Norepinephrine release from spinal synaptosomes - Auto-alpha(2)-adrenergicreceptor modulation

Background Clonidine produces analgesia after spinal injection by activatin g alpha(2)-adrenergic receptors. Recently, clonidine has been demonstrated to increase spinal release of norepinephrine (NE) in vivo, in contrast to t hat anticipated by classic presynaptic autoinhibition, The purpose of the c urrent study was to determine if clonidine could inhibit release of NE in a preparation of spinal cord tissue lacking synaptic circuits. Methods: Crude synaptosomes were prepared from male Sprague-Dawley rat spin al cord, loaded with [H-3]NE, and stimulated by potassium chloride to relea se [H-3]NE. Samples were incubated with clonidine in the absence or presenc e of various inhibitors. To study the effect of alpha(2a)-adrenergic recept or subtypes, some animals were pretreated with an oligodeoxynucleotide (ODN ) composed of a sense or antisense sequence to a portion of this receptor. Results: Potassium chloride produced a concentration-dependent increase In [H-3]NE release, and this release was inhibited by clonidine with a concent ration producing 50% maximal inhibition (IC50) of 1.3 mu M. The effect of c lonidine was inhibited by the alpha(2)-adrenergic antagonists, yohimbine an d idazoxan, but not by alpha(1)-adrenergic, muscarinic, or opioid antagonis ts. Intrathecal pretreatment with antisense ODN to alpha(2A)-adrenergic rec eptors reduced alpha(2A)-adrenergic receptor protein expression compared wi th sense ODN control and also reduced clonidine-induced inhibition of [H-3] NE release. Conclusions These data demonstrate the existence of classic autoinhibitory alpha(2)-adrenergic receptors in the spinal cord probably of the alpha(2A) subtype. They further suggest that clonidine-induced stimulation of spinal NE release must occur from indirect actions, presumably due to activation o f a spinal circuit.