A reply to "Relevant factors in the eutrophication of the Uruguay River and the Río Negro".

A recent paper by Beretta-Blanco and Carrasco-Letelier (2021) claims that agricultural eutrophication is not one of the main causes for cyanobacterial blooms in rivers and artificial reservoirs. By combining rivers of markedly different hydrological characteristics e.g., presence/absence and number of dams, river discharge and geological setting, the study speculates about the role of nutrients for modulating phytoplankton chlorophyll-a. Here, we identified serious flaws, from erratic and inaccurate data manipulation. The study did not define how erroneous original dataset values were treated, how the variables below the detection/quantification limit were numerically introduced, lack of mandatory variables for river studies such as flow and rainfall, arbitrary removal of pH > 7.5 values (which were not outliers), and finally how extreme values of other environmental variables were included. In addition, we identified conceptual and procedural mistakes such as biased construction/evaluation of model prediction capability. The study trained the model using pooled data from a short restricted lotic section of the (large) Uruguay River and from both lotic and reservoir domains of the Negro River, but then tested predictability within the (small) Cuareim River. Besides these methodological considerations, the article shows misinterpretations of the statistical correlation of cause and effect neglecting basic limnological knowledge of the ecology of harmful algal blooms (HABs) and international research on land use effects on freshwater quality. The argument that pH is a predictor variable for HABs neglects overwhelming basic paradigms of carbon fluxes and change in pH because of primary productivity. As a result, the article introduces the notion that HABs formation are not related to agricultural land use and water residence time and generate a great risk for the management of surface waterbodies. This reply also emphasizes the need for good practices of open data management, especially for public databases in view of external reproducibility.

Experimental evidence on the effects of temperature and salinity in morphological traits of the Microcystis aeruginosa complex.

Microcystis aeruginosa complex (MAC) encompasses noxious colonial bloom forming cyanobacteria. MAC representatives bloom in eutrophic freshwater and brackish ecosystems with stagnant water, were temperature and salinity are the main variables modulating their distribution, biomass and toxicity. Cell abundance and biovolume of MAC colonies define regulatory standards for public health. These variables depend upon colony size that in turn changes with environmental conditions. Here, we conducted two series of experiments to evaluate the response of MAC colonies morphological traits (length, volume, mucilage and number of cells) to temperature and salinity. In two series of experiments in the laboratory, we exposed natural MAC communities to three different temperatures (10, 21 and 30 °C) and four salinity levels (0, 5, 10 and 25 ppt) typically found in estuaries. We found that average colony length, volume and mucilage thickness did not change with temperature, but the cell-free space inside the colonies was smaller at the highest evaluated temperature (30 °C). Salinity fostered an increase in colony length, volume and mucilage thickness, while cell-free space diminished, resulting in higher cell density. The number of cells per colony was significantly related to colony size (length and volume) and both, temperature and salinity, affected the parameters of the relationships. Based on present results we propose statistical models to predict cell number per colony based on length and volume and accounting for the effect of salinity and temperature on these traits. This is applicable to ecological studies and to the monitoring of estuarine aquatic environments, by means of a fast and more accurate estimation of cell numbers to define MAC toxic populations early warning systems. A protocol is suggested for its application while the analysis of the interaction of temperature and salinity, as well as the variability in natural environments are objectives for future researches.

Impact of nutritional stress on the honeybee colony health.

Honeybees Apis mellifera are important pollinators of wild plants and commercial crops. For more than a decade, high percentages of honeybee colony losses have been reported worldwide. Nutritional stress due to habitat depletion, infection by different pests and pathogens and pesticide exposure has been proposed as the major causes. In this study we analyzed how nutritional stress affects colony strength and health. Two groups of colonies were set in a Eucalyptus grandis plantation at the beginning of the flowering period (autumn), replicating a natural scenario with a nutritionally poor food source. While both groups of colonies had access to the pollen available in this plantation, one was supplemented with a polyfloral pollen patty during the entire flowering period. In the short-term, colonies under nutritional stress (which consumed mainly E. grandis pollen) showed higher infection level with Nosema spp. and lower brood and adult bee population, compared to supplemented colonies. On the other hand, these supplemented colonies showed higher infection level with RNA viruses although infection levels were low compared to countries were viral infections have negative impacts. Nutritional stress also had long-term colony effects, because bee population did not recover in spring, as in supplemented colonies did. In conclusion, nutritional stress and Nosema spp. infection had a severe impact on colony strength with consequences in both short and long-term.

Improved biovolume estimation of Microcystis aeruginosa colonies: A statistical approach.

The Microcystis aeruginosa complex (MAC) clusters many of the most common freshwater and brackish bloom-forming cyanobacteria. In monitoring protocols, biovolume estimation is a common approach to determine MAC colonies biomass and useful for prediction purposes. Biovolume (µm3 mL-1) is calculated multiplying organism abundance (orgL-1) by colonial volume (µm3org-1). Colonial volume is estimated based on geometric shapes and requires accurate measurements of dimensions using optical microscopy. A trade-off between easy-to-measure but low-accuracy simple shapes (e.g. sphere) and time costly but high-accuracy complex shapes (e.g. ellipsoid) volume estimation is posed. Overestimations effects in ecological studies and management decisions associated to harmful blooms are significant due to the large sizes of MAC colonies. In this work, we aimed to increase the precision of MAC biovolume estimations by developing a statistical model based on two easy-to-measure dimensions. We analyzed field data from a wide environmental gradient (800 km) spanning freshwater to estuarine and seawater. We measured length, width and depth from ca. 5700 colonies under an inverted microscope and estimated colonial volume using three different recommended geometrical shapes (sphere, prolate spheroid and ellipsoid). Because of the non-spherical shape of MAC the ellipsoid resulted in the most accurate approximation, whereas the sphere overestimated colonial volume (3-80) especially for large colonies (MLD higher than 300 µm). Ellipsoid requires measuring three dimensions and is time-consuming. Therefore, we constructed different statistical models to predict organisms depth based on length and width. Splitting the data into training (2/3) and test (1/3) sets, all models resulted in low training (1.41-1.44%) and testing average error (1.3-2.0%). The models were also evaluated using three other independent datasets. The multiple linear model was finally selected to calculate MAC volume as an ellipsoid based on length and width. This work contributes to achieve a better estimation of MAC volume applicable to monitoring programs as well as to ecological research.

16K prolactin induces NF-kappaB activation in pulmonary fibroblasts.

The amino-terminal 16 kDa fragment of prolactin (16K PRL) promotes the expression of the inducible isoform of nitric oxide synthase (iNOS) accompanied by the production of nitric oxide (NO) by rat pulmonary fibroblasts. The present study was designed to elucidate whether the mechanism by which 16K PRL promotes iNOS expression involves the activation of nuclear factor-kappa B (NF-kappaB), a key transcription factor for iNOS induction. 16K PRL stimulated DNA-binding activity of NF-kappaB in pulmonary fibroblasts as demonstrated by gel shift assays. Likewise, fluorescence immunocytochemistry showed that 16K PRL promotes nuclear translocation of the p65 subunit of NF-kappaB. Finally, treatment with 16K PRL induced the degradation of the NF-kappaB inhibitor kappaB-beta (IkappaB-beta), and such degradation was prevented by blocking IkappaB-beta phosphorylation. Altogether, these results show that 16K PRL activates NF-kappaB nuclear translocation via the phosphorylation and degradation of IkappaB-beta. These findings are consistent with NF-kappaB being part of the signal transduction pathway activated by 16K PRL to induce iNOS expression.

Roles of prolactin and related members of the prolactin/growth hormone/placental lactogen family in angiogenesis.

Prolactin, growth hormone and placental lactogen are members of a family of polypeptide hormones which share structural similarities and biological activities. Numerous functions have been attributed to these hormones, among which stand out their recently discovered effects on angiogenesis, the process by which new blood vessels are formed from the pre-existing microvasculature. Prolactin, growth hormone and placental lactogen, along with two non-classical members of the family, proliferin and proliferin-related protein, can act both as circulating hormones and as paracrine/autocrine factors to either stimulate or inhibit various stages of the formation and remodeling of new blood vessels, including endothelial cell proliferation, migration, protease production and apoptosis. Such opposing actions can reside in similar but independent molecules, as is the case of proliferin and proliferin-related protein, which stimulate and inhibit angiogenesis respectively. The potential to exert opposing effects on angiogenesis can also reside within the same molecule as the parent protein can promote angiogenesis (i.e. prolactin, growth hormone and placental lactogen), but after proteolytic processing the resulting peptide fragment acquires anti-angiogenic properties (i.e. 16 kDa prolactin, 16 kDa growth hormone and 16 kDa placental lactogen). The unique properties of the peptide fragments versus the full-length molecules, the regulation of the protease responsible for specific protein cleavage, the selective expression of specific receptors and their associated signal transduction pathways are issues that are being investigated to further establish the precise contribution of these hormones to angiogenesis under both physiological and pathological situations. In this review article, we summarize the known and speculative issues underlying the effects of the prolactin, growth hormone and placental lactogen family of proteins on angiogenesis, and address important remaining enigmas in this field of research.

Regulation of gonadotropin-releasing hormone secretion: insights from GT1 immortal GnRH neurons.

The study of the mammalian GnRH system has been greatly advanced by the development of immortalized cell lines. Of particular relevance are the so-called GT1 cells. Not only do they exhibit many of the known physiologic characteristics of GnRH neurons in situ, but in approximately one decade have yielded new insights regarding the intrinsic physiology of individual cells and networks of GnRH neurons, as well as the nature of central and peripheral signals that directly modulate their function. For instance, valuable information has been generated concerning intrinsic properties of the system such as the inherent pulsatile pattern of secretion displayed by networks of GT1 cells. Concepts regarding feedback regulation and autocrine feedback of GnRH neurons have been dramatically expanded. Likewise, the nature of the receptors and of the proximal and distal signal transduction mechanisms involved in the actions of multiple afferent signals has been identified. Understanding this neuronal system allows a better comprehension of the hypothalamic-pituitary-gonadal axis and of the regulatory influences that ultimately control reproductive competence.

GABA inhibition of immortalized gonadotropin-releasing hormone neuronal excitability involves GABA(A) receptors negatively coupled to cyclic adenosine monophosphate formation.

Gamma-aminobutyric acid (GABA) has been implicated in the regulation of reproduction, particularly in the developmental modulation of gonadotropin-releasing hormone (GnRH) secretion. GnRH neurons are innervated by GABA-containing processes, and the administration of GABA stimulates and inhibits GnRH secretion in vivo and in vitro. We have previously shown that GABA can exert both of these actions in sequence, by acting directly on immortalized GnRH neurons. While the stimulation is the result of a GABA(A) receptor-mediated depolarization of the plasma membrane, the mechanism involved in the delayed inhibition is the subject of the present investigation. GABA (1 nM-10 microM) decreased the intracellular concentration of cyclic adenosine monophosphate (cAMP) in a dose- and time-dependent fashion. This effect was blocked by bicuculline and mimicked by muscimol but not by baclofen. To analyze the effect of GABA on cellular excitability, we used fura-2 loaded GT1-7 cells. Activation of voltage-sensitive calcium channels by high K+-induced depolarization (35 mM) increased [Ca2+]i. GABA (10 microM) and muscimol (10 microM) reduced the amplitude of K+-induced [Ca2+]i transients. This inhibition was blocked by forskolin (20 microM) or 8-Br-cAMP (1 mM). Altogether, these results show that GABA(A) receptors mediate a sustained inhibitory effect of GABA on GnRH neurons, and suggest the involvement of the cAMP pathway decreasing cellular excitability.

Molecular heterogeneity of prolactin in the plasma of patients with systemic lupus erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) has been associated with high levels of prolactin in the circulation of some patients. Although prolactin stimulates immune responses, the relationship between hyperprolactinemia and the pathophysiology of SLE remains controversial. This study was undertaken to investigate whether circulating bioactive prolactin isoforms are associated with the activity of SLE. METHODS: The molecular heterogeneity of prolactin was studied in the plasma of patients with active and inactive SLE and in healthy volunteers by radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Nb2-cell bioassay, and immunoprecipitation-Western blots. The specificity of the bioassay determinations was assessed by neutralization of growth-promoting effects with antiserum to human prolactin. RESULTS: Significantly higher prolactin levels were detected by bioassay and by ELISA than by RIA in both subsets of SLE patients and in normal individuals. Plasma prolactin levels in the SLE patients were significantly greater than those in the normal controls when measured by ELISA, but not by RIA or bioassay. The bioassay:ELISA and bioassay:RIA ratios were similar between SLE patients and controls, suggesting that prolactin biopotency was not altered with the disease, and none of the 3 assays detected a difference in prolactin levels between patients with active SLE and those with inactive SLE. However, the prolactin detected in plasma was associated with immunoreactive proteins of 130 kd and 23 kd, and the concentration of the 130-kd prolactin-like species was 10-fold higher in inactive SLE versus active SLE patients. CONCLUSION: Discrepancies among assays substantiate the molecular heterogeneity of circulating prolactin. The prolactin isotype that is found in association with inactive SLE could be of potential use as a marker for the inactive form of the disease and as an index for the efficacy of treatment.

Human umbilical vein endothelial cells express multiple prolactin isoforms.

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.

Proteolytic cleavage confers nitric oxide synthase inducing activity upon prolactin.

Prolactin (PRL), originally associated with milk secretion, is now known to possess a wide variety of biological actions and diverse sites of production beyond the pituitary. Proteolytic cleavage is a common post-translational modification that can either activate precursor proteins or confer upon the peptide fragment unique biological actions not exerted by the parent molecule. Recent studies have demonstrated that the 16-kDa N-terminal proteolytic cleavage product of PRL (16K-PRL) acts as a potent inhibitor of angiogenesis. Despite previous demonstrations of 16K-PRL production in vivo, biological functions beyond its antiangiogenic actions remain unknown. Here we show that 16K-PRL, but not full-length PRL, acts to promote the expression of the inducible isoform of nitric oxide synthase (iNOS) and nitric oxide (*NO) production by pulmonary fibroblasts and alveolar type II cells with potency comparable with the proinflammatory cytokines interleukin-1beta, interferon gamma, and tumor necrosis factor alpha. The differential effect of 16K-PRL versus PRL occurs through a receptor distinct from known PRL receptors. Additionally, pulmonary fibroblasts express the PRL gene and endogenously produce 16K-PRL, suggesting that this pathway may serve both autocrine and paracrine roles in the regulation of *NO production. These results reveal that proteolytic cleavage of PRL confers upon this classical hormone potent iNOS inducing activity, suggesting its role in inflammatory/immune processes.

Male reproductive condition is the limiting factor of efficiency in the male effect during seasonal anestrus in female goats.

Two experiments were conducted to determine whether the failure of males to induce sexual activity in goats during seasonal anestrus is due to unresponsiveness of females to male stimulus or insufficient stimulation from males. In the first study, one group of males (sexually inactive, SI; n = 4) was kept under natural photoperiod while the other (sexually active, SA; n = 4) was subjected to 2.5 mo of long days (16L:8D) and received 2 s.c. implants of melatonin. Two mo later, 2 different flocks of anovulatory goats previously separated from bucks were exposed to either SI (n = 34) or SA (n = 40) bucks. Progesterone assays and estrous behavior were used to determine ovarian and behavioral responses of the females to teasing. Of the goats exposed to SI males, only 2 ovulated, and none showed estrous behavior during the 35 days of the study. In contrast, all females (40 of 40) in contact with SA males ovulated and showed at least one estrous behavior during the first 11 days following male introduction (P < 0.001). Overall, 38 of 40 females stimulated with SA bucks were diagnosed pregnant at Day 35, according to progesterone assay (versus 0 in SI-treated group: P < 0.001). To control for a possible difference of responsiveness between flocks, the experiment was repeated 1 yr later using a single flock of goats divided into 2 groups. Again, over the first 14 days, 1 of 33 goats showed estrous behavior in the SI-treated group versus 27 of 33 in the SA-treated group (P < 0.001). Therefore, treating bucks with long days and melatonin increased their teasing capacity to induce sexual activity in females during anestrus. These results indicate that the absence of response to teasing at this time of the year is not due to female unresponsiveness, but to insufficient stimulation from the male.

Inhibition of rat corneal angiogenesis by 16-kDa prolactin and by endogenous prolactin-like molecules.

PURPOSE: The cornea is an avascular organ, where induction of new blood vessels involves the turn-on of proangiogenic factors and/or the turn-off of antiangiogenic regulators. Prolactin (PRL) fragments of 14 kDa and 16 kDa bind to endothelial cell receptors and inhibit angiogenesis. This study was designed to determine whether antiangiogenic PRL-like molecules are involved in cornea avascularity. METHODS: Sixteen-kDa PRL and basic fibroblast growth factor (bFGF) or anti-PRL antibodies were placed into rat cornea micropockets and neovascularization evaluated by the optical density associated with capillaries stained by the peroxidase reaction and by the number of vessels growing into the implants. Prolactin receptors in corneal epithelium were investigated by immunocytochemistry. RESULTS: bFGF induced a dose-dependent stimulation of corneal neovascularization. This effect was inhibited by coadministration of 16-kDa PRL, as indicated by a 65% reduction in vessel density and a 50% decrement in the incidence of angiogenic responses. Corneal angiogenic reactions of different intensities were induced by implantation of polyclonal and monoclonal anti-PRL antibodies. Corneal epithelial cells were labeled by several anti-PRL receptor monoclonal antibodies. CONCLUSIONS: These findings show that exogenous 16-kDa PRL inhibits bFGF-induced corneal neovascularization and suggest that PRL-like molecules with antiangiogenic actions function in the cornea. PRL receptors in the corneal epithelium may imply that PRL in the cornea derives from lacrimal PRL internalized through an intracellular pathway. These observations are consistent with the notion that members of the PRL family are potential regulators of corneal angiogenesis.

Potentiation of prolactin secretion following lactotrope escape from dopamine action. I. Dopamine withdrawal augments l-type calcium current.

Hypothalamic dopamine (DA) tonically inhibits prolactin (PRL) release from the anterior pituitary gland. Transient escapes from this DA tone elicit a pronounced potentiation of the PRL-releasing action of secretagogues such as thyrotropin-releasing hormone (TRH). Previous evidence has suggested that modulation of Ca(2+) channels can be involved in this potentiation. With a lactotropic cell line (GH(4)C(1)) expressing human D(2)-DA receptors, we tested the hypothesis that a brief escape from the tonic inhibitory action of DA triggers a facilitation of Ca(2+) influx through Ca(2+) channels. We initially found that in these cells, DA effectively and reversibly inhibited PRL secretion, and reversibly enhanced an inwardly rectifying K(+) current. The effects of DA administration and withdrawal on Ca(2+) currents were examined using the patch-clamp technique in the whole-cell configuration and Ba(2+) as a divalent charge carrier through Ca(2+) channels. Macroscopic Ba(2+) currents were significantly decreased by short term (1-10 min) applications of DA (500 nM), which further declined following 24 h of constant exposure to DA. After DA removal, a biphasic facilitation of the density of Ba(2+) currents was observed. An initial 2-fold enhancement of conductance was detected between 10 and 40 min, followed by a second facilitation of the same magnitude observed 24 h after DA withdrawal. The present results directly demonstrate that dissociation of DA from D(2)-receptors expressed in GH(4)C(1) lactotrope cells causes an increase of high-voltage-activated Ca(2+) channel function, which may play an important role in the cross-talking amplification of endocrine cascades such as that involved in the TRH-induced PRL-release potentiating action of DA withdrawal.

Changes in the expression of neurohypophyseal prolactins during the estrous cycle and after estrogen treatment.

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.

Functional uncoupling between intracellular calcium dynamics and secretion in the alphaT3-1 gonadotropic cell line.

Gonadotropin releasing hormone (GnRH) stimulates both transcription and secretion of the alpha subunit of the gonadotropins in a Ca2+-dependent fashion. In this study, we examined the role of Ca2+ as the signal coupling agonist occupancy of GnRH receptors to hormone secretion using the gonadotropic cell line alphaT3-1. Treatment of alphaT3-1 cells for 60 min with GnRH (0.1-100 nM), veratridine (50 microM) or high K+ (56 mM) was completely ineffective in stimulating secretion. The lack of effect occurred in spite of a robust, specific, and dose-dependent biphasic [Ca2+]i response consisting of a rapid peak sensitive to thapsigargin (200 nM) followed by a smaller plateau sensitive to the extracellular application of EGTA (5 mM). On the other hand, treatment of alphaT3-1 cells with the Ca2+ ionophore ionomycin resulted in a significant dose-dependent stimulation of secretion and [Ca2+]i responses comparable to those elicited by GnRH. Binding assays revealed the presence of Ins(1,4,5)P3 receptors (Kd = 3.2 nM, Bmax = 50.5 fmol/mg protein) but not ryanodine receptors in alphaT3-1 cell membranes. Together, these results show a functional uncoupling between the [Ca2+]i response and secretion in this cell line, suggesting that the increase in [Ca2+]i triggered by GnRH and depolarization may be necessary but not sufficient to stimulate exocytosis.

Expression of prolactin mRNA and of prolactin-like proteins in endothelial cells: evidence for autocrine effects.

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.

Immunoreactive prolactin forms colocalize with vasopressin in neurons of the hypothalamic paraventricular and supraoptic nuclei.

The prolactin (PRL) gene is expressed in the hypothalamo-neurohypophyseal system as revealed by the detection of the PRL mRNA and of PRL-like immunoreactive and biologically active proteins in hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and in the neurohypophysis. We have investigated the distribution of cells containing PRL-like molecules in the PVN and SON by immunocytochemistry with a specific antiserum directed against the 16-kD N-terminal fragment of PRL. PRL-positive cells were found to be concentrated throughout the ventral SON and in the lateroposterior region of the PVN. The cellular distribution of PRL-immunoreactive cells resembled more closely that of vasopressin (VP) than that of oxytocin magnocellular neurons. Moreover, double immunofluorescence labelling, followed by confocal microscopy, indicated the coexistence of PRL- and VP-related antigens within the same neurons of the PVN and SON. Pre-embedding immunoperoxidase on the ultrastructural level showed a PRL-like product in granular-type particles within the neural soma and projections in the SON and PVN. These findings are consistent with the expression and secretion of PRL-like molecules by vasopressinergic neurons of the hypothalamo-neurohypophyseal system.

Differential effects of basic fibroblast growth factor, epidermal growth factor, transforming growth factor-alpha, and insulin-like growth factor-I on a hypothalamic gonadotropin-releasing hormone neuronal cell line.

Recent studies in several neuronal lineages suggest that extrinsic factors such as polypeptide growth factors regulate various stages of neuronal development, from initial commitment of multipotent progenitors to induction of specific gene expression that is characteristic of terminal neuronal differentiation. In the present study, immortalized hypothalamic neurons of the GT1-1 lineage were used to analyze proliferative, as well as morphological and molecular differentiation actions of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and insulin-like growth factor-I (IGF-I). These effects were compared with those induced by specific activators of protein kinase A and C pathways, which potently inhibited cell proliferation and gonadotropin-releasing hormone (GnRH) gene expression, but stimulated morphological neuronal maturation as determined by the length and number of neurite outgrowth. bFGF exerted a broad spectrum of stimulatory effects, increasing the rate of proliferation measured both by the incorporation of 3H-thymidine and by cell number, and parameters of terminal differentiation, such as neurite outgrowth and induction of gene expression. bFGF stimulated the expression of the hybrid transgene-containing portions of the rat GnRH promoter. In contrast, EGF, TGF-alpha, and IGF-I inhibited cell proliferation, while having subtle effects on neurite outgrowth. Thus, GT1-1 cells appear to be differentially responsive to distinct neurotrophic factors, providing a model for studying the specific effects of neurotrophic factors on functional differentiation, migration, and connectivity of hypothalamic neurons.

The catecholaminergic stimulation of gonadotropin-releasing hormone release by GT1-1 cells does not involve phosphoinositide hydrolysis.

Gonadotropin-releasing hormone (GnRH) secretion is modulated by a large number of neuromediators, among which catecholamines play a central role. Previous results have shown that both dopamine (DA) and norepinephrine (NE) stimulate GnRH secretion in GT1 neuronal cell lines. These stimulatory effects appear to involve D1-dopaminergic and beta 1-adrenergic receptors positively coupled to adenylate cyclase. However, in spite of a similar efficacy of these catecholamines to stimulate GnRH secretion, DA is two-fold more efficacious than NE to stimulate the formation of cyclic AMP. This rises the possibility that other signaling pathways and other receptor subtypes could be involved in the catecholaminergic stimulation of GnRH release. Since the signaling pathway triggered by phosphoinositide hydrolysis is a potent stimulator of GnRH secretion and appears to mediate the releasing actions of neuromediators such as histamine and endothelin, we investigated if this signaling pathway was also involved in the catecholaminergic stimulation of GnRH release in GT1 cells. Both DA and NE stimulated inositol phosphates production in GT1-1 cells with a very low potency and long latency with respect to GnRH secretion. Inositol phosphates production was stimulated by DA and NE only at a concentration of 100 microM, i.e. two to three orders of magnitude higher than the effective concentrations to maximally stimulate GnRH secretion. The effects of both catecholamines do not appear to be secondary to the stimulation of cyclic AMP production, since treatment of GT1-1 cells with forskolin did not affect inositol phosphates production. The effects of DA and NE on inositol phosphates production were blocked by specific antagonists such as SCH-23390, spiroperidol and phentolamine. However, specific dopaminergic agonists such as SKF-38393 and bromocriptine, or adrenergic agonists such as clonidine, methoxamine and isoproterenol were not capable of stimulating inositol phosphates production. Thus, due to the low potency and apparent non-specificity of these effects, we conclude that inositol phosphates production is not involved in the catecholaminergic stimulation of GnRH release.