THE SUBCELLULAR DISTRIBUTION OF SUBSTANCE PIN THE NERVOUS TISSUES

Abstract

1. Rabbit's brain and spinal cord were homogenated in 0.32M sucrose and fractionated into six subcellular fractions (nuclear, mitochondrial, vesicular, amorphous, microsomal and cytoplasmic) by applying WHITTAKER'S fractional centrifugation in a density gradient, and the activity of substance P in these subcellular fraction was compared.
2. A greater part of SP activity (about 50-70%) was found in the vesicular and cytoplasmic supernatant. So the neuronal SP activity would be chiefly of particulate origin.
3. Such particulate SP was released not so easily by treating with 0.32M sucrose at neutral pH in the cold. But it was liberated with relative rapidity by raising temperature (60-100°C), acidification (pH3-4) and hypotonicity.
4. On incubation with the microsomal fraction of neuronal tissues the particulate SP activity as well as extracted SP were inactivated, but other fractions showed hardly any inactivating effect. Such an inactivating power of the supernatant fraction was suppressed by adding strychnine or LSD-25 into the incubation media, while it was completely destroyed by boiling but not lost by dialysis. Hence this action would be of enzymic nature.
5. Electron-microscopical observations were also made on these subcellular fractions and it was confirmed that the vesicular fraction was chiefly composed of smaller particles of 200-3000Å in diameter, while the neuronal microsomes could be differentiated into two categories; solid and membrane-like ones. The microsomal SP and SP inactivating enzyme would be probably present in some particles in the latter.