Karnam R. Purushotham

Utilization of the non-obese diabetic (NOD) mouse as an animal model for the study of secondary Sjögren's syndrome.

Sjogren’s syndrome (S.S.) in the human patient population is an autoimmune inflammatory disease presenting clinical symptoms of xerophthalmia and xerostomia1. This condition predominantly affects women. Most diagnoses of S.S. is made in association with autoimmune connective tissue diseases such as rheumatoid arthritis or systemic lupus erythematosus.2 However, it can also be obseved as an isolated phenomenon, described as primary S.S.

Detection of insulin and insulin-like growth factors I and II in saliva and potential synthesis in the salivary glands of mice: Effects of type 1 diabetes mellitus

The salivary glands of mammals synthesize and secrete a number of peptide growth factors that play important roles in cell/tissue homeostasis and embryonic development. Using a radioimmunoassay, insulin, insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II) were detected in saliva from mice. Unlike epidermal growth factor (EGF), there was no sexual dimorphism in the concentrations of the insulin growth factor family. Immunohistochemical localization of IGF-I and IGF-II was confined to the duct cells of both the parotid and the submandibular glands. Reverse transcriptase-polymerase chain reaction amplification of total RNA from parotid and submandibular glands confirmed the presence of all three hormone/growth factor mRNAs in both glands. The levels of insulin and IGF-I were higher in saliva from an animal model for autoimmune type 1 diabetes, the non-obese diabetic (NOD) mouse, than in a second inbred strain, BALB/c. In contrast, the IGF-II levels were decreased relative to the BALB/c strain. With the onset of diabetes in NOD mice, insulin levels declined, while IGF-I and IGF-II levels showed trends toward lower levels of these growth factors when compared with non-diabetic animals. These changes were reflected in the concentrations from parotid and submandibular gland cell lysates.

Desipramine induced changes in salivary proteins, cultivable oral microbiota and gingival health in aging female NIA Fischer 344 rats

Cyclic antidepressants are still a dominating group of psychotherapeutic drugs used in the treatment of depression. One of their major side effect is salivary gland dysfunction (oral dryness, xerostomia), leading in humans to increased oral disease and dysfunction of speech, chewing, swallowing and taste. The purpose of this study was to assess the effects of the long-term administration of the tricyclic antidepressant desipramine and the reversibility of this treatment following a 15 d washout period on specific salivary proteins, composition of oral microbiota, and oral health (gingivitis) of aging female F344 rats. Total salivary proteins showed decreased concentrations with age and desipramine. Similar SDS/PAGE protein profiles appeared in all phases but in different relative amounts with age and treatment. While certain proteins maintained steady levels (lactoferrin) or decreased with age and treatment (amylase), the synthesis of proline-rich proteins, high molecular weight mucin-type glycoproteins, and lysozyme was induced with desipramine and age. The oral microbiota was significantly changed with age and the administration of the antidepressant. The incidence of gingivitis with desipramine was highest in the oldest animals, For the different parameters measured, recovery was delayed with age. These data indicate, that desipramine has profound effects on salivary protein secretion. This may partially explain the changes in microbiota and the increased incidence of gingivitis.

Reconstituted Cl- pump protein: a novel ion(Cl-)-motive ATPase.

Cl− absorption by theAplysia californica foregut is effected through an active Cl− transport mechanism located in the basolateral membrane of the epithelial absorptive cells. These basolateral membranes contain both Cl−-stimulated ATPase and ATP-dependent Cl− transport activities which can be incorporated into liposomes via reconstitution. Utilizing the proteoliposomal preparation, it was demonstrated that ATP, and its subsequent hydrolysis, Mg2+, Cl−, and a pH optimum of 7.8 were required to generate maximal intraliposomal Cl− accumulation, electrical negativity, and ATPase activity. Additionally, an inwardly-directed valinomycininduced K+ diffusion potential, making the liposome interior electrically positive, enhanced both ATP-driven Cl− accumulation and electrical potential while an outwardly-directed valinomycininduced K+ diffusion potential, making the liposome interior electrically negative, decreased both ATP-driven Cl− accumulation and electrical potential compared with proteoliposomes lacking the ionophore. Either orthovanadate orp-chloromercurobenzene sulfonate inhibited both the ATP-dependent intraliposomal Cl− accumulation, intraliposomal negative potential difference, and also Cl−-stimulated ATPase activity. Both aspects of Cl− pump transport kinetics and its associated catalytic component kinetics were the first obtained utilizing a reconstituted transporter protein. These results strongly support the hypothesis that Cl−-ATPase actively transports Cl− by an electrogenic process.

The Role of Phosphotyrosine Signaling Pathway in Parotid Gland Proliferation and Function

Tyrosine phosphorylation and the intracellular signaling processes associated with it have been the focus of intense study due to its importance in the regulation of biological processes as diverse as cell proliferation and cell differentiation. While much of what we now understand has been derived from the study of cell lines and tumor cells, the salivary glands provide a model to examine the effects of tyrosine kinases and tyrosine phosphatases in a normal differentiated tissue. This review will focus, therefore, on the role tyrosine kinases and phosphatases play in inducing the transition from stasis to active proliferation and their potential role in mediating secretory function of the salivary glands.

Alterations in the subcellular distribution of p21ras-GTPase activating protein in proliferating rat acinar cells

Rat parotid acinar cells undergo transient proliferation in response to chronic administration of the beta-adrenergic agonist isoproterenol or epidermal growth factor (EGF). Treatment with these agents caused an increase in tyrosine phosphorylation of p21ras-GTPase activating protein (GAP). This phosphorylation event was accompanied by a redistribution of the protein from the plasma membrane to internal membrane compartments. Separation of subcellular membranes revealed increased GAP associated with a low density population of vesicles concomitant with growth stimulation as well as to the nuclear membrane, but not the nucleoplasm. Upon cessation of hyperplasia induced by isoproterenol, phosphorylated GAP present in the plasma membrane returned to control cell levels.

Role of protein phosphorylation and inositol phospholipid turnover in rat parotid gland proliferation

The involvement of protein phosphorylation in isoproterenol (ISO)-mediated proliferation in the rat parotid gland was investigated by labeling the cells with [32P] orthophosphate. An increased (4–6 fold) incorporation of the radiolabel was noted in the total parotid gland homogenates of ISO-treated animals when compared to controls. Plasma membrane, nuclear membrane and cytoplasm were isolated, the proteins separated by SDS/PAGE and the phosphoproteins detected by autoradiography. Two phosphoproteins with apparent Mr of 45 and 170 kDa were identified in the cytoplasm while the 170 kDa phosphoprotein also appeared as part of plasma membrane. Transfer of these proteins to nitrocellulose followed by Western blot detection with an antiphosphotyrosine monoclonal antibody showed reactivity with the 170 kDa region of the plasma membrane and cytoplasm. Separate in vitro studies involving incubations of rat parotid slices with 0.2 mM ISO and [3H] myo-inositol for 1 min induced inositol phosphate hydrolysis resulting in a significant increase in inositol-bis and -tris phosphate production. Inositol phosphate production can be blocked by pre-incubation with a mixed β-adrenergic receptor antagonist but not with physiological concentrations of α- or β1-specific adrenergic receptor antagonists, indicating the ISO effects are mediated through the β2-adrenergic receptors. The inclusion of calmodulin antagonists along with ISO prevented the expression of cell-surface galactosyltransferase and retarded gland hypertrophy and hyperplasia. These results suggest that ISO treatment leads to the phosphorylation of target proteins which may be involved in signal transduction pathways leading to cell proliferation.

Cell surface galactosyltransferase acts as a general modulator of rat acinar cell proliferation

SummarySeveral physiological parameters were examined for inducing acinar cell proliferation and corresponding increased expression of β1–4 galactosyltransferase. In this study, dietary changes causing acinar cell proliferation included the following: the introduction of animals to a liquid diet (causing gland atrophy) followed by reintroduction of solid chow, gustatory stimulation provided by the introduction of 0.5% citric acid to animal drinking water, and removal of the submandibular gland with subsequent reliance on the parotid gland for salivary protein. Alterations in growth factor levels were produced by injecting animals with a chronic (3 day) regimen of either nerve growth factor or epidermal growth factor. Parotid gland proliferation could be blocked in all cases except EGF by the injection of propranolol, a β-adrenoceptor antagonist, or the galactosyltransferase specific modifier protein, α-lactalbumin. EGF-induced proliferation could, however, be prevented by treating the animals with monoclonal antibody to EGF receptor or galactosyltransferase modifier protein a-lactalbumin. These results for normal acinar cell proliferation suggest a direct role for cell surface β1–4 galactosyltransferase in signalling and maintaining active cell growth.

ALTERATIONS IN THE LEVEL OF PHOSPHOTYROSINE SIGNAL TRANSDUCTION CONSTITUENTS IN HUMAN PAROTID TUMORS

Abstract Human parotid tumors were evaluated for the activation of the phosphotyrosine signaling pathway by Western blot, enzyme activity assay, and reverse transcriptase-polymerase chain reaction. Warthins tumor and mucoepidermoid carcinomas had the greatest level of tyrosine phosphorylated proteins identified in plasma membrane fractions. These tumors, along with pleomorphic adenocarcinoma, showed high levels of membrane expression of the tyrosine kinase receptor, c-erbB-2, and phosphatidyiinositol-3-kinase. Expression of the epidermal growth factor receptor was confined to normal tissue. The level of mRNA for c-erb was elevated only in mucoepidermoid carcinomas. Messenger RNA levels for ras were unchanged from control levels in all tumors, while the level of src mRNA was higher in the tumor samples than the normal parotid tissue. The activities of several signal transduction kinases, including protein kinase A and C were elevated in tumor tissue (7.7- to 18.9-and 0.4- to 3.7-fold higher, respectively), relative to surrounding normal tissue. While the level of glandular amylase was reduced (22%-0% of normal levels) in the tumor tissue, epidermal growth factor (EGF) and transforming growth factor-α (TGFα) content was dramatically higher in the neoplastic tissue (10- to 170-fold and 4.6- to 6.0-fold, respectively). These results suggest that with the presence of elevated levels of EGF, TGFα, and the oncoprotein receptor c-erbB-2 in the membrane of parotid tumors, cell proliferation and activation of the phosphotyrosine signal transduction pathway may involve autocrine stimulation through the expression of high levels of growth factor and receptor in the same tissue.

Down-regulation of cellular proto-oncogenes during inhibition of rat parotid acinar cell proliferation

The role of cell surface galactosyltransferase in mediating isoproterenol-induced parotid gland hypertrophy and hyperplasia was examined in rat parotid gland acinar cells. Introduction of the transferase modifier, alpha-lactalbumin, or galactosyltransferase-associated kinase inhibitor trifluoperazine, into beta-agonist-treated rats prevented acinar cell proliferation as determined by [3H]thymidine incorporation after 96 h of treatment. However, [3H]thymidine incorporation into DNA after 24 h of treatment, with injection of a combination of isoproterenol/alpha-lactalbumin or isoproterenol/trifluoperazine, was similar to injections of isoproterenol alone; suggesting that acinar cells could be stimulated to undergo a single round of DNA synthesis. Northern blot analysis of myc and fos expression followed a similar pattern of down-regulation to control levels after 96 h but not after 24 h. Hybridization with erb B showed little change with proliferation, confirming previous observations on protein levels of the EGF-receptor in acinar cells. Western blot analysis of nuclear protein expression of myc revealed that isoproterenol caused an increase in a 62-kDa protein which was again down-regulated with inhibition of cell proliferation. Analysis of protein levels of Rb110 protein showed no change in protein level in the nucleus with cell proliferation, but did show an associated increase in protein phosphorylation in response to growth stimulation.