bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterotetramer formation and charybdotoxin sensitivity of two K+ channels cloned from smooth muscle
Authors: Russell S. N. , Overturf K. E. , Horowitz B.
Published: December 2017

Delayed rectifier K+ channels are involved in the electrical activity of all excitable cells. The relationship between native K+ currents recorded from these cells and cloned K+ channel cDNAs has been difficult to ascertain partly because of contradictions in pharmacological characteristics between native and expressed currents. Through the study of the charybdotoxin (CTX) pharmacology of two cloned smooth muscle delayed rectifier K+ channels (cKv 1.2 and cKv1.5) expressed in oocytes, evidence for heterotetramer formation was obtained. We have shown that the presence of even a single CTX-insensitive subunit renders the heterotetrameric channel insensitive to CTX. The two K+ channel clones differ in an amino acid at the mouth of the pore region, which may be in a position to block the access of CTX to its binding site and hence determine CTX sensitivity of the heterotetrameric channel. These results may explain discrepancies reported between native and cloned smooth muscle K+ channels.
Mechanotransduction in vertebrate hair cells: structure and function of the stereociliary bundle
Authors: Hackney C. M. , Furness D. N.
Published: December 2017

The mechanosensitive hair cells of the vertebrate acousticolateralis system have an apical bundle of stereocilia, deflections of which control the opening of mechano-electrical transduction channels and thus generate receptor potentials in the cell below. This review describes current theories of hair cell function in the light of recent immunocytochemical and ultrastructural findings; in particular, the location and operation of the transduction channels are considered. The most widely accepted hypothesis of mechanotransduction by hair cells is that fine extracellular links that run between the tips of shorter stereocilia and the sides of taller ones operate the transduction channels. However, the fact that the transduction channels are amiloride sensitive has led to labeling experiments using antibodies to the amiloride-sensitive epithelial Na+ channel from kidney which suggest that the mechanotransduction channels may not be directly associated with the tip links. Instead, they appear to be located near a junctionlike structure at the point of contact between the shorter and taller stereocilia. The implications of these findings for the tip link hypothesis are discussed.
The iodide channel of the thyroid. II. Selective iodide conductance inserted into liposomes
Authors: Golstein P. E. , Sener A. , Beauwens R.
Published: December 2017

An iodide channel has been previously identified in the plasma membrane of bovine throcytes [Golstein et al., Am. J. Physiol. 263 (Cell Physiol. 32): C590-C597, 1992]. The plasma membrane proteins were solubilized and ultrafiltered, and the protein fraction collected above 100 kDa was inserted in liposomes. Voltage-sensitive uptake of radiolabeled I- by these proteoliposomes was studied. To this end, an outward I- gradient was set up by loading the proteoliposomes with KI and removing extraliposomal I-. I- exit from the proteoliposome induces an inside positive membrane potential, which leads to the uptake of 125I- added to the incubation medium. This uptake was abolished by valinomycin, which in the presence of K+ short circuits the liposomal membrane potential, demonstrating the conductive nature of this uptake. A double reciprocal plot of I- influx over I- concentration suggests the existence of a single population of channels in these proteoliposomes with a Michaelis-Menten constant for I- of approximately 9 microM. When the proteoliposomes were loaded with KCl or KSCN instead of I-, no conductive uptake occurred anymore, suggesting that these anions are unable to diffuse through the I- conductance, hence do not generate a diffusion potential. I- uptake by KI-loaded proteoliposomes was not inhibited in the presence of a 1,000-fold excess of extraliposomal Cl- but was completely inhibited by a 1,000-fold excess of extraliposomal SCN-, indicating that Cl- does not permeate the I- channel, whereas SCN- inhibits it. SCN- and flufenamate were both shown to be competitive inhibitors of the I- channel with an inhibitor constant of approximately 10 and 750 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Localization of the Na/Ca exchange-dependent Ca compartment in cultured neonatal rat heart cells
Authors: Langer G. A. , Wang S. Y. , Rich T. L.
Published: December 2017

It has been previously established, in both adult and cultured neonatal cardiac cells, that there is a discrete Na/Ca exchange-dependent Ca compartment. It has been proposed that a component of junctional sarcoplasmic reticulum (JSR) Ca and Ca bound to the apposed inner sarcolemmal leaflet represent together the subcellular locus of the compartment. The present study examines this proposal. The amount of Ca in the total compartment is measured isotopically in intact functional cells (using the on-line "scintillation disk" technique) under a variety of perfusion conditions. Under identical labeling conditions, sarcolemmal membranes are rapidly (within a few hundred milliseconds) isolated from another set of intact cells by "gas dissection," and the amount of Ca bound to the membranes is measured. Probes that specifically decrease SR Ca content (thapsigargin, caffeine, low-dose ryanodine) decrease total cell content and sarcolemmal binding proportionally. High-dose ryanodine (producing closure of SR channels) markedly reduces sarcolemmal binding relative to total content of the compartment. The sarcolemmal sites saturate between 1 and 2 mM extracellular Ca ([Ca]o), whereas the total compartment saturates between 4 and 6 mM [Ca]o. Below 1 mM [Ca]o, sarcolemmal binding is maintained relative to total compartment content. Finally, the total compartment increases after reversal of the intracellular Na to extracellular Na ([Na]i/[Na]o) gradient with sarcolemmal content-to-total content ratio dependent on the method used to reverse the [Na]i/[Na]o ratio. The results are consistent with localization of the Na/Ca exchange-dependent compartment to the subsarcolemmal region ("cleft") where JSR Ca is in equilibrium with anionic inner sarcolemmal leaflet Ca binding sites.
Role of protein kinase C and tyrosine kinase activity in IFN-gamma-induced expression of the class II MHC gene
Authors: Lee Y. J. , Panek R. B. , Huston M. , Benveniste E. N.
Published: December 2017

Astrocytes are induced by interferon-gamma (IFN-gamma) to express class II major histocompatibility complex (MHC) antigens. Our previous studies demonstrated that IFN-gamma-initiated signaling events important for class II expression include activation of protein kinase C (PKC) and the Na+/H+ antiporter. We have extended these studies and found that protein tyrosine kinase (PTK) activity is also required for class II expression. Treatment of astrocytes with inhibitors specific for PKC and PTK blocked INF-gamma-induced class II gene transcription, mRNA expression, and protein expression. Immunoblotting and immunoprecipitation experiments demonstrated that IFN-gamma induced tyrosine phosphorylation of the p91 component of ISGF3, which is blocked by preincubation of cells with PTK inhibitors. Treatment of astrocytes with IFN-gamma and either PKC and PTK inhibitors changed the mobility and intensity of a nuclear factor, IFN-gamma-enhanced factor X, which binds to the X box of the class II MHC promoter. Taken together, these data provide evidence that activation of both PTK and PKC is required for IFN-gamma-induced expression of the class II gene.
Phospholipase A2 activation in human neutrophils requires influx of extracellular Ca2+ and leukotriene B4
Authors: Reddy S. , Bose R. , Rao G. H. , Murthy M.
Published: December 2017

We have demonstrated that phospolipase A2 (PLA2) activation in human neutrophils requires both the influx of extracellular Ca2+ and leukotriene B4 (LTB4). Surprisingly, the eicosanoids (LTB4 and its omega-oxidation products) formed were quantitatively very similar in both thapsigargin (Thap)- and A-23187-stimulated neutrophils. In contrast, Thap had very little effect on the activation of PLA2 when 5-lipoxygenase (5-LO) was blocked by BW755C or MK-886, whereas A-23187 caused a substantial activation. The lack of PLA2 activation in Thap-stimulated neutrophils results from the inhibition of LTB4 formation in the presence of 5-LO inhibitors. It appears that A-23187 activates both LTB4-dependent and -independent PLA2, whereas Thap activates LTB4-dependent PLA2. Experiments with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid demonstrated that activation of Thap-sensitive PLA2 and 5-LO requires the influx of Ca2+. Neither the transient elevation of cytosolic Ca2+ from intracellular stores nor the sustained Ca2+ influx alone without LTB4 appears sufficient to cause the activation of LTB4-dependent PLA2. We suggest that the activation of LTB4-dependent PLA2 involves 1) a sustained elevation of cytosolic Ca2+ coupled to the influx of extracellular Ca2+ and 2) a coupling between LTB4 and its receptor. We conclude that LTB4-dependent PLA2 plays an important role in the poststimulatory formation of lipid mediators such as prostaglandins, leukotrienes, and platelet-activating factor.
Ca(2+)-insensitive sustained contraction of skinned smooth muscle after acidic ADP treatment
Authors: Morimoto S. , Ogawa Y.
Published: December 2017

After an acidic treatment in the presence of ADP, Triton X-100-skinned rabbit aortic smooth muscle strips were found to develop a large sustained, Ca(2+)-insensitive tension when returned to a relaxing solution with neutral pH. The presence of ADP during treatment was essential for the manifestation of the Ca(2+)-insensitive contraction. This contraction was reversibly eliminated by withdrawal of MgATP or addition of vanadate and was found to be accompanied by an extraordinarily high level of 20-kDa myosin light-chain (MLC20) phosphorylation. The rate constant for dephosphorylation of MLC20 in treated strips was about one-twenty-fifth that in untreated control, when determined after removal of Ca2+, Mg2+, and ATP. Two-dimensional phosphopeptide mapping of tryptic digests of MLC20 showed that most incorporated phosphate was in the peptides which would be phosphorylated by myosin light-chain kinase. These results provide strong evidence that ADP inactivates myosin light-chain phosphatase under acidic conditions.
Thin filament regulation of force activation is not essential in single vascular smooth muscle cells
Authors: Brozovich F. V. , Yamakawa M.
Published: December 2017

To investigate thin filament regulation of force activation in smooth muscle, we recorded force and stiffness of alpha-toxin-permeabilized single smooth muscle cells. At pCa 9, the rigor state was characterized by high in-phase stiffness, low force, and low quadrature stiffness, suggesting that the attachment of rigor cross bridges does not depend on either Ca2+ or myosin light chain (MLC) phosphorylation, and cross bridges can enter a rigor state without producing force. At pCa 4, 20 microM ATP increased force, in-phase stiffness, and quadrature stiffness, while 20 microM CTP did not increase any of these parameters, suggesting that although MLC phosphorylation is not required for the formation of rigor cross bridges, MLC phosphorylation is required for detached cross bridges to attach to actin and undergo a force-producing isomerization. These results also suggest that for smooth muscle, force activation is regulated by myosin light-chain kinase. From rigor, 20 microM ATP (pCa 9) increased force and quadrature without changing in-phase stiffness. This force increase could be explained if in rigor solution both actomyosin (AM) and AM.ADP cross bridges exist (2, 32), and ATP-induced detachment of AM cross bridges is accompanied by AM.ADP cross bridges undergoing a force-producing isomerization in combination with cooperative cross-bridge reattachment (36). Thus results of our experiments suggest that thin filament-based regulation of force activation is not essential in smooth muscle, and a population of cross bridges must begin in an attached state for force to be produced in the absence of MLC phosphorylation.
Angiotensin-converting enzyme inhibition, intracellular Na+, and Na(+)-K+ pumping in cardiac myocytes
Authors: Hool L. C. , Whalley D. W. , Doohan M. M. , Rasmussen H. H.
Published: December 2017

Angiotensin-converting enzyme (ACE) inhibitors can reduce cardiac mass in both clinical and experimental cardiac hypertrophy. Because cytoplasmic Na+ and pH have been implicated as regulators of cell growth, we examined the effect of treatment with an ACE inhibitor on intracellular Na+ activity (alpha iNa) and pH (pHi) in the heart. After treatment of rabbits with captopril for 8 days alpha iNa was reduced relative to controls (3.6 +/- 0.4, n = 8, vs. 8.2 +/- 0.4 mM, n = 9, P < 0.001), whereas pHi was unchanged. To account for the difference in alpha iNa we measured electrogenic Na(+)-K+ pump activity in single isolated myocytes. Treatment with captopril increased pump currents at near-physiological levels of intracellular Na+ but had no effect at near-saturating levels of Na+. A similar increase in Na(+)-K+ pump activity occurred in rabbits treated with another ACE inhibitor, enalapril, but not with the vasodilator, hydralazine. We speculate that a decrease in alpha iNa after treatment with captopril may contribute to the well-documented ability of ACE inhibitors to reduce cardiac mass.
Permeation and gating properties of a cloned renal K+ channel
Authors: Chepilko S. , Zhou H. , Sackin H. , Palmer L. G.
Published: December 2017

The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.
Osmotic regulation of synthesis of glycerophosphocholine from phosphatidylcholine in MDCK cells
Authors: Kwon E. D. , Jung K. Y. , Edsall L. C. , Kim H. Y. , Garcia-Perez A. , Burg M. B.
Published: December 2017

Glycerophosphocholine (GPC) is osmotically regulated in renal medullary cells and in cultured Madin-Darby canine kidney (MDCK) cells. Previously, it was shown that a high extracellular concentration of urea or NaCl causes these cells to accumulate large amounts of GPC. GPC is known to be a product of phosphatidylcholine (PC) catabolism. The purpose of the present experiments was to examine the role of changes in the rate of GPC synthesis from PC in hyperosmotically induced GPC accumulation. When 1-palmitoyl-2-lysophosphatidyl-[methyl-3H]choline ([3H]LPC) is added to the medium, it is taken up by the cells and most of it is rapidly converted to PC. During a chase, 3H lost from PC appears almost exclusively in GPC and sphingomyelin. The rate of catabolism of PC is twofold greater in cells exposed to high NaCl (200 mosmol/kgH2O, added for 2 days) than in control or high-urea medium. Increased PC catabolism in NaCl-treated cells is associated with a 2.6-fold increase in GPC synthesis from PC; sphingomyelin synthesis decreases, and total cell PC does not change. Also, neither total mass nor specific radioactivity of lysophosphatidylcholine changes. PC catabolism is unaffected by short (2 h) exposure to high NaCl or urea. To investigate the enzymatic basis for the increased PC catabolism in response to high NaCl, phospholipase activity was measured in cell homogenates with 1-palmitoyl-2-[1-14C]palmitoyl-PC as a substrate. Exposure of cells to high NaCl for 2 days (but not 2 h) increases activity 2.8-fold compared with control or high-urea medium. Lysophospholipase activity (measured with [3H]LPC as the substrate) is unchanged. The increased phospholipase activity occurs with dipalmitoyl PC, but not sn-2-arachidonyl PC, as a substrate. Collectively, these data suggest a role for a phospholipase, unrelated to the arachidonyl-selective enzyme, in the regulation of PC catabolism during accumulation of GPC induced by prolonged exposure to high extracellular NaCl.
Sensitive detection of myosin heavy chain composition in skeletal muscle under different loading conditions
Authors: Fauteck S. P. , Kandarian S. C.
Published: December 2017

Different loading conditions were employed to study changes in myosin heavy chain (MHC) composition with the aid of a sensitive approach for separating and detecting MHC isoforms. Separation and detection of MHCs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were achieved to a degree such that MHC composition is consistent with previous reports on functional and mRNA data. Neonatal MHC was detected at low levels in control, 14-day hindlimb unweighted (HU), and 28-day HU soleus muscles. Type IIa MHC remained unchanged in all groups, representing approximately 9% of total MHC present. Type IIb MHC was not detected in control but represented 3% of total MHC at both 14 and 28 days of HU. Type IIx MHC also was not detected in control but represented 6% of total MHC at 14 days HU, and increased to 14% of total MHC at 28 days HU (P < 0.05). Type I MHC decreased from 89% in control to 72% at 28 days HU (P < 0.05). Furthermore, the type I MHC band was separated into two bands of approximately equal content in all groups when low amounts of protein were loaded on gels. The decrease in type I MHC with HU could be attributed entirely to a decrease in the percentage of the band in the type I region with lower mobility, which corresponds to beta-MHC. In addition, hypertrophied plantaris muscles demonstrated a fast-to-slow shift in MHC composition as evidenced by increased I, IIa, and IIx MHC and decreased IIb MHC expression.
Regulation of Na+/H+ exchange activity by recruitment of new Na+/H+ antiporters: effect of calyculin A
Authors: Guizouarn H. , Borgese F. , Pellissier B. , Garcia-Romeu F. , Motais R.
Published: December 2017

The Na+/H+ antiporter of trout red blood cells, beta-NHE, is activated by agonists of the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A (PKA) and by those of protein kinase C (PKC). beta-NHE, once activated, shifts into a refractory state, accounting for its desensitization. It had previously been shown that desensitization is blocked and reversed by the protein phosphatase inhibitor okadaic acid (OA). In this study we examined the effect of another protein phosphatase inhibitor, calyculin A (CIA). CIA was at least 10 times more potent than OA in blocking beta-NHE desensitization, suggesting that desensitization is controlled by phosphatase-1. Furthermore, CIA alone induced a large Na+/H+ exchange in unstimulated red blood cells, a property not shared by OA. The characteristics of ClA-induced Na+/H+ exchange are very different from those of the exchange triggered by activation of beta-NHE by PKA or PKC agonists, i.e., a flat pH dependence and total insensitivity to PKA and PKC inhibitors. Simultaneous addition of maximal concentrations of ClA and catecholamine produced an additive stimulation of the Na+/H+ exchange, consistent with the interpretation that these agents act on two distinct pools of exchangers. Screening of different cDNA libraries suggested that only one isoform of antiporter exists in the trout red blood cell; it therefore seems likely that regulation of the Na+/H+ antiporter beta-NHE involves a recycling mechanism. The reasons why intracellular beta-NHE show different properties from membrane beta-NHE are discussed.
Changes in contractile protein mRNA accumulation in response to spaceflight
Authors: Esser K. A. , Hardeman E. C.
Published: December 2017

Ten rats were exposed to 9 days of zero gravity aboard the National Aeronautics and Space Administration SLS-1 space mission (June 1991). Levels of fast and slow isoform mRNAs from six contractile protein gene families were quantified in the flight soleus and extensor digitorum longus (EDL) muscles. The gene families studied were myosin light chain-1 (MLC-1), myosin light chain-2 (MLC-2), troponin (Tn) T, TnI, TnC, and tropomyosin. In the EDL muscle there was no change in slow mRNA levels with a general increase in fast mRNA levels from 23 to 232%. Changes in slow mRNA levels were seen in the flight soleus muscle with TnCslow and TnTslow levels increasing slightly, and MLC-1slow a, MLC-1slow b, TnIslow, alpha-Tmslow, and MLC-2slow levels decreasing. All fast mRNA levels increased in the flight soleus muscle from 170 to 1,100%. We can conclude that exposure to zero gravity results in 1) a general increase in fast mRNA levels in both fast and slow muscles and 2) differing directional changes in slow mRNA accumulation in the soleus muscle.
Depletion and filling of intracellular calcium stores in vascular smooth muscle
Authors: Blatter L. A.
Published: December 2017

In vascular smooth muscle, binding of vasoactive substances to surface membrane receptors leads to a rise of intracellular cytoplasmic Ca2+ and to contraction. Cytoplasmic free Ca2+ concentration ([Ca2+]i) increases through release of Ca2+ from intracellular stores and Ca2+ entry through surface membrane ion channels. Membrane-permeant and membrane-impermeant forms of fura 2 were used to distinguish changes in intracellularly stored Ca2+ ([Ca2+]s) from changes in [Ca2+]i. The spatiotemporal patterns of the movement of Ca2+ between these two cellular compartments in cultured vascular smooth muscle cells (A7r5 cell line) were visualized with digital imaging fluorescence microscopy. The Ca2+ stores were localized by double staining with a fluorescent organelle-specific dye and the Ca2+ indicator. [Ca2+]s was measured after accumulation of the membrane-permeant form of fura 2 inside the stores and quenching of the fura 2 fluorescence in the cytoplasmic compartment with manganese. Stimulation with vasopressin led to a transient increase of [Ca2+]i and a concomitant decrease of [Ca2+]s. After stimulation with vasopressin, [Ca2+]i returned rapidly to normal resting levels, whereas the recovery of [Ca2+]s occurred on a much slower time scale. The refilling pathway of depleted stores involved Ca2+ entry into the bulk cytoplasmic compartment before uptake into the stores.
Molecular determinants of ion conduction and inactivation in K+ channels
Authors: Kukuljan M. , Labarca P. , Latorre R.
Published: December 2017

K+ channel-forming proteins can be grouped into three families that differ by the number of potential membrane-spanning segments. The largest of these families is composed of tetrameric channels with subunits containing six putative membrane-spanning segments (S1-S6). Inward rectifiers comprise a second family of K+ channels with subunits having two transmembrane domains (M1, M2). Monomers in the third family are proteins containing only one membrane-spanning segment, and they give origin to minK+ channels. Joining together segments S5 and S6 in the case of voltage-gated K+ channels and M1 and M2 in inward rectifiers, there is a highly conserved region with a hairpin shape called the H5 or P region. The P region, the loop connecting the S4 and S5 domains and the S6 transmembrane segment in Shaker-type K+ channels and the COOH-terminal in inward rectifiers, appears to play crucial roles in ion conduction. In Shaker K+ channels the NH2-terminal has been identified as responsible for fast inactivation (N-type inactivation). If the fast-inactivation gate is removed, a slower inactivation process persists, and its rate can be altered by mutations of amino acid residues forming part of the region in the neighborhood of the COOH-terminal (C-type inactivation). In this review we discuss the strategies followed to identify the different structures of K+ channels involved in ion conduction and inactivation processes and how they interplay.
Lipoxygenase inhibitors block PDGF-induced mitogenesis: a MAPK-independent mechanism that blocks fos and egr
Authors: Beno D. W. , Mullen J. , Davis B. H.
Published: December 2017

Hepatic Ito cells proliferate during liver injury and fibrogenesis. Platelet-derived growth factor (PDGF)-induced [3H]thymidine incorporation was studied as Ito cells express the PDGF receptor after injury and activation. Pretreatment with either the nonspecific lipoxygenase inhibitor (nordihydroguaiaretic acid) or specific inhibitors of 5-lipoxygenase (SC-41661 and ICI-230487) inhibited PDGF-induced mitogenesis. Ito cells predominantly produce the leukotriene (LT) C4 >> LTB4. The PDGF-induced signal transduction cascade was studied to determine the potential mechanism of action of the lipoxygenase inhibitors. It was found that PDGF receptor abundance and receptor activation were not altered by lipoxygenase inhibition, suggesting that a postreceptor mechanism was involved. The two-key cytoplasmic serine-threonine kinases Raf and MAPK (mitogen-activated protein kinase), which are induced by PDGF and transmit the signal to the nucleus, were also not altered. Because Raf and MAPK can independently induce nuclear signaling, this suggests that the mechanism of action lies parallel or distal to these secondary messengers. Lipoxygenase inhibition did result in the suppression of PDGF-induced fos and egr expression. Collectively, this work suggests that lipoxygenase inhibition leads to the suppression of mitogenesis in part by disrupting the nuclear signaling that is required for protooncogene transcription at a step distal or parallel to MAPK activation.
Angiotensin II decreases inducible nitric oxide synthase expression in rat astroglial cultures
Authors: Chandler L. J. , Kopnisky K. , Richards E. , Crews F. T. , Sumners C.
Published: December 2017

Consistent with stimulation of expression of an inducible form of nitric oxide synthase (iNOS), exposure of rat astroglial cultures to lipopolysaccharide (LPS) caused a time-dependent increase in the accumulation of nitrite in the culture media. Addition of the peptide angiotensin II (ANG II) with LPS decreased subsequent formation of nitrite in a concentration-dependent manner (concentration inhibiting 50% of maximal response approximately 1 nM). The ANG II effect could be blocked by the ANG II type 1 (AT1 receptor antagonist losartan but not by the ANG II type 2 (AT2) receptor antagonist PD-123177. ANG II had no effect on nitrite formation stimulated by a combination of inflammatory cytokines (interleukin-1 beta, tumor necrosis factor-alpha, and interferon-gamma). A brief 10-min exposure to ANG II was sufficient to cause an approximately 30% inhibition of the LPS response, with maximal inhibition of approximately 65% after 3 h, and occurred only when ANG II was added during the iNOS induction phase. Consistent with partial inhibition of LPS-stimulated expression of iNOS, ANG II reduced the levels of both iNOS mRNA and iNOS protein. These results demonstrate that ANG II can decrease LPS-stimulated NO production in astroglia by inhibiting induction of iNOS expression.
Physiological roles and properties of potassium channels in arterial smooth muscle
Authors: Nelson M. T. , Quayle J. M.
Published: December 2017

This review examines the properties and roles of the four types of K+ channels that have been identified in the cell membrane of arterial smooth muscle cells. 1) Voltage-dependent K+ (KV) channels increase their activity with membrane depolarization and are important regulators of smooth muscle membrane potential in response to depolarizing stimuli. 2) Ca(2+)-activated K+ (KCa) channels respond to changes in intracellular Ca2+ to regulate membrane potential and play an important role in the control of myogenic tone in small arteries. 3) Inward rectifier K+ (KIR) channels regulate membrane potential in smooth muscle cells from several types of resistance arteries and may be responsible for external K(+)-induced dilations. 4) ATP-sensitive K+ (KATP) channels respond to changes in cellular metabolism and are targets of a variety of vasodilating stimuli. The main conclusions of this review are: 1) regulation of arterial smooth muscle membrane potential through activation or inhibition of K+ channel activity provides an important mechanism to dilate or constrict arteries; 2) KV, KCa, KIR, and KATP channels serve unique functions in the regulation of arterial smooth muscle membrane potential; and 3) K+ channels integrate a variety of vasoactive signals to dilate or constrict arteries through regulation of the membrane potential in arterial smooth muscle.
Protein kinase C regulates the magnitude and stability of CFTR currents in pancreatic duct cells
Authors: Winpenny J. P. , McAlroy H. L. , Gray M. A. , Argent B. E.
Published: December 2017

Activation of protein kinase C (PKC) inhibits adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated fluid secretion in rat pancreatic ducts (N. Ashton, R. L. Evans, and B. E. Argent. J. Physiol. Lond. 452: 99P, 1992). Using the patch-clamp technique, we have investigated whether this inhibition of fluid secretion results from an effect of PKC on cystic fibrosis transmembrane conductance regulator (CFTR) Cl channels. Exposure to 100 nM 4 beta-phorbol 12,13-dibutyrate (PDBu) had no effect on CFTR current density in unstimulated duct cells, but caused a 31% increase in the magnitude of CFTR currents recorded from cells stimulated with cAMP. Furthermore, prolonged (2-4 h) exposure of stimulated duct cells to 100 nM PDBu (a condition that should downregulate PKC) significantly slowed the rate at which CFTR currents run down after establishing a whole cell recording. A similar effect was observed with calphostin C (500 nM), a specific inhibitor of PKC. Thus, although inhibition of ductal fluid secretion by PDBu is unlikely to be explained by an effect on CFTR, modulation of PKC activity can affect both the magnitude and stability of CFTR currents in pancreatic duct cells.
Regulation of skeletal alpha-actin promoter in young chickens during hypertrophy caused by stretch overload
Authors: Carson J. A. , Yan Z. , Booth F. W. , Coleman M. E. , Schwartz R. J. , Stump C. S.
Published: December 2017

Anterior latissimus dorsi (ALD) muscles of 3-wk-old male chickens were injected with plasmids containing various lengths of the chicken skeletal alpha-actin promoter (ranging from -2,090 to -77 relative to the transcription start site) driving luciferase. Hypertrophy of the left ALD muscle was induced by attaching a weight (11% of body wt) to the left wing of each chicken, with the unweighted contralateral wing serving the control. Six days of stretch overload significantly increased muscle mass 110%. Luciferase activity from the -2,090 actin-luciferase chimeric gene increased 127% compared with the contralateral control ALD muscle. Luciferase activities driven by the -424, -202, and -99 actin promoters were 179, 134, and 378% higher, respectively, in the stretched ALD muscle than in the contralateral control ALD muscle. Luciferase activity from the -77 deletion construct was not different between stretched and control muscles. These data indicate that the gene region responding to stretch is downstream of -99 and imply, but do not conclusively prove, that the region between -99 and -77, which contains serum response element 1, contributes to the stretch-induced increase in skeletal alpha-actin promoter activity in the ALD muscle.
Adaptive differentiation of avian exocrine cells alters their pHi response to mAChR activation
Authors: Bentz C. , Hildebrandt J. P.
Published: December 2017

Muscarinic receptor-mediated changes in intracellular pH (pHi) were measured in isolated 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-loaded cells, suspended in bicarbonate-containing media, from the exocrine nasal gland of freshwater-fed ducklings (Anas platyrhynchos). The pHi recovery from an acid load was sensitive to amiloride, required sodium ions in the external medium, and was independent of added bicarbonate. These findings are consistent with the hypothesis that the pHi recovery was mediated by a Na+/H+ exchanger. Muscarinic activation of cells resulted in a sustained cytosolic alkalinization that was sensitive to atropine and that was blocked by amiloride. Activation of protein kinase C (PKC) or inhibition of protein phosphatases mimicked the effect of receptor activation on pHi, whereas inhibitors of PKC blocked the response, indicating that phosphorylation of a major pHi control mechanism results in a shift of pHi to more alkaline values. In contrast, fully differentiated salt gland cells isolated from nasal glands of salt-stressed ducklings responded to muscarinic receptor activation with a transient cytosolic acidification. These findings raise the question whether the cytosolic alkalinization in muscarinic acetylcholine receptor-activated naive cells may serve as a signal or a permissive factor for the initiation of adaptive growth and/or differentiation processes observed in the salt glands of salt-stressed birds.
C-type natriuretic peptide neuromodulates via "clearance" receptors
Authors: Trachte G. J. , Kanwal S. , Elmquist B. J. , Ziegler R. J.
Published: December 2017

A recently discovered endogenous autacoid, C-type natriuretic peptide, was tested in a pheochromocytoma (PC12) cell line for effects on 1) catecholamine release induced by a depolarizing stimulus, 2) guanylyl and adenylyl cyclase activities, and 3) specific 125I-labeled atrial natriuretic peptide (ANP) binding. C-type natriuretic peptide suppressed evoked neurotransmitter release in the absence of guanylyl cyclase activation or adenylyl cyclase inhibition; however, both a "clearance" (ANP-C) receptor binding agent, des-[Gln18Ser19Gly20Leu21Gly22]-ANF-(4-23)-NH2 (cANF), and pertussis toxin prevented this neuromodulatory effect. The C-type natriuretic peptide preferentially bound to receptors that also bound cANF. The results suggest that C-type natriuretic peptide suppressed evoked neurotransmitter efflux by binding to ANP-C receptors coupled to a pertussis toxin-sensitive process; furthermore, the neuromodulatory effect of C-type natriuretic peptide occurred independently of guanylyl cyclase activation or adenylyl cyclase inhibition. The novel aspects of these findings are 1) neuromodulatory effects of C-type natriuretic peptide, 2) guanylyl cyclase-independent actions of C-type natriuretic peptide, and 3) ANP-C receptors mediating C-type natriuretic peptide actions.
Ca2+-insensitive sustained contraction of skinned smooth muscle after acidic ADP treatment
Authors: Morimoto S. , Ogawa Y.
Published: December 2017

Page C27: S. Morimoto and Y. Ogawa. “Ca2+-insensitive sustained contraction of skinned smooth muscle after acidic ADP treatment.” Between lines 14 and 15, the following equation was inadvertently omitted by the printer:

P = Po exp(-kt)
Multifunctional transporter models: lessons from the transport of water, sugars, and ring compounds by GLUTs
Authors: Fischbarg J. , Vera J. C.
Published: December 2017

Facilitative glucose transporters (GLUTs) have recently been shown to be multifunctional, transporting substrates other than sugars, such as water and ring compounds as large as nitrobenzene-diazol-aminoglucose. Other membrane proteins, including transporters and cystic fibrosis transmembrane conductance regulator, have also revealed a finite permeability to water. We compare the alpha-helical and beta-barrel models for the structure of GLUTs, discuss recent evidence, and argue that a beta-barrel fold explains it better. We show a model for GLUTs consisting of a relatively rigid beta-barrel translocation unit ("channel") of diameter ample enough to allow permeation of the above substrates (approximately 20 A) but gated shut by mobile loops at both ends. Such gates would open only after aromatic interactions would lead to binding of the ring substrates for GLUTs; water would, however, traverse crevices in the closed gates. Using the insights gained from GLUTs, we propose that other transporters may share with GLUTs the motif of a beta-barrel channel and would be permeable to water due to the presence of such channels together with similarly behaving gates.
TNF modulates endothelial properties by decreasing cAMP
Authors: Koga S. , Morris S. , Ogawa S. , Liao H. , Bilezikian J. P. , Chen G. , Thompson W. J. , Ashikaga T. , Brett J. , Stern D. M. , et al.
Published: December 2017

Tumor necrosis factor-alpha (TNF-alpha), a monokine that contributes to vascular dysfunction accompanying the host response to gram-negative sepsis, has been shown to increase vascular permeability in vivo and to diminish the barrier function of cultured endothelial cell (EC) monolayers. The studies reported here indicate that a mechanism through which TNF alters EC barrier function involves a reduction in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) content, due in part to increased cyclic nucleotide phosphodiesterase (CNPDE) activities. TNF increased the diffusional transit of [3H]sorbitol, [3H]inulin, and 125I-labeled albumin across confluent bovine aortic EC monolayers. This effect of TNF was both time and dose dependent and occurred in parallel with a fall in EC cAMP. cAMP analogues, such as dibutyryl cAMP (DBcAMP), prevented TNF-induced perturbation of EC barrier function. TNF also mediated another important alteration in the EC phenotype, in that both mRNA and activity of the anticoagulant cofactor thrombomodulin were reduced after exposure of EC to TNF and were normalized by the addition of DBcAMP. EC monolayers exposed to TNF-alpha showed increased cAMP levels when exposed to 3-isobutyl-1-methylxanthine, a nonspecific CNPDE inhibitor. Ion exchange chromatography of cytosol derived from TNF-treated EC consistently showed an approximately 245% increase in phosphodiesterase (PDE) IV (high-affinity, cAMP-specific PDE) activity as identified by rolipram inhibition. PDE II activity was increased by 150% after TNF-alpha treatment of early passage EC, which was identified by cGMP-activated hydrolysis of cAMP. Western and Northern analyses, as well as activity studies, revealed that TNF treatment did not change the amount of PDE IV protein or mRNA but rather increased the specific activity of the isozyme, suggesting that a posttranslational modification had occurred. These data indicate that activation of EC CNPDE activity and decreased intracellular cAMP may represent a mechanism by which TNF increases EC permeability and promotes a procoagulant EC phenotype.
Effects of Ba2+ and Cs+ on apical membrane K+ conductance in toad retinal pigment epithelium
Authors: Hughes B. A. , Shaikh A. , Ahmad A.
Published: December 2017

Intracellular microelectrode techniques were employed to characterize the blocker sensitivity of the K+ conductance (gK) at the apical membrane of the toad retinal pigment epithelium (RPE). Increasing the K+ concentration in the apical bath ([K+]o) from 2 to 5 mM produced a rapid depolarization of the apical membrane potential (VA). The addition of 0.5 mM Ba2+ or 5 mM Cs+ to the apical bath rapidly depolarized VA and increased the transepithelial resistance and ratio of apical-to-basolateral membrane resistance. In the presence of apical Ba2+ or Cs+, the response of VA to delta [K+]o was markedly reduced, indicating that these ions are effective blockers of apical gK. The Ba(2+)- and Cs(+)-induced decreases in the apparent apical-to-basolateral membrane conductance ratio were concentration dependent, with apparent dissociation constants of 17 microM and 0.5 mM, respectively. The apparent blocker sensitivity of apical gK is similar to that previously demonstrated for the inwardly rectifying K+ conductance in isolated toad RPE cells, suggesting that the inwardly rectifying K+ conductance comprises much of apical gK.
Nonselective cation channel activation during wound healing in the corneal endothelium
Authors: Watsky M. A.
Published: December 2017

Rabbit corneas were injured by mechanical or thermal trauma. At several time points after wounding, corneal endothelial cells were isolated and their ion channels examined using standard and amphotericin perforated-patch whole cell patch-clamp configurations. Within 15-24 h after mechanical or thermal trauma, a nonselective cation current was observed in 79% of the cells examined that was not present in unwounded or sham-wounded corneas. By 73 h postwounding, the current was present in only 10% of the cells examined. The wound healing-induced current is outwardly rectifying, activates at depolarized voltages, shows no sign of inactivation, and is inhibited by flufenamic acid, quinidine, and acetate. In addition to this new current, it was observed that endothelial cells from freeze-wounded corneas no longer expressed the transient K+ current seen in control, sham, and mechanically wounded corneas. Corneal endothelial superfusion experiments found no significant difference in swelling rates between control and flufenamic acid-superfused wounded corneas, indicating that the wound healing-induced channel is not involved in the stromal hydration maintenance function of the corneal endothelium.
Biphasic regulation of paracellular permeability in human cervical cells by two distinct nucleotide receptors
Authors: Gorodeski G. I. , Hopfer U. , De Santis B. J. , Eckert R. L. , Rorke E. A. , Utian W. H.
Published: December 2017

The effects of extracellular ATP (ATPe) were characterized in human cervical cells grown on filters. ATPe changed the transepithelial conductance (GT) in a biphasic manner with an initial acute increase of 13 +/- 3% (phase I), lasting approximately 2 min, followed by a sustained decrease of 49 +/- 17% (phase II). ATPe also effected decreases of permeabilities to pyranine and to sucrose, which correspond to the phase II decrease in GT. Both phase I and II effects appear to be mediated by apical nucleotide receptors. However, the phase I effect differed from the phase II effect as follows: 1) a higher 50% effective concentration for ATPe, 22 vs. 3 microM; 2) different nucleotide specificity; 3) lack of influence of pretreatment with pertussis toxin; 4) independence from time in culture after seeding; and 5) involvement of cytosolic calcium, rather than diacylglycerol, as intracellular messenger. These differences suggest the presence of two different types of nucleotide receptors that use different intracellular signaling systems and have opposite effects on the paracellular permeability and electrical conductance. These results suggest that ATPe could regulate cervical mucus production in vivo by regulating the paracellular permeability. Depending on the specific receptors present, ATPe could either increase or decrease mucus production.
Nuclear proteins bind a cis-acting element in the smooth muscle alpha-actin promoter
Authors: McNamara C. A. , Thompson M. M. , Vernon S. M. , Shimizu R. T. , Blank R. S. , Owens G. K.
Published: December 2017

Identification of the regulators of smooth muscle cell (SMC) gene expression is critical to understanding SMC differentiation and alterations in SMC phenotype in vascular disease. Previous studies revealed positive transcriptional activity within the chicken smooth muscle (SM) alpha-actin promoter region from -209 to -257. In the present study, transient transfections of wild-type and mutant chicken SM alpha-actin promoter/reporter gene constructs into rat aortic SMC demonstrated that the positive transcriptional activity of this region was abolished with a two base pair mutation in a conserved sequence motif at -225 to -233 (TGTTTATC to TACTTATC). Electrophoretic mobility shift assays revealed that nuclear factors bound promoter fragments containing this sequence and that specific mutations in the TGTTTATC motif abolished nuclear factor binding. Studies thus provide evidence for binding of a nuclear factor to a positive cis-acting element within the SM alpha-actin promoter. Further characterization of this factor may contribute to a better understanding of the molecular mechanisms that regulate differentiation of SMC in vascular disease.
Chorda tympani taste response of rat to hydrochloric acid subject to voltage-clamped lingual receptive field
Authors: DeSimone J. A. , Callaham E. M. , Heck G. L.
Published: December 2017

The chorda tympani nerve response of the rat to HCl was obtained with the lingual receptive field under voltage clamp. Unlike NaCl responses, HCl responses were not affected by inside positive voltage perturbations. However, HCl responses under negative voltage clamp were suppressed in contrast to NaCl responses, which were enhanced. Unlike NaCl responses, HCl responses were amiloride insensitive. HCl rinsing from the tongue produced a large off-response. At zero current clamp the off-response coincided with an anomalous increased positive potential. The paracellular resistance was also higher for HCl relative to the same concentration of NaCl. This is evidence that H+ binds to the normally fixed anionic sites of the paracellular pathway rendering it anion selective. It is postulated that release of bound H+ from surface buffer sites is responsible for the second burst of neural activity upon rising HCl. Acids stimulate primarily through the paracellular pathway, which also furnishes buffering sites that regulate H+ concentration, thereby protecting the sensory apparatus from hyperacidic conditions.
Ca2+-insensitive sustained contraction of skinned smooth muscle after acidic ADP treatment
Authors: Morimoto S. , Ogawa Y.
Published: December 2017

Page C27: S. Morimoto and Y. Ogawa. “Ca2+-insensitive sustained contraction of skinned smooth muscle after acidic ADP treatment.” Between lines 14 and 15, the following equation was inadvertently omitted by the printer:

(See PDF)
Regulatory volume decrease in frog retinal pigment epithelium
Authors: Adorante J. S.
Published: December 2017

Pages C89–C1OO: J. S. Adorante.“Regulatory volume decrease in frog retinal pigment epithelium.” The origin line should read: School of Optometry, University of California, Berkeley, California 94720. On page C99, the following sentence should be added to the acknowledgment: This work was supported by National Eye Institute Grants EY-02205 (to S. S. Miller) and Core Grant EY-03176 and National Research Service Award EY-05968 (to J. S. Adorante).