With ageing, smooth muscle cell (SMC) iNOS production of NO, possibly stimulated by an imbalance in pro- and anti-oxidant systems, plays an important role in circulatory control. ^{[2, 3]} In vitro incubation of aortic rings provide a model to study such phenomena. We studied the effect of age on the response of aortic rings to 3 days' incubation in culture medium, and the mechanism of this response

.Five-mm rings of the thoracic aorta from 3- and 12-mo rats were incubated (I) or not (NI) in culture medium and isometric contraction, in the presence and absence of endothelium, evoked by norepinephrine (NE) or high [K⁺]_e determined. Experiments were repeated in the presence of propranolol (0.3 µM), polymixin B, an endotoxin antagonist (50 µM), [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of NO-stimulated guanylate cyclase (5 µM), anti-oxidant (pyrrolidine dithiocarbamate, PDTC, 50 µM or glutathion, GSH, 3mM) during incubation and/or during measurement of contraction. iNOS mRNA was determined by real-time PCR in pooled rings (n=4), catalase (CAT) and GSH reductase (GSSR) activities by published methods. ^{[1, 4]}

Incubation did not change high [K⁺]_e-evoked contraction but reduced that evoked by NE. The reduction was greater in rings from 12-mo rats; endothelium removal had no effect (% mean square variance 6% vs 36% for incubation). The reduction in NE-evoked contraction was not due to endotoxin contamination or beta-adrenoceptor-mediated dilation (Emax NE 12-mo I 1.9±0.3, + polymixin B 1.5±0.4, + propronolol 1.5±0.2 g, P>0.05). ODQ fully restored contractility (Emax NE 3-mo NI 3.3±0.2, 3-mo I 1.3±0.1, 3-mo I + ODQ 3.4±0.2 g, P<0.05). In rings from 12-mo rats, L-NAME partially restored, and further addition of anti-oxidants fully restored, NE-evoked contraction. iNOS expression was higher in rings from 12-mo rats and increased upon incubation. CAT fell with age, GSSR increased upon incubation in rings from 3-mo donors only.

	NE	[K+]e	iNOS	CAT (x103)	GSSR
3-mo NI	3.7±0.2	4.7±0.3	x1	7.9±0.5	23±2
3-mo I	2.3±0.2*	4.7±0.9	x20	6.6±0.5	36±3*
12-mo NI	4.6±0.3	5.1±0.2	x3	4.9±0.9$	25±3
12-mo I	0.9±0.1*$	5.1±0.5	x21	5.2±0.3$	23±2
	NE, +L-NAME 2.4±0.3*, +PDTC 3.8±0.2, +GSH 3.7±0.3

Table 1: Emax (g), iNOS mRNA (ratio vs 3-mo NI), CAT and GSSR (nmol.min^-1.mg protein^-1) of endothelium-denuded aortic rings (6-12, each from a different rat, per group; *: P < 0.05 vs NI, $ vs 3-mo, ANOVA + Bonferroni test).

Incubation of the aorta produces a specific reduction in NE-evoked contraction, which is greater in rings from older animals. This involves a decrease in anti-oxidant capacity with stimulation of guanylate cyclase and fall in contraction; induction of SMC iNOS is partially implicated.

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