Katsuji Oguchi

An investigation of monoamine receptors involved in antinociceptive effects of antidepressants.

We attempted to determine which monoamine re-ceptor subtypes are predominantly involved in antidepressant-induced antinociception. Antinociceptive effects were evaluated by using formalin tests with rats. Antidepressants acting as potent inhibitors of norepinephrine reuptake (nisoxetine, nortriptyline, and maprotiline) or inhibiting reuptake of both norepinephrine and serotonin (5-HT) (imipramine and milnacipran) induced dose-dependent antinociception. Simultaneous intraperitoneal administration of antidepressants and either prazosin (&agr;1 antagonist) or ketanserin (5-HT2 antagonist) significantly antagonized antinociceptive effects. Fluvoxamine (selective serotonin reuptake inhibitor) induced antinociception less potently than other antidepressants and was significantly antagonized by ketanserin, but not prazosin. Ondansetron (5-HT3 antagonist) significantly antagonized antinociception by 10 mg/kg of imipramine. In contrast, SDZ-205,557 (5-HT4 antagonist) markedly enhanced antinociception by small-dose (2.5 mg/kg) imipramine. Imipramine-induced antinociception was significantly antagonized by intracerebroventricular administration of prazosin or ketanserin, but not by yohimbine (&agr;2 antagonist) or ondansetron, and was significantly enhanced by intracerebroventricularly administered SDZ-205,557. These findings suggest that &agr;1 adrenoceptors and 5-HT2 receptors in the brain are involved in antidepressant-induced antinociception. In addition, the results suggested functional interactions between noradrenergic and serotonergic neurons as mechanisms for antidepressant-induced antinociception.

TNF-α Drives Human CD14+ Monocytes to Differentiate into CD70+ Dendritic Cells Evoking Th1 and Th17 Responses

Many mechanisms involving TNF-α, Th1 responses, and Th17 responses are implicated in chronic inflammatory autoimmune disease. Recently, the clinical impact of anti-TNF therapy on disease progression has resulted in re-evaluation of the central role of this cytokine and engendered novel concept of TNF-dependent immunity. However, the overall relationship of TNF-α to pathogenesis is unclear. Here, we demonstrate a TNF-dependent differentiation pathway of dendritic cells (DC) evoking Th1 and Th17 responses. CD14+ monocytes cultured in the presence of TNF-α and GM-CSF converted to CD14+ CD1alow adherent cells with little capacity to stimulate T cells. On stimulation by LPS, however, they produced high levels of TNF-α, matrix metalloproteinase (MMP)-9, and IL-23 and differentiated either into mature DC or activated macrophages (Mφ). The mature DC (CD83+ CD70+ HLA-DR high CD14low) expressed high levels of mRNA for IL-6, IL-15, and IL-23, induced naive CD4 T cells to produce IFN-γ and TNF-α, and stimulated resting CD4 T cells to secret IL-17. Intriguingly, TNF-α added to the monocyte culture medium determined the magnitude of LPS-induced maturation and the functions of the derived DC. In contrast, the Mφ (CD14highCD70+CD83−HLA-DR−) produced large amounts of MMP-9 and TNF-α without exogenous TNF stimulation. These results suggest that the TNF priming of monocytes controls Th1 and Th17 responses induced by mature DC, but not inflammation induced by activated Mφ. Therefore, additional stimulation of monocytes with TNF-α may facilitate TNF-dependent adaptive immunity together with GM-CSF-stimulated Mφ-mediated innate immunity.

Asthma-related environmental fungus, Alternaria, activates dendritic cells and produces potent Th2 adjuvant activity.

Asthma is thought to result from dysregulated Th2-like airway inflammatory responses to the environment. Although the etiology of asthma is not fully understood in humans, clinical and epidemiological evidence suggest a potential link between exposure to environmental fungi, such as Alternaria, and development and/or exacerbation of asthma. The goal of this project was to investigate the mechanisms of airway Th2 responses by using Alternaria as a clinically relevant model for environmental exposure. Airway exposure of naive animals to an experimental Ag, OVA, or a common allergen, short ragweed pollen, induced no or minimal immune responses to these Ags. In contrast, mice developed strong Th2-like immune responses when they were exposed to these Ags in the presence of Alternaria extract. Extracts of other fungi, such as Aspergillus and Candida, showed similar Th2 adjuvant effects, albeit not as potently. Alternaria stimulated bone marrow-derived dendritic cells (DCs) to express MHC class II and costimulatory molecules, including OX40 ligand, in vitro. Importantly, Alternaria inhibited IL-12 production by activated DCs, and DCs exposed to Alternaria enhanced Th2 polarization of CD4+ T cells. Furthermore, adoptive airway transfer of DCs, which had been pulsed with OVA in the presence of Alternaria, showed that the recipient mice had enhanced IgE Ab production and Th2-like airway responses to OVA. Thus, the asthma-related environmental fungus Alternaria produces potent Th2-like adjuvant effects in the airways. Such immunogenic properties of certain environmental fungi may explain their strong relationships with human asthma and allergic diseases.

Effects of various dopamine uptake inhibitors on striatal extracellular dopamine levels and behaviours in rats

In vivo central effects of some dopamine uptake inhibitors were evaluated in both brain microdialysis and behavioural studies in rats, and compared with their in vitro affinities to dopamine uptake sites. IC50 values of GBR12909 (1-[2- bis(4-fluorophenyl)methoxy]ethyl]-4-(3- phenylpropyl)piperazine), diclofensine, mazindol, amfonelic acid and nomifensine for inhibiting 1 nM [3H]GBR12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) binding to rat striatal membrane were 7.0, 36, 81, 187 and 290 nM, respectively. In the brain microdialysis study, dopamine levels in the striatal dialysates were increased to 16.3- (GBR12909), 14.1- (nomifensine), 4.8- (diclofensine) and 1.9-fold (amfonelic acid) the respective basal levels 40-60 min after i.p. administration (0.1 mmol/kg) and thereafter decreased slowly but remained at the elevated levels for a further 3 h, while mazindol gradually increased dopamine levels though less pronouncedly than others (1.7-fold 200 min after administration). Remarkable and comparable stereotyped behaviours (licking and forepaw treading) were continuously observed at least for 3 h after administration of GBR12909, nomifensine and amfonelic acid, while stereotypies induced by diclofensine and mazindol were moderate and marginal, respectively. In vivo potencies of dopamine uptake inhibitors to increase the extracellular dopamine levels in the striatum tended to correlate with their in vitro affinities to dopamine uptake sites except in the case of nomifensine, and correlated significantly with their potencies to induce stereotyped behaviours except in the case of amfonelic acid. Based on these findings, pharmacological characteristics of these dopamine uptake inhibitors are discussed.

Evidence for a depressive-like state induced by repeated saline injections in Fischer 344 rats.

We investigated the behavioral changes induced by mild stress in animals that may be relatively susceptible to a depressive-like state, the Fischer 344 rat strain. The mild stress of repeated handling and intraperitoneal (i.p.) injections with saline (2 ml/kg, twice a day for 14 days) elicited a moderate suppression of body weight gain, a decrease in open field activity, and a prolonged immobility during the tail suspension test in Fischer 344 rats compared with Sprague-Dawley rats. Chronic treatment of Fischer 344 rats with imipramine (10 mg/kg i.p., twice a day for 14 days) effectively suppressed open field activity and prolonged immobility. These results suggest that repeated saline injections may be a mild stressor in these rats. In the Fischer 344 strain, which may be vulnerable to the effects of mild stressors, repeated saline injections might induce a depressive-like state and could presumably represent an experimental model for depression.

Nitric oxide‐induced calcium release via ryanodine receptors regulates neuronal function

Mobilization of intracellular Ca2+ stores regulates a multitude of cellular functions, but the role of intracellular Ca2+ release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca2+ release from intracellular stores of central neurons, and thereby promote prolonged Ca2+ signalling in the brain. Reversible S‐nitrosylation of type 1 RyR (RyR1) triggers this Ca2+ release. NO‐induced Ca2+ release (NICR) is evoked by type 1 NO synthase‐dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO‐induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.

Antinociceptive efficacy of antidepressants: assessment of five antidepressants and four monoamine receptors in rats

AbstractPurpose. For assessment of the antinociceptive potency of antidepressants, we compared the antinociceptive effects of serotonin selective reuptake inhibitors (SSRIs) and classical tricyclic antidepressants (TCAs) in rats. We also attempted to elucidate the monoamine receptor subtypes predominantly involved in the antinociceptive effect of antidepressants. Methods. Male Wistar rats received SSRIs (sertraline, fluvoxamine, and citalopram) or TCAs (imipramine and desipramine) intraperitoneally, and the reaction time until pain response in the hot plate test and licking time in the formalin test were measured 60 min later. We also observed the effects of prazosin (an α1 antagonist), WB-4101 (a selective α1A antagonist), yohimbine (an α2 antagonist), WAY-100635 (a selective 5-HT1A antagonist), and ketanserin (a 5-HT2 antagonist), which were simultaneously administered with imipramine or desipramine, on the antidepressant-induced antinociceptive effect in the formalin test. Results. In the hot plate test, desipramine, 20 mg·kg−1, but not imipramine or sertraline, produced a significant increase in reaction time. In the formalin test, desipramine and imipramine produced significant reductions in the licking time at over 5 mg·kg−1 and at over 10 mg·kg−1, respectively. These reductions were nearly complete at 20 mg·kg−1. On the other hand, both SSRIs induced significant reductions in the licking time only at 20 mg·kg−1. Prazosin, WB-4101, and ketanserin significantly antagonized the antinociceptive effect of 10 mg·kg−1 of imipramine. However, imipramine-induced antinociception was not affected by yohimbine and WAY-100635. Prazosin and ketanserin also significantly suppressed antinociception by 5 mg·kg−1 of desipramine. Conclusion. These findings suggest that classical TCAs are likely to have a therapeutic advantage over SSRIs for pain control. In addition, it is likely that central α1 adrenoceptors and 5-HT2 receptors are predominantly involved in imipramine- and desipramine-induced antinociception.

Ca2+-dependent enhancement of [3H]noradrenaline uptake in PC12 cells through calmodulin-dependent kinases

Ca(2+)-dependent regulation of [3H]noradrenaline ([3H]NA) uptake through the NA transporter was studied using PC12 cells. Preincubation for 10 min in the presence of 0.3-10 mM ca2+ in Krebs-Ringer (KR) buffer induced marked enhancement of the uptake (at 1 mM Ca2+, 6.6 times greater than that observed in the absence of Ca2+), which reflected both an increase in Vmax and a decrease in K(m) of the uptake process. Preincubation with 1 mM Ca2+ also induced a significant increase in the Bmax and Kd of [3H]desipramine binding. The uptake was still enhanced after washing cells with Ca(2+)-free buffer following preincubation with 1 mM Ca2+. 1-[N, O-bis(5-Isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), 2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-c hlo rocinnamyl) -N-methylbenzylamine (KN-93) (inhibitors of Ca2+/calmodulin-dependent kinase II), N-(6-aminohexyl)-5-chloro-1-naphthalenesulonamide (W-7) (a calmodulin antagonist), wortmannin (a myosin light chain kinase inhibitor) significantly reduced Ca(2+)-dependent enhancement of the uptake. Mycalolide B (an inhibitor of actin-myosin interaction) also inhibited the enhancement. Although calphostin C (a protein kinase C (PKC) inhibitor) did not affect the enhancement, 12-o-tetradecanoylphorbol 13-acetate (TPA) inhibited the uptake. A synthetic peptide with a sequence (KKVIYKFFS579 IRGSLW) contained in the intracellular COOH-terminal domain of a rat NA transporter was phosphorylated by purified brain Ca2+/calmodulin-dependent protein kinase II. These results suggest that Ca(2+)-dependent enhancement of the [3H]NA uptake in PC12 cells are mediated by activation of calmodulin-dependent protein kinases, probably through stimulation of translocation of the NA transporter to the plasma membrane and/or direct phosphorylation of the transporter itself.

PHARMACOLOGICAL CHARACTERIZATION OF DOPAMINE TRANSPORT IN CULTURED RAT ASTROCYTES

The effects of GBR-12909 (selective DA uptake inhibitor), zimelidine (selective 5-HT uptake inhibitor) and nisoxetine (selective NE uptake inhibitor) on the uptake of 30 nM [3H]DA into cultured rat astrocytes were examined. [3H]DA uptake was inhibited by approximately 50% by GBR-12909 or zimelidine in a concentration-dependent manner (100 nM to approximately 10 microM). Furthermore, the inhibition curves of GBR-12909 were biphasic, and uptake was completely inhibited by a high concentration of GBR-12909 (100 microM). [3H]DA uptake was also inhibited by approximately 50% by nisoxetine in a concentration-dependent manner (0.1 to approximately 100 nM), and nisoxetine was more potent than GBR-12909 or zimelidine. The inhibitory potencies were in the order nisoxetine > GBR-12909 > zimelidine. The uptake of [3H]DA under Na+-free conditions was approximately 50% of that under normal conditions. Thus, DA was taken up by both Na+-dependent and Na+-independent mechanisms. Nisoxetine (100 nM), zimelidine (100 microM) and GBR-12909 (10 microM) inhibited [3H]DA uptake into astrocytes only in the presence of Na+. On the other hand, this uptake was completely inhibited by a high concentration of GBR-12909 (100 microM) in the absence of Na+. The present data suggest that the Na+-dependent uptake of [3H]DA in cultured rat astrocytes may occur in the NE uptake system. Furthermore, astrocytes express the extraneuronal monoamine transporter (uptake2), which is an Na+-independent system, and this transporter is involved in the inactivation of centrally released DA.

The Influence of Droperidol, Diazepam, and Physostigmine on Ketamine-induced Behavior and Brain Regional Glucose Utilization in Rat

Diazepam and droperidol are used clinically with ketamine anes-thesia to reduce emergence hallucinations, vivid unpleasant dreams, and hyperexcitability. Also, there are reports that the recovery time from ketamine anesthesia is shortened after administration of physostigmine. The authors investigated the influence of diazepam, droperidol, and physostigmine pretreatment on ketamine anesthesia by measuring the brain regional activity and behavioral responses in rat. The 2-deoxyglucose brain local metabolic mapping method was used to determine regional brain functional activity. The recovery of tail flick response and righting reflex from ketamine anesthesia were prolonged by diazepam and by droperidol pretreatment, but the duration of agitation was shortened; physostigmine caused no significant change in any of these responses. Ketamine alone caused a statistically significant (P < 0.05) increase in the rate of glucose utilization along the hippocampal molecular layer (control 87 μmol·100 g-1·min-1; ketamine 166 μmol·100 g-1·min-1) and a decrease in medial geniculate (25%), inferior colliculus (37%), and lateral habenula (18%). Diazepam, droperidol, and physostigmine pretreatment did not significantly alter any ketamine-induced glucose use changes, except for a decreased activity in hippocampal molecular layer with diazepam pretreatment (20%) and an increased activity in the lateral habenula with droperidol pretreatment (94%, P < 0.05). These findings corroborate the “epileptogenic” character of ketamine anesthesia and implicate the hippocampus as a major focus. The reduced activity in the hippocampus induced by diazepam pretreatment and the increased activity in the lateral habenula induced by droperidol pretreatment may be factors in the clinical reduction of ketamine hyperexcitability and hallucination by these drugs.