Br. J. Pharmaol. (1994), 113, 1275-1280 f;." Mamillan Press Ltd, 1994 The relative importane of nitri oide and nitri oide-independent mehanisms in aetylholine-evoked dilatation of the rat mesenteri bed Sarah J.W. Parsons, *Anthony Hill, Gareth J. Waldron, 'Franes Plane & Christopher J. Garland Department of Pharmaology, University Walk, Bristol BS8 ltd and *Department of Physiology and Pharmaology, University of Southampton, Southampton S09 3TU 1 The relative ontribution of nitri oide (NO) to aetylholine-indued smooth musle relaation was investigated in the rat perfused mesenteri vasulature and in isolated segments of seond, third and fourth order arterial branhes. 2 The EC50 values and maimal relaation to aetylholine were not signifiantly different in the sequential arterial branhes, being approimately 0.05 JiM and 85%, respetively. 3 The NO synthase inhibitor L-NW-nitro-L-arginine methyl ester (L-NAME; 100 pm) redued aetylholine-evoked endothelium-dependent dilatation and relaation in the perfused mesenteri bed and in isolated arterial segments. The maimum response to aetylholine in both preparations was redued by between 35% to 40% while the EC", values were inreased by 5-6 fold. L-NAME had no effet on basal smooth musle tone in either ase. 4 In ontrast, endothelium-dependent dilatation of the perfused mesenteri bed evoked by A23187 (0.002-20 nmol), was unaffeted by eposure to L-NAME. The ECM values and maimal responses eliited by A23187 (20 nmol) before and after eposure to L-NAME were 0.96 ± 0.5 nmol and 67.0 + 7.0% (n = 4), and 0.7 ± 0.4 nmol and 70.0 ± 5.0% (n = 4; P > 0.01), respetively. 5 Perfusion of the isolated mesenteri bed with raised K+-Krebs buffer (25 mm) had no effet on basal tone, but redued the amplitude of both aetylholine- and A23187-evoked dilatation. The maimum responses to aetylholine (2 jlmol) and A23187 (20 nmol) were redued from 67.5 ± 7.3% and 65.4±8.2% to 18.9±11.0% (n=5; P<0.01) and 13.5±12.0% (n=4; P<0.01), respetively. 6 Eposure of the mesenteri bed to L-NAME in the presene of raised K+-Krebs further redued the maimal response eliited by aetylholine to only 8.9 ± 2.8% (n =4; P< 0.01). 7 These results indiate that aetylholine-evoked vasodilatation of the rat mesenteri vasulature is mediated by both NO-dependent and -independent mehanisms. The relative ontribution made by these mehanisms does not appear to differ in sequential branhes of the mesenteri artery. In ontrast, A23187-evoked vasodilatation appears to be mediated predominantly by a NO-independent mehanism whih is sensitive to inreases in the etraellular potassium onentration and may reflet the ation of endothelium-derived hyperpolarizing fator (EDHF). Keywords: Aetylholine; nitri oide; rat mesenteri bed; endothelium; EDHF; Nnitro-L-arginine methyl ester (L-NAME) Introdution Endothelium-derived relaing fator (EDRF) has been identified as nitri oide (NO), or a losely related moleule, whih is synthesized from L-arginine by the enzyme NO synthase (reviewed by Monada et al., 1991). Strutural analogues of L-arginine suh as Nnitro-L-arginine (L- NOARG) and Nnitro-L-arginine methyl ester (L-NAME) have been used to blok this pathway and inhibit relaation mediated by the release of endothelium-derived NO (Rees et al., 1989). However, in several blood vessels, suh as the porine oronary artery and rabbit femoral artery, endothelium-dependent relaations are not inhibited by NO synthase inhibitors, indiating that other endothelium-derived fators, distint from NO, may ontribute to the loal regulation of smooth musle tone (Chen et al., 1988; Taylor & Weston, 1988; Nagao & Vanhoutte, 1992; Plane et al., 1992). Endothelium-dependent relaation evoked by agents suh as aetylholine is aompanied by smooth musle hyperpolarization. Although eogenous NO an evoke smooth musle hyperpolarization in some vessels under ertain onditions, there is now onsiderable evidene that a separate endothelium-derived hyperpolarizing fator (EDHF) is also ' Author for orrespondene. released and ontributes to redutions in smooth musle tone, aounting for the relaation not mediated by NO (Chen et al., 1988; Tare et al., 1990; Garland & MPherson, 1992; Nagao & Vanhoutte, 1992). Furthermore, when endothelium-dependent hyperpolarization is abolished by inreased etraellular potassium onentration, the remaining relaation is ompletely bloked by inhibitors of NO synthase (Nagao & Vanhoutte, 1992; Waldron et al., 1993). The relative ontribution of NO and EDHF to endothelium-dependent relaation appears to vary with both different vessels and agonists (Palmer et al., 1988; Rees et al., 1989; Plane et al., 1992). A omparison of various isolated arteries from the rat indiates that the NO-independent omponent of relaation may be more prominent in small arteries. In ontrast, NO is probably the predominant fator released from the endothelium in large onduit arteries (Nagao et al., 1992). Several investigations in the miroirulation also suggest a heterogeneous distribution of endothelial ell funtion, with the ontribution made by NO varying along the vasular network (Tang & Joyner, 1992; Hester et al., 1993). In the perfused rat mesenteri bed, aetylholine-evoked relaations have been reported to be ompletely inhibited by L-arginine analogues suh as L-NOARG and L-NAME, suggesting that the redutions in tone are mediated solely by
1276 S.J.W. PARSONS et al. endothelium-derived NO (Moore et al., 1990). However, this ontrasts with studies in isolated arterial segments from this vasular bed, where aetylholine-evoked relaations were found to be largely resistant to NO synthase inhibitors (Garland & MPherson, 1992; Bennett et al., 1992; Waldron et al., 1993). In third order arterial branhes from Wistar- Kyoto rats, both the relaation and hyperpolarization indued by aetylholine were found to be insensitive to L-arginine analogues, suggesting a major ontribution from EDHF (Garland & MPherson, 1992; Waldron et al., 1993). The membrane potential hange evoked by aetylholine, and a substantial omponent of the relaation, are inhibited by 25 mm potassium, a onentration whih has no effet on relaation to eogenous or endothelium-derived NO (Parsons et al., 1991; Plane & Garland, 1993; Waldron et al., 1993). In the absene of a hange in membrane potential, the remaining omponent of the relaation to aetylholine an be bloked by L-NAME. These observations show that inreasing the eternal potassium onentration to 25 mm in the mesenteri irulation an be used to inhibit the tension hange to hyperpolarization, and presumably to differentiate between the relative ontribution made by eah of these mehanisms. In the present study, we have attempted to resolve the apparently onfliting observations made in the perfused mesenteri preparation and in isolated arterial segments. We have investigated the relative importane of NO-dependent and -independent mehanisms in endothelium-dependent dilatation of the isolated, perfused mesenteri bed of the rat to aetylholine and the alium ionophore A23187. In addition, the possibility that the relative ontribution from NO and NO-independent mehanisms varied between seond, third and fourth order branh arteries from this vasular bed was also eamined. Some of these results have been presented in preliminary form to the British Pharmaologial Soiety (Parsons et al., 1993). Methods Rat isolated mesenteri arteries Endothelium-dependent relaations of isolated segments of rat mesenteri arteries were studied as previously desribed (Garland & MPherson, 1992). Briefly, arterial segments (2 mm in length) from either seond, third or fourth order branhes from the superior mesenteri artery were mounted in a small vessel myograph (model 400A, J.P. Trading, Denmark) and tension hanges displayed via a Kipp & Zonen pen reorder. Vessels were allowed to equilibrate under zero fore for 30 min before the onstrution of a passive diameter-tension urve (Mulvany & Halpern, 1977). From this urve the effetive transmural pressure was alulated. The vessels were set at tensions equivalent to those generated at 0.9 times the diameter of the vessel at 100 mmhg. Nonlinear urve fitting of the passive diameter-tension urve was ahieved using a ustom written programme for the IBM PC (NORMALIZE, G.A. MPherson). Vessel diameters at an equivalent transmural pressure of 100 mmhg (D13,0) are given in the tet. After an initial equilibration period of 60 min, tissues were pre-ontrated with a submaimal onentration of noradrenaline (1-1OI1M) and relaed to umulative onentrations of aetylholine (1 nm-30 PM). Following washout, NO synthase inhibitors were pre-inubated in the organ bath for 360 min before the ontration-relaation yle was repeated. Rat isolated perfused mesentery Rat mesentery preparations were isolated as desribed by Moore et al. (1990). Briefly, the mesenteri vasular bed was annulated via the superior mesenteri artery, separated from the intestine, and perfused at 5 ml min-' with warmed (37 C) Krebs buffer whih had been bubbled with 95% 02/CO2. Perfusion pressure was monitored ontinuously by a Bell & Howell pressure transduer onneted to a pen reorder. To indue tone, noradrenaline (150 gm) was added to the Krebs reservoir at a onentration suffiient to inrease perfusion pressure by 890 mmhg, representing approimately 780% of the maimum response. Bolus doses of aetylholine (0.02 nmol-2 pmol), A23187 (0.002-20 nmol) or sodium nitroprusside (0.002 nmol-22tmol) were injeted in a small volume (100 gl) to prevent injetion artifats. In some eperiments, preparations were perfused for 1 min with 3-(3-holamiidopropyl) 1-propanesulphonate (CHAPS; 4.7 mg ml-') to remove the endothelial ell layer. Following eposure to CHAPS, responses to aetylholine were abolished, whereas dilatation evoked by sodium nitroprusside was unaltered. Inhibitors of NO biosynthesis were added to the Krebs reservoir and allowed to perfuse the tissues for at least 30 min before pre-onstrition. Solutions and drugs Tissues were maintained in Krebs buffer of the following omposition (mm): NaCl 119, NaHCO3 25.0, KCl 4.7, MgSO4 1.2, CaC12 2.5, KH2PO4 1.18, disodium EDTA 0.027, and gluose 11.0. Raised K+-Krebs solution (25 mm KCI) was prepared by diret replaement of NaCl with KCl. Drugs used were A23187 (Sigma), aetylholine hloride (BDH), L-NAME (Sigma), L-NOARG (Sigma), noradrenaline bitartrate (arterenol, Sigma) and sodium nitroprusside (Sigma). All drugs were dissolved in Krebs buffer eept A23 187, the stok solution of whih was dissolved in dimethylsulphoide (DMSO). Analysis of data In all eperiments, relaations were epressed as a perentage derease in the indued level of tone. All data are epressed as mean ± s.e.mean. The signifiane between mean values was alulated by Student's t test, with rejetion of the null hypothesis at the 5% level (P< 0.05). In some eperiments, analysis of variane (ANOVA) was used to ompare the effets of L-NAME and raised K+ when used together. In eperiments on isolated arterial segments, EC50 values are epressed as - log M whereas in the perfused preparation, where bolus doses of drugs were added, EC50 values are quoted in lmol. Results Aetylholine-evoked relaation of isolated segments from the mesenteri bed Aetylholine (1 nm-10 LM) evoked dose-dependent relaation in noradrenaline-ontrated segments of seond (D,0, 454±15 = m; n=7), third (D,00=349±18 Lm; n=14) and fourth (D1,o = 316 ± 12 tm; n = 6) order branhes of rat mesenteri arteries. The EC50 values and maimal relaation evoked by aetylholine were not signifiantly different between the arterial segments from the different branhes (Table 1). Conentration-response urves for aetylholineevoked relaations in the different arterial branhes are shown in Figure 1 (a, b and ). Pre-inubation of tissues with L-NAME (100I1M; 30min) did not alter the resting tension of any of the segments from the different arterial branhes. However, following eposure to L-NAME, aetylholine-evoked relaations were depressed to a similar etent in eah arterial branh (Figure 1). In eah ase there was a 5 to 6 fold inrease in the ECm onentration and the maimal response was redued by approimately 40% (Table 1). Inreasing the inubation time with
ACETYLCHOLINE-EVOKED RELAXATIONS 1277 Table 1 Comparison of % maimal relaation of indued tone (RP,.) and ECv values (-log M) for aetylholine in isolated arterial segments in the presene and absene of NG-nitro-L-arginine methyl ester (L-NAME, 100 1M) Branh 2nd 3rd 4th D100 (.Lm) 454 ± 15 349 ± 18 316 ± 12 Control R., 88.3 ± 2.3 80.6 ± 4.2 84.7 ± 5.0 Control EC50 7.15 (7.34-6.96) 7.25 (7.57-6.97) 7.44 (7.76 ± 7.11) L-NAME (100 JAM) R. 58.0 ± 15.5 49.2 ± 12.0 57.7 ± 10.4 L-NAME (100 FM) ECu0 6.72 (6.86-6.57) 6.74 (6.98-6.49) 6.83 (7.25-6.41) a o 4 ' 60-10 b o 4 r._ I-1 C 0-8-6 2.2 4 60 - -9-8 -7 log [AChl M 3-9 -8-7 log [ACh] M -6-5 -6- -5 L-NAME to 60 min did not further depress the aetylholineevoked responses (data not shown). Indomethain (10 ytm) was without effet on aetylholineevoked relaations in the presene or absene of L-NAME (n = 3). Effet of L-NAME on endothelium-dependent dilatation of the isolated perfused mesenteri bed Bolus injetions of aetylholine (0.2 nmol- 2 mol), evoked transient, dose-dependent dereases in perfusion pressure of tissues pre-onstrited with noradrenaline (150ItM). These responses were abolished following removal of the endothelium with CHAPS. The maimal response evoked by aetylholine (2 pmol) was 77.2 ± 7.6% (n = 12) relaation of indued tone and the EC50 value was 32.7 ± 13.1 nmol (n= 12). When added to tissues pre-onstrited with noradrenaline, L-NAME (100 pm) and L-NOARG (10011M) aused a small, rapid, endothelium-dependent inrease in perfusion pressure of 11.8 ± 2.4 mmhg (n = 4), whih then delined to the previous level within 8-O min. In the absene of indued tone, infusion of the L-arginine analogues for up to 30 min had no effet on basal perfusion pressure, and did not enhane the ontrations evoked by noradrenaline. Aetylholine-evoked responses were signifiant depressed in 80% of preparations following eposure to the NO synthase inhibitors. In the remainder, relaation was not modified. In the tissues in whih NO synthase inhibitors redued relaation, the maimum response to aetylholine (2 imol) was redued by 35.4 ± 3.0% (n = 6; P<0.01). A similar redution was observed in the presene of L-NOARG. Dose-response urves for aetylholine-indued dilatation in the presene and absene of L-NAME are shown in Figure 2. Removal of L-NAME from the Krebs buffer did not restore aetylholine-evoked dilatation over the 45 min reovery period of the eperiments, although responses were partially restored by the infusion of 1I00 JM L-arginine (71% of maimal response; n = 2). Bolus injetions of A23187 (0.002-20 nmol) also evoked transient, dose-dependent redutions in the perfusion pressure of noradrenaline-onstrited preparations whih were abolished by removal of the endothelium. The maimal response evoked by A23187 (0.02 limol) was 65.44 ± 8.2% (n = 4) relaation of indued tone and the ECm value was 0.96 ± 0.48 nmol (n = 4). However, in ontrast to aetylholine, A23187-evoked responses were not depressed and in some ases were inreased, by prior eposure to L-NAME (100 gm) for up to 60 min. The maimal response evoked by A23187 in the presene of L-NAME was 68.14 ± 5.1% (n =4; P>0.05) and the ECu0 value was 0.7 ± 0.4 nmol 8 I -10 inn -9-8 -7 log [AChl M -6-5 Figure 1 Mean onentration-response urves for aetylholineevoked relaation in isolated segments of seond (a), third (b) and fourth () order branhes of the rat mesenteri bed, in the presene (0) and absene (0) of Nnitro-L-arginine methyl ester (100 #M). Points are the mean with s.e.mean from 3 or 4 separate eperiments, *P<0.01.
1278 S.J.W. PARSONS et al. (n =4; P> 0.05). Figure 3 shows dose-response urves for A23187-indued relaation of noradrenaline-onstrited tissues in the presene and absene of L-NAME. Indomethain (10 M) was without effet on responses to either aetylholine or A23187, in either the presene or the absene of L-NAME (data not shown). The nitrovasodilator agent, sodium nitroprusside (0.002-20 nmol), evoked dose-dependent redutions in noradrenaline-indued tone, and these relaations were unaffeted by removal of the endothelium or eposure to L-NAME (100 tm; 30 min). The maimal response evoked by sodium nitroprusside in the presene and absene of L- NAME was 77.5 ± 3.9% and 79.0 ± 5.0% (n = 3; P>0.01). Effet of raised K+ Krebs buffer on endothelium-dependent dilatation of the isolated, perfused mesenteri bed Perfusion of isolated mesenteri preparations with raised K+- Krebs buffer had no effet on basal perfusion pressure and did not enhane noradrenaline-evoked onstrition. However, in the presene of raised K+-Krebs buffer, the dilatation of noradrenaline-indued tone evoked by aetylholine or A23187 was signifiantly redued. In the presene of raised K+-Krebs buffer, the responses to low doses of aetylholine were not signifiantly inhibited, but the maimal response was redued from 77.2 ± 9.2 to 18.9 ± 11.1% (n = 5; P<0.01) ompared to ontrol. Subsequent eposure to L-NAME (100 pm), in the ontinued presene of raised K+-Krebs buffer, did not further depress the aetylholine-evoked dilatation (maimal response 8.9 ± 2.8%; n = 4; ANOVA). Dose-response urves for aetylholine-evoked responses in the presene of raised K+-Krebs buffer, alone or together with L-NAME, are shown in Figure 4. In the presene of raised K+-Krebs buffer, the responses to all doses of A23187 (0.002-20 nmol) were signifiantly inhibited by 890%, ompared to ontrol responses in normal Krebs buffer. The maimal response to A23 187 (20 nmol) in the presene of raised K+-Krebs buffer was 0 20 0-0 " 4 6 8 0.01 0.01 0.1 1 10 100 Dose ACh (nmol) 1000 10000 Figure 2 Mean dose-response urves for aetylholine-evoked dilatation of the rat isolated perfused mesenteri bed pre-onstrited with noradrenaline (1-10 sm). Points show dilatation in the presene (@) and absene (0) of Nnitro-L-arginine methyl ester (100pM), and are the mean with s.e.mean from 6 separate eperiments. 0.01 0.1 1 10 100 Dose ACh (nmol) 1000 10000 Figure 4 Mean dose-response urves for aetylholine-evoked dilatation of the rat isolated perfused mesenteri bed pre-onstrited with noradrenaline (1-1OIM). Points show dilatation in the absene (0) and presene of 25 mm K+-Krebs buffer (M), or 25mM K+- Krebs buffer and Nnitro-L-arginine methyl ester (100 tm; 0). Points are the mean with s.e.mean from 4 separate eperiments, *P<0.01. o 4 2 o 4 '- 6 0-8~ C o 4 r._ ' 0 8.60 8~ 0.001 0.01 0.1 1 10 100 Dose A23187 (nmol) Figure 3 Mean dose-response urves for A23187-evoked dilatation of the rat isolated perfused mesenteri bed pre-onstrited with noradrenaline (1-10 pm). Points show dilatation in the presene (0) and absene (0) of Nnitro-L-arginine methyl ester (100 pm), and are the mean with s.e.mean from 4 separate eperiments, *P<0.01. 0.001 0.01 0.1 1 Dose A23187 (nmol) 10 100 FIgure 5 Mean dose-response urves for A23187-evoked dilatation of the rat isolated perfused mesenteri bed pre-onstrited with noradrenaline (1-101&M). Points show dilatation in the absene (0) and presene of 25 mm K+-Krebs buffer (M), and are the mean with s.e.mean from 4 separate eperiments, *P<0.01.
ACETYLCHOLINE-EVOKED RELAXATIONS 1279 13.5 ± 11.9% (n =4; P< 0.01). Dose-response urves for A23187 in normal and raised K+-Krebs buffer are shown in Figure 5. Relaations evoked by the nitrovasodilator, sodium nitroprusside, were unaltered in the presene of high K+- Krebs buffer (results not shown), and the ylo-oygenase inhibitor, indomethain (10 ILM) did not alter responses to either aetylholine or A23187. Disussion These data indiate that endothelium-dependent dilatation of the rat mesenteri vasulature is mediated by both NOdependent and -independent mehanisms. The relative ontribution made by these separate mehanisms appears to vary with aetylholine or A23187, and as in studies of other arteries, the NO-independent mehanisms appear to make a larger ontribution in the responses evoked by A23 187 (Nagao & Vanhoutte, 1992; Plane et al., 1992). In the rat perfused mesenteri vasulature, L-NAME and L-NOARG attenuated the reversal of noradrenaline-indued tone evoked by the musarini agonist, aetylholine, reduing the maimum response by around 35% and shifting the onentration-response urve to the right. However, in the presene of raised K+-Krebs buffer, the maimum response to aetylholine was redued by 72% and, subsequent eposure to L-NAME almost abolished the relaation to aetylholine. These results are in agreement with previous observations on isolated third order branhes of the mesenteri bed, whih indiated that aetylholine-evoked relaation was mediated by the release of NO and, at higher onentrations, a hyperpolarizing fator distint from NO and inhibited by 25 mm potassium (Waldron et al., 1993). As in the isolated segments, the potassium-sensitive pathway appears to make a very signifiant ontribution to aetylholine-evoked smooth musle relaation. In ontrast, A23187-evoked dilatation was not inhibited by eposure to the L-arginine analogue L-NAME, indiating that NO is probably not important in smooth musle relaation to this agent in the rat mesenteri vasulature. Interestingly, at some doses of A23187 the dilatation was inreased following eposure to L-NAME. We do not have an eplanation for this observation and it was not investigated further. However, as with aetylholine, the relaation to A23187 was signifiantly redued in the presene of a raised onentration of potassium, suggesting that A23187 may indue relaation by a mehanism whih is dependent on inreased potassium ondutane. Previous studies have also demonstrated that fators distint from NO ontribute to endothelium-dependent relaation in this vasular bed. For eample, methylene blue and oyhaemoglobin are muh less effetive in bloking aetylholine-evoked dilatation in the perfused mesentery, in onentrations whih ompletely inhibit equivalent responses in other arteries suh as the rabbit aorta (Khan et al., 1992). In addition, oyhaemoglobin and methylene blue were found to inhibit ompletely relaation to low onentrations of aetylholine in the rat mesenteri bed, but were less effetive against higher onentrations, when up to 58% relaation persisted (Khan et al., 1992). This profile was very similar to the responses to aetylholine obtained in the presene of L-NAME in the present study, and imply that relaation to low onentrations of aetylholine predominantly reflets the release of NO, while at higher onentrations, an additional fator, probably EDHF, makes a major ontribution. The identity of the mediator of NO-independent relaation is unlear although, in some vessels, suh as the rabbit and bovine pulmonary artery, endothelium-dependent relaation is mediated by the release of both NO and produts of arahidoni aid metabolism (Chand et al., 1987; Ignarro et al., 1987). In the rabbit oronary artery, bradykinin-evoked relaation whih persists in the presene of L-NOARG is mediated by prostaylin ating through glibenlamidesensitive potassium hannels (Jakson et al., 1993). A role for prostanoids in the NO-independent responses to aetylholine and A23187 in the rat mesentery is unlikely, as indomethain was without effet in this and a previous study (Garland & MPherson, 1992). L-NAME and L-NOARG did not affet the resting tone in either the perfused mesenteri preparation or in isolated arteries, indiating that the basal release of NO is low in the mesenteri bed. A transient endothelium-dependent ontration was observed when L-NAME was added to perfused preparations stimulated with noradrenaline, whih may suggest that NO release is stimulated during arterial ontration. However, following eposure to the NO synthase inhibitor, noradrenaline-indued ontrations were not enhaned indiating that NO release does not have an important modulatory role in this vasular bed. Another reent study has also reported that L-NAME inreased the basal perfusion pressure in this bed, but only in the presene of inreased etraellular potassium (Adeagbo & Triggle, 1993). These workers suggested that EDHF rather than NO is the priniple regulator of basal perfusion pressure, the ontribution of NO only beoming apparent after the ation of EDHF had been abolished (Adeagbo & Triggle, 1993). In the rabbit aorta, elevated potassium onentrations have been shown to inhibit the relaation to both aetylholine and sodium nitroprusside, by inhibiting the formation and ation of guanosine 3':5'-yli monophosphate (yli GMP) in the vasular smooth musle (Collins et al., 1988). As vasodilatation of the perfused mesenteri bed evoked by the nitrovasodilator, sodium nitroprusside, was unaltered in the presene of 25mM potassium in the mesenteri bed, it is unlikely that this potassium onentration has a signifiant inhibitory ation on the formation or the ellular ations of yli GMP. This is in agreement with other studies whih have also found that this onentration of potassium does not inhi6it either the release or ations of endothelium-derived NO or the smooth musle responses to eogenous NO (Parsons et al., 1991; Plane & Garland, 1993; Waldron et al., 1993). In other studies, higher onentrations of potassium (up to 60 mm) had no effet on the size of relaation to endothelium-derived NO, indiating that inhibition of either the release or the ations of NO are unlikely to aount for the redued relaation to aetylholine and A23187 observed in the presene of 25 mm K+-Krebs buffer (Parsons et al., 1991; Plane et al., 1992; Plane & Garland, 1993). In fat, in the perfused preparation, the presene of 25 mm K+ had little effet on the relaation to the lowest dose of aetylholine, but signifiantly depressed vasodilatation stimulated by higher doses of either aetylholine or A23187. This supports the idea that the release of an additional fator, whih is sensitive to inreases in the etraellular potassium onentration, depends on the level of intraellular alium within the endothelium. If this is the ase, then it appears that EDHF prodution is more sensitive to alium influ than is the synthesis of NO. In a previous study on the perfused mesenteri bed, L- NOARG (200 gm) was found to abolish aetylholine-evoked dilatation (Moore et al., 1990). In ontrast, in our eperiments L-NAME (100 LM) and L-NOARG (100 1M) redued the maimum response to aetylholine in both the perfused preparation and isolated arterial segments by only 35%; This inhibition was not improved by inreasing the onentration of the NO synthase inhibitors to 200 gm, or by inreasing the eposure time to 60 min. Furthermore, in 20% of the perfused preparations, aetylholine-evoked responses were totally resistant to the bloking ation of NO synthase inhibitors. The reasons for the disrepany between these studies is unlear, but in the light of our observations, it does not appear to be due to a variation in the relative ontribution of NO to relaation down the vasular bed, as L-NAME redued aetylholine-evoked relaation to a similar etent in
1280 S.J.W. PARSONS et al. eah of the seond, third and fourth order branhes of the mesenteri artery. In onlusion, these data indiate that in the rat mesenteri vasulature, endothelium-dependent vasodilatation is mediated by the release of both NO and an additional fator whih is probably EDHF. The relative ontribution made by these two mehanisms to the overall relaation appears to vary with different agonists. Relaation to low doses of aetylholine appears to be mediated by NO, whereas higher onentrations ativate an additional mehanism. Relaation to A23187 appears to be independent of NO prodution, and mediated by a potassium-sensitive mehanism alone, implying a major role for EDHF. This work was made possible by finanial support from the Wellome Trust. Referenes ADEAGBO, A.S.O. & TRIGGLE, C.R. (1993). Varying etraellular [K+]: a funtional approah to separating EDHF- and EDNOrelated mehanisms in perfused rat mesenteri arterial bed. J. Cardiovas. Pharmaol., 21, 423-429. BENNETT, M.A., WATT, P.A.C. & THURSTON, H. (1992). Endothelium-dependent modulation of resistane vessel ontration: studies with Nnitro-L-arginine methyl ester and Nnitro-Larginine. Br. J. Pharmaol., 107, 616-621. CHAND, N., MAHONEY, T.P., DIAMANTIS, W. & SOFIA, R.D. (1987). Pharmaologial modulation of bradykinin-, aetylholine- and alium ionophore A23187-indued relaation of rabbit pulmonary arterial segments. Eur. J. Pharmaol., 137, 173-177. CHEN, G., SUZUKI, H. & WESTON, A.H. (1988). Aetylholine releases endothelium-derived hyperpolarizing fator and EDRF from rat blood vessels. Br. J. Pharmaol., 95, 1165-1174. COLLINS, P., HENDERSON, A.H., LANG, D. & LEWIS, M.J. (1988). Endothelium-derived relaing fator and nitroprusside ompared in noradrenaline and K+-ontrated rabbit and rat aortae. J. Physiol., 400, 395-400. GARLAND, C.J. & MCPHERSON, G.A. (1992). Evidene that nitri oide does not mediate the hyperpolarization and relaation to aetylholine in the rat small mesenteri artery. Br. J. Pharmaol., 105, 429-435. HESTER, R.L., ERASLAN, A. & SAITO, Y. (1993). Differenes in EDNO ontribution to arteriolar diameters at rest and during funtional dilation in striated musle. Am. J. Physiol., 256, H146-HI51. IGNARRO, L.J., BYRNS, R.E., BUGA, G.M. & WOOD, K.S. (1987). Mehanisms of endothelium-dependent vasular smooth musle relaation eliited by bradykinin and VIP. Am. J. Physiol., 253, H 1074-H 1082. JACKSON, W.F., KONIG, A.M., DAMBACHER, T. & BUSSE, R. (1993). Prostaylin-indued vasodilation in the rabbit heart is mediated by ATP-sensitive potassium hannels. Am. J. Physiol., 264, H238-243. KHAN, M.T., JOTHIANANDAN, D., MATSUNAGA, K. & FURCH- GOTT, R.F. (1992). Vasodilation indued by aetylholine and by glyeryl trinitrate in rat aorti and mesenteri vasulature. J. Vas. Res., 29, 228. MONCADA, S., PALMER, R.M.J. & HIGGS, E.A. (1991). Nitri oide: physiology, pharmaology and pathophysiology. Pharmaol. Rev., 43, 109-142. MOORE, P.K., AL-SWAYEH, O.A., CHONG, N.W.S., EVANS, R.A. & GIBSON, A. (1990). L-NG-nitro arginine (L-NOARG), a novel, L-arginine-reversible inhibitor of endothelium-dependent vasodilatation in vitro. Br. J. Pharmaol., 99, 408-412. MULVANY, M.J. & HALPERN, W. (1977). Contratile responses of small resistane vessels in spontaneously hypertensive and normotensive rats. Cir. Res., 41, 19-26. NAGAO, T., ILLIANO, S. & VANHOUTTE, P.M. (1992). Heterogeneous distribution of endothelium-dependent relaations resistant to NG-nitro-L-arginine in rats. Am. J. Physiol., 263, H109H1094. NAGAO, T. & VANHOUTTE, P.M. (1992). Hyperpolarization as a mehanism for endothelium-dependent relaation in the porine oronary artery. J. Physiol., 445, 355-367. PALMER, R.M.J., REES, D.D., ASHTON, D.S. & MONCADA, S. (1988). L-arginine is the physiologial preursor for the formation of nitri oide in endothelium-dependent relaation. Biohem. Biophys. Res. Commun., 153, 1251-1256. PARSONS, A.A., SCHILLING, L. & WAHL, M. (1991). Analysis of aetylholine-indued relaation of rabbit isolated middle erebral artery: effets of inhibitors of nitri oide synthesis, Na,K- ATPase and ATP-sensitive K hannels. J. Cereb. Blood Flow Metab., 11, 70704. PARSONS, S., GARLAND, C.J. & PLANE, F. (1993). The role of nitri oide in aetylholine-evoked dilatation of the rat isolated mesenteri bed. Br. J. Pharmaol., 110, 86P. PLANE, F. & GARLAND, C.J. (1993). Differential effets of aetylholine, nitri oide and levromakalim on smooth musle membrane potential and tone in the rabbit basilar artery. Br. J. Pharmaol., 110, 651-656. PLANE, F., PEARSON, T. & GARLAND, C.J. (1992). Differenes between endothelium-dependent relaation to aetylholine and A23187 in the rabbit isolated femoral artery. Br. J. Pharmaol., 107, 201P. REES, D.D., PALMER, R.M.J., HODSON, H.F. & MONCADA, S. (1989). A speifi inhibitor of nitri oide formation from L-arginine attenuates endothelium-dependent relaation. Br. J. Pharmaol., 96, 418-424. TANG, T. & JOYNER, W.L. (1992). Differential role of endothelial funtion on vasodilator responses in series-arranged arterioles. Mirovas. Res., 44, 61-72. TARE, M., PARKINGTON, H.C., COLEMAN, H.A., NEILD, T.O. & DUSTING, G.J. (1990). Hyperpolarization and relaation of arterial smooth musle aused by nitri oide derived from the endothelium. Nature, 346, 69-71. TAYLOR, S.G. & WESTON, A.H. (1988). Endothelium-derived hyperpolarizing fator: a new endogenous inhibitor from the vasular endothelium. Trends Pharmaol. Si., 9, 272-274. WALDRON, G.J., MCPHERSON, G.A. & GARLAND, C.J. (1993). Aetylholine-indued relaation in the rat isolated mesenteri artery: relative importane of smooth musle hyperpolarization Br. J. Pharmaol., 108, 3P. (Reeived Marh 31, 1994 Revised July 21, 1994 Aepted August 18, 1994)