Measurement of inositol(1,4,5)trisphosphate using a stereospecific radioreceptor mass assay.

Inositol(1,4,5)trisphosphate [Ins(1,4,5)P(3)] is an important second messenger that activates its cognate Ins(1,4,5)P(3) receptor to release Ca(2+) from intracellular stores. The assay described in this chapter uses the Ins(1,4,5)P(3) receptor (essentially as a binding protein) to measure the biologically active trisphosphate (specifically from other trisphosphates). The binding protein (Ins(1,4,5)P3 receptor) is prepared from Bovine adrenal glands and this is mixed with [(3)H]-labeled and unlabeled (generated from biological samples or standards) Ins(1,4,5)P(3). Using the same principles as for radioimmunoassay/ELISA the mass of Ins(1,4,5)P(3) in biological samples can be estimated.

Characterization of SB-705498, a potent and selective vanilloid receptor-1 (VR1/TRPV1) antagonist that inhibits the capsaicin-, acid-, and heat-mediated activation of the receptor.

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.

Discovery of SB-705498: a potent, selective and orally bioavailable TRPV1 antagonist suitable 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.

Measurement of inositol(1,4,5)trisphosphate using a stereospecific radioreceptor mass assay.

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.

Cannabinoid CB2 receptor activation inhibits mechanically evoked responses of wide dynamic range dorsal horn neurons in naïve rats and in rat models of inflammatory and neuropathic pain.

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.

Evidence for biological effects of exogenous LPA on rat primary afferent and spinal cord neurons.

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<0.001) and frequency of spinal neuronal firing (P<0.01), compared to vehicle. Intraplantar injection of LPA (1 microg/5 microl) did not significantly alter innocuous and noxious mechanically evoked responses of spinal neurons, but a higher dose of LPA (5 microg) significantly (P<0.05) attenuated mechanically evoked responses of spinal neurons. Calcium imaging studies demonstrated that LPA (0.001-3 microM) increases intracellular calcium concentration in adult DRG neurons, suggesting that LPA can produce direct effects on. Incubation of spinal cord sections with LPA (1 microM) significantly (P<0.001) increased [(35)S]GTPgammaS binding in the superficial laminae of the dorsal horn of the spinal cord, suggesting that LPA may also have biological effects at this level. These data provide further evidence that exogenous LPA can modulate nociceptive processing and suggest that this may be mediated by a direct effect on primary afferent nociceptors.

TRPV1 and CB(1) receptor-mediated effects of the endovanilloid/endocannabinoid N-arachidonoyl-dopamine on primary afferent fibre and spinal cord neuronal responses in the rat.

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.

Discovery of small molecule antagonists of TRPV1.

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 Ca(2+) 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.

Cannabidiol lacks the vanilloid VR1-mediated vasorespiratory effects of capsaicin and anandamide in anaesthetised rats.

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.

Noladin ether, a putative endocannabinoid, attenuates sensory neurotransmission in the rat isolated mesenteric arterial bed via a non-CB1/CB2 G(i/o) linked receptor.

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% (P<0.05, 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.

Inhibition of C6 glioma cell proliferation by anandamide, 1-arachidonoylglycerol, and by a water soluble phosphate ester of anandamide: variability in response and involvement of arachidonic acid.

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 Ca(2+) 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 Ca(2+) 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.

Effects of central hypocretin-1 administration on hemodynamic responses in young-adult and middle-aged rats.

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.

The effects of local and intravenous anesthetics on recombinant rat VR1 vanilloid receptors.

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.

Lack of an interaction between orexinergic and opioid/nociceptinergic systems in rat cerebrocortical slices.

We have recently reported that orexins (OXs) selectively evoke norepinephrine release from rat cerebrocortical slices. In the present study, we have examined orexin-opioid interactions in OXA (100 nM) and K(+) (40 mM)-evoked norepinephrine release. OXA-evoked norepinephrine release was reversed approximately 90% by SB-334867 (OX(1)-receptor antagonist) (10 microM) but not naloxone (10 microM). [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE) (DOP-agonist) and nociceptin/orphanin-FQ (N/OFQ) also failed to affect OXA-evoked release. [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) (MOP-agonist) and spiradoline (KOP-agonist) significantly reduced OXA-evoked release with the concentration producing 50% of the maximal inhibition (EC(50)) [maximal inhibition (E(max))] of 3.2 microM [41.8%] and 4.3 microM [54.9%] respectively. The effects of DAMGO and spiradoline were naloxone (10 microM)-insensitive. In contrast, naloxone significantly antagonized the inhibitory effects of DAMGO and spiradoline on K(+)-evoked release. We conclude that opioid receptors (DOP and KOP) are involved in K(+) but not OXA-evoked release. Moreover, we have failed to demonstrate an interaction between orexinergic and opioid/N/OFQ-ergic systems in this system.

N-Morpholino- and N-diethyl-analogues of palmitoylethanolamide increase the sensitivity of transfected human vanilloid receptors to activation by anandamide without affecting fatty acid amidohydrolase activity.

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.

Activation of vanilloid receptor 1 by resiniferatoxin mobilizes calcium from inositol 1,4,5-trisphosphate-sensitive stores.

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.

Pharmacology of vanilloids at recombinant and endogenous rat vanilloid receptors.

This study compared the actions of members of five different chemical classes of vanilloid agonists at the recombinant rat vanilloid VR1 receptor expressed in HEK293 cells, and at endogenous vanilloid receptors on dorsal root ganglion cells and sensory nerves in the rat isolated mesenteric arterial bed. In mesenteric beds, vanilloids elicited dose-dependent vasorelaxation with the rank order of potency: resiniferatoxin>>capsaicin=olvanil>phorbol 12-phenyl-acetate 13-acetate 20-homovanillate (PPAHV)>isovelleral. Scutigeral was inactive. Responses were abolished by capsaicin pretreatment and inhibited by ruthenium red. In VR1-HEK293 cells and dorsal root ganglion neurones, Ca(2+) responses were induced by resiniferatoxin>capsaicin=olvanil>PPAHV; all four were full agonists. Isovelleral and scutigeral were inactive. The resiniferatoxin-induced Ca(2+) response had a distinct kinetic profile. Olvanil had a Hill coefficient of approximately 1 whilst capsaicin, resiniferatoxin and PPAHV had Hill coefficients of approximately 2 in VR1-HEK293 cells. The capsaicin-induced Ca(2+) response was inhibited in a concentration-dependent manner by ruthenium red>capsazepine>isovelleral. These data show that resiniferatoxin, capsaicin, olvanil and PPAHV, but not scutigeral and isovelleral, are agonists at recombinant rat VR1 receptors and endogenous vanilloid receptors on dorsal root ganglion neurones and in the rat mesenteric arterial bed. The vanilloids display the same relative potencies (resiniferatoxin>capsaicin=olvanil>PPAHV) in all of the bioassays.

Cannabinoid modulation of sensory neurotransmission via cannabinoid and vanilloid receptors: roles in regulation of cardiovascular function.

Capsaicin-sensitive sensory nerves are widely distributed in the cardiovascular system. They are activated by a variety of physical and chemical stimuli, characteristically by capsaicin acting via the vanilloid receptor VR1, and have a role in the regulation of peripheral vascular resistance and maintenance of homeostasis via their afferent and efferent functions. Cannabinoids, a recently discovered family of extracellular signalling molecules, can act at cannabinoid (CB) receptors expressed on sensory nerves, to cause inhibition of sensory neurotransmitter release. There is recent evidence, however, that anandamide, an endogenous cannabinoid, can activate VR1, coexpressed with CB receptors on the same sensory nerve terminals, causing a release of sensory neurotransmitter, vasorelaxation and hypotension. Hence, anandamide can elicit opposite actions, inhibition via CB receptors and excitation via VR1, on sensory neurotransmission. The possible biological significance of this is discussed.

The physiology and pharmacology of the orexins.

Orexin-A and orexin-B are two peptides derived by proteolytic cleavage from a 130 amino acid precursor prepro-orexin, which recently were isolated from the rat hypothalamus. Orexin-A is fully conserved across mammalian species, whilst rat and human orexin-B differ by 2 amino acids. These peptides bind to two G(q)-coupled receptors, termed OX(1) and OX(2). The receptors are 64% homologous and highly conserved across species. Orexin-A is equipotent at OX(1) and OX(2), whilst orexin-B displays moderate ( approximately 10-fold) selectivity for OX(2). Prepro-orexin is found in the hypothalamus and, to a markedly lesser extent, the testes, adrenals, and myenteric plexus. However, orexin-A and orexin-B are found throughout the CNS, due to extrahypothalamic projections, as well as in the adrenals and small intestine. OX(1) is expressed mainly in the hypothalamus and locus coeruleus, as well as other brain regions and the spinal cord. OX(2) is expressed in the hypothalamus, cortex, spinal cord, and a few discrete brain nuclei. Both receptors are also expressed in the gut. The orexins modulate feeding behaviour and energy homeostasis, as well as associated drinking behaviours, and also regulate the sleep-wake cycle. Moreover, disruption of prepro-peptide expression or mutations in the gene encoding OX(2) result in a narcoleptic phenotye in various animal models, whilst several clinical studies have linked disruption of the orexin system to narcolepsy in humans. The orexins also have cardiovascular and neuroendocrine effects. This review further details the pharmacology and localisation of these peptides and summarises the evidence for their role in the physiology outlined above.