Effect of intrathecally administered local anesthetics on protein phosphorylation in the spinal cord

To elucidate the biochemical mechanisms of spinal anesthesia, we studied the effects of procaine and tetracaine on protein phosphorylation in the mouse spinal cord. Mice were injected intrathecally with either procaine, tetracaine (67 mM/approximately 2%, 10 microL, N = 5/drug), or saline (N = 4/group). Five minutes after injection, animals were killed with a guillotine, and the spinal cord was removed. The caudal 3-cm cord segment was homogenized and centrifuged, and an aliquot of the supernatant was used for phosphorylation assays. Calcium-dependent phosphorylation was initiated by incubating the samples in buffer containing [gamma-32P]ATP at 37 degrees for 30 min. The proteins were electrophoresed using slab gel and two-dimensional electrophoresis, and phosphorylated proteins were visualized by autoradiography. The data demonstrated that spinal anesthesia changes the phosphorylation state of five endogenous substrate proteins with apparent molecular masses of 130 (protein-a), 105 (protein-b), 55 (protein-c), 47 (protein-d), and 33 (protein-e) kDa. In two-dimensional electrophoresis, protein-a resolved into two proteins (a1 and a2). Analysis of variance of the densitometric data suggested a significant effect for the treatment (F(2,16) 735, P < 0.00005). Post hoc comparisons with the saline-treated controls, using the Newman-Keuls test, indicated that local anesthetics significantly affected phosphoproteins (P < 0.05) except for protein-al in the tetracaine-treated group. Further characterization of these phosphoproteins should aid in determining their role in the signal transduction cascade affected by spinal anesthesia.