Imidazoline Receptors Research Articles

Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

ABSTRACTMacroautophagy/autophagy is the main intracellular catabolic pathway in neurons that eliminates misfolded proteins, aggregates and damaged organelles associated with ageing and neurodegeneration. Autophagy is regulated by both MTOR-dependent and -independent pathways. There is increasing evidence that autophagy is compromised in neurodegenerative disorders, which may contribute to cytoplasmic sequestration of aggregation-prone and toxic proteins in neurons. Genetic or pharmacological modulation of autophagy to promote clearance of misfolded proteins may be a promising therapeutic avenue for these disorders. Here, we demonstrate robust autophagy induction in motor neuronal cells expressing SOD1 or TARDBP/TDP-43 mutants linked to amyotrophic lateral sclerosis (ALS). Treatment of these cells with rilmenidine, an anti-hypertensive agent and imidazoline-1 receptor agonist that induces autophagy, promoted autophagic clearance of mutant SOD1 and efficient mitophagy. Rilmenidine administration to mutant SOD1G93A mice upregulated autophagy and mitophagy in spinal cord, leading to reduced soluble mutant SOD1 levels. Importantly, rilmenidine increased autophagosome abundance in motor neurons of SOD1G93A mice, suggesting a direct action on target cells. Despite robust induction of autophagy in vivo, rilmenidine worsened motor neuron degeneration and symptom progression in SOD1G93A mice. These effects were associated with increased accumulation and aggregation of insoluble and misfolded SOD1 species outside the autophagy pathway, and severe mitochondrial depletion in motor neurons of rilmenidine-treated mice. These findings suggest that rilmenidine treatment may drive disease progression and neurodegeneration in this mouse model due to excessive mitophagy, implying that alternative strategies to beneficially stimulate autophagy are warranted in ALS.

A Biomarker to Differentiate between Primary and Cocaine-Induced Major Depression in Cocaine Use Disorder: The Role of Platelet IRAS/Nischarin (I1-Imidazoline Receptor).

The association of cocaine use disorder (CUD) and comorbid major depressive disorder (MDD; CUD/MDD) is characterized by high prevalence and poor treatment outcomes. CUD/MDD may be primary (primary MDD) or cocaine-induced (CUD-induced MDD). Specific biomarkers are needed to improve diagnoses and therapeutic approaches in this dual pathology. Platelet biomarkers [5-HT2A receptor and imidazoline receptor antisera selected (IRAS)/nischarin] were assessed by Western blot in subjects with CUD and primary MDD (n = 16) or CUD-induced MDD (n = 9; antidepressant free, AD−; antidepressant treated, AD+) and controls (n = 10) at basal level and/or after acute tryptophan depletion (ATD). Basal platelet 5-HT2A receptor (monomer) was reduced in comorbid CUD/MDD subjects (all patients: 43%) compared to healthy controls, and this down-regulation was independent of AD medication (decreases in AD−: 47%, and in AD+: 40%). No basal differences were found for IRAS/nischarin contents in AD+ and AD− comorbid CUD/MDD subjects. The comparison of IRAS/nischarin in the different subject groups during/after ATD showed opposite modulations (i.e., increases and decreases) in response to low plasma tryptophan levels with significant differences discriminating between the subgroups of CUD with primary MDD and CUD-induced MDD. These specific alterations suggested that platelet IRAS/nischarin might be useful as a biomarker to discriminate between primary and CUD-induced MDD in this dual pathology.

The protective effect of 2-(2-benzonfu-ranyl)-2-imidazoline against oxygen-glucose deprivation in cultured rat cortical astrocytes

Astrocytes play a pivotal role in neuronal survival in the setting of post-ischemic brain inflammation, but the astrocyte-derived mediators of ischemic brain injury remain to be defined. 2-(2-Benzofu-ranyl)-2-imidazoline (2-BFI) is a newly discovered ligand for high-affinity imidazoline I2 receptors (I2Rs) mainly located on the mitochondrial outer membrane in astrocytes. We previously reported that in a rat model of cerebral ischemia-reperfusion injury, 2-BFI limits infarct volume, reduces neurological impairment scores, and inhibits neuronal apoptosis in the ischemic penumbra. This study was performed to clarify the underlying mechanism in an astrocyte oxygen-glucose deprivation (OGD) model. The results show that 2-BFI reduces lipid peroxidation and inhibits mitochondria apoptotic pathways.

Peripheral versus central effect of intravenous moxonidine on rat carotid sinus baroreflex-mediated sympathetic arterial pressure regulation

AimsMoxonidine is a centrally acting antihypertensive agent with a selectivity to I1-imidazoline receptors higher than that to α2-adrenergic receptors. The present study aimed to quantify a peripheral effect of moxonidine on carotid sinus baroreflex-mediated sympathetic arterial pressure (AP) regulation separately from its central effect. Main methodsIn eight anesthetized Wistar rats, changes in efferent sympathetic nerve activity (SNA) and AP in response to a carotid sinus pressure input were compared before and during an intravenous administration of moxonidine (100μgkg−1 bolus followed by a continuous infusion at 200μg·kg−1·h−1). Key findingsMoxonidine significantly narrowed the range of the AP response (55.3±5.8 to 39.1±6.1mmHg, P<0.05) without changing the minimum AP (77.2±6.4 to 80.7±5.1mmHg, not significant). In the neural arc, moxonidine reduced the minimum SNA (56.6±5.9 to 29.7±6.2%, P<0.05) without affecting the range of the SNA response (45.3±5.5 to 40.2±5.0%, not significant). In the peripheral arc, moxonidine increased the intercept (3.0±8.5 to 51.1±7.2mmHg, P<0.01) and reduced the slope (1.28±0.06 to 0.92±0.15mmHg/%, P<0.05). SignificanceMoxonidine increased AP at any given SNA, suggesting that the peripheral vasoconstrictive effect is stronger than generally recognized. The peripheral vasoconstrictive effect of moxonidine may partly offset the vasodilatory effect attained by centrally-mediated sympathoinhibition.

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.

Role of intracellular Ca2+ signaling in the antinociceptive and discriminative stimulus effects of the imidazoline I2 receptor agonist 2-BFI in rats.

Recent research has established the imidazoline I2 receptor as a promising target for the development of novel analgesics. However, despite an increasing understanding of imidazoline I2 receptor-mediated behavioral effects, little is known about post-I2-receptor signaling mechanisms. This study examined the effects of several inhibitors of Ca2+ signaling mechanisms on two behavioral effects of the prototypical imidazoline I2 receptor ligand 2-(2-benzofuranyl)-2-imidazoline (2-BFI). The von Frey filament test was used to examine the antinociceptive effects of 2-BFI in complete Freund's adjuvant (CFA)-induced inflammatory pain in rats. A two-lever drug discrimination paradigm in which rats were trained to discriminate 5.6mg/kg (intraperitoneally) 2-BFI from its vehicle was used to examine the discriminative stimulus effects of 2-BFI. The L-type Ca2+ channel blockers verapamil and nimodipine, the calmodulin antagonist W-7, and the internal Ca2+ release inhibitor ryanodine all attenuated the antinociceptive effects of 2-BFI. Oxycodone- and acetaminophen-induced antinociception was unaffected by pretreatment with the Ca2+ channel blockers. Rats learned to reliably discriminate 5.6mg/kg 2-BFI from saline. The I2 receptor agonists BU224, RS45041, tracizoline, and CR4056 all fully substituted for 5.6mg/kg 2-BFI while idazoxan, S22687, 2,5-dimethoxy-4-methylamphetamine (DOM), and phenyzoline produced partial or no substitution. Verapamil, nimodipine, and W-7 did not alter the discriminative stimulus effects of 2-BFI. These results indicate that the antinociceptive effects of 2-BFI involve intracellular Ca2+ elevation and/or downstream Ca2+/calmodulin signaling, whereas the discriminative stimulus effects of 2-BFI are mediated by a distinct, independent mechanism.

Agmatine: multifunctional arginine metabolite and magic bullet in clinical neuroscience?

Agmatine, the decarboxylation product of arginine, was largely neglected as an important player in mammalian metabolism until the mid-1990s, when it was re-discovered as an endogenous ligand of imidazoline and α2-adrenergic receptors. Since then, a wide variety of agmatine-mediated effects have been observed, and consequently agmatine has moved from a wallflower existence into the limelight of clinical neuroscience research. Despite this quantum jump in scientific interest, the understanding of the anabolism and catabolism of this amine is still vague. The purification and biochemical characterization of natural mammalian arginine decarboxylase and agmatinase still are open issues. Nevertheless, the agmatinergic system is currently one of the most promising candidates in order to pharmacologically interfere with some major diseases of the central nervous system, which are summarized in the present review. Particularly with respect to major depression, agmatine, its derivatives, and metabolizing enzymes show great promise for the development of an improved treatment of this common disease.

Pharmacological analysis of the inhibition produced by moxonidine and agmatine on the vasodepressor sensory CGRPergic outflow in pithed rats

Calcitonin gene-related peptide (CGRP) plays a role in several (patho)physiological functions, and modulation of its release is considered a therapeutic target. In this respect, electrical spinal (T9­-T12) stimulation of the perivascular sensory outflow in pithed rats produces vasodepressor responses mediated by CGRP release. This study investigated the role of imidazoline I1 and I2 receptors in the inhibition by moxonidine and agmatine of these vasodepressor responses. Male Wistar pithed rats (pretreated i.v. with 25mg/kg gallamine and 2mg/kg⋅min hexamethonium) received i.v. continuous infusions of methoxamine (20μg/kg⋅min) followed by physiological saline (0.02ml/min), moxonidine (1, 3, 10 or 30μg/kg⋅min) or agmatine (1000 or 3000μg/kg⋅min). Under these conditions, electrical stimulation (0.56–5.6Hz; 50V; 2ms) of the spinal cord (T9-T12) produced frequency-dependent vasodepressor responses which were: (i) unchanged during saline infusion; and (ii) inhibited during the above infusions of moxonidine or agmatine. Moreover, using i.v. administrations, the inhibition by 3μg/kg⋅min moxonidine or 3000μg/kg⋅min agmatine (which failed to inhibit the vasodepressor responses by α-CGRP; 0.1–1µg/kg) was: (i) unaltered after saline (1ml/kg), rauwolscine (300μg/kg; α2-adrenoceptor antagonist) or BU224 (300μg/kg; imidazoline I2 receptor antagonist); and (ii) reversed after AGN 192403 (3000μg/kg; imidazoline I1 receptor antagonist). This reversion was relatively more pronounced after AGN 192403 plus rauwolscine. These blocking doses of antagonists lacked any effects on the electrically-induced vasodepressor responses. Therefore, the inhibition of the vasodepressor sensory CGRPergic outflow by moxonidine and agmatine is mainly mediated by prejunctional imidazoline I1 receptors on perivascular sensory nerves.

Identification of morin as an agonist of imidazoline I-3 receptor for insulin secretion in diabetic rats.

Morin is a flavonoid contained in guava that is known to reduce hyperglycemia in diabetics. Morin has been demonstrated to increase plasma insulin. However, the mechanism(s) remains unknown. The present study is designed to investigate the effect of morin on the imidazoline receptor (I-R) that regulates insulin secretion. We used Chinese hamster ovary (CHO) cells transfected with an I-R expression construct (NISCH-CHO-K1 cells) to identify the direct effect of morin on the I-R. Moreover, the imidazoline I3 receptor (I-3R) is known to be present in pancreatic β cells and involved in insulin secretion. Therefore, we applied a specific antagonist (KU14R) to block I-3R in diabetic rats. Additionally, the effect of morin on insulin secretion was characterized in isolated pancreatic islets. Morin decreased blood glucose levels by increasing plasma insulin levels in diabetic rats. In CHO cells expressing an I-R, morin increased calcium influx in a dose-dependent manner. Additionally, KU14R dose-dependently inhibited the morin-induced effects, including hypoglycemia and the increase in insulin secretion and plasma C-peptide levels, in diabetic rats. Furthermore, morin enhanced insulin secretion from isolated pancreatic islets, and this effect was also dose-dependently inhibited by KU14R. Phospholipase C (PLC) is known to couple with the I-R, and a PLC inhibitor dose-dependently attenuated the insulin secretion induced by morin in isolated pancreatic islets. Taken together, these data suggest that morin can activate I-3R to enhance insulin secretion. Therefore, it would be useful to develop morin into a treatment for diabetic disorders.

Diosmin, a Citrus Nutrient, Activates Imidazoline Receptors to Alleviate Blood Glucose and Lipids in Type 1-Like Diabetic Rats.

Diosmin is a nutrient that is widely contained in citrus and that has been indicated to improve glucose metabolism in diabetic disorders. Recently, we demonstrated that diosmin induces β-endorphin to lower hyperglycemia in diabetic rats. However, the mechanisms of diosmin in opioid secretion were unclear. Therefore, we focused on the secretion of opioids from isolated adrenal glands induced by diosmin. The changes in the released β-endorphin-like immunoreactivity (BER) were determined using ELISA. Diosmin increased the BER level in a dose-dependent manner, and this effect was markedly reduced in the absence of calcium ions. Activation of the imidazoline I-2 receptor (I-2R) has been introduced to induce opioid secretion. Interestingly, we observed that diosmin activates CHO cells expressing I-R. Additionally, diosmin-increased BER was inhibited by the blockade of I-2R in isolated adrenal glands. Additionally, an antagonist of I-2R blocked diosmin-induced effects, including the reduction in hyperglycemia and the increase in plasma BER in streptozotocin-induced diabetic rats (STZ-diabetic rats). Repeated treatment of STZ-diabetic rats with diosmin for one week induced changes in hepatic glycogen, lipid levels, and the expression of phosphoenolpyruvate carboxykinase (PEPCK). Furthermore, an antagonist of I-2R blocked the diosmin-induced changes. Additionally, plasma lipids modified by diosmin were also reversed by the blockade of I-2R in STZ-diabetic rats. Taken together, we suggest that diosmin may activate I-2R to enhance the secretion of β-endorphin from adrenal glands and to influence metabolic homeostasis, resulting in alleviation of blood glucose and lipids in STZ-diabetic rats.

Upregulation of IRAS/nischarin (I1-imidazoline receptor), a regulatory protein of μ-opioid receptor trafficking, in postmortem prefrontal cortex of long-term opiate and mixed opiate/cocaine abusers

Imidazoline receptor antisera-selected (IRAS)/nischarin, a putative I1-imidazoline receptor, has recently been shown to regulate μ-opioid receptor (OR) trafficking and resensitisation. To study a possible involvement of this μ-OR regulator in opiate dependence, the present study assessed by Western blot analysis the contents of IRAS/nischarin and μ-OR in total homogenates and subcellular preparations of postmortem human prefrontal cortex (PFC/BA9) of long-term opiate and mixed opiate/cocaine abusers as well as of matched healthy control subjects. In the PFC/BA9 of long-term opiate/cocaine abusers (all subjects together) IRAS/nischarin content was increased (+67%, p < 0.01, n = 11) when compared with matched controls (n = 10). Similar increases were found for the subgroups of opiate (+72%, n = 6) and mixed opiate/cocaine (+61%, n = 5) abusers. IRAS/nischarin immunocontents were also found increased in subcellular membrane preparations (+61%, p < 0.05, n = 10) of PFC/BA9 from opiate addicts. In the same brain samples, the levels of μ-OR were not different to those in control subjects. Based on the increased contents in brains of opiate abusers and the reported function as μ-OR regulator, IRAS/nischarin could represent a new promising target for treatment of opiate use disorder.

Dexmedetomidine relieves formaldehyde-induced pain in rats through both α2 adrenoceptor and imidazoline receptor

Dexmedetomidine (DEX) is a highly selective α2 adrenergic receptor agonist. In this study, we investigated the analgesic effect and the underlying mechanisms of DEX on inflammatory visceral pain in rats. Twenty-five male Sprague Dawley (SD) rats were randomly divided into 5 groups, including control, sham, low dose DEX, medium dose DEX and high dose DEX group. Pain was induced with 10% formalin and scored every 15min till 2 h-post the induction. Hematoxylin-eosin (HE) staining was used to evaluate the toxicity of DEX on spinal cord neurons. Acetycholine (Ach) and noradrenaline (NA) levels were determined by using ELISA method. The expressions of natural nitric oxide synthase (nNOS), protein kinase γ (PKCγ) and protease-activated receptor 2 (PAR2) were determined by using western blot. DEX treatment relieved formaldehyde-induced pain in rats in a dose-dependent manner. Furthermore, DEX showed little neuro-toxicity on the spinal cord neurons, even at the highest dosage used in our study. Ach level was significantly increased in Sham group compared with control group. DEX treatment decreased NA levels and increased Ach levels in the incubation medium of spinal cord sections. Western blot analysis showed that the expression of nNOS, PKCγ and PAR2 was significantly decreased in DEX group compared with Sham group, whereas these effects of DEX on nNOS, PKCγ and PAR2 were blocked by both yohimbine and idazoxan, indicating that the analgesic effect of DEX is mediated by both α2 adrenergic receptor and imidazoline receptor. Yohimbine and idazoxan treatment significantly enhanced pain scores compared to DEX group, and which antagonizes the effects DEX. In conclusion, our study demonstrated that DEX could inhibit formaldehyde-induced pain by inhibiting nNOS, PKCγ and PAR2 expression through α2 adrenergic receptor and imidazoline receptor.

Activation of imidazoline I1 receptor by moxonidine regulates the progression of liver fibrosis in the Nrf2-dependent pathway

Imidazoline I1 receptor (I1R) has been recognized as a promising target in the treatment of many diseases, but little is known about its function in liver fibrogenesis. This study aimed to investigate the effect of I1R activation on the development and progression of liver fibrosis. The results showed that I1R expression was decreased in the livers of both patients and mice with liver fibrosis, and in TGF-β-treated hepatic stellate cells (HSCs). Activation of I1R by moxonidine (MOX) significantly inhibited the progression of liver fibrosis in carbon tetrachloride-induced mice and attenuated the activation of HSCs and kupffer cells. MOX also suppressed the activation of TLR4/NF-κB and TGF-β/Smad signaling, however, knockdown of I1R abrogated the inhibitory effects of MOX. Additionally, MOX activated Nrf2 signaling in vivo and in vitro, but knockout or knockdown of Nrf2 ameliorated the anti-inflammatory and anti-fibrotic effects of MOX. Taken together, activation of I1R negatively regulates the progression of liver fibrosis in the Nrf2-dependent pathway, which suggests that specifically targeting I1R may be a potential therapeutic strategy for the treatment of liver fibrosis.

Comparative pharmacodynamic analysis of imidazoline compounds using rat model of ocular mydriasis with a test of quantitative structure–activity relationships

Imidazol(in)e derivatives, having the chemical structure similar to clonidine, exert diverse pharmacological activities connected with their interactions with alpha2-adrenergic receptors, e.g. hypotension, bradycardia, sedation as well as antinociceptive, anxiolytic, antiarrhythmic, muscle relaxant and mydriatic effects. The mechanism of pupillary dilation observed after systemic administration of imidazol(in)es to rats, mice and cats depends on the stimulation of postsynaptic alpha2-adrenoceptors within the brain. It was proved that the central nervous system (CNS)-localized I1-imidazoline receptors are not engaged in those effects. It appeared interesting to analyze the CNS-mediated pharmacodynamics of imidazole(in)e agents in terms of their chromatographic and calculation chemistry-derived parameters.In the present study a systematic determination and comparative pharmacometric analysis of mydriatic effects in rats were performed on a series of 20 imidazol(in)e agents, composed of the well-known drugs and of the substances used in experimental pharmacology. The eye pupil dilatory activities of the compounds were assessed in anesthetized Wistar rats according to the established Koss method. Among twenty imidazol(in)e derivatives studied, 18 produced diverse dose-dependent mydriatic effects. In the quantitative structure–activity relationships (QSAR) analysis, the pharmacological data (half maximum mydriatic effect – ED50 in μmol/kg) were considered along with the structural parameters of the agents from molecular modeling. The theoretically calculated lipophilicity parameters, CLOGP, of imidazol(in)es, as well as their lipophilicity parameters from HPLC, logkw, were also considered. The attempts to derive statistically significant QSAR equations for a full series of the agents under study were unsuccessful. However, for a subgroup of eight apparently structurally related imidazol(in)es a significant relationship between log(1/ED50) and logkw values was obtained. The lack of “predictive” QSAR for the whole series of the structurally diverse agents is probably due to a complex mechanism of the ligand–alpha2-adrenergic receptor interactions, which are predominantly of a highly structurally specific polar nature. Such interactions are difficult to quantify with the established chemical structural descriptors, contrary to the less specific, molecular bulkiness-related interactions.

Harmane suppresses microglial neuroinflammatory response and induce antidepressant-like effect in rats

ObjectiveHarmane is a beta-carboline, which binds to imidazoline receptors and it has been previously shown that it may have an antidepressant effect when administered acutely. This study is planned to investigate the effect of harmane on chronic unpredictable mild stress (CUMS) model and microglial (Iba-1) immunoreactivity in the same model as markers of neuroinflammation.MethodsMale Wistar Albino rats (290–360 g) were divided into groups such as control (saline), CUMS, CUMS + Imipramine (20 mg/kg; i.p.), CUMS + Harmane5 (5 mg/kg; i.p.), CUMS + Harmane10 (10 mg/kg; i.p.) groups (n = 10–12 in each). In CUMS model, various stressors were applied for 40 days. On day 20, harmane administration was started for 20 days. At the end, sucrose preference and forced swimming tests were performed. Then, brains were removed with paraformaldehyde perfusion for Iba-1 immunohistochemical analysis in hippocampus. One-way analysis of variance and Tukey's test were used for statistical analysis.ResultsThe time of immobility in forced swim test was significantly reduced while sucrose preference was increased in Imipramine and CUMS + harmane10 groups. In immunohistochemical experiments, Iba-1 were overexpressed in CUMS group and Harmane significantly reduced the overexpression of Iba-1.ConclusionOur results suggest that chronic administration of harmane has an antidepressant-like activity in chronic stress model of depression. These results support the notion of imidazoline receptors involvement in depression by modulating neuroinflammation and at least a part of its antidepressant effect might be through modulating microglial activation as a reflection of neuroinflammation.This research was supported by Marmara University, Scientific Research Projects – SAG-C-YLP-110915-0415 and SAG-E-120613-0233.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Effects of I2-imidazoline receptor (IR) alkylating BU99006 in the mouse brain: Upregulation of nischarin/I1-IR and μ-opioid receptor proteins and modulation of associated signalling pathways

5-isothiocyanato-2-benzofuranyl-2-imidazoline (BU99006) was shown to selectively and irreversibly bind to I2-imidazoline receptors (IRs) in vitro and in vivo, but cell signalling consequences of I2-IR alkylation have not been reported previously. This study assessed by Western blot the effects of BU99006 on nischarin (candidate I1-IR), μ-OR (regulated by nischarin) and associated signalling mediators in the mouse brain. Acute treatment with BU99006 (20 mg/kg, i.p., 1–3 h) led to fast (peak at 1 h) and shortlasting (decline up to 3 h) upregulation of nischarin and μ-OR contents in the hippocampus (less or non-significant effects in cortex) and altered the expression of cytoskeletal β-actin (reduced contents at 3 h). In the same hippocampal samples of BU99006-treated mice, an inhibition of the MAPK species MEK, ERK and JNK was detected at 1 and/or 2 h after drug administration, which was paralleled by enhanced calpain activity (increased contents of p25 and spectrin breakdown products). Correlation analysis indicated the involvement of cdk5/p25 in MEK/ERK inhibition. These neurochemical effects of I2-alkylating BU99006 show a close relation between I1- and I2-IRs expressed in the mouse brain and between these receptors and the μ-OR, accompanied by cytoskeletal alterations and differential effects on multifunctional MAPK and cdk5 signalling pathways.

Tolerance and cross-tolerance to the antinociceptive effects of oxycodone and the imidazoline I2 receptor agonist phenyzoline in adult male rats.

Emerging evidence suggests the potential utility of combining opioids with imidazoline I2 receptor agonists for chronic pain. However, chronic pain management requires prolonged pharmacotherapy, and the consequence of such combination therapy remains unclear. This study examined the anti-hyperalgesic effect of the opioid oxycodone, the selective I2 receptor agonist phenyzoline, alone and in combination, during prolonged treatment. Von Frey filament test was used to examine the anti-hyperalgesic effect of drugs in complete Freund's adjuvant (CFA)-induced inflammatory pain or chronic constriction injury (CCI)-induced neuropathic pain in rats. Twice-daily treatment with oxycodone and phenyzoline, alone or in combination, was continued until the development of significant tolerance (oxycodone) or as long as 19days passed (phenyzoline). In rats receiving CFA or CCI manipulation, mechanical hyperalgesia was dose-dependently reversed by oxycodone and phenyzoline. Twice-daily treatment with 2×ED50 dose of oxycodone for 7days led to significant antinociceptive tolerance to oxycodone but not cross-tolerance to phenyzoline. Similarly, twice-daily treatment with 2×ED50 dose of phenyzoline for 19days led to significant antinociceptive tolerance to phenyzoline but not cross-tolerance to oxycodone. Twice-daily treatment with the combined oxycodone and phenyzoline using different ratios (1:3, 1:1 and 3: 1) at the doses that were functionally equivalent to the treatment doses of oxycodone and phenyzoline for 13-19days generally led to delayed antinociceptive tolerance. Combination therapy with oxycodone and I2 receptor agonists maintains prolonged antinociceptive effectiveness with reduced propensity to develop tolerance.

Imidazoline I2 receptors: An update.

Since first introduced more than two decades ago, the research in imidazoline I2 receptors has been steadily increasing. This review provides an update on the current status of I2 receptor pharmacology. Imidazoline I2 receptors or I2 binding sites refer to several (at least four) different proteins that bind to [3H]-idazoxan and [3H]-2-BFI with high affinity. The molecular identities of the proteins remain elusive. One of the proteins (45kD) seems to be consistent with the identity of brain creatine kinase. The biological functions of I2 receptors have been primarily unveiled by the studies of selective I2 receptor ligands. Accumulating evidence suggests that I2 receptor ligands are effective analgesics for persistent and chronic painful conditions such as inflammatory, neuropathic and postoperative pain. One selective I2 receptor ligand, CR4056, has been advanced to phase II clinical trial with the therapeutic indication of chronic inflammatory pain (osteoarthritis). The expansion to the treatment of other chronic pain conditions should be expected if CR4056 could eventually be approved as a new drug. I2 receptor ligands also demonstrate robust discriminative stimulus activity and induce a characteristic discriminative cue in animals. Biochemical and preclinical in vivo investigations also suggest that I2 receptor ligands have neuroprotective activity and modulate body temperature. The emerging discrepancies of a range of purported selective I2 receptor ligands suggest different pharmacological effects mediated by discrete I2 receptor components which likely attribute to the I2 receptor-related proteins. It is proposed that the I2 receptors represent an emerging drug target for the treatment of neurological disorders such as pain and stroke, and deserve more research attention to translate preclinical findings to pharmacotherapies.

Further analysis of the inhibition by agmatine on the cardiac sympathetic outflow: Role of the α2-adrenoceptor subtypes

This study has investigated the role of the α2-adrenoceptor subtypes involved in the inhibition of the cardiac sympathetic outflow induced by intravenous (i.v) infusions of agmatine. Therefore, we analysed the effect of an i.v. bolus injections of the selective antagonists BRL 44408 (300μg/kg; α2A), imiloxan (3000μg/kg; α2B), and JP-1302 (300μg/kg; α2C) given separately, and their combinations: BRL 44408 plus Imiloxan, JP 1302 plus imiloxan, BRL 44408 plus JP-1302, BRL 44408 plus imiloxan plus JP-1302 on the cardiac sympatho-inhibition of agmatine. Also, the effect of the combination BRL 44408 plus JP-1302 plus AGN 192403 (3000μg/kg; I1 antagonist) was evaluated. In this way, i.v. infusions of 1000μg/kg min of agmatine, but not 300, inhibited the tachycardic response induced by electrical stimulation. Furthermore, the antagonists used or their combinations had no effect on the electrically-induced tachycardic response. On the other hand, the inhibitory response of agmatine was: (1) partially antagonized by BRL 44408 or JP-1302 given separately, a similar response was observed when we administered their combination with imiloxan, but not by imiloxan alone, (2) antagonized in greater magnitude by the combination BRL 44408 plus JP-1302 or the combination BRL 44408 plus imiloxan plus JP-1302, and (3) abolished by the combination BRL 44408 plus JP-1302 plus AGN 192403. Taken together, these results demonstrate that the α2A- and α2C-adrenoceptor subtypes and I1-imidazoline receptors are involved in the inhibition of the cardiac sympathetic outflow induced by agmatine.

Imidazoline I2 receptor inhibitor idazoxan regulates the progression of hepatic fibrosis via Akt-Nrf2-Smad2/3 signaling pathway.

Liver fibrosis is a global health problem and its relationship with imidazoline I2 receptor has not been reported. This study aimed to investigate the effects and underlying mechanisms of imidazoline I2 receptor (I2R) inhibitor idazoxan (IDA) on carbon tetrachloride (CCl4)-induced liver fibrosis. In vivo liver fibrosis in mice was induced by intraperitoneally injections of CCl4 for eight weeks, and in vitro studies were performed on activated LX2 cells treated with transforming growth factor-β (TGF-β). Our results showed that IDA significantly improved liver inflammation, ameliorated hepatic stellate cells activation and reduced collagen accumulation by suppressing the pro-fibrogenic signaling of TGF-β/Smad. Further investigation showed that IDA significantly balanced oxidative stress through improving the expressions and activities of anti-oxidant and detoxifying enzymes and activating Nrf2-the key defender against oxidative stress with anti-fibrotic potentials. Even more impressively, knock out of Nrf2 or suppression of Akt by perifosine (PE) eliminated the anti-oxidant and anti-fibrotic effects of IDA in vivo and in vitro, suggesting that Akt/Nrf2 constitutes a critical component of IDA's protective functions. Taken together, IDA exhibits potent effects against liver fibrosis via Akt-Nrf2-Smad2/3 signaling pathway, which suggests that specifically targeting I2R may be a potentially useful therapeutic strategy for liver fibrosis.