Eileen L. Watson

Ca2+-activated membrane ATPase: Selective inhibition by ruthenium red

1. 1. Ca2+-ATPase, (Na+-K+)-ATPase and Mg2+-ATPase activities were determined in isolated red blood cell membranes. 2. 2. The hexavalent dye, ruthenium red, produced concentration-dependent inhibition of Ca2+-ATPase (I50 ≅ 10−5M) without significantly affecting (Na+-K+)-ATPase or Mg2+-ATPase. 3. 3. We suggest that ruthenium red may be a useful tool for selective inhibition of Ca2+ transport and/or Ca2+-ATPase.

Human oral epithelial cell culture. I, improved conditions for reproducible culture in serum-free medium

SummaryGingival tissue from healthy adult human donors was used as a source of epithelial cells for culture. An overnight incubation of this tissue with dispase facilitated the mechanical separation of the surface epithelium from the underlying fibrous connective tissue. This step minimized culture contamination with fibroblasts. The epithelium was then trypsinized to prepare a single cell suspension. The cell pellets were collected by centrifugation and resuspended in keratinocyte growth medium, incubated at 37° C and 5% CO2 in a humid atmosphere. Primary cultures grew in small islands that coalesced at confluency. Immunohistochemistry demonstrated uniform staining of the cells with antibodies to keratins of stratified squamous epithelium. Ultrastructurally, the cells contained distinct intermediate filaments. When cells were grown in media with low calcium (0.15 mM), cell-to-cell contacts were via interlacing papillary projections with no desmosomes. However, when cells were grown under physiologic calcium (1.2 mM), desmosomes were prominent and well developed. Cells were maintained in culture for over 100 d (7 passages).

Mechanisms of non-electrolyte penetration across dog and rabbit oral mucosa in vitro

Abstract Permeability coefficients were determined for 20 14 C-labelled compounds using canine and rabbit lingual frenulum as the test tissue. The oil-to-water distribution coefficients of these compounds differed by over 5 × 10 5 -fold. A linear relationship between permeability coefficients and oil-to-water distribution coefficients was found for those compounds with oil-to-water distribution coefficient greater than that of water, demonstrating the importance of lipid solubility on the rate of transfer across oral mucosa. The data further indicate that hydrogen bonding, cylindrical radius and chemical differences contribute to penetration rates. The plot of permeability coefficient versus molecular volume for compounds with oil-to-water distribution coefficients less than that of water is biphasic, suggesting two different routes of permeation for these compounds. This indicates that oil-soluble compounds traverse oral mucosa via solvation, whereas water-soluble molecules with a molecular volume of less than 80 cm 3 /mol cross primarily through membrane pores; larger water-soluble molecules pass extracellularly.

Ryanodine Receptor Type III (Ry3R) Identification In Mouse Parotid Acini PROPERTIES AND MODULATION OF [3H]RYANODINE-BINDING SITES

Immunoblot analysis and [3H]ryanodine binding were used to characterize and identify ryanodine receptors (RyRs) in nonexcitable mouse parotid acini. Western analysis revealed ryanodine receptor type III (Ry3R) to be the only detectable isoform in parotid microsomal membranes. Binding of [3H]ryanodine to microsomal fractions was dependent on Ca2+, salt, pH, and temperature. At 23 °C, and in the presence of 0.5 m KCl and 100 μm Ca2+, [3H]ryanodine bound specifically to membranes with high affinity (K d = 6 nm); maximum binding capacity (B max) was 275 fmol/mg protein. Mg2+ and ruthenium red inhibited [3H]ryanodine binding (IC50 = 1.4 mm and 0.5 μm, respectively). 4-Chloro-3-ethylphenol enhanced the binding of [3H]ryanodine 2.5-fold; whereas ATP and caffeine were much less efficacious toward activating Ry3R (56% and 18% maximal enhancement, respectively). Bastadin, a novel modulator of the 12-kDa FK506 binding protein·RyR complex, increased [3H]ryanodine binding 3–4-fold by enhancingK d . The immunosuppressant FK506 enhanced [3H]ryanodine receptor occupancy at >100 μm and antagonized the action of bastadin, suggesting that an immunophilin modulates Ry3R in parotid acini. These results suggest that Ry3R may play an important role in Ca2+ homeostasis in mouse parotid acini.

Arachidonic acid regulates two Ca2+ entry pathways via nitric oxide

Several regulated Ca2+ entry pathways have been identified, with capacitative Ca2+ entry (CCE) being the most characterized. In the present study, we examined Ca2+ entry pathways regulated by arachidonic acid (AA) in mouse parotid acini. AA induced Ca2+ release from intracellular stores, and increased Ca2+ entry. AA inhibited thapsigargin (Tg)-induced CCE, whereas AA activated Ca2+ entry when CCE was blocked by gadolinium (Gd3+). AA-induced Ca2+ entry was associated with depletion of calcium from ryanodine-sensitive stores; both AA-induced Ca2+ release and Ca2+ entry were inhibited by tetracaine and the nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI). The nitric oxide (NO) donor, 1,2,3,4-ox-triazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162), but not 8-bromo-cGMP, mimicked the effects of AA in inhibiting CCE. Results suggest that AA acts via nitric acid to inhibit the CCE pathway that is selective for Ca2+, and to activate a second Ca2+ entry pathway that is dependent on depletion of Ca2+ from ryanodine-sensitive stores.

GTP-Binding Proteins and Regulated Exocytosis

Regulated exocytosis, which occurs in response to stimuli, is a two-step process involving the docking of secretory granules (SGs) at specific sites on the plasma membrane (PM), with subsequent fusion and release of granule contents. This process plays a crucial role in a number of tissues, including exocrine glands, chromaffin cells, platelets, and mast cells. Over the years, our understanding of the proteins involved in vesicular trafficking has increased dramatically. Evidence from genetic, biochemical, immunological, and functional assays supports a role for ras-like monomeric GTP-binding proteins (smgs) as well as heterotrimeric GTP-binding protein (G-protein) subunits in various steps of the vesicular trafficking pathway, including the transport of secretory vesicles to the PM. Data suggest that the function of GTP-binding proteins is likely related to their localization to specific cellular compartments. The presence of both G-proteins and smgs on secretory vesicles/granules implicates a role for these proteins in the final stages of exocytosis. Molecular mechanisms of exocytosis have been postulated, with the identification of a number of proteins that modify, regulate, and interact with GTP-binding proteins, and with the advent of approaches that assess the functional importance of GTP-binding proteins in downstream, exocytotic events. Further, insight into vesicle targeting and fusion has come from the characterization of a SNAP receptor (SNARE) complex composed of vesicle, PM, and soluble membrane trafficking components, and identification of a functional linkage between GTP-binding and SNARES.

Nitric oxide acts independently of cGMP to modulate capacitative Ca2+ entry in mouse parotid acini

Carbachol- and thapsigargin-induced changes in cGMP accumulation were highly dependent on extracellular Ca2+ in mouse parotid acini. Inhibition of nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC) resulted in complete inhibition of agonist-induced cGMP levels. NOS inhibitors reduced agonist-induced Ca2+ release and capacitative Ca2+ entry, whereas the inhibition of sGC had no effect. The effects of NOS inhibition were not reversed by 8-bromo-cGMP. The NO donor GEA-3162 increased cGMP levels blocked by the inhibition of sGC. GEA-3162-induced increases in Ca2+ release from ryanodine-sensitive stores and enhanced capacitative Ca2+ entry, both of which were unaffected by inhibitors of sGC but reduced by NOS inhibitors. Results support a role for NO, independent of cGMP, in agonist-mediated Ca2+release and Ca2+ entry. Data suggest that agonist-induced Ca2+influx activates a Ca2+-dependent NOS, leading to the production of NO and the release of Ca2+ from ryanodine-sensitive stores, providing a feedback loop by which store-depleted Ca2+ channels are activated.

Capacitative Ca2+ entry is involved in cAMP synthesis in mouse parotid acini.

Muscarinic receptor interaction leading to augmentation of isoproterenol-stimulated cAMP accumulation in mouse parotid acini involves Ca2+ (28). The effectiveness of capacitative Ca2+entry and intracellular Ca2+release on this response was determined in time course studies by using three independent tools to manipulate the free intracellular Ca2+ concentration: the muscarinic agonist carbachol, thapsigargin, and ionomycin. Time course studies revealed that Ca2+ release from intracellular stores by carbachol produced an early rapid increase (0.25-0.5 min) in stimulated cAMP levels, whereas capacitative Ca2+ entry resulted in a sustained increase in stimulated cAMP levels that was blocked by La3+. Capacitative Ca2+ entry, alone, was involved in thapsigargin and ionomycin augmentation of stimulated cAMP accumulation. The inability of phosphodiesterase inhibitors, 3-isobutyl-1-methylxanthine and milrinone, to prevent agonist augmentation of cAMP levels, as well as the finding that the type VIII adenylyl cyclase (ACVIII) is expressed in parotid acini, suggests that capacitative Ca2+ entry augments stimulated cAMP accumulation, at least in part, via activation of this adenylyl cyclase isoenzyme.

Phosphorylation of rabbit parotid microsomal protein occurs only with β-adrenergic stimulation

Summary β-adrenergic stimulation of rabbit parotid gland secretion is associated with phosphorylation of a 34,000 (major) and a 30,000 (minor) molecular weight protein located in the microsomal fraction. Phosphorylation of the same proteins is not observed with either carbachol or phenylephrine despite the fact that both stimulate amylase release. The evidence is consistent with the hypothesis that phosphorylation of specific proteins in the plasma membrane is involved in β-adrenergic stimulated exocytosis.

Immunolocalization of rap1 in the rat parotid gland: detection on secretory granule membranes.

The objective of this study was to localize rap1 in the rat parotid gland. Rap1 is a small GTP-binding protein that has been linked to phagocytosis in neutrophils and various functions in platelets. In this study, we used [α-32 P]-GTP-blot overlay analysis, immunoblot analysis, and immunohistochemistry to identify rap1 in rat parotid gland. The immunohistochemical techniques included immunoperoxidase and widefield microscopy with image deconvolution. Rap1 was identified in the secretory granule membrane (SGM), plasma membrane (PM), and cytosolic (CY) fractions, with the largest signal being in the SGM fraction. The tightly bound vs loosely adherent nature of SGM-associated rap1 was determined using sodium carbonate, and its orientation on whole granules was assessed by trypsin digestion. Rap1 was found to be a tightly bound protein rather than a loosely adherent contaminant protein of the SGM. Its orientation on the cytosolic face of the secretory granule (SG) is of significance in postulating a function for rap1 because exocytosis involves the fusion of the cytoplasmic face of the SG with the cytoplasmic face of the PM, with subsequent release of granule contents (CO). Therefore, the localization and high concentration of rap1 on the SGM and its cytosolic orientation suggest that it may play a role in the regulation of secretion.