Ms23 On Vascular Smooth Muscle Contraction, Blood Pressure And Hemodynamic Effects

Cyclic adenosine Y,5'-monophosphate(cAMP)is synthesized from ATP by at least 9 closely related isoforms of adenylyl cyclases.Cyclic AMP,as a "second messenger",play a key role in biological signal transmissions in many living cells,especially in the myocardial and various smooth muscles.A variety of biological active substances elicit an intracellular cAMP elevation followed by a cascade of special and sophisticated biological reactions,and eventually produce corresponding observed effects.In response to intracellular elevation of cAMP,vascular and respiratory smooth muscles relax,while the myocardial cells exhibit excitation.Cyclic AMP is degraded by at least 11 closely related isoforms of cyclic nucleotide phosphodiesterases (PDEs).These PDEs specifically cleave the 3',5'-cyclic phosphate moiety of cAMP and/or cGMP to produce the corresponding inert 5'-nucleotide and terminate second messenger activity of cyclic nucleotides.In the cardiovascular system,the main isoforms of PDEs are PDE1,PDE2, PDE3,PDE4 and PDES.An overwhelming and increasing amount of studies show that these PDEs play crucial roles in the control and regulation of the cardiovascular system.Therefore, agents which inhibits PDE or PDEs may exert a succession of pronounced effects on various cells.PDEs inhibitors have been clinically used to treat chronic heart failure,conditions with increased peripheral vascular resistance,chronic obstructive pulmonary diseases,anandria, allergy and depressive disorders.MS23 is a brand new compound,synthesized by Department of Basic Pharmaceutical Science of Pharmacy College of University of South Carolina.MP01 is an analog of MS23,with fewer and milder toxical effects.Previous studies indicated that MS23 concentrationdependently inhibited cAMP degradation,without effect on cGMP in guinea pig brain. Enzymogical studies verified that MS23 specially inhibited PDE4 and PDE3 without significant effect on other PDEs.Previous studies also suggested that MS23 has relaxation effect on vascular and respiratory smooth muscle,and that it might be used to treat hypertension and chronic obstructive pulmonary diseases.Therefore,it is necessary to clear out pharmacological profiles of MS23 on vascular smooth muscle.The present studies were designed to observe and compare the effects of MS23 and MP01 on the contractions induced by noradrenaline,60 mmol/L KCl,thromboxane A₂ analog U46619 and phenylephrine in rat isolated thoracic aortic rings and coronary,middle cerebral,renal and mesenteric artery rings.Relaxation on precontractions in rat isolated thoracic aortic rings.Male Wistar rats weighing 200～250g were killed by cervical dislocation and thoracic aortas were removed and placed in chilled(4℃)physiological salt solution(PSS)which was gassed with 100%O₂.The blood vessels were cleaned of connective tissue and fat,and cut into 3-4mm long rings.Extreme care was exercised during preparation of the rings to preserve the integrity of the endothelium.The rings were mounted in water-jacketed tissue baths containing 10.0 ml of PSS(bubbled with 100%O₂ and maintained at 37℃)for measurement of isometric tension. Passive tension was adjusted to 2.0 g and all subsequent measurements represent force generated above this baseline.A 2h equilibration period was allowed before any experimental intervention, and during equilibration,the bath was flushed every 20 minutes with the fresh PSS.After equilibration,the rings were activated 2～5 times with PSS containing 60 mmol/L KC1 for 10 minutes.The integrity of endothelium was presumed by observation that the relaxation induced by 10^-5mol/L acetylcholine on 60 mmol/L KC1 contraction was greater than 30%.When the contraction induced by 60 mmol/L KC1 and the relaxation induced by 10^-5mol/L acetylcholine were repeatable(the change was less than 10%between successive contractions or relaxation), the effects of the drugs to be tested were observed.The contraction tensions induced by 60mmol/L KCl and 10^-5mol/L NA were 2.34±0.94g and 1.58±0.62g,respectively.In concentration range of 10^-6～3×10^-5mol/L, MS23,MP01 and aminophylline concentration- dependently relaxed the precontraction induced by 60 mmol/L KCI in rat isolated thoracic aortic rings.The maximum relaxation were 90.5±8.2 %,94.5±14.6%,55.2±27.6%;the calculated EC5o were 9.19×10^-6,7.26×10^-6,2.67×10^-5 mol/L respectively.The maximal relaxation of MS23,MP01 and aminophylline in the precontraction induced by noradrenaline(10^-5mol/L)were 76.2±15.4%,86.6±28.4%, 53.6±19.8%;EC₅₀were 1.44×10^-5,6.71×10^-6,2.69×10^-5mol/L respectively. Preincubation with MS23 or MP01 shifted the noradrenaline concentration- contraction curve in a nonparallel manner to the right with the maximal response depressed.The calculated IC₅₀for MS23 and MP01 were 4.84×10^-6mol/L and 1.97×10^-6mol/L respectively.These results showed that MS23,MP01 and aminophylline have direct relaxation effects on KCl- and noradrenaline- induced contractions without any preference between these two stimulators;and that the relaxation potency of MS23 is nearly equal to MP01,but stronger than aminophylline.0.1 mmol/L NG-nitro-L-arginine methylester ester(L-NAME),a nitric oxide(NO) synthetase inhibitor,had no effects on MS23 or MP01 induced relaxation,while abolished acetylcholine induced relaxation.Deprivation of endothelium did not effect the relaxation induced by MS23 or MP01.These results indicated that NO production and endothelium are not involved in the mechanism of relaxation induced by MS23 or MP01;and these results are also agree with the previous conclusion that MS23 is a PDE inhibitor.Relaxation of MS23 on precontractions in rat isolated coronary,middle cerebral,renal,and mesenteric artery ringsTo study and compare the relaxation effects of MS23,MP01 and aminophylline on precontractions in rat isolated small artery with different origin,rat coronary,middle cerebral, renal,and mesenteric arteries were isolated.The third-order branches of the superior mesenteric artery and renal arteries,middle cerebral artery and coronary artery were isolated and cut into 2mm-long rings.The rings were mounted on a wire myograph(Multi Myograph System-610M,Danish Myo Technology A/S,Denmark).The rings were normalized according to standard procedures and stretched to a state equal to 100mmHg,80mmHg,80mmHg, 80mmHg,respectively.The rings were equilibrated for at least for 1h,and then contracted and relaxed as above mentioned in the experiments with thoracic aortic rings before use of any relaxants to be tested.First,we studied and compared the effects of MS23,MP01 and aminophylline on the precontraction induced by 60 mmol/L KCl in rat isolated coronary,middle cerebral,renal, and mesenteric artery rings.The contraction tensions induced by 60 mmol/L KCl in rat isolated coronary,middle cerebral,renal,and mesenteric artery rings were 8.67±6.73 mN, 2.16±1.1 mN,4.05±2.13 mN,8.89±4.34 mN,respectively.MS23,MP01 and aminophylline concentration- dependently relaxed this contraction,and these three relaxants showed no preference among the four arteries with different origin.The relaxation values of EC₅₀on 60 mmol/L KCl induced contraction in these small arteries with different origin were around 10^-5 mol/L for MS23 and MP01.The maximal relaxation response produced by aminophylline(3×10⁵ mol/L)was only 35.2%.By comparison,it was found that the relaxation effects of both MS23 and MP01 on these small artery rings were comparably potent to their effects on aortic rings, while the relaxation effect of aminophylline on these small artery rings were much weaker than its effects on aortic tings.Secondly,we compared the effects of MS23,MP01 and aminophylline on the precontraction induced by 60 mmol/L KCl,noradrenaline(10μmol/L)and thromboxane A₂ analog U46619(1μmol/L)in rat isolated renal artery rings.The contraction tensions induced by 60 mmol/L KCl,noradrenaline and U46619 in rat renal artery rings were 4.05±2.12mN,2.42±2.55mN,7.02±3.45mN,respectively.60 mmol/L KCl and U46619 induced contractions were more sensitive to the relaxation of MS23,MP01 and aminophylline,while noradrenaline-induced contraction was more resistant to these three relaxants.These three relaxants,at maximal concentration(3×10^-5mol/L),only produced relaxations of less than 30%on noradrenaline-induced contraction.These results suggested that relaxation effects of MS23,MP01 and aminophylline have selectivity over the stimulators.The significance of the selectivity needs further clarification.Finally,we observed the effect of MS23 on isoprenaline induced relaxation in rat isolated renal artery rings.Isoprenaline(3×10^-8mol/L)concentration dependently relaxed the 60 mmol/L KCl induced contraction in most of rat isolated renal artery rings,with the maximal relaxation response being(42.8±16.4)%.MS23(1μmol/L)itself produced no significant relaxation.However,the relaxation response of isoprenaline was significantly increased(76.8±19.8 vs 42.8±16.4)%(P＜0.01)in presence of MS23(1μmol/L) Presence of MS23 decreased EC₄₀of isoprenaline by nearly 20 fold.This result suggested that MS23 ameliorate vascular relaxation effects of isoprenaline,and also given us a clue that MS23 may improve vascular relaxation state.CONCLUSIONS1.MS23,its analog MP01 and aminophylline concentration dependently relax the precontractions induced by 60 mmol/L KCl and noradrenaline in rat isolated thoracic aortic rings.The relaxation effects of the three tested relaxants manifest no selectivity between the two stimulators.NO production and presence of endothelium are not involved in mechanism of the relaxation.2.MS23 and MP01 have profound relaxation effects on 60 mmol/L KC1 induced contraction in rat isolated thoracic aortic rings,and coronary,middle cerebral,renal and mesenteric artery rings,without any preference among these five vascular beds tested;while the relaxation potency of aminophylline on the small arteries is much weaker than its effect on thoracic aortic rings.3.60 mmol/L KCl and U46619 induced contractions are more sensitive to the relaxation of the three tested PDE inhibitors,while noradrenaline- induced contractions is more resistant to them in rat isolated renal artery rings.4.MS23 enhances vascular relaxation effects of isoprenaline in rat isolated renal artery rings. BACKGROUND:Cyclic AMP,as an important intracellular second messenger,plays key role in regulating the functions of the myocytes and vascular smooth muscle.Increased intracellular cAMP levels are followed by a cascade of biological sophisticated,subtle and refined reactions,and eventually result in observed effects.For instance,cardiac excitations,such as enhanced contractility,accelerated heart rate and other positive effects are exhibited,whereas vascular contraction inhibition or relaxation are manifected in response to increased intracellular cAMP. The effects of cAMP is terminated by degradation which is carried out by cyclic nucleotide phosphodiesterases(PDEs),and therefore,inhibition of PDEs would produce marked pharmacological effects to the cardiovascular system.Actually,PDE inhibitors are now widely used clinically to relieve various diseases such as chronic heart failure,diseases with increased peripheral vascular resistance,chronic obstructive pulmonary diseases,anandria etc.Clinical studies reveal that short term use of PDE inhibitors such as aminophylline and milrinone produces beneficial therapeutic effects in treating chronic heart failure,but chronic use of them may bring about severe adverse effects such as myocardial hypertrophy,arrhythemia,cardiac dysfunctions.Elimination or extenuation of the harmful stimulatory effects and reservation of the beneficial effects of PDE inhibitors on the heart would make PDE inhibitor safer,more applicable and more effective.MS23 was designed to inhibit PDE4 and to cure chronic obstructive pulmonary diseases, because most of PDE4 inhibitors have anti-inflammatory effects and relax bronchial smooth muscle.Enzymological studies showed that MS23 selective inhibited PDE4,and also had some inhibitory effects on PDE3.MS23 was found to relax bronchial and vascular smooth muscle,and to inhibit myocardial Ca²⁺influx in previous studies.Toxicological studies showed that MS23 could cause severe toxic effects.A series of modifications on the molecule of MS23 were made, and systemic screening on the synthesized analog candidates gave birth to MP01.Enzymological studies showed that the inhibitory effects of MP01 on PDE4 and PDE3 were comparable to those of MS23,but toxic effects were much fewer and milder.Previous studies revealed that MS23 and MP01 had remarkable relaxation effects on rings of arteries isolated from various organs,and had inhibitory effects on myocardial Ca²⁺;influx,and therefore,their effects of cardiovascular system in intact animal deserve further investigation.AIM:To study and compare effects of MS23,MP01 and aminophylline on artery blood pressure and hemodynamic parameters in anaesthetized normotensive and spontaneous hypertensive rats; to observe the effects of MS23 and MP01 on systolic and diastolic artery blood pressure in conscious unrestrained spontaneous hypertensive rats.METHODS AND RESULTS:â‘ Hemodynamical effects in anaesthetized normotensive rats:Male normotensive Wistar rats weighing 240～260g were anaesthetized with intraperitoneal injection of urethane 1.0g/kg.Catheters were inserted into left ventricle and common carotid artery to record left ventricle pressure and artery blood pressure.The pressures were recorded by Powerlab system through the pressure transducers.The recorded or calculated parameters were left ventricle systolic pressure(LVSP),left ventricle end diastolic pressure(LVEDP),the maximal rise rate of left ventricle pressure(+dp/dt_max),the maximal decline rate of left ventricle pressure(-dp/dt_max),mean artery pressure(MAP)and heart rate(HR).The electrocardiogram was recorded also.The differences of above parameters between before the injection and 1,5,10, 20,30,60,90,120 min after the drug administration were calculated,respectively.MAP was dose-dependently reduced following intravenous administration(iv)of MS23 0.1～1.0 mg/kg.The maximal decline of MAP was 53.1±14.7 mmHg,appeared at 20～30min after intravenous injection,and the hypotensive effect persisted for about 90min.MP01 and aminophylline had similar hypotensive effects.MS23 1.0 mg/kg iv and MP01 1.0 mg/kg iv markedly reduced HR,237.6±26.8 vs 204.8±31.5 beats/min(P＜0.05)for MS23,and 230.2±32.6 vs 204.5±22.4 beats/rain(P＜0.05)for MP01,respectively.The PR interval was significantly prolonged in electrocardiogram in MS23- or MP01- treated rats.Contrarily,aminophylline 10 mg/kg iv accelerated the HR with shortened PR interval,and the maximal HR increase was 28.8±5.7 beats/min.MS23,MP01 and aminophylline showed no significant effects on LVEDP and -dp/dt_max. â‘¡Hemodynamical effects in anaesthetized spontaneous hypertensive rats:Male spontaneous hypertensive rats weighing 220-260g,with MAP being 164.2±12.6 mmHg were used.The operation and parameter recording procedures were same as above mentioned.In anaesthetized spontaneous hypertensive rats,MS23 0.3mg/kg and MP01 0.3mg/kg iv significantly lowered MAP.The hypotensive effect was appeared at 1min, culminated at around 60min after the injection.MAP was reduced by 52.3±15.8 mmHg for MS23 and 54.6±18.6 mmHg for MP01,respectively.The hypotensive effect persisted for nearly 120min.The maximal MAP reduction induced by aminophylline 10mg/kg was 30.3±17.4mmHg, appeared at 10min after the medication.The persistence time of hypotensive effects in aminophylline-treated rats was much shorter,as compared with MS23 or MP01.MS23 0.3 mg/kg iv and MP01 0.3 mg/kg iv significantly reduced HR by 39.1±9.8 beats/min for MS23,and 50.2±4.9 beats/min for MP01,respectively.Contrarily,aminophylline 10 mg/kg iv increased the HR by 29.4±4.8 beats/min,with shortened PR interval.MS23 and MP01 decreased,but aminophylline increased LVSP and +dp/dt_max.These three phosphodiesterases showed no significant effects on LVEDP and -dp/dt_max.â‘¢Effects of MS23 and MP01 on SBP,DBP and HR in conscious unrestrained spontaneous hypertensive rats.Effects of MS23 and MP01 on arterial systolic blood pressure(SBP),arterial diastolic blood pressure(DBP)and HR were observed in conscious unrestrained male spontaneous hypertensive rats,with MAP being 166.5±10.7mmHg,weighing 230～250g.The animals were anaesthetized with pentobarbital 40mg/kg iv and a fine catheter was inserted into abdominal aorta and connected to a pressure transducer implanter which transduces blood pressures into an electric signals and wirelessly emit the signals.The implanter was fixed in the abdominal cavity.The animals were allowed to recover for 7 days from the operation.The signals of SBP and DBP were received and analyzed with a telemetry blood pressure measure system(Data Science International,DSI).When the SBP and SBP were stable,MS23(1.0mg/kg)or MP01(1.0mg/kg) or equal volume of normal salinewere intragastrically administrated.Declines of SBP and DBP began at 30min,culminated at about 4h after medications,and hypotensive effects of both MS23 and MP01 persisted for about 6h.DBP was more pronounced reduced as compared with SBP. Neither MS23 nor MP01 showed any significant effects on HR.CONCLUSION: 1.MS23 reduces MAP,LVSP,and +dp/dtmax,and decrease HR,but had no significant effects on LVEDP and -dp/dt_maxin anaesthetized normotensive and spontaneous hypertensive rats.2.In anaesthetized normotensive and spontaneous hypertensive rats,the pharmacologic features and effect potency of MP01 are Similar to MS23;however,aminophylline has different pharmacologic profile in that it reduces MAP,but accelerates HR,enlarged LVSP and +dp/dt_max,with no significant effects on LVEDP and -dp/dt_max.3.MS23 and MP01 lower SBP and DBP,and have more pronounced hypotensive effect on DBP,without significant effect on the HR.