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ブタ副腎髄質細胞の電位依存性カルシウムチャネル : サブタイプと脱分極によるfacilitationについて
| Title: | ブタ副腎髄質細胞の電位依存性カルシウムチャネル : サブタイプと脱分極によるfacilitationについて |
| Other Titles: | Voltage-Dependent Calcium Channels in Poecine Adrenal Chromaffin Cells : Channel Subtypes and Mechanisms of Their Faclitaion |
| Authors: | 北村, 直樹1 Browse this author |
| Authors(alt): | Kitamura, Naoki1 |
| Issue Date: | 25-Mar-1998 |
| Publisher: | Hokkaido University |
| Abstract: | To study the characteristics of voltage-dependent calcium channels in porcine adrenal chromafiin cells, calcium (barium) currents, rise of intracellular calcium concentration ([Ca2+]i) and catecholamine release responses induced by stimulation with high K+ (60 mM) were measured by whole-cell voltage clamp technique, microfluorometry and HPLC-ECD method, respectively. The results obtained were as follows: 1. Voltage-current relationship of calcium current indicated that porcine adrenal chromaffin cells possess only high voltage-activated type of calcium channels. The calcium current was inhibited by ω-conotoxin GVIA, nifedipine and ω-agatoxin IVA dose-dependently, though the magnitudes of inhibition were various. The degree of inhibition by maximal doses of these three agents was 78%, 15% and 6%, respectively. When these three agents were applied onto the same cell, calcium current was inhibited additively. 2. When 1-s depolarizing pulses were applied in the absence and presence of calcium channel blockers, ω-conotoxin GV IA-sensitive currents inactivated faster than nifedipine or ω-agatoxin IVA-sensitive currents. 3. Rises in [Ca2+]i and catecholamine release in response to stimulation by high K+ were inhibited to about 50% by either ω-conotoxin GVIA (1μM) or nifedipine (10μM) but not by ω-agatoxin IVA (0.1μM). In addition, these responses were almost abolished by the combined application of ω-conotoxin GVIA and nifedipine. 4. A strong depolarizing pulse (a prepulse to +100mV) applied prior to a test pulse caused about 20% increase of amplitude of barium current evoked by the test pulse (facilitation of barium currents). The degree of the facilitation of barium currents was increased with the increase in the voltage (in a range over +20 mV) and duration of the prepulses. Moreover the facilitation of barium currents was decreased with increase in intervals between the prepulses and the test pulses. 5. The application of 8-Bromo-cAMP (1 mM) or forskolin (10 μM) decreased the amplitudes of barium currents without affecting the degree of facilitation of barium currents by the prepulses. In addition, an intracellular application of Rp-cAMPS, an inhibitor of PKA, did not have any effects on the amplitudes of barium currents and the degree of facilitation of barium currents. 6. The intracellular application of GTPγS (100 μM) decreased the amplitudes of barium currents, but not affected those in the presence of prepulses. On the other hand, the application of GDPIβS (100μM) caused a slight increase in the amplitudes of barium currents but had no effects on the amplitudes of barium currents in the presence of prepulses. Consequently, the degree of facilitation increased in the presence of GTPγS and decreased in the presence of GDPβS. 7. GTPγS-sensitive component of barium current was sensitive to ω-conotoxin GVIA but not to nifedipine. The facilitation of barium currents by the prepulses was abolished by ω-conotoxin GVIA but not by either ω-agatoxin IVA or nifedipine. Furthermore, Bay K 8644 caused about 80% increase of barium currents in amplitude but showed no effect on the facilitation by prepulses. 8. GTPγS applied intracellularly slowed the time course of activation of barium current (kinetic slowing). The kinetic slowing of barium currents by GTPγS was abolished by ω-conotoxin GVIA or the depolarizing prepulses. 9. Pertussis toxin did not affect the amplitude of barium currents and the degree of facilitation of barium currents. Cholera toxin, however, increased the degree of facilitation of barium currents without effects on the amplitudes of barium currents. Based on these results, it is clarified that porcine adrenal chromaffin cells possess ω-conotoxin GVIA-sensitive N- and nifedipine-sensitive L- and ω-agatoxin IVA-sensitive P/Q-type calcium channels and that L- and N-type channels mainly contribute to the rise in [Ca2+]i and catecholamine release by depolarizing the cells. N-type calcium channels are mainly involved in the depolarizing prepulse-induced facilitation of barium currents. The facilitatidn seems to result from the prepulse-induced relief of tonic inhibition on calcium channels by G-protein but not from PKA-induced phosphorylation of channels during the prepulse. Moreover, Gs-type but not Gi-type G-protein may be involved in this mechanism. |
| Conffering University: | 北海道大学 |
| Degree Report Number: | 甲第4459号 |
| Degree Level: | 博士 |
| Degree Discipline: | 獣医学 |
| Type: | theses (doctoral) |
| URI: | http://hdl.handle.net/2115/51477 |
| Appears in Collections: | 学位論文 (Theses) > 博士 （獣医学）
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Submitter: 北村 直樹
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