EFFECT OF INTRATHECALLY ADMINISTERED LOCAL-ANESTHETICS ON PROTEIN-PHOSPHORYLATION IN THE SPINAL-CORD

To elucidate the biochemical mechanisms of spinal anesthesia, we studi ed the effects of procaine and tetracaine on protein phosphorylation i n the mouse spinal cord. Mice were injected intrathecally with either procaine, tetracaine (67 mM/similar to 2%, 10 mu L, N = 5/drug), or sa line (N = 4/group). Five minutes after injection, animals were killed with a guillotine, and the spinal cord was removed. The caudal 3-cm co rd segment was homogenized and centrifuged, and an aliquot of the supe rnatant was used for phosphorylation assays. Calcium-dependent phospho rylation was initiated by incubating the samples in buffer containing [gamma-P-32]ATP at 37 degrees for 30 min. The proteins were electropho resed using slab gel and two-dimensional electrophoresis, and phosphor ylated proteins were visualized by autoradiography. The data demonstra ted that spinal anesthesia changes the phosphorylation state of five e ndogenous substrate proteins with apparent molecular masses of 130 (pr otein-a), 105 (protein-b), 55 (protein-c), 47 (protein-d), and 33 (pro tein-e) kDa. In two-dimensional electrophoresis, protein-a resolved in to two proteins (a1 and a2). Analysis of variance of the densitometric data suggested a significant effect for the treatment (F-2,F-16 735, P < 0.00005). Post hoc comparisons with the saline-treated controls, u sing the Newman-Keuls test, indicated that local anesthetics significa ntly affected phosphoproteins (P < 0.05) except for protein-a1 in the tetracaine-treated group. Further characterization of these phosphopro teins should aid in determining their role in the signal transduction cascade affected by spinal anesthesia. (C) 1997 Elsevier Science Inc.