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PLA₂ and TRPV4 channels regulate endothelial calcium in cerebral arteries

Departments of ¹Anesthesiology, ²Molecular Physiology and Biophysics, and ³Graduate Program in Cardiovascular Sciences, Baylor College of Medicine, Houston; and ⁴Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, Texas

Submitted 13 September 2006 ; accepted in final form 22 October 2006

	ABSTRACT

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We previously demonstrated that endothelium-derived hyperpolarizing factor (EDHF)-mediated dilations in cerebral arteries are significantly reduced by inhibitors of PLA₂. In this study we examined possible mechanisms by which PLA₂ regulates endothelium-dependent dilation, specifically whether PLA₂ is involved in endothelial Ca²⁺ regulation through stimulation of TRPV4 channels. Studies were carried out with middle cerebral arteries (MCA) or freshly isolated MCA endothelial cells (EC) of male Long-Evans rats. Nitro-L-arginine methyl ester (L-NAME) and indomethacin were present throughout. In pressurized MCA, luminally delivered UTP produced increased EC intracellular Ca²⁺ concentration ([Ca²⁺]_i) and MCA dilation. Incubation with PACOCF₃, a PLA₂ inhibitor, significantly reduced both EC [Ca²⁺]_i and dilation responses to UTP. EC [Ca²⁺]_i was also partially reduced by a transient receptor potential vanilloid (TRPV) channel blocker, ruthenium red. Manganese quenching experiments demonstrated Ca²⁺ influx across the luminal and abluminal face of the endothelium in response to UTP. Interestingly, PLA₂-sensitive Ca²⁺ influx occurred primarily across the abluminal face. Luminal application of arachidonic acid, the primary product of PLA₂ and a demonstrated activator of certain TRPV channels, increased both EC [Ca²⁺]_i and MCA diameter. TRPV4 mRNA and protein was demonstrated in the endothelium by RT-PCR and immunofluorescence, respectively. Finally, application of 4-phorbol 12,13-didecanoate (4PDD), a TRPV4 channel activator, produced an increase in EC [Ca²⁺]_i that was significantly reduced in the presence of ruthenium red. We conclude that PLA₂ is involved in EC Ca²⁺ regulation through its regulation of TRPV4 channels. Furthermore, the PLA₂-sensitive component of Ca²⁺ influx may be polarized to the abluminal face of the endothelium.

fura 2; manganese quenching; transient receptor potential; endothelium-derived hyperpolarizing factor; phospholipase A₂; brain

In addition, our group recently demonstrated a critical role for cytoplasmic phospholipase A₂ (PLA₂) in EDHF-mediated dilation in rat MCA (33). In that study, we found that inhibitors of cytoplasmic PLA₂ (cPLA₂) significantly reduced EDHF-mediated dilations to the purinergic receptor agonist uridine triphosphate (UTP). The specific role of PLA₂ in EDHF-mediated dilation, however, was not determined.

The purpose of the present study was to examine the mechanism by which PLA₂ contributes to EDHF-mediated dilation. We evaluated two hypotheses regarding endothelial Ca²⁺ regulation and EDHF-mediated dilation. Our first hypothesis states that PLA₂ is critical for normal endothelial Ca²⁺ regulation following UTP stimulation. In our proposed model, the critical role of PLA₂ activation would be to promote the sustained or plateau component of endothelial [Ca²⁺]_i to sufficient levels for IK_Ca activation (17). To examine the mechanism of Ca²⁺ regulation further, we examined the role of transient receptor potential (TRP) channels in mediating Ca²⁺ influx in MCA endothelium. Our second hypothesis states that TRPV4 channels contribute to endothelial Ca²⁺ influx and are regulated by products generated subsequent to activation of PLA₂. For these studies we measured endothelial [Ca²⁺]_i and Ca²⁺ influx in pressurized MCA, [Ca²⁺]_i in isolated endothelial cells, and message for candidate TRP channels by cell-specific PCR.

	METHODS

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Isolated/pressurized artery preparation. Rat MCA were harvested and mounted in a pressurized vessel chamber as described previously (14, 17). MCA were pressurized to a mean transmural pressure of 85 mmHg, and flow was established through the lumen of the artery (100 µl/min) with PSS of the following composition (in mM): 119 NaCl, 4.7 KCl, 21 NaHCO₃, 1.18 KH₂PO₄, 1.17 MgSO₄, 0.026 EDTA, 5.5 glucose, and 1.6 CaCl₂. PSS was warmed (37°C) and gassed with 5% CO₂-20% O₂-75% N₂ to maintain a pH of 7.4. Nitro-L-arginine methyl ester (L-NAME) and indomethacin (10 µM each) were included in all experiments to inhibit NO synthase (NOS) and COX, respectively (18). The vessel chamber was custom-manufactured for performing fluorescence measurements in pressurized arteries (ChuelTech, Houston, TX).

In this preparation, compounds could be delivered via the abluminal or luminal perfusate to target them to the smooth muscle side or luminal endothelial side, respectively. Abluminal perfusion delivers compounds to the smooth muscle cells as well as the basolateral face of the endothelium. Luminal perfusion provides for delivery of compounds to the apical face of the endothelium. Because of the tight junctions between the endothelial cells of the MCA, hydrophilic compounds are prevented from passing across the endothelial layer (16).

Endothelial calcium measurements. [Ca²⁺]_i was selectively measured in the endothelium of pressurized arteries with fura-2 as described previously (14, 17). In brief, fura-2 AM was delivered through the lumen of the artery for a short period of time to selectively load the endothelium. For the isolated endothelial cell studies, cells were allowed to settle on a glass coverslip before incubation in the perfusion chamber with fura-2 AM (0.35 µM) plus Pluronic F-127 (0.01%) for 12–15 min. Cells were then washed with fresh PSS for 15 min to remove excess fura-2 AM and to allow complete deesterification of the dye. The fura-2 emission (520/40 nm) was measured from three excitation wavelengths (340/15, 360/15, and 380/15 nm) at a frequency of 4 Hz. The 360-nm excitation wavelength reflects the Ca²⁺-insensitive isosbestic point for fura-2. The autofluorescence/background was subtracted before the ratios were calculated. In the isolated cells, we calculated the ratio of 360/380 emissions instead of the 340/380 ratio as in intact arteries because of the lower intensity of the 340-nm emission in the cell preparation. Fura-2 loading and experiments with isolated cells took place at room temperature in PSS of the following composition (in mM): 137 NaCl, 5.6 KCl, 1.6 CaCl₂, 1 MgCl₂, 10 glucose, and 10 HEPES, with pH adjusted to 7.4 with NaOH.

Manganese quenching measurements. Manganese (Mn²⁺) quenching is a technique that is used to evaluate Ca²⁺ influx into fura-2-loaded cells (9, 23). Mn²⁺ acts as a Ca²⁺ surrogate or tracer and is used in the absence or presence of Ca²⁺. Mn²⁺ differs from Ca²⁺ in that its binding to fura-2 results in a quenching of fura-2 fluorescence. Since the intracellular concentration of Mn²⁺ is negligible, the decay of fura-2 fluorescence in the presence of added extracellular Mn²⁺ reflects Mn²⁺ (and Ca²⁺) entry. The initial rate of quench was used as an index of Ca²⁺ entry.

We administered MnCl₂ either luminally or abluminally to evaluate endothelial Ca²⁺ influx across the apical and basolateral membrane surface, respectively. The two membrane surfaces are separated by tight junctions that form a barrier between luminal and abluminal compartments (16). Fura-2 loading was restricted to the endothelium (14); therefore, the measured fluorescence quenching is reflective of endothelial Mn²⁺ influx. For luminal Mn²⁺ delivery, 300 µM MnCl₂ was added to the luminal perfusate and delivered simultaneously with UTP. For abluminal Mn²⁺ delivery, 150 µM MnCl₂ was administered to the abluminal perfusate 2 min before UTP delivery. This time lag was used to allow the Mn²⁺ sufficient time to reach the basolateral surface of the endothelium before UTP reached the luminal surface. The lower concentration of MnCl₂ was used so that quenching due to basal Mn²⁺/Ca²⁺ influx would not dominate the measurement.

Cell digestion protocol. Rat MCA (left and right) were removed from the brain, cleaned of connective tissue, and cut into small rings. Our digestion protocol was modified from a protocol initially established by Jackson et al. (10). The artery segments were incubated for 10 min in a wash buffer solution containing (in mM) 137 NaCl, 5.6 KCl, 0.1 CaCl₂, 1 MgCl₂, 10 glucose, 10 HEPES, 0.005 verapamil, and 0.1% BSA, with pH adjusted to 7.35 with NaOH. The wash solution was carefully removed and replaced with digestion buffer containing papain (26 U/ml) plus dithioerythritol (1 mg/ml) and incubated for 35 min at 37°C. At the end of the incubation period, the papain solution was carefully removed and the vessel segments were washed with the wash buffer solution. The solution was then replaced with a second digestion solution containing collagenase (Sigma blend; 1.4 mg/ml), elastase (43 U/ml), and soybean trypsin inhibitor (0.9 mg/ml) and incubated for 8 min at 37°C. The artery segments were then washed twice with wash buffer and triturated with a P-200 pipette tip to disperse smooth muscle cells and small endothelial cell clusters. Endothelial cells were found in multi-cell planar sheets among individual smooth muscle cells. The digestion buffer consisted of (in mM) 135 NaCl, 5 KCl, 1.5 MgCl₂, 0.42 Na₂HPO₄, 0.44 NaH₂PO₄, 4.17 NaHCO₃, and 10 HEPES, with pH adjusted to 7.25 with NaOH. The papain, dithioerythritol, and collagenase were purchased from Sigma; elastase was purchased from Calbiochem; and Soybean trypsin inhibitor was purchased from Worthington.

RT-PCR. RT-PCR with freshly isolated endothelial and smooth muscle cells was based on the protocol described by Kohler et al. (11) In brief, endothelial or smooth muscle cells were harvested into a new glass pipette (50-µm tip opening) that had the tip prefilled with 1 µl of RNAlater solution. Smooth muscle cells were identified on the basis of their classic cigar shape morphology. Endothelial cells were harvested as multi-cell planar sheets containing 5–50 cells. Harvested cells were expelled into a PCR tube and frozen in liquid N₂. The following components of the first-strand synthesis kit (Invitrogen) were then added to each tube: 5x first-strand buffer (2 µl), 10 mM dNTP (0.5 µl), 50 µM random hexamer (1 µl), 0.1 mM DTT (1 µl), 10 U/µl RNase inhibitor (1 µl), and diethyl pyrocarbonate H₂O (1.5 µl). Two rapid freeze-thaw cycles were performed to rupture cells and gain access to the RNA before the addition of 1 µl of Superscript II reverse transcriptase. Samples were incubated at 37°C for 1 h and then stored at –20°C for later use.

Our initial assignment of cell type was based on morphological characteristics. This cell assignment was confirmed by a PCR screening of cell-specific products. The final assignment of endothelial cells required that the cell preparation demonstrate PCR amplicons for endothelial NOS (eNOS) but not for SM22 (and vice versa for smooth muscle cells). Only cell preparations that demonstrated no contamination from the other cell type were used.

PCR was performed for 35–50 cycles using the following temperature protocol: 94°C (30 s), 53–55°C (60 s), and 72°C (30 s), with an initial step at 94°C (2 min) to activate the platinum Taq polymerase (Invitrogen). Gene-specific primers were as follows: ATGGATGAGCCAACTCAAGG (forward) and CCAGCTCTGTCCTCAGAAGG (reverse) for eNOS; GGCAGCTGAGGATTATGGAG (forward) and GCTGGCCTTCCCTTTCTAAC (reverse) for SM22; TGACTACCGGTGGTGTTTCA (forward) and TGATCCCTGCATAGTGTCCA (reverse) for TRPV1; and ATCAACTCGCCCTTCAGAGA (forward) and GGTGTTCTCTCGGGTGTTGT (reverse) for TRPV4. The predicted size of amplicons were 356, 325, 330, and 339 bp for eNOS, SM22, TRPV1, and TRPV4, respectively. Primers for TRPV1 and TRPV4 were a gift from Drs. Roger O'Neil and Rachel Brown at the University of Texas Health Science Center at Houston.

Immunohistochemistry. A section of brain containing the MCA was removed and quickly frozen in embedding compound. The MCA/brain was sectioned in 10-µm slices and placed on glass slides for immunohistochemistry. In this series of experiments, we evaluated immunoreactivity to an anti-TRPV4 antibody (a gift from Dr. Stefan Heller, Stanford University School of Medicine). Control experiments were performed using either 1) preimmune serum or 2) block solution in place of TRPV4 antibody.

Frozen sections were fixed with 4% paraformaldehyde for 30 min and then washed with PBS (3 x 5 min). Sections were then permeabilized with Triton X-100 (0.2%; 15 min) and blocked with PBS containing 5% goat serum (60 min). Incubation with anti-TRPV4 antibody (or control) was performed overnight at 4°C at 1:300 dilution. The antibody and preimmune solutions were prepared in PBS containing 1% goat serum. Sections were next washed with PBS (3 x 5 min) and incubated with a Cy2-conjugated donkey anti-rabbit IgG secondary (Jackson ImmunoResearch, West Grove, PA). The secondary detection antibody was incubated covered at room temperature (60 min) at 3.8 µg/ml final concentration. The sections were washed with PBS (5 x 10 min) and stained with 4,6-diamidino-2-phenylindole (1:10,000 dilution) before coverslips were applied. Imaging was performed using a Nikon TE-2000 fluorescence microscope with deconvolution capabilities. Sections were acquired at 0.17-µm intervals using a x60 oil objective (NA 1.40).

Statistical methods. Comparisons between concentration-response curves between two groups were performed using two-way repeated-measures ANOVA followed by either a Holm-Sidak or Student-Newman-Keuls test to compare individual points. Unpaired comparisons between two groups were evaluated using a t-test (SigmaStat). Significance was defined as P < 0.05.

	RESULTS

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Initial experiments were performed with pressurized MCA to determine whether the endothelial [Ca²⁺]_i in response to luminal UTP was altered following PLA₂ inhibition. Note that inhibitors of NOS and COX (L-NAME and indomethacin, 10 µM each) were present throughout to focus on the EDHF-mediated component of dilation. Some experiments were performed in the additional presence of PACOCF₃, an inhibitor of PLA₂. Mean baseline diameter in the presence or absence of PACOCF₃ was 192 ± 8 and 183 ± 10 µm, respectively. Mean maximum diameter for the two groups was 291 ± 6 and 281 ± 4 µm, respectively. There was no significant difference between control and PACOCF₃ groups for either baseline or maximum diameter (P > 0.05; t-test).

Figure 1 shows representative diameter and endothelial [Ca²⁺]_i measurements in pressurized MCA in response to luminally delivered UTP (10^–7–10^–5 M). Experiments were performed with vehicle control (1:1,000 EtOH) or with 20 µM PACOCF₃, an inhibitor of PLA₂. Note that whereas 10^–5 M UTP produced sustained maximal dilation in the control arteries, it produced only submaximal oscillations in the PACOCF₃-treated arteries. The endothelial [Ca²⁺]_i responses to UTP were likewise attenuated following PLA₂ inhibition. The steady-state mean values are summarized in Fig. 2.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Representative traces of middle cerebral artery (MCA) diameter (A and C) and endothelial cytosolic free Ca2+ concentration ([Ca2+]i) (B and D) in response to UTP in the presence and absence of a PLA2 inhibitor, PACOCF3. UTP was delivered luminally at the time and concentration indicated by the arrows. R340/380 reflects the endothelial [Ca2+]i measured with fura-2 in pressurized arteries. Nitro-L-arginine methyl ester (L-NAME) and indomethacin (10–5 M each) were present throughout.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Summary of endothelial [Ca2+]i (A) and MCA diameter responses (B) to luminal UTP in the absence and presence of PACOCF3. Data are presented as means ± SE. The groups were significantly different for both endothelial [Ca2+]i and diameter (2-way repeated-measures ANOVA; n = 5 each). Individual points were compared using the Holm-Sidak method; significant differences are indicated by asterisks.

It is apparent from the results shown in Figs. 1 and 2 that global endothelial [Ca²⁺]_i responses were reduced following PLA₂ inhibition. We next sought to determine whether this reduced [Ca²⁺]_i response was due to reduced Ca²⁺ influx. We used the Mn²⁺ quenching technique to evaluate Ca²⁺ influx from both luminal and abluminal compartments. In this technique, we supplemented the perfusing solution with 150–300 µM MnCl₂. Mn²⁺ acts as a Ca²⁺ surrogate and enters the cells along the same pathways as Ca²⁺. However, when Mn²⁺ binds to fura-2 in the endothelial cell, it causes the quenching of fura-2 fluorescence. Therefore, decay of fura-2 fluorescence measured at the isosbestic wavelength (360 nm) indicates Mn²⁺ (and thus Ca²⁺) entry.

In our initial experiments, we administered MnCl₂ (300 µM) luminally with UTP (10 µM), based on the assumption that significant Ca²⁺ influx occurred across the luminal/apical membrane of the endothelium. Surprisingly, the Mn²⁺ quenching was similar between control and PACOCF₃ groups. The quench rate was –21 ± 1 and –24 ± 2%/min, respectively (P > 0.05, n = 5 each). These results suggest that UTP-stimulated Ca²⁺ influx across the luminal membrane is not altered by PLA₂ inhibition.

We next applied MnCl₂ (150 µM) to the abluminal compartment to evaluate Ca²⁺ influx across the basolateral membrane of the endothelium. The addition of MnCl₂ alone produced some quenching (Fig. 3A), similar to when we administered MnCl₂ alone luminally (n = 3, not shown). The slope of quenching with MnCl₂ alone (reflected by the region between MnCl₂ addition and UTP addition) was –20.9 ± 2.6 and –19.3 ± 4.7%/min for control and PACOCF₃ groups, respectively. The basal Ca²⁺ influx, reflected by the addition of MnCl₂ alone, did not differ between groups (P = 0.78, t-test). The subsequent luminal delivery of UTP produced significantly less quenching in the PACOCF₃ group compared with the control group (Fig. 3, A and B). Curves were significantly different with individual differences at 450 and 550 s (2-way repeated-measures ANOVA with Holm-Sidak method for individual comparisons). The initial slope of quenching was –28.1 ± 2.3 and –14.2 ± 1.6%/min for control and PACOCF₃ groups, respectively (P < 0.01; t-test). Therefore, it appeared that Ca²⁺ influx across the basolateral membrane was reduced following PLA₂ inhibition.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Endothelial Ca2+ influx was significantly reduced across the basolateral membrane following PLA2 inhibition. Ca2+ influx was evaluated using the Mn2+ quenching technique in pressurized rat MCA. A: means of individual fluorescence (F360) traces are presented for MnCl2 delivered abluminally. UTP (10–5 M) and MnCl2 (150 µM) were administered over the time courses indicated. The slope of Mn2+ quenching was determined from the early portion of the UTP-mediated response. The region from which the slope was derived is indicated by the shaded lines. B: summary of slope data for abluminal MnCl2 delivery. The F360 quenching curves (A) and quenching rates (B) were significantly different (2-way repeated-measures ANOVA and ANOVA, respectively). L-NAME and indomethacin (10 µM each) were present throughout.

View larger version (9K): [in this window] [in a new window]   Fig. 4. Arachidonic acid (AA) produced a concentration-dependent increase in endothelial [Ca2+]i (A) and diameter (B) in pressurized rat MCA. AA (10–100 µM) was administered luminally in the presence of PACOCF3 (20 µM); PACOCF3 was added to inhibit endogenous production of AA. The maximum artery diameter (corresponding to the diameter in Ca2+-free buffer) is indicated by the open circle in B. L-NAME and indomethacin (10 µM each) were present throughout.

View larger version (33K): [in this window] [in a new window]   Fig. 5. A: whole MCA and freshly isolated endothelial cells expressed message for TRPV4 but not TRPV1. RT-PCR was performed from whole MCA and freshly isolated MCA endothelial cells. The predicted PCR amplicon sizes for TRPV1 and TRPV4 are 330 and 339 bp, respectively. The marker lanes () contain a 50-bp ladder. Data are representative of 3 separate preparations. B: evaluation of endothelial cell preparation purity using RT-PCR. The cDNA generated from harvested endothelial cell samples was evaluated for expression of an endothelial marker (endothelial nitric oxide synthase, eNOS) and lack of smooth muscle cell marker (SM22). EC, endothelial cells; H2O, water controls.

View larger version (142K): [in this window] [in a new window]   Fig. 6. Endothelial and smooth muscle cells expressed TRPV4 channel protein. A: anti-TRPV4 channel immunoreactivity (green) was prevalent on endothelial cells (Endo) as well as in the smooth muscle layers (SMC). The nuclei were stained with 4,6-diamidino-2-phenylindole (blue), and the internal elastic lamina (IEL) was visualized by autofluorescence on the red channel (red). The IEL forms the boundary between the endothelium and the smooth muscle layers. B: the preimmune control was acquired and processed identically to the anti-TRPV4 sections.

View larger version (10K): [in this window] [in a new window]   Fig. 7. Endothelial [Ca2+]i response to UTP is reduced in the presence of ruthenium red (RuR; 3 µM). The point between baseline and 10–7 M UTP represents the effect of adding RuR alone. The groups were significantly different (2-way repeated-measures ANOVA; n = 4 each). Individual points were compared using the Holm-Sidak method; significant differences are indicated by asterisks. L-NAME and indomethacin (10 µM each) were present throughout.

View larger version (20K): [in this window] [in a new window]   Fig. 8. Administration of 4-phorbol 12,13-didecanoate (4-PDD; 3 µM) produces a significant increase in endothelial [Ca2+]i that is reversed by RuR (1 µM) in freshly isolated MCA endothelial cells. Representative (A) and summary R360/380 data (B) are presented. The 4-PDD response was significantly different from baseline and post-RuR levels (1-way repeated-measures ANOVA). Individual points were compared using the Student-Newman-Keuls method. *Significant difference from baseline. , Significant difference from 4-PDD.

	DISCUSSION

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Endothelium-derived hyperpolarizing factor. Early studies speculated that EDHF was a factor that originated in the endothelium and then diffused to the smooth muscle. Although there is evidence for a transferable factor in certain coronary and renal beds, other vascular beds appear to lack evidence for transferable factors (1). In rat MCA, it appears that hyperpolarization originates in the endothelium and is then conducted to the surrounding smooth muscle (25). Endothelial cell hyperpolarization results from elevated [Ca²⁺]_i that must reach a critical threshold before activating small- and/or intermediate-conductance K_Ca channels (15, 17). The transfer of hyperpolarization between endothelium and smooth muscle then likely occurs via intercellular gap junctions between the two tissue types (25). Once the innermost layer of smooth muscle is hyperpolarized, the hyperpolarization can be amplified and propagated to outer layers via the activation of smooth muscle inwardly rectifying K⁺ channels (4).

Endothelial Ca²⁺ regulation is PLA₂ sensitive. Activation of PLA₂ liberates AA from membrane phospholipids. The resulting AA can then act directly on target proteins or be metabolized to one of several other bioactive metabolites. The evidence for a direct effect of AA on Ca²⁺ influx in endothelial cells comes from the finding that both AA and nonmetabolized AA metabolites promote Ca²⁺ influx in intact endothelial preparations (7, 19). In addition, application of AA and analogs to the cytosolic face of inside-out patches stimulates opening of nonselective cation channels. In this latter preparation, the metabolizing enzymes should have been washed away. Evidence also has been presented supporting a role for AA metabolites of the cytochrome P-450 monooxygenases: epoxyeicosatrienoic acids (EETs). Several EET products have been demonstrated to trigger Ca²⁺ influx in cultured endothelial cells, whereas the inhibition of cytochrome P-450 enzymes results in diminished Ca²⁺ influx (8, 27, 30).

A previous study from our laboratory demonstrated that activation of endothelial PLA₂ was critical for EDHF-mediated dilations (33). Given our current understanding of the EDHF-dependent mechanism in rat cerebral arteries, it seemed unlikely that PLA₂ would liberate an endothelial product that must then diffuse to the smooth muscle before exerting a hyperpolarizing effect. Instead, we postulated that PLA₂ acts as a critical modulator of some step in the EDHF-dependent mechanism. Given the above-mentioned findings regarding AA and AA metabolites on Ca²⁺ influx, we evaluated a role for PLA₂/AA on MCA endothelial Ca²⁺ regulation.

As shown in Figs. 1 and 2, inhibition of PLA₂ resulted in a profound reduction in endothelial [Ca²⁺]_i responses to UTP. This endothelial [Ca²⁺]_i reflects the sum of Ca²⁺ release from internal stores and Ca²⁺ influx from the extracellular milieu. Since Ca²⁺ influx accounts for the sustained [Ca²⁺]_i response as well as our proposed pathway by which PLA₂/AA modulates endothelial [Ca²⁺]_i, we examined Ca²⁺ influx using the Mn²⁺ quenching technique. Interestingly, Mn²⁺/Ca²⁺ influx across the luminal/apical membrane was unaltered by PLA₂ inhibition. In these experiments, we added MnCl₂ to the luminal perfusate only. In pressurized cerebral arteries, the endothelial cells are tightly coupled and do not readily allow passage of hydrophilic compounds (16). In contrast to influx across the apical membrane, Mn²⁺/Ca²⁺ influx was reduced across the basolateral membrane following PLA₂ inhibition. In this later experiment, MnCl₂ was delivered to the basolateral surface of the endothelium by being added to the abluminal perfusate. Together, these data suggest that the PLA₂-dependent Ca²⁺ influx occurs primarily across the basolateral membrane of MCA endothelial cells in response to UTP.

Does endothelial Ca²⁺ influx demonstrate apical-basolateral polarity? It is well established that endothelial cells in the brain, like many other epithelial cells, exhibit apical/basolateral polarity of expression and function (3, 13, 20, 21, 24). This means that the apical membranes (blood facing) can be quite different from the basolateral membranes (smooth muscle facing) in both protein composition and function. Our study adds to the already extensive list of polarized proteins and functions of cerebral endothelium. In particular, our studies suggest that the extracellular milieu on the adventitial side provides the source of Ca²⁺ for PLA₂-sensitive Ca²⁺ influx. This is not to say that all Ca²⁺ influx in response to UTP stimulation occurs across the abluminal membrane. UTP did indeed stimulate Ca²⁺ influx across the luminal/apical membrane above the basal levels, but not by a PLA₂-sensitive mechanism. Thus it appears that more than one Ca²⁺ influx mechanism exists to promote Ca²⁺ entry into MCA endothelium, at least one of which is PLA₂ dependent. The identity of the channels involved in mediating Ca²⁺ influx in this tissue remains to be resolved and is discussed further below.

TRPV4 channels are expressed and functional in the endothelium. From our study, we cannot conclusively state which Ca²⁺ influx channels are involved in the PLA₂-sensitive and -insensitive Ca²⁺ entry mechanism. However, we do provide evidence indicating a likely involvement of TRPV4 channels. TRPV1 and TRPV4 are logical candidates for Ca²⁺ influx channels on the basis of a demonstrated presence in other endothelial preparations (6, 12, 30, 31), moderate Ca²⁺ conductance (2), and known activation by either AA or AA metabolites (2, 28, 30). We found no evidence for TRPV1 channel mRNA in either whole MCA or isolated endothelial cells (Fig. 5). In addition, application of 10 µM capsaicin (a TRPV1 activator) failed to promote an increase in endothelial [Ca²⁺]_i (data not shown). However, using PCR, we did find evidence for TRPV4 message in both whole artery and isolated endothelial cells. Furthermore, TRPV4 protein was identified in both smooth muscle and endothelium by immunohistochemistry (Fig. 6). The presence of TRPV4 mRNA in cerebral smooth muscle cells was recently described (5). Thus our demonstration of TRPV4 protein in MCA smooth muscle was an expected finding. However, we believe the present study to be the first to report TRPV4 mRNA and protein in MCA endothelium. We also found functional evidence for TRPV4 channels in MCA endothelial cells. Application of a selective TRPV4 channel activator, 4-PDD, promoted a significant increase in [Ca²⁺]_i from freshly isolated MCA endothelial cells. The [Ca²⁺]_i response was rapidly reduced by subsequent delivery of a TRPV channel blocker, RuR, suggesting that the response to 4-PDD occurred by stimulating Ca²⁺ influx. In addition, luminal application of AA produced an increase in endothelial [Ca²⁺]_i (Fig. 4A). These latter data demonstrate that AA or an AA metabolite stimulates increased [Ca²⁺]_i in MCA endothelium. The metabolism of AA could conceivably occur via any of the pathways other than COX, which was inhibited in the present studies. If metabolism of AA is required, the most likely pathway would be via cytochrome P-450 epoxygenases to generate EETs. It should be noted that in our laboratory, we have not found a role for the EETs in UTP-mediated dilation (32). Rather, it appears that AA itself acts as the signaling molecule in the rat MCA endothelial cells.

An interesting explanation of our findings is that TRPV4 channels are expressed preferentially on the basolateral membrane. Although we are not aware of any studies that have reported polarity to TRP channel expression, TRP channels have been shown to be rapidly shuttled to the plasma membrane in response to various stimuli (22). Additional studies are required to evaluate the possibility that TRPV4 channel expression is restricted to the basolateral membrane in rat MCA.

In summary, we have demonstrated that endothelial [Ca²⁺]_i and diameter responses to UTP are significantly reduced following PLA₂ inhibition. This reduced [Ca²⁺]_i response results from decreased Ca²⁺ influx across the basolateral membrane but not across the apical membrane. Furthermore, we demonstrate that TRPV4 channels are present and appear to contribute to endothelial Ca²⁺ influx and EDHF-mediated dilations in rat MCA.

	GRANTS

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This work was supported by American Heart Association Grants 0230353N (to S. P. Marrelli), 0665100Y (to S. P. Marrelli), and 0270110N (to R. M. Bryan) and National Institute of Neurological Disorders and Stroke Grant R01 NS-46666 (to R. M. Bryan).

	ACKNOWLEDGMENTS

 
We thank Dr. Stefan Heller (Stanford University School of Medicine) for generously providing the TRPV4 channel antibody and preimmune control serum.

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. P. Marrelli, Baylor College of Medicine, One Baylor Plaza, Suite 434-D, Houston, TX 77030 (e-mail: marrelli{at}bcm.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

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