Activation of alpha-2 adrenergic receptors inhibits norepinephrine release by a pertussis toxin-insensitive pathway independent of changes in cytosolic calcium in cultured rat sympathetic neurons.

Whole-cell electrophysiological studies suggest that sympathetic nerve alpha-2 adrenergic receptors are coupled to voltage-dependent N-type calcium channels through the Gi family 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 microM) and oxymetazoline (0.1-10 microM), and the N-type calcium channel blocker, omega-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 omega-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 omega-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.