The binding affinities of muscarinic antagonists were compared with their abilities to block carbachol (CCh)-mediated stimulation of Ca2+ mobilization and inhibition of isoproterenol-elicited adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in rat parotid cells. The binding of [3H]quinuclidinyl benzilate (QNB) to membranes was inhibited by antagonists with the following potencies (dissociation constant, nM): atropine (1.1) approximately 4-diphenylacetoxy-N-methylpiperidine methbromide (4-DAMP) (1.6) much greater than pirenzepine (136) greater than 11-[[2-[(diethylamino)methyl-1-piperidinyl]-acetyl]acetyl]-5,11- dihydro-6H-pyrido[2,3-b][1,4]-benzodiazepine-6-one (AF-DX 116) (5,293). AF-DX 116 blocked Ca2+ mobilization and inhibition of cAMP accumulation with low affinities [inhibitory concentration at 50% (IC50) = 3150 and 6,528 nM, respectively], whereas 4-DAMP blocked these responses with considerably higher affinities (IC50 = 4.3 and 11.4 nM, respectively). Schild plots of 4-DAMP and AF-DX 116 antagonism of CCh-stimulated inositol trisphosphate accumulation showed inhibitor constant (Ki) values of 0.85 and 1,585 nM, respectively, whereas Schild plots of 4-DAMP, AF-DX 116, and methoctramine antagonism of CCh-induced inhibition of cAMP accumulation showed Ki values of 1.3, 1,585, and 2,754 nM, respectively. Preincubation of cells with 0.1 mM 3-isobutyl-1-methylxanthine did not prevent the capacity of CCh to inhibit cAMP accumulation. Pertussis toxin blocked the CCh-elicited and Gi-mediated inhibition of cAMP formation. Northern blot analysis showed the presence of mRNA for the M3, but not for the M2, subtype in parotid gland. An immunochemical procedure using m1-m5 specific antibodies was performed in parotid membranes and showed that the m3 receptor accounts for 93% of precipitable receptors. These data suggest that M3 receptors in the rat parotid are coupled to both the stimulation of Ca2+ mobilization and the inhibition of cAMP accumulation.

To date, there is a major effort in deciphering the role of complex microbial communities, especially the oral and gut microbiomes, in the pathogenesis of various diseases. Increasing evidence indicates a key role for the oral microbiome in autoimmune diseases. In this review article, we discuss links of the oral microbiota to a group of autoimmune diseases, that is, Sjögren's syndrome (SS), systemic lupus erythematosus (SLE), Crohn's disease (CD), and rheumatoid arthritis (RA). We particularly focus on factors that affect the balance between the immune system and the composition of microbiota leading to dysbiosis, loss of tolerance and subsequent autoimmune disease progression and maintenance.

In vulnerable populations, such as infants and children, saliva makes the perfect diagnostic medium because of its noninvasive collection, easy handling and storage of samples. Its unique biomarker profiles help tremendously in the diagnosis of many diseases and conditions. In fact, saliva genomics, proteomics, transcriptomics, metabolomics and microbiome-based discoveries have led to complementary and powerful diagnostic information. In children and neonates, saliva is the preferred medium not only for diagnosis of caries and aggressive periodontitis but also for a number of systemic conditions, metabolic diseases, cognitive functions, stress assessment and evaluation of immunological and inflammatory responses to vaccination. In this review, we provide an overview of current and future applications of saliva diagnostics to various diseases and conditions and highlight studies in paediatrics across the "omic" spectrum. Emerging frontiers in salivary diagnostics research that may significantly advance the field are also highlighted.

We have investigated the possible role of a cAMP-mediated protein-phosphorylation event(s) as the key regulatory mechanism in .3-adrenoreceptor-stimulated activation of mannosylphosphodolichol (Man-P-Dol) synthase (GDPmannose:dolichyl-phosphate O-,-D-mannosyltransferase, EC 2.4.1.83) in rat parotid acinar cells. Microsomal membranes isolated from these cells pretreated with 10 1AM isoproterenol for 60 min showed -40-80% enhanced Man-P-Dol synthase activity compared to the untreated controls. This change in enzyme activity was not associated with a significant alteration in apparent Km for GDP-mannose, but the V.,, was enhanced 2-fold. When microsomal membranes isolated from control cells were phosphorylated in vitro by a cAMP-dependent protein kinase, an increase in Man-P-Dol synthase activity, similar to that with membranes from isoproterenol-treated cells, was observed (i.e., a moderate change in Km for GDPmannose but a 2-fold higher V.). Furthermore, treatment of in vitro phosphorylated microsomal membranes by alkaline phosphatase led to a substantial reduction in Man-P-Dol synthase activity. Increased Man-P-Dol synthesis (m30-40%) was also observed in bovine brain and hen oviduct microsomal membranes after in vitro protein phosphorylation. In aggregate, these results strongly suggest that agents that increase cAMP in cells may modulate protein N-glycosylation in those cells by activating this key glycosyltransferase of the dolichol cascade by a cAMP-dependent protein kinase-mediated protein phosphorylation/dephosphorylation cycle.The biosynthesis of oligosaccharides and their attachment to protein asparagine residues is a multistep process that has received considerable attention in the last decade (1). The individual enzymes in this cascade pathway have been documented (2-6), but information concerning mechanism(s) by which cells may regulate the extent of N-linked protein glycosylation is relatively limited. However, several reports have suggested that extracellular signaling may contribute to the regulation of events leading to protein N-glycosylation (7-11).Earlier we demonstrated (12) that f3-adrenoreceptor stimulation markedly enhances protein N-glycosylation through a cAMP-mediated mechanism in rat parotid acinar cells. This appears to be due to increased enzymatic transfer of Nacetylglucosamine 1-phosphate, mannose (Man), and glucose from their respective nucleotides to dolichol monophosphate (Dol-P) (13). Since isoproterenol, a, -adrenergic agonist, activates cAMP-dependent protein kinases (14-16), it was hypothesized that enhancement of glycosyltransferase activities seen in microsomal membranes from cells pretreated with isoproterenol may be due in part to a cyclic AMP-dependent protein kinase-mediated phosphorylation of these enzymes.In work reported here, we tested this hypothesis by utilizing an in vitro phosphorylating system and found evidence supporting protein phosphorylation as a likely mechanistic event leading to enhanced mannosylphosphodolichol (Man-P-Dol) synthase (GDP-mannose:...