Galanin receptors and their second messengers in the lumbar dorsal spinal cord

The ligand binding properties of galanin receptors were examined in crude synaptosomal fraction preparations of lumbar dorsal spinal cord, using chloramin-T mono-iodinated porcine Tyr26 galanin as ligand. The equilibrium binding of [125I]galanin showed the presence of a single population of high-affinity binding sites with KD = 0.6 +/- 0.2 nM in a concentration of 55 +/- 15 fmol mg-1 protein (Bmax). The N-terminal fragments galanin (1-16) and galanin (1-12) fully displaced specific [125I]galanin binding from membranes with IC50 values 6 nM and 4 microM, respectively. The C-terminal fragment galanin (17-29) did not displace [125I]galanin when applied in the concentration range 10(-11)-10(-4) M. GTP inhibited the specific binding of [125I] galanin in a concentration dependent manner, with 54% inhibition at 1 mM, suggesting that the galanin receptor found in lumbar dorsal spinal cord is G-protein coupled. Second messenger systems, through which the galanin receptor in lumbar dorsal spinal cord may exert its effect, were also studied. A galanin (10 microM) produced inhibition (58%) of the depolarization induced cGMP increase was found, whereas galanin (10 microM) did not inhibit the noradrenalin (100 microM) activated cAMP synthesis or phosphoinositide turnover in tissue slices of the spinal cord. Bilateral transection of the sciatic nerve at midthigh level 14 days prior to the binding experiment was performed, a treatment which is known to cause a dramatic increase of galanin-like immunoreactivity in the superficial layers of the dorsal spinal cord, dorsal root ganglion and in galanin mRNA levels, but no significant effect on Bmax or KD of the galanin receptor was found.