Localization of protein kinase C subspecies in the rabbit retina

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Neuroscience Letters 177 (1994) 15-18

HEUROSCIINC[ LETTERS

Localization of protein kinase C subspecies in the rabbit retina J a r i K o i s t i n a h o a'b'*, S t e p h e n M . S a g a r b aDepartment of Biomedical Sciences, University of Tampere, PO Box 607, FIN-33101 Tampere, Finland bDepartment of Neurology (127), University of California, San Francisco and Department of Veterans Affairs Medical Center, 4150 Clement Street, San Francisco, CA 94121, USA Received 4 April 1994; Revised version received 19 May 1994; Accepted 19 May 1994

Abstract

The localization of PKC subspecies ~, r, y, e and ( was studied immunocytochemicallyin the rabbit retina. Conventional, Ca2+-sensitive PKC subtypes ~, r, y were all localized in different neuronal populations. The ~'-subspecies, which does not require Ca2÷for activation, was colocalizedwith PKC-~. PKC-e, which is independent of Ca2÷and DAG, was colocalizedwith PKC-fl. Some populations of neurons, including cone bipolar cells, contained none of the PKC-subspecies studied. These results imply a cellular segregation of different signaling pathways in mammalian retina. Key words: Bipolar cell; Horizontal cell; Amacrine cell; Signal transduction

In the central nervous system, protein kinase C (PKC) participates in the response to neurotransmitters, growth factors and hormones [7]. The enzyme is activated by diacylglycerol produced by agonist-induced hydrolysis of phospholipids [7]. Molecular cloning has revealed ten subspecies of PKC (or,ill, ~II, Y, £~, e, ~, 0, ( , ~) [7], seven of which are found in the brain. Since the subspecies show different enzymological properties, tissue distribution, and intracellular localization, individual members of the PKC family likely have different functions in signal transduction [7,9]. Previous studies have shown the presence of several PKC subspecies in the retinas of lower vertebrates and rodents [1,2,10,16]. Here we have applied an immunocytochemical technique to the rabbit retina to explore the cellular localization of 0~,fl, 7, e and (-subtypes of PKC. Adult, male New Zealand white rabbits, kept on a 12:12 h light,lark cycle, were taken to a lighted room 6 h prior to sacrificed. Posterior eye cups were fixed in freshly prepared 20% methanol, 2% paraformal* Corresponding author: Neural Injury Research Laboratory, Department of Biomedical Sciences, University of Tampere, P.O. Box 607, FIN-33101 Tampere, Finland. Fax: 358-31-2156170; E-mail: [email protected] 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0304-3940(94)00412-4

dehyde, 0.1 M phosphate buffer, pH 7.4, for 2 h at 4 ° C. The samples were immersed for 12 h in 20% sucrose, 0.1 M phosphate buffer (PB) and cryosectioned at 20/lm. The sections were rinsed in PB and incubated in PB-G (0.1% bovine serum albumin, 0.2% Triton X-100, 2% normal goat serum in PB) when polyclonal rabbit antibodies were used or in PB-H (0.1% bovine serum albumin, 0.2% Triton X-100, 2% normal horse serum in PB) when monoclonal antibodies were used for 30 min. Rabbit PKC-0~, -e and -( antibodies (GIBCO BRL, Gaithersburg, MD), raised against peptides corresponding to amino acids 313-326, 726-737, and 577-592, respectively, were added at a dilution of 1:100 in PB-G. An affinity-purified PKC-fl antibody (provided by Dr. Michael Iadarola) raised to a synthetic peptide corresponding to amino acids 307-327 [12] was added at a dilution of 1:300. Monoclonal PKC-c~,fl (clone MC5, Amersham, Arlington Heights, IL), PKC-fl (MC-2a, Seikagaku, Tokyo, Japan) and PKC-?' antibodies (UBI, Lake Placid, NY), which were raised against purified whole proteins, were used at dilutions of 1:100-250 in PB-H. Previous studies have demonstrated specificity of the antibodies in Western blotting experiments [5,11,12]. The sections were incubated at room temperature for 24 h; and, following washing in PB, sites of antibody

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J. Koistinaho, S.M. Sagar/Neuroscience Letters 177 (1994) 15 1,~

Fig. 1. Retinal cross-section immunostained for P K C - ~ (A), PKC-fl with the monoclonal (B) and polyclonal (C) antibody, and for PKC-e (D). IN, inner nuclear layer. One arrow points to an immunoreactive neuron in the ganglion cell layer and the double arrow points to an immunoreactive neuron in the INL. Bar = 25/lm.

binding were visualized using FITC- and rhodamineconjugated IgG (1:40, Boehringer Mannheim, Indianapolis, IN) secondary antibodies. Sections were mounted in PB-glycerol, and examined using a Leitz fluorescence microscope. Both PKC-~ and MC5 (PKC-~,fl) antibodies displayed strong immunoreactivity to a subpopulation of bipolar cells; double-labelling experiments showed that these antibodies were colocalized in the same cells (Figs. I A and 2A). These observations are consistent with previous studies that have shown that MC5 antibody and PKC-~ antibody stain rod bipolar cells in mammalian retinas [6,11,16]. In addition, MC5 antibody displayed immunoreactivity (IR) to the inner segment of photoreceptors and some amacrine cells in the inner nuclear

layer (INL). These structures were only very slightly stained with PKC-~ antibody. Monoclonal antibody to PKC-fl strongly labelled a sparse population of amacrine cells (Fig. 1B). These cells were in the inner sublayer of the INL and sent a dense meshwork of processes in the inner plexiform layer (IPL) throughout the retina. The polyclonal PKC-fl antibody also faintly labelled a few neuronal bodies in the INL, but more strongly labelled horizontal cells, nerve fibers in the IPL, some neuronal cell bodies in the ganglion cell layer (GCL) and bundles of ganglion cell axons in the nerve fiber layer (Fig. 1C). A strong PKC-e-IR was seen in horizontal cells, in a few amacrine cells, in the inner sublayer of the INL and in some neurons in the GCL (Fig. 1D). In addition, two

J. Koistinaho, S.M. Sagar / Neuroscience Letters 177 (1994) 15-18

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Fig. 2. A retinal cross-sectiondouble-stainedfor PKC-ctwith a monoclonal MC5 antibody (A) and PKC-~"(B) as described in the text. The subspecies are colocalizedin rod bipolar cells. C: PKC-~ immunoreactivity is found in a population of small amacrine cells (arrows). ON, outer nuclear layer; IN, inner nuclear layer. Bar = 25/tm.

separate bands of horizontally running nerve fibers in the I N L and bundles of ganglion cell axons in the G C L were stained with PKC-e-antibody. Although antibodies to P K C - e and PKC-fl demontrated similar staining patterns, double-label experiments could not be done because both antibodies were raised in the rabbit. PKC-~'-IR was seen in a subpopulation of bipolar cells. Since the labelling pattern resembled the staining with PKC-~ and MC5 antibodies, retinal sections were double-labelled with P K C - ( and MC5 antibodies. Fig. 2A,B. demonstrates that PKC-~'-IR is localized in the same subpopulation of bipolar cells as PKC-~. P K C - y IR was seen as a bright rim on the outer membrane of small cells in the middle of the inner nuclear layer (2C). PKC-~'-IR cells were more numerous than PKC-fl-IR cells in the I N L and they did not send out immunoreactive processes. Preabsorbtion controls performed for ~, fl, e and ( antibodies were devoid of IR. Exclusion of PKC-7 or PKC-~,fl antibodies from incubation medium eliminated specific immunostaining. P K C subtypes have been categorized according to ' their enzymological properties [7]. ~, fli, fl1~ and 7/PKCs (Group A) are conventional subtypes, which are activated by Ca z+, D A G and phospholipids [7]. G r o u p B consists of newly discovered PKCs ~, e, 0 and r/, which do not require Ca z+ for activation [7]. Subtypes of both Group A and G r o u p B can be activated by phorbol esters [7]. Subtypes ( and 2 are atypical PKCs (Group C) because they cannot be activated by DAG, Ca 2+ or phorbol esters [7]. The present study shows that all these PKC categories are present in the rabbit retina. The subtype, which can be activated without D A G and Ca 2÷, was localized in rod bipolar cells with the c~-subtype, which requires D A G and Ca 2+ to be activated. Both ~" and ~ subtypes were found in cell bodies, dendrites, axons and nerve terminals of these cells. Similarly, fl and e subspecies were colocalized in the same retinal structures. Our data suggest that several P K C second messenger pathways coexist in same neurons with a similar subcellular localization. In the rabbit retina, there are two types of cone bipolar cells: ON cone bipolar cells, which, similar to rod bipolar cells, depolarize in the response to light, and O F F cone bipolar cells, which hyperpolarize in response to light [14]. The presynaptic neurotransmitter of all bipolar cells is glutamate, but rod bipolar cells and O N cone bipolar cells are thought to have different types of glutamate receptors than O F F cone bipolar cells [14]. The present

results demonstrate the presence of two PKC subspecies, and ~" in rod bipolar cells. The lack of PKC enzymes in cone bipolar cells indicates that the subtypes of retinal bipolar cells differ substantially in signal transduction pathways.

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J. Ko&tinaho, S.M. Sagar/Neuroscience Letters 177 I 1994) 15 I,~

Monoclonal PKC-fl antibody recognizes a subpopulation of amacrine cells, whereas polyclonal PKC-fl antibody also stains horizontal cells and ganglion cell axons. Using another polyclonal antibody, Osborne et al. [10] found PKC-fl immunoreactivity in amacrine and/or ganglion cells of the rabbit retina. PKC-fl mRNA has been shown to be differentially spliced to yield fl~ and #If subtypes, which differ only in a short sequence in their carboxyl-terminal regions [19], but which show distinct localization in the brain [8]. Because the polyclonal antibody is directed to amino acids shared by each of these differentially spliced PKCs, it would be expected to cross-react with both fl~ and fin subtypes [12]. The antigen for the monoclonal PKC-fl antibody was purified from the brain, which has been shown to have fl~ and fl~l subspecies in a 8:55 ratio [13]. It is likely that both PKCfl antibodies applied in the present study demonstrate PKC-fl proteins, but the monoclonal antibody may have greater affinity to #if subtype than to #I" In the cerebellum, PKC-7, but not PKC-~ or PKC-fl, mediates GABA release from Purkinje cells [13]. In the rabbit retina, the ~' subtype was localized to numerous amacrine cells in the INL, where the majority of the neurons contain GABA [14]. Even though PKC-~' was not found in nerve terminals it may be present in them in undetectable concentrations. Another possibility is that PKC-7 is not present in nerve terminals, and is not therefore directly associated with neurotransmitter release. PKC-e is enriched in the brain [5], but there are no reports of its cellular localization. PC12 cells have been reported to contain all PKC subtypes, including PKC-e [5]. The localization of the PKC-e subtype in horizontal, amacrine and ganglion cells indicates that PKC-e has an important role in neuronal signal transduction in the retina. These observations demonstrate that each PKC subtype has a specific, but not necessarily unique, cellular distribution in retinal neurons. This implies a cellular segregation of different signaling pathways in mammalian retina. The anatomic segregation of the various PKC isozymes into different retinal cell populations may facilitate the study of their function in neuronal signal transduction. This study was supported by the National Instute of Neurological Diseases and Stroke (NS27488).

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