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Research was conducted to examine the hypothesis that a rat parotid gland cell line, Par-C10, displays neurotransmitter-regulated transepithelial anion secretion. The cell line was developed on permeable supports and evaluated for development of transcellular resistance, polarization and changes in short-circuit current in reaction to relevant receptor agonists. Results demonstrate that Par-C10 cells are the first of its kind to exhibit its usefulness in transcellular ion secretion experiments in Ussing chambers.

An experiment was conducted to study the changes in the functions of extracellular receptors (P2) of salivary glands that will provide evidence for the expression of these receptors both in vitro and in vivo. Results indicated that the receptors have a role in the regulation of salivary gland function as reflected in the upregulation of receptor activity and mRNA expression both in vitro and in vivo. It was also observed that possible expression of various P2 receptor subtypes can occur with the regulation of P2 receptor expression and activity.

The expression and distribution P2Y G protein-coupled receptors in rat salivary gland cells were analyzed by reverse transcriptase-polymerase chain reaction. The expression of P2Y receptors in rat salivary glands was developmentally regulated via postranslational and translational modification. Furthermore, salivary gland cells from older rats exhibited lower levels of P2Y receptor messenger RNA expression compared to the salivary gland cells of postnatal rats.

The P(sub 2U) purinoceptor agonist, uridine 5'-triphosphate (UTP) induces the regulatory volume decrease (RVD) in HSG-PA human salivary gland duct cells, in response to low osmolarity. The UTP-treated cells have increased Ca2+ mobilization, hyperpolarization and K+ transport. RVD is associated with the net efflux of KCl. Increase in Ca2+ opens the Ca2+ -activated K+ channels through which the K+ ions move out. The receptor-mediated mechanism of RVD is sensitive to extracellular control.

Nucleotides such as ATP and ADP act as intercellular messengers and exert a widespread influence on cellular function by acting on a variety of cell surface receptors. Until recently, progress has been restrained, in part, by a lack of cloned receptors. Now, however, the successful coloning of variety of [P.sub.2] purinoceptors is holding out the prospect of rapid advances in the understanding of this diverse group of receptors and the potent therapeutic resource they represent. In this article, Michael Boarder and colleagues summarize the findings of recent cloning studies, and assess the impact of these on the understanding of the function of the G protein-couple [P.sub.2] purinoceptors in several types of cells and tissues.

Analysis of a cDNA, cloned from human airway epithelial (CF/T43) cells that encode a protein having characteristics of a P2U nucleotide receptor, provided insights into the sequence and functional expression. Expression of the human P2U receptor in 1321N1 cells confirmed autocine ATP secretion and activation of transduced receptors.

The prototype strain (MDR1.3) is derived from the gene cryIA(c), which is obtained from Bacillus thuringienesis subsp kurstaki when inserted into the chromosome Clavibacter xyli subsp cynodontis, and this bacterial endophyte exhibits activity against the European corn borer. Such genetically engineered bacterial endophytes are useful for controlling insect pests. The European corn borer insect is found throughout most of the US corn belt.

P2U purinergic receptor circular DNA expresses itself for a brief period of time in the K-562 mammalian cell line deficient in the receptor. This expression system serves as a reference frame for biochemical and molecular pharmacological characterization of wildtype and recombinant P2U receptors. Agonist selectivity and signaling characteristics of the cloned P2U receptor is analyzed using 3'-O-(4-benzoylbenzoyl)adenosine 5'-(alpha-32P)triphosphate (alpha-32P)-BzATP, a photoaffinity ATP analog and a polyhistidine coupled P2U receptor. This facilitates the identification of the receptor as a 53-kDa plasma membrane protein.

The [P2Y.sub.2] nucleotide receptor ([P2Y.sub.2R]) contains the integrin-binding domain arginine-glycine-aspartic acid (RGD) in its first extracellular loop, raising the possibility that this G protein-coupled receptor interacts directly with an integrin. Binding of a peptide corresponding to the first extracellular loop of the [P2Y.sub.2R] to K562 erythroleukemia cells was inhibited by antibodies against [[Alpha].sub.V][[Beta].sub.5] integrins and the integrin-associated thrombospondin receptor, CD47. Immuno-fluorescence of cells transfected with epitope-tagged [P2Y.sub.2Rs] indicated that [[Alpha].sub.V] integrins colocalized 10-fold better with the wild-type [P2Y.sub.2R] than with a mutant [P2Y.sub.2R] in which the RGD sequence was replaced with RGE. Compared with the wild-type [P2Y.sub.2R], the RGE mutant required 1,000-fold higher agonist concentrations to phosphorylate focal adhesion kinase, activate extracellular signal-regulated kinases, and initiate the PLC-dependent mobilization of intracellular [Ca.sup.2+]. Furthermore, an anti-[[Alpha].sub.V] integrin antibody partially inhibited these signaling events mediated by the wild-type [P2Y.sub.2R]. Pertussis toxin, an inhibitor of [G.sub.i/o] proteins, partially inhibited [Ca.sup.2+] mobilization mediated by the wild-type [P2Y.sub.2R], but not by the RGE mutant, suggesting that the RGD sequence is required for [P2Y.sub.2R] mediated activation of [G.sub.o], but not [G.sub.q]. Since CD47 has been shown to associate directly with [G.sub.i/o] family proteins, these results suggest that interactions between [P2Y.sub.2Rs], integrins, and CD47 may be important for coupling the [P2Y.sub.2R] to [G.sub.o]. Key words: purinergic receptors * cell surface receptors * integrins * GTP-binding proteins * signal transduction

Ahn, Jae Suk, Jean M. Camden, Ann M. Schrader, Robert S. Redman, and John T. Turner. Reversible regulation of P2[Y.sub.2] nucleotide receptor expression in the duct-ligated rat submandibular gland. Am J Physiol Cell Physiol 279: C286-C294, 2000.--Ligation of the main excretory duct of the rat submandibular gland (SMG) produces a pronounced atrophy that is reversed upon ligature removal. Based on previous studies by our group and others suggesting that P2[Y.sub.2] nucleotide receptors are upregulated in response to tissue damage, we hypothesized that P2[Y.sub.2] receptor activity and mRNA levels would increase after duct ligation and return to control levels after ligature removal. Our results support this hypothesis. Intracellular [Ca.sup.2+] mobilization in response to the P2[Y.sub.2] receptor agonist UTP in SMG cells was increased significantly after ligation periods of 1.5 to 7 days, whereas no significant response was observed in the contralateral, nonligated gland. P2[Y.sub.2] receptor mRNA, as measured by semiquantitative RT-PCR, increased about 15-fold after 3 days of ligation. These increases reverted to control levels by 14 days after ligature removal. In situ hybridization revealed that the changes in P2[Y.sub.2] receptor mRNA abundance occurred mostly in acinar cells, which also were more adversely affected by ligation, including an increase in the appearance of apoptotic bodies. These findings support the idea that P2[Y.sub.2] receptor upregulation may be an important component of the response to injury in SMG and that recovery of normal physiological function may signal a decreased requirement for P2[Y.sub.2] receptors. salivary glands; tissue damage and regeneration; receptor regulation

Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium. Am J Physiol Gastrointest Liver Physiol 279: G132-G138, 2000.--The loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial HCO[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] secretion contributes to the pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF) patients. Recent studies have investigated [P2Y.sub.2] nucleotide receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by stimulating [Ca.sup.2+]-activated Cl[sup.-] secretion. However, the value of this treatment in facilitating transepithelial [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] secretion is unknown. Gallbladder mucosae from CFTR knockout mice were used to isolate the [Ca.sup.2+]-dependent anion conductance during activation of luminal [P2Y.sub.2] receptors. In Ussing chamber studies, UTP stimulated a transient peak in short-circuit current ([I.sub.sc]) that declined to a stable plateau phase lasting 30-60 min. The plateau [I.sub.sc] after UTP was [Cl.sup.-] independent, HCO[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] dependent, insensitive to bumetanide, and blocked by luminal DIDS. In pH stat studies, luminal UTP increased both [I.sub.sc] and serosal-to-mucosal HCO[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] flux ([J.sup.s[right arrow]m]) during a 30-min period. Substitution of [Cl.sup.-] with gluconate in the luminal bath to inhibit [C1.sup.-]/HCO[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]exchange did not prevent the increase in [J.sup.s[right arrow]m] and [I.sub.sc] during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in [J.sup.s[right arrow]m and [I.sub.sc]. We conclude that [P2Y.sub.2] receptor activation results in a sustained (30-60 rain) increase in electrogenic HCO3[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] secretion that is mediated via an intracellular [Ca.sup.2+]-dependent anion conductance in CF gallbladder. cystic fibrosis; biliary system; [P2Y.sub.2] receptor; nucleotide receptor; purinoceptor; chloride

The desensitization of UTP-stimulated transepithelial anion secretion was examined using the murine gallbladder as a model epithelium. Murine gallbladder was used because this tissue responds to UTP stimulation with Ca2+-activated anion secretion. The methods used were cell culture, bioelectric, intracellular Ca2+, total IP production measurements and the RT-PCR. The murine gallbladder responded to UTP with an abrupt increase in the Isc.

This article describes the application of an acoustic reflectometry technique to detect blockages in long lengths of gas-filled pipelines. The use of the technique in laboratory pipelines has been previously reported. However, in this article, the technique is applied to a series of pipelines that provide the ideal test bed to thoroughly assess the capabilities of using acoustic reflectometry to detect and locate defects in industrial gas-filled pipelines. In particular, the use of the technique for detecting the early formation of hydrates in subsea pipelines is evaluated. Four very different pipelines were used for this evaluation. These pipelines varied in inside diameter from 0.06 to 0.6 m, were operated at pressures of up to 10 MPa and contained both flowing and static gases. In all tests, the acoustic reflectometry technique was able to accurately detect and locate the blockages within the pipeline. The results suggest that acoustic reflectometry is capable of detecting full and partial blockages in pipelines with lengths of up to 10 km. [ABSTRACT FROM AUTHOR]