High Prolactin Excretion in Patients with Diabetes Mellitus and Impaired Renal Function.

BACKGROUND: The metabolic clearance of prolactin (PRL) is partially executed by the kidney. Here, we investigate the urine excretion of PRL in patients with Diabetes Mellitus and renal impairment. METHODS: Serum and urine samples were collected from male, mestizo patients in central Mexico employing a cross-sectional study design. Ninety-eight individuals had either no diabetes and normal renal function (control), diabetes and normal renal function, or diabetes with impaired renal function. PRL was determined by a chemiluminescent immunometric assay; protein, albumin, and creatinine were evaluated using quantitative colorimetric assays. The results were analyzed using ANOVA-testing. RESULTS: Patients with Diabetes Mellitus and renal impairment had significantly higher urine PRL levels than patients with Diabetes Mellitus and normal renal function and control patients. Higher urine PRL levels were associated with lower glomerular filtration rates, higher serum creatinine, and higher urinary albumin-to-creatinine ratios (UACR). Urine PRL levels correlated positively with UACR. Serum PRL levels were similar among groups. CONCLUSIONS: Patients with Diabetes Mellitus and impaired renal function demonstrate a high urinary PRL excretion. Urinary PRL excretion in the context of proteinuria could contribute to PRL dysregulation in renal impairment.

Surveillance Web System and Mouthwash-Saliva qPCR for Labor Ambulatory SARS-CoV-2 Detection and Prevention.

This study provides a safe and low-cost in-house protocol for RT-qPCR-based detection of SARS-CoV-2 using mouthwash-saliva self-collected specimens to achieve clinical and epidemiological surveillance in a real-time web environment applied to ambulatory populations. The in-house protocol comprises a mouthwash-saliva self-collected specimen, heat virus inactivation, and primers to target virus N-gene region and the human RPP30-gene. Aligning with 209 SARS-CoV-2 sequences confirmed specificity including the Alpha variant from the UK. Development, validation, and statistical comparison with official nasopharyngeal swabbing RT-qPCR test were conducted with 115 specimens of ambulatory volunteers. A web-mobile application platform was developed to integrate a real-time epidemiological and clinical core baseline database with mouthwash-saliva RT-qPCR testing. Nine built-in algorithms were generated for decision-making on testing, confining, monitoring, and self-reports to family, social, and work environments. Epidemiological and clinical follow-up and SARS-CoV-2 testing generated a database of 37,351 entries allowing individual decision-making for prevention. Mouthwash-saliva had higher sensitivity than nasopharyngeal swabbing in detecting asymptomatic and mild symptomatic cases with 720 viral copy number (VCN)/mL as the detection limit (Ct = 37.6). Cycling threshold and viral loading were marginally different (p = 0.057) between asymptomatic (35 Ct ± 2.8; 21,767.7 VCN/mL, range 720-77,278) and symptomatic (31.3 Ct ± 4.5; 747,294.3 VCN/mL, range 1433.6-3.08 × 106). We provided proof-of-concept evidence of effective surveillance to target asymptomatic and moderate symptomatic ambulatory individuals based on integrating a bio-safety level II laboratory, self-collected, low-risk, low-cost detection protocol, and a real-time digital monitoring system. Mouthwash-saliva was effective for SARS-CoV-2 sampling for the first time at the community level.

17-Beta-estradiol directly regulates the expression of adrenergic receptors and kisspeptin/GPR54 system in GT1-7 GnRH neurons.

Estradiol plays a critical role in the feedback regulation of reproduction, in part by modulating the neurosecretory activity of gonadotropin-releasing hormone (GnRH) neurons. While indirect effects of estradiol on GnRH neurons have been clearly demonstrated, direct actions are still controversial. In the current study, we examined direct effects of 17beta-estradiol upon the expression of receptors for afferent signals at the level of the GnRH neuron, using immortalized GT1-7 cells. Using RT-PCR, we confirmed the expression of mRNA for the adrenergic receptors (AR) alpha(1)A-, alpha(1)B-, alpha(1)D-, alpha(2)A-, alpha(2)C-, and beta(1)-AR, and showed for the first time that mRNAs for alpha(2)B-, beta(2)- and beta(3)-AR, for kisspeptin and its receptor GPR54 and for the novel estrogenic receptor GPR30 are expressed in GT1-7 cells. After treatment with 10 nM 17beta-estradiol, alpha(1)B-AR mRNA was significantly increased (14-fold) after 6 h as determined by real-time PCR, while alpha(1)B- and alpha(1)D-AR mRNA were significantly increased (19- and 23-fold, respectively) after 24 h. The expression of KiSS-1 and GPR54 mRNAs were also significantly increased (8- and 6-fold, respectively) after 24 h treatment of GT1-7 cells with estradiol. GPR30 mRNA expression was not affected by estradiol. Our data also showed that kisspeptin-10 (1-10 nM) can significantly stimulate GnRH release and GnRH mRNA expression in GT1-7 cells. These results suggest that the complex physiologic effects of estradiol on the function of the reproductive axis could be mediated partly through direct modulation of the expression of receptors for afferent signals in GnRH neurons.

Prolactins are natural inhibitors of angiogenesis in the retina.

PURPOSE: Disruption of the normally antiangiogenic environment of the retina leads to aberrant angiogenesis, the major cause of vision loss throughout the world. Prolactin (PRL), the hormone originally associated with milk production, can be proteolytically processed to 16K-PRL, a 16 kDa N-terminal PRL fragment with potent antiangiogenic and vasoconstrictive actions. This study was conducted to determine whether 16K-PRL is found naturally in the retina and plays a role in angiogenesis and vasodilation. METHODS: Expression of PRL mRNA in rat retina was determined by RT-PCR and in situ hybridization. Western blot was used to examine the expression of PRL and derived fragments in retinal homogenates. The role of PRL and 16K-PRL in the retina was studied by intravitreal injection of either antibodies against PRL or small interfering RNAs (siRNA), to suppress expression of retinal PRL mRNA. RESULTS: Rat retina expressed PRL mRNA in the outer nuclear, outer plexiform, inner nuclear, and ganglion cell layers. Both full-length PRL and N-terminal 16K-PRL were detected in retinal homogenates by polyclonal and monoclonal antibodies. The intravitreal injection of antibodies able to neutralize the actions of 16K-PRL increased the number of retinal blood vessels and capillary area by threefold. Furthermore, siRNA-mediated inhibition of PRL mRNA increased retinal neovascularization threefold and resulted in a significant increase in vasodilation. CONCLUSIONS: These results demonstrate that PRL is synthesized and cleaved to antiangiogenic 16K-PRL by retinal tissue and that these molecules play a key role in preventing angiogenesis in the healthy retina.

Cytokine induction of prolactin receptors mediates prolactin inhibition of nitric oxide synthesis in pulmonary fibroblasts.

Prolactin (PRL) has been implicated as a modulator of immune function, and some of its actions may be linked to NO synthesis. Because NO acts as a mediator of inflammation, we speculated that an inflammatory milieu could unmask pathways by which PRL could affect NO synthesis. Here, we show that pro-inflammatory cytokines induce the expression of PRL receptors in pulmonary fibroblasts, allowing PRL to inhibit cytokine-induced NO production and the expression of the inducible nitric oxide synthase (iNOS). Inhibition of iNOS expression by PRL correlates with the phosphorylation of STAT-5b (signal transducer and activator of transcription 5b) and the suppression of expression of IRF-1 (interferon regulatory factor 1), a transcription factor for iNOS. These results reveal previously unrecognized mechanisms by which PRL and PRL receptors may play significant modulatory roles during immune-inflammatory processes.

Prolactin in eyes of patients with retinopathy of prematurity: implications for vascular regression.

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.

Treatment of major depression in individuals with spinal cord injury.

OBJECTIVE: This study evaluated the effects of treating major depression in individuals with spinal cord injury (SCI), focusing on the degree of improvement and correlated changes that could be expected in 6 months of treatment. DESIGN: A pretreatment-posttreatment design was used. Random assignment to a nontreatment group could not be implemented ethically. Therefore, this study compared participants who declined treatment to persons who accepted treatment over a 24-month period. SETTING AND PARTICIPANTS: Participants were outpatients of a large urban rehabilitation center in southern California. Twenty-eight participants who accepted treatment were assigned to a treatment group; 15 individuals who declined treatment were assigned to a nontreatment group. The age of the participants ranged from 20 to 74 years. Varying levels of SCI dysfunction were represented. INTERVENTIONS: A 6-month combination of psychotherapy and antidepressant medication. OUTCOME MEASURES: A depression inventory, a community activities checklist, and a life satisfaction scale. RESULTS: A significant (P < 0.001) 57% reduction in depressive symptoms occurred in the treatment group, whereas there was no significant change in the nontreatment group. At the end of 6 months, 30% of participants had no depression, 42% had minor depression, and 29% still had major depression, but to a lesser degree. Community activities increased significantly over the treatment period, as did life satisfaction. CONCLUSION: The results suggest that depression is treatable in this population, although 6 months may not be sufficient to reach maximum benefit in all cases. This study further identified obstacles that limited the ability to randomize participants into treatment arms and made it difficult to deliver services to all those in need. Complications related to SCI, such as difficulties in transportation, likely restrict the ability to implement needed services to many individuals with SCI.

Expression and cellular localization of prolactin and the prolactin receptor in mammalian retina.

Prolactin (PRL), originally associated with milk secretion, is known to have a wide variety of biological actions and diverse sites of production beyond the pituitary gland. Recent studies have demonstrated that PRL is synthesized in retinal tissue. To gain insights into the functional role of PRL in the mammalian retina, we mapped the distribution of the PRL protein and the expression and localization of the PRL receptor (PRLR) in the retina of adult rats and green monkeys. PRL was examined in retinal sections by double immunolabeling combining anti-PRL antibodies with antibodies specific for glutamine synthetase (labeling Müller cells), glial fibrillary acidic protein (labeling astrocytes), or neuronal nuclei protein (labeling neurons). PRL was detected throughout the rat retina: in the photoreceptor outer segments, Müller cells, interneurons, ganglion cells, and astrocytes. The PRLR was examined by RT-PCR, in situ hybridization, immunohistochemistry, and Western blot. The long isoform of the PRLR was localized in the photoreceptor nuclear layer, inner nuclear layer, and ganglion cell layer of rat retina. The monkey retina showed a similar distribution of PRL and PRLR immunoreactivities. These findings suggest that PRL functions as a local regulator of various cell types in the mammalian retina.

GABA inhibition of cyclic AMP production in immortalized GnRH neurons is mediated by calcineurin-dependent dephosphorylation of adenylyl cyclase 9.

The neurotransmitter gamma-aminobutyric acid (GABA) is an important modulator of gonadotropin-releasing hormone (GnRH), and consequently of reproduction. GABA, acting via ionotropic GABAA receptors, exerts a biphasic effect on GnRH secretion in immortalized GnRH cells. The initial increase in GnRH secretion is triggered by a sharp rise in [Ca2+]i, while the progressive decline of GnRH levels that follows is paralleled by reduced levels of intracellular cAMP. The experiments described here were designed to explore the potential signaling pathways involved in this novel GABAA ionotropic inhibition of cAMP synthesis in GT1-7 cells. Using RT-PCR and real-time PCR, we found that GT1-7 cells express 8 of 9 known membrane adenylyl cyclase (AC) isoforms, including a large proportion of AC3 and AC9, as well as AC5 and AC6, all of which are negatively regulated by increases in [Ca2+]i. In contrast, isoforms of AC that are positively regulated by [Ca2+]i were barely detectable (AC1) or undetectable (AC8). Pharmacological activation of L-type voltage-operated calcium channels with BayK 8644 produced a decrease in [cAMP]i similar to that induced by GABA, while blocking these calcium channels with verapamil reversed the effect of GABA on cAMP synthesis. Furthermore, blocking calcineurin with deltamethrin, FK-506 or cyclosporin A blocked the inhibitory effect of GABA on [cAMP]i, supporting the involvement of AC9 in this effect. In addition, blocking Ca2+/calmodulin-dependent protein kinase II (CamKII) with KN-62 partially reversed the action of GABA, suggesting that AC3 may also be involved in this effect. Finally, GABA increased phosphatase activity in a calcium-dependent manner, an effect blocked by calcineurin inhibitors. Collectively, our results show that the ionotropic action of GABA via the activation of GABAA receptors can decrease AC activity in immortalized GnRH neurons, and that the effect of GABA appears to be mediated by a transient increase in [Ca2+]i followed by activation of calcineurin and CamKII, leading to dephosphorylation of AC9 and phosphorylation of AC3, respectively, and subsequently reducing the synthesis of cAMP.

Matrix metalloproteases from chondrocytes generate an antiangiogenic 16 kDa prolactin.

The 16 kDa N-terminal fragment of prolactin (16K-prolactin) is a potent antiangiogenic factor. Here, we demonstrate that matrix metalloproteases (MMPs) produced and secreted by chondrocytes generate biologically functional 16K-prolactin from full-length prolactin. When incubated with human prolactin at neutral pH, chondrocyte extracts and conditioned medium, as well as chondrocytes in culture, cleaved the Ser155-Leu156 peptide bond in prolactin, yielding - upon reduction of intramolecular disulfide bonds - a 16 kDa N-terminal fragment. This 16K-prolactin inhibited basic fibroblast growth factor (FGF)-induced endothelial cell proliferation in vitro. The Ser155-Leu156 site is highly conserved, and both human and rat prolactin were cleaved at this site by chondrocytes from either species. Conversion of prolactin to 16K-prolactin by chondrocyte lysates was completely abolished by the MMP inhibitors EDTA, GM6001 or 1,10-phenanthroline. Purified MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 cleaved human prolactin at Gln157, one residue downstream from the chondrocyte protease cleavage site, with the following relative potency: MMP-8 > MMP-13 > MMP-3 > MMP-1= MMP-2 > MMP-9. Finally, chondrocytes expressed prolactin mRNA (as revealed by RT-PCR) and they contained and released antiangiogenic N-terminal 16 kDa prolactin (detected by western blot and endothelial cell proliferation). These results suggest that several matrix metalloproteases in cartilage generate antiangiogenic 16K-prolactin from systemically derived or locally produced prolactin.

Prolactin stimulates integrin-mediated adhesion of circulating mononuclear cells to endothelial cells.

Attachment of leukocytes to endothelial cells is an essential step for the extravasation and recruitment of cells at sites of inflammation. The pituitary hormone prolactin (PRL) is involved in the inflammatory process. Here, we show that treatment with PRL of human peripheral blood mononuclear cells (PBMC) stimulates their adhesion to human umbilical vein endothelial cells (HUVEC) activated by interleukin-1beta. Stimulation of adhesion by PRL is mediated via integrins leukocyte functional antigen-1 (LFA-1) and very late antigen-4 (VLA-4), because immunoneutralization of both integrins prevents PRL action. Also, PRL promotes the adhesion of PBMC to immobilized intercellular adhesion molecule-1 and fibronectin, ligands for LFA-1 and VLA-4, respectively. Stimulation of integrin-mediated cell adhesion by PRL may involve the activation of chemokine receptors, because PRL upregulates the expression of the G-protein-coupled chemokine receptor CXCR3 in PBMC, and pertussis toxin, a specific G-protein inhibitor, blocks PRL stimulation of PBMC adhesion to HUVEC. In addition, PRL stimulates tyrosine phosphorylation pathways leading to leukocyte adhesion. PRL triggered the tyrosine phosphorylation of Janus kinase-2, of signal transducer and activator of transcription-3 and 5, and of the focal adhesion protein paxillin. Furthermore, genistein, a tyrosine kinase inhibitor, blocked PRL-stimulated adhesion of PBMC and Jurkat T-cells to HUVEC. These results suggest that PRL promotes integrin-mediated leukocyte adhesion to endothelial cells via chemokine receptors and tyrosine phosphorylation signaling pathways.

Increased hypothalamic expression of prolactin in lactation: involvement in behavioural and neuroendocrine stress responses.

Prolactin (PRL) has recently been shown to exert an anxiolytic effect in male and virgin female rats, as well as an inhibitory tone on hypothalamic-pituitary-adrenal (HPA) axis activity. Reduced emotional and neuroendocrine stress responses have been described in lactation, a time of high blood PRL levels. Here we tested brain PRL-receptor (PRL-R)-mediated effects on anxiety, maternal behaviour, HPA axis and oxytocin stress responses in lactating rats. Chronic intracerebroventricular (i.c.v.) infusion of antisense oligonucleotides against the long form of the PRL-R (AS; osmotic minipump, 0.5 microg/0.5 microL/h) in order to downregulate brain PRL-R expression increased the anxiety-related behaviour on the elevated plus maze (P < 0.01) compared with mixed bases- and vehicle-treated rats. Also, PRL-R AS treatment impaired maternal behaviour (P < 0.05), whereas physiological parameters of lactation (weight gain of the litter, number of milk ejection reflexes during a 20-min suckling period) were not affected. PRL-R AS treatment further evoked an increase (P < 0.05) in the stress-induced adrenocorticotropin release, demonstrating an inhibitory role of PRL on HPA axis responses in lactation. Inhibition of stress responses of the oxytocin system by brain PRL was evidenced by higher stress-induced (P < 0.05) plasma oxytocin concentration in PRL-R AS-treated lactating rats and, in contrast, decreased stress-induced oxytocin release (P < 0.01) in chronic i.c.v. ovine PRL-treated (1 microg/0.5 microL/h) virgin rats. Finally, an increased expression of the hypothalamic PRL gene was seen by RT-PCR in pregnancy and lactation, suggesting an activated state of the brain PRL system during the peripartum period. In summary, activation of the brain PRL system in the peripartum period significantly contributes to emotional and neuroendocrine adaptations, including downregulation of the responsiveness of the HPA axis and oxytocin systems to stressors seen at this time.

In vivo release and gene upregulation of brain prolactin in response to physiological stimuli.

Although prolactin (PRL) actions and expression in the brain have been shown, dynamic changes in its intracerebral release and gene expression have still not been demonstrated. Using push-pull perfusion, the in vivo release of PRL was monitored within the paraventricular nucleus (PVN) and medial preoptic area (MPOA) of virgin female, lactating and male rats in response to various stimuli. Perfusion with a depolarizing medium (56 mm K(+)) increased local release of PRL within both the PVN (P < 0.05) and MPOA (P < 0.05) of urethane-anaesthetized rats, indicating release from excitable neuronal structures. The PRL in perfusates was verified by radioimmunoassay, Nb2 cell bioassays and western blot. Systemic osmotic stimulation (3 m NaCl i.p., 8 mL/kg b.w.) raised PRL concentration in plasma (P < 0.01) but not within the PVN, suggesting independent release from the pituitary and in distinct brain regions. Immobilization for 30 min increased PRL release within the PVN (P < 0.05) and the MPOA (P < 0.01) of virgin female and male (P < 0.05 each) rats and increased hypothalamic PRL mRNA expression (P = 0.008) after 30 and 90 min as revealed by real-time polymerase chain reaction. This indicates a stress-induced activation of both PRL release from and synthesis in hypothalamic neurons. Additionally, PRL was significantly released within, but not outside, the PVN (P < 0.01) and the MPOA (P < 0.05) of lactating rats during suckling and this was accompanied by a significant increase of PRL mRNA (P < 0.05) in the hypothalamus 60 min after suckling. This is the first demonstration of stimulus-induced, locally restricted release and gene upregulation of PRL within the brain, emphasizing the involvement of this 'novel' neuropeptide in various brain functions.