Characterization of prejunctional 5-HT1 receptors that mediate the inhibition of pressor effects elicited by sympathetic stimulation in the pithed rat

1. A study was made of the effects of 5-carboxamidotryptamine (5-CT) on pressor responses induced in vivo by electrical stimulation of the sympathetic outflow from the spinal cord of pithed rats. All animals had been pretreated with atropine. Sympathetic stimulation (0.1, 0.5, 1 and 5 Hz) resulted in frequency-dependent increases in blood pressure. Intravenous infusion of 5-CT at doses of 0.01, 0.1 and 1 μg kg⁻¹ min⁻¹ reduced the pressor effects obtained by electrical stimulation. The inhibitory effect of 5-CT was significantly more pronounced at lower frequencies of stimulation. In the present study we characterized the pharmacological profile of the receptors mediating the above inhibitory effect of 5-CT.
2. The inhibition induced by 0.01 μg kg⁻¹ min⁻¹ of 5-CT on sympathetically-induced pressor responses was partially blocked after i.v. treatment with methiothepin (10  μg kg⁻¹), WAY-100,635 (100 μg kg⁻¹) or GR127935T (250 μg kg⁻¹), but was not affected by cyanopindolol (100 μg kg⁻¹).
3. The selective 5-HT_1A receptor agonist 8-OH-DPAT and the selective 5-HT_1B/1D receptor agonists sumatriptan and L-694,247 inhibited the pressor response, whereas the 5-HT_1B receptor agonists CGS-12066B and CP-93,129 and the 5-HT_2C receptor agonist m-CPP did not modify the pressor symapthetic responses.
4. The selective 5-HT_1A receptor antagonist WAY-100,635 (100 μg kg⁻¹) blocked the inhibition induced by 8-OH-DPAT and the selective 5-HT_1B/1D receptor antagonist GR127935T (250 μg kg⁻¹) abolished the inhibition induced either by L-694,247 or sumatriptan.
5. None of the 5-HT receptor agonists used in our experiments modified the pressor responses induced by exogenous noradrenaline (NA).
6. These results suggest that the presynaptic inhibitory action of 5-CT on the electrically-induced pressor response is mediated by both r-5-HT_1D and 5-HT_1A receptors.

     

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.

     

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.

     

Characterization of putative 5-HT7 receptors mediating tachycardia in the cat

1. It has been suggested that the tachycardic response to 5-hydroxytryptamine (5-HT) in the spinal-transected cat is mediated by ‘5-HT₁-like'' receptors since this effect, being mimicked by 5-carboxamidotryptamine (5-CT), is not modified by ketanserin or MDL 72222, but it is blocked by methiothepin, methysergide or mesulergine. The present study was set out to reanalyse this suggestion in terms of the IUPHAR 5-HT receptor classification schemes proposed in 1994 and 1996.
2. Intravenous (i.v.) bolus injections of the tryptamine derivatives, 5-CT (0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μg kg⁻¹), 5-HT (3, 10 and 30 μg kg⁻¹) and 5-methoxytryptamine (3, 10 and 30 μg kg⁻¹) as well as the atypical antipsychotic drug, clozapine (1000 and 3000 μg kg⁻¹) resulted in dose-dependent increases in heart rate, with a rank order of agonist potency of 5-CT >> 5-HT > 5-methoxytryptamine >> clozapine.
3. The tachycardic effects of 5-HT and 5-methoxytryptamine were dose-dependently antagonized by i.v. administration of lisuride (30 and 100 μg kg⁻¹), ergotamine (100 and 300 μg kg⁻¹) or mesulergine (100, 300 and 1000 μg kg⁻¹); the highest doses of these antagonists used also blocked the tachycardic effects of 5-CT. Clozapine (1000 and 3000 μg kg⁻¹) did not affect the 5-HT-induced tachycardia, but attenuated, with its highest dose, the responses to 5-methoxytryptamine and 5-CT. However, these doses of clozapine as well as the high doses of ergotamine (300 μg kg⁻¹) and mesulergine (300 and 1000 μg kg⁻¹) also attenuated the tachycardic effects of isoprenaline. In contrast, 5-HT-, 5-methoxytryptamine- and 5-CT-induced tachycardia were not significantly modified after i.v. administration of physiological saline (0.1 and 0.3 ml kg⁻¹), the 5-HT_1B/1D receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (500 μg kg⁻¹) or the 5-HT_3/4 receptor antagonist, tropisetron (3000 μg kg⁻¹).
4. Intravenous injections of the 5-HT₁ receptor agonists, sumatriptan (30, 100 and 300 μg kg⁻¹) and indorenate (300 and 1000 μg kg⁻¹) or the 5-HT₄ receptor (partial) agonist cisapride (300 and 1000 μg kg⁻¹) were devoid of effects on feline heart rate per se and failed to modify significantly 5-HT-induced tachycardic responses.
5. Based upon the above rank order of agonist potency, the failure of sumatriptan, indorenate or cisapride to produce cardioacceleration and the blockade by a series of drugs showing high affinity for the cloned 5-ht₇ receptor, the present results indicate that the 5-HT receptor mediating tachycardia in the cat is operationally similar to other putative 5-HT₇ receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine external carotid and coronary arteries, rat systemic vasculature and guinea-pig ileum). Since these responses represent functional correlates of the 5-ht₇ gene product, the 5-HT₇ receptor appellation is reinforced. Therefore, the present experimental model, which is not complicated by the presence of other 5-HT receptors, can be utilized to characterize and develop new drugs with potential agonist and antagonist properties at functional 5-HT₇ receptors.

     

Signalling pathways in bradykinin- and nitric oxide-induced hypotension in the normotensive rat; role of K+-channels

1. Bradykinin and nitric oxide (NO) are potent hypotensive agents. In the present study, the role of K⁺-channels in the signalling pathways responsible for their hypotensive action was investigated in normotensive, anaesthetized rats. The rats were treated with ion-channel inhibitors before administration of bradykinin (2.8, 5.6, 28 and 56 nmol kg⁻¹, i.v.) followed in some of the protocols by nitroprusside (1.1, 3.5, 7, 14, and 28 nmol kg⁻¹, i.v.).
2. No attenuation of the hypotensive response to bradykinin was detected for inhibitors of the Na-K-Cl-cotransporter (30 μmol kg⁻¹ furosemide), the ATP-sensitive K⁺-channel (40 μmol kg⁻¹ glibenclamide), high conductance Ca²⁺-activated K⁺-channel (180 μmol kg⁻¹ tetraethylammonium, 54 μmol kg⁻¹ tetrabutylammonium, 35 nmol kg⁻¹ iberiotoxin, 35 nmol kg⁻¹ charybdotoxin) or the low conductance Ca²⁺-activated K⁺-channel (74 nmol kg⁻¹ apamin).
3. However, the voltage-sensitive K⁺-channel (I_A) inhibitor 4-aminopyridine (4.05–40.5 μmol kg⁻¹) induced a concentration-dependent (P<0.0001) attenuation of the hypotensive response (P<0.0001). Bradykinin had no effect on heart rate in anaesthetized rats and this observation was not altered by pretreatment with 4-aminopyridine.
4. 4-Aminopyridine (53 μmol kg⁻¹) also significantly attenuated the hypotensive response to nitroprusside (P<0.0003) without altering the heart rate concentration-response curve. Of the two Ca²⁺-activated K⁺-channel inhibitors tested on nitroprusside-induced hypotension, tetrabutylammonium induced a slight attenuation (P<0.0101), whereas iberiotoxin had no effect.
5. We therefore concluded that, although the acute hypotensive response to bradykinin in the normotensive rat is not mediated through nitric oxide synthesis, the hypotensive response to both agents was mediated through opening of voltage-sensitive K⁺-channels (I_A), resulting in a decrease in peripheral vascular resistance.

     

Pharmacokinetic-pharmacodynamic modelling of the anti-lipolytic and anti-ketotic effects of the adenosine A1-receptor agonist N6-(p-sulphophenyl)adenosine in rats

1. The purpose of this study was to develop and validate an integrated pharmacokinetic-pharmacodynamic model for the anti-lipolytic effects of the adenosine A₁-receptor agonist N⁶-(p-sulphophenyl)adenosine (SPA). Tissue selectivity of SPA was investigated by quantification of haemodynamic and anti-lipolytic effects in individual animals.
2. After intravenous infusion of SPA to conscious normotensive Wistar rats, arterial blood samples were drawn for determination of blood SPA concentrations, plasma non-esterified fatty acid (NEFA) and β-hydroxybutyrate levels. Blood pressure and heart rate were monitored continuously.
3. The relationship between the SPA concentrations and the NEFA lowering effect was described by the indirect suppression model. Administration of SPA at different rates and doses (60 μg kg⁻¹ in 5 min and 15 min, and 120 μg kg⁻¹ in 60 min) led to uniform pharmacodynamic parameter estimates. The averaged parameters (mean±s.e., n=19) were E_max: −80±2% (% change from baseline), EC₅₀: 22±2 ng ml⁻¹, and Hill factor: 2.2±0.2.
4. In another group, given 400 μg kg⁻¹ SPA in 15 min, pharmacodynamic parameters for both heart rate and anti-lipolytic effect were derived within the same animal. The reduction in heart rate was directly related to blood concentration on the basis of the sigmoidal E_max model. SPA inhibited lipolysis at concentrations lower than those required for an effect on heart rate. The EC₅₀ values (mean±s.e., n=6) were 131±31 ng ml⁻¹ and 20±3 ng ml⁻¹ for heart rate and NEFA lowering effect, respectively.
5. In conclusion, the relationship between blood SPA concentrations and anti-lipolytic effect was adequately described by the indirect suppression model. For SPA a 6 fold difference in potency was observed between the effects on heart rate and NEFAs, indicating some degree of tissue selectivity in vivo.

     

Opposite effects of CCKB agonists in grooming behaviour in rats: further evidence for two CCKB subsites

1. The hypothesis of the existence of two CCK_B receptor subsites, CCK_B1 and CCK_B2 corresponding probably to different coupling states of CCK_B receptors, was studied by measuring grooming behaviour in rats.
2. The B1 receptor agonist, BC197 (300 μg kg⁻¹, i.p.) produced a 45–50% decrease in grooming activity, which was prevented by both the CCK_B receptor antagonists CI-988 (20 μg kg⁻¹ i.p.) and L-365,260 (200 μg kg⁻¹, i.p.).
3. In contrast, 3, 10 and 30 μg kg⁻¹, i.p., of the potent B2 receptor agonist, BC264, enhanced grooming (150–190%). This effect was prevented by previous injection of 75 μg kg⁻¹ of L-365,260 while higher doses (200 μg kg⁻¹, i.p.) produced only a partial antagonism. Moreover, CI-988 (20 μg kg⁻¹, i.p.), showed an opposite effect in potentiating the responses induced by BC264. However, 200 μg kg⁻¹ of CI-988 tended to suppress the increase of grooming induced by BC264.
4. The effects of BC264 were prevented by the D₁ receptor (SCH 23390) and D₂ receptor (sulpiride) antagonists, while those of BC197 were only antagonized by sulpiride, emphasizing the existence of a link between peptidergic (CCK) and dopaminergic systems.
5. This study brings additional evidence for the existence of the two CCK_B receptor subsites and suggests that particular attention should be focused on the selectivity of CCK_B receptor agonists, notably to explain the fact that some compounds such as Boc-CCK4 induce anxiogenic-like effects while others, including BC264, are devoid of these effects.

     

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.

     

Facilitation and inhibition of male rat ejaculatory behaviour by the respective 5-HT1A and 5-HT1B receptor agonists 8-OH-DPAT and anpirtoline,as evidenced by use of the corresponding new and selective receptor antagonists NAD-299 and NAS-181

1. Ejaculatory problems and anorgasmia are well-known side-effects of the SSRI antidepressants, and a pharmacologically induced increase in serotonergic neurotransmission inhibits ejaculatory behaviour in the rat. In the present study the role of 5-HT_1A and 5-HT_1B receptors in the mediation of male rat ejaculatory behaviour was examined by use of selective agonists and antagonists acting at these 5-HT receptor subtypes.
2. The 5-HT_1A receptor agonist 8-OH-DPAT (0.25–4.00 μmol kg⁻¹ s.c.) produced an expected facilitation of the male rat ejaculatory behaviour, and this effect was fully antagonized by pretreatment with the new selective 5-HT_1A receptor antagonist (R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R) tartrate monohydrate (NAD-299) (1.0 μmol kg⁻¹ s.c.). NAD-299 by itself (0.75–3.00 μmol kg⁻¹ s.c.) did not affect the male rat ejaculatory behaviour.
3. The 5-HT_1B receptor agonist anpirtoline (0.25–4.00 μmol kg⁻¹ s.c.) produced a dose-dependent inhibition of the male rat ejaculatory behaviour, and this effect was fully antagonized by pretreatment with the 5-HT_1B receptor antagonist isamoltane (16 μmol kg⁻¹ s.c.) as well as by the new and selective antagonist (R)-(+)-2-(3-morpholinomethyl-2H-chromene-8-yl)oxymethylmorpholino methansulphonate (NAS-181) (16 μmol kg⁻¹ s.c.). Isamoltane (1.0–16.0 μmol kg⁻¹ s.c.) and NAD-181 (1.0–16.0 μmol kg⁻¹ s.c.) had no, or weakly facilitatory effects on the male rat ejaculatory behaviour. The non-selective 5-HT₁ receptor antagonist (−)-pindolol (8 μmol kg⁻¹ s.c.), did not antagonize the inhibition produced by anpirtoline.
4. The present results demonstrate opposite effects, facilitation and inhibition, of male rat ejaculatory behaviour by stimulation of 5-HT_1A and 5-HT_1B receptors, respectively, suggesting that the SSRI-induced inhibition of male ejaculatory dysfunction is due to 5-HT_1B receptor stimulation.

     

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.

     

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.

     

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.

     

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.

     

A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [³H]-cyclic adenosine monophosphate ([³H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The α₁-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca²⁺ channel blocker, nifedipine (10 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml⁻¹ 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7±0.9 fold).
2. In the presence of phenylephrine (1 μM), NECA and the A₁ adenosine receptor selective agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC₅₀ values 8.18±0.19, 7.79±0.29 and 8.15±0.43 respectively). The A₃ adenosine receptor agonists N⁶-iodobenzyl-5′-N-methyl-carboxamido adenosine (IBMECA) and N⁶-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pK_B 8.75±0.88) and the maximal response to NECA was reduced.
3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A_2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC₅₀ 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A_2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM).
4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 μM)-stimulated accumulation of [³H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17±6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [³H]-cyclic AMP in preparations of epididymis (pEC₅₀ values 5.35±0.35 and 6.42±0.40 respectively), the NECA-induced elevation of [³H]-cyclic AMP was antagonised by XAC (apparent pK_B 6.88±0.88) and also by the A_2A adenosine receptor antagonist, ZM 241385 (apparent pK_B 8.60± 0.76).
5. These studies are consistent with the action of stable adenosine analogues at post-junctional A₁ and A₂ adenosine receptors in the epididymis of the guinea-pig. A₁ Adenosine receptors potentiate α₁-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A₂ adenosine receptors, possibly of the A_2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

     

Modulation of the firing activity of noradrenergic neurones in the rat locus coeruleus by the 5-hydroxtryptamine system

1. The aim of the present study was to investigate the putative modulation of locus coeruleus (LC) noradrenergic (NA) neurones by the 5-hydroxytryptaminergic (5-HT) system by use of in vivo extracellular unitary recordings and microiontophoresis in anaesthetized rats. To this end, the potent and selective 5-HT_1A receptor antagonist WAY 100635 (N-{2-[4(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarboxamide trihydroxychloride) was used.
2. In the dorsal hippocampus, both local (by microiontophoresis, 20 nA) and systemic (100 μg kg⁻¹, i.v.) administration of WAY 100635 antagonized the suppressant effect of microiontophorectically-applied 5-HT on the firing activity of CA₃ pyramidal neurones, indicating its antagonistic effect on postsynaptic 5-HT_1A receptors.
3. WAY 100635 and 5-HT failed to modify the spontaneous firing activity of LC NA neurones when applied by microiontophoresis. However, the intravenous injection of WAY 100635 (100 μg kg⁻¹) readily suppressed the spontaneous firing activity of LC NA neurones.
4. The lesion of 5-HT neurones with the neurotoxin 5,7-dihydroxytryptamine increased the spontaneous firing activity of LC NA neurones and abolished the suppressant effect of WAY 100635 on the firing activity of LC NA neurones.
5. In order to determine the nature of the 5-HT receptor subtypes mediating the suppressant effect of WAY 100635 on NA neurone firing activity, several 5-HT receptor antagonists were used. The selective 5-HT₃ receptor antagonist BRL 46470A (10 and 100 μg kg⁻¹, i.v.), the 5-HT_1D receptor antagonist GR 127935 (100 μg kg⁻¹, i.v.) and the 5-HT_1A/1B receptor antagonist (−)-pindolol (15 mg kg⁻¹, i.p.) did not prevent the suppressant effect of WAY 100635 on the firing activity of LC NA neurones. However, the suppressant effect of WAY 100635 was prevented by the non-selective 5-HT receptor antagonists spiperone (1 mg kg⁻¹, i.v.) and metergoline (1 mg kg⁻¹, i.v.), by the 5-HT₂ receptor antagonist ritanserin (500 μg kg⁻¹, i.v.). It was also prevented by the 5-HT_1A receptor/α_1D-adrenoceptor antagonist BMY 7378 (1 mg kg⁻¹, i.v.) and by the α₁-adrenoceptor antagonist prazosin (100 μg kg⁻¹, i.v.).
6. These data support the notion that the 5-HT system tonically modulates NA neurotransmission since the lesion of 5-HT neurones enhanced the LC NA neurones firing activity and the suppressant effect of WAY 100635 on the firing activity of NA neurones was abolished by this lesion. However, the location of the 5-HT_1A receptors involved in this complex circuitry remains to be elucidated. It is concluded that the suppressant effect of WAY 100635 on the firing activity of LC NA neurones is due to an enhancement of the function of 5-HT neurones via a presynaptic 5-HT_1A receptor. In contrast, the postsynaptic 5-HT receptor mediating this effect of WAY 100635 on NA neurones appears to be of the 5-HT_2A subtype.

     

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.

     

Negative modulation of nitric oxide production by neurotensin as a putative mechanism of the diuretic action of SR 48692 in rats

1. We investigated the effect of the non-peptide neurotensin (NT) antagonist SR 48692 on renal function in rats and the involvement of nitric oxide (NO) in the diuretic action of this compound.
2. In fed animals, SR 48692 dose-dependently (0.5 to 12.5 mg kg⁻¹, p.o., 0.03 to 1 mg kg⁻¹, i.p. and 0.1 to 1 μg/rat, i.c.v.) increased urine output and urinary excretion of Na⁺, K⁺ and Cl⁻ and reduced urine osmolality. The diuretic activity was also evident in water-deprived, fasted animals and in fasted, water-loaded rats.
3. NT (0.1 μg/rat, i.c.v.) had no effect on urine output in fed rats, but reduced the diuretic action of SR 48692 (1 μg/rat, i.c.v.). The opposite result was obtained in fasted, water-loaded animals: NT dose-dependently (0.01 and 0.1 μg/rat, i.c.v.) inhibited diuresis and this effect was significantly inhibited by i.c.v. SR 48692. In this experimental condition, SR 48692 did not further increase the on-going diuresis.
4. The NO synthesis inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME; 30 mg kg⁻¹, i.p.) alone had no effect on urine output in fed rats but prevented the diuretic action of i.c.v. or i.p. SR 48692; L-arginine (1 g kg⁻¹, i.p.) but not D-arginine (1 g kg⁻¹, i.p.) restored the SR 48692-dependent increase in diuresis. L-NAME had no effect on furosemide-stimulated diuresis.
5. Systemically administered L-NAME or i.c.v. NT in fasted, water-loaded rats significantly reduced water diuresis but this effect was no longer seen in animals given i.p. L-arginine. Rats receiving i.c.v. NT, whose diuresis was significantly reduced, also excreted less nitrates and nitrites in urine.
6. Increased diuresis after central or systemic administration of SR 48692 to fed rats was paralleled by increased urinary excretion of nitrates and nitrites, this being consistent with peripheral enhancement of NO production after NT-receptor blockade by SR 48692. The increase in diuresis after furosemide also involved an increase of nitrates and nitrites in urine, but this effect was about half that attained with an equipotent diuretic dose of SR 48692.
7. In fed rats, the NO donor isosorbide-dinitrate, reduced systolic blood pressure (unlike SR 48692 which did not affect blood pressure) but also dose-dependently (1 and 5 mg kg⁻¹, i.p.) stimulated urine output.
8. The overall effects of SR 48692 strongly support a link between the actions of endogenous NT, AVP and peripheral NO production in the modulation of renal excretion of water, Na⁺, K⁺ and Cl⁻.

     

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.

     

Local neurogenic regulation of rat hindlimb circulation: CO2-induced release of calcitonin gene-related peptide from sensory nerves

1. The mechanism of release of calcitonin gene-related peptide (CGRP) from sensory nerves in response to skeletal muscle contraction was investigated in the rat hindlimb in vivo and in vitro.
2. In the anaesthetized rat, sciatic nerve stimulation at 10 Hz for 1 min caused a hyperaemic response in the hindlimb. During the response, partial pressure of CO₂ in the venous blood effluent from the hindlimb significantly increased from 43±3 to 73±8 mmHg, whereas a small decrease in pH and no appreciable change in partial pressure of O₂ were observed.
3. An intra-arterial bolus injection of NaHCO₃ (titrated to pH 7.2 with HCl), which elevated PCO₂ of the venous blood, caused a sustained increase in regional blood flow of the iliac artery. Capsaicin (0.33 μmol kg⁻¹, i.a.) and a specific calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP(8–37), (100 nmol kg⁻¹ min⁻¹, i.v.) significantly suppressed the hyperaemic response to NaHCO₃. Neither ND_Ω-nitro-L-arginine methyl ester (1 μmol kg⁻¹ min⁻¹, i.v.) nor indomethacin (5 mg kg⁻¹, i.v.) affected the response.
4. The serum level of CGRP-like immunoreactivity in the venous blood was significantly increased by a bolus injection of NaHCO₃ (pH=7.2) from 50±4 to 196±16 fmol ml⁻¹.
5. In the isolated hindlimb perfused with Krebs-Ringer solution, a bolus injection of NaHCO₃ (pH=7.2) caused a decrease in perfusion pressure which was composed of two responses, i.e., an initial transient response and a slowly-developing long-lasting one. CGRP(8–37) significantly inhibited the latter response by 73%.
6. These results suggest that CO₂ liberated from exercising skeletal muscle activates capsaicin-sensitive perivascular sensory nerves locally, which results in the release of CGRP from their peripheral endings, and then the released peptide causes local vasodilatation.