Modulation of retinal glutamate transport during ischaemia by protein kinase C

Purpose: To investigate the role of isoform-selective modulators of protein kinase C (PKC) activity upon the function of retinal glutamate transporters during periods of simulated ischaemia. Methods: Adult pigmented rats were euthanased by an overdose of sodium pentobarbitone (200mg/kg; i.p.). Isolated retinas were preincubated for 15 minutes in oxygenated Lockes buffer containing the PKC modulators before exposure to a simulated ischaemic insult (15 minutes). Ischaemia was simulated in vitro by omitting both glucose and oxygen from the incubation media. 3H-D-aspartate (1µCi) was included in the incubation media (2ml) during the ischaemic insult and retinal uptake was quantified by liquid scintillation. Results: Rat retinas accumulate 3H-D-aspartate in a time dependent manner in vitro. Immunohistochemistry for D-aspartate shows that it is the glial Müller cells that exclusively take-up the glutamate analogue under control conditions. Simulated ischaemia for 15 minutes resulted in a significant decrease in the accumulation of 3H-D-aspartate from 455 nmoles/mg protein to 251 nmoles/mg. The pan-isoform-selective PKC inhibitor, chelerytherine (25µM) and the PKCδ-selective inhibitor, rottlerin (20µM) (but not the PKCα,ß,γ inhibitor Gö6976 (1µM)) caused a further significant reduction in 3H-D-aspartate uptake under ischaemic conditions to 121 nmoles/mg and 201 nmoles/mg respectively. Stimulation of PKC with the phorbol esters PMA (1µM) and PDBu (1µM), or the PKCδ,ϵ-selective activator, ingenol (300µM), under ischaemic conditions did not alter significantly the accumulation of 3H-D-aspartate compared to ischaemia alone. Conclusion: These data suggest that the activity of the Müller cell glutamate transporter, GLAST, is compromised during retinal ischaemia. Moreover, that activity of GLAST can be modulated by inhibitors of PKC, particularly the PKCδ isoform, under ischaemic conditions. This suggest that the targeting of specific glutamate transporters with isoform-specific modulators of PKC activity may have significant implications for the understanding of neurodegenerative conditions arising from compromised glutamate homeostasis.