bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms of sodium pump regulation
Authors: Therien Alex G. , Blostein Rhoda
Published: December 2017

The Na+-K+-ATPase, or sodium pump, is the membrane-bound enzyme that maintains the Na+ and K+ gradients across the plasma membrane of animal cells. Because of its importance in many basic and specialized cellular functions, this enzyme must be able to adapt to changing cellular and physiological stimuli. This review presents an overview of the many mechanisms in place to regulate sodium pump activity in a tissue-specific manner. These mechanisms include regulation by substrates, membrane-associated components such as cytoskeletal elements and the γ-subunit, and circulating endogenous inhibitors as well as a variety of hormones, including corticosteroids, peptide hormones, and catecholamines. In addition, the review considers the effects of a range of specific intracellular signaling pathways involved in the regulation of pump activity and subcellular distribution, with particular consideration given to the effects of protein kinases and phosphatases.
Impairment of NF-κB activation and modulation of gene expression by calpastatin
Authors: Chen Fei , Demers Laurence M. , Vallyathan Val , Lu Yongju , Castranova Vincent , Shi Xianglin
Published: December 2017

To address the involvement of the calpain system in both basal and silica-induced nuclear factor (NF)-κB activation, several human bronchial epithelial cell lines were established in which an intracellular inhibitor of calpain, calpastatin, was stably expressed. Reduced basal and silica-induced inhibitor (IκBα) degradation and NF-κB activation were observed in cells stably overexpressing calpastatin. In addition, the cells in which calpain was constitutively inhibited by the overexpression of calpastatin exhibited a notable morphological change. Whereas empty vector-transfected cells displayed a morphology indistinguishable from that of parental cells, cells overexpressing calpastatin exhibited a mosaic morphological change with reduced formation of lamella 30 min after the cells were seeded. Genefilter microarray experiments, in which 3,965 human genes can be evaluated for their expression at the same time, showed that calpastatin downregulated genes encoding several membrane-associated proteins or nuclear proteins and upregulated genes of collagen α2, DAZ, and mitochondrial capsule selenoprotein. These results suggest that, in addition to their proteolytic activities on cytoskeletal proteins and other cellular regulatory proteins, calpain-calpastatin systems can also affect the expression levels of genes encoding structural or regulatory proteins.
ATP induces dephosphorylation of myosin light chain in endothelial cells
Authors: Noll T. , Schäfer M. , Schavier-Schmitz U. , Piper H. M.
Published: December 2017

In cultured porcine aortic endothelial monolayers, the effect of ATP on myosin light chain (MLC) phosphorylation, which controls the endothelial contractile machinery, was studied. ATP (10 μM) reduced MLC phosphorylation but increased cytosolic Ca2+ concentration ([Ca2+]i). Inhibition of the ATP-evoked [Ca2+]i rise by xestospongin C (10 μM), an inhibitor of the inositol trisphosphate-dependent Ca2+ release from endoplasmic reticulum, did not affect the ATP-induced dephosphorylation of MLC. MLC dephosphorylation was prevented in the presence of calyculin A (10 nM), an inhibitor of protein phosphatases PP-1 and PP-2A. Thus ATP activates MLC dephosphorylation in a Ca2+-independent manner. In the presence of calyculin A, MLC phosphorylation was incremented after addition of ATP, an effect that could be abolished when cells were loaded with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid acetoxymethyl ester (10 μM). Thus ATP also activates a Ca2+-dependent kinase acting on MLC. In summary, ATP simultaneously stimulates a functional antagonism toward both phosphorylation and dephosphorylation of MLC in which the dephosphorylation prevails. In endothelial cells, ATP is the first physiological mediator identified to activate MLC dephosphorylation by a Ca2+-independent mechanism.
Multiple receptor activation elicits synergistic IP formation in nonpigmented ciliary body epithelial cells
Authors: Cilluffo Marianne C. , Esqueda Evette , Farahbakhsh Nasser A.
Published: December 2017

We have examined the interaction between muscarinic and α2-adrenergic receptor activation on inositol phosphate (IP) formation in the nonpigmented cells of the ciliary body epithelium (NPE cells) of the rabbit. We have compared these changes with those previously observed in the intracellular free Ca2+ concentration. Whereas muscarinic receptor activation causes an increase in intracellular Ca2+ and IP formation, activation of α2-receptors does not significantly increase either intracellular Ca2+ or IPs over basal levels. However, simultaneous activation of muscarinic and α2-adrenergic receptors with the specific agonists carbachol and UK-14304 produces massive Ca2+ increases and results in a synergistic increase in IP formation. This synergistic IP formation is inhibited by both muscarinic and α2-adrenergic receptor antagonists as well as by pertussis toxin and an inhibitor of phospholipase C. IP formation is predominantly independent of intracellular Ca2+, because it is decreased but not prevented by blocking the entry of Ca2+ with LaCl3 or chelating intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid. Thus synergistic IP formation underlies, at least in part, the synergistic increase in intracellular Ca2+ resulting from simultaneous activation of muscarinic and α2-adrenergic receptors.
In vitro system to study realistic pulsatile flow and stretch signaling in cultured vascular cells
Authors: Peng Xinqi , Recchia Fabio A. , Byrne Barry J. , Wittstein Ilan S. , Ziegelstein Roy C. , Kass David A.
Published: December 2017

We developed a novel real-time servo-controlled perfusion system that exposes endothelial cells grown in nondistensible or distensible tubes to realistic pulse pressures and phasic shears at physiological mean pressures. A rate-controlled flow pump and linear servo-motor are controlled by digital proportional-integral-derivative feedback that employs previously digitized aortic pressure waves as a command signal. The resulting pressure mirrors the recorded waveform and can be digitally modified to yield any desired mean and pulse pressure amplitude, typically 0–150 mmHg at shears of 0.5–15 dyn/cm2. The system accurately reproduces the desired arterial pressure waveform and cogenerates physiological flow and shears by the interaction of pressure with the tubing impedance. Rectangular glass capillary tubes [1-mm inside diameter (ID)] are used for real-time fluorescent imaging studies (i.e., pHi, NO, Ca2+), whereas silicon distensible tubes (4-mm ID) are used for more chronic (i.e., 2–24 h) studies regarding signal transduction and gene expression. The latter have an elastic modulus of 12.4 · 106 dyn/cm2 similar to in vivo vessels of this size and are studied with the use of a benchtop system. The new approach provides the first in vitro application of realistic mechanical pulsatile forces on vascular cells and should facilitate studies of phasic shear and distension interaction and pulsatile signal transduction.
p27Kip1 is an inducer of intestinal epithelial cell differentiation
Authors: Quaroni Andrea , Tian Jean Q. , Seth Prem , Ap Rhys Colette
Published: December 2017

Constant renewal of the intestinal epithelium is a highly coordinated process that has been subject to intense investigation, but its regulatory mechanisms are still essentially unknown. In this study, we have demonstrated that forced expression of the cyclin-dependent kinase inhibitors (CKIs) p27Kip1 and p21Cip1/WAF1 in human intestinal epithelial cells led to expression of differentiation markers at both the mRNA and protein levels. Cell differentiation was temporally dissociated from inhibition of retinoblastoma protein phosphorylation and growth arrest, already established 1 day after infection with recombinant adenoviruses. p27Kip1 proved significantly more efficient than p21Cip1/WAF1 in induction of cell differentiation. In contrast, forced expression of p16INK4a resulted in growth arrest without induction of differentiation markers. These results implicate both p27Kip1 and p21Cip1/WAF1 in the differentiation-timing process, but p21Cip1/WAF1 may act indirectly by increasing p27Kip1 levels. These results also suggest that induction of intestinal epithelial cell differentiation by CKIs is not related to their effects on the cell cycle and may involve interactions with cellular components other than cyclins and cyclin-dependent kinases.
Intra- and extracellular measurement of reactive oxygen species produced during heat stress in diaphragm muscle
Authors: Zuo Li , Christofi Fievos L. , Wright Valerie P. , Liu Cynthia Yu , Merola A. John , Berliner Lawrence J. , Clanton Thomas L.
Published: December 2017

Skeletal muscles are exposed to increased temperatures during intense exercise, particularly in high environmental temperatures. We hypothesized that heat may directly stimulate the reactive oxygen species (ROS) formation in diaphragm (one kind of skeletal muscle) and thus potentially play a role in contractile and metabolic activity. Laser scan confocal microscopy was used to study the conversion of hydroethidine (a probe for intracellular ROS) to ethidium (ET) in mouse diaphragm. During a 30-min period, heat (42°C) increased ET fluorescence by 24 ± 4%, whereas in control (37°C), fluorescence decreased by 8 ± 1% compared with baseline ( P < 0.001). The superoxide scavenger Tiron (10 mM) abolished the rise in intracellular fluorescence, whereas extracellular superoxide dismutase (SOD; 5,000 U/ml) had no significant effect. Reduction of oxidized cytochrome c was used to detect extracellular ROS in rat diaphragm. After 45 min, 53 ± 7 nmol cytochrome c · g dry wt−1 · ml−1 were reduced in heat compared with 22 ± 13 nmol · g−1 · ml−1 in controls ( P < 0.001). SOD decreased cytochrome creduction in heat to control levels. The results suggest that heat stress stimulates intracellular and extracellular superoxide production, which may contribute to the physiological responses to severe exercise or the pathology of heat shock.
Potent inhibition of the aortic smooth muscle maxi-K channel by clinical doses of ethanol
Authors: Walters F. S. , Covarrubias M. , Ellingson J. S.
Published: December 2017

We investigated the effects of clinically relevant ethanol concentrations (5–20 mM) on the single-channel kinetics of bovine aortic smooth muscle maxi-K channels reconstituted in lipid bilayers (1:1 palmitoyl-oleoyl-phosphatidylethanolamine: palmitoyl-oleoyl-phosphatidylcholine). Ethanol at 10 and 20 mM decreased the channel open probability ( P o) by 75 ± 20.3% mainly by increasing the mean closed time (+82 to +960%, n = 7). In some instances, ethanol also decreased the mean open time (−40.8 ± 22.5%). The P o-voltage relation in the presence of 20 mM ethanol exhibited a rightward shift in the midpoint of voltage activation (Δ V ½ ≅ 17 mV), a slightly steeper relationship (change in slope factor, Δ k, ≅ −2.5 mV), and a decreased maximum P o (from ∼0.82 to ∼0.47). Interestingly, channels inhibited by ethanol at low Ca2+ concentrations (2.5 μM) were very resistant to ethanol in the presence of increased Ca2+ (≥ 20 μM). Alcohol consumption in clinically relevant amounts may alter the contribution of maxi-K channels to the regulation of arterial tone.
Cloning and characterization of a type III Na-dependent phosphate cotransporter from mouse intestine
Authors: Bai Liqun , Collins James F. , Ghishan Fayez K.
Published: December 2017

Intestinal and renal absorption of inorganic phosphate (Pi) is critical for phosphate homeostasis in mammals. We have isolated a cDNA that encodes a type III Na-dependent phosphate cotransporter from mouse small intestine (mPit-2). The nucleotide sequence of mPit-2 predicts a protein of 653 amino acids with at least 10 putative transmembrane domains. Kinetic studies, carried out in Xenopus oocytes, showed that mPit-2 cRNA induces significant Na-dependent Piuptake with an apparent Michaelis constant ( K m) for phosphate of 38 μM. The transport of phosphate by mPit-2 is inhibited at high pH. Northern blot analysis demonstrated the presence of mPit-2 mRNA in various tissues, including intestine, kidney, heart, liver, brain, testis, and skin. The highest expression of mPit-2 in the intestine was found in the jejunum. In situ hybridization revealed that mPit-2 mRNA is expressed throughout the vertical crypt-villus axis of the intestinal epithelium. The presence of mPit-2 in the mouse intestine and its unique transport characteristics suggest that multiple Na-dependent cotransporters may contribute to phosphate absorption in the mammalian small intestine.
Volume-regulated chloride conductance in the LNCaP human prostate cancer cell line
Authors: Shuba Y. M. , Prevarskaya N. , Lemonnier L. , Van Coppenolle F. , Kostyuk P. G. , Mauroy B. , Skryma R.
Published: December 2017

Patch-clamp recordings were used to study ion currents induced by cell swelling caused by hypotonicity in human prostate cancer epithelial cells, LNCaP. The reversal potential of the swelling-evoked current suggested that Cl− was the primary charge carrier (termed I Cl,swell). The selectivity sequence of the underlying volume-regulated anion channels (VRACs) for different anions was Br−≈I− > Cl− > F− > methanesulfonate ≫ glutamate, with relative permeability numbers of 1.26, 1.20, 1.0, 0.77, 0.49, and 0.036, respectively. The current-voltage patterns of the whole cell currents as well as single-channel currents showed moderate outward rectification. Unitary VRAC conductance was determined at 9.6 ± 1.8 pS. Conventional Cl− channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 μM) and DIDS (100 μM) inhibited whole cell I Cl,swell in a voltage-dependent manner, with the block decreasing from 39.6 ± 9.7% and 71.0 ± 11.0% at +50 mV to 26.2 ± 7.2% and 14.5 ± 6.6% at −100 mV, respectively. Verapamil (50 μM), a standard Ca2+ antagonist and P-glycoprotein function inhibitor, depressed the current by a maximum of 15%. Protein tyrosine kinase inhibitors downregulated I Cl,swell(genistein with an IC50 of 2.6 μM and lavendustin A by 60 ± 14% at 1 μM). The protein tyrosine phosphatase inhibitor sodium orthovanadate (500 μM) stimulated I Cl,swell by 54 ± 11%. We conclude that VRACs in human prostate cancer epithelial cells are modulated via protein tyrosine phosphorylation.
Adaptive responses to peroxynitrite: increased glutathione levels and cystine uptake in vascular cells
Authors: Buckley Barbara J. , Whorton A. Richard
Published: December 2017

We and others recently demonstrated increased glutathione levels, stimulated cystine uptake, and induced γ-glutamylcysteinyl synthase (γ-GCS) in vascular cells exposed to nitric oxide donors. Here we report the effects of peroxynitrite on glutathione levels and cystine uptake. Treatment of bovine aortic endothelial and smooth muscle cells with 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, resulted in transient depletion of glutathione followed by a prolonged increase beginning at 8–9 h. Concentration-dependent increases in glutathione of up to sixfold occurred 16–18 h after 0.05–2.5 mM SIN-1. Responses to SIN-1 were inhibited by copper-zinc superoxide dismutases and manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride, providing evidence for peroxynitrite involvement. Because glutathione synthesis is regulated by amino acid availability, we also studied cystine uptake. SIN-1 treatment resulted in a prolonged increase in cystine uptake beginning at 6–9 h. Increases in cystine uptake after SIN-1 were blocked by inhibitors of protein and RNA synthesis, by extracellular glutamate but not by extracellular sodium. These studies suggest induction of the xc − pathway of amino acid uptake. A close correlation over time was observed for increases in cystine uptake and glutathione levels. In summary, vascular cells respond to chronic peroxynitrite exposure with adaptive increases in cellular glutathione and cystine transport.
Heterologous desensitization of response mediated by selective PKC-dependent phosphorylation of Gi-1 and Gi-2
Authors: Murthy K. S. , Grider J. R. , Makhlouf G. M.
Published: December 2017

This study examined the ability of protein kinase C (PKC) to induce heterologous desensitization by targeting specific G proteins and limiting their ability to transduce signals in smooth muscle. Activation of PKC by pretreatment of intestinal smooth muscle cells with phorbol 12-myristate 13-acetate, cholecystokinin octapeptide, or the phosphatase 1 and phosphatase 2A inhibitor, calyculin A, selectively phosphorylated Gαi-1 and Gαi-2, but not Gαi-3 or Gαo, and blocked inhibition of adenylyl cyclase mediated by somatostatin receptors coupled to Gi-1 and opioid receptors coupled to Gi-2, but not by muscarinic M2 and adenosine A1 receptors coupled to Gi-3. Phosphorylation of Gαi-1 and Gαi-2 and blockade of cyclase inhibition were reversed by calphostin C and bisindolylmaleimide, and additively by selective inhibitors of PKCα and PKCɛ. Blockade of inhibition was prevented by downregulation of PKC. Phosphorylation of Gα-subunits by PKC also affected responses mediated by βγ-subunits. Pretreatment of muscle cells with cANP-(4–23), a selective agonist of the natriuretic peptide clearance receptor, NPR-C, which activates phospholipase C (PLC)-β3 via the βγ-subunits of Gi-1 and Gi-2, inhibited the PLC-β response to somatostatin and [d-Pen2,5]enkephalin. The inhibition was partly reversed by calphostin C. Short-term activation of PKC had no effect on receptor binding or effector enzyme (adenylyl cyclase or PLC-β) activity. We conclude that selective phosphorylation of Gαi-1 and Gαi-2 by PKC partly accounts for heterologous desensitization of responses mediated by the α- and βγ-subunits of both G proteins. The desensitization reflects a decrease in reassociation and thus availability of heterotrimeric G proteins.
Volume overload cardiac hypertrophy exhibits decreased expression of Gsα and not of Giα in heart
Authors: Di Fusco Francesco , Hashim Shehla , Anand-Srivastava Madhu B.
Published: December 2017

We have recently reported enhanced levels of Giα proteins in genetic and other experimentally induced models of hypertension, whereas the levels of Gsα were decreased in hypertensive rats expressing cardiac hypertrophy. The present studies were undertaken to investigate whether the decreased levels of Gsα are associated with cardiac hypertrophy per se and used an aortocaval fistula (AV shunt; volume overload) rat model that exclusively expresses cardiac hypertrophy. Cardiac hypertrophy in Sprague-Dawley rats (200–250 g) was induced under anesthesia, and, after a period of 10 days, the hearts were used for adenylyl cyclase activity determination, protein quantification, and mRNA level determination. A temporal relationship between the expression of Gsα proteins and cardiac hypertrophy was also examined on days 2, 3, 7, and 10 after induction of AV shunt in the rat. The heart-to-body-weight ratio (mg/g) was significantly increased in AV shunt rats after 3, 7, and 10 days of induction of AV shunt compared with sham-operated controls, whereas arterial blood pressure was not different between the two groups. Guanosine 5′- O-(3-thiotriphosphate) (GTPγS) stimulated adenylyl cyclase activity in a concentration-dependent manner in heart membranes from both groups; however, the degree of stimulation was significantly decreased in AV shunt rats. In addition, the stimulatory effects of isoproterenol were also diminished in AV shunt rats compared with control rats, whereas glucagon-stimulated adenylyl cyclase activity was not different in the two groups. The inhibitory effects of oxotremorine (receptor-dependent Gi functions) and low concentrations of GTPγS on forskolin-stimulated adenylyl cyclase activity (receptor-independent Gi functions) were not different in the two groups. In addition forskolin and NaF also stimulated adenylyl cyclase activity to a lesser degree in AV shunt rats compared with control rats. The levels of Giα-2 and Giα-3 proteins and mRNA, as determined by immunoblotting and Northern blotting, respectively, were not different in both groups; however, the levels of Gsα45 and Gsα47, and not of Gsα52, proteins were significantly decreased in AV shunt rats by days 7 and 10 compared with control rats, whereas no change was observed on days 2 and 3 after induction of AV shunt. These results suggest that the decreased expression of Gsα proteins may not be the cause but the effect of hypertrophy and that the diminished responsiveness of adenylyl cyclase to GTPγS, isoproterenol, NaF, and forskolin in hearts from AV shunt rats may partly be due to the decreased expression of Gsα. It can be concluded from these studies that the decreased expression of Gsα may be associated with cardiac hypertrophy and not with arterial hypertension.
Tom20-mediated mitochondrial protein import in muscle cells during differentiation
Authors: Grey Janice Y. , Connor Michael K. , Gordon Joseph W. , Yano Masato , Mori Masataka , Hood David A.
Published: December 2017

Mitochondrial biogenesis is accompanied by an increased expression of components of the protein import machinery, as well as increased import of proteins destined for the matrix. We evaluated the role of the outer membrane receptor Tom20 by varying its expression and measuring changes in the import of malate dehydrogenase (MDH) in differentiating C2C12 muscle cells. Cells transfected with Tom20 had levels that were twofold higher than in control cells. Labeling of cells followed by immunoprecipitation of MDH revealed equivalent increases in MDH import. This parallelism between import rate and Tom20 levels was also evident as a result of thyroid hormone treatment. Using antisense oligodeoxynucleotides, we inhibited Tom20 expression by 40%, resulting in 40–60% reductions in MDH import. In vitro assays also revealed that import into the matrix was more sensitive to Tom20 inhibition than import into the outer membrane. These data indicate a close relationship between induced changes in Tom20 and the import of a matrix protein, suggesting that Tom20 is involved in determining the kinetics of import. However, this relationship was dissociated during normal differentiation, since the expression of Tom20 remained relatively constant, whereas imported MDH increased 12-fold. Thus Tom20 is important in determining import during organelle biogenesis, but other mechanisms (e.g., intramitochondrial protein degradation or nuclear transcription) likely also play a role in establishing the final mitochondrial phenotype during normal muscle differentiation.
Hyposmolality stimulates Na+/H+ exchange and HCO3 − absorption in thick ascending limb via PI 3-kinase
Authors: Good David W. , Di Mari John F. , Watts Bruns A.
Published: December 2017

The signal transduction mechanisms that mediate osmotic regulation of Na+/H+ exchange are not understood. Recently we demonstrated that hyposmolality increases HCO3 − absorption in the renal medullary thick ascending limb (MTAL) through stimulation of the apical membrane Na+/H+ exchanger NHE3. To investigate the mechanism of this stimulation, MTALs from rats were isolated and perfused in vitro with 25 mM HCO3 −-containing solutions. The phosphatidylinositol 3-kinase (PI 3-K) inhibitors wortmannin (100 nM) and LY-294002 (20 μM) blocked completely the stimulation of HCO3 − absorption by hyposmolality. In tissue strips dissected from the inner stripe of the outer medulla, the region of the kidney highly enriched in MTALs, hyposmolality increased PI 3-K activity 2.2-fold. Wortmannin blocked the hyposmolality-induced PI 3-K activation. Further studies examined the interaction between hyposmolality and vasopressin, which inhibits HCO3 −absorption in the MTAL via cAMP and often is involved in the development of plasma hyposmolality in clinical disorders. Pretreatment with arginine vasopressin, forskolin, or 8-bromo-cAMP abolished hyposmotic stimulation of HCO3 − absorption, due to an effect of cAMP to inhibit hyposmolality- induced activation of PI 3-K. In contrast to their effects to block stimulation by hyposmolality, PI 3-K inhibitors and vasopressin have no effect on inhibition of apical Na+/H+ exchange (NHE3) and HCO3 − absorption by hyperosmolality. These results indicate that hyposmolality increases NHE3 activity and HCO3 − absorption in the MTAL through activation of a PI 3-K-dependent pathway that is inhibited by vasopressin and cAMP. Hyposmotic stimulation and hyperosmotic inhibition of NHE3 are mediated through different signal transduction mechanisms.
Molecular determinants for the distinct pH sensitivity of Kir1.1 and Kir4.1 channels
Authors: Xu Haoxing , Yang Zhenjiang , Cui Ningren , Giwa Lande R. , Abdulkadir Latifat , Patel Monika , Sharma Puja , Shan George , Shen Wangzhen , Jiang Chun
Published: December 2017

Kir1.1 (ROMK1) is inhibited by hypercapnia and intracellular acidosis with midpoint pH for channel inhibition (p K a) of ∼6.7. Another close relative, Kir4.1 (BIR10), is also pH sensitive with much lower pH sensitivity (p K a ∼6.0), although it shares a high sequence homology with Kir1.1. To find the molecular determinants for the distinct pH sensitivity, we studied the structure-functional relationship using site-directed mutagenesis. An NH2-terminal residue (Lys-53) was found to be responsible for the low pH sensitivity in Kir4.1. Mutation of this lysine to valine (K53V), a residue seen at the same position in Kir1.1, markedly increased channel sensitivity to CO2/pH. Reverse mutation on Kir1.1 (V66K) decreased the CO2/pH sensitivities. Interestingly, mutation of these residues to glutamate greatly enhanced the pH sensitivity in both channels. Other contributors to the distinct pH sensitivity were histidine residues in the COOH terminus, whose numbers are fewer in Kir4.1 than Kir1.1. Mutation of two of these histidine residues in Kir1.1 (H342Q/H354N) reduced CO2/pH sensitivities, whereas the creation of two histidines (S328H/G340H) in Kir4.1 increased the CO2/pH sensitivities. Combined mutations of the lysine and histidine residues in Kir4.1 (K53V/S328H/G340H) gave rise to a channel that had CO2/pH sensitivities almost identical to those of the wild-type Kir1.1. Thus the residues demonstrated in our current studies are likely the molecular basis for the distinct pH sensitivity between Kir1.1 and Kir4.1.
Differential expression of cell-cell adhesion proteins and cyclin D in MEK1-transdifferentiated MDCK cells
Authors: Marschitz Ingrid , Lechner Judith , Mosser Irene , Dander Martina , Montesano Roberto , Schramek Herbert
Published: December 2017

Overexpression of a constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 (caMEK1) in epithelial Madin-Darby canine kidney (MDCK)-C7 cells disrupts morphogenesis, induces an invasive phenotype, and is associated with a reduced rate of cell proliferation. The role of cell-cell adhesion molecules and cell cycle proteins in these processes, however, has not been investigated. We now report loss of E-cadherin expression as well as a marked reduction of β- and α-catenin expression in transdifferentiated MDCK-C7 cells stably expressing caMEK1 (C7caMEK1) compared with epithelial mock-transfected MDCK-C7 (C7Mock1) cells. At least part of the remaining α-catenin was coimmunoprecipitated with β-catenin, whereas no E-cadherin was detected in β-catenin immunoprecipitates. In both cell types, the proteasome-specific protease inhibitors N-acetyl-Leu-Leu-norleucinal (ALLN) and lactacystin led to a time-dependent accumulation of β-catenin, including the appearance of high-molecular-weight β-catenin species. Quiescent as well as serum-stimulated C7caMEK1 cells showed a higher cyclin D expression than epithelial C7Mock1 cells. The MEK inhibitor U-0126 inhibited extracellular signal-regulated kinase phosphorylation and cyclin D expression in C7caMEK1 cells and almost abolished their already reduced cell proliferation rate. We conclude that the transdifferentiated and invasive phenotype of C7caMEK1 cells is associated with a diminished expression of proteins involved in cell-cell adhesion. Although β-catenin expression is reduced, C7caMEK1 cells show a higher expression of U-0126-sensitive cyclin D protein.
Impaired [Ca2+]i and pHiresponses to κ-opioid receptor stimulation in the heart of chronically hypoxic rats
Authors: Pei Jian-Ming , Zhou Jing-Jun , Bian Jin-Song , Yu Xiao-Chun , Fung Man-Lung , Wong Tak-Ming
Published: December 2017

κ-Opioid receptor (κ-OR) stimulation with U50,488H, a selective κ-OR agonist, or activation of protein kinase C (PKC) with 4-phorbol 12-myristate 13-acetate (PMA), an activator of PKC, decreased the electrically induced intracellular Ca2+ ([Ca2+]i) transient and increased the intracellular pH (pHi) in single ventricular myocytes of rats subjected to 10% oxygen for 4 wk. The effects of U50,488H were abolished by nor-binaltorphimine, a selective κ-OR antagonist, and calphostin C, a specific inhibitor of PKC, while the effects of PMA were abolished by calphostin C and ethylisopropylamiloride (EIPA), a potent Na+/H+exchange blocker. In both right hypertrophied and left nonhypertrophied ventricles of chronically hypoxic rats, the effects of U50,488H or PMA on [Ca2+]i transient and pHi were significantly attenuated and completely abolished, respectively. Results are first evidence that the [Ca2+]i and pHi responses to κ-OR stimulation are attenuated in the chronically hypoxic rat heart, which may be due to reduced responses to PKC activation. Responses to all treatments were the same for right and left ventricles, indicating that the functional impairment is independent of hypertrophy. κ-OR mRNA expression was the same in right and left ventricles of both normoxic and hypoxic rats, indicating no regional specificity.
Differential regulation of Ca2+ sparks and Ca2+ waves by UTP in rat cerebral artery smooth muscle cells
Authors: Jaggar Jonathan H. , Nelson Mark T.
Published: December 2017

Uridine 5′-triphosphate (UTP), a potent vasoconstrictor that activates phospholipase C, shifted Ca2+ signaling from sparks to waves in the smooth muscle cells of rat cerebral arteries. UTP decreased the frequency of Ca2+ sparks and transient Ca2+-activated K+ (KCa) currents and increased the frequency of Ca2+ waves. The UTP-induced reduction in Ca2+ spark frequency did not reflect a decrease in global cytoplasmic Ca2+, Ca2+influx through voltage-dependent Ca2+ channels (VDCC), or Ca2+ load of the sarcoplasmic reticulum (SR), since global Ca2+ was elevated, blocking VDCC did not prevent the effect, and SR Ca2+ load did not decrease. However, blocking protein kinase C (PKC) with bisindolylmaleimide I did prevent UTP reduction of Ca2+ sparks and transient KCacurrents. UTP decreased the effectiveness of caffeine, which increases the Ca2+ sensitivity of ryanodine-sensitive Ca2+ release (RyR) channels, to activate transient KCa currents. This work supports the concept that vasoconstrictors shift Ca2+ signaling modalities from Ca2+ sparks to Ca2+ waves through the concerted actions of PKC on the Ca2+ sensitivity of RyR channels, which cause Ca2+ sparks, and of inositol trisphosphate (IP3) on IP3 receptors to generate Ca2+ waves.
Sustained membrane depolarization and pulmonary artery smooth muscle cell proliferation
Authors: Platoshyn Oleksandr , Golovina Vera A. , Bailey Colleen L. , Limsuwan Alisa , Krick Stefanie , Juhaszova Magdalena , Seiden Jan E. , Rubin Lewis J. , Yuan Jason X.-J.
Published: December 2017

Pulmonary vasoconstriction and vascular medial hypertrophy greatly contribute to the elevated pulmonary vascular resistance in patients with pulmonary hypertension. A rise in cytosolic free Ca2+ ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) triggers vasoconstriction and stimulates cell growth. Membrane potential ( E m) regulates [Ca2+]cyt by governing Ca2+influx through voltage-dependent Ca2+ channels. Thus intracellular Ca2+ may serve as a shared signal transduction element that leads to pulmonary vasoconstriction and vascular remodeling. In PASMC, activity of voltage-gated K+(Kv) channels regulates resting E m. In this study, we investigated whether changes of Kv currents [ I K(V)], E m, and [Ca2+]cyt affect cell growth by comparing these parameters in proliferating and growth-arrested PASMC. Serum deprivation induced growth arrest of PASMC, whereas chelation of extracellular Ca2+ abolished PASMC growth. Resting [Ca2+]cyt was significantly higher, and resting E m was more depolarized, in proliferating PASMC than in growth-arrested cells. Consistently, whole cell I K(V) was significantly attenuated in PASMC during proliferation. Furthermore, E mdepolarization significantly increased resting [Ca2+]cyt and augmented agonist-mediated rises in [Ca2+]cyt in the absence of extracellular Ca2+. These results demonstrate that reduced I K(V), depolarized E m, and elevated [Ca2+]cyt may play a critical role in stimulating PASMC proliferation. Pulmonary vascular medial hypertrophy in patients with pulmonary hypertension may be partly caused by a membrane depolarization-mediated increase in [Ca2+]cyt in PASMC.
Distinct mechanisms of action of selective estrogen receptor modulators in breast and osteoblastic cells
Authors: Nuttall Mark E. , Stroup George B. , Fisher Paul W. , Nadeau Daniel P. , Gowen Maxine , Suva Larry J.
Published: December 2017

Raloxifene and idoxifene are selective estrogen receptor modulators (SERMs) that exhibit tissue-specific agonist or antagonist properties via interactions with the estrogen receptor (ER). Both compounds are similarly osteoprotective in the ovariectomized rat in vivo as assessed by measurement of bone mineral density, urinary pyridinium cross-links, and serum osteocalcin, suggesting a similar mechanism of action. However, we have identified a fundamental difference in this mechanism via the estrogen response element (ERE) in osteoblast-like cells. With the use of ERE-luciferase reporter constructs, raloxifene, like the complete ER-antagonist ICI-182780, acts as an antagonist via the ERE in osteoblastic cells. In contrast, idoxifene, like 17β-estrogen itself and 4-OH-tamoxifen, acts as an agonist in osteoblastic cells via an ER/ERE-mediated mechanism. Both ICI-182780 and raloxifene inhibited the ERE-dependent agonist activity of 17β-estradiol and idoxifene in osteoblastic cells. In contrast, in breast cells, raloxifene, idoxifene, 4-OH-tamoxifen, and ICI-182780 had no agonist activity and, indeed, raloxifene and idoxifene were potent antagonists of ERE-mediated 17β-estradiol action, indicating an ERE-dependent mode of action in these cells. Although these SERMs exhibit a similar antagonist activity profile in breast cells, they can be distinguished mechanistically in osteoblastic cells.
In squid nerves intracellular Mg2+promotes deactivation of the ATP-upregulated Na+/Ca2+ exchanger
Authors: DiPolo Reinaldo , Berberián Graciela , Beaugé Luis
Published: December 2017

We investigated the role of intracellular Mg2+(Mgi 2+) on the ATP regulation of Na+/Ca2+ exchanger in squid axons and bovine heart. In squid axons and nerve vesicles, the ATP-upregulated exchanger remains activated after removal of cytoplasmic Mg2+, even in the absence of ATP. Rapid and complete deactivation of the ATP-stimulated exchange occurs upon readmission of Mgi 2+. At constant ATP concentration, the effect of intracellular Mg2+ concentration ([Mg2+]i) on the ATP regulation of exchanger is biphasic: activation at low [Mg2+]i, followed by deactivation as [Mg2+]i is increased. No correlation was found between the above results and the levels of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5) P 2] measured in nerve membrane vesicles. Incorporation of PtdIns(4,5) P 2 into membrane vesicles activates Na+/Ca2+ exchange in mammalian heart but not in squid nerve. Moreover, an exogenous phosphatase prevents MgATP activation in squid nerves but not in mammalian heart. It is concluded that 1) Mgi 2+ is an essential cofactor for the deactivation part of ATP regulation of the exchanger and 2) the metabolic pathway of ATP upregulation of the Na+/Ca2+ exchanger is different in mammalian heart and squid nerves.
Expression and localization of Na+-HCO3 − cotransporter in bovine corneal endothelium
Authors: Sun Xing Cai , Bonanno Joseph A. , Jelamskii Sergey , Xie Qiang
Published: December 2017

Functional studies support the presence of the Na+-HCO3 −cotransporter (NBC) in corneal endothelium and possibly corneal epithelium; however, molecular identification and membrane localization have not been reported. To test whether NBC is expressed in bovine cornea, Western blotting was performed, which showed a single band at ∼130 kDa for freshly isolated and cultured endothelial cells, but no band for epithelium. Two isoforms of NBC have recently been cloned in kidney (kNBC) and pancreas (pNBC). RT-PCR was run using cultured and fresh bovine corneal endothelial and fresh corneal epithelial total RNA and specific primers for kNBC and pNBC. RT-PCR analysis for pNBC was positive in endothelium and weak in epithelium. The RT-PCR product was subcloned and confirmed as pNBC by sequencing. No specific bands for kNBC were obtained from corneal cells. Indirect immunofluorescence and confocal microscopy indicated that NBC locates predominantly to the basolateral membrane in corneal endothelial cells. Furthermore, Na+-dependent HCO3 − fluxes and HCO3 −-dependent cotransport with Na+ were elicited only from the basolateral side of corneal endothelial cells. Therefore, we conclude that pNBC is present in the basolateral membrane of both fresh and cultured bovine corneal endothelium and weakly expressed in the corneal epithelium.
β-Adrenergic receptor/cAMP-mediated signaling and apoptosis of S49 lymphoma cells
Authors: Yan Lizhen , Herrmann Volker , Hofer Jason K. , Insel Paul A.
Published: December 2017

β-Adrenergic receptor (βAR) activation and/or increases in cAMP regulate growth and proliferation of a variety of cells and, in some cells, promote cell death. In the current studies we addressed the mechanism of this growth reduction by examining βAR-mediated effects in the murine T-lymphoma cell line S49. Wild-type S49 cells, derived from immature thymocytes (CD4+/CD8+) undergo growth arrest and subsequent death when treated with agents that increase cAMP levels (e.g., βAR agonists, 8-bromo-cAMP, cholera toxin, forskolin). Morphological and biochemical criteria indicate that this cell death is a result of apoptosis. In cyc− and kin− S49 cells, which lack Gsα and functional protein kinase A (PKA), respectively, βAR activation of Gsα and cAMP action via PKA are critical steps in this apoptotic pathway. S49 cells that overexpress Bcl-2 are resistant to cAMP-induced apoptosis. We conclude that βAR activation induces apoptosis in immature T lymphocytes via Gsα and PKA, while overexpression of Bcl-2 prevents cell death. βAR/cAMP/PKA-mediated apoptosis may provide a means to control proliferation of immature T cells in vivo.
Determinants of the contractile properties in the embryonic chicken gizzard and aorta
Authors: Ogut Ozgur , Brozovich Frank V.
Published: December 2017

Smooth muscle is generally grouped into two classes of differing contractile properties. Tonic smooth muscles show slow rates of force activation and relaxation and slow speeds of shortening ( V max) but force maintenance, whereas phasic smooth muscles show poor force maintenance but have fast V max and rapid rates of force activation and relaxation. We characterized the development of gizzard and aortic smooth muscle in embryonic chicks to identify the cellular determinants that define phasic (gizzard) and tonic (aortic) contractile properties. Early during development, tonic contractile properties are the default for both tissues. The gizzard develops phasic contractile properties between embryonic days ( ED) 12 and 20, characterized primarily by rapid rates of force activation and relaxation compared with the aorta. The rapid rate of force activation correlates with expression of the acidic isoform of the 17-kDa essential myosin light chain (MLC17a). Previous data from in vitro motility assays (Rover AS, Frezon Y, and Trybus KM. J Muscle Res Cell Motil 18: 103–110, 1997) have postulated that myosin heavy chain (MHC) isoform expression is a determinant for V max in intact tissues. In the current study, differences in V max did not correlate with previously published differences in MHC or MLC17a isoforms. Rather, V max was increased with thiophosphorylation of the 20-kDa regulatory myosin light chain (MLC20) in the gizzard, suggesting that a significant internal load exists. Furthermore, V max in the gizzard increased during postnatal development without changes in MHC or MLC17 isoforms. Although the rate of MLC20 phosphorylation was similar at ED 20, the rate of MLC20 dephosphorylation was significantly higher in the gizzard versus the aorta, correlating with expression of the M130 isoform of the myosin binding subunit in the myosin light chain phosphatase (MLCP) holoenzyme. These results indicate that unique MLCP and MLC17 isoform expression marks the phasic contractile phenotype.
BKCa channel activation by membrane-associated cGMP kinase may contribute to uterine quiescence in pregnancy
Authors: Zhou Xiao-Bo , Wang Ge-Xin , Ruth Peter , Hüneke Bernd , Korth Michael
Published: December 2017

We investigated the influence of pregnancy on large-conductance calcium-activated potassium channel (BKCa) activity ( NP o) and on channel expression in membranes of isolated human myometrial smooth muscle cells. NP o in inside-out patches was higher in pregnant myometria (PM) compared with nonpregnant myometria (NPM), and the half-maximal activation potential was shifted by 39 mV to more negative potentials. This effect was not due to an enhanced BKCachannel expression. In the presence of cAMP kinase (PKA) or cGMP kinase (PKG), NP o increased in patches from PM but decreased in those from NPM. Western blot analysis and use of a specific PKG inhibitor (1 μM KT-5823) verified the existence of a partially active membrane-associated PKG. Inhibition of PKA by 100 nM PKI, the inhibitory peptide of PKA, had no effect on NP o. 8- p-Chlorophenylthio-cGMP (8-pCPT-cGMP) hyperpolarized cells from PM. This effect was abolished by iberiotoxin, a specific blocker of BKCachannels. It is concluded that an endogenous, membrane-bound PKG in myometrial cells specifically enhances BKCa channel activity during pregnancy and thus may contribute to uterine quiescence during pregnancy.
Ca2+ current activity decreases during meiotic progression in bovine oocytes
Authors: Tosti Elisabetta , Boni Raffaele , Cuomo Annunziata
Published: December 2017

By using the whole cell voltage-clamp technique, we studied changes in plasma membrane permeability at different meiotic stages of bovine oocytes. Follicular oocytes were matured in vitro and activated by Ca2+ ionophore. Oocytes at germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), and meiosis exit were used for electrophysiological recording. By clamping the oocytes at −30 mV, we found that the L-type voltage-dependent Ca2+ channels were active at the GV stage and that their activity decreased after the GVBD stage. Furthermore, the resting potential decreased from the GV to the MI stage and increased again at MII. A significant decrease of the steady-state conductance occurred from the GV to the MI stage, followed by a sharp increase at the MII stage. With the addition of organic L-type Ca2+ channel blockers (nifedipine and verapamil), we inhibited the Ca2+ currents. However, only in the case of verapamil was there a decrease of in vitro maturation efficiency. Our results suggest that, in addition to the cumulus-oocyte junctions, the plasma membrane channels provide another mode of Ca2+ entry into bovine oocytes during meiosis.
Arginine transport through system y+L in cultured human fibroblasts: normal phenotype of cells from LPI subjects
Authors: Dall'Asta Valeria , Bussolati Ovidio , Sala Roberto , Rotoli Bianca Maria , Sebastio Gianfranco , Sperandeo Maria Pia , Andria Generoso , Gazzola Gian C.
Published: December 2017

In lysinuric protein intolerance (LPI), impaired transport of cationic amino acids in kidney and intestine is due to mutations of the SLC7A7 gene. To assess the functional consequences of the LPI defect in nonepithelial cells, we have characterized cationic amino acid (CAA) transport in human fibroblasts obtained from LPI patients and a normal subject. In both cell types the bidirectional fluxes of arginine are due to the additive contributions of two Na+-independent, transstimulated transport systems. One of these mechanisms, inhibited by N-ethylmaleimide (NEM) and sensitive to the membrane potential, is identifiable with system y+. The NEM- and potential-insensitive component, suppressed by l-leucine only in the presence of Na+, is mostly due to the activity of system y+L. The inward and outward activities of the two systems are comparable in control and LPI fibroblasts. Both cell types express SLC7A1 (CAT1) and SLC7A2 (CAT2B and CAT2A) as well as SLC7A6 (y+LAT2) and SLC7A7 (y+LAT1). We conclude that LPI fibroblasts exhibit normal CAA transport through system y+L, probably referable to the activity of SLC7A6/y+LAT2.
Properties of CFTR activated by the xanthine derivative X-33 in human airway Calu-3 cells
Authors: Bulteau Laurence , Dérand Renaud , Mettey Yvette , Métayé Thierry , Morris M. Rachel , McNeilly Ceinwen M. , Folli Chiara , Galietta Luis J. V. , Zegarra-Moran Olga , Pereira Malcolm M. C. , Jougla Chantal , Dormer Robert L. , Vierfond Jean-Michel , Joffre Michel , Becq Frédéric
Published: December 2017

The pharmacological activation of the cystic fibrosis gene protein cystic fibrosis transmembrane conductance regulator (CFTR) was studied in human airway epithelial Calu-3 cells, which express a high level of CFTR protein as assessed by Western blot and in vitro phosphorylation. Immunolocalization shows that CFTR is located in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novel synthesized xanthine derivative 3,7-dimethyl-1-isobutylxanthine (X-33) is an activator of the CFTR channel in Calu-3 cells. Whole cell current activated by X-33 or IBMX is linear, inhibited by glibenclamide and diphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene. Intracellular cAMP was not affected by X-33. An outwardly rectifying Cl− current was recorded in the absence of cAMP and X-33 stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the use of short-circuit recordings, X-33 and IBMX were able to stimulate a large concentration-dependent CFTR transport that was blocked by glibenclamide but not by DIDS. Our results show that manipulating the chemical structure of xanthine derivatives offers an opportunity to identify further specific activators of CFTR in airway cells.