Nargeot J
J Pharmacol. 1985;16 Suppl 1:89-118.
We have shown in the first part of this work, how pharmacological technics can be associated to electrophysiological measurements, to study the action of neurotransmitters on the excitation-contraction coupling in cardiac cells. However, to better understand their mechanism of action, it is necessary to have informations about the link between the activation of the receptor and the conductance changes. Kinetics measurements have been very useful to investigate this problem at the nicotinic cholinergic receptor. This study was performed with technics such as iontophoresis, voltage relaxations, noise analysis and recently patch clamp measurements. Nicotinic responses are very fast (in the order of the msec) and are however the best known. The binding of the agonist to the receptor would lead to the opening of a channel only by molecular conformation change. By comparison, beta-adrenergic and muscarinic responses in heart or smooth muscle can be considered as "slow responses" because they are thought to be mediated by intracellular biochemical steps. We have developed a new technique using photosensitive compounds, which allows to jump the extra- or intracellular concentration of some active molecules. The experiments have been performed on frog atrial fibers, in current or voltage clamp conditions using a double sucrose gap technique. Various photochemical reactions have been exploited. The photoisomerization of an azobenzene derivative (Bis-Q) allows to study the muscarinic-induced potassium conductance. Bis-Q binds to muscarinic receptors and blocks the effect of agonists. These drugs have the additional property that their cis and trans configuration have different potencies. The photoisomerization cis----trans or trans----cis is obtained a few msec after a 1 msec duration light flash (respectively visible or UV), and then induces a concentration jump of antagonist near the receptor. The results show that the potassium conductance change has a slow relaxation time constant (tau = 500 msec.) and this suggest rather successive intracellular steps than the opening of a channel directly linked to the receptor. Cyclic nucleotides are considered as the intracellular messengers for various neurotransmitters or hormones. Our photochemical technique allows to jump the intracellular concentration of cAMP and cGMP in a few msec. cAMP (or cGMP) orthonitrobenzyl derivatives are able to penetrate cell membrane without injection and have no effect up to 100 microM. A UV flash (1 msec duration) induces a photolysis of the derivative and generates instantaneously cAMP (or cGMP) inside the cell.(ABSTRACT TRUNCATED AT 400 WORDS)
在本研究的第一部分，我们展示了药理学技术如何与电生理测量相结合，以研究神经递质对心肌细胞兴奋 - 收缩偶联的作用。然而，为了更好地理解它们的作用机制，有必要了解受体激活与电导变化之间的联系。动力学测量对于研究烟碱型胆碱能受体的这一问题非常有用。这项研究采用了离子电泳、电压松弛、噪声分析以及最近的膜片钳测量等技术。烟碱型反应非常迅速（以毫秒计），但却是最为人所熟知的。激动剂与受体的结合仅通过分子构象变化就会导致通道开放。相比之下，心脏或平滑肌中的β - 肾上腺素能和毒蕈碱型反应可被视为“缓慢反应”，因为它们被认为是由细胞内生化步骤介导的。我们开发了一种使用光敏化合物的新技术，该技术能够瞬间改变某些活性分子的细胞外或细胞内浓度。实验是在蛙心房纤维上进行的，采用双蔗糖间隙技术，在电流钳或电压钳条件下进行。利用了各种光化学反应。一种偶氮苯衍生物（双Q）的光异构化可用于研究毒蕈碱诱导的钾电导。双Q与毒蕈碱受体结合并阻断激动剂的作用。这些药物具有额外的特性，即它们的顺式和反式构型具有不同的效力。在持续1毫秒的闪光（分别为可见光或紫外线）后几毫秒内可实现顺式 - 反式或反式 - 顺式的光异构化，然后在受体附近诱导拮抗剂浓度的瞬间变化。结果表明，钾电导变化具有缓慢的松弛时间常数（τ = 500毫秒），这表明涉及一系列细胞内步骤，而非直接与受体相连的通道开放。环核苷酸被认为是各种神经递质或激素的细胞内信使。我们的光化学技术能够在几毫秒内瞬间改变细胞内cAMP和cGMP的浓度。cAMP（或cGMP）的邻硝基苄基衍生物无需注射就能穿透细胞膜，在浓度达到100微摩尔之前没有作用。一次紫外线闪光（持续1毫秒）会诱导该衍生物的光解，并在细胞内瞬间产生cAMP（或cGMP）。（摘要截选至400字）
