Modification of Noradrenaline Release in Pithed Spontaneously Hypertensive Rats by I1-Binding Sites in Addition to 2-Adrenoceptors

It is known that moxonidine acts as an agonist at presynaptic 2-adrenoceptors of the postganglionic sympathetic nerve terminals and leads to a reduction in noradrenaline release. In addition, it is conceivable that I1-binding sites located in other regions of the preand postganglionic sympathetic neurons are involved in this effect. Our aim was to investigate whether and to what extent activation of the I1-binding sites contributes to the moxonidine-induced inhibition of noradrenaline release. Noradrenaline release was induced in pithed spontaneously hypertensive rats (pretreated with phenoxybenzamine/desipramine at 10/0.5 mg/kg) by stimulation of sympathetic overflow from the spinal cord. Noradrenaline overflow was reduced using moxonidine (0.18, 0.6, and 1.8 mg/kg) by 39.4, 70.4, or 78.7%, respectively, even when all 1-/ 2-adrenoceptors were blocked effectively by phenoxybenzamine. In contrast, the I1antagonist efaroxan (0.1, 1, and 3 mg/kg) increased noradrenaline overflow from 453 (control) to 1710, 1999, or 2754 pg/ml, suggesting an autoreceptor-like function of I1-binding sites. In consequence, moxonidine (0.18, 0.6, and 1.8 mg/kg) reduced the increase in noradrenaline overflow in efaroxan-treated animals (1 mg/kg) by 22.7, 41.7, and 50.5%, respectively. Agmatine (6 and 60 mg/kg), an endogenous agonist at I1-binding sites, reduced noradrenaline overflow ( 36 or 53%), even under 2-adrenoceptor blockade. When 2-endo-amino-3-exoisopropylbicyclo[2.2.1]heptane (AGN192403) (10 mg/kg) was injected, a selective blocker of I1-binding sites, noradrenaline overflow was not influenced by agmatine. It is concluded that moxonidine reduces noradrenaline overflow by acting at I1binding sites in addition to its agonistic property at 2-adrenoceptors. The exact location of the I1-binding sites on the preor postsynaptic sympathetic neurons is unknown, but the location in the preor postsynaptic membrane of the sympathetic ganglion is the most plausible explanation. Clonidine and the related compounds moxonidine and rilmenidine that induce inhibition of noradrenaline release from sympathetic neurons are second line antihypertensives. Recently, the presynaptic 2-adrenoceptor mediating an inhibition of noradrenaline release from sympathetic nerves at which these compounds act was subclassified as the 2Aand 2C-adrenoceptor (Altman et al., 1999). In addition, clonidine and other imidazolines have been suggested to modulate noradrenaline release via non-I1-/non-I2-presynaptic imidazoline binding sites, which were identified on the sympathetic axon terminals of rabbit, rat, guinea pig, and human cardiovascular tissue (Göthert et al., 1999; Molderings and Göthert, 1999). In contrast, investigations by other authors revealed that these drugs inhibit noradrenaline release exclusively by activating prejunctional 2-adrenoceptors (Bohmann et al., 1994; Gaiser et al., 1999). Whether imidazoline binding sites are involved (in addition to 2-adrenoceptors) in noradrenaline release may be dependent on stimulation conditions (Molderings et al., 1999a) and/or species differences (Molderings et al., 2000). Such results have been based mainly on in vitro studies appropriate for the identification of a drug’s presynaptic site of action. However, to what extent do such in vitro results apply in vivo? Studies in conscious and pithed rabbits have supported the view that central sympathetic inhibition is mediated only via 2-adrenoceptors (Urban et al., 1995; Bock et al., 1999; Szabo et al., 1999). In pithed, spontaneously hypertensive rats (SHR) and rabbits, rilmenidine and moxonidine decreased the stimulated overflow of noradrenaline (Häuser et al., 1995; Urban et al., 1995; Szabo et al., 1999). Because imidazoline derivatives were still able to reduce noradrenaline overflow dose dependently after rauwolscine pretreatment, an 2-adrenoceptor-independent mechanism was suggested (Häuser et al., 1995). However, it seems not unlikely that clonidine, moxonidine, or rilmenidine induces sympathetic inhibitory effects via 2-adrenoceptors even in the presence of rauwolscine, because this 2-blocker was Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.044966. ABBREVIATIONS: SHR, spontaneously hypertensive rats; KO, knockout; PE, polyethylene; nAch, nicotinic acetylcholine. 0022-3565/03/3043-1063–1071$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 304, No. 3 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 44966/1043805 JPET 304:1063–1071, 2003 Printed in U.S.A. 1063 at A PE T Jornals on O cber 8, 2017 jpet.asjournals.org D ow nladed from characterized to be a competitive antagonist (Bock et al., 1999), which would strengthen the idea of an imidazoline binding site-independent regulation of noradrenaline release. The best way to show in vivo whether imidazoline binding sites would have some impact in regulating noradrenaline release is to use 2-adrenergic receptor knockout (KO) mice (Hein, 2001). However, the residual 2-mediated effect in the 2A/D-KO mice suggests that the 2C-adrenoceptor also functions as a presynaptic autoreceptor for inhibiting noradrenaline release (Altman et al., 1999). Because only the 2A/C-double KO mouse (an animal model that was not available to us) would serve as a suitable experimental tool to answer this question, we decided to overcome the limitation of a competitive blockade of 2-adrenoceptors by performing experiments on reduced noradrenaline overflow evoked by clonidine-like substances (e.g., moxonidine) under irreversible 2-adrenoceptor blockade in pithed SHR. This rat strain is a well established model of sympathetic hyperactivity, which represents another rationale for using SHR instead of mice. Using an irreversible blocker of 2-adrenoceptors (phenoxybenzamine) and a specific ligand for imidazoline binding sites (2-endo-amino-3-exo-isopropylbicyclo[2.2.1]heptane; AGN192403), the aim of this study was to determine 1) whether imidazoline binding sites contribute to the moxonidine-induced modification of noradrenaline release in vivo and, if so, 2) to specify the subtype of imidazoline binding sites involved in this effect. Materials and Methods Animal Preparation. The present study was conducted according to the declaration of Helsinki, following the guidelines for the care and use of laboratory animals as adopted by the Ministerium für Natur und Umwelt des Landes Schleswig Holstein, Deutschland, animal protocol no. 9/A4/91. Male, spontaneously hypertensive rats (Charles River, Sulzfeld, Germany), weighing 200 to 250 g, were pithed under ether anesthesia using a steel rod (1.5 mm diameter) coated with enamel except for the length of the thoracolumbar spinal cord (Th4-Th12 segments) as described by Gillespie and Muir (1967). A steel cannula was inserted as an indifferent electrode into the dorsal subcutis located near the lumbar vertebral column. Both vagal nerves were cut at the neck, and neuromuscular junctions were blocked by d-tubocurarine (3 mg/kg). Polyethylene catheters were inserted into both femoral veins (PE-10) for drug administration and into both carotid arteries (PE-50) for measuring blood pressure and collecting blood samples. The polyethylene catheter (PE-50), which was inserted into the left carotid artery, was connected to a Statham P23 Db pressure transducer (Hellige, Freiburg, Germany). Blood pressure and heart rate were recorded continuously and sampled