浙江大学 文本 Increased vasoconstriction to noradrenaline by 1400W, inhibitor of iNOS_生理学

clencedirect.com p 可SEVIER y4404263-26 .com/p Increased vasoconstriction to noradrenaline by 1400W,inhibitor of iNOS, in rats with streptozotocin-induced diabetes Xing Cheng.Catherine C.Y.Pang of 3 weeks The 616.45 122×10 ats with carly st ib1400 1.Introduction ic phases of diabete s in cLeod,2 lins are e rats a Th tal on t line at th hase (3 wreks)of strepto on to induced diabetes is due t

Increased vasoconstriction to noradrenaline by 1400W, inhibitor of iNOS, in rats with streptozotocin-induced diabetes Xing Cheng, Catherine C.Y. Pang* Department of Pharmacology and Therapeutics, Faculty of Medicine, The University of British Columbia, 2176 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3 Received 29 October 2003; accepted 4 November 2003 Abstract There is evidence that inducible nitric oxide synthase (iNOS) is activated at the acute phase of diabetes. We examined if selective inhibition of iNOS by 1400W (N-3-aminomethyl-benzyl-acetamidine) increases vascular response to noradrenaline in rats with streptozotocin (60 mg/kg i.v.)-induced diabetes for a duration of 3 weeks. The effects of noradrenaline on mean arterial pressure (MAP; 6, 16, 45 and 122
10 9 mol/kg/min) and mean circulatory filling pressure (MCFP; 16 and 45
10 9 mol/kg/min) were obtained in conscious and unrestrained diabetic rats and control rats before as well as after treatment with 1400W (3 mg/kg followed by 3 mg/kg/h, i.v.). Rats with early streptozotocin-induced diabetes had decreased mean arterial pressure and mean circulatory filling pressure responses to noradrenaline. Treatment with 1400W did not affect responses in the control rats but increased maximum pressor response to noradrenaline (from 46 F 3 to 63 F 5) and mean circulatory filling pressure response to the high dose (45 nmol/kg/min) of noradrenaline (from 1.0 F 0.2 to 3.8 F 0.3 mmHg) in the diabetic rats. Thus, selective inhibition of iNOS by 1400W increases arterial and venous constriction to noradrenaline in conscious rats with streptozotocin-induced diabetes. D 2003 Elsevier B.V. All rights reserved. Keywords: Capacitance; Mean circulatory filling pressure; Noradrenaline; Vasoconstriction 1. Introduction Diabetes is associated with functional abnormalities in the cardiovascular system. In vitro and in vivo studies have shown that pressor response and arterial constriction to vasoactive agents such as noradrenaline, angiotensin and endothelins are depressed in diabetic rats at 2 weeks after the injection of streptozotocin (Hebden et al., 1987; Makino and Kamata, 1998; Bardell and MacLeod, 2001; Misurski et al., 2001; Cheng et al., 2003a). In addition, rats with streptozotocin-induced diabetes had depressed venocon￾striction to noradrenaline in vivo at 2 weeks after injection of streptozotocin (Cheng et al., 2003a). It is unclear what was the primary cause of reduced vasoconstriction at the acute phase of diabetes. Several studies have shown that the inducible isoform of nitric oxide synthase (iNOS) is activated at both the acute and chronic phases of diabetes. Indeed, iNOS was detected in the mesenteric artery of rats with streptozotocin-induced diabetes at 12 –14 weeks (Bardell and MacLeod, 2001), cardiac myocytes from diabetic rats at 8 weeks after injection of streptozotocin (Smith et al., 1997) and platelets from patients with type I and II diabetes (Tannous et al., 1999). The total activities of iNOS and endothelium nitric oxide synthase (eNOS) and the level of mRNA encoding these isoforms of nitric oxide synthase are increased in the hearts of rats with streptozotocin-induced diabetes for a duration of 4 to 6 weeks after induction (Stockklauser￾Fa¨rber et al., 2000). Moreover, in vitro inhibition of iNOS by S-ethyl-isothiourea increased the potency (reduced EC50) of contraction to noradrenaline in endothelium-denuded mesenteric arteries from rats rendered diabetic for 12 – 14 weeks (Bardell and MacLeod, 2001). There is as yet no published studies which examined if induction of iNOS contributes to the reduced arterial and venous constriction to noradrenaline at the acute phase of diabetes. The present study examined if attenuated in vivo arterial and venous vasoconstriction to noradrenaline at the early phase (3 weeks) of streptozotocin-induced diabetes is due to 0014-2999/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2003.11.002 * Corresponding author. Tel.: +1-604-822-2039; fax: +1-604-822- 6012. E-mail address: ccypang@interchange.ubc.ca (C.C.Y. Pang). www.elsevier.com/locate/ejphar European Journal of Pharmacology 484 (2004) 263 – 268

264 X Cheng.C.C.Poz/E 044280425-26对 23.Eperimental protocol n of INOS (G sure that a8m ame (6, g et al., prior to a dings wer 2.Materials and method 145×10 Each d 2..E tal animals and induction of diabetes from ou at the 2.4.Mear datory filling pressure n ements Central venous pr MeNeill.1999).Pls was m as fi plateau value whi re).M as fol 2.5.Drugy (1400w m(San Diep

activation of iNOS. Pressor and mean circulatory filling pressure (MCFP; index of body venous tone) responses to noradrenaline were measured in vehicle-treated control rats and streptozotocin-induced diabetic rats prior to and follow￾ing the administration of 1400W (N-3-aminomethyl-benzyl￾acetamidine), a selective inhibitor of iNOS (Garvey et al., 1997; Alderton et al., 2001). Mean circulatory filling pressure is the driving force of venous return and is experimentally the equilibrium pressure that exists in the circulation immediately after an abrupt cessation of blood flow (Guyton, 1963; Guyton et al., 1954; Rothe, 1993; Pang, 2000). It has been shown that 1400W is>1000-fold (in vitro) and >50-fold (in vivo) more selective in inhibiting iNOS than eNOS (Garvey et al., 1997) and is able to reverse hypotension (Wray et al., 1998) and augment cardiac output (Cheng et al., 2003b) in rats with Escherichia coli lipopoly￾saccharide-induced sepsis, a condition associated with the activation of iNOS. 2. Materials and methods 2.1. Experimental animals and induction of diabetes Male Wistar rats (300 – 350 g) were obtained from Charles River Canada. The rats were maintained under 12:12 h light:dark cycle (lights on from 7 a.m. to 7 p.m.) and supplied with standard laboratory chow diet (PMI Feeds) and water ad libitum. The rats were injected with streptozotocin (60 mg/kg, 1 ml/kg, i.v.) or an equal volume of vehicle (0.9% NaCl) via the tail vein under light halothane anaesthesia. The rats were considered to be diabetic and used for the study if they had hyperglycemia (>15 mM) at 48 h after injection of strepto￾zotocin as detected by AccuSoft (Hoffmann-La Roche) test strips (Sambandam et al., 2000; McNeill, 1999). Plasma glucose was measured by the glucose oxidase method (Sigma, Trinder 100 kit) via the use of a Spectrarainbow (ART F039039, Austria). The rats were studied at 3 weeks after injection of streptozotocin or the vehicle. 2.2. Surgical preparation The streptozotocin-induced diabetic and control rats were surgically prepared under halothane anaesthesia. A saline￾filled, balloon-tipped catheter was inserted into the right atrium through the right external jugular vein. Cannulae were also inserted into an iliac artery for the measurement of mean arterial pressure (MAP) by a pressure transducer (P23DB, Gould Statham, Oxnard, CA), into the right iliac vein for the withdrawal of blood (0.1 ml) for the measure￾ment of plasma glucose and infusion of drugs, and into the inferior vena cava via the left iliac vein for the measurement of central venous pressure by another pressure transducer (P23DB, Gould Statham). All cannulae were tunnelled subcutaneously to the back of the neck and exteriorised. The rats were recovered from surgery and anaesthesia for at least 6 h prior to the study. 2.3. Experimental protocol The diabetic and control rats (n = 6 or 7 each) were placed in a small cage and allowed to wander freely during the study. After equilibration for 1 h, the rats were first pretreated with propranolol (8
10 7 mol/kg i.v. bolus followed by continuous infusion at 3.4
10 7 mol/kg/ min) to block h-adrenoceptors. At 15 min after the start of administration of propranolol, dose – response curves of single doses of noradrenaline (6, 16, 45 and 122
10 9 mol/kg/min) were constructed in the diabetic and control rats. Mean arterial pressure and heart rate (HR) measure￾ments were taken prior to and at 10 min after the start of infusion of propranolol, and at the plateau phase of response to noradrenaline (3 to 10 min after the start of infusion), whereas mean circulatory filling pressure readings were taken at the baseline and the plateau phase of response to the 2nd and 3rd doses of noradrenaline (16 and 45
10 9 mol/kg/min). Each dose of noradrenaline was followed by a recovery period of 10– 15 min. After recovery of the response to the last dose of noradrenaline, the rats were given 1400W (3 mg/kg followed by 3 mg/kg/h, i.v.). At 1 h later, a 2nd dose– response curve of noradrenaline was constructed in both groups of rats. Haemodynamic measure￾ments were again taken at the baseline condition (5 min prior to the infusion of noradrenaline) and at the plateau phase of response to noradrenaline. 2.4. Mean circulatory filling pressure measurements Central venous pressure was measured after transiently stopping the circulation through injection of a small volume of fluid into the right atrial balloon. Within 5 s following inflation of the balloon, mean arterial pressure decreased to a plateau value (referred to as final arterial pressure), while central venous pressure increased to a plateau value (referred to as venous plateau pressure). Mean circulatory filling pressure was calculated as follows: mean circulatory filling pressure = venous plateau pressure + 1/60 (final arterial pres￾sure venous plateau pressure), using 1/60 as the ratio of arterial to venous compliance (Yamamoto et al., 1980). 2.5. Drugs N-3-aminomethyl-benzyl-acetamidine (1400W) was obtained from Calbiochem (San Diego, CA). Noradrenaline and propranolol were from Sigma (USA). All drugs were dissolved in normal saline (0.9% NaCl). 2.6. Statistical analyses Data were log-transformed prior to statistical analysis to obtain ED50 values to noradrenaline using the GraphPad 264 X. Cheng, C.C.Y. Pang / European Journal of Pharmacology 484 (2004) 263–268

X Comng C.C.I.Pong Earprau furxal af Plarunwlogy 44 (2004)241-168 2M5 Tahie 1 Banite vuo of body weiaH,plastr glactoc.tan kral peooarc (MAP hea te (H)and mean cialakry filling pam (CPP)in corsciots.dabetie rats and coutrdl nats premreeted wih proprarolol a 3 etea体N)respectively Cenmrol Disberi yet填ep 436±21 35现±10 Maatu gluwe (m 61±03 24A+2 MAP fmHig) I实bascline 1土6 10g土2 2pdl bewGN 113±6 106±2 aecf和xIrW ● cratd afr l4ToW H设nstU ◇ d aw4 hon 104 te hauline 59±$ 39±1 ◆ 2nd haseline 365⊥4 6⊥1” 于Pw包 【s bascline 7.3±03 68±04 2nd bawEne 67±02 68±05 -123 valaes of MAP.HR ud MCFP were ubtained u S min prior lo the stat uf noradeeaaline irfcra,both benm flt haclire)and afer (nd hwrline) he administoton of 1400'(makg follwed by 3 mgksh i.v.) 180 Noradrenaline (mulikg min),log mean circulatory filling pressure and heart rate (1102, 6.30.1 mmlg and 37413 beats/min,respectively) Pg1.aa-moree crrves fer the rf作sman土号EM,w=6-7r clative to the control rats(119±5,6.5±0.2 mmHg and proup)of aorodienaliae en (A)mcan anerial poessare (AMAP)and (B) 402土7 beats/min. hart nal:4HR)befw[oya园（口习dfAr●）nd■]h Pretreatmnt with progranoll docreased the heart rate in admerestrto ef1400'13 mokg followed by 3 mokgh 1v.)in comscous the diabetic(-35±7 beats/min）刻well ts control rats irgectnd with中tronencin0mokg门}mi量刘r白e vehicle (-33±6 eats/mnin】asa10 mmin after the start of 0%NaL,IOad●月E3 oeks prioe初c奖2s.b. infusion but did not significantly alrer the mean arterinl pressure and mean cireulatory filling pressure.After treat- Prism program.ED and E vales before and after the ment with propranolol,the heart rae was slightly lower in administration of 1400W were analysed by one way the diabetic rats relstive to the control rats,but mean arterial analysis of variance (ANOVA)followed hy the Tukey pressure and mean circulatory filling pressure remained Test.Dose-response relationships of mean arterial pres- slightly but insignificantly lower in the diabetic rats relative sure (Fig.1)were analysed hy one-way (berween groups) o the controls (Ist bascline,Table 1).The administration of ad two-wny (within the same group)repemed measures 1400W did not signiticantly aflect the mea arkrial pres- ANOVA followed by the Tukey test (SigmaStat stmtistical sure.heirt rate and mean circulatory filling pressure in software).In nll cases,n probability of emror (P<0.0l5)was cither the dsabetic or control rats (2nd basline,Table 1). selected as the criterion for statistical significance Tabie 2 EDs and for dose-response curves of the changes in mean ameral 3.Results preou:(MAP)sueadrtadine befere ua afer the ulainistriou uf 14iw行m fnileand hy1 mgph i￥)自hec rs and on se=6=7 per gcup)o铺33线s followine Lv.is6 out of strepo0uei 3.1.Baseline vules (60 mgkgl and he vehicle (09%NaCT.nopactively The rats had higher plasma concentration of plocose at EDsa (nmolkg) 48 h after i.v.injection of strepozotocin relative to the Crtel Dabctic Cretel Dihesc vehicle-.treated control rats21.0±1.5v&s.7±02mM SAP besre 1400W 5+02 23.6+37 6+ 46±3 &3+06 163+65 61±2 respectively). At 3 wecks afer the indoction.the diabetic rats had All value an nea:上8.EMa=i-了gnup Al dhia of EDa were lower body weight and higher plasma glucose than the log-trarstoened prior to statistical anabss. Dunko sianicard diffeeace frum cuool (P005 control rats (Table 1).At this time.the diabetic rats had slightly (insignificant)lower basetine mean arterial pressure. 14w4P<05队

Prism program. ED50 and Emax values before and after the administration of 1400W were analysed by one way analysis of variance (ANOVA) followed by the Tukey Test. Dose – response relationships of mean arterial pres￾sure (Fig. 1) were analysed by one-way (between groups) and two-way (within the same group) repeated measures ANOVA followed by the Tukey test (SigmaStat statistical software). In all cases, a probability of error (P < 0.05) was selected as the criterion for statistical significance. 3. Results 3.1. Baseline values The rats had higher plasma concentration of glucose at 48 h after i.v. injection of streptozotocin relative to the vehicle-treated control rats (21.0 F 1.5 vs. 5.7 F 0.2 mM, respectively). At 3 weeks after the induction, the diabetic rats had lower body weight and higher plasma glucose than the control rats (Table 1). At this time, the diabetic rats had slightly (insignificant) lower baseline mean arterial pressure, mean circulatory filling pressure and heart rate (110 F 2, 6.3 F 0.1 mmHg and 374 F 13 beats/min, respectively) relative to the control rats (119 F 5, 6.5 F 0.2 mmHg and 402 F 7 beats/min). Pretreatment with propranolol decreased the heart rate in the diabetic ( 35 F 7 beats/min) as well as control ( 33 F 6 beats/min) rats at 10 min after the start of infusion but did not significantly alter the mean arterial pressure and mean circulatory filling pressure. After treat￾ment with propranolol, the heart rate was slightly lower in the diabetic rats relative to the control rats, but mean arterial pressure and mean circulatory filling pressure remained slightly but insignificantly lower in the diabetic rats relative to the controls (1st baseline, Table 1). The administration of 1400W did not significantly affect the mean arterial pres￾sure, heart rate and mean circulatory filling pressure in either the diabetic or control rats (2nd baseline, Table 1). Fig. 1. Dose – response curves for the effects (mean F S.E.M, n = 6 – 7 per group) of noradrenaline on (A) mean arterial pressure (DMAP), and (B) heart rate (DHR) before [(o) and (5)] and after [(.) and (n)] the administration of 1400W (3 mg/kg followed by 3 mg/kg/h i.v.) in conscious rats injected with streptozotocin [60 mg/kg, (5) and (n)] or the vehicle [0.9% NaCl, (o) and (.)] at 3 weeks prior to these experiments. Table 1 Baseline values of body weight, plasma glucose, mean arterial pressure (MAP), heart rate (HR) and mean circulatory filling pressure (MCFP) in conscious, diabetic rats and control rats pretreated with propranolol at 3 weeks following the administration of streptozotocin (60 mg/kg i.v.) or the vehicle (0.9% NaCl), respectively Control Diabetic Body weight (g) 436 F 21 332 F 10a Plasma glucose (mM) 6.1 F 0.3 24.4 F 2.4a MAP (mmHg) 1st baseline 118 F 6 109 F 2 2nd baseline 113 F 6 105 F 2 HR (beats/min) 1st baseline 369 F 5 339 F 10a 2nd baseline 365 F 4 336 F 13a MCFP (mmHg) 1st baseline 7.3 F 0.3 6.8 F 0.4 2nd baseline 6.7 F 0.2 6.8 F 0.5 All values are means F S.E.M. (n = 6 – 7 per group). a Denotes the significant difference from controls ( P < 0.05). Baseline values of MAP, HR and MCFP were obtained at 5 min prior to the start of noradrenaline infusion, both before (1st baseline) and after (2nd baseline) the administration of 1400W (3 mg/kg followed by 3 mg/kg/h i.v.). Table 2 ED50 and Emax for dose – response curves of the changes in mean arterial pressure (MAP) to noradrenaline before and after the administration of 1400W (3 mg/kg followed by 3 mg/kg/h i.v.) in diabetic rats and control rats (n = 6 – 7 per group) at 3 weeks following i.v. injection of streptozotocin (60 mg/kg) and the vehicle (0.9% NaCl), respectively ED50 (nmol/kg) Emax (mmHg) Control Diabetic Control Diabetic MAP before 1400W 6.8 + 0.2 23.6 + 3.7a 61 F 3 46 F 3a MAP after 1400W 6.3 + 0.6 16.3 + 6.5a 62 F 2 63 F 5b All values are means F S.E.M. (n = 6 – 7 per group). All data of ED50 were log-transformed prior to statistical analysis. a Denotes significant difference from control ( P < 0.05). b Denotes significant difference from before the administration of 1400W ( P < 0.05). X. Cheng, C.C.Y. Pang / European Journal of Pharmacology 484 (2004) 263–268 265

26 YCimg CCY Pang /Funggvas lumdl gf Phorsurology ( 3.2.Response to morndnenalme 3.3E5g时Pwea4了400 W on rexponses to poradrenalme Noradrenaline bad caused a dose-dependent inerease in mean aterial pressure and decreases in heart rale in the Pretrealment (60 min)with 1400W did not signilicantly comrol and diabetic rats.Curve analyses show that the afect the mean arterial pressure,heart rale (Fig.1)and mean changes in mean arterial pressure and decreases in heart circulatory filling pressure (Fig 2A)responses to nordren- rate in the diabetie rats were sgnificnmly smaller than aline in the control group.The 1400W treatment in the those of'the controls (Fie.1A,B).The diabetie mats had diabetic rats mmgmented the noendrenaline-induca increases lower poreney (higher EDsa)nnd lwer efieacy (F)of in mean arerial pressure (Fie.IA)such that the maximam response to noradrenaline than the comtrols (Tahle 2) mean arterial pressure was restorod,and potency of response Noradrenaline had (16 and 45 nmol/kgfmin)increased the to norad山2 line was slightly(insignificantly）)ncreased mean circulatory filling pressure in the diabetic and (Table 2)Pretrestment with 1400W slightly (insignificant- control rats,and both changes were significantly and ly)increased the mean circulsory filling pressure response markedly less in the disbelic than the controls rats o the low dose of noradreraline but restored the mean 下ig.2A.B circulatory filling pressure response to the high dose of noradrenaline relative o the corresponding respomse in the control rats (Fig.2B) 5 7A.Contral naSwe 14rOW Afxer 1400W 4.Discusxion At 3 weeks after the induction of diabetes with strep ozotocin.the rats had reduced potency (higher ED)and efbcacy (.of pressor response as well as attenuated the mean circulatory filling pressure response to noradren- aline.These results were similar to those reported previ- 2 ously for mts with streptogntocin-induced diabetes for a durntion of 2 weeks (Cheng et al.,2003a)and are indicntive of genemlised depression of aadrenoceptor- mediated vasoconstriction at the acute phase of dinhetes. Both the diabetie and control rats had dose-deperdent 61B.Diahetes bradycardic response o poradrenaline that was likely due to reflex parasympathetic activation and not sympathetic Be14团 Aet 140707 withdrawal because the rats were pretreated with propran olol to bleek the chronotropic and ino国ogie《-lsp ar-modited)effeets of noradrenaline.Reduced pressor response to noradrenaline has hoen reported in pithod rats at 2 wecks (Foy and Iacas,1976 IJeas,1985).and in 3 consciois rats at 4 to$weeks (Jackson and Carnier,1983) and 6 weeks (Yu and MeNeill,1992)following the induction with streptovotocin.However,unlike the present study,these nats were not pretreated with B-adrenoceptor antigonist It is unclear what was the primary cause of attenualed vasoconstriction.There is evidence that iNOS is activalod at the acule phase of diabetes (see Introduction).Furthermore, 165 1645 iNOS induction in diabetes may be a consequenee of Noradrenaline (nmolkg'min) hyperglyeemis.The expooure of'aortic endothelial cells to hight ghucose for 7-10 days was reported to inerease the release of peroxynitrite,a highly reactive and cytotoxie 45 nranlitpminl an the ch九msnn和irltory lic nrexen0 oxidant derived fiom the interncrion of NO with the free (AMCFP)befure aad efer the adrinotratos of 1400W (3 mpke followed radical superoxide (Zou et al,2002).Moreover,2 h perfu- y3 kgh iv.}口comrio rate inected wih(A)he(a%是C or IB)strepeoaotocin mpkgl ot 3 weeks pror to these cxperimerts. sion of hesrts with a solution containing a high concentra "Sigtilicarly differeal P005)fomn omeponding poptse pundl tion of glucose in vitro was associated with an increase in Al in the cormol rore given the whicle "Sipnificxrly diffemet fiom the expression of the iNOS (but not eNOS)gene.the esporses a the diobesie rors before the oda由ution of L4O间W gereration of nitnc oxide and superuxide.and the apoptosis

3.2. Response to noradrenaline Noradrenaline had caused a dose-dependent increase in mean arterial pressure and decreases in heart rate in the control and diabetic rats. Curve analyses show that the changes in mean arterial pressure and decreases in heart rate in the diabetic rats were significantly smaller than those of the controls (Fig. 1A,B). The diabetic rats had lower potency (higher ED50) and lower efficacy (Emax) of response to noradrenaline than the controls (Table 2). Noradrenaline had (16 and 45 nmol/kg/min) increased the mean circulatory filling pressure in the diabetic and control rats, and both changes were significantly and markedly less in the diabetic than the controls rats (Fig. 2A,B). 3.3. Effects of pretreatment with 1400W on responses to noradrenaline Pretreatment (60 min) with 1400W did not significantly affect the mean arterial pressure, heart rate (Fig. 1) and mean circulatory filling pressure (Fig. 2A) responses to noradren￾aline in the control group. The 1400W treatment in the diabetic rats augmented the noradrenaline-induced increases in mean arterial pressure (Fig. 1A), such that the maximum mean arterial pressure was restored, and potency of response to noradrenaline was slightly (insignificantly) increased (Table 2). Pretreatment with 1400W slightly (insignificant￾ly) increased the mean circulatory filling pressure response to the low dose of noradrenaline but restored the mean circulatory filling pressure response to the high dose of noradrenaline relative to the corresponding response in the control rats (Fig. 2B). 4. Discussion At 3 weeks after the induction of diabetes with strep￾tozotocin, the rats had reduced potency (higher ED50) and efficacy (Emax) of pressor response as well as attenuated the mean circulatory filling pressure response to noradren￾aline. These results were similar to those reported previ￾ously for rats with streptozotocin-induced diabetes for a duration of 2 weeks (Cheng et al., 2003a) and are indicative of generalised depression of a-adrenoceptor￾mediated vasoconstriction at the acute phase of diabetes. Both the diabetic and control rats had dose-dependent bradycardic response to noradrenaline that was likely due to reflex parasympathetic activation and not sympathetic withdrawal because the rats were pretreated with propran￾olol to block the chronotropic and inotropic (h-adrenocep￾tor-mediated) effects of noradrenaline. Reduced pressor response to noradrenaline has been reported in pithed rats at 2 weeks (Foy and Lucas, 1976; Lucas, 1985), and in conscious rats at 4 to 5 weeks (Jackson and Carrier, 1983) and 6 weeks (Yu and McNeill, 1992) following the induction with streptozotocin. However, unlike the present study, these rats were not pretreated with h-adrenoceptor antagonist. It is unclear what was the primary cause of attenuated vasoconstriction. There is evidence that iNOS is activated at the acute phase of diabetes (see Introduction). Furthermore, iNOS induction in diabetes may be a consequence of hyperglycemia. The exposure of aortic endothelial cells to high glucose for 7 –10 days was reported to increase the release of peroxynitrite, a highly reactive and cytotoxic oxidant derived from the interaction of NO with the free radical superoxide (Zou et al., 2002). Moreover, 2 h perfu￾sion of hearts with a solution containing a high concentra￾tion of glucose in vitro was associated with an increase in the expression of the iNOS (but not eNOS) gene, the generation of nitric oxide and superoxide, and the apoptosis Fig. 2. Effects (mean F S.E.M, n = 6 – 7 per group) of noradrenaline (16 and 45 nmol/kg/min) on the changes in mean circulatory filling pressure (DMCFP) before and after the administration of 1400W (3 mg/kg followed by 3 mg/kg/h i.v.) in conscious rats injected with (A) vehicle (0.9% NaCl), or (B) streptozotocin (60 mg/kg) at 3 weeks prior to these experiments. *Significantly different ( P < 0.05) from corresponding responses (in panel A) in the control rats given the vehicle. # Significantly different from responses in the diabetic rats before the administration of 1400W. 266 X. Cheng, C.C.Y. Pang / European Journal of Pharmacology 484 (2004) 263–268

I Ctmng.Cr Pong Fargpeau Aursol af Ptarunnlogy 454 (004)241-168 2M行 of cnrdiac cells (Ceriella et nl.,2002)The expression of and mean circulatory filling pressure of anesthetised mts eNOS,in comtrast to that of iNOS,is decreased in retinal (Ng and Pang,1998).An important mechanism by which vascular codothelial cells incubated with high glucose and nitric oxide mediates vasodilatalion is through the inhibition glycated proteins (Chakravarthy et al.19981.There is also of noradrenaline-modialod vasoconstriction (Zanzinger et a indication that,in addition lo causing ular damage.an a.1941 overproduction of nitric oxide thtough iNOS indudion can In summary.the present study is the first lo demonstrate case eardiovascular depression.Indood,hypotersion in that the seleetive inhibition of iNOS by 1400W inereases lpopolysaecharide-induced sopticaemin is associalod with c-adrenocepor-malined constriction in arterial resistanee NOS induction,and the blocknde of iNOS by 140W in as well as cmacitaree vessels at the aeue phnse of diabetes this eondition reversed hypotension (Wray et al.1998)and Our findings suggest that seleetive inhihitors of iNOS may augmented the peripheral vascular resistance and cardiac be a viable therapeutic opticn in restoring vascular contrac cutput (Cheng et al.,2003b).It is therefore imperative to tion in diabetes. determine if the blockade of iNOS restores vasoconstriction in diabetes. It is well known that a small amount of nitric oxide.as Acknowledgements synthesizod by the constitutive enzymes eNOS and nNOS. is impartant for normal physiological function.To cluckdale Supported by the Heart and Stroke Foundation of RC. the vascular role of iNOS in diabetes.it is therefore and Yukon,and Doctoral award to X.Cheng from the Heart important to selectively inhibit the activity of iNOS.Among the inhibitors of nitric oxide synthases,1400W is by far the and Struke Foundation of Canada.The scientific and most selective for inhibiting the activity of iNOS:its ratio of technical advice of Dr.Brian Rodrigues (University of selectivity for iNOS vs.eNOS is4000 fold,in contrast to British Columbia)is gratefully appreciaked.We acknowl- edge Ms.Su Lin Lim for technical assistance. those of aminoguanidine (II-fuld N iminoethylL omi thine (49-fold:Alderton et al.,2001)and isothioureas (two-to sixfuld:Garvey et al.,1991)In addition,the in vitro potency of 1400W in inhibiting iNOS is 135 and 19 References times those of aminoguanidine and N'-iminocthyl-tomi- thine,respectively (Alderton et al,2001).There is also Aldemo.WK..Cooper,C.E_Knowks,R.G.2001.Nitrie codide syn. evidence that 1400W is effective in reversing vascular thaasc ainature,function and intibiun.Bouchin.J.357 (P1.3) 59-春15Ru nhnormalities in pathophysiological conditions known to Barddll,AL MacLood KM..2001.Evideace fur inducille nimric-onile be associated with the induction of iNOS.Indeed.n single ymhu星ad图yna4山里3dh围业fn甲 injection (0.3 mgkg s.c.,ED.o dose)of 1400W reversed the o3ctn-dhgo rats.人.Phammacol.上p.TncL2%.252-259 delayed lipopalysaccharide (iNOS)-induced vasculr injury CerieDu,A..Qurliru,L.D'Amio,M,Di Filippu.C.Marfdla.R in rats (Garvey et al.,1997).Furthermore,1400W (3 mg/kg Nappo.P.Berino.1..Rosi.B.Canglono,D.2002 Acte hyger zlyccme indeces sitrocyrsine fomation annd tpopooss it perfasod followed by 3 mp/kg/h iv.)restored the blood pressure of heart frum rat.Di hte 51,1006-1082. tats with lipopolysuccharide-indueed endotoxic shock,and Chakrvanty.U.layes,R.Gi.Sar.A.W.MeAuley.E.Archer.DB. the drug did not affeet the blood pressure of comtrol rats I9月线.Curluive silrie uile syulne esprsaut i田ctiaal vio (Wrny t al.1998;Chang et al,20036). In the present study,1400W treatment restored the mnd predacts Dibetex 4),045-952. Cheng.X..Learg.S.W.Lin.S.L..Pane.CC.Y.2003a.Anematod ane effiency (maximum response)and insignificnntly incresed u山6 tion in wu进kiUn2bh2山alr:plucm-a the potency (reduced EDse)of menn anerial pressure dnred dishetex Fir J Phxrool.1K.299-t04 response to noradrenaline,and it also significanly incresed Cheng.X.,Leutg S.W.Lo.L..Pang.C.C.Y,2003b.Selctive vessus the mean circulatory filling pressure respoose to a high dose n国-dectiv wappreaien of aitri家oide wyrtheoe国yiotal hamno of noradrenaline.Furtherore.1400W did not affect any dynomics in rars wrh or withou LPS-irdicod endotaxcaome.Nyuryt Sdeniolebeng Ardh.Phanskel.367,372-379. teasured variables in the control rals.This ability of 1400W f可y小M,aPD,i.c nf espermmbl divhetor,fhod depn- to sclctively restore vascular response to noradrenaline in ￥通ard gencti obesry on th2 sersiivty of,imed rats to aunooom the diabctic rats shows that overproduction of nitric oxide wB红1.%ac52,219-234 by iNOS plays an important role in depressing o-adreno- Girten.A C.1903.Venons retem.In:Ilamihor,W.I.Dow.P.(lds 1 opor-modised arterial and venous constriction at the acue Hu book of Paysiolky.vel.IL Arctioun Paysiokpictl Socity. Wanhingoor.0C甲1所-111 phase of diabels.This interpretation is in line with well- Giuyen.A.C.Polizo,D.Amsorg G.G,1954.Mea circalation filling known clinical snd experimental observations tht trtric esse m8 d immedately afer cessan3 n oe heart pom写heAm人 oxide isa potent and efficacicus ditr of arterial resistunce Ful179.201-207. as well as capacitance vessels.Furthermore.the exogenous Garvey EP.Oplinger.JA..Tanory.G1.Sherran PA.Pawkr.M. Marshal.S.Hamton.MF.Poith.JE.Furine.E.S.1994.Potert supply of nitric oxide through the administration of nitro- ndclodiv ikibilion of hanen aitrie oxak:sythao Innibilin by vasodilators,such as acetylpenicillamine or sodium nitro- nonvmio acid inthiotras I.Iol Chem 204.20664-21676 prusside,has been shown to reduce mean arterial pressure Giuver.EP.,Opliager.JA.Farfne.ES..Kift RJ.,Laszo,F.Whiuk

of cardiac cells (Ceriello et al., 2002). The expression of eNOS, in contrast to that of iNOS, is decreased in retinal vascular endothelial cells incubated with high glucose and glycated proteins (Chakravarthy et al., 1998). There is also an indication that, in addition to causing cellular damage, an overproduction of nitric oxide through iNOS induction can cause cardiovascular depression. Indeed, hypotension in lipopolysaccharide-induced septicaemia is associated with iNOS induction, and the blockade of iNOS by 1400W in this condition reversed hypotension (Wray et al., 1998) and augmented the peripheral vascular resistance and cardiac output (Cheng et al., 2003b). It is therefore imperative to determine if the blockade of iNOS restores vasoconstriction in diabetes. It is well known that a small amount of nitric oxide, as synthesized by the constitutive enzymes eNOS and nNOS, is important for normal physiological function. To elucidate the vascular role of iNOS in diabetes, it is therefore important to selectively inhibit the activity of iNOS. Among the inhibitors of nitric oxide synthases, 1400W is by far the most selective for inhibiting the activity of iNOS; its ratio of selectivity for iNOS vs. eNOS is>4000-fold, in contrast to those of aminoguanidine (11-fold), N5 -iminoethyl-L-orni￾thine (49-fold; Alderton et al., 2001) and isothioureas (two- to sixfold; Garvey et al., 1994). In addition, the in vitro potency of 1400W in inhibiting iNOS is 135 and 19 times those of aminoguanidine and N5 -iminoethyl-L-orni￾thine, respectively (Alderton et al., 2001). There is also evidence that 1400W is effective in reversing vascular abnormalities in pathophysiological conditions known to be associated with the induction of iNOS. Indeed, a single injection (0.3 mg/kg s.c., ED50 dose) of 1400W reversed the delayed lipopolysaccharide (iNOS)-induced vascular injury in rats (Garvey et al., 1997). Furthermore, 1400W (3 mg/kg followed by 3 mg/kg/h i.v.) restored the blood pressure of rats with lipopolysaccharide-induced endotoxic shock, and the drug did not affect the blood pressure of control rats (Wray et al., 1998; Cheng et al., 2003b). In the present study, 1400W treatment restored the efficacy (maximum response) and insignificantly increased the potency (reduced ED50) of mean arterial pressure response to noradrenaline, and it also significantly increased the mean circulatory filling pressure response to a high dose of noradrenaline. Furthermore, 1400W did not affect any measured variables in the control rats. This ability of 1400W to selectively restore vascular response to noradrenaline in the diabetic rats shows that overproduction of nitric oxide by iNOS plays an important role in depressing a-adreno￾ceptor-mediated arterial and venous constriction at the acute phase of diabetes. This interpretation is in line with well￾known clinical and experimental observations that nitric oxide is a potent and efficacious dilator of arterial resistance as well as capacitance vessels. Furthermore, the exogenous supply of nitric oxide through the administration of nitro￾vasodilators, such as acetylpenicillamine or sodium nitro￾prusside, has been shown to reduce mean arterial pressure and mean circulatory filling pressure of anaesthetised rats (Ng and Pang, 1998). An important mechanism by which nitric oxide mediates vasodilatation is through the inhibition of noradrenaline-mediated vasoconstriction (Zanzinger et al., 1994). In summary, the present study is the first to demonstrate that the selective inhibition of iNOS by 1400W increases a-adrenoceptor-mediated constriction in arterial resistance as well as capacitance vessels at the acute phase of diabetes. 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