Glutamate is the principle excitatory neurotransmitter within the central nervous system (CNS). However, elevated extracellular glutamate concentrations induce excitotoxic neuronal death mediated by excessive stimulation of neuronal glutamate receptors. Therefore, precise regulation of the extracellular glutamate concentration is critical to facilitate neurotransmission and to prevent excitotoxic neuronal damage. Low extracellular glutamate concentrations are maintained by the activity of high-affinity glutamate transporters located on glial and neuronal cell membranes. Modulation of glutamate transporter activity is essential as perturbation of glutamate homeostasis, and resultant excitotoxicity, is implicated in many CNS pathologies. Protein kinase C (PKC), a ubiquitous intracellular signalling messenger, is reported to modulate the activity of high-affinity glutamate transporters in cell culture, though conflicting effects have been reported. This study aimed to investigate PKC-mediated modulation of glutamate transporter activity in situ
and to determine whether PKC-mediated regulation of glutamate transporter activity confers neuroprotection against an ischaemic insult.
The retina is an excellent model of the CNS. Four high-affinity glutamate transporters are expressed in the rat retina: the EAAC-1, EAAT-5 and GLT-1 subtypes are expressed by neuronal populations while the glial Müller cells exclusively express the GLAST subtype. Under physiological conditions, GLAST dominates extracellular glutamate clearance and plays a key role in retinal glutamate homeostasis.
A decrease in retinal PKC activity inhibited retinal glutamate transporter activity. Panisoform and 5-isoform selective PKC inhibition reduced the accumulation of tritiated D-aspartate, a non-metabolisable glutamate analogue, in isolated retinas and dissociated retinal cultures in vitro. Immunohistochemical analysis revealed that inhibition of PKC activity suppressed GLAST activity and blocked Miiller cell glutamate uptake. This effect was mediated by the Müller cell specific PKC6 isoform. Interestingly, a population of bipolar neurones retained the ability to accumulate glutamate following PKC inhibition. The PKC-mediated suppression of glutamate transporter activity resulted in excitotoxic damage. In contrast, stimulation of PKC activity did not influence glutamate transporter activity or affect retinal histology.
PKC-mediated regulation of glutamate transporter activity was also studied in isolated retinas subjected to in vitro ischaemic conditions. Pan-isoform PKC inhibition and PKC8-selective inhibition suppressed the transporter activity in the ischaemic retina and exacerbated histological damage. PKC activation did not influence ischaemic glutamate transporter activity or affect immediate retinal outcome following a simulated in vitro ischaemic insult.
Functional and histological experiments were performed to determine whether the modulation of PKC in vivo affects retinal ischaemic outcome. In vivo retinal ischaemia was induced by raised intra-ocular pressure. PKC inhibition did not affect retinal function as assessed by electroretinography. PKC activation did, however, suppress retinal function, possibly by interfering with the phototransduction cascade. Moreover, glutamate and GABA immunohistochemistry suggested that PKC modulation in vivo did not influence glutamate transporter activity. During the in vivo ischaemic insult, PKC inhibition did not significantly influence retinal damage or functional outcome. Interestingly, the selective activation of the Müller cell PKC5 isoform offered neuroprotection to photoreceptors within the ischaemic retina in vivo. This suggests that it may be possible to enhance glutamate transporter activity by the pharmacological manipulation of regulating enzymes or proteins, thereby providing neuroprotection against excitotoxic damage.