The vanilloid receptor 1 (VR1, TRPV1), which is a member of the transient receptor potential (TRP) superfamily, is highly localized on peripheral and central processes of nociceptive afferent fibers. Activation of TRPV1 contributes to the pronociceptive effects of capsaicin, protons, heat, and various endogenous lipid agonists such as anandamide and N-arachidonoyl-dopamine. A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)urea] is a novel potent and selective antagonist at both human and rat TRPV1 receptors. In vivo, A-425619 dose dependently reduced capsaicininduced mechanical hyperalgesia (ED 50 ϭ 45 mol/kg p.o.). A-425619 was also effective in models of inflammatory pain and postoperative pain. A-425619 potently reduced complete Freund's adjuvant-induced chronic inflammatory pain after oral administration (ED 50 ϭ 40 mol/kg p.o.) and was also effective after either i.t. administration or local injection into the inflamed paw. Furthermore, A-425619 maintained efficacy in the postoperative pain model after twice daily dosing p.o. for 5 days. A-425619 also showed partial efficacy in models of neuropathic pain. A-425619 did not alter motor performance at the highest dose tested (300 mol/kg p.o.). Taken together, the present data indicate that A-425619, a potent and selective antagonist of TRPV1 receptors, effectively relieves acute and chronic inflammatory pain and postoperative pain.The vanilloid receptor VR1 or TRPV1 is a nonselective cation channel that is activated by exogenous vanilloid compounds such as capsaicin (Caterina and Julius, 2001). Anatomical and functional studies have shown that TRPV1 receptors are expressed on peripheral nociceptors (for review, see Cortright and Szallasi, 2004). Recently, the analgesic potential of TRPV1 receptor blockade has been demonstrated by various approaches including gene disruption, neutralizing antibodies, or receptor antagonism (Caterina et al., 2000;Davis et al., 2000;Kamei et al., 2001;Walker et al., 2003). Although TRPV1 gene-disrupted mice showed mostly normal behavioral responses to noxious heat, they did not develop thermal hyperalgesia to mustard oil or complete Freund's adjuvant (CFA;Caterina et al., 2000). These results suggest that TRPV1 receptors are required for responses to noxious thermal stimuli under inflammatory conditions but that other mechanisms are in part responsible for normal sensation of noxious heat. Consistent with this conclusion, Davis et al. (2000) showed that TRPV1 knockout mice did not develop thermal hyperalgesia in response to carrageenan but showed normal responses to noxious heat. However, TRPV1 knockout mice did develop mechanical allodynia in response to CFA and mustard oil, showed normal responses to formalin, and developed both thermal hyperalgesia and mechanical allodynia after partial nerve injury (Caterina et al., 2000). A role for TRPV1 in thermal hypersensitivity has also been described in diabetic mice. Following i.t. administration of a TRPV1-neutralizing antibody, a partial reduction in thermal hypersen...

Prolonged activation of opioid receptors leads to their phosphorylation, desensitization, internalization, and down-regulation. To elucidate the relationship between -opioid receptor (MOR) phosphorylation and the regulation of receptor activity, a series of receptor mutants was constructed in which the 12 Ser/Thr residues of the COOH-terminal portion of the receptor were substituted to Ala, either individually or in combination. All these mutant constructs were stably expressed in human embryonic kidney 293 cells and exhibited similar expression levels and ligand binding properties. to Ala accelerated MOR internalization kinetics. The present data show that the basal phosphorylation of MOR could play a role in modulating agonist-induced receptor internalization kinetics. Furthermore, even though -receptors and ␦-opioid receptors have the same motif encompassing agonist-induced phosphorylation sites, the different agonist-induced internalization properties controlled by these sites suggest differential cellular regulation of these two receptor subtypes.Opioid alkaloids, as well as endogenous opioid peptides, exert their multiple biological effects on target tissues by interacting with specific cell surface receptors including the ␦-, -, and -opioid receptors (1). These opioid receptors belong to the superfamily of G protein-coupled receptors (GPCRs).1 The -opioid receptor (MOR) serves as the principle physiological target for most clinically important opioid analgesics, such as morphine and fentanyl (2, 3). Although many opioid alkaloids exert their pharmacological effects via MOR, their binding affinity for the receptor and potency to activate the receptor do not always correspond to their abilities to induce tolerance (4 -8). This suggests that other cellular processes that modulate MOR responsiveness, such as receptor desensitization and internalization, may contribute to opioid tolerance and dependence. Like many other GPCRs, opioid receptors are regulated by agonist-dependent processes and undergo receptor phosphorylation, desensitization, internalization, and down-regulation (1). Interestingly, in addition to the subtype-specific regulation of opioid receptors (9 -12), individual opioid receptors are differentially regulated by distinct opioid agonists (5-8, 13, 14). In the case of MOR, opioid agonists demonstrating equivalent ability to activate receptor signaling exhibit remarkable differences in their abilities to functionally desensitize (5, 6) and induce internalization of the receptor in both transfected cells and neurons (7,8,(13)(14)(15). However, the detailed molecular events underlying this differential regulation of MOR by distinct agonists remain unclear. Using chimeric, truncated, or mutated opioid receptors, several studies reported the crucial role of the C-tail of opioid receptors in regulating their activities and trafficking (11, 16 -20). Whereas several potential phosphorylation sites were suggested to be involved in regulating the activity and trafficking of opioid receptors, the actual ...

It is well established that the vanilloid receptor, VR1, is an important peripheral mediator of nociception. VR1 receptors are also located in several brain regions, yet it is uncertain whether these supraspinal VR1 receptors have any influence on the nociceptive system. To investigate a possible nociceptive role for supraspinal VR1 receptors, capsaicin (10 nmol in 0.4 microl) was microinjected into either the dorsal (dPAG) or ventral (vPAG) regions of the periaqueductal gray. Capsaicin-related effects on tail flick latency (immersion in 52 degrees C water) and on neuronal activity (on-, off-, and neutral cells) in the rostral ventromedial medulla (RVM) were measured in lightly anesthetized rats. Administration of capsaicin into the dPAG but not the vPAG caused an initial hyperalgesic response followed later by analgesia (125 +/- 20.96 min postinjection). The tail flick-related burst in on-cell activity was triggered earlier in the hyperalgesic phase and was delayed or absent during the analgesic phase. Spontaneous activity of on-cells increased at the onset of the hyperalgesic phase and decreased before and during the analgesic phase. The tail flick-related pause in off-cell activity as well as spontaneous firing for these cells was unchanged in the hyperalgesic phase. During the analgesic phase, off-cells no longer paused during noxious stimulation and had increased levels of spontaneous activity. Neutral cell firing was unaffected in either phase. Pretreatment with the VR1 receptor antagonist, capsazepine (10 nmol in 0.4 microl), into the dPAG blocked the capsaicin-induced hyperalgesia as well as the corresponding changes in on- and off-cell activity. VR1 receptor immunostaining was observed in the dPAG of untreated rats. Microinjection of capsaicin likely sensitized and then desensitized dPAG neurons affecting nocifensive reflexes and RVM neuronal activity. These results suggest that supraspinal VR1 receptors in the dPAG contribute to descending modulation of nociception.

The vanilloid receptor transient receptor potential type V1 (TRPV1) integrates responses to multiple stimuli, such as capsaicin, acid, heat, and endovanilloids and plays an important role in the transmission of inflammatory pain. Here, we report the identification and in vitro characterization of A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea], a novel, potent, and selective TRPV1 antagonist. A-425619 was found to potently block capsaicin-evoked increases in intracellular calcium concentrations in HEK293 cells expressing recombinant human TRPV1 receptors (IC 50 ϭ 5 nM). A-425619 showed similar potency (IC 50 ϭ 3-4 nM) to block TRPV1 receptor activation by anandamide and N-arachidonoyl-dopamine. Electrophysiological experiments showed that A-425619 also potently blocked the activation of native TRPV1 channels in rat dorsal root ganglion neurons (IC 50 ϭ 9 nM). When compared with other known TRPV1 antagonists, A-425619 exhibited superior potency in blocking both naive and phorbol estersensitized TRPV1 receptors. Like capsazepine, A-425619 demonstrated competitive antagonism (pA 2 ϭ 2.5 nM) of capsaicinevoked calcium flux. Moreover, A-425619 was 25-to 50-fold more potent than capsazepine in blocking TRPV1 activation. A-425619 showed no significant interaction with a wide range of receptors, enzymes, and ion channels, indicating a high degree of selectivity for TRPV1 receptors. These data show that A-425619 is a structurally novel, potent, and selective TRPV1 antagonist.

Chronic activation of the -opioid receptor (MOR1TAG) results in the loss of agonist response that has been attributed to desensitization and down-regulation of the receptor. It has been suggested that opioid receptor phosphorylation is the mechanism by which this desensitization and down-regulation occurs. When MOR1TAG was stably expressed in both neuroblastoma neuro2A and human embryonic kidney HEK293 cells, the opioid agonist [D-Ala 2 ,MePhe 4 ,Gly 5 -ol]enkephalin (DAMGO) induced a time-and concentration-dependent phosphorylation of the receptor, in both cell lines, that could be reversed by the antagonist naloxone. Protein kinase C can phosphorylate the receptor, but is not involved in DAMGO-induced MOR1TAG phosphorylation. The rapid rate of receptor phosphorylation, occurring within minutes, did not correlate with the rate of the loss of agonist-mediated inhibition of adenylyl cyclase, which occurs in hours. This lack of correlation between receptor phosphorylation and the loss of response was further demonstrated when receptor phosphorylation was increased by either calyculin A or overexpression of the G-protein receptor kinases. Calyculin A increased the magnitude of MOR1TAG phosphorylation without altering the DAMGO-induced loss of the adenylyl cyclase response. Similarly, when -and ␦-opioid (DOR1TAG) receptors were expressed in the same system, overexpression of ␤-adrenergic receptor kinase 2 elevated agonist-induced phosphorylation for both receptors. However, in the same cell lines under the same conditions, overexpression of ␤-adrenergic receptor kinase 2 and ␤-arrestin 2 accelerated the rate of DPDPE-but not DAMGO-induced receptor desensitization. Thus, these data show that phosphorylation of MOR1TAG is not an obligatory event for the DAMGO-induced loss in the adenylyl cyclase regulation by the receptor.Phosphorylation of membrane receptors, such as tyrosine kinase receptors or G-protein coupled receptors (GPCRs), 1 leads to an alteration in the receptor activities (1). While autophosphorylation of the tyrosine kinase receptors by the associated kinases results in the initiation of the signal cascades, agonist-induced phosphorylation of the GPCRs by protein kinases, recruited to the vicinity of the receptors, blunts the signal cascades. Rapid phosphorylation of these GPCRs, by specific protein kinases, i.e. G-protein-coupled receptor kinases, GRKs, and the subsequent binding of arrestins, has been considered to be a mechanism for the agonist-induced homologous desensitization of these receptors (2, 3).From the cloning of the receptors, it is clear that -, ␦-, and -opioid receptors belong to the superfamily of GPCRs (4, 5). As with other GPCRs, prolonged agonist treatment results in an attenuation of effector responses and down-regulation of the receptor, both in clonal cell lines that express the opioid receptor endogenously (6, 7) or in cell lines that stably express cloned opioid receptors (8, 9). Chronic exposure to opioid agonist results in a reduced receptor affinity for the agonist, as well as...

Intact colicin N and various colicin derivatives, including a natural fragment lacking the first 36 amino-acid residues, a chymotryptic fragment lacking the first 66 amino acids and a thermolytic fragment comprising residues 183-387, were used to locate the regions involved in colicin-N uptake by sensitive Escherichia coli cells. Two separate domains of the molecule participate in colicin-N entry. Specific binding to OmpF receptor site requires a region located between residues 67-182. A N-terminal domain, located between residues 17-66, is involved during the translocation step after binding to receptor. Two sub-regions, residues 17-36 and residues 37-36, can be defined in this domain. The location and interactions between these domains are discussed in comparison to other colicins which use similar cell components for their uptake.

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The transient receptor potential vanilloid (TRPV) 1 receptor, a nonselective cation channel expressed on peripheral sensory neurons and in the central nervous system, plays a key role in pain. TRPV1 receptor antagonism is a promising approach for pain management. In this report, we describe the pharmacological and functional characteristics of a structurally novel TRPV1 antagonist, (R)-(5-tert-butyl-2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-4-yl)-urea (ABT-102), which has entered clinical trials. At the recombinant human TRPV1 receptor ABT-102 potently (IC 50 ϭ 5-7 nM) inhibits agonist (capsaicin, N-arachidonyl dopamine, anandamide, and proton)-evoked increases in intracellular Ca 2ϩ levels. ABT-102 also potently (IC 50 ϭ 1-16 nM) inhibits capsaicin-evoked currents in rat dorsal root ganglion (DRG) neurons and currents evoked through activation of recombinant rat TRPV1 currents by capsaicin, protons, or heat. ABT-102 is a competitive antagonist (pA 2 ϭ 8.344) of capsaicin-evoked increased intracellular Ca 2ϩ and shows high selectivity for blocking TRPV1 receptors over other TRP receptors and a range of other receptors, ion channels, and transporters. In functional studies, ABT-102 blocks capsaicin-evoked calcitonin gene-related peptide release from rat DRG neurons. Intraplantar administration of ABT-102 blocks heat-evoked firing of wide dynamic range and nociceptive-specific neurons in the spinal cord dorsal horn of the rat. This effect is enhanced in a rat model of inflammatory pain induced by administration of complete Freund's adjuvant. Therefore, ABT-102 potently blocks multiple modes of TRPV1 receptor activation and effectively attenuates downstream consequences of receptor activity. ABT-102 is a novel and selective TRPV1 antagonist with pharmacological and functional properties that support its advancement into clinical studies.The transient receptor potential vanilloid (TRPV) 1 receptor is a nonselective cation channel that is expressed on peripheral and central terminals of small and medium-sized primary sensory neurons and plays a key role in the detection and modulation of nociceptive stimuli (Caterina and Julius, 2001). In the central nervous system, the channel is expressed presynaptically in lamina I and postsynaptically in lamina II of the spinal cord dorsal horn and at supraspinal sites in pain pathways (Valtschanoff et al., 2001; Article, publication date, and citation information can be found at