Regulation of corneal noradrenaline release and topography of sympathetic innervation: Functional implications for adrenergic mechanisms in the human cornea.

Having established a main neuronal origin for noradrenaline (NA) in the cornea, we set out to study the physiologic determinants of its release and to correlate functional findings with sympathetic nerve density and overall topography. Whole corneas were obtained from 3 to 4 month-old rabbits and human donors. Study of prejunctional effects was carried out after incubation with radiolabelled NA (3H-NA). Corneas were superfused with warm aerated amine-free medium with cocaine and hydrocortisone to block subsequent neuronal and extraneuronal NA uptake. Samples were collected every 5 min. Four periods of transmural electrical stimulation were applied to assess evoked release of 3H-NA in the absence and in the presence of alpha-2 adrenoceptor antagonists. Catecholamines were extracted with alumina from the superfusate collected and quantified by high pressure liquid chromatography with electrochemical detection (HPLC-ED). Corneal nerve morphology was studied by immunofluorescence staining with monoclonal antibodies and subsequent confocal microscopy. Corneal lamellar sections were also produced (epithelium, stroma, endothelium) and endogenous NA and adrenaline (AD) were quantified by HPLC-ED. Results are means ±â€¯SEM. ANOVA and t-tests were used for statistical analysis. Ratios between enzymatic end products and their substrates were calculated. In both rabbit and human corneas, electrical stimulation increased the outflow of 3H-NA per minute and per shock. Addition of the alpha-2 adrenoceptor antagonist rauwolscine further increased the electrically-evoked overflow of 3H-NA in a concentration-dependent manner. Immunofluorescence revealed particular staining patterns for sensory and sympathetic fibres, epithelial cells and stromal keratocytes. In human corneal lamellar sections only NA was identified, particularly in the endothelium and epithelium. In the rabbit, concentration of NA was ten times that of AD. Electrically-evoked overflow reflects action potential-induced NA release by sympathetic nerves in the cornea and an alpha-2 adrenoceptor-mediated mechanism for its release is presented. Sympathetic innervation has similar functional relevance in both rabbit and human corneas.

Validation of [(11) C]ORM-13070 as a PET tracer for alpha2c -adrenoceptors in the human brain.

This study explored the use of the α2C -adrenoceptor PET tracer [(11) C]ORM-13070 to monitor α2C -AR occupancy in the human brain. The subtype-nonselective α2 -AR antagonist atipamezole was administered to eight healthy volunteer subjects to determine its efficacy and potency (Emax and EC50 ) at inhibiting tracer uptake. We also explored whether the tracer could reveal changes in the synaptic concentrations of endogenous noradrenaline in the brain, in response to several pharmacological and sensory challenge conditions. We assessed occupancy from the bound-to-free ratio measured during 5-30 min post injection. Based on extrapolation of one-site binding, the maximal extent of inhibition of striatal [(11) C]ORM-13070 uptake (Emax ) achievable by atipamezole was 78% (95% CI 69-87%) in the caudate nucleus and 65% (53-77%) in the putamen. The EC50 estimates of atipamezole (1.6 and 2.5 ng/ml, respectively) were in agreement with the drug's affinity to α2C -ARs. These findings represent clear support for the use of [(11) C]ORM-13070 for monitoring drug occupancy of α2C -ARs in the living human brain. Three of the employed noradrenaline challenges were associated with small, approximately 10-16% average reductions in tracer uptake in the dorsal striatum (atomoxetine, ketamine, and the cold pressor test; P < 0.05 for all), but insulin-induced hypoglycemia did not affect tracer uptake. The tracer is suitable for studying central nervous system receptor occupancy by α2C -AR ligands in human subjects. [(11) C]ORM-13070 also holds potential as a tool for in vivo monitoring of synaptic concentrations of noradrenaline, but this remains to be further evaluated in future studies.

GV16 acupoint stimulation with bee venom reduces peripheral hypersensitivity via activation of α2 adrenoceptors in a nitroglycerin-induced migraine mouse model.

Background: Peripheral hypersensitivities develop in the face and hindpaws of mice with nitroglycerin (NTG)-induced migraine. We evaluated whether diluted bee venom (DBV) injections at acupoints prevented these peripheral hypersensitivities and c-Fos expression in the trigeminal nucleus caudalis (TNC). Methods: NTG (10 mg/kg, intraperitoneal, i.p.) was administered every other day for nine days. DBV (0.1 mg/kg) was subcutaneously injected into the ST36 (Zusanli), LI4 (Hegu), or GV16 (Fengfu) acupoints 75 min after each NTG injection. Mice were pretreated with naloxone (5 mg/kg, i.p.) or yohimbine (5 mg/kg, i.p.) 30 min before the DBV injections. Results: NTG injection caused facial cold allodynia, hindpaw mechanical allodynia, and increased c-Fos-immunoreactive (ir) cells in the TNC. Repetitive DBV injections at GV16, but not the ST36, or LI4 acupoints, suppressed NTG-induced hindpaw mechanical allodynia and facial cold allodynia. The number of c-Fos-ir cells also decreased in response to DBV injections at the GV16 acupoint. Remarkably, pretreatment with yohimbine reversed the anti-allodynic effects of DBV injections and attenuated the decreased c-Fos expression in response to GV16 DBV treatment. Naloxone did not block the effects of GV16 DBV stimulation. Conclusion: These findings demonstrate that repetitive DBV treatment at the GV16 acupoint relieves NTG-induced facial and hindpaw hypersensitivities and decreases in c-Fos expression in the TNC via activation of the alpha-2 adrenoceptors, but not the opioid receptors.

Role of alpha-2 adrenergic and kappa opioid receptors in the effects of alcohol gavage-induced dependence on alcohol seeking.

Noradrenaline and alpha-2 receptors are implicated in the neuroadaptive changes in alcohol dependence leading to increased alcohol seeking. Preclinical methods often used to induce dependence, such as alcohol vapor, require long exposure periods. Another method, gavage with alcohol, induces dependence in a shorter time frame (4-6 d), but its effects on relapse are less well understood. We examined the role of alpha-2 receptors in alcohol self-administration (ASA) and relapse in male and female rats made alcohol dependent by gavage. The influence of these variables on the inhibitory effects of the alpha-2 agonist guanfacine on ASA, and on reinstatement induced by the alpha-2 antagonist yohimbine were determined. We also extended this analysis to relapse induced by the kappa opioid receptor agonist U50,488. Male and female Sprague Dawley rats, trained to self-administer alcohol were treated with intragastric vehicle or alcohol (12 g/kg/d for 5 d). In Exp. 1 we examined the effects of alcohol gavage on reinstatement induced by yohimbine (0.625-1.25 mg/kg) and U50,488 (1.25-2.5 mg/kg). In Exp. 2 we determined the effects of a longer period of alcohol gavage on guanfacine (0.25-0.75 mg/kg)-induced reductions in ASA and on yohimbine (0.625-2.5 mg/kg)-induced reinstatement. Our key findings are that alcohol dependence induced by gavage produces sex-specific effects on reinstatement. Non-dependent females had greater reinstatement than males, but dependence reduced reinstatement in females. In males, dependence modestly enhanced yohimbine-induced reinstatement, while U50-induced reinstatement was absent irrespective of dependence condition. Alcohol dependence did not modify the inhibitory effects of guanfacine on ASA in males or females.

Dexmedetomidine elevates the lethal dose threshold of bupivacaine in rats: A dosing study.

Dexmedetomidine (DMED), an alpha-2 adrenoreceptor agonist, has been widely used in regional anesthesia procedures. However, the effect of DMED on local anesthetic cardiotoxicity has not been well delineated. This study consisted of two experiments. In experiment A, 42 Sprague-Dawley (SD) rats were randomly divided into 6 groups (n = 7), each group was pretreated with DMED 0 µg kg-1 (D0 group), 1 µg kg-1 (D1 group), 3 µg kg-1 (D3 group), 6 µg kg-1 (D6 group), 12 µg kg-1 (D12 group), and 24 µg kg-1 (D24 group), administered through the right femoral vein. In experiment B, 20 SD rats were randomly divided into 4 groups (n = 5), such as control group, DMED group, yohimbine (YOH) group, and DMED + YOH group. Each subgroup in experiment B was also pretreated similarly as in experiment A. After pretreatment of rats as described above (in experiments A and B), bupivacaine 2.5 mg kg-1 min-1 was infused to induce cardiac arrest. In experiment A, the lethal dose threshold of bupivacaine and plasma bupivacaine concentration in D3 and D6 group were higher than the other groups. In experiment B, there was no interaction between DMED and YOH in lethal dose threshold, arrhythmia time, plasma concentration of bupivacaine, and myocardial content of bupivacaine. DMED doses of 3-6 µg kg-1 elevated the lethal dose threshold of bupivacaine without involvement of the alpha-2 adrenoceptors.

Impairment of natriuresis and diuresis induced by intrarenal adrenoceptor mechanisms in an experimental model of cirrhosis in rats.

The contribution of intrarenal alpha-2 adrenergic receptors in mediating the enhanced renal excretory responses evoked by the alpha-2-agonist xylazine was examined in a model of cirrhosis in rats. In sham-operated rats, xylazine (0.2 mg/kg, i.v.) increased diuresis and natriuresis (urine flow, control: 78 ± 12.1, 10 min: 155 ± 17, 20 min: 194 ± 19, 30 min: 146 ± 16, 40 min: 114 ± 13, 50 min: 95 ± 10.5 µl/min/g; urinary sodium excretion, control: 6.75 ± 2.08, 10 min: 7.12 ± 2.1, 20 min: 13.4 ± 4.6, 30 min: 14.6 ± 4.02, 40 min: 12.05 ± 2.35, 50 min: 12.7 ± 2.45 µeq/min/g), which was accompanied by a significant reduction in renal sympathetic nerve activity (RSNA) (control: 100, 10 min: 39.5 ± 5.8, 20 min: 53 ± 8.8, 30 min: 72 ± 7.0, 40 min: 83 ± 5.0, 50 min: 94 ± 6.1 AU). Xylazine (0.2 mg/kg) in cirrhotic animals, despite resulting in a significant reduction in RSNA (control: 100, 10 min: 73 ± 4.3*, 20 min: 70 ± 5.0*, 30 min: 76 ± 7.0*, 40 min: 85 ± 5.5*, 50 min: 92 ± 4.8* AU), was unable to increase natriuresis. A higher dose (20 mg/kg) of xylazine was not capable of increasing natriuresis and diuresis, even in the presence of a robust reduction in RSNA. Renal denervation did not alter the onset and time course of cirrhosis. The results indicated that during the development of cirrhosis, there is an adaptive process that disables the intrarenal alpha-2 adrenoceptor mechanisms that selectively promote water and urinary sodium excretion via a sympathetic renal nerve-independent mechanism. Thus, in cirrhotic rats, the diuresis/natriuresis induced by xylazine is independent on RSNA. Intrarenal and/or hormonal changes are probably involved in the impairment of xylazine-induced diuresis/natriuresis in cirrhosis.

Dexmedetomidine enhances tolerance to bupivacaine cardiotoxicity in the isolated rat hearts: alpha 2 adrenoceptors were not involved.

BACKGROUND: Dexmedetomidine was proved to mitigate bupivacaine-induced cardiotoxicity but mechanism of this ability is still unclear. This study was designed to investigate the direct effects of dexmedetomidine on cardiotoxicity induced by bupivacaine on Langendorff rat heart preparation and the role of alpha 2 adrenoceptors in this process was explored. METHODS: Hearts of rat were isolated, mounted on a Langendorff system. Five experimental groups were assessed after 10 min Krebs-Henseleit buffer (KHB) infusions as follow: (1) Group Con, only KHB was perfused; (2) Group Dex, KHB was perfused for 5 min, then dexmedetomidine (10 nmol/L) was added; (3) Group Bupi, KHB was perfused for 25 min, then bupivacaine (50 µmol/L) was added; (4) Group Bupi + Dex, KHB was perfused for 5 min, then the dexmedetomidine (10 nmol/L) was added for 20 min, at last a mixture of KHB + dexmedetomidine + bupivacaine were perfused; (5) Group Bupi + Dex + Yoh, a combination of KHB + yohimbine (alpha 2 adrenoceptor antagonists, 1 µmol/L) was perfusion for 5 min, then dexmedetomidine (10 nmol/L) was added for 20 min, at last a mixture of KHB + yohimbine + dexmedetomidine + bupivacaine was perfused. The experimental perfusion was maintained for 35 min in group Con and group Dex, and the experimental perfusion was sustained until asystole in the other three groups. RESULTS: Compared with group Bupi, dexmedetomidine significantly increased the time to first arrhythmia (P <  0.001) and time to asystole (P <  0.001) in group Bupi + Dex. In addition, dexmedetomidine also significantly increased the time to 25, 50 and 75% reductions in heart rate (P <  0.001) and the time to 25, 50 and 75% reductions in rate-pressure product (P <  0.001) in group Bupi + Dex. Dexmedetomidine increased the cardiac tissue bupivacaine content when asystole (Bupi + Dex vs. Bupi, 58.5 ± 6.3 vs. 46.8 ± 5.6 nmol/g, P = 0.003). The benefit of dexmedetomidine on bupivacaine-induced cardiotoxicity were not eliminated by yohimbine. CONCLUSIONS: Dexmedetomidine could delay the occurrence of bupivacaine-induced arrhythmia and asystole in the isolated rat hearts, but the alpha 2 adrenoceptors were not involved in this process.

Role of cortical alpha-2 adrenoceptors in alcohol withdrawal-induced depression and tricyclic antidepressants.

INTRODUCTION: Although a role for alpha-2 adrenoceptors (alpha-2 ARs) in alcohol use disorder (AUD) and depression is suggested, very little information on a direct interaction between alcohol and these receptors is available. METHODS: In this study adult female Wistar and Wistar-Kyoto (WKY) rats, a putative animal model of depression, were exposed to alcohol vapor 3h daily for 10days (blood alcohol concentration ∼150mg%) followed by daily injection of 10mg/kg of imipramine (IMP, a selective norepinephrine NE/serotonin reuptake inhibitor) or nomifensine (NOMI, a selective NE/dopamine reuptake inhibitor). On day 11 animals were tested for open field locomotor activity (OFLA) and forced swim test (FST) and were sacrificed 2h later for measurement of alpha-2 ARs densities in the frontal cortex and hippocampus using [3H]RX 821002 as the specific ligand. RESULTS: Chronic alcohol treatment increased the immobility in the FST, without affecting OFLA in both Wistar and WKY rats, suggesting induction of depressive-like behavior in Wistar rats and an exacerbation of this behavior in WKY rats. Alcohol treatment also resulted in an increase in cortical but not hippocampal alpha-2 ARs densities in both Wistar and WKY rats. The behavioral effects of alcohol were completely blocked by IMP and NOMI and the neurochemical effects (increases in alpha-2 ARs) were significantly attenuated by both drugs in both strains. CONCLUSIONS: The results suggest a role for cortical alpha-2 ARs in alcohol withdrawal-induced depression and that selective subtype antagonists of these receptors may be of adjunct therapeutic potential in AUD-depression co-morbidity.

Nitric oxide is involved in ibuprofen preemptive analgesic effect in the plantar incisional model of postsurgical pain in mice.

Control of postoperative pain is far from satisfactory. Yet, non-steroidal anti-inflammatory drugs (NSAIDs) remain an important choice. The production of nitric oxide (NO), which plays an important role in the development and maintenance of inflammatory hyperalgesia, is inhibited by NSAIDs. Monoamines also play a key role in the modulation of nociception. The aim of the present work is to study the involvement of NO and monoamines in the antinociceptive mechanism of ibuprofen in postsurgical pain in mice. Surgical incision resulted in mechanical allodynia and increased spinal NO levels. The nitric oxide synthase inhibitor l-NAME (50mg/kg), administered intraperitoneally (i.p.), 30min before the incision decreased the development of postsurgical mechanical allodynia and reduced spinal NO levels. Ibuprofen (100 and 300mg/kg, i.p.), administered 30min before the incision, dose-dependently decreased both spinal NO levels and the development of mechanical allodynia. Administration of ibuprofen (100mg/kg i.p.), 20min following surgery, did not significantly reduce spinal NO level and resulted in a smaller antiallodynic effect. l-Arginine (600mg/kg i.p.), administered 20min before ibuprofen administration, restored both spinal NO level and mechanical allodynia in ibuprofen-treated mice. The selective alpha-2 adrenoceptor blocker yohimbine (4mg/kg i.p.), administered 30min before ibuprofen, also blocked ibuprofen effect on both mechanical allodynia and spinal NO level. These results suggest that inhibition of NO synthesis is involved in the analgesic activity of ibuprofen in post-surgical pain. Alpha-2 adrenoceptors are also involved in the analgesic activity of ibuprofen and NO may be involved in this mechanism.

GHB for cataplexy: Possible mode of action.

The sleep disorder narcolepsy is caused by the loss of orexinergic neurones in the lateral hypothalamus. A troublesome symptom of narcolepsy is cataplexy, the sudden loss of muscle tone in response to strong emotions. It can be alleviated by antidepressants and sodium oxybate (γ-hydroxybutyric acid (GHB)). It is likely that the noradrenergic nucleus locus coeruleus (LC) is involved since it is essential for the maintenance of muscle tone, and ceases to fire during cataplectic attacks. Furthermore, alpha-2 adrenoceptors proliferate in the LC in cataplexy, probably due to 'heterologous denervation supersensitivity' resulting from the loss/weakening of the orexinergic input to the LC. This would lead to the sensitization of the autoinhibition mechanism of LC neurones mediated by inhibitory alpha-2 adrenoceptors ('autoreceptors'). Thus the excitatory input from the amygdala to the LC, activated by an emotional stimulus, would lead to the 'switching off' of LC activity via the supersensitive auto-inhibition mechanism. GHB is an agonist at both γ-aminobutyric acid (GABA) GABA (B) and GHB receptors that may be a subtype of an extrasynaptic GABA(A) receptor. GHB may prevent a cataplectic attack by dampening the tone of LC neurones via the stimulation of inhibitory extrasynaptic GABA receptors in the LC, and thus increasing the threshold for autoinhibition.

Differential effects of an alpha-2 agonist on wakefulness and paradoxical sleep in the rat: A polygraphic and biochemical approach.

The purpose of this experiment was to determine the sensitivity of wakefulness and paradoxical sleep to the α2-agonist, c1onidine. The drug inhibited both wakefulness and paradoxical sleep but the smallest dose necessary to inhibit wakefulness was 64 times larger than the smallest dose inhibiting paradoxical sleep. The effect on paradoxical sleep was inhibitory for all the clonidine doses but wakefulness was enhanced transiently after the four largest doses used. The time between injection and maximum wakefulness enhancement was highly correlated with the dose of c1onidine. The brain level measured after these four different doses at the moment of maximum wakefulness enhancement was the same, suggesting that this effect occurred only when a critical concentration of the drug was attained in the brain and not when the concentration was higher or lower. These data suggest that different α2-adrenoceptors are involved in these two states of vigilance or, alternatively, that their sensitivity is modulated physiologically. In addition, a sensitivity imbalance between different α2-adrenoceptors may exist in wakefulness but not in paradoxical sleep.

Glutamate requires NMDA receptors to modulate alpha2 adrenoceptor in medulla oblongata cultured cells of newborn rats.

α2 Adrenoceptors (α2-ARs) are important in regulating the central control of blood pressure in medulla oblongata. However, it is unclear how this receptor is modulated by different receptors, especially the glutamatergic. In the present study, we studied the influence of ionotropic glutamatergic receptors over the α2-ARs in cultured cells of the medulla oblongata of newborn rats. For this purpose, the protein level of the α2-ARs was assessed after administration to the cultured cells of glutamate (glu), the agonists NMDA and kainate (KA), the NMDA receptor antagonist MK801 and the KA receptor antagonist DNQX. Results indicate that the α2-AR protein levels were increased after the treatments with glu and NMDA, and the addition of MK801 to this treatment thwarted this increase. Notwithstanding the fact that KA did not alter the receptor protein level, the combined treatment of DNQX with glu prevented the α2-AR protein modulation. In conclusion, the present study suggests that ionotropic glutamatergic receptors could be related to the α2-AR protein regulation in the medulla oblongata.

The role of alpha-2 adrenoceptors in the anticonvulsant effects of adenosine on pentylenetetrazole-induced seizure threshold in mice.

Adenosine has anticonvulsant effects in various models of seizures. Alpha-2 adrenoceptors have also demonstrated different effects in different models of epilepsy. In this study, the role of alpha-2 adrenoceptors in the anticonvulsant effects of adenosine in mice was determined according to the method of intravenous pentylenetetrazole-induced seizure. In this study, N(6)-cyclohexyladenosine (CHA) (a selective A1 receptor agonist), clonidine (an alpha-2 adrenoceptors agonist), yohimbine (an alpha-2 adrenoceptors antagonist) and 8-cyclopentyl-1,3-dimethylxanthine (8-CPT) (a selective A1 receptor antagonist) were used. CHA at doses of 0.5, 1 and 2mg/kg significantly increased seizure threshold with the maximum anticonvulsant effect at 2mg/kg. Yohimbine (0.1, 1 and 10mg/kg), clonidine (0.1, 0.5, 1 and 2mg/kg) and 8-CPT (0.5, 1, 2 and 4mg/kg) had no effect on seizure by itself. Combination of yohimbine (10mg/kg) and CHA (0.25mg/kg) increased clonic seizure latency showing that yohimbine and CHA have an additive effect. Increasing the seizure threshold created by combining ineffective doses of yohimbine (10mg/kg) and CHA (0.25mg/kg) was completely inhibited by 8-CPT (4mg/kg) or clonidine (1 and 2mg/kg). Clonidine (0.5, 1 and 2mg/kg) inhibited the anticonvulsant effects of CHA (2mg/kg). Combination of 8-CPT (1mg/kg) and clonidine (0.5mg/kg) which completely inhibited the anticonvulsant effect of CHA (2mg/kg) indicates that 8-CPT and clonidine have an additive effect. In conclusion, adenosine and yohimbine exhibit an additive effect on the enhancement of the pentylenetetrazole-induced seizure threshold in mice, indicating the interaction of alpha-2 adrenoceptors and A1 adenosine receptors.

Enhancing central noradrenergic function in depression: is there still a place for a new antidepressant?

Noradrenaline has long played a key role in the way the etiology of depression is conceptualized and in the mechanism of action of many current antidepressants. Tricyclic antidepressants (TCAs), monoamine oxidase inhibitors (MAOIs), serotonin-noradrenaline reuptake inhibitors (SNRIs), selective noradrenaline reuptake inhibitors (NRIs), the noradrenergic and specific serotonergic antidepressant (NaSSA) mirtazapine, and many atypicals, like mianserin and bupropion, influence, at least in part, central noradrenergic function. Enhancement of noradrenergic function may be particularly helpful in patients with melancholia. However, while noradrenaline will continue to be a target for research into the etiology and treatment of depression, it is unlikely that antidepressants acting solely on noradrenaline will be pursued.

Alpha-2 Adrenoceptor Binding Profile of Natural Products Used to Treat Psychotic illnesses / 대한정신약물학회지

Natural products have long been major sources of pharmaceutical products in medicine. We have undertaken a series of studies designed to characterize the pharmacologic profiles of natural products used to treat psychotic illnesses in Korean traditional medicine. In our previous screening assays, we found that three of plant extracts (Coptidis j, Citrus u, and Phellodendron a) contain active components which show relatively potent binding to alpha-2 adrenoceptors. The present study is to explore pharmacologic activities of extracts as agonism or antagonism by G-protein modulations in radioligand receptor binding study. We have found that two extracts(Coptidis j and Phellodendron a) contain active ingredients which have antagonistic properties to alpha-2 adrenoceptors, whereas one plant extract (Citrus u) has agonistic properties. The demonstration of three plant extracts used to treat psychotic illnesses in Korea may help in the elucidation or their pharmacologic characteristics and provide insights for the development of new psychotropic drugs.