No abstract

The present study examined the effects of 20-hydroxyeicosatetraenoic acid (20-HETE) and 17-octadecynoic acid (17-ODYA), an inhibitor of the metabolism of arachidonic acid by P-450, on K(+)-channel activity in vascular smooth muscle cells (VSM) isolated from renal arterioles of the rat. Two types of K+ channels were characterized using inside-out excised membrane patches. One channel exhibited a large conductance (250.3 +/- 5 pS), was activated by membrane depolarization and elevations in cytoplasmic Ca2+ concentration, and was blocked by low concentrations (< 1 mM) of tetraethylammonium (TEA). The other K+ channel exhibited an intermediate conductance (46.3 +/- pS), was activated by membrane depolarization but not by changes in intracellular Ca2+ concentration, and was blocked by 4-aminopyridine (5 mM). Addition of 20-HETE to the bath (1-100 nM), reduced the frequency of opening of the large-conductance Ca(2+)-activated K+ channel recorded using cell-attached patches on VSM. It had no effect on the intermediate-conductance K+ channel: 17-ODYA (1 microM) increased the activity of the large-conductance Ca(2+)-activated K+ channel, and this effect was reversed by 20-HETE (10 nM). 20-HETE (1-1000 nM) reduced the diameter of isolated perfused small renal arteries of the rat by approximately 15% TEA (1 mM) blocked the vasoconstrictor response to 20-HETE (100 nM). These studies suggest that 20-HETE is an endogenously formed vasoconstrictor that acts in part by inhibiting the opening of the large-conductance Ca(2+)-activated K+ channel in renal arteriolar VSM.

Blood flow interactions with the vascular endothelium represent a specialized example of mechanical regulation of cell function that has important physiological and pathological cardiovascular consequences. The endothelial monolayer in vivo acts as a signal transduction interface for forces associated with flowing blood (hemodynamic forces) in the acute regulation of artery tone and chronic structural remodeling of arteries, including the pathology of atherosclerosis. Mechanisms related to spatial relationships at the cell surfaces and throughout the cell that influence flow-mediated endothelial mechanotransduction are discussed. In particular, flow-mediated ion channel activation and cytoskeletal dynamics are considered in relation to topographic analyses of the luminal and abluminal surfaces of living endothelial cells.

The present study examined the effects of 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) on the diameter of small renal arteries of the rat and assessed their action on K(+)-channel activity in vascular smooth muscle (VSM) cells isolated from these vessels. The R,S-isomer of 11,12-EET (1, 10, and 100 nM) increased the diameter of small renal arteries preconstricted with phenylephrine; however, the S,R-isomer was inactive. Both the R,S- and S,R-isomers of 14,15-EET had little effect on the diameter of these vessels even at a high concentration (100 nM). The vasodilator effect of 11(R),12(S)-EET was attenuated by tetraethylammonium (TEA, 1 mM) and iberiotoxin (100 nM), selective inhibitors of the large-conductance Ca(2+)-activated K+ (KCa) channel. In contrast, apamin (100 nM) and 4-aminopyridine (2 mM), which are inhibitors of other types of K+ channels, had no effect on the vasodilatory effect of 11,12-EET. In patch-clamp experiments, 100 nM racemic 11,12-EET increased outward K+ currents in VSM cells. Addition of the R,S-isomer or racemic 11,12-EET (1-100 nM), but not the S,R-isomer, increased the activity of KCa channel recorded from renal VSM cells with cell-attached patches. However, racemic EET had no effect on this channel when added to the internal (inside-out) or external (outside-out) face of excised membrane patches. These results suggest that 11,12-EET is a potent dilator of small renal arteries and that the R,S-isomer is the active enantiomer. The vasodilator effect of 11,12-EET appears to involve activation of KCa channel.

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. Cat cerebral VSMCs incubated with [14C]arachidonic acid ([14C]AA) produced 20‐HETE (3.9 ± 1.1 pmol min−1 (mg protein)−1). 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. 20‐HETE (1–300 nM) induced a concentration‐dependent constriction of cat cerebral arteries, which was inhibited by nifedipine. 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. 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.