PREJUNCTIONAL ALPHA(2)-ADRENOCEPTORS AND ADENYLYL-CYCLASE REGULATION IN THE RABBIT IRIS-CILIARY BODY

Agents that elevate intracellular cyclic AMP (cAMP) have been found to enhance the synaptic discharge of norepinephrine (NE) from sympatheti c nerve terminals in the rabbit iris-ciliary body and other peripheral tissues. We explored the hypothesis that prejunctional alpha(2)-adren ergic receptors that mediate feedback inhibition of NE release may be coupled to adenylyl cyclase inhibition. To indirectly monitor cAMP cha nges in sympathetic axon terminals, we analyzed the cAMP-mediated acti vation of tyrosine hydroxylase, a sympathetic marker protein that unde rgoes acute phosphorylation and activation by cAMP-dependent protein k inase A. Tyrosine hydroxylase activity was assayed in situ by incubati on of rabbit iris-ciliary body tissue segments in buffered Krebs-Ringe r solution containing the substrate tyrosine (100 mu M) and the DOPA d ecarboxylase inhibitor brocresine (30 mu M). Intraneuronal DOPA accumu lation was quantified by HPLC with electrochemical detection. Tyrosine hydroxylase activity was increased approximately 2 fold by incubation with forskolin (10 mu M) plus IBMX (0.5 mM) or with 8-Bromo-cAMP (3 m M). Simultaneous addition of the alpha(2)-adrenergic agonist clonidine (1 mu M) attenuated the response to forskolin/IBMX, but had no effect on the response to 8-Br-cAMP. Clonidine-mediated inhibition of the fo rskolin/IBMX response was abolished by treatment of tissues with N-eth yhnaleimide (NEM), an alkylating agent that inactivates pertussis toxi n-sensitive G proteins (G(i)) that couple receptors to adenylyl cyclas e inhibition. These findings suggest that prejunctional alpha(2)-adren oceptors in the rabbit iris-ciliary body are negatively coupled to ade nylyl cyclase. This mechanism may contribute to autofeedback regulatio n of NE biosynthesis and release.