Cloning,expression and pharmacology of a truncated splice variant of the human 5-HT7 receptor (h5-HT7(b))

1. The rat 5-hydroxytryptamine (5-HT)₇ receptor displays two splice variations, a long form, and a truncated splice isoform, arising from the introduction of a stop codon near the carboxy-terminus. The human 5-HT₇ receptor gene contains at least two introns and encodes a 445 amino acid 5-HT receptor.
2. A truncated splice variation in the human 5-HT₇ receptor was isolated from a human placental cDNA library. In accordance with current NC-IUPHAR nomenclature guidelines, it is suggested that this receptor be denoted as the h5-HT_7(b) receptor and the long form of the receptor as h5-HT_7(a).
3. The h5-HT_7(b) receptor was stably expressed in HEK 293 cells and ligand affinities were determined by displacement of [³H]-5-carboxyamidotryptamine (5-CT; K_d=0.28±0.06 nM, B_max=7.3±1.7 pmol mg⁻¹ protein). The rank order of affinities (pK_i) for a series of ligands was: 5-carboxamidotryptamine (5-CT, 9.65)>5-hydroxytryptamine (5-HT, 9.41)>methiothepin (8.87)>mesulergine (7.87)>8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT, 6.85)>ketanserin (6.44).
4. The h5-HT_7(b) receptor coupled positively to adenylyl cyclase in HEK 293 cells. This response was elicited by a number of agonists with the following order of potency (pEC₅₀): 5-CT (8.7±0.11)>5-MeOT (5-methoxytryptamine; 8.1±0.20)>5-HT (7.5±0.13)>tryptamine (5.6±0.36)>8-OH-DPAT (5.3±0.28)>5-methoxytryptamine (5.0±0.06). This rank order was comparable to that observed in the radioligand binding studies.
5. In a similar fashion to that described for the 5-HT_7(a) receptor, PCR studies suggested that the 5-HT_7(b) receptor mRNA is found in great abundance throughout the brain, in the small intestine and aorta.
6. It is concluded that the h5-HT₇ receptor, like the rat receptor, exists as splice variants exhibiting similar pharmacology, signal transduction and distribution. It is thus likely that there exists a complex physiological role for alternate splicing products of the 5-HT₇ receptor gene.

     

Importance of the C-terminus of the human 5-HT3A receptor subunit

Amongst the family members of Cys-loop LGICs, the atypical ability of the 5-HT3A subunit to form functional homomeric receptors allowed a direct investigation of the role of the C-terminus. Deletion of the three C-terminal amino acids (ΔGln⁴⁵³-ΔTyr⁴⁵⁴-ΔAla⁴⁵⁵) from the h5-HT3A subunit prevented formation of a specific radioligand binding site as well as expression within the cell membrane. Removal of merely the C-terminal residue (ΔAla⁴⁵⁵) reduced specific radioligand binding (to 4 ± 1% relative to the wild-type; cells grown at 37 °C) and also cell membrane expression; these reductions were less evident when the ΔAla⁴⁵⁵ expressing cells were grown at 27 °C (specific radioligand binding levels 27 ± 5% relative to wild-type also grown at 27 °C). Mutation of the h5-HT3A C-terminal amino acid, alanine, for either glycine (Ala⁴⁵⁵Gly), valine (Ala⁴⁵⁵Val) or leucine (Ala⁴⁵⁵Leu) reduced specific radioligand binding levels by 24 ± 23%, 32 ± 12% and 88 ± 1%, respectively; the latter mutant also displaying reduced membrane expression. In contrast, mutation to alanine of the two amino acids preceding the C-terminal alanine (Gln⁴⁵³Ala and Tyr⁴⁵⁴Ala) had no detrimental effects on specific radioligand binding or cell membrane expression levels. The present study demonstrates an important role for the C-terminus in the formation of the functional h5-HT₃A receptor. The partial restoration of 5-HT₃ receptor binding and cell membrane expression when cells expressing C-terminal mutant 5-HT3A subunits were grown at a lower temperature (27 °C) suggests that the C-terminus stabilises the 5-HT₃ receptor allowing subunit folding and subsequent maturation.     

Targeted gene deletion of the 5-HT3A receptor subunit produces an anxiolytic phenotype in mice

Anxiety disorders are the most common psychiatric disorders. Typical medications used to treat patients are benzodiazepines or antidepressants that target serotonin (5-HT) activity. The ionotropic 5-HT(3) receptor has emerged as a potential therapeutic target because selective antagonist compounds reduce anxiety in rodents, primates, and humans. 5-HT binds to the extracellular N-terminus of the 5-HT(3A) receptor subunit, but receptor activation is also enhanced by distinct allosteric sites. It is not known if specific molecular subunits of the 5-HT(3) receptor modulate anxiety. To address this issue, we characterized anxiety-like behavior of mice with a targeted deletion of the 5-HT(3A) receptor subunit gene in the light/dark box, elevated plus maze, and novelty interaction animal models of anxiety. 5-HT(3A) null mice exhibited an anxiolytic behavioral phenotype that was highly correlated across behavioral measures. This evidence indicates that the 5-HT(3A) molecular subunit influences anxiety-like behavior. Pharmacotherapy that targets specifically the 5-HT(3A) receptor subunit may provide a novel treatment for anxiety disorders.     

Acute and chronic tryptophan depletion differentially regulate central 5-HT1A and 5-HT2A receptor binding in the rat

Rationale Tryptophan depletion is used to reduce central serotonergic function and to investigate its role in psychiatric illness. Despite widespread clinical use, its effects on serotonin (5-HT) receptors have not been well characterized. Objective The aim of this study was to examine the effect of acute (ATD) and chronic tryptophan depletion (CTD) on free-plasma tryptophan (TRP), central TRP and 5-HT and brain 5-HT_1A and 5-HT_2A receptor binding in the rat. Methods TRP and 5-HT were measured by high-performance liquid chromatography and receptor levels determined by homogenate radioligand binding and in-vitro receptor autoradiography. Results Free-plasma TRP, central TRP and central 5-HT levels were significantly and similarly reduced by ATD and 1- and 3-week CTD compared to controls. ATD significantly reduced 5-HT_1A binding in the dorsal raphe (14%) but did not significantly alter postsynaptic 5-HT_1A binding (frontal cortex, remaining cortex and hippocampus) or 5-HT_2A binding (cortex and striatum). One-week CTD did not significantly alter cortical 5-HT_2A binding or postsynaptic 5-HT_1A binding. Furthermore, 3-week CTD did not significantly alter 5-HT_1A binding but significantly increased cortical 5-HT_2A binding without affecting striatal or hippocampal levels. In the CTD 1 and 3-week groups, rat body weight was significantly decreased as compared to controls. However, weight loss was not a confounding factor for decreased cortical 5-HT_2A-receptor binding. Conclusion ATD-induced reduction in somatodendritic 5-HT_1A autoreceptor binding may represent an intrinsic ‘homeostatic response’ reducing serotonergic feedback in dorsal raphe projection areas. In contrast, the increase in 5-HT_2A receptor after CTD may be a compensatory response to a long-term reduction in 5-HT.     

Characterization of the contraction to 5-HT in the canine colon longitudinal muscle

1. Although conscious dogs have often been used for colonic motility studies with 5-hydroxytryptamine (5-HT), the effects of 5-HT on the isolated colon have not been thoroughly characterized yet. The current study was undertaken to characterize the response to 5-HT of the canine isolated colon longitudinal muscle.
2. Longitudinal strips of canine midcolon deprived of (sub)mucosa were prepared for isotonic measurement. 5-HT induced contractions from 3 nM onwards, which were not affected by selective inhibition of 5-HT re-uptake, monoamine oxidase or blockade of α-adrenoceptors. Tetrodotoxin (0.3 μM) did not affect the responses to 5-HT, suggesting that smooth muscle 5-HT receptors are involved. The selective 5-HT₄ receptor antagonist SB 204070 (10 nM) slightly enhanced contractions to 5-HT and therefore it was included in the organ bath solution in all further experiments. The 5-HT₁ and 5-HT₂ receptor antagonist methysergide (0.1 μM) depressed the curve to 5-HT, but the selective 5-HT₃ receptor antagonist granisetron (0.3 μM) had no effect.
3. Besides 5-HT, α-methyl-5-HT (α-Me-5-HT), 5-methoxytryptamine (5-MeOT), 2-methyl-5-HT (2-Me-5-HT) and 5-carboxamidotryptamine (5-CT) also induced contractions, with the following rank order of potency (pEC₅₀ values in parentheses): 5-HT (6.9)=α-methyl-5-HT (6.9)>2-Me-5-HT (5.8)=5-MeOT (5.7)=5-CT (5.6), indicative of 5-HT₂ receptor involvement. α-Me-5-HT produced a bell-shaped curve, which was not affected by α-adrenoceptor blockade. 5-HT, 5-MeOT, 2-Me-5-HT and 5-CT produced a monophasic concentration-response curve, consistent with an interaction with a single receptor site. 8-Hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and tryptamine only induced contractions at a concentration exceeding 1 μM.
4. The selective 5-HT_2B receptor antagonist SB 204741 (0.3 μM) did not affect the curve to 5-HT. Ketanserin, cisapride and spiroxatrine behaved as competitive antagonists with pK_b values of, respectively, 8.4, 8.1 and 6.7. Spiroxatrine (1 μM) shifted the curve to 5-MeOT rightward yielding an apparent pA₂ of 7.1. Other antagonists at 5-HT_2A receptors also surmountably inhibited the contractions to 5-HT (apparent pA₂ value in parentheses): mesulergine (8.2), cinanserin (8.2), yohimbine (6.2) and mianserin (8.6). However, as well as a rightward shift, methiothepin (8.3), pizotifen (8.6) and spiperone (8.8) also caused a depression of the curve, indicative of ‘pseudo-irreversible'' antagonism. Taken together, the above mentioned affinity estimates most closely corresponded to literature affinity values for 5-HT_2A receptors.
5. It was concluded that 5-HT induces contractions of the canine midcolon longitudinal muscle primarily by stimulation of smooth muscle 5-HT_2A receptors. The presence of inhibitory 5-HT₄ receptors cannot be ruled out.

     

BIMT 17, a 5-HT2A receptor antagonist and 5-HT1A receptor full agonist in rat cerebral cortex

In the search for antidepressant agents with a rapid onset of action, we have found that compound BIMT 17 (1-[2-[4-(3-trifluoromethylphenyl)piperazin1-yl]ethyl]benzimidazol-[1H]-2-one) shows a good affinity for cerebral cortical 5-HT_1A (pK _i = 7.72) and 5-HT_2A (pK _i = 6.90) receptors, with no appreciable affinity for the other 5-HT receptor subtypes, including 5-HT_2C. BIMT 17 reduced forskolin-stimulated cAMP accumulation in the cerebral cortex (pEC₅₀ = 6.09) and in the hippocampus (pEC₅₀ = 6.50), and antagonized 5-HT-induced phosphatidylinositol turnover (pK _i = 6.96) in the cerebral cortex. The effect on cAMP accumulation was blocked by the 5-HT_1A receptor antagonist tertatolol. Buspirone, 8-OH-DPAT and S 14671 {1-[2-(2-thenoylamino)ethyl]-4[1-(7-methoxynaphtyl)]piperazine, claimed to be 5-HT_1A receptor agonists, did not reduce forskolin-stimulated cAMP formation in the cerebral cortex.On the basis of these data, it was concluded that BIMT 17 was the only compound that behaved as a full agonist with respect to the CAMP response in the cortex, while exerting concurrent agonism at 5-HT_1A receptors and antagonism at 5-HT_2A receptors. These characteristics might explain the peculiar behaviour of BIMT 17 in mimicking the inhibitory action of 5-HT on the basal firing rate of the cortical neurons (see accompanying paper).     

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.

     

Anxiolytic-like effect of a serotonergic ligand with high affinity for 5-HT1A, 5-HT2A and 5-HT3 receptors

S-(-)-2-[[4-(napht-1-yl)piperazin-1-yl]methyl]-1,4-dioxoperhydropyrrolo[1,2-alpha]-pyrazine (CSP-2503) is a serotonin (5-HT) receptor ligand with selectivity and high affinity for 5-HT1A, 5-HT2A and 5-HT3 receptors. CSP-2503 reduced rectal temperature and 5-HT neuronal hypothalamic activity in mice, decreased electrical activity of raphe nuclei cells in rats and blocked the enhancement of adenylate cyclase activity induced by forskolin in HeLa cells transfected with the human 5-HT1A receptor. This compound also blocked head-twitches induced by the 5-HT(2A/2C) receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI). Contractions of guinea pig ileum induced by the 5-HT3 receptor agonist 2-methyl-5-HT were prevented by CSP-2503. Moreover, it reduced the bradycardia reflex induced by 2-methyl-5-HT in anaesthetized rats. In the light/dark box and social interaction tests, CSP-2503 presented anxiolytic activity, an action shared by 5-HT1 agonists and 5-HT3 antagonists. Taken together, these results suggest that CSP-2503 is a new 5-HT1 receptor agonist with 5-HT2A and 5-HT3)receptor antagonist activities that might be useful in a number of conditions associated with anxiety.     

Effects of triiodothyronine and imipramine on basal 5-HT levels and 5-HT(1) autoreceptor activity in rat cortex

Clinical studies have shown that triiodothyronine (T3) both augments and accelerates the therapeutic response to antidepressant drugs, particularly tricyclics. There is evidence that this effect is mediated by the serotonergic system. We show here that T3 administered daily for 7 days over the range 0.02-0.5 mg/kg increases basal serotonin (5-hydroxytryptamine, 5-HT) levels, as measured by in vivo microdialysis in rat cortex, in a dose-dependent fashion. All the doses of T3 examined reduced 5-HT(1A) autoreceptor activity, as measured by the effect of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 0.05 mg/kg s.c.) to decrease 5-HT levels in frontal cortex. T3 administered daily for 14 days at 0.02 mg/kg also reduced 5-HT(1B) autoreceptor activity, as measured by the effect of locally administered 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP 93129, 10 microM) to decrease 5-HT levels. In animals administered imipramine (10 mg/kg/day by osmotic minipump) concurrently with T3 injections, no further changes in either 5-HT(1A) or 5-HT(1B) autoreceptor activity were seen. We suggest that the effect of T3 to accelerate the therapeutic actions of antidepressant drugs may be due to a combination of the actions of T3 at autoreceptors and the actions of the drugs at postsynaptic 5-HT(1A) receptors.     

Development of Fluorescent Ligands for the Human 5-HT1A Receptor

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Functional interactions between 5-HT2A and presynaptic 5-HT1A receptor-based responses in mice genetically deficient in the serotonin 5-HT transporter (SERT)

Background and purpose:

Despite decreased presynaptic 5-HT_1A and altered 5-HT_2A receptor function in genetically-deficient serotonin (5-HT) transporter (SERT) mice, the 5-HT_1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate salt (WAY 100635) still induced head twitches in these mice, a well-established 5-HT_2A receptor-mediated response.

Experimental approach:

Interactions between 5-HT_1A and 5-HT_2A receptors were assessed using the head-twitch response following 5-HT_1A and 5-HT_2A receptor agonists and antagonists in SERT wild-type (+/+), heterozygous (+/−), and knockout (−/−) mice. The role of brain 5-HT availability in WAY 100635 induced head twitches was also examined.

Key results:

WAY 100635 induced head twitches in a SERT gene-dose dependent manner, inducing 5-fold more head twitches in SERT −/− versus SERT +/+ mice. In SERT −/− mice, inhibition of 5-HT synthesis with p-chlorophenylalanine (PCPA) markedly depleted tissue 5-HT in all five brain areas examined and abolished WAY 100635 induced head twitches. Further, the selective 5-HT reuptake inhibitor fluvoxamine increased WAY 100635 induced head twitches in SERT +/+ and +/− mice. Head twitches following the 5-HT_2A receptor agonist (+/−)-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI) were robust in SERT +/+ and +/− mice but much reduced in SERT −/− mice. DOI-induced head twitches were decreased by the 5-HT_1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) in SERT +/+ and +/− mice. All drug-induced head twitches were blocked by the 5-HT_2A receptor antagonist a-Phenyl-1-(2-phenylethyl)-4-piperidinemethanol (MDL 11,939).

Conclusions and implications:

These data show that indirect activation of 5-HT_2A receptors via blockade of presynaptic 5-HT_1A receptors potentiated head-twitch responses, suggesting functional interactions between these receptors, interactions affected by altered 5-HT availability. Our findings strongly support the correlation of WAY 100635 induced head twitches with increased 5-HT availability, induced genetically or pharmacologically.     

Purification of 5-hydroxytryptamine3 receptors from porcine brain

1. We demonstrate, for the first time, the purification of the 5-hydroxytryptamine₃ (5-HT₃) receptor from a native tissue source, pig cerebral cortex.
2. From a range of detergents, the non-ionic detergent Triton X-100 was demonstrated to exhibit the least inhibition of [³H]-(S)-zacopride binding to membrane bound 5-HT₃ receptors from pig cerebral cortex at concentrations above its critical micellular concentration (CMC). This detergent was therefore selected to solubilize 5-HT₃ binding sites from homogenates of pig cerebral cortex. Maximum yield (43.8±3.7%, mean±s.e.mean, n=13) was obtained with Triton X-100 at 0.4% (22.1×CMC). Radioligand binding studies with [³H]-(S)-zacopride indicated that the solubilized 5-HT₃ receptor displayed near identical pharmacology to the membrane bound receptor (the correlation coefficient (r) between the pK_i values of structurally unrelated compounds competing for [³H]-(S)-zacopride binding in the membrane bound and solubilized 5-HT₃ receptor preparations was 0.99, B_max=20.7±4.2 fmol mg⁻¹ protein, K_d=1.57±0.53 nM, mean±s.e.mean, n=6).
3. Solubilized (0.4% Triton X-100) 5-HT₃ receptors were affinity purified using Affi-Gel 15 coupled to the high affinity 5-HT₃ receptor ligand GR119566X. Radioligand binding studies indicated that the pharmacological profile of the affinity purified 5-HT₃ receptor, assessed using ligands with a range of affinities spanning 3 orders of magnitude, was similar to that in both crude homogenates (r=0.85) and solubilized 5-HT₃ receptor sites (r=0.85) from pig brain. The specific activity for the purified 5-HT₃ receptor overlapped the theoretical specific activity of the receptor (B_max=3.27±1.41 and 5.35±2.33 nmol mg⁻¹ protein, assessed by saturation and competition studies respectively, mean±s.e.mean, n=3–4), which indicated a 60 000–100 000 fold purification of the membrane bound receptor.
4. Under non-reducing conditions, samples of the affinity purified protein failed to enter a 10% separating gel in SDS–PAGE analysis, indicating a molecular mass for the receptor complex of >200 kDa. Further investigation of the non-reduced purified protein with a 7.5% separating gel gave a mass for the complex of ∼279 kDa. Under reducing conditions, SDS–PAGE analysis of the affinity purified 5-HT₃ receptor resulted in 3–6 silver stained bands at apparent molecular masses of 37, 44–50, 52, 57–61, 63 and 65–71 kDa (n=12). Unlike protein bands at 45, 50, 60 and 66 kDa, the bands corresponding to proteins of 52, 57, 63 and 71 kDa consistently gave no reaction with an antiserum specific for the cloned A subunit of the 5-HT₃ receptor in both a modified dot blot procedure and a Western blot procedure (n=2–5).
5. We conclude that we have purified the 5-HT₃ receptor from pig brain to homogeneity and suggest this may contain non-5-HT₃-A receptor subunit(s).

     

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.

     

On the role of brain 5-HT7 receptor in the mechanism of hypothermia: comparison with hypothermia mediated via 5-HT1A and 5-HT3 receptor

Intracerebroventricular administration of selective agonist of serotonin 5-HT₇ receptor LP44 (4-[2-(methylthio)phenyl]-N-(1,2,3,4-tetrahydro-1-naphthalenyl)-1-pyperasinehexanamide hydrochloride; 10.3, 20.5 or 41.0 nmol) produced considerable hypothermic response in CBA/Lac mice. LP44-induced (20.5 nmol) hypothermia was significantly attenuated by the selective 5-HT₇ receptor antagonist SB 269970 (16.1 fmol, i.c.v.) pretreatment. At the same time, intraperitoneal administration of LP44 in a wide range of doses 1.0, 2.0 or 10.0 mg/kg (2.0, 4.0, 20.0 μmol/kg) did not cause considerable hypothermic response. These findings indicate the implication of central, rather than peripheral 5-HT₇ receptors in the regulation of hypothermia. The comparison of LP44-induced (20.5 nmol) hypothermic reaction in eight inbred mouse strains (DBA/2J, CBA/Lac, C57BL/6, BALB/c, ICR, AKR/J, C3H and Asn) was performed and a significant effect of genotype was found.In the same eight mouse strains, functional activity of 5-HT_1A and 5-HT₃ receptors was studied. The comparison of hypothermic responses produced by 5-HT₇ receptor agonist LP44 (20.5 nmol, i.c.v.) and 5-HT_1A receptor agonist 8-OH-DPAT 1.0 mg/kg, i.p. (3.0 μmol/kg), 5-HT₃ receptor agonist m-CPBG (40.0 nmol, i.c.v.) did not reveal considerable interstrain correlations between 5-HT₇ and 5-HT_1A or 5-HT₃ receptor-induced hypothermia. The selective 5-HT₇ receptor antagonist SB 269970 (16.1 fmol, i.c.v.) failed to attenuate the hypothermic effect of 8-OH-DPAT 1.0 mg/kg, i.p. (3.0 μmol/kg) and m-CPBG (40.0 nmol, i.c.v.) indicating that the brain 5-HT₇ receptor is not involved in the hypothermic effects of 8-OH-DPAT or m-CPBG. The obtained results suggest that the central 5-HT₇ receptor plays an essential role in the mediation of thermoregulation independent of 5-HT_1A and 5-HT₃ receptors.     

Functional coupling of a recombinant Human 5-HT5A receptor to G-proteins in HEK-293 cells

1. We have cloned, expressed and pharmacologically characterized the Human 5-HT_5A receptor.
2. We have shown that ligand activation of the Human 5-HT_5A receptor results in functional coupling to G-proteins in HEK-293 cells.
3. Stimulation of the receptor with 5-CT (5-carboxamidotryptamine) resulted in a dose-dependent increase in the % [³⁵S]-GTPγS binding over the basal level. This is the first study to describe such G-protein activation for the Human 5-HT_5A receptor in any cell.
4. A dose-dependent inhibition of cyclic AMP accumulation was observed in the recombinant Human 5-HT_5A receptor cell line, suggesting a functional coupling to a Gαi, G-protein in the HEK-293 cell line.
5. A ligand-stimulated reduction in the detectable level of the catalytic domain of protein kinase A (PKA) in nuclear extracts isolated from Human 5-HT_5A expressing cells was observed. This observation was consistent with the reduction in the level of cyclic AMP accumulation, in response to receptor activation.

     

Pharmacological characterization of the 5-HT1A, 5-HT2 and 5-HT3 receptors in the bovine ciliary muscle

We aimed to investigate the effects of serotonin (5-hydroxytryptamine, 5-HT) on the bovine ciliary muscle and subsequently to characterize and identify the subtypes of 5-HT receptors involved in the serotonin-evoked contractility muscle. The binding of [3H]ketanserin, [3H]granisetron and [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) was analyzed. All labelled compounds bound with high affinity to a single site in the membrane preparations studied. The affinity (K(d)) of the binding site was 7.5+/-1.2 nM for [3H]ketanserin, 6.9+/-0.8 nM for [3H]granisetron and 4.4+/-0.31 nM for [3H]8-OH-DPAT. The density of receptors (B(max)) was 1062+/-43.0 fmol/mg protein for [3H]ketanserin, 566+/-2.32 fmol/mg protein for [3H]granisetron and 205+/-4.63 fmol/mg protein for [3H]8-OH-DPAT. The serotonin-induced contraction appeared to be competitively antagonized by ketanserin (0.1, 1 and 10 microM) and ondansetron (0.1, 10 and 100 microM) which produced a pA(2) value of 8.5+/-0.12 and 8.0+/-0.19, respectively. 8-OH-DPAT and 5-carboxamidotryptamine (5-CT) proved to be completely ineffective. We conclude that serotonin induces bovine ciliary muscle contraction via 5-HT(2) and 5-HT(3) receptors while the 5-HT(1A) receptors, although present, do not mediate the contractile response.     

5-Chloroindole: a potent allosteric modulator of the 5-HT3 receptor

Background and Purpose

The 5-HT₃ receptor is a ligand-gated ion channel that is modulated allosterically by various compounds including colchicine, alcohols and volatile anaesthetics. However the positive allosteric modulators (PAMs) identified to date have low affinity, which hinders investigation because of non-selective effects at pharmacologically active concentrations. The present study identifies 5-chloroindole (Cl-indole) as a potent PAM of the 5-HT₃ receptor.

Experimental Approach

5-HT₃ receptor function was assessed by the increase in intracellular calcium and single-cell electrophysiological recordings in HEK293 cells stably expressing the h5-HT₃A receptor and also the mouse native 5-HT₃ receptor that increases neuronal contraction of bladder smooth muscle.

Key Results

Cl-indole (1–100 μM) potentiated agonist (5-HT) and particularly partial agonist [(S)-zacopride, DDP733, RR210, quipazine, dopamine, 2-methyl-5-HT, SR57227A, meta chlorophenyl biguanide] induced h5-HT₃A receptor-mediated responses. This effect of Cl-indole was also apparent at the mouse native 5-HT₃ receptor. Radioligand-binding studies identified that Cl-indole induced a small (∼twofold) increase in the apparent affinity of 5-HT for the h5-HT₃A receptor, whereas there was no effect upon the affinity of the antagonist, tropisetron. Cl-indole was able to reactivate desensitized 5-HT₃ receptors. In contrast to its effect on the 5-HT₃ receptor, Cl-indole did not alter human nicotinic α7 receptor responses.

Conclusions and Implications

The present study identifies Cl-indole as a relatively potent and selective PAM of the 5-HT₃ receptor; such compounds will aid investigation of the molecular basis for allosteric modulation of the 5-HT₃ receptor and may assist the discovery of novel therapeutic drugs targeting this receptor.

Linked Articles

Recent reviews on allosteric modulation can be found at:Kenakin, T (2013). New concepts in pharmacological efficacy at 7TM receptors: IUPHAR Review 2. British Journal of Pharmacology 168: 554–575. doi: 10.1111/j.1476-5381.2012.02223.xRoche D, Gil D and Giraldo J (2013). Mechanistic analysis of the function of agonists and allosteric modulators: reconciling two-state and operational models. British Journal of Pharmacology 169: 1189–1202. doi: 10.1111/bph.12231     

5-HT2A and 5-HT2C receptor antagonists have opposing effects on a measure of impulsivity: interactions with global 5-HT depletion

Rationale Global serotonin (5-HT) depletion increases the number of premature responses made on the five-choice serial reaction time task (5CSRT) in rats. In contrast, the 5-HT_2A receptor antagonist M100907 decreases this measure of impulsivity. Mounting evidence suggests that 5-HT_2A and 5-HT_2C receptors have opposing effects on behaviour, and that the 5-HT_2C receptor antagonist SB 242084 produces a pattern of behaviour similar to 5-HT depletion.Objectives To assess the effects of 5-HT_2A and 5-HT_2C receptor antagonists on performance of the 5CSRT, to directly compare the effects of these drugs with those of ICV 5,7-dihydroxytryptamine (5,7-DHT) lesions and to investigate whether 5-HT depletion affects the action of these agents.Methods The effects of M100907 (0, 0.01, 0.03, 0.1 mg/kg IP) and SB 242084 (0, 0.1, 0.25, 0.5 mg/kg IP) were investigated on performance of the 5CSRT in both ICV 5,7-DHT-lesioned and sham-operated rats.Results ICV 5,7-DHT lesions, which significantly decreased forebrain levels of 5-HT by around 90%, increased levels of premature responding, decreased omissions and the latency to respond correctly, yet did not affect performance accuracy. M100907 decreased premature responding in sham-operated controls but not in 5-HT-depleted rats. In contrast, SB 242084 increased premature responding in all animals, and also decreased the latency to make a correct response in sham-operated controls.Conclusions These data support the view that serotonergic regulation of impulsive behaviour through different members of the 5-HT₂ receptor family is functionally heterogeneous. Although both 5-HT_2A and 5-HT_2C receptors participate in controlling this form of impulsive action, their relative contribution may depend on the endogenous state of the 5-HT system.