Evidence that urocortin I acts as a neurohormone to stimulate alpha MSH release in the toad Xenopus laevis.

We have raised the hypothesis that in the South African clawed toad Xenopus laevis, urocortin 1 (UCN1), a member of the corticotropin-releasing factor (CRF) peptide family, functions not only within the brain as a neurotransmitter/neuromodulator but also as a neurohormone, promoting the release of alpha-melanophore-stimulating hormone (alphaMSH) from the neuroendocrine melanotrope cells in the intermediate lobe of the pituitary gland. This hypothesis has been investigated by (1) assessing the distribution of UCN1 and CRF by light immunocytochemistry, (2) determining the subcellular presence of UCN1 in the neural lobe of the pituitary gland by immuno-electron microscopy applying high-pressure freezing and cryosubstitution, and (3) testing the effect of UCN1 on MSH release from toad melanotrope cells using in vitro superfusion. In the X. laevis brain, the main site of UCN1-positive somata was found to be the Edinger-Westphal nucleus. UCN1 immunoreactivity (ir) also occurs in the nucleus posteroventralis tegmenti, central gray, nucleus reticularis medius, nucleus motorius nervi facialis, and nucleus motorius nervi vagi. UCN1 occurs together with CRF in the nucleus motorius nervi trigemini, and in the magnocellular nucleus, which send a UCN1- and CRF-containing fiber tract to the median eminence. Strong UCN1-ir and CRF-ir were found in the external zone of the median eminence. From the internal zone of the median eminence, UCN1-ir fibers, but few CRF-ir fibers, were found to project to the pituitary neural lobe, where they form numerous neurohemal axon terminals. Ultrastructurally, two types of terminal containing UCN1-ir secretory granules were distinguished: type A contains large, moderately electron-dense, round secretory granules and type B is filled with smaller, strongly electron-dense, ellipsoid secretory granules. In vitro superfusion studies showed that UCN1 stimulated the release of alphaMSH from melanotrope cells in a dose-dependent manner. Our results support the hypothesis that in X. laevis, UCN1 released from neurohemal axon terminals in the pituitary neural lobe functions as a stimulatory neurohormone for alphaMSH release from melanotrope cells of the pituitary intermediate lobe.

Role of cortical filamentous actin in the melanotrope cell of Xenopus laevis.

In secretory cells filamentous actin (f-actin) is mostly present subjacent to the plasma membrane, referred to as cortical actin. While the function of cortical actin in the secretory processes has been extensively studied, little attention has been given to the role of actin in signal transduction and intracellular second messenger dynamics. Analysis with the fluorescent f-actin probe Alexa-phalloidin shows that Xenopus laevis pituitary melanotrope cells possess a thick cortical actin ring. This cell is a good model to study the possible function(s) of f-actin in signal transduction processes. Regulation of the release of alpha-MSH from this cell involves a convergence of various receptor mechanisms to regulate the activity of voltage-operated Ca2+ channels. We have considered three potential functions for the cortical actin ring in the signaling process of the melanotrope: (1) it functions as a barrier for access of secretory granules to the membrane for exocytosis, (2) it is involved in anchoring components of the Ca2+ signalling machinery of the cell, and/or (3) it helps to form a scaffold for components of the signal transduction machinery used by the various neurotransmitters and neuropeptides that regulate the activity of the cell. To test these possibilities we have examined the effect of the f-actin depolymerising toxin latrunculin B on Ca2+ signaling, signal transduction and alpha-MSH secretion in the melanotrope. We show that while the toxin is effective in disrupting the cortical actin ring, this treatment has no effect on either Ca2+ signaling or the signal transduction processes studied. The toxin does induce an increase in alpha-MSH release, indicating that the cortical actin ring acts as a barrier for secretory granule access to the membrane.