Stimulation by calcitonin gene-related peptide of atrial natriuretic peptide secretion in vitro and its mechanism of action

Calcitonin gene-related peptide (CGRP) is present in nerve fibers within atrial tissue, raising the possibility that CGRP release may influence atrial natriuretic peptide (ANP) secretion. We, therefore, examined the effect of CGRP on immunoreactive ANP (ANP-IR) secretion. Isolated rat left atria paced at 2 Hz were superfused with 0.1 microM CGRP. A biphasic 2-fold increase in ANP-IR secretion occurred in response to CGRP. We next examined the mechanism of CGRP-stimulated secretion. The biphasic ANP-IR secretory response to CGRP was similar to that induced by superfusion with the beta-adrenergic agonist isoproterenol and (Bu)2cAMP, but distinct from that of the non-cAMP dependent stimuli phenylephrine, ouabain, and Bay K 8644. Superfusion with 0.1 microM CGRP for 4 min with 100 microM isobutylmethylxanthine increased atrial cAMP content from 4.29 +/- 1.21 to 10.32 +/- 2.14 pmol/mg atrial weight (P less than 0.001). Atria were next superfused with methacholine, an inhibitor of adenylyl cyclase activation. The addition of 0.1 microM isoproterenol or 0.1 microM CGRP to the superfusate containing 10 microM methacholine failed to stimulate ANP-IR secretion and lowered cAMP accumulation by 70%. Superfusion with 10 microM atropine negated the inhibitory effects of methacholine. We conclude that 1) CGRP stimulates ANP-IR secretion in vitro; and 2) CGRP-stimulated secretion appears to be mediated by cAMP. Thus, ANP-IR secretion may be modulated by atrial nerve fibers containing CGRP in vivo.     

Inhibition of decidual prolactin release by a decidual peptide

Conditioned media from cultures of human decidual explants and aqueous extracts of human decidual tissue contain a factor that causes a reversible dose-dependent inhibition of decidual PRL release in vitro. Decidual explants incubated for 30 min in medium containing 50, 100, and 250 micrograms/ml of a dialyzed and lyophilized preparation of decidual conditioned medium (DCM) released 32.4 +/- 2.7%, 70.9 +/- 4.5%, and 100.0%, respectively, less PRL than control explants. DCM, however, had no measurable effect on the synthesis of decidual PRL or the synthesis and release of trichloroacetic acid-precipitable 35S-labeled proteins. The effect was of short duration and completely reversible. The inhibition of decidual PRL release was not due to PRL, since 500 micrograms/ml human pituitary PRL (a PRL concentration 40 times that in the minimal effective dose of DCM) added to the incubation medium of decidual explants had no effect on the synthesis or release of decidual PRL or trichloroacetic acid-precipitable 35S-labeled decidual proteins. The inhibitory activity eluted from Sephadex G-200 with an apparent molecular weight of 38,000-45,000 daltons, was heat labile, was destroyed by treatment with trypsin, and was unaffected by extraction with acetone-ethanol. These results strongly suggest that the release of decidual PRL is under local control, regulated in part by a factor(s) other than PRL that is released by the decidua.     

A milk-borne factor inhibits mammotrope differentiation in the neonatal rat

Within the first few days of neonatal life in the rat, a milk-borne peptide is transferred to the neonatal circulation and transported to the pituitary gland where it acts directly to induce final differentiation of mammotropes. As we were attempting to purify this stimulatory peptide, we separated an antagonistic activity that serves as the focus of the present study. Milk obtained on days 2–3 of lactation was subjected to pH fractionation followed by acetone precipitation to yield two fractions that stimulated and inhibited, respectively, mammotrope differentiation in cultures of neonatal pituitary cells. The stimulatory agent more than doubled the proportion of prolactin secretors in those cultures, whereas the inhibitory agent exerted the opposite effect when tested alone. Moreover, the inhibitory agent severely attenuated mammotrope differentiation evoked by the stimulatory fraction or by basic FGF, an established inducer of this developmental phenomenon. The discovery of a milk-borne inhibitor, coupled with the previously described milk stimulatory factor, indicates that maternal control of mammotrope differentiation is considerably more sophisticated than previously believed.     

Stimulation of B cell differentiation by adherent mononuclear cells in systemic lupus erythematosus

We studied B cell proliferation and differentiation in response to factors released by adherent monocytes in patients with systemic lupus erythematosus (SLE). Adherent cell supernatants (ACS) were added to peripheral blood mononuclear cells, and the effects on IgG synthesis, the number of Ig-secreting cells (ISC), and proliferation were determined. Exposure of SLE mononuclear cells to autologous ACS caused an increase (approximately two-fold) in IgG production and ISC numbers. In contrast, exposure of normal mononuclear cells to autologous ACS did not significantly increase IgG production or ISC numbers. Addition of SLE ACS to cultures of normal mononuclear cells did not stimulate ISC production. There was no significant level of 3H-thymidine uptake by cultures of SLE or normal mononuclear cells in response to either SLE or normal ACS. In the presence of an excess number of autologous T cells, ACS stimulation of IgG synthesis was further enhanced. These findings indicate that adherent monocytes contribute to B cell hyperactivity in SLE by stimulating B cell differentiation. SLE mononuclear cells appear to be more responsive to ACS stimulation than are normal mononuclear cells.     

Stimulation by leumorphin of prolactin secretion from the pituitary in rats

The effect of leumorphin (LM), one of big leu-enkephalins derived from preproenkephalin B, on PRL secretion was studied in the rat in vivo and in vitro. Intracerebroventricular injection of synthetic porcine LM (0.06-6 nmol/rat) caused a dose-related increase in plasma PRL levels in urethane-anesthetized male rats and in conscious freely moving rats. Intravenous injection of LM (3 nmol/100 g BW) also raised plasma PRL levels in these animals. The plasma PRL response to intracerebroventricular LM (0.6 nmol/rat) was blunted by naloxone (125 micrograms/100 g BW, iv). The stimulating effect of LM on PRL release was the most potent among the peptides derived from preproenkephalin B. In in vitro studies, PRL release from superfused anterior pituitary cells was stimulated in a dose-related manner by LM (10(-9)-10(-6) M), and the effect was blunted by naloxone (10(-5) M). These results suggest that LM has a potent stimulating effect on PRL secretion from the pituitary in the rat by acting, at least in part, directly at the pituitary through an opiate receptor.     

Autocrine control of prolactin secretion by vasoactive intestinal peptide

The functions of vasoactive intestinal polypeptide (VIP) and the many other neuropeptides that are localized in the anterior pituitary gland remain unknown although VIP of hypothalamic origin is established to act as a PRL-releasing factor. Evidence is presented here that locally-produced VIP acts in an autocrine fashion to stimulate PRL release. VIP antibodies or a VIP antagonist profoundly but reversibly suppressed PRL secretion in primary cultures of rat pituitary cells or the GH3 cell line. This evidence was obtained with the use of a reverse hemolytic plaque assay for microscopic demonstration of PRL release from individual cells under conditions precluding cell-cell interaction. We suggest that most of the high rate of "spontaneous" PRL secretion attributed to lactotropes deprived of hypothalamic influence is due in fact to the stimulatory effects of VIP acting in an autocrine fashion.     

Stimulation of human eosinophilopoiesis by hydrocortisone in vitro

Excess hydrocortisone (HC) evokes neutrophilia and eosinopenia in man. In addition, the hormone enhances human granulopoiesis in vitro at physiological as well as pharmacological concentrations. This study addressed the prospect that HC exerts opposite effects on the precursors of neutrophils and eosinophils, stimulating the former and inhibiting the latter. Experiments conducted on unseparated bone marrow (BM) cells demonstrated an increase in eosinophil clonogenesis in vitro with the addition of HC to the culture system. Secondary cultures, established from such primary harvests, revealed that HC had a direct impact on the clonogenic cells. Furthermore, administration of HC to normal subjects, at a dose which resulted in consistent eosinopenia, prompted an increase in the generation of eosinophil clones from peripheral blood cells ex vivo. Thus the hormone stimulates production of both neutrophils and eosinophils. Previous reports of opposite effects appear to have resulted from deficiencies of growth factors in the cell cultures. The contrasting effects of HC on neutrophil and eosinophil concentrations in the peripheral blood are more likely due to opposite effects on the distribution of these terminally differentiated cells in the circulation and extravascular tissues.     

Synthesis of prolactin by human decidua in vitro.

To determine whether human decidua and/or chorion synthesizes and secretes prolactin, explants of decidua obtained at Caesarian section and explants of chorion from the membranes separating dizygotic twins were cultured for periods of up to 6 days. The decidual explants released 366 +/- 37 ng prolactin/100 mg tissue (mean +/- S.D.) during each day in culture and incorporated 3H-labelled amino acids into immunoprecipitable prolactin. In the radioimmunoassay for prolactin, serial dilutions of incubation medium displaced 125I-labelled prolactin parallel to the displacement by pituitary prolactin and the prolactin in the medium eluted from Sephadex G-150 in a position indentical to that of pituitary prolactin. Chorionic explants released prolactin into the incubation medium during day 1 of culture only and did not incorporate 3H-labelled amino acids into prolactin. These results demonstrate that prolactin is synthesized by the decidua and not by the chorion and suggest that the decidua is the source of prolactin in amniotic fluid.     

Extracellular matrix-driven alveolar epithelial cell differentiation in vitro

During homeostasis and in response to injury, alveolar type II (AT2) cells serve as progenitor cells to proliferate, migrate, differentiate, and re-establish both alveolar type I (AT1) and AT2 cells into a functional alveolar epithelium. To understand specific changes in cell differentiation, we monitored morphological characteristics and cell-specific protein markers over time for isolated rat AT2 cells cultured on combinations of collagen, fibronectin and/or laminin-5 (Ln5). For all matrices tested, cultured AT2 cells displayed reduced expression of AT2 cell-specific markers from days 1 to 4 and increased expression of AT1-specific markers by day 3, with continued expression until at least day 5. Over days 5 to 7 in culture, cells took on an AT1-like phenotype (on collagen/fibronectin alone; collagen alone; or Ln5 alone), an AT2-like phenotype (on collagen/fibronectin/Ln5; or collagen/Ln5), or both AT1-like and AT2-like phenotypes (on collagen/fibronectin matrix with a subsaturating amount of Ln5). Cells transferred between matrices at day 4 of culture retained the ability to alter day 7 phenotype. We conclude that in vitro, (1) AT2 cells exhibited phenotype plasticity that included an intermediate cell type with both AT1 and AT2 cell characteristics independent of day 7 phenotype; (2) both collagen and Ln5 were needed to promote the development of an AT2-like phenotype at day 7; and (3) components of the extracellular matrix (ECM) contribute to phenotypic switching of alveolar cells in culture. The described tissue culture models provide accessible models for studying changes in alveolar epithelial cell physiology from AT2 cell progenitors to the establishment of alveolar epithelial monolayers that represent AT1-like, AT2-like, or a mix of AT1- and AT2-like cells.     

Stimulation of clonal tumor cell growth in vitro by inhibiting the serum polyamine oxidase activity

Summary Several tumors are characterized by elevated levels of polyamines involved in vital cell proliferation processes. Polyamine oxidases (PAO), present in ruminant and particularily in fetal calf serum (FCS), degrade polyamines to polyaminoaldehydes and other products that inhibit cell proliferation. Since most in vitro assays for cloning tumor stem cells use FCS as an essential supplement of the nutrient media, we examined the effects of specifically inhibiting the PAO activity on the clonal growth of leukemic cells and the following normal lymphocytes: the W 25 rat chloroleukemia, the M1 mouse myeloblastic and the L 1210 rat lymphoblastic leukemia, a primary human acute myeloblastic leukemia (AML) and acute lymphocytic leukemia (ALL) as well as normal human PHA-stimulated lymphocytes. In the presence od horse serum, nontoxic doses of the PAO inhibitor 1-hydroxybenzyloxyamine did not affect colony growth of either cell type. However, in the presence of FCS, clonal growth of W 25, ALL, AML, and PHA lymphocytes was significantly stimulated by the enzyme inhibitor. Our data suggest (a) that poor cell proliferation of several tumors in vitro may result from the reaction of polyamines (from cells) and PAO (from serum), (b) that this can be easily tested by means of a specific PAO inhibitor, and (c) that the growth conditions can be optimized by adding nontoxic doses of the enzyme inhibitor or by exchanging FCS for another serum.     

Stimulation of prolactin and growth hormone secretion by muscimol, a gamma-aminobutyric acid agonist.

The alteration in circulating levels of PRL, GH, TSH, and cortisol was studied after the oral administration of muscimol (3-hydroxy-5-aminomethylisoxazole) to human subjects with Huntington's disease (n = 4) and chronic schizophrenia (n = 5). PRL levels rose significantly in a dose-dependent fashion within a 120-min time interval. GH rose significantly but modestly over the same time interval, whereas TSH and cortisol levels remained unchanged. Since muscimol is thought to be a potent and specific gamma-aminobutyric acid agonist, these data indicate that gamma-aminobutyric acid-mediated neural transmission may function to stimulate the release of plasma PRL and GH in human subjects.     

异戊烯焦磷酸刺激γδT细胞增殖及体外抗HBV作用

目的采用异戊烯焦磷酸(isopentenyl pyrophosphate,IPP)刺激人外周血单个核细胞(peripheral bloodmononuclear cells,PBMCs)中γδT细胞特异性增殖,观察其体外对HBV复制和表达的抑制作用。方法体外用IPP和rhIL-2刺激人PBMCs培养10天,流式细胞术检测培养前后γδT细胞含量;将γδT细胞与HepG2.2.15细胞按一定比例共孵育培养后,ELISA法测定上清中HBsAg,HBeAg的表达,荧光定量PCR检测HBVDNA的含量。结果采用IPP和rhIL-2刺激人PBMCs,可以使人外周血γδT细胞选择性激活和扩增,由4.34%±1.79%。增加至55.65%±6.88%。所扩增的γδT细胞,能够部分抑制HepG2.2.15细胞中HBVDNA复制和HBeAg的表达,对HB-sAg的表达没有明显抑制作用。结论采用IPP刺激PBMCs,能使γδT细胞选择性增殖,增殖的细胞能够降低HepG2.2.15细胞中HBV的复制和HBeAg表达,在乙型肝炎的过继性免疫治疗中具有潜在的临床应用价值。     

Stimulation in vivo of the secretion of prolactin and growth hormone by beta-endorphin.

Morphine sulfate (MS) and the opioid peptide beta-endorphin beta-LPH-(61-91) stimulate prolactin and growth hormone release in steroid-primed and non-treated male rats when injected intravenously or intracisternally. On a molar basis beta-endorphin is at least 20 times more potent than MS, whereas Met5-enkephalin (beta-LPH-(61-65)) and alpha-endorphin (beta-LPH-(61-76)) are devoid of activity at the dose injected (300 mug). The in vivo stimulatory effects of beta-endorphin on prolactin secretion are reversed by the opiate antagonist naloxone. The absence of in vitro effect of MS and beta-endorphin on prolactin and growth hormone secretion by cultured rat pituitary cells suggest that they have a central nervous system site of action.