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Vanilloid receptor-1 (TRPV1) is a nonselective cation channel, predominantly expressed by sensory neurons, which plays a key role in the detection of noxious painful stimuli such as capsaicin, acid, and heat. TRPV1 antagonists may represent novel therapeutic agents for the treatment of a range of conditions including chronic pain, migraine, and gastrointestinal disorders. Here we describe the in vitro pharmacology of N-(2-bromophenyl)-N'-[((R)-1-(5-trifluoromethyl-2-pyridyl)pyrrolidin-3-yl)]urea (SB-705498), a novel TRPV1 antagonist identified by lead optimization of N-(2-bromophenyl)-N'-[2-[ethyl(3-methylphenyl)amino]ethyl]urea (SB-452533), which has now entered clinical trials. Using a Ca(2+)-based fluorometric imaging plate reader (FLIPR) assay, SB-705498 was shown to be a potent competitive antagonist of the capsaicin-mediated activation of the human TRPV1 receptor (pK(i) = 7.6) with activity at rat (pK(i) = 7.5) and guinea pig (pK(i) = 7.3) orthologs. Whole-cell patch-clamp electrophysiology was used to confirm and extend these findings, demonstrating that SB-705498 can potently inhibit the multiple modes of receptor activation that may be relevant to the pathophysiological role of TRPV1 in vivo: SB-705498 caused rapid and reversible inhibition of the capsaicin (IC(50) = 3 nM)-, acid (pH 5.3)-, or heat (50 degrees C; IC(50) = 6 nM)-mediated activation of human TRPV1 (at -70 mV). Interestingly, SB-705498 also showed a degree of voltage dependence, suggesting an effective enhancement of antagonist action at negative potentials such as those that might be encountered in neurons in vivo. The selectivity of SB-705498 was defined by broad receptor profiling and other cellular assays in which it showed little or no activity versus a wide range of ion channels, receptors, and enzymes. SB-705498 therefore represents a potent and selective multimodal TRPV1 antagonist, a pharmacological profile that has contributed to its definition as a suitable drug candidate for clinical development.

Small molecule antagonists of the vanilloid receptor TRPV1 (also known as VR1) are disclosed. Pyrrolidinyl ureas such as 8 and 15 (SB-705498) emerged as lead compounds following optimisation of the previously described urea SB-452533. Pharmacological studies using electrophysiological and FLIPR-Ca2+-based assays showed that compounds such as 8 and 15 were potent antagonists versus the multiple chemical and physical modes of TRPV1 activation (namely capsaicin, acid and noxious heat). Furthermore, 15 possessed suitable developability properties to enable progression of this compound into in vivo studies and subsequently clinical development.

Peripheral cannabinoid 2 receptors (CB2 receptors) modulate immune responses and attenuate nociceptive behaviour in models of acute and persistent pain. The aim of the present study was to investigate whether peripheral CB2 receptors modulate spinal processing of innocuous and noxious responses and to determine whether there are altered roles of CB2 receptors in models of persistent pain. Effects of local administration of the CB2 receptor agonist JWH-133 (5 and 15 microg/50 microL) on mechanically evoked responses of spinal wide dynamic range (WDR) neurons in noninflamed rats, rats with carrageenan-induced hindpaw inflammation, sham operated rats and spinal nerve-ligated (SNL) rats were determined in anaesthetized rats in vivo. Mechanical stimulation (von Frey filaments, 6-80 g) of the peripheral receptive field evoked firing of WDR neurons. Mechanically evoked responses of WDR neurons were similar in noninflamed, carrageenan-inflamed, sham-operated and SNL rats. Intraplantar injection of JWH-133 (15 microg), but not vehicle, significantly (P < 0.05) inhibited innocuous and noxious mechanically evoked responses of WDR neurons in all four groups of rats. In many cases the selective CB2 receptor antagonist, SR144528 (10 microg/50 microL), attenuated the inhibitory effects of JWH-133 (15 microg) on mechanically evoked WDR neuronal responses. The CB1 receptor antagonist, SR141716A, did not attenuate the inhibitory effects of JWH-133 on these responses. Intraplantar preadministration of JWH-133 also inhibited (P < 0.05) carrageenan-induced expansion of peripheral receptive fields of WDR dorsal horn neurons. This study demonstrates that activation of peripheral CB2 receptors attenuates both innocuous- and noxious-evoked responses of WDR neurons in models of acute, inflammatory and neuropathic pain.

There is growing behavioural evidence that the phospholipid growth factor lysophosphatidic acid (LPA) modulates nociceptive responses in vivo. The present study investigated further the effects of LPA on peripheral nociceptive processing. Effects of intraplantar injection of LPA on ongoing and peripheral mechanically evoked responses of spinal neurons were studied in vivo. In addition, LPA-evoked responses of adult rat dorsal root ganglion (DRG) neurons were studied with calcium imaging. To determine whether LPA may also act at the level of the spinal cord, LPA receptor G-protein coupling in lumbar spinal cord sections was studied with in vitro autoradiography of guanylyl 5'-[g-[(35)S]thio]triphosphate ([(35)S]GTPgammaS) binding. Intraplantar injection of LPA (5 microg/5 microl) significantly increased the duration (P

N-arachidonoyl-dopamine (NADA) is an endogenous ligand at TRPV1 and CB(1) receptors, which are expressed on primary afferent nociceptors. The aim of this study was to determine contributions of proposed pronociceptive TRPV1 and antinociceptive CB(1) receptors to effects of peripheral NADA on primary afferent fibre function. Effects of NADA on primary afferent nociceptor function, determined by whole cell patch clamp and calcium imaging studies of adult dorsal root ganglion (DRG) neurons, were determined. Application of NADA (1 microm) to DRG neurons depolarized the resting membrane potential (Vm) from -58 +/- 1 to -44 +/- 3 mV (P < 0.00001) and evoked a significant increase (P < 0.0001) in intracellular calcium (74 +/- 11% of response to 60 mm KCl), compared to basal. The TRPV1 receptor antagonist capsazepine abolished NADA-evoked depolarization of Vm (P < 0.0001) and NADA-evoked calcium responses (P < 0.001), which were also blocked by the CB(1) receptor antagonist SR141716A (P < 0.001). Effects of NADA (1.5 microg and 5 microg/50 microL) on mechanically evoked responses of dorsal horn neurons in anaesthetized Sprague-Dawley rats were studied. Intraplantar injection of the higher dose of NADA (5 microg/50 microL) studied significantly inhibited innocuous (8, 10 g) mechanically evoked responses of dorsal horn neurons compared to vehicle, effects blocked by intraplantar injection of SR141716A. Higher weight (26-100 g) noxious-evoked responses of dorsal horn neurons were also significantly inhibited by NADA (5 microg/50 microL), effects blocked by intraplantar injection of the TRPV1 antagonist, iodo-resiniferatoxin. NADA has a complex pattern of effects on DRG neurons and primary afferent fibres, which is likely to reflect its dual site of action at TRPV1 and CB(1) receptors and the differential expression of these receptors by primary afferent fibres.

Noladin ether has recently been reported to be an endocannabinoid, with selectivity for the cannabinoid (CB) CB1 receptor. In the present study, we investigated the effects of noladin ether in the rat isolated mesenteric arterial bed, cultured dorsal root ganglia (DRG) cells and human vanilloid (TRPV1)-receptor-expressing HEK293 cells (TRPV1-HEK293 cells). Electrical field stimulation of the mesenteric bed evoked frequency-dependent vasorelaxation due to the action of calcitonin gene-related peptide (CGRP) released from sensory nerves. Noladin ether (0.1–3 μM) attenuated sensory neurogenic relaxation in a concentration-dependent manner. Noladin ether (1 μM) reduced vasorelaxation at a submaximal frequency (8 Hz), from 57.3±6.8 to 23.3±3.8% (P

Vanilloid receptor-1 (TRPV1) is a non-selective cation channel, predominantly expressed by peripheral sensory neurones, which is known to play a key role in the detection of noxious painful stimuli, such as capsaicin, acid and heat. To date, a number of antagonists have been used to study the physiological role of TRPV1; however, antagonists such as capsazepine are somewhat compromised by non-selective actions at other receptors and apparent modality-specific properties. SB-366791 is a novel, potent, and selective, cinnamide TRPV1 antagonist isolated via high-throughput screening of a large chemical library. In a FLIPR-based Ca(2+)-assay, SB-366791 produced a concentration-dependent inhibition of the response to capsaicin with an apparent pK(b) of 7.74 +/- 0.08. Schild analysis indicated a competitive mechanism of action with a pA2 of 7.71. In electrophysiological experiments, SB-366791 was demonstrated to be an effective antagonist of hTRPV1 when activated by different modalities, such as capsaicin, acid or noxious heat (50 degrees C). Unlike capsazepine, SB-366791 was also an effective antagonist vs. the acid-mediated activation of rTRPV1. With the aim of defining a useful tool compound, we also profiled SB-366791 in a wide range of selectivity assays. SB-366791 had a good selectivity profile exhibiting little or no effect in a panel of 47 binding assays (containing a wide range of G-protein-coupled receptors and ion channels) and a number of electrophysiological assays including hippocampal synaptic transmission and action potential firing of locus coeruleus or dorsal raphe neurones. Furthermore, unlike capsazepine, SB-366791 had no effect on either the hyperpolarisation-activated current (I(h)) or Voltage-gated Ca(2+)-channels (VGCC) in cultured rodent sensory neurones. In summary, SB-366791 is a new TRPV1 antagonist with high potency and an improved selectivity profile with respect to other commonly used TRPV1 antagonists. SB-366791 may therefore prove to be a useful tool to further study the biology of TRPV1.

Orexins (OXs) regulate sleep with possible interactions with brain noradrenergic neurons. In addition, noradrenergic activity affects barbiturate anesthesia. As we have also recently reported that OXs selectively evoke norepinephrine release from rat cerebrocortical slices we hypothesized that barbiturate anesthesia may result from of an interaction with central orexinergic systems. To test this hypothesis, we performed a series of in vivo and in vitro studies in rats. In vivo, the effects of i.c.v. OX A, B and SB-334867-A (OX1 receptor antagonist) on pentobarbital, thiopental or phenobarbital-induced anesthesia times (loss of righting reflex) was assessed. In vitro effects of barbiturates and SB-334867-A on OX-evoked norepinephrine release from cerebrocortical slice was examined. In Chinese hamster ovary cells expressing human OX1/OX2 receptors OX A- and B-evoked increases in intracellular Ca2+ were measured with and without barbiturates. OX A and B significantly decreased pentobarbital, thiopental and phenobarbital anesthesia times by 15-40%. SB-334867-A increased thiopental-induced anesthesia time by approximately by 40%, and reversed the decrease produced by OX A. In vitro, all anesthetic barbiturates inhibited OX-evoked norepinephrine release with clinically relevant IC50 values. A GABAA antagonist, bicuculline, did not modify the inhibitory effects of thiopental and the GABAA agonist, muscimol, did not inhibit norepinephrine release. In addition there was no interaction of barbiturates with either OX1 or OX2 receptors. Collectively our data suggest that orexinergic neurons may be an important target for barbiturates, and GABAA, OX1 and OX2 receptors may not be involved in this interaction.

UNLABELLED Capsaicin, acting at the vanilloid 1 receptor (VR1), may potentiate local anesthetic activity, and as a ligand-gated ion channel of the transient receptor potential family, may also be a target for IV general anesthetics. We have examined whether local (lidocaine, prilocaine, and procaine 0.1-10 mM; 10 mM represents 0.25%-0.27% wt/vol) or IV anesthetics (propofol 10 micro M, thiopental 100 micro M, and ketamine 100 micro M) interact with recombinant rat VR1 expressed in human embryonic kidney (HEK293) cells (VR1-HEK293). We have assessed receptor interaction functionally by monitoring intracellular Ca(2+) ([Ca(2+)](i)) in Fura2-loaded cells at 37 degrees C. The addition of capsaicin (60 nM) produced a time-dependent biphasic increase in [Ca(2+)](i) amounting to 50-100 nM above than basal, which was inhibited by capsazepine 10 micro M and was absent in wild type HEK293 cells. Lidocaine and prilocaine alone (e.g., at 10 mM) significantly increased [Ca(2+)](i) by 67 +/- 6 nM and 33 +/- 7 nM, respectively, and concentration-dependently inhibited the capsaicin response. The effects of procaine were obscured by anesthetic-induced quenching of Fura2. In wild type HEK293 cells, lidocaine (10 mM) alone produced a small increase in [Ca(2+)](i). All IV anesthetics failed to modify capsaicin-increased [Ca(2+)](i). In conclusion, the present data suggest that local but not IV general anesthetics interact with recombinant rat VR1 receptors with the former anesthetics having antagonistic activity. IMPLICATIONS Vanilloid receptors (VR1) are activated by capsaicin, the pain-producing component of hot chili peppers. We suggest that local (but not IV general) anesthetics may have inhibitory actions on this receptor.

The abilities of 19 analogues of palmitoylethanolamide and two analogues of oleoylethanolamide to affect the Ca(2+) influx into human embryonic kidney cells expressing the human vanilloid receptor (hVR1-HEK293 cells) in response to anandamide (AEA) have been investigated using a FLIPR assay and a bovine serum albumin-containing assay medium. Only palmitoylethanolamide produced any effect in the absence of AEA. The ability of palmitoylethanolamide to potentiate the response to AEA was retained when the N-CH(2)CH(2)OH group was replaced by N-CH(2)CH(2)Cl,whereas replacement with N-alkyl substituents [from -H up to -(CH(2))(12)CH(3)] resulted either in a reduction or in a complete loss of this activity. The tertiary amide N-(CH(2)CH(3))(2) (19) and N-morpholino (20) analogues of palmitoylethanolamide potentiated the response to 1 microM AEA to a greater degree than the parent compound, whereas the N-(CH(3))(2) analogue was inactive. 19 and 20 produced leftward shifts in the dose-response curve for AEA activation of Ca(2+) influx into hVR1-HEK293 cells. EC(50) values for AEA to produce Ca(2+) influx into hVR1-HEK293 cells were 1.1, 1.1, 0.54 and 0.36 microM in the presence of 0, 1, 3 and 10 microM 19, respectively. The corresponding values for 20 were 1.5, 1.3, 0.77 and 0.17 microM, respectively. The compounds did not affect the dose-response curves to capsaicin. The ability of oleoylethanolamide to potentiate AEA is retained by the N-CH(2)CH(3) and N-CH(CH(3))(2) analogues (22 and 23, respectively). 22 and 23 produced a small ( approximately 25%) inhibition of the binding of [(3)H]-CP55,940 and [(3)H]-WIN 55,212-2 to CB(1) and CB(2) receptors, respectively, expressed in CHO cells. The compounds inhibited the metabolism of 2 microM [(3)H]-AEA by rat brain fatty acid amidohydrolase with IC(50) values of 5.6 and 11 microM, respectively. In contrast, 19 and 20 were without effect on either binding to CB receptors or fatty acid amidohydrolase activity. Minor reductions in the accumulation of 10 microM [(3)H]-AEA into C6 glioma cells were seen at 10 microM concentrations of 19 and 20. It is concluded that 19 and 20 selectively enhance AEA effects upon VR1 receptors without potentially confounding effects upon CB receptors or fatty acid amidohydrolase activity.

1 Capsaicin and resiniferatoxin (RTX) stimulate Ca2+ influx by activating vanilloid receptor 1 (VR1), a ligand-gated Ca2+ channel on sensory neurones. We investigated whether VR1 activation could also trigger Ca2+ mobilization from intracellular Ca2+ stores. 2 Human VR1-transfected HEK293 cells (hVR1-HEK293) were loaded with Fluo-3 or a mixture of Fluo-4 and Fura Red and imaged on a fluorometric imaging plate reader (FLIPR) and confocal microscope respectively. 3 In Ca2+ -free media, RTX caused a transient elevation in intracellular free Ca2+ concentration in hVR1-HEK293 cells (pEC(50) 6.45+/-0.05) but not in wild type cells. Capsaicin (100 microM) did not cause Ca2+ mobilization under these conditions. 4 RTX-mediated Ca2+ mobilization was inhibited by the VR1 receptor antagonist capsazepine (pIC(50) 5.84+/-0.04), the Ca2+ pump inhibitor thapsigargin (pIC(50) 7.77+/-0.04), the phospholipase C inhibitor U-73122 (pIC(50) 5.35+/-0.05) and by depletion of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores by pretreatment with the acetylcholine-receptor agonist carbachol (20 microM, 2 min). These data suggest that RTX causes Ca2+ mobilization from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in hVR1-HEK293 cells. 5 In the presence of extracellular Ca2+, both capsaicin-mediated and RTX-mediated Ca2+ rises were attenuated by U-73122 (10 microM, 30 min) and thapsigargin (1 microM, 30 min). We conclude that VR1 is able to couple to Ca2+ mobilization by a Ca2+ dependent mechanism, mediated by capsaicin and RTX, and a Ca2+ independent mechanism mediated by RTX alone.

Vanilloid receptor-1 (VR1, also known as TRPV1) is a thermosensitive, nonselective cation channel that is expressed by capsaicin-sensitive sensory afferents and is activated by noxious heat, acidic pH and the alkaloid irritant capsaicin. Although VR1 gene disruption results in a loss of capsaicin responses, it has minimal effects on thermal nociception. This and other experiments--such as those showing the existence of capsaicin-insensitive heat sensors in sensory neurons--suggest the existence of thermosensitive receptors distinct from VR1. Here we identify a member of the vanilloid receptor/TRP gene family, vanilloid receptor-like protein 3 (VRL3, also known as TRPV3), which is heat-sensitive but capsaicin-insensitive. VRL3 is coded for by a 2,370-base-pair open reading frame, transcribed from a gene adjacent to VR1, and is structurally homologous to VR1. VRL3 responds to noxious heat with a threshold of about 39 degrees C and is co-expressed in dorsal root ganglion neurons with VR1. Furthermore, when heterologously expressed, VRL3 is able to associate with VR1 and may modulate its responses. Hence, not only is VRL3 a thermosensitive ion channel but it may represent an additional vanilloid receptor subunit involved in the formation of heteromeric vanilloid receptor channels.

1 The abilities of a series of saturated N-acyl ethanolamines and related compounds to affect the ability of anandamide (AEA) to produce a Ca2+ influx into human embryonic kidney cells expressing the human vanilloid receptor (hVR1-HEK293 cells) has been investigated. 2 The C3:0, C4:0, C6:0 and C10:0 ethanolamides neither affected basal Ca2+-influx, nor the influx in response to a submaximal concentration of AEA (1 muM). In contrast, the C12:0, C17:0, C18:0 ethanolamides and the monounsaturated compound oleoylethanolamide (C18:1) greatly potentiated the response to AEA. Palmitoylethanolamide (C16:0) produced both a response per se and an augmentation of the response to AEA. 3 Lauroylethanolamide (C12:0) produced a leftward shift in the dose-response curve for AEA. EC50 values for AEA to produce Ca2+ influx into hVR1-HEK293 cells were 1.8, 1.5, 1.1 and 0.22 muM in the presence of 0, 1, 3 and 10 muM lauroylethanolamide, respectively. Lauroylethanolamide did not affect the dose-response curves to capsaicin. 4 Palmitoylethylamide was synthesized and found to be a mixed-type inhibitor (K-i(slope) 4.1 muM, K-i(intercept) 66 muM) of [H-3]-AEA metabolism by rat brain membranes. 5 The -amide, -ethylamide, -isopropylamide, -butylamide, -cyclohexamide and -trifluoromethyl ketone analogues of palmitoylethanolamide had little or no effect on the Ca2+ influx response to 1 muM AEA. 6 There was no obvious relation between the abilities of the compounds to enhance the Ca2+-influx response to 1 muM EA into hVR1-HEK293 cells and to prevent the hydrolysis of AEA by rat brain membranes. 7 It is concluded that although palmitoylethanolamide has entourage-like effects at VR1 receptors expressed on hVR1-HEK293 cells, other N-acyl ethanolamines have even more dramatic potentiating effects. It is possible that they may play an important role under conditions where their synthesis is increased, such as in severe inflammation.

We have studied activation by phorbol derivatives of TRPV4 channels, the human VRL-2, and murine TRP12 channels, which are highly homologous to the human VR-OAC, and the human and murine OTRPC4 channel. 4alpha-Phorbol 12,13-didecanoate (4alpha-PDD) induced an increase in intracellular Ca(2+) concentration, [Ca(2+)](i), in 1321N1 cells stably transfected with human VRL-2 (hVRL-2.1321N1) or HEK-293 cells transiently transfected with murine TRP12, but not in nontransfected or mock-transfected cells. Concomitantly with the increase in [Ca(2+)](i), 4alpha-PDD activated an outwardly rectifying cation channel with an Eisenman IV permeation sequence for monovalent cations that is Ca(2+)-permeable with P(Ca)/P(Na) = 5.8. Phorbol 12-myristate 13-acetate also induced an increase in [Ca(2+)](i) but was approximately 50 times less effective than 4alpha-PDD. EC(50) for Ca(2+) increase and current activation was nearly identical (pEC(50) approximately 6.7). Similar effects were observed in freshly isolated mouse aorta endothelial cells which express TRP12 endogenously. By using 4alpha-PDD as a tool to stimulate TRP12, we showed that activation of this channel is modulated by [Ca(2+)](i); an increase in [Ca(2+)](i) inhibits the channel with an IC(50) of 406 nm. Ruthenium Red at a concentration of 1 microm completely blocks inward currents at -80 mV but has a smaller effect on outward currents likely indicating a voltage dependent channel block. We concluded that the phorbol derivatives activate TRPV4 (VR-OAC, VRL-2, OTRPC4, TRP12) independently from protein kinase C, in a manner consistent with direct agonist gating of the channel.

Reverse transcription polymerase chain reaction (RT-PCR) demonstrated the presence of bombesin BB2 receptor mRNA but not bombesin BB1 receptor or bombesin BB3 receptor mRNA in cultured human astrocytes. Neuromedin C hyperpolarised human astrocytes in whole-cell current and voltage clamp recordings and increased the intracellular free Ca 2+ ion concentration ([Ca 2+ ] i ) in single astrocytes. Treatment with neuromedin C caused larger and more frequent increases in [Ca 2+ ] i than those triggered by neuromedin B, with 96% and 78% of cells responding, respectively. The stimulatory effects of neuromedin C were inhibited significantly by treatment with U73122 or the bombesin BB2 receptor antagonist [ d -Phe 6 , des-Met 14 ]bombesin-(6–14) ethylester. A Fluorometric Imaging Plate Reader (FLIPR) was used to measure [Ca 2+ ] i in cell populations. Neuromedin C was approximately 50-fold more potent than neuromedin B in elevating [Ca 2+ ] i in astrocytes and Chinese hamster ovary (CHO) cells expressing human bombesin BB2 receptors (hBB2-CHO). However, in CHO cells expressing the bombesin BB1 receptor hBB1-CHO, neuromedin B was 32-fold more potent than neuromedin C. [ d -Phe 6 , des-Met 14 ]bombesin-(6–14) ethylester was a partial agonist in hBB1-CHO cells ( E max =55%) but was a noncompetitive antagonist in both hBB2-CHO cells and astrocytes. These studies report the first identification of functional bombesin receptors on cultured human astrocytes and have demonstrated that the bombesin BB2 receptor contributes significantly to astrocyte physiology.

Undifferentiated bipolar CG-4 cell line oligodendrocytes provide a model system for the O-2A progenitor cell from which oligodendrocytes are derived both in vivo and in vitro. The exchange of neuroblastoma conditioned basal media for basal media causes differentiation of undifferentiated bipolar CG-4 cells into multipolar oligodendrocyte-like cells whilst replacement with basal media containing 20% foetal bovine serum favours the formation of type-2 astrocyte-like cells. Here, we demonstrate that activation of these differentiation pathways correlates with distinct changes both in cell metabolism and in signal transduction. Exchange of neuroblastoma conditioned media for basal media correlates with stimulation of basal metabolic activity, reduced phosphorylation of p44/42 MAP kinase and reduced phophorylation of the transcription factor CREB. In contrast, differentiation with basal medium containing 20% foetal bovine serum (FBS), into type 2 astrocyte-like cells, correlates with reduction in basal metabolic activity, increased phosphorylation of p44/42 MAP kinase and increased phophorylation of the transcription factor CREB. Inhibition of protein kinase C blocked both the metabolic and morphological changes associated with differentiation towards mature multipolar oligodendrocyte-like cells. Inhibition of PKA and MEK did not effect metabolic activity. The rapid return of neuroblastoma conditioned basal media to cells treated with basal media, increased phosphorylation of CREB and MAP kinase. These results demonstrate that protein kinase C and p44/42 MAP kinase signalling pathways are modulated during bipolar CG-4 cell differentiation and demonstrate that the transcription factor CREB may play a pivotal role in differentiation along oligodendrocyte-or astrocyte-lineages.

This communication reports SARs for the first orexin-1 receptor antagonist series of 1-aryl-3-quinolin-4-yl and 1-aryl-3-naphthyridin-4-yl ureas. One of these compounds, 31 (SB-334867), has excellent selectivity for the orexin-1 receptor, blood-brain barrier permeability and shows in vivo activity following ip dosing.

The effects of three structurally related cannabinoids on human and rat recombinant vanilloid VR1 receptors expressed in human embryonic kidney (HEK293) cells and at endogenous vanilloid receptors in the rat isolated mesenteric arterial bed were studied. In the recombinant cells, all three were full agonists, causing concentration-dependent increases in [Ca(2+)](i) (FLIPR), with a rank order of potency relative to the vanilloids capsaicin and olvanil, of olvanil> or =capsaicin>AM404 ((allZ)-N-(4-hydroxyphenyl)-5,8,11,14-eicosatetraenamide)>anandamide>methanandamide. These responses were inhibited by the vanilloid VR1 receptor antagonist, capsazepine. In the mesenteric arterial bed, vasorelaxation was evoked by these ligands with a similar order of potency. The AM404-induced vasorelaxation was virtually abolished by capsaicin pretreatment. AM404 inhibition of capsaicin-sensitive sensory neurotransmission was blocked by ruthenium red, but not by cannabinoid CB(1) and CB(2) receptor antagonists. AM404 had no effect on relaxations to calcitonin gene-related peptide. These data demonstrate that the vasorelaxant and sensory neuromodulator properties of AM404 in the rat isolated mesenteric arterial bed are mediated by vanilloid VR1 receptors.

The pharmacology of various peptide and non-peptide ligands was studied in Chinese hamster ovary (CHO) cells stably expressing human orexin-1 (OX1) or orexin-2 (OX2) receptors by measuring intracellular calcium ([Ca2+]i) using Fluo-3AM. Orexin-A and orexin-B increased [Ca2+]i in CHO-OX1 (pEC50=8.38±0.04 and 7.26±0.05 respectively, n=12) and CHO-OX2 (pEC50=8.20±0.03 and 8.26±0.04 respectively, n=8) cells. However, neuropeptide Y and secretin (10 pM – 10 μM) displayed neither agonist nor antagonist properties in either cell-line. SB-334867-A (1-(2-Methyylbenzoxanzol-6-yl)-3-[1,5]naphthyridin-4-yl-urea hydrochloride) inhibited the orexin-A (10 nM) and orexin-B (100 nM)-induced calcium responses (pKB=7.27±0.04 and 7.23±0.03 respectively, n=8), but had no effect on the UTP (3 μM)-induced calcium response in CHO-OX1 cells. SB-334867-A (10 μM) also inhibited OX2 mediated calcium responses (32.7±1.9% versus orexin-A). SB-334867-A was devoid of agonist properties in either cell-line. In conclusion, SB-334867-A is a non-peptide OX1 selective receptor antagonist. British Journal of Pharmacology (2001) 132, 1179–1182; doi:10.1038/sj.bjp.0703953

Prompted by conflicting literature, this study compared the pharmacology of human 5-hydroxytryptamine2 (5-HT2) receptors expressed in SH-SY5Y cells using a fluorometric imaging plate reader (FLIPR) based Ca2+ assay. 5-Hydroxytryptamine (5-HT) increased intracellular calcium concentration ([Ca2+]i) at 5-HT2A, 5-HT2B and 5-HT2C receptors (pEC(50)=7.73+/-0.03, 8.86+/-0.04 and 7.99+/-0.04, respectively) and these responses were inhibited by mesulergine (pKB=7.42+/-0.06, 8.77+/-0.10 and 9.52+/-0.11). A range of selective agonists and antagonists displayed the expected pharmacology at each receptor subtype. Sodium butyrate pretreatment increased receptor expression in SH-SY5Y/5-HT2B (15-fold) and SH-SY5Y/5-HT2C cells (7-fold) and increased agonist potencies and relative efficacies. In contrast, sodium butyrate pretreatment of SH-SY5Y/5-HT(2A) cells did not affect receptor expression. The present study provides a direct comparison of agonist and antagonist pharmacology at 5-HT(2) receptor subtypes in a homogenous system and confirms that agonist potency and efficacy varies with the level of receptor expression.

Rationale: Orexins A and B have recently been discovered and shown to be derived from prepro-orexin, primarily expressed in the rat hypothalamus. Orexin-A has been ascribed a number of in vivo functions in the rat after intracerebroventricular (ICV) administration, including hyperphagia, neuroendocrine modulation and, most recently, evidence for a behavioural response characterised by an increase in grooming. Objectives: Here, we have investigated the orexin-receptor subtypes involved in the grooming response to orexin-A (3 μg, ICV) in the rat. Methods: Male rats, habituated to clear Perspex behavioural observation boxes, were pretreated with antagonists with mixed selectivity for OX1, OX2, 5-HT2B and 5-HT2C receptor subtypes prior to the administration of orexin-A and the intense grooming response elicited by this peptide assessed. Results: Pretreatment of rats with a mixed OX1/5-HT2B/2C receptor antagonist 1-(4-methylsulfanylphenyl)-3-quinolin-4-ylurea (SB-284422), revealed a significant, but incomplete, blockade of orexin-A-induced grooming. Despite the low potency of orexin-A at 5-HT2B and 5-HT2C receptors in vitro (pKi

Analogues of the nonselective bombesin receptor synthetic agonist H-D-Phe-Gln-Trp-Ala-Val-βAla-His-Phe-Nle-NH2 were prepared and their biological activity assessed at the NMB-preferring/bombesin receptor (NMB-R; BB1), the GRP-preferring/bombesin receptor (GRP-R; BB2) and the orphan receptor bombesin receptor subtype-3 (BRS-3; BB3). Progressive N-terminal deletions identified the minimum C-terminal sequences required for maintaining a significant agonist effect, whilst an alanine scan, targeted changes in stereochemistry and other pertinent substitutions identified key side-chain and stereochemical requirements for activation. Key structural elements required for functional potency at BB1 BB2 and BB3, and for selectivity between these receptor subtypes were established. Synthetic peptides were discovered, which were highly potent agonists at BB2 and extremely selective over both BB1 and BB3. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.

Bombesin and its receptors have been shown to have a role regulating circadian rhythms in the hamster suprachiasmatic and dorsal raphe nuclei and have been implicated in the regulation of sleep. We have identified and characterised a bombesin receptor endogenously expressed in a Chinese hamster ovary cell line (CHO/DG44). Using a range of bombesin-like peptides, we demonstrate that this receptor displays bombesin BB2 receptor-like pharmacology. We also show that this receptor signals through inositol-[1,4,5]-trisphosphate and protein kinase C and thus provides a useful model system to aid in the interpretation of hamster suprachiasmatic nucleus studies of mammalian circadian rhythm.

The vanilloid receptor (VR1) is a ligand-gated ion channel, which plays an important role in nociceptive processing. Therefore, a pharmacological characterization of the recently cloned rat VR1 (rVR1) was undertaken. HEK293 cells stable expressing rVR1 (rVR1-HEK293) were loaded with Fluo-3AM and then incubated at 25 degrees C for 30 min with or without various antagonists or signal transduction modifying agents. Then intracellular calcium concentrations ([Ca(2+)](i)) were monitored using FLIPR, before and after the addition of various agonists. The rank order of potency of agonists (resiniferatoxin (RTX)>capsaicin>olvanil>PPAHV) was as expected, and all were full agonists. The potencies of capsaicin and olvanil, but not RTX or PPAHV, were enhanced at pH 6.4 (pEC(50) values of 7.47+/-0.06, 7.16+/-0.06, 8.19+/-0.06 and 6.02+/-0.03 respectively at pH 7.4 vs 7.71+/-0.05, 7.58+/-0.14, 8.10+/-0.05 and 6.04+/-0.08 at pH 6.4). Capsazepine, isovelleral and ruthenium red all inhibited the capsaicin (100 nM)-induced Ca(2+) response in rVR1-HEK293 cells, with pK(B) values of 7.52+/-0.08, 6.92+/-0.11 and 8.09+/-0.12 respectively (n=6 each). The response to RTX and olvanil were also inhibited by these compounds. None displayed any agonist-like activity. The removal of extracellular Ca(2+) abolished, whilst inhibition of protein kinase C with chelerythrine chloride (10 microM) partially (approximately 20%) inhibited, the capsaicin (10 microM)-induced Ca(2+) response. However, tetrodotoxin (3 microM), nimodipine (10 microM), omega-GVIA conotoxin (1 microM), thapsigargin (1 microM), U73122 (3 microM) or H-89 (3 microM) had no effect on the capsaicin (100 nM)-induced response. In conclusion, the recombinant rVR1 stably expressed in HEK293 cells acts as a ligand-gated Ca(2+) channel with the appropriate agonist and antagonist pharmacology, and therefore is a suitable model for studying the effects of drugs at this receptor.

The endogenous cannabinoid anandamide was identified as an agonist for the recombinant human VR1 (hVR1) by screening a large array of bioactive substances using a FLIPR-based calcium assay. Further electrophysiological studies showed that anandamide (10 or 100 microM) and capsaicin (1 microM) produced similar inward currents in hVR1 transfected, but not in parental, HEK293 cells. These currents were abolished by capsazepine (1 microM). In the FLIPR anandamide and capsaicin were full agonists at hVR1, with pEC(50) values of 5. 94+/-0.06 (n=5) and 7.13+/-0.11 (n=8) respectively. The response to anandamide was inhibited by capsazepine (pK(B) of 7.40+/-0.02, n=6), but not by the cannabinoid receptor antagonists AM630 or AM281. Furthermore, pretreatment with capsaicin desensitized the anandamide-induced calcium response and vice versa. In conclusion, this study has demonstrated for the first time that anandamide acts as a full agonist at the human VR1.

A functional tachykinin NK3 receptor was cloned from the rabbit iris sphincter muscle and its distribution investigated in ocular tissues. Standard polymerase chain reaction (PCR) techniques were used to clone a full length rabbit NK3 receptor cDNA consisting of 1404 nucleotides. This cDNA encoded a protein of 467 amino acids with 91 and 87% homology to the human and rat NK3 receptors respectively. In CHO-K1 cells transiently expressing the recombinant rabbit NK3 receptor, the relative order of potency of NKB>>NKASP to displace [125I]-[MePhe7]-NKB binding and to increase intracellular calcium, together with the high affinity of NK3 selective agonists (e.g. senktide, [MePhe7]-NKB) and antagonists (e.g. SR 142801, SB 223412) in both assays was consistent with NK3 receptor pharmacology. In binding and functional experiments, agonist concentration response curves were shallow (0.7–0.8), suggesting the possibility of multiple affinity states of the receptor. Quantitative real time PCR analysis revealed highest expression of rabbit NK3 receptor mRNA in iris sphincter muscle, lower expression in retina and iris dilator muscle, and no expression in lens and cornea. In situ hybridization histochemistry revealed discrete specific localization of NK3 receptor mRNA in the iris muscle and associated ciliary processes. Discrete specific labelling of NK3 receptors with the selective NK3 receptor agonist [125I]-[MePhe7]-NKB was also observed in the ciliary processes using autoradiography. Our study reveals a high molecular similarity between rabbit and human NK3 receptor mRNAs, as predicted from previous pharmacological studies, and provide the first evidence that NK3 receptors are precisely located on ciliary processes in the rabbit eye. In addition, there could be two affinity states of the receptor which may correspond to the typical and ‘atypical’ NK3 receptor subtypes previously reported. British Journal of Pharmacology (1999) 128, 627–636; doi:10.1038/sj.bjp.0702854

Endomorphin-1 and -2 (E-1/E-2) have been proposed as endogenous ligands for the μ-opioid receptor. The aims of this study are to characterize the binding of E-1/E-2 and the subsequent effects on cyclic AMP formation and [Ca2+]i levels in SH-SY5Y and Chinese hamster ovary (CHO) cells expressing endogenous and recombinant μ-opioid receptors. E-1 displaced [3H]-diprenorphine ([3H]-DPN) binding in CHOμ and SH-SY5Y membranes with pKi values of 8.02±0.09 and 8.54±0.13 respectively. E-2 displaced [3H]-DPN binding in CHOμ and SH-SY5Y cells with pKi values of 7.82±0.11 and 8.43±0.13 respectively. E-1/E-2 bound weakly to CHOδ and CHOκ membranes, with IC50 values of greater than 10 μM. In CHOμ cells, E-1/E-2 inhibited forskolin (1 μM) stimulated cyclic AMP formation with pIC50 values of 8.03±0.16 (Imax=53.0±9.3%) and 8.15±0.24 (Imax=56.3±3.8%) respectively. In SH-SY5Y cells E1/E2 inhibited forskolin stimulated cyclic AMP formation with pIC50 values of 7.72±0.13 (Imax=46.9±5.6%) and 8.11±0.31 (Imax=40.2±2.8%) respectively. E-1/E-2 (1 μM) increased [Ca2+]i in fura-2 loaded CHOμ cell suspensions in a thapsigargin sensitive and naloxone reversible manner. Mean increases observed were 106±28 and 69±6.7 nM respectively. In single adherent cells E-1/E-2 (1 μM) increased [Ca2+]i with a mean 340/380 ratio change of 0.81±0.09 and 0.40±0.08 ratio units respectively. E-1/E-2 failed to increase intracellular calcium in CHOδ, CHOκ and SH-SY5Y cells. These data show that E-1/E-2 bind with high affinity and selectivity to μ-opioid receptors and modulate signal transduction pathways typical of opioids. This provides further evidence that these two peptides may be endogenous ligands at the μ-opioid receptor. British Journal of Pharmacology (1999) 128, 472–478; doi:10.1038/sj.bjp.0702798

We have assessed the utility of the Cytosensor microphysiometer for studying the pharmacology of recombinant CRF receptors. Chinese hamster ovary cells stably expressing the human CRF1 or CRF2 receptor were perfused in the Cytosensor with bicarbonate-free Hams F12 (pH 7.4) containing 0.2% bovine serum albumin. The rank order of potencies of agonist peptides were CRF = sauvagine = urocortin = urotensin at CRF1 (pEC50 values 11.16 +/- 0.17, 11.37 +/- 0.14, 11.43 +/- 0.09 and 11.46 +/- 0.13; n = 4), and urocortin = sauvagine > urotensin > CRF at CRF2 (pEC50 values 10.88 +/- 0.12, 10.44 +/- 0.05, 9.36 +/- 0.12 and 8.53 +/- 0.07; n = 7-9). alpha-Helical CRF (9-41) was a competitive antagonist at the CRF2 receptor (pK(B) = 6.99 +/- 0.08, n = 4), but was a partial agonist at the CRF1 receptor (pEC50 = 6.85 +/- 0.08, Emax = 33%, n = 3). CP 154,526 was a competitive antagonist at the CRF1 receptor (pK(B) = 8.17 +/- 0.05, n = 6), but was inactive at the CRF2 receptor. These data are consistent with established CRF receptor pharmacology and show that the Cytosensor is a viable method for assessing the functional activity of CRF-receptor agonists and antagonists.

Nociceptin(NC) is the endogenous ligand for the opioid receptor like-1 receptor (NC-receptor). [Phe1ΨC(CH2-NH)Gly2]Nociceptin(1–13)NH2 ([F/G]NC(1–13)NH2) has been reported to antagonize NC actions in peripheral guinea-pig and mouse tissues. In this study, we investigated the effects of a range of NC C-terminal truncated fragments and [F/G]NC(1–13)NH2 on NC receptor binding, glutamate release from rat cerebrocortical slices (rCX), inhibition of cyclic AMP accumulation in CHO cells expressing the NC receptor (CHONCR) and electrically evoked contractions of the rat vas deferens (rVD). In radioligand binding assays, a range of ligands inhibited [125I]-Tyr14-NC binding in membranes from rCX and CHONCR cells. As the peptide was truncated there was a general decline in pKi. [F/G]NC(1–13)NH2 was as potent as NC(1–13)NH2. The order of potency for NC fragments to inhibit cyclic AMP accumulation in whole CHONCR cells was NCNH2NC=NC(1–13)NH2>NC(1–12)NH2>>NC(1–11)NH2. [F/G]NC(1–13)NH2 was a full agonist with a pEC50 value of 8.65. NCNH2 and [F/G]NC(1–13)NH2 both inhibited K+ evoked glutamate release from rCX with pEC50 and maximum inhibition of 8.16, 48.5±4.9% and 7.39, 58.9±6.8% respectively. In rVD NC inhibited electrically evoked contractions with a pEC50 of 6.63. Although [F/G]NC(1–13)NH2, displayed a small (instrinsic activity α=0.19) but consistent residual agonist activity, it acted as a competitive antagonist (pA2 6.76) in the rVD. The differences between [F/G]NC(1–13)NH2 action on central and peripheral NC signalling could be explained if [F/G]NC(1–13)NH2 was a partial agonist with high strength of coupling in the CNS and low in the periphery. An alternative explanation could be the existence of central and peripheral receptor isoforms. British Journal of Pharmacology (1999) 127, 123–130; doi:10.1038/sj.bjp.0702539

1 The aim of the present study was to validate the Cytosensor microphysiometer, a novel system that measures the extracellular acidification rate as a reliable index of the integrated functional response to receptor activation, as a method for studying NK3 receptor pharmacology, and then to use this system to assess the functional activity of novel compounds at this receptor. 2 The selective NK3 agonist senktide caused reproducible, concentration-related increases in acidification ratein CHO-NK3 cells, with a pEC50 value of 8.72±0.11 (n=15). [β-Ala8]NKA(4–10), the selective NK2 agonist, elicited a much weaker response (pEC50=6.68±0.08, n=4), while the NK1-selective agonist substance P methylester only caused a very weak response at concentrations 3 μM (n=2). The rank order of potency for the endogenous tachykinins NKB>NKA>substance P (n=3) confirmed the response was mediated by the NK3 receptor. Moreover, the actual potencies obtained were consistent with affinities measured in radioligand binding studies. 3 The novel compounds PD156319-121 (0.3–1 μM), PD161182 (10–300 nM), PD168001 (10–100 nM) and PD168073 (10–100 nM) all acted as surmountable antagonists of the senktide-induced acidification response, with pA2 values of 7.49, 8.67, 9.17 and 9.25 respectively (n=3–5). In comparison the known NK3 antagonist SR142801 (10–100 nM) had a pA2 value of 8.83 (n=8) for the interaction with senktide. Again, these values are consistent with the radioligand binding data. 4 Amiloride (1 mM) inhibited the senktide-induced acidification response by 68.3±3.3 (n=4), indicating that the Na+/H+ antiporter plays an important role in this response, and this is consistent with the importance of this antiporter in other acidification responses. 5 Inhibition of protein kinase C with staurosporine (0.1 μM), or depletion of the intracellular Ca2+ stores with thapsigargin (1 μM), both resulted in a reduction in the maximum response to senktide (63.3±1.7 and 68.9±3.2% respectively, n=3–5), and co-application of these inhibitors abolished the response (n=3). This strongly suggested that the NK3 receptor was coupling via phospholipase C (PLC), as would be expected, although this could not be confirmed by the use of the putative PLC/PLA2 inhibitor U73122. 6 In conclusion, we have demonstrated the utility of the Cytosensor in the characterization of functional responses to agonists, and assessment of the affinities of antagonists in CHO cells expressing the human NK3, and have shown that our series of novel compounds are non-peptide NK3 antagonists of high affinity, as exemplified by PD168073. British Journal of Pharmacology (1998) 125, 761–766; doi:10.1038/sj.bjp.0702156

Nociceptin/orphanin FQ is the endogenous agonist of the orphan receptor ORL-1. In this study, we sought to examine any possible regional differences of nociceptin binding using [ 125 I]Tyr 14 -nociceptin, and of agonist induced inhibition of cAMP formation in membranes prepared from cerebrocortex, cerebellum and brainstem. The binding of [ 125 I]Tyr 14 -nociceptin was concentration-dependent and saturable, with B max and p K d (pM) values of 179.7±15.3 fmol/mg protein and 10.26±0.09 (60.0), 12.4±1.8 fmol/mg protein and 10.44±0.07 (37.0), 52.3±0.8 fmol/mg protein and 10.16±0.08 (74.0) in cerebrocortical, cerebella and brainstem membranes, respectively. In all preparations, nociceptin up to 1 μ M failed to inhibit basal and forskolin stimulated cAMP formation. In all tissues forskolin stimulated and nabilone (acting at the central cannabinoid receptor) inhibited cAMP formation. Collectively these data report regional differences in ORL-1 receptor expression and that these receptors uncouple during membrane preparation.

The rat μ-opioid receptor has recently been cloned, yet its second messenger coupling remains unclear. The endogenous μ-opioid receptor in SH-SY5Y cells couples to phospholipase C (PLC), increases [Ca2+]i and inhibits adenylyl cyclase (AC). We have examined the effects of μ-opioid agonists on inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), [Ca2+]i and adenosine 3′: 5′-cyclic monophosphate (cyclic AMP) formation in Chinese hamster ovarian (CHO) cells transfected with the cloned μ-opioid receptor. Opioid receptor binding was assessed with [3H]-diprenorphine ([3H]-DPN) as a radiolabel. Ins(1,4,5)P3 and cyclic AMP were measured by specific radioreceptor assays. [Ca2+]i was measured fluorimetrically with Fura-2. Scatchard analysis of [3H]-DPN binding revealed that the Bmax varied between passages. Fentanyl (10 pm–1 μm) dose-dependently displaced [3H]-DPN, yielding a curve which had a Hill slope of less than unity (0.6±0.1), and was best fit to a two site model, with pKi values (% of sites) of 9.97±0.4 (27±4.8%) and 7.68±0.07 (73±4.8%). In the presence of GppNHp (100 μm) and Na+ (100 mm), the curve was shifted to the right and became steeper (Hill slope=0.9±0.1) with a pKi value of 6.76±0.04. Fentanyl (0.1 nm–1 μm) had no effect on basal, but dose-dependently inhibited forskolin (1 μm)-stimulated, cyclic AMP formation (pIC50=7.42±0.23), in a pertussis toxin (PTX; 100 ng ml−1 for 24 h)-sensitive and naloxone-reversible manner (Ki=1.7 nm). Morphine (1 μm) and [d-Ala2, MePhe4, gly(ol)5]-enkephalin (DAMGO, 1 μm) also inhibited forskolin (1 μm)-stimulated cyclic AMP formation, whilst [d-Pen2, d-Pen5], enkephalin (DPDPE, 1 μm) did not. Fentanyl (0.1 nm–10 μm) caused a naloxone (1 μm)-reversible, dose-dependent stimulation of Ins(1,4,5)P3 formation, with a pEC50 of 7.95±0.15 (n=5). PTX (100 ng ml−1 for 24 h) abolished, whilst Ni2+ (2.5 mm) inhibited (by 52%), the fentanyl-induced Ins(1,4,5)P3 response. Morphine (1 μm) and DAMGO (1 μm), but not DPDPE (1 μm), also stimulated Ins(1,4,5)P3 formation. Fentanyl (1 μm) also caused an increase in [Ca2+]i (80±16.4 nm, n=6), reaching a maximum at 26.8±2.5 s. The increase in [Ca2+]i remained elevated until sampling ended (200 s) and was essentially abolished by the addition of naloxone (1 μm). Pre-incubation with naloxone (1 μm, 3 min) completely abolished fentanyl-induced increases in [Ca2+]i. In conclusion, the cloned μ-opioid receptor when expressed in CHO cells stimulates PLC and inhibits AC, both effects being mediated by a PTX-sensitive G-protein. In addition, the receptor couples to an increase in [Ca2+]i. These findings are consistent with the previously described effector-second messenger coupling of the endogenous μ-opioid receptor. British Journal of Pharmacology (1997) 120, 1165–1171; doi:10.1038/sj.bjp.0701012

Opioids have stimulatory as well as the traditional inhibitory effects on neurotransmission, but the underlying mechanisms are poorly understood. Here, Darren Smart and David Lambert review the stimulatory effects of opioids on second messengers, including inositol (1,4,5)-trisphosphate (IP3), protein kinase C (PKC), Ca2+, and cAMP, and propose that these coordinated changes at the cellular level underlie the facilitatory effects of opioids on neurotransmission. The evidence for a possible role for these stimulatory effects, particularly the activation of PKC by opioids, in the development of tolerance is also discussed.

We have recently reported that, in SH-SY5Y cells, mu-opioid receptor occupancy activates phospholipase C via a pertussis toxin-sensitive G-protein. In the present study we have further characterized the mechanisms involved in this process. Fentanyl (0.1 microM) caused a monophasic increase in inositol 1,4,5-trisphosphate mass formation, with a peak (20.5 +/- 3.6 pmol/mg of protein) at 15 s. Incubation in Ca(2+)-free buffer abolished this response, while Ca2+ replacement 1 min later restored the stimulation of inositol 1,4,5-trisphosphate formation (20.1 +/- 0.6 pmol/mg of protein). In addition, nifedipine (1 nM-0.1 mM), an L-type Ca(2+)-channel antagonist, caused a dose-dependent inhibition of inositol 1,4,5-trisphosphate formation, with an IC50 of 60.3 +/- 1.1 nM. Elevation of endogenous beta/gamma subunits by selective activation of delta-opioid and alpha 2 adrenoceptors failed to stimulate phospholipase C. Fentanyl also caused a dose-dependent (EC50 of 16.2 +/- 1.0 nM), additive enhancement of carbachol-induced inositol 1,4,5-trisphosphate formation. In summary, we have demonstrated that in SH-SY5Y cells activation of the mu-opioid receptor allows Ca2+ influx to activate phospholipase C. However, the possible role of this mechanism in the process of analgesia remains to be elucidated.

Inositol(1,4,5)trisphosphate [Ins(1,4,5)P(3)] is an intracellular second messenger that plays an important role in calcium homeostasis and, thus, many diverse cellular processes including neuronal signaling, smooth muscle contraction, fertilization, and sensory perception. Ins(1,4,5)P(3) formation is triggered by the activation of a wide variety of seven-transmembrane, G protein-linked receptors, e.g., muscarinic, glutamate, dopamine, and opioid receptors (1-3), as well as by the activation of the tyrosine kinase-linked growth factor receptors. Ins(1,4,5)P(3) is produced by the phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate, and is metabolized by 3-kinase and 5-phosphatase, with the actual intracellular concentration of Ins(1,4,5)P(3) being dependent on the balance between formation and metabolism. Ins(1,4,5)P(3) in turn binds to the Ins(1,4,5)P(3) receptor on the smooth endoplasmic reticulum, causing a conformational change that opens the intrinsic calcium channel in the receptor, thus allowing the efflux of calcium ions from the intracellular stores. For further details, see the reviews by Berridge and Furuichi and Mikoshiba.

The irreversible opioid receptor antagonists naloxonazine and beta-funaltrexamine have been used to determine whether multiple mu-opioid receptors exist on undifferentiated SH-SY5Y human neuroblastoma cells. Naloxonazine binds irreversibly to the mu 1-opioid receptor subtype and reversibly to the mu 2-opioid receptor subtype. On SH-SY5Y cells naloxonazine afforded a Ki of 3.4 +/- 0.7 nM, and was fully reversible, indicating the mu-opioid receptor population on SH-SY5Y cells was solely of the mu 2-opioid receptor subtype. The alkylating agent beta-funaltrexamine was maximally able to alkylate only 60% of the mu-opioid receptor sites on SH-SY5Y cells, labelled with [3H]diprenorphine or [3H][D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO). The reversible binding of naloxonazine and the insensitivity of a percentage of the mu-opioid receptor sites to alkylation by beta-funaltrexamine suggests that differences do exist in the mu 2-opioid receptor population on undifferentiated SH-SY5Y cells. This may indicate further heterogeneity or the inability of beta-funaltrexamine to alkylate all relevant nucleophilic groups in a single population of receptors.

The cellular mechanisms underlying the clinical effects of volatile anaesthetics remain unknown, although the plasma membrane and its associated proteins are likely targets. One such protein is the enzyme phospholipase C (PLC), which catalyses the formation of the second messenger inositol(1,4,5)triphosphate [Ins(1,4,5)P3]. Using SH-SY5Y human neuroblastoma cells we have demonstrated that halothane (0.50, 0.75 and 1.00%) enhances basal Ins(1,4,5)P3 mass formation approximately 1.8-fold. Halothane also caused a dose-dependent enhancement of carbachol-stimulated biphasic Ins(1,4,5)P3 formation at both the peak (half-maximal stimulation, ec 50 = 0.76% ) and plateau ( ec 50 = 0.74% ) phases. At 1%, halothane did not alter the affinity for carbachol at either the peak ( ic 50: air = 9.4 ± 1.5, halothane = 12.7 ± 1.0 μM) or plateau ( ec 50: air = 11.7 ± 1.2, halothane = 11.6 ± 1.0 μM) phase, but did increase the maximum Ins(1,4,5)P3 response at both phases (air vs halothane: peak, 79.9 ± 0.5 vs 124.8 ± 2.5; plateau, 33.2 ± 0.5 vs 47.9 ± 0.6 pmol/mg protein). Isoflurane (2%) also enhanced basal and carbachol-stimulated Ins(1,4,5)P3 formation 2-fold and 1.5-fold, respectively. In summary, clinically relevant doses of the volatile anaesthetics halothane and isoflurane enhance basal and carbachol-stimulated Ins(1,4,5)P3 formation. Thus, activation of PLC, and subsequent potential Ins(1,4,5)P3-mediated rises in intracellular calcium, could play a part in the cellular mechanisms of volatile agent-induced anaesthesia.

The pharmacology of the orexin-like peptides, hypocretin-1 and hypocretin-2, was studied in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX1) or orexin-2 (OX2) receptors by measuring intracellular calcium ([Ca2+]i) using Fluo-3AM. Orexin-A and orexin-B increased [Ca2+]i in CHO-OX1 (pEC50=7.99±0.05 and 7.00±0.10 respectively, n=8) and CHO-OX2 (pEC50=8.30±0.05 and 8.21±0.07 respectively, n=5). However, hypocretin-1 and hypocretin-2 were markedly less potent, with pEC50 values of 5.31±0.04 and 5.41±0.04 respectively in CHO-OX2 cells (n=5). In CHO-OX1 cells 10 μM hypocretin-1 only elicited a 37.5±3.4% response whilst 10 μM hypocretin-2 elicited a 18.0±2.1% response (n=8). Desensitisation of OX1 or OX2 with orexin-A (100 nM) abolished the response to orexin-A (10 nM) and the hypocretins (10 μM), but not to UTP (3 μM). In conclusion, the hypocretins are only weak agonists at the orexin receptors. British Journal of Pharmacology (2000) 129, 1289–1291; doi:10.1038/sj.bjp.0703257

The cellular mechanisms underlying the physiological effects of the orexins are poorly understood. Therefore, the pharmacology of the recombinant human orexin receptors was studied using FLIPR. Intracellular calcium ([Ca2+]i) was monitored in Chinese hamster ovary (CHO) cells stably expressing orexin-1 (OX1) or orexin-2 (OX2) receptors using Fluo-3AM. Orexin-A and orexin-B increased [Ca2+]i in a concentration dependent manner in CHO-OX1 (pEC50=8.03±0.08 and 7.30±0.08 respectively, n=5) and CHO-OX2 (pEC50=8.18±0.10 and 8.43±0.09 respectively, n=5) cells. This response was typified as a rapid peak in [Ca2+]i (maximal at 6–8 s), followed by a gradually declining secondary phase. Thapsigargin (3 μM) or U73122 (3 μM) abolished the response. In calcium-free conditions the peak response was unaffected but the secondary phase was shortened, returning to basal values within 90 s. Calcium (1.5 mM) replacement restored the secondary phase. In conclusion, orexins cause a phospholipase C-mediated release of calcium from intracellular stores, with subsequent calcium influx. British Journal of Pharmacology (1999) 128, 1–3; doi:10.1038/sj.bjp.0702780

Inositol(1,4,5)trisphosphate [Ins(1,4,5)P(3)] is an intracellular second messenger that plays an important role in calcium homeostasis and, thus, many diverse cellular processes including neuronal signaling, smooth muscle contraction, fertilization, and sensory perception. Ins(1,4,5)P(3) formation is triggered by the activation of a wide variety of seven-transmembrane, G protein-linked receptors, e.g., muscarinic, glutamate, dopamine, and opioid receptors (1-3), as well as by the activation of the tyrosine kinase-linked growth factor receptors. Ins(1,4,5)P(3) is produced by the phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate, and is metabolized by 3-kinase and 5-phosphatase, with the actual intracellular concentration of Ins(1,4,5)P(3) being dependent on the balance between formation and metabolism. Ins(1,4,5)P(3) in turn binds to the Ins(1,4,5)P(3) receptor on the smooth endoplasmic reticulum, causing a conformational change that opens the intrinsic calcium channel in the receptor, thus allowing the efflux of calcium ions from the intracellular stores. For further details, see the reviews by Berridge and Furuichi and Mikoshiba.

Nociceptin, an endogenous ligand for the orphan receptor ORL1, has recently been described. In this study we have shown that nociception inhibits 46 mM K(+)-stimulated glutamate release from rat perfused cerebrocortical slices with an IC50 of 51 nM. At 100 nM the inhibition amounted to 68 +/- 14% and was naloxone (10 microM)-insensitive excluding an activation of mu, delta and kappa opioid receptors. These data demonstrate the functional coupling of ORL1 in glutamatergic neurones and implicates a role for nociceptin in glutamatergic neurotransmission.

Classically, opioids inhibit Ca2+ influx, but recent reports suggest opioids may also stimulate Ca2+ entry. Therefore, we have measured the effect of opioids on intracellular Ca2+ ([Ca2+]i), fluorimetrically, in Fura-2-loaded SH-SY5Y cells. Fentanyl 0.3 mumol litre-1 caused a mean increase in [Ca2+]i of 18.8 (SEM 2.1) nmol litre-1 in some (30.3%) batches of SH-SY5Y cells. In responding cells, the fentanyl-induced increase in [Ca2+]i was dose-dependent, with an EC50 of 0.73 mumol litre-1. This response was naloxone-reversible, and the delta opioid agonist [D-Pen2,5]enkephalin had no effect on [Ca2+]i, suggesting the fentanyl-induced Ca2+ response was entirely mediated by the mu opioid receptor. Fentanyl 0.3 mumol litre-1 increased [Ca2+]i without preactivation of phospholipase C by another agonist, and this was markedly reduced by Ni2+ 2.5 mmol litre-1. These data suggest that mu opioids directly increase [Ca2+]i by stimulating Ca2+ influx in SH-SY5Y cells.

Peripheral cannabinoid 2 receptors (CB2 receptors) modulate immune responses and attenuate nociceptive behaviour in models of acute and persistent pain. The aim of the present study was to investigate whether peripheral CB2 receptors modulate spinal processing of innocuous and noxious responses and to determine whether there are altered roles of CB2 receptors in models of persistent pain. Effects of local administration of the CB2 receptor agonist JWH-133 (5 and 15 microg/50 microL) on mechanically evoked responses of spinal wide dynamic range (WDR) neurons in noninflamed rats, rats with carrageenan-induced hindpaw inflammation, sham operated rats and spinal nerve-ligated (SNL) rats were determined in anaesthetized rats in vivo. Mechanical stimulation (von Frey filaments, 6-80 g) of the peripheral receptive field evoked firing of WDR neurons. Mechanically evoked responses of WDR neurons were similar in noninflamed, carrageenan-inflamed, sham-operated and SNL rats. Intraplantar injection of JWH-133 (15 microg), but not vehicle, significantly (P0.05) inhibited innocuous and noxious mechanically evoked responses of WDR neurons in all four groups of rats. In many cases the selective CB2 receptor antagonist, SR144528 (10 microg/50 microL), attenuated the inhibitory effects of JWH-133 (15 microg) on mechanically evoked WDR neuronal responses. The CB1 receptor antagonist, SR141716A, did not attenuate the inhibitory effects of JWH-133 on these responses. Intraplantar preadministration of JWH-133 also inhibited (P0.05) carrageenan-induced expansion of peripheral receptive fields of WDR dorsal horn neurons. This study demonstrates that activation of peripheral CB2 receptors attenuates both innocuous- and noxious-evoked responses of WDR neurons in models of acute, inflammatory and neuropathic pain.

1 Noladin ether has recently been reported to be an endocannabinoid, with selectivity for the cannabinoid (CB) CB1 receptor. In the present study, we investigated the effects of noladin ether in the rat isolated mesenteric arterial bed, cultured dorsal root ganglia (DRG) cells and human vanilloid (TRPV1)-receptor-expressing HEK293 cells (TRPV1-HEK293 cells). 2 Electrical field stimulation of the mesenteric bed evoked frequency-dependent vasorelaxation due to the action of calcitonin gene-related peptide (CGRP) released from sensory nerves. Noladin ether (0.1-3 microm) attenuated sensory neurogenic relaxation in a concentration-dependent manner. Noladin ether (1 microm) reduced vasorelaxation at a submaximal frequency (8 Hz), from 57.3+/-6.8 to 23.3+/-3.8% (P0.05, n=4). 3 The inhibitory effects of noladin ether were unaffected by the CB1 antagonists SR141716A and LY320135, and the CB2 antagonist SR144528 (1 microm). 4 Noladin ether had no effect on vasorelaxation elicited by exogenous CGRP or capsaicin. These data suggest that noladin ether is acting at a prejunctional site and no interaction with TRPV1 is involved. 5 In mesenteric beds from pertussis toxin (PTX)-pretreated rats, the inhibitory actions of noladin ether on sensory neurotransmission were abolished, indicating the involvement of G(i/o) protein-coupled receptors. 6 Noladin ether evoked a concentration-dependent increase in intracellular Ca2+ concentration in TRPV1-HEK293 cells at 10 microm (36.5+/-3.2% of maximal capsaicin-induced response), but it was a less potent agonist than both capsaicin and anandamide and at 1 microm it was essentially inactive. Noladin ether (1 microm) had no effect on capsaicin-evoked Ca2+ responses in DRG cells, and produced no response alone, indicating it neither modulates nor acts directly on TRPV1 receptors. 7 These data demonstrate that noladin ether attenuates sensory neurotransmission in rat mesenteric arteries via a non-CB1 non-CB2 PTX-sensitive prejunctional site, independently of TRPV1 receptors.

Small molecule antagonists of the vanilloid receptor 1 (TRPV1, also known as VR1) are disclosed. Ureas such as 5 (SB-452533) were used to explore the structure activity relationship with several potent analogues identified. Pharmacological studies using electrophysiological and FLIPR Ca2+ based assays showed compound 5 was an antagonist versus capsaicin, noxious heat and acid mediated activation of TRPV1. Study of a quaternary salt of 5 supports a mode of action in which compounds from this series cause inhibition via an extracellularly accessible binding site on the TRPV1 receptor.

The results of vasorespiratory studies in rats anaesthetised with pentobarbital show that (+/-) cannabidiol, a cannabinoid that lacks psychotropic actions and is inactive at cannabinoid (CB) receptors, does not affect respiration or blood pressure when injected (1-2000 microg; 3.2-6360 nmol i.a.). Cannabidiol in doses up to 2 mg (6360 nmol) i.a. or i.v. did not affect the fall in mean blood pressure or the increase in ventilation (respiratory minute volume) caused by capsaicin and high doses of anandamide, responses that are mediated by activation of vanilloid VR1 (TRPV1) receptors in this species. Similar results were obtained with (-) cannabidiol (30-100 microg i.a.; 95-318 nmol). It has previously been shown using human embryonic kidney (HEK) cells over-expressing vanilloid human VR1 (hVR1) receptors that cannabidiol is a full agonist at vanilloid VR1 receptors in vitro. However, in the intact rat cannabidiol lacked vanilloid VR1 receptor agonist effects. We conclude that there are substantial functional differences between human and rat vanilloid VR1 receptors with respect to the actions of cannabidiol as an agonist at vanilloid VR1 receptors. Studies in vivo show that cannabidiol lacks any significant effect on mean blood pressure or respiratory minute volume when injected i.a. or i.v., and that this cannabinoid does not modulate the vanilloid VR1 receptor-mediated cardiovascular and ventilatory changes reflexly evoked by capsaicin or anandamide in rats anaesthetised with pentobarbital.

It has previously been shown that the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit the proliferation of C6 glioma cells in a manner that can be prevented by a combination of capsazepine (Caps) and cannabinoid (CB) receptor antagonists. It is not clear whether the effect of 2-AG is due to the compound itself, due to the rearrangement to form 1-arachidonoylglycerol (1-AG) or due to a metabolite. Here, it was found that the effects of 2-AG can be mimicked with 1-AG, both in terms of its potency and sensitivity to antagonism by Caps and CB receptor antagonists. In order to determine whether the effect of Caps could be ascribed to actions upon vanilloid receptors, the effect of a more selective vanilloid receptor antagonist, SB366791 was investigated. This compound inhibited capsaicin-induced Ca2+ influx into rVR1-HEK293 cells with a pK(B) value of 6.8 +/- 0.3. The combination of SB366791 and CB receptor antagonists reduced the antiproliferative effect of 1-AG, confirming a vanilloid receptor component in its action. 1-AG, however, showed no direct effect on Ca2+ influx into rVR1-HEK293 cells indicative of an indirect effect upon vanilloid receptors. Identification of the mechanism involved was hampered by a large inter-experimental variation in the sensitivity of the cells to the antiproliferative effects of 1-AG. A variation was also seen with anandamide, which was not a solubility issue, since its water soluble phosphate ester showed the same variability. In contrast, the sensitivity to methanandamide, which was not sensitive to antagonism by the combination of Caps and CB receptor antagonists, but has similar physicochemical properties to anandamide, did not vary between experiments. This variation greatly reduces the utility of these cells as a model system for the study of the antiproliferative effects of anandamide. Nevertheless, it was possible to conclude that the antiproliferative effects of anandamide were not solely mediated by either its hydrolysis to produce arachidonic acid or its CB receptor-mediated activation of phospholipase A(2) since palmitoyltrifluoromethyl ketone did not prevent the response to anandamide. The same result was seen with the fatty acid amide hydrolase inhibitor palmitoylethylamide. Increasing intracellular arachidonic acid by administration of arachidonic acid methyl ester did not affect cell proliferation, and the modest antiproliferative effect of umbelliferyl arachidonate was not prevented by a combination of Caps and CB receptor antagonists. (C) 2003 Elsevier Inc. All rights reserved.

The prevalence of hypertension in middle age correlates with impaired autonomic regulation and as norepinephrinergic neurons decline with increasing age, and this reduction may contribute to this impairment. Central hypocretin-activated norepinephrinergic neurons contribute to sympathetic regulation. In the present study we compared sympathoadrenal effects of intracerebroventricular (i.c.v.) hypocretin-1(5 nmol) between young-adult (12–14 weeks) and middle-aged (12–14 months) rats. Arterial blood pressure, heart rate and plasma catecholamines were assessed under pentobarbital anesthesia. In addition, we compared hypocretin-1 and K+-evoked norepinephrine release from the cerebrocortical slices prepared from young-adult and middle-aged rats. We also examined whether the novel hypocretin receptor-1 antagonist (SB-334867) could reverse these hypocretin-1 effects both in vivo and in vitro. I.c.v. hypocretin-1 significantly increased blood pressure by some 7%, heart rate by 9% and plasma norepinephrine concentrations by 100% in young-adult rats. In middle-aged rats these parameters did not change. Plasma epinephrine did not increase in either group. There was a significant correlation between changes in mean arterial pressure and plasma norepinephrine. Similarly, hypocretin-1 evoked norepinephrine release from cerebrocortical slices prepared from young-adult rats was significantly higher than that of middle-aged rats whilst K+-evoked release did not differ between the groups. SB-334867 significantly attenuated hypocretin-1-increased blood pressure and both in vivo and in vitro norepinephrine release. The present data suggest that hypocretinergic neurons may contribute to the regulation of central but not adrenal sympathetic activity. Moreover, sympathetic regulation by hypocretinergic neurones may disappear in middle-age in rats.