Losartan improves impaired nitric oxide synthase-dependent dilatation of cerebral arterioles in type 1 diabetic rats

Abstract

We examined whether activation of angiotensin-1 receptors (AT1R) could account for impaired responses of cerebral arterioles during type 1 diabetes (T1D). First, we measured responses of cerebral arterioles in nondiabetic rats to eNOS-dependent (acetylcholine and adenosine diphosphate (ADP)) and -independent (nitroglycerin) agonists before and during application of angiotensin II. Next, we examined whether losartan could improve impaired responses of cerebral arterioles during T1D. In addition, we harvested cerebral microvessels for Western blot analysis of AT1R protein and measured production of superoxide anion by brain tissue under basal conditions and in response to angiotensin II in the absence or presence of losartan. We found that angiotensin II specifically impaired eNOS-dependent reactivity of cerebral arterioles. In addition, while losartan did not alter responses in nondiabetics, losartan restored impaired eNOS-dependent vasodilatation in diabetics. Further, AT1R protein was higher in diabetics compared to nondiabetics. Finally, superoxide production was higher in brain tissue from diabetics compared to nondiabetics under basal conditions, angiotensin II increased superoxide production in nondiabetics and diabetics, and losartan decreased basal (diabetics) and angiotensin II-induced production of superoxide (nondiabetics and diabetics). We suggest that activation of AT1R during T1D plays a critical role in impaired eNOS-dependent dilatation of cerebral arterioles.

Figure 1

Responses of cerebral arterioles to acetylcholine, ADP and nitroglycerin in nondiabetic rats under control conditions (open bars), following a 2-hour superfusion with angiotensin II (1.0 μM; closed bars) and following a 2-hour superfusion with angiotensin II in the presence of apocynin (1.0 mM; hatched bars). Values are means ± SE. * p < 0.05 versus response under basal conditions and in the presence of apocynin.

Figure 2

Responses of cerebral arterioles to acetylcholine in nondiabetic rats before (open bars; n=6) and during (left hatched bars; n=6) application of losartan (0.1 mM), and in diabetic rats before (closed bars; n=9) and during (cross hatched bars; n=9) application of losartan. Values are means ± SE. * p < 0.05 versus response in nondiabetic rats and diabetic rats after treatment with losartan.

Figure 3

Responses of cerebral arterioles to ADP in nondiabetic rats before (open bars; n=6) and during (left hatched bars; n=6) application of losartan (0.1 mM), and in diabetic rats before (closed bars; n=9) and during (cross hatched bars; n=9) application of losartan. Values are means ± SE. * p < 0.05 versus response in nondiabetic rats and diabetic rats after treatment with losartan.

Figure 4

Responses of cerebral arterioles to nitroglycerin in nondiabetic rats before (open bars; n=6) and during (left hatched bars; n=6) application of losartan (0.1 mM), and in diabetic rats before (closed bars; n=9) and during (cross hatched bars; n=9) application of losartan. Values are means ± SE.

Figure 5

Superoxide anion production by parietal cortex tissue in nondiabetic (open bars; n=15) and diabetic (closed bars; n=26) rats under basal conditions (Basal), during exposure to angiotensin II (AII; 1.0 μM), in the presence of losartan (Losartan; 0.1mM), and during exposure to angiotensin II in the presence of losartan (AII+Losartan). Values are means±SE. a p<0.05 versus basal production of superoxide anion in nondiabetic rats, b p<0.05 versus basal levels of superoxide anion, c p<0.05 versus response to angiotensin II in nondiabetic rats, d p<0.05 versus basal production of superoxide anion in diabetic rats, and e p<0.05 versus production of superoxide anion during exposure to angiotensin II.

Figure 6

AT1R protein in cerebral microvessels obtained from nondiabetic and diabetic rats. The upper panel shows Western immunoblot of AT1R protein in cerebral cortex microvessels (upper lanes are microvessels from nondiabetic rats and lower lanes are microvessels from diabetic rats). The lower panel shows the quantified data for AT1R protein in nondiabetic (open bars) and diabetic (closed bars) rats. Optical density of AT1R protein positive bands was quantified by scanning densitometry and plotted relative to nondiabetic rats. Values are means ± SE. *p<0.05 versus nondiabetic rats.