P0 phosphorylation in nerves from normal and diabetic rats: Role of protein kinase C and turnover of phosphate groups

The effects of phorbol ester and forskolin on the net phosphorylation and turnover of P₀ phosphate groups was studied in normal and exprimentally diabetic rats. In sciatic nerve segments isolated from normal rats and incubated with [³²P]-inorganic phosphate, phosphorylation of the major peripheral myelin protein, P₀, was increased 2–5 fold in a time and dose-dependent manner by phorbol 12,13 dibutyrate (PDB). This increase was blocked by the protein kinase inhibitors, H-7 and staurosporine. Both the basal and PDB-stimulated phosphorylation of P₀ were significantly greater in segments of sciatic nerve from streptozotocin-induced diabetic rats. Prolonged exposure of nerve segments to PDB abolished the stimulated phosphorylation of P₀ and immunoblots of nerve proteins revealed a decrease in the content of the protein kinase C -isoform. The adenylate cyclase activator, forskolin, had no affect on the PDB-stimulated phosphorylation of P₀ in normal nerve but decreased phosphorylation in diabetic nerve. To measure turnover of P₀ phosphate groups, nerves were incubated with³²P and incorporated label was then chased in radioactivity-free medium for up to 4 hours. P₀ from normal nerve prelabeled under basal conditions lost 25% of its radioactivity during this time. In contrast, nearly all of the additional phosphate groups prelabeled in the presence of PDB disappeared after 2 hours of chase. P₀ phosphate groups from diabetic nerve displayed similar turnover kinetics. When forskolin was added to the chase medium, the turnover of P₀ phosphate moieties was accelerated in normal, but not in diabetic nerve. These findings clearly establish a prominent role for protein kinase C in P₀ phosphorylation, provide evidence for heterogeneous turnover of P₀ phosphate groups and suggest that cyclic AMP-mediated processes may modulate P₀ phosphorylation. Further, these results indicate that the metabolism of P₀ phosphate moieties is perturbed in nerve from diabetic animals.Special issue dedicated to Dr. Marjoris B. Lees.     

Differential influence of increased polyol pathway on protein kinase C expressions between endoneurial and epineurial tissues in diabetic mice

To explore the relationship between polyol pathway and protein kinase C (PKC), we examined PKC activities and expressions of PKC isoforms separately in endoneurial and vessel-rich epineurial tissues in diabetic mice transgenic for human aldose reductase (Tg). Tg and littermate control mice (Lm) were made diabetic by streptozotocin at 8 weeks of age and treated orally with aldose reductase inhibitor (ARI) (fidarestat 3-5 mg/kg/day) or placebo for 12 weeks. At the end, compared with non-diabetic state, sorbitol contents were increased 6.4-fold in endoneurium and 5.1-fold in epineurium in diabetic Tg, whereas the increase was detected only in endoneurium in diabetic Lm. Endoneurial PKC activity was significantly reduced in diabetic Tg. By contrast, epineurial PKC activity was increased in both diabetic Lm and diabetic Tg and there was no significant difference between the two groups. These changes were all corrected by ARI treatment. Consistent with the changes of PKC activities, diabetic Tg showed decreased expression of PKC alpha in endoneurium, whereas there was an increased expression of PKC beta II in epineurium in both diabetic Tg and diabetic Lm. These findings suggest the presence of dichotomous metabolic pathway between neural and vascular tissues in the polyol-PKC-related pathogenesis of diabetic neuropathy.     

In situ regulation of cell-cell communication by the cAMP-dependent protein kinase and protein kinase C

The effects of cAMP-dependent protein kinase A and protein kinase C on cell-cell communication have been examined in primary ovarian granulosa cells microinjected with purified components of these two regulatory cascades. These cells possess connexin43 ( 1)-type gap junctions, and are well-coupled electrotonically and as judged by the cell-to-cell transfer of fluorescent dye. Within 2–3 min after injection of the protein kinase A inhibitor (PKI) communication was sharply reduced or ceased, but resumed in about 3 min with the injection of the protein kinase A catalytic subunit. A similar resumption also occurred in PKI-injected cells after exposure to follicle stimulating hormone. Microinjection of the protein kinase C inhibitor protein caused a transient cessation of communication that spontaneously returned within 15–20 min. Treatment of cells with activators of protein kinase C, TPA or OAG for 60 min caused a significant reduction in communication that could be restored within 2–5 min by the subsequent injection of either the protein kinase C inhibitor or the protein kinase A catalytic subunit. With a longer exposure to either protein kinase C activator communication could not be restored and this appeared to be related to the absence of aggregates of connexin43 in membrane as detected immunologically. In cells injected with alkaline phosphatase communication stopped but returned either spontaneously within 20 min or within 2–3 min of injecting the cell with either the protein kinase A catalytic subunit or with protein kinase C. When untreated cells were injected with protein kinase C communication diminished or ceased within 5 min. Collectively these results demonstrate that cell-cell communication is regulated by both protein kinase A and C, but in a complex interrelated manner, quite likely by multiple phosphorylation of proteins within or regulating connexin-43 containing gap junctions.Abbreviations C catalytic subunit of protein kinase A - CKI protein kinase C inhibitor protein - Cx connexin protein - dbcAMP N⁶,2-O-dibutyryladenosine 3:5-cyclic monophosphate - OAG 1-oleoyl-2-acetyl-sn-glycerol - protein kinase A cAMP-dependent protein kinase - protein kinase C Ca²⁺-sensitive phospholipid-dependent protein kinase - PKI protein kinase A inhibitor protein - R regulatory subunit of protein kinase A - TRA 12-O-tetradecanoylphorbol-13-acetate - 8Br-cAMP 8-bromoadenosine 3:5 cyclic monophosphate     

Membrane protein kinase C activity rapidly increases in quiescent tsRSV-infected NRK cells upon reactivation of the mitogenic v-src protein kinase

The viral src protein kinase, pp60^v-src, is a powerful intracellular mitogen which can initiate and maintain the proliferation of quiescent cells in the absence of any exogenous growth factors. In an attempt to understand how pp60^v-src induces proliferation, we examined the early events in the G₀ to G₁ transition caused by the activation of a thermolabile v-src protein in quiescent, serum-starved tsRSV-transformed NRK cells. The reactivation of pp60^v-src, in the presence of exogenous growth factors, triggered a rapid biphasic surge of membrane-associated protein kinase C (PKC) activity. Unlike TPA-stimulated PKC activity, the pp60^v-src-induced increase in PKC was readily extracted from membranes by EGTA. The down-regulation of PKC activity in these quiescent cells by prolonged exposure to TPA strongly inhibited the ability of the reactivated v-src protein to stimulate DNA replication in serum-deficient medium, suggesting that PKC plays a role in the initial signal by which the viral enzyme induces the G₀ to G₁ transition in NRK cells.     

Regulation of protein kinase C by the anti-Parkinson drug, MAO-B inhibitor, rasagiline and its derivatives, in vivo

We have recently shown that the anti-Parkinson-propargyl-containing monoamine oxidase B (MAO-B) inhibitor drug, rasagiline [N-propargyl-(1R)-aminoindan], and its cholinesterase inhibitor derivatives TV3326 and TV3279, regulate amyloid precursor protein (APP) processing by a protein kinase C (PKC)-dependent mechanism in SH-SY5Y neuroblastoma and PC12 cells. In the present study, we investigated the effect of rasagiline and its derivatives on the regulation of the PKC-dependent mechanism and APP processing under in vivo conditions. Administration of rasagiline (0.1 mg/kg) to male C57/BL mice for 14 days significantly decreased membrane-bound holoprotein APP levels in the hippocampus. Additionally, we observed that rasagiline up-regulated p-PKC levels and the expression of alpha and epsilon PKC isozymes in the hippocampus, indicating that the mechanism by which rasagiline affects APP processing may be related to PKC-associated signalling. The results also demonstrate that rasagiline treatment significantly elevated the levels of phosphorylated myristoylated alanine-rich C kinase substrate (p-MARCKS), a major substrate for PKC, as well as the levels of receptors for activated C kinase 1 (RACK1). Similar effects on APP and PKC levels were also demonstrated for the two cholinesterase inhibitor derivatives of rasagiline, TV3326 and TV3279. These results indicate that rasagiline and its derivatives regulate PKC-dependent mechanisms and APP processing. The activation and induction of PKC and MARCKS by these drugs may have a crucial role not only in their neuroprotective activity, but also in their ability to affect neuronal plasticity and spatial learning processes.     

Regulation of guanylate cyclase activity by atrial natriuretic factor and protein kinase C

Summary The putative second messenger of certain atrial natriuretic factor (ANF) signal transductions is cyclic GMP. Recently, we purified a 180-kDa protein, apparently containing both ANF receptor and guanylate cyclase activities, and hypothesized that this is one of the cyclic GMP transmembrane signal transducers. The enzyme is ubiquitous and appears to be conserved. Utilizing the 180-kDa membrane guanylate cyclase, we now show that the 180-kDa guanylate cyclase is regulated in opposing fashions by two receptor signals—ANF stimulating it and protein kinase C inhibiting it. Furthermore, protein kinase C phosphorylates the 180-kDa enzyme. This suggests a novel switch on and switch off mechanism of the cyclic GMP signal transduction. Switch off represents the phosphorylation while switch on the dephosphorylation of the enzyme.     

Phosphorylation of HMG-I by protein kinase C attenuates its binding affinity to the promoter regions of protein kinase C gamma and neurogranin/RC3 genes

A 20-kDa DNA-binding protein that binds the AT-rich sequences within the promoters of the brain-specific protein kinase C (PKC) gamma and neurogranin/RC3 genes has been characterized as chromosomal nonhistone high-mobility-group protein (HMG)-I. This protein is a substrate of PKC alpha, beta, gamma, and delta but is poorly phosphorylated by PKC epsilon and zeta. Two major (Ser44 and Ser64) and four minor phosphorylation sites have been identified. The extents of phosphorylation of Ser44 and Ser64 were 1:1, whereas those of the four minor sites all together were <30% of the major one. These PKC phosphorylation sites are distinct from those phosphorylated by cdc2 kinase, which phosphorylates Thr53 and Thr78. Phosphorylation of HMG-I by PKC resulted in a reduction of DNA-binding affinity by 28-fold as compared with 12-fold caused by the phosphorylation with cdc2 kinase. HMG-I could be additively phosphorylated by cdc2 kinase and PKC, and the resulting doubly phosphorylated protein exhibited a >100-fold reduction in binding affinity. The two cdc2 kinase phosphorylation sites of HMG-I are adjacent to the N terminus of two of the three predicted DNA-binding domains. In comparison, one of the major PKC phosphorylation sites, Ser64, is adjacent to the C terminus of the second DNA-binding domain, whereas Ser44 is located within the spanning region between the first and second DNA-binding domains. The current results suggest that phosphorylation of the mammalian HMG-I by PKC alone or in combination with cdc2 kinase provides an effective mechanism for the regulation of HMG-I function.     

The connexin43 gap junction protein is phosphorylated by protein kinase A and protein kinase C: In vivo and in vitro studies

There is general agreement that the connexin43 gap junction protein is a substrate for phosphorylation by protein kinase C but there is no similar consensus regarding the action of protein kinase A. Our previous studies demonstrated that channels formed by connexin43 were reversibly gated in response to microinjected protein kinase A and protein kinase C, but we did not determine whether these effects involved direct action on the connexin43 protein. Using a combination of in vivo metabolic labeling and in vitro phosphorylation of recombinant protein and synthetic peptides, we now find that connexin43 is a relatively poor substrate for purified protein kinase A compared to protein kinase C, but that phosphorylation can be accelerated by 8-Br-cAMP (8-bromoadenosine 3,5-cyclic monophosphate) which also enhances connexin43 synthesis but at a much slower rate than phosphorylation. Phosphorylation of a critical amino acid, Ser364, by protein kinase A, appears to be necessary for subsequent multiple phosphorylations by protein kinase C. However, protein kinase C can phosphorylate connexin43 at a reduced level in the absence of prior phosphorylation. The results suggest that the correct regulation of channels formed by connexin43 may require sequential phosphorylations of this protein by protein kinase A and protein kinase C.     

A phorbol ester and phospholipid-activated, calcium-unresponsive protein kinase in mouse epidermis: characterization and separation from protein kinase C

The phosphorylation of an Mr 82,000 protein (p82) in the Triton X-100 extract of the particulate fraction of mouse epidermis is dependent on the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or diacylglycerol and phospholipid and, contrary to protein kinase C (PKC)-catalyzed phosphorylation, cannot be activated by calcium plus phospholipid. The novel p82 kinase differs also from PKC in many other respects, such as substrate specificity, turnover rate, and sensitivity to inhibitors. The p82 kinase can be separated from PKC by chromatography on phenyl sepharose and does not react with a polyclonal PKC antiserum. Like PKC, the novel kinase phosphorylates its substrate on threonine and serine, but not on tyrosine. Similar to PKC, the epidermal p82-kinase system is down-modulated after TPA treatment of mouse skin, with a half-life of around 5 h. Down-modulation is also accomplished by the phorbol ester RPA, but not by the Ca2+ ionophore A23187, and it is inhibited by the immunosuppressive agent cyclosporin A. In addition to down-modulation, TPA treatment of the animals activates a phosphatase that dephosphorylates phosphorylated p82 in the extract of the particulate fraction.     

Ligation of Fc receptor of macrophages stimulates protein kinase C and antileishmanial activity

Fc receptors are known to express on the surface of mature monocytes macrophages and lymphocytes. In this study a ligand e.g. liposomal IgG (human IgG coupled to PE-liposome via carbodimide reaction) was developed to ligate the Fc receptor of macrophages. When liposomal IgG was incubated with macrophages at 37°C for 5 min, it induced the macrophage activation which suppress the parasite burden approximatley to an extent of 60%, 50% and 45%, when macrophages were infected with UR6, AG83 and GE1 strains of L-donovani respectively. Superior efficacy of liposomal IgG were achieved compared to the treatment with free IgG and free liposomes. The activity of protein kinase C (PKC) has been found to be higher in the Fc receptor targeted macrophage membrane fraction, suggesting its translocation from the cytosol. Staurosporine, a potent inhibitor of the enzyme protein kinase C (PKC) has been found to protect the parasite inside the macrophage indicating the role of PKC in the signaling process. The liposomal IgG treatment has been found to induce the generation of significant amount of superoxide and hydrogen peroxide which helped to suppress the parasite burden. Further when liposomal IgG were incubated with IFN- primed, LPS activated macrophages, a significant amount of NO release was also noticed, indicating its role in parasite killing. The above results suggest that Fc receptor mediated activation by liposomal IgG may be used as an alternative approach to kill parasites intracellularly.     

Differential regulation of GLAST immunoreactivity and activity by protein kinase C: evidence for modification of amino and carboxyl termini

Many neurotransmitter transporters, including the GLT-1 and EAAC1 subtypes of the glutamate transporter, are regulated by protein kinase C (PKC) and these effects are associated with changes in cell surface expression. In the present study, the effects of PKC activation on the glutamate aspartate transporter (GLAST) subtype of glutamate transporter were examined in primary astrocyte cultures. Acute (30 min) exposure to the phorbol 12-myristate 13-acetate (PMA) increased (approximately 20%) transport activity but had the opposite effect on both total and cell surface immunoreactivity. Chronic treatment (6 or 24 h) with PMA had no effect on transport activity but caused an even larger decrease in total and cell surface immunoreactivity. This loss of immunoreactivity was observed using antibodies directed against three different cytoplasmic epitopes, and was blocked by the PKC antagonist, bisindolylmaleimide II. We provide biochemical and pharmacological evidence that the activity observed after treatment with PMA is mediated by GLAST. Two different flag-tagged variants of the human homolog of GLAST were introduced into astrocytes using lentiviral vectors. Although treatment with PMA caused a loss of transporter immunoreactivity, flag immunoreactivity did not change in amount or size. Together, these studies suggest that activation of PKC acutely up-regulates GLAST activity, but also results in modification of several different intracellular epitopes so that they are no longer recognized by anti-GLAST antibodies. We found that exposure of primary cultures of neurons/astrocytes to transient hypoxia/glucose deprivation also caused a loss of GLAST immunoreactivity that was attenuated by the PKC antagonist, bisindolylmaleimide II, suggesting that some acute insults previously thought to cause a loss of GLAST protein may mimic the phenomenon observed in the present study.     

Zinc deficiency in rats decreases thrombin-stimulated platelet aggregation by lowering protein kinase C activity secondary to impaired calcium uptake

Aggregation of rat platelets, when stimulated by adenosine diphosphate (ADP) or fluoride, is impaired by zinc deficiency, and the defect is associated with a decreased uptake of external Ca²⁺. Zinc deficiency also impairs the aggregatory response of platelets to phorbol myristate acetate (PMA), an activator of protein kinase C, but low zinc status decreases the PMA response only when calcium is added to the external medium. The purpose of this study was to determine the role of protein kinase C in rat platelet function and its relationship to the zinc deficiency pathology observed in platelets stimulated by thrombin (THR). The percent of maximal aggregation and the concentration of cytosolic-free Ca²⁺ were measured in washed platelets stimulated by THR and PMA. For the protein kinase C experiments platelets were obtained from rats fed a grain-based diet, and for the thrombin experiments they were from rats fed purified diets. In the latter experiments, immature male rats were fed for 2 weeks a low zinc diet (<1 mg/kg) ad libitum or a zinc adequate (100 mg/kg) diet either ad libitum or pair-fed. Zinc deficiency impaired the aggregation of platelets stimulated by 0.045 U/mL of THR by approximately 40%, and the external calcium uptake (0.03 U/mL of THR) was decreased by approximately 30%. Staurosporine, a protein kinase C inhibitor, decreased thrombin-induced aggregation in a concentration-dependent manner, but it had no effect on the external calcium uptake. While PMA had a synergistic effect with thrombin in the stimulation of platelet aggregation, it actually decreased the cytosolic-free calcium response to thrombin. It is concluded that zinc deficiency impairs thrombin-stimulated platelet aggregation and calcium uptake and that protein kinase C activity is essential for rat platelet aggregation. Protein kinase C does not stimulate calcium uptake and must act downstream of the calcium uptake defect. A model of rat platelet activation is presented depicting impaired Ca²⁺ uptake as the primary defect in zinc deficiency.     

Phosphorylation of MP26, a lens junction protein,is enhanced by activators of protein kinase C

Summary MP26, a protein thought to form gap junctional channels in the lens, and other lens proteins were phosphorylated under conditions that activate protein kinase C. Phosphorylation was detected both in lens fiber cell fragments in an in vivo labeling procedure with³²P-phosphate and in cell homogenates with³²P-ATP. In these experiments, both calcium and 12-O-tetradecanoylphorbol 13-acetate (TPA) were necessary for maximal phosphorylation of MP26. Calcium stimulated the phosphorylation of MP26 approximately fourfold and TPA with calcium led to a sevenfold increase. If TPA was present, 1 m calcium was sufficient for maximal labeling. Phosphoamino acid analysis demonstrated approximately 85% phosphoserine, 15% phosphothreonine, and no phosphotyrosine when MP26 was phosphorylated in lens homogenates in the presence of TPA and calcium and then electrophoretically purified. Phosphorylation occurred near the cytoplasmic, C-terminal of MP26. The possible involvement of other kinases was also examined. The Walsh inhibitor, which affects cAMP-dependent protein kinases, had no influence on the TPA-mediated increase in phosphorylation. In studies with isolated membranes and added kinases, MP26 was also found to not be a substrate for calcium/calmodulindependent protein kinase II. Thus, protein kinase C may have phosphorylated MP26 in a direct manner.     

The relationship between the bilayer to hexagonal phase transition temperature in membranes and protein kinase C activity

A number of substances affect the activity of protein kinase C. Among uncharged and zwitterionic compounds, those which activate protein kinase C also lower the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine while substances which inhibit protein kinase C raise this transition temperature. Using this criteria, we have identified 3-chloro-5-cholestene, 5-cholan-24-ol and eicosane as new protein kinase C activators and have shown that Z-Ser-Leu-NH₂, Z-Gly-Leu-NH₂, Z-Tyr-Leu-NH₂, cyclosporin A and cholestan-3, 5, 6-triol are protein kinase C inhibitors.     

甲状腺素对大鼠心脏细胞蛋白激酶C信号途径的影响

目的 :探讨甲状腺素对新生大鼠心脏细胞中蛋白激酶C(proteinkinaseC ,PKC)信号途径的影响。 方法 :培养新生大鼠心肌细胞及成纤维细胞 ,用 1%血清培养基或血管紧张素Ⅱ(angiotensinⅡ ,AngⅡ)处理细胞 2 4h后 ,加入甲状腺素(三碘甲状腺素原氨酸 ,triiodothyronine,T3 )继续培养 4 8h后 ,用PKC活性检测试剂盒检测细胞中PKC活性 ,用West ernblot的方法检测细胞中PKCα及PKCε的表达。结果 :在 1%血清培养基中 ,T3 能明显抑制心肌细胞中PKC活性 ,使心肌细胞中PKCε表达下降 ,对PKCα的表达却没有显著的影响 ;在心肌成纤维细胞中 ,无论是PKC活性还是PKCα及PKCε的表达均未观察到T3 的调控作用。预先用AngⅡ处理 2 4h后 ,心肌细胞及心肌成纤维细胞中PKC活性明显增加 ,PKCε的表达显著增加 ,随后用T3 处理后 ,心肌细胞中PKC活性及PKCε的表达明显降低 ;而心肌成纤维细胞中PKC活性没有发生显著性的变化。结论 :甲状腺素能明显抑制心肌细胞中PKC活性及PKCε亚型的表达 ,其对心肌细胞中PKC信号途径的调控作用可能在心肌的多种病理生理过程中起着重要的作用。     

PKC在慢性低氧大鼠肺动脉重构中的作用

目的 :探讨蛋白激酶C(PKC)在慢性低氧大鼠肺动脉重构中的作用。方法 :采用透射电镜、放射活性测定法、免疫组化、图像分析等方法综合进行评价。结果 :①肺动脉平均压 (mPAP)、右心室重量比 (RV LV S)显著高于对照组 (P <0 .0 1) ;②光镜下肺细小动脉管壁面积 管总面积 (WA TA)、肺细小动脉中膜平滑肌细胞核密度 (SMC)显著高于对照组 (P <0 .0 1) ;电镜显示肺动脉中膜平滑肌细胞增生 ,胶原纤维较对照组明显为多 ;③肺组织PKC总活性(PKCt)、胞膜PKC活性 (PKCm)、胞浆PKC活性 (PKCc)及PKCm PKCt的百分比显著高于对照组 (P <0 .0 1) ;④免疫组化显示肺细小动脉 (直径约 10 0～ 2 0 0 μm)PKC含量、Ⅰ型胶原含量显著高于对照组 (P <0 .0 1) ,Ⅲ型胶原组间无明显差异 (P >0 .0 5 ) ;⑤肺组织PKCt、PKCm、PKCm PKCt和肺动脉管壁PKC的表达与肺细小动脉中膜平滑肌细胞核密度 (SMC)、肺动脉管壁Ⅰ型胶原的表达均呈正相关。结论 :PKC参与慢性低氧肺动脉平滑肌细胞增殖、管壁胶原表达的调控 ,从而参与了低氧性肺动脉重构的过程     

Modulation of protein kinase C activity by NaF in bone marrow derived macrophages

Stimulation of murine bone marrow derived macrophages with NaF, prelabeled with [1-¹⁴C]oleate and [³H]inositol, increased the production of inositol phosphates and the release of 1,2-[¹⁴C]diacylglycerol (DAG). Moreover, NaF also induced activation of protein kinase C. These results indicate that bone marrow derived macrophages exhibit a phosphatidyl-4,5-bisphosphate phospholipase C activity, sensitive to NaF, which might be modulated by G-proteins. Activation of protein kinase C could have been mediated by NaF-induced release of DAG.     

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), -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 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. However, the electrophoresis/blotting experiments of tissues exposed for 5 hours (but not one hour) to kainate established clearly that , , and PKC are translocated from cytosolic and membrane compartments.