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Increased tyrosine phosphorylation mediates the cooling-induced contraction and increased vascular reactivity of Raynaud's disease.

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Vol. 50, No. 5, May 2004, pp 1578–1585
DOI 10.1002/art.20214
© 2004, American College of Rheumatology
Increased Tyrosine Phosphorylation Mediates the
Cooling-Induced Contraction and Increased Vascular Reactivity
of Raynaud’s Disease
Philip B. Furspan, Soumya Chatterjee, and Robert R. Freedman
Objective. Increased levels of protein tyrosine
kinase (PTK) are mechanistically associated with increased contractile responsiveness to cooling. This
study tests the hypothesis that increased PTK activity
mediates the increased vascular reactivity to agonists
and cooling associated with primary Raynaud’s disease
Methods. The response of dermal arterioles isolated from control (n ⴝ 29) and RD (n ⴝ 29) subjects to
contractile and dilatory agents at 37°C and 31°C was
characterized using the microvessel perfusion technique. Fluorescence immunohistochemistry was used to
measure tyrosine phosphorylation.
Results. At 37°C, arteries from RD patients exhibited similar sensitivity to the specific ␣2-adrenergic
agonist UK 14,304, to serotonin, and to angiotensin II.
At 31°C, however, the response to all 3 agonists was
greater in the arterioles from the RD patients than in
those from the control subjects. Agonist-induced contraction at both temperatures was reversed by cumulative addition of the PTK inhibitors genistein (1–30 ␮M)
and tyrphostin 47 (0.1–10 ␮M). All arterioles from
control subjects relaxed slightly in response to cooling,
whereas more than half of those from RD patients
contracted. This cooling-induced contraction was reversed by the cumulative addition of genistein. The 3
agonists elicited large increases in tyrosine phosphorylation only in arterial segments from RD patients at
31°C. Cooling from 37°C to 31°C elicited a large increase
in tyrosine phosphorylation in arterioles from RD pa-
tients, but not those from control subjects. All increases
in tyrosine phosphorylation could be prevented by
Conclusion. Increased tyrosine phosphorylation
mediates cooling-induced contraction and the increased
vascular reactivity of skin arterioles from individuals
with RD.
Raynaud’s disease (RD) is characterized by episodic digital vasospasms that are provoked by cold
and/or emotional stress. Lewis (1) hypothesized that a
“local fault” caused cold hypersensitivity of the digital
blood vessels.
A possible mechanistic link between cooling and
vasoconstriction/vasospasm is suggested by recent studies investigating the biochemical basis of cold-induced
modification of vascular response. These studies have
strongly implicated the protein tyrosine kinase (PTK)
signal transduction pathway as the mediator of coldinduced vasoconstriction in a variety of blood vessels
(2–4). During the last decade, PTK signal transduction
pathways have been revealed to play an important role
in the mediation of agonist- and growth factor–initiated
contractile activity in vascular smooth muscle (5).
Wagerle et al (2) demonstrated that the coldinduced contraction of the middle cerebral artery of the
lamb could be reversed completely by 2 structurally
diverse inhibitors of PTK, genistein and tyrphostin 47,
but not by staurosporine, a protein kinase C inhibitor.
Conversely, sodium orthovanadate (SOV), an inhibitor
of protein tyrosine phosphatase, potentiated the coldinduced contraction. In a subsequent study from the
same group, it was observed that whereas rat tail artery
did not normally contract in response to cooling from
37°C to 24°C, rat tail artery pretreated with SOV did
contract in response to cooling (6). We have corroborated these results (4). Dahdah et al (3) observed that
contraction in lamb coronary artery was associated with
Supported by a grant from the American Heart Association,
Midwest Affiliate (51302Z).
Philip B. Furspan, PhD, Soumya Chatterjee, MD, Robert R.
Freedman, PhD: Wayne State University, Detroit, Michigan.
Address correspondence and reprint requests to Philip B.
Furspan, PhD, C. S. Mott Center, Wayne State University, 275 East
Hancock Avenue, Detroit, MI 48201. E-mail:
Submitted for publication September 5, 2003; accepted in
revised form January 23, 2004.
increased tyrosine phosphorylation when the temperature was reduced from 37°C to 7°C. These results suggest
that increased levels of PTK activity are mechanistically
associated with increased contractile responsiveness to
cold. This conclusion is supported by evidence indicating
that cooling enhances the contractile effect of
G-protein–coupled agonists that activate PTK signal
transduction pathways (5,7). PTK pathways have also
been implicated in the mediation of vasospasm in other
vascular diseases, for example, stroke, heart disease, and
atherosclerosis (8–10).
The present study was designed to examine the
role played by PTK activity in the cooling- and agonistinduced contraction of arterioles from patients with
Raynaud’s disease (RD) and from healthy control subjects. Because the reactivity to a number of different
agonists has been reported to be enhanced in blood
vessels from RD patients compared with those from
controls, we have examined the actions of 3 of these
agonists. We found that the enhanced reactivity does not
result from a defect in the interaction between a specific
agonist and its receptor, but rather from an alteration in
the PTK signal transduction pathway that mediates their
Characteristics of the study subjects. Raynaud’s disease was classified using the Allen and Brown criteria (11):
bilateral digital (must include fingers) color changes (2 of the
following 3: blanching, cyanosis, rubor), provoked by cold
and/or emotional stress, for at least 2 years, in the absence of
any identifiable disease process (e.g., scleroderma, rheumatoid
arthritis, etc.). Additionally, the 29 premenopausal women
with RD (ages 21–54 years) were required to be negative for
antinuclear and anticentromere antibodies (on HEp-2 cell
substrate). Twenty-nine healthy, age-matched (ages 21–50
years) women with no evidence of any physical disorder were
also evaluated in this study.
Each study subject was medication-free (at least 2
weeks), had related their medical history, and had completed
an extensive symptom questionnaire used in our previous
studies (12,13) to ensure the absence of any disorder. All
subjects were menstruant. Subjects did not undergo a biopsy at
a specific point in their menstrual cycle, since hormone status
does not appear to affect the incidence or severity of attacks of
vasospasm (14,15). Smokers were not included because smoking impairs vascular function (16). All patients and normal
volunteers were required to be free of any cardiovascular
disease (e.g., hypertension, heart disease) or other disease that
might affect vascular function (e.g., diabetes).
All procedures followed in this study were in accordance with Wayne State University guidelines for studies
involving humans. An institutional review committee approved
this study, and all subjects gave written informed consent.
Vessel preparation. Using lidocaine (without norepinephrine) as a local anesthetic, skin biopsies (6 mm in diameter) were taken from the medial forearm of all study subjects.
Arterioles of usable size (mean ⫾ SEM pressurized diameter
136 ⫾ 11 ␮m and 137 ⫾ 10 ␮m in control subjects and RD
patients, respectively) were dissected out from the dermal–
subcutaneous boundary. Arterioles were used immediately or
were stored overnight at 4°C in Eagle’s minimum essential
medium (Sigma, St. Louis, MO). Preliminary experiments
found no difference in reactivity between vessels that were
used the day of the biopsy and vessels from the same biopsy
tissue that were used the next day. Similar results have been
reported in the literature (17,18).
Arterioles were attached to the proximal micropipette
using individual strands from 3-0 nylon braided suture and
were superfused with physiologic salt solution (118 mM NaCl,
4.7 mM KCl, 1.18 mM KH2PO4, 1.17 mM MgSO4䡠7H2O, 1.6
mM CaCl2䡠2H2O, 25.0 mM NaHCO3, 5.5 mM dextrose, and 1.2
mM CaNa2–EDTA) and aerated with 95% O2 and 5% CO2. A
perfusion pressure of ⬃20 mm Hg was used to clear the lumen
of blood, followed by attachment of the distal end of the artery
segment to the closed distal pipette. The superfusate was
warmed to, and maintained at, 37°C. Transmural pressure was
controlled with a pressure-servo system. After an equilibration
period of 45 minutes at 20 mm Hg, the pressure was increased
to 40 mm Hg (19). Precise and rapid (⬍2 minutes) temperature reduction was accomplished by cooling the superfusate as
it flowed through a jacketed temperature-exchange coil connected to a second circulating heating/cooling waterbath that
was maintained at 31°C. Agents to be evaluated were added
directly to the superfusate. Superfusate replacement occurred
in less than 2 minutes.
Contractile activity of the blood vessels was monitored
with a video camera and quantified with a video dimension
analyzer (Living Systems Instrumentation, Burlington, VT)
that was connected to a video monitor. Changes in diameter
were recorded on videotape and a computer-based dataacquisition system (DATAQ Instruments, Akron, OH) and
were expressed as the percentage of change from baseline.
Experimental protocols. Perfused microvessels. After
equilibration, arterioles were exposed to 95 mM K⫹–
physiologic salt solution (equimolar substitution of KCl for
NaCl). The presence of an intact endothelium was determined
by exposing arterioles that had been precontracted with norepinephrine (10⫺6M) to increasing concentrations of acetylcholine (10⫺9–10⫺6M). Concentration-response curves were
constructed for the agonists tested: UK 14,304, a specific
␣2-adrenergic agonist (10⫺10–10⫺8M), serotonin (10⫺9–
10⫺6M), and angiotensin II (10⫺10–10⫺8M). All of these
agonists have been reported to exert their contractile effects, at
least in part, via the PTK pathway (4,20). Responses were
measured at 37°C and at 31°C. A low temperature of 31°C was
chosen because it represents the temperature reached by the
fingers shortly after exposure to moderately cold temperature
(10°C) (21) and because preliminary results indicated that
lower temperatures significantly inhibited contraction in response to these agonists (i.e., 31°C was the lowest temperature
at which responses of arterioles from normal individuals did
not exhibit a decrease in responsiveness to agonists). Results
Figure 1. Contraction of skin arteriole segments from control subjects (solid line) and patients
with Raynaud’s disease (broken line) induced by the agonists UK 14,304, serotonin, and
angiotensin II at 37°C (open symbols) and 31°C (solid symbols). Values are the mean ⫾ SEM of
6 samples per group. ⴱⴱ ⫽ P ⬍ 0.01; ⴱ ⫽ P ⬍ 0.05.
were expressed as the percentage of change in the lumen
diameter from baseline.
The effect of PTK inhibition on agonist-induced contraction was determined by cumulatively adding genistein
(1–100 ␮M) or tyrphostin 47 (0.1–10 ␮M), both of which are
inhibitors of PTK activity, to the bath after contraction (⬃60%
decrease in diameter) in response to an agonist had reached a
plateau. The order of exposure to agonist, antagonist, and
temperature was randomized.
The response of vessels to cooling from 37°C to 31°C
was determined in the absence and presence of SOV (100 ␮M),
an inhibitor of tyrosine phosphatase. If contraction occurred,
genistein (1–100 ␮M) was added. SOV is able to inhibit
tyrosine phosphatase in part because of a similarity in size and
charge that it shares with inorganic phosphatase (22). In
addition, SOV is able to assume a trigonal bipyramidal coordination that is thought to resemble a phosphate group (22).
Fluorescence immunohistochemistry (23). Freshly obtained dermal arteries measuring 1–3 mm in length and
100–200 ␮m in diameter were cleaned of adventitial tissue and
cut into pieces ⬃200 ␮m wide. Segments were placed in wells
containing 50 ␮l of phosphate buffered saline (PBS) composed
of 2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, and 8 mM
Na2HPO4. After incubation in PBS at 37°C on a thermostatically controlled block for 30 minutes, the pieces were transferred to wells containing PBS and the agents of interest. If
indicated, the temperature was reduced to 31°C by adding an
equivalent volume of 25°C PBS and the agent of interest. After
5 minutes at the appropriate temperature, the vessel segments
were transferred to PBS containing 2% paraformaldehyde for
10 minutes to fix the tissues (this and subsequent steps were
performed at room temperature).
Next, the tissue was incubated with 0.1% Triton X-100
in PBS for 10 minutes. After a wash in PBS, the pieces were
incubated in a solution containing 0.9% sodium citrate, 2%
goat serum, 1% bovine serum albumin, 0.05% Triton X-100,
0.025% NaNO3, and tetramethyl rhodamine isothiocyanate–
labeled phosphotyrosine monoclonal antibody (1:50 dilution)
for 45 minutes. After incubation, the vessel pieces were washed
with 0.02% Triton X-100 and 0.9% sodium citrate in PBS and
then in PBS alone.
The tissue segments were then mounted on glass slides
and examined under a microscope equipped with an epifluorescence optical system. Measurement of fluorescence intensity as an indicator of tyrosine phosphorylation was performed
using SimplePCI, a Compix (Cranberry Township, PA) image
analysis program.
Reagents. All drugs and chemicals were obtained from
Sigma (St. Louis, MO). Genistein and tyrphostin 47 were
dissolved in DMSO.
Statistical analysis. Concentration-response curves
were calculated as geometric means. Paired and unpaired
t-tests and analysis of variance were performed. When necessary, modified analyses were used to allow for multiple testing
procedures (e.g., Bonferroni correction for multiple compari-
Figure 2. Effect of genistein and tyrphostin 47 on skin arteriole segments maintained at 31°C from
control subjects (solid line) and Raynaud’s disease patients (broken line). Values are the mean ⫾
SEM of 6 samples per group. ⴱ ⫽ P ⬍ 0.05, ⴱⴱ ⫽ P ⬍ 0.01.
sons). In all cases, P values less than 0.05 were considered
significant. All results are expressed as the mean ⫾ SEM.
The contraction of arterioles from RD patients
and control subjects in response to UK 14,304, serotonin, and angiotensin II was not significantly different
when measured at 37°C (Figure 1). At 31°C, however,
the curves for all 3 agonists applied to arterioles from
RD patients were shifted to the left compared with those
from control subjects (Figure 1). The maximum contraction of arterioles from RD patients at 31°C was not only
greater than that of control subjects at both 37°C and
31°C, but it was also greater than the maximum contraction of arterioles from RD patients at 37°C (Figure 1).
Contraction in response to 95 mM KCl was equivalent in
both groups and at both temperatures (data not shown).
The involvement of PTK in the mediation of
contraction in response to the 3 agonists is suggested by
the relaxation induced by the cumulative addition of the
PTK inhibitors genistein (1–30 ␮M) and tyrphostin 47
(0.1–10 ␮M). At 37°C, the response to genistein was
similar irrespective of the subject group or the agonist
evaluated (data not shown). When applied to arterioles
maintained at 31°C, those from RD patients exhibited
greater sensitivity to the relaxant effect of both genistein
and tyrphostin 47 (Figure 2). Vehicle (DMSO; 1:10,000
to 1:1,000 dilution) was without effect. Genistein and
tyrphostin 47 were not effective in reversing the contraction caused by 95 mM KCl (data not shown).
In response to cooling the superfusate from 37°C
to 31°C, none (0 of 15) of the arterioles from control
subjects exhibited contraction (Figure 3). Of the arterioles from RD patients, however, more than half (8 of
15) contracted in response to cooling (Figure 3). The
contraction in response to cooling was reversed by the
cumulative addition of genistein (1–30 ␮M) (Figure 3).
After washout of genistein, the vessel diameter remained at the baseline size. Pretreating the arterioles
with SOV (100 ␮M) produced a small contraction (9.2 ⫾
5.1%, n ⫽ 6) in arterioles from control subjects but did
not alter the response of those from RD patients.
Concentrations of UK 14,304, serotonin, and
angiotensin II that caused maximal contraction of arte-
Figure 3. Change in lumen diameter of skin arteriole segments (only
those that responded; see text for details) from control subjects and
Raynaud’s disease (RD) patients in response to a reduction in the bath
temperature from 37°C to 31°C, and effect of various concentrations of
genistein on the contraction induced in the arterioles from the RD
patients. Values are the mean and SEM of 15 control samples and 8
RD samples per group. ⴱⴱ ⫽ P ⬍ 0.01 versus control group; # ⫽ P ⬍
0.05 versus untreated RD group.
rioles also caused increases in tyrosine phosphorylation,
as indicated by increases in fluorescence intensity (Figure 4). The pattern of increases in tyrosine phosphorylation was similar for the 3 agonists: RD patient arterioles at 31°C ⬎⬎ control subject arterioles at 31°C ⬎
RD patient arterioles at 37°C ⬎ control subject arterioles at 37°C. Only the increases in tyrosine phosphorylation elicited at 31°C in arteriole segments from RD
Figure 4. Change in tyrosine phosphorylation (as indicated by fluorescence intensity) of skin arteriole segments from control subjects and
Raynaud’s disease (RD) patients in response to 3 different agonists,
UK 14,304, serotonin, and angiotensin II, at 37°C and at 31°C, and in
segments from RD patients exposed to each of the agonists at 31°C in
the presence of 30 ␮M genistein. Values are the mean and SEM of 5
samples per group. ⴱⴱⴱ ⫽ P ⬍ 0.01 versus control group at 31°C.
Figure 5. Change in tyrosine phosphorylation (as indicated by fluorescence intensity) of skin arteriole segments from control subjects and
Raynaud’s disease (RD) patients in response to a decrease in the bath
temperature from 37°C to 31°C in the absence and presence of 100 ␮M
sodium orthovanadate (SOV). Values are the mean and SEM of 5
samples per group. ⴱ ⫽ P ⬍ 0.05 versus untreated control group.
Values for the RD groups were not significantly different from each
patients were significantly greater than the other values.
Genistein (30 ␮M) was able to prevent the increases in
tyrosine phosphorylation in all cases. (Only the results of
its effect on arteriole segments from RD patients at 31°C
are shown in Figure 4.) Tyrphostin 47 (3 ␮M) had a
similar effect on tyrosine phosphorylation (data not
Cooling in the absence of agonists caused a small
increase in tyrosine phosphorylation in arteriole segments from control subjects and a large increase in
segments from RD patients (Figure 5). The presence of
SOV (100 ␮M) increased the level of tyrosine phosphorylation in arteriole segments from control subjects, but
did not increase it significantly in segments from RD
patients (Figure 5).
Because some studies have reported impaired
endothelium-mediated vasodilation in individuals with
RD (24,25), we examined the response of precontracted
(with 10⫺6M norepinephrine) arterioles from RD patients and control subjects to acetylcholine (10⫺8–
10⫺6M). At 37°C, there was no difference in the response to acetylcholine between arterioles from RD
patients and those from control subjects (Figure 6).
Surprisingly, at 31°C, arterioles from control subjects
exhibited a reduced sensitivity and maximal relaxation to
acetylcholine compared with those from the other
groups (Figure 6). A decrease in sensitivity, but not
maximal relaxation, to bradykinin (10⫺11–10⫺8M) was
Figure 6. Response of precontracted (with 10⫺6M norepinephrine)
skin arterioles from control subjects (solid line) and Raynaud’s disease
(RD) patients (broken line) to the cumulative addition of acetylcholine (ACh) at 37°C (open symbols) and 31°C (solid symbols). Values
are the mean ⫾ SEM of 7 samples per group. ⴱ ⫽ P ⬍ 0.05 and ⴱⴱ ⫽ P
⬍ 0.01 for the control value at 31°C compared with the RD value at
31°C; ## ⫽ P ⬍ 0.01 for the control value at 31°C compared with the
control value at 37°C.
also observed in arterioles from control subjects at 31°C
(data not shown).
These experiments strongly suggest a causative
relationship between increased tyrosine phosphorylation and the increased vascular reactivity at reduced
temperature and cooling-induced contraction associated
with RD. Contraction in response to the 3 agonists
tested and in response to cooling was reversed by the
cumulative addition of the PTK inhibitors genistein and
tyrphostin: contraction in response to 95 mM KCl was
not. The same agonists also elicited increases in tyrosine
phosphorylation that could be prevented by genistein
and tyrphostin 47. PTK mediation of contraction induced by G-protein–coupled receptor agonists such as
those used in this study is well established (5). Thus, the
increased tyrosine phosphorylation revealed in the
present study may underlie reports of increased vascular
reactivity to a variety of agonists in vivo in RD patients
(12,13,26). The increased vascular reactivity associated
with increased tyrosine phosphorylation may be related
to the reported enhancement of myofilament calcium
sensitivity in vascular smooth muscle by PTK (27,28).
The PTK signal transduction system also has been
implicated in the pathophysiology of other vascular
diseases characterized by increased vascular reactivity
and vasospasm, such as hypertension (29) and arteriosclerosis (10).
Of course, the hallmark of RD is cooling-induced
vasospasm of digital blood vessels. Although we did not
observe cooling-induced vasospasm (i.e., complete closure of the lumen) in the arterioles we tested, we did
observe cooling-induced contraction in more than onehalf the arterioles from RD patients but not control
subjects. The lack of a vasospastic response to cooling in
all isolated vessels may relate to the absence of conditions in vitro that are required for vasospasm to occur in
response to cooling. The inconsistent occurrence of the
cooling-induced contractile response suggests that specific conditions may be required for this response to
occur, perhaps relating to the balance between contractile and relaxing influences existing at the time the vessel
is exposed to the reduction in temperature. Even in vivo,
vasospasm in response to laboratory cooling occurs only
80–90% of the time in individuals with RD (30,31).
The involvement of PTK in the cooling-induced
contraction of arterioles from RD patients is suggested
by its reversal by genistein and tyrphostin 47 and by the
large increase in tyrosine phosphorylation measured in
arteriole segments. The inhibition of an increase in
tyrosine phosphorylation by these PTK inhibitors correlates with their reversal of contraction in isolated arterioles (Figures 4 and 2). Although cooling elicited an
increase in tyrosine phosphorylation in untreated and
SOV-treated arteriole segments from control subjects,
only those treated with SOV exhibited contraction.
These results suggest that there may be a threshold level
of tyrosine phosphorylation that is required for contraction to occur and/or that other conditions need to exist
in addition to a specific level of tyrosine phosphorylation. In a previous study, we found that rat tail artery
cooled from 37°C to 25°C did not contract, whereas
those previously exposed to SOV, the protein tyrosine
phosphatase inhibitor, did contract (4). Tyrosine phosphorylation of human skin arterioles uniformly increases
in response to cooling. However, the fact that they do
not always contract or exhibit vasospasm (either in vitro
or in vivo) suggests that tyrosine phosphorylation by
itself is required, but is not sufficient, for contraction to
Other investigators have reported a role for PTK
in the mediation of other vasospastic conditions. Fujikawa et al (9) reported that stimulation of the tyrosine
kinase pathway(s) of canine basilar artery by subarachnoid hemorrhage leads to vasospasm. Ito et al (10)
observed that the specific PTK inhibitor ST 638 blocked
coronary vasospasm induced by chronic interleukin-1
treatment in pigs.
Because some studies have suggested that impaired endothelium-dependent vasodilation plays a role
in the increased vascular reactivity that is characteristic
of RD (24,25), we examined the response of precontracted arterioles to acetylcholine. We found that the
response to acetylcholine is impaired at 31°C in arterioles from control subjects, whereas the response of
arterioles from RD patients at 31°C did not differ from
the response at 37°C. This finding is consistent with a
report of an enhanced dilatory response, as measured by
fingertip blood flow after methacholine infusion, in RD
patients compared with control subjects (32). The absence of impairment in the vasodilatory response may
represent a compensatory adjustment to the greater
vascular reactivity at this temperature. This adjustment
may occur as a consequence of an increase in PTK
activity in endothelial cells from RD patients paralleling
that in vascular smooth muscle cells. A number of studies
have suggested that PTK plays a role in the mediation of
endothelium-dependent relaxation (33–35).
In the present study, we have presented evidence
for a role of increased tyrosine phosphorylation in the
mediation of cooling-induced contraction and the increased vascular reactivity of skin arterioles from individuals with Raynaud’s disease. The identity of the
protein(s) that is phosphorylated by PTK in this preparation remains to be identified.
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phosphorylation, increase, contractile, induced, raynaud, vascular, tyrosine, reactivity, disease, cooling, mediated
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