Recurrence of Breast Cancer After Primary Treatment: A Matched Comparison Study of Disease-Free Survival in Women Who Do and Do Not Receive Adjunctive Naturopathic Oncology Care.

PURPOSE: To compare disease free survival experienced by women who received usual oncologic care compared to a cohort of women who received naturopathic oncology care in addition to usual care. METHODS: Women with breast cancer who received naturopathic oncology (NO) care in Western Washington State (WA) (N = 176) were recruited to a prospective study of clinical health-related quality of life outcomes and then matched to women who received usual care (UC) only (N = 334). RESULTS: Among 510 women with breast cancer stages 1 to 3, a total of 50 women (10%) experienced a disease-free survival (DFS) ending event within the observation period; 23 (6.8% of those in the UC cohort, and 27 (15.3% of those in the NO cohort (P < .05). Although, women in the 2 cohorts received similar surgical, chemotherapy, and radiation treatment, women with breast cancer who received naturopathic oncology adjunctive care were less likely to use anti-estrogen therapy, and experienced poorer DFS (logrank test, P < .05). However, differences in DFS could not be shown to be due to cohort differences in anti-estrogen therapy, baseline HRQOL, or naturopathic oncology therapies prescribed. The stage 3 women in the naturopathic oncology group had more advanced disease at diagnosis. They were more likely to have 5 or more metastatic lymph nodes at baseline (18.5%) compared to their usual care matched control group (13%). Women in the naturopathic oncology group also had higher grade tumors at diagnosis. CONCLUSIONS: Results show that recurrence of breast cancer was associated with more advanced malignant lymph node involvement; and that naturopathic oncology services provided in 2009-2015 did not improve disease-free survival in these high-risk breast cancer patients.

Generalized Titarenko's algorithm for ring artefacts reduction.

A fast algorithm for ring artefact reduction in high-resolution micro-tomography with synchrotron radiation is presented. The new method is a generalization of the one proposed by Titarenko and collaborators, with a complete sinogram restoration prior to reconstruction with classical algorithms. The generalized algorithm can be performed in linear time and is easy to implement. Compared with the original approach, with an explicit solution, this approach is fast through the use of the conjugate gradient method. Also, low/high-resolution sinograms can be restored using higher/lower-order derivatives of the projections. Using different order for the derivative is an advantage over the classical Titarenko's approach. Several numerical results using the proposed method are provided, supporting our claims.

Fluvastatin and lovastatin inhibit granulocyte macrophage-colony stimulating factor-stimulated human eosinophil adhesion to inter-cellular adhesion molecule-1 under flow conditions.

BACKGROUND: Eosinophil accumulation in the lung is an important feature of airway inflammation in asthma. There is therefore much interest in developing novel therapies to prevent this process. Accumulating evidence suggests that statins have anti-inflammatory properties, including inhibition of leucocyte accumulation. We therefore assessed the ability of five statins to inhibit human eosinophil adhesion to recombinant human inter-cellular adhesion molecule (rhICAM)-1 under physiologically relevant flow conditions. METHODS: Purified eosinophils were pre-treated with a panel of statins before elucidation of the adhesion profiles of resting and granulocyte macrophage-colony stimulating factor (GM-CSF)-stimulated cells to rhICAM-1-coated microchannels at a flow rate of 0.5 dynes/cm(2). Images were recorded in real-time at 1 min intervals and analysed using Ducocell software. RESULTS: Fluvastatin and lovastatin (both 10 nm) significantly inhibited GM-CSF-stimulated eosinophil adhesion to rhICAM-1 after 2 min (34.4+/-3.0% inhibition and 37.8+/-12.6% inhibition, respectively, n=4, P<0.05) but had no significant inhibitory effect on unstimulated eosinophil adhesion. Mevastatin, simvastatin, and pravastatin (all 10 nm) had no significant effect on GM-CSF-stimulated eosinophil adhesion to rhICAM-1. A concentration range of fluvastatin and lovastatin inhibited GM-CSF stimulated eosinophil adhesion with significant (P<0.05) inhibition observed at low concentrations of 1 nm for both drugs. Mevalonate (100 nm) reversed fluvastatin-mediated but not lovastatin-mediated inhibition of eosinophil adhesion. CONCLUSIONS: Inhibition of eosinophil adhesion to ICAM-1 by fluvastatin and lovastatin under physiological shear stress represent novel actions by these drugs that may inform the development of anti-inflammatory therapy for allergic disease.

Upregulation of PIP3-dependent Rac exchanger 1 (P-Rex1) promotes prostate cancer metastasis.

Excessive activation of G-protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK) pathways has been linked to prostate cancer metastasis. Rac activation by guanine nucleotide exchange factors (GEFs) plays an important role in directional cell migration, a critical step of tumor metastasis cascades. We found that the upregulation of P-Rex1, a Rac-selective GEF synergistically activated by Gbetagamma freed during GPCR signaling, and PIP3, generated during either RTK or GPCR signaling, strongly correlates with metastatic phenotypes in both prostate cancer cell lines and human prostate cancer specimens. Silencing endogenous P-Rex1 in metastatic prostate cancer PC-3 cells selectively inhibited Rac activity and reduced cell migration and invasion in response to ligands of both epidermal growth factor receptor and G-protein-coupled CXC chemokine receptor 4. Conversely, expression of recombinant P-Rex1, but not its 'GEF-dead' mutant, in non-metastatic prostate cancer cells, such as CWR22Rv1, increased cell migration and invasion through Rac-dependent lamellipodia formation. More importantly, using a mouse xenograft model, we showed that the expression of P-Rex1, but not its mutant, induced lymph node metastasis of CWR22Rv1 cells without an effect on primary tumor growth. Thus, by functioning as a coincidence detector of chemotactic signals from both GPCRs and RTKs, P-Rex1-dependent activation of Rac promotes prostate cancer metastasis.

Regulator of G-protein signaling 2 (RGS2) inhibits androgen-independent activation of androgen receptor in prostate cancer cells.

Hormones acting through G protein-coupled receptors (GPCRs) can cause androgen-independent activation of androgen receptor (AR) in prostate cancer cells. Regulators of G-protein signaling (RGS) proteins, through their GTPase activating protein (GAP) activities, inhibit GPCR-mediated signaling by inactivating G proteins. Here, we identified RGS2 as a gene specifically downregulated in androgen-independent prostate cancer cells. Expression of RGS2, but not other RGS proteins, abolished androgen-independent AR activity in androgen-independent LNCaP cells and CWR22Rv1 cells. In LNCaP cells, RGS2 inhibited G(q)-coupled GPCR signaling. Expression of exogenous wild-type RGS2, but not its GAP-deficient mutant, significantly reduced AR activation by constitutively activated G(q)Q209L mutant whereas silencing endogenous RGS2 by siRNA enhanced G(q)Q209L-stimulated AR activity. RGS2 had no effect on RGS-insensitive G(q)Q209L/G188S-induced AR activation. Furthermore, extracellular signal-regulated kinase 1/2 (ERK1/2) was found to be involved in RGS2-mediated regulation of androgen-independent AR activity. In addition, RGS2 functioned as a growth suppressor for androgen-independent LNCaP cells whereas androgen-sensitive LNCaP cells with RGS2 silencing had a growth advantage under steroid-reduced conditions. Finally, RGS2 expression level was significantly decreased in human prostate tumor specimens. Taken together, our results suggest RGS2 as a novel regulator of AR signaling and its repression may be an important step during prostate tumorigenesis and progression.

Molecular and pharmacological characterization of muscarinic receptor subtypes in a rat parotid gland cell line: comparison with native parotid gland.

The molecular and pharmacological characteristics of muscarinic receptor subtypes in the rat parotid acinar cell line, PAR-C5, were determined and compared with native rat parotid glands to evaluate the PAR-C5 cell line as a model to study receptor-mediated secretion. Reverse transcription-polymerase chain reaction (RT-PCR) identified mRNAs for M(3), M(4), and M(5) receptor subtypes in both PAR-C5 cells and parotid glands. Specific [N-methyl-(3)H]scopolamine binding in PAR-C5 and parotid membranes was to a single class of sites with mean K(D) values of 0.38 and 0.64 nM, respectively. Binding affinities (K(I) values) of muscarinic receptor subtype-selective drugs were obtained in side-by-side experiments comparing PAR-C5 cells with parotid glands. Nonlinear regression analysis indicated that competition binding curves for drugs in PAR-C5 cells and parotid glands fit best to a one-site binding model. K(I) values (nM) in PAR-C5 cells and parotid glands, respectively, for atropine (1.0, 2.1), darifenacin (1.2, 2.0), 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (2.9, 2.4), tripitramine (220, 180), pirenzepine (320, 720), and methoctramine (1400, 1700) were consistent with their known affinities at the M(3) receptor subtype. Affinities (K(B) values) of muscarinic receptor subtype-selective drugs for blocking methacholine-stimulated Ca(2+) mobilization were determined to show which subtype mediates Ca(2+)-dependent secretion in Fura-2-loaded PAR-C5 cells. K(B) values (nM) for atropine (0.44), 4-DAMP (0.38), pirenzepine (140), and methoctramine (320) for blocking Ca(2+) responses correlated well with their known affinities at the M(3) receptor (r(2) = 0.99). These results show that at the level of mRNA, receptor protein and function, PAR-C5 cells and parotid glands are similar, establishing PAR-C5 cells as an important model for muscarinic receptor-mediated secretion.

Saliva and dental caries.

A study of saliva and its tooth-protective components reveals at least four important functions of saliva: (1) buffering ability, (2) a cleansing effect, (3) antibacterial action, and (4) maintenance of a saliva supersaturated in calcium phosphate. Several salivary constituents subserve one or more of these functions. Research has yielded important information about organic and inorganic secretory products. It is also clear that saliva as a unique biologic fluid has to be considered in its entirety to account fully for its effects on teeth. Saliva is greater then the sum of its parts. One reason for this is that salivary components display redundancy of function, each often having more than one function. This redundancy, however, does not imply that proteins that share functional roles all contribute to the same degree. For instance, when comparing proteins that inhibit calcium phosphate precipitation, statherin and acidic proline-rich proteins are most potent, whereas histatins, cystatins, and mucins appear to play lesser roles. The complex interaction between proteins is another major factor contributing to saliva's function. In this regard, heterotypic complexes of various proteins have been shown to form on hydroxyapatite. Mucin binding to other salivary proteins, including proline-rich proteins, histatins, cystatins, and statherin, is well documented. The complexes, whether adsorbed to the tooth surface or in saliva, have important implications for bacterial clearance, selective bacterial aggregation on the tooth surface, and control of mineralization and demineralization. Finally, proteolytic activity of saliva generates numerous products whose biologic activities are often different from their parent compounds. Fluoride is another important component of saliva that is discussed separately in other articles in this issue. The ability of saliva to deliver fluoride to the tooth surface constantly makes salivary fluoride an important player in caries protection largely by promoting remineralization and reducing demineralization. Some key properties of salivary components discussed in this article are listed in Table 1. Saliva is well adapted to protection against dental caries. Saliva's buffering capability; the ability of the saliva to wash the tooth surface, to clear bacteria, and to control demineralization and mineralization; saliva's antibacterial activities; and perhaps other mechanisms all contribute to its essential role in the health of teeth. The fact that the protective function of saliva can be overwhelmed by bacterial action indicates the importance of prevention and therapy as in other infectious diseases. The knowledge of functional properties of saliva as well as those of its separate components may permit a better assessment of dental caries susceptibility. Future research is essential to characterize more fully salivary components and their interactions and how these affect the caries process. With such knowledge, the use of modified oral molecules as therapeutic agents may become a reality. Equally intriguing is the prospect of influencing the secretion of salivary components by greater knowledge and control over the secretory processes responsible for the delivery of those components.

Inhibition of rat parotid ecto-ATPase activity.

The inhibitory profile of several known and suspected ecto-ATPase inhibitors was compared on ecto-ATPase activity in rat parotid plasma membranes. Those chemicals with high IC50 (above 130 microM) were the nucleotides alpha,beta-methylene ATP, beta,gamma-methylene ATP, 2-methylthio ATP, inosine triphosphate, 5'-p-fluorosulphonylbenzoyladenosine, the sulphonates, 1-amino-2-naphthol-4-sulphonic acid, Coomassie brilliant blue G, and the stilbene disulphonates, DIDS and SITS. Those agents with low IC50 were: Coomassie brilliant blue R (114 microM), ATPgammaS (49 microM), suramin (72 microM) and Reactive blue 2 (28 microM). The last three inhibitors have similar potencies as inhibitors of ATP hydrolysis by whole parotid acinar cells. ARL67156, a selective inhibitor of ecto-ATPase, had an IC50 of approx. 120 microM. Suramin displayed non-competitive inhibition of ecto-ATPase whereas the inhibitory effects of ATPgammaS and Reactive blue 2 were curvilinear on Dixon plots. These results define the effects of various agents on ecto-ATPase in an exocrine tissue that has been shown to respond to extracellular ATP.

Identification of muscarinic receptor subtypes in mouse parotid gland.

Immunoprecipitation of muscarinic receptors from mouse parotid membranes by specific subtype antisera showed that M3 and M1 receptors represented 75 and 15% of the total number of precipitable receptors, respectively. [N-methyl-3H]methylscopolamine (NMS) labeled a single class of high-affinity binding sites in membranes from parotid glands with a dissociation constant of 0.67 +/- 0.02 nM and a maximum binding capacity of 176 +/- 15 fmol/mg protein. Competition curves for NMS, atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and para-fluoro-hexahydro-sila-difenidol fit best to a one-site binding model, whereas pirenzepine and methoctramine fit best to a two-site binding model, indicating 76-90% M3 receptors. Results from the use of pirenzepine indicated that the second mouse parotid receptor subtype, unlike that of the submandibular gland, has atypical characteristics for an M1 receptor. The rank order of potency of muscarinic antagonists in inhibiting phosphoinositide turnover and biphasic effects of carbachol on isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was atropine > or = 4-DAMP >> pirenzepine > AF-DX 116. A specific M1 antagonist, m1-toxin, had no effect on carbachol augmentation or inhibition of isoproterenol responses. Results suggest that M3 receptors couple to both augmentation and inhibition of stimulated cAMP levels.

Metabolism of extracellular ATP by rat parotid cells.

ATP hydrolysis and the products of ATP metabolism were measured in intact rat parotid acini. The purpose was to determine the contribution of extracellular enzymes in metabolizing ATP and its metabolites. The total enzyme activity accounting for extracellular ATP breakdown was at least 75% dependent on added divalent cations, consistent with the presence of ectoATPase. Approximately 50% of the added ATP was hydrolysed in 1 h by the cells and this percentage was independent of cell protein concentration from 80 to 296 micrograms/ml and independent of ATP concentration from 4 to 80 microM. ADP. AMP and adenosine were identified as metabolites. Cell adenosine uptake was not a factor in controlling the levels of extracellular adenosine. Generation of adenosine was limited under conditions of higher rates of ATP hydrolysis. Studies in parotid cell membranes showed that very little feedback inhibition of ectoATPase was observed. 5' Nucleotidase was present at levels of activity of 0.06-0.19 mumol/mg protein/h in intact acini. The results confirm the presence of ectonucleotidases which can generate ADP, AMP and adenosine. Ectonucleotidase could contribute to reducing the effect of extracellular ATP on the parotid cell.

Enhanced hypo-osmoregulation induced by warm-acclimation in antarctic fish is mediated by increased gill and kidney Na+/K(+)-ATPase activities.

Serum osmolality and serum inorganic ion concentrations were studied in two antarctic fish species, Trematomus bernacchii and T. newnesi, during 5 weeks of acclimation to 4 degrees C and compared with control values for groups acclimated to -1.5 degrees C. Acclimation to 4 degrees C significantly decreased the serum osmolality of both species, thereby increasing their seawater-to-extracellular fluid (ECF) osmotic gradient. The decline in osmolality with acclimation to 4 degrees C was accompanied by significant and rapid losses of Na+ and Cl- during the first 14 days of acclimation and was maintained throughout the study period. At day 35 of acclimation, the lipid composition and microsomal Na+/K(+)-ATPase specific activities at 4 degrees C and 37 degrees C were determined in membranes from gill, kidney, liver and muscle tissues. No warm-induced decrease in fatty acid unsaturation was found in the tissues of either species. In the gills and kidneys of both species, the Na+/K(+)-ATPase activities assayed at 4 degrees C were increased after acclimation to 4 degrees C. The Na+/K(+)-ATPase activities at 37 degrees C increased at the higher acclimation temperature in T. newnesi kidneys and T. bernacchii gills, but in both species there was no compensation to temperature in the liver, regardless of assay temperature. Muscle Na+/K(+)-ATPase activity decreased in response to warm-acclimation in T. bernacchii and T. newnesi assayed at 4 degrees C and 37 degrees C, respectively. During acclimation to 4 degrees C, the discontinuity in the Arrhenius plot of the Na+/K(+)-ATPase activities of T. newnesi gill moved to a lower temperature, whereas that of kidney remained unchanged. The results indicate that acclimation to 4 degrees C induced a decrease in serum osmolality which resulted from the positive compensation of Na+/K(+)-ATPase in osmoregulatory tissues. The enhancement in Na+/K(+)-ATPase activity at 4 degrees C suggests that energy expenditure in antarctic fish may be lessened, in part, by maintaining a reduced seawater-to-ECF osmotic gradient.

Localization of an ecto-ATPase/cell-CAM 105 (C-CAM) in the rat parotid and submandibular glands.

We investigated an ecto-ATPase/cell-CAM 105 (C-CAM), previously shown to be distinct from the Ca2+ pump, in rat parotid and submandibular glands. Polyclonal antibodies raised against the enzyme were employed using indirect immunofluorescence, peroxidase-anti-peroxidase (PAP), and electron microscopic immunogold labeling procedures to visualize the location of the enzyme. In the PAP-stained sections and with immunofluorescence, labeling was observed on the luminal and lateral surfaces and the intercellular canaliculi of the acinar cells of both glands. The luminal surface of the intercalated ducts was brightly stained, whereas those of the striated and excretory ducts were less prominently labeled. The basal surface of the acinar cells in the parotid gland and the lateral and basal surfaces of the duct cells were not labeled. Apparent labeling was observed on the basal surface of the submandibular acinar cells. Electron microscopy revealed that for both glands the enzyme was primarily localized along the luminal border of the acinar cells, mainly in association with microvilli, with slightly less reactivity along the intercellular canaliculi and lateral borders and relatively little along the basolateral membranes. Gold labeling was also observed on the luminal borders of the intercalated and striated ducts. Possible functions of the C-CAM include breakdown of ATP, stabilization of the microvillar membranes, cell adhesion, and involvement in secretory mechanisms.

Biphasic effects of carbachol on stimulated cAMP accumulation in mouse parotid acini.

Carbachol (0.1-10 microM) augmented the isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by approximately 50% in mouse parotid acini; at carbachol concentrations > 10 microM the stimulatory trend was reduced. These effects were time dependent. In the presence of 3-isobutyl-1-methylxanthine (IBMX), the overall response to carbachol was an inhibition of the isoproterenol response. Pretreatment of acini with pertussis toxin failed to reverse this inhibition, suggesting that the effects of carbachol were not related to effects on the GTP binding protein, Gi. A-23187 mimicked the effects of carbachol on isoproterenol-stimulated cAMP accumulation in the presence and absence of IBMX. In the presence of IBMX, carbachol failed to inhibit isoproterenol-stimulated cAMP accumulation when calcium was absent from the extracellular media and depleted from intracellular stores by thapsigargin. By contrast, in the absence of IBMX, removal of calcium abolished augmentation of isoproterenol responses by low concentrations of carbachol, whereas at higher carbachol concentrations isoproterenol responses were significantly inhibited; the time to maximal cAMP accumulation was decreased approximately eightfold. The results show that the mechanisms underlying the effects of carbachol on cAMP metabolism involve both the enzymes that synthesize and degrade cAMP.

Localization and characterization of a parotid Ca(2+)-dependent ecto-ATPase.

Parotid acini were isolated and tested to further establish the presence of ecto-ATPase in the intact cells. Inhibitors were used to determine if the inhibitor profile of the ATPase was similar to that of a Ca(2+)-ATPase from parotid membranes identified previously as an ecto-ATPase. The Ca(2+)-ATPase of intact cells was insensitive to oligomycin (10 micrograms/ml), N-ethylmaleimide (NEM) (0.1 mM), ruthenium red (0.1 mM), sodium azide (1 mM), and was inhibited approximately 22% by sodium orthovanadate (Na3VO4) (1 mM). This profile was similar to the Ca(2+)-ATPase of intact cells. Trifluoperazine (TFP) (0.1 mM) inhibited the enzyme in intact cells by approximately 32%. The nucleotide substrate specificity of the enzyme also reflected very closely the pattern seen in isolated membranes.

Atypical alpha-1 adrenergic receptors on the rat parotid gland acinar cell.

Subtypes of alpha-1 adrenergic receptors on rat parotid gland acinar cell membranes were characterized using subtype selective alpha adrenergic receptor antagonists. The alpha-1 adrenergic receptor antagonist beta-iodo-[125I]-4-hydroxyphenyl-ethyl-aminomethyl-tetralone (125IBE) had an equilibrium dissociation constant for specific binding sites on these membranes of 0.241 +/- 0.03 nM and a total number of specific radioligand binding sites of 41 +/- 4 fmol bound/mg of protein. Displacement of 125IBE binding by subtype-selective alpha-1 adrenergic receptor antagonists 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane HCl (WB4101) and 5-methylurapidil fit best to biphasic competition curves. The high- and low-affinity inhibition equilibrium dissociation constant for WB4101 were 0.45 +/- 0.1 and 27 +/- 6 nM, respectively. Similarly, the high- and low-affinity inhibition equilibrium dissociation constants for 5-methylurapidil were 0.16 +/- 0.03 and 71 +/- 20 nM, respectively. These affinities for 125IBE binding sites suggest the presence of alpha-1A and alpha-1B adrenergic receptor subtypes on acinar cell membranes. The irreversible alpha-1 adrenergic receptor antagonist chloroethylclonidine was used to inactivate alpha-1B adrenergic receptors on acinar cell membranes. After treatment with chloroethylclonidine, saturation binding analysis demonstrated no change in the total number of 125IBE binding sites. In addition, competition curves for WB4101 and 5-methylurapidil again showed two sites of 125IBE displacement, with no change in antagonist affinities in membranes treated with chloroethylclonidine.(ABSTRACT TRUNCATED AT 250 WORDS)

A Ca(2+)-ATPase from rat parotid gland plasma membranes has the characteristics of an ecto-ATPase.

A Ca(2+)-ATPase with an apparent Km for free Ca2+ = 0.23 microM and Vmax = 44 nmol Pi/mg/min was detected in a rat parotid plasma membrane-enriched fraction. This Ca(2+)-ATPase could be stimulated without added Mg2+. However, the enzyme may require submicromolar concentrations of Mg2+ for its activation in the presence of Ca2+. On the other hand, Mg2+ could substitute for Ca2+. The lack of a requirement for added Mg2+ distinguished this Ca(2+)-ATPase from the Ca(2+)-transporter ATPase in the plasma membranes and the mitochondrial Ca(2+)-ATPase. The enzyme was not inhibited by several ATPase inhibitors and was not stimulated by calmodulin. An antibody which was raised against the rat liver plasma membrane ecto-ATPase, was able to deplete this Ca(2+)-ATPase activity from detergent solubilized rat parotid plasma membranes, in an antibody concentration-dependent manner. Immunoblotting analysis of the pellet with the ecto-ATPase antibody revealed the presence of a 100,000 molecular weight protein band, in agreement with the reported ecto-ATPase relative molecular mass. These data demonstrate the presence of a Ca(2+)-ATPase, with high affinity for Ca2+, in the rat parotid gland plasma membranes. It is distinct from the Ca(2+)-transporter, and immunologically indistinguishable from the plasma membrane ecto-ATPase.

Calcium uptake in rat parotid gland secretory granules.

45Ca2+ uptake in isolated rat parotid secretory granules was examined in the presence of oxalate. Uptake of calcium was dependent on time, with the maximum occurring at 15 min. The uptake of calcium was dependent on adenosine-5'-triphosphate (ATP), and substitution of ATP with beta, gamma-methylene-ATP did not stimulate calcium uptake. Enzyme marker analysis indicated that mitochondria accounted for no greater than 3.0 +/- 0.2% of the observed ATP-dependent calcium uptake. Calcium uptake was blocked by the ATPase inhibitors tributyltin, IC50 = 12.2 +/- 0.6 nmol/L and 4-acetamido-4'-isothiocyano-2,2'-stilbene disulphonic acid (SITS), IC50 = 3.0 +/- 0.3 mumol/L. These results indicate that in the parotid secretory granule there is a calcium uptake mechanism that is dependent on the hydrolysis of ATP and is suppressed by two inhibitors of granule ATPase.

The effect of HCO3- on anion-stimulated ATPase from rat parotid granules.

ATPase from isolated secretory granules was stimulated in a concentration-dependent manner by HCO3- above 0.9 mM. Maximal stimulation was found at about 16 mM HCO3- and was about half of that with sulphite (SO3(2-)). The activation site(s) appeared to be similar to at least one class of SO3(2-) sites, HCO3(-)-stimulate ATPase was inhibited by SITS. Furthermore, maximal stimulation with SO3(2-) was not enhanced with HCO3-. At low Mg2+ concentrations, Ca2+ stimulated granule ATPase. At higher concentrations of Mg2+ (0.5 mM and above), Ca2+ at 0.1 mM or less had little effect on HCO3(-)-ATPase, and Ca2+ at 4 mM inhibited HCO3(-)-ATPase. At concentrations of Ca2+ above 0.44 mM, the enzyme was partially stimulated in the absence of Mg2+ and presence of HCO3-. Mitochondrial contamination did not account for the presence of ATPase in the isolated granule fraction. The granule ATPase may be regulated by HCO3- and calcium and this could be related to changes in the granule environment during exocytosis.

Characteristics of anion-stimulated Mg-ATPase from rat parotid gland secretory granules.

Magnesium-dependent adenosine triphosphatase (Mg-ATPase) was assayed in highly purified secretory granules. The enzyme was stimulated by sulphite and isethionate, unaffected by chloride and inhibited by fluoride and thiocyanate. Inhibition was not related to the permeant properties of the anion, but the relative inhibitory potency of the anions was similar to that in some other studies of secretory granule ATPases. Maximum contribution to the anion-stimulated ATPase by contaminating mitochondria was estimated at 9.3%. The enzyme was inhibited by the stilbene disulphonic acid inhibitor, 4-acetamido-4'-isothiocyano-2,2'-stilbene disulphonic acid (SITS). The IC50 was 0.16 mM in the absence of sulphite and increased in the presence of sulphite. The relation of the inhibition by SITS to sulphite was complex. Both Vmax and Km parameters were changed by SITS. Furthermore the data are consistent with the presence of two anion-stimulated ATPases. The ATPase was sensitive to tributyltin, dicyclohexylcarbodiimide (DCCD) and oligomycin, only moderately sensitive to azide, probenecid and N-ethylmaleimide (NEM) and rather insensitive to carbonylcyanide m-chlorophenylhydrazone (CCCP) and sulphisoxazole. ATPase activity was stimulated by calcium both in the presence and absence of magnesium. These findings suggest that the ATPase(s) present in parotid secretory granules is unique among secretory granule ATPases.