ABSTRACT
We have previously shown that the melanotrope population of the pituitary intermediate lobe of Rana ridibunda is composed of two subpopulations, of low (LD) and high density (HD), that show distinct ultrastructural features and display different synthetic and secretory rates. To investigate whether LD and HD melanotrope cells also differ in proopiomelanocortin (POMC) processing, we have analyzed the POMC-end products in single cells from both subpopulations by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The mass spectra revealed the presence of 8 POMC-derived peptides in HD and LD melanotrope cells, indicating a similar processing of the precursor in both subpopulations. However, the relative abundance of three POMC-end products (i.e. lys-gamma1-MSH, acetyl-alpha-MSH, and CLIP fragment) was higher in the HD subset. Moreover, two peptides with molecular weights of 1030 and 1818 Da, respectively, were detected that could not be assigned to any product deduced from the frog POMC sequence. The relative amount of the 1030 Da peptide was higher in LD melanotrope cells. Taken together, our results suggest that POMC processing is differentially regulated in the two melanotrope cell subsets.
Subject(s)
Pituitary Gland/chemistry , Pro-Opiomelanocortin/analysis , Animals , Male , Mass Spectrometry , Pituitary Gland/cytology , Radioimmunoassay , Rana ridibunda , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
The frog intermediate lobe consists of a single endocrine cell type, the melanotrope cells, which are under the tonic inhibitory control of dopamine. Separation of dispersed pars intermedia cells in a Percoll density gradient has revealed the existence of two melanotrope cell subpopulations, referred to as high-density (HD) and low-density (LD) cells. The aim of the present study was to investigate the effects of dopamine on each of these melanotrope cell subsets. Increasing doses of dopamine, ranging from 10(-9)-10(-6) M, inhibited the release of alpha-melanocyte-stimulating hormone (alpha-MSH) in LD (but not in HD) melanotrope cells. In addition, dopamine provoked a significant reduction of the rate of acetylation of alpha-MSH in LD cells but not in HD cells. Similarly, dopamine significantly decreased the accumulation of POMC messenger RNA in LD cells, whereas it did not affect POMC gene expression in the HD melanotrope subset. On the other hand, microfluorimetric studies revealed that dopamine induced a significant reduction of KCl-stimulated cytosolic free calcium concentration in both LD and HD cells. The present study provides additional evidence for functional heterogeneity of melanotrope cells in the frog pars intermedia. Because dopamine plays a pivotal role in the regulation of alpha-MSH secretion, these data suggest the involvement of cell heterogeneity in the physiological process of background color adaptation in amphibians.
Subject(s)
Dopamine/pharmacology , Pituitary Gland/drug effects , Pro-Opiomelanocortin/genetics , alpha-MSH/metabolism , Acetylation , Animals , Calcium/metabolism , Cells, Cultured , Centrifugation, Density Gradient , Chromatography, High Pressure Liquid , In Situ Hybridization , Male , Pituitary Gland/cytology , Pituitary Gland/metabolism , Pro-Opiomelanocortin/biosynthesis , Pro-Opiomelanocortin/metabolism , RNA, Messenger/metabolism , Radioimmunoassay , Rana ridibundaABSTRACT
Cell heterogeneity designates the phenomenon by which a particular cell type is composed of morphologically and physiologically distinct cell subpopulations. We have previously isolated two subsets of melanotrope cells in the intermediate lobe of the frog pituitary by means of a separation procedure based on a Percoll density gradient High density (HD) melanotrope cells were found to exhibit a more granulated cytoplasm and a lower secretory rate than low density (LD) cells. In the present study, we have investigated the biochemical and functional characteristics of each melanotrope cell subpopulation by using various approaches, including chromatographic analysis for the measurement of the proportion of acetylated alpha MSH, microfluorimetric measurement of the cytosolic free calcium concentration ([Ca2+]i) and in situ hybridization for quantification of POMC messenger RNA (mRNA). Under basal conditions, LD melanotrope cells showed higher secretory activity, acetylation rate, [Ca2+]i, and POMC mRNA content compared to HD cells. Incubation of the cells with 100 nM TRH for 2 h induced a more pronounced activation of alpha MSH secretion, [Ca2+]i mobilization, and POMC mRNA accumulation in LD than in HD melanotrope cells. Conversely, TRH increased the rate of acetylation of alpha MSH in HD cells, but did not affect acetylation in LD cells. Taken together, these results demonstrate that the frog intermediate lobe is composed of two subsets of endocrine cells with distinct biochemical and functional characteristics. The coexistence of two cell subpopulations in the frog pars intermedia is consistent with the idea of a cell secretory cycle, in which each melanotrope subset represents a specific state of cellular activity.
