Search PubMed⌕ Search

Biomedical subjects

A Habib

Publications and source records attributed to A Habib.

At least 37 records · Page 2Linked to original sources
PubMed1999

Tyrosine phosphorylation of cortactin associated with Syk accompanies thromboxane analogue-induced platelet shape change.

Thromboxane A(2) (TxA(2)) is a potent vasoconstrictor and platelet agonist. Pharmacological studies have defined two classes of thromboxane receptors (TPs) in human platelets; sites that bind the agonist 1S-(1,2(5Z),3-(1E,3S),4)-7- 3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-2.2. 1-heptan-2-yl-5-heptenoic acid (I-BOP) with high affinity support platelet shape change, whereas low affinity sites that bind irreversibly the antagonist GR 32191 transduce platelet aggregation. As the mechanisms of signal transduction involved in platelet aggregation begin to be elucidated, few results concern those involved in platelet shape change, which is independent of the engagement of GPIIb/IIIa. To elucidate the respective role of the two classes of pharmacological binding sites of TPs in shape change, platelets were incubated with I-BOP at low concentrations or stimulated by I-BOP at high concentrations after pretreatment with GR 32191 or activated with low concentrations of 8-epi-prostaglandin F(2)alpha. Under these three conditions, there is a rapid stimulation of protein tyrosine phosphorylation of the 80/85-kDa doublet identified as the cytoskeletal protein cortactin. Tyrosine phosphorylation of cortactin is kinetically correlated with the occurrence of shape change. These biochemical and morphological events are both inhibited by SQ 29548, a TP antagonist, indicating the specificity of the signal. Since tyrosine kinase Syk was activated early during platelet activation, we examined the possibility that cortactin is a potential substrate of Syk in TxA(2)-induced platelet shape change. p72 Syk phosphorylation and kinase activity took place during the period when platelets were changing shape upon low concentrations of I-BOP stimulation. Furthermore, cortactin was associated with Syk, and this association increases along with the level of phosphorylation. These data suggest a novel pathway for a G protein-coupled TxA(2) high affinity receptor to the protein-tyrosine kinase Syk, which is associated with cortactin in the very early steps of platelet activation.

Blood Platelets↗
PubMed1999

Differential signaling by the thromboxane receptor isoforms via the novel GTP-binding protein, Gh.

Thromboxane A2 acts via G protein-coupled receptors; two splice variants of the thromboxane A2 receptor (TPalpha and TPbeta) have been cloned. It is unknown whether they differ in their capacity to activate intracellular signaling pathways. Recently, a high molecular weight G protein, Gh, that can also function as a tissue transglutaminase, has been described. We investigated whether Gh functions as a signaling protein in association with thromboxane receptors. First, we sought Gh expression in cells known to express TPs. Reverse transcription-polymerase chain reaction and immunoblotting demonstrated Gh expression in platelets, megakaryocytic cell lines, and endothelial and vascular smooth muscle cells. Second, immunoprecipitation of both TPalpha and TPbeta in transfected COS-7 cells resulted in the co-immunoprecipitation of Gh, indicating that TPs may associate Gh in vivo. Finally, agonist activation of TPalpha, but not of TPbeta, resulted in stimulation of phospholipase C-mediated inositol phosphate production in cells cotransfected with Gh. By contrast, agonist activation of both TP isoforms resulted in Gq-mediated inositol phosphate signaling. Gh is expressed in platelets and vascular cells and may associate with both TP isoforms. However, stimulation of TP isoforms results in differential activation of downstream signaling pathways via this novel G protein.

Animals↗
PubMed1999

Phosphorylation of the thromboxane receptor alpha, the predominant isoform expressed in human platelets.

A single gene encodes the human thromboxane receptor (TP), of which there are two identified splice variants, alpha and beta. Both isoforms are rapidly phosphorylated in response to thromboxane agonists when overexpressed in human embryonic kidney 293 cells; this phenomenon is only slightly altered by inhibitors of protein kinase C. Pharmacological studies have defined two classes of TP in human platelets; sites that bind the agonist I-BOP with high affinity support platelet shape change. Low affinity sites, which irreversibly bind the antagonist GR 32191, transduce platelet activation and aggregation. Isoform-specific antibodies permitted detection of TPalpha, but not TPbeta, from human platelets, although mRNA for both isoforms is present. A broad protein band of 50-60 kDa, reflecting the glycosylated receptor, was phosphorylated upon activation of platelets for 2 min with I-BOP. This was a rapid ( approximately 30 s) and transient (maximum, 2-4 min) event and was inhibited by TP antagonists. Both arachidonic acid and low concentrations of collagen stimulated TPalpha phosphorylation, which was blocked by cyclooxygenase inhibition or TP antagonism. Blockade of the low affinity TP sites with GR 32191 prevented I-BOP-induced TPalpha phosphorylation. This coincided with agonist-induced platelet aggregation and activation but not shape change. Also, activation of these sites with the isoprostane iPF2alpha-III induced platelet shape change but not TPalpha phosphorylation. Heterologous TP phosphorylation was observed in aspirin-treated platelets exposed to thrombin, high concentrations of collagen, and the calcium ionophore A 23187. Both homologous and heterologous agonist-induced phosphorylation of endogenous TPalpha was blocked by protein kinase C inhibitors. TPalpha was the only isoform detectably translated in human platelets. This appeared to correspond to the activation of the low affinity site defined by the antagonist GR 32191 and not activated by the high affinity agonist, iPF2alpha-III. Protein kinase C played a more important role in agonist-induced phosphorylation of native TPalpha in human platelets than in human embryonic kidney 293 cells overexpressing recombinant TPalpha.

Arachidonic Acid↗
PubMed1999

Enteral nutrition access devices.

During the past decade a concerted effort has been made to use enteral nutrition instead of parenteral nutrition for hospitalized patients. Enteral nutrition has major advantages over parenteral nutrition in terms of cost and fewer serious complications. A clinician interested in initiating enteral nutrition may be limited by a lack of familiarity with the variety of options for enteral access and the difficulty of choosing among them. This paper reviews the different enteral access routes and devices available to the clinician.

Enteral Nutrition↗
PubMed1998

Platelet-derived growth factor-BB and thrombin generate positive and negative signals for human hepatic stellate cell proliferation. Role of a prostaglandin/cyclic AMP pathway and cross-talk with endothelin receptors.

Proliferation of myofibroblastic hepatic stellate cells (HSC) in response to growth factors is essential for the development of liver fibrosis. We have reported that prostaglandins (PG) and cyclic AMP (cAMP) inhibit growth of human HSC. This PG/cAMP pathway transduces the endothelin (ET) B-mediated antiproliferative effect of endothelin-1 (ET-1) and up-regulates ETB receptors. Here, we show that platelet-derived growth factor (PDGF)-BB and thrombin, although mitogenic, generate growth inhibitory PGE2 in myofibroblastic human HSC. The two peptides elicit early PGE2 and cAMP synthesis, and also promote delayed induction of cyclooxygenase (COX)-2. Both early and delayed production of PGE2 counteract the mitogenic effect of PDGF-BB and thrombin because: (i) pretreatment with the COX inhibitor ibuprofen markedly enhances the mitogenic effect of both peptides; (ii) blocking early synthesis of PGE2 greatly enhances extracellular signal-regulated kinase (ERK) activation by both growth factors; (iii) enhancement of DNA synthesis by ibuprofen is only lost when the inhibitor is added after COX-2 induction has occurred. Finally, PDGF-BB and thrombin raise ETB receptors through the PG pathway. Thus, ibuprofen blunts growth factor-induced increase in ETB receptors. Up-regulation of the growth inhibitory ETB receptors by both mitogens may enhance the antiproliferative effect of ET-1 and thereby establish a negative feedback of their mitogenic effect. Our results shed light on novel growth inhibitory signals evoked by two mitogenic growth factors expressed during liver injury.

Adipocytes↗
PubMed1998

Role of NF-kappaB in the antiproliferative effect of endothelin-1 and tumor necrosis factor-alpha in human hepatic stellate cells. Involvement of cyclooxygenase-2.

During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype and proliferate and synthesize fibrosis components. Endothelin-1 (ET-1), which inhibited the growth of human myofibroblastic HSC, increased the formation of two NF-kappaB DNA binding complexes; this effect was also observed with tumor necrosis factor-alpha (TNF-alpha). The complexes were identified as the p50/p50 and p50/p65 NF-kappaB dimers. Activation of NF-kappaB was associated with the degradation of the inhibitory protein IkappaB-alpha; no IkappaB-beta was detected. Activation of NF-kappaB and degradation of IkappaB-alpha were prevented by the NF-kappaB inhibitors sodium salicylate and MG-132. In addition to cyclooxygenase-1 (COX-1), COX-2 is also constitutively expressed in human HSC, and the use of dexamethasone and of SC-58125, a selective COX-2 inhibitor, revealed that COX-2 accounts for basal COX activity. Moreover, COX-2 mRNA and protein were up-regulated by ET-1 and TNF-alpha, whereas COX-1 was unaffected. Induction of COX-2 and stimulation of COX activity by ET-1 and TNF-alpha were prevented by sodium salicylate and MG-132, suggesting that activation of NF-kappaB by either factor is needed for stimulation of COX-2. Finally, SC-58125 and dexamethasone reduced the growth inhibitory effect of ET-1 and TNF-alpha, indicating that activation of COX-2 is required for inhibition of HSC proliferation. Taken together, our results suggest that NF-kappaB, by inducing COX-2 expression, may play an important role in the negative regulation of human myofibroblastic HSC proliferation.

Adipocytes↗
PubMed1998

Activation of human aortic smooth-muscle cells is inhibited by PPARalpha but not by PPARgamma activators.

Peroxisome proliferator-activated receptors (PPARs) are key players in lipid and glucose metabolism and are implicated in metabolic disorders predisposing to atherosclerosis, such as dyslipidaemia and diabetes. Whereas PPARgamma promotes lipid storage by regulating adipocyte differentiation, PPARalpha stimulates the beta-oxidative degradation of fatty acids. PPARalpha-deficient mice show a prolonged response to inflammatory stimuli, suggesting that PPARalpha is also a modulator of inflammation. Hypolipidaemic fibrate drugs are PPARalpha ligands that inhibit the progressive formation of atherosclerotic lesions, which involves chronic inflammatory processes, even in the absence of their atherogenic lipoprotein-lowering effect. Here we show that PPARalpha is expressed in human aortic smooth-muscle cells, which participate in plaque formation and post-angioplasty re-stenosis. In these smooth-muscle cells, we find that PPARalpha ligands, and not PPARgamma ligands, inhibit interleukin-1-induced production of interleukin-6 and prostaglandin and expression of cyclooxygenase-2. This inhibition of cyclooxygenase-2 induction occurs transcriptionally as a result of PPARalpha repression of NF-kappaB signalling. In hyperlipidaemic patients, fenofibrate treatment decreases the plasma concentrations of interleukin-6, fibrinogen and C-reactive protein. We conclude that activators of PPARalpha inhibit the inflammatory response of aortic smooth-muscle cells and decrease the concentration of plasma acute-phase proteins, indicating that PPARalpha in the vascular wall may influence the process of atherosclerosis and re-stenosis.

Acute-Phase Proteins↗
PubMed1998

Compliance with disposition to primary care physicians and psychiatrists in elderly homebound mentally ill patients.

In a naturalistic study, the authors evaluated homebound mentally ill elderly patients as part of a psychiatric outreach team in two rural/semi-urban regions. Of the 80 patients evaluated in the two counties, 15 patients were admitted to nursing homes and were excluded from the analyses of follow-up data. Of the remaining 65 patients, 36 were referred to a primary care physician and 29 to a psychiatrist. The follow-up rate with primary care physicians (75%) was significantly greater than with psychiatrists (41% [P < 0.006]). At 3- to 6-month follow-up by telephone, patients who were compliant showed significantly greater clinical improvement than those who were not compliant (P < 0.0001). Overall, the higher follow-up rate for primary care physicians, compared with psychiatrists, reinforces the importance of educating and training primary care physicians in the diagnosis and management of mental disorders in elderly patients.

Aged↗
PubMed1997

Regulation of the expression of cyclooxygenase-2 by nitric oxide in rat peritoneal macrophages.

Activation of rat peritoneal macrophages by LPS resulted in time-dependent production of nitric oxide and enhancement of cyclooxygenase (Cox) activity. This stimulation was accompanied by increased expression of inducible enzymes, NO synthase, and Cox-2, contrasting with no variation in constitutive Cox-1. Inhibition of NO production in LPS-treated macrophages by either the L-arginine analogue N(G)-monomethyl-L-arginine (L-NMMA) or aminoguanidine was accompanied by an additional enhancement of Cox activity parallel to the expression of Cox-2 protein analyzed by Western blot. Addition of NO donors (3-morpholinosydnonimine (Sin-1), sodium nitroprusside, or S-nitrosoglutathione) reversed the effects of L-NMMA, confirming the role of NO on Cox-2 expression. Specific immunoprecipitation of Cox-2 showed a pattern of protein expression similar to that observed in intact cells. Enzyme activity tested on the immunoprecipitates was correlated with the enzyme mass. In contrast, there was no variation in immunoprecipitated Cox-1 protein or in activity. Levels of mRNA for Cox-2 were increased in macrophages stimulated by LPS in the presence of L-NMMA compared with LPS alone. Metabolic labeling using [35S]methionine showed that inhibition of NO formation resulted in enhanced de novo synthesis of the 35S-labeled Cox-2. The amount of Cox-2 protein induced in the presence or absence of L-NMMA did not change for at least 6 h, suggesting that NO does not modify the t1/2 of the enzyme. These results provide evidence that NO participates in PG production by negative regulation of Cox-2 expression.

Animals↗
PubMed1997

Rapid, agonist-dependent phosphorylation in vivo of human thromboxane receptor isoforms. Minimal involvement of protein kinase C.

Thromboxane A2 (TxA2) is a potent vasoconstrictor and platelet agonist. Its biological function is tightly regulated. G protein-coupled membrane receptors transduce the effects of TxA2. However, although a single thromboxane receptor (TP) gene has been identified, two splice variants have been cloned from human placenta and megakaryocytic lines (TPalpha) and from human endothelial cells (TPbeta). These differ in the length of their carboxyl-terminal extensions (15 versus 79 residues), which contain multiple potential sites for receptor phosphorylation. Given that TP agonists activate protein kinase C (PKC), it would seem possible that PKC-dependent phosphorylation of TPs might play a central role in homologous desensitization of these receptors. To determine if the TP isoforms were differentially phosphorylated in response to agonist in vivo, human embryonic kidney (HEK) 293 cells were stably transfected with TPalpha and TPbeta. Isoform-specific anti-peptide antibodies were developed and used to immunoprecipitate the phosphorylated receptors. U46619, a PGH2/TxA2 mimetic, induced specific phosphorylation of both isoforms. Phosphorylation of the two isoforms was similar in dose and time dependence, reaching a plateau at around 100 nM U46619. Inhibition of PKC with either GF 109203X (5 microM) or RO 31-8220 (5 microM) or of protein kinase A with H-89 (50 microM) marginally influenced agonist-dependent phosphorylation of either isoform and failed to modulate homologous desensitization of agonist-induced stimulation of inositol phosphate formation. Similar results were obtained when PKC was down-regulated by long term incubation with the phorbol ester, phorbol myristate acetate. Although short term stimulation with phorbol myristate acetate caused PKC-dependent phosphorylation of TPs in vivo, thrombin stimulation of the TP-transfected HEK cells in vivo failed to phosphorylate either of the TP isoforms. Thus, despite the capacity of PKC to phosphorylate TPs in HEK 293 cells and the likely activation of PKC by TP stimulation, this enzyme, like protein kinase A, contributes marginally to rapid, agonist-induced phosphorylation of either TP isoform.

Cell Line↗
PubMed1997

Cyclooxygenase-2 and synthesis of PGE2 in human bronchial smooth-muscle cells.

The purpose of this study was to determine the mechanism of enhanced prostaglandin synthesis in cultured human bronchial smooth-muscle cells challenged with interleukin-1 beta (IL-1 beta). Cells were incubated with IL-1 beta (10 to 50 U/ml) for 0 to 24 h. Prostaglandin E2 (PGE2) production was evaluated through the conversion of exogenous (14C)-arachidonic acid and specific enzyme immunoassay of endogenous products. IL-1 beta enhanced PGE2 formation in a concentration- and time-dependent manner, reaching its peak at 6 to 8 h and fading at 18 to 24 h. Immunoblot analysis showed that the inducible cyclooxygenase enzyme (COX-2) was expressed only in IL-1 beta treated cells, whereas the constitutive isoform of cyclooxygenase (COX-1) remained unaltered. COX-2 expression and PGE2 formation were inhibited by dexamethasone (2 microM), cycloheximide (10 microM), and IL-1-receptor antagonist (IL-1 ra) (250 ng/ml), independently. PGE2 synthesis was significantly reduced by compound SC-58125, a specific COX-2 inhibitor. The close parallelism between the kinetics of COX-2 protein expression and PGE2 accumulation, as well as the constitutive nature of COX-1 isoform, indicate that IL-1 beta-driven PGE2 formation in human bronchial smooth-muscle cells is mediated by de novo expression of COX-2 enzyme.

Arachidonic Acid↗
PubMed1996

Regulation of cyclooxygenases by protein kinase C. Evidence against the importance of direct enzyme phosphorylation.

Cyclooxygenases (COXs) are key prostaglandin biosynthetic enzymes. While COX-1 expression is largely constitutive, COX-2 is highly regulated by cytokines, growth factors, and tumor promoters, such as the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA). While phosphorylation of transcription factors may regulate COX transcription, the existence of PKC consensus sequences suggests that direct enzyme phosphorylation might also regulate differential expression of the enzymes. Nevertheless, phosphorylation of both human recombinant COX-1 and COX-2 by rat brain PKC in vitro was minimal, as was phosphorylation of peptides based on PKC consensus sequences in COX-1 (less than 4% of the phosphorylation of the PKC-alpha pseudosubstrate peptide). Similarly, phosphorylation of the corresponding COX-2 peptides was not observed using either the phosphocellulose paper absorption method or electrospray mass spectrometry. MEG-01 and NIH 3T3 cells were labeled with [32P]orthophosphate to investigate COX phosphorylation in vivo. COX-2 synthesis was induced by PMA (100 nM) or serum stimulation in NIH 3T3 cells. COX-1 was expressed constitutively in MEG-01 cells. Specific polyclonal antibodies raised against sequences of human COX-1 (Ala24-Cys35) and COX-2 (Asn580-Lys598) were used for immunoprecipitation. Neither COX-1 nor COX-2 was phosphorylated in vivo, irrespective of the presence of a phosphatase inhibitor (1 microM okadaic acid). Although COX-1 and COX-2 are differentially regulated, no differences were observed in terms of susceptibility to phosphorylation by PKC either in vitro or in vivo. Despite regulated expression of COX-2 by PMA and the existence of consensus sequences for PKC phosphorylation, it appears that it is an unfavorable substrate for this enzyme.

3T3 Cells↗
PubMed1996

Cyclooxygenase-1 and -2 of endothelial cells utilize exogenous or endogenous arachidonic acid for transcellular production of thromboxane.

The presence of prostaglandin (PG) H2 in the supernatant of human umbilical vein endothelial cells (HUVEC) stimulated by thrombin restores the capacity of aspirin-treated platelets to generate thromboxane (TX) B2. Induction of cyclooxygenase-2 (Cox-2) by interleukin (IL)-1alpha or a phorbol ester increases this formation. HUVEC treated with aspirin lost their capacity to generate PGs but recovery occurred after 3- or 6-h induction of Cox-2 with phorbol ester or IL-1alpha. Enzyme activity of the newly synthesized Cox-2 in aspirin-treated cells, evaluated after immunoprecipitation, was similar to untreated cells but after 18 h of cell stimulation only 50-60% recovery of Cox-1 was observed. The use of SC58125, a selective Cox-2 inhibitor, confirmed these findings in intact cells. Cyclooxygenase activity was related to the amount of Cox proteins present in the cells, but after induction of Cox-2, contribution of the latter to PG production was 6-8-fold that of Cox-1. Aspirin-treated or untreated cells were incubated in the absence or presence of SC58125 and stimulated by thrombin, the ionophore A23187, or exogenous arachidonic acid. The production of endogenous (6-keto-PGF1alpha, PGE2, PGF2alpha) versus transcellular (TXB2) metabolites was independent of the inducer, the source of arachidonic acid and the Cox isozyme. However, in acetylsalicylic acid-treated cells, after 6-h stimulation with IL-1alpha, newly synthesized Cox-2 produced less TXB2 than 6-keto-PGF1alpha compared to untreated cells. At later times (>18 h), there was no metabolic difference between the cells. These studies suggest that in HUVEC, Cox compartmentalization occurring after short-term activation may selectively affect transcellular metabolism, but not constitutive production, of PGs.

Arachidonic Acid↗
PubMed1996

Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation.

Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

Animals↗
PubMed1995

Involvement of tyrosine kinases in the induction of cyclo-oxygenase-2 in human endothelial cells.

In addition to a constitutive cyclo-oxygenase (Cox-1), human endothelial cells also possess an inducible cyclo-oxygenase (Cox-2) which plays an important role in the regulation of the synthesis of prostacyclin (prostaglandin I2). Cox-2 is regulated and expressed in large quantities upon activation of the cells by inducers such as phorbol myristate acetate (PMA), an activator of protein kinase C (PKC), or interleukin-1 alpha. We have investigated the involvement of protein tyrosine kinases in Cox-2 expression by human endothelial cells upon activation by these inducers. PMA or interleukin-1 alpha provoke an increase in the phosphorylation of substrates of 110 and 120 kDa and additional phosphorylations for a broad band of multiple substrates in the 70 kDa range. This stimulation was accompanied by the induction of Cox-2 protein, detectable after stimulation for 1 h, which is consistent with an increase in activity reflected by prostacyclin synthesis; no variation in the expression of Cox-1 could be observed. Three distinct inhibitors of protein tyrosine kinases, genistein, herbimycin or AG-213, reduced tyrosine phosphorylation of cell substrates, consistently with their pharmacological effects. Under these conditions, there was selective reduction of Cox-2 expression without modification of Cox-1. Regulation of Cox-2 induction is also dependent on the activation of PKC since Ro 31-8220 or PKC depletion by PMA prevented its induction. Our results suggest that within the time-frame of our experiments these effects on kinases are specific for Cox-2 rather than Cox-1.

Benzoquinones↗
PubMed1995

Immunological studies of human constitutive cyclooxygenase (COX-1) using enzyme immunometric assay.

Polyclonal antisera and six distinct monoclonal antibodies (mAbs) were raised against constitutive cyclooxygenase (COX-1) purified from ram seminal vesicles. Immunoblotting experiments revealed that the polyclonal antisera and 4 of the mAbs strongly recognized human COX in platelet extracts. Different two-site immunometric assays of ram COX-1 were established using different combinations of mAbs. The assays were performed in 96-well microtiter plates coated with one mAb, with another mAb (covalently labeled with acetylcholinesterase (AChE)) as tracer. One combination (solid phase CX-101 + CX-105-AChE) exhibited the best sensitivity, with significant detection of concentrations as low as 23 pg/ml (0.3 fmol/ml of sheep COX-1). Unfortunately, this assay poorly cross-reacted with human COX-1 from platelet extracts. Another combination (solid phase CX-111 + CX-110-AChE) exhibited good recognition of human COX-1 but poor cross-reactivity with ram COX-1. Finally, purified anti-COX-1 IgG coated and CX-110-AChE were chosen as the best compromise since both good sensitivity (limit of detection, 113 pg/ml of ram COX-1) and significant cross-reactivity between COX-1 from both species were observed. In parallel, polyclonal antibodies were raised in rabbits against a peptide of 12 amino acids corresponding to the aminoterminal part of human COX-1. These polyclonal antibodies were affinity-purified and used in development of another two-site immunometric assay of COX-1 with CX-110-AChE as tracer. These two assays were used to analyze the COX-1 content of human platelets and cultured human umbilical vein cells (HUVEC). The results obtained with each assay were compared in terms of sensitivity and specificity. The validity of both assays was checked by analyzing platelets and HUVEC extracts previously fractionated by molecular sieve chromatography.

Amino Acid Sequence↗
PubMed1995

Differential measurement of constitutive (COX-1) and inducible (COX-2) cyclooxygenase expression in human umbilical vein endothelial cells using specific immunometric enzyme immunoassays.

We have produced and characterized monoclonal antibodies (mAbs) directed against a specific carboxyterminal sequence of human cyclooxygenase-2 (residues 580-598). A rabbit polyclonal antiserum was also raised against another sequence of 10 amino acids (residues 570-581) not present in human constitutive cyclooxygenase-1. Affinity-purified polyclonal antibodies, coated on microtiter plates, were used as capture antibodies in a two-site immunometric assay, with an mAb-acetylcholinesterase conjugate used as tracer. The detection limit was 500 fmol/ml of peptide C3-COX2 (residues 570-595). The assay was specific for the cyclooxygenase-2 (COX-2) isoform, since no immunoreactivity could be detected in platelet extracts known to be rich in cyclooxygenase-1 (COX-1). In contrast, extracts from cultured human umbilical vein endothelial cells challenged with 20 nM phorbol myristate acetate (PMA) showed an increase in COX-2 immunoreactivity related both to the increase in enzyme activity and the variations observed by Western blot analysis. Under these conditions, analysis of the same cell lysates with another immunometric assay specific for COX-1 revealed insignificant variation of this enzyme. The specificity of detection was further assessed by measuring the immunoreactivity of the fractions obtained after molecular sieve chromatography of control and stimulated cell extracts, and corroborated the marked enhancement of COX-2 by comparison with COX-1. Treatment of PMA-activated cells with H-7 or actinomycin D totally abolished the COX-2 signal and had little effect on COX-1. No significant variation in COX-2 immunoreactivity was observed using the inactive isomer 4 alpha-PMA, even at 100 nM. These assays constitute the first quantitative analysis of constitutive COX-1 and of inducible COX-2 in nucleated cells at the protein level.

Amino Acid Sequence↗