Apoptosis and mitosis of parenchymal cells in the duct-ligated rat submandibular gland

Apoptosis and proliferation of parenchymal cells during atrophy of rat submandibular gland induced by double duct ligation were investigated using immunohistochemistry for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labelling (TUNEL) and transmission electron microscopy (TEM). At 2 and 3 days after ligation, increased PCNA positive cells and mitoses were seen in ducts; thereafter PCNA positive cells decreased in number. At 3 and 4 days, the acinar cell population rapidly decreased, with many remaining TUNEL positive acinar cells. During this period, TEM showed typical apoptotic acinar cells that were phagocytosed by adjacent acinar cells or intraepithelial macrophages. After 7 days, most acinar cells had disappeared, leaving prominent residual ducts; a few acinar cells remained, especially at the lobule periphery. Submandibular gland duct ligation thus induced marked depletion of acinar cell by apoptosis and a concurrent short-lived cycle of duct cell proliferation.     

Involvement of apical P2Y2 receptor-regulated CFTR activity in muscarinic stimulation of Cl(-) reabsorption in rat submandibular gland

Previously, we presented in vivo evidence for a physiological significance of cAMP-regulated CFTR Cl(-) channels in Ca(2+)-activated Cl(-) reabsorption in the ductal system of the rat submandibular gland. Here, we address the mechanism by which basal CFTR activation contributes to the transepithelial Cl(-) movement evoked by muscarinic stimulation. The Cl(-) concentration ([Cl(-)]) was increased in the final saliva from rat submandibular gland during pilocarpine stimulation when a small interfering RNA for CFTR or a specific CFTR inhibitor, CFTR(inh)-172, was injected retrogradely into the gland's own duct, indicating that basal CFTR activation is involved in Cl(-) reabsorption. Systemically administered propranolol failed to alter the [Cl(-)], suggesting little involvement of a beta-adrenergic pathway in the Cl(-) movement that occurs through basal CFTR activation. Intraductal injection of suramin (a nonspecific P2-receptor antagonist) increased the salivary [Cl(-)], indicating the existence of endogenous purinergic activation. Upon separate intraductal injection, ATP and a P2Y(2)-receptor agonist, UTP, decreased the salivary [Cl(-)] almost equipotently. CFTR(inh)-172 and suramin each prevented these effects, whereas 2',3'-O-(4-benzoylbenzoyl)-ATP (Bz-ATP), a P2X(7) agonist, had no specific effect. Pilocarpine stimulation evoked ATP secretion into the salivary fluid. Immunohistochemistry revealed the partial coexistence of CFTR and P2Y(2) receptors on the luminal surface of epithelial cells in the striated ducts of this gland. These results raise the possibility that muscarinic stimulation-induced Cl(-) reabsorption occurs through basal CFTR activity and that this is regulated by P2Y(2) receptors in the ductal epithelium via stimulation by ATP secreted into the salivary fluid.     

The P2Y(2) nucleotide receptor mediates tissue factor expression in human coronary artery endothelial cells

The discovery of the role of P2Y(12) receptor in platelet aggregation leads to a new anti-thrombotic drug Plavix; however, little is known about non-platelet P2Y receptors in thrombosis. This study tested the hypothesis that endothelial P2Y receptor(s) mediates up-regulation of tissue factor (TF), the initiator of coagulation cascade. Stimulation of human coronary artery endothelial cells (HCAEC) by UTP/ATP increased the mRNA level of TF but not of its counterpart-tissue factor pathway inhibitor, which was accompanied by up-regulation of TF protein and cell surface activity. RT-PCR revealed a selective expression of P2Y(2) and P2Y(11) receptors in HCAEC. Consistent with this, TF up-regulation was inhibited by suramin or by siRNA silencing of P2Y(2) receptor, but not by NF-157, a P2Y(11)-selective antagonist, suggesting a role for the P2Y(2) receptor. In addition, P2Y(2) receptor activated ERK1/2, JNK, and p38 MAPK pathways without affecting the positive NF-κB and negative AKT regulatory pathways of TF expression. Furthermore, TF up-regulation was abolished or partially suppressed by inhibition of p38 or JNK but not ERK1/2. Interestingly, blockade of the PLC/Ca(2+) pathway did not affect P2Y(2) receptor activation of p38, JNK, and TF induction. However, blockade of Src kinase reduced phosphorylation of p38 but not JNK, eliminating TF induction. In contrast, inhibition of Rho kinase reduced phosphorylation of JNK but not p38, decreasing TF expression. These findings demonstrate that P2Y(2) receptor mediates TF expression in HCAEC through new mechanisms involving Src/p38 and Rho/JNK pathways, possibly contributing to a pro-thrombotic status after vascular injury.     

Molecular determinants of P2Y2 nucleotide receptor function

In the mammalian nervous system, P2 nucleotide receptors mediate neurotransmission, release of proinflammatory cytokines, and reactive astrogliosis. Extracellular nucleotides activate multiple P2 receptors in neurons and glial cells, including G protein-coupled P2Y receptors and P2X receptors, which are ligand-gated ion channels. In glial cells, the P2Y2 receptor subtype, distinguished by its ability to be equipotently activated by ATP and UTP, is coupled to pro-inflammatory signaling pathways. In situ hybridization studies with rodent brain slices indicate that P2Y2 receptors are expressed primarily in the hippocampus and cerebellum. Astrocytes express several P2 receptor subtypes, including P2Y2 receptors whose activation stimulates cell proliferation and migration. P2Y2 receptors, via an RGD (Arg-Gly-Asp) motif in their first extracellular loop, bind to alphavbeta3/beta5 integrins, whereupon P2Y2 receptor activation stimulates integrin signaling pathways that regulate cytoskeletal reorganization and cell motility. The C-terminus of the P2Y2 receptor contains two Src-homology-3 (SH3)-binding domains that upon receptor activation, promote association with Src and transactivation of growth factor receptors. Together, our results indicate that P2Y2 receptors complex with both integrins and growth factor receptors to activate multiple signaling pathways. Thus, P2Y2 receptors present novel targets to control reactive astrogliosis in neurodegenerative diseases.     

Agonist-induced phosphorylation and desensitization of the P2Y2 nucleotide receptor

Purification of HA-tagged P2Y₂ receptors from transfected human 1321N1 astrocytoma cells yielded a protein with a molecular size determined by SDS-PAGE to be in the range of 57–76 kDa, which is typical of membrane glycoproteins with heterogeneous complex glycosylation. The protein phosphatase inhibitor, okadaic acid, attenuated the recovery of receptor activity from the agonist-induced desensitized state, suggesting a role for P2Y₂ receptor phosphorylation in desensitization. Isolation of HA-tagged P2Y₂ nucleotide receptors from metabolically [³²P]-labelled cells indicated a (3.8 ± 0.2)-fold increase in the [³²P]-content of the receptor after 15 min of treatment with 100 μM UTP, as compared to immunoprecipitated receptors from untreated control cells. Receptor sequestration studies indicated that ∼40% of the surface receptors were internalized after a 15-min stimulation with 100 μM UTP. Point mutation of three potential GRK and PKC phosphorylation sites in the third intracellular loop and C-terminal tail of the P2Y₂ receptor (namely, S243A, T344A, and S356A) extinguished agonist-induced receptor phosphorylation, caused a marked reduction in the efficacy of UTP to desensitize P2Y₂ receptor signalling to intracellular calcium mobilization, and impaired agonist-induced receptor internalization. Activation of PKC isoforms with phorbol 12-myristate 13-acetate that caused heterologous receptor desensitization did not increase the level of P2Y₂ receptor phosphorylation. Our results indicate a role for receptor phosphorylation by phorbol-insensitive protein kinases in agonist-induced desensitization of the P2Y₂ nucleotide receptor. (Mol Cell Biochem xxx: 35–45, 2005)     

Developmental expression of metabotropic P2Y(1) and P2Y(2) receptors in freshly isolated astrocytes from rat hippocampus

There are at least three subtypes of cloned metabotropic P2 receptors linked to intracellular Ca(2+) rises in rat brain cells, namely, P2Y(1), P2Y(2) and P2Y(4). In this study we explore the subtypes of the metabotropic P2 receptors seen in freshly isolated astrocytes (FIAs) from P8-P25 rats. We found by single cell RT-PCR that in process-bearing FIAs from hippocampi of P8-P12 rats, 31% of the glial fibrillary acidic protein (GFAP) mRNA (+) cells expressed P2Y(1) mRNA while only 5% of the cells tested expressed P2Y(2) mRNA. The expression of P2Y(1) receptor mRNA was not changed in FIAs from the hippocampi of P18-P25 rats, but 38% of the GFAP mRNA (+) cells in the P18-P25 age group then showed P2Y(2) mRNA. We also studied whether the mRNA was expressing functional receptor protein by measuring Ca(2+) responses to specific agonists for P2Y(1) and P2Y(2). We found that similar proportions of GFAP mRNA (+) FIAs responded to ATP or UTP as showed mRNAs for P2Y (1) and P2Y(2,) respectively. Total tissue RNA from P9 and P24 rat hippocampus showed a 2.8-fold increase in P2Y(2) mRNA levels from P9 to P24 with a decrease in P2Y(1) mRNA. Thus, this study shows a marked up-regulation of mRNA for P2Y(2) from 9 to 24 days in rat hippocampus, and some of this increase is likely due to the protoplasmic astrocytes which is being translated into functional receptor protein in these cells.     

Proteinic prorenin-releasing-stimulator (PRS) in the rat submandibular gland

The release of prorenin as well as renin from rat renal slices was confirmed by a rat prorenin-prosegment ELISA system and an assay system for determining the renin activity. A significant increase of the prorenin release was found by adding rat submandibular gland extract to the slice medium, indicating the existence of a prorenin-releasing stimulator (PRS) in the extract. The pI and molecular mass of PRS were 8.5-8.7 and 28-30 kDa, respectively. The PRS was completely inactivated by boiling or a proteinase treatment.     

Current knowledge on the nucleotide agonists for the P2Y2 receptor

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. There are eight mammalian P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14). P2Y2 receptors are widely expressed and play important roles in multiple functionalities. Diquafosol tetrasodium, known as INS365, which was the first P2Y2 receptor agonists that had been approved in April 2010 and launched in Japan by Santen Pharmaceuticals. Besides, a series of similar agonists for the P2Y2 receptor are undergoing development to cure different diseases related to the P2Y2 receptor. This article illustrated the structure and functions of the P2Y2 receptor and focused on several kinds of agonists about their molecular structures, research progress and chemical synthesis methods. Last but not the least, we summarized the structures-activity relationship (SAR) of agonists for the P2Y2 receptor and expected more efficient agonists for the P2Y2 receptor.     

Immunolocalisation of P2X and P2Y nucleotide receptors in the rat nasal mucosa

Purinoceptor subtypes were localised to various tissue types present within the nasal cavity of the rat, using immunohistochemical methods. P2X₃ receptor immunoreactivity was localised in the primary olfactory neurones located both in the olfactory epithelium and vomeronasal organs (VNO) and also on subepithelial nerve fibres in the respiratory region. P2X₅ receptor immunoreactivity was found in the squamous, respiratory and olfactory epithelial cells of the rat nasal mucosa. P2X₇ receptor immunoreactivity was also expressed in epithelial cells and colocalised with caspase 9 (an apoptotic marker), suggesting an association with apoptosis and epithelial turnover. P2Y₁ receptor immunoreactivity was found within the respiratory epithelium and submucosal glandular tissue. P2Y₂ receptor immunoreactivity was localised to the mucus-secreting cells within the VNO. The possible functional roles of these receptors are discussed.     

Distribution and regulation of mitochondrial hexokinase in rat submandibular gland

1. In rat submandibular gland, hexokinase was distributed not only in cytosol fraction but also in mitochondrial fraction. 2. Glucose-6-phosphate and ATP were most effective substances on releasing hexokinase from mitochondria. However, all the hexokinase in mitochondria could not be extracted with these substances. 3. Concentrations of glucose-6-phosphate and ATP were decreased with the administration of epinephrine in vivo. 4. Increase of the amount of mitochondria-bound hexokinase was observed for 5 min with epinephrine administration, and it returned to the control level after 10 min. 5. In rat submandibular gland, mitochondrial hexokinase may reversibly bind to and release from mitochondria as observed in brain.     

Voluntary exercise increases IgA concentration and polymeric Ig receptor expression in the rat submandibular gland

Salivary IgA—a primary factor in local immunity of the oral cavity—plays an important role in maintaining local immune function in the oral cavity and prevent upper respiratory tract infections. Oral IgA levels are known to fluctuate in an exercise-dependent manner; thus, we investigated the effects of voluntary exercise on salivary IgA secretion in rats to better understand the mechanism by which this occurs. Six-week-old male Wistar rats were placed in individual cages with or without access to exercise wheels for three weeks. Notably, animals who engaged in voluntary exercise demonstrated significant increases in IgA concentration in saliva and submandibular gland tissue, as well as a markedly higher salivary IgA flow rate. Moreover, active rats also exhibited elevated polymeric Ig receptor (pIgR) mRNA expression in submandibular gland tissue. Collectively, these results suggest that voluntary exercise may increase salivary IgA concentration and boost immune function in the oral cavity.     

Fructose 2,6-bisphosphate-dependent regulation of phosphofructokinase in rat submandibular gland

1. Regulation of phosphofructokinase in rat submandibular gland was non-Michaelis-Menten type at physiological pH. 2. At pH 7.3, ATP played a dual role on phosphofructokinase acting as a substrate and inhibitor at high concentration of ATP. 3. The activator of phosphofructokinase was present in cytosol fraction, and its properties were resemble to those of fructose 2,6-bisphosphate. 4. Both the activator and authentic fructose 2,6-bisphosphate relieved the inhibition of phosphofructokinase by ATP, and increased the affinity for fructose 6-phosphate. 5. Concentration of fructose 2,6-bisphosphate in rat submandibular gland was 8.22 nmol/g tissue, and which was about the half of that in liver. 6. Phosphofructokinase in rat submandibular gland was found to be regulated synergistically by ATP, fructose 6-phosphate and fructose 2,6-bisphosphate.     

Nitric oxide synthase/PGE(2) cross-talk in rat submandibular gland

It is known that nitric oxide modulates the prostaglandin generation. However, little is known about the regulatory action of prostaglandin on nitric oxide production. There is a molecular cross-talk between nitric oxide and prostaglandin. Here, we examined biochemical signalling pathways coupled to the prostaglandin E(2) (PGE(2)) receptor related to nitric oxide synthase stimulation in rat submandibular gland. PGE(2) through the stimulation of its own receptor, triggered activation of phosphoinositide turnover (IPs), translocation of protein kinase C (PKC), stimulation of nitric oxide synthase activity (NOS) and increased production of cyclic GMP (cGMP). PGE(2) stimulation of NOS and cGMP production was blunted by agents interfering with calcium influx, calcium/calmodulin and phospholipase C (PLC) activities; while PKC inhibitor was able to stimulate PGE(2) effects. PGE(2) did not evoke amylase release, indicating that NOS/ cGMP pathway were not associated with this enzyme secretion. Our results suggest that this prostanoid could act as vasoactive chemical mediator through its ability to activate NOS-cGMP pathway via own gland membrane receptor.     

Characterization and channel coupling of the P2Y(12) nucleotide receptor of brain capillary endothelial cells

Rat brain capillary endothelial (B10) cells express an unidentified nucleotide receptor linked to adenylyl cyclase inhibition. We show that this receptor in B10 cells is identical in sequence to the P2Y(12) ADP receptor ("P2Y(T)") of platelets. When expressed heterologously, 2-methylthio-ADP (2-MeSADP; EC(50), 2 nm), ADP, and adenosine 5'-O-(2-thio)diphosphate were agonists of cAMP decrease, and 2-propylthio-D-beta,gamma-difluoromethylene-ATP was a competitive antagonist (K(B), 28 nm), as in platelets. However, 2-methylthio-ATP (2-MeSATP) (EC(50), 0.4 nm), ATP (1.9 microm), and 2-chloro-ATP (190 nm), antagonists in the platelet, were also agonists. 2-MeSADP activated (EC(50), 0.1 nm) GIRK1/GIRK2 inward rectifier K(+) channels when co-expressed with P2Y(12) receptors in sympathetic neurons. Surprisingly, P2Y(1) receptors expressed likewise gave that response; however, a full inactivation followed, absent with P2Y(12) receptors. A new P2Y(12)-mediated transduction was found, the closing of native N-type Ca(2+) channels; again both 2-MeSATP and 2-MeSADP are agonists (EC(50), 0.04 and 0.1 nm, respectively). That action, like their cAMP response, was pertussis toxin-sensitive. The Ca(2+) channel inhibition and K(+) channel activation are mediated by beta gamma subunit release from a heterotrimeric G-protein. G alpha subunit types in B10 cells were also identified. The presence in the brain capillary endothelial cell of the P2Y(12) receptor is a significant extension of its functional range.     

Alpha-2 adrenergic regulation of norepinephrine release in the rat submandibular gland as measured by HPLC-EC

In many tissues, norepinephrine appears to inhibit its own release through an interaction at alpha adrenergic receptors. We have developed an assay for measuring the release of endogenous norepinephrine based on HPLC and have studied the regulation of release in the rat submandibular gland by alpha adrenergic antagonists. The method uses electrochemical detection to quantitate norepinephrine released from tissue slices and does not require preloading of the tissue with [3H]norepinephrine. Yohimbine, an alpha-2 adrenergic antagonist, potentiates by 50% the release caused by potassium induced depolarization with an EC50 of 0.14 microM. Prazosin, an alpha-1 antagonist, has a similar effect, but is less potent with an EC50 of 0.77 microM. Thus, the alpha adrenergic receptor mediating the regulation of norepinephrine release is of the alpha-2 subtype. The observed equal efficacies and lack of additivity of release potentiation by yohimbine and prazosin at maximal doses suggest that both drugs act at the same receptor. The five-fold difference in potency between prazosin and yohimbine is consistent with the recent observations indicating species differences between rodent and non-rodent alpha-2 adrenergic receptors.     

P2Y(6) nucleotide receptor mediates monocyte interleukin-8 production in response to UDP or lipopolysaccharide

Extracellular nucleotides are autocrine and paracrine cellular mediators that signal through P2 nucleotide receptors. Monocytic cells express several P2Y receptors but the role of these G protein-coupled receptors in monocytes is not known. Here, we present evidence that P2Y(6) regulates chemokine production and release in monocytes. We find that UDP, a selective P2Y(6) agonist, stimulates interleukin (IL)-8 release in human THP-1 monocytic cells whereas other nucleotides are relatively inactive. P2 receptor antagonists or P2Y(6) antisense oligonucleotides inhibit IL-8 release induced by UDP. Furthermore, UDP specifically activated IL-8 production in astrocytoma 1321N1 cells transfected with human P2Y(6). Since lipopolysaccharide has been suggested to activate P2 receptors via nucleotide release, we tested whether IL-8 production stimulated by lipopolysaccharide might result from P2Y(6) activation. P2 antagonists or apyrase, an enzyme which hydrolyzes nucleotides including UDP, inhibit IL-8 production induced by lipopolysaccharide but not by other stimuli. Furthermore, IL-8 gene expression activated by lipopolysaccharide is enhanced by P2Y(6) overexpression and inhibited by P2Y(6) antisense oligonucleotides. Thus, UDP activates IL-8 production via P2Y(6) in monocytic cells. Furthermore, lipopolysaccharide mediates IL-8 production at least in part by autocrine P2Y(6) activation. These findings indicate a novel role for P2Y(6) in innate immune defenses.     

The P2Y2 nucleotide receptor mediates vascular cell adhesion molecule-1 expression through interaction with VEGF receptor-2 (KDR/Flk-1)

UTP stimulates the expression of pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells through activation of the P2Y(2) nucleotide receptor P2Y(2)R. Here, we demonstrated that activation of the P2Y(2)R induced rapid tyrosine phosphorylation of vascular endothelial growth factor receptor (VEGFR)-2 in human coronary artery endothelial cells (HCAEC). RNA interference targeting VEGFR-2 or inhibition of VEGFR-2 tyrosine kinase activity abolishes P2Y(2)R-mediated VCAM-1 expression. Furthermore, VEGFR-2 and the P2Y(2)R co-localize upon UTP stimulation. Deletion or mutation of two Src homology-3-binding sites in the C-terminal tail of the P2Y(2)R or inhibition of Src kinase activity abolished the P2Y(2)R-mediated transactivation of VEGFR-2 and subsequently inhibited UTP-induced VCAM-1 expression. Moreover, activation of VEGFR-2 by UTP leads to the phosphorylation of Vav2, a guanine nucleotide exchange factor for Rho family GTPases. Using a binding assay to measure the activity of the small GTPases Rho, we found that stimulation of HCAEC by UTP increased the activity of RhoA and Rac1 (but not Cdc42). Significantly, a dominant negative form of RhoA inhibited P2Y(2)R-mediated VCAM-1 expression, whereas expression of dominant negative forms of Cdc42 and Rac1 had no effect. These data indicate a novel mechanism whereby a nucleotide receptor transactivates a receptor tyrosine kinase to generate an inflammatory response associated with atherosclerosis.