Differential addressing of 5-HT1A and 5-HT1B receptors in epithelial cells and neurons.
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The 5-HT1A and 5-HT1B serotonin receptors are expressed in a variety of neurons in the central nervous system. While the 5-HT1A receptor is found on somas and dendrites, the 5-HT1B receptor has been suggested to be localized predominantly on axon terminals. To study the intracellular addressing of these receptors, we have used in vitro systems including Madin-Darby canine kidney (MDCK II) epithelial cells and primary neuronal cultures. Furthermore, we have extended these studies to examine addressing in vivo in transgenic mice. In epithelial cells, 5-HT1A receptors are found on both apical and basolateral membranes while 5-HT1B receptors are found exclusively in intracellular vesicles. In hippocampal neuronal cultures, 5-HT1A receptors are expressed on somatodendritic membranes but are absent from axons. In contrast, 5-HT1B receptors are found on both dendritic and axonal membranes, including growth cones where they accumulate. Using 5-HT1A and 5-HT1B knockout mice and the binary tTA/tetO system, we generated mice expressing these receptors in striatal neurons. These in vivo experiments demonstrate that, in striatal medium spiny neurons, the 5-HT1A receptor is restricted to the somatodendritic level, while 5-HT1B receptors are shipped exclusively toward axon terminals. Therefore, in all systems we have examined, there is a differential sorting of the 5-HT1A and 5-HT1B receptors. Furthermore, we conclude that our in vivo transgenic system is the only model that reconstitutes proper sorting of these receptors. (+info)
Precontraction with elevated concentrations of extracellular potassium enables both 5-HT1B and 5-HT2A "silent" receptors in rabbit ear artery.
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The present study was conducted to determine the effect of a small (<10%) K+-induced precontraction on the response to vasoconstrictors in the rabbit aorta and ear artery rings. In both tissues, 15 mM K+ shifted the methoxamine concentration response curve (CRC) approximately 2.4-fold to the left. There was no change in the sensitivity of the control and amplified CRCs to the alpha1 adrenoceptor antagonist prazosin (100 nM). In the aorta, the CRC for serotonin was shifted 4.5-fold to the left in the presence of 15 mM K+, and both the control and amplified CRCs were antagonized equally by the 5-HT2A antagonist ketanserin (10 nM). In contrast, 16 and 20 mM K+ caused up to an approximately 60-fold leftward shift of the serotonin CRC in the rabbit ear artery. This effect of 16 mM K+ was not altered by mechanical removal of the endothelium or by in vitro denervation using 6-hydroxydopamine. The K+-amplified CRC was insensitive to 100 nM prazosin at serotonin concentrations below 3 microM, but was significantly antagonized by 10 nM ketanserin, suggesting that 5-HT2A receptors are involved in the K+-amplified response. The 5-HT1B-selective antagonist, GR 127935, did not affect control responses to serotonin, but significantly blocked the K+-amplified response. Furthermore, the combination of ketanserin and GR 127935 produced a significantly greater blockade of the amplified response than either antagonist alone, supporting the conclusion that both 5-HT2A and 5-HT1B receptors mediate the K+-amplified response to serotonin in the rabbit ear artery. (+info)
Canine external carotid vasoconstriction to methysergide, ergotamine and dihydroergotamine: role of 5-HT1B/1D receptors and alpha2-adrenoceptors.
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The antimigraine drugs methysergide, ergotamine and dihydroergotamine (DHE) produce selective vasoconstriction in the external carotid bed of vagosympathectomized dogs anaesthetized with pentobarbital and artificially respired, but the receptors involved have not yet been completely characterized. Since the above drugs display affinity for several binding sites, including alpha-adrenoceptors and several 5-HT1 and 5-HT2 receptor subtypes, this study has analysed the mechanisms involved in the above responses. Intracarotid (i.c.) infusions during 1 min of methysergide (31-310 microg min(-1)), ergotamine (0.56-5.6 microg min(-1)) or DHE (5.6-31 microg min(-1)) dose-dependently reduced external carotid blood flow (ECBF) by up to 46+/-4, 37+/-4 and 49+/-5%, respectively. Blood pressure and heart rate remained unchanged. The reductions in ECBF by methysergide were abolished and even reversed to increases in animals pre-treated with GR127935 (10 microg kg(-1), i.v.). The reductions in ECBF by ergotamine and DHE remained unchanged in animals pre-treated (i.v.) with prazosin (300 microg kg(-1)), but were partly antagonized in animals pre-treated with either GR127935 (10 or 30 microg kg(-1)) or yohimbine (1000 microg kg(-1)). Pre-treatment with a combination of GR127935 (30 microg kg(-1)) and yohimbine (1000 microg kg(-1)) abolished the responses to both ergotamine and DHE. The above doses of antagonists were shown to produce selective antagonism at their respective receptors. These results suggest that the external carotid vasoconstrictor responses to methysergide primarily involve 5-HT1B/1D receptors, whereas those to ergotamine and DHE are mediated by 5-HT1B/1D receptors as well as alpha2-adrenoceptors. (+info)
Pharmacological diversity between native human 5-HT1B and 5-HT1D receptors sited on different neurons and involved in different functions.
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The releases of [3H]5-hydroxytryptamine ([3H]5-HT) and of endogenous glutamic acid and their modulation through presynaptic h5-HT1B autoreceptors and h5-HT1D heteroreceptors have been investigated in synaptosomal preparations from fresh neocortical samples obtained from patients undergoing neurosurgery. The inhibition by 5-HT of the K+ (15 mM)-evoked overflow of [3H]5-HT was antagonized by the 5-HT1B/5-HT1D receptor ligand GR 127935, which was ineffective on its own; this drug was previously found to behave as a full agonist at the h5-HT1D heteroreceptor regulating glutamate release. The recently proposed selective h5-HT1B receptor ligand SB-224289 also prevented the effect of 5-HT at the autoreceptor, being inactive on its own; in contrast, SB-224289, at 1 microM, was unable to interact with the h5-HT1D heteroreceptor. The inhibitory effect of 5-HT on the K+-evoked overflow of glutamate was antagonized by the h5-HT1D receptor ligand BRL-15572; added in the absence of 5-HT the compound was without effect. BRL-15572 (1 microM) was unable to modify the effect of 5-HT at the autoreceptor regulating [3H]5-HT release. The selective 5-HT1A receptor antagonist (+)-WAY 100135, previously found to be an agonist at the h5-HT1D heteroreceptor regulating glutamate release, could not interact with the h5-HT1B autoreceptor when added at 1 microM. It is concluded that native h5-HT1B and h5-HT1D receptors exhibit a hitherto unexpected pharmacological diversity. (+info)
Regulation of calcitonin gene-related peptide secretion by a serotonergic antimigraine drug.
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We have investigated the regulation of calcitonin gene-related peptide (CGRP) release from trigeminal neurons by the serotonergic antimigraine drug sumatriptan. Serum levels of the neuropeptide CGRP are elevated during migraine. Treatment with the drug sumatriptan returns CGRP levels to normal coincident with the alleviation of headache. However, despite this clinical efficacy, the cellular target and mechanism of sumatriptan action are not well understood beyond the pharmacology of its recognition of the 5-HT1 class of serotonin receptors. We have used cultured trigeminal neurons to demonstrate that sumatriptan can directly repress CGRP secretion from sensory neurons. The stimulated secretion in response to depolarization or inflammatory agents was inhibited, but not the basal secretion rate. Unexpectedly, sumatriptan did not lower cAMP levels, in contrast to the classical role ascribed to the 5-HT1 receptors. Instead, activation of 5-HT1 receptors caused a slow and remarkably prolonged increase in intracellular calcium. The inhibition of CGRP secretion is attenuated by the phosphatase inhibitor okadaic acid, suggesting that sumatriptan action is mediated by calcium-recruited phosphatases. These results suggest that 5-HT1 agonists may block a deleterious feedback loop in migraine at the trigeminal neurons and provide a general mechanism by which this class of drugs can attenuate stimulated neuropeptide release. (+info)
Dural vasodilation causes a sensitization of rat caudal trigeminal neurones in vivo that is blocked by a 5-HT1B/1D agonist.
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1. Migraine headache pain is thought to result from an abnormal distention of intracranial, extracerebral blood vessels and the consequent activation of the trigeminal nervous system. Migraine is also often accompanied by extracranial sensory disturbances from facial tissues. These experiments investigate whether meningeal dilation produces central sensitization of neurones that receive convergent input from the face. 2. Single unit extracellular activity was recorded from the trigeminal nucleus caudalis of anaesthetized rats in response to either noxious stimulation of the dura mater, innocuous stimulation of the vibrissae or to a transient dilation of the meningeal vascular bed. 3. Rat alpha-CGRP (calcitonin gene-related peptide; 1 microg kg(-1), i.v.) caused a dilation of the middle meningeal artery and facilitated vibrissal responses by 36+/-7%. 4. The 5-HT1B/1D agonist, L-741,604 (3 mg kg(-1), i.v.), inhibited responses to noxious stimulation of the dura mater (16+/-7% of control) and, in a separate group of animals, blocked the CGRP-evoked facilitation of vibrissal responses. 5. L-741,604 (3 mg kg(-1), i.v.) also inhibited responses to innocuous stimulation of the vibrissa (14+/-10% of control) with neurones that received convergent input from the face and from the dura mater, but not with cells that received input only from the face (70+/-12% of control). 6. These data show that dilation of meningeal blood vessels causes a sensitization of central trigeminal neurones and a facilitation of facial sensory processing which was blocked by activation of pre-synaptic 5-HT1B/1D receptors. 7. Sustained dural blood vessel dilation during migraine may cause a sensitization of trigeminal neurones. This may underlie some of the symptoms of migraine, such as the headache pain and the extracranial allodynia. Inhibition of this central sensitization may therefore offer a novel strategy for the development of acute and/or prophylactic anti-migraine therapies. (+info)
5-hydroxytryptamine stimulation of phospholipase D activity in the rabbit isolated mesenteric artery.
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1. The involvement of phospholipase D (PLD) in the 5-hydroxytryptamine 5-HT1B/5-HT1D-signalling pathway was assessed in the rabbit isolated mesenteric artery. 2. RT-PCR analysis of mesenteric smooth muscle cells revealed a strong signal corresponding to mRNA transcript for the 5-HT1B receptor. The PCR fragment corresponded to the known sequence for the 5-HT1B receptor. No signal corresponding to 5-HT1D mRNA was detected. 3. Neither 5-HT (3 microM) nor KCl (45 mM) individually stimulated any significant increase in the smooth muscle concentration of [33P]-PtdBut to reflect PLD activity. However, in the presence of KCl (45 mM), 5-HT evoked a concentration-dependent increase in [33P]-PtdBut, to a maximum of 84% with 5-HT (3 microM). 4. [33P]-PtdBut accumulation evoked by 5-HT in the presence of KCl was abolished in nominally calcium-free Krebs-Henseleit Buffer (KHB) or with the selective protein kinase C inhibitor, Ro-31 8220 (10 microM, 20 min). 5. 5-HT (3 microM) in the presence of KCl (45 mM) failed to increase either the accumulation of [33P]-phosphatidic acid in the presence of butanol, or total [3H]-inositol phosphates ([3H]-InsP) in the presence of LiCl (10 mM). 6. 5-HT (0.1-1 microM) abolished forskolin (1 microM) stimulated increases in cyclic AMP (15 fold increase), an action which was pertussis toxin-sensitive. 7. Therefore, in the presence of raised extracellular potassium 5-HT can stimulate PLD via 5-HT1B receptors in the rabbit mesenteric artery. This action requires extracellular calcium and the activation of protein kinase C. These characteristics are identical to the profile for 5-HT1B/5-HT1D-receptor evoked contraction in vascular smooth muscle cells, suggesting a role for PLD in this response to 5-HT. (+info)
Identification of mRNA for 5-HT1 and 5-HT2 receptor subtypes in human coronary arteries.
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OBJECTIVE: Although pharmacological studies have indicated that serotonin (5-HT)-evoked contraction of the human coronary artery is mediated by 5-HT1-like and 5-HT2 receptors, the gene expression of 5-HT receptors is still unclear. We examined mRNA expression of 5-HT1 and 5-HT2 receptor subtypes in human coronary arteries. METHODS: Total RNA was extracted from human coronary arteries of 14 patients at autopsy by the guanidine method. Reverse transcription-polymerase chain reaction (RT-PCR) and ribonuclease protection assays were performed to identify 5-HT1 and 5-HT2 receptor mRNA expression in human coronary artery. RESULTS: By RT-PCR, 5-HT1b, 5-HT2A and 5-HT2B mRNAs were detected in all of the 14 patients. 5-HT1A, 5-HT1D, and 5-HT1E mRNAs were detected in only some patients. However, neither 5-HT1F mRNA nor 5-HT2C mRNA was detected in any patient. By ribonuclease protection assay, 5-HT1B and 5-HT2A signals were detected in all patients examined, but neither 5-HT1A, 5-HT1D nor 5-HT2B signal was detected in any patient. CONCLUSIONS: Of 5-HT1/2 receptor subtypes, 5-HT1B and 5-HT2A receptor mRNAs were predominantly expressed in human coronary arteries. Our finding provides molecular evidence that the 5-HT1B receptor may be the 5-HT1-like receptor which mediates constriction of human coronary arteries. (+info)