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The localization of serotonin-7 (5-HT(7)) receptors and the biological activity of ligands have suggested that 5-HT(7) receptors might be involved in pain, migraine, epilepsy, anxiety, depression, memory, and sleep. In the present study, the potential involvement of 5-HT(7) receptors in epilepsy and other seizure disorders was assessed by comparing the seizures produced by three types of electrical stimulation and three chemical convulsants in 5-HT(7) receptor-deficient (knockout, KO) mice to those seizures observed in wild-type (WT) mice. Thresholds for producing electroshock-induced clonic seizures did not differ between KO versus WT mice. However, thresholds for producing electroshock-induced tonic seizures were significantly lower in KO than in WT mice. Seizures produced by pentylenetetrazole (PTZ, a GABA(A) receptor antagonist), N-methyl-d-aspartate (NMDA, an agonist at NMDA-type glutamate receptors), and cocaine (an inhibitor of monoamine uptake) were also studied. PTZ was more potent in inducing seizures in 5-HT(7) KO mice than in wild-type mice. Likewise, cocaine was more potent in inducing seizures in 5-HT(7) KO than in WT mice; moreover, death resulted from cocaine administration in 5-HT(7) KO mice but not in WT mice. There was a similar trend for NMDA that did not reach statistical significance. The present findings point to the potential for a generalized reduction in seizure threshold with constitutive deletion of the 5-HT(7) receptor gene. Since seizures have not been reported with pharmacological blockade of the receptor, the findings suggest that adaptive changes may play a role in the low seizure thresholds in these mice. In addition, the data suggest that the lower thresholds for seizures produced by diverse mechanisms should be taken into account when interpreting other aspects of the phenotype and behavioral pharmacology of this mouse.

We previously demonstrated that 5-hydroxytryptamine 2A (5-HT 2A ) receptor-mediated rat arterial contraction was dependent on activation of tyrosine kinases, including mitogen-activated protein kinase (MAPK) kinase. In the current study, we examined arterial smooth muscle for the presence of serotonin (5-hydroxytryptamine, 5-HT) 5-HT 1B, 5-HT 1D, 5-HT 1F, 5-HT 2A, 5-HT 2B, and 5-HT 7 receptor mRNA and hypothesized that, if present, activation of these receptors would stimulate the extracellular signal-regulated kinase (Erk) MAPK pathway and an Erk MAPK-dependent contraction. RT-PCR analyses of rat aortic smooth muscle cells, cultured and fresh, indicated the presence of 5-HT 1B, 5-HT 1D, 5-HT 1F, 5-HT 2A, 5-HT 2B, and 5-HT 7 receptor mRNA. The 5-HT 1B agonists RU24969 and CGS12066B, 5-HT 1B/1D/1F receptor agonist sumatriptan, and 5-HT 2B receptor agonist BW723C86 (10(-9) - 10(-4) M ) did not contract the aorta, nor did the 5-HT 7 receptor antagonist LY215840 leftward shift 5-HT-induced contraction. The 5-HT 1E/1F receptor agonist BRL54443 induced contraction, but this was abolished by the 5-HT 2A/2C receptor antagonist ketanserin (10 nM ); contraction was not observed with a different 5-HT 1F receptor agonist, LY344864. 5-HT and alpha-methyl-5-HT produced a concentration-dependent increase in Erk MAPK activity in cultured aortic smooth muscle cells and in aorta contracted with these agonists. All other agonists were inactive; a high concentration of BRL54443 (10 microM ) stimulated Erk MAPK activation (150% basal). Thus, while mRNA and possibly protein for multiple 5-HT receptors are present in aortic smooth muscle, only the 5-HT 2A receptor plays a significant role in directly modulating contractility and activating the Erk MAPK pathway.

[3H]5-HT revealed the presence of serotonin receptors in cultured rat sensory neurons. [3H]5-CT binding was inhibited by cyanopindolol with an IC50 of 0.87 +/- 0.30 nM, suggesting the expression of the 5-HT1B receptor in these neurons. The presence of 5-HT1B receptors was confirmed by the displacement of [125I]Iodocyanopindolol binding by cyanopindolol with an IC50 of 2.43 +/- 0.81 nM. 5-HT1B receptors are the predominant type of serotonin receptors labeled by [3H]5-HT in cultured DRG neurons, representing approximately 60% of the specific [3H]5-HT binding sites. In addition, 5-HT1D and 5-HT2A receptor binding was also found in these neurons. RT-PCR analysis of RNA isolated from embryonic sensory neurons in culture confirmed the expression of 5-HT1B, 5-HT1D and 5-HT2A receptor mRNA. It also demonstrated the presence of 5-HT1F, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A and 5-HT5B receptor mRNA and the absence of 5-HT1A, 5-HT1E, 5-HT2B, 5-HT6 and 5-HT7 mRNA. The identification of multiple subtypes of serotonin receptors expressed in cultured embryonic sensory neurons suggests that DRG neuronal cultures may be an excellent model to examine the direct effects of serotonin on the activity of these sensory neurons.

In previous reports, [3H]5-HT has been used to characterize the pharmacology of the rat and human 5-HT2B receptors. 5-HT, the native agonist for the 5-HT2B receptor, has a limitation in its usefulness as a radioligand since it is difficult to study the agonist low-affinity state of a G protein-coupled receptor using an agonist radioligand. When using [3H]5-HT as a radioligand, rauwolscine was determined to have relatively high affinity for the human receptor (Ki human = 14.3+/-1.2 nM, compared to Ki rat = 35.8+/-3.8 nM). Since no known high affinity antagonist was available as a radioligand, these studies were performed to characterize [3H]rauwolscine as a radioligand for the cloned human 5-HT2B receptor expressed in AV12 cells. When [3H]rauwolscine was initially tested for its usefulness as a radioligand, complex competition curves were obtained. After testing several alpha2-adrenergic ligands, it was determined that there was a component of [3H]rauwolscine binding in the AV12 cell that was due to the presence of an endogenous alpha2-adrenergic receptor. The alpha2-adrenergic ligand efaroxan was found to block [3H]rauwolscine binding to the alpha2-adrenergic receptor without significantly affecting binding to the 5-HT2B receptor and was therefore included in all subsequent studies. In saturation studies at 37 degrees C, [3H]rauwolscine labeled a single population of binding sites, Kd = 3.75+/-0.23 nM. In simultaneous experiments using identical tissue samples, [3H]rauwolscine labeled 783+/-10 fmol of 5-HT2B receptors/mg of protein, as compared to 733+/-14 fmol of 5-HT2B receptors/mg of protein for [3H]5-HT binding. At 0 degrees C, where the conditions for [3H]5-HT binding should label mostly the agonist high affinity state of the human 5-HT2B receptor, [3H]rauwolscine (Bmax = 951+/-136 fmol/mg), again labeled significantly more receptors than [3H]5-HT (Bmax = 615+/-34 fmol/mg). The affinity of [3H]rauwolscine for the human 5-HT2B receptor at 0 degrees C did not change, Kd = 4.93+/-1.27 nM, while that for [3H]5-HT increased greatly (Kd at 37 degrees C = 7.76+/-1.06 nM; Kd at 0 degrees C = 0.0735+/-0.0081 nM). When using [3H]rauwolscine as the radioligand, competition curves for antagonist structures modeled to a single binding site, while agonist competition typically resulted in curves that best fit a two site binding model. In addition, many of the compounds with antagonist structures displayed higher affinity for the 5-HT2B receptor when [3H]rauwolscine was the radioligand. Typically, approximately 85% of [3H]rauwolscine binding was specific binding. These studies display the usefulness of [3H]rauwolscine as an antagonist radioligand for the cloned human 5-HT2B receptor. This should provide a good tool for the study of both the agonist high- and low-affinity states of the human cloned 5-HT2B receptor.

Two mutations of the rat serotonin 5-HT2A receptor were made, expressed and examined for their ability to bind and be stimulated by certain tryptamines as well as their ability to bind antagonists. Mutation of Ser207 to an Ala (S207A) resulted in no substantial changes in binding of either 5-HT2A antagonists or agonists. In contrast, mutation of Ser239 to an Ala (S239A) resulted in significant changes in the 5-HT2A receptor with some but not all agonists and antagonists examined. Specifically, 5-HT had decreased affinity for the S239A mutated 5-HT2A receptor, showing over a 10-fold decrease in receptor-binding displacement, while still being capable of stimulating IP3 formation. However, the agonists tryptamine, 5-methoxytryptamine (5-MeOT), and N-1-isopropyl-5-methoxytryptamine; and the antagonists ketanserin, LY 86057, and LY 53857 were significantly less affected by a S239A mutation. These results suggest that while 5-HT might have a direct interaction with the Ser239 of the 5-HT2A receptor, tryptamine and 5-MeOT interact with this receptor in a different manner.

One of the most profound increases in vascular responsiveness in hypertension has been observed for serotonin (5-hydroxytryptamine, 5-HT). This study investigates the hypothesis that the increase in vascular responsiveness to 5-HT is the result of altered 5-HT receptor signal transduction. Mesenteric arteries were dissected from deoxycorticosterone- (DOCA) salt hypertensive and sham-normotensive rats for use in isolated tissue experiments. Agonist contractile potencies indicated that a 5-HT2 receptor mediates contraction to 5-HT in both sham and DOCA-salt arteries. In arteries from sham rats, ketanserin (5-HT2A/5-HT2C selective), LY53857 (5-HT2 selective) and spiperone (5-HT2A/5-HT2C selective) shifted contraction to 5-HT (pKB = 8.58, 8.35 and 9.52, respectively) indicating that a 5-HT2A receptor mediates contraction in arteries from normotensive rats. By contrast, ketanserin and spiperone did not shift contraction to 5-HT in DOCA-salt mesenteric arteries (pKB > 6.52, > 7.52, respectively). LY53857 did shift the response to 5-HT in DOCA-salt mesenteric arteries (pKB = 7.85). Thus, contraction in arteries from DOCA-salt rats is predominantly mediated by 5-HT2B receptors. Unlike the 5-HT receptor in the sham mesenteric artery and aorta (5-HT2A receptor), the 5-HT receptor in DOCA-salt mesenteric arteries and stomach fundus (5-HT2B receptor) were relatively insensitive to phenoxybenzamine (10-300 nM). These data suggest that the 5-HT2B receptor is insensitive to phenoxybenzamine, is increased in number or, alternatively, has increased G protein coupling. DOCA-salt mesenteric arteries were more sensitive to contraction by the direct G protein stimulator AIF4- (-log EC50 [M]: DOCA-salt = 2.82 +/- 0.04; sham = 2.55 +/- 0.03, P < .05). PCR analyses indicated an increase in mRNA for the 5-HT2B receptor in mesenteric arteries of DOCA-salt hypertensive arteries, supporting an increase in receptor number. Taken together these studies demonstrate significant changes in 5-HT receptor signal transduction in DOCA-salt hypertension, both at the level of the receptor and G protein and may provide one reason why ketanserin has proved to be a relatively ineffective antihypertensive agent in some forms of hypertension.

The 5-Hydroxytryptamine2B (5-HT2B) receptor was cloned originally from rat stomach fundus and its pharmacology was determined to be consistent with that of the receptor responsible for contraction of rat fundal tissue in response to 5-HT. Recently, the cloning of the human homolog of the 5-HT2B receptor has been reported and, in this study, we report a detailed pharmacological characterization of this human receptor. The cloned human 5-HT2B receptor has high affinity for [3H]5-HT (Kd = 10.6 +/- 1.5 nM), and the pharmacology of this receptor matches closely the rat 5-HT2B receptor, consistent with the structural relatedness of these two proteins. Most compounds tested show no difference in affinity for the human or rat receptors. There were, however, groups of compounds that discriminated between the human and rat 5-HT2B receptors. Examples include certain ergolines such as methysergide and mesulergine, which have higher affinity for the human than for the rat receptor. Similarly, certain benzoylpiperidines, e.g., ketanserin, pirenperone and pipamperone, and the antipsychotics clozapine and olanzapine have higher affinity for the human 5-HT2B receptor. These pharmacological findings reinforce the desirability of having the human forms of receptors when considering drug actions.

The 5-Hydroxytryptamine (5-HT)2C receptor (originally known as the 5-HT1C receptor) is a member of the 5-HT2 subfamily of G protein coupled receptors, which is known to couple to phospholipase C. Within the 5-HT2 subfamily, only the 5-HT2C receptor also coupled to inhibition of forskolin-stimulated cAMP production when expressed at high density (12 pmol/mg membrane protein) in stably transformed AV12 cells. The 5-HT2C receptor coupled with high efficacy to both phospholipase C as measured by IP3 (inositol 1,4,5-trisphosphate) production and to inhibition of forskolin-stimulated cAMP production (EC50 = 2.98 nM +/- 0.9 and IC50 = 47.99 nM +/- 10.25 respectively). The 5-HT2A and 5-HT2B receptors, while coupling to phospholipase C with high affinity (EC50s of 19.24 nM +/- 6.44 and 1.24 nM +/- 0.136 respectively), did not decrease adenylyl cyclase activity. The 5-HT2C receptor actions in both systems showed the expected pharmacology for the 5-HT2C receptor, e.g., mesulergine antagonized the effects of 5-HT and spiperone did not. Preincubation of cells with PTX showed that the G protein coupling of the 5-HT2C receptor to phospholipase C is PTX insensitive, while the G protein coupling to inhibition of adenylyl cyclase is PTX sensitive, even to concentrations as low as 20 ng/ml of PTX. PTX pretreatment of the 5-HT2C bearing cells also unmasked a small stimulatory effect on adenylyl cyclase. When expressed at low density the 5-HT2C receptor potentiated forskolin-stimulated cAMP production by 2 fold while still maintaining its ability to enhance PI hydrolysis. A more modest potentiation of cAMP production was noted with low density expression of the 5-HT2B receptor. Thus the ability of the 5-HT2C receptor to interact with several effectors through at least two different G proteins is, in part, receptor subtype specific but also influenced by receptor density.

Over the last several years the use of molecular cloning technology has revealed a vast diversity among serotonin (5-HT) receptors, whereby what was previously thought to be a family of three pharmacologically defined classes of 5-HT receptors is actually composed of seven distinct subfamilies designated 5-HT1-7. The 5-HT1, 5-HT2, and 5-HT5 subfamilies currently consist of five, three and two subtypes respectively while the 5-HT3, 5-HT4, 5-HT6, and 5-HT7 "subfamilies" have at present one subtype each. Fourteen separate genes encode 13 receptors which fall in the superfamily of G protein-coupled receptors and one ligand-gated ion channel receptor. Our lab has contributed to the elucidation of this subtype diversity by cloning the cDNAs from both rat and human encoding the 5-HT2B receptor. This receptor subtype is equally homologous (approximately 70%) to the 5-HT2A and 5-HT2C receptors when amino acids comprising the transmembrane domains are compared and is clearly the third member of the 5-HT2 subfamily. The 5-HT2B receptor has been shown to couple to phosphoinositide hydrolysis as do the other two members of this subfamily when expressed in AV12-664 cells. Limited pharmacological analyses indicated that both rat and human 5-HT2B receptors are similar but distinguishable. With one tantalizing exception, the mRNAs for these receptors appear to be similarly distributed within rat and human. The 5-HT2B receptor mRNA is not found in rat brain, whereas in human brain it has been identified in multiple regions. This later finding suggests that the 5-HT2B receptor may be serving a unique CNS function in man that is absent in rat.

Pig and rhesus monkey 5-HT2A receptor cDNA clones were isolated. The pig and rhesus monkey clones encode proteins that share a 94% and 95% homology, respectively, with the rat 5-HT2A receptor, and a 97% and > 99% homology, respectively, with the human 5-HT2A receptor. Within the transmembrane regions of the pig and monkey receptors, the deduced amino acid shows only three differences compared to that of the rat and are identical to the human 5-HT2A receptor clone.

Currently three subtypes of the 5-HT2 receptor have been identified through binding studies and/or molecular biology studies, 5-HT2A, 5-HT2B, and 5-HT2C. The identification of these receptor subtypes in the spinal cord of humans and experimental animal species is incomplete. In the present study, polymerase chain reaction with Southern hybridization analysis was used to determine whether the mRNA for the 5-HT2A, 5-HT2B and 5-HT2C receptors could be detected in spinal cord tissue from rats, cats, monkeys and humans. Brain tissue from rat, cat and monkey were also examined for comparison. 5-HT2A and 5-HT2B receptor mRNAs were amplified and localized with rat 5-HT2A and 5-HT2B primers and probes, respectively, in tissue from all species. In rats, 5-HT2C receptor mRNA was amplified and localized with rat 5-HT2C primer and mouse 5-HT2C probe, respectively, while in cat, monkey, and human tissue, human 5-HT2C primers and probes were used. Spinal cord from all four species contained mRNA for the 5-HT2A receptor. Brain tissue from rat, cat and monkey also contained 5-HT2A mRNA. Spinal cord tissue from all four species contained 5-HT2B message. Brain tissue from cat and monkey contained 5-HT2B mRNA, but this was not detected in rat brain. 5-HT2C mRNA was detected in spinal cord and brain tissue from rat, monkey and human, but was only detected in brain tissue of the cat (i.e. no 5-HT2C mRNA was detected in cat spinal cord).(ABSTRACT TRUNCATED AT 250 WORDS)

The serotonin 2c (5-HT2C) receptor mediates its cellular effects by interacting with heterotrimeric guanine nucleotide binding proteins (G proteins). To characterize which G proteins are involved in functional coupling to the receptor, a mouse 5-HT2C receptor was expressed in Xenopus oocytes, and antisense oligoncleotides complementary to the mRNA sequence of the endogenous Xenopus G protein alpha subunits were used to inhibit G protein synthesis. Antisense oligonucleotide against the Xenopus G(o) alpha subunit inhibited the 5-HT2C receptor function, and coexpression of a rat G(o) alpha subunit reversed the inhibition by the anti-Xenopus G(o) oligonucleotide. Furthermore, antisense oligonucleotides against both the G(o) and Gi1 alpha subunits inhibited the electrophysiologic response induced by stimulation of the 5-HT2C receptor. These data suggest that both G(o) and Gi1 are involved in functional coupling of the 5-HT2C receptor to phospholipase C in Xenopus oocytes.

Clones encoding a portion of the human 5-hydroxytryptamine (5-HT)2B receptor gene were isolated from a human placental genomic library. Based on distribution studies of 5-HT2B receptor mRNA, human uterus cDNA libraries were constructed and screened, resulting in the isolation of several full-length cDNA clones. These clones harbored a common single open reading frame encoding a protein of 481 amino acids. The deduced amino acid sequence of the human 5-HT2B receptor displayed 91.5% identity within the transmembrane domains and 82% identity overall with the rat 5-HT2B receptor. The human 5-HT2B receptor stably expressed in AV12-664 cells demonstrated high affinity (Kd = 10.18 +/- 1.60 nM), saturable [3H]serotonin binding, similar to that previously described for the rat 5-HT2B receptor. The pharmacological profile of the human 5-HT2B receptor was virtually identical to that of the rat 5-HT2B receptor, with the exceptions of the 5-HT2A receptor antagonists ketanserin and spiperone. Both compounds exhibited higher affinity at the human 5-HT2B receptor (ketanserin, Ki = 376 +/- 58 nM; spiperone, Ki = 697 +/- 54 nM) than at the rat 5-HT2B receptor (ketanserin, Ki = 3559 +/- 175 nM; spiperone, Ki = 3278 +/- 92 nM). Functional coupling of the human 5-HT2B receptor was also demonstrated in AV12-664 cells, where 5-HT produced a dose-dependent increase in phosphatidylinositol hydrolysis (EC50 = 27 +/- 12 nM) analogous to that seen with the rat 5-HT2B receptor. Reverse transcription-polymerase chain reaction studies revealed human 5-HT2B receptor mRNA to be expressed in many tissues, including the central nervous system. The presence of 5-HT2B receptor mRNA in human brain and not in rat brain raises the possibility that the 5-HT2B receptor may be of significance in higher brain function.

Heterogeneity of the 5-hydroxytryptamine2 (5-HT2) receptor across species has been implicated in several pharmacological and physiological studies. Although 5-HT2 receptors in the rat have been linked to increases in phosphoinositide (PI) hydrolysis, little evidence exists to support the association of guinea pig 5-HT2 receptors with PI hydrolysis, the second messenger generally linked with 5-HT2 receptors. In the present study, we have taken a molecular and biochemical approach to determining whether species differences in brain 5-HT2 receptors exist between rat and guinea pig. First, we isolated partial cortical 5-HT2 receptor cDNA clones that encompassed the third intracellular loop, a receptor area putatively important in receptor-effector coupling. The amino acid sequences deduced from the cDNA clones for rat and guinea pig brain 5-HT2 receptor were 97% homologous. However, the guinea pig 5-HT2 receptor had two tandem substitutions that disrupted a potential alpha helix in the region of the third cytoplasmic loop, which theoretically could alter the intracellular coupling of the guinea pig cortical 5-HT2 receptor. Because of these molecular differences, we examined further the pharmacological activation of the brain 5-HT2 receptor from guinea pig. 5-HT and the 5-HT2 receptor agonist alpha-methyl-5-HT increased PI hydrolysis in guinea pig cortical slices whereas the 5-HT1C receptor agonist 5-methyltryptamine was significantly less potent. In addition, the 5-HT2 receptor antagonists LY53857, ketanserin, and spiperone blocked 5-HT-stimulated PI hydrolysis. These pharmacological data suggested that activation of the 5-HT2 receptor in guinea pig cortical slices was associated with PI hydrolysis. Thus, although areas of the guinea pig brain 5-HT2 receptor that influence receptor-effector coupling were different from the rat, such differences were not critical to receptor-effector coupling because, as in the rat, guinea pig brain 5-HT2 receptors were also coupled to PI hydrolysis.

Previous work has suggested that species differences in the structure-activity relationship for ergolines and tryptamines at the 5-hydroxytryptamine (5-HT)2A (formerly known as the 5-HT2) receptor are related to aliphatic substitution at the N1-position on the indole nucleus. The present work has confirmed these findings by examining the rat and human cloned 5-HT2A receptors. As previously found, N1-substitution of ergolines or tryptamines had no effect or increased affinity for the rat 5-HT2A receptor but decreased affinity for the human receptor. Also, the N1-unsubstituted analogues had higher affinity for the human 5-HT2A receptor, whereas the N1-alkyl analogues had a higher affinity for the rat receptor. By mutating the rat 5-HT2A receptor, the importance of the Ala/Ser242 species variation in amino acid sequence was examined in relation to this structure-activity relationship. Three mutations of the rat 5-HT2A receptor were made, i.e., A242S, A242V, and A242T. All three mutations resulted in functional (able to stimulate inositol phosphate hydrolysis) 5-HT2A receptors with high affinity for [3H]ketanserin and 1-(2,5-dimethoxy-4-[125I]iodophenyl)isopropylamine. The A242S mutation resulted in a pharmacological profile that was almost identical to that of the human 5-HT2A receptor but differed significantly from that of the wild-type rat receptor. This strongly suggests that the Ala/Ser242 species variation accounts for the differences in the structure-activity relationship. The A242V and A242T mutations resulted in differing but profound effects on affinity for the different ergolines and tryptamines. The results are discussed in terms of the importance of position 242 in the binding of these ligands to 5-HT2A receptors. In addition, arguments are presented that suggest that a hydrogen-bonding interaction occurs between the human 5-HT2A receptor at Ser242 and the N1-hydrogen of N1-unsubstituted ergolines and tryptamines and may serve as an important contact point in the receptor.

The identity of the serotonin (5-HT) receptor(s) that mediate(s) contraction in canine coronary artery and saphenous vein remains controversial. Ring segments of endothelium-denuded coronary artery and helical strips of saphenous vein were suspended in organ chambers for measurement of isometric force. 5-HT, alpha Me-5-HT and sumatriptan contracted both coronary artery and saphenous vein and the non-selective 5-HT receptor antagonist 1-naphthylpiperazine (100nM) blocked 5-HT- and sumatriptan-induced contraction in both tissues. The agonist rank order potency for contraction (5-HT > sumatriptan > alpha Me5-HT > 5-MeOT > 5-MeT) was similar in both tissues and was consistent with that for a 5-HT1D receptor. Oligonucleotide primers specific for the 5-HT1D receptor sequence were designed for use in a polymerase chain reaction (PCR). cDNA derived from total RNA or mRNA from canine tissues was used in the PCR. PCR resulted in the amplification of a 632 base pair sequence in both canine coronary artery and saphenous vein; consistent with that expected for the 5-HT1D receptor. Southern blot analysis, with an oligonucleotide probe internal to the sequence amplified by the PCR primers, confirmed that the sequence amplified by PCR was the 5-HT1D receptor. Thus, the 5-HT1D receptor is expressed in canine coronary artery and saphenous vein and taken together with the pharmacological data, supports the possibility that a 5-HT1D-like receptor mediates contraction in these two tissues.

The present studies document marked differences in contractile responsiveness to serotonin in trachea and aorta between guinea pig and rat. For example, the guinea pig trachea and rat aorta markedly contract in response to serotonin via activation of 5-HT2A receptors. In contrast, the rat trachea and guinea pig aorta only modestly contract to serotonin. The availability of 5-HT2A receptor selective cDNA clones from brain of both guinea pig and rat permitted molecular probes to be designed and PCR amplification studies initiated to identify and quantify 5-HT2A receptor specific mRNA in these tissues. For trachea, 3-fold higher concentrations of 5-HT2A receptor specific mRNA were found in guinea pig relative to rat trachea. These data are consistent with the more profound contractile response to serotonin in guinea pig versus rat trachea and suggest that differences in tracheal contractility to serotonin correlate with the density of 5-HT2A receptor mRNA. In contrast, although rat aorta contracted more dramatically to serotonin than guinea pig aorta, rat aorta possessed a similar concentration of 5-HT2A receptor specific mRNA as compared to guinea pig aorta. Thus, for the aorta, differences in the concentration of 5-HT2A receptor mRNA are not sufficient to explain the observed differences in contractility between tissues from guinea pig and rat. These studies documenting 5-HT2A receptor mRNA in rat trachea and guinea pig aorta, two tissues that do not markedly contract in response to serotonin indicate that 5-HT2A receptor mRNA although present, has not resulted in a receptor capable of mediating a contractile response in these tissues.

The rat 5-hydroxytryptamine (5-HT)2F (serotonin2F) receptor is a newly cloned member of the 5-HT2/1C receptor family. The pharmacology of the 5-HT2F receptor was explored using a variety of structurally different compounds in a radioligand binding assay. In addition, the 5-HT2F receptor was shown to stimulate production of inositol 1,4,5-trisphosphate in the transformed cells. Based on the affinities of the compounds tested, their known affinities for certain of the other 5-HT receptors, and the fact that activation of the cloned 5-HT2F receptor stimulates inositol 1,4,5-trisphosphate production, the 5-HT2F receptor was determined to be a novel receptor and a member of the 5-HT2/1C receptor family. In addition, several agonists and partial agonists were evaluated for contractile activity in the rat stomach fundus, and these activities were correlated with their binding affinities at the 5-HT2F receptor. A highly significant correlation was found, providing additional evidence that is consistent with the 5-HT2F receptor being the stomach fundal contractile receptor. [3H]5-HT had high affinity for this receptor both at 37 degrees and at 0 degree (Kd = 7.87 +/- 0.55 and 0.12 +/- 0.02 nM, respectively). The difference in affinity for [3H]5-HT at the two temperatures prompted an investigation of potential temperature-dependent differences in the binding affinities of agonists versus antagonists. Agonists such as 5-HT, 5-methoxytryptamine, etc., showed higher affinity for the 5-HT2F receptor at 0 degree than at 37 degrees, whereas antagonists such as methysergide, 1-naphthylpiperazine, etc., showed no difference in affinity for this receptor at the two different temperatures. Therefore, the affinity of a compound for the 5-HT2F receptor at 37 degrees versus 0 degree was shown to be useful for predicting agonist or antagonist activity. Additionally, information is provided about some of the structural requirements for the affinity of certain tryptamines at the 5-HT2F receptor.

Using the polymerase chain reaction amplification technique in conjunction with conventional cloning techniques, we have isolated a novel member of the serotonin [5-hydroxytryptamine (5-HT)] 1C/2 receptor subfamily (designated 5-HT2F) from rat stomach fundus. Two DNA fragments were amplified from cDNA synthesized from rat stomach fundus poly(A)+ RNA using the polymerase chain reaction technique with degenerate oligonucleotide primers derived from sequence comparisons of the second, third, and sixth putative transmembrane domains of known 5-HT receptors. These fragments were used as hybridization probes to isolate full length cDNA clones from rat stomach fundus cDNA libraries. Full length cDNA clones contained one open reading frame encoding a 479-amino acid protein with seven hydrophobic domains, characteristic of members of the guanine nucleotide-binding protein-coupled receptor superfamily. Within these seven putative transmembrane domains, the 5-HT2F receptor shared greatest homology with the rat 5-HT1C and 5-HT2 receptor subtypes (70% and 68%, respectively). Cell lines stably expressing the 5-HT2F receptor were established and demonstrated functional coupling to phosphatidylinositol hydrolysis upon 5-HT stimulation analagous to that observed for the 5-HT1C and 5-HT2 receptors. Membranes from the stably transfected cell lines (but not the untransfected parental lines) exhibited high affinity (Kd = 7.9 nM), saturable binding of [3H]5-HT. Maximum binding ranged from 0.1 to 2.4 pmol/mg of protein, depending on the clonal isolate. Using [3H]5-HT as the basis for a radioligand binding assay, the relative affinities of several tryptamine and piperazine derivatives for the cloned 5-HT2F receptor correlated with their relative potencies to contract the rat stomach fundus. These data suggest a probable relationship between this novel 5-HT2F receptor and the serotonin contractile receptor of the rat stomach fundus.

The receptor mediating contraction in response to serotonin in the rat stomach fundus has not been characterized in light of the currently acceptable serotonergic receptor classification scheme. Several biochemical and pharmacological approaches to a characterization of this receptor have demonstrated nonidentity with the 5-hydroxytryptamine2 (5HT2), 5HT3, 5HT1A, and 5HT1B receptors, as defined by radiolabeled ligand binding studies in brain cortical membranes. Although there have been reports suggesting that the receptor in the rat stomach fundus may be analogous to the 5HT1C receptor, other pharmacological and biochemical studies have not been consistent with this idea. The present study utilized high affinity ligands for the 5HT1C receptor and the recently derived 5HT1C receptor cDNA clone to provide a more definitive approach to the examination of the relationship between the 5HT1C receptor and the serotonergic contractile receptor in the rat stomach fundus. Using three ligands with high affinity at the 5HT1C receptor, LY53857, ritanserin, and SCH23390, the contractile response to serotonin was inhibited by all three ligands. However, inhibition did not appear competitive nor was the inhibitory potency of these ligands consistent with their affinity at 5HT1C binding sites in brain cortical membranes. We further showed that SCH23390, unlike LY53857 and ritanserin, was also a partial agonist, producing a maximal contraction that was approximately 50% of the maximal response to serotonin in the rat stomach fundus. Thus, the use of these ligands did not support the contention that the receptor mediating serotonin-induced contractions in the rat stomach is identical to the 5HT1C receptor. In more definitive studies using a 5HT1C receptor cDNA probe, we were unable to detect hybridization of the probe with any mRNA species from the rat stomach fundus, whereas the 5HT1C receptor cDNA probe did hybridize to the 5HT1C receptor mRNA in rat brain. Because the cathepsin-D cDNA probe hybridized equally in rat brain and stomach fundus, ensuring the integrity of the RNA preparation from both tissues, the absence of measurable quantities of the 5HT1C receptor mRNA in the rat stomach was probe specific and not an artifact. Furthermore, primers specific for the rat 5HT1C receptor sequence did not detect significant levels of receptor mRNA in rat fundus, although the target sequence was amplified a minimum of 10(5)-fold in a polymerase chain reaction. These studies do not support the contention that the receptor mediating contractile responses to serotonin in the rat stomach fundus is identical to the 5HT1C receptor.(ABSTRACT TRUNCATED AT 400 WORDS)