© 2000 American Heart Association, Inc.
Integrative Physiology |
Opposite Effects of Pressurized Steady Versus Pulsatile Perfusion on Vascular Endothelial Cell Cytosolic pH
Role of Tyrosine Kinase and Mitogen-Activated Protein Kinase Signaling
From the Division of Cardiology, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Md.
Correspondence to David A. Kass, MD, Halsted 500, Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287. E-mail dkass{at}bme.jhu.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—Endothelial cytosolic pH (pHi) modulates ion channel function, vascular tone, and cell proliferation. Steady shear induces rapid acidification in bicarbonate buffer. However, in vivo shear is typically pulsatile, potentially altering this response. We tested effects and mechanisms of pHi modulation by flow pulsatility, comparing pressurized steady versus pulse-flow responses in bovine aortic endothelial cells cultured within glass capillary tubes. Cells were loaded with the fluorescent pHi indicator carboxy seminaphthorhodafluor-1 and perfused with physiological pulsatile pressure and flow generated by a custom servo-control system. Raising mean pressure from 0 to 90 mm Hg at 0.5 mL/min steady flow in bicarbonate buffer induced sustained acidification (-0.33±0.09 pH units, P<0.01). A subsequent increase in steady flow resulted in further acidification. In contrast, if mean pressure and flow were unchanged but perfusion made pulsatile, pHi rose +0.3±0.03 (P<0.0001) over 30 to 60 minutes. HCO3- removal and use of acid/base exchange inhibitors 5-(N-ethyl-N-isopropyl)amiloride or diisothiocyanato stilbene disulfonic acid identified both extracellular Na+–independent Cl--HCO3- and Na+-H+ exchangers as activated by static pressure, whereas pulsatility activated extracellular Na+–dependent Cl--HCO3- and Na+-H+ exchangers to raise pHi. Pulse-perfusion alkalinization occurred with or without flow reversal and increased 1.6-fold in Ca2+-free buffer. Inhibition of c-Src tyrosine kinase (4-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo [3,4-d]pyrimidine; PP2) or MEK-1 (mitogen-activated protein kinase [MAP]/extracellular signal–regulated kinase [ERK]–1) (PD98059, blocking ERK1/2) blocked or reversed the pulsatile-flow pHi change to acidification. In contrast, PP2 had no effect on steady flow acidification, whereas MEK-1 inhibition converted it to alkalinization. Thus, pulsatile and steady flow trigger opposite effects on endothelial pHi by differential activation of acid/base exchangers linked to c-Src and MAP kinase phosphorylation, but not to Ca2+. These data highlight specific signaling responses triggered by phasic shear profiles.
Key Words: endothelium • acid-base • pulsatile shear • mitogen-activated protein kinase • tyrosine kinase
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Vascular endothelial cells respond to increased shear stress by various intracellular signal transduction pathways. Whereas most prior in vitro studies have studied the effects of steady laminar flow and shear, growing evidence suggests that flow pulsatility can induce differential and/or independent effects on cell signaling. For example, cyclic shear stress markedly elevates endothelial NO synthase activity1 and is associated with more sustained cytosolic calcium transients.2 Sustained exposure to phasic elevated hydrostatic pressure stimulates endothelial secretion of an antiproliferative factor.3 In vivo, perfusion pulse frequency rather than mean flow rate appears to dominate NO-mediated epicardial dilation,4 whereas selective enhancement of coronary flow pulsatility at constant mean pressure and cardiac workload augments mean coronary flow.5
These and related observations have stimulated a growing interest in identifying cell-signaling mechanisms differentially activated by steady versus pulsatile shear stress. Cytosolic pH (pHi) plays an important role in endothelial mechanoreception.6 7 We previously showed that abrupt increases in steady laminar flow decrease endothelial pHi by activating the extracellular Na+ (Na+o)–independent Cl--HCO3- exchanger,6 whereas pHi rises when laminar fluid shear stress is abruptly reduced after a period of sustained steady flow.7 Thus, there may be opposing inputs to pHi regulation during pulsatile flow as a result of the cyclic rises and falls of shear stress. pHi may serve as an intracellular signaling mechanism linking shear stress to altered vascular tone. For example, endothelial NO synthase activation is sustained by intracellular alkalinization,8 whereas NO production in response to laminar shear is attenuated by inhibition of the Na+-H+ exchanger.9
The present study tested the hypothesis that endothelial pHi is differentially influenced by steady laminar versus pulsatile shear stress. We also examined the mechanisms involved in pulse perfusion–induced pHi changes, including the importance of flow (and shear) reversal, the dependence on Ca2+, and the role of c-Src family tyrosine kinase and mitogen-activated protein (MAP) kinase activation. c-Src has been shown to modulate a variety of endothelial shear responses9 10 11 and can influence pHi in cardiomyocytes.12 Steady shear stress–induced c-Src is linked to activation of extracellular signal–regulated kinase (ERK1/2),10 which can induce various signaling responses, including transcriptional changes13 and activation of the Na+-H+ exchanger.14 15 16
To achieve these aims, we developed a novel servo-perfusion system to expose cultured endothelial cells to physiological pulsatile pressure and flow stimuli while simultaneously enabling fluorescence microscopy to quantify pHi alterations.17 The results provide evidence that flow pulsatility induces directionally opposite pHi changes from that observed with constant flow by a c-Src– and MAP kinase–dependent but not extracellular Ca2+–dependent pathway.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Cell Culture and pHi Measurement
Bovine aortic endothelial cells (BAECs, Corriell Institute, passages 4 to 12) were cultured in DMEM with 5% FCS, penicillin (5 mg/mL), streptomycin (5 mg/mL), and neomycin (10 mg/mL) at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were washed, loaded, and grown to confluence on 1 side of 1% gelatin-coated glass rectangular capillary tubes.7 Endothelial pHi was measured by c-seminaphthorhodafluor (SNARF) 1/acetoxymethyl ester fluorescence, as previously described.6 7 18
Pulse-Pressure Servopump System
Pulsatile perfusion was generated using a novel, custom-designed
computer-controlled servo system, which was recently described in
detail.17 Mean flow was generated by a nonpulsatile flow
pump (MC-Z, Ismatec), and realistic pulse-perfusion waveforms
were superimposed by an electromagnetic servo motor (Applied
Engineering) controlled by digital feedback. The servo-command signal
was a previously recorded aortic pressure wave, which could be
modified to achieve any desired pulse amplitude and mean. An inline
flowmeter (1N, Transonic) placed just upstream of the capillary tube
recorded phasic flow. Effluent passed via a downstream resistor to
vary mean pressure at any given flow.
Protocols
BAECs were generally perfused first with nonpulsatile steady
flow at 0.5 mL/min with either bicarbonate buffer or nonbicarbonate
HEPES buffer.6 Because pulse perfusion was generated by
pressure feedback control, positive pressurization of the
perfusate was required. We used 90 mm Hg to reflect in
vivo conditions. As no prior data existed regarding effects of
pressurization alone on pHi, studies were also
conducted to determine this response. For these studies, BAECs were
first stabilized at 0.5 mL/min flow and 0 mm Hg perfusion
pressure and then pressurized to 90 mm Hg at constant
flow.
To test the effects of flow pulsatility on pHi, cells were first perfused at constant flow (0.5 mL/min) and 90 mm Hg mean pressure, and after stabilization, pulsatility (75 mm Hg) was initiated and sustained for 60 to 90 minutes. pHi was continuously monitored by c-SNARF 1 fluorescence.
To test the contribution of specific acid-base extruders, studies were performed with 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 50 µmol/L, Molecular Probes), to inhibit the Na+-H+ exchanger, or diisothiocyanato stilbene disulfonic acid (DIDS) (100 µmol/L x 60 minutes pretreatment, Sigma), to inhibit HCO3--dependent exchangers.
Because increasing flow pulsatility at 0.5 mL/min flow resulted in bidirectional shear, studies were performed to test the role of flow reversal on pHi by using higher mean flows (3 or 6 mL/min) and/or reduced pulsatility (35 versus 75 mm Hg).
To test the importance of Ca2+-dependent signaling, studies were performed in Ca2+-free buffer with 1 mmol/L EGTA. Cells were exposed to the EGTA-containing buffer for at least 15 minutes before the experiments were initiated. To test the influence of c-Src, cells were incubated for 15 minutes with 50 µmol/L 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo (3,4-d)pyrimidine (PP2; CalBiochem). PP2 was maintained at the same concentration during the study as well. Lastly, the role of ERK1/2 activation was studied by preincubating with the MAP/ERK kinase (MEK)–1 inhibitor PD98059 for 30 minutes at 50 to 75 µmol/L, with this concentration maintained during the study.
Analysis
Changes in pHi before and after increases
in mean pressure, flow, or flow pulsatility at constant mean pressure
and flow were compared by paired Student t test. Data
examining the role of specific signaling mechanisms were contrasted
with the appropriate controls by a nonpaired t test. Data
are presented as mean±SEM, with P<0.05 considered
statistically significant.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Servo Perfusion Waveforms
Figure 1A
shows
representative waveforms for pressure, flow (and
shear), and rate of change of shear as generated by the servo system.
Pressures closely duplicated the command signal, and
physiological flow resulted from the interaction of
pressure with the perfusion tubing impedance. At a mean flow of 0.5
mL/min and 75 mm Hg pulse pressure, peak flow was 9.1 mL/min.
Shear stress (
) was calculated as =4
µQ/
r,3 where µ is
fluid viscosity, Q is flow rate, and r is the
internal radius of the capillary tube (0.05 cm). This yielded a mean
shear of 0.83 dyne/cm2 for steady and pulsatile
flow, with peak shears of 20 dyne/cm2 with flow
pulsatility. As is often true in vivo, pulsatile shear was asymmetrical
(lower panel, Figure 1), with an early rapid rise and decline
followed by a lower stable negative shear (-4.3
dyne/cm2). Figures 1B and 1C display the
two alternative stimuli used in studies regarding the role of flow
reversal (see below).
|
Effect of an Abrupt Increase in Static Mean Pressure on
Endothelial pHi
Figure 2A displays example tracings
of pHi in response to a sudden rise in luminal
pressure from 0 to 90 mm Hg, in either
HCO3- or HEPES buffer. In
HCO3- buffer, increasing
pressure induced a consistent pHi decline
of –0.33±0.09 pH units (n=7; P<0.01). Acidification began
within 5 minutes after increasing pressure and continued until a steady
state was reached 33±4 minutes later. pHi did
not recover even after 45 minutes as long as flow remained
pressurized.
|
To test whether the pHi decline by pressurization
was mediated by activation of the
Na+o-independent
Cl--HCO3-
exchanger, studies were also performed in HEPES buffer and with
selective acid/base exchanger inhibitors. In
HCO3--free HEPES buffer,
increasing pressure elevated rather than lowered
pHi (n=4, P=0.013; P=0.004
versus HCO3-, Figure 2A
). Similar alkalinization was observed in cells pretreated
with DIDS in HCO3- buffer
(P=0.022; P=0.002 versus
HCO3-, Figure 2B). The
latter alkalinization was abolished by further addition of EIPA (n=3,
P=0.005 versus
HCO3-+DIDS, Figure 2B)
to block the Na+-H+
exchanger. Summary data are shown in Figure 2C. Thus, exposure
of endothelial cells to pressurized steady flow in
physiological
HCO3- buffer activated
both the Na+o-independent
Cl--HCO3-
exchanger and the Na+-H+
exchanger, with the net effect being a gradual sustained intracellular
acidification.
Effect of Pulsatile Flow on pHi
Figure 3 contrasts effects of
increasing steady versus pulsatile flow on pHi,
both measured in the presence of 90 mm Hg mean pressure.
Consistent with our previous studies,6 7
increasing steady flow from 0.5 to 2 mL/min (0.83 to 3.3
dyne/cm2) resulted in abrupt acidification
(Figure 3A). Importantly, this was observed in cells already
acidified by pressurization, and the steady flow–induced response was
considerably faster than from pressure alone. In contrast, when flow
was made pulsatile while the same mean pressure and flow were
maintained, pHi changed in the opposite direction
(Figure 3B). In HCO3-
buffer, flow pulsatility raised pHi by +0.3±0.03
(n=9; P<0.0001). This started 5 to 10 minutes after
initiating pulsatility and peaked after 49±8 minutes. In 75% of the
monolayers, continuation of pulsatility led to complete or partial
recovery to baseline pHi 23±4 minutes after
maximal alkalinization. The remaining monolayers showed no recovery
during the observation period.
|
Addition of EIPA to HCO3-
buffer resulted in similar but somewhat temporally delayed
pHi rise as compared with
HCO3- alone
(P=0.029, Figure 4A). However,
in HEPES buffer, the response fell 56% (P=0.01 versus
HCO3-, Figure 4B).
Similarly, when BAECs were perfused with
HCO3- buffer containing DIDS,
pulsatile shear raised pHi by about half that
observed with HCO3- buffer
alone. Although EIPA alone in
HCO3- buffer had little effect,
EIPA in HEPES buffer fully inhibited 
pHi with
pulsatile flow (P=0.007 versus HEPES alone, Figure 4C). Figure 4D provides summary results. Thus, in
physiological
HCO3- buffer, pulsatile shear
caused alkalinization by activation of both the
Na+o-dependent
Cl--HCO3-
exchanger and the EIPA-sensitive
Na+-H+ exchanger.
|
50%. C,
Combination of EIPA with a HEPES buffer essentially eliminated any
pHi change with pulsatile perfusion. In each panel, arrow
indicates time at which pulsatile flow/pressure was initiated. D,
Summary data for these experiments. *P<0.05.
Effects of Modifying Phasic Flow Waveform
Increasing pulsatility at a mean flow of 0.5 mL/min resulted in
flow reversal at a low shear rate (Figure 1A). Purely
symmetrical (sinusoidal) flow reversal can markedly reduce
endothelial Ca2+ and NO
signaling.1 2 To test whether the flow reversal associated
with our more realistic waveforms was critical to the differential
pHi response, studies were performed at higher
mean flows at the same or reduced pulsatility to prevent flow reversal.
At 6 mL/min and addition of 75 mm Hg (Figure 1B),
pulsatility still resulted in significant alkalinization but to a
lesser degree (+0.15±0.05, n=5, P=0.04; P=0.02
versus low-flow studies). However, at half this flow and pulsatility,
pHi was +0.21±0.05 (P<0.05), not
significantly different from that in studies with flow reversal. Thus,
part of the pHi rise with pulsatility may be
offset by greater mean flow-dependent signaling, but flow reversal per
se is not required to generate the response.
Role of Ca2+ and c-Src in pHi Response to
Pulse Perfusion
Figures 5A and 5C display
results from experiments performed in Ca2+-free
HCO3- buffer with EGTA. Rather
than being inhibited, pHi elevation with flow
pulsatility was enhanced by removal of Ca2+ in
the buffer, rising +0.54±0.09 units (n=5, P=0.003,
P<0.01 versus HCO3-
with Ca2+).
|
In contrast, inhibition of c-Src with PP2 profoundly inhibited
endothelial alkalinization by pulse perfusion (Figure 5B), with no change or a slight decline in
pHi (–0.11±0.06 units, P<0.0001
versus HCO3- without PP2).
Interestingly, PP2 did not affect the acidification from an acute rise
in steady flow (Figure 5C). Thus, c-Src appeared selectively
involved in the pulse-perfusion pHi response.
Role of ERK1/2 on Pulsatile and Steady Flow–Induced
pHi
Figure 6 displays example and group
data for studies using the MEK-1 inhibitor PD98059.
Incubation with PD98059 did not alter basal pHi
(7.13±0.09 versus 7.15±0.15 with or without PD98059, respectively).
However, subsequent pHi changes to both steady
and pulsatile flow were directionally reversed. With steady flow,
PD98059 converted the acidification to an alkalinization of
approximately the same magnitude (P<0.01), whereas for
pulsatile flow, alkalinization converted to an acidification
(P<0.005, compared 50 minutes after the onset of pulsatile
flow). Thus, unlike c-Src, activation of ERK1/2 contributed to both
steady and pulsatile pHi responses.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This study reveals several novel findings about the differential responses of endothelial pHi to steady and pulsatile shear stress. First, increasing static mean pressure with constant laminar shear caused sustained intracellular acidification. Second, pulsatile perfusion at a constant mean flow and pressure induced intracellular alkalinization. The latter finding is intriguing, given that increasing steady laminar flow in nonpressurized or pressurized perfusate induced the opposite pHi change.6 7 Although pulsatile and steady shear stress are known to trigger different extents of endothelial responses in intracellular Ca2+,2 19 NO synthesis,20 21 the activities of NADH oxidase, heme oxygenase-1, and levels of Cu/Zn superoxide dismutase mRNA and protein,22 in none of these instances was signaling from one directionally opposite that of the other. This pHi response depended on differential effects of phasic versus steady shear on the principal acid-base exchangers. Whereas steady flow–induced acidosis was related to activation of the Na+o-independent Cl--HCO3- exchanger, pulsatile flow–induced alkalinization resulted from activation of both the Na+-H+ exchanger and the Na+o-dependent Cl--HCO3- exchanger. Differential signaling was further linked to pulse-flow activation of c-Src and ERK1/2, although the latter was involved with steady-flow pHi change as well.
Effects of Perfusion Pressure
Sumpio et al23 previously reported that similar
levels of constant pressure (up to 80 or 120 mm Hg) trigger BAEC
proliferation associated with changes in actin and microtubular
networks. More recently, this group found that somewhat higher static
and phasic pressures (135 mm Hg) inhibited cell
growth.3 Both sets of studies were performed in the
absence of flow. Pressure can also induce
endothelium-dependent
vasoconstriction24 25 by inhibiting NO
release26 and enhancing endothelin-1
release.27 These pressure-dependent changes appear
mediated at least in part by phospholipase C and protein kinase C
activation. It remains unknown whether pressure-induced intracellular
acidification, as observed in the present study, is coupled with
such signaling. However, regulatory effects of phospholipase C and
protein kinase C on both
HCO3--dependent acid/base
exchangers28 29 and the
Na+-H+
exchanger30 31 have been described.
Effects of Flow Pulsatility
In contrast to steady laminar flow, sustained intracellular
alkalinization induced by pulsatile flow was linked to both the
Na+o-dependent
Cl--HCO3-
exchanger and the Na+-H+
exchanger. Differential signaling associated with flow pulsatility has
been previously reported for Ca2+ and
NO,1 2 with a marked reduction in both responses to
oscillating flow, ie, fully symmetrical (sinusoidal) flow reversal. The
more physiological flow waveforms used in the
present study displayed typical asymmetrical patterns, with lower
reversing shears during what would be the diastolic phase.
This raised the possibility that the differential
pHi changes were related to flow reversal.
However, we observed similar results when higher mean flows and reduced
pulsatility prevented flow reversal. Further, flow elevation resulted
in less alkalinization. This may be due to a greater effect of mean
shear–dependent acidification or flow-dependent saturation of a
component of this response.
Prior studies have revealed a complex role of calcium in shear-induced signaling cascades. For example, Ayajiki et al9 reported that initial NO-induced vasorelaxation is Ca2+ dependent, whereas more sustained effects are Ca2+ independent. Shear stress activation of ERK1/2, which involves c-Src–tyrosine kinase,10 is reportedly Ca2+ independent,32 whereas c-Src–dependent phosphorylation of the p130 Crk-associated substrate (Cas), which may play a role in focal adhesions, is abolished by intracellular Ca2+ chelation.11 MAP kinase activation in response to steady shear stress is also reportedly Ca2+ independent.33 The finding that in Ca2+-free buffer with EGTA, pulse flow raised endothelial pHi even more than in the presence of normal Ca2+ suggests that although the latter may contribute to countering an acidification signal, it is not central to alkalinization.
Role of c-Src and MAP Kinase (ERK1/2)
The present data establish an important role for both
c-Src–family tyrosine kinases in pulsatile-flow
pHi signaling and ERK1/2 MAP kinase in both
pulsatile flow– and steady flow–induced pHi
changes. Activation of MAP kinase by steady shear has been linked to
upstream signaling via c-Src and
ß1-integrins/focal adhesion
kinases.11 34 35 MAP kinase can activate the
Na+-H+ exchanger to
contribute to alkalinization, and this can be triggered in
cardiomyocytes by mechanical stretch,14
endothelin,15 or hydrogen peroxide.16 Similar
activation due to pulsatile flow is supported by the present
findings, because both c-Src and MAP kinase inhibition prevented the
attendant alkalinization. However, the latter also resulted in
acidification, and furthermore, ERK1/2 but not c-Src inhibition
prevented or reversed acidification by steady flow. This suggests that
alternate pathways link the mechanical stimulus to ERK1/2 that can
critically determine its net effect on pHi. In
addition to the Na+-H+
exchanger, the Na+o-independent
Cl--HCO3-
exchanger can also be activated in cardiomyocytes
by ATP phosphorylation modulated by
c-Src.12 Neither of these pathways, to our knowledge, has
previously been shown to play a role in pHi
modulation due to steady or pulsatile shear stress.
Experimental Issues
Cellular alkalinization generally reversed despite continued
exposure to pulsatile flow, and this might suggest a limited influence
of such signals in physiological systems
continuously exposed to phasic shear. However, in vivo shear undergoes
frequent dynamic change from sympathetic stimulation, exertional
demand, etc. We also imposed pulsatility only after cells were already
acclimatized to steady flow and pressure. This is unlike most other in
vitro studies of shear stress signaling, in which different mechanical
stresses are generally imposed de novo on previously non–mechanically
stimulated cells.2 9 10 11 Because the
pHi signaling reported in the present study
occurs in cells already flow and pressure adapted, our findings may be
very relevant to the often-varying in vivo situation. Shear
stress–induced changes in pHi may be important
in vivo, given that pHi modulates NO release
stimulated by bradykinin or shear stress.8 9
pHi changes are also linked to vascular
proliferation in part via endothelial
production of smooth muscle cell mitogens such as
platelet-derived growth factor.36
Conclusion
The effects of pulsatile shear are only beginning to be realized
and yet may be more directly relevant to many
physiological and
pathophysiological conditions than the more
often–studied effects of steady laminar fluid shear stress. The
present study demonstrates for the first time that
pHi signaling is differentially modulated by
phasic versus steady shear stress and links this to a differential
signaling via c-Src tyrosine kinase and ERK1/2-kinase activity. Further
studies are needed to elucidate the specific nature of mechanical
stress required for this discrimination and the impact that such
changes ultimately have on endothelial physiology.
|
Footnotes |
|---|
1 Both authors contributed equally to this study.
Received December 14, 1999; accepted April 28, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Noris M, Morigi M, Donadelli R, Aiello S, Foppolo M, Todeschini M, Orisio S, Remuzzi G, Remuzzi A. Nitric oxide synthesis by cultured endothelial cells is modulated by flow conditions. Circ Res. 1995;76:536–543.
2.
Helmlinger G, Berk BC, Nerem RM. Calcium responses of
endothelial cell monolayers subjected to pulsatile and
steady laminar flow differ. Am J Physiol. 1995;269:C367–C375.
3. Vouyouka AG, Powell RJ, Ricotta J, Chen H, Dudrick DJ, Sawmiller CJ, Dudrick SJ, Sumpio BE. Ambient pulsatile pressure modulates endothelial cell proliferation. J Mol Cell Cardiol. 1998;30:609–615.[Medline] [Order article via Infotrieve]
4.
Canty JM, Schwartz JS. Nitric oxide mediates
flow-dependent epicardial coronary vasodilation to changes in
pulse frequency but not mean flow in conscious dogs.
Circulation. 1994;89:375–384.
5.
Recchia FA, Senzaki H, Saeki A, Byrne BJ, Kass DA.
Pulse pressure-related changes in coronary flow in vivo are
modulated by nitric oxide and adenosine. Circ Res. 1996;79:849–856.
6.
Ziegelstein RC, Cheng L, Capogrossi MC. Flow-dependent
cytosolic acidification of vascular endothelial cells.
Science. 1992;258:656–659.
7.
Ziegelstein RC, Blank PS, Cheng L, Capogrossi MC.
Cytosolic alkalinization of vascular endothelial cells
produced by an abrupt reduction in fluid shear stress. Circ
Res. 1998;82:803–809.
8.
Fleming I, Hecker M, Busse R. Intracellular
alkalinization induced by bradykinin sustains activation of the
constitutive nitric oxide synthase in endothelial
cells. Circ Res. 1994;74:1220–1226.
9.
Ayajiki K, Kindermann M, Hecker M, Fleming I, Busse R.
Intracellular pH and tyrosine phosphorylation but not
calcium determine shear stress-induced nitric oxide production
in native endothelial cells. Circ Res. 1996;78:750–758.
10. Takahashi M, Berk BC. Mitogen-activated protein kinase (ERK1/2) activation by shear stress and adhesion in endothelial cells: essential role for a herbimycin-sensitive kinase. J Clin Invest. 1996;98:2623–2631.[Medline] [Order article via Infotrieve]
11.
Okada M, Takahashi M, Suero J, Murry CE, Traub O,
Kawakatsu H, Berk BC. Shear stress stimulation of p130(cas) tyrosine
phosphorylation requires calcium-dependent c-Src
activation. J Biol Chem. 1999;274:26803–26809.
12.
Puceat M, Roche S, Vasort G. Src family tyrosine kinase
regulates intracellular pH in cardiomyocytes. J
Cell Biol. 1998;141:1637–1646.
13. Thomson S, Mahadevan LC, Clayton AL. MAP kinase-mediated signalling to nucleosomes and immediate-early gene induction. Semin Cell Dev Biol. 1999;10:205–214.[Medline] [Order article via Infotrieve]
14.
Yamazaki T, Komuro I, Kudoh S, Zou Y, Nagai R, Aikawa
R, Uozumi H, Yazaki Y. Role of ion channels and exchangers in
mechanical stretch-induced cardiomyocyte
hypertrophy. Circ Res. 1998;82:430–437.
15.
Moor AN, Fliegel L. Protein kinase-mediated regulation
of the Na+/H+ exchanger in
the rat myocardium by mitogen-activated protein
kinase-dependent pathways. J Biol Chem. 1999;274:22985–22992.
16.
Sabri A, Byron KL, Samarel AM, Bell J, Lucchesi PA.
Hydrogen peroxide activates mitogen-activated protein
kinases and Na+-H+ exchange in neonatal rat
cardiac myocytes. Circ Res. 1998;82:1053–1062.
17. Xingqi P, Recchia FA, Byrne, BJ, Wittstein IS, Ziegelstein RC, Kass DA. A novel in vitro system for generating realistic arterial pressure/flow waveforms to study pulse-perfusion signaling of endothelial cells. Am J Physiol. In press.
18.
Blank PS, Silverman HS, Chung OY, Hogue BA, Stern MD,
Hansford RG, Lakatta EG, Capogrossi MC. Cytosolic pH measurements in
single cardiac myocytes using carboxy-seminaphthorhodafluor-1.
Am J Physiol. 1992;263:H276–H284.
19. Helmlinger G, Berk BC, Nerem RM. Pulsatile and steady flow-induced calcium oscillations in single cultured endothelial cells. J Vasc Res. 1996;33:360–369.[Medline] [Order article via Infotrieve]
20.
Ziegler T, Bouzourene K, Harrison VJ, Brunner HR, Hayoz
D. Influence of oscillatory and unidirectional flow environments on the
expression of endothelin and nitric oxide synthase in cultured
endothelial cells. Arterioscler Thromb Vasc
Biol. 1998;18:686–692.
21.
Ziegler T, Silacci P, Harrison VJ, Hayoz D. Nitric
oxide synthase expression in endothelial cells exposed
to mechanical forces. Hypertension. 1998;32:351–355.
22.
De Keulenaer GW, Chappell DC, Ishizaka N, Nerem RM,
Alexander RW, Griendling KK. Oscillatory and steady laminar shear
stress differentially affect human endothelial redox
state: role of a superoxide-producing NADH oxidase. Circ
Res. 1998;82:1094–1101.
23. Sumpio BE, Widmann MD, Ricotta J, Awolesi MA, Watase M. Increased ambient pressure stimulates proliferation and morphologic changes in cultured endothelial cells. J Cell Physiol. 1994;158:133–139.[Medline] [Order article via Infotrieve]
24.
Harder DR, Sanchez-Ferrer C, Kauser K, Stekiel WJ,
Rubanyi GM. Pressure releases a transferable
endothelial contractile factor in cat cerebral
arteries. Circ Res. 1989;65:193–198.
25.
Rubanyi GM. Endothelium-dependent
pressure-induced contraction of isolated canine carotid arteries.
Am J Physiol. 1988;255:H783–H788.
26. Hishikawa K, Nakaki T, Suzuki H, Saruta T, Kato R. Transmural pressure inhibits nitric oxide release from human endothelial cells. Eur J Pharmacol. 1992;215:329–331.[Medline] [Order article via Infotrieve]
27.
Hishikawa K, Nakaki T, Marumo T, Suzuki H, Kato R,
Saruta T. Pressure enhances endothelin-1 release from cultured human
endothelial cells. Hypertension. 1995;25:449–452.
28. Ludt J, Tonnessen TI, Sandvig K, Olsnes S. Evidence for involvement of protein kinase C in regulation of intracellular pH by Cl-/HCO3- antiport. J Membr Biol. 1991;119:179–186.[Medline] [Order article via Infotrieve]
29. Ruiz OS, Qiu YY, Cardoso LR, Arruda JA. Regulation of the renal Na/HCO3 cotransporter, VII: mechanism of the cholinergic stimulation. Kidney Int. 1997;51:1069–1077.[Medline] [Order article via Infotrieve]
30. Kitazono T, Takeshige K, Cragoe EJ Jr, Minakami S. Involvement of calcium and protein kinase C in the activation of the Na+/H+ exchanger in cultured bovine aortic endothelial cells stimulated by extracellular ATP. Biochim Biophys Acta. 1989;1013:152–158.[Medline] [Order article via Infotrieve]
31.
Yasutake M, Haworth RS, King A, Avkiran M. Thrombin
activates the sarcolemmal
Na+-H+ exchanger. Evidence
for a receptor-mediated mechanism involving protein kinase C.
Circ Res. 1996;79:705–715.
32.
Tseng, H, Peterson T, Berk BC. Fluid shear stress
stimulates mitogen-activated protein kinases in bovine aortic
endothelial cells. Circ Res. 1995;77:869–878.
33. Stamatas GN, Patrick CW Jr, McIntire LV. Intracellular pH changes of human aortic smooth muscle cells in response to fluid shear stress. Tissue Eng. 1997;3:391–403.[Medline] [Order article via Infotrieve]
34.
Ishida T, Peterson TE, Kovach NL, Berk BC. MAP kinase
activation by flow in endothelial cells: role of
ß1 integrins and tyrosine kinases. Circ Res. 1996;79:310–316.
35.
Li S, Kim M, Hu YL, Jalali S, Schlaepfer DD, Hunter T,
Chien S, Shyy JYJ. Fluid shear stress activation of focal adhesion
kinase. J Biol Chem. 1997;272:30455–30462.
36.
Shankar S, de la Motte CA, Dicorleto PE. Thrombin
stimulates PDGF production and monocyte adhesion through
distinct intracellular pathways in human endothelial
cells. Am J Physiol. 1992;262:C199–C206.
This article has been cited by other articles:
 |
|
 |
|
  C. J. Taylor, P. A. Nicola, S. Wang, M. A. Barrand, and S. B. Hladky Transporters involved in regulation of intracellular pH in primary cultured rat brain endothelial cells J. Physiol., November 1, 2006; 576(3): 769 - 785. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-P. Qiu, Q. Hu, N. Paolocci, R. C. Ziegelstein, and D. A. Kass Differential effects of pulsatile versus steady flow on coronary endothelial membrane potential Am J Physiol Heart Circ Physiol, June 5, 2003; 285(1): H341 - H346. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Peng, F. A. Recchia, B. J. Byrne, I. S. Wittstein, R. C. Ziegelstein, and D. A. Kass In vitro system to study realistic pulsatile flow and stretch signaling in cultured vascular cells Am J Physiol Cell Physiol, September 1, 2000; 279(3): C797 - C805. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||