Effects of the novel antidepressant S-adenosyl-methionine on α1- and β-adrenoceptors in rat brain

α₁- and β-adrenoceptors were studied ex vivo in the brains of rats receiving repeated daily treatment with the standard antidepressant imipramine or the atypical antidepressant S-adenosyl-L-methionine (SAM), which has minimal effects on monoamine reuptake or turnover. Consistent with past studies, a decrease in the density of β receptors at three weeks and an increase in the affinity of α₁ receptors for the agonist phenylephrine at one week of treatment was observed with imipramine. By comparison, an increase in the density of β receptors and a decrease in the affinity of α₁ receptors for phenylephrine was observed at one week of treatment with SAM. These changes were no longer apparent at three weeks of treatment. The results suggest that treatment with SAM does lead to changes in adrenergic neurotransmission, but that down regulation of β receptors or increased agonist affinity of α₁ receptors may not be necessary for the production of antidepressant effects.     

Enhanced expression of G protein-coupled receptor kinase 2 selectively increases the sensitivity of A2A adenosine receptors to agonist-induced desensitization

1. G protein-coupled receptor kinases (GRKs) are thought to be important in mediating the agonist-induced phosphorylation and consequent desensitization of G protein-coupled receptor (GPCR) responses. We have previously shown that stable expression of a dominant negative mutant G protein- coupled receptor kinase 2 (GRK2) construct in NG108-15 mouse neuroblastoma x rat glioma cells suppresses the agonist-induced desensitization of A_2A and A_2B adenosine receptor-stimulated adenylyl cyclase activity (Mundell et al., 1997). To further determine the role of GRK2 in agonist-induced desensitization of these adenosine receptors, we stably overexpressed wild type GRK2 in NG108-15 cells.
2. In homogenates prepared from cells overexpressing GRK2, the acute stimulation of adenylyl cyclase by activation of A_2A and A_2B adenosine receptors was markedly reduced, but could be reversed by pretreating the cells with AD (adenosine deaminase), to remove extracellular adenosine from the medium. On the other hand, acute stimulation of adenylyl cyclase by secretin, iloprost, NaF and forskolin was the same in GRK2 overexpressing cells and plasmid-transfected control cells.
3. Cells overexpressing GRK2 were more sensitive to adenosine receptor agonist-induced desensitization than plasmid-transfected control cells. This effect was selective since the agonist sensitivity of desensitization for secretin and IP-prostanoid receptor-stimulated adenylyl cyclase activity was not affected by GRK2 overexpression.
4. These results further implicate GRK2 as the likely mechanism by which A₂ adenosine receptors undergo short-term desensitization in NG108-15 cells, and indicate that even when overexpressed, GRK2 retains its substrate specificity for native receptors in intact cells. Furthermore, the susceptibility of GPCRs to desensitization appears to depend on the level of GRK expression, such that in cells that express high levels of GRK2, low agonist concentrations may be sufficient to trigger GRK-mediated desensitization.

     

α-Methyl-5-HT, a 5-HT2 receptor agonist, stimulates β2-adrenoceptors in guinea pig airway smooth muscle

Alpha-methyl-5-HT is widely used as a high-affinity 5-HT(2) receptors agonist, though some studies have postulated that this drug also activates other serotonergic receptors. In the present work, we found that a wide range of concentrations of alpha-methyl-5-HT induced biphasic responses (contraction followed by relaxation) in guinea pig tracheal rings. The relaxing phase caused by 32microM alpha-methyl-5-HT was blocked by 0.1microM propranolol. Furthermore, during an ongoing histamine-induced contraction, alpha-methyl-5-HT (0.1-100microM) produced a concentration-dependent relaxation starting at 10microM. This relaxation was fully abolished by 0.1microM propranolol or 1microM ICI 118,551 (a selective beta(2)-adrenoceptor antagonist). Additionally, in electrophysiological recordings, 32microM alpha-methyl-5-HT also enhanced the membrane K(+) currents of single tracheal myocytes, an effect reverted by propranolol and ICI 118,551, and mimicked by 0.1microM salbutamol. Thus, we concluded that alpha-methyl-5-HT activates beta(2)-adrenoceptors in guinea pig tracheal smooth muscle at concentrations >or=10microM. This effect must be taken into account when this drug is used in airway smooth muscle and in other tissues expressing beta(2)-adrenoceptors.     

Electroconvulsive shock but not antidepressant drugs increases alpha 1-adrenoceptor binding sites in rat brain

The present experiments served to compare the effects of the 3 5-HT₁ agonists, 8-OH-DPAT, 5-MeODMT and TFMPP on the blood pressure and heart rate of normotensive anaesthetized rats. All the agonists induced, after i.v. injection, a decrease in blood pressure and heart rate. The hypotensive effects of 5-MeODMT and TFMPP were preceded by an increase, suppressed by both ketanserin and methysergide. The decrease in blood pressure induced by 5-MeODMT and 8-OH-DPAT was not antagonized by ketanserin, cocaine (and methysergide for 8-OH-DPAT) but was antagonized by methysergide (for 5-MeODMT) and spiroxatrine (for both). Bradycardia was not susceptible to ketanserin and cocaine (for 5-MeODMT) or to ketanserin and methysergide (for 8-OH-DPAT) but to methysergide and spiroxatrine (for 5-MeODMT) and cocaine and spiroxatrine (for 8-OH-DPAT). These results suggested that the hypotension and bradycardia induced by 5-MeODMT and 8-OH-DPAT are due to the stimulation of ‘5-HT₁-like’ receptors and probably to the 5-HT_1A subtype; the 5-MeODMT-induced hypertension being ascribed to the stimulation of 5-HT₂ receptors.     

Selective destruction of the serotonergic fibers of the fornix-fimbria and cingulum bundle increases 5-HT1 but not 5-HT2 receptors in rat midbrain

Selected and localized lesions of serotonergic (5-HT) neurons were made by microinjection of 5,7-dihydroxytryptamine (5,7-DHT), after pretreatment with desipramine, into the cingulum bundle and fornix-fimbria; these are the major serotonergic hippocampal inputs from the median raphe nucleus. Two weeks after the lesion, the binding of [³H]5-HT (5-HT₁ receptor) was determined in the hippocampus which receives the afferent terminals and, in addition, in the septum/hypothalamus and midbrain from where the fibers originate. Scatchard analysis showed there was no significant change in binding parameters in the hippocampus; however, a significant increase was observed in the B_max in the midbrain (38%) with no change in the K_D. The caudate which receives 5-HT inputs via other 5-HT tracts was not affected by the lesion. The changes in 5-HT₁ receptor number or affinity were not observed 6 days or 5 weeks after the lesion. The binding of the ligands [³H]spiroperidol and [³H]ketanserin to the 5-HT₂ receptor population was also determined in the same brain areas; no changes in receptor binding occurred two weeks after the lesion. These experiments demonstrate that a selective lesion of the serotonergic system can increase 5-HT₁ receptors in the midbrain, which contains the serotonin cell bodies. In addition, as 5-HT₂ binding is not altered, this further supports the hypothesis that 5-HT₁ and 5-HT₂ receptors are distinct populations of receptors.     

Effects of castration on α1-adrenoceptor subtypes in the rat aorta

The expression of α₁-adrenoceptor subtypes in several tissues is regulated by gonadal hormones. In this study, we investigated whether castration regulates the α₁-adrenoceptor subtypes mediating the contractions of the aorta from male rats to noradrenaline. Noradrenaline induced similar concentration-dependent contractions in the aorta from control and castrated rats. Treatment of the aorta from both control and castrated rats with the α_1B/α_1D-adrenoceptor alkylating agent chloroethylclonidine resulted in ≈1600-fold rightward shift in the concentration–response curves to noradrenaline. The pA₂ values found for WB 4101, benoxathian (α_1A-selective) and BMY 7378 (α_1D-selective) indicate that α_1D-adrenoceptors are involved in the contractions of the aorta from control and castrated rats to noradrenaline. However, there was a 15-fold difference between the pK_B estimated through the lowest effective concentrations of the α_1A-adrenoceptor selective antagonist 5-methyl-urapidil in the aorta from control and castrated rats. The pK_B estimated in aorta from control rats is consistent with the interaction with α_1D-adrenoceptors (7.58±0.06), while that calculated in organs from control rats is consistent with α_1A-adrenoceptors (8.76±0.09). These results suggest that castration induces plasticity in the α₁-adrenoceptor subtypes involved in the contractions of the aorta to noradrenaline.     

In vivo pharmacology of irindalone,a 5-HT2 receptor antagonist with predominant peripheral effects

The in vivo effects of irindalone, a newly developed serotonin₂ (5-HT₂) antagonist, have been investigated in comparison with a series of reference compounds. Irindalone potently antagonizes the pressor response induced by 5-HT in pithed rats, but has a 173 times weaker effect against the α₁-adrenoceptor agonist phenylephrine. Irindalone is relatively weak in rat models detecting central 5-HT₂ antagonism, that is, inhibition of quipazine- or I-5-HTP plus citalopram-induced head twitches, inhibition of I-5-HTP plus citalopram-induced increases of flexor reflexes, and inhibition of the discriminative stimulus properties induced by d-LSD. Furthermore, it displaces in vivo ³H-ketanserin binding in frontal cortex. Irindalone weakly antagonizes the flexor reflex stimulated by the α₁-adrenoceptor agonist St 587. No dopamine receptor inhibition is detected in the methylphenidate gnawing test in mice. High bioavailability is indicated by the identical ED₅₀ values obtained in the head twitch model after s.c. and p.o. administration. The activity profile of irindalone resembles that of ketanserin except in two characteristics: ketanserin has greater potency than irindalone as an antagonist in the 5-HTP-induced flexor reflex, but has a shorter duration of action. The effect of irindalone is stereoselective, since its opposite enantiomer Lu 21-099 is almost inactive in the models for central and peripheral 5-HT₂ receptor antagonism. Finally, the effect of repeated treatment with irindalone (18 μmol/kg, p.o., twice daily for 2 weeks) on inhibition of quipazine-induced head twitches was studied. Two days after the last dose, the potency for inhibiting quipazine was unchanged, indicating that no tolerance to 5-HT₂ receptor antagonism develops using this dose regimen. It is concluded that irindalone is a potent 5-HT₂ antagonist with preferential effects at peripheral sites.     

Evidence from binding studies for α2-adrenoceptors directly associated with glomeruli from rat kidney

Binding of the α₂-adrenoceptor radioligands [³H]clonidine and [³H]rauwolscine but not the α₁-adrenoceptor radioligand [³H]prazosin was enhanced in membranes prepared from rat isolated renal glomeruli. [³H]Rauwolscine binding to glomeruli was stereoselective with respect to the (−)-isomer of noradrenaline and the order of potency of a series of antagonists for displacement of binding indicated that the α₂-adrenoceptors in this preparation differ somewhat from those in some other species and tissues. Chemical sympathectomy produced no significant change in the number of sites labelled by [³H]rauwolscine indicating that few of the α₂-adrenoceptors in glomerular membranes are associated with sympathetic nerve terminals.     

Chronic fluoxetine induces opposite changes in G protein coupling at pre and postsynaptic 5-HT1A receptors in rat brain

Chronic treatment with the antidepressant fluoxetine may lead to changes in the properties of pre- and postsynaptic 5-HT(1A) receptors due to modifications in the receptor-G protein coupling process. We have evaluated, in rats, the effect of chronic fluoxetine (10 mg/kg/day) at brain 5-HT(1A) receptors using different techniques. The density of 5-HT(1A) receptors was unchanged in fluoxetine-treated rats vs. vehicle group. Stimulation of [(35)S]GTPgammaS binding induced by (+/-)8-OH-DPAT was significantly attenuated in dorsal raphe nucleus after fluoxetine (+3.7 vs. +31.2% in vehicle). The inhibition of dorsal raphe firing by (+/-)8-OH-DPAT (ED(50) in vehicle = 2.1 microg/kg, i.v.) was also attenuated in rats treated with fluoxetine (ED(50)=4.7 microg/kg). In contrast, a significant increase on (+/-)8-OH-DPAT-induced stimulation of [(35)S]GTPgammaS binding was observed in CA(1) (+53.4 vs.+20.2% in vehicle) and dentate gyrus (+105.7 vs. +52.6% in vehicle) but not in entorhinal cortex. Our data demonstrate that fluoxetine-induced desensitization of 5-HT(1A) autoreceptors occurs at G protein level. Moreover, a relevant finding is the region-specific hypersensitivity of postsynaptic 5-HT(1A) receptors, in the hippocampus but not in entorhinal cortex, following chronic fluoxetine. These differential adaptive changes in brain 5-HT(1A) receptors could underlie the mechanism of action of antidepressants and also contribute to their clinical effects.     

Relaxant β-Receptors in the Trachea,but not Prejunctional α2-Receptors in the Tracheal Cholinergic Neurones,are Subjected to Homologous Desensitization

Abstract: Guinea-pigs were pretreated with either isoprenaline, terbutaline or the α₂ agonist B-HT 920 in order to asses the hypothesis that β- and α₂ receptors in trachea are subjected to homologous desensitization. In these experiments the β receptor activity was investigated on tracheal ring preparations contracted with carbacholine. Both in the isoprenaline and the terbutaline pretreated group the relaxant responses to β agonists were diminished. Pretreatment with B-HT 920 did not affect the sensitivity of the trachea to β stimulation. In order to asses the responsiveness of α₂ receptors the trachea was contracted by electrical field stimulation in the presence of propranolol. During these conditions contractions were mediated by activation of cholinergic neurones and inhibitory effects of alpha stimulation were due to inhibition of the cholinergic neurotransmission by stimulation of prejunctional α₂ receptors. In these tests neither isoprenaline, terbutaline nor B-HT 920 pretreatment affected the responsiveness of α stimulation to inhibit the electrically induced contractions.     

Pharmacological characterization of a rat 5-hydroxytryptamine type3 receptor subunit (r5-HT3A(b)) expressed in Xenopus laevis oocytes

1. The present study has utilized the two electrode voltage-clamp technique to examine the pharmacological profile of a splice variant of the rat orthologue of the 5-hydroxytryptamine type 3A subunit (5-HT_3A(b)) heterologously expressed in Xenopus laevis oocytes.
2. At negative holding potentials, bath applied 5-HT (300 nM–10 μM) evoked a transient, concentration-dependent (EC₅₀=1.1±0.1 μM), inward current. The response reversed in sign at a holding potential of −2.1±1.6 mV.
3. The response to 5-HT was mimicked by the 5-HT₃ receptor selective agonists 2-methyl-5-HT (EC₅₀=4.1±0.2 μM), 1-phenylbiguanide (EC₅₀=3.0±0.1 μM), 3-chlorophenylbiguanide (EC₅₀=140± 10 nM), 3,5-dichlorophenylbiguanide (EC₅₀=14.5±0.4 nM) and 2,5-dichlorophenylbiguanide (EC₅₀= 10.2±0.6 nM). With the exception of 2-methyl-5-HT, all of the agonists tested elicited maximal current responses comparable to those produced by a saturating concentration (10 μM) of 5-HT.
4. Responses evoked by 5-HT at EC₅₀ were blocked by the 5-HT₃ receptor selective antagonist ondansetron (IC₅₀=231±22 pM) and by the less selective agents (+)-tubocurarine (IC₅₀=31.9± 0.01 nM) and cocaine (IC₅₀=2.1±0.2 μM).
5. The data are discussed in the context of results previously obtained with the human and mouse orthologues of the 5-HT_3A subunit. Overall, the study reinforces the conclusion that species differences detected for native 5-HT₃ receptors extend to, and appear largely explained by, differences in the properties of homo-oligomeric receptors formed from 5-HT_3A subunit orthologues.

     

Differential effects of the novel antidepressant agomelatine (S 20098) versus fluoxetine on 5-HT1A receptors in the rat brain

Agomelatine (S 20098) is a novel antidepressant drug with melatonin receptor agonist and 5-HT(2C) receptor antagonist properties, but actual mechanisms underlying its antidepressant action are unknown. Because functional desensitization of 5-HT(1A) autoreceptors in the dorsal raphe nucleus (DRN) occurs after chronic administration of several classes of antidepressants, we investigated whether this adaptive change could also be induced by agomelatine. Neither acute nor chronic treatment with agomelatine (10 mg/kg i.p. for 14 days or 50 mg/kg i.p. for 21 days) changed the density of 5-HT(1A) receptors and their coupling with G proteins in the DRN and the hippocampus in rats. Moreover, these treatments did not affect the basal electrophysiological characteristics and the responses to 5-HT(1A) receptor stimulation of DRN and hippocampal neurons in brain slices. Parallel experiments with melatonin (10 mg/kg i.p. for 14 days) and fluoxetine (5 mg/kg i.p. for 14 days) as reference compounds showed that the former was unable to affect 5-HT(1A) receptors whereas the latter decreased both the 5-HT(1A) receptor-mediated [(35)S]GTP-gamma-S binding and the potency of ipsapirone, a 5-HT(1A) receptor agonist, to inhibit neuronal firing in the DRN. These data indicate that the antidepressant action of agomelatine is not mediated through the same mechanisms as SSRIs or tricyclics.     

Synergism between fluoxetine and the mGlu2/3 receptor agonist, LY379268, in an in vitro model for antidepressant drug-induced neurogenesis

We examined the interaction between the selective serotonin reuptake inhibitor, fluoxetine, and group-II metabotropic glutamate (mGlu) receptors using progenitor cells isolated from cultured cerebellar granule cells, considered as an in vitro model of antidepressant-drug induced neurogenesis. These cells expressed mGlu3 receptors negatively coupled to adenylyl cyclase. A 72-h treatment with either fluoxetine or low concentrations of mGlu2/3 receptor agonists (LY379268 or 2R,4R-APDC) enhanced cell proliferation. The action of fluoxetine was mediated by the activation of 5-HT(1A) receptors. We found a strong synergism between fluoxetine and LY379268 in enhancing cell proliferation and inhibiting cAMP formation. The increased cell proliferation induced by fluoxetine+LY379268 was abrogated by the cAMP analogue, 8-Br-cAMP, as well as by drugs that inhibit the mitogen-activated protein kinase and phosphatidyilinositol-3-kinase pathways. Interestingly, fluoxetine and LY379268 also acted synergistically in promoting neuronal differentiation when progenitor cells were incubated in the presence of serum. These data support the hypothesis that a combination between classical antidepressants and mGlu2/3 receptor agonists may be helpful in the experimental treatment of depression.     

Urethane,but not pentobarbitone,attenuates presynaptic receptor function in rats: a contribution to the choice of anaesthetic

Background and purpose:

We examined whether cannabinoid CB₁ and histamine H₃ receptors resemble α₂-adrenoceptors in that their presynaptically mediated cardiovascular effects are less marked in urethane- than in pentobarbitone-anaesthetized pithed rats.

Experimental approach:

Effects of the cannabinoid agonist CP-55,940 and the H₃ receptor agonist imetit on electrically induced tachycardic and vasopressor responses, respectively, was compared in pithed rats anaesthetized with urethane or pentobarbitone. The affinity of urethane for the three receptors was measured by radioligand binding studies in rat brain cortex membranes and its potency assessed in superfused mouse tissues preincubated with ³H-noradrenaline.

Key results:

The neurogenic tachycardic response was less markedly inhibited by CP-55,940 in urethane- than in pentobarbitone-anaesthetized pithed rats. Imetit inhibited the neurogenic vasopressor response after pentobarbitone but not after urethane. The catecholamine-induced tachycardic and vasopressor response did not differ between rats anaesthetized with either compound. Urethane 10 mM (plasma concentration reached under anaesthesia) did not affect binding to CB₁ or H₃ receptors and α₂ adrenoceptors, nor did it alter the inhibitory effect of agonists at the three receptors on electrically evoked ³H-noradrenaline release.

Conclusions and implications:

Urethane, but not pentobarbitone, abolished the H₃ receptor-mediated vascular response in pithed rats and attenuated the CB₁ receptor-mediated cardiac response much more than pentobarbitone. The weaker effects of CB₁, H₃ and α₂ receptor agonists cannot be explained by antagonism by urethane at the three receptors in vitro. Pentobarbitone, but not urethane, is suitable as an anaesthetic for investigations of inhibitory presynaptic receptor function in pithed and anaesthetized rats.     

Evidence that 5-HT2C receptor antagonists are anxiolytic in the rat Geller-Seifter model of anxiety

Four non-selective 5-HT_2C/5-HT_2A receptor antagonists, mianserin (2–8 mg/kg), 1-naphthyl piperazine (1-NP) (0.5–1 mg/kg), ICI 169,369 (20 mg/kg) and LY 53857 (5 mg/kg), increased punished responding for a food reward in the rat Geller-Seifter test 30 min after subcutaneous (SC) administration. This property was shared by the benzodiazepine anxiolytic chlordiazepoxide (5 mg/kg SC). However, the selective 5-HT_2A receptor antagonists ketanserin (0.2–1 mg/kg SC) and altanserin (0.5, 1 mg/kg SC) had little effect. The 5-HT_1A, 5-HT_1B and-adrenergic receptor antagonists pindolol and cyanopindolol (6 mg/kg SC) did not affect punished responding either, nor did the 5-HT_1D receptor partial agonist and ₂ adrenergic receptor antagonist yohimbine (2.5 mg/kg SC) or the histamine H₁ receptor antagonist mepyramine (1 mg/kg SC). Unpunished responding was also modestly increased after some doses of the 5-HT_2C/5-HT_2A receptor antagonists. However, this effect was inconsistent and was also seen after chlordiazepoxide. Furthermore, it was not associated with the increase in punished responding observed in rats orally treated with mianserin (10, 20 mg/kg), 1-NP (10, 20 mg/kg) or ICI 169,369 (50 mg/kg). The action of the 5-HT_2C/5-HT_2A receptor antagonists tested is therefore consistent with anxiolysis. The results also strongly suggest that this effect is mediated by blockade of the 5-HT_2C receptor, although the possibility of 5-HT_2B receptor mediation is discussed.     

The rostroventromedial medulla is not involved in α2-adrenoceptor-mediated antinociception in the rat

The aim of the current study was to investigate the role of the rostroventromedial medulla (RVM) in α₂-adrenoceptor-mediated antinociception. Medetomidine or clonidine, selective α₂-adrenoceptor agonists were microinjected into the RVM in unanesthetized rats with a chronic guide cannula. The antinociceptive effects were evaluated using the tail-flick and hot-plate tests. For comparison, medetomidine was microinjected into the cerebellum or the periaqueductal gray (PAG). To study the role of medullospinal pathways, the tail-flick latencies were also measured in spinalized rats. The reversal of the antinociception induced by intracerebral microinjections of medetomidine was attempted by s.c. atipamezole, a selective α₂-adrenoceptor antagonist. The reversal of the antinociception induced by systemic administration of medetomidine was attempted by microinjections of 5% lidocaine or atipamezole into the RVM. When administered into the RVM, medetomidine produced a dose-dependent (1–30 μg) antinociception in the tail-flick and hot-plate tests, which antinociceptive effect was completely reversed by atipamezole (1 mg/kg, s.c.). Also clonidine produced a dose-dependent (3–30 μg) antinociception following microinjection into the RVM. Microinjections of medetomidine into the cerebellum or the PAG produced an identical dose-response curve in the tail-flick test as that obtained following microinjection into the RVM. In spinalized rats the antinociceptive effect (tail-flick test) induced by medetomidine microinjected into the RVM was not less effective than in intact rats. Lidocaine (5%) or atipamezole (5 μg) microinjected into the RVM did not attenuate the antinociception induced by systemically administered medetomidine (100 μg/kg, s.c.). The adapting skin temperature of the tail was increased in a nonmonotonic fashion following medetomidine. The results indicate that the RVM is not a site which is critical for the α₂-adrenergic antinociception. The antinociception following intracerebral microinjections of medetomidine into the RVM, PAG or the cerebellum in the current study can be explained by a spread of the α₂-adrenoceptor agonist into the spinal level to activate directly spinal α₂-adrenoceptors. Also, the antinociception following systemic administration of medetomidine can be explained by spinal α₂-adrenergic mechanisms. The medetomidine-induced increase of the adapting skin temperature may have attenuated the medetomidine-induced increases in the response latencies to noxious heat.     

Chronic dexamethasone administration decreases noradrenaline-stimulated, but not serotonin-stimulated, phosphoinositide metabolism in the rat brain

The present study was undertaken to investigate the effects of chronic administration of dexamethasone on the noradrenaline- and serotonin-stimulated (5-HT-stimulated) phosphoinositide metabolism in hippocampus and frontal cortex of the rat brain. For determination of phosphoinositide metabolism, slices from selected regions of the rat brain (hippocampus or frontal cortex) were loaded with myo- [³H] inositol and stimulated with the agonists (noradrenaline or 5-HT) in the presence of LiCl (7.5 mM). Administration of dexamethasone (1 mg/kg/day) every 2nd day for 14 days markedly reduced the noradrenaline-stimulated phosphoinositide metabolism in the rat hippocampus (IP₁: 60% of the control value). In the rat frontal cortex, the noradrenaline-stimulated phosphoinositide metabolism was less depressed by the chronic administration of dexamethasone (IP₁: 84% of the control value). However, the chronic administration of dexamethasone did not affect the 5-HT-stimulated phosphoinositide metabolism in the rat brain. The binding characteristics of ₁-adrenoceptors and 5-HT_2A receptors were unaffected by the chronic treatment with dexamethasone. These results indicate that chronic administration of dexamethasone induces regional and neurotransmitter-specific changes of phosphoinositide metabolism in rat brain. The results suggest that the reduction of noradrenaline-stimulated phosphoinositide metabolism is due do modification of the intracellular signal transduction system.     

5-HT3 receptor-mediated photic-like responses of the circadian clock in the rat

Serotonin (5-HT) and 5-HT agonists have various resetting effects on the master clock, located in the suprachiasmatic nucleus (SCN), depending on the species. In rats, they induce photic-like effects on both locomotor activity rhythms and gene expression in the SCN. The 5-HT receptor(s) mediating these effects at circadian time 22 are localized in the SCN, most likely at a presynaptic level, on the retinohypothalamic terminals (RHT) known to convey photic information by releasing glutamate. Indeed, RHT degeneration blocks photic-like effects of a non-specific 5-HT agonist, quipazine. However, the 5-HT receptor subtype(s) involved is still unknown, although 5-HT(3) receptor activation is known to induce glutamate release. We thus analyzed the effects of selective 5-HT(3) agonist and antagonist, as well as a specific NMDA receptor antagonist, on different parameters of the clock. This study shows that the 5-HT(3) receptor mediates the resetting effects of quipazine on locomotor activity rhythms. The 5-HT(3) receptor is only partially implicated in quipazine-induced expression of c-FOS, while NMDA receptor inhibition blocks quipazine photic-like effects on both parameters. Taken together, photic-like responses produced by 5-HT stimulation in rats are likely mediated by (presynaptic?) 5-HT(3) receptor activation followed by NMDA receptor activation.     

Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABAA receptors but with the opposite functionality

1. Imidazoquinoxaline PNU-97775 and imidazoquinoline PNU-101017 are benzodiazepine site ligands with a second low affinity binding site on GABA_A receptors, the occupancy of which at high drug concentrations reverses their positive allosteric activity via the benzodiazepine site, and may potentially minimize abuse liability and physical dependence.
2. In this study we discovered, with two imidazoquinoxaline analogues, that the functionality of the second site was altered by the nitrogen substituent on the piperazine ring moiety: PNU-100076 with a hydrogen substituent on the position produced a negative allosteric effect via the second low affinity site, like the parent compounds, while PNU-100079 with a trifluoroethyl substituent produced a positive allosteric response.
3. These functional characteristics were monitored with Cl⁻ currents measurements in cloned rat αxβ2γ2 subtypes of GABA_A receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABA_A receptors with binding of [³⁵S]-TBPS, which is a high affinity ligand specific for GABA_A receptors with exquisite sensitivity to allosteric modulations.
4. This structure-functional relationship could be exploited to further our understanding of the second allosteric site of imidazoquinoxaline analogues, and to develop more effective benzodiazepine site ligands without typical side effects associated with those currently available on the market.

     

Interaction of morphine but not fentanyl with cerebral α2‐adrenoceptors in α2‐adrenoceptor knockout mice

Objectives α₂‐Adrenergic and μ‐opioid receptors belong to the rhodopsin family of G‐protein coupled receptors and mediate antinociceptive effects via similar signal transduction pathways. Previous studies have revealed direct functional interactions between both receptor systems including synergistic and additive effects. To evaluate underlying mechanisms, we have studied whether morphine and fentanyl interacted with α₂‐adrenoceptor‐subtypes in mice lacking one individual α₂‐adrenoceptor‐subtype (α₂‐adrenoceptor knockout). Methods Opioid interaction with α₂‐adrenoceptors was investigated by quantitative receptor autoradiography in brain slices of α_2A‐, α_2B‐ or α_2C‐adrenoceptor deficient mice. Displacement of the radiolabelled α₂‐adrenoceptor agonist [¹²⁵I]paraiodoclonidine from α₂‐adrenoceptors in different brain regions by increasing concentrations of morphine, fentanyl and naloxone was analysed. The binding affinity of both opioids to α₂‐adrenoceptor subtypes in different brain regions was quantified. Key findings Morphine but not fentanyl or naloxone provoked dose‐dependent displacement of [¹²⁵I]paraiodoclonidine from all α₂‐adrenoceptor subtypes in the brain regions analysed. Binding affinity was highest in cortex, medulla oblongata and pons of α_2A‐adrenoceptor knockout mice. Conclusions Our results indicated that morphine interacted with α₂‐adrenoceptors showing higher affinity for the α_2B and α_2C than for the α_2A subtype. In contrast, fentanyl and naloxone did not show any relevant affinity to α₂‐adrenoceptors. This effect may have an impact on the pharmacological actions of morphine.