Kitamura Go, Ohta Toshio, Kai Takahiko, Kon Yasuhiro, Ito Shigeo
Laboratory of Pharmacology, Department of Biomedical Science, Hokkaido University, Sapporo 0600818, Japan.
Brain Res. 2002 Jun 28;942(1-2):11-22. doi: 10.1016/s0006-8993(02)02648-3.
The inhibitory effects of opioids on voltage-dependent calcium channels (VDCCs) were investigated in cultured porcine adrenal chromaffin cells using whole-cell patch clamp technique. The effects of the opioid on Ca(2+) increase and catecholamine secretion induced by high K(+) were also examined in single cells by fura-2 microfluorimetry and amperometry. A depolarizing pulse to 0 mV (test pulse) from a holding potential of -80 mV evoked an inward barium current (I(Ba)), which was reversibly inhibited by methionine-enkephalin. This inhibitory effect of methionine-enkephalin was abolished by naloxone. Selective agonists of opioid receptor subtypes (DAMGO: mu, DPDPE: delta, U50488: kappa) dose-dependently inhibited I(Ba). In inhibitory potency, the order was DAMGO>U50488>DPDPE. These agonists applied sequentially produced a reversible I(Ba) inhibition in the same cells. The inhibitory effect of DAMGO on I(Ba) almost disappeared in the presence of omega-conotoxin GVIA but not omega-agatoxin IVA plus nifedipine. Application of a conditioning prepulse to +100 mV prior to the test pulse partly retrieved the I(Ba) inhibition by DAMGO, suggesting the involvement of voltage-sensitive components in opioid-induced VDCC inhibition. Intracellular application of GDPbetaS or GTPgammaS as well as pretreatment with pertussis toxin significantly reduced the extent of I(Ba) inhibition induced by DAMGO. DAMGO reversibly inhibited the Ca(2+) increase and catecholamine release induced by high K(+). RT-PCR revealed the expression of mu-, delta- and kappa-opioid receptor mRNAs in cultured adrenal chromaffin cells. These results suggest that porcine adrenal chromaffin cells possess mu-, delta- and kappa-opioid receptors and activation of opioid receptors mainly inhibits N-type VDCCs via pertussis toxin-sensitive G-proteins.
采用全细胞膜片钳技术，在培养的猪肾上腺嗜铬细胞中研究了阿片类药物对电压依赖性钙通道（VDCCs）的抑制作用。还通过fura-2显微荧光测定法和安培法在单细胞中检测了阿片类药物对高钾诱导的[Ca(2+)]i升高和儿茶酚胺分泌的影响。从-80 mV的 holding 电位去极化至0 mV（测试脉冲）可诱发内向钡电流（I(Ba)），甲硫氨酸脑啡肽可对其产生可逆性抑制。纳洛酮可消除甲硫氨酸脑啡肽的这种抑制作用。阿片受体亚型的选择性激动剂（DAMGO：μ，DPDPE：δ，U50488：κ）剂量依赖性地抑制I(Ba)。在抑制效力方面，顺序为DAMGO>U50488>DPDPE。这些激动剂依次应用可在同一细胞中产生可逆的I(Ba)抑制。在存在ω-芋螺毒素GVIA的情况下，DAMGO对I(Ba)的抑制作用几乎消失，但在存在ω-阿加毒素IVA加硝苯地平的情况下则不然。在测试脉冲之前施加至+100 mV的条件预脉冲可部分恢复DAMGO对I(Ba)的抑制作用，提示电压敏感成分参与了阿片类药物诱导的VDCC抑制。细胞内应用GDPβS或GTPγS以及百日咳毒素预处理可显著降低DAMGO诱导的I(Ba)抑制程度。DAMGO可逆性抑制高钾诱导的[Ca(2+)]i升高和儿茶酚胺释放。RT-PCR显示培养的肾上腺嗜铬细胞中存在μ-、δ-和κ-阿片受体mRNA的表达。这些结果表明，猪肾上腺嗜铬细胞具有μ-、δ-和κ-阿片受体，阿片受体的激活主要通过百日咳毒素敏感的G蛋白抑制N型VDCCs。
