Evaluating the Completeness of ClinicalTrials.gov.

Background: To date, although studies have been conducted to assess compliance with listing clinical trial information, to our knowledge there is nothing in the literature examining the completion and accuracy of clinical trial site information on ClinicalTrials.gov .

Methods: We compared clinical trial information originating from ClinicalTrials.

Characterization and localization of adenosine A2 receptors in bovine rod outer segments.

Previous work from this laboratory has shown that retinal adenosine A2 binding sites are localized over outer and inner segments of photoreceptors in rabbit and mouse retinal sections. In the present study, adenosine receptor binding has been characterized and localized in membranes from bovine rod outer segments (ROS). Saturation studies with varying concentrations (10-150 nM) of 5'-(N-[2,8-3H]ethylcarboxamido)adenosine ([3H]NECA) and 100 micrograms of ROS membrane protein show a single site with a KD of 103 nM and a Bmax of 1.

Characterization of adenosine A2 receptors in bovine retinal pigment epithelial membranes.

The pharmacological characteristics of adenosine A2 receptors are described for membranes prepared from bovine retinal pigmented epithelial (RPE) cells. RPE cells were isolated after removal of retina, lysed by freeze-thawing, and membranes separated from cytoplasmic components. A single population of adenosine binding sites is present in RPE membranes, as determined from saturation analysis and competition binding assays.

Characterization of adenosine A2 receptors in bovine retinal membranes.

Two classes of extracellular receptors for adenosine, A1 and A2, have been demonstrated in the mammalian retina. Our laboratory has previously reported the pharmacological characteristics of the mammalian retinal A1 receptors. We now report our characterization of retinal A2 receptors based on data obtained from both adenylate cyclase assays and radioligand binding studies.

Type I calmodulin-sensitive adenylyl cyclase is neural specific.

The distribution of type I calmodulin-sensitive adenylyl cyclase in bovine and rat tissues was examined by northern blot analysis and in situ hybridization. Northern blot analysis using poly(A)(+)-selected RNA from various bovine tissues indicated that mRNA for type I adenylyl cyclase was found only in brain, retina, and adrenal medulla, suggesting that this enzyme is neural specific. In situ hybridization studies using bovine, rabbit, and rat retina indicated that mRNA for type I adenylyl cyclase is found in all three nuclear layers of the neural retina and is particularly abundant in the inner segment of the photoreceptor cells.

The determination of total cGMP levels in rod outer segments from intact toad photoreceptors in response to light superimposed on background and to consecutive flashes: a second light flash accelerates the dark recovery rate of cGMP levels in control media, but not in Na(+)-free, low Ca2+ medium.

In previous experiments we established that a light flash reduced cGMP levels of toad rod outer segments within the transduction time interval, but that recovery of the dark level of cGMP occurred more slowly than reported electrophysiological recovery of membrane potential. We now report that a second light flash accelerates the recovery rate of total cGMP following an initial flash, but that this acceleration is blocked in a medium which is both sodium and calcium deficient. We also noted that calcium deficiency only elevated cGMP levels when sodium was present.

Muscarinic receptor stimulated GTPase activity in synaptic membranes from bovine retina.

GTPase activity has been measured in synaptic membranes from bovine retina, with and without muscarinic receptor stimulation. Maximal stimulation above basal levels was achieved with 5 microM oxotremorine and 100 microM carbachol. (4-Hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride, which is selective for the M1 muscarinic receptor, failed to stimulate GTPase activity.

Evidence for the action of endogenous adenosine in the rabbit retina: modulation of the light-evoked release of acetylcholine.

Much evidence has accumulated supporting the hypothesis that the purine nucleoside adenosine may indeed function as a neuromodulator in the mammalian retina, but to date no reports have directly illustrated a physiological role for this nucleoside. In other regions of the CNS, adenosine agonists decrease transmitter release, whereas antagonists increase release. A similar role for adenosine in the retina is now apparent.

Adenosine in vertebrate retina: localization, receptor characterization, and function.

1. The uptake of [3H] adenosine into specific populations of cells in the inner retina has been demonstrated. In mammalian retina, the exogenous adenosine that is transported into cells is phosphorylated, thereby maintaining a gradient for transport of the purine into the cell.

Characterization of adenosine A1-receptor binding sites in bovine retinal membranes.

Using retinas prepared from freshly dissected bovine eyes, we have characterized the binding of the A1-selective agonist, [3H]PIA (N6-R-[3H](2-phenylisopropyl)adenosine). Specific binding was linear over a range of membrane protein concentrations from 0.10 to 1.

GAP-43-like immunoreactivity in the adult retina of several species.

The localization of GAP-43-like immunoreactivity has been determined in retinas from adult toad, snake, rat, rabbit, cow and human. Specific labeling was conspicuous in discrete sublaminae within the inner plexiform layer of all mammalian species tested. In contrast, the toad retina exhibited punctate labeling in the outer plexiform layer, while the snake retina had little or no GAP-43-like immunoreactivity.

Localization of 2-[125I]iodomelatonin binding sites in mammalian retina.

Specific melatonin binding sites were localized in the mammalian retina using the selective radioligand 2-[125I]iodomelatonin. Frozen sections obtained from both pigmented and albino rabbit eyes and albino mouse eyes were incubated with 2-[125I]iodomelatonin in the absence and presence of competing agents. In eyecups from albino rabbits, the highest density of specific 2-[125I]iodomelatonin binding sites was localized over the inner plexiform layer.

The accumulation of [3H]phenylisopropyl adenosine ([3H]PIA) and [3H]adenosine into rabbit retinal neurons is inhibited by nitrobenzylthioinosine (NBI).

Uptake of [3H]adenosine and [3H]R-phenylisopropyladenosine (R-PIA) into retinal cells was assessed autoradiographically, in the presence and absence of the purine nucleoside transport inhibitor, nitrobenzylthioinosine (NBI). Under control conditions, both purine nucleosides were accumulated in cell bodies localized to the ganglion cell layer, and the inner nuclear layer. In the presence of NBI, significantly less accumulation of nucleosides within cell bodies was observed, particularly within the inner nuclear layer, suggesting that most of the uptake occurred via the transport of both substrates.

Acetylcholine release from the rabbit retina mediated by kainate receptors.

The cholinergic amacrine cells of the rabbit retina may be labeled with 3H-choline (3H-Ch), and the activity of the cholinergic population may be monitored by following the release of 3H-ACh. Glutamate analogs caused massive ACh release, up to 50 times the basal efflux, with the following rank order of potency: alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) greater than quisqualate (QQ) = kainate (KA) much greater than NMDA (in magnesium-free medium) much greater than glutamate greater than aspartate. In contrast, the release of 3H-Ch was unchanged.

Discrete distributions of adenosine receptors in mammalian retina.

Binding sites for both the adenosine A1 receptor agonists [3H]phenylisopropyladenosine and [3H]cyclohexyladenosine and the mixed A1-A2 agonist N-[3H]ethylcarboxamidoadenosine [( 3H]NECA) were localized in rabbit and mouse retinas using autoradiographic techniques. These two classes of agonists bound to very different regions of mammalian retinas. A1 agonist binding was localized to the inner retina, particularly over the inner plexiform layer.

Regulation of cyclic AMP levels in mammalian retina: Effects of depolarizing agents and transmitters.

Cellular depolarization in brain results in a modulation of cAMP levels by releasing neurotransmitters having receptors linked via GTP-binding proteins to adenylate cyclase. In order to determine the transmitters regulating cAMP during cellular depolarization in mammalian retina, the modulation of cAMP by depolarizing media was investigated. Cyclic AMP levels in light adapted retinas increased following exposure to depolarizing media, but levels in dark adapted retinas remained unaltered.

Dopamine and its agonists reduce a light-sensitive pool of cyclic AMP in mouse photoreceptors.

The exposure to bright light of dark-adapted (DKA) mouse retinas incubated in the dark (DI) in IBMX-containing medium causes a marked loss of cyclic AMP. This light response also occurs if the medium contains 10 mM aspartate or cobaltous ion, agents believed to confine the effects of light to photoreceptors. Thus, the action of light in the presence of either of these agents defines a light-sensitive pool of cyclic AMP in photoreceptors.

Adenosine: autoradiographic localization and electrophysiologic effects in the cat retina.

Autoradiography with 3H-adenosine was used to localize cells that accumulate adenosine in the cat retina. Electrophysiologic effects elicited by adenosine on DC-electroretinograms (ERG) and optic nerve responses (ONR) were studied in isolated, arterially perfused cat eyes. Subpopulations of cells localized in the ganglion cell layer and inner nuclear layer showed clear labeling for adenosine.

Displaced cholinergic, GABAergic amacrine cells in the rabbit retina also contain adenosine.

It is generally accepted that the purine nucleoside, adenosine, plays a neuromodulatory role in the central nervous system (CNS) (Daly et al., 1981; Phillis & Wu, 1983; Williams, 1986; Williams, 1987; Snyder, 1985). Adenosine is thought to exert its primary effects presynaptically, by inhibiting the release of neurotransmitters including gamma-aminobutyric acid (GABA) and acetylcholine (ACh) (Phillis & Barraco, 1985; Proctor & Dunwiddie, 1987).

Comparison of adenosine uptake and endogenous adenosine-containing cells in mammalian retina.

Autoradiographic techniques were used to label [3H]-adenosine and [3H]-cyclohexyladenosine accumulating cells in rabbit, mouse, and ground squirrel retinas. Immunohistochemical methods revealed the distribution of cells that stained for endogenous adenosine. Comparisons of these two markers revealed for all three species that the distribution of specific subpopulations of retinal cells that store or accumulate the purine nucleoside, adenosine, is similar.

Light-induced losses and dark recovery rates of guanosine 3',5'-cyclic monophosphate in rod outer segments of intact amphibian photoreceptors.

We used an apparatus in which pieces of dark-adapted amphibian retinas (Rana pipiens, Bufo marinus) obtained under infrared illumination were exposed to precise intervals of 500-nm illuminations, and then frozen by contact of their outer segment surface with a liquid helium-cooled copper mirror. Sections of the frozen outer segment layer were obtained in a cryostat and then assayed for total extractable cyclic 3',5'-guanosine monophosphate (cGMP). Significant losses of cGMP with respect to the dark level were evident as early as 60 ms after light onset.

Light induces a rapid and transient increase in inositol-trisphosphate in toad rod outer segments.

The sub-second time course of changes in the content of [3H]inositol-1,4,5-trisphosphate was determined in rod outer segments from very rapidly frozen Bufo retinas that had been incubated with [3H]inositol. Rod outer segments were cut off frozen specimens with a cryostat microtome and the water soluble extracts were analyzed. The content of [3H]inositol-1,4,5-trisphosphate rose after approximately 250 msec of bright illumination, but returned to the unstimulated level after 1 sec, whether the stimulus remained on or not.

Adenosine A1 receptor-mediated inhibition of adenylate cyclase in rabbit retina.

The purine adenosine has been postulated as playing a role in CNS neurotransmission or modulation. Evidence is now provided that inhibition of adenylate cyclase in rabbit retinal homogenates is mediated via adenosine A1 receptors. Nanomolar concentrations of the A1 receptor agonists, cyclohexyladenosine (CHA) and phenylisopropyladenosine (PIA), significantly inhibited the activity of forskolin-stimulated adenylate cyclase in preparations in which endogenous adenosine was destroyed by pretreatment with adenosine deaminase.

Tryptamine and some related molecules block the accumulation of a light-sensitive pool of cyclic AMP in the dark-adapted, dark-incubated mouse retina.

Dark-adapted retinas of mice (C57BL/6J) incubated in the dark in media containing 1 mM 3-isobutylmethylxanthine (IBMX) or 5 mM Co2+ accumulate cyclic AMP (cAMP). A portion of this pool is light sensitive, as light can prevent or reverse its accumulation. Similarly, tryptamine, serotonin, 5-methoxytryptamine, bufotenine, and 5-methoxydimethyltryptamine can block the accumulation of the light-sensitive pool of cAMP, whereas tryptophan, melatonin, N-acetylserotonin, 5-methoxytryptophol, and tetrahydro-beta-carbolines are inactive.

Rapid declines in cyclic GMP of rod outer segments of intact frog photoreceptors after illumination.

Considerable disagreement has resulted from experiments designed to test whether light-induced falls in cGMP in outer segment (OS) of photoreceptors precede their light-induced electrical responses. Different studies have reported initial declines at 50 ms, at s, or not at all for physiological stimuli. Such studies have employed whole retinas, isolated rod OS, or isolated rod OS with attached inner segments and involved a variety of techniques.