Cytochrome P450 4A expression and arachidonic acid omega-hydroxylation in the kidney of the spontaneously hypertensive rat.
20-Hydroxyeicosatetraenoic acid (20-HETE) is a major arachidonate metabolite in the kidney of the spontaneously hypertensive rat (SHR). The increase in its synthesis has been associated with the elevation of blood pressure in the SHR. The omega-hydroxylation of arachidonic acid is an activity associated with members of the CYP4A gene family which, in the rat, comprises three major isoforms: 4A1, 4A2 and 4A3. 20-HETE displays potent and diverse biological activities which can affect pro- and anti-hypertensive mechanisms dependent upon where, when and by which isoform it has been produced. Therefore, it is important to identify and characterize its biosynthetic system. We compared CYP4A mRNA and protein expression to patterns of 20-HETE synthesis in the SHR kidney. The reverse transcription/polymerase chain reaction (RT/PCR) technique was used to amplify CYP4A mRNA in microdissected nephron segments. Southern blot hybridization of PCR products obtained from nephron segments with the CYP4A1 cDNA probe demonstrated strong signals in S2 and S3 segments of the proximal tubule. Immunoblots of nephron segments using a polyclonal anti-rat liver CYP4A1 antibody which cross-reacts with CYP4A2 and CYP4A3, and 14C-arachidonic acid metabolism, confirmed that arachidonic acid omega-hydroxylation, i.e., 14C-20HETE formation, and CYP4A proteins were also localized mainly in the S2 and S3 segments. Correlation also existed between the age-dependent increase in arachidonate omega-hydroxylation in the kidney and CYP4A mRNA levels as measured by Northern hybridization of total RNA using the CYP4A1 cDNA probe. Immunoblot analysis revealed that at 7 weeks, where 20-HETE production is at its maximum, all three proteins are expressed. CYP4A3 and 4A1 immunoreactive proteins appeared at 3 weeks, showed maximum levels at 5 and 7 weeks, respectively, and gradually decreased to lower levels at 13 and 20 weeks, whereas CYP4A2 levels were undetectable at 3, 5 and 7 weeks but appeared at 13-20 weeks. Additional immunoblots indicated that renal cortical CYP4A1 protein levels were higher in SHR compared to Sprague-Dawley and Wistar-Kyoto rats. The increased levels of CYP4A1-immunoreactive band at 7 weeks corresponded to the maximal activity of arachidonate omega-hydroxylation. Thus, CYP4A1 might play a significant role in contributing to the increased cortical/proximal production of 20-HETE seen in 7-week-old SHR. However, given the high homology among members of the CYP4A gene family and the lack of specific tools to discern among these isoforms, additional studies have to be carried out to substantiate our findings.
Schwartzman
M L
ML
Department of Pharmacology, New York Medical College, Valhalla 10595, USA.
da Silva
J L
JL
Lin
F
F
Nishimura
M
M
Abraham
N G
NG
D000375:Aging / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D015152:Blotting, Northern; D015139:Blotting, Southern; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D006900:Hydroxylation; D006973:Hypertension / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D007668:Kidney / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D006899:Mixed Function Oxygenases / Q000096:biosynthesis*; D009399:Nephrons / Q000033:anatomy & histology / Q000201:enzymology / Q000648:ultrastructure; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D051381:Rats; D011918:Rats, Inbred SHR
D006893:Hydroxyeicosatetraenoic Acids; D015345:Oligonucleotide Probes; D016718:Arachidonic Acid; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A