Rapid effects of 17beta-estradiol and progesterone on sheep visceral and parietal pleurae via a nitric oxide pathway.

We investigated the effects of 17beta-estradiol and progesterone on transepithelial electrical resistance (R(TE)) in sheep visceral and parietal pleurae. Specimens of intact pleurae from adult female sheep were used. The samples were transferred to the laboratory within 30 min after death of the animal in a Krebs-Ringer solution at 4 degrees C. The pleura was then mounted as a planar sheet in Ussing-type chambers, and electrical measurements were made. There was an increase in R(TE) in all of the samples examined after addition of 17beta-estradiol and progesterone in visceral and parietal pleurae. This increase was rapid within 1 min, lasted for ~15 min, returned to the basal level within 30-45 min, and was dose dependent. Tamoxifen, an estrogen receptor antagonist, did not significantly eliminate the effect of 17beta-estradiol. Furthermore, no steroid receptors were identified in cytosolic preparations of visceral and parietal pleura with ligand binding assays. The estrogen- and progesterone-induced increase in R(TE) in both visceral and parietal pleurae was affected by addition of an inhibitor of nitric oxide synthase. Indeed, previous administration of N(omega)-nitro-L-arginine methyl ester prevented the increase in R(TE) by 17beta-estradiol and progesterone. These results suggest that 17beta-estradiol and progesterone induce an increase in R(TE) in both visceral and parietal pleura and thus alter the transepithelial permeability. The effect of steroids may be accounted for by rapid release of nitric oxide in pleura.

Opioids are non-competitive inhibitors of nitric oxide synthase in T47D human breast cancer cells.

Opioids and nitric oxide (NO) interact functionally in different systems. NO-generating agents decrease the activity of opioid agonists, prevent opioid tolerance, and are used in opioid withdrawal syndromes. There exist, however, few reports indicating a direct interaction of the two systems. T47D human breast cancer cells in culture express opioid receptors, and opioid agonists inhibit their growth, while they release high amounts of the NO-related molecules NO(2-)/NO(3-)to the culture medium. We have used this system to assay a possible direct interaction of opiergic and nitric oxide systems. Our results show that delta- or mu-acting opioid agonists do not modify the release of NO(2-)/NO(3-). In contrast, kappa-acting opioid agonists (ethylketocyclazocine, and alpha(S1)-casomorphine) decrease the release of NO(2-)/NO(3-), in a time- and dose-dependent manner. The general opioid antagonist diprenorphine (10(-6) M) produce a similar NO(2-)/NO(3-)release inhibition, indicating a possible non-opioid-receptor mediated phenomenon. In addition, ethylketocyclazocine, alpha(S1)-casomorphin and diprenorphine directly inhibit NOS activity: agonists, interact with both calcium-dependent and independent NOS-isoforms, while the antagonist diprenorphine modifies only the activity of the calcium-dependent fraction of the enzyme. Analysis of this interaction revealed that opioids modify the dimeric active form of NOS, through binding to the reductase part of the molecule, acting as non-competitive inhibitors of the enzyme. This interaction opens interesting new possibilities for tumor biology and breast cancer therapy.

Somatostatin and opioid receptors in mammary tissue. Role in cancer cell growth.

Somatostatin and opioid systems, are the two main inhibitory systems in mammals. Both classes of substances have been identified in normal and malignant mammary gland, as well as their cognitive receptors. They have been implied in the inhibition of cell growth of cancer cells and cell lines, in a dose-dependent and reversible manner. Somatostatin acts through homologous receptors (SSTRs), belonging to five distinct classes (SSTR1-5). We, and others have identified SSTR2 and 3 as been the only SSTRs present in the breast. Furthermore, opioids act through the three classes of opioid receptors (mu, delta,kappa). In the breast, kappa opioid receptor subtypes (kappa 1-kappa 3) are the most widely expressed. We further have shown that opioids, in addition to their binding to opioid receptors, compete for binding to SSTRs. This functional interaction, together with other identified modes of opioid action in the breast (modulation of steroid receptors, proteases' secretion, interaction with cytoskeletal elements), will be discussed, taking into consideration also the possible local production of casomorphins (casein-derived opioids), which are very potent antiproliferative agents.

TNF receptor family member BCMA (B cell maturation) associates with TNF receptor-associated factor (TRAF) 1, TRAF2, and TRAF3 and activates NF-kappa B, elk-1, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase.

BCMA (B cell maturation) is a nonglycosylated integral membrane type I protein that is preferentially expressed in mature B lymphocytes. Previously, we reported in a human malignant myeloma cell line that BCMA is not primarily present on the cell surface but lies in a perinuclear structure that partially overlaps the Golgi apparatus. We now show that in transiently or stably transfected cells, BCMA is located on the cell surface, as well as in a perinulear Golgi-like structure. We also show that overexpression of BCMA in 293 cells activates NF-kappa B, Elk-1, the c-Jun N-terminal kinase, and the p38 mitogen-activated protein kinase. Coimmunoprecipitation experiments performed in transfected cells showed that BCMA associates with TNFR-associated factor (TRAF) 1, TRAF2, and TRAF3 adaptor proteins. Analysis of deletion mutants of the intracytoplasmic tail of BCMA showed that the 25-aa protein segment, from position 119 to 143, conserved between mouse and human BCMA, is essential for its association with the TRAFs and the activation of NF-kappa B, Elk-1, and c-Jun N-terminal kinase. BCMA belongs structurally to the TNFR family. Its unique TNFR motif corresponds to a variant motif present in the fourth repeat of the TNFRI molecule. This study confirms that BCMA is a functional member of the TNFR superfamily. Furthermore, as BCMA is lacking a "death domain" and its overexpression activates NF-kappa B and c-Jun N-terminal kinase, we can reasonably hypothesize that upon binding of its corresponding ligand BCMA transduces signals for cell survival and proliferation.

Potent inhibitory action of red wine polyphenols on human breast cancer cells.

Breast cancer (one of the most common malignancy in Western societies), as well as esophagus, stomach, lung, bladder, and prostate cancer, depend on environmental factors and diet for growth and evolution. Dietary micronutriments have been proposed as effective inhibitory agents for cancer initiation, progression, and incidence. Among them, polyphenols, present in different foods and beverages, have retained attention in recent years. Red wine is a rich source of polyphenols, and their antioxidant and tumor arresting effects have been demonstrated in different in vitro and in vivo systems. In the present study, we have measured the antiproliferative effect of red wine concentrate, its total polyphenolic pool, and purified catechin, epicatechin, quercetin, and resveratrol, which account for more than 70% of the total polyphenols in red wine, on the proliferation of hormone sensitive (MCF7, T47D) and resistant (MDA-MB-231) breast cancer cell lines. Our results indicate that polyphenols, at the picomolar or the nanomolar range, decrease cell proliferation in a dose- and a time-dependant manner. In hormone sensitive cell lines, a specific interaction of each polyphenol with steroid receptors was observed, with IC(50)s lower than previously described. Interaction of polyphenols with steroid receptors cannot fully explain their inhibitory effect on cell proliferation. In addition, discrete antioxidant action on each cell line was detected under the same concentrations, both by modifying the toxic effect of H(2)O(2), and the production of reactive oxygen species (ROS), after phorbol ester stimulation. Our results suggest that low concentrations of polyphenols, and consecutively, consumption of wine, or other polyphenol-rich foods and beverages, could have a beneficial antiproliferative effect on breast cancer cell growth.

Wine antioxidant polyphenols inhibit the proliferation of human prostate cancer cell lines.

The effect of different wine antioxidant polyphenols (catechin, epicatechin, quercetin, and resveratrol) on the growth of three prostate cancer cell lines (LNCaP, PC3, and DU145) was investigated. A dose- and time-dependent inhibition of cell growth by polyphenols was found at nanomolar concentrations. The proliferation of LNCaP and PC3 cells was preferentially inhibited by flavonoids (catechin, epicatechin, and quercetin), whereas resveratrol was the most potent inhibitor of DU145 cell growth. Possible mechanisms of action were investigated: 1) The competition of polyphenols for androgen binding in LNCaP cells revealed significant interaction only in the case of high concentrations of quercetin, at least at five orders of magnitude higher than the concentrations needed for cell growth inhibition. All other phenols showed low interactions. 2) Oxygen species production after mitogen stimulation and H2O2 sensitivity of these cell lines did not correlate with the observed antiproliferative effects, ruling out such a mode of action. 3) NO production revealed two different patterns: LNCaP and DU145 cells produced high concentrations of NO, whereas PC3 cells produced low concentrations. Phorbol ester stimulation of cells did not reveal any additional effect in LNCaP and DU145 cells, whereas it enhanced the secretion of NO in PC3 cells. Polyphenols decreased NO secretion. This effect correlates with their antiproliferative action and the inhibition of inducible NO synthase. It is therefore proposed that the antiproliferative effect of polyphenols is mediated through the modulation of NO production. In conclusion, our data show a direct inhibitory effect of low concentrations of antioxidant wine phenols on the proliferation of human prostate cancer cell lines mediated by the production of NO, further suggesting potential beneficial effects of wine and other phenol-containing foods or drinks for the control of prostate cancer cell growth.

Opioid agonists modify breast cancer cell proliferation by blocking cells to the G2/M phase of the cycle: involvement of cytoskeletal elements.

Opioids decrease cell proliferation in different systems including breast, prostate, lung, kidney, and intestine, through an interaction with opioid as well as other membrane-receptor systems (somatostatin, cholinergic), through an unidentified mechanism. Recently, we have reported an interaction of taxol with opioid membrane sites (BBRC 235, 201-204, 1997), and an involvement of opioids to the modification of actin cytoskeleton in renal OK cells (J Cell Biochem. [19981 70:60-69), indicating a possible action of the opioid effect. In the present work, we have examined the effect of two general opioid agonists (ethylketocyclazocine and etorphine) on the cell cycle, in human breast cancer T47D cells, as well as a possible modification of the cellular cytoskeleton under their action, in order to explain the antiproliferative effect of these agents. These two opioids produce a dose-dependent and reversible decrease of the proliferation of T47D cells, with a maximum attained at 10(-8) M. The addition of 10(-8) M of either opioid produced a significant increase of the number of cells arrested in the G2/M phase. Confocal laser microscopy revealed a modification of the actin and tubulin microfilaments, with a clear redistribution at the periphery of the cell, reversed by the addition of the general opioid antagonist diprenorphine. Furthermore, differences between the two opioids were obvious, attributed to the different receptor affinity of each agent. The observed redistribution of actin and tubulin cytoskeletal elements gives therefore a possible answer of the antiproliferative action of opioids. The modification of the cytoskeleton, directly involved to cell division, might provoke a "mechanical" obstacle, which could be the reason of the antiproliferative effect of these agonists. Furthermore, the observed tubulin-opioid interaction by opioids provides a possible explanation of the arrest at the G2/M phase of T47D cells under opioid treatment. Nevertheless, although the observed interaction of opioids with cytoskeletal elements gives a plausible answer of the antiproliferative effects of the agents, this might not be the only action of these agents in cell proliferation. Other, direct or indirect, genomic actions, which which remains to be elucidated, might be taken into consideration.

Kappa1-opioid binding sites are the dominant opioid binding sites in surgical specimens of human pheochromocytomas and in a human pheochromocytoma (KAT45) cell line.

The adrenal medulla produces opioids which exert paracrine effects on adrenal cortical and chromaffin cells and on adrenal splanchnic nerves, via specific binding sites. The opioid binding sites in the adrenals are detectable mainly in the medullary part of it and differ in type between species. Thus, the bovine adrenal medulla contains mostly kappa-opioid binding sites and fewer delta- and mu-opioid binding sites while primate adrenals contain mainly delta sites and few kappa-opioid binding sites. Most chromaffin cell tumors, the pheochromocytomas, produce opioids which suppress catecholamine production by the tumor. The aim of the present work was to identify the types of opioid binding sites in human pheochromocytomas. For this purpose, we characterized the opioid binding sites on crude membrane fractions prepared from 14 surgically excised pheohromocytomas and on whole KAT45 cells, a recently characterized human pheochromocytoma cell line. Our data showed that human pheohromocytomas are heterogeneous, as expected, with regard to the production of catecholamines and the distribution and profile of their opioid binding sites. Indeed, only one out of the 14 pheochromocytomas expressed exclusively delta and mu opioid sites, while in the remaining 13 tumors kappa-type binding sites were dominant. The KAT45 cell line possessed a significant number of kappa1 binding sites, fewer kappa2-opioid binding sites and kappa3-opioid binding sites, and minimal binding capacity for delta- and mu-opioid receptor agonists sites. More specifically, the kappa1 sites/cell were approximately 18,000, the kappa2 4500/cell and the kappa3 sites 2000/cell. Our findings for the surgical specimens and the cell line combined with previously published pharmacological data obtained from KAT45 cells suggest that kappa sites appear to be the most prevalent opioid binding sites in pheochromocytomas. Finally, in normal bovine adrenals the profile of opioid binding sites differs in adrenaline and noradrenaline producing chromaffin cells. To test the hypothesis that the type of catecholamine produced by a pheochromocytoma depends on its cell of origin, we compared our binding data with the catecholamine content of each pheochromocytoma examined. We found no correlation between the type of the predominant catecholamine produced and the opioid binding profile of each tumor suggesting that this hypothesis may not be valid.

The characterization of murine BCMA gene defines it as a new member of the tumor necrosis factor receptor superfamily.

The BCMA gene is a new gene discovered by the molecular analysis of a t(4;16) translocation, characteristic of a human T cell lymphoma. It has no significant similarity with any known protein or motif, so that its function was unknown. This report describes the cloning of murine BCMA cDNA and its genomic counterpart. The mouse gene is organized into three exons, like the human gene, and lies in murine chromosome 16, in the 16B3 band, the counterpart of the human chromosome 16p13 band, where the human gene lies. Murine BCMA cDNA encodes a 185 amino acids protein (184 residues for the human), has a potential central transmembrane segment like the human protein and is 62% identical to it. The murine BCMA mRNA is found mainly in lymphoid tissues, as is human BCMA mRNA. Alignment of the murine and human BCMA protein sequences revealed a conserved motif of six cysteines in the N-terminal part, which strongly suggests that the BCMA protein belongs to the tumor necrosis factor receptor (TNFR) superfamily. Human BCMA is the first member of the TNFR family to be implicated in a chromosomal translocation.

Modulation of the estrogen-regulated proteins cathepsin D and pS2 by opioid agonists in hormone-sensitive breast cancer cell lines (MCF7 and T47D): evidence for an interaction between the two systems.

In many cancer cell lines, including breast, prostate, lung, brain, head and neck, retina, and the gastrointestinal tract, opioids decrease cell proliferation in a dose-dependent and reversible manner. Opioid and/or other neuropeptide receptors mediate this decrease. We report that only the steroid-hormone-sensitive cell lines MCF7 and T47D respond to opioid growth inhibition in a dose-dependent manner. Therefore, an interaction of the opioid and steroid receptor system might exist, as is the case with insulin. To investigate this interaction, we have assayed two estrogen-inducible proteins (pS2 and the lysosomal enzyme cathepsin D) in MCF7 and T47D cells. When cells were grown in the presence of FBS (in which case a minimal quantity of estrogens and/or opioids is provided by the serum), we observed either no effect of etorphine or ethylketocyclazocine (EKC) or an increase of secretion and/or production of pS2 and cathepsin D. However, when cells were cultured in charcoal-stripped serum and in the absence of phenol red, the effect of the two opioids is different: EKC decreased the production and/or secretion of pS2 and cathepsin D, whereas etorphine increased their synthesis and/or secretion. The differential effect of the two general opioids was attributed to their different receptor selectivity. Furthermore, the variations of the ratio of secreted/produced protein and the use of cycloheximide indicate that opioids selectively modify the regulatory pathway of each protein discretely. In conclusion, through the interaction with opioid and perhaps other membrane-receptor sites, opioid agonists modify in a dose-dependent manner the production and the secretion of two estrogen-regulated proteins. Opioids may therefore disturb hormonal signals mediated by the estrogen receptors. Hence, these chemicals may have potential endocrine disrupting activities.

Opioid alkaloids and casomorphin peptides decrease the proliferation of prostatic cancer cell lines (LNCaP, PC3 and DU145) through a partial interaction with opioid receptors.

Opioid agonists (ethylketocyclazocine, etorphine, [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Ala2, N-Me-Phe4-Gly-ol]enkephalin (DAGO), [D-Ser2,Leu5]enkephalin-Thr6 (DSLET) and morphine were found to inhibit the proliferation of human prostate cancer cell lines (LNCaP, DU145, and PC3), in a dose-dependent manner. The 50% inhibitory concentrations (IC50) were in the picomolar range. In many cases, this effect was antagonized by the general opioid antagonist, diprenorphine, indicating the existence of specific opioid binding sites. Saturation binding experiments with selective ligands and effectors showed no opioid sites on the LNCaP cell line, kappa1 and mu sites on the PC3 cell line, and kappa1, kappa3 and mu sites on the DU145 cell line. In other cases, the opioid effect was not antagonized by diprenorphine, indicating that the action of opioids might be mediated through other membrane receptors. Furthermore, casomorphin peptides, issued from bovine alpha- (alpha-casein-90-95 and alpha-casein-90-96) and beta-caseins (beta-casomorphin and beta-casomorphin-1-5), and human alphaS1-casein (alphas -casomorphin and alphaS1-casomorphin amide) inhibited cell proliferation of human prostate cell lines, also by a mechanism partly involving opioid receptors. As opioid neurons can be found in the prostate gland, and casomorphin peptides might reach the gland through the general circulation, the above findings indicate a putative role of opioids in prostate cancer cell growth.

Taxol inhibits opioid binding on T47D human breast cancer cells.

In the T47D human breast cancer cell line, Taxol was found to compete for ethylketocyclazocine opioid binding (IC50 3.3 pM). In contrast, no interaction of the drug with [3H]diprenorphine binding occurred. Binding was multiphasic, in the absence of colchicine (10[-6] M), but monophasic in its presence, indicating an involvement of the cytoskeleton in this process. Alignment of Taxol binding domains on alpha and beta tubulin with the kappa opioid site revealed homology of these sites with the first extracellular loop of the receptor. These results indicate a possible new action of Taxol, indicating for the first time a membrane action of the agent.

Identification and characterization of opioid and somatostatin binding sites in the opossum kidney (OK) cell line and their effect on growth.

Opioids and somatostatin analogs have been implicated in the modulation of renal water handling, but whether their action is accomplished through central and/or peripheral mechanisms remains controversial. In different cell systems, on the other hand, opioids and somatostatin inhibit cell proliferation. In the present study, we have used an established cell line, derived from opossum kidney (OK) proximal tubules, in order to characterize opioid and somatostatin receptors and to investigate the action of opioids and somatostatin on tubular epithelial tissue. Our results show the presence of one class of opioid binding sites with kappa, selectivity (KD 4.6 +/- 0.9 nM, 57,250 sites/cell), whereas delta, mu, or other subtypes of the kappa site were absent. Somatostatin presents also a high affinity site on these cells (KD 24.5 nM, 330,000 sites/cell). No effect of either opioids or somatostatin on the activity of the NA+/Pi cotransporter was observed, indicating that these agents do not affect ion transport mechanisms. However, opioid agonists and somatostatin analogs decrease OK cell proliferation in a dose-dependent manner; in the same nanomolar concentration range, they displayed reversible specific binding for these agents. The addition of diprenorphine, a general opioid antagonist, reversed the effects of opioids, with the exception of morphine. Furthermore, morphine interacts with the somatostatin receptor in this cell line too, as was the case in the breast cancer T47D cell line. Our results indicate that in the proximal tubule opioids and somatostatin do not affect transport, but they might have a role in the modulation of renal cell proliferation either during ontogenesis or in kidney repair.

Identification of a novel opioid peptide (Tyr-Val-Pro-Phe-Pro) derived from human alpha S1 casein (alpha S1-casomorphin, and alpha S1-casomorphin amide).

A new casomorphin pentapeptide (alpha S1-casomorphin) has been isolated from the sequence of human alpha S1-casein [alpha S1-casein-(158-162)], with the sequence Tyr-Val-Pro-Phe-Pro. This peptide was found to bind with high affinity to all three subtypes of the kappa-opioid receptor (kappa 1-kappa 2). When amidated at the C-terminus, alpha S1-casomorphin amide binds to the delta- and kappa 3-opioid sites. Both alpha S1-casomorphin and its amide inhibit in a dose-dependent and reversible manner the proliferation of T47D human breast cancer cells. This anti-proliferative activity was greater for alpha S1-casomorphin, which was the most potent opioid in inhibiting T47D cell proliferation. In T47D breast cancer cells, other casomorphins have been found to bind to somatostatin receptors in addition to opioid sites. In contrast, alpha S1-casomorphin and its amide do not interact with somatostatin receptors in our system.

Antiproliferative and receptor binding properties of alpha- and beta-casomorphins in the T47D human breast cancer cell line.

In previous studies, we have shown that opioid agonists ([D-Ala2, D-Leu5]enkephalin (DADLE), [D-Ser2, Leu5]enkephalin-Thr6 (DSLET), ethylketocyclazocine and etorphine) bind to opioid binding sites and decrease cell proliferation of human T47D breast cancer cells. Furthermore, we provided evidence about a cross-reaction, also in the T47D human breast cancer cell line, of mu-acting opioids with type-II somatostatin receptors. Since a potential source of opioid activity in the breast might be casomorphin peptides (produced by the enzymatic degradation of alpha-casein and beta-casein), we investigated the antiproliferative action of five different casomorphin peptides: alpha-casein-(90-95), alpha-casein-(90-96), beta-casomorphin, beta-casomorphin-(1-5) and morphiceptin. We show that all five peptides decreased, in a dose-dependent manner, cell proliferation. The general antagonist diprenorphine produced only a partial reversal of their action. Furthermore, we provide evidence that all peptides (except for morphiceptin) bind to delta- and kappa-opioid binding sites of T47D cells with different selectivity. Finally, we show that these peptides are also partial competitors at the somatostatin receptors present in the same cell line.

The antiproliferative effect of opioid receptor agonists on the T47D human breast cancer cell line, is partially mediated through opioid receptors.

In the present study, we investigated the action of opioid receptor agonists on the proliferation of cells of the T47D human breast cancer cell line, grown in the absence of exogenously added steroids and growth factors. We found that the opioid receptor agonists ethylketocyclazocine, morphine, [D-Ala2,D-Leu5]enkephalin (DADLE), [D-Ser2,Leu5]enkephalin-Thr6 (DSLET) and etorphine inhibit dose dependently cell proliferation. The opioid receptor antagonist diprenorphine had no significant effect per se, but it was able to reverse the action of all opioid receptor agonists except morphine. In order to investigate the mechanism of action of opioids on T47D cells, we characterised the opioid receptors present on this cell line, by saturation binding, using radiolabelled [D-Ala2,N-Me-Phe4-Gly5-ol]enkephalin (DAGO, mu-opioid receptor agonist), ethylketocyclazocine (kappa 1-, kappa 2-, mu- and delta-opioid receptor agonist), diprenorphine (kappa 2-, kappa 3-, delta- and mu-opioid receptor antagonist), DADLE (delta- and mu-opioid receptor agonist), and effectors. We identified opioid binding sites belonging mainly to the kappa-type (kappa 1, kappa 2 and kappa 3), a few delta-opioid receptor sites, but no mu-opioid receptors. Our results indicate that the inhibitory effect of opioids on T47D cell growth is mediated through kappa- and delta-opioid receptors. The effect of mu-acting morphine might not be mediated through opioid receptors.

Identification, characterization and localization of corticotropin-releasing hormone receptors in human placenta.

The purpose of this work was to identify, characterise, and localise specific CRH binding sites in whole vaginally delivered term human placental membranes as well as in dispersed and purified trophoblastic membranes. We have found that whole placenta membranes contained specific and saturable CRH-binding sites. Maximum specific binding was obtained at pH 7.2-7.4, 22 C, for 2 h. The order of potency of CRH analogs was h/rCRH > alpha-helCRH > oCRH. Scatchard analysis revealed a single population of high affinity binding sites with a dissociation constant of 1.25 nM and maximum binding capacity of 119 fmol/mg of protein. Purified syncytiotrophoblast membranes contained high affinity CRH binding sites exhibiting the same dissociation constant and maximum binding capacity as whole placental membranes, suggesting that the CRH binding sites are expressed almost exclusively by the syncytiotrophoblast. Our findings indicate that CRH binding sites in the human placenta are exhibit similar characteristics to that described for anterior pituitary, brain and adrenals.

Morphine cross-reacts with somatostatin receptor SSTR2 in the T47D human breast cancer cell line and decreases cell growth.

In a previous study, we found that morphine decreases, in a dose-dependent manner, the cell growth of T47D human breast cancer cells, despite the lack of mu opioid receptors and an interaction of morphine with other opioid sites. We have therefore examined a possible interaction of morphine with other membrane receptor systems of the cell. The present study describes for the first time an interaction between mu-acting opioid drugs and the somatostatinergic system. We have found that [125I]Tyr11-somatostatin binds with high affinity to T47D cells. Analysis of the binding data showed the presence of two components: one with high affinity but low capacity (Kd, 0.145 nM; 1450 sites/cell), and another of lower affinity but higher capacity (Kd, 1.192 nM; 11920 sites/cell). Somatostatin-14 and somatostatin-28 showed multiphasic displacement curves, indicating heterogeneity of binding sites. The latter was confirmed by reverse transcription-PCR, which revealed the existence of the somatostatin receptor subtypes 2 and 3 (SSTR2 and SSTR3), with a relative mRNA concentration of 85 and 15%, respectively. Morphine and the morphinomimetic peptide morphiceptine (Tyr-Pro-Phe-Pro-NH2) displace somatostatin from its binding sites. Further analysis indicated that mu-acting opioids interact with the SSTR2 receptor subtype.

Identification and characterization of opioid-binding sites present in the Ishikawa human endometrial adenocarcinoma cell line.

Normal epithelial cells of human endometrium, and Ishikawa human endometrial adenocarcinoma cells (an in vitro model for the study of steroid hormone effects on human endometrium) have been found to express and secrete opioid peptides deriving from proenkephalin, prodynorphin, and proopiomelanocortin. These opioids may act locally, affecting the uterine tissues. In the present study, we identified and characterized opioid-binding sites on the Ishikawa cell line, producing evidence for the mechanism of local opioid action. We used an acid shock before the receptor assay to dissociate any endogenously bound peptide. The acidification improved specific binding by 2- to 4.5-fold. Characterization of opioid binding using different radiolabeled opioids and effectors has shown the existence of a low concentration of delta-sites (Kd, 6.20 nmol/L; 4,890 sites/cell), no mu-sites, low affinity kappa 1-sites (Kd, 10.8 nmol/L; 276,000 sites/cell), kappa 2-sites with high affinity for ethylketocyclazocine (Kd, approximately 1 nmol/L) and low affinity for diprenorphine (Kd, approximately 8 nmol/L) at a concentration of 93,000 sites/cell, and high affinity kappa 3-sites (Kd, 3.6 nmol/L; 77,000 sites/cell). In conclusion, our report characterizes opioid sites in a particular and homogeneous cell type of human endometrium, i.e. in epithelial cells. The coexistence of opioid sites and their endogenous ligands in the Ishikawa cell line makes these cells a good model for the study of autocrine/paracrine interactions of opioids in nonneural tissues.

Estrogen and progesterone receptors in breast cancer microsamples simultaneously quantified by enzyme-ligand immunoassay.

A new method (enzyme-ligand immunoassay, ELIA) is described for the estimation of estrogen (ER) and progesterone (PR) receptors in microsamples of human breast cancer tissue. The technique, based on the nonisotopic measurement of receptor-bound estradiol and progesterone, involves three steps: (a) simultaneous saturation of active receptors with their respective authentic ligands, (b) heat treatment of the cytosol to release the steroids from their cognate receptors before or after absorption with dextran-coated charcoal, and (c) measurement of both steroids present in the cytosol by a modified competitive-inhibition enzyme immunoassay. The useful range of the method was 10-4000 pmol/L for ER and 6.5-1000 pmol/L for PR. The correlation coefficient (r) between the one-point and Scatchard plot analysis was 0.95 for ER and 0.99 for PR. Comparison of the one-point ELIA and expected values with the radioligand binding assay (RLBA) results for EORTC samples gave r = 0.88 and 0.99 for ER and PR, respectively. Further comparison of the one-point ELIA with RLBA and with a commercial enzyme immunoassay, in blind testing of cancer tissue microsamples from 70 patients, gave good agreement for ER with r = 0.95-0.97 and concordance of 92.9-94.4% (cutoff, 15 pmol/g protein) against the other two methods. The results were more disperse in all three methods for PR estimation, the assay correlating perhaps better with the enzyme immunoassay (r = 0.90) at a concordance of 89.4% (same cutoff value).