Cyclic AMP-receptor proteins in human salivary glands.

In mammalian species, cyclic AMP receptor proteins (cARP) are the regulatory (R) subunits of cyclic AMP-dependent protein kinase (PKA), the cellular effector of cyclic AMP-mediated signal transduction. An isoform of the PKA type II R subunit (RII), cARP, is a polyfunctional protein, present in most tissues and cells. It is expressed in salivary and other glands of rodents, and secreted into the saliva of rats and Man. The aim of the present study was to determine the expression of cARP in human salivary glands using immunoelectron microscopy. Thin sections of normal salivary glands embedded in LR Gold resin were labeled with anti-cARP primary antibody, then with gold-conjugated secondary antibody. Labeling was present in the secretory granules and cytoplasm of parotid, submandibular (SMG) and sublingual gland serous cells. Quantitative analysis showed considerable variability in granule labeling from sample to sample, indicating shifts in expression and cellular location of cARP. Unlike rodent salivary glands, the granules of intercalated and striated duct cells also were labeled. The cytoplasm and granules of mucous cells of the SMG and sublingual glands were unlabeled, while the Golgi complex and filamentous bodies in these cells showed moderate reactivity. Mitochondria and nuclei of both serous and mucous cells were unlabeled. Labeling also was present in the connective tissue adjacent to the epithelial cells. The results indicate that serous cells of the parotid and SMG are the major source of salivary cARP. They also reveal significant species differences in the glandular distribution of RII. RII binds to cytoskeletal and nuclear proteins, and may function to regulate extracellular cyclic AMP levels. Thus, the tissue and cellular distribution of RII may serve as an index of regulation of gene expression and cell differentiation.

Expression and distribution of parotid secretory proteins in experimental diabetes.

Previous studies of experimental diabetes have demonstrated changes in the levels of specific salivary proteins. The present study is part of a larger effort aimed at elucidating the mechanism(s) by which insulin regulates salivary protein expression in the rat parotid gland. Diabetes was induced in 2-3-month-old male Fischer 344 rats by injection of streptozotocin (STZ). After 30 days one group of rats was given insulin for 7 days. Untreated rats served as controls. As previously observed, parotid acinar cells from diabetic rats accumulated lipid and contained occasional crystalloid lysosomes. Quantitative immunogold labeling of secretory granules in diabetic glands revealed decreases of 30-60% for proline-rich-proteins (PRPs), amylase and parotid secretory protein (PSP), but labeling for acidic epididymal glycoprotein (AEG) was unchanged. The response to insulin treatment was variable: amylase and PSP labeling were partly restored, but PRP and AEG labeling showed little change. Photoaffinity labeling of cyclic AMP receptor proteins (cARP) showed changes in several tissues including a consistent increase in the diabetic parotid gland. Immunogold labeling of secretory granules with antibody to cARP was similar in control and diabetic parotids, but nuclear and cytoplasmic label was decreased in diabetic acinar cells. These results indicate that STZ-diabetes and insulin reconstitution cause variable changes in the expression of parotid secretory proteins. Changes in cARP levels suggest that the insulin and cyclic AMP pathways may interact in regulating expression of salivary secretory proteins.

Cyclic AMP-receptor responses to hypergravity.

BACKGROUND: Altered gravity (G) encountered during spaceflight causes physiologic changes in humans and in experimental animals. In addition to weightlessness (0G) in space, sharply increased G forces are exerted on the spacecraft during the lift-off and reentry phases. Previous studies showed major changes in cAMP-associated activity of rat heart muscle after spaceflight, indicating that (hormone) signaling pathways may have been affected. HYPOTHESIS: The present study was designed to test the hypothesis that cAMP-related cellular responses of exocrine glands after simulated hypergravity (centrifugation at 1.7G) differ from the effects of 0G. METHODS: A portion of the parotid and lachrymal gland tissue was fixed for morphologic and immunocytochemical study, and another was used for biochemical determinations. A short-term tissue culture was established from each gland to determine the effects of stimulation by norepinephrine. Heart muscle (ventricle) was also studied. Soluble and particulate fraction extracts of tissue homogenates were prepared, photoaffinity labeled with the [32P]8-N3-analog of cAMP, proteins separated by electrophoresis and the cAMP-reactive proteins (cARP) identified by autoradiography. RESULTS: Differences were seen in protein banding patterns of the gland extracts and in altered cARP distribution in the 1.7G samples of heart ventricle and exocrine gland tissues, when compared with 1G controls. In the heart, cARP increased in the soluble fraction, while the particulate fraction extract showed no change. In acinar cells of the parotid, labeled cARP had accumulated, but decreased after stimulation to the level of the 1G controls. Immunogold labeling showed an increased content of amylase in the secretory granules of the 1.7G animals, while morphologic observation revealed few changes in the structure of parotid acinar cells. The response in the lachrymal gland was translocation of an isoform of cARP from the particulate to the cytoplasmic compartment. CONCLUSIONS: Changes distinct from those due to 0G, but specific for hyper-G were found in cARP activity, protein synthesis, as well as in an apparent inhibition of regulated secretion.

Cyclic AMP-receptor proteins in the enamel matrix.

Cyclic AMP receptor proteins (cARP) are present in a variety of cell types. Intracellularly, they are the regulatory (R) subunits of type II cyclic AMP-dependent protein kinase (PKA: E.C.2.7.1.37). Additionally, cARP are secretory products of several cell types.[1] That cARP are present in and secreted by ameloblasts into the enamel matrix of the rat incisor was demonstrated by photoaffinity labeling, Western blotting and immunogold cytochemistry. Gold particles were present over cytoplasmic regions including Tomes' Processes of secretory ameloblasts, secretory granules and in the Golgi region. Specific RII labeling was seen in the enamel matrix, but not in dentin. The enamel matrix was more reactive during early maturation compared to the secretory stage of amelogenesis. Nuclear labeling with the RII antibody showed higher intensity in maturation than in secretory ameloblasts. These results demonstrate that cARP are expressed in ameloblasts and secreted into the enamel matrix. The role(s) of cARP in enamel matrix mineralization and the involvement of PKA-regulated pathways in enamel protein synthesis and secretion remain to be determined.

Quantitative immunocytochemistry of Ca(2+)-Mg2+ ATPase in ameloblasts associated with enamel secretion and maturation in the rat incisor.

Our previous studies revealed intense membrane-associated labeling for Ca(2+)-Mg2+ ATPase (Ca(2+)-pump) in secretory and maturation ameloblasts in the rat incisor, both by enzyme cytochemistry and by immunohistochemical techniques. The purpose of the present study was to map the distribution of Ca(2+)-pump protein at the cellular and subcellular levels by means of a Ca(2+)-pump-specific monoclonal antibody and electron microscopic immunogold cytochemistry. Tissue specimens were dissected from secretory, early, and late enamel maturation zones. We quantified results by comparing gold particle densities over ameloblast lateral and distal plasma membrane regions, supranuclear cytoplasm, regions of the ruffled borders, and nuclei. The highest concentration of gold particles was seen over the distal membranes of early-maturation ameloblasts relative to those in late-maturation and secretory stages. Cytoplasmic labeling was less than that of the distal and lateral membranes, and gold particles located over nuclei were considered to be due to non-specific binding. These results are consistent with our earlier findings and suggest a role for the plasma membrane Ca(2+)-pump in the regulation of calcium availability to mineralizing enamel.

Localization of plasma membrane Ca2+ pump mRNA and protein in human ameloblasts by in situ hybridization and immunohistochemistry.

The distribution of the plasma membrane Ca(2+)-pump (PMCA) proteins in human ameloblasts was examined immunohistochemically using monoclonal antibodies JA8 and 5F10. Further, the distribution of mRNA transcripts derived from two PMCA genes, PMCA-1 and PMCA-4 was examined using in situ hybridization. In rats, the PMCA-1 gene is purported to code for PMCA proteins with a role in maintaining the intracellular Ca2+ levels in nonepithelial cells. Other genes including the PMCA-4 gene may code for PMCA proteins characteristic of Ca2+ transporting epithelia. The present results show immunohistochemical staining in the Tomes processes and plasma membranes of human ameloblasts. Our studies also demonstrate a gradation of expression of the PMCA-1 and PMCA-4 mRNA transcripts which parallels the onset and progression of enamel mineralization. These studies suggest that PMCA proteins in human ameloblasts may function both in intracellular Ca2+ homeostasis and in regulating the vectorial Ca2+ influx into mineralizing enamel.

Cyclic AMP-reactive proteins in human saliva.

This study was conducted to compare cyclic AMP-reactive proteins (cARP), the secretory form of regulatory (R) subunits of cyclic AMP-dependent protein kinase (PKA), and cyclic nucleotide phosphodiesterase (PDE) activity in human whole saliva with that of parotid fluid. Additionally, experiments were done to determine whether secretory cARP is altered by environmental stimuli. Earlier work showed that R subunits are present in parotid fluid and in salivary glands of rats. No previous information is available about secretory PDE in saliva. Whole and parotid ductal saliva samples were collected by a non-invasive procedure from healthy volunteers. After photoaffinity labelling with [32P]-8-N3-cAMP, the R subunits were identified by autoradiography. Cyclic nucleotide PDE activity was measured as a function of the conversion of the cyclic nucleotide to the tritiated 5'-nucleotide. The results showed that R of the type II cAPK, RII (M(r) 50-54 kDa) and/or a slower-moving isoform (M(r) 54-56 kDa, RIIa) were present in all parotid saliva samples tested. Whole saliva was positive for RII in more than 95% of the samples tested (n = 62), but with 50-90% reduction in concentration compared to parotid fluid. Both female and male subjects exposed to controlled auditory (60-80 dB) stimuli responded by a two- to five-fold increase in photoaffinity labelling of cARP (salivary RII, RIIa and RIIfr). There was considerable individual variability, but in all cases the differences in the results were significant (p < 0.05, n = 20). Whole saliva showed measurable PDE activity in fresh or frozen samples, whereas no PDE activity was detected in parotid fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoaffinity labeling of regulatory subunits of protein kinase A in cardiac cell fractions of rats.

The regulatory (R) subunits of adenosine 3',5',-cyclic monophosphate-dependent protein kinase were measured by photoaffinity labeling in heart tissue of rats flown on COSMOS 2044. Densitometric analyses showed a significant decrease of RII subunits in the particulate cell fraction extract (S2; P less than 0.05 in all cases) when extracts of tissue samples from vivarium controls were compared with those from flight animals. Photoaffinity labeling of the soluble fraction (S1) was unaffected by spaceflight or any of the simulation conditions. This was previously observed in heart muscle of rats flown on Spacelab 3 and COSMOS 1887 missions. A change in either the number or reactivity of the R subunits apparently results from some aspect of spaceflight. Proteins of the S2 fraction constitute a minor (less than 10%) component of the total, whereas the S1 fraction contained most of the cell proteins. No changes were seen when total incorporation of photoaffinity label was calculated on the basis of adrenal gland weights. A negative correlation resulted on comparison of controls with flight, synchronous control, and a tail-suspended simulation group of animals when incorporation of total counts due to azido labeling was based on body weights. Environmental factors that influence organismic responses may alter individual hormonal responses and may be reflected on the molecular level of organization. Conversely, changes in a relatively minor aspect of adenosine 3',5'-cyclic monophosphate-mediated reactions may be representative of a metabolic effect on an organismic level.

Growth of sebaceous cells in monolayer culture.

Rat preputial cells were grown in an epithelial cell primary monolayer culture system identical to that used for culturing epidermal cells, which were studied for comparison. Despite similar appearance when observed by phase contrast microscopy, other features identified the preputial cells as a unique epithelial cell population. Preputial cells grew as a relatively small number of large colonies, formed domes before confluence, and expressed a specific acinar keratin, K4, which had previously been found in human sebaceous glands. In addition, preputial cells formed fewer cornified envelopes than epidermal cells, too few to discern the reduction of envelope formation by retinoic acid treatment in vitro which was found in epidermal cells. Rat preputial cells in monolayer culture, therefore, are a promising model for studying the effects of hormones on sebaceous cell growth and differentiation.

Cyclic AMP-receptor proteins in heart muscle of rats flown on Cosmos 1887.

A frequent cellular response to organismal stress is the increase in ligand binding by beta-adrenergic receptors. The extracellular signal is amplified by intracellular increases in cyclic AMP and the ensuing activation of cyclic AMP-dependent protein kinase (cAPK). The molecular mechanisms involve the binding of cyclic AMP to regulatory (R) subunits of cAPK, thus freeing the catalytic subunit for protein phosphorylation. This study was carried out to determine the cellular compartmentalization of the cyclic AMP-receptor proteins in heart ventricular tissue obtained from rats flown on the Cosmos 1887 mission. Photoaffinity labeling of soluble and particulate cell fractions with an [32P]-8-azido analog of cyclic AMP was followed by electrophoretic separation of the proteins and by autoradiographic identification of the labeled isoforms of cAPK R subunits. The results showed that RII in the particulate subcellular fraction was significantly decreased in heart cells from rats in the flight group when compared to controls. Protein banding patterns in both the cytoplasmic fraction and in a fraction enriched in chromatin-bound proteins showed some variability in tissues of individual animals, but exhibited no changes that could be directly attributed to flight conditions. No significant change was apparent in the distribution of RI or RII cyclic AMP binding in the soluble fractions. These findings indicate that the cardiac cell integrity or its protein content is not compromised under flight conditions. There is, however, what appears to be an adaptive molecular response which can be detected using microanalytical methods, indicating that a major hormone regulated mechanism may be affected during some phase of travel in space.

Cyclic AMP-receptor protein activity in rat preputial cells.

Cells from the rat preputial gland--a type of sebaceous gland--exhibited specific responsiveness of cyclic 3',5'-adenosine monophosphate (AMP) dependent protein kinase to stimulation by agents that elevate intracellular cyclic AMP. Electron microscopy shows that the rat preputial gland resembles the human sebaceous gland, not only in terms of containing a sebocyte-like population of cells in an acinar arrangement at different maturational stages, but also in the morphology of its organelles such as abundant and sometimes atypical mitochondria, many perinuclear lysosomes with crystalline inclusions, lipid droplets of various sizes, and peroxisomes. Other cell types, among them duct and inflammatory cells, were evident in the tissue sections, but constituted a minor component. Responses to stimulation of the adenylate cyclase-protein kinase pathways were determined using preputial cells that had been both freshly dispersed and grown in monolayer culture. Stimulation with isoproterenol (IPR) or forskolin (FS) resulted in both cases in an increase of cyclic AMP binding of the regulatory (R) subunits of cyclic AMP-dependent protein kinase, as determined by photoaffinity labeling of R subunits with an azido analog of cyclic AMP ([32P]-8-azido cyclic AMP). Cells from the epidermis under comparable conditions responded to a lesser degree and with a different distribution of R subunit isoforms. There are, therefore, differences in receptor activity as well as in the transduction pathways between the two types of epithelial cell populations. These results indicate that the preputial gland contains precursor cells that differentiate in culture to retain specific molecular mechanisms of action mediated via cyclic AMP.

Regulatory subunits of cyclic AMP-dependent protein kinase: presence in granules and secretion by exocrine and endocrine cells.

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, 32P-labeled-8-azido-cyclic AMP, revealed the presence 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.

Site-selective 8-Cl-cAMP which causes growth inhibition and differentiation increases DNA (CRE)-binding activity in cancer cells.

Control mechanisms of normal differentiation are disrupted in cancer cells but can be restored by treatment with site-selective cAMP analogs. The cellular events associated with such changes entail compartmental redistribution of the cAMP-dependent protein kinase type II regulatory subunit, RII beta. The results of this study indicate that the molecular mechanisms of action involve changes in specific DNA-binding activity of putative transcription factors. Gel retardation analyses revealed that nuclear extracts from cells of various human cancer cell lines [colon cancer (LS-174T), gastric cancer (TMK-1), and leukemia (K-562)] and rodent pheochromocytoma (PC12) show a concentration-dependent increase in binding activity to a synthetic DNA that contained the cAMP-responsive element 5'-TGACGTCA-3' after treatment with 8-Cl-cAMP. Such an increase in cAMP-responsive element binding activity was not observed in the 8-C1-cAMP-unresponsive MKN-1 gastric cancer cells. These findings indicate that the antitumor activity of site-selective cAMP analogs may reside in the induction of transcription factors that restore normal gene regulation in cancer cells.

Immunogold localization of the type II regulatory subunit of cyclic AMP-dependent protein kinase. Monoclonal antibody characterization and RII distribution in rat parotid cells.

A mouse monoclonal antibody of the IgM class, MAb BB1, specific for the type II regulatory subunit (RII) of cyclic AMP-dependent protein kinase (cAPK), was produced using a purified subcellular protein fraction from rat parotid gland as the original antigen. The antibody immunoprecipitated radioactivity labeled RII from bovine heart cAPK, and from rat and human parotid saliva. Western blot analysis revealed specific binding of the antibody to proteins of 52 and 54 KD in extracts of rat parotid tissue, parotid saliva, and bovine heart cAPK. Immunogold labeling of thin sections of rat parotid gland revealed specific labeling of acinar cell nuclei (especially the heterochromatin), cytoplasm (particularly in areas containing granular endoplasmic reticulum), and the content of secretory granules. Labeling was greatly reduced (approximately 84%) when the antibody was pre-absorbed with an excess of bovine heart cAPK. In duct cells the cytoplasm and nuclei were also labeled, but few gold particles were present over secretory granules. These results provide additional evidence for the presence of nuclear cAPK in rat parotid cells, and confirm previous observations on the presence of cAPK regulatory subunits in acinar secretory granules and saliva. The hybridoma reagent will be used for studies of stimulus responses in the parotid and for immunocytochemical analyses of RII distribution in other secretory tissues.

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.

Cardiac muscle ultrastructure and cyclic AMP reactions to altered gravity conditions.

Morphological and biochemical analyses of heart muscle of rats subjected to microgravity on Spacelab 3 (SL-3) flight and rats born and reared under increased gravity (1.7 G) conditions were compared with 1-G controls. Electronmicroscopic studies showed an increase in the number of lipid droplets and in areas of glycogen storage. Distribution changes of microtubules and cytoskeletal elements from both SL-3 and 1.7-G groups were observed. The high Km cyclic AMP phosphodiesterase activity was lower (P less than 0.05) in SL-3 heart muscle, and low Km activity was lower in 1.7-G males but was unaltered in females. Cyclic AMP-dependent protein kinase (cA-PK) activity was decreased in subcellular fractions of heart muscle of SL-3 animals. Recompartmentalization of cA-PK activity occurred in particulate tissue fraction of 1.7-G animals (70.3% of total for 1.7 G vs. 35.9% for controls). Phosphorylation of endogenous low-mobility proteins increased in SL-3 heart-soluble fractions. Photoaffinity labeling (18 h, 4 degrees C) decreased in type II cA-PK regulatory (R) subunits in both SL-3 and in 1.7-G male heart tissue particulate fractions. The 1.7-G female heart R subunit distribution did not differ from controls. These findings indicate that in heart muscle altered gravity conditions influenced physiological reactions similar to catecholamine-induced receptor-mediated hormonal responses.

Salivary gland ultrastructure and cyclic AMP-dependent reactions in Spacelab 3 rats.

Environmental stimuli influencing catecholamine levels induce changes in cyclic AMP-dependent reactions and cell morphology in the rat parotid. Responses of salivary glands to spaceflight were determined by measurement of cyclic AMP-mediated reactions in fresh-frozen salivary glands and by microscopic evaluation of ultrastructure in fixed parotid glands. Decreased cell-free protein phosphorylation occurred in parotid glands in three of five flight animals. Protein kinase activity ratios were decreased in the soluble and increased in the particulate fractions of Spacelab 3 (SL-3) rat sublingual glands, compared with ground controls. Biochemical analyses show that effects of space flight on salivary glands are similar to those induced experimentally by physiological manipulation or alteration of catecholamine levels. Morphological evaluation of three SL-3 rat parotid glands showed increased numbers of lysosomes, autophagic vacuoles containing degenerating secretory product, and accumulation of lipid droplets. Since these animals lost weight, consistent with disruption of food and water consumption, morphological changes may in part be due to decreased masticatory stimulation, as occurs with reduced food intake or a liquid diet. The observed changes may reflect physiological responses of the gastrointestinal and autonomic systems to effects of spaceflight.

Exocytosis in rat parotid acini after in vitro treatment with forskolin is accompanied by cellular redistribution of regulatory subunits of cyclic AMP-dependent protein kinase.

Incubation of rat parotid acinar tissue with 1 mumol/L forskolin resulted in progressive exocytosis which was virtually complete after a 30-minute incubation period. Cyclic AMP binding to protein kinase (cA-PK) regulatory (R) subunits, determined by photo-affinity labeling with [32P]-8-azido cyclic AMP, was found to increase in a time-dependent manner in the 10,000-g supernatant fraction of a broken cell preparation. An apparent redistribution of protein kinase R subunits took place in the 600-g supernatant after in vitro treatment of cells with forskolin. In control cells, RI and RII subunits and a 35-to-40-kdal fragment were present in approximately equal amounts throughout the incubation. Azido labeling of RII appeared either to increase or to remain unchanged, while that of RI decreased in the 600-g pellet. Only type I isozyme R subunits were found in the 600-g pellet in either the absence or presence of forskolin. These finding indicate that a temporal relationship exists between stimulated protein exocytosis and cyclic AMP-dependent protein kinase activation. Forskolin stimulation of adenylate cyclase in salivary gland cells therefore provides a defined system for the study of cyclic AMP-mediated protein secretion.