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.

     

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.

     

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.

     

Pharmacological analysis of the haemodynamic effects of 5-HT1B/D receptor agonists in the normotensive rat

1. The receptors involved in mediating the haemodynamic effects of three 5-HT_1B/D receptor agonists were investigated in pentobarbitone anaesthetized rats (n=6–17 per group).
2. Cumulative intravenous (i.v.) infusions of rizatriptan and sumatriptan (from 0.63 to 2500 μg kg⁻¹; each dose over 5 min) induced dose-dependent and marked hypotension (−42±6 and −34±4 mmHg at the highest dose, respectively; both P<0.05 vs vehicle: +5±3 mmHg) and bradycardia (−85±16 and −44±12 beats min⁻¹ at the highest dose, respectively; both P<0.05 vs vehicle: +16±6 beats min⁻¹). Zolmitriptan evoked only moderate hypotension at the highest dose (−19±9 mmHg; P<0.05 vs vehicle).
3. A high dose of the 5-HT_1B/D receptor antagonist, GR 127935 (0.63 mg kg⁻¹, i.v.), failed to antagonize the hypotension and bradycardia evoked by sumatriptan (−35±6 mmHg and −52±19 beats min⁻¹, respectively; both not significant vs sumatriptan in untreated rats), but moderately reduced the hypotension and bradycardia evoked by rizatriptan (−20±5 mmHg and −30±17 beats min⁻¹, respectively; both P<0.05 vs vehicle and vs rizatriptan in untreated rats).
4. The selective 5-HT_1A receptor antagonist, WAY 100635 (0.16 and 0.63 mg kg⁻¹, i.v.), dose-dependently attenuated the haemodynamic responses evoked by rizatriptan and sumatriptan, which were almost abolished by the higher dose of WAY 100635 (−4±3 mmHg and −15±8 beats min⁻¹; both not significant vs vehicle and P<0.05 vs rizatriptan in untreated rats). A slight but statistically significant reduction in mean arterial pressure (MAP) persisted at the highest dose of sumatriptan (−13±4 mmHg following the higher dose of WAY 100635; P<0.05 vs vehicle).
5. In pithed rats with MAP normalized by angiotensin II, rizatriptan failed to induce hypotension or bradycardia (+5±4 mmHg and −6±16 beats min⁻¹, respectively; both NS vs vehicle and P<0.05 vs rizatriptan in untreated rats). Similarly, sumatriptan failed to induce bradycardia in pithed rats (+5±6 beats min⁻¹; not significant vs vehicle and P<0.05 vs sumatriptan in untreated rats), whereas a slight but statistically significant reduction in MAP, compared to controls, occurred at the highest dose (−9±9 mmHg; P<0.05 vs both vehicle and sumatriptan in untreated rats).
6. In bilaterally vagotomized and atropine-treated (1 mg kg⁻¹, i.v.) rats, the reductions in MAP and heart rate evoked by rizatriptan (−31±4 mmHg and −64 ±9 beats min⁻¹, respectively; both P<0.05 vs vehicle and not significant vs rizatriptan in controls) and sumatriptan (−47±8 mmHg and −56±10 beats min⁻¹, respectively; both P<0.05 vs vehicle and not significant vs sumatriptan in controls) were not statistically significantly different from those observed in controls.
7. In conclusion, the 5-HT_1B/D receptor agonists, rizatriptan and sumatriptan, elicit hypotension and bradycardia in the normotensive anaesthetized rat predominantly via activation of central 5-HT_1A receptors, and a consequent reduction in sympathetic outflow.

     

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.

     

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.

     

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.

     

Effect of the selective 5-HT1A receptor antagonist WAY 100635 on the inhibition of e.p.s.ps produced by 5-HT in the CA1 region of rat hippocampal slices

1. The actions of N-(2-(-4(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635), a novel and selective 5-hydroxytryptamine_1A (5-HT_1A) antagonist, on excitatory postsynaptic potentials (e.p.s.ps) were investigated by use of intracellular recordings in pyramidal cells of the CA1 region of rat hippocampal slices.
2. WAY 100635 (10 nM) did not affect any of the investigated parameters of cell excitability such as membrane potential, total input resistance (R_in), firing threshold, action potential amplitude, action potential frequency adaptation, and slow afterhyperpolarization (sAHP) which follows repetitive firing of action potentials. WAY 100635 did not have any effect on either the slope or the amplitude of e.p.s.ps evoked by stimulation of the CA1 stratum radiatum.
3. Bath application of either 5-hydroxytryptamine (5-HT, 10–30 μM) or 5-carboxamidotryptamine (5-CT, 300 nM) hyperpolarized the membrane potential (ΔV_m=−4.1±0.9 and −6.0±0.9 mV, respectively), and reduced R_in (−25±8% and −18±1%, respectively). 5-HT blocked the action potential frequency adaptation and significantly reduced the amplitude of the sAHP that follows repetitive firing of action potentials.
4. 5-HT significantly decreased the amplitude of evoked e.p.s.ps (−14±6%). This effect was greater in the presence of the GABA_A receptor antagonist bicuculline (10 μM, −45±12%) and was mimicked by 5-CT (−49±5%). Both AMPA and NMDA components of e.p.s.ps were significantly reduced in amplitude by 5–HT (−38±8%, n=6, and −29±12%, n=3, respectively; P<0.05).
5. WAY 100635 fully antagonized the hyperpolarization, the reduction of R_in, and the decrease in amplitude of e.p.s.ps elicited by 5-HT, while it did not affect the action of 5-HT on the action potential frequency adaptation. In the presence of WAY 100635, 5-HT elicited a depolarization which was blocked by 10–30 μM RS 23597-190, a selective 5-HT₄ receptor antagonist.
6. Our data demonstrate that WAY 100635 is devoid of direct effects on CA1 pyramidal cell excitability and on evoked e.p.s.ps, while it fully antagonizes the effects of 5-HT on excitatory synaptic transmission and on hyperpolarization, without affecting the 5-HT₄ receptor-mediated response. Since WAY 100635 selectively antagonizes 5-HT_1A receptor-mediated actions of 5-HT, our data also demonstrate that the inhibitory action of 5-HT on excitatory synaptic transmission in CA1 is mediated by 5-HT_1A receptors.

     

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.

     

The effect of the selective 5-HT1A agonists alnespirone (S-20499) and 8-OH-DPAT on extracellular 5-hydroxytryptamine in different regions of rat brain

1. We have examined the effects of the systemic administration of the selective 5-HT_1A agonist alnespirone (S-20499) on in vivo 5-hydroxytryptamine (5-HT) release in the dorsal raphe nucleus, the median raphe nucleus and four forebrain areas innervated differentially by both (dorsal striatum, frontal cortex, ventral hippocampus and dorsal hippocampus).
2. Alnespirone (0.1–3 mg kg⁻¹, s.c.) dose-dependently reduced extracellular 5-HT in the six areas examined. In forebrain, the maximal reductions occurred in striatum and frontal cortex (maximal reduction to 23 and 29% of baseline, respectively). Those in dorsal and ventral hippocampus were more moderate (to ca 65% of baseline). In contrast, the decrease in 5-HT elicited in the median raphe nucleus was more marked than that in the dorsal raphe nucleus (to ca 30 and 60% of baseline, respectively). The selective 5-HT_1A antagonist WAY-100635 (0.5 mg kg⁻¹, s.c.) prevented the decrease in 5-HT induced by alnespirone (0.3 mg kg⁻¹, s.c.) in frontal cortex.
3. 8-OH-DPAT (0.025, 0.1 and 0.3 mg kg⁻¹, s.c.) also reduced extracellular 5-HT in a regionally-selective manner (e.g., to 32% of baseline in striatum and to 69% in dorsal hippocampus at 0.1 mg kg⁻¹, s.c.). In midbrain, 8-OH-DPAT reduced the dialysate 5-HT slightly more in the median than in the dorsal raphe nucleus at all doses examined.
4. Doses of both compounds close to their respective ED₅₀ values (0.3 mg kg⁻¹ alnespirone, 0.025 mg kg⁻¹ 8-OH-DPAT) reduced 5-HT to a comparable extent in all regions examined. However, the reductions attained at higher doses were more pronounced for 8-OH-DPAT.
5. These data show that the reduction of 5-HT release elicited by alnespirone and 8-OH-DPAT is more important in forebrain areas innervated by 5-hydroxytryptaminergic neurones of the dorsal raphe nucleus. This regional selectivity seems unlikely to be accounted for by differences in the sensitivity of 5-HT_1A autoreceptors controlling 5-HT release, given the dissimilar effects of these two 5-HT_1A agonists in regions rich in cell bodies and nerve terminals. This suggests the presence of complex mechanisms of control of 5-HT release by 5-HT_1A 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.

     

Agonistic properties of alniditan,sumatriptan and dihydroergotamine on human 5-HT1B and 5-HT1D receptors expressed in various mammalian cell lines

1. Alniditan, a novel migraine abortive agent, is a potent 5-HT_1B/5-HT_1D receptor agonist of nM affinity. We compared the agonistic properties of alniditan, sumatriptan and dihydroergotamine on the cloned human 5-HT_1B receptor expressed at 200 fmol mg⁻¹ protein (B_max) in non-induced L929sA cells, at 740 fmol mg⁻¹ protein in HEK 293 and at 2300 fmol mg⁻¹ protein in mIFNβ-induced L929sA cells, and on the human cloned 5-HT_1D receptor expressed in C6 glioma cells (B_max 780 fmol mg⁻¹ protein).
2. Sodium butyrate treatment increased the expression level of human (h)5-HT_1B receptors in HEK 293 cells and h5-HT_1D receptors in C6 glioma cells approximately 3 fold, the binding affinities of [³H]-5-HT and [³H]-alniditan were unaffected.
3. Agonistic properties were evaluated based on inhibition of cyclic AMP accumulation in the cells after stimulation of adenylyl cyclase by forskolin or isoproterenol. Alniditan, sumatriptan and dihydroergotamine were full agonists at the h5-HT_1B receptor (IC₅₀ values were 1.7, 20 and 2 nM, respectively in HEK 293 cells) and h5-HT_1D receptors (IC₅₀ values of 1.3, 2.6 and 2.2 nM, respectively). At the h5-HT_1B receptor the agonist potency of the compounds slightly increased with higher receptor density. The opposite was seen for antagonists (ocaperidone, risperidone and ritanserin).
4. This comparative study demonstrated that alniditan was 10 times more potent than sumatriptan at the h5-HT_1B receptor, and twice as potent at the h5-HT_1D receptor. Dihydroergotamine was more potent an agonist at the h5-HT_1B receptor when expressed at high and low level in L929sA cells (but not in HEK 293 cells), and was less potent at the h5-HT_1D receptor.

     

Characterization of 5-HT receptors mediating constriction of porcine carotid arteriovenous anastomoses; involvement of 5-HT1B/1D and novel receptors

1. It was previously shown that porcine cranial arteriovenous anastomoses (AVAs) constrict to 5-hydroxytryptamine (5-HT), ergotamine, dihydroergotamine, as well as sumatriptan and that sumatriptan acts exclusively via 5-HT_1B/1D receptors. The present study was devoted to establish the contribution of 5-HT_1B/1D receptors in the constriction of AVAs elicited by 5-HT (in presence of 0.5 mg kg⁻¹ ketanserin), ergotamine and dihydroergotamine in anaesthetized pigs.
2. Intracarotid infusion of 5-HT (2 μg kg⁻¹ min⁻¹) and intravenous doses of ergotamine (2.5–20 μg kg⁻¹) and dihydroergotamine (3–100 μg kg⁻¹) reduced AVA and increased nutrient blood flows and vascular conductances. The vasodilator response to 5-HT, observed mainly in the skin and ear, was much more prominent than that of the ergot alkaloids.
3. Treatment with the 5-HT_1B/1D receptor antagonist {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (0.5 mg kg⁻¹, i.v.) significantly attenuated both ergot-induced AVA constriction and arteriolar dilatation, whereas {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 only slightly affected the carotid vascular effects of 5-HT.
4. The results suggest that 5-HT constricts carotid AVAs primarily via receptors, which seem to differ from those (5-HT_1B/1D) stimulated by sumatriptan. The ergot alkaloids produce AVA constriction for a substantial part via 5-HT_1B/1D receptors, but also stimulate unidentified receptors. Both these non-5-HT_1B/1D receptors may be targets for the development of novel antimigraine drugs.
5. The moderate vasodilator response to the ergot derivatives seems to be mediated, at least in part, by 5-HT_1B/1D receptors, whereas the arteriolar dilatation caused by 5-HT may be mediated by other, possibly 5-HT₇ receptors.

     

The 5-HT4 receptor antagonist ML10375 inhibits the constitutive activity of human 5-HT4(c) receptor

Transient expression in COS-7 cells of the recombinant human 5-hydroxytryptamine (5-HT) h5-HT_4(c) receptor isoform led to constitutive activity of the receptor. The 5-HT₄ receptor antagonist 2-(cis-3,5-dimethylpiperidino)ethyl 4-amino-5-chloro-2-methoxybenzoate (ML10375) at 1 μM completely abolished the 5-HT (1 μM)-mediated increase in adenylyl cyclase activity in COS-7 cells expressing the h5-HT_4(c) receptor. Moreover, ML10375 also reduced basal cAMP levels in cells over-expressing the receptor, even in the absence of agonist. The inhibitory effect of ML10375 on basal adenylyl cyclase activity was not modified by pre-treatment of the cells with pertussis toxin, indicating that ML10375 acts through inactivation of spontaneously active h5-HT_4(c) receptors rather than through a G_i/G_o regulatory pathway. We conclude that ML10375 acts as an inverse agonist on the h5-HT_4(c) receptor.     

Modulation by 5-HT1A receptors of the 5-HT2 receptor-mediated tachykinin-induced contraction of the rat trachea in vitro

1. In the Fisher 344 rat, tachykinins have been shown to cause the release of 5-hydroxytryptamine (5-HT) from airway mast cells, which then causes direct smooth muscle activation as well as the release of acetylcholine from cholinergic nerves. The aim of the present study was to examine the modulatory effects of 5-HT receptors on the neurokinin A (NKA)-induced release of endogenous 5-HT and airway smooth muscle contraction in the isolated Fisher 344 rat trachea.
2. The selective 5-HT₂ receptor antagonist ketanserin (0.1 μM) produced an almost complete inhibition of the contractions caused by NKA (n=4, P<0.0001, two-way ANOVA), and a significant rightward shift of the concentration-response curve to 5-HT (n=8, P<0.001, two-way ANOVA).
3. The partial agonist for 5-HT_1A receptors, 8-OH-DPAT (1 μM), and the full agonist for 5-HT₁ receptors, 5-CT (0.3 μM), potentiated the submaximal contractions induced by the 5-HT₂ receptor agonist α-methyl-5-HT (0.1 μM) (n=4; P<0.005 and P<0.05, respectively). 8-OH-DPAT (1 μM), as well as the 5-HT_1A receptor antagonists pMPPI, SDZ 216525 and NAN-190 (0.1 μM each), caused significant inhibition of the tracheal contractions induced both by NKA (10  nM–3  μM) and 5-HT (10 nM–10 μM) (n=4–10). This suggests that activation of 5-HT_1A receptors potentiates the 5-HT₂ receptor-mediated contractions.
4. SDZ 216525 (0.1 μM) significantly reduced the maximal contraction produced by 1 μM NKA (n=10, P<0.001), without affecting the release of endogenous 5-HT. These data rule out the involvement of a 5-HT_1A receptor-mediated positive feedback mechanism of the 5-HT release from mast cells.
5. Even in the presence of atropine (1 μM), 8-OH-DPAT (1 μM) further reduced the maximal NKA-induced contraction (n=4, P<0.0001), while the contractions of the rat isolated trachea induced by electrical field stimulation and the concentration-response curve to carbachol were unaffected by pMPPI (0.1 μM), SDZ 216525 (0.1 μM), NAN-190 (0.1 μM) and 8-OH-DPAT (1 μM) (n=4–6). These data demonstrate that the 5-HT_1A receptor-mediated potentiation of contractile responses is not due to non-specific inhibition of airway smooth muscle contraction or to modulation of postganglionic nerve activation.
6. The selective 5-HT_1B/1D receptor antagonist GR 127935, the selective 5-HT₃ receptor antagonist tropisetron and the selective 5-HT₄ receptor antagonists SB 204070 and GR 113808 (0.1 μM each) had no effect on the concentration-response curve for NKA (n=6–10), ruling out the involvement of 5-HT_1B/1D, 5-HT₃ and 5-HT₄ receptors.
7. The α-adrenoreceptor antagonist phentolamine (1 μM) had no effect on the 5-HT-induced contractions (n=4), ruling out the involvement of α-adrenoreceptors.
8. In conclusion, the tachykinin-induced contraction of the F334 rat isolated trachea is mediated by the stimulation of 5-HT₂ receptors. Activation of 5-HT_1A receptors located on airway smooth muscle potentiates the direct contractile effects of 5-HT₂ receptor activation. The 5-HT_1B/1D, 5-HT₃ and 5-HT₄ receptors are not involved in the NKA-induced contraction of rat airways.

     

Recombinant saphenous vein 5-HT1B receptors of the rabbit: comparative pharmacology with human 5-HT1B receptors

1. The rabbit recombinant saphenous vein 5-hydroxytryptamine_1B (rb 5-HT_1B) receptor stably transfected in rat C6-glial cells was characterized by measuring adenosine 3′:5′-cyclic monophosphate (cyclic AMP) formation upon exposure to various 5-HT receptor ligands. The effects of agonists and antagonists were compared with their effects determined previously at the human cloned 5-HT_1B (h 5-HT_1B) receptor under similar experimental conditions.
2. Intact C6-glial cells expressing rb 5-HT_1B receptors exhibited [³H]-5-carboxamidotryptamine (5-CT) binding sites with a K_d of 0.80±0.13 nM and a B_max between 225 to 570 fmol mg⁻¹ protein. The binding affinities of a series of 5-HT receptor ligands determined in a membrane preparation with [³H]-5-CT or [³H]-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide (GR 125,743) were similar. With the exception of ketanserin, ligand affinities were comparable to those determined at the cloned h 5-HT_1B receptor site.
3. rb 5-HT_1B receptors were negatively coupled to cyclic AMP formation upon stimulation with 5-HT agonists. Of the several 5-HT agonists tested, 5-CT was the most potent, the potency rank order being: 5-CT>5-HT>zolmitriptan>naratriptan>rizatriptan>sumatriptan>R(+)-8-(hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The maximal responses of these agonists were similar to those induced by 5-HT. The potency of these agonists showed a positive correlation (r²=0.87; P<0.002) with their potency at the cloned h 5-HT_1B receptor subtype.
4. 2′-Methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide (GR 127,935), methiothepin and ketanserin each behaved as silent, competitive antagonists at rb 5-HT_1B receptors; pK_B values were 8.41, 8.32 and 7.05, respectively when naratriptan was used as an agonist. These estimates accorded with their binding affinities and the potencies found on 5-HT and/or sumatriptan-mediated contraction of isolated rabbit saphenous vein segments.
5. In conclusion, the recombinant saphenous vein 5-HT_1B receptor of the rabbit shares important pharmacological similarities with the cloned h 5-HT_1B receptor. However, ketanserin is a more potent antagonist of rb 5-HT_1B receptors.

     

Comparative desensitization of the human 5-HT2A and 5-HT2C receptors expressed in the human neuroblastoma cell line SH-SY5Y

1. We have used previously characterized clones of the human neuroblastoma cell line, SH-SY5Y, constitutively expressing either the human 5-HT_2A or 5-HT_2C receptor to compare their desensitization profiles after exposure to 5-HT.
2. 5-HT stimulated [³H]-inositol phosphate ([³H]-IP_x) production at both the 5-HT_2C (pEC₅₀=8.03±0.15) and 5-HT_2A receptors (pEC₅₀=7.15±0.08), with maximal responses occurring after exposure to 1 μM and 10 μM 5-HT, respectively.
3. Exposure of cells to maximally effective concentrations of 5-HT caused time- and concentration-dependent desensitization of [³H]-IP_x formation. The 5-HT_2A response desensitized slower (t_1/2=110 min) and with lower sensitivity than that of the 5-HT_2C receptor (t_1/2=12.5 min). In each case, desensitization was blocked by co-administration of a specific receptor antagonist. Following exposure to 10 μM 5-HT for 2 h, both receptors exhibited extensive desensitization, with subsequent responses to 5-HT reduced by more than 80%.
4. 5-HT stimulated Ins(1,4,5)P₃ production with a potency similar to that for [³H]-IP_x production at each receptor. In both cases Ins(1,4,5)P₃ levels peaked rapidly then returned to basal level within a short time. This peak consistently occurred earlier for the 5-HT_2C receptor (5 s) than for the 5-HT_2A receptor (20 s).
5. Prior exposure of SH-SY5Y/5-HT_2C cells to 5-HT (1 μM/15 min) caused a significant decrease in the 5-HT-stimulated peak in Ins(1,4,5)P₃ levels whereas no such change occurred in SH-SY5Y/5-HT_2A cells following exposure to 10 μM 5-HT for 15 min.
6. These results indicate that the human 5-HT_2A and 5-HT_2C receptors both exhibit desensitization at the level of inositol phosphate formation when expressed in the same cellular environment, with the 5-HT_2C receptor being more sensitive to 5-HT-mediated desensitization than the 5-HT_2A receptor.

     

5-HT1B receptor-mediated contractions in human temporal artery: evidence from selective antagonists and 5-HT receptor mRNA expression

1. In the human temporal artery both 5-HT_1-like and 5-HT_2A receptors mediate the contractile effects of 5-hydroxytryptamine (5-HT) and we have suggested that the 5-HT_1-like receptors resemble more closely recombinant 5-HT_1B than 5-HT_1D receptors. To investigate further which subtype is involved, we investigated the blockade of 5-HT-induced contractions by the 5-HT_1B-selective antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2-methyl-4′[(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-yl] carbonyl} furo[2,3-f]indole-3-spiro-4′-piperidine oxalate) and the 5-HT_1D-selective antagonist BRL-15572 (1-phenyl-3[4-3-chlorophenyl piperazin-1-yl] phenylpropan-2-ol). We also used RT-PCR to search for the mRNA of 5-HT_1B, 5-HT_1D and other 5-HT receptors.
2. The contractile effects of 5-HT in temporal artery rings were partially antagonized by SB-224289 (20, 200 nM) (apparent K_B=1 nM) and ketanserin (1 μM) but not by BRL-15572 (500 nM).
3. Sumatriptan evoked contractions (EC₅₀, 170 nM) that were resistant to blockade by BRL-15572 (500 nM) but antagonized by SB-224289 (20, 200 nM).
4. The potency of 5-HT (EC₅₀) was estimated to be 94 nM for the ketanserin-sensitive receptor and 34 nM for the SB-224289-sensitive receptor. The fraction of maximal 5-HT response mediated through SB-224289-sensitive receptors was 0.20–0.67, the remainder being mediated through ketanserin-sensitive receptors.
5. We detected arterial receptor mRNA for the following receptors (incidence): 5-HT_1B (8/8), 5-HT_1D (2/8), 5-HT_1F (0/4), 5-HT_2A (0/8), 5-HT_2B (0/8), 5-HT_2C (0/8), 5-HT₄ (4/8) and 5-HT₇ (4/8).
6. We conclude that the ketanserin-resistant fraction of the 5-HT effects and the effects of sumatriptan are mediated by 5-HT_1B receptors. The lack of antagonism by BRL-15572 rules out 5-HT_1D receptors as mediators of the contractile effects of 5-HT and sumatriptan.

     

Involvement of 5-HT6 receptors in nigro-striatal function in rodents

1. 4-Amino-N-(2,4 bis-methylamino-pyrimidin-4-yl) benzene sulphonamide (Ro 04-6790) is a potent, selective and competitive antagonist for the 5-HT₆ receptor which can be detected in the cerebro-spinal fluid (CSF) of rats following intraperitoneal administration. Since 5-HT₆ receptor mRNA and 5-HT₆ receptor-like immunoreactivity have been shown to be present in the striatum, the purpose of the present study was to evaluate the effect of 5-HT₆ receptor antagonism on haloperidol- and SCH 23390-induced catalepsy in mice and on the turning behaviour of rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle.
2. Ro 04-6790 (3, 10 and 30 mg kg⁻¹ i.p.) did not induce catalepsy and had no effect on catalepsy induced by either haloperidol or SCH 23390.
3. Ro 04-6790 (3, 10 and 30 mg kg⁻¹ i.p.) did not itself induce rotational behaviour in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle nor did it affect the rotational behaviour induced by either L-Dopa or amphetamine.
4. 5-HT₆ receptor antagonism inhibited the rotational behaviour of 6-OHDA lesioned rats induced by treatment with the muscarinic antagonists scopolamine and atropine.
5. The data support earlier conclusions from experiments with antisense oligonucleotides that the 5-HT₆ receptor is involved in the control of acetylcholine neurotransmission in the rat brain.

     

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

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