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Agonist-induced calcium transients in cultured smooth muscle cells: Measurements with fura-2 loaded monolayers
Vol.
136.
May
14,
No. 3, 1986
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1986
927-934
AGONIST-INDUCED CALCIUM TRANSIENTS IN CULTURED SMOOTH MUSCLE CELLS: MEASUREMENTS WITH PURA-P LOADED MONOLAYERS Elwood E. Reynolds’
’
and George R. Dubyak’
Departments of Pharmacology’ and Biochemistry/Biophysics3, University of Pennsylvania, Philadelphia, PA 19104 Received
March
18,
1986
Elevation of cytosolic Ca+2 in response to depolarization and various receptor agonists was measured in several types of cultured smooth muscle cells (DDTl, AlO, rabbit aorta) loaded with the either quin-2 or fura-2, and assayedeither in suspensionor in monolayer cultures attached to plastic cover sli s. Agonists (norepinephrine, vaso ressin) induced both the re easeof intracellular CaA and the influx of extracellular Ca+P. Agonist-induced Ca+!l +j i flux was not blocked by dihydropyridines, and depolarization did not induce Ca influx. However, in fura- load?! monolayers of PC12 cells, depolarization did induce dihydropyridine-sensitive C influx. Thus cultured smooth muscle cells a$ear to express receptor-operated Ca+I channels, but not functional voltage-operated Ca channels. W 1986 Academic Press, Inc.
We have previously described a system in which it is possible to measure aladrenergic receptor (al-R) mediated increases in cytosolic calcium (Ca+2)i in a smooth muscle cell line (DDT1) using the Ca+2-sensitive fluorescent probe quin-2 (1).
In
suspensioncultures of DDTl cells, norepinephrine (NE) stimulated a rapid increase in (Ca+2)i
by
distinct
two
mechanisms: 1) release of Ca+2 from intracellular
sites
(mobilization), and 2) influx of extracellular Ca+2 (Ca+2)ex. The influx component was not inhibited nifedipine.
by
the voltage-operated
Ca+2 channel (VOC) blockers diltiazem or
Depolarization of DDTl cells, however, did not induce Ca+2 influx.
These
results suggested that the al-R on DDTl smooth muscle cells are coupled to receptoroperated (ROC) Ca+2 channels in the plasma membrane,end that there are no functional VOC on these cells. However, the lack of depolarization-induced Ca+2 influx and the insensitivity of NE-stimulated Ca+2 influx to organic Ca+2 channel blockers raised the possibility that the properties of the Ca+2 channels on DDTl cells may be dramatically altered by
’
This work was supported by the American Heart Association, Southeastern Pennsylvania Chapter (E.E.R.), and NIH grant HL-15835 (G.R.D.) 0006-291X/86
927
All
Copyrighr 0 1986 rights of reproduction
$1.50
by Academic Press. Inc. in any form reserved.
BIOCHEMICAL
AND
several aspects of the assay conditions.
First,
Vol.
136,
No. 3, 1986
the cells was 1.0-1.5 mM, and this represents capacity.
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
the cytosolic
concentration
a very large intracellular
of quin-2 in Ca+2 buffer
Second, the DDTl cells were grown and assayed in suspension, rather than as
attached cells in monolayer.
Third, it was possible that DDTl cells have aberrant
channels.
The purpose of this study was to assess each of these potential
measuring
changes in (Ca+2)i in 1) DDTl
cells loaded with
Ca+2
problems by
the new Ca +2 -sensitive
fluorescent probe, fura-2, which has a higher quantum yield than quin-2 and can thus be used at much lower cytosolic concentrations (2), 2) in DDTl cells attached to plastic cover slips and assumingtheir normal morphology, 3) in another smooth muscle cell line (AlO) and in primary cultures of rabbit aorta, and 4) in PC12 cells, which are known to have functional VOC (3). MATERIALS AND METHODS: DDTl smooth muscle cells, derived from a transplantable leiomyosarcoma tumor from Syrian hamster ductus deferens (4) were grown in suspensionculture as previously described (5). A10 cells, derived from rat thoracic aorta, were grown on plastic tissue culture dishesas previously described (6). For suspensionassays, A10 cells were washed with balanced salt solution (BSS) containing 120 mM NaCl, 5 mM KCl, 1.5 mM MgC12, 1.0 mM CaC12,25 mM Na-HEPES 10 mM D-glucose, 0.1 % BSA, pH 7.4, then incubated for 5 minutes with Ca+2- and Mg+’ -free BSS containing 1 mM EDTA, after which the cells were dislodged, centrifuged, then resuspended in Leibowitz’s L15 medium (pH 7.4) containing 0.1 % BSA. PC12 cells were grown and treated with NGF (100 rig/ml) as previously described (7). Primary cultures of rabbit aorta were prepared and maintained as previously described (8). Monolayer cultures were prepared by seeding cells on c ver slips (14 x 16 mm) made of ACLAR plastic at a density of 40,000-60,000 cells/emB in DMEM supplementedwith 20% fetal bovine serum, 100 units/ml penicillin, and 100 ug/ml streptomycin, and the monolayer cultures were assayed2 days later. Cell suspensionswere loaded w\th quin-2 as previously described (1). To load cells with fura-2, cell suspensions(l-2 x 10 cells/ml) were incubated in BSScontaining 2 uM fura- AM for 15 minutes at 37’C centrifuged, resuspendedin fresh BSSand incubated an additional 15-20 minutes at 37dC to completely hydrolyze the entrapped ester. The cells were recentrifuged, resuspendedin fresh BSS, and stored on ice. Under these conditions the cells were loaded with 50-100 nM cytosolic fura-2. The intracellular fura2 signals were calibrated as previously described (1) subsequelt to permgabilization of the cells with 25 pg/ml digitonin. Cell monolayers (l-2 x 10 cells/cm ) were loaded with fura- by incubating the individual ACLAR coverslips in L15 containing 2-4 uM fura- AM for 40 minutes at 37’C. The monolayers were then transferred to fresh BSS and incubated an additional 20 minutes at 37OC. The loaded monolayers were stored on ice until being used. Under these condit’ons, the cell monolayers routinely accumulated 200-300 uM cytosolic fura-2. The (Ca+A). was calibrated by exposing the monolayer to 4-10 uM ionomycin followed by addition od 6.6 mM EGTA and alkalinization of the media to pH 8.5. Chemicals and supplies were obtained from the following sources: Quin-2 AM and ionomycin from Calbiochem (La Jolla, CA); fura- AM from Molecular Probes (Junction City, Oregon); L-norepinephrine, penicillin, streptomycin, carbachol, serotonin, ATP, arginine-vasopressin, angiotensin II, and BSA from Sigma Chemical Co. (St. Louis, MO); nitrendipine from Miles Pharmaceuticals (West Haven, CT); DMEM and L15 media from GIBCO (Grand Island, NY); fetal bovine serum from HyClone Laboratories (Logan, UT); ACLAR plastic from Allied Chemical Corp (Pottsville, PA). 928
BIOCHEMICAL
Vol. 136, No. 3, 1986
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION: Addition of 10 uM NE to either quin-2 loaded (1.5 mM) or fura- loaded (150 uM) cell suspensionsresulted in a rapid increase in (Ca+2)i which was mediated by al-R
DDTl
(1) and composedof two components (Fig 1). Addition of La+3 (Figs lA, 1C) or chelation of (Ca+2)ex with EGTA (Figs lB, 1D) abolished the second component and only slighty reduced the first component, suggesting that the first component represents release of Ca+2 from intracellular stores (mobilization) and the second component represents the influx of (Ca+2)ex through Ca+2 channels in the plasma membrane. The primary difference between NE-induced Ca+2 transients in quin-2 and fura-
loaded cells is that
the magnitude and kinetics of the Ca+2 influx component are different.
The larger,
slower, and prolonged influx seen in quin-2 loaded cells is most likely due to the much larger Ca+2 buffering capacity intrinsically associated with the use of high cytosolic concentrations (1.5 mM) of such a Ca+2 chelator. DDT, Single m
Cell q
1.5 mM Intracellular Owl 2 1.5 mM Extracellular Co’+ Control
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Ca+2transients in singlecell suspensions of DDT either 1.5 mM intracellular quin-2 (A,B) or 150 JIM fura- (C,DJf was measured in dye-loadedDDT cells in either normalBSS(A,C) or in BSSwhich wassupplemented with 2 mM EGTA f&D) or 25 uM LaC12 (A$) for 0.5 min
prior to the addition of 10 uM NE.
Figure 2: Norepinephrine-induced Ca+2 transients in monolayer cultf$es of DDTL cells transients were grown and assayed on ACLAR plastic cover slips. Cytosolic Ca measured in fura- loaded DDT cells in either normal BSS (top transient) or in BSS which was supplemented with 4 mM EGTA (bottom transient) 0.5 min prior to the addition of 10 uM NE. 929
Vol. 136, No. 3, 1986
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUIJICATIONS
Fig 2 shows NE-induced Ca+2 transients measured in fura-
loaded DDTl cells
which are attached to ACLAR cover slips and assayedin monolayer cultures rather than in suspension. NE causesa rapid increase in (Ca+2)i from 140n&l to 500 nM, followed by a decline to basal levels in about 3 minutes (Fig 2). Prior addition of 2 mM EGTA causes a small reduction in the initial phase of NE-stimulated increase in (Cae2)i and a rapid decline of (Ca+2)l to basal levels within one minute, suggesting that the persistant elevation of (Ca+2)i in the absence of EGTA is maintained by the influx of (Ca+2)ex. Thus NE induces both Ca+2 mobilization and Ca+2 influx in monolayer cultures of DDTl cells, but this Ca+2 influx is more transient than in suspensioncultures (Fig 1C). This may be due to 1) the rapid “desensitization” of either al-R or Ca+2 channels, or 2) more efficient Ca“2 extrusion/sequestration in attached DDTl cells. It was of considerable interest to determine whether ROC or VOC could be observed on a smooth muscle cell line other than DDT1. A10 cells, derived from rat thoracic aorta, were used for these studies. These cells do not express aIR, but have
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Figure 3: Arginine-J,sopressin(AVP) - inducedCa+2 transientsin fura- loadedA10 cells. Cytosolic Ca wasmeasuredin (A) singlecells suspensions of A10 cell< or (B) monolayercultures of A10 ceils assayedin either normal BSS(uppertransient) or BSS supplementedwith 3 mM EGTA 0.5 min prior to the addition of 10 nM AVP (lower transient).
(AVP)- and depolarizationinduced Ca+2 transients in Figure 4: Arginine-vasopressin fura- loaded A10 cells. Cytosolic Ca+2 wasmeasuredin (A) singlecell suspensions of
AI0 cells, or (B) monolayercultures of Al0 cells in normal BSS. In (B), 0.66 uM nitrendipinewasaddedprior to the additionof 8.3 nM AVP. 930
Vol.
136,
81octiiM1cA~
No. 3, 1986
vasopressin
receptors
B’IOPHYSICAL
coupled to phosphoinositide
cells detached from their growth vasopressin
AND
surface
RESEARCH
hydrolysis
(9).
In fura-
in (Ca+2)i, after which (Ca+2)i declines’to
plateau phase which is about 100 nM above basal levels (Fig 3A). loaded A10 cells grown on ACLAR
cultures
(Pig 3B). Addition
induced
increase
elevation
of EGTA causes a substantial
in (Ca+2)i and abolishes
A similar pattern
reduction
is
in the initial AVP-
the second phase representing
a sustained
of (Ca+2)i (Fig 3B). Thus in both suspension and monolayer cultures and Ca+2 influx.
of fura-
Depolarization
loaded A10 cells in suspension (Fig 4A) or in monolayer (Fig 4B) with 50mM or
45 mM KC1 did not cause any increase in (Ca+2)i. Ca+2 influx blocker
in A10 monolayer cultures
nitrendipine
at a concentration
Furthermore,
was not affected
presence
of Ca+2 channels
dihydropyridines,
which
(0.66 uM) which normally
are activated
block
with regard to the and not blocked
by
and which are not activated by depolarization.
cells might conceivably
demonstrated
will completely
to DDTI
by receptors
The failure to detect depolarization-induced
was of therefore
the AVP-stimulated
by the organic Ca’2 channel
VOC (Fig 4B). Thus A10 smooth muscle cells are similar
PC12
a
cover slips and assayed in monolayer
loaded A10 cells, AVP induces both Ca+2 mobilization of fura-
loaded A10
by EDTA and assayed in suspension, arginine-
(AVP) causes a rapid elevation
seen in fura-
COMMUNICATIONS
increases in (Ca+2)i in DDTl and A10
by due to some inhibitory
of interest
to determine
effect
if depolarization-induced
in any other type of cell loaded with
pheochromocytoma
cells
In fura-
dihydropyridines.
assayed in monolayer cultures,
functional
furad. VOC
loaded PC12 cells attached depolarization Addition
in (Ca+2)i (Figs 5A, SD, SE). depolarization-induced
have
of quin-2 or fura-
Caq2 influx could
Toll (3) has reported which
are
to ACLAR
caused a rapid and transient
of 40 mM NaCl had no effect
increase in (Ca+2)l was completely
per se. It
inhibited
be
that by
cover slips and large increase (Fig 5B).
This
inhibited by 3 uM nitrendipine
(Fig 5D) or 25 uM La+3 (Fig 5E). Finally,
we investigated
observed in primary
cultures
and loaded with fura-2. similar to the NE-induced DDTl cells (Fig 2).
whether
receptor-mediated
of rabbit aorta grown
elevation of (Ca+2)i could be
and assayed on ACLAR
cover slips
Addition of NE caused an increase in (Ca+2)i (Fig 6A) which was Ca +2 transient
Angiotensin
observed in fura-
II, serotonin, 931
loaded monolayer cultures of
and ATP caused an elevation of (Ca+2)i
Vol.
136,
No. 3, 1986
BIOCHEMICAL
AND
PC 12 A
BIOPHYSICAL
RESEARCH
Monolayers
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~-
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40
[D
mM NaCl
A Control
w’ 40
t 5 mM Carbachol
+ 3 PM
Medium
t
3pM
nitrendipine 40
Control
R 5mM 25pM 40
x
,149
i
nitrendipine
I mM KCI
+25pM
Medium
:::I
replaced
mM KCI
IE
COMMUNICATIONS
LaClS
replaced K++ La3+
>w40
mM KCI
t mM
KCI
Figure 5: De@arization - inducedCa+2 transientsin monolayercultures of PC12cells. Cytosolic Ca wasmeasuredin fura- loadedmonolayercultures of NGP-treated PC12 cells in normalBSSand stimulatedwith either 40mM KC1(A,D,E), 40mM NaCl (B), or 5 mM carbachol(Cl. In (D) and(El, the mediumwasaspiratedafter the first additionof 40 mM KC1and replacedwith normalBSScontainingeither 3 uM nitrendipine(D) or 25 uM LaC13(El. which was of larger magnitude and longer duration than the NE-induced Ca+2 transient (Fig 6). AVP and depolarization (40 mM KC11had no effect on (Ca+2)i when added either after NE and angiotensin II (Fig 6) or alone with no drug pretreatment (not shown). The results of this study suggest that the properties of al-R-mediated Ca+2 transients observed in our previous study with DDTl cells were not artifacts of high cytosolic
concentrations of quin-2, or of the
suspension. Qualitatively
cells being
grown and assayed in
similar NE-induced Ca+2 transients were observed in DDTl
cells loaded with fura-2, and in cells assayed as monolayer cultures on ACLAR cover slips. Similar Ca+2 transients were also observed in fura-
loaded A10 smooth muscle
cells (suspended and monolayer) stimulated with AVP.
AVP induced both Ca+2
932
Vol. 136, No. 3, 1986
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
2016 522 224 96 25 (40mM)
(IOpM)
-fSerotonin (5pM)
2016 522 224 96 25
1
Figure 6: Caq2 transients in fura- loaded monolayersof primary cultures of rabbit aorta smoothmusclecells. All transientsare from the samebatch of fura- loadedcells assayedin normalBSS,andeachtransient(A,B,C,D) representsan individualcover slip.
mobilization and Ca+2 influx, and this Ca+2 influx was not blocked by nitrendipine. Furthermore, depolarization did not increase (Ca+2)i in A10 cells, suggesting either that 1) these cells do not have functional VOC, or 2) the number of functional VGC is insufficient
to promote Ca‘2 influx
at a rate exceeding the net rate of Ca+2
sequestration/extrusion. Thus, the apparent discrepancy between our results and those of Kongsamut -et al (6), which demonstrated depolarization-induced 45Ca+2 influx in A10 cells which could by inhibited by dihydropyridines, may be due to the inherent differences in the two methods used to measure Ca+2 influx.
However, a more serious reason for
this discrepancy was the possibility that quin-2 and/or fura-
may inhibit the function of
VOC.
However, this is not the case because depolarization-induced Ca+2 transients, which were blocked by both nitrendipine and La +3, were observed in fura- loaded PC12 cells.
Furthermore, several previous reports have demonstrated that depolarization-
induced Ca+2 transients can be observed in quin-2 loaded GH3 (10) and PC12 (11) cells. It is possiblethat 45Ca+2 accumulation studies can detect small increasesin Ca+2 influx which are insufficient to exceed the Ca+2 homeostatic capacity of cells. If so, there would not be a large enough change in (Ca+‘)i to be detected with dyes such as quin-2 and fura-2. Finally, no depolarization-induced Ca+2 transients were observed in fura-
loaded
monolayer primary cultures of rabbit aorta, although substantial increases in (Ca+2)i were induced by several receptor agonists. The failure to detect depolarization-induced 933
Vol.
Ca+2
136,
3,
No.
1986
BIOCHEMICAL
influx in DDTl,
cultured
AND
ROC.
RESEARCH
AlO, and rabbit aorta cells using fluorescent
smooth muscle cells do not retain
functional
BIOPHYSICAL
Caponi --et al (12) reported
functional
dyes suggests
that
VOC, although they do retain
that depolarization
changes in (Ca+2)i in quin-2 loaded monolayers
COMMUNICATIONS
of primary
elicited only very small
rat thoracic
aorta smooth
muscle cells which responded to angiotensin II and AVP with large increases in (Ca+2)i. The technique represents
of measuring
an excellent
Ca+2 transients
model system
with
fura-
in monolayer
with which to study the properties
cultures
of receptor-
operated Ca+2 channels in smooth muscle cells in their normal morphology with minimal perturbation. vascular
The successful
smooth
vasoconstrictor investigate
muscle
application
(rabbit
aorta)
of this technique to primary which
respond
agents will provide an excellent
the transduction
mechanisms
involved
cultures
to a number
model system
with
in receptor-mediated
of adult
of important
which
to further
elevation
of
(Ca’2)i in vascular smooth muscle.
REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Reynolds, E. E., and Dubyak, G. R. (1985) Biochem. Biophys. Res. Comm. 130: 627632. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260: 3440-3450. Toll, L. (1982) J. Biol. Chem. 257: 13189-13192. Norris, J.S., Gorski, J., and Kohler, P.O. (1974) Nature 2.48: 422-424. Cornett, L.E., and Norris, J.S. (1982) J. Biol. Chem. 25734-697. Kongsamut, S., Freedman, S.B., and Miller, R.J. TivS) Biochem. Biophys. Res. Comm. 127: 71-79. VincentxL.M., Ambrosini, A., DiVirgilio, F., Pozzan, T., and Melolesi, J. (1985) J. Cell Biol. 100: 1330-1333. Colucci, Wx, Brock, T.A., Gimbrone, M.A. Jr., and Alexander, R.W. (1985) Mol. Pharm. 27: 517-524. Aiyar, NTNambi, P., Stassen, F.L., and Crooke, S.T. (1985) The Pharmacologist -27: 222. Albert, P.R. and Tashjian, A.H. (1984) J. Biol. Chem. 259: 15350-15363. DiVirgilio, F., Pozzan, T., Wollheim, C.B., VincentinixM., and Meldolesi, J. (1986) J. Biol. Chem. 261: 32-35. Capponi, A., LeTP.D., Vallotton, M.B. (1985) J. Biol. Chem. 2260. 7836-7842.
934
136.
May
14,
No. 3, 1986
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS Pages
1986
927-934
AGONIST-INDUCED CALCIUM TRANSIENTS IN CULTURED SMOOTH MUSCLE CELLS: MEASUREMENTS WITH PURA-P LOADED MONOLAYERS Elwood E. Reynolds’
’
and George R. Dubyak’
Departments of Pharmacology’ and Biochemistry/Biophysics3, University of Pennsylvania, Philadelphia, PA 19104 Received
March
18,
1986
Elevation of cytosolic Ca+2 in response to depolarization and various receptor agonists was measured in several types of cultured smooth muscle cells (DDTl, AlO, rabbit aorta) loaded with the either quin-2 or fura-2, and assayedeither in suspensionor in monolayer cultures attached to plastic cover sli s. Agonists (norepinephrine, vaso ressin) induced both the re easeof intracellular CaA and the influx of extracellular Ca+P. Agonist-induced Ca+!l +j i flux was not blocked by dihydropyridines, and depolarization did not induce Ca influx. However, in fura- load?! monolayers of PC12 cells, depolarization did induce dihydropyridine-sensitive C influx. Thus cultured smooth muscle cells a$ear to express receptor-operated Ca+I channels, but not functional voltage-operated Ca channels. W 1986 Academic Press, Inc.
We have previously described a system in which it is possible to measure aladrenergic receptor (al-R) mediated increases in cytosolic calcium (Ca+2)i in a smooth muscle cell line (DDT1) using the Ca+2-sensitive fluorescent probe quin-2 (1).
In
suspensioncultures of DDTl cells, norepinephrine (NE) stimulated a rapid increase in (Ca+2)i
by
distinct
two
mechanisms: 1) release of Ca+2 from intracellular
sites
(mobilization), and 2) influx of extracellular Ca+2 (Ca+2)ex. The influx component was not inhibited nifedipine.
by
the voltage-operated
Ca+2 channel (VOC) blockers diltiazem or
Depolarization of DDTl cells, however, did not induce Ca+2 influx.
These
results suggested that the al-R on DDTl smooth muscle cells are coupled to receptoroperated (ROC) Ca+2 channels in the plasma membrane,end that there are no functional VOC on these cells. However, the lack of depolarization-induced Ca+2 influx and the insensitivity of NE-stimulated Ca+2 influx to organic Ca+2 channel blockers raised the possibility that the properties of the Ca+2 channels on DDTl cells may be dramatically altered by
’
This work was supported by the American Heart Association, Southeastern Pennsylvania Chapter (E.E.R.), and NIH grant HL-15835 (G.R.D.) 0006-291X/86
927
All
Copyrighr 0 1986 rights of reproduction
$1.50
by Academic Press. Inc. in any form reserved.
BIOCHEMICAL
AND
several aspects of the assay conditions.
First,
Vol.
136,
No. 3, 1986
the cells was 1.0-1.5 mM, and this represents capacity.
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
the cytosolic
concentration
a very large intracellular
of quin-2 in Ca+2 buffer
Second, the DDTl cells were grown and assayed in suspension, rather than as
attached cells in monolayer.
Third, it was possible that DDTl cells have aberrant
channels.
The purpose of this study was to assess each of these potential
measuring
changes in (Ca+2)i in 1) DDTl
cells loaded with
Ca+2
problems by
the new Ca +2 -sensitive
fluorescent probe, fura-2, which has a higher quantum yield than quin-2 and can thus be used at much lower cytosolic concentrations (2), 2) in DDTl cells attached to plastic cover slips and assumingtheir normal morphology, 3) in another smooth muscle cell line (AlO) and in primary cultures of rabbit aorta, and 4) in PC12 cells, which are known to have functional VOC (3). MATERIALS AND METHODS: DDTl smooth muscle cells, derived from a transplantable leiomyosarcoma tumor from Syrian hamster ductus deferens (4) were grown in suspensionculture as previously described (5). A10 cells, derived from rat thoracic aorta, were grown on plastic tissue culture dishesas previously described (6). For suspensionassays, A10 cells were washed with balanced salt solution (BSS) containing 120 mM NaCl, 5 mM KCl, 1.5 mM MgC12, 1.0 mM CaC12,25 mM Na-HEPES 10 mM D-glucose, 0.1 % BSA, pH 7.4, then incubated for 5 minutes with Ca+2- and Mg+’ -free BSS containing 1 mM EDTA, after which the cells were dislodged, centrifuged, then resuspended in Leibowitz’s L15 medium (pH 7.4) containing 0.1 % BSA. PC12 cells were grown and treated with NGF (100 rig/ml) as previously described (7). Primary cultures of rabbit aorta were prepared and maintained as previously described (8). Monolayer cultures were prepared by seeding cells on c ver slips (14 x 16 mm) made of ACLAR plastic at a density of 40,000-60,000 cells/emB in DMEM supplementedwith 20% fetal bovine serum, 100 units/ml penicillin, and 100 ug/ml streptomycin, and the monolayer cultures were assayed2 days later. Cell suspensionswere loaded w\th quin-2 as previously described (1). To load cells with fura-2, cell suspensions(l-2 x 10 cells/ml) were incubated in BSScontaining 2 uM fura- AM for 15 minutes at 37’C centrifuged, resuspendedin fresh BSSand incubated an additional 15-20 minutes at 37dC to completely hydrolyze the entrapped ester. The cells were recentrifuged, resuspendedin fresh BSS, and stored on ice. Under these conditions the cells were loaded with 50-100 nM cytosolic fura-2. The intracellular fura2 signals were calibrated as previously described (1) subsequelt to permgabilization of the cells with 25 pg/ml digitonin. Cell monolayers (l-2 x 10 cells/cm ) were loaded with fura- by incubating the individual ACLAR coverslips in L15 containing 2-4 uM fura- AM for 40 minutes at 37’C. The monolayers were then transferred to fresh BSS and incubated an additional 20 minutes at 37OC. The loaded monolayers were stored on ice until being used. Under these condit’ons, the cell monolayers routinely accumulated 200-300 uM cytosolic fura-2. The (Ca+A). was calibrated by exposing the monolayer to 4-10 uM ionomycin followed by addition od 6.6 mM EGTA and alkalinization of the media to pH 8.5. Chemicals and supplies were obtained from the following sources: Quin-2 AM and ionomycin from Calbiochem (La Jolla, CA); fura- AM from Molecular Probes (Junction City, Oregon); L-norepinephrine, penicillin, streptomycin, carbachol, serotonin, ATP, arginine-vasopressin, angiotensin II, and BSA from Sigma Chemical Co. (St. Louis, MO); nitrendipine from Miles Pharmaceuticals (West Haven, CT); DMEM and L15 media from GIBCO (Grand Island, NY); fetal bovine serum from HyClone Laboratories (Logan, UT); ACLAR plastic from Allied Chemical Corp (Pottsville, PA). 928
BIOCHEMICAL
Vol. 136, No. 3, 1986
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RESULTS AND DISCUSSION: Addition of 10 uM NE to either quin-2 loaded (1.5 mM) or fura- loaded (150 uM) cell suspensionsresulted in a rapid increase in (Ca+2)i which was mediated by al-R
DDTl
(1) and composedof two components (Fig 1). Addition of La+3 (Figs lA, 1C) or chelation of (Ca+2)ex with EGTA (Figs lB, 1D) abolished the second component and only slighty reduced the first component, suggesting that the first component represents release of Ca+2 from intracellular stores (mobilization) and the second component represents the influx of (Ca+2)ex through Ca+2 channels in the plasma membrane. The primary difference between NE-induced Ca+2 transients in quin-2 and fura-
loaded cells is that
the magnitude and kinetics of the Ca+2 influx component are different.
The larger,
slower, and prolonged influx seen in quin-2 loaded cells is most likely due to the much larger Ca+2 buffering capacity intrinsically associated with the use of high cytosolic concentrations (1.5 mM) of such a Ca+2 chelator. DDT, Single m
Cell q
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Ca+2transients in singlecell suspensions of DDT either 1.5 mM intracellular quin-2 (A,B) or 150 JIM fura- (C,DJf was measured in dye-loadedDDT cells in either normalBSS(A,C) or in BSSwhich wassupplemented with 2 mM EGTA f&D) or 25 uM LaC12 (A$) for 0.5 min
prior to the addition of 10 uM NE.
Figure 2: Norepinephrine-induced Ca+2 transients in monolayer cultf$es of DDTL cells transients were grown and assayed on ACLAR plastic cover slips. Cytosolic Ca measured in fura- loaded DDT cells in either normal BSS (top transient) or in BSS which was supplemented with 4 mM EGTA (bottom transient) 0.5 min prior to the addition of 10 uM NE. 929
Vol. 136, No. 3, 1986
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUIJICATIONS
Fig 2 shows NE-induced Ca+2 transients measured in fura-
loaded DDTl cells
which are attached to ACLAR cover slips and assayedin monolayer cultures rather than in suspension. NE causesa rapid increase in (Ca+2)i from 140n&l to 500 nM, followed by a decline to basal levels in about 3 minutes (Fig 2). Prior addition of 2 mM EGTA causes a small reduction in the initial phase of NE-stimulated increase in (Cae2)i and a rapid decline of (Ca+2)l to basal levels within one minute, suggesting that the persistant elevation of (Ca+2)i in the absence of EGTA is maintained by the influx of (Ca+2)ex. Thus NE induces both Ca+2 mobilization and Ca+2 influx in monolayer cultures of DDTl cells, but this Ca+2 influx is more transient than in suspensioncultures (Fig 1C). This may be due to 1) the rapid “desensitization” of either al-R or Ca+2 channels, or 2) more efficient Ca“2 extrusion/sequestration in attached DDTl cells. It was of considerable interest to determine whether ROC or VOC could be observed on a smooth muscle cell line other than DDT1. A10 cells, derived from rat thoracic aorta, were used for these studies. These cells do not express aIR, but have
A
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2016 096 522 336 224 149 96 56 nitrendipine
AVP
Figure 3: Arginine-J,sopressin(AVP) - inducedCa+2 transientsin fura- loadedA10 cells. Cytosolic Ca wasmeasuredin (A) singlecells suspensions of A10 cell< or (B) monolayercultures of A10 ceils assayedin either normal BSS(uppertransient) or BSS supplementedwith 3 mM EGTA 0.5 min prior to the addition of 10 nM AVP (lower transient).
(AVP)- and depolarizationinduced Ca+2 transients in Figure 4: Arginine-vasopressin fura- loaded A10 cells. Cytosolic Ca+2 wasmeasuredin (A) singlecell suspensions of
AI0 cells, or (B) monolayercultures of Al0 cells in normal BSS. In (B), 0.66 uM nitrendipinewasaddedprior to the additionof 8.3 nM AVP. 930
Vol.
136,
81octiiM1cA~
No. 3, 1986
vasopressin
receptors
B’IOPHYSICAL
coupled to phosphoinositide
cells detached from their growth vasopressin
AND
surface
RESEARCH
hydrolysis
(9).
In fura-
in (Ca+2)i, after which (Ca+2)i declines’to
plateau phase which is about 100 nM above basal levels (Fig 3A). loaded A10 cells grown on ACLAR
cultures
(Pig 3B). Addition
induced
increase
elevation
of EGTA causes a substantial
in (Ca+2)i and abolishes
A similar pattern
reduction
is
in the initial AVP-
the second phase representing
a sustained
of (Ca+2)i (Fig 3B). Thus in both suspension and monolayer cultures and Ca+2 influx.
of fura-
Depolarization
loaded A10 cells in suspension (Fig 4A) or in monolayer (Fig 4B) with 50mM or
45 mM KC1 did not cause any increase in (Ca+2)i. Ca+2 influx blocker
in A10 monolayer cultures
nitrendipine
at a concentration
Furthermore,
was not affected
presence
of Ca+2 channels
dihydropyridines,
which
(0.66 uM) which normally
are activated
block
with regard to the and not blocked
by
and which are not activated by depolarization.
cells might conceivably
demonstrated
will completely
to DDTI
by receptors
The failure to detect depolarization-induced
was of therefore
the AVP-stimulated
by the organic Ca’2 channel
VOC (Fig 4B). Thus A10 smooth muscle cells are similar
PC12
a
cover slips and assayed in monolayer
loaded A10 cells, AVP induces both Ca+2 mobilization of fura-
loaded A10
by EDTA and assayed in suspension, arginine-
(AVP) causes a rapid elevation
seen in fura-
COMMUNICATIONS
increases in (Ca+2)i in DDTl and A10
by due to some inhibitory
of interest
to determine
effect
if depolarization-induced
in any other type of cell loaded with
pheochromocytoma
cells
In fura-
dihydropyridines.
assayed in monolayer cultures,
functional
furad. VOC
loaded PC12 cells attached depolarization Addition
in (Ca+2)i (Figs 5A, SD, SE). depolarization-induced
have
of quin-2 or fura-
Caq2 influx could
Toll (3) has reported which
are
to ACLAR
caused a rapid and transient
of 40 mM NaCl had no effect
increase in (Ca+2)l was completely
per se. It
inhibited
be
that by
cover slips and large increase (Fig 5B).
This
inhibited by 3 uM nitrendipine
(Fig 5D) or 25 uM La+3 (Fig 5E). Finally,
we investigated
observed in primary
cultures
and loaded with fura-2. similar to the NE-induced DDTl cells (Fig 2).
whether
receptor-mediated
of rabbit aorta grown
elevation of (Ca+2)i could be
and assayed on ACLAR
cover slips
Addition of NE caused an increase in (Ca+2)i (Fig 6A) which was Ca +2 transient
Angiotensin
observed in fura-
II, serotonin, 931
loaded monolayer cultures of
and ATP caused an elevation of (Ca+2)i
Vol.
136,
No. 3, 1986
BIOCHEMICAL
AND
PC 12 A
BIOPHYSICAL
RESEARCH
Monolayers
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t 5 mM Carbachol
+ 3 PM
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nitrendipine 40
Control
R 5mM 25pM 40
x
,149
i
nitrendipine
I mM KCI
+25pM
Medium
:::I
replaced
mM KCI
IE
COMMUNICATIONS
LaClS
replaced K++ La3+
>w40
mM KCI
t mM
KCI
Figure 5: De@arization - inducedCa+2 transientsin monolayercultures of PC12cells. Cytosolic Ca wasmeasuredin fura- loadedmonolayercultures of NGP-treated PC12 cells in normalBSSand stimulatedwith either 40mM KC1(A,D,E), 40mM NaCl (B), or 5 mM carbachol(Cl. In (D) and(El, the mediumwasaspiratedafter the first additionof 40 mM KC1and replacedwith normalBSScontainingeither 3 uM nitrendipine(D) or 25 uM LaC13(El. which was of larger magnitude and longer duration than the NE-induced Ca+2 transient (Fig 6). AVP and depolarization (40 mM KC11had no effect on (Ca+2)i when added either after NE and angiotensin II (Fig 6) or alone with no drug pretreatment (not shown). The results of this study suggest that the properties of al-R-mediated Ca+2 transients observed in our previous study with DDTl cells were not artifacts of high cytosolic
concentrations of quin-2, or of the
suspension. Qualitatively
cells being
grown and assayed in
similar NE-induced Ca+2 transients were observed in DDTl
cells loaded with fura-2, and in cells assayed as monolayer cultures on ACLAR cover slips. Similar Ca+2 transients were also observed in fura-
loaded A10 smooth muscle
cells (suspended and monolayer) stimulated with AVP.
AVP induced both Ca+2
932
Vol. 136, No. 3, 1986
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
2016 522 224 96 25 (40mM)
(IOpM)
-fSerotonin (5pM)
2016 522 224 96 25
1
Figure 6: Caq2 transients in fura- loaded monolayersof primary cultures of rabbit aorta smoothmusclecells. All transientsare from the samebatch of fura- loadedcells assayedin normalBSS,andeachtransient(A,B,C,D) representsan individualcover slip.
mobilization and Ca+2 influx, and this Ca+2 influx was not blocked by nitrendipine. Furthermore, depolarization did not increase (Ca+2)i in A10 cells, suggesting either that 1) these cells do not have functional VOC, or 2) the number of functional VGC is insufficient
to promote Ca‘2 influx
at a rate exceeding the net rate of Ca+2
sequestration/extrusion. Thus, the apparent discrepancy between our results and those of Kongsamut -et al (6), which demonstrated depolarization-induced 45Ca+2 influx in A10 cells which could by inhibited by dihydropyridines, may be due to the inherent differences in the two methods used to measure Ca+2 influx.
However, a more serious reason for
this discrepancy was the possibility that quin-2 and/or fura-
may inhibit the function of
VOC.
However, this is not the case because depolarization-induced Ca+2 transients, which were blocked by both nitrendipine and La +3, were observed in fura- loaded PC12 cells.
Furthermore, several previous reports have demonstrated that depolarization-
induced Ca+2 transients can be observed in quin-2 loaded GH3 (10) and PC12 (11) cells. It is possiblethat 45Ca+2 accumulation studies can detect small increasesin Ca+2 influx which are insufficient to exceed the Ca+2 homeostatic capacity of cells. If so, there would not be a large enough change in (Ca+‘)i to be detected with dyes such as quin-2 and fura-2. Finally, no depolarization-induced Ca+2 transients were observed in fura-
loaded
monolayer primary cultures of rabbit aorta, although substantial increases in (Ca+2)i were induced by several receptor agonists. The failure to detect depolarization-induced 933
Vol.
Ca+2
136,
3,
No.
1986
BIOCHEMICAL
influx in DDTl,
cultured
AND
ROC.
RESEARCH
AlO, and rabbit aorta cells using fluorescent
smooth muscle cells do not retain
functional
BIOPHYSICAL
Caponi --et al (12) reported
functional
dyes suggests
that
VOC, although they do retain
that depolarization
changes in (Ca+2)i in quin-2 loaded monolayers
COMMUNICATIONS
of primary
elicited only very small
rat thoracic
aorta smooth
muscle cells which responded to angiotensin II and AVP with large increases in (Ca+2)i. The technique represents
of measuring
an excellent
Ca+2 transients
model system
with
fura-
in monolayer
with which to study the properties
cultures
of receptor-
operated Ca+2 channels in smooth muscle cells in their normal morphology with minimal perturbation. vascular
The successful
smooth
vasoconstrictor investigate
muscle
application
(rabbit
aorta)
of this technique to primary which
respond
agents will provide an excellent
the transduction
mechanisms
involved
cultures
to a number
model system
with
in receptor-mediated
of adult
of important
which
to further
elevation
of
(Ca’2)i in vascular smooth muscle.
REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
Reynolds, E. E., and Dubyak, G. R. (1985) Biochem. Biophys. Res. Comm. 130: 627632. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260: 3440-3450. Toll, L. (1982) J. Biol. Chem. 257: 13189-13192. Norris, J.S., Gorski, J., and Kohler, P.O. (1974) Nature 2.48: 422-424. Cornett, L.E., and Norris, J.S. (1982) J. Biol. Chem. 25734-697. Kongsamut, S., Freedman, S.B., and Miller, R.J. TivS) Biochem. Biophys. Res. Comm. 127: 71-79. VincentxL.M., Ambrosini, A., DiVirgilio, F., Pozzan, T., and Melolesi, J. (1985) J. Cell Biol. 100: 1330-1333. Colucci, Wx, Brock, T.A., Gimbrone, M.A. Jr., and Alexander, R.W. (1985) Mol. Pharm. 27: 517-524. Aiyar, NTNambi, P., Stassen, F.L., and Crooke, S.T. (1985) The Pharmacologist -27: 222. Albert, P.R. and Tashjian, A.H. (1984) J. Biol. Chem. 259: 15350-15363. DiVirgilio, F., Pozzan, T., Wollheim, C.B., VincentinixM., and Meldolesi, J. (1986) J. Biol. Chem. 261: 32-35. Capponi, A., LeTP.D., Vallotton, M.B. (1985) J. Biol. Chem. 2260. 7836-7842.
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