Effects Of Rho Kinase And PDE On Cyclic Nucleotide-mediated Vasodilation

The second messengers,cyclic adenosine monophosphate(cAMP) and cyclic guanosine monophosphate(cGMP),are cyclic nucleotides and they play a key role in mediating a variety of cellular functions.In blood vessels,the intracellular level of both messengers is directly related to the relaxation of vascular smooth muscle cells(SMCs). The production of cAMP is catalyzed by adenylyl cyclase from ATP.Stimulation of theβ-adrenoceptor,a G-protein-coupled receptor on cell membrane,or an adenylyl cyclase activator forskolin leads to an increased accumulation of intracellular cAMP.Isoprenaline is a potentβ-adrenoceptor agonist which stimulates increase in the cAMP level and thus relaxes vascular smooth muscle of blood vessels.On the other hand,growing evidence shows that vascular endothelium cells also expressβ-adrenoceptors and their activation results in cAMP accumulation causing endothelium-dependent vascular relaxation through stimulation of endothelial NO biosynthesis.Similar to cAMP,guanylyl cyclase is the enzyme which catalyses the formation of cGMP from GTP and can be activated by endogenous NO and NO donors.In conduit arteries,NO production is mediated by endothelial NO synthase and NO is released from endothelial cells in response to acetylcholine and other endothelium-dependent dilators.Endothelium-derived NO diffuses readily to the adjacent vascular smooth muscle cells,where it stimulates soluble guanylyl cyclase and hence increases cGMP levels.NO donors such as sodium nitroprusside(SNP) and nitroglycerin(NTG) can release NO directly.Both cAMP and cGMP decrease the intracellular calcium concentration or calcium sensitivity in vascular smooth muscle cells, resulting in vasorelaxation.Phosphodiesterases(PDEs) are a diverse family of enzymes that hydrolyze cyclic nucleotides and inhibition of the PDE activity enhances vasodilatation mediated by cyclic nucleotides.NO donors are widely used to treat cardiovascular diseases such as arteriosclerosis, hypertension,coronary artery disease.However,the development of nitrate tolerance limits therapeutic value of nitrate drugs.Therefore,it is necessary to understand the mechanisms involved in the development of nitrate tolerance in order to attenuate or prevent nitrate tolerance.It is known that the production of thromboxane A₂ can be greatly enhanced in response to both physiological and pathological stimuli and this prostanoid activates the Rho/Rho kinase pathway through TP receptor,leading to a robust calcium-sensitizing effect or reduced NO release and hence increased vascular tone.However,it is unclear whether the activation of TP receptor could also negatively affect the cAMP-mediated vasodilatation.In the present study,rat carotid arteries from both sides were isolated and suspended in myograph for the measurement of isometric force and rat aortas were suspended in organ baths for recording changes in vessel tension.Primary culture of rat aortic endothelial cells was performed and changes in NO production and intracellular calcium concentration were measured using fluorescent dyes.Levels of relevant proteins in vascular tissues were detected by Western blot analysis.The tissue content of cyclic nucleotides in arteries were chemically assayed by using ELISA kits.We aimed to examine:(1) whether activation of TP receptors attenuates endothelium-derived NO-dependent relaxation induced by theβ-adrenoceptor isoprenaline in rat carotid arteries via a Rho kinase-dependent mechanism, (2) whether the activation of TP receptors inhibits endothelium-independent relaxation in response to cAMP-elevating agents in rat carotid arteries via the increased activity of type 3 or type 4 phosphodiesterase(PDE3,PDE4),and(3) whether the acute development of nitrate tolerance in isolated rat aortic rings exposed to NTG can be ameliorated by a new potent PDE5 inhibitor,TO 156 via increase of intracellular cGMP.The present study may be helpful to probe into the mechanism of vascular dysfunction in response to both physiological and pathological stimuli and provide theoretical significance for PDE5 inhibitors to increase therapeutic value of nitrate drugs by prevention of nitrate tolerance.The present study demonstrated that:1) Isoprenaline induces endothelial-dependent vasorelaxation,which is blocked by eNOS inhibitor(L-NAME) and sGC inhibitor(ODQ). The relaxation to isoprenaline was concentration-dependently attenuated in rings pre-contracted with U46619(3*10^-8M,1*10^-7TM,3*10^-7M).Treatment with TP receptor antagonist(S18886,0.3μM) completely normalized isoprenaline-induced relaxation in the presence of U46619.In rings with endothelium contracted by phenylephrine plus U46619, treatment with Y27632 or HA 1077 partially,but significantly improved isoprenaline-induced relaxation.However,the inhibitors failed to influence relaxation in endothelium-denuded rings.Both inhibitors(Y27632 and HA 1077) failed to influence isoprenaline-evoked relaxation of rings with endothelium after inhibition of NO production by L-NAME or inhibition of sGC by ODQ.Imaging results with rat aortic endothelium cells showed that exposure to isoprenaline(1*10^-7 M) caused a slow but constant rise in dichlorofluorescein fluorescence intensity which was almost abolished after pre-exposure to L-NAME.However,exposure to isoprenaline(1*10^-7 M) caused a quicker rise in dichlorofluorescein fluorescence intensity and it was totally abolished in Ca2+ free PSS solution.But it increased sharply and significantly when CaC12(lmM) were administered. The rise in fluorescence intensity of both NO and Ca2+ was significantly inhibited by pretreatment with U46619(1*10^-7 M),which were totally abolished by S18886(0.3μM) pretreatment.Pre-stimulation with Rho kinase inhibitor,Y27632 reversed the inhibitory effect on the dichlorofluorescein fluorescence intensity of NO and Ca2+ by U46619 in endothelial cells.Western blotting data demonstrated isoprenaline(1*10^-7 M) induced phosphorylation of e-NOS at Ser1177 site which was significantly inhibited by U46619 (1*10^-7 M).Pre-exposure to Y27632 partially reversed the inhibited phosphorylation.2) Cyclic AMP-elevating agents,isoprenaline and forskolin,induce concentration-dependently relaxation in carotid artery rings without endothelium.The relaxation to both agents was concentration-dependently attenuated in rings pre-contracted with U46619 compared to phenylephrine-constricted artery rings.The attenuated relaxation was totally blocked by pretreatment with TP receptor antagonist,S 18886(0.3μM).In rings contracted by phenylephrine plus U46619,treatment with Y27632 partially,but significantly improved cAMP-elevating agents-induced relaxation.Similarly,both PDE3 inhibitor cilostazol and PDE4 inhibitor rolipram and nonselective PDEs inhibitor IBMX improved the vascular relaxation in response to isoprenaline or forskolin.Inhibition of PDE5 with T0156 did not affect relaxation induced by the two cAMP-elevating agents. However,pretreatment with cilostazol or rolipram failed to alter the vasodilation to isoprenaline in endothelium-denuded rings contracted with only phenylephrine,cAMP level was much lower in U46619-pretreated carotid arteries in response to both isoprenaline and forskolin.PDE3,PDE4 and Rho kinase inhibitor(cilostazol,rolipram and Y 27632 respectivley) reversed the inhibited cAMP production by U46619.3) A vascular tolerance(attenuated relaxation) to the NO donor,nitroglycerin can be readily induced by pre-exposure to NTG in rat isolated thoracic arteries with and without endothelium.The inhibition of PDE5 with T0156 improved the reduced relaxation to NTG in tolerant arteries with much higher extent than in non-tolerant arteries,cGMP level was much lower in tolerant arteries than in non-tolerant arteries and T0156 increased cGMP level in response to 1μM NTG,11.6-fold in tolerant rings,6.6-fold in non-tolerant rings. Nitrate tolerance was not associated with changes in the activity of thromboxane A₂ receptors,COX-derived prostanoids,or endothelin A receptor under current experimental condition.In summary,the above-mentioned results demonstrate that:1) Activation of TP receptor attenuated the cAMP-mediated endothelium-dependent relaxation through stimulation of Rho kinase,which inhibited the activity of eNOS through reducing calcium influx and eNOS phosphorylation,thus leading decreased production of NO.2) Stimulation of TP receptor impairs vascular endothelium-independent relaxation to cAMP-elevating agents.This inhibited relaxation induced appeared to be through activation of Rho kinase by increasing activity of cAMP-hydrolyzing PDEs,leading to decreased cAMP level.3) The present pharmacological results also showed a significant contributing role of the increased PDE5 activity in the acute development of nitrate tolerance in rat aortas in vitro.T0156 was proved to be a useful drug in maintaining vasodilator responses to nitroglycerin,the effect of which are frequently attenuated upon continuous administration.