Proteins regulating vesicular docking and fusion : histochemical studies on their presence and regulation in endocrine, neuroendocrine and neuronal cells

Abstract

Regulated transmitter release involves docking and fusion of transmitter-containing vesicles with the plasma membrane. Vesicular docking and fusion events are mediated by specific exocytotic proteins located at the vesicle membrane, in the cytoplasm or at the plasma membrane. This thesis focuses on the distribution and regulation of exocytotic proteins and their isoforms in endocrine, neuroendocrine and neuronal tissues. In the endocrine pancreas, anterior pituitary and the histamine-producing enterochromaffin-like (ECL) cells of the stomach, vesicle-associated membrane protein-2 (VAMP-2), mammalian homologue of unc-18 (munc-18), synaptosomal-associated protein of 25 kDa (SNAP-25) and syntaxin mRNA and/or protein were demonstrated in all cell types. Synaptotagmin l/II was present in somatostatin-containing cells of the endocrine pancreas and all hormone-containing cells of the anterior pituitary, with the exception of gonadotropes, whereas no synaptotagmin I/II could be detected in stomach ECL cells. Moreover, cysteine string protein (CSP) and synaptotagmin III mRNA and/or protein were demonstrated in the anterior pituitary and in stomach ECL cells. Estrogen replacement of ovariectomized rats showed regulation of SNAP-25a and SNAP-25b mRNA, but not VAMP-2,munc-18 or Hrs-2 mRNA. SNAP-25, VAMP-2 and munc-18 mRNA were down-regulated in the pituitary gland of aged male rats.

Incubation of cultured anterior pituitary cells with botulinum neurotoxin F, a VAMP-specific endopeptidase, reduces Ca2+-stimulated secretion of GH. In hypothalamic magnocellular neurosecretory neurons, VAMP-2 and SNAP-25a mRNA, but not VAMP-I and SNAP-25b mRNA were detected. After osmotic challenge (2% NaCI in the drinking water), a significant increase in VAMP-2, munc-18, Hrs-2 and SNAP-25a mRNA was demonstrated in magnocellular neurons. After sciatic nerve injury (axotomy), there was a decrease in VAMP-I, SNAP-25a and SNAP-25b and an increase in VAMP-2 mRNA levels in lumbar spinal a-motoneurons. The distinct alterations in VAMP-I and VAMP-2 mRNA levels could to some extent be correlated to changes in the acetylcholine-synthesizing enzyme, choline acetyl transferase (ChAT) and a-calcitoningene-related peptide (a-CGRP) mRNA levels, respectively.

In spinal motoneurons, dorsal root ganglion cells, as well as in somatomotoric cranial nerve nuclei, SNAP-25a RNA transcript labeling was nuclear, whereas the SNAP-25b RNA transcript labeling was cytoplasmic. In the parasympathetic cranial nerve nuclei Edinger-Westphal nucleus and dorsal motor nucleus of the vagus nerve, SNAP-25a RNA transcript labeling was cytoplasmic, whereas SNAP-25b RNA labeling was undetectable. Taken together, the results show that several exocytotic proteins and their isoforms, originally identified in the nervous system also are present in endocrine and neuroendocrine cells. The results also show an isoform-specific expression of exocytotic proteins in endocrine, neuroendocrine and neuronal cells. Levels of mRNA for several of the exocytotic proteins are changed after hormonal influence or experimental conditions that involve altered secretion.