COCULTURE OF EMBRYONIC CHICK HEART-CELLS AND CILIARY GANGLIA INDUCES PARASYMPATHETIC RESPONSIVENESS IN EMBRYONIC CHICK HEART-CELLS

We have developed a system for the co-culture of embryonic chick heart cells obtained from embryos at 3.5 days in ovo with ciliary ganglia f rom chick embryos at 7 days in vivo. After 3 days of co-culture, remov al of the ciliary ganglia resulted in complete degeneration of axons w ithin 6-8 h, leaving the post-innervated heart cell culture devoid of neurons. Embryonic chick heart cells at 3.5 days in ovo are unresponsi ve to muscarinic stimulation. However, following 3 days of co-culture with ciliary ganglia, the heart cells developed a negative chronotropi c response to muscarinic stimulation (paired t test, P < 0.02) which p ersisted for at least 24 h after removal of the ciliary ganglion. The development of muscarinic responsiveness was associated with an increa se in the levels of specific alpha-subunits of the guanine nucleotide binding proteins (G-proteins), with a 3-fold increase in the level of alpha39 (39 kDa subunit) and a 2.5-fold increase in the level of alpha 41. The level of the G-protein subunit alpha(s) remained unchanged. Cu lture of embryonic chick heart cells at 3.5 days in ovo with medium co nditioned by the growth of embryonic chick heart cells and ciliary gan glia had an effect on the chronotropic response to muscarinic stimulat ion and on alpha39 and alpha41. levels identical to that of co-culture . These data suggest that a soluble factor released during the co-cult ure of embryonic chick heart cells and ciliary ganglia is capable of i nducing muscarinic responsiveness. These studies suggest that innervat ion of the heart may induce parasympathetic responsiveness by increasi ng the availability of G-proteins which couple the muscarinic receptor to a physiological response.