Calyculin-A disrupts subplasmalemmal junction and recurring Ca 2+ waves in vascular smooth muscle

Abstract

Excitation–contraction coupling (E–C coupling) in phenylephrine(PE)-stimulated rabbit inferior vena cava (IVC) depends on the generation of asynchronous recurring Ca 2+ waves in the in situ vascular smooth muscle cells (VSMC). Previous studies by our group have implicated a putative non-selective cationic store-operated channel and the reverse-mode Na +–Ca 2+ exchange in refilling of the intracellular Ca 2+ store via the sarco/endoplasmic reticulum ATPase (SERCA) and the maintenance of the recurring Ca 2+ waves. We hypothesize that for the proper functioning of these three Ca 2+ translocators in the process of SR refilling, the plasma membrane (PM) and the underlying superficial sarcoplasmic reticulum (SR) form specialized PM–SR junctions, which are essential for the maintenance of the recurring Ca 2+ waves. In order to test this hypothesis, calyculin-A, a serine/threonine phosphatase inhibitor that has been demonstrated to result in the disruption of the PM–SR junctions was used. In the control rabbit IVC, electron microscopy of the in situ VSMC indicates that 14.2 ± 0.7% of the PM is closely apposed by the prominent superficial SR network, forming numerous flattened PM–SR junctional cytoplasmic spaces. In the control IVC stimulation with 5 μM PE resulted in sustained recurring Ca 2+ waves with a frequency of 0.42 ± 0.02 Hz. In calyculin-A treated rabbit IVC, a concentration-dependent dissociation of the superficial SR and loss of PM–SR junctions was observed. This progressive loss of the PM–SR junctions occurs over the same concentration range as the inhibition of PE-induced recurring Ca 2+ waves. These findings offer support for the hypothesis that the presence of the PM–SR junctions is required for the generation of asynchronous recurring Ca 2+ waves, which underlie excitation–contraction coupling in the VSMC of the rabbit IVC.