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N N Osborne

Publications and source records attributed to N N Osborne.

At least 73 records · Page 4Linked to original sources
PubMed1997

Pharmacologic evidence for 5-HT1A receptors associated with human retinal pigment epithelial cells in culture.

PURPOSE: The authors investigate the possible presence of 5-hydroxytryptamine (5-HT1) type serotonin receptors negatively coupled to adenylate cyclase activity in cultured human retinal pigment epithelial (RPE) cells. METHODS: Adenylate cyclase activity was assessed by the determination of cellular adenosine 3':5' cyclic monophosphate (cAMP) levels and the effects of serotonin on both basal and forskolin-stimulated cAMP levels studied. RESULTS: Serotonin at 100 microM had no effect on the basal levels of cAMP in cultured human RPE cells, but attenuated by 43.6% the stimulation in cAMP production induced by forskolin (5 microM). This effect was dose dependent for serotonin with half-maximal inhibition (EC50) occurring at approximately 1.5 x 10(-9) M. The 5-HT1 receptor agonist 8-hydroxy [2-di-n-propylamino] tetralin (8-OH DPAT), buspirone, 5-carboxyamidotryptamine, and RU24969 mimicked the inhibitory effect of serotonin in a dose-dependent manner. The actions of serotonin and 8-OH DPAT (10 microM) were dose-dependently attenuated by the serotonergic antagonists spiroxatrine, propranolol, and spiperone. Pretreatment of RPE cell cultures with pertussis toxin abolished the serotonin-induced reduction of forskolin-elevated cAMP levels. Stimulation of cAMP production by the beta-adrenoceptor agonist isoproterenol at 0.1 microM, but not at 10 microM or 100 microM, also was attenuated by serotonin (100 microM), whereas cAMP production induced by the adenosine receptor agonist 5'-[N-ethyl]-carboxamidoadenosine (NECA) at 1 microM, 10 microM, and 100 microM was unaffected. Serotonin and 8-OH DPAT dose-dependently inhibited isoproterenol-stimulated (0.1 microM) cAMP production with EC50 values of approximately 10 microM, and pertussis toxin pretreatment partially blocked these effects. CONCLUSIONS: Cultured human RPE cells possess 5-HT1A receptors negatively coupled to cAMP production through a pertussis toxin-sensitive G protein. These receptors show differential effects on forskolin-, isoproterenol-, and NECA-stimulated cAMP production, which may reflect a unique spatial distribution of receptor proteins or the phenotypic heterogeneity of RPE cells that is the result of or that is preserved in culture.

Adenylate Cyclase Toxin↗
PubMed1997

Induction of apoptosis in cultured human retinal pigment epithelial cells is counteracted by flupirtine.

PURPOSE: The aim of the study was to determine whether flupirtine can counteract the induction of apoptosis in cultured retinal pigment epithelium (RPE) cells. METHODS: Confluent cultures were subjected to experimental ischemia (medium free of serum, glucose, and oxygen) with or without various substances for specific periods. The cells were then examined for breakdown of DNA by the TUNEL procedure and agarose gel electrophoresis. Moreover cells were processed for the localization of oncogene proteins (bcl-2, TIAR, ICH-1t) associated with apoptosis. The effect of flupirtine on reactive oxygen species also was determined. RESULTS: When RPE cells were subjected to ischemia for 72 hours approximately 65% of cells remained attached to the coverslips and approximately 65% of their nuclei showed clear fragmentation of DNA by TUNEL. Most of the cells exhibited a shrunken appearance typical of apoptosis. Fragmentation of the DNA from cells given ischemia for 72 hours was also confirmed by agarose gel electrophoresis. Inclusion of flupirtine (flupirtine gluconate, 100 microM) or 10% fetal calf serum in the medium prevented ischemia-induced apoptosis occurring after 72 hours. Neither N-methyl-D-aspartate (NMDA) (100 microM) nondeferoxamine (100 microM) nor the NMDA antagonists dextromethorphan (100 microM), memantine (100 microM), and MK-801 (10 microM) had a similar effect. NMDA, and to a lesser extent memantine, induced apoptosis independently. Treatment of RPE cells in serum-free medium with flupirtine (flupirtine gluconate, 100 microM) for 72 hours caused an upregulation of bcl-2 protein. In contrast, the oncogene proteins for TIAR and ICH-1t, were lower in flupirtine-treated cells than in control cells. Flupirtine, like deferoxamine, prevents iron-ascorbate-induced reactive oxygen species formation in retinal cells, but only flupirtine prevents ischemia-induced apoptosis in RPE cells. CONCLUSIONS: The combined data demonstrate that flupirtine is an effective agent in preventing death by apoptosis. Flupirtine reduces formation of reactive oxygen species in retinal dissociates and causes changes in various oncogene products in RPE cultures, which may explain its action in preventing apoptosis induced by ischemia. The current results also suggest that NMDA receptors are not involved in the induction of ischemia-induced apoptosis in RPE, cells.

Aged↗
PubMed1996

The ocular blood flow tonograph: a new instrument for the measurement of intraocular pressure in rabbits.

The Ocular Blood Flow Tonograph (OBFT) and Digilab Pneumatonograph (PTG) were calibrated for use on New Zealand White rabbits using a pressure manometer and performing tonometry at varying known pressures. The calibration experiments showed that a good correlation exists between OBFT measurements and manometric readings of IOP. Moreover, no difference between open and closed stopcock measurements were observed. Corrective equations for conversion of OBFT readings to mm Hg are documented. The OBFT was compared with the PTG and the results showed there was no significant difference between the IOP measured by either instrument in a group of rabbits displaying a range of different IOPs. Regression analysis of the data gave a slope of 0.88 and a correlation co-efficient of 0.78. The OBFT proved easier to use than the PTG under restricted lighting conditions. The OBFT was also used to measure the circadian variation in IOP and to measure the IOP after the topical application of prostaglandin E2 and various serotonergic agonists. The combined data demonstrated that the OBFT is an accurate and reliable tonometer for the measurement of IOP in rabbits.

Animals↗
PubMed1996

The occurrence of protein kinase C theta and lambda isoforms in retina of different species.

The localization and immunochemical identification of the novel protein kinase C theta (nPKC theta) and the atypical protein kinase C lambda (aPKC lambda) isoforms in retinas of different species were analyzed by immunohistochemistry and SDS-PAGE/Western blotting. nPKC theta immunoreactivity is associated with bipolar cells of mammalian (rabbit, rat and guinea pig) retinas but not the non-mammalian goldfish retina which has a lower concentration of nPKC theta. However, SDS-PAGE and Western blotting data indicate the antigen recognized by the nPKC theta monoclonal antibody in the retina is of a lower molecular weight than that expected for nPKC theta. This would suggest nPKC theta is more susceptible to degradation/breakdown than other PKC isoforms found in the retina or that the nPKC theta antibody may be recognizing an unknown retinal antigen. A comparison of nPKC theta and cPKC alpha immunoreactivities in bipolar cells shows unique distributions exist for the two isoforms. nPKC theta is present in the developing retina at an earlier stage than cPKC alpha. The typical 'transport' of cPKC alpha toward axonal terminals by phorbol-12,13-dibutyrate does not occur for nPKC theta yet both are translocated from the cytosolic to membrane compartments. The inner plexiform layer and the inner nuclear layer (putative horizontal cells) of all species examined (rabbit, rat, guinea pig and goldfish) exhibited positive immunoreactivity for aPKC lambda as confirmed by SDS-PAGE/Western blotting.

Animals↗
PubMed1996

Antigens associated with specific retinal cells are affected by ischaemia caused by raised intraocular pressure: effect of glutamate antagonists.

Raising the rat's intraocular pressure above the systolic blood pressure for 60 min followed by a reperfusion of 7-10 days caused an ischaemic insult to the retina. The b-wave of the electroretinogram was abolished and the retinal thickness was much reduced, the greatest influence being associated with the inner retinal layers. No obvious histological damage was apparent at the light microscopy level. Immunocytochemistry, however, revealed a clear change in the nature of certain antigens associated with specific cell-types. Thy-1 antigen located to ganglion cell membranes was much reduced, suggesting that the ganglion cells are affected by ischaemia. Calretinin-immunoreactivity associated with amacrine cells is drastically reduced by ischaemia. In contrast, Ret-P1, located to the outer segments of the photoreceptors is unaffected by ischaemia. Ischaemia also caused GFAP-immunoreactivity to be expressed in the Müller cells, which is normally only associated with astrocytes in the ganglion/nerve fibre layer. Injection of a mixture of CNQX and MK-801, kainate and NMDA receptor antagonists, respectively, into the eye just before ischaemia failed to reverse the changes induced by the insult. However, analysis 3 days after reperfusion revealed that when the ischaemic insult was reduced to 45, rather than 60 min, the changes in the calretinin-immunoreactivity were reversed. The results show that immunocytochemistry provides a powerful way of following biochemical changes associated with specific cell types caused by ischaemia.

Animals↗
PubMed1996

Do beta-adrenoceptors and serotonin 5-HT1A receptors have similar functions in the control of intraocular pressure in the rabbit?

Serotonin 5-HT1A-type and beta-adrenergic receptors have similar molecular characteristics, which explains why certain substances like propranolol have affinities for both receptor types. Activation of 5-HT1A receptors leads to a decrease in intracellular cAMP levels whereas the opposite occurs for beta-adrenergic receptors. Both 5-HT1A and beta-adrenergic receptors exist in the rabbit ciliary processes. Topical application of the 5-HT1A agonist, 8-hydroxydipropylaminotetralin (8-OH-DPAT) reduces intraocular pressure (IOP). beta-Antagonists are also known to reduce IOP. It is postulated that substances which have an affinity for 5-HT1A and/or beta-adrenergic receptors and lead to a reduction in intracellular cAMP levels in the ciliary processes diminish IOP.

Adrenergic beta-Antagonists↗
PubMed1996

Protection of rabbit retina from ischemic injury by flupirtine.

PURPOSE: The aim of this study was to determine wether flupirtine can slow down the changes seen in the rabbit retina after ischemia--reperfusion. METHODS: A suction-cup procedure, which raises intraocular pressure, was used to give an ischemic insult to the rabbit retina. Electroretinograms were recorded before ischemia and at different periods after ischemia. In some instances, flupirtine was injected into the eye before ischemia. Immunohistochemistry was used to study the effect of ischemia-reperfusion on the gamma-aminobutyric acid (GABA) immunoreactivity and uptake of serotonin by the retina. The effect of flupirtine and ischemia on retinal adenosine triphosphate (ATP) levels were determined in in vivo and in vitro experiments. RESULTS: Ischemia for 75 minutes causes a change in the nature of normal GABA immunoreactivity and reduction in the b-wave of the electroretinogram. When flupirtine is injected into the vitreous humor at the onset of ischemic insult, the changes in GABA immunoreactivity are reduced and the recovery of the reduced b-wave of the electroretinogram after defined reperfusion times is enhanced significantly. Rat retinas incubated in vitro in physiological solution containing flupirtine caused a significant rise in the tissues' ATP content compared with control samples. However, incubation of the tissue in physiological solution saturated with nitrogen caused a drop in retinal ATP levels. Addition of flupirtine prevented this decrease from taking place. Serotonin injected into the vitreous humor of the rabbit eye is taken up by certain amacrine cells. The amount of serotonin taken up is reduced greatly in retinas, as judged by immunohistochemistry when tissues are subjected to ischemia. Because the ischemia was shown to cause a drop in the tissue ATP level, it is concluded that this is the cause of the reduced uptake of exogenous serotonin. Injection of flupirtine into the vitreous humor during ischemia enhanced the uptake of serotonin. CONCLUSIONS: Combined data show that flupirtine is a neuroprotective agent in retinal ischemia and that one mode of its mechanism of action is to influence ATP levels. Flupirtine may lower the activity of NMDA receptors, thus causing ATP levels to be less affected in the presence of the drug as a secondary effect.

Adenosine Triphosphate↗
PubMed1996

Involvement of adenosine in retinal ischemia. Studies on the rat.

PURPOSE: The aim of this study was to determine whether adenosinergic agents can be used to slow down the changes seen in the rat retina after ischemia-reperfusion. METHODS: Ischemia-reperfusion injury to the rat retina was induced by raising the intraocular pressure above the systolic blood pressure for 45 minutes, followed by reperfusion for 3 days. This insult caused a reduction of the b-wave of the electroretinogram (54% +/- 5%, n = 23) relative to the contralateral control retina, an expression of glial fibrillary acidic protein (GFAP) in the Müller cells, and an alteration in the "staining" pattern of the calretinin immunoreactivity. The normal two to three bands of calretinin immunoreactivity in the inner plexiform layer appeared as a single band. Elevation of the intraocular pressure for 60 minutes, followed by reperfusion of 2 weeks, caused a 40% reduction in the thickness of the inner nuclear and plexiform layers. No statistically significant changes in the other retinal layers were recorded. RESULTS: When the adenosine deaminase inhibitor erythro-9-(2-hydroxyl-3-nonyl)adenine (EHNA) was injected into the eye just before ischemia, the ischemia-reperfusion changes in the b-wave and calretinin immunoreactivity were largely prevented. Similar results were observed when the adenosine A1 receptor agonist, R-N6-(2-phenylisopropyl)adenosine (R-PLA), was administered intraperitoneally just before ischemia. Injection of adenosine deaminase into the eye before ischemia seemed to potentiate the ischemia-reperfusion effect because the reduction of the b-wave was almost complete (8% +/- 4%, n = 6). The ischemia-reperfusion-induced expression of GFAP in the Müller cells was not reduced by any of the adenosinergic agents tested. This suggests that GFAP expression in the Müller cells is not related to a reduction in the b-wave. An injection of EHNA into the eye before ischemia reduced the thinning of the inner plexiform and nuclear retinal layers so that no significant difference between them and the control retinas existed. However, an injection of R-PIA just before ischemia did not reduce the thinning of the retinal layers in a statistically significant way, possibly because the R-PIA protective effect is less than that of EHNA and is difficult to detect when thickness of the retinal layers is measured. It may be necessary to use higher concentrations of R-PIA to observe a protective effect. CONCLUSIONS: The combined data show that substances resulting in the activation of adenosine A1 receptors protect the retina against changes induced by ischemia-reperfusion.

Adenine↗
PubMed1995

Redistribution of GABA immunoreactivity following central retinal artery occlusion.

gamma-Aminobutyric acid (GABA) is normally primarily in amacrine cells in the rat retina. Immediately after an ischaemic insult, attained by occlusion of the central retinal artery for 60 min, GABA is then found to be associated with Müller cells. During subsequent reperfusion, the distribution of GABA immunoreactivity gradually reverts from the glial cells back into neuronal elements of the retina. Twenty-four hours after ischaemia, GABA staining is indistinguishable from that seen in control animals. It is suggested that during central retinal artery occlusion, Müller cell energy levels are sufficient to allow the active uptake of released GABA, but insufficient to metabolise it to glutamine. The normal cycle of GABA metabolites from Müller cells to neurones is thus inhibited. Restoration of blood flow and the consequent increase in retinal energy levels, as indicated by a slight recovery of the electroretinogram b-wave, facilitates glutamine shunting between glial cells and amacrine cells, resulting in the synthesis of neuronal GABA.

Animals↗
PubMed1995

The effect of kainate on protein kinase C, GABA, and the uptake of serotonin in the rabbit retina in vivo.

The purpose of this study was to investigate the effect of kainate on protein kinase C (PKC), gamma-aminobutyrate (GABA) and serotonin uptake in the rabbit retina. Kainate when injected into the vitreous humour produces a change in the GABA immunoreactivity within 6 hours. After 3 days, remnants of the normal GABA immunoreactivity still persist and additionally astrocyte and microglia-like elements "stain" positively for GABA. After 7 days exposure to kainate none of the normal GABA immunoreactivity is apparent, instead a number of round-shaped elements which may be reactive astrocytes and/or microglia stain positively for GABA. During these stages kainate does not affect the alpha PKC immunoreactivity associated with the on-bipolar cells. Six hours following kainate treatment the ability of certain GABA amacrine cells to take up exogenous serotonin is unaffected. After three days only a few of these cells can still take up exogenous serotonin and then not avidly. After seven days the GABA/serotonin amacrine cells cannot take up exogenous serotonin suggesting that all of these neurons are irreversibly damaged. One hour after treatment with kainate both calcium-dependent and -independent PKC species are translocated from the cytosolic to membrane compartments. After 5 hours and 7 days there was also evidence from the enzyme assay experiments that kainate caused the calcium-dependent and -independent PKC enzymes to be translocated but because the total enzyme activity was reduced due perhaps to down-regulation of the enzyme this was difficult to assess precisely.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

The influence of experimental ischaemia on protein kinase C and the GABAergic system in the rabbit retina.

Pressure-induced ocular ischaemia followed by 25-28 hr of reperfusion to the rabbit retina drastically reduces or eliminates the b-wave of the electroretinogram and results in all the GABA from the amacrine cells being released, as judged by immunohistochemistry. Some of these GABA cells have the capacity to take-up exogenous serotonin and these GABA/serotonin cells have kainate/AMPA receptors. Previous studies have shown that an ischaemic insult causes these receptors to be stimulated to produce a release of the cells' GABA. The majority of the GABA/serotonin cells are also incapable of taking-up exogenous serotonin after ischaemia, which suggests that they are irreversibly damaged. However, there was still a minority of the cells which accumulated serotonin, which shows that neurones containing kainate/AMPA receptors are not irreversibly damaged at the same rate by ischaemia. The "staining" patterns for GABAA-receptor and GABA immunoreactivities in the rabbit retina are very similar and following ischaemia the GABAA-receptor immunoreactivity was reduced in intensity and became patchy in nature. It is not known whether this result reflects a down-regulation of the GABAA-receptors caused by the released GABA or a destruction of cells containing the GABAA-receptors. The ischaemic conditions used caused patchy, irregular and inconsistent signs of histological damage to the retina, even in areas of similar eccentricity, suggesting this parameter should be used with caution when judging the severity of an ischaemic insult. alpha-Protein kinase C (alpha PKC) present in the on-bipolar cells which have glutamate metabotropic APB receptors is both reduced or down-regulated and translocated by ischaemia. This is also the case for delta PKC which is absent from the on-bipolar cells. These data were established by a combination of immunohistochemistry and electrophoresis/blotting experiments. Enzyme analysis also showed that all PKC calcium-dependent and -independent isoenzymes, are translocated and reduced by ischaemia making it difficult to judge whether PKC inhibitors may be appropriate anti-ischaemic agents.

Animals↗
PubMed1995

Agonist-induced effects on cyclic AMP metabolism are affected in pigment epithelial cells of the Royal College of Surgeons rat.

Recent work has demonstrated that stimulation of cAMP production via A2-adenosine receptors is reduced in cultured retinal pigment epithelial cells from the RCS rat. Cultured rat RPE cells are also shown to possess beta 2-adrenergic receptors positively coupled to cAMP production. Isoproterenol and salbutamol both stimulate cAMP levels with half maximal (EC50) values of 0.5 and 0.2 microM, respectively. Isoproterenol action is attenuated most effectively by the beta 2-antagonist, ICI 118551, while the beta 1-antagonist, CGP 20712A, is only partially effective. Isoproterenol-stimulated cAMP production is markedly reduced in the RCS rat RPE when compared to control cultures. In passaged RCS rat RPE cells cAMP stimulation by 10 microM isoproterenol was 6.4% of that by control cultures and in primary cultures it was around 75% of controls. The observed EC50 values were 0.4 and 1.3 microM for passaged control and RCS rat RPE cells, respectively. Melatonin negatively influences cAMP production in the RPE via Gi-proteins. Melatonin attenuated the action of forskolin by 51.1% in control rat RPE but only by 18.6% in the RCS rat RPE. The dose-response curve for melatonin shows an approximate 1000-fold shift in potency in the RCS rat. bFGF also has an inhibitory effect on rat RPE cells. bFGF (50ng/ml) attenuated forskolin-stimulated cAMP levels by 61.9% in control rat RPE but had not effect on the action of forskolin in RCS rat RPE. Serotonin (100 microM) potentiates the forskolin-induced stimulation of cAMP by 140.1%. However, unlike isoproterenol, melatonin and bFGF, the action of serotonin on adenylate cyclase appears normal in the RCS rat RPE. We conclude that the defect in the RCS rat RPE is likely to be due to impaired coupling of the components of the adenylate cyclase system and that this is most probably an abnormal interaction of adenylate cyclase with G-protein alpha-subunits.

Adrenergic beta-Agonists↗
PubMed1995

Prolonged bilateral carotid artery occlusion induces electrophysiological and immunohistochemical changes to the rat retina without causing histological damage.

Reduction of the retinal blood flow by occlusion of both common carotid arteries suppressed the b-wave of the rat's electroretinogram. Transient occlusion of the carotids for 45 min reduced the b-wave by 46% without affecting the amplitude of the a-wave. The normal ERG activity returned 30 min after restoration of blood flow. Prolonged carotid occlusion for 7 days totally abolished the b-wave but enhanced the a-wave amplitude. Although b-wave amplitude suppression has been considered as an indicator of retinal ischaemia, no histological changes were seen in retinas of rats subjected to 45 min or 7 days of two-vessel occlusion, when observed by light microscopy. Moreover, GABA, GABAA receptor, calretinin and PKC-alpha immunoreactivities were unaltered. Carotid artery occlusion did, however, induce the expression of the cytoskeletal protein, glial fibrillary acidic protein (GFAP), in retinal Müller cells. The increase in the Müller cell GFAP immunoreactivity was related to how long the carotids were occluded as well as the reperfusion time. Prolonged occlusion for 7 days resulted in a 356% increase in retinal GFAP. These findings show that a reduction of retinal blood flow by occlusion of the carotids causes a metabolic stress to the retina and elicits events associated with gliosis without resulting in 'ischaemic-like' morphological changes.

Animals↗
PubMed1995

Characteristics of [3H]5-hydroxytryptamine binding to iris-ciliary body tissue of the rabbit.

PURPOSE: This study sought to identify, characterize, and localize subtypes of 5-hydroxytryptamine (HT) receptors in rabbit iris-ciliary body. METHODS: Radioligand binding assays were performed with [3H]5-hydroxytryptamine on membranes prepared from rabbit iris-ciliary bodies and on tissue sections subsequently developed by autoradiography. RESULTS: [3H]5-HT appeared to bind to a single population of receptors in membrane preparations of rabbit iris-ciliary body. The apparent affinity of the ligand (KD) was 2.19 nM, and the density of binding sites was 58.3 fmol/mg protein. Binding of [3H] 5-HT exhibited guanosine-5-triphosphate sensitivity. Competitive inhibition experiments were performed to differentiate between 5-HT receptor subtypes. A relative potency order of 5-CT > 5-HT = 8-OH-DPAT > ipsapirone > RU24969 > sumatriptan > ritanserin > ketanserin was demonstrated. The apparent inhibitory constants for the ligands tested fit with the profile expected of binding to 5-HT1A receptors. Inhibition studies with [3H] 5-HT plus 100 nM 8-OH-DPAT (which inhibits binding to 5-HT1A receptors only) representing total binding, indicated that no further displacement occurred when ligands preferentially selective for 5-HT1B, 5-HT1D alpha,1D beta, or 5-HT2C were tested. Total binding of [3H] 5-HT in tissue sections developed by autoradiography was displaced completely by 100 nM 8-OH-DPAT. Melatonin showed little affinity for the [3H] 5-HT binding sites. CONCLUSIONS: A population of 5-HT1A receptors is present in rabbit ciliary processes. There is no evidence to suggest the presence of 5-HT1D alpha, 5-HT1D beta, 5-HT2B, or 5-HT2C receptors in the iris-ciliary body.

Animals↗
PubMed1995

Influence of excitatory amino acids and ischemia on rat retinal choline acetyltransferase-containing cells.

PURPOSE: To compare the effects of glutamate agonists and different types of ischemic insult on choline acetyltransferase (ChAT) immunoreactivity in the rat retina. METHODS: Rat retinas were exposed to different glutamate agonists in vivo or in vitro for specific periods of time, and the retinas were then fixed and processed for the localization of ChAT immunoreactivity. In other experiments, rats were administered an ischemic insult either by ligaturing the carotids (two-vessel occlusion [2-VO] procedure), cannulating the anterior chamber, and raising the intraocular pressure (high intraocular pressure [HIOP] procedure) or placing a ligature around the optic nerve sufficiently tightly to prevent blood flow through the central retinal artery (vascular ligation [VL] procedure). The electroretinogram was recorded, and, after a specific period of time, reperfusion was allowed to occur. Thirty to 36 hours after reperfusion, the retinas were dissected and processed for the localization of ChAT, as well as for parvalbumin, Thy-1, and alpha PKC immunoreactivities. RESULTS: Of the glutamate agonists tested, only kainate reduced ChAT immunoreactivity significantly in vivo and in vitro. This effect of kainate could be counteracted by the antagonist CNQX (6-cyano-2,3-dihydroxy-7-nitroquinoxaline-2,3-dione). The ChAT immunoreactivity was unaffected in retinas in which ischemia was induced by the 2-VO procedure. In contrast, ChAT immunoreactivity was obliterated in retinas in which the HIOP was used and drastically reduced when the VL procedure was used. Interestingly, neither alpha PKC nor Thy-1 immunoreactivities were affected in retinas subjected to HIOP or VL methods. However, parvalbumin immunoreactivity was reduced in the HIOP model but only slightly altered in the VL model. CONCLUSIONS: The current results suggest that kainate receptors are associated with the cholinergic retinal neurones in the rat retina. Activation of these receptors by kainate causes a reduction in the neurones' ChAT content. This effect can be mimicked by subjecting the retina to a sufficiently harsh ischemic insult, as occurs in the VL and HIOP procedures. When the ischemic insult is mild, as in the 2-VO procedure, no obvious change in ChAT immunoreactivity is apparent. The HIOP procedure for inducing an ischemic insult was found to be the most severe of the three procedures used, because ChAT immunoreactivity was obliterated and clear changes in the parvalbumin immunoreactivity also were recorded. Interestingly, neither the HIOP nor the VL procedures caused a change in the Thy-1 and alpha PKC immunoreactivities.

Animals↗
PubMed1995

Pertussis toxin-sensitive melatonin receptors negatively coupled to adenylate cyclase associated with cultured human and rat retinal pigment epithelial cells.

PURPOSE: Studies were conducted to investigate the influence of melatonin on adenylate cyclase activity in cultured human and rat retinal pigment epithelial (RPE) cells. METHODS: Adenylate cyclase activity was assessed by measurement of cAMP levels in cultured RPE cells using a specific cAMP-binding protein isolated from bovine adrenal cortex to detect cellular cAMP by competition with a standard amount of tritiated cAMP. The effects of melatonin on basal cAMP levels and those induced by the direct activator of adenylate cyclase, forskolin, were studied. RESULTS: Exposure of human RPE cells to 100 microM of melatonin had no effect on basal cAMP levels, but it caused a 41% reduction in the forskolin (5 microM) stimulation of cAMP. This melatonin-induced reduction in forskolin-stimulated adenylate cyclase activity was dose dependent, with half-maximal (EC50) reduction at 4.2 x 10(-10) M. 2-iodomelatonin, 6-chloromelatonin, and 6-hydroxymelatonin mimicked the melatonin effect with EC50 values of 3.5 x 10(-10) M, 4.3 x 10(-9)M, and 1.9 x 10(-7)M, respectively. Preexposure of cells to pertussis toxin (100 ng/ml) for 18 hours completely attenuated the ability of melatonin to influence the forskolin stimulation of cAMP levels. Propranolol did not influence the action of melatonin but did antagonize the ability of serotonin to reduce the forskolin-elevated cAMP levels. Thus, melatonin receptors are distinct from serotonin receptors. Melatonin receptors negatively linked to cAMP metabolism are also associated with cultured hooded rat RPE cells. Melatonin and iodomelatonin caused dose-dependent reductions in forskolin-stimulated cAMP production with half-maximal values of 2.4 x 10(-9)M and 3.28 x 10(-9)M, respectively. CONCLUSIONS: The results show that human and rat RPE cells possess melatonin receptors negatively coupled to adenylate cyclase and sensitive to pertussis toxin.

Adenylate Cyclase Toxin↗
PubMed1994

Immunohistochemical evidence for flupirtine acting as an antagonist on the N-methyl-D-aspartate and homocysteic acid-induced release of GABA in the rabbit retina.

When rabbit retinas are exposed in vitro to specific excitatory amino acid receptor agonists certain GABAergic amacrine cells are activated to cause a release of GABA. The GABA that is not released can be detected by immunohistochemistry. Exposure of tissues to kainate or NMDA each caused a characteristic change in the GABA immunoreactivity. CNQX antagonised the kainate effect specifically while MK-801 counteracted the influence of NMDA. The effect produced by kainate was mimicked by domoic acid while the influence of homocysteic acid was identical with NMDA. Flupirtine alone did not influence the nature of the GABA immunoreactivity and so did not act as a kainate or NMDA agonist. However, flupirtine counteracted the influence produced by NMDA and homocysteic acid but had no effect on the kainate and domoic acid responses. Thus in this system flupirtine acts as an NMDA antagonist.

6-Cyano-7-nitroquinoxaline-2,3-dione↗