Involvement of cytochromeP-450 enzyme activity in the control of microvascular permeability in canine lung

Abstract

Products of cytochrome P-450 enzymes may play a role in capacitative Ca2+ entry in endothelial cells, which can promote a rise in vascular permeability. Thapsigargin (150 nM) stimulated capacitative Ca2+ entry and increased the capillary filtration coefficient ( K f,c) in isolated normal canine lung lobes. Pretreatment of the lobes with cytochrome P-450 inhibitors clotrimazole (10 μM) or 17-octadecynoic acid (5 μM) abolished the thapsigargin-induced increases in K f,c. Because clotrimazole also blocks Ca2+-activated K+ channels, the K+-channel blocker tetraethylammonium (10 mM) was used to ensure that permeability was not influenced by this mechanism. Tetraethylammonium did not affect thapsigargin-induced permeability. The effects of the cytochrome P-450 arachidonic acid metabolite 5,6-epoxyeicosatrienoic acid (EET) were also investigated in lobes taken from control dogs and dogs with pacing-induced heart failure (paced at 245 beats/min for 4 wk). 5,6-EET (10 μM) significantly increased K f,c in lobes from the control but not from the paced animals. We conclude that cytochrome P-450 metabolites are involved in mediating microvascular permeability in normal canine lungs, but an absence of 5,6-EET after heart failure does not explain the resistance of lungs from these animals to permeability changes.

Role of EETs in regulation of endothelial permeability in rat lung

“…EETs have been shown to evoke hyperpolarization in EC via activation of Ca 2ϩ -dependent potassium channels, an effect that would enhance the electrochemical gradient for Ca 2ϩ entry (16). Although we have shown that potassium channel activation did not play a role in the canine lung (18), it remains possible that this is a critical step in rat lung EC. Potential targets that might plausibly be involved in the Ca 2ϩ -independent regulation of EC permeability by EETs include tyrosine kinases and the extracellular regulated protein kinases (ERK).…”

“…In the canine lung, we have shown that the store depletion-induced increase in lung EC permeability can be completely blocked by using 17-octadecynoic acid (17-ODYA) and clotrimazole to inhibit P-450 epoxygenases (18), an observation that supports the notion that EETs play an important role in regulation of EC permeability. However, there is no evidence at this point that this regulatory mechanism is an obligate participant in regulation of EC permeability regardless of the source or the injury paradigm.…”

“…The epoxygenase metabolites of AA are synthesized as a family of four regioisomers, including 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET (32). EETs have been proposed as mediators of acute lung injury in canine lung, although only the effect of 5,6-EET has been reported (18,38). We evaluated the effect of each EET regioisomer independently on K f,c in the isolated rat lung.…”

“…Because of the ability of Gd 3ϩ to bind to albumin (3), lungs used for these experiments were perfused throughout with 1% albumin-3% clinical grade dextran, a combination that does not alter K f,c in the lung (30 (1,13). Thus we tested whether TG-induced EC injury in rat lung was dependent on EET synthesis, as previously shown for canine lung (18). First, the effect of TG alone was documented (150 nM, n ϭ 12).…”

“…To block activation of PLA2, we utilized mepacrine (100 M, n ϭ 5), a nonspecific PLA2 inhibitor, or the more selective inhibitor methyl arachidonyl fluorophosphonate (MAFP, 2.5 M, n ϭ 4) (31,48). In separate studies, we tested whether AA metabolism via the P-450 pathway was involved, using the P-450 inhibitor 17-ODYA (5 M, n ϭ 8) or the specific epoxygenase inhibitor propargyloxyphenyl hexanoic acid (PPOH, 50 M, n ϭ 9) (18,46). To test whether EET metabolism by cyclooxygenase or epoxide hydrolase was a limiting factor, the cyclooxygenase inhibitor ibuprofen (30 M) and the epoxide hydrolase inhibitor dicyclohexylurea (DCU, 3 M) were added in combination (n ϭ 5) (7,50).…”