Effects of an Oral CRTh2 Antagonist (AZD1981) on Eosinophil Activity and Symptoms in Chronic Spontaneous Urticaria.

BACKGROUND: Approximately 50% of patients with chronic spontaneous urticaria (CSU) experience symptoms that are not fully controlled by antihistamines, indicating an unmet clinical need. OBJECTIVE: To evaluate the effects of the selective CRTh2 antagonist AZD1981 on symptoms and targeted leukocytes in adults with persistent CSU despite treatment with H1-antihistamines. METHODS: We performed a single-center, randomized, placebo-controlled study involving adult CSU subjects with symptoms despite daily antihistamines. The subjects underwent a 2-week placebo run-in and 4 weeks of double-blinded therapy with either AZD1981 40 mg TID or placebo, followed by a 2-week placebo washout. The primary objective was to assess the effect of AZD1981 on CSU signs and symptoms. Secondary objectives included the effects of AZD1981 on prostaglandin D2 (PGD2)-induced eosinophil shape change, circulating leukocyte subsets, CRTh2 expression on blood leukocytes, and total blood leukocyte histamine content. RESULTS: Twenty-eight subjects were randomized to AZD1981 or placebo, with 26 subjects completing the study. The urticaria activity scores declined during the treatment phase in both groups, and they were significantly reduced in the AZD1981 group at the end of washout. AZD1981 treatment increased circulating eosinophils and significantly impaired PGD2-mediated eosinophil shape change. CRTh2 surface expression rose significantly on blood basophils during active treatment. No serious adverse events were observed. CONCLUSIONS: This is the first study to examine the efficacy of a CRTh2 antagonist in antihistamine-refractory CSU. AZD1981 treatment was well tolerated, effectively inhibited PGD2-mediated eosinophil shape change, shifted numbers of circulating eosinophils, and reduced weekly itch scores more than hives during treatment and into washout. Further studies are needed to determine whether inhibition of the PGD2/CRTh2 pathway will be an -effective treatment for CSU.

The effects of the stromal cell-derived cyclooxygenase-2 metabolite prostaglandin E2 on the proliferation of colon cancer cells.

It is well known that tumor-surrounding stromal tissues support tumor development through secreting soluble factors such as various cytokines, chemokines, and growth factors. It has also been suggested that tumor-associated fibroblast and immune cells have a high expression of cyclooxygenase-2 (COX-2) and produce and secrete several prostaglandins (PGs) to adjacent cancer tissues. From these findings, we assumed that COX-2 inhibition might have an anticancer effect on cancer cells even without COX-2 expression in COX-2-dependent mechanisms through blocking the effect of stroma-derived PGs. Here, because of the complex involvement of various factors in vivo, we investigated this possibility with an in vivo-mimicking model using a Transwell system. To test our hypothesis, we used COX-2-transfected cell lines as stromal cells in our model. When we cocultured cancer cells (LS174T cells without COX-2 expression) with COX-2-high stromal cells in the Transwell membrane system, we observed that the proliferation of cancer cells was promoted and vascular endothelial growth factor synthesis was up-regulated significantly. These effects were blocked completely by COX-2 inhibitors and phosphoinositide-3-kinase inhibitors and partially by the PG E(2) receptor 4 antagonist. Even if some cancer cells did not express COX-2, they were found to have expression of PG receptors and PG-related downstream signaling molecules associated with cell viability. Our observation suggests that these cells can be influenced by PGs derived from stromal tissues. These findings also suggest that COX-2 inhibitors can be used to control the interaction between cancer and surrounding stromal tissues and suppress the proliferation of cancer cells regardless of the expression of COX-2 in cancer cells.

Comprehensive expression analysis of prostanoid enzymes and receptors in the human endometrium across the menstrual cycle.

Prostanoids are well-described primary mediators of inflammatory processes and are essential for the normal physiological function of the female reproductive system. The aim of this study was to determine the temporal expression of the prostanoid biosynthetic enzymes (PTGS1, PTGS2, PTGES, PTGES2, PTGES3, AKR1B1, AKR1C3, CBR1, HPGDS, PTGDS, PTGIS, TBXAS1 and HPGD) and the prostanoid receptors (PTGER1, PTGER2, PTGER3, PTGER4, PTGFR, PTGDR, GPR44, PTGIR and TBXA2R) in the human endometrium throughout the menstrual cycle. The analysis identified PTGFR to have a distinct expression profile compared with other components of the prostanoid system, as expression is maximal during the proliferative phase. Immunohistochemical analysis for PTGER1 suggests a dual function for this receptor depending on its temporal (proliferative versus secretory) and spatial (nuclear versus cell membrane) expression. The expression profiles of the PGF(2α) synthases identified AKR1B1 and CBR1 as the likely regulators of PGF(2α) production during the menstrual phase. Immunohistochemical analysis for AKR1B1, CBR1 and AKR1C3 suggest expression to be in the glandular epithelium and vasculature. This study represents the first comprehensive analysis of the components of prostanoid biosynthetic and signalling pathway in the human endometrium. The expression profiles described have the potential to identify specific prostanoid components that may be dysregulated in inflammatory-associated disorders of the endometrium.

CRTH2 expression on T cells in asthma.

Mast cell-derived prostaglandin D2 (PGD2) is the major prostanoid found within the airway of asthmatics immediately following allergen challenge. PGD2 has been shown to have chemokinetic effects on eosinophils and T helper type 2 (Th2) cells in vitro. This occurs through the interaction of PGD2 with the G-protein-coupled chemokine receptor homologous molecule expressed on Th2 lymphocytes (CRTH2). The expression of CRTH2 has been shown to be highly selective for Th2 cells. Using flow cytometry we have studied the expression of CRTH2 on T cells in blood and bronchoalveolar lavage fluid in asthmatics and normal subjects. CRTH2 expression was confined to a small percentage of blood T cells in asthmatics (1.8%+/-0.2) and normal (1.6%+/-0.2) subjects. CRTH2 was enriched significantly on interleukin (IL)-4+/IL-13+ T cells compared to interferon (IFN)-gamma+ T cells (P<0.001). There was a small population of CRTH2+ T cells in the bronchoalveolar lavage (BAL) of asthmatics (2.3%+/-0.6) and normal subjects (0.3%+/-0.1), and there was a significant difference between the two groups (P<0.05). There were similar amounts of PGD2 in the BAL of asthma and normal subjects. Within paired blood-BAL samples from the same subject there was no increase in CRTH2+ T cells in the BAL compared to blood in asthmatics. Enrichment of CRTH2 on IL-4+ and IL-13+ T cells compared to IFN-gamma+ T cells was also seen in BAL from asthmatics (P<0.001). CRTH2 is expressed preferentially by IL-4+/IL-13+ T cells compared to IFN-gamma+ T cells. However, given their small numbers they are unlikely to have a significant involvement in the pathogenesis of asthma. CRTH2 antagonism may not diminish T cell accumulation in the asthmatic lung.

Expression and regulation of prostaglandin receptors in the human placenta and fetal membranes at term and preterm.

Prostaglandins (PGs) play an important role in parturition in many species, including humans. The present study examined the distribution of PG receptor subtypes (EP1-4 and FP) in intrauterine tissues at term and preterm birth. Placentas and fetal membranes were collected from patients at term in labour (n = 12) or not in labour (n = 12). Preterm tissue was collected from three different groups of patients: (1) idiopathic preterm labour (PTL) without chorioamnionitis or betamethasone (BM) treatment (n = 9), (2) idiopathic PTL that received BM with no chorioamnionitis (PTL-BM; n = 9) and (3) pregnancies that were complicated with chorioamnionitis and had no BM (PTL-CHA; n = 6). EP1-4 and FP receptors were localised and levels of expression were determined by western blot analysis. All EP receptors and FP were localised to the amnion, placenta and choriodecidua. Moreover, isolated amnion mesenchymal, amnion epithelial, chorion trophoblast and syncytiotrophoblast cells in primary culture also expressed PG receptors. A significant increase was observed in EP1, EP3 and FP expression in placenta, chorion and amnion with labour. Maternal betamethasone treatment increased EP1, EP3 and FP receptor protein expression and chorioamnionitis decreased expression in all the receptor subtypes. These changes in PG receptors in the fetal membranes are consistent with the development of a feed-forwards cascade mediated through PG action that may contribute to the birth process.

Niacin ameliorates ulcerative colitis via prostaglandin D2-mediated D prostanoid receptor 1 activation.

Niacin, as an antidyslipidemic drug, elicits a strong flushing response by release of prostaglandin (PG) D2 However, whether niacin is beneficial for inflammatory bowel disease (IBD) remains unclear. Here, we observed niacin administration-enhanced PGD2 production in colon tissues in dextran sulfate sodium (DSS)-challenged mice, and protected mice against DSS or 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in D prostanoid receptor 1 (DP1)-dependent manner. Specific ablation of DP1 receptor in vascular endothelial cells, colonic epithelium, and myeloid cells augmented DSS/TNBS-induced colitis in mice through increasing vascular permeability, promoting apoptosis of epithelial cells, and stimulating pro-inflammatory cytokine secretion of macrophages, respectively. Niacin treatment improved vascular permeability, reduced apoptotic epithelial cells, promoted epithelial cell update, and suppressed pro-inflammatory gene expression of macrophages. Moreover, treatment with niacin-containing retention enema effectively promoted UC clinical remission and mucosal healing in patients with moderately active disease. Therefore, niacin displayed multiple beneficial effects on DSS/TNBS-induced colitis in mice by activation of PGD2/DP1 axis. The potential efficacy of niacin in management of IBD warrants further investigation.

Cyclooxygenase-2 and prostaglandin F2 alpha in Syrian hamster Leydig cells: Inhibitory role on luteinizing hormone/human chorionic gonadotropin-stimulated testosterone production.

We have previously found that cyclooxygenase-2 (COX-2), a key enzyme in the biosynthesis of prostaglandins (PGs), is present in the testicular interstitial cells of infertile men, whereas it is absent in human testes with no evident morphological changes or abnormalities. To find an animal model for further investigating COX-2 and its role in testicular steroidogenesis, we screened testes from adult species ranging from mice to monkeys. By using immunohistochemical assays, we found COX-2 expression only in Leydig cells of the reproductively active (peripubertal, pubertal, and adult) seasonal breeder Syrian hamster. COX-2 expression in hamster Leydig cells was confirmed by RT-PCR. In contrast, COX-1 expression was not detected in hamster testes. Because COX-2 expression implies PG synthesis, we investigated the effect of various PGs on testosterone production and found that PGF2 alpha stood out because it significantly reduced human chorionic gonadotropin-stimulated testosterone release from isolated hamster Leydig cells in a dose-dependent manner. This mechanism involves a decreased expression of testicular steroidogenic acute regulatory protein and 17beta-hydroxysteroid dehydrogenase. Testicular concentration and content of PGF2 alpha in reproductively active hamsters as well as production of PGF2 alpha from isolated hamster Leydig cells were also determined. Moreover, PGF2 alpha receptors were localized in Leydig cells of hamsters and testicular biopsies from patients with Sertoli cell only and germ arrest syndromes. Thus, in this study, we described a COX-2-initiated pathway that via PGF2 alpha production, PGF2 alpha receptors, steroidogenic acute regulatory protein, and 17beta-hydroxysteroid dehydrogenase represents a physiological local inhibitory system of human chorionic gonadotropin-stimulated testosterone production in the Syrian hamster testes.

Loss of prostaglandin E receptors during progression of rat mammary tumors from hormonal dependence to autonomy.

Prostaglandin E (PGE) receptors and PGE-adenylate cyclase responsiveness were measured in tumor samples from a hormone-dependent subline of the transplantable MTW9 rat mammary tumor and from an autonomous subline derived from the hormone-dependent tumor. Scatchard analysis of the equilibrium binding data suggested that the hormone-dependent, slow-growing (MTW9A) tumors contain two major types of binding sites for PGE2: a high-affinity component (Kd less than 10(-9) M) and a low-affinity component [Kd greater than 10(-8) M]. The hormone-autonomous, fast-growing tumors (MTW9D), however, have lost more than 80% of the PG binding sites and exhibited mainly a predominant PGE lower affinity component (Kd greater than 10(-8) M). Loss of PGE receptors in autonomous tumors was not due to in vivo down-regulation of these receptors by excessive production of PGE, since both the hormone-dependent and autonomous tumors endogenously produce and release approximately the same amounts of PGE. Incubation of tumor tissues in vitro with PGE caused a significant stimulation of adenylate cyclase activity in the MTW9A tumors, whereas adenylate cyclase activity was not stimulated in the MTW9D tumors even in the presence of the nonhydrolyzable analogue of GTP, Gpp[NH]p. The results suggest that loss of PGE receptors and PGE-adenylate cyclase responsiveness occurs during progression of mammary tumors from hormonal dependence to autonomy and that the subsequent loss of cyclic AMP is associated with the uncontrolled growth characteristics of the autonomous tumors.

High-affinity binding site for leukotriene C4 in human erythrocytes.

A specific, high-affinity binding site for leukotriene C4 was identified in human erythrocyte particulate fraction and in vesicle preparation. The binding was saturable, reversible and specific. Vesicle preparations showed that binding sites were localized on the outside of the plasma membrane. The dissociation constant and site density were found to be Kd = 15.9 +/- 3.2 nmol X 1(-1) and N = 152 +/- 35 sites per cell, respectively, as calculated from Scatchard analysis. The effect of leukotriene C4 did not modify the calcium influx and did not inhibit the ATPase-dependent calcium efflux. In this paper, the physiological significance of these sites is discussed.

Long-term agonist stimulation of IP prostanoid receptor depletes the cognate G(s)alpha protein in membrane domains but does not change the receptor level.

Iloprost (IP) stimulation (1 microM, 2 h) of Flag-epitope-tagged human IP prostanoid receptor (FhIPR) expressed in HEK293 cells resulted in specific decrease of endogenous G(s)alpha protein in detergent-insensitive, caveolin-enriched, membrane domains (DIMs). Receptor protein FhIPR, caveolin, G(i)alpha and GPI-linked, domain markers CD55 and CD59 were unchanged. The same result was obtained in HEK293 cells expressing FhIPR-G(s)alpha fusion protein. The endogenous G(s)alpha decreased, but the level of Flag-hIPR-G(s)alpha protein did not change. The specific depletion of domain-bound pool of G(s)alpha as consequence of iloprost stimulation was also demonstrated in membrane domains prepared according to alkaline treatment plus sonication protocol (detergent-free procedure of Song et al.). Our data further indicated that in control, quiescent cells only a very small amount of IP prostanoid receptor was present in DIMs together with large amount of its cognate G(s)alpha protein. Expressed in quantitative terms, DIMs contained 30-40% of the total cellular amount of G proteins whereas the content of IP prostanoid receptors was 1-3%. The dominant portion (>95%) of FhIPR as well as FhIPR-G(s)alpha was localised in high-density area of the gradient containing detergent-solubilised proteins. FhIPR and FhIPR-G(s)alpha distribution was similar to that of transmembrane plasma membrane (PM) markers (CD147, MHCI, CD29, Tapa1, the alpha subunit of Na,K-ATPase, transmembrane form of CD58 and CD44). All these proteins are known to be fully solubilised by detergent and thus unable to float in density gradient. Our data indicate that (i) long-term agonist stimulation of IP prostanoid receptor is associated with preferential decrease of its cognate G protein G(s)alpha from membrane domains; receptor level is unchanged. (ii) Very small fraction (1-3%) of total cellular amount of receptors is recovered in DIMs together with roughly 40% of G proteins. These data suggest a "supra-stoichiometric" arrangement of G proteins and corresponding receptors in DIMs.

Letrozole is more effective neoadjuvant endocrine therapy than tamoxifen for ErbB-1- and/or ErbB-2-positive, estrogen receptor-positive primary breast cancer: evidence from a phase III randomized trial.

PURPOSE: Expression of ErbB-1 and ErbB-2 (epidermal growth factor receptor and HER2/neu) in breast cancer may cause tamoxifen resistance, but not all studies concur. Additionally, the relationship between ErbB-1 and ErbB-2 expression and response to selective aromatase inhibitors is unknown. A neoadjuvant study for primary breast cancer that randomized treatment between letrozole and tamoxifen provided a context within which these issues could be addressed prospectively. PATIENTS AND METHODS: Postmenopausal patients with estrogen- and/or progesterone receptor-positive (ER+ and/or PgR+) primary breast cancer ineligible for breast-conserving surgery were randomly assigned to 4 months of neoadjuvant letrozole 2.5 mg daily or tamoxifen 20 mg daily in a double-blinded study. Immunohistochemistry (IHC) for ER and PgR was conducted on pretreatment biopsies and assessed by the Allred score. ErbB-1 and ErbB-2 IHC were assessed by intensity and completeness of membranous staining according to published criteria. RESULTS: For study biopsy-confirmed ER+ and/or PgR+ cases that received letrozole, 60% responded and 48% underwent successful breast-conserving surgery. The response to tamoxifen was inferior (41%, P =.004), and fewer patients underwent breast conservation (36%, P =.036). Differences in response rates between letrozole and tamoxifen were most marked for tumors that were positive for ErbB-1 and/or ErbB-2 and ER (88% v 21%, P =.0004). CONCLUSION: ER+, ErbB-1+, and/or ErbB-2+ primary breast cancer responded well to letrozole, but responses to tamoxifen were infrequent. This suggests that ErbB-1 and ErbB-2 signaling through ER is ligand-dependent and that the growth-promoting effects of these receptor tyrosine kinases on ER+ breast cancer can be inhibited by potent estrogen deprivation therapy.

Temporal analysis of prostaglandin F2α receptor, caspase 3, and cyclooxygenase 2 messenger RNA expression and prostaglandin F2α receptor and cyclooxygenase 2 protein expression in endometrial tissue from multiparous Nelore (Bos taurus indicus) cows treated with cloprostenol sodium during puerperium.

The use of cloprostenol sodium in puerperium is questionable, as both favorable and unfavorable responses during the uterine involution process have been reported in the literature. This study is based on the hypothesis that cloprostenol sodium promotes modifications in the prostaglandin F2α receptor (FP), caspase 3 (CASP-3), and cyclooxygenase 2 (COX-2) mRNA expression that may favor the process of postpartum uterine involution in multiparous Nelore (Bos taurus indicus) females. Additionally, we aimed to describe the presence and immunolocalization of the FP and COX-2 protein in endometrial tissue at different postpartum time points in these females. Multiparous Nelore cows (n = 24) were treated with cloprostenol sodium (n = 12) or saline solution (n = 12) on postpartum Days 1 and 4 (Day 0 = birth), and endometrial biopsies were performed with a Yomann biopsy instrument and collected on Days 1, 7, 14, 28, and 42 postpartum. The mRNA expression from samples on the Days 1, 7, 14, 28, and 42 and the protein expression from samples on the Days 1, 14, 28, and 42 were then analyzed. The treated cows had altered FP and CASP-3 mRNA expression, and FP and COX-2 protein were observed in the endometrial surface epithelium, the stroma, and the glandular epithelium, with cytoplasmic immunolocalization. Although we attribute the change in CASP-3 mRNA expression to physiological phenomena, the results obtained for FP mRNA expression opens new doors for the study of hormonal protocols associated with cloprostenol sodium in the puerperium of Zebu females.

The monoclonal antibodies 18d7/91f2 recognize a receptor regulatory protein on mouse bone marrow stromal cells.

Two monoclonal antibodies 18D7 and 91F2 were developed by immunizing rats with the mouse bone marrow-derived osteogenic cell line MN7. Hybridomas secreting rat antibodies against MN7 cell surface markers were selected by flow cytometry analysis. Both the monoclonal antibody 18D7 and the monoclonal antibody 91F2 are directed against the same cell surface antigen present on MN7 cells. Here, we report on the immunopurification of the 18D7/91F2 antigen and its identification as the prostaglandin F2 alpha receptor regulatory protein (FPRP). FPRP is expressed as a single messenger RNA (mRNA) of approximately 6 kilobases (kb) in MN7 cells and is differentially expressed in developing osteogenic cultures of bone marrow cells of the mouse. However, addition of the monoclonal antibodies 18D7 and 91F2 to these cultures did not inhibit bone formation in vitro. Both monoclonal antibodies reacted with mouse stromal cell lines established from bone marrow, thymus, spleen, and mandibular condyles. Immunohistochemical analysis of mature tibia of mice using the monoclonal antibody 18D7 revealed the presence of a distinct population of bone marrow cells close to trabecular and endosteal bone surfaces. In the central bone marrow, hardly any positive cells were found. In 17-day-old fetal mouse radius 18D7 immunoreactivity was restricted to cells in the periosteum in close vicinity to the bone collar. Mature osteoblasts, osteoclasts, osteocytes, growth plate chondrocytes, and mature macrophages were all negative. Taken together, these results suggest that FPRP plays a role in the osteogenic differentiation process.

Regulation of prostaglandin F2 alpha receptor expression in cultured human granulosa-luteal cells.

PGF2 alpha is a metabolite of arachidonic acid that triggers regression of the corpus luteum. Recent animal studies have indicated that PGF2 alpha (FP) receptor messenger ribonucleic acid (mRNA) is expressed in the corpus luteum. To understand the regulation of the FP receptor in the ovary we have cloned a partial complementary DNA (cDNA) sequence of the FP receptor from human granulosa cells obtained from women undergoing in vitro fertilization. The sequence of this cDNA is identical to the previously reported FP receptor sequences obtained from human uterine and placental cDNA libraries. Low levels of the FP receptor mRNA were observed in freshly isolated granulosa cells or in cultured granulosa-luteal (GL) cells, as detected by reverse transcriptase-PCR. hCG and 8-bromo-cAMP increased the steady state levels of the FP receptor mRNAs after incubation for 24-48 h, as detected by Northern blot hybridization. The stimulatory effect of hCG was concentration and culture stage dependent. Further, hCG and 8-bromo-cAMP increased binding of radiolabeled PGF2 alpha to intact GL cells. In contrast, phorbol 12-myristate 13-acetate inhibited basal as well as hCG- and 8-bromo-cAMP-induced FP receptor mRNA expression and binding of the radiolabeled ligand. In summary, hCG, 8-bromo-cAMP, and phorbol 12-myristate 13-acetate modulate the expression of the FP receptor in human GL cells, which may represent a mechanism to regulate the responsiveness of the ovary to PGF2 alpha.

Secretion of monocyte chemoattractant protein-1 by endothelial cells of the bovine corpus luteum: regulation by cytokines but not prostaglandin F2alpha.

Information regarding the regulation of monocyte chemoattractant protein-1 (MCP-1) in regression of the corpus luteum (CL) is limited. This study tested the hypothesis that endothelial cells derived from bovine CL are a source of MCP-1, and that proinflammatory cytokines, prostaglandin F2alpha (PGF2alpha), and progesterone regulate MCP-1 expression. Endothelial cells were treated without (Control) or with PGF2alpha (1 micro M), TNFalpha (100 ng/ml), interferon-gamma (IFNgamma, 200 IU/ml), and TNFalpha + IFNgamma for 24 and 48 h in the absence or presence of progesterone (P4, 250 ng/ml). Increases in MCP-1 mRNA and protein were observed in response to TNFalpha within 24 and 48 h of culture, respectively (P < 0.05). Interferon-gamma stimulated (P < 0.05) both MCP-1 mRNA and protein after 24 h of culture, and this effect was also sustained through 48 h of culture (P < 0.05). Cotreatment of cultures with TNFalpha + IFNgamma lead to further increases (P < 0.05) in MCP-1 in both 24- and 48-h cultures. Surprisingly, neither PGF2alpha nor P4 affected MCP-1 production. Subsequent experiments revealed that the endothelial cells lacked prostaglandin F2alpha receptor mRNA, and the MAPK pathway, although present and responsive to growth factor stimulation, was unresponsive to PGF2alpha stimulation. In summary, endothelial cells derived from bovine CL respond to TNFalpha and IFNgamma stimulation with an increase in MCP-1 secretion. In contrast, neither PGF2alpha nor P4 directly influenced endothelial expression of MCP-1. These results suggest that cytokines stimulate the synthesis of MCP-1 observed during PGF2alpha-induced luteal regression.

Host factors and breast cancer growth characteristics.

The rate of growth and spread of breast cancer varies considerably from patient to patient. An observational study was undertaken to identify possible associations between breast cancer growth characteristics and a wide variety of host factors, including demographic, anthropometric, hormonal and dietary variables in 91 patients with breast cancer. Increasing age was associated with favourable growth characteristics, while previous tonsillectomy was associated with adverse growth characteristics. There were no significant associations in anthropometric variables. For postmenopausal women, increasing bioavailability of oestradiol was associated with favourable growth characteristics, while increasing prolactin concentration was associated with adverse growth characteristics. Increasing consumption of sugar, fibre, fruit and vegetables and vitamins was associated with favourable growth characteristics. Consumption of fat (monounsaturated and saturated) was associated with adverse characteristics when adjustment was made for total energy intake. The host environment may play a role in the control of breast cancer growth. In particular, the associations with oestrogen and progesterone receptor status indicate that nutrients may be of value as biological response modifiers in patients having hormonal therapy. This requires further investigation to assess therapeutic potential.

Demonstration of specific E-type prostaglandin receptors using enriched preparations of canine parietal cells and [3H]misoprostol free acid.

High-affinity, E-type prostaglandin binding sites in enriched canine parietal cell preparations were identified with [3H] misoprostol free acid, a prostaglandin E1 analogue. Saturable, reversible, and highly stereospecific binding was identified, with approximately 8,000 binding sites per cell. Prostaglandin I and F bound weakly, and cimetidine and histamine did not bind. The results indicate that [3H] misoprostol free acid binds to E-type prostaglandin receptors, which suggests that the ulcer-healing inhibition of gastric acid secretion by misoprostol results from its interaction with a specific E-type prostaglandin receptor.

Prostaglandin E2 binding sites in bovine iris-ciliary body.

In the eye, prostaglandins (PGs), in particular PGE2 and PGF2 alpha, may induce vasodilation, disruption of the blood-aqueous barrier, and biphasic effects on intraocular pressure, depending on the species. The initial event leading to many of these physiologic responses is the interaction between the PG and a receptor. We have explored the specificity and selectivity of PGE2 receptors in bovine iris-ciliary body (ICB) membrane preparations. Pigment-free bovine ICB membranes were prepared by high-speed sucrose density-gradient centrifugation. Membranes were incubated with 1 nM 3H-PGE2 in the presence or absence of varying concentrations of unlabeled PGE2 or F2 alpha. Binding of 3H-PGE2 to membranes at 37 degrees C increased linearly with protein concentration, and binding reached equilibrium in 30 min. Specific PGE2 binding represented 80% of total 3H-PGE2 binding. Studies with unlabeled PGE2 or F2 alpha, as competing ligands, showed a dose-dependent inhibition of 3H-PGE2 specific binding. The IC50 for unlabeled PGE2 and F2 alpha was 3 and 379 nM, respectively, which suggests a 100-fold greater selectivity of the binding sites for PGE2 over F2 alpha. Scatchard analysis of saturation data revealed a mean Kd value of 13.3 nM with a Bmax of 156 fmoles bound/mg protein. The general linearity of our Scatchard plots tends to suggests a single class of binding sites for PGE2, although more than a single binding site could be present. These results indicate that binding sites selective for PGE2 exist in the bovine ICB.

Localization of EP(1) and FP receptors in human ocular tissues by in situ hybridization.

PURPOSE: To examine the expression and localization of EP(1) and FP receptor mRNAs in normal human ocular tissues by in situ hybridization. METHODS: Digoxigenin-labeled human EP(1) and FP receptor antisense and sense riboprobes were used for in situ hybridization on paraffin sections of normal human eye tissue. RESULTS: In situ hybridization revealed the presence of high levels of both EP(1) and FP receptor mRNA transcripts in the blood vessels of iris, ciliary body, and choroid. Both the endothelial and smooth muscle cells of blood vessels demonstrated intense hybridization signals corresponding to EP(1) receptor mRNA transcript. EP(1) receptor hybridization signals were present in all the muscle fibers of the ciliary body. In the retina, hybridization signals for EP(1) receptors were observed in photoreceptors and both nuclear layers and in ganglion cells. The hybridization signals corresponding to FP receptor transcript were similar to those of EP(1) receptors in the iris tissues. In the ciliary muscle, FP receptor mRNA transcript was predominantly present in the circular muscle and in the collagenous connective tissues; no hybridization signal for this receptor was observed in the retina. CONCLUSIONS: The wide distribution of EP(1) and FP receptor mRNAs in human ocular tissues appears to be localized in the functional sites of the respective receptor agonists. Selective localization of FP receptor mRNA in the circular muscles and collagenous connective tissues of the ciliary body suggests their involvement in the increased uveoscleral outflow of aqueous humor by PGF(2alpha).

Localization of the prostaglandin F2 alpha receptor messenger RNA and protein in the cynomolgus monkey eye.

PURPOSE: To determine the distribution of the prostaglandin F2 alpha (FP) receptor within the monkey eye. METHODS: The expression and localization of the FP receptor was studied by in situ hybridization and immunohistochemistry. Cryosections of the eye were hybridized with a 35S-labeled FP receptor riboprobe, and paraffin sections were immunostained with polyclonal antibodies against an FP receptor peptide. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed on cultured cell populations from the eye. RESULTS: Results of the three methods largely correlated with each other. The highest expression of FP receptor mRNA and protein was found in the corneal, conjunctival, and iridial epithelium, the ciliary muscle, and ciliary processes. Iridial and choroidal melanocytes, the retina, and the optic nerve expressed lower levels of both FP receptor message and protein. Using immunohistochemistry, the FP receptor protein was found in connective tissue fibroblasts, the corneal endothelium, and the vasculature; however, FP receptor expression was not detected using in situ hybridization. With RT-PCR, cultured retinal pigment epithelial and ciliary muscle cells from the cynomolgus monkey eye were found to express the FP receptor. CONCLUSIONS: The FP receptor was found to be distributed widely in the ocular tissues, suggesting an array of autocrine and paracrine functions of PGF2 alpha in the eye.