Muscarinic Receptor Activation of Arachidonate-mediated Ca<sup>2+</sup> Entry in HEK293 Cells Is Independent of Phospholipase C

Receptor-enhanced entry of Ca2+in non-excitable cells is generally ascribed to a capacitative mechanism in which the activation of the entry pathway is specifically dependent on the emptying of agonist-sensitive intracellular Ca2+stores. Although such entry can be clearly demonstrated under conditions of maximal or near-maximal stimulation, it is uncertain whether such a mechanism can operate during the oscillatory [Ca2+]signals that are frequently seen following stimulation with low concentrations of agonists. In this study, we report that the stimulation of human m3 muscarinic receptors stably transfected into HEK293 cells results in the appearance of a novel arachidonate-mediated Ca2+entry pathway. We show that the generation of arachidonic acid and the activation of this pathway are specifically associated with stimulation at the low agonist concentrations that typically give rise to oscillatory [Ca2+]signals. At such agonist concentrations, however, the generation of arachidonic acid is independent of the simultaneous activation of the phospholipase C-inositol 1,4,5-trisphosphate pathway. We further show that the arachidonate-mediated Ca2+entry demonstrates characteristics that distinguish it from the corresponding capacitative pathway in the same cells and therefore is likely to represent an entirely distinct pathway that is specifically responsible for the receptor-enhanced entry of Ca2+during [Ca2+]oscillations.

在非兴奋性细胞中，受体增强的Ca2+内流通常归因于一种容量性机制，在此机制中，内流途径的激活特别依赖于对激动剂敏感的细胞内Ca2+库的排空。尽管在最大或接近最大刺激的条件下可以清楚地证明这种内流，但在低浓度激动剂刺激后经常出现的振荡性[Ca2+]信号期间，这种机制是否能起作用还不确定。在本研究中，我们报道稳定转染到人HEK293细胞中的人m3毒蕈碱受体受到刺激后，会出现一种新的花生四烯酸介导的Ca2+内流途径。我们表明，花生四烯酸的产生以及该途径的激活与低激动剂浓度下的刺激特异性相关，这种低浓度刺激通常会产生振荡性[Ca2+]信号。然而，在这种激动剂浓度下，花生四烯酸的产生与磷脂酶C - 肌醇1,4,5 - 三磷酸途径的同时激活无关。我们进一步表明，花生四烯酸介导的Ca2+内流表现出的特征使其有别于同一细胞中相应的容量性途径，因此它可能代表一种完全不同的途径，该途径专门负责在[Ca2+]振荡期间受体增强的Ca2+内流。