2؉ , independently of CaM, inhibited isoproterenol-stimulated AC. Data suggest that agonist augmentation of stimulated cAMP levels is due to activation of AC8 in mouse parotid acini, and strongly support a role for AC5/6 in the inhibition of stimulated cAMP levels.To date, 10 different ACs, 1 each with distinct regulatory properties, have been cloned; their existence suggests that they may be differentially regulated. The enzymes exhibit type specific stimulatory and inhibitory regulation by G-protein ␣ and ␤␥ subunits, Ca 2ϩ , CaM, forskolin, P-site inhibitors, protein kinases A and C (PKC) (2-6), and calcineurin (7) entry plays an important role in promoting AC synthesis. These data, combined with findings that AC8 is expressed in mouse parotid acini (1) and that Ca 2ϩ /CaM stimulates AC and augments the effects of forskolin on cyclase activity in membrane fractions and intact cells (21,22), are consistent with results obtained in HEK 293 cells expressing AC8 (8, 11).Interpretation of the mechanism(s) involved in the cross-talk that occurs between the Ca 2ϩ and cAMP signaling pathways in cells is complex and requires not only identification of AC subtypes expressed, but also tools that provide definitive answers as to regulation of AC synthesis in specific cell types. Thus, the goal of the present study was to determine the involvement of AC8 in agonist-induced augmentation of stimulated cAMP levels in mouse parotid acini by examining the effects of carbachol and the microsomal Ca 2ϩ -ATPase inhibitor, thapsigargin, on isoproterenol-induced cAMP accumulation in acini from AC8-KO mice. Our data show that carbachol and thapsigargin augmented stimulated cAMP accumulation in acini from wild type (WT) mice as previously reported (1), whereas these agents not only prevented augmentation, but inhibited isoproterenol-induced cAMP accumulation in AC8-KO mice. Augmentation of stimulated cAMP accumulation, however, was not affected in acini from AC1-KO mice. Agonist-induced inhibition of stimulated cAMP accumulation was reversed in a nominally Ca 2ϩ -free buffer and in the presence of lanthanum (La 3ϩ ), but not by KN-62, an inhibitor of CaM kinase, or by the CaM antagonist, calmidazolium. Studies with isolated parotid membranes revealed that Ca 2ϩ , independently of CaM, inhibits AC activity in a concentration-dependent manner, consistent with the expression of the Ca 2ϩ -inhibited AC5/6 isoforms in parotid gland. Results demonstrate that capacitative Ca 2ϩ entry is associated with the activation of AC8 in mouse parotid acini and support an involvement of AC5/6 in the inhibition of cAMP synthesis.

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response to bacteria. Human gingival epithelial cells (GECs) were transfected with siRNA specific for PAR1 or PAR2, then stimulated with periopathogen Porphyromonas gingivalis, bridging organism between pathogens and non-pathogens Fusobacterium nucleatum, or non-pathogen Streptococcus gordonii. PAR1 or PAR2 knock-down resulted in up-regulated NOD1 and NOD2 expression with P. gingivalis or F. nucleatum stimulation (p<0.01), as well as enhanced TLR2 and TLR4 expression when cells were stimulated by bacteria that utilize TLR2 or TLR4, respectively. Involvement of PARs for induction of CC chemokine ligand 20 (CCL20), a cytokine with antimicrobial properties, was observed following stimulation of the three bacterial species. Furthermore, results from multiple cytokine ELISA array showed receptors utilized in the induction of various innate immune markers are tailored to individual bacterium tested. Our data suggest complex interplay of several receptors is required for appropriate innate immune responses to the different types of bacteria present within the oral cavity and that receptor expression itself is altered depending on which organism the cell encounters. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptImmunol Lett. Author manuscript; available in PMC 2011 July 8. Published in final edited form as:Immunol Lett. Materials and Methods Human epithelial cells and bacterial culture conditionsHealthy gingival samples were obtained from patients undergoing third-molar extraction at the Department of Oral Surgery, School of Dentistry, University of Washington. Tissue was prepared as described earlier, and subsequently isolated GECs were grown in Keratinocyte Basal Media supplemented with Keratinocyte Growth Media (Cambrex, Walkersville, MD) [21]. Cells were grown in media containing 0.15 mM calcium (Ca ++ ). P. gingivalis (ATCC 33277) cells were cultured in anaerobic conditions (85% N 2 , 10% H 2 , 5% CO 2 ) at 37°C in Trypticase soy broth (BBL, Sparks, MD) supplemented with 1 g of yeast extract, 5 mg of hemin and 1 mg of menadione per liter. Streptococcus gordonii (Challis DL1) was grown in Trypticase soy broth at 37°C under static conditions. Fusobacterium nucleatum (ATCC 25586) was grown in Todd-Hewitt broth supplemented with 1 g of yeast extract per 100 ml at 37°C in anaerobic conditions. All bacterial species used in this study are from our laboratory stock. Bacterial numbers were estimated by density in a GENios Multi-detection Reader (Phenix, Hayward, CA). Transfection of keratinocytes with siRNASpecific siRNAs were custom-synthesized...

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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.