bluuebunny/crossref_metadata_2025 huggingface dataset:
α2-Adrenergic receptors increase cell migration and decrease F-actin labeling in rat aortic smooth muscle cells
Authors: Richman Jeremy G. , Regan John W.
Published: December 2017

Vascular wound healing and such pathologies as atherosclerosis and restenosis are characterized by migration and proliferation of the smooth muscle cells of the media after denudation of the intima. To explore possible roles that α2-adrenergic receptors (α2-ARs) might have in these cellular responses, we characterized the α2-ARs present in explant-derived cultures of rat aortic smooth muscle (RASM) cells. The results of immunofluorescence microscopy and reverse transcription followed by the polymerase chain reaction indicated that all three α2-AR subtypes (α2A, α2B, and α2C) were initially present. Mitogen-activated protein kinase activity in the RASM cells was stimulated fivefold over basal by the α2-selective agonist dexmedetomidine (Dex) and was blocked by coincubation with the α2-selective antagonist rauwolscine (RW) or by preincubation of the cells with the Gi/Go-protein inhibitor pertussis toxin. α2-AR activation by Dex did not promote cell proliferation, as measured by the incorporation of [3H]thymidine. However, Dex significantly increased RASM cell migration, and antagonist blocked this effect. Incubation of RASM cells with Dex also produced a marked decrease in F-actin labeling, which again was prevented by coincubation with RW. The evidence clearly reveals the presence of functional α2-ARs in RASM cells. The involvement of α2-AR activation with cytoskeletal changes and cell migration is novel and indicates a potential role of these receptors in vascular wound healing and pathogenesis.
A basolateral sorting signal is encoded in the α-subunit of Na-K-ATPase
Authors: Muth T. R. , Gottardi C. J. , Roush D. L. , Caplan M. J.
Published: December 2017

Na-K-ATPase and H-K-ATPase are highly homologous ion pumps that exhibit distinct plasma membrane distributions in epithelial cells. We have studied the α-subunits of these heterodimeric pumps to identify the protein domains responsible for their polarized sorting. A chimeric α-subunit construct (N519H) was generated in which the first 519 amino acid residues correspond to the Na-K-ATPase sequence and the remaining 500 amino acids are derived from the H-K-ATPase sequence. In stably transfected LLC-PK1 cell lines, we found that the N519H chimera is restricted to the basolateral surface under steady-state conditions, suggesting that residues within the NH2-terminal 519 amino acids of the Na-K-ATPase α-subunit contain a basolateral sorting signal. H-K-ATPase β-subunit expressed alone in LLC-PK1 cells accumulates at the apical surface. When coexpressed with N519H, the H-K-ATPase β-subunit assembles with this chimera and accompanies it to the basolateral surface. Thus the NH2-terminal basolateral signal in the Na-K-ATPase α-subunit masks or is dominant over any apical sorting information present in the β-polypeptide. In gastric parietal cells, the H-K-ATPase β-subunit targets the H-K-ATPase to an intracellular vesicular compartment which fuses with the plasma membrane in response to secretagogue stimulation. To test whether the chimera-H-K-ATPase β-subunit complex is directed to a similar compartment in LLC-PK1cells, we treated transfected cells with drugs that raise intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) levels. Elevation of cytosolic cAMP increased the surface expression of both the N519H chimera and the H-K-ATPase β-subunit. This increase in surface expression, however, appears to be the result of transcriptional upregulation and not recruitment of chimera to the surface from a cAMP-inducible compartment.
Human orbital fibroblasts are activated through CD40 to induce proinflammatory cytokine production
Authors: Sempowski Gregory D. , Rozenblit Julia , Smith Terry J. , Phipps Richard P.
Published: December 2017

CD40 is an important signaling and activation antigen found on certain bone marrow-derived cells. Recently, CD40 has also been shown to be expressed by nonhematopoietic cells, including certain human fibroblasts, but not others. Little is known about the function of CD40 on fibroblasts. The current study investigates the hypothesis that CD40 is expressed on orbital fibroblasts and represents a pathway for interaction between these fibroblasts and CD40 ligand-expressing cells, such as T lymphocytes and mast cells. We report here that orbital connective tissue fibroblasts, obtained from normal donors and from patients with severe thyroid-associated ophthalmopathy (TAO), express functional CD40. CD40 is upregulated ∼10-fold by interferon-γ (500 U/ml) treatment for 72 h. These fibroblasts become activated through triggering of CD40 with CD40 ligand (CD40L). This is evidenced by nuclear translocation of nuclear factor-κB and induction of the proinflammatory and chemoattractant cytokines interleukin-6 and interleukin-8, respectively. These data support the concept that cognate interactions between orbital fibroblasts and infiltrating T lymphocytes, via the CD40-CD40L pathway, may promote the tissue remodeling observed in TAO and other inflammatory diseases of the orbit. Disruption of the CD40-CD40L interaction may represent a therapeutic intervention to reduce the inflammatory components of TAO, which remains a vexing clinical problem.
Ontogeny of malate-aspartate shuttle capacity and gene expression in cardiac mitochondria
Authors: Scholz Thomas D. , Koppenhafer Stacia L. , Teneyck Cynthia J. , Schutte Brian C.
Published: December 2017

Developmental downregulation of the malate-aspartate shuttle has been observed in cardiac mitochondria. The goals of this study were to determine the time course of the postnatal decline and to identify potential regulatory sites by measuring steady-state myocardial mRNA and protein levels of the mitochondrial proteins involved in the shuttle. By use of isolated porcine cardiac mitochondria incubated with saturating concentrations of the cytosolic components of the malate-aspartate shuttle, shuttle capacity was found to decline by ∼50% during the first 5 wk of life (from 921 ± 48 to 531 ± 53 nmol ⋅ min−1 ⋅ mg protein−1). Mitochondrial aspartate aminotransferase mRNA levels were greater in adult than in newborn myocardium. mRNA levels of mitochondrial malate dehydrogenase in adult cardiac tissue were 224% of levels in newborn tissue, whereas protein levels were 54% greater in adult myocardium. Aspartate/glutamate carrier protein levels were also greater in adult than in newborn tissue. mRNA and protein levels of the oxoglutarate/malate carrier were increased in newborn myocardium. It was concluded that 1) myocardial malate-aspartate shuttle capacity declines rapidly after birth, 2) divergence of mitochondrial malate dehydrogenase mRNA and protein levels during development suggests posttranscriptional regulation of this protein, and 3) the developmental decline in malate-aspartate shuttle capacity is regulated by decreased oxoglutarate/malate carrier gene expression.
Nitric oxide inhibits superoxide production by inflammatory polymorphonuclear leukocytes
Authors: Ródenas Jesús , Mitjavila M. Teresa , Carbonell Teresa
Published: December 2017

Nitric oxide (NO ⋅) has a complex role in the inflammatory response. In this study, we modified the levels of endogenous NO ⋅ in vivo in an acute model of inflammation and evaluated the interactions between NO ⋅ and superoxide anion ([Formula: see text]) produced by polymorphonuclear leukocytes (PMNs) accumulated in the inflamed area. We injected phosphate-buffered saline (control group), 6 μmol ofl- N 5-(1-iminoethyl)ornithine (l-NIO group), or 6 μmol ofl-arginine (l-arginine group) into the granuloma pouch induced by carrageenan in rats.[Formula: see text] plus[Formula: see text] (indicative of NO ⋅ generation) was 188 nmol in the exudate of the control group, but it decreased in the l-NIO group ( P < 0.05) and increased in thel-arginine group ( P < 0.05). When PMNs from treated rats were incubated in vitro, the production of superoxide anion ([Formula: see text]) decreased by ∼46% in thel-arginine group. Furthermore,[Formula: see text] was inhibited in PMNs whenl-arginine was added to the incubation medium before phorbol 12-myristate 13-acetate stimulation but not when added simultaneously. Our results suggest a protective role for NO ⋅ in inflammation, through the inactivation of NADPH oxidase and the consequent impairment of[Formula: see text] production for cell-mediated injury.
Age-dependent reductions in A1 adenosine receptor expression in rat testes
Authors: Bhat Satyanarayan G. , Wilson Marty , Ramkumar Vickram
Published: December 2017

The rat testis expresses high levels of A1 adenosine receptors (A1 AR) that couple to the inhibition of adenylyl cyclase activity. However, the physiological role of these receptors in the testis is not clear. Previous studies have documented a number of changes in the testis associated with the aging process. The goal of this study was to assess whether alteration in the expression and function of the testicular A1 AR occurs in aging, using the Fischer 344 rats as an aging model. Quantitation of A1 AR expression by radioligand binding of [3H]1,3-dipropyl-8-cyclopentylxanthine, an antagonist radioligand, indicates reductions in receptor number by 35 ± 13.3 and 53 ± 18.2% in 18- and 25-mo-old rats, respectively, compared with 3-mo-old rats. Similar reductions in A1 AR expression were determined using Western blotting and receptor autoradiography. Quantitation of the Gi proteins using selective antibodies indicate age-dependent reductions in the levels of αi-1,2-, αi-3- and β-subunits. Furthermore, the modulatory influences of guanosine 5′- O-(3-thiotriphosphate) on the binding of agonist and antagonist radioligands to the A1 AR were substantially reduced. Northern blotting analysis of rat testicular poly(A)+ RNA indicates both a 3.4-kb transcript and a 5.6-kb transcript that hybridized to the canine A1 AR cDNA probe. The levels of the 5.6-kb transcript were decreased by 24 ± 18 and 52 ± 3% in the 18- and 25-mo-old rats, respectively, compared with the 3-mo-old rats. These results indicate age-dependent deficits in the A1 AR signal transduction pathway in the testes and predict concomitant reductions in the action of adenosine.
Model of β-cell mitochondrial calcium handling and electrical activity. II. Mitochondrial variables
Authors: Magnus Gerhard , Keizer Joel
Published: December 2017

In the preceding article [ Am. J. Physiol. 274 ( Cell Physiol. 43): C1158–C1173, 1998], we describe the development of a kinetic model for the interaction of mitochondrial Ca2+ handling and electrical activity in the pancreatic β-cell. Here we describe further results of those simulations, focusing on mitochondrial variables, the rate of respiration, and fluxes of metabolic intermediates as a function of d-glucose concentration. Our simulations predict relatively smooth increases of O2consumption, adenine nucleotide transport, oxidative phosphorylation, and ATP production by the tricarboxylic acid cycle asd-glucose concentrations are increased from basal to 20 mM. On the other hand, we find that the active fraction of pyruvate dehydrogenase saturates, due to increases in matrix Ca2+, near the onset of bursting electrical activity and that the NADH/NAD+ ratio in the mitochondria increases by roughly an order of magnitude as glucose concentrations are increased. The mitochondrial ATP/ADP ratio increases by factor of <2 between thed-glucose threshold for bursting and continuous spiking. According to our simulations, relatively small changes in mitochondrial membrane potential (∼1 mV) caused by uptake of Ca2+ are sufficient to alter the cytoplasmic ATP/ADP ratio and influence ATP-sensitive K+ channels in the plasma membrane. In the simulations, these cyclic changes in the mitochondrial membrane potential are due to synchronization of futile cycle of Ca2+ from the cytoplasm through mitochondria via Ca2+ uniporters and Na+/Ca2+exchange. Our simulations predict steady mitochondrial Ca2+concentrations on the order of 0.1 μM at low glucose concentrations that become oscillatory with an amplitude on the order of 0.5 μM during bursting. Abrupt increases in mitochondrial Ca2+concentration >5 μM may occur during continuous electrical activity.
Elevated growth hormone increases the Ca2+ sensitivity of slow- and fast-twitch skeletal muscle of female rats
Authors: Xu Xiaoping , Forrer Janet , Bechtel Peter J. , Best Philip M.
Published: December 2017

To determine the effect of plasma growth hormone (GH) on skeletal muscle function, we measured the free Ca2+concentration-tension relationship of slow-twitch (soleus) and fast-twitch (peroneus longus) muscles isolated from rats undergoing acromegaly in response to implanted, GH-secreting tumors. Muscles from adult (9 mo) and aged rats (24 mo) were studied after the tumor-bearing rats weighed over 50% more than their age-matched controls. Ca2+-activated isometric tension was recorded from skinned muscle fibers. For soleus muscles, the free Ca2+ concentration producing 50% of maximal tension ([Ca2+]50) was 2.0 μM for rats with tumors and 3.4–3.6 μM for controls. For peroneus longus fibers, [Ca2+]50shifted from 6.1–6.7 μM in controls to 3.5 μM after tumors were introduced into either adult or aged rats. Soleus muscle fibers from neonatal rats (14 days) were less sensitive to Ca2+ than those isolated from adult rats, having a [Ca2+]50of 7.3 μM. The Ca2+ sensitivity of peroneus longus fibers did not change with age. We conclude that significant increases in myofibrillar Ca2+ sensitivity occur in skeletal muscles undergoing rapid growth induced by GH-secreting tumors.
Evidence for Na+/Ca2+exchange in intact single skeletal muscle fibers from the mouse
Authors: Balnave Christopher D. , Allen David G.
Published: December 2017

The myoplasmic free Ca2+concentration ([Ca2+]i) was measured in intact single fibers from mouse skeletal muscle with the fluorescent Ca2+ indicator indo 1. Some fibers were perfused in a solution in which the concentration of Na+ was reduced from 145.4 to 0.4 mM (low-Na+solution) in an attempt to activate reverse-mode Na+/Ca2+exchange (Ca2+ entry in exchange for Na+ leaving the cell). Under normal resting conditions, application of low-Na+ solution only increased [Ca2+]iby 5.8 ± 1.8 nM from a mean resting [Ca2+]iof 42 nM. In other fibers, [Ca2+]iwas elevated by stimulating sarcoplasmic reticulum (SR) Ca2+ release with caffeine (10 mM) and by inhibiting SR Ca2+ uptake with 2,5-di( tert-butyl)-1,4-benzohydroquinone (TBQ; 0.5 μM) in an attempt to activate forward-mode Na+/Ca2+exchange (Ca2+ removal from the cell in exchange for Na+ influx). These two agents caused a large increase in [Ca2+]i, which then declined to a plateau level approximately twice the baseline [Ca2+]iover 20 min. If the cell was allowed to recover between exposures to caffeine and TBQ in a solution in which Ca2+ had been removed, the increase in [Ca2+]iduring the second exposure was very low, suggesting that Ca2+ had left the cell during the initial exposure. Application of caffeine and TBQ to a preparation in low-Na+ solution produced a large, sustained increase in [Ca2+]iof ∼1 μM. However, when cells were exposed to caffeine and TBQ in a low-Na+ solution in which Ca2+ had been removed, a sustained increase in [Ca2+]iwas not observed, although [Ca2+]iremained higher and declined slower than in normal Na+ solution. This suggests that forward-mode Na+/Ca2+exchange contributed to the fall of [Ca2+]iin normal Na+ solution, but when extracellular Na+ was low, a prolonged elevation of [Ca2+]icould activate reverse-mode Na+/Ca2+exchange. The results provide evidence that skeletal muscle fibers possess a Na+/Ca2+exchange mechanism that becomes active in its forward mode when [Ca2+]iis increased to levels similar to that obtained during contraction.
Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia
Authors: Helman Sandy I. , Liu Xuehong , Baldwin Kieron , Blazer-Yost Bonnie L. , Els Willem J.
Published: December 2017

To study and define the early time-dependent response (≤6 h) of blocker-sensitive epithelial Na+channels (ENaCs) to stimulation of Na+ transport by aldosterone, we used a new modified method of blocker-induced noise analysis to determine the changes of single-channel current ( i Na) channel open probability ( P o), and channel density ( N T) under transient conditions of transport as measured by macroscopic short-circuit currents ( I sc). In three groups of experiments in which spontaneous baseline rates of transport averaged 1.06, 5.40, and 15.14 μA/cm2, stimulation of transport occurred due to increase of blocker-sensitive channels. N T varied linearly over a 70-fold range of transport (0.5–35 μA/cm2). Relatively small and slow time-dependent but aldosterone-independent decreases of P o occurred during control (10–20% over 2 h) and aldosterone experimental periods (10–30% over 6 h). When the P o of control and aldosterone-treated tissues was examined over the 70-fold extended range of Na+ transport, P o was observed to vary inversely with I sc, falling from ∼0.5 to ∼0.15 at the highest rates of Na+ transport or ∼25% per 3-fold increase of transport. Because decreases of P o from any source cannot explain stimulation of transport by aldosterone, it is concluded that the early time-dependent stimulation of Na+ transport in A6 epithelia is due exclusively to increase of apical membrane N T.
Activation of Ca2+-activated K+(maxi-K+) channel by angiotensin II in myocytes of the guinea pig ileum
Authors: Romero Fernando , Silva Bagnólia A. , Nouailhetas Viviane L. A. , Aboulafia Jeannine
Published: December 2017

We investigated the regulation of the Ca2+-activated K+(maxi-K+) channel by angiotensin II (ANG II) and its synthetic analog, [Lys2]ANG II, in freshly dispersed intestinal myocytes. We identified a maxi-K+ channel population in the inside-out patch configuration on the basis of its conductance (257 ± 4 pS in symmetrical 150 mM KCl solution), voltage and Ca2+ dependence of channel opening, low Na+-to-K+and Cl−-to-K+permeability ratios, and blockade by external Cs+ and tetraethylammonium chloride. ANG II and [Lys2]ANG II caused an indirect, reversible, Ca2+- and dose-dependent activation of maxi-K+ channels in cell-attached experiments when cells were bathed in high-K+ solution. This effect was reversibly blocked by DUP-753, being that it is mediated by the AT1 receptor. Evidences that activation of the maxi-K+ channel by ANG II requires a rise in intracellular Ca2+concentration ([Ca2+]i) as an intermediate step were the shift of the open probability of the channel-membrane potential relationship to less positive membrane potentials and the sustained increase in [Ca2+]iin fura 2-loaded myocytes. The preservation of the pharmacomechanical coupling of ANG II in these cells provides a good model for the study of transmembrane signaling responses to ANG II and analogs in this tissue.
Frequency modulation of Ca2+sparks is involved in regulation of arterial diameter by cyclic nucleotides
Authors: Porter Valerie A. , Bonev Adrian D. , Knot Harm J. , Heppner Thomas J. , Stevenson Andra S. , Kleppisch Thomas , Lederer W. J. , Nelson Mark T.
Published: December 2017

Forskolin, which elevates cAMP levels, and sodium nitroprusside (SNP) and nicorandil, which elevate cGMP levels, increased, by two- to threefold, the frequency of subcellular Ca2+ release (“Ca2+ sparks”) through ryanodine-sensitive Ca2+ release (RyR) channels in the sarcoplasmic reticulum (SR) of myocytes isolated from cerebral and coronary arteries of rats. Forskolin, SNP, nicorandil, dibutyryl-cAMP, and adenosine increased the frequency of Ca2+-sensitive K+(KCa) currents [“spontaneous transient outward currents” (STOCs)] by two- to threefold, consistent with Ca2+ sparks activating STOCs. These agents also increased the mean amplitude of STOCs by 1.3-fold, an effect that could be explained by activation of KCa channels, independent of effects on Ca2+ sparks. To test the hypothesis that cAMP could act to dilate arteries through activation of the Ca2+spark→KCa channel pathway, the effects of blockers of KCachannels (iberiotoxin) and of Ca2+sparks (ryanodine) on forskolin-induced dilations of pressurized cerebral arteries were examined. Forskolin-induced dilations were partially inhibited by iberiotoxin and ryanodine (with no additive effects) and were entirely prevented by elevating external K+. Forskolin lowered average Ca2+ in pressurized arteries while increasing ryanodine-sensitive, caffeine-induced Ca2+ transients. These experiments suggest a new mechanism for cyclic nucleotide-mediated dilations through an increase in Ca2+ spark frequency, caused by effects on SR Ca2+ load and possibly on the RyR channel, which leads to increased STOC frequency, membrane potential hyperpolarization, closure of voltage-dependent Ca2+ channels, decrease in arterial wall Ca2+, and, ultimately, vasodilation.
Proteinase-activated receptors: novel mechanisms of signaling by serine proteases
Authors: Déry Olivier , Corvera Carlos U. , Steinhoff Martin , Bunnett Nigel W.
Published: December 2017

Although serine proteases are usually considered to act principally as degradative enzymes, certain proteases are signaling molecules that specifically regulate cells by cleaving and triggering members of a new family of proteinase-activated receptors (PARs). There are three members of this family, PAR-1 and PAR-3, which are receptors for thrombin, and PAR-2, a receptor for trypsin and mast cell tryptase. Proteases cleave within the extracellular NH2-terminus of their receptors to expose a new NH2-terminus. Specific residues within this tethered ligand domain interact with extracellular domains of the cleaved receptor, resulting in activation. In common with many G protein-coupled receptors, PARs couple to multiple G proteins and thereby activate many parallel mechanisms of signal transduction. PARs are expressed in multiple tissues by a wide variety of cells, where they are involved in several pathophysiological processes, including growth and development, mitogenesis, and inflammation. Because the cleaved receptor is physically coupled to its agonist, efficient mechanisms exist to terminate signaling and prevent uncontrolled stimulation. These include cleavage of the tethered ligand, receptor phosphorylation and uncoupling from G proteins, and endocytosis and lysosomal degradation of activated receptors.
Glucagon-mediated Ca2+signaling in BHK cells expressing cloned human glucagon receptors
Authors: Hansen Lars H. , Gromada Jesper , Bouchelouche Pierre , Whitmore Ted , Jelinek Laura , Kindsvogel Wayne , Nishimura Erica
Published: December 2017

From video imaging of fura 2-loaded baby hamster kidney (BHK) cells stably expressing the cloned human glucagon receptor, we found the Ca2+ response to glucagon to be specific, dose dependent, synchronous, sensitive to pertussis toxin, and independent of Ca2+ influx. Forskolin did not elicit a Ca2+response, but treatment with a protein kinase A inhibitor, the Rp diastereomer of 8-bromoadenosine-3′,5′-cyclic monophosphothioate, resulted in a reduced glucagon-mediated Ca2+ response as well as Ca2+ oscillations. The specific phospholipase C inhibitor U-73122 abolished the Ca2+ response to glucagon, and a modest twofold increase in inositol trisphosphate (IP3) production could be observed after stimulation with glucagon. In BHK cells coexpressing glucagon and muscarinic (M1) acetylcholine receptors, carbachol blocked the rise in intracellular free Ca2+ concentrations in response to glucagon, whereas glucagon did not affect the carbachol-induced increase in Ca2+. Furthermore, carbachol, but not glucagon, could block thapsigargin-activated increases in intracellular free Ca2+concentration. These results indicate that, in BHK cells, glucagon receptors can activate not only adenylate cyclase but also a second independent G protein-coupled pathway that leads to the stimulation of phospholipase C and the release of Ca2+ from IP3-sensitive intracellular Ca2+ stores. Finally, we provide evidence to suggest that cAMP potentiates the IP3-mediated effects on intracellular Ca2+ handling.
Dynamics of myosin light chain phosphorylation at Ser19 and Thr18/Ser19in smooth muscle cells in culture
Authors: Sakurada Katsuhiko , Seto Minoru , Sasaki Yasuharu
Published: December 2017

Using the specific antibodies pLC1 and pLC2 for mono- and diphosphorylated 20-kDa myosin light chain (MLC20) at Ser19 and at both Thr18 and Ser19, respectively, we visualized the dynamics of the MLC20phosphorylation in rabbit aortic smooth muscle cells (cell line SM-3) stimulated with PGF2α. In the resting state, the diphosphorylated form was located in the peripheral region of the cell, such as the leading edge or the adhesion plaque, and the monophosphorylated form was located not only in the peripheral region but also on a discontinuous fibrillary structure along the long axis of the cell. After stimulation with 30 μM PGF2α, although localization of the monophosphorylated form changed little, the content of the diphosphorylated form increased and the distribution spread along the fibrillary structure to an extent the same as or similar to that of the monophosphorylated form, which colocalized with actin filament bundles. The diphosphorylation of MLC20 was more sensitive to protein kinase inhibitors, HA-1077, HA-1100, staurosporine, wortmannin, and ML-9, than was the monophosphorylation. In light of these observations, we propose that MLC20 diphosphorylation and monophosphorylation are regulated by different mechanisms.
Voltage dependence of Ca2+sparks in intact cerebral arteries
Authors: Jaggar Jonathan H. , Stevenson Andrá S. , Nelson Mark T.
Published: December 2017

Ca2+ sparks have been previously described in isolated smooth muscle cells. Here we present the first measurements of local Ca2+ transients (“Ca2+ sparks”) in an intact smooth muscle preparation. Ca2+sparks appear to result from the opening of ryanodine-sensitive Ca2+ release (RyR) channels in the sarcoplasmic reticulum (SR). Intracellular Ca2+ concentration ([Ca2+]i) was measured in intact cerebral arteries (40–150 μm in diameter) from rats, using the fluorescent Ca2+ indicator fluo 3 and a laser scanning confocal microscope. Membrane potential depolarization by elevation of external K+ from 6 to 30 mM increased Ca2+ spark frequency (4.3-fold) and amplitude (∼2-fold) as well as global arterial wall [Ca2+]i(∼1.7-fold). The half time of decay (∼50 ms) was not affected by membrane potential depolarization. Ryanodine (10 μM), which inhibits RyR channels and Ca2+ sparks in isolated cells, and thapsigargin (100 nM), which indirectly inhibits RyR channels by blocking the SR Ca2+-ATPase, completely inhibited Ca2+ sparks in intact cerebral arteries. Diltiazem, an inhibitor of voltage-dependent Ca2+ channels, lowered global [Ca2+]iand Ca2+ spark frequency and amplitude in intact cerebral arteries in a concentration-dependent manner. The frequency of Ca2+sparks (<1 s−1 ⋅ cell−1), even under conditions of steady depolarization, was too low to contribute significant amounts of Ca2+ to global Ca2+ in intact arteries. These results provide direct evidence that Ca2+ sparks exist in quiescent smooth muscle cells in intact arteries and that changes of membrane potential that would simulate physiological changes modulate both Ca2+ spark frequency and amplitude in arterial smooth muscle.
A luciferase-engineered cell line for study of cAMP regulation in endothelial cells
Authors: Xavier-Neto J. , Pereira A. C. , Motoyama A. H. , Krieger J. E.
Published: December 2017

cAREL is a cAMP-responsive endothelial cell line carrying a luciferase reporter gene introduced by stable transfection of a luciferase enhancer trap into rabbit aortic endothelial cells. Luciferase gene expression in cAREL was stimulated 233-fold by 8-BrcAMP. Treatment with the β-adrenoceptor agonist isoproterenol induced a 7.0-fold increase in luciferase expression, which was partially blocked by either β1- or β2-adrenoceptor antagonists and totally blocked by propranolol and by a combination of β1- plus β2-adrenoceptor antagonists. Receptor stimulation was mimicked by cholera toxin, forskolin, 8-BrcAMP, and isobutylmethylxanthine but not by 8BrcGMP, dexamethasone, or phorbol 12-myristate 13-acetate. Stimulation by isoproterenol was completely blocked by H-89, a protein kinase A inhibitor. cAREL was also stimulated by A-23187, and this effect was abrogated by EGTA and H-89. cAREL is the first cAMP-sensitive endothelial cell line described, and it can be useful as a positive control, as a model for cAMP regulation, as a background to genetic introduction of receptors, as an indicator of intracellular pathway activation, and as a tool to investigate cAMP effects on other signaling pathways.
Eosinophil major basic protein increases membrane permeability in mammalian urinary bladder epithelium
Authors: Kleine Teri J. , Gleich Gerald J. , Lewis Simon A.
Published: December 2017

The eosinophil granule protein major basic protein (MBP) is toxic to a wide variety of cell types, by a poorly understood mechanism. To determine whether the action of MBP involves an alteration in membrane permeability, we tested purified MBP on rabbit urinary bladder epithelium using transepithelial voltage-clamp techniques. Addition of nanomolar concentrations of MBP to the mucosal solution caused an increase in apical membrane conductance only when the voltage across the apical membrane was cell interior negative. The magnitude of the MBP-induced conductance was a function of MBP concentration, and the rate of the initial increase in conductance was a function of the transepithelial voltage. The MBP-induced conductance was nonselective for K+ and Cl−. Mucosal Ca2+ reversed the induced conductance, whereas mucosal Mg2+partially blocked the induced conductance and slowed the rate of the increase in conductance. The induced conductance was partially reversed by changing the voltage gradient across the apical membrane to cell interior positive. Prolonged exposure resulted in an irreversible loss of the barrier function of the urinary bladder epithelium. These results suggest that an increase in cell membrane ion permeability is an initial step in MBP-induced loss of barrier function.
Glibenclamide blocks volume-sensitive Cl− channels by dual mechanisms
Authors: Liu Yan , Oiki Shigetoshi , Tsumura Takehiko , Shimizu Takahiro , Okada Yasunobu
Published: December 2017

To study the mechanisms of glibenclamide actions on volume-sensitive Cl−channels, whole cell patch-clamp studies were performed at various pH levels in human epithelial Intestine 407 cells. Extracellular application of glibenclamide reversibly suppressed volume-sensitive Cl− currents in the entire range of voltage examined (−100 to +100 mV) and accelerated the depolarization-induced inactivation at pH 7.5. When glibenclamide was applied from the intracellular side, in contrast, no effect was observed. At acidic pH, at which the weak acid glibenclamide exists largely in the uncharged form, the instantaneous current was, in a voltage-independent manner, suppressed by the extracellular drug at micromolar concentrations without significantly affecting the depolarization-induced inactivation. At alkaline pH, at which almost all of the drug is in the charged form, glibenclamide speeded the inactivation time course and induced a leftward shift of the steady-state inactivation curve at much higher concentrations. Thus it is concluded that glibenclamide exerts inhibiting actions on swelling-activated Cl−channels from the extracellular side and that the uncharged form is mainly responsible for voltage-independent inhibition of instantaneous currents, whereas the anionic form facilitates voltage-dependent channel inactivation in human epithelial Intestine 407 cells.
l-Arginine inhibits vasopressin-stimulated mesangial cell Ca2+
Authors: Balsam Leah , Nikbakht Nasrin
Published: December 2017

l-Arginine (l-Arg) affects various parameters that modulate the progression of renal disease. These same factors [e.g., glomerular filtration rate, changes in mesangial cell (MC) tension, and production of NO] are all controlled at least in part by changes in MC intracellular Ca2+concentration ([Ca2+]i). We therefore evaluated the effect of l-Arg on MC [Ca2+]i. We found thatl-Arg inhibits the vasopressin-stimulated rise in MC [Ca2+]i both in rat and murine cell cultures. This effect does not appear to be due to metabolism of l-Arg to either NO or l-ornithine (l-Orn). Blocking the metabolism of l-Arg with N ω-monomethyl-l-arginine, an NO synthase inhibitor, or with 20 mM l-valine (l-Val), an inhibitor of Orn formation, does not reverse the inhibition. However, other cationic amino acids, as well guanidine, the functional group ofl-Arg, all inhibit the vasopressin-stimulated rise in [Ca2+]i, consistent with a structural basis for this effect. We conclude that 1)l-Arg inhibits vasopressin-stimulated murine and rat MC [Ca2+]irise, 2) this inhibition is not mediated by metabolism of l-Arg to either NO or l-Orn, and 3) the effect ofl-Arg is due to its cationic functional group, guanidine.
Uptake and metabolism of biotin by human peripheral blood mononuclear cells
Authors: Zempleni Janos , Mock Donald M.
Published: December 2017

We studied the uptake of biotin into human peripheral blood mononuclear cells (PBMC) using [3H]biotin and studied the catabolism of biotin in PBMC using [14C]biotin. Over 30 min, [3H]biotin uptake was greater at 37°C than at 25°C ( K T = 2.6 ± 0.4 nM, maximal velocity = 2.9 ± 0.2 fmol ⋅ 106cells−1 ⋅ 30 min−1). Ouabain reduced [3H]biotin uptake to 65% of control values, suggesting that biotin uptake is Na-K-ATPase dependent. Unlabeled biotin and biotin analogs reduced the uptake of [3H]biotin to 22–70% of control values, suggesting the presence of a competition for a structurally specific biotin transporter. When endocytosis by PBMC was stimulated by various acyl glycerols, [3H]biotin uptake was 40–73% of control values; these data are consistent with the hypothesis that stimulated endocytosis reduces biotin transporter density on the cell surface. During a 168-h incubation, PBMC did not catabolize [14C]biotin.
Differential inhibition of Na+/Ca2+exchanger isoforms by divalent cations and isothiourea derivative
Authors: Iwamoto Takahiro , Shigekawa Munekazu
Published: December 2017

We compared the properties of three mammalian Na+/Ca2+exchanger isoforms, NCX1, NCX2, and NCX3, by analyzing the effects of Ni2+ and other cations as well as the recently identified inhibitor isothiourea derivatives on intracellular Na+-dependent45Ca2+uptake into CCL-39 (Dede) fibroblasts stably expressing each isoform. All these NCX isoforms had similar affinities for the extracellular transport substrates Ca2+ and Na+. Ni2+ inhibited45Ca2+uptake by competing with Ca2+ for the external transport site, with 10-fold less affinity in NCX3 than in NCX1 or NCX2. Ni2+ and Co2+ were most efficient in such discrimination of NCX isoforms, although their inhibitory potencies were less than those of La3+ and Cd2+. The monovalent cation Li+ stimulated45Ca2+uptake rate by all NCX isoforms similarly with low affinity, although the extent of stimulation was somewhat smaller in NCX1. On the other hand, the isothiourea derivative KB-R7943 was threefold more inhibitory to NCX3 than to NCX1 or NCX2. Thus distinct differences in the kinetic and pharmacological properties were detected between NCX3 and the other two isoforms.
Neuropeptide Y inhibits ciliary beat frequency in human ciliated cells via nPKC, independently of PKA
Authors: Wong Lid B. , Park C. Lucy , Yeates Donovan B.
Published: December 2017

The intracellular mechanisms whereby the inhibitory neurotransmitter neuropeptide Y (NPY) decreases ciliary beat frequency (CBF) were investigated in cultured human tracheal and bronchial ciliated cells. CBF was measured by nonstationary analysis laser light scattering. NPY at 1 and 10 μM decreased CBF from a baseline of 6.7 ± 0.5 ( n = 12) to 6.1 ± 0.5 ( P < 0.05) and 5.8 ± 0.4 ( P < 0.01) Hz, respectively. Prior application of PYX-1, an NPY antagonist, prevented the decreases of CBF induced by both doses of NPY. Two broad protein kinase C (PKC) kinase inhibitors, staurosporine and calphostin C, also abolished the NPY-induced decrease in CBF. The NPY-induced decrease in CBF was abolished by GF 109203X, a novel PKC (nPKC) isoform inhibitor, whereas this decrease in CBF was not attenuated by Gö-6976, a specific inhibitor of conventional PKC isoforms. Because pretreatment with NPY did not block the stimulation of CBF by forskolin and pretreatment with forskolin did not abolish the NPY-induced inhibition of CBF, this NPY receptor-mediated signal transduction mechanism appears to be independent of the adenylate cyclase-protein kinase A (PKA) pathway. Inhibition of Ca2+-ATPase by thapsigargin also prevented the suppression of CBF induced by subsequent application of NPY. These novel data indicate that, in cultured human epithelia, NPY decreases CBF below its basal level via the activation of an nPKC isoform and Ca2+-ATPase, independent of the activity of PKA. This is consistent with the proposition that NPY is an autonomic efferent inhibitory neurotransmitter regulating mucociliary transport.
p38 Mitogen-activated protein kinase expression and activation in smooth muscle
Authors: Hedges Jason C. , Yamboliev Ilia A. , Ngo Mai , Horowitz Burton , Adam Leonard P. , Gerthoffer William T.
Published: December 2017

There is relatively little known about expression and activation of p38 mitogen-activated protein kinases (MAPKs) through G protein-linked, seven-transmembrane-spanning (STM) receptors in mammalian smooth muscle. To investigate the role of p38 MAPK in smooth muscle, we cloned and sequenced the p38 MAPK expressed in canine smooth muscles. A full-length clone of the canine p38 MAPK expressed in colonic smooth muscle was obtained by RT-PCR. The deduced amino acid sequence revealed 99% identity to the human p38 MAPK and differed from the human enzyme in only two conservative substitutions. The deduced molecular mass of the canine p38 MAPK is 41.2 kDa, with a calculated isoelectric point of 5.41. Canine p38 MAPK was found to be expressed in colonic, tracheal, and vascular smooth muscles and underwent increased tyrosine phosphorylation in response to motor neurotransmitters, acetylcholine (ACh) and neurokinin A (NKA), in colonic smooth muscle. There was an eightfold increase in p38 MAPK phosphorylation after a 10-min incubation with ACh and a threefold increase with NKA. We also identified a p38 immunoreactive kinase activity isolated from colonic smooth muscle homogenate by Mono Q chromatography. Partially purified p38 MAPK and activated recombinant p38 MAPK (Mpk2) phosphorylated both the known p38 MAPK substrate ATF2, as well as porcine stomach h-caldesmon in vitro. The results suggest that elements of the “stress-response” pathway may be coupled to transcriptional control as well as to cytoskeletal and possibly contractile protein phosphorylation in mammalian smooth muscle.
Myosin heavy chain isoform expression in rat smooth muscle development
Authors: White Sheryl L. , Zhou Ming Yuan , Low Robert B. , Periasamy Muthu
Published: December 2017

Smooth muscle myosin heavy chains (MHCs), the motor proteins that power smooth muscle contraction, are produced by alternative splicing from a single gene. The smooth muscle MHC gene is capable of producing four isoforms by utilizing alternative splice sites located at the regions encoding the carboxy terminus and the junction of the 25- and 50-kDa tryptic peptides. These four isoforms, SM1A, SM1B, SM2A, and SM2B, are a combination of one of two heavy chains containing different carboxy-terminal tails (1 or 2) without (A) or with (B) an additional motif in the myosin head. In the present study, using RNA analysis and isoform-specific antibodies, we demonstrate the expression patterns of MHC isoforms during development in rat smooth muscle tissues. RNase protection analysis indicates that the mRNAs for SMA and SMB isoforms, which differ by a 21-nucleotide insertion in the region encoding the S1 head region of the myosin molecule, are differentially expressed during development in a highly tissue-specific manner. Smooth muscle MHC transcripts are first detectable in developing rat smooth muscle tissues at 17 days of fetal development. The SMB mRNA is shown to be expressed in smooth muscle from fetal bladder, intestine, and stomach and from neonatal aorta; however, it is not expressed in cultured smooth muscle cells from rat aorta. The SMA mRNA is also present at all stages of development in the smooth muscles examined; however, it is much less abundant than SMB mRNA in most fetal smooth muscles. We show here that the SMB isoform, which contains a unique seven-amino acid insertion at the junction of the 25- and 50-kDa tryptic peptides, is present in conjunction with SM1 and SM2 tails on immunoblots of smooth muscle from stomach, intestine, bladder, and uterus and is expressed during development in a pattern distinct from that of the SM1 and SM2 tail isoforms.
Two MCAT elements of the SM α-actin promoter function differentially in SM vs. non-SM cells
Authors: Swartz Ellen A. , Johnson A. Daniel , Owens Gary K.
Published: December 2017

Transcriptional activity of the smooth muscle (SM) α-actin gene is differentially regulated in SM vs. non-SM cells. Contained within the rat SM α-actin promoter are two MCAT motifs, binding sites for transcription enhancer factor 1 (TEF-1) transcriptional factors implicated in the regulation of many muscle-specific genes. Transfections of SM α-actin promoter-CAT constructs containing wild-type or mutagenized MCAT elements were performed to evaluate their functional significance. Mutation of the MCAT elements resulted in increased transcriptional activity in SM cells, whereas these mutations either had no effect or decreased activity in L6 myotubes or endothelial cells. High-resolution gel shift assays resolved several complexes of different mobilities that were formed between MCAT oligonucleotides and nuclear extracts from the different cell types, although no single band was unique to SM. Western blot analysis of nuclear extracts with polyclonal antibodies to conserved domains of the TEF-1 gene family revealed multiple reactive bands, some that were similar and others that differed between SM and non-SM. Supershift assays with a polyclonal antibody to the TEF-related protein family demonstrated that TEF-1 or TEF-1-related proteins were contained in the shifted complexes. Results suggest that the MCAT elements may contribute to cell type-specific regulation of the SM α-actin gene. However, it remains to be determined whether the differential transcriptional activity of MCAT elements in SM vs. non-SM is due to differences in expression of TEF-1 or TEF-1-related proteins or to unique (cell type specific) combinatorial interactions of the MCAT elements with other cis-elements and trans-factors.
Using gadolinium to identify stretch-activated channels: technical considerations
Authors: Caldwell Ray A. , Clemo Henry F. , Baumgarten Clive M.
Published: December 2017

Gadolinium (Gd3+) blocks cation-selective stretch-activated ion channels (SACs) and thereby inhibits a variety of physiological and pathophysiological processes. Gd3+ sensitivity has become a simple and widely used method for detecting the involvement of SACs, and, conversely, Gd3+insensitivity has been used to infer that processes are not dependent on SACs. The limitations of this approach are not adequately appreciated, however. Avid binding of Gd3+ to anions commonly present in physiological salt solutions and culture media, including phosphate- and bicarbonate-buffered solutions and EGTA in intracellular solutions, often is not taken into account. Failure to detect an effect of Gd3+ in such solutions may reflect the vanishingly low concentrations of free Gd3+ rather than the lack of a role for SACs. Moreover, certain SACs are insensitive to Gd3+, and Gd3+ also blocks other ion channels. Gd3+ remains a useful tool for studying SACs, but appropriate care must be taken in experimental design and interpretation to avoid both false negative and false positive conclusions.
GLP-1 action in L6 myotubes is via a receptor different from the pancreatic GLP-1 receptor
Authors: Yang H. , Egan J. M. , Wang Y. , Moyes C. D. , Roth J. , Montrose M. H. , Montrose-Rafizadeh C.
Published: December 2017

The incretin hormone glucagon-like peptide-1 (GLP-1)-(7—36) amide is best known for its antidiabetogenic actions mediated via a GLP-1 receptor present on pancreatic endocrine cells. To investigate the molecular mechanisms of GLP-1 action in muscle, we used cultured L6 myotubes. In L6 myotubes, GLP-1 enhanced insulin-stimulated glycogen synthesis by 140% while stimulating CO2 production and lactate formation by 150%. In the presence of IBMX, GLP-1 diminished cAMP levels to 83% of IBMX alone. In L6 myotubes transfected with pancreatic GLP-1 receptor, GLP-1 increased cAMP levels and inhibited glycogen synthesis by 60%. An antagonist of pancreatic GLP-1 receptor, exendin-4-(9—39), inhibited GLP-1-mediated glycogen synthesis in GLP-1 receptor-transfected L6 myotubes. However, in parental L6 myotubes, exendin-4-(9—39) and GLP-1-(1—36) amide, an inactive peptide on pancreatic GLP-1 receptor, displaced125I-labeled GLP-1 binding and stimulated glycogen synthesis by 186 and 130%, respectively. These results suggest that the insulinomimetic effects of GLP-1 in L6 cells are likely to be mediated by a receptor that is different from the GLP-1 receptor found in the pancreas.
Induction of Mn SOD in human monocytes without inflammatory cytokine production by a mutant endotoxin
Authors: Tian Licheng , White Julie E. , Lin Hung-Yun , Haran Visa S. , Sacco Joseph , Chikkappa G. , Davis Faith B. , Davis Paul J. , Tsan Min-Fu
Published: December 2017

Endotoxin selectively induces monocyte Mn superoxide dismutase (SOD) without affecting levels of Cu,Zn SOD, catalase, or glutathione peroxidase. However, little is known about the structure-activity relationship and the mechanism by which endotoxin induces Mn SOD. In this study we demonstrated that a mutant Escherichia coli endotoxin lacking myristoyl fatty acid at the 3′ R-3-hydroxymyristate position of the lipid A moiety retained its full capacity to coagulate Limulus amoebocyte lysate compared with the wild-type E. coli endotoxin and markedly stimulated the activation of human monocyte nuclear factor-κB and the induction of Mn SOD mRNA and enzyme activity. However, in contrast to the wild-type endotoxin, it failed to induce significant production of tumor necrosis factor-α and macrophage inflammatory protein-1α by monocytes and did not induce the phosphorylation and nuclear translocation of mitogen-activated protein kinase. These results suggest that 1) lipid A myristoyl fatty acid, although it is important for the induction of inflammatory cytokine production by human monocytes, is not necessary for the induction of Mn SOD, 2) endotoxin-mediated induction of Mn SOD and inflammatory cytokines are regulated, at least in part, through different signal transduction pathways, and 3) failure of the mutant endotoxin to induce tumor necrosis factor-α production is, at least in part, due to its inability to activate mitogen-activated protein kinase.