Yingzhan Zhang

Involvement of cyclo‐oxygenase‐1‐mediated prostacyclin synthesis in the vasoconstrictor activity evoked by ACh in mouse arteries

This study was to determine whether the endothelium of mouse major arteries produces prostacyclin (PGI2) and, if so, to determine how PGI2 affects vasomotor reactivity and whether cyclo‐oxygenase‐1 (COX‐1) contributes to PGI2 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 PGI2 metabolite 6‐keto‐PGF1α 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‐PGF1α, which was abolished by COX‐1−/−, but not by COX‐2−/−. Interestingly, COX‐1−/− enhanced the dilatation in response to ACh, while PGI2, 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 PGI2 in mesenteric arteries. Real‐time PCR showed that the PGI2 (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 PGI2 synthesis and suggest that in given mouse arteries, PGI2 mediates not dilatation but rather vasoconstrictor activity, possibly due to a low expression or functional presence of IP receptors, which enables PGI2 to act mainly on TP receptors.

Role of cyclooxygenase-1-mediated prostacyclin synthesis in endothelium-dependent vasoconstrictor activity of porcine interlobular renal arteries.

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.

Vasoconstrictor role of cyclooxygenase-1-mediated prostacyclin synthesis in non-insulin-dependent diabetic mice induced by high-fat diet and streptozotocin

This study tested the hypothesis that in diabetic arteries, cyclooxygenase (COX)-1 mediates endothelial prostacyclin (PGI2) synthesis, which evokes vasoconstrictor activity under the pathological condition. Non-insulin-dependent diabetes was induced to C57BL/6 mice and those with COX-1 deficiency (COX-1(-/-) mice) using a high-fat diet in combination with streptozotocin injection. In vitro analyses were performed 3 mo after. Results showed that in diabetic aortas, the endothelial muscarinic receptor agonist ACh evoked an endothelium-dependent production of the PGI2 metabolite 6-keto-PGF1α, which was abolished in COX-1(-/-) mice. Meanwhile, COX-1 deficiency or COX-1 inhibition prevented vasoconstrictor activity in diabetic abdominal aortas, resulting in enhanced relaxation evoked by ACh. In a similar manner, COX-1 deficiency increased the relaxation evoked by ACh in nitric oxide synthase-inhibited diabetic renal arteries. Also, in diabetic abdominal aortas and/or renal arteries, both PGI2 and the COX substrate arachidonic acid evoked contractions similar to those of nondiabetic mice. However, the contraction to arachidonic acid, but not that to PGI2, was abolished in vessels from COX-1(-/-) mice. Moreover, we found that 3 mo after streptozotocin injection, systemic blood pressure increased in diabetic C57BL/6 mice but not in diabetic COX-1(-/-) mice. These results explicitly demonstrate that in the given arteries from non-insulin-dependent diabetic mice, COX-1 remains a major contributor to the endothelial PGI2 synthesis that evokes vasoconstrictor activity under the pathological condition. Also, our data suggest that COX-1 deficiency prevents or attenuates diabetic hypertension in mice, although this could be related to the loss of COX-1-mediated activities derived from both vascular and nonvascular tissues.

Concomitant activation of functionally opposing prostacyclin and thromboxane prostanoid receptors by cyclo‐oxygenase‐1‐mediated prostacyclin synthesis in mouse arteries

This study aimed to determine whether cyclo‐oxygenase‐1 (COX‐1) mediates dilatation of mouse arteries via synthesis of prostacyclin (PGI2) and, if so, how PGI2 (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 PGI2 metabolite 6‐keto‐PGF1α was analysed with high‐performance liquid chromatography–mass spectroscopy or enzyme immunoassay, respectively. Results showed that AA, which evoked endothelium‐dependent 6‐keto‐PGF1α 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‐PGF1α 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 PGI2 in the carotid artery. Also, we noted that COX‐1−/− mice had a reduced basal plasma 6‐keto‐PGF1α level. These results demonstrate an explicit vasodilator role for COX‐1‐mediated endothelial PGI2 synthesis and suggest that the functionally opposing IP and TP receptors concomitantly mediate the vasomotor reaction to PGI2, with the dilator activity of IP receptors being compromised by the vasoconstrictor effect of TP receptors and vice versa.

Cyclo‐oxygenase‐1 or ‐2‐mediated metabolism of arachidonic acid in endothelium‐dependent contraction of mouse arteries

•  What is the central question of this study? To determine the specific cyclo‐oxygenase (COX) isoform(s) involved in endothelium‐dependent contraction and whether prostaglandin I2, a mediator of endothelium‐derived vasoconstrictor activity, can be generated in medial smooth muscle from prostaglandin H2 that might diffuse from the endothelium. •  What is the main finding and its importance? Our results demonstrate a predominant role for COX‐1 in arachidonic acid metabolism and suggest that in the given mouse arteries, metabolites from either COX isoform cause contraction. Moreover, our results imply that some of the prostaglandin I2 involved in the vasoconstrictor activity of endothelial COX‐mediated metabolism could possibly be generated from prostaglandin H2 in the medial smooth muscle. These findings add to our current understanding of mechanisms for endothelium‐dependent contraction.

Effect of celecoxib on cyclooxygenase-1-mediated prostacyclin synthesis and endothelium-dependent contraction in mouse arteries.

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 ~0.364 μM. 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 30 mM 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 30 mM 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.

Role of cyclooxygenase-1 and -2 in endothelium-dependent contraction of atherosclerotic mouse abdominal aortas.

The objective of this study was to determine the role of cyclooxygenase (COX)‐1 or ‐2 in endothelium‐dependent contraction under atherosclerotic conditions. Atherosclerosis was induced in apoE knockout (apoE−/−) mice and those with COX‐1−/− (apoE−/−‐COX‐1−/−) by feeding with high fat and cholesterol food. Aortas (abdominal or the whole section) were isolated for functional and/or biochemical analyses. As in non‐atherosclerotic conditions, the muscarinic receptor agonist acetylcholine (ACh) evoked an endothelium‐dependent, COX‐mediated contraction following NO synthase (NOS) inhibition in abdominal aortic rings from atherosclerotic apoE−/− mice. Interestingly, COX‐1 inhibition not only abolished such a contraction in rings showing normal appearance, but also diminished that in rings with plaques. Accordingly, only a minor contraction (<30% that of apoE−/− counterparts) was evoked by ACh (following NOS inhibition) in abdominal aortic rings of atherosclerotic apoE−/−‐COX‐1−/− mice with plaques, and none was evoked in those showing normal appearance. Also, the contraction evoked by ACh in apoE−/−‐COX‐1−/− abdominal aortic rings with plaques was abolished by non‐selective COX inhibition, thromboxane‐prostanoid (TP) receptor antagonism, or endothelial denudation. Moreover, it was noted that ACh evoked a predominant production of the prostacyclin (PGI2, which mediates abdominal aortic contraction via TP receptors in mice) metabolite 6‐keto‐PGF1α, which was again sensitive to COX‐1 inhibition or COX‐1−/−. Therefore, in atherosclerotic mouse abdominal aortas, COX‐1 can still be the major isoform mediating endothelium‐dependent contraction, which probably results largely from PGI2 synthesis as in non‐atherosclerotic conditions. In contrast, COX‐2 may have only a minor role in such response limited to areas of plaques under the same pathological condition.

A vasoconstrictor response to COX-1-mediated prostacyclin synthesis in young rat renal arteries that increases in prehypertensive conditions.

This study aimed to determine whether prostacyclin (PGI2) functions as an endothelium-derived contracting factor (EDCF) in young rat renal arteries, and, if so, we wanted to examine the underlying mechanism(s) and how it changes in prehypertensive conditions. Vessels from Wistar-Kyoto (WKY) and prehypertensive spontaneously hypertensive rats (SHRs) of 25-28 days of age were isolated for functional and biochemical analyses. Result showed that following NO synthase (NOS) inhibition PGI2 and the thromboxane-prostanoid (TP) receptor agonist U-46619 evoked contractions in young WKY renal arteries that were similar to those in prehypertensive SHRs. Meanwhile, the endothelial muscarinic receptor agonist ACh evoked an endothelium-dependent contraction under NOS-inhibited conditions and a production of the PGI2 metabolite 6-keto-PGF1α; both were sensitive to cyclooxygenase (COX) and/or COX-1 inhibition but higher in prehypertensive SHRs than in young WKYs. Interestingly, in WKY renal arteries PGI2 did not evoke relaxation even after TP receptor antagonism that diminished the contraction evoked by the agonist. Indeed, PGI2 (IP) receptors were not detected in the vessel with Western blot. Moreover, we noted that treatment with the nonselective COX inhibitor indomethacin, which was started at the prehypertensive stage, blunted the elevation of systolic blood pressure and reduced the heart-to-body ratio in SHR within 2 mo of treatment. These results demonstrate that due to scarcity of IP receptors, PGI2, which is derived mainly from COX-1-mediated metabolism, acts as an EDCF in young WKY renal arteries, and it increases in prehypertensive conditions. Also, our data revealed that COX inhibition starting from the prehypertensive stage has an antihypertensive effect in young SHRs.

Role of E-type prostaglandin receptor EP3 in the vasoconstrictor activity evoked by prostacyclin in thromboxane-prostanoid receptor deficient mice

Prostacyclin, also termed as prostaglandin I2 (PGI2), evokes contraction in vessels with limited expression of the prostacyclin receptor. Although the thromboxane-prostanoid receptor (TP) is proposed to mediate such a response of PGI2, other unknown receptor(s) might also be involved. TP knockout (TP−/−) mice were thus designed and used to test the hypothesis. Vessels, which normally show contraction to PGI2, were isolated for functional and biochemical analyses. Here, we showed that the contractile response evoked by PGI2 was indeed only partially abolished in the abdominal aorta of TP−/− mice. Interestingly, further antagonizing the E-type prostaglandin receptor EP3 removed the remaining contractile activity, resulting in relaxation evoked by PGI2 in such vessels of TP−/− mice. These results suggest that EP3 along with TP contributes to vasoconstrictor responses evoked by PGI2, and hence imply a novel mechanism for endothelial cyclooxygenase metabolites (which consist mainly of PGI2) in regulating vascular functions.

Increased role of E prostanoid receptor-3 in prostacyclin-evoked contractile activity of spontaneously hypertensive rat mesenteric resistance arteries

This study aimed to determine whether E prostanoid receptor-3 (EP3) is involved in prostacyclin (PGI2)-evoked vasoconstrictor activity of resistance arteries and if so, how it changes under hypertensive conditions. Mesenteric resistance arteries from Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) were isolated for functional and biochemical studies. Here we show that in vessels from WKYs, PGI2 or the endothelial muscarinic agonist ACh (which stimulates in vitro PGI2 synthesis) evoked vasoconstrictor activity, which increased in SHRs. The thromboxane-prostanoid receptor (TP) antagonist SQ29548 partially removed the vasoconstrictor activity, and an increased contractile activity of PGI2 resistant to SQ29548 was observed in SHRs. Interestingly, L798106, an antagonist of EP3 (whose expression was higher in SHRs than in WKYs), not only added to the effect of SQ29548 but also caused relaxation to PGI2 more than that obtained with SQ29548. In accordance, EP3 deletion, which reduced PGI2–evoked contraction, together with SQ29548 resulted in relaxation evoked by the agonist in mouse aortas. These results thus demonstrate an explicit involvement of EP3 in PGI2-evoked vasoconstrictor activity in rat mesenteric resistance arteries and suggest that up-regulation of the receptor contributes significantly to the increased contractile activity evoked by PGI2 under hypertensive conditions.