Systemic morphine produce antinociception mediated by spinal 5-HT7, but not 5-HT1A and 5-HT2 receptors in the spinal cord

BACKGROUND AND PURPOSE: The serotonergic system within the spinal cord have been proposed to play an important role in the analgesic effects of systemic morphine. Currently, seven groups of 5-HT receptors (5-HT1-7) have been characterized. One of the most recently identified subtypes of 5 HT receptor is the 5-HT7 receptor. We aimed to examine the role of spinal 5-HT7 receptors in the antinociceptive effects of systemic morphine. EXPERIMENTAL APPROACH: The involvement of spinal 5-HT7 receptor in systemic morphine antinociception was compared to that of the 5-HT1A and 5-HT2 receptors by using the selective 5-HT7 receptor antagonist, SB-269970, the selective 5-HT1A receptor antagonist, WAY 100635, the selective 5-HT2 antagonist ketanserin as well as the non-selective 5-HT1,2,7 receptor antagonist, metergoline. Nociception was evaluated by the radiant heat tail-flick test. KEY RESULTS: I.t. administration of SB-269970 (10 microg) and metergoline (20 microg) completely blocked the s.c. administered morphine-induced (1, 3, 5 and 10 mg kg(-1)) antinociception in a time-dependent manner. Additionally, i.t. administration of SB-269970 (1, 3, 10 and 20 microg) and metergoline (1, 5, 10 and 20 microg) dose dependently inhibited the antinociceptive effects of a maximal dose of morphine (10 mg kg(-1), s.c.). I.t. administration of WAY 100635 (20 microg) or ketanserine (20 microg) did not alter morphine-induced (1, 3, 5 and 10 mg kg(-1), s.c.) antinociception. CONCLUSION AND IMPLICATIONS: These findings indicate that the involvement of spinal 5-HT7, but not of 5-HT1A or of 5-HT2 receptors in the antinociceptive effects of systemic morphine.     

Delta(9)-tetrahydrocannabinol prolongs the immobility time in the mouse forced swim test: involvement of cannabinoid CB(1) receptor and serotonergic system

In the present study, we investigated the effect of Delta(9)-tetrahydrocannabinol (THC), the principal psychoactive component of marijuana, on immobility time during the forced swim test. THC (2 and 6 mg/kg, i.p.) significantly prolonged the immobility time. In addition, THC at the same doses did not significantly affect locomotor activity in the open-field test. The selective cannabinoid CB(1) receptor antagonist rimonabant (3 mg/kg, i.p.) significantly reduced the enhancement of immobility by THC (6 mg/kg). Similarly, the selective serotonin (5-HT) reuptake inhibitor (SSRI) citalopram (10 mg/kg, i.p.) and 5-HT(1A/7) receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 0.3 mg/kg, i.p.) significantly reduced this THC-induced effect. Moreover, the selective 5-HT(1A) receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide dihydrochloride (WAY100635, 1 mg/kg, i.p.) and the postsynaptic 5-HT(1A) receptor antagonist MM-77 (0.1 mg/kg, i.p.) reversed this reduction effect of 8-OH-DPAT (0.3 mg/kg). In contrast, the selective 5-HT(7) receptor antagonist (R)-3-[2-[2-(4-methylpiperidin-1-yl)ethyl]pyrrolidine-1-sulfonyl]phenol hydrochloride (SB269970) had no effect on this reduction effect of 8-OH-DPAT. WAY100635 (1 mg/kg) also reversed the reduction effect of citalopram (10 mg/kg). These findings suggest that the 5-HT(1A) receptors are involved in THC-induced enhancement of immobility.     

The effect of SB-269970, a 5-HT(7) receptor antagonist, on 5-HT release from serotonergic terminals and cell bodies

1. The presence of 5-HT(7) receptor mRNA and protein in 5-HT neurons suggests that this receptor may act as a 5-HT autoreceptor. In this study, the effect of the 5-HT(7) receptor antagonist, SB-269970 ((R)-1-[3-hydroxy phenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine), was investigated on 5-HT release in the guinea-pig and rat cortex and the rat dorsal raphe nucleus (DRN), using the techniques of in vitro [(3)H]-5-HT release or fast cyclic voltammetry, respectively. 2. Cortical slices were loaded with [(3)H]-5-HT and release was evoked by electrical stimulation. 5-CT inhibited the evoked release of [(3)H]-5-HT in a concentration-dependent manner. SB-269970 had no significant effect on [(3)H]-5-HT release while the 5-HT(1B) receptor antagonist, SB-224289 significantly potentiated [(3)H]-5-HT release. In addition, SB-269970 was unable to attenuate the 5-CT-induced inhibition of release while SB-224289 produced a rightward shift of the 5-CT response, generating estimated pK(B) values of 7.8 and 7.6 at the guinea-pig and rat terminal 5-HT autoreceptors respectively. 3. Rat DRN slices were electrically stimulated and the evoked 5-HT efflux detected by voltammetric analysis. 8-OH-DPAT inhibited evoked 5-HT efflux and was fully reversed by WAY 100635. SB-269970 had no effect on either 5-HT efflux per se or 8-OH-DPAT-induced inhibition of 5-HT efflux. In addition, 5-CT inhibited 5-HT efflux in a concentration-dependent manner. SB-269970 was unable to attenuate the 5-CT-induced inhibition of 5-HT efflux. 4. In conclusion, we were unable to provide evidence to suggest a 5-HT autoreceptor role for 5-HT(7) receptors. However, investigations with more selective 5-HT(7) receptor agonists are needed to confirm the data reported here.     

GABAergic modulation of 5-HT7 receptor-mediated effects on 5-HT efflux in the guinea-pig dorsal raphe nucleus

5-HT(7) receptor mRNA and protein are localised in the dorsal raphe nucleus (DRN) on non-serotonergic neurones. The effect of 5-HT(7) receptor antagonism on 5-HT efflux was measured from guinea-pig DRN slices, using the technique of fast cyclic voltammetry. The 5-HT(7) receptor antagonist, SB-269970-A, significantly inhibited 5-HT efflux. The GABA(A) receptor agonist, muscimol, significantly inhibited 5-HT efflux, to a similar degree as SB-269970-A. In contrast, the GABA(A) receptor antagonist, bicuculline, significantly increased 5-HT efflux and attenuated the muscimol-induced inhibition. The muscimol and SB-269970-A effects were not additive and in the presence of bicuculline the SB-269970-A-induced inhibition of 5-HT efflux was attenuated. These data suggest that 5-HT(7) receptor antagonist-induced inhibition of 5-HT efflux occurs indirectly via activation of GABA(A) receptors. That is, 5-HT(7) receptors may be located on GABA interneurones and when activated decrease GABA release and hence decrease the inhibitory tone on 5-HT neurones, increasing 5-HT efflux in the DRN. Therefore, in the presence of GABAergic tone 5-HT(7) receptor antagonists would decrease 5-HT release from the DRN.     

5-Hydroxytryptamine potentiates neurogenic contractions of rat isolated urinary bladder through both 5-HT(7) and 5-HT(2C) receptors

Serotonin (5-HT) enhances the neurogenic contractile response induced by electrical field stimulation (EFS) in the rat isolated urinary bladder. The aim of this study was to functionally characterize the receptors involved in this effect by using a range of 5-HT receptor subtype selective agonists and antagonists. 5-HT produced a concentration-dependent potentiation of contractile responses to EFS with a pEC₅₀ value of 6.86 ± 0.24. SB-269970 (0.01, 0.1 and 1 μM), a selective 5-HT₇ receptor antagonist, caused a concentration-dependent rightward shift of the 5-HT-induced response. The pA₂ value was 8.16 with a slope of 0.46 ± 0.08. Neither ketanserine nor SB-204741, 5-HT_2A and 5-HT_2B receptors antagonists, respectively, affected the concentration–response curve to 5-HT. However, 5-HT response was antagonized by the selective 5-HT_2C receptor antagonist SB-242084 (0.1 and 1 μM). In the presence of 1 μM of both antagonists SB-269970 and SB-242084, 5-HT response was almost fully inhibited. 5-CT, a 5-HT₇ receptor agonist, induced a biphasic concentration-dependent potentiation of neurogenic contractions. SB-269970 concentration-dependently antagonized the first phase of 5-CT response with a pA₂ value of 8.77 and a slope not significantly different from unity (0.91 ± 0.11) that suggests a competitive antagonism. WAY-161503, a 5-HT_2C receptor agonist (0.01–10 μM), induced a concentration-dependent potentiation of contractile response to EFS while DOI (a selective 5-HT_2A agonist) had no effect. SB-242084 (0.1 and 1 μM) antagonized the effect of WAY-161503 in a concentration-dependent manner. The current results demonstrate that 5-HT potentiates neurogenic contractions of rat isolated detrusor muscle through both 5-HT₇ and 5-HT_2c receptors.     

The hypothermic effect of 5-CT in mice is mediated through the 5-HT7 receptor

The 5-HT(7) receptor is a recent addition to the 5-HT receptor family and to date there is no clear idea as to its potential role in the CNS. The receptor has been mapped by in situ hybridization and 5-HT(7)-like immunoreactivity and has been detected in discrete areas of the brain including the hypothalamus (Oliver et al., 1999). This suggests the receptor may be involved in temperature regulation and have shown that a selective 5-HT(7) receptor antagonist reverses the hypothermic effect of 5-CT in guinea-pigs. The current study confirmed that the 5-HT(7) receptor antagonists, SB-269970 (1-30 mg/kg, i.p.) and SB-258719 (5-20 mg/kg, i.p.), but not the 5-HT(1A) receptor antagonist, WAY 100635(0.1-1 mg/kg, s.c.), or the 5-HT(1B/D) antagonist, GR127935 (1.25-5 mg/kg, i.p.), reversed the hypothermic effect of 5-CT in mice. In addition the effect of 5-CT on body temperature was examined on 5-HT(7) receptor null mutant mice. 5-CT (0.1-1 mg/kg, i.p.) significantly reduced rectal temperature in wildtype but not 5-HT(7) receptor knockout mice. This suggests that the hypothermic effects of 5-CT are mediated through the 5-HT(7) receptor. All procedures were carried out in accordance with the UK Animals (Scientific Procedures) Act (1986).     

Enhancement of 5-HT1B and 5-HT1D receptor antagonist effects on extracellular 5-HT levels in the guinea-pig brain following concurrent 5-HT1A or 5-HT re-uptake site blockade.

The effects of selective serotonin re-uptake inhibitor (SSRI), paroxetine, and 5-HT1A, 5-HT1B and 5-HT1B/1D receptor antagonists on in vivo extracellular 5-HT levels in the guinea-pig frontal cortex and dorsal hippocampus were investigated using the technique of microdialysis. The aim of the study was to further investigate the autoreceptor roles of the 5-HT1A, 5-HT1B and 5-HT1D receptors in the median vs dorsal raphe nuclei. In the frontal cortex, 5-HT1A (WAY 100635, 1 mg/kg i.p.) or 5-HT1B (SB-224289, 4 mg/kg i.p.) receptor antagonists had no effect on extracellular levels of 5-HT, whilst the mixed 5-HT1B/1D receptor antagonist (GR 127935, 0.3 mg/kg i.p) produced a significant decrease in extracellular 5-HT levels. Paroxetine (10 microM) significantly increased extracellular 5-HT levels when perfused locally into the cortex. Administration of SB-224289, followed 120 min later by WAY 100635, had no effect on extracellular 5-HT levels. In contrast, sequential administration of either WAY 100635 and GR 127935, or SB-224289 and paroxetine significantly increased extracellular 5-HT levels. In the dorsal hippocampus, whilst 5-HT1A receptor antagonism elicited by administration of WAY 100635 had no effect, both 5-HT1B and mixed 5-HT1B/1D receptor blockade significantly increased extracellular 5-HT levels. Administration of SB-224289 followed 120 min later with WAY 100635, or WAY 100635 followed 30 min later with GR 127935, potentiated the effect of the three compounds alone, significantly increasing extracellular 5-HT levels. These data demonstrate that to simultaneously increase extracellular 5-HT in both frontal cortex and dorsal hippocampus of the guinea-pig brain concurrent 5-HTA1A, 5-HT1B and 5-HT1D receptor blockade is required. Whereas in the dorsal hippocampus, 5-HT1B receptor blockade is sufficient to elicit an increase in extracellular 5-HT levels.     

Evidence for the involvement of central 5-HT7 receptors in the micturition reflex in anaesthetized female rats

(1) The effects of the selective 5-HT7 receptor antagonists SB-269970 (3-300 microg kg-1; n=5-6) and SB-656104 (30 microg kg-1; n=5) administered centrally (i.c.v.) were investigated on the 'micturition reflex' in the urethane anaesthetized female rat. (2) In cystometric recordings, SB-269970 caused significant increases in volume of 58+/-15 and 138+/-33% and pressure of 140+/-46 and 149+/-60% thresholds at 10 and 30 microg kg-1. These changes were associated with significant decreases in distension-induced bladder contraction of 62+/-14 and 60+/-11%, respectively. However, there was no change in residual volume. At the higher doses, SB-269970 blocked the micturition reflex. SB-656104 had similar effects to SB-269970 but in addition significantly increased the residual volume. (3) SB-269970 (10 microg kg-1; n=5) given i.v. had no effect on the micturition reflex. (4) SB-269970 (30 microg kg-1; n=4) given intrathecally (i.t.) had no effect on micturition reflex, although the selective 5-HT1A receptor antagonist WAY-100635 given i.t. after SB-269970 caused a significant increase in the volume threshold. (5) Using an isovolumetric method in which urethral changes were measured, SB-269970 (30 microg kg-1; n=4; i.c.v.) failed to have any effect on these urethral-evoked changes although they significantly reduced the amplitude of the bladder contraction. (6) These data demonstrate that 5-HT7 receptors located supraspinally in the rat are involved in the control of micturition.     

5-HT7 receptor subtype as a mediator of the serotonergic regulation of luteinizing hormone release in the zona incerta

5-Hydroxytryptamine (5-HT) and the 5-HT(1A/7) receptor agonist (+)-8-hydroxy-2-(di-n-propylamino) tetralinHBr (8-OH-DPAT), injected into the zona incerta (an area in the dorsal hypothalamus) of the female rat, inhibit the release of luteinizing hormone (LH) and the effects of both are blocked by the 5-HT(2/7) receptor antagonist, ritanserin. As both 8-OH-DPAT and ritanserin have moderate activity at the 5-HT7 receptor subtype, the possibility that this subtype might mediate their effects in the zona incerta has been investigated. Ovariectomised rats were primed with 5 microg oestradiol benzoate followed at 48 h by 0.5 mg progesterone, which induces an LH surge. 5-Carboxamidotryptamine (5-CT), a potent but non-selective agonist at 5-HT7 receptors, like 5-HT and 8-OH-DPAT, inhibited the LH surge at 5 and 1.25 nmol injected bilaterally into the zona incerta. The non-selective 5-HT(2/7) receptor antagonist ritanserin and the selective 5-HT7 receptor antagonist, (R)-3-(2-(2-(4-methyl-piperidin-1-yl)-pyrrolidine-1-sulfonyl)-phenol (SB-269970-A) at 0.5 microg/side blocked all three receptor agonists when injected concurrently into the zona incerta. However, lower (0.2 microg) and higher doses (2 and 5 microg) of SB-269970-A were less effective, indicating a bell-shaped dose-response curve. SB-269970-A was also inhibitory when administered systemically (1 mg/kg intraperitoneally (i.p.)). When LH release was suppressed by 5 microg oestradiol benzoate, SB-269970-A (0.5 and 2 microg) did not elevate levels, indicating it is unlikely that 5-HT7 receptors mediate a tonic inhibition on release but rather are involved in terminating the pre-ovulatory LH surge. These data demonstrate that 5-HT7 receptors play a role in the regulation of LH by the zona incerta in rat brain.     

Role of uptake inhibition and autoreceptor activation in the control of 5-HT release in the frontal cortex and dorsal hippocampus of the rat

1. Using brain microdialysis, we compared the relative role of 5-hydroxytryptamine (5-HT; serotonin) blockade and somatodendritic 5-HT(1A) and/or terminal 5-HT(1B) autoreceptor activation in the control of 5-HT output. 2. Fluoxetine (10 mg kg(-1) i.p.) doubled the 5-HT output in frontal cortex and dorsal hippocampus. The 5-HT(1A) receptor antagonist WAY 100635, (0.3 mg kg(-1) s.c.) potentiated the effect of fluoxetine only in frontal cortex (to approximately 500 % of baseline). 3. Methiothepin (10 mg kg(-1) s.c.) further enhanced the 5-HT rise induced by fluoxetine+WAY 100635, to 835+/-179% in frontal cortex and 456+/-24% in dorsal hippocampus. Locally applied, methiothepin potentiated the fluoxetine-induced 5-HT rise more in the former area. 4. The selective 5-HT(1B) receptor antagonist SB-224289 (4 mg kg(-1) i.p.) enhanced the effect of fluoxetine (10 mg kg(-1) i.p.) in both areas. As with methiothepin, SB-224289 (4 mg kg(-1) i.p.) further enhanced the 5-HT increase produced by fluoxetine+WAY 100635 more in frontal cortex (613+/-134%) than in dorsal hippocampus (353+/-59%). 5. Locally applied, fluoxetine (10 - 300 microM; EC(50)=28 - 29 microM) and citalopram (1 - 30 microM; EC(50)=1.0 - 1.4 microM) increased the 5-HT output two to three times more in frontal cortex than in dorsal hippocampus. These data suggest that the comparable 5-HT increase produced by systemic fluoxetine in frontal cortex and dorsal hippocampus results from a greater effect of reuptake blockade in frontal cortex that is offset by a greater autoreceptor-mediated inhibition of 5-HT release. As a result, 5-HT autoreceptor antagonists preferentially potentiate the effect of fluoxetine in frontal cortex.     

SB-699551-A (3-cyclopentyl-N-[2-(dimethylamino)ethyl]-N-[(4'-{[(2-phenylethyl)amino]methyl}-4-biphenylyl)methyl]propanamide dihydrochloride), a novel 5-ht5A receptor-selective antagonist, enhances 5-HT neuronal function: Evidence for an autoreceptor role for the 5-ht5A receptor in guinea pig brain

This study utilised the selective 5-ht(5A) receptor antagonist, SB-699551-A (3-cyclopentyl-N-[2-(dimethylamino)ethyl]-N-[(4'-{[(2-phenylethyl)amino]methyl}-4-biphenylyl)methyl]propanamide dihydrochloride), to investigate 5-ht5A receptor function in guinea pig brain. SB-699551-A competitively antagonised 5-HT-stimulated [35S]GTPgammaS binding to membranes from human embryonic kidney (HEK293) cells transiently expressing the guinea pig 5-ht5A receptor (pA2 8.1+/-0.1) and displayed 100-fold selectivity versus the serotonin transporter and those 5-HT receptor subtypes (5-HT(1A/B/D), 5-HT2A/C and 5-HT7) reported to modulate central 5-HT neurotransmission in the guinea pig. In guinea pig dorsal raphe slices, SB-699551-A (1 microM) did not alter neuronal firing per se but attenuated the 5-CT-induced depression in serotonergic neuronal firing in a subpopulation of cells insensitive to the 5-HT1A receptor-selective antagonist WAY-100635 (100 nM). In contrast, SB-699551-A (100 or 300 nM) failed to affect both electrically-evoked 5-HT release and 5-CT-induced inhibition of evoked release measured using fast cyclic voltammetry in vitro. SB-699551-A (0.3, 1 and 3 mg/kg s.c.) did not modulate extracellular levels of 5-HT in the guinea pig frontal cortex in vivo. However, when administered in combination with WAY-100635 (0.3 mg/kg s.c.), SB-699551-A (0.3, 1 or 3 mg/kg s.c.) produced a significant increase in extracellular 5-HT levels. These studies provide evidence for an autoreceptor role for the 5-ht5A receptor in guinea pig brain.     

Autoradiographic characterisation of [35S]GTPgammaS binding stimulation mediated by 5-HT1B receptor in postmortem human brain

G-protein activation mediated by 5-HT1B receptors was studied in human brain by [35S]GTPgammaS autoradiographic methods. 5-HT (10 microM) increased [35S]GTPgammaS binding in caudate-putamen nucleus, globus pallidus, dentate gyrus, CA1, entorhinal cortex and substantia nigra. In basal ganglia and midbrain, this effect was blocked by GR 127935 (5-HT(1B/1D) antagonist). In contrast, WAY 100635 (selective 5-HT1A antagonist) reversed the effect of 5-HT in hippocampus and entorhinal cortex. Therefore, a detailed pharmacological study was carried out in basal ganglia and substantia nigra using 5-HT and the 5-HT(1B/1D) agonists GTI and CP 93129. In these areas, these agonists stimulated [35S]GTPgammaS binding in a concentration-dependent manner, with no significant differences in the potency for a given structure. Furthermore, GTI was more potent in the putamen than in globus pallidus. In caudate-putamen, the three agonists showed the same efficacy, while in globus pallidus and substantia nigra the efficacy of 5-HT was higher than GTI and CP 93129. The selective 5-HT1B antagonist SB-224289 inhibited GTI- and CP 93129-stimulated [35S]GTPgammaS binding in basal ganglia and substantia nigra, while coincubation with BRL 15572 (selective 5-HT1D antagonist) did not result in any significant change. Here we report the anatomical pattern of distribution of 5-HT1B-dependent functionality by using specific pharmacological tools in human brain sections.     

In vivo evidence that 5-HT(2C) receptors inhibit 5-HT neuronal activity via a GABAergic mechanism

BACKGROUND AND PURPOSE: Recent evidence suggests that 5-HT(2C) receptor activation may inhibit midbrain 5-HT neurones by activating neighbouring GABA neurones. This hypothesis was tested using the putative selective 5-HT(2C) receptor agonist, WAY 161503. EXPERIMENTAL APPROACH: The effect of WAY 161503 on 5-HT cell firing in the dorsal raphe nucleus (DRN) was investigated in anaesthetised rats using single unit extracellular recordings. The effect of WAY 161503 on DRN GABA neurones was investigated using double label immunohistochemical measurements of Fos, glutamate decarboxylase (GAD) and 5-HT(2C) receptors. Finally, drug occupancy at 5-HT(2A) receptors was investigated using rat positron emission tomography and ex vivo binding studies with the 5-HT(2A) receptor radioligand [(11)C]MDL 100907. KEY RESULTS: WAY 161503 caused a dose-related inhibition of 5-HT cell firing which was reversed by the 5-HT(2) receptor antagonist ritanserin and the 5-HT(2C) receptor antagonist SB 242084 but not by the 5-HT(1A) receptor antagonist WAY 100635. SB 242084 pretreatment also prevented the response to WAY 161503. The blocking effects of SB 242084 likely involved 5-HT(2C) receptors because the drug did not demonstrate 5-HT(2A) receptor occupancy in vivo or ex vivo. The inhibition of 5-HT cell firing induced by WAY 161503 was partially reversed by the GABA(A) receptor antagonist picrotoxin. Also, WAY 161503 increased Fos expression in GAD positive DRN neurones and DRN GAD positive neurones expressed 5-HT(2C) receptor immunoreactivity. CONCLUSIONS AND IMPLICATIONS: These findings indicate that WAY 161503 inhibits 5-HT cell firing in the DRN in vivo, and support a mechanism involving 5-HT(2C) receptor-mediated activation of DRN GABA neurones.     

WAY100635 prevents the changes induced by fluoxetine upon the 5-HT1A receptor functionality

5-HT1A receptor-mediated signalling in rat brain was evaluated after chronic administration (14 days; s.c.) of the selective serotonin reuptake inhibitor (SRRI) fluoxetine (10 mg/kg/day) alone, or in combination with the 5-HT1A receptor antagonist WAY100635 (0.1 mg/kg/day). The density of 5-HT1A binding sites was unchanged following fluoxetine, WAY100635, or the combination of fluoxetine and WAY100635. However, the net stimulation of [35S]GTPgammaS binding induced by the 5-HT1A agonist 8-OH-DPAT was significantly attenuated in dorsal raphe nucleus (DRN), but not in hippocampus, after chronic fluoxetine. Moreover, depending of the area analysed, the basal binding of [35S]GTPgammaS was differentially affected by this treatment: increased in DRN and decreased in hippocampal dentate gyrus. Interestingly, the changes in [35S]GTPgammaS basal binding and on 5-HT1A receptors functionality were prevented by the concomitant administration of WAY100635. The inhibition of dorsal raphe firing by 8-OH-DPAT was also attenuated in fluoxetine-treated rats (ED50 = 2.12 +/- 0.32 microg/kg and 4.34 +/- 0.09 microg/kg, for vehicle and fluoxetine respectively), an effect which was also prevented by the concomitant administration of WAY100635 (ED50 = 2.10 +/- 0.58 microg/kg). Chronic administration of WAY100635 alone did not affect the 5-HT1A receptor-induced stimulation of [35S]GTPgammaS binding, nor the 8-OH-DPAT-induced inhibition of 5-HT neuron firing. These results demonstrate that the concomitant blockade of 5-HT1A receptors when administering fluoxetine prevents those adaptive changes of 5-HT1A receptor function associated with the chronic administration of this antidepressant. These findings could be relevant from the therapeutic point of view, and further support the potential benefit of treatments with a SSRI/5-HT1A receptor antagonist combination.     

Characterization of the receptors involved in the 5-HT-induced excitation of canine antral longitudinal muscle

1. We aimed to characterize the 5-HT receptors involved in the 5-HT-induced effect on electrically induced contractions of dog antrum longitudinal muscle in vitro. 2. In the presence of L-NOARG (0.1 mM), electrical field stimulation (EFS) induced atropine- and tetrodotoxin-sensitive contractions. Tetrodotoxin or atropine left any agonist tested ineffective. These EFS-induced contractions were on average enhanced by 5-HT (0.3 microM), however, pronounced variation in the response to 5-HT was observed. There were non-significant trends of the selective 5-HT3 receptor antagonist granisetron (1 microM), and methysergide (1 microM; preventing interactions of 5-HT with 5-HT1, 5-HT2, 5-ht5, 5-HT6 and 5-HT7 receptors) to increase the response to 5-HT. The selective 5-HT4 receptor antagonist GR 113808 (0.1 microM) displayed a non-significant trend to inhibit the 5-HT-induced increase. 3. Combination experiments with methysergide (1 microM), granisetron (1 microM) and GR 113808 (0.1 microM) revealed that the 5-HT (0.3 microM)-induced response consisted of (1) an excitatory component blocked by GR 113808, (2) excitatory and inhibitory components both blocked by methysergide. 4. The selective 5-HT4 receptor agonist prucalopride (0.3 microM) increased EFS-induced contractions, an effect prevented by GR 113808 (0.1 microM). 5. The increase of EFS-induced contractions by the preferential 5-HT2 receptor agonist alpha-Me-5-HT (0.3 microM) was antagonized by 5-HT2B receptor antagonists. 6. The 5-HT1/5-HT7 receptor agonist 5-carboxamidotryptamine (5-CT; 0.3 microM) inhibited EFS-induced contractions. This was prevented by methysergide (1 microM), the 5-HT7 receptor antagonist mesulergine (0.3 microM) and the selective 5-HT7 receptor antagonist SB-269970 (0.3 microM). 7. In the presence of GR 113808 (0.1 microM), alpha-Me-5-HT (1 microM) increased EFS-induced contractions. The 5-HT (0.3 microM)-induced inhibition of the stimulation by alpha-Me-5-HT was prevented by SB-269970 (0.3 microM). 8. In conclusion, dog antral longitudinal muscle is endowed with (1) excitatory neuronal 5-HT4 receptors and 5-HT2B receptors and (2) inhibitory smooth muscle 5-HT7 receptors.     

Serotonin inhibition of the NMDA receptor/nitric oxide/cyclic GMP pathway in human neocortex slices: involvement of 5-HT(2C) and 5-HT(1A) receptors

The NMDA receptor/nitric oxide (NO)/cyclic GMP pathway and its modulation by 5-hydroxytryptamine (5-HT) was studied in slices of neocortical samples obtained from patients undergoing neurosurgery. The cyclic GMP elevation produced by 100 microM NMDA was blocked by 100 microM of the NO synthase inhibitor N(G)-nitro-L-arginine (L-NOARG) or by 10 microM of the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-alpha] quinoxaline-1-one (ODQ). The NMDA effect was prevented by 5-HT or by the 5-HT(2) agonist (+/-)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane ((+/-)-DOI; EC(50)=22 nM). The (+/-)-DOI inhibition was insensitive to the 5-HT(2A) receptor antagonist MDL 100907 or the 5-HT(2B) antagonist rauwolscine; it was largely prevented by 1 microM of the non-selective 5-HT(2C) antagonists mesulergine (5-HT(2A,B,C)), ketanserin (5-HT(2A,C)) or SB 200646A (5-HT(2B,C)); it was completely abolished by 0.1 microM of the selective 5-HT(2C) receptor antagonist SB 242084. The NMDA-induced cyclic GMP elevation also was potently inhibited by the selective 5-HT(2C) agonist RO 60-0175 and by the antidepressant trazodone, both added at 1 microM, in an SB 242084-sensitive manner. Finally, the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; 1 microM) inhibited the NMDA-evoked cyclic GMP response, an effect blocked by the selective 5-HT(1A) receptor antagonist WAY 100635. In conclusion, the NMDA receptor/NO/cyclic GMP pathway in human neocortex slices can be potently inhibited by activation of 5-HT(2C) or 5-HT(1A) receptors.     

5-HT7 receptor-mediated dilatation in the middle meningeal artery of anesthetized rats

Topical administration of 5-carboxamidotryptamine (5-CT; 0.01-1000 microM) to the exposed dura mater encephali of anesthetized rats produced decreases in blood pressure and dilatation in the middle meningeal artery. Pretreatment with the 5-HT(1B/1D) receptor antagonist, N-[4-methoxy-3-(4-methyl-1-piperazinyl) phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl) [1,1-biphenyl]-4-carboxamide hydrochloride monohydrate (GR-127935; 1 mg/kg, i.v.), unmasked meningeal dilator responses to lower concentrations of 5-CT, and attenuated those to higher concentrations; GR-127935 also inhibited 5-CT-induced hypotension. The 5-HT7 receptor antagonist, (R)-1-{(3-hydroxyphenyl)sulfonyl}-2-{2-(2-(4-methyl-1-piperidinyl) ethyl} pyrrolidine (SB-269970; 1 mg/kg, i.v.), strongly inhibited dilator and hypotensive responses to 5-CT; the combination of GR-127935+SB-269970 (1 mg/kg, i.v., each) further inhibited meningeal and hypotensive responses. Thus, 5-CT may produce dilatation in the middle meningeal artery via 5-HT7 receptors; complex effects appear to involve 5-HT(1B/1D) receptors.     

Repeated administration of citalopram and imipramine alters the responsiveness of rat hippocampal circuitry to the activation of 5-HT7 receptors

The effects of a selective serotonin reuptake inhibitor, citalopram, and a tricyclic antidepressant drug, imipramine, administered repetitively for 14 days, were investigated ex vivo in rat hippocampal slices. Spontaneous epileptiform bursts were recorded from the CA3 area in nominally Mg(2+)-free incubation conditions. 5-carboxamidotryptamine (5-CT) dose-dependently increased bursting frequency in the presence of N-[2-[4-(2-methoxyphenyl)-1 piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide (WAY 100635). This effect could be dose-dependently blocked by (2R)-1-[(3-Hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine hydrochloride (SB 269970), thus implicating the involvement of 5-HT(7) receptors. Repeated treatment with citalopram or imipramine resulted in an attenuation of the excitatory effects of the activation of hippocampal 5-HT(7) receptor.     

Contractile effects of 5-hydroxytryptamine and 5-carboxamidotryptamine in the equine jejunum

The use of human prokinetic drugs in colic horses leads to inconsistent results. This might be related to differences in gastrointestinal receptor populations. The motor effects of 5-hydroxytryptamine (5-HT; serotonin) on the equine mid-jejunum were therefore studied. Longitudinal muscle preparations were set up for isotonic measurement. 5-HT induced tonic contractions with superimposed phasic activity; these responses were not influenced by tetrodotoxin and atropine, suggesting a non-neurogenic, non-cholinergic pathway. The 5-HT receptor antagonists GR 127935 (5-HT(1B,D)), ketanserin (5-HT(2A)), SB 204741 (5-HT(2B)), RS 102221 (5-HT(2C)), granisetron (5-HT(3)), GR 113808 (5-HT(4)) and SB 269970 (5-HT(7)) had no influence on the 5-HT-induced response; the 5-HT(1A) receptor antagonists NAN 190 (pK(b)=8.13+/-0.06) and WAY 100635 (pK(b)=8.69+/-0.07), and the 5-HT(1,2,5,6,7) receptor antagonist methysergide concentration-dependently inhibited the 5-HT-induced contractile response. The 5-HT(1,7) receptor agonist 5-carboxamidotryptamine (5-CT) induced a contractile response similar to that of 5-HT; its effect was not influenced by tetrodotoxin and atropine, and SB 269970, but antagonised by WAY 100635. 8-OHDPAT, buspiron and flesinoxan, which are active at rat and human 5-HT(1A) receptors, had no contractile influence. These results suggest that the contractile effect of 5-HT in equine jejunal longitudinal muscle is due to interaction with muscular 5-HT receptors, which cannot be characterised between the actually known classes of 5-HT receptors.