Attenuated 5-Hydroxytryptamine Receptor-Mediated Responses in Aortae from Streptozotocin-Induced Diabetic Rats G.M

Br. J. Pharmacol. (1994), 111, 370-376 '." Macmillan Press Ltd, 1994

Attenuated 5-hydroxytryptamine receptor-mediated responses in aortae from streptozotocin-induced diabetic rats G.M. James, 1W.C. Hodgson, E.A. Davis & 2J.M. Haynes

Department of Pharmacology, Monash University, Clayton, Victoria, Australia, 3168

1 This study was designed to examine further the attenuated contractile responses to 5-hydroxytryptamine (5-HT) previously observed in aortae from diabetic rats. 2 Cumulative concentration-response curves to 5-HT, and the 5-HT receptor agonists, at-methyl 5-HT (a-Me-5-HT, 5-HT21lc agonist), (± )-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, 5-HT21IC agonist) and 5-carboxamidotryptamine (5-CT, 5-HTIA/1B/1D agonist), were examined in endothelium- intact and -denuded aortae from 2-week streptozotocin (STZ)-diabetic and control rats. 3 In endothelium-intact and -denuded aortae from diabetic rats, maximum responses to 5-HT and a-Me-5-HT were significantly reduced compared to those of aortae from control rats. Responses to these agonists were inhibited by the 5-HT21lc receptor antagonist, ketanserin (0.1 jAM). 4 The attenuated responses to 5-HT of aortae from diabetic rats were normalized by chronic insulin treatment of the rats (5 units day-', s.c.), but not by altering the glucose concentration of the bathing fluid. 5 The nitric oxide synthase inhibitor N-nitro-L-arginine (NOLA, 0.1 mM) significantly potentiated responses to both 5-HT and a-Me-5-HT in endothelium-intact aortae. However, the difference between maximum responses of aortae from diabetic and control rats was still evident in the presence of NOLA. 6 Endothelium-intact rings, in the presence of ketanserin (0.1 tiM) and preconstricted with the thromboxane A2-mimetic, U46619 (0.1-0.3jIM), from control and diabetic rats, did not relax to cumulative additions of 5-HT (1 nM-30 jM). 7 Contractile responses to DOI were obtained only in endothelium-denuded aortae, and in endothelium-intact aortae in the presence of NOLA, from control rats. 8 Contractile responses to 5-CT were obtained only in endothelium-denuded aortae from both control and diabetic rats, and in endothelium-intact aortae in the presence of NOLA, from control rats. 9 [3H]-ketanserin binding studies showed that there was no significant change in the affinity or density of [3H]-ketanserin for binding sites in membrane preparations of aortae from control and diabetic rats. 10 These results suggest that 5-HT contracts aortae from rats via 5-HT2,,c receptor activation. However, the simultaneous release of EDRF from endothelial cells in response to 5-HT does not appear to be receptor-mediated. The attenuated contractile responses observed to 5-HT in aortae from 2-week diabetic rats do not appear to be mediated by changes in either endothelial cell function or an alteration in 5-HT receptor affinity or density. Keywords: Diabetes mellitus; rat aortae; endothelium; 5-hydroxytryptamine; N-nitro-L-arginine

Introduction Methods Previous work from our laboratory has shown that contrac- Male Wistar rats (300-420 g) were treated with STZ (60 mg tile responses to the 5-hydroxytryptamine (5-HT) receptor kg-', i.v.) or vehicle (50 mM citrate buffer) under 4% halo- agonists ( ± )-1-(2,5-dimethoxy-4-iodophenyl)-2-aminoprop- thane anaesthesia (2: 1 02/N20). The animals were then ane (DOI) and 5-HT are attenuated in aortic rings from housed in treatment pairs, being allowed free access to food streptozotocin (STZ)-diabetic rats (Sikorski et al., 1993). This and water at all times. Rat body weights and blood glucose latter finding supported the earlier work of Hagen et al. levels were measured on the day of STZ or citrate buffer (1985) and Head et al. (1987). However, the mechanism(s) administration and again after 2 weeks. Only rats displaying behind this abnormality has yet to be elucidated. We have elevated blood glucose levels (> 16 mM, Ames Minilab 1) postulated that functional and/or structural changes of the after 2 weeks were considered to be diabetic. Vehicle control endothelium of vessels from diabetic rats are unlikely to be rats had blood glucose levels of between 3-8 mM when tested responsible for these changes but that a downregulation of at the same time. After 2 weeks, a STZ-induced diabetic and the 5-HT receptor population in aortae from diabetic rats a control animal were killed, and 5 mm rings cut from each may contribute (Sikorski et al., 1993). The aim of the present descending thoracic aorta. Where indicated, endothelial cells study was to identify further the mechanisms underlying the were removed from aortae by rubbing the intimal surface attenuated response to 5-HT receptor agonists of isolated with thin wire. Rings were placed in 15 ml organ baths aortae from 2-week STZ-diabetic rats. This included examin- containing Krebs solution of the following composition (mM): ing the effects of altering the glucose level of the physiolog- NaCl 118.4, KCl 4.7, CaC12 2.5, NaHCO3 25.0, KH2PO4 1.2, ical solution, chronic insulin administration and radioligand MgSO4.7H20 1.2 and glucose 11.1 Where indicated, high- binding studies. glucose Krebs consisted of Krebs solution with 30 mM glucose. Physiological solutions were maintained at 37°C, and bubbled with 5% CO2 in 02. Rings were placed under 10 g resting tension throughout the experiment, as this approx- Author for correspondence. imates the physiological wall tension (Fulton et al., 1991; 2 Present address: Department of Medicine, Repatriation Hospital, Sikorski et al., 1993). After 1 h equilibration, a submaximal Bundoora, Victoria, Australia, 3083. concentration of phenylephrine (0.3 fiM) was added to the ATTENUATED 5-HT CONTRACTILE RESPONSES IN DIABETES 371 bath. At the plateau of contraction, acetylcholine (10 LM) Binding analysis was added. The presence of functional endothelial cells was indicated by subsequent relaxation. Following this procedure, Data from both saturation and competition studies were a single cumulative concentration-response curve was con- analysed initially using the computer programme EBDA structed, on each tissue, either in the presence or absence of (McPherson, 1983), to obtain estimates of BmaX KD and Ki antagonists/inhibitors. NOLA was allowed to equilibrate for and then LIGAND (Munson & Rodbard, 1980), to obtain 15 min before the addition of any agonist. Ketanserin and final parameter estimates ( s.e.mean) of B.., KD and Ki. indomethacin were allowed 30 min equilibration. Drugs Insulin treatment The following drugs were used: acetylcholine chloride (Sigma), Where indicated, STZ-treated rats were injected with a single 5-carboxamidotryptamine maleate (Glaxo), (± )-l-(2,5-dime- daily dose of Lente MC insulin zinc suspension (5 units thoxy4-iodophenyl)-2-aminopropane hydrochloride (Research day-', s.c.) commencing on the second day after STZ ad- Biochemicals Inc.), 5-hydroxytryptamine creatine sulphate ministration (Hodgson & King, 1992). (Sigma), iproniazid P04 (Sigma), indomethacin (Sigma), insulin (Lente MC zinc suspension, CSL-Novo), ketanserin tartrate (Janssen), x-methyl-5-hydroxytryptamine maleate (Research Binding studies Biochemicals Inc.), methysergide maleate (Sandoz), N-nitro- L-arginine (Sigma), phenylephrine HCl (ICN Pharmaceuti- Tissue preparation The remaining segments of thoracic aor- cal), streptozotocin (Sigma), U-0521 (3',4'-dimethyl-2-methyl- tae, obtained from control and diabetic rats at the time of propiophenone; Upjohn), U46619 ((l5S)-hydroxy-1 la,9a- killing, were denuded of endothelial cells (as above) and the (epoxymethano) prosta-5Z, 13E-dienoic acid; Upjohn), 2-(2, tissues stored at - 80C until required. 6-dimethoxyphenoxyethyl) aminomethyl-1, 4-benzodioxane hydrochloride (WB-4101 HC1, Research Biochemicals Inc.). Phenylephrine was dissolved in catecholamine diluent Radioligand binding (0.312 g NaH2PO4 and 0.08 g ascorbic acid per litre of 0.9% (w/v) saline). Ketanserin was dissolved in dimethyl sulphox- Aortae from 8-10 diabetic or control rats were weighed and ide. Indomethacin was dissolved in 1% NaCO3. U46619 was suspended in 10 ml ice-cold Krebs phosphate buffer (KPB) of prepared in ethanol, which was evaporated under a stream of composition, (mM): NaCl 119, KCl 4.8, MgSO4 1.2, CaCl2 N2, and redissolved in distilled water. Further dilutions of 2.5, glucose 11.7, NaH2PO4 1.3 and Na2HPO4 8.7. The tissues these drugs, and stock solutions of all other drugs (except for were crudely chopped before being homogenized (12 s) in a phenylephrine), were prepared in distilled water. polytron (Kinematica PT 10-35). The chopped tissues were For binding experiments WB-4101, methysergide and 5-HT then homogenized in a glass homogenizer (4-6 strokes) prior were diluted in KPB containing 0.4% ascorbic acid, 40 tiM to centrifugation (30 g, 7 min, 40C; Sorvall model RC5C), iproniazid and 120 !LM of the catechol-O-methyl transferase passed through a double layer of gauze, centrifuged again inhibitor, U-0521 (Henseling, 1983). (45,000 g, 15 min, 4C), and the supernatant removed. The membrane pellet was resuspended (10 ml KPB) and allowed Statistics to equilibrate (O min, 37°C, to promote the breakdown of endogenous catecholamines). The suspension was cooled in Comparisons between responses in aortae from STZ-treated an ice-bath and spun again (45,000 g, 15 min, 4°C). The and control rats were made by two-way analysis of variance membrane pellets were resuspended in KPB (8-10 ml). Mem- (ANOVA). Multiple comparisons were analysed by Tukey's brane binding to [3H]-ketanserin (specific activity 60.0 Ci test. Values shown are means ± s.e.mean. Shifts between mmol', NEN Research Products) was carried out by incu- linear portions of concentration-response curves were cal- bating 100ILI of tissue homogenate (30-63 jg protein) with culated by the Apple Ile COMPAR programme. ECm values [3HJ-ketanserin for 10 min (37°C) in a final volume of 200 gIL. were determined from the Em.. of each individual curve [3H]-ketanserin reaches binding equilibrium with 5-HT2-re- (Apple Ile LINUS) and the geometric mean (i.e. mean of the ceptors within 5 min and remains stable for at least a further log values) determined. In all cases, statistical significance is 15 min (Leysen et al., 1982). Methysergide (30 gM) was used indicated by P <0.05. to estimate non-specific binding (as previously described by Leysen et al. (1982)). The level of specific binding was approximately 30%. The reaction was stopped with 5 ml ice-cold Results KPB, the reaction mixture was then drawn, under vacuum, through Gelman A/E filter papers using a Brandel M-24 cell As shown in Table 1, body weights of 2-week diabetic rats harvester. Filter papers were washed five times with 4 ml were significantly less than those of age-matched controls. In ice-cold KPB and then partially dried under vacuum. The addition, although control rats gained weight in the 2 weeks filter papers were placed in scintillation vials, 4 ml PCS following vehicle injection, weights of diabetic rats decreased (Amersham) was added and c.p.m. were recorded over a significantly. The reduction in body weight observed in 5.5 min period. Protein determinations were made by the diabetic rats was not evident in insulin-treated diabetic rats. modified Lowry method (Schacterle & Pollack, 1973). Blood glucose levels of diabetic rats 2 weeks after STZ- treatment were elevated significantly compared to those of Saturation studies control rats and compared to their pre-injection levels. How- ever, blood glucose levels of insulin-treated diabetic rats were For saturation studies eight concentrations of [3H]-ketanserin no different from those of control rats (Table 1). (0.05-28 nM) were run in triplicate. The dry weights of aortae from diabetic rats were significantly less than those of control rats and insulin-treated diabetic rats (Table 1). Competition studies As shown in Figure 1, 5-HT (0.1 tM-0.1 mM) produced concentration-dependent contractile responses in aortae from For competition studies eight concentrations of methysergide, both control and diabetic rats. Removal of the endothelium WB-4101 and 5-HT (0.1 nM-0.1 mM) were used to displace significantly potentiated maximum responses to 5-HT in aor- [3H]-ketanserin (2.4 nM) from binding sites. Competition stu- tae from both control and diabetic rats (Table 2). Maximum dy points were run in triplicate. responses to 5-HT of endothelium-intact and -denuded aor- 372 G.M. JAMES et al.

Table 1 Body weights, blood glucose levels and aortic ring dry weights of control and streptozotocin (STZ)-treated rats Body weights Blood glucose levels Aortic ring dry weights (g) (mM) (mg) (n) Initial Final Initial Final With E Without E Control 59 353 ± 3 399 ± 4t 5.5 ± 0.2 6.3 ± 0.1 1.98 ± 0.02 1.97 ± 0.03 STZ 62 343 ± 3 308 ± 4*t 5.7 ± 0.2 22.0 ± 0.6*t 1.78 ± 0.03* 1.84 ± 0.02* STZ/insulin 6 328±8 366 ± 4*t§ 6.2 ± 0.4 5.0±0.3§ 2.02 ± 0.02§ 2.01 ± 0.04§

Initial measurements were made at the time of STZ or vehicle injection, and final measurements made 2 weeks later. E = endothelial cells. *Significantly different from corresponding value in control group, P<0.05 ANOVA. tSignificantly different from initial value in same treatment group, P<0.05 ANOVA. §Significantly different from corresponding value in diabetic group, P<0.05 ANOVA.

(2.6 ± 0.2 g without indomethacin; 1.9 ± 0.1 g with indomethacin, P < 0.05). In the presence of ketanserin (0.1 M), 5-HT concentration- response curves were shifted to the right in endothelium-denuded aortae from both control and diabetic rats by factors of 42 (26, 71, 95% confidence limits (c.l.); 30 degrees of freedom (d.f.)) and 29 (15, 54, 95% c.l.; 30 d.f.), respectively (Figure 2b). In the presence of ketanserin (0.1 g.M), endothelium-intact aortae, from both groups of rats, did not respond to increasing concentrations of 5-HT (n = 6, data not shown). responses to 5-HT (10 mM) were obtained C However, gmM-0.3 0 in endothelium-intact aortae, from both control and diabetic C -7 -6 -5 -4 rats, in the combined presence of ketanserin (0.1 gM) and C4 NOLA (0.1 mM). In the presence of these two agents, max- b imum responses of aortae from diabetic rats were still ,x 5 significantly reduced compared to those of aortae from control rats (P<0.05, ANOVA; Figure 2a). 4 DOI (10 nM- 10 4M) failed to produce contractile responses in aortae (with or without endothelial cells) from 3 T diabetic rats (Figure 3), and produced only small responses in endothelium-intact aortae from control rats (Figure 3a). However, marked contractile responses were obtained in 12- endothelium-denuded aortae (Figure 3b), and in endothelium-intact aortae in the presence of NOLA, from control 2 0 -, rats (Figure 3a). a-Me-5-HT (0.1 M-0.1 mM) produced concentration-de- -7 -6 -5 -4 pendent constriction in aortae from both control and diabetic rats (Figure 4). Maximum responses to a-Me-5-HT were log [5-Hydroxytryptaminel (M) significantly potentiated by the addition of NOLA to endothelium-intact aortae from both control and diabetic rats Figure 1 Responses of rat aortic rings to 5-hydroxytryptamine (P< 0.05, ANOVA, Figure 4a). Maximum responses were (n=5-6). (a) Diabetic (0-0; 0-0 30 mm glucose), insulin- significantly attenuated in aortae from diabetic rats, in the treated diabetic (A) and control (0-0; 0-0 30mm glucose) absence or presence of NOLA, compared to those obtained with endothelium. (b) Diabetic (U-U; U-- 30mm glucose), in aortae from control rats (P< 0.05, ANOVA, Figure 4a). insulin-treated diabetic (A) and control (@-@; *--* 30mM In the presence of ketanserin (0.1 a-Me-5-HT concentra- glucose) without endothelium. Values indicate mean ± s.e.mean. gM), tion-response curves were shifted to the right in endothelium- denuded aortae from both control and diabetic rats by factors of 422 (210, 1046, 95% c.l.; 36 d.f.) and 257 (92, 1017, 95% c.l.; 36 d.f.), respectively (Figure 4b). However, in the pre- tae from diabetic rats were significantly attenuated compared sence of ketanserin (0.1 tM) endothelium-intact aortae, from to those of control rats (Table 2). Maximum responses to both groups of rats, did not respond to increasing concentra- 5-HT of aortae from insulin-treated diabetic rats were not tions of a-Me-5-HT (n =4, data not shown). significantly different from those of aortae from control rats 5-CT (0.1 sM-0.3 mM) produced concentration-dependent (Table 2). When experiments were performed on aortae contractile responses in endothelium-denuded aortae from bathed in 30 mM glucose Krebs solution, maximum responses different control and diabetic rats (Figure Sb) and in endo- of endothelium-denuded aortae from control rats, but not thelium-intact aortae, in the presence of NOLA (0.1 mM), diabetic rats, were significantly reduced compared to those of from control rats (Figure Sa). NOLA had no significant aortae bathed in normal Krebs solution. However, the effect on responses to 5-CT in endothelium-intact aortae difference between responses of aortae from control and from diabetic rats (Figure Sa). Maximum responses to 5-CT diabetic rats was still significant (Table 2). in endothelium-denuded aortae from diabetic rats were In the presence of indomethacin (10pM), maximum res- significantly attenuated compared to those obtained in aortae ponses to 5-HT of endothelium-intact or -denuded aortae from control rats (Table 2). Ketanserin (0.1 M) completely from diabetic rats were still significantly reduced compared to abolished responses to 5-CT (0.1IM-0.3 mM) in endothe- controls (Figure 2). However, a comparison of Figures lb lium-denuded aortae from both control and diabetic rats and 2b shows that maximum responses of endothelium- (n = 3, data not shown). denuded aortae from diabetic rats were significantly lower When the above results were expressed as tension devel- than those obtained to 5-HT in the absence of indomethacin oped per mg of tissue weight, contractile responses to 5-HT, ATTENUATED 5-HT CONTRACTILE RESPONSES IN DIABETES 373

Table 2 Maximum responses and EC5u values for agonists on aortae from streptozotocin (STZ)-treated and control (C) rats with ( + ) and without (-) endothelial cells (E) C+E STZ + E C-E STZ - E 5-HT Maximum (g) 3.06 ± 0.17 1.30 ± 0.07* 4.14 ± 0.24t 2.60 ± 0.16*t EC5u (- logM) 5.54 ± 0.12 5.07 ± 0.19* 6.04 ± 0.25t 5.36 ± 0.21 5-HT/ Maximum (g) 2.72 ± 0.20 0.98 ± 0.15* 3.48 ± 0.09*t 2.45 ± 0.33*t 30 mM glucose EC50 (-logM) 5.97 ± 0.06 5.22 ± 0.15* 6.74 ± 0.28t 5.67 ± 0.15*t 5-HT/insulin Maximum (g) NA 3.17 ± 0.26§ NA 3.56±0.22§ ECu (- logM) 5.85 ± 0.12§ 6.53 ± 0.30t§ DOI Maximum (g) 0.43 ± 0.09 0.04 ± 0.02* 2.14 ± 0.12t 0.08 ± 0.03* EC5u (-logM) 5.59 ± 0.36 NA 7.06 ± 0.16 NA a-Me-5-HT Maximum (g) 2.80 ± 0.21 0.64 ± 0.14* 3.32 ± 0.36 1.40 ± 0.37* EC5u (-logM) 6.30 ± 0.23 5.73±0.09 7.49 ± 0.17t 6.04 ± 0.06*t 5-CT Maximum (g) 0.40 ± 0.16' 0.30 ± 0.23' 3.25 ± 0.07t 1.57 ± 0.23'*t EC5u (-logM) NA NA 4.64 ± 0.22 NA Values are mean ± s.e.mean; n = 5-6. *Significantly different from corresponding value in control group, P<0.05 unpaired t test. tSignificantly different from corresponding value in same treatment group with endothelial cells, P <0.05 unpaired t test. §Significantly different from corresponding value in diabetic group, P< 0.05, ANOVA. NA = not applicable. For other abbreviations, see text. 'As these responses were not maximum, EC5u values have not been calculated.

5,-a 3,1a 4- 2 3. 7"? I I U- __u o--o-, ' -..- 2 1 1 o1 11 6'-- / - -- C o/ C - -6 0 0 .6 0 ._4 ._a b _ _- . C C -8 -7 -6 -5 c) -7 -6 -5 -4 -3 .) C C a) b 4) b Cl) 5, 4-C,) (a 3 a1)C 41) C. 4. C 3. / 2 -

2- 0//zs,^e~~~0/ /r 1. 7 / / Ai --a -- 0 -7 -6 -5 -4 -3 -8 -7 -6 -5 log [5-Hydroxytryptamine] (M) log [DOI] (M)

Figure 2 Responses of rat aortic rings to 5-hydroxytryptamine in Figure 3 Responses of rat aortic rings to (± )- 1-(2,5-dimethoxy-4- the presence of indomethacin, ketanserin,@~~~~or ketanserin and N-nitro- iodophenyl)-2-aminopropane (DOI) alone, in the presence of N- L-arginine (NOLA) combined (n = 5). (a) Diabetic (0-0* indo- nitro-L-arginine (NOLA), indomethacin or indomethacin and NOLA methacin; 0---0 ketanserin and NOLA) and control (0-0 combined (n = 5). (a) Diabetic (0-0; 0---0 with NOLA) and indomethacin; 0---0 ketanserin and NOLA) with endothelium. control (0-0; 0-0 with NOLA) with endothelium. (b) (b) Diabetic (U-U indomethacin; U--U ketanserin) and control Diabetic (U) and control (0) without endothelium. Values indicate (@-* indomethacin; *---@ ketanserin) without endothelium. mean ± s.e.mean. Values indicate mean ± s.e.mean. *P<0.05, significantly different from maximal value in corresponding control group, ANOVA. #P<0.05, significantly different from without indomethacin (Figure lb), ANOVA.

The relaxant effects of 5-HT were examined in endothelium-intact rings, in the presence of ketanserin (0.1 JiM), x-Me-5-HT, DOI and 5-CT of aortae from diabetic rats were preconstricted with a submaximal concentration of the significantly attenuated compared to those obtained in aortae thromboxane (Tx)A2-mimetic, U46619 (0.1-0.3 piM). 5-HT from controls (n = 5-6 all groups, data not shown). (1 nM-30 JtM) did not induce significant relaxation (n = 3, NOLA (0.1 mM) had no significant effect on basal tone in data not shown) in aortae from control or diabetic rats. In unconstricted endothelium-intact aortic rings from control or fact at higher concentrations of 5-HT (3-30 jM) some addi- diabetic rats. tional contraction was observed. 374 G.M. JAMES et al.

a 5,1 4a

4. 3- 3. 0- - 0 2 ,1 1 2 / / #0 1 0, 11 0) IT/ .0,,I6 # c C 0 ._h .a0 -6 -5 -4 -3 -7 -6 -5 -4 -3 7 -6 -5 -4 -3 a) C .) -)U) b (A 5, 4-a) 0) 0 0) C

-6 -5 -4 -3 -6 -5 -3 log [a-methyl-5-HT] (M) log [5-CT] (M)

Figure 4 Responses of rat aortic rings to a-methyl-5-HT alone and Figure 5 Responses of rat aortic rings to 5-carboxamidotryptamine in the presence of N-nitro-L-arginine (NOLA) or ketanserin (n = (5-CT) alone or in the presence of N-nitro-L-arginine (NOLA) 5-6). (a) Diabetic (0-0; 0---0 with NOLA) and control (n = 4-6). (a) Diabetic (0-0; 0-0 with NOLA) and control (0-0; 0--O with NOLA) with endothelium. (b) Diabetic (0-0; 0-0 with NOLA) with endothelium. (b) Diabetic (U) (U-U; *---- with ketanserin) and control (0-0; 0 0 and control (0) without endothelium. Values indicate mean ± s.e. with ketanserin) without endothelium. *P<0.05, significantly differ- mean. ent from corresponding group without NOLA, ANOVA. #P<0.05, significantly different from corresponding control, ANOVA. Values indicate mean ± s.e.mean.

glucose concentration in the bathing medium. These results suggest that transient changes in blood glucose levels may Radioligand binding saturation studies contribute to the altered responsiveness to 5-HT but that other factors are more important. We have previously shown In membrane preparations of aortae from control and dia- that contractile responses to endothelin-1, in aortae from betic rats, [3H]-ketanserin bound to single saturable binding control rats, are also attenuated in high glucose Krebs solu- sites (KD 1.33 (0.76,2.34; 95% c.l.) nM, B.a. 17.4 ± 3.6 (fmol tion (Hodgson & King, 1992). mg ' protein), n = 3 and (KD 2.31 (1.02,5.23; 95% c.l.)nM, It has been postulated that the majority of the contractile Bmax 16.3 ± 5.2, n = 3; respectively). response to 5-HT in rat aortae is mediated by 5-HT2 receptors (Cohen et al., 1981). Results of the present study, using Radioligand binding competition studies the 5-HT2 receptor antagonist, ketanserin, appear to confirm this hypothesis for aortae from both control and diabetic In membrane preparations of aortae from control and dia- rats. However, the fact that indomethacin significantly inhib- betic rats, both WB-4101 and methysergide competed for two ited responses to 5-HT in endothelium-intact aortae from [3H]-ketanserin binding sites (Table 3). In both treatment diabetic rats suggests that a contractile metabolite of arachi- groups 5-HT competed for one [3H]-ketanserin binding site donic acid, possibly TxA2, may be contributing to the re- (Table 3). sponse in this tissue. This finding supports the earlier work of Hagen et al. (1985) who used the cyclo-oxygenase inhibitor, meclofenamic acid. However, they found that meclofenamic Discussion acid also produced a significant reduction in 5-HT responses in aortae from control rats. The present study confirms earlier reports that contractile Contractile responses to the 5-HT receptor agonists, a-Me- responses to 5-HT in aortae from diabetic rats are attenuated 5-HT, DOI and 5-CT were also diminished in aortae from (Owen & Carrier, 1979; Hagen et al., 1985; Head et al., 1987; diabetic rats. Responses to these agonists were still Sikorski et al., 1993). This decrease in responsiveness was no significantly reduced in endothelium-denuded aortae and longer apparent in aortae from insulin-treated diabetic rats when EDRF production was inhibited by NOLA. This sug- suggesting that the changes observed were not due to the gests that altered EDRF release, as has been previously diabetogen per se but linked to the metabolic imbalance observed in aortae from diabetic rats (Oyama et al., 1986; produced by insulin deficiency. However, responses to 5-HT Kamata et al., 1989), was not responsible for the attenuated obtained in aortae bathed in high glucose Krebs, from 5-HT receptor-mediated constriction. However, it does diabetic rats, were still attenuated compared to those obtain- appear from the present study that the magnitude of the ed in aortae from controls. This was despite the fact that contractile response to 5-HT, in aortae from both diabetic maximum responses to 5-HT in endothelium-denuded aortae and control rats, is attenuated by the simultaneous release of from control rats were significantly reduced by altering the EDRF as responses were potentiated by the removal of the ATTENUATED 5-HT CONTRACTILE RESPONSES IN DIABETES 375

Table 3 Mean molar inhibition constants, K1 for competition by WB-4101, methysergide and 5-hydroxytryptamine at [3H]-ketanserin binding sites in membrane preparations of rat aortae Control Diabetic KiH 0.16 (0.09, 0.28) nM (41%) 0.21 (0.01, 4.00) nM (38%)* WB-4101 KiL 4.68 (2.39, 9.15) 1tM (59%) 0.55 (0.14, 2.18)gM (62%)* KiH 1.40 (0.15, 13.20) nM (43%) 10.36 (2.32, 46.24) nM (46%) Methysergide KiL 7.85 (0.41, 150.0) !LM (57%) 54.5 (12.26, 243.3) JAM (54%) 5-HT K1 268 (89, 804) nM 150 (34, 662)nM

n = 3 (except *, n = 2). Values shown are from three competition study replicates with 95% confidence limits and percentage of high and low affinity binding sites. WB-4101 and methysergide competed for two [3H]-ketanserin binding sites, 5-HT competed for one site only. endothelium and by the presence of NOLA. The exact We have previously suggested (Sikorski et al., 1993) that in mechanism responsible for this release of EDRF is unclear. aortae from diabetic rats, peripheral 5-HT2 receptors may be However, there appears to be three likely options. These are; downregulated, as activation of these receptors promotes (1) basal release, (2) 5-HT receptor-mediated release and (3) hyperglycaemia (Chaouloff et al., 1990). However, results of stimulated release due to smooth muscle contraction. As the present study suggest that there is no significant change NOLA had no effect on resting tone in aortae from either in the density or affinity of [3H]-ketanserin for binding sites group of rats it does not appear that there was any in membrane preparations of aortae from control and dia- significant basal EDRF release. 5-HT receptor-mediated betic rats, nor is there a significant change in the affinity of release of EDRF has been demonstrated in canine and pig 5-HT for these sites. However, the binding studies indicate isolated coronary arteries via 5-HT1-like receptor activation that both methysergide and WB4101 compete with [3H]- (Cocks & Angus, 1983; Angus, 1989). However, in the pres- ketanserin at two binding sites. [3H]-ketanserin has been ent study, relaxant responses to 5-HT could not be obtained shown to bind significantly with ml-adrenoceptors in human in rat aortae preconstricted with U46619, when the 5-HT and pig frontal cortex membrane preparations (Hoyer et al., receptors mediating contraction were blocked with 1987). In the present study, it is likely that [3H]-ketanserin is ketanserin. This finding is in agreement with the previous also binding to ax-adrenoceptors present in rat aortae (Wen- work of Martin et al. (1986) and indicates that in rat aortae ham & Marshall, 1992). The high and low affinity binding 5-HT is probably causing the release of EDRF by producing sites observed for methysergide may then represent 5-HT2 an increase in smooth muscle tone (Vargas et al., 1990; Vo et and al-adrenoceptor binding, respectively. Conversely, WB- al., 1991). However, this alternative does not adequately 4101 may be competing with low affinity at 5-HT2 and high explain why contractile responses to 5-HT in endothelium- affinity at a,-adrenoceptors. intact aortae, in the presence of ketanserin, could be obtained Aortic ring dry weights from diabetic rats were significant- only in the additional presence of NOLA because in these ly decreased compared to rings from control rats. However, experiments there was no induced tone. Also, responses to this difference in tissue weight is unlikely to be responsible the more specific agonists DOI and 5-CT were obtained only for the altered responsiveness observed in aortae from dia- in endothelium-intact rings, from control rats, when NOLA betic rats as when results were expressed as tension developed was present. Therefore, the exact mechanism (or stimulus) per mg of tissue, responses to all the contractile agents used for the release of the EDRF remains to be elucidated. were still attenuated compared to responses obtained in aor- The reason/s for the absence of responses to DOI and tae from control rats. 5-CT in endothelium-intact rings, even in the presence of It has been shown that 5-HT-induced contraction in rat NOLA, from diabetic rats is still unclear. However, if, as aorta can be analysed into two distinct components (i.e. indicated by our binding studies, there is no significant phasic and tonic). The phasic component appears to be change in receptor affinity/density between aortae from con- mediated by calcium influx via voltage-dependent channels trol and diabetic rats, it is possible that the alteration is due and the tonic component by phosphoinositide hydrolysis pro- to post-receptor changes. ducts (Nakaki et al., 1985). We have previously shown that Interestingly, in endothelium-denuded aortae from both responses to KCI are diminished in the aortae of 2-week control and diabetic rats, the 5-HT2 receptor antagonist, diabetic rats (Fulton et al., 1991), indicating that there is ketanserin, produces an approximately 10 fold greater shift in likely to be an impairment in the influx of calcium through concentration-response curves to the 5-HT2 receptor agonist, voltage-operated channels. However, although this impair- a-Me-5-HT than to 5-HT. This suggests that in rat aortae, ment probably contributes to the changes observed in the 5-HT may be acting at more than one 5-HT receptor sub- present study, the changes observed in 5-HT receptor-media- type. ted responses are markedly greater than those observed to It would appear also that in the present study the 5-HTIA/B/,ID KCl. Therefore, it is likely that there is also an alteration in receptor agonist 5-CT (Fozard, 1987) produces vasocon- the second messenger system (IP3/DAG). This hypothesis is striction via the activation of 5-HT2/1c receptors. This is likely supported by previous work from our laboratory which has as responses to 5-CT were abolished by ketanserin. Indeed, it shown that responses to endothelin-1 (Fulton et al., 1991; has been shown previously that high concentrations of 5-CT Hodgson & King, 1992), an agonist which utilizes the same can interact with 5-HT2 receptors in rabbit aorta (Feniuk et second messenger system are also attenuated during diabetes. al., 1985). In conclusion, in aortae of rats with diabetes of 2-weeks Previous work in our laboratory has shown that contractile duration, 5-HT receptor-mediated contractile responses are responses to DOI can be obtained only in aortae from con- attenuated compared to controls. The decreased responses do trol rats, and only when the endothelial cells have been not appear to be due to altered EDRF release since this removed or a nitric oxide synthase inhibitor present (Sikorski difference was still apparent in the presence of NOLA but et al., 1993). This finding was confirmed in the present study. may be due to alterations in the second messenger system. In contrast, contractile responses to another 5-HT2/1c agonist, a-Me-5-HT, were obtained in aortae, with and without endothelial cells, from both control and diabetic rats. The reason for the differing results between these two agonists is un- We thank Glaxo Group Research (U.K.) and Upjohn (U.S.A.) for known. the generous gifts of 5-CT and U46619, respectively. 376 G.M. JAMES et al.

References

ANGUS, J.A. (1989). 5-HT receptors in the coronary artery circula- LEYSEN, J.E., NIEMEGEERS, C.J.E., VAN NUETEN, J.M. & LADURON, tion. Trends Pharmacol. Sci., 10, 89-90. P.M. (1982). [3H]ketanserin (R 41 468), a selective 3H-ligand for CHAOULOFF, F., LAUDE, D. & BAUDRIE, V. (1990). Effects of the serotonin2 receptor binding sites. Binding properties, brain distri- 5-HT1c/5-HT2 receptor agonists DOI and a-methyl-5-HT on bution, and functional role. Mol. Pharmacol., 21, 301-314. plasma glucose and insulin levels in the rat. Eur. J. Pharmacol., MARTIN, W., FURCHGOTT, R.F., VILLANI, G.M. & JOTHIANAN- 187, 435-443. DAN, D. (1986). Depression of contractile responses in rat aorta COCKS, T.M. & ANGUS, J.A. (1983). Endothelium-dependent relaxa- by spontaneously released endothelium-derived relaxing factor. J. tion of coronary arteries by noradrenaline and serotonin. Nature, Pharmacol. Exp. Ther., 237, 529-538. 305, 627-630. MCPHERSON, G.A. (1983). A practical computer based approach to COHEN, M.L., FULLER, R.W. & WILEY, K.S. (1981). Evidence for the analysis of radioligand binding experiments. Comp. Prog. 5-HT2 receptors mediating contraction in vascular smooth mus- Biomed., 17, 107-114. cle. J. Pharmacol. Exp. Ther., 218, 421-425. MUNSON, P.J. & RODBARD, D. (1980). Ligand: a versatile com- FENIUK, W., HUMPHREY, P.P.A., PERREN, M.J. & WATTS, A.D. puterized approach for characterization of ligand-binding sys- (1985). A comparison of 5-hydroxytryptamine receptors media- tems. Analyt. Biochem., 107, 662-666. ting contraction in rabbit aorta and dog saphenous vein: evidence NAKAKI, T., ROTH, B.L., CHUANG, D.-M. & COSTA, E.K. (1985). for different receptor types obtained by use of selective agonists Phasic and tonic components in 5-HT2 receptor-mediated rat and antagonists. Br. J. Pharmacol., 86, 697-704. aorta contraction: participation of Ca"+ channels and phos- FOZARD, J.R. (1987). 5-HT: The enigma variations. Trends Phar- pholipase C. J. Pharmacol. Exp. Ther., 234, 442-446. macol. Sci., 8, 501-506. OWEN, M.P. & CARRIER, G.O. (1979). Alterations in vascular smooth FULTON, D.J.R., HODGSON, W.C., SIKORSKI, B.W. & KING, R.G. muscle sensitivity to vasoactive agents by streptozotocin induced (1991). Attenuated responses to endothelin-1, KCl and CaCl2, but diabetes. Proc. West. Pharmacol., 22, 363-366. not noradrenaline, of aortae from rats with streptozotocin- OYAMA, Y., KAWASAKI, H., HATTORI, Y. & KANNO, M. (1986). induced diabetes mellitus. Br. J. Pharmacol., 104, 928-932. Attenuation of endothelium-dependent relaxation in aorta from HAGEN, A.A., SHIRASAWA, Y. & WHITE, R.P. (1985). Experimental diabetic rats. Eur. J. Pharmacol., 131, 75-78. diabetes: reduction of serotonin-induced vasoconstriction by mec- SCHACTERLE, G.R. & POLLACK, R.L. (1973). A simplified method lofenamic acid in vitro. Pharmacology, 30, 197-204. for the quantitive assay of small amounts of protein in biological HEAD, R.J., LONGHURST, P.A., PANEK, R.L. & STITZEL, R.E. (1987). material. Anal. Biochem., 51, 654-655. A contrasting effect of the diabetic state upon contractile res- SIKORSKI, B.W., JAMES, G.M., GLANCE, S.D., HODGSON, W.C. & ponses of aortic preparations from rat and rabbit. Br. J. Phar- KING, R.G. (1993). Effect of endothelium on diabetes-induced macol., 91, 275-286. changes in constrictor responses mediated by 5-HT in rat aorta. HENSELING, M. (1983). Accumulation of 3H-(-)noradrenaline in the J. Cardiovasc. Pharmacol., 22, 423-430. rabbit aorta not related to uptake, and uptake2, but sensitive to VARGAS, H.M., IGNARRO, L.J. & CHAUDHURI, G. (1990). Physio- 3,4-dihydroxy-2-methylpropiophenone (U-0521) and oxytetracyc- logical release of nitric oxide is dependent on the level of vascular line. Naunyn-Schmied. Arch. Pharmacol., 323, 121-127. tone. Eur. J. Pharmacol., 190, 393-397. HODGSON, W.C. & KING, R.G. (1992). Effects of glucose, insulin or VO, P.A., REID, J.J. & RAND, M.J. (1991). Endothelial nitric oxide aldose reductase inhibition on responses to endothelin-1 of aortic attenuates vasoconstrictor responses to nerve stimulation and rings from streptozotocin-induced diabetic rats. Br. J. Phar- noradrenaline in the rat tail artery. Eur. J. Pharmacol., 199, macol., 106, 644-649. 123-125. HOYER, D., VOS, P., CLOSSE, A., PAZOS, A., PALACIOS, J.M. & WENHAM, D. & MARSHALL, I. (1992). ae-Adrenoceptor subtype(s) in DAVIES, H. (1987). [3H]Ketanserin labels 5-HT2 receptors and rat thoracic aorta defined using abanoquil and other selective a,-adrenoceptors in human and pig brain membranes. Naunyn- antagonists. Br. J. Pharmacol., 107, 375P. Schmied. Arch. Pharmacol., 335, 226-230. KAMATA, K., MIYATA, N. & KASUYA, Y. (1989). Impairment of (Received July 26, 1993 endothelium-dependent relaxation and changes in levels of cyclic Revised September 28, 1993 GMP in aorta from streptozotocin induced diabetic rats. Br. J. Accepted October 8, 1993) Pharmacol., 97, 614-618.