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 G(i) family of proteins to inhibit neurot ransmitter release. Because most nerve terminals are too small for dir ect electrophysiological recordings, the aim of this study was to exam ine the relationship between alpha-2 adrenergic receptor-mediated inhi bition of norepinephrine release and the rise in cytosolic calcium in neurites from cultured sympathetic neurons. In cultured rat superior c ervical ganglion neurons, the alpha-e adrenergic receptor agonists, UK -14304 (0.01-10 mu M) and oxymetazoline (0.1-10 mu M), and the N-type calcium channel blocker, omega-conotoxin GVIA (0.1-10 nM), inhibited t he release of tritiated norepinephrine in response to electrical stimu lation (1 Hz, 30 pulses, 0.1 ms, 70 V). The inhibitory effect of the a lpha-2 adrenergic receptor agonists was not altered by pretreatment wi th 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 processe s. 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 ele ctrically stimulated rise in cytosolic calcium. These data suggest tha t blockade of N-type calcium channels with omega-conotoxin GVIA inhibi ts stimulated norepinephrine release and cytosolic calcium measured wi th fura-2 at similar concentrations, whereas activation of alpha-2 adr energic receptor inhibits norepinephrine release by a pathway that is insensitive to pertussis toxin and changes in cytosolic calcium in neu rites from cultured rat superior cervical ganglion cells.