Acti Ons of Epoxygenase M Eta Bolites O N the Preglomerular Vasculature1
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Acti ons of Epoxygenase M eta bolites o n the Preglomerular Vasculature1 John D. lmig,2 L.G. Navar, Richard J. Roman, K. Kishta Reddy, and John P. Falck methacin. In contrast, the vasoconstrictor response to J.D. Imig. L.G. Navar. Department of Physiology. Tu- 5,6-EET was abolished by both removal of the endo- lane University School of Medicine, New Orleans. Lou- thellum or cyclooxygenase inhibition. The thrombox- isiana ane/enderoperoxide receptor inhibitor, SQ 29,548, R.J. Roman. Department of Physiology. Medical Col- resulted in a 60% attenuation of the afferent arteriolar lege of Wisconsin, Milwaukee. Wisconsin vasconstriction to 5,6-EEl. These results indicate that the preglomerular vasoconstriction to 5,6-EEl is cyclo- K.K. Reddy. JR. Falck. Department of Molecular Ge- oxygenase dependent and requires an intact endo- netics University of Texas Southwestern Medical Center Dallas. Texas thelium, whereas the vasodilation to 1 1,12-EET is ste- reoselective and is the result of direct action of the (J. Am. Soc. Nephrol. 1996; 7:23o4-2370) epoxide on the preglomerular vascular smooth mus- cle. ABSTRACT Key Words: Epoxyeicosatrienoic acid. kidney. afferent arte- Epoxygenase metabolites of arachidonic acid are riole. endothellum. cyclooxygenase produced by the kidney and have been implicated in the control of renal blood flow. This study examined A rachidonic acid metabolites of the cytochrome the preglomerular actions of various epoxyeicosatrie- P450 pathway include epoxyeicosatnienoic acids noic acids (EET). By use of the in vitro blood-perfused (EET) and dihydroxyeicosatrienoic acids (DIHETE) juxtamedullary nephron preparation, interlobular formed via epoxygenase enzymes and hydnoxyeicosa- and afferent arteriolar diameter responses to 5,6-EEl, traenoic acids (HETE) formed via P450 w-hydroxybase 8,9-EET, 1 1,12-EEl, and 14,15-EEl were determined. Di- enzymes. The kidney produces all four regioisomenic EET (5,6-; 8,9-; 11,12-; 14,15-), corresponding DI- ameters of interlobular and afferent arterioles precon- HETE, HETE (16-, 17-, 18, 19-, 20-), and 2O-COOH stricted with 0.5 M norepinephrine averaged 24 ± 1 anachidonic acid ( 1-3). Stereoselective production of m (N = 27) and 17 ± 1 m (N = 32), respectively, at various regioisomenic EET (4,5) occurs in the kidney. a renal perfusion pressure of 100 mm Hg. Superfusion In the human kidney, the predominant epoxide with 0.0 1 to 100 nM 1 1 , 1 2-EEl caused graded in- formed Is 14, 15-EET and 66% of this regiolsomer is in creases in diameters of the interlobular and afferent the (R,S) configuration (5), whereas 14, 15(S,R)-EET is arterioles. At a dose of 100 nM, 1 1,12-EET increased the the predominant stereoisomer and 1 1 , 1 2(R,S)-EET is diameters of the interlobular and afferent arterioles by the major epoxide produced in the rat kidney (4). Alterations in the renal production of cytochnome 18 ± 2% (N = 10) and 20 ± 3% (N = 9), respectively. P450 metabobites occurs during the development of The vasodilatory response to 11, 12-EEl was stereose- hypertension ( 1 ,6), after increased dietary salt intake lective because 1 1, 12(R,S)-EET but not 1 1, 12(S,R)-EET (7,8), after uninephrectomy (9), and after induction of increased the diameters of the interlobular and affer- diabetes mellitus ( 1 0). EET have been reported to alter ent arterioles. 14,15-EEl had a much smaller effect renal vascular tone (3, 1 1) and tubular transport (12- and increased the diameters of the these vessels by 14). Endotheliab cells synthesize epoxygenase metab- 10%; 8,9-EET did not significantly affect vascular diam- obites from arachidonic acid (15-17) and these metab- eters. In contrast, 5,6-EEl constricted the interlobular olites have been reported to be vasodilators or and afferent arterioles by 16 ± 3% (N = 6) and 21 ±3% vasoconstrictors depending on the route of adminis- (N = 7), respectively. The corresponding diols, 5,6- tration (18). DIHETE and 1 1, 12-DIHETE, had no effect on diameters We have recently demonstrated that arachidonic of the interlobular and afferent arterioles at concen- acid decreased the diameter of the affenent arteriole without affecting the postgbomerular vasculature (19). trations up to 1 M. The vasodilatory response to Infusion of arachidonic acid in the presence of indo- 1 1 ,12-EET was not affected by removal of the endo- methacin resulted in vasodibation of the pregbomeru- thelium or by inhibition of cyclooxygenase with indo- bar vasculature that was attributed to an endotheliab- derived cytochrome P450 epoxygenase metabolite Received March 18. 1996. Accepted July 8, 1996. ( 1 9). Depending on the experimental conditions and 2 Correspondence to Dr. J.D. Imig, Department of Physiology, SL39, Tulane species studied, either renal vasoconstnictive (3,20) or University School of Medicine, 1430 Tulane Avenue. New Orleans, LA 70112. vasodibatory ( 1 1 , 2 1 ) responses to epoxygenase metab- 1046.6673/071 1-2364$03.00/0 olites have been observed. Infusion of 5,6-EET or Journal of the American Society of Nephrology Copyright C 1996 by the American Society of Nephrology 8,9-EET into the renal artery of the rat was reported to 2364 Volume 7 . Number 1 1 - 1996 Imig et al increase renal vascular resistance. The vasoconstric- reconstituted blood, and renal artery perfusion pressure tion observed may be related to the formation of a measured at the tip of the cannuba was set to 100 mm Hg. cycbooxygenase compound because 5,6-EET in- The organ chamber was warmed and the tissue surface was continuously superfused with a Tyrode’s solution containing creased renal blood flow during cyclooxygenase inhi- 1 % albumin at 37#{176}C. bition (3). In contrast, 5,6-EET, 8,9-EET, and 1 1, 12- Interbobular and afferent arteriobar diameters were mea- EET dilated the isolated perfused rabbit kidney sured via videomicroscopy techniques. The tissue was trans- preconstnicted with phenylephnlne, and the vasodiba- illuminated on the fixed stage of a Leitz Laborlux microscope tion to 5,6-EET was abolished by cycbooxygenase in- equipped with a 75-watt xenon lamp and a water immersion hibition (1 1). In the rat, 1 1,12(R,S)-EET has been objective (40 x ). Video images of the tissue under study were shown to dilate renal arteries preconstricted with generated by a Newvlcon camera (Model NC-67 M; Dage-MTI, phenybephnine and to activate Ca2-activated K Michigan City, IN), passed through a time date generator channels in vascular smooth muscle cells isolated (Model WV-810; Panasonic, Secaucus, NJ) and image en- from renal arteries (22). Collectively, these studies hancer (MFJ Enterprises, Starkville, MS), displayed on a monitor (Model WV-5410, Panasonic), and videotaped for suggest that renal blood flow is affected by epoxygen- later analysis (super VHS VCR, Panasonic). The final magni- ase metabolites; however, the intrarenal sites of action fication at the video monitor was X950 to X 1200. of these metabolites and their ability to exert direct effects on the pregbomerular vasculature have not Vascular Response to Epoxygenase been demonstrated directly. Accordingly, this study was undertaken to Investigate the renal pregbomerubar Metabolites actions of epoxygenase metabolites. Preglomerular After a 20-mm equilibration period, preglomerular inter- vascular responses to superfusion of all four regiolso- lobular and afferent arterioles were chosen for study. Mea- menic EET were observed by using the in vitro blood- surements of interbobubar and afferent arteriolar diameters perfused juxtamedublary nephron preparation. Ad- were made at beast 50 m from any branch points. Norepi- ventitial administration to the renal microvasculatune nephnine (0.5 M) was added to the perfusate to elevate basal permits investigation of the panacnine actions of ep- vascular tone. The diameters of interlobubar arterioles and afferent arterioles decreased from 29 ± 1 j.tm to 24 ± 1 m oxygenase metabolites. Cycbooxygenase, thrombox- and from 20 ± 1 m to 16 ± 1 m, respectively, after ane/enderoperoxide (TP) receptor, and endothelium noreplnephrine administration. After the diameter measure- dependency of the interlobulan and affenent arterioban ment in the presence of norepinephrine, cumulative concen- vasodilation to 1 1 , 1 2-EET and vasoconstriction to tration response curves during superfusion of either 5,6- 5,6-EET were also investigated. EET, 8,9-EET, 11,12-EET, or 14,15-EET (0.01 to 100 nM) were obtained. Superfusion directly over the adventitial side MATERIALS AND METHODS of pregbomerular vessels permits investigation of the para- Vascular Preparation crime actions of epoxygenase metabolites. Four regioisomeric EET were synthesized according to the method of Falck et al. Experiments were performed on male Sprague Dawley (25), and the stereoisomers were separated by chiral phase CD-VAF rats (Charles River Laboratories, Wilmington, MA) high-performance liquid chromatography (26) in the labora- weighing an average of 339 ± 5 g. All experiments were tory of Dr. Falck at the University of Texas Southwestern approved by the Tulane University Animal Care and Use Medical Center. Stock solutions of epoxygenase metabolites Committee. The rats were anesthetized with sodium pento- in ethanol were kept in sealed vials and stored under nitro- barbital (40 mg/kg body wt intraperitoneably lip)), the right gen at -80#{176}Cuntil the experiment. Immediately before use, carotid artery was cannulated, and a midline abdominal the stock solution was added to superfusion solutions and incision was made. The right renal artery of the kidney was the final concentration of ethanol vehicle was <0.05% (vol/ cannulated via the superior mesenteric artery and perfusion vob). A previous study has demonstrated that 5,6-EET is of the kidney was initiated immediately. The kidney was unstable and undergoes extensive conversion to 5,6-DIHETE perfused with a Tyrode’s solution containing 6% albumin and its corresponding 8-lactone (3).