ exchanger 3 (NHE3) by facilitating the formation of a multiprotein signaling complex. With the use of antibodies specific for NHERF and NHERF2, immunocytochemical analysis of rat kidney was undertaken to determine the nephron distribution of both proteins and their colocalization with other transporters and with ezrin. NHERF was most abundant in apical membrane of proximal tubule cells, where it colocalized with ezrin and NHE3. NHERF2 was detected in the glomerulus and in other renal vascular structures. In addition, NHERF2 was strongly expressed in collecting duct principal cells, where it colocalized with ROMK. These results indicate a striking difference in the nephron distribution of NHERF and NHERF2 and suggests NHERF is most likely to be the relevant biological regulator of NHE3 in the proximal tubule, while NHERF2 may interact with ROMK or other targets in the collecting duct. The finding that NHERF isoforms occur in different cell types suggests that NHERF and NHERF2 may subserve different functions in the kidney.
α1H T-type Ca2+ channel is the predominant subtype expressed in bovine and rat zona glomerulosa
Authors: Schrier Andrew D. , Wang Hongge , Talley Edmund M. , Perez-Reyes Edward , Barrett Paula Q.
Published: December 2017

The low voltage-activated (T-type) Ca2+ channel has been implicated in the regulation of aldosterone secretion from the adrenal zona glomerulosa by extracellular K+ levels, angiotensin II, and ACTH. However, the identity of the specific subtype mediating this regulation has not been determined. We utilized in situ hybridization to examine the distribution of three newly cloned members of the T-type Ca2+ channel family, α1G, α1H, and α1I, in the rat and bovine adrenal gland. Substantial expression of only the mRNA transcript for the α1H-subunit was detected in the zona glomerulosa of both rat and bovine. A much weaker expression signal was detected for the α1H transcript in the zona fasciculata of bovine. Whole cell recordings of isolated bovine adrenal zona glomerulosa cells showed the native low voltage-activated current to be inhibited by NiCl2 with an IC50 of 6.4 ± 0.2 μM. Because the α1H subtype exhibits similar NiCl2 sensitivity, we propose that the α1Hsubtype is the predominant T-type Ca2+ channel present in the adrenal zona glomerulosa.
Voltage dependence of the coupling of Ca2+ sparks to BKCa channels in urinary bladder smooth muscle
Authors: Herrera Gerald M. , Heppner Thomas J. , Nelson Mark T.
Published: December 2017

Large-conductance Ca2+-dependent K+(BKCa) channels play a critical role in regulating urinary bladder smooth muscle (UBSM) excitability and contractility. Measurements of BKCa currents and intracellular Ca2+ revealed that BKCa currents are activated by Ca2+ release events (Ca2+ sparks) from ryanodine receptors (RyRs) in the sarcoplasmic reticulum. The goals of this project were to characterize Ca2+ sparks and BKCa currents and to determine the voltage dependence of the coupling of RyRs (Ca2+ sparks) to BKCachannels in UBSM. Ca2+ sparks in UBSM had properties similar to those described in arterial smooth muscle. Most Ca2+ sparks caused BKCa currents at all voltages tested, consistent with the BKCa channels sensing ∼10 μM Ca2+. Membrane potential depolarization from −50 to −20 mV increased Ca2+ spark and BKCacurrent frequency threefold. However, membrane depolarization over this range had a differential effect on spark and current amplitude, with Ca2+ spark amplitude increasing by only 30% and BKCa current amplitude increasing 16-fold. A major component of the amplitude modulation of spark-activated BKCa current was quantitatively explained by the known voltage dependence of the Ca2+ sensitivity of BKCa channels. We, therefore, propose that membrane potential, or any other agent that modulates the Ca2+sensitivity of BKCa channels, profoundly alters the coupling strength of Ca2+ sparks to BKCa channels.