Rusin K I, Moises H C
Department of Physiology, University of Michigan Medical School, Ann Arbor 48109-0622, USA.
Neuroscience. 1998 Aug;85(3):939-56. doi: 10.1016/s0306-4522(97)00674-x.
Whole-cell patch-clamp recordings were obtained from nodose ganglion neurons acutely dissociated from 10-30-day-old rats to characterize the Ca2+ channel types that are modulated by GABA(B) and mu-opioid receptors. Five components of high-threshold current were distinguished on the basis of their sensitivity to blockade by omega-conotoxin GVIA, nifedipine, omega-agatoxin IVA and omega-conotoxin MVIIC. Administration of the mu-opioid agonist H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol (0.3-1 mM) or the GABA(B) agonist baclofen in saturating concentrations suppressed high-threshold Ca2+ currents by 49.9+/-2.4% (n=69) and 18.7+/-2.1% (n=35), respectively. The inhibition by H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol exceeded that by baclofen in virtually all neurons that responded to both agonists (67%), and occlusion experiments revealed that responses to mu-opioid and GABA(B) receptor activation were not linearly additive. In addition, administration of staurosporine, a non-selective inhibitor of protein kinase A and C, did not affect the inhibitory responses to either agonist or prevent the occlusion of baclofen-induced current inhibition by H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol. Blockade of N-type channels by omega-conotoxin GVIA eliminated current suppression by baclofen in all cells tested (n=11). Mu-opioid-induced inhibition in current was abolished by omega-conotoxin GVIA in 12 of 30 neurons tested, but was only partially reduced in the remaining 18 neurons. In the latter cells administration of omega-agatoxin IVA reduced, but did not eliminate the mu-opioid sensitive current component that persisted after blockade of N-type channels. This residual component of mu-opioid-sensitive current was blocked completely by omega-conotoxin MVIIC in nine neurons, whereas responses to H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol were still recorded in the remaining cells after administration of these Ca2+ channel toxins and nifedipine. Dihydropyridine-sensitive (L-type) current was not affected by activation of mu-opioid or GABA(B) receptors in any of the neurons. These data indicate that in nodose ganglion neurons mu-opioid receptors are negatively coupled to N-, P- and Q-type channels as well as to a fourth, unidentified toxin-resistant Ca2+ channel. In contrast, GABA(B) receptors are coupled only to N-type channels. Furthermore, the results do not support a role for either protein kinase C or A in the modulatory pathway(s) coupling mu-opioid and GABA(B) receptors to Ca2+ channels, but rather lend credence to the notion that the signalling mechanisms utilized by these two receptors might simply compete for inhibitory control of a common pool of N-type channels.
采用全细胞膜片钳记录技术，从10 - 30日龄大鼠急性分离的结状神经节神经元中获取数据，以表征受γ-氨基丁酸B（GABA(B)）和μ-阿片受体调节的钙离子通道类型。根据它们对ω-芋螺毒素GVIA、硝苯地平、ω-阿加毒素IVA和ω-芋螺毒素MVIIC阻断作用的敏感性，区分出高阈值电流的五个成分。给予μ-阿片激动剂H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol（0.3 - 1 mM）或饱和浓度的GABA(B)激动剂巴氯芬，分别使高阈值钙离子电流抑制49.9±2.4%（n = 69）和18.7±2.1%（n = 35）。在几乎所有对两种激动剂都有反应的神经元中（67%），H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol的抑制作用超过巴氯芬，并且阻断实验表明，对μ-阿片和GABA(B)受体激活的反应不是线性相加的。此外，给予星形孢菌素（一种蛋白激酶A和C的非选择性抑制剂），既不影响对任何一种激动剂的抑制反应，也不能阻止H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol对巴氯芬诱导的电流抑制的阻断作用。ω-芋螺毒素GVIA阻断N型通道，消除了所有测试细胞（n = 11）中巴氯芬对电流的抑制作用。在30个测试神经元中，ω-芋螺毒素GVIA消除了12个神经元中μ-阿片诱导的电流抑制，但在其余18个神经元中仅部分降低。在后者细胞中，给予ω-阿加毒素IVA减少但未消除在N型通道阻断后持续存在的μ-阿片敏感电流成分。在9个神经元中，这种μ-阿片敏感电流的残余成分被ω-芋螺毒素MVIIC完全阻断，而在给予这些钙离子通道毒素和硝苯地平后，其余细胞中仍记录到对H-Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol的反应。在任何神经元中，二氢吡啶敏感的（L型）电流均不受μ-阿片或GABA(B)受体激活的影响。这些数据表明，在结状神经节神经元中，μ-阿片受体与N型、P型和Q型通道以及第四种未鉴定的抗毒素钙离子通道负性偶联。相比之下，GABA(B)受体仅与N型通道偶联。此外，结果不支持蛋白激酶C或A在将μ-阿片和GABA(B)受体与钙离子通道偶联的调节途径中起作用，而是支持这两种受体利用的信号机制可能只是竞争对共同的N型通道池进行抑制控制的观点。
