Bioscienee Reports, Vol. 7, No. 10, 1987
The Role of Lysolecithin in the Relaxation of
Vascular Smooth Muscle
Richard J. Bing and M a y t h e m Saeed
Received October 15, 1987
KEY WORDS: endothelium; vascular relaxation; lysolecithin; phospholipids.
The effect of lysolecithin (lysophosphatidylcholine) on the relaxation of rabbit aortic
strip closely resembled that produced by acetylcholine (ACh) which releases the
endothelium-derived relaxing factor (EDRF). Relaxation induced by lysolecithin
depended on the presence of endothelium and was inhibited by hemoglobin and
methylene blue. It appeared to be mediated by the second messenger, c-GMP.
Lysolecithin induced relaxation was slower but more persistent than that resulting
from the endothelium-derived relaxing factor (EDRF) produced by acetylcholine
(ACh). Like lysolecithin, Triton X-100, a non-ionic detergent, also preferentially
relaxed aortic strips with intact endothelium. The results demonstrate the importance
of phospholipids derived from cell membranes in vascular smooth muscle relaxation.
Endothelium-derived relaxing factors appear as a group of heterogeneous substances.
INTRODUCTION
Local factors have been implicated in the maintenance of vascular smooth muscle tone
with the vascular endothelium playing an important role. For example, endothelial
cells release an endothelium-derived relaxing factor (EDRF) which is inhibited by
lipoxygenase and phospholipase inhibitors and by substances which block guanylate
cyclase (1-3). Cyclooxygenase blockage does not inhibit relaxation. EDRF has a short
half-life of six seconds (4) and is potentiated by free radical scavengers such as
superoxide dismutase (SOD) (5). In addition to relaxing isolated preconstricted
vascular strips, EDRF also dilates coronary arteries of perfused hearts (6) and of hearts
in ,fftu and regulates arterial tone (7). Recently EDRF has been identified as nitric oxide
(NO) (8). Like EDRF, NO induces relaxation of precontracted strips and the
Huntington Medical ResearchInstitutes, Pasadena, California.
783
0144-8463/87/1000-0783505.00/0 9 1987 Plenum Publishing Corporation
784
Bing and Saeed
relaxation is inhibited by hemoglobin and methylene blue (9) and potentiated by
superoxide dismutase (SOD) (5,10).
In a previous publication we furnished evidence of the role of phospholipids in
vascular smooth muscle relaxation and production of c-GMP (11). Prior to this,
Huang and Lee (12) had obtained a dose dependent relaxation with PLAz in
precontracted intact and rubbed aortic preparations, and F6stermann and Neufang
(13) discovered a smooth muscle relaxing effect in the unrubbed preparation by
melittin, an activator of PLA 2. In addition, thimerasol which prevents conversion of
lysolecithin to lecithin relaxed the preparation (14). We found that addition of PLA 2 to
both unrubbed (intact) and rubbed (endothelium-deprived) precontracted rabbit
aortic strips caused relaxation (11). In the rubbed strip relaxation was dose dependent
and was inhibited by methylene blue and hemoglobin. Indomethacin had no effect.
Phospholipase C (PLC) caused a prolonged increase in tension, while phospholipase
D was ineffective. A significant increase in c-GMP occurred in the strips following
onset of relaxation. In the unrubbed strip PLA2 also caused relaxation which was
potentiated by SOD and inhibited by hemoglobin.
The present report demonstrates that lysolecithin (lysophosphatidylcholine) is a
phospholipid involved in the relaxation of vascular smooth muscle. It is entirely
possible that other enzymatic products of lecithin possess similar relaxing properties.
Relaxation depends on the presence of endothelium and is strongly inhibited by
methylene blue, hemoglobin, partially inhibited by nordihydroguiaretic-acid
(NDGA), but not by indomethacin; it is potentiated by superoxide dismutase.
MATERIALS AND METHODS
A total of 20 rabbits were used; seven aortic strips were obtained from each animal
for the study of the effect of lysolecithin and its inhibitors. The effect of lysolecithin was
tested by bioassay of rabbit aortic strips suspended in oxygenated Krebs-Henseleit
solution at a resting tension of 1.5 g. Male white New Zealand rabbits weighing 2.43.1 kg were anesthetized with pentobarbital (30mg/kg) and heparinized with
500 IU/kg I.V. Tracheostomy was performed and the animals were ventilated with a
respirator (Bird Mark 10, Space Tech., Palm Springs, CA) to assure sufficient oxygen
supply. Median sternotomy was performed and the thoracic aorta was removed and
immersed in ice cold Krebs-Henseleit solution (15). After removal of adjacent
superficial connective and adipose tissue, the aorta was cut in rings of about 3 mm in
width. These rings were cut into transverse strips (15). Endothelium was removed by
gently rubbing the intimal surface with moistened filter paper wrapped around a
wooden stick.
Strips were mounted in an organ chamber of 20ml capacity with both ends
fastened (15). One end was tied to the bottom of the chamber, the other end was
attached to an isometric pressure transducer (UL-20-Gr, Shinkoh, Minebea Co. Ltd.,
Tokyo, Japan). The chambers were carefully oxygenated with 95 ~o Oz and 5 ~o CO2 by
slow bubbling to prevent foaming. Strips were allowed to equilibrate for 60 minutes,
and basal tension of the strips was adjusted to 1.5 g.
Tension development was induced by addition of histamine (10 -5 M) to the
Smooth Muscle Relaxation
785
organ chamber. After a steady state was reached, acetylcholine (ACh) (10 - 6 M) was
added to induce EDRF release by the endothelial cells. In four experiments lysolecithin
was dissolved in Triton X-100 (10 -6 M) and control observations were carried out
with Triton X-100 alone. After it was found that Triton X-100 alone caused relaxation
mainly of the unrubbed precontracted strip, lysolecithin was dissolved in DMSO.
Suspension was prepared by placing lysolecithin powder (5 rag) on a Watch glass
adding 100 pl DMSO plus 0.5 ml Krebs-Henseleit gradually and stirring continuously
wJ:th a glass rod until all solid particles had disappeared. Lysolecithin (10-7, 10-6 and
10-5 M) in DMSO (heretofore referred as lysolecithin) was then added to the muscle
bath. Hemoglobin (10- 6 and 10- 5 M) or methylene blue (10- 5 M) were added during
lysolecithin induced relaxation while indomethacin (t0 -5 M) and (3x10 -5 M)
NDGA were added 20 minutes prior to precontraction with histamine (11).
Superoxide dismutase (SOD, 150 U/ml) (10) was administered after the addition of
lysolecithin.
All chemicals were purchased from Sigma Chemical Co., St Louis, MO.
Lysolecithin is prepared by PLA 2 from egg L-c~-phosphatidylcholine. It contains
primarily palmitic and stearic acids.
RESULTS
The effect of lysolecithin (10 -7, 10 -6 and 10 .5 M). on the precontracted
unrubbed aortic strip is shown in Fig. 1. Lysolecithin (10 -6 M) resulted in a slight
Onrubbed Str~p
renswn
~ mm
Z~ &
.:,
L;,~,,,.,,,~
O5
Twns~on
;00 ~B
~50u/m~
-~
Rt~bbeOStrip
,2. ,;.
,o'.
,0",;~ ,;....... ,;'.
Fig. 1. The effect of lysolecitbin, superoxide dismutase
(SOD) and methylene blue (Mt3) on reIaxation of intact and
endothelium-deprived aortic strips is shown. In the intact
(unrubbed) preparation lysolecithin induced relaxation was
inhibited by methylene blue and potentiated by SOD. In the
endothelium-deprived (rubbed) preparation, the effect of
lysolecithin and methylene blue was attenuated. The
comparatively rapid relaxation with acetylcholine is shown.
All preparations were precontracted with histamine (HA).
786
Bing and Saeed
Unrubbed
Strip
3 rain
Ten|lOt1
(9)
,& ,0-~,
w
"7~176
~
-
,,
-~
mm
Fig. 2. The effect of hemoglobin (Hb) and nordihydroguiareticacid 0NDGA) on lysolecithin induced relaxation in the unrubbed
aortic strip are illustrated. NDGA partially inhibits relaxation,
while hemoglobin inhibition is more complete. Comparative
effects of acetylcholine are also shown. All preparations were
precontracted with histamine (HA).
decrease in tension, while 10 .5 M lysolecithin caused a marked decline in tension
which is comparable to the relaxation induced by ACh (10- 6 M) (Fig. 1). As compared
to the relaxation following ACh (10 -6 M), the fall in tension with lysolecithin was
more gradual. As shown in Figs 1 and 2 both hemoglobin (10 -6 and 10 -5 M) and
methylene blue (10-5 M) completely inhibited relaxation. Indomethacin (10- 5 M)
had no effect on relaxation induced by lysolecithin. Nordihydroguiaretic-acid
(NDGA) partially inhibited relaxation (Fig. 2). Relaxation was slightly potentiated by
superoxide dismutase (SOD) (Fig. 1). The addition of DMSO alone to the bath had no
effect on tension, while Triton X-100 caused marked relaxation of both unrubbed and
rubbed preparations (Fig. 3).
In the rubbed strip the effect of lysolecithin was markedly reduced (Fig. 1). The
decline in tension was very slight and extended over several minutes. As expected, ACh
had no effect on developed tension in the rubbed strip. Hemoglobin did not alter
tension, while methylene blue caused a gradual increase (Fig. 1).
DISCUSSION
Lysolecithin (lysophosphatidylcholine) is a smooth muscle relaxant in the
unrubbed rabbit aortic strip (Figs 1 and 2). In the rubbed preparation, relaxation is
considerably reduced (Fig. i). In the unrubbed strip, hemoglobin (Fig. 2) and
methylene blue (Fig. 1) inhibit relaxation of lysolecithin. Hemoglobin is known to
Smooth MuscleRelaxation
re,,$1on
787
Un~bbed str~p
3 mm
r (q)
Rubbed Steep
3 rain
T,;;....
:]/
4
-
~
',Z ....
4
M'A
rrlr~ x- ~O0
Fig. 3. The effect of the detergent Triton X-100 on the
unrubbed and rubbed aorta is shown. Relaxation with
7.5 x 10 .4 M of Triton X-100 is more pronounced in the
unrubbed preparation. For comparative purposes, the
effect of acetylcholine is also shown. All preparations
were precontracted with histamine (HA).
block almost completely the relaxation and increase in cyclic GMP induced by
acetylcholine, by binding to EDRF; methylene blue mediates oxidation of a
component ofguanylate cyclase after entering the cell thus inhibiting relaxation. These
substances also abolish the coronary arterial relaxation elicited by NO (16). The
difference in response to rubbed to unrubbed preparation may be the result of
diminished c-GMP content in the rubbed preparation, since the c-GMP of unrubbed
strips exceeds that of endothelium-deprived preparations (11,17). The possibility must
be considered that the action oflysolecithin may be in some way related to that of nitric
oxide (NO). One apparent difference is that the acetylcholine induced relaxation which
is due to EDRF causes rapid relaxation (15 seconds), while relaxation with lysolecithin
is greatly prolonged (10 minutes). However definite answers to this question cannot be
obtained with certainty until cross tolerance experiments between nitroglycerine and
lysolecithin suggested by Molina (24) have been performed.
The effect of lysolecithin in eliciting smooth muscle relaxation is not unexpected
on l~hebasis of findings by Huang and Lee (12), by F6stermann and Neufang (13) and
by Bing and Saeed (1 i). The latter workers demonstrated that PLA 2 releases a relaxing
factor in unrubbed and rubbed precontracted rabbit aortic strips (11). In the former,
788
Bing and Saeed
PLA 2 induced relaxation is inhibited by hemoglobin, methylene blue and
parabromphenylbromide (PBPB), and is slightly potentiated by superoxide dismutase
(11). Relaxation is accompanied by a rise in c-GMP. In the unrubbed preparation,
PLA 2 causes a biphasic response in tension but no elevation in c-GMP, probably
because the rise in c-GMP is delayed by the contraction. In the rubbed strip, methylene
blue and hemoglobin have little or no effect in inhibiting relaxation. This is in line with
the finding by Ignarro (17), that endothelium-denuded rings are less sensitive to
methylene blue than endothelium-intact rings. Huang and Lee (12) observed dose
dependent relaxation in precontracted aortic rings in both rubbed and unrubbed
preparations. These workers found no effect of indomethacin, but methylene blue
inhibited PLA 2 induced relaxation. F6stermann and Neufang (13) discovered that the
PLA 2 activator, melittin, relaxes unrubbed aortic strips; furthermore, an inhibitor of
LAT (acyl-CoA-lysolecithin acyltransferase) which converts lysolecithin to lecithin
such as thimerasol causes concentration-dependent relaxation if the endothelium is
intact.
The relaxing effect of Triton X-100 (Fig. 3) on the bioassay preparation deserves
some comments. Triton X-100 has been reported to increase guanylate cyclase activity
in mammary gland homogenates by several fold (18). Fujimoto and Okabayashi (19)
found that Triton X-100, similar to phospholipase A 2 acts primarily on particulate
guanylate cyclase, and that perturbation of membrane architecture rather than
solubilization account for stimulation of guanylate cyclase (19). On the other hand,
Zwiller found that the effect of lysolecithin was not identical to that of Triton X-100,
since solubilization of guanylate cyclase activity was much lower with lysolecithin (20).
In any case, the similarity in response to Triton X-100 and lysolecithin, both
detergents, suggests a common action on the cell membrane.
Lysolecithin is a widely distributed and naturally occurring detergent (21). Like
PLA 2, it activates guanylate cyclase (20). Shier et al. (23) observed that the guanylate
cyclase activity in membrane preparations from mouse fibroblasts is stimulated 5-fold
by lysolecithin at concentrations of 100/~g/ml and above.
Our findings illustrate that lysolecithin in the presence of endothelium is a
consistent and powerful relaxant of rabbit aortic strips. The rate of relaxation is much
more gradual than that resulting from the addition of acetylcholine. Similar to EDRF
(NO) relaxation is inhibited by methylene blue and hemoglobin slightly potentiated by
SOD and partially inhibited by NDGA. Indomethacin has no effect. Therefore
lysolecithin, similar to PLA 2 and EDRF causes relaxation through formation of cGMP. Apparently NO mediated relaxation is not the only endothelium-derived
mechanism inducing relaxation. Endothelial relaxing factors appear as a group of
heterogeneous substances some of them originating in membranes of endothelial cells.
ACKNOWLED GEMENTS
Supported by grants from The Council for Tobacco Research-USA, Inc., New
York, New York; Margaret W. and Herbert Hoover Jr Foundation, Pasadena,
California; The Sam S. Stein and Rose Stein Charitable Trust, Sandusky, Ohio and the
Patron Saint Foundation, Pasadena, California. We thank Dr Dougherty from the
California Institute of Technology for advice and discussion.
Smooth Muscle Relaxation
789
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