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-hydroxy- tryptamine (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 throm- boxane 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 endo- thelium-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 glu- cose. 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), insu- lin (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.