An Arg-Gly-Asp peptide stimulates constriction in rat afferent
arteriole
Kay-Pong
Yip and
Donald J.
Marsh
Department of Molecular Pharmacology, Physiology and
Biotechnology, Brown University, Providence, Rhode Island 02912
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ABSTRACT |
The potential role of integrins in the myogenic mechanism was
studied in the rat afferent arteriole (AA) by fluorescence
immunolocalization and microperfusion of isolated AA. Confocal
fluorescence images were acquired from frozen sections of rat kidney
after indirect immunostaining for various integrin
- and
-subunits. The
1-,
3-,
3-,
5-, and
V-integrins
were found on the plasma membrane in smooth muscle of AA, providing the
morphological basis for participation of integrins in
mechanotransduction. With 1 mM nitro-L-arginine methyl
ester (L-NAME) in the luminal perfusate to inhibit
endogenous nitric oxide (NO) production from AA, the hexapeptide GRGDSP
(10
7-10
3 M) induced immediate
vasoconstriction. The constriction was dose dependent and specific for
peptides with arginine-glycine-aspartic acid (RGD) motifs, commonly
found on the binding sites of extracellular matrix to integrins. In
controls, the hexapeptide GRGESP induced no constriction. GRGDSP, 1 mM,
induced a 21.6 ± 2.6% decrease (P < 0.05,
n = 6) in lumen diameter for 30 s and an 18.3 ± 4.1% increase (P < 0.05, n = 6) in smooth muscle
intracellular calcium concentration for 18 s, as measured by the
emission ratio of Fluo-3/Fura Red. Binding of exogenous RGD motifs with
exposed integrins on AA smooth muscle therefore triggers
calcium-dependent vasoconstriction. However, the dose response to RGD
was not sensitive to the myogenic tone of the vessel, which suggests
that the integrin-mediated vasoconstriction is different from myogenic
constriction.
confocal fluorescence microscopy; mechanotransduction; intracellular calcium
 |
INTRODUCTION |
A MYOGENIC RESPONSE and tubuloglomerular feedback (TGF)
are the two major mechanisms responsible for renal blood flow
autoregulation. The sensor responsible for the role of TGF in
regulating afferent arteriolar (AA) resistance is known (2), but the
signal transduction in myogenic response in AA is not elucidated.
Studies of myogenic mechanism from other vascular beds suggest that an
increase of transmural pressure activates stretch-sensitive cation
channels in vascular smooth muscle cells (VSMC), which open
voltage-gated calcium channels by depolarization (9, 16). The influx of extracellular Ca2+, plus the release from endogenous
Ca2+ stores, triggers the initial myogenic constriction
(8). Stretch-sensitive mechanisms can account for the trigger of
myogenic constriction. However, the stretch resulting from an increase
of transmural pressure will diminish once the vessel contracts. The
signal for maintaining the myogenic constriction after the initial
response to stretch is not identified.
The transmembrane heterodimeric proteins, integrins, have been shown to
mediate the transduction of mechanical force from an extracellular site
into the cell via their cytoplasmic connections with the cytoskeleton
(30). Evidence of integrin involvement in stretch-related phenomena has
been reported in the motor nerve terminal. Chen and Grinnell (4) have
found that stretch-enhanced release of neurotransmitter from the frog
motor terminal is calcium dependent and can be inhibited by either
integrin antibodies or peptides containing the sequence
arginine-glycine-aspartic acid (RGD). RGD is a common motif found in
many extracellular matrix proteins that bind to integrins. Stretch
resulting from changes in the transmural pressure of arterioles will
alter the attachment between the extracellular matrix and the integrins
of vascular smooth muscle. Exogenous RGD peptides have been found to
alter smooth muscle intracellular Ca2+ concentrations
([Ca2+]i) in skeletal arterioles (7). Soluble
RGD peptide inhibited the activity of L-type calcium channel in
isolated smooth muscle cells, whereas fibronectin-coated beads
increased the Ca2+ current (32).
[Ca2+]i in smooth muscle is known to be the
primary determinant of myogenic stimulation (16, 33). These
observations suggested that changes in the interactions between
integrins and extracellular matrix due to stretch might be involved in
myogenic response by altering smooth muscle
[Ca2+]i.
If integrins are involved in stretch-related signal transduction in rat
AA, then integrins should be localized in the plasma membrane of smooth
muscle. Binding of exogenous RGD to integrins on smooth muscle might
alter the arteriole's contractile state, probably by altering smooth
muscle [Ca2+]i. The effects of exogenous RGD
on the contractile state arteriole response should be sensitive to the
myogenic tone of the vessel. In a relaxed state, exogenous RGD peptide
might cause contraction of vascular smooth muscle, but in a contracted
state as would be present with increased transmural pressure gradient,
RGD peptide might cause relaxation (17). The goals of the present study were therefore 1) to test for the presence of integrins on the vascular smooth muscle of AA with indirect immunofluorescence microscopy, 2) to test whether exogenous peptides containing
the RGD sequence can alter the contractile state of AA and whether this
response is dependent on the underlying myogenic tone, and 3)
to investigate whether the exogenous RGD sequence-containing peptides
can induce changes in smooth muscle [Ca2+]i
in AA.
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MATERIALS AND METHODS |
Tissue preparation for immunofluorescence.
Sections of kidney for immunostaining were obtained from male
Sprague-Dawley rats (180-250 g, Harlan). Rats were anesthetized with halothane. Kidneys were perfusion fixed with a mixture of 4%
paraformaldehyde and 4% sucrose in phosphate-buffered saline (PBS) by
retrograde perfusion via a cannula inserted into the descending aorta
distal to the renal artery. The kidneys were then removed, and blocks
of tissue were cut into 4-mm cubes. Blocks of tissue were postfixed
overnight in the same fixative at 4°C, washed with 0.1 M aqueous
NH4Cl solution for 30 min, and cryoprotected by incubation
in 2.3 M sucrose in PBS for 1 h. The tissue blocks were next snap
frozen in optimum cutting temperature compound (OCT, Miles Laboratory)
with liquid nitrogen-cooled isopentane. Cryosections (7 µm) were cut
and transferred to Fisher Superfrost Plus-charged glass slides.
Sections were first incubated with 1% sodium dodecyl sulfate (SDS) in
PBS for 5 min for antigen retrieval as suggested by Brown et al. (3).
After SDS was removed by washing in PBS, the sections were blocked with
20% donkey serum in PBS for 20 min and then incubated with a mixture
of anti-smooth muscle
-actin antibodies and antibodies specific to
each integrin subunit for double labeling. After 2-h incubation, the
sections were washed three times with fresh PBS followed by incubation with the appropriate CY5-conjugated secondary antibodies for 60 min.
All incubations were carried out in a moistened chamber at room
temperature. The sections were then rinsed with fresh PBS three times,
mounted in glycerol, and examined with a ×63 plan-apochromat objective (NA 1.4, oil immersion) on a Zeiss Axiovert model 100TV inverted microscope. Glycerol is used as a mounting medium to minimize
the effects of refractive index mismatch during the confocal image
acquisition, which will cause significant aberration in three-dimensional image reconstruction or merging of images in multiple-labeling studies if not corrected (12, 14). The microscope is
coupled to a model MRC-1000 confocal scanning unit (Bio-Rad) equipped
with a krypton-argon laser, three photomultiplier detectors, and one
transmitted light detector. Frozen sections stained with only the
secondary antibody were used as negative controls.
Antibodies.
Rabbit polyclonal anti-rat
1 antibody was kindly
provided by Dr. S. Adler (1). Rabbit polyclonal
3 and
5 antibodies were purchased from Chemicon (Temecula,
CA), and mouse monoclonal immunoglobulin G (IgG) antibody to
V was purchased from GIBCO (Gaitherburg, MD). Mouse
monoclonal IgM antibody to
3 was purchased from
Transduction Laboratories (Lexington, KY). The anti-
3
antibody can recognize the extracellular domain of integrin, whereas
the others recognize the cytoplasmic domain of integrins. Fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal IgG to smooth muscle
-actin was purchased from Sigma Chemical (St. Louis, MO). Dilution
for all primary antibodies was determined prior to the experiment. The
primary antibodies for integrins were selectively purchased from
different vendors to optimize their specificity. All secondary
conjugated antibodies were affinity purified and were designed for
multiple labeling study. Secondary antibodies were diluted at 1:400 for
all studies (Jackson Immunoresearch Laboratory, West Grove, PA).
Microperfusion of AA.
Experiments were conducted in AA isolated from rat juxtamedullary
nephrons as reported previously (33). In brief, a segment of afferent
arteriole (300-400 µm) just proximal to a glomerulus was
dissected, cannulated, and perfused in a temperature-controlled perfusion chamber (Vestavia, AL) mounted on a Zeiss Axiovert 100TV inverted microscope. The intraluminal pressure of the vessel was initially set at 80 mmHg. Vessels were discarded if there was fluid
leakage. The effects of the peptides GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), cyclic GRGDSP (cyclic Gly-Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys-Ala), and
GRGESP (Gly-Arg-Gly-Glu-Ser-Pro) on the AA lumen diameter were then
monitored when the peptides were introduced into the perfusion chamber.
Transmitted light images of the perfused vessel were acquired by the
confocal scanning unit with a ×63 plan-apochromat objective (NA 1.4).
All peptides were purchased from GIBCO. The maximal response (change in
lumen diameter) was used to construct the dose-response curve.
Measurement of intracellular calcium and luminal diameter.
The temporal relationship of changes in luminal diameter and
[Ca2+]i in VSMC was determined from the
confocal fluorescence images of the perfused vessel as described
previously (13, 33). In brief, a mixture of 20 µM Fluo-3-AM and 40 µM Fura Red-AM (Molecular Probes, Eugene OR) was loaded the into the
VSMC from the bathing solution at room temperature for 30 min. The
vessel was then washed and incubated at 35°C for another 30 min
before measurements were begun. The images were acquired from the
midplane of the perfused AA with the 488-nm laser line. Emissions were
collected simultaneously with a band-pass filter of 522/35 nm and a
long-pass filter 580LP on two separate photomultiplier detectors at 1 Hz and stored digitally. The [Ca2+]i changes
in VSMC following the introduction of RGD peptides into the perfusion
chamber were monitored in smooth muscle cells during playback of the
stored fluorescence image. The Fluo-3/Fura Red emission ratio from a
particular region of the fluorescence image was calculated
retrospectively after subtraction of the dark current and background
fluorescence at each emission wavelength. The emission ratio was then
used as a ratiometric index of the relative change in
[Ca2+]i (13, 33). Changes of luminal diameter
were determined at the same sites where the ratio measurements were
made. Emission ratio was calculated by the software (Time
Course/Ratiometric Software Module) supplied by Bio-Rad. Lumen diameter
was measured automatically from the stored image with an edge-detecting
algorithm based on covariance (15). The algorithm was implemented with a Matrox IP-8 imaging board (28, 33).
Results are shown as means ± SE; n is the number of vessels
studied. Student's t-test was used when applicable.
P < 0.05 was considered statistically significant.
Only one vessel was used from each animal.
Solutions.
The composition of the dissecting solution consisted of (in mM) 115 NaCl, 25 NaHCO3, 2.5 K2HPO4, 1.2 MgSO4, 1.8 CaCl2, 5.5 glucose, 2.0 pyruvic
acid, and 1 g/dl dialyzed bovine serum albumin (BSA, fraction V,
Calbiochem). The luminal perfusate and bathing solution were identical
to the dissecting solution except that no BSA was added to the bathing
solution, and 1 mM nitro-L-arginine methyl ester
(L-NAME) was included in the luminal perfusate to inhibit
nitric oxide (NO) production when required. BSA was excluded in the
bathing solution to prevent bacteria growth in the perfusion chamber.
All solutions were gassed with 5% CO2 before use, and pH
was adjusted to 7.4.
 |
RESULTS |
Immunohistochemical localization.
Immunofluorescence
of integrins from arterioles was weaker than that of tubules in
general. The two most common
-integrins,
1 and
3, were both found on the basolateral plasma membrane of
smooth muscle cells in AA (Fig. 1). The
signal on the apical side of the arterioles was the result of the
integrins on the endothelial cells (23). The strongest signal of
1-integrin is found on the brush border of proximal
tubules and glomeruli (Fig. 1A). Thick ascending limbs and
distal convoluted tubules were also stained. No
3-integrin was found on the brush border of proximal
tubules (Fig. 1C). The signals of
3 on
glomeruli and AA were weaker than those of
1. Three
other
-integrin subunits,
3,
5, and
V, were also localized on the basolateral plasma membrane of AA smooth muscle cells. The strongest signal of
3 was found on the glomeruli (Fig.
2a). A strong signal of
5 was found on the thick ascending limbs (Fig.
2C), whereas the signal from the AA was barely visible. The
strongest signal of
V was recognized in thick ascending
limbs (Fig. 3). These observations confirmed the presence of diversified integrins in the smooth muscle of
AA, which could provide the morphological basis for integrin to
participate in myogenic response. Negative controls, made by omitting
the primary antibodies in the staining procedures, demonstrated no
detectable signal in arterioles. The control indicates successful
blocking as well as the specificity of the secondary antibodies. The
strong background in the localization of FITC-conjugated smooth muscle
-actin was due to the autofluorescence from proximal tubules. The
intensity of autofluorescence in proximal tubules is wavelength
dependent. Autofluorescence from proximal tubules was minimal when
fluorescence was collected with CY5 filter set (excitation 647 nm)
compared to FITC filter set (excitation 488 nm).

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Fig. 1.
Colocalization of 1-integrin (A),
3-integrin (C), and smooth muscle -actin
(B and D) in afferent arterioles (AA) with double
labeling. The 1- and 3-integrins were
labeled with CY5-conjugated secondary antibodies. Arrows, basolateral
and apical side of afferent arteriole. PT, proximal tubule; TAL, thick
ascending limb.
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Fig. 2.
Colocalization of 3-integrin (A),
5-integrin (C), and smooth muscle -actin (B
and D) in AA with double labeling. The 3- and
5-integrins were labeled with CY5-conjugated secondary
antibodies. Symbols used are the same as in Fig. 1.
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Fig. 3.
Immunofluorescence of V-integrin in renal cortex
(A) and the corresponding transmitted light image
(B). The V-integrin was labeled with
CY5-conjugated secondary antibodies. Symbols used are the same as in
Fig. 1.
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To demonstrate the abundance of integrins on the plasma membrane of
smooth muscle cells and on the endothelial cells, which could not be
revealed by immunofluorescence on cryosections, immunolocalization of
integrins was also performed in a few isolated AA with
anti-
3 antibody. This antibody can recognize the
extracellular domain of
3-integrin. Figure 4, A
and B, shows confocal fluorescence micrographs of integrin
3-subunit on AA. Spindle-shaped smooth muscle cells with
immunofluorescence on the plasma membrane could be recognized (Fig.
4A). The exposed endothelium was
heavily stained by the integrin antibodies (Fig. 4B).

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Fig. 4.
A: immunofluorescence of 3-integrin on the
surface of a cannulated AA perfused at 80 mmHg. Arrows, edges of
positively stained single smooth muscle cell. B:
immunofluorescence of 3-integrin on the luminal surface
of an AA. Glomerulus and some smooth muscle cells in the distal end
were removed to expose the endothelium. Arrows, stained endothelial
cells. Vessels were incubated with primary antibody for 20 min.
CY5-conjugated secondary antibody was used to label the
anti- 3 antibody.
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Effects of RGD-containing peptides in perfused AA.
There was no consistent significant change of lumen diameter in AA when
GRGDSP (10
5-10
3 M) was introduced
into the perfusion chamber (data not shown). This observation suggested
that either the exogenous GRGDSP peptides had no effect on the
contraction of AA or the effects induced by the exogenous RGD peptide
were masked by some other mechanism. One possibility is that the
continuous accumulation of endogenous NO from AA endothelium (20) masks
the constriction induced by the GRGDSP peptide. To test this
hypothesis, 1 mM L-NAME was included in the luminal
perfusate to inhibit the NO synthase before the GRGDSP was introduced
into perfusion chamber. Impairment of NO production by
L-NAME was suggested by a reduced lumen diameter and the
ability of 1 mM L-arginine to reverse the constriction. The
mean diameters of AA before and after NO synthase blockade were 22.3 ± 1.6 and 16.3 ± 1.4 µm (P < 0.05, n = 12),
respectively. With 1 mM L-NAME in the luminal perfusate,
addition of GRGDSP to the bathing solution elicited a persistent
vasoconstriction in AA. The constriction was dependent on the dose of
GRGDSP over a range of concentrations from 10
7 to
10
3 M (Fig. 5). The peptide
GRGESP, which does not carry the RGD motif, failed to elicit
constriction (Fig. 5), indicating that constriction was specific to the
RGD motif. Another RGD peptide, cyclic GRGDSP, was used to test whether
the dose-dependent constriction was sensitive to the steric
conformation of the RGD motif. The dose-response curves of these two
RGD-containing peptides were similar (Fig. 5), suggesting that the
induced constriction was not sensitive to the steric conformation of
the RGD motif.

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Fig. 5.
Dose-response curves for constriction induced by different RGD
sequence-containing peptides and the inactive control.
Solid triangles ( ) and solid circles ( ) indicate
that the constriction is significant (P < 0.05). Mean
diameter in the baseline is 16.3 ± 1.4 µm (n = 12).
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The average time course of variations in lumen diameters of AA induced
by 1 mM GRGDSP is shown in Fig. 6. The mean
basal diameter was 16.6 ± 2.1 µm (n = 8). The
constriction was immediate and reached a maximum within 6 s and
remained significantly constricted for another 35 s
(P < 0.05). The original vessel diameter was fully restored
after the removal of the peptides by washing the perfusion chamber. The
average constriction was 19.7 ± 2.1% (P < 0.05, n = 8) of the baseline diameter in the first 30 s after the
application of the peptide. To determine whether the constriction
triggered by the RGD sequence-containing peptides is associated with a
rise in VSMC [Ca2+]i, the simultaneous
variations of luminal diameter and VSMC
[Ca2+]i in the perfused vessels were
determined in a separate study. Addition of 1 mM GRGDSP to the bath
triggered an immediate constriction in the vessel as observed
previously (Fig. 7B).
Constriction was completed after 5 s, and the average constriction for
the first 30 s was 21.6 ± 2.6% (P < 0.05,
n = 6) of the baseline. The emission ratio of Fluo-3/Fura
Red, a ratiometric index of the relative change of VSMC
[Ca2+]i, increased immediately after the
addition of 1 mM GRGDSP to the bath (Fig. 7A). The emission
ratio reached its peak value after 5 s of RGD peptide application and
remained elevated for 18 s. Both the increase in emission ratio and
decrease in lumen diameter are significantly different
(P < 0.05) from the baseline 2 s after the peptide
application. These observations indicate that the exogenous
RGD-containing peptide triggers the constriction by elevating smooth
muscle [Ca2+]i.

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Fig. 6.
Normalized time course of changes in AA lumen diameter when exposed to
1 mM of GRGDSP. RGD peptide was added to perfusion chamber at time
0. Dotted lines are SE. Solid circles ( ) indicate that the
constriction is significant (P < 0.05, n = 8).
Mean diameter in the baseline is 16.6 ± 2.1 µm (n = 8).
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Fig. 7.
Normalized time course of changes in AA smooth muscle
[Ca2+]i (A) and the corresponding
change in lumen diameter when the vessels were exposed to 1 mM GRGDSP
(B). RGD peptide was added to the perfusion chamber at
time 0. Dotted lines are SE. Solid circles ( ) indicate that
the constriction is significant (P < 0.05,
n = 6). Mean diameter in the baseline is 18.1 ± 0.5 µm
(n = 6).
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To test the effect of myogenic tone of AA on the GRGDSP induced
vasoconstriction, the responses of the luminal diameter to variations
in dose were determined at perfusion pressures of 80, 120, and 160 mmHg, respectively, in a separate study. Significant myogenic
constriction (P < 0.05, n = 5, Table
1) was found when perfusion pressure was
increased. However, there was no significant difference in the
constriction induced by GRGDSP when the myogenic tone of the vessels
was increased (Table 1). The myogenic constriction and GRGDSP-induced
constriction were additive. If integrins were involved in the myogenic
mechanism, then there should have been an effect of underlying vascular
tone on the response to exogenous RGD. No such interaction was
observed. These observations suggested that RGD-induced constriction
was not related to myogenic constriction, despite the fact that
exogenous RGD-containing peptides could induce vasoconstriction via an
elevation of smooth muscle [Ca2+]i.
 |
DISCUSSION |
There is only a limited amount of literature available regarding the
distribution of integrins in the afferent arterioles (1). To pursue the
hypothesis that integrins might be involved in regulating the tone in
AA, indirect immunofluorescence microscopy was employed to establish
their presence in the AA as well as their distribution along the
nephron. The
1-,
3-,
3-,
5-, and
V-integrins were found on the
basolateral plasma membrane of smooth muscle cell in AA. The apical
immunofluorescence signal from AA was most likely the result of
integrins on the endothelial cells rather than on smooth muscle cells,
because the immunofluorescence signal did not colocalize with the
smooth muscle
-actin and the endothelium of isolated AA was positive
labeled by integrin antibodies. These observations were also consistent
with the report of identification of RGD peptide binding sites on
endothelium of renal vasculature (23).
The
- and
-integrin subunits found in AA are similar to those
found in rat tail and mesenteric arteries (29), except that no
3 was found in the latter two vessels. Because the
availability of antibodies for immunolocalization was limited by
species specificity, it is possible that other integrin subunits, which
have not been tested, might also be present in the smooth muscle of AA.
Two of these untested integrins are
4 and
6, which have been reported in human resistance vessels
(6). The
1- and
3-integrins are the two
most widely expressed
-subunits through which the extracellular matrix is connected to cytoskeleton (5). Although not all
- and
-integrin subunits in the AA have been identified, the presence of
1 and
3 in AA is sufficient to provide
the morphological basis required to transmit a mechanical signal from
the extracellular matrix into the cytosol of smooth muscle, which could
be an integral part of myogenic response in AA.
The next hypothesis tested was whether exogenous RGD
sequence-containing peptides could alter the vessel's tone. If
integrins were involved in regulating the smooth muscle tone, then
binding of exogenous RGD-containing peptide to the exposed integrins in smooth muscle cells might alter the contractile state of AA. In the
presence of L-NAME to inhibit endothelial NO production,
exogenous GRGDSP peptides induced a dose-dependent constriction in
perfused AA. NO is known to be released continuously from endothelium
to modulate the tone of renal arterioles (20). The accumulation of NO
is particularly significant in perfused arterioles, because the
continuous release of NO is unopposed by the NO scavenging property of
hemoglobin (33). The requirement for NO synthase blockade to unmask
constriction of RGD peptide need not imply interactions between
integrins and NO. GRGDSP in the micromolar range induced the shortening
of freshly isolated renal VSMC in the absence of L-NAME
(unpublished observation). Since the control peptide, GRGESP, failed to
induce constriction at any dosage tested, the constriction was specific
for peptides with RGD motifs. These observations are consistent with
the hypothesis that binding of integrins on smooth muscle with
exogenous RGD peptides could alter the vascular tone.
The binding of exogenous RGD peptides to some heterodimers of integrins
(for example,
V
3) is known to be
sensitive to the steric conformation of the RGD motif (17, 18). The
possibility that the RGD-induced constriction was sensitive to the
steric constraint of the RGD motif was tested by constructing the
dose-response curve using a cyclic GRGDSP and comparing the
dose-response curve to that of its linear counterpart, GRGDSP. However,
there was no significant difference between the dose-response curves of these two peptides. These observations indicated that the
integrin-mediated constriction in AA was not sensitive to the steric
constraint of the RGD motif. Interestingly, Mogford et al. (17) showed that cyclic GRGDSP is more powerful than the linear GRGDSP in changing
the vascular tone of arterioles isolated from rat cremaster muscle. The
V
3-integrin was suggested to
be the binding site for the cyclic GRGDSP in this preparation (17).
The time course of change in lumen diameter showed that the
constriction induced by the RGD was rapid and reversible. The onset of
constriction was almost immediate and reached a maximum after 6 s. The
vessels started to recover toward the baseline after 35 s, but full
recovery was achieved only when the RGD peptides were removed from the
perfusion chamber. These finding indicated that either the signal
generated by the exogenous RGD motifs was transient in nature or that
the vessel was desensitized very rapidly to that signal. However,
repeated applications of RGD peptides induced similar vasoconstrictions
(data not shown), showing no sign of desensitization. The signal
generated by the binding of exogenous RGD to the integrins in AA was
probably diminished once the vessel was contracted. Contraction of the
vessel would reduce the surface area exposed to the exogenous RGD,
thereby reducing the ratio of integrins bound by exogenous RGD to the
extracellular matrix and thus the impact of exogenous RGD on the
vessel's tone.
[Ca2+]i-dependent activation of myosin light
chain kinase and its phosphorylation of the 20-kDa light chain of
myosin is generally considered the primary mechanism responsible for
regulation of contractile force in arterial smooth muscle (22). One
possible mechanism by which RGD peptide induces vasoconstriction is by elevating the [Ca2+]i of AA smooth muscle.
This hypothesis was tested by simultaneously monitoring the smooth
muscle [Ca2+]i and vessel diameter by using
confocal fluorescence microscopy coupled to digital image processing
technique. Collecting the fluorescence image of the vessel confocally
enabled us to measure the lumen diameter simultaneously with smooth
muscle [Ca2+]i without the complication of
out-focus fluorescence, e.g., fluorescence arising from the endothelium
(33). Smooth muscle [Ca2+]i was measured by
the emission ratio of Fluo-3/Fura Red. The emission ratio is
independent of the changes of local dye concentration that occur during
the constriction of the vessel (13, 33). Exposure of the perfused AA to
RGD peptides induced an immediate increase of smooth muscle
[Ca2+]i, which then decreased gradually after
18 s. The time course of change in smooth muscle
[Ca2+]i coincided for the most part with the
change of lumen diameter. The initially elevated smooth muscle
[Ca2+]i was restored toward the baseline
after 18 s, whereas the vessel diameter became not significantly
different from the baseline after 30 s. The relaxation in AA was
delayed compared to the smooth muscle [Ca2+]i
profile. This delay might reflect the latch phenomenon in stimulated smooth muscle or increased sensitivity of calcium-dependent
phosphorylation in smooth muscle (11, 22).
The mechanism of the RGD-induced increase of smooth muscle
[Ca2+]i is not addressed in this study. In
cremaster arteriole smooth muscle, RGD peptides inhibited the
Ca2+ current, whereas fibronectin-coated beads increased
the Ca2+ current (32). However, RGD peptides induced
dilatation in cremaster muscle arteriole (17) but constriction in AA.
The mechanisms of altering smooth muscle
[Ca2+]i might be different in these two
vascular beds. Elevation of [Ca2+]i in
integrin-mediated attachment between cultured cells and their substrate
was well documented (21, 25-27, 34). In Madin-Darby canine kidney
cells, binding with RGD peptide-coated beads increases the
[Ca2+]i (27). Integrin antibodies immobilized
on surface can also increase [Ca2+]i in
endothelial cells (24). The transient increase of
[Ca2+]i in smooth muscles cells induced by
RGD-containing peptides is consistent with these observations. There is
no consensus on how the occupancy and clustering of integrins could
elevate the [Ca2+]i in these cells.
Mobilization of intracellular calcium stores was suggested in rat
osteoclasts (34), whereas the voltage-independent calcium channel was
implicated in human endothelial cells (24).
Myogenic constriction of AA is associated with an sustained increase in
smooth muscle [Ca2+]i (33). GRGDSP-induced
constriction is also associated with increased
[Ca2+]i. If the signaling mechanisms of these
two constrictions have certain elements in common, then activation of
myogenic constriction might alter the response induced by GRGDSP. One
potential common element is the interactions of integrin with RGD
motifs from extracellular matrix and GRGDSP peptides. However, the dose
response of GRGDSP seemed to be insensitive to the myogenic tone of the
vessel. The effects of transmural pressure and RGD peptide were
additive, which suggested that the RGD-induced vasoconstriction was not related to myogenic constriction. An alternative explanation was that
the myogenic constriction and RGD-induced vasoconstriction were
mediated by two separate populations of integrins.
Although RGD peptides induce vasoconstriction by increasing the smooth
muscle [Ca2+]i in renal arterioles, the
effects seemed to be vascular bed specific. Fragments of collagen and
RGD peptides were shown to induce endothelium-independent dilation
rather than constriction in rat cremaster skeletal first-order
arterioles (17). The dilation was associated with a decrease of smooth
muscle [Ca2+]i and rhythmic calcium spikes
(7). The renal vasculature is well known for its uniqueness in its
response to vasoactive agents. It is not a surprise to find renal
vasculature responding to the same agent in an opposite direction
compared with other vascular beds. Another example is adenosine, which
causes constriction in renal arterioles but dilatation in other
vascular beds (31). The finding of RGD-induced vasoconstriction in AA
might have profound effects in the potential use of RGD peptides in
ameliorating the tubular obstruction and leukocyte infiltration in
acute renal failure, which counts on exogenous RGD peptides given
intravenously to occupy the exposed integrins on the luminal surface of
renal epithelial cells (10, 19). The RGD-induced constriction in AA
might cause hypoperfusion and reduction of glomerular filtration rate
while attempting to rescue tubular function. Whether RGD-induced constriction will increase the AA resistance in vivo will depend on the
rate of NO released from the renal endothelium. Interestingly RGD
binding sites were also demonstrated in renal endothelium during
ischemic acute renal failure, which suggested abnormality of integrin
receptors in vascular endothelial cells in acute renal failure (23).
In summary, in this study we used indirect immunofluorescence
microscopy to demonstrate the presence of a variety of integrin subunits in the AA of rat kidney. The exogenous RGD-containing peptide
induced a dose-dependent constriction in the isolated AA. This induced
constriction was specific to the RGD motif, and the constriction was
associated with an increase in the smooth muscle
[Ca2+]i.
 |
ACKNOWLEDGEMENTS |
The technical assistance of D. Friedel is appreciated.
 |
FOOTNOTES |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-15968 and by the National Kidney
Foundation.
Address for reprint requests: K.-P. Yip, Dept. of Physiology, Box G-B
397, Brown Univ., Providence, RI 02912.
Received 11 March 1997; accepted in final form 25 June 1997.
 |
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