IN-VIVO AND IN-VITRO EVIDENCE OF ALTERED NITRIC-OXIDE METABOLISM IN THE SPONTANEOUSLY DIABETIC, INSULIN-DEPENDENT BB EDINBURGH RAT/

1 Altered vasoreactivity may contribute significantly to the pathogene sis of diabetic vascular complications. This study investigated the ef fect of (a) insulin-treated diabetes, and (b) chronic in vivo administ ration of N-omega-nitro-L-arginine methyl ester (L-NAME), a nitric oxi de (NO) synthase inhibitor, on mean arterial pressure and in vitro vas cular reactivity to noradrenaline in mesenteric arterial bed preparati ons from spontaneously diabetic, insulin-dependent and treated BB rats , the best animal model of insulin-dependent mellitus (IDDM) currently available. Four groups of animals from the Edinburgh colony (BB/E) of spontaneously diabetic BB rats were studied: age-matched (mean+/-s.e. mean=156+/-2d) non-diabetic (glycated haemoglobin=3.8+/-0.1%) and insu lin-treated diabetic (glycated haemoglobin=6.2+/-0.5%; duration of dia betes=56+/-4d) groups were either L-NAME treated (oral dose=27+/-1 mg kg(-1) d(-1); duration of treatment from 30 until 153 days of age) or untreated. Although our diabetic BB/E rats do not achieve overall norm oglycaemia, individual adjustment of the daily insulin dose administer ed to every diabetic rat achieves better glycaemic control than previo us groups studying altered vascular reactivity and endothelial dysfunc tion in this animal model of diabetes. 2 Mean arterial pressure (measu red directly via indwelling carotid arterial cannulae) was not signifi cantly different between non-diabetic (116+/-3 mmHg; n=10) and diabeti c (122+/-2 mmHg; n=12) BB/E rats. L-NAME treatment significantly (P<0. 001) increased mean arterial pressure in both groups (165+/-6 mmHg; n= 9 and 142+/-4 mmHg; n=6 respectively) but the degree of hypertension o bserved in L-NAME-treated diabetic rats was significantly (P<0.01) att enuated compared to nondiabetic rats treated with L-NAME. 3 Mesenteric arterial bed preparations were cannulated under anaesthesia, excised and intralumenally perfused ex vivo with noradrenaline (0.2-20 mu M). Basal perfusion pressures were not significantly different in mesenter y preparations from non-diabetic (27.0+/-2.6 mmHg) and diabetic (27.1/-3.2 mmHg) BB/E rats. There was no significant difference in maximal response above basal perfusion pressure (MAX) or pEC(50), defined as t he negative log of the agonist concentration required to give 50% of t he maximal response above basal perfusion pressure, to noradrenaline i n untreated non-diabetic (166+/-7 mmHg and 5.74+/-0.05 respectively) a nd diabetic (170+/-11 mmHg and 5.59+/-0.05) BB/E rats. 4 In vivo treat ment of non-diabetic and diabetic BB/E rats with L-NAME had no signifi cant effect on basal perfusion pressure (25.9+/-4.8 mmHg and 28.5+/-3. 9 mmHg respectively). L-NAME treatment in vivo increased (P<0.001) MAX to noradrenaline of non-diabetic rats (224+/-8 mmHg) but did not affe ct the value for diabetic rats (178+/-14 mmHg). L-NAME treatment did n ot alter the pEC(50) values in either group (5.71+/-0.05 and 5.65+/-0. 05). 5 Consistent with previous studies using vascular preparations fr om spontaneously diabetic BB rats, mesentery preparations from diabeti c BB/E rats (n=12) exhibited a significantly reduced vasodilator respo nse to acetylcholine (F value=4.4, P<0.05) across the concentration ra nge studied compared to non-diabetic BB/E rats (n=12) although there w as no significant difference in maximal relaxation (diabetic 53.1+/-4. 3% vs non-diabetic 55.7+/-5.5%) or pEC(50), (diabetic 6.92+/-0.25 vs n on-diabetic 7.49+/-0.22). There was no significant (F value=0.8, P>0.1 ) difference in the response to GTN between preparations from non-diab etic and diabetic rats (maximal relaxation: 49.6+/-3.7% vs 48.5+/-4.3% ; pEC(50): 7.84+/-0.12 vs 7.89+/-0.22 respectively). 6 In conclusion, vascular responsiveness to noradrenaline is not impaired in spontaneou sly diabetic BB/E rats with significantly better glycaemic control tha n those used in previous studies. However, following chronic L-NAME tr eatment, diabetic BB/E rats exhibit attenuated hypertension and an abs ence of enhanced vascular responsiveness to noradrenaline in vitro com pared to similarly treated non-diabetic rats. These results, together with the significantly impaired endothelium-dependent vasodilatation a nd unchanged endothelium-independent vasodilatation in vitro of prepar ations from diabetic BB/E rats, are consistent with the hypothesis tha t functional changes in the synthesis and metabolism of NO (rather tha n altered vascular responsiveness to NO) occur in diabetes. Our result s indicate that good glycaemic control alone is insufficient to preven t these abnormalities in NO availability and further studies to charac terize the origin of these changes are necessary.