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• W2022292199 abstract "Whole-cell patch-clamp electrophysiological investigation of endothelial cells cultured from human saphenous vein (HSVECs) has identified a voltage-gated Na+ current with a mean peak magnitude of −595 ± 49 pA (n = 75). This current was inhibited by tetrodotoxin (TTX) in a concentration-dependent manner, with an IC50value of 4.7 μm, suggesting that it was of the TTX-resistant subtype. An antibody directed against the highly conserved intracellular linker region between domains III and IV of known Na+ channel α-subunits was able to retard current inactivation when applied intracellularly. This antibody identified a 245-kDa protein from membrane lysates on Western blotting and positively immunolabeled both cultured HSVECs and intact venous endothelium. HSVECs were also shown by reverse transcription-polymerase chain reaction to contain transcripts of the hH1 sodium channel gene. The expression of Na+ channels by HSVECs was shown using electrophysiology and cell-based enzyme-linked immunosorbent assay to be dependent on the concentration and source of human serum. Together, these results suggest that TTX-resistant Na+ channels of the hH1 isoform are expressed in human saphenous vein endothelium and that the presence of these channels is controlled by a serum factor. Whole-cell patch-clamp electrophysiological investigation of endothelial cells cultured from human saphenous vein (HSVECs) has identified a voltage-gated Na+ current with a mean peak magnitude of −595 ± 49 pA (n = 75). This current was inhibited by tetrodotoxin (TTX) in a concentration-dependent manner, with an IC50value of 4.7 μm, suggesting that it was of the TTX-resistant subtype. An antibody directed against the highly conserved intracellular linker region between domains III and IV of known Na+ channel α-subunits was able to retard current inactivation when applied intracellularly. This antibody identified a 245-kDa protein from membrane lysates on Western blotting and positively immunolabeled both cultured HSVECs and intact venous endothelium. HSVECs were also shown by reverse transcription-polymerase chain reaction to contain transcripts of the hH1 sodium channel gene. The expression of Na+ channels by HSVECs was shown using electrophysiology and cell-based enzyme-linked immunosorbent assay to be dependent on the concentration and source of human serum. Together, these results suggest that TTX-resistant Na+ channels of the hH1 isoform are expressed in human saphenous vein endothelium and that the presence of these channels is controlled by a serum factor. Vascular endothelial cells form the primary interface between the blood and the underlying tissue. These cells not only provide a barrier of varying permeability between the blood and the smooth muscle of the vessel wall, but are a major contributor to the processes of vascular growth and repair, vascular autoregulation, and control of vascular tone by secretion of both relaxant and contractile factors (1Jaffe E.A. Ann. N. Y. Acad. Sci. 1985; 454: 279-291Crossref PubMed Scopus (90) Google Scholar, 2Vanhoutte P.M. Rubanyi G.M. Miller V.M. Houston D.S. Annu. Rev. Physiol. 1986; 48: 307-320Crossref PubMed Scopus (566) Google Scholar). Endothelial cells are known to possess a broad spectrum of ion channels that open in response to a variety of stimuli, including membrane potential, receptor occupation, elevation of [Ca2+]i, and mechanical deformation induced by flow (3Revest P.A. Abbott N.J. Trends Pharmacol. Sci. 1992; 13: 404-407Abstract Full Text PDF PubMed Scopus (42) Google Scholar). Levels of [Ca2+]i are an important factor in the control of endothelial cell function (4Marsden P.A. Goligorsky M.S. Brenner B.M. J. Am. Soc. Nephrol. 1991; 1: 931-948PubMed Google Scholar), and ion channels, with their ability to allow both Ca2+ entry either directly or indirectly, via control of membrane potential, are critical to this process (5Adams D.J. Barakeh J. Laskey R. van Breemen C. FASEB J. 1989; 3: 2389-2400Crossref PubMed Scopus (378) Google Scholar). Definitive data regarding the exact repertoire of ion channels expressed by endothelial cells are still sparse, particularly in venous endothelium. In this study, we report the presence of a voltage-gated Na+ current present in human saphenous vein endothelial cells (HSVECs). 1The abbreviations used are: HSVECshuman saphenous vein endothelial cellsTTXtetrodotoxinPBSphosphate-buffered salineTEAtetraethylammoniumRT-PCRreverse transcription-polymerase chain reaction. This type of channel is normally only expressed by classically excitable cells that generate action potentials such as neurons and cardiac and skeletal muscle. Voltage-gated Na+ channels are characterized by their kinetics; voltage dependence; and sensitivity to the guanidinium toxin, tetrodotoxin (TTX). TTX-sensitive Na+ channels are blocked by nanomolar concentrations of TTX and are found in tissues such as mature skeletal muscle (6Cohen S.A. Barchi R.L. Int. Rev. Cytol. 1993; 137C: 55-103PubMed Google Scholar). In contrast, TTX-resistant channels have a substantially lower affinity for the toxin, requiring 0.1–10 μm for inhibition (7Hille B. Ionic Channels of Excitable Membranes. Sinauer Associates, Inc., Sunderland, MA1992Google Scholar). TTX-resistant channels are found in a wide variety of tissue types, including cardiac cells (8Brown A.M. Lee K.S. Powell T. J. Physiol. 1981; 318: 479-500Crossref PubMed Scopus (213) Google Scholar) and denervated or developing skeletal muscle (9Kallen R.G. Sheng Z.H. Yang J. Chen L.Q. Rogart R.B. Barchi R.L. Neuron. 1990; 4: 232-242Abstract Full Text PDF Scopus (230) Google Scholar) and corneal endothelium (10Watsky M.A. Cooper K. Rae J.L. Pfluegers Arch. Eur. J. Physiol. 1991; 419: 454-459Crossref PubMed Scopus (23) Google Scholar). A third class of voltage-gated Na+ channels, expressed by embryonic cardiac cells (11Bkaily G. Jaques D. Sculptoreanu A. Yamamoto T. Carrier D. Vigneault D. Sperelakis N. J. Mol. Cell. Cardiol. 1991; 23: 25-39Abstract Full Text PDF PubMed Scopus (20) Google Scholar) and dorsal root ganglion neurons (12Kostyuk P.G. Veselovsky N.S. Tsyndrenko A.Y. Neuroscience. 1981; 6: 2423-2430Crossref PubMed Scopus (318) Google Scholar), remain unblocked by TTX concentrations in excess of 100 μm and are classified as TTX-insensitive. The voltage-gated sodium current we describe here in HSVECs is TTX-resistant and appears to result from expression of the cardiac Na+ channel gene (hH1). human saphenous vein endothelial cells tetrodotoxin phosphate-buffered saline tetraethylammonium reverse transcription-polymerase chain reaction. HSVECs were obtained by enzymatic release from saphenous vein harvested during high ligation of varicose veins or bypass surgery. After removal of any residual external connective tissue, the vein was carefully opened along its longitudinal axis with a scalpel blade. HSVECs were obtained by placing the vein luminal face down in a shallow Petri dish containing Ca2+- and Mg2+-free phosphate-buffered saline (PBS; 150 mm NaCl, 2 mm NaH2PO4, and 10 mm Na2HPO4) and 1 mg/ml collagenase (Type II, Sigma) and incubating at room temperature (20–22 °C) for 30 min. Cells were placed in culture on fibronectin-coated dishes or flasks as appropriate and grown in M199 medium supplemented with heparin, endothelial cell growth supplement, antibiotic solution (200 units/ml penicillin and 200 μg/ml streptomycin), and 10% (v/v) heat-inactivated human serum. Serum was obtained either from non-diabetic patients with peripheral arterial disease (>65 years old) or from healthy donors (<30 years old). Cultures, characterized by positive immunostaining for von Willebrand factor, were maintained at 37 °C in humidified CO2 in air atmosphere and used in experiments at passages 0–3. Experiments were performed at room temperature (20–22 °C) using the whole-cell configuration of the patch-clamp technique (13Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth F.J. Pfluegers Arch. J. Eur. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15172) Google Scholar) on subconfluent HSVECs grown in 35-mm diameter Petri dishes. These were placed on the stage of an inverted microscope (Diaphot 200, Nikon, Tokyo, Japan), visualized with phase-contrast optics, and continuously superfused at 2 ml/min with extracellular solution. The standard pipette and extracellular solutions were designed to isolate INa (pipette: 120 mm CsCl, 10 mm EGTA, 2 mmMgCl2, 5 mm NaCl, 5 mm HEPES, 2 mm Na2ATP, and 0.5 mmNa2GTP; extracellular: 120 mm NaCl, 4 mm KCl, 2 mm MgCl2, 2 mm CaCl2, 10 mm TEA-Cl, and 10 mm HEPES (pH 7.3) with CsOH), although all current-clamp and some preliminary experiments employed “quasiphysiological” solutions (pipette: 140 mm KCl, 2 mmMgCl2, 1 mm CaCl2, 0.05 mm EGTA, 20 mm HEPES, 2 mmNa2ATP, and 0.5 mm Na2GTP; extracellular: 135 mm NaCl, 5 mm KCl, 2 mm MgCl2, 2 mm CaCl2, 11 mm glucose, and 10 mm HEPES (pH 7.3) with NaOH). Patch-clamp pipettes were manufactured from borosilicate glass (GC150-15TF, Clark Electromedical, Reading, United Kingdom) using a two-stage puller (PB7, Narashige, Tokyo, Japan) and fire-polished to give final resistances of 1–3 megaohms when filled with pipette solution. Whole-cell membrane currents (voltage-clamp) and potentials (current-clamp) were recorded using an Axopatch 200A patch clamp amplifier (Axon Instruments Inc., Foster City, CA) with analogue cell capacitance (mean cell capacitance = 35.1 ± 1.4 picofarads,n = 131) and series resistance (routinely <5 megaohms) maximally compensated. Signals were low pass-filtered with an 8-pole Bessel-type filter at either 2 or 5 kHz prior to digitization at 10 kHz by a Digidata 1200 interface (Scientific Solutions, Solon, OH) and storage on a computer hard disk (486DX2, Opus Technology). Analysis was performed using pClamp 6 software (Axon Instruments Inc.), which was also employed to generate voltage step protocols. When recordingINa linear leakage, currents were subtracted using a 4-subpulse (P/4) method (14Bezanilla F. Armstrong C.M. J. Gen. Physiol. 1977; 70: 549-566Crossref PubMed Scopus (645) Google Scholar), and INaamplitude was measured as the difference between the maximal inward current and the holding current level. Junction potentials were measured as described previously (15Fenwick E.M. Marty A. Neher E. J. Physiol. (Lond.). 1982; 331: 577-597Crossref Scopus (580) Google Scholar), although only determinations of the INa reversal potential were corrected. Total cellular RNA was extracted from HSVECs grown to confluence in T75 culture flasks by incubation with 6 ml of RNAzol reagent (Cinna/Biotecx Labs Inc.) at 4 °C for 10 min. Contaminating DNA was removed (Message Clean kit, GenHunter Corp.) prior to reverse transcription (Reverse Transcriptor kit, R&D Systems Ltd., Abingdon, UK), both according to the manufacturers' protocols. Sodium channel cDNA was amplified by PCR using oligonucleotide primers directed against the 3′-untranslated region of the human cardiac hH1channel (16Gellens M.E. George A.L. Chen L. Chahine M. Horn R. Barchi R.L. Kallen R.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 554-558Crossref PubMed Scopus (560) Google Scholar). The primer sequences used were 5′-GACCTGTGACCTGGTCTGGT-3′ and 5′-CCATGTCCATGGAAAAATCC-3′ (Perkin-Elmer, Warrington, UK). HSVEC RNA (1 μg) was used for PCR amplification using 1 μmprimers for 30 cycles (94 °C, 1 min; 50 °C, 1 min; and 72 °C, 1 min) at a MgCl2 concentration of 1 mm. The resulting PCR fragments were analyzed on a 2.5% (w/v) agarose gel containing ethidium bromide and sequenced using an ABI 373 automated sequencer to confirm fragment identity. Immunohistochemical analysis for the presence of voltage-gated Na+ channels in HSVECs was performed on intact tissue sections and cultured HSVECs using a polyclonal antibody raised in rabbits against the highly conserved cytosolic linker region between domains III and IV (peptide sequence TEEQKKYYNAMKKLGSKKP, amino acids 1490–1508) of known Na+ channel α-subunits (“anti-Naα”; TCS Biologicals, Botolph Claydon, UK). Small lengths of human saphenous vein were fixed overnight in Zamboni's solution (consisting of 1.7% (w/v) paraformaldehyde and 15% (v/v) saturated picric acid in PBS) and washed daily in PBS/sucrose solution (PBS supplemented with 440 mm sucrose) for 7 days prior to mounting in OCT compound (Miles Inc.). Veins were cryosectioned using a microtome (Bright Instruments, Huntingdon, UK) to produce 8–10-μm thick transverse sections, which were transferred to 3-aminopropyltriethoxysilane-coated slides prior to overnight incubation at 4 °C with primary antibody (anti-Naα, 7.5 μg/ml in PBS buffer supplemented with 1% (w/v) bovine serum albumin and 1% (v/v) human serum). Specific binding was visualized by alkaline phosphatase staining (Vector Red®, Vector Labs, Peterborough, UK) subsequent to primary antibody detection using a biotinylated secondary antibody (goat anti-rabbit, 1:200 dilution, 1 h) and a tertiary streptavidin-alkaline phosphatase conjugate incubation (1:200 dilution, 1 h; Dako, High Wycombe, UK). The presence of endothelium on intact vein sections was verified by using a mouse monoclonal antibody directed against thrombomodulin. A protocol similar to that described above was employed for cultured HSVECs, which were, however, fixed by incubation in 100% methanol at 4 °C for 2 min. Controls for both intact vein sections and HSVECs were prepared by omitting either the primary or secondary antibodies. HSVECs, grown to confluence in T25 culture flasks, were lysed for 30 min at 4 °C using 1 ml of lysis buffer that consisted of 140 mm NaCl, 10 mm Tris, 1 mm phenylmethylsulfonyl fluoride, 1 mm sodium metavanadate, 1 mm EDTA, 0.2 mg/ml aprotinin, 2 μg/ml pepstatin A, 2 μg/ml leupeptin, and 2.5% (v/v) Nonidet P-40. Lysed samples were centrifuged at 6000 × g for 10 min to separate membrane (particulate) and cytosolic (supernatant) fractions, which were suspended in loading buffer (40 μl for particulate and 80 μl for cytosolic fractions). Aliquots (16 μl) of sample lysates were fractionated by SDS-polyacrylamide gel electrophoresis using an 8–25% gradient gel (PhastGel, Pharmacia, St. Albans, UK) and transferred to polyvinylidene difluoride filter membranes (Immobilon-P, Millipore, Amersham, UK) for Western blotting. Membranes were incubated in blocking solution containing 5% (w/v) milk powder, 5% (w/v) bovine serum albumin, and 2% (v/v) human serum in Tris-buffered saline/Tween 20 solution (20 mm Tris base, 137 mm NaCl, and 0.05% (v/v) Tween 20 (pH 7.4) with HCl) for 6 h at room temperature and washed prior to overnight incubation at 4 °C in primary antibody solution (5 μg/ml anti-Naα antibody) prepared in Tris-buffered saline/Tween 20 solution and 1% (w/v) bovine serum albumin. After washing, membranes were incubated at room temperature for 30 min with a biotinylated secondary antibody (goat anti-rabbit, 1:1000) and a tertiary streptavidin-horseradish peroxidase conjugate (1:1000; Dako). Bound antibodies were detected using enhanced chemiluminescence (ECL, Amersham, Amersham, UK). Using this method, we could routinely detect protein levels as low as 0.5 pg/sample. HSVECs (105/well) were seeded onto 24-well plates and grown to confluence over 48 h. HSVECs were fixed by incubation in 100% methanol at 4 °C for 2 min and washed with Tris-buffered saline supplemented with 0.5% (w/v) bovine serum albumin. Cells were incubated with the anti-Naα antibody (2 μg/ml) for 40 min at 37 °C, washed, and subjected to two further incubations (30 min each at 37 °C) with a biotinylated secondary antibody (1:500) and a final streptavidin-horseradish peroxidase conjugate (1:500). Cells were thoroughly washed prior to assessment of anti-Naα antibody binding by colorimetric assay using o-phenylenediamine as the substrate, and the optical density was measured at 492 nm. Data are expressed as mean ± S.E. (n = number of observations). Normalized activation curves for INa were calculated by dividing conductances (gNa), derived from peak currents divided by the Na+ driving force (Vm − ENa), by the largest conductances measured. Steady-state inactivation curves (h∞) and activation curves (m∞) were fitted with a Boltzmann function, where V0.5 is the midpoint andKV is the slope factor:I/Imax = 1/1 +exp((V− V0.5)/KV). Kinetic analysis of INa was only performed if the peak current exceeded 500 pA, and time constants for current activation and inactivation were derived by fitting a Hodgkin-Huxley model (17Hodgkin A.L. Huxley A.F. J. Physiol. (Lond.). 1952; 117: 500-544Crossref Scopus (14521) Google Scholar) to the data as described elsewhere (18Sontheimer H. Waxman S.G. J. Neurophysiol. 1992; 68: 1001-1011Crossref PubMed Scopus (112) Google Scholar). The 50% inhibitory concentration (IC50) for TTX was calculated by fitting the concentration inhibition curve to a logistic plot incorporating Hill coefficients (nH) using MicroCal Origin (MicroCal Inc., Northampton, MA): bound = [drug]nH/[drug]nH+ IC50. Reversal potentials (Erev) were obtained by fitting a second-order polynomial to the current-potential (I-V) plots over the appropriate voltage regions (usually +20 to +80 mV). Where appropriate, results were tested for significance using Student's unpairedt test. Culture materials were obtained from Gibco Laboratories (Paisley, UK), and the thrombomodulin antibody was a gift from Dr. J. Amiral (Serbio Research, Paris, France). TTX, purchased from Calbiochem (Nottingham, UK), was dissolved in water prior to addition to the appropriate solution. Unless indicated, all other chemicals were obtained from Sigma (Poole, UK). Under whole-cell current clamp using quasiphysiological K+-containing solutions, the resting membrane potential of single HSVECs was found to be −28 ± 6 mV (n = 24, range of −3 to −71 mV). Hyperpolarizing voltage-clamp pulses from a holding potential of −50 mV produced small inward currents in 14 of the 24 cells (58%) investigated (Fig.1 A). These currents showed marked time-dependent inactivation at strongly negative potentials, inwardly rectified, conducting little outward current, and reversed close to the potassium equilibrium potential (Fig.1 B). Currents were fully eliminated by substituting Cs+ for K+ in the pipette solution and by the addition of 10 mm TEA in the extracellular solution, suggesting that they were carried by potassium ions. Depolarizing voltage steps from a potential of −120 mV to more positive potentials (−40 to +60 mV) elicited transient inward currents in HSVECs (Fig.2 A). These currents were present in 10 of the 24 cells (42%) investigated using K+-containing intracellular solutions, but unlike the inward current described above, these currents remained even when intracellular K+ was substituted with Cs+. All further electrophysiological experiments performed in this study used the Cs+/TEA-containing solutions to isolate this transient inward current and to avoid contamination from K+currents. With the use of the Cs+/TEA solutions, the current was found to be present in 75 of the 131 cells (57%) investigated, with a peak inward amplitude that varied between −70 and −1700 pA (mean of −595 ± 49 pA). The current was voltage-gated, activating at −50 mV and reaching a peak near −10 mV (Fig. 2, A(inset) and B), and had a mean reversal potential of +68 ± 4.2 mV (n = 36), close to the calculated Nernst potential for Na+ of +63 mV (ENa) for these solutions at 22 °C. The fast activation and inactivation kinetics of the inward current and its reversal close to ENa suggested that this was likely to be a Na+ current. To confirm this, extracellular Na+ was replaced with equimolar choline chloride, which totally abolished the inward current (n = 6) (data not shown). These results indicate that the rapidly activating and inactivating inward current in HSVECs is a voltage-gated Na+ current, which we have designated asINa. To facilitate comparison of INa in HSVECs with other known Na+ currents, we determined the sensitivity of the current to the guanidinium toxin, TTX. TTX has been shown to block a wide range of Na+ channels of different origins (7Hille B. Ionic Channels of Excitable Membranes. Sinauer Associates, Inc., Sunderland, MA1992Google Scholar), which are classified as TTX-sensitive, TTX-resistant, or TTX-insensitive based on the IC50 value for TTX blockade. In HSVECs, as the concentration of TTX in the extracellular solution was increased,INa decreased, with 100% blockade occurring at 30 μm (Fig. 2). An equal reduction in the amplitude ofINa was observed across the entire voltage range of activation, suggesting that TTX binding was not voltage-dependent (Fig. 2 B). A logistic plot fitted to the concentration inhibition curve yielded an IC50 value for TTX of 4.7 μm (Fig.2 C), suggesting that INa belongs to the TTX-resistant classification of Na+ channels (19Sculptoreanu A. Morton M. Gartside C.L. Hauschka S.D. Catterall W.A. Scheuer T. Am. J. Physiol. 1992; 262: C724-C730Crossref PubMed Google Scholar). The steady-state activation and inactivation properties of INa were assessed by the construction of normalized m∞ and h∞curves. Fits to the normalized activation curves (m∞; Fig. 3), generated by conversion of theI-V relationship to conductances, gave a mean half-activation voltage (Vm) of −29.2 ± 1.2 mV (n = 17) and an average K value (slope factor) of 7.2 ± 0.3 mV. The steady-state inactivation parameter (h∞) of INawas measured using a standard two-pulse protocol. Cells, clamped at −120 mV, were conditioned with a 1-s prepulse to potentials between −120 and −20 mV prior to a 30-ms test pulse to −10 mV, the potential that routinely evoked the maximal current (Fig. 3 A). These data show that significant inactivation was observed at −80 mV and that INa was inactivated completely at −40 mV. h∞ curves were calculated by normalizing the peak current recorded during the test pulse to the maximum current measured on stepping from −120 mV to the test potentials and were plotted as a function of the prepulse level. The averaged data indicate that INa was half-inactivated at −75.4 ± 1.3 mV (Vh; n = 17), with a slope factor of 5.7 ± 0.2 mV (Fig. 3 B). There were not any areas of significant overlap between m and hcurves. The time dependence of recovery from inactivation also was evaluated using a double-pulse protocol. Cells were stepped from a holding potential of −120 mV to 0 mV for 20 ms to elicit and inactivate INa. The cells were then clamped at −120, −100, or −80 mV for a variable duration of between 4 and 64 ms in 4-ms increments, prior to a second test pulse to 0 mV (Fig.4 A). Recovery from inactivation was found to be strongly voltage-dependent, with complete recovery requiring potentials more negative than −80 mV (Fig. 4 B). By fitting a single exponential function to the data, the recovery time constants were calculated to be 3.3 ± 0.3, 9.4 ± 1.2, and 32.2 ± 1.7 ms for cells held at −120, −100, and −80 mV, respectively (n = 4). Time constants for activation (τm) and inactivation (τh) were obtained by fitting a Hodgkin-Huxley model (see “Experimental Procedures”) to the inward currents. Both τm and τh were voltage-dependent, becoming more rapid as the test potential became increasingly more depolarized (Fig.5). This was much more marked with τh, which was reduced from 3.77 ms at −30 mV to 0.43 ms at +50 mV. The hyperpolarized half-maximal inactivation potential (Vh) and the inactivation time course are consistent with those reported for the cardiac isoform of Na+ channels (7Hille B. Ionic Channels of Excitable Membranes. Sinauer Associates, Inc., Sunderland, MA1992Google Scholar, 20Chahine M. Deschene I. Chen L.Q. Kallen R.G. Am. J. Physiol. 1996; 271: H498-H506Crossref PubMed Google Scholar).Figure 4Recovery from inactivation ofINa. A, series of current traces elicited by a double-pulse protocol. The cell was clamped at −120 mV, and a 20-ms voltage step to 0 mV was used to elicit and inactivateINa. The cell was then clamped at −100 mV for 4–64 ms in 4-ms increments before a second test pulse to 0 mV. B, recovery from inactivation occurs as a function of the holding potential (Vhold). Plots are of the ratio of the amplitude of the second and first current pulses as a function of the interval between the two. The lines represent a single exponential curve fitted to the data. Time constants estimated from these fits were 3.6 ms at −120 mV, 9.7 ms at −100 mV, and 34.6 ms at −80 mV.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Analysis of the time course ofINa in HSVECs. A, open circles show the best fit ofINa (continuous line) obtained by a voltage pulse to 0 mV from a holding potential of −120 mV by the Hodgkin-Huxley equation (see “Data Analysis and Curve Fitting”). τm = 0.43 ms; τh = 0.82 ms. B, averaged activation (τm) and inactivation (τh) time constants for INa across the voltage range −30 to +50 mV. Error bars indicate the S.E. (n = 14).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The addition of an antibody directed against the cytosolic linker region between domains III and IV of known Na+ channel α-subunits (anti-Naα) to the pipette solution (10 μg/ml) produced substantial slowing of Na+ current inactivation. In four cells, τh at 0 mV was increased from 0.71 ms to 1.25 ms within 15 min of establishing whole-cell configuration without any effect on peak current amplitude (data not shown). On the basis of electrophysiological data, particularly the current kinetics and sensitivity to TTX, the voltage-gated Na+ current in HSVECs appeared to closely resemble the human cardiac sodium channel, hH1. To test this hypothesis, mRNA isolated from HSVECs was reverse-transcribed, and the resulting cDNA was amplified (RT-PCR) with specific primers targeted against the 3′-untranslated region of the hH1 cDNA (16Gellens M.E. George A.L. Chen L. Chahine M. Horn R. Barchi R.L. Kallen R.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 554-558Crossref PubMed Scopus (560) Google Scholar). The expected product of 180 base pairs was produced only in those samples that had been reverse-transcribed. In the absence of a RT step, no product was present after PCR (Fig. 6). DNA sequencing of the RT-PCR product confirmed that it was identical to the 3′-untranslated region of the human hH1 sodium channel. Using Western blotting and employing the anti-Naα antibody, we were able to detect Na+ channel protein in the membrane (but not the cytosolic) fraction of HSVECs with guinea pig cardiac myocytes acting as a positive control (Fig.7). The antibody routinely recognized a single protein band that had an apparent molecular mass of 242 ± 9 kDa when separated by SDS-polyacrylamide gel electrophoresis (n = 5). This value is close to the molecular mass of the human hH1 α-subunit of 230 kDa as calculated from the deduced amino acid sequence. Subconfluent HSVECs, which were electrophysiologically confirmed to be expressing INa, also immunostained positively with the anti-Naα antibody (Fig. 8). This antibody was used to demonstrate the presence of Na+channels in the endothelium of freshly excised human saphenous vein. In intact saphenous vein endothelium, the immunostaining for Na+ channel α-subunits was intermittent, with not all endothelial cells being stained (Fig.9).Figure 9Immunohistochemical detection of voltage-gated sodium channels in intact human saphenous vein sections. A, vein section immunostained with thrombomodulin antibody to confirm the presence of intact endothelium; B, serial section as in A, showing positive immunoreactivity to the anti-Naα antibody. Note that not all cells staining positive for thrombomodulin expression are expressing Na+channels.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To assess the effects of serum on expression of INa by subconfluent HSVECs, we measured the magnitude and prevalence ofINa in cells that had been incubated for 48 h in growth medium that was serum-free or supplemented with 10% (v/v) human serum either from peripheral arterial disease patients 65 years of age and over (“aged”) or from healthy donors under 30 years of age (“young”). Under serum-free conditions and in medium supplemented with aged serum, INa was of small magnitude and was found in relatively few cells (TableI). However, medium supplemented with young serum was found to increase significantly both the magnitude ofINa and the number of HSVECs in whichINa was observed (Table I). The stimulatory effect of serum upon INa expression was confirmed by cell-based enzyme-linked immunosorbent assay of confluent HSVECs using the anti-Naα antibody. Inclusion of 2 or 10% (v/v) young serum in the incubation medium of HSVECs for 6 h increased the relative concentration of sodium channel protein 2- and 4-fold, respectively. The absorbance increased from 0.10 ± 0.03 (n = 8) in serum-free medium to 0.19 ± 0.03 (n = 7) and 0.36 ± 0.06 (n = 6) for HSVECs cultured in 2 and 10% (v/v) young sera, respectively.Table IEffects of growth conditions upon presence of INa in HSVECs as assessed by electrophysiologyGrowth conditionsINaoccurrenceINa magnitude (mean ± S.E.)pAM199 + 10% (v/v) aged serum7/18 (39%)−153 ± 22M199 + 10% (v/v) young serum19/33 (58%)−559 ± 119aSignificantly different from serum-free M199 medium (p < 0.01, Student's unpaired t test).Serum-free M1996/18 (33%)−196 ± 36Subconfluent HSVECs were cultured for 48 h under three growth conditions" @default.
• W2022292199 created "2016-06-24" @default.
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• W2022292199 date "1998-08-01" @default.
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• W2022292199 title "Human Saphenous Vein Endothelial Cells Express a Tetrodotoxin-resistant, Voltage-gated Sodium Current" @default.
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