Differential effects of wortmannin on the release of substance P and aminoacids from the isolated spinal cord of the neonatal rat

1 Effects of wortmannin, an inhibitor of myosin light chain kinase, on the release of substance P and amino acids, GABA and glutamate, were investigat ed in the isolated spinal cord preparation of the neonatal rat. 2 Wortmannin at 0.5-10 mu M depressed the release of substance P evoked by high-K+ (90 mM) medium from the spinal cord (IC50=1.1 mu M). Wortmannin als o depressed the high-K+ (70 mM)-evoked release of substance P from cultured dorsal root ganglion neurons of neonatal rats. In contrast, the high-K+ (9 0 mM)-evoked release of GABA and glutamate from the spinal cord was not aff ected by wortmannin (0.1-10 mu M). 3 Upon stimulation of a dorsal root, a monosynaptic reflex and a subsequent slow ventral root depolarization were evoked in the ipsilateral ventral ro ot of the same segment in the isolated spinal cord preparation. The magnitu de of the slow ventral root depolarization was depressed gradually to about 70% of the control during the course of 30 min under wortmannin (1 mu M). In contrast, the monosynaptic reflex was unaffected by wortmannin. 4 Immunofluorescent staining revealed that immunoreactivities of substance P and myosin II were colocalized at presynaptic terminals in the dorsal hor n of the neonatal rat spinal cord. 5 The present results suggest that myosin phosphorylation by myosin light c hain kinase may play a crucial role in the release of substance P, but not in the release of GABA and glutamate in the neonatal rat spinal cord. This may reflect a difference in the exocytic mechanisms of substance P-containi ng large dense core vesicles and amino acid-containing small clear vesicles .