Neuroscience Letters, 22 (1981) 119-124 119 © Elsevier/North-Holland Scientific Publishers Ltd.

I M M U N O F L U O R E S C E N T L O C A L I Z A T I O N OF C Y C L I C G M P ,

C A L M O D U L I N A N D C Y C L I C G M P - D E P E N D E N T P R O T E I N

T H E C H O R O I D P L E X U S KINASE IN

RICHARD CUMMING*, WAI YIU CHEUNG, ROBERT W. WALLACE and ALTON L. STEINER**

Department of Medicine, University o f North Carolina, Chapel Hill, NC 27514 (U.S.A .)

and

(IV. Y.C. and R. IV. 14/.) Department of Biochemistry, St. Jude Children's Research Hospital and the University o f Tennessee Center for Health Sciences, Memphis, TN 38101 (U.S.A .)

(Received September 30th, 1980; Revised version received November 20th, 1980; Accepted December 9th, 1980)

An immunofluorescent technique has demonstrated that tissue-bound pools of cyclic GMP, calmodulin and cyclic GMP-dependent protein kinase are localized within the cytoplasm of the epithelial cells of the choroid plexus. In all other cell types and regions of the central nervous system previously examined, however, these molecules have shown contrasting immunofluorescent localization. These results suggest that the interaction in the choroid plexus may be related to the specialized physiological functions of the neuroglial epithelial cell.

The role o f cyclic G M P in the central nervous system is currently not well unders tood . It has been demons t ra ted in brain slices, however, that there is an

absolute requirement for Ca 2+ in order for neurotransmit ters and other agents to st imulate soluble guanyla te cyclase [7]. The impor tance o f Ca 2+ for both

maintaining basal cyclic G M P levels and eliciting responses in vitro, has also been observed in a wide variety o f tissues outside the central nervous system [19].

Calmodul in , a low molecular weight, high-affini ty, calcium-binding protein, has

recently been found to play an essential role in regulating a variety o f enzyme

systems in the central nervous system. These enzymes include phosphodiesterase [2],

synaptosomal C a / M g A T P a s e [20], and one class o f adenylate cyclase [26]. Calmodul in , funct ioning as an intraceUular calcium-receptor protein, may therefore

potential ly regulate cyclic G M P by modi fy ing calc ium-dependent enzymes. It has

*Present address: Department of Neurology, University of North Carolina, Chapel Hill, NC 27514, U.S.A. **Present address: Division of Endocrinology, University of Texas School of Medicine, Houston, TX 77025, U.S.A.

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been show, for example, that the degradation of cyclic GMP is catalyzed by a calmodulin-regulated low Km phosphodiesterase [11]. Employing specific and sensitive immunofluorescent techniques in a variety of systemic tissues, this laboratory has previously demonstrated localization of cyclic GMP and calmodulin in similar cellular compartments [8, 22]. Possible functional relationships between these molecules have been suggested from these data [9]. Studies in the central nervous system, however, have demonstrated contrasting cellular localization using the same antibodies, with cyclic GMP observed in the processes of fibrous astrocytes [3], and calmodulin distributed at neuronal sites (ref. 28 and unpublished observations).

In this paper we report that in one specialized cell type in the central nervous system, the non-neuronal epithelial cells of the choroid plexus, cyclic GMP and calmodulin show identical immunofluorescent localization. In addition, we have employed an antibody directed against cyclic GMP-dependent protein kinase, which has demonstrated localization at the same site. These observations are discussed with regard to the roles of cyclic GMP, Ca z+ and calmodulin in ion transport/secre- tory processes in these cells.

Immunofluorescent procedures were carried out essentially as described previously [3, 21]. Male Charles River CD rats of approximately 200 g were employed in the study. Antibodies against calmodulin, cyclic GMP and cyclic GMP- dependent protein kinase, have previously been characterized by immunodiffusion analysis and radioimmunoassay, and selected for immunocytochemical studies in our laboratory [1, 8, 9, 22, 23, 25]. Sagittal frozen sections of cerebellum and underlying brain stem were employed for studies on the choroid plexus of the IV ventricle. Coronal sections at the level of the caudate were used for study of the choroid plexus of the lateral ventricles.

To ensure specificity of staining patterns, application of buffer instead of rabbit antibodies produced no staining. Substitution of non-immune immunoglobulin at the same protein concentrations as the first rabbit antibodies, resulted in minimal non-specific staining at the apex of the ependymal cells or choroid plexus epithelial cells (see Fig. 1 and ref. 27). Liquid-phase absorption of antibodies with their respective antigens [21] was employed as a final test of specificity.

Comparison of the immunofluorescent localization of calmodulin (Fig. 1A and B) with that of cyclic GMP (Fig. 1C and D), demonstrates strikingly similar localizations of these molecules. Intense staining is shown within the cytoplasm of the cuboidal cells of choroid plexus epithelium, from either the IV or lateral ventricles. Fig. 1C demonstrates that staining is concentrated towards the apex of the cells, and is absent from a 'brush-border' membrane [17]. The immunofluorescent staining of cyclic GMP-dependent protein kinase (Fig. 1F), though diffuse in contrast to cyclic GMP and calmodulin, is found at the same site. In addition, staining with this antibody is observed within the epithelial cell nuclei and internal vasculature. Liquid-phase absorption of the antibodies (employing

a

I

E

i i I

G

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Fig. 1. Immunofluorescent localization of calmodulin, cyclic GMP and cyclic GMP-dependent protein kinase in the choroid plexus. A, B and E: choroid plexus of the lateral ventricle. C, D, F and G: choroid plexus of the IV ventricle. A: calmodulin immunofluorescence in the epithelial cells of the convoluted choroid plexus. Lines of dots represent non-specific immunofluorescence. × 160. B: calmodulin immunofluorescence within the cytoplasm of the cuboidal epithelial cells. × 640. C: cyclic GMP immunofluorescence within two epithelial cells. The intense perinuclear staining is distributed towards the apex of the cells. × 2160. D: cyclic GMP immunofluorescence within the cytoplasm of the cuboidal epithelial cells, x 640. E: liquid-phase absorption control of calmodulin antibody with bovine brain calmodulin. Dots represent non-specific immunofluorescence, x 640. F: cyclic GMP-dependent protein kinase immunofluorescence in the cytoplasm and nuclei of the epithelial cells, and the internal vasculature. × 640. G: liquid-phase absorption control of cyclic GMP-dependent protein kinase antibody with the purified enzyme from bovine lung. x 640. (All reduced by one-third in production.)

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optimal ratios of antibody to antigen) is shown in Fig. 1E and G for calmodulin and cyclic GMP-dependent protein kinase, respectively.

Previous studies from this laboratory have demonstrated markedly similar immunofluorescent staining patterns between cyclic GMP and calmodulin in somatic tissues such as liver, intestine and muscle [8, 9, 22]. The results in this paper confirm these observations within the cytoplasm of the epithelial cells of the choroid plexus. However, in all other regions and cell types of the central nervous system examined, immunofluorescent staining with the cyclic GMP antibody has been restricted to the processes of fibrous astrocytes [3]. This is in contrast to the neuronal distribution of calmodulin, which is primarily localized at post-synaptic sites (ref. 28 and unpublished data). The identical localization of both of these molecules within one cell type in the central nervous system may therefore be related to specialized functions at this site.

A further indication that the parallel localization may be of functional significance, is the observation that the neuroglial ependymal cells which border the ventricles (and are therefore also in contact with cerebrospinal fluid) are unstained for cyclic GMP and calmodulin. These cells are derived embryologically from the same cell type as the epithelial cell of the choroid plexus, but they have not become specialized for both ionic transport and secretion of cerebrospinal fluid [6]. We have previously demonstrated immunofluorescent localization of cyclic GMP to the subependymal glial fiber plexus [3], which is formed from basal projections of the ependymal cells. An example of structural specialization in the choroid plexus is the loss of these basal projections from the epithelial cells.

The discrete cytoplasmic distribution of cyclic GMP and calmodulin may be associated with structures involved in secretion, such as the Golgi apparatus and RNA granules, which are particularly abundant there [15]. The asymmetric cytoplasmic staining (see Fig. l C for example) may, however, represent association of these molecules with mitochondria, which are frequently distributed towards the apex of the epithelial cell l17]. Application of the immunocytochemical technique to the ultrastructural level will be required to further characterize the subcellular localization of these molecules.

Cyclic GMP has recently been demonstrated using biochemical techniques in the rabbit choroid plexus in vitro. Stimulation by cholinergic agonists caused elevation of cyclic GMP levels and release of the nucleotide into the medium [18]. Cyclic GMP has also been implicated in secretory processes in other tissues, employing both biochemical and immunocytochemical techniques [12]. The possible function of calmodulin in the choroid plexus may be to influence ATP-dependent Ca 2+ transport and anion fluxes, as demonstrated in other tissues [10, 13, 24].

Employing an antibody directed against cyclic GMP-dependent protein kinase, we have observed specific immunofluorescence in the epithelial cells of the choroid plexus. Although the staining is neither as intense nor discrete as for cyclic GMP or calmodulin, this localization suggests that cyclic GMP is bound to this specific

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receptor protein in the cytoplasm of the epithelial cells [23]. The presence of cyclic GMP-dependent protein kinase at other sites in the choroid plexus may be due to functions of the protein unrelated to interaction with cyclic GMP. Alternatively, the soluble pool of cyclic GMP or calmodulin may be lost during buffer washing of the unfixed tissue sections [4]. We have observed that a variety of fixatives employed to prevent possible losses, however, have eliminated staining. This presumably results from denaturation of the antigenic determinants of the molecules [22].

We have recently demonstrated that the regulatory and catalytic subunits of cyclic AMP-dependent protein kinase are also detected in the choroid plexus by immunofluorescence. These subunits show different patterns of subcellular localization from cyclic GMP-dependent protein kinase, however [5].

In conclusion, this study has demonstrated potential sites of interaction between tissue-bound pools of cyclic GMP, calmodulin and cyclic GMP-dependent protein kinase. This interaction in the central nervous system, may be restricted to the epithelial cells of the choroid plexus. Future studies will require manipulation of the neural inputs to the choroid plexus [14, 16] in order to examine functional relationships in this specialized cell type.

This study was supported in part by USPHS Grant AM 19438. We are grateful to Dr. J.A. Beavo for the antibody to cyclic GMP-dependent protein kinase.

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