Effect Of Endothelin 1 (ET-1) On The Calcium Dynamics Of Rat Retinal Astrocytes

Abstract

Purpose: : ET-1 is a vasoactive peptide that may be elevated in the aqueous, vitreous and plasma of patients with glaucoma or diabetic retinopathy and in related animal models. ET-1 has been shown to increase intracellular calcium ([Ca²⁺]_i) and the proliferation of type I optic nerve astrocytes in vitro. We have examined the effect of ET-1 on type I astrocyte [Ca²⁺]_i in ex vivo retina to assess ET-1-induced calcium dynamics in an intact tissue preparation and to determine if retinal astrocytes could be a target of ET-1. We also assessed the potential effect of ET-1 on [Ca²⁺]_i in two other potential retinal targets, Müller cells and neurons in the ganglion cell layer (GCL).

Methods: : Cells in isolated adult rat retinas were labeled by incubation in fluo-4 or -8 AM (astrocytes), X-Rhod-1 AM (Müller cells) or loaded by electroporation of fura-2 salt (GCL neurons). In some cases it was possible to simultaneously image retinal vessels. Loaded cells were then imaged and exposed to ET-1 (0.1 nM - 1 µM). To assess the involvement of specific endothelin receptors, the effect of ET-1 was tested in the presence of ET_A (BQ610, 1 µM) and/or ET_B (BQ788, 1 µM) antagonists and in a mutant rat (sl/sl) that does not express functional ET_B receptors. The effect of an ET_B agonist (IRL-1620, 0.01 nM-10 nM) was also tested.

Results: : Application of ET-1 produced a dose-dependent (≥1 nM; EC₅₀ 40 nM) increase of retinal astrocyte [Ca²⁺]_i. IRL-1620 (≥2.5 nM) also increased astrocyte [Ca²⁺]_i but with a similar response at each effective concentration (2.5-10 nM). ET-1 did not affect blood vessel diameter at ≤10 nM but produced contraction at ≥50 nM (18 ± 8% reduction, mean ± SD; 200 nM: 34 ± 13%). ET-1 had no apparent effect on Müller cell or GCL neuron [Ca²⁺]_i. The effect of 50 nM ET-1 on astrocyte [Ca²⁺]_i was not affected by BQ610 or BQ877 alone (1 µM) and there was no difference between the [Ca²⁺]_i increase produced by 200 nM ET-1 in astrocytes from wildtype and sl/sl rats. However, the astrocyte [Ca²⁺]_i increase in response to 50 nM ET-1 was reduced (86 ± 10%) when BQ610 and BQ788 were combined.

Conclusions: : ET-1 induces an increase of [Ca²⁺]_i in retinal astrocytes through either ET- receptor type and, at higher concentration, can induce the contraction of the retinal blood vessels. ET-1 did not alter [Ca²⁺]_i in Müller cells or neurons in the GCL suggesting the lack of endothelin receptors in these retinal cell types, or an action incapable of altering [Ca²⁺]_i, and that alterations of Müller cell or GCL neuron [Ca²⁺]_i are not a consequence of ET-1-induced increases of retinal astrocyte [Ca²⁺]_i.

Keywords: astrocyte • calcium • retina