More than 25 years ago we began studying the control of norepinephrine release by using a perfused neuroeffector model and extensively characterized the physiological, pharmacological and biochemical properties of transmitter release. We extended investigations to include mechanisms at the cellular level by developing a model of cultured sympathetic neurons (SN) from chick embryo. We have established that NGF-supported SN take up tritiated norepinephrine (3H-NE), which can be released by physiologically relevant electrical stimulation. SN express classical ion channels, membrane receptors coupled to second messenger systems and Ca2+-dependent, regulated transmitter release. Agents which affect voltage-dependent Ca2+ entry or second messenger levels affect 3H-NE release in the expected manner. Thus, chick SN in culture are an excellent model of SN in vivo. However, we have noted three surprising and important differences in release properties in SN in culture compared to their counterparts in perfused organs. First, background release of transmitter in the non-stimulation period is low in sympathetic effector organs, but relatively high in cultured SN. Second, electrically evoked release is proportional to stimulation frequency in intact organs, but in cultured SN maximum release occurs at low frequency and does not increase with frequency. Third, the K+ channel blocker tetraethylammonium (TEA) greatly enhances release in neuroeffector organs, but has little effect on evoked release of 3H-NE in cultured SN. However, these abnormal release properties can be corrected when SN are co-cultured with cardiac cells. We have further demonstrated that cardiac cells cause a change in voltage-dependent Ca2+ entry and/or handling in neuritic regions of SN (sites of transmitter release) and modulate K+ currents in a manner which accounts for the changes in 3H-NE release properties. Most recently, we have discovered that chronic treatment of SN with adenosine (Ado) mimicks some effects of cardiac cells on SN release properties. Thus, Ado may well be the agent released by cardiac cells and responsible for modulation of Ca2+ handling and transmitter release in co-cultured SN. This proposal is intended to test this hypothesis and establish a new physiological role for Ado as a trophic molecule necessary for functional maturation of SN during development.
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