Tamoxifen enhances myoepithelial cell suppression of human breast carcinoma progression in vitro by two different effector mechanisms.

Our previous studies have indicated that myoepithelial cells surrounding ductal and acinar epithelium of glandular organs, such as the breast, exert multiple paracrine suppressive effects on incipient and developing cancers that arise from this epithelium. Myoepithelial cells and derived cell lines (HMS 1-6) exert these effects through the secretion of a number of different effector molecules that exert anti-invasive, anti-proliferative, and anti-angiogenic activities. Since previous basic and clinical studies have examined the role of estrogen agonists and antagonists on human breast cancer cells and because issues of hormone replacement therapy (HRT) and tamoxifen chemoprevention are such timely issues in breast cancer, we wondered whether or not hormonal manipulations might affect myoepithelial cells in vitro as far as their paracrine suppressive activities on breast cancer were concerned. The present in vitro study demonstrates that treatment of myoepithelial cells with tamoxifen but not 17beta-estradiol increases both maspin secretion and invasion-blocking ability. Furthermore tamoxifen but not 17beta-estradiol increases inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by myoepithelial cells when they are co-cultured with conditioned media from or breast carcinoma cells directly. This increased myoepithelial NO exerts both autocrine and paracrine antiproliferative effects which can be blocked by inhibition of iNOS. 17beta-Estradiol, however, competes with all of these suppressive effects of tamoxifen suggesting that the mechanism of tamoxifen action is estrogen receptor mediated. Myoepithelial cells lack ER-alpha but express ER-beta. Tamoxifen, but not 17beta-estradiol, increases AP-1 CAT but not ERE-CAT activity. Again, 17beta-estradiol competes with the transcription-activating effects of tamoxifen. These experiments collectively suggest that the actions of tamoxifen on the increased secretion of maspin and increased production of NO by myoepithelial cells are mediated through ER-beta and the transcription-activation of an ER-dependent AP-1 response element.

A meningioma-mimicking tumor caused by Mycobacterium avium complex in an immunocompromised patient.

Intracranial tuberculomas manifesting radiologically as typical dural-based "meningiomas" have been reported, most frequently in immunosuppressed patients. Their incidence is high in developing countries; they are only sporadically observed in Western Europe and North America, usually in patients with acquired immunodeficiency syndrome (AIDS). According to published reports, intracranial tuberculomas are always due to infection by Mycobacterium tuberculosis. We report a case of a 50-year-old woman with systemic lupus erythematosus (SLE) who presented with a dural based, meningioma-like mass in the right frontal region, resulting from a localized infection by Mycobacterium avium complex. Histologically, the mass resembled a meningioma in being composed of spindly cells arranged in a fascicular pattern. Immunohistochemical stains showed this tumor to consist of a large aggregate of AFB-laden histiocytes without caseating necrosis or multinucleated giant cells.

Nitric oxide donors induce extrusion of cyclic GMP from isolated human blood platelets by a mechanism which may be modulated by prostaglandins.

In the presence of 3-isobutyl-methylxanthine (IBMX), induction of cyclic 3',5'-guanosine monophosphate (GMP) production in human washed platelets (HWP) by nitric oxide donors (NOD) is followed by its accumulation in the surrounding medium in a time- and concentration-dependent manner. Thirty minutes incubation of HWP with 3-morpholino-sydonimine (SIN-1, 10 microM) at 37 degrees C resulted in a 4.6-fold increase of cyclic GMP in platelets, whereas in the extracellular medium the increase was 17.6-fold. Similar results were obtained when other NOD such as S-nitroso-N-acetylpenicyllamine (SNAP) and 3-(2-methoxy-5-chlorophenyl)oxatriazol-5-imine (GEA 3184) and the selective phosphodiesterase inhibitor, zaprinast (M&B 22948, 10 microM), were used. Probenecid (1-300 microM), an inhibitor of organic anion transport, or ouabain (1-300 microM), an inhibitor of Na+/K+ adenine triphosphate (ATP)-ase had no effect on cyclic GMP production or extrusion after stimulation with SIN-1. Significantly prostaglandin A1 (PGA1) and prostaglandin D2 (PGD2) inhibited the efflux of cyclic GMP from platelets induced by SNAP (10 microM) in a concentration-dependent fashion, with an IC50 of 63 +/- 16 and 143 +/- 17 microM, respectively. These studies suggest that the extrusion of cyclic GMP from human platelets after activation of soluble guanylate cyclase by NOD may contribute to the control of cyclic GMP levels in platelets with potential physiological and therapeutic consequences.

Plasma ANP and cyclic GMP levels versus left ventricular performance at different AV delays in AV sequential pacing.

Eleven resting patients with an implanted DDD pacemaker were studied. After 30 minutes of AV sequential pacing at a rate of 80 beats/min with three consecutive atrioventricular delays (AVDs; 100, 150, and 200 msec) peripheral venous blood was drawn for further analyses by specific radioimmunoassays of atrial natriuretic peptide (ANP) and the ANP second messenger, cyclic guanosine monophosphate (cGMP). Relative changes in left ventricular (LV) stroke volume following alterations of AVD were assessed by means of pulsed-Doppler echocardiography through measurement of LV outflow time-velocity integrals (TVI). The optimal AVD (oAVD) was defined in individual patients as that which was associated with the greatest TVI and with improvement over both other AVDs of more than 4%. The oAVD was found in nine patients. For these nine patients no significant differences in either plasma ANP or cGMP between various AVDs were observed. However, we found such differences with respect to values measured at oAVD; both ANP and cGMP levels were lowest at oAVD. Pooling together the data obtained in 11 patients at three AVDs, a positive correlation between ANP and cGMP levels was found (r = 0.7, P < 0.0001, n = 33). Moreover, changes of plasma ANP and cGMP induced by every AVD increment of 50 msec were also correlated (r = 0.6, P < 0.01, n = 22). It is concluded that in AV sequential pacing at rest plasma ANP reaches minimal levels at the AVD, which provides the best LV performance. Although levels of cGMP changed in parallel with those of ANP, low relative values of cGMP differences may limit the usefulness of cGMP assays in optimization of the AVD.

Carboxyebselen a potent and selective inhibitor of endothelial nitric oxide synthase.

Ebselen (Ebs) a glutathione peroxidase like agent has been recently described as an inhibitor of nitric oxide synthase (NOS). Presently, we report that carboxyebselen (HOOC-Ebs), a hydrophyllic derivative of Ebs inhibits NOS present in enzymatic preparations from bovine endothelium, porcine cerebella, and murine spleen, however, it is both more potent and more selective for the constitutive endothelial NOS than Ebs. Unlike Ebs, HOOC-Ebs (0.1-30 microM) causes a concentration-dependent endothelium-independent relaxations of rings of rabbit aorta. The mechanism of this relaxation remains unknown and it is attenuated by glutathione (GSH, 30-300 microM) and N-acetyl-L-cysteine (NAC, 30-300 microM). The vasorelaxant activity of acetylcholine (Ach, 0.1-1 microM) in aortic rings exposed to low concentrations of HOOC-Ebs (0.1-1 microM) or rings exposed to 10 microM HOOC-Ebs after their pretreatment with GSH or NAC (30-300 microM) remained unchanged. The lack of activity of HOOC-Ebs as a NOS inhibitor in intact endothelial cells contrasts the effectiveness of Ebs in this respect.

Inhibition of endothelial nitric oxide synthase by ebselen. Prevention by thiols suggests the inactivation by ebselen of a critical thiol essential for the catalytic activity of nitric oxide synthase.

NO synthase (NOS) is a unique P-450-type enzyme containing both a reductase and a heme domain on a single polypeptide. We show that ebselen [Ebs, 2-phenyl-1,2-benzisoselenazol-3-(2H) one], a nontoxic selenoorganic compound known to break a cysteine thiolate/Fe bond of some of P-450 enzymes, is a relatively selective inhibitor of endothelial isoform of NOS. In rings of rabbit aorta, Ebs irreversibly blocked both the basal as well as acetylcholine- or calcium ionophore A23187-stimulated release of nitric oxide with an IC50 of 6 microM. In homogenates of bovine aortic endothelial cells, Ebs inhibited the activity of NOS, assayed by monitoring conversion of L-[2,3-3H]arginine to L-[2,3-3H]citrulline, with an IC50 of 8.5 microM. The inhibitory action of Ebs was prevented by glutathione, N-acetyl-L-cysteine or dithiothreitol (30-500 microM). The prevention by thiols of Ebs-induced inhibition of NOS suggests that these are competing with a thiol group of NOS that is essential for the catalytic activity of the enzyme. The consequence of the presence of thiols is the "trapping" of Ebs in the form of inactive selenyl sulfides. Consistent with the proposed mechanism of action of Ebs is lack of activity of diselenide of Ebs, which also demonstrates that the action of Ebs is independent of its glutathione peroxidase-like activity. In comparison to endothelial preparations, IC50 values of Ebs for inhibition of soluble isoforms of NOS present in homogenates of porcine cerebellum and of spleens obtained from lipopolysaccharide-treated rats were more than 30-fold higher.

Plasma ANP and cyclic GMP after physical exercise in patients with mitral valve disease and in healthy subjects.

Plasma levels of both atrial natriuretic peptide (ANP) and cyclic GMP are elevated in patients with various heart diseases as compared to healthy subjects. In this study patients with advanced mitral valve disease (Group A) and healthy subjects (Group B) were exposed to symptom-limited upright stepwise physical exercise on a cycle ergometer. Concentrations of ANP and cyclic GMP were measured in plasma at rest (20 min in supine position) or 5 min after physical exercise by specific radioimmunoassays. Here we show that short dynamic exercise caused a significant increase in plasma levels of ANP and cyclic GMP, in both groups. In Group A strong correlation between plasma ANP and cyclic GMP was found at rest (r = 0.91, P < 0.001, n = 11) and after physical exercise (r = 0.85, P < 0.001, n = 11). In contrast, there was no correlation between plasma concentrations of ANP and cyclic GMP in Group B at rest (r = -0.16, P > 0.05, n = 10) or after exercise loading (r = 0.14, P > 0.05, n = 10). Absolute increases in circulating levels of both substances were not found to correlate in either group. These data suggest that exercise-induced elevations in plasma cyclic GMP may be due not only to ANP release but also to an as yet undetermined factor, possibly EDRF/NO.

NG-hydroxy-L-arginine and hydroxyguanidine potentiate the biological activity of endothelium-derived relaxing factor released from the rabbit aorta.

We investigated the effects of NG-hydroxy-L-arginine (L-HOArg) and hydroxyguanidine (HOG) on the synthesis and vasorelaxant activity of endothelium-derived relaxing factor (NO) released from the rabbit aortic endothelium. Both L-HOArg (10 microM) and HOG (10 microM) equally potentiated the vasorelaxant activity of NO released by Ach (0.1 or 0.3 microM) from the luminally perfused rabbit aorta and bioassayed using the superfused strips of the endothelium-denuded rabbit aorta. This potentiation was caused by the generation of a more stable vasodilator during the chemical reaction of L-HOArg or HOG with NO and it was abolished by NG-nitro-L-arginine methyl ester (L-NO2Arg, 10 microM). In contrast, in organ baths, L-HOArg (10 microM) or HOG (10 microM) did not affect the relaxations of intact rabbit aortic rings induced by Ach (0.01-1 microM). At concentrations higher than 10 microM, both L-HOArg and HOG were endothelium-independent vasorelaxants. However, L-HOArg (100 microM) prevented the inhibition by L-NO2Arg (10 microM) of Ach-induced relaxations of bathed aortic rings which indicates that L-HOArg is still a substrate for the NO synthase in the endothelium of the rabbit aorta.

Prostaglandin E2 (PGE2) differentially regulates the production of IL-2 and IL-3 by murine immune T-cells.

Eicosanoids are important mediators of inflammation, and have been shown to have potent, and usually suppressive immunoregulatory activities. In the paper, we have examined the role of prostaglandin (PGE2) production in the regulation of two cytokines, IL-2 and IL-3, which both play a key role in contact sensitivity and delayed type hypersensitivity reactions. In agreement with previous studies, we demonstrate that prostaglandins down-regulate IL-2 production in the system. Unexpectedly, however, IL-3 levels are enhanced in the presence of the prostaglandin PGE2 and conversely, are inhibited by treatment with aspirin, a potent inhibitor of prostaglandin metabolism. The implications of this result in terms of the immunoregulatory role of PGs will be discussed.

Diphenylene iodonium, an inhibitor of free radical formation, inhibits platelet aggregation.

Diphenylene iodonium is an inhibitor of the enzyme NADPH-oxidase and prevents the generation of oxygen-derived free radicals in neutrophils (Cross and Jones, 1986). Here we show that diphenylene iodonium (0.25-2 microM) inhibited, according to the dose, thrombin-induced platelet-aggregation in human washed platelets and ADP-induced platelet aggregation in platelet-rich plasma. At the concentrations which inhibited platelet aggregation diphenylene iodonium did not alter platelet concentrations of cAMP or cGMP but enhanced the anti-platelet activity of iloprost, sodium nitroprusside or cultured endothelial cells. These findings highlight the importance of free radicals as platelet pro-aggregatory agents.

The use of oxyhaemoglobin to explore the events underlying inhibition of platelet aggregation induced by NO or NO-donors.

1. Full inhibition of thrombin-induced platelet aggregation was elicited by the least maximal platelet inhibitory concentrations of nitric oxide (NO; 7 +/- 1 microM) or NO-donors which included sodium nitroprusside (NaNp; 80 +/- 13 microM) 3-morpholinosydnonimine (SIN-1; 3 +/- 0.1 microM) or endothelial cells (EC; 2.36 +/- 0.12 x 10(5) added 1 min before thrombin. Oxyhaemoglobin (oxyHb; 10 microM) administered 30s to 10 min after stimulation with thrombin caused a time-dependent reversal of the inhibition induced by these agents. OxyHb was ineffective when these agents were co-incubated with the non-selective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 0.05 mM). 2. OxyHb did not reverse the platelet inhibition with IBMX (0.2 mM) or that caused by a selective guanosine 3'; 5'-cyclic monophosphate (cyclic GMP) phosphodiesterase inhibitor 2-O-propoxyphenyl-8-azapurin-6-one, (M & B 22948; 20 microM). In addition, oxyHb did not reverse the inhibition with iloprost (1 nM) which inhibits platelet aggregation through stimulation of adenylate cyclase. 3. The inhibition of platelet aggregation by NO (7 +/- 1 microM) or NaNp (80 +/- 13 microM) was accompanied by a 13 fold increase in cyclic GMP levels occurring within 15 s of addition of these agents. In the continued presence of NO or NaNp, the reversing effect of oxyHb given 1 min after thrombin was associated with a pronounced decrease in cyclic GMP levels. 4. We conclude that the inhibition of platelet aggregation by activators of guanylate cyclase depends in the first few minutes on continuous stimulation of the enzyme in order to maintain intracellular concentrations of cyclic GMP, except when its breakdown is inhibited. 5. The addition of agents such as oxyHb after the inhibition of platelet aggregation offers another way of investigating the biochemical changes involved in maintaining platelets in an inactive state.

Vasorelaxant and platelet-suppressant potencies of four NO-donors.

Rabbit aortic strips were arranged in a Vane's cascade and superfused with Krebs buffer which contained phenylephrine hydrochloride (100 nM) and indomethacin (5 microM). EC50 (nM) for vasorelaxant action of NO-donors on rabbit aortic strips were as follows: glyceryl trinitrate--64.0, sodium nitroprusside--124.5, S-nitroso-N-acetylpenicillamine--305.7, SIN-1 (metabolite of molsidomine)--510.0. On the other hand IC50 (microM) for antiaggregatory action of NO-donors in human platelet rich plasma aggregated with threshold concentrations of collagen (0.5-1 micrograms/ml) were as follows: S-nitroso-N-acetylpenicillamine--7,1, SIN-1--13.6, sodium nitroprusside--28.0. Glyceryl trinitrate did not inhibit platelet aggregation at concentrations up to 1 mM. Sodium nitroprusside was more specific (EC50/IC50 = 0.0043) than SNAP and SIN-1 (EC50/IC50 = 0.043 and 0.037, respectively) as a vasorelaxant than as a platelet suppressant. Thus relative potencies of four c-GMP stimulants as vasorelaxants and platelet-suppressants differed considerably and several possible explanations for these differences were put forward.

Quantification of the potencies of EDRF-releasers from isolated rabbit aortic strips.

We have compared several known releasers of endothelium-derived relaxing factor (EDRF)(13) in respect to their potencies to generate EDRF by endothelium of rabbit aortic strips (RbA) superfused with Krebs' buffer. The vasorelaxation by EDRF which is equivalent to 10 pmoles of GTN was evoked by 0.7 pmoles of substance P(SP), 50 pmoles of acetylcholine (Ach), 521 pmoles of calcium ionophore A 23187, 2720 pmoles of ADP. Threshold potencies of these agonists are inversely proportional to the maximum amount of EDRF released. Phospholipase C (PLC) from Clostridium perfringens at a dose of 0.1 U caused the relaxation of a similar magnitude. Phospholipase A2 (1 U), thrombin (1 U), bradykinin (30 nmoles) and serotonin (10 pmoles) did not release EDRF. It is concluded that endothelial cells of RbA differ from endothelial cells of other species in their susceptibility to release EDRF in response to various agonists.

Endothelium-derived relaxing factor (EDRF) from cultured and fresh endothelial cells.

Porcine or bovine endothelial cells cultured on microcarrier beads, packed into adapted chromatographic columns, perfused with Krebs' buffer and activated with appropriate stimuli (e.g. bradykinin, ADP or phospholipase C) release EDRF and prostacyclin into the perfusing fluid. In the effluent EDRF and prostacyclin might be bio-assayed using the Vane's superfusion cascade (rabbit aortic strips and bovine coronary artery strips, respectively) against nitroglycerine (GTN) and synthetic prostacyclin standards. Prostacyclin might be also quantified as 6-keto-PGF1 alpha by RIA. A spatial separation of the generator (endothelial cells) from the effector (vascular smooth muscle) has allowed to prove that EDRF is nitric oxide, that its activity is inhibited by superoxide anions and by chemicals which act via free radicals, finally, that the release of EDRF and prostacyclin is coupled by a receptor-mediated activation of phospholipase C. Although so successful, the above technique suffers from its essentials, i.e. from using cultured cells instead of fresh intact endothelial cells. Cultured endothelial cells are not responsive to many receptor agonists including acetylcholine, substance P and 5-hydroxytryptamine. Unlike fresh intact endothelial preparations the cultured cells which are perfused with Krebs' buffer generate superoxide anions at such concentrations that it might be obligatory infusing superoxide dismutase in order to detect EDRF. Nonetheless, a couple of data obtained with the cultured endothelial cells have been reproduced in the fresh cell preparations, e.g. release of EDRF by ADP and ATP, a coupled release of EDRF and prostacyclin by phospholipase C or a paradoxical augmentation of the sodium-nitroprusside-induced vasorelaxation by methylene blue.(ABSTRACT TRUNCATED AT 250 WORDS)