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Enzymatic synthesis of UTP γ S, a potent hydrolysis resistant agonist of P 2U ‐purinoceptors
Author(s) -
Lazarowski Eduardo R.,
Watt William C.,
Stutts M. Jackson,
Brown H. Alex,
Boucher Richard C.,
Harden T. Kendall
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb15175.x
Subject(s) - uridine triphosphate , gtp' , biochemistry , adenosine , autotaxin , purinergic receptor , chemistry , uridine diphosphate , biology , microbiology and biotechnology , nucleotide , lysophosphatidic acid , receptor , enzyme , gene
1 The defective Cl − secretion characteristic of cystic fibrosis airway epithelial cells can be bypassed by an alternative Ca 2+ dependent Cl − secretory pathway that is activated by extracellular nucleotides, e.g. uridine‐5′ triphosphate (UTP), acting on P 2U purinoceptors. Since UTP is susceptible to hydrolysis by nucleotidases and phosphatases present in the airways, the identification of stable P 2U ‐purinoceptor agonists would be of therapeutic relevance. 2 Uridine‐5′‐0‐(3‐thiotriphosphate) (UTP γ S) was synthesized by nucleoside diphosphate kinasecatalyzed transfer of the γ‐phosphorothioate from guanosine‐5′‐0‐(3‐thiotriphosphate) (GTP γ S) or adenosine‐5′=O‐(3‐thiotriphosphate) (ATP γ S) to UDP. Formation of UTP γ S was illustrated by observation of transfer of 35 S from [ 35 S]‐GTP γ S and transfer of 3 H from [ 3 H]‐UDP. The chemical identity of high performance liquid chromatography (h.p.l.c.)‐purified UTP γ S was confirmed by nuclear magnetic resonance analysis. 3 Human 1321N1 astrocytoma cells stably expressing the phospholipase C‐coupled human P 2U ‐purinoceptor were utilized to test the activity of UTP γ S. UTP γ S (EC 50 = 240 nM) was essentially equipotent to UTP and ATP for stimulation of inositol phosphate formation. 4 Unlike [ 3 H]‐UTP, [ 3 H]‐UTP γ S was not hydrolyzed by alkaline phosphatase, acid phosphatase, or apyrase. Moreover, no hydrolysis was detected during a 1 h incubation with human nasal epithelial cells. 5 UTP γ S was equally potent and efficacious with UTP for stimulation of Cl − secretion by human nasal epithelium from both normal donors and cystic fibrosis patients. Based on its high potency and resistance to hydrolysis, UTP γ S represents a promising compound for treatment of cystic fibrosis.