Beta-adrenergic Receptor Agonists and Antagonists Protect Retinal Cells in Culture by Different Mechanisms

Abstract

Abstract: : Purpose: To determine whether both ß-adrenergic receptor agonists and antagonists can prevent retinal cell death in culture and subsequently to compare the protective mechanisms of these classes of compounds. Methods: Studies were carried out on mixed rat retinal cultures that had been prepared either 6 days (young cultures) or 20 days (old cultures) previously. Young cultures consisted of both GFAP/vimentin-positive glia and PGP 9.5/GABA-positive neurones, whereas old cultures were devoid of neurones. Cultures were exposed to bacterial endotoxin (lipopolysaccharide; LPS) for defined periods of time and their supernatants analysed for nitrite content (measure of nitric oxide synthase or NOS activity) or lactate dehydrogenase (LDH) release (measure of cell death). In addition, cultures were analysed by immunocytochemistry for changes in inducible NOS (iNOS)-, GFAP- and GABA-immunoreactivities and also for DNA breakdown and cell death by the TUNEL method. The influences of both ß-adrenergic receptor agonists and antagonists on these processes were determined. Results: LPS stimulated nitrite production in a dose-dependent manner in both young and old cultures; an increase in iNOS-immunoreactivity was solely associated with glial cells. LPS also caused an increase in cell death as indexed by LDH release and increased TUNEL-labelling of nuclei in young cultures but there was no such effect in old cultures. The cells that underwent death were predominantly neurones. The effects of LPS (10µg/ml) were attenuated by L-NAME, indomethacin and dexamethasone. Moreover, although both ß-adrenergic receptor agonists (arterenol, salbutamol, isoproterenol; all 100µM) and ß-adrenergic receptor antagonists (betaxolol, timolol, ICI-118551, metipranolol; all 100µM) were able to significantly prevent LPS-induced retinal cell death in the young cultures, only the agonists attenuated LPS-induced nitrite production and increases in iNOS-immunoreactivity in young and old cultures. Conclusions: The results show that LPS induces an increase in glial iNOS expression and nitrite production which leads to retinal neuronal death. ß-adrenergic receptor agonists prevent such increases in iNOS activity and expression and consequently reduce the incidences of neurone death. In contrast, ß-adrenergic receptor antagonists counteract neurone death by a process that is independent of the activation of glial iNOS.

Keywords: retinal culture • neuroprotection • nitric oxide