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Biomedical subjects

R T Fletcher

Publications and source records attributed to R T Fletcher.

At least 37 records · Page 2Linked to original sources
PubMed1984

Effects of diamide on cyclic nucleotide levels in rat retina.

The concentrations of guanosine 3',5' monophosphate (cyclic GMP) and adenosine 3',5' monophosphate (cyclic AMP) were measured in rat retinas incubated under control conditions and in retinas incubated with diamide, a relatively specific glutathione oxidant. Retinas incubated with either glucose or pyruvate as the substrate for 30 min in the dark contained about 50 picomoles cyclic GMP/mg protein and 7 picomoles cyclic AMP/mg protein. Light-exposed control retinas contained about 70% less cyclic GMP (14.4 picomoles/mg protein) and 20% less cyclic AMP (5.4 picomoles/mg protein). Addition of diamide to the incubation medium at concentrations between 0.1 and 1.0 mM produced a concentration-dependent decrease in the dark level of cyclic GMP, but did not affect its concentration in the light or the concentration of cyclic AMP in dark-maintained and light-exposed retinas. The major effect of diamide was to reduce the normal dark-light difference in the concentration of cyclic GMP. Oxidizing conditions thus appear to alter selectively the light-sensitive compartment of retinal cyclic GMP.

Animals↗
PubMed1982

Retinal pigment epithelium contains a distinctive strychnine-binding site.

Membranes prepared from the retinal pigment epithelium of several species possess a specific site which binds [3H]strychnine. This binding has a somewhat lower affinity and a much greater density than the corresponding interaction in the hindbrain or neural retina. Binding is not greatly altered in the presence of 10(-3) M glycine, L-alanine, beta-alanine, taurine, or serine. Thus, the receptor does not resemble the classical glycine receptor of the hindbrain and spinal cord. This new type of binding site appears to be confined to the pigment epithelial layer of the retina.

Amino Acids↗
PubMed1982

Induction of dark-adaptive retinomotor movement (cell elongation) in teleost retinal cones by cyclic adenosine 3','5-monophosphate.

In the teleost retina, the photoreceptors and retinal pigment epithelium (RPE) undergo extensive movements (called retinomotor movements) in response to changes in light conditions and to an endogenous circadian rhythm. Photoreceptor movements serve to reposition the light-receptive outer segments and are effected by changes in inner segment length. Melanin granule movements within the RPE cells provide a movable melanin screen for rod outer segments. In the dark (night), cones elongate, rods contract, and pigment granules aggregate to the base of the RPE cell; in the light (day), these movements are reversed. We report here that treatments that elevate cytoplasmic cyclic adenosine 3',5'-monophosphate (cAMP) provoke retinomotor movements characteristic of nighttime dark adaptation, even in bright light at midday. To illustrate this response, we present a quantitative description of the effects of cyclic nucleotides on cone length in the green sunfish, Lepomis cyanellus. Cone elongation is induced when light-adapted retinas are exposed to exogenous cAMP analogues accompanied by phosphodiesterase (PDE) inhibitors (either by intraocular injection or in retinal organ culture). Cone movements is not affected by cyclic GMP analogies. Dose-response studies indicate that the extent, but not the rate, of cone elongation is proportional to the concentration of exogenous cAMP and analogue presented. As has been reported for other species, we find that levels of cAMP are significantly higher in dark- than in light-adapted green sunfish retinas. On the basis of these observations, we suggest that cAMP plays a role in the light and circadian regulation of teleost cone length.

Animals↗
PubMed1980

Differential control of protein kinase activities of the retinal photoreceptor. Cation effects on phosphorylation by adenosine and guanosine 5'-triphosphates.

ATP-kinase activity in photoreceptor membranes is maximal at equimolar amounts of ATP and Mg2+. GTP-kinase activity is maximal with high concentrations of Mn2+. Under these conditions, calcium ion markedly inhibits phosphorylation with ATP but not with GTP. GTP-kinase activity is maximal at pH 6.5 and decreases at higher pH; little change is seen in ATP-kinase activity over the pH range of 6.0-7.5. GTP-kinase activity is inhibited by ATP and other adenine compounds; ATP-kinase activity is less sensitive, although I mM adenosine decreases activity by about threefold. No marked differences in the protein moieties phosphorylated by ATP or GTP were seen in bovine or frog outer segment membranes. Fluxes in cation and metabolite concentrations as well as availability of ATP and GTP could thus exert a major influence on protein kinase activity in the photoreceptor unit, affording the possibility of differential phosphorylation under various physiological conditions.

Adenosine↗
PubMed1980

Cyclic nucleotides vary by area in the retina and pigmented epithelium of the human and monkey.

Cyclic GMP and cyclic AMP are present in lower concentrations in the central (macular) region of the neural retina of the human and monkey than in other areas. This pattern approximates the distribution of rod photoreceptor cells. Surprisingly, an inverse gradient of cyclic GMP concentration is observed in the pigmented epithelium. Levels in the central region are over fourfold higher than in cells in the periphery, offering the first evidence of biochemical differences in this embryologically uniform cell type.

Adolescent↗
PubMed1978

Cyclic nucleotides and protein kinase systems in the developing chick retina and pigment epithelium.

During embryonic development of the chick neural retina, cyclic nucleotide levels are relatively uniform but rise abruptly at the time of hatching. The rise is thus not temporally correlated with features of morphological development such as outer segment elongation but rather with the onset of actual visual function. In the pigment epithelium, the cyclic AMP level declines throughout the embryonic period studied and does not rise at hatching. Cyclic GMP levels are much lower in both retina and pigment epithelium but rise several-fold at hatching. A binding protein is observed for cyclic AMP in the retina prior to outer segment development; cyclic GMP binding is considerably lower. Retinal ATP-kinase activity is high throughout the embryonic period studied and is stimulated up to 6-fold by 1 muM cyclic AMP and by 100 muM cyclic GMP. The major rise in GTP-kinase activity correlates temporally with photoreceptor outer segment development and may be involved intimately in the visual process.

Animals↗
PubMed1978

Rod-cone dysplasia in Irish setters: a defect in cyclic GMP metabolism in visual cells.

An abnormality in retinal guanosine 3,5-monophosphate (cyclic GMP) metabolism is demonstrated in the inherited rod-cone dysplasis of Irish Setter dogs. Affected visual cells are deficient in cyclic GMP phosphodiesterase activity and have elevated levels of cyclic GMP. The biochemical abnormalities observed in affected retinas of Irish Setters are similar to those in the retinas of mice with inherited retinal degeneration before visual cell degeneration begins. A defect in cyclic GMP metabolism may be characteristic of early-onset degenerative diseases of the retina, possibly including those that affect humans.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1978

Characterization of guanylate cyclase of rod outer segments of the bovine retina.

Guanylate cyclase (GTP pyrophosphate-lyse (cyclizing), EC 4.6.1.2.) of bovine retinal rod outer segments is almost completely particulate, i.e. associated with rod outer segment membranes. In contrast to particulate guanylate cyclase in other tissues, treatment of rod outer segments with Triton X-100 does not solublize the enzyme but inhibits it. Enzyme activity is dependent on the presence of divalent cation, especially Mn2+ with only poor activation by Mg2+ (10-fold lower) and no activation seen with other cation. Ezpression of maximal activity required Nm2+ and GTP in equimolar concentrations with an apparent Km of 8 . 10(-4) M and V of 10 nmol/min per mg protein. Excess of Mn2+ over that required for the formation of the Mn . GTP complex was inhibitory. Ca2+, Ba2+ and Co2+ inhibited enzyme activity when assayed with the Mn . GTP substrate complex. In the presence of a fixed concentration of 1mM Mn2+, the enzyme exhibited strong negative cooperative interactions with GTP, characterized by an intermediary plateau region in the substrate vs. enzyme activity curve, a curve of downward concavity in the double reciprocal plot and a Hill coefficient of 0.5. Nucleotides such as ITP, ATP and UTP at higher concentrations (1 mM) stimulates activity by 40%. NaN3 has no effect on the guanylate cyclase. It is thus possible that the guanylate cyclase may be regulated in vivo by both the metal : GTP substrate ratio and the free divalent cation concentration as well as by the ATP concentration and thus play an important but yet undefined role in the visual process.

Animals↗
PubMed1976

Differential phosphorylation by GTP and ATP in isolated rod outer segments of the retina.

Isolated bovine rod outer segment protein is phosphorylated with GTP-gamma-32P and ATP-gamma 32P and to a much lesser extent by CTP-gamma-32P and UTP-gamma-32P. Phosphorylation with both GTP (GTP-kinase activity) and ATP (ATP-kinase activity) is markedly stimulated by light; phosphorylation with GTP is lower in dark-adapted and higher in light-adapted rod outer segments than is phosphorylation with ATP. Km values of 20 and 200 muM and Vmax values of 2.1 and 5.9 nmol/(mg min(-1)) were calculated using ATP and GTP, respectively, in light-adapted outer segments. When outer segments are incubated with GTP-gamma-32P under the usual conditions employed in these experiments, no formation of ATP-gamma-32P was detected by the techniques of high-pressure liquid chromatography and thin-layer chromatography. In intact, light-bleached outer segments, GTP appears to specifically phosphorylate rhodopsin. Histone and phosvitin are not phosphorylated to any appreciable extent by GTP. Histone appears to block rhodopsin phosphorylation by GTP while histone and, to some extent, phosvitin, both act as substrates for ATP-kinase activity. Cyclic AMP and other adenine derivates have a marked inhibitory effect on GTP-kinase activity. Phosphate also inhibits GTP-kinase activity but stimulates ATP-kinase activity. Such differences in phosphorylation with GTP and ATP indicate that these activities are either due to separate enzyme systems or, if only one enzyme is involved, the activities are under separate physiological control in the photoreceptor unit.

Adenosine↗
PubMed1975

Studies on the differentiation of retinal pigmented epithelium cells in culture.

Sephadex G-25 fractions of chick embryo extract affect the differentiation of cloned embryonic pigmented epithelial cells (PE) in culture. Both tyrosinase activity and amounts of melanin are markedly affected by the extract fractions. 5-Hydroxyindole decreased melanin accumulation in PE cells while alpha-methyl-p-tyrosine had no apparent effect on the process. Melanin disposal as well as biosynthesis may be important in controlling the accumulation of pigment in PE cells under culture conditions. Triiodothyronine greatly increased visible pigmentation and affected the morphology of cultured PE cells.

Animals↗