Expression of prolactin gene and secretion of prolactin by rat retinal capillary endothelial cells.

PURPOSE: Prolactin fragments inhibit blood vessel formation, whereas anti-prolactin antibodies induce angiogenesis in the cornea. Endothelial cells from brain capillaries and the umbilical vein produce prolactin, and this study was undertaken to determine whether retinal capillary endothelial cells could be a source for prolactin in the eye. METHODS: Primary cultures of rat retinal endothelial cells were investigated for the expression of prolactin mRNA by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis and by in situ hybridization. The prolactin protein was analyzed by immunocytochemistry, enzyme-linked immunoabsorbent assay, Western blot analysis, and the Nb2-cell bioassay. The effect of prolactin and the 16-kDa prolactin fragment on retinal endothelial cell proliferation was investigated, and the expression of the cloned prolactin receptor was analyzed by RT-PCR and Southern blot analysis. RESULTS: Retinal endothelial cells expressed prolactin mRNA and full-length 23-kDa prolactin. Prolactin was observed in the cytoplasm of cells and in their conditioned medium at levels 300 times those described in endothelial cells from other vessels and species. Exogenous 16-kDa prolactin inhibited rat retinal endothelial cell proliferation, whereas 23-kDa prolactin was inactive. No evidence was obtained for the expression of the cloned prolactin receptor in these cells, but the prolactin receptor was amplified in whole rat retina. CONCLUSIONS: Endothelial cells from the microcirculation of rat retina produce and release prolactin. That the cloned prolactin receptor was not expressed in these cells argues against direct autocrine effects of prolactin. Possible paracrine effects are suggested by the expression of the prolactin receptor in retinal tissue.

Potentiation of prolactin secretion following lactotrope escape from dopamine action. II. Phosphorylation of the alpha(1) subunit of L-type, voltage-dependent calcium channels.

Modulation of Ca(2+) channels has been shown to alter cellular functions. It can play an important role in the amplification of signals in the endocrine system, including the pleiotropically regulated pituitary lactotropes. Prolactin (PRL) secretion is tonically inhibited by dopamine (DA), the escape from which triggers acute episodes of hormone secretion. The magnitude of these episodes is explained by a potentiation of the PRL-releasing action of secretagogues such as thyrotropin-releasing hormone (TRH). While the mechanisms of this potentiation are not fully understood, it is known to be mimicked by the dihydropyridine, L-type Ca(2+) channel agonist Bay K 8644 and blocked by nifedipine and methoxyverapamil. The potentiation is also blocked by inhibitors of cyclic AMP-dependent protein kinase and protein kinase C. We recently described that the escape from tonic actions of DA results in increased macroscopic Ca(2+) currents in GH(4)C(1) lactotropic clonal cells transfected with a cDNA encoding the long form of the human D(2)-DA receptor. Here we show that the withdrawal from DA potentiates the PRL-releasing action of TRH in GH(4)C(1)/D(2)-DAR cells to the same extent as in enriched lactotropes in primary culture. In both experimental models a low density of dihydropyridine receptors was shown by (+)-[(3)H]PN200-110 binding. Photoaffinity labelling with the dihydropyridine [(3)H]azidopine revealed a protein consistent with the alpha(1) subunit of L-type Ca(2+) channels of 165-170 kDa. In both experimental models, the facilitation of TRH action triggered by the escape from DA was correlated with an enhanced rate of phosphorylation of this putative alpha(1) subunit, the nature of which was further supported by immunoprecipitation with selective antibodies directed against the alpha(1C) and alpha(1D) subunit of voltage-gated calcium channels. We propose that PKA- and PKC-dependent phosphorylation of the alpha(1) subunit of high voltage activated, L-type Ca(2+) channels is responsible for the facilitation of Ca(2+) currents in lactotropes, and hence for the potentiation of secretagogue-mediated PRL secretion. Thus, phosphorylation of Ca(2+) channels in endocrine cells may be a mechanism for the regulation of various functions including amplification of hormone secretion.

Immunoreactive prolactins of the neurohypophyseal system display actions characteristic of prolactin and 16K prolactin.

We have described the expression of the prolactin (PRL) gene and the occurrence of PRL-like immunoreactive proteins in the hypothalamic-neurophypophyseal system of the rat. Here, we investigated the nature of neurohypophyseal PRL-like antigens, by studying the biological activity of medium conditioned by incubated neurohypophyses in specific bioassays for PRL and for the 16 kDa N-terminal fragment of PRL (16K PRL). Neurohypophyseal conditioned medium (NHCM) obtained after incubating neurohyphyseal lobes (1 h at 37°C) was enriched with proteins of 14 kDa and 23 kDa, that crossreacted with PRL-and 16K PRL-directed antisera. The NHCM stimulated in a dose-dependent fashion the proliferation of Nb2-lymphoma PRL-dependent cells. This effect paralleled that of PRL and 16K PRL standards and was neutralized by different dilutions of both PRL-and 16K PRL-antisera. Also, the NHCM inhibited the proliferation of endothelial cells in culture, an antiangiogenic-effect exerted by 16K PRL. The antiangiogenic effect of the NHCM was parallel to that of 16K PRL standard and neutralized by 16K PRL antiserum in a dose-dependent fashion. These results indicate that NHCM contains proteins that share receptor activation properties as well as antigenic determinants with both PRL and 16K PRL.

Phosphoinositide hydrolysis in response to the withdrawal of dopamine inhibition in enriched lactotrophs in culture.

The formation of inositol phosphates and hydrolysis of polyphosphoinositides in response to DA addition or removal was studied in cultured anterior pituitary cells from E2-treated rats. The cells were incubated for 24 h with [3H]inositol to label the polyphosphoinositides either with or without DA (500 nM) in the medium. [3H]inositol phosphate and [3H]polyphosphoinositide levels were determined by anion-exchange chromatography after KOH deacylation of the latter. Acute (5 min) administration of DA to cells cultured without DA resulted in no change in the levels of [3H]inositol phosphates but a significant increase (50 +/- 10%) in the levels of [3H]polyphosphoinositides. Similarly, tonic exposure to DA for 40 h resulted in an increase (127 +/- 9%) of [3H]polyphosphoinositide levels. Acute withdrawal of DA from the medium for 5 min induced a 77 +/- 9% increase in the levels of [3H]inositol phosphate and a 27 +/- 3% decrease in [3H]polyphosphoinositide levels. These results suggest that the inositol phosphate messenger system of lactotrophs is negatively coupled to DA receptors. The efficiency of dopamine removal to activate this pathway is consistent with the kinetics of DA binding to its receptor and observations that DA removal is an important physiological signal for PRL release.