Formation of new capillary blood vessels, termed angiogenesis, is essential for the growth and development of tissues and underlies a variety of diseases including tumor growth. Members of the prolactin hormonal family bind to endothelial cell receptors and have direct effects on cell proliferation, migration and tube formation. Because many angiogenic and antiangiogenic factors are produced by endothelial cells, we investigated whether endothelial cells expressed the prolactin gene. Here we show that bovine brain capillary endothelial cells (BBCEC) in culture express the full-length prolactin messenger RNA, in addition to a novel prolactin transcript, lacking the third exon of the gene. In addition cultures of BBCEC synthesize and secrete prolactin-like immunoreactive proteins with apparent molecular masses of 23, 21 and 14 kDa. The prolactin-like nature of these proteins in supported by the observation that Nb2-cells, a prolactin-responsive cell line, were stimulated to proliferate when co-cultured with endothelial cells and this stimulation was neutralized with prolactin-directed antibodies. Finally, consistent with a possible autocrine effect of endothelial-derived prolactins, polyclonal and monoclonal prolactin antibodies specifically inhibited basal and basis fibroblast growth-factor-stimulated growth of endothelial cells. Taken together, the present findings support the hypothesis of the prolactin gene being expressed in endothelial cells as proteins that could act in an autocrine fashion to regulate cell proliferation.

Purpose. Disruption of the anti-angiogenic environment of the retina leads to neovascular eye diseases, including retinopathy of prematurity (ROP). Prolactin (PRL), the hormone originally associated with milk secretion, is proteolytically processed to 16K-PRL, a fragment with potent antiangiogenic, proapoptotic effects. Whether 16K-PRL is produced in eyes of patients with ROP and promotes the regression of intraocular blood vessels associated with this disease was investigated. methods. PRL was quantified in the aqueous humor, subretinal fluid, and serum from patients with stage 5 ROP and in patients with non-neovascular eye disorders. Intraocular expression of PRL was evaluated by RT-PCR, in situ hybridization, and Western blot analysis. AntiPRL antibodies were injected intravitreously in neonatal rats, and apoptosis of hyaloid vessels determined by TUNEL and ELISA. results. PRL was elevated in ocular fluids and serum from ROP patients. There was no correlation between PRL in ocular fluids and its level in serum, whereas PRL in aqueous humor and subretinal fluid were significantly correlated. PRL mRNA was expressed in blood vessels and leukocytes within retrolental fibrovascular membranes of ROP patients, and these membranes contained a 16 kDa immunoreactive PRL. The 16K-PRL isoform was more concentrated in subretinal fluid than in serum and was generated from PRL by subretinal fluid proteases. Intravitreous injection of neutralizing antiPRL antibodies inhibited the apoptosis of hyaloid vessels in neonatal rats. conclusions. 16K-PRL derived from PRL internalized from the circulation or synthesized intraocularly can stimulate apoptosis-induced vascular regression and contribute to the development and progression of ROP.

About a decade ago, cell membranes from the electric organ of Torpedo and from the rat brain were transplanted to frog oocytes, which thus acquired functional Torpedo and rat neurotransmitter receptors. Nevertheless, the great potential that this method has for studying human diseases has remained virtually untapped. Here, we show that cell membranes from the postmortem brains of humans that suffered Alzheimer's disease can be microtransplanted to the plasma membrane of Xenopus oocytes. We show also that these postmortem membranes carry neurotransmitter receptors and voltage-operated channels that are still functional, even after they have been kept frozen for many years. This method provides a new and powerful approach to study directly the functional characteristics and structure of receptors, channels, and other membrane proteins of the Alzheimer's brain. This knowledge may help in understanding the basis of Alzheimer's disease and also help in developing new treatments.

Different models of rodent maternal separation (MS) have been used to investigate long-term neurobiological and behavioral changes, associated with early stress. However, few studies have involved the analysis of sex-related differences in central anxiety modulation. This study investigated whether MS during breastfeeding affected adult males and females in terms of anxiety and brain GABA-A receptor-alpha-subunit immunoreactivity. The brain areas analyzed were the amygdale (AM), hippocampus (HP), medial prefrontal cortex (mPFC), medial preoptic area (POA) and paraventricular nucleus (PVN). Rats were housed under a reversed light/dark cycle (lights off at 7∶00 h) with access to water and food ad libitum. Animals underwent MS twice daily during the dark cycle from postnatal day 1 to postnatal day 21. Behavior was tested when rats were 65–70 days old using the elevated plus maze and after brains were treated for immunohistochemistry. We found that separated females spent more time in the open arms and showed more head dipping behavior compared with controls. The separated males spent more time in the center of the maze and engaged in more stretching behavior than the controls. Immunohistochemistry showed that separated females had less immunostained cells in the HP, mPFC, PVN and POA, while separated males had fewer immunolabeled cells in the PFC, PVN and AM. These results could indicate that MS has gender-specific effects on anxiety behaviors and that these effects are likely related to developmental alterations involving GABA-A neurotransmission.

Members of the prolactin (PRL) hormonal family have direct effects on endothelial cell proliferation, migration and tube formation. Moreover, isoforms of PRL may function as autocrine regulators of endothelial cells. Bovine brain capillary endothelial cells (BBCEC) express the PRL gene, while anti-PRL antibodies inhibit BBCEC proliferation. Here, we show the expression of the PRL gene into various PRL isoforms in endothelial cells from the human umbilical vein. Reverse transcription-polymerase chain reaction of total RNA from human umbilical vein endothelial cells (HUVEC) detected the full-length PRL mRNA as well as a 100 bp smaller PRL transcript similar to the one previously reported in BBCEC. HUVEC were positive to PRL immunocytochemistry. In addition, various PRL immunoreactive proteins were detected in HUVEC extracts and HUVEC conditioned media by metabolic labelling immunoprecipitation analysis. These PRL immunorelated proteins had apparent molecular masses of 60, 23, 21, 16 and 14 kDa. In contrast to previous findings in BBCEC, HUVEC conditioned media contained very little PRL bioactivity as determined by the selective bioassay of Nb2 cell proliferation. Moreover, some polyclonal or monoclonal antibodies directed against PRL stimulated HUVEC proliferation, in contrast to the inhibitory effect seen in BBCEC. The present findings extend the previous observations about the expression of PRL gene in endothelial cells from bovine brain capillaries to human cells of the umbilical vein, implicating that endothelium from different types of vessels and species share the expression of PRL gene but may differ in the putative autocrine role of the PRL isoforms expressed.

Estrogens are recognized regulators of the expression of neurohypophyseal hormones and of anterior pituitary prolactin (PRL). Here we have investigated whether the levels of PRL mRNA and of 23 and 14 kDa PRL variants present in the hypothalamo-neurohypophyseal system change during the estrous cycle or in response to estrogen treatment. The reverse transcription polymerase chain reaction (RT-PCR) was performed to examine PRL mRNA expression in isolated paraventricular (PVN) and supraoptic (SON) hypothalamic nuclei. In both nuclei PRL mRNA levels appeared higher in cycling females than in male rats, with the highest level occurring at estrus. This increase may involve estrogen action, since estrogen administration to ovariectomized rats was associated with apparently higher PRL mRNA levels in both the PVN and SON. Expression of the PRL gene at these sites may occur via both transcriptional factor Pit-1-dependent and -independent mechanisms. RT-PCR detected the mRNA for Pit-1 in the PVN but only at estrus. The concentration of the 23 kDa immunoreactive PRL determined in the neurohypophysis was significantly higher during estrus and after estrogen treatment. However, no difference was detected in the levels of the neurohypophyseal 14 kDa PRL-like fragment along the estrous cycle nor after estrogen administration. This lack of parallelism between neurohypophyseal PRLs could relate to an estrogen-induced inhibition of the proteolysis of 23 kDa PRL at this site, since estrogen treatment reduced the activity of neurohypophyseal proteolytic enzymes able to cleave PRL. Altogether our results are consistent with estrogens having a stimulatory effect on PRL gene expression in the hypothalamo-neurohypophyseal system and a concomitant inhibitory action on PRL proteolysis at this site.