Cloning and Functional Characterization of Two Murine Uridine Nucleotide Receptors Reveal a Potential Target for Correcting Ion Transport Deficiency in Cystic Fibrosis Gallbladder

  1. Eduardo R. Lazarowski1,
  2. Lori G. Rochelle1,
  3. Wanda K. O'Neal1,
  4. Carla M. P. Ribeiro1,
  5. Barbara R. Grubb1,
  6. Vivian Zhang3,
  7. T. Kendall Harden2 and
  8. Richard C. Boucher1
  1. Departments of 1Medicine (E.R.L., L.G.R., W.K.O., C.M.P.R., B.R.G., R.C.B.) and 2Pharmacology (T.K.H.), University of North Carolina School of Medicine, Chapel Hill, North Carolina; and 3Inspire Pharmaceuticals, Durham, North Carolina (V.Z.)

    Abstract

    Extracellular nucleotides regulate transepithelial ion secretion via multiple receptors. The P2Y2 receptor is the predominant transducer of chloride transport responses to nucleotides in the airways, but the P2 receptors that control ion transport in gastrointestinal epithelia have not been identified. UTP and UDP promote chloride secretion in mouse jejuna and gallbladder epithelia, respectively, and these responses were unaffected by P2Y2receptor gene disruption. Pharmacological data suggested the involvement of P2Y4 and P2Y6 receptors in gastrointestinal responses. To identify the P2Y receptors responsible for the gastrointestinal actions of UTP and UDP, we have cloned the murine P2Y4 and P2Y6 receptors and have stably expressed each in a null cell line to examine the nucleotide-promoted inositol phosphate formation and intracellular Ca2+mobilization. The (m)P2Y4 receptor was potently, but not selectively, activated by UTP (UTP ≥ ATP >ITP > GTP > CTP), and it was not activated by UDP or ADP. The (m)P2Y6 receptor was highly selective for UDP (UDP ≫ ADP = GDP). The nucleotide selectivities observed with the recombinant (m)P2Y4 and (m)P2Y6 receptors resemble those for nucleotide-promoted chloride transport in murine P2Y2(−/−) jejuna and gallbladder epithelial cells, respectively. Ion transport responses to nucleotide additions were examined in freshly excised tissues from cystic fibrosis transmembrane regulator-deficient mice. Although the effect of UTP on jejunal short-circuit current (Isc) was impaired in the CF mouse, UDP-promoted Isc changes were not affected in CF gallbladder epithelium, suggesting that the P2Y6 receptor is a target for treatment of CF gallbladder disease.

    Footnotes

    • Send reprint requests to: Dr. Eduardo R. Lazarowski, CB# 7248, 7017 Thurston-Bowles Bldg., Cystic Fibrosis/Pulmonary Research and Treatment Center, Department of Medicine, University of North Carolina, Chapel Hill, NC 27599-7248. E-mail:Eduardo_Lazarowski{at}med.unc.edu

    • This study was supported by National Institutes of Health Grant HL34322 and the Cystic Fibrosis Foundation (CFF Lazaro99GO). This work was presented in part as an abstract to the 3rd International Symposium on Nucleosides and Nucleotides, Purines 2000, Madrid, Spain; July 9–13, 2000.

    • Abbreviations:
      CFTR
      cystic fibrosis transmembrane conductance regulator
      CF
      cystic fibrosis
      RT
      reverse transcriptase
      h
      human
      r
      rat
      m
      mouse
      PCR
      polymerase chain reaction
      bp
      base pair
      [Ca2+]i
      intracellular calcium
      Isc
      short-circuit current
      • Received September 29, 2000.
      • Accepted December 7, 2000.
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    1. JPET April 1, 2001 vol. 297 no. 1 43-49
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