Intracellular pH (pHi) regulation in human umbilical vein endothelial cells (HUVEC) was investigated. The pHi was recorded using seminaphthorhodafluor-1 (SNARF-1). Cells were intracellularly acid loaded with NH4Cl prepulse. In HEPES-buffered Tyrode (nominally free), pHi recovery from acid load was inhibited by 1.5 mM amiloride or Na+-free solution. Additionally, in -buffered Tyrode, a -dependent pHi recovery from acidosis was evident in the presence of 1.5 mM amiloride, which mediated complete recovery of pHi (7.26). In Na+-free solution, the -dependent acid extruder mediated pHi recovery after an acid load but only back to 7.09. These results suggest that there are two -dependent acid extruders in the HUVEC. One is Na+ dependent, and the other is Na+ independent. The former was further shown to be completely inhibited by 0.5 mM DIDS, whereas the latter was only inhibited by 24.6%. In Cl−-free solution, both of the -dependent pathways were inhibited. In conclusion, one -dependent acid extruder in the HUVEC resembles the Na+-dependent Cl−/ exchange found in other tissues, and the other is Cl− dependent but Na+ independent.
- intracellular pH
- human umbilical vein endothelial cells
- sodium/hydrogen exchange
- chloride/bicarbonate exchange
endothelium is an essential component of the vascular wall and plays a fundamental role in hemostasis. Particularly, endothelial cells play an important role in maintaining vascular tone by releasing vasodilators such as endothelium-derived relaxing factor or nitric oxide (8, 19) and prostacyclin (18), as well as releasing vasoconstrictors such as endothelin (30). Changes of intracellular pH (pHi) affect the enzyme activity of nitric oxide synthase, endothelin-converting enzyme in endothelial cells, and further affect their release (1, 17). pH was also found to mediate channel activities in endothelial plasma membrane, including the opening of calcium channels and calcium-activated potassium channels (24, 28), as well as affect calcium homeostasis inside the cells (32). Because of direct contact with flowing blood, endothelial cells are exposed to a wide diversity of physical and chemical stimuli, and pHi is constantly affected.
It has been shown that hemodynamic shear stress can lead to a decrease in pHi in rat aortic endothelial cells (33). Reducing the pH of perfusion solution can also cause a decrease in pHi in cultured human umbilical vein endothelial cells (HUVEC; unpublished observation). It is therefore important to understand the pHi regulation mechanisms in endothelial cells. Previous studies have demonstrated the existence of Na+/H+exchange as an acid extruder in HUVEC (7, 9) and Na+-independent Cl−/ exchange as an acid loader in rat aortic endothelial cells (33). -dependent acid extrusion pathways were found to play an important role in pHi regulation in many other cell types (3, 12, 27). Recently, Na+-dependent Cl−/ exchange as an acid extruder has been shown to function in piglet cerebral microvascular endothelial cells (10) and rat aortic endothelial cells (31). However, little is known about -dependent acid extrusion mechanisms in the HUVEC. We therefore studied acid efflux of cultured HUVEC in as well as in nominally -free HEPES buffers in the present study.
Isolation and culture of HUVEC.
HUVEC were obtained by collagenase (Sigma Chemical, Poole, UK) digestion of human umbilical vein using the method described previously (26). The cells were cultured in medium 199 with Earle’s salts (ICN Biochemicals), containing 20% FCS, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 0.01% heparin. HUVEC were subcultured using trypsin-EGTA, and seeded on gelatin-coated coverslips (6-mm diam, Chance Propper). Fourth to eighth passaged HUVEC were used for pHi measurements. Twenty-four hours before the measurement, culture medium containing 20% FCS was removed and replaced by 1% FCS to minimize growth factor-mediated Na+/H+exchange activity (20).
Measurement of pHi.
The pHi of a single HUVEC was measured using carboxyseminaphthorhodafluor-1 (SNARF-1), a dual-emission fluoroprobe (4). One coverslip with HUVEC was placed at the bottom of the perfusion chamber, and cells were loaded by incubation in a 5 μM solution of SNARF-1-acetoxymethyl ester at room temperature for 15 min. Perfusion was then started at a constant flow rate of ∼2.3 ml/min. The chamber temperature was maintained at 37°C. Individual cells were excited at 540 ± 12 nm, and fluorescence was measured simultaneously at 590 ± 5 and 640 ± 5 nm using an inverted microscope (Nikon Diaphot) converted for epifluorescence. The signals were then digitized at 0.5 kHz (CED1401). The emission ratio of 590/640 was calculated and converted to a linear pH scale using in situ calibration data from the nigericin technique (4). Finally, the pHi signal was averaged over 0.5-s intervals.
HEPES-buffered Tyrode contained (in mM) 140 NaCl, 4.5 KCl, 2.5 CaCl2, 1 MgCl2, 11 glucose, and 20 HEPES, pH adjusted to 7.4 at 37°C with NaOH. In -buffered Tyrode, NaCl concentration was reduced to 117 mM and HEPES was replaced with 23 mM NaHCO3. After initial review, more experiments were carried out in the -buffered Tyrode plus 12 mM NaCl to maintain constant osmolarity between the HEPES-buffered and -buffered Tyrode. The data obtained in this osmolarity- compensated buffer [e.g., resting pHi 7.27 ± 0.02,n = 7; H+ efflux (J H) 2.80 ± 0.48, n = 4] showed no significant difference from those in original buffer (seeresults). In Na+-free Tyrode, Na+ was replaced withN-methyl-d-glucamine. In Cl−-free buffer, Na-gluconate, K-gluconate, Ca-gluconate (12 mM), and MgSO4 were used. All -buffered solutions were equilibrated with 5% CO2-95% air and had a pH of 7.4 at 37°C. NH4Cl, amiloride (or dimethyl amiloride, DMA), and DIDS (Sigma) were added to the solutions without osmotic compensation.
In nominally -free HEPES-buffered Tyrode (extracellular pH 7.4), steady-state pHi of a single HUVEC used in the study was 7.21 ± 0.01 (n = 57) at 37°C. After intracellular acid loads, induced by the NH4Cl-removal method (21), pHi in the cell recovered to resting level in a pHi-dependent manner. The recovery was inhibited by 1.5 mM amiloride (n = 8; Fig.1 A), a classical Na+/H+exchanger inhibitor (6), or in Na+-free Tyrode (n = 15; Fig.2 A), consistent with Na+/H+exchange activity in these cells as reported previously (7, 9).
In 5% CO2, 23 mM -buffered Tyrode, the steady-state pHi in individual HUVEC was 7.26 ± 0.02 (n = 25), slightly more alkaline than that in HEPES buffer (7.21 ± 0.01). These values are similar to those of rat aortic endothelial cells reported in a previous study (7.27 ± 0.02 and 7.22 ± 0.03, respectively) (33). This suggests that -dependent pH regulation mechanisms, like in many other tissues, may play a major role in maintaining resting pHilevel in endothelial cells under physiological conditions. Incubating the cells in Na+-free buffer decreased the resting pHi by 0.09 ± 0.02 (restabilized at 7.16 ± 0.02, n = 5; data not shown). Figure 1 A shows one of eight experiments where pHirecovery after an acid load was inhibited by 1.5 mM amiloride in HEPES-buffered Tyrode. However, after the perfusion solution was changed to -buffered Tyrode, pHi started to recover in the presence of 1.5 mM amiloride following a transient drop [due to hydration of diffused CO2 into the cell. This process equilibrated quickly, as shown in Fig. 1, because of the presence of carbonic anhydrase in the HUVEC (15,16)]. This suggests that there are also -dependent acid extruders in the HUVEC, as found in other types of cells (3, 12, 27). pHi recovery through the -dependent pathway was able to bring pHi back to resting level.
To assess and compareJ H by different pathways, the intracellular intrinsic buffering power (βi) of individual cultured HUVEC was estimated using a method of stepwise reduction of external (from 20 mM,n = 7) (12). βi (in mM) equals Δ[ ]i/ΔpHi, where [ ]iequals ([H+]i× [ ]o)/[H+]o(i means intracellular and o means extracellular for these concentrations.). The βi in HUVEC was plotted as a function of pHi and fitted by linear equation (least squares; r = 0.95). βi equals −3.65 pHi plus 41.10.
The pHi recovery in HEPES buffer (through Na+/H+exchange) and in buffer in the presence of amiloride (through -dependent acid extruders) in the experiment shown in Fig. 1 A was further plotted withJ H vs. pHi.J H was calculated according to the equation used in a previous study (J H = βtotal ⋅ dpHi/dt, where dpHi/dtis change in pHi over time; see details in legend) (12). From the linear-fitted data shown in Fig.1 B, theJ H through -dependent pathways was faster than that of Na+/H+exchange over the entire pHirange. Pooled data from a total of six similar experiments showed thatJ H through -dependent pathways was on average nearly 1.5-fold greater than Na+/H+exchange (2.73 ± 0.36 vs. 1.85 ± 0.36 meq/min, respectively) at mean pHi of 7.04. These data suggested that under physiological conditions -dependent acid extruders may contribute more than one-half in pHi recovery from intracellular acidosis in the HUVEC.
In the absence of extracellular Na+, dependent pHi recovery after an acid load was not fully inhibited (Fig. 2); pHi recovered but only back to 7.09 ± 0.03 (n = 20). For the experiments as shown in Fig. 2 B, pHi was allowed to restabilize at resting level after switching to buffer before acid loading was applied, so that the experiments were carried out in a steady state for equilibrium. These results suggest that there are probably two -dependent acid extrusion pathways in HUVEC. One is dependent on extracellular Na+ and can mediate the pHi recovery fully back to resting level (7.26 ± 0.02), and the other is independent of extracellular Na+ but can only mediate partial pHi recovery. The acid efflux (J H) through the latter was calculated to be 1.94 ± 0.47 meq/min at pHi of 7.0, compared with 2.92 ± 0.67 meq/min for total -dependent acid extrusion pathways (n = 4).
DIDS, a stilbene-derived anion exchanger inhibitor, has been shown to inhibit to a varying extent Cl−/ exchange (12), Na+-dependent Cl−/ exchange (23), and Na+- symport (12). We studied the effect of DIDS on the -dependent acid extruders in HUVEC. DIDS (0.5 mM) completely inhibited pHi recovery, at pHi of 7.08 in -buffered Tyrode, in the presence of amiloride or DMA [a more potent and relatively selective amiloride analog for the inhibition of Na+/H+exchange (14)], suggesting inhibition of the Na+-dependent pathway (n = 3; Fig.3). The same concentration of DIDS, however, inhibited pHi recovery after an acid load by only 24.61 ± 0.42% at mean pHi of 6.97 in Na+-free -buffered Tyrode (n = 5, data not shown). This suggests that the Na+-independent pathway in HUVEC is less sensitive to DIDS.
We next tested the Cl−dependency of the dependent acid extrusion pathways in the HUVEC. As shown in Fig.4 A, intracellular alkalinization occurred after the perfusion solution was changed to Cl−-free Tyrode (probably due to activation of influx in exchange of Cl−efflux via Cl−/ exchange). pHirecovery mediated by the -dependent acid extruders in HUVEC was gradually slowed during consecutive acid loads, as intracellular Cl− was depleted; the pHi after each recovery was gradually elevated. The same results were obtained in nine other experiments. After extracellular Cl− was returned to its normal concentration, pHi recovery speeded up as intracellular Cl− was replenished. These results suggest that both -dependent acid extrusion pathways in HUVEC depend on intracellular Cl−. Furthermore, after a long incubation in both Na+- and Cl−-free buffer, in which only the Na+-independent pathway works, pHi recovery after an acid load was fully inhibited (Fig. 4 B,n = 5). This indicates that the Na+-independent -dependent acid extrusion pathway is also Cl− dependent. Finally, it was found that this novel Na+-independent but Cl−- and -dependent pathway was inhibited by 88.04 ± 4.76% in 145 mM K+Tyrode solution (n = 7).
In the present study we demonstrated that in addition to Na+/H+exchange there are two dependent acid extrusion pathways in the HUVEC. Both Na+/H+exchange and -dependent acid extrusion pathways may play a major role in pHi recovery in intracellular acidosis and in maintaining basal level pHi under physiological conditions. The Na+- and Cl−-dependent pathway, which can bring pHi back to resting levels after an acid load, resembles the Na+-dependent Cl−/ exchange found in squid giant axons (3), snail neurons (27), and the chicken heart (13). The other -dependent acid extruder in the HUVEC, which is Cl−dependent but Na+ independent, has not been documented.
It has been reported in ureter smooth muscle cells that under extreme intracellular acidosis, conductance could contribute to pHi recovery (2). However, it is unlikely that the Cl−- and -dependent but Na+-independent pHi recovery we observed in the HUVEC could be due to influx through channels. The resting membrane potential of HUVEC has been estimated at 55.3 mV using perforated patch-clamp technique (22). At this membrane potential, the calculated pHi equilibrium for is pH 6.50, which means that influx of can only occur at pHi below this value. On the contrary, the Na+-independent acid extrusion pathway in HUVEC can actually bring pHi back to 7.09 ± 0.03. Furthermore, if conductance could function as a base loader in the HUVEC, it should be enhanced by depolarization of membrane potential in a high-K+ solution. Instead, it was found that raising extracellular K+ inhibited the pHi recovery. Therefore, the possibility of influx through a channel to work as an alkaline loader in HUVEC can be ruled out.
As previously shown in sheep cardiac Purkinje strands (29) and ureter smooth muscle cells (2), Cl−/ exchange was not reversed to contribute to acid extrusion. Taking typical values of 150 mM for extracellular Cl−, 30 mM for intracellular Cl− [the intracellular Cl− concentration in HUVEC was calculated to be ∼35 mM according to Cl− reversal potential (22), although no data are available from direct measurement], and 7.40 for extracellular pH, the predicted equilibrium pHi is 6.70 for Cl−/ exchange. Thus Cl−/ exchange would not be at equilibrium in a resting HUVEC with normal extracellular pH and pHi>6.70. It would be energized to mediate efflux in exchange for Cl− influx to act as an acid loader as reported previously in rat aortic endothelial cells (33). Actually, what we have found was that pHi recovered back to 7.09 ± 0.03 after an acid load through the Na+-independent and -dependent acid extrusion pathway. Only in the absence of extracellular Cl−, as shown in Fig. 4, did the reversal of Cl−/ exchange occur, promoting Cl− efflux and influx and causing alkalization. Therefore, it is less likely that Cl−/ exchange works in a reversed mode as a possible mechanism for the Na+-independent but Cl−- and -dependent pHi recovery in HUVEC.
Recently, we have observed the involvement of carbonic anhydrase in rapid local pHi regulation in cardiac myocyte, using a fast flow exchange perfusion chamber (unpublished observation). Vascular endothelial cells including HUVEC have been known to express several types of carbonic anhydrase in and/or on the surface of the cells (15, 16). Kurtz (11) demonstrated a plasma membrane H+-ATPase in rabbit S3 proximal tubule, which can regulate pHi in a Na+-independent manner but requires intracellular Cl−. Chen and Boron (5) showed that CO2/ can stimulate a H+ pump in the same tissue, which explained the pHi recovery mediated by a Na+-independent pathway in buffer. We have recently reported that lowering extracellular pH can mediate a fall of pHi in guinea pig ventricular myocyte through a novel Cl−-dependent but Na+-independent and DIDS-insensitive pathway (25). Whether the same or a similar mechanism as mentioned previously operates in the HUVEC to account for the Cl−-dependent but Na+-independent pHi recovery remains unclear. Further studies are needed to confirm the properties of the - and Cl−-dependent but Na+-independent acid extrusion pathway found in HUVEC in this study.
This work was supported in part by the British Heart Foundation.
Address for reprint requests and other correspondence: B. Sun, Thrombosis and Vascular Biology, Maryland Research Laboratories, Otsuka America Pharmaceutical, 9900 Medical Center Dr., Rockville, MD 20850 (E-mail:).
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- Copyright © 1999 the American Physiological Society