NORADRENALINE SYNTHESIS AFTER SYMPATHETIC-NERVE ACTIVATION IN RAT ATRIA AND ITS DEPENDENCE ON CALCIUM BUT NOT CAM KINASE-II AND PROTEIN-KINASE-A OR PROTEIN-KINASE-C

1 The biosynthesis of noradrenaline following sympathetic nerve activa tion was investigated in rat atria. In particular the time course of n oradrenaline synthesis changes, the relationship of changes in synthes is to transmitter release and the possible roles of second messengers and protein kinases were examined. 2 Rat atria incubated with the prec ursor [H-3]-tyrosine synthesized [H-3]-noradrenaline, Synthesis was en hanced following pulsatile electrical field stimulation (3 Hz for 5 mi n) with the bulk of the increase occurring in the first 45 min after t he commencement of electrical stimulation. In separate experiments rat atria were pre-incubated with [H-3]-noradrenaline and the radioactive outflow in response to electrical field stimulation (3 Hz for 5 min) was taken as an index of noradrenaline release. 3 Stimulation-induced (S-I) noradrenaline synthesis was significantly correlated to S-I nora drenaline release for a variety of procedures which modulate noradrena line release by mechanisms altering Ca2+ entry into the neurone (r(2)= 0.99): those which decreased release: tetrodotoxin (0.3 mu M), Ca2+-fr ee medium, lowering the frequency of nerve activation to 1 Hz, and tho se which increased release, tetraethylammonium (0.3 mM), phentolamine (1 mu M) and the combination of phentolamine (1 mu M) and adenosine (1 0 mu M). On the strength of this relationship we suggest that Ca2+ ent ry is a determining factor in S-I synthesis changes rather than the am ount of noradrenaline released. Indeed the reduction in noradrenaline release with the calmodulin-dependent protein (CAM) kinase II inhibito r KN-62 (10 mu M) which acts subsequent to Ca2+ entry, did not affect S-I synthesis. 4 The cell permeable cyclic AMP analogue, 8-bromoadenos ine 3',5'-monophosphate (BrcAMP, 90 and 270 mu M), dose-dependently in creased basal [H-3]-noradrenaline synthesis in unstimulated rat atria. This effect was antagonized by the selective protein kinase A (PKA) a ntagonist, Rp-8-chloroadenosine 3', 5-'cyclic monophosphorothioate (RC lcAMPS, 300 mu M), suggesting that PKA activation enhances basal norad renaline biosynthesis in sympathetic nerve terminals. 5 The protein ki nase inhibitors, KN-62 (CAM kinase II, 10 mu M), RClcAMPS (PKA, 300 mu M), polymyxin B (protein kinase C (PKC), 21 mu M) and staurosporine ( PKC, PKA and CAM kinase II, 0.1 mu M) did not affect S-I synthesis, al though KN-62, polymyxin B and staurosporine decreased S-I release. We conclude that S-I synthesis is triggered by Ca2+ entering the neurone but that the signalling pathway does not involve classical protein kin ases and appears distinct from the steps involved in transmitter relea se.