| Background and ObjectiveVascular endothelial cells are key to regulate vascular tension and maintain cardiovascular homeostasis by releasing a series of small molecular substances with vasoactivities.Under the conditions of hypertension and aging,dysfunction of endothelial cells will impair vascular structure and functions.One of the main causes of endothelial dysfunction is the imbalance of endothelium-derived vasoactive factors,and among them,prostanoids produced by cyclooxygenase（COX）-mediated arachidonic acid（AA）pathway play important roles.Prostacyclin（PGI2）,one of the main products of AA metabolism in endothelium,has long been considered as an endothelial relaxing factor,which evokes vasodilation by acting on IP receptor.However,emerging studies have shown that PGI2 can also function as endothelium-derived contracting factor（EDCF）to mediate vasoconstriction by acting on TP and/or EP3 receptors.Endothelium-dependent contractions（EDCs）are exacerbated by aging and vascular diseases.It has been demonstrated that EDCs are more prominent in aging rats or age-matched hypertensive adult rats due to enhanced production of COX products and/or alterations in downstream receptors.Spontaneously hypertensive rats（SHRs）,which blood pressure is still comparable with that of Wistar-Kyoto rats（WKYs）at about 4 weeks of age,become hypertensive at about 6 weeks of age and establish hypertension at about 3-4 months of age,are widely used as a model of human hypertension with WKYs as normotensive controls.However,how EDCs change and the underlying mechanism（s）during the transition period from adolescence to adulthood when SHRs develop hypertension and may undergo considerable alterations in vessels,remain largely unclear.Iliac arteries provide blood to pelvic organs and legs and are an object of clinical intervention.Hence,we aim to clearly elucidate how EDCs changes during the development of hypertension in adolescent SHRs and the underlying mechanism（s）,thus providing experimental basis in terms of vascular biology for the control of the increase of blood pressure in this period.Moreover,one of the methods for prevention and treatment of hypertension is to control blood pressure by relaxing blood vessels and/or inhibiting vasoconstriction.Our previous studies have shown that antagonizing TP receptor suppresses vasoconstriction to PGI2 in some arteries,thus improving vasodilation,and further blocking EP3 receptor can better improve the relaxation.In blood vessels such as rat mesenteric artery,L-798106,an antagonist of EP3,not only adds to the effect of the TP antagonist SQ29548 but also causes relaxation to PGI2 more than that obtained with SQ29548.There is a pressing need to demonstrate the effect of L-798106 on EDCs in iliac arteries and how it affects the development of hypertension in SHRs.Therefore,in this study,the WKY and SHR iliac,renal and abdominal arteries of different ages（4 weeks:adolescent;12-16 weeks:adult）were isolated for biochemical and/or functional analyses to solve the above issues.Materials and MethodsIn this study,male WKY or SHR progenies 4 weeks（adolescent）and 12-16 weeks（adult）of age with specific pathogen free（SPF）were obtained.The WKY and SHR iliac,renal and abdominal arteries were isolated for biochemical and/or functional analyses.The systolic blood pressure and the heart rate of the conscious WKY and SHR were measured by a noninvasive blood pressure measurement system.Vasomotor reactions were analyzed by isometric force measurement in the isolated WKY and SHR iliac and/or renal arteries.The PGI2 metabolite 6-keto-PGF1?,the TxA2 metabolite TxB2,PGE2,PGD2 or PGF2?produced in adult WKY and SHR iliac arteries was detected with HPLC-MS.Measurement of the PGI2 metabolite 6-keto-PGF1?was performed with ELISA.Expressions of EP3,TP receptors,and?β-actin（internal controls）in the iliac aortas were detected by Western blot.The expression of IP receptor m RNAs was analyzed were detected by real-time PCR.The hearts were obtained to evaluate the heart-to-body weight ratio.Results1.ACh evoked vasoconstrictions in adolescent SHR iliac arteries in the presence of L-NAME,and the contractions were however abolished in endothelium-denuded vessels,so the effect was EDCs.The EDCs were comparable in adolescent WKY and SHR iliac arteries（P>0.05）,which were declined in adult vessels（P<0.01）.Interestingly,the decrease of vasoconstriction to ACh with age was to a lesser extent in SHR iliac arteries compared with that in WKYs（P<0.01）.2.The signal of 6-keto-PGF1αproduction in adult WKY and SHR iliac arteries was clearly detected with HPLC-MS.In both WKY and SHR iliac arteries,ACh evoked an increase in levels of the PGI2 metabolite 6-keto-PGF1αas against basal conditions（in PSS only,unstimulated with ACh）（P<0.01）.Interestingly,either the basal 6-keto-PGF1αor that after ACh stimulation was reduced with age in both WKY and SHR arteries（P<0.01）.Also,the increase of 6-keto-PGF1αevoked by ACh was reduced in adolescent SHR iliac compared with that in adolescent WKYs（P<0.01）.In addition,vasoconstriction induced by PGI2,the TP agonist U46619,or the EP3agonist sulprostone was attenuated in adult iliac arteries compared with those in the adolescents（P<0.01）.3.TP or EP3 receptor protein expression was clearly detected in iliac arteries with Western blot.There were comparable levels of TP receptor expression in the adolescent WKY and SHR vessels（P>0.05）,which were declined in adults（P<0.01）.However,the extent of reduction was less in SHRs（P<0.05）.In the meantime,the expression of EP3 receptor in the vessels was lower in adolescent SHRs than in WKY counterparts（P<0.05）,which was downregulated（P<0.01）to a similar level in adults of both strains（P>0.05）.The level of IP receptor m RNAs in iliac arteries,which did not change with age in WKYs（P>0.05）,was upregulated in adult SHRs than in adolescent counterparts（P<0.01）.4.Under the baseline conditions,the vasoconstriction evoked by ACh or PGI2 was reduced by either SQ29548 or L-798106 in the adolescent SHR iliac arteries（P<0.01）.L-798106suppressed the contraction to a greater extent than SQ29548（P<0.01）.In arteries precontracted with PE the relaxation evoked by ACh was blunted by a biphasic force development,which only tended to decrease in the presence of SQ29548（P>0.05）.However,L-798106 abolished the biphasic force and resulted in better relaxation of the arteries（P<0.01）.Similarly,in both adolescent and adult iliac arteries precontracted with PE,the contraction evoked by PGI2 only tended to be blunted by SQ29548（P>0.05）,but L-798106 reversed it into a relaxation（P<0.01）.5.U46619 evoked a potent concentration-dependent contraction in the adolescent SHR iliac arteries,which was significantly reduced in the presence of L-798106（P<0.01）.6.ACh evoked vasoconstrictions in adolescent SHR renal arteries or abdominal aortas in the presence of L-NAME,which were declined in adult vessels（P<0.01）.Similar results were obtained in WKY renal arteries（P<0.01）.7.SBP in control SHRs was steadily increased with age.The elevation of SBP in L-798106treated SHRs was blunted within 15 days of treatment（P<0.01）.However,the body weight,heart rate and heart-to-body weight ratio were not affected by L-798106 treatment（P>0.05）.Conclusions1.There exists comparable EDCs in both adolescent WKY and SHR iliac arteries,which was decreased with the increase of age and SHR blood pressure during the transition from adolescence to adulthood.2.The decrease of EDCs was lesser extent with age in SHR iliac arteries than that of WKYs due to differential downregulation of TP and EP3 receptor,which manifested that relatively larger EDCs in those of adult SHRs compared with those of adult WKYs.3.EDCs in other blood vessels,such as renal arteries and abdominal aortas,also showed similar changes as those in iliac arteries during the transition from adolescence to adulthood.4.L-798106,which partial antagonizes the contraction induced by TP receptor activation in addition to its antagonism on EP3 receptor but retains the vasodilation effect of IP receptor activation and thus blunts the development of hypertension in adolescent SHRs. |
