Opposite Effects of Pressurized Steady Versus Pulsatile Perfusion on Vascular Endothelial Cell Cytosolic pH
Role of Tyrosine Kinase and Mitogen-Activated Protein Kinase Signaling
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.
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
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.
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 × 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.
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.
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 (τ) was calculated as τ=4 μQ/πr,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 ΔpHi 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.
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, ΔpHi 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.
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 β1-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.
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
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.
↵1 Both authors contributed equally to this study.
- Received December 14, 1999.
- Accepted April 28, 2000.
- © 2000 American Heart Association, Inc.
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