Mitragynine, bioactive alkaloid of kratom, reduces chemotherapy-induced neuropathic pain in rats through α-adrenoceptor mechanism.

BACKGROUND AND PURPOSE: Kratom is a coffee-like plant containing compounds that cause opioid and stimulant effects. The most prevalent bioactive alkaloid of kratom is mitragynine (MG). Opioid effects of MG are apparent (e.g. antinociception and nanomolar affinity for µ, κ and δ opioid receptors), but effects encompassing interactions with additional systems, such as adrenergic and dopaminergic, remain undefined. Given that enhanced adrenergic transmission is a mechanism common to most first-line neuropathic pain medications, we tested the hypothesis that MG reduces chemotherapy-induced neuropathic pain through a mechanism involving α-adrenoceptor activation. METHODS: Rats were injected once with oxaliplatin (6 mg/kg IP) to induce allodynia and then treated with MG (0, 1, 5, 10 mg/kg IP) for 5-7 days. To investigate receptor mechanisms, a fixed dose of MG (5 mg/kg IP) was injected with yohimbine (5 mg/kg IP, α2-adrenoceptor antagonist), prazosin (5 mg/kg IP, α1-adrenoceptor antagonist), or naltrexone (5 mg/kg IP, opioid antagonist). KEY RESULTS: MG (5, 10 mg/kg) dose-dependently reduced mechanical sensitivity in oxaliplatin-injected rats. Anti-allodynic effects of MG were completely inhibited by yohimbine, and significantly reduced by prazosin and naltrexone. MG produced modest hyperlocomotion but only at a dose (30 mg/kg) higher than those required to reduce allodynia. CONCLUSION AND IMPLICATION: The finding that MG reduced neuropathic pain through a mechanism requiring active α-adrenoceptors indicates that the pharmacological profile of MG includes activation of adrenergic, as well as opioid, systems.

Analgesic Effect of Melittin on Oxaliplatin-Induced Peripheral Neuropathy in Rats.

Oxaliplatin is a chemotherapeutic agent used for metastatic colon and other advanced cancers. Most common side effect of oxaliplatin is peripheral neuropathy, manifested in mechanical and cold allodynia. Although the analgesic effect of bee venom has been proven to be effective against oxaliplatin-induced peripheral neuropathy, the effect of its major component; melittin has not been studied yet. Thus, in this study, we investigated whether melittin has an analgesic effect on oxaliplatin-induced allodynia. Intraperitoneal single injection of oxaliplatin (6 mg/kg) induced mechanical and cold allodynia, resulting in increased withdrawal behavior in response to von Frey filaments and acetone drop on hind paw. Subcutaneous melittin injection on acupoint ST36 (0.5 mg/kg) alleviated oxaliplatin-induced mechanical and cold allodynia. In electrophysiological study, using spinal in vivo extracellular recording, it was shown that oxaliplatin-induced hyperexcitation of spinal wide dynamic range neurons in response to peripheral stimuli, and melittin administration inhibited this neuronal activity. In behavioral assessment, analgesic effect of melittin was blocked by intrathecal α1- and α2- adrenergic receptor antagonists administration. Based on these results, we suggest that melittin could be used as an analgesic on oxaliplatin-induced peripheral neuropathy, and that its effect is mediated by activating the spinal α1- and α2-adrenergic receptors.

The adenosine A2A receptor antagonist SCH58261 reduces macrophage/microglia activation and protects against experimental autoimmune encephalomyelitis in mice.

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS) affecting more than 2.5 million individuals worldwide. In the present study, myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) mice were treated with adenosine receptor A2A antagonist SCH58261 at different periods of EAE development. The administration of SCH58261 at 11-28 days post-immunization (d.p.i.) with MOG improved the neurological deficits. This time window corresponds to the therapeutic time window for MS treatment. SCH58261 significantly reduced the CNS neuroinflammation including reduced local infiltration of inflammatory cells, demyelination, and the numbers of macrophage/microglia in the spinal cord. Importantly, SCH58261 ameliorated the EAE-induced neurobehavioral deficits. By contrast, the SCH58261 treatment was ineffective when administered at the beginning of the onset of EAE (i.e., 1-10 d.p.i). The identification of the effective therapeutic window of A2A receptor antagonist provide insight into the role of A2A receptor signaling in EAE, and support SCH58261 as a candidate for the treatment of MS in human.

Dorsal BNST α2A-Adrenergic Receptors Produce HCN-Dependent Excitatory Actions That Initiate Anxiogenic Behaviors.

Stress is a precipitating agent in neuropsychiatric disease and initiates relapse to drug-seeking behavior in addicted patients. Targeting the stress system in protracted abstinence from drugs of abuse with anxiolytics may be an effective treatment modality for substance use disorders. α2A-adrenergic receptors (α2A-ARs) in extended amygdala structures play key roles in dampening stress responses. Contrary to early thinking, α2A-ARs are expressed at non-noradrenergic sites in the brain. These non-noradrenergic α2A-ARs play important roles in stress responses, but their cellular mechanisms of action are unclear. In humans, the α2A-AR agonist guanfacine reduces overall craving and uncouples craving from stress, yet minimally affects relapse, potentially due to competing actions in the brain. Here, we show that heteroceptor α2A-ARs postsynaptically enhance dorsal bed nucleus of the stria terminalis (dBNST) neuronal activity in mice of both sexes. This effect is mediated by hyperpolarization-activated cyclic nucleotide-gated cation channels because inhibition of these channels is necessary and sufficient for excitatory actions. Finally, this excitatory action is mimicked by clozapine-N-oxide activation of the Gi-coupled DREADD hM4Di in dBNST neurons and its activation elicits anxiety-like behavior in the elevated plus maze. Together, these data provide a framework for elucidating cell-specific actions of GPCR signaling and provide a potential mechanism whereby competing anxiogenic and anxiolytic actions of guanfacine may affect its clinical utility in the treatment of addiction.SIGNIFICANCE STATEMENT Stress affects the development of neuropsychiatric disorders including anxiety and addiction. Guanfacine is an α2A-adrenergic receptor (α2A-AR) agonist with actions in the bed nucleus of the stria terminalis (BNST) that produces antidepressant actions and uncouples stress from reward-related behaviors. Here, we show that guanfacine increases dorsal BNST neuronal activity through actions at postsynaptic α2A-ARs via a mechanism that involves hyperpolarization-activated cyclic nucleotide gated cation channels. This action is mimicked by activation of the designer receptor hM4Di expressed in the BNST, which also induces anxiety-like behaviors. Together, these data suggest that postsynaptic α2A-ARs in BNST have excitatory actions on BNST neurons and that these actions can be phenocopied by the so-called "inhibitory" DREADDs, suggesting that care must be taken regarding interpretation of data obtained with these tools.

Suppressive Effects of Bee Venom Acupuncture on Paclitaxel-Induced Neuropathic Pain in Rats: Mediation by Spinal α2-Adrenergic Receptor.

Paclitaxel, a chemotherapy drug for solid tumors, induces peripheral painful neuropathy. Bee venom acupuncture (BVA) has been reported to have potent analgesic effects, which are known to be mediated by activation of spinal α-adrenergic receptor. Here, we investigated the effect of BVA on mechanical hyperalgesia and spinal neuronal hyperexcitation induced by paclitaxel. The role of spinal α-adrenergic receptor subtypes in the analgesic effect of BVA was also observed. Administration of paclitaxel (total 8 mg/kg, intraperitoneal) on four alternate days (days 0, 2, 4, and 6) induced significant mechanical hyperalgesic signs, measured using a von Frey filament. BVA (1 mg/kg, ST36) relieved this mechanical hyperalgesia for at least two hours, and suppressed the hyperexcitation in spinal wide dynamic range neurons evoked by press or pinch stimulation. Both melittin (0.5 mg/kg, ST36) and phospholipase A2 (0.12 mg/kg, ST36) were shown to play an important part in this analgesic effect of the BVA, as they significantly attenuated the pain. Intrathecal pretreatment with the α2-adrenergic receptor antagonist (idazoxan, 50 µg), but not α1-adrenergic receptor antagonist (prazosin, 30 µg), blocked the analgesic effect of BVA. These results suggest that BVA has potent suppressive effects against paclitaxel-induced neuropathic pain, which were mediated by spinal α2-adrenergic receptor.

The role of α1- and α2-adrenoceptor subtypes in the vasopressor responses induced by dihydroergotamine in ritanserin-pretreated pithed rats.

BACKGROUND: Dihydroergotamine (DHE) is an acute antimigraine agent that displays affinity for dopamine D2-like receptors, serotonin 5-HT1/2 receptors and α1/α2-adrenoceptors. Since activation of vascular α1/α2-adrenoceptors results in systemic vasopressor responses, the purpose of this study was to investigate the specific role of α1- and α2-adrenoceptors mediating DHE-induced vasopressor responses using several antagonists for these receptors. METHODS: For this purpose, 135 male Wistar rats were pithed and divided into 35 control and 100 pretreated i.v. with ritanserin (100 µg/kg; to exclude the 5-HT2 receptor-mediated systemic vasoconstriction). Then, the vasopressor responses to i.v. DHE (1-3100 µg/kg, given cumulatively) were determined after i.v. administration of some α1/α2-adrenoceptor antagonists. RESULTS: In control animals (without ritanserin pretreatment), the vasopressor responses to DHE were: (i) unaffected after prazosin (α1; 30 µg/kg); (ii) slightly, but significantly, blocked after rauwolscine (α2; 300 µg/kg); and (iii) markedly blocked after prazosin (30 µg/kg) plus rauwolscine (300 µg/kg). In contrast, after pretreatment with ritanserin, the vasopressor responses to DHE were: (i) attenuated after prazosin (α1; 10 and 30 µg/kg) or rauwolscine (α2; 100 and 300 µg/kg); (ii) markedly blocked after prazosin (30 µg/kg) plus rauwolscine (300 µg/kg); (iii) attenuated after 5-methylurapidil (α1A; 30-100 µg/kg), L-765,314 (α1B; 100 µg/kg), BMY 7378 (α1D; 30-100 µg/kg), BRL44408 (α2A; 100-300 µg/kg), imiloxan (α2B; 1000-3000 µg/kg) or JP-1302 (α2C; 1000 µg/kg); and (iv) unaffected after the corresponding vehicles (1 ml/kg). CONCLUSION: These results suggest that the DHE-induced vasopressor responses in ritanserin-pretreated pithed rats are mediated by α1- (probably α1A, α1B and α1D) and α2- (probably α2A, α2B and α2C) adrenoceptors. These findings could shed light on the pharmacological profile of the vascular side effects (i.e. systemic vasoconstriction) produced by DHE and may lead to the development of more selective antimigraine drugs devoid vascular side effects.

Chronic citalopram administration desensitizes prefrontal cortex but not somatodendritic α2-adrenoceptors in rat brain.

Selective serotonin reuptake inhibitors (SSRIs) regulate brain noradrenergic neurotransmission both at somatodendritic and nerve terminal areas. Previous studies have demonstrated that noradrenaline (NA) reuptake inhibitors are able to desensitize α2-adrenoceptor-mediated responses. The present study was undertaken to elucidate the effects of repeated treatment with the SSRI citalopram on the α2-adrenoceptor sensitivity in locus coeruleus (LC) and prefrontal cortex (PFC), by using in vivo microdialysis and electrophysiological techniques, and in vitro stimulation of [35S]GTPγS binding autoradiography. Repeated, but not acute, treatment with citalopram (5 mg/kg, i.p., 14 days) increased extracellular NA concentration selectively in PFC. The α2-adrenoceptor agonist clonidine (0.3 mg/kg, i.p.), administered to saline-treated animals (1 ml/kg i.p., 14 days) induced NA decrease in LC (Emax = -44 ± 4%; p < 0.001) and in PFC (Emax = -61 ± 5%, p < 0.001). In citalopram chronically-treated rats, clonidine administration exerted a lower decrease of NA (Emax = -25 ± 7%; p < 0.001) in PFC whereas the effect in LC was not different to controls (Emax = -36 ± 4%). Clonidine administration (0.625-20 µg/kg, i.v.) evoked a dose-dependent decrease of the firing activity of LC noradrenergic neurons in both citalopram- (ED50 = 3.2 ± 0.4 µg/kg) and saline-treated groups (ED50 = 2.6 ± 0.5 µg/kg). No significant differences between groups were found in ED50 values. The α2-adrenoceptor agonist UK14304 stimulated specific [35S]GTPγS binding in brain sections containing LC (144 ± 14%) and PFC (194 ± 32%) of saline-treated animals. In citalopram-treated animals, this increase did not differ from controls in LC (146 ± 22%) but was lower in PFC (141 ± 8%; p < 0.05). Taken together, long-term citalopram treatment induces a desensitization of α2-adrenoceptors acting as axon terminal autoreceptors in PFC without changes in somatodendritic α2-adrenoceptor sensitivity.

Activation of the trigeminal α2-adrenoceptor produces sex-specific, estrogen dependent thermal antinociception and antihyperalgesia using an operant pain assay in the rat.

Higher prevalence of several pain disorders in women and sexual dimorphism in G-protein coupled receptor-induced analgesia has been reported. We have previously shown that α2-adrenoceptor-induced antinociception is sex-specific and attenuated by estrogen in the female rat. However, this evidence was obtained using reflexive withdrawal-based nociceptive assays conducted on restrained animals that may not involve cerebral processing. Hence, we evaluated whether activation of the trigeminal α2-adrenoceptor produces sex-specific antinociceptive and antihyperalgesic effects in the orofacial region of the rat using a reward conflict-based operant paradigm in which animals must tolerate nociceptive thermal stimulation to be rewarded. Male and ovariectomized (OVX) Sprague-Dawley rats were implanted intracisternally with a PE10 cannula for drug injections. A group of OVX rats (OVX+E) was administered subcutaneously with estradiol 48h before the test. Effect of clonidine, an α2-adrenoceptor agonist, was determined on the operant pain assay using a fully automated Orofacial Pain Assessment Device. Number of spout licks, thermode contacts, and amount of reward intake were automatically recorded by the ANY-maze software. Using acute pain modeling, clonidine produced a dose-dependent increase in all three parameters in male and OVX groups, however, it was ineffective in the OVX+E group. Similarly, using inflammatory pain modeling, clonidine significantly increased these parameters in carrageenan-treated male and OVX groups but not in the OVX+E group. Thus, α2-adrenoceptor activation produces sex-specific antinociception and antihyperalgesia and estrogen attenuates these effects in female rats using an operant pain assay. These findings may help the discovery of effective analgesics for each sex.

Analgesic effect of electroacupuncture on paclitaxel-induced neuropathic pain via spinal opioidergic and adrenergic mechanisms in mice.

This study was designed to determine the antinociceptive effect and related neuronal mechanism of electroacupuncture (EA) on paclitaxel (PTX)-induced neuropathic pain in mice. PTX (4 mg/kg, i.p.) was administered once a day for 5 consecutive days to induce neuropathic pain. EA stimulation (2 mA, 2 Hz, 30 min) was applied at the ST36 acupoint bilaterally once in every 2 days. Repeated EA stimulation significantly attenuated PTX-induced mechanical allodynia and thermal hyperalgesia. In a separate set of experiment, the antinociceptive effect of a single EA stimulation 8 days after PTX treatment was reduced by intrathecal pretreatment with naloxone (opioid receptor antagonist), idazoxan (alpha2-adrenoceptor antagonist) or propranolol (beta-adrenoceptor antagonist), but not prazosin (alpha1-adrenoceptor antagonist). Moreover, EA remarkably suppressed the PTX-enhanced phosphorylation of the NMDA receptor NR2B subunit in the spinal dorsal horn, and intrathecal pretreatment of naloxone, idazoxan (IDA) or propranolol blocked the effect of EA. In conclusion, EA stimulation at the ST36 acupoint significantly diminished PTX-induced neuropathic pain in mice via the mediation of spinal opioid receptor, alpha2- and beta-adrenoceptors.

On the role of A2A and D2 receptors in control of cocaine and food-seeking behaviors in rats.

Recent studies indicate that adenosine may influence dopamine neurotransmission via A2A receptors which antagonistically interact with D2 receptor-mediated signaling in the brain. We examined the effects of selective A2A receptor ligands such as the agonist CGS 21680 and the antagonists KW 6002 or SCH 58261 as well as of the D2-like receptor antagonist raclopride on reinstatement of cocaine seeking induced by cocaine, the cocaine-conditioned cue, or the D2-like receptor agonist quinpirole in rats. For comparison, effects of the A2A receptor ligands on reinstatement of food seeking were also studied. CGS 21680 significantly attenuated the reinstatement of cocaine (ip) seeking, and even more potently it reduced quinpirole (ip) or the cue-induced relapse of cocaine seeking as well as cue-induced food seeking. A potent reduction toward the cocaine-, quinpirole-, or cue-induced reinstatement of cocaine seeking was seen with raclopride. Pretreatment with KW 6002 or SCH 58261 reinstated cocaine seeking, and such increases were blocked by raclopride. In the higher doses, KW 6002 or SCH 58261 evoked food-seeking. In combination with the subthreshold dose of cocaine (2.5 mg/kg) or with the cue, low doses of KW 6002 but not SCH 58261 reinstated cocaine-seeking behavior, while none of the A2A receptor antagonists affected the cue-induced food-seeking behavior. The results indicate that A2A activation and D2-like receptor blockade counteract cocaine and food relapse. It is proposed that A2A receptor- and D2 receptor-mediated adenosine and dopamine signaling antagonistically interact in the striato-pallidal GABA neurons to regulate cocaine and food-seeking behavior.

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.

Effect of the norepinephrine transporter (NET) inhibition on µ-opioid receptor (MOR)-induced anti-nociception in a bone cancer pain model.

Although norepinephrine transporter (NET) inhibition has an additional effect on µ-opioid receptor (MOR)-mediated anti-nociception in inflammatory and neuropathic pain, its effect on cancer pain is not well characterized. We investigated the additional effect of NET inhibition on MOR activation using a mouse femur bone cancer (FBC) pain model by comparing the anti-nociceptive effect of the dual-acting opioids tramadol and tapentadol and the clinically used MOR-targeted opioids oxycodone and morphine. The anti-nociceptive effects of subcutaneously administered opioids were assessed using the von-Frey filament test. Oxycodone (1 - 10 mg/kg) and morphine (5 - 50 mg/kg) dose-dependently exhibited potent anti-nociceptive effects, whereas tramadol (10 - 56 mg/kg) and tapentadol (10 - 30 mg/kg) exhibited partial effects. Rota-rod analyses of tapentadol at a higher dose (> 30 mg/kg) showed a significant decrease in motor coordination, which was partially recovered by pretreatment with MOR or α(1)-adrenoceptor antagonists. The partial anti-nociceptive effect of tapentadol (30 mg/kg) was completely suppressed by a MOR antagonist, but not by α(1)- or α(2)-adrenoceptor antagonists, suggesting that neither α(1)-adrenoceptor- nor α(2)-adrenoceptor-mediated pathways are involved in anti-nociception in the FBC model. We conclude that addition of NET inhibition does not contribute to MOR-mediated anti-nociception in bone cancer pain.

Preventive and alleviative effect of tramadol on neuropathic pain in rats: roles of α2-adrenoceptors and spinal astrocytes.

The acute analgesic effect of tramadol has been extensively investigated; however, its long-term effect on neuropathic pain has not been well clarified. In this study, we examined the effects of repeated administration of tramadol on partial sciatic nerve ligation-induced neuropathic pain in rats. Each drug was administered once daily from 0 - 6 days (preventive effect) or 7 - 14 days (alleviative effect) after the surgery. Mechanical allodynia was evaluated just before (preventive or alleviative effect) and 1 h after (analgesic effect) drug administration. Like morphine, first administration of tramadol (20 mg/kg) showed an acute analgesic effect on the developed mechanical allodynia, which was diminished by naloxone. Like amitriptyline, repeated administration of tramadol showed preventive and alleviative effects on the mechanical allodynia that was diminished by yohimbine, but not naloxone. The alleviative effects of tramadol lasted even after drug cessation or in the presence of yohimbine. Repeated administration of tramadol increased the dopamine ß-hydroxylase immunoreactivity in the spinal cord. Furthermore, tramadol inhibited the nerve ligation-induced activation of spinal astrocytes, which was reduced by yohimbine. These results suggest that tramadol has both µ-opioid receptor-mediated acute analgesic and α2-adrenoceptor-mediated preventive and alleviative effects on neuropathic pain, and the latter is due to α2-adrenoceptor-mediated inhibition of astrocytic activation.

Nucleus tractus solitarii A(2a) adenosine receptors inhibit cardiopulmonary chemoreflex control of sympathetic outputs.

Previously we have shown that stimulation of inhibitory A1 adenosine receptors located in the nucleus tractus solitarii (NTS) attenuates cardiopulmonary chemoreflex (CCR) evoked inhibition of renal, adrenal and lumbar sympathetic nerve activity and reflex decreases in arterial pressure and heart rate. Activation of facilitatory A2a adenosine receptors, which dominate over A1 receptors in the NTS, contrastingly alters baseline activity of regional sympathetic outputs: it decreases renal, increases adrenal and does not change lumbar nerve activity. Considering that NTS A2a receptors may facilitate release of inhibitory transmitters we hypothesized that A2a receptors will act in concert with A1 receptors differentially inhibiting regional sympathetic CCR responses (adrenal>lumbar>renal). In urethane/chloralose anesthetized rats (n=38) we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of serotonin 5HT3 receptor agonist, phenylbiguanide, (1-8µg/kg) before and after selective stimulation, blockade or combined blockade and stimulation of NTS A2a adenosine receptors (microinjections into the NTS of CGS-21680 0.2-20pmol/50nl, ZM-241385 40pmol/100nl or ZM-241385+CGS-21680, respectively). We found that stimulation of A2a adenosine receptors uniformly inhibited the regional sympathetic and hemodynamic reflex responses and this effect was abolished by the selective blockade of NTS A2a receptors. This indicates that A2a receptor triggered inhibition of CCR responses and the contrasting shifts in baseline sympathetic activity are mediated via different mechanisms. These data implicate that stimulation of NTS A2a receptors triggers unknown inhibitory mechanism(s) which in turn inhibit transmission in the CCR pathway when adenosine is released into the NTS during severe hypotension.

Effects of electrical stimulation of autonomic nerves to the ovary on the ovarian testosterone secretion rate in rats.

Previously, we demonstrated that electrical stimulation of the superior ovarian nerve (SON), but not the ovarian nerve plexus (ONP), reduces the secretion rate of estradiol from the ovary via activation of alpha 2-adrenoceptors in rats. The inhibitory effect of SON on estradiol secretion may be due to reduced production of testosterone, a direct precursor of estradiol. Here, we examined the effects of electrical stimulation of the SON and the ONP on ovarian testosterone secretion in rats. On the day of estrous, ovarian venous blood samples were collected intermittently from the ovarian vein. The secretion rate of testosterone from the ovary was calculated from the difference in the testosterone concentration between ovarian venous plasma and systemic arterial blood plasma, and the rate of ovarian venous plasma flow. Stimulation of either the SON or ONP reduced the secretion rate of testosterone from the ovary. The reduction of the testosterone secretion rate by SON stimulation was not influenced by an alpha 2-adrenoceptor antagonist (yohimbine), but it was abolished by an alpha 1-adrenoceptor antagonist (prazosin). Our results show that ovarian nerves have an inhibitory role in ovarian testosterone secretion, via activation of alpha 1-adrenoceptors, but not alpha 2-adrenoceptors. This, therefore, indicates that the reduction of estradiol secretion by SON stimulation is independent of the reduction of testosterone secretion.

Diluted bee venom injection reduces ipsilateral mechanical allodynia in oxaliplatin-induced neuropathic mice.

Oxaliplatin, which is used as one of anti-cancer drugs, commonly induces peripheral neuropathic pain. We have previously reported that an injection of diluted bee venom (DBV) produced a significant anti-nociceptive effects in several pain models of mice or rats. In this study, we evaluated time- and dose-dependent development of oxaliplatin-induced mechanical allodynia in bilateral hind paws of mice, and investigated the effect of DBV injection on this mechanical allodynia. DBV (0.1 mg/kg) was subcutaneously injected into the Zusanli acupoint 2 weeks after oxaliplatin (10 mg/kg) injection. One hour after DBV injection, we observed a significant reduction of mechanical allodynia in the ipsilateral hind paw, but not in the contralateral hind paw to DBV injection site. We subsequently examined whether this effect of DBV was related to the activation of peripheral nerves in DBV injected site, and then whether it was mediated by the activation of spinal cord alpha-2 adrenoceptors or opioid receptors. Subcutaneous pre-injection of 2% lidocaine (40 mg/kg) into the Zusanli acupoint completely blocked the anti-allodynic effect of DBV. Intrathecal pretreatment with yohimbine (25 µg/mouse), an alpha-2 adrenoceptor antagonist, also prevented the anti-allodynic effect of DBV, whereas pretreatment with naloxone (20 µg/mouse), an opioid receptor antagonist, did not block the effect of DBV. Taken together, these findings demonstrate that DBV injection into the Zusanli acupoint significantly reduces ipsilateral mechanical allodynia generated by oxaliplatin in mice, and also suggest that this anti-allodynic effect is dependent on the peripheral nerve activation in injected site and spinal cord alpha-2 adrenoceptors.

Neuronal α1/2-adrenergic stimulation of IFN-γ, IL-6, and CXCL-1 in murine spleen in late experimental arthritis.

OBJECTIVE: Functional cross-talk exists between sympathetic nerve fibers and cytokine-producing splenic cells in early collagen type II-induced arthritis (CIA) (day 32). These earlier experiments demonstrated exclusively neuronal sympathetic regulation of IFN-γ, CXCL1, IL-6, and TGF-ß. However, in late arthritis, the sympathetic influence might change due to loss of sympathetic nerve fibers and appearance of neurotransmitter-producing cells. We aimed to investigate neurotransmitter-dependent regulation of IFN-γ, CXCL1, IL-6, and TGF-ß in murine spleen in late CIA. METHODS: Spleens were removed when animals reached day 58 (46-68) after immunization to generate 0.35 mm-thick spleen slices, which were transferred to superfusion microchambers to electrically induce release of neurotransmitters. Using respective neurotransmitter antagonists, effects of released neurotransmitters on cytokine secretion were investigated. RESULTS: There was electrically induced inhibition of IFN-γ, CXCL1, and IL-6, and stimulation of TGF-ß, which was much less pronounced than in early CIA. There existed ß adrenergic inhibition of IFN-γ, IL-6, and TGF-ß (and stimulation of CXCL1) independent of electrical stimulation (interpreted as non-neuronal). However, there was a neuronal α1/2 adrenergic stimulation of IFN-γ, CXCL1, and IL-6 and, we observed neuronal A1-adenosinergic stimulation of TGF-ß. CONCLUSIONS: In the late phase of CIA, non-neuronal modulation of cytokine secretion increases while neuronal regulation strikingly decreases. Particularly, ß-adrenergic effects are non-neuronal while α1/2-adrenergic effects are clearly neuronal. We suggest that alterations in sympathetic innervation of the spleen fundamentally change the functional neuroimmune interplay in the spleen of arthritic mice.

Blockade of platelet alpha2B-adrenergic receptors: a novel antiaggregant mechanism.

BACKGROUND: Platelets play a vital role in hemostasis and thrombosis. Catecholamines have a profound effect on platelet aggregation and atherothrombosis but the exact mechanism involved is insufficiently understood. In this report, we demonstrate the existence and role of alpha2B-adrenergic receptors (α2B-ARs) in normal human platelets. METHODS: Sixteen healthy individuals were recruited as donors of normal blood from which platelets were isolated. The presence of α2B-ARs in platelets was proven by Western blot analysis. In order to investigate their function, we performed light transmittance aggregometry and platelet function activity tests by examining the inhibitory effects of specific α2B-AR antibodies and of the selective α2B-AR antagonist ARC 239. RESULTS: Pretreatment of human platelets with agents that selectively block α2B-ARs showed a substantial inhibition in platelet aggregation that had been induced by adenosine diphosphate (ADP), by epinephrine and by arachidonic acid. The percent aggregation decreased from 81.5 ± 1.7% to 35.8 ± 5% and to 24 ± 4.6% for ADP with α2B-Abs and ARC 239 respectively, from 72.2 ± 1.9% to 25.5 ± 4.3% and to 8.8 ± 1.7% for epinephrine with α2B-Abs and ARC 239 respectively, and from 87 ± 2.1% to 47.9 ± 6.2% and to 61.2 ± 5.7% for arachidonic acid with α2B-Abs and ARC 239 respectively, p<0.05 for all. Additionally, collagen/epinephrine closure time increased from 120.8 ± 6.1s to 189.5 ± 39.5s (p=0.001). CONCLUSIONS: Our results reveal that contrary to previous knowledge, the α2B-AR subtype does exist in platelets and is an important regulator of aggregation. Inhibition of α2B-ARs in platelets may offer a novel therapeutic opportunity in the prevention of atherothrombotic events.

Dexmedetomidine administration before, but not after, ischemia attenuates intestinal injury induced by intestinal ischemia-reperfusion in rats.

BACKGROUND: Intestinal ischemia-reperfusion (I/R) injury is a devastating complication in the perioperative period. Dexmedetomidine is commonly applied in the perioperative period. The authors aimed to determine the effects of different doses of dexmedetomidine (given before or after intestinal ischemia) on intestinal I/R injury and to explore the underlying mechanisms. METHODS: Intestinal I/R injury was produced in rat by clamping the superior mesenteric artery for 1 h followed by 2 h reperfusion. Intravenous infusion of dexmedetomidine was performed at 2.5, 5, and 10 µg · kg(-1) · h(-1) for 1 h before or after ischemic insult. In addition, yohimbine hydrochloride was administered intravenously to investigate the role of α2 adrenoreceptor in the intestinal protection conferred by dexmedetomidine. RESULTS: Intestinal I/R increased mortality of rats and caused notable intestinal injury, as evidenced by statistically significant increases in Chiu's scores; serum diamine oxidase and tumor necrosis factor-α concentration, accompanied by increases in the intestinal mucosal malondialdehyde concentration; myeloperoxidase activity; and epithelial cell apoptosis (all P < 0.05 vs. Sham). Except malondialdehyde and myeloperoxidase, all changes were improved by the administration of 5 µg · kg(-1) · h(-1) dexmedetomidine before ischemia (all P < 0.05 vs. Injury) but not after ischemia. Infusion of 2.5 µg · kg(-1) · h(-1) dexmedetomidine before or after ischemia produced no beneficial effects, and infusion of 10 µg · kg(-1) · h(-1) dexmedetomidine led to severe hemodynamic suppression. Yohimbine abolished the intestinal protective effect of the 5 µg · kg(-1) · h(-1) dexmedetomidine infusion before ischemia and was accompanied by the disappearance of its antiapoptotic and antiinflammatory effect. CONCLUSION: Dexmedetomidine administration before, but not after, ischemia dose-dependently protects against I/R-induced intestinal injury, partly by inhibiting inflammatory response and intestinal mucosal epithelial apoptosis via α2 adrenoreceptor activation.

Prominent contribution of L-type Ca2+ channels to cutaneous neurovascular transmission that is revealed after spinal cord injury augments vasoconstriction.

In patients with spinal cord injury (SCI), somatosympathetic reflexes produce exaggerated decreases in skin blood flow below the lesion. This hypoperfusion appears to result from an increased responsiveness of cutaneous arterial vessels to neural activation. Here we investigated the mechanisms that underlie SCI-induced enhancement of neurovascular transmission in a cutaneous vessel, the rat tail artery. Isometric contractions of arterial segments from T11 spinal cord transected and sham-operated rats were compared 6 wk postoperatively. SCI more than doubled the amplitudes of contractions of arteries in response to moderate frequencies of nerve stimulation (0.1 to 1 Hz). In arteries from SCI rats, but not those from sham-operated rats, the L-type Ca(2+) channel blocker nifedipine (1 µM) reduced the amplitudes of nerve-evoked contractions. Furthermore, while the sensitivity to the agonists phenylephrine (α(1)-adrenoceptor selective) and clonidine (α(2)-adrenoceptor selective) did not differ significantly between arteries from SCI and sham-operated rats, nifedipine had a greater inhibitory effect on contractions to both agents in arteries from SCI rats. Although sensitivity to clonidine was unchanged, SCI selectively reduced the contribution of postjunctional α(2)-adenceptors to nerve-evoked contractions. In arteries from unoperated rats, the L-type channel agonist BAY K 8644 (0.1 µM) produced a similar enhancement of nerve-evoked contraction to that produced by SCI and also selectively reduced the contribution of α(2)-adrenceptors to these responses. Together the findings demonstrate that the SCI-induced enhancement of neurovascular transmission in the rat tail artery can largely be accounted for by an increased contribution of L-type Ca(2+) channels to activation of the vascular smooth muscle.