[Frontiers in Bioscience E2, 441-448, January 1, 2010]
The influence of constitutive cox-2 in smooth muscle tissue on the contractile effect of phenylephrine in the rat abdominal aorta
Maria del Carmen Castillo-Hernandez1, Gustavo Guevara-Balcazar1, Pedro Lopez-Sanchez1, Juan Asbun-Bojalil2, Enrique Castillo-Henkel1, Carlos Castillo-Henkel2
1
TABLE OF CONTENTS
1. ABSTRACT
Prostanoids are involved in the phenylephrine-induced contraction of the aorta. Here, we examined whether or not constitutive cyclooxygenase-2 (phosholipases C and A2) is the source of prostanoids in the smooth muscle of the arterial wall of the thoracic and abdominal aorta. Both cyclooxygenase isoforms (COX-1 and COX-2) were expressed in the two aortic segments, but their expression was not altered by phenylephrine, the protein synthesis inhibitor cycloheximide, or the phospholipase A2 inhibitors arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosponate. Indomethacin and NS398, which are a non-selective and selective COX-2 inhibitor, respectively, but not SC-560, which is a COX-1-selective inhibitor, inhibited the effect of phenylephrine on the abdominal, but not the thoracic, aorta. Similarly, U73122, which is a phospholipase C inhibitor, and RHC80267, which is a diacylglycerol lipase inhibitor, inhibited the effect of phenylephrine. These findings suggest that prostanoids, which are produced by constitutively active COX-2, influence the contractile response of the abdominal aorta and that the production of arachidonic acid relies on phospholipase C and diacylglycerol lipase.
2. INTRODUCTION
The effect of alpha1-adrenergic agonists on the vascular tone is the result of a complex network of direct and indirect actions, which are influenced by many factors, including species (1), gender (2) and local or regional characteristics of the vascular bed. These characteristics may be modified by aging (3,4) or the development of diseases such as hypertension, diabetes or arteriosclerosis (5,6,7). Accordingly, studies of the contractile effects of norepinephrine in arteries from rabbits (8) as well as in both arteries and veins from canines (9,10) demonstrated that alpha1-adrenoceptor-mediated arterial and vein sensitivity varied by species and region. Quantitative and qualitative regional variations in the alpha1-adrenergic vasocontractile effects may be due to differences in the number, affinity or subtype of alpha1-adrenergic receptors, as well as differences in the predominant intracellular pathways of distinct vascular regions (11,12,13,14). In addition to phosphoinositide turnover and calcium signaling (14), prostaglandins may also be involved in the vasocontractile effect that is mediated by alpha1- adrenoceptors. Studies suggest that cyclooxygenase-2 (COX-2) is the COX isoform that is responsible for the contractile effects of prostanoids. According to some authors, this enzyme is virtually absent in the healthy vasculature and only expressed in pathological conditions, such as atherosclerosis or diabetes (15,16,17). However, other researchers suggest that COX-2 is present in the healthy vasculature, mainly in the vascular endothelium, and upregulated in pathological conditions such as hypertension (18). Given the current controversy over the role of COX-2 in the vasculature, we decided to evaluate the capacity of rat aortic smooth muscle cells to produce contractile prostaglandins in response to phenylephrine (Phen), as well as the role of constitutively active COX-2 and the influence of the vascular region in this process. We also investigated the role of phospolipases C and A2 in the production of arachidonic acid, which is necessary to produce the COX-2-derived prostanoids.
3. MATERIALS AND METHODS
The experiments were conducted using protocols approved by the Animal Care Committee of our institution, which are in agreement with the UK Animals (Scientific Procedures) Act of 1986.
3.1. Preparation of the aortic rings and measurement of tension
Male Wistar rats (250 to 300g) were kept in the animal colony until time of sacrifice. The animals were maintained on a 12/12 hrs light-dark cycle at a constant temperature (22 ± 2oC), with food and water freely available in their home cages.
The animals were euthanized by decapitation, and the aortic tissues were immediately excised, placed in cold solution Krebs, cleaned and freed from the surrounding connective tissue. Taking the diaphragm as a reference, the abdominal and thoracic segments of the aorta were split, and each segment was cut into rings (4-5 mm long) that were then placed in 10 ml chambers filled with Krebs-bicarbonate solution of the following composition (mM): NaCl, 118; KCl, 4.7; KH2PO4, 1.2; MgSO4.7H2O, 1.2; CaCl2 .2H2O, 2.5; NaHCO3, 25; dextrose, 11.7; and calcium disodium EDTA, 0.026.
Tissue baths were maintained at 37oC, pH 7.4 and bubbled with a mixture of 95% O2 and 5% CO2. Rings were mounted on two stainless steel hooks in order to fix them to the bottom of the chamber and to a Grass FTO3 force displacement transducer connected to a 7D Grass Polygraph (Grass Instrument Co., Quincy MA, U.S.A), which was used to record the isometric tension developed by the aortic rings. The rings were given 2 g of initial tension and allowed to equilibrate for 2 h. Thirty minutes after setting up the organ bath, tissues were first exposed to Phen (10-6 M) to test their contractile responses and then rinsed three times with Krebs solution to restore tension to the precontracted level. Optimal ring tension was selected from preliminary experiments in which the rings were stretched to elicit the greatest response to Phen (10-6 M). We employed denuded aortic rings that were prepared by gently turning the rings several times on the distal portion of a pair of small forceps. The lack of endothelial integrity was pharmacologically assessed by acetylcholine-induced vasodilatation (10-6 M). Segments showing no relaxation were considered to be endothelium-denuded.
3.2. Phenylephrine concentration-response curves in the presence or absence of different antagonists.
3.2.1. Phenylephrine concentration-response curves
After the equilibration period, we obtained Phen concentration-response curves (10-9 to 10-5 M) for aortic rings without endothelium from the thoracic and abdominal segments, in order to determine if there were regional differences in the contractile response to this drug.
3.2.2. Phenylephrine concentration-response curves in the presence of indomethacin
In order to evaluate if prostanoids are involved in the phenylephrine-induced contraction and if this contractile effect is specific to the vascular region, the contractile response of the thoracic and abdominal aortic rings to Phen was analyzed in the absence and presence of indomethacin (10-5 M). This drug was added 30 minutes before obtaining the Phen concentration-response curve.
3.2.3. Phenylephrine concentration-response curves in the presence of NS-398 and SC-560
To determine the enzymatic source of the prostanoids involved in the phenylephrine-induced contraction, we tested the response of the aortic rings to phenylephrine in the presence of selective COX-1 (SC-560, 10-6 M) and COX-2 (NS-398 10-7 M) inhibitors. We also used immunoblot analysis to detect the presence of COX-1 and COX-2 in the endothelium-denuded thoracic and abdominal aortic segments.
3.2.4. Phenylephrine concentration-response curves in the presence of cycloheximide
To evaluate if the de novo synthesis of COX is involved in the contractile effect of Phen, we pretreated aortic rings with cycloheximide (10-5 M), which is an inhibitor of protein synthesis, 30 minutes before obtaining a Phen concentration-response curve.
3.2.5. Phenylephrine concentration-response curves in the presence of AATFMK and MAPF
To determine whether cytosolic and intracellular phospholipase A2 (cPLA2 and iPLA2) were sources of the arachidonic acid needed to produce the prostanoids involved in the contractile effect of Phen, aortic rings were pretreated with arachidonyl trifluoromethyl ketone (AATFMK; 1.6 10-5 M) and methyl arachidonyl fluorophosponate (MAPF; 10-6 M) 30 minutes before determining the Phen concentration-response curve of these rings.
3.2.6. Phenylephrine concentration-response curves in the presence of U73122 and RHC80267
We also evaluated phosoholipase C (PLC) and diacylglycerol lipase as possible sources of arachidonic acid in the production of the contractile prostanoids. To do this, we incubated abdominal aortic rings for 30 min with either U73122, which is an inhibitor of PLC, or RHC80267, which is an inhibitor of diacylglycerol lipase, before examining these rings Phen concentration-response curves.
3.3. COX-1 and COX-2 immunoblot analysis
Briefly, samples were homogenized in Tris-HCl, pH 7.4 with a protease cocktail (MiniComplete-EDTA free, Roche, Mannheim, Germany), and the total protein content of the sample was analyzed by the Lowry method (Lowry et al., 1951). Immunoblots were performed in duplicate using 50 �g of protein per lane on a 10% SDS-polyacrylamide gel. The samples were then transferred onto a polyvinylidene fluoride (PVDF) membrane (Hybond-P, Amersham Biosciences, UK). The PVDF membrane was blocked with TBS containing 5% skim milk and 0.05% Tween for 2 hours at room temperature, followed by an overnight incubation at 4�C with a 1:400 dilution of a polyclonal antibody against COX-1 or COX-2 (Santa Cruz Biotechnology, CA, USA). After the primary incubation, the membrane was washed and incubated with the corresponding secondary (anti-goat or anti-rabbit) HRP-labeled antibody (Zymed, USA), diluted 1:10,000 in the blocking solution, for two hours at room temperature. The blots were washed and developed using an ECL detection system (Luminol, Santa Cruz Biotechnology). The blots were then stripped and, as a control, beta-actin levels were determined using a polyclonal antibody. Images from films were digitally acquired, and a densitometry analysis was performed using the Quantity One Image Acquisition and Analysis Software (BioRad, Hercules, CA. USA). Data are expressed as normalized optical density (OD) arbitrary units.
3.4. Drugs
Phenylephrine HCl, acetylcholine HCl, NS-398, SC-560, indomethacin, arachidonyl trifluoromethyl ketone (AATFMK), methyl arachidonyl fluorophosponate (MAFP), U72122, RHC 80267 and cycloheximide were purchased from Sigma Chemical Co. (St. Louis, MO, USA.). NS 398 was purchased from ICN Biomedicals Inc. (Irvine, CA. USA). The majority of the drugs were prepared daily in deionized water and kept on ice until use. NS-398 aliquots (10-4 M) were prepared in dimethyl sulfoxide and kept frozen at -200C until use. RHC 80267 and SC-560 were also diluted in dimethyl sulfoxide. Indomethacine was dissolved in 3% Na2CO3. Cycloheximide was diluted in ethanol and kept frozen. Control rings that were treated with corresponding concentrations of the solvent were studied simultaneously.
3.5. Data and statistical analysis
Data are presented as the mean +/- SEM throughout the paper. In all experiments, n equals the number of rats from which vessel segments were obtained. Non-linear regression curve fits were calculated for the concentration-response curves, and the -log EC50 and Emax were obtained. Statistical comparisons were performed by ANOVA in order to determine the differences in the data, followed by a post hoc test. In all cases, a p-value less than 0.05 was considered to be statistically significant.
4.-RESULTS
4.1. Phenylephrine concentration-response curves of thoracic and abdominal aortic rings without endothelium
Phen (10-9 -10-5 M) elicited a concentration-dependent contractile effect in aortic rings; the contractions were of similar magnitudes for both the thoracic and abdominal segments. The maximal responses to Phen were 1.67 +/- 0.12 g and 1.42 +/- 0.23 g, while the -log CE50 was 7.03 +/- 0.08 and 6.85 +/- 0.11 M in the thoracic and abdominal segments, respectively. Similar results were obtained using aortic rings with endothelium or using another contractile agent, such as serotonin or angiotensin II, instead of Phen (data not shown).
4.2. Effect of indomethacin on the contractile response to phenylephrine
The role of prostanoids in the contractile effect of Phen was determined by using indomethacin, which is a nonspecific inhibitor of COX. Pretreatment with indomethacin (10-5 M) significantly depressed the contractile response to Phen in the abdominal, but not thoracic, rings (Figure 1). For the abdominal rings, the maximal effect changed from 1.42 +/- 0.23 g to 0.70 +/- 0.03 g in the presence of indomethacin, while the -log EC50 decreased from 6.85 +/- 0.11 to 6.52 +/- 0.07 M when pretreated with indomethacin.
4.3. Effect of SC-560 on the contractile response to phenylephrine
Pretreatment with the selective COX-1 inhibitor SC-560 (10-6 M) did not modify the contractile response of the abdominal aortic rings to Phen (Figure 2a).
4.4. Effect of NS 398 on the contractile response to phenylephrine
Pretreatment with the selective COX-2 inhibitor NS 398 (10-6 and 10-5 M) depressed the contractile response of the abdominal aortic rings to Phen in a concentration-dependent manner (Figure 2b). The presence of NS 398 decreased the maximal response to Phen from 1.06 g +/- 0.087g to 0.63 +/- 0.050 at 10-5 M and 0.39 +/- 0.041 g at 10-6 M. The -log EC50 response to Phen was 6.6 +/- 0.22 M, 6.6 +/- 0.21 M and 6.2 +/- 0.10 M for no NS 398 pretreatment, pretreatment with 10-5 M NS 398 and pretreatment with 10-6 M NS 398, respectively (Figure 2b).
4.5. Immunoblot analysis of COX-1 and COX-2
The protein expression of the COX-1 and COX-2 receptors was assayed using specific antibodies against each receptor. These antibodies had been previously tested to ensure that they did not cross-react with a different enzyme subtype. COX-1 and COX-2 were expressed in both aortic segments, with higher relative expression in the thoracic aorta. When Phen was added to both tissues, the relative expression of both COX-1 and COX-2 did not change significantly (Figure 3).
4.6. Evaluation of the effect of cycloheximide on contractile response to phenylephrine
Pretreatment with cycloheximide did not influence the contractile effect of Phen in the abdominal rings (Figure 4). Similar results were seen for the thoracic rings (data not shown).
4.7. Effect of AATFMK and MAFP on the contractile response to phenylephrin
Pretreatment with AATFMK (1.6 x 10-5 M) or MAFP (10-6 M), which are both potent inhibitors of the non-secretory forms of PLA2 (cPLA2 and iPL2), did not affect the contractile response of the abdominal aortic rings to Phen (Figure 5). The maximal responses to Phen were 1.47 +/- 0.055 g and 1.54 +/- 0.062 g in the absence or presence of AATFMK, respectively, and 1.33 +/- 0.024 g and 1.31 +/- 0.03 g in the absence or presence of MAFP, respectively.
4.8. Effects of U73122 and RHC80267 on the contractile response to phenylephrine.
Pretreatment with U73122, which is an inhibitor of PLC, completely eliminated the contractile effect of Phen on the abdominal aortic rings (Figure 6a). In contrast, pretreatment with RHC80267, which is an inhibitor of diacylglycerol lipase, significantly shifted the concentration-response curve of Phen to the right (Figure 6b).
5.-DISCUSSION
The results of the present study did not show quantitative differences in the contractile effect of Phen in the different regions of the aorta of male Wistar rats. The magnitude of the contraction was similar in the thoracic and abdominal segments of the aorta, either in the absence or presence of endothelium. Similar results were observed using serotonin or angiotensin II as the contractile agents instead of Phen, which indicated, the region of the aorta was not a significant factor in the magnitude of the contractile effect and that the magnitude was independent of the drug employed. There are qualitative differences, however, since the mechanism of contraction in the rat abdominal aorta is different from that of the thoracic aorta. Prostanoids other than thromboxane A2 are involved in the contraction elicited by the adrenergic amines in the abdominal, but not the thoracic, aorta12. Our results confirm those of Lamb et al (1994), since the non-selective COX inhibitor indomethacin inhibited the contractile effect of the α1-adrenergic agonist Phen only in the abdominal segment of the rat aorta.
Phen is a non-selective agonist of all three subtypes of α1-adrenergic receptors (alpha1A, alpha1B and alpha1D), which are coupled through Gq/11 proteins and phospholipase C to phosphoinositide turnover and calcium signaling (14). Activation of this pathway explains the vasocontractile effect of α1-adrenergic receptor agonists. However, this signaling pathway does not completely explain all of the alpha1-adrenergic actions. Multiple signaling pathways are clearly involved in the activity of alpha1-adrenergic receptors, which seem to utilize different G proteins and depend on the specific signaling pathways expressed by different cell types (14,19,20,21). In addition to phospholipase C (PLC), both phospholipase A2 (PL A2 ) (22,23) and phospholipase D (PLD) (24) appear to be involved in alpha1-adrenergic activation. These three enzymes cause the release of arachidonic acid, which is the source of several vasoactive compounds. Differences in the distribution of the alpha1-adrenoceptors subtypes between
vessels, or even along the same vessel, could explain the variation in the mechanisms underlying the contractile response to alpha1-adrenergic agonists (25). In the case of the rat aorta, however, this hypothesis does not explain our results, because alpha1D is the predominant subtype of alpha1-adrenergic receptor in both segments of the aorta (14,26).
The regional differences in the mechanism of Phen in the aorta could also be related to the isoform of COX that is present in the different segments. Of the two well-known COX isoforms, COX-1 is considered to be constitutively expressed and involved in a variety of physiological processes. COX-2 is thought to be an inducible isoform that is upregulated by inflammatory citokines (27). However, recent reports have shown that both COX isoforms are expressed in many organs, including the lung, kidney and heart (28). COX-2 mediates the response to the prostanoid precursor, arachidonic acid, in the canine coronary vascular bed and in the rat lung, whereas COX-1 is the dominant isoform in the cerebral vascular bed of the mouse (29,30,31). Given the variability in the distribution of these COX isoforms, we analyzed the relative participation of the COX isoenzymes in the response to Phen by quantifying the levels of COX-1 and COX-2 expression. Although pretreatment of endothelium-denuded abdominal segments with SC-560 did not modify the contractile response to Phen, the selective COX-2 inhibitor NS 398 inhibited this effect in a concentration-dependent manner in the abdominal segment only. Given the similar potencies of NS 398 and indomethacin in the rat abdominal aorta, it seems likely that these agents act to prevent the formation of vasoconstrictor prostaglandins by COX-2 in vascular smooth muscle. Since we used endothelial-denuded aortic rings, the COX-2 that is involved in the contractile effect of Phen is likely located in the smooth muscle layer and perhaps the outer layer of the aorta as well. Our results differ from those of Alvarez et al (2005), who concluded that the COX-2 that is involved in the contractile effect of Phen is located in the endothelial layer of the aorta. These differences could be explained by the different ages and strains of rats used in the two studies (6-month old SHR and WKY rats vs. 3-month old WKY rats in the current study). In addition, the immunoblot results further support our conclusion, confirming the presence of COX-2 in the endothelial-denuded abdominal segments. The fact that this isoform is also found in the thoracic segments suggests there is an undiscovered mechanism within the abdominal, but not thoracic, segment of the aorta that couples the α1-adrenergic signal transduction pathway to the COX-2 system and that the presence of COX-2 is not enough to induce this effect.
Cycloheximide is an inhibitor of protein synthesis that helps determine whether these processes are necessary for various physiological or cellular adaptations. This drug prevented the production of prostaglandins in some tissues within 10 minutes of exposure, suggesting that prostaglandin production requires the production of a new polypeptide and that other metabolic processes are not affected (32). This result does not fully address the question of whether the COX-2 that is involved in the production of the Phen-induced contractile prostanoids in the abdominal aortic segment was inducible or constitutively active. Given
that the presence of Phen did not modify the expression of COX-2 and that cycloheximide did not affect the contractile response, we believe that the COX-2 involved in this response is constitutively active. Since pretreating the two aortic segments with cycloheximide did not modify the contractile effect of Phen, we conclude that the trauma of aortic dissection or endothelium removal likely did not induce COX-2 expression.
The generation of free arachidonic acid following receptor stimulation can result from the activity of a variety of different phospholipases, including PLA2, PLC and PLD (33). PLA2 has many subtypes with diverse functions, structures and regulatory mechanisms (34). Types I-III and V PLA2 enzymes are secreted, do not show specificity for the fatty acid moiety at the sn-2-position of phosphatidylcholine and require millimolar concentrations of Ca for activity (35). Types IV and VI PLA2 enzymes are cytosolic and show a preference for the hydrolysis of phosphatidylcholine, which possesses an arachidonyl moiety at the sn-2-position that leads to the release of arachidonic acid (35). Both type IV PLA2 (cPLA2) and type VI PLA2 (iPLA2) are activated following agonist binding to receptors, which results in the release of arachidonic acid. Type VI PLA2 is considered to be principally involved in the restructuring of membranes by reincorporating free arachidonic acid into phospholipids (36), whereas, type IV PLA2 is generally thought to be the principal enzyme that is involved in agonist-induced arachidonic acid generation.
Accordingly, agonist binding to various cell surface receptors has been shown to lead to the activation of cPLA2 and the generation of arachidonic acid in several different cell types (21,37). Nevertheless, we can rule out the participation of the non-secretory forms of PLA2 (IV and VI PLA2) in the production of the arachidonic acid, since neither the inhibitor of IV PLA2, AATFMK, nor the inhibitor of VI PLA2, MAPF, affected the activity of Phen on the abdominal aorta.
Even if PLA2 is the major pathway of arachidonic acid release, PLC or PLD can also produce this acid. Activation of PLC results in the formation of IP3 and diacylglycerol, and the hydrolysis of the latter by diacylglycerol lipase may serve as a source of arachidonic acid (38,39). PLD can hydrolyze phosphatidylcholine to form phosphatidic acid and choline. The resulting phosphatidic acid can then be acted upon by phosphatidic acid phosphohydrolase to yield diacylglycerol, from which arachidonic acid can be released via the action of diacylglycerol and monoacylglycerol lipases (35).
The total inhibition of Phen-induced contraction by U73122 demonstrates that the activation of PLC plays an effect in both the prostanoid-dependent and -independent contractile response of the rat abdominal aorta to this adrenergic drug. In addition, the inhibition of this effect by RHC80267, which is an inhibitor of diacylglycerol lipase (40), supports the idea that diacylglycerol may be the source of the arachidonic acid that is necessary to synthesize the prostanoids involved in the contractile response of the rat abdominal aorta to Phen. As a consequence, the arachidonic acid required to synthesize the prostanoids involved in this response to Phen could be obtained from diacylglycerol, which may be a direct or indirect product of PLC. This enzyme directly produces diacylglycerol by acting on phospholipids in the cellular membrane, and may indirectly produce it through the activation of PLD.
6. Acknowledgments
This work was supported by a research grant from the Coordinación General de Posgrado e Investigación del I.P.N. and partially by grant 46217 to PLS from CONACYT.
7. References
1, Bevan JA, Bevan RD, Kite K, Oriowo MA. Species difference in sensitivity of aortae to norepinephrine are related to á-adrenoceptor affinity. Trends Pharmacol Sci 9,87-89 (1988)
doi:10.1016/0165-6147(88)90173-3
PMid:2854313
2. Luksha L, Poston L, Gustafsson JA, Aghajanova L, Kublickiene K. Gender-specific alterations of adrenergic responses in small femoral arteries from estrogen receptor-â knockout mice. Hypertension 46, 1163-1168 (2005)
doi:10.1161/01.HYP.0000185648.48498.c1
PMid:16216990
3. Vila E, Vivas NM, Tabernero A. Jesús Giraldo, Arribas SM. á1-Adrenoceptor vasoconstriction in the tail artery during ageing. Br J Pharmacol 121(5), 1017-1023 (1997)
doi:10.1038/sj.bjp.0701193
PMid:9222562 PMCid:1564759
4. Smith EG, Voyles WF, Kirby BS, Markwald RR, Dinenno FA. Ageing and leg postjunctional á-adrenergic vasoconstrictor responsiveness in healthy men. J Physiol 582, 63-71 (2007)
doi:10.1113/jphysiol.2007.130591
PMid:17463044 PMCid:2075284
5. Kähönen, M., Tolvanen J-P, Sallinen,K., Wu, X., Pörsti, I. Influence of gender on control of arterial tone in experimental hypertension. Am J Physiol Heart Circ Physiol 275, H15-H22 (1998)
6. Stepp DW, Frisbee JC. Augmented adrenergic vasoconstriction in hypertensive diabetic obese Zucker rats. Am. J. Physiol. Heart Circ Physiol 282, H816-820 (2002)
7. Baumgart D, Haude M, Görge G, Liu F, Ge J, Große-Eggebrecht C, Erbel R, Heusch G. Augmented á-adrenergic constriction of atherosclerotic human coronary arteries. Circulation. 99, 2090-2097 (1999)
8. Takayanagi I, Onozuka S, Koike K. Variation in sensitivity of á1-adrenoceptor stimulants and á1-adrenoceptor mechanisms in rabbit arteries. Japan J Pharmacol 55, 513-522 (1991)
doi:10.1254/jjp.55.513
9. Takayanagi I, Koike K, Noguchi R, Ogishima M, Takiguchi F, Sato T. A regional difference in á -adrenoceptor mechanism in in canine veins. Arch Int Pharmacodyn Ther 289, 106-117 (1987)
10. Takayanagi I, Koike K, Kawano K, Onozuka S. A regional difference in á1-adrenoceptors in canine arteries. Arch Int Pharmacodyn Ther 294, 175-184 (1988)
11. Colucci WS, Gimbrone MA, Alexander RW. Regulation of myocardial and vascular alpha-adrenergic receptor affinity. Effects of guanine nucleotides, cations, estrogen, and catecholamine depletion. Circ Res 55, 78-88 (1984)
12. Lamb VL, Schwartz AJ, Rohn WR, Kaiser L. Cyclooxygenase inhibitors depress norepinephrine constriction of rat abdominal, but not thoracic, aorta. Eur J Pharmacol 256, 221-226 (1994)
doi:10.1016/0014-2999(94)90250-X
PMid:7914169
13. Griendling KK, Sastre A, Milnor WR. Regional differences in alpha 1- adrenoceptor numbers and responses in canine aorta. Am J Physiol Heart Circ Physiol 247, H928-H935 (1984)
14. J Garcia, J Vazquez, R Villalobos. á1-adrenoceptors: subtypes, signaling, and roles in health and disease. Arch Med Res 30, 449-458 (1999)
doi:10.1016/S0188-0128(99)00059-7
PMid:10714357
15. Hermann M. Cyclooxygenase-2 and nitric oxide. J Cardiovasc Pharmacol 47(1), S21-S25 (2006)
doi:10.1097/00005344-200605001-00005
PMid:16785825
16. Schonbeck U, Sukhova GK, Graber P, Coulter S, Libby P. Augmented expression of cyclooxygenase-2 in human atherosclerotic lesions. Am J Pathol 155, 1281-1291 (1999)
17. Guo Z, Su W, Allen S, Pang H, Daugherty A, Smart E, Gong MC. Cox-2 up-regulation and vascular smooth muscle contractile hyperrreactivity in spontaneous diabetic db/db mice. Cardiovasc Res 67, 723-735 (2005)
doi:10.1016/j.cardiores.2005.04.008
PMid:15885672
18. Alvarez Y, Briones AM, Balfagon G, Alonso MJ, Salaices M. Hypertension increases the participation of vasoconstrictor prostanoids from cyclooxygenase-2 in phenylephrine responses. J Hypert 23(4), 767-777 (2005)
doi:10.1097/01.hjh.0000163145.12707.63
PMid:15775781
19. Perez DM, DeYoung MB, Graham RM. Coupling of the expressed á1B-and á1D-adrenergic receptors to multiple signal pathways is both G protein and cells type specific. Mol Pharmacol 44, 784-795 (1993)
20. Morgan NG, Charest R, Blackmore PF, Exton JH. Potentiation of á1-adrenergic responses in rat liver by a cyclic AMP-dependent mechanism. Proc Natl Acad Sci 81, 4208-4212 (1984)
doi:10.1073/pnas.81.13.4208
21. LaBelle EF, Polyak E. Norepinephrine stimulates arachidonic acid release from vascular smooth muscle via activation of cPLA2. Am J Physiol Cell Physiol 274, 1129-1137 (1998)
22. Burch RM, Luini A, Axelrod J. Phospholipase A2 and phospholipase C are activated by distinct GTP-binding proteins in response to á1-adrenergic stimulation in FRTL5 thyroid cells. Proc Natl Acad Sci 83, 7201-7205 (1986)
doi:10.1073/pnas.83.19.7201
23. Perez D, Hwa J, Gaivin R, Mathur M, Brown F, Graham RM. Constitutive activation states of a G protein-coupled-receptor. Mol Pharmacol 49, 112-122 (1996)
24. Balboa MA, Insel PA. Stimulation of phospholipase D via á1-adrenergic receptors in Madin-Darby canine cells is independent of PKCá and -å activation. Mol Pharmacol 53, 221-227 (1998)
25. Richardson J, Chatwin H, Hirasawa A, Tsujimoto G, Evans PD. Agonist-specific coupling of a cloned human alpha 1A-adrenoceptor to differente second messenger pathways. Naunyn Schmiedebergs Arch Pharmacol 367(4), 333-341 (2003)
doi:10.1007/s00210-003-0703-x
PMid:12690424
26. J Asbun, E Castillo, B Escalante, C Castillo. Does segmental difference in alpha 1-adrenoceptor subtype explain contractile difference in rat abdominal and thoracic aorta? Vasc Pharmacol 38, 169-175 (2002)
doi:10.1016/S1537-1891(02)00164-7
PMid:12402516
27. Vane JR, Bakhle YS, Botting RM. Cyclooxygenase 1 and 2. Annu Rev Pharmacol Toxicol 38, 97-120 (1998)
doi:10.1146/annurev.pharmtox.38.1.97
PMid:9597150
28. Asano K, Lilly CM, Drazen JM. Prostaglandin G/H synthase-2 is the constitutive and dominant isoform inncultured human lung epithelial cells. Am J Physiol 271, L126-L181 (1996)
29. Hennan JK, Huang J, Barret TD, Driscoll EM, Willen DE, Park AM, Crofford LJ, Lucchesi BR. Effects of selective cyclooxygenase- 2 inhibition on vascular responses and thrombosis in canine coronary arteries. Circ 104(7), 820-825 (2001)
doi:10.1161/hc3301.092790
PMid:11502709
30. Niwa K, Haensel C, Ross ME, Iadecola C. Cyclooxygenase-1 participates in selected vasodilator responses of the cerebral circulation. Circ Res 88(6), 600-8 (2001)
31. Baber SR, Champion HC, Bivalacqua TJ, Hyman AL, Kadowitz PJ. Role of cyclooxygenase-2 in the generation of vasoactive prostanoids in the rat pulmonary and systemic vascular beds. Circ 108(7), 896-901 (2003)
doi:10.1161/01.CIR.0000084536.87322.BB
PMid:12900348
32. Fagan JM, Goldberg AL. Inhibitors of protein and RNA aynthesis cause a rapid block in prostaglandins production at the prostaglandin synthasa step. Proc Natl Acad Sci 83, 2771-2775 (1986)
doi:10.1073/pnas.83.8.2771
33. Exton JH. Signaling through phosphatidylcholine breakdown. J Biol Chem 265,1-4 (1990)
34. Dennis EA. The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem Sci 22, 1-2 (1997)
doi:10.1016/S0968-0004(96)20031-3
PMid:9020581
35. Osterhout JL, Shuttleworth TJ. A Ca2+-independent activation of a type IV cytosolic phospholipase A2 underlies the receptor stimulation of arachidonic acid-dependent noncapacitative calcium entry. J Biol Chem 275, 8248-8254 (2000)
doi:10.1074/jbc.275.11.8248
PMid:10713151
36. Balsinde J, Balboa MA, Dennis EA. Antisense inhibition of group VI Ca2+-independent phospholipase A2 blocks phospholipid fatty acid remodeling in murine P388D1 macrophages. J Biol Chem 272, 29317-29321 (1997)
doi:10.1074/jbc.272.46.29317
PMid:9361012
37. Shuttleworth TJ, Thompson JL. Muscarinic receptor activation of arachidonate-mediated Ca2+ entry in HEK293 cells is independent of phospholipase C J Biol Chem 273, 32636-32643 (1998)
doi:10.1074/jbc.273.49.32636
PMid:9830003
38. Balsinde J, Balboa MA, Insel PA, Dennis EA. Regulation and inhibition of phospholipase A2. Ann Rev Pharmacol Toxicol 39, 175-189 (1999)
doi:10.1146/annurev.pharmtox.39.1.175
PMid:10331081
39. Konrad RJ, Major CD,Wolf BA. Diacylglycerol hydrolysis to arachidonic acid is necessary for insulin secretion from isolated pancreatic islets: sequential action of diacylglycerol and monoacylglycerol lipases. Biochemistry 33, 13284-13294 (1994)
doi:10.1021/bi00249a015
PMid:7947736
40. Tu P, Kunert-Keil C, Lucke S, Brinkmeier H, Bouron A. Diacylglycerol analogues activate second messenger-operated calcium channels exhibiting TRPC-like properties in cortical neurons. J Neurochem 108(1), 126-138 (2009)
doi:10.1111/j.1471-4159.2008.05752.x
PMid:19094061
Abbreviations: COX: Cyclooxigenase, COX-1: cyclooxigenase 1, COX-2: cyclooxigenase-2, Phen: phenylephrine, AATFMK: arachidonyl trifluoromethyl ketone, MAPF: methyl arachidonyl fluorophosponate, PLC: phosoholipase C
Key Words: Phenyleprhine, Thoracic Aorta, Abdominal Aorta, Cyclooxygenase 2, Cyclooxygenase 1, COX-2, COX-1, Vascular Smooth Muscle
Send correspondence to: P Carlos Castillo-Henkel, Laboratorio de Farmacologia Cardiovascular, Departamento de Posgrado e Investigacion, Escuela Superior de Medicina IPN, Plan de San Luis y Diaz Miron s/n, Col. Casco Sto. Tomas, Del. Miguel Hidalgo, Mexico, D.F. CP 11340, Tel: 52-557296300- 62827, Fax: 52-557296300-62806, E-mail:drcarloscastillo@hotmail.com