William P. Clarke

Functional Selectivity and Classical Concepts of Quantitative Pharmacology

The concept of intrinsic efficacy has been enshrined in pharmacology for half of a century, yet recent data have revealed that many ligands can differentially activate signaling pathways mediated via a single G protein-coupled receptor in a manner that challenges the traditional definition of intrinsic efficacy. Some terms for this phenomenon include functional selectivity, agonist-directed trafficking, and biased agonism. At the extreme, functionally selective ligands may be both agonists and antagonists at different functions mediated by the same receptor. Data illustrating this phenomenon are presented from serotonin, opioid, dopamine, vasopressin, and adrenergic receptor systems. A variety of mechanisms may influence this apparently ubiquitous phenomenon. It may be initiated by differences in ligand-induced intermediate conformational states, as shown for the β2-adrenergic receptor. Subsequent mechanisms that may play a role include diversity of G proteins, scaffolding and signaling partners, and receptor oligomers. Clearly, expanded research is needed to elucidate the proximal (e.g., how functionally selective ligands cause conformational changes that initiate differential signaling), intermediate (mechanisms that translate conformation changes into differential signaling), and distal mechanisms (differential effects on target tissue or organism). Besides the heuristically interesting nature of functional selectivity, there is a clear impact on drug discovery, because this mechanism raises the possibility of selecting or designing novel ligands that differentially activate only a subset of functions of a single receptor, thereby optimizing therapeutic action. It also may be timely to revise classic concepts in quantitative pharmacology and relevant pharmacological conventions to incorporate these new concepts.

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.

The elusive nature of intrinsic efficacy

In the discipline of pharmacology, drugs (ligands) are used as tools to elucidate the processes of biological systems. Because of this, pharmacologists strive to delineate all characteristics of drugs. Decades of research have resulted in the proposal that ligands possess two properties that are intrinsic to the ligand and are invariant of the system in which their effects are investigated. These properties are affinity (the capacity of a drug to bind to a receptor) and intrinsic efficacy (the capacity of a drug to activate or inactivate a receptor). Although affinity is a relatively easy parameter to measure with a variety of techniques, ways of quantifying intrinsic efficacy have remained elusive ever since its inception. Furthermore, recent evidence suggests that intrinsic efficacy might not be a single, ligand-dependent parameter but that agonists might have multiple intrinsic efficacies. William Clarke and Richard Bond discuss several reasons why the claim that intrinsic efficacy is a ligand-dependent parameter should be questioned, and the possible impact of these findings.

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.

Spiperone differentiates multiple 5-hydroxytryptamine responses in rat hippocampal slices in vitro.

The predominant effect of bath perfusion of 5-HT on CA1 population spikes in rat dorsal hippocampal slices in vitro is a dose dependent decrease in amplitude (Beck and Goldfarb, 1985; Rowan and Anwyl, 1985). This is often preceded by a smaller transient increase. The 5-HT induced decrease has an ECs0 of 3.2 #M and a slope index of 2.1 (Beck and Goldfarb, 1985). The effects of 5-HT agonists and antagonists, described below, suggest that the decrease in amplitude is mediated by a receptor with characteristics similar to the 5-HTaA binding site, whereas the receptor(s) mediating the increase do not correspond to any of the 5-HT~ or 5-HT 2 sites. The experimental methods were as described (Beck and Goldfarb, 1985) except that the perfusion fluid contained 24 mM NaHCO 4 and 2.5 mM CaC12. Monopolar stimulation of CA1 stratum radiatum was used. Antagonists were present in the perfusion fluid throughout the experiment, except as noted in fig. 1 The 5-HT analog 5-carboxyamidotryptamine (5-CONH2-T) has nanomolar affinity for [3H]5HT binding sites in brain membranes (Engel et al., 1983); these have been shown to be both 5-HT1A and 5-HTIB sites (Ebersole et al., in preparation). 5-CONH2-T produced only a decrease in population spike amplitude. The effect was rapidly reversible and concentration dependent (fig. 1A). The ECs0 was 20 _+ 2 nM with a slope index of 2.1 +_ 0.2 (mean _+ S.E.M. of 3 slices). 8-Hydroxy-2-(di-n-propylamine)tetralin (8OH-DPAT), a 5-HT agonist that selectively binds

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.

Serotonin decreases population spike amplitude in hippocampal cells through a pertussis toxin substrate

Activation of the serotonin1A receptor decreases CA1 population spike amplitude and inhibits forskolin-stimulated adenylate cyclase in rat hippocampus. Pretreatment of rats with pertussis toxin blocked both responses. Because the electrophysiological and biochemical responses to serotonin were correlated after pertussis toxin treatment, we conclude that both responses are mediated by a common regulatory protein, presumably Gi.