Photoaffinity labeling of parotid saliva, pancreatic and seminal fluids with the cyclic AMP analogue,. 32P-labeled-8-azido-cyclic AMP, revealed the pres- ence of ...
Regulatory subunits of cyclic AMP-dependent protein kinase: presence in granules and secretion by exocrine and endocrine cells
A. R. HAND* National Institute of Dental Research, National Institutes of Health, Building 10, Room 1AI3, Bethesda, Maryland 20892, USA
and M. I. MEDNIEKS Department of Pediatrics, University of Chicago, Wyler Childrens Hospital, Chicago, Illinois 60637, USA •Author for correspondence
Summary
Cyclic AMP-dependent protein kinase (cAPK) is the intracellular mediator of signal transduction events involving the adenylate cyclase-cyclic AMP system. A monoclonal antibody (MAb BB1) to the type II regulatory subunit (RII) of cAPK was used in a post-embedding immunogold-labeling procedure to determine the ultrastructural localization of RII in several different secretory cells of the rat. Label was present in nuclei, especially over the heterochromatin, and in the cytoplasm, particularly in areas containing rough endoplasmic reticulum. Immunolabeled RII -was also present in secretory granules of the parotid gland, exocrine and endocrine pancreas, seminal vesicle, anterior and intermediate pituitary, and intestinal endocrine cells. Photoaffinity labeling of parotid saliva, pancreatic and seminal fluids with the cyclic AMP analogue, 32 P-labeled-8-azido-cyclic AMP, revealed the pres-
Introduction Cyclic AMP-dependent protein kinase (cAPK) participates in the regulation of various cellular activities (see reviews by Flockhart & Corbin, 1982; Lohman & Walter, 1984). These include both nuclear and cytoplasmic events; among the latter, in many glandular tissues, is the process of exocytosis. The precise mechanisms involved in determining the specificity of cAPK actions are not well known. Intracellular redistribution of the holoenzyme or of its subunits may be critically important in cyclic AMP-mediated responses of cells to hormonal stimulation. Immunocytochemical experiments and subcellular fractionation have shown that the catalytic (C) and regulatory (R) subunits are present in the nuclei and cytoplasm of many cell types (for references see Kuettel et al. 1985; Mednieks et al. 1987), and occasionally are associated with other organelles such as the Golgi apparJournal of Cell Science 93, 675-681 (1989) Printed in Great Britain © The Company of Biologists Limited 1989
ence of cyclic AMP-binding proteins •with electrophoretic mobilities similar to those of authentic cAPK regulatory subunits. These results confirm our previous observations on the localization of cAPK regulatory subunits in the rat parotid using polyclonal antibodies, and extend them to a number of other exocrine and endocrine cells. The apparent widespread occurrence of cAPK subunits in secretory granules and secretory fluids suggests that cAPK may be involved in specific intragranular regulatory and/or phosphorylation events, or that it has an unidentified extracellular function.
Key words: cyclic AMP-dependent protein kinase, immunocytochemistry, protein A-gold, rat, exocrine and endocrine cells, secretion.
atus (Nigg et al. 1985; De Camilli et al. 1986), cytoskeletal elements (Vallee et al. 1981; Browne et al. 1982; De Camilli et al. 1986) and the acrosome (Pariset et al. 1986). The results of our previous immunocytochemical studies of the distribution of cAPK R subunits in rat parotid acinar cells showed that in addition to their expected localization in the nucleus and in the cytoplasm, both the type I (RI) and type II (RII) regulatory subunits were present in the contents of the secretory granules (Mednieks et al. 1987). Earlier, we demonstrated that cyclic AMP-binding proteins secreted in response to /J-adrenergic stimulation of the parotid gland can be detected in ductal saliva by photoaffinity labeling with 32P-labeled-8-azido-cyclic AMP (Mednieks & Hand, 1984). In this report, we demonstrate that immunoreactive cAPK R subunits are present in the secretory granules of several other rat exocrine and endocrine cells, 675
and that the secretory fluids of the exocrine pancreas and the seminal vesicle also contain cyclic AMP-binding proteins. Materials and methods Immunological reagents A mouse monoclonal antibody (MAb) to RII from the rat parotid gland was prepared as described (Siraganian et al. 1983; Mednieks et al. 1989). The 40-70K (K = \&MT) chromatographic peak of a secretory granule preparation was used as the original antigen and the hybrid cells were screened against type II cAPK from bovine heart (Sigma Chemical Co., St Louis, MO). The IgM antibodies secreted by the clone BBl IIB1 (MAb BBl) were inhibited in competitive enzyme-linked immunosorbent assays (ELISA) by preincubation with either
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the original antigen or with bovine heart cAPK. MAb BBl immunoprecipitated labeled proteins with the same electrophoretic mobility as RII from I-labeled total soluble proteins of rat and liver homogenates (Hand & Jungmann, 1989), as well as RII from type II cAPK (bovine heart; Sigma) and from rat and human parotid saliva that had been photoaffinity labeled with 32P-labeled-8-azido-cyclic AMP (Mednieks et al. 1989). This MAb reacted with protein bands of 52 and 54K (electrophoretically distinct RII isoforms) on immunoblots of parotid gland extracts and parotid saliva (Mednieks et al. 1989). Antigen-absorbed antibody was prepared by incubating the reconstituted ascites fluid (diluted 1: 30) overnight at 4°C with a calculated tenfold excess of bovine heart cAPK or with rat parotid saliva, then diluting to the final concentration before immunostaining. Unlabeled secondary antibody (affinity-purified goat anti-mouse IgM) was obtained from Cappel (West Chester, PA), and 5 and 15 nm protein A-gold solutions were
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Fig. 1. Inimunogold labeling of thin sections of exocrine cells with MAb BBl. A. Nucleus (n) and secretory granules (sg) of parotid acinar cell labeled with MAb BBl. Few gold particles are seen over euchromatin; mitochondria (in) are unlabeled. The electron-lucent appearance of the granules is due to omission of OsC>4 post-fixation. X27000. B. Zymogen granules of pancreatic acinar cell labeled with MAb BBl. X22000. C. Apical portion of seminal vesicle cell labeled with MAb BBl. Dense cores of secretory granules and luminal content (/) are heavily labeled. X390O0. Bars, 0-5 /*m.
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purchased from Janssen Life Sciences Products (Beerse, Belgium). Tissue preparation and immunogold labeling Tissues were obtained from adult male Sprague-Dawley rats after fixation of anesthetized (chloral hydrate, 400mgkg~ , i.p.) animals by vascular perfusion with 1% phosphatebuffered glutaraldehyde. Parotid glands, pancreas, seminal vesicle, duodenum and pituitary glands were fixed for 1 h, rinsed in 0-1 M-phosphate buffer, pH7 - 4, containing 5 % sucrose, dehydrated in ethanol, substituted with acetone or propylene oxide, and embedded in Epon. Thin sections were collected on nickel grids and immunostained by a modified protein A-gold method as described (Mednieks et al. 1989). Briefly, the sections were etched with sodium metaperiodate, treated with 1 % bovine serum albumin (BSA) in Tris-buffered saline, incubated for 16-24 h at 4°C with reconstituted MAb BB1 ascites diluted in 0-02M-Tris-HCl, p H 7 4 , containing 0-5 M-NaCl and 0-1 % Tween 20, incubated for 1 h at 25°C with unlabeled, affinity-purified goat anti-mouse IgM, and finally incubated for 30min at 25°C with protein A-gold. The sections were rinsed with Tris-buffered saline after each incubation, and with distilled water prior to staining with uranyl acetate and lead citrate. The sections were observed and photographed in a Zeiss EM 10A transmission electron microscope. Quantitative analysis of relative labeling intensities was performed as described (Mednieks et al. 1987, 1989) on micrographs of cells labeled with 15 nm gold particles using a ZIDAS digitizing system (Carl Zeiss, Inc., Thornwood, NY) interfaced with a microcomputer. Statistical analyses (
