Characterization of Store–Operated Ca2+ Influx in Trabecular Meshwork Cells

Abstract

Purpose: : In non–excitable cells, the major Ca²⁺ entry pathway is the store–operated (capacitative) Ca²⁺ influx, which activates after the emptying of intracellular Ca²⁺ stores. Store–operated Ca²⁺ influx participates in cell Ca²⁺ homeostasis as well as in different processes related to cell growth and differentiation, enzyme control or exocytosis. Moreover, store–operated Ca²⁺ influx appears to be involved in cellular contractility and regulation of vascular smooth muscle tone. Since trabecular meshwork (TM) contractility modulates outflow facility, we studied the presence and characteristics of store–operated Ca²⁺ influx in TM cells as a possible regulator of this tissue function.

Methods: : Primary cultures of bovine TM cells were used. Intracellular calcium concentration ([Ca²⁺]_i) was measured in TM cells loaded with Fura–2. Store–operated calcium currents were characterized using the whole–cell configuration of the patch clamp technique.

Results: : Bradykinin evoked a biphasic increase in [Ca²⁺]_i: an initial Ca²⁺ release from intracellular stores followed by a sustained extracellular Ca²⁺ influx. Ca²⁺ influx was greatly reduced when extracellular Ca²⁺ was removed or in the presence of 2–APB, a blocker of store–operated Ca²⁺ channels. Similar results were obtained with endothelin–1 stimulation. After intracellular stores depletion with thapsigargin (in Ca²⁺–free solution), reintroduction of extracellular Ca²⁺ produced a secondary Ca²⁺ influx that could be prevented by preincubation with 2–APB or La³⁺. In patch–clamp recordings, depletion of intracellular stores with IP₃ + EGTA or with thapsigargin induced a non–voltage–gated inward current with the properties described for store–operated Ca²⁺ currents.

Conclusions: : TM cells possess store–operated Ca²⁺ currents that appears to participate in the intracellular Ca²⁺ increments induced by different hormones or neurotransmitters. Block of Ca²⁺ influx by 2–APB and La³⁺ suggests the involvement of TRPC channels. Their participation in TM outflow regulation is under current study.

Keywords: trabecular meshwork • calcium • outflow: trabecular meshwork