Jean M. Camden

Src homology 3 binding sites in the P2Y2 nucleotide receptor interact with Src and regulate activities of Src, proline-rich tyrosine kinase 2, and growth factor receptors.

Many G protein-coupled receptors activate growth factor receptors, although the mechanisms controlling this transactivation are unclear. We have identified two proline-rich, SH3 binding sites (PXXP) in the carboxyl-terminal tail of the human P2Y2 nucleotide receptor that directly associate with the tyrosine kinase Src in protein binding assays. Furthermore, Src co-precipitated with the P2Y2 receptor in 1321N1 astrocytoma cells stimulated with the P2Y2 receptor agonist UTP. A mutant P2Y2 receptor lacking the PXXP motifs was found to stimulate calcium mobilization and serine/threonine phosphorylation of the Erk1/2 mitogen-activated protein kinases, like the wild-type receptor, but was defective in its ability to stimulate tyrosine phosphorylation of Src and Src-dependent tyrosine phosphorylation of the proline-rich tyrosine kinase 2, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor. Dual immunofluorescence labeling of the P2Y2 receptor and the EGFR indicated that UTP caused an increase in the co-localization of these receptors in the plasma membrane that was prevented by the Src inhibitor PP2. Together, these data suggest that agonist-induced binding of Src to the SH3 binding sites in the P2Y2 receptor facilitates Src activation, which recruits the EGFR into a protein complex with the P2Y2 receptor and allows Src to efficiently phosphorylate the EGFR.

P2Y2 Nucleotide Receptors Enhance α-Secretase-dependent Amyloid Precursor Protein Processing

The amyloid precursor protein (APP) is proteolytically processed by β- and γ-secretases to release amyloid β, the main component in senile plaques found in the brains of patients with Alzheimer disease. Alternatively, APP can be cleaved within the amyloid β domain by α-secretase releasing the non-amyloidogenic product sAPPα, which has been shown to have neuroprotective properties. Several G protein-coupled receptors are known to activate α-secretase-dependent processing of APP; however, the role of G protein-coupled nucleotide receptors in APP processing has not been investigated. Here it is demonstrated that activation of the G protein-coupled P2Y2 receptor (P2Y2R) subtype expressed in human 1321N1 astrocytoma cells enhanced the release of sAPPα in a time- and dose-dependent manner. P2Y2 R-mediated sAPPα release was dependent on extracellular calcium but was not affected by 1,2-bis(2-aminophenoxy)ethane-N,N,N,-trimethylammonium salt, an intracellular calcium chelator, indicating that P2Y2 R-stimulated intracellular calcium mobilization was not involved. Inhibition of protein kinase C (PKC) with GF109203 or by PKC down-regulation with phorbol ester pre-treatment had no effect on UTP-stimulated sAPPα release, indicating a PKC-independent mechanism. U0126, an inhibitor of the mitogen-activated protein kinase pathway, partially inhibited sAPPα release by UTP, whereas inhibitors of Src-dependent epidermal growth factor receptor transactivation by P2Y2 Rs had no effect. The metalloprotease inhibitors phenanthroline and TAPI-2 and the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone also diminished UTP-induced sAPPα release. Furthermore, small interfering RNA silencing of an endogenous adamalysin, ADAM10 or ADAM17/TACE, partially suppressed P2Y2R-activated sAPPα release, whereas treatment of cells with both ADAM10 and ADAM17/TACE small interfering RNAs completely abolished UTP-activated sAPPα release. These results may contribute to an understanding of the non-amyloidogenic processing of APP.

Proinflammatory cytokines tumor necrosis factor-α and interferon-γ alter tight junction structure and function in the rat parotid gland Par-C10 cell line

Sjögrens syndrome (SS) is an autoimmune disorder characterized by inflammation and dysfunction of salivary glands, resulting in impaired secretory function. The production of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) is elevated in exocrine glands of patients with SS, although little is known about the effects of these cytokines on salivary epithelial cell functions necessary for saliva secretion, including tight junction (TJ) integrity and the establishment of transepithelial ion gradients. The present study demonstrates that chronic exposure of polarized rat parotid gland (Par-C10) epithelial cell monolayers to TNF-alpha and IFN-gamma decreases transepithelial resistance (TER) and anion secretion, as measured by changes in short-circuit current (I(sc)) induced by carbachol, a muscarinic cholinergic receptor agonist, or UTP, a P2Y(2) nucleotide receptor agonist. In contrast, TNF-alpha and IFN-gamma had no effect on agonist-induced increases in the intracellular calcium concentration [Ca(2+)](i) in Par-C10 cells. Furthermore, treatment of Par-C10 cell monolayers with TNF-alpha and IFN-gamma increased paracellular permeability to normally impermeant proteins, altered cell and TJ morphology, and downregulated the expression of the TJ protein, claudin-1, but not other TJ proteins expressed in Par-C10 cells. The decreases in TER, agonist-induced transepithelial anion secretion, and claudin-1 expression caused by TNF-alpha, but not IFN-gamma, were reversible by incubation of Par-C10 cell monolayers with cytokine-free medium for 24 h, indicating that IFN-gamma causes irreversible inhibition of cellular activities associated with fluid secretion in salivary glands. Our results suggest that cytokine production is an important contributor to secretory dysfunction in SS by disrupting TJ integrity of salivary epithelium.

Development and characterization of SV40 immortalized rat parotid acinar cell lines.

SummaryRat parotid salivary gland acinar cells were transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 30 clonal cell lines, 2 were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the β-adrenergic agonist (isoproterenol), vasoactive intestinal peptide prostaglandin E1, and forskolin were effective activators of intracellular cyclic adenosine 3′:5′-cyclic monophosphate production. Phenylephrine, carbamylcholine, and UTP were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without affect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express the SV40 large T antigen. Electron microscopic evaluation documented moderate to high levels of cytodifferentiation with the maintenance of tripartite junctional complexes, cellular polarization, and presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 22 and 36 h, respectively.

Development and characterization of SV40 immortalized rat submandibular acinar cell lines

SummaryRat submandibular salivary gland acinar cells were transfected by CaPO4 precipitation using a plasmid containing a replication-defective simian virus (SV40) genome. Out of 27 clonal cell lines, two were shown to have moderate to high levels of cytodifferentiation and salivary gland acinar cell function. Functional studies with the two cell lines indicated that the β-adrenergic agonist, isoproterenol, vasoactive intestinal peptide, and prostaglandin E1 were effective activators of intracellular cyclic AMP production. Epinephrine, norepinephrine, phenylephrine, acetylcholine, and P2U-purinoceptor agonists were effective in increasing inositol phosphate production and intracellular free calcium levels, whereas substance P was without effect. Utilizing indirect immunofluorescence analysis, both cell lines were shown to express glutamine/glutamic acid-rich proteins, a submandibular acinar cell specific secretory protein family. Electron microscopic evaluation documented the maintenance of tripartite junctional complexes, cellular polarization, and the presence of moderate amounts of secretory granules and rough endoplasmic reticulum. The two cell lines had doubling times of 25 h.

Mechanisms of agonist-dependent and -independent desensitization of a recombinant P2Y2 nucleotide receptor

UTP activates P2Y2 receptors in both 1321N1 cell transfectants expressing the P2Y2 receptor and human HT-29 epithelial cells expressing endogenous P2Y2 receptors with an EC50 of 0.2- 1.0 μM. Pretreatment of these cells with UTP diminished the effectiveness of a second dose of UTP (the IC50 for UTP-induced receptor desensitization was 0.3 - 1.0 μM for both systems). Desensitization and down-regulation of the P2Y2 nucleotide receptor may limit the effectiveness of UTP as a therapeutic agent. The present studies investigated the phenomenon of P2Y2 receptor desensitization in human 1321N1 astrocytoma cells expressing recombinant wild type and C-terminal truncation mutants of the P2Y,2 receptor. In these cells, potent P2Y2 receptor desensitization was observed after a 5 min exposure to UTP. Full receptor responsiveness returned 5-10 min after removal of UTP. Thapsigargin, an inhibitor of Ca2+-ATPase in the endoplasmic reticulum, induced an increase in the intracellular free calcium concentration, [Ca2+]i, after addition of desensitizing concentrations of UTP, indicating that P2Y2 receptor desensitization is not due to depletion of calcium from intracellular stores. Single cell measurements of increases in [Ca2+]i induced by UTP in 1321N1 cell transfectants expressing the P2Y2 receptor indicate that time- and UTP concentration-dependent desensitization occurred uniformly across a cell population. Other results suggest that P2Y2 receptor phosphorylation/dephosphorylation regulate receptor desensitization/resensitization. A 5 min preincubation of 1321N1 cell transfectants with the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), reduced the subsequent response to UTP by about 50% whereas co-incubation of PMA with UTP caused a greater inhibition in the response. The protein phosphatases - 1 and -2A inhibitor, okadaic acid, partially blocked resensitization of the receptor. Furthermore, C-terminal truncation mutants of the P2Y2 receptor that eliminated several potential phosphorylation sites including two for PKC were resistant to UTP-, but not phorbol ester-induced desensitization. Down regulation of protein kinase C isoforms prevented phorbol ester-induced desensitization but had no effect on agonist-induced desensitization of wild type or truncation mutant receptors. These results suggest that phosphorylation of the C-terminus of the P2Y2 receptor by protein kinases other than protein kinase C mediates agonist-induced receptor desensitization. A better understanding of the molecular mechanisms of P2Y2 nucleotide receptor desensitization may help optimize a promising cystic fibrosis pharmacotherapy based on the activation of anion secretion in airway epithelial cells by P2Y2 receptor agonists.

Autonomic receptors in the developing submandibular glands of neonatal rats

The functional maturation of the gland during the neonatal period involves specific temporal sequences in the appearance of autonomic membrane receptors and associated changes in the secretory response to receptor stimulation. The density and affinity of cholinergic muscarinic and of α- and β-adrenergic receptors were determined in the glands of 1-, 7-, 14- and 21-day-old rats, using the radioligands [3H]-quinuclidinyl benzylate, [3H]-prazosin and [3H]-dihydroalprenolol for the measurement of muscarinic cholinergic, α1-adrenergic and β-adrenergic receptors, respectively. The density of binding sites followed similar developmental courses, whether expressed as pmolg tissue or pmolg protein. The densities (pmolg protein) of β-adrenergic and muscarinic receptors were low in 1-day-old animals, but increased rapidly; adult levels were reached by 2 and 3 weeks of age, respectively. α1-Adrenergic receptor binding was barely detectable at birth, increased slightly during the first week, dramatically by 14 days and approached adult levels by 21 days. The number of receptors per gland for these 3 autonomic receptor-binding sites increased 50 – 100-fold during the first 3 weeks of postnatal development. The affinities (KD) for each of the three ligands did not differ significantly with age. Surprisingly, the α2-adrenergic receptor density ([3H]-p-aminoclonidine binding) was high in 1-day-old animals and increased significantly during the first 2 weeks of life. The binding declined after 3 weeks and was nearly undetectable by 6 weeks. Frozen submandibular glands had markedly lower α2-adrenergic binding compared to fresh glands. These findings suggest that each of the autonomic receptors in rat submandibular glands follows its own specific developmental pattern, and that the appearance of individual receptor types may correlate with the ability of the developing gland to respond to specific stimulants. A K+-release response can be elicited by epinephrine at 2 weeks of age, when α1 receptor binding sites appear, while both the receptor binding sites and the response to muscarinic agonists are present at birth. As β-adrenergic receptor-binding sites may develop simultaneously with isoproterenol-stimulated adenylate-cyclase activity, it seems that receptor density is important in this response. The functional significance of the high density of α2-adrenergic receptors in the submandibular gland of neonatal animals is not clear.

P2 Receptors for Extracellular Nucleotides in the Central Nervous System: Role of P2X7 and P2Y2 Receptor Interactions in Neuroinflammation

Extracellular nucleotides induce cellular responses in the central nervous system (CNS) through the activation of ionotropic P2X and metabotropic P2Y nucleotide receptors. Activation of these receptors regulates a wide range of physiological and pathological processes. In this review, we present an overview of the current literature regarding P2X and P2Y receptors in the CNS with a focus on the contribution of P2X7 and P2Y2 receptor-mediated responses to neuroinflammatory and neuroprotective mechanisms.

Cftr and ENaC ion channels mediate NaCl absorption in the mouse submandibular gland

Cystic fibrosis is caused by mutations in CFTR, the cystic fibrosis transmembrane conductance regulator gene. Disruption of CFTR‐mediated anion conductance results in defective fluid and electrolyte movement in the epithelial cells of organs such as the pancreas, airways and sweat glands, but the function of CFTR in salivary glands is unclear. Salivary gland acinar cells produce an isotonic, plasma‐like fluid, which is subsequently modified by the ducts to produce a hypotonic, NaCl‐depleted final saliva. In the present study we investigated whether submandibular salivary glands (SMGs) in ΔF508 mice (CftrΔF/ΔF) display ion transport defects characteristic of cystic fibrosis in other tissues. Immunolocalization and whole‐cell recordings demonstrated that Cftr and the epithelial Na+ (ENaC) channels are co‐expressed in the apical membrane of submandibular duct cells, consistent with the significantly higher saliva [NaCl] observed in vivo in CftrΔF/ΔF mice. In contrast, Cftr and ENaC channels were not detected in acinar cells, nor was saliva production affected in CftrΔF/ΔF mice, implying that Cftr contributes little to the fluid secretion process in the mouse SMG. To identify the source of the NaCl absorption defect in CftrΔF/ΔF mice, saliva was collected from ex vivo perfused SMGs. CftrΔF/ΔF glands secreted saliva with significantly increased [NaCl]. Moreover, pharmacological inhibition of either Cftr or ENaC in the ex vivo SMGs mimicked the CftrΔF/ΔF phenotype. In summary, our results demonstrate that NaCl absorption requires and is likely to be mediated by functionally dependent Cftr and ENaC channels localized to the apical membranes of mouse salivary gland duct cells.

P2Y2 nucleotide receptor-mediated responses in brain cells.

Acute inflammation is important for tissue repair; however, chronic inflammation contributes to neurodegeneration in Alzheimers disease (AD) and occurs when glial cells undergo prolonged activation. In the brain, stress or damage causes the release of nucleotides and activation of the Gq protein-coupled P2Y2 nucleotide receptor subtype (P2Y2R) leading to pro-inflammatory responses that can protect neurons from injury, including the stimulation and recruitment of glial cells. P2Y2R activation induces the phosphorylation of the epidermal growth factor receptor (EGFR), a response dependent upon the presence of a SH3 binding domain in the intracellular C terminus of the P2Y2R that promotes Src binding and transactivation of EGFR, a pathway that regulates the proliferation of cortical astrocytes. Other studies indicate that P2Y2R activation increases astrocyte migration. P2Y2R activation by UTP increases the expression in astrocytes of αVβ3/5 integrins that bind directly to the P2Y2R via an Arg-Gly-Asp (RGD) motif in the first extracellular loop of the P2Y2R, an interaction required for Go and G12 protein-dependent astrocyte migration. In rat primary cortical neurons (rPCNs) P2Y2R expression is increased by stimulation with interleukin-1β (IL-1β), a pro-inflammatory cytokine whose levels are elevated in AD, in part due to nucleotide-stimulated release from glial cells. Other results indicate that oligomeric β-amyloid peptide (Aβ1-42), a contributor to AD, increases nucleotide release from astrocytes, which would serve to activate upregulated P2Y2Rs in neurons. Data with rPCNs suggest that P2Y2R upregulation by IL-1β and subsequent activation by UTP are neuroprotective, since this increases the non-amyloidogenic cleavage of amyloid precursor protein. Furthermore, activation of IL-1β-upregulated P2Y2Rs in rPCNs increases the phosphorylation of cofilin, a cytoskeletal protein that stabilizes neurite outgrowths. Thus, activation of pro-inflammatory P2Y2Rs in glial cells can promote neuroprotective responses, suggesting that P2Y2Rs represent a novel pharmacological target in neurodegenerative and other pro-inflammatory diseases.