skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Transendothelial transport and metabolism of adenosine and inosine in the intact rat aorta

Abstract

This study was aimed at defining the role of vascular endothelium in the transport and metabolism of adenosine. For this purpose, endothelium-intact and endothelium-denuded isolated rat aortas, perfused at constant flow (2 ml/min), were prelabeled with 3H-adenosine or 3H-inosine for 10 minutes at concentrations of 0.012-100 microM. Sequestration of adenosine by endothelium was determined from radioactivity recovered during selective endothelial cell removal with deoxycholic acid (0.75% for 15 seconds). In the physiological concentration range of adenosine (0.012-1 microM), fractional sequestration by endothelium was 90-92% of the total adenosine incorporation by the aorta. Endothelial sequestration of inosine at 0.1 microM was 85%. At 100 microM adenosine or inosine, fractional sequestration by aortic endothelium was 33% and 39%, respectively. Analysis of the specific radioactivity of adenine nucleotides extracted from prelabeled aortas indicated that most of the adenosine was incorporated into endothelial adenine nucleotides. Incorporation of inosine into endothelial ATP was approximately 15% that of adenosine. Inhibition of aortic adenosine deaminase with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not influence sequestration of 0.1 microM adenosine, but resulted in a 49% reduction of total endothelial incorporation at 100 microM adenosine. Transfer of radioactive purines from the endothelium to underlying smooth muscle after prelabeling was equivalent to onlymore » 1%/hr of total endothelial radioactivity.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Univ. of Duesseldorf (Germany, F.R.)
OSTI Identifier:
5879012
Resource Type:
Journal Article
Resource Relation:
Journal Name: Circ. Res.; (United States); Journal Volume: 64:6
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ADENOSINE; MEMBRANE TRANSPORT; METABOLISM; INOSINE; ADENINES; AORTA; ATP; ENDOTHELIUM; HYDROLASES; PERFUSED TISSUES; PURINES; RATS; TRACER TECHNIQUES; TRITIUM COMPOUNDS; AMINES; ANIMAL TISSUES; ANIMALS; ANTIMETABOLITES; AROMATICS; ARTERIES; AZAARENES; BLOOD VESSELS; BODY; CARDIOVASCULAR SYSTEM; DRUGS; ENZYMES; HETEROCYCLIC COMPOUNDS; ISOTOPE APPLICATIONS; LABELLED COMPOUNDS; MAMMALS; NUCLEOSIDES; NUCLEOTIDES; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANS; RIBOSIDES; RODENTS; TISSUES; VERTEBRATES; 550501* - Metabolism- Tracer Techniques

Citation Formats

Kroll, K., Kelm, M.K., Buerrig, K.F.S., and Schrader, J.. Transendothelial transport and metabolism of adenosine and inosine in the intact rat aorta. United States: N. p., 1989. Web. doi:10.1161/01.RES.64.6.1147.
Kroll, K., Kelm, M.K., Buerrig, K.F.S., & Schrader, J.. Transendothelial transport and metabolism of adenosine and inosine in the intact rat aorta. United States. doi:10.1161/01.RES.64.6.1147.
Kroll, K., Kelm, M.K., Buerrig, K.F.S., and Schrader, J.. 1989. "Transendothelial transport and metabolism of adenosine and inosine in the intact rat aorta". United States. doi:10.1161/01.RES.64.6.1147.
@article{osti_5879012,
title = {Transendothelial transport and metabolism of adenosine and inosine in the intact rat aorta},
author = {Kroll, K. and Kelm, M.K. and Buerrig, K.F.S. and Schrader, J.},
abstractNote = {This study was aimed at defining the role of vascular endothelium in the transport and metabolism of adenosine. For this purpose, endothelium-intact and endothelium-denuded isolated rat aortas, perfused at constant flow (2 ml/min), were prelabeled with 3H-adenosine or 3H-inosine for 10 minutes at concentrations of 0.012-100 microM. Sequestration of adenosine by endothelium was determined from radioactivity recovered during selective endothelial cell removal with deoxycholic acid (0.75% for 15 seconds). In the physiological concentration range of adenosine (0.012-1 microM), fractional sequestration by endothelium was 90-92% of the total adenosine incorporation by the aorta. Endothelial sequestration of inosine at 0.1 microM was 85%. At 100 microM adenosine or inosine, fractional sequestration by aortic endothelium was 33% and 39%, respectively. Analysis of the specific radioactivity of adenine nucleotides extracted from prelabeled aortas indicated that most of the adenosine was incorporated into endothelial adenine nucleotides. Incorporation of inosine into endothelial ATP was approximately 15% that of adenosine. Inhibition of aortic adenosine deaminase with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not influence sequestration of 0.1 microM adenosine, but resulted in a 49% reduction of total endothelial incorporation at 100 microM adenosine. Transfer of radioactive purines from the endothelium to underlying smooth muscle after prelabeling was equivalent to only 1%/hr of total endothelial radioactivity.},
doi = {10.1161/01.RES.64.6.1147},
journal = {Circ. Res.; (United States)},
number = ,
volume = 64:6,
place = {United States},
year = 1989,
month = 6
}
  • A new method, based on computer-assisted kinetic analysis of /sup 45/Ca efflux data, was used to measure calcium contents and fluxes for extracellular and intracellular compartments in intact segments of rabbit aorta. After a 1-hour loading period, efflux data were collected for 8 hours using a flow-through tissue chamber. These long-term effluxes were necessary because information on intracellular calcium metabolism was concentrated in the slow components of the efflux curves while earlier components appeared to be dominated by washout of extracellular calcium. Intracellular compartments were identified as those whose calcium contents were altered by 10 microM phenylephrine. This method complementsmore » previous approaches by providing simultaneous estimates of compartmental calcium contents and fluxes without requiring the assumption of isotopic equilibrium and without recourse to standard wash techniques for removal of extracellular calcium. In normal, calcium-containing, bicarbonate-buffered physiological salt solution these compartments contained a total of approximately 300 nmol Ca/g wet aorta. Of this total, 55 nmol/g were associated with the slowest resolvable compartment whose turnover time was 170 minutes and whose exchange flux was 0.32 nmol min-1g-1. Two other intracellular compartments had turnover times of 30 minutes. One of these was phenylephrine releasable and contained 145 nmol/g; it exchanged calcium at 4.9 nmol min-1g-1. In normal physiological salt solution the plasma membrane was, surprisingly, not rate limiting for Ca efflux; and in 10 microM phenylephrine the membrane Ca flux was even greater, increasing 3.5-fold compared to control.« less
  • Kinetic alpha-deuterium isotope effects have been measured for the purine nucleoside phosphorylase-catalyzed phosphorolysis of adenosine and inosine by a competitive double label technique at saturating concentrations of the second substrate, phosphate. Under these conditions the observed isotope effect, kH/kD, is on the second order rate constant, Vmax/Km, for reaction of nucleoside with the Michaelis complex of enzyme and phosphate. For adenosine, at neutral pH, the isotope effect is unity. For inosine, kH/kD was determined as a function of pH. These values suggest a mechanism for purine nucleoside phosphorylase involving a change in rate-limiting step from one at pH values nearmore » neutrality for which cleavage of the nucleoside C-N bond is not rate limiting to a step at extremes of pH with a transition state having considerable oxocarbonium ion-like character.« less
  • ly es but It is suggested that, if a radiobiological change is enhanced by
  • The authors have used directly combined high-performance liquid chromatography-mass spectrometry (LC/MS) to examine the mechanism of the reaction catalyzed by the double-stranded RNA unwinding/modifying activity. A double-stranded RNA substrate in which all adenosines were uniformly labeled with {sup 13}C was synthesized. An LC/MS analysis of the nucleoside products from the modified, labeled substrate confirmed that adenosine is modified to inosine during the unwinding/modifying reaction. Most importantly, they found that no carbons are exchanged during the reaction. By including H{sub 2} {sup 18}O in the reaction, they showed that water serves efficiently as the oxygen donor in vitro. These results aremore » consistent with a hydrolytic deamination mechanism and rule out a base replacement mechanism. Although the double-stranded RNA unwinding/modifying activity appears to utilize a catalytic mechanism similar to that of adenosine deaminase, coformycin, a transition-state analogue, will not inhibit the unwinding/modifying activity.« less