Cat cerebral arterial smooth muscle cells express cytochrome P450 4A2 enzyme and produce the vasoconstrictor 20-HETE which enhances L-type Ca2+ current.
1. Cerebral arteries express cytochrome P450 4A enzymes (P450 4A) and produce 20- hydroxyeicosatetraenoic acid (20-HETE), a potent constrictor of pial arterioles. It is not known which cell type in the vessel wall is responsible for the formation of 20-HETE. We examined whether freshly isolated cerebral arterial muscle cells (VSMCs) express P450 4A and produce 20-HETE. We also studied the effect of 20-HETE on pressurized cerebral arteries and on whole-cell L-type Ca2+current (ICa) recorded in cat cerebral VSMCs. 2. Cat cerebral VSMCs incubated with [14C]arachidonic acid ([14C]AA) produced 20-HETE (3.9 +/- 1.1 pmol min-1 (mg protein)-1). 3. Reverse transcription-polymerase chain reaction studies revealed that cat cerebral VSMCs express mRNA for P450 4A which metabolizes AA to 20-HETE. Cloning and sequencing of the cDNA amplified from mRNA isolated from VSMCs showed > 96 % amino acid homology to the rat and human P450 4A2 and 4A3. 4. 20-HETE (1-300 nM) induced a concentration-dependent constriction of cat cerebral arteries, which was inhibited by nifedipine. 5. Addition of 10 and 100 nM 20-HETE to the bath increased peak ICa by 50 +/- 3 and 100 +/- 10 %, respectively. This effect was not influenced by altering the frequency of depolarization. 20-HETE (100 nM) failed to increase ICa in the presence of nifedipine. 6. These results demonstrate that cat cerebral VSMCs express P450 4A enzyme, and produce 20-HETE which activates L-type Ca2+ channel current to promote cerebral vasoconstriction.
Gebremedhin
D
D
Department of Physiology and The Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI and The Clement Zablocki Medical Center, Milwaukee, WI 53226, USA.
Lange
A R
AR
Narayanan
J
J
Aebly
M R
MR
Jacobs
E R
ER
Harder
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002415:Cats; D002536:Cerebral Arteries / Q000378:metabolism / Q000502:physiology*; D003001:Cloning, Molecular; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry; D017931:DNA Primers; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism* / Q000494:pharmacology*; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism / Q000502:physiology*; D009543:Nifedipine / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology*; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D017931:DNA Primers; D006893:Hydroxyeicosatetraenoic Acids; D012333:RNA, Messenger; D014662:Vasoconstrictor Agents; 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; D009543:Nifedipine