Small Molecule Targets TMED9 and Promotes Lysosomal Degradation to Reverse Proteinopathy.

Intracellular accumulation of misfolded proteins causes toxic proteinopathies, diseases without targeted therapies. Mucin 1 kidney disease (MKD) results from a frameshift mutation in the MUC1 gene (MUC1-fs). Here, we show that MKD is a toxic proteinopathy. Intracellular MUC1-fs accumulation activated the ATF6 unfolded protein response (UPR) branch. We identified BRD4780, a small molecule that clears MUC1-fs from patient cells, from kidneys of knockin mice and from patient kidney organoids. MUC1-fs is trapped in TMED9 cargo receptor-containing vesicles of the early secretory pathway. BRD4780 binds TMED9, releases MUC1-fs, and re-routes it for lysosomal degradation, an effect phenocopied by TMED9 deletion. Our findings reveal BRD4780 as a promising lead for the treatment of MKD and other toxic proteinopathies. Generally, we elucidate a novel mechanism for the entrapment of misfolded proteins by cargo receptors and a strategy for their release and anterograde trafficking to the lysosome.

Agmatine Inhibits Behavioral Sensitization to Ethanol Through Imidazoline Receptors.

BACKGROUND: Locomotor sensitization to repeated ethanol (EtOH) administration is proposed to play a role in early and recurring steps of addiction. The present study was designed to examine the effect of agmatine on EtOH-induced locomotor sensitization in mice. METHODS: Mice received daily single intraperitoneal injection of EtOH (2.5 g/kg, 20 v/v) for 7 consecutive days. Following a 3-day EtOH-free phase, the mice were challenged with EtOH on day 11 with a single injection of EtOH. Agmatine (10 to 40 µg/mouse), endogenous agmatine enhancers (l-arginine [80 µg/mouse], arcaine [50 µg/mouse], aminoguanidine [25 µg/mouse]), and imidazoline receptor agonist/antagonists were injected (intracerebroventricular [i.c.v.]) either daily before the injection of EtOH during the 7-day development phase or on days 8, 9, and 10 (EtOH-free phase). The horizontal locomotor activity was determined on days 1, 3, 5, 7, and 11. RESULTS: Agmatine (20 to 40 µg/mouse) administration for 7 days (development phase) significantly attenuated the locomotor sensitization response of EtOH challenge on day 11. Further, the agmatine administered only during EtOH-free period (days 8, 9, and 10) also inhibited the enhanced locomotor activity on the 11th day to EtOH challenge as compared to control mice indicating blockade of expression of sensitization. Daily treatment (i.c.v.) with endogenous agmatine enhancers like l-arginine (80 µg/mouse) or arcaine (50 µg/mouse) and aminoguanidine (25 µg/mouse) restrained the development as well as expression of sensitization to EtOH. Imidazoline I1 receptor agonist, moxonidine, and I2 agonist, 2-BFI, not only decreased the development and expression of locomotor sensitization but also potentiated the effect of agmatine when employed in combination. Importantly, I1 receptor antagonist, efaroxan, and I2 antagonist, idazoxan, blocked the effect of agmatine, revealing the involvement of imidazoline receptors in agmatine-mediated inhibition of EtOH sensitization. CONCLUSIONS: Inhibition of EtOH sensitization by agmatine is mediated through imidazoline receptors and project agmatine and imidazoline agents in the pharmacotherapy of alcohol addiction.

Antidepressant-like action of agmatine in the acute and sub-acute mouse models of depression: a receptor mechanism study.

Previous studies have shown that agmatine, a potential neuromodulator or co-transmitter, exhibited antidepressant-like action in animal models, yet its mechanism, especially the receptor mechanism, remains unclear. In the present study, using efaroxan, a preferential antagonist of I1 imidazoline receptor (I1R) and yohimbine, an antagonist of α2 adrenergic receptor (α2AR), we investigated the roles of I1R and α2AR in agmatine's antidepressant-like effect in acute and sub-acute depression models in mice. We found that in the tail-suspension test (TST) and the forced swimming test (FST), acute administration of agmatine (20 and 40 mg/kg, p.o.) significantly shortened the immobility time. Concurrent administration of efaroxan (1 mg/kg, i.p.) completely abolished the antidepressant-like effects of agmatine (40 mg/kg, p.o.) whereas yohimbine (5 mg/kg, i.p.) failed to exert similar effects, suggesting that the acute antidepressant-like effects of agmatine was mainly mediated by I1R but not α2AR. Additionally, in the learned helplessness (LH) test, repeated administration of agmatine (20 mg/kg, p.o., q.d.) for 5 days significantly decreased the escape latency and the number of escape failure, and these effects were respectively abolished by concurrent administration of efaroxan (0.5 mg/kg,i.p., q.d.) and yohimbine (3 mg/kg, i.p., q.d.) for 5 days, suggesting that the antidepressant-like action of agmatine in the LH test was achieved via the activation of both I1R and α2AR. In summary, we found that the antidepressant-like effects of agmatine in the TST and the FST were mediated by activating I1R and in the sub-acute LH test were mediated by activating both I1R and α2AR.

Alpha-2 adrenoceptors and imidazoline receptors in cardiomyocytes mediate counterbalancing effect of agmatine on NO synthesis and intracellular calcium handling.

Evidence suggests that intracellular Ca(2+) levels and contractility of cardiomyocytes can be modulated by targeting receptors other than already identified adrenergic or non-adrenergic sarcolemmal receptors. This study uncovers the presence in myocardial cells of adrenergic α2 (α2-AR) and imidazoline I1 (I1R) receptors. In isolated left ventricular myocytes generating stationary spontaneous Ca(2+) transients in the absence of triggered action potentials, the prototypic agonist of both receptors agmatine can activate corresponding signaling cascades with opposing outcomes on nitric oxide (NO) synthesis and intracellular Ca(2+) handling. Specifically, activation of α2-AR signaling through PI3 kinase and Akt/protein kinase B stimulates NO production and abolishes Ca(2+) transients, while targeting of I1R signaling via phosphatidylcholine-specific phospholipase C (PC-PLC) and protein kinase C (PKC) suppresses NO synthesis and elevates averaged intracellular Ca(2+). We identified that endothelial NO synthase (eNOS) is a major effector for both signaling cascades. According to the established eNOS transitions between active (Akt-dependent) and inactive (PKC-dependent) conformations, we suggest that balance between α2-AR and I1R signaling pathways sets eNOS activity, which by defining operational states of myocellular sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) can adjust Ca(2+) re-uptake and thereby cardiac inotropy. These results indicate that the conventional catalog of cardiomyocyte sarcolemmal receptors should be expanded by the α2-AR and I1R populations, unveiling previously unrecognized targets for endogenous ligands as well as for existing and potential pharmacological agents in cardiovascular medicine.

Behavioral effects of the imidazoline I(2) receptor ligand BU99006 in rats.

The imidazoline I2 receptor ligand BU99006 binds to and attenuates effects mediated by I2 receptors in vitro, although its effects in vivo have not been studied previously. This study examined the effects of BU99006 in two behavioral assays in rats: hypothermia and 2-BFI discrimination. BU99006 (3.2-15 mg/kg, intraperitoneally) produced a dose-dependent hypothermic effect (rectal temperature), which was antagonized by the I2 receptor antagonist idazoxan. BU99006 (3.2 or 10 mg/kg administered 10 min or 2 h before the session, respectively) did not significantly alter hypothermia produced by the I2 receptor agonist 2-BFI (10 mg/kg). In rats discriminating 5.6 mg/kg 2-BFI, BU99006 (1.78-17.8 mg/kg, intraperitoneally) produced 40 and 82% responding on the 2-BFI-associated lever when it was administered immediately or 2 h before the test sessions, respectively. BU99006 enhanced the discriminative stimulus and rate-suppressing effects of 2-BFI. Collectively, these data suggest that BU99006 is an imidazoline I2 receptor agonist with no evidence of I2 receptor antagonism in rats.

[Practice of antihypertensive treatment in diabetic patients with hypertension in Hungary]. / Diabeteses hypertoniás betegek vérnyomáscsökkento kezelésének hazai gyakorlata.

Diabetes mellitus as comorbidity is present in 20-25% of patients suffering from high blood pressure. Because simultaneous presence of these two diseases results in a significant increase of cardiovascular risk, various guidelines chiefly focus on the antihypertensive treatment of patients with diabetes. Combined drug therapy is usually required to achieve the blood pressure target value of <140/85 mmHg defined for patients with diabetes, which must be based on angiotensin converting enzyme-inhibitors or angiotensin receptor blockers. These can be/must be combined with low dose, primarily thiazide-like diuretics, calcium channel blockers with neutral metabolic effect, and further options include the addition of beta blockers, imidazoline-l-receptor antagonists, or alpha-1-adrenoreceptor blockers. Evidence-based guidelines are obviously present in local practice. Although most of the patients receive angiotensin converting enzyme-inhibitor+indapamide or angiotensin converting enzyme-inhibitor+calcium channel blocker combined therapy with favorable metabolic effects, yet the use of angiotensin converting enzyme-inhibitors containing hydrochlorothiazide having diabetogenic potential, and angiotensin receptor blocker fixed combinations is still widespread. Similarly, interesting therapeutic practice can be observed with the use of less differentiated beta blockers, where the 3rd generation carvedilol and nebivolol are still in minority.

Mediation of AMP kinase in the increase of glucose uptake in L6 cells induced by activation of imidazoline I-2 receptors.

Recent work using radioactive tracer indicates that activation of imidazoline I2 receptor (I2R) by guanidinium derivatives may increase the glucose uptake in the skeletal muscle. However, the effect of I2R activation on nonradioactive glucose uptake is still unknown. The ability of glucose uptake in cultured L6 cells is then determined using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) as a fluorescence indicator. The changes in 5'-AMP-activated protein kinase (AMPK) expression were also identified by Western blot analysis. In the present study, 2-(2-benzofuranyl)-2-imidazoline (2-BFI) is used to stimulate I2R while 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) is applied to activate AMPK directly. Both compounds can increase 2-NBDG in L6 cells in a concentration-dependent manner. Meanwhile, compound C at concentrations sufficient to inhibit AMPK blocked this increase of glucose uptake by 2-BFI or AICAR. However, only 2-BFI-induced glucose uptake action was dose-dependently blocked by BU224, a specific I2R antagonist, in L6 cells. Moreover, AMPK phosphorylation was markedly increased by 2-BFI or AICAR in L6 cells. Similarly, only the effect of 2-BFI was attenuated by BU224 in L6 cells. Thus, we suggest that AMPK is mediated in I2R activation for increase of glucose uptake in the skeletal muscle cell and I2R will be a new target for diabetic therapy.

Agmatine attenuates acquisition but not the expression of ethanol conditioned place preference in mice: a role for imidazoline receptors.

The present study investigated the effect of agmatine on acquisition and expression of ethanol conditioned place preference (CPP) and its modulation by imidazoline agents. Swiss albino mice were treated intraperitoneally with saline or agmatine (20-40 mg/kg) before injection of ethanol (1.25 mg/kg) during conditioning days or on a test day (20-120 mg/kg), to observe the effect on acquisition or expression of CPP, respectively. Agmatine inhibited the acquisition but not the expression of ethanol CPP. Furthermore, both the I1 receptor antagonist, efaroxan (9 mg/kg) and the I2 receptor antagonist, BU224 (5 mg/kg) attenuated the agmatine-induced inhibition of the ethanol CPP acquisition. In contrast, the I2 receptor agonist, 2-BFI (5 mg/kg) and I1 receptor agonist, moxonidine (0.4 mg/kg) alone, or a combination of their subeffective doses, significantly attenuated the effect of agmatine (20 mg/kg) on acquisition of ethanol CPP. Agmatine or imidazoline agents alone produced neither place preference nor aversion, and at the doses used in the present study did not affect locomotor activity. Thus, agmatine attenuates the acquisition of ethanol CPP at least in part by imidazoline (I1 or I2) receptors. In future studies, agmatine or agents acting at the imidazoline receptors could be explored for their therapeutic potential in ethanol dependence.

Plasma glucose-lowering action of allantoin is induced by activation of imidazoline I-2 receptors in streptozotocin-induced diabetic rats.

Allantoin, an active principle of yam, is documented to lower plasma glucose in diabetic rats. However, action mechanisms of allantoin remain obscure. It has been indicated that metformin shows ability to activate imidazoline I-2 receptors (I-2R) to lower blood sugar. Allantoin has also a chemical structure similar to metformin; both belong to guanidinium derivative. Thus, it is of special interest to know the effect of allantoin on I-2R. In the present study, the marked plasma glucose-lowering action of allantoin in streptozotocin-induced type-1 like diabetic rats was blocked by specific I-2R antagonist, BU224, in a dose-dependent manner. Also, the increase of ß-endorphin release by allantoin was blocked by BU224 in the same manner. Otherwise, amiloride at the dose sufficient to block I-2AR abolished the allantoin-induced ß-endorphin release and inhibited the blood glucose-lowering action of allantoin markedly but not completely. The direct effect of allantoin on glucose uptake in isolated skeletal muscle was also blocked by BU224. Also, the phosphorylation of AMPK in isolated skeletal muscle was raised by allantoin in a concentration-dependent manner. More-over, insulin sensitivity in diabetic rats was markedly increased by allantoin and this action was also blocked by BU224. These results suggest that allantoin has an ability to activate imidazoline I-2R while I-2AR is linked to the increase of ß-endorphin release and I-2BR is related to other actions including the influence in skeletal muscle for lowering of blood glucose in type-1 like diabetic rats. Thus, allantoin can be developed to treat diabetic disorders in the future.

Stimulatory effect of allantoin on imidazoline I1 receptors in animal and cell line.

Allantoin is known as the agonist of imidazoline receptor, especially the I2 subtype. Effect of allantoin on imidazoline I1 receptor (I1R) relating to reduction of blood pressure and its merit in steatosis are still obscure. Also, farnesoid X receptor (FXR) plays an important role in lipid homeostasis related to I1R activation. Thus, we administered allantoin into high fat diet (HFD)-fed mice showing hypertriglyceridemia and hypercholesterolemia. Allantoin significantly improved hyperlipidemia in HFD mice after 4 weeks of administration. Pretreatment with efaroxan, at a dose sufficient to inhibit I1R activation, attenuated the action of allantoin. In addition, in cultured HepG2 cells, allantoin increased the expression of farnesoid X receptor (FXR). The allantoin-induced FXR expression was blocked by efaroxan. Similar changes were observed in the expressions of FXR-targeted genes. Otherwise, allantoin also lowered systolic blood pressure (SBP) in HFD mice that can be blocked by efaroxan. Taken together, allantoin has an ability to activate I1R for improvement of metabolic disorders.

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.

In vivo and in vitro imaging of I2 imidazoline receptors in the monkey brain.

I2 imidazoline receptors (I2Rs) are associated with depression, Alzheimer's disease, and Huntington's disease. However, in vivo imaging of I2Rs in the monkey brain has not been reported until now. We performed in vitro and in vivo imaging of (I2Rs) in the monkey brain using ¹¹C-labeled 2-(3-fluoro-4-tolyl)-4,5-dihydro-1H-imidazole ([¹¹C]FTIMD) which has high and selective affinity of I2Rs. In an auto-radiography (ARG) study, the distribution pattern of [¹¹C]FTIMD in the monkey brain was similar to that of [³H]idazoxan binging to I2Rs in the human brain, which was previously described. The specific binding of [¹¹C]FTIMD accounted for >97% of total binding in brain regions existing I2 Rs. In positron emission tomography (PET) studies, the radioactivity was accumulated in brain regions existing I2Rs ligand BU224, the accumulated radioactivity was decreased to approximately 66%-75% of the baseline measurement at 15-45 min after injection of [¹¹C]FTIMD. These results suggest that [¹¹C]FTIMD shows the specific-binging to I2Rs in the monkey brain as depicted by PET and ARG. We performed the first in vivo imaging of I2Rs using [¹¹C]FTIMD in the monkey brain.

Activation of imidazoline I-2B receptor by metformin to increase glucose uptake in skeletal muscle.

Metformin (dimethylbiguanide) belongs to guanidinium-derivative and is widely used for treatment of diabetic disorders in clinic. Metformin lowers blood glucose in diabetic animals through increase of glucose uptake into skeletal muscle. Recent evidence indicates that activation of imidazoline I2B receptor (I2BR) by guanidinium-derivatives also increased glucose uptake; however, the effect of metformin on I2BR is still unknown. The blood glucose levels were determined by a glucose kit. The ability of glucose uptake into isolated skeletal muscle or cultured C2C12 cells was determined using 2-[14C]-deoxyglucose as tracer. The expressions of 5' AMP-activated protein kinase (AMPK) and glucose transporter 4 (GLUT-4) were identified by Western blotting analysis. The metformin-induced blood glucose-lowering action was dose-dependently blocked by BU224, a specific I2R antagonist, in Wistar rats. Also, similar reversion by BU224 was observed in isolated skeletal muscle regarding the metformin-induced glucose uptake. Moreover, AMPK phosphorylation by metformin was concentration-dependently reduced by BU224 in isolated skeletal muscle. In addition, signals for metformin increased glucose uptake were identified via I2R/PI3K/PKC/AMPK dependent pathway in C2C12 cells. Thus, we suggest that metformin can activate I2BR to increase glucose uptake and I2BR will be a new target for diabetic therapy.

Assessment of the analgesic effect of centhaquin in mouse tail flick and hot-plate tests.

BACKGROUND: Centhaquin is a centrally acting hypotensive drug like clonidine. Clonidine also produces analgesia and hypothermia in mice and potentiates morphine analgesia. Clonidine analgesia is blocked by idazoxan and naloxone while it is potentiated by BQ123 and sulfisoxazole. This study was conducted to determine the analgesic and hypothermic properties of centhaquin, and to assess whether it potentiates morphine analgesia. Yohimbine (α(2)-adrenergic antagonist), idazoxan (imidazoline/α(2)-adrenergic antagonist), naloxone (opioid antagonist), and BQ123 and sulfisoxazole (endothelin ET(A) antagonists) were used to study the involvement of these receptors in centhaquin analgesia and hypothermia. METHODS: Analgesic (tail flick and hot-plate tests) latencies and body temperatures were measured in male Swiss Webster mice treated with vehicle plus centhaquin, antagonists plus centhaquin or centhaquin plus morphine. RESULTS: Centhaquin produced dose-dependent analgesia which was partially blocked by yohimbine, idazoxan and naloxone. BQ123 and sulfisoxazole did not affect centhaquin analgesia. Morphine analgesia was not potentiated by centhaquin. Centhaquin produced mild hypothermia which was not blocked by yohimbine, idazoxan, naloxone, BQ123 or sulfisoxazole. CONCLUSIONS: This is the first report demonstrating the analgesic activity of centhaquin. The α(2)-adrenergic, imidazoline and opioid receptors are involved in mediating centhaquin analgesia. Endothelin ET(A) receptors do not play a role in centhaquin analgesia; centhaquin does not augment morphine analgesia.

Benzofuranyl-2-imidazoles as imidazoline I2 receptor ligands for Alzheimer's disease.

Recent findings unveil the pharmacological modulation of imidazoline I2 receptors (I2-IR) as a novel strategy to face unmet medical neurodegenerative diseases. In this work, we report the chemical characterization, three-dimensional quantitative structure-activity relationship (3D-QSAR) and ADMET in silico of a family of benzofuranyl-2-imidazoles that exhibit affinity against human brain I2-IR and most of them have been predicted to be brain permeable. Acute treatment in mice with 2-(2-benzofuranyl)-2-imidazole, known as LSL60101 (garsevil), showed non-warning properties in the ADMET studies and an optimal pharmacokinetic profile. Moreover, LSL60101 induced hypothermia in mice while decreased pro-apoptotic FADD protein in the hippocampus. In vivo studies in the familial Alzheimer's disease 5xFAD murine model with the representative compound, revealed significant decreases in the protein expression levels of antioxidant enzymes superoxide dismutase and glutathione peroxidase in hippocampus. Overall, LSL60101 plays a neuroprotective role by reducing apoptosis and modulating oxidative stress.

Novel targets for Huntington's disease in an mTOR-independent autophagy pathway.

Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases such as Huntington's disease. Autophagy induction with the mTOR inhibitor rapamycin accelerates clearance of these toxic substrates. As rapamycin has nontrivial side effects, we screened FDA-approved drugs to identify new autophagy-inducing pathways. We found that L-type Ca2+ channel antagonists, the K+ATP channel opener minoxidil, and the G(i) signaling activator clonidine induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, in which cAMP regulates IP3 levels, influencing calpain activity, which completes the cycle by cleaving and activating G(s)alpha, which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced, and we provide proof of principle for therapeutic relevance in Huntington's disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca2+ (like excitotoxicity) inhibit autophagy, thus retarding clearance of aggregate-prone proteins.

Agmatine reverses memory deficits induced by Aß1-42 peptide in mice: A key role of imidazoline receptors.

Agmatine is a biogenic amine synthesized following decarboxylation of L-arginine by the enzyme arginine decarboxylase and exhibits favourable outcome in neurodegenerative disorders. Present study was designed to examine the relationship between agmatine and the imidazoline receptors in memory deficits induced by Aß1-42 peptide in mice. Mice were treated with single intracerebroventricular (i.c.v.) injection of Aß1-42 peptide (3 µg) and evaluated for learning and memory in Morris water maze (MWM) and subjected to Aß1-42, TNF-α and IL-6 and BDNF immunocontent analysis within the hippocampus. While the learning and memory impairment was evident in the mice subjected to MWM test following Aß1-42 peptide administration, there was a significant increase in Aß1-42, TNF-α and IL-6 and reduction in BDNF immunocontent within the hippocampus. Daily intraperitoneal (i.p.) treatment with agmatine (10 and 20 mg/kg); imidazoline I1 receptor agonist, moxonidine and imidazoline I2 receptor agonist, 2-BFI starting from day 8 to 27 post-Aß1-42 injection, significantly prevented the cognitive deficits and normalized the Aß1-42 peptide, IL-6, TNF-α and BDNF immunocontent in hippocampus. On the other hand, pre-treatment with imidazoline I1 receptor antagonist, efaroxan and imidazoline I2 receptor antagonist, BU 224 attenuated the effects of agmatine on learning and memory in MWM, IL-6, TNF-α and BDNF content. In conclusion, the present study provides functional evidence for the involvement of the imidazoline receptors in agmatine induced reversal of Aß1-42 induced memory deficits in mice. The data projects agmatine and imidazoline receptor agonists as a potential therapeutic target for the treatment of AD.

Moxonidine inhibits excitatory inputs to airway vagal preganglionic neurons via activation of both α2-adrenoceptors and imidazoline I1 receptors.

As an imidazoline I1 receptor agonist with very weak binding affinity for α2-adrenoceptors, moxonidine is commonly used in the treatment of hypertension. Moxonidine also has been implicated to act centrally to reduce airway vagal outflow. However, it is unknown at which central sites moxonidine acts to affect airway vagal activity, and how moxonidine takes effect at synaptic and receptor levels. In this study, airway vagal preganglionic neurons (AVPNs) were retrogradely labeled in neonatal rats from the intrathoracic trachea; retrogradely labeled AVPNs in the external formation of the nucleus ambiguus (NA) were identified in rhythmically active medullary slices using whole-cell patch-clamp techniques; and the effects of moxonidine on the spontaneous excitatory postsynaptic currents (EPSCs) of AVPNs were observed at synaptic level. The results show that moxonidine (10 µmol·L-1) significantly inhibited the frequency of spontaneous EPSCs in both inspiratory-activated and inspiratory-inhibited AVPNs. This effect was partially blocked by SKF-86466 (10 µmol·L-1), a highly selective antagonist of α2-adrenoceptors, or AGN-192403, a selective antagonist of imidazoline I1 receptors, and was completely blocked by efaroxan (10 µmol·L-1), an antagonist of both α2-adrenoceptors and imidazoline I1 receptors. These results demonstrate that moxonidine inhibits the excitatory inputs to AVPNs via activation of both α2-adrenoceptors and imidazoline I1 receptors, and suggest that physiologically both of these two types of receptors are involved in the central regulation of airway vagal activity at preganglionic level. Moxonidine might be potentially useful in diseases with aberrant airway vagal activity such as asthma and chronic obstructive diseases.

Low dose of moxonidine within the rostral ventrolateral medulla improves the baroreflex sensitivity control of sympathetic activity in hypertensive rat.

AIM: To determine the effects of the centrally antihypertensive drug moxonidine injected into the rostral ventrolateral medulla (RVLM) on baroreflex function in spontaneously hypertensive rats (SHR). METHODS: Baroreflex sensitivity control of renal sympathetic nerve activity (RSNA) and barosensitivity of the RVLM presympathetic neurons were determined following application of different doses of moxonidine within the RVLM. RESULTS: Three doses (0.05, 0.5, and 5 nmol in 50 nL) of moxonidine injected bilaterally into the RVLM dose-dependently reduced the baseline blood pressure (BP) and RSNA in SHR. At the highest dose (5 nmol) of moxonidine injection, the maximum gain (1.24%+/-0.04%/mmHg) of baroreflex control of RSNA was significantly decreased. However, the lower doses (0.05 and 0.5 nmol) of moxonidine injection into the RVLM significantly enhanced the baroreflex gain (2.34%+/-0.08% and 2.01%+/-0.07%/mmHg). The moxonidine-induced enhancement in baroreflex function was completely prevented by the imidazoline receptor antagonist efaroxan but not by the alpha(2)-adrenoceptor antagonist yohimbine. A total of 48 presympathetic neurons were recorded extracellularly in the RVLM of SHR. Iontophoresis of applied moxonidine (30-60 nA) dose-dependently decreased the discharge of RVLM presympathetic neurons but also significantly increased the barosensitivity of RVLM presympathetic neurons. CONCLUSION: These data demonstrate that a low dose of moxonidine within the RVLM has a beneficial effect on improving the baroreflex function in SHR via an imidazoline receptor-dependent mechanism.

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.