Early upregulation of kinin B1 receptors in retinal microvessels of the streptozotocin-diabetic rat

Abstract

(1) Retinal microvessel responses to kinin B1 and B2 receptor agonists and antagonists were investigated in streptozotocin (STZ)-diabetic rats and age-matched controls. In addition, quantitative in vitro autoradiography was performed on retinas from control and STZ-diabetic rats with radioligands specific for B2 ([125I]HPP-Hoe 140), and B1 receptors ([125I]HPP-[des-Arg10]-Hoe 140). (2) In control rats, the B2 receptor agonist bradykinin (BK, 0.1-50 nm) vasodilated retinal vessels in a concentration and time-dependent manner. This effect was completely blocked by the B2 receptor antagonist Hoe140 (1 microm). In contrast, the B1 receptor agonist des-Arg9-BK (0.1-50 nm) was without effect. (3) Des-Arg9-BK was able to produce a concentration-dependent vasodilatation as early as 4 days after STZ injection, and the effect of 1 nm des-Arg9-BK was inhibited by the B1 receptor antagonist des-Arg10-Hoe140 (1 microm). Low-level B1 receptor binding sites were detected in control rats, but densities were 256% higher in retinas from 4- to 21-day STZ-diabetic rats. (4) In control rats, the vasodilatation in response to 1 nm BK involved neither calcium influx nor nitric oxide (NO) as GdCl3 and l-NAME were without effect. However, the vasodilatation did involve intracellular calcium mobilization as well as products of the cyclooxygenase-2 (COX-2) pathway as 2,5-di-t-butylhydroquinone (BHQ), cADP ribose and l-745 337 inhibited this response. The vasodilatation response was blocked by trans-2-phenyl cyclopropylamine (TPC) demonstrating that prostacyclins mediate this response. (5) In STZ-diabetic rats, the vasodilatation in response to des-Arg9-BK involved both calcium influx and intracellular calcium mobilization from stores both IP3 sensitive and non-IP3 sensitive. Indeed, the effect was blocked by GdCl3, BHQ and cADP ribose. Furthermore, NO production and products of the COX-2 pathway including prostacyclin are involved as the response was inhibited by l-NAME, l-745 377 and TPC. (6) Vasodilatation in response to either 1 nm BK or 1 nm des-Arg9-BK were blocked by NF023 demonstrating that a Go/Gi G-protein transduces both these effects. (7) This is the first report on the retinal circulation which provides evidence for vasodilator B2 receptors and the upregulation of B1 receptors very early following induction of diabetes with STZ rats. These results suggest that kinin receptors may be potential targets for therapeutics to treat retinopathies.

Figure 1

BK induces rat retinal vessel dilation. (a) Kinetic of the effect of BK (1 nM) and des-Arg⁹-BK (1 nM) up to 30 min. (b) Concentration–response effect of BK or des-Arg⁹-BK. Retinal vessel diameter was recorded after a serial (10 min) increasing topical application of BK or des-Arg⁹-BK (0.1–50 nM). (c) B₂ receptors mediate BK-induced retinal vessel dilation. Retinal vessel diameter was recorded after a topical application of Hoe 140 (1 μM) or des-Arg¹⁰-Hoe 140 (1 μM) followed 15 min later by the application of BK (1 nM). Responses are expressed as per cent change in the outer diameter of the vessel from baseline (n=5–7). Statistical comparison to time zero (a) or in the absence of agonist (b) or to U-46619 (1 μM) (c) is indicated by (*) or to BK (c) is indicated by (†), where **, P<0.01; *** and †††, P<0.001.

Figure 2

Induction of kinin B₁ receptor expression in STZ-diabetic rat retinal vessels. Vessel diameter of retina from 1, 4, 7 and 21 days STZ-treated rats was recorded after a serial (10 min) topical application of des-Arg⁹-BK (0.01–1 nM). Moreover, retinal vessel diameter to des-Arg⁹-BK (1 nM) was recorded 15 min after the application of des-Arg¹⁰-Hoe 140 (1 μM). Responses are expressed as per cent change in the outer diameter of the vessel from baseline (n=3–6). Statistical comparison to U-46619 is indicated by (*) or to des-Arg⁹-BK is indicated by (1 nM) (†), where *, P<0.05; **, and ††, P<0.01.

Figure 3

Pharmacology of the effects of BK and des-Arg⁹-BK on retinal vessel tone. Vessel diameter of retina from control (a) or STZ-diabetic rats at 21 days (b) was recorded after topical application of NF023 (100 μM), L-NAME (100 μM), indomethacin (1 μM), L-745 337 (1 μM), TPC (5 μM), GdCl₃ (10 mM), BHQ (10 μM) or cADP ribose (10 μM) followed by the application of BK (1 nM) (a), or des-Arg⁹-BK (1 nM) (b). The responses are expressed as per cent change in the outer diameter of the vessel from baseline (n=4–8). Statistical comparison to U-46619 is indicated by (*) or to agonist alone is indicated by (†), where †, P<0.05; **, and ††, P<0.01.

Figure 4

Quantification of specific binding sites with [¹²⁵I]-HPP-Hoe 140 (B₂ receptors) and [¹²⁵I]-HPP-[des-Arg¹⁰]-Hoe 140 (B₁ receptors) in the retinal tissue of control (c) and STZ-diabetic rats at 4, 7 and 21 days. Values represent the means±s.e.m. of four rats in each group. Statistical comparison to control is indicated by **P<0.01.