Co-culture 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 from chick embryos at 7 days in vivo. After 3 days of co-culture, removal of the ciliary ganglia resulted in complete degeneration of axons within 6-8 h, leaving the post-innervated heart cell culture devoid of neurons. Embryonic chick heart cells at 3.5 days in ovo are unresponsive to muscarinic stimulation. However, following 3 days of co-culture with ciliary ganglia, the heart cells developed a negative chronotropic response to muscarinic stimulation (paired t test, P < 0.02) which persisted for at least 24 h after removal of the ciliary ganglion. The development of muscarinic responsiveness was associated with an increase in the levels of specific alpha-subunits of the guanine nucleotide binding proteins (G-proteins), with a 3-fold increase in the level of alpha 39 (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. Culture of embryonic chick heart cells at 3.5 days in ovo with medium conditioned by the growth of embryonic chick heart cells and ciliary ganglia had an effect on the chronotropic response to muscarinic stimulation and on alpha 39 and alpha 41 levels identical to that of co-culture. These data suggest that a soluble factor released during the co-culture of embryonic chick heart cells and ciliary ganglia is capable of inducing muscarinic responsiveness. These studies suggest that innervation of the heart may induce parasympathetic responsiveness by increasing the availability of G-proteins which couple the muscarinic receptor to a physiological response.