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N N Osborne

Publications and source records attributed to N N Osborne.

At least 55 records · Page 3Linked to original sources
PubMed1999

Photoreceptors are preferentially affected in the rat retina following permanent occlusion of the carotid arteries.

Carotid artery occlusion (two vessel occlusion; 2-VO) for 3 or 9 months causes a suppression of the electroretinogram. However, after 3 months the retinal morphology appears unaffected judging from the localisation of GABA, ChAT, alpha PKC, Thy-1 and GFAP immunoreactivities. Moreover, no difference in NMDA-R1, opsin or Thy-1 mRNA levels were detected. In contrast, after 9 months 2-VO photoreceptor degeneration occurred as indicated by thinning of the outer nuclear layer and reduced Ret-P1 immunoreactivity. All other immunoreactivities appeared normal. These findings were supported by analysis of retinal mRNA levels. We conclude that the major effect of prolonged 2-VO is photoreceptor degeneration.

Animals↗
PubMed1999

The potential of neuroprotection in glaucoma treatment.

Visual field loss in glaucoma is due to death of retinal ganglion cells. Reducing or slowing down the loss of ganglion cells in glaucoma, a concept known as neuroprotection, would appear to be the only way forward. This does not imply that treatment of risk factors, such as elevated intraocular pressure, must not be continuously implemented. In this paper we point out that very little is known about the mechanisms of ganglion cell death in glaucoma and that data derived from studies on the "ideal animal model for glaucoma" must not be overemphasized. We also propose that the death processes of neurones in various diseases are fundamentally the same but vary in cause. Experimental data show that the death rate of neuronal populations is dependent on the impact of the insult and that neuroprotectants are more likely to benefit a patient in diseases in which the neurones die slowly, as in glaucoma, than in a disease in which the death of a set of neurones is rapid. We conclude that if a putative neuroprotectant can be administered in such a way that it reaches the retina in appropriate amounts and has insignificant side effects, it is likely to attenuate ganglion cell death and thus benefit the glaucoma patient.

Animals↗
PubMed1999

Assessment of Thy-1 mRNA levels as an index of retinal ganglion cell damage.

PURPOSE: Thy-1 is primarily, if not entirely, expressed by the ganglion cells within the retina. This knowledge was used to index ganglion cell death after ischemia and excitotoxicity by studying changes in Thy-1 mRNA levels. METHODS: Insults to the rat retina were delivered either by elevation of intraocular pressure for 60 minutes or by intravitreal injection of N-methyl-D-aspartate (NMDA). After a defined period, changes in Thy-1 immunoreactivity and mRNA levels of Thy-1 and NR1 (NMDA receptor subunit) were used to index ganglion cell sensitivity to damage. Opsin mRNA levels were used as an internal control because photoreceptors lack NMDA receptors. RESULTS: Retinal Thy-1 immunoreactivity, associated with the ganglion cell and inner plexiform layers, is reduced by ischemia or intravitreal injections of NMDA in a dose-dependent manner. Using a semi-quantitative polymerase chain reaction (reverse transcription-polymerase chain reaction) methodology, the levels of total retinal Thy-1 and NR1 mRNAs were shown to be dramatically reduced after both transient ischemia and intravitreal injection of NMDA. The effect of NMDA was found to be both time- and dose-dependent. In contrast, no change occurred in the levels of opsin mRNA unless high levels of NMDA (200 nmoles) were administered. CONCLUSIONS: Ischemia and NMDA-induced excitotoxicity caused retinal ganglion cell destruction, but the photoreceptors were unaffected. Measurement of total retinal Thy-1 mRNA levels provides a useful way of following ganglion cell death especially when combined with immunohistochemical localization of Thy-1. Additionally, the effect on other retinal cell types such as the photoreceptors can be followed in concert using this technique.

Animals↗
PubMed1998

Flupirtine, a nonopioid centrally acting analgesic, acts as an NMDA antagonist.

1. Flupirtine (Katadolon) is a member of a class of triaminopyridines and is used as a nonopioid analgesic agent with muscle relaxant properties. 2. In situ experiments have revealed that flupirtine protects against ischemic-induced insults to the retina and brain. 3. Data derived from in vitro and in vivo studies suggest that flupirtine functions as a weak N-methyl-D-aspartate (NMDA) antagonist with little evidence that it acts on AMPA-kainate type glutamate receptors. 4. No evidence could be found from binding studies to suggest that flupirtine has an affinity for any of the characterized binding sites associated with the NMDA receptor. 5. Studies on cultured cortical neurons show that the NMDA-induced influx of 45Ca2+ is more readily decreased by flupirtine when a reducing agent (dithiothreitol) is present. However, when N'-ethylmaleimide, which is thought to alkylate the NMDA receptor redox site, is present, no obvious effect on the NMDA-induced influx of 45Ca2+ is produced by flupirtine. 6. Flupirtine is also known to counteract the production of reactive oxygen species caused by ascorbate/iron as well as to prevent apoptosis in cells lacking NMDA receptors induced by oxidative stress. 7. To explain all the experimental data, it is suggested that flupirtine affects the redox state/pH/electrons in the cell. The specific way by which flupirtine antagonizes the NMDA receptor might be by an action on the known redox site of the receptor.

Aminopyridines↗
PubMed1998

The localization of endogenous zinc and the in vitro effect of exogenous zinc on the GABA immunoreactivity and formation of reactive oxygen species in the retina.

1. Endogenous zinc is localized mainly in the retinal photoreceptors and retinal pigment epithelial cells in the mammalian retina. No other types of retinal neurons contain large amounts of zinc. 2. Low concentrations of exogenous zinc, like the N-methyl-D-aspartate (NMDA) antagonist MK-801, counteract the NMDA-induced changes in the gamma-aminobutyric acid (GABA) immunoreactivity in the rabbit retina. However, greater concentrations of zinc exacerbate the effects of NMDA and ischemic-like insults (lack of glucose and oxygen) on GABA immunoreactivity. The data suggest that low concentrations of zinc are neuroprotective, but higher concentrations of zinc have a negative effect. 3. When low concentrations of zinc are present during ischemic-like insults to the retina, the GABA immunoreactivity is localized to the Müller cells, suggesting that the metabolism of GABA in the Müller glial cells is prevented. 4. Ascorbate/iron-induced generation of reactive oxygen species (ROS) in the retina is prevented by deferoxamine but not by zinc. High concentrations of zinc potentiate the ascorbate/iron induced formation of ROS.

Animals↗
PubMed1998

Immunohistochemical distribution of neurotrophins and their receptors in the rat retina and the effects of ischemia and reperfusion.

1. Neurotrophins are molecules that regulate the survival, development and maintenance of specific functions in different populations of nerve cells. 2. In the present work, we studied the localization, at the cellular level, of the different neurotrophins and their receptors within the rat retina in control and after ischemia-reperfusion of the retina. We found variations in the localization of some of these molecules depending on the reperfusion time of the retina after the ischemic lesion. 3. Thus it is suggested that the changes in the distribution and concentration of neurotrophins and their receptors caused by ischemia are protective reactions related to neuronal damage and synaptic reorganization.

Animals↗
PubMed1998

Localization of 5-hydroxytryptamine1A and 5-hydroxytryptamine7 receptors in rabbit ocular and brain tissues.

Serotonin is thought to play a physiological role in various tissues of the rabbit eye, yet little is known about the relative distribution of the different serotonin receptors. Demonstration of the receptor subtypes present in the various ocular tissues is essential in order to understand the function of serotonin in the eye. Using a combination of in situ hybridization histochemistry, in vitro receptor autoradiography and polymerase chain reaction studies, we have explored the distribution of the 5-hydroxytryptamine1A and 5-hydroxytryptamine7 receptors in the rabbit eye. As these receptors have not been sequenced in the rabbit, we initially established the suitability of the oligonucleotide probes by analysis of brain tissue. The distributions of 5-hydroxytryptamine1A and 5-hydroxytryptamine7 receptor messenger RNAs in rabbit brain correlated well with those in other species, confirming the specificity of the probes for detection of the messenger RNAs in rabbit tissues. In the eye, the expression of 5-hydroxytryptamine1A receptors appears to be restricted to the epithelial cell layer of the ciliary processes, although very low levels may appear in the retina. In contrast, the expression of 5-hydroxytryptamine7 receptor messenger RNA is more widespread with positive signals evident in the ciliary processes, retina and iris. The results confirm the existence of 5-hydroxytryptamine1A receptors in the ciliary body and their localization in the ciliary epithelium supports the hypothesis that they are involved in the secretion of aqueous humour. Unexpectedly, there was little evidence to support the idea that 5-hydroxytryptamine1A receptors are present in the retina and iris sphincter. However, the subsequent finding of 5-hydroxytryptamine7 receptor messenger RNA in the retina and iris may explain the apparent absence of 5-hydroxytryptamine1A receptors in these tissues. The presence of both 5-hydroxytryptamine1A and 5-hydroxytryptamine7 receptors in the ciliary processes may account for the complex intraocular pressure response of the rabbit to serotonin.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1998

Prevention of glutathione depletion-induced apoptosis in cultured human RPE cells by flupirtine.

We have recently reported that the non-opiate analgesic, flupirtine, counteracts apoptosis in cultures of human retinal pigmented epithelial (RPE) cells induced by deprivation of serum, oxygen and glucose (experimental ischaemia). In the present study, human RPE cells grown on coverslips were treated with buthionine sulphoxamine (BSO), a compound that inhibits glutathione biosynthesis. BSO caused a dose-dependent reduction in culture density and an increase in the number of cell nuclei that were positively labelled by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) procedure. These data show that reduction of glutathione levels causes apoptosis in the RPE cultures. When flupirtine gluconate was co-incubated with BSO, it dose-dependently prevented the induction of apoptosis. The most effective concentration of flupitine found to inhibit cell death caused by BSO (1 micro M - 1 mM) was 100 micro M. The presence of serum (2% or 10%) in the culture medium did not have any effect on the outcome of apoptosis and overall cell death caused by BSO. Futhermore, melatonin, also known to reduce experimental ischaemia-induced overall cell death and apoptosis of cultured RPE cells had only a mild protective effect at 1 mM. The combined data suggest that flupirtine prevents apoptosis by increasing the cellular levels of reduced glutathione and/or protects the cells against the damaging effects of reactive oxygen species (ROS) that are produced subsequent to inhibition of glutathione production.

Journal Article↗
PubMed1998

Expression of protein kinase C isoenzymes in cultured hooded rat retinal pigmented epithelial cells: comparison with dystrophic Royal College of Surgeons rat.

PURPOSE: To determine the isoenzymes of Protein Kinase C (PKC) that are present in cultured retinal pigmented epithelial (RPE) cells from both the Lister hooded rat and the Royal College of Surgeons (RCS) rat. METHODS: PKC isoenzymes were determined using immunocytochemistry and electrophoresis / Western blotting. RESULTS: Analyses showed the presence of six isoenzymes of PKC (PKCalpha, beta1, beta2, delta, epsilon and zeta) within cultured RPE cells derived from Lister hooded rats. Significantly, however, cultures of cells derived from dystrophic Royal College of Surgeons rats differed in PKCdelta and PKCalpha isoenzyme expression as compared with cultured hooded rat RPE cells. CONCLUSIONS: Rat RPE cells express several PKC isoenzymes. Differences in expression of PKCalpha and PKCdelta in RCS rat RPE cells may relate to the decreased phagocytic capability of these cells as compared with those of the Lister hooded rat.

Animals↗
PubMed1998

Melatonin counteracts ischemia-induced apoptosis in human retinal pigment epithelial cells.

PURPOSE: To investigate whether the neurohormone melatonin can prevent apoptosis caused by deprivation of oxygen, glucose, and serum (experimental ischemia) in cultured human retinal pigment (RPE) cells. METHODS: Cultures of human RPE cells established from a variety of donors were grown to passage four and then subjected to experimental ischemia, with or without various substances, for up to 72 hours. Cells were examined for morphologic changes and breakdown of DNA, assessed by TdT-dTUP terminal nick-end labeling (TUNEL) and agarose gel electrophoresis. Changes in transcription and translation of various proto-oncogenes (bcl-2, TIAR, ICH-1S/1) were assessed by analysis of mRNA and protein levels, respectively. The effect of various substances on the iron-ascorbate-induced formation of reactive oxygen species (ROS) in chick retinal dissociates was also investigated. RESULTS: Cultured human RPE cells on coverslips that were incubated in serum-free medium, glucose, and oxygen remained viable for up to 40 hours. Thereafter, there was a steady decrease in cell numbers and an increase in the number of cells labeled by the TUNEL method. By 72 hours 65% of cells remained attached to the coverslips, of which approximately 65% were TUNEL positive. Furthermore, most of the experimental ischemia-treated cells exhibited a shrunken appearance typical of apoptosis. Fragmentation of the DNA from cells in which ischemia was induced for 72 hours was also confirmed by agarose gel electrophoresis. Inclusion of 100 microM melatonin significantly decreased the amount of apoptotic cell nuclei after ischemia, but the effect was mild compared with that of fetal calf serum, which almost completely counteracted cell death. The action of melatonin was not prevented by 0.01 mM to 1 mM luzindole, a melatonin receptor antagonist. In addition, 100 microM ascorbate did not counteract ischemia-induced apoptosis. Treatment of RPE cells with 100 microM flupirtine gluconate for 72 hours caused an upregulation of the proto-oncogene protein Bcl-2 and a decrease in TIAR and ICH-1L proteins compared with that in control cells. Melatonin at 100 microM had no such effect. The levels of the mRNA transcripts for ICH-1L relative to those for ICH-1S were significantly decreased in cultures treated with 100 microM flupirtine or 100 microM melatonin when compared with levels in control cells. However, the effect of flupirtine was greater than that of melatonin. Ten micromolar ascorbate and 5 microM iron stimulated the formation of ROS in chick retinal cell dissociates. Ascorbate, melatonin, and flupirtine (all at 100 microM) blunted this response in the order flupirtine > melatonin >> ascorbate. Luzindole had no effect, alone or in the presence of melatonin. CONCLUSIONS: The presented data show that melatonin counteracted ischemia-induced apoptosis in human RPE cells by a process that seemed to be independent of melatonin receptors. Moreover, melatonin and flupirtine counteracted iron-ascorbate-induced ROS formation and decreased the ratio of mRNA for ICH-1L and ICH-1S. However, melatonin was less potent than flupirtine in its action in each case, which suggests that either the two compounds act on different signaling pathways or that they act on the same pathway with differing potency. The failure to detect an effect of melatonin on the levels of Bcl-2, ICH-1L, and TIAR proteins when compared with the effect of flupirtine was probably caused by the sensitivity of the procedures. It is suggested that substances that can prevent ROS formation can potentially nullify apoptotic cell death, but this is difficult to detect experimentally when the substance has only a mild effect, such as in the case of ascorbate.

Aged↗
PubMed1997

Melatonin protects primary cultures of rat cortical neurones from NMDA excitotoxicity and hypoxia/reoxygenation.

Studies on rat cortical cultures show that glutamate (10 microM) or hypoxia followed by reoxygenation causes damage to the cells as indexed by a release of lactate dehydrogenase (LDH). These effects could be counteracted by the N-methyl-D-aspartate (NMDA) antagonist MK-801 (2 microM) but not by the kainate/AMPA antagonist CNQX (100 microM). These data favour the view that the damage caused to the cells by glutamate and hypoxia/reperfusion is mediated via NMDA receptors. The damage to the cells could also be prevented by melatonin (100 microM). The melatonin effect is not mediated by specific receptors because it was not blunted by the melatonin antagonist, luzindole. Moreover, NMDA stimulated an accumulation of 45Ca2+ by cortical neurones, but although this effect was counteracted by MK-801, melatonin was ineffective, which showed that the neuroprotective effect of melatonin is not elicited by direct action with NMDA receptors. Ascorbate and iron stimulated the production of free radicals in a retinal cell preparation. Chelation of the iron with deferoxamine prevented this process as did melatonin while MK-801 had no effect. The combined findings suggest that melatonin counteracts the in vitro destructive effects of NMDA or hypoxia/reperfusion by preventing accumulation of excessive free radicals.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1997

Retinal neurones containing kainate receptors are influenced by exogenous kainate and ischaemia while neurones lacking these receptors are not -- melatonin counteracts the effects of ischaemia and kainate.

The present experiments were carried out on three types of neurone in primary rabbit retinal cultures. One cell-type, bipolar neurones, have glutamate APB-type metabotropic receptors and can be identified by the presence of thetaPKC-immunoreactivity. The other two cell-types are primarily amacrine cells and can be 'stained' for the localisation of GABA immunoreactivity or for serotonin taken up from the medium. Most of the serotonin-accumulating and GABA-containing neurones contain glutamate kainate-type receptors. Exposure of the cultures to treatment of kainate (50 microM) or experimental ischaemia (8 h followed by 16 h reoxygenation) produced essentially similar findings. The serotonin-accumulating and GABA cells were affected as they were drastically reduced in numbers while the numbers of thetaPKC-containing cells were unaffected. Inclusion of the kainate/AMPA antagonist CNQX (100 microM) or melatonin (100 microM) to the medium during kainate or ischaemia treatments largely prevented the detrimental influences on the serotonin-accumulating and GABA cells. It is concluded that during experimental ischaemia excessive glutamate is released to influence cells which contain kainate and APB-type receptors. However, only the neurones containing the kainate receptors are negatively affected with the generation of free radicals. Melatonin or CNQX protects against this effect by scavenging free radicals or acting at the receptor level, respectively.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1997

In vivo and in vitro experiments show that betaxolol is a retinal neuroprotective agent.

The aim of the study was to determine whether betaxolol is a neuroprotective agent and can therefore slow down the changes seen in the retina following ischaemia/reperfusion. Ischaemia was induced in one rat eye by raising the intraocular pressure for 45 min. Three days later electroretinograms were recorded from both eyes and the retinas were examined immunohistochemically for the localisation of calretinin and choline acetyltransferase (ChAT) immunoreactivities. The effect of glutamate agonists, hypoxia or experimental ischaemia was examined on the GABA immunoreactivity, lactate dehydrogenase (LDH) and internal calcium levels ([Ca2+]i) of the isolated rabbit retina, rat cortical cultures and chick retinal cell cultures respectively. Betaxolol was tested to see whether it can attenuate the influence of the glutamate agonists, hypoxia or experimental ischaemia. Ischaemia for 45 min causes a change in the nature of the normal calretinin immunoreactivity, an obliteration of the ChAT immunoreactivity and a drastic reduction in the b-wave of the electroretinogram after 3 days of reperfusion. When betaxolol was injected i.p. into the rats before ischaemia and on the days of reperfusion the changes to the calretinin and ChAT immunoreactivities were reduced and the reduction of the b-wave was prevented. Rabbit retinas incubated in vitro in physiological solution lacking oxygen/glucose or containing the glutamate agonists kainate or NMDA caused a change in the nature of the GABA immunoreactivity. Inclusion of betaxolol partially prevented the changes caused by NMDA and lack of oxygen/glucose. Rat cortical cultures exposed to glutamate or hypoxia/reoxygenation resulted in a release of LDH. The release of the enzyme was almost completely attenuated when betaxolol was included in the culture medium. Kainate increased the [Ca2+]i in chick retinal cultures, as measured with Indo-1. In a medium with sodium, this kainate-induced elevation of [Ca2+]i was significantly reduced by betaxolol. The combined data show that betaxolol is a neuroprotective agent and attenuates the effects on the retina induced by raising the intraocular pressure to simulate an ischaemic insult as may occur in glaucoma.

Animals↗
PubMed1997

Protein kinase C activation by serotonin potentiates agonist-induced stimulation of cAMP production in cultured rat retinal pigment epithelial cells.

Serotonin stimulates inositol phosphate production and intracellular calcium mobilization in cultured rat retinal pigment epithelial (RPE) cells through interaction with 5-HT2A receptors, but decreases cAMP production in cultured human RPE cells via 5-HT1A receptors. Studies were therefore undertaken to investigate the effect of serotonin on the cAMP system in rat RPE cells. Exposure of cultured rat RPE cells to serotonin (100 microM) for 10 minutes had no effect on the basal levels of cAMP. However, a 5 minute preincubation with serotonin potentiated the production of cAMP induced by a 5 minute exposure to forskolin (5 microM), isoproterenol (1 microM) and 5'-[N-ethylcarboxamido]-adenosine (10 microM) by 133.0%, 296.8% and 651.9%, respectively. This effect of serotonin was dose-dependent on forskolin and 5'-[N-ethylcarboxamido]-adenosine with half-maximal effects close to those reported for its action on inositol phosphates production. The antagonists ketanserin, methysergide and spiperone attenuated the action of serotonin, while yohimbine and spiroxatrine were ineffectual, thus indicating that the potentiating effect was through the 5-HT1A receptor. Incubation of cultured rat RPE cells with bradykinin stimulates inositol phosphates production with half-maximal effect observed at 1 nM. Bradykinin also potentiates the action of forskolin, isoproterenol and 5'-[N-ethylcarboxamido]-adenosine on cAMP production in a dose-dependent manner with little effect on basal levels. RPE cells exposed to serotonin (500 microM) or phorbol 12-13 dibutyrate (1 microM) for 30 minutes showed translocation of protein kinase C to the membrane from the cytosol, with 53.3% and 29.4% increases in membrane activity, respectively. Forskolin- and 5'-[N-ethylcarboxamido]-adenosine-induced cAMP production was potentiated by phorbol 12-13 dibutyrate (1 microM) treatment. The effect of both serotonin and phorbol 12-13 dibutyrate on forskolin-induced cAMP production was attenuated by pretreatment of cell cultures with the protein kinase C antagonists staurosporin and calphostin C at 1 microM. Thus, the production of cAMP in cultured rat RPE cells is potentiated by 5-HT2A receptors through activation of protein kinase C. This effect is, however, not specific since bradykinin, which stimulates inositol phosphates turnover, also potentiates stimulated cAMP production.

Adenosine↗
PubMed1997

Antagonism of muscarinic receptors in the rabbit iris-ciliary body by 8-OH-DPAT and other 5-HT1A receptor agonists.

Physiological studies have shown that serotonin and 5-HT1A agonists can influence muscarinic function in the rabbit iris-ciliary body (ICB). The purpose of this study was to examine whether a direct interaction exists between muscarinic and 5-HT1A receptors in the ICB. At high concentrations, the 5-HT1A agonist 8-OH-DPAT attenuated the carbachol-induced stimulation of inositol phosphates (InsPs) production, but this was not blocked by the presence of 5-HT1A antagonists. In contrast, serotonin failed to influence carbachol-induced InsPs formation. Moreover, 8-OH-DPAT but not serotonin displayed affinity for [3H]QNB binding sites in the ICB. The combined data suggest that activation of 5-HT1A receptors in the ICB does not cause a modulation of muscarinic receptor-stimulated phosphoinositide turnover. The data instead suggest that, at high concentrations, 8-OH-DPAT acts as an antagonist at muscarinic receptors and in this way influences muscarinic receptor function. The mechanism of 5-HT-induced modulation of muscarinic function in the ICB therefore remains to be elucidated.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1997

Retinal protein kinase C.

The protein kinase C (PKC) family of serine/threonine kinase isoenzymes are universally expressed in vertebrate tissues where they control vital cellular functioning. PKC comprises twelve currently identified mammalian isoenzymes, described in three distinct groups according to their need for different effector stimulation. Immunological localisation studies in various vertebrate retinas have indicated the presence, so far, of eight of the PKC subspecies, each with a unique cellular distribution in this tissue. Use of these immunological probing techniques with antibodies raised to the individual PKC family members by immunohistochemistry and western blotting, along with biochemical tools such as the potent activators, the tumour-promoting phorbol esters can hopefully lead to elucidation of the roles of these enzymes in the neural retina. Research work to date has pinpointed a number of roles for PKC in this tissue including control of dopamine release, modulation of glutamate receptor function (probably by a process of direct receptor phosphorylation), phosphorylatory modulation of GABAC-receptor function, an involvement in the retinal ischaemic cascade process (the relevance of which is unknown as yet), involvement in control of cytoskeletal interactions by cytoskeletal element-kinase action and feedback control of enzymes involved in the process of inositol phosphate signalling. PKC has been shown to have an important regulatory role in the process of phototransduction: many of the enzymes and proteins making up the phototransduction cascade act as in vitro and in vivo substrates for PKC-dependent phosphorylation and can have their normal function modified in this way. Also, PKC has been implicated in the control of spinule formation in the retina, a process involved in retinal synaptic plasticity and functioning. All of this work has been described, herein. Collation and utilisation of knowledge of all of the work described here may help us to determine the exact roles for individual isoenzymes in the retina. This in turn may help us to understand and further to prevent pathological conditions leading to inappropriate retinal functioning and possible blindness. Furthermore, understanding the roles of PKC in the neural retina may lead us to vital clues in the understanding of the functioning of this important group of enzymes in the nervous system as a whole and eventually to the prevention of many major neuropathological disorders.

Animals↗
PubMed1997

Induction of apoptosis in cultured human retinal pigmented epithelial cells: the effect of protein kinase C activation and inhibition.

The presence of the non-selective protein kinase C (PKC) inhibitors, staurosporine (100 nM) and polymyxin B (100 microM) in cultured human RPE cells for more than 24 h triggers apoptotic death. Apoptosis is characterized by a diminishing number of cells, a labelling of nuclei by the TUNEL method and by observable morphological changes. An inhibitor of PKC and cyclic nucleotide-dependent protein kinases, 1-(5-isoquinolinesulphonyl)-2-methyl piperazine (H-7; 100 microM), was without effect, as was the specific PKC inhibitor, calphostin C (100 nM). The PKC-activating phorbol esters, phorbol-12-myristate-13-acetate (PMA; 1 microM) and phorbol-12,13-dibutyrate (PDB; 1 microM) and the non-tumour-promoting phorbol ester, 4 alpha-PMA (1 microM) were without effect, as was the diacyl glycerol analogue, 1,2-dioctanoyl-snglycerol (DOG; 10 microM). The PKC activators did not attenuate the apoptosis induced by staurosporine or polymyxin B. Furthermore, deprivation of glucose and oxygen (simulated ischemia) for 72 h induced apoptosis: this could be prevented by inclusion of 10% (v/v) foetal bovine serum (FBS) but not by a variety of PKC activators. Six PKC isoenzymes were shown to be present in RPE cells (alpha, beta 1, beta 2, delta, epsilon, E) and only the calcium-dependent cPKC levels changed after treatment with staurosporine or simulated ischaemia. Since only the less selective inhibitors of PKC induced apoptosis, it is suggested that PKC is not involved directly in the induction process of apoptosis in RPE cells. It is possible that the staurosporine and polymyxin B-induced effects of apoptosis in RPE cells are triggered by an unknown kinase-dependent pathway, but whether the 'ischaemia'-induced death is related to this same process remains to be elucidated.

Adult↗