Cyclic AMP elevating agents and nitric oxide modulate angiotensin II-induced leukocyte-endothelial cell interactions in vivo

Abstract

Angiotensin (Ang-II) is a key molecule in the development of cardiac ischaemic disorders and displays proinflammatory activity in vivo. Since intracellular cyclic nucleotides elevating agents have proved to be effective modulators of leukocyte recruitment, we have evaluated their effect on Ang-II-induced leukocyte-endothelial cell interactions in vivo using intravital microscopy within the rat mesenteric microcirculation. Pretreatment with iloprost significantly inhibited (1 nM) Ang-II-induced increase in leukocyte rolling flux, adhesion and emigration at 60 min by 96, 92 and 90% respectively, and returned leukocyte rolling velocity to basal levels. Pretreatment with salbutamol or co-superfusion with forskolin exerted similar effects. When theophylline was administered, leukocyte rolling flux, adhesion and emigration elicited by Ang-II were significantly attenuated by 81, 89 and 71% respectively. Rolipram administration caused similar reduction of Ang-II-induced leukocyte responses. Co-superfusion of Ang-II with the NO-donor, spermine-NO, or 8-Br-cyclic GMP, or pretreatment with a transdermal nytroglycerin patch, resulted in a significant reduction of the leukocyte-endothelial cell interactions elicited by Ang-II. Salbutamol preadministration did not modify leukocyte-endothelial cell interactions elicited by either L-NAME or L-NAME+Ang-II, indicating that the inhibitory leukocyte effects caused by cyclic AMP-elevating agents are mediated through NO release. In conclusion, we have provided evidence that cyclic AMP elevating agents and NO donors, are potent inhibitors of Ang-II-induced leukocyte-endothelial cell interactions. Thus, they could constitute a powerful therapeutical tool in the control of the leukocyte recruitment characteristic of the vascular lesions that occur in cardiovascular disease states where Ang-II plays a critical role.

Figure 1

Effect of the prostacyclin analogue iloprost and the β₂-receptor agonist salbutamol on Ang-II-induced leukocyte rolling flux (A), leukocyte adhesion (B) and leukocyte emigration (C) in rat mesenteric postcapillary venules. Parameters were measured 0, 15, 30 and 60 min after superfusion with Ang-II (1 nM) in animals untreated (n=5) or pretreated with iloprost (3 μg kg⁻¹, i.v., n=5) or with salbutamol (1 mg kg⁻¹, i.v., n=5). Results are represented as mean±s.e.mean. *P<0.05 or **P<0.01 relative to the control value (0 min) in the untreated group. +P<0.05 or ++P<0.01 relative to the untreated group.

Figure 2

Effect of the adenylyl cyclase activator forskolin on Ang-II-induced leukocyte rolling flux (A), leukocyte adhesion (B) and leukocyte emigration (C) in rat mesenteric postcapillary venules. Parameters were measured 0, 15, 30 and 60 min after superfusion with Ang-II (1 nM) in animals untreated (n=5) or treated with forskolin (0.1 μM, n=5). Results are represented as mean±s.e.mean. *P<0.05 or **P<0.01 relative to the control value (0 min) in the untreated group. +P<0.05 or ++P<0.01 relative to the untreated group.

Figure 3

Effect of the PDE IV inhibitor rolipram and the non-specific PDE inhibitor theophylline on Ang-II-induced leukocyte rolling flux (A), leukocyte adhesion (B) and leukocyte emigration (C) in rat mesenteric postcapillary venules. Parameters were measured 0, 15, 30 and 60 min after superfusion with Ang-II (1 nM) in animals untreated (rolipram vehicle, n=5) or pretreated with rolipram (8 mg kg⁻¹, i.p., n=5) and in animals untreated (n=5) or pretreated with theophylline (5 mg kg⁻¹, i.v., n=5). Results are represented as mean±s.e.mean. *P<0.05 or **P<0.01 relative to the control value (0 min) in the untreated group. +P<0.05 or ++P<0.01 relative to the untreated group.

Figure 4

Effect of the NO donor spermine-NO and the cyclic GMP analog 8-Br-cyclic GMP on Ang-II-induced leukocyte rolling flux (A), leukocyte adhesion (B) and leukocyte emigration (C) in rat mesenteric postcapillary venules. Parameters were measured 0, 15, 30 and 60 min after superfusion with Ang-II (1 nM) in animals untreated (n=5) or co-superfused with spermine-NO (100 μM, n=5) or with 8-Br-cyclic GMP (100 μM, n=5). Results are represented as mean±s.e.mean. *P<0.05 or **P<0.01 relative to the control value (0 min) in the untreated group. +P<0.05 or ++P<0.01 relative to the untreated group.

Figure 5

Effect of the nitric oxide donor nitroglycerin on Ang-II-induced leukocyte rolling flux (A), leukocyte adhesion (B) and leukocyte emigration (C) in rat mesenteric postcapillary venules. Parameters were measured 0, 15, 30 and 60 min after superfusion with Ang-II (1 nM) in animals pretreated with placebo patch (n=5) or with nitroglycerin patch (160 ng min⁻¹ rat⁻¹, n=5). Results are represented as mean±s.e.mean. *P<0.05 or **P<0.01 relative to the control value (0 min) in the untreated group. +P<0.05 or ++P<0.01 relative to the untreated group.

Figure 6

Effect of salbutamol pretreatment on L-NAME and L-NAME+Ang-II-induced leukocyte rolling flux (A), leukocyte adhesion (B) and leukocyte emigration (C) in the rat mesenteric postcapillary venules. Parameters were measured 0, 15, 30 and 60 min after superfusion with L-NAME (100 μM) or L-NAME+Ang-II (1 nM) in animals untreated (n=5) or pretreated with salbutamol (1 mg kg⁻¹, n=5 in both groups). Results are presented as mean±s.e.mean. *P<0.05 or **P<0.01 relative to the control value (0 min) in the untreated group.