Regular brief interruptions to sitting after a high-energy evening meal attenuate glycemic excursions in overweight/obese adults.

BACKGROUND AND AIMS: Modern Western lifestyles are characterized by consumption of approximately 45% of total daily energy intake at the evening meal, followed by prolonged sitting while watching television (TV), which may deleteriously impact glycemic control. After a high-energy evening meal (dinner), we examined whether regular, brief activity bouts during TV commercial breaks could acutely lower postprandial glucose and insulin responses in overweight/obese adults, compared to prolonged uninterrupted sitting. METHODS AND RESULTS: Nine overweight/obese adults (29.7 ± 4.06 kg m-2; aged 32 ± 3 years; 5 male) completed two laboratory-based conditions of three and a half hours: prolonged sitting during TV viewing (SIT); and, prolonged sitting interrupted every 20 min with 3 min of light-intensity body-weight resistance activities (active commercial breaks; ACBs). Venous postprandial glucose and insulin responses to dinner were calculated as positive incremental area under the curve (iAUC) from baseline. Interstitial glucose was measured using a continuous glucose monitor and quantified as total AUC (tAUC). Compared to SIT, plasma glucose iAUC was reduced by 33% [3.4 ± 1.0 vs 5.1 ± 1.0 (mean ± SEM) mmol h·L-1, p = 0.019] and plasma insulin iAUC by 41% (813 ± 224 vs 1373 ± 224, p = 0.033 pmol h·L-1) for the ACB condition. During the ACB condition there was a significant reduction in interstitial glucose tAUC (24.4 ± 5.2 vs 26.9 ± 5.2 mmol h·L-1, p < 0.001), but this did not persist beyond the laboratory observation period. CONCLUSIONS: Regular brief light-intensity activity bouts can attenuate glycemic responses during television viewing time following a high-energy evening meal in overweight/obese adults.

Modulation of TRPV4 by diverse mechanisms.

Transient receptor potential ion channels (TRP) are a superfamily of non-selective ion channels which are opened in response to a diverse range of stimuli. The TRP vanilloid 4 (TRPV4) ion channel is opened in response to heat, mechanical stimuli, hypo-osmolarity and arachidonic acid metabolites. However, recently TRPV4 has been identified as an ion channel that is modulated by, and opened by intracellular signalling cascades from other receptors and signalling pathways. Although TRPV4 knockout mice show relatively mild phenotypes, some mutations in TRPV4 cause severe developmental abnormalities, such as the skeletal dyplasia and arthropathy. Regulated TRPV4 function is also essential for healthy cardiovascular system function as a potent agonist compromises endothelial cell function, leading to vascular collapse. A better understanding of the signalling mechanisms that modulate TRPV4 function is necessary to understand its physiological roles. Post translational modification of TRPV4 by kinases and other signalling molecules can modulate TRPV4 opening in response to stimuli such as mechanical and hyposmolarity and there is an emerging area of research implicating TRPV4 as a transducer of these signals as opposed to a direct sensor of the stimuli. Due to its wide expression profile, TRPV4 is implicated in multiple pathophysiological states. TRPV4 contributes to the sensation of pain due to hypo-osmotic stimuli and inflammatory mechanical hyperalsgesia, where TRPV4 sensitizaton by intracellular signalling leads to pain behaviors in mice. In the vasculature, TRPV4 is a regulator of vessel tone and is implicated in hypertension and diabetes due to endothelial dysfunction. TRPV4 is a key regulator of epithelial and endothelial barrier function and signalling to and opening of TRPV4 can disrupt these critical protective barriers. In respiratory function, TRPV4 is involved in cystic fibrosis, cilary beat frequency, bronchoconstriction, chronic obstructive pulmonary disease, pulmonary hypertension, acute lung injury, acute respiratory distress syndrome and cough.In this review we highlight how modulation of TRPV4 opening is a vital signalling component in a range of tissues and why understanding of TRPV4 regulation in the body may lead to novel therapeutic approaches to treating a range of disease states.

The tyrosine kinase inhibitor bafetinib inhibits PAR2-induced activation of TRPV4 channels in vitro and pain in vivo.

BACKGROUND AND PURPOSE: Protease-activated receptor 2 (PAR2) is expressed on nociceptive neurons, and can sensitize transient receptor potential (TRP) ion channels to amplify neurogenic inflammation and pain. The mechanisms by which this occurs are not fully understood. PAR2 causes receptor-operated activation of TRPV4 channels and TRPV4 null mice have attenuated PAR2-stimulated neurogenic inflammation and mechanical hyperalgesia. Here we investigate the intracellular signalling mechanisms underlying PAR2-induced TRPV4 channel activation and pain. EXPERIMENTAL APPROACH: Responses of non-transfected and TRPV4-transfected HEK293 cells to agonists of PAR2 (trypsin and SLIGRL) and TRPV4 channels (GSK1016790A) were determined using calcium imaging. Inhibitors of TRPV4 channels (HC067047), sarcoendoplasmic reticulum calcium transport ATPase (thapsigargin), Gαq (UBO-QIC), tyrosine kinases (bafetinib and dasatinib) or PI3 kinases (wortmannin and LY294002) were used to investigate signalling mechanisms. In vivo effects of tyrosine kinase inhibitors on PAR2 -induced mechanical hyperalgesia were assessed in mice. KEY RESULTS: In non-transfected HEK293 cells, PAR2 activation transiently increased intracellular calcium ([Ca(2+) ]i ). Functional expression of TRPV4 channels caused a sustained increase of [Ca(2+) ]i , inhibited by HC067047, bafetinib and wortmannin; but not by thapsigargin, UBO-QIC, dasatinib or LY294002. Bafetinib but not dasatinib inhibited PAR2-induced mechanical hyperalgesia in vivo. CONCLUSIONS AND IMPLICATIONS: This study supports a role for tyrosine kinases in PAR2-mediated receptor-operated gating of TRPV4 channels, independent of Gαq stimulation. The ability of a tyrosine kinase inhibitor to diminish PAR2-induced activation of TRPV4 channels and consequent mechanical hyperalgesia identifies bafetinib (which is in development in oncology) as a potential novel analgesic therapy.

Transient receptor potential (TRP) channels in the airway: role in airway disease.

Over the last few decades, there has been an explosion of scientific publications reporting the many and varied roles of transient receptor potential (TRP) ion channels in physiological and pathological systems throughout the body. The aim of this review is to summarize the existing literature on the role of TRP channels in the lungs and discuss what is known about their function under normal and diseased conditions. The review will focus mainly on the pathogenesis and symptoms of asthma and chronic obstructive pulmonary disease and the role of four members of the TRP family: TRPA1, TRPV1, TRPV4 and TRPM8. We hope that the article will help the reader understand the role of TRP channels in the normal airway and how their function may be changed in the context of respiratory disease.

A propofol binding site on mammalian GABAA receptors identified by photolabeling.

Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABAA (γ-aminobutyric acid type A) receptors, but where it binds to this receptor is not known and has been a matter of some debate. We synthesized a new propofol analog photolabeling reagent whose biological activity is very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin that have been identified using X-ray crystallography. Using a combination of protiated and deuterated versions of the reagent to label mammalian receptors in intact membranes, we identified a new binding site for propofol in GABAA receptors consisting of both ß3 homopentamers and α1ß3 heteropentamers. The binding site is located within the ß subunit at the interface between the transmembrane domains and the extracellular domain and lies close to known determinants of anesthetic sensitivity in the transmembrane segments TM1 and TM2.

High and persistent excretion of hepatitis A virus in immunocompetent patients.

The duration and level of virus excretion in blood and faeces of patients with hepatitis A virus (HAV) infection were studied in relation to levels of alanine aminotransferase (ALT), disease severity and HAV genotype. Clinical data, blood and faeces were collected from 27 patients with acute hepatitis A (median age: 33 years) for a maximum of 26 weeks. Single blood donations from 55 other patients with acute HAV (median age: 32 years) were also used. Virus loads were quantified by competitive nested RT-PCR. HAV was excreted in faeces for a median period of 81 days after disease onset, with 50% of patients still excreting high levels at Day 36 (2 x 10(6) - 2 x 10(8) copies/ml faeces suspension). Viraemia was detected, but not quantifiable, for a median period of 42 days. In the first 10 days of illness, higher ALT levels were correlated with higher viraemia levels. Comparison of patients infected with genotype 1a with those infected with type 1b did not differ significantly in terms of the duration of HAV excretion or jaundice. In conclusion, faecal excretion of HAV is at a high titre in the first month, perhaps making patients infectious for a longer period than assumed currently. Blood banks should be aware that viraemia may be present for more than 1 month, and genotype did not affect the duration of virus excretion or jaundice.

Altered visual experience and acute visual deprivation affect predatory targeting by infrared-imaging Boid snakes.

Boid and Crotaline snakes use both their eyes and infrared-imaging facial pit organs to target homeothermic prey. These snakes can target in complete darkness, but the eyes can also effectively direct predatory strikes. We investigated the behavioral correlates of boid snakes' simultaneous use of two imaging systems by testing whether congenital unilateral visual deprivation affects targeting performance. Normally sighted Burmese pythons exhibited average targeting angle of zero (on the midline axis of the head), but three unilaterally anophthalmic Burmese pythons targeted preferentially on the sighted side. A unilaterally anophthalmic amethystine python also targeted on the sighted side, and a unilaterally anophthalmic Brazilian rainbow boa tended to target on the sighted side, though its mean targeting angle was not significantly different from zero. When unilaterally anophthalmic Burmese pythons were temporarily blinded, mean strike angle changed to that of normally sighted snakes. These results show that while infrared-imaging snakes can shift between visual and infrared information under acute experimental conditions, loss of part of the visual field during development results in abnormal predatory targeting behavior. In contrast, normally sighted snakes subjected to temporary unilateral blinding do not target preferentially on the sighted side. Therefore, while loss of part of the visual field may be compensated for by infrared input in normal snakes, partial absence of visual input during development may alter central organization of visual information. Conversely, absence of half the visual field during development does not alter targeting performance based upon infrared input alone, suggesting that organization of the central infrared map does not depend upon normal organization of visual input.

Prey targeting by the infrared-imaging snake Python molurus: effects of experimental and congenital visual deprivation.

Boid and crotaline snakes possess two distinct types of organ evolved to image radiant electromagnetic energy: the lateral eye, which responds to visible light, and the pit organ, which responds to infrared radiation. While infrared imaging may allow accurate predatory targeting in complete absence of visual information, both infrared and visual information are probably normally involved in prey targeting. We examined the roles of vision and infrared imaging in Python molurus predatory performance under conditions of (1) high visual contrast; (2) very low visual contrast; (3) complete blinding; (4) experimental monocular occlusion; and (5) congenital monocularity. Normally sighted pythons were equally successful at targeting white (BALB/c) and black (C57BL6/J) mice (Mus domesticus) against a black background. Binocularly occluded snakes exhibited strike angles and distances similar to non-occluded snakes, but exhibited lower strike success, suggesting that high visible contrast is not required for accurate targeting, but that precise targeting depends to some degree upon visual information. Strike angles, distances and latencies were indistinguishable between snakes subjected to experimental monocular occlusion and normally sighted snakes. However, snakes congenitally lacking one eye preferentially targeted on the sighted side. Thus, accurate targeting of highly mobile homeothermic prey by Python can be accomplished with little or no visual information, but performance can be affected by complete visual deprivation or by alteration of visual input during development. The developmental effects of early visual deprivation in this system provide a novel opportunity to investigate the neural integration of two electromagnetic radiation-imaging systems in a single animal.

The role of Clock in the developmental expression of neuropeptides in the suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the dominant circadian pacemaker in mammals. To understand better the ontogeny of mouse SCN and the role of the pacemaker in peptide expression, the authors examined the distribution of cells that were immunoreactive for vasopressin (AVP) or vasoactive intestinal polypeptide (VIP) in wild type and Clock mutant mice at two developmental stages. Clock homozygous mice failed to show the dramatic increase in the number of VIP-immunoreactive (VIP-ir) neurons from postnatal day 6 (P6) to P30 that was found in the SCN of wild type mice. The number of AVP-ir neurons was relatively constant in the postnatal SCN but was significantly reduced in Clock/Clock mice. The effects of the Clock mutation varied with position in the SCN for both peptides. Densitometry of immunolabeled brains indicated that the Clock mutation reduced AVP expression specifically in the SCN and not in other brain areas. The SCN did not significantly change shape or size with age or Clock genotype. Taken together, these results indicate that the neonatal mouse SCN has its full complement of cells, some of which are not yet mature in their neuropeptide content. Furthermore, the observation that the Clock mutation appears to act on a subset of AVP and VIP cells suggests heterogeneity within these cell classes in the SCN.

Circadian control of photoreceptor outer segment membrane turnover in mice genetically incapable of melatonin synthesis.

Vertebrate retinal photoreceptors periodically shed membrane from their outer segment distal tips; this material is phagocytosed and degraded by the retinal pigmented epithelium. Both a circadian oscillator and the daily light-dark cycle affect disk shedding, and the effects of both may be mediated by melatonin. To clarify melatonin's role in this process, we asked whether endogenous melatonin is required for rhythmic disk shedding in mouse retina. We analyzed disk shedding in two mouse strains: C3H, which produce melatonin in retina and pineal under the control of circadian oscillators, and C57BL/6, which do not produce melatonin. In cyclic light, both strains exhibited a robust cycle of disk phagosome content in the pigmented epithelium. Peak shedding occurred just after dawn, and trough levels occurred during the middle of the dark phase. In constant darkness, mice exhibited circadian rhythms of locomotor activity, the characteristics of which were similar between strains. Both strains also exhibited rhythmic disk shedding in constant darkness, although amplitudes of the rhythms were damped. Exogenous melatonin delivered once per day failed to reestablish high-amplitude cyclic shedding in mice held in constant darkness. Our results show that, while disk shedding in cyclic light is robustly rhythmic, neither rhythmic production of melatonin nor the circadian oscillator responsible for rhythmic locomotor activity is sufficient to drive high-amplitude rhythmic shedding in constant darkness. More importantly, melatonin is required neither for cyclic changes in the rate of disk shedding in cyclic light, nor for the circadian rhythm of disk shedding in constant darkness.

Surface ultrastructure of pit organ, spectacle, and non pit organ epidermis of infrared imaging boid snakes: A scanning probe and scanning electron microscopy study.

Boid snakes possess unique infrared imaging pit organs. The ultrastructure of the surfaces of these organs scatter or reflect electromagnetic radiation of specific wavelengths. Pit organ epidermal surfaces of boid snakes are covered with arrays of pore-like structures called micropits. In order to determine the dimensions of this complicated surface structure, we have performed the first ultrastructural analysis on snake epidermis by high-resolution microscopy techniques. Using scanning probe microscopy and scanning electron microscopy, we found that the epidermis of pit organ, maxillary non pit organ, spectacle, and ventral scales contain arrays of micropits. These scale surfaces also contain major surface features of overlapping plate-like structures. Pit organ micropits averaged 319 nm in diameter and 46 nm in depth and were spaced an average of 808 nm from each other. These micropits were significantly deeper, of greater diameter, and spaced at greater distances apart than those of the other scales. Plate structures of the pit organs had a mean distance between plates of 3.5 microm and a mean plate step height of 151 nm. These differences serve to strengthen the argument that arrays of micropit and plate surface structures function as spectral filters or anti-reflective coatings with respect to incident electromagnetic radiation.

Nanoscale design of snake skin for reptation locomotions via friction anisotropy.

Multi-mode scanning probe microscopy is employed to investigate the nanostructure of dermal samples from three types of snakes. Sophisticated friction modifying nanostructures are described. These include an ordered microfibrillar array that can function to achieve mission adaptable friction characteristics. Significant reduction of adhesive forces in the contact areas caused by the 'double-ridge' nanoscale microfibrillar geometry provides ideal conditions for sliding in forward direction with minimum adhesive forces and friction. Low surface adhesion in these local contact points may reduce local wear and skin contamination by environmental debris. The highly asymmetric, 'pawl-like' profile of the microfibrillar ends with radius of curvature 20-40 nm induces friction anisotropy in forward backward motions and serves as an effective stopper for backward motion preserving low friction for forward motion. The system of continuous micropores penetrating through the snake skin may serve as a delivery system for lubrication/anti-adhesive lipid mixture that provides for boundary lubrication of snake skins.

The Python pit organ: imaging and immunocytochemical analysis of an extremely sensitive natural infrared detector.

The Python infrared-sensitive pit organ is a natural infrared imager that combines high sensitivity, ambient temperature function, microscopic dimensions, and self-repair. We are investigating the spectral sensitivity and signal transduction process in snake infrared-sensitive neurons, neither of which is understood. For example, it is unknown whether infrared receptor neurons function on a thermal or a photic mechanism. We imaged pit organs in living Python molurus and Python regius using infrared-sensitive digital video cameras. Pit organs were significantly more absorptive and/or emissive than surrounding tissues in both 3-5 microns and 8-12 microns wavelength ranges. Pit organs exhibited greater absorption/emissivity in the 8-12 microns range than in the 3-5 microns range. To directly test the relationship between photoreceptors and pit organ infrared-sensitive neurons, we performed immunocytochemistry using antisera directed against retinal photoreceptor opsins. Retinal photoreceptors were labeled with antisera specific for retinal opsins, but these antisera failed to label terminals of infrared-sensitive neurons in the pit organ. Infrared-receptive neurons were also distinguished from retinal photoreceptors on the basis of their calcium-binding protein content. These results indicate that the pit organ absorbs infrared radiation in two major atmospheric transmission windows, one of which (8-12 microns) matches emission of targeted prey, and that infrared receptors are biochemically distinct from retinal photoreceptors. These results also provide the first identification of prospective biochemical components of infrared signal transduction in pit organ receptor neurons.

The tau mutation shortens the period of rhythmic photoreceptor outer segment disk shedding in the hamster.

The outer segments of vertebrate retinal photoreceptors undergo periodic shedding of membrane from their distal tips. This circadian rhythm of disk shedding persists with a period of about 24 h in the absence of external time cues. A circadian oscillator controlling photoreceptor disk shedding may exist in the eye, but in addition, the circadian clock in the hypothalamic suprachiasmatic nucleus (SCN) may also influence ocular rhythms including that of disk shedding. The tau mutation directly affects the SCN, and shortens the period of locomotor activity from 24 h in wild-type hamsters to 20 h in homozygous mutants. Here we show that homozygous tau-mutant hamsters in a 20-h light/dark cycle exhibit a 20-h oscillation in the rate of disk shedding, with peak phagosome numbers in the retinal pigmented epithelium occurring just after light onset. The numbers of phagosomes are significantly elevated from mid-dark levels prior to light onset, indicating that the disk shedding cycle anticipates dawn. Under conditions of constant darkness, the disk shedding rhythm in tau-mutant hamsters persists with a period of approximately 20 h. These results indicate that a rhythm of retinal photoreceptor outer segment disk shedding exists in the hamster eye, and that the period of this rhythm is shortened by the tau mutation.

Light perception in the vertebrate brain: an ultrastructural analysis of opsin- and vasoactive intestinal polypeptide-immunoreactive neurons in iguanid lizards.

Recent biochemical and immunocytochemical evidence indicates that a population of circadian and reproductive rhythm-entraining photoreceptors lies in the basal diencephalon of iguanid lizards. Here, we report the results of correlated light and electron microscopy of opsin-immunoreactive cells in the basal brain, and we discuss their ultrastructural relationship to known photoreceptors. Cerebrospinal fluid (CSF)-contacting bipolar neurons in the lizards Anolis carolinensis and Iguana iguana were immunolabeled with antisera generated against vertebrate retinal opsins and vasoactive intestinal polypeptide (VIP). Within the brain, opsin-immunoreactive cells were found exclusively in the ependyma of the basal region of the lateral ventricles (adjacent to nucleus paraolfactorius/nucleus ventromedialis and neostriatum/paleostriatum). Cells in the same anatomical location and with the same morphology were labeled with anti-VIP antisera. These cells possessed a dendritic process that extended toward the lateral ventricle, ending in a bulbous terminal that protruded into the ventricle. Axonal processes travelled ventrally and caudally. The entire cell, including the axonal process, exhibited opsin-like and VIP-like immunoreactivity. By light microscopy, opsin-like immunostaining appeared punctate, with immunoreactivity greatest in the bulbous terminal. Opsin- and VIP-immunostained thick sections were resectioned, and individual cells observed by light microscopy were then characterized using electron microscopy. We found that all immunostained cells were morphologically similar and that they were morphologically distinct from neighboring nonimmunoreactive cells. CSF-contacting opsin- and VIP-immunoreactive cells lacked the membranous stacks characteristic of retinal photoreceptors but were ciliated and contained numerous large electron-dense vesicles. Multiple synaptic contacts were made on the soma and putative dendritic processes of opsin- and VIP-immunoreactive CSF-contacting neurons. Our results provide the first ultrastructural characterization of opsin-immunostained encephalic CSF-contacting neurons in a vertebrate animal, and they indicate that these putative photoreceptors share structural features with pineal photoreceptors and with certain invertebrate extraretinal photoreceptors, but they are morphologically and biochemically distinct from visual photoreceptors of the retina.

Melatonin deacetylase activity in the pineal gland and brain of the lizards Anolis carolinensis and Sceloporus jarrovi.

Melatonin modulates a variety of rhythmic processes in vertebrates, and is synthesized in both the retina and pineal gland. We have shown previously that retinal melatonin is deacetylated generating 5-methoxytryptamine, which is then deaminated by monoamine oxidase, producing 5-methoxyindoleacetic acid and 5-methoxytryptophol. This process occurs within the eyes of a variety of vertebrates including the iguanid lizard Anolis carolinensis. To determine whether melatonin deacetylase activity also occurs in the pineal organ or in other parts of the lizard brain, pineals and brains of Anolis carolinensis and Sceloporus jarrovi were cultured in the presence of [3H-methoxy]-melatonin. High-performance liquid chromatography of the resulting culture media and tissues revealed the generation of radiolabeled 5-methoxytryptamine and 5-methoxyindoleacetic acid. These two methoxyindoles were the only radiolabeled metabolites detectable, and together accounted for all melatonin lost. Both the loss of melatonin and the production of melatonin metabolites were inhibited by inclusion of 100 microM eserine, an inhibitor of the melatonin deacetylase. Pargyline, a monoamine oxidase inhibitor, reduced the production of 5-methoxyindoleacetic acid and increased the production of 5-methoxytryptamine relative to control incubations. Similar effects of eserine and pargyline were seen in eyecup, brain and pineal gland, but the specific activity of melatonin deacetylation in cultured pineal glands was much greater than in either brains or eyecups. These results indicate that pineal glands of both Anolis carolinensis and Sceloporus jarrovi can rapidly catabolize melatonin by a mechanism very similar to that in the eye, that the melatonin deacetylation pathway exists elsewhere in the iguanid brain, and also extend our previous observations of ocular melatonin deacetylation to an additional species.

Identification of vertebrate deep brain photoreceptors.

Since the beginning of this century evidence has accumulated which demonstrates that nonmammalian vertebrates possess photoreceptors situated deep within the brain. These photoreceptors have been implicated in several different areas of physiology, but in all species examined, they play a critical role in the regulation of circadian and reproductive responses to light. Many attempts have been made to localize these sensory cells over the past 50 years, but until recently all attempts have failed. As a result, this important sensory system remains largely unexplored. Recent attempts to localize these photoreceptors, in a range of vertebrates, using combined antibody and biochemical approaches has met with some success. However, inconsistencies have emerged. Published and preliminary data raise the possibility of several types of encephalic photoreceptor photopigment (cone-like, rod-like or different from both), and depending on species at least two types of photoreceptor cell: CSF-contacting neurons (larval lamprey, reptiles and birds) and classical neurosecretory neurons within the nucleus magnocellularis preopticus (NMPO)(fish and amphibians).

Solubilization and biochemical characterization of the melatonin deacetylase from Xenopus laevis retina.

Melatonin deacetylase, an enzyme activity recently discovered in the Xenopus laevis retina, regulates local melatonin levels. The deacetylase occurs in retina, retinal pigment epithelium, and skin, all sites of melatonin action, and is widely distributed among vertebrates. We have solubilized the enzyme from Xenopus retina and pigment epithelium using nonionic detergents, and have developed a specific enzyme assay. We have characterized the enzyme and now report that the deacetylase is relatively specific for melatonin and is inhibited by the melatonin precursor N-acetylserotonin and the product of the deacetylase, 5-methoxytryptamine. Inhibition of deacetylase activity by eserine (physostigmine) suggests a relationship between deacetylase and cholinesterase activities. However, among a variety of cholinesterase inhibitors tested, only eserine inhibits the deacetylase. Furthermore, eserine is much less potent as an inhibitor of the deacetylase than the cholinesterases, and purified cholinesterases failed to deacetylate melatonin. We also show that melatonin deacetylase and aryl acylamidase (an enzyme related to cholinesterases) activities are differentially extractable from Xenopus ocular tissues, and that they exhibit different pH optima and inhibition profiles. Our results provide an initial characterization of the Xenopus retinal melatonin deacetylase, and indicate that deacetylase activity is distinct from cholinesterase and aryl acylamidase activities.

Rhythmic regulation of retinal melatonin: metabolic pathways, neurochemical mechanisms, and the ocular circadian clock.

1. Current knowledge of the mechanisms of circadian and photic regulation of retinal melatonin in vertebrates is reviewed, with a focus on recent progress and unanswered questions. 2. Retinal melatonin synthesis is elevated at night, as a result of acute suppression by light and rhythmic regulation by a circadian oscillator, or clock, which has been localized to the eye in some species. 3. The development of suitable in vitro retinal preparations, particularly the eyecup from the African clawed frog, Xenopus laevis, has enabled identification of neural, cellular, and molecular mechanisms of retinal melatonin regulation. 4. Recent findings indicate that retinal melatonin levels can be regulated at multiple points in indoleamine metabolic pathways, including synthesis and availability of the precursor serotonin, activity of the enzyme serotonin N-acetyltransferase, and a novel pathway for degradation of melatonin within the retina. 5. Retinal dopamine appears to act through D2 receptors as a signal for light in this system, both in the acute suppression of melatonin synthesis and in the entrainment of the ocular circadian oscillator. 6. A recently developed in vitro system that enables high-resolution measurement of retinal circadian rhythmicity for mechanistic analysis of the circadian oscillator is described, along with preliminary results that suggest its potential for elucidating general circadian mechanisms. 7. A model describing hypothesized interactions among circadian, neurochemical, and cellular mechanisms in regulation of retinal melatonin is presented.

Melatonin deacetylation: retinal vertebrate class distribution and Xenopus laevis tissue distribution.

Deacetylation is a rapid clearance mechanism for ocular melatonin. We have studied the distribution of retinal melatonin deacetylase activity among vertebrate classes. Exogenous radiolabeled melatonin is metabolized by ocular tissue prepared from the amphibian Xenopus laevis, the reptile Anolis carolinensis, the teleost fish Carassius auratus, and the bird Gallus domesticus. In contrast, we were unable to detect ocular melatonin breakdown in rat or pig. In each species exhibiting ocular melatonin breakdown, melatonin is first deacetylated to 5-methoxytryptamine, which is deaminated, producing 5-methoxyindoleacetic acid and 5-methoxytryptophol. Deacetylation of melatonin is inhibited by eserine (physostigmine), causing a reduction in the levels of all 3 metabolites. Deamination of 5-methoxytryptamine is inhibited by the monoamine oxidase inhibitor pargyline, such that 5-methoxyindoleacetic acid and 5-methoxytryptophol levels are decreased while levels of 5-methoxytryptamine are increased. Incubation with the deacetylase inhibitor eserine increases endogenous melatonin levels in Xenopus and Carassius eyecups, indicating that endogenous melatonin is metabolized via the deacetylase. We also studied the tissue distribution of the deacetylase in Xenopus laevis. Melatonin deacetylation occurs in retina, retinal pigment epithelium, and skin, all of which are sites of melatonin action. These results indicate that among non-mammalian vertebrates, deacetylation is a common clearance mechanism for ocular melatonin, and may degrade melatonin at other sites of action as well. Melatonin deacetylation may help regulate local melatonin concentration, and generates other biologically active methoxyindoles.