Comparison of the effects of sibutramine and other monoamine reuptake inhibitors on food intake in the rat

1. The effects of the potent 5-hydroxytryptamine (5-HT) and noradrenaline reuptake inhibitor (serotonin-noradrenaline reuptake inhibitor, SNRI), sibutramine, on the cumulative food intake of freely-feeding male Sprague-Dawley rats during an 8 h dark period were investigated and compared to those of the selective 5-HT reuptake inhibitor (selective serotonin reuptake inhibitor, SSRI), fluoxetine; the selective noradrenaline reuptake inhibitor, nisoxetine; the 5-HT and noradrenaline reuptake inhibitors, venlafaxine and duloxetine; and the 5-HT releaser and 5-HT reuptake inhibitor, (+)-fenfluramine.
2. Sibutramine (3 and 10 mg kg⁻¹, p.o.) and (+)-fenfluramine (1 and 3 mg kg⁻¹, p.o.) produced a significant, dose-dependent decrease in food intake over the 8 h dark period. These responses became apparent within the first 2 h following drug administration.
3. Fluoxetine (3, 10 and 30 mg kg⁻¹, p.o.), and nisoxetine (3, 10 and 30 mg kg⁻¹, p.o.) had no significant effect on food intake during the 8 h dark period. However, a combination of fluoxetine and nisoxetine (30 mg kg⁻¹, p.o., of each) significantly decreased food intake 2 and 8 h after drug administration.
4. Venlafaxine (100 and 300 mg kg⁻¹, p.o.) and duloxetine (30 mg kg⁻¹, p.o.) also significantly decreased food intake in the 2 and 8 h following drug administration.
5. The results of this study demonstrate that inhibition of 5-HT and noradrenaline reuptake by sibutramine, venlafaxine, duloxetine, or by a combination of fluoxetine and nisoxetine, markedly reduces food intake in freely-feeding rats and suggest that this may be a novel approach for the treatment of obesity.

     

Role of α2-adrenoceptors in the regulation of intestinal water transport

1. The influence of the sympathetic nervous system on intestinal fluid transport by the jejunum and ileum of the anaesthetized rat was investigated under basal conditions and during active secretion induced by intra-arterial infusion of vasoactive intestinal peptide (VIP).
2. Intra-arterial infusion of noradrenaline (3, 10, 30 nmol min⁻¹, i.a.) and i.v. injection of the selective α₂-adrenoceptor agonist UK 14,304 (1 μmol kg⁻¹, i.v.) increased the rate of basal fluid absorption. The effect of UK 14,304 was blocked by yohimbine (10 μmol kg⁻¹, i.v). However, the selective α₁-adrenoceptor agonist phenylephrine (5 μmol kg⁻¹, i.v.) did not alter either the jejunal or ileal absorption rate.
3. The α₂-adrenoceptor antagonists yohimbine (0.3, 1.0, 3 and 10 μmol kg⁻¹, i.v.) and rauwolscine (10 μmol kg⁻¹, i.v.) decreased the basal absorption rate, while the α₁-adrenoceptor antagonist prazosin (3 μmol kg⁻¹, i.v.) was without effect. Intracerebroventricular injection of yohimbine (3 μmol kg⁻¹) caused a significant antiabsorptive effect in the jejunum but not ileum.
4. Peripheral chemical sympathectomy induced by pretreating animals with 6-hydroxydopamine (150 mg kg⁻¹, i.p., total dose) induced a trend towards impaired absorption in the jejunum and ileum.
5. The findings provide evidence that the sympathetic nervous system exerts tonic control on intestinal fluid transport and that the effect is mainly through peripheral α₂-adrenoceptors.
6. The subtype determination of α₂-adrenoceptors in modulating intestinal fluid transport was assessed by determining the effects of α₂-adrenoceptor agents on intestinal fluid secretion induced by i.a. infusion of VIP (0.8 μg min⁻¹).
7. Intravenous administration of UK 14,304 caused a dose-dependent reversal of the secretory phase of the VIP-induced response, but failed to restore fluid transport to the control level of net absorption. EC₅₀ values were 0.17 μmol kg⁻¹ in the jejunum and 0.22 μmol kg⁻¹ in the ileum.
8. The effect of UK 14,304 was blocked by the selective α_2A/D antagonist BRL 44408 and the non-selective α₂ antagonist yohimbine (each 10 μmol kg⁻¹). The selective α_2B/C antagonist ARC 239 (10 μmol kg⁻¹) did not affect the antisecretory action of UK 14,304. It is suggested that the α₂-adrenoceptors in the rat intestinal epithelium are the α_2D or α_2A-like subtype.

     

The role of α2-adrenoceptor antagonism in the anti-cataleptic properties of the atypical neuroleptic agent,clozapine, in the rat

1. The mechanism underlying the anticataleptic properties of the atypical neuroleptic agent, clozapine, has been investigated in the rat.
2. The close structural analogues of clozapine, loxapine (0.1 mg kg⁻¹ s.c.) and iso-clozapine (1 and 3 mg kg⁻¹ s.c.) induced catalepsy in rats. In contrast, clozapine and the regio-isomer of loxapine, iso-loxapine (up to 10 mg kg⁻¹ s.c.) did not produce catalepsy, but at a dose of 1 mg kg⁻¹ significantly inhibited catalepsy induced by loxapine (0.3 mg kg⁻¹ s.c.).
3. Radioligand binding assays showed that cataleptogenic potential was most clearly predicted by the D₂/5-HT_1A, D₂/5-HT_1B/1D and D₂/α₂-receptor affinity (K_D) ratios: i.e. 30–100-fold higher ratios were calculated for loxapine and iso-clozapine, whereas the ratios were less than 1 for clozapine and iso-loxapine. The ratios of affinities for D₂ to 5-HT_2A, 5-HT_2C or D₁ did not reflect the grouping of cataleptic and non-cataleptic compounds.
4. Co-treatment with the α₂-adrenoceptor antagonists, yohimbine (1–10 mg kg⁻¹ s.c.), RX 821002 (1–10 mg kg⁻¹ s.c.) and MK-912 (0.3 and 1 mg kg⁻¹ s.c.) dose-dependently inhibited the cataleptic response to loxapine (0.3 mg kg⁻¹). Yohimbine (1–10 mg kg⁻¹ s.c.) also dose-dependently inhibited the cateleptic response to haloperidol (0.3 mg kg⁻¹ s.c.). The α₂-adrenoceptor antagonists had no effect per se.
5. Neither yohimbine (10 mg kg⁻¹) nor RX821002 (3 mg kg⁻¹) altered the cataleptic response to the D₁ receptor antagonist, SCH 23390 (1 mg kg⁻¹ s.c.), while, like clozapine, both compounds abolished the response to the 5-HT_2A receptor antagonist, MDL 100,151 (3 mg kg⁻¹ s.c.).
6. The present data strongly implicate α₂-adrenoceptor blockade in the anticataleptic properties of clozapine and suggest that its lack of extrapyramidal side effects in the clinic may also be a consequence of this property.

     

Antidepressant-like effects of endogenous histamine and of two histamine H1 receptor agonists in the mouse forced swim test

1. Effects of substances which are able to alter brain histamine levels and two histamine H₁ receptor agonists were investigated in mice by means of an animal model of depression, the forced swim test.
2. Imipramine (10 and 30 mg kg⁻¹, i.p.) and amitriptyline (5 and 15 mg kg⁻¹, i.p.) were used as positive controls. Their effects were not affected by pretreatment with the histamine H₃ receptor agonist, (R)-α-methylhistamine, at a dose (10 mg kg⁻¹, i.p.) which did not modify the cumulative time of immobility.
3. The histamine H₃ receptor antagonist, thioperamide (2–20 mg kg⁻¹, s.c.), showed an antidepressant-like effect, with a maximum at the dose of 5 mg kg⁻¹, which was completely prevented by (R)-α-methylhistamine.
4. The histamine-N-methyltransferase inhibitor, metoprine (2–20 mg kg⁻¹, s.c.), was effective with an ED₅₀ of 4.02 (2.71–5.96) mg kg⁻¹; its effect was prevented by (R)-α-methylhistamine.
5. The histamine precursor, L-histidine (100–1000 mg kg⁻¹, i.p.), dose-dependently decreased the time of immobility [ED₃₀ 587 (499–712) mg kg⁻¹]. The effect of 500 mg kg⁻¹ L-histidine was completely prevented by the selective histidine decarboxylase inhibitor, (S)-α-fluoromethylhistidine (50 mg kg⁻¹, i.p.), administered 15 h before.
6. The highly selective histamine H₁ receptor agonist, 2-(3-trifluoromethylphenyl)histamine (0.3–6.5 μg per mouse, i.c.v.), and the better known H₁ agonist, 2-thiazolylethylamine (0.1–1 μg per mouse, i.c.v.), were both dose-dependently effective in decreasing the time of immobility [ED₅₀ 3.6 (1.53–8.48) and 1.34 (0.084–21.5) μg per mouse, respectively].
7. None of the substances tested affected mouse performance in the rota rod test at the doses used in the forced swim test.
8. It was concluded that endogenous histamine reduces the time of immobility in this test, suggesting an antidepressant-like effect, via activation of H₁ receptors.

     

Gabapentin (neurontin) and S-(+)-3-isobutylgaba represent a novel class of selective antihyperalgesic agents

1. Gabapentin (neurontin) is a novel antiepileptic agent that binds to the α₂δ subunit of voltage-dependent calcium channels. The only other compound known to possess affinity for this recognition site is the (S)-(+)-enantiomer of 3-isobutylgaba. However, the corresponding (R)-(−)-enantiomer is 10 fold weaker. The present study evaluates the activity of gabapentin and the two enantiomers of 3-isobutylgaba in formalin and carrageenan-induced inflammatory pain models.
2. In the rat formalin test, S-(+)-3-isobutylgaba (1–100 mg kg⁻¹) and gabapentin (10–300 mg kg⁻¹) dose-dependently inhibited the late phase of the nociceptive response with respective minimum effective doses (MED) of 10 and 30 mg kg⁻¹, s.c. This antihyperalgesic action of gabapentin was insensitive to naloxone (0.1–10.0 mg kg⁻¹, s.c.). In contrast, the R-(−)-enantiomer of 3-isobutylgaba (1–100 mg kg⁻¹) produced a modest inhibition of the late phase at the highest dose of 100 mg kg⁻¹. However, none of the compounds showed any effect during the early phase of the response.
3. The s.c. administration of either S-(+)-3-isobutylgaba (1–30 mg kg⁻¹) or gabapentin (10–100 mg kg⁻¹), after the development of peak carrageenan-induced thermal hyperalgesia, dose-dependently antagonized the maintenance of this response with MED of 3 and 30 mg kg⁻¹, respectively. Similar administration of the two compounds also blocked maintenance of carrageenan-induced mechanical hyperalgesia with MED of 3 and 10 mg kg⁻¹, respectively. In contrast, R-(−)-3-isobutylgaba failed to show any effect in the two hyperalgesia models.
4. The intrathecal administration of gabapentin dose-dependently (1–100 μg/animal) blocked carrageenan-induced mechanical hyperalgesia. In contrast, administration of similar doses of gabapentin into the inflamed paw was ineffective at blocking this response.
5. Unlike morphine, the repeated administration of gabapentin (100 mg kg⁻¹ at start and culminating to 400 mg kg⁻¹) over 6 days did not lead to the induction of tolerance to its antihyperalgesic action in the formalin test. Furthermore, the morphine tolerance did not cross generalize to gabapentin. The s.c. administration of gabapentin (10–300 mg kg⁻¹), R-(−) (3–100 mg kg⁻¹) or S-(+)-3-isobutylgaba (3–100 mg kg⁻¹) failed to inhibit gastrointestinal motility, as measured by the charcoal meal test in the rat. Moreover, the three compounds (1–100 mg kg⁻¹, s.c.) did not generalize to the morphine discriminative stimulus. Gabapentin (30–300 mg kg⁻¹) and S-(+)-isobutylgaba (1–100 mg kg⁻¹) showed sedative/ataxic properties only at the highest dose tested in the rota-rod apparatus.
6. Gabapentin (30–300 mg kg⁻¹, s.c.) failed to show an antinociceptive action in transient pain models. It is concluded that gabapentin represents a novel class of antihyperalgesic agents.

     

Citalopram-induced hypophagia is enhanced by blockade of 5-HT1A receptors: role of 5-HT2C receptors

1. The selective 5-hydroxytryptamine reuptake inhibitor citalopram (10 and 20 mg kg⁻¹, i.p.) significantly reduced food intake in male rats (CD-COBS) habituated to eat their daily food during a 4-h period.
2. The 5-HT_1A receptor antagonist WAY100635 (0.3 mg kg⁻¹) administered systemically did not modify feeding but significantly potentiated the reduction in food intake caused by 10 mg kg⁻¹ i.p. citalopram. The dose of 5 mg kg⁻¹ i.p. citalopram was not active in animals pretreated with vehicle but significantly reduced feeding in animals pretreated with WAY100635.
3. WAY100635 (0.1 μg 0.5 μl⁻¹) injected into the dorsal raphe significantly potentiated the hypophagic effect of 10 mg kg⁻¹ citalopram.
4. WAY100635 (1.0 μg 0.5 μl⁻¹) injected into the median raphe did not modify feeding or the hypophagic effect of 10 mg kg⁻¹ citalopram.
5. The 5-HT_2B/2C receptor antagonist SB206553 (10 mg kg⁻¹, p.o.) slightly reduced feeding by itself but partially antagonized the effect of WAY100635 administered systemically (0.3 mg kg⁻¹, s.c.) or into the dorsal raphe (0.1 μg 0.5 μl⁻¹) in combination with 10 mg kg⁻¹ i.p. citalopram. The hypophagic effect of 10 mg kg⁻¹ i.p. citalopram alone was not significantly modified by SB206553.
6. Brain concentrations of citalopram and its metabolite desmethylcitalopram in rats pretreated with SB206553, WAY100635 and their combination were comparable to those of vehicle-pretreated rats, 90 min after citalopram injection.
7. The hypophagic effect of citalopram was potentiated by blocking 5-HT_1A receptors. Only the effect of the WAY100635/citalopram combination seemed to be partially mediated by central 5-HT_2C receptors.

     

Electrophysiological and neurochemical evidence that pindolol has agonist properties at the 5-HT1A autoreceptor in vivo

1. It has been hypothesized that 5-HT_1A autoreceptor antagonists may enhance the therapeutic efficacy of SSRIs and other antidepressants. Although early clinical trials with the β-adrenoceptor/5-HT₁ ligand, pindolol, were promising, the results of recent more extensive trials have been contradictory. Here we investigated the actions of pindolol at the 5-HT_1A autoreceptor by measuring its effect on 5-HT neuronal activity and release in the anaesthetized rat.
2. Pindolol inhibited the electrical activity of 5-HT neurones in the dorsal raphe nucleus (DRN). This effect was observed in the majority of neurones tested (10/16), was dose-related (0.2–1.0 mg kg⁻¹, i.v.), and was reversed by the 5-HT_1A receptor antagonist, WAY 100635 (0.1 mg kg⁻¹, i.v.), in 6/7 cases tested.
3. Pindolol also inhibited 5-HT neuronal activity when applied microiontophoretically into the DRN in 9/10 neurones tested. This effect of pindolol was current-dependent and blocked by co-application of WAY 100635 (3/3 neurones tested).
4. In microdialysis experiments, pindolol caused a dose-related (0.8 and 4 mg kg⁻¹, i.v.) fall in 5-HT levels in dialysates from the frontal cortex (under conditions where the perfusion medium contained 1 μM citalopram). In rats pretreated with WAY 100635 (0.1 mg kg⁻¹, i.v.), pindolol (4 mg kg⁻¹, i.v.) did not decrease, but rather increased 5-HT levels.
5. We conclude that, under the experimental conditions used in this study, pindolol displays agonist effects at the 5-HT_1A autoreceptor. These data are relevant to previous and ongoing clinical trials of pindolol in depression which are based on the rationale that the drug is an effective 5-HT_1A autoreceptor antagonist.

     

Infarct size-reducing effect of heat stress and α1 adrenoceptors in rats

1. Noradrenaline (NA), which is abundantly released during heat stress (HS), is known to induce both delayed cardioprotection and heat stress protein (HSP) 72 expression by the mediation of α₁ adrenoceptors. Therefore, we have investigated the implication of α₁ adrenoceptors in HS-induced resistance to myocardial infarction, in the isolated rat heart model.
2. Rats were pretreated with prazosin (1 mg kg⁻¹, i.p., Praz) or 5-methylurapidil (3 mg kg⁻¹, i.v, 5MU) or chloroethylclonidine (3 mg kg⁻¹, i.v., CEC) or vehicle (V) in order to selectively antagonize α₁, α_1A and α_1B adrenoceptors. They were then either heat stressed (42°C for 15 min) or sham anaesthetized. Twenty-four hours later, their hearts were isolated, retrogradely perfused, and subjected to a 30 min occlusion of the left coronary artery followed by 120 min of reperfusion.
3. Infarct-to-risk ratio was significantly reduced in HS+V (15.4±1.8%) compared to Sham+V (35.7±1.3%) hearts. This effect was abolished in Praz-treated (29.1±1.6% in HS+Praz vs 34.1±4.0% in Sham+Praz), 5MU-treated (34.5±2.2% in HS+5MU vs 31.2±2.0% in Sham+5MU) and CEC-treated (33.4±3.0% in HS+CEC vs 32.4±1.3% in Sham+CEC) groups. Western blot analysis of myocardial HSP72 showed an HS-induced increase of this protein, which was not modified by Praz, 5MU and CEC pretreatments.
4. We conclude that both α_1A and α_1B adrenoceptor subtypes appear to play a role in the heat stress-induced cardioprotection, independently of the HSP72 level. Further investigations are required to elucidate the precise role of HSPs in this adaptative response.

     

Risperidone inhibits 5-hydroxytryptaminergic neuronal activity in the dorsal raphe nucleus by local release of 5-hydroxytryptamine

1. The effects of risperidone on brain 5-hydroxytryptamine (5-HT) neuronal functions were investigated and compared with other antipsychotic drugs and selective receptor antagonists by use of single cell recording and microdialysis in the dorsal raphe nucleus (DRN).
2. Administration of risperidone (25–400 μg kg⁻¹, i.v.) dose-dependently decreased 5-HT cell firing in the DRN, similar to the antipsychotic drug clozapine (0.25–4.0 mg kg⁻¹, i.v.), the putative antipsychotic drug amperozide (0.5–8.0 mg kg⁻¹, i.v.) and the selective α₁-adrenoceptor antagonist prazosin (50–400 μg kg⁻¹, i.v.).
3. The selective α₂-adrenoceptor antagonist idazoxan (10–80 μg kg⁻¹, i.v.), in contrast, increased the firing rate of 5-HT neurones in the DRN, whereas the D₂ and 5-HT_2A receptor antagonists raclopride (25–200 μg kg⁻¹, i.v.) and MDL 100,907 (50–400 μg kg⁻¹, i.v.), respectively, were without effect. Thus, the α₁-adrenoceptor antagonistic action of the antipsychotic drugs might, at least partly, cause the decrease in DRN 5-HT cell firing.
4. Pretreatment with the selective 5-HT_1A receptor antagonist WAY 100,635 (5.0 μg kg⁻¹, i.v.), a drug previously shown to antagonize effectively the inhibition of 5-HT cells induced by risperidone, failed to prevent the prazosin-induced decrease in 5-HT cell firing. This finding argues against the notion that α₁-adrenoceptor antagonism is the sole mechanism underlying the inhibitory effect of risperidone on the DRN cells.
5. The inhibitory effect of risperidone on 5-HT cell firing in the DRN was significantly attenuated in rats pretreated with the 5-HT depletor PCPA (p-chlorophenylalanine; 300 mg kg⁻¹, i.p., day⁻¹ for 3 consecutive days) in comparison with drug naive animals.
6. Administration of risperidone (2.0 mg kg⁻¹, s.c.) significantly enhanced 5-HT output in the DRN.
7. Consequently, the reduction in 5-HT cell firing by risperidone appears to be related to increased availability of 5-HT in the somatodendritic region of the neurones leading to an enhanced 5-HT_1A autoreceptor activation and, in turn, to inhibition of firing, and is probably only to a minor extent caused by its α₁-adrenoceptor antagonistic action.

     

α1A-Adrenoceptor mediated contraction of rat prostatic vas deferens and the involvement of ryanodine stores and Ca2+ influx stimulated by diacylglycerol and PKC

1. The present study has investigated the α₁-adrenoceptor subtype mediating contraction of the rat isolated prostatic vas deferens and the possible effector mechanisms involved in this response by use of functional experiments.
2. Contractions to noradrenaline in the rat isolated prostatic vas deferens were antagonized by prazosin (9.4, 1.04±0.19, pA₂ and Schild plot slope), 5-methyl urapidil (8.9, 1.10±0.13), BMY 7378 (6.4, 1.53±0.07) and RS 17053 (8.3, 1.13±0.18). These affinities are consistent with the response being mediated by the α_1A-adrenoceptor subtype.
3. The contraction to noradrenaline at 37°C consisted of an initial phasic response, composed of many rhythmic contractile spikes and a more slowly developing tonic contraction. When the temperature was lowered to 25°C the phasic contraction became a smooth single response which was increased in magnitude.
4. In Ca²⁺-free Krebs solution the tonic contraction to noradrenaline (10⁻⁴ M) was abolished, suggesting that this response was dependent on influx of extracellular Ca²⁺. After 2 min in Ca²⁺-free Krebs solution at 37°C and 25°C the phasic response to noradrenaline (10⁻⁴ M) was 38±2% and 91±4%, respectively, compared with the phasic contraction to noradrenaline (10⁻⁴ M in normal Krebs solution) and after 30 min it was abolished at 37°C and was 7±1% at 25°C. Ryanodine abolished the noradrenaline response in Ca²⁺-free Krebs solution for 2 min at 25°C, while cyclopiazonic acid reduced it to 36±2%.
5. In normal Krebs solution at 25°C the protein kinase C inhibitor calphostin C reduced the tonic contraction to noradrenaline (10⁻⁵ M) from 36±8% to 14±3% compared with the phasic contraction to noradrenaline (10⁻⁴ M). The DAG kinase inhibitor R 59022 increased the contraction following the initial phasic response to a maximum of 107±17% after 35 s, before dropping down to a well maintained contraction which was still greater in magnitude compared with the control. Nifedipine (3×10⁻⁷ M) reduced the tonic contraction from 49±6% to 7±1% but did not reduce the phasic response. Ryanodine (10⁻⁴ M) reduced the phasic contraction from 50±2% to 7±1% and the tonic response from 47±5% to 27±5%.
6. The phorbol ester phorbol-12,13-dibutyrate at 25°C produced a transient contraction of the rat prostatic vas deferens, maximum response (10⁻⁵ M) 48±4%, compared with the maximum tonic response to noradrenaline. The contraction to PDBu (10⁻⁵ M) was reduced to 23±2% by calphostin C (10⁻⁶ M) and to 15±1% by nifedipine (3×10⁻⁷ M) and was abolished after 2 min in Ca²⁺-free Krebs solution.
7. In conclusion, the α_1A-adrenoceptor mediated contraction to noradrenaline of the rat prostatic vas deferens appears to consist of an initial phasic component due to the release of intracellular Ca²⁺ from ryanodine-sensitive stores. These stores are depleted in the absence of extracellular Ca²⁺ and this depletion is slower at 25°C than at 37°C. The phasic contraction is followed by a tonic contraction involving activation of protein kinase C by diacylglycerol and influx of Ca²⁺ through nifedipine-sensitive channels.

     

Effect of acute and chronic tramadol on [3H]-5-HT uptake in rat cortical synaptosomes

1. Tramadol hydrochloride is a centrally acting opioid analgesic, the efficacy and potency of which is only five to ten times lower than that of morphine. Opioid, as well as non-opioid mechanisms, may participate in the analgesic activity of tramadol.
2. [³H]-5-hydroxytryptamine (5-HT) uptake in rat isolated cortical synaptosomes was studied in the presence of tramadol, desipramine, fluoxetine, methadone and morphine. Methadone and tramadol inhibited synaptosomal [³H]-5-HT uptake with apparent K_is of 0.27±0.04 and 0.76±0.04 μM, respectively. Morphine essentially failed to inhibit [³H]-5-HT uptake (K_i 0.50±0.30 M).
3. Methadone, morphine and tramadol were active in the hot plate test with ED₅₀s of 3.5, 4.3 and 31 mg kg⁻¹, respectively. At the highest tested dose (80 mg kg⁻¹) tramadol produced only 77±5.3% of the maximal possible effect.
4. When [³H]-5-HT uptake was examined in synaptosomes prepared from rats 30 min after a single dose of morphine, methadone or tramadol, only tramadol (31 mg kg⁻¹, s.c., equal to the ED₅₀ in the hot plate test) and methadone (35 mg kg⁻¹, s.c., equal to the ED₉₀ in the hot plate test) decreased uptake.
5. Animals were chronically treated for 15 days with increasing doses of tramadol or methadone (5 to 40 mg kg⁻¹ and 15 to 120 mg kg⁻¹, s.c., respectively). Twenty-four hours after the last drug injection, a challenge dose of methadone (35 mg kg⁻¹, s.c.) or tramadol (31 mg kg⁻¹, s.c.) was administered. [³H]-5-HT uptake was not affected in synaptosomes prepared from rats chronically-treated with methadone, whereas chronic tramadol was still able to reduce this parameter by 42%.
6. Rats chronically-treated with methadone showed a significant increase in [³H]-5-HT uptake (190%) 72 h after drug withdrawal. In contrast, [³H]-5-HT uptake in rats chronically-treated with tramadol (110%) did not differ significantly from control animals.
7. These results further support the hypothesis that [³H]-5-HT uptake inhibition may contribute to the antinociceptive effects of tramadol. The lack of tolerance development of [³H]-5-HT uptake, together with the absence of behavioural alterations after chronic tramadol treatment, suggest that tramadol has an advantage over classical opioids in the treatment of pain disorders.

     

Pharmacological characterization of MT-1207, a novel multitarget antihypertensive agent

Hypertension is a serious public health problem worldwide. MT-1207, chemically named 3-(4-(4-(1H-benzotriazole-1-yl)butyl)piperazine-1-yl) benzisothiazole hydrochloride, is a new chemical entity that has entered into clinical trial as antihypertensive agent in China. In this paper we report the pharmacological profile of MT-1207 regarding its acute, subacute, and long-term effects on hypertensive animal models, and its actions on isolated organs in vitro as well as its molecular targets. Blood pressure (BP) was measured in conscious animals; amlodipine was taken as a positive control drug. We showed that both single dose of MT-1207 (1.25−20 mg/kg, ig) in spontaneously hypertensive rats (SHR) and MT-1207 (0.25−6 mg/kg, ig) in two-kidney one-clip (2K1C) dogs dose-dependently decreased BP. MT-1207 quickly decreased BP within 5 min after administration; the hypotensive effect lasted for 8 and 12 h, respectively, in SHR and 2K1C dogs without reflex increase in heart rate. Multiple doses of MT-1207 (5 mg · kg⁻¹ · d⁻¹ in SHR; 2 mg · kg⁻¹ · d⁻¹ in 2K1C dogs, for 7 days) significantly decreased BP, slightly reduced heart rate, and both of them recovered after withdrawal. Long-term administration of MT-1207 (10 mg · kg⁻¹ · d⁻¹ for 4 months or more time) produced a stable BP reduction, improved baroreflex sensitivity, reduced renal and cardiovascular damage in SHR, and delayed stroke occurrence and death in stroke-prone SHR. In isolated rat aortic rings precontracted by adrenaline, KCl, noradrenaline or 5-hydroxytryptamine (5-HT), MT-1207 (10⁻⁹–10⁻⁴ M) caused concentration-dependent relaxation. In a panel of enzyme activity or radioligand binding assays of 87 molecular targets, MT-1207 potently inhibited adrenergic α_1A, α_1B, α_1D, and 5-HT_2A receptors with K_i < 1 nM. The antagonism of MT-1207 against these receptors was confirmed in isolated rabbit arteries. We conclude that MT-1207 is a novel and promising single-molecule multitarget agent for hypertension treatment to reduce hypertensive organ damage and stroke mortality.     

Evidence for the involvement of 5-HT4 receptors in the 5-hydroxytryptamine-induced pattern of migrating myoelectric complex in sheep

1. The effects induced by 5-hydroxytryptamine (5-HT) on gastrointestinal myoelectric activity in conscious sheep were recorded through electrodes chronically implanted and analysed by computer. The 5-HT receptors and the cholinergic neuronal pathways involved in these actions were investigated.
2. The intravenous (i.v.) administration of 5-HT (2, 4 and 8 μg kg⁻¹ min⁻¹, 5 min) induced an antral inhibition concomitant with a duodenal activity front that migrated to the jejunum, followed by a period of intestinal inactivity. This myoelectric pattern closely resembled that observed in the phases III and I of the migrating myoelectric complex (MMC) in sheep. The 0.5 μg kg⁻¹ min⁻¹ dose evoked the same pattern in only two out of the six animals used. Likewise, the 1 μg kg⁻¹ min⁻¹ dose similarly affected four of the six animals. In addition, a transient stimulation was observed in the antrum and jejunum when the two highest doses were used.
3. The 5-HT₁ antagonist, methiothepin (0.1 mg kg⁻¹), the 5-HT₂ antagonists, ritanserin (0.1 mg  kg⁻¹) and ketanserin (0.3 mg  kg⁻¹), the 5-HT₃ antagonists, granisetron (0.2 mg kg⁻¹) and ondansetron (0.5 mg kg⁻¹), as well as the 5-HT₄ antagonist, {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808 (0.2 mg kg⁻¹), did not modify the spontaneous gastrointestinal myoelectric activity. However, the cholinoceptor antagonists, atropine (0.2 mg kg⁻¹) and hexamethonium (2 mg kg⁻¹), inhibited gastrointestinal activity.
4. When these antagonists were injected i.v. 10 min before 5-HT (2 or 4 μg kg⁻¹ min⁻¹, 5 min), only {"type":"entrez-nucleotide","attrs":{"text":"GR113808","term_id":"238362519","term_text":"GR113808"}}GR113808, atropine and hexamethonium were able to modify the 5-HT-induced actions, all of them being completely blocked by the three antagonists.
5. Our data show that 5-HT initiates a MMC-like pattern in the gastrointestinal area in sheep through 5-HT₄ receptors. Furthermore, these actions are mediated by cholinergic neural pathways involving muscarinic and nicotinic receptors. However, our results do not indicate a role for either 5-HT₁, 5-HT₂ or 5-HT₃ receptors in the 5-HT-induced effects.

     

Acute dilation to α2-adrenoceptor antagonists uncovers dual constriction and dilation mediated by arterial α2-adrenoceptors

Background and purpose:

In mouse tail arteries, selective α₂-adrenoceptor antagonism with rauwolscine caused powerful dilation during constriction to the α₁-adrenoceptor agonist phenylephrine. This study therefore assessed phenylephrine''s selectivity at vascular α-adrenoceptors and the mechanism(s) underlying dilation to rauwolscine.

Experimental approach:

Mouse isolated tail arteries were assessed using a pressure myograph.

Key results:

The α₂-adrenoceptor agonist UK14,304 caused low-maximum constriction that was inhibited by rauwolscine (3 × 10⁻⁸ M) but not by the selective α₁-adrenoceptor antagonist prazosin (10⁻⁷ M). Concentration–effect curves to phenylephrine, cirazoline or noradrenaline were unaffected by rauwolscine but were inhibited by prazosin, which was more effective at high compared with low levels of constriction. In the presence of prazosin, rauwolscine inhibited the curves and was more effective at low compared with high levels of constriction. Although rauwolscine alone did not affect concentration–effect curves to phenylephrine, noradrenaline or cirazoline, it caused marked transient dilation when administered during constriction to these agonists. Dilation was mimicked by another α₂-adrenoceptor antagonist (RX821002, 3 × 10⁻⁸ M), was dependent on agonist selectivity, and did not occur during adrenoceptor-independent constriction (U46619). During constriction to UK14,304 plus U46619, rauwolscine or rapid removal of UK14,304 caused transient dilation that virtually abolished the combined constriction. Endothelial denudation reduced these dilator responses.

Conclusions and implications:

Inhibition of α₂-adrenoceptors caused transient dilation that was substantially greater than the contribution of α₂-adrenoceptors to the constriction. This reflects a slowly reversing α₂-adrenoceptor-mediated endothelium-dependent dilation and provides a rapid, sensitive test of α₂-adrenoceptor activity. This approach also clearly emphasizes the poor selectivity of phenylephrine at vascular α-adrenoceptors.     

Mediation of 5-HT-induced external carotid vasodilatation in GR 127935-pretreated vagosympathectomized dogs by the putative 5-HT7 receptor

1. The vasodilator effects of 5-hydroxytryptamine (5-HT) in the external carotid bed of anaesthetized dogs with intact sympathetic tone are mediated by prejunctional sympatho-inhibitory 5-HT_1B/1D receptors and postjunctional 5-HT receptors. The prejunctional vasodilator mechanism is abolished after vagosympathectomy which results in the reversal of the vasodilator effect to vasoconstriction. The blockade of this vasoconstrictor effect of 5-HT with the 5-HT_1B/1D receptor antagonist, GR 127935, unmasks a dose-dependent vasodilator effect of 5-HT, but not of sumatriptan. Therefore, the present study set out to analyse the pharmacological profile of this postjunctional vasodilator 5-HT receptor in the external carotid bed of vagosympathectomized dogs pretreated with GR 127935 (20 μg kg⁻¹, i.v.).
2. One-minute intracarotid (i.c.) infusions of 5-HT (0.330 μg min⁻¹), 5-carboxamidotryptamine (5-CT; 0.010.3 μg min⁻¹), 5-methoxytryptamine (1100 μg min⁻¹) and lisuride (31000 μg min⁻¹) resulted in dose-dependent increases in external carotid blood flow (without changes in blood pressure or heart rate) with a rank order of agonist potency of 5-CT>>5-HT⩾5-methoxytryptamine>lisuride, whereas cisapride (1001000 μg min⁻¹, i.c.) was practically inactive. Interestingly, lisuride (mean dose of 85±7 μg kg⁻¹, i.c.), but not cisapride (mean dose of 67±7 μg kg⁻¹, i.c.), specifically abolished the responses induced by 5-HT, 5-CT and 5-methoxytryptamine, suggesting that a common site of action may be involved. In contrast, 1 min i.c. infusions of 8-OH-DPAT (33000 μg min⁻¹) produced dose-dependent decreases, not increases, in external carotid blood flow and failed to antagonize (mean dose of 200±33 μg kg⁻¹, i.c.) the agonist-induced vasodilator responses.
3. The external carotid vasodilator responses to 5-HT, 5-CT and 5-methoxytryptamine were not modified by intravenous (i.v.) pretreatment with either saline, (±)-pindolol (4 mg kg⁻¹) or ritanserin (100 μg kg⁻¹) plus granisetron (300 μg kg⁻¹), but were dose-dependently blocked by i.v. administration of methiothepin (10 and 30 μg kg⁻¹, given after ritanserin plus granisetron), mesulergine (10 and 30 μg kg⁻¹), metergoline (1 and 3 mg kg⁻¹), methysergide (1 and 3 mg kg⁻¹) or clozapine (0.3 and 1 mg kg⁻¹). Nevertheless, the blockade of the above responses, not significant after treatment with the lower of the two doses of metergoline and mesulergine, was nonspecific after administration of the higher of the two doses of methysergide and clozapine.
4. Based upon the above rank order of agonist potencies and the antagonism produced by a series of drugs showing high affinity for the cloned 5-ht₇ receptor, our results indicate that the 5-HT receptor mediating external carotid vasodilatation in GR 127935-pretreated vagosympathectomized dogs is operationally similar to the putative 5-HT₇ receptor mediating relaxation of vascular and non-vascular smooth muscles (e.g. rabbit femoral vein, canine coronary artery, rat systemic vasculature and guinea-pig ileum) as well as tachycardia in the cat.

     

Catecholamine modulatory effects of nepicastat (RS-25560-197), a novel,potent and selective inhibitor of dopamine-β-hydroxylase

1. Inhibitory modulation of sympathetic nerve function may have a favourable impact on the progression of congestive heart failure. Nepicastat is a novel inhibitor of dopamine-β-hydroxylase, the enzyme which catalyses the conversion of dopamine to noradrenaline in sympathetic nerves. The in vitro pharmacology and in vivo catecholamine modulatory effects of nepicastat were investigated in the present study.
2. Nepicastat produced concentration-dependent inhibition of bovine (IC₅₀=8.5±0.8 nM) and human (IC₅₀=9.0±0.8  nM)dopamine-β-hydroxylase. The corresponding R-enantiomer (RS-25560-198) was approximately 2–3 fold less potent than nepicastat. Nepicastat had negligible affinity (>10 μM) for twelve other enzymes and thirteen neurotransmitter receptors.
3. Administration of nepicastat to spontaneously hypertensive rats (SHRs) (three consecutive doses of either 3, 10, 30 or 100 mg kg⁻¹, p.o.; 12 h apart) or beagle dogs (0.05, 0.5, 1.5 or 5 mg kg⁻¹, p.o.; b.i.d., for 5 days) produced dose-dependent decreases in noradrenaline content, increases in dopamine content and increases in dopamine/noradrenaline ratio in the artery (mesenteric or renal), left ventricle and cerebral cortex. At the highest dose studied, the decreases in tissue noradrenaline were 47%, 35% and 42% (in SHRs) and 88%, 91% and 96% (in dogs) in the artery, left ventricle and cerebral cortex, respectively. When tested at 30 mg kg⁻¹, p.o., in SHRs, nepicastat produced significantly greater changes in noradrenaline and dopamine content, as compared to the R-enantiomer (RS-25560-198), in the mesenteric artery and left ventricle.
4. Administration of nepicastat (2 mg kg⁻¹, b.i.d, p.o.) to beagle dogs for 15 days produced significant decreases in plasma concentrations of noradrenaline and increases in plasma concentrations of dopamine and dopamine/noradrenaline ratio. The peak reduction (52%) in plasma concentration of noradrenaline and the peak increase (646%) in plasma concentration of dopamine were observed on day-6 and day-7 of dosing, respectively.
5. The findings of this study suggest that nepicastat is a potent, selective and orally active inhibitor of dopamine-β-hydroxylase which produces gradual modulation of the sympathetic nervous system by inhibiting the biosynthesis of noradrenaline. This drug may, therefore, be of value in the treatment of cardiovascular disorders associated with over-activation of the sympathetic nervous system, such as congestive heart failure.

     

The influence of antibodies to TNF-α and IL-1β on haemodynamic responses to the cytokines,and to lipopolysaccharide,in conscious rats

1. Male, Long Evans rats (350–450 g) were anaesthetized and had pulsed Doppler probes and intravascular catheters implanted to allow monitoring of regional (renal, mesenteric and hindquarters) haemodynamics in the conscious state. Our main objectives were to:- assess the effects of administering human recombinant tumour necrosis factor (TNF)-α and human recombinant interleukin-1 (IL-1)β, alone and together; determine the influence of pretreatment with a mixture of antibodies to TNF-α and IL-1β on responses to co-administration of the cytokines; ascertain if pretreatment with a mixture of the antibodies to TNF-α and IL-1β had any influence on the responses to lipopolysaccharide (LPS).
2. TNF-α (10, 100 and 250 μg kg⁻¹, in separate groups, n=3, 9 and 8, respectively) caused tachycardia (maximum Δ, +101±9 beats min⁻¹) and modest hypotension (maximum Δ, −10±2 mmHg), accompanied by variable changes in renal and mesenteric vascular conductance, but clear increases in hindquarters vascular conductance; only the latter were dose-related (maximum Δ, +6±6, +27±9, and +61±12% at 10, 100 and 250 μg kg⁻¹, respectively).
3. IL-1β (1, 10, and 100 μg kg⁻¹ in separate groups, n=8, 8 and 9, respectively) evoked changes similar to those of TNF-α (maximum Δ heart rate, +69±15 beats min⁻¹; maximum Δ mean blood pressure, −14±2 mmHg; maximum Δ hindquarters vascular conductance, +49±17%), but with no clear dose-dependency.
4. TNF-α (250 μg kg⁻¹) and IL-1β (10 μg kg⁻¹) together caused tachycardia (maximum Δ, +76±15 beats min⁻¹) and hypotension (maximum Δ, −24±2 mmHg) accompanied by increases in renal, mesenteric and hindquarters vascular conductances (+52±6%, +23±8%, and +52±11%, respectively). Thereafter, blood pressure recovered, in association with marked reductions in mesenteric and hindquarters vascular conductances (maximum Δ, −50±3% and −58±3%, respectively). Although bolus injection of LPS (3.5 mg kg⁻¹) caused an initial hypotension (maximum Δ, −27±11 mmHg) similar to that seen with co-administration of the cytokines, it did not cause mesenteric or hindquarters vasodilatation, and there was only a slow onset renal vasodilatation. The recovery in blood pressure following LPS was less than after the cytokines, and in the former condition there was no mesenteric vasoconstriction. By 24 h after co-administration of TNF-α and IL-1β or after bolus injection of LPS, the secondary reduction in blood pressure was similar (−16±2 and −13±3 mmHg, respectively), but in the former group the tachycardia (+117±14 beats min⁻¹) and increase in hindquarters vascular conductance (+99±21%) were greater than after bolus injection of LPS (+54±16 beats min⁻¹ and +43±9%, respectively).
5. Pretreatment with antibodies to TNF-α and IL-1β (300 mg kg⁻¹) blocked the initial hypotensive and mesenteric and hindquarters vasodilator responses to co-administration of the cytokines subsequently. However, tachycardia and renal vasodilatation were still apparent. Premixing antibodies and cytokines before administration prevented most of the effects of the latter, but tachycardia was still present at 24 h.
6. Pretreatment with antibodies to TNF-α and IL-1β before infusion of LPS (150 μg kg⁻¹ h⁻¹ for 24 h) did not affect the initial fall in blood pressure, but suppressed the hindquarters vasodilatation and caused a slight improvement in the recovery of blood pressure. However, pretreatment with the antibodies had no effect on the subsequent cardiovascular sequelae of LPS infusion.
7. The results indicate that although co-administration of TNF-α and IL-1β can evoke cardiovascular responses which, in some respects, mimic those of LPS, and although antibodies to the cytokines can suppress most of the cardiovascular effects of the cytokines, the antibodies have little influence on the haemodynamic responses to LPS, possibly because, during infusion of LPS, the sites of production and local action of endogenous cytokines, are not accessible to exogenous antibodies.

     

Essential role for endothelin ETB receptors in fever induced by LPS (E. coli) in rats

1. The influence of endothelin receptor antagonists on febrile responses to E. coli lipopolysaccharide (LPS), interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α) and endothelin-1 (ET-1) was assessed in conscious rats.
2. Intravenous (i.v.) LPS (5.0 μg kg⁻¹) markedly increased rectal temperature to a peak of 1.30°C over baseline at 2.5 h. Pretreatment with the mixed endothelin ET_A/ET_B receptor antagonist bosentan (10 mg kg⁻¹, i.v.) or the selective endothelin ET_B receptor antagonist BQ-788 (N-cis-2,6-dimethyl-piperidinocarbonyl-L-γ-methylleucyl-D-1-methoxycarboyl-D-norleucine; 3 pmol, into a lateral cerebral ventricle–i.c.v.) reduced the peak response to LPS to 0.90 and 0.75°C, respectively. The selective endothelin ET_A receptor antagonist BQ-123 (cyclo[D-Trp-D-Asp-Pro-D-Val-Leu]; 3 pmol, i.c.v.) was ineffective.
3. Increases in temperature caused by IL-1β (180 fmol, i.c.v.), TNF-α (14.4 pmol, i.c.v.) or IL-1β (150 pmol kg⁻¹, i.v.) were unaffected by BQ-788 (3 pmol, i.c.v.).
4. Central injection of endothelin-1 (0.1 to 3 fmol, i.c.v.) caused slowly-developing and long-lasting increases in rectal temperature (starting 2 h after administration and peaking at 4–6 h between 0.90 and 1.15°C) which were not clearly dose-dependent. The response to endothelin-1 (1 fmol, i.c.v.) was prevented by BQ-788, but not by BQ-123 (each at 3 pmol, i.c.v.). Intraperitoneal pretreatment with the cyclo-oxygenase inhibitor indomethacin (2 mg kg⁻¹), which partially reduced LPS-induced fever, did not modify the hyperthermic response to endothelin-1 (3 fmol, i.c.v.).
5. Therefore, central endothelin(s) participates importantly in the development of LPS-induced fever, via activation of a prostanoid-independent endothelin ET_B receptor-mediated mechanism possibly not situated downstream from IL-1β or TNF-α in the fever cascade.

     

Respiratory and cardiovascular effects of the μ-opioid receptor agonist [Lys7]dermorphin in awake rats

1. Changes in respiratory variables, arterial blood pressure and heart rate were studied in awake rats after injection of the opioid peptide [Lys⁷]dermorphin and its main metabolites, [1-5]dermorphin and [1-4]dermorphin.
2. Fifteen minutes after injection, doses of [Lys⁷]dermorphin producing antinociception (i.c.v., 36–120 nmol; s.c., 0.12–4.7 μmol kg⁻¹) significantly increased respiratory frequency and minute volume of rats breathing air or hypoxic inspirates. This respiratory stimulation was reversed to depression by the 5-HT receptor antagonist ritanserin (2 mg kg⁻¹, s.c.), was blocked by naloxone (0.1 mg kg⁻¹, s.c.), significantly reduced by the μ₁ opioid receptor antagonist naloxonazine (10 mg kg⁻¹, s.c., 24 h before) but unaffected by peripherally acting opioid antagonist naloxone methyl bromide (3 mg kg⁻¹, s.c.). Forty five minutes after injection, doses of the peptide producing catalepsy (s.c., 8.3–14.2 μmol kg⁻¹, i.c.v., 360 nmol) significantly reduced respiratory frequency and volume of rats breathing air and blocked the hypercapnic ventilator response of rats breathing from 4% to 10% CO₂. I.c.v. administration of [1-5]dermorphin and [1-4]dermorphin (from 36 to 360 nmol) never stimulated respiration but significantly reduced basal and CO₂-stimulated ventilation. Opioid respiratory depression was only antagonized by naloxone.
3. In awake rats, [Lys⁷]dermorphin (0.1–1 mg kg⁻¹, s.c.) decreased blood pressure. This hypotensive response was abolished by naloxone, reduced by naloxone methyl bromide and unaffected by naloxonazine.
4. In conclusion, the present study indicates that analgesic doses of [Lys⁷]dermorphin stimulate respiration by activating central μ₁ opioid receptors and this respiratory stimulation involves a forebrain 5-hydroxytryptaminergic excitatory pathway.

     

Effect of chronic m-CPP on locomotion,hypophagia, plasma corticosterone and 5-HT2C receptor levels in the rat

1. The present study examined 5-HT_2C receptor agonist-induced behavioural tolerance and 5-HT_2C receptor down-regulation in adult rat brain. The effect of chronic subcutaneous infusion of the 5-HT_2C receptor agonist, m-chlorophenylpiperazine (m-CPP, 10 mg kg⁻¹, day⁻¹), for 14 days was examined on daily food intake, the ability of acute m-CPP (2.5 mg kg⁻¹, i.p.) to induce hypolocomotion in a novel arena and elevate plasma corticosterone levels and on ex vivo cortical [³H]-mesulergine binding and hippocampal 5-HT_2C receptor protein levels.
2. Before chronic infusion, m-CPP (2.5 mg kg⁻¹, i.p.) attenuated the number of turns and rears made in a novel open field arena. In contrast, while m-CPP still elicited this hypolocomotion following 14 days, saline infusion, no such hypolocomotion occurred in rats given chronic m-CPP (10 mg kg⁻¹ day⁻¹), indicating that almost complete tachyphylaxis of this behaviour occurred with chronic 5-HT_2C receptor agonist injection.
3. During chronic infusion of m-CPP, rats consumed less food per day than saline-treated controls. Acute challenge with m-CPP following two weeks, treatment still attenuated food intake over the next four hours (by 43% and 30%, respectively from that on the previous day) in saline and m-CPP infusion groups, showing that only partial tolerance to 5-HT_2C receptor agonist-induced hypophagia occurred.
4. In naive home cage rats, plasma corticosterone was elevated in a dose-dependent manner 35 min after m-CPP injection (0.5, 1 and 3 mg kg⁻¹, i.p.) but levels were comparable to control values 16 h after m-CPP (2, 5 and 10 mg kg⁻¹, i.p.). Sixteen hours after a single m-CPP injection (2.5 mg kg⁻¹, i.p.), plasma corticosterone levels were comparable in a group of rats which had received 14 days infusion of m-CPP or saline. However, following a similar acute m-CPP injection (2.5 mg kg⁻¹, i.p., −16 h) in rats previously infused for 14 days with m-CPP, plasma corticosterone levels were lower than those in a separate group which received no chronic infusions (but only acute m-CPP injection), even though the plasma m-CPP levels were comparable in both groups. The data are consistent with the proposal that chronic m-CPP induced some down-regulation of hypothalamic 5-HT_2C receptors which contribute, in a tonic manner, to plasma corticosterone secretion under the conditions investigated.
5. Chronic m-CPP infusion reduced the amount of [³H]-mesulergine binding (by 27%, without altering the K_D) in membranes prepared from parietal/occipital/temporal cortex (under conditions to exclude binding to 5-HT_2A receptors) and 5-HT_2C receptor protein-like immunoreactive levels measured by radioimmunoassay in the hippocampus by 38%, confirming that 5-HT_2C receptor down-regulation had occurred.
6. Even after 14 days m-CPP infusion only partial behavioural tolerance and 5-HT_2C receptor down-regulation were observed, which may vary in different brain regions of the rat. Thus the hypophagia produced by m-CPP may involve activation of 5-HT_2C receptors in the hypothalamus, where there is a greater receptor reserve or which are more resistant to agonist-induced down-regulation than 5-HT_2C receptors in limbic areas (striatum and nucleus accumbens) mediating m-CPP-induced hypolocomotion.