PURINE AND PYRIMIDINE NUCLEOTIDE-SENSITIVE PHOSPHOINOSITIDASE-C IN AMPULLA FROM FROG SEMICIRCULAR CANAL

A microassay was developed to screen the abilities of ATP analogues to stimulate phosphoinositidase C in single ventral regions (including d ark cells and sensory cells) of ampullas microdissected from posterior vertical semicircular canals of Rana ridibunda and labeled with myo-[ H-3]inositol. ATP induced a dose-dependent and saturable increase of t otal [H-3]inositol phosphate production accompanied by an equivalent d ecrease in the [H-3]phosphoinositide pool. The rank order of analogues revealing agonistic potencies for phosphoinositidase C activation was as follows: uridine 5'-triphosphate greater than or equal to adenosin e 5'-O-[3-thiophosphate] tetralithium greater than or equal to adenosi ne 5'-O-[2-thiodiphosphate] trilithium greater than or equal to ATP gr eater than or equal to ADP = inosine 5'-triphosphate greater than or e qual to guanosine 5'-triphosphate greater than or equal to 2-methylthi o-adenosine 5'-triphosphate tetrasodium greater than or equal to 2'-de soxy-thymidine 5'-triphosphate greater than or equal to cytidine 5'-tr iphosphate = (alpha,beta>)-methyl ATP > AMP, whereas adenosine 3',5'-c yclic monophosphate and adenosine were almost devoid of activity. For antagonists, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was far more active than suramin for competitive inhibition of ATP-induced enz yme stimulation, whereas reactive blue 2 acted as a noncompetitive inh ibitor. Results indicate that the putative P-2 receptors triggering ph osphoinositidase C activation in ventral ampullary epithelium from fro g semicircular canal exhibit mainly the functional properties of P-2Y and P-2U receptors.