The effects of disruption of A kinase anchoring protein-protein kinase A association on protein kinase A signalling in neuroendocrine melanotroph cells of Xenopus laevis.

The secretory activity of melanotroph cells from Xenopus laevis is regulated by multiple neurotransmitters that act through adenylyl cyclase. Cyclic adenosine monophosphate (cAMP), acting on protein kinase A (PKA), stimulates the frequency of intracellular Ca(2+) oscillations and the secretory activity of the melanotroph cell. Anchoring of PKA near target proteins is essential for many PKA-regulated processes, and the family of A kinase anchoring proteins (AKAPs) is involved in the compartmentalisation of PKA type II (PKA II) regulatory subunits. In the present study, we determined to what degree cAMP signalling in Xenopus melanotrophs depends on compartmentalised PKA II. For this purpose, a membrane-permeable stearated form of Ht31 (St-Ht31), which dislodges PKA II from AKAP (thus disrupting PKA II signalling), was used. The effect of St-Ht31 on both secretion of radiolabelled peptides and intracellular Ca(2+) signalling by superfused Xenopus melanotrophs was assessed. St-Ht31 stimulated secretion but had no effect on Ca(2+) signalling. We conclude Xenopus melanotrophs possess a St-Ht31-sensitive PKA II that is associated with the exocytosis machinery and, furthermore, that Ca(2+) signalling is regulated by an AKAP-independent signalling system. Moreover, our results support a recent proposal that AKAP participates in regulating PKA activity independently from cAMP.

Calcium influx through voltage-operated calcium channels is required for proopiomelanocortin protein expression in Xenopus melanotropes.

Melanotrope cells of Xenopus laevis generate transitory increases in intracellular Ca(2+), known as Ca(2+) oscillations. These oscillations arise from the influx of Ca(2+) through voltage-operated Ca(2+) channels (VOCCs). Such oscillations are the driving force for secretion of a-melanophore-stimulating hormone (alpha-MSH) from the cell. The influx of Ca(2+) through VOCCs initiates the mobilization of intracellular Ca(2+) to generate a Ca(2+) wave. The function of the Ca(2+) wave in the melanotrope is unknown, but its presence in the nucleus suggests a role in the regulation of gene expression, perhaps that of proopiomelanocortin (POMC), the precursor protein for alpha-MSH. To determine the possible function of Ca(2+) waves in Xenopus melanotropes, we addressed whether functional VOCCs, which are an established requirement for both secretion and Ca(2+) wave initiation, are also required to maintain POMC gene expression.

Expression and characterization of the extracellular Ca(2+)-sensing receptor in melanotrope cells of Xenopus laevis.

The extracellular Ca(2+)-sensing receptor (CaR) is expressed in many different organs in various species, ranging from mammals to fish. In some of these organs, this G protein-coupled receptor is involved in the control of systemic Ca(2+) homeostasis, whereas in other organs its role is unclear (e.g. in the pituitary gland). We have characterized the CaR in the neuroendocrine melanotrope cell of the intermediate pituitary lobe of the South African clawed toad Xenopus laevis. First, the presence of CaR mRNA was demonstrated by RT-PCR and in situ hybridization. Then it was shown that activation of the CaR by an elevated extracellular Ca(2+) concentration and different CaR-activators, including L-phenylalanine and spermine, stimulates both Ca(2+) oscillations and secretion from the melanotrope. Furthermore, it was revealed that activation of the receptor stimulates Ca(2+) oscillations through opening of voltage-operated Ca(2+) channels in the plasma membrane of the melanotropes. Finally, it was shown that the CaR activator L-phenylalanine could induce the biosynthesis of proopiomelanocortin in the intermediate lobe. Thus, in this study it is demonstrated that the CaR is present and functional in a defined cell type of the pituitary gland, the amphibian melanotrope cell.