Identification of vasotocin-like immunoreactivity in chromaffin cells of the frog adrenal gland: effect of vasotocin on corticosteroid secretion.
The presence of neurohypophyseal nonapeptides in the adrenal gland of nonmammalian vertebrates and the possible action of these regulatory peptides on corticosteroid secretion have never been investigated. We have applied the indirect immunofluorescence technique to examine whether vasotocin (AVT) and/or mesotocin (MT) are located in frog adrenal (interrenal) tissue. Using antisera against AVT and tyrosine hydroxylase, we found that all chromaffin cells contain an AVT-like peptide. Labeling of consecutive sections with phenylethanolamine-N-methyltransferase or AVT antibodies showed that both noradrenaline- and adrenaline-storing cells contain AVT-like immunoreactivity. In contrast no labeling of frog adrenal slices was observed using a MT antiserum. At the ultrastructural level, the immunogold technique revealed that the AVT-immunoreactive peptide is sequestered in chromaffin granules with varying electron densities. Filtration of frog adrenal tissue extracts on Sep-Pak C-18 cartridges showed that the elution profile of the AVT-like peptide was similar to that of synthetic AVT. The apparent concentration of AVT in the adrenal was 2.7 ng/g tissue. Since chromaffin cells represent approximately one third of all interrenal cells, the actual concentration of AVT in chromaffin tissue was about 8 ng/g tissue. The role of AVT in the regulation of frog adrenal steroidogenesis was studied in vitro using perifused frog interrenal slices. Graded doses of AVT (10(-10)-10(-7) M) induced a dose-dependent stimulation of both corticosterone and aldosterone secretion. The other neurohypophyseal peptides (vasopressin, oxytocin, and MT) were also able to enhance corticosteroid secretion, but AVT was by far the most potent stimulator of steroidogenesis. Prolonged administration (4 h) of AVT induced a rapid increase in corticosterone and aldosterone output, followed by a gradual decline of corticosteroid secretion. These results show that an AVT-like peptide is stored in chromaffin granules of frog adrenal gland. Our data also indicate that synthetic AVT is a potent stimulator of corticosteroid secretion by frog interrenal cells. Since in amphibians adrenocortical and chromaffin cells are intimately intermingled, these results suggest that AVT produced by chromaffin cells may regulate corticosteroid release locally, through a cell to cell mode of communication.