Extracellular ATP (>3 x 10-5 M) elicits a transient, biphasic contraction of the vas deferens. The specific P(2x)-purinoceptor photoaffinity label antagonist, arylazido aminopropionyl ATP (ANAPP3), inhibits the first contractile phase. The second phase, which is increased for adenosine 5'-O-(3-thio-triphosphate)(ATPγS) and attenuated for 5'-anhydride-substituted ATP analogs, has been hypothesized to be initiated by phosphate chain cleavage, and is inhibited irreversibly by periodate-oxidized ATP (P-ATP), an affinity label. We examined whether phosphorylation occurs during contraction to ATPγS and ATP, and whether the irreversible inhibition of the second phase of contraction by P-ATP results from its covalent incorporation. After incubation of intact tissues with [35S]ATPγS (3 mM), homogenization, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, 35SPO23-(35S) was incorporated predominantly into a single, 19 to 21 kD protein. The incorporation was transient and paralleled the time course of the transient contraction to ATPγS (peak by 5 to 10 sec, then rapid decline during 60 sec). Treatment with P-ATP (10 mM; 5 min) or ANAPP3 (10-4 M) inhibited 35S incorporation at 60 sec. 32PO43- (32P) from [γ-32P] ATP (3 mM) also was incorporated rapidly into the protein; removal of 32P, however, was faster than removal of 35S. [3H]P-ATP was incorporated into several proteins, but not into the 19 to 21 kD protein nor the ones labeled by photolyzed ANAPP3. Incorporation of 35S from [35S]ATPγS, and [3H]P-ATP, were not affected by incubation in the presence of histamine or norepinephrine (10-4-10-3 M), whereas ATP (10-2 M) reduced incorporation of [3H]P-ATP (10-5-10-2 M). The results support the hypothesis that the second phase of contraction to ATP involves a transductive phosphoryl transfer involving an ecto-kinase, which is inhibited by P-ATP.
|Original language||English (US)|
|Number of pages||10|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|State||Published - 1991|
All Science Journal Classification (ASJC) codes
- Molecular Medicine