The I1-imidazoline receptor: from binding site to therapeutic target in cardiovascular disease

Abstract

Objective: To review previous work and present additional evidence characterizing the I1-imidazoline receptor and its role in cellular signaling, central cardiovascular control, and the treatment of metabolic syndromes. Second-generation centrally-acting antihypertensives inhibit sympathetic activity mainly via imidazoline receptors, whereas first-generation agents act via alpha2-adrenergic receptors. The I1 subtype of imidazoline receptor resides in the plasma membrane and binds central antihypertensives with high affinity.

Methods and results: Radioligand binding assays have characterized I1-imidazoline sites in the brainstem site of action for these agents in the rostral ventrolateral medulla. Binding affinity at I1-imidazoline sites, but not at other classes of imidazoline binding sites, correlates closely with the potency of central antihypertensive agents in animals and in human clinical trials. The antihypertensive action of systemic moxonidine is eliminated by the I1/alpha2-antagonist efaroxan, but not by selective blockade of alpha2-adrenergic receptors. Until now, the cell signaling pathway coupled to I1-imidazoline receptors was unknown. Using a model system lacking alpha2-adrenergic receptors (PC12 pheochromocytoma cells) we have found that moxonidine acts as an agonist at the cell level and I1-imidazoline receptor activation leads to the production of the second messenger diacylglycerol, most likely through direct activation of phosphatidylcholine-selective phospholipase C. The obese spontaneously hypertensive rat (SHR; SHROB strain) shows many of the abnormalities that cluster in human syndrome X, including elevations in blood pressure, serum lipids and insulin. SHROB and their lean SHR littermates were treated with moxonidine at 8 mg/kg per day. SHROB and SHR treated with moxonidine showed not only lowered blood pressure but also improved glucose tolerance and facilitated insulin secretion in response to a glucose load. Because alpha2-adrenergic agonists impair glucose tolerance, I1-imidazoline receptors may contribute to the multiple beneficial effects of moxonidine treatment.

Conclusion: The I1-imidazoline receptor is a specific high-affinity binding site corresponding to a functional cell-surface receptor mediating the antihypertensive actions of moxonidine and other second-generation centrally-acting agents, and may play a role in countering insulin resistance in an animal model of metabolic syndrome X.

Fig. 1

Radioligand binding experiments in bovine rostral ventrolateral medulla (RVLM) membranes demonstrating the presence of non-adrenergic binding sites specific for imidazolines. Inhibition of radioligand binding by increasing concentrations of clonidine (circles) or (–)-norepinephrine (squares) is shown. Each point represents the mean of three to seven separate experiments conducted in triplicate. Data are expressed as a percentage of total specific binding and replotted and reanalyzed from separate reports from laboratories in New York City [12] and Strasbourg [16] obtained using different radioligands: [³H]-p-NH₂-clonidine (PAC) [12] and [³H]-clonidine [16]. Non-specific binding was defined in the presence of 10 and 100 μM phentolamine for [³H]-PAC and [³H]-clonidine, respectively. The bovine RVLM was used because of its large size and ready availability with a short post-mortem delay. Affinity values (K_i) are as follows: clonidine versus [³H]-PAC, 17 ± 2 nM; clonidine versus [³H]-clonidine, 17 ± 4 nM; norepinephrine versus [³H]-PAC, 99 ± 29 nM for 62 ± 2% of sites; norepinephrine versus [³H]-clonidine, 32 ± 9 nM for 69 ± 3% of sites. Data were analyzed and competition curves generated using the Prism program (GraphPAD, San Diego, California, USA). Note the close agreement between data from two separate labs [12,16] using different radioligands.

Fig. 2

Radioligand binding experiments in bovine rostral ventrolateral medulla (RVLM) membranes demonstrating the selectivity of the anesthetic/analgesic agents detomidine and medetomidine for α₂-adrenergic receptors relative to I₁-imidazoline receptors. Inhibition of [³H]-p-NH₂-clonidine binding by increasing concentrations of detomidine (squares) or medetomidine (circles) is shown. Data represent the mean ± SE from four experiments conducted in triplicate, are expressed as a percentage of total specific binding, and are reported here for the first time. Non-specific binding was defined in the presence of 10 μM phentolamine. Data were analyzed and competition curves generated using the Prism program (GraphPAD, San Diego, California, USA). Note that detomidine and medetomidine bind with high affinity only to about half the specific binding sites labeled by [³H]-p-NH₂-clonidine in the RVLM. The remaining half shows low affinity. The sites displaying low affinity for detomidine and medetomidine appear to be I₁-imidazoline sites. Relative to Fig. 1, a greater proportion of [³H]-p-NH₂-clonidine binding sites in the RVLM are I₁-imidazoline sites (49 versus 36%), probably because of optimization of assay conditions [26].

Fig. 3

Hypothetical model of signaling mechanisms coupled to I₁-imidazoline receptor (I₁R) activation. PC-PLC, phosphatidylcholine-selective phospholipase C; PC, phosphatidylcholine; Choline PO₄, choline phosphate; DAG, diacylglyceride; PKC, protein kinase C; PGE₂, prostaglandin E₂.

Fig. 4

Relationship between vasodepressor potency of compounds upon direct microinjection into the rostral ventrolateral medulla (RVLM) of Sprague–Dawley rats and their potency in binding assays in RVLM membranes of (a) high-affinity I₁-imidazoline receptors [52] or (b) low-affinity imidazoline sites [16,56]. Shown on the y-axis is the net fall in mean arterial pressure, relative to vehicle control, of a standardized microinjection of a single dose (1 nmol) into the RVLM of normotensive Sprague–Dawley rats under urethane anesthesia (1.0 g/kg) [,, 103]. Shown on the x-axis in (a) is the negative log of the binding affinity (pK_i) at high-affinity I₁-imidazoline sites in bovine RVLM [52]. Shown on the x-axis in (b) is the pK_i at low-affinity imidazoline sites in post-mortem samples of human RVLM [16,56]. Note that vasodepressor potency correlates with relative ranking at high-affinity I₁-imidazoline sites (r = 0.82) but not at low-affinity imidazoline sites (r = –0.18). Note also that the binding affinities in (b) are 1–3 log units lower than those in (a), consistent with the designation of the former as low-affinity sites.

Fig. 5

Effect of increasing cumulative doses of the α₂-antagonist rauwolscine and subsequent treatment with the I₁/α₂-antagonist efaroxan on mean arterial blood pressure in an spontaneously hypertensive rats (SHR) implanted with an osmotic minipump delivering moxonidine. Three SHR were treated with moxonidine at a dose of 8 mg/kg daily by implantation for 28 days with an osmotic minipump (Alzet) containing either moxonidine free base in 20% dimethylsulfoxide/80% 0.1 M acetic acid. On day 28, SHR were anesthetized with urethane (1 g/kg intraperitoneally) and the femoral artery was cannulated for direct measurement of arterial blood pressure and intra-arterial administration of antagonists. Mean arterial pressure was recorded every 2 min. Data are shown from a single animal, representative of the results from three rats. The dashed line indicates basal level of mean arterial pressure determined during a 10 min baseline period. Doses indicated in the figure are in mg/kg bodyweight. Administration of the α₂-adrenergic antagonist rauwolscine at a dose of 0.1 mg/kg immediately lowered mean arterial pressure from 105 to 64 mmHg. By the time a cumulative dose of rauwolscine of 0.5 mg/kg had been administered, mean arterial pressure had returned to its starting level. At 30 min after the first dose of rauwolscine, the I₁-imidazoline antagonist efaroxan was administered at a dose of 0.2 mg/kg. Mean arterial pressure promptly rose to the level of vehicle-treated SHR. The failure of a potent α₂-adrenergic antagonist to reverse the antihypertensive action of moxonidine, and the effectiveness of an I₁-imidazoline antagonist support a role in the antihypertensive actions of moxonidine for I₁-imidazoline receptors.

Fig. 6

Dose-dependent sedative action of moxonidine relative to clonidine following oral administration in rats. Female Sprague–Dawley rats weighing 100–125 g were given increasing doses of clonidine, moxonidine or vehicle (1% Tylose, 0.2% Tween-80) by gavage at 10 ml/kg bodyweight. One hour later, hexobarbital (10% in 1.0 M NaOH) was administered intraperitoneally at a dose of 34 mg/kg. This dose of hexobarbital is known to be just below the threshold for sleep induction. Rats were tested for the loss of righting reflex during the first 1 min after hexobarbital administration. Rats which did not right themselves were counted as sedated. Ten rats were tested in each group, and each rat was tested only once. Data were analyzed by non-linear regression fitting to a logistic equation of variable slope using the Prism program (GraphPAD, San Diego, California, USA). The dose–response curves were also generated by the program and represent the best fit. On a molar basis, clonidine was 165-fold more potent than moxonidine in potentiating the sedative action of hexobarbital: median effective dose (ED₅₀) clonidine, 0.31 ± 0.1 μmol/kg; ED₅₀ moxonidine, 51 ± 3 μmol/kg. The slopes of the dose-response curves (n_H) did not differ between drugs: clonidine, 1.14 ± 0.53; moxonidine, 1.37 ± 0.15.

Fig. 7

Oral glucose tolerance tests in (a, c) spontaneously hypertensive rats (SHR) and (b, d) obese SHR (SHROB) with and without moxonidine treatment (8 mg/kg daily for 90 days). SHROB and SHR were treated with moxonidine at a dose of 8 mg/kg daily in their drinking water for 90 days. The concentration of moxonidine in each rat’s water bottle was adjusted weekly according to changes in fluid consumption and body weight. Saccharin (0.1%) was added to the moxonidine solution to maintain palatability. For oral glucose tolerance testing, rats were fasted for 18 h and then given 12 g of glucose per kg body weight by gavage. Blood (0.7 ml) was obtained from a cut on the tail at the times shown and glucose was measured by colorimetric glucose oxidase assay (One-Touch, Lifescan, Milpitas, California, USA). A radioimmunoassay kit (Linco Research, St Charles, Missouri, USA) was used with rat insulin standard and antibodies directed against rat insulin. Assays were conducted in duplicate and the intra-assay coefficient of variation was less than 5%. All values are given as means ± SE obtained from seven animals in each group of SHR, and eight animals in each group of SHROB.

Fig. 8

Area under the curve for glucose tolerance tests in lean spontaneously hypertensive rats (SHR) and obese SHR (SHROB) phenotypes with and without chronic moxonidine treatment for (a) glucose and (b) insulin. Glucose tolerance curves presented were analyzed for area using the trapezoidal method (Prism program, GraphPAD). Note that insulin areas under the curve are plotted on a logarithmic scale to facilitate comparisons between groups. Data were analyzed by using a two-way analysis of variance by phenotype and drug treatment and Newman–Keuls post-hoc analysis. *Significant effect of phenotype, with SHROB > SHR in all cases except glucose area under the curve after moxonidine treatment, where SHROB < SHR. ^†Significant effect of moxonidine treatment, with glucose area under the curve being significantly decreased by moxonidine in SHROB and insulin area under the curve increased by moxonidine in both SHROB and in SHR.