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Characterization of cytochrome P-450-dependent arachidonic acid metabolism in rabbit intestine
C Macica1, M Balazy, J R Falck
1Department of Pharmacology, New York Medical College, Valhalla 10595.
Abstract:
We characterized cytochrome P-450-dependent arachidonate (P-450-AA) metabolism throughout the intestinal tract, since some metabolites derived via this pathway modify epithelial ion transport and regional blood flow. Microsomes (0.3 mg/ml) were prepared from each region of the intestines of anesthetized New Zealand White male rabbits and incubated with [14C]AA (7 microM) for 30 min at 37 degrees C. In the presence of NADPH (1 mM), ileal microsomes exhibited the greatest P-450-AA metabolism, whereas duodenal microsomes exhibited little or no activity. For jejunal, ileal, and cecal microsomes, AA metabolism was reduced in the absence of NADPH and by boiling microsomes, was unaffected by indomethacin (10 microM) and BW-755C (50 microM), but was significantly attenuated by the P-450 enzyme inhibitors, 7-ethoxyresorufin (1 microM) and SKF-525A (100 microM). However, colonic (ascending, transverse, and descending) microsomal activity was inhibited by both P-450 and lipoxygenase inhibitors. Analysis of ileal AA metabolites by high-pressure liquid chromatography and negative ion chemical ionization gas chromatography-mass spectrometry revealed products corresponding to monohydroxyeicosatetraenoic acids (HETEs). Semiquantitative analysis showed that 20-, 19-, 18-, 17-, and 16-HETEs were present in a ratio of 6.2:3.3:0.3:0.1:0.1, respectively. Furthermore, ileal P-450-HETEs dilated the isolated perfused mesenteric bed, as did 20-HETE, the predominant ileal AA metabolite. Because 20-HETE was also shown to affect epithelial ion transport, we suggest that P-450-AA metabolites may make important contributions to intestinal function.
Insights
Cytochrome P-450-dependent arachidonic acid (AA) metabolism is highest in the ileum, producing metabolites like 20-HETE that influence intestinal blood flow and ion transport.
Area of Science:
- Biochemistry
- Physiology
Background:
- Cytochrome P-450 (P-450) dependent metabolism of arachidonic acid (AA) produces bioactive compounds.
- These metabolites are known to influence epithelial ion transport and regional blood flow in the intestines.
Purpose of the Study:
- To characterize P-450-AA metabolism across different regions of the rabbit intestinal tract.
- To identify the specific metabolites produced and their potential physiological roles in intestinal function.
Main Methods:
- Microsomal preparations from various rabbit intestinal regions (duodenum, jejunum, ileum, colon) were incubated with [14C]AA.
- Metabolism was assessed by measuring product formation in the presence and absence of NADPH and specific inhibitors.
- Metabolite identification was performed using high-pressure liquid chromatography and gas chromatography-mass spectrometry.
Main Results:
- Ileal microsomes showed the highest P-450-AA metabolic activity, while duodenal activity was minimal.
- Metabolism was NADPH-dependent and inhibited by P-450 inhibitors but not by indomethacin or BW-755C in the jejunum, ileum, and cecum.
- Colonic activity was inhibited by both P-450 and lipoxygenase inhibitors.
- The predominant ileal metabolite identified was 20-hydroxyeicosatetraenoic acid (20-HETE).
- Ileal P-450-HETEs, including 20-HETE, caused vasodilation in the isolated perfused mesenteric bed and affected epithelial ion transport.
Conclusions:
- P-450-AA metabolism significantly contributes to intestinal function, particularly in the ileum.
- The metabolite 20-HETE plays a key role in regulating intestinal blood flow and ion transport.
- These findings highlight the importance of P-450-derived metabolites in maintaining intestinal homeostasis.