(Received for publication, June 23, 1995; and in revised form, September 29, 1995)
From the
To elucidate the intracellular Ca (Ca
) transient
responsible for nitric oxide (NO) production in endothelial cells (ECs)
and the subsequent Ca
reduction
in vascular smooth muscle cells (VSMCs), we administrated four agonists
with different Ca
-mobilizing
mechanisms for both cells in iso- or coculture. We monitored the
Ca
of both cells by
two-dimensional fura-2 imaging, simultaneously measuring NO production
as NO
. The order of potency of the
agonists in terms of the peak Ca
in ECs was bradykinin (100 nM) > ATP (10 µM)
> ionomycin (50 nM) > thapsigargin (1 µM).
In contrast, the order in reference to both the extent of
Ca
reduction in cocultured VSMCs
and the elevation in NO production over the level of basal release in
ECs completely matched and was ranked as thapsigargin > ionomycin
> ATP > bradykinin. Treatment by N
-monomethyl-L-arginine monoacetate but
not indomethacin or glybenclamide restored the
Ca
response in cocultured VSMCs
to the isoculture level. In ECs, when the Ca
influx
was blocked by Ni
or by chelating extracellular
Ca
, all four agonists markedly decreased NO
production, the half decay time of the
Ca
degenerating phase, and the
area under the Ca
curve. The
amount of produced NO hyperbolically correlated to the half decay time
and the area under the Ca
curve
but not to the Ca
peak level.
Thus, the sustained elevation of Ca
in ECs, mainly a result of Ca
influx,
determines the active NO production and subsequent
Ca
reduction in adjacent VSMCs.
Furthermore, L-arginine but not D-arginine or L-lysine at high dose (5 mM) without agonist enhanced
the NO production, weakly reduced the Ca
in ECs, and markedly decreased the
Ca
in VSMCs, demonstrating the
autocrine and paracrine effects of NO (Shin, W. S., Sasaki, T., Kato,
M., Hara, K., Seko, A., Yang, W. D., Shimamoto, N., Sugimoto, T., and
Toyo-oka, T.(1992) J. Biol. Chem. 267, 20377-20382).
Endothelial cells (ECs) ()modulate the contractility
of underlying vascular smooth muscle cells (VSMCs), secreting several
vasoconstrictors and vasorelaxants(1, 2) . The
principle endothelium-derived relaxing factors have been identified as
nitric oxide (NO) (3) and prostacyclin. Both are regulated by
intracellular calcium ions
(Ca
)(4) . In the absence
of extracellular Ca
(Ca
), NO production is
greatly reduced(5, 6) . Recently, we have presented
evidence that NO affects the handling of Ca
by an autocrine action in NO-producing ECs and by a
paracrine action in adjacent VSMCs(7) . However, little
quantitative information is available on the relationship between the
Ca
in ECs and NO production.
There is also a lack of information regarding the amount of NO produced
in ECs and its action to reduce the Ca
levels in neighboring VSMCs.
The
Ca in VSMCs is crucial, because
it is a primary factor in the regulation of muscle
contractility(8) . Most Ca
transients in ECs induced by agents that cause the release
of NO consist of a peak followed by a degeneration phase(5) .
The peak originates from the release of Ca
from
endoplasmic reticulum. The influx of Ca
from the
extracellular medium accounts for the maintenance of the subsequent
portion of degeneration phase. To determine which component of the
Ca
transients of ECs is most
significant in indicating the production of NO and to monitor the
biological action of NO on VSMCs, we simultaneously measured the stable
NO metabolite, nitrite (NO
), in the
medium and the Ca
of both ECs
and VSMCs in coculture by two-dimensional image analysis. We report
here a unique communication between the Ca
in ECs and the Ca
in
VSMCs mainly mediated by NO.
To assess the biological effect of NO on the
Ca in VSMCs, we cultured ECs and VSMCs
together(7) . Three days after the seeding of VSMCs, at a
density of 1.5
10
/cm
, the culture
dishes were semiconfluent. ECs were then added to the culture at the
same initial density of VSMCs, 1.5
10
/cm
. Under these conditions, both VSMCs and
ECs grew symbiotically.
In a pilot study
for dose determination, we searched for suitable agonists and their
appropriate concentrations that make it possible to analyze
Ca transients using different
receptor/effector coupling systems. Iso- or cocultured ECs or VSMCs
were stimulated by ATP (10 µM) or BK (100 nM) for
3 min. In the cases of TG (1 µM) and IM (50 nM),
the stimulation time was extended to 40 min because the
Ca
response to these agonists was much
more gradual than ATP or BK. To quantify the Ca
responses in ECs, we measured three parameters: the peak level,
the half decay time (HDT), and the area under the
Ca
curve (AUC) by computer-assisted
planimetry as shown in Fig. 1. To examine the reproducibility of
Ca
responses to the same agonist, a
second stimulation was performed after a 30-min equilibration period
following the washout of the first application of the agonist.
Figure 1: Measurement of peak, HDT, and AUC.
For the simultaneous measurement of
both Ca and NO content, 0.8 ml of medium
was sampled from the glass dishes at 3 min in ATP and BK studies and at
40 min in TG and IM studies after completion of the
Ca
measurement. Released NO was
completely oxidized to NO
when left
standing overnight 4 °C in a sealed Eppendorf tube. In a
preliminary study, the amount of sealed NO
was shown to be stable under this condition until assay. The
amount of NO
was determined by the
modified method of Yui et al.(12) . In short,
NO
was measured by colorimetry after
Griess reaction. To enhance the sensitivity, light absorbancy was
detected by spectrometer of HPLC at the maximum sensitivity, and full
scale was set to 0.01 absorbance. Lumen of all tubings was coated by
Teflon to prevent acid corrosion.
To further verify the results of
NO measurement, we performed a bioassay
of NO by measuring the Ca
reduction to
below basal levels or the blunted Ca
rise
in VSMCs caused by the native NO from cocultured ECs(7) . When
indicated, the substrate for NO synthase (NOS), L-arginine (5
mM), the NOS inhibitor, L-NMMA (500 µM),
the cyclooxygenase inhibitor, indomethacin (50 µM), or the
ATP-sensitive K
channel blocker, glybenclamide (10
µM), was added to ascertain what factor modifies the
Ca
handling in iso- or cocultures.
Figure 2:
Time course of NO production determined as
NO from bovine aortic ECs. ECs were
treated by control phosphate-buffered medium without agonist (Ctl,
), with ATP (10 µM,
), with
bradykinin (BK, 100 nM,
, A), with
thapsigargin (TG, 1 µM,
) or with ionomycin (IM, 50 nM,
, B). Each point denotes
the mean ± S.E. (n = 6-10). For the
details on sampling and measurement of NO, see under ``Materials
and Methods.''
Figure 3:
Dependency of NO production on
Ca influx or substrate in bovine aortic ECs. Control
samples obtained from phosphate-buffered medium were analyzed to
precisely determine relative effect. ATP (10 µM), BK (100
nM), TG (1 µM), or IM (50 nM) was added
in the presence of 1 mM extracellular Ca
,
Ca
(+); 1 mM EGTA
in place of Ca
,
Ca
(-), or
Ca
(+) plus Ni
(1 mM, A). *, p < 0.05 versus control; #, p < 0.05 versus Ca
(+). Agonist was
added to ECs with or without pretreatment by L-NMMA (500
µM) or L-NMMA plus L-arginine (5
mM, B). Each column denotes the mean ± S.E. *, p < 0.05 versus control; #, p < 0.05 versus agonists (n =
5-8).
To verify the agonist-induced NO production, we used the NO synthase inhibitor, L-NMMA. Pretreatment by L-NMMA (500 µM, 30 min) inhibited NO production over the basal level induced by all four agonists. However, the addition of L-arginine (5 mM) negated this inhibition and increased the production of NO after administration of all four agonists, ranging from 2-fold in the case of BK to 8-fold in the case of TG (Fig. 3B). These results indicate that NO was synthesized from L-arginine.
Figure 4:
Ca dynamics in isocultured ECs induced by four kinds of agonist
with different Ca
-mobilizing
actions. At the arrow, ATP (10 µM,
), BK
(100 nM,
), TG (1 µM,
), or IM (50
nM,
) was added in the presence of 1 mM Ca
, and the
Ca
was monitored (A; (7) ). To show the later phase after stimulation by TG or IM,
the recording time was extended to 2400 s (B). Each point
denotes the mean ± S.E. (n =
130-147).
In cocultured ECs, the
Ca exhibited essentially the same
transients as those in isocultured ECs (see Fig. 6Fig. 7Fig. 8Fig. 9Fig. 10).
All three parameters, including the peak Ca
level, HDT, and AUC, in coculture were comparable with those in
isoculture (Table 1). These results suggest that the
Ca
handling system in ECs is not under
the influence of cocultured VSMCs.
Figure 6:
Two-dimensional images of the
Ca response of ECs and VSMCs in
coculture (
200) induced by ATP with or without
Ca
and the
Ca
dynamics in both cells. The
experiments show the positions of ECs and VSMCs (A),
fura-2-loaded cultured cells in the resting state at an excitation
wavelength of 380 nm (B), and peak F340/F380 divided by
resting F340/F380 ratio image in the presence (C) or the
absence (D) of Ca
after
stimulation by ATP (10 µM). At the arrow, ATP was
added. The second dose of ATP was applied 30 min after the first dose.
The Ca
transient of ECs (
)
and VSMCs (
) with (E) or without (F)
Ca
. Each point denotes the mean
± S.E. (n = 6-8). The bar represents 100 µm.
Figure 7:
Two-dimensional images of the
Ca response of ECs and VSMCs in
coculture (
200) induced by BK and the
Ca
dynamics in both cells. The
experiments show fura-2-loaded cultured cells in the resting state at
an excitation wavelength of 380 nm (A), the F340/F380 divided
by resting F340/F380 ratio image at 15 (peak, B) or 150 s (C) after the addition of BK (100 nM), and the
Ca
transient (D) in ECs
(
) and VSMCs (
). At the arrow, BK was added. Each
point denotes the mean ± S.E. (n = 6-10).
The bar represents 100 µm.
Figure 8:
Two-dimensional images of the
Ca response of ECs and VSMCs in
coculture (
200) induced by TG and the
Ca
dynamics in both cells. The
experiments show fura-2-loaded cultured cells in the resting state at
an excitation wavelength of 380 nm (A), the F340/F380 divided
by resting F340/F380 ratio image at 120 (B) or 2400 s (C) after stimulation by TG (1 µM), and the
Ca
transient (D) in ECs
(
) and VSMCs (
). At the arrow, TG was added. Each
point denotes the mean ± S.E. (n = 9-11).
The bar represents 100 µm.
Figure 9:
Two-dimensional images of the
Ca response of ECs and VSMCs in
coculture (
200) induced by TG with pretreatment by L-NMMA and the Ca
dynamics in both cells. The experiments show fura-2-loaded
cultured cells in the resting state at an excitation wavelength of 380
nm (A), the F340/F380 divided by resting F340/F380 ratio image
at 480 (B), 2100 (C), or 2400 s (D) after
stimulation by TG, following pretreatment with L-NMMA (500
µM), and the Ca
transient (E) in ECs (
) and VSMCs (
). At
the arrow, TG was added. Each point denotes the mean ±
S.E. (n = 12-14). The bar represents 100
µm.
Figure 10:
Two-dimensional images of the
Ca response of ECs and VSMCs in
coculture (
200) induced by IM and the
Ca
dynamics in both cells. The
experiments show fura-2-loaded cultured cells in the resting state at
an excitation wavelength of 380 nm (A), the F340/F380 divided
by resting F340/F380 ratio image at 180 (B) or 2400 s (C) after the stimulation by IM (50 nM), and the
Ca
transient (D) in ECs
(
and VSMCs (
). At the arrow, IM was added. Each
point denotes the mean ± S.E. (n = 8-15).
The bar represents 100 µm.
Figure 5:
Ca dynamics in isocultured VSMCs by four kinds of agonist. At
the arrow, ATP (10 µM,
) or BK (100
nM,
) was added in the presence of 1 mM Ca
, and the
Ca
was monitored (A; (7) ). To show the later phase after stimulation by TG (1
µM,
) or IM (50 nM,
), the recording
time was extended to 2400 s (B). Each point denotes the mean
± S.E. (n =
45-78).
When VSMCs were
cocultured with ECs, these Ca changes in
VSMCs were strikingly modified (Fig. 6Fig. 7Fig. 8Fig. 9Fig. 10and Table 2). The Ca
in VSMCs after ATP
stimulation did not increase but decreased to below basal level
(-21 ± 3% at 3 min; Fig. 6). In addition, the
ATP-induced Ca
transient in cocultured
VSMCs was completely dependent on the Ca
.
The Ca
in VSMCs exhibited a significant
reduction below the basal level in the presence of
Ca
, whereas in the absence of
Ca
, the VSMCs showed a
Ca
rise to a lower peak than those in
isoculture, followed by a gradual decline (Fig. 6F). BK
also reduced the Ca
in VSMCs but to a
lesser extent (-5 ± 1%) than ATP ( Fig. 7and Table 2). TG caused a large Ca
reduction in VSMCs (-24 ± 3% at 10 min; Fig. 8). This reduction was sustained up to 40 min (-25
± 3%) after the treatment. Unlike the other three agonists, IM
did not decrease the Ca
below the basal
level in VSMCs but significantly attenuated the rise of
Ca
(Fig. 10) compared with the
Ca
rise in isoculture. The
Ca
increase started at 1 min (9 ±
1% over the basal level), peaked at 5 min (21 ± 1%), and
remained above the baseline at 40 min (10 ± 1%). As a control
study by phosphate-buffered saline, in isocultured VSMCs as well as
cocultured VSMCs and ECs, Ca
showed no
significant changes during 40 min of follow-up (data not shown).
Figure 11:
Two-dimensional images of the
Ca response of ECs and VSMCs in
coculture (
200) induced by L-arginine alone and the
Ca
dynamics in both cells. The
experiments show fura-2-loaded cultured cells in the resting state at
an excitation wavelength of 380 nm (A), the F340/F380 divided
by resting F340/F380 ratio control image (B), and 360 s (C) after the addition of L-arginine (5 mM). D, the Ca
transient in
ECs (
) and VSMCs (
). E, NO production in
isocultured ECs with (
) or without (
)
Ca
. At the arrow, L-arginine (5 mM) was added. Each point denotes the
mean ± S.E. (n = 10-12). The bar represents 100 µm.
As summarized in Fig. 12A, the NO production increased sharply as the
HDT rose to 200 s. After that time, further increments of
NO production became reduced. The AUC was
plotted in Fig. 12B. The amount of
NO
sharply increased until the AUC
reached 20,000%
s and then gradually increased. Accordingly, the
amount of NO
hyperbolically correlated to
both the HDT of Ca
(r =
0.90, p < 0.001) and the AUC (r = 0.86, p < 0.001) but did not correlate to the peak
Ca
in ECs (r = 0.17, p > 0.05; Fig. 12C). These results suggest
that the NO production rate was saturated by a long sustained
Ca
augmentation.
Figure 12:
Correlation between the HDT of
Ca in the degeneration phase and
NO production (A), AUC and NO production (B), or the
peak Ca
level and NO production (C). Each point denotes the mean ± S.E. (n = 12).
This is the first report to describe that (i) NO is released
from ECs at the basal level even without the presence of agonist, (ii)
the maintenance of Ca level, which is
supported by Ca
influx, determines the rate of NO
production over the basal release in ECs, (iii) NO production is not
related to the peak Ca
but dependent on
the sustained elevation of Ca
, as
represented by the HDT or AUC of Ca
in
ECs, (iv) the production of NO reduces the Ca
in cocultured VSMCs in a paracrine manner, dependent on the level
of NO production, and (v) L-arginine alone enhances NO
production, which has both autocrine and paracrine actions on ECs and
VSMCs in coculture, respectively.
Previously, two indirect methods have been employed to quantify the
NO effect on vessels. One involves the measurement of the isometric
tension developed in vessel strips in the presence or the absence of
ECs and the subsequent comparison of the extent of the vessel
relaxation. However, the NO effect cannot be evaluated correctly in
this measurement, because the vessel does not contract in the absence
of Ca. Another method relies on the
measurement of intracellular cGMP(17, 18) , which is
synthesized after the activation of guanylate cyclase. cGMP does not,
however, explain all actions of NO(19) . Both methods cannot
simultaneously measure the Ca
dynamics in
ECs and/or VSMCs. No data have been reported concerning the extent of
Ca
elevation in ECs relative to NO
production rate or the Ca
reduction in
VSMCs.
Present NO measurement was used
to assay NO production mediated by inducible NOS(12) , where
the amount of released NO was much larger than that from eNOS and
accordingly can be easily assayed. The amount of
NO
in the culture medium of ECs is not
measurable by a simple spectrophotometry, because of its insufficient
sensitivity. NO
determination by HPLC,
with enhancing the sensitivity after the oxidation of NO to
NO
(20) , has made it possible to
document the trace quantities of NO released from ECs mediated by eNOS
for the first time. In this study, ATP and BK induced no fold increase
in NO production. However, fold increase in Ca
or cGMP induced by ATP, BK, or A23187 as identified by Schmidt et al.(21) did not mean that the rise of NO
production must be also in fold. In contrast, there was a marked and
continuous rise in NO production by TG and IM in spite of the lower
Ca
rise. These results proved that our
method is reliable, although the NO measurements still required
multiple sampling to minimize variation between experiments.
In
addition to the NO determination, we employed biological action of
native NO produced in ECs, because NO reduces the
Ca in cocultured VSMCs in a paracrine
manner (7) , dependent on the level of NO production. Using
both the NO determination and the bioassay, we succeeded for the first
time in developing the quantitative aspect of
Ca
component responsible for NO
production but also Ca
reduction in VSMCs
that leads to the muscle relaxation.
The doses of these
agonists were adjusted to appropriately raise the
Ca in ECs and produce NO within the
measurable range of our system. For those results, we selected the
doses determined in a preliminary study according to the following
three criteria: (i) The maximum dose was not used to avoid eliciting a
saturated response in both ECs and VSMCs and to accurately evaluate L-NMMA or indomethacin. The EC
for each agonist
varied between ECs and VSMCs. For example, BK induced a larger response
in ECs (EC
= 30 nM) than in VSMCs
(EC
= 12 µM). BK at 100 nM caused a significant response in ECs but a weak response in VSMCs.
In contrast, the response to IM was stronger in VSMCs (EC
= 7 nM) than in ECs (EC
= 24
nM). The appropriate range of concentrations of IM was narrow
for the Ca
measurement. (ii) Agonists
should homogeneously excite ECs. All ECs responded to TG and IM, but
only 80-85% of the cells responded to ATP at 100 nM (EC
= 400 nM) or BK at 10
nM. The lower the concentration of ATP or BK, the fewer the
number of cells able to respond. The latent time from agonist
application to the onset of the Ca
rise
was more variable at the lower doses. (iii) The agonist concentration
chosen should not induce the Ca
oscillation that makes the HDT measurement difficult. Low
concentrations of ATP causes the oscillation, as reported by Lynch et al.(27) . We used 10 µM ATP that
produced an exponential degeneration after the peak. BK rarely induced
the oscillation, and TG or IM caused no oscillation at all.
Furthermore, the quantification of
sustained Ca elevation by the HDT or AUC
revealed that the NO production rate was hyperbolically correlated to
these two parameters but not to the peak Ca
level at all (Fig. 12). The simple
Ca
-calmodulin (CaM) scheme would not be applicable
for the eNOS activation, because the scheme assumes a homogenous
distribution of Ca
, CaM, and eNOS. In the
Ca
signaling mediated by inositol
1,4,5-trisphosphate where ATP and BK are concerned, the peak
Ca
is supplied from the release of
Ca
from internal stores, independent of the
Ca
influx ( Table 1and Fig. 6F). After TG and IM stimulation, the peak
Ca
would not be formed by the release of
Ca
from internal stores but chiefly by the entry of
extracellular Ca
, as confirmed the delay of peak time
between in the presence (Fig. 4) and the absence of
Ca
or the Ni
treatment. (
)The Ca
release from internal stores
accounts for only
-
of the
Ca
entry, as shown by the AUC (Table 1). These
results also suggest that the Ca
release from
internal stores play a less significant role in elevating the
Ca
than the Ca
influx
from extracellular source in the cases of TG and IM stimulation.
Compartment of either the Ca
or NOS enzymes might
explain the dissociation between peak Ca
and NO production; eNOS is located on the cytoplasmic
membrane(28) , where the entered Ca
may
directly activate the enzyme by coupling with CaM.
The activation of
eNOS in ECs as well as constitutive NOS in brain is strictly
Ca-CaM-dependent. Compared with the binding of
inducible NOS to CaM, the binding of eNOS to CaM is loose and
reversible(29) . The Ca
-dependent
down-regulation of NOS mediated by the phosphorylation of NOS protein
and the resultant decrease in the activity (30) or the dual
regulation of constitutive NOS activity by Ca
(31) is not likely, because the Ca
concentration they employed (10-2000 µM)
exceeded the physiological Ca
range.
Furthermore, we should be very careful if biochemical study in
cell-free system does take place in vivo also.
The
Ca reduction in cocultured VSMCs became
stable 3 min after ATP or BK stimulation (Fig. 5, 6 and 7), and
it was sustained for up to 40 min after TG or IM stimulation. The long
lasting action of TG and IM is compatible with a continuous production
of NO (Fig. 2), because the biological lifetime of NO is very
short(3) . In the late phase of TG-induced
Ca
transients, the low
Ca
level, which was completely supported
by the Ca
influx, stimulated ECs to produce large
amounts of NO. These results suggest that Ca
influx
might trigger NO production and that the activation of NOS does not
require high concentrations of Ca
to
sustain the NO production.
In addition, the NO production induced by L-arginine was not dependent on Ca (Fig. 11). The L-arginine study cannot be
explained by the Ca
-CaM theory and suggests that a
basal or even lowered Ca
level within ECs
is sufficient to activate eNOS, if a high dose of substrate is
supplied. No biochemical data are available on the relationship between
NOS activation and the substrate concentration. After the NO is
produced, the L-arginine concentration might not significantly
reduce, because it is supplied from the glutamine or recycled from L-citrulline within a cell(32) . However, several
clinical reports (33, 34) indicated that L-arginine might potentiate the NO production. Present studies
gave direct evidence for that.
Our previous data with the
measurement of cGMP (35) have suggested that NO is basically
released in vivo without agonist stimulation, just under shear
stress. The basal release of NO evident in this study could also be
present in vivo(36) , because vascular ECs are usually
under shear stress. The Ca in ECs is
dependent on the blood flow rate (35) , which could stimulate
basal NO synthesis. The subsequent NO production determined by
physiological flow could serve to inhibit the proliferation of ECs (18) or VSMCs (37) and prevent platelet adhesion (38) , suppressing the progression of arteriosclerosis or
thrombus formation in the vessel wall.
NO might have several routes to reduce the
Ca in VSMCs as follows: (i) NO activates
a guanylate cyclase in the soluble fraction of VSMCs and increases the
intracellular concentration of cGMP, which accelerates the efflux of
Ca
(39) , (ii) NO may directly inhibit
Ca
entry through voltage-dependent Ca
channels (40) or may enhance the outward K
current by activating Ca
-dependent K
channels that cause hyperpolarization(19) , and (iii) NO
could potentiate the Na
-Ca
exchange(41) .
The exogenous administration of NO gas
is imprecise to exactly control NO concentration without oxidation to
NO in the incubation medium. NO donors,
such as sodium nitroprusside and s-nitroso-n-acetyl-D,L-penicillamine,
are also inadequate for this quantitative purpose because the rate of
NO supply is uncertain. This is the first report that for the most part
resolves these problems by using NO naturally produced from ECs. By
combining NO determination with NO bioassay measuring the
Ca
transients of ECs and VSMCs in
coculture, we have succeeded both in identifying the significant
component of the Ca
responsible for NO
production in ECs and in quantifying the relationship between NO
release and the subsequent Ca
reduction
in VSMCs.
The present methods of coculturing different cell types, of measuring intracellular information by two-dimensional imaging of each cell, and of simultaneously determining released factors are of great value not only in the understanding of vascular biology but also in the examination of the relationships between a wide variety of cell populations that coexist in close proximity and require intracellular communication to regulate their mutual interaction.