BACKGROUND
the attenuation of endothelium-dependent vasodilatation elicited by hormones or shear stress is a condition observed in cardiovascular diseases.
this condition is often referred to as endothelial dysfunction.
it is thought that endothelial dysfunction is an early development in the time course of cardiovascular diseases.
clinical tests of patients for endothelial dysfunction include measuring the vasodilator responses of blood vessels to an agonist of the endothelium e.g.
cholinergic agonist acetylcholine.
protease-activated receptor  <dig>  is a g protein coupled receptor which can be activated by trypsin-like serine proteases and par2-activating peptides   <cit> .
there are a few variations in the mechanisms that have been attributed to the par2-ap mediated vasodilatation mechanisms during endothelial dysfunction.
these mechanisms have included the selective activation of sk <dig>   <cit> , endothelial nitric oxide synthase   <cit> , and cyclooxygenases   <cit> .
in non-obese diabetes models of endothelial dysfunction, increased par <dig> expression was reported  <cit> .
the uncertainty in the mechanisms of par <dig> vasodilatation in endothelial dysfunction may be due to the choice of experimental models particularly the reliance on genetic strains of rodents.
human diseases represent complex phenotypes so it is important to investigate par <dig> in multiple experimental models.
chronic angiotensin ii  infusion produces a model of acquired hypertension and endothelial dysfunction in animals.
it is also linked to pro-inflammation signaling pathways involving p <dig> mitogen activated protein kinase and nuclear factor-κb.
these transcription pathways are proposed to partly link ang ii receptor signalling to changes in endothelial cell phenotype, which include induction of cyclooxygenase .
in endothelial cell culture conditions the p <dig> mitogen activated protein kinase and nuclear factor-κb pathways link cytokines to induction of par <dig> expression and are activated by par2-ap  <cit> .
interestingly, the induction of cox- <dig> in endothelial cells in culture enabled par <dig> to stimulate cells to produce pgi <dig>  which was proposed as being vasculoprotective  <cit> .
to date the effect of chronic ang ii-induced endothelial dysfunction on par2-ap vasodilatation is unknown.
we have described a trend for higher blood pressures in par <dig> gene  knockout mice  after two weeks of ang ii infusion  <cit> .
these results may be consistent with the proposal of par2-mediated protection of blood vessels against the negative effects of chronic ang ii.
the primary goal of this study was to determine the effects of ang ii-induced endothelial dysfunction on vasodilatation by par2-ap .
we also tested whether par <dig> gene deficiency was protective against chronic ang ii-induced endothelial dysfunction in the vasculature.
our findings provide new and significant additions to understanding vascular pharmacology and important interactions with cardiovascular pathology.
also, these findings highlight the potential for sk <dig>  to be a potential pharmaceutical target for hypertension.
RESULTS
preserved par2-mediated relaxations of mesenteric arteries in ang ii c57
to determine whether chronic ang ii treatment attenuated the vasodilator effectiveness of par <dig>  we measured relaxations by the par <dig> activating peptide 2fly of mesenteric arteries contracted submaximally by α1-adrenoceptor agonist .
chronic ang ii did not reduce the effectiveness of par <dig> to cause vasodilatation of arteries .
2fly-mediated relaxations of arteries of angii c <dig> vs. saline c <dig> were the same .
these data indicated that par2-mediated vasodilatation by 2fly was protected against treatment by chronic ang ii.
as we have reported elsewhere  <cit>  par <dig> was confirmed as being the specific target of 2fly in the bioassay by our finding that concentrations up to  <dig> μm of this peptide had no effect on arteries from saline par2-/- and ang ii par2-/- .
values are mean, n = number of mice.
variables were determined by curve fitting data points from cumulative drug concentration-responses relationships to a four parameter logistic function.
treatments of arteries included antagonists of cox- <dig> , cox- <dig> , cox-1/ <dig> , nos , prostaglandin e2receptors , prostaglandin i2receptor  and thromboxane a2receptor .
comparisons were made by two-way anova   followed by bonferroni post hoc tests: gp <  <dig> , and hp <  <dig> , compared to same inhibitor treatment in saline pump group ; ip <  <dig> , jp <  <dig> , and kp <  <dig> , controls compared to inhibitor within same pump treatment .
c <dig>  c57bl/6j; emax, maximum relaxation response where 100% is complete reversal of contraction.
contributions of nos to preserved par2-mediated relaxations of mesenteric arteries in ang ii c57
in normal  mice it is well-established that enos contributes by a small extent to par2-mediated vasodilatation of small mesenteric arteries  <cit> .
to determine whether an increased contribution by nos to par <dig> activity could be attributed to the preserved vasodilator effectiveness of par <dig>  we compared 2fly-induced relaxations in the absence  and presence of l-name .
treatment of arteries with l-name inhibited 2fly-induced relaxations compared with controls to the same extents in saline and ang ii c <dig> .
these data indicated there was not a significant change in endothelium-derived no elicited by 2fly in ang ii c <dig>
contributions of cox to preserved par2-mediated relaxations of mesenteric arteries in ang ii c57
inhibitors of cox have no effect on par2-mediated vasodilatations of c <dig> mesenteric arteries  <cit> .
however, chronic in vivo treatment with ang ii has been reported to induce expression of cyclooxygenases in vascular tissues.
to determine whether de novo contribution of cox to par <dig> activity could be attributed to preserved vasodilator effectiveness of par <dig>  we compared 2fly-induced relaxations in the absence  and presence of cox inhibitors .
as expected, the 2fly crc of untreated arteries from saline c <dig>  were not different in the presence of cox inhibitors .
the potency of 2fly in ang ii c <dig>  was reduced by either nonselective or selective cox isoform inhibitors .
indomethacin  in the absence  and presence of l-name  caused rightward shifts of the 2fly crc in ang ii vs. saline c <dig>  similarly, cox- <dig> inhibitor  rightward shifted the 2fly crc in ang ii c <dig> vs. saline c <dig>  cox- <dig> inhibitor  reduced the steepness  of the 2fly crc relationship in ang ii c <dig> vs. saline c <dig>  and thus, inhibited the effectiveness of 2fly at the low to middle range of concentrations .
to investigate whether a single metabolite of cox activity could be attributed to the potency changes for 2fly, we compared responses in the absence and presence of various receptor antagonists for prostanoids and thromboxane a <dig>  the results of these experiments  did not support a role for any specific metabolite in the actions of cox inhibitors on 2fly potency in ang ii c <dig>  2fly crc in ang ii c <dig> vs. saline c <dig>  were not different in the presence of antagonists for pgi <dig> receptor , pge <dig> receptors , and thromboxane a <dig> receptor .
contributions of sk <dig>  to preserved par-mediated relaxations of mesenteric arteries in ang ii c57
it has been demonstrated numerous times  <cit>  that combined inhibition of nos, sk <dig> , and sk <dig> / <dig>  is required to block vasodilatation of normal mouse mesenteric arteries by par2-activating peptides; and acetylcholine.
recent work by us  <cit>  indicated a primary role for sk <dig>  in contributing to preserved par <dig> vasodilatation in obese diabetic mouse mesenteric arteries.
to determine the contribution of sk <dig>  to the vasodilatation mediated by par <dig> in ang ii c <dig>  we investigated 2fly-induced relaxations in the presence of specific sk <dig>  inhibitor tram- <dig>  which was done in the absence and presence of nos inhibitor l-name.
indeed treatment of arteries by sk <dig>  inhibitor reduced the effectiveness of 2fly in ang ii mice  and when combined with pretreatment by l-name the 2fly crc in ang ii c <dig> was blocked compared with controls .
ach mediated vasodilatations in saline vs. ang ii and c <dig> vs. par2-/-
we determined ach mediated vasodilatations of arteries from saline and ang ii treatments of both c <dig> and par2-/- to reproduce previous reports of the negative effects of ang ii treatment on endothelial cell function.
these experiments also assessed the protection attributed to par2-/- genotype against the negative effects of ang ii.
par2-/- genotype did not afford protection for untreated arteries against the negative effects on endothelial function of ang ii treatment , table 2).
vasodilatation by ach of untreated  arteries was decreased in ang ii vs. saline treatments to the same extent in c <dig> vs. par2-/- .
values are mean , n = number of mice.
variables were determined by curve fitting data points from cumulative drug concentration-responses relationships to a four parameter logistic function.
treatments of arteries included antagonists of cox- <dig> , cox- <dig> , cox-1/ <dig> , nos , prostaglandin e2receptors , prostaglandin i2receptor .
comparisons were made by two-way anova   followed by bonferroni post hoc tests: dp <  <dig> , ang ii c <dig> controls compared to saline c <dig> controls; ep <  <dig> , inhibitor group compared with saline c <dig> controls; fp <  <dig> , inhibitor group compared with saline par2-/- controls; gp <  <dig> , group compared with ang ii c <dig> controls; hp <  <dig> , inhibitor group compared with ang ii par2-/- controls; i data from par2-/- were included as artery treatment factor for analyses.
c <dig>  c57bl/6j; emax, maximum relaxation response where 100% is complete reversal of contraction; par2-/-, protease-activated receptor  <dig> gene knockout mice.
we determined ach mediated vasodilatations in the absence and presence of l-name in c <dig> and par2-/- to investigate the involvement of enos in these responses.
while enos contribution to ach-mediated vasodilatation was the same in saline c <dig> vs. saline par2-/- , there was less enos contribution to ach mediated relaxations in ang ii c <dig> vs. ang ii par2-/- .
this was found in the observation that l-name caused no further attenuation of ach relaxations in ang ii par2-/-  compared with untreated arteries of ang ii par2-/- .
we tested ach-mediated vasodilatations in the absence and presence of cox inhibitors to determine the selectivity of their actions on the potency of endothelium-dependent agonists in ang ii c <dig>  these experiments indicated that the cox inhibitors effects in ang ii c <dig> were affecting selectively 2fly in our bioassays.
treatments of arteries by indomethacin in the absence  or presence of l-name  compared with controls  did not differentially affect ach crc in saline- vs. ang ii-treated mice.
in addition, ach crc were not significantly affected by selective antagonists of cox- <dig>  cox- <dig>  pgi <dig> receptor, and pge <dig> receptors .
we determined the effects of combined inhibition of enos, sgc, sk <dig> , sk <dig> , and sk <dig>  to confirm the involvement of these elements in the residual ach-mediated vasodilatations.
a combination of l-name, odq, apamin plus tram- <dig> was sufficient to block ach crc .
nitroprusside relaxations of arteries
to test the sensitivity of vascular smooth muscle to no, nitroprusside-induced vasodilatations were determined.
indeed nitroprusside, which spontaneously releases no and bypasses the endothelium, was slightly less potent in ang ii c <dig> vs. saline c <dig> .
the nitroprusside crc was shifted to the right in ang ii c <dig> vs. saline c <dig> .
surprisingly, we found that par <dig> -/- genotype was protective against the potency shift caused by angii.
the nitroprusside crc were not different in ang ii par2-/- vs. saline par2-/- .
values are mean , n = number of mice.
variables were determined by curve fitting data points from cumulative drug concentration-responses relationships to a four parameter logistic equation.
treatments of arteries included antagonists of cox- <dig> , cox- <dig> , prostaglandin e <dig> receptors , prostaglandin i <dig> receptor .
comparisons were made by two-way anova   followed by bonferroni post hoc tests: bp <  <dig> , control compared to saline pump treatment; cdata of control arteries from par2-/- were included as a treatment factor for analyses.
c <dig>  c57bl/6j mice; emax, maximum relaxation response where 100% is complete reversal of contraction; par2-/-, protease-activated receptor  <dig> gene knockout mice.
to test for potential off-target effects of the various inhibitors of cox on vascular smooth muscle sensitivity to no, nitroprusside-induced vasodilatations in the presence and absence of these inhibitors were determined.
the various inhibitors of cox and pgi2/pge <dig> receptors did not affect vascular smooth muscle sensitivity to no of the arteries.
nitroprusside crc were not affected by the presence of fr <dig>  ns <dig>  cay <dig> and the combination of ah <dig>  l <dig> and l <dig> .
endothelium-dependence of vasodilators in saline c <dig> and ang ii c <dig> arteries
to determine whether ach- or 2fly-induced relaxations were due to direct activation of vascular smooth muscle cells, the inner linings of mesenteric arteries were damaged and then the effectiveness of the agonists were assayed.
as expected from previous work in various mouse arteries  <cit> , in both saline and ang ii c <dig> the ach- and 2fly-induced relaxations were reduced to effective  <dig> after being subjected to endothelium removal, but nitroprusside relaxed these arteries .
contraction of arteries by cirazoline in par2-/- vs. c57
we determined the contractions of arteries by α1- adrenergic receptor agonist cirazoline after saline and ang ii treatment of c <dig> and par2-/- to investigate the effect of par2-/- genotype on vasoconstrictor agonist effectiveness.
these experiments also investigated the effects of ang ii on arteries responses to cirazoline.
surprisingly, cirazoline was a more potent vasoconstrictor in par2-/- than in c <dig>  ang ii treatments did not increase the vasoconstrictor effectiveness of cirazoline in arteries of either strain.
in saline par2-/-  and ang ii par2-/-  the cirazoline crc were shifted to the left  of saline c <dig>  and ang ii c <dig> , respectively .
par2-ap 2fly and acetylcholine under baseline conditions in c <dig> and par2-/-
to determine whether the chronic ang ii treatment induced a potential for vasoconstrictor activity by 2fly and ach, we measured isometric tension of mesenteric arteries after exposing tissues to agonists.
we found no evidence of vasoconstrictor activity for these agonists in the mesenteric arteries.
there were no observed changes in tension at baseline after the addition of either 2fly  or acetylcholine  to arteries from saline c <dig> , ang ii c <dig> , saline par2-/-  and ang ii par2-/- .
mrna expression of cox- <dig>  cox- <dig>  and par2
to determine whether mrna expression of cox- <dig>  cox- <dig> and par <dig> were increased by ang ii treatment of mice, we measured the mrna expression pattern of these genes by real-time pcr.
cox- <dig> mrna expression  was significantly upregulated in ang ii c <dig> .
cox- <dig> and par <dig> mrna expression  were not significantly different between treatments.
given the effectiveness of 2fly was reduced by cox inhibitors in ang ii c <dig>  and these par <dig> mediated relaxations were endothelium-dependent, we also normalized the mrna for cox- <dig> and cox- <dig> relative to par <dig> reference to relate mrna expression to the functional observations.
according to this analyses, cox- <dig> and cox- <dig> mrna  were significantly upregulated in ang ii c <dig> .
mrna expression in mesenteric arterial cascades from mice was determined by real-time pcr and normalised to triad housekeeping, and to protease-activated receptor  <dig> genes .
angiotensin ii  c <dig>  n = 12; saline c <dig>  n =  <dig>
values reported are the mean and se range .
ap <  <dig> , bp <  <dig> , cp <  <dig> , gene upregulated in ang ii c <dig> compared with saline c <dig>  see methods for details of statistical analyses.
to determine whether the par2-/- genotype affected the constitutive expression of cox-1/ <dig> we compared the gene expression pattern in c <dig> with par2-/-.
cox- <dig> and cox- <dig> mrna were not significantly different between par2-/- and c <dig> .
protein expression of cox isoforms in mesenteric arterial beds
to determine whether upregulated cox mrna expression patterns and effectiveness of cox inhibitors were matched to increased levels of cox proteins in arteries, we measured the levels of cox proteins by western blot.
the ratio of cox- <dig> to cox- <dig> protein expression in mesenteric arterial beds was significantly higher in ang ii c <dig> vs. saline c <dig> .
discussion
our main finding was the par2-ap vasodilatation of small caliber arteries was protected against the negative effects of chronic ang ii infusion.
activation of sk <dig>  was critical to the mechanism of par <dig> mediated relaxations in ang ii mice.
ang ii infusion in mice attenuated no-dependent relaxations by ach and increased the ratio of cox- <dig> to cox- <dig> protein expression.
inhibiting either cox- <dig> or cox- <dig> decreased the potency of 2fly in ang ii-treated mice.
ang ii treatment did not increase par <dig> expression in arteries.
the par2-ap-mediated acute vasodilatation during ang ii infusion involves components of smooth muscle relaxation pathways that are resistant to the negative effects of endothelial dysfunction in cardiovascular disease.
these components may be potential therapeutic targets for protection against the consequences of cardiovascular disease.
inhibition of sk <dig>  and endothelial no synthase was sufficient to block par2-ap mediated vasodilatation.
however, we found that chronic ang ii infusion of mice also led to a change in par <dig> mediated relaxations of vascular smooth muscle from indomethacin-insensitive to indomethacin-sensitive.
these finding suggest cox is part of a minor supplementary mechanism of action for par <dig> after ang ii treatment.
in human volunteers, aspirin treatment decreased the par2-ap vasodilatation of forearm blood vessels  <cit> .
in non-obese diabetic mice, a selective cox- <dig> inhibitor decreased the effectiveness of par2-ap on aortas  <cit> .
this attenuation of par2-ap became larger as the effectiveness of ach decreased and the mice developed hyperglycemia with age  <cit> .
under cell culture conditions, exposure to par <dig> agonists increase cox- <dig> mrna, protein and pgi <dig>  <cit>  in endothelial cells.
we found either fr <dig>  or ns <dig>  was sufficient to decrease the potency of 2fly in ang ii-treated mice.
antagonists of pgi <dig> and pge <dig> receptors in our study did not replicate the effects of cox inhibitors.
therefore, it appeared that pgi <dig> and pge <dig> receptors were not involved with the supplementary mechanism.
we speculate that par <dig> activates cox-dependent production of vasodilator reactive oxygen species, which would be independent of prostaglandin receptors.
in our study, ang ii infusion reduced the potency of nitroprusside in arteries.
other studies have attributed the attenuation of nitroprusside to quenching of the no by free radicals.
in human vein endothelial cells, par2-ap increased reactive oxygen species production from mitochondrial complex iii and lesser amounts from cox- <dig>  <cit> .
it has been reported that cox-dependent reactive oxygen species  <cit>  could activate big kca which is expressed on vascular smooth muscle.
this would be consistent with our interpretation that cox inhibitors had an indirect effect on the par <dig> relaxations.
our study highlights that the experimental model of cardiovascular disease impacts the mechanisms of par <dig> action in blood vessels  <cit> .
albeit our study focused on a small caliber artery which is used to model the resistance vasculature and thus, uses less no than large caliber conductance type  for endothelium-dependent relaxations by par <dig>  <cit> .
in other genetic hypertension models, the par <dig> vasodilatation of small caliber arteries was not linked to cox  <cit> .
also, our current and previous studies indicate that persistent par <dig> vasodilatation is entirely endothelium-dependent.
however, in non-obese diabetic mice aortas and spontaneously hypertensive rat basilar arteries the par2-ap vasodilatations were endothelium-independent  <cit> .
on the other hand, the central role of sk <dig>  has been highlighted as a common finding in all of our studies on small caliber arteries.
we reported that in obese diabetic mice that sk <dig>  was the key link in the preserved par <dig> mediated vasodilation  <cit> .
recent studies have demonstrated the utility of targeting sk <dig> , sk <dig>  and sk <dig>  agonists for modulating vascular tone, presumably by endothelium-dependent hyperpolarization, and lowering blood pressure in ang ii-induced hypertensive mice  <cit> .
the protection of the par <dig> vasodilatation against ang ii-acquired hypertension suggests that endothelial kca activation by selected membrane receptors could also provide the vasculature with the ability to compensate or replace no during disease.
some investigators have reported that uncoupled endothelial no synthase may contribute to a superoxide-mediated vasodilatation of aortas during endothelial dysfunction in diabetes, which was also proposed to be linked to ang ii  <cit> .
however, inhibition of uncoupled endothelial no synthase by l-name remains a point of controversy  <cit>  so it is uncertain whether a similar connection between par <dig> and uncoupled endothelial no synthase exists in the ang ii infusion model.
the results of experiments in par2-/- indicate that par <dig> null genotype had mixed effects on the negative outcomes of ang ii infusions on blood vessels.
first, there was an increased sensitivity of arteries to adrenergic receptor stimulation in par2-/-.
second, there was less l-name sensitive endothelial no-derived relaxation activity  in par2-/- after treatment with ang ii.
third, the potency of nitroprusside in ang ii par2-/- was not decreased as in ang ii c <dig>  the first of the effects above would be expected to increase systolic and pulse pressures in par2-/-.
the second and third effects mentioned could be interpreted as protective compensatory mechanisms that may limit increases in blood pressures in par2-/-.
our radiotelemetry study of blood pressures indicated that by the end of treatment period, ang ii produced larger systolic and pulse pressure changes in par2-/- than in c <dig>  <cit> .
a limitation to the scope of our study was the focus on disruption by ang ii of endothelium-dependent vasodilatation.
we examined only one vasoconstrictor system, necessarily to establish conditions for bioassay, so it was possible that par2-/- may have affected vasoconstrictor systems other than α <dig> adrenergic receptors e.g.
5-hydroxytryptamine, thromboxane a <dig>  <cit> .
in the past  <dig> years, the literature weighs heavily with evidence linking activated par <dig> to pro-inflammatory signal transduction in endothelial cells  <cit> .
there is also a broad literature contending that chronic elevated levels of ang ii elicits increased pro-inflammatory signalling by cytokines and pro-thrombotic conditions that contribute to the disruption of normal endothelial and vascular smooth muscle cell biology  <cit> .
in wild-type animals, it is possible that increased activity of coagulation factors and or other serine proteinases during chronic ang ii infusion leads to par <dig> activation and thus, the production of cytokines that cause vascular dysfunction.
accordingly, it would be expected that the production of pro-inflammatory mediators elicited by ang ii in par <dig> null mice may be attenuated and thus, the disruptions by ang ii of the endothelium and vascular smooth muscle are reduced.
CONCLUSIONS
par2-ap effectiveness is protected against the negative effects of ang ii.
par <dig> null expression had mixed effects in vitro on the negative outcomes of ang ii hypertension.
the preserved par <dig> vasodilatation which is mediated largely via sk <dig>  may lead to discovery of other endothelial cell signaling that is resistant to vascular diseases.

 the attenuation of endothelium-dependent vasodilatation elicited by hormones or shear stress is a condition observed in cardiovascular diseases. this condition is often referred to as endothelial dysfunction. it is thought that endothelial dysfunction is an early development in the time course of cardiovascular diseases. clinical tests of patients for endothelial dysfunction include measuring the vasodilator responses of blood vessels to an agonist of the endothelium e.g. cholinergic agonist acetylcholine. protease-activated receptor is a g protein coupled receptor which can be activated by trypsin-like serine proteases and par2-activating peptides. there are a few variations in the mechanisms that have been attributed to the par2-ap mediated vasodilatation mechanisms during endothelial dysfunction. these mechanisms have included the selective activation of sk, endothelial nitric oxide synthase, and cyclooxygenases. in non-obese diabetes models of endothelial dysfunction, increased par expression was reported. the uncertainty in the mechanisms of par vasodilatation in endothelial dysfunction may be due to the choice of experimental models particularly the reliance on genetic strains of rodents. human diseases represent complex phenotypes so it is important to investigate par in multiple experimental models. chronic angiotensin ii infusion produces a model of acquired hypertension and endothelial dysfunction in animals. it is also linked to pro-inflammation signaling pathways involving p mitogen activated protein kinase and nuclear factor-κb. these transcription pathways are proposed to partly link ang ii receptor signalling to changes in endothelial cell phenotype, which include induction of cyclooxygenase. in endothelial cell culture conditions the p mitogen activated protein kinase and nuclear factor-κb pathways link cytokines to induction of par expression and are activated by par2-ap. interestingly, the induction of cox- in endothelial cells in culture enabled par to stimulate cells to produce pgi which was proposed as being vasculoprotective. to date the effect of chronic ang ii-induced endothelial dysfunction on par2-ap vasodilatation is unknown. we have described a trend for higher blood pressures in par gene knockout mice after two weeks of ang ii infusion. these results may be consistent with the proposal of par2-mediated protection of blood vessels against the negative effects of chronic ang ii. the primary goal of this study was to determine the effects of ang ii-induced endothelial dysfunction on vasodilatation by par2-ap. we also tested whether par gene deficiency was protective against chronic ang ii-induced endothelial dysfunction in the vasculature. our findings provide new and significant additions to understanding vascular pharmacology and important interactions with cardiovascular pathology. also, these findings highlight the potential for sk to be a potential pharmaceutical target for hypertension.  preserved par2-mediated relaxations of mesenteric arteries in ang ii c57 to determine whether chronic ang ii treatment attenuated the vasodilator effectiveness of par we measured relaxations by the par activating peptide 2fly of mesenteric arteries contracted submaximally by α1-adrenoceptor agonist. chronic ang ii did not reduce the effectiveness of par to cause vasodilatation of arteries. 2fly-mediated relaxations of arteries of angii c vs. saline c were the same. these data indicated that par2-mediated vasodilatation by 2fly was protected against treatment by chronic ang ii. as we have reported elsewhere par was confirmed as being the specific target of 2fly in the bioassay by our finding that concentrations up to μm of this peptide had no effect on arteries from saline par2-/- and ang ii par2-/-. values are mean, n = number of mice. variables were determined by curve fitting data points from cumulative drug concentration-responses relationships to a four parameter logistic function. treatments of arteries included antagonists of cox-, cox-, cox-1/, nos, prostaglandin e2receptors, prostaglandin i2receptor and thromboxane a2receptor. comparisons were made by two-way anova followed by bonferroni post hoc tests: gp <, and hp <, compared to same inhibitor treatment in saline pump group; ip <, jp <, and kp <, controls compared to inhibitor within same pump treatment. c c57bl/6j; emax, maximum relaxation response where 100% is complete reversal of contraction. contributions of nos to preserved par2-mediated relaxations of mesenteric arteries in ang ii c57 in normal mice it is well-established that enos contributes by a small extent to par2-mediated vasodilatation of small mesenteric arteries. to determine whether an increased contribution by nos to par activity could be attributed to the preserved vasodilator effectiveness of par we compared 2fly-induced relaxations in the absence and presence of l-name. treatment of arteries with l-name inhibited 2fly-induced relaxations compared with controls to the same extents in saline and ang ii c. these data indicated there was not a significant change in endothelium-derived no elicited by 2fly in ang ii c contributions of cox to preserved par2-mediated relaxations of mesenteric arteries in ang ii c57 inhibitors of cox have no effect on par2-mediated vasodilatations of c mesenteric arteries. however, chronic in vivo treatment with ang ii has been reported to induce expression of cyclooxygenases in vascular tissues. to determine whether de novo contribution of cox to par activity could be attributed to preserved vasodilator effectiveness of par we compared 2fly-induced relaxations in the absence and presence of cox inhibitors. as expected, the 2fly crc of untreated arteries from saline c were not different in the presence of cox inhibitors. the potency of 2fly in ang ii c was reduced by either nonselective or selective cox isoform inhibitors. indomethacin in the absence and presence of l-name caused rightward shifts of the 2fly crc in ang ii vs. saline c similarly, cox- inhibitor rightward shifted the 2fly crc in ang ii c vs. saline c cox- inhibitor reduced the steepness of the 2fly crc relationship in ang ii c vs. saline c and thus, inhibited the effectiveness of 2fly at the low to middle range of concentrations. to investigate whether a single metabolite of cox activity could be attributed to the potency changes for 2fly, we compared responses in the absence and presence of various receptor antagonists for prostanoids and thromboxane a the results of these experiments did not support a role for any specific metabolite in the actions of cox inhibitors on 2fly potency in ang ii c 2fly crc in ang ii c vs. saline c were not different in the presence of antagonists for pgi receptor, pge receptors, and thromboxane a receptor. contributions of sk to preserved par-mediated relaxations of mesenteric arteries in ang ii c57 it has been demonstrated numerous times that combined inhibition of nos, sk, and sk / is required to block vasodilatation of normal mouse mesenteric arteries by par2-activating peptides; and acetylcholine. recent work by us indicated a primary role for sk in contributing to preserved par vasodilatation in obese diabetic mouse mesenteric arteries. to determine the contribution of sk to the vasodilatation mediated by par in ang ii c we investigated 2fly-induced relaxations in the presence of specific sk inhibitor tram- which was done in the absence and presence of nos inhibitor l-name. indeed treatment of arteries by sk inhibitor reduced the effectiveness of 2fly in ang ii mice and when combined with pretreatment by l-name the 2fly crc in ang ii c was blocked compared with controls. ach mediated vasodilatations in saline vs. ang ii and c vs. par2-/- we determined ach mediated vasodilatations of arteries from saline and ang ii treatments of both c and par2-/- to reproduce previous reports of the negative effects of ang ii treatment on endothelial cell function. these experiments also assessed the protection attributed to par2-/- genotype against the negative effects of ang ii. par2-/- genotype did not afford protection for untreated arteries against the negative effects on endothelial function of ang ii treatment, table 2). vasodilatation by ach of untreated arteries was decreased in ang ii vs. saline treatments to the same extent in c vs. par2-/-. values are mean, n = number of mice. variables were determined by curve fitting data points from cumulative drug concentration-responses relationships to a four parameter logistic function. treatments of arteries included antagonists of cox-, cox-, cox-1/, nos, prostaglandin e2receptors, prostaglandin i2receptor. comparisons were made by two-way anova followed by bonferroni post hoc tests: dp <, ang ii c controls compared to saline c controls; ep <, inhibitor group compared with saline c controls; fp <, inhibitor group compared with saline par2-/- controls; gp <, group compared with ang ii c controls; hp <, inhibitor group compared with ang ii par2-/- controls; i data from par2-/- were included as artery treatment factor for analyses. c c57bl/6j; emax, maximum relaxation response where 100% is complete reversal of contraction; par2-/-, protease-activated receptor gene knockout mice. we determined ach mediated vasodilatations in the absence and presence of l-name in c and par2-/- to investigate the involvement of enos in these responses. while enos contribution to ach-mediated vasodilatation was the same in saline c vs. saline par2-/-, there was less enos contribution to ach mediated relaxations in ang ii c vs. ang ii par2-/-. this was found in the observation that l-name caused no further attenuation of ach relaxations in ang ii par2-/- compared with untreated arteries of ang ii par2-/-. we tested ach-mediated vasodilatations in the absence and presence of cox inhibitors to determine the selectivity of their actions on the potency of endothelium-dependent agonists in ang ii c these experiments indicated that the cox inhibitors effects in ang ii c were affecting selectively 2fly in our bioassays. treatments of arteries by indomethacin in the absence or presence of l-name compared with controls did not differentially affect ach crc in saline- vs. ang ii-treated mice. in addition, ach crc were not significantly affected by selective antagonists of cox- cox- pgi receptor, and pge receptors. we determined the effects of combined inhibition of enos, sgc, sk, sk, and sk to confirm the involvement of these elements in the residual ach-mediated vasodilatations. a combination of l-name, odq, apamin plus tram- was sufficient to block ach crc. nitroprusside relaxations of arteries to test the sensitivity of vascular smooth muscle to no, nitroprusside-induced vasodilatations were determined. indeed nitroprusside, which spontaneously releases no and bypasses the endothelium, was slightly less potent in ang ii c vs. saline c. the nitroprusside crc was shifted to the right in ang ii c vs. saline c. surprisingly, we found that par -/- genotype was protective against the potency shift caused by angii. the nitroprusside crc were not different in ang ii par2-/- vs. saline par2-/-. values are mean, n = number of mice. variables were determined by curve fitting data points from cumulative drug concentration-responses relationships to a four parameter logistic equation. treatments of arteries included antagonists of cox-, cox-, prostaglandin e receptors, prostaglandin i receptor. comparisons were made by two-way anova followed by bonferroni post hoc tests: bp <, control compared to saline pump treatment; cdata of control arteries from par2-/- were included as a treatment factor for analyses. c c57bl/6j mice; emax, maximum relaxation response where 100% is complete reversal of contraction; par2-/-, protease-activated receptor gene knockout mice. to test for potential off-target effects of the various inhibitors of cox on vascular smooth muscle sensitivity to no, nitroprusside-induced vasodilatations in the presence and absence of these inhibitors were determined. the various inhibitors of cox and pgi2/pge receptors did not affect vascular smooth muscle sensitivity to no of the arteries. nitroprusside crc were not affected by the presence of fr ns cay and the combination of ah l and l. endothelium-dependence of vasodilators in saline c and ang ii c arteries to determine whether ach- or 2fly-induced relaxations were due to direct activation of vascular smooth muscle cells, the inner linings of mesenteric arteries were damaged and then the effectiveness of the agonists were assayed. as expected from previous work in various mouse arteries, in both saline and ang ii c the ach- and 2fly-induced relaxations were reduced to effective after being subjected to endothelium removal, but nitroprusside relaxed these arteries. contraction of arteries by cirazoline in par2-/- vs. c57 we determined the contractions of arteries by α1- adrenergic receptor agonist cirazoline after saline and ang ii treatment of c and par2-/- to investigate the effect of par2-/- genotype on vasoconstrictor agonist effectiveness. these experiments also investigated the effects of ang ii on arteries responses to cirazoline. surprisingly, cirazoline was a more potent vasoconstrictor in par2-/- than in c ang ii treatments did not increase the vasoconstrictor effectiveness of cirazoline in arteries of either strain. in saline par2-/- and ang ii par2-/- the cirazoline crc were shifted to the left of saline c and ang ii c, respectively. par2-ap 2fly and acetylcholine under baseline conditions in c and par2-/- to determine whether the chronic ang ii treatment induced a potential for vasoconstrictor activity by 2fly and ach, we measured isometric tension of mesenteric arteries after exposing tissues to agonists. we found no evidence of vasoconstrictor activity for these agonists in the mesenteric arteries. there were no observed changes in tension at baseline after the addition of either 2fly or acetylcholine to arteries from saline c, ang ii c, saline par2-/- and ang ii par2-/-. mrna expression of cox- cox- and par2 to determine whether mrna expression of cox- cox- and par were increased by ang ii treatment of mice, we measured the mrna expression pattern of these genes by real-time pcr. cox- mrna expression was significantly upregulated in ang ii c. cox- and par mrna expression were not significantly different between treatments. given the effectiveness of 2fly was reduced by cox inhibitors in ang ii c and these par mediated relaxations were endothelium-dependent, we also normalized the mrna for cox- and cox- relative to par reference to relate mrna expression to the functional observations. according to this analyses, cox- and cox- mrna were significantly upregulated in ang ii c. mrna expression in mesenteric arterial cascades from mice was determined by real-time pcr and normalised to triad housekeeping, and to protease-activated receptor genes. angiotensin ii c n = 12; saline c n = values reported are the mean and se range. ap <, bp <, cp <, gene upregulated in ang ii c compared with saline c see methods for details of statistical analyses. to determine whether the par2-/- genotype affected the constitutive expression of cox-1/ we compared the gene expression pattern in c with par2-/-. cox- and cox- mrna were not significantly different between par2-/- and c. protein expression of cox isoforms in mesenteric arterial beds to determine whether upregulated cox mrna expression patterns and effectiveness of cox inhibitors were matched to increased levels of cox proteins in arteries, we measured the levels of cox proteins by western blot. the ratio of cox- to cox- protein expression in mesenteric arterial beds was significantly higher in ang ii c vs. saline c. discussion our main finding was the par2-ap vasodilatation of small caliber arteries was protected against the negative effects of chronic ang ii infusion. activation of sk was critical to the mechanism of par mediated relaxations in ang ii mice. ang ii infusion in mice attenuated no-dependent relaxations by ach and increased the ratio of cox- to cox- protein expression. inhibiting either cox- or cox- decreased the potency of 2fly in ang ii-treated mice. ang ii treatment did not increase par expression in arteries. the par2-ap-mediated acute vasodilatation during ang ii infusion involves components of smooth muscle relaxation pathways that are resistant to the negative effects of endothelial dysfunction in cardiovascular disease. these components may be potential therapeutic targets for protection against the consequences of cardiovascular disease. inhibition of sk and endothelial no synthase was sufficient to block par2-ap mediated vasodilatation. however, we found that chronic ang ii infusion of mice also led to a change in par mediated relaxations of vascular smooth muscle from indomethacin-insensitive to indomethacin-sensitive. these finding suggest cox is part of a minor supplementary mechanism of action for par after ang ii treatment. in human volunteers, aspirin treatment decreased the par2-ap vasodilatation of forearm blood vessels. in non-obese diabetic mice, a selective cox- inhibitor decreased the effectiveness of par2-ap on aortas. this attenuation of par2-ap became larger as the effectiveness of ach decreased and the mice developed hyperglycemia with age. under cell culture conditions, exposure to par agonists increase cox- mrna, protein and pgi in endothelial cells. we found either fr or ns was sufficient to decrease the potency of 2fly in ang ii-treated mice. antagonists of pgi and pge receptors in our study did not replicate the effects of cox inhibitors. therefore, it appeared that pgi and pge receptors were not involved with the supplementary mechanism. we speculate that par activates cox-dependent production of vasodilator reactive oxygen species, which would be independent of prostaglandin receptors. in our study, ang ii infusion reduced the potency of nitroprusside in arteries. other studies have attributed the attenuation of nitroprusside to quenching of the no by free radicals. in human vein endothelial cells, par2-ap increased reactive oxygen species production from mitochondrial complex iii and lesser amounts from cox-. it has been reported that cox-dependent reactive oxygen species could activate big kca which is expressed on vascular smooth muscle. this would be consistent with our interpretation that cox inhibitors had an indirect effect on the par relaxations. our study highlights that the experimental model of cardiovascular disease impacts the mechanisms of par action in blood vessels. albeit our study focused on a small caliber artery which is used to model the resistance vasculature and thus, uses less no than large caliber conductance type for endothelium-dependent relaxations by par. in other genetic hypertension models, the par vasodilatation of small caliber arteries was not linked to cox. also, our current and previous studies indicate that persistent par vasodilatation is entirely endothelium-dependent. however, in non-obese diabetic mice aortas and spontaneously hypertensive rat basilar arteries the par2-ap vasodilatations were endothelium-independent. on the other hand, the central role of sk has been highlighted as a common finding in all of our studies on small caliber arteries. we reported that in obese diabetic mice that sk was the key link in the preserved par mediated vasodilation. recent studies have demonstrated the utility of targeting sk, sk and sk agonists for modulating vascular tone, presumably by endothelium-dependent hyperpolarization, and lowering blood pressure in ang ii-induced hypertensive mice. the protection of the par vasodilatation against ang ii-acquired hypertension suggests that endothelial kca activation by selected membrane receptors could also provide the vasculature with the ability to compensate or replace no during disease. some investigators have reported that uncoupled endothelial no synthase may contribute to a superoxide-mediated vasodilatation of aortas during endothelial dysfunction in diabetes, which was also proposed to be linked to ang ii. however, inhibition of uncoupled endothelial no synthase by l-name remains a point of controversy so it is uncertain whether a similar connection between par and uncoupled endothelial no synthase exists in the ang ii infusion model. the results of experiments in par2-/- indicate that par null genotype had mixed effects on the negative outcomes of ang ii infusions on blood vessels. first, there was an increased sensitivity of arteries to adrenergic receptor stimulation in par2-/-. second, there was less l-name sensitive endothelial no-derived relaxation activity in par2-/- after treatment with ang ii. third, the potency of nitroprusside in ang ii par2-/- was not decreased as in ang ii c the first of the effects above would be expected to increase systolic and pulse pressures in par2-/-. the second and third effects mentioned could be interpreted as protective compensatory mechanisms that may limit increases in blood pressures in par2-/-. our radiotelemetry study of blood pressures indicated that by the end of treatment period, ang ii produced larger systolic and pulse pressure changes in par2-/- than in c. a limitation to the scope of our study was the focus on disruption by ang ii of endothelium-dependent vasodilatation. we examined only one vasoconstrictor system, necessarily to establish conditions for bioassay, so it was possible that par2-/- may have affected vasoconstrictor systems other than α adrenergic receptors e.g. 5-hydroxytryptamine, thromboxane a. in the past years, the literature weighs heavily with evidence linking activated par to pro-inflammatory signal transduction in endothelial cells. there is also a broad literature contending that chronic elevated levels of ang ii elicits increased pro-inflammatory signalling by cytokines and pro-thrombotic conditions that contribute to the disruption of normal endothelial and vascular smooth muscle cell biology. in wild-type animals, it is possible that increased activity of coagulation factors and or other serine proteinases during chronic ang ii infusion leads to par activation and thus, the production of cytokines that cause vascular dysfunction. accordingly, it would be expected that the production of pro-inflammatory mediators elicited by ang ii in par null mice may be attenuated and thus, the disruptions by ang ii of the endothelium and vascular smooth muscle are reduced.  par2-ap effectiveness is protected against the negative effects of ang ii. par null expression had mixed effects in vitro on the negative outcomes of ang ii hypertension. the preserved par vasodilatation which is mediated largely via sk may lead to discovery of other endothelial cell signaling that is resistant to vascular diseases. 
in conditions of endothelial dysfunction, angiotensin ii induction of cyclooxygenases increases sensitivity to par agonist and the preserved vasodilatation mechanism involves activation of sk. par2-mediated vasodilatation of resistance type arteries is protected against the negative effects of angiotensin ii-induced vascular dysfunction in mice. under conditions of cardiovascular dysfunction, protease-activated receptor agonists maintain vasodilatation activity, which has been attributed to increased cyclooxygenase- nitric oxide synthase and calcium-activated potassium channel activities. protein and mrna expression of cyclooxygenase- and - were increased, and cyclooxygenase activity increased the sensitivity of arteries to 2fly in only angiotensin ii-treated wt.
 under conditions of cardiovascular dysfunction, protease-activated receptor agonists maintain vasodilatation activity, which has been attributed to increased cyclooxygenase- nitric oxide synthase and calcium-activated potassium channel activities. protease-activated receptor agonist mediated vasodilatation is unknown under conditions of dysfunction caused by angiotensin ii. the main purpose of our study was to determine whether par2-induced vasodilatation of resistance arteries was attenuated by prolonged angiotensin ii treatment in mice. we compared the vasodilatation of resistance-type arteries from angiotensin ii-treated par wild-type mice induced by par agonist 2-furoyl-ligrlo-amide to the responses obtained in controls. we also investigated arterial vasodilatation in angiotensin ii-treated par deficient mice.  2fly-induced relaxations of untreated arteries from angiotensin ii-treated wt were not different than saline-treated wt. treatment of arteries with nitric oxide synthase inhibitor and sk inhibitor blocked 2fly in angiotensin ii-treated wt. protein and mrna expression of cyclooxygenase- and - were increased, and cyclooxygenase activity increased the sensitivity of arteries to 2fly in only angiotensin ii-treated wt. these protective vasodilatation mechanisms were selective for 2fly compared with acetylcholine- and nitroprusside-induced relaxations which were attenuated by angiotensin ii; par2-/- were protected against this attenuation of nitroprusside.  par2-mediated vasodilatation of resistance type arteries is protected against the negative effects of angiotensin ii-induced vascular dysfunction in mice. in conditions of endothelial dysfunction, angiotensin ii induction of cyclooxygenases increases sensitivity to par agonist and the preserved vasodilatation mechanism involves activation of sk. 