Liu X, Farley J M
Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, USA.
J Pharmacol Exp Ther. 1996 May;277(2):796-804.
We demonstrated previously that acetylcholine (ACh) induces Ca++ oscillations in tracheal myocytes. These oscillations, as measured with fluo3-loaded cells and confocal microfluorimetry, correlated with Ca++-dependent Cl- current (Clca) oscillations measured by whole-cell voltage-clamp recording. In the current study, we investigated the role of inositol 1,4,5-trisphosphate (IP3) in ACh-induced oscillations in Ca++ and membrane currents. Both an IP3 receptor monoclonal antibody (5 micrograms/ml) and an IP3 receptor antagonist, heparin (5 mg/ml), directly introduced into the cells via the patch pipette, reduced or abolished oscillations in Clca induced by ACh. In addition, IP3 (1-100 microM) applied intracellularly, elicited concentration-dependent Clca oscillations that resembled those induced by ACh. Increasing external Ca++ concentration enhanced IP3-induced Clca oscillations, whereas verapamil (10 microM), a voltage-operated Ca++ channel blocker, attenuated IP3-induced Clca oscillations as well as both control and IP3-enhanced spontaneous transient outward currents. However, neither 5 microgram/ml IP3 receptor monoclonal antibody nor 5 mg/ml heparin altered the caffeine-induced transient Clca. Caffeine (10 mM) reversibly eliminated IP3-induced Clca oscillations as well as IP3-enhanced spontaneous transient outward currents, which indicates that caffeine releases Ca++ via a mechanism independent of the IP3 receptor. The findings are consistent with the hypothesis that ACh-induced Ca++ oscillations can arise and be sustained via IP3-induced Ca++ release pathways.
我们先前已证明，乙酰胆碱（ACh）可诱导气管肌细胞中的Ca++振荡。这些振荡，通过用fluo3负载细胞和共聚焦显微荧光测定法测量，与通过全细胞电压钳记录测量的Ca++依赖性Cl-电流（Clca）振荡相关。在当前研究中，我们研究了肌醇1,4,5-三磷酸（IP3）在ACh诱导的Ca++和膜电流振荡中的作用。通过膜片吸管直接将IP3受体单克隆抗体（5微克/毫升）和IP3受体拮抗剂肝素（5毫克/毫升）引入细胞，可减少或消除ACh诱导的Clca振荡。此外，细胞内应用IP3（1-100微摩尔）可引发浓度依赖性的Clca振荡，类似于ACh诱导的振荡。增加细胞外Ca++浓度可增强IP3诱导的Clca振荡，而维拉帕米（10微摩尔），一种电压门控Ca++通道阻滞剂，可减弱IP3诱导的Clca振荡以及对照和IP3增强的自发瞬时外向电流。然而，5微克/毫升的IP3受体单克隆抗体和5毫克/毫升的肝素均未改变咖啡因诱导的瞬时Clca。咖啡因（10毫摩尔）可逆地消除IP3诱导的Clca振荡以及IP3增强的自发瞬时外向电流，这表明咖啡因通过一种独立于IP3受体的机制释放Ca++。这些发现与以下假设一致，即ACh诱导的Ca++振荡可通过IP3诱导的Ca++释放途径产生并维持。
