Osteoclasts and their circulating precursors in rheumatoid arthritis: Relationships with disease activity and bone erosions.

Patients with rheumatoid arthritis (RA) have very different outcomes, particularly with regard to bone erosions. Since osteoclasts are responsible for bone destruction adjacent to rheumatoid synovium, profiling osteoclasts from circulating precursors in RA could help identify patients at risk for bone destruction. In this study, we sought to determine whether the functional characteristics of osteoclasts generated from their blood precursors were modified by RA activity or were intrinsic to osteoclasts and associated with the RA phenotype (erosive or not). Osteoclasts were generated in vitro from peripheral blood mononuclear cells (PBMCs) of subjects with RA (n = 140), as well as sex- and age-matched healthy controls (n = 101). Osteoclastic parameters were analyzed at baseline and during the follow-up for up to 4 years, with regular assessment of RA activity, bone erosions, and bone mineral density (BMD). As a validation cohort, we examined RA patients from the Early Undifferentiated PolyArthritis (EUPA) study (n = 163). The proportion of CD14+ PBMC was higher in RA than in control subjects, but inversely correlated with the 28-joint disease activity score (DAS28). Also surprisingly, in osteoclast cultures from PBMCs, active RA was associated with lower osteoclastogenic capacity, while in vitro bone resorption per osteoclast and resistance to apoptosis were similar in both active and quiescent RA. In a small subgroup analysis, osteoclasts from subjects with recent RA that had progressed at four years to an erosive RA exhibited at baseline greater resistance to apoptosis than those from patients remaining non-erosive. Our findings establish that when RA is active, circulating monocytes have a reduced potential to generate osteoclasts from PBMCs in vitro. In addition, osteoclasts associated with erosive disease had resistance to apoptosis from the start of RA.

Monocytes from male patients with ankylosing spondylitis display decreased osteoclastogenesis and decreased RANKL/OPG ratio.

The present study investigates the osteoclastogenic capacity of peripheral blood mononuclear cells (PBMCs) in male patients with ankylosing spondylitis (AS). We demonstrated that monocytes from these patients display a lower capacity to generate osteoclasts compared to cells from healthy controls, and osteoclastogenesis was negatively correlated with disease duration. INTRODUCTION: Ankylosing spondylitis (AS) is a disease characterized by new bone growth that leads to syndesmophyte formation but AS patients frequently present with low bone mineral density/fractures. Osteoclastogenesis in AS patients is poorly studied and controversial. The aim of this study is to determine if the osteoclastogenic capacity of PBMCs is different in AS patients compared to controls and the relationship between osteoclastogenesis and clinical/laboratory parameters. METHODS: PBMCs from 85 male AS patients and 59 controls were tested for CD16+ cells and induced to differentiate into osteoclasts over 3 weeks in vitro. Serum levels of RANKL, osteoprotegerin (OPG), C-terminal telopeptide of type I collagen (CTX), and amino-terminal pro-peptide of type I collagen (P1NP) were also evaluated. RESULTS: PBMCs from AS patients had fewer CD16+ cells and produced fewer osteoclasts compared to controls. Apoptosis occurred less frequently in osteoclasts obtained from AS patients than in osteoclasts from the controls. A lower RANKL/OPG and CTX/P1NP were observed in AS patients compared to controls. AS patients taking NSAIDs presented no difference regarding the number of OCs produced and the percentage of CD16+ cells compared to controls. However, patients taking TNF inhibitors (TNFi) presented lower OC numbers than controls. A negative correlation was demonstrated between the number of osteoclasts generated from PBMCs of AS patients and disease duration. CONCLUSION: Monocytes from male AS patients display a lower capacity to generate osteoclasts in vitro compared to cells from controls. Osteoclastogenesis was negatively correlated with disease duration. This finding supports the idea that osteoclasts play a role in the physiopathology of bone disease in AS patients.

The increased in vitro osteoclastogenesis in patients with rheumatoid arthritis is due to increased percentage of precursors and decreased apoptosis - the In Vitro Osteoclast Differentiation in Arthritis (IODA) study.

Increases in local and systemic bone resorption are hallmarks of rheumatoid arthritis (RA). Osteoclasts are implicated in these processes and their enhanced differentiation may contribute to bone destruction. We observed that in vitro osteoclastogenesis varies among healthy individuals and hypothesized that increased osteoclastogenesis could be a marker for the presence of RA. Our objective in the present study was to determine if in vitro osteoclastogenesis from peripheral blood mononuclear cells (PBMCs) was different in patients with RA compared to healthy controls and osteoarthritis (OA) patients. Expression of CD14 in PBMCs was quantified and PBMCs were incubated for 21 days in the presence of the osteoclastogenic cytokines M-CSF and RANKL. Differentiation on cortical bone slices permitted the analysis of bone resorption while apoptotic potential was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. In vitro osteoclastogenesis was higher in PBMCs from RA patients compared to controls, and a similar increase was observed in the percentage of osteoclast precursors in RA patients. Osteoclasts from RA patients showed lower apoptotic rates than osteoclasts from healthy controls. No difference was observed in bone resorption activity between RA patients and controls. Interestingly, the difference in osteoclast number and apoptosis rate allowed the implementation of an algorithm capable of distinguishing patients with RA from controls. In conclusion, our study shows that osteoclast differentiation from PBMCs is enhanced in patients with RA, and this difference can be explained by both a higher percentage of osteoclast precursors in the blood and by the reduced apoptotic potential of mature osteoclasts.

Temporal expression and signalling of prostacyclin receptor in the human endometrium across the menstrual cycle.

Prostacyclin (PGI(2)) synthesis and function in the human uterus has been implicated in the regulation of the process of normal and dysfunctional menstruation. PGI(2) synthesis is elevated during normal menstruation and is also associated with blood loss in women who suffer from heavy menses. This study was designed to outline further the role of PGI(2) in menstruation by investigating the temporal pattern and site of expression of prostaglandin I synthase (PGIS) and the prostacyclin receptor (IP receptor) in the non-pregnant human endometrium across the menstrual cycle. Quantitative RT-PCR demonstrated increased expression of PGIS and IP receptor during the menstrual phase of the cycle compared with all other phases (P < 0.05). Furthermore, PGIS and IP receptor were localised to the glandular epithelium, stromal and endothelial cells in the basal and functional layers of the endometrium. Functionality of the IP receptor in the human endometrium was assessed by measuring cAMP generation following treatment with 100 nmol l(-1) of the PGI(2) analogue, iloprost. cAMP generation was significantly higher in endometrial tissue collected during the proliferative compared with the secretory phase of the menstrual cycle (P < 0.05). In conclusion, this study has confirmed increased expression and signalling of PGIS and IP receptor during the menstrual phase and outlines a potential autocrine/paracrine role for PGI(2) on several cellular compartments in the endometrium including the endothelium. This may underscore a pivotal role for PGI(2) receptor signalling in normal and dysfunctional menstruation.

Immunolocalization of the prostaglandin E2 receptor subtypes in human bone tissue: differences in foetal, adult normal, osteoporotic and pagetic bone.

PGE(2) is an important mediator of bone metabolism, but the precise localization of its receptors in human bone remains unknown. The present study used specific antibodies against EP(1), EP(2), EP(3) and EP(4) receptors for immunolocalization in normal, osteoporotic and pagetic human adult bone and in human foetal bone. No labelling was obtained for the EP(1) and EP(2) receptors. The EP(3) receptor was detected in foetal osteoclasts, osteoblasts and osteocytes, but only in osteoclasts and some osteoblasts from adult bone. The EP(4) receptor was detected in foetal osteoclasts, osteoblasts and osteocytes and in adult osteoclasts and osteoblasts, but not in adult osteocytes. Our results show differences in PGE(2) receptor expression in foetal and adult human bone but no difference in adult normal compared to pathologic bone. Finally, these results show that the distribution of EP receptors in human osteoblasts in bone corresponds in part to what we recently described in human osteoblasts in culture.

A comparative study of the antipyretic effects of indomethacin and dipyrone in rats.

OBJECTIVE: Compare the antipyretic effects of dipyrone and indomethacin. MATERIALS AND METHODS: Fever was induced in rats by i. v. LPS or i. c. v. interleukins (IL), prostaglandins (PG), arachidonic acid (AA), pre-formed pyrogenic factor (PFPF), tumour necrosis factor-alpha (TNF-alpha) or corticotrophin releasing hormone (CRH). Dipyrone and indomethacin were administered i.p., arginine vasopressin V1-receptor antagonist, d(CH2)5 Tyr(Me)AVP, into the ventral septal area. Cyclooxygenase (COX-1/-2) blocking activity was assessed in transfected COS-7 cells. CRH release from isolated hypothalami was determined by ELISA. RESULTS: Indomethacin or dipyrone reduced LPS, IL-1beta, IL-6 or TNF-alpha induced fever and CRH release from rat hypothalamus. Only dipyrone inhibited IL-8, PFPF or PGF2alpha fever. Only indomethacin inhibited fever induced by AA or IL-1beta, plus AA. Neither antipyretic affected fever caused by PGE2 or CRH. d(CH2)5Tyr(Me)AVP only blocked antipyresis induced by indomethacin. Dipyrone at a very high concentration (10 mM) inhibited only COX-1, while indomethacin (0.1 microM) blocked COX-1 and COX-2 in COS-7 cells. CONCLUSION: The antipyretic effect of dipyrone differs from that of indomethacin in that it does not depend on AVP release or inhibition of PG synthesis.

Immunohistochemical localization of the prostacyclin receptor (IP) human bone.

Prostacyclin (PGI(2)) is an important mediator implicated in bone metabolism. Among the natural prostaglandins it is the most potent inhibitor of bone resorption and mediates bone modelling and remodelling induced by strain changes. The effects of prostacyclin depend on its interaction with a specific receptor (IP). Despite its well documented effects on bone the localization and distribution of the IP receptor in human bone remain unknown. The present study used specific antipeptide antibodies to IP receptor for immunolocalization of the IP receptor in normal, osteoporotic and Pagetic human adult bone and in human fetal bone. The IP receptor was detected in fetal and adult osteoclasts and osteoblasts. Fetal osteocytes also expressed IP receptor but not adult osteocytes. Interestingly, the expression of IP receptor in adult osteoblasts was gradually lost as these cells were trapped in the matrix and became osteocytes. The IP receptor showed a perinuclear distribution within the cells, but in multinuclear osteoclasts not all nuclei were positive. Our results showed differences in IP receptor expression in fetal and adult human bone and, in adult bone, with the differentiation of osteoblasts into osteocytes. They also showed that there is no difference on the expression of prostacyclin receptors in Pagetic, osteoporotic and normal human bone, and they confirm the presence of the IP receptor in human osteoblasts as had been demonstrated by our previous study with human osteoblasts in culture.

Characterization of the prostaglandin receptors in human osteoblasts in culture.

Prostaglandins have complex actions on bone metabolism that depend on interactions with different types and subtypes of receptors. Our objective was to characterize the prostaglandins receptors present in primary cultures of human osteoblasts. RT-PCR analysis revealed the presence of DP, EP(4), IP, FP and TP receptor mRNA in primary cultures of human osteoblasts. FP receptor mRNA was detected only after 3 weeks of confluency, all the others were detected at every culture time tested. To verify the functionality of these receptors we challenged the cells with the prostanoids and synthetic analogues and determined the intracellular levels of cAMP. All receptors found by RT-PCR were coupled to second messengers except for the DP subtype. These results clearly show the presence of functional EP(4), IP, FP and TP receptors in human osteoblasts in culture.

Anti-inflammatory effects of the products from Wilbrandia ebracteata on carrageenan-induced pleurisy in mice.

Wilbrandia ebracteata Cogn. (Cucurbitaceae) is commonly known in Brazil as "Taiuia". The roots are employed in folk medicine for the treatment of several diseases, such as rheumatic disease. This study has evaluated the anti-inflammatory action of dicloromethane fraction (F-DCM), purified fraction (PFIII) and Cucurbitacin B extracted from crude extract of W. ebracteata in experimental models in vivo. The F-DCM (0.3 to 10 mg.kg(-1), i.p. or 3 to 30 mg.kg(-1) p.o.) produced significant but not dose-dependent inhibition of the carrageenan-induced cell influx and exsudate leakage in the pleural cavity of mice. The F-DCM 0.01 to 10 mg.kg(-1), i.p. or 0.1 to 10 mg.kg(-1) p.o.) decreased the levels of PGE2 in the exsudate leakage induced by carrageenan in the pleural cavity after 4 h with a calculated ID50 of 0.01 (0.002-0.09, i.p.) and 0.29 (0.05-1.45, p.o.) mg.kg(-1). The PFIII (3 mg.kg(-1), i.p.) inhibited 80% of cell migration (1.50 +/- 0.09 x 10(6) cells/cavity) and exsudate leakage by about 50% (3.09 +/- 0.71 microg/ml) in relation to the control group. Cucurbitacin B (0.1 mg.kg(-1), i.p.), the main compound of PFIII, reduced significantly the levels of PGE2 in the exsudate leakage by 40.7% (10.41 +/- 2.67 ng.ml(-1)). These data show that the active principle(s) present in the F-DCM of W. ebracteata elicited pronounced anti-inflammatory effects when assessed by i.p. or p.o. routes, as well as PFIII. The F-DCM was also able to prevent PGE2 formation in exsudate leakage induced by carrageenan, as well as Cucurbitacin B, its active principle. These results indicate that the anti-inflammatory activity of Wilbrandia ebracteata can be related with the inhibition of the production of PGE2.

Systematic pharmacological approach to the characterization of NSAIDs.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory diseases. NSAIDs inhibit cyclooxygenase (COX), the rate limiting enzyme responsible for the conversion of arachidonic acid into prostaglandins. Recent studies have shown the existence of two isoforms of cyclooxygenase: COX-1, now often referred to as the constitutive form, and COX-2, an inducible form which is the major isoenzyme involved in prostaglandin synthesis in inflammation and other pathological situations. Since inhibition of prostaglandin production in tissues where they play a physiological role leads to important side effects, a COX-2 preferential inhibitor would present therapeutical advantages. In the present study, we evaluated the inhibitory properties of cyclooxygenase inhibitors on human COX-1 and COX-2 using a heterologous expression system. We investigated instantaneous inhibition and pre-incubation inhibition as well as time recovery of cyclooxygenase activity assays with the aid of four NSAIDs: mefenamic acid, indomethacin, aspirin and NS-398. Our results demonstrate that instantaneous inhibition assays have little correlation with clinical results. Inhibition assays using pre-incubation with the drugs tested, however, more closely resemble the data from in vivo studies. Cyclooxygenase recovery assays enabled better characterization of simple competitive inhibitors, competitive reversible time-dependent inhibitors and irreversible time-dependent inhibitors. The data illustrate the usefulness of our system in allowing a better determination of the pharmacological characteristics of NSAIDs as well as permitting a comparison among different drugs.

Regulation of cyclooxygenase-2 expression in bovine chondrocytes in culture by interleukin 1alpha, tumor necrosis factor-alpha, glucocorticoids, and 17beta-estradiol.

OBJECTIVE: To determine the effects of interleukin 1alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha), dexamethasone, and 17beta-estradiol on the expression of cyclooxygenase-1 (COX-1) and COX-2 in bovine chondrocytes. METHODS: Northern blot analysis was used to quantify COX-1 and COX-2 mRNA expression in primary cultures of bovine chondrocytes and prostaglandin production to evaluate COX activity. RESULTS: IL-1alpha and TNF-alpha increased the expression of COX-2. This effect was independent of de novo protein synthesis and dependent on increased mRNA stability in the case of IL-1alpha. Dexamethasone inhibited the effects of both cytokines. 17beta-estradiol inhibited COX-2 mRNA expression in basal conditions, but had no effect on COX-2 expression induced by cytokines. The specific COX-2 inhibitor compound NS 398 prevented the increase in prostaglandin E2 (PGE2) production induced by the cytokines. COX-1 levels remained stable with all treatments. CONCLUSION: Increase in mRNA stability is a mechanism implicated in the induction of COX-2 by some cytokines. The effects of IL-1alpha and TNF-alpha on PGE2 production are mainly due to an increase in COX-2 activity as shown by the effect of compound NS 398. 17beta-estradiol inhibits COX-2 mRNA expression in basal conditions, suggesting that estrogens could be implicated in the control of cartilage metabolism.

Functional IL-2 receptors are expressed by rat lung type II epithelial cells.

Interferon (IFN)-gamma-induced inhibition of type II epithelial cell thymidine incorporation (45% decrease vs. control) was restored by cocultures with mitogen-activated peripheral blood mononuclear cells (PBMC) and conditioned media (CM) from mitogen-activated PBMC. Successive exposure of type II cells to IFN-gamma and interleukin (IL)-2 produced similar alterations in thymidine incorporation. Given that IL-2 is a powerful pleiotropic cytokine produced by lymphoid and myeloid cells, the presence of IL-2 receptors (IL-2R) was assessed in primary cultures of rat type II pneumocytes (TIIP) and the nontransformed alveolar type II epithelial cell line L2. The presence of IL-2R membrane protein on rat type II cells was established by immunodetection assays. The expression of all three murine IL-2R alpha-, beta-, and gamma-chain RNA transcripts in primary TIIP cultures and L2 cells was highlighted by reverse transcriptase-polymerase chain reaction analysis. Overall, these experiments demonstrate, for the first time, that type II epithelial cells can express functional IL-2R, confirming TIIP as a potential "partner" in the lung immune system. Consequently, it can be speculated that TIIP are new cellular targets for lymphokines using (IL-2R) gamma-chain-bearing receptors in lung distal air-spaces.

Expression of recombinant human cyclooxygenase isoenzymes in transfected COS-7 cells in vitro and inhibition by tenoxicam, indomethacin and aspirin.

The recent discovery of cyclooxygenase-2 (COX-2), an isoenzyme associated mainly with inflammation created the need to reevaluate cyclooxygenase inhibitors with reliable screening methods. In the present study we standardized a technique to determine the IC50S of cyclooxygenase inhibitors on recombinant human COX-1 and COX-2 expressed in mammalian cells and used it to study the compounds tenoxicam, aspirin and indomethacin. The IC50S of aspirin, indomethacin and tenoxicam for human COX-1 were 0.41 +/- 0.07 microgram/ml, 0.008 +/- 0.003 microgram/ml, and 7.94 +/- 3.28 micrograms/ml, respectively, and for human COX-20.64 +/- 0.16 microgram/ml, 0.09 +/- 0.05 microgram/ml, and 10.61 +/- 1.50 micrograms/ml, for aspirin, indomethacin, and tenoxicam. Tenoxicam had the lowest IC50hCOX-2/IC50hCOX-1 ratio (1.34), followed by aspirin (1.53) and indomethacin (10.82). The system described in the present study provides a simple and efficient way to determine the specificity of NSAID inhibition for each of the human cyclooxygenase isoenzymes separately.

Induction of cyclooxygenase-2 by parathyroid hormone in human osteoblasts in culture.

OBJECTIVE: Parathyroid hormone (PTH) induced bone resorption by osteoclasts depends on the presence of osteoblasts. PTH induced production of prostaglandins by osteoblasts and induction of bone resorption by prostaglandins suggest that these autacoids may be implicated in the effects of PTH on bone. Our objective was to determine if the increase in prostaglandin production induced in human osteoblasts by PTH is due to an increase in cyclooxygenase-2 (COX-2) expression. METHODS: Primary cultures of human osteoblasts were obtained from specimens of trabecular bone. Confluent cells were treated with PTH, dexamethasone or compound NS-398, a specific COX-2 inhibitor. The concentration of prostaglandin E2 (PGE2) in the supernatants was determined by radioimmunoassay and COX-2 mRNA levels evaluated by Northern blot. RESULTS: PTH induced COX-2 mRNA expression and PGE2 production. These effects were time and concentration dependent and were inhibited by dexamethasone. Compound NS-398 reduced PGE2 production to the same extent as dexamethasone, and neither compound had an additive effect on this variable. CONCLUSION: These results show that PTH induces COX-2 expression in human osteoblasts in culture and suggest that this isoenzyme is the main factor in the control of prostaglandin synthesis in these experimental conditions.

Characterization of the PGE2 receptor subtype in bovine chondrocytes in culture.

1. Prostaglandin E2 (PGE2) is an autacoid that decreases proteoglycan synthesis, increases metalloprotease production by cultured chondrocytes, and can modulate some of the actions of interleukin-1 on cartilage. The objective of the present study was to characterize the subtype of prostaglandin E2 receptor present in bovine chondrocytes in culture. 2. Primary cultures of articular chondrocytes were prepared from slices of bovine carpal cartilage by sequential digestion with type III hyaluronidase, trypsin, type II collagenase, followed by overnight incubation in Dulbecco's Modified Eagle's Medium (DMEM) with type II collagenase, washing, and seeding at a density of 2 x 10(5) cells cm-2 in DMEM with 10% foetal bovine serum. 3. PGE2 and carbaprostacyclin induced dose-dependent increases in intracellular cyclic AMP in bovine chondrocytes in culture. The potencies of these compounds were different, and maximal doses of PGE2 and carbaprostacyclin had an additive effect. PGD2 induced a small increase in intracellular cyclic AMP only at a high concentration (10(-5) M). 4. PGE2 was more potent that the EP2 agonist 11-deoxy-PGE1 at inducing increases in intracellular cyclic AMP. The EP2 agonist butaprost, however, induced only a small increase at a concentration of 10(-5)M. 17-Phenyl-PGE2 (EP1 agonist), sulprostone and MB 28767 (15S-hydroxy-9-oxo-16-phenoxy-omega-tetranorprost-13E-enoic acid) (EP3 agonists) did not induce an increase in intracellular cyclic AMP at concentrations up to 10(-5)M. 5. The EP4 antagonist AH 23848B ([1 alpha(Z),2 beta, 5 alpha]-(+/-) -7-[5-[[(1,1'-biphenyl)-4-yl]methoxyl-2-(4-morpholinyl) -3-oxocyclopentyl]-5-heptenoic acid) antagonized PGE2 but not carbaprostacyclin effects on intracellular cyclic AMP. The Schild plot slope was different from 1 but this could be due to an interaction of PGE2 with IP receptors in high doses. The exact nature of the antagonism by compound AH 23848B could not be definitely established in these experimental conditions. 6. Neither PGE2 nor any of its analogues inhibited the increase in intracellular cyclic AMP induced by forskolin, and pertussis toxin did not alter the response to PGE2, suggesting that no Gi-coupled PGE2 receptors are present in these cells. Stimulation with PGE2 did not induce significant increases in intracellular inositol-trisphosphate levels nor increases in intracellular free calcium as determined by confocal microscopy, suggesting the absence of phospholipase-C-coupled or of calcium channel-coupled PGE2 receptors in bovine chondrocytes in these experimental conditions. 7. These results show for the first time that bovine chondrocytes in culture present a functional PGE2 receptor that has some pharmacological characteristics of an EP4 subtype, as well as an IP receptor.

Leukotriene receptors in HL-60 cells differentiated into eosinophils, monocytes and neutrophils.

The expression of leukotriene B4 (LTB4) and leukotriene D4 (LTD4) receptors was determined, by binding assay, in HL-60 cells differentiated into the monocyte/macrophage, neutrophil, and eosinophil lineages. Monocyte/ macrophage- and neutrophil-differentiated cells developed specific LTB4 receptors with high affinities (Kd = 1.27 nM and 2.65 nM, respectively) and low affinities (Kd = 26.41 nM and 55.63 nM, respectively). These receptors were functional and specific as indicated by the ability of LTB4 to elicit an increase in intracellular calcium concentration antagonised by specific antagonists. Eosinophil-differentiated cells developed mainly LTD4 receptors (Kd = 41.91 nM), and stimulation with LTD4 induced an increase in intracellular calcium that was antagonised by a specific LTD4 antagonist. These results show, for the first time, that eosinophil-differentiated HL-60 cells express specific functional LTD4 receptors. These cells could be used for the study of the actions of peptidoleukotrienes on eosinophils, and for studies on the molecular mechanisms regulating LTD4 receptor expression.

Mutations of two adjacent amino acids generate inactive and constitutively active forms of the human platelet-activating factor receptor.

We have mutated two residues, Ala230 and Leu231, in the C-terminal portion of the third intracellular loop of the human platelet-activating factor (PAF) receptor into Glu230 and Arg231, respectively. The Leu231 --> Arg231 substitution led to two major modifications: 1) increased constitutive activity of the PAF receptor resulting in agonist-independent production of inositol phosphates and 2) increased affinity of the receptor for binding PAF (agonist) but not WEB2086 (antagonist). The L231R mutant was able to adopt at least two conformations: (i) a higher affinity state than the corresponding state of the wild-type receptor (WT), dependent on G protein coupling, and (ii) a low affinity state, higher than the one for the uncoupled WT receptor. The Ala230 --> Glu230 substitution also resulted in two major modifications: 1) unresponsiveness in terms of phosphatidylinositol hydrolysis in response to PAF and 2) a marked decrease in affinity of the receptor for binding the agonist but not the antagonist. Competition binding studies of transient receptor expression in COS-7 cells and the inability of guanosine 5 -O-(3-thiotriphosphate) to modulate the decrease in affinity of a stable A230E mutant in Chinese hamster ovary cells suggest an inherent low affinity conformation for this mutant. Alternatively, mutation of Ala230 to Gln230 suggested that the residue 230 has a fundamental effect on receptor affinity and its charge is determinant in G protein coupling of the PAF receptor. In this report, we show that substitution of two immediately adjacent residues of the PAF receptor, Ala230 and Leu231, surprisingly leads to an inactive and a constitutively active phenotype, respectively. These results further support the concept of constitutively active G protein-coupled receptors as adopting ''active'' state conformations similar to those induced by agonist binding to WT receptors.

Effect of PAF on rat lung vascular permeability: role of platelets and polymorphonuclear leucocytes.

1. The objectives of the present experiments were to assess the contribution of polymorphonuclear leucocytes (PMNLs), platelets and their products such as thromboxane A2 (TxA2), histamine and 5-hydroxytryptamine to platelet activating factor (PAF)-mediated protein extravasation in rat lungs. 2. Intravenous injection of PAF (1.0 and 5.0 micrograms kg-1) increased dose-dependently (up to 7.5 fold) the vascular permeability of the trachea, upper and lower bronchi to Evans blue dye (EB), a marker of albumin extravasation. The permeability of the pulmonary parenchyma was not affected significantly by PAF. 3. Thrombocytopenia induced by administration of the IgG fraction of goat anti-rat platelet serum (APS; 15 mg 100 g-1, i.p., 16-18 h) reduced by 55, 58 and 40% the effects of the lower dose of PAF (1.0 microgram kg-1) and by 31, 23 and 15% the effects of the higher dose of PAF (5.0 micrograms kg-1) on the permeability of the trachea, upper and lower bronchi respectively to albumin. 4. PMNL depletion induced by administration of rabbit anti-rat polymorphonuclear serum (ANS; 2 mg kg-1, i.v., 24 h) did not reduce significantly the effects of the lower dose of PAF (1.0 microgram kg-1) on the airways, however the effects of the higher dose of PAF (5.0 micrograms kg-1) on the permeability of the trachea, upper and lower bronchi to albumin were reduced by 43, 25 and 23% respectively. 5. The injection of both the anti-platelet and the anti-PMNL sera reduced by 61, 62 and 96% the effects of the lower dose of PAF (1.0 microg kg-1) and by 44, 39 and 47% the effects of the higher dose of PAF (5.0 microg kg-1) on the permeability of the trachea, upper and lower bronchi respectively.6. The combined injection of the TxA2-mimetic (U-44069; 5.0 microg kg-1) and PAF (1.0 and 5.0 microg kg-1)in thrombocytopenic rats overcame the vascular permeability decrease induced by APS treatment.7. Pretreatment of the animals with a combination of antagonists to histamine (mepyramine;3.0 mg kg-1) and 5-hydroxytryptamine (methysergide; 2.5 mg kg-1) did not cause a significant inhibition of the effect of PAF (1.0 and 5.0 microg kg-1) on EB extravasation in the airways.8. These data show that the effect of intravenous PAF on rat vascular permeability is partly modulated by polymorphonuclear leucocyte and platelet activation. Our results suggest that following its release,TxA2 could increase postcapillary hydrostatic pressure by inducing a venoconstriction and potentiate the extravasation elicited by PAF. These results do not suggest a major role for histamine and/or 5-hydroxytryptamine on PAF-induced albumin extravasation.

Expression of prostaglandin endoperoxide synthase-1 and prostaglandin endoperoxide synthase-2 in human osteoblasts.

A Prostaglandin endoperoxide synthase isoenzyme was recently identified in several cell lines. Osteoblasts possess Prostaglandin endoperoxide synthase activity, but it is not known which isoenzymes are present in these cells. Our objective was to identify these isoenzymes in human osteoblasts. Resting cells in culture did not produce measurable amounts of PGE2 and did not express Prostaglandin endoperoxide synthase-1 or Prostaglandin endoperoxide synthase-2 mRNAs detectable by Northern blot. Treatment with rhIL-1 alpha or rhTNF alpha induced both the expression of Prostaglandin endoperoxide synthase-2 mRNA and the synthesis of PGE2, rhIL-1 alpha being more potent on an equimolar basis than rhTNF alpha. Dexamethasone inhibited the increase in Prostaglandin endoperoxide synthase-2 mRNA and the production of PGE2 induced by both cytokines. These results suggest that Prostaglandin endoperoxide synthase-2 may be the relevant isoenzyme for prostanoid production in human osteoblasts in culture.