Nitric Oxide and Mitochondria Regulate Cytosolic Ca2+ Signaling in Sheared Vascular Endothelial Cells

Abstract

Vascular endothelial cell (EC) exposure to arterial-level fluid mechanical shear stress is known to cause an increase in cytosolic calcium levels ([Ca2+]c). The [Ca2+]c increase is mediated by both extracellular Ca2+ influx and endoplasmic reticulum (ER)-stored Ca2+ release. ECs are exposed to shear stress under physiological conditions and shear stress, via the [Ca2+]c increase, activates the endothelial nitric oxide synthase (eNOS) that produces nitric oxide (NO). Despite progress in understanding Ca2+ signaling, the exact intracellular pathways that determine Ca2+ homeostasis during mechanotransduction still need to be determined. In particular, it is not known whether NO has an effect on Ca2+ signaling and whether the mitochondria play a role in shaping the Ca2+ signal. The close proximity of mitochondria to the ER is thought to cause the mitochondria to experience higher local Ca2+ levels than the cytosol, which suggests that they could be involved in [Ca2+]c signaling possibly by helping to refill the ER. To answer some of these questions, we preincubated cultured human ECs with the Ca2+-sensitive fluorophore fluo-4 and discovered that shear-induced [Ca2+]c shows a different spatiotemporal profile in the presence of the eNOS inhibitor L-NAME, compared to its absence. Under control conditions, 30% of ECs transiently increase their [Ca2+]c within the first min of shear exposure followed by oscillations at a frequency of ~1.55/min. In the presence of L-NAME, 90% of ECs transiently increase their [Ca2+]c within the first min of shear exposure, but the oscillatory response is dampened to ~1.45/min. The NO-dependent [Ca2+]c response may be due to effects of NO on the cGMP-PKG-IP3R and may involve changes in the mitochondrial buffering capacity. Understanding endothelial Ca2+ homeostasis is important, since deregulation of Ca2+ signaling is the hallmark of endothelial dysfunction and cardiovascular disease.