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beta-Adrenergic receptor (beta-AR) concentration can be measured in vivo using positron emission tomography (PET) and the high-affinity antagonist [18F]-(S)-fluorocarazolol {[18F]-(S)-FCZ}. However, the influence of endogenous catecholamines on the in vivo binding properties of [18F]-(S)-FCZ should be measured to aid in selection of the model used to estimate receptor concentration based on PET data. Herein we addressed the questions "What is the influence of endogenous catecholamines on the [18F]-(S)-FCZ binding in the heart?" and "In what range are the in vivo concentrations of endogenous beta-AR ligands?" In PET studies, 3 drug regimens were used to manipulate the levels of endogenous catecholamines. The time courses of myocardial concentration of [18F]-(S)-FCZ were compared before and after drug administration. In vitro binding assays and computer simulations were performed to complement the in vivo studies. Despite the large changes of endogenous catecholamines, no significant changes were observed in the [18F]-(S)-FCZ myocardial concentration. In vitro assays showed that (S)-FCZ has an affinity for beta-receptors that is 3900 and 9500 times higher than those of norepinephrine (NE) and epinephrine (EPI), respectively. Computer simulations support the hypothesis that the binding affinities relative to ligand concentrations in vivo are sufficient to explain the apparent lack of effect of endogenous catecholamines on [18F]-(S)-FCZ myocardial concentration. Increased levels of catecholamines in the physiological range do not affect the myocardial concentration of [18F]-(S)-FCZ as measured by PET. This lack of effect suggests that the myocardial concentration of NE at the synaptic sites cannot be higher than 300 nM.

The I1-imidazoline receptor is a novel neurotransmitter receptor found mainly in the brainstem, adrenal medulla and kidney. The actions of moxonidine are described at the level of individual biomolecules, cells, tissues, organs and finally with integrative functions. The receptor functions at the cellular level works through arachidonic acid and phospholipid signaling cascades in neuronal cells with the net result of inhibiting sympathetic premotor neurons.

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.

Moxonidine is a second-generation, centrally acting antihypertensive drug with a distinctive mode of action. Moxonidine activates I1-imidazoline receptors (I1-receptors) in the rostroventrolateral medulla (RVLM), thereby reducing the activity of the sympathetic nervous system. Moxonidine leads to a pronounced and long-lasting blood pressure reduction in different animal models of hypertension, e.g., spontaneously hypertensive rats, renal hypertensive rats, and renal hypertensive dogs. Blood pressure reduction with moxonidine is usually accompanied by a reduction in heart rate which, however, in most studies is of shorter duration and lesser magnitude than the fall in blood pressure. Chronic administration of moxonidine to SHRs with established hypertension causes normalization of myocardial fibrosis, capillarization, and regressive changes in myocytes, in parallel with the reduction of blood pressure. Left ventricular hypertrophy and renal glomerulosclerosis are also significantly reduced. After withdrawal of chronic moxonidine treatment, blood pressure gradually rises to pretreatment values. Direct injection of moxonidine into the vertebral artery of cats elicits a more pronounced fall in blood pressure compared with i.v. injection of an equivalent dose. This observation and others clearly indicate that moxonidine's antihypertensive activity is centrally mediated. The RVLM is the site of action within the CNS that mediates pronounced blood pressure reduction after direct administration of moxonidine into the RVLM of anesthetized SHRs. Selective I1-receptor antagonists introduced into this area abolish the action of systemic moxonidine. Receptor binding studies have shown high and selective affinity of moxonidine for I1-receptors vs. alpha(2)-adrenergic receptors. In vivo studies using a variety of selective I1 or alpha(2)-adrenergic agonists and antagonists have confirmed the primary role of I1-receptors in blood pressure regulation by moxonidine. In addition to lowering blood pressure, moxonidine possesses further properties that appear likely to be relevant in its therapeutic application in the hypertensive syndrome. Moxonidine increases urine flow rate and sodium excretion after central and direct intrarenal administration. It is active against ventricular arrhythmias in a variety of experimental settings. It lacks the respiratory depressant effect attributed to central alpha 2 activation. It exerts beneficial effects on glucose metabolism and blood lipids in genetically hypertensive obese rats. It exhibits anti-ulcer activity. And, finally, moxonidine lowers intraocular pressure, suggesting a possible benefit in glaucoma. Therefore, moxonidine, by its novel mode of action, represents a new therapeutic principle in the treatment of hypertension. Because of its unique profile, moxonidine may prove to be effective in slowing progression of the disease by providing protective effects beyond merely blood pressure reduction. Further studies are needed to verify this potential.

The rostral ventrolateral medulla (RVLM) is the primary region maintaining vasomotor tone, and a site of action for central antihypertensive agents. In vitro [125I]p-iodoclonidine binding studies showed that moxonidine was selective for I1-imidazoline over alpha 2-adrenergic receptors in the RVLM. We identified efaroxan and SK&F 86466 as selective I1- and alpha 2-antagonists, respectively. We tested moxonidine's action within the RVLM of spontaneously hypertensive rats (SHRs) on I1-imidazoline or alpha 2-adrenergic receptors, and determined whether the RVLM mediates the action of systemic moxonidine. SHRs were anesthetized, paralyzed, and ventilated and the RVLM was localized by testing for a pressor response to 2 nmol glutamate. To test whether I1 or alpha 2 mediates hypotensive effects of moxonidine, the I1/alpha 2 antagonist efaroxan (4 nmol) or the alpha 2-blocker SK&F 86466 (10 nmol) was administered 15 min before 4 nmol moxonidine. Efaroxan elevated blood pressure and abolished the action of moxonidine, whereas alpha 2-blockade with SK&F 86466 slightly lowered blood pressure and only partially attenuated moxonidine's effect. The depressor effect of intravenous moxonidine (40 micrograms/kg) was reversed within 10 min by microinjection of 10 nmol efaroxan into the RVLM. Prior bilateral microinjections of efaroxan (10 nmol in 80 nl/site) into the RVLM prevented the hypotensive action of moxonidine given i.v. (40 micrograms/kg). Pharmacokinetic studies showed that at the peak vasodepressor response (8 min post-injection), [3H]moxonidine spread less than 1 mm from the injection site. Moxonidine is a centrally acting antihypertensive with a selective action on I1-imidazoline receptors in RVLM.

Polyclonal antisera raised against para-aminoclonidine coupled to haemocyanin exhibit high affinity for para-aminoclonidine, clonidine and chloroethylclonidine (IC50 less than 100 nmol/l). Anti-para-aminoclonidine antibodies also cross-react with naphazoline, oxymetazoline and tolazoline at moderate concentrations (IC50, 300-500 mumol/l); the phenyl-imidazoles detomidine, medetomidine and MPV830 are weakly cross-reactive (IC50 greater than 0.2 mmol/l). All of these compounds bind with high affinity to both imidazole and alpha 2-adrenergic receptors. Compounds which are imidazole- or alpha 2-specific do not cross-react with anti-para-aminoclonidine antibodies (IC50 greater than 1 mmol/l). Anti-para-aminoclonidine also recognizes an endogenous clonidine-displacing substance in the brain. Thus, (a) binding to anti-para-aminoclonidine antibodies defines a subset of phenyl-imidazol(in)e ligands which bind to both imidazole and alpha 2-adrenergic receptors, suggesting that anti-para-aminoclonidine recognition sites resemble a hybrid of the two receptor types; (b) antibodies to imidazole- or alpha 2-specific agents may be useful as models for differentiating between these types; (c) since clonidine-displacing substance is recognized by anti-para-aminoclonidine antibodies, it may have phenyl and imidazole rings as parts of its chemical structure.

Clonidine acts within the rostral ventrolateral medulla to lower arterial pressure. The receptor mechanism for this action is unknown. In the rostral ventrolateral medulla clonidine binds not only to the alpha 2-adrenergic receptor but also to a novel class of sites which are specific for imidazolines and imidazoles and are distinct from adrenergic or histaminergic receptors. In order to distinguish whether a putative imidazole receptor or the alpha 2-receptor mediates the clonidine hypotensive response, a series of clonidine analogues were tested (1) in radioligand binding assays of their affinity at imidazole and alpha 2-adrenergic receptors, and (2) by microinjection into the rostral ventrolateral medulla of anaesthetized rats to measure their capacity to lower arterial pressure. The hypotensive response elicited by the test agents was strongly correlated with affinity at imidazole sites (r = 0.92) but not with alpha 2-adrenergic affinity (r = 0.36). An imidazole receptor in the rostral ventrolateral medulla may participate in the hypotensive action of clonidine.