Imidazoline receptors associated with noradrenergic terminals in the rostral ventrolateral medulla mediate the hypotensive responses of moxonidine but not clonidine.

We determined whether the cardiovascular actions of central anti-hypertensive agents clonidine and moxonidine are dependent on noradrenergic or serotonergic innervation of the rostral ventrolateral medulla (RVLM) in conscious rabbits. 6-Hydroxydopamine (6-OHDA) or 5,6-dihydroxytriptamine (5,6-DHT) was injected into the RVLM to deplete noradrenergic and serotonergic terminals respectively. One, 2 and 4 weeks later, responses to fourth ventricular (4V) clonidine (0.65 microg/kg) and moxonidine (0.44 microg/kg) were examined. Destruction of noradrenergic pathways in the RVLM by 6-OHDA reduced the hypotensive response to 4V moxonidine to 62%, 47% and 60% of that observed in vehicle treated rabbits at weeks 1, 2 and 4 respectively. The moxonidine induced bradycardia was similarly attenuated (to 46% of vehicle). Conversely, 6-OHDA had no effect on the hypotensive or bradycardic effects of 4V clonidine. Efaroxan (I(1)-imidazoline receptor/alpha(2)-adrenoceptor antagonist; 3.5, 11, 35 microg/kg) and 2-methoxyidazoxan (alpha(2)-adrenoceptor antagonist; 0.3, 0.9, 3 microg/kg) equally reversed the hypotension to 4V clonidine, suggesting a mainly alpha(2)-adrenoceptor mechanism. Efaroxan preferentially reversed responses to moxonidine in both vehicle and 5,6-DHT groups and in the 1st week after 6-OHDA, suggesting a mechanism involving mainly I(1)-imidazoline receptors. This selectivity was subsequently lost in the 2nd and 4th weeks when the remaining hypotension was mainly mediated by alpha(2)-adrenoceptors. Depletion of serotonergic terminals did not alter the responses to either agonist nor did it change the relative effectiveness of the antagonists. Western blots of RVLM tissues probed with imidazoline and alpha(2)-adrenoceptor antisera showed a pattern of bands close to that reported in other species. The main effect of 6-OHDA was an 18% lower level of the 42 kDa imidazoline protein (P<0.05). We conclude that the hypotensive and bradycardic actions of moxonidine but not clonidine are mediated through imidazoline receptors and are dependent on intact noradrenergic pathways within the RVLM. Furthermore, the noradrenergic innervation may be associated with a 42 kDa imidazoline receptor protein.

Relevance of imidazoline receptors and agmatine to psychiatry: a decade of progress.

The cardiovascular relevance of imidazoline receptors (IR) has received tremendous attention since their discovery in 1984. However, evidence also has accumulated for the relevance of IR and an endogenous ligand, agmatine, to psychiatric disease. Emphasis has been placed on altered levels of the I(1)-imidazoline site on human platelets and in human postmortem brain tissue from depressed patients. Attempts at exploring the molecular nature of the I(1) protein have led to the cloning of a protein, IRAS. Based on transfection studies, IRAS seems to be involved in neuronal plasticity events. The I(2) site also appears linked to psychiatric research since some of these sites are localized to a specific domain on monoamine oxidases. Different peptides have been identified by means of an imidazoline-receptor-binding-protein (IRBP) antiserum, and these peptides, some of which appear to be fragments derived from IRAS, undergo changes in platelets and brain commensurate with altered mood states of the subject, notably depressive symptomatology. The search for an endogenous ligand for imidazoline receptor(s) also has led to agmatine, a decarboxylated derivative of arginine. Research on agmatine has mushroomed over the past several years and its measurement in the blood and brain has opened new research opportunities. This novel neurotransmitter interacts with a variety of receptors and has been implicated in mediation of stress responses, analgesia, drug addiction and withdrawal, convulsions, and neuroprotection. Given that IR and agmatine appear involved in a multitude of neurophysiologic and pathologic functions, the potential for new drug development is intriguing.

Structure-activity analysis of guanidine group in agmatine for brain agmatinase.

To identify a selective inhibitor of mammalian agmatinase, screening was performed on four analogues of agmatine with modifications directly to the guanidine group, six analogues with modifications to the carbon-amine chain, and one analogue with modifications at both ends of the molecule. Control compounds were aminoguanidine and 7-nitroindazole, known inhibitors of the three isoforms (i, e, n) of nitric oxide synthase (NOS), and arcaine, a known inhibitor of the glutamate NMDA receptor. These compounds were compared for inhibition of rat agmatinase and arginine decarboxylase (ADC) activities. Results were studied by ab initio Hartee-Fock descriptors based on optimized geometries and van der Waals radii. Linear correlations were obtained using various geometric and electronic descriptors of the carbon (C), nitrogen (N), and hydrogen (H) atoms in the guanidine moiety. The best fit equation for percent activity remaining of rat agmatinase was = 0.3225 D + 72.76 D1916 + 64.97 D1920 - 192.58 H21 - 253.09 (r = 0.89), where D is the calculated dipole moment, D1916 and D1920 are the N19-N16 and N19-N20 distances, respectively, and H21 is the charge on H21. This agmatinase equation is distinct from the equations fit for ADC, the three NOS isoforms, and inhibition of NMDA receptor binding.

Agmatine crosses the blood-brain barrier.

The question of whether agmatine crosses the blood-brain barrier has not been directly addressed, even though peripheral injection of this compound has produced behavioral responses in drug withdrawal, antidepressant, and anti-anxiety paradigms. Two models were used in this investigation. In the first, mice were injected intraperitoneally (i.p.) with agmatine (10, 50, or 300 mg/kg body weight) or arginine (600 mg/kg). After 1 or 3 hours, the animals were killed under gas anesthesia by perfusing their brains with ice-cold saline, and whole-brain agmatine was measured by HPLC. In parallel studies, a rhesus monkey was injected under gas anesthesia either intravenously (i.v.) with agmatine (30 mg/kg) or arginine (150 mg/kg), or intracerebroventricularly (i.c.v.) with agmatine (0.3 mg/kg i.c.v.). At varying times thereafter, cisterna magna cerebrospinal fluid (CSF) and blood plasma were collected and analyzed for agmatine levels. A rise in mouse brain agmatine was apparent after doses of 50 and 300 mg/kg i.p. Monkey CSF agmatine peaked in parallel with plasma agmatine 15 minutes following intravenous (i.v.) agmatine injection and at one sixth the level of the plasma peak. Monkey CSF agmatine peaked 43 minutes after i.v. arginine injection. The ventricular injection of agmatine resulted in a threefold sustained rise in blood plasma agmatine for at least 24 hours after injection. Therefore, agmatine and its precursor, arginine, cross the blood-brain barrier. CSF agmatine may be newly synthesized from peripherally injected arginine.

Atypical [(3)H]clonidine binding sites in human caudate and platelets on cryostat-cut sections.

Pharmacological characterization is described for a human imidazoline binding site (I-site) labeled by [(3)H]clonidine using standard autoradiographic method. Under conditions that mask alpha(2)-adrenergic sites, only a single high affinity site was observed in human caudate and blood platelet sections. Affinity constants (K(i)) were highly correlated between the two tissues (r = 0.90, P = 0.0003). This site is dissimilar to classical I(1) and I(2) sites, even though both tissues possess abundant I(1) and I(2) sites by filtration binding methods. It is suggested that the isotonic buffer conditions inherent to the procedure alter drug affinities to the classical I(1) site.

Association between I(2) binding sites and monoamine oxidase-B activity in platelets.

An I(2) imidazoline binding site on monoamine oxidase-B (MAO-B) is known to be encoded by a noncatalytic part of the enzyme, different from that which recognizes mechanism-based inhibitors. Herein, the relationship between I(2)-imidazoline binding sites and MAO-B activity has been assessed using a semi-purified source of MAO-B: platelet mitochondrial membranes. A positive correlation between I(2) sites and MAO-B activity was observed (r = 0.61, P = 0.0016) among 24 human subjects. Nevertheless, the variance in MAO-B activity cannot be completely accounted for in relation to I(2) sites. Therefore, I(2) density and MAO-B activity are only weakly correlated in platelets.

Cell signaling by imidazoline-1 receptor candidate, IRAS, and the nischarin homologue.

IRAS transfection into Chinese hamster ovary (CHO) or pheochromocytoma (PC-12) cell lines leads to the appearance of nonadrenergic binding sites for radiolabeled-clonidine. Nischarin is the mouse homologue of IRAS. IRAS seems to be a cytosolic protein that is anchored to the intracellular side of plasma membranes by a POX domain. Previous studies of IRAS-transfected HEK293 cells, and Nischarin-transfected 3T3 cells have shown this protein can intrinsically mediate cell growth and differentiation independent of imidazoline drugs through binding to insulin receptor substrates (HEK293 cells) and fibronectin receptors (3T3 cells). Herein, a growth-arrested PC-12 cell line stably transfected with IRAS is shown to express lower basal and nerve growth factor-stimulated levels of the activated form of extracellular receptor kinase than found in a vector-only transfected control cell line treated similarly. These findings suggest that IRAS is a membrane-associated mediator of receptor signaling.

IRAS splice variants.

The human I(1)-imidazoline receptor candidate gene, iras, has previously been cloned and mapped to locus 3p21.1-9 (also known as Nischarin; accession No. AC006208). By comparison to a database of expressed sequence tags (ESTs), three alternatively spliced transcripts have been deduced. A map of 21 exons was constructed for the medium-length transcript (IRAS-M) containing 5,232 base pairs (bp) and encoding 1,504 amino acids (aas). Introns 13B and 13C are inserted into the two alternative transcripts, forming IRAS-S and IRAS-L mRNA (short and long isoforms). Northern blots confirmed the existence of these mRNA isoforms. In most brain regions the order of mRNA abundance was IRAS-M > IRAS-L > IRAS-S mRNA. Although aas 1 through 510 are theoretically identical, truncated proteins could be derived from IRAS-S (2,678 bp transcript yields 515 aas) and IRAS-L (9,457 bp transcript yields 583 aas). Because exon-16 of the iras gene has been proposed to encode the functional domains of imidazoline and a-5 integrin binding, only IRAS-M is expected to possess I(1) receptor properties. Subtype-selective cDNA expression constructs were therefore generated and used to transfect CHO cells. High-affinity I(1) binding was endowed by IRAS-M and IRAS-L, but not by IRAS-S transfection.

Imidazoline receptor proteins are decreased in the hippocampus of individuals with major depression.

BACKGROUND: A downregulation of I(2)-imidazoline binding sites has been reported in frontal cortices of depressed suicide victims, according to I(2)-radioligand binding and confirmed by Western blotting. We now report Western blots of imidazoline receptor proteins in hippocampi of subjects with and without depression at the time of death. METHODS: Postmortem diagnoses were obtained from 17 cases of Axis I major depressive disorder and 17 cases without Axis I psychopathology. No psychotropic compounds were found in body fluids. Hippocampi were removed, sectioned, and assessed histologically. Throughout the analysis, each major depressive disorder sample was paired with a sample from a psychiatrically healthy subject based on equivalent life spans and postmortem delays. The antiserum was identical to that used in previous studies that reported a downregulation of cortical 29/30-kd imidazoline receptor-binding proteins in depression. RESULTS: A triad of imidazoline receptor-binding protein bands (40-50 kd) was detected in the human hippocampus. Subjects with major depressive disorder had significantly less intensity in each imidazoline receptor-binding proteins band compared with control subjects (p =. 01 for overall bands). CONCLUSIONS: The present results can be aligned with previous reports of downregulation of I(2)-radioligand binding sites in both cortices and platelets of depressed patients.

Imidazoline and alpha(2a)-adrenoceptor binding sites in postmenopausal women before and after estrogen replacement therapy.

BACKGROUND: Platelet alpha(2A)-adrenoceptors (alpha(2A)AR) and imidazoline binding sites (subtype I(1)) have been proposed as peripheral markers of brain stem receptors that mediate sympathetic outflow and are reported to be elevated in major depression. METHODS: In our study, p[(125)I]-iodoclonidine was used to assess platelet alpha(2A)AR and I(1) binding sites in healthy postmenopausal women (n = 34) compared with healthy women of reproductive age (n = 26). Receptor determinations were repeated in 19 postmenopausal women following 59-60 days of estrogen replacement therapy (ERT; 0.1 mg estradiol transdermal patches). RESULTS: I(1) binding sites were twofold higher in platelets of postmenopausal women compared with women of reproduction age but were down-regulated (normalized) after 59-60 days of ERT. All other binding parameters, including platelet alpha(2A)AR density, were not different between groups nor were they changed after ERT. Platelet I(1) densities after 59-60 days of ERT were positively correlated with plasma luteinizing hormone concentrations. CONCLUSIONS: It is suggested that increased imidazoline binding sites might be associated with mood and behavioral changes in postmenopausal women.

Autoradiographic comparison of [3H]-clonidine binding to non-adrenergic sites and alpha(2)-adrenergic receptors in human brain.

UNLABELLED: Clonidine is a partial agonist at brain alpha(2)-adrenoceptors (alpha(2)AR), but also has high affinity (K(D) = 51 nM) in homogenate binding assays for non-adrenergic imidazoline-binding sites (I-sites; imidazoline receptors). Herein, an autoradiographic comparison of [3H]-clonidine binding to I-sites and alpha(2)AR in sections of human brain is reported. For I-sites, the adrenergic component of 50 nM [3H]-clonidine binding was masked with either 60 microM norepinephrine (NE; alpha(2)AR agonist) or 12.5 microM methoxy-idazoxan (MIDX; selective alpha(2)AR antagonist), whereas the remaining non-adrenergic sites were studied by displacement with 20 microM cirazoline. Levels of [3H]-clonidine binding to alpha(2)AR and I-sites, determined in adjacent tissue sections, were positively correlated across 27 brain regions (p = 0.0003; r(2) = 0.385). The principal olivary nucleus and the rostral portion of the ventrolateral medulla had highest ratios of I-sites: alpha(2)AR (>4:1). Quantitative transepts drawn across hippocampal images revealed alpha(2)AR enrichments in the CA-1 and inner molecular layers of the dentate gyrus-areas not enriched in I-sites. Competition curves were generated for I-sites in caudate sections using 10 ligands known to distinguish between I(1) and I(2) subtypes. The rank-order of affinities were cirazoline > harmane > BDF6143 > idazoxan = tizanidine (affinities of agmatine, efaroxan, moxonidine, NE, and oxymetazoline were too low to be reliable). Only the endogenous I-site ligand, harmane, had a monophasic displacement curve at the non-adrenergic sites (Ki = 521 +/- 12 nM). IN CONCLUSION: 1) the distribution of non-adrenergic [3H]-clonidine binding sites in human brain sections was correlated with, but distinct from alpha(2)AR; and 2) the affinities of these sites was distinct from alpha(1)AR, alpha(2)AR, I(1) or I(2) sites as previously defined in membrane binding assays. The properties of this non-adrenergic [3H]-clonidine binding site are consistent with I-sites previously labeled by [3H]-cirazoline in rat brain.

Imidazoline receptor antisera-selected (IRAS) cDNA: cloning and characterization.

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.

Elevated P-selectin on platelets in depression: response to bupropion.

Increased platelet activation has been suggested as a possible reason for the increased vulnerability of depressed patients to ischemic heart disease (IHD). Translocation of P-selectin, an integral alpha-granule membrane protein, to the platelet surface is a measure of platelet activation. Herein, western blots of platelet plasma membranes containing P-selectin were quantified in patients with major depression (n=19; mean age=39 +/- 2 years) and healthy comparison subjects (n=17; mean age=36 +/- 2 years). None evidenced clinical signs of IHD, and only two patients had a lifestyle IHD risk factor (smoking). Blood was obtained from all 19 depressed patients before treatment, and 15 returned after 6-8 weeks of open-label bupropion treatment. Bupropion was chosen as the antidepressant because it did not elevate plasma norepinephrine or serotonin, endogenous agonists that can induce platelet degranulation. Western blotting revealed more P-selectin immunoreactivity (75 kD band) in depressed patients compared to healthy controls (P=0.003). After bupropion treatment, P-selectin remained high in depressed patients. beta3-Integrin, a reference plasma membrane protein that does not translocate during activation, was of equivalent density in depressed patients and healthy control subjects, and was unchanged after treatment with bupropion. P-Selectin failed to correlate with severity of illness based on the Hamilton Depression scale, or with the post-treatment plasma concentration of bupropion. The results suggest an elevation in P-selectin on platelet plasma membranes might be a trait marker for depression.

Imidazoline receptor antisera-selected cDNA clone and mRNA distribution.

A novel cDNA, designated Imidazoline Receptor Antisera-Selected cDNA-1 (iras-1), encodes a 167-kD protein. Two of its predicted peptides (42-43 kD) are immunologically consistent with a previously reported 1(1)-imidazoline binding protein. In the present study, two forms of iras mRNA (6.0 and 9.5 kb) were quantified across fresh rat tissues. Highest levels were found in brain (almost exclusively 6.0 kb in size), followed by liver and lung (9.5 > or = 6.0 kb iras mRNA), kidney (6.0 > 9.5 kb), heart (6.0 kb), spleen (6.0 > or = 9.5 kb), testes (6.0 > 9.5 kb), and skeletal muscle (6.0 > 9.5 kb). A correlation exists (p = 0.71, p = 0.05) between total (6.0 + 9.5 kb) iras mRNA and I1 BMAX values across rat tissues, corrected for housekeeping gene expression. Thus, total iras mRNA appears to be roughly proportional to the density of I1-imidazoline binding sites.

Effect of bupropion on immunodensity of putative imidazoline receptors on platelets of depressed patients.

A substantial number of studies have demonstrated increased imidazoline receptors (I1 binding sites) on platelets of depressed patients and downregulation following antidepressant treatments. Herein, imidazoline receptor binding protein (IRBP) antiserum was used to quantify imidazoline receptors on platelets of depressed patients before and after treatment with the atypical aminoketone antidepressant, bupropion. Western blots revealed an increase in IRBP-immunodensity (p = 0.01, two-tailed) in a 33 kDa protein band in untreated depressed patients (n = 21) as compared with controls (n = 17). This band has been positively correlated with I1 binding sites on platelets. Following 6 weeks' treatment with bupropion, IRBP-immunodensity was downregulated in depressed patients (p = 0.03, paired t-test); predominantly in responders (p = 0.005). Patients non-responsive to bupropion (n = 5) were significantly different from responders (p = 0.05) by exhibiting no elevation in IRBP-immunodensity at pre-treatment and no downregulation of the 33 kDa band after treatment. IRBP-immunodensity was negatively correlated (r = -0.79, p = 0.01) with plasma concentrations of bupropion and its metabolites at week-4 of BUP treatment. Thus, a 33-kDa IRBP on platelet plasma membranes is elevated in depression and normalized in responders to bupropion.

Plasma agmatine and platelet imidazoline receptors in depression.

Plasma agmatine concentrations are elevated significantly in depressed patients compared to healthy controls. Treatment with the antidepressant bupropion normalized plasma agmatine levels. Correlational evidence is presented that a change in plasma agmatine levels may lead to similar changes in platelet I1 imidazoline receptors.

Characterization of a partial cDNA clone detected by imidazoline receptor-selective antisera.

A cDNA clone has been isolated from a human hippocampal cDNA expression library by relying on the selectivity of two antisera that are specific for imidazoline binding proteins. A 1789 bp cDNA clone was sequenced and shown to contain a single open-reading frame that predicts a 66 kDa polypeptide, but it is truncated based on its lack of a stop codon and poly-A+ tail. Two regions of homology exist for the predicted amino acid sequence in common with chromogranin-A and B proteins, a zinc finger protein, and the ryanodine receptor. Northern blot analyses of poly-A+ mRNA from 36 human tissues indicated two differentially expressed transcripts of 6.0 and 9.5 kb. The 6.0 kb mRNA form was enriched in brain and endocrine tissues as compared to other tissues, but not in strict concordance with I1-imidazoline binding sites. The highest overall amounts of the combined transcripts were found in pituitary. In situ hybridization histochemistry revealed an enrichment of the message in neuronal cell bodies of the rat hippocampus and cerebellar cortex. This clone has some of the properties expected of an imidazoline receptor.