Knockout model reveals the role of Nischarin in mammary gland development, breast tumorigenesis and response to metformin treatment.

Previously, our lab discovered the protein Nischarin and uncovered its role in regulating cell migration and invasion via its interactions with several proteins. We subsequently described a role for Nischarin in breast cancer, in which it is frequently underexpressed. To characterize Nischarin's role in breast tumorigenesis and mammary gland development more completely, we deleted a critical region of the Nisch gene (exons 7-10) from the mouse genome and observed the effects. Mammary glands in mutant animals showed delayed terminal end bud formation but did not develop breast tumors spontaneously. Therefore, we interbred the animals with transgenic mice expressing the mouse mammary tumor virus-polyoma middle T-antigen (MMTV-PyMT) oncogene. The MMTV-PyMT mammary glands lacking Nischarin showed increased hyperplasia compared to wild-type animal tissues. Furthermore, we observed significantly increased tumor growth and metastasis in Nischarin mutant animals. Surprisingly, Nischarin deletion decreased activity of AMPK and subsequently its downstream effectors. Given this finding, we treated these animals with metformin, which enhances AMPK activity. Here, we show for the first time, metformin activates AMPK signaling and inhibits tumor growth of Nischarin lacking PyMT tumors suggesting a potential use for metformin as a cancer therapeutic, particularly in the case of Nischarin-deficient breast cancers.

Agmatine Protects Against the Progression of Sepsis Through the Imidazoline I2 Receptor-Ribosomal S6 Kinase 2-Nuclear Factor-κB Signaling Pathway.

OBJECTIVES: The knowledge that agmatine is found in the human body has existed for several years; however, its role in sepsis has not yet been studied. In the present study, we investigate the role of agmatine in the progression and treatment of sepsis. DESIGN: Clinical/laboratory investigations. SETTING: Medical centers/University-based research laboratory. SUBJECTS: Elective ICU patients with severe sepsis and healthy volunteers; C57BL/6 mice weighing 18-22 g. INTERVENTIONS: Serum agmatine level and its associations with inflammatory markers were assessed in patients with sepsis. Agmatine was administered intraperitoneally to mice before a lipopolysaccharide challenge. Human peripheral blood mononuclear cells and murine macrophages were pretreated with agmatine followed by lipopolysaccharide stimulation. MEASUREMENTS AND MAIN RESULTS: Serum agmatine levels were significantly decreased in patients with sepsis and lipopolysaccharide-induced mice, and correlated with Acute Physiology and Chronic Health Evaluation II score, procalcitonin, tumor necrosis factor-α, and interleukin-6 levels. In a therapeutic experiment, exogenous agmatine attenuated the cytokine production of peripheral blood mononuclear cells from patients with sepsis and healthy controls. Agmatine also exerted a significant beneficial effect in the inflammatory response and organ damage and reduced the death rate in lipopolysaccharide-induced mice. Imidazoline I2 receptor agonist 2-benzofuran-2-yl blocked the pharmacological action of agmatine; whereas, other imidazoline receptor ligands did not. Furthermore, agmatine significantly impaired the inflammatory response by inactivating nuclear factor-κB, but not protein 38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and inducible nitric oxide synthase signaling in macrophages. Activation of imidazoline I2 receptor or knockdown of ribosomal S6 kinase 2 counteracted the effects of agmatine on phosphorylation and degradation of inhibitor of nuclear factor-κBα. CONCLUSIONS: Endogenous agmatine metabolism correlated with the progression of sepsis. Supplemental exogenous agmatine could ameliorate the lipopolysaccharide-induced systemic inflammatory responses and multiple organ injuries through the imidazoline I2 receptor-ribosomal S6 kinase 2-nuclear factor-κB pathway. Agmatine could be used as both a clinical biomarker and a promising pharmaconutrient in patients with severe sepsis.

Imidazoline 2 binding sites reflecting astroglia pathology in Parkinson's disease: an in vivo11C-BU99008 PET study.

Astroglia are multifunctional cells that regulate neuroinflammation and maintain homeostasis within the brain. Astroglial α-synuclein-positive cytoplasmic accumulations have been shown post-mortem in patients with Parkinson's disease and therefore astroglia may play an important role in the initiation and progression of Parkinson's disease. Imidazoline 2 binding sites are expressed on activated astroglia in the cortex, hippocampus, basal ganglia and brainstem; therefore, by measuring imidazoline 2 binding site levels we can indirectly evaluate astrogliosis in patients with Parkinson's disease. Here, we aimed to evaluate the role of astroglia activation in vivo in patients with Parkinson's disease using 11C-BU99008 PET, a novel radioligand with high specificity and selectivity for imidazoline 2 binding sites. Twenty-two patients with Parkinson's disease and 14 healthy control subjects underwent 3 T MRI and a 120-min 11C-BU99008 PET scan with volume of distribution (VT) estimated using a two-tissue compartmental model with a metabolite corrected arterial plasma input function. Parkinson's disease patients were stratified into early (n = 8) and moderate/advanced (n = 14) groups according to disease stage. In early Parkinson's disease, increased 11C-BU99008 VT uptake was observed in frontal (P = 0.022), temporal (P = 0.02), parietal (P = 0.026) and occipital (P = 0.047) cortical regions compared with healthy controls. The greatest 11C-BU99008 VT increase in patients with early Parkinson's disease was observed in the brainstem (52%; P = 0.018). In patients with moderate/advanced Parkinson's disease, loss of 11C-BU99008 VT was observed across frontal (P = 0.002), temporal (P < 0.001), parietal (P = 0.039), occipital (P = 0.024), and insula (P < 0.001) cortices; and in the subcortical regions of caudate (P < 0.001), putamen (P < 0.001) and thalamus (P < 0.001); and in the brainstem (P = 0.018) compared with healthy controls. In patients with Parkinson's disease, loss of 11C-BU99008 VT in cortical regions, striatum, thalamus and brainstem correlated with longer disease duration (P < 0.05) and higher disease burden scores, measured with Movement Disorder Society Unified Parkinson's Disease Rating Scale (P < 0.05). In the subgroup of patients with moderate/advanced Parkinson's disease, loss of 11C-BU99008 VT in the frontal (r = 0.79; P = 0.001), temporal (r = 0.74; P = 0.002) and parietal (r = 0.89; P < 0.001) cortex correlated with global cognitive impairment. This study demonstrates in vivo the role of astroglia in the initiation and progression of Parkinson's disease. Reactive astroglia observed early in Parkinson's disease could reflect a neuroprotective compensatory mechanisms and pro-inflammatory upregulation in response to α-synuclein accumulation. However, as the disease progresses and significant neurodegeneration occurs, astroglia lose their reactive function and such loss in the cortex has clinical relevance in the development of cognitive impairment.

Responsiveness of α2-adrenoceptor/I1-imidazoline receptor in the rostral ventrolateral medulla to cardiovascular regulation is enhanced in conscious spontaneously hypertensive rat.

Stimulation of α2-adrenoceptor/I1-imidazoline receptors in the rostral ventrolateral medulla decreases the blood pressure via sympathoinhibition. However, alteration of receptor responses in genetically hypertensive rats remains unclear. We examined cardiovascular responses of α2-adrenoceptor/I1-imidazoline receptor agonist and antagonists microinjected into the rostral ventrolateral medulla of conscious spontaneously hypertensive rats and normotensive Wistar Kyoto rats. Injection of 2-nmol clonidine-an α2-adrenoceptor/I1-imidazoline receptor agonist-unilaterally into the rostral ventrolateral medulla decreased the blood pressure, heart rate, and renal sympathetic nerve activity; the responses were significantly enhanced in spontaneously hypertensive rats than in Wistar Kyoto rats. Co-injection of 2-nmol 2-methoxyidazoxan (a selective α2-adrenoceptor antagonist) or 2-nmol efaroxan (an I1-receptor antagonist) with 2 nmol of clonidine attenuated the hypotensive and bradycardic effects of clonidine-only injection. Injection of 2-methoxyidazoxan alone increased the blood pressure and heart rate in spontaneously hypertensive rats, but not in Wistar Kyoto rats. These results suggest enhanced responsiveness of α2-adrenoceptor/I1-imidazoline receptors in the rostral ventrolateral medulla of spontaneously hypertensive rats.

Dexmedetomidine Prevents Cognitive Decline by Enhancing Resolution of High Mobility Group Box 1 Protein-induced Inflammation through a Vagomimetic Action in Mice.

BACKGROUND: Inflammation initiated by damage-associated molecular patterns has been implicated for the cognitive decline associated with surgical trauma and serious illness. We determined whether resolution of inflammation mediates dexmedetomidine-induced reduction of damage-associated molecular pattern-induced cognitive decline. METHODS: Cognitive decline (assessed by trace fear conditioning) was induced with high molecular group box 1 protein, a damage-associated molecular pattern, in mice that also received blockers of neural (vagal) and humoral inflammation-resolving pathways. Systemic and neuroinflammation was assessed by proinflammatory cytokines. RESULTS: Damage-associated molecular pattern-induced cognitive decline and inflammation (mean ± SD) was reversed by dexmedetomidine (trace fear conditioning: 58.77 ± 8.69% vs. 41.45 ± 7.64%, P < 0.0001; plasma interleukin [IL]-1ß: 7.0 ± 2.2 pg/ml vs. 49.8 ± 6.0 pg/ml, P < 0.0001; plasma IL-6: 3.2 ± 1.6 pg/ml vs. 19.5 ± 1.7 pg/ml, P < 0.0001; hippocampal IL-1ß: 4.1 ± 3.0 pg/mg vs. 41.6 ± 8.0 pg/mg, P < 0.0001; hippocampal IL-6: 3.4 ± 1.3 pg/mg vs. 16.2 ± 2.7 pg/mg, P < 0.0001). Reversal by dexmedetomidine was prevented by blockade of vagomimetic imidazoline and α7 nicotinic acetylcholine receptors but not by α2 adrenoceptor blockade. Netrin-1, the orchestrator of inflammation-resolution, was upregulated (fold-change) by dexmedetomidine (lung: 1.5 ± 0.1 vs. 0.7 ± 0.1, P < 0.0001; spleen: 1.5 ± 0.2 vs. 0.6 ± 0.2, P < 0.0001), resulting in upregulation of proresolving (lipoxin-A4: 1.7 ± 0.2 vs. 0.9 ± 0.2, P < 0.0001) and downregulation of proinflammatory (leukotriene-B4: 1.0 ± 0.2 vs. 3.0 ± 0.3, P < 0.0001) humoral mediators that was prevented by α7 nicotinic acetylcholine receptor blockade. CONCLUSIONS: Dexmedetomidine resolves inflammation through vagomimetic (neural) and humoral pathways, thereby preventing damage-associated molecular pattern-mediated cognitive decline.

Relationship between central behavioral effects and peripheral sympathetic neurotransmission functionality during acute cocaine withdrawal syndrome in adult rats.

BACKGROUND: Acute cocaine withdrawal syndrome (ACWS) is characterized as a set of organic alterations triggered by abrupt discontinuation of chronic cocaine consumption, usually occurring at 24-40 hours after withdrawal. However, little is known about the relationship between central and peripheral sympathetic neurotransmission during ACWS. OBJECTIVE AND METHODS: We investigated the mechanisms involved in central and peripheral sympathetic neurotransmission and how ACWS affects the sympathetic functionality. Cocaine was administered twice daily for 5 days in Wistar rats (at least 5 in each group): on the first and second day, 15 mg/kg/i.p.; third day, 20 mg/kg/i.p.; and finally in the last two days, 30 mg/kg/i.p. Subsequently, at 1, 24, 48 and 120 h after cocaine administration the following experiments were done: (i) at the central level, behavioral tests of open-field and elevated plus maze; and (ii) at the peripheral level, tests of catecholamine release, function of α2-adrenergic receptors (α2-ARs), imidazoline receptors (I(1,2)-Rs), L-type voltage-gated (Ca(v1.2)) Ca(2+) channels and α1-ARs. RESULTS: During ACWS, rats showed hypolocomotion and exacerbation of anxiogenic-effects 24 h after cocaine withdrawal. Likewise, a decrease in the catecholamine release and activity of α2-ARs/I(1,2)-Rs at 24-48 h after cocaine withdrawal was observed. A decrease in Ca(v1.2) channels and α1-ARs function at 48 h after cocaine withdrawal was observed. CONCLUSIONS: The relationship of central and peripheral sympathetic neurotransmission during ACWS possibly due to a failure in activation and/or inactivation of presynaptic α2-ARs/I(1,2)-Rs, may offer a potential target for attenuating ACWS.

Imidazoline receptor antisera-selected/Nischarin regulates the effect of agmatine on the development of morphine dependence.

Agmatine, an endogenous ligand for imidazoline receptor, has been shown to prevent opioid dependence, but not much is known about the mechanisms of the effect of agmatine. In the present study, we investigated the function of I1 imidazoline receptor and its candidate protein imidazoline receptor antisera-selected (IRAS)/Nischarin in morphine dependence as well as in the effect of agmatine inhibiting morphine dependence by pharmacological and molecular approaches. Results showed that inhibition of IRAS or Nischarin did not change the development of morphine dependence in vitro and in vivo under the basal condition. Agmatine could reduce the cyclic 3', 5' adenosine monophosphate (cAMP) overshoot at the concentration of 0.01-10 µM in the primary cultured rat hippocampal neurons and attenuated the withdrawal signals and the elevation of FosB and ΔFosB at the dose of 5 mg/kg in the morphine-dependent mice. The effect of agmatine was inhibited by efaroxan (I1 imidazoline receptor non-specific antagonist) and the RNA interference against IRAS or Nischarin. These findings indicate that I1 imidazoline receptor or IRAS/Nischarin mediates the effect of agmatine on morphine dependence and provide evidence that I1 imidazoline receptor may be a new target for treating morphine dependence.

Inhibitory influence of agmatine in ethanol withdrawal-induced depression in rats: Behavioral and neurochemical evidence.

Although ethanol withdrawal depression is one of the prominent reasons for ethanol consumption reinstatement and ethanol dependence, its neurochemical basis is not clearly understood. The present study investigated the role of the agmatinergic system in ethanol withdrawal-induced depression using the forced swim test (FST) in rats. Chronic exposure of animals to ethanol for 21 days and its abrupt withdrawal produced depression-like behavior, as evidenced by increased immobility time in the FST, compared to the pair-fed control animals. The ethanol withdrawal-induced depression was significantly attenuated by agmatine (20-40 µg/rat, i.c.v. [intracerebroventricularly]), moxonidine (50 µg/rat, i.c.v.), 2-BFI (20 µg/rat, i.c.v.), L-arginine (80 µg/rat, i.c.v.), amino-guanidine (25 µg/rat, i.c.v.), and arcaine (50 µg/rat, i.c.v.) by their once-daily administration during the withdrawal phase (Days 21, 22, and 23). The antidepressant effect of agmatine in ethanol-withdrawn rats was potentiated by the imidazoline receptor I1 agonist moxonidine (25 µg/rat, i.c.v.) and the imidazoline receptor I2 agonist, 2-BFI (10 µg/rat, i.c.v.) at their sub-effective doses. On the other hand, it was completely blocked by the imidazoline receptor I1 antagonist, efaroxan (10 µg/rat, i.c.v.) and the imidazoline receptor I2 antagonist, idazoxan (4 µg/rat, i.c.v.). In addition, agmatine levels were significantly reduced in brain samples of ethanol-withdrawn rats as compared to the pair-fed control animals. In conclusion, the present study suggests the importance of the endogenous agmatinergic system and the imidazoline receptors system in ethanol withdrawal-induced depression. The data project agmatine as a potential therapeutic target for the alcohol withdrawal-induced depression.

Modulation of stress by imidazoline binding sites: implications for psychiatric disorders.

In this review, we present evidence for the involvement of imidazoline binding sites (IBS) in modulating responses to stress, through central control of monoaminergic and hypothalamo-pituitary-adrenal (HPA) axis activity. Pharmacological and physiological evidence is presented for differential effects of different IBS subtypes on serotoninergic and catecholaminergic pathways involved in control of basal and stress-stimulated HPA axis activity. IBS ligands can modulate behavioural and neuroendocrine responses in animal models of stress, depression and anxiety, and a body of evidence exists for alterations in central IBS expression in psychiatric patients, which can be normalised partially or fully by treatment with antidepressants. Dysfunction in monoaminergic systems and the HPA axis under basal and stress-induced activation has been extensively reported in psychiatric illnesses. On the basis of the literature, we suggest a potential therapeutic role for selective IBS ligands in the treatment of depression and anxiety disorders.

Chronic ethanol attenuates centrally-mediated hypotension elicited via alpha(2)-adrenergic, but not I(1)-imidazoline, receptor activation in female rats.

AIMS: This study dealt with the effect of chronic ethanol administration on hemodynamic responses elicited by alpha(2)-adrenergic (alpha-methyldopa) or I(1)-imidazoline (rilmenidine) receptor activation in telemetered female rats. MAIN METHODS: The effects of alpha-methyldopa or rilmenidine on blood pressure (BP), heart rate (HR) and their variability were investigated in rats that received liquid diet without or with ethanol (5% w/v) for 12 weeks. To evaluate the effect of each drug on cardiovascular autonomic control (BP and HR variability) in the absence or presence of ethanol, three time-domain indices of hemodynamic variability were measured: (i) standard deviation of mean arterial pressure (SDMAP), (ii) standard deviation of beat-to-beat intervals, and (iii) root mean square of successive differences in R-R intervals. KEY FINDINGS: In liquid diet-fed control rats, i.p. rilmenidine (600 microg/kg) or alpha-methyldopa (100 mg/kg) reduced BP along with decreases and increases, respectively, in HR. Both drugs had no effect on HR variability but reduced BP variability (SDMAP), suggesting a reduced vasomotor sympathetic tone. Ethanol feeding attenuated reductions in BP and SDMAP evoked by alpha-methyldopa but not by rilmenidine. SIGNIFICANCE: We conclude that chronic ethanol preferentially compromises alpha(2)- but not I(1)-receptor-mediated hypotension in female rats probably via modulation of vasomotor sympathetic activity. These findings highlight the adequacy of rilmenidine use to lower BP in hypertensive alcoholic females.

Effects of spinal alpha(2)-adrenoceptor and I(1)-imidazoline receptor activation on hindlimb movement induction in spinal cord-injured mice.

A partial recovery of locomotor functions has been shown in spinal cord-transected (Tx) cats after regular treadmill training and repeated administration of clonidine, an alpha(2)-adrenoreceptor agonist. However, clonidine has generally failed to show prolocomotor effects in other models (e.g., rat or mudpuppy in vitro-isolated spinal cord preparations). The reasons for this discrepancy remain unclear, but they may suggest condition- or species-specific effects induced by clonidine. This study is aimed at examining both the acute (at 6 or 41 days post-Tx) and chronic effects of repeated (once a week for one month) clonidine administration (0.25-5.0 mg/kg i.p.) on hindlimb movement generation in Tx mice (thoracic segment9/10). Locomotor-like (LM) and nonlocomotor movements (NLM) were assessed both in open-field and treadmill conditions. The results show that clonidine consistently failed, in both conditions, to induce LM and NLM at all time points even though control experiments revealed hindlimb movements steadily induced by 8-hydroxy-2-(di-N-propylamino)-tetralin (8-OH-DPAT), a serotonin receptor agonist. In turn, clonidine acutely suppressed (I(1)-imidazoline receptor-mediated) the frequency of spontaneously occurring LM and NLM but apparently increased spinal excitability over time, because the frequency of spontaneous LM and NLM was significantly greater in clonidine-treated (before an injection) than vehicle-treated animals after repeated administration for a few weeks. The results clearly show that clonidine can not acutely induce hindlimb movements in untrained and otherwise nonstimulated (e.g., no tail or perineal pinching) Tx mice, although repeated administration may progressively facilitate the expression of spontaneous hindlimb movements.

G-protein inwardly rectifying potassium channels are involved in the hypotensive effect of I1-imidazoline receptor selective ligands.

OBJECTIVE: The present study examined the role of G-protein inwardly rectifying potassium (GIRK) channels in the depressor responses elicited by intracisternal injections of imidazoline-like drugs in anesthetized rabbits. METHODS AND RESULTS: Intracisternal injections of the I1-imidazoline receptor (I1R) selective ligands LNP509 (30 microg/kg) and LNP640 (2 microg/kg) (subthreshold doses), and of the GIRK channel opener flupirtine (30 microg/kg) did not affect mean arterial blood pressure (MAP). LNP509 and LNP640, however, elicited substantial depressor responses in rabbits pretreated with flupirtine (-17 +/- 2 and -18 +/- 1 mmHg, respectively, P < 0.05). Injection of higher doses of LNP509 (200 microg/kg) or LNP640 (10 microg/kg) elicited substantial reductions in MAP (-45 +/- 3 and -39 +/- 2 mmHg, respectively, P < 0.05) in naive rabbits. The depressor responses elicited by the higher doses of LNP509 or LNP640 were markedly diminished by pretreatment with the GIRK channel blocker tertiapin-Q (10 microg/kg) (-23 +/- 3 and -26 +/- 2 mmHg, respectively, P < 0.05 compared with nonpretreated rabbits), whereas tertiapin-Q (10 microg/kg) did not affect MAP by itself. Maximal-specific binding (Bmax) of the I1R ligand [I]LNP911 to PC12 cell membranes (296 +/- 59 fmol/mg protein) was enhanced by flupirtine pretreatment whereas it was reduced by tertiapin-Q pretreatment (687 +/- 122 and 68 +/- 21 fmol/mg protein, respectively, P < 0.05 vs. control binding). CONCLUSION: These findings demonstrate that the modulation of GIRK channels affects I1R's function and raise the possibility that GIRK channels, and I1Rs are parts of a single proteic complex.

Dexmedetomidine reduces long-term potentiation in mouse hippocampus.

BACKGROUND: Dexmedetomidine (Precedex; Abbott Laboratories, Abbott Park, IL) is a selective alpha2-adrenergic agonist that also has affinity for imidazoline receptors. In clinical studies, dexmedetomidine has sedative effects and impairs memory, but the action of dexmedetomidine on synaptic plasticity in the brain has yet to be established. In the present study, the authors investigated the effects of dexmedetomidine on two forms of synaptic plasticity-long-term potentiation (LTP) and paired-pulse facilitation-in the CA1 region of mouse hippocampal slices. METHODS: The authors recorded Schaffer collateral-evoked field excitatory postsynaptic potentials from mouse hippocampal slices in CA1 stratum radiatum. The slope of the rising phase of the field excitatory postsynaptic potential was used to estimate the strength of synaptic transmission. RESULTS: Application of dexmedetomidine for 20 min before "theta burst" stimulation dose-dependently attenuated LTP, and half-inhibitory concentration of dexmedetomidine was 28.6 +/- 5.7 nm. The inhibitory effect of dexmedetomidine on LTP was not abolished by an alpha2-adrenoceptor antagonist (yohimbine), an imidazoline type 1 receptor and alpha2-adrenoceptor antagonist (efaroxan), an alpha1-adrenoceptor antagonist (prazosin), or a gamma-aminobutyric acid type A receptor antagonist (picrotoxin). However, an imidazoline type 2 receptor and alpha2-adrenoceptor antagonist (idazoxan) completely blocked the dexmedetomidine-induced attenuation. Furthermore, 2-benzofuranyl-2-imidaloline, a selective imidazoline type 2 receptor ligand, reduced LTP. 2-(4,5-dihydroimidaz-2-yl)-quinoline, another imidazoline type 2 receptor ligand, abolished the 2-benzofuranyl-2-imidaloline-induced attenuation, but the inhibitory effect of dexmedetomidine on LTP was not abolished by 2-(4,5-dihydroimidaz-2-yl)-quinoline. Dexmedetomidine did not affect paired-pulse facilitation. CONCLUSION: Dexmedetomidine impairs LTP in area CA1 of the mouse hippocampus via imidazoline type 2 receptors and alpha2-adrenoceptors.

Dexmedetomidine increases hippocampal phosphorylated extracellular signal-regulated protein kinase 1 and 2 content by an alpha 2-adrenoceptor-independent mechanism: evidence for the involvement of imidazoline I1 receptors.

BACKGROUND: Dexmedetomidine is a potent and selective alpha2-adrenoceptor (alpha2AR) agonist that exhibits a broad pattern of actions, including sedation, analgesia, and neuroprotection. Recent studies have emphasized the role of phosphorylated extracellular signal-regulated protein kinases (pERK1 and 2) in coupling rapid events such as neurotransmitter release and receptor stimulation long-lasting changes in synaptic plasticity and cell survival. Here, the authors hypothesized that dexmedetomidine increases pERK1 and 2 content and examined the mechanisms involved in this effect. METHODS: The effects of dexmedetomidine and their sensitivity to various pharmacologic agents on expression of pERK1 and 2 were studied by Western blots in hippocampal slices obtained from rats, wild-type mice, and mice carrying targeted deletions of the alpha2AR subtypes. RESULTS: Dexmedetomidine induced a concentration-related increase in the expression of pERK1 and 2 in rat hippocampal slices (EC50 [95% confidence interval] for pERK1, 0.97 [0.68-1.37] microm; for pERK2, 1.15 [0.62-2.14] microm). This effect was insensitive to the inhibitors of the alpha2AR-mediated signaling pathway, to prazosin, and to PP2, an inhibitor of the focal adhesion kinase-Src kinases. In contrast, it was still present in mice deleted for each of the alpha2AR subtypes and was markedly decreased by the antagonist of the I1-imidazoline receptors efaroxan, by phospholipase C and protein kinase C inhibitors, and by PD 098059, a direct inhibitor of ERK1 and 2 phosphorylation. CONCLUSION: Dexmedetomidine increases the expression of pERK1 and 2 via mechanisms independent of alpha2AR activation. The I1-imidazoline receptors likely contribute to these effects. The results may be relevant to some long-term effects (e.g., neuroprotective) of dexmedetomidine in the brain.

Activation of I(2)-imidazoline receptors may ameliorate insulin resistance in fructose-rich chow-fed rats.

Agmatine, an endogenous ligand of imidazoline receptors, was employed to screen the effect on insulin resistance in rats induced by a diet containing 60% fructose. Single intravenous (i.v.) injection of agmatine sulfate for 30min decreased the plasma glucose concentrations in a dose-dependent manner from 0.5mg/kg to 3mg/kg in rats received 4-week fructose-rich chow without an alteration of systolic blood pressure. The plasma glucose lowering action of agmatine (1mg/kg, i.v.) was abolished by the pretreatment with BU224 (1mg/kg, i.v.) at sufficient dosage to block I(2)-imidazoline receptors. In addition, the value of glucose-insulin index, the areas under the curve of glucose and insulin during the intraperitoneal glucose tolerance test, showing an index of in vivo insulin sensitivity was reversed by the same treatment with agmatine in fructose-rich chow-fed rats; this action was also blocked by BU224. Our results suggest that activation of I(2)-imidazoline receptor to improve insulin action on glucose disposal can be considered for targeting glucose metabolism under insulin-resistant state.

[Imidazoline receptors].

An increasing number of studies suggest that the pharmacology and therapeutic potential of a family of imidazoline receptors continues to generate substantial interest of investigators. This review analyzes the functional role of imidazoline receptors, their subpopulation, distribution in the central and peripheral nervous system and action of related ligands. Besides to their brainstem location where I1-receptor sites play a significant role to regulate and modulate blood pressure, they also are found in different parts of brain with the highest densities in the striatum, pallidum, hippocampus, amygdala, substantia nigra, while I3-receptor sites were revealed in pancreas which enhances insulin secretion, I2-receptors are widely distributed in interpeduncular nucleus, arcuate and pineal gland and take a part in monoamine turnover. It is conclusion that imidazoline receptor in near future can become a therapeutic target in the treatment of diabetes, stroke, mood disorders and hyperalgesic condition.

Sympathoexcitation of moxonidine in the caudal ventrolateral medulla is dependent on I1-imidazoline receptors in anesthetized rats.

Moxonidine is a second-generation centrally acting antihypertensive drug that has a high affinity for I(1)-imidazoline receptors (I(1)R). The caudal ventrolateral medulla (CVLM), an important region involved in cardiovascular activity, contains binding sites for centrally acting drugs. Our study aimed to determine the effects of moxonidine injected into the CVLM on cardiovascular activity in anesthetized rats. Unilateral microinjection of moxonidine (0.4 and 4 nmol) into the CVLM dose-dependently increased blood pressure (BP) by 8+/-2 and 18+/-2 mmHg and renal sympathetic nerve activity (RSNA) by 19+/-3 and 48+/-5% without modifying heart rate. Microinjection of the I(1)R/alpha(2)-adrenoceptor antagonist efaroxan (4 nmol) into the CVLM produced significant decreases in baseline BP and RSNA, but also completely abolished the increases in BP (2+/-1 versus 18+/-2 mmHg, P<0.01) and RSNA (3+/-2 versus 45+/-10%, P<0.01) evoked by subsequent injection of moxonidine (4 nmol). However, prior injection of yohimbine (500 pmol), a selective antagonist of alpha(2)-adrenoceptors, into the CVLM had no significant (P>0.05) effect on the moxonidine-induced increase in BP (18+/-2 versus 17+/-3 mmHg) and RSNA (45+/-10 versus 42+/-7%). The current data suggest that moxonidine injection into the CVLM has an excitatory effect on cardiovascular activity, which is mediated by an I(1)R dependent mechanism.

Characterization of an antiproliferative effect of imidazoline receptor ligands on PC12 cells.

The present study aimed at investigating the influence of imidazoline receptor ligands on the proliferation of PC12 cells and the involvement of the sphingosine-1-phosphate (S1P) signaling system in this effect. In cultured PC12 cells, S1P (0.3-100 nM) and the I(1) imidazoline receptor ligands moxonidine (0.3 and 1 mM), agmatine (1 mM), idazoxan (10-100 microM) and efaroxan (1-100 microM) concentration-dependently reduced protein contents which were used as estimates for cell number. The antiproliferative effects elicited by the compounds were abolished after knock-down of S1P(1), S1P(2) and S1P(3) receptors by RNA interference indicating an involvement of S1P receptors. In conclusion, the present data add further evidence to the recent finding that effects of imidazoline receptor ligands in PC12 cells are mediated by homo- and heterodimers of members of the S1P receptor family.

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

RATIONALE: 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. OBJECTIVE: This study examined the anti-hyperalgesic effect of the opioid oxycodone, the selective I2 receptor agonist phenyzoline, alone and in combination, during prolonged treatment. METHODS: 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 19 days passed (phenyzoline). RESULTS: 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 7 days led to significant antinociceptive tolerance to oxycodone but not cross-tolerance to phenyzoline. Similarly, twice-daily treatment with 2 × ED50 dose of phenyzoline for 19 days 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 13-19 days generally led to delayed antinociceptive tolerance. CONCLUSIONS: Combination therapy with oxycodone and I2 receptor agonists maintains prolonged antinociceptive effectiveness with reduced propensity to develop tolerance.

Constructing the ecstasy of MDMA from its component mental organs: Proposing the primer/probe method.

The drug MDMA, commonly known as ecstasy, produces a specific and distinct open hearted mental state, which led to the creation of a new pharmacological class, "entactogens". Extensive literature on its mechanisms of action has come to characterize MDMA as a "messy" drug with multiple mechanisms, but the consensus is that the distinctive entactogenic effects arise from the release of neurotransmitters, primarily serotonin. I propose an alternative hypothesis: The entactogenic mental state is due to the simultaneous direct activation of imidazoline-1 (I1) and serotonin-2 (5-HT2) receptors by MDMA. This hypothesis emerges from "mental organ" theory, which embodies many hypotheses, the most relevant of which are: "Mental organs" are populations of neurons that all express their defining metabotropic receptor, and each mental organ plays a distinct role in the mind, a role shaped by evolution as mental organs evolve by duplication and divergence. Mental organs are the mechanism by which evolution sculpts the mind. Mental organs can be in or out of consciousness. In order for a mental organ to enter consciousness, three things must happen: The mental organ must be activated directly at its defining receptor. 5-HT2 must be simultaneously activated. One of the functions of activated 5-HT2 is to load other simultaneously activated mental organs fully into consciousness. In some cases THC must be introduced to remove long-term blocks mediated by the cannabinoid system. I propose the "primer/probe" method to test these hypotheses. A "primer" is a drug that selectively activates 5-HT2 (e.g. DOB or MEM) or serotonin-1 (5-HT1) and 5-HT2 (e.g. DOET or 2C-B-fly). A "probe" is a drug that activates a receptor whose corresponding mental organ we wish to load into consciousness in order to understand its role in the mind. The mental organ is loaded into consciousness when the primer and probe are taken together, but not when taken separately. For example, the blood pressure medications rilmenidine and moxonidine are selective for imidazoline-1 and can be used to test the hypothesis that the entactogenic mental effects of MDMA are due to loading the imidazoline-1 mental organ into consciousness. The primer/probe method is not limited to testing the specific hypothesis about MDMA and imidazoline, but is a general method for studying the role of mental organs in the mind. For example, the role of dopamine mental organs can be studied by using Parkinson's drugs such as ropinirole or pramipexole as probes.