Vranesic I, Staub C, Knöpfel T
Brain Research Institute, University of Zürich, Switzerland.
Neurosci Res. 1993 Mar;16(3):209-15. doi: 10.1016/0168-0102(93)90125-a.
The responses of slice-cultured Purkinje cells to trans-DL-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD) were examined by intracellular recording techniques and fura-2 microfluorometry. Bath-application of t-ACPD (100 microM, 30 s), a selective agonist of metabotropic glutamate receptors (mGluRs), to Purkinje cells voltage-clamped near their resting potential -65 to -60 mV) consistently induced a transient inward current, followed by a slower outward current (Iout). This outward current was characterized by a linear current-voltage relationship in the range from -130 to -60 mV and accompanied by a significant decrease in membrane conductance. The extrapolated reversal potential of Iout was positive to 0 mV. When t-ACPD was applied for 60 s or more it became apparent that Iout emerged in parallel to the wash-out of t-ACPD. Microfluorometric fura-2 measurements in combination with electrophysiological recordings were used to assess the relation between Iout and intracellular free calcium concentration ([Ca2+]i). In contrast to the inward current that was associated with a transient elevation in [Ca2+]i. Iout was not correlated with an elevated [Ca2+]i. When t-ACPD was applied in the presence of caffeine (5 mM), Iout was reversibly enhanced in amplitude. Caffeine affected neither the t-ACPD-induced calcium signal nor the resting [Ca2+]i. While longer applications of caffeine alone induced outward currents with a current-voltage relationship similar to that of Iout, short applications (30 s) of caffeine had no detectable effect per se but still were effective in enhancing Iout when applied in conjunction with t-ACPD. 3-Isobutyl-1-methylxanthine (IBMX, 0.5 mM), a more selective and potent phosphodiesterase inhibitor than caffeine, exhibited caffeine-like effects at a 10-fold lower concentration. We propose that Iout is generated by a transient inhibition of an inward current that is tonically active at rest and largely voltage-independent in the range tested. Our observations provide evidence for an involvement of cyclic nucleotide second messenger systems in the regulation of this current.
采用细胞内记录技术和fura - 2显微荧光测定法，研究了脑片培养的浦肯野细胞对反式-DL-1-氨基-1,3-环戊烷二羧酸（t-ACPD）的反应。将代谢型谷氨酸受体（mGluRs）的选择性激动剂t-ACPD（100 microM，30 s）浴式应用于静息电位在-65至-60 mV附近电压钳制的浦肯野细胞时，持续诱发一个短暂的内向电流，随后是一个较慢的外向电流（Iout）。该外向电流的特征是在-130至-60 mV范围内呈线性电流-电压关系，并伴有膜电导的显著降低。Iout的外推反转电位为正于0 mV。当t-ACPD应用60 s或更长时间时，很明显Iout与t-ACPD的洗脱同时出现。结合电生理记录的fura - 2显微荧光测量用于评估Iout与细胞内游离钙浓度（[Ca2+]i）之间的关系。与与[Ca2+]i短暂升高相关的内向电流相反，Iout与[Ca2+]i升高无关。当在咖啡因（5 mM）存在的情况下应用t-ACPD时，Iout的幅度可逆性增强。咖啡因既不影响t-ACPD诱导的钙信号，也不影响静息[Ca2+]i。虽然单独长时间应用咖啡因会诱发具有与Iout相似电流-电压关系的外向电流，但短时间应用（30 s）咖啡因本身没有可检测到的影响，但与t-ACPD联合应用时仍能有效增强Iout。3-异丁基-1-甲基黄嘌呤（IBMX，0.5 mM）是一种比咖啡因更具选择性和强效的磷酸二酯酶抑制剂，在浓度低10倍时表现出类似咖啡因的作用。我们提出，Iout是由对静息时持续活动且在所测试范围内基本与电压无关的内向电流的短暂抑制产生的。我们的观察结果为环核苷酸第二信使系统参与该电流的调节提供了证据。
