No abstract

The subcellular storage of 5-HT was studied in sheep thyroid parafollicular cells. These cells are neural crest derivatives and were investigated as a serotonergic model system. Light and electron microscopic immunocytochemistry was used to examine the distributions of 5-HT, 45 and 56 kDa forms of 5-HT binding protein (SBP), and calcitonin. A single type of parafollicular cell was found that contained calcitonin, 5-HT, and 45 kDa SBP but not 56 kDa SBP. The secretory granules of parafollicular cells all displayed calcitonin immunoreactivity, and many were also immunoreactive for 5-HT and 45 kDa SBP. Granules were isolated, first by size and then by density, on successive metrizamide gradients. These provided a granular fraction that was enriched in calcitonin, endogenous 5-HT, and 45 kDa SBP. Immunoblots confirmed the presence of 45 kDa SBP in the isolated granules and in suspensions of parafollicular cells that were purified by an affinity chromatographic technique. Parafollicular cell granules did not appear to contain substantial stores of ATP. Granules isolated on Percoll gradients were morphologically homogeneous and took up 3H-5-HT. The specificity of this uptake was confirmed by quantitative electron microscopic radioautography. The granular uptake of 3H-5-HT was inhibited by reserpine (10 microM). It is concluded that parafollicular cell granules are different from other amine-storing vesicles that do contain ATP; nevertheless, since parafollicular cell granules store 5-HT and have the same 45 kDa SBP as is found in serotonergic axon terminals, parafollicular cell granules may be analogous to the synaptic vesicles of serotonergic neurons.

The thyroid parafollicular cell is an endocrine cell derived from the neural crest that stores 5-hydroxytryptamine (5-HT). In common with serotonergic neurons, but in contrast to 5-HT-storing cells that are not neurectodermal derivatives, parafollicular cells also contain a specific 5-HT binding protein. Despite this similarity to serotonergic neurons, parafollicular cells in situ were found to express an endocrine phenotype with few neural characteristics. Thus, the cells costore 5-HT with calcitonin, not calcitonin gene-related peptide (CGRP), which is the product of the calcitonin gene expressed in neurons, and they do not contain neurofilaments. The ability of adult parafollicular cells to respond to microenvironmental perturbations by expressing neuronal characteristics was examined. Sheep thyroid glands were dissociated, and parafollicular cells were purified by affinity chromatography. The purified parafollicular cells were grown in culture on a variety of substrates in the presence or absence of the beta subunit of nerve growth factor (beta-NGF). Parafollicular cells survived in culture for at least a week but retained a roughly spherical shape. Nevertheless, a subset of the cultured parafollicular cells began to display CGRP immunoreactivity. The addition of beta-NGF to the cultured parafollicular cells induced a number of them to extend neurites and increased the proportion of cells in which CGRP immunoreactivity could be found. Neurite-bearing parafollicular cells appeared not to survive for more than 2 d. While their survival was not enhanced when they were grown on collagen, polylysine, laminin, or reconstituted basal lamina, parafollicular cells that had extended neurites in response to beta-NGF survived for at least a week when cocultured with an explant of aneuronal chick hindgut. The effect of the gut was local and only those neurite-bearing parafollicular cells that were growing in direct contact with the explant survived. The thyroid parafollicular cell therefore resembles another crest-derived endocrine cell, the adrenal chromaffin cell, in being able to manifest neural properties in culture. For the parafollicular cell these neural properties include the processing of RNA encoded by the calcitonin gene to express CGRP and neurite outgrowth in response to beta-NGF.