Monoclonal antibodies identify two novel proteins associated with vasopressin secretory granules of the rat neurohypophysis

Abstract

Immunocytochemical and immunoblotting technique have been used to characterize the antigens recognized by two monoclonal antibodies (MAbs C6 and D5) produced against dissociated cells from punches of neonatal supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei of the rat. Peroxidase immunocytochemistry revealed that both MAbs label magnocellular perikarya in the adult and neonatal SON and PVN as well as smaller neurons in the suprachiasmatic nucleus. Axons of the hypothalamo-neurohypophysial tract are also immunolabeled within the hypothalamus and zona interna of the median eminence, and C6 and D5 each bind specifically to both the adult and neonatal neurohypophysis. Dual-label immunofluorescence experiments employing C6 or D5 simultaneously with rabbit antisera specific for either oxytocin, neurophysin or vasopressin neurophysin revealed that C6 binds only to vasopressinergic magnocellular perikarya in the SON, while D5 labels both vasopressinergic and a small subset of oxytocinergic magnocellular neurons. Post-embedding immunogold analysis of MAb binding to the neurohypophysis at the ultrastructural level showed that both C6 and D5 recognize antigens associated with large dense core neurosecretory granules in a subset of neurosecretory axons. Initial biochemical characterization of the antigens recognized by C6 and D5 was performed using SDS-PAGE and Western immunoblotting. MAbs C6 and D5 label single protein bands with apparent molecular weights of 38 and 68 kDa, resp., in blots of reduced extracts from the adult neurointermediate lobe. No cross-reactivity between C6 and D5 and the neurophysins was apparent, nor did anti-neurophysin sera recognize the bands identified by C6 and D5. We have therefore designated these novel antigens as VPGP38 and VPGP68 for VasoPressin Granule Proteins.