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).

     

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.

     

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.

     

Characteristics of the actions by which 5-hydroxytryptamine affects electrical and mechanical activities in rabbit jugular vein graft

BACKGROUND AND PURPOSE

The vasomodulating actions of 5-HT in vein grafts, and the underlying mechanisms, remain to be fully clarified. Here, we characterized the actions by which 5-HT affects electrical and mechanical activities in rabbit autologous jugular vein grafts.

EXPERIMENTAL APPROACH

Smooth muscle cell (SMC) membrane potential and isometric tension were measured in vein grafts 4 weeks after implantation into carotid arteries. Changes in the expression of 5-HT receptor subtypes and in myosin heavy chain isoforms (SM1, SM2 and SMemb) were examined by immunohistochemistry and Western blot analysis.

KEY RESULTS

The walls of grafted veins displayed massive increases in the number of SM1- and SM2-positive SMCs. 5-HT induced a large depolarization and contraction that were each reduced by both 5-HT_2A- and 5-HT_1B/1D-receptor antagonists. The 5-HT-induced contraction was not modified by a 5-HT₇-receptor antagonist. The 5-HT₇-receptor-selective agonist AS 19 did not induce relaxation during the contraction to prostaglandin F_2α. Immunohistochemical and Western blot analyses revealed that immunoreactive responses against 5-HT_2A and 5-HT_1B/1D receptors were increased in the vein graft.

CONCLUSIONS AND IMPLICATIONS

5-HT is able to induce a large contraction in rabbit autologous jugular vein grafts through (i) an increased number of differentiated contractile SMCs; (ii) an increased number of SMCs expressing contractile 5-HT_2A- and 5-HT_1B/1D receptors; and (iii) a down-regulation of the function of the relaxant SMC 5-HT₇ receptors. These changes in the vein graft may help it to resist the higher pressure present on the arterial side of the circulation.     

5-hydroxytryptamine induced relaxation in the pig urinary bladder neck

Background and purpose

5-Hydroxytryptamine (5-HT) is one of the inhibitory mediators in the urinary bladder outlet region. Here we investigated mechanisms involved in 5-HT-induced relaxations of the pig bladder neck.

Experimental approach

Urothelium-denuded strips of pig bladder were mounted in organ baths for isometric force recordings of responses to 5-HT and electrical field stimulation (EFS).

Key results

After phenylephrine-induced contraction, 5-HT and 5-HT receptor agonists concentration-dependently relaxed the preparations, with the potency order: 5-carboxamidotryptamine (5-CT) > 5-HT = RS67333 > (±)-8-hydroxy-2-dipropylaminotetralinhydrobromide > m-chlorophenylbiguanide > α-methyl-5-HT > ergotamine. 5-HT and 5-CT relaxations were reduced by the 5-HT₇ receptor antagonist (2R)-1-[(3-hydroxyphenyl)sulphonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine hydrochloride and potentiated by (S)-N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide dihydrochloride (WAY 100135) and cyanopindolol, 5-HT_1A and 5-HT_1A/1B receptor antagonists respectively. Inhibitors of 5-HT_1B/1D, 5-HT₂, 5-HT_2B/2C, 5-HT₃, 5-HT₄, 5-HT_5A and 5-HT₆ receptors failed to modify 5-HT responses. Blockade of monoamine oxidase A/B, noradrenergic neurotransmission, α-adrenoceptors, muscarinic and purinergic receptors, nitric oxide synthase, guanylate cyclase and prostanoid synthesis did not alter relaxations to 5-HT. Inhibitors of Ca²⁺-activated K⁺ and ATP-dependent K⁺ channels failed to modify 5-HT responses but blockade of neuronal voltage-gated Na⁺-, Ca²⁺-and voltage-gated K⁺ (K_v)-channels potentiated these relaxations. Adenylyl cyclase activation and cAMP-dependent protein kinase (PKA) inhibition potentiated and reduced, respectively, 5-HT-induced responses. Under non-adrenergic, non-cholinergic, non-nitrergic conditions, EFS induced neurogenic, frequency-dependent, relaxations which were resistant to WAY 100135 and cyanopindolol.

Conclusions and implications

5-HT relaxed the pig urinary bladder neck through muscle 5-HT₇ receptors linked to the cAMP-PKA pathway. Prejunctional 5-HT_1A receptors and K_v channels modulated 5-HT-induced relaxations whereas postjunctional K⁺ channels were not involved in such responses. 5-HT₇ receptor antagonists could be useful in the therapy of urinary incontinence produced by intrinsic sphincter deficiency.     

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.     

Evidence that 5-hydroxytryptamine7 receptors play a role in the mediation of afferent transmission within the nucleus tractus solitarius in anaesthetized rats

Background and purpose:

Central 5-hydroxytryptamine (5-HT)-containing pathways utilizing 5-HT₇ receptors are known to be critical for the mediation of cardiovascular reflexes. The nucleus tractus solitarius (NTS) is a site involved in the integration of cardiovascular afferent information. The present experiments examined the involvement of the 5-HT₇ receptor in the processing of cardiovascular reflexes in the NTS.

Experimental approach:

In anaesthetized rats extracellular recordings were made from 104 NTS neurones that were excited by electrical stimulation of the vagus nerve and/or activation of cardiopulmonary afferents. Drugs were applied ionophoretically in the vicinity of these neurones.

Key results:

The non-selective 5-HT₇ receptor agonist 5-carboxamidotryptamine maleate (5-CT) applied to 78 neurones increased the firing rate in 18 by 59% and decreased it in 38 neurones by 47%. Similarly, the 5-HT_1A agonist 8-OH-DPAT applied to 20 neurones had an excitatory (8), inhibitory (7) or no effect (5) on the 20 neurones tested. In the presence of the 5-HT₇ antagonist SB 258719 the 5-CT excitation was attenuated. Furthermore, the excitatory response of NTS neurones evoked by electrical stimulation of the vagus nerve or activation of cardiopulmonary afferents with intra atrial phenylbiguanide was attenuated by SB 258719. The inhibitory action of 5-CT was unaffected by SB 258719 and the 5-HT_1A antagonist WAY-100635. WAY-100635 failed to have any effect on 5-CT and vagal afferent-evoked excitations.

Conclusions and implications:

Vagal afferent-evoked excitation of NTS neurones can be blocked by SB 258719, a selective 5-HT₇ antagonist. This observation further supports the involvement of 5-HT neurotransmission in NTS afferent processing.     

Blockade of human and porcine myocardial 5-HT4 receptors by SB 203186

We investigated the blockade of the positive inotropic effects of 5-hydroxytryptamine (5-HT) by SB 203 186 (piperidinoethyl-indole-3-carboxylate hydrochloride) and its affinity for 5-HT₄ receptors of human right atrium and piglet left atrium. We also compared the blocking effects of SB 203 186 against 5-HT-evoked tachycardia in anaesthetised adult Yucatan minipigs as well as new-born Camborough piglets.SB 203 186 caused competitive antagonism of the positive inotropic effects of 5-HT in electrically paced atrial preparations of man (pK _B = 8.9) and piglet (pK _B = 8.5) at concentrations (up to 0.3 mo]/l) which were devoid of depressant or stimulant effects. The affinity of SB 203 186 for atrial 5-HT₄ receptors was 30–160 times higher than that of tropisetron. 5-HT caused tachycardia with similar potency and efficacy in Yucatan minipigs and new-born Camborough piglets. SB 203 186 (0.1–3 mg/kg, i.v.) surmountably antagonised 5-HT-evoked tachycardia in anaesthetised Yucatan minipigs or new-born Camborough piglets with similar potency. The blocking potency of SB 203 186 in Yucatan minipigs was 17 times higher than that of tropisetron. Intraduodenally administered SB 203 186 (0.3–3 mg/kg) to new-born Camborough piglets produced blockade of 5-HT-evoked tachycardia which was maximal after 20 min and lasted for more than 3 h with 0.3 mg/kg. The antagonism produced by the SB 203 186 administration in new-born Camborough piglets was dose-related and threefold greater through the intravenous route than through the intraduodenal route.We conclude that SB 203 186 is an antagonist with nanomolar affinity for both human and porcine atrial 5-HT₄ receptor. The in vivo results demonstrate that the sinoatrial 5-HT₄ receptors function is similar in new-born Camborough piglets and adult Yucatan minipigs. Both porcine breeds are valid models for human atrial 5-HT₄ receptors as demonstrated with the antagonist SB 203186.     

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.

     

Molecular structural basis of ligand selectivity for 5-HT2 versus 5-HT1C cortical receptors

Summary A molecular structural criterion of ligand selectivity for the 5-HT₂ versus 5-HT_1C receptor was hypothesized on the basis of radioligand binding data. Despite the large number of compounds which have been tested at both receptors, analysis of published data led to the identification of only five agents which are greater than 10-fold selective for the 5-HT₂ versus the 5-HT_1C receptor. Comparison of the two-dimensional structures revealed that, although these five compounds represent three distinct structural classes, they share a common structural feature located in the region hypothesized to be involved in receptor binding: a carbonyl or carboxyl oxygen interposed spatially between an aromatic ring and nitrogen atom. This structural feature was used to predict the relative selectivity of compounds that had not previously been analyzed at both the 5-HT₂ and 5-HT_1C receptors.All six drugs tested which contain the identified reactive carbonyl or carboxyl group were found to be selective for the 5-HT₂ versus the 5-HT_1C receptor with selectivity ratios ranging from 26 to 380. By contrast, three agents which are structurally similar but do not contain the reactive carbonyl or carboxyl group displayed equally high affinity for both receptor binding sites. Since the physiological roles of the 5-HT₂ and 5-HT_1C receptor are markedly different, it would be of potential clinical and scientific value to utilize this molecular structural feature to further identify chemical compounds which would selectively interact with only one of the two receptors. Send offprint requests to S. J. Peroutka at the current address     

5-HT7, but not 5-HT2B, receptors mediate hypotension in vagosympathectomized rats

This study evaluated the possible involvement of 5-HT(2B) receptors in long-lasting hypotension to 5-hydroxytryptamine (5-HT), which is predominantly mediated by 5-HT7 receptors, in anaesthetised vagosympathectomized rats. Intravenous injections of 5-HT and 5-carboxamidotryptamine (5-CT) elicited a dose-dependent hypotension that was dose-dependently antagonised by (R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl) ethyl] pyrrolidine (SB-269970; a selective 5-HT7 receptor antagonist), but not by saline. Interestingly, alpha-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine (BW723C86; a 5-HT(2B) receptor agonist) produced vasopressor responses without affecting hypotension to 5-HT. These results suggest that hypotension to 5-HT and 5-CT is mainly mediated by 5-HT7 receptors, whilst the role of 5-HT(2B) receptors seems unlikely.     

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.

     

Evidence for mediation by 5-HT2 receptors of 5-hydroxytryptamine-induced contraction of canine basilar artery

Summary The agonist potencies of 8 indole derivatives and the potencies of 19 recognized antagonists to inhibit constrictor responses to 5-hydroxytryptamine (5-HT) of canine basilar artery were established. In addition the affinities of the indole derivatives for [³H]5-hydroxytryptamine ([³H]5-HT) binding sites and the affinities of the antagonists for [¹²⁵Iodo]LSD ([¹²⁵I]LSD) binding sites in rat brain cortex membranes were determined. Comparison was also made between the potencies of the antagonists on canine basilar artery and the K _D values published for displacement of [³H]ketanserin binding (Leysen et al. 1982).There was a good correlation between the affinities of the antagonists for 5-HT₂ binding sites labelled by both [¹²⁵I]LSD and [³H]ketanserin and the affinity parameters calculated for inhibition of constrictor responses to 5-HT of canine basilar artery. No correlation could be found between the affinities of the indole derivatives for 5-HT₁ binding sites labelled by [³H]5-HT and their potencies to constrict canine basilar artery.It is concluded that constrictor responses to 5-HT of canine basilar artery are mediated by 5-HT₂-like receptors.     

Activation of submucosal 5-HT3 receptors elicits a somatostatin-dependent inhibition of ion secretion in rat colon

Background and purpose:

5-Hydroxytryptamine (5-HT) is a key regulator of the gastrointestinal system and we have shown that submucosal neuronal 5-HT₃ receptors exerted a novel inhibitory effect on colonic ion transport. The aim of the present study was to investigate the precise mechanism(s) underlying this inhibitory effect.

Experimental approach:

Mucosa/submucosa or mucosa-only preparations from rat distal colon were mounted in Ussing chambers for measurement of short-circuit current (I_sc) as an indicator of ion secretion. Somatostatin release was determined with radioimmunoassay. Intracellular cAMP content was measured with enzyme-linked immunoadsorbent assay (elisa). Immunohistochemical techniques were used to study the expression of 5-HT₃ receptors, somatostatin and somatostatin receptors in colonic tissue.

Key results:

In rat distal colonic mucosa/submucosa preparations, pretreatment with 5-HT₃ receptor antagonists enhanced 5-HT-induced increases in I_sc. However, in mucosa-only preparations without retained neural elements, pretreatment with 5-HT₃ receptor antagonists inhibited 5-HT-induced ΔI_sc. Pretreatment with a somatostatin-2 (sst₂) receptor antagonist in mucosa/submucosa preparations augmented 5-HT-induced ΔI_sc. Combination of sst₂ and 5-HT₃ receptor antagonists did not cause further enhancement of 5-HT-induced ΔI_sc. Moreover, both sst₂ and 5-HT₃ receptor antagonists enhanced 5-HT-induced increase in intracellular cAMP concentration in the mucosa/submucosa preparations. 5-HT released somatostatin from rat colonic mucosa/submucosa preparations, an effect prevented by pretreatment with 5-HT₃ receptor antagonists. Immunohistochemical staining demonstrated the presence of 5-HT₃ receptors on submucosal somatostatin neurons and of sst₂ receptors on colonic mucosa.

Conclusion and implications:

Activation of neuronal 5-HT₃ receptors in the submucosal plexus of rat colon suppressed 5-HT-induced ion secretion by releasing somatostatin from submucosal neurons.     

Modulation by 5-HT3 and 5-HT4 receptors of the release of 5-hydroxytryptamine from the guinea-pig small intestine

Summary The effects of agonists and antagonists of 5-hydroxytryptamine (5-HT) receptors on the release of endogenous 5-HT from enterochromaffin cells were studied in the vascularly perfused isolated guinea-pig small intestine. The experiments were done in the presence of tetrodotoxin in order to exclude a neuronally mediated influence on 5-HT release.The 5-HT₃ receptor agonist 2-methyl-5-HT increased 5-HT release, and this effect was antagonized by 1 nmol/l tropisetron. Nanomolar concentrations of tropisetron, MDL 72 222 and granisetron decreased 5-HT release. Ondansetron (0.1 and 1 mol/1) did not modify 5-HT release.5-Methoxytryptamine, BIMU8 and cisapride concentration-dependently inhibited 5-HT release. BIMU8 was more potent than 5-methoxytryptamine. Micromolar concentrations of tropisetron (1 and 10 mol/1) enhanced the release, whilst methiothepine (0.1 mol/l) did not affect the release of 5-HT.The results suggest that enterochromaffin cells of the guinea-pig ileum do not contain 5-HT₁ and 5-HT₂ receptors, but are endowed with 5-HT₃ and 5-HT₄ autoreceptors. Activation of the 5-HT₃ receptors triggers a positive feedback mechanism leading to an increase of 5-HT release. The 5-HT₃ receptors on the enterochromaffin cell differ from neuronal 5-HT3 receptors on guinea-pig myenteric plexus by their high affinity for tropisetron and MDL 72 222, and their very low affinity for ondansetron. Stimulation of 5-HT₄ receptors causes inhibition of release; the inhibitory 5-HT₄ receptor mechanism appears to predominate.Correspondence to H. Kilbinger at the above address     

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.

     

Characterization of an endothelial 5-hydroxytryptamine (5-HT) receptor mediating relaxation of the porcine coronary artery

Summary The pharmacological properties of the endothelial 5-hydroxytryptamine (5-HT) receptors involved in relaxation of vascular smooth muscle were determined in rings of pig coronary artery contracted with 10 nmol/1 of the thromboxane A₂ receptor agonist 9,11-dideoxy-11,9-epoxy-methano-prostaglandin F₂ (U 46619).(1) In the presence of 10 mol/l ketanserin, relaxation was obtained with: 5-HT (apparent pD₂ value 7.00), 5-carboxamidotryptamine (5-CONH₂-T; 6.42), 5-aminotryptamine (5-NH₂-T; 5.96), 5-methoxytryptamine (5-OCH₃-T; 5.92), tryptamine, 7-trifluoromethyl-4(4-methyl-1-piperazinyl)-pyrrolo(1,2-a)quinoxaline maleate (CGS 12066 A) and 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole succinate (RU 24969). The maximum relaxation obtainable with the agonists was about 40–60% of the U 46619-induced contraction and the concentration-response curves for 5-HT, 5-NH₂-T and 5-OCH₃-T were bell-shaped. The endothelium-dependence of this effect (i. e. the failure to relax the artery in endothelium-denuded preparations) was demonstrated for 5-HT, 5-CONH₂-T, RU 24969, CGS 12066A and tryptamine.(2) 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), 4-hydroxytryptamine, quipazine and yohimbine were ineffective in decreasing the tension of arteries with or without endothelium. Ipsapirone elicited full relaxation of U 46619-induced contraction, but this effect was not endothelium-dependent.(3) Metitepine (0.03-1 mol/l), 6-chloro-2-(1-piperazinyl)pyrazine (MK 212; 10 mol/l), methysergide (1 gmol/l) and cyanopindolol (0.1 mol/l) antagonized the relaxing effect of 5-HT in a non-surmountable manner, whereas metergoline (0.1 mol/l), quipazine (10 mol/l), yohimbine (1 mol/l), propranolol (1 mol/l) and (3-tropanyl)-1H-indole-3-carboxylic acid ester (ICS 205-930; 0.1 mol/l) did not. However, spiroxatrine (0.1 mol/l) and mesulergine (10 mol/l) enhanced the 5-HT-induced relaxation. The endothelium-dependent relaxation induced by 5-CONH₂-T was also inhibited by metitepine 1 gmol/l.(4) The 5-HT-induced relaxation was probably mediated by release of an endothelium-derived relaxing factor (EDRF). Gossypol, an inhibitor of EDRF, virtually abolished the 5-HT-induced relaxation while indometacin, an inhibitor of cyclooxygenase and accordingly of PGI₂ formation, did not.In conclusion, the failure of ketanserin and ICS 205–930 to counteract the relaxant effect of 5-HT receptor agonists excludes the involvement of 5-HT₂ and 5-HT₃ receptors, respectively, in the endothelium-dependent relaxation of the porcine coronary artery. The rather high potency of 5-CONH₂-T and the ability of certain 5-HT receptor antagonists, such as metitepine, methysergide and cyanopindolol, to counteract the effect of 5-HT are compatible with a 5-HT₁ character of the endothelial receptor. However, on the basis of the present data, no final classification, in particular with respect to the known 5-HT₁ receptor subtypes, is possible. Classification is also hampered by the bell-shaped character of the concentration-response curves for 5-HT receptor agonists and by their property to produce only partial relaxation. Send offprint requests to M. Gothert at the above address     

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.     

Genetic knockout and pharmacological blockade studies of the 5-HT7 receptor suggest therapeutic potential in depression

The affinity of several antidepressant and antipsychotic drugs for the 5-HT7 receptor and its CNS distribution suggest potential in the treatment of psychiatric diseases. However, there is little direct evidence of receptor function in vivo to support this. We therefore evaluated 5-HT7 receptors as a potential drug target by generating and assessing a 5-HT7 receptor knockout mouse. No difference in assays sensitive to potential psychotic or anxiety states was observed between the 5-HT7 receptor knockout mice and wild type controls. However, in the Porsolt swim test, 5-HT7 receptor knockout mice showed a significant decrease in immobility compared to controls, a phenotype similar to antidepressant treated mice. Intriguingly, treatment of wild types with SB-258719, a selective 5-HT7 receptor antagonist, did not produce a significant decrease in immobility unless animals were tested in the dark (or active) cycle, rather than the light, adding to the body of evidence suggesting a circadian influence on receptor function. Extracellular recordings from hypothalamic slices showed that circadian rhythm phase shifts to 8-OH-DPAT are attenuated in the 5-HT7 receptor KO mice also indicating a role for the receptor in the regulation of circadian rhythms. These pharmacological and genetic knockout studies provide the first direct evidence that 5-HT7 receptor antagonists should be investigated for efficacy in the treatment of depression.     

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.