bcl-2 expression in the spectrum of preinvasive breast lesions

This study was designed to define more precisely the relationship between specific angiotensin receptors and the growth of vascular smooth muscle cells in response to angiotensin II. These experiments employed quiescent A10 cells which were characterized as smooth muscle by the expression of specific contractlle proteins. Cell growth was monitored by measuring the incorporation of metabolic precursors into RNA or DNA. The treatment of A10 cells with angiotensin II (1 microM) stimulated a hypertrophic response as indicated by an increase in RNA synthesis and protooncogene expression in the absence of DNA synthesis. This increase in RNA synthesis could be blocked by PD123319, an AT2 antagonist, but not by losartan, an AT1 antagonist. RT-PCR analysis demonstrated that quiescent A10 cells express only the AT2 receptor while proliferating A10 cells express the AT1a and AT1b receptors in addition to the AT2 receptor. In addition, induction of AT2 receptor-mediated RNA synthesis was prevented by indomethacin, a cyclooxygenase inhibitor. These studies therefore support a direct connection between the AT2 receptor and smooth muscle growth that is mediated, in part, by prostaglandin synthesis.     

Halogen-substituted trimetoquinol analogs as thromboxane A2 receptor antagonists in platelets and aorta

Trimetoquinol (TMQ) is a non-prostanoid compound that blocks prostaglandin H2/thromboxane A2 (TXA2) receptor-mediated responses initiated by a prostaglandin (PG) H2 analog, U46619, in human platelets and rat aorta. Ring fluorine-substituted TMQ analogs selectively antagonized PG-dependent human platelet activation induced by U46619, arachidonic acid, collagen, ADP or epinephrine; and were about 300-fold less potent as inhibitors of PG-independent responses mediated by thrombin or bacterial phospholipase C. For each inducer of the PG-dependent pathway, the rank order of inhibitory potency was identical (TMQ > 8-fluoro-TMQ > 5-fluoro- TMQ). Iodine substitution yielded a similar rank order of antagonism against U46619-induced platelet activation (TMQ > 8-iodo-TMQ > 5-iodo-TMQ), and all TMQ analogs inhibited platelet aggregation in whole blood as well as in platelet-rich plasma. Inhibition of specific [3H]SQ 29,548 binding by TMQ analogs was highly correlated with inhibition of functional responses to U46619. Radioligand binding experiments using TMQ analogs with rat platelets showed no interspecies difference in comparison with human platelets. The rank order of inhibitory potencies for the fluorinated (but not iodinated) TMQ analogs changed in rat thoracic aorta with 8-fluoro-TMQ > TMQ > or = 5-fluoro-TMQ as antagonists of U46619-induced vascular contraction. These findings demonstrate that the primary mechanism of antiplatelet action of TMQ analogs is related to a blockade of TXA2 receptor sites, and ring-halogenated TMQ analogs distinguish between TXA2-mediated functional responses in vascular smooth muscle and platelets.     

Molecular biology of prostaglandin E receptors--expression of multi-function by PGE receptor subtypes and isoforms

We reviewed the recent advance in the molecular characterization of prostaglandin E(PGE) receptor. PGE exerts versatile actions in diverse tissues and cells through specific cell surface receptors. PGE receptors(EP) consist of 4 subtypes(EP1, EP2, EP3 and EP4), which differ in the couple of signal transduction. In addition, at least three isoforms of EP3 are produced through alternative RNA splicing from a single gene and differ only in the efficiency of G protein activation and in the specificity of coupling to G proteins. These subtypes and isoforms express and function in specific tissues and cells in various organs, in where they mediate a variety of PGE2-induced physiological responses.     

Distribution of prostaglandin E receptors in the rat gastrointestinal tract

AIMS: In order to study the role of prostaglandin in the regulation of the gastrointestinal functions, gene expression of prostaglandin receptors along the rat gastrointestinal tracts were investigated. METHODS: Rats were used for the study. The combination of counterflow elutriation separation of mucosal cells and Northern blot analysis was used to detect the gene expression of prostaglandin receptors in gastrointestinal tracts. RESULTS: In small intestine and colon, prostaglandin E2 EP1 and EP3 receptor mRNAs were mainly localized in the deeper intestinal wall containing muscle layers. EP4 receptor gene expression, on the other hand, was detected in the intestinal mucosal layer. In the stomach, EP1 mRNA was detected in gastric muscle layers, whereas EP3 and EP4 receptor gene expression was mainly present in the gastric mucosal layer containing epithelial cells. In gastric epithelial cells, parietal cells were found to have both EP3 and EP4 receptors. At lower concentrations, prostaglandin E2 inhibited gastric acid secretion by parietal cells probably through EP4 receptors. At higher concentrations, however, it stimulated it. On the other hand, mucous cells possessed only EP4 receptor mRNA. CONCLUSIONS: Thus, it is suggested that prostaglandin E2 modulates gastrointestinal functions through at least three different prostaglandin receptors (EP1, EP3, and EP4), each of which has a distinct contribution in the gastrointestinal tract.     

Platelets inhibit the induction of nitric oxide synthesis by interleukin-1 beta in vascular smooth muscle cells

We have investigated the role of platelets in regulating the hemostatic and vasomotor properties of vascular smooth muscle. Experiments were performed to examine the effect of the releasate from activated platelets on the production of nitric oxide from interleukin-1 beta (IL-1 beta)-treated cultured rat aortic smooth muscle cells. Treatment of vascular smooth muscle cells with IL-1 beta resulted in significant accumulation of nitrite in the culture media and in marked elevation of intracellular cyclic guanosine monophosphate (GMP) levels. The releasate from collagen-aggregated platelets blocked the IL-1 beta-mediated production of nitrite and the accumulation of cyclic GMP in smooth muscle cells in a platelet number-dependent manner. In functional assays, the perfusates from columns containing IL-1 beta-treated smooth muscle cells relaxed detector blood vessels without endothelium and the addition of IL-1 beta-treated smooth muscle cells to suspensions of platelets inhibited their thrombin-induced aggregation. The simultaneous treatment of smooth muscle cells with IL-1 beta and the platelet releasate abolished both the vasorelaxing activities of the perfusates and the inhibition of platelet aggregation. Platelet releasates treated with a neutralizing antibody to platelet-derived growth factor (PDGF) failed to block IL-1 beta-induced nitric oxide production by the smooth muscle cells, as measured by both biochemical and functional assays. The platelet releasate from a patient with gray platelet syndrome likewise failed to block IL-1 beta-induced nitrite release by smooth muscle cells. These results demonstrate that platelets downregulate the production of nitric oxide by IL-1 beta-treated vascular smooth muscle cells through the release of PDGF. This effect may represent a novel mechanism by which platelets regulate vasomotor tone and thrombus formation at sites of vascular injury.     

Expression, function, and regulation of E-type prostaglandin receptors (EP3) in the nonischemic and ischemic pig heart

The action of prostacyclin, prostaglandin E1 (PGE1), and their mimetics on myocardial function includes changes in contractility, electrophysiological properties, and protection from injury caused by transient myocardial ischemia. This study was undertaken to investigate the basic properties of myocardial E-type prostaglandin (EP) receptors. Ligand binding studies using an enriched preparation of sarcolemmal membranes prepared from pig hearts revealed a single class of binding sites for [3H]PGE1, with a Kd of 3.7 nmol/L and a Bmax of 92 fmol/mg protein. Competition experiments indicated highest affinity for EPs, suggesting an EP receptor. In addition, the EP receptor subtype-selective agonists sulprostone (EP1 and EP3) and M&B 28.767 (EP3) were active, suggesting the presence of an EP3 receptor subtype. PGE1 stimulated sarcolemmal GTPase and inhibited sarcolemmal adenylyl cyclase activity, indicating EP3 receptor coupling to an inhibitory G protein (Gi). Additional in vivo experiments showed that intracoronary infusion of PGE1 (1 nmol/min) decreased isoprenaline-stimulated left ventricular contractile activity without altering systemic vascular resistance. This inhibition of beta-adrenergic effects is compatible with the known myocardial anti-ischemic action of prostaglandins. Further experiments examined EP3 receptor density and G-protein coupling in sarcolemma from ischemic and reperfused ischemic myocardium. In anesthetized open-chest minipigs, occlusion of the left anterior descending coronary artery for 60 minutes increased EP3 receptor density by 50%, whereas receptor affinity was unchanged. This upregulation was prevented by pretreatment with colchicine (2 mg/kg i.v.), indicating microtubule-dependent receptor externalization. Northern hybridization showed comparable EP3 receptor mRNA expression in control and ischemic myocardium. The increase of receptor protein was reversed during 60 minutes of reperfusion. G-protein coupling proved to be intact in ischemic and reperfused ischemic myocardial tissue, as shown by preserved GTP-gamma-S-induced decrease of [3H]PGE1 binding. These data demonstrate for the first time that myocardial receptors for PGE1 belong to the EP3 subtype. The properties of this receptor include inhibition of adenylyl cyclase and upregulation during regional myocardial ischemia, suggesting an involvement in the anti-ischemic activity of E- and I-type prostaglandins.     

Potentiation of retinoic acid-induced differentiation of HL-60 cells by prostaglandin EP2 receptor

Human promyeloid HL-60 cells are differentiated by all-trans retinoic acid (RA) to granulocytes, and prostaglandin (PG) E2 potentiates the RA-induced differentiation. Here we examined which subtype of PGE receptors was involved in this potentiating activity of PGE2. Northern blot analysis demonstrated that HL-60 cells expressed three subtypes of PGE receptor, EP2, EP3, and EP4. Among various EP agonists, and EP2-selective agonist, butaprost, preferentially potentiated the RA-induced differentiation of HL-60 cells. Butaprost not only decreased the half-maximal concentration of RA but also increased the maximal level of the differentiation. Butaprost concentration-dependently stimulated the cAMP formation, and 8-Br-cAMP strongly potentiated the RA-induced differentiation. These results demonstrate that the EP2 receptor enhances the RA-induced differentiation of HL-60 cells via stimulation of adenylate cyclase.     

Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes

We examined the contribution of specific EP receptors in regulating cell growth. By RT-PCR and northern hybridization, adult human keratinocytes express mRNA for three PGE2 receptor subtypes associated with cAMP signaling (EP2, EP3, and small amounts of EP4). In actively growing, non-confluent primary keratinocyte cultures, the EP2 and EP4 selective agonists, 11-deoxy PGE1 and 1-OH PGE1, caused complete reversal of indomethacin-induced growth inhibition. The EP3/EP2 agonist (misoprostol), and the EP1/EP2 agonist (17-phenyl trinor PGE2), showed less activity. Similar results were obtained with agonist-induced cAMP formation. The ability of exogenous dibutyryl cAMP to completely reverse indomethacin-induced growth inhibition support the conclusion that growth stimulation occurs via an EP2 and/or EP4 receptor-adenylyl cyclase coupled response. In contrast, activation of EP3 receptors by sulprostone, which is virtually devoid of agonist activity at EP2 or EP4 receptors, inhibited bromodeoxyuridine uptake in indomethacin-treated cells up to 30%. Although human EP3 receptor variants have been shown in other cell types to markedly inhibit cAMP formation via a pertussis toxin sensitive mechanisms, EP3 receptor activation and presumably growth inhibition was independent of adenylyl cyclase, suggesting activation of other signaling pathways.     

Coronary artery and cultured aortic smooth muscle cells express mRNA for both the classical estrogen receptor and the newly described estrogen receptor beta

Estrogens exhibit potent anti-atherogenic effects through mechanisms which may involve direct effects on the artery. The existence of the classical estrogen receptor (ERalpha) in vascular tissues has been established. Recently a new estrogen receptor (ERbeta) has been discovered which represents a distinct gene product with homology to the classical ERalpha. The purpose of the present study was to determine if ERbeta mRNA is expressed in vascular tissues of female and male primates. Oligonucleotide primers were developed for the specific RT-PCR amplification of ERalpha or ERbeta mRNA. RT-PCR products of the appropriate size for ERalpha and for ERbeta were observed after amplification of RNA isolated from coronary arteries of both male and female cynomolgus monkeys. Similar results were obtained from cultured aortic smooth muscle cells and from monkey reproductive tissues such as ovary and uterus. The relative expression of ERbeta to ERalpha mRNA was greatest in ovary, on the same order of magnitude in monkey vascular tissues and uterus, while the human breast cancer cell line MCF-7 exhibited a very low level of ERbeta relative to ERalpha. Sequence analysis of isolated RT-PCR products showed >95% similarity between the monkey and the published human sequences for both ERalpha and ERbeta. These findings suggest that estrogen may influence vascular gene expression not only through classical ERalpha but also through the newly described ERbeta. These findings also demonstrate the potential for targeting of these receptors in males for prevention or treatment of heart disease.     

The expression of the lectin-like oxidized low-density lipoprotein receptor (LOX-1) on human vascular smooth muscle cells and monocytes and its down-regulation by lovastatin

Accumulation of oxidatively modified low-density lipoprotein (oxLDL) in the vascular wall is a characteristic feature of atherosclerosis. oxLDL can be taken up into monocytes, smooth muscle cells, and endothelial cells by several known scavenger receptors such as scavenger receptor class A I and II, CD36, and CD68. A new lectin-like oxLDL receptor (LOX-1) was recently found in bovine and human endothelial cells. We studied whether LOX-1 is also expressed in other cells present in the atherosclerotic lesion and whether its expression can be modified. We found LOX-1 expression in human blood monocytes, umbilical smooth muscle and endothelial cells, and 3T3 fibroblasts. LOX-1 mRNA expression in monocytes could be significantly suppressed by lovastatin. Thus, LOX-1 expression is not restricted to endothelial cells and its down-regulation by HMG-CoA reductase inhibitors could contribute to the clinical benefits of these drugs.     

Aggressiveness, hypoalgesia and high blood pressure in mice lacking the adenosine A2a receptor

Adenosine is released from metabolically active cells by facilitated diffusion, and is generated extracellularly by degradation of released ATP. It is a potent biological mediator that modulates the activity of numerous cell types, including various neuronal populations, platelets, neutrophils and mast cells, and smooth muscle cells in bronchi and vasculature. Most of these effects help to protect cells and tissues during stress conditions such as ischaemia. Adenosine mediates its effects through four receptor subtypes: the A1, A2a, A2b and A3 receptors. The A2a receptor (A2aR) is abundant in basal ganglia, vasculature and platelets, and stimulates adenylyl cyclase. It is a major target of caffeine, the most widely used psychoactive drug. Here we investigate the role of the A2a receptor by disrupting the gene in mice. We found that A2aR-knockout (A2aR-/-) mice were viable and bred normally. Their exploratory activity was reduced, whereas caffeine, which normally stimulates exploratory behaviour, became a depressant of exploratory activity. Knockout animals scored higher in anxiety tests, and male mice were much more aggressive towards intruders. The response of A2aR-/- mice to acute pain stimuli was slower. Blood pressure and heart rate were increased, as well as platelet aggregation. The specific A2a agonist CGS 21680 lost its biological activity in all systems tested.     

Prostaglandin and kallikrein-kinin systems]

We reviewed the recent advances in the molecular characterization of prostanoid and bradykinin receptors. Prostanoids and bradykinin exert versatile actions in diverse tissues and cells through specific cell surface receptors. Molecular biological studies revealed the primary structure of eight types and subtypes of prostanoid receptors from various species. These include the thromboxane A2 receptor, prostacyclin receptor, prostaglandin (PG) F receptor, PGD receptor and four subtypes of PGE receptor (EP1, EP2, EP3 and EP4). There are four subtypes of bradykinin receptor (BK1, BK2, BK3 and BK4), but it is still unknown about the detail of BK3 and BK4. These results also have achieved the remarkable development in the field of human hypertension.     

Lymphatic endothelial tumors induced by intraperitoneal injection of incomplete Freund's adjuvant

Endothelial cells form the inner lining of blood and lymphatic vessels. In mice, only tumors of the blood vessel endothelium (haemangiomas) have been thus far reported. Here we describe a highly reproducible method for the induction of benign tumors of the lymphatic endothelial cells (lymphangiomas) in mice by intraperitoneal injection of incomplete Freund's adjuvant. Morphological and histopathological studies of the lesions revealed the presence of cells at various levels of vascular development. The lymphangiomas developed in the peritoneal cavity and expressed the endothelial markers CD31/PECAM (platelet endothelial cell adhesion molecule), CD54/ICAM-1 (InterCellular Adhesion Molecule-1), and CD102/ICAM-2, as well as the vascular endothelial growth factor (VEGF) receptor Flk-1, the endothelial cell specific receptors Tie-1 and Tie-2 and the lymphatic endothelial cell specific Flt4 receptor as shown by in situ hybridization. The Flk-1 and Flt4 receptors were also identified in immunoblots of the tumors and in cells cultured from them. When induced in beta-galactosidase knock-in Flt4(+/-) mice, the tumor endothelia could be stained blue in a number of tumor cells although the staining was of lower intensity than in normal lymphatic vessels. The tumor-derived cells could be propagated in vitro and they spontaneously differentiated, forming vessel-like structures. Murine lymphangiomas thus represent a highly reproducible and convenient source of lymphatic endothelial cells.     

Suppression of granulopoiesis by vesnarinone

The diverse intrarenal effects of the prostaglandins (PG) are mediated by distinct guanine nucleotide regulatory protein (G-protein)-coupled receptors. The cDNA for these receptors have been cloned, their signal transduction mechanisms determined, and their intrarenal distribution mapped. PGE2, the major intrarenal prostaglandin, interacts with at least three distinct E-prostanoid (EP) receptors that are highly expressed in specific regions of the kidney. Each EP receptor not only selectively binds PGE2, but also preferentially couples to different signal transduction pathways, including: stimulation of cAMP generation, via Gq (EP2 and EP4 receptors); inhibition of cAMP generation, via Gi (EP3 receptors); and activation of phosphatidylinositol hydrolysis (EP1 receptor), via one of the Gq family members. Activation of each these EP receptors is responsible for a distinct renal effect of PGE2, including its well-described renal hemodynamic and transport effects along the nephron. Other intrarenal prostanoid receptors include the PGF2 alpha receptor (FP), the thromboxane A2 receptor (TP) and the prostacyclin receptor (IP). Knowledge about localization of these receptors and their affinities for receptor-selective agonists and antagonists should aid in the understanding of renal disease and the development of therapeutic strategies for the use of these prostaglandin analogs in select renal diseases.     

The P2X1 receptor, an adenosine triphosphate-gated cation channel, is expressed in human platelets but not in human blood leukocytes

Extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP) activate multiple types of P2-nucleotide receptors expressed in platelets or leukocytes. Electrophysiological and biochemical studies have indicated expression of the P2X1 receptor, an ATP-gated cation channel, in human and rat platelets, rat basophilic leukemia (RBL) cells, and phorbol myristate acetate (PMA)-differentiated HL-60 myeloid cells. Although these findings suggest that P2X1 receptors are present in both blood leukocytes and blood platelets, the relative levels of P2X1 receptor expression and function in human blood leukocytes and platelets have not been directly characterized. On the basis of both immunoblot analysis and functional assays of P2X1 receptor-mediated ionic fluxes, we report that there is significant expression of P2X1 receptors in human platelets, but not in neutrophils, monocytes, or blood lymphocytes. Thus, unlike platelets and myeloid progenitor cell lines, fully differentiated human blood leukocytes do not express functionally significant numbers of P2X1 receptors, suggesting the downregulation of P2X1 receptor gene expression during the differentiation of phagocytic leukocytes. By contrast, P2X1 receptor expression is strongly maintained during megakaryocytic differentiation and platelet release. Immunoblot analysis indicated that the platelet P2X1 receptor migrates as an approximately 60-kD protein during SDS-electrophoresis under reducing or nonreducing conditions. Treatment of platelet membranes with endoglycosidase-F causes the P2X1 receptor band to migrate as a 46-kD protein, verifying the highly glycosylated nature of the mature receptor protein. Additional studies of nucleotide-induced changes in Ca2+ influx/mobilization demonstrated that the platelet P2X1 receptors are pharmacologically distinct from the well-characterized ADP receptors of these cells. This finding suggests a unique role for these ATP-gated ion channels during hemostasis or thrombosis.     

Decreased type II/type I TGF-beta receptor ratio in cells derived from human atherosclerotic lesions. Conversion from an antiproliferative to profibrotic response to TGF-beta1

Atherosclerosis and postangioplasty restenosis may result from abnormal wound healing. The present studies report that normal human smooth muscle cells are growth inhibited by TGF-beta1, a potent wound healing agent, and show little induction of collagen synthesis to TGF-beta1, yet cells grown from human vascular lesions are growth stimulated by TGF-beta1 and markedly increase collagen synthesis. Both cell types increase plasminogen activator inhibitor-1 production, switch actin phenotypes in response to TGF-beta1, and produce similar levels of TGF-beta activity. Membrane cross-linking of 125I-TGF-beta1 indicates that normal human smooth muscle cells express type I, II, and III receptors. The type II receptor is strikingly decreased in lesion cells, with little change in the type I or III receptors. RT-PCR confirmed that the type II TGF-beta1 receptor mRNA is reduced in lesion cells. Transfection of the type II receptor into lesion cells restores the growth inhibitory response to TGF-beta1, implying that signaling remains responsive. Because TGF-beta1 is overexpressed in fibroproliferative vascular lesions, receptor-variant cells would be allowed to grow in a slow, but uncontrolled fashion, while overproducing extracellular matrix components. This TGF-beta1 receptor dysfunction may be relevant for atherosclerosis, restenosis and related fibroproliferative diseases.