Zolmitriptan stimulates a Ca(2+)-dependent K(+) current in C6 glioma cells stably expressing recombinant human 5-HT(1B) receptors.

Stimulation of a Ca(2+)-dependent K(+) current by zolmitriptan, a 5-HT(1B/1D) receptor partial agonist, was investigated in C6 glioma cells stably expressing recombinant human 5-HT(1B) receptors. Outward K(+) currents (I(K)) were examined in non-transfected C6 glioma cells and in cells expressing cloned human 5-HT(1B) receptors using the patch-clamp technique in the whole-cell configuration. In C6 glioma cells expressing recombinant human 5-HT(1B) receptor, zolmitriptan increased I(K) in a concentration-dependent manner (maximum increase 16.3+/-7.8%, n=5, p<0.001) with a pD(2) value (geometric mean with 95% confidence intervals) of 7.03 (7.90-6.10). Zolmitriptan failed to elicit increases in I(K) in non-transfected C6 cells. In the presence of the mixed 5-HT(1B/1D) receptor antagonist, N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2(-methyl-4(5-methyl-1 ,2,4)-oxadiazol-3-yl)[1,1-biphenyl]-4-carboxamide 2HCl (GR 127935, 0. 1 mcM), zolmitriptan (1 mcM) failed to significantly increase I(K) in C6 cells expressing human 5-HT(1B) receptors confirming that zolmitriptan-evoked responses were indeed mediated by human 5-HT(1B) receptors. In C6 cells expressing cloned human 5-HT(1B) receptors, zolmitriptan-induced increases in I(K) were prevented by the calcium chelator, EGTA (5 mM) when included in the patch pipette (maximum increase -3.3+/-4.2%, n=4, P=NS). The Ca(2+)-dependent K(+) channel blockers, iberiotoxin (0.1 mcM) and tetraethylammonium (TEA, 1 mM), abolished zolmitriptan-induced increases in I(K) (4.5+/-7.3%, n=4 and -0.8+/-1.7%, n=4, respectively, P=NS in each case) in C6 cells expressing human 5-HT(1B) receptors, confirming the involvement of Ca(2+)-dependent K(+) channels. In conclusion, the 5-HT(1B/1D) receptor partial agonist, zolmitriptan, stimulates I(K/Ca) in C6 glioma cells stably transfected with human 5-HT(1B) receptors suggesting an increase of hyperpolarizing current.

Activation of recombinant h 5-HT1B and h 5-HT1D receptors stably expressed in C6 glioma cells produces increases in Ca2+-dependent K+ current.

The putative coupling between stably expressed recombinant h 5-HT1B or h 5-HT1D receptors and K+ channels which regulate excitability was investigated in C6 glioma cells. Outward K+ currents (IK) were examined in nontransfected C6 glioma cells and in cells expressing cloned h 5-HT1B or h 5-HT1D 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-HT1B or h 5-HT1D 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 EC50 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-HT1B and h 5-HT1D receptors in transfected C6 cells. In the presence of the mixed 5-HT1B/D receptor antagonist GR 127935 (0.1 microM), sumatriptan (1 microM) failed to significantly increase IK in C6 cells expressing h 5-HT1B receptors (-7.5+/-3.5%, P=NS, n=6), although a higher concentration of GR 127935 (1 microM) was required to significantly inhibit sumatriptan-evoked increases in IK in C6 cells expressing h 5-HT1D receptors (-1.8+/-3.5%, P=NS, n=6), confirming that sumatriptan-evoked responses were indeed mediated by h 5-HT1B and h 5-HT1D receptors, respectively. In C6 cells expressing either cloned h 5-HT1B or h 5-HT1D 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-HT1B receptors, sumatriptan (1 microM) similarly failed to significantly increase IK in the presence of dibutyryl cAMP (10 microM) or when a nominally Ca2+-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 Ca2+-dependent K+ channel blockers iberiotoxin (0.1 microM) 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-HT1B receptors, confirming the involvement of Ca2+-dependent K+ channels. In C6 cells expressing cloned h 5-HT1B receptors, sumatriptan (1 microM) similarly failed to significantly increase IK after 30-min incubation with thapsigargin (1 microM) 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-HT1B and h 5-HT1D receptors stably transfected in C6 glioma cells are positively coupled to Ca2+-dependent K+ channels, and the outward hyperpolarizing current mediated by these channels is dependent upon IP3 receptor-mediated intracellular Ca2+ release.