* Microcirculation Research Institute and Departments of Medical Physiology, Pathology and Laboratory Medicine,
Texas A & M University Health Science Center, College Station, Texas 77843-1114
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Abstract |
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Vasoactive effects of soluble matrix proteins
and integrin-binding peptides on arterioles are mediated by v
3 and
5
1 integrins. To examine the underlying mechanisms, we measured L-type Ca2+ channel
current in arteriolar smooth muscle cells in response to
integrin ligands. Whole-cell, inward Ba2+ currents were
inhibited after application of soluble cyclic RGD peptide, vitronectin (VN), fibronectin (FN), either of two
anti-
3 integrin antibodies, or monovalent
3 antibody.
With VN or
3 antibody coated onto microbeads and
presented as an insoluble ligand, current was also inhibited. In contrast, beads coated with FN or
5 antibody
produced significant enhancement of current after bead
attachment. Soluble
5 antibody had no effect on current but blocked the increase in current evoked by FN-coated beads and enhanced current when applied in
combination with an appropriate IgG. The data suggest
that
v
3 and
5
1 integrins are differentially linked
through intracellular signaling pathways to the L-type Ca2+ channel and thereby alter control of Ca2+ influx in
vascular smooth muscle. This would account for the vasoactive effects of integrin ligands on arterioles and
provide a potential mechanism for wound recognition
during tissue injury.
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Introduction |
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INTEGRINS are heterodimeric receptors (,
) that mediate cell-extracellular matrix (ECM)1 and cell-cell
adhesion events. The cytoskeleton is mechanically linked to the ECM by integrins so that cytoskeletal stiffening increases in direct proportion to applied stress (Wang
et al., 1993
). Integrins can therefore serve as mechanochemical transducers (Ingber, 1991
). Integrins can also
function as signaling receptors that transduce biochemical
signals both into and out of cells (Clark and Brugge, 1995
;
Sjaastad and Nelson, 1997
). Intracellular signals known to
be linked to integrins include pH, Ca2+, protein kinase C
activation, and protein tyrosine phosphorylation (Schwartz
et al., 1991a
; Schwartz, 1993
).
Integrin signaling pathways are generally believed to be
initiated by integrin clustering through interactions with
insoluble ECM ligands (Clark and Brugge, 1995). These
signals are initiated by cell interactions with ECM-coated
substrates or with beads coated with ECM proteins or antiintegrin antibodies (Miyamoto et al., 1995b
; Plopper et
al., 1995
). When soluble ECM protein or antibody is
added, minimal or no signaling is thought to occur, but if
soluble antibody is followed by a cross-linking antibody, signaling pathways are activated (Yamada and Geiger,
1997
). A widely studied recognition site on ECM proteins,
including vitronectin (VN) and fibronectin (FN) (Schwarzbauer, 1991
), is the tripeptide Arg-Gly-Asp (RGD), which
is recognized by a common subset of integrins, including
v
3,
5
1,
v
5, and
IIb
3. RGD peptides are known to
disrupt integrin-dependent cell adhesive events (Akiyama,
1996
) as well as produce inhibitory effects on major cellular processes such as platelet aggregation and angiogenesis
(Weiss et al., 1997
). For this reason, RGD peptides are potential therapeutic agents for thrombotic diseases and cancer. One important, unresolved issue is whether RGD
peptides act solely by disrupting cell-ECM contacts or
whether they provide direct signals to cells by binding to
unoccupied integrins. Recent data from our laboratories
suggest that soluble RGD peptides may provide vasoactive signals to cells in the vascular wall (Mogford et al.,
1996
, 1997
; D'Angelo et al., 1997
). Thus, RGD peptides
may be capable of directly stimulating integrin-dependent
intracellular signaling pathways.
In rat cremaster muscle arterioles, integrin-binding RGD
peptides and fragments of denatured collagen type I cause
dilation through an interaction with the v
3 integrin on
vascular smooth muscle (Mogford et al., 1996
). Dilation is
associated with a decrease in intracellular Ca2+ concentration ([Ca2+]i) (D'Angelo et al., 1997
) and can be prevented
by a function-blocking antibody specific for the
3 integrin
(Mogford et al., 1996
). In addition to these prolonged effects, RGD peptides also cause a transient, endothelium-independent constriction of arterioles, mediated by the
5
1 integrin (Mogford et al., 1997
). In rat afferent arterioles, RGD peptide causes a sustained constriction that is associated with an increase in smooth muscle cell [Ca2+]i
(Yip and Marsh, 1997
). The signaling mechanisms downstream from integrin-ligand binding are poorly understood, particularly in vascular smooth muscle cells (SMCs).
We hypothesized that the L-type, voltage-gated calcium
channel was involved in the vasoactive responses of arterioles since this channel is known to be a major pathway for
calcium entry into vascular SMCs. To test this hypothesis, we isolated single SMCs from rat cremaster arterioles and
selectively measured whole-cell calcium current before
and after application of integrin ligands in both soluble
and insoluble form.
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Materials and Methods |
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Cell Isolation Techniques
Male Sprague-Dawley rats (120-200 g) were anesthetized with intraperitoneal injection of pentobarbital sodium (120 mg/kg). All animal handling procedures followed institutional guidelines. The two cremaster muscles were excised and pinned flat for vessel dissection in a 4°C silastic-coated Plexiglas chamber containing Ca2+-free saline solution. The composition was (in mM) 147 NaCl, 8.6 KCl, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 D-glucose, 2.0 pyruvate, 0.02 EDTA, and 3 MOPS (pH adjusted to 7.4 with NaOH), with BSA (0.1 mg/ml; Amersham Life Science, Arlington Heights, IL) added to maintain cell integrity. Dissected segments of first- and second-order arterioles were transferred to a tube of low-Ca2+ saline solution containing (in mM) 144 NaCl, 5.6 KCl, 0.1 CaCl2, 1.0 MgCl2, 0.42 Na2HPO4, 0.44 NaH2PO4, 10 Hepes, 4.17 NaHCO3, and 1 mg/ml BSA (pH adjusted to 7.4 with NaOH) at room temperature for 10 min. After allowing the vessels to settle to the bottom of the tube, the solution was decanted and replaced with low-Ca2+ saline containing 26 U/ml papain (Sigma Chemical Co., St. Louis, MO) and 1 mg/ml dithioerythritol (Sigma Chemical Co.). The vessels were incubated for 30 min at 37°C with occasional agitation, after which vessel fragments were transferred to low-Ca2+ saline solution containing 1.95 collagenase (FALGPA U/ml; Sigma Chemical Co.), 1 mg/ml soybean trypsin inhibitor (Sigma Chemical Co.), and 75 U/ml elastase (Calbiochem, La Jolla, CA) for 15 min at 37°C. After further digestion, the remaining fragments were rinsed two times with low-Ca2+ saline solution and gently triturated using a fire-polished Pasteur pipette to release single cells.
Patch Clamp Techniques
Perforated, whole-cell recordings were made as described previously (Rae
and Fernandez, 1991). Micropipettes were pulled from 1.5-mm glass tubing (Corning No. 8161; Warner Instruments, Hamden, CT) on a programmable puller and fire polished. Pipette resistances ranged from 1 to 3 M
.
The pipettes were dipped for 2-3 s in Cs+ pipette solution (high Cs+) containing (in mM) 110 CsCl, 20 TEA chloride, 10 EGTA, 2 MgCl2, 10 Hepes, and 1 CaCl2 (pH adjusted to 7.2 with CsOH) and then backfilled
with the same solution containing 240 µg/ml amphotericin B. An EPC-7
amplifier (HEKA, Darmstadt-Eberstadt, Germany) was used to record
current, and hydraulic manipulators (model M0-102; Narishige, Tokyo,
Japan) were used for fine control of the micropipettes. Analog to digital
conversions were made using a TL-1 DMA interface (Axon Instruments,
Foster City, CA) and stored on a Pentium computer for subsequent analysis. Data were sampled at 5-10 kHz and filtered at 1-2 kHz using an eight-pole Bessel filter. Series resistance varied from 2 to 6 M
. Current records
were analyzed using pClamp (version 6.0.3; Axon Instruments). Currents
through the L-type calcium channel were elicited by voltage ramps (from
100 mV to +80 mV, duration = 200 ms) or by voltage steps (from
80
to +60 mV in 10 mV increments, duration = 300 ms). All experiments
were performed at 22°C.
A suspension of freshly dispersed cells was plated onto a thin glass coverslip in a recording chamber on the stage of an inverted microscope. The
coverslip was not usually treated, but in some experiments it was coated
with FN (120 kD, 20 µg/ml) before addition of cells (Schwartz, 1993). Current recordings were made from individual cells between 30 min and 3 h
after plating. Cells harvested using the digestion procedure were elongated with tapered ends in physiological saline solution (PSS), refractile
under interference contrast optics, and contractile in solutions containing
140 mM K+ or 20 mM Ba2+. At the beginning of each experiment, the recording chamber was suffused with PSS from a gravity-fed reservoir at a
rate of 1.5 ml/min. PSS had the following composition (in mM): 136 NaCl,
5.9 KCl, 10 Hepes, 1.16 NaH2PO4, 1.2 MgCl2, 1.8 CaCl2, 18 D-glucose, 0.02 EGTA, and 2 pyruvate (pH adjusted to 7.4 with NaOH). To record whole-cell current through the calcium channel, Ba2+ (20 mM) was used
as the charge carrier in place of K+ and Na+ in the bath solution. This procedure is known to increase the size of the inward currents elicited by depolarization, and to minimize calcium-dependent inactivation of these
currents (Griffith et al., 1994
). The Ba2+ bath solution (20 Ba2+) contained
(in mM) 20 BaCl2, 124 choline chloride, 10 Hepes, and 15 D-glucose (pH
adjusted to 7.4 with TEA-OH).
Both ramp and step voltage protocols elicited inward, whole-cell Ba2+
currents (IBa) that peaked at +30 mV (range = 3.0-10.4 pA/pF); typically,
these currents were stable for more than 30 min. Since current-voltage
(I-V) relations for the ramp and step protocols were nearly identical, the
average of five voltage ramps was used to measure IBa in most experiments. The activation portion of the I-V curve (from 60 to +30 mV) increased smoothly to a single maximum with no secondary "hump" in its
voltage dependence, which is the pattern consistent with activation of only
a single type Ca2+ channel (L-type) in this tissue (Nelson et al., 1990
; Cox
et al., 1992
). As noted previously (Hill et al., 1996
), the entire I-V relation
was shifted about 30 mV to the right in 20 mM Ba2+ solution. This behavior is typical for voltage-gated calcium channels because of the fact that
the equilibrium potential for the permeable ion shifts to the right with increasing extracellular ion concentration. When physiological Ca2+ is used
as the charge carrier, the peak of the I-V curve occurs between
10 mV
and 0 mV, and the activation threshold occurs at approximately
50 mV,
as demonstrated in other SMC preparations (Aaronson et al., 1988
).
Ligand Application
VN, FN (120 kD), lyophilized cyclic GPenGRGDSPCA (cRGD, with Pen
indicating penicillamine), and the control GRGESP peptide (RGE) were
obtained from GIBCO-BRL (Gaithersburg, MD). The anti-3 integrin
function-blocking antibodies (F11; anti-rat monoclonal), 2C9.G2 (monoclonal), and the anti-
5 integrin function-blocking antibody (HM
5-1;
anti-rat monoclonal raised in Armenian hamster) were obtained from
PharMingen (San Diego, CA). Anti-rat MHC class I monoclonal antibody (MHC; clone R4-8B1) was obtained from Seikagaku Inc. (Tokyo,
Japan). Anti-Armenian hamster monoclonal IgG was obtained from
Sigma Chemical Co. Monovalent antibodies were made by digesting F11
(in stock solution) with papain, followed by subsequent extraction of Fc
fragments using a column of anti-mouse Fc coupled to Sephadex. The resulting Fab digest displayed a prominent band at 50 kD with no evidence
of intact F11 at 150 kD.
For application to single cells, each agent was added to 20 Ba2+ solution and ejected from a picospritzer pipette (General Valve Corp., Fairfield, NJ) positioned ~50 µm away from a cell (Fig. 1 A).
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Application of Protein-coated Beads
Streptavidin-coated microspheres (3.2 µm in diameter) were obtained
from Bangs Laboratories (Fishers, IN). Before each experiment, the
beads were coated with protein using a biotinylation procedure. Biotinylated FN, F11, VN, HM5-1, and MHC were prepared using a method
similar to that described previously (Hnatowich et al., 1987
; Larson et al.,
1992
). The molar ratio of NHS-LC-Biotin (Pierce Chemical Co., Rockford, IL) to protein (10 µg/ml) was 20:1. To remove unreacted biotin, ultrafree-MC filters were used (Millipore Corp., Bedford, MA). Nonspecific
sites on the beads were blocked by incubation with 0.1% heat-denatured
BSA in PSS. A dilute suspension of beads in Ba2+ bath solution was then
used to backfill micropipettes for application to single cells. These pipettes
were positioned 5-10 µm away from the cells and fashioned so that their
tip diameters were approximately twice the diameter of the beads; gentle
pressure from a glass syringe (<2 cm H2O) was used to eject the beads
(Fig. 1 B).
Data Analysis
Whole-cell recordings were made from cells with capacitances varying from 4 to 16 pF. We used data only from cells in which stable gigaseals were maintained. In most analyses, the raw current value was normalized to cell capacitance (an index of cell size) and expressed as current density (pA/pF). Statistical comparisons were performed with repeated-measures analysis of variance followed by post hoc tests, or with an independent two-tail t test, as appropriate. Averaged values are expressed as mean ± SEM. Values of P < 0.05 were considered to be statistically significant.
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Results |
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Effect of cRGD on IBa
The effect of soluble cRGD peptide (100 µM for 1 min) on
inward Ba2+ current is shown in Fig. 2. This dose of peptide was reported to produce near-maximal dilation of isolated cremaster arterioles (Mogford et al., 1996). Currents
from single arteriolar myocytes were elicited every 15 s by
a depolarizing pulse to +30 mV (300-ms duration) from a
holding potential of
80 mV. The time course of the response from a representative cell is shown on the left side
of Fig. 2 A, and individual current traces at the indicated time points are shown on the right side. Before peptide application, peak current ranged from
76 pA to
77 pA.
Within 15 s after application of soluble cRGD peptide
(100 µM) from a picospritzer pipette, current was inhibited (to
60 pA) and maximal inhibition (to
51 pA) was
achieved 45 s after cRGD application. Nearly complete recovery from inhibition (to
75 pA) was observed within
30 s after peptide washout.
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The average response of nine cells to soluble RGD peptide is summarized in Fig. 2 B, where the data for each cell have been normalized to the peak Ba2+ current recorded just before peptide application. On average, 100 µM cRGD produced 22% inhibition of IBa at +30 mV (measurements taken immediately before peptide washout). Also illustrated in the bar graph are the effects of vehicle, RGE peptide (which does not interact with integrin receptors), and nifedipine, a dihydropyridine calcium channel blocker. Neither vehicle nor RGE peptide (80 µM; n = 4) had a significant effect on IBa. Nifedipine (1 mM; n = 7) produced nearly 100% inhibition of current at this dose, which is consistent with the behavior of an L-type Ca2+ channel. A comparison of current-voltage relationships recorded before and during cRGD application (Fig. 2 C) indicates that inhibition of IBa occurred across the entire range of voltages associated with activation of the L-type Ca2+ channel. Thus, there appeared to be no significant effect of RGD peptide on the threshold or reversal potential of the current.
Effect of Vitronectin on IBa
VN is known to interact with several integrins, including
v
3 (the VN receptor). Fig. 3 A illustrates the effect of
soluble VN on IBa. Before application, peak current in this
representative cell was stable between
86 and
87 pA.
Within 15 s after ejection of soluble VN (0.04 µM) from
the picospritzer pipette, IBa decreased to
69 pA, with a
further inhibition to
49 pA at 60 s after application. Recovery of current was complete within 60 s after VN washout. The bar graph in Fig. 3 A summarizes results from
seven cells. On average, this concentration of soluble VN
inhibited current by 39 ± 5%. Although not illustrated in
this figure, inhibition of IBa by VN was sustained during
longer periods of application (48 ± 7% inhibition at 4 min).
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Fig. 3 B shows the effect of VN-coated beads on IBa. The top trace shows the time course of changes in current before (time = 0 min) and after attachment of four beads to a representative cell. Note that both peak and steady-state currents were inhibited within 1 min of bead attachment, remained inhibited for ~5 min, and then gradually returned toward control levels even though the beads appeared to remain attached. Data from six cells are summarized in the lower portion of Fig. 3 B. On average, a 20% inhibition of IBa was observed in response to bead attachment. As a control for nonspecific mechanical effects associated with bead application, the response to uncoated beads was also tested (open circles); no significant changes in IBa were noted with uncoated beads (n = 5) or with BSA-coated beads (n = 4).
Inhibition of IBa after attachment of VN-coated beads
was proportional to the number of beads that attached to a
given cell, a process over which we had only partial control. Regression analysis of the percent inhibition of IBa as
a function of the number of attached beads gave a correlation coefficient of 0.86 (IBa =
0.8 pA
5.2 × No. of
beads). For the purpose of determining the average responses of cells to coated beads in this and subsequent protocols, data were therefore pooled from cells to which
between two and five beads attached.
Effect of 3 Antibody on IBa
To test the hypothesis that the effects of cRGD and VN
were mediated through the v
3 receptor, a function-blocking, monoclonal antibody to the rat
3 integrin (F11)
was applied to the cells. F11 is known to block the dilatory
effects of cRGD peptide on isolated arterioles (Mogford
et al., 1996
).
3 integrins are known to associate with two
different
subunits (Hemler, 1990
), but only one of those,
v, has been identified in vascular smooth muscle (Yip and
Marsh, 1997
). Fig. 4 A shows the time course of changes in
IBa after application of soluble F11 (0.03 µM) to a representative cell. In this cell, soluble F11 inhibited current from
70 pA to
45 pA by 1 min after application. Data
from nine cells are summarized in the bar graph of Fig. 4 A
and show that this dose of soluble F11 inhibited IBa by an
average of 33 ± 5%. We also tested the effect of a second
3 integrin antibody, 2C9.G2, which is reported to block
adhesion (Schultz and Armant, 1995
). After 60 s of application, soluble 2C9.G2 (0.03 µM) inhibited IBa by 22 ± 4.5% (n = 8). In addition, we made Fab fragments of F11
to test the effect of a monovalent integrin ligand on Ca2+
current. After dilution to 0.03 µM in PSS, Fab fragments
caused a 29 ± 5% inhibition of IBa 1 min after application
(n = 7). As a control for nonspecific effects of antibody, a
nonintegrin binding antibody (anti-rat MHC, 0.2 µM) was
also tested; MHC had no significant effect on current (n = 4), as shown in the right portion of Fig. 4 A.
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When F11-coated beads were applied to cells, IBa was inhibited (Fig. 4 B, closed circles). IBa was reduced to 61% of control at 1.5 min after F11 bead attachment. The inhibition lasted ~5 min, after which current gradually and spontaneously returned toward control values, even though the beads remained attached. As a control for nonspecific effects of antibody-coated beads, we tested the responses of cells to MHC-coated beads, which had no significant effect on IBa (Fig. 4 B, open circles).
Effect of Fibronectin on IBa
Next, we examined the effect of FN on current. FN is
known to interact with both v
3 and
5
1 receptors
present in this cell type, as well as with a number of other
integrins (Hynes, 1992
). Fig. 5 A shows the time course of
changes in IBa in response to soluble FN (0.1 µM). In this
cell, soluble FN inhibited IBa from
80 pA to
56 pA
within 60 s after application. The response of seven cells to
soluble FN is summarized by the bar graph in Fig. 5 A. On
average, this concentration of soluble FN reduced IBa to
75% of control at 1 min. The inhibition was maintained for at least 10 min, when current was still reduced to 80 ± 5%
(n = 5; data not shown).
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To test the effect of insoluble FN on IBa, FN-coated beads were applied to single cells. The top trace in Fig. 5 B shows the response of a representative cell to attachment of three FN-coated beads. Interestingly, FN-coated beads had the opposite effect on current compared with VN-coated beads or F11-coated beads. Attachment of FN-coated beads led to an enhancement of IBa as early as 1 min after bead attachment. This enhancement peaked at 2 min (~135% of control), remained stable for 10 min, and then declined gradually by 16 min, even though the beads remained attached. For reference, the time course of changes in IBa in response to BSA-coated beads (which had no significant effect on current) is shown.
Effect of 5 Antibody on IBa
The fact that insoluble VN and insoluble FN had opposite
effects on current suggests that an integrin other than v
3
might mediate the enhancement of IBa in response to FN-coated beads. Experiments by Mogford et al. (1997)
also
suggest a role for the
5
1 receptor in vasoactive responses
of arterioles because RGD peptide-mediated dilation was
converted to constriction after blockade of
3 integrins:
the steady-state portion of that constriction was mediated
by endothelin and blocked by
5 antibody, but the initial
transient constriction was an endothelium-independent response.
To test for the involvement of the 5
1 integrin in our
preparation, we used the anti-rat
5 antibody, HM
5-1.
The
5 subunit is known to associate only with
1, making
this antibody specific for the
5
1 heterodimer (Hynes,
1992
). Fig. 6 A shows the effect of applying soluble HM
5-1
to a representative cell: no significant change in IBa was
observed. The bar graph in Fig. 6 A summarizes the response of nine cells to application of soluble HM
5-1,
which on average produced less than a 2% change in IBa.
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However, when beads coated with HM5-1 were applied to cells, a large and significant increase in IBa was
consistently observed, as summarized in Fig. 6 B (left).
Within the first minute after attachment of
5-coated
beads, IBa had increased to 158% of control. IBa peaked at
170% of control ~3 min after bead application and then
progressively declined toward control; however, IBa did
not completely recover even by 17 min after
5-coated
bead attachment. Individual current recordings before and
after HM
5-1 application are shown in Fig. 6 B (right).
The two sets of tracings represent currents evoked from a
holding potential of
80 mV (top) or
40 mV (bottom)
before and after attachment of HM
5-1-coated beads. As
is evident from these recordings, the current stimulated by HM
5-1 was completely inhibited by nifedipine (1 µM),
which is consistent with the conclusion that it flowed
through L-type calcium channels. Although there is no selective blocker of T-type calcium channels, the possibility
that some current might be contributed by T-type channels is ruled out by the fact that the time course of the current recordings evoked from the two different holding potentials are virtually identical.
Fig. 6 C compares the current-voltage relationships for
control current and current stimulated by bound HM5-1.
There appeared to be no significant effect on either the
threshold or reversal potential of the current.
Effect of Antibody Pretreatment on IBa Response to Coated Beads
To test the idea that clustering of receptors was required
to initiate signaling through the 5
1 integrin, soluble
5
antibody was first applied to cells, and then anti-hamster
IgG was subsequently added. As shown in Fig. 7 A, there
was no response to either agent alone, but when both
agents were applied in combination, a significant enhancement in current was noted. The time course of this enhancement was approximately the same as that seen in response to insoluble
5 antibody (compare to Fig. 6 B).
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If the response of arteriolar smooth muscle cells to FN-coated beads involves interaction of insoluble FN with
both v
3 and
5
1 integrins, we predicted that pretreatment with antibody specific to one integrin would result in
changes in current characteristic of selective activation of
the other integrin. To test this hypothesis, cells were
treated with either F11 to block
3 or HM
5-1 to block
5
before application of FN-coated beads. Fig. 7, B-D, shows
the results of these experiments.
In Fig. 7 B, application of soluble F11 caused a 30% inhibition of IBa, an effect which is comparable to that observed previously (compare to Fig. 4 A). From this new
baseline, application of FN-coated beads increased current
from 70% of control to 116% of control. Although interpretation of this response is complicated by the shift in
baseline, the absolute change in IBa appeared to be larger
than that observed in response to FN-coated beads alone
(average increase = 35%; Fig. 5 B) and nearly as large as that produced by insoluble HM5-1 (Fig. 6 B).
In Fig. 7 C, application of soluble HM5-1 again produced no change in IBa, but subsequent application of FN-coated beads caused a significant inhibition of current,
which is the opposite response observed to FN-coated
beads alone (Fig. 5). This observation is consistent with
the hypothesis that insoluble FN activates both
3 and
5
integrins in these cells. Importantly, it also indicates that
soluble HM
5-1 was indeed interacting with the
5
1 receptor even though no change in IBa was noted in response to soluble HM
5-1 alone. Interestingly, simultaneous application of F11 and HM
5-1 resulted in a 34 ± 8% decrease in current (n = 5), and little change in current was
noted after subsequent application of FN-coated beads
(Fig. 7 D).
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Discussion |
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To investigate the mechanisms underlying the vasoactive
effects of ECM proteins and integrin-specific peptides on
rat skeletal muscle arterioles (Mogford et al., 1996, 1997
),
we measured the response of L-type Ca2+ channel current
in arteriolar myocytes to integrin ligands. Soluble
v
3
ligands (cRGD, VN, FN, bivalent or monovalent
3 antibodies) caused significant inhibition of calcium current, as
did beads coated with VN or
3 antibody. In contrast,
beads coated with
5
1 ligands (FN or
5 antibody) caused
significant enhancement of current. Soluble
5 antibody
alone had no effect on current but blocked the increase in
current evoked by FN-coated beads and enhanced current
when applied in combination with an appropriate IgG.
This is the first electrophysiological evidence for regulation of a Ca2+ channel by integrin-ligand interactions and
demonstrates that
v
3 and
5
1 integrins in smooth muscle are differentially linked through intracellular signaling
pathways to the L-type calcium channel.
The implications of our findings are threefold: (a) part
of the resting current through L-type Ca2+ channels in vascular smooth muscle, and therefore blood vessel tone, is
dependent on integrin-matrix interactions; (b) bidirectional regulation of Ca2+ influx in this cell type can be
achieved through preferential ligation of v
3 or
5
1 integrins; (c) soluble integrin ligands can initiate signaling
through the
v
3 receptor. Since ECM protein denaturation and fragmentation can provide soluble integrin-specific signals to cells (Davis, 1992
), this mechanism is likely
to be important in the microvascular response to injury
(Mogford et al., 1996
). Inhibition of smooth muscle cell
Ca2+ current could account for integrin-mediated vasodilation of arterioles (Mogford et al., 1996
).
Integrin Signaling in Response to Tissue Injury
Local vasodilation is one of the initial responses to tissue
injury, resulting in an increase in blood flow to the affected area. This response is mediated primarily by arterioles,
which are the strategic control point for local regulation of
pressure and flow in every tissue. Increased flow contributes to injury repair by enhancing delivery of inflammatory cells to the injured site. Classic mediators of injury-induced arteriolar dilation include reactive oxygen species
(Wei et al., 1981), tachykinins, and histamine (Treede et
al., 1990
). Recently, Mogford et al. (1996)
described an additional mechanism by which RGD-containing peptides
induce vasodilation by interacting with the
v
3 integrin on smooth muscle cells of rat skeletal muscle arterioles.
Involvement of the
v
3 integrin was implicated by the
findings that (a) cRGD and GRGDSP peptide were more
potent vasodilators than GRGDNP peptide (enhancement of RGD potency by cyclization implicates the involvement of
v integrins [Pierschbacher and Ruoslahti,
1987
]) and (b) dilations were attenuated in the presence of
a function-blocking
3 monoclonal antibody (Mogford et
al., 1996
). In addition to synthetic peptides, fragments of
denatured collagen type I were potent vasodilators of arterioles (Mogford et al., 1996
). While RGD sequences are not exposed in native collagen, cryptic RGD sites become
exposed after collagen denaturation and proteolysis, allowing for their interaction with RGD-binding integrins.
Exposure of cryptic RGD sites has been proposed to be a
potential wound recognition signal during tissue injury
(Davis, 1992
). Thus, a certain proportion of the
v
3 receptors may normally be unoccupied on vascular smooth muscle, and after tissue injury, generation of RGD peptide
signals that bind the receptor result in decreased Ca2+ current, arteriolar dilation, and increased blood flow to the injured tissue.
Arteriolar dilations to RGD-containing peptides and
proteins are mediated by direct effects on vascular smooth
muscle integrins rather than on endothelial cell integrins
(Mogford et al., 1996). However, none of the downstream
signaling mechanisms in smooth muscle have been identified, except that dilation to soluble cyclo-RGD peptide is
preceded by a significant decrease in smooth muscle [Ca2+]i (D'Angelo et al., 1997
). Our finding that cRGD
caused an inhibition of current through the L-type Ca2+
channel in the same cell type (Fig. 2) is consistent with
data from intact vessels. In isolated arterioles (Mogford et
al., 1996
; D'Angelo et al., 1997
), dilation was the result of
inhibition of myogenic tone, which in resistance vessels
(Nelson et al., 1990
; Hill and Meininger, 1994
) is dependent on basal influx of Ca2+ through L-type Ca2+ channels
and can be antagonized by dihydropyridines (Hill and Meininger, 1994
). We confirmed that the dihydropyridine
nifedipine completely blocked current in our cells (Figs. 2
B and 6 B). Mogford et al. (1996)
found that cRGD peptide also inhibited phenylephrine- and KCl-induced vascular tone, but the primary actions of both agents are known
to be mediated by Ca2+ influx through voltage-gated Ca2+
channels as well (Nelson et al., 1988
).
Integrin-mediated [Ca2+]i Signaling
Integrin-mediated [Ca2+]i signaling has been demonstrated in a number of cell types, including endothelium
(Schwartz and Denninghoff, 1994) and vascular smooth
muscle (McNamee et al., 1993
). Integrins including
IIb
3,
v
6,
v
5,
5
1, and
v
3 (Hynes, 1992
) are known to be
involved in [Ca2+]i signaling responses; these integrins also
recognize the RGD sequence common to many ECM (FN,
osteopontin, and collagens) and plasma proteins (FN, VN,
and fibrinogen). Thus, our finding that Ca2+ channel current (and by direct extension Ca2+ influx [Ganitkevich and
Isenberg, 1991
]) is modulated after
v
3 and
5
1 receptor
ligation is consistent with previous reports in the literature.
Changes in [Ca2+]i initiated by integrin ligation involve a
number of mechanisms that result in Ca2+ release from intracellular stores and/or Ca2+ influx (McNamee et al.,
1993; Somogyi et al., 1994
; Sjaastad et al., 1996
). In endothelial cells,
v integrins mediate a rise in [Ca2+]i after adhesion to FN (Schwartz and Denninghoff, 1994
). The mechanism underlying this response was not determined,
but [Ca2+]i increases did not occur in the absence of extracellular Ca2+. Likewise, both Ca2+ release and Ca2+ influx
contributed to the [Ca2+]i rise after adhesion of MDCK
cells to RGD-coated beads (Sjaastad et al., 1996
), but the
influx component was more important for feedback regulation of integrin-mediated adhesion. No mechanism for
integrin-mediated Ca2+ influx in nonexcitable cells has
been identified, although a role for a 50-kD integrin-associated protein, not yet characterized electrophysiologically, has been postulated (Brown, 1993
; Schwartz et al.,
1993
).
Our data represent the first electrophysiological evidence that integrin ligation can modulate a plasma membrane Ca2+ channel. To make these measurements, Ba2+
was used instead of Ca2+ to carry current through the
L-type Ca2+ channel because (a) Ba2+ is more permeable
than Ca2+ through this channel, resulting in larger current;
(b) Ba2+ blocks the large, outward K+ current that normally masks Ca2+ current in these cells; and (c) Ba2+ currents do not exhibit the rapid inactivation observed when Ca2+ is used (Griffith et al., 1994). Nifedipine, a dihydropyridine that is a selective antagonist of L-type calcium
channels (as opposed to other types of voltage-gated calcium channels [Birnbaumer et al., 1994
]) at concentrations
less than 10
5 M, produced essentially a complete block
of basal Ca2+ current (Fig. 2 B) as well as inhibited the
enhanced current in response to insoluble
5-antibody
(Fig. 6 B).
Although we have not directly measured [Ca2+]i in our
preparation, it is highly likely that any treatment causing a
significant change in IBa would lead to a similar directional
change in [Ca2+]i; this relationship has been clearly demonstrated for visceral (Ganitkevich and Isenberg, 1991)
and vascular (Fleischmann et al., 1994
) smooth muscle.
The previously reported decrease in arteriolar smooth
muscle [Ca2+]i in response to soluble RGD peptide (D'Angelo et al., 1997
) is consistent with inhibition of IBa by soluble RGD peptide (Fig. 2). In another vascular bed, RGD
peptide caused a constriction that was associated with an
increase in SMC [Ca2+]i (Yip and Marsh, 1997
).
The direct effect of cRGD, VN, FN, and F11 on Ca2+
channel current in isolated SMCs provides strong support
for the concept that interaction of v
3 with soluble
ligands transduces an intracellular signal in this cell type. It
remains to be determined if
v
3 expressed in other cell
types, such as endothelium, delivers a similar or different
signal. However, endothelial cells (with one exception
[Bossu et al., 1989
]) lack voltage-gated calcium channels
and, in some ways, use opposite mechanisms of controlling calcium entry than smooth muscle. Therefore, it is not surprising that ligation of
3 integrins might lead to increases
in endothelial cell [Ca2+]i (Schwartz and Denninghoff,
1994
) but opposite changes in SMC [Ca2+]i.
Effects of Soluble and Insoluble Integrin Ligands on Ba2+ Current
A number of possible explanations may account for the
differences between the effects of soluble and insoluble integrin ligands on Ca2+ channel current. An obvious possibility is that inhibition of current by soluble FN may be
mediated by competitive antagonism of existing integrin-
matrix interactions, as suggested for other systems (Poole
and Watson, 1995). This would require constitutive phosphorylation of the channel through an integrin-dependent
pathway. Indeed, the L-type calcium channel in vascular
smooth muscle has been shown to require tyrosine phosphorylation for normal function (Wijetunge et al., 1992
;
Wijetunge and Hughes, 1996
), but whether integrins regulate this pathway is not known. If they do, then disruption
of existing integrin-matrix interactions by soluble ligands
would produce inhibition of current while clustering of receptors by insoluble ligands (Altieri et al., 1990
; Schwartz, 1993
), including antibodies (Miyamoto et al., 1995a
),
would produce enhancement of current. In our system,
soluble ligands of the
v
3 receptor did produce inhibition
of current; however, insoluble
3 ligands (VN and F11)
also produced inhibition of current (Figs. 3 and 4). Thus, it
seems likely that these effects resulted from activation of a
signaling pathway rather than competition for existing
3-
matrix interactions. Likewise, since insoluble
5 caused enhancement of current, the competition hypothesis would predict that soluble
5 should reduce current, which it
did not.
A more tenable explanation for our results is the possibility that v
3 and
5
1 integrins provide distinct and opposing signals to regulate calcium current. As illustrated in
Fig. 8 A, we propose that selective ligands of the
v
3 receptor (F11, 2C9.G2, VN) cause inhibition of current, selective ligands of the
5
1 receptor (HM
5-1) cause enhancement of current, and ligands for both receptors (FN)
cause an intermediate response. Our hypothesis requires
that several conditions be met: (a)
1 and
3 integrins must
signal through different mechanisms in smooth muscle
cells. This is supported by the different responses of current to selective ligands of the two respective integrins
(Fig. 4 B vs. Fig. 6 B). In endothelial cells as well (Leavesley et al., 1993
),
1 and
3 integrins play different roles in
regulating Ca2+ entry (Leavesley et al., 1993
). Our hypothesis also requires that (b) the
5
1 integrin can only be activated by insoluble ligands. This is consistent with the observation that soluble
5 antibody had no effect on current
(Fig. 6 A), yet was an effective blocker of the response to
insoluble FN (Fig. 7 C). Experiments by other groups have
also shown that soluble
5 antibody failed to increase pHi
unless it was cross-linked with a secondary antibody to induce integrin clustering (Schwartz et al., 1991b
). Our hypothesis requires that (c) the
v
3 integrin must be capable
of signaling when ligands are supplied in either a soluble
or insoluble form. In support of this is the observation that
six different soluble
3 ligands (cRGD, VN, FN, bivalent
F11, monovalent F11, and 2C9.G2 antibody) all caused inhibition of IBa, as did two different insoluble
3 ligands
(VN and F11). According to our hypothesis, (d) soluble signals must be transmitted only through the
v
3 integrin
and not the
5
1 integrin. This is supported by the observation that soluble FN (a proposed ligand only for
v
3)
inhibited current, while insoluble FN (a known ligand for
both
v
3 and
5
1) enhanced current. Finally, our hypothesis predicts that (e) selective ligands of the
5
1 receptor should produce a larger enhancement in current than a common ligand for both integrins (Fig. 8 B). Accordingly, the magnitude of the increased current was
nearly twofold greater when cells were presented with
5
antibody-coated beads compared with FN-coated beads
(compare Figs. 5 and 6). Collectively, our data are consistent with the hypothesis that
v
3 ligation leads to inhibition of the Ca2+ channel, whereas
5
1 ligation leads to
stimulation of the Ca2+ channel. Further work will be
needed to thoroughly test this hypothesis and to determine if other integrins in vascular smooth muscle are also
linked to this channel.
|
Mechanisms of Calcium Current Modulation
The mechanisms by which v
3 and
5
1 ligands modulate
this calcium channel are not yet clear, but the possibility
that the ligands exert a direct effect on the channel seems
unlikely for several reasons: (a) selective antibodies for
3
and
5 integrins modulate Ca2+ current, suggesting that
regulation occurs in a signaling pathway upstream from
the channel rather than at the channel itself; (b) there is no
reported RGD binding sequence in the structure of
1c,
the L-type subunit found in vascular smooth muscle (Koch et al., 1990
); and (c) antagonists such as dihydropyridines
inhibit Ca2+ channels within seconds, and divalent cations
block within a fraction of a second (Dolphin, 1995
;
Hughes, 1995
), while inhibition of current by soluble
v
3
ligands (Figs. 2-5) required ~60 s to achieve >90% of its
maximal effect.
In terms of mechanisms, a more likely possibility is that
modulation of current after integrin ligation involves clustering of integrin receptors, recruitment of cytoskeletal
proteins, and tyrosine phosphorylation of cytoplasmic signaling molecules, such as FAK, Src, or paxillin, as they are
brought into close proximity (Clark and Brugge, 1995).
One difference between the effects of soluble and insoluble ligands in our experiments is that soluble ligands had
sustained effects on current (soluble FN inhibited current
for at least 10 min), while insoluble ligands elicited changes in current that lasted between 6 and 14 min followed by spontaneous recovery. The latter observation
would be consistent with a phosphorylation-dependent
signaling step that is subject to negative feedback control.
This could occur at the level of the receptor, at the channel, or at an intermediate step. In this regard, the affinity
of the
5
1 integrin for ligand has been shown to be controlled by the Ca2+-dependent phosphatase CaMKII
(Bouvard et al., 1998
), such that inhibition of CaMKII preserves the high affinity state of
5
1. A link between integrin signaling and CaMKII has also been demonstrated in
vascular smooth muscle (Bilato et al., 1997
). Activation of CaMKII after Ca2+ influx through L-type channels could
reduce
5
1 affinity and reverse the enhancement of current stimulated by
5
1 ligation. However, other possibilities for initiating signals downstream from integrin ligation
may also exist, including pathways involving phospholipase C and protein kinase C (Somogyi et al., 1994
).
It is likely that v
3 and
5
1 integrins associate directly
with one of the L-type Ca2+ channel subunits (e.g.,
1c) or
with another protein that controls gating or modulates
channel activity. A number of cytoplasmic signaling molecules are potential candidates to interact with the calcium
channel. Data from recent experiments on the L-type Ca2+
channel in visceral smooth muscle (Hu et al., 1998
) have
shown that PDGF, which activates a receptor tyrosine
kinase, enhances L-type Ca2+ current and this effect is
blocked after dialysis of the cells with anti-FAK or anti-Src antibodies. Furthermore,
1c coprecipitates with c-Src
in that tissue and has a potential tyrosine phosphorylation site (Koch et al., 1990
). Dialysis of SMCs with c-Src (Wijetunge and Hughes, 1995
) or with a peptide that activates
c-Src (Wijetunge and Hughes, 1996
) results in enhancement of Ca2+ current. Taken together, these results and
our own preliminary data showing that tyrosine kinase inhibitors reverse the enhancement of current in response to
insoluble FN (Wu, X., G.A. Meininger, G.E. Davis, J.E.
Mogford, S.H. Platts, and M.J. Davis. 1997. Microcirculation. 4:136a) suggest that the pore-containing subunit of
the L-type Ca2+ channel may be tyrosine phosphorylated
by c-Src, which in turn is regulated by integrin ligation.
Additional experiments will be needed to directly test this
idea.
![]() |
Footnotes |
---|
Received for publication 9 January 1998 and in revised form 26 August 1998.
Address all correspondence to Michael J. Davis, Department of Medical
Physiology, 336 Reynolds Medical Building, Texas A & M University
Health Science Center, College Station, TX 77843-1114. Tel.: (409) 845-7816. Fax: (409) 847-8635. E-mail: mjd{at}tamu.edu
The authors thank Judy A. Davidson for technical assistance and Drs. Cindy Meininger and Emily Wilson for advice on various aspects of the experimental design.
This study was supported by National Institutes of Health grants HL-46502 to M.J. Davis and HL-33324 and HL-55050 to G.A. Meininger. Address for reprint requests: M.J. Davis, Dept. of Medical Physiology, 346 Reynolds Medical Building, Texas A & M University Health Science Center, College Station, TX 77843-1114.
![]() |
Abbreviations used in this paper |
---|
ECM, extracellular matrix;
F11, 3 integrin monoclonal antibody;
FAK, focal adhesion kinase;
FN, fibronectin;
HM
5-1,
5 integrin monoclonal antibody;
IBa, whole-cell Ba2+ current;
MHC, anti-rat IgG monoclonal antibody;
PSS, physiological saline solution;
SMC, smooth muscle cell(s);
VN, vitronectin.
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