Fatty acid binding protein 5 inhibition attenuates pronociceptive cytokine/chemokine expression and suppresses osteoarthritis pain: A comparative human and rat study.

OBJECTIVE: Osteoarthritis (OA) is often accompanied by debilitating pain that is refractory to available analgesics due in part to the complexity of signaling molecules that drive OA pain and our inability to target these in parallel. Fatty acid binding protein 5 (FABP5) is a lipid chaperone that regulates inflammatory pain; however, its contribution to OA pain has not been characterized. DESIGN: This combined clinical and pre-clinical study utilized synovial tissues obtained from subjects with end-stage OA and rats with monoiodoacetate-induced OA. Cytokine and chemokine release from human synovia incubated with a selective FABP5 inhibitor was profiled with cytokine arrays and ELISA. Immunohistochemical analyses were conducted for FABP5 in human and rat synovium. The efficacy of FABP5 inhibitors on pain was assessed in OA rats using incapacitance as an outcome. RNA-seq was then performed to characterize the transcriptomic landscape of synovial gene expression in OA rats treated with FABP5 inhibitor or vehicle. RESULTS: FABP5 was expressed in human synovium and FABP5 inhibition reduced the secretion of pronociceptive cytokines (interleukin-6 [IL6], IL8) and chemokines (CCL2, CXCL1). In rats, FABP5 was upregulated in the OA synovium and its inhibition alleviated incapacitance. The transcriptome of the rat OA synovium exhibited >6000 differentially expressed genes, including the upregulation of numerous pronociceptive cytokines and chemokines. FABP5 inhibition blunted the upregulation of the majority of these pronociceptive mediators. CONCLUSIONS: FABP5 is expressed in the OA synovium and its inhibition suppresses pronociceptive signaling and pain, indicating that FABP5 inhibitors may constitute a novel class of analgesics to treat OA.

Monoacylglycerol Lipase and Cyclooxygenase-2 Expression in Osteoarthritic Human Knees.

Background: Osteoarthritis (OA) is a progressive degenerative joint disease that presents with significant pain and functional disability. The endocannabinoid 2-arachidonoylglycerol activates cannabinoid receptors to reduce pain while its hydrolysis by the enzyme monoacylglycerol lipase (MAGL) generates arachidonic acid, the direct precursor to proalgesic eicosanoids synthesized by cyclooxygenase-2 (COX-2), highlighting the potential for crosstalk between MAGL and COX-2. While COX-2 expression in human OA cartilage has been described, the distribution of MAGL in knee osteochondral tissue has not been reported and was the goal of the current study. Methods: MAGL and COX-2 expression in International Cartilage Repair Society grade II and grade IV knee osteochondral tissue obtained from male and female subjects with OA was investigated through immunohistochemistry. Immunolocalization of both proteins was investigated within articular cartilage and subchondral bone. Results: MAGL is expressed throughout the cartilage of grade II arthritic tissue, with prominent distribution in the superficial and deep zones. Elevated expression of MAGL was evident in grade IV samples, with additional distribution observed in subchondral bone. COX-2 expression followed a similar pattern, with uniform distribution in cartilage and increased expression in grade IV tissue. Conclusions: This study establishes MAGL expression in arthritic cartilage and subchondral bone of subjects with OA. The proximity between MAGL and COX-2 suggests the potential for crosstalk between endocannabinoid hydrolysis and eicosanoid signaling in the maintenance of OA pain.

FABP5 deletion in nociceptors augments endocannabinoid signaling and suppresses TRPV1 sensitization and inflammatory pain.

The endocannabinoid anandamide (AEA) produces antinociceptive effects by activating cannabinoid receptor 1 (CB1). However, AEA also serves as an agonist at transient receptor potential vanilloid receptor 1 (TRPV1) in nociceptive sensory neurons, which may exacerbate pain. This potential functional duality is highlighted by the failure of an inhibitor of the AEA catabolic enzyme fatty acid amide hydrolase (FAAH) to afford pain relief in a clinical trial. Consequently, it remains to be determined whether elevating AEA levels in nociceptors leads to antinociceptive or pro-nociceptive effects. Fatty acid binding protein 5 (FABP5) is an intracellular carrier that mediates AEA transport to FAAH for inactivation. Leveraging the abundant expression of FABP5 in TRPV1+ nociceptors, we employed a conditional knockout strategy to demonstrate that FABP5 deletion in nociceptors augments AEA levels, resulting in the emergence of antinociceptive effects mediated by CB1. Mechanistically, FABP5 deletion suppresses inflammation- and nerve growth factor-mediated TRPV1 sensitization via CB1, an effect mediated by calcineurin. Unexpectedly, inhibition of FAAH failed to blunt TRPV1 sensitization, uncovering functionally distinct outputs resulting from FABP5 and FAAH inhibition. Collectively, our results demonstrate that FABP5 serves a key role in governing endocannabinoid signaling in nociceptors to disrupt TRPV1 sensitization and pain, and position FABP5 as a therapeutic target for the development of analgesics.

FABP5 coordinates lipid signaling that promotes prostate cancer metastasis.

Prostate cancer (PCa) is defined by dysregulated lipid signaling and is characterized by upregulation of lipid metabolism-related genes including fatty acid binding protein 5 (FABP5), fatty acid synthase (FASN), and monoacylglycerol lipase (MAGL). FASN and MAGL are enzymes that generate cellular fatty acid pools while FABP5 is an intracellular chaperone that delivers fatty acids to nuclear receptors to enhance PCa metastasis. Since FABP5, FASN, and MAGL have been independently implicated in PCa progression, we hypothesized that FABP5 represents a central mechanism linking cytosolic lipid metabolism to pro-metastatic nuclear receptor signaling. Here, we show that the abilities of FASN and MAGL to promote nuclear receptor activation and PCa metastasis are critically dependent upon co-expression of FABP5 in vitro and in vivo. Our findings position FABP5 as a key driver of lipid-mediated metastasis and suggest that disruption of lipid signaling via FABP5 inhibition may constitute a new avenue to treat metastatic PCa.

FABP1 controls hepatic transport and biotransformation of Δ9-THC.

The increasing use of medical marijuana highlights the importance of developing a better understanding of cannabinoid metabolism. Phytocannabinoids, including ∆9-tetrahydrocannabinol (THC), are metabolized and inactivated by cytochrome P450 enzymes primarily within the liver. The lipophilic nature of cannabinoids necessitates mechanism(s) to facilitate their intracellular transport to metabolic enzymes. Here, we test the central hypothesis that liver-type fatty acid binding protein (FABP1) mediates phytocannabinoid transport and subsequent inactivation. Using X-ray crystallography, molecular modeling, and in vitro binding approaches we demonstrate that FABP1 accommodates one molecule of THC within its ligand binding pocket. Consistent with its role as a THC carrier, biotransformation of THC was reduced in primary hepatocytes obtained from FABP1-knockout (FABP1-KO) mice. Compared to their wild-type littermates, administration of THC to male and female FABP1-KO mice potentiated the physiological and behavioral effects of THC. The stark pharmacodynamic differences were confirmed upon pharmacokinetic analyses which revealed that FABP1-KO mice exhibit reduced rates of THC biotransformation. Collectively, these data position FABP1 as a hepatic THC transport protein and a critical mediator of cannabinoid inactivation. Since commonly used medications bind to FABP1 with comparable affinities to THC, our results further suggest that FABP1 could serve a previously unrecognized site of drug-drug interactions.

Fatty-acid-binding protein 5 controls retrograde endocannabinoid signaling at central glutamate synapses.

Endocannabinoids (eCBs) are lipid-signaling molecules involved in the regulation of numerous behaviors and physiological functions. Released by postsynaptic neurons, eCBs mediate retrograde modulation of synaptic transmission and plasticity by activating presynaptic cannabinoid receptors. While the cellular mechanisms by which eCBs control synaptic function have been well characterized, the mechanisms controlling their retrograde synaptic transport remain unknown. Here, we demonstrate that fatty-acid-binding protein 5 (FABP5), a canonical intracellular carrier of eCBs, is indispensable for retrograde eCB transport in the dorsal raphe nucleus (DRn). Thus, pharmacological inhibition or genetic deletion of FABP5 abolishes both phasic and tonic eCB-mediated control of excitatory synaptic transmission in the DRn. The blockade of retrograde eCB signaling induced by FABP5 inhibition is not mediated by impaired cannabinoid receptor function or reduced eCB synthesis. These findings indicate that FABP5 is essential for retrograde eCB signaling and may serve as a synaptic carrier of eCBs at central synapses.

Fatty acid-binding protein 5 controls microsomal prostaglandin E synthase 1 (mPGES-1) induction during inflammation.

Fatty acid-binding proteins (FABPs) are intracellular lipid carriers that regulate inflammation, and pharmacological inhibition of FABP5 reduces inflammation and pain. The mechanism(s) underlying the anti-inflammatory effects associated with FABP5 inhibition is poorly understood. Herein, we identify a novel mechanism through which FABP5 modulates inflammation. In mice, intraplantar injection of carrageenan induces acute inflammation that is accompanied by edema, enhanced pain sensitivity, and elevations in proinflammatory cytokines and prostaglandin E2 (PGE2). Inhibition of FABP5 reduced pain, edema, cytokine, and PGE2 levels. PGE2 is a major eicosanoid that enhances pain in the setting of inflammation, and we focused on the mechanism(s) through which FABP5 modulates PGE2 production. Cyclooxygenase 2 (COX-2) and microsomal prostaglandin E synthase 1 (mPGES-1) are enzymes up-regulated at the site of inflammation and account for the bulk of PGE2 biosynthesis. Pharmacological or genetic FABP5 inhibition suppressed the induction of mPGES-1 but not COX-2 in carrageenan-injected paws, which occurred predominantly in macrophages. The cytokine interleukin 1ß (IL-1ß) is a major inducer of mPGES-1 during inflammation. Using A549 cells that express FABP5, IL-1ß stimulation up-regulated mPGES-1 expression, and mPGES-1 induction was attenuated in A549 cells bearing a knockdown of FABP5. IL-1ß up-regulates mPGES-1 via NF-κB, which activates the mPGES-1 promoter. Knockdown of FABP5 reduced the activation and nuclear translocation of NF-κB and attenuated mPGES-1 promoter activity. Deletion of NF-κB-binding sites within the mPGES-1 promoter abrogated the ability of FABP5 to inhibit mPGES-1 promoter activation. Collectively, these results position FABP5 as a novel regulator of mPGES-1 induction and PGE2 biosynthesis during inflammation.

Fatty-acid-binding protein inhibition produces analgesic effects through peripheral and central mechanisms.

Background Fatty-acid-binding proteins (FABPs) are intracellular carriers for endocannabinoids, N-acylethanolamines, and related lipids. Previous work indicates that systemically administered FABP5 inhibitors produce analgesia in models of inflammatory pain. It is currently not known whether FABP inhibitors exert their effects through peripheral or central mechanisms. Here, we examined FABP5 distribution in dorsal root ganglia and spinal cord and examined the analgesic effects of peripherally and centrally administered FABP5 inhibitors. Results Immunofluorescence revealed robust expression of FABP5 in lumbar dorsal root ganglia. FABP5 was distributed in peptidergic calcitonin gene-related peptide-expressing dorsal root ganglia and non-peptidergic isolectin B4-expressing dorsal root ganglia. In addition, the majority of dorsal root ganglia expressing FABP5 also expressed transient receptor potential vanilloid 1 (TRPV1) and peripherin, a marker of nociceptive fibers. Intraplantar administration of FABP5 inhibitors reduced thermal and mechanical hyperalgesia in the complete Freund's adjuvant model of chronic inflammatory pain. In contrast to its robust expression in dorsal root ganglia, FABP5 was sparsely distributed in the lumbar spinal cord and intrathecal administration of FABP inhibitor did not confer analgesic effects. Administration of FABP inhibitor via the intracerebroventricular (i.c.v.) route reduced thermal hyperalgesia. Antagonists of peroxisome proliferator-activated receptor alpha blocked the analgesic effects of peripherally and i.c.v. administered FABP inhibitor while antagonism of cannabinoid receptor 1 blocked the effects of peripheral FABP inhibition and a TRPV1 antagonist blocked the effects of i.c.v. administered inhibitor. Although FABP5 and TRPV1 were co-expressed in the periaqueductal gray region of the brain, which is known to modulate pain, knockdown of FABP5 in the periaqueductal gray using adeno-associated viruses and pharmacological FABP5 inhibition did not produce analgesic effects. Conclusions This study demonstrates that FABP5 is highly expressed in nociceptive dorsal root ganglia neurons and FABP inhibitors exert peripheral and supraspinal analgesic effects. This indicates that peripherally restricted FABP inhibitors may serve as a new class of analgesic and anti-inflammatory agents.

Light pulse duration differentially regulates mouse locomotor suppression and phase shifts.

Brief exposure of mice to nocturnal light causes circadian rhythm phase shifts, simultaneously inducing locomotor suppression, a drop in body temperature, and associated sleep. The exact nature of the relationship between these light-induced responses is uncertain, although locomotor suppression and phase shift magnitudes are related to stimulus irradiance. Whether stimulus duration has similar effects is less clear. Here, the relationship between stimulus duration and response magnitude was evaluated further using 100 µW/cm(2) white light-emitting diode pulses administered for 30, 300, 1200, or 3000 sec. The results show that, in general, shorter pulses yielded smaller responses and larger pulses yielded larger responses. However, the 300-sec pulse failed to augment locomotor suppression compared with the effect of a 30-sec pulse (44.7 ± 4.8 vs 40.6 ± 2.0 min) but simultaneously induced much larger phase shifts (1.28 ± 0.20 vs 0.52 ± 0.11 h). The larger phase shifts induced by the 300-sec stimulus did not differ from those induced by either the 1200- or 3000-sec pulses (1.43 ± 0.10 and 1.30 ± 0.17 h, respectively). The results demonstrate differential photic regulation of the two response types. Pulses ranging from 300 to 3000 sec produce equal phase shifts (present data); pulses ranging from 30 to 600 sec produce equal locomotor suppression levels. Greater suppression can occur additively in response to pulses of 1200 sec or more (present data), but this is not true for phase shifts. Nocturnal light appears to trigger a fixed duration event, locomotor suppression, or phase shift, with the latter followed by a light-refractory interval during which locomotor suppression can additively increase. The results also provide further support for the view that temporal integration of photic energy applies, at best, across a limited set of stimulus durations for both light-induced locomotor suppression/sleep and phase shift regulation.

Retinofugal projections in the mouse.

The laboratory mouse is increasingly a subject for visual system investigation, but there has been no comprehensive evaluation of this species' visual projections. Here, projections were visualized and mapped following intraocular injection of cholera toxin B subunit. Tissue was processed using standard procedures applied to 30 µm free-floating sections with diaminobenzidine as the chromogen. The mouse retina projects to ~46 brain regions, including 14 not previously described in this species. These include two amygdaloid nuclei, the horizontal limb of the diagonal band, the paraventricular hypothalamic nucleus, several visual thalamic nuclei, the paranigral nucleus, several pretectal nuclei, and the dorsal cortex of the inferior colliculus. Dense retinal patches were also observed in a narrow portion of the ipsilateral intermediate layer of the superior colliculus. The superior fasciculus of the accessory optic tract, which innervates the medial terminal nucleus, was also determined to be a terminal zone throughout its length. The results are compared with previous descriptions of projections from mouse intrinsically photoreceptive retinal ganglion cells, and with data from the hamster, Nile grass rat, and laboratory rat. The retinal projection patterns are similar in all four species, although there are many differences with respect to the details. The specific visual functions of most retinorecipient areas are unknown, but there is substantial convergence of retinal projections onto regions concerned with olfaction and audition.

Brief light stimulation during the mouse nocturnal activity phase simultaneously induces a decline in core temperature and locomotor activity followed by EEG-determined sleep.

Light exerts a variety of effects on mammals. Unexpectedly, one of these effects is the cessation of nocturnal locomotion and the induction of behavioral sleep (photosomnolence). Here, we extend the initial observations in several ways, including the fundamental demonstration that core body temperature (T(c)) drops substantially (about 1.5°C) in response to the light stimulation at CT15 or CT18 in a manner suggesting that the change is a direct response to light rather than simply a result of the locomotor suppression. The results show that 1) the decline of locomotion and T(c) begin soon after nocturnal light stimulation; 2) the variability in the magnitude and onset of light-induced locomotor suppression is very large, whereas the variability in T(c) is very small; 3) T(c) recovers from the light-induced decline in advance of the recovery of locomotion; 4) under entrained and freerunning conditions, the daily late afternoon T(c) increase occurs in advance of the corresponding increase in wheel running; and 5) toward the end of the subjective night, the nocturnally elevated T(c) persists longer than does locomotor activity. Finally, EEG measurements confirm light-induced sleep and, when T(c) or locomotion was measured, show their temporal association with sleep onset. Both EEG- and immobility-based sleep detection methods confirm rapid induction of light-induced sleep. The similarities between light-induced loss of locomotion and drop in T(c) suggest a common cause for parallel responses. The photosomnolence response may be contingent upon both the absence of locomotion and a simultaneous low T(c).

Neurons identified by NeuN/Fox-3 immunoreactivity have a novel distribution in the hamster and mouse suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) has several structural characteristics and cell phenotypes shared across species. Here, we describe a novel feature of SCN anatomy that is seen in both hamster and mouse. Frozen sections through the SCN were obtained from fixed brains and stained for the presence of immunoreactivity to neuronal nuclear protein (NeuN-IR) using a mouse monoclonal antibody which is known to exclusively identify neurons. NeuN-IR did not identify all SCN neurons as medial NeuN-IR neurons were generally not present. In the hamster, NeuN-IR cells are present rostrally, scattered in the dorsal half of the nucleus. More caudally, the NeuN-IR cells are largely, but not exclusively, scattered inside the lateral and dorsolateral border. At mid- to mid-caudal SCN levels, a dense group of NeuN-IR cells extends from the dorsolateral border ventromedially to encompass the central subnucleus of the SCN (SCNce). The pattern is similar in the mouse SCN. NeuN-IR does not co-localize with either cholecystokinin- or vasoactive intestinal polypeptide, but does with vasopressin-IR in the caudal SCN. In the hamster SCNce, numerous cells contain both calbindin- and NeuN-IR. The distribution of NeuN-IR cells in the SCN is unique, especially with regard to its generally lateral location through the length of the nucleus. The distribution of NeuN-IR cells is not consistent with most schemas representing SCN organization or with terminology referring to its widely accepted subdivisions. NeuN has recently been identified as Fox-3 protein. Its function in the SCN is not known, nor is it known why a large proportion of SCN cells do not contain NeuN-IR.

Two components of nocturnal locomotor suppression by light.

In nocturnal rodents, millisecond light ("flash") stimuli can induce both a large circadian rhythm phase shift and an associated state change from highly active to quiescence followed by behavioral sleep. Suppression of locomotion ("negative masking") is an easily measured correlate of the state change. The present mouse studies used both flashes and longer light stimuli ("pulses") to distinguish initiation from maintenance effects of light on locomotor suppression and to determine whether the locomotor suppression exhibits temporal integration as is thought to be characteristic of phase shift responses to pulse, but not flash, stimuli. In experiment 1, locomotor suppression increased with irradiance (0.01-100 microW/cm( 2)), in accordance with previous reports. It also increased with stimulus duration (3-3000 sec), but interpretation of this result is complicated by the ability of light to both initiate and maintain locomotor suppression. In experiment 2, an irradiance response curve was determined using a stimulus series of 10 flashes, 2 msec each, with total flash energy varying from 0.0025 to 110.0 J/m(2). This included a test for temporal integration in which the effects of two equal energy series of flashes that differed in the number of flashes per series (10 vs 100), were compared. The 10 flash series more effectively elicited locomotor suppression than the 100 flash series, a result consistent with prior observations involving flash-induced phase shifts. In experiment 3, exposure of mice to an 11-h light stimulus yielded irradiance-dependent locomotor suppression that was maintained for the entire stimulus duration by a 100-microW/cm(2) stimulus. Light has the ability to initiate a time-limited (30-40 min) interval of locomotor suppression (initiation effect) that can be extended by additional light (maintenance effect). Temporal integration resembling that seen in phase-shifting responses to light does not exist for either phase shift or locomotor suppression responses to flashes or for locomotor suppression responses to light pulses. The authors present an alternative interpretation of data thought to demonstrate temporal integration in the regulation of phase shift responses to light pulses.

Targeted destruction of photosensitive retinal ganglion cells with a saporin conjugate alters the effects of light on mouse circadian rhythms.

Non-image related responses to light, such as the synchronization of circadian rhythms to the day/night cycle, are mediated by classical rod/cone photoreceptors and by a small subset of retinal ganglion cells that are intrinsically photosensitive, expressing the photopigment, melanopsin. This raises the possibility that the melanopsin cells may be serving as a conduit for photic information detected by the rods and/or cones. To test this idea, we developed a specific immunotoxin consisting of an anti-melanopsin antibody conjugated to the ribosome-inactivating protein, saporin. Intravitreal injection of this immunotoxin results in targeted destruction of melanopsin cells. We find that the specific loss of these cells in the adult mouse retina alters the effects of light on circadian rhythms. In particular, the photosensitivity of the circadian system is significantly attenuated. A subset of animals becomes non-responsive to the light/dark cycle, a characteristic previously observed in mice lacking rods, cones, and functional melanopsin cells. Mice lacking melanopsin cells are also unable to show light induced negative masking, a phenomenon known to be mediated by such cells, but both visual cliff and light/dark preference responses are normal. These data suggest that cells containing melanopsin do indeed function as a conduit for rod and/or cone information for certain non-image forming visual responses. Furthermore, we have developed a technique to specifically ablate melanopsin cells in the fully developed adult retina. This approach can be applied to any species subject to the existence of appropriate anti-melanopsin antibodies.

Diurnal changes in exocytosis and the number of synaptic ribbons at active zones of an ON-type bipolar cell terminal.

The number and morphology of synaptic ribbons at photoreceptor and bipolar cell terminals has been reported to change on a circadian cycle. Here we sought to determine whether this phenomenon exists at goldfish Mb-type bipolar cell terminals with the aim of exploring the role of ribbons in transmitter release. We examined the physiology and ultrastructure of this terminal around two time points: midday and midnight. Nystatin perforated-patch recordings of membrane capacitance (C(m)) revealed that synaptic vesicle exocytosis evoked by short depolarizations was reduced at night, even though Ca(2+) currents were larger. The efficiency of exocytosis (measured as the DeltaC(m) jump per total Ca(2+) charge influx) was thus significantly lower at night. The paired-pulse ratio remained unchanged, however, suggesting that release probability was not altered. Hence the decreased exocytosis likely reflects a smaller readily releasable vesicle pool at night. Electron microscopy of single sections from intact retinas averaged 65% fewer ribbons at night. Interestingly, the number of active zones did not change from day to night, only the probability of finding a ribbon at an active zone. Additionally, synaptic vesicle halos surrounding the ribbons were more completely filled at night when these on-type bipolar cells are more hyperpolarized. There was no change, however, in the physical dimensions of synaptic ribbons from day to night. These results suggest that the size of the readily releasable vesicle pool and the efficiency of exocytosis are reduced at night when fewer ribbons are present at bipolar cell terminal active zones.

Endocannabinoids in the intact retina: 3 H-anandamide uptake, fatty acid amide hydrolase immunoreactivity and hydrolysis of anandamide.

There is much evidence for an endocannabinoid system in the retina. However, neither the distribution of endocannabinoid uptake, the regulation of endocannabinoid levels, nor the role of endocannabinoid metabolism have been investigated in the retina. Here we focused on one endocannabinoid, anandamide (AEA), and its major hydrolyzing enzyme, fatty acid amide hydrolase (FAAH), in the goldfish retina. Immunoblots of FAAH immunoreactivity (IR) in goldfish retina, brain and rat retina, and brain homogenates showed a single band at 61 kDa that was blocked by preadsorption with peptide antigen. Specific FAAH IR (blocked by preadsorption) was most prominent over Müller cells and cone inner segments. Weaker label was observed over some amacrine cells, rare cell bodies in the ganglion cell layer, and in four lamina in the inner plexiform layer. FAAH activity assays showed that goldfish-retinal and brain homogenates hydrolyzed AEA at rates comparable to rat brain homogenate, and the hydrolysis was inhibited by methyl arachidonyl fluorophosphonate (MAFP) and N-(4 hydroxyphenyl)-arachidonamide (AM404), with IC(50)s of 21 nM and 1.5 microM, respectively. Cellular 3H-AEA uptake in the intact retina was determined by in vitro autoradiography. Silver-grain accumulation at 20 degrees C was most prominent over cone photoreceptors and Müller cells. Uptake was significantly reduced when retinas were incubated at 4 degrees C, or preincubated with 100 nM MAFP or 10 microM AM404. There was no differential effect of blocking conditions on the distribution of silver grains over cones or Müller cells. The codistribution of FAAH IR and 3H-AEA uptake in cones and Müller cells suggests that the bulk clearance of AEA in the retina occurs as a consequence of a concentration gradient created by FAAH activity. We conclude that endocannabinoids are present in the goldfish retina and underlay the electrophysiological effects of cannabinoid ligands previously shown on goldfish cones and bipolar cells.

Vanilloid receptor like 1 (VRL1) immunoreactivity in mammalian retina: colocalization with somatostatin and purinergic P2X1 receptors.

The distribution of vanilloid receptor like1 immunoreactivity (VRL1-IR) in the retinas of rat, cat, and monkey was studied by single- and double-labeling immunocytochemistry. The patterns were similar for all three species in that VRL1-IR was most prominent in the inner plexiform layer, with scattered compact projections to the outer plexiform layer (OPL). VRL1-immunoreactive cell bodies were present throughout the rat retina, represented by amacrine cells in the inner nuclear layer and ganglion cell layer (GCL). In cat and monkey retinas, VRL1-immunoreactive cell bodies were restricted to the GCL in the inferior retina. Occasional cell bodies were associated with retinal blood vessels, but their identity as pericytes, glia, or neurons is uncertain. All VRL1-immunoreactive cells and processes colocalized with somatostatin and purinergic P2X1 receptor-IR but not with tyrosine hydroxylase-IR. VRL1-immunoreactive processes in the OPL did not label with antisera against synaptic vesicle 2 (SV2), suggesting that they were dendritic and did not derive from interplexiform cells. However, VRL1-immunoreactive processes in the far periphery toward the pars plana labeled for SV2, suggesting that these processes were presynaptic. The VRL1-immunoreactive cell bodies in the monkey GCL were not calbindin-immunoreactive, demonstrating that they were not displaced H2 horizontal cells. The VRL1-immunoreactive cells in cat and monkey could represent biplexiform and/or associational ganglion cells that receive input in the OPL throughout the retina and direct output to the far periphery. The presence of P2X1 receptors and vanilloid receptor like 1 protein on somatostatin-containing neurons in mammalian retina adds to the growing complexity regarding the chemical control of retinal function that is likely to include the microcirculation.

Evidence against the presence of an anandamide transporter.

On the basis of temperature dependency, saturability, selective inhibition, and substrate specificity, it has been proposed that an anandamide transporter exists. However, all of these studies have examined anandamide accumulation at long time points when downstream effects such as metabolism and intracellular sequestration are operative. In the current study, we have investigated the initial rates (<1 min) of anandamide accumulation in neuroblastoma and astrocytoma cells in culture and have determined that uptake is not saturable with increasing concentrations of anandamide. However, anandamide hydrolysis, after uptake in neuroblastoma cells, was saturable at steady-state time points (5 min), suggesting that fatty acid amide hydrolase (FAAH) may be responsible for observed saturation of uptake at long time points. In general, arvanil, olvanil, and N-(4-hydroxyphenyl)arachidonylamide (AM404) have been characterized as transport inhibitors in studies using long incubations. However, we found these "transport inhibitors" did not inhibit anandamide uptake in neuroblastoma and astrocytoma cells at short time points (40 sec or less). Furthermore, we confirmed that these inhibitors in vitro were actually inhibitors of FAAH. Therefore, the likely mechanism by which the transport inhibitors raise anandamide levels to exert pharmacological effects is by inhibiting FAAH, and they should be reevaluated in this context. Immunofluorescence has indicated that FAAH staining resides mainly on intracellular membranes of neuroblastoma cells, and this finding is consistent with our observed kinetics of anandamide hydrolysis. In summary, these data suggest that anandamide uptake is a process of simple diffusion. This process is driven by metabolism and other downstream events, rather than by a specific membrane-associated anandamide carrier.