HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Supplemental Data All Versions of this Article: 18/6/757 03-0609fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SCHILDKNECHT, S. Articles by ULLRICH, V. Search for Related Content PubMed PubMed Citation Articles by SCHILDKNECHT, S. Articles by ULLRICH, V.

COX-2 inhibitors selectively block prostacyclin synthesis in endotoxin-exposed vascular smooth muscle cells ¹

Department of Biology, University of Konstanz, Konstanz, Germany

²Correspondence: Fakultät für Biologie, Universität Konstanz, Fach X910-Sonnenbühl, 78457 Konstanz, Germany. E-mail: volker.ullrich{at}uni-konstanz.de

SPECIFIC AIMS

We have earlier shown nitration and inhibition of prostacyclin (PGI₂) synthase by the action of endotoxin in the vascular endothelium. This process models the first phase of endothelial dysfunction in endotoxemia. Since high levels of 6-keto-PGF₁ as the stable degradation product of PGI₂ were found in septic patients, we investigated its possible synthesis by vascular smooth muscle cells, which have high amounts of constitutive PGI₂ synthase but are devoid of significant cyclooxygenase (COX) activity under resting conditions.

Therefore, the involvement of inducible COX-2 in the formation of vasoactive eicosanoids by bovine smooth muscle cells was investigated as a model for a second phase of endotoxic shock in which smooth muscle may replace the superoxide-inactivated endothelium by coupling newly synthesized COX-2 to constitutive PGI₂ synthase. We were especially interested in whether the inducible process was blocked by parallel induction of NO synthase-2.

PRINCIPAL FINDINGS

1. LPS causes predominant up-regulation of 6-keto-PGF₁ as an indicator for PGI₂ synthase activity
Primary cultures of bovine smooth muscle cells showed a two- to threefold increase in 6-keto-PGF₁ and PGE₂ formation over controls when treated with 10 ng/mL IL-1ß or TNF-. In contrast, 10 µg/mL LPS evoked an 300 fold higher release specifically of 6-keto-PGF₁ whereas formation of PGE₂ was relatively weak, with an increase of 3- to 5-fold. No increase in eicosanoid formation was detected in the absence of proinflammatory stimuli (Fig. 1 ).

View larger version (13K): [in this window] [in a new window]   Figure 1. Formation of 6-keto-PGF1 by smooth muscle cells. A) Time course of LPS-stimulated 6-keto-PGF1 release (end point detection). Smooth muscle cells were treated with and without LPS (10 µg/mL; E. coli). Without LPS, no formation of 6-keto-PGF1 was observed (time course coincides with abscissa). When LPS was added, the first 2 h show nearly no 6-keto-PGF1 formation. After this period, an increase of up to 30 ng/mL was observed after 10 h. B) Activity of prostacyclin synthase (relative formation). LPS (10 µg/mL) was added to smooth muscle cells at t = 0. Medium was removed when indicated, cells were washed twice, and new medium + LPS was added for another 30 min. Data reflect formation of PGI2 in the 30 min. A maximum in activity was observed at 8 h. Formation of 6-keto-PGF1 was standardized to values at t = 0. 6-Keto-PGF1 level at t = 0 was 100 pg/mL and defined as 1. Prostanoid formation was analyzed by EIA. Values are mean ± SD (n=4). *P< 0.05 vs. t = 0.

2. 6-keto-PGF₁ formation follows expression of COX-2
An increase of 6-keto-PGF₁ formation was observed after 4 h of incubation with LPS with a maximum of PGI₂ synthesis after 8 h, followed by a decline until 12 h. The observed rise correlated with an up-regulation of inducible COX-2 observed on mRNA and protein levels. Under the detection limit before the addition of LPS, COX-2 mRNA peaked at 4 h, followed by a rapid decline whereas the corresponding COX-2 protein showed a maximum at 5–6 h. COX-1 mRNA expression was not affected by LPS. (Fig. 2 )

View larger version (21K): [in this window] [in a new window]   Figure 2. A) Protein expression of COX-2 and prostacyclin synthase in bovine smooth muscle cells. Cells were stimulated with LPS (10 µg/mL) for the periods indicated. Levels of PGI2 synthase (52 kDa) remained on the same constant level at all times. Within the first 2 h, COX-2 protein (72 kDa) was not detectable. A peak in protein expression can be observed at 5 or 6 h. From 8 to 12 h, a weaker steady-state expression was detected. The blot shows representative results of 4 independent experiments. B) Induction of cyclooxygenase-2 (COX-2) mRNA in smooth muscle cells as a function of time after exposure to LPS (10 µg/mL). Without stimulation, COX-2 mRNA was under the detection limit. The message peaks at 4 h, then rapidly declines to a weaker constant expression. C) Expression of cyclooxygenase-1 (COX-1) mRNA in smooth muscle cells as a function of time. Expression of COX-1 did no change significantly. Note small changes of expression compared with COX-2. Data were normalized with respect to GAPDH. Data are mean ± SD from 3 independent experiments. *P < 0.05 vs. t = 0.

3. Effects of COX inhibitors
To further confirm COX-2 dependent 6-keto-PGF₁ formation, COX inhibitors were used. The isoform unspecific inhibitor aspirin effectively blocked formation of both PGI₂ and PGE₂. COX-2 specific inhibitor DuP-697 completely inhibited formation of PGI₂ at low concentrations (10 nM) whereas PGE₂ levels remained at about one-third that of control after 3 h of incubation. COX-1-specific inhibitor SC-560 revealed no significant reduction in 6-keto-PGF₁ formation and reduced PGE₂ by 25%. Therefore release of PGI₂ by smooth muscle cells was completely dependent on the expression of COX-2 whereas PGE₂ formation seemed to involve both COX isoforms.

4. LPS does not cause early induction of NO synthase in bovine smooth muscle cells
Formation of ^•NO as a precursor of peroxynitrite generation was measured. NOS activity assay revealed no change in the activity of NO synthases in bovine smooth muscle cells in the time frame of 0–12 h. Accumulation of nitrite as another indicator for ^•NO formation did not exceed 0.5 µM after 12 h of incubation with LPS. A gradual increase of iNOS mRNA was observed, but remained low compared with the induction in mesangial cells or adventitial fibroblasts.

CONCLUSIONS AND SIGNIFICANCE

Our data indicate that after nitration and inhibition of endothelial prostacyclin synthase under septic conditions, vascular smooth muscle cells can take over regulation of vascular tone in an autocrine manner. Induction of the early immediate gene COX-2 causes specific up-regulation of prostacyclin formation, exerting vasodilatory properties. Release of PGI₂ by smooth muscle cells may be the dominating source of vasorelaxing mediators responsible for the severe hypotension observed in septic patients. Since we have shown inhibition of PGI₂ synthase in the endothelium under septic conditions in previous work, the question was asked why smooth muscle PGI₂ synthase was not affected by peroxynitrite. It was shown that endogenous formation of ^•NO as one precursor for peroxynitrite formation responsible for nitration and inhibition of PGI₂ synthase was very weak in the period investigated and thus can explain the lack of inhibition. Inactivation of the enzyme would otherwise antagonize the beneficial effects of PGI₂ with regard to vasorelaxation and inhibition of platelet aggregation. Our data warrant caution in using COX-2 inhibitors clinically in sepsis and lead us to speculate whether ^•NO formation by adventi-tial fibroblasts and superoxide release from leukocytes could be deleterious in the end stage of septic shock.

View larger version (109K): [in this window] [in a new window]   Figure 3. Vascular smooth muscle in the development of septic shock. Under physiological conditions, the endothelium maintains an equilibrium of vascular tone by a basal formation of eNOS-derived •NO and COX-1-derived PGI2 among a variety of other mediators. Exposure to endotoxin leads to an activation of the endothelium. Increased formation of •O2– traps •NO to form peroxynitrite, which in turn nitrates and inactivates prostacyclin synthase. The lack of •NO and PGI2 favors vasoconstriction, which is further triggered by the activation of the TxA2/ PGH2 receptor. This Phase I of endothelial activation (1–2 h after LPS) is followed by the induction of COX-2, leading to PGI2 synthesis in smooth muscle and vessel relaxation (Phase II). High concentrations of endotoxin present during sepsis may lead to a loss of endothelial barrier integrity or even complete loss of the endothelium. Exposure of the smooth muscle layer to LPS and cytokines results in the induction of COX-2 and PGI2 release to provide anti-aggregatory and anti-adhesive properties. NOS = NO synthase; COX = cyclooxygenase; PGI2= prostacyclin; PGH2= prostaglandin H2; PGIS = prostacyclin synthase; IP = prostacyclin receptor; TP = PGH2/TxA2 receptor; ONOO– = peroxynitrite.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0609fje; doi: 10.1096/fj.03-0609fje

This article has been cited by other articles:

				N. Sandbo, S. Taurin, D. M. Yau, S. Kregel, R. Mitchell, and N. O. Dulin Downregulation of smooth muscle {alpha}-actin expression by bacterial lipopolysaccharide Cardiovasc Res, May 1, 2007; 74(2): 262 - 269. [Abstract] [Full Text] [PDF]

				M.-E. Gendron, N. Thorin-Trescases, L. Villeneuve, and E. Thorin Aging associated with mild dyslipidemia reveals that COX-2 preserves dilation despite endothelial dysfunction Am J Physiol Heart Circ Physiol, January 1, 2007; 292(1): H451 - H458. [Abstract] [Full Text] [PDF]

				P. Nieri, C. Martinelli, C. Blandizzi, N. Bernardini, R. Greco, C. Ippolito, M. Del Tacca, and M. C. Breschi Role of Cyclooxygenase Isoforms and Nitric-Oxide Synthase in the Modulation of Tracheal Motor Responsiveness in Normal and Antigen-Sensitized Guinea Pigs J. Pharmacol. Exp. Ther., November 1, 2006; 319(2): 648 - 656. [Abstract] [Full Text] [PDF]

				R. Nasrallah and R. L. Hebert Prostacyclin signaling in the kidney: implications for health and disease Am J Physiol Renal Physiol, August 1, 2005; 289(2): F235 - F246. [Abstract] [Full Text] [PDF]

				T. Lu, X.-L. Wang, T. He, W. Zhou, T. L. Kaduce, Z. S. Katusic, A. A. Spector, and H.-C. Lee Impaired Arachidonic Acid-Mediated Activation of Large-Conductance Ca2+-Activated K+ Channels in Coronary Arterial Smooth Muscle Cells in Zucker Diabetic Fatty Rats Diabetes, July 1, 2005; 54(7): 2155 - 2163. [Abstract] [Full Text] [PDF]

				M. M. Bachschmid and B. van der Loo A New "Sunshine" in the Vasculature? Circulation, April 5, 2005; 111(13): 1571 - 1573. [Full Text] [PDF]

This Article Full Text (PDF) Supplemental Data All Versions of this Article: 18/6/757 03-0609fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SCHILDKNECHT, S. Articles by ULLRICH, V. Search for Related Content PubMed PubMed Citation Articles by SCHILDKNECHT, S. Articles by ULLRICH, V.