Complementary mechanisms of enhanced oxytocin-stimulated prostaglandin E2 synthesis in rabbit amnion at the end of gestation.

The up-regulation of oxytocin (OT) receptors in rabbit amnion at the end of gestation is associated with a large increase in the ability of OT to stimulate PGE2 synthesis. The purpose of these investigations was to determine what other factors contribute to this increase. OT enhanced PGE2 synthesis at several levels. The concentrations of cytosolic phospholipase A2, which generates arachidonic acid for PGE2 synthesis, and PGH endoperoxide synthases (types 1 and 2), which catalyze the conversion of arachidonic acid to prostanoids, rose substantially in rabbit amnion at term. OT stimulated translocation of cytosolic phospholipase A2 to the cell particulate fraction, presumably by a Ca2+-mediated process, and phosphorylation of cytosolic phospholipase A2 via the extracellular regulated protein kinase 2/1-mediated pathway. OT-stimulated increases in intracellular Ca2+ concentrations and extracellular regulated protein kinase 2/1 phosphorylation were both mediated by G(q/11) activation. OT also increased the expression of PGH endoperoxide synthase-2 after treatment of amnion cells in culture for 2 h; however, PGE2 release in response to OT was virtually immediate. These findings show that the rapid stimulation of PGE2 synthesis by OT occurs through cytosolic phospholipase A2 activation and PGH endoperoxide synthase-1 activity, both of which, along with OT receptor concentrations, are considerably up-regulated in the amnion at the end of gestation.

Functional oxytocin receptors discovered in human osteoblasts.

Undifferentiated or differentiated human trabecular bone cells with osteogenic capacity in primary culture express oxytocin receptors (OTRs). OTR expression then persists upon differentiation to an osteoblast phenotype. A human epithelial osteosarcoma cell line, Saos-2, also expresses OTRs. Expression was determined both at mRNA and protein levels. Functional OTRs are evidenced by an increase in intracellular calcium concentration, [Ca2+]i, in response to 10 nM oxytocin (OT). An oxytocin antagonist (OTA) blocked this effect, demonstrating specificity for OT. OT also stimulated prostaglandin E2 (PGE2) synthesis in both confluent undifferentiated and differentiated human trabecular bone cells. This is the first report of OTR mRNA and protein expression and of prescribed OT signal pathways in osteoblastic cells. Since PGE2 has been shown to increase bone turnover in favor of bone formation, OT may be a new class of a bone anabolic agent.

Multiple protein kinase pathways are involved in gastrin-releasing peptide receptor-regulated secretion.

Gastrin-releasing peptide (GRP) and its amphibian homolog, bombesin, are potent secretogogues in mammals. We determined the roles of intracellular free Ca(2+) ([Ca(2+)](i)), protein kinase C (PKC), and mitogen-activated protein kinases (MAPK) in GRP receptor (GRP-R)-regulated secretion. Bombesin induced either [Ca(2+)](i) oscillations or a biphasic elevation in [Ca(2+)](i). The biphasic response was associated with peptide secretion. Receptor-activated secretion was blocked by removal of extracellular Ca(2+), by chelation of [Ca(2+)](i), and by treatment with inhibitors of phospholipase C, conventional PKC isozymes, and MAPK kinase (MEK). Agonist-induced increases in [Ca(2+)](i) were also inhibited by dominant negative MEK-1 and the MEK inhibitor, PD89059, but not by an inhibitor of PKC. Direct activation of PKC by a phorbol ester activated MAPK and stimulated peptide secretion without a concomitant increase in [Ca(2+)](i). Inhibition of MEK blocked both bombesin- and phorbol 12-myristate 13-acetate-induced secretion. GRP-R-regulated secretion is initiated by an increase in [Ca(2+)](i); however, elevated [Ca(2+)](i) is insufficient to stimulate secretion in the absence of activation of PKC and the downstream MEK/MAPK pathways. We demonstrated that the activity of MEK is important for maintaining elevated [Ca(2+)](i) levels induced by GRP-R activation, suggesting that MEK may affect receptor-regulated secretion by modulating the activity of Ca(2+)-sensitive PKC.

Demonstration of functional oxytocin receptors in human breast Hs578T cells and their up-regulation through a protein kinase C-dependent pathway.

Oxytocin (OT) receptors (OTRs) have been demonstrated in a number of human breast tumors and tumor cells, but it was not clear whether the receptors were functional. We examined the regulation and function of OTR in a tumor cell line, Hs578T, derived from human breast. These cells expressed moderate levels of OTR when cultured in 10% FBS, as demonstrated by RT-PCR and binding analyses. Serum deprivation resulted in the loss of OTRs, with no effect on cell viability. Restoration of serum and addition of 1 microM dexamethasone (DEX) increased OTR levels by about 9-fold. Up-regulation was blocked by the addition of phospholipase C and PKC inhibitors. Serum/DEX treatment also increased steady state OTR messenger RNA levels. OT increased intracellular Ca2+ in a time- and dose-responsive manner, and the effects of OT were lost when OTRs were down-regulated by serum starvation. Serum/DEX up-regulation of OTR restored the responsiveness to OT. OT also stimulated ERK-2 (extracellular signal-regulated protein kinase) phosphorylation and PGE2 synthesis in Hs578T cells. In addition to showing that OTRs in the breast tumor cells are functional, these studies show that Hs578T cells can be used to study molecular regulation of OTR gene expression and intracellular signaling pathways stimulated by OT.

Parathyroid hormone-related peptide production and action in a myoepithelial cell line derived from normal human breast.

Physiological roles for PTH-related peptide (PTHrP) appear varied, but remain to be clarified. The peptide is present in large amounts in milk, and PTHrP mRNA has been shown to be present in high amounts in lactating mammary gland. Because PTHrP can cause smooth muscle relaxation, we hypothesized that the peptide might affect the contractility of the breast myoepithelial cell and thereby affect milk ejection. To test this idea, we asked whether PTHrP might affect second messenger responses in a human mammary gland myoepithelial-cell line (Hs578Bst) derived originally from normal breast tissue. To verify the presumed origin of these cells, we also examined the effects of oxytocin. Cells were grown in culture in multiwell plates and exposed to test peptides for 15 min in buffer containing 1 mM isobutylmethylxanthine, and intracellular cAMP was measured by RIA. Both PTHrP-(1-34) and PTH-(1-34) increased cAMP in a dose-related fashion (ED50, 5 nM), with a maximal effect (3-fold) occurring at 100 nM. The ability of PTHrP to stimulate cAMP was inhibited by a 10- to 100-fold molar excess of the specific inhibitors, PTH-(3-34) or PTHrP-(7-34). Inhibitors alone did not alter cAMP. Oxytocin also produced an increase in cAMP, but the effect was inconsistent and occurred only with high doses (0.1-1 microM). Using cells grown on coverslips and loaded with fura-2AM, intracellular Ca2+ was monitored in cells exposed to test peptides. Oxytocin (0.2-20 nM) produced rapid dose-related increases in intracellular Ca2+, with a peak and plateau characteristic of initial mobilization of intracellular Ca2+, followed by entry of extracellular Ca. The plateau was eliminated by the Ca channel antagonist La3+ or by perfusion of cells with Ca-free medium. PTHrP (10-100 nM) altered the intracellular Ca2+ response to oxytocin in 66% of 39 preparations tested. PTHrP inhibited the Ca2+ response when given before oxytocin or transiently decreased the plateau phase of the Ca2+ response when given after oxytocin. Analysis of cellular mRNA using reverse transcription polymerase chain reaction indicated that these cells express the gene for PTHrP, and immunohistochemistry using antiserum to PTHrP revealed positive staining of cells. Measurement of immunoreactive PTHrP in conditioned medium confirmed that these cells can synthesize and secrete the peptide. The finding of a response of this cell line to oxytocin provides functional evidence of their myoepithelial derivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential response to gamma radiation of human stomach cancer cells in vitro.

In vitro effects of radiation were studied in two permanent cell lines (AGS and SII) from two patients with adenocarcinoma of the stomach and three permanent sublines from each cell line. Radiation survival parameters for AGS and SII parent cell lines and sublines were determined after in vitro irradiation of their cells with 0.5 to 10 Gy of 60Co gamma rays. The AGS and SII cell lines had different growth properties, DNA contents and radiation survival curves. Surviving fractions of SII parent cells (76 chromosomes) after 2.0 and 10 Gy were 1.22 and 17.8 times greater, respectively, than values for AGS parent cells (47 chromosomes). Sensitivities (D0) were 1.08 and 1.45 Gy for AGS and SII parent lines, respectively. The D0 values for AGS parent cells and sublines were similar (1.01 to 1.08 Gy), but SII parent cells and sublines had D0 values of 1.45, 1.36, 1.37 and 1.12 Gy (for SII-A). Also, the SII parent cells had survival fractions after 2.0 and 10 Gy that were 1.3 and 11.3 times greater, respectively, than values for the SII-A cells. These data show differences in radiation responses among stomach cancer cell lines and sublines that may relate to DNA content, but there was no consistent correlation between radiation response and a particular cell characteristic.