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ABSTRACT: This study aimed to determine whether the cyclooxygenase (COX) substrate arachidonic acid (AA) evokes endothelium-dependent contraction and if so, the specific (COX) isoform(s) involved and whether prostacyclin (prostaglandin I2; PGI2), a mediator of endothelium-derived vasoconstrictor activity, can be generated in medial smooth muscle from the intermediate COX product PGH2 that might diffuse from the endothelium. Aortas and/or carotid arteries were isolated from C57BL/6 mice or those lacking one of the two COX isoforms (COX-1(-/-) or COX-2(-/-)) for functional and/or biochemical analyses. Results showed that in vessels from C57BL/6 mice, exogenous AA evoked not only endothelium-dependent production of the PGI2 metabolite 6-keto-PGF1α, but also contractions reduced by thromboxane-prostanoid receptor antagonism or endothelial denudation. The minimal concentration for AA to evoke contraction was 0.3 μM, a level thought to activate only COX-2. However, neither the contraction nor 6-keto-PGF1α production was altered in vessels from COX-2(-/-) mice, while both were reduced in COX-1(-/-) counterparts. In vessels from COX-1(-/-) mice, AA also caused minor contractions that were sensitive to non-selective COX inhibition. Real-time PCR showed that like COX-1, COX-2 mainly existed in the endothelium, but it unaltered in COX-1(-/-) mice. Also, we noted that in endothelium-denuded aortas, PGH2 generated PGI2 as in intact vessels. These results demonstrate a predominant role for COX-1 and suggest that in the given mouse arteries, metabolites from either COX isoform cause contraction. Moreover, our results imply that some of the PGI2 involved in vasoconstrictor activity of endothelial COX-mediated metabolism could possibly be generated from PGH2 in medial smooth muscle.
Experimental physiology 03/2013; · 3.17 Impact Factor
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ABSTRACT: This study aimed to determine whether a cyclooxygenase-2 (COX-2) inhibitor celecoxib influences endothelium-dependent contraction independent of its action on COX-2 and, if so, the underlying mechanism(s). Abdominal aortas and/or carotid arteries from C57BL/6 mice or those with genetic COX-2 deficiency (COX-2(-/-)) were isolated for functional and/or biochemical analyses. Result showed that following NO synthase inhibition celecoxib not only reduced the contraction evoked by acetylcholine in C57BL/6 abdominal aorta, but also that in COX-2(-/-) mice showing a comparable magnitude. Notably, the IC(50) of celecoxib obtained in COX-2(-/-) abdominal aorta was only ~0.364μM. Also, celecoxib exhibited a similar effect on COX-2(-/-) carotid arteries. Interestingly, celecoxib was not only found to inhibit the production of the prostacyclin (PGI(2)) metabolite 6-keto-PGF(1α) in COX-2(-/-) aortas, but also caused a reduction in the contraction evoked by PGI(2), by the α(1)-adrenergic agonist phenylephrine, or by 30mM K(+)-induced depolarization in COX-2(-/-) and/or C57BL/6 abdominal aorta. Moreover, N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS398), another COX-2 inhibitor, also reduced the contraction evoked by acetylcholine or by 30mM K(+)-induced depolarization in COX-2(-/-) mice. These results demonstrate explicitly that in mouse arteries celecoxib not only inhibits COX-1-mediated synthesis of PGI(2) and probably some other prostanoids, but also causes a reduction in vessel contractility that is independent of either COX-2 or COX-1, leading to an inhibition of COX-1-mediated endothelium-dependent contraction with an IC(50) value far below that of it considered for COX-1. Also, our data suggest that such effects of celecoxib could be possibly shared by some other COX-2 inhibitors, such as NS398.
European journal of pharmacology 11/2012; · 2.59 Impact Factor
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ABSTRACT: This study aimed to determine whether cyclo-oxygenase-1 (COX-1) mediates dilatation of mouse arteries via synthesis of prostacyclin (PGI(2)) and, if so, how PGI(2) (IP) receptors contribute and whether thromboxane prostanoid (TP) receptors are implicated in the process. Mesenteric arteries were isolated from wild-type mice or mice with COX-1 deficiency (COX-1(-/-)). The vasomotor reaction to the COX substrate arachidonic acid (AA) was determined with isometric force measurement, while the in vitro production or the plasma level of the PGI(2) metabolite 6-keto-PGF(1α) was analysed with high-performance liquid chromatography-mass spectroscopy or enzyme immunoassay, respectively. Results showed that AA, which evoked endothelium-dependent 6-keto-PGF(1α) production, elicited relaxation that was inhibited or enhanced by antagonizing IP or TP receptors, respectively. Also, IP receptor blockade resulted in contraction in response to AA (following NO synthase inhibition), which was prevented by a concomitant TP receptor antagonism. Meanwhile, COX-1(-/-) or COX-1 inhibition abolished the in vitro 6-keto-PGF(1α) production and reduced the relaxation or contraction observed with AA. Real-time PCR showed that whereas TP receptor mRNAs were detected at similar levels, IP receptor mRNAs were present at higher levels in the branches than in the main stem of the mesenteric artery. In addition, antagonizing the IP receptors enhanced the contraction evoked by PGI(2) in the carotid artery. Also, we noted that COX-1(-/-) mice had a reduced basal plasma 6-keto-PGF(1α) level. These results demonstrate an explicit vasodilator role for COX-1-mediated endothelial PGI(2) synthesis and suggest that the functionally opposing IP and TP receptors concomitantly mediate the vasomotor reaction to PGI(2), with the dilator activity of IP receptors being compromised by the vasoconstrictor effect of TP receptors and vice versa.
Experimental physiology 03/2012; 97(7):895-904. · 3.17 Impact Factor
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ABSTRACT: This study aimed to determine whether PGI(2) would be evoked by the endogenous endothelial B(2) receptor agonist bradykinin (BK) in the porcine interlobular renal artery and, if so, to determine how it would influence the vasomotor reaction, and the specific cyclooxygenase (COX) isoform(s) involved in its synthesis. The production of the PGI(2) metabolite 6-keto-PGF(1α) was analyzed with HPLC-mass spectroscopy, while vasomotor reaction to PGI(2) or BK was determined with isometric force measurement. Results showed that BK evoked an increase in the production of 6-keto-PGF(1α), which was abolished by endothelial denudation that removed COX-1 expression, or was reduced by COX-1 inhibition. Interestingly, PGI(2) evoked a potent contraction, which was prevented by antagonizing thromboxane-prostanoid (TP) receptors and was not enhanced by antagonizing the vasodilator PGI(2) (IP) receptors. The IP receptor agonists MRE-269 and iloprost did not induce any relaxation. Moreover, iloprost, which is also a PGI(2) analog, caused a contraction, which was sensitive to TP receptor antagonism, but was to a significantly lesser extent than that of PGI(2). Indeed, IP receptors were not detected by RT-PCR or Western blotting in the vessel. Following nitric oxide synthase (NOS) inhibition, BK also evoked an endothelium-dependent contraction, which was blocked by TP receptor antagonism. In addition, inhibition of COX-1 (but not COX-2) impeded the vasoconstrictor activity of BK and expedited the relaxation induced by the agonist in NOS-intact vessels. These results demonstrate that in the porcine interlobular renal artery BK evokes endothelial COX-1-mediated PGI(2) synthesis, which mainly leads to the activation of TP receptors and a vasoconstrictor response, possibly due to a scarcity of vasodilator activity mediated by IP receptors. Also, our data suggested that the effect of a PGI(2) analog on TP receptors could be reduced compared with that of PGI(2) due to modified structure as with iloprost.
AJP Renal Physiology 02/2012; 302(9):F1133-40. · 4.42 Impact Factor
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ABSTRACT: This study was to determine whether the endothelium of mouse major arteries produces prostacyclin (PGI(2)) and, if so, to determine how PGI(2) affects vasomotor reactivity and whether cyclo-oxygenase-1 (COX-1) contributes to PGI(2) synthesis. Abdominal aortas, carotid and femoral arteries were isolated from wild-type mice and/or those with COX-1 or -2 deficiency (COX-1(-/-); COX-2(-/-)) for biochemical and/or functional analyses. The PGI(2) metabolite 6-keto-PGF(1α) was analysed with high-performance liquid chromatography-mass spectroscopy, while vasoreactivity was determined with isometric force measurement. Results showed that in the abdominal aorta, ACh evoked endothelium-dependent production of 6-keto-PGF(1α), which was abolished by COX-1(-/-), but not by COX-2(-/-). Interestingly, COX-1(-/-) enhanced the dilatation in response to ACh, while PGI(2), which evoked relaxation of the mesenteric artery, caused contraction that was abolished by antagonizing thromboxane prostanoid (TP) receptors in the abdominal aorta. However, the TP receptor agonist U46619 evoked similar contractions in the abdominal aorta and mesenteric artery. Also, antagonizing TP receptors enhanced the relaxation in response to PGI(2) in mesenteric arteries. Real-time PCR showed that the PGI(2) (IP) receptor mRNA level was lower in the abdominal aorta than in mesenteric arteries. In addition, COX-1(-/-) not only abolished the contraction in response to ACh following NO inhibition in abdominal aorta, but also those in the carotid and femoral arteries. These results demonstrate an explicit role for endothelial COX-1 in PGI(2) synthesis and suggest that in given mouse arteries, PGI(2) mediates not dilatation but rather vasoconstrictor activity, possibly due to a low expression or functional presence of IP receptors, which enables PGI(2) to act mainly on TP receptors.
Experimental physiology 11/2011; 97(2):277-89. · 3.17 Impact Factor
