LNP 906, the first high-affinity photoaffinity ligand selective for I1 imidazoline receptors

Abstract

1 The hypotensive effect of imidazoline-like drugs, such as clonidine, was attributed both to alpha2-adrenergic receptors and nonadrenergic imidazoline receptors, which are divided into I1, I2 and I3 subtypes. 2 We have recently synthesized a derivative of (2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), the first high-affinity and selective ligand for I1 receptors (I1R), with a photoactivable function (LNP 906). 3 This work aims to test whether this derivative retained the binding properties of LNP 911 and bound irreversibly to I1R. 4 Binding studies showed that LNP 906 exhibited nanomolar affinity for I1R and was selective for I1R over I2 receptors and alpha2-adrenergic receptors (alpha2Ars). 5 Upon exposure to u.v. light, LNP 906 irreversibly blocked the binding of [125I]-paraiodoclonidine (PIC) to I1R, time- and dose-dependently, on PC12 cell membranes and interacted with I1R in a reversible and competitive manner in the absence of light. Pharmacological studies showed that this blockade was prevented by the concomitant presence of rilmenidine (a well-known I1 agonist), but not by rauwolscine (an alpha2 antagonist). 6 Finally, LNP 906 clearly antagonized the decrease in forskolin-stimulated cAMP level induced by rilmenidine, but not by melatonin. 7 These results indicate that LNP 906 is the first high-affinity and selective photoaffinity ligand for I1R and that it behaves as an I1R antagonist.

Figure 1

Test of the specific binding of LNP 906 for I₁ and I₂ receptors. Competition assays are performed as described in Methods either with 0.2 nM [¹²⁵I]-LNP 911 or with 5 nM [¹²⁵I]-PIC in PC12 membranes with increasing concentrations of LNP 906; assays on I₂ receptors were performed with 5 nM [³H]-idazoxan in rabbit kidney membranes. In these studies, nonspecific bindings were determined either by 100 μM PIC for [¹²⁵I]-LNP 911 and [¹²⁵I]-PIC or by 100 μM cirazoline for [³H]-idazoxan. Each point is the mean±s.e.m. of three to 11 experiments performed in triplicate and using different membrane preparations. Curves were analysed using the iterative nonlinear least-squares curve-fitting program GraphPad.

Figure 2

Influence of LNP 906 on the kinetic dissociation of [¹²⁵I]-LNP 911 binding to PC12 cell membranes at 25°C. Kinetic dissociation at 25°C of the radioligand was performed in the absence or presence of 50 μM LNP 906. Dissociation was started with the addition of LNP 911 (100 μM) after 60 min incubation at 25°C (as described in Methods). Nonspecific binding of [¹²⁵I] LNP 911 was determined by 100 μM PIC in triplicate at different times. The apparent rate constants of dissociation (k₋₁) were 0.019 and 0.033 for the dissociation with LNP 911 or with LNP 911 in the presence of LNP 906, respectively. Binding at t=0 (100%) represented, respectively, 3917 and 4185 c.p.m. in the absence and presence of LNP 906. The result shown is representative of four separate experiments.

Figure 3

Effect of varying exposure to ultraviolet irradiation on irreversible binding of LNP 906 to PC12 membranes. Membranes (20–50 μg) were preincubated for 60 min in the dark in the absence or presence of 20 μM LNP 906, then irradiated at 25°C for 5–45 min, followed by washing and determination of [¹²⁵I]-PIC binding. Control membranes were free of LNP 906 and were not irradiated. Specific binding of [¹²⁵I]-PIC was determined with 100 μM PIC at different times indicated. Means±s.e.m. of four independent experiments performed in triplicate. Significance of the differences between control experiments (dark, without LNP 906) and photolysed membranes treated with LNP 906 are given. ^**P<0.01, ^***P<0.005.

Figure 4

Concentration dependence for the photoinactivation of I₁ receptors by LNP 906 following exposure to short-wavelength ultraviolet light. Membranes (20–50 μg) were incubated as described in experimental procedures, without and with increasing concentrations of LNP 906 (2–200 μM) for a period of 60 min, and then irradiated at 25°C for 15 min. Then, the binding of [¹²⁵I]-PIC was assessed. Control membranes were free of LNP 906 and were not irradiated. Specific binding of [¹²⁵I]-PIC was defined with 100 μM PIC in triplicate. Values are means means±s.e.m. of five independent experiments performed in triplicate. ^**P<0.01.

Figure 5

Specificity of the photolabelling of I₁ receptors by LNP 906. Membranes were preincubated for 60 min, with 20 μM LNP 906, in the presence or absence of competitors (rilmenidine, rauwolscine) in a final volume of 0.25 ml. The competitors and the final concentrations used are listed in the figure. Following preincubation, samples were irradiated for 15 min and the remaining I₁ receptor activity was then assayed by the specific binding of [¹²⁵I]-PIC. Control membranes were free of LNP 906 and were not irradiated. Controls represent identical samples of I₁ receptors, which were carried through the same preincubation and binding steps as those samples, which were preincubated with LNP 906 plus competitor. Specific binding of [¹²⁵I]-PIC was defined with 100 μM PIC in triplicate. Each point is the mean of four±s.e.m. experiments performed in triplicate. ^**P<0.01.

Figure 6

Antagonist activity of LNP 906 on the forskolin-stimulated cAMP level response of I₁R agonists in PC12 cells. Cells (10⁵) were first preincubated in the presence of LNP 906 (10⁻⁵ M) for 10 min at 37°C, then increasing concentrations of rilmenidine (a) or melatonin (b) were added with 1 μM forskolin for a period of 30 min at the same temperature in the dark. cAMP level was measured as described in Methods. Results represent the average of at least four experiments performed in triplicate, and are expressed as the percentage of inhibition±s.e.m. of the control (100% represents the value in the presence of forskolin only, which was 222±19 fmol min⁻¹/10⁵ cells, full column). ^* or ^#P<0.05, ^** or ^##P<0.01 (^* represents the significance of the differences between cells treated with or free of LNP 906; # represents the significance between cells treated with rilmenidine or melatonin and controls (free of rilmenidine)).