The vanilloid receptor VR1 is a nonselective cation channel that is most abundant in peripheral sensory fibers but also is found in several brain nuclei. VR1 is gated by protons, heat, and the pungent ingredient of ''hot'' chili peppers, capsaicin. To date, no endogenous compound with potency at this receptor comparable to that of capsaicin has been identified. Here we examined the hypothesis, based on previous structure-activity relationship studies and the availability of biosynthetic precursors, that N-arachidonoyl-dopamine (NADA) is an endogenous ''capsaicin-like'' substance in mammalian nervous tissues. We found that NADA occurs in nervous tissues, with the highest concentrations being found in the striatum, hippocampus, and cerebellum and the lowest concentrations in the dorsal root ganglion. We also gained evidence for the existence of two possible routes for NADA biosynthesis and mechanisms for its inactivation in rat brain. NADA activates both human and rat VR1 overexpressed in human embryonic kidney (HEK)293 cells, with potency (EC50 Ϸ 50 nM) and efficacy similar to those of capsaicin. Furthermore, NADA potently activates native vanilloid receptors in neurons from rat dorsal root ganglion and hippocampus, thereby inducing the release of substance P and calcitonin gene-related peptide (CGRP) from dorsal spinal cord slices and enhancing hippocampal paired-pulse depression, respectively. Intradermal NADA also induces VR1-mediated thermal hyperalgesia (EC50 ‫؍‬ 1.5 ؎ 0.3 g). Our data demonstrate the existence of a brain substance similar to capsaicin not only with respect to its chemical structure but also to its potency at VR1 receptors. V anilloid receptors of type 1 (VR1) are nonselective cation channels, expressed in peripheral sensory C and A␦ fibers and gated by nociceptive stimuli such as low pH, heat, and some plant toxins, of which capsaicin, the pungent principle of chili peppers, is the best known example (1-4). Evidence obtained by several laboratories and using different techniques (5-10) showed that VR1 is present also in the central nervous system, where it is unlikely to be the target of noxious heat and low pH, thus suggesting the existence of brain endogenous agonists for this receptor (11). Indeed, lipid mediators previously known to serve other functions in the brain, i.e., the endocannabinoid anandamide and some lipoxygenase derivatives, activate VR1, albeit with a potency considerably lower than that of capsaicin (12)(13)(14). The antinociceptive effects of VR1 blockers in two models of inflammatory hyperalgesia (15, 16) suggest that ''endovanilloids'' might be produced also by peripheral tissues and act in concert with locally enhanced temperature and acidity during inflammation.If an endovanilloid did exist, what would be the structural prerequisites that would allow for an optimal interaction with vanilloid receptors? Structure-activity relationship studies for vanilloid receptors have indicated that both the vanillyl-amine moiety and a long, unsaturated acyl chain are necessary to...

Acute stress suppresses pain by activating brain pathways that engage opioid or non-opioid mechanisms. Here we show that an opioid-independent form of this phenomenon, termed stressinduced analgesia 1 , is mediated by the release of endogenous marijuana-like (cannabinoid) compounds in the brain. Blockade of cannabinoid CB 1 receptors in the periaqueductal grey matter of the midbrain prevents non-opioid stress-induced analgesia. In this region, stress elicits the rapid formation of two endogenous cannabinoids, the lipids 2-arachidonoylglycerol 2 (2-AG) and anandamide 3 . A newly developed inhibitor of the 2-AG-deactivating enzyme, monoacylglycerol lipase 4,5 , selectively increases 2-AG concentrations and, when injected into the periaqueductal grey matter, enhances stress-induced analgesia in a CB 1 -dependent manner. Inhibitors of the anandamide-deactivating enzyme fatty-acid amide hydrolase 6 , which selectively elevate anandamide concentrations, exert similar effects. Our results indicate that the coordinated release of 2-AG and anandamide in the periaqueductal grey matter might mediate opioid-independent stress-induced analgesia. These studies also identify monoacylglycerol lipase as a previously unrecognized therapeutic target.Stress activates neural systems that inhibit pain sensation. This adaptive response, referred to as stress-induced analgesia (SIA), depends on the recruitment of brain pathways that project from the amygdala to the midbrain periaqueductal grey matter (PAG) and descend to the brainstem rostroventromedial medulla and dorsal horn of the spinal cord 7 . Endogenous opioid peptides have key functions in this process 1,8 , but other as yet unidentified neurotransmitters are also known to be involved 1 . We proposed that endocannabinoids might be implicated in stress analgesia for two reasons. First, agonists of CB 1 receptors-the predominant cannabinoid receptor subtype present in the brain 9,10 -exert profound antinociceptive effects 7 and suppress activity in nociceptive neurons 11-14 . Second, CB 1 antagonists increase the activity of nociceptive rostroventromedial medulla neurons 14 and enhance sensitivity to noxious stimuli 15 , indicating that an intrinsic endocannabinoid tone might regulate descending antinociceptive pathways 7 . To study non-opioid SIA we delivered brief, continuous electric foot shock to rats and quantified their sensitivity to pain after stress by using the tail-flick test. As demonstrated previously 1,16 , this stimulation protocol caused a profound antinociceptive effect that was not affected by intraperitoneal (i.p.) injection of the opiate antagonist naltrexone (14 mg kg 21 ) (Fig. 1a). However, the response was almost abolished by administration of the CB 1 antagonist rimonabant (SR141617A, 5 mg kg

Hair bundles of the inner ear have a unique structure and protein composition that underlies their sensitivity to mechanical stimulation. Using mass spectrometry, we identified and quantified >1100 proteins, present from a few to 400,000 copies per stereocilium, from purified chick bundles; 336 of these were significantly enriched in bundles. Bundle proteins that we detected have been shown to regulate cytoskeleton structure and dynamics, energy metabolism, phospholipid synthesis, and cell signaling. Three-dimensional imaging using electron tomography allowed us to count the number of actin-actin crosslinkers and actin-membrane connectors; these values compared well to those obtained from mass spectrometry. Network analysis revealed several hub proteins, including RDX (radixin) and SLC9A3R2 (NHERF2), which interact with many bundle proteins and may perform functions essential for bundle structure and function. The quantitative mass spectrometry of bundle proteins reported here establishes a framework for future characterization of dynamic processes that shape bundle structure and function.

L-type voltage gated calcium channels (LTCs) play an important role in neuronal development by promoting dendritic growth and arborization1–3. A point mutation in CaV1.2 causes Timothy Syndrome (TS)4, a neurodevelopmental disorder associated with autism spectrum disorders (ASD). We report that channels with the TS mutation cause activity-dependent dendrite retraction in rodent neurons and in induced pluripotent stem cell (iPSCs)– derived neurons from individuals with TS. Dendrite retraction is independent of calcium permeation through the mutant channel, is associated with ectopic activation of RhoA and is inhibited by over-expression of the channel associated GTPase Gem. These results suggest that CaV1.2 can activate RhoA signaling independently of Ca2+ and provide novel insights into the cellular basis of TS and other ASDs.

. Sex and hormonal cycle differences in rat brain levels of pain-related cannabimimetic lipid mediators. Am J Physiol Regul Integr Comp Physiol 291: R349 -R358, 2006. First published March 23, 2006 doi:10.1152/ajpregu.00933.2005.-One important function of endocannabinoids and related lipid mediators in mammalian central nervous system is modulation of pain. Evidence obtained during the last decade shows that altered levels of these compounds in the brain accompany decreases in pain sensitivity. Such changes, if sexually dimorphic, could account for sex differences in pain and differences that occur during different phases of the hormonal cycle in females. To examine this possibility, we measured the levels of the pain-modulatory lipids anandamide, 2-arachidonoyl glycerol, N-arachidonoyl glycine, N-arachidonoyl gamma amino butyric acid, and N-arachidonoyl dopamine in seven different brain areas (pituitary, hypothalamus, thalamus, striatum, midbrain, hippocampus, and cerebellum) in male rats, and in female rats at five different points in the estrous cycle. The cerebellum did not demonstrate a change in endocannabinoid production across the estrous cycle, whereas all other areas tested showed significant differences in at least one of the compounds measured. These changes in levels occurred predominantly within the 36-h time period surrounding ovulation and behavioral estrus. Differences between males and females were measured as either estrous cycle-independent (all estrous cycles combined) or cycle-dependent (comparisons of males to each estrous cycle). In cycle-independent analyses, small sex differences were observed in the pituitary, hypothalamus, cerebellum, and striatum, whereas no differences were observed in the thalamus, midbrain, and hippocampus. In cycle-dependent analyses, the hypothalamus and pituitary showed largest sex differences followed by the striatum, midbrain, and hippocampus, whereas no sex differences were measured in thalamus and cerebellum. These data provide a basis for investigations into how differences in sex and hormonal status play a role in mechanisms regulating endocannabinoid production and pain. estrous; anandamide; 2-arachidonoyl glycerol; N-arachidonoyl glycine ENDOGENOUS CANNABINOIDS are involved in a myriad of physiological processes, including immune function, feeding regulation, vascular tone, memory, mood, reproduction, and pain (for reviews, see Refs. 29,31,34,35,[44][45][46]. Sex differences in the cannabinoid signaling system have been reported, these being limited mainly to examinations of sex-and cycle-related differences in the efficacy of the exogenous cannabinoid delta-9 tetrahydrocannabinol (THC; reviewed in Ref. 6). The antinoceptive effects of THC were significantly greater in female rats vs. male rats in a model of acute peripheral pain (39). Prenatal THC exposure produced a sex-dependent effect on proenkephalin mRNA levels in several brain regions in which females had significantly higher expression than males (28). Also, the cannabinoid receptor agonist CP...

Label-free quantitation of proteins analyzed by tandem mass spectrometry uses either integrated peak intensity from the parent-ion mass analysis (MS1) or features from fragment-ion analysis (MS2), such as spectral counts or summed fragment-ion intensity. We directly compared MS1 and MS2 quantitation by analyzing human protein standards diluted into Escherichia coli extracts on an Orbitrap mass spectrometer. We found that summed MS2 intensities were nearly as accurate as integrated MS1 intensities, and both outperformed MS2 spectral counting in accuracy and linearity. We compared these results to those obtained from two low-resolution ion-trap mass spectrometers; summed MS2 intensities from LTQ and LTQ Velos instruments were similar in accuracy to those from the Orbitrap. Data from all three instruments are available via ProteomeXchange with identifier PXD000602. Abundance measurements using MS1 or MS2 intensities had limitations, however. While measured protein concentration was on average well correlated with the known concentration, there was considerable protein-to-protein variation. Moreover, not all human proteins diluted to a mole fraction of 10−3 or lower were detected, with a strong fall-off below 10−4 mole fraction. These results show that MS1 and MS2 intensities are simple measures of protein abundance that are on average accurate, but should be limited to quantitation of proteins of intermediate to higher fractional abundance.

Actin-rich structures like stereocilia and microvilli are assembled with precise control of length, diameter, and relative spacing. We found that developmental widening of the second-tallest stereocilia rank (row 2) of mouse inner hair cells correlated with the appearance of mechanotransduction. Correspondingly, Tmc1 KO/KO ;Tmc2 KO/KO or Tmie KO/KO hair cells, which lack transduction, have significantly altered stereocilia lengths and diameters. EPS8 and the short splice isoform of MYO15A, identity markers for row 1 (tallest), lost their row exclusivity in transduction mutants, a result that was mimicked by block of transduction channels. Likewise, the heterodimeric capping protein subunit CAPZB and its partner TWF2 lost their row 2 tip localization in mutants, and GNAI3 failed to accumulate at row 1 tips. Redistribution of marker proteins was accompanied by increased variability in stereocilia height. Transduction channels thus specify and maintain row identity and control addition of new actin filaments to increase stereocilia diameter.

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