Activation of recombinant h 5-HT1B and h 5-HT1D receptors stably expressed in C6 glioma cells produces increases in Ca2+-dependent K+ current
Abstract The putative coupling between stably expressed recombinant h 5-$ HT_{1B} $ or h 5-$ HT_{1D} $ receptors and $ K^{+} $ channels which regulate excitability was investigated in C6 glioma cells. Outward $ K^{+} $ currents (IK) were examined in non-transfected C6 glioma cells and in cells expressing cloned h 5-$ HT_{1B} $ or h 5-$ HT_{1D} $ receptors using the patch-clamp technique in the whole-cell configuration. IK was elicited by a depolarizing step from a holding potential of –60 mV. In C6 glioma cells expressing either recombinant h 5-$ HT_{1B} $ or h 5-$ HT_{1D} $ receptors, sumatriptan similarly increased IK in a concentration-dependent manner (maximum increase 19.4±7.2%, n=8, P<0.05 and 25.1±3.9%, n=6, P<0.001, respectively) with $ EC_{50} $ values (geometric mean with 95% confidence intervals in parentheses) of 56.3 nM (7.9–140 nM) and 68.7 nM (16–120 nM), respectively. Sumatriptan failed to elicit increases in IK in non-transfected cells, confirming a specific involvement of the respective membrane h 5-$ HT_{1B} $ and h 5-$ HT_{1D} $ receptors in transfected C6 cells. In the presence of the mixed 5-$ HT_{1B/D} $ receptor antagonist GR 127935 (0.1 µM), sumatriptan (1 µM) failed to significantly increase IK in C6 cells expressing h 5-$ HT_{1B} $ receptors (–7.5±3.5%, P=NS, n=6), although a higher concentration of GR 127935 (1 µM) was required to significantly inhibit sumatriptan-evoked increases in IK in C6 cells expressing h 5-$ HT_{1D} $ receptors (–1.8±3.5%, P=NS, n=6), confirming that sumatriptan-evoked responses were indeed mediated by h 5-$ HT_{1B} $ and h 5-$ HT_{1D} $ receptors, respectively. In C6 cells expressing either cloned h 5-$ HT_{1B} $ or h 5-$ HT_{1D} $ receptors, sumatriptan-induced increases in IK were prevented by the calcium chelator EGTA (5 mM) when included in the patch pipette (maximum increase 0.57±0.6%, n=3, P=NS and –2.8±1.6%, n=5, P=NS, respectively). In C6 cells expressing cloned h 5-$ HT_{1B} $ receptors, sumatriptan (1 µM) similarly failed to significantly increase IK in the presence of dibutyryl cAMP (10 µM) or when a nominally $ Ca^{2+} $-free medium was included in the patch pipette (–19.4±5.1%, n=5 and –5.2±4.3%, n=5, respectively, P=NS in each case). In addition, the $ Ca^{2+} $-dependent $ K^{+} $ channel blockers iberiotoxin (0.1 µM) and tetraethylammonium (TEA, 1 mM) abolished sumatriptan-induced increases in IK (–0.5±1.0%, n=4 and –3.9±3.1%, n=4, respectively, P=NS in each case) in C6 cells expressing h 5-$ HT_{1B} $ receptors, confirming the involvement of $ Ca^{2+} $-dependent $ K^{+} $ channels. In C6 cells expressing cloned h 5-$ HT_{1B} $ receptors, sumatriptan (1 µM) similarly failed to significantly increase Ik after 30-min incubation with thapsigargin (1 µM) or when heparin (2 mg/ml) was included in the patch pipette (1.1±0.4%, n=5 and 1.2±2.4%, n=5, respectively, P=NS). In conclusion, evidence is provided that both recombinant h 5-$ HT_{1B} $ and h 5-$ HT_{1D} $ receptors stably transfected in C6 glioma cells are positively coupled to $ Ca^{2+} $-dependent $ K^{+} $ channels, and the outward hyperpolarizing current mediated by these channels is dependent upon $ IP_{3} $ receptor-mediated intracellular $ Ca^{2+} $ release..
Medienart: |
Artikel |
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Erscheinungsjahr: |
1998 |
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Erschienen: |
1998 |
Enthalten in: |
Zur Gesamtaufnahme - volume:358 |
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Enthalten in: |
Naunyn-Schmiedeberg's archives of pharmacology - 358(1998), 6 vom: Dez., Seite 608-615 |
Sprache: |
Englisch |
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Beteiligte Personen: |
Grand, Bruno Le [VerfasserIn] |
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Links: |
Volltext [lizenzpflichtig] |
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Anmerkungen: |
© Springer-Verlag Berlin Heidelberg 1998 |
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doi: |
10.1007/PL00005301 |
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funding: |
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Förderinstitution / Projekttitel: |
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PPN (Katalog-ID): |
OLC2039932984 |
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245 | 1 | 0 | |a Activation of recombinant h 5-HT1B and h 5-HT1D receptors stably expressed in C6 glioma cells produces increases in Ca2+-dependent K+ current |
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520 | |a Abstract The putative coupling between stably expressed recombinant h 5-$ HT_{1B} $ or h 5-$ HT_{1D} $ receptors and $ K^{+} $ channels which regulate excitability was investigated in C6 glioma cells. Outward $ K^{+} $ currents (IK) were examined in non-transfected C6 glioma cells and in cells expressing cloned h 5-$ HT_{1B} $ or h 5-$ HT_{1D} $ receptors using the patch-clamp technique in the whole-cell configuration. IK was elicited by a depolarizing step from a holding potential of –60 mV. In C6 glioma cells expressing either recombinant h 5-$ HT_{1B} $ or h 5-$ HT_{1D} $ receptors, sumatriptan similarly increased IK in a concentration-dependent manner (maximum increase 19.4±7.2%, n=8, P<0.05 and 25.1±3.9%, n=6, P<0.001, respectively) with $ EC_{50} $ values (geometric mean with 95% confidence intervals in parentheses) of 56.3 nM (7.9–140 nM) and 68.7 nM (16–120 nM), respectively. Sumatriptan failed to elicit increases in IK in non-transfected cells, confirming a specific involvement of the respective membrane h 5-$ HT_{1B} $ and h 5-$ HT_{1D} $ receptors in transfected C6 cells. In the presence of the mixed 5-$ HT_{1B/D} $ receptor antagonist GR 127935 (0.1 µM), sumatriptan (1 µM) failed to significantly increase IK in C6 cells expressing h 5-$ HT_{1B} $ receptors (–7.5±3.5%, P=NS, n=6), although a higher concentration of GR 127935 (1 µM) was required to significantly inhibit sumatriptan-evoked increases in IK in C6 cells expressing h 5-$ HT_{1D} $ receptors (–1.8±3.5%, P=NS, n=6), confirming that sumatriptan-evoked responses were indeed mediated by h 5-$ HT_{1B} $ and h 5-$ HT_{1D} $ receptors, respectively. In C6 cells expressing either cloned h 5-$ HT_{1B} $ or h 5-$ HT_{1D} $ receptors, sumatriptan-induced increases in IK were prevented by the calcium chelator EGTA (5 mM) when included in the patch pipette (maximum increase 0.57±0.6%, n=3, P=NS and –2.8±1.6%, n=5, P=NS, respectively). In C6 cells expressing cloned h 5-$ HT_{1B} $ receptors, sumatriptan (1 µM) similarly failed to significantly increase IK in the presence of dibutyryl cAMP (10 µM) or when a nominally $ Ca^{2+} $-free medium was included in the patch pipette (–19.4±5.1%, n=5 and –5.2±4.3%, n=5, respectively, P=NS in each case). In addition, the $ Ca^{2+} $-dependent $ K^{+} $ channel blockers iberiotoxin (0.1 µM) and tetraethylammonium (TEA, 1 mM) abolished sumatriptan-induced increases in IK (–0.5±1.0%, n=4 and –3.9±3.1%, n=4, respectively, P=NS in each case) in C6 cells expressing h 5-$ HT_{1B} $ receptors, confirming the involvement of $ Ca^{2+} $-dependent $ K^{+} $ channels. In C6 cells expressing cloned h 5-$ HT_{1B} $ receptors, sumatriptan (1 µM) similarly failed to significantly increase Ik after 30-min incubation with thapsigargin (1 µM) or when heparin (2 mg/ml) was included in the patch pipette (1.1±0.4%, n=5 and 1.2±2.4%, n=5, respectively, P=NS). In conclusion, evidence is provided that both recombinant h 5-$ HT_{1B} $ and h 5-$ HT_{1D} $ receptors stably transfected in C6 glioma cells are positively coupled to $ Ca^{2+} $-dependent $ K^{+} $ channels, and the outward hyperpolarizing current mediated by these channels is dependent upon $ IP_{3} $ receptor-mediated intracellular $ Ca^{2+} $ release. | ||
700 | 1 | |a Panissié, Anne |4 aut | |
700 | 1 | |a Pauwels, Petrus J. |4 aut | |
700 | 1 | |a John, G. W. |4 aut | |
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