Abstract

Whole-cell electrophysiological studies suggest that sympathetic nervealpha-2 adrenergic receptors are coupled to voltage-dependent N-type calcium channels through the G_ifamily of proteins to inhibit neurotransmitter release. Because most nerve terminals are too small for direct electrophysiological recordings, the aim of this study was to examine the relationship between alpha-2 adrenergic receptor-mediated inhibition of norepinephrine release and the rise in cytosolic calcium in neurites from cultured sympathetic neurons. In cultured rat superior cervical ganglion neurons, the alpha-2 adrenergic receptor agonists, UK-14304 (0.01–10 μM) and oxymetazoline (0.1–10 μM), and the N-type calcium channel blocker, ω-conotoxin GVIA (0.1–10 nM), inhibited the release of tritiated norepinephrine in response to electrical stimulation (1 Hz, 30 pulses, 0.1 ms, 70 V). The inhibitory effect of the alpha-2 adrenergic receptor agonists was not altered by pretreatment with pertussis toxin (200 ng/ml, 18 h), although pertussis toxin blocked the inhibition of forskolin-stimulated cAMP accumulation by UK-14304. In fura-2 loaded cells, electrical stimulation (1 Hz, 30 pulses, 0.1 ms, 70 V) increased cytosolic calcium in sympathetic neuronal processes. Blockade of N-type calcium channels with ω-conotoxin (1 and 10 nM) reduced the rise in cytosolic calcium by 25 ± 3% and 52 ± 6%, respectively, whereas UK-14304 and oxymetazoline did not alter the electrically stimulated rise in cytosolic calcium. These data suggest that blockade of N-type calcium channels with ω-conotoxin GVIA inhibits stimulated norepinephrine release and cytosolic calcium measured with fura-2 at similar concentrations, whereas activation of alpha-2 adrenergic receptor inhibits norepinephrine release by a pathway that is insensitive to pertussis toxin and changes in cytosolic calcium in neurites from cultured rat superior cervical ganglion cells.