Integrin mobilizes intracellular Ca2+ in renal
vascular smooth muscle cells
Wah-Lun
Chan1,
N.-H.
Holstein-Rathlou2, and
Kay-Pong
Yip3
1 Department of Molecular Pharmacology, Physiology, and
Biotechnology, Brown University, Providence, Rhode Island 02912;
3 Department of Physiology and Biophysics, University of
South Florida, Tampa, Florida 33612; and 2 Department of Medical
Physiology, The University of Copenhagen, DK-2200 Copenhagen N,
Denmark
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ABSTRACT |
Peptides with the Arg-Gly-Asp (RGD) motif
induce vasoconstriction in rat afferent arterioles by increasing the
intracellular Ca2+ concentration
([Ca2+]i) in vascular smooth muscle cells
(VSMC). This finding suggests that occupancy of integrins on the plasma
membrane of VSMC might affect vascular tone. The purpose of this study
was to determine whether occupancy of integrins by exogenous RGD
peptides initiates intracellular Ca2+ signaling in cultured
renal VSMC. When smooth muscle cells were exposed to 0.1 mM hexapeptide
GRGDSP, [Ca2+]i rapidly increased from
91 ± 4 to 287 ± 37 nM and then returned to the baseline
within 20 s (P < 0.05, 34 cells/5 coverslips). In
controls, the hexapeptide GRGESP did not trigger Ca2+
mobilization. Local application of the GRGDSP induced a regional increase of cytoplasmic [Ca2+]i, which
propagated as Ca2+ waves traveling across the cell and
induced a rapid elevation of nuclear [Ca2+]i.
Spontaneous recurrence of smaller-amplitude Ca2+ waves were
found in 20% of cells examined after the initial response to
RGD-containing peptides. Blocking dihydropyridine-sensitive Ca2+ channels with nifedipine or removal of extracellular
Ca2+ did not inhibit the RGD-induced Ca2+
mobilization. However, pretreatment of 20 µM ryanodine completely eliminated the RGD-induced Ca2+ mobilization.
Anti-
1 and anti-
3-integrin antibodies
with functional blocking capability simulate the effects of GRGDSP in
[Ca2+]i. Incubation with
anti-
1- or
3-integrin antibodies
inhibited the increase in [Ca2+]i induced by
GRGDSP. We conclude that exogenous RGD-containing peptides induce
release of Ca2+ from ryanodine-sensitive Ca2+
stores in renal VSMC via integrins, which can trigger cytoplasmic Ca2+ waves propagating throughout the cell.
confocal microscopy; immunofluorescence; calcium; wave
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INTRODUCTION |
THE ARG-GLY-ASP (RGD)
motif is a common motif found in many extracellular matrix proteins
that bind to integrins. RGD-containing peptides induce vasodilatation
in cremaster muscle arterioles by lowering the intracellular
Ca2+ concentration ([Ca2+]i; see
Ref. 8). L-type Ca2+ channels
(24), K+ channels (18), and
v
3-integrins were suggested to be
involved (15). However,
5
1-integrins mediate an increase of the
Ca2+ current in smooth muscle cells isolated from the same
vascular bed (24). In rat renal afferent arterioles,
RGD-containing peptides induce vasoconstriction rather than dilatation.
The constriction is associated with a pronounced increase in the
[Ca2+]i in the smooth muscle cells as
measured by confocal fluorescence microscopy (27). The
[Ca2+]i in renal vascular smooth muscle is
the major determinant in the myogenic mechanism of renal autoregulation
(19), in which renal arterioles constrict when the
transmural pressure is increased. Extracellular mechanical stimuli can
be transduced into the cytosol via interactions between the
cytoskeleton and the cytoplasmic domain of integrins (23).
It is hypothesized that variations of transmural pressure in renal
arterioles will alter the interactions between integrins and the
extracellular matrix, which contributes to the mechanotransduction in
renal autoregulation (20, 27). It has been demonstrated
that the myogenic component in renal autoregulation is oscillating at
0.1-0.2 Hz (4, 25). These oscillations can be driven
by a temporally and spatially coordinated release of intracellular
Ca2+ in the form of Ca2+ wave or
[Ca2+]i oscillation in afferent arteriolar
vascular smooth muscle. [Ca2+]i oscillation
can be considered as a recurrence of fast-propagating intracellular
Ca2+ waves. We hypothesized that occupancy of integrins
would trigger spatial and temporal variations of
[Ca2+]i in afferent arteriolar vascular
smooth muscle cells. The present study was performed to test these
hypotheses in cultured renal vascular smooth muscle cells using
confocal fluorescence microscopy.
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MATERIALS AND METHODS |
Isolation and culture of rat renal preglomerular smooth muscle
cells.
Renal preglomerular smooth muscle cells were isolated from
Sprague-Dawley rats by an iron oxide sieving method (10, 13, 29). Briefly, rats were anesthetized with halothane and
laparotomized, and the kidneys were exposed. The abdominal aorta distal
to the renal arteries was cannulated, and 1% iron oxide
(Fe3O4) in calcium-free Hanks' balanced salts
solution (HBSS) was perfused into the kidney. The kidneys were then
removed, and the cortex was harvested. The tissue was minced and
transferred to HBSS. Iron oxide-containing tissue was isolated from the
suspension with a side-pull magnet (Perseptive Diagnostics) and was
resuspended in HBSS. The suspension was passed through needles of
decreasing size (18, 20, and 23 G) and was filtered on a 200-mesh sieve
(Sigma). The iron oxide-containing tissue on the sieve was then
digested with collagenase (type 1, 2 mg/ml; Worthington) in HBSS (with
calcium chloride) at 37°C with gentle shaking for 30 min. The
remaining iron oxide-containing tissue was removed by a magnetic
method, and smooth muscle cells in the supernatant were centrifuged
down and resuspended in 10 ml of DMEM with 10% FBS. The cells were
seeded onto collagen I-coated T25 culture flasks (Becton-Dickinson) and
incubated at 37°C in 5% CO2 and 95% air at 98%
humidity. The cells were allowed to seed for 2 days before the medium
was changed. The medium was changed every 2-3 days until the cells
had grown to confluence. The cells were passed approximately every 5 days and were passed onto collagen I-coated coverslips
(Becton-Dickinson) during the sixth passage. Cells were grown close to
confluence for the immunohistochemistry and Ca2+
measurement studies. Cells from at least three different preparations (rats) were studied in each experimental protocol.
Measurement of
[Ca2+]i in cultured renal
smooth muscle cells.
The [Ca2+]i of cultured renal preglomerular
smooth muscle cells grown on coverslips was measured with fluo 3 from
the fluorescence images acquired with an MRC-1000 (Bio-Rad) confocal
scanning unit, which was mounted on a Zeiss Axiovert 100TV inverted
microscope. Renal preglomerular smooth muscle cells at sixth passage
were grown on collagen I-coated coverslips for 3-4 days and used
for measurement before they become fully confluent. The cells on the coverslips were loaded with 2 µM of fluo 3-AM (Molecular Probes) in
DMEM for 20 min at 37°C. The excess dye was washed away by HBSS (with
calcium) and incubated in the same buffer for 15 min. The coverslip was
then inserted in the bottom of a perfusion chamber (Vestavia) and
mounted on the inverted microscope. The changes in the smooth muscle
cell [Ca2+]i were measured at room
temperature. Fluorescence was excited with the 488-nm line of the
krypton-argon laser. Emission was collected through a band-pass filter
522/32 nm at 1 Hz and stored digitally. All fluorescence images were
acquired with a Zeiss plan-apochromat objective [63 × at numeric
aperture (NA) 1.4 or 40 × at NA 1.2]. Residence time of the
laser on the coverslip is 0.29 s. The changes of
[Ca2+]i in each cell after exposure to 0.1 mM
of either hexapeptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) or GRGESP
(Gly-Arg-Gly-Glu-Ser-Pro) were obtained during the retrospective
analysis of the stored fluorescence images with software (Time
Course/Ratiometeric Software Module) supplied by Bio-Rad. A testing
dose of 0.1 mM of the peptides was chosen so that the increase in
fluorescence intensity does not saturate the detector during the image
acquisition. A similar dosage was used in smooth muscle cells isolated
from cremaster muscle arterioles (24). RGD peptides were
administered in the perfusion chamber as a bolus in most studies. RGD
peptides were present in the chamber throughout the recording period.
When RGD was required to be applied locally on the coverslip, a
micropipette (5-10 µm diameter) attached to a microperfusion
pump was used (Hampel).
Calibration of fluorescence emission was performed using the
nonfluorescent Ca2+ ionophore 4-bromo-A-23187
(10
5 M) in the presence of extracellular Ca2+
to saturate the intracellular dye with Ca2+ and thereby
obtain maximal fluorescence (Fmax). The minimal
fluorescence (Fmin) was measured after addition of
Ca2+-free HBSS containing EGTA (4 mM). Fluorescence
intensity (F) was converted to [Ca2+]i using
the equation (2).
where Kd is the dissociation constant
(320 nM; provided by Molecular Probes). Because fluo 3 is a
nonratiometric dye, photobleaching and leakage during repeated image
acquisitions will render calibrations unreliable. Consequently,
calibrations and conversion of the fluorescent signal to
[Ca2+]i were only performed in the studies
that involved a single exposure of the cells to the RGD-containing peptide.
To determine the role of dihydropyridine-sensitive Ca2+
channels in the RGD-induced increase of
[Ca2+]i, smooth muscle cells were incubated
with nifedipine (1 µM) at room temperature for 5 min in darkness
before exposure to 0.1 mM GRGDSP. To study the effects of depleting
extracellular Ca2+ in the RGD-induced elevation of
[Ca2+]i, smooth muscle cells were washed
three times (3 min each) with Ca2+-free HBSS containing
EGTA (4 mM) before GRGDSP was introduced into the perfusion chamber.
To test whether ryanodine-sensitive Ca2+ stores are
involved in RGD peptide-induced Ca2+ mobilization, smooth
muscle cells were incubated with ryanodine (20 µM) for 20 min in
Ca2+-free HBSS before the cells were exposed to GRGDSP. To
test whether inositol trisphosphate (IP3)-sensitive
Ca2+ stores are involved in RGD-induced Ca2+
mobilization, xestospongin C (50 µM) was used instead of ryanodine.
To test whether the RGD-induced [Ca2+]i
elevation is mediated by specific integrins, the cells were incubated
with functional blocking anti-
1-, or -
2-,
or -
3-integrin antibodies (50 µg/ml) for 20 min before
exposure to GRGDSP hexapeptide. Changes in
[Ca2+]i during the incubation of
anti-integrin were also monitored.
2-Integrins are
exclusively expressed in leukocytes but not in smooth muscle cells and
thus served as negative controls (21).
Immunofluorescence of
-integrin subunits in cultured smooth
muscle cells.
To determine whether incubation of functional blocking anti-integrin
antibodies induces any redistribution of integrin in the smooth muscle
cells, the immunofluorescence of
1 and
3
was compared when cells were fixed by using two different fixation protocols. First, preglomerular smooth muscle cells grown on collagen I-coated coverslips were fixed in 2% paraformaldehyde for 10 min. The
coverslips were blocked with 20% donkey serum in PBS for 60 min and
then incubated with a mixture of anti-smooth muscle
-actin antibodies and antibodies specific to
1- or
3-integrin subunits for double-labeling studies. After
2 h of incubation, the sections were washed three times with fresh
PBS followed by incubation with the appropriate Cy3- or Cy5-conjugated
secondary antibodies for 60 min. Cy3 (indocarbocyanine, absorption peak
550, emission peak 570) and Cy5 (indodicarbocyanine, absorption peak
650, emission peak 670) are cyanine-based fluorophores, which are
brighter and more photostable than the widely used fluorescein-based
fluorophores. All incubations were carried out in a moistened chamber
at room temperature. The coverslips were then rinsed with fresh PBS
three times, mounted in Gel/Mount (Fisher), and examined with a
confocal microscope. Cultured cells incubated with
anti-
2-integrin antibodies only were used as negative
controls. All antibodies were diluted with PBS containing 20% donkey serum.
To study the effects of anti-integrin antibodies on the distribution of
integrins in live cells, a separate study was performed where cultured
cells were incubated with functional blocking anti-integrin antibodies
(50 µg/ml,
1 or
3) for 20 min before
the fixation with paraformaldehyde. Antibodies were diluted with DMEM
in all functional blocking studies.
Antibodies.
Mouse monoclonal IgM antibody to
3 was purchased from
Transduction Laboratories (Lexington, KY). Functional blocking
antibodies to
1 (hamster, monoclonal IgM, FITC conjugate
or nonconjugated)-,
2 (mouse, monoclonal IgG)-, and
3-integrins (mouse, monoclonal IgG, nonconjugated) were
purchased from PharMingen (San Diego, CA). Cy3-conjugated and
nonconjugated mouse monoclonal IgG to smooth muscle
-actin were
purchased from Sigma Chemical (St. Louis, MO). Dilution for all primary
antibodies was determined before the experiment. All secondary
conjugated antibodies purchased have been solid-phase adsorbed to
optimize the signal for the multiple labeling study. Secondary
antibodies were diluted at 1:400 for all studies (Jackson
Immunoresearch Laboratory).
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RESULTS |
Effect of RGD-containing peptide on
[Ca2+]i.
Exposing renal smooth muscle cells loaded with fluo 3 to 0.1 mM
hexapeptide GRGDSP in HBSS elicited an immediate increase of
fluorescence emission intensity, indicating an elevation of smooth
muscle [Ca2+]i. The increase of mean
[Ca2+]i reached a peak value of 287.0 ± 37.2 nM from a baseline of 91.4 ± 3.9 nM within 8 s and then
returned to baseline within 20 s (P < 0.05, 34 cells/5 coverslips; Fig. 1). There were
two types of responses in terms of the change in
[Ca2+]i profile in the individual cells.
Eighty percent of the cells examined displayed a rapid increase of
[Ca2+]i and then simply returned to the
baseline level similar to the profile shown in Fig. 1. In 20% of the
cells studied, there were recurrences of smaller-amplitude
Ca2+ waves after the initial response to the RGD peptide.
The propagation of the Ca2+ wave could be
visualized by the time delays between the
[Ca2+]i increases in the different regions of
the cell (Fig. 2). The mean propagation
velocity was 24.4 ± 1.7 µm/s (n = 6). As a
result of these spontaneous recurrent Ca2+ waves, the mean
[Ca2+]i of the whole cell was set to
oscillate. Hexapeptide GRGESP (0.1 mM) in HBSS or HBSS alone did not
induce any change in [Ca2+]i (Fig. 1). This
confirmed our previous observation from isolated afferent arterioles
that the increase of smooth muscle [Ca2+]i is
triggered by the RGD motif.

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Fig. 1.
Time course of change in intracellular Ca2+
concentration ([Ca2+]i) when cells were
exposed to either 10 4 M of GRGDSP or 10 4 M
of GRGESP. The peptide was added to the perfusion chamber at time
0. Dotted lines are SE. , Increase of
[Ca2+]i is significant (P < 0.05) compared with the baseline.
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Fig. 2.
Recurrence of spontaneous Ca2+ waves induced by
10 4 M of GRGDSP. Fluorescence intensity of fluo 3 is
measured simultaneously in 6 regions across a single smooth muscle
cell. Each sampling region is a 12 µm × 12 µm square. The
Ca2+ wave propagates from region 1 to
region 6. The peptide is added to the perfusion chamber at
time 0 as a bolus. The Ca2+ wave is propagating
at 24 µm/s.
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Local application of 0.1 mM GRGDSP within the perfusion chamber through
a micropipette induced a cytoplasmic Ca2+ wave that
propagated across the cells (Fig. 3). The
cytoplasmic Ca2+ wave triggered a rapid increase of
nuclear [Ca2+]i when it propagated through
the nucleus (Fig. 4). The mean
Ca2+ wave propagation velocity was 27.6 ± 1.4 µm/s (n = 6), which is not significantly different
from the spontaneous recurrent Ca2+ waves. In some
occasions, locally induced Ca2+ waves were observed to
spread into the adjacent cells.

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Fig. 3.
Propagation of Ca2+ wave in a single renal
vascular smooth muscle cell. A: projection of 10 images
collected before the appearance of Ca2+ wave. Arrow
indicates the nucleus. B-L: consecutive images
collected at 1 Hz to display the propagation of calcium wave. GRGDSP
(0.1 mM) was applied locally on the coverslip with a micropipette.
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Fig. 4.
Reversal of the nucleocytoplasmic Ca2+ gradient when a
Ca2+ wave passes through the nucleus in the vascular smooth
muscle cell shown in Fig. 3. Fluorescence intensity of fluo 3 is
measured simultaneously in three regions, a cytoplasmic region anterior
to the nucleus, a nuclear region, and a cytoplasmic region posterior to
the nucleus. The area of each region is 12 µm × 12 µm. The
Ca2+ wave is propagating at a velocity of 27 µm/s.
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Effects of nifedipine and external
Ca2+ depletion on
[Ca2+]i response.
To determine whether the increase of [Ca2+]i
triggered by RGD-containing peptides in renal vascular smooth muscle
cells is due to an influx of extracellular Ca2+ through
voltage-gated dihydropyridine-sensitive Ca2+ channels, the
effects of nifedipine on the [Ca2+]i were
determined. Smooth muscle cells loaded with fluo 3 were first exposed
to 0.1 mM of GRGDSP to confirm that these cells were responding to RGD
motifs. The smooth muscle cells were then incubated with nifedipine (1 µM) for 5 min. There was no change in the fluo 3 emission intensity
when the cells were exposed to 0.3 M KCl in HBSS, indicating that
dihydropyridine-sensitive Ca2+ channels were successfully
blocked by nifedipine (data not shown). Incubation with nifedipine did
not inhibit the increase of [Ca2+]i induced
by GRGDSP or the induction and propagation of Ca2+ waves.
The peaks of normalized fluo 3 emission intensity before and after the
treatment with nifedipine were 2.04 ± 0.13 and 1.73 ± 0.23, respectively (29 cells/3 coverslips, Fig.
5A).

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Fig. 5.
Normalized time course of the change in fluo 3 fluorescence
intensity when cells were exposed to 10 4 M GRGDSP after
incubation with 1 µM nifedipine, 29 cells/3 coverslip (A)
or in the absence of extracellular Ca2+, 24 cells/3
coverslips (B). The peptide was added to the perfusion
chamber at time 0. Dotted lines are SE. ,
Fluorescence intensity is significantly different (P < 0.05) from the baseline.
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Removal of extracellular Ca2+ from the bathing
medium by including 4 mM EGTA in Ca2+-free buffer did not
inhibit the increase of [Ca2+]i or the
propagation of Ca2+ waves induced by the RGD-containing
peptide. The peaks of normalized fluo 3 emission intensity before and
after the removal of extracellular Ca2+ were 1.77 ± 0.12 and 1.78 ± 0.19, respectively (24 cells/3 coverslips, Fig.
5B). These observations suggest that the Ca2+
mobilization and propagation of Ca2+ wave do not depend on
influx of extracellular Ca2+. However, the increase of
[Ca2+]i triggered by RGD peptides in
individual cell was not simultaneous. A delayed onset of the
[Ca2+]i increase was observed in a
substantial number of cells. The variable time courses for the response
to the RGD-containing peptide also resulted in an increased variability
for the recovery of [Ca2+]i in Fig.
5B. Inclusion of 1 mM Mg2+ in the
Ca2+-free solution reduced the heterogeneity in the onset
of rising [Ca2+]i (data not shown). These
observations are consistent with the observation that binding of RGD
motifs to integrins is affected by the extracellular divalent ions
(17).
Effects of IP3 receptor blocker and ryanodine on
[Ca2+]i response in the
absence of extracellular Ca2+.
Smooth muscle cells loaded with fluo 3 were first exposed to 0.1 mM of
GRGDSP to confirm that these cells were responding to RGD motifs. The
smooth muscle cells were incubated with 50 µM xestospongin C for 30 min in the absence of extracellular Ca2+. There was a small
transient increase of fluo 3 emission during the first few minutes of
incubation, which was most likely due to the inhibiting effect of
xestospongin C in endoplasmic reticulum Ca2+ pumps at this
concentration (9). There was no change in fluo 3 emission
intensity when the cells were exposed to 1 µM of phenylephrine after
xestospongin C incubation, which indicated that the IP3 receptors were blocked. However, 0.1 mM of GRGDSP could still induce
mobilization of intracellular Ca2+ as reflected in an
increase of fluo 3 emission (Fig.
6A). The peaks of normalized
fluo 3 emission intensity before and after the incubation of
xestospongin C were 1.76 ± 0.06 and 1.52 ± 0.08, respectively (58 cells/4 coverslips). On the contrary, preincubation of
20 µM of ryanodine for 20 min totally abolished the Ca2+
mobilization effect of 0.1 mM of GRGDSP (Fig. 6B).
Phenylephrine (1 µM) could induce an increase of fluo 3 emission
after the pretreatment of ryanodine. These observations suggest that
the Ca2+ mobilization induced by RGD-containing peptides
involves the ryanodine-sensitive Ca2+ stores but not the
IP3-sensitive Ca2+ stores.

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Fig. 6.
Normalized time course of the change in fluo 3 fluorescence intensity when cells were exposed to 10 4 M
GRGDSP after incubation with 50 µM xestospongin C, 58 cells/4
coverslip (A) or after the incubation of 20 µM ryanodine,
106 cells/6 coverslips (B). The peptide was added to the
perfusion chamber at time 0. Dotted lines are SE.
and , Fluorescence intensity is
significantly different (P < 0.05) from the baseline.
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Effects of anti-integrin antibodies on the
[Ca2+]i of smooth muscle
cells.
To determine whether the Ca2+ mobilization induced by RGD
peptides is mediated by integrins (15), cells loaded with
fluo 3 were first exposed to 0.1 mM of GRGDSP to confirm that these
cells were responsive to the RGD motifs. The cells were then incubated with functional blocking anti-
1-,
anti-
2-, or anti-
3-integrin antibodies
for 20 min. Preincubation of the cells with either anti-
1- or anti-
3-integrin antibodies
completely inhibited the elevation of [Ca2+]i
induced by GRGDSP (Fig. 7).
Anti-
2-integrin antibodies did not inhibit the increase
of [Ca2+]i induced by GRGDSP (Fig.
8). The latter observation was expected because
2-integrins are exclusively expressed in
leukocytes (21). These observations suggest that the
increase of [Ca2+]i triggered by RGD motifs
is mediated by specific binding of RGD-containing peptides to
functional
-integrin and that the interactions are mediated by more
than one class of integrin heterodimers. To test whether the ligation
of
1- and
3-integrins by the functional blocking antibodies can trigger Ca2+ mobilization, the
changes in emission intensity of fluo 3 were monitored when smooth
muscle cells were exposed to these antibodies. Both
anti-
1- and anti-
3-integrin antibodies
could induce an increase in [Ca2+]i in smooth
muscle cells when applied separately (Fig.
9). During multiple exposure of the
smooth muscle cells to the same antibody, an increase of
[Ca2+]i was only detected in the first
exposure. During sequential application of these two antibodies into
the smooth muscle cells, each antibody could induce a rise in
[Ca2+]i no matter which antibody was
administered first. These observations indicate that these two
antibodies ligate two different classes of integrin and that the
ligation of either
1- or
3-integrin alone
can mobilize intracellular Ca2+.

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Fig. 7.
Normalized time course of change in fluo 3 fluorescence
intensity when cells were exposed to 10 4 M GRGDSP after
incubation with functional blocking anti- 1 integrin
antibody, 24 cells/4 coverslips (A) or after incubation of
functional blocking anti- 3-integrin antibody, 27 cells/4
coverslips (B). , Fluorescence intensity is
significantly different (P < 0.05) from baseline.
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Fig. 8.
Normalized time course of change in fluo 3 fluorescence intensity when cells were exposed to 10 4 M
GRGDSP before (A) and after (B) incubation with
functional blocking anti- 2-integrin antibodies (41 cells/3 coverslips). The peptide was added to the perfusion chamber at
time 0. Dotted lines are SE. and
, Fluorescence intensity is significantly different
(P < 0.05) from the baseline.
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Fig. 9.
Normalized time course of change in fluorescence
intensity when cells were exposed to anti- 1-integrin
antibodies (30 cells/3 coverslips) and anti- 3-integrin
antibodies (27 cells/3 coverslips). The concentration of antibodies
used was 50 µg/ml. The peptide was added to the perfusion chamber at
time 0. Dotted lines are SE. and
, Fluorescence intensity is significantly different
(P < 0.05) from the baseline.
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Immunofluorescence of integrin
-subunits.
Figure 10 shows the immunofluorescence
after double labeling for
1- and
-actin.
1-Integrin was found mainly along the stress fibers and
on the periphery of nuclei. The staining for
-actin confirmed that
the cells were smooth muscle cells. When functional-blocking anti-
1-integrin antibodies were incubated for 20 min
with smooth muscle cells before fixation, the immunofluorescence signal
of
1-integrin along the stress fibres disappeared, and
only the signal on the periphery of nuclei remained (Fig. 10,
C and D). These observations suggest that
incubation with functional-blocking anti-integrin antibodies in living
cells induces a redistribution of integrins from the plasma membrane.
3-Integrin showed a fibrillar distribution with more
signal near the edge of the cells and the periphery of nuclei in
controls (Fig. 11A). A
redistribution of
3-integrin was also found when
functional blocking antibodies of
3-integrin were
incubated with the cultured cells before the cell fixation (Fig.
11C). Treatment of functional blocking
anti-
1- or anti-
3-integrin antibodies
before paraformaldehyde fixation did not result in cell detachment. In
the controls, no immunofluorescence of
2 was detected in
the vascular smooth muscle cells as expected (image not shown).

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Fig. 10.
Colocalization of 1-integrin (A and
C) and smooth muscle -actin (B and
D) in preglomerular smooth muscle cells. Cellular fixation
was performed before (A and B) and after
(C and D) incubation with functional-blocking
antibodies. In C and D, cells were incubated with
functional blocking anti- 1-antibodies (hamster
monoclonal IgG) for 20 min before fixation.
Anti- 1-integrin antibody was conjugated to FITC.
Anti- -actin antibody was conjugated to Cy3.
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Fig. 11.
Colocalization of 3-integrin (A and
C) and smooth muscle -actin (B and
D) in preglomerular smooth muscle cells. Cellular fixation
was performed before (A and B) and after
(C and D) incubation with functional blocking
antibodies. In C and D, cells were incubated with
functional blocking anti- 3-antibodies (mouse monoclonal
IgG) for 20 min before fixation. A mouse monoclonal IgM
anti- 3-integrin antibody was used to label
3-integrin after fixation. Anti- -actin-antibody
(mouse monoclonal IgG) was conjugated to Cy3. 3-Integrin
was visualized with Cy5-conjugated secondary antibody.
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DISCUSSION |
The utility of isolated afferent arterioles for studying the
subcellular variations of [Ca2+]i is limited
by the geometry of blood vessels, where the individual smooth muscle
cell wraps around the vessel (26). Refined methodology is
now available for isolating vascular smooth muscle cells from preglomerular resistance vessels for acute studies (10,
29). Because of the enzymatic digestion process used in the
isolation of the smooth muscle cells, the integrity of integrins on
freshly isolated smooth muscle cells might be compromised. We took an alternative approach by culturing preglomerular smooth muscle cells on
coverslips so that the subcellular spatial and temporal variations in
intracellular Ca2+ could be monitored by confocal
fluorescence microscopy. We first demonstrated that exposing the cells
to the hexapeptide GRGDSP resulted in an increase of
[Ca2+]i. In contrast, no response was
observed when the hexapeptide GRGESP was used. These observations are
in agreement with previous studies performed in isolated afferent
arterioles (27). The baseline
[Ca2+]i in the cultured smooth muscle cells
is 91 ± 4 nM, which is consistent with reports from another
laboratory (29). GRGDSP (0.1 mM) increased the
[Ca2+]i to 287 ± 37 nM within 8 s,
and the values were restored to the baseline within 20 s. The
time-course profile of the [Ca2+]i increase
is similar to that in isolated afferent arterioles (27).
These observations suggest that the machinery required for
RGD-dependent mobilization of intracellular Ca2+ in intact
arteriolar smooth muscle is also present in the primary cultures.
Local application of RGD peptides on the coverslip triggered
propagation of Ca2+ waves traveling across the smooth
muscle cell (Figs. 3 and 4). Because wave propagation did not depend on
extracellular Ca2+, it suggests that the propagation is due
to a regenerative release of Ca2+ from intracellular stores
(22, 28). The propagation velocity of intracellular
Ca2+ waves in the present study is in the range of
24-27 µm/s, which is faster than those observed in human vein
smooth muscle cells (7.5 µm/s, induced by histamine; see Ref.
16), in the smooth muscle cell line A7r5 (16 µm/s,
induced by vasopressin; see Ref. 1), and in human
bronchial smooth muscle cells (18 µm/s, induced by arachidonic acid;
see Ref. 6). This is the first report of integrin-mediated
Ca2+ waves in smooth muscle cells. No other comparable data
are available in the literature. The baseline fluorescence signal in
the nucleus is in general lower than that of the cytoplasm, which
indicates the existence of a nucleocytoplasmic Ca2+
gradient. The gradient was reversed when the cells were stimulated with
RGD peptides. Similar observations of reversing the nucleocytoplasmic Ca2+ gradient were also reported in a smooth muscle cell
line when stimulated by pharmacological agonists (12).
Because the increase of nuclear [Ca2+]i was
initiated when the cytoplasmic Ca2+ wave reached the
nucleus, it is likely that the increase of nuclear [Ca2+]i is also the result of
Ca2+-induced Ca2+ release. It is known that a
mechanical signal can be transduced from the plasma membrane to the
nucleus via the integrins and the cytoskeleton (14). If
ligand binding on integrins had triggered a nuclear
[Ca2+]i increase directly, the rise of
nuclear [Ca2+]i would most likely have
preceded the arrival of the cytoplasmic Ca2+ wave. The
propagation of integrin-initiated Ca2+ waves was not
limited to an individual cell. In some cases, it was observed that the
wave propagated into the adjacent smooth muscle cells. The recurrent
Ca2+ waves resulted in local oscillation of
[Ca2+]i. The coupling of smooth muscles cells
by Ca2+ oscillations might allow a population of cells to
operate in unison to regulate myogenic tone (16).
Interestingly, we have found that there are myogenic oscillations
(vasomotion) in afferent arterioles, and an increase of transmural
pressure induced by acute hypertension enhances the power of the
vasomotion (25). These observations are consistent with
the notion that there are oscillations of
[Ca2+]i in renal vascular smooth muscle in
vivo. Together with our previous observation that RGD-containing
peptides can induce vasoconstriction in isolated afferent arterioles by
increasing the vascular smooth muscle [Ca2+]i
(27), the present study strongly suggests that integrins might be part of the signaling mechanism of the myogenic response in
renal autoregulation.
Dihydropyridine-sensitive Ca2+ channels are abundant in
renal arterioles (11) and are known to mediate
Ca2+ influx in renal vascular smooth muscle. A patch-clamp
study in smooth muscle cells isolated from cremaster muscle arterioles has suggested that exogenous RGD peptides modulate the activity of
Ca2+ channels (24). Inhibition or stimulation
of the Ca2+ current (measured by Ba2+ current)
depends on which integrin heterodimers are ligated. However, the
inability of nifedipine and depletion of extracellular Ca2+
to inhibit the increase of [Ca2+]i induced by
GRGDSP in the present study suggests that the increase of
[Ca2+]i is not the result of an extracellular
Ca2+ influx but is due to the release of Ca2+
from intracellular stores. It is not surprising that smooth muscle cells from cremaster muscle arterioles and renal arterioles could employ very different Ca2+ signaling mechanism when ligands
bind to integrins. RGD-containing peptides induce constriction in
afferent arterioles but dilatation in cremaster skeletal arterioles
(15, 27). Blocking of the IP3 receptor with
xestospongin C had no effects on Ca2+ mobilization induced
by RGD-containing peptides, but ryanodine completely abolished it.
These observations strongly suggest that the increase of
[Ca2+]i is due to the release of
Ca2+ from ryanodine-sensitive Ca2+ stores and
that the Ca2+ wave observed is due to
Ca2+-induced Ca2+ release through ryanodine
receptors. In the vascular smooth muscle cell line A7r5, the
vasopressin-induced Ca2+ waves are mediated by
IP3-sensitive Ca2+ stores (1).
The next hypothesis tested was whether specific interactions between
RGD peptides and integrins are required for the elevation of
[Ca2+]i. Antibodies against
1-
and
3-integrins were chosen to test this hypothesis
because these are the two most widely expressed integrin
-subunits
through which the extracellular matrix is connected to the cytoskeleton
(5). Furthermore, a study in smooth muscle cells isolated
from cremaster muscle arterioles has shown that exogenous RGD motifs
increase the Ca2+ current via
5
1-integrins and decrease the
Ca2+ current via
v
3-integrins
(24). Immunofluorescence evidence from the present study
indicates that these two
-integrin subunits are also abundant in the
primary cultures of renal vascular smooth muscle cells. Pretreatment
with anti-
1- or anti-
3-integrin
functional blocking antibodies totally abolished the elevation of
[Ca2+]i triggered by the hexapeptide GRGDSP
(Fig. 7). The inhibition could be due to the blocking of specific
interactions between integrins and RGD motifs or deterioration of
cellular physiological conditions. However, 1 µM phenylephrine still
triggered an increase of [Ca2+]i even though
the cells no longer responded to RGD peptides (data not shown), which
indicates that the cells could still respond to a pharmacological
agonist with mobilization of Ca2+. There was a transient
decrease in the fluo 3 emission intensity when RGD peptides were
introduced into the perfusion chamber (Fig. 7). This transient decrease
(4-5 s) in emission intensity was due to the temporary dislocation
of the coverslip from the focal plane during solution switching.
Ligation of either
1- or
3-integrin seems
to be sufficient to induce Ca2+ mobilization. It is
expected that blocking of both
1- and
3-integrin subunits is required to completely inhibit
the effects of RGD peptides in [Ca2+]i. Our
observations indicate that functional blocking of either
1 or
3 is sufficient to inhibit that. It
might be because soluble RGD peptide is less efficient to ligate
integrins compared with anti-integrin antibodies and/or the binding of
functional blocking antibodies to
1-integrin reduces the
binding affinity of
3 to soluble RGD peptides and vice
versa. The observation that sequential application of these two
antibodies both can trigger Ca2+ signaling is consistent
with this notion. Recognition of integrin by anti-integrin antibodies
does not depend on the RGD binding motifs.
Incubation of the smooth muscle cells with anti-
1- or
anti-
3-integrin antibodies before cellular fixation
resulted in very different immunofluorescence patterns of integrins
when compared with preparations with cellular fixation before
incubation with antibodies. The difference in immunofluorescence
patterns suggests that there is a redistribution/clustering of
integrins on the plasma membrane as a result of the occupancy by the
functional-blocking antibodies. The redistribution and clustering of
integrins in the plasma membrane might result in conceding the binding
sites for RGD or changing the microenvironment of the RGD binding
sites, which prevents the interactions of RGD with the
-integrins.
All of the above observations indicate that specific interactions between RGD peptides and integrins are required for the increase of
[Ca2+]i in renal vascular smooth muscle
cells. It is not certain whether the changes in
[Ca2+]i are the result of the RGD-containing
peptides interacting with free integrin on the cell surface or the
result of the RGD peptides acting through inhibition of the attachment
of the vascular smooth muscle cells to the collagen matrix
(7). It can be a combination of both. These two events are
not mutually exclusive. The end result of either mechanism is the
formation of new ligand-integrin interactions. The
anti-
1- or anti-
3-integrin antibodies can trigger Ca2+ mobilization in the presence of 0.1 mM GRGDSP
(data not shown). By assuming that all free RGD binding sites have been
occupied by GRGDSP at this condition, this observation indicates that
inhibition of smooth muscle cell attachment is capable of mobilizing
intracellular Ca2+.
Integrins are 
-heterodimers. The integrin
-subunits
associated with the
1- and
3-subunits to
mediate the intracellular Ca2+ mobilization were not
identified in this study. Wu et al. (24) have reported
that Ca2+ current (measured by Ba2+ current)
was enhanced by
5
1-agonists and was
reduced by
v
3-agonists in smooth
muscle cells isolated from cremaster muscle arterioles. However, our
observations indicate that the Ca2+ mobilization in renal
vascular smooth muscle does not depend on an influx of extracellular
Ca2+.
1-Integrin can pair up a wide range of
-subunits,
1 to
9 and
v
(3).
3-Integrin is most commonly paired
with
v and
IIb (3).
Identification of which heterodimer of integrins is involved in this
signaling process will be the next step to elucidate the possible role
of integrins in the mechanotransduction process of renal autoregulation
(15, 27).
In summary, we have demonstrated that the hexapeptide GRGDSP
induces an increase of [Ca2+]i in cultured
renal vascular smooth muscle cells similar to that observed in smooth
muscle cells of isolated afferent arterioles. Local application of
GRGDSP could trigger a regenerative Ca2+ wave that
propagates across the cell. The increase of intracellular [Ca2+]i depends on the
Ca2+ released through ryanodine receptors and requires
interactions of the RGD motif and integrins. Finally, the study is the
first to show that RGD-containing peptides can trigger subcellular
spatial and temporal variations in intracellular Ca2+.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institutes of Health Grants
DK-15968, HL-45623, and HL-59156.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: K.-P.
Yip, Dept. of Physiology and Biophysics, College of Medicine, University of South Florida, MDC 8, 12901 Bruce B. Downs Blvd., Tampa,
FL 33612 (E-mail: dyip{at}hsc.usf.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 August 1999; accepted in final form 20 September 2000.
 |
REFERENCES |
1.
Blatter, LA,
and
Wier WG.
Agonist-induced [Ca2+]i waves and Ca2+-induced Ca2+ releases in mammalian vascular smooth muscle cells.
Am J Physiol Heart Circ Physiol
263:
H576-H586,
1992[Abstract/Free Full Text].
2.
Burner, M,
Centeno G,
Burki E,
and
Brunner HR.
Confocal microscopy to analyse cytosolic and nuclear calcium in cultured vascular cell.
Am J Physiol Cell Physiol
266:
C1118-C1127,
1994[Abstract/Free Full Text].
3.
Cheresh, DA,
and
Mecham RP.
Integrins Molecular and Biological Responses to the Extracellular Matrix. New York: Academic, 1994.
4.
Chon, KH,
Chen YM,
Marmarelis VZ,
Marsh DJ,
and
Holstein-Rathlou N-H.
Detection of interactions between myogenic and TGF mechanisms using nonlinear analysis.
Am J Physiol Renal Fluid Electrolyte Physiol
267:
F160-F173,
1994[Abstract/Free Full Text].
5.
Clark, EA,
and
Brugge JS.
Integrins and signal transduction pathways: the road taken.
Science
268:
233-239,
1995[ISI][Medline].
6.
Colliard-Rouiller, C,
and
Durand J.
Arachidonic acid-induced calcium signalling in human airway smooth muscle cells.
Respir Physiol
107:
263-273,
1997[ISI][Medline].
7.
D'Angelo, G,
and
Adam LP.
Integrin-dependent modulation of vascular smooth muscle function (Abstract).
Biophys J
78:
113A,
2000.
8.
D'Angelo, G,
Mogford JE,
Davis GE,
Davis MJ,
and
Meininger GA.
Integrin-mediated reduction in vascular smooth muscle [Ca2+]i induced by RGD-containing peptide.
Am J Physiol Heart Circ Physiol
272:
H2065-H2070,
1997[Abstract/Free Full Text].
9.
De Smet, P,
Parys JB,
Callewaert G,
Weidema AF,
Hill E,
De Smedt H,
Erneux C,
Sorrentino V,
and
Missiaen L.
Xestospongin C is an equally potent inhibitor of the inositol 1,4,5-triphosphate receptor and the endoplasmic-reticulum Ca2+ pumps.
Cell Calcium
26:
9-13,
1999[ISI][Medline].
10.
Gebremedhin, D,
Kaldunski M,
Jacobs ER,
Harder DR,
and
Roman RJ.
Coexistence of two types of Ca2+-activated K+ channels in rat renal arterioles.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F69-F81,
1996[Abstract/Free Full Text].
11.
Goligorsky, MS,
Colflesh D,
Gordienko D,
and
Moore LC.
Branching points of renal resistance vessels are enriched in L-type calcium channels and initiate vasoconstriction.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F251-F257,
1995[Abstract/Free Full Text].
12.
Himpens, B,
Smedt HD,
Droogmans G,
and
Casteels R.
Differences in regulation between nuclear and cytoplasmic Ca2+ in cultured smooth muscle cells.
Am J Physiol Cell Physiol
263:
C95-C105,
1992[Abstract/Free Full Text].
13.
Inscho, EW,
Belott TP,
Mason MJ,
Smith JB,
and
Navar JG.
Extracellular ATP increased cytosolic calcium in cultured rat renal arterial smooth muscle cells.
Clin Exp Pharmacol Physiol
23:
503-507,
1997[ISI].
14.
Maniotis, AJ,
Chen CS,
and
Ingber DE.
Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilized nuclear structure.
Proc Natl Acad Sci USA
94:
849-854,
1997[Abstract/Free Full Text].
15.
Mogford, JE,
Davis GE,
Platts SH,
and
Meininger GA.
Vascular smooth muscle
v
3 integrin mediates arteriolar vasodilation in response to RGD peptides.
Circ Res
79:
821-826,
1996[Abstract/Free Full Text].
16.
Neylon, CB,
Mason WT,
and
Irvine RF.
Histamine-induced calcium oscillations in human vascular smooth muscle: temporal sequence and spatial organization in single cells.
Clin Exp Pharm Physiol
18:
299-302,
1991[ISI][Medline].
17.
Phillips, DR,
Charo IF,
and
Scarborough RM.
GPIIb-IIIa: The responsive integrin.
Cell
65:
359-362,
1991[ISI][Medline].
18.
Platts, SH,
Mogford JE,
Davis MJ,
and
Meininger GA.
Role of K+ channels in arteriolar vasodilation mediated by integrin interaction with RGD-containing peptide.
Am J Physiol Heart Circ Physiol
275:
H1449-H1454,
1998[Abstract/Free Full Text].
19.
Roman, RJ,
and
Harder DR.
Cellular and ionic signal transduction mechanisms for the mechanical activation of renal arterial vascular smooth muscle.
J Am Soc Nephrol
4:
986-996,
1993[Abstract].
20.
Sanderson, MJ,
Charles AC,
Boitano S,
and
Dirksen ER.
Mechanisms and function of intercellular calcium signaling.
Mol Cell Endocrinol
98:
173-187,
1994[ISI][Medline].
21.
Springer, TA.
Adhesion receptors of the immune system.
Nature
346:
425-434,
1990[ISI][Medline].
22.
Tsien, RW,
and
Tsien RY.
Calcium channels, stores, and oscillations.
Annu Rev Cell Biol
6:
715-760,
1990[ISI].
23.
Wang, N,
Butler JP,
and
Ingber DE.
Mechanotransduction across the cell surface and through the cytoskeleton.
Science
260:
1124-1127,
1993[ISI][Medline].
24.
Wu, X,
Mogford JE,
Platts SH,
Davis GE,
Meininger GA,
and
Davis MJ.
Modulation of calcium current in arteriolar smooth muscle by
v
3 and
5
1 integrin ligands.
J Cell Biol
143:
241-252,
1998[Abstract/Free Full Text].
25.
Yip, K-P,
Holstein-Rathlou N-H,
and
Marsh DJ.
Mechanisms of temporal variation in single nephron blood flow in the rat.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F427-F434,
1993[Abstract/Free Full Text].
26.
Yip, K-P,
and
Marsh DJ.
[Ca2+]i in rat afferent arteriole during constriction measured with confocal fluorescence microscopy.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F1004-F1011,
1996[Abstract/Free Full Text].
27.
Yip, K-P,
and
Marsh DJ.
An Arg-Gly-Asp peptide stimulates constriction in rat afferent arterioles.
Am J Physiol Renal Fluid Electrolyte Physiol
273:
F768-F776,
1997[Abstract/Free Full Text].
28.
Young, SH,
Ennes HS,
and
Mayer EA.
Propagation of calcium waves between colonic smooth muscle cells in culture.
Cell Calcium
20:
257-271,
1996[ISI][Medline].
29.
Zhu, Z,
and
Arendshorst WJ.
Angiotensin II-receptor stimulation of cytosolic calcium concentration in cultured renal resistance arterioles.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F1239-F1247,
1996[Abstract/Free Full Text].
Am J Physiol Cell Physiol 280(3):C593-C603
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