MS 2512, pp. 253-265 Journal of Physiology (1994), 477.

2 25,3

Mechanisms of vasoconstriction induced by endothelin- 1 in

smooth muscle of rabbit mesenteric artery
Makoto Yoshida, Akito Suzuki and Takeo Itoh*
Department of Pharmacology, Faculty of Medicine, Kyushu University,
Fukuoka 812, Japan
1. The mechanism underlying the vasoconstriction induced by endothelin-1 (ET-1) was
investigated by measuring the intracellular concentration of Ca2+ ([Ca2`]1), isometric
force and phosphorylation of the myosin light chain (MLC) in endothelium-denuded
unskinned and ,b-escin-treated skinned smooth muscle from resistance vessels of the
rabbit mesentery. The role of protein kinase C (PKC) in the action of ET-1 was studied in
skinned smooth muscle using a synthetic peptide, PKC19-36, which corresponds to the
autoinhibitory domain of PKC.
2. ET-1 (> 01 nM) induced slowly developing, maintained increases in [Ca2+]i and force.
Nicardipine completely blocked the ET-1-induced increase in [Ca2+]1. BQ-123 (an inhibitor
of the ETA receptor) blocked the ET-1-induced contraction but IRL 1620 (Suc-[Glu9,
Ala`"5]-ET-1(8-21), an agonist of the ETB receptor) failed to induce contraction.
3. In ionomycin- and 70 mm K+-treated strips, ET-1 shifted the [Ca2+]1-force relationship to
the left and enhanced the maximum amplitude of contraction induced by 2-6 mm Ca2+.
In skinned smooth muscle treated with ionomycin, Ca21 (041-3 uM) increased both force
and MLC phosphorylation, in a concentration-dependent manner. ET-1 with GTP
shifted both the Ca2+-force and Ca2+-MLC phosphorylation relationships to the left
without significant changes in the maximum responses. ET-1 with GTP did not change
the relationship between force and MLC phosphorylation. Similar effects were observed
with phorbol 12,13-dibutyrate (PDBu, an activator of PKC). These results indicate that
the sensitivity of MLC phosphorylation to Ca2+ is enhanced both by ET-1 with GTP and
by PDBu.
4. PKC19-36 (an inhibitor of PKC) modified neither the contraction nor MLC
phosphorylation induced by 0 3 /IM Ca2+ but blocked the PDBu-induced enhancement of
both these Ca2+-induced responses. However, PKC1936 only partly inhibited the
enhancement produced by ET-1 with GTP on the Ca2+-induced responses. PKC19 36did
not modify the relationship between force and MLC phosphorylation in the presence
either of ET-1 with GTP or of PDBu. By contrast, BQ-123, neomycin and guanosine 5'-O-
(2-thiodiphosphate) (GDP/JS) each abolished the ET-1-induced enhancement of the
contraction induced by 0 3 /SM Ca2+.
5. These results suggest that ET-1 acts on the ETA receptor and increases Ca2+ influxes
through an activation of the dihydropyridine-sensitive Ca2" channel, causing a long-
lasting and maintained contraction in resistance vessels of the rabbit mesentery. ET-1
increases Ca2+-induced MLC phosphorylation through an activation of both PKC-
dependent and -independent mechanisms and thus causes an increase in the sensitivity
of contractile proteins to Ca2+.

Endothelin is one of a family of peptides which includes a locally acting paracrine hormone rather than a
endothelin-1 (ET-1), endothelin-2, endothelin-3 (ET-3) and circulating hormone (Haynes & Webb, 1993). The
sarafotoxins, and ET-1 is known to be one of the most immunoreactive concentration of ET-1 is increased in
potent naturally occurring vasoconstrictors (Yanagisawa et response to various types of stimuli, such as shear stress,
al. 1988; Yanagisawa & Masaki, 1989). ET-1 is thought to be hypoxia, growth factors and so on. Circulating
*To whom all correspondence should be addressed.
254 M. Yoshida, A. Suzuki and T Itoh
concentrations of endothelin-like immunoreactivity in
venous plasma are estimated to be 0-25-20 pg ml' and this
immunoreactivity comprises proendothelin-1 (65 %), ET-1
(25 %) and ET-3 (10 %). Circulating concentrations of ET-1
are approximately tenfold lower than those which cause
vasoconstriction, although the local concentrations of ET-1
are expected to be higher (for a review see Haynes & Webb,
1993).
Two receptors for endothelins are known: a selective
receptor for ET-1 (ETA receptor) and a non-selective ETB
receptor (Arai, Hori, Aramori, Ohkubo & Nakanishi, 1990;
Sakurai et al. 1990). It was originally thought that,
whereas the ETA receptor mediates the ET-1-induced
vasoconstriction, the vasodilating effects are mediated by
the ETB receptor on endothelial cells. It has now been
suggested that both endothelin receptors may mediate the
vasoconstriction induced by endothelins in some vascular
tissues. For example, ET-1 induces vasoconstriction
through an activation of a non-A subtype of ET receptors
in rabbit pulmonary artery and saphenous vein (Ihara et
al. 1992b; Moreland, McMullen, Delaney, Lee & Hunt,
1992b). A possible role for ET-1 has been suggested in the
pathogenesis of hypertension on the basis of its potent
vasoconstricting activity, but it has not yet been
determined which receptors contribute to the ET-1-induced
vasoconstriction in small-diameter resistance vessels.
Recently, agents that act selectively on ETA and ETB
receptors have been synthesized: thus, BQ-123 is a selective
ETA receptor antagonist (Ihara et al. 1992a) while IRL 1620
(Suc-[Glu9, Ala11"5]-ET-1(8-21)) is a selective ETB receptor
agonist (Takai et al. 1992). These peptides should help the
pharmacological characterization of the endothelin
receptors distributed in small resistance vessels.
When ET-1 binds to its receptor, it promotes the
hydrolysis of phosphatidylinositol 4,5-bisphosphate,
forming inositol 1,4,5-trisphosphate (IP3) and diacyl-
glycerol (DAG) (Griendling, Tsuda & Alexander, 1989;
Ohlstein, Horohonich & Hay, 1989). IP3 releases Ca2" from
the cellular storage sites and DAG activates protein kinase
C (PKC). ET-1 also activates Ca2" influxes through an
activation of dihydropyridine-sensitive Ca2" channels in
vascular smooth muscle cells (Goto et at. 1989; Inoue, Oike,
Nakao, Kitamura & Kuriyama, 1990). Thus, it has been
suggested that, in vascular smooth muscle cells, ET-1
increases the intracellular concentration of Ca2+ ([Ca2+]i)
through an activation of both Ca2+ influx and Ca2+ release
and thus causes vasoconstriction. However, the relative
importance of these Ca2+-mobilization mechanisms in the
ET-1-induced contraction in small resistance vessels has
not been established.
It was recently found that Ca2"-mobilizing agonists
increase the sensitivity of contractile proteins to Ca21
through an activation of unidentified GTP-binding
proteins in skinned smooth muscle (Kitazawa, Kobayashi,
Horiuti, Somlyo & Somlyo, 1989; Itoh, Kajikuri &
Kuriyama, 1992). ET-1, like other Ca2+-mobilizing agonists,
J. Physiol. 477.2
increases the sensitivity of contractile proteins to Ca21
(Rembold, 1990; Nishimura, Moreland, Ahn, Kawase,
Moreland & van Breemen, 1992). It has been suggested that
PKC may play a primary role in this ET-1-induced Ca21
sensitization in vascular tissue (Ohlstein et al. 1989;
Nishimura et al. 1992). However, evidence to the contrary
has also been reported (Moreland, Cilea & Moreland, 1992a;
Shimamoto, Shimamoto, Kwan & Daniel, 1992). Thus, at
present, the mechanism of the ET-1-induced Ca2+
sensitization in vascular smooth muscle remains unclear.
In the present experiments, using very small resistance
vessels of the rabbit mesentery, we first tried to
characterize the receptors which contribute to the ET-1-
induced contraction. In order to determine the
contribution of dihydropyridine-sensitive Ca2+ influx to
the ET-1-induced contraction, we then studied the effect of
nicardipine (a dihydropyridine-type Ca21 channel blocker)
on the ET-1-induced increases in Ca2+ mobilization and
force. Thirdly, we studied the mechanism that underlies
the ET-1-induced increase in the sensitivity of contractile
proteins to Ca21 in ionomycin-treated, unskinned and in
,-escin-skinned smooth muscles. Finally, to study the role
of PKC in the ET-1-induced enhancement of Ca2+-induced
contraction, the effect was determined of a selective
inhibitor of PKC (PKC19-36, which corresponds to the
autoinhibitory region of PKC; House & Kemp, 1987) on the
contraction and myosin light chain (MLC) phosphorylation
that were both induced by Ca2+ in the presence and absence
either of ET-1 with GTP or of phorbol 12,13-dibutyrate
(PDBu; an activator of PKC) in skinned smooth muscle.
METHODS
Male albino rabbits, weighing 1-9-2 5 kg, were anaesthetized
with pentobarbitone sodium (40 mg kg-', i.v.) and then
exsanguinated. A segment of the third branch of the
mesenteric artery distributing to the region of the ileum
(diameter around 70 ,um) was excised immediately and cleaned
by removal of connective tissue in Krebs solution at room
temperature.
Experiments on unskinned smooth muscle
To enable the simultaneous recording of [Ca2+], and isometric
force, fine circularly cut strips of the vessel (0 3-0 5 mm long,
0-04-0-05 mm wide, 0-02-0-03 mm thick) were prepared as
previously described (Itoh et al. 1992). Endothelial cells were
removed by the gentle rubbing of the internal surface of the
vessel using small knives. The absence of endothelial cells was
confirmed by the inability of acetylcholine (1 /SM) to cause
relaxation during contractions induced by noradrenaline (NA),
as described previously (Itoh et al. 1992). The strip was
transferred into a chamber of 0-2 ml volume and mounted
horizontally on an invert-microscope (Diaphoto TMD with
special optics for epifluoresence; Nikon). The resting force was
adjusted so as to obtain a maximal contraction in 128 mm K+.
To enable loading of fura-2 into smooth muscle cells of the
strip, 1 /M acetoxymethyl ester of fura-2 (fura-2 AM) dissolved
by dry dimethyl sulphoxide (1 mm stock solution) was applied
J. Physiol. 477.2 Effect of PKC on ET-induced vasoconstriction
for 1 h in Krebs solution at room temperature (20 C), as
reported previously (Itoh et al. 1992). Two alternative
excitation wavelengths, 340 and 380 nm (each slit 5 nm) were
applied by a spectrofluorimeter (Spex, Edison, NJ, USA) and
the data analysed using customized software provided by Spex
(DM-3000CM). The ratio of the fura-2 fluorescence intensities
excited by light of wavelength 340 or 380 nm was calculated
after subtraction of the background fluorescence. Background
fluorescence (including the autofluorescence of the strip) as
excited by 340 and 380 nm UV light was measured following
application of a solution containing 50/M ionomycin, 20 mM
MnCl2, 110 mm KCl and 10 mm 3-(N-morpholino)propane-
sulphonic acid (Mops) after the experiment. Under these
conditions, the background fluorescence intensity was 10-15 %
of the fura-2 signals in smooth muscle strips at either
excitation wavelength. Cytosolic Ca2+ concentrations were
calculated using the formula described by Grynkiewicz, Poenie
& Tsien (1985) and using in vitro calibration (Itoh et al. 1992).
Experiments on chemically skinned smooth muscle
Chemically skinned smooth muscle strips were made using
/?-escin (Itoh et al. 1992). The methods used to make skinned
muscles and the composition of the solutions have been
described elsewhere (Itoh, Kanmura & Kuriyama, 1986). When
the Ca2"-force relationship was to be determined, the
concentration of EGTA in the solution was 4 mm and 1/SM
ionomycin was applied to avoid spurious effects due to Ca2"
release from intracellular storage sites in the skinned muscle.
To prevent deterioration of the Ca2+-induced contraction,
0-1/SM calmodulin was applied throughout the experiments, as
described previously (Itoh et at. 1986). Various concentrations
of Ca21 were cumulatively applied from low to high
concentration. The amplitudes of the contractions induced by
various concentrations of Ca2+ were normalized with respect to
that induced by 10/SM Ca21 in the same strip.
Measurements of myosin light chain phosphorylation
Muscle strips (0-2-0-3 mm long, 1P2 mm wide, 0-02-0-03 mm
thick) were suspended in a relaxing solution for over 10 min.
Then the tissues were skinned using a relaxing solution
containing 30 /zM /-escin with 1/M ionomycin for 25 min and
washed again with the relaxing solution (containing no fl-escin).
Each skinned muscle strip was then suspended in solutions
containing various concentrations of Ca21 for 15 min in the
presence or absence either of 10 nM ET-1 with 100/SM GTP or of
0-1 /M PDBu. The strips were quick-frozen with 10 %
trichloroacetic acid (TCA) in acetone-dry ice containing 10 mM
dithiothreitol and allowed to reach room temperature. The
strips were then washed 3 times with acetone to remove
residual TCA. Protein, including myosin light chain (MLC),
was extracted in a lysing solution containing 8 M urea, 20 mM
Tris, 23 mm glycine, 10 mm dithiothreitol, 01004% Bromo-
phenol Blue and saturated sucrose (pH 8 6) (Persechini, Kamm
& Stull, 1986).
Non-phosphorylated, monophosphorylated and diphos-
phorylated forms of MLC were separated by urea-glycerol
polyacrylamide gel electrophoresis followed by electrophoretic
transfer of the proteins to nitrocellulose paper and
quantification of the relative amounts of each form by an
immunoblot procedure, as reported previously (Persechini et
at. 1986). Antibody against bovine tracheal MLC was used as a
first antibody and biotin-labelled goat anti-rabbit IgG
(Histofine, Seikagaku Kogyo, Tokyo, Japan) as a second
antibody. Alkaline phosphatase-labelled streptavidine was
255
then applied. Regions containing MLC were visualized as dark
blue bands after incubation with the colour development
reagents, 5-bromo-4-chloro-3-indolyl phosphate toluidine and
p-nitroblue tetrazolium chloride (alkaline phosphatase
substrate kit, Vector Laboratories, Burlingame, CA, USA).
Relative amounts of non-phosphorylated, monophosphorylated
and diphosphorylated MLC were quantified densitometrically
by scanning the blot using a chromatoscanner (CS-930,
Shimadzu, Japan). The phosphorylation of MLC was expressed
in moles of phosphate per mole of MLC.
Calculation of Hill coefficient
The slope of the concentration-response relationship for the
effect of Ca2" on force and MLC phosphorylation is shown as
the Hill coefficient (n) and mid-point position (pK= -log K,
where K is the dissociation constant). These were obtained by
fitting the data points for each curve to eqn (1) by a non-linear
least-squares method:
F/Fo = (C/K)n/ [1 + (C/K)n], (1)
where C represents the concentration of Ca2+, F is the
amplitude of contraction at any given concentration of Ca2",
and Fo is the maximum response evoked by 10/SM Ca2"
expressed as a relative force of 1-0.
Solutions
The ionic composition of the Krebs solution was as follows
(mM): Na+, 137-5; K+, 5 9; Mg2", 1-2; Ca2", 2-6; HC03-, 15-5;
H2PO4-, 1-2; Cl-, 134-3; glucose, ll*5. The concentration of K+
was modified by replacing NaCl with KCl, isosmotically. To
prevent both NA outflow from sympathetic nerve terminals
and /J-adrenoceptor stimulation by exogenously applied NA,
3/SM guanethidine (Mishima, Miyahara & Suzuki, 1984) and
1/SM propranolol were added to the Krebs solution throughout
the experiment. Ca2+-free Krebs solution was made by
substituting an equimolar concentration of MgCl2 for CaCl2
and adding 2 mm EGTA. The solutions were bubbled with
95% 02-5 % C02, and their pH maintained at 7 3-7A4.
The calibration solution for Ca21 measurement in intact
strips contained 11 mm EGTA, 110 mm KCl, 1 mM MgCl2, 2 /M
fura-2 and 20 mm N-2-hydroxyethylpiperazine-N'-2-ethane-
sulphonic acid (Hepes) (pH 7-1) with or without 11 mm CaCl2.
For experiments on skinned muscle, the composition of the
relaxing solution was 87 mm potassium methanesulphonate
(KMS), 20 mm piperazine-N-N'-bis(2-ethanesulphonic acid)
(Pipes), 5*1 mm Mg(MS)2, 5-2 mm ATP, 5 mm phosphocreatine
and 4 mm ethyleneglycol-bis-(f-aminoethylether)-N,N,NN'-
tetraacetic acid (EGTA). Various Ca21 concentrations were
prepared by adding appropriate amounts of Ca(MS)2 to 4 mM
EGTA, based on the calculation reported previously (Itoh et al.
1986). The pH of the solution was adjusted to 741 at 25 C with
KOH and the ionic strength was standardized at 0-2 M by
changing the amount of KMS added.
Drugs
Drugs used were ET-1 (Peptide Institute, Minoh, Japan),
fura-2, fura-2 AM, EGTA, Pipes and Hepes (Dojin,
Kumamoto, Japan), PKC19-36 (Peninsula Laboratories,
Taylor, CA, USA), calmodulin, NA, GTP and ,J-escin (Sigma,
St Louis, MO, USA), guanethidine (Tokyo Kasei, Tokyo,
Japan), ATP (Na salt; Kojin, Tokyo, Japan), PDBu (Wako
Pure Chemical, Tokyo, Japan), propranolol (Nacalai, Kyoto,
Japan) and ionomycin (free acid; Calbiochem, La Jolla, CA,
USA). BQ-123 and IRL 1620 (Suc-[Glu9, Ala" 15]-ET-1(8-21)
256 M. Yoshida, A. Suzuki and T Itoh
were kindly provided by Banyu Pharmaceutical Co. Ltd
(Tsukuba, Japan) and Ciba-Geigy Ltd (Takarazuka, Japan),
respectively. The antibody against bovine tracheal MLC was
provided by Dr J. T. Stull (Department of Physiology and
Pharmacology, University of Texas Southwestern Medical
Center, Dallas, TX, USA).
Statistics
The values recorded were expressed as means + S.D. and
statistical significance determined using Student's paired or
unpaired t test. Probabilities less than 5% (P< 0-05) were
considered significant.
RESULTS
The ETA receptor mediates ET-1-induced
contraction
Figure IA compares the effects of 128 mm K+, 10 /M
noradrenaline (NA) and 10 nM ET-1 on mechanical activity
in a small and very thin smooth muscle strip of the rabbit
mesenteric artery. High K+ and NA each produced a
phasic, followed by a tonic contraction. ET-1 (10 nM)
induced a slowly developing, maintained contraction. The
vasoconstricting activity of ET-1 was observed to occur at
concentrations from 01 to 3 nm, in a concentration-
dependent ,nanner (not shown). The maximum amplitudes
of contraction induced by 10,UM NA and 10 nM ET-1 were
respectively 0'68 + 0415 and 0 93 + 0-28 times that induced
by 128 mm K+ (n = 5). IRL 1620 (1 /M, an ETB selective
agonist) did not produce contraction, but subsequently
5 mg
128 mM K+ 10OuM NA 10 nM ET-1
B Figure 1. Effects of 128 mm K+, 10 /bM noradrenaline (NA)
5 mg
5 min
1 sM IRL 1620 10 nM ET-1
C Each agent was applied as indicated by the bar.
5 mg
10 min
0-1 ltM BQ-123
J. Physiol. 477.2
applied 10 nm ET-1 did (Fig. 1B). BQ-123 (041SM, an ETA
receptor antagonist) did not produce contraction, but
completely blocked the contraction induced by ET-1. The
action of BQ-123 was reversible: following wash-out of this
antagonist, ET-1 produced contraction (Fig. 1C).
ET-1 increases [Ca2+]i through an activation of
the dihydropyridine-sensitive Ca2" channel
Figure 2A shows the effects of 128 mm K+, 10 ,UM NA and
10 nM ET-1 on increases in [Ca2+]i and force in a thin
smooth muscle strip of the rabbit mesenteric artery. The
resting [Ca2+]i and force in the muscle strips were
123-4 + 2741 nm and 0-8 + 0 3 mg, respectively (n = 4). High
K+ produced a phasic, followed by a tonic increase in both
[Ca2+]i and force. The maximum increases in [Ca2+]i and
force were 80341 + 315-8 nm and 11-1 + 4-3 mg, respectively
(n = 4). NA produced a large phasic, followed by a small
tonic increase in [Ca2+]i and force. NA induced phasic and
tonic increases in [Ca2+]i (the latter measured 2 min after
the application) of 5231 + 191P2 and 179-5 + 25-6 nM,
respectively (n = 4). The NA-induced phasic and tonic
contractions took the level to 8-9 + 3-6 and 4-8 + 2-9 mg,
respectively (n = 4). ET-1 slowly increased both [Ca2+], and
force, the effects being maintained for over 30 min. The
maximum increases in [Ca2+]i and force induced by ET-1
were 174-1 + 23-3 nm and 6-7 + 2-0 mg, respectively. When
1 FM nicardipine was applied during the ET-1-induced
maintained contraction, the increase in [Ca2+]i induced by
10 nM ET-1 was completely blocked, but 10-20 % of the
ET-1-induced force remained.
and 10 nM endothelin-1 (ET-1) on mechanical activity in
smooth muscle strips of rabbit mesenteric artery
A, stimulants were applied at 10 min intervals in a single
smooth muscle strip. B, IRL 1620 (1 FM) was applied for 10 min
and washed out for 8 min, then ET-1 was applied. C, BQ-123
(0-1 uM) was pre-treated for 20 min and ET-1 applied in the
presence of BQ-123 followed by a wash-out of BQ-123 only.
J. Physiol 477.2 Effect of PKC on ET-induced vasoconstriction2 257

Figure 2B shows the relationship between [Ca2+]i and
force during the tonic phase induced by 128 mm K+, 10 /M
NA or 10 nm ET-1. The relationships were obtained 2 min
after application of high K+ or NA and after 10 in
application of ET-1. ET-1 produced a larger contraction
with a smaller increase in [Ca2+]i when compared to high
K+.
ET- 1 increases the sensitivity of contractile
proteins to Ca`+ in unskinned smooth muscle
In our study of the effects of ET-1 on the [Ca2+]i-force
relationship, ionomycin (04/1M) was applied for 30 min in
Krebs solution and was present throughout the
experiment to eliminate the function of intracellular Ca2"
storage sites. Ca2+-free solution containing 5-9 mm K+ and
2 mm EGTA was then applied for 1 min, followed by a
2 min application of Ca2+-free solution containing 70 mM
K+ with 2 mm EGTA. At this point, various concentrations
of Ca2+ (016-2-6 mM) were cumulatively applied from low
to high in the presence of 70 mm K+. After application of
Ca2+-free solution with 5 9 mm K+ for 1 min, the resting
[Ca2+]i and force decreased slightly to 95-9 + 16 nm and
0 4 + 0 3 mg, respectively (n = 4). Subsequent application
of Ca2+-free solution with 70 mm K+ did not increase [Ca2+]j
(9541 + 3-4 nM) or force (0 4 + 0 3 mg).
Figure 3 shows the effects of 10 nm ET-1 on the
[Ca2+]i-force relationship derived from the use of various
concentrations of Ca2+ in solutions containing 70 mm K+ in
ionomycin-treated muscle strips. ET-1 slightly lowered the
increases in [Ca2+]i induced by low concentrations of Ca2"
(0416 and 0 33 mM) but slightly enhanced the increase in
[Ca2+]i induced by higher concentrations (1P3 and 2-6 mM
Ca2"). ET-1 shifted the [Ca2+]i-force relationship to the left
and increased the maximum amplitude of contraction
induced by 2-6 mm Ca2". The values of [Ca2"], for the half-
maximum contraction (ED50) were 235-8 + 450 nm and
183-7 + 33-4 nM in the absence and presence of 10 nM ET-1,
respectively. These values are significantly different
(P < 0 05; paired t test).
However, since [Ca2+]i was measured in muscle strips
(multicellular preparations), the signal represents the mean
[Ca2+]i within each smooth muscle cell in the strip. Thus,
the mean tension at given [Ca2+]i in the presence of NA or
ET-1 may be underestimated if spatial and temporal
inhomogeneities of changes in [Ca2+]i following the agonist
stimulation occur in individual cells.
ET- 1 enhances Ca2+-induced contraction with
a corresponding increase in MLC
phosphorylation
To study the effects of ET-1 on contractile proteins more
directly, its effects on the contraction and MLC
phosphorylation induced by various concentrations of Ca2+
were observed in ionomycin- and fl-escin-treated skinned
smooth muscle strips. In the skinned muscle, the minimum
concentration of Ca2+ needed to produce contraction was
01/SM and the maximum contraction was obtained at 3/M
Ca2+. Figure 4A shows the effects of 10 nM ET-1 + 100 /M

A
800
E 600 .2
S 2min
400k-
X 200 -

Figure 2. Effects of high K+ (128 mm), 10j/M NA

and 10 nm ET-1 on [Ca2+]i and force oL High K+ NA ET-1 Nicardipine
In A, each agent was applied as indicated by the
bar. [Ca2+]i, upper trace; force, lower trace. - 12 [ t g
Nicardipine (1/SM) was applied (as indicated by the
bar) during the steady- state contraction induced by
ET-1. The results illustrated were obtained from a 04 ~~~~~~~2
mi
single smooth muscle strip and were reproducible in
another 3 strips. B, the relationship between [Ca21]i High K+ NA ET-1 Nicardipine
and force during the tonic phase of the response to
each stimulant. Data were obtained from the tonic B
responses 2 min after application of 128 mm K+ and 1-0
10/SM NA and 10 min after 10 nm ET-1. The 0-8
maximum amplitude of contraction induced by 0)am 10 nM ET-1

128 mm K+ was normalized as a relative force of 1P0 .2 0-6

for each strip. Results shown are each the mean of 4 a)

observations with S.D. shown by vertical and < 0Q4 10 FM NA 128 mm K+

horizontal bars. 0-2 I
-

0-0 L
100 150 200 250 300 350
[Ca2+], (nm)
258 M. Yoshida, A. Suzuki and T Itoh J.PlysioL 477.2

A ET-1
/~~~
4020
4300 Controll20

Control

10 E 6 7 2 mmn[ 142+] (nM)

_ _ 4 5 6D
cm 200 82/
50 100 150 200 250 300 350

2 3 2

Figure 3. Effects of ET-11 on [Ca2+]-force relationship in ionomyci-treated smooth muscle strips

The muscle strips were treated with 04 4uM ionomycin for 30 min in Krebs solution. A, actual tracings
of [Ca2+]1 (upper records) and force (lower records). Ca2+-free solution containing 2 miM EGTA with
5 9 mM K+was applied for 1 min (shown a.s application 1) followed by a 2 min application of Ca2+-free
solution containing 2 mMi EGTA with 70 mM K+ (application 2) in the presence or absence of 10 nM
ET-1. Finally, solutions containing various concentrations of Ca2+from 0*t6 to 2*6 miM with 70 mr K+
were cumulatively applied from low to high in the presence or absence of 10 nM ET-1. These solutions
contained the following: 0M16 mMi Ca2+ (application 3), 0 33 miM Ca2+ (application 4), 0 65 mMi Ca2+
808
(application 5), 1P3 mxv Ca2+ (application 6)Conro
and 2M6 mMi Ca2+ (application 7). B, summary of the effects of
ET-1 on [Ca2+]1-force relationship
010 nM--I 7, in ionomycin-treated smcoth muscle strips. 0, control; 0, in
the presence of ET-1. The maximum amplitude of contraction induced by 128 mM K+ in Krebs solution
was normalized as a relative force of 1~0 for each strip. Each symbol represents the mean of 5
observations with S.D. shown by vertical and horizontal bars.
U_[C2] M [Ca2+], (nM)

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Figure 3. Effects of ET-1 wit GTP B reaionomyinstreatweensmoothmucle(A)rand
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tepeecofE1.Temaximum 'D 128MC
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0.0 0.0 **

[Ca2+J (M) [Ca2+] (M)

Figure 4. Effects of ET-1 with GTP and of PDBu on relationships between Ca"~-force (A) and
Ca"-myosmn light chain (MLO) phosphorylation (B) in f6-escin-treated skinned smooth muscle
strips
Ca2' at any given concentration was applied for 15 min in a relaxing solution containing 4 mm~EGTA
in the absence (0) or presence of either 10 nm ET-1 with 100 /Sm GTP (0) or 0-1 /Im PDBu (A). In A, the
maximum amplitude of contraction induced by 10/tm Ca21 was normalized as a relative force of 1P0 for
each strip. The curves for the effects of Ca21 on force and MLC phosphorylation were obtained by
fitting the data points to eqn (1) by a non-linear least-squares method (see Methods). Each data point
shown is the mean of 4-8 observations with S.D. shown by a vertical bar.
J. Physiol. 477.2 Effect of PKC on ET-induced vasoconstriction 259

Table 1. Effects of PKC19-36 (0'1 mm) on the contraction and MLC phosphorylation
(mol phosphate (mol MLC)-') induced by 0 3 jSm Ca2" in the presence and absence of 10 nm
ET-1 with 100 jSm GTP or 01 FSM PDBu in fl-escin-treated skinned muscle strips

Relative force MLC phosphorylation

Control 0-25 + 0-03 0-16 + 0-06
Control + PKC19-36 0-24 + 0-05 0-17 + 0-05
ET-1 + GTP 0-49 + 0.05* 0-23 + 0.02*
ET-1 + GTP + PKC19-36 0-41 + 0.07* 0-21 + 0.03*
PDBu 0-58 + 0.05* 029 + 0.07*
PDBu + PKC19-36 0-31 + 0-08 0-18 + 005

*Significant difference from the control without PKC19-36. The maximum amplitude of
contraction induced by 10/SM Ca2" was normalized as a relative tension of 1P0 for each strip.

GTP and of 041/M PDBu on contraction induced by
various concentrations of Ca2" (0 03-10 /M). ET-1 + GTP
and PDBu each shifted the Ca2"-force relation curve to the
left without a change in the maximum amplitude of
contraction induced by 10/M Ca2+. The threshold
concentration of Ca2+ needed to produce contraction was
slightly lower in the presence of either ET-1 + GTP
(0-03,uM) or PDBu (0-03 /SM) than in the control (04/1M).
The concentration of Ca2+ required for half-maximum
force (ED50) was 0-60 + 015 /bM in the control (n = 6),
0-25 + 0 04 /uM in the presence of ET-1 + GTP (n = 6) and
0414 + 0 07 /#M in PDBu (n = 6). Hill coefficients were
241+ 0-3 in the control, 1P4 + 0-2 in the presence of
ET-1 + GTP and 1P9 + 0 9 in PDBu.
Figure 4B shows the effects of 10 nM ET-1 + 100 /M GTP
and of 0 1 /M PDBu on the MLC phosphorylation induced
by various concentrations of Ca2" (0 03-10 /M). In Ca2"-free
solution containing 4 mm EGTA, MLC phosphorylation
was 0 05 + 0 03 mol phosphate (mol MLC)' (n = 6) and it
was increased by Ca2+ (003-10 /sM) in a concentration-
dependent manner. Under these conditions, Ca2+
(0 03-10 /SM) mainly increased the monophosphorylated

1-2

1*0

a 0-8
0

co
rf( 0-4
0-2

00
0 0-1 0-2 0-3 0-4 0-5 0-6 0-7 0-8
MLC phosphorylation
(mol phosphate (mol MLC)-1)

Figure 5. Relationship between relative force and myosin light chain (MIC) phosphorylation in
the presence of various concentrations of Ca2" (0 03-10 EM) with or without ET-1 plus GTP and
with or without PDBu in fl-escin-treated skinned smooth muscle strips
0, control; 0, in the presence of 10 nM ET-1 with 100 fM GTP; A, in 0 1/sM PDBu. The curve was
obtained by fitting the data points to eqn (1) by a non-linear least-squares method according to the
equation F/Fo = (C/K)l/[1 + (C/K)"]. n, Hill coefficient; K, dissociation constant. C and F/Fo represent
MLC phosphorylation (mol phosphate (mol MLC)1) and relative force, respectively, at any given
concentration of Ca2" in the absence (0) or presence of either ET-1 plus GTP (0) or PDBu (A). The fitted
parameters of n and K were 2-5 and 0-28 mol phosphate (mol MLC)', respectively (r = 0 99). The data
shown in Fig. 4 were used for this analysis. Results shown are each the mean of 4-8 observations with
S.D. shown by vertical and horizontal bars.
260 M. Yoshida, A. Suzuki and T Itoh J. PhysioL. 477.2

A a B
0-7
= PDBu
0-6 M ET-1 + GTP

a1)
o
0-5
ciu

a)
0-4
b Err
0-3

0-2
Control 10 /IM 30juM 100auM
PKC 1g-36
PDBu or ET-1 + GTP

03,uM Ca2+

Figure 6. Effects of a protein kinase C inhibitor, PKC19_36, on contraction induced by 0 3 /m COa2+

in the presence of PDBu or of ET-1 with GTP in fl-escin-treated skinned smooth muscle strips
A, after the muscle strip was skinned by application of 30 uM /-escin for 25 min, 0 3 /M Ca2 was
applied. PDBu (0 1,UM, A a) or ET-1 (10 nM) with GTP (100 uM, A b) was applied during the steady-state
contraction induced by 0 3 AM Ca2". PKC19-36 (041 mM) was then applied during the tonic phase of the
Ca2"-induced contraction in the presence of PDBu or of ET-1 with GTP. After recording the recovery
of the response following the wash-out of PKC19 by a solution containing Ca2" and either PDBU or
36

ET-1 with GTP, 10/uM Ca2" was finally applied to determine the maximum Ca2`-induced contraction
in each strip. Each agent was applied as indicated by the bar. The results shown are typical traces and
were reproducible in another 3 strips. B, concentration-dependent effects of PKC,9-36 on contraction
induced by 0 3 FM Ca21 in the presence of 10 nm ET-1 with 100 /M GTP or of 01 FuM PDBu. Fl, in the
presence of 041/M PDBu; X, in 10 nM ET-1 with 100 /M GTP. PKC,9-36 was cumulatively applied from
a low to a high concentration during the steady-state contraction induced by 0-3 /M Ca21 in the
presence either of PDBu or of ET-1 with GTP. The maximum amplitude of contraction induced by
10j/M Ca2+ was normalized as a relative force of 1 0 for each strip. The relative force induced by 0-3 uM
Ca2' in the absence of ET-1 or PDBu was 0-25 + 0-02 and this was not modified by 01 mm PKC,9-36
(0-24 + 0 03). Results shown are each the mean of 4 observations with S.D. shown by a vertical bar.
*Significant difference from the corresponding control.

form of MLC (over 95 %) in the presence or absence of
ET-1 + GTP or of PDBu. PDBu and ET-1 + GTP each
shifted the Ca2`-MLC phosphorylation (monophos-
phorylated form) relation curve to the left without affecting
the maximum level of MLC phosphorylation obtained with
10/SM Ca2+. Figure 5 shows the relationship between relative
force and the monophosphorylated form of MLC in the
presence of various concentrations of Ca2+ with or without
either PDBu or ET-1 + GTP. Neither ET-1 + GTP nor
PDBu induced a detectable change in this relationship.
ET- 1 increases MLC phosphorylation by both
PKC-dependent and -independent
mechanisms
PKC19-36 which corresponds to a pseudosubstrate region
of PKC, selectively inhibits PKC (House & Kemp, 1987).
PKC19-36 (01 mM) modified neither the contraction nor the
MLC phosphorylation induced by 0 3 /SM Ca2' alone
(Table 1). The effects of PKC19-36 were then observed on
contractions induced by 0 3 ,UM Ca2" in the presence either
of 10 nm ET-1 + 100 /SM GTP or of 01 ,CM PDBu (Fig. 6A).
PKC19-36 (10-100/SM) attenuated the PDBu-induced
enhancement of the Ca2`-induced contraction in a
concentration-dependent manner and, at 0-1 mm, blocked
the action of PDBu. PKC peptide (0-1 mM) also blocked the
PDBu-induced enhancement of the MLC phosphorylation
induced by 0 3 /SM Ca2` (Table 1). By contrast, PKC1936
only partly inhibited the ET-1-induced enhancement of
both the contraction and MLC phosphorylation induced by
0 3 /SM Ca2+ (Table 1). PKC19_36 did not modify the
relationship between relative force and MLC
phosphorylation (Fig. 7).
BQ-123 (04/1M) did not itself modify the contraction
induced by 0 3 /SM Ca2", but completely blocked the ET-1-
induced enhancement of the Ca2+-induced contraction in
skinned strips. IRL 1620 (1/ M) did not modify the
contraction induced by 0 3 ,uM Ca2` either in the presence
or absence of ET-1 + GTP (not shown).
J. Physiol. 477.2 Effect of PKC on ET-induced vasoconstriction 261

0-7 r

0-6 -

0)
0 0-5 I
a)

0)
0-4 I

0-3 F
0-2 g I I I I I

0-10 0-15 0-20 0-25 0-30 0 35 0-4

MLC phosphorylatfon
(mol phosphate (mol MLC)-1)

Figure 7. Effects of PKC19,36 on relationship between relative force and myosin light chain
(MLC) phosphorylation in the presence and absence of ET-1 with GTP or of PDBu in fl-escin-
treated skinned smooth muscle strips
*, 03 FuM Ca2+ alone; triangles, in the presence of 10 nm ET-1 plus 100 /M GTP without (A) or with (A)
01 mm PKC19 36; squares, in 01/FM PDBu without (U) or with (5) 0.1 mm PKC19 36. The curve was
obtained by fitting the data points by a non-linear least-squares method using the parameters obtained
in Fig. 5 (r = 0 98). The maximum amplitude of contraction induced by 10/SM Ca2` was normalized as a
relative force of 1P0 in each strip. Results shown are each the mean of 4 observations with S.D. shown by
vertical and horizontal bars.

ET-1 enhances Ca2"-induced contraction with
the mediation of GTP-binding proteins
Neomycin interacts with polyphosphoinositides and
inhibits the activation of phospholipase C, the enzyme
which causes hydrolysis of phosphatidylinositol 4,5-bis-
phosphate (Cockcroft & Comperts, 1985). Neomycin
(01 mM) did not itself modify the contraction induced by
0 3 ,UM Ca2", but it completely blocked the enhancement of
the Ca2"-induced contraction induced by ET-1 + GTP, but
not that induced by GTPyS (Fig. 8A). The extent of the
GTPyS-induced enhancement of the Ca"+-induced
contraction was similar whether neomycin was present
(2-2 + 0-2 times control, n = 4) or absent (2-3 + 0 4 times
control, n = 4). Figure 8B shows the effects of GDP/JS on
the ET-1-induced enhancement of the contraction induced
by 0'3 /LM Ca2+: GDP,BS (1 mM) completely blocked this
effect of ET-1.

A B

10 mg

ET-1 + GTP GTPyS

0-1 mM neomycin
1 0 #m Ca2+
0-3 #M Ca2+ 0.3 FM Ca2+

Figure 8. Effects of neomycin (A) and GDP,6S (B) on contraction induced by 03 /SM Ca2" in the
presence of ET- 1 with GTP in fl-escin-treated skinned smooth muscle strips
After the muscle strip had been skinned by an application of 30/SM /)-escin for 25 min, 0 3 uM Ca2+ was
first applied. In A, 0.1 mm neomycin was applied during the steady state of the Ca2"-induced
contraction and 10 nm ET-1 with 100/uM GTP then applied in the presence of 03 /SM Ca2` and 0 1 mM
neomycin. GTPyS (10/SM) was subsequently applied in a solution containing 03 /SM Ca2" plus 0-1 mM
neomycin. Finally, 10/SM Ca2` was applied to obtain the maximum Ca2+-induced contraction for that
strip. B, ET-1 (10 nM) with GTP (100/SM) was applied on the steady state of the contraction induced by
03 /SM Ca2". GDPflS (1 mM) was then applied in a solution containing 03 /SM Ca2" and ET-1 with GTP.
Each agent was applied as indicated by the bar. The results shown are typical traces and were
reproducible in another 2 strips.
262 M. Yoshida, A. Suzuki and T Itoh
DISCUSSION
ET-1-induced Ca21 mobilization
The existence of two distinct endothelin receptor subtypes,
ETA and ETB, has been reported: ETA receptors are
abundant in vascular smooth muscle cells whereas ETB
receptors, which mediate vasodilatation through the
release of vasorelaxing factors, are plentiful in the
endothelial cells (Arai et al. 1990; Sakurai et al. 1990).
However, recent pharmacological evidence suggests that
ET-1 produces vasoconstriction through an activation of a
non-A subtype of ET receptors in some vessels, such as the
rabbit pulmonary artery and saphenous veins from various
species (Ihara et al. 1992b; Moreland et al. 1992b). In the
present experiments, the vasoconstriction induced by ET-1
was completely blocked by an ETA selective antagonist,
BQ-123, and the ETB agonist IRL 1620 failed to produce
any contraction even at very high concentrations. Further,
in fl-escin-treated skinned smooth muscle, BQ-123 did not
itself modify the contraction induced by 0 3 /LM Ca2" but it
did block the ET-1-induced enhancement of Ca2"-induced
contractions. By contrast, IRL 1620 did not modify the
contraction induced by 0-3 /SM Ca2+ in the presence or
absence of ET-1 in the skinned strips. Since cellular pH,
ionic conditions and concentrations of Ca2+ are well
clamped in skinned smooth muscle, any non-specific
actions of BQ-123 would be minimized. These results
suggest that ET-1 produces contraction through an
activation of the ETA receptor subtype in resistance vessels
of very small diameter in the rabbit mesentery.
On the basis of isometric tension recording, it was
originally suggested that in porcine coronary artery ET-1
activates the voltage-dependent, dihydropyridine-
sensitive Ca2+ channel and that, therefore, the contraction
was supported by an influx of extracellular Ca2+
(Yanagisawa et al. 1988). Later, it was confirmed that in
patch-clamp experiments, ET-1 activates both the
dihydropyridine-sensitive and -resistant (but inhibitable)
Ca2" channels in vascular smooth muscle cells (Goto et al.
1989; Inoue et al. 1990). However, it has also been reported
that the ET-1-induced contraction is not inhibited by
organic Ca2+ channel blockers, including dihydropyridines,
in some arterial tissues (mainly conduit arteries; Ohlstein et
al. 1989), suggesting that species and regional differences
exist in the mechanisms responsible for the ET-1-induced
contractions seen in various types of vascular bed.
ET-1 activates the hydrolysis of phosphatidylinositol
4,5-bisphosphate (PIP2) and generates inositol 1,4,5-tris-
phosphate (1P3) which causes release of Ca2+ from the
intracellular storage sites in various types of vascular
smooth muscle (Ohlstein et al. 1989). However, in our
preliminary experiments on smooth muscle of the rabbit
mesenteric artery, we found that at concentrations below
10 nM, ET-1 did not increase [Ca2+]i in Ca2+-free solution
containing 2 mm EGTA whereas, at concentrations over
10 nm, this peptide produced a rapid and transient increase
J. Phy8iol. 477.2
in [Ca2"], in Ca2+-free solution. In the present experiments,
the concentrations of ET-1 used were below 10 nm and we
did not observe an initial rapid increase in [Ca2+]i following
the application of ET-1 in eleven out of twelve muscle
strips obtained from six animals, and nicardipine
completely blocked the ET-1-induced sustained increase in
[Ca2+]i in small-diameter resistance vessels (70 ,um) of the
rabbit mesentery. These results suggest that the role of the
dihydropyridine-sensitive Ca2" channel on contraction
induced by ET-1 (<10 nM) is of greater importance in
resistance vessels than in conduit vessels.
In the present experiments, although nicardipine
completely inhibited the ET-1-induced increase in [Ca2+]i,
a portion (10-20 %) of the ET-1-induced contraction
remained in the presence of nicardipine. Since nicardipine
did not lower [Ca2+]i below the resting level in the presence
of ET-1, and since ET-1 enhanced the force at any given
concentration of [Ca2+]i in unskinned muscle strips, the
nicardipine-resistant component of the ET-1 contraction
might be due to an ET-1-induced increase in the sensitivity
of the contractile proteins to [Ca2+]i under resting
conditions.
ET-1-induced increase in the sensitivity of
contractile proteins to Ca2+
Myosin light chain (MLC) phosphorylation by the
Ca2+-calmodulin-MLC kinase complex plays a pivotal role
in the initiation of smooth muscle contraction (Hartshorne,
1987). In unskinned smooth muscle, ET-1 shifted the
[Ca2+]i-force relationship to the left and enhanced the
maximum contraction induced by an application of 2'6 mM
Ca21 in solutions containing high K+, suggesting that ET-1
increases the efficacy with which [Ca2+]i produces
contraction. In /J-escin-treated skinned muscle strips,
ET-1 + GTP and PDBu alone (an activator of PKC) each
enhanced the contraction induced by lower concentrations
of Ca2+ (0 03-1 /iM) in parallel with increases in MLC
phosphorylation. These results suggest that both ET-1 and
PDBu increase the phosphorylation of MLC at any given
low concentration of Ca2+ (0 03-1 /bM) and thereby enhance
the Ca2+-induced contraction.
ET-1 greatly enhances the production of diacylglycerol
(DAG), which activates PKC, through a breakdown of
membrane phospholipids in various types of smooth muscle
(Griendling et al. 1989). Moreover, ET-1 induces sustained
translocation of PKC from cytosol to the membrane in the
bovine carotid artery (Haller, Smallwood & Rasmussen,
1990) and, furthermore, H-7 or staurosporine, an inhibitor
of PKC, attenuates the ET-1-induced contraction (Ohlstein
et al. 1989; Nishimura et al. 1992). These results suggest
that PKC may play a primary role in the ET-1-induced
contraction in some vascular tissues. However, since
staurosporine and H-7 each inhibit PKC at its ATP binding
site, a region with a high degree of sequence homology in
most kinases (Hidaka, Inagaki, Kawamoto & Sasaki, 1984;
Edelman, Blumenthal & Krebs, 1987), these agents may
J. Phy8iol. 477.2 Effect of PKC on ET-induced vasoconstriction
have some non-specific actions, especially at high
concentrations (Hidaka et al. 1984; Riiegg & Burgess, 1989).
In our preliminary experiments, high concentrations of
H-7 (over 10,uM) inhibited both the contraction and MLC
phosphorylation induced by 10 /SM Ca2" in a concentration-
dependent manner without a change in the relationship
between tension and MLC phosphorylation in fl-escin-
treated skinned smooth muscle, suggesting that high
concentrations of H-7 may inhibit MLC kinase as well as
PKC.
Recently, Moreland et al. (1992a) reported that, when a
concentration of staurosporine that selectively inhibits
PKC was used, this agent failed to alter the amplitude of
contraction induced by ET-1 in swine carotid artery. More
recently, using calphostin C (a new and more selective type
of PKC inhibitor), Shimamoto et al. (1992) reported a minor
contribution (13 %) of PKC to the ET-1-induced contraction
in rat aorta. These findings are in good agreement with our
present results: PKC peptide (PKC19-36), a novel inhibitor
of PKC (House & Kemp, 1987), completely inhibited the
PDBu-induced enhancement of both the contraction and
MLC phosphorylation induced by 0 3 uM Ca2", but only
partly attenuated the ET-1-induced enhancement of these
two components of the response. These results suggest that
PKC is unlikely to play a major role in the ET-1-induced
increase.in the sensitivity of the contractile proteins to
Ca2" in the smooth muscle of the rabbit mesenteric artery.
However, we cannot deny the possibility that our results
using fl-escin-treated skinned smooth muscles may under-
estimate the role of PKC in ET-1-induced contraction,
because some unidentified proteins which contribute to the
ET-1-induced contraction may leak out from skinned
muscle cells.
It has been reported that in vitro phosphorylation of
heavy meromyosin by PKC does not stimulate actin-
activated myosin MgATPase activity, but, rather, reduces
the rate of phosphorylation of MLC by MLC kinase,
causing a decrease in the actin-activated MgATPase
activity of heavy meromyosin which has been pre-
phosphorylated by MLC kinase (Nishikawa, Sellers,
Adelstein & Hidaka, 1984). Further, in glycerine-treated
porcine carotid artery, phosphorylation of MLC by PKC
neither provokes contraction nor modifies the contraction
induced by Ca2+-calmodulin (Sutton & Haeberle, 1990).
These results suggest that MLC phosphorylation by PKC
either has no effect or indeed causes an inhibition of Ca2+-
induced contraction in smooth muscle. In the present
experiments, PDBu enhanced both the contraction and
MLC phosphorylation induced by Ca2" in ,J-escin-treated
skinned muscles. Under these conditions, Ca2+ (0 03-1 ,uM)
increased, in a concentration-dependent manner, the
monophosphorylated, but not the diphosphorylated, form
of MLC in the presence or absence of PDBu. Furthermore,
the relationship between force and MLC phosphorylation
was not changed in the presence or absence of PDBu, and,
in the presence of PDBu, the force-MLC phosphorylation
263
relationship was not modified by the novel PKC inhibitor,
PKC19-36. Although we did not try to determine whether
or not PKC directly phosphorylates MLC, all these results
suggest that PKC may indirectly activate the
phosphorylation of MLC by MLC kinase. Phosphorylation
of MLC kinase by PKC causes a decrease in its affinity for
the Ca2+-calmodulin complex (Nishikawa, Shirakawa &
Adelstein, 1985) and MLC phosphorylation is balanced
between MLC kinase and MLC phosphatase in smooth
muscle cells. Thus, it may be that PKC activated by PDBu
increases MLC phosphorylation through a direct or indirect
inhibition of MLC phosphatase.
It was recently hypothesized that PKC may increase
Ca2"-induced contraction through the phosphorylation of
caldesmon (Walsh, 1990) and/or calponin (Nakamura,
Mino, Yamamoto, Naka & Tanaka, 1993), which are thin-
filament-linked regulatory proteins in smooth muscle.
However, the physiological role of these proteins in smooth
muscle has not been clarified, especially in the response to
agonist stimulation and their part, if any, in the
physiological actions of PKC is unclear.
GTP-binding proteins mediate ET-1-induced
Ca2" sensitization in contractile proteins
In smooth muscle, agonist-induced Ca2+ sensitization of the
contractile proteins requires GTP and is inhibited by
GDP/1S, suggesting that GTP-binding proteins (G-proteins)
may play a role in the agonist-induced increase in the
sensitivity of the contractile proteins to Ca2" (Kitazawa et
al. 1989). It has been hypothesized that agonist with GTP
inhibits MLC phosphatase through an activation of
unidentified G-proteins and then enhances Ca2"-induced
MLC phosphorylation, causing an increase in the
amplitude of the Ca2+-induced contraction in smooth
muscle (Kitazawa, Gaylinn, Denney & Somlyo, 1991). In
the present experiments, ET-1 increased the sensitivity of
both the contraction and MLC phosphorylation to Ca2" in
the presence of GTP and the action of ET-1 on the Ca2+-
induced contraction was abolished by either GDP/?S or
neomycin. It is known that neomycin interacts with
polyphosphoinositides and inhibits phospholipase C
(Cockcroft & Comperts, 1985). This action of neomycin is
not due to its binding to IP3 (Prentki, Deeney, Matschinski
& Joseph, 1986) because, in the present experiments,
skinned muscle strips were treated with ionomycin in a
solution containing a relatively high concentration of
EGTA (4 mM) to prevent Ca2+ release from the storage
sites. A direct inhibitory action of neomycin on
unidentified G-proteins is unlikely, since neomycin did not
affect the GTPyS-induced enhancement of the Ca2+-
induced contraction. Neomycin might inhibit the
activation of PKC through a reduction in DAG production
via its inhibitory action on phospholipase C. However, this
is unlikely to be the case because there is little contribution
of PKC to ET-1-induced Ca2+ sensitization of the
contractile proteins in .-escin-treated skinned strips. At
264 M.
Yoshida, A.
present, the sites of action of neomycin on ET-1-induced
enhancement of the sensitivity of the contractile proteins
to Ca2" remain unclear.
There are many G-protein families known (Bourne,
Sanders & McCormick, 1990) and it was recently suggested
that small G-proteins, like rho p21 (Hirata et al. 1992)
and/or ras p21 (Pfitzer & Satoh, 1993), may contribute to
the agonist-induced increase in the sensitivity of the
contractile proteins to Ca2". However, it has not yet been
clarified how agonist-receptor signals couple to these small
G-proteins. Thus, it might be reasonable to speculate that
membrane phospholipids which interact with neomycin
may play a role in the coupling between the ET-1-ETA
receptor complex and unidentified G-proteins, which leads
to an increase in the sensitivity of the contractile proteins
to Ca2.
In conclusion, ET-1 binds to the ETA receptor and
increases Ca2+ influx through an activation of
dihydropyridine-sensitive Ca2+ influxes, causing a long-
lasting and maintained contraction in resistance vessels of
the rabbit mesentery. ET-1 increases the phosphorylation
of MLC induced by lower concentrations of Ca2+ through
an activation of both PKC-dependent and -independent
mechanisms and thus enhances the Ca2+-induced
contraction. It is suggested that unidentified GTP-binding
proteins could play a role in the ET-1-induced increase in
the sensitivity of contractile proteins to Ca2'.
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Acknowledgements
We thank Dr R. J. Timms for the language editing. We
gratefully acknowledge Dr J. T. Stull (University of Texas,
Dallas, TX, USA) for providing an antibody against bovine
tracheal myosin light chain. BQ-123 and IRL 1620 were gifts
from Banyu Pharmaceutical Co. Ltd (Japan) and Ciba-GCeigy
Ltd (Japan), respectively. This work was partly supported by
a grant-in-aid from the Ministry of Education of Japan.

Received 28 June 1993; accepted 19 October 1993.