Imidazoline Receptor Proteins Research Articles

Investigation of morin-induced insulin secretion in cultured pancreatic cells.

Morin is a flavonoid contained in guava that is known to reduce hyperglycemia in diabetes. Insulin secretion has been demonstrated to increase following the administration of morin. The present study is designed to investigate the potential mechanism(s) of morin-induced insulin secretion in the MIN6 cell line. First, we identified that morin induced a dose-dependent increase in insulin secretion and intracellular calcium content in MIN6 cells. Morin potentiated glucose-stimulated insulin secretion (GSIS). Additionally, we used siRNA for the ablation of imidazoline receptor protein (NISCH) expression in MIN6 cells. Interestingly, the effects of increased insulin secretion by morin and canavanine were markedly reduced in Si-NISCH cells. Moreover, we used KU14R to block imidazoline I3 receptor (I-3R) that is known to enhance insulin release from the pancreatic β-cells. Without influence on the basal insulin secretion, KU14R dose-dependently inhibited the increased insulin secretion induced by morin or efaroxan in MIN6 cells. Additionally, effects of increased insulin secretion by morin or efaroxan were reduced by diazoxide at the dose sufficient to open KATP channels and attenuated by nifedipine at the dose used to inhibit L-type calcium channels. Otherwise, phospholipase C (PLC) is introduced to couple with imidazoline receptor (I-R). The PLC inhibitor dose-dependently inhibited the effects of morin in MIN6 cells. Similar blockade was also observed in protein kinase C (PKC) inhibitor-treated cells. Taken together, we found that morin increases insulin secretion via the activation of I-R in pancreatic cells. Therefore, morin would be useful to develop in the research and treatment of diabetic disorders.

Immunodetection and subcellular distribution of imidazoline receptor proteins with three antibodies in mouse and human brains: Effects of treatments with I1- and I2-imidazoline drugs

Various imidazoline receptor (IR) proteins have been proposed to mediate the effects of selective I1- and I2-IR drugs. However, the association of these IR-binding proteins with classic I1- and I2-radioligand binding sites remains somewhat controversial. In this study, three IR antibodies (anti-NISCH and anti-nischarin for I1-IRs; and anti-IRBP for I1/I2-IRs) were used to immunodetect, characterize and compare IR protein patterns in brain (mouse and human; total homogenate, subcellular fractionation, grey and white matter) and some cell systems (neurones, astrocytes, human platelets). Various immunoreactive IRs (specific molecular weight bands coincidently detected with the different antibodies) were related to I1-IR (167 kDa, 105/115 kDa and 85 kDa proteins) or I2-IR (66 kDa, 45 kDa and 30 kDa proteins) types. The biochemical characterization of cortical 167 kDa protein, localized in the membrane/cytosol but not in the nucleus, indicated that this I1-IR also forms part of higher order nischarin-related complexes. The contents of I1-IR (167 kDa, 105/115 kDa, and 85 kDa) proteins in mouse brain cortex were upregulated by treatment with I1-drugs (moxonidine, efaroxan) but not with I2-drugs (BU-224, LSL 61122). Conversely, the contents of I2-IR (66 kDa, 45 kDa and 30 kDa) proteins in mouse brain cortex were modulated by treatment with I2-drugs (decreases after BU-224 and LSL 61122, and increases after idazoxan) but not with I1-drugs (with the exception of moxonidine). These findings further indicate that brain immunoreactive IR proteins exist in multiple forms that can be grouped in the already known I1- and I2-IR types, which are expressed both in neurones and astrocytes.

Moxonidine as an Insulin Sensitizing Agent

Oral medications used to treat type 2 diabetes have important adverse effects. Novel agents with fewer risks are needed. Published studies indicate that moxonidine can have insulin sensitizing effects. The objective of this study is to provide insight into the mechanism(s) by which moxonidine acts as an insulin sensitizing agent. The acute and chronic effects of moxonidine on calcium handling by islet cells were determined in vitro using isolated mouse pancreatic islets. Time‐course and dose‐response studies were also carried out in cells, followed by Western blotting. Moxonidine lowered calcium levels in islet cells in a manner that was dose‐dependent up to 10nM, reversible and independent of the KATP channel. Moxonidine [1μM] caused an increased phosphorylation of both protein kinase B (PKB) and protein dependent kinase (PDK) by greater than 3 fold in HepG2 and HEK293 cells. The increased PKB phosphorylation could be blocked by efaroxan (10μM). Cells overexpressing the imidazoline receptor protein IRAS, showed greater phosphorylation of PKB compared to control, following moxonidine treatment. Moxonidine caused a time‐dependent increase in glucose uptake in HepG2 cells of up to 2.5 fold, and this could be blocked by efaroxan and by phosphatidyl inositol‐3‐ kinase inhibitor LY294002. These data suggests that moxonidine may regulate islet insulin release while sensitizing tissues to the action of insulin.These studies were supported in part by NIH1R15DK084468–01 A1and the Center for Health Disparities at Loma Linda University

Pharmacological Properties of the Central Antihypertensive Agent, Moxonidine

The sympathetic nervous system plays a central role in the pathophysiology not only of hypertension and other cardiovascular diseases but also metabolic disorders including disturbances of glucose and lipid homeostasis. A centrally acting sympathetic agent is therefore attractive not only for lowering blood pressure, but also intervening with multiple disease processes. Older agents such as clonidine and guanabenz have numerous side effects, including sedation and dry mouth that limit their acceptability to patients. Moxonidine and the related agent rilmenidine have greatly reduced side effects, because they have reduced activity at the α(2) -adrenergic receptors that mediate these undesirable actions. Instead, moxonidine and rilmenidine act primarily through a novel cellular site, termed the I(1) -imidazoline receptor. The molecular biology of the I(1) -imidazoline receptor protein has recently been described, and the cell signaling pathways linked to this protein have been characterized. Moxonidine has unique effects on a number of cell types through this unusual cellular site of action. There are multiple therapeutic implications of these cellular actions, especially for metabolic syndrome and its associated derangements in glucose and lipid metabolism. Finally, the clinical trials that seemed to identify an unfavorable outcome in severe heart failure are dissected and critiqued. We conclude that moxonidine and future successors to this agent could be of great value in treating multiple chronic diseases.

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 μg/kg) and moxonidine (0.44 μg/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/α 2-adrenoceptor antagonist; 3.5, 11, 35 μg/kg) and 2-methoxyidazoxan (α 2-adrenoceptor antagonist; 0.3, 0.9, 3 μg/kg) equally reversed the hypotension to 4V clonidine, suggesting a mainly α 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 α 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 α 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.

In vivo effects of the I(2)-alkylating agent BU99006 on the immunodensity of imidazoline receptor proteins in the mouse brain.

The binding sites for imidazol(ine)/guanidine drugs (identified inter alia with [(3)H]-clonidine and [(3)H]-idazoxan) are heterogeneous in nature, and various pharmacologic types of imidazoline receptors (IRs) have been characterized (I(1)R, I(2A)R, I(2B)R, and I(3)R). IR-receptor proteins have also been immunodetected using an antibody raised against an approximately 70-kD idazoxan/clonidine binding protein, which probably recognizes all types of IRs. In this study, the in vivo effects of the selective I(2)-alkylating agent BU99006 (5-isothiocyanato-2-benzofuranyl-2-imidazoline) on immunoreactive IR proteins were assessed in the mouse brain to unravel the molecular nature of the I(2)R subtypes. In mouse tissues (cerebral cortex, liver, testis, and kidney) this antibody revealed the presence of 30-, 43-, 45-, 66-, and/or 85-kD IR proteins. Treatment with BU99006 (20 mg/kg, intraperitoneally, for 4 hours) significantly decreased the immunodensity of specific IR proteins in the brain (30 kD: 249%; 45 kD: 237%; 66 kD: 218%). In contrast, the immunoreactivities of 43-kD and 85-kD IR proteins were not altered after I(2)R alkylation. Prolonged treatment with BU99006 (20 mg/kg, intraperitoneally. for 8 hours) resulted in modest but significant increases in the expression of all the immunodetected IR proteins in the mouse brain (20%-36%), suggesting compensatory increases in IR protein synthesis after alkylation of I(2) sites. The results indicate that the 30-kD and 45-kD proteins, but not the 43-kD and 85-kD proteins, immunodetected in the mouse brain are related to the I(2)R.

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.

Densities of I2-imidazoline receptors, imidazoline receptor proteins, and MAO-B sites in human gliomas and pituitary adenomas.

Densities of I2-imidazoline receptors, imidazoline receptor proteins, and MAO-B sites in human gliomas and pituitary adenomas.

Pharmacologic and molecular discrimination of I2-imidazoline receptor subtypes.

I2-imidazoline receptors (I2-IR) are characterized by their high affinity for imidazolines and guanidines and medium affinity for imidazolidines. The differential recognition of I2-IR by amiloride led to subtype these sites as amiloride-sensitive (I2A-IR) and amiloride-insensitive (I2B-IR). I2-IR labeled with [3H]idazoxan or [3H]2-BFI in the rabbit cerebral cortex (I2A-IR) displayed higher affinities for amiloride and amiloride analogs than in the rat cerebral cortex (I2B-IR). Other drugs tested displayed biphasic curves in competition experiments, indicating the existence of high and low affinity sites for both I2-IR subtypes. The drugs (+)- and (-)-medetomidine, bromoxidine, moxonidine, and clorgyline were more potent on the high and/or low affinity sites of I2B-IR than on I2A-IR. Preincubation (30 min at 25 degrees C) with 10(-6) M isothiocyanatobenzyl imidazoline (IBI) or with 10(-6) M clorgyline reduced by 40% and 26%, respectively, the binding of [3H]2-BFI to I2B-IR, but it did not alter the binding of the radioligand to I2A-IR. These results indicated that the I2-IR subtypes differ in their pharmacologic profiles and in the nature of the imidazoline binding site involved in clorgyline and IBI alkylation. In rat cortical membranes, western blot detection of immunoreactive imidazoline receptor proteins revealed a double band of approximately 29/30 kD and three less intense bands of approximately 45, approximately 66, and approximately 85 kD. In rabbit cortical membranes the antibody detected proteins of approximately 30, approximately 57, approximately 66, and approximately 85 kD. It is suggested that I2-IR may be related to more than one receptor protein and that I2-IR subtypes differ in the nature of the proteins implicated.

Lack of effect of reserpine on immunoreactive imidazoline receptor proteins in rat brain.

Annals of the New York Academy of SciencesVolume 881, Issue 1 p. 61-64 Lack of Effect of Reserpine on Immunoreactive Imidazoline Receptor Proteins in Rat Braina ANTONIO MIRALLES, ANTONIO MIRALLES Laboratory of Neuropharmacology, Associate Unit of the Institute of Neurobiology Ramón y Cajal/CSIC, Department of Biology, University of Balearic Islands, Cra. Valldemossa km 7.5, E-07071 Palma de Mallorca, SpainSearch for more papers by this authorANDRÉS OZAITA, ANDRÉS OZAITA Laboratory of Neuropharmacology, Associate Unit of the Institute of Neurobiology Ramón y Cajal/CSIC, Department of Biology, University of Balearic Islands, Cra. Valldemossa km 7.5, E-07071 Palma de Mallorca, SpainSearch for more papers by this authorCATALINA RIBAS, CATALINA RIBAS Laboratory of Neuropharmacology, Associate Unit of the Institute of Neurobiology Ramón y Cajal/CSIC, Department of Biology, University of Balearic Islands, Cra. Valldemossa km 7.5, E-07071 Palma de Mallorca, SpainSearch for more papers by this authorJESÚS A. GARCÍA-SEVILLA, JESÚS A. GARCÍA-SEVILLA Laboratory of Neuropharmacology, Associate Unit of the Institute of Neurobiology Ramón y Cajal/CSIC, Department of Biology, University of Balearic Islands, Cra. Valldemossa km 7.5, E-07071 Palma de Mallorca, SpainSearch for more papers by this author ANTONIO MIRALLES, ANTONIO MIRALLES Laboratory of Neuropharmacology, Associate Unit of the Institute of Neurobiology Ramón y Cajal/CSIC, Department of Biology, University of Balearic Islands, Cra. Valldemossa km 7.5, E-07071 Palma de Mallorca, SpainSearch for more papers by this authorANDRÉS OZAITA, ANDRÉS OZAITA Laboratory of Neuropharmacology, Associate Unit of the Institute of Neurobiology Ramón y Cajal/CSIC, Department of Biology, University of Balearic Islands, Cra. Valldemossa km 7.5, E-07071 Palma de Mallorca, SpainSearch for more papers by this authorCATALINA RIBAS, CATALINA RIBAS Laboratory of Neuropharmacology, Associate Unit of the Institute of Neurobiology Ramón y Cajal/CSIC, Department of Biology, University of Balearic Islands, Cra. Valldemossa km 7.5, E-07071 Palma de Mallorca, SpainSearch for more papers by this authorJESÚS A. GARCÍA-SEVILLA, JESÚS A. GARCÍA-SEVILLA Laboratory of Neuropharmacology, Associate Unit of the Institute of Neurobiology Ramón y Cajal/CSIC, Department of Biology, University of Balearic Islands, Cra. Valldemossa km 7.5, E-07071 Palma de Mallorca, SpainSearch for more papers by this author First published: 06 February 2006 https://doi.org/10.1111/j.1749-6632.1999.tb09342.x a This study was supported by DGICYT Grant PB94-0002-Mod C from MEC, Madrid, Spain. J.A.G.-S. is a member of the Institut d'Estudis Catalans. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Volume881, Issue1IMIDAZOLINE RECEPTORS AND THEIR ENDOGENOUS LIGANDS: CURRENT CONCEPTS AND THERAPEUTIC POTENTIALJune 1999Pages 61-64 RelatedInformation

Pharmacologic characterization of imidazoline receptor proteins identified by immunologic techniques and other methods.

Biochemical and pharmacologic evidence supports the heterogeneous nature of imidazoline receptors (IRs). However, only monoamine oxidase (MAO) (55- and 61-kD) isozymes have been identified as imidazoline binding site-containing proteins. Idazoxan-binding proteins of approximately 70- and approximately 45-kD of unknown amino acid sequences have been isolated from chromaffin cells and rat brain, respectively. Other proteins of approximately 27-30 to > 80 kD have been visualized by immunologic and photoaffinity labeling techniques in different tissues and species. The specific antiserum that recognizes the approximately 70-, approximately 45-, and approximately 29-kD IR proteins, but not MAO, was used to quantitate these proteins in the rat brain cortex. Treatments (7 days) with the I2-selective imidazoline drugs idazoxan (10 mg/kg), cirazoline (1 mg/kg), and LSL 60101 ([2-(2-benzofuranyl) imidazole; 10 mg/kg]) induced differential changes in these proteins: levels of the approximately 29-kD IR were increased by idazoxan and LSL 60101 (23%), levels of the approximately 45-kD protein only by cirazoline (44%), and those of the approximately 66-kD protein only by idazoxan (50%). These treatments also increased the densities of [3H]-idazoxan (I2) binding sites (32-42%). Chronic treatment with efaroxan, RX821002, and yohimbine (10 mg/kg), which possess very low affinity for I2-IRs, did not alter either their immunoreactivities or the density of I2 sites. Chronic treatment with MAO inhibitors clorgyline and phenelzine (10 mg/kg) and acute treatment with EEDQ (1.6 mg/kg, 6 h) induced decreases in the levels of these IR proteins (17-47%) and I2 sites (31-57%). Significant correlations were found when the mean percentage changes in immunoreactivity of IR proteins were related to the mean percentage changes in the density of I2 sites after treatment with the foregoing drug (r = 0.92, r = 0.69, and r = 0.75 for the approximately 29-, approximately 45-, and approximately 66-kD proteins, respectively). These results indicate that in the rat cerebral cortex, the I2 sites labeled by [3H]idazoxan are heterogeneous and that the related immunoreactive IR proteins contribute differently to the modulation of I2 sites after drug treatment.

Imidazoline receptor proteins in brains of patients with Alzheimer's disease

Imidazoline receptors (29/30- and 45-kDa proteins) were quantitated in postmortem brains of patients with Alzheimer's disease (AD) by using immunoblotting techniques and a specific antiserum. Increased levels of the 29/30-kDa protein (30%), 45-kDa protein (36%) and glial fibrillary acidic protein (88%) were found in the frontal cortex of AD patients. These findings are in line with the reported higher density of imidazoline receptors labelled by [ 3H]idazoxan in AD brains, suggesting that these imidazoline receptor proteins are related to the I 2-imidazoline receptor located in mitochondria of glial (astrocyte) cells.

Density of Imidazoline Receptors in Platelets of Euthymic Patients with Bipolar Affective Disorder and in Brains of Lithium-Treated Rats

Background: Platelet imidazoline receptors have been shown to be up-regulated in patients with unipolar major depression. This study examines the status of imidazoline receptor proteins in platelets of euthymic bipolar patients and in brains of lithium-treated rats. Methods: Platelets were collected from 12 bipolar patients (lithium-treated or drug-free) and brains from chronic lithium-treated rats. Imidazoline receptors were quantitated by immunoblotting, using a specific antiserum, and/or radioligand binding. Results: No changes in platelet imidazoline receptors (35-kDa and 45-kDa proteins) were found. Lithium treatment did not alter brain imidazoline receptors (29/30-kDa, 45-kDa, and 66-kDa proteins or density/affinity of [ 3H]-idazoxan binding sites). Conclusions: Imidazoline receptor proteins are not altered in platelets of euthymic patients with bipolar affective disorder.

Imidazoline receptor proteins are regulated in platelet-precursor MEG-01 cells by agonists and antagonists

The I 1-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 (B max) of platelet I 1 binding sites (I 1 sites). I 1-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 B max values for I 1 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 I 1 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 I 2 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 receptorsensitive ligands; moxonidine, idazoxan and agmatine (10 μM 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 α 2-antagonist, off-set its induction of 85 kDa protein by reducing the 33 kDa band. Yohimbine, a non-imidazoline α 2-antagonist, was ineffective alone, or in combination with moxonidine (up to 40 μM), 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.

Co-detection by two imidazoline receptor protein antisera of a novel 85 kilodalton protein

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 I 1 binding sites. Furthermore, the immunodensity of the 85 kDa band, against anti-idiotypic antiserum, was linearly correlated with reported I 1 site radioligand B max values ( r 2 = 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.

Immunocytochemical localization of an imidazoline receptor protein in the central nervous system

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 CA 1 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 α 2 adrenoreceptors and are heavily represented in sensory processing centers and the visceral nervous system.

Densities of I 2-imidazoline receptors, α2-adrenoceptors and monoamine oxidase B in brains of suicide victims

In the frontal cortex of suicide victims, the density of I 2-imidazoline receptors labeled by [ 3H]idazoxan was lower (40%) than that in healthy subjects. In the same brains, the immunoreactivity of a 29 30 kDa imidazoline receptor protein was also found to be significantly decreased (19%) and it showed a positive correlation with the density of I 2-imidazoline receptors. Also in the same brains, the density of α 2-adrenoceptor antagonist binding sites labeled by [ 3H]RX821002 (2-methoxy idazoxan) was found to be unchanged. The density of brain I 2-imidazoline receptors, correlated with aging in control subjects but not in suicides. The density of brain MAO-B sites labeled by [ 3H]Ro 19-6327 (lazabemide) in suicides was no different to that in age-matched controls. As expected, the density of brain MAO-B sites in suicides showed a positive correlation with age, but it did not correlate with the density of I 2-imidazoline receptors in the same brains. The data indicate that the I 2-imidazoline receptors labeled by [ 3H]idazoxan in the brain of suicides is related to a 29 30 kDa imidazoline receptor protein identified by immunoblot analysis. The data also inddicate that the brain I 2-imidazoline receptor cannot be identified with the MAO-B isoenzyme. The decreased density of I 2-imidazoline receptors in the brain of suicide victims might play a role in the pathogenesis of major depression.

Pharmacological and molecular discrimination of brain I2-imidazoline receptor subtypes.

I2-imidazoline receptors labelled with [3H]-idazoxan in the rabbit and rat brains displayed high and low affinity, respectively, for the guanidide amiloride; reinforcing the previous definition of I2A-imidazoline receptors expressed in the rabbit brain and I2B-imidazoline receptors expressed in the rat brain. Other drugs tested displayed biphasic curves in competition experiments, indicating the existence of high and low affinity sites for both subtypes of I2-imidazoline receptors. Among the drugs studied, bromoxidine, moxonidine, (+)- and (-)-medetomidine and clorgyline were more potent on the high and/or low affinity sites of I2B-than on their corresponding of I2A-imidazoline receptors (KiH ratios 20 to 65). No correlation was found for the potencies of the drugs tested at the low affinity sites of both I2-imidazoline receptor subtypes. Preincubation (30 min at 25 degrees C) with 10(-6) M clorgyline reduced by 60% the Bmax of [3H]-idazoxan binding to I2B-imidazoline receptors in the rat brain, but it did not affect the binding parameters of the radioligand saturation curves to I2A-imidazoline receptors in the rabbit brain. These results indicated that I2A- and I2B-imidazoline receptor subtypes differ in the pharmacological profiles of their high and low affinity sites and in the ability to irreversibly bind clorgyline. In rat cortical membranes western blot detection of immunoreactive imidazoline receptors proteins revealed a double band of approximately 29/30 kDa and two less intense bands of approximately 45 and approximately 66 kDa. In rabbit cortical membranes the antibody used detected proteins of approximately 30, approximately 57 and approximately 66 kDa. It is suggested that different imidazoline receptor proteins (approximately 45 vs approximately 57 kDa) may account for the different pharmacological profiles of I2-imidazoline receptor subtypes.

Immunodetection and quantitation of imidazoline receptor proteins in platelets of patients with major depression and in brains of suicide victims.

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. 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. 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. 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.

Pharmacological modulation of immunoreactive imidazoline receptor proteins in rat brain: relationship with non-adrenoceptor [3H]-idazoxan binding sites.

1. The densities of various imidazoline receptor proteins (with apparent molecular masses of approximately 29/30-45- and 66-kDa) were quantitated by immunoblotting in the rat cerebral cortex after various drug treatments. The modulation of these imidazoline receptor proteins was then compared with the changes in the density of non-adrenoceptor [3H]-idazoxan binding sites (I2-sites) induced by the same drug treatments. 2. Chronic treatment (7 days) with the I2-selective imidazol(in)e drugs idazoxan (10 mg kg-1), cirazoline (1 mg kg-1) and LSL 60101 (10 mg kg-1) differentially increased the immunoreactivity of imidazoline receptor proteins. The levels of the 29/30-kDa protein were increased by idazoxan and LSL 60101 (23%), the levels of the 45-kDa protein only by cirazoline (44%) and those of the 66-kDa protein only by idazoxan (50%). These drug treatments also increased the density of I2-sites (32-42%). 3. Chronic treatment (7 days) with efaroxan (10 mg kg-1), RX821002 (10 mg kg-1) and yohimbine (10 mg kg-1), which possess very low affinity for I2-imidazoline receptors, did not alter either the immunoreactivity of imidazoline receptor proteins or the density of I2-sites. 4. Chronic treatment (7 days) with the monoamine oxidase (MAO) inhibitors clorgyline (10 mg kg-1) and phenelzine (10 mg kg-1) decreased the immunoreactivity of the 29/30-kDa (17-24%), 45-kDa (19%) and 66-kDa (23-31%) imidazoline receptor proteins. The alkylating agent N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (1.6 mg kg-1, 6 h) also decreased the levels of the three imidazoline receptor proteins (20-47%). These drug treatments consistently decreased the density of I2-sites (31-57%). 5. Significant correlations were found when the mean percentage changes in immunoreactivity of imidazoline receptor proteins were related to the mean percentage changes in the density of I2-sites after the various drug treatments (r = 0.92 for the 29/30-kDa protein, r = 0.69 for the 45-kDa protein and r = 0.75 for the 66-kDa protein). 6. In the rat cerebral cortex the I2-imidazoline receptor labelled by [3H]-idazoxan is heterogeneous in nature and the related imidazoline receptor proteins (29/30-, 45- and 66-kDa) detected by immunoblotting contribute differentially to the modulation of I2-sites after drug treatment.