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Abstract

Very little is known regarding the mechanism of action for the endothelium-derived hyperpolarizing factor (EDHF) response in cerebral vessels. The authors tested two hypotheses: (1) activation of the cytoplasmic form of phospholipase A₂ (cPLA₂) is involved with EDHF-mediated dilations in rat middle cerebral arteries; and (2) activation of the cPLA₂ involves an increase in endothelial Ca²⁺ through activation of phospholipase C. Middle cerebral arteries were isolated from the rat, pressurized to 85 mm Hg, and luminally perfused. The EDHF response was elicited by luminal application of uridine triphosphate (UTP) after NO synthase and cyclooxygenase inhibition (10⁻⁵ mol/L N-nitro-l-arginine methyl ester and 10⁻⁵ mol/L indomethacin, respectively). AACOCF₃ and PACOCF₃, inhibitors of cPLA₂ (Ca²⁺-sensitive) and Ca²⁺-insensitive PLA₂ (iPLA₂), dose dependently attenuated the EDHF response. A selective inhibitor for iPLA2, haloenol lactone suicide substrate, had no effect on the EDHF response. The EDHF response elicited by UTP was accompanied by an increase in endothelial Ca²⁺ (144 to 468 nmol/L), and the EDHF dilation was attenuated with U73122, a phospholipase C inhibitor. The authors conclude that the EDHF response elicited by luminal UTP in rat middle cerebral arteries involved activation of phospholipase C, an increase in endothelial Ca²⁺, and activation of cPLA₂.

Keywords

EDHF Endothelium-derived hyperpolarizing factor Cerebrovascular circulation Endothelium Phospholipase C Calcium

In arteries and arterioles there is at least one factor or mechanism, not involving nitric oxide/endothelium-derived relaxing factor (NO/EDRF) or cyclooxygenase metabolites (most often prostacyclin), that is responsible for dilations when endothelial receptors are stimulated. The factor or mechanism is termed endothelium-derived hyperpolarizing factor (EDHF) and is characterized by the following: (1) it requires endothelium, (2) it is distinct from both NO/EDRF and prostacyclin, (3) it relaxes by hyperpolarizing the vascular smooth muscle, and (4) it involves calcium-activated potassium channels (K_Ca) (Golding et al., 2002). The EDHF response can be elicited in cerebral vessels by ATP, ADP, UTP, substance P, A23187 (Ca²⁺ ionophore), and acetylcholine (see Golding et al., 2002 for a brief review).

Very little is known regarding the mechanism of action of the EDHF response in cerebral vessels. There are, however, three observations that support the idea that the EDHF mechanism is different in cerebral and peripheral vessels. First, the EDHF-mediated relaxations in guinea pig middle cerebral arteries (MCAs) and mesenteric resistance vessels were differently affected by a number of pharmacological inhibitors, including those that inhibit cytochrome P450 enzymes and potassium channels (Dong et al., 2000). Second, the presence of basal concentrations of NO attenuated the EDHF response by 50% to 100%, depending on the agonist and concentration in peripheral arteries (Bauersachs et al., 1996). In rat MCAs, however, basal or above basal concentrations of NO had no effect on the EDHF response (Schildmeyer and Bryan Jr., 2002). Third, estrogen downregulated the EDHF response in cerebral vessels (Golding and Kepler, 2001, Xu et al., 2001) while it upregulated the response in peripheral vessels (Tagawa et al., 1997; McCulloch and Randall, 1998). Therefore, the mechanism for the EDHF response in cerebral vessels is unique and cannot be extrapolated from peripheral mechanisms. It must be determined independently from that in peripheral vessels.

The purpose of the present study was to determine whether activation of phospholipase A₂ (PLA₂) is involved with EDHF-mediated dilations. One important pathway for the activation of cytoplasmic PLA₂ (cPLA₂) involves an increase in Ca²⁺ through the activation of phospholipase C (PLC) and subsequent liberation of inositol 1,4,5-triphosphate (IP₃). After determining that activation of PLA₂ is involved, subsequent goals were to determine whether the PLC and an increase in cytoplasmic Ca²⁺ were also steps in the EDHF response.

MATERIALS AND METHODS

The Animal Protocol Review Committee at Baylor College of Medicine approved the experimental protocol. MCAs were harvested from male Long Evans rats (250 to 350 g) after isoflurane (3%) anesthesia (Bryan Jr. et al., 1996). Micropipettes were inserted into the ends of each MCA segment, and the vessel was secured in place using nylon ties. Each MCA was bathed luminally and abluminally in a 37°C physiologic salt solution (PSS), which was equilibrated with a gas mixture of 20% O₂/5% CO₂/balance N₂. The pH of the PSS was 7.4, Pco₂ was 35 mm Hg, and Po₂ was ∼130 mm Hg.

MCAs were pressurized to 85 mm Hg by raising PSS-containing reservoirs, connected to the micropipettes by tubing, above the vessel. Pressure transducers on either side of the MCA allowed measurement of the perfusion pressure across the vessel. Luminal flow was adjusted to 150 μL/min by setting inflow and outflow reservoirs at different heights (Bryan Jr. et al., 1996). Each MCA was visualized after magnification using a video monitor. Diameters were continuously measured using Optimus image-analysis software (Bothell, WA, U.S.A.).

After warming and pressurizing to 85 mm Hg, MCAs developed spontaneous tone over the course of an hour by constricting to ∼75% of their initial diameters.

The EDHF response was elicited by the luminal application of UTP after inhibition of nitric oxide synthase and cyclooxygenase with 10⁻⁵ mol/L N-nitro-l-arginine methyl ester (l-NAME) and 10⁻⁵ mol/L indomethacin, respectively. Each MCA was subjected to only one concentration response curve.

Measurement of Ca²⁺ in endothelium

Ca⁺ concentrations in the cytoplasm of the endothelium and vessel diameter were simultaneously measured as previously described (Marrelli, 2000, 2001). Briefly, fura-2 AM (0.67 μmol/L final concentration) was added to the luminal perfusate (5-minute exposure). Addition of fura-2 AM through the lumen at the above concentration and duration selectively loads the endothelium. For Ca²⁺ measurements, the vessels were illuminated with excitation light alternating between wavelengths of 340 and 380 nm using a xenon arc lamp, appropriate filters, and a filter changer (Intracellular Imaging, Cincinnati, OH, U.S.A.). Additionally, red light from a separate lamp was used to trans-illuminate the vessels for diameter measurements. The light was collected with a quartz objective (Nikon 10X, NA = 0.5) and subsequently split with a dichroic mirror to separate the fluorescence and red light signals. The red light was diverted to a CCD for diameter measurements, and the remainder of the light continued to a photomultiplier after passing through a 510-nm narrow bandpass filter. Intensities of the 510-nm fluorescence light were used to quantitate intracellular Ca ⁺ according to the following equation:

{[{Ca}^{2 +}]}_{i} = β \cdot K_{d} \cdot (R - R_{min}) / (R_{max} - R)

(1)

where [Ca²⁺]i was the intracellular Ca²⁺ concentration in the endothelium, β was the ratio of the 380-nm fluorescence intensity for Ca²⁺-unbound fura 2 over Ca²⁺-bound fura 2, R was the ratio of light intensity at 510 nm when excited at 340 nm to the intensity when excited at 380 nm (340/380 ratio) at a given condition (i.e., luminal addition of UTP), R_min was the 340/380 ratio with zero [Ca²⁺]_i, R_max was the 340/380 ratio when [Ca²⁺]_i was sufficiently high to saturate fura 2, and K_d was 282 nmol/L. β, R_min and R_max were determined in a separate group of vessels as previously described (Marrelli, 2000).

Drugs and reagents

UTP, l-NAME, and indomethacin were purchased from Sigma. AACOCF₃ (arachidonyltrifluoromethyl ketone), AACOCH₃ (arachidonylmethyl ketone), HELSS [haloenol lactone suicide substrate or E-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (also known as BEL)], ETYA (5,8,11,14-eicosatetraynoic acid), PACOCF₃ (palmitoyl trifluoromethyl ketone), PACOCH₃ (heptadeca-2-one), RHC-80267 [1,6-bis(cyclohexyloximinocarbonylamino) hexane], U73122 [1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1 H-pyrrole-2,5-dione], and U73343 [1-[6-[[(17β)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-pyrrolidine-2,5-dione], an analog of U73122, were purchased from Biomol. S-nitroso-N-acetylpenicillamine (SNAP) was purchased from RBI/Sigma. Fura2-AM and pluronic were purchased from Teflabs (Austin, TX, U.S.A.).

AACOCF₃, AACOCH₃, PACOCF₃, PACOCH₃, and ETYA were dissolved in ethanol; U73122, U73343, HELSS, and RHC-80267 were dissolved in dimethyl sulfoxide (DMSO). Fura 2-AM (50 μg) was dissolved in 75 μL of DMSO (containing 14% pluronic). Indomethacin was prepared by dissolving it in a 15-mmol/L Na₂CO₃ solution. Other reagents were dissolved in distilled water.

All blockers were administered 30 minutes prior to the addition of UTP. The composition of the PSS was as previously described (Bryan Jr. et al., 1996).

Statistical analysis

All data are reported as mean ± standard deviation. For concentration-response curves, the results are presented as the percentage of the maximum diameter and calculated by the following equation:

\begin{array}{l} % maximum diameter = & [(D_{UTP} - D_{base}) / (D_{max} - D_{base})] \\ \times 100 \end{array}

(2)

where D_UTP was the diameter after luminal administration of UTP, D_base was the baseline diameter before addition of UTP, and D_max was the maximum diameter at 85 mm Hg (measured using Ca-free/EGTA buffer).

For statistical analysis, one- or two-way repeated measures ANOVA was used followed by Tukey test for individual comparisons when appropriate. P < 0.05 defined the acceptable level of significance.

RESULTS

A total of 141 MCAs were studied. At a pressure of 85 mm Hg, the maximum outside diameter (Ca²⁺-free/EGTA buffer) was 290 ± 20 μm and, after developing spontaneous tone, was 221 ± 23 μm. The diameter after spontaneous tone represented 76 ± 5% of the maximum diameter.

In the presence of NO synthase and cyclooxygenase inhibitors, 10⁻⁵ mol/L l-NAME and 10⁻⁵ mol/L indomethacin, respectively, the luminal addition of UTP produced a dose-dependent dilation (ETOH control in Figs. 1A and 1B). This l-NAME/indomethacin-resistant component of the dilation is the EDHF response (Marrelli et al., 1999; You et al., 1999a, b ; Golding and Kepler, 2001; Marrelli, 2001). Addition of either AACOCF₃ or PACOCF₃, inhibitors of the cytoplasmic (Ca²⁺ sensitive, cPLA₂) and Ca²⁺-insensitive (iPLA₂) forms of PLA₂, produced dose-dependent inhibitions of the EDHF-mediated dilations to UTP (l-NAME/indomethacin present) when added to both the luminal and abluminal compartments (Fig. 1). PACOCF₃ completely inhibited the EDHF response at a concentration of 2 × 10⁻⁵ mol/L (Fig. 1B). AACOCH₃ and PACOCH₃ are analogs of AACOCF₃ and PACOCF₃, respectively and were used as control since they have little or no blocking effect on PLA₂. Neither AACOCH₃ nor PACOCH₃ had an effect on the dilation to UTP when compared to the ethanol (ETOH) control (Figs. 1A and 1B).

Fig. 1.

The effects of AACOCF₃ (A) and PACOCF₃ (B), inhibitors of the cytoplasmic (Ca²⁺ sensitive) and Ca²⁺-insensitive forms of phospholipase A₂, on the endothelium-derived hyperpolarizing factor-mediated dilations to UTP in the rat middle cerebral artery. All experiments were conducted in the presence of 10⁻⁵ mol/L l-NAME and 10⁻⁵ mol/L indomethacin. (A) AACOCF₃ was administered luminally (lu), abluminally (ab), or both (lu/ab). The number of observations (n) was 22 for the vehicle controls (ETOH), 6 for the AACOCF₃ (3 × 10⁻⁶ mol/L, lu/ab) group, 8 for AACOCH₃, and 5 for the remaining three groups. AACOCH₃ is an analog of AACOCF₃ and was used as a negative control. There was a significant group difference (P < 0.001), a significant concentration difference (P < 0.001), and a significant interaction between groups and concentration (P < 0.001) (two-way repeated measures analysis of variance [ANOVA]). *P < 0.05 compared to the corresponding point in the ETOH control group or the group receiving AACOCH₃ (Tukey test). (B) PACOCF₃ was administered both luminally (lu), and abluminally (ab). The number of observation (n) was 14 for the vehicle controls (ETOH), 6 for the PACOCF₃ (10⁻⁵ mol/L, lu/ab) group, 7 for the PACOCF₃ (2 × 10⁻⁵ mol/L, lu/ab) group, and 5 for the PACOCH₃ (2 × 10⁻⁵ mol/L, lu/ab). There was a significant group difference (P < 0.001), a significant concentration difference (P < 0.001), and a significant interaction between groups and concentration (P < 0.001) (two-way repeated measures ANOVA). *P < 0.05 compared to the corresponding point in the ETOH control group or to the group receiving PACOFH₃ (Tukey test). UTP, uridine triphosphate.

When 10⁻⁵ mol/L AACOCF₃ was added to the luminal compartment, the EDHF response was attenuated to the same degree as when the same concentration was added to both the luminal and abluminal compartments. However, the addition of 10⁻⁵ mol/L AACOCF₃ to the abluminal compartment alone had no effect on the EDHF response (Fig. 1A). The addition of pharmacological agents to the luminal compartment has preference to endothelium, while addition to the abluminal compartment has preference to vascular smooth-muscle cells.

The selective inhibitor of the Ca²⁺ insensitive form of PLA₂ (_iPLA2), HELSS had no effect on the EDHF response (i.e, l-NAME and indomethacin present) when it was added luminally and abluminally at a concentration of 3 × 10⁻⁷ and 10⁻⁶ mol/L (Fig. 2). At a concentration of 1.5 × 10⁻⁵ mol/L, HELSS attenuated the dilation at 10⁻⁴ mol/L UTP but not at any other UTP concentration.

Fig. 2.

The effects of haloenol lactone suicide substrate (HELSS), a selective inhibitor of the Ca²⁺-insensitive form of phospholipase A₂, on the endothelium-derived hyperpolarizing factor-mediated dilations to UTP in the rat middle cerebral artery. All experiments were conducted in the presence of 10⁻⁵ mol/L l-NAME and 10⁻⁵ mol/L indomethacin (indo). HELSS was administered both luminally and abluminally; UTP was administered luminally to the vehicle controls (DMSO [dimethyl sulfoxide], n = 8), HELSS (3 × 10⁻⁷ mol/L) (n = 3), HELSS (10⁻⁶ mol/L) (n = 4), and HELSS (1.5 × 10⁻⁵ mol/L) (n = 5). There was no group difference (P = 0.24), a significant concentration difference (P < 0.001), and a significant interaction between groups and concentration (P= 0.002) (two-way repeated measures analysis of variance). *P < 0.05 compared to the corresponding point in the DMSO control group (Tukey test). l-NAME, N-nitro-l-arginine methyl ester; UTP, uridine triphosphate.

In the presence of l-NAME and indomethacin, the luminal and abluminal application of ETYA (2 × 10⁻⁵ mol/L) inhibited the dilation to UTP by 50% to 60% (P < 0.001, Fig. 3). ETYA inhibits PLA₂s as well as arachidonic acid specific and nonspecific acyl-CoA synthesis, cyclooxygenase, and all lipoxygenases (Tobias and Hamilton, 1979; Chi et al., 1982). This degree of inhibition of the EDHF response is slightly more in the rat MCA compared to porcine coronary arteries (Pomposiello et al., 1999).

Fig. 3.

The effects of 2 × 10⁻⁵ mol/L ETYA, an inhibitor of phospholipase A₂, cyclooxygenase, and lipoxygenases, on the endothelium-derived hyperpolarizing factor-mediated dilations to UTP in the rat middle cerebral artery. All experiments were conducted in the presence of 10⁻⁵ mol/L l-NAME and 10⁻⁵ mol/L indomethacin. ETYA was administered both luminally and abluminally. The number of observations (n) was 22 for the ETOH control and 6 for ETYA group. There was a significant group difference (P < 0.001), a significant concentration difference (P < 0.001), and a significant interaction between groups and concentration (P < 0.001) (two-way repeated measures analysis of variance). *P < 0.05 compared to the corresponding point in the ETOH control group (Tukey test). ETYA, 5,8,11,14-eicosatetraynoic acid; l-NAME, N-nitro-l-arginine methyl ester; UTP, uridine triphosphate.

The diacylglycerol lipase inhibitor, RHC-80267 (10⁻⁵ mol/L) (Sutherland and Amin, 1982), had minimal effects on the EDHF response (n = 8 for DMSO controls and 6 for the RHC-80267 group, data not shown). In the presence of l-NAME and indomethacin, RHC-80267 attenuated the dilator response by 8% at a UTP concentration of 10⁻⁵ mol/L; RHC-80267 was without an effect at all other UTP concentrations.

Addition of the PLC inhibitor, U73122 (10⁻⁶ mol/L), to both the luminal and abluminal compartments of the MCA preparation significantly attenuated the EDHF response compared to the DMSO control or after the addition of 10⁻⁶ mol/L U73343, an analog of U73122 that only slightly inhibits PLC (Fig. 4). Maximum dilations for the U73343 control and the U73122 group were 100 ± 0.6% and 42 ± 40% respectively. The concentration of UTP required to elicit one half of the maximum response was 2.6 ± 1.2 μmol/L for the U73343 control and 19 ± 18 μmol/L for the U73122 group. Two-way repeated measures ANOVA revealed a group difference (P = 0.001), a concentration difference (P < 0.001), and a significant interaction between group and concentration (P = 0.011).

Fig. 4.

The effects of 10⁻⁶ mol/L U73122, a phospholipase C inhibitor, on the endothelium-derived hyperpolarizing factor-mediated dilations to UTP in the rat middle cerebral artery. All experiments were conducted in the presence of 10⁻⁵ mol/L l-NAME and 10⁻⁵ mol/L indomethacin. U73122 and U73343 (an analog of U73122 that only slightly inhibits phospholipase C), were administered both luminally and abluminally. UTP was administered luminally to the vehicle controls (DMSO [dimethyl sulfoxide], n = 8), U73343 (n = 5), and the U73122 (n = 6) groups. There was a significant group difference (P = 0.001), a significant concentration difference (P < 0.001), and a significant interaction between groups and concentration (P = 0.011) (two-way repeated measures analysis of variance). *P < 0.05 compared to all other groups (Tukey test). l-NAME, N-nitro-l-arginine methyl ester; UTP, uridine triphosphate.

The effects of luminally administered UTP on the concentration of free Ca²⁺ in endothelium and diameter are shown in Fig. 5. Figure 5A shows endothelial Ca²⁺ and diameter measured simultaneously from an individual MCA after inhibition of NO synthase and cyclooxygenase. Note that the addition of 10⁻⁶ mol/L UTP increased endothelial Ca²⁺ by approximately 100 nmol/L over baseline with very little effect on diameter. With the addition of 10⁻⁵ mol/L UTP, Ca²⁺ further increased to greater than 500 nmol/L and the MCA dilated. Figure 5B shows the summary data from 5 MCAs. The group data further demonstrate that 10⁻⁶ mol/L UTP increased endothelial Ca²⁺ from 154 ± 33 to 246 ± 34 nmol/L (n = 5) with little effect on the MCA diameter. Application of 10⁻⁵ mol/L UTP increased Ca²⁺ to 468 ± 113 nmol/L and produced a near-maximal dilation of the MCA.

Fig. 5.

(A) Endothelial Ca²⁺ concentration and diameter, measured simultaneously from a single middle cerebral artery (MCA), during the luminal infusion of UTP at the concentrations noted (log M). The experiment was conducted in the presence of 10⁻⁵ mol/L l-NAME and 10⁻⁵ mol/L indomethacin. (B) Mean endothelial Ca²⁺ and diameter from five rat MCAs during the luminal infusion of UTP. All experiments were conducted in the presence of 10⁻⁵ mol/L l-NAME and 10⁻⁵ mol/L indomethacin. There was a significant change in Ca²⁺ (P < 0.001) and diameter (expressed as a percent of the maximum diameter (P < 0.001, one-way repeated analysis of variance). *P < 0.05 compared to the corresponding value before UTP was administered (Tukey test). l-NAME, N-nitro-l-arginine methyl ester; UTP, uridine triphosphate.

In order to determine whether the attenuation of the EDHF response by blockers as described above was a general inhibition of dilation, the dilator response to SNAP, an NO donor, was studied in the presence of AACOCF₃, ETYA, and U73122. The inhibitors were added to both the luminal and abluminal compartments and were added at the highest concentration used in the studies described above. In the presence of l-NAME and indomethacin, 10⁻⁶ mol/L U73122 had no significant effect on the dilation to SNAP compared to the DMSO control (n = 3 for each group, P = 0.28 for group effect, P < 0.001 for concentration effect, and P = 0.57 for an interaction between group and concentration, data not shown). Similarly, in the presence of l-NAME and indomethacin, 10⁻⁵ mol/L AACOCF₃ and 2 × 10⁻⁵ mol/L ETYA had no significant effect on the dilation to SNAP compared to the ETOH control (n = 3 for each group, P = 0.47 for group effect, P < 0.001 for concentration effect, and P = 0.51 for an interaction between group and concentration, two-way repeated measures ANOVA, data not shown).

DISCUSSION

ATP and UTP, which are potent agonists for P2Y₂ receptors, are found in plasma in physiologically relevant concentrations (Bryan Jr., 2002). There is strong evidence that these agonists are important regulators of blood flow during normal physiologic states and during pathologic conditions. Administration of UTP or ATP, P2Y₂ agonists, to the lumen of rat cerebral arteries and arterioles dilates in part by the EDHF response (You et al., 1999a, b ; Bryan Jr. et al., 2001a,b). In the present study, we have demonstrated that the EDHF response in rat MCAs involves activation of cPLA₂. We further provide evidence that cPLA₂ activation is accompanied by activation of PLC and an increase in endothelial Ca²⁺.

Involvement of cytoplasmic form of phospholipase A₂ in the endothelium-derived hyperpolarizing factor response

PLA₂, a lipase that catalyzes the hydrolysis of the sn−2 linkage of diacyl glycerophosphates, can be subdivided into three major classes: (1) sPLA₂ or the secreted form, which requires millimolar concentrations of Ca²⁺ for activation; (2) cPLA₂, which is activated by submicromolar concentrations of Ca²⁺, and (3) iPLA₂, which is calcium insensitive. cPLA₂ and iPLA₂ are active within the cell whereas, as the name suggests, the sPLA₂ is secreted and its catalytic activity occurs extracellularly. Because of the fact that increases in endothelial Ca²⁺ appeared to be involved with the EDHF response (Marrelli, 2001), we suspected that cPLA₂ might be involved. Our results from this series of studies demonstrated that our hypothesis was valid.

AACOCF₃ and PACOCF₃, inhibitors of iPLA₂ and cPLA₂ (Conde-Frieboes et al., 1996; Balsinde and Dennis, 1997), dose dependently blocked the EDHF response in rat MCAs (Fig. 1). For both inhibitors, a concentration of 10⁻⁵ mol/L (or 10 μmol/L) blocked approximately one half of the dilation produced by UTP. We obtained a complete inhibition of the dilation with 2 × 10⁻⁵ mol/L PACOCF₃ (Fig. 1B). Inhibition of the EDHF dilation in our studies is consistent with reported values for the IC₅₀, the inhibitor concentration necessary to inhibit 50% of the PLA₂ activation. IC₅₀ values reported for AACOCF₃ range from 5 to 50 μmol/L for cPLA₂ and 0.5 to 15 μmol/L for iPLA₂. IC₅₀ reported for PACOCF₃ was 45 μmol/L for cPLA₂ and 3 μmol/L for iPLA₂ (Ackermann et al., 1995; Conde-Frieboes et al., 1996; Handlogten et al., 2001).

In order to determine which form of the PLA₂ enzyme, iPLA₂ or cPLA₂, was involved, we further used HELSS, a selective inhibitor of iPLA₂. HELSS, which is 1,000-fold more selective for iPLA₂ over cPLA₂, has IC₅₀ values for iPLA₂ of 1 μmol/L or less (Hazen et al., 1991; Ackermann et al., 1995). HELSS had no effect at 10⁻⁶ mol/L (1 μmol/L) and at a concentration of 1.5 × 10⁻⁵ mol/L (15 μmol/L) attenuated the EDHF response at a UTP concentration of 10⁻⁴ mol/L but not at 10⁻⁵ mol/L UTP (Fig. 2). We have consistently observed that the vasomotor response produced by 10⁻⁴ mol/L UTP is often difficult to interpret since UTP, which has a constrictor response when in direct contact with smooth muscle, could be gaining access to the smooth muscle at this higher concentration. Note that the HELSS did not inhibit the EDHF-mediated dilations at 10⁻⁵ mol/L UTP. We consider this lack of inhibition at 10⁻⁵ mol/L UTP to be more insightful than the apparent inhibition at 10⁻⁴ mol/L UTP. Regardless of whether our interpretation regarding the apparent attenuation of the response is correct or not, iPLA₂ does not seem to be involved at UTP concentrations up to and including 10⁻⁵ mol/L UTP. We therefore conclude that the predominant, if not the exclusive, form of PLA₂ involved in the EDHF response is cPLA₂.

Although these studies cannot conclusively distinguish between endothelial and smooth-muscle cPLA₂, our data would suggest the endothelium as the site of PLA₂ activation. Intraluminal application of the inhibitor AACOCF₃ alone inhibited the EDHF response to the same degree as the combination of the luminal/extra-luminal application of the inhibitor at the same concentration. When AACOCF₃ was administered only abluminally, no inhibition occurred (Fig. 1A). As noted earlier, the luminal application of a drug has preferential access to the endothelium and the abluminal application has preferential access to the vascular smooth muscle. While these data support the idea that cPLA₂ in endothelium is involved with the EDHF response, additional studies are required to support this conclusion.

AACOCF₃ has been reported to abolish or inhibit the EDHF response in some peripheral arteries but failed to have an effect in others (Fulton et al., 1996; Adeagbo and Henzel, 1998; Hutcheson et al., 1999; Drummond et al., 2000; Hutcheson and Griffith, 2000). The above studies implicating PLA₂ are supported by inhibition of the EDHF response with quinacrine, a less specific PLA₂ inhibitor (Bauersachs et al., 1994; Fukao et al., 1997). Interestingly, Dong et al. (2000) reported that 1 μmol/L AACOCF₃ had no effect on guinea pig mesenteric arteries but inhibited the EDHF response in guinea pig MCAs by 48%. These latter authors did not conduct further studies to distinguish between cPLA₂ and iPLA₂. Similar to the guinea pig MCA and other peripheral vessels, the EDHF response in rat MCAs appears to rely heavily, if not exclusively, on the activation of PLA₂ (specifically cPLA₂).

Involvement of phospholipase C

A possible involvement of PLC was tested using the inhibitor, U73122. U73122 (10⁻⁶ mol/L or 1 μmol/L) produced a 50% to 60% inhibition of the EDHF response (Fig. 4). This attenuation is consistent with reported IC₅₀ concentrations in the μmol/L range (Bleasdale et al., 1990) and previously reported concentrations for inhibition of the EDHF response in the porcine coronary artery (20 μmol/L abolished the EDHF response) (Weintraub et al., 1995) and rabbit mesenteric artery (0.5 μmol/L produced greater than 50% inhibition) (Hutcheson et al., 1999). In guinea pig, 1 μmol/L U73122 had no effect on the EDHF response in the mesenteric artery but produced a 39% inhibition in the MCA (Dong et al., 2000). Thus, while PLC, as determined by inhibitor studies with U73122, appears to be involved with the EDHF response in a number of arteries including the rat MCA, it may not be involved with the EDHF response in all vascular systems.

Involvement of Ca²⁺ in the endothelium-derived hyperpolarizing factor response

The regulation of cPLA₂ is both complex and interesting. While this enzyme is catalytically active under resting conditions, it can be further activated through serine phosphorylation (MAP kinase) and/or possibly tyrosine phosphorylation (Leslie, 1997; Balsinde et al., 1999; Hirabayashi and Shimizu, 2000). Although increases in Ca²⁺ do not catalytically activate cPLA₂, Ca²⁺ increases the efficiency of the reaction by promoting cPLA₂ binding to the membrane, the site of the phospholipid substrate (Balsinde et al., 1999; Hirabayashi and Shimizu, 2000; Leslie, 1997). Phosphatidylinositol 4,5-bisphosphate (PIP₂) may also promote PLA₂ binding to membrane, but it has not been as extensively studied as Ca²⁺ (Balsinde et al., 1999; Hirabayashi and Shimizu, 2000).

Although circumstantial evidence has indicated a second messenger role for Ca²⁺ in the EDHF response, direct proof of that has only been recently demonstrated (Marrelli, 2001). The endothelial Ca²⁺ concentration necessary to elicit the EDHF response was ∼330 nmol/L in the rat MCA (Marrelli, 2001). This concentration is 100 nmol/L greater than the Ca²⁺ threshold for activation of NO synthase (Marrelli, 2001). Consistent with these previous studies, we demonstrate that the EDHF response does not occur at an endothelial Ca²⁺ concentration of 246 nmol/L but appears to be fully active at 468 nmol/L (Fig. 5).

This magnitude for the Ca²⁺ increase is consistent with the concentration of Ca²⁺ necessary to promote cPLA₂ binding to the membrane. For example, at a concentration of 450 nmol/L, 70% of the cPLA₂ was lost from the soluble fraction in a macrophage cell line indicating binding to the membrane. This loss in the soluble fraction was accompanied by activation of cPLA₂ to a similar magnitude (Channon and Leslie, 1990). Not only does our study demonstrate that endothelial Ca²⁺ increases in the MCA upon application of UTP, but also that it increases to levels that are required for activation of cPLA₂.

Events downstream of cytoplasmic form of phospholipase A₂ in the endothelium-derived relaxing factor response

A critical step that remains to be determined regarding the EDHF response in rat MCAs, is the event(s) downstream from PLA₂. The product of the cPLA₂ reaction could be the EDHF itself, could be metabolized into the EDHF, or could simply be a step in the signal process in the EDHF response.

There are a number of possibilities for the product of the cPLA₂ reaction being involved in the EDHF response. Arachidonic acid immediately comes to mind since it has been the most studied product of the enzymatic reaction and PLA₂ has a preference for arachidonic acid containing phospholipids. Metabolites of arachidonic acid, through the lipoxygenase or epoxygenase pathway, have been implicated in the EDHF response in peripheral arteries (Campbell et al., 1996; Pfister et al., 1998; Fisslthaler et al., 1999; Zink et al., 2001). In line with peripheral vessels, arachidonic acid dilated the rat basilar artery through activation of K_Ca and hyperpolarization of the vascular smooth muscle (Faraci et al., 2001). The dilation produced by addition of the arachidonic acid appeared to be due to conversion to 12(S)-hydroxyeicosatetraenoic acid, a metabolite of the lipoxygenase pathway.

The products of these pathways could be synthesized on demand following activation of cPLA₂ and the subsequent liberation of arachidonic acid. Alternatively, these products could be stored in the phospholipid pool and liberated directly from the sn−2 position of the glycerol backbone by the action of cPLA₂. Metabolites of arachidonic acid through the epoxygenase and lipoxygenase pathway (epoxyeicosatrienoic acids, hydroxyeicosatetraenoic acids, and others) can be incorporated into the sn−2 position of the phospholipid and released upon activation of PLA₂ (Richards et al., 1986; VanRollins et al., 1993; Weintraub et al., 1997, 1999; Zink et al., 2001; Fleming, 2001).

We must also consider, however, that there are fatty acids, other than arachidonic acid, or metabolites in the sn−2 position of the glycerol phosphate backbone that, when cleaved by PLA₂, could be involved with the EDHF process in the rat MCA. Often overlooked in the PLA₂ enzymatic reaction are lysophospholipids, the molecule remaining after catalytic action at the sn−2 position of the glycerol backbone. Lysophospholipids can be converted to lysophosphatidic acid by action of phospholipase D. Lysophosphatidic acid is rapidly emerging as an important signaling molecule (Hla et al., 2001) and appears to be a dilator in the rat MCA (unpublished observation).

Although the product of the cPLA₂ reaction, which is involved with EDHF dilations, is not known, we must point out that our studies do not preclude proposed mechanisms for EDHF such as K⁺ from endothelium or gap junctions (Edwards et al., 1998; Chaytor et al., 1998). The product of the cPLA₂ reaction could be the EDHF itself, could be metabolized into the EDHF, or could be a step in the signaling process leading to release of K⁺ from endothelium or gap junction involvement.

In summary, we have demonstrated that activation of PLC, cPLA₂, and an increase in endothelial Ca²⁺ are key steps in mediating the EDHF response in rat MCAs. The increase in endothelial Ca²⁺ was sufficient to promote binding of cPLA₂ to the membrane, the location of the phospholipid substrate.

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Role of Cytoplasmic Phospholipase A 2 in Endothelium-Derived Hyperpolarizing Factor Dilations of Rat Middle Cerebral Arteries

Abstract

Keywords

MATERIALS AND METHODS

Measurement of Ca2+ in endothelium

Drugs and reagents

Statistical analysis

RESULTS

DISCUSSION

Involvement of cytoplasmic form of phospholipase A2 in the endothelium-derived hyperpolarizing factor response

Involvement of phospholipase C

Involvement of Ca2+ in the endothelium-derived hyperpolarizing factor response

Events downstream of cytoplasmic form of phospholipase A2 in the endothelium-derived relaxing factor response

References

Measurement of Ca²⁺ in endothelium

Involvement of cytoplasmic form of phospholipase A₂ in the endothelium-derived hyperpolarizing factor response

Involvement of Ca²⁺ in the endothelium-derived hyperpolarizing factor response

Events downstream of cytoplasmic form of phospholipase A₂ in the endothelium-derived relaxing factor response