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Creating a robust and unbiased assay for the study of current and novel analgesics has been a daunting task. Traditional rodent models of pain and inflammation typically rely on a negative reaction to various forms of evoked stimuli to elicit a pain response and are subject to rater interpretation. Recently, models such as weight bearing and gait analysis have been developed to address these drawbacks while detecting a drug's analgesic properties. We have recently developed the Reduction of Spontaneous Activity by Adjuvant (RSAA) model as a quick, unbiased method for the testing of potential analgesics. Rats, following prior administration of an activity-decreasing inflammatory insult, will positively increase spontaneous locomotor exploration when given single doses of known analgesics. The RSAA model capitalizes on a rat's spontaneous exploratory behavior in a novel environment with the aid of computer tracking software to quantify movement and eliminate rater bias.

Purpose: We examined the potential for the pro-inflammatory complement proteins C5a and C3a to increase VEGF expression in ARPE-19 cells., Materials and Methods: Expression of complement receptors in ARPE-19 cells was evaluated by RT-PCR. VEGF secretion from ARPE-19 cells treated with C5a or C3a was determined by ELISA., Results: C5a and C3a receptor, but not C5L2, were detected in human eye tissue and ARPE-19 cells. C5a, but not C3a, treatment increased VEGF secretion from ARPE-19 cells, an effect inhibited by the C5aR antagonist, NDT 9513727., Conclusions: C5a receptor mediates increased VEGF secretion from ARPE-19 cells, suggesting a role for the C5a receptor in the pathogenesis of macular degeneration.

The somatosensory effects of natural products such as capsaicin, mustard oil, and menthol have been long recognized. Over the last decade, the identification of transient receptor potential (TRP) channels in primary sensory neurons as the targets for these agents has led to an explosion of research into the roles of "thermoTRPs" TRPV1, TRPV2, TRPV3, TRPV4, TRPA1, and TRPM8 in nociception. In concert, through the efforts of many industrial and academic teams, a number of agonists and antagonists of these channels have been discovered, paving the way for a better understanding of sensory biology and, potentially, for novel treatments for diseases.

Preclinical research has identified an array of ion channels in sensory neurons involved in the generation and transduction of pain as potential targets for pharmacological intervention. Paramount among these new targets is the family of thermosensitive transient receptor potential channels, referred to as "thermoTRPs". We detect a wide range of noxious stimuli via a limited number (as of today, six) of thermoTRP channels, four of which (TRPV1-TRPV4) respond to heat and two (TRPA1 and TRPM8) are sensitive to cold. Targeting these thermoTRP channels represents a new and logical strategy in pain relief. Unlike traditional analgesic drugs that either suppress inflammation (e.g. NSAIDs and COX-2 inhibitors) or block pain transmission (e.g. opiates), TRP channel inhibitors aim to prevent pain by blocking a receptor where pain is generated. The archetypal thermoTRP is the vanilloid (capsaicin) receptor TRPV1. TRPV1 has a dynamic threshold of activation. Agents in inflammatory soup, including endogenous TRPV1 agonists (so-called "endovanilloids"), act in concert to reduce the heat activation threshold of TRPV1. In patients, the expression of TRPV1 is up-regulated in a number of painful inflammatory disorders. TRPV1 as a pain target has been validated by genetic deletion and pharmacological inhibition experiments. This area of drug development has been moving rapidly. It took less than a decade from the cloning of TRPV1 to clinical trials with potent small molecule TRPV1 antagonists. This review evaluates current evidence that supports particular TRP channels as targets for novel analgesic drugs, along with potential adverse effects that may limit drug development.

The complement system represents an innate immune mechanism of host defense that has three effector arms, the C3a receptor, the C5a receptor (C5aR), and the membrane attack complex. Because of its inflammatory and immune-enhancing properties, the biological activity of C5a and its classical receptor have been widely studied. Because specific antagonism of the C5aR could have therapeutic benefit without affecting the protective immune response, the C5aR continues to be a promising target for pharmaceutical research. The lack of specific, potent and orally bioavailable small-molecule antagonists has limited the clinical investigation of the C5aR. We report the discovery of NDT 9513727 [N,N-bis(1,3-benzodioxol-5-ylmethyl)-1-butyl-2,4-diphenyl-1H-imidazole-5-methanamine], a small-molecule, orally bioavailable, selective, and potent inverse agonist of the human C5aR. NDT 9513727 was discovered based on the integrated use of in vitro affinity and functional assays in conjunction with medicinal chemistry. NDT 9513727 inhibited C5a-stimulated responses, including guanosine 5'-3-O-(thio)triphosphate binding, Ca(2+) mobilization, oxidative burst, degranulation, cell surface CD11b expression and chemotaxis in various cell types with IC(50)s from 1.1 to 9.2 nM, respectively. In C5a competition radioligand binding experiments, NDT 9513727 exhibited an IC(50) of 11.6 nM. NDT 9513727 effectively inhibited C5a-induced neutropenia in gerbil and cynomolgus macaque in vivo. The findings suggest that NDT 9513727 may be a promising new entity for the treatment of human inflammatory diseases.

Recent evidence suggests that the P2X(7) receptor may play a role in the pathophysiology of preclinical models of pain and inflammation. Therefore, pharmacological agents that target this receptor may potentially have clinical utility as anti-inflammatory and analgesic therapy. We investigated and characterized the previously reported P2X(7) antagonist N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, hydrochloride salt (AACBA; GSK314181A). In vitro, AACBA was a relatively potent inhibitor of both human P2X(7)-mediated calcium flux and quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triemethylammonio)propyl]-diiodide (YO-PRO-1) uptake assays, with IC(50) values of approximately 18 and 85 nM, respectively. Compared with the human receptor, AACBA was less potent at the rat P2X(7) receptor, with IC(50) values of 29 and 980 nM in the calcium flux and YO-PRO-1 assays, respectively. In acute in vivo models of pain and inflammation, AACBA dose-dependently reduced lipopolysaccharide-induced plasma interleukin-6 release and prevented or reversed carrageenan-induced paw edema and mechanical hypersensitivity. In chronic in vivo models of pain and inflammation, AACBA produced a prophylactic, but not therapeutic-like, prevention of the clinical signs and histopathological damage of collagen-induced arthritis. Finally, AACBA could not reverse L(5) spinal nerve ligation-induced tactile allodynia when given therapeutically. Consistent with previous literature, these results suggest that P2X(7) receptors do play a role in animal models of pain and inflammation. Further study of P2X(7) antagonists both in preclinical and clinical studies will help elucidate the role of the P2X(7) receptor in pain and inflammatory mechanisms and may help identify potential clinical benefits of such molecules.

Background: Translating promising analgesic compounds into reliable pain therapeutics in humans is made particularly challenging by the difficulty in measuring the pain quantitatively. This problem is manifest not only in clinical settings in which patient pain assessments involve mostly subjective measures but also in preclinical settings wherein laboratory animals, most commonly rodents, are typically evaluated in stimulus-evoked response tests., Objective: Given the limitations of traditional pain tests, we sought out new approaches to measure pain, and analgesia, in laboratory animals., Methods: We reviewed the peer reviewed literature to identify pain tests that could be utilized in preclinical settings to understand the effects of new and established analgesics., Results/conclusions: The tests identified include weight bearing differential, suppression of feeding, reduction in locomotor activity, gait analysis, conditioning models and functional MRI. Although the pharmacology of known and new analgesics has not been broadly established in these models, they hold the promise of better predictive utility for the discovery of pain relievers.

Since the molecular identification of the capsaicin receptor, now known as TRPV1, transient receptor potential (TRP) channels have occupied an important place in the understanding of sensory nerve function in the context of pain. Several TRP channels exhibit sensitivity to substances previously known to cause pain or pain-like sensations; these include cinnamaldehyde, menthol, gingerol, and icillin. Many TRP channels also exhibit significant sensitivity to increases or decreases in temperature. Some TRP channels are sensitized in vitro by the activation of other receptors such that these channels may be activated by processes, such as inflammation that result in pain. TRP channels are suggested to be involved in processes as diverse as sensory neuron activation events, neurotransmitter release and action in the spinal cord, and release of inflammatory mediators. These functions strongly suggest that specific and selective inhibition of TRP channel activity will be of use in alleviating pain.

Thermosensitive TRP channels display unique thermal responses, suggesting distinct roles mediating sensory transmission of temperature. However, whether relative expression of these channels in dorsal root ganglia (DRG) is altered in nerve injury is unknown. We developed a multiplex ribonuclease protection assay (RPA) to quantify rat TRPV1, TRPV2, TRPV3, TRPV4, TRPA1, and TRPM8 RNA levels in DRG. We used the multiplex RPA to measure thermosensitive TRP channel RNA levels in DRG from RTX-treated rats (300 microg/kg) or rats with unilateral sciatic nerve chronic constriction injury (CCI). TRPV1 and TRPA1 RNA were significantly decreased in DRG from RTX-treated rats, indicating functional colocalization of TRPA1 and TRPV1 in sensory nociceptors. In DRG from CCI rats, TRPA1, TRPV2, and TRPM8 RNA showed slight but significant increases ipsilateral to peripheral nerve injury. Our findings support the hypothesis that increased TRP channel expression in sensory neurons may contribute to mechanical and cold hypersensitivity.

The clinical use of TRPV1 (transient receptor potential vanilloid subfamily, member 1; also known as VR1) antagonists is based on the concept that endogenous agonists acting on TRPV1 might provide a major contribution to certain pain conditions. Indeed, a number of small-molecule TRPV1 antagonists are already undergoing Phase I/II clinical trials for the indications of chronic inflammatory pain and migraine. Moreover, animal models suggest a therapeutic value for TRPV1 antagonists in the treatment of other types of pain, including pain from cancer. We argue that TRPV1 antagonists alone or in conjunction with other analgesics will improve the quality of life of people with migraine, chronic intractable pain secondary to cancer, AIDS or diabetes. Moreover, emerging data indicate that TRPV1 antagonists could also be useful in treating disorders other than pain, such as urinary urge incontinence, chronic cough and irritable bowel syndrome. The lack of effective drugs for treating many of these conditions highlights the need for further investigation into the therapeutic potential of TRPV1 antagonists.

The majority of rodent models used to evaluate analgesic drug effects rely on evoked measures of nociceptive thresholds as primary outcomes. These approaches are often time-consuming, requiring extensive habituation sessions and repeated presentations of eliciting stimuli, and are prone to false-positive outcomes due to sedation or tester subjectivity. Here, we describe the reduction of spontaneous activity by adjuvant (RSAA) model as an objective and quantifiable behavioral model of inflammatory pain that can predict the analgesic activity of a variety of agents following single-dose administration. In the RSAA model, activity was measured in nonhabituated rats using standard, photocell-based monitors. Bilateral inflammation of the knee joints by complete Freund's adjuvant (CFA) reduced the normal level of activity (horizontal locomotion and vertical rearing) by approximately 60% in a novel environment. This reduction in activity was dose-dependently reversed by ibuprofen, rofecoxib, celecoxib, piroxicam, and dexamethasone, whereas gabapentin and amitriptyline were inactive. Morphine significantly reversed the activity-suppressing effects of CFA, at 1 mg/kg s.c., but at higher doses locomotor activity progressively declined, coincident with the induction of sedation. In contrast to morphine and anti-inflammatory therapies, amphetamine did not affect vertical rearing, even though it increased horizontal locomotion. Thus, unlike standard measures of analgesia such as alteration in thermal or mechanical sensitivity, the RSAA model operationally defines analgesia as a drug-induced increase in spontaneous behavior (vertical rearing in a novel environment). We conclude that the RSAA model is valuable as an objective measure of analgesic efficacy that is not dependent on an evoked stimulus response.

Background: Distinct pathways of leukocyte activation during simulated cardiopulmonary bypass are mediated by the complement C5a anaphylatoxin. We hypothesized that a human C5a receptor antagonist would specifically inhibit the inflammatory response of neutrophils to simulated extracorporeal circulation, while preserving the C5b-9 pathway for innate immunity., Methods: An in vitro extracorporeal circuit recirculated fresh heparinized whole blood through a membrane oxygenator with and without addition of a small molecule human C5a receptor antagonist. Samples were periodically drawn over 90 minutes for complement and leukocyte activation studies., Results: Addition of the C5a receptor antagonist to simulated extracorporeal circulation abrogated both neutrophil CD11b upregulation and interleukin 8 release (p < 0.01 for both), despite full generation of C3a and C5b-9; however, elastase release from neutrophils was unaffected. Although C5a receptor blockade only trended toward inhibiting monocyte CD11b upregulation (p = 0.09), circuit clearance of both monocytes (p = 0.04) and neutrophils (p = 0.01) was significantly decreased. In addition, the C5a receptor antagonist completely blocked both neutrophil-platelet and monocyte-platelet conjugate formation (p < 0.001 for both), without affecting platelet P-selectin expression., Conclusions: C5a receptor blockade during simulated extracorporeal circulation completely blocked neutrophil beta2 integrin upregulation and induction of plasma interleukin 8, suggesting an acute downregulatory effect on neutrophil chemotaxis-related pathways, while preserving terminal complement generation and neutrophil elastase release. Inhibition of leukocyte-platelet conjugate formation suggests a novel function for leukocyte adhesive receptors, possibly related to preservation of elastase generation.

Neuronal hyperexcitability in both injured and adjacent uninjured neurons is associated with states of chronic injury and pain and is likely subject to neuroinflammatory processes. Chronic inflammatory responses are largely orchestrated by chemokines. One chemokine, monocyte chemoattractant protein-1 (MCP-1), in the presence of its cognate receptor, the beta chemokine receptor 2 (CCR2), produces neural activity in dissociated neuronal cultures of neonatal dorsal root ganglion (DRG) neurons. Using a neuropathic pain model, chronic compression of the DRG (CCD), we compared anatomically separate populations of noncompressed lumbar DRG (L3/L6) with compressed lumbar DRG (L4/L5) for changes in the gene expression of CCR2. In situ hybridization revealed that CCR2 mRNA was up-regulated in neurons and nonneuronal cells present in both compressed L4/L5 and ipsilateral noncompressed L3/L6 DRGs at postoperative day 5 (POD5). The total percentages of compressed and noncompressed neurons exhibiting CCR2 mRNA transcripts in L3, L5, and L6 DRG were 33 +/- 3.5%, 49 +/- 6.2%, and 41 +/- 5.6%, respectively, and included cell bodies of small, medium, and large size. In addition, the preferred CCR2 ligand, MCP-1, was up-regulated by POD5 in both compressed L4/L5 and noncompressed L3/L6 DRG neurons. Application of MCP-1 to the cell bodies of the intact formerly compressed DRG in vitro produced potent excitatory effects not observed in control ganglia. MCP-1/CCR2 signaling is directly involved with a chronic compression injury and may contribute to associated neuronal hyperexcitability and neuropathic pain.

A subset of transient receptor potential (TRP) channels exhibits activity that is highly sensitive to temperature changes and is expressed in sensory tissues, such as nociceptors and skin. Some of these thermosensitive TRP channels, such as TRPV1, TRPV4 and TRPA1, are activated or sensitized by molecules generated by inflammation and/or cell damage. TRPV1, also known as the capsaicin receptor, is particularly important in mediating hyperalgesic responses in inflammatory pain states, as demonstrated by research in knockout animals and with small-molecule antagonists. It is anticipated that TRPV1 antagonists, and perhaps antagonists at other thermosensitive TRP channels, will provide new therapeutic options with which to treat clinical pain.

There is mounting evidence that the vanilloid (capsaicin) receptor; transient receptor potential channel, vanilloid subfamily member 1 (TRPV1), is subjected to multiple interacting levels of control. The first level is by reversible phosphorylation catalyzed by intrinsic kinases (e.g. protein kinase A and C) and phosphatases (e.g. calcineurin), which plays a pivotal role in receptor sensitization vs. tachyphylaxis. In addition, this mechanism links TRPV1 to intracellular signaling by various important endogenous as well as exogenous substances such as bradykinin, ethanol, nicotin and insulin. It is not clear, however, whether phosphorylation per se is sufficient to liberate TRPV1 under the inhibitory control of phosphatydylinositol-4,5-bisphosphate. The second level of control is by forming TRPV1 heteromers and their association with putative regulatory proteins. The next level of regulation is by subcellular compartmentalization. The membrane form of TRPV1 functions as a nonselective cation channel. On the endoplasmic reticulum, TRPV1 is present in two differentially regulated forms, one of which is inositol triphosphate-dependent whereas the other is not. These three TRPV1 compartments provide a versatile regulation of intracellular Ca(2+) levels. Last, there is a complex and poorly understood regulation of TRPV1 activity via control of gene expression. Factors that downregulate TRPV1 expression include vanilloid treatment and growth factor (notably, nerve growth factor) deprivation. By contrast, TRPV1 appears to be upregulated during inflammatory conditions. Interestingly, following experimental nerve injury and in animal models of diabetic neuropathy TRPV1 is present on neurons that do not normally express TRPV1. Combined, these findings imply an important role for aberrant TRPV1 expression in the development of neuropathic pain and hyperalgesia. In humans, disease-related changes in TRPV1 expression have already been described (e.g. inflammatory bowel disease and irritable bowel syndrome). The mechanisms that regulate TRPV1 gene expression under pathological conditions are unknown but a better understanding of these pathways has obvious implications for rational drug development.

Substance P (SP) has been widely studied as a mediator of nociception. The release of SP from primary afferent neurons is increased during nociception, and SP activates neurokinin-1 (NK-1) receptors in the spinal cord and periphery. Nociception-evoked alterations in NK-1 receptor gene expression have been studied in rat models of persistent pain but have not been characterized in any murine models of peripheral inflammation. This study assessed behavioral responses and NK-1 receptor mRNA gene expression in mice receiving formalin or Freund's complete adjuvant (CFA) as an inflammatory stimulus. Mechanical withdrawal thresholds were measured before injection of formalin or CFA and hind paw licking/biting timed during the late-phase of the formalin response. Two and 24 hours after formalin or CFA injection, mechanical withdrawal thresholds were measured and the mice euthanized. Solution hybridization-nuclease protection assays were used to quantify NK-1 receptor mRNA levels. Results demonstrated that inflamed hind paws were edematous, and the withdrawal thresholds of the inflamed hind paws were significantly lower after formalin or CFA injection. Neurokinin-1 receptor mRNA levels in the ipsilateral dorsal spinal cords of mice were higher at 24 h after formalin injection or 4 days after CFA injection. These results confirm that mice are hyperalgesic at late time points after formalin or adjuvant injection when NK-1 receptor gene expression is elevated in the dorsal spinal cord. This supports the hypothesis that increased NK-1 receptor gene expression contributes to the development and maintenance of a hyperalgesic state.

The irritant action of capsaicin is mediated by the vanilloid receptor, VR1, which is expressed in sensory neurons termed nociceptors. Capsaicin also desensitizes nociceptors and, thus, is useful clinically as an analgesic. Given the potential importance of VR1 in pain, we have cloned the human capsaicin receptor, hVR1, from a human dorsal root ganglia (DRG) cDNA library. Human VR1 protein is 85% identical to the rat VR1 and many of the amino acid differences are concentrated at the amino and carboxyl termini. VR1 is expressed in DRG as an approximately 4.2 kilobase RNA, and is also expressed in the central nervous system and in the kidney. Capsaicin (EC(50) = 853 nM), low pH (<5.5), and noxious heat (44 degrees C) activate hVR1 expressed in Xenopus oocytes. Subthreshold pH (6.4) sensitizes VR1 to capsaicin (EC(50) = 221 nM). This study demonstrates the similarity of human and rat VR1 in integrating multiple noxious stimuli., (Copyright 2001 Academic Press.)

Corticotropin-releasing hormone (CRH) plays a key role in the regulation of responses to stress. The presence of a high affinity binding protein for CRH (CRH-BP) has been reported in mammals. We have characterized the biochemical properties and expression of CRH-BP in the South African clawed frog, Xenopus laevis. Apparent inhibition constants (K(i[app])) for different ligands were determined by competitive binding assay. Xenopus CRH-BP (xCRH-BP) exhibited a high affinity for xCRH (K(i[app])=1.08 nM) and sauvagine (1.36 nM). Similar to rodent and human CRH-BPs, the frog protein binds urotensin I and urocortin with high affinity, and ovine CRH with low affinity. RT-PCR analysis showed that xCRH-BP is expressed in brain, pituitary, liver, tail, and intestine. Brain xCRH-BP mRNA is expressed at a relatively constant level throughout metamorphosis and increases slightly in the metamorphic frog. By contrast, the gene is strongly upregulated in the tail at metamorphic climax. Thus, regulation of xCRH-BP gene expression is tissue specific. Because xCRH-BP binds CRH-like peptides with high affinity the protein may regulated, the bioavailability of CRH in amphibia as it does in mammals.

The vanilloid (capsaicin) receptor, VR1, is expressed in dorsal root ganglion and mediates the sensory response to vanilloids and other noxious stimuli. There is evidence for VR1 expression in CNS regions as well, but its function in these tissues is unknown. The identification of a rat VR1 5' splice variant and the rat stretch inhibitable channel, which are also expressed in dorsal root ganglia and CNS, raises the possibility that these and/or other VR1 variants may regulate VR1 activity. We have used a quantitative ribonuclease protection assay to characterize the central and peripheral expression of VR1 and VR1 variant RNA in the rat. The data confirm that VR1 is widely expressed in CNS, with highest RNA levels found in cerebral cortex, hippocampus, and cerebellum. VR1 RNA expression in dorsal root ganglia is approximately 28 times greater than in any other tissue sample studied. VR1 5' splice variant RNA is expressed at levels 12 times lower than VR1 in dorsal root ganglia, but at similar levels to VR1 in all other tissues examined. A VR1-related RNA expressed at high levels in kidney was detected, and was distinct from VR1 or stretch inhibitable channel. Our results also show that peripheral inflammation does not change VR1 RNA levels in rat dorsal root ganglia. Systemic resiniferatoxin administration, however, decreases VR1 expression in dorsal root ganglia by 65-80%, an effect that persists for at least 2 months. This study demonstrates that VR1 is expressed at high levels in dorsal root ganglia relative to other tissues and that VR1 5' splice variant is expressed at low levels in dorsal root ganglia compared to VR1. VR1 gene expression in dorsal root ganglia is regulated in response to systemic resiniferatoxin but not peripheral inflammation.

The cloned vanilloid receptor VR1 has attracted recent attention as a molecular integrator of painful stimuli on primary sensory neurons. The existence of vanilloid-sensitive neurons in the brain is, however, controversial. In this study, we have used an antibody and a complementary RNA probe to explore the distribution of neurons that express VR1 in rat and in certain areas of human brain. In the rat, we observed VR1-expressing neurons throughout the whole neuroaxis, including all cortical areas (in layers 3 and 5), several members of the limbic system (e.g., hippocampus, central amygdala, and both medial and lateral habenula), striatum, hypothalamus, centromedian and paraventricular thalamic nuclei, substantia nigra, reticular formation, locus coeruleus, cerebellum, and inferior olive. VR1-immunopositive cells also were found in the third and fifth layers of human parietal cortex. Reverse transcription-PCR performed with rat VR1-specific primers verified the expression of VR1 mRNA in cortex, hippocampus, and hypothalamus. In the central nervous system, neonatal capsaicin treatment depleted VR1 mRNA from the spinal nucleus of the trigeminal nerve, but not from other areas such as the inferior olive. The finding that VR1 is expressed not only in primary sensory neurons but also in several brain nuclei is of great importance in that it places VRs in a much broader perspective than pain perception. VRs in the brain (and putative endogenous vanilloids) may be involved in the control of emotions, learning, and satiety, just to name a few exciting possibilities.

Tachykinins and their receptors are involved in the amplification of inflammation in the airways. We analyzed the expression of preprotachykinin-A (PPT-A) and neurokinin-1 (NK-1) receptor genes by intrinsic airway neurons in the rat. We also tested the hypothesis that PPT-A-encoded peptides released by these neurons fulfill the requisite role of substance P in immune complex injury of the lungs. We found that ganglion neurons in intact and denervated airways or in primary culture coexpress PPT-A and NK-1 receptor mRNAs and their protein products. Denervated ganglia from tracheal xenografts (nu/nu mice) or syngeneic lung grafts had increased PPT-A mRNA contents, suggesting preganglionic regulation. Formation of immune complexes in the airways induced comparable inflammatory injuries in syngeneic lung grafts, which lack peptidergic sensory fibers, and control lungs. The injury was attenuated in both cases by pretreatment with the NK-1 receptor antagonist LY-306740. We conclude that tachykinins released by ganglia act as a paracrine or autocrine signal in the airways and may contribute to NK-1 receptor-mediated amplification of immune injury in the lungs.

1 Although the cloned rat vanilloid receptor VR1 appears to account for both receptor binding and calcium uptake, the identification of vanilloids selective for one or the other response is of importance because these ligands may induce distinct patterns of biological activities. 2 Phorbol 12,13-didecanoate 20-homovanillate (PDDHV) evoked 45Ca(2+)-uptake by rat dorsal root ganglion neurons (expressing native vanilloid receptors) in culture with an EC50 of 70 nM but inhibited [3H]-resiniferatoxin (RTX) binding to rat dorsal root ganglion membranes with a much lower potency (Ki>10,000 nM). This difference in potencies represents a more than 100 fold selectivity for capsaicin-type pharmacology. 3 45Ca2+ influx by PDDHV was fully inhibited by the competitive vanilloid receptor antagonist capsazepine, consistent with the calcium uptake occurring via vanilloid receptors. 4 PDDHV induced calcium mobilization in CHO cells transfected with the cloned rat vanilloid receptor VR1 with an EC50 of 125 nM and inhibited [3H]-RTX binding to these cells with an estimated Ki of 10,000 nM. By contrast, PDDHV failed to evoke a measurable calcium response in non-transfected CHO cells, confirming its action through VR1. 5 We conclude that PDDHV is two orders of magnitude more potent for inducing calcium uptake than for inhibiting RTX binding at vanilloid receptors, making this novel vanilloid a ligand selective for capsaicin-type pharmacology. These results emphasize the importance of monitoring multiple endpoints for evaluation of vanilloid receptor structure-activity relations. Furthermore, PDDHV now provides a tool to explore the biological correlates of capsaicin-type vanilloid pharmacology.

[(3)H]Resiniferatoxin (RTX) binding and calcium uptake by rat dorsal root ganglion (DRG) neurons show distinct structure-activity relations, suggestive of independent vanilloid receptor (VR) subtypes. We have now characterized ligand binding to rat VR1 expressed in human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells and compared the structure-activity relations with those for calcium mobilization. Human embryonic kidney cells (HEK293/VR1 cells) and Chinese hamster ovary cells transfected with VR1 (CHO/VR1 cells) bound [(3)H]RTX with affinities of 84 and 103 pM, respectively, and positive cooperativity (Hill numbers were 2.1 and 1.8). These parameters are similar to those determined with rat DRG membranes expressing native VRs (a K(d) of 70 pM and a Hill number of 1.7). The typical vanilloid agonists olvanil and capsaicin inhibited [(3)H]RTX binding to HEK293/VR1 cells with K(i) values of 0.4 and 4.0 microM, respectively. The corresponding values in DRG membranes were 0.3 and 2.5 microM. HEK293/VR1 cells and DRG membranes also recognized the novel vanilloids isovelleral and scutigeral with similar K(i) values (18 and 20 microM in HEK293/VR1 cells; 24 and 21 microM in DRGs). The competitive vanilloid receptor antagonist capsazepine inhibited [(3)H]RTX binding to HEK293/VR1 cells with a K(i) value of 6.2 microM and binding to DRG membranes with a K(i) value of 8.6 microM. RTX and capsaicin induced calcium mobilization in HEK293/VR1 cells with EC(50) values of 4.1 and 82 nM, respectively. Thus, the relative potencies of RTX (more potent for binding) and capsaicin (more potent for calcium mobilization) are similar in DRG neurons and cells transfected with VR1. We conclude that VR1 can account for both the ligand binding and calcium uptake observed in rat DRG neurons.

CRH is the primary hypothalamic regulator of the stress response in higher organisms, where it acts as the key mediator of ACTH release in the hypothalamus-pituitary-adrenal axis. The 37-kDa CRH-binding protein (CRH-BP) is known to bind CRH and antagonize CRH-induced ACTH release in vitro. The expression of this protein in anterior pituitary corticotrophs suggests a role for CRH-BP in modulation of the stress response. To investigate the in vivo role of rat CRH-BP, the regulation of pituitary CRH-BP gene expression by acute restraint stress and/or adrenalectomy was examined using ribonuclease protection assays. After restraint stress, steady-state levels of CRH-BP transcripts increase two to three times over basal level and remain significantly higher than basal levels for 120 min after the start of restraint. Adrenalectomy decreases CRH-BP messenger RNA steady-state levels to 8% of control levels. These results demonstrate that pituitary CRH-BP messenger RNA levels are increased in response to acute restraint stress and that glucocorticoids play a significant role in this positive regulation. These data also suggest that increased CRH-BP levels, in response to stress, may modulate the endocrine stress response by providing an additional feedback mechanism to maintain homeostasis of the hypothalamus-pituitary-adrenal axis.

The CRH-binding protein (CRH-BP) antagonizes the ACTH-releasing activity of the neuropeptide CRH in vitro. However, the function of CRH-BP in vivo and the molecular mechanisms that regulate CRH-BP expression are not well understood. In this study, the rat CRH-BP gene was characterized, and CRH-BP promoter sequences were identified. The rat CRH-BP gene spans almost 12 kilobases and contains 7 exons. Ribonuclease protection experiments indicate that transcription of the CRH-BP gene initiates at multiple sites in rat cerebral cortex. Transfection experiments with CRH-BP-reporter constructs, containing 88-3500 bp 5' flanking and 66 bp 5' untranslated DNA from the rat CRH-BP gene, demonstrate basal promoter activity in multiple cell lines. CRH-BP-reporter constructs also demonstrate positive regulation of promoter activity by cAMP in a variety of cell lines and by CRH in cells expressing the CRH receptor. The DNA sequences between -341 and -88 bp, including the cAMP response element-like sequence at -127 bp, are required for maximal cAMP and CRH regulation of CRH-BP promoter activity. These studies suggest that CRH-BP transcription in vivo may be positively regulated by cAMP and CRH.

A 37 kDa corticotropin-releasing hormone-binding protein (CRH-BP), distinct from the CRH receptor, is expressed in rat anterior pituitary corticotrophs and many regions of the brain, suggesting that CRH-BP may modulate the biological activity of CRH. In these studies a mouse brain CRH-BP (mCRH-BP) cDNA has been isolated and characterized. The 1666 nucleotide mCRH-BP cDNA is expressed in brain and pituitary and encodes a 322 amino acid protein that is highly homologous to human and rat CRH-BPs. Recombinant mCRH-BP, expressed in cultured mammalian cells, binds human CRH (Kd(app) = 0.56 nM and Ki(app) = 0.37 nM) and the alpha-helical (9-41) CRH antagonist (Ki(app) = 0.28 nM) with high affinity, but exhibits much weaker affinity for ovine CRH (Ki(app) = 206 nM). Recombinant mCRH-BP also blocks CRH-induced adrenocorticotropin release from AtT-20 cells. Additional biochemical characterization of the binding activity of mCRH-BP indicates that CRH-BP and CRH receptor utilize different molecular interactions to bind CRH.