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The imidazoline-1 receptor (IR1) is considered a novel target for drug discovery. Toward cloning an IR1, a truncated cDNA clone was isolated from a human hippocampal lambda gt11 cDNA expression library by relying on the selectivity of two antisera directed against candidate IR proteins. Amplification reactions were performed to extend the 5' and 3' ends of this cDNA, followed by end-to-end PCR and conventional cloning. The resultant 5131-basepair molecule, designated imidazoline receptor-antisera-selected (IRAS) cDNA, was shown to encode a 1504-amino acid protein (IRAS-1). No relation exists between the amino acid sequence of IRAS-1 and proteins known to bind imidazolines (e.g., it is not an alpha2-adrenoceptor or monoamine oxidase subtype). However, certain sequences within IRAS-1 are consistent with signaling motifs found in cytokine receptors, as previously suggested for an IR1. An acidic region in IRAS-1 having an amino acid sequence nearly identical to that of ryanodine receptors led to the demonstration that ruthenium red, a dye that binds the acidic region in ryanodine receptors, also stained IRAS-1 as a 167-kD band on SDS gels and inhibited radioligand binding of native I1 sites in untransfected PC-12 cells (a source of authentic I1 binding sites). Two epitope-selective antisera were also generated against IRAS-1, and both reacted with the same 167-kD band on Western blots. In a host-cell-specific manner, transfection of IRAS cDNA into Chinese hamster ovary cells led to high-affinity I1 binding sites by criteria of nanomolar affinity for moxonidine and rilmenidine. Thus, IRAS-1 is the first protein discovered with characteristics of an IR1.

Agmatine, an amine and organic cation, is an endogenous ligand at alpha 2-adrenergic and imidazoline (I-) receptors, to which it binds with high affinity. In addition, agmatine has properties of an endogenous neurotransmitter. Thus, agmatine (a) is locally synthesized in brain by a specific enzyme, arginine decarboxylase; (b) is stored in a large number of neurons with selective distribution in the CNS; (c) is associated with small vesicles in axon terminals that, at least in hippocampus, make synaptic asymmetric (excitatory) synapses on pyramidal cells; (d) is released from synaptosomes in a Ca(2+)-dependent manner; (e) can be enzymatically degraded by agmatinase in synaptosomes; (f) can be inactivated by selective reuptake; (g) blocks the ligand-gated NMDA receptor channel at sites distinct from ligand-binding and polyamine sites; and (h) has systemic actions when administered intraventricularly. Additionally, (i) agmatine is a precursor of brain putrescine and, hence, of higher polyamines, and (j) it competitively inhibits the activity of all isozymes of nitric oxide synthase. Agmatine meets most criteria to establish it as a novel neurotransmitter/neuromodulator in the CNS. However, agmatine differs from forms of clonidine displacing system with respect to distribution, bioactivity, and capacity to interact with antibodies raised to imidazoline-like drugs. Thus, there are multiple endogenous ligands of the imidazoline receptors, one of which is agmatine.

We have shown that cultured vascular smooth muscle cells (VSMC) and brain astroglial cells express I-receptors of the I2 subtype. While imidazoline agents are anti-proliferative in smooth muscle cells, they increase the expression of glial fibrillary acidic protein (GFAP) in astrocytes. Because increases in GFAP suppress the induction of calcium-independent, inducible nitric oxide synthase (NOS-2), we measured whether idazoxan and related imidazolines and agmatine would also suppress the expression of NOS-2. Cultured astrocytes and macrophages, RAW 264.7 cell line, were incubated with lipopolysaccharide (LPS, 1 microgram/ml) or cytokine mixture in the presence of 1-100 microM of idazoxan, agmatine, or other imidazoline agents. Idazoxan potently (IC50 10 microM) decreased the activity of NOS-2 in astrocytes, but was less potent in RAW 264.7 cells. By contrast, agmatine was most potent in RAW 264.7 cells (IC50, 10 microM) but less potent in glial cells and VSMC. Both idazoxan and agmatine decreased the activity of NOS-2 by reducing the levels of enzyme protein as measured by immunoblot and immunocytochemistry. No specific binding of [3H]-idazoxan was observed in RAW 264.7 cell membranes. We conclude that idazoxan, agmatine, and selected imidazoline agents inhibit the expression of NOS-2 and proliferation in primary glial cells and VSMC. While the antiproliferative actions appear mediated by I-receptors of the I2 type, the anti-inflammatory response is probably not mediated by I-receptors but possibly by direct actions on signal transduction enzymes.

Agmatine, which in other life forms serves as a metabolic intermediate for polyamine biosynthesis, appears to have properties in mammals consistent with its actions as a neurotransmitter/neuromodulator. Thus, agmatine is synthesized unequally in brain by arginine decarboxylase (ADC); is stored in neurons and axon terminals with a heterogeneous distribution; is released from synaptosomes by depolarization; is enzymatically converted by agmatinase to putrescine; interacts not only with alpha2-adrenergic and I-receptors in the CNS, but also may selectively block NMDA receptor channels; and, when administered centrally, has several potent biological actions. Clarification of its role in normal brain function, however, has not yet been fully established, in part because of the absence of agents that selectively affect its biosynthesis or degradation.

Imidazoline receptors (I-receptors) are considered as potential therapeutic targets for a spectrum of stress-induced illnesses. Yet, I-receptors remain poorly defined at the molecular level. In this study, candidate imidazoline receptor proteins were compared using two imidazoline receptor-selective antisera of diverse origins. One antiserum was derived from affinity-purified imidazoline-binding protein. The second antiserum was produced as an anti-idiotypic antiserum, from purified IgG selective for imidazolines. Despite such diverse origins, both antisera co-identified an 85 kDa band on western blots from a variety of tissues. The integrity of the 85 kDa band was dependent on protection by eight different protease inhibitors. Other proteolytic breakdown products (obtained after homogenization with only one protease inhibitor) were comparable in size to previously reported smaller immunoreactive bands. The full-size 85 kDa band was also enriched in plasma membrane fractions and abundant in rat PC12 cells and brain regions known to be abundant in I1 binding sites. Furthermore, the immunodensity of the 85 kDa band, against anti-idiotypic antiserum, was linearly correlated with reported I1 site radioligand Bmax values (r2 = 0.8736, P = 0.0002) across nine rat tissues. Therefore, a possible candidate for the full-length imidazoline receptor(s) appears to be an 85 kDa protein.

The I1-imidazoline receptor is a novel brainstem modulator of sympathetic outflow that is elevated on platelets and in brains of depressed patients. A positive correlation has been reported (accompanying manuscript) between plasma norepinephrine (NE) concentrations and the densities (Bmax) of platelet I1 binding sites (I1 sites). I1-candidate proteins of 33 kDa and 85 kDa are now identified on Western blots probed with anti-imidazoline receptor antiserum (IRBP antiserum), that correlate with Bmax values for I1 sites. Furthermore, a human megakaryoblastoma cell line (MEG-01) has been used to study the regulation of these proteins on megakaryocytic cells, while bovine adrenal chromaffin cells provide a standard I1 cell type for comparison. Both the 33 kDa and 85 kDa IRBP-immunoreactive bands were enriched in plasma membrane fractions. IRBP antiserum did not cross-react with I2 imidazoline binding sites located on platelet mitochondrial membranes. The 85 kDa band was enhanced under conditions lacking fetal bovine serum (FBS) from the culture medium 6 h prior to harvesting. Conversely, 33 kDa protein was enhanced on MEG-01 cells grown in the presence of 10% FBS; suggesting that a precursor (85 kDa) and product (33 kDa) relationship might be induced by serum. The 85 kDa band was robustly up-regulated in response to imidazoline receptor-sensitive ligands; moxonidine, idazoxan and agmatine (10 microM each for 6 h). NE also up-regulated the 85 kDa IRBP-immunoreactive protein on MEG-01 membranes, but to a lesser extent. Idazoxan, an imidazoline alpha 2-antagonist, off-set its induction of 85 kDa protein by reducing the 33 kDa band. Yohimbine, a non-imidazoline alpha 2-antagonist, was ineffective alone, or in combination with moxonidine (up to 40 microM), but yohimbine blocked NE's induction of the 85 kDa band. Therefore, a rise in either plasma NE and/or endogenous I-site ligands (i.e. agmatine) could explain an elevation of imidazoline receptors observed in depression.

Imidazoline (I) receptors have been implicated in the regulation of arterial blood pressure and behavior although their distribution in the central nervous system (CNS) remains in question. Presumptive I- receptor sites were detected in the rat central nervous system with a polyclonal antibody to an imidazoline receptor protein (IRP) with binding characteristics of the native receptor. IRP-like immunoreactivity (LI) was detected in neurons and glia by light and electron microscopy. Spinal cord: processes were heavily labeled in superficial laminae I and II of the dorsal horn, lateral-cervical and -spinal nuclei and sympathetic cell column. Medulla: label was concentrated in the area postrema, rostral, subpostremal and central subnuclei of nucleus tractus solitarii, spinal trigeminal nucleus caudalis, and inferior olivary subnuclei. Visceromotor neurons in the dorsal vagal and ambigual nuclei were surrounded by high concentrations of immunoreactive processes. In reticular formation, label was light, though predominant in the intermediate reticular zone and ventrolateral medulla. Pons: label was detected in the neuropil of the periventricular gray, concentrated in the dorsal- and external-lateral subnuclei of lateral parabrachial nucleus, and present intracellularly in the mesencephalic trigeminal nucleus. Midbrain: IRP-LI was most heavily concentrated in the interpeduncular nucleus, nuclei interfascicularis and rostral-linearis, the subcommissural organ, central gray, and in glia surrounding the cerebral aqueduct. Diencephalon: high densities were detected in the medial habenular nucleus, nucleus paraventricularis thalami, other midline-intralaminar thalamic nuclei, the supramammillary and mediobasal hypothalamic nuclei. In the median eminence, immunolabeled processes were restricted to the lamina interna and lateral subependymal zone. Telencephalon: IRP-LI was concentrated in the central amygdaloid nucleus, bed nucleus of stria terminalis and globus pallidus, followed by moderate labeling of the medial amygdaloid nucleus, amygdalostriatal zone and caudoputamen, the hilus of the dentate gyrus, and stratum lacunosum-moleculare of field CA1 of Ammon's horn. The subfornical organ and organum vasculosum lamina terminalis were filled with diffuse granular immunoreactivity. Ultrastructural studies identified IRP-LI within glia and neurons including presynaptic processes. I-receptor(s) localize to a highly restricted network of neurons in the CNS and circumventricular regions lying outside of the blood-brain barrier. Putative imidazoline receptors have a unique distribution pattern, show partial overlap with alpha 2 adrenoreceptors and are heavily represented in sensory processing centers and the visceral nervous system.

Clonidine, moxonidine, and rilmenidine are centrally acting antihypertensive agents that lower arterial pressure by inhibiting the tonic activity of sympathoexcitatory neurons in the rostral ventrolateral medulla. Competing hypotheses have been put forward by different investigators to explain the sympathoinhibition evoked by "imidazoline drugs": either via central actions at alpha 2-adrenergic receptors or novel I1-imidazoline receptors. These different perspectives are presented in the accompanying reviews.

Vascular smooth muscle cells of rat aorta express imidazoline receptors whose stimulation, by drugs or an endogenous ligand, agmatine, inhibits serum-stimulated proliferation. We investigated whether imidazoline receptors are expressed in human vascular smooth muscle cells if their stimulation is antiproliferative. Cultured human coronary artery vascular smooth muscle cells express a nonadrenergic binding site for 3H-idazoxan and an imidazoline receptor-binding protein as revealed by immunocytochemical and immunoblot analyses with a specific antibody. Idazoxan and agmatine dose-dependently inhibited serum-stimulated proliferation of these cells as measured by the incorporation of 3H-thymidine (IC50: 5 and 70 micromol/L, respectively) and serum-stimulated expression of proliferating cell nuclear antigen and cell numbers. The agents inhibited proliferation of human and rat (aorta) smooth muscle cells stimulated by either norepinephrine (6560+/-440 disintegrations per minute norepinephrine versus 3345+/-220 norepinephrine and idazoxan), angiotensin II (7680+/-335 disintegrations per minute angiotensin II versus 3769+/-450 angiotensin II and idazoxan), or platelet-derived growth factor (IC50: 3 micromol/L for idazoxan and 40 micromol/L for agmatine), indicating inhibition of mitosis mediated by G-protein or receptor tyrosine kinase pathways. We conclude that human vascular smooth muscle cells express imidazoline-receptors whose activation inhibits proliferation by interacting at a distal step in an intracellular signal cascade common to G-protein and receptor tyrosine kinase mitogenic pathways. Agmatine, synthesized in endothelium, may act as a paracrine regulator of vascular smooth muscle cell proliferation through imidazoline receptors, and agents acting at this site may be useful in treating vascular hyperplasia.

Background: Imidazoline receptors are a newly discovered family of receptors, some of which, like alpha 2-adrenoceptors, have a presynaptic inhibitory effect on the release of norepinephrine. The aim of this study was to identify by immunodetection imidazoline receptor proteins in human platelets and the brain to assess their status in depression and suicide., Methods: Platelets were collected from 26 drug-free depressed patients and 26 controls. Specimens of frontal cortex (Brodmann area 9) were collected from 13 suicide victims and 11 controls. Levels of imidazoline receptor proteins were assessed by immunoblotting techniques. Solubilized imidazoline receptors were separated by gel electrophoresis, transferred to nitrocellulose membranes, labeled with a specific anti-imidazoline receptor antiserum, and quantitated by image analysis., Results: Platelet and brain membranes expressed similar 45-kd imidazoline receptor proteins, and their mean +/- SEM immunoreactivities were found to be increased in depressed patients (platelets, 40% +/- 5%) and suicide victims (brain, 51% +/- 14%). Platelets also expressed a 35-kd imidazoline receptor protein that was also found to be up-regulated in depressed patients (21% +/- 4%). In contrast, brain membranes did not express this 35-kd protein but revealed a 29/30-kd imidazoline receptor protein that was found to be down-regulated in suicide victims (19% +/- 3%). In a subset of depressed patients who underwent antidepressant treatment, a change in the immunoreactivity of the up-regulated 45-kd platelet imidazoline receptor protein (-35% +/- 5%), but not of the 35-kd protein, was observed., Conclusion: The results support a role for the newly discovered imidazoline receptors (mainly the 45-kd receptor expressed in the brain and platelets) in the pathogenesis of depression.

We investigated whether vascular smooth muscle and endothelial cells express imidazoline (I-) receptors, their endogenous ligand agmatine and/or its biosynthetic enzyme arginine decarboxylase (ADC), and if I-receptors regulate smooth muscle proliferation. Membranes of cultured rat aortic smooth muscle or bovine pulmonary artery endothelial cells bind 3H-idazoxan. Binding was inhibited by: idazoxan > cirazoline > UK 14,304 > naphazoline > tolazoline > guanabenz > amiloride > clonidine = phentolamine > > epinephrine. Agmatine competitively inhibited binding of 3H-idazoxan (Ki of 240 +/- 25 nM). Smooth muscle and endothelial cells were immunostained in vitro and in situ by antibodies to an I-receptor binding protein and by antibodies to agmatine. Rat aorta also contained substantial amounts of agmatine measured by HPLC (8.69 +/- 1.1 ng/g). Membranes of rat aorta and cultured endothelial but not smooth muscle cells expressed substantial amounts of ADC. The incorporation of 3H-thymidine and numbers of smooth muscle cells stimulated by fetal calf serum was inhibited > 90% by: idazoxan > UK 14,304 > naphazoline > cirazoline > agmatine highly correlating (r= .996; P < .01) with affinities for 3H-idazoxan binding site. Tolazoline, but not rauwolscine, blocked the antiproliferative action of idazoxan. We conclude that (1) vascular smooth muscle and endothelium contain imidazoline receptors of the I2 subclass; (2) stimulation of the receptors inhibits vascular smooth muscle proliferation; (3) agmatine, synthesized in endothelium by ADC may be an endogenous agonist of I2 receptors to inhibit vascular growth and (4) I2 receptors may be functionally active.

Clonidine, idazoxan, and related imidazoline adrenergic drugs bind to non-adrenergic sites in brain and several peripheral tissues. These sites, termed imidazoline receptors, appear to exist in two major subclasses, I1 sites labeled by clonidine and I2 sites labeled by idazoxan. In this study, we investigated whether rat prostate expresses imidazoline receptors and, if so, whether their expression can be regulated by circulating testosterone. Studies in rat ventral prostate membrane revealed that [3H]idazoxan, but not [3H]p-aminoclonidine, bound to non-adrenergic sites. The binding of [3H]idazoxan was saturable (Bmax: 941 +/- 105 fmol/mg protein) and high affinity (KD: 16.4 +/- 2.3 nM). The rank order of the inhibition of binding by imidazoline ligands was cirazoline > clonidine > UK 14,304 > guanabenz, indicating an I2 subclass of imidazoline receptors. Bilateral orchiectomy increased the number of binding sites (Bmax) for [3H]idazoxan without changing the affinity (KD). Testosterone replacement, while completely restoring the plasma testosterone levels, only partially reversed the increase in Bmax. In contrast, the binding of [3H]idazoxan to prostate membranes of rats in different age groups (4, 7, and 16 months) revealed a progressive decrease in the Bmax without any change in KD. We conclude that the rat prostate expresses the I2 subclass of imidazoline receptors and that the expression is regulated by circulating testosterone.

Imidazoline (I) receptors constitute a family of nonadrenergic high-affinity binding sites for clonidine, idazoxan, and allied drugs. One major subclass, the I1 receptors, whose subcellular distribution and signal transduction mechanisms are uncertain, partly mediates the central hypotensive actions of clonidine-like drugs. The I2 receptors, another subclass, are mitochondrial, not G protein coupled, and have diversified functions. Several endogenous ligands for I receptors, collectively termed clonidine-displacing substances (CDSs), have been detected in tissues and serum, but the structure of only one, agmatine (decarboxylated arginine), is known. Agmatine, widely distributed and bioactive, binds, like clonidine, to alpha 2-adrenergic and I receptors of all subclasses. The presence of agmatine and its biosynthetic enzyme in synaptosomes and specific neuronal pathways as well as serum suggests that it may be a novel neurotransmitter/hormone. Another CDS that binds to I receptors and to antibodies to imidazoline drugs has been detected, but its structure is unknown.

A group of clinically useful antihypertensive agents, including clonidine, moxonidine, and rilmenidine, are all ligands at alpha(2)-adrenergic and imidazoline (I-) receptors, the latter principally of the I1-subclass. These agents all lower blood pressure by reducing the activity of tonically active sympathoexcitatory reticulospinal neurons of the C1 area of the rostroventrolateral medulla (RVL). They tonically excite preganglionic sympathetic neurons in the spinal cord by release of L-glutamate and mediate most reflexes influencing blood pressure. The RVL contains alpha(2)-adrenergic and I1-receptors, and there is evidence to suggest that both receptors may participate in the hypotensive actions of the drugs. However, because only activation of the alpha(2)-adrenergic receptors appears responsible for somnolence, the imidazoline-receptor agonists moxonidine and rilmenidine, both relatively selective for I-receptors, may have superior clinical utility in antihypertensive therapy, since they are sympatholytic and also suppress the generation of angiotensin II.

Imidazoli(di)nes bind to molecular entities different from alpha 2-adrenoceptors: the so-called imidazoline receptors (IRs). Two main types of IRs have been described, the clonidine- and the idazoxan-preferring types, as well as other IRs whose pharmacological properties do not fit either type, but little is known about the molecular features of these receptors. In this study, IR proteins have been solubilized from the rat brain, using the zwitterionic detergent CHAPS, and analyzed by pharmacological and immunological means two of the four peak discriminated by gel filtration chromatography using [3H]idazoxan binding and a specific antibody. The IR eluted in the first peak accounted for 80% of the specific binding of [3H]idazoxan to solubilized brain membranes, and its pharmacological features corresponded to the non-adrenoceptor component of [3H]idazoxan binding in rat brain native membranes. The elution volume of this peak corresponded to a 130-140-kDa protein, but immunoblot analysis with a specific anti-IR antiserum showed the presence of a approximate 45-kDa IR protein, suggesting that this receptor is either an oligomeric protein complex or that it is associated with other proteins. This result was in agreement with the isolation and immunodetection of a 45-kDa peptide by affinity chromatography, which supported the relationship between this protein and a rat brain imidazoline binding site. The second peak, accounting for 15% of the specific binding of [3H]idazoxan to solubilized membranes, had a Mr of approximately 65-70,000, as determined by gel filtration chromatography and immunoblotting.(ABSTRACT TRUNCATED AT 250 WORDS)

As adrenal medullary chromaffin cells express imidazoline binding sites in the absence of alpha 2-adrenergic receptors, these cells provide an ideal system in which to determine whether imidazolines can influence catecholamine gene expression through nonadrenergic receptors. This study evaluates the ability of clonidine and related drugs to regulate expression of the gene for the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) in the rat adrenal gland and in bovine adrenal chromaffin cell cultures. In vivo, PNMT and tyrosine hydroxylase (TH) mRNA levels increase in rat adrenal medulla after a single injection of clonidine. Clonidine also dose-dependently stimulates PNMT mRNA expression in vitro in primary cultures of bovine chromaffin cells, with a threshold dose of 0.1 microM. Other putative imidazoline receptor agonists, including cimetidine, rilmenidine, and imidazole-4-acetic acid, likewise enhance PNMT mRNA production showing relative potencies that correlate with their binding affinities at chromaffin cell I1-imidazoline binding sites. The effects of clonidine on PNMT mRNA appear to be distinct from and additive with those exerted by nicotine. Moreover, neither nicotinic antagonists nor calcium channel blockers, which attenuate nicotine's influence on PNMT mRNA production, diminish clonidine's effects on PNMT mRNA. Although 100 microM clonidine diminishes nicotine-stimulated release of epinephrine and norepinephrine in chromaffin cells, this effect appears unrelated to stimulation of imidazoline receptor subtypes. This is the first report to link imidazoline receptors to neurotransmitter gene expression.

We sought to determine if smooth muscle and endothelial cells of blood vessels express imidazoline receptors. Membranes of cultured smooth muscle cells specifically bind with high affinity to alpha 2-adrenergic ligands, [3H]p-aminoclonidine, [3H]rauwolscine, and [3H]idazoxan. All of [3H]rauwolscine and [3H]p-aminoclonidine but less than 10% of [3H]idazoxan binding was displaced by 10 microM epinephrine, indicating a nonadrenergic binding site for [3H]idazoxan. [3H]Idazoxan binding was inhibited with a rank order of potency: cirazoline > idazoxan > naphazoline >> guanabenz > amiloride > clonidine = phentolamine. Agmatine, an endogenous ligand for I-receptors, inhibited binding with a Ki of 240 +/- 25 nM. The binding of [3H]idazoxan to membranes of pulmonary artery endothelial cells was to both alpha 2-adrenergic and imidazoline receptors. Cultured smooth muscle cells, as well as rat carotid arterioles, were specifically immunostained by antibodies to an I-receptor-associated protein. We conclude that vascular smooth muscle and endothelial cells express not only alpha 2-adrenergic receptors but also I-receptors of the I2 subclass with high affinity for agmatine. Since serum contains an endogenous ligand for I-receptors, possibly agmatine, the results suggest the presence of a novel receptor mechanism on vascular smooth muscle which may regulate vascular tone.

1. There are several endogenous ligands that bind to I-receptors of both the I1 and I2 subclass. These include: (a) classic CDS, a partially purified entity isolated by the criteria that it displaces binding ligands to alpha 2- and I-receptors; (b) immunoreactive (ir)-CDS, a moiety that binds to antibodies raised against clonidine, para-amino-clonidine, or idazoxan; and (c) agmatine. 2. Classic-CDS, not yet defined structurally, binds to I1, I2, and alpha 2-adrenergic receptors, is neither a peptide nor a catecholamine, and has purportedly a molecular weight of 588 Da. By ligand binding assays, it was found in brain, serum, CSF, and placenta and in a neural-glial cell line. Partially purified classic CDS is bioactive. Like clonidine, it contracts aorta and vas deferens and inhibits platelet aggregation, effects largely attributable to agonism at alpha 2-adrenergic receptors. Unlike clonidine, it contracts rat gastric fundus and releases catecholamines from chromaffin cells, effects attributable to actions at I-receptors. Injected into the RVL, classic CDS alters arterial pressure, but the direction of change of pressure has differed between groups of investigators. However, in the absence of structure, it is possible that ligand binding and bioactivity may be attributable to different molecules. 3. Ir-CDS, also of unknown structure, is a material(s) that binds to antibodies raised against clonidine, PAC, or idazoxan. Ir-CDS, measured by radioimmunoassay, is unevenly distributed in brain with highest concentrations in the hypothalamus, midbrain, and dorsal medulla. It is contained in the gastric fundus, adrenal gland, heart, kidney, and serum in amounts substantially higher than found in brain. Ir-CDS may be elevated in the serum of some patients with hypertension and in the CSF of patients with structural brain disease. The concentration of ir-CDS and bioactivity on gastric fundus directly correlates, suggesting that it may share similarities with classic-CDS. However, until the structure of classic and ir-CDS is determined, the possibility that ligand binding and antibody recognition are properties of different molecules must be considered. 4. Agmatine (decarboxylated arginine) is the only endogenous molecule that, like CDS, binds to alpha 2- and I-receptors of both classes. It and its biosynthetic enzyme arginine decarboxylase are present in brain, and agmatine is widely distributed throughout the body. However, the distribution of agmatine and ir-CDS differs, whereas the biological actions of agmatine do not mimic those of classic CDS. Its presence raises the possibility of an alternative pathway for polyamine biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

I-receptors can be localized immunocytochemically in rat nervous system with polyclonal antibodies to an IRBP. I-receptors are cytoplasmic and detected in neuronal perikarya, processes, and glia. Labeled neuronal perikarya in the CNS are uncommon and localized to the mesencephalic trigeminal nucleus. I-receptors are heavily represented in primary sensory systems including: somatosensory systems (spinal and trigeminal) and visceral afferent systems (NTS), in central networks subserving autonomic regulation, neuroendocrine control and emotional behaviors, in circumventricular (neurohaemal) organs and in nonneuronal cells including astrocytes with regional densities paralleling neuronal innervation. The distributions of I-receptors and alpha 2-adrenergic receptors partially differ. I-receptors in the CNS appear to relate broadly to the visceral brain and its afferent inputs, particularly pain. Its functions may relate to regulation of integrative behaviors related to stress.

Rilmenidine and idazoxan reduce the volume of focal ischemic infarctions produced by occlusion of the middle cerebral artery in the rat by 33% and 29%, respectively, by preserving neurons within the ischemic penumbra. In contrast, the alpha 2-selective antagonist SKF-86466 is without effect. The neuroprotective action of rilmenidine is dose dependent and parallels its antihypertensive actions. Neuroprotection cannot be attributed to changes in cerebral blood flow. We conclude that the neuroprotection produced by rilmenidine is attributable to an interaction with imidazoline receptors (IRs). However, the mechanism of action is not obvious. If it results from an action within the penumbra (direct), it is mediated by mitochondrial I-2 receptors on astrocytes, since cortical neurons are devoid of IRs. Neuroprotection might occur by selectively stimulating Ca2+ uptake into astrocytes, and thereby reducing Ca2+ uptake into neurons. Alternatively, rilmenidine may act indirectly to activate pathways in the brain that are neuroprotective. Neuroprotection may be a therapeutic target for rilmenidine and allied agents that act at central IRs.

A specific anti-imidazoline receptor (IR) protein antiserum was used to detect these putative receptors in brain and other tissues. In immunoblotting experiments, various brain areas and tissues expressed a double band of 29-30 kDa, and some of them bands of 47 and 66 kDa. The human medulla oblongata also showed bands of 44 and 50 kDa; and the rat adrenal medulla, bands of 58 and 86 kDa. Total immunoreactivity correlated with the density of IR binding sites in the various tissues. This is first evidence of heterogeneity of immunoreactive IRs in the mammalian brain.

We sought to characterize the non-adrenergic binding site for imidazolines, the imidazoline receptor in whole membranes and subcellular compartments of chromaffin cells of bovine adrenal medulla. [3H]Idazoxan exhibited saturable and high affinity (KD = 5 nM) binding to chromaffin cell membranes fully displaceable by idazoxan and cirazoline but not by epinephrine or rauwolscine. Binding sites were highly enriched in mitochondrial but not plasma membranes and absent from nuclear fractions. The rank order of potency for displacement of [3H]idazoxan from mitochondrial membranes was: cirazoline > idazoxan > naphazoline > amiloride > detomedine > clonidine >> phentolamine > cimetidine = imidazole 4-acetic acid > p-iodoclonidine = epinephrine = norepinephrine = rauwolscine. Binding was also inhibited with high affinity by the purported endogenous ligand clonidine-displacing substance and by K+ and the K(+)-channel antagonists 4-aminopyridine and tetraethylammonium bromide but not Na+. We conclude that: (a) adrenal chromaffin cells express imidazoline receptors but not alpha 2-adrenergic receptors; (b) these sites are predominantly localized to adrenal medullary mitochondria; and (c) imidazoline receptors conform to an idazoxan preferring (I-2) rather than the clonidine preferring (I-1) subclass and are amiloride sensitive. The data support the view that alpha 2-adrenergic and imidazoline receptors are distinct receptor species and that adrenal chromaffin cells would be a useful cultured cell system, expressing only imidazoline receptors, for further molecular and functional studies of the receptors.

Clonidine and related imidazoline agents, beside binding to alpha 2-adrenergic receptors, have been shown to bind to a non-adrenergic site (imidazoline sites) in brain and peripheral tissues. However, which cell types in brain, namely neurons or glia, express this binding site and the cellular effects of activation of this site are not known. We investigated the cellular localization of imidazoline binding sites in cultured rat cortical astrocytes and neurons. Membranes prepared from cultured astrocytes showed specific, high affinity binding (KD: 4 nM) for 3H-idazoxan with about tenfold higher number of binding sites than alpha 2-adrenergic sites (Bmax: 220 vs. 20 fmol/mg protein). Displacement studies exhibited the rank order of potency: cirazoline > idazoxan > amiloride > clonidine >>> epinephrine = ruawolscine defining this site as I-2a subtype of imidazoline binding sites. Moreover, the binding was inhibited by K+ but not by Na+, another characteristic of imidazoline binding sites. In contrast, membranes prepared from cultured neurons showed fewer binding sites for 3H-idazoxan that were completely displayed by adrenergic agents. Incubation of astrocytes with idazoxan, but not rauwolscine, resulted in a concentration-dependent increase in the levels of mRNA for the astrocyte specific molecule glial fibrillary acidic protein. We conclude that (a) the non-adrenergic imidazoline binding sites are expressed in astrocytes but not in neurons in rat cerebral cortex and (b) these "receptors" may influence astrocyte physiology by regulating the levels of GFAP.

Polyclonal antibodies were raised in rabbits against a 70-kDa ligand-binding protein of the imidazoline receptor purified from solubilized bovine adrenal chromaffin cell membranes by ligand affinity chromatography. The antibodies labeled a single protein (approximately 70 kDa) in Western blots of bovine adrenal chromaffin cell membranes, inhibited 40% of specific [3H]idazoxan binding to imidazoline receptors in chromaffin cell membranes, and specifically immunoprecipitated 75% of all imidazoline-binding activity of solubilized chromaffin cell membrane proteins. The antibodies specifically immunostained heterogeneous subsets of cultured bovine chromaffin cells. They stained subpopulations of chromaffin cells of rat adrenal medulla but not the cells of adrenal cortex. We conclude that the antibodies recognize with high specificity and selectivity a approximately 70-kDa binding protein associated with or representing the imidazoline receptor that is expressed in mammalian species. Highly specific antibodies against the imidazoline receptor protein will permit mapping of the distribution of imidazoline receptors in brain and periphery and also may be useful as probes in cloning genes encoding the imidazoline receptors.

We sought to isolate and partially purify proteins corresponding to the binding element of the imidazoline receptor (IR) from adrenal chromaffin cell membranes. These cells express IRs of the I-2 subclass and not alpha 2-adrenergic receptors. Proteins were solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate-containing buffer and were assayed by binding of [3H]idazoxan, an imidazoline radioligand. Two ligand affinity resins, p-aminoclonidine-Trisacryl GF-2000 (PAC-ReactiGel) and idazoxan-PharmaLink agarose (IDA-agarose), were synthesized. These allowed purification by single-step affinity chromatography of a major receptor binding protein component of 70 kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and [3H]idazoxan binding assay. The purified imidazoline-binding proteins from IDA-agarose and PAC-ReactiGel had similar affinities for the radioligand [3H]idazoxan (Kd = 3.7 and 4.9 nM, respectively) and a displacement profile, showing sensitivity to imidazoline agents (cirazoline > clonidine) and insensitivity to catecholamines and adrenergic agents (epinephrine approximately rauwolscine), that was similar to that of the intact membrane receptor. The imidazoline-binding protein did not bind to concanavalin A, suggesting that it may not be glycosylated or that the sugar moieties present are not recognized by this lectin. The results indicate that IR and alpha 2 receptor proteins may be biochemically distinct and that IDA-agarose and PAC-ReactiGel columns are useful for purification of sufficient quantities of imidazoline-binding proteins to allow for structural and functional studies of the IR.

Clonidine, idazoxan, rilmenidine, and comparable agents bind to imidazol(in)e (IR), as well as alpha 2-adrenergic, receptors. Interaction with IRs mediates the hypotension elicited by these drugs at their site of action in the rostral ventrolateral medulla oblongata (RVL) and probably the neuroprotection in focal ischemic cerebral infarction. Unlike alpha 2-adrenergic receptors, IRs are not coupled to G-proteins. Their native ligand may be clonidine-displacing substance (CDS), a potent, partially purified adrenomedullary secretagogue, distributed regionally in brain and some peripheral organs. IRs and CDS may be important in the genesis, expression, and/or therapy of hypertension and stroke.

Identification of nonadrenergic binding sites for clonidine and related imidazolines in brain and peripheral tissues and partial purification of an endogenous ligand for these sites have led to the postulation of a novel transmitter/receptor system. The receptors seem to be present in adrenal medulla and to regulate chromaffin cell function. The present study was undertaken to test the ability of the putative endogenous ligand clonidine-displacing substance (CDS) to displace [3H]idazoxan binding to adrenal chromaffin cell membranes and to release catecholamines from cultured chromaffin cells. CDS potently displaces [3H]idazoxan binding to chromaffin cell membranes, with an IC50 of 5 units. The displacement of [3H]idazoxan binding by CDS was not modified by guanosine 5'-(beta, gamma-imido)triphosphate, suggesting that the imidazoline binding sites may not be GTP-binding protein-coupled receptors. CDS produced a large release of catecholamines from chromaffin cells, and the release was partially blocked by cobalt, a calcium channel blocker. The calcium-dependent release reached a plateau above 5 units of CDS, with a maximal response at 15 min. It is concluded that endogenous CDS, prepared from brain, regulates the secretion of catecholamines from adrenal chromaffin cells, probably by activating imidazole receptors.

Clonidine and related imidazoline compounds bind to alpha 2-adrenergic as well as to newly described non-adrenergic imidazole/imidazoline receptors in brain and peripheral tissues. The present study was undertaken to identify the signal transduction mechanism coupled to this new class of receptors (imidazole receptors) using bovine adrenal chromaffin cells. Clonidine did not modify the basal or forskolin-stimulated production of cyclic AMP (cAMP), suggesting the absence of functionally active alpha 2-adrenergic receptors in adrenal chromaffin cells. Clonidine also failed to modify the basal and GTP gamma S- or carbachol-stimulated increase in phosphoinositide hydrolysis. However, clonidine increased significantly the production of cyclic GMP (cGMP) as well as the uptake of 45Ca2+. The cGMP response to clonidine was slower (peak at 15 min) and smaller (only about 50% over control) than the response to acetylcholine and was not shared by other agents that bind to imidazole receptors. In contrast, all agents that bind to imidazole receptors increased the influx of 45Ca2+ into chromaffin cells. It is concluded that (a) alpha 2-adrenergic and imidazole receptors are functionally distinct and linked to different signal transduction mechanisms; (b) the classical G-protein coupled soluble second messenger systems are not coupled to imidazole receptors; (c) clonidine may increase cGMP by a non-receptor-mediated intracellular action; and (d) imidazole receptors may regulate intracellular calcium levels through an ion regulating system that may be different from calcium channels.

We sought to determine the site of action and receptor type responsible for the antihypertensive actions of rilmenidine, an oxazoline analogue of clonidine. In anesthetized paralyzed rats decerebration did not alter the dose dependent reductions in arterial pressure and heart rate elicited by i.v. drug. Rilmenidine microinjected bilaterally into the C1 area of the rostral ventrolateral medulla (RVL), but not nucleus tractus solitarii (NTS) nor caudal ventrolateral medulla (CVL), elicited dose-dependent falls in arterial pressure and heart rate at doses an order of magnitude less than required systemically. Prior microinjection into the C1 area of the selective alpha 2-adrenoceptor antagonist SKF-86466, even at high doses, failed to modify the hypotension to i.v. rilmenidine. However, microinjection of 3- to 10-fold lower doses of idazoxan, a ligand for imidazole as well as alpha 2-adrenoceptors, blocked the effects. Rilmenidine also competed with the clonidine analogue [3H]p-aminoclonidine ([3H]PAC) at specific binding sites in membranes of bovine ventrolateral medulla and frontal cortex. In RVL rilmenidine competed with binding to imidazole and alpha 2-adrenergic binding sites with a 30-fold selectivity for the imidazole binding sites. In frontal cortex binding was of lower affinity and restricted to alpha 2-adrenergic sites. We conclude that rilmenidine, like clonidine, acts to lower arterial pressure by an action on imidazole receptors in the C1 area of RVL. The higher selectivity of rilmenidine for imidazole to alpha 2-adrenoceptors as compared to clonidine may explain the lower sedative effects of rilmenidine.

The rostral ventrolateral medulla is the primary site of action for clonidine, a centrally acting antihypertensive. In the rostral ventrolateral medulla, clonidine binds not only to alpha-2 adrenergic receptors but also to specific imidazole sites. In order to determine whether a putative imidazole receptor mediates the hypotensive action of clonidine, a series of compounds was tested 1) in vitro for binding affinity at imidazole and alpha-2 sites and 2) in vivo for ability to lower arterial pressure and heart rate when microinjected directly into the rostral ventrolateral medulla. Hypotensive potency was correlated with affinity at imidazole sites (r = 0.84), but not with alpha-2 affinity (r = -0.05). The bradycardia elicited by this series of compounds also correlated with affinity at imidazole receptors (r = 0.89), but not with alpha-2 affinity (r = 0.10). Furthermore, the imidazole idazoxan selectively reversed the fall in arterial pressure elicited by clonidine, whereas SKF-86466, an alpha-2 antagonist which is not an imidazole, failed to attenuate clonidine's action. An imidazole receptor in the rostral ventrolateral medulla appears to mediate the central hypotensive actions of clonidine and related centrally acting imidazoles and may participate in the regulation of arterial pressure and heart rate.

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.