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Biomedical subjects

C R Gardner

Publications and source records attributed to C R Gardner.

At least 55 records · Page 3Linked to original sources
PubMed1992

Epidermal growth factor suppresses nitric oxide and hydrogen peroxide production by keratinocytes. Potential role for nitric oxide in the regulation of wound healing.

In the skin, wounding initiates a complex array of physiological processes mediated by growth factors and inflammatory mediators which stimulate tissue repair and protect against infection. We report that primary cultures of human keratinocytes and a mouse keratinocyte cell line respond to the inflammatory stimuli gamma-interferon and lipopolysaccharide or tumor necrosis factor-alpha by producing nitric oxide and hydrogen peroxide, two reactive mediators that are important in nonspecific host defense. Nitric oxide is produced by the l-arginine- and NADPH-dependent enzyme, nitric oxide synthase. In murine keratinocytes, optimal enzymatic activity was found to be dependent on Ca2+ and calmodulin as well as on glutathione. Inflammatory mediators were also found to inhibit the growth of keratinocytes, an effect that could be reversed by a nitric oxide synthase inhibitor. Epidermal growth factor (EGF), which promotes wound healing by stimulating cellular proliferation, was found to be a potent antagonist of reactive nitrogen and reactive oxygen intermediate production by keratinocytes. EGF also reversed the growth inhibitory actions of the inflammatory mediators. These data suggest that nitric oxide produced by keratinocytes is important in the control of cellular proliferation during wound healing. Our findings that EGF effectively regulates the production of free radicals by keratinocytes may represent an important pathway by which this growth factor not only stimulates epidermal cell proliferation but also facilitates the resolution of inflammation following wounding.

Amino Acid Oxidoreductases↗
PubMed1992

Discriminative stimulus properties of RU 33965, a benzodiazepine receptor weak partial inverse agonist.

Rats were trained to discriminate the low-efficacy benzodiazepine receptor inverse agonist RU 33965 from vehicle in a two-lever discrimination task on a fixed ratio (FR) 20 schedule. Consistent discrimination was obtained at 0.5 mg/kg PO RU 33965. Both leptazol and stronger inverse agonists (FG7142, S-135, RU 34000) substituted for the cue. The weak inverse agonists/antagonists RU 33094, RU 34030, Ro 15-1788, and ZK 93426 also substituted for the cue with the latter two compounds being particularly potent. The agonist and partial agonists diazepam, RU 33203, and RU 39419 did not substitute for the RU 33965 cue but RU 39419 antagonised it. The full agonists diazepam and loprazolam only consistently antagonised the cue when given IP 5 min pretest. These data suggest that the RU 33965 cue results from its weak inverse agonist activity at benzodiazepine receptors, but kinetic factors must be considered when interpreting drug effects in discrimination studies.

Animals↗
PubMed1992

A review of recently-developed ligands for neuronal benzodiazepine receptors and their pharmacological activities.

There has recently been a large increase in the number of new benzodiazepine receptor ligands, some with benzodiazepine structures, but many with other chemical structures. The pharmacological activities of these ligands have been interpreted on the basis of a continuum of efficacy ranging from full agonist through different degrees of partial agonism to antagonist and through partial to full inverse agonists. Inconsistencies with this hypothesis are considered in terms of alternative hypotheses, particularly the existence of functionally separable receptor subtypes. The potential of partial agonists as non-sedative anxiolytic agents with reduced potential of dependence and of weak partial inverse agonists as pro-cognitive agents is discussed. A pharmacophore for benzodiazepine receptors is proposed and supporting evidence presented.

Animals↗
PubMed1992

Effects of RU33368, a low affinity ligand for neuronal benzodiazepine receptors, on rodent behaviours and GABA-mediated synaptic transmission in rat cerebellar slices.

1. The effects of the low affinity benzodiazepine receptor ligand RU33368 were studied on rodent behaviours and on GABA-mediated synaptic transmission in rat cerebellar slices. 2. RU33368 inhibited stress induced ultrasounds in rat pups without inducing marked muscle relaxation. RU33368 also enhanced operant responding in rats that had been suppressed by mild footshock. These effects of RU33368 in these two models of anxiety were both blocked by the benzodiazepine antagonist Ro15-1788 (flumazonil). 3. In cerebellar slices RU33368 enhanced stimulus-induced synaptic inhibition of Purkinje layer cells with a minimal effective concentration in the order of 1 microM. The classical benzodiazepine agonist RU32007 was approx. 10 times more potent. This action of RU33368 was blocked by Ro15-1788. 4. The minimal effective concentration of RU33368 fully blocked the effect of RU32007 in 2 of 4 cells tested and partially antagonized it in a third cell. 5. These data suggest that RU33368 is a partial agonist at benzodiazepine receptors and this, at least in part, explains its non-sedative anxiolytic behavioural profile.

Animals↗
PubMed1991

Receptor sub-types involved in responses of Purkinje cell to exogenous excitatory amino acids and local electrical stimulation in cerebellar slices in the rat.

The effects of the NMDA receptor antagonist, 2-amino-5-phosphonovalerate (APV) and non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on responses of Purkinje cells to exogenous excitatory amino acids and to electrical stimulation of the parallel fibres, were investigated in slices of the cerebellum of the rat. Glutamate, aspartate, kainate and quisqualate all induced excitation of Purkinje cells. Responses to kainate and quisqualate were blocked by CNQX (10 microM) but not by APV (10 microM). N-Methyl-D-aspartate induced biphasic excitatory-inhibitory responses, both components of which were blocked by APV but not by CNQX. The inhibitory component was less sensitive to blockade by APV but was totally blocked by bicuculline, the GABAA receptor antagonist. Parallel fibre stimulation most commonly induced inhibition of Purkinje cells, with or without preceding excitation. This inhibition was blocked by APV and excitatory responses were often revealed. A less commonly-observed predominantly excitatory response was blocked by CNQX but not by APV and inhibition tended to be revealed. These data suggest that parallel fibre-Purkinje cell synapses possess non-NMDA postsynaptic receptors, while the parallel fibre-inhibitory interneuron synapses possess functional NMDA receptors.

2-Amino-5-phosphonovalerate↗
PubMed1991

Modulation of GABA-mediated inhibition in rat cerebellar slices by benzodiazepine receptor ligands.

1. Extracellular recordings were made from the Purkinje cell layer of rat cerebellar slices. Compounds were perfused over the slice and bipolar stimulating electrodes placed in the external layer of the slice close to the recording electrode. 2. Stimulus-evoked inhibition of Purkinje layer cell activity was sensitive to bicuculline methiodide and picrotoxin, suggesting it was gamma-aminobutyric acid (GABA) mediated. The benzodiazepine ligands RU 32007 and Ro 19-0528 reversibly increased the period of inhibition, as did pentobarbital. This benzodiazepine effect was antagonised by Ro 15-1788. 3. Five inverse agonists all reduced the period of stimulated inhibition and this effect was reversed by Ro 15-1788, suggesting the involvement of benzodiazepine receptors. 4. It is concluded that this system provides a convenient physiological and possibly quantitative model for studying the action of benzodiazepine receptor ligands.

Animals↗
PubMed1991

2-(oxadiazolyl)- and 2-(thiazolyl)imidazo[1,2-a]pyrimidines as agonists and inverse agonists at benzodiazepine receptors.

Oxadiazoles, like the benzoyl group in a series of imidazo[1,2-a]pyrimidines, have been found to be metabolically stable alternatives to ester groups in benzodiazepine-receptor ligands. This change has lead to a number of compounds which bind to the receptors and which exhibit potent agonist activity in a food-motivated conflict test thought to predict anxiolytic properties. Compounds 4, 5, and 13 were equipotent with chlordiazepoxide but showed little or no myorelaxant effects. Replacing the oxadiazole group by thiazole gave compounds such as 23 which binds to the benzodiazepine receptor but exhibits the intrinsic activity of a partial inverse agonist in vivo.

Animals↗
PubMed1991

Investigation of the role of metalloproteinases in recombinant human interleukin-1 beta-induced degradation of rat femoral head cartilage.

The mechanism of proteoglycan (GAG) loss from rat femoral articular cartilage (FHC) induced by recombinant human interleukin-1 beta (rhIL-1 beta) in vitro has been investigated. The metalloproteinase inhibitor 1,10-phenanthroline, the serine proteinase inhibitor N alpha-p-tosyl-l-lysine chloromethyl ketone (TLCK), the activator of latent metalloproteinase p-aminophenylmercuric acid (APMA), and an inhibitory metalloproteinase substrate analogue U27391 were tested for their ability to modulate rhIL-1 beta-induced GAG loss and GAG synthesis ([35S]O4 uptake) inhibition. As expected 1,10-phenanthroline inhibited GAG loss, however [35S]O4 incorporation was significantly reduced. TLCK was without effect, and APMA inhibited both parameters. U27391 reversed both the inhibition of [35S]O4 incorporation and GAG loss. It is concluded that the adverse effects on proteoglycan metabolism explain the inhibitory actions of 1,10-phenanthroline and APMA, whilst the action of TLCK may indicate that serine proteinases are not involved in the activation of latent metalloproteinase. U27391 exhibited chondroprotective activity and confirmed the induction of either metalloproteinases such as stromelysin or collagenase by rhIL-1 beta.

Animals↗
PubMed1991

Changes in sulfated proteoglycan production after activation of rat liver macrophages.

Production of extracellular matrix proteins-in particular, the proteoglycans-by macrophages is important in many of their functions, including cell-cell recognition, adhesion and phagocytosis. In this study, we characterized changes in sulfated proteoglycan production by hepatic macrophages following in vivo activation with lipopolysaccharide. We found that both resident Kupffer cells and liver macrophages from lipopolysaccharide-treated rats incorporated [35S]sulfate into proteoglycans. Lipopolysaccharide-activated macrophages incorporated two to three times more of the label than did resident Kupffer cells. In addition, although both cell types produced chondroitin sulfate and heparan sulfate, resident Kupffer cells synthesized more chondroitin sulfate whereas lipopolysaccharide-activated cells produced more heparan sulfate. Using specific antibodies and flow cytometry, we also found that hepatic macrophages produced chondroitin-4-sulfate, chondroitin-6-sulfate and chondroitin-O-sulfate. Lipopolysaccharide-activated macrophages contained more chondroitin-4-sulfate and chondroitin-O-sulfate and less heparan sulfate than did resident Kupffer cells. Both tunicamycin and beta-D-xylosides, inhibitors of sulfated proteoglycan biosynthesis, were found to block phagocytosis by the cells. Taken together, these results suggest that sulfated proteoglycans are important in activation and functional responsiveness of liver macrophages.

Animals↗
PubMed1991

Liver macrophage-mediated cytotoxicity toward mastocytoma cells involves phagocytosis of tumor targets.

Macrophage-mediated cytotoxicity toward tumor cells usually involves extracellular lysis of the targets. In this study, we report that liver macrophages from rats treated with lipopolysaccharide (5 mg/kg, intravenous) also kill certain tumor cell targets by phagocytosis. Liver macrophages were coincubated with P815 mouse mastocytoma cells for 24 to 72 hr at an effector/target ratio of 10:1. Macrophage phagocytosis was characterized by flow cytometry and by light and electron microscopy. For flow-cytometric studies, P815 cells were prelabeled with the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate. We found that coincubation of macrophages with labeled targets resulted in a time-dependent increase in macrophage-associated fluorescence, reaching a maximum at 72 hr. This correlated with light-microscopic observations of increased numbers of tumor cells in the macrophages and enhanced macrophage surface area and density. Electron microscopic studies revealed that the initial event in the phagocytic process involved the capture of P815 cells by the pseudopodia of the macrophages. Target cells were then surrounded by lamellipodia, internalized in phagosomes and destroyed. These data, together with previous studies, provide evidence for multiple mechanisms of cytotoxicity mediated by activated liver macrophages.

Animals↗
PubMed1990

Fluorescence assay for per-cell estimation of cytochrome P-450-dependent monooxygenase activities in keratinocyte suspensions and cultures.

An assay was characterized that facilitated per-cell estimation of cytochrome P-450-dependent monooxygenase activities in whole-cell suspensions and cultures of murine epidermal keratinocytes (MEKs). 7-Ethoxycoumarin O-deethylase (7-ECD), 7-ethoxyresorufin O-deethylase (7-ERD), and 7-pentoxyresorufin O-deethylase (7-PRD) activities were monitored by fluorescent detection of their products. MEKs were made permeable by a freeze-thaw cycle, and xenobiotic metabolism occurred in situ. Analyses of cultured MEKs were made with the cells attached to the culture dishes. Product formation was proportional with MEK cell number and assay time and was dependent upon a NADPH-generating system. The three monooxygenase activities were inhibited to various degrees, in a dose-dependent manner, by the P-450 inhibitors alpha-naphthoflavone and metyrapone. The number of MEKs obtained from a single mouse was sufficient for multiple analyses. The assay was also used to determine monooxygenase activities in whole-cell suspensions of rat hepatocytes. Constitutive per hepatocyte 7-ECD, 7-PRD, and 7-ERD activities were 357-, 96-, and 1926-fold greater, respectively, than the activities measured in suspensions of dorsal MEKs.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1990

Differential interactions of Ro 15-4513 with benzodiazepines, ethanol and pentobarbital.

The effects of the imidazobenzodiazepine Ro 15-4513 in combination with three CNS depressants (ethanol, benzodiazepine agonists and pentobarbital) were examined in three different experiments. Full antagonism of classical benzodiazepines by Ro 15-4513 was seen in all three situations. Partial antagonism of ethanol occurred in the pull up test of muscle relaxation in rats, but not in the inhibition of ultrasounds produced in rat pups by mild stress. The depressant effect of ethanol on twitching of the urethane-anaesthetised rat suprahyoid muscles was reversed. No attenuation of the effects of pentobarbital was seen in any test.

Anesthesia↗
PubMed1990

Quantitative analysis of gamma-aminobutyric acid (GABA) receptors of Purkinje cell layer from rat cerebellar slices.

1. Extracellular recordings were made from Purkinje cell layer of rat cerebellar slices. Cell activity was sensitive to both magnesium and manganese ions. 2. Glutamic and aspartic acids both excited cell activity while gamma-aminobutyric acid (GABA), muscimol, taurine, beta-alanine and delta-aminolaevulinic acid all inhibited activity. 3. The sensitivity to GABA varied with depth while no such effect was observed with muscimol. 4. Bicuculline methiodide, picrotoxin and pitrazepin blocked the action of muscimol with pA2 values of 5.92, 5.97 and 5.71 respectively. 5. The benzodiazepines flurazepam and RU 32007 both potentiated the GABA inhibition and this potentiation was blocked by Ro 15-1788, a benzodiazepine antagonist.

Animals↗
PubMed1989

Discriminative stimulus properties of CL218872 and chlordiazepoxide in the rat.

Rats were trained to discriminate either CL218872 (5 mg/kg PO) or chlordiazepoxide (5 mg/kg PO) from vehicle in a 2-lever discrimination task on an FR20 schedule. The discriminative cues produced by these two drugs generalised to a range of benzodiazepine receptor agonists and partial agonists. Nitrazepam, diazepam, RU32698 and RU32514 were less potent in substituting for the CL218872 cue than the chloridiazepoxide cue. Zopiclone, RU31719 and RU43028 substituted for both cues with similar potency, whilst zolpidem and CL218872 were clearly more potent in substituting for the CL218872 cue. Chlordiazepoxide substituted only partially for the CL218872 cue, even at doses which decreased the rate of responding. CGS9896 substituted partially for both cues, but was less effective with the CL218872 cue. RU39419 substituted for the chlordiazepoxide cue, but antagonised the CL218872 cue. CGS8216 and FG7142 antagonised both cues. The contributions of benzodiazepine receptor subtypes or partial agonism to the generation of the CL218872 cue is discussed.

Animals↗
PubMed1989

The discriminative stimulus properties of the 5HT1 agonist RU24969.

Using standard operant procedures, rats were trained to discriminate the 5-HT agonist RU24969 (0.5 mg/kg IP) from saline. Stable responding was established and tests of stimulus generalisation and stimulus antagonism were performed with a range of 5-HT receptor ligands. The RU24969 cue was selective as neither 5-HT receptor ligands MK212, DPAT, ipsapirone, GR38032F or the 5-HT releasing agent, fenfluramine nor yohimbine were able to substitute for RU24969 in tests of generalisation. However, TFMPP, CPP and, of particular interest, propranolol substituted for the RU24969 stimulus, although full substitution only occurred with doses that disrupted responding. The RU24969 stimulus was not antagonised by propranolol, metergoline, ritanserin or GR38032F. This pharmacological profile suggests that the RU24969 stimulus is mediated via a subtype of 5-HT1 sites different from 5-HT1A and that propranolol may be an agonist at this site.

Animals↗
PubMed1989

Controlled gastric emptying. II. In vitro erosion and gastric residence times of an erodible device in beagle dogs.

An erodible gastric retention device fabricated from various polymeric blends was examined in vitro for its dissolution properties and in vivo in fasting dogs for assessment of its gastric retention potential. Dissolution studies were conducted with extruded rods of polymer blends to assess their potential as candidates for the erodible component of a gastrically retained device. Based on results from dissolution studies, rods of poly(ortho ester)/polyethylene blends (POE/PE) (45% erosion at pH 1.5 and 24 hr) were used to fabricate arms for tetrahedron-shaped devices. Corners for the tetrahedral device were fabricated from Silastic 382 loaded with 15% barium sulfate for X-ray visualization. Beagle dogs were dosed with tetrahedron-shaped test devices administered in gelatin capsules and gastric retention monitored by X ray over a 24-hr period. A comparison of in vitro erosion rates and in vivo performance of various polymer blends indicated a definite trend for increased gastric retention of devices made from the more slowly eroding blends. The results indicate that the blending of erodible and nonerodible polymers is a valid approach for obtaining materials that will provide the necessary structural properties to achieve gastric retention yet lose integrity within a desired time.

Animals↗
PubMed1988

Potential use of drugs modulating 5HT activity in the treatment of anxiety.

1. It has been long suggested that central 5HT-mediated systems may be involved in modulation of anxiety and in the anxiolytic effect of benzodiazepines. However, recent evidence has questioned this hypothesis, particularly with respect to the mode of action of benzodiazepines. 2. Development of 5HT agonists and antagonists selective for different 5HT receptor sub-types (5HT1A, 5HT1B, 5HT2, 5HT3) has opened a new avenue for investigation of the potential role of 5HT in anxiety. 3. Buspirone is clinically active in the treatment of anxiety and it, and other anxiolytic candidates, gepirone and isapirone, may act as agonists (or perhaps partial agonists) on 5HT1A receptors. 4. The prototype 5HT1A agonist 8OH-DPAT may also have potential anxiolytic effects. 5HT1A agonists may act to suppress the activity of 5HT neurones as a major part of their action. 5. Although there is some supporting evidence, there is no clear indication of anxiolytic activity with agonists with some selectivity for 5HT1B sites (RU24969, mCPP, TMPP). 6. A selective 5HT2 antagonist, ritanserin, has anxiolytic effects in clinical studies but, like the 5HT1A agonists, does not show a similar profile to benzodiazepines in models of anxiety. 7. This raises the question of clinical predictivity of the various models used. 8. A recently developed 5HT3 antagonist, GR38032F, has been claimed to possess potential anxiolytic activity but its mode of action in this respect requires further elucidation.

8-Hydroxy-2-(di-n-propylamino)tetralin↗