Reconstitution of neurotransmission by determining communication between differentiated PC12 pheochromocytoma and HEL 92.1.7 erythroleukemia cells

Neurotransmitter release was monitored using fura-2-loaded HEL 92.1.7 cells dispersed among differentiated PC12 cells (loaded with another Ca2+ indica tor fluo-3) and immobilised using transparent polycarbonate membrane filter s with uniform pore size. Depolarisation with K+ caused a rapid rise in Ca2 + concentration in the PC12 eels, followed by a delayed secondary Ca2+ resp onse in simultaneously monitored nearby HEL cells. There was a lag period o f about 20 s between the responses of the two cell types. Voltage-gated Ca2 + channels in PC12 cells were inhibited by the P/Q-type (omega -conotoxin M VIIC, omega -agatoxin IVA), N-type (omega -conotoxinGVIA) and L-type channe l blockers (nifedipine) as determined using fura-2 or whole-cell patch-clam p recordings. The communication between the cell types on the other hand wa s sensitive to P/Q- and N-type but not to L-type channel blockers. This sug gests that, as in neurons, P/Q- and N-type Ca2+ channels mediate the releas e of neurotransmitters acting on HEL cells. Theoretically, the procedure em ployed should be sensitive enough to detect single exocytotic events. Our r esults demonstrate that a random distribution between effector and target c ells is sufficient to allow communication between cells in a manner similar to extrasynaptic transmission.