Kelly A. Berg

Constitutive Activity of the Serotonin2C Receptor Inhibits In Vivo Dopamine Release in the Rat Striatum and Nucleus Accumbens

Numerous research has pointed out that serotonin2c (5-HT2C) receptor, a subtype of 5-HT receptors belonging to the G-protein-coupled receptor superfamily, modulates the activity of mesencephalic dopamine (DA) neurons, the dysfunction of which is involved in devastating diseases such as schizophrenia, Parkinsons disease, and drug addiction. In the present study, using in vivo intracerebral microdialysis and Chinese hamster ovary (CHO) cells expressing 5-HT2C receptors to identify appropriate 5-HT2C receptor ligands, we sought to determine whether the property of 5-HT2C receptors to spontaneously activate intracellular signaling pathways in vitro (constitutive activity) participates in the tonic inhibitory control that they exert on DA release in the rat striatum and nucleus accumbens in vivo. In CHO cells, the purported antagonist 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f] indole hydrochloride (SB 206553), but not 6-chloro-5-methyl-1-[6-(2-methylpiridin-3-yloxy)pyridin-3-yl carbamoyl] indoline (SB 242084), decreased basal inositol phosphate accumulation, thus behaving as a 5-HT2C inverse agonist. Its effect was prevented by SB 242084. In vivo, SB 206553 (1–10 mg/kg) elicited a dose-dependent and clear-cut increase in accumbal and striatal DA release compared with SB 242084 (1–10 mg/kg), and the 5-HT2C agonist S-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine hydrochloride (Ro-60-0175) (0.3–3 mg/kg) inhibited DA release. Pretreatment by SB 242084 reversed the change in DA release elicited by Ro-60-0175 and SB 206553. Furthermore, SB 206553-stimulated DA release was insensitive to reduction of 5-HT neuronal function induced by the 5-HT1A agonist (±)-8-hydroxy-2-dipropylaminotetralin or intra-raphe injections of 5,7-dihydroxytryptamine neurotoxin. The obtained results provide the first in vivo evidence that constitutive activity of the 5-HT2C receptor tonically inhibits mesencephalic DA neurons and underscore the need for a better understanding of the pathophysiological role of constitutive receptor activity.

Bradykinin-Induced Functional Competence and Trafficking of the δ-Opioid Receptor in Trigeminal Nociceptors

Peripheral opioid analgesia is increased substantially after inflammation. We evaluated the hypothesis that an inflammatory mediator, bradykinin (BK), evokes functional competence of the δ-opioid receptor (DOR) for inhibiting trigeminal ganglia (TG) sensory neurons. We also evaluated whether BK evokes trafficking of the DOR to the plasma membrane. Rat TG cultures were pretreated with BK (10 μm) or vehicle, and the effects of DOR agonists ([d-Pen2,5]-enkephalin or [d-Ala2, d-Leu5]-enkephalin) on BK (10μm)/prostagladin E2 (PGE2; 1 μm)-stimulated immunoreactive calcitonin gene-related peptide (iCGRP) release or PGE2 (1 μm)-stimulated cAMP accumulation were measured. The effect of BK treatment on opioid receptor trafficking was evaluated by DOR immunohistochemistry, cell-surface DOR biotinylation, and live imaging of neurons transfected with mDOR–green fluorescent protein. BK pretreatment rapidly and significantly increased DOR agonist inhibition of evoked iCGRP release and cAMP accumulation. These effects of BK pretreatment were blocked by a B2 receptor antagonist (HOE-140; 10μm) or a protein kinase C (PKC) inhibitor [bisindolymaleimide (BIS); 1μm]. Moreover, BK treatment rapidly and significantly increased the accumulation of DOR in the plasma membrane. However, BK-induced trafficking of DOR was not reversed by pretreatment with BIS, nor was trafficking evoked by application of a PKC activator PMA (200 nm). These data suggest that BK, in a PKC-dependent manner, induces rapid functional competence of DOR for inhibiting TG nociceptors and in a PKC-independent manner rapidly induces trafficking of DOR to the plasma membrane. These findings indicate that exposure to certain inflammatory mediators rapidly alters the signaling properties and neuronal localization of DOR, possibly contributing to peripheral opioid analgesia.

RNA-editing of the 5-HT2C receptor alters agonist-receptor-effector coupling specificity

The serotonin2C (5‐HT2C) receptor couples to both phospholipase C (PLC)‐inositol phosphate (IP) and phospholipase A2 (PLA2)‐arachidonic acid (AA) signalling cascades. Agonists can differentially activate these effectors (i.e. agonist‐directed trafficking of receptor stimulus) perhaps due to agonist‐specific receptor conformations which differentially couple to/activate transducer molecules (e.g. G proteins). Since editing of RNA transcripts of the human 5‐HT2C receptor leads to substitution of amino acids at positions 156, 158 and 160 of the putative second intracellular loop, a region important for G protein coupling, we examined the capacity of agonists to activate both the PLC‐IP and PLA2‐AA pathways in CHO cells stably expressing two major, fully RNA‐edited isoforms (5‐HT2C‐VSV, 5‐HT2C‐VGV) of the h5‐HT2C receptor. 5‐HT increased AA release and IP accumulation in both 5‐HT2C‐VSV and 5‐HT2C‐VGV expressing cells. As expected, the potency of 5‐HT for both RNA‐edited isoforms for both responses was 10 fold lower relative to that of the non‐edited receptor (5‐HT2C‐INI) when receptors were expressed at similar levels. Consistent with our previous report, the efficacy order of two 5‐HT receptor agonists (TFMPP and bufotenin) was reversed for AA release and IP accumulation at the non‐edited receptor thus demonstrating agonist trafficking of receptor stimulus. However, with the RNA‐edited receptor isoforms there was no difference in the relative efficacies of TFMPP or bufotenin for AA release and IP accumulation suggesting that the capacity for 5‐HT2C agonists to traffic receptor stimulus is lost as a result of RNA editing. These results suggest an important role for the second intracellular loop in transmitting agonist‐specific information to signalling molecules.

Fine-tuning Serotonin2C Receptor Function in the Brain: Molecular and Functional Implications

The serotonin(2C) receptor (5-HT(2C)R) is a member of the serotonin(2) family of 7-transmembrane-spanning (7-TMS) receptors, which possesses unique molecular and pharmacological properties such as constitutive activity and RNA editing. The 5-HT(2C)R is widely expressed within the central nervous system, where is thought to play a major role in the regulation of neuronal network excitability. In keeping with its ability to modulate dopamine (DA) neuron function in the brain, the 5-HT(2C)R is currently considered as a major target for improved treatments of neuropsychiatric disorders related to DA neuron dysfunction, such as depression, schizophrenia, Parkinsons disease or drug addiction. The aim of this review is to provide an update of the functional status of the central 5-HT(2C)R, covering molecular, cellular, anatomical, biochemical and behavioral aspects to highlight its distinctive regulatory properties, the emerging functional significance of constitutive activity and RNA editing in vivo, and the therapeutic potential of inverse agonism.

Functional Selectivity of Hallucinogenic Phenethylamine and Phenylisopropylamine Derivatives at Human 5-Hydroxytryptamine (5-HT)2A and 5-HT2C Receptors

2,5-Dimethoxy-4-substituted phenylisopropylamines and phenethylamines are 5-hydroxytryptamine (serotonin) (5-HT)2A/2C agonists. The former are partial to full agonists, whereas the latter are partial to weak agonists. However, most data come from studies analyzing phospholipase C (PLC)-mediated responses, although additional effectors [e.g., phospholipase A2 (PLA2)] are associated with these receptors. We compared two homologous series of phenylisopropylamines and phenethylamines measuring both PLA2 and PLC responses in Chinese hamster ovary-K1 cells expressing human 5-HT2A or 5-HT2C receptors. In addition, we assayed both groups of compounds as head shake inducers in rats. At the 5-HT2C receptor, most compounds were partial agonists for both pathways. Relative efficacy of some phenylisopropylamines was higher for both responses compared with their phenethylamine counterparts, whereas for others, no differences were found. At the 5-HT2A receptor, most compounds behaved as partial agonists, but unlike findings at 5-HT2C receptors, all phenylisopropylamines were more efficacious than their phenethylamine counterparts. 2,5-Dimethoxyphenylisopropylamine activated only the PLC pathway at both receptor subtypes, 2,5-dimethoxyphenethylamine was selective for PLC at the 5-HT2C receptor, and 2,5-dimethoxy-4-nitrophenethylamine was PLA2-specific at the 5-HT2A receptor. For both receptors, the rank order of efficacy of compounds differed depending upon which response was measured. The phenylisopropylamines were strong head shake inducers, whereas their phenethylamine congeners were not, in agreement with in vitro results and the involvement of 5-HT2A receptors in the head shake response. Our results support the concept of functional selectivity and indicate that subtle changes in ligand structure can result in significant differences in the cellular signaling profile.

PAR-2 agonists activate trigeminal nociceptors and induce functional competence in the delta opioid receptor

&NA; The role of protease activated receptor‐2 (PAR‐2) activation in trigeminal nociception and in induction of functional competence in the delta opioid receptor (DOR) is not known. In this study, we evaluated whether agonists of PAR‐2 activate the capsaicin‐sensitive subclass of trigeminal nociceptors in a PLC–PKC‐dependent manner and induce functional competence in the DOR. Adult male rat trigeminal ganglion (TG) cultured neurons were treated with the PAR‐2 agonist (SL‐NH2) or an enzyme activator of PAR (trypsin) and the activation of TG nociceptors was assessed using three independent methods: neuropeptide release, calcium influx, and whole cell patch‐clamp. The specificity of SL‐NH2 and trypsin responses was evaluated using TG cultures transfected with siRNA against PAR‐2. The in vivo role of PAR‐2 activation was determined measuring SL‐NH2 and trypsin‐evoked nocifensive behavior and increase in blood flow. Trigeminal neurons were treated with SL‐NH2/vehicle and then the DOR agonist to determine DOR inhibition of evoked neuropeptide release and cAMP accumulation. The results showed that SL‐NH2 (100 &mgr;M) and trypsin (1–600 nM) activate TG nociceptors, which is partly reversible by the PKC inhibitor bisindolylmaleimide (500 nM) and by ruthenium red (10 &mgr;M). In cultures treated with siRNA against PAR‐2, both SL‐NH2 and trypsin responses were significantly diminished. Both SL‐NH2 and trypsin evoke nocifensive behavior and increases in blood flow in an orofacial pain model. Application of SL‐NH2 rapidly produced functional competence of DOR for inhibiting nociceptor function. In inflamed tissue, endogenous proteases may activate TG nociceptors and generate pain. Moreover, activation of PAR‐2 can also induce functional competence in DOR.

Physiological and therapeutic relevance of constitutive activity of 5-HT2A and 5-HT2C receptors for the treatment of depression

Serotonin(2A) (5-HT(2A)) and 5-HT(2C) receptors are highly homologous members of the serotonin(2) family of 7-transmembrane-spanning (7-TMS) receptors. Both of these receptor subtypes have been implicated in the aetiology and/or treatment of affective disorders such as anxiety and depression. Regulation of dopaminergic neurotransmission by 5-HT(2A) and 5-HT(2C) receptor systems has been well established. In general, agonist activation of 5-HT(2A) receptors can facilitate stimulated dopamine (DA) release, whereas 5-HT(2C) agonists inhibit dopaminergic neural activity and DA release under both basal and activated conditions. However, recent experimental evidence suggests that 5-HT(2A) and 5-HT(2C) receptors can be constitutively active (agonist-independent activity) in vivo. Alterations in the constitutive activity of 5-HT(2A) and 5-HT(2C) receptor systems could be involved in the mechanisms underlying anxiety and depression or exploited for therapeutic benefit. Consequently, drugs with inverse agonist properties may have more activity in vivo to regulate DA neurotransmission than that afforded by simple competitive antagonism.

Rapid Modulation of μ-Opioid Receptor Signaling in Primary Sensory Neurons

Management of pain by opioid analgesics is confounded by central adverse effects that limit clinical dosages. Consequently, there is considerable interest to understand peripheral analgesic effects of opioids. The actions of opioids on peripheral sensory neurons have been difficult to study because of a general lack of effect of opioid agonists on nociceptor function in culture despite documented presence of opioid receptors. In this study, the μ-opioid receptor agonist, [d-Ala2,N-MePhe4,Gly-ol5]-enkephalin (DAMGO), did not alter guanosine 5′-O-(3-[35S]thio)-triphosphate (GTPγ[35S]) binding, adenylyl cyclase activity, or neuropeptide release in primary cultures of rat trigeminal ganglion (TG). However, after brief exposure to bradykinin (BK), DAMGO stimulated GTPγ[35S] binding and inhibited both prostaglandin E2 (PGE2)-stimulated adenylyl cyclase activity and BK/PGE2-stimulated neuropeptide release. The effect of BK was blocked by the B2 antagonist HOE 140 [d-Arg[Hyp3,Thi5,d-Tic7,Oic8]-bradykinin], but not by the B1 antagonist, Lys-[Leu8]des-Arg9-BK, and was mimicked by the protease-activated receptor-2 agonist, Ser-Leu-Ile-Gly-Arg-Leu-NH2, and by activation of protein kinase C (PKC) or by administration of arachidonic acid (AA). The enhanced responsiveness of μ-opioid receptor signaling by BK priming was blocked by both cyclooxygenase and PKC inhibitors; however, the effect of AA was blocked only by a cyclooxygenase inhibitor. The results indicate that μ-opioid receptor signaling in primary sensory TG neurons is enhanced by activation of phospholipase C-coupled receptors via a cyclooxygenase-dependent AA metabolite that is downstream of PKC.

A Conservative, Single-Amino Acid Substitution in the Second Cytoplasmic Domain of the Human Serotonin2C Receptor Alters Both Ligand-Dependent and -Independent Receptor Signaling

The post-transcriptional process of mRNA editing changes up to three amino acids in the second intracellular domain (i2) of the serotonin2C (5-HT2C) receptor and alters some signaling characteristics of the receptor. Here, we report that the substitution of valine for isoleucine (I156V; 5-HT2C-VNI), which occurs naturally as a result of mRNA editing, alters both ligand-dependent and -independent signaling. Agonist functional selectivity at the 5-HT2C-VNI receptor differed from the nonedited 5-HT2C-INI receptor. Ligands with selectivity for phospholipase C (PLC) signaling in 5-HT2C-INI cells retained this selectivity in 5-HT2C-VNI-expressing cells. However, ligands with selectivity for phospholipase A2 (PLA2) signaling in 5-HT2C-INI cells lost the capacity for preferential PLA2 activation in 5-HT2C-VNI cells. Maximal PLC responses elicited by 5-HT (full agonist) and lysergic acid diethylamide and 2,5-dimethoxy-4-iodophenylisopropylamine (partial agonists) at edited receptors (5-HT2C-VNI, 5-HT2C-VSV, and 5-HT2C-VGV) were not different from 5-HT2C-INI receptors, suggesting that the capacity of the agonist-occupied receptor to couple to Gq/11 proteins was not different. Ligand-independent (i.e., constitutive) receptor activity toward PLC for the 5-HT2C-VNI receptor was markedly reduced to a level similar to that for the fully edited 5-HT2C-VSV isoform. However, there was no difference in the thermal stability of the edited receptors, suggesting that mRNA editing does not alter the capacity of receptors to adopt active conformations. These results indicate that a conservative change in one amino acid (I156V) located in i2 of the 5-HT2C receptor produces profound changes in receptor function that differ depending upon whether the receptor is unoccupied or occupied by agonist.

Peripheral delta opioid receptors require priming for functional competence in vivo

Although centrally acting opioid analgesics produce profound antinociception under basal conditions, the antinociceptive properties of peripherally restricted opioid analgesics are generally only detectable after inflammation or injection of inflammatory mediators. Despite considerable research, the cellular mechanisms regulating the functional competence of peripheral opioid receptor systems for inhibition of nociception remain unclear. Recent work has demonstrated that brief pre-treatment (priming) with bradykinin, arachidonic acid, protease-activated receptor-2 agonists, or direct activators of protein kinase C (PKC) are capable of inducing the functional competence of the opioid receptor system in cultures of primary sensory neurons in vitro. Here we report that the peripheral delta opioid receptor system also requires PKC-dependent priming to inhibit prostaglandin E(2) (PGE(2))-induced thermal allodynia in the rat. Peripheral hindpaw injection of [D-Pen(2,5)]-enkephalin (DPDPE), a selective delta opioid receptor agonist, did not alter PGE(2)-induced thermal allodynia. However, following priming (15 min) with bradykinin or arachidonic acid, DPDPE produced a significant reduction in allodynia that was antagonist reversible, peripherally restricted, and exhibited a typical dose-response relationship. Furthermore, the bradykinin priming effect was blocked by the PKC inhibitors, bisindolylmaleimide I and chelerythrine. Collectively, these data support prior in vitro findings that, although present on primary sensory neurons, peripheral opioid receptor systems are functionally inactive under basal conditions and require activation of a PKC- and arachidonic acid-dependent signaling pathway to develop functional competence in vivo.