Endothelin-induced calcium responses in human vascular smooth muscle cells JEFFREY P. GARDNER, GORO TOKUDOME, HARUO TOMONARI, ELIZABETH MAHER, DAVID HOLLANDER, AND ABRAHAM AVIV Department of Pediatrics and Hypertension Research Center, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark 07103; and Department of Obstetrics and Gynecology, St. Barnabas Medical Center, Livingston, New Jersey 07039 Gardner, Jeffrey P., Goro Tokudome, Haruo Tomonari, Elizabeth Maher, David Hollander, and Abraham Aviv. Endothelin-induced calcium responses in human vascular smooth muscle cells. Am. J. Physiol. 262 (CeZZ Physiol. 31): C148-Cl55 1992-The effects of endothelin-1 (ET) on the cytosolic free Ca (Cai) and cytosolic pH (pHi) were examined in primary cultures of human umbilical artery (HUA) vascular smooth muscle cells (VSMCs), respectively, loaded with furaand 2’,7’-bis(carboxyethyl)-5(6)-carboxyfluorescein. In 1 mM Ca, ET produced a dose-dependent, biphasic increase in the signal with a maximal effect at 400 nM ET. At this concentration, ET produced a Cai transient (mean ~fr SE; a rise from basal Ca; of 86 t 16 to 216 t 33 nM) that lasted for -50-60 S. The Ca; transient was followed by a slow but sustained increase in Ca;. Both ET-induced Cai transient and posttransient Cai were attenuated in Ca deficient medium or by verapamil and nicardipine. In contrast to ET, thrombin elicited only a monaphasic Cai response in HUA VSMCs. This response was also partially sensitive to Ca removal or verapamil. KC1 (45 mM) depolarization did not elicit a Cai response. However, the presence of voltage sensitive Ca channels in HUA VSMCs was demonstrated by enhanced Mn uptake in cells depolarized with KCl. Both ET and thrombin treatment did not alter pHi. HUA VSMCs demonstrated a single class of ET receptors (-13,000 sites/cell) with an equilibrium dissociation constant of 0.34 nM. Nicardipine did not alter ET binding. These observations suggest a dual effect of ET on the Ca; profile in HUA VSMCs that is mediated by Ca mobilization and Ca entry through Ca channels. The Ca entry could include influx through receptoroperated Ca channels, voltage-sensitive Ca channels, or both, but without a direct interaction between ET and these channels. human umbilical artery; endothelin; hydrogen antiport
calcium
channels;
sodium-
ADMINISTRATION ofendothelin(ET)produces increased blood pressure, peripheral vascular resistance, diminished cardiac output, and sodium excretion as well as multiple hormonal changes, including a rise in the circulating levels of the atria1 natriuretic peptide, vasopressin, aldosterone, and plasma renin activity (10, 24). Although, in part, these effects may be mediated through specific interactions of ET with different cells (e.g., zona glomerulosa cells and renal mesangial cells; Refs. 2 and 7), the main target cell of ET appears to be the vascular smooth muscle cell (VSMC). ET is a potent vasoconstrictor, exerting its effect primarily via the cytosolic free Ca (Ca;) messenger system (12, 16, 23, 26, 29, 35, 36) and phosphatidylinositol breakdown (23, 26, 29, 30, 35, 36). Information concerning the effect of ET on the VSMC has been derived primarily from cells originating from laboratory animals. Resink et al. (30) have demonstrated that ET stimulates phospholipase C in human VSMCs,
SYSTEMIC
Cl48
0363-6143/92
$2.00 Copyright
and Clozel and co-workers (6) have shown that specific receptors for ET are present in the human umbilical vein VSMCs. However, little is known of the nature and origin of ET-induced Ca; signals in the human VSMC. The presence of immunoreactive endothelin in human plasma (4) suggests that ET functions as a hormone. However, its major role is likely to be the local regulation of VSMC and as such it may play a central role in the physiology or pathophysiology of the cardiovascular system. In the present work we have characterized the ET-induced Ca; response and ET-binding kinetics in VSMCs obtained from the human umbilical artery (HUA). In addition, we examined whether ET exerts an effect on the Na-H antiport in these cells. METHODS Materiak. Synthetic ET-l was from Cambridge Biochemicals (Valley Stream, NY), and 12’I-ET-l was from New England Nuclear (Wilmington, DE). Fetal bovine serum (FBS), cu-minimal essential medium (ar-MEM), and collagenase were from Hazelton Biologics (Lenexa, KS). Fura-2/acetoxymethyl ester (AM) and 2’,7’-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)/AM were from Molecular Probes (Eugene, OR). HHF35 muscle actin-specific monoclonal antibody was from Organon Teknika (Westchester, PA). 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was provided by E. Cragoe, Jr. All other chemicals, including phorbol 12-myristate 13-acetate (PMA), verapamil, nicardipine, and human thrombin (catalog No. T-9135), were from Sigma (St. Louis, MO). IsoZation and growth of primary VSMCs. VSMCs were obtained from umbilical arteries of 16 normal male and female newborns. Each umbilical artery was grossly cleaned and immersed in a-MEM plus antibiotics (30 pg/ml streptomycin and 30 units/ml penicillin). It was cut longitudinally, and, using needles, the resulting rectangle was fastened to the bottom of a tissue culture dish covered with a thick layer of silicone rubber. The intima was scraped and a transverse slit across the short axis of the vascular rectangle was made (with the aid of a dissecting microscope) to -5O-80% of the thickness of the media. The line of cleavage was used as a starting point for stripping the media as a single sheath. To assure complete removal of the endothelium, the preparation was suspended for 5 min in cw-MEM containing 0.1% collagenase. The media was rinsed three times, and fragments (-0.5 x 0.5 mm) were sandwiched between glass cover slips (17 x 30 mm), which were placed into culture dishes. Cultures were maintained in cyMEM, 15% FBS, 5% CO,-95% air plus antibiotics. Growth extensions of VSMCs from explants were observed in -2 wk. In contrast to fibroblasts, HUA VSMCs grew very slowly and confluency on the cover slips was reached in about 2 mo. Thus any preparation demonstrating rapid growth was discarded. Indirect immunofluorescence of the cells with HHF35 muscle
0 1992 the American
Physiological
Society
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ENDOTHELIN
AND
VASCULAR
actin-specific monoclonal antibody were performed as described (34) to ascertain the purity of the VSMC preparations. Antibiotics were removed 3 days prior to experiments, which were performed in monolayers of primary cultures. Before loading the cells with the fluorescent dyes, cells on cover slips were washed twice with N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) buffered solution (HBS) containing (in mM) 140 NaCl, 5 KCl, 1 CaClz, 1 MgClz, 10 glucose, and 20 HEPES [pH 7.4 for Ca; measurements and pH 7.1 for intracellular pH (pHi) measurements]. Experiments were performed in 1 mM Ca or Ca-free HBS. Measurements of Cai. Cellswere incubated for 60 min with 5 PM fura-2/AM in 3 ml (37°C) of HBS plus 0.1% bovine serum albumin (BSA). They were washedtwice with HBS, and the slideswere securedin a quartz cuvette in a SPEX CM3 fluorescencespectrometer equipped with a thermostatically controlled (37°C) cell holder, a stirrer, and a suction device for rapidly removing solutions. Excitation wavelengths were set at 340 and 380 nm and emissionwavelength at 505 nm. Fluorescencewas monitored for l-3 min until the Ca; signal stabilized and basalCai measurementwasobtained. Thereafter, cellswere subjectedto specific agonists(e.g., ET and thrombin) or experimental perturbations, and Cai signals were recorded for an additional 150-300 s. Cai calibration was performed on each cover slip by exposing cells to 3 mM ethylene glycol-his@aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) with 10 PM ionomycin, followed by 3 mM Ca. Autofluorescence was determined by subjecting cells to 2 mM Mn and 10 PM ionomycin. Autofluorescence was measuredfor each slide and subtracted from the Cai signal. Mn uptake. For the Mn uptake experiments, cells were treated in a similar fashion asthoseprepared for measurements of Ca;. They were suspendedin HBS plus 1 mM MnCl,. Mn uptake by the cells was monitored at 360 nm. This is the isosbesticwavelength for fura-2. Thus changesin Cai do not alter the fura- emission,whereasMn uptake results in quenching of the fluorescence. Any factor enhancing Mn uptake is expected to acceleratethe rate of fura- quenching (5, 13, 31). Measurement of pHi. Cover slips were incubated at 37°C for 60 min in HBS with 2 PM BCECF/AM. After two washeswith HBS, they were placed in the spectrometer (excitation wavelengths 440 and 503 nm and emissionwavelength 530 nm) and monitored for 7-12 min until the pHi signal stabilized. Cells were then treated with specific agonists or experimental perturbations, and the pHi was followed for a period up to 250 s. Whenever necessary, Na was isosmotically replaced by Nmethyl-D-glucamine. Calibration of fluorescent intensity of intracellular BCECF against pHi was performed at the end of each experiment by subjecting cells to 5 ,ug/ml of nigericin in HBS with 150mM KC1 (pH 6.4-7.6). ET binding. 12’I-ET binding kinetics wereperformed at 22°C. In preliminary studies, we showedthat the binding of 12”1-ET (33.8 PM) reached a plateau at 120 min. All further binding experiments were performed for 150 min in HBS, 0.2% BSA, and 100 kU/ml aprotinin in the presenceof 33.8 pM 12’I-ET (specific activity = 2,200Ci/mmol) and varying concentrations of unlabeled ET (0.015-3 nM). At the end of each experiment, cells were washed three times with HBS, 1251was extracted with 5% tetrachloroacetic acid (TCA), and radioactivity was counted in a gammacounter. Total binding was 1.7 t 1.4% of total activity in the binding solution and nonspecific binding (measuredin the presenceof 100 nM unlabeled ET) was 27.7 $- 7.6% of the total binding. Aliquots of trypsinized cells were usedfor determination of cell number (measuredin a Coulter Counter ZBI). Data analysis. Mn quenching of fura- wasbest fitted to an
Cl49
MUSCLE
exponential function with two compartments A = al x eAklxt + a2 x e-hzxt
(I)
where A is the cellular pool at time t, al and a2are compartments (expressedas percentage of the cellular pool; i.e., fura- fluorescencebefore treatments with KC1 or ET), and k, and k2 are rate constants. Parametersof Na activation of Na-H antiport were based on the change (A) in pHi in the first 5 s after activation of the exchanger in nigericin acidified cells, as described previously (15). The model u = V,,,,, x [Na]“/& + [Na]” was used, where u is reaction velocity, Vmax is maximal reaction velocity, K0..5is concentration for half-maximal stimulation, and n is the Hill coefficient. For the displacementof 1251-ETby unlabeledET, curves were fit to the data according to the model B = B,,, X [L/&[ 1 + (i/K$] + L], as previously described (28). In this model, B is specific binding, B,,, is maximal specific binding, & is equilibrium dissociation constant, L is concentration of ‘““I-ET, i is concentration of unlabeled ET, and n is the Hill coefficient. Nonlinear regressionanalysesfor computations of parameters of the Mn quenching of fura-2, Na activation of Na-H antiport, and 12’1-ET-bindingkinetics were performed with an IBM compatible PC (SAS REG and GLM programs, SAS Institute, Cary, NC). Basal Ca; levels, peak Cai transients, and posttransient Cai were compared using the Student’s t test. Statistical analysesof parametersderived from the Mn quenching were performed using weighted least squaresaccording to the method describedby Johnson and Milliken (18). Data are presentedas meansor meanst SE. RESULTS
Figure 1 depicts staining of primary cultures of HUA VSMCs with HHF35 muscle actin-specific antibody. Stress fibers could be easily seen transversing the length of the VSMCs. The same appearance has been demonstrated by others with respect to this antibody staining of rat VSMCs (34) or immunofluorescence with cysmooth muscle actin of human umbilical vein VSMCs (6) ET increased Cai in a dose-response manner (Fig. 2). A Cai response could be easily discerned at 4 nM. Maximal effect was demonstrated at 400 nM. At this concentration, in the presence of 1 mM Ca, basal Cai rose from 86 t 16 to 216 -t 33 nM (n = 4) at 10 s postexposure. The effect of ET on Cai was biphasic; after the return to basal level, the Cai started to rise again -50-60 s after the initial exposure. This secondary increase in Cai was not due to dye leakage (checked by the addition of Mn). ET-induced Cai responses were substantially attenuated in Ca deficient medium (0.3 mM EGTA substituted for Ca). Cells were maintained in this medium for l-3 min for measurements of basal Cai and then treated with ET. At 400 nM, ET increased Cai from 53 & 9 to 75 t 10 nM at 20 s postexposure without a subsequent elevation in Cai (Fig. 3). Verapamil (15 PM), a phenylalkylamine derivative, produced a marked effect on the ET-induced Cai signal (Fig. 4). It lowered the peak Ca; signal from 247 t 35 nM to 106 t 8 nM at 20 s (P < 0.01) and ablated the posttransient Ca; rise (n - 5). In further experiments, we examined the effect of nicardipine, a dihydropyridine derivative, on the Ca; signals induced by ET (Table 1). Maximal effect of nicardipine (-72% inhibition of the peak Ca; response) was attained at 6
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Cl50
ENDOTHELIN
AND VASCULAR MUSCLE 300
ET (4x10-%) c
225 P .= 150 ts 75
n
0l-l
0 -
50
OL
200
--.---
0e
200
TIME keel
TIME (WI
250
s
TIME kec)
150
+ C
T
a
I
&T
I
I
5
1
l
“g ‘OOi-I 50
t OL
0
I
I
20
40
I
I
60 80 TIME (set)
I 100
I
I
I
120
140
160
Fig. 2. Endothelin (ET)-induced intracellular Ca (CaJ signals in 1 mM Ca-containing medium. Insets: cellular response to different concentrations of ET.
0
Olr
0
I
I
20
40
I
50
I
60 80 TIME kec)
”
”
too I50 200 TIME (see)
250
1
300
I
I
I
I
100
120
140
160
Fig. 1. Immunofluorescence with HHF35 muscle actin-specific monoclonal antibody. A: HUA VSMCs (magnification at ~1,250); I?: human skin fibroblasts (magnified at ~1,250); C: monolayer of confluent HUA VSMCs (magnification at x100). Stress fibers transverse length of VSMCs. Nonspecific staining is observed in fibroblasts.
Fig. 3. Effect of 400 nM ET on Cai in Ca-free medium. Inset: specific illustration showing that a repeat treatment with ET after 5 washings did not further alter Ca signal.
PM. Nicardipine also attenuated the posttransient Cai response. For instance, at 150 s postexposure to 100 nM ET, the mean Cai level in the presence of 10 PM nicardipine was 30% of that in cells treated with ET alone (not shown). Inasmuch as verapamil and nicardipine inhibit voltage-sensitive Ca channels (a), the presence of these channels was examined by treatment with 45 mM KC1 (substituting for an equivalent concentration of NaCl). Paradoxically, there was no effect of depolarization by KC1 on the Cai signal. Because Ca and Mn appear to share the same pathways for entry into the cell, the effects of KC1 depolarization and ET on Mn quenching of fura(at wavelength of 360 nm) were examined (Fig. 5, Table 2). The fura- quenching profile for control cells was best fit by a biexponential function (as described under METHODS). It is apparent, however, that for these cells,
compartment a2 was of a very small magnitude. The quenching profiles of fura- in cells treated with KC1 or ET were also described by biexponential functions with compartments al -70% and a2 -30%. Compared with control cells, the rate constant kl, associated with compartment al, demonstrated a marked increase in KC1 depolarized cells and a further increase in ET-treated cells (Table 2). These alterations were reflected by similar increases in Mn-evoked fura- quenching and suggest divalent cation entry occurring through voltage-sensitive Ca channels. VSMCs treated with thrombin (0.5 NIH U/ml) in Cacontaining HBS (pH 7.4) demonstrated a rise in Cai from basal levels of 113 + 5 to 187 + 17 nM (n = 6) at 25 s after exposure (Fig. 6). There was no posttransient increase in Cai in VSMCs subjected to thrombin. Cells maintained in Ca-free medium for l-3 min and subse-
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-w-------w
VW*
l3NUU’l’Hl3LlN
ET (4~10-~
! -
OLI
0
.
WV-
VASCULAR
ANU
M)
I
I
I
I
20
40
60
80
I 100
I
I
I
120
140
160
TIME bed Fig. 4. Effect of 15 PM verapamil on ET-induced Cai signals. Closed circles, VSMCs treated with 400 nM ET; open triangles, VSMCs treated with same dose of ET but in presence of verapamil.
Table 1. Inhibition by nicardipine of ET-evoked Cai transient n
4 5 4 5 4
Nicardipine,
PM
Peak
0.3 1.0 3.0 6.0 10.0
Ca;, % of control
101.5~13.8 60.5t22.1 38.3zk6.5 24.1tl0.9 27.6t9.2
Data are means t SE and expressed as percentage of control cells treated with 100 nM ET. n, no. of observations for each nicardipine concentration.
100
150
200
250
300
TIME bed Fig. 5. Mn quenching of fura-2. Circles, control; squares, 45 mM KC1 depolarization; triangles, 100 nM ET. Treatment with KC1 or ET was performed at t = 0 by replacing the HBS with either KC1 or ET HBS with 1 mM Mn. Curves were fit to data according to exponential model described in METHODS.
quently treated with thrombin showed a rise from basal Cai levels of 46 t 4 to 117 t 12 nM (n = 4) at 30 s (Fig. 6). In both Ca-containing and Ca-free media, Ca; returned to basal level within 80-150 s after exposure to thrombin. Both basal Cai and peak Cai transient levels
MUSCLE
Cl51
after thrombin treatment were significantly lower (P c 0.01) in Ca-free medium than in 1 mM Ca medium. Because many agonists activate the Na-H antiport in concert with raising Ca;, we measured the effect of ET and thrombin on the HUA VSMC pHi profile. Neither ET nor thrombin exerted any influence on the pHi under basal condition (pHi = 7.1-7.25) or in Na-free medium. PMA (100 nM) also did not influence the pHi profile (not shown). However, when quiescent VSMCs (deprived of serum for 24 h) were subjected to 10% FBS in Nacontaining HBS (pH 7.4), a biphasic change in pHi was observed (n = 4); an immediate acidification was followed by sustained alkalinization (Fig. 7A). In Na-deficient medium (n = 5), exposure to 10% FBS produced only sustained acidification (Fig. 7B). That a Na-H antiport exists in HUA VSMCs was also demonstrated by monitoring the Na-dependent, EIPAsensitive recovery from cellular acidification (Fig. 8). Cells were acidified with nigericin to pHi ~6.6, washed three times in Na-free HBS (pH = 7.4), and then subjected to different Na concentrations in HBS (pH = 7.4). The pHi recovery was Na dependent and sensitive to EIPA. The following parameters were obtained for the Na-activation kinetics of the antiport, according to the model described in METHODS: Vmax= 0.12 pH U/5 s, Ko5 = 50 mM, and n = 2.0. ET or thrombin exerted no effect on the rate of Na-dependent recovery from nigericininduced acidification. The presence of specific ET receptors on HUA VSMCs was confirmed by lz51-ET-binding experiments (Fig. 9). There was no effect of nicardipine on ET binding to its receptors (Fig. 9, inset). Data derived from these experiments (n = 5) were used to calculate a B,,, = 12,722 sites/cell and & = 0.34 nM for ET in these cells. DISCUSSION
In this study, we have demonstrated that the ETinduced Cai response in HUA VSMCs involves a combination of Ca mobilization from intracellular stores and Ca influx from the extracellular compartment. In fact, Ca influx was the main component of the ET-induced Cai response, as shown by the Ca; signals and Mn quenching of fura- (Figs. 3-5 and Tables 1 and 2). Although both ET and thrombin produced a rise in Cai, subtle differences were observed in the cellular response to both agonists. Whereas the thrombin-evoked Cai signal was essentially monophasic, i.e., a brisk Ca; transient rapidly returning to basal Cai level, a biphasic profile in the ETinduced Cai response was observed in Ca-containing medium. Two major changes were noted in the ETinduced Cai response of VSMCs suspended in Ca-free medium or treated with verapamil or nicardipine. First, the amplitudes of the Cai transient and posttransient Ca; were substantially diminished in the presence of nicardipine, and second, the biphasic profile of the Cai signal became monophasic in the presence of verapamil or in Ca-free medium. From these observations, we suggest that the second phase of the ET-induced Cai response relates to the influx of extracellular Ca, while the Ca; transient results from both extracellular Ca influx and intracellular Ca mobilization. Recently, we have arrived
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Cl52 Table
ENDOTHELIN
2. Quenching
of fura-
AND
Control 45 mM KC1 100 nM ET
0.0128t1.35 0.0278t0.0007 0.0350t0.0006*
Values are means t SE. al and a2, compartments each treatment). Statistical analyses were performed ET treatments significantly differ from control at
r
r THROMBIN
P
al
k2, s-l
99.90t0.0003 70.35+1.45 71.14t0.80
-0.0034t1.19 0.0057t0.0002 0.0061~0.0002
I 40
I 60
I 80
I 100
I
1
120
140
160
TIME (set)
Fig. 6. Effect free medium.
of 0.5 U/ml
NIH
0
I, 1
50
of thrombin
100 150 TIME (set)
I
1 1
80
,-
12OI Ill, 1
,
in 1 mM
200
1
Ca and in Ca-
250
1 I
I ,
160
200
' 240 ' TIME Ised
260
+0.50 7.001
III ,,I
0
50
1 80 1
40
100 150 TIME (set)
III I
I1
120
-0.10 Fig. 7. pHi containing
I 160
250
I
II 200 I
I
I 240 II
I
280
TIME (set)
i -0.50
200 I1
I i T-
profile
of HUA
(A) and Na-deficient
VSMCs (B)
a2 3.281kO.0015 31.59t1.56 29.88t0.85
(expressed as percentage of the cellular pool); rlz2 and kp, rate constants; t, time (n = 6 for using Johnson and Milliken (18) procedure, as indicated under METHODS. Both KC1 and different from KC1 treatment at P < 0.001. < 0.001. * Significantly
(0.5 U/ml 1
I 20
0
MUSCLE
by Mn kl, s-l
250
VASCULAR
subjected to 10% FBS in Namedia. Insets: specific tracings.
at a similar conclusion with respect to the action of ET on human myometrial cells (22). Yanagisawa and co-workers (37) proposed that the mode of action of ET is primarily through the dihydropyridine-sensitive Ca channels. This idea is supported by the striking homology between ET and cr-scorpion toxins, which act on tetradotoxin-sensitive Na channels. The Ca and Na channels belong to a broad category of voltage-dependent channels. Results of subsequent studies, performed primarily using cultured rat VSMCs, demonstrated a spectrum of ET-induced Ca; responses. Some workers showed that ET-induced Cai responses were observed only in Ca-containing medium and attenuated by Ca channel blockers (12, 36), thereby supporting the original concept introduced by Yanagisawa et al. (37). Others have shown that ET, like vasoconstrictors such as angiotensin II and vasopressin, exerts its effect through both Ca; mobilization and Ca entry (19, 29, 35). Yet other groups have concluded that in the rat VSMC, the ET-induced Ca; signal solely depends on mobilization from intracellular stores without involvement of the dihydropyridine-sensitive Ca channels (26, 27). It is clear, however, that ET exerts some of its cellular effect in HUA VSMCs through Ca entry. As shown by the ETbinding experiments for these and other cells, this action is not because it is an agonist for the dihydropyridinesensitive Ca channels (7, 16). Rather, ET stimulation of Ca channels is likely to be an indirect one (12, 35, 36). We monitored fura- quenching by Mn as an indicator of Mn uptake to demonstrate the presence of voltagesensitive Ca channels in HUA VSMCs. Mn has been used previously to characterize Ca transport in a variety of cells (5, 13, 22, 25, 31). It has substantially greater affinity than Ca to fluorescent probes such as quin2 and fura-2. Moreover, Mn is a poor substrate for the endoplasmic reticulum Ca pump [Ca-adenosinetriphosphatase (ATPase)] (12, 25). These features of Mn suggest an explanation why KC1 depolarization did not produce a detectable alteration in Cai, yet it produced a significant acceleration of the rate of Mn quenching of fura-2. We propose that the density or activity of voltage-sensitive Ca channels in cultured HUA VSMCs is relatively small. Thus depolarization produced only a small increase in Ca uptake, and the entering Ca was either taken up by the endoplasmic reticulum or recycled back to the medium without an apparent effect on the Cai signal. However, because of the aforementioned features of Mn, its increased entry by KC1 depolarization or ET could be easily detected. In this regard, the difference between the apparent E& (-40 nM) for ET-evoked Ca; response and the & of 3.4 nM for ET binding can be explained by the same process. At lower concentrations, ET may
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ENDOTHELIN No+
I
6.52 I" 0
VASCULAR
MUSCLE
Cl53
hM)
J 200
100
AND
6501 .
0
100 TIME bed
TIME (sec)
200 Fig. 8. Na-dependent, &(N-ethyl-N-isopropyl)amiloride (EIPA)-sensitive recovery of HUA VSMCs from nigericin-induced acidification. Cells were preincubated with HBS containing 0.5 pg/ml nigericin (pH 6.8) for 5 min. Insets: Na dependency (left) and EIPA sensitivity (right) of pHi recovery. Figure demonstrates kinetics of rate of Na-dependent recovery as a function of Na concentration. Line was fit to data according to model described in METHODS.
0.15
0
20
40
100
60 Na+(mM)
r
1400
s- 1000 & 800 *s U’ 600 GI$ -z1 au 400 v>o E L- 200 A 04
1200 A = \8 z 51000 s -E
g
10-l'
10-10
10-g
10-e
800
P E E 600 E k t; 400 A tc 200
0
10-8 10-g ET(M) Fig. 9. Displacement of 1251-ET by unlabeled ET. Inset: displacement of 1251-ET by unlabeled ET in presence (open circles) or absence (closed circles) of 100 nM nicardipine. Lines were fit to data according to model described in METHODS. IO-"
I O-10
accelerate Ca entry and enhance its mobilization. However, these alterations may not be sufficient to raise the Cai. Because verapamil and nicardipine attenuated the ET-evoked Cai response, voltage-sensitive Ca channels may be indirectly activated by ET and their function may be more apparent after ET-induced Ca mobilization, perhaps to refill cellular Ca stores. Alternatively, our two observations (the presence of voltage-dependent, divalent cation influx and nicardipine-verapamil blockade of the ET-induced Cai signal) could be mutually exclusive.
ET-induced Ca influx could occur through other pathways, including receptor-operated Ca channels. In this regard, Zschauer et al. (38) demonstrated stimulation of voltage-independent Ca channels by thrombin in human platelets. Although these channels shared several characteristics with voltage-sensitive Ca channels, they differed with respect to sensitivity to dihydropyridines. The biological equivalents of compartments al and a2, described by the Mn-induced fura- quenching model, are not readily apparent. It is possible that al represents a cytosolic compartment that includes the submembrane domain. In this compartment, fura- is rapidly quenched by the accelerated Mn influx after KC1 or ET treatment. Compartment a2 is less readily available to Mn, and it may represent a deeper portion of the cytosol as well as cellular organelles that probably do not include the endoplasmic reticulum. The cellular action of many vasoactive agents is mediated via phospholipase C activation and the stimulation of protein kinase C dependent and independent pathways, leading to activation of the Na-H antiport (3, 14, 17). Although ET activates phospholipase C in rat (1, 26, 33, 35) and human (30) VSMCs, ET-induced phosphatidylinositol breakdown in rat VSMCs is less pronounced than that of thrombin (26), angiotensin II (l), and vasopressin (35). Moreover, Badr et al. (2) and Koh et al. (20), respectively, showed that it produces intracellular alkalinization in mesangial cells and rat VSMCs. We, however, could not demonstrate ET-induced intracellular alkalinization in HUA VSMCs. This inability to activate the Na-H antiport appears not to relate specifically to ET, inasmuch as thrombin (17) and PMA (14), which readily activate the antiport in rat VSMCs, were also incapable of eliciting intracellular alkalinization in HUA VSMCs. The underlying mecha-
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Cl54
ENDOTHELIN
AND
nisms for these observations are not apparent, because the Na-H antiport in HUA VSMCs could be activated by serum and by acidification. In Na-containing medium, cells subjected to serum showed an initial acidification followed by alkalinization. The acidification presumably represents activation of the Ca-ATPase (a Ca-H exchanger) due to the rapid rise in Ca; (14). The subsequent alkalinization reflects the Na-H antiport activity. This latter phase was ablated in Na-deficient medium. The Na-dependent recovery from acidification showed a Hill coefficient value of -2. A Hill coefficient of -2 has been shown by others for human erythrocytes (32) and by us for cultured human fibroblasts (9) and myometrial cells (17). It might indicate positive cooperativity on more than one population of the Na-H antiport. Finally, that the ET action on the HUA VSMCs is exerted through specific receptors has been demonstrated by the lz51-ET-binding experiments. These experiments demonstrate a single class of ET receptors with B,,, and & values that are quite similar to those reported for rat thoracic aorta (16) and human umbilical vein (6) VSMCs. They appear not to relate to the dihydropyridine-sensitive Ca channel receptors. This work was supported by National Heart, Lung, and Blood Institute Grant HL-34807 and by a grant-in-aid from the American Heart Association (New Jersey Affiliate). E. Maker and 9. P. Gardner were research fellows of the American Heart Association (New Jersey Affiliate). Address for reprint requests: J. P. Gardner, Dept. of Pediatrics and Hypertension Research Ctr., Room F-464, MSB, Univ. of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 S. Orange Ave., Newark, NJ 07103. Received
15 November
1989; accepted
in final
form
29 July
1991.
VASCULAR
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
REFERENCES 1. Araki, S., Y. Kawahara, K. Kariya, M. Sunako, H. Fukuzaki, and Y. Takai. Stimulation of phospholipase C-mediated hydrolysis of phosphoinositides by endothelin in cultured rabbit aortic smooth muscle cells. Biochem. Biophys. Res. Commun. 159: 1072-1079,1989. 2. Badr, K., J. Murray, M. Breyer, K. Takahashi, T. Inagami, and R. Harris. Mesangial cell, glomerular and renal vascular response to endothelin in the rat kidney. Elucidation of signal transduction pathways. J. CZin. Inuest. 83: 336-342, 1989. 3. Berk, B. C., M. S. Aronow, T. A. Brock, E. Cragoe, Jr., M. A. Gimbrone, Jr., and R. W. Alexander. Angiotensin IIstimulated Na’/H’ exchange in cultured vascular smooth muscle cells. J. BioZ. Chem. 262: 5057-5064, 1987. 4. Cernacek, P., and D. J. Stewart. Immunoreactive endothelin in human plasma: marked elevation in patients with cardiogenic shock. Biochem. Biophys. Res. Commun. 161: 562-567, 1989. 5. Chien, W. W., R. Mohabir, and W. T. Clusin. Effect of thrombin on calcium homeostasis in chick embryo heart cells: receptor operated calcium entry with inositol trisphosphate and pertussis toxin-sensitive G proteins as second messengers. J. CZin. Inuest. 85: 1436-1443, 1990. 6. Clozel, M., W. Fischli, and C. Guilly. Specific binding of endothelin on human vascular smooth muscle cells in culture. J. CZin. Inuest. 83: 1758-1761, 1989. E. N., C. E. Gomez-Sanchez, M. F. Foecking, and S. 7. Cozza, Chiou. Endothelin binding to cultured calf adrenal zona glomerulosa cells and stimulation of aldosterone secretion. J. CZin. Inuest. 84: 1032-1035, 1989. S. F. Calcium antagonists and vascular smooth muscle. In: 8. Flaim, Recent Advances in ArteriaZ Diseases, edited by T. N. Tulenko and R. H. Cox. New York: Liss, 1986. 9. Gardner, J. P., G. Tokudome, H. Tomonari, M. Aladjem, E.
20.
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27.
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J. Cragoe, Jr., and A. Aviv. Refined estimation of kinetic parameters of the Na’/H’ antiport in human fibroblasts and platelets. Proc. Sot. Exp. BioZ. Med. 194: 165-171, 1990. Goetz, K. L., B. C. Wang, J. B. Madwed, J. L. Zhu, and R. J. Leadley, Jr. Cardiovascular, renal and endocrine responses to intravenous endothelin in conscious dogs. Am. J. Physiol. 255 (Regulatory Integrative Comp. Physiol. 24): Rl064-R1068, 1988. Gomez, D. A., A. G. Costa, and V. M. C. Madeira. Magnesium and manganese ions modulate Ca2+ uptake and its energetic coupling in sarcoplasmic reticulum. Arch. Biochem. Biophys. 249: 199206,1986. Goto, K., Y. Kasuya, N. Matsuki, Y. Takuwa, H. Kurihara, T. Ishikawa, S. Kimura, M. Yanagisawa, and T. Masaki. Endothelin activates the dihydropyridine sensitive, voltage-dependent Ca2+ channel in vascular smooth muscle. Proc. NatZ. Acad. Sci. USA 86: 3915-3918,1989. Hallam, T. J., and T. J. Rink. Agonists stimulate divalent cation channels in the plasma membrane of human platelets. FEBS Lett. 186: 175-179, 1985. Hatori, N., B. Fine, A. Nakamura, E. Cragoe, Jr., and A. Aviv. Angiotensin II effect on cytosolic pH in cultured rat vascular smooth muscle cells. J. BioZ. Chem. 262: 5073-5078, 1987. Hatori, N., J. Gardner, H. Tomonari, B. P. Fine, and A. Aviv. Na+-H+ antiport activity in skin fibroblasts from blacks and whites. Hypertension 15: 140-145, 1990. Hirata, Y., H. Yoshimi, S. Takata, T. Watnabe, S. Kumagi, K. Nakajima, and S. Sakakibara. Cellular mechanism of action by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 154: 868875,1988. Huang, C.-L., M. G. Cogan, E. J. Cragoe, and H. E. Ives. Thrombin activation of the Na+-H+ exchanger in vascular smooth muscle cells: evidence for a kinase C-independent pathway which is Ca2+ dependent and pertussis toxin-sensitive. J. BioZ. Chem. 262: 14134-14140,1987. Johnson, P., and G. A. Milliken. A simple procedure for testing linear hypotheses about the parameters of a nonlinear model using weighted least squares. Commun. Stat. Sim. Comput. 12: 135-145, 1983. Kai, H., H. Kanaide, and M. Nakamura. Endothelin-sensitive intracellular Ca2’ store overlaps with caffeine-sensitive one in rat aortic smooth muscle cells in primary culture. Biochem. Biophys. Res. Commun. 158: 235-243,1989. Koh, E., S. Morimoto, S. Kim, T. Nabata, Y. Miyashita, and T. Ogihara. Endothelin stimulates Na+/H’ exchange in vascular smooth muscle cells. Biochem. Int. 20: 375-380, 1990. Komuro, I., H. Kurihara, T. Sugiyama, F. Takaku, and Y. Yazaki. Endothelin stimulates c-fos and c-myc expression and proliferation of vascular smooth muscle cells. FEBS Lett. 238: 249252,1988. Maher, E., A. Bardequez, J. P. Gardner, L. Goldsmith, G. Weiss, M. Mascarina, and A. Aviv. Endothelin and oxytocin, calcium signaling in cultured human myometrial cells. J. CZin. Inuest. 87: 1251-1258, 1991. Marsden, P., N. Danthuluri, B. Brenner, B. Ballermann, and T. Brock. Endothelin action on vascular smooth muscle involves inositol triphosphate and calcium mobilization. Biochem. Biophys. Res. Commun. 158: 80-93, 1989. Miller, W. L., M. M. Redfield, and J. C. Burnett, Jr. Integrated cardiac, renal and endocrine actions of endothelin. J. CZin. Inuest. 83: 317-320, 1989. Missiane, L., I. Declerck, G. Droogmans, L. Plessers, H. De Smedt, L. Raeymaekers, and R. Casteels. Agonist-dependent Ca2+ and Mn2+ entry dependent on state of filling of Ca2’ stores in aortic smooth muscle cells of the rat. J. Physiol. Lond. 427: 171186,199O. Mitsuhashi, T., R. C. Morris, Jr., and H. E. Ives. Endothelininduced increases in vascular smooth muscle Ca2+ do not depend on dihydropyridine-sensitive Ca2+ channels. J. CZin. Inuest. 84: 635-639,1989. Muldoon, L. L., K. D. Rodland, M. L. Forsythe, and B. E. Magum. Stimulation of phosphatidyl inositol hydrolysis, diacylglycerol release, and gene expression in response to endothelin, a potent new agonist for fibroblasts and smooth muscle cells. J. BioZ. Chem. 264: 8529-8536,1989.
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AND
28. Nakamura, M., A. Nakamura, B. Fine, and A. Aviv. Blunted cGMP response to ANF in vascular smooth muscle cells of SHR. Am. J. Physiol. 255 (Cell Physiol. 24): C573-C580, 1988. 29. Niasiro, N., H. Yamamoto, H. Kanaide, and M. Nakamura. Does endothelin mobilize calcium from intracellular store sites in rat aortic vascular smooth muscle cells in primary culture? Biochem. Biophys. Res. Commun. 156: 312-317, 1988. 30. Resink, T. J., T. Scott-Burden, and F. R. Buhler. Endothelin stimulates phospholipase C in cultured vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 157: 1360-1368, 1988. 31. Sage, S. O., R. Reast, and T. J. Rink. ADP evokes biphasic Ca2+ influx in furaloaded human platelets: evidence for Ca2+ entry regulated by the intracellular Ca2’ store. Biochem. J. 265: 675-680, 1990. 32. Semplicini, A., A. Spalvins, and M. Canessa. Kinetics and stoichiometry of human red cell Na+/H+ exchange. J. Membr. Biol. 107: 219-228, 1989. 33. Sugiura, M., T. Inagami, M. T. Gregory, and J. A. Jones. Endothelin action: inhibition by a protein kinase C inhibitor and involvement of phosphoinositols. Biochem. Biophys. Res. Commun.
VASCULAR
MUSCLE
Cl55
158: 170-176, 1989. 34. Tsukada, T., D. Tippens, D. Gordon, and A. M. Gown. HHF35, a muscle-actin-specific monoclonal antibody. Am. J. Pathol. 126: 51-60, 1987. 35. Van Renterghem, C. P. Vigne, J. Barhanin, A. SchmidAlliana, C. Frelin, and M. Lazdunski. Molecular mechanism of action of the vasoconstrictor peptide endothelin. Biochem. Biophys. Res. Commun. 157: 977-985, 1988. 36 Xuan, Y. T., A. R. Whorton, and W. T. Watkins. Inhibition by nicardipine on endothelin-mediated inositol phosphate formation and Ca2’ mobilization in smooth muscle cell. Biochem. Biophys. Res. Commun. 160: 758-764, 1989. 37. Yanagisawa, M., H. Kurihara, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Mitsui, Y. Yazaki, K. Goto, and T. Masaki. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature Lord. 332: 411-415, 1988. 38. Zschauer, A. C,, C. van Breeman, F. R. Buhler, and M. T. Nelson. Calcium channels in thrombin-activated human platelet membrane. Nature Land. 334: 703-705.1988.
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calcium
channels;
sodium-
ADMINISTRATION ofendothelin(ET)produces increased blood pressure, peripheral vascular resistance, diminished cardiac output, and sodium excretion as well as multiple hormonal changes, including a rise in the circulating levels of the atria1 natriuretic peptide, vasopressin, aldosterone, and plasma renin activity (10, 24). Although, in part, these effects may be mediated through specific interactions of ET with different cells (e.g., zona glomerulosa cells and renal mesangial cells; Refs. 2 and 7), the main target cell of ET appears to be the vascular smooth muscle cell (VSMC). ET is a potent vasoconstrictor, exerting its effect primarily via the cytosolic free Ca (Ca;) messenger system (12, 16, 23, 26, 29, 35, 36) and phosphatidylinositol breakdown (23, 26, 29, 30, 35, 36). Information concerning the effect of ET on the VSMC has been derived primarily from cells originating from laboratory animals. Resink et al. (30) have demonstrated that ET stimulates phospholipase C in human VSMCs,
SYSTEMIC
Cl48
0363-6143/92
$2.00 Copyright
and Clozel and co-workers (6) have shown that specific receptors for ET are present in the human umbilical vein VSMCs. However, little is known of the nature and origin of ET-induced Ca; signals in the human VSMC. The presence of immunoreactive endothelin in human plasma (4) suggests that ET functions as a hormone. However, its major role is likely to be the local regulation of VSMC and as such it may play a central role in the physiology or pathophysiology of the cardiovascular system. In the present work we have characterized the ET-induced Ca; response and ET-binding kinetics in VSMCs obtained from the human umbilical artery (HUA). In addition, we examined whether ET exerts an effect on the Na-H antiport in these cells. METHODS Materiak. Synthetic ET-l was from Cambridge Biochemicals (Valley Stream, NY), and 12’I-ET-l was from New England Nuclear (Wilmington, DE). Fetal bovine serum (FBS), cu-minimal essential medium (ar-MEM), and collagenase were from Hazelton Biologics (Lenexa, KS). Fura-2/acetoxymethyl ester (AM) and 2’,7’-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)/AM were from Molecular Probes (Eugene, OR). HHF35 muscle actin-specific monoclonal antibody was from Organon Teknika (Westchester, PA). 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was provided by E. Cragoe, Jr. All other chemicals, including phorbol 12-myristate 13-acetate (PMA), verapamil, nicardipine, and human thrombin (catalog No. T-9135), were from Sigma (St. Louis, MO). IsoZation and growth of primary VSMCs. VSMCs were obtained from umbilical arteries of 16 normal male and female newborns. Each umbilical artery was grossly cleaned and immersed in a-MEM plus antibiotics (30 pg/ml streptomycin and 30 units/ml penicillin). It was cut longitudinally, and, using needles, the resulting rectangle was fastened to the bottom of a tissue culture dish covered with a thick layer of silicone rubber. The intima was scraped and a transverse slit across the short axis of the vascular rectangle was made (with the aid of a dissecting microscope) to -5O-80% of the thickness of the media. The line of cleavage was used as a starting point for stripping the media as a single sheath. To assure complete removal of the endothelium, the preparation was suspended for 5 min in cw-MEM containing 0.1% collagenase. The media was rinsed three times, and fragments (-0.5 x 0.5 mm) were sandwiched between glass cover slips (17 x 30 mm), which were placed into culture dishes. Cultures were maintained in cyMEM, 15% FBS, 5% CO,-95% air plus antibiotics. Growth extensions of VSMCs from explants were observed in -2 wk. In contrast to fibroblasts, HUA VSMCs grew very slowly and confluency on the cover slips was reached in about 2 mo. Thus any preparation demonstrating rapid growth was discarded. Indirect immunofluorescence of the cells with HHF35 muscle
0 1992 the American
Physiological
Society
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ENDOTHELIN
AND
VASCULAR
actin-specific monoclonal antibody were performed as described (34) to ascertain the purity of the VSMC preparations. Antibiotics were removed 3 days prior to experiments, which were performed in monolayers of primary cultures. Before loading the cells with the fluorescent dyes, cells on cover slips were washed twice with N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) buffered solution (HBS) containing (in mM) 140 NaCl, 5 KCl, 1 CaClz, 1 MgClz, 10 glucose, and 20 HEPES [pH 7.4 for Ca; measurements and pH 7.1 for intracellular pH (pHi) measurements]. Experiments were performed in 1 mM Ca or Ca-free HBS. Measurements of Cai. Cellswere incubated for 60 min with 5 PM fura-2/AM in 3 ml (37°C) of HBS plus 0.1% bovine serum albumin (BSA). They were washedtwice with HBS, and the slideswere securedin a quartz cuvette in a SPEX CM3 fluorescencespectrometer equipped with a thermostatically controlled (37°C) cell holder, a stirrer, and a suction device for rapidly removing solutions. Excitation wavelengths were set at 340 and 380 nm and emissionwavelength at 505 nm. Fluorescencewas monitored for l-3 min until the Ca; signal stabilized and basalCai measurementwasobtained. Thereafter, cellswere subjectedto specific agonists(e.g., ET and thrombin) or experimental perturbations, and Cai signals were recorded for an additional 150-300 s. Cai calibration was performed on each cover slip by exposing cells to 3 mM ethylene glycol-his@aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) with 10 PM ionomycin, followed by 3 mM Ca. Autofluorescence was determined by subjecting cells to 2 mM Mn and 10 PM ionomycin. Autofluorescence was measuredfor each slide and subtracted from the Cai signal. Mn uptake. For the Mn uptake experiments, cells were treated in a similar fashion asthoseprepared for measurements of Ca;. They were suspendedin HBS plus 1 mM MnCl,. Mn uptake by the cells was monitored at 360 nm. This is the isosbesticwavelength for fura-2. Thus changesin Cai do not alter the fura- emission,whereasMn uptake results in quenching of the fluorescence. Any factor enhancing Mn uptake is expected to acceleratethe rate of fura- quenching (5, 13, 31). Measurement of pHi. Cover slips were incubated at 37°C for 60 min in HBS with 2 PM BCECF/AM. After two washeswith HBS, they were placed in the spectrometer (excitation wavelengths 440 and 503 nm and emissionwavelength 530 nm) and monitored for 7-12 min until the pHi signal stabilized. Cells were then treated with specific agonists or experimental perturbations, and the pHi was followed for a period up to 250 s. Whenever necessary, Na was isosmotically replaced by Nmethyl-D-glucamine. Calibration of fluorescent intensity of intracellular BCECF against pHi was performed at the end of each experiment by subjecting cells to 5 ,ug/ml of nigericin in HBS with 150mM KC1 (pH 6.4-7.6). ET binding. 12’I-ET binding kinetics wereperformed at 22°C. In preliminary studies, we showedthat the binding of 12”1-ET (33.8 PM) reached a plateau at 120 min. All further binding experiments were performed for 150 min in HBS, 0.2% BSA, and 100 kU/ml aprotinin in the presenceof 33.8 pM 12’I-ET (specific activity = 2,200Ci/mmol) and varying concentrations of unlabeled ET (0.015-3 nM). At the end of each experiment, cells were washed three times with HBS, 1251was extracted with 5% tetrachloroacetic acid (TCA), and radioactivity was counted in a gammacounter. Total binding was 1.7 t 1.4% of total activity in the binding solution and nonspecific binding (measuredin the presenceof 100 nM unlabeled ET) was 27.7 $- 7.6% of the total binding. Aliquots of trypsinized cells were usedfor determination of cell number (measuredin a Coulter Counter ZBI). Data analysis. Mn quenching of fura- wasbest fitted to an
Cl49
MUSCLE
exponential function with two compartments A = al x eAklxt + a2 x e-hzxt
(I)
where A is the cellular pool at time t, al and a2are compartments (expressedas percentage of the cellular pool; i.e., fura- fluorescencebefore treatments with KC1 or ET), and k, and k2 are rate constants. Parametersof Na activation of Na-H antiport were based on the change (A) in pHi in the first 5 s after activation of the exchanger in nigericin acidified cells, as described previously (15). The model u = V,,,,, x [Na]“/& + [Na]” was used, where u is reaction velocity, Vmax is maximal reaction velocity, K0..5is concentration for half-maximal stimulation, and n is the Hill coefficient. For the displacementof 1251-ETby unlabeledET, curves were fit to the data according to the model B = B,,, X [L/&[ 1 + (i/K$] + L], as previously described (28). In this model, B is specific binding, B,,, is maximal specific binding, & is equilibrium dissociation constant, L is concentration of ‘““I-ET, i is concentration of unlabeled ET, and n is the Hill coefficient. Nonlinear regressionanalysesfor computations of parameters of the Mn quenching of fura-2, Na activation of Na-H antiport, and 12’1-ET-bindingkinetics were performed with an IBM compatible PC (SAS REG and GLM programs, SAS Institute, Cary, NC). Basal Ca; levels, peak Cai transients, and posttransient Cai were compared using the Student’s t test. Statistical analysesof parametersderived from the Mn quenching were performed using weighted least squaresaccording to the method describedby Johnson and Milliken (18). Data are presentedas meansor meanst SE. RESULTS
Figure 1 depicts staining of primary cultures of HUA VSMCs with HHF35 muscle actin-specific antibody. Stress fibers could be easily seen transversing the length of the VSMCs. The same appearance has been demonstrated by others with respect to this antibody staining of rat VSMCs (34) or immunofluorescence with cysmooth muscle actin of human umbilical vein VSMCs (6) ET increased Cai in a dose-response manner (Fig. 2). A Cai response could be easily discerned at 4 nM. Maximal effect was demonstrated at 400 nM. At this concentration, in the presence of 1 mM Ca, basal Cai rose from 86 t 16 to 216 -t 33 nM (n = 4) at 10 s postexposure. The effect of ET on Cai was biphasic; after the return to basal level, the Cai started to rise again -50-60 s after the initial exposure. This secondary increase in Cai was not due to dye leakage (checked by the addition of Mn). ET-induced Cai responses were substantially attenuated in Ca deficient medium (0.3 mM EGTA substituted for Ca). Cells were maintained in this medium for l-3 min for measurements of basal Cai and then treated with ET. At 400 nM, ET increased Cai from 53 & 9 to 75 t 10 nM at 20 s postexposure without a subsequent elevation in Cai (Fig. 3). Verapamil (15 PM), a phenylalkylamine derivative, produced a marked effect on the ET-induced Cai signal (Fig. 4). It lowered the peak Ca; signal from 247 t 35 nM to 106 t 8 nM at 20 s (P < 0.01) and ablated the posttransient Ca; rise (n - 5). In further experiments, we examined the effect of nicardipine, a dihydropyridine derivative, on the Ca; signals induced by ET (Table 1). Maximal effect of nicardipine (-72% inhibition of the peak Ca; response) was attained at 6
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Cl50
ENDOTHELIN
AND VASCULAR MUSCLE 300
ET (4x10-%) c
225 P .= 150 ts 75
n
0l-l
0 -
50
OL
200
--.---
0e
200
TIME keel
TIME (WI
250
s
TIME kec)
150
+ C
T
a
I
&T
I
I
5
1
l
“g ‘OOi-I 50
t OL
0
I
I
20
40
I
I
60 80 TIME (set)
I 100
I
I
I
120
140
160
Fig. 2. Endothelin (ET)-induced intracellular Ca (CaJ signals in 1 mM Ca-containing medium. Insets: cellular response to different concentrations of ET.
0
Olr
0
I
I
20
40
I
50
I
60 80 TIME kec)
”
”
too I50 200 TIME (see)
250
1
300
I
I
I
I
100
120
140
160
Fig. 1. Immunofluorescence with HHF35 muscle actin-specific monoclonal antibody. A: HUA VSMCs (magnification at ~1,250); I?: human skin fibroblasts (magnified at ~1,250); C: monolayer of confluent HUA VSMCs (magnification at x100). Stress fibers transverse length of VSMCs. Nonspecific staining is observed in fibroblasts.
Fig. 3. Effect of 400 nM ET on Cai in Ca-free medium. Inset: specific illustration showing that a repeat treatment with ET after 5 washings did not further alter Ca signal.
PM. Nicardipine also attenuated the posttransient Cai response. For instance, at 150 s postexposure to 100 nM ET, the mean Cai level in the presence of 10 PM nicardipine was 30% of that in cells treated with ET alone (not shown). Inasmuch as verapamil and nicardipine inhibit voltage-sensitive Ca channels (a), the presence of these channels was examined by treatment with 45 mM KC1 (substituting for an equivalent concentration of NaCl). Paradoxically, there was no effect of depolarization by KC1 on the Cai signal. Because Ca and Mn appear to share the same pathways for entry into the cell, the effects of KC1 depolarization and ET on Mn quenching of fura(at wavelength of 360 nm) were examined (Fig. 5, Table 2). The fura- quenching profile for control cells was best fit by a biexponential function (as described under METHODS). It is apparent, however, that for these cells,
compartment a2 was of a very small magnitude. The quenching profiles of fura- in cells treated with KC1 or ET were also described by biexponential functions with compartments al -70% and a2 -30%. Compared with control cells, the rate constant kl, associated with compartment al, demonstrated a marked increase in KC1 depolarized cells and a further increase in ET-treated cells (Table 2). These alterations were reflected by similar increases in Mn-evoked fura- quenching and suggest divalent cation entry occurring through voltage-sensitive Ca channels. VSMCs treated with thrombin (0.5 NIH U/ml) in Cacontaining HBS (pH 7.4) demonstrated a rise in Cai from basal levels of 113 + 5 to 187 + 17 nM (n = 6) at 25 s after exposure (Fig. 6). There was no posttransient increase in Cai in VSMCs subjected to thrombin. Cells maintained in Ca-free medium for l-3 min and subse-
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-w-------w
VW*
l3NUU’l’Hl3LlN
ET (4~10-~
! -
OLI
0
.
WV-
VASCULAR
ANU
M)
I
I
I
I
20
40
60
80
I 100
I
I
I
120
140
160
TIME bed Fig. 4. Effect of 15 PM verapamil on ET-induced Cai signals. Closed circles, VSMCs treated with 400 nM ET; open triangles, VSMCs treated with same dose of ET but in presence of verapamil.
Table 1. Inhibition by nicardipine of ET-evoked Cai transient n
4 5 4 5 4
Nicardipine,
PM
Peak
0.3 1.0 3.0 6.0 10.0
Ca;, % of control
101.5~13.8 60.5t22.1 38.3zk6.5 24.1tl0.9 27.6t9.2
Data are means t SE and expressed as percentage of control cells treated with 100 nM ET. n, no. of observations for each nicardipine concentration.
100
150
200
250
300
TIME bed Fig. 5. Mn quenching of fura-2. Circles, control; squares, 45 mM KC1 depolarization; triangles, 100 nM ET. Treatment with KC1 or ET was performed at t = 0 by replacing the HBS with either KC1 or ET HBS with 1 mM Mn. Curves were fit to data according to exponential model described in METHODS.
quently treated with thrombin showed a rise from basal Cai levels of 46 t 4 to 117 t 12 nM (n = 4) at 30 s (Fig. 6). In both Ca-containing and Ca-free media, Ca; returned to basal level within 80-150 s after exposure to thrombin. Both basal Cai and peak Cai transient levels
MUSCLE
Cl51
after thrombin treatment were significantly lower (P c 0.01) in Ca-free medium than in 1 mM Ca medium. Because many agonists activate the Na-H antiport in concert with raising Ca;, we measured the effect of ET and thrombin on the HUA VSMC pHi profile. Neither ET nor thrombin exerted any influence on the pHi under basal condition (pHi = 7.1-7.25) or in Na-free medium. PMA (100 nM) also did not influence the pHi profile (not shown). However, when quiescent VSMCs (deprived of serum for 24 h) were subjected to 10% FBS in Nacontaining HBS (pH 7.4), a biphasic change in pHi was observed (n = 4); an immediate acidification was followed by sustained alkalinization (Fig. 7A). In Na-deficient medium (n = 5), exposure to 10% FBS produced only sustained acidification (Fig. 7B). That a Na-H antiport exists in HUA VSMCs was also demonstrated by monitoring the Na-dependent, EIPAsensitive recovery from cellular acidification (Fig. 8). Cells were acidified with nigericin to pHi ~6.6, washed three times in Na-free HBS (pH = 7.4), and then subjected to different Na concentrations in HBS (pH = 7.4). The pHi recovery was Na dependent and sensitive to EIPA. The following parameters were obtained for the Na-activation kinetics of the antiport, according to the model described in METHODS: Vmax= 0.12 pH U/5 s, Ko5 = 50 mM, and n = 2.0. ET or thrombin exerted no effect on the rate of Na-dependent recovery from nigericininduced acidification. The presence of specific ET receptors on HUA VSMCs was confirmed by lz51-ET-binding experiments (Fig. 9). There was no effect of nicardipine on ET binding to its receptors (Fig. 9, inset). Data derived from these experiments (n = 5) were used to calculate a B,,, = 12,722 sites/cell and & = 0.34 nM for ET in these cells. DISCUSSION
In this study, we have demonstrated that the ETinduced Cai response in HUA VSMCs involves a combination of Ca mobilization from intracellular stores and Ca influx from the extracellular compartment. In fact, Ca influx was the main component of the ET-induced Cai response, as shown by the Ca; signals and Mn quenching of fura- (Figs. 3-5 and Tables 1 and 2). Although both ET and thrombin produced a rise in Cai, subtle differences were observed in the cellular response to both agonists. Whereas the thrombin-evoked Cai signal was essentially monophasic, i.e., a brisk Ca; transient rapidly returning to basal Cai level, a biphasic profile in the ETinduced Cai response was observed in Ca-containing medium. Two major changes were noted in the ETinduced Cai response of VSMCs suspended in Ca-free medium or treated with verapamil or nicardipine. First, the amplitudes of the Cai transient and posttransient Ca; were substantially diminished in the presence of nicardipine, and second, the biphasic profile of the Cai signal became monophasic in the presence of verapamil or in Ca-free medium. From these observations, we suggest that the second phase of the ET-induced Cai response relates to the influx of extracellular Ca, while the Ca; transient results from both extracellular Ca influx and intracellular Ca mobilization. Recently, we have arrived
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Cl52 Table
ENDOTHELIN
2. Quenching
of fura-
AND
Control 45 mM KC1 100 nM ET
0.0128t1.35 0.0278t0.0007 0.0350t0.0006*
Values are means t SE. al and a2, compartments each treatment). Statistical analyses were performed ET treatments significantly differ from control at
r
r THROMBIN
P
al
k2, s-l
99.90t0.0003 70.35+1.45 71.14t0.80
-0.0034t1.19 0.0057t0.0002 0.0061~0.0002
I 40
I 60
I 80
I 100
I
1
120
140
160
TIME (set)
Fig. 6. Effect free medium.
of 0.5 U/ml
NIH
0
I, 1
50
of thrombin
100 150 TIME (set)
I
1 1
80
,-
12OI Ill, 1
,
in 1 mM
200
1
Ca and in Ca-
250
1 I
I ,
160
200
' 240 ' TIME Ised
260
+0.50 7.001
III ,,I
0
50
1 80 1
40
100 150 TIME (set)
III I
I1
120
-0.10 Fig. 7. pHi containing
I 160
250
I
II 200 I
I
I 240 II
I
280
TIME (set)
i -0.50
200 I1
I i T-
profile
of HUA
(A) and Na-deficient
VSMCs (B)
a2 3.281kO.0015 31.59t1.56 29.88t0.85
(expressed as percentage of the cellular pool); rlz2 and kp, rate constants; t, time (n = 6 for using Johnson and Milliken (18) procedure, as indicated under METHODS. Both KC1 and different from KC1 treatment at P < 0.001. < 0.001. * Significantly
(0.5 U/ml 1
I 20
0
MUSCLE
by Mn kl, s-l
250
VASCULAR
subjected to 10% FBS in Namedia. Insets: specific tracings.
at a similar conclusion with respect to the action of ET on human myometrial cells (22). Yanagisawa and co-workers (37) proposed that the mode of action of ET is primarily through the dihydropyridine-sensitive Ca channels. This idea is supported by the striking homology between ET and cr-scorpion toxins, which act on tetradotoxin-sensitive Na channels. The Ca and Na channels belong to a broad category of voltage-dependent channels. Results of subsequent studies, performed primarily using cultured rat VSMCs, demonstrated a spectrum of ET-induced Ca; responses. Some workers showed that ET-induced Cai responses were observed only in Ca-containing medium and attenuated by Ca channel blockers (12, 36), thereby supporting the original concept introduced by Yanagisawa et al. (37). Others have shown that ET, like vasoconstrictors such as angiotensin II and vasopressin, exerts its effect through both Ca; mobilization and Ca entry (19, 29, 35). Yet other groups have concluded that in the rat VSMC, the ET-induced Ca; signal solely depends on mobilization from intracellular stores without involvement of the dihydropyridine-sensitive Ca channels (26, 27). It is clear, however, that ET exerts some of its cellular effect in HUA VSMCs through Ca entry. As shown by the ETbinding experiments for these and other cells, this action is not because it is an agonist for the dihydropyridinesensitive Ca channels (7, 16). Rather, ET stimulation of Ca channels is likely to be an indirect one (12, 35, 36). We monitored fura- quenching by Mn as an indicator of Mn uptake to demonstrate the presence of voltagesensitive Ca channels in HUA VSMCs. Mn has been used previously to characterize Ca transport in a variety of cells (5, 13, 22, 25, 31). It has substantially greater affinity than Ca to fluorescent probes such as quin2 and fura-2. Moreover, Mn is a poor substrate for the endoplasmic reticulum Ca pump [Ca-adenosinetriphosphatase (ATPase)] (12, 25). These features of Mn suggest an explanation why KC1 depolarization did not produce a detectable alteration in Cai, yet it produced a significant acceleration of the rate of Mn quenching of fura-2. We propose that the density or activity of voltage-sensitive Ca channels in cultured HUA VSMCs is relatively small. Thus depolarization produced only a small increase in Ca uptake, and the entering Ca was either taken up by the endoplasmic reticulum or recycled back to the medium without an apparent effect on the Cai signal. However, because of the aforementioned features of Mn, its increased entry by KC1 depolarization or ET could be easily detected. In this regard, the difference between the apparent E& (-40 nM) for ET-evoked Ca; response and the & of 3.4 nM for ET binding can be explained by the same process. At lower concentrations, ET may
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ENDOTHELIN No+
I
6.52 I" 0
VASCULAR
MUSCLE
Cl53
hM)
J 200
100
AND
6501 .
0
100 TIME bed
TIME (sec)
200 Fig. 8. Na-dependent, &(N-ethyl-N-isopropyl)amiloride (EIPA)-sensitive recovery of HUA VSMCs from nigericin-induced acidification. Cells were preincubated with HBS containing 0.5 pg/ml nigericin (pH 6.8) for 5 min. Insets: Na dependency (left) and EIPA sensitivity (right) of pHi recovery. Figure demonstrates kinetics of rate of Na-dependent recovery as a function of Na concentration. Line was fit to data according to model described in METHODS.
0.15
0
20
40
100
60 Na+(mM)
r
1400
s- 1000 & 800 *s U’ 600 GI$ -z1 au 400 v>o E L- 200 A 04
1200 A = \8 z 51000 s -E
g
10-l'
10-10
10-g
10-e
800
P E E 600 E k t; 400 A tc 200
0
10-8 10-g ET(M) Fig. 9. Displacement of 1251-ET by unlabeled ET. Inset: displacement of 1251-ET by unlabeled ET in presence (open circles) or absence (closed circles) of 100 nM nicardipine. Lines were fit to data according to model described in METHODS. IO-"
I O-10
accelerate Ca entry and enhance its mobilization. However, these alterations may not be sufficient to raise the Cai. Because verapamil and nicardipine attenuated the ET-evoked Cai response, voltage-sensitive Ca channels may be indirectly activated by ET and their function may be more apparent after ET-induced Ca mobilization, perhaps to refill cellular Ca stores. Alternatively, our two observations (the presence of voltage-dependent, divalent cation influx and nicardipine-verapamil blockade of the ET-induced Cai signal) could be mutually exclusive.
ET-induced Ca influx could occur through other pathways, including receptor-operated Ca channels. In this regard, Zschauer et al. (38) demonstrated stimulation of voltage-independent Ca channels by thrombin in human platelets. Although these channels shared several characteristics with voltage-sensitive Ca channels, they differed with respect to sensitivity to dihydropyridines. The biological equivalents of compartments al and a2, described by the Mn-induced fura- quenching model, are not readily apparent. It is possible that al represents a cytosolic compartment that includes the submembrane domain. In this compartment, fura- is rapidly quenched by the accelerated Mn influx after KC1 or ET treatment. Compartment a2 is less readily available to Mn, and it may represent a deeper portion of the cytosol as well as cellular organelles that probably do not include the endoplasmic reticulum. The cellular action of many vasoactive agents is mediated via phospholipase C activation and the stimulation of protein kinase C dependent and independent pathways, leading to activation of the Na-H antiport (3, 14, 17). Although ET activates phospholipase C in rat (1, 26, 33, 35) and human (30) VSMCs, ET-induced phosphatidylinositol breakdown in rat VSMCs is less pronounced than that of thrombin (26), angiotensin II (l), and vasopressin (35). Moreover, Badr et al. (2) and Koh et al. (20), respectively, showed that it produces intracellular alkalinization in mesangial cells and rat VSMCs. We, however, could not demonstrate ET-induced intracellular alkalinization in HUA VSMCs. This inability to activate the Na-H antiport appears not to relate specifically to ET, inasmuch as thrombin (17) and PMA (14), which readily activate the antiport in rat VSMCs, were also incapable of eliciting intracellular alkalinization in HUA VSMCs. The underlying mecha-
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Cl54
ENDOTHELIN
AND
nisms for these observations are not apparent, because the Na-H antiport in HUA VSMCs could be activated by serum and by acidification. In Na-containing medium, cells subjected to serum showed an initial acidification followed by alkalinization. The acidification presumably represents activation of the Ca-ATPase (a Ca-H exchanger) due to the rapid rise in Ca; (14). The subsequent alkalinization reflects the Na-H antiport activity. This latter phase was ablated in Na-deficient medium. The Na-dependent recovery from acidification showed a Hill coefficient value of -2. A Hill coefficient of -2 has been shown by others for human erythrocytes (32) and by us for cultured human fibroblasts (9) and myometrial cells (17). It might indicate positive cooperativity on more than one population of the Na-H antiport. Finally, that the ET action on the HUA VSMCs is exerted through specific receptors has been demonstrated by the lz51-ET-binding experiments. These experiments demonstrate a single class of ET receptors with B,,, and & values that are quite similar to those reported for rat thoracic aorta (16) and human umbilical vein (6) VSMCs. They appear not to relate to the dihydropyridine-sensitive Ca channel receptors. This work was supported by National Heart, Lung, and Blood Institute Grant HL-34807 and by a grant-in-aid from the American Heart Association (New Jersey Affiliate). E. Maker and 9. P. Gardner were research fellows of the American Heart Association (New Jersey Affiliate). Address for reprint requests: J. P. Gardner, Dept. of Pediatrics and Hypertension Research Ctr., Room F-464, MSB, Univ. of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 S. Orange Ave., Newark, NJ 07103. Received
15 November
1989; accepted
in final
form
29 July
1991.
VASCULAR
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
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ENDOTHELIN
AND
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