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.

     

Investigation of the mechanisms underlying the hypophagic effects of the 5-HT and noradrenaline reuptake inhibitor,sibutramine, in the rat

1. Sibutramine is a novel 5-hydroxytryptamine (5-HT) and noradrenaline reuptake inhibitor (serotonin- noradrenaline reuptake inhibitor, SNRI) which is currently being developed as a treatment for obesity. Sibutramine has been shown to decrease food intake in the rat. In this study we have used a variety of monoamine receptor antagonists to examine the pharmacological mechanisms underlying sibutramine-induced hypophagia.
2. Individually-housed male Sprague-Dawley rats were maintained on reversed phase lighting with free access to food and water. Drugs were administered at 09 h 00 min and food intake was monitored over the following 8 h dark period.
3. Sibutramine (10 mg kg⁻¹, p.o.) produced a significant decrease in food intake during the 8 h following drug administration. This hypophagic response was fully antagonized by the α₁-adrenoceptor antagonist, prazosin (0.3 and 1 mg kg⁻¹, i.p.), and partially antagonized by the β₁-adrenoceptor antagonist, metoprolol (3 and 10 mg kg⁻¹, i.p.) and the 5-HT receptor antagonists, metergoline (non-selective; 0.3 mg kg⁻¹, i.p.); ritanserin (5-HT_2A/2C; 0.1 and 0.5 mg kg⁻¹, i.p.) and SB200646 (5-HT_2B/2C; 20 and 40 mg kg⁻¹, p.o.).
4. By contrast, the α₂-adrenoceptor antagonist, RX821002 (0.3 and 1 mg kg⁻¹, i.p.) and the β₂-adrenoceptor antagonist, ICI 118,551 (3 and 10 mg kg⁻¹, i.p.) did not reduce the decrease in food intake induced by sibutramine.
5. These results demonstrate that β₁-adrenoceptors, 5-HT_2A/2C-receptors and particularly α₁-adrenoceptors, are involved in the effects of sibutramine on food intake and are consistent with the hypothesis that sibutramine-induced hypophagia is related to its ability to inhibit the reuptake of both noradrenaline and 5-HT, with the subsequent activation of a variety of noradrenaline and 5-HT receptor systems.

     

The role of A3 adenosine receptors in central regulation of arterial blood pressure

1. Pharmacological studies have suggested that A₃ receptors are present on central neurons. Recently this adenosine receptor subtype has been identified in the rat and its presence in the central nervous system has been confirmed.
2. In this study we investigated the effects of acute intracerebroventricular (i.c.v.) injections of N⁶-2-(4-aminophenyl)-ethyladenosine (APNEA), a non-selective A₃ adenosine receptor agonist, on arterial blood pressure (ABP) and heart rate (HR), after treatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective antagonist of A₁ adenosine receptors.
3. Anaesthetized rats, after DPCPX (12 μg⁻¹ kg i.c.v.), were treated with APNEA (0.4–4 μg kg⁻¹ i.c.v.) resulting in a transitory and dose-dependent decrease in arterial blood pressure without a change in heart rate. APNEA also induced hypotensive responses after i.c.v. pretreatment with aminophylline, at a dose of 20 μg kg⁻¹. In contrast, pretreatment 48 h before, with 4 μg kg⁻¹ i.c.v. of pertussis toxin reduced the hypotensive effect induced by APNEA. Administration of APNEA at a higher dose (20 μg kg⁻¹ i.c.v.), after DPCPX, induced a decrease in ABP of −66±5.4 mmHg and after 3 min a decrease in heart rate of −62±6.0 beats min⁻¹. Transection of the spinal cord abolished this significant fall in ABP, but not the decrease of HR.
4. These results suggest that a population of A₃-receptors is present in the CNS, whose activation induces a decrease in blood pressure with no change of heart rate.

     

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.

     

Vasodilator effects of adrenomedullin on small pulmonary arteries and veins in anaesthetized cats

1. This study was conducted to determine adrenomedullin (AM) action sites in the pulmonary vascular bed and the relation between its vasodilator effects and vascular tone. Moreover, an examination was made into whether calcitonin gene-related peptide (CGRP) receptors mediate pulmonary vasodilatations induced by AM. To this end, we directly measured internal diameter (i.d.) changes in small pulmonary arteries and veins (100–1100 μm i.d.) by use of an X-ray televison system on the in vivo cat lung.
2. Under control (resting vascular tone) conditions, AM injections into the left main pulmonary artery caused dose-related i.d. increases in both small arteries and veins. The mean i.d. increase of the 100–1100 μm arteries (4±1, 11±2, and 17±2% with 0.01, 0.1, and 1 nmol kg⁻¹ AM, respectively) was significantly larger than that for the veins (1±1, 5±2, and 7±2% with 0.01, 0.1 and 1 nmol kg⁻¹ AM, respectively) whatever the injected dose of AM.
3. When unilobar hypoxia (5% O₂) had decreased the i.d. of the 100–1100 μm arteries and veins by 16±3 and 6±3%, respectively, AM (0.1 nmol kg⁻¹) was able to induce significantly larger i.d. increases in the arteries (28±3%) and veins (11±3%) than those under control conditions.
4. The AM-induced i.d. response pattern in the serially connected pulmonary arteries was quite different from that induced by CGRP; AM caused a greater increase in smaller vessels (100–500 μm) than in larger vessels (500–1100 μm). In the case of CGRP, a greater increase was observed in the larger vessels.
5. CGRP_8–37 (100 nmol kg⁻¹, i.v., followed by a continuous infusion of 0.2 nmol kg⁻¹ min⁻¹) had no significant effect on the i.d. increase induced by AM (0.1 nmol kg⁻¹) in any serial segments of the arteries and veins.
6. The results indicate that, in the cat, AM induces greater vasodilatation in small pulmonary arteries and lesser vasodilatation in small veins, the maximum dilatation being in the more peripheral arterial segment (100–500 μm). The vasodilator effect of AM was enhanced when vascular tone was elevated. The data suggest that the AM-induced pulmonary vasodilatation is not mediated by CGRP receptors but by its own specific receptor.

     

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.

     

Differential agonist activity of somatostatin and L-362855 at human recombinant sst4 receptors

1. The operational characteristics of somatostatin (SRIF) sst₄ receptors are poorly understood. In this study, we have characterized human recombinant sst₄ receptors expressed in CHO cells (CHOsst₄) by radioligand binding and microphysiometry.
2. Increasing concentrations SRIF or other SRIF receptor ligands inhibited specific [¹²⁵I]-Tyr¹¹-SRIF binding in CHOsst₄ cell membranes with respective pIC₅₀ values of SRIF (8.82), L-362855 (7.40), BIM-23027 (<5.5) and MK-678 (<5.5).
3. These ligands displayed agonist activity, producing concentration-dependent increases in rates of extracellular acidification (EAR) with pEC₅₀ values of SRIF (9.6) and L-362855 (8.0), respectively. BIM-23027 and MK-678 were at least 1000 times weaker than SRIF. The SRIF maximum was about 40% of that observed with L-362855.
4. In the presence of SRIF (0.1–1 nM), concentration-effect curves to L-362855 were displaced to the right with a progressive reduction in the L-362855 maximum.
5. When cells were only exposed to a single maximally effective concentration of SRIF or L-362855, there was no difference in the magnitude of the agonist-induced increase in EAR. However, a second agonist challenge, 30 min later showed that responses to SRIF but not L-362855 were markedly desensitized.
6. When concentration-effect curves to SRIF and L-362855 were obtained by combining data from cells exposed to only a single agonist concentration, SRIF (pEC₅₀ 9.2) was approximately 20 times more potent than L-362855 (pEC₅₀ 8.0) but the maxima were the same. Responses to both SRIF and L-362855 were abolished by pertussis toxin.
7. SRIF and L-362855-induced increases in EAR were inhibited by N-ethyl isopropyl amiloride (10 μM) but were not modified by inhibitors of PKC (Go-6976), MAP kinase (PD-98059), tyrosine kinase (genistein) or tyrosine phosphatase (sodium orthovanadate).
8. The results suggest that SRIF-induced increases in EAR in CHOsst₄ cells involved activation of the Na⁺/H⁺ antiporter and were mediated via Gi/Go G proteins. Responses to SRIF, but not L-362855, were subject to marked desensitization which may be a consequence of differential activation of receptor-effector coupling pathways.

     

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.

     

Differential effects of the neuropeptide galanin on striatal acetylcholine release in anaesthetized and awake rats

1. In the present study the mechanisms were examined by which the neuropeptide galanin modulates the extracellular concentrations of striatal acetylcholine (ACh) in enflurane anaesthetized and in freely moving male rats by use of in vivo microdialysis and high performance liquid chromatography.
2. The perfusion of galanin through the microdialysis probe (0.3 nmol μl⁻¹, flow rate: 2 μl min⁻¹) caused a statistically significant increase in the basal striatal ACh levels in anaesthetized but a decrease in awake animals. No significant effect was revealed after a low dose (0.1 nmol μl⁻¹, flow rate: 2 μl min⁻¹) of galanin perfusion. Both the stimulating and inhibitory effects of galanin on basal ACh release were reversible.
3. The muscarinic antagonist scopolamine (0.1 mg kg⁻¹, subcutaneously (s.c.)) caused a significant increase in ACh release in both anaesthetized and awake animals.
4. The combination of galanin plus scopolamine attenuated the stimulant effect on ACh release caused by scopolamine alone in awake animals.
5. The putative galanin receptor antagonist M35 at 0.3 nmol μl⁻¹ but not at 0.1 nmol μl⁻¹ caused a significant reduction (20%) in ACh release, supporting the view that M35 at higher concentrations behaves as a partial agonist at the galanin receptor. When M35 (0.1 nmol μl⁻¹) was co-infused with galanin (0.3 nmol μl⁻¹) the galanin-evoked decrease in ACh release was completely blocked.
6. Taken together, these results indicate that galanin affects basal ACh release via stimulation of galanin receptors within the striatum. The mechanism involved is dependent on the anaesthesia procedure which may act via enhancement of γ-aminobutyric acid_A (GABA_A) mediated transmission within striatal and/or output neurones. In addition, anaesthesia may also decrease the activity of glutamatergic striatal afferents. The results with M35 indicate that the role of galanin perfused in striatum is permissive in the normal rat. Furthermore, galanin is a potent inhibitory modulator of basal ACh release also in the striatum, as recently was shown in the ventral hippocampus in awake animals.

     

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.

     

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.

     

Selectivity of action of 8-alkylamino analogues of N6-cyclopentyladenosine in vivo: haemodynamic versus anti-lipolytic responses in rats

1. A₁ adenosine receptor agonists with reduced intrinsic activity may be therapeutically useful as result of an increased selectivity of action. In this study the tissue selectivity of three 8-alkylamino substituted analogues of N⁶-cyclopentyladenosine (CPA) was investigated for haemodynamic and anti-lipolytic effects using an integrated pharmacokinetic-pharmacodynamic approach.
2. Chronically instrumented male Wistar rats received intravenous infusions of 4.0 mg kg⁻¹ 8-methylaminoCPA (8MCPA), 12.0 mg kg⁻¹ 8-ethylaminoCPA (8ECPA), 20.0 mg kg⁻¹ 8-butylaminoCPA (8BCPA) or vehicle during 15 min. During experimentation, serial arterial blood samples were drawn for the determination of agonist concentrations and plasma non-esterified fatty acid (NEFA) levels. Blood pressure and heart rate were monitored continuously. In addition to the CPA analogues, each rat received a rapid bolus infusion of CPA to determine the maximal effects of the full agonist.
3. The concentration-time profiles of the CPA analogues could be described by a bi-exponential function. Values for clearance, volume of distribution at steady state and elimination half-life were 44±5, 48±6 and 39±2 ml min⁻¹ kg⁻¹, 0.97±0.09, 0.84±0.10 and 1.05±0.07 1 kg⁻¹ and 25±2, 28±2 and 40±2 min for 8MCPA, 8ECPA and 8BCPA, respectively (mean±s.e.mean, n=6–8).
4. Different models were used to derive the concentration-effect relationships for heart rate and NEFA, yielding estimates of potency (EC₅₀) and instrinsic activity (E_max) for both effects of the compounds in vivo. On heart rate the compounds acted as partial agonists, with E_max values of −173±14, −131±11 and −71±6 beats min⁻¹ for 8MCPA, 8ECPA and 8BCPA, respectively. These E_max values were significantly lower than the maximal effect of CPA (−208±8 beats min⁻¹). With regard to the anti-lipolytic effect all three compounds were full agonists and lowered NEFA levels to the same extent as CPA (69%). The estimated E_max values were 63±5, 63±4 and 68±2%, respectively.
5. Furthermore, the compounds were more potent in causing anti-lipolytic than cardiovascular effects. The EC₅₀ values for the NEFA and heart rate lowering effects were 37±15, 68±22 and 659±108 ng ml⁻¹ and 164±22, 341±76 and 975±190 ng ml⁻¹ for 8MCPA, 8ECPA and 8BCPA, respectively (mean±s.e.mean, n=6–8).
6. This study demonstrates that partial agonists for the A₁ adenosine receptor have increased selectivity of action in vivo. The 8-alkylamino analogues of CPA may be useful anti-lipolytics with less pronounced haemodynamic side effects.

     

Characterization of human recombinant somatostatin sst5 receptors mediating activation of phosphoinositide metabolism

1. We have functionally characterized the human recombinant somatostatin (SRIF) sst₅ receptor in Chinese hamster ovary-K1 (CHOsst₅) cells by measuring total [³H]-inositol phosphate ([³H]-InsPx) accumulation, in the presence of 10 mM LiCl, in cells labelled with [³H]-myo-inositol.
2. In CHOsst₅ cells, SRIF, SRIF-28 and the cyclic hexapeptide, L-362,855, produced time- and concentration-related increases in [³H]-InsPx accumulation, with similar potency (pEC₅₀ values of 6.5, 6.8 and 7.2, respectively). L-362,855 behaved as a partial agonist, producing approximately 30% of the SRIF maximum response. The other peptide analogues of SRIF, BIM-23027 and BIM-23056, were inactive as agonists.
3. Increasing concentrations of L-362,855 increased [³H]-InsPx accumulation and simultaneously produced rightward shifts of SRIF concentration-effect curves, with an estimated pK_p value of 7.6, confirming that it was acting as a partial agonist.
4. BIM-23056, but not BIM-23027, potently antagonized SRIF-induced [³H]-InsPx accumulation, with an estimated pK_B value of 7.4. BIM-23056 did not antagonize [³H]-InsPx accumulation induced by uridine 5′-triphosphate (UTP).
5. SRIF- but not UTP-induced [³H]-InsPx accumulation was inhibited by increasing concentrations of pertussis toxin (0.01–100 ng ml⁻¹), indicating the involvement of pertussis toxin-sensitive G-proteins.
6. These findings show that the human recombinant sst₅ receptor, when stably expressed in CHO-K1 cells, is able to mediate activation of phosphoinositide metabolism in a pertussis toxin-sensitive manner. In this system L-362,855 behaved as a partial agonist while BIM-23056 was a specific antagonist. These agents should provide useful tools for functionally characterizing endogenous SRIF receptors.

     

PYY-preferring receptor in the dorsal vagal complex and its involvement in PYY stimulation of gastric acid secretion in rats

1. Microinjection of peptide YY (PYY, 7–46 pmol) into the dorsal vagal complex (DVC) stimulated gastric acid secretion in urethane-anaesthetized rats. Using a variety of neuropeptide Y (NPY) and PYY derivatives, we characterized the pharmacological profile of the receptor mediating the acid secretory response to PYY.
2. [Pro³⁴]rat(r)/porcine(p)PYY and [Pro³⁴]human(h)PYY (23–117 pmol), microinjected unilaterally into the DVC resulted in a similar maximal increase in net acid secretion reaching 68±11 and 89±31 μmol 90 min⁻¹ respectively.
3. Rat/hNPY and pNPY (47 pmol) microinjected into the DVC induced a similar net gastric acid secretion (27±8 and 23±8 μmol 90 min⁻¹ respectively) and a higher dose (116 pmol) tended to reduce the response.
4. Pancreatic polypeptide (PP, 4–46 pmol), [Leu³¹,Pro³⁴]r/hNPY (47 and 117 pmol) and the Y2 selective agonists, hPYY_3-36, pNPY_5-36 and pNPY_13-36 (25–168 pmol) microinjected into the DVC failed to influence basal gastric acid secretion.
5. The rank order of potency of PYY⩾[Pro³⁴]r/pPYY=[Pro³⁴]hPYY>r/hNPY=pNPY to stimulate gastric acid secretion upon injection into the DVC and the ineffectiveness of PP, [Leu³¹,Pro³⁴]NPY and C-terminal NPY/PYY fragments suggest that a PYY-preferring receptor subtype may be involved in mediating the stimulating effect.

     

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.

     

Noradrenaline release from rat sympathetic neurones triggered by activation of B2 bradykinin receptors

1. The role of bradykinin receptors in the regulation of sympathetic transmitter release was investigated in primary cultures of neurones dissociated from superior cervical ganglia of neonatal rats. These cultures were loaded with [³H]-noradrenaline and the outflow of radioactivity was determined under continuous superfusion.
2. Bradykinin (100 nmol l⁻¹ applied for 10 min) caused a transient increase in tritium outflow that reached a peak within four minutes after the beginning of the application and then declined towards the baseline, despite the continuing presence of the peptide. ATP (100 μmol l⁻¹) and nicotine (10 μmol l⁻¹) caused elevations in ³H outflow with similar kinetics, whereas outflow remained elevated during a 10 min period of electrical field stimulation (0.5 ms, 50 mA, 50 V cm⁻¹, 1.0 Hz).
3. When bradykinin was applied for periods of 2 min, the evoked ³H overflow was half-maximal at 12 nmol l⁻¹ and reached a maximum of 2.3% of cellular radioactivity. The preferential B₁ receptor agonist des-Arg⁹-bradykinin failed to alter ³H outflow. The B₂ receptor antagonists, [D-Phe⁷]-bradykinin (1 μmol l⁻¹) and Hoe 140 (10 nmol l⁻¹), per se did not alter ³H outflow, but shifted the concentration-response curve for bradykinin-evoked ³H overflow to the right by a factor of 7.9 and 4.3, respectively.
4. Bradykinin-induced overflow was abolished in the absence of extracellular Ca²⁺ and in the presence of either 1 μmol l⁻¹ tetrodotoxin or 300 μmol l⁻¹ Cd²⁺, as was electrically-induced overflow. Activation of α₂-adrenoceptors by 1 μmol l⁻¹ UK 14,304 reduced both bradykinin- and electrically-triggered overflow. The Ca²⁺-ATPase inhibitor thapsigargin (0.3 μmol l⁻¹) failed to alter either type of stimulated overflow. Caffeine (10 mmol l⁻¹) enhanced bradykinin-induced overflow, but reduced overflow triggered by electrical field stimulation.
5. Inclusion of Ba²⁺ (0.1 to 1 mmol l⁻¹) in the superfusion medium enhanced electrically induced overflow by approximately 100% and potentiated bradykinin-triggered overflow by almost 400%. Application of 1 mmol l⁻¹ Ba²⁺ for periods of 2 min triggered ³H overflow, and this overflow was abolished by 1μmol l⁻¹ tetrodotoxin and enhanced by 10 mmol l⁻¹ caffeine. In contrast, inclusion of tetraethylammonium (0.1 to 1 mmol l⁻¹) in the superfusion buffer caused similar increases of bradykinin- and electrically evoked ³H overflow (by about 100%), and tetraethylammonium, when applied for 2 min, failed to alter ³H outflow.
6. Treatment of cultures with 100 ng ml⁻¹ pertussis toxin caused a significant increase in bradykinin-, but not in electrically-, evoked tritium overflow. Treatment with 100 ng ml⁻¹ cholera toxin reduced both types of stimulated ³H overflow.
7. These data reveal bradykinin as a potent stimulant of action potential-mediated and Ca²⁺-dependent transmitter release from rat sympathetic neurones in primary cell culture. This neurosecretory effect of bradykinin involves activation of B₂-receptors, presumably linked to pertussis- and cholera toxin-insensitive G proteins, most likely members of the Gq family. Results obtained with inhibitors of muscarinic K⁺ (K_M) channels, like caffeine and Ba²⁺, indicate that the secretagogue action of bradykinin probably involves inhibition of these K⁺ channels.

     

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.

     

Differential effects of somatostatin and angiopeptin on cell proliferation

1. Somatostatin (SRIF) exerts antiproliferative effects, and angiopeptin (an sst₂/sst₅ receptor-selective analogue) has recently been evaluated in clinical trials for the prophylaxis of restenosis following coronary angioplasty. Using an in vitro model of cell growth we have examined the effects of SRIF and angiopeptin on cell proliferation in CHO-K1 cells stably transfected with the human or rat recombinant sst₂ or sst₅ receptor and compared these with their effects on rat aortic vascular smooth muscle cells (VSMC) expressing endogenous somatostatin receptors.
2. In CHO-K1 cells, expressing either human or rat recombinant sst₂ or sst₅ receptors, or in rat aortic VSMC, SRIF and angiopeptin (0.1–1000 nM) had no effect on basal re-growth of cells into a denuded area of a previously confluent monolayer. In contrast, basic fibroblast growth factor (bFGF, 10 ng ml⁻¹) stimulated re-growth of these cells.
3. SRIF (0.1–1000 nM) caused a concentration-dependent inhibition of the bFGF-stimulated re-growth in CHO-K1 cells expressing human sst₂ (h sst₂) or sst₅ (h sst₅) receptors (pIC₅₀=8.05±0.03 and 8.56±0.12, respectively). In contrast, angiopeptin (0.1–1000 nM) acted as a partial agonist at the h sst₂ receptor (44.6±2.7% inhibition of the bFGF-stimulated re-growth at 100 nM; pIC₅₀=8.69±0.25) but was devoid of any agonist activity at the h sst₅ receptor.
4. In CHO-K1 cells stably expressing rat recombinant sst₂ (r sst₂) or sst₅ (r sst₅) receptors, SRIF (0.1–1000 nM) was able to inhibit the bFGF-stimulated re-growth (pIC₅₀=7.98±24 and 8.50±0.12, respectively). Angiopeptin (0.1–1000 nM) caused a concentration-dependent inhibition of bFGF-stimulated re-growth at the r sst₂ receptor (pIC₅₀=8.08±0.24) but acted as a partial agonist at the r sst₅ receptor (maximum response=57.7±3.6% inhibition of bFGF-stimulated re-growth at 100 nM; pIC₅₀=8.60±0.16).
5. Although angiopeptin was inactive as an agonist at the h sst₅ receptor, 100 nM angiopeptin potently antagonized the SRIF-induced inhibition of proliferation in CHO h  sst₅ (estimated pK_B=10.4±0.3). 5-Hydroxytryptamine (0.1 nM–10 μM) also inhibited bFGF-stimulated re-growth (pIC₅₀=8.36±0.11) and angiopeptin had no effect on this response (pK_B<7).
6. SRIF (0.1–1000 nM) caused a concentration-dependent (pIC₅₀=8.04±0.08) inhibition of bFGF-stimulated re-growth in VSMC, whereas angiopeptin displayed weak agonist activity, only inhibiting bFGF-stimulated re-growth at concentrations greater than 100 nM. Angiopeptin (100 nM) caused a rightward displacement of the concentration-effect curve to SRIF with an estimated pK_B value of 7.70±0.12.
7. These findings suggest that the low intrinsic activity of angiopeptin at the h sst₂ receptor, combined with its lack of agonist activity at the h sst₅ receptor, may explain the poor clinical efficacy of angiopeptin in trials for coronary artery restenosis, which contrasts with encouraging data found in equivalent in vivo animal studies.

     

Induction of nitric oxide synthase in vivo and cell injury in rat duodenal epithelium by a water soluble extract of Helicobacter pylori

1. Helicobacter pylori (Hp) infection, which involves the gastric antrum and duodenal mucosa, may be involved in peptic ulceration by stimulating the local release of cytoxic or pro-inflammatory factors.
2. Nitric oxide (NO) is known to be cytotoxic at high concentration. The aim of the present study was therefore to investigate the ability of a water soluble extract of Hp to induce NO synthase in duodenal mucosa and epithelial cells following its administration in vivo in rats and determine its association with cell damage.
3. Administration of Hp water extract (4 ml kg⁻¹) led to the expression of the calcium-independent inducible nitric oxide synthase (iNOS) after 4 h in the duodenum, determined as [¹⁴C]-arginine conversion to citrulline.
4. This iNOS activity was not reduced by pretreatment with anti-neutrophil serum (0.4 ml kg⁻¹, i.p., 3 h before challenge). However, dexamethasone pretreatment (1 mg kg⁻¹, i.v., 2 h before the extract), or administration of the NO synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME, 5 mg kg⁻¹, i.v., 2.5 h after the extract) reduced this activity.
5. Furthermore, iNOS was expressed in duodenal isolated epithelial cells 4 h after the i.v. challenge with the extract, at a time when the cellular viability was also reduced, as assessed by trypan blue exclusion.
6. Dexamethasone pretreatment, administration of L-NAME, or pretreatment with polymyxin B (1 mg kg⁻¹, i.v.) which binds endotoxin, reduced both the iNOS activity and epithelial cell damage.
7. The induction of NO synthase by the Hp extract thus results in duodenal epithelial cell injury and such actions could play a role in pathogenesis of peptic ulcer disease.

     

Effect of YC-1, an NO-independent,superoxide-sensitive stimulator of soluble guanylyl cyclase,on smooth muscle responsiveness to nitrovasodilators

1. We studied the effects of 3-(5′-hydroxymethyl-2′furyl)-1-benzyl indazole (YC-1) on the activity of purified soluble guanylyl cyclase (sGC), the formation of guanosine-3′ : 5′ cyclic monophosphate (cyclic GMP) in vascular smooth muscle cells (VSMC), and on the tone of rabbit isolated aortic rings preconstricted by phenylephrine (PE). In addition, we assessed the combined effect of YC-1, and either NO donors, or superoxide anions on these parameters.
2. YC-1 elicited a direct concentration-dependent activation of sGC (EC₅₀ 18.6±2.0 μM), which was rapid in onset and quickly reversible upon dilution. YC-1 altered the enzyme kinetics with respect to GTP by decreasing K_M and increasing V_max. Activation of sGC by a combination of sodium nitroprusside (SNP) and YC-1 was superadditive at low and less than additive at high concentrations, indicating a synergistic activation of the enzyme by both agents. A specific inhibitor of sGC, 1H-(1,2,4)-oxdiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (NS 2028), abolished activation of the enzyme by either compound.
3. YC-1 induced a concentration-dependent increase in intracellular cyclic GMP levels in rat cultured aortic VSMC, which was completely inhibited by NS 2028. YC-1 applied at the same concentration as SNP elicited 2.5 fold higher cyclic GMP formation. Cyclic GMP-increases in response to SNP and YC-1 were additive.
4. YC-1 relaxed preconstricted endothelium-denuded rabbit aortic rings in a concentration-dependent manner (50% at 20 μM) and markedly increased cyclic GMP levels. Relaxations were inhibited by NS 2028. A concentration of YC-1 (3 μM), which elicited only minor effects on relaxation and cyclic GMP, increased the vasodilator potency of SNP and nitroglycerin (NTG) by 10 fold and markedly enhanced SNP- and NTG-induced cyclic GMP formation.
5. Basal and YC-1-stimulated sGC activity was sensitive to inhibition by superoxide (O₂⁻) generated by xanthine/xanthine oxidase, and was protected from this inhibition by superoxide dismutase (SOD). YC-1-stimulated sGC was also sensitive to inhibition by endogenously generated (O₂⁻ in rat preconstricted endothelium-denuded aortic rings. Relaxation to YC-1 was significantly attenuated in aortae from spontaneously hypertensive rats (SHR), which generated O₂⁻ at a higher rate than aortae from normotensive Wistar Kyoto rats (WKY). SOD restored the vasodilator responsiveness of SHR rings to YC-1.
6. In conclusion, these results indicate that YC-1 is an NO-independent, O₂⁻-sensitive, direct activator of sGC in VSMC and exerts vasorelaxation by increasing intracellular cyclic GMP levels. The additive or even synergistic responses to NO-donors and YC-1 in cultured VSMC and isolated aortic rings apparently reflect the direct synergistic action of YC-1 and NO on the sGC. The synergism revealed in this in vitro study suggests that low doses of YC-1 may be of therapeutic value by permitting the reduction of nitrovasodilator dosage.