12-Hydroxyeicosatetraenoic acid participates in angiotensin II afferent arteriolar vasoconstriction by activating L-type calcium channels.

The lipoxygenase (LO) metabolite, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], constricts renal vessels, contributes to the vascular response to angiotensin, and has been implicated in cardiovascular and renal diseases. The current studies were performed to determine if renal microvascular 12(S)-HETE production is stimulated by angiotensin and the contribution of L-type calcium channels to the vasoconstriction elicited by 12(S)-HETE. Angiotensin increased renal microvascular 12(S)-HETE production by 64%, whereas cyclooxygenase metabolite production was not altered. Renal microvessels also expressed platelet-type 12-LO and leukocyte-type 12-LO. In the juxtamedullary preparation, afferent arteriolar diameter averaged 21 +/- 1 microm and 12(S)-HETE caused a graded decrease in vessel caliber. The afferent arteriolar response to 12(S)-HETE was abolished during L-type calcium channel inhibition. Renal microvascular smooth muscle cells were studied using fluorescence microscopy. Renal myocyte [Ca2+]i averaged 93 +/- 5 nmol/l. The 12(S)-HETE (5 micromol/l) increased myocyte [Ca2+]i to a peak value of 340 +/- 55 nmol/l. The peak [Ca2+]i response following exposure to 12(S)-HETE was greatly attenuated in the absence of extracellular Ca2+ or calcium channel blockade. These results demonstrate that renal microvascular 12(S)-HETE production is increased in response to angiotensin, and activation of L-type calcium channels is an important mechanism responsible for the afferent arteriolar vasoconstriction elicited by 12(S)-HETE.

The arachidonate lipoxygenase (LO) product, 12(S)hydroxyeicosatetraenoic acid (12(S)-HETE), has been implicated as a key contributor to the pathogenesis of ath-erosclerosis, hypertension, and diabetic nephropathy (1)(2)(3)(4). 12-LO expression has been detected in renal vascular and glomerular cells, and these cells have the capacity to produce 12(S)-HETE (5)(6)(7)(8). In addition to its involvement in glomerular inflammation and vascular smooth muscle cell growth, 12(S)-HETE is a renal vasoconstrictor (5,(8)(9)(10). 12(S)-HETE decreases renal blood flow and glomerular filtration independent of cyclooxygenase (COX) activity (9). Additionally, the action of angiotensin II on the renal vasculature involves participation of the LO pathway. A previous study by our laboratory demonstrated that the afferent arteriolar constriction to angiotensin II is attenuated in the presence of 12-LO inhibition (11). In contrast, norepinephrine-mediated renal microvascular vasoconstriction was unaffected by 12-LO inhibition (11). One aim of the current study was to determine renal microvascular production of COX metabolites and 12(S)-HETE in response to angiotensin II and norepinephrine.
Although the importance of 12(S)-HETE in renal and cardiovascular disease is now established, the mechanism by which 12(S)-HETE constricts the preglomerular vasculature remains to be identified. It is known that 12(S)-HETE causes depolarization of the vascular smooth muscle cell membrane and may act through activation of protein kinase C to constrict blood vessels (9). Depolarization of vascular smooth muscle should activate L-type calcium channels. Therefore, a second aim of the present study was to determine the contribution of L-type calcium channel activation to the 12(S)-HETE mediated renal vasoconstriction. exposed to permit cannulation of the abdominal aorta via the superior mesenteric artery. Ligatures were placed around the abdominal aorta at sites proximal and distal to the left and right renal arteries, respectively. The kidneys were cleared of blood by perfusion of the isolated aortic segment with an ice-cold, low-calcium physiological salt solution (PSS; pH 7.35) of the following composition (in mmol/l): 125 NaCl, 5.0 KCl, 1.0 MgCl 2 , 10.0 glucose, 20.0 HEPES ( N -2-hydroxyethylpiperazine-N Ј -2-ethanesulfonic acid), 0.1 CaCl 2 , and 6% BSA. The kidneys were resected and decapsulated, and the renal medullary tissue was removed. Cortical tissue was pressed through a sieve (180 m mesh), and the sieve retentate was washed repeatedly with ice-cold low-calcium PSS. Renal microvessels were incubated (37 Њ C) with gentle agitation for 1-1.5 h in a Tyrode's solution containing 0.2 mg/ml each of dithiothreitol, collagenase, trypsin inhibitor, and albumin. Microvessels were transferred to a 100 m nylon mesh and rinsed. Purified microvessels were examined under a stereomicroscope, and short segments were collected for subsequent use. Purity of the collected microvascular sample was assessed by alkaline phosphatase activity measurement as previously described (12).
We confirmed the presence of L-12-LO protein expression in renal microvessels with a commercially available antibody. Renal microvessels and kidney cortex were harvested and processed as previously described (12). Samples were separated by electrophoresis on a 10% stacking Tris-glycine gel, and proteins were transferred electrophoretically to a nitrocellulose membrane. The primary antibody used was rabbit anti-murine L-12-LO poly-clonal antibody (1:1,000; Cayman Chemical Co.). The blots were then washed in a PBS-0.1% Tween 20 solution and incubated with the secondary antibody (anti-rabbit 1:100,000), conjugated to horseradish peroxidase for 1 h at room temperature, and washed. Detection was accomplished using enhanced chemiluminescence Western blotting (Amersham Corp.), and blots were exposed to X-ray film (Hyperfilm-ECL, Amersham Corp.).

Afferent arteriolar response to 12(S)-HETE
Experiments were performed on male Sprague Dawley rats (Charles River Laboratories, Wilmington, MA) weighing an average of 350-450 g. Tulane University and Medical College of Georgia Animal Care and Use Committees approved the experimental procedures. The rats were anesthetized with sodium pentobarbital (40 mg/kg body weight ip), the right carotid artery was cannulated, and a midline abdominal incision was made. The right renal artery of the kidney was cannulated via the superior mesenteric artery, and the kidney was immediately perfused with a Tyrode's solution containing 6% albumin and a mixture of L-amino acids (11).
Blood was collected through the carotid artery cannula into a heparinized syringe (2,000 U). Erythrocytes were separated from plasma and leukocytes by centrifugation and were reconstituted in Tyrode's solution containing 6% albumin to yield a hematocrit of 20%. The reconstituted red blood cell containing solution was filtered and stirred continuously in a closed reservoir that was pressurized by a 95% O 2 -5% CO 2 tank. The kidney was removed and maintained in an organ chamber at room temperature throughout the isolation and dissection procedure. The juxtamedullary microvasculature was isolated for study as previously described (11). The Tyrode's solution was replaced by reconstituted blood, and renal artery perfusion pressure was set to 100 mmHg. The organ chamber was warmed and the tissue surface was continuously superfused with a Tyrode's solution containing 1% albumin at 37 Њ C. Following a 20 min equilibration period, an afferent arteriole was chosen for study, and baseline diameter was measured using video-microscopy techniques as previously described (11). Afferent arteriolar inside diameters were measured at 15 s intervals using a digital image-shearing monitor. The image-shearing device is accurate to within 0.2% of the screen width or 0.2 m and measurement reproducibility is within 0.5 m.
Following the equilibration period, baseline diameter measurements of the afferent arteriole were made. The arteriole was subsequently exposed to increasing concentrations of 12(S)-HETE, 12(R)-HETE, and 15(S)-HETE (0.01-5 mol/l), and diameter changes were monitored for 3 min at each concentration. The afferent arteriolar diameter 12(S)-HETE response in the presence of diltiazem was determined to assess the involvement of L-type calcium channels. The kidney was exposed to 10 mol/l diltiazem for a minimum of 20 min, and the afferent arteriole diameter response to 12(S)-HETE (5 mol/l) was assessed. Steady-state diameter was attained by the end of the second minute, and the average diameter of the third minute of each treatment period was utilized for statistical analysis.

Signaling mechanisms involved in the renal microvascular smooth muscle cell calcium response to 12(S)-HETE
rum, 100 U/ml penicillin, and 200 g/ml streptomycin. Renal microvascular cell suspensions were stored on ice until use.
Experiments were performed using a standard microscopebased fluorescence spectrophotometry system. The excitation wavelengths were set at 340 nm and 380 nm, and the emitted light was collected at 510 Ϯ 10 nm. Measurements of fluorescence intensity were collected at five data points per second, and the data were collected and analyzed with the aid of Photon Technology International software. Calibration of the fluorescence data was accomplished as previously described (14).
Measurement of [Ca 2 ϩ ] i in single microvascular smooth muscle cells was performed as previously described (14). Suspensions of freshly isolated renal microvascular cells loaded with the calcium sensitive fluorescent probe, fura 2 acetoxymethyl ester (4.0 mol/l). An aliquot of cell suspension was transferred to the perfusion chamber and mounted to the stage of a Nikon Diaphot inverted microscope. The cells were continuously superfused (1. Studies were performed to determine the role of extracellular calcium on the increase in [Ca 2 ϩ ] i induced by 12(S)-HETE. The contribution of calcium influx to the response was determined by exposing single cells to 12(S)-HETE (5 mol/l) while being bathed in nominally calcium-free PSS. Previous studies have shown that [Ca 2 ϩ ] i remains unchanged when preglomerular smooth muscle cells are subjected to strong depolarizing conditions while being bathed in nominally calcium-free conditions. These responses were compared with responses obtained from similar cells challenged in normal-calcium PSS. The role of L-type calcium channels in 12(S)-HETE-mediated calcium responses was assessed with 10 mol/l diltiazem. We have previously reported that diltiazem prevented the depolarization-induced increase in preglomerular smooth muscle [Ca 2 ϩ ] i in response to 90 mmol/l KCl (14). Addition of vehicle to the superfusate had no effect on the [Ca 2 ϩ ] i response elicited by 12(S)-HETE (n ϭ 10 cells from three dispersions).

Statistics
Data are presented as mean Ϯ SEM. The significance of differences in renal microvessel COX metabolite and 12(S)-HETE production was evaluated using an unpaired t -test. Differences in mean afferent arteriolar diameters between groups were evaluated with a two-way ANOVA for repeated measures followed by Duncan's multiple range test. Differences within and between groups of renal microvascular smooth muscle cell [Ca 2 ϩ ] i values were analyzed by

Renal microvessel COX and 12(S)-HETE production in response to angiotensin II
We have previously reported that the 12-LO inhibitor, baicalein, attenuated the afferent arteriolar constriction to angiotensin II but did not alter the vasoconstrictor response to norepinephrine (11). In the current study, we provide additional biochemical evidence for the involvement of 12(S)-HETE in the renal microvascular response to angiotensin II. As depicted in Fig. 1 , angiotensin II significantly increased renal microvessel 12(S)-HETE gener-ation by 64%. In contrast, 12(S)-HETE production by renal microvessels was unaltered when incubated with norepinephrine. Production of the COX metabolites TXB 2 , 6-keto-PGF 1 ␣ , or PGE 2 was not changed when renal microvessels were incubated with angiotensin II or norepinephrine. These results provide support for the postulate that 12(S)-HETE is an important mediator of angiotensin II actions on the renal microvasculature. Another set of experiments was conducted to begin identifying the cell signaling pathways influenced by 12(S)-HETE.

Renal microvascular expression of 12-LO and 15-LO
The identity of the LO localized to the renal microvasculature is not known. We demonstrate the presence of at least three LO enzymes in renal microvessels. As depicted in Fig. 2A , platelet and leukocyte 12-LO, as well as type 2 15-LO mRNA expression, was detected in the renal cortex and microvessels. Next, we determined the presence of L-12-LO with a commercially available antibody to confirm that mRNA expression translated to protein expression. L-12-LO protein expression was detected in four renal cortical and renal microvascular samples (Fig. 2B). Thus, the renal microvasculature is capable of metabolizing arachidonic acid to 12-HETE and 15-HETE.  Downloaded from cular constriction. During calcium channel blockade with diltiazem, the afferent arteriolar vasoconstrictor response to 12(S)-HETE was significantly (P Ͻ 0.05) attenuated. Afferent arteriolar diameter averaged 22.2 Ϯ 1.8 m and decreased by 1 Ϯ 1% in response to 12(S)-HETE (Fig. 4).

Renal microvascular smooth muscle cell calcium response to 12(S)-HETE
A total of 125 single renal microvascular smooth muscle cells prepared from 22 tissue dispersions were examined in the present study. The baseline [Ca 2ϩ ] i in cells treated with 12(S)-HETE averaged 93 Ϯ 5 nmol/l (n ϭ 125), and no significant difference was evident in resting [Ca 2ϩ ] i between any of the treatment groups.
The first series of experiments determined the effects of 12(S)-HETE on [Ca 2ϩ ] i in freshly isolated rat renal microvascular smooth muscle cells. We evaluated the role of Ca 2ϩ influx in the 12(S)-HETE increase in [Ca 2ϩ ] i in single renal microvascular smooth muscle cells. Renal microvascular smooth muscle cells were exposed to a solution containing 5 mol/l 12(S)-HETE while they were being bathed in Ca 2ϩ -free solution. As shown in representative traces, the 12(S)-HETE-mediated increase in [Ca 2ϩ ] i was markedly attenuated in cells bathed in Ca 2ϩ -free medium (Figs. 6, 8). Resting [Ca 2ϩ ] i averaged 98 Ϯ 19 nmol/l in the absence of extracellular Ca 2ϩ and increased to 179 Ϯ 69 nmol/l after the administration of 12(S)-HETE. The steady-state response to 5 mol/l 12(S)-HETE was also noticeably reduced when cells were bathed in Ca 2ϩ -free solution and averaged 9 Ϯ 7 nmol/l. Next, we determined the effect of calcium channel blockade with diltiazem on the 12(S)-HETE-mediated increase in [Ca 2ϩ ] i . As depicted in Figs. 7 and 8

DISCUSSION
The importance of the 12-LO metabolite, 12(S)-HETE, as a significant component in cardiovascular and kidney pathologies has been well established (1)(2)(3)(4)(5)15). Increased cultured vascular smooth muscle and endothelial cell 12(S)-HETE production is elevated by high glucose (2,16). The 12-LO pathway or 12(S)-HETE levels are elevated in a diabetic pig model, ischemic preconditioned rat hearts, the spontaneously hypertensive rat (SHR), and patients with essential hypertension (1)(2)(3)17). The increased 12(S)-HETE levels in hypertension and diabetes appear to play a key role in mediating the smooth muscle cell hypertrophy, and atherosclerotic and vascular constrictor actions of angiotensin II (3,8,(18)(19)(20)(21). In the kidney, 12(S)-HETE decreases renal blood flow, constricts renal vessels, and is involved in the afferent arteriolar response to angiotensin II (5,9,11). Although the renal vascular actions of 12(S)-HETE have been long recognized, the intercellular signaling mechanisms responsible for 12(S)-HETE activity remain poorly defined. The current study provides further First, we evaluated renal microvessel COX metabolite and 12(S)-HETE production in response to angiotensin II or norepinephrine. The main finding of this set of experiments was that angiotensin II but not norepinephrine increased renal microvascular 12(S)-HETE generation. In addition, we determined the LO enzymes that could be responsible for renal microvascular 12(S)-HETE production. Renal microvessels contained platelet-and leukocyte-type 12-LO, as well as type 2 15-LO. The finding of L-12-LO mRNA and protein expression in renal microves-sels is consistent with previous findings in the porcine aorta and coronary arteries (2,22). 12(S)-HETE generation could also possibly be due to the presence of P-12-LO, which has been shown to be an endothelial arachidonate 12-LO (23). Interestingly, we detected mRNA expression of 15-LO2 in renal cortical and microvascular tissue. This finding is in contrast to the first description of this enzyme that was unable to detect 15-LO2 in the human kidney (24). 15-LO2 converts arachidonic acid to 15(S)-HETE but is a poor metabolizer of linoleic acid (24). This suggested that, in addition to 12(S)-HETE, other arachidonate LO metabolites could contribute to angiotensin II-mediated renal vasoconstriction.
We compared the effects of 12(S)-HETE, 12(R)-HETE,   (4,18,21,23). In contrast, 12(R)-HETE is a more active constrictor of small canine renal arteries (9). The reason for the differences may depend on the species studied, route of administration, subsequent metabolism of the parent compound, or size of the vessel studied. Nevertheless, the vasoconstrictor actions of 12(S)-HETE do appear to require a slightly higher dose when compared with its angiogenic properties (4,18,23,25). A dose of 0.1 M 12(S)-HETE is near maximal for the vascular growth activities, and a dose between 1 M and 5 M 12(S)-HETE was required for maximal actions on the afferent arteriolar diameter. Overall, the findings of the present study support the concept that 12(S)-HETE is the primary LO metabolite involved in the control of renal vascular resistance. These findings, combined with the previous observation that inhibition of the 12-LO pathway attenuates the afferent arteriolar response to angiotensin II, provide convincing evidence that 12(S)-HETE is importantly involved in the renal vascular actions of angiotensin II. There are a number of studies that provide corroborating evidence for the view that 12(S)-HETE is a mediator for the vascular intracellular actions of angiotensin II (3,8,(18)(19)(20)(21). The involvement of 12(S)-HETE in the renal vasoconstrictor response to angiotensin II has been demonstrated in several studies (11,21,26). Bell-Quilley et al. (26) demonstrated that in the isolated perfused rat kidney, inhibition of the 12-LO pathway attenuated the angiotensin II-mediated decrease in renal blood flow. In contrast, LO inhibition attenuated the renal arcuate artery vasoconstrictor response to norepinephrine and KCl but had no effect on the vascular response to ET-1 (27). Attenuation of the vasoconstrictor response to angiotensin II but not norepinephrine has also been demonstrated for the aorta and large arteries of the skeletal muscle and pulmonary vasculatures (19)(20)(21)(28)(29)(30). 12(S)-HETE also directly potentiates angiotensin II-mediated contraction of hamster aorta and SHR aorta (3,31). The concentration of 12(S)-HETE required to potentiate angiotensin-mediated vascular contraction appears to be lower than that required to directly constrict the afferent arteriole (31). Enhanced vascular actions of angiotensin II in the presence of 12(S)-HETE appear to involve increased intracellular calcium signaling in the SHR (3). Even though a link between angiotensin II and 12-(S)-HETE is well established, the mechanism by which 12(S)-HETE contracts the vasculature remains unknown.
Since renal vascular responses to angiotensin II are reported to involve activation of calcium influx pathways (32)(33)(34) and 12(S)-HETE depolarizes renal vessels isolated from dogs (9), we postulated that the afferent arteriolar constriction elicited by 12(S)-HETE involves activation of L-type calcium channels. Afferent arteriolar diameter decreased in response to 12(S)-HETE in a dose-  Downloaded from dependent manner. The L-type calcium channel inhibitor, diltiazem, abolished the afferent arteriolar constriction elicited by 12(S)-HETE. Diltiazem was administered at a dose that completely blocks KCl-mediated afferent arteriolar constriction (35). These initial functional studies suggest that L-type calcium channel activation contributes to the 12(S)-HETE-mediated constrictor response. These results are in agreement with studies in renal canine arcuate arteries that demonstrate vascular smooth muscle cell depolarization (9). In contrast, recent studies in the coronary and cerebral vasculatures have provided evidence that 12(S)-HETE is a vasodilator and acts by activating large-conductance Ca 2ϩ -activated K ϩ channels (17,(36)(37)(38). The reason for this apparent discrepancy is not known, but studies performed in renal myocytes demonstrate that 12(S)-HETE increased intracellular calcium levels to a degree consistent with the observed afferent arteriolar constriction.
We performed additional studies to determine the relative contributions of agonist-induced calcium mobilization from intracellular stores and calcium influx to the 12(S)-HETE increases in renal myocyte intracellular calcium. In the studies presented here, the contribution of voltage-dependent L-type calcium channels is demonstrated by the pronounced attenuation of the 12(S)-HETE-induced increase in intracellular calcium by 84%. This finding suggests that calcium influx through L-type calcium channels accounts for a majority of the increase in cell calcium observed during exposure to 12(S)-HETE. We also considered the relative contribution of agonistinduced mobilization from intracellular stores to the 12(S)-HETE-induced increases in intracellular calcium in renal myocytes. The 12(S)-HETE-mediated increase in intracellular calcium was markedly attenuated in cells bathed in nominally calcium-free medium. Interestingly, the response obtained in the absence of extracellular calcium closely resembles the response obtained during blockade of L-type calcium channels. These findings establish that 12(S)-HETE elevates intracellular calcium through two different mechanisms. The renal myocyte response to 12(S)-HETE involves a small intracellular calcium mobilization component, and a large portion involves the influx of extracellular calcium via L-type calcium channels.
Interestingly, the cell signaling pathways that contribute to the 12(S)-HETE-mediated afferent arteriolar constriction are the same pathways utilized by angiotensin II (32-34, 39, 40). The afferent arteriolar response to angiotensin II is largely dependent on influx of calcium through L-type calcium channels, and mobilization of intracellular calcium contributes to a smaller extent (32-34, 39, 40). These parallels between angiotensin II and 12(S)-HETE can be likened to the hypertrophic and matrix protein production actions in vascular smooth muscle cells. Although the angiotensin II cell-signaling pathways for growth and matrix production are different than the pathways responsible for vascular constriction, the fact that 12(S)-HETE appears to mediate the vascular growth and constrictor actions of angiotensin II is worthy of further discussion. A number of studies have provided evidence that 12(S)-HETE activation of the Ras-MAPK pathway to the vascular smooth muscle growth and matrix gene expression mediates the hypertrophic actions of angiotensin II (4,8). These actions of 12(S)-HETE are consistent with the postulate that angiotensin II activation of the 12-LO pathway plays a key role in atherosclerosis in diabetes and other renal and cardiovascular diseases. 12(S)-HETE is elevated in hypertension and contributes to the pressor actions of angiotensin II (1,3,29). Consistent with this possibility, reports have demonstrated that 12(S)-HETE contributes to the angiotensin II-induced vascular constrictor response (19)(20)(21)(27)(28)(29)(30). In this regard, 12(S)-HETE has been postulated to be a mediator for the intracellular actions of angiotensin II (3,18); however, the cell-signaling mechanisms by which 12(S)-HETE increased intracellular calcium were not investigated. In the current report we provide evidence that 12(S)-HETE constricts the afferent arteriole by mobilizing small amounts of intracellular calcium and causing a large influx of extracellular calcium through L-type calcium channels.
In summary, we observed an increase in renal microvessel 12(S)-HETE production in response to angiotensin II but not norepinephrine. This finding is in agreement with our previous report that the afferent arteriolar constriction in response to angiotensin II but not norepinephrine involves activation of the 12-LO pathway. We also determined that the 12(S)-HETE-induced afferent arteriolar constrictor response was dependent on activation of L-type calcium channels. Next, we evaluated renal myocyte intracellular calcium regulation in response to 12(S)-HETE. 12(S)-HETE increased intracellular calcium primarily through activation of L-type calcium channels. Taken together, these findings support the postulate that 12(S)-HETE is part of the cell-signaling pathway responsible for the angiotensin II-mediated activation of L-type calcium channels and afferent arteriolar vasoconstriction.