Structure‐activity relationship of a pyrimidine receptor in the rat isolated superior cervical ganglion

Abstract

1 The effects of pyrimidines and purines on the d.c. potential of the rat isolated superior cervical ganglion (SCG) have been examined by a grease-gap technique to determine the structure-activity requirements of the receptor activated by pyrimidines, i.e. a pyrimidinoceptor. 2 5-Aminoimidazole4-carboxamide-l-f-D-ribofuranosyl (ZTP), the pyrimidines, cytidine 5'-triphosphate (CTP), uridine 5'-triphosphate (UTP) and thymidine 5'-triphosphate (TTP) and the purines, adenosine 5'-triphosphate (ATP; in the presence of an Al-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (1 Mm)), adenosine 5'-0-(3-thiotriphosphate) (ATPyS), guanosine 5'-triphosphate (GTP), inosine 5'-triphosphate (ITP) depolarized ganglia in a concentration-dependent manner.The relative order of ZTP and purine 5'-triphosphates in depolarizing ganglia was ZTP>ATPyS > >ATP? ITP=GTP, and for the pyrimidine 5'-triphosphates UTP>TTP>CTP. Depolarizations evoked by ATPyS were followed by concentration-dependent hyperpolarizations at 100 and 1000 MM. 3 At concentrations of between 0.1 Mm and 1 mm, uridine 5'-diphosphate (UDP), uridine 5'-diphosphoglucose (UDPG) and uridine 5'-diphosphoglucuronic acid (UDPGA) evoked significant and concentration-dependent depolarizations, whereas uridine 5'-monophosphate (UMP), uridine and uracil were inactive or produced small (<45 pY) depolarizations. The relative order of potency of uridine analogues in depolarizing ganglia was UDP> UTP > UDPG > UDPGA > > uracil > UMP = pseudouridine uridine. At 3 and 10 mM, uridine produced concentration-dependent hyperpolarizations. Nikkomycin Z, a nucleoside resembling UTP (viz. the triphosphate chain at the 5'-position on the ribose moiety being replaced by a peptide), was inactive between 1 uM and 1 mM. Generally, a concentration of 10 mm was required before thymidine, 6-azathymine, 6-azauracil or 6-azauridine depolarized ganglia. 4 Suramin (300 Mm), a P2-purinoceptor antagonist, significantly depressed depolarizations evoked by a,fi-methylene-ATP (a,fi-MeATP; 100 Mm), ATPyS (100 Mm), CTP (1 mM), GTP (1 mM), ZTP (30 MM) and ATP (300 MM) in the presence of DPCPX (1 yM). Suramin reversed a small depolarization evoked by UMP (1 mM) into a small hyperpolarization. In contrast depolarizations evoked by UDP, UTP, UDPG (all at 100 Mm) and TTP (300 Mm) were unaltered or enhanced by suramin. 5 It is concluded that the rat SCG contains distinct nucleotide receptors including a P2-purinoceptor (activated by a,f,-MeATP, ATP, GTP, ITP and ZTP) and a pyrimidinoceptor (activated by UTP, UDP, UDPG, UDPGA and TTP). The pyrmidinoceptor on rat SCG neurones had specific structure activity requirements with the di-and triphosphates of uridine being the most effective depolarizing agonists examined.

A G Protein-coupled Receptor for UDP-glucose

“…The first expressed KIAA0001 in conjunction with the endogenous yeast G␣ protein, Gpalp (21). The second expressed KIAA0001 and a hybrid of Gpalp and the promiscuous mammalian G␣ protein, G␣ 16 (22). A control yeast strain transformed with empty vector did not respond to UDP-glucose nor did yeast strains transfected with the same G proteins and a number of other receptors.…”

“…Activation of KIAA0001 in Mammalian Cells-In parallel studies, we transiently co-transfected mammalian HEK-293 cells with both KIAA0001 and G␣ 16 to promote coupling to intracellular Ca 2ϩ . We screened these cells against the same ligand bank in an intracellular Ca 2ϩ assay, and UDP-glucose was the only substance observed to clearly elicit KIAA0001-mediated Ca 2ϩ responses.…”

“…Briefly, strain CY10560 (FUS1-HIS3 GPA1-3907 ade2⌬3447 ade8⌬345 can1 far1⌬1442 sst2⌬1056 ste14::trp1::LYS2 ste18␥6 -3841 ste3⌬1156 trp1 his3 leu2 lys2 ura3) containing the native G␣ gene, GPA1, and a hybrid G␥ gene encoding Ste18p residues 1-92/human ␥ 2 residues 60 -67/ Ste18p residues 106 -110, was transformed with Cp1584 (ARS-2 bla TRP1 FUS1-lacZ) and either Cp5004 (KIAA0001 carried on Cp3700, a derivative of Cp1651) or the empty vector, Cp3700. Similar strains were created in which GPA1 was replaced with human G␣ sequences encoding G␣ i2 (7), G␣ s , containing a D229S mutation, or G␣ 16 , in which the N-terminal 41 amino acids of G␣ 16 are replaced with those of Gpalp and an S270P mutation is incorporated.…”

“…A single clone responded most sensitively to UDP-glucose in a GTP-␥-S assay and was used in all other experiments. Stable clonal HEK-293 cells co-expressing G␣ 16 and KIAA0001 were generated by transfecting cells with a derivative of pCDN-G␣ 16 in which the neomycin coding sequence had been replaced with a histidinol selectable marker (10). A single histidinol-resistant clone (mRNA-positive for G␣ 16 by Northern analysis) was transfected with KIAA0001 as described above, and a single G418-resistant clonal cell line most sensitive to UDP-glucose in an intracellular Ca 2ϩ assay was used in all further work.…”

“…Receptors and G␣ 16 were subcloned into the mammalian expression vector pCDN (9) and transiently transfected into HEK-293 cells using lipofectAMINE Plus (Life Technologies, Inc.), according to the manufacturer's instructions with minor modifications. Thus, a mixture of 15 g of receptor DNA and 15 g of G␣ 16 DNA was used to transfect a single 175-cm 2 culture flask of cells at 80% confluency. For transient transfection of receptor DNA alone, 30 g of plasmid DNA was used.…”