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• W2115802065 abstract "Voltage-dependent K+ channels (VDPC) are expressed in most mammalian cells and involved in the proliferation and activation of lymphocytes. However, the role of VDPC in macrophage responses is not well established. This study was undertaken to characterize VDPC in macrophages and determine their physiological role during proliferation and activation. Macrophages proliferate until an endotoxic shock halts cell growth and they become activated. By inducing a schedule that is similar to the physiological pattern, we have identified the VDPC in non-transformed bone marrow-derived macrophages and studied their regulation. Patch clamp studies demonstrated that cells expressed outward delayed and inwardly rectifying K+ currents. Pharmacological data, mRNA, and protein analysis suggest that these currents were mainly mediated by Kv1.3 and Kir2.1 channels. Macrophage colony-stimulating factor-dependent proliferation induced both channels. Lipopolysaccharide (LPS)-induced activation differentially regulated VDPC expression. While Kv1.3 was further induced, Kir2.1 was down-regulated. TNF-α mimicked LPS effects, and studies with TNF-α receptor I/II double knockout mice demonstrated that LPS regulation mediates such expression by TNF-α-dependent and -independent mechanisms. This modulation was dependent on mRNA and protein synthesis. In addition, bone marrow-derived macrophages expressed Kv1.5 mRNA with no apparent regulation. VDPC activities seem to play a critical role during proliferation and activation because not only cell growth, but also inducible nitric-oxide synthase expression were inhibited by blocking their activities. Taken together, our results demonstrate that the differential regulation of VDPC is crucial in intracellular signals determining the specific macrophage response. Voltage-dependent K+ channels (VDPC) are expressed in most mammalian cells and involved in the proliferation and activation of lymphocytes. However, the role of VDPC in macrophage responses is not well established. This study was undertaken to characterize VDPC in macrophages and determine their physiological role during proliferation and activation. Macrophages proliferate until an endotoxic shock halts cell growth and they become activated. By inducing a schedule that is similar to the physiological pattern, we have identified the VDPC in non-transformed bone marrow-derived macrophages and studied their regulation. Patch clamp studies demonstrated that cells expressed outward delayed and inwardly rectifying K+ currents. Pharmacological data, mRNA, and protein analysis suggest that these currents were mainly mediated by Kv1.3 and Kir2.1 channels. Macrophage colony-stimulating factor-dependent proliferation induced both channels. Lipopolysaccharide (LPS)-induced activation differentially regulated VDPC expression. While Kv1.3 was further induced, Kir2.1 was down-regulated. TNF-α mimicked LPS effects, and studies with TNF-α receptor I/II double knockout mice demonstrated that LPS regulation mediates such expression by TNF-α-dependent and -independent mechanisms. This modulation was dependent on mRNA and protein synthesis. In addition, bone marrow-derived macrophages expressed Kv1.5 mRNA with no apparent regulation. VDPC activities seem to play a critical role during proliferation and activation because not only cell growth, but also inducible nitric-oxide synthase expression were inhibited by blocking their activities. Taken together, our results demonstrate that the differential regulation of VDPC is crucial in intracellular signals determining the specific macrophage response. Immune system responses to an antigen involve a complex network of several cell types. Among them, the mononuclear phagocyte family comprises numerous cell types, including tissue macrophages, Kupffer cells, dermal Langerhans cells, osteoclasts, microglia, and perhaps some of the interdigitating and follicular dendritic cells from lymphoid organs (1Ogawa M. Blood. 1993; 81: 2844-2853Crossref PubMed Google Scholar). Macrophages perform critical functions in the immune system, acting as regulators of homeostasis and as effector cells in infection, wounding, and tumor growth. In response to different growth factors and cytokines, macrophages can proliferate, become activated or differentiate. As monocytes differentiate into mature, non-proliferating macrophages they can produce a large variety of responses, including chemotaxis, phagocytosis, and secretion of numerous cytokines and other substances. To elicit the appropriate physiological response plasma membrane protein expression changes dramatically from proliferation to activation (2Celada A. Nathan C. Immunol. Today. 1993; 15: 100-102Abstract Full Text PDF Scopus (140) Google Scholar). Voltage-dependent potassium channels (VDPC) 1The abbreviations used are: VDPCvoltage-dependent potassium channelsBMDMbone marrow-derived macrophagesiNOSinducible nitric-oxide synthaseLPSlipopolysaccharideM-CSFmacrophage-colony stimulating factorMgTxrMargatoxinTNF-αtumor necrosis factor αILinterleukinRTreverse transcriptasePBSphosphate-buffered salineANOVAanalysis of variance.1The abbreviations used are: VDPCvoltage-dependent potassium channelsBMDMbone marrow-derived macrophagesiNOSinducible nitric-oxide synthaseLPSlipopolysaccharideM-CSFmacrophage-colony stimulating factorMgTxrMargatoxinTNF-αtumor necrosis factor αILinterleukinRTreverse transcriptasePBSphosphate-buffered salineANOVAanalysis of variance. are a group of plasma membrane ion channels with a key role in controlling repolarization and resting membrane potential in electrically excitable cells. K+ channels are also involved in the maintenance of vascular smooth muscle tone, glucose-stimulated insulin release by β-pancreatic cells, cell volume regulation, and cell growth (3Hille B. Ion Channels of Excitable Membranes. 3rd Ed. Sinauer Associates, Sunderland, MA2001Google Scholar). Leukocytes express a number of voltage-gated and/or second messenger-modulated ion channels, and the electrophysiological properties of many of these channels are known (4Gallin E.K. Livengood D.R. Am. J. Physiol. 1981; 241: C9-C17Crossref PubMed Google Scholar, 5Gallin E.K. Biophys. J. 1984; 46: 821-825Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 6Ypey D.L. Clapham D.E. Proc. Natl. Acad. U. S. A. 1984; 81: 3083-3087Crossref PubMed Scopus (97) Google Scholar, 7Gallin E.K. Sheehy P.A. J. Physiol. 1985; 369: 475-499Crossref PubMed Scopus (66) Google Scholar, 8Gallin E.K. McKinney L.C. J. Membr. Biol. 1988; 103: 55-66Crossref PubMed Scopus (61) Google Scholar, 9McKinney L.C. Gallin E.K. J. Membr. Biol. 1988; 103: 41-53Crossref PubMed Scopus (65) Google Scholar, 10Gallin E.K. Am. J. Physiol. 1989; 257: C77-C85Crossref PubMed Google Scholar, 11McKinney L.C. Gallin E.K. J. Membr. Biol. 1992; 130: 265-276Crossref PubMed Scopus (22) Google Scholar, 12Judge S.I. Montcalm-Mazzilli E. Gallin E.K. Am. J. Physiol. 1994; 267: C1691-C1698Crossref PubMed Google Scholar, 13DeCorsey T.E. Kim S.Y. Silver M.R. Quandt F.N. J. Membr. Biol. 1996; 152: 141-157Crossref PubMed Scopus (80) Google Scholar, 14Eder C. Klee R. Heinemann U. Naumyn-Schmiedeberg's Arch. Pharmacol. 1997; 356: 233-239Crossref PubMed Scopus (33) Google Scholar, 15Schmid-Antomarchi H. Schmid-Alliana A. Romey G. Ventura M.A. Breittmayer V. Millet M.A. Husson H. Moghrabi B. Lazdunski M. Rossi B. J. Immunol. 1997; 159: 6209-6215PubMed Google Scholar, 16Colden-Stanfield M. Gallin E.K. Am. J. Physiol. 1998; 275: C267-C277Crossref PubMed Google Scholar, 17Qiu M.R. Campbell T.J. Breit S.N. Clin. Exp. Immunol. 2002; 130: 67-74Crossref PubMed Scopus (30) Google Scholar). Despite considerable progress, important questions remain unsolved, the relationship of these proteins to cell function being one of the most relevant (18Gallin E.K. Physiol. Rev. 1991; 71: 775-811Crossref PubMed Scopus (180) Google Scholar, 19Eder C. Am. J. Physiol. 1998; 275: C327-C342Crossref PubMed Google Scholar, 20DeCoursey T.E. Grinstein S. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. Lippincott Willians & Wilkins, Philadelphia, PA1999: 141-157Google Scholar). VDPC are associated with macrophage functions such as migration, proliferation, activation, and cytokine production (see Refs. 18Gallin E.K. Physiol. Rev. 1991; 71: 775-811Crossref PubMed Scopus (180) Google Scholar and 19Eder C. Am. J. Physiol. 1998; 275: C327-C342Crossref PubMed Google Scholar for reviews). Although microglia appears to express most neuronal channels, circulating macrophages have a number of VDPC yet to be defined (19Eder C. Am. J. Physiol. 1998; 275: C327-C342Crossref PubMed Google Scholar). These proteins have been studied in various cellular models, and outward delayed and inwardly rectifying K+ currents have been identified. Furthermore, the presence of the shaker-like Kv1.3 and Kir2.1 channels has been detected in some studies. However, the use of either activated or transformed macrophage cell lines has led to controversial results (4Gallin E.K. Livengood D.R. Am. J. Physiol. 1981; 241: C9-C17Crossref PubMed Google Scholar, 5Gallin E.K. Biophys. J. 1984; 46: 821-825Abstract Full Text PDF PubMed Scopus (75) Google Scholar, 6Ypey D.L. Clapham D.E. Proc. Natl. Acad. U. S. A. 1984; 81: 3083-3087Crossref PubMed Scopus (97) Google Scholar, 7Gallin E.K. Sheehy P.A. J. Physiol. 1985; 369: 475-499Crossref PubMed Scopus (66) Google Scholar, 8Gallin E.K. McKinney L.C. J. Membr. Biol. 1988; 103: 55-66Crossref PubMed Scopus (61) Google Scholar, 9McKinney L.C. Gallin E.K. J. Membr. Biol. 1988; 103: 41-53Crossref PubMed Scopus (65) Google Scholar, 10Gallin E.K. Am. J. Physiol. 1989; 257: C77-C85Crossref PubMed Google Scholar, 11McKinney L.C. Gallin E.K. J. Membr. Biol. 1992; 130: 265-276Crossref PubMed Scopus (22) Google Scholar, 12Judge S.I. Montcalm-Mazzilli E. Gallin E.K. Am. J. Physiol. 1994; 267: C1691-C1698Crossref PubMed Google Scholar, 13DeCorsey T.E. Kim S.Y. Silver M.R. Quandt F.N. J. Membr. Biol. 1996; 152: 141-157Crossref PubMed Scopus (80) Google Scholar, 14Eder C. Klee R. Heinemann U. Naumyn-Schmiedeberg's Arch. Pharmacol. 1997; 356: 233-239Crossref PubMed Scopus (33) Google Scholar, 15Schmid-Antomarchi H. Schmid-Alliana A. Romey G. Ventura M.A. Breittmayer V. Millet M.A. Husson H. Moghrabi B. Lazdunski M. Rossi B. J. Immunol. 1997; 159: 6209-6215PubMed Google Scholar, 16Colden-Stanfield M. Gallin E.K. Am. J. Physiol. 1998; 275: C267-C277Crossref PubMed Google Scholar, 17Qiu M.R. Campbell T.J. Breit S.N. Clin. Exp. Immunol. 2002; 130: 67-74Crossref PubMed Scopus (30) Google Scholar, 18Gallin E.K. Physiol. Rev. 1991; 71: 775-811Crossref PubMed Scopus (180) Google Scholar, 19Eder C. Am. J. Physiol. 1998; 275: C327-C342Crossref PubMed Google Scholar, 20DeCoursey T.E. Grinstein S. Gallin J.I. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. Lippincott Willians & Wilkins, Philadelphia, PA1999: 141-157Google Scholar). Thus, while a high-conductance Ca2+-dependent K+ channel has been clearly identified as an early step in transmembrane signal transduction in macrophages (21Blunck R. Scheel O. Müller M. Brandenburg K. Seitzer U. Seydel U. J. Immunol. 2001; 166: 1009-1015Crossref PubMed Scopus (123) Google Scholar), the physiological role of VDPC in either proliferation or activation is not known. Primary culture of bone marrow-derived macrophages (BMDM) is a unique non-transformed model in which proliferation and activation can be studied separately, mimicking physiological processes that occur in the body (22Soler C. García-Manteiga J. Valdés R. Xaus J. Comalada M. Casado F.J. Pastor-Anglada M. Celada A. Felipe A. FASEB J. 2001; 15: 1979-1988Crossref PubMed Scopus (97) Google Scholar). Macrophages are generated in the bone marrow and, through the bloodstream, reach all tissues, stop proliferation, and become activated (2Celada A. Nathan C. Immunol. Today. 1993; 15: 100-102Abstract Full Text PDF Scopus (140) Google Scholar). Macrophage colony-stimulating factor (M-CSF) is the specific growth factor for this cell type (23Stanley E.R. Berg K.L. Einstein D.B. Lee P.S. Pixley F.J. Wang Y. Yeung Y.G. Mol. Reprod. Dev. 1997; 46: 4-10Crossref PubMed Scopus (340) Google Scholar). On the other hand, lipopolysaccharide (LPS) is a major component of the outer Gram-negative bacteria membrane, which interacts with monocytes/macrophages and induces a variety of intracellular signaling cascades, finally leading to the release of endogenous mediators such as TNF-α, IL-1, and IL-6 (24Sweet M.J. Hume D.A. J. Leukocyte Biol. 1996; 60: 8-26Crossref PubMed Scopus (698) Google Scholar). Furthermore, LPS triggers cellular activation and apoptosis by an early TNF-α-dependent mechanism (25Xaus J. Comalada M. Valledor A.F. Lloberas J. López-Soriano F.J. Argilés J.M. Bogdan C. Celada A. Blood. 2000; 95: 3823-3831Crossref PubMed Google Scholar). voltage-dependent potassium channels bone marrow-derived macrophages inducible nitric-oxide synthase lipopolysaccharide macrophage-colony stimulating factor rMargatoxin tumor necrosis factor α interleukin reverse transcriptase phosphate-buffered saline analysis of variance. voltage-dependent potassium channels bone marrow-derived macrophages inducible nitric-oxide synthase lipopolysaccharide macrophage-colony stimulating factor rMargatoxin tumor necrosis factor α interleukin reverse transcriptase phosphate-buffered saline analysis of variance. Several VDPC candidates could be present in macrophages and our first interest was to identify these channels in a primary culture of BMDM. Macrophages mainly expressed the outward delayed Kv1.3 and the inwardly rectifying Kir2.1 potassium channels. Because VDPC could be involved not only in proliferation but also in activation, our second goal was to determine their specific role by inducing a schedule that is similar to the physiological pattern in BMDM. M-CSF-dependent proliferation led to an up-regulation of VDPC generating an increase in potassium current densities without changes in current kinetics. When cells were further incubated with LPS the electrophysiological properties changed dramatically. Thus, Kv1.3 was further increased, whereas Kir2.1 was down-regulated. We also show that TNF-α partially mimicked the response to LPS, suggesting that there are TNF-α-dependent and -independent mechanisms mediating LPS-induced VDPC modulation in macrophages. Our results have physiological relevance and indicate that VDPC expression is important as an early regulatory step, and fine-tuning modulation of their expression is crucial to the specific membrane signaling that triggers the appropriate immune response. Animals and Cell Culture—BMDM from 6- to 10-week-old BALB/c or C57/BL6 mice (Charles River Laboratories) were used. Cells were isolated and cultured as described elsewhere (22Soler C. García-Manteiga J. Valdés R. Xaus J. Comalada M. Casado F.J. Pastor-Anglada M. Celada A. Felipe A. FASEB J. 2001; 15: 1979-1988Crossref PubMed Scopus (97) Google Scholar). Briefly, animals were killed by cervical dislocation, and both femurs were dissected removing adherent tissue. The ends of bones were cut off and the marrow tissue was flushed by irrigation with medium. The marrow plugs were passed through a 25-gauge needle for dispersion. The cells were cultured in plastic dishes (150 mm) in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum and 30% supernatant of L-929 fibroblast (L-cell) conditioned media as a source of M-CSF. Macrophages were obtained as a homogeneous population of adherent cells after 7 days of culture and maintained at 37 °C in a humidified 5% CO2 atmosphere. For experiments, they were cultured with the same tissue culture differentiation medium (Dulbecco's modified Eagle's medium, 20% fetal bovine serum, 30% L-cell medium) or arrested at G0 by M-CSF deprivation in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for at least 18 h. G0-arrested cells were further incubated in the absence or presence of recombinant murine M-CSF (1200 units/ml), with or without LPS (100 ng/ml) or TNF-α (100 ng/ml), for the indicated times. In some experiments, cells were exposed to rMargatoxin (MgTx), BaCl2, cycloheximide, and actinomycin D as previously described (26Garcia-Calvo M. Leonard R.J. Novick J.N. Stevens S.P. Schmalhofer W. Kaczorowski G.J. Garcia M.L. J. Biol. Chem. 1993; 268: 18866-18874Abstract Full Text PDF PubMed Google Scholar, 27Kalman K. Pennington M.W. Lanigan M.D. Nguyen A. Rauer H. Mahnir V. Paschetto K. Kem W.R. Grissmer S. Gutman G.A. Christian E.P. Cahalan M.D. Norton R.S. Chandy K.G. J. Biol. Chem. 1998; 273: 32697-32707Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 28Lepple-Wienhues A. Berweck S. Böhmig M. Leo C.P. Meyling B. Garbe C. Wiederholt M. J. Membr. Biol. 1996; 151: 149-157Crossref PubMed Scopus (94) Google Scholar, 29Celada A. Klemsz M.J. Maki R.A. Eur. J. Immunol. 1989; 19: 1103-1109Crossref PubMed Scopus (65) Google Scholar, 30Cullel-Young M. Barrachina M. López-López C. Goñalons E. Lloberas J. Soler C. Celada A. Immunogenetics. 2001; 53: 136-144Crossref PubMed Scopus (28) Google Scholar). The TNF-α receptor I/II double knockout mice (C57/BL6) used in this study were generated and characterized as previously reported (25Xaus J. Comalada M. Valledor A.F. Lloberas J. López-Soriano F.J. Argilés J.M. Bogdan C. Celada A. Blood. 2000; 95: 3823-3831Crossref PubMed Google Scholar, 31Bruce A.J. Boling W. Kindy M.S. Peschon J. Kraemer P.J. Carpenter M.K. Holtsberg F.W. Mattson M.P. Nat. Med. 1996; 2: 788-794Crossref PubMed Scopus (837) Google Scholar). All animal handling was approved by the ethics committee of the University of Barcelona in accordance with EU regulations. DNA Synthesis—DNA synthesis was measured as the incorporation of [3H]thymidine (Amersham Biosciences) to DNA, as described elsewhere (22Soler C. García-Manteiga J. Valdés R. Xaus J. Comalada M. Casado F.J. Pastor-Anglada M. Celada A. Felipe A. FASEB J. 2001; 15: 1979-1988Crossref PubMed Scopus (97) Google Scholar). Briefly, macrophages (5 × 104) were seeded in 24-well plates in 1 ml of medium without M-CSF for at least 18 h. Cells were then cultured for a further 24 h in the absence or presence of M-CSF with/without LPS, TNF-α, MgTx, or BaCl2 (1 mm). Finally, the medium was removed and replaced with 0.5 ml of the same medium containing 1 μCi/ml [3H]thymidine. After three additional hours of incubation, cells were fixed in 70% methanol, washed three times in ice-cold 10% trichloroacetic acid, and solubilized in 1% SDS and 0.3% NaOH. The whole content of the well was used for counting radioactivity. RNA Isolation and RT-PCR Analysis—Total RNA from mouse macrophages, brain, and liver was isolated using the Tripure reagent (Roche Diagnostics), following the manufacturer's instructions. Samples were further treated with the DNA-free kit from Ambion Inc. to remove DNA. Ready-to-Go RT-PCR beads (Amersham Biosciences) were used in a one-step RT-PCR as described elsewhere (22Soler C. García-Manteiga J. Valdés R. Xaus J. Comalada M. Casado F.J. Pastor-Anglada M. Celada A. Felipe A. FASEB J. 2001; 15: 1979-1988Crossref PubMed Scopus (97) Google Scholar, 32Fuster G. Vicente R. Coma M. Grande M. Felipe A. Methods Find. Exp. Clin. Pharmacol. 2002; 24: 253-259Crossref PubMed Scopus (17) Google Scholar, 33Coma M. Vicente R. Busquets S. Carbó N. Tamkun M.M. López-Soriano F.J. Argilés J.M. Felipe A. FEBS Lett. 2003; 536: 45-50Crossref PubMed Scopus (21) Google Scholar, 34Grande M. Suàrez E. Vicente R. Cantó C. Coma M. Tamkun M.M. Zorzano A. Gumà A. Felipe A. J. Cell. Physiol. 2003; 195: 187-193Crossref PubMed Scopus (24) Google Scholar). Total RNA and selected primers at 1 μm were added to the beads. The RT reaction was initiated by incubating the mixture at 42 °C for 30 min. Once the first-strand cDNA had been synthesized, the conditions were set for further PCR: 92 °C for 30 s, either 55 °C (Kv1.3 and 18S) or 60 °C (Kir2.1) for 1 min and 72 °C for 2 min. These settings were applied for 40 cycles. Every 10 cycles, 10 μl of the total reaction was collected in a separate tube for further electrophoresis and analysis. A range of dilutions of RNA from each independent sample was performed to obtain an exponential phase of amplicon production (not shown) as described previously (32Fuster G. Vicente R. Coma M. Grande M. Felipe A. Methods Find. Exp. Clin. Pharmacol. 2002; 24: 253-259Crossref PubMed Scopus (17) Google Scholar). The same independent RNA aliquot was used to analyze the VDPC mRNA expression and the respective amount of 18 S rRNA. Primer sequences and accession numbers were: Kv1.3 (accession number M30441), forward, 5′-CTCATCTCCATTGTCATCTTCTGA-3′ (base pairs 741–765) and reverse, 5′-TTGAATTGGAAACAATCAC-3′ (base pairs 1459–1440); Kir2.1 (accession number AF021136), forward, 5′-TGGCTGTGTGTTTTGGTTGATAGC-3′ (base pairs 297–320) and reverse, 5′-CTTTGCCATCTTCGCCATGACTGC-3′ (base pairs 555–532); and 18 S (accession number X00686), forward, 5′-CGCAGAATTCCCACTCCCGACCC-3′ (base pairs 482–498) and reverse, 5′-CCCAAGCTCCAACTACGAGC-3′ (base pairs 694–675). Kv1.5 and other Kv mRNA expression was analyzed by PCR as previously described (33Coma M. Vicente R. Busquets S. Carbó N. Tamkun M.M. López-Soriano F.J. Argilés J.M. Felipe A. FEBS Lett. 2003; 536: 45-50Crossref PubMed Scopus (21) Google Scholar). In all cases negative controls were performed in the absence of the RT reaction. Once the exponential phase of the amplicon production had been determined the specificity of each product was confirmed in test RT-PCR using the appropriate cDNA probe in a Southern blot analysis. PCR-generated VDPC cDNA probes from mouse brain were subcloned using the pSTBlue-1 acceptor vector kit (Novagen) and the sequences were confirmed using the Big Dye Terminator Cycle sequencing kit and an ABI 377 sequencer (Applied Biosystems). EcoRI-digested [α-32P]CTP random primer-labeled cDNAs were used as probes as previously described (34Grande M. Suàrez E. Vicente R. Cantó C. Coma M. Tamkun M.M. Zorzano A. Gumà A. Felipe A. J. Cell. Physiol. 2003; 195: 187-193Crossref PubMed Scopus (24) Google Scholar). At least three different filters were made from independent samples and representative blots are shown. Results were analyzed with Phoretix software (Nonlinear Dynamics). Protein Extracts and Western Blot—Cells were washed twice in cold phosphate-buffered saline (PBS) and lysed on ice with lysis solution (1% Nonidet P-40, 10% glycerol, 50 mmol/liter HEPES, pH 7.5, 150 mmol/liter NaCl) supplemented with 1 μg/ml aprotinin, 1 μg/ml leupeptin, 86 μg/ml iodoacetamide, and 1 mm phenylmethylsulfonyl fluoride as protease inhibitors. Sample protein concentration was determined by Bio-Rad protein assay. The proteins from cell lysates (100 μg) were boiled at 95 °C in Laemmli SDS-loading buffer and separated on 10% SDS-PAGE. They were transferred to nitrocellulose membranes (Immobilon-P, Millipore), and blocked in 5% dry milk-supplemented 0.2% Tween 20 PBS prior to immunoreaction. To monitor Kv1.3 and Kir2.1 expression, rabbit polyclonal antibodies (Alomone Labs) were used. To study the expression of inducible nitric-oxide synthase (iNOS), a rabbit antibody against mouse iNOS (Santa Cruz Biotechnology) was used. The rabbit polyclonal anti-Kv1.5 antibody was a kind gift from Dr. M. M. Tamkun (Colorado State University). As a loading and transfer control, a monoclonal anti-β-actin antibody (Sigma) was used. Electron Microscopy—Cell monolayers on Petri dishes were scraped and collected into PBS buffer. BMDM were cryofixed by high-pressure freezing using an EMPact (Leica). Freeze substitution was performed in an “Automatic Freeze Substitution system” (AFS) from Leica, using acetone containing 0.5% of uranyl acetate, for 3 days at –90 °C. On the fourth day, the temperature was slowly increased, 5 °C/h, to –50 °C. At this temperature samples were rinsed in acetone and then infiltrated and embedded in Lowicryl HM20. Ultrathin sections were picked up on Formvar-coated copper-palladium grids. For immunogold localization, samples were blocked with 10% fetal calf serum in PBS for 20 min and incubated at room temperature for 1 h with polyclonal anti-Kv1.3 or anti-Kir2.1 (1:200). Washes were performed with PBS prior to adding goat anti-rabbit conjugated to 10 nm colloidal gold (BioCell Research Laboratory) for 1 h at room temperature. Finally, samples were washed and contrasted with 2% uranyl acetate for 30 min and observed in a Hitachi 600AB electron microscope. Electrophysiological Recordings—Whole cell currents were measured using the patch clamp technique. An EPC-9 (HEKA) with the appropriate software was used for data recording and analysis. Currents were filtered at 2.9 kHz. Series resistance compensation was always above 70%. Patch electrodes of 2–4 Mohms were fabricated in a P-97 puller (Sutter Instruments Co.) from borosilicate glass (outer diameter 1.2 mm and inner diameter 0.94 mm; Clark Electromedical Instruments Co.). Electrodes were filled with the following solution (in mm): 120 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, 11 EGTA, 20 d-glucose, adjusted to pH 7.3 with KOH. The extracellular solution contained (in mm): 120 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 25 d-glucose, adjusted to pH 7.4 with NaOH. After establishing the whole cell configuration of the patch clamp technique macrophages were clamped to a holding potential of –60 mV. To evoke voltage-gated currents all cells were stimulated with 200-ms square pulses ranging from –150 to +50 mV in 10-mV steps. All recordings were routinely subtracted for leak currents. The pharmacological characterization of the inward rectifier K+ current was performed by adding to the external solution BaCl2 and CsCl at various concentrations (28Lepple-Wienhues A. Berweck S. Böhmig M. Leo C.P. Meyling B. Garbe C. Wiederholt M. J. Membr. Biol. 1996; 151: 149-157Crossref PubMed Scopus (94) Google Scholar). To block the outward current, MgTx and ShK-Dap22 were added to the external solution (26Garcia-Calvo M. Leonard R.J. Novick J.N. Stevens S.P. Schmalhofer W. Kaczorowski G.J. Garcia M.L. J. Biol. Chem. 1993; 268: 18866-18874Abstract Full Text PDF PubMed Google Scholar, 27Kalman K. Pennington M.W. Lanigan M.D. Nguyen A. Rauer H. Mahnir V. Paschetto K. Kem W.R. Grissmer S. Gutman G.A. Christian E.P. Cahalan M.D. Norton R.S. Chandy K.G. J. Biol. Chem. 1998; 273: 32697-32707Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Before experiments, toxins were reconstituted to 10 μm in Tris buffer (0.1% bovine serum albumin, 100 mm NaCl, 10 mm Tris, pH 7.5). All recordings were done at room temperature (20–23 °C). Reagents—Recombinant murine TNF-α was obtained from Prepo-Tech EC. Recombinant murine M-CSF was from R&D Systems. Cycloheximide, actinomycin D, LPS, CsCl, and BaCl2 were purchased from Sigma, and MgTx from Alomone Labs. ShK-Dap22 was from Bachem Biosciences Inc. Other reagents were of analytical grade. Analysis and Statistics—According to the solutions used, the calculated equilibrium potential for potassium was –79 mV (EK) using the Nernst equation. The normalized G/Gmaxversus voltage curve was fitted using Boltzmann's equation: G/Gmax = 1/(1 + exp((V[1,2]–V)/k)), where V½ is the voltage at which the current is half-activated and k is the slope factor of the activation curve. Values are expressed as the mean ± S.E. The significance of differences was established by either Student's t test or one way ANOVA (Graph Pad, PRISM 3.0) for either two-group or two-factor comparison, respectively. A value of p < 0.05 was considered significant. Macrophages Express Outward and Inward K+Currents: Pharmacology and Molecular Characterization of Kv1.3 and Kir2.1—Cells (n = 80) plated in the presence in L-cell-conditioned medium expressed outward delayed and inward rectifier potassium currents (Fig. 1A). Following a train of 200-ms depolarizing pulses to +50 mV at 400-ms intervals, the outward current showed a characteristic cumulative inactivation phenomenon (Fig. 1B). Fig. 1C shows the effect of MgTx and ShK-Dap22 on the outward conductance. The IC50 for inhibition were ∼5 and ∼3 pm for MgTx and ShK-Dap22, respectively. These results indicated that Kv1.3 would be the main channel responsible for the outward potassium current. On the other hand, the high sensitivity to Ba2+ (Fig. 1D) and Cs+ (Fig. 1E), together with the closed state above 0 mV of the inward current, indicated that the channel was Kir2.1. To identify K+ channels at the molecular level, we performed RT-PCR analyses. Mouse brain and liver RNAs were used as positive and negative controls, respectively. Fig. 2A shows that macrophages expressed Kv1.3 and Kir2.1 mRNA to a similar extent to that observed in the brain. In addition, specific Kv1.3 and Kir2.1 signals were obtained by Western blot analysis in brain and BMDM protein samples (Fig. 2B). The presence of other VDPC (Kv1.1, Kv1.2, Kv1.6, and Kv3.1) analyzed by RT-PCR was negative (Fig. 2C). However, BMDM expressed Kv1.5 mRNA but the protein expression was below detection levels analyzed by Western blot (Fig. 2C and data not shown).Fig. 2Voltage-dependent K+ channel expression in macrophages.A, mRNA expression of Kv1.3 and Kir2.1 in the mouse brain and macrophages but not in the liver. 1 μg of total RNA was used in RT-PCR reactions as described under “Experimental Procedures.” B, Kv1.3 and Kir2.1 protein expression in the mouse brain and BMDM. Western blot analysis were performed in the presence and the absence of the control antigen peptide. C, VDPC expression in BMDM. RT-PCR was set as described under “Experimental Procedures” with oligonucleotides from Kv1.1 (accession number NM_010595; base pairs 1102–1807), Kv1.2 (accession number NM_008417, base pairs 841–1691), Kv1.5 (accession number AF302768," @default.
• W2115802065 created "2016-06-24" @default.
• W2115802065 creator A5006867583 @default.
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• W2115802065 date "2003-11-01" @default.
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