Subcellular Distribution of Opioid Peptides in Rat Hypothalamus and Pituitary

Homogenates of rat anterior lobe (AL) and neurointermediate lobe (NIL) pituitary and rat hypothalamus were subjected to subcellular fractionation and density gradient centrifugation. The subcellular distribution of immunoreactive dynorophin A (ir‐Dyn A) in NIL was found to be similar to that of ir‐arginine vasopressin (ir‐AVP). ir‐Dyn A migrated as a discrete band on sucrose density gradients, which corresponded in sedimentation rate to that of ir‐AVP, suggesting that these two peptides are stored within organelles of similar size and density. Two other products of prodynorphin, ir‐α‐neoendorphin ir‐α‐nEND) and ir‐Dyn A‐(1–8) also comigrated with ir‐AVP. ir‐[Leu5]‐enkephalin (ir‐LE), which may be a product of prodynorphin or proenkephalin, was also found to migrate in this region of the gradient. When a homogenate of rat hypothalamus was prepared using a method that has been developed for synaptosome isolation, ir‐Dyn A was found to comigrate with Na+/K+‐activated adenosine triphosphatase (Na/K‐ATPase), a synaptosomal marker enzyme. Using a more concentrated homogenate ir‐Dyn A was found to migrate to a less dense region where peptide‐containing synaptic vesicles have previously been localized. When a synaptosomal preparation was lysed in hypotonic solution a shift was seen in the migration rate of (ir‐Dyn A to this region of the gradient (containing putative synaptic vesicles). Thus the bulk of hypothalamic dynorphin appears to be present within synaptosotne‐like structures which, upon lysis, release a less dense, smaller subcellular organelle corresponding in sedimentation characteristics to other types of peptide‐containing synaptic vesicles. Using either method ir‐AVP migrated to a position that was different from that of ir‐Dyn A and was unaffected by lysis conditions. These results indicate that the majority of the dynorphin in the hypothalamus is not stored with vasopressin at the subcellular level. Immunoreactivities associated with Dyn A‐(4–8), α‐nEND, LE, and [Met5]‐en‐kephalin‐Arg6‐Phe7 (ME‐RF) in hypothalamus were all found to migrate with ir‐Dyn A and all are shifted to approximately the same location by lysis. Thus all of the prodynorphin and proenkephalin products examined appear to be stored within synaptic vesicle‐like organelles present primarily within nerve terminals in the hypothalamus.