Bacterial cell wall-induced hepatic granulomas. An in vivo model of T cell-dependent fibrosis.

In vitro studies implicate a molecular link between inflammatory mononuclear cells and alterations in fibroblast growth and function. We have extended these observations in an experimental animal model in which we document the T cell-dependence of fibrosis that occurs after activation of the cell-mediated immune system by specific antigen. Chronic granulomatous lesions were induced in the livers of susceptible rats by the intraperitoneal injection of group A streptococcal cell walls (SCW). The development of granulomas that are composed primarily of lymphocytes and macrophages was associated with the recruitment and proliferation of connective tissue cells. Furthermore, this expanded population of fibroblasts generated a collagenous structure consisting primarily of types I and III collagen around the granuloma. The progression of these chronic inflammatory lesions leads to the formation of fibrotic nodules throughout the livers of the treated animals. Intact granulomas, as well as mononuclear cells derived from the granulomas, spontaneously elaborated a soluble factor(s) that stimulates fibroblast proliferation. Physicochemical analysis revealed that the primary granuloma-derived peak of fibroblast growth activity corresponded to an apparent Mr of 40,000, which is consistent with a previously described T lymphocyte--derived fibroblast-activating factor (FAF) in guinea pig and human. Furthermore, the fibrosis that occurs in the granuloma is apparently T cell--dependent, since no fibrotic lesions developed in SCW-injected athymic nude rats nor in SCW-injected animals treated with the T cell inhibitor, cyclosporin A (CsA). Mononuclear cells from neither of these functionally T cell--deficient animals could generate FAF activity. These data show a role for T lymphocyte--derived cytokines in the development of hepatic fibrosis in SCW-injected rats.

Rapid onset synovial inflammation and hyperplasia induced by transforming growth factor beta.

After intraarticular injection of TGF-beta 1 or TGF-beta 2, marked swelling and erythema of the injected joints were apparent within 12-24 h. On a scale of 0 to 4, by day 3, the TGF-beta-treated joints had articular indices (AI) of 3.6 +/- 0.5 to 4.0 +/- 0.0 compared with no response for the vehicle-injected contralateral joints. Histopathologic evaluation revealed a predominantly mononuclear phagocyte infiltrate with some neutrophils and T lymphocytes, consistent with active inflammation. The monocytic pattern of leukocyte infiltration at 2-3 d was comparable to that seen in animals with antigen-induced arthritis after 2-3 wk. Extensive synovial fibroblast hyperplasia became apparent within 48 h, likely as a result of TGF-beta induction of growth factor synthesis by the accumulating monocytes. TGF-beta 2, a homologue of TGF-beta 1, was found to induce a similar level of synovitis and synovial hyperplasia consistent with its parallel monocyte and fibroblast chemotactic properties and ability to induce transcription and translation of monocyte/macrophage-derived growth factors. These data suggest that TGF-beta, released by platelets and activated inflammatory cells, may play a direct role in leukocyte recruitment and activation in arthritic and other chronic inflammatory lesions.

Immunocytochemical localization of MG1 mucin in human bulbourethral glands.

Salivary mucins MG1 and MG2 have been found in the oral cavity where they perform several functions such as the formation of the mucous layer covering the oral mucosa and teeth. Recent studies have demonstrated their presence in other organs and tissues. The aim of this study was to determine their expression in human bulbourethral (Cowper's) glands. Normal bulbourethral glands were obtained at surgery and fixed in a mixture of 1% paraformaldehyde-1.25% glutaraldehyde in 0.1 M cacodylate buffer and embedded in Epon resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells were intensely reactive with anti-MG1, whereas no labeling was detected for MG2. These results indicate that MG1 is not exclusively a salivary component and furthermore show that bulbourethral glands represent a significant source of the MG1 detected in human seminal plasma.

The fine structure of von Ebner's gland of the rat.

The fine structure of von Ebner's gland was studied in untreated rats and rats stimulated to secrete by fasting-refeeding or injection of pilocarpine. Cytological features were similar to those reported for pancreas and parotid gland. Abundant granular endoplasmic reticulum filled the basal portion of the cell, a well-developed Golgi complex was located in the vicinity of the nucleus, and the apical portion of the cell was filled with dense secretory granules. Dense heterogeneous bodies resembling lysosomes were closely associated with the Golgi complex. Coated vesicles were seen in the Golgi region and also in continuity with the cell membrane. Granule discharge occurred by fusion of the granule membrane with the cell membrane at the secretory surface. Successive fusion of adjacent granules to the previously fused granule formed a connected string of granules in the apical cytoplasm. Myoepithelial cells were present within the basement membrane, and nerve processes were seen adjacent to acinar and myoepithelial cells. Duct cells resembled the intercalated duct cells of the major salivary glands.

Relationship between the Golgi apparatus, GERL, and secretory granules in acinar cells of the rat exorbital lacrimal gland.

The method of secretory granuleformation in the acinar cells of the rat exorbital lacrimal gland was studied by electron microscope morphological and cytochemical techniques. Immature secretory granules at the inner face of the Golgi apparatus were frequently attached to a narrow cisternal structure similar to GERL as described in neurons by Novikoff et al. (Novikoff, P. M., A. B. Novikoff, N. Quintana, and J.-J. Hauw. 1971. J. Cell Bio. 50:859-886). In the lacrimal gland. GERL was located adjacent to the inner Golgi saccule, or separated from it by a variable distance. Portions of GERL were often closely paralleled by modified cisternae of rough endoplasmic reticulum (RER), which lacked ribosomes on the surface adjacent to GERL. Diaminobenzidine reaction product of the secretory enzyme peroxidase was localized in the cisternae of the nuclear envelope, RER, peripheral Golgi vesicles, Golgi saccules, and immature and mature secretory granules. GERL was usually free of peroxidase reaction product or contained only a small amount. Thiamine pyrophosphatase reaction product was present in two to four inner Golgi saccules; occasionally, the innermost saccule was dilated and fenestrated, and contained less reaction product than the next adjacent saccule. Acid phosphatase (AcPase) reaction product was present in GERL, immature granules, and, rarely, in the innermost saccule, but not in the rest of the Golgi saccules. Thick sections of AcPase preparations viewed at 100 kV revealed that GERL consisted of cisternal, and fenestrated or tublular portions. The immature granules were attached to GERL by multiple connections to the tublular portions. These results suggest that, in the rat exorbital lacrimal gland, the Golgi saccules participate in the transport of secretory proteins, and that GERL is involved in the formation of secretory granules.

Uptake and fate of luminally administered horseradish peroxidase in resting and isoproterenol-stimulated rat parotid acinar cells.

The formation and fate of apical endocytic vesicles in resting and isoproterenol-stimulated rat parotid acinar cells were studied using luminally administered horseradish peroxidase (HRP) to mark the vesicles. The tracer was taken up from the lumen by endocytosis in small, smooth-surfaces "c"- or ring-shaped vesicles. About 1 h after HRP administration the vesicles could be found adjacent to the Golgi apparatus. At later times HRP reaction product was localized in multivesicular bodies and lysosomes; in isoproterenol-stimulated cells it was also present in autophagic vacuoles. HRP reaction product was never localized in any structure associated with secretory granule formation. These results suggest that the apical endocytic vesicles play a role in membrane recovery, but that they are degraded and not reutilized directly in secretory granule formation. Additionally, it was found that when isoproterenol was injected before HRP administration, the apical junctional complexes became permeable to the tracer, allowing it to gain access to the lateral and basal intercellular spaces. This permeability may provide an additional route whereby substances in the extracellular fluid could reach the saliva.

The structural organization of the septate and gap junctions of Hydra.

The septate junctions and gap junctions of Hydra were studied utilizing the extracellular tracers lanthanum hydroxide and ruthenium red. Analysis of the septate junction from four perspectives has shown that each septum consists of a single row of hexagons sharing common sides of 50-60 A. Each hexagon is folded into chair configuration. Two sets of projections emanate from the corners of the hexagons. One set (A projections) attaches the hexagons to the cell membranes at 80-100-A intervals, while the other set (V projections) joins some adjacent septa to each other. The septate junctions generally contain a few large interseptal spaces and a few septa which do not extend the full length of the junction. Basal to the septate junctions the cells in each layer are joined by numerous gap junctions. Gap junctions also join the muscular processes in each layer as well as those which connect the layers across the mesoglea. The gap junctions of Hydra are composed of rounded plaques 0.15-0.5 micro in diameter which contain 85-A hexagonally packed subunits. Each plaque is delimited from the surrounding intercellular space by a single 40-A band. Large numbers of these plaques are tightly packed, often lying about 20 A apart. This en plaque configuration of the gap junctions of Hydra contrasts with their sparser, more widely separated distribution in many vertebrate tissues. These studies conclude that the septate junction may possess some barrier properties and that both junctions are important in intercellular adhesion. On a morphological basis, the gap junction appears to be more suitable for intercellular coupling than the septate junction.

Alteration of tight junctional permeability in the rat parotid gland after isoproterenol stimulation.

The permeability of junctional complexes to ultrastructural tracers of different molecular weight and the freeze-fracture appearance of junctional structure were investigated in the resting and stimulated rat parotid gland. Tracers were administered retrogradely via the main excretory duct, and allowed to flow by gravity (16 mmHg) into the gland for 15-60 min. Secretion was induced in some animals by intraperitoneal injection of isoproterenol. In resting glands, the tracers microperoxidase , cytochrome c, myoglobin, tyrosinase (subunits), and hemoglobin were restricted to the luminal space of the acini and ducts. In glands stimulated 1-4 h before tracer administration, reaction product for microperoxidase , cytochrome c, myoglobin, and tyrosinase was found in the intercellular and interstitial spaces, whereas hemoglobin was usually retained in the lumina. In contrast, horseradish peroxidase and lactoperoxidase appeared to penetrate the tight junctions and reaction product was localized in the extracellular spaces in both resting and stimulated glands. Diffuse cytoplasmic staining for horseradish peroxidase and lactoperoxidase was frequently observed in acinar and duct cells. The distribution of horseradish peroxidase was similar in both Sprague-Dawley and Wistar-Furth rats, and at concentrations of 0.1-10 mg/ml in the tracer solution. Freeze-fracture replicas of stimulated acinar cells revealed an increased irregularity of the tight junction meshwork, but no obvious gaps or discontinuities were observed. These findings indicate that (a) tight junctions in the resting rat parotid gland are impermeable to tracers of molecular weight greater than or equal to 1,900; (b) stimulation with isoproterenol results in a transient increase in junctional permeability allowing passage of tracers of molecular weight less than or equal to 34,500; (c) junctional permeability cannot be directly correlated with junctional structure; and (d) the behavior of horseradish peroxidase and lactoperoxidase in the rat parotid gland is inconsistent with their molecular weights. Cell membrane damage due to the enzymatic activity or binding of these two tracers may account for the observed distribution.

Stimulation of Xenopus oocyte maturation by inhibition of the G-protein alpha S subunit, a component of the plasma membrane and yolk platelet membranes.

Oocytes of Xenopus laevis undergo maturation when injected with an affinity-purified antibody against the COOH-terminal decapeptide of the alpha subunit of the G-protein Gs, an antibody that inhibits Gs activity. Germinal vesicle breakdown, chromosome condensation, and polar body formation occur, with a time course similar to that for oocytes treated with progesterone. The alpha S antibody-injected oocytes also acquire the ability to be activated by sperm. Coinjection of the catalytic subunit of cAMP-dependent protein kinase, or incubation with cycloheximide, inhibits maturation in response to injection of the alpha S antibody; these experiments show that the alpha S antibody acts at an early point in the pathway leading to oocyte maturation, before formation of maturation promoting factor, and like progesterone, its action requires protein synthesis. Immunogold electron microscopy shows that alpha S is present in the yolk platelet membranes as well as the plasma membrane. These results support the hypothesis that progesterone acts by inhibiting alpha S, and suggest that the target of progesterone could include yolk platelet membranes as well as the plasma membrane.

Binding and uptake of 125I-insulin into rat liver hepatocytes and endothelium. An in vivo radioautographic study.

Electron microscope radioautography has been used to study hormone-receptor interaction. At intervals of 3, 10, and 20 min after the injection of 125I-insulin, free hormone was separated from bound hormone by whole body perfusion with modified Ringer's solution. The localization of bound hormone, fixed in situ by perfusion with glutaraldehyde, was determined. At 3 min, 125I-insulin has been shown to be exclusively localized to the hepatocyte plasmalemma (Bergeron et al., 1977, Proc. Natl. Acad. Sci. U. S. A., 74:5051--5055). In the present study, quantitation indicated that 10(5) receptors were present per cell and distributed equally along the sinusoidal and lateral segments of the hepatocyte plasmalemma. At later times, label was found in the Golgi region. At 10 min, both secretory elements of the Golgi apparatus and lysosome-like vacuoles were labeled, and at 20 min the label was especially concentrated over the latter vacuoles. Acid phosphatase cytochemistry showed that the vacuoles did not react and therefore were presumed not to be lysosomal. These Golgi vacuoles may constitute a compartment involved in the initial degradation and/or site of action of the hormone. Control experiments were carried out at all time intervals and consisted of parallel injections of radiolabeled insulin with excess unlabeled hormone. At all times in controls, label was diminished over hepatocytes and was found primarily over endothelial cells and within the macropinocytotic vesicles and dense bodies of these cells. Kupffer cells and lipocytes were unlabeled after the injection of 125I-insulin with or without excess unlabeled insulin.

Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1.

The sphingolipid metabolite sphingosine-1-phosphate (SPP) has been implicated as a second messenger in cell proliferation and survival. However, many of its biological effects are due to binding to unidentified receptors on the cell surface. SPP activated the heterotrimeric guanine nucleotide binding protein (G protein)-coupled orphan receptor EDG-1, originally cloned as Endothelial Differentiation Gene-1. EDG-1 bound SPP with high affinity (dissociation constant = 8.1 nM) and high specificity. Overexpression of EDG-1 induced exaggerated cell-cell aggregation, enhanced expression of cadherins, and formation of well-developed adherens junctions in a manner dependent on SPP and the small guanine nucleotide binding protein Rho.

Automated tracking of migrating cells in phase-contrast video microscopy sequences using image registration.

Analysis of in vitro cell motility is a useful tool for assessing cellular response to a range of factors. However, the majority of cell-tracking systems available are designed primarily for use with fluorescently labelled images. In this paper, five commonly used tracking systems are examined for their performance compared with the use of a novel in-house cell-tracking system based on the principles of image registration and optical flow. Image registration is a tool commonly used in medical imaging to correct for the effects of patient motion during imaging procedures and works well on low-contrast images, such as those found in bright-field and phase-contrast microscopy. The five cell-tracking systems examined were Retrac, a manual tracking system used as the gold standard; CellTrack, a recently released freely downloadable software system that uses a combination of tracking methods; ImageJ, which is a freely available piece of software with a plug-in for automated tracking (MTrack2) and Imaris and Volocity, both commercially available automated tracking systems. All systems were used to track migration of human epithelial cells over ten frames of a phase-contrast time-lapse microscopy sequence. This showed that the in-house image-registration system was the most effective of those tested when tracking non-dividing epithelial cells in low-contrast images, with a successful tracking rate of 95%. The performance of the tracking systems was also evaluated by tracking fluorescently labelled epithelial cells imaged with both phase-contrast and confocal microscopy techniques. The results showed that using fluorescence microscopy instead of phase contrast does improve the tracking efficiency for each of the tested systems. For the in-house software, this improvement was relatively small (<5% difference in tracking success rate), whereas much greater improvements in performance were seen when using fluorescence microscopy with Volocity and ImageJ.

Impact of feeding method on overall activity of indoor, client-owned dogs.

OBJECTIVE: To compare the total daily activity time, walking time and running time using food-dispensing toys versus bowls in a group of client-owned, primarily indoor dogs. MATERIALS AND METHODS: A two-way, two period, randomised repeated measures mixed-effects crossover study performed on 26 client-owned, primarily indoor dogs. RESULTS: Toy feeding increased average daily total activity time by 12% and walking time by 26%. Average daily total activity time and walking time were reduced by 8 and 7% respectively with each increase in year of age. Gender, body condition and muscle condition had no significant effect on average daily total activity or walking time. Toy feeding, time, their interaction, age, gender, body condition and muscle condition had no significant effect on average daily running time. CLINICAL SIGNIFICANCE: Feeding toys may be helpful during weight loss programs to achieve the goal of increasing daily exercise duration in dogs that need to lose weight.

Changes in organization of the endoplasmic reticulum during Xenopus oocyte maturation and activation.

The organization of the endoplasmic reticulum (ER) in the cortex of Xenopus oocytes was investigated during maturation and activation using a green fluorescent protein chimera, immunofluorescence, and electron microscopy. Dense clusters of ER developed on the vegetal side (the side opposite the meiotic spindle) during maturation. Small clusters appeared transiently at the time of nuclear envelope breakdown, disappeared at the time of first polar body formation, and then reappeared as larger clusters in mature eggs. The appearance of the large ER clusters was correlated with an increase in releasability of Ca(2+) by IP(3). The clusters dispersed during the Ca(2+) wave at activation. Possible relationships of ER structure and Ca(2+) regulation are discussed.

Trafficking, assembly, and function of a connexin43-green fluorescent protein chimera in live mammalian cells.

To examine the trafficking, assembly, and turnover of connexin43 (Cx43) in living cells, we used an enhanced red-shifted mutant of green fluorescent protein (GFP) to construct a Cx43-GFP chimera. When cDNA encoding Cx43-GFP was transfected into communication-competent normal rat kidney cells, Cx43-negative Madin-Darby canine kidney (MDCK) cells, or communication-deficient Neuro2A or HeLa cells, the fusion protein of predicted length was expressed, transported, and assembled into gap junctions that exhibited the classical pentalaminar profile. Dye transfer studies showed that Cx43-GFP formed functional gap junction channels when transfected into otherwise communication-deficient HeLa or Neuro2A cells. Live imaging of Cx43-GFP in MDCK cells revealed that many gap junction plaques remained relatively immobile, whereas others coalesced laterally within the plasma membrane. Time-lapse imaging of live MDCK cells also revealed that Cx43-GFP was transported via highly mobile transport intermediates that could be divided into two size classes of <0.5 microm and 0.5-1.5 microm. In some cases, the larger intracellular Cx43-GFP transport intermediates were observed to form from the internalization of gap junctions, whereas the smaller transport intermediates may represent other routes of trafficking to or from the plasma membrane. The localization of Cx43-GFP in two transport compartments suggests that the dynamic formation and turnover of connexins may involve at least two distinct pathways.

Histological and biochemical characterization of von Ebner's glands in the Syrian hamster; comparison with rat von Ebner's glands.

We report here for the first time a morphological description and observations on some of the secretory proteins of the von Ebner's lingual salivary glands (VEG) of the Syrian hamster. Hamster VEG were macroscopically less distinct, but histologically similar to rat VEG. VEG extracts of hamster and rat were assayed for lipase, alpha-amylase and peroxidase activities. Unlike rat VEG, which is rich in lipase activity, hamster VEG extract had no detectable lipase activity and did not react with antibodies to either rat lingual lipase or human gastric lipase in Western blots. Immunohistochemical reactions with the anti-rat lingual lipase antibody were very weak in hamster VEG and strong in rat VEG. Moderate alpha-amylase enzyme activities and immunohistochemical reactions were demonstrated in both hamster and rat VEG. Peroxidase activity was negligible in the VEG, unlike the high activity in the submandibular glands of both species. An 18 kDa von Ebner's gland protein (VEGP), a member of the lipocalin superfamily of hydrophobic ligandbinding proteins, was abundant in rat VEG, but not detected in hamster VEG. Thus, hamster VEG differs from rat VEG in macroscopic appearance and the absence of lipase and VEGP. It is similar to rat VEG histologically and with regard to the presence of alpha-amylase and absence of peroxidase.

Structure-activity studies of rapamycin analogs: evidence that the C-7 methoxy group is part of the effector domain and positioned at the FKBP12-FRAP interface.

BACKGROUND: Rapamycin is an immunosuppressant natural product, which blocks T-cell mitogenesis and yeast proliferation. In the cytoplasm, rapamycin binds to the immunophilin FKBP12 and the complex of these two molecules binds to a recently discovered protein, FRAP. The rapamycin molecule has two functional domains, defined by their interaction with FKBP12 (binding domain) or with FRAP (effector domain). We previously showed that the allylic methoxy group at C-7 of rapamycin could be replaced by a variety of different substituents. We set out to examine the effects of such substitutions on FKBP12 binding and on biological activity. RESULTS: Rapamycin C-7-modified analogs of both R and S configurations were shown to have high affinities for FKBP12, yet these congeners displayed a wide range of potencies in splenocyte and yeast proliferation assays. The X-ray crystal structures of four rapamycin analogs in complexes with FKBP12 were determined and revealed that protein and ligand backbone conformations were essentially the same as those observed for the parent rapamycin-FKBP12 complex and that the C-7 group remained exposed to solvent. We then prepared a rapamycin analog with a photoreactive functionality as part of the C-7 substituent. This compound specifically labeled, in an FKBP12-dependent manner, a protein of approximately 250 kDa, which comigrates with recombinant FRAP. CONCLUSIONS: We conclude that the C-7 methoxy group of rapamycin is part of the effector domain. In the ternary complex, this group is situated in close proximity to FRAP, at the interface between FRAP and FKBP12.

Effective distribution of domestic wastewater effluent between percolation trenches in on-site treatment systems.

On-site treatment systems discharging to groundwater rely on the effective distribution of effluent across a percolation area to provide an appropriate loading rate for the subsoil. In Ireland, this is achieved in a distribution box which splits the effluent evenly between the requisite number of percolation pipes. The flow regime experienced at four different distribution boxes was monitored continuously over twelve month periods which established that the most common flow rates at the distribution unit were in the range 1-4 litres/minute for a four to five person dwelling. In addition, the average flow rate from the four sites was only 100 litres per person per day, compared to recommended design value of 180 litres per person per day. Two distribution boxes were also tested in the laboratory to assess their distribution efficiency over a range of loading rates. The most commonly installed unit was found to significantly favour two out of the four trenches and both units were shown to perform particularly poorly at a range of different off-level installation angles. Modifications to the boxes were also tested, involving plastic V-notch inserts which were shown to greatly improve the hydraulic distribution and make the unit much less sensitive to off-level installation or subsidence.

Regulation of fibroblast growth factor 2 and fibroblast growth factor receptors by transforming growth factor beta in human osteoblastic MG-63 cells.

Fibroblast growth factor 2 (FGF-2) and its receptors (FGFRs) are important regulators of bone cell function. Although FGF-2 is a major modulator of bone cell function, its expression and regulation in human osteoblasts have not been investigated. We examined FGF-2 messenger RNA (mRNA) expression and regulation in the human osteosarcoma MG-63 cells. Northern analysis revealed that MG-63 cells expressed FGF-2 mRNA transcripts of 7, 4, 2.2, and 1.3 kilobases (kb). In the absence of serum, treatment with transforming growth factor beta (TGF-beta; 0.1-10 ng/ml) increased all FGF-2 mRNA transcripts. Maximal increase was seen with 1 ng/ml of TGF-beta. TGF-beta increased FGF-2 mRNA expression within 2 h and this was sustained for 24 h. Phorbal myristate acetate (PMA; 1 microM) also increased FGF-2 mRNA at 6 h. Time course studies showed that TGF-beta did not significantly alter FGFR1 or FGFR2 mRNA expression in MG-63 cells. Western blotting with anti-human FGF-2 revealed that MG-63 cells synthesize three isoforms of FGF-2 protein of approximately 18, 22/23, and 24 kDa, which were increased after either 6 h or 24 h of treatment with TGF-beta. Increased FGF-2 mRNA and protein expression in response to TGF-beta was markedly reduced by the protein kinase A (PKA) inhibitor H-89. Immunogold labeling of MG-63 cells treated with TGF-beta showed increased labeling for FGF-2 and FGFR2 in the nuclei. In contrast, TGF-beta treatment significantly decreased FGFR1 labeling in the nuclei. These data show that TGF-beta regulates FGF-2 gene expression in human osteosarcoma cells. Furthermore, TGF-beta modulates the cellular localization of FGF-2 and its receptors.

Ultrastructural immunocytochemical localization of cyclic AMP-dependent protein kinase regulatory subunits in rat parotid acinar cells.

Polyclonal antibodies to types I and II regulatory (R) subunits of cyclic AMP-dependent protein kinase (cA-PK) were utilized in a post-embedding immunogold-labeling procedure to localize these proteins in rat parotid acinar cells. Both RI and RII were present in the nuclei, cytoplasm, rough endoplasmic reticulum (RER), Golgi apparatus, and secretory granules. In the nuclei, gold particles were mainly associated with the heterochromatin. In the cytoplasm, the label was principally found in areas of RER. Most gold particles were located between adjacent RER cisternae or over their membranes and attached ribosomes; occasional particles were also present over the cisternal spaces. Labeling of the Golgi apparatus was significantly greater than background, although it was slightly lower than that over the RER cisternae. In secretory granules, gold particles were present over the granule content; no preferential localization to the granule membrane was observed. Morphometric analysis revealed equivalent labeling intensities for RI and RII in the cytoplasm-RER compartment. Labeling intensities for RII in the nuclei and secretory granules were about 50% greater than in the cytoplasm-RER, and 3 to 4-fold greater than values for RI in these two compartments. Electrophoresis and autoradiography of the postnuclear parotid-tissue fraction, the contents of purified secretory granules and saliva collected from the main excretory duct, after photoaffinity labeling with [32P]-8-azido-cyclic AMP, revealed the presence of R subunits. Predominantly RII was present in the granule contents and saliva, while both RII and RI were present in the cell extracts. Additionally, R subunits were purified from saliva by affinity chromatography on agarose-hexane-cyclic AMP. These findings confirm the localization of cA-PK in parotid cell nuclei and establish the acinar secretory granules as the source of the cyclic AMP-binding proteins in saliva.