Role of GAD peptides p217 and p290 in the repair of INS receptor in salivary tissues of type 1 diabetic mice.

Glutamate decarboxylase or glutamic acid decarboxylase (GAD) is a protein associated with autoimmune diseases, including type-1 diabetes. This disease is primarily associated with the occurrence of a specific isoform: GAD65. Conversely, some specific peptides of this protein may block autoimmunity in diabetes. In this respect, understanding the relationship between GAD and the development of diabetes is important, and it is necessary to understand the role of each GAD peptide to design effective autoimmune diabetes treatments. The purpose of the present study was to analyze the effects of treatment with GAD-derived peptides p217 and p290 on INS receptors in the salivary epithelium of nonobese diabetic (NOD) animals. Three groups of 7 mice each were studied: I, BALB/c mice (control); II, NOD mice; and III, NOD mice treated with peptides p290 and p217. Groups I and II only received buffered saline solution. Glucose levels were measured daily during the 21 days of the experiment. After the study, the animals were euthanized and the parotid and submandibular glands were removed for the analysis of INS-R by fluorescence microscopy. Therapy with two peptides together was associated with reduced glucose levels in NOD mice and intense INS-R expression in both salivary organs. Our approach of combining GAD p217 and p290 peptides contributed to hormonal balance and promoted the repair of INS-R.

The small GTPase Rab33A participates in regulation of amylase release from parotid acinar cells.

Amylase is released from exocrine parotid acinar cells via typical exocytosis. Exocytosis of amylase-containing granules occurs through several steps, including formation, maturation, and transport of granules. These steps are thought to be regulated by members of the small GTPase Rab family. We previously demonstrated that Rab27 and its effectors mediate amylase release from parotid acinar cells, but the functional involvement of other Rab proteins in exocrine granule exocytosis remains largely unknown. Here, we studied isoproterenol (IPR)-induced amylase release from parotid acinar cells to investigate the possible involvement of Rab33A, which was recently suggested to regulate exocytosis in hippocampal neurons and PC12 cells. Rab33A was endogenously expressed in parotid acinar cells and present in secretory granules and the Golgi body. Functional ablation of Rab33A with anti-Rab33A antibody or a dominant-negative Rab33A-T50N mutant significantly reduced IPR-induced amylase release. Our results indicated that Rab33A is a novel component of IPR-stimulated amylase secretion from parotid acinar cells.

Effects of isoproterenol on aquaporin 5 levels in the parotid gland of mice in vivo.

In the membrane fraction of mouse parotid gland (PG), the protein level of aquaporin 5 (AQP5), a member of the water channel family, was increased by injection (ip) of isoproterenol (IPR), a ß-adrenergic agonist, at 1 h, and stayed at high levels until 6 h; this change occurred simultaneously as amylase secretion. The AQP5 level then decreased and returned toward the original level at 12-48 h. After IPR injection, the AQP5 mRNA gradually increased and reached a maximum at 24 h. The facts suggest a rapid appearance of AQP5 at plasma membrane by IPR and subsequent degradation/metabolism by activation of proteolytic systems. Pretreatment of animals with two calpain inhibitors, N-Ac-Leu-Leu-methininal (ALLM) and calpeptin, as well as a protein synthesis inhibitor, cycloheximide (CHX), significantly suppressed the IPR-induced AQP5 degradation in the PG membrane fraction; such suppression was not observed by two proteasome inhibitors, MG132 and lactacystin, or the lysosome denaturant chloroquine, although most of these inhibitors increased AQP5 protein levels in unstimulated mice. The AQP5 protein was also degraded by µ-calpain in vitro. Furthermore, we demonstrated that µ-calpain was colocalized with AQP5 in the acinar cells by immunohistochemistry, and its activity in the PG was increased at 6 h after IPR injection. These results suggest that the calpain system was responsible for IPR-induced AQP5 degradation in the parotid gland and that such a system was coupled to the secretory-restoration cycle of amylase in the PG.

Laser irradiation affects enzymatic antioxidant system of streptozotocin-induced diabetic rats.

The aim of the present study was to analyze the effect of low-power laser irradiation in the antioxidant enzymatic system of submandibular (SMG) and parotid (PG) salivary glands of streptozotocin-induced diabetic rats. The animals were randomly divided into six groups: three diabetic groups (D0, D5, and D20) and three non-diabetic groups (C0, C5, and C20), according to laser dose received (0, 5, and 20 J/cm(2), respectively). Areas of approximately 1 cm(2) were demarcated in the salivary glands (each parotid and both submandibular glands) and after irradiated according to Simões et.al. (Lasers Med Sci 24:202-208, 2009). A diode laser (660 nm/100 mW) was used, with laser beam spot of 0.0177 cm(2). The group treated with 5 J/cm(2) laser dose was subjected to irradiation for 1 min and 4 s (total irradiation time) and the group treated with 20 J/cm(2) laser dose was subjected to irradiation for 4 min and 16 s. Twenty-four hours after irradiation the animals were euthanized and the salivary glands were removed for biochemical analysis. The total antioxidant values (TA), the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase enzymes were determined. SOD and CAT activities, as well as TA were higher in SMG of irradiated diabetic rats. However, in SMG of non-diabetic rats, laser irradiation decreased TA values and led to an increase in the CAT activity. In addition, there was a decrease in the activity of CAT in PG of diabetic and non-diabetic animals after laser irradiation. According to the results of the present study, low-power laser irradiation can affect the enzymatic antioxidant system of salivary glands of streptozotocin-induced diabetic rats.

Effects of extracorporeal shock-wave lithotripsy directed at the parotid gland on oxidative stress parameters and some trace element levels in facial nerve of rats.

INTRODUCTION: This study was designed to assess the effect of extracorporeal shock-wave lithotripsy (ESWL) exposure of the parotid gland on oxidative stress and some trace element levels in the facial nerves of rats. METHODS: Twelve male Wistar albino rats were divided into two groups, each consisting of 6 animals. The rats in the first group served as controls. The left parotid glands of animals in the second group were treated with 1000 18-kV shock waves while anesthetized with ketamine. The animals in both groups were euthanized 72 h after the ESWL treatment, and the right facial nerve was harvested for determination of oxidant/antioxidant status and trace element levels. RESULTS: Lipid peroxidation product malondialdehyde (MDA) and antioxidant glutathione (GSH) levels increased, and the activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), decreased in the facial nerves of ESWL-treated rats. The levels of iron, lead, manganese, and cobalt increased, and magnesium, cadmium, and copper levels decreased. CONCLUSIONS: ESWL treatment of the parotid gland may increase lipid peroxidation and decrease antioxidant enzyme activity in adjacent tissues such as the facial nerve. It also causes a decrease or increase in many mineral levels of the facial nerve, which is an undesirable condition for normal physiological function.

Exocyst subunits are involved in isoproterenol-induced amylase release from rat parotid acinar cells.

Exocytosis of secretory granules in parotid acinar cells requires multiple events: tethering, docking, priming, and fusion with a luminal plasma membrane. The exocyst complex, which is composed of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) that are conserved in yeast and mammalian cells, is thought to participate in the exocytotic pathway. However, to date, no exocyst subunit has been identified in salivary glands. In the present study, we investigated the expression and function of exocyst subunits in rat parotid acinar cells. The expression of mRNA for all eight exocyst subunits was detected in parotid acinar cells by RT-PCR, and Sec6 and Sec8 proteins were localized on the luminal plasma membrane. Sec6 interacted with Sec8 after 5 min of stimulation with isoproterenol. In addition, antibodies to-Sec6 and Sec8 inhibited isoproterenol-induced amylase release from streptolysin O-permeabilized parotid acinar cells. These results suggest that an exocyst complex of eight subunits is required for amylase release from parotid acinar cells.

Chitinase expression in parotid glands of non-obese diabetic mice.

OBJECTIVE: This investigation was a basal study that used a mouse model of xerostomia to identify protein biomarkers of xerostomia in saliva. We identified genes expressed differently in parotid glands from non-obese diabetic mice with diabetes and those from control mice; subsequently, we investigated expression of the proteins encoded by these genes in parotid glands and saliva. MATERIALS AND METHODS: DNA microarray and real-time PCR analyses were performed to detect differences between NOD/ShiJcl and C57BL/6JJcl (control) female mice in gene expression from parotid glands or parotid acinar cells. Subsequently, protein expression was assessed using immunoblotting and immunohistochemistry. Similarly, enzyme activity in saliva was assessed using zymography. RESULTS: Based on gene expression analyses, Chia expression was higher in diabetic mice than non-diabetic mice and control mice; similarly, expression of chitinase, the protein encoded by Chia, was higher in diabetic mice. Saliva from NOD/ShiJcl mice had more chitinase than saliva from control mice. CONCLUSIONS: Chitinase was highly expressed in parotid acinar cells from diabetic mice compared with non-diabetic and control mice. Increased chitinase expression and enzyme activity may characterize the autoimmune diabetes in mice; however, further investigation is required to assess its use as a biomarker of xerostomia in humans.

[Effect of sijunzi decoction on salivary amylase secretion disorder and VIP-cAMP signaling pathway in splenasthenic rats].

OBJECTIVE: To observe the effect of Sijunzi Decoction on secretion disorder of salivary amylase in splenasthenic rat and its mechanism. METHODS: The model group rats received reserpine 0.5 mg/kg through subcutaneous injection while the control group rats received the same volume of saline for 8 days. After being modeled, the model group were divided into treatment group and model control group, treatment group were given orally Sijunzi Decoction, model control group and normal group were fed the same amount of distilled water for 4 weeks. The animal were anaesthetized and the left parotid was removed, the wounds were sutured. When the animals were awake but drowsy, 20 microL 10% glacial acetic acid was applied on the apex of the tongue once a minute for 30 minutes, removed the right parotid gland of the animals. The samples were frozen and amylase activity and VIP, cyclic adenosine monophosphate (cAMP) content and VAMP-8, SNAP-23 protein expression in the parotid glands were detected. RESULTS: Change of sAA in parotid acinar was not significantly different between treatment group and normal groups, but higher in model control groups after acid stimulation. The VIP and PKA contents were not significantly different among three groups. VIP, cAMP content and PKA activity increased significantly in normal group while VIP increased slightly, cAMP and PKA activity decreased in model control groups, which returned to some degrees in treatment group after acid stimulation. Expression of VAMP-8 protein was not significantly different between treatment group and model control groups, while expression of SNAP-23 was lower in model control groups, expression of VAMP-8 and SNAP-23 was higher in treatment group than which in model control groups. CONCLUSION: Sijunzi Decoction has a certain effect on secretion disorder of salivary amylase in splenasthenic rat, which mechanism may be related to recover changes of VIP-cAMP signal pathway in the splenasthenic rat's parotid gland cells,including increase VIP content and expression of VAMP-8 and SNAP-23.

Regulation and identification of Na,K-ATPase alpha1 subunit phosphorylation in rat parotid acinar cells.

The stimulation of fluid and electrolyte secretion in salivary cells results in ionic changes that promote rapid increases in the activity of the Na,K-ATPase. In many cell systems, there are conflicting findings concerning the regulation of the phosphorylation of the Na,K-ATPase α subunit, which is the catalytic moiety. Initially, we investigated the phosphorylation sites on the α1 subunit in native rat parotid acinar cells using tandem mass spectrometry and identified two new phosphorylation sites (Ser(222), Ser(407)), three sites (Ser(217), Tyr(260), Ser(47)) previously found from large scale proteomic screens, and two sites (Ser(23), Ser(16)) known to be phosphorylated by PKC. Subsequently, we used phospho-specific antibodies to examine the regulation of phosphorylation on Ser(23) and Ser(16) and measured changes in ERK phosphorylation in parallel. The G-protein-coupled muscarinic receptor mimetic carbachol, the phorbol ester phorbol 12-myristate 13-acetate, the Ca(2+) ionophore ionomycin, and the serine/threonine phosphatase inhibitor calyculin A increased Ser(23) α1 phosphorylation. Inhibition of classical PKC proteins blocked carbachol-stimulated Ser(23) α1 subunit phosphorylation but not ERK phosphorylation, which was blocked by an inhibitor of novel PKC proteins. The carbachol-initiated phosphorylation of Ser(23) α1 subunit was not modified by ERK or PKA activity. The Na,K-ATPase inhibitor ouabain reduced and enhanced the carbachol-promoted phosphorylation of Ser(23) and Ser(16), respectively, the latter because ouabain itself increased Ser(16) phosphorylation; thus, both sites display conformational-dependent phosphorylation changes. Ouabain-initiated phosphorylation of Ser(16) α1 was not blocked by PKC inhibitors, unlike carbachol- or phorbol 12-myristate 13-acetate-initiated phosphorylations, suggesting that this site was also a substrate for a kinase other than PKC.

Evidence for amylase release by cyclin-dependent kinase 5 in the rat parotid.

Cyclin-dependent kinase 5 (Cdk5) plays no apparent role in cell cycle regulation, and Cdk5 is not activated by cyclins but only p35 or p39. Although the enzymatic activity of Cdk5 is highest in the central nervous system, recent reports indicate that it also has important functions in non-neuronal cells. In the present study, we investigated whether Cdk5 and its activators are expressed in rat parotid acinar cells, whether a ß-adrenergic agonist enhances the expression of Cdk5, and whether Cdk5 mediates amylase release. We found that Cdk5 and its activator, cyclin I, were expressed in rat parotid acinar cells, and that the expression of Cdk5 was enhanced by treatment of the cells with isoproterenol. Amylase release stimulated by isoproterenol was depressed by the addition of olomoucine, a Cdk5 inhibitor, or by the introduction of an anti-Cdk5 antibody. Cdk5 activity was enhanced by treatment with isoproterenol and this enhanced activity was attenuated by the addition of olomoucine. Olomoucine also attenuated both phosphorylation of Munc18c and translocation of Munc18c from the plasma membrane induced by isoproterenol. These results indicated that ß-stimulation of rat parotid acinar cells enhanced the expression of Cdk5, and that this Cdk5 activation may mediate amylase release through phosphorylation of Munc18c.

The contribution of AKAP5 in amylase secretion from mouse parotid acini.

A-kinase (PKA) anchoring proteins (AKAPs) are essential for targeting type II PKA to specific locales in the cell to control function. In the present study, AKAP5 (formerly AKAP150) and AKAP6 were identified in mouse parotid acini by type II PKA regulatory subunit (RII) overlay assay and Western blot analysis of mouse parotid cellular fractions, and the role of AKAP5 in mouse parotid acinar cell secretion was determined. Mice were euthanized with CO(2). Immunofluorescence staining of acinar cells localized AKAP5 to the basolateral membrane, whereas AKAP6 was associated with the perinuclear region. In functional studies, amylase secretion from acinar cells of AKAP5 mutant [knockout (KO)] mice treated with the beta-adrenergic agonist, isoproterenol, was reduced overall by 30-40% compared with wild-type (WT) mice. In contrast, amylase secretion in response to the adenylyl cyclase (AC) activator, forskolin, and the cAMP-dependent protein kinase (PKA) activator, N(6)-phenyl-cAMP, was not statistically different in acini from WT and AKAP5 KO mice. Treatment of acini with isoproterenol mimicked the effect of the Epac activator, 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT-2'-O-Me-cAMP), in stimulating Rap1. However, in contrast to isoproterenol, treatment of acini with 8-pMeOPT-2'-O-Me-cAMP resulted in stimulation of amylase secretion from both AKAP5 KO and WT acinar cells. As a scaffolding protein, AKAP5 was found to coimmunoprecipitate with AC6, but not AC8. Data suggest that isoproterenol-stimulated amylase secretion occurs via both an AKAP5/AC6/PKA complex and a PKA-independent, Epac pathway in mouse parotid acini.

Evidence of a generalized defect of acinar cell function in Shwachman-Diamond syndrome.

BACKGROUND: : Because the acinar cells of the exocrine pancreas in patients with Shwachman-Diamond syndrome (SDS) are severely depleted, we hypothesized that a similar deficiency may be present in acinar cells of the parotid gland. PATIENTS AND METHODS: : We determined serum pancreatic isoamylase and parotid amylase activities in 16 patients with SDS, 13 healthy controls, and 13 disease controls (cystic fibrosis or fibrosing pancreatitis). Parotid amylase and electrolyte concentrations were measured in stimulated parotid gland secretions. Starch digestion was assessed by breath hydrogen testing in patients with SDS (with and without enzyme supplements) and healthy controls. RESULTS: : Serum pancreatic and parotid isoamylase values were lower in the patients with SDS than in the healthy controls (P < 0.0001 and P = 0.0002, respectively). Serum pancreatic isoamylase, but not parotid isoamylase, was significantly lower in the disease controls than in the healthy controls (P < 0.0001 and P = 0.17, respectively). Secreted parotid gland amylase concentration (units per milligram of protein) in patients with SDS was lower than that in the healthy controls (P = 0.04), whereas the disease controls were comparable to the healthy subjects (P = 0.09). Secreted parotid chloride concentration was inversely correlated with amylase concentration in the patients with SDS (P = 0.01), but no correlation was seen in the healthy controls or disease controls. When patients with SDS ingested starch without enzyme supplementation, their breath hydrogen excretion was significantly higher than that in the healthy controls (P = 0.009). Following starch ingestion with enzymes, breath hydrogen in the patients with SDS was lower (P < 0.05) than with no enzyme treatment, and no different from controls (P = 0.37). CONCLUSIONS: : Mutations in the SBDS gene cause a generalized functional abnormality of exocrine acinar cells.

Anti-inflammatory action of cholecystokinin and melatonin in the rat parotid gland.

OBJECTIVE: To define the influence of cholecystokinin and melatonin on the inflammatory response of the lipopolysaccharide-exposed rat parotid gland. MATERIALS AND METHODS: Bacterial lipopolysaccharide was infused retrogradely into the parotid duct. The degree of inflammation three hours postadministration was estimated from the activity of myeloperoxidase, reflecting glandular neutrophil infiltration. RESULTS: The myeloperoxidase activity of the lipopolysaccharide-exposed gland was 10-fold greater than that of the contralateral gland. Combined with sulphated cholecystokinin-8 (10 or 25 µg kg(-1) , given twice intraperitoneally) or melatonin (10 or 25 mg kg(-1) x 2) the lipopolysaccharide-induced response was elevated 4.6- and 3.5-folds at the most. The cholecystokinin-A receptor antagonist lorglumide reduced the inhibitory effect of cholecystokinin-8, while the melatonin 2-preferring receptor antagonist luzindole had no effect on the melatonin-induced inhibition. Unselective nitric oxide-synthase inhibition abolished the increase in myeloperoxidase activity, whereas inhibition of inducible or neuronal nitric oxide-synthase (of non-nervous origin) halved the inflammatory response. CONCLUSION: Some hormones may contribute to anti-inflammatory action in salivary glands in physiological conditions. They are potential pharmacological tools for treating gland inflammation. The inflammation, as judged from the myeloperoxidase activity, was entirely dependent on nitric oxide-synthase activity, indicating that the hormones directly or indirectly reduced the generation of nitric oxide.

Increase nitric oxide synthase activity in parotid glands from rats with experimental periodontitis.

OBJECTIVE: In this study we investigated the activity of the nitric oxide synthase (NOS) in parotid glands from rats with experimental periodontitis and controls. METHODS: Periodontitis was produced by a ligature placed around the cervix of the two lower first molar. Experiments were carried out 22 days after the ligature. RESULTS: Ligation caused an increase in parotid NOS activity. The selective blocker of the inducible isoform of the enzyme partially inhibited its activity in parotid glands from rat with ligature. In controls, the activity was partially inhibited by the antagonists of the selective neural and endothelial isoforms. NOS activity in rats with ligature was cyclic adenosine monophosphate (cAMP)-dependent while in controls it was calcium-dependent. Prostaglandin E2 concentration was increased in parotid gland from rats with ligature. The inhibitor of prostaglandin production, FR 122047, diminished both, prostaglandin production and NOS activity. In rats with ligature unstimulated amylase released is increased. Both, prostaglandin and NOS were involved in the increment of amylase release. CONCLUSION: It can be concluded that in parotid glands from ligated rats, prostaglandin E2 production is increased and, through cAMP accumulation, activates the inducible NOS isoform. The increment of nitric oxide production participates in the increase in basal amylase release.

Expression of matrix metalloproteinases MMP-2, MMP-9 and their tissue inhibitors TIMP-1 and TIMP-2 in the epithelium and stroma of salivary gland pleomorphic adenomas.

AIMS: The balance between matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) is involved in the morphogenesis of normal salivary gland as well as in the mechanisms of tumour invasion and metastasis. The role of MMPs and TIMPs in pleomorphic adenoma has not been elucidated sufficiently. Our aim was to analyse the mRNA and protein expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 in the epithelium and stroma of pleomorphic adenoma and to evaluate their roles. METHODS AND RESULTS: In each sample from six patients, cells from the epithelium and stroma were obtained by laser microdissection. The mRNA expression of MMPs and TIMPs was determined by real-time quantitative reverse transcriptase-polymerase chain reaction and protein expression was confirmed by immunohistochemistry. Results showed that mRNA expression of MMPs and TIMPs was significantly higher in stroma than in epithelium in most patients. MMPs and TIMPs were immunoreactive mainly in epithelium rather than in stroma. CONCLUSIONS: Our results provide preliminary evidence that stromal myoepithelium may be the primary source of MMPs and that the stroma has the potential to play a more important role than ductal epithelium in biological behaviour of pleomorphic adenomas. These findings seem worthy of further investigation.

Androgen ablation leads to an upregulation and intranuclear accumulation of deoxyribonuclease I in rat prostate epithelial cells paralleling their apoptotic elimination.

After androgen ablation by castration, the epithelial cells of the rat ventral prostate are eliminated by apoptosis. The number of cells showing apoptotic chromatin degradation increases with time up to day 3 after castration as verified by in situ end labeling of fragmented DNA. Apoptotic chromatin degradation is catalyzed by a Ca2+, Mg2+-dependent endonuclease. Recently, evidence has been presented that suggests deoxyribonuclease I (DNase I) is identical or very closely related to the apoptotic endonuclease (Peitsch, M.C., B. Polzar, H. Stephan, T. Crompton, H.R. MacDonald, H.G. Mannherz, and J. Tschopp. 1993. EMBO [Eur. Mol. Biol. Organ.] J. 12:371-377). Therefore, the expression of DNase I in the ventral prostate of the rat was analyzed before and after androgen ablation at the level of protein, enzymatic activity, and gene transcripts using immunohistochemical and biochemical techniques. DNase I immunoreactivity was detected only in a few single epithelial cells before androgen ablation. After castration, a time-dependent increase in DNase I immunoreactivity was observed within the epithelial cells. It first appeared after about 12 h in the apical region of a large number of epithelial cells. Up to day 3 after castration, the intracellular DNase I antigenicity continuously increased, and the cell nuclei gradually became DNase I positive. At day 5, almost all nuclei of the epithelium were stained by anti-DNase I. DNase I immunoreactivity was particularly concentrated in cells showing morphological signs of apoptosis, like nuclear fragmentation, and in many cases was found to persist in apoptotic bodies. DNase I gene transcripts were detected in control animals using dot and Northern blotting as well as RNase protection assay. After androgen ablation, the amount of DNase I gene transcripts in total extractable RNA was found unchanged or only slightly decreased up to day 5. Their exclusive localization within the epithelial cells was verified by in situ hybridization. Before castration, the DNase I gene transcripts were homogeneously distributed in all epithelial cells. At day 3, DNase I-specific mRNA was found to be highly concentrated in cells of apoptotic morphology. Using the zymogram technique, a single endonucleolytic activity of about 32 kD was detected in tissue homogenates before castration. After androgen ablation, the endonucleolytic activity increased about four- to sevenfold up to day 3. At day 5, however, it had dropped to its original level. At day 1, three new endonucleolytic variants of higher molecular mass were expressed. At day 3, the predominant endonucleolytic activity exhibited an apparent molecular mass of 32 kD. Enzymatic analysis of the endonucleases present in prostate homogenates before and after castration demonstrated properties identical to DNase I. They were inhibited by chelators of divalent cations, Zn2+ ions and monomeric actin. Immunodepletion was achieved by immobilized antibodies specific for rat parotid DNase I. A polyclonal antibody raised against denatured DNase I was shown by Western blotting to stain a 32-kD band after enrichment of the endonuclease from day 0 and 3 homogenates by preparative gel electrophoresis. The data thus indicate that androgen ablation leads to translational upregulation of an endonucleolytic activity with properties identical to DNase I in rat ventral prostate, followed by its intracellular retention and final nuclear translocation in those epithelial cells that are destined to apoptotic elimination.

Subcellular location of stimulus-affected endogenous phosphoproteins in the rat parotid gland.

Rat parotid minces were labeled with [32P]Pi, stimulated with isoproterenol, homogenized in sucrose, and fractionated on continuous sucrose density gradients. We analyzed the resulting fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiograms were made from the gels. Comparison of fractions from control and isoproterenol-stimulated minces revealed seven phosphoproteins that were affected by isoproterenol. The subcellular location of these proteins was determined by comparing their distribution in the sucrose gradients with that of a number of enzymes that are characteristic of specific organelles. Isoproterenol decreased the phosphorylation of two cytoplasmic proteins (Mr 16,000 and 18,000) and increased the phosphorylation of a third (Mr 14,000). The phosphorylation of two endoplasmic reticulum proteins was increased by isoproterenol (Mr 20,500 and 22,500), as was an Mr 31,000 protein which was probably the S6 ribosomal protein. The phosphorylation of a secretory granule protein (Mr 24,000) was decreased by isoproterenol. We then developed a purification scheme for parotid secretory granules. By using this method, we demonstrated that the phosphorylation of the Mr 24,000 was also decreased by carbamylcholine. Granules purified by this method also contained a small number of other phosphoproteins whose phosphorylation was increased only by isoproterenol. Secretory granule-associated stimulus-affected phosphoproteins were found in the particulate fraction when the granules were hypotonically lysed, and were not extracted from the particulate fraction by washing with 0.6 M KCl.

Two novel peripheral membrane proteins, pasin 1 and pasin 2, associated with Na+,K(+)-ATPase in various cells and tissues.

Purification of pig kidney Na+,K(+)-ATPase at low concentrations of SDS (0.5%) allowed copurification of several peripheral membrane proteins. Some of these associated proteins were identified as components of the membrane cytoskeleton. Here we describe two novel globular proteins of of Mr 77,000 (pasin 1) and Mr 73,000 (pasin 2) which copurify and coimmunoprecipitate with Na+,K(+)-ATPase and can be stripped off Na+,K(+)-ATPase microsomes by 1 M KCl. Pasin 1 and pasin 2 were detected by immunoblot analysis in various cells and tissues including erythrocytes and platelets. Immunostaining revealed colocalization of pasin 1 and Na+,K(+)-ATPase along the basolateral cell surface of epithelial cells of kidney tubules and parotid striated ducts (titers of pasin 2 antibodies were too weak for immunocytochemistry). In erythrocytes, pasin 1 and pasin 2 are minor components bound to the cytoplasmic surface of the plasma membrane. Pasin 1 showed the same electrophoretic mobility as protein 4.1b. However, both proteins have different isoelectric points (pasin 1, pI 6; protein 4.1, pI 7), different chymotryptic fragments, and are immunologically unrelated. Short pieces of sequence obtained from pasin 1 and pasin 2 were not found in any other known protein sequence. The occurrence of pasin 1 and pasin 2 in diverse cells and tissues and their association with Na+,K(+)-ATPase suggests a general role of these proteins in Na+,K(+)-ATPase function.

Secretion granules of the rabbit parotid gland. Isolation, subfractionation, and characterization of the membrane and content subfractions.

A fraction of secretion granules has been isolated from rabbit parotid by a procedure which was found to be especially effective in reducing contamination resulting from aggregation and/or cosedimentation of granules with other cell particulates. The fraction, representing 15 percent (on the average) of the total tissue amylase activity, was homogeneous as judged by electron microscopy and contaminated to exceedingly low levels by other cellular organelles as judged by marker enzymatic and chemical assays. Lysis of the granules was achieved by their gradual exposure to hypotonic NaHCO3, containing 0.5 mM EDTA. The content and the membranes separated by centrifugation of the granule lysate were characterized primarily by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis which indicated that the content was composed of a limited number of molecular weight classes of polypeptides of which three bands (having approximate mol wt 58,000, 33, 000, and 12,000) could be considered major components. The gel profile of the membrane subfraction was characterized by 20-30 Coomassie brilliant blue-staining bands of which a single species of mol wt 40,000 was the conspicuous major polypeptide. Two types of experiments employing gel electrophoretic analysis were carried out for identifying and assessing the extent of residual secretory protein adsorbed to purified granule membranes: (a) examination of staining and radioactivity profiles after mixing of radioactive secretion granule extract with nonradioactively labeled granule membranes and (b) comparison of gel profiles of secretion granule extract and granule membranes with those of unlysed secretion granules and secretory protein dischraged from lobules in vitro or collected by cannulation of parotid ducts, the last two samples being considered physiologic secretory standards. The results indicated that the membranes were contaminated to a substantial degree by residual, poorly extractable secretory protein even though assays of membrane fractions for a typical secretory enzyme activity (amylase) indicated quite through separation of membranes and content. Hence, detailed examination of membrane subfractions for residual content species by gel electrophoresis points to the general unity and sensitivity of this technique as a means for accurately detecting a defined set of polypeptides occurring as contaminants in cellular fractions or organelle subfractions.

Radioautographic analysis of the secretory process in the parotid acinar cell of the rabbit.

Intracellular transport of secretory proteins has been studied in the parotid to examine this process in an exocrine gland other than the pancreas and to explore a possible source of less degraded membranes than obtainable from the latter gland. Rabbit parotids were chosen on the basis of size (2-2.5 g per animal), ease of surgical removal, and amylase concentration. Sites of synthesis, rates of intracellular transport, and sites of packaging and storage of newly synthesized secretory proteins were determined radioautographically by using an in vitro system of dissected lobules capable of linear amino acid incorporation for 10 hr with satisfactory preservation of cellular fine structure. Adequate fixation of the tissue with minimal binding of unincorporated labeled amino acids was obtained by using 10% formaldehyde-0.175 M phosphate buffer (pH 7.2) as primary fixative. Pulse labeling with leucine-(3)H, followed by a chase incubation, showed that the label is initially located (chase: 1-6 min) over the rough endoplasmic reticulum (RER) and subsequently moves as a wave through the Golgi complex (chase: 16-36 min), condensing vacuoles (chase: 36-56 min), immature granules (chase: 56-116 min), and finally mature storage granules (chase: 116-356 min). Distinguishing features of the parotid transport apparatus are: low frequency of RER-Golgi transitional elements, close association of condensing vacuoles with the exit side of Golgi stacks, and recognizable immature secretory granules. Intracelular processing of secretory proteins is similar to that already found in the pancreas, except that the rate is slower and the storage is more prolonged.