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Title: Ectonucleotidase activity in smooth muscle preparations of the rat and guinea-pig
Author: Tennant, Jason P.
ISNI:       0000 0001 3516 1427
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 2000
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1. The technique of high performance liquid chromatography (h.p.l.c.) was employed to determine the degree of influence of ectonucleotidases, responsible for the extracellular degradation of purine and pyrimidine mono- and dinucleotides, on the levels of these pharmacologically active compounds in smooth muscle tissues which have previously been shown to possess receptors for both these compounds and/or their breakdown products. 2. The rat colon muscularis mucosae, a tissue which contains receptors for both ATP, UTP and adenosine, was shown to degrade ATP, UTP and adenine dinucleotides, although the latter to a much lesser extent. The P2 receptor antagonist, suramin, resulted in an inhibition of this degradation in all cases except that of ATP, with an accompanying increase in the half-life of each substrate. 3. The PI receptor antagonist, 8-SPT, the adenosine deaminase inhibitor EHNA, the potent inhibitor of nucleoside membrane transport mechanisms NBTI and the xanthine oxidase inhibitor allopurinol had no significant effects on the half-life for the degradation of ATP by this tissue. However preincubation with, EHNA resulted in the observation of adenosine accumulation in the incubation medium, which is completely absent in the presence of control tissues. In contrast NBTI resulted in a significant accumulation of AMP in the incubation buffer although, interestingly, no adenosine was detected in the presence of these tissues. A decrease in the pH of the extracellular environment of the rat colon muscularis mucosae results in a decrease in the levels of AMP detected in the incubation medium with a simultaneous increase in the amount of inosine detected in these tissues, which appears to indicate that the lower levels of AMP observed in these tissues is due to an increased rate of inosine production from AMP, presumably via rapid deamination of adenosine, as opposed to a decrease in the rate of production of AMP from ATP via ADP. 4. The rat vas deferens was shown to rapidly degrade extracellular ATP with a half-life of 5.83±0.4 minutes. The major breakdown products initially were ADP and AMP, with inosine as the ultimate product. Little or no adenosine was detected in the incubation buffer. The half-life of ATP was unaffected by preincubation with NA, A23187, forskolin, sodium nitroprusside, glibenclamide or glipizide. However, preincubation with both forskolin and sodium nitroprusside resulted in a significant increase in the levels of AMP detected in the buffer. In addition, sodium nitroprusside also resulted in a simultaneous decrease in the level of inosine detected in the buffer, although there was no corresponding change in the adenosine levels observed with these tissues. 5. In the case of the guinea-pig vas deferens ATP, Ap4A and Ap5A were all degraded, resulting in the formation of ADP, AMP, adenosine and inosine from ATP; ATP, ADP, AMP, adenosine and inosine from Ap4A and AP4, ADP, AMP, adenosine and inosine from Ap5A. In addition, the breakdown of ATP, Ap4A and Ap5A was inhibited by preincubation of tissues with suramin. 6. The bisected guinea-pig vas deferens degraded extracellular ATP, with a similar pattern of breakdown products detectable in the incubation medium as in the presence of the whole tissue. Although the half-lives for ATP in the presence of the prostatic and epididymal portions were significantly different, when the relative weights of the two halves were taken into consideration the rate constants for each of these reactions did not show any significant differences. Interestingly, there was a significant correlation between tissue weight and the extracellular half-life for ATP in the presence of the epididymal, but not the prostatic portion of the guinea-pig vas deferens, indicating that an additional factor other than tissue weight may influence the rate of extracellular ATP by the prostatic portion of this tissue. 7. Preincubation of the rat whole duodenum with A3’P5’PS caused a dose-dependent relaxation of the carbachol precontracted tissue, which was inhibited by suramin. Both ATP and A3’P5’PS were degraded by the rat duodenum, although A3’P5’PS to a much lesser extent. The degradation product of A3’P5’PS metabolism was A3’P5’P, with no other metabolite detected in the incubation buffer. Preincubation with suramin resulted in no detectable degradation of A3’P5’PS.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Pharmacology & pharmacy & pharmaceutical chemistry