A Novel Single-Photon Counting Technique Applied to Highly Sensitive Measurement of [Ca2+]i Transient in Human Aortic Smooth Muscle Cells

Abstract

This study demonstrates that aequorin, a luminescent natural dye, is useful for vascular cell intracellular Ca2+ concentration ([Ca2+]i) determination. A new single-photon counting technique was developed to resolve the effects of fluid flow shear stress on [Ca2+]i in human aortic smooth muscle cells (HASMCs). Confluent HASMCs were grown on petri dishes loaded with aequorin. Then the dishes were placed in a luminometer chamber after the physiological level of shear stress was applied to the HASMC surfaces. The chamber was housed inside a highly sensitive photomultiplier tube. It detected ultraweak photon emission in response to the [Ca2+]i transient. In the presence of 2.0mM extracellular Ca2+, a shear stress of 12dyn∕cm2, applied for 60s to the top surface of the HASMC monolayer, elicited a sharp increase in [Ca2+]i.