bluuebunny/crossref_metadata_2025 huggingface dataset:
PKC regulates agonist-induced force enhancement in single alpha-toxin-permeabilized vascular smooth muscle cells
Authors: Brozovich F. V.
Published: December 2017

To determine whether activation of protein kinase C (PKC) is involved in the mechanism of agonist-induced force enhancement, force and stiffness were measured in both Ca(2+)- and agonist-stimulated contractions of single isolated alpha-toxin-permeabilized smooth muscle cells. PKC function was inhibited with the pseudosubstrate inhibitor (residues 19-31) of PKC (PKI). For Ca2+ activation, PKI did not change (P > 0.05) steady-state force or stiffness. However, for agonist activation at pCa 7 (n = 13), PKI depressed force by 28.7 +/- 4.5% (P < 0.05), in-phase stiffness by 35.4 +/- 4.0% (P < 0.05), and quadrature stiffness by 25.6 +/- 4.4% (P < 0.05), and for agonist activation at pCa 4 (n = 7), PKI depressed force by 25.8 +/- 2.9% (P < 0.05), in-phase stiffness by 35.6 +/- 5.6% (P < 0.05), and quadrature stiffness by 20.3 +/- 4.1% (P < 0.05). These results suggest that the agonist-induced force enhancement in alpha-toxin-permeabilized smooth muscle is due to the activation of PKC.
Intercellular metabolic coupling in canine colon musculature
Authors: Farraway L. , Ball A. K. , Huizinga J. D.
Published: December 2017

Intercellular communication within the musculature of the canine colon was studied by examining the results of neurobiotin diffusion after injection of the tracer into smooth muscle cells at different locations within the muscle layer. Circular muscle at the submucosal surface, circular muscle adjacent to the myenteric plexus, and longitudinal muscle demonstrated different degrees of time-dependent tracer spread. At the submucosal surface, tracer spread was rapid, extensive, and unimpeded by connective tissue septa. At the myenteric side, tracer spread was also extensive but was much slower and confined to bundles of cells bordered by septa. In contrast to previous studies that suggest an absence of gap junctions at the myenteric side of the circular muscle, the neurobiotin spread indicates full metabolic coupling of all circular smooth muscle cells. Furthermore, in contrast to the belief that longitudinal muscle is completely devoid of gap junctions, tracer spread occurred between cells in this layer, although neurobiotin diffusion was very limited, nonuniform, and slow. In each area of the musculature studied, tracer spread was inhibited by octanol. When very long injection and wait times were implemented at the submucosal surface of the circular muscle, neurobiotin was observed to cross septa through the network of interstitial cells of Cajal, indicating that it is this network that provides communication between lamellae.
Identification and function of A1 adenosine receptors in normal and cystic fibrosis human airway epithelial cells
Authors: McCoy D. E. , Schwiebert E. M. , Karlson K. H. , Spielman W. S. , Stanton B. A.
Published: December 2017

A role for adenosine in the regulation of ion transport in pulmonary epithelial cells has recently been proposed. Although evidence exists documenting the presence and function of adenosine A2 receptors in airway epithelia, the presence of adenosine A1 receptors remains controversial. The present study used reverse transcriptase-polymerase chain reaction (PCR) and whole cell patch-clamp analysis to investigate A1 receptor presence and function in normal and cystic fibrosis (CF) human airway epithelial cells. Oligonucleotide primers complementary to the human brain A1 receptor sequence generated a PCR product of the predicted size (311 bp) in normal tracheal (9HTEo-) and CF submucosal (2CFSMEo-) airway cell lines and in primary cultures of CF nasal polyp epithelial cells. An oligonucleotide probe internal to the PCR primers hybridized with the 311-bp cDNAs by Southern blot analysis. cDNA sequencing demonstrated that the normal and CF airway cell PCR products are 100% identical to the corresponding sequence of the human brain adenosine A1 receptor. Northern blot analysis of 9HTEo-and 2CFSMEo- poly(A)+ RNA revealed the presence of two bands of approximately 3.0 and approximately 5.5 kb corresponding to the A1 receptor. Whole cell patch-clamp analyses demonstrated that 8-cyclopentyl-1,3-dipropylxanthine, a specific A1 receptor antagonist, increases adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- conductance in 9HTEo-airway cells and allows cAMP to increase Cl- conductance in 2CFSMEo- CF airway cells and CF nasal polyp epithelial cells in primary culture. These results provide evidence for the presence and function of A1 receptors in normal and CF airway epithelial cells and provide support for a role of adenosine A1 receptors in modulating airway epithelial cell Cl- transport.
Developmental regulation of membrane protein sorting in Drosophila embryos
Authors: Shiel M. J. , Caplan M. J.
Published: December 2017

We have examined the process of membrane protein targeting in the polarized cells of the developing Drosophila melanogaster embryo. Human placental alkaline phosphatase (PLAP) is a glycosylphosphatidyl inositol-linked protein that accumulates at the apical membranes of mammalian epithelial cells. A chimeric construct composed of the transmembrane and cytosolic portions of the vesicular stomatitis virus G protein coupled to the ectodomain of PLAP, termed PLAPG, has been found to behave as a basolateral protein (D. A. Brown, B. Crise, and J. K. Rose. Science Wash. DC 232: 34-47, 1989). The subcellular distributions of these proteins were examined in the epithelial and neuronal tissues of transgenic Drosophila embryos. In the surface ectoderm, both PLAP and PLAPG were restricted to the basolateral membranes throughout development. Internal epithelia derived from the surface ectoderm accumulated PLAP at their apical surfaces, whereas PLAPG retained its basolateral distribution. The redistribution of PLAP from the basolateral to the apical plasma membrane was found to be coincident with the invagination of the surface epithelium to form internal structures, suggesting that the sorting pathways that function in the epithelium of the Drosophila embryo are developmentally regulated.
Functional coupling of Na(+)-K(+)-2Cl- cotransport and Ca(2+)-dependent K+ channels in vascular endothelial cells
Authors: O'Neill W. C. , Steinberg D. F.
Published: December 2017

To determine whether the activation of Na(+)-K(+)-2Cl- cotransport by Ca(2+)-mobilizing agonists is a direct effect of Ca2+ or is secondary to activation of Ca(2+)-dependent K+ channels [via cell shrinkage or decreased intracellular Cl- concentration ([Cl-]), we measured K+ fluxes in aortic endothelial cells in response to ATP and bradykinin. With either agonist there was an immediate bumetanide-insensitive efflux inhibitable by the K+ channel blockers tetrabutylammonium (TBA, 23 mM) and quinidine (1 mM), followed several minutes later by increased bumetanide-sensitive efflux or influx (Na(+)-K(+)-2Cl- cotransport). ATP induced a loss of cell K+ that was prevented by TBA and augmented by bumetanide. Both TBA and quinidine prevented the stimulation of cotransport by agonists but not by hypertonic shrinkage. Raising medium [K+] to prevent K+ loss also blocked activation of cotransport by agonists. The results indicate that the stimulation of Na(+)-K(+)-2Cl- cotransport by Ca2+ is not direct but instead is indirect via activation of Ca(2+)-dependent K+ channels and a resulting decrease in cell volume and intracellular [Cl-]. This suggests that at least one role of Na(+)-K(+)-2Cl- cotransport in endothelial cells is to maintain cell volume and intracellular [Cl-] during agonist stimulation.
Determination of intracellular calcium in vivo via fluorine-19 nuclear magnetic resonance spectroscopy
Authors: Song S. K. , Hotchkiss R. S. , Neil J. , Morris P. E. , Hsu C. Y. , Ackerman J. J.
Published: December 2017

Fluorine-19-nuclear magnetic resonance (19F-NMR) spectroscopic detection of the NMR-active Ca2+ indicator 5-fluoro-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA) is one method for measuring cytosolic free Ca2+ concentration ([Ca2+]i) and has been used previously to measure [Ca2+]i in isolated cells and perfused organs. The aim of the present investigation was to demonstrate the feasibility of determining [Ca2+]i in vivo and in situ using 19F-NMR and 5F-BAPTA. Experiments were performed on male Sprague-Dawley rats with a surface-coil antenna employed for NMR interrogation. The Ca2+ indicator, 5F-BAPTA, was infused either intravenously (kidney, spleen) or intraventricularly (brain) as a 100 mg/ml solution of the cell-permeant acetoxymethyl ester (5F-BAPTA-AM) in dimethyl sulfoxide. Rats tolerated intravenous infusion without evident change in mean arterial blood pressure. In all tissues examined, kidney, spleen, and brain, [Ca2+]i was approximately 200 nM. To our knowledge, these results represent the first in vivo and in situ determinations of [Ca2+]i employing 19F-NMR.
Glucose transporter expression and functional role of hexokinase in insulin biosynthesis in mouse beta TC3 cells
Authors: Nagamatsu S. , Nakamichi Y. , Sawa H.
Published: December 2017

It was previously reported that insulin biosynthesis in mouse beta TC3 cells was regulated by glucose (Nagamatsu, S., and D. F. Steiner. Endocrinology 130: 748-754, 1992). In the present study, we examined the effect of glucose on the glucose transporter expression and hexokinase activities and determined the relationship between them and glucose-stimulated insulin biosynthesis in beta TC3 cells. Reverse transcriptase-polymerase chain reaction and Northern blot analysis revealed that beta TC3 cells expressed GLUT-1 and GLUT-3 glucose transporter mRNAs, but not GLUT-2. The levels of GLUT-1 and GLUT-3 mRNAs were not affected by glucose (0 or 11 mM glucose) over a period of 48 h. Immunoprecipitation of metabolically labeled beta TC3 cells with specific antibodies against GLUT-1 or GLUT-3 proteins revealed no effect of glucose on the biosynthesis of glucose transporters. Hexokinase [low Michaelis constant (Km) hexokinase] activity from cells incubated in 11 mM glucose for 48 h increased nearly twofold compared with cells maintained in 0 mM glucose, although the amount of cellular hexokinase protein detected by immunoblot analysis was unchanged between 0 and 11 mM glucose conditions. Glucokinase (high Km hexokinase) activity, in contrast, was not affected by glucose. Preincubation of beta TC3 cells with 2-deoxyglucose to inhibit hexokinase, thereby inhibiting all glycolysis, resulted in the decrease of glucose-stimulated insulin biosynthesis. Thus, in mouse beta TC3 cells that do not express GLUT-2, there is a close relationship between hexokinase activity and glucose-stimulated insulin biosynthesis, but not between the glucose transporter and glucose-stimulated insulin biosynthesis.
Immunohistochemical localization of pH-sensitive K+ channel, RACTK1
Authors: Suzuki M. , Takigawa T. , Kimura K. , Koseki C. , Imai M.
Published: December 2017

RACTK1 cDNA encodes a pH-sensitive K+ channel from the renal cortical collecting tubule cells, which is only localized in the kidney by Northern blots (Suzuki, M., K. Takahashi, M. Ikeda, H. Hayakawa, A. Ogawa, Y. Kawaguchi, and O. Sakai. Nature Lond. 367: 642-645, 1994). We further investigated the localization of the channel by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Immunohistochemical analysis showed that the signal was detected in various epithelia, including the luminal membrane of the cortical collecting tubules, artery of the kidney, and pancreatic ducts. Interestingly, the signal was also detected in the coronary vascular smooth muscle and cerebral artery. These tissue distributions were confirmed by localization of mRNA determined by RT-PCR. These findings suggest that RACTK1 encoding pH-sensitive K+ channels is widely distributed in epithelia as well as in vascular smooth muscle cells.
Relative expression of the human epithelial Na+ channel subunits in normal and cystic fibrosis airways
Authors: Burch L. H. , Talbot C. R. , Knowles M. R. , Canessa C. M. , Rossier B. C. , Boucher R. C.
Published: December 2017

The availability of the newly cloned subunits (alpha, beta, gamma) of the epithelial Na+ channel (ENaC) permits molecular studies of the pathogenesis of the abnormal Na+ transport rates of cystic fibrosis (CF) airway epithelia. Northern analyses of airway epithelia showed that both normal and CF airway epithelia express ENaC subunit mRNAs in a ratio of alpha > beta > gamma. In situ hybridization studies revealed expression of all three ENaC subunits in the superficial epithelium and the alpha- and beta-subunits in the gland ductular and acinar epithelium of both normal and CF airways. Ribonuclease protection assays revealed that the steady-state levels of alpha-, beta-, and gamma-ENaC mRNAs were similar in CF and normal airway superficial epithelia. These findings indicate that 1) Na+ transport defects in CF airways disease may be expressed in glandular acinar and ductal epithelium as well as superficial epithelium, and 2) the molecular pathogenesis of Na+ hyperabsorption in CF airways does not reflect increased levels of Na+ channel mRNAs, and probably number, but reflects an absence of the normal inhibitory regulation of Na+ channels by CF transmembrane conductance regulator proteins.
Relationships between mesangial cell proliferation and types I and IV collagen mRNA levels in vitro
Authors: He C. J. , Striker L. J. , Tsokos M. , Yang C. W. , Peten E. P. , Striker G. E.
Published: December 2017

Changes in the composition of the mesangial extracellular matrix (ECM) and cell turnover are present in glomerular disease. To determine if ECM changes play a role in perpetuating mesangial cell dysfunction, we examined a line of mouse mesangial cells cultured on films or gels of several ECM components and also on methyl cellulose, an inert substrate that prevents attachment. Cells on films of fibronectin or type IV or I collagen had persistently high growth rates and high levels of alpha 1-I and alpha 1-IV collagen mRNAs. In contrast, on gels of type IV or I collagen or matrigel, the growth rate was low. The alpha 1-IV collagen mRNA levels were low on type IV collagen gel or matrigel, whereas the alpha 1-I collagen mRNA levels remained high. In contrast, the alpha 1-I collagen mRNA levels were low on type I collagen gel, and the alpha 1-IV collagen mRNA levels were high. Cells on methyl cellulose formed floating aggregates, did not proliferate, and had a 5- to 10-fold decrease in both alpha 1-I and alpha 1-IV collagen mRNA levels. These phenotypic changes were largely reversible. Finally, when matrigel was layered over cells on fibronectin films, alpha 1-IV collagen mRNA levels decreased, but alpha 1-I collagen mRNA levels and proliferation remained high. Thus proliferation and alpha 1-I and alpha 1-IV collagen mRNA levels in mesangial cells were independently regulated and depended on attachment and the nature of the adjacent matrix.
ATP depletion inhibits capacitative Ca2+ entry in rat thymic lymphocytes
Authors: Marriott I. , Mason M. J.
Published: December 2017

The present study investigates the requirement for cellular ATP in the increase in plasma membrane Ca2+ permeability activated by the release of Ca2+ from intracellular stores in rat thymic lymphocytes (capacitative Ca2+ entry). The permeability state of this pathway following activation with thapsigargin was probed in control and ATP-depleted cells using fluorometric measurements of intracellular Ca2+, Mn2+ entry, and membrane potential, and unidirectional measurements of Ca2+ uptake using 45Ca2+. The capacitative Ca(2+)-entry pathway was markedly inhibited in cells depleted of ATP by incubation in glucose-free solution containing oligomycin, antimycin A, and 2-deoxy-D-glucose. These data cannot be explained on the basis of a loss of the transmembrane electrochemical gradient for Ca2+, alterations in intracellular pH or cellular Na+ content, a direct effect of the inhibitors of ATP production on the capacitative Ca(2+)-entry pathway, or the ability of thapsigargin to release Ca2+ from intracellular stores. Rather, these data are consistent with a requirement for ATP or a high-energy phosphate donor in the activation and/or maintained activation of capacitative Ca2+ entry.
Head-down tilt increases rat cardiac muscle eIF-2 alpha phosphorylation
Authors: Menon V. , Thomason D. B.
Published: December 2017

We previously demonstrated that head-down tilt in rats decreases heart polypeptide initiation rate and proposed a mechanism whereby redistribution of the chaperone heat-shock cognate/heat-shock protein-70 (HSC/HSP-70) facilitates the phosphorylation of eukaryotic initiation factor-2 alpha (eIF-2 alpha). In this study, two-dimensional gel electrophoretic analysis of eIF-2 alpha showed no phosphorylation in control hearts. At 8 h of head-down tilt, there was a 45% increase in total eIF-2 alpha, and 79% was phosphorylated. At 18 h, eIF-2 alpha increased to 142% of control, of which 4% was phosphorylated. This is consistent with the previous study where, at 8 h, there was a 78% increase in polysomal HSC/HSP-70 and a shift in the polysome center-of-mass to lighter polysomes (indicating decreased initiation). After 18 h of suspension, polysomal HSC/HSP-70 levels were 24% relative to control, and the center-of-mass returned toward control. We conclude that the decrease in polypeptide initiation during head-down tilt is mediated by HSC/HSP-70 via phosphorylation of eIF-2 alpha.
Primary structure and functional expression of the mouse and frog α-subunit of the gastric H+-K+-ATPase
Authors: Mathews Paul M. , Claeys Dirk , Jaisser Frédéric , Geering Käthi , Horisberger Jean-Daniel , Kraehenbuhl Jean-Pierre , Rossier Bernard C.
Published: December 2017

Pages C1207-C1214: Paul M. Mathews, Dirk Claeys, Frédéric Jaisser, Käthi Geering, Jean-Daniel Horisberger, Jean-Pierre Kraehenbuhl, and Bernard C. Rossier. “Primary structure and functional expression of the mouse and frog agr-subunit of the gastric H+-K+-ATPase.” Page C1214: the following sentence was inadvertently omitted from the acknowledgments: Paul M. Mathews and Dirk Claeys contributed equally to this study.
Effects of okadaic acid and intracellular Cl- on Na(+)-K(+)-Cl- cotransport
Authors: Altamirano A. A. , Breitwieser G. E. , Russell J. M.
Published: December 2017

The Na(+)-K(+)-Cl- cotransporter of the squid giant axon requires ATP and is inhibited by intracellular Cl- (Cli-) in a concentration-dependent manner ([Cl-]i > or = 200 mM completely inhibits the cotransporter). In the present study we address the question of whether inhibition of cotransport by Cli- is due to a Cl(i-)-induced increase of protein phosphatase activity. Intracellular dialysis was used to apply the phosphatase inhibitor okadaic acid (OKA) under conditions of [Cl-]i at 0, 150, or 300 mM during measurement of cotransporter-mediated unidirectional Cl- influx into axons. At 0 mM [Cl-]i, the application of 250 nM OKA had no effect on the cotransport-mediated Cl- influx when axons were dialyzed with the normal intracellular ATP concentration ([ATP]i = 4 mM). Reduction of [ATP] to 50 microM resulted in a significant decrease of the bumetanide-sensitive CL- influx, which could be partially reversed by OKA treatment. Similarly, in ATP-limited axons with [Cl-]i at 150 mM, cotransporter influx was partially stimulated by treatment with OKA. However, axons dialyzed with 300 mM [Cl-]i ([ATP]i = 50 microM) had no measurable cotransport influx, nor was subsequent treatment with OKA able to induce a cotransport-mediated Cl- influx. We conclude that the inhibition of cotransport caused by Cli- is not the result of an increase in the OKA-sensitive protein phosphatase activity.
cAMP evokes a rise in intracellular Na+ mediated by Na+ pump inhibition in rat aortic smooth muscle cells
Authors: Borin M. L.
Published: December 2017

The effect of adenosine 3',5'-cyclic monophosphate (cAMP) on intracellular Na+ concentration ([Na+]i) was studied in primary cultured vascular smooth muscle cells from rat aorta. [Na+]i was measured using digital imaging of cells loaded with the Na(+-)sensitive fluorescent dye sodium-bonding benzofuran isophthalate. The cAMP level was raised by 1) the membrane-permeable cAMP derivative 8-bromoadenosine 3',5'-cyclic monophosphate, 2) the combination of the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, and 3) the beta-adrenoceptor agonist isoproterenol. All three methods caused a dose-dependent continuous rise in [Na+]i during 40-60 min of observations. A rise in [Na+]i may be caused by stimulation of the Na+ influx and/or inhibition of Na+ efflux; therefore, the involvement of both mechanisms was studied. Elevation of the cAMP level had no effect on Na+ influx, measured as the rate of rise of [Na+]i when Na+ efflux was inhibited with 1 mM ouabain. In contrast, elevation of the cAMP level attenuated Na+ efflux, measured as the rate of decline of [Na+]i in Na(+)-loaded cells exposed to Na(+)-free medium. cAMP-induced inhibition of Na+ efflux was not observed when the Na+ pump was inhibited; therefore, cAMP inhibits the Na+ pump-mediated component of Na+ efflux. Agents that raise the cAMP level also inhibited, in a dose-dependent fashion, ouabain-sensitive 86Rb uptake in rat aortic rings. The latter observation confirms that the cAMP-induced inhibition of the Na+ pump occurs both in cultured cells and in the native tissue.(ABSTRACT TRUNCATED AT 250 WORDS)
Na+/H+ exchange modulates acidification of early rat liver endocytic vesicles
Authors: Van Dyke R. W.
Published: December 2017

Endocytic vesicles are acidified by an electrogenic vacuolar H(+)-ATPase. These studies examined whether rat liver endosomes also exhibit Na+/H+ exchange and whether this transporter alters acidification. Extravesicular Na+ caused saturable proton efflux from acidified endosomes with a Michaelis constant for Na+ of 7.6 mM, whereas an in-to-out Na+ gradient caused endosome acidification without MgATP and accelerated acidification with MgATP. Na(+)-driven proton fluxes were little altered by valinomycin or carbonyl cyanide m-chlorophenylhydrazone. Na+/H+ exchange was inhibited by Li+ but was not affected by K+, Cl-, amiloride (1 mM), or 5-(N,N-dimethyl) amiloride (0.1 mM). Na+/H+ exchange was detected in “early” but not in “late” liver endosomes or in lysosomes. These data suggest that early rat liver endosomes exhibit Na+/H+ exchange that, immediately after endosome formation, may accelerate vesicular acidification. Because of its insensitivity to amiloride, this exchanger may be a pharmacologically altered form of Na+/H+ exchanger-1 or a new isoform.
Functional characterization of phosphoinositide-specific phospholipase C-beta 1 and -beta 3 in intestinal smooth muscle
Authors: Murthy K. S. , Makhlouf G. M.
Published: December 2017

Soluble and membrane phosphoinositide-specific phospholipases obtained separately from dispersed circular and longitudinal intestinal muscle cells were characterized for substrate specificity and G protein dependence using selective antibodies to various isoforms of phospholipase C (PLC) and G protein subunits. Western blot analysis disclosed the presence of the main PLC isozymes, PLC-gamma 1, PLC-delta 1, and PLC-beta 1. Soluble PLC from circular and longitudinal muscle was stimulated by guanosine 5'-O-(3-thiophosphate) and inhibited by PLC-beta 1 antibody (80-90%) and PLC-beta 3 antibody (approximately 25%) but not by G protein antibodies. Membrane PLC from circular and longitudinal muscle was stimulated by cholecystokinin octapeptide (CCK-8) and inhibited selectively by PLC-beta 1 antibody (85%), PLC-beta 3 antibody (15%), and G alpha q/11 antibody (90%). CCK-8-induced contraction in permeabilized circular muscle cells was also selectively inhibited by PLC-beta 1 antibody (76%), PLC-beta 3 antibody (24%), and G alpha q/11 antibody (86%). The combined effects of PLC-beta 1 and PLC-beta 3 antibodies on PLC activity and muscle contraction were additive, causing complete inhibition. Soluble and membrane PLC from circular and longitudinal muscle were immunologically similar but functionally different. The enzymes from circular muscle preferentially hydrolyzed endogenous and exogenous phosphatidylinositol 4,5-biphosphate (PIP2), confirming previous findings of preferential hydrolysis of PIP2 in dispersed intestinal circular muscle cells
Human ATP1AL1 gene encodes a ouabain-sensitive H-K-ATPase
Authors: Modyanov N. N. , Mathews P. M. , Grishin A. V. , Beguin P. , Beggah A. T. , Rossier B. C. , Horisberger J. D. , Geering K.
Published: December 2017

The cDNA for ATP1AL1, the fifth member of the human Na-K-adenosinetriphosphatase (ATPase)/H-K-ATPase gene family, was recently cloned (A. V. Grishin, V. E. Sverdlov, M. B. Kostina, and N. N. Modyanov. FEBS Lett. 349: 144-150, 1994). The encoded protein (ATP1AL1) has all the primary structural features common to the catalytic alpha-subunit of ion-transporting P-type ATPases and is similar (63-64% identity) to the Na-K-ATPase alpha-subunit isoforms and the gastric H-K-ATPase alpha-subunit. In this study, ATP1AL1 was expressed in Xenopus laevis oocytes in combination with the beta-subunit of rabbit gastric H-K-ATPase. The functional properties of the stable alpha/beta-complex were studied by 86Rb+ uptake and demonstrated that ATP1AL1 is a novel human K(+)-dependent ATPase [apparent half-constant activation/(K1/2) for K+ approximately 375 microM)]. ATP1AL1-mediated inward K+ transport was inhibited by ouabain (inhibition constant approximately 13 microM) and was found to be inhibited by high concentrations of SCH-28080 (approximately 70% at 500 microM). ATP1AL1 expression resulted in the alkalinization of the oocytes' cytoplasm and ouabain-sensitive proton extrusion, as measured with pH-sensitive microelectrodes. These data argue that ATP1AL1 is the catalytic alpha-subunit of a human nongastric P-type ATPase capable of exchanging extracellular potassium for intracellular protons.
Responsiveness of mutants of NHE1 isoform of Na+/H+ antiport to osmotic stress
Authors: Bianchini L. , Kapus A. , Lukacs G. , Wasan S. , Wakabayashi S. , Pouyssegur J. , Yu F. H. , Orlowski J. , Grinstein S.
Published: December 2017

Hypertonic activation of NHE1, the ubiquitous Na+/H+ exchanger, plays a central role in cell volume regulation, yet little is known about the underlying mechanism. We probed the osmotic responsiveness of full-length and truncated constructs of NHE1 transfected into cells lacking endogenous antiport activity. The hypertonic stimulation of NHE1 was preserved after heterologous transfection of the full-length NHE1 or of constructs truncated at positions 698 or 703. In contrast, mutants truncated at position 635 (delta 635) failed to respond to osmotic challenge. Transfectants (delta 635) behaved as if constitutively activated, having a permanently elevated cytosolic pH (pHi) under isotonic, unstimulated conditions. The delta 635 mutant displayed H+ binding with high affinity and low cooperativity. Constructs delta 582 or delta 566 had a reduced H+ sensitivity and were therefore inactive at resting pHi. Such cells were unresponsive to osmotic stress near physiological pHi but could be activated by shrinking after an acid load. Jointly, these results suggest that the H+ affinity and high cooperativity of the antiporter, earlier attributed to a single “modifier site,” can be varied independently and are probably controlled by different regions of the molecule. The data indicate that volume or osmolarity-sensitive site(s) exist between the NH2-terminus and residue 566. This putative volume-sensitive site is therefore different from the site(s) postulated to mediate the stimulatory effects of calcium and growth factors.
Inhibition and acceleration of Na+/Ca2+/K+ exchange fluxes by Ag+ in bovine retinal rod outer segments
Authors: Schnetkamp P. P. , Szerencsei R. T. , Tucker J. E. , Van den Elzen P.
Published: December 2017

The effect of Ag+ on Ca2+ fluxes mediated by the retinal rod Na+/Ca2+/K+ exchanger was investigated in intact bovine rod outer segments (ROS). Intracellular Na+ concentration ([Na+]in)-dependent Ca2+ influx and extracellular Na+ concentration ([Na+]out)-dependent Ca2+ efflux were monitored by changes in cytosolic free Ca2+ measured with the fluorescent Ca(2+)-indicating dye fluo 3. Ag+ was the most effective inhibitor of Na+/Ca2+/K+ exchange fluxes described to date, with half-maximal inhibition observed at 2-8 microM Ag+. Inhibition by Ag+ could be reversed by addition of beta-mercaptoethanol but not by addition of cysteine. Reversal by beta-mercaptoethanol resulted in a marked acceleration of [Na+]out-dependent lowering of cytosolic free Ca2+ but not of [Na+]in-dependent Ca2+ influx. We suggest that Ag+ inhibits and accelerates Na+/Ca2+/K+ exchange fluxes by binding to cysteine residues on the cytosolic surface of the exchanger protein.
Effects of fluoride and cholera and pertussis toxins on sensory transduction in the carotid body
Authors: Cachero T. G. , Rocher A. , Rigual R. J. , Gonzalez C.
Published: December 2017

The regulation of the chemoreceptor cell function by G proteins has been studied by measuring the release of 3H-labeled catecholamines ([3H]CA) in carotid bodies (CBs) treated with fluoride, cholera toxin (CTX), and pertussis toxin (PTX). Fluoride augmented the basal release of [3H]CA in a dose- (5-20 mM) and Ca(2+)-dependent manner. Nisoldipine (1 microM) and ethylisopropyl amiloride (EIPA; 10 microM) inhibited this effect by approximately 60%, and both drugs combined inhibited it in full. BAY K 8644 (1 microM) doubled the effect of fluoride. The effects of fluoride on the stimulus-evoked release of [3H]CA varied with the type of stimulus and the duration of the treatment. Simultaneous application of fluoride with the stimulus increased by five times the release evoked by hypoxia and by two times that by K+ and dinitrophenol (DNP). Preincubation with fluoride for 1 h caused an inhibition (approximately 70%) of the release evoked by high K+ and veratridine, whereas that evoked by DNP and low PO2 was still augmented (approximately 2 times). Preincubation (4 h) of the CBs with CTX (3 micrograms/ml) reduced by 54% the release of [3H]CA evoked by 35 mM K+ but did not affect that evoked by low PO2 or DNP. A similar treatment with PTX (1 microgram/ml) affected only the release of [3H]CA evoked by DNP, reducing it by 65%. The data show that fluoride, CTX, and PTX have different effects on the release of [3H]CA evoked by high external K+, DNP, and low PO2, indicating that the stimulus-secretion coupling process for each stimulus is differently regulated by G proteins.
Dexamethasone enhances expression of mitochondrial oxidative phosphorylation genes in rat distal colon
Authors: Rachamim N. , Latter H. , Malinin N. , Asher C. , Wald H. , Garty H.
Published: December 2017

Dexamethasone and aldosterone are major activators of Na+ reabsorption in tight epithelia. The genes whose expression mediates the steroid actions are mostly unknown. To identify such genes, we performed differential screening of a rat colon cDNA library with total 32P-labeled cDNA probes reverse transcribed from steroid-stimulated and steroid-depleted poly(A)+ RNA. Several cDNAs whose corresponding mRNA is enhanced two- to threefold after dexamethasone injection were identified. Partial sequencing indicated that four of them code for subunits of cytochrome-c oxidase and 16S mitochondrial mRNA. The dexamethasone-induced increase in mitochondrial RNA abundance could not be mimicked by a low-salt diet, found to increase plasma aldosterone from 1.0 +/- 0.1 to 12.8 +/- 1.4 nM. Induction of mitochondrial genes by adrenal steroids may serve to prevent limitation of transport by the ATP supply to the Na(+)-K+ pump under conditions of maximal stimulation of Na+ transport.
Ins(1,4,5)P3 activates Drosophila cation channel Trpl in recombinant baculovirus-infected Sf9 insect cells
Authors: Dong Y. , Kunze D. L. , Vaca L. , Schilling W. P.
Published: December 2017

The trp-like (trpl) gene product (Trpl) is thought to form a nonselective cation channel important for signal transduction in Drosophila photoreceptor cells. This channel may be the insect homologue of mammalian channels involved in Ca2+ signal transduction. To determine the mechanism of receptor-mediated activation of Trpl, whole cell membrane currents were examined in Sf9 insect cells after infection with recombinant baculovirus. Stimulation by bradykinin increased whole cell Trpl currents three- to fivefold. Similar activation of Trpl was observed by inclusion of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the pipette solution during whole cell recordings. These currents were 1) not seen in noninfected cells or in cells expressing only the B2 receptor, 2) mimicked by D-myo-inositol 2,4,5-trisphosphate, and 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate, 3) not seen with D-myo-inositol 1,4-bisphosphate or D-myo-inositol 1,3,4,5-tetrakisphosphate, and 4) blocked by heparin, but not by de-N-sulfated heparin. In contrast, Trpl currents were unaffected by thapsigargin. These results demonstrate that the Trpl cation channel is activated by Ins(1,4,5)P3 in a heparin-sensitive fashion. Regulation of channel activity by Ins(1,4,5)P3 may occur by a number of mechanisms, including direct binding of Ins(1,4,5)P3 to the Trpl channel or direct physical interaction between the Ins(1,4,5)P3 receptor/Ca(2+)-release channel of the endoplasmic reticulum and the Trpl protein.
Unitary conductance of Na+ channel isoforms in cardiac and NB2a neuroblastoma cells
Authors: Baumgarten C. M. , Dudley S. C. , Rogart R. B. , Fozzard H. A.
Published: December 2017

Unitary conductances of native Na+ channel isoforms (gamma Na) have been determined under a variety of conditions, making comparisons of gamma Na difficult. To allow direct comparison, we measured gamma Na in cell-attached patches on NB2a neuroblastoma cells and rabbit ventricular myocytes under identical conditions [pipette solution (in mM): 280 Na+ and 2 Ca2+, pH 7.4; 10 degrees C]. gamma Na of NB2a channels, 22.9 +/- 0.9 pS, was 21% greater than that of cardiac channels, 18.9 +/- 0.9 pS. In contrast, respective extrapolated reversal potentials, +62.4 +/- 4.6 and +57.9 +/- 5.1 mV, were not significantly different. Several kinetic differences between the channel types were also noted. Negative to -20 mV, mean open time (MOT) of the NB2a isoform was significantly less than that of cardiac channels, and, near threshold, latency to channel opening decayed more rapidly in NB2a. On the basis of analysis of MOT between -60 and 0 mV, the rate constants at 0 mV for the open-to-closed (O-->C) and open-to-inactivated (O-->I) transitions were 0.42 +/- 0.11 and 0.47 +/- 0.11 ms-1 in NB2a and 0.10 +/- 0.06 and 1.19 +/- 0.07 ms-1 in myocytes. The slope factors were -38.9 +/- 8.7 and +10.7 +/- 6.1 mV in NB2a and -27.3 +/- 7.1 and +23.7 +/- 4.9 mV in myocytes. Transition rate constants were significantly different in NB2a and cardiac cells, but voltage dependence was not.
Alterations in reactive oxygen, pH, and calcium in astrocytoma cells during lethal injury
Authors: Wu Y. , Taylor B. M. , Sun F. F.
Published: December 2017

Exposure of cultured human astrocytoma cells to iodoacetic acid results in rapid depletion of cellular ATP and cell death. Pathophysiological changes in the injured cells, including formation of reactive oxygen species (ROS), cell viability, glutathione, pH, and cytosolic calcium, were characterized at the cellular level via fluorescence microscopy. After iodoacetic acid treatment, cellular ATP and intracellular glutathione fell sharply to undetectable levels within 2 h. ROS, as detected by the oxidation of dichlorofluorescein, appeared in 20 min and reached a maximum before the loss of membrane integrity. Cells became acidotic within 10 min. Cytosolic free calcium concentration exhibited a slow increase and then a sharp influx shortly before the rupture of the cell membrane. The addition of lipophilic antioxidants, nordihydroguaiaretic acid or the troloxamine U-78517F, eliminated the accumulation of ROS and delayed the onset of cell death without affecting other parameters observed in the early phase of the injury. We conclude that ROS is formed and may play important roles during lethal cell injury caused by energy depletion.
Shrinkage activates a nonselective conductance: involvement of a Walker-motif protein and PKC
Authors: Nelson D. J. , Tien X. Y. , Xie W. , Brasitus T. A. , Kaetzel M. A. , Dedman J. R.
Published: December 2017

The ability of all cells to maintain their volume during an osmotic challenge is dependent on the regulated movement of salt and water across the plasma membrane. We demonstrate the phosphorylation-dependent gating of a nonselective conductance in Caco-2 cells during cellular shrinkage. Intracellular application of exogenous purified rat brain protein kinase C (PKC) resulted in the activation of a current similar to that activated during shrinkage with a Na(+)-to-Cl- permeability ratio of approximately 1.7:1. To prevent possible PKC- and/or shrinkage-dependent activation of cystic fibrosis transmembrane regulator (CFTR), which is expressed at high levels in Caco-2 cells, a functional anti-peptide antibody, anti-CFTR505-511, was introduced into the cells via the patch pipette. Anti-CFTR505-511, which is directed against the Walker motif in the first nucleotide binding fold of CFTR, prevented the PKC/shrink-age current activation. The peptide CFTR505-511 also induced current inhibition, suggesting the possible involvement of a regulatory element in close proximity to the channel that shares sequence homology with the first nucleotide binding fold of CFTR and whose binding to the channel is required for channel gating.
Transcriptional regulation of endothelial constitutive PGHS-1 expression by phorbol ester
Authors: Xu X. M. , Tang J. L. , Hajibeigi A. , Loose-Mitchell D. S. , Wu K. K.
Published: December 2017

Human endothelial cells contain two isoforms of prostaglandin H synthase (PGHS). PGHS-1 is constitutively expressed, whereas PGHS-2 is inducible. To determine whether expression of PGHS-1 is regulated, we treated cultured human umbilical vein endothelial cells (HUVEC) with phorbol 12-myristate 13-acetate (PMA) or its inactive analogue and measured PGHS-1 mRNA levels by Northern analysis and competitive polymerase chain reaction. PMA increased PGHS-1 mRNA levels determined by both techniques in a time- and concentration-dependent manner. The mRNA level was increased about twofold over the basal level after 4-6 h of PMA (10-50 nM) treatment. The level of PGHS-1 protein was similarly increased by PMA. Stimulation of PGHS-1 mRNA levels was abrogated by cycloheximide, actinomycin D, staurosporine, or calphostin C. The 5'-promoter activity of human PGHS-1 gene was increased twofold over the basal level by PMA in NS-20 cells. These results indicate that the constitutive PGHS-1 in HUVEC is transcriptionally stimulated by PMA in a protein kinase C-dependent manner.
Role of phosphodiesterase II in cross talk between cGMP and cAMP in human neuroblastoma NB-OK-1 cells
Authors: Delporte C. , Poloczek P. , Winand J.
Published: December 2017

Cyclic nucleotides levels and cyclic nucleotide phosphodiesterase (PDE) activities were measured in human neuroblastoma NB-OK-1 cells possessing atrial natriuretic peptide (ANP) receptors of the A type and pituitary adenylate cyclase activating polypeptide (PACAP)-preferring receptors. Adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) degradation were interrelated since the increase in cGMP, induced by ANP-(99-126), stimulated the hydrolysis of cAMP by PDE isoenzyme II. In intact NB-OK-1 cells, the levels of cAMP and cGMP attained in the presence of, respectively, 1 nM PACAP-(1-27) and 10 nM ANP-(99-126), and in the absence or presence of PDE inhibitors, strongly suggested that cAMP hydrolysis was mainly achieved by isoenzyme IV, and to a lesser extent by isoenzymes I, II, and III, while cGMP was degraded by isoenzymes I, II, III, and V. More than one-half of total cAMP- and cGMP-hydrolyzing activities was present in the membrane-bound fraction. Cyclic nucleotide PDE activities separated by anion-exchange chromatography showed that isoenzymes III and IV were mainly present in the membrane fraction, while isoenzymes I, II, and V were in the cytosolic fraction.
Physiologically regulated alternative splicing patterns of fast troponin T RNA are conserved in mammals
Authors: Briggs M. M. , Schachat F.
Published: December 2017

NH2-terminal isoforms of fast troponin T (TnT) are generated by alternative splicing of fast TnT RNA transcripts. Significantly different estimates for the number of isoforms have been obtained by nucleic acid and protein chemical studies. To resolve this controversy and to determine whether specific 5'-splicing patterns correlate with fiber phenotype, we generated representative populations of 5'-TnT cDNAs from the TnT mRNAs expressed in a set of physiologically and anatomically diverse skeletal muscles. Sequencing and restriction enzyme analyses revealed a total of nine cDNAs that encode the six adult and three perinatal NH2-terminal TnT variants previously identified. Three major 5'-splicing pathways (the TnT1f, TnT2f, and TnT3f patterns) account for more than 90% of the TnT mRNAs and proteins in adult rabbit skeletal muscle. Comparative studies in rats, mice, and humans show that these splicing patterns are conserved and that fast-twitch fibers that are primarily glycolytic utilize the TnT1f and TnT2f patterns preferentially, whereas fast-twitch fibers that are primarily oxidative use the TnT1f and TnT3f patterns preferentially.
Cloning of an aquaporin homologue present in water channel containing endosomes of toad urinary bladder
Authors: Siner J. , Paredes A. , Hosselet C. , Hammond T. , Strange K. , Harris H. W.
Published: December 2017

Regulation of total body water balance in amphibians by antidiuretic hormone (ADH) contributed to their successful colonization of terrestrial habitats approximately 200-300 million years ago. In the mammalian kidney, ADH modulates epithelial cell apical membrane water permeability (Pf) by fusion and retrieval of cytoplasmic vesicles containing water channel proteins called aquaporins (AQPs). To determine the role of AQPs in ADH-elicited Pf in amphibians, we have identified and characterized a unique AQP from Bufo marinus called AQP toad bladder (AQP-TB). AQP-TB possesses many structural features common to other AQPs, AQP-TB is expressed abundantly in ADH-responsive tissues, including toad urinary bladder and skin as well as lung, skeletal muscle, kidney, and brain. In a manner identical to that reported for the mammalian ADH-elicited water channel AQP2, AQP-TB expression is increased significantly by intervals of dehydration or chronic ADH stimulation. However, expression of AQP-TB protein in Xenopus laevis oocytes does not significantly increase oocyte Pf. The lack of expression of functional AQP-TB water channels in oocytes may result from intracellular sequestration of AQP-TB due to the presence of a YXRF sequence motif present in its carboxyterminal domain.
T cell-monocyte interactions regulate epithelial physiology in a coculture model of inflammation
Authors: McKay D. M. , Croitoru K. , Perdue M. H.
Published: December 2017

We have examined the effect of T cell activation, with or without monocytes, on epithelial electrolyte transport and barrier functions. Confluent monolayers of human T84 epithelial cells were cocultured (1-3 days) with peripheral blood mononuclear cells (PBM) or T cells activated by anti-CD3 antibody. Monolayers were then mounted in Ussing chambers, and changes in ion transport (indicated by short-circuit current, Isc) and barrier (indicated by resistance and radiolabeled probe fluxes) functions were assessed. Coculture with activated PBM or conditioned medium from these cells altered the transport (decreased Isc responses to carbachol and forskolin) and barrier (decreased resistance and increased fluxes of [3H]mannitol and 51Cr-EDTA) properties of T84 monolayers. In contrast, coculture with equal numbers of T cells activated in the absence of monocytes did not significantly affect epithelial physiology. Monocytes treated with conditioned media from activated T cells evoked epithelial abnormalities similar to those caused by culture with activated PBM. Total correction of epithelial abnormalities was achieved only by treating T cell-conditioned medium with anti-interferon-gamma (IFN-gamma) before addition to monocytes, as well as addition of anti-tumor necrosis factor-alpha (TNF-alpha) to the coculture. Exogenous recombinant IFN-gamma and TNF-alpha added to T84 monolayers did not mimic the physiological changes induced by immune cells; addition of these cytokines to monocytes did reproduce the effects. We conclude that T cell-derived IFN-gamma activates monocytes to release TNF-alpha and other soluble mediators, resulting in epithelial dysfunction.
Transmembrane chloride currents in human atrial myocytes
Authors: Li G. R. , Feng J. , Wang Z. , Nattel S.
Published: December 2017

The present study was designed to evaluate the presence of basal, swelling-induced, and cAMP-dependent Cl- currents in human atrial myocytes studied with the whole cell patch-clamp technique. Under basal conditions, a small outwardly rectifying background conductance was noted that reversed close to 0 mV and was not altered by Cl- replacement. Isoproterenol (1 microM), forskolin (3 microM), and 8-bromoadenosine 3',5'-cyclic monophosphate (50 microM) did not increase membrane conductance, even when responsiveness to isoproterenol was confirmed by an increase in Ca2+ current and when perforated-patch techniques (nystatin) were used. Exposure to hyposmotic solutions increased cell volume and induced a whole cell conductance that showed outward rectification, was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (100 microM), and responded to changes in Cl- gradient in a fashion consistent with a Cl(-)-selective conductance, with estimated relative permeabilities of 1, 0.25, and 0.07 for Cl-, methanesulfonate, and aspartate, respectively. The results suggest that human atrial cells lack basal and adenosine 3',5'-cyclic monophosphate-dependent Cl- current but manifest a substantial Cl- conductance in the presence of cell swelling.
Insulin sensitizes beta-agonist and forskolin-stimulated lipolysis to inhibition by 2',5'-dideoxyadenosine
Authors: Gokmen-Polar Y. , Coronel E. C. , Bahouth S. W. , Fain J. N.
Published: December 2017

In isolated rat adipocytes incubated in the absence of insulin, 2',5'-dideoxyadenosine blocked the increase in total adenosine 3',5'-cyclic monophosphate (cAMP) accumulation due to beta 1- or beta 3-catecholamine agonists and forskolin without affecting their stimulation of lipolysis. The inhibition of cAMP accumulation by 2',5'-dideoxyadenosine was not reflected in the total cytosolic cAMP-dependent protein kinase A activity, suggesting that the inhibition of cAMP occurred in cellular compartments distinct from those involved in the regulation of bulk protein kinase A activity. However, there was a good correlation between effects of lipolytic agents on cytosolic protein kinase A activity in fat cell extracts and lipolysis. Furthermore, it was possible to see an inhibition of the increase due to beta-agonists in cAMP accumulation, protein kinase A activity, and lipolysis by 2',5'-dideoxyadenosine in the presence of insulin. These data suggest that the readily measurable accumulation of cAMP seen with catecholamines in the absence of insulin is in a compartment separate from that involved in protein kinase A activation.