Noradrenaline synthesis after sympathetic nerve activation in rat atria and its dependence on calcium but not CAM kinase II and protein kinases A or C.
1. The biosynthesis of noradrenaline following sympathetic nerve activation was investigated in rat atria. In particular the time course of noradrenaline synthesis changes, the relationship of changes in synthesis to transmitter release and the possible roles of second messengers and protein kinases were examined. 2. Rat atria incubated with the precursor [3H]-tyrosine synthesized [3H]-noradrenaline. Synthesis was enhanced following pulsatile electrical field stimulation (3 Hz for 5 min) with the bulk of the increase occurring in the first 45 min after the commencement of electrical stimulation. In separate experiments rat atria were pre-incubated with [3H]-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 noradrenaline release for a variety of procedures which modulate noradrenaline release by mechanisms altering Ca2+ entry into the neurone (r2 = 0.99): those which decreased release: tetrodotoxin (0.3 microM), Ca(2+)-free medium, lowering the frequency of nerve activation to 1 Hz, and those which increased release, tetraethylammonium (0.3 mM), phentolamine (1 microM) and the combination of phentolamine (1 microM) and adenosine (10 microM). On the strength of this relationship we suggest that Ca2+ entry is a determining factor in S-I synthesis changes rather than the amount of noradrenaline released. Indeed the reduction in noradrenaline release with the calmodulin-dependent protein (CAM) kinase II inhibitor KN-62 (10 microM) which acts subsequent to Ca2+ entry, did not affect S-I synthesis. 4. The cell permeable cyclic AMP analogue, 8-bromoadenosine 3',5'-monophosphate (BrcAMP, 90 and 270 microM), dose-dependently increased basal [3H]-noradrenaline synthesis in unstimulated rat atria. This effect was antagonized by the selective protein kinase A (PKA) antagonist, Rp-8-chloroadenosine 3',5'-cyclic monophosphorothioate (RClcAMPS, 300 microM), suggesting that PKA activation enhances basal noradrenaline biosynthesis in sympathetic nerve terminals. 5. The protein kinase inhibitors, KN-62 (CAM kinase II, 10 microM), RClcAMPS (PKA, 300 microM), polymyxin B (protein kinase C (PKC), 21 microM) and staurosporine (PKC, PKA and CAM kinase II, (0.1 microM) did not affect S-I synthesis, although 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 kinases and appears distinct from the steps involved in transmitter release.
Kotsonis
P
P
Prince Henry's Institute of Medical Research, Clayton, Victoria, Australia.
Binko
J
J
Majewski
H
H
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006321:Heart / Q000187:drug effects* / Q000294:innervation*; D008297:Male; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D009638:Norepinephrine / Q000096:biosynthesis* / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology; D013564:Sympathetic Nervous System / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D013873:Thionucleotides / Q000494:pharmacology
D004791:Enzyme Inhibitors; D013873:Thionucleotides; D015124:8-Bromo Cyclic Adenosine Monophosphate; C016957:adenosine-3',5'-cyclic phosphorothioate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium; D009638:Norepinephrine