Mechanism of Peroxide-Induced Potentiation of Sympathetic Neurotransrnission in Bovine Irides: Role of Extracellular Calcium

Abstract

Hydrogen peroxide (H₂O₂) and enzymes that regulate its metabolism are present in tissues of the anterior segment of the eye. We have previously shown that in vitro, H₂O₂ can enhance sympathetic neurotransmis-sion in irides from several mammalian species. In the present study, we investigated the role of extracellular calcium in H₂O₂-_-induced potentiation of sympathetic neurotransmission in the bovine isolated iris. Isolated bovine hemiirides were incubated in a bicarbonate-buffered, carbogen-gassed Krebs buffer solution containing [³H]-norepinephrine ([³H]NE) for 60min. After incubation, tissues were prepared for studies of [³H]NE release using the superfusion method. Release of [³H]NE was elicited by consecutive trains of electrical field stimulation. Removal of calcium from the buffer solution attenuated field-stimulated [³H]NE overflow in isolated, superfused bovine irides without affecting basal tritium efflux. H₂O₂ (1 mM) enhanced evoked [³H]NE release to the same extent in tissues exposed to buffer solutions containing normal calcium (1.3 mM) as in those containing low calcium (0.13 mM) or zero calcium. However, in the presence of zero-calcium buffer solution containing the chelator, EDTA (lmM), H₂O₂ (1 mM) caused a gradual and sustained increase in basal tritium efflux. In buffer solutions containing high calcium (1.95 mM), the magnitude of H₂O₂-_-induced increase in field-stimulated [³H]NE release was significantly (P < 0.05) attenuated. Although the neuronal calcium channel antagonist w-conotoxin (20 nM) inhibited [³H]NE by 25%, it had no effect on H₂O₂ (1 mM)-induced potentiation of evoked [³H]NE overflow. We conclude that while trace amounts of extracellular calcium are necessary for H₂O₂-_-induced enhancement of sympathetic neurotransmission, increasing extracellular (buffer) calcium concentration impaired peroxide-induced enhancement of [³H]NE release. Furthermore, voltage-activated calcium channels may not be directly involved in peroxide-induced alteration of adrenergic neurosecre-tion in bovine isolated irides.

Key Words:

• Hydrogen peroxide
• extracellular calcium
• bovine iris
• norepinephrine release
• calcium channel antagonists
• oxidative stress