© 1995 American Heart Association, Inc.
Articles |
Lysophosphatidylcholine Inhibits Relaxation of Rabbit Abdominal Aorta Mediated by Endothelium-Derived Nitric Oxide and Endothelium-Derived Hyperpolarizing Factor Independent of Protein Kinase C Activation
From the Division of Biochemistry, Glaxo Wellcome Research and Development, Research Triangle Park, NC.
Correspondence to Conrad L. Cowan, PhD, Department of Receptor Biochemistry, Glaxo Wellcome Research and Development, 5 Moore Dr, Research Triangle Park, NC.
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Abstract |
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Abstract Hypercholesterolemia is associated with increased oxidized LDL and impaired endothelium-dependent relaxation (EDR). An inhibitory component of oxidized LDL is lysophosphatidylcholine (LPC). To determine the effect and mechanism(s) of action of LPC on EDR mediated by endothelium-derived nitric oxide (EDNO) and endothelium-derived hyperpolarizing factor (EDHF), rabbit abdominal aortic rings were suspended for measurement of isometric tension and studied under three conditions: control; with 25 mmol/L K+ buffer to isolate relaxation mediated by EDNO; and in rings treated with N
-nitro-L-arginine methyl ester
(L-NAME, 30 µmol/L) to isolate relaxation mediated by EDHF.
Incubation with LPC (10 and 30 µmol/L) for 30 minutes inhibited EDR
in a concentration-dependent manner. LPC (30 µmol/L)
significantly inhibited maximal relaxation to acetylcholine in control,
25 mmol/L K+–, and L-NAME–treated rings (77.1±7.8%,
42.1±8.9%, and 3.4±7.7%) compared with untreated rings
(99.0±0.9%, 90.9±2.2%, and 54.7±4.7%, P<.05).
Inhibition of relaxation was specific to
endothelium-dependent responses in that relaxation
to direct smooth muscle vasodilators (papaverine, 8-bromo-cGMP, and
sodium nitroprusside) were unaltered by LPC. The inhibition by LPC (30
µmol/L) was not due to cytotoxicity, because EDR returned to normal
levels after repeated washing with physiological
salt solution containing 0.1% albumin. Coincubation with
protein kinase C inhibitors, staurosporine (20
nmol/L) or calphostin C (1 µmol/L), had no effect on the EDR
inhibition by LPC (30 µmol/L). Furthermore, LPC continued to inhibit
EDR in rings in which protein kinase C was downregulated by incubation
for 18 hours with 1 µmol/L phorbol 12-myristate 13-acetate
(PMA). The inhibition of EDR to the receptor-independent agonist
A23187 by LPC (30 µmol/L) but not by PMA (30 nmol/L) further supports
a lack of effect of LPC on protein kinase C. Thus, the
inhibitory effect of LPC on EDR is not limited to EDNO but
also inhibits relaxation mediated by EDHF. Also, the inhibition of
relaxation to EDNO and EDHF is not mediated by activation of protein
kinase C.
Key Words: endothelium-derived nitric oxide • rabbit • endothelium-derived hyperpolarizing factor • protein kinase C • lysophosphatidylcholine
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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EDR is inhibited early in the atherosclerotic process, even before histological evidence of plaque formation.1 2 Ox-LDL, a product of endothelial and/or macrophage oxidation of native LDL and present in atherosclerotic lesions,3 is primarily responsible for this inhibition.4 5 6 7 In turn, the inhibitory action of ox-LDL is attributed to LPC, which accumulates in the LDL particle during the oxidative process such that LPC concentrations were found to be eightfold greater in aorta of squirrel monkeys fed an atherogenic diet compared with those fed a normal diet.8 Removal of LPC from ox-LDL with phospholipase B or albumin renders ox-LDL inactive,9 10 11 and conversely, direct treatment of arterial rings with LPC mimics the inhibitory effects of ox-LDL on EDR.9 10 11 12 The effect of LPC on EDR coupled with other actions of LPC, such as acting as a chemoattractant of mononuclear leukocytes and inducing adhesion molecule expression (ICAM-1 and VCAM-1) and monocyte adhesion,13 14 suggests that LPC is likely to play an important role in promoting the atherogenic process.
Previous studies showing LPC inhibition of EDR have reported the total endothelium-dependent response or have focused on the action of LPC on nitric oxide production and bioavailability. However, at least two components account for nonprostanoid EDR: one is nitric oxide or a related nitrosyl compound that acts on the vascular smooth muscle to stimulate guanylate cyclase; the other is distinct from nitric oxide and involves membrane hyperpolarization.15 16 17 18 19 The latter component is mediated by an as yet unidentified factor called EDHF. The contribution of EDHF to EDR may be altered in cardiovascular disease. Thus, while the contribution of EDHF-mediated relaxation was shown to be decreased in hypertension,20 21 the contribution of EDHF was found to be increased in hypercholesterolemic rabbit carotid arteries.22
The effects of LPC in the vascular wall may be related to the effects of LPC on various membrane-associated enzymes that play an important role in intracellular signaling, including activation of protein kinase C23 and guanylate cyclase24 and inhibition of adenylate cyclase.24 Two recent studies showed that pretreatment or coincubation with staurosporine or calphostin C attenuated LPC inhibition of EDR in pig coronary artery and rabbit aorta.25 26 Therefore, the present study investigated the action of LPC on endothelial relaxation mediated independently by EDNO and EDHF. Furthermore, this study addressed the involvement of protein kinase C activation in the effect of LPC on relaxation mediated by each of these factors.
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Methods |
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Male New Zealand White rabbits were killed by exsanguination after anesthesia with sodium pentobarbital (200 mg) and anticoagulation with heparin (1000 U). The abdominal aorta was quickly dissected free and placed in PSS of the following composition (in mmol/L): NaCl 118.0, KCl 4.5, CaCl2 2.5, KH2PO4 1.1, MgSO4 1.2, NaHCO3 25.0, and D-glucose 11.0. Solutions of high-K+ PSS were made by equimolar replacement of NaCl with KCl. Adhering perivascular tissue was carefully removed from the arteries, which were then cut into 3- to 4-mm rings. Removal of the endothelium in desired rings was performed by inserting the tip of a forceps into the lumen and gently rolling the ring on moistened filter paper.
Organ Chamber Studies
For measurement of circumferential isometric force, rings were
suspended on triangular stainless steel wires, one stationary and the
other connected to a strain-gauge transducer (Grass model FT03,
Grass Instruments, Inc) coupled to a polygraph recorder (Grass
model 7D), and electronically collected and analyzed (Modular
Instruments Inc, Biowindow version 1.13 and Bioreport version 1.06).
Rings were placed in 10-mL organ baths containing PSS maintained at
37°C and continuously bubbled with 95% O2/5%
CO2. The rings were stretched in a stepwise fashion to a
basal tension of 6 g. After 30 minutes, during which the bathing
solution was changed every 10 minutes, the rings were contracted with
50 mmol/L K+ PSS for 6 to 10 minutes. The rings were then
washed every 10 minutes for 30 minutes, during which time the passive
tension was adjusted to 6 g. Rings were contracted a second time to 50
mmol/L K+ PSS. Again, the rings were washed every 10
minutes for 40 minutes, during which time the passive tension was
adjusted to 6 g. All vessels were treated with
indomethacin (1 µmol/L) to inhibit prostanoid
production by cyclooxygenase. To test the
vasodilatory activity of compounds, rings were contracted with
phenylephrine and relaxed by cumulative half-log
increases in agonist concentration. The phenylephrine
concentration was adjusted among rings to produce tone 40% to 60%
that of the second K+ contraction. An initial ACh
concentration response was determined in all rings to evaluate
endothelial integrity. The rings were then washed
repeatedly with PSS for 30 to 40 minutes. During the final rinse, the
bathing solution of some rings was replaced with PSS containing 25
mmol/L K+. Rings were exposed to agents as indicated in the
following manner: L-NAME and indomethacin were
present for at least 30 minutes before agonist concentration
response; LPC and PMA were added 30 and 15 minutes before
phenylephrine contraction, respectively; and
staurosporine (20 nmol/L) and calphostin C (1 µmol/L)
were added to the baths 30 minutes before LPC. Equivalent volumes of
vehicle (DMSO) were added to control baths. Experiments with calphostin
C were done under normal fluorescent lighting.27 A
second concentration response with various vasodilators was then
determined to evaluate the effect of these treatments on
endothelium-dependent and -independent relaxation.
Control and treated rings within a group were handled in parallel to
eliminate time- or exposure-dependent effects on the responses.
PMA Contraction
After stepwise stretching to a basal tension of 6 g and two
contractions to 50 mmol/L K+ PSS, rings were treated with
staurosporine (20 nmol/L), calphostin C (0.1 or 1
µmol/L), or vehicle (DMSO, 0.1% maximum) for 30 minutes before
contraction with PMA (1 and 10 µmol/L). Maximal contraction was
reached 90 to 120 minutes after addition of PMA. This experiment was
done in parallel with rings from the same animals as the experiments to
determine the effects of staurosporine and calphostin C on
the inhibitory effects of LPC.
Albumin Wash
After determination of the ACh concentration response of
LPC-treated rings, the rings were washed three times with PSS. The
rings were then washed three times with PSS containing 0.1%
albumin, followed each time by two washes with normal PSS.
Gassing was interrupted while the baths contained the albumin
PSS (3 to 4 minutes) and restored during washing with PSS. Control ACh
concentration responses were determined in rings washed with PSS
containing albumin but not treated with LPC.
PMA Incubation
Rings were placed in DMEM (with 100 U/mL penicillin and 100
µg/mL streptomycin) containing either PMA (1 µmol/L), 4
-PMA (1
µmol/L), or vehicle (DMSO, 0.01%) and incubated for 18 hours at
37°C in a humidified incubator. Agents were not sterilized before
addition to DMEM. For removal of DMEM and incubation, the rings were
washed twice before being placed into the organ chambers for
measurement of isometric tension as described above.
Materials
The following compounds were purchased from Sigma Chemical Co
and prepared daily in saline unless otherwise indicated: ACh
chloride, albumin, calcium ionophore (A23187),
cromakalim, indomethacin (50 mmol/L
Na2CO3 buffered to pH 7.4),
L--LPC palmitoyl, L-NAME,
L-phenylephrine hydrochloride, PMA, SNP,
staurosporine, calphostin C (Kamiya Biomedical Co), and
papaverine (RBI). DMEM, penicillin, and streptomycin were obtained from
GIBCO BRL. PMA, calphostin C, and staurosporine were
dissolved in DMSO, divided into aliquots, and stored frozen until use.
Cromakalim was dissolved in 1 part DMSO and diluted with 2 parts water,
with subsequent dilution in water. A23187 was dissolved in ethanol,
stored at -4°C, and diluted for concentration response in
water. LPC was prepared in PSS and sonicated for 10 minutes before
addition to baths.
Statistical Analysis
Responses to vasodilator agents were determined as the maximum
relaxation after addition of each concentration of agent and calculated
as a percent of phenylephrine-induced tone. Because
of differences in maximal relaxation within the concentration range
studied for a given agonist, the IC50 or IC30
was determined as the negative log molar concentration of agonist that
reduced tone 50% or 30% of initial
phenylephrine-induced tone, respectively. Data are
expressed as mean±SEM; geometric means of drug concentrations were
analyzed. All data are paired, with comparison of groups being
made from rings from the same animal. In all experiments, n equals the
number of animals from which rings were taken. Statistical evaluation
of concentration-response curves was performed by two-way ANOVA
for repeated measures (SAS). IC50 or
IC30, phenylephrine concentration, and
developed tone were compared by Student's t test for paired
comparisons. Results were considered to be significantly different from
control when P<.05.
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Results |
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Endothelium-Dependent Relaxation
ACh caused concentration-dependent relaxation of abdominal aorta under control conditions and in the presence of 25 mmol/L K+ or L-NAME (30 µmol/L) with IC30s of 7.7±0.0, 7.5±0.1, and 6.6±0.1 -log mol/L and maximal relaxations of 99.0±0.9%, 90.9±2.2%, and 54.7±4.7%, respectively (n=12, 12, and 15, grouped controls, Fig 1
). Incubation
of the rings with LPC (10 or 30 µmol/L) for 30 minutes caused a
concentration-dependent inhibition of ACh-induced relaxation.
LPC (10 µmol/L) significantly shifted to the right the
concentration-response curves of control and 25 mmol/L
K+–treated rings and significantly decreased the maximal
relaxation of 25 mmol/L K+–treated rings (Fig 1). LPC (10
µmol/L) had no significant effect on relaxation of L-NAME–treated
rings. LPC (30 µmol/L) significantly shifted to the right the
concentration-response curves of control, 25 mmol/L
K+–treated, and L-NAME–treated rings (Fig 1). Maximal
relaxation to ACh was significantly inhibited by LPC (30
µmol/L) in control, 25 mmol/L K+–treated, and
L-NAME–treated rings (77.1±7.8%, 42.1±8.9%, and 3.4±7.7%, n=9,
9, and 9, P<.05 versus untreated paired controls, Fig 1).
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A23187 caused concentration-dependent relaxation with an
IC50 of 7.4±0.2 -log mol/L and maximal relaxation of
96.9±1.9%. LPC (10 µmol/L) had no significant effect on relaxation
to A23187. LPC (30 µmol/L) significantly increased the
IC50 and inhibited the maximal relaxation (6.6±0.2
-log mol/L and 68.1±6.1%, P<.05 versus untreated
rings, n=7, Fig 2).
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In a separate group of rings, relaxation to ACh, which had been
inhibited by LPC (30 µmol/L) in 25 mmol/L K+– and
L-NAME–treated rings, was completely restored by repetitive washing
with PSS containing 0.1% albumin such that relaxation of rings
treated with LPC and then washed with albumin was not different
from that of control rings that were washed with albumin but
had not been treated with LPC (n=4 or 5, Fig 3).
Furthermore, scanning electron microscopy of rings used in this
experiment showed no difference in appearance of the
endothelium between rings treated with LPC (30
µmol/L) compared with untreated rings.
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Endothelium-Independent Relaxation
LPC (30 µmol/L) had no effect on relaxation to
8-bromo-cGMP or papaverine (Table 1). LPC (30
µmol/L) significantly inhibited the concentration response to
cromakalim, slightly inhibiting the IC50 without
significantly inhibiting maximal relaxation (Table 1).
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The concentration response to SNP of intact rings was significantly
inhibited in a concentration-dependent manner by LPC (ANOVA,
untreated versus LPC-treated, 10 and 30 µmol/L, P<.05,
Fig 4). LPC (10 and 30 µmol/L) inhibited
IC50 and maximal relaxation compared with untreated rings
(IC50, 7.6±0.1 and 7.3±0.1 versus 7.8±0.1
-log mol/L, respectively, P<.05, n=7; maximal
relaxation, 98.7±1.4% and 89.9±4.3% versus 100.6±0.6%,
respectively, P<.05, n=7). Interestingly, this inhibition
was endothelium dependent, because removal of the
endothelium increased the sensitivity of the rings to
SNP and abolished the inhibitory effect of LPC (n=4, Fig 4).
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Protein Kinase C Involvement
PMA (30 nmol/L) inhibited receptor- and
endothelium-dependent relaxation of rabbit
abdominal aorta to ACh, significantly decreasing maximal relaxation
from 99.6±0.5% to 94.8±1.3% and shifting the IC50 from
-7.5±0.05 to -6.9±0.09 (P<.05, n=8). However,
PMA (30 nmol/L) did not alter endothelium-dependent
receptor-independent relaxation to A23187 (n=5, Fig 2
).
Incubation of the rings for 30 minutes with either
staurosporine (20 nmol/L) or calphostin C (0.1 or 1.0
µmol/L) inhibited protein kinase C activation, as demonstrated by the
inhibition of PMA-induced contraction (Table 2).
However, neither staurosporine (20 nmol/L) nor calphostin C
(1.0 µmol/L) affected the inhibitory action of 30
µmol/L LPC on relaxation to ACh of 25 mmol/L K+– or
L-NAME–treated rings (Figs 5 and 6).
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Incubation of rings for 18 hours with PMA (1 µmol/L) blocked
contraction to subsequent 1 and 10 µmol/L PMA compared with rings
incubated with the inactive phorbol ester 4-PMA (1 µmol/L PMA,
-1.6% versus 21.1%; 10 µmol/L PMA, 3.4% versus 56.5% of
tone during contraction 50 mmol/L K+. Relaxation to ACh of
rings incubated with PMA (1 µmol/L) was inhibited compared with those
incubated in 4-PMA (maximum relaxation, 61.6% versus 92.2%,
respectively); however, LPC (30 µmol/L) continued to inhibit
relaxation of rings incubated with PMA (19.2% maximum relaxation, Fig 7). This qualitative (n=2) demonstration of LPC
inhibition of relaxation to ACh in control rings incubated with PMA to
downregulate protein kinase C was also observed for 25 mmol/L
K+– or L-NAME–treated groups (data not shown).
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Discussion |
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The present findings demonstrate that treatment of rabbit abdominal aorta with LPC for 30 minutes nearly abolishes relaxation mediated by EDHF and provides additional support for inhibition of EDNO-mediated relaxation by LPC. Inhibition of EDR by LPC is due to alterations of the endothelium but is not due to cytotoxicity. Finally, in contrast to previous reports, several lines of evidence demonstrate that inhibition of EDR by LPC is not due to stimulation of protein kinase C activity.
Accumulation of ox-LDL and hence LPC has been linked to the atherogenic process. Although both ox-LDL and LPC have been measured in atherosclerotic arterial walls, the actual subendothelial concentration of LPC has not been determined and may reach substantially elevated concentrations compared with that in plasma.6 To study the direct acute effects of LPC on arterial responses, LPC is added to organ chambers containing buffer. Phospholipids such as LPC exist in aqueous buffer as free monomers at concentrations below, and as micelles at concentrations above, the critical micelle concentration.28 29 The critical micelle concentration of LPC in Krebs-Henseleit buffer at pH 7.4 was determined spectrophotometrically to be 40 to 50 µmol/L.28 The action of LPC on cells is related to micelle formation such that alterations of cellular functions occur at concentrations less than, and cytotoxicity occurs at concentrations greater than, the critical micelle concentration.28 30 31 Studies in bovine aortic endothelial cells found that markers of cellular viability, such as dye exclusion, LDH activity, and leucine incorporation, were not altered by concentrations less than, but were altered by concentrations greater than, 40 to 50 µmol/L LPC.30 31 In the present study, LPC (30 µmol/L) did not inhibit smooth muscle vasodilation to papaverine, 8-bromo-cGMP, or SNP (in endothelium-denuded rings), suggesting that this concentration was not cytotoxic to vascular smooth muscle cells. Also, the inhibitory effect of LPC on EDR was completely reversed by washing the LPC-treated rings with PSS containing albumin, suggesting that LPC was not cytotoxic to the endothelial cells. The lack of cytotoxicity of LPC (30 µmol/L) was further supported by scanning electron microscopy, which showed no difference in endothelial integrity between LPC-treated and -untreated rings. That LPC inhibited relaxation to the endothelium-dependent vasodilators ACh and calcium ionophore but did not inhibit smooth muscle vasodilation suggests that the concentration of LPC used in the present study had endothelium-specific effects that were not due to cytotoxicity.
Nitric oxide–independent relaxation probably mediated by EDHF has been demonstrated in rabbit abdominal aorta.32 To study EDR mediated solely by either EDNO or EDHF, elevated extracellular potassium is used to inhibit hyperpolarization17 or an arginine analogue is used to block production of nitric oxide, respectively.33 A combination of elevated external potassium and an arginine analogue is required to nearly abolish endothelium-dependent relaxation.19 32 It was anticipated that investigation of the effects of LPC on relaxation mediated by either mechanism independently would reveal differences that would provide insight into the chemical nature of EDHF or differences in intracellular signaling between EDNO and EDHF. Currently, receptor activation leading to increased intracellular calcium results in release of both EDNO and EDHF. The inhibition by LPC of relaxation mediated by both EDNO and EDHF in the present study suggests that LPC may be acting at a common point in the signal transduction pathways of EDNO and EDHF. In contrast to the present findings of the acute effects of LPC on EDNO and EDHF, a long-term study of the effects of hypercholesterolemia on EDR of rabbit carotid artery found that while the magnitude of relaxation was not altered by cholesterol feeding, the relative contributions of EDNO and EDHF to the relaxation were altered.22 EDR, which was not sensitive to charybdotoxin in the absence of L-NAME in the normal artery, became entirely sensitive to charybdotoxin after 10 weeks of cholesterol feeding, suggesting that the normal contribution of nitric oxide to EDR was abolished in this artery by cholesterol feeding and that the EDHF-mediated relaxation increased to compensate for this loss. The results of the present study suggesting that LPC directly inhibits the production and/or the effect of EDHF (as well as EDNO) could be considered contrary to the previous findings; however, (1) in the hypercholesterolemia study, the increased role for charybdotoxin-sensitive relaxation was seen in the carotid artery but not the abdominal aorta, and (2) the altered response of the cholesterol-feeding study could indicate that an adaptive process takes place over time that compensates for the effects of LPC and can restore or enhance the EDHF response. Thus, the acute effect of LPC, as seen in the present study, inhibits relaxation mediated by both EDNO and EDHF, possibly through inhibition of some common signal transduction mechanism rather than through diverse postproduction destructive actions such as superoxide oxidation of nitric oxide.
Receptor-dependent intracellular signaling of
endothelial cells involves protein kinase C. Activation
of protein kinase C by phorbol esters such as PMA inhibits
endothelium-dependent relaxation.34 35
In the present study, PMA inhibited relaxation of the rabbit
abdominal aorta to ACh, indicating that this tissue has a
PMA-stimulated isoform of protein kinase C. This inhibition may be a
result of a negative feedback response in which protein kinase C
inhibits further receptor stimulation.36 Alternatively,
protein kinase C activation may stimulate superoxide anion
production in endothelial cells,37
which could lead to increased nitric oxide conversion to
peroxynitrite,38 thereby reducing the bioavailability of
nitric oxide. LPC has been shown to alter the activity of several
membrane-associated intracellular signaling enzymes, including
activation of protein kinase C.23 25 26 37 Support for
inhibition of EDR by LPC through activation of protein kinase C was
recently found in rabbit thoracic aortas.26 37 Ohara et
al,37 using a bioassay system, showed that relaxation of a
detector ring (dog coronary artery) to ACh, which was inhibited
by incubating the donor artery (rabbit thoracic aorta) with LPC (10
µmol/L) for 30 minutes, could be substantially restored by
coincubation with calphostin C (50 nmol/L). Ohgushi et
al26 found that coincubation with
staurosporine (20 nmol/L) partially restored relaxation
inhibited by LPC (10 µmol/L), shifting the relaxation toward control
and restoring maximal relaxation to control. In the present study,
staurosporine (20 nmol/L) and calphostin C (1 µmol/L)
nearly abolished contraction to PMA, indicating that treatment of the
arteries with these inhibitors successfully inhibited
activation of protein kinase C. However, in contrast to the studies
cited above, neither staurosporine nor calphostin C
affected inhibition of EDR by LPC. To further investigate whether
protein kinase C activation was required for LPC inhibition of EDR,
rings were incubated for 18 hours with 1 µmol/L PMA, which
downregulates protein kinase C enzyme. After several hours of washing
with normal PSS, rings incubated in PMA did not contract to subsequent
PMA compared with control rings incubated with the inactive phorbol
ester 4-PMA, indicating that incubation in PMA substantially reduced
the protein kinase C enzyme activity. However, LPC (30 µmol/L)
continued to substantially inhibit EDR of rings incubated in PMA.
Although these studies were qualitative because of the effect of PMA
incubation itself on control relaxation, the continued inhibition of
EDR by LPC further indicates that protein kinase C activation did not
play a role in the inhibition of EDR by LPC in the present study. A
third line of evidence dissociating protein kinase C from inhibition of
EDR by LPC is the differing effects of LPC and PMA on relaxation to
calcium ionophore. EDR to calcium ionophore (A23187) occurs
independently of receptor proteins. Therefore, inhibitors
of EDR that act specifically on the proteins involved in receptor
signaling (ie, the receptor, G proteins, PLC, protein kinase C, etc)
and are proximal to the rise in intracellular calcium would not inhibit
EDR to calcium ionophore. Thus, PMA inhibits EDR to
receptor-mediated agonists34 35 but, as shown in the
present study, does not inhibit relaxation to calcium ionophore. In
contrast, LPC (30 µmol/L) inhibited relaxation to
receptor-dependent and -independent EDR, suggesting that the
mechanism of LPC does not involve alteration of proteins involved in
the receptor-operated signal transduction, including protein kinase
C. Collectively, these results rule out protein kinase C involvement in
LPC inhibition of EDR.
There are several differences between the studies that found a role for protein kinase C activation in the inhibitory action of LPC and the present study that might offer some explanation for the contrasting results. Ohara et al37 and Ohgushi et al26 used thoracic aorta (stated and inferred from data, respectively), and Ohara et al used a bioassay system; both factors limit investigation to responses mediated solely by nitric oxide. In those previous studies, 10 µmol/L LPC was used and found to substantially inhibit EDR; in the present study, however, this concentration of LPC had little to no effect on EDR. Increasing the concentration of LPC to 30 µmol/L increased the inhibition of EDR to levels comparable to those previously reported and comparable to inhibition by ox-LDL or hypercholesterolemia. The difference in the effective concentration of LPC may provide another explanation for the differing results regarding the role of protein kinase C. Using a cell-free system, Ohgushi et al26 showed that purified protein kinase C from bovine aortic endothelial cells was stimulated by low (0.5 µmol/L) and inhibited by higher (5 or 10 µmol/L) concentrations of LPC. Another group also reported the biphasic effect of LPC on protein kinase C activity, finding, however, that concentrations of LPC <20 µmol/L stimulated while those >30 µmol/L inhibited activity of protein kinase C.39 This may suggest that studies that find specific effects with low concentrations of LPC are stimulating protein kinase C and that perhaps in the present study, in which slightly higher concentrations of LPC were needed to get substantial inhibition, protein kinase C was inhibited and therefore was not involved.
Another interesting finding of the present study is that in intact but not denuded rings, LPC (10 and 30 µmol/L) inhibited relaxation to SNP. Removal of the endothelium increased the sensitivity of the rings to SNP and removed the inhibitory effect of LPC, suggesting that LPC was acting through the endothelium to inhibit relaxation to SNP. Increased potency of SNP has been reported previously after removal of the endothelium or inhibition of nitric oxide production or action.40 41 42 This effect is thought to be due to the basal nitric oxide release and elevated basal cGMP levels, which depress subsequent nitrovasodilator responses. LPC (1 to 100 µmol/L) has been shown to increase nitric oxide production in a concentration-dependent manner.43 Furthermore, LPC itself was shown to relax rabbit thoracic aorta from normal but not atherosclerotic animals.44 45 These relaxations to LPC were endothelium dependent, were sensitive to hemoglobin and methylene blue, and were correlated to increased cGMP content. In this context, it seems likely that increased basal nitric oxide release mediated the concentration- and endothelium-dependent inhibition by LPC of relaxation to SNP. Thus, a component of the inhibition of EDNO response to ACh may be an endothelium-dependent depression of vascular smooth muscle responsiveness to nitric oxide; however, the small amount of inhibition of SNP concentration response by LPC suggests that LPC inhibits endothelium-dependent relaxation primarily by decreasing agonist-stimulated nitric oxide release.
LPC has been shown to have broad and diverse effects on intracellular signaling processes, including inhibition of inositol 1,4,5-triphosphate production and calcium elevation,25 30 inhibition of G protein–mediated signal transduction,46 47 stimulation of membrane-bound guanylate cyclase from 3T3 mouse fibroblasts,24 inhibition of sodium fluoride stimulation of adenylate cyclase,24 stimulation of nitric oxide synthase,45 48 and stimulation/inhibition of protein kinase C.23 25 26 37 39 Rather than having a direct action on a specific protein, LPC could act to alter the membrane environment with which these proteins are associated and thereby alter their affinity for substrate or cofactors. Alteration of membrane fluidity by LPC could provide an explanation for the diverse reported effects of LPC.39 In a related fashion, the effect of cholesterol on ion channels (Ca2+ and K+) has been linked to alteration of membrane fluidity.49 50 51 52
The results of the present study show that LPC inhibits endothelium-dependent relaxation mediated by both EDHF and EDNO in a manner independent of protein kinase C activation. It is interesting to speculate that the effect of LPC is due to alteration of endothelial cell membrane fluidity, as is suggested by its amphiphilic characteristics and its effects on diverse membrane-associated proteins.
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Selected Abbreviations and Acronyms |
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Received May 24, 1995; accepted October 11, 1995.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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