Involvement of ecto-phosphoryl transfer in contractions of the smooth muscle of the guinea pig vas deferens to adenosine 5'-triphosphate

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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, 35SPO2 3-(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. 32PO4 3- (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 languageEnglish (US)
Pages (from-to)339-348
Number of pages10
JournalJournal of Pharmacology and Experimental Therapeutics
Volume258
Issue number1
StatePublished - 1991

Fingerprint

Vas Deferens
Smooth Muscle
Guinea Pigs
Adenosine Triphosphate
Proteins
Photoaffinity Labels
Affinity Labels
Purinergic Receptors
Anhydrides
periodate-oxidized adenosine 5'-triphosphate
Autoradiography
Sodium Dodecyl Sulfate
Histamine
Polyacrylamide Gel Electrophoresis
Norepinephrine
Phosphotransferases
Phosphates
Phosphorylation

All Science Journal Classification (ASJC) codes

  • Pharmacology

Cite this

@article{2a6f900aafe34c92acf7d039bd99a783,
title = "Involvement of ecto-phosphoryl transfer in contractions of the smooth muscle of the guinea pig vas deferens to adenosine 5'-triphosphate",
abstract = "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, 35SPO2 3-(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. 32PO4 3- (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.",
author = "Lamport-Vrana, {S. J.} and Vrana, {K. E.} and Fedan, {J. S.}",
year = "1991",
language = "English (US)",
volume = "258",
pages = "339--348",
journal = "Journal of Pharmacology and Experimental Therapeutics",
issn = "0022-3565",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "1",

}

TY - JOUR

T1 - Involvement of ecto-phosphoryl transfer in contractions of the smooth muscle of the guinea pig vas deferens to adenosine 5'-triphosphate

AU - Lamport-Vrana, S. J.

AU - Vrana, K. E.

AU - Fedan, J. S.

PY - 1991

Y1 - 1991

N2 - 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, 35SPO2 3-(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. 32PO4 3- (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.

AB - 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, 35SPO2 3-(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. 32PO4 3- (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.

UR - http://www.scopus.com/inward/record.url?scp=0025895455&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025895455&partnerID=8YFLogxK

M3 - Article

C2 - 2072306

AN - SCOPUS:0025895455

VL - 258

SP - 339

EP - 348

JO - Journal of Pharmacology and Experimental Therapeutics

JF - Journal of Pharmacology and Experimental Therapeutics

SN - 0022-3565

IS - 1

ER -