The Effect of Extracellular pH Changes on Intracellular pH and Nitric Oxide Concentration in Endothelial and Smooth Muscle Cells from Rat Aorta

Abstract

Aims

It has been known for more than a century that pH changes can alter vascular tone. However, there is no consensus about the effects of pH changes on vascular response. In this study, we investigated the effects of extracellular pH (pH_o) changes on intracellular pH (pH_i) and intracellular nitric oxide concentration ([NO]_i) in freshly isolated endothelial cells and cross sections from rat aorta.

Main Methods

The HCl was used to reduce the pH_o from 7.4 to 7.0 and from 7.4 to 6.5; the NaOH was used to increase the pH_o from 7.4 to 8.0 and from 7.4 to 8.5. The fluorescent dyes 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate (SNARF-1) and diaminofluorescein-FM diacetate (DAF-FM DA) were employed to measure the pH_i and [NO]_i, respectively. The fluorescence intensity was measured in freshly isolated endothelial cells by flow cytometry and in freshly obtained aorta cross sections by confocal microscopy.

Key Findings

The endothelial and vascular smooth muscle pH_i was increased at pH_o 8.5. The extracellular acidification did not change the endothelial pH_i, but the smooth muscle pH_i was reduced at pH_o 7.0. At pH_o 8.5 and pH_o 6.5, the endothelial [NO]_i was increased. Both extracellular alkalinization and acidification increased the vascular smooth muscle [NO]_i.

Significance

Not all changes in pH_o did result in pH_i changes, but disruption of acid-base balance in both directions induced NO synthesis in the endothelium and/or vascular smooth muscle.

Introduction

It has been known for more than a century that pH changes can alter vascular tone and thereby influence the circulation and blood pressure. Gaskell was probably the first to show the importance of pH in modulating the vascular tone. Studying mylohyoid muscle arteries and mesenteric arteries from frog, he demonstrated that acidification increased the vascular diameter while alkalinization decreased this diameter [1]. It has also been known that both extracellular pH (pH_o) and intracellular pH (pH_i) can alter vascular tone and that they influence each other [2], [3]. However, the following areas remain unclear: the expected vascular response to pH reduction or augmentation; the mechanisms responsible for pH-induced vasodilation or constriction; whether pH_o changes pH_i; and which of the compartment pH is the major modulator of the vascular tone.

Considering there is no consensus about the effects of pH changes on vascular response we have developed a research strategy to address this issue and have demonstrated that extracellular alkalinization causes endothelium-dependent relaxation through the nitric oxide (NO) pathway in rat aorta [4]. On the other hand, rat aorta response to extracellular acidification is more complex and involves endothelium-independent relaxation through NO and hyperpolarization pathways [5]. In an attempt to better understand these previous findings showing the role of NO in the relaxation induced by changes in pH_o, the present study was carried out to investigate the effects of pH_o changes on pH_i and intracellular NO concentration ([NO]_i) in freshly isolated endothelial cells and freshly obtained cross sections from rat aorta.

Materials and Methods

1. Materials

HEPES, NaOH, nigericin and poly-L-lysine were purchased from Sigma (St. Louis, MO, USA). The probes 5-(and-6)-carboxy SNARF-1, acetoxymethyl ester, acetate (SNARF-1) and diaminofluorescein-FM diacetate (DAF-FM DA) were acquired from Invitrogen (Carlsbad, CA, USA). HCl was purchased from Zilquímica (Ribeirão Preto, SP, Brazil). Thiopental sodium was purchased from Cristália (São Paulo, SP, Brazil). All the other salts were obtained from Vetec Química Fina (Duque de Caxias, RJ, Brazil). All drugs were prepared with distilled water.

2. Experimental design

Freshly isolated endothelial cells and freshly obtained cross sections from rat aorta were exposed to pH_o changes for analyzing pH_i and [NO]_i by flow cytometry and confocal microscopy. The experimental protocol was designed to mimic metabolic alkalosis or acidosis, as we have previously done [4], [5]. Then, the extracellular alkalinization was induced by NaOH, whilst the extracellular acidification was induced by HCl. The most popular method for measuring pH_i has involved the use of pH-sensitive fluoroprobes [6], and we choose SNARF-1. DAF-FM DA, a selective NO fluorescent probe, was chosen because it exhibits a stable fluorescence intensity in a large range of pH (above pH 5.8) [7].

3. Animals

The experimental procedures and animal handling were reviewed and approved by the Institutional Committee for Animal Care and Use of the School of Medicine of Ribeirão Preto, University of São Paulo, and were in accordance with the Directive 2010/63/EU (European Commission). Rats were housed under standard laboratory conditions (12 h light/dark cycle at 21°C), with free access to food and water.

Male Wistar rats (230–280 g) were anesthetized with thiopental sodium (40 mg/kg, intraperitoneal injection), and underwent laparotomy for exsanguination via the abdominal aorta and thoracotomy for thoracic aorta harvesting. The thoracic aorta was carefully dissected free of connective tissue and immediately immersed in Hanks solution (composition [mM]: NaCl 145.0, KCl 5.0, CaCl₂ 1.6, NaH₂PO₄ 0.5, MgCl₂ 0.5, dextrose 10.0, HEPES 10.0; pH 7.4) to perform cytofluorographic and confocal microscopy analyses.

4. Endothelial pH_i and [NO]_i measurement by flow cytometry

The thoracic aorta, immersed in Hanks solution, was longitudinally opened, and the endothelial cells were isolated by gentle rake friction. The Hanks solution containing the isolated cells was centrifuged at 200 g for 2 min, and the cells were resuspended in 1 mL of Hanks buffer. The cells were then loaded with SNARF-1 (10 µM) or DAF-FM DA (5 µM) and maintained in a humidified 37°C incubator gassed with 5% CO₂ for 30 or 20 min, respectively. The cytofluorographic analysis was performed using a FACScan (Becton-Dickinson, San Jose, CA, USA): the fluorescence was excited with the 488 nm line of an argon ion laser for both dyes, and the emitted fluorescence was measured at 580 nm for SNARF-1 in the acidification experiments, 640 nm for SNARF-1 in the alkalinization experiments, and 515 nm for DAF-FM DA. The fluorescence intensity was evaluated using CellQuest 1.2 software (Becton-Dickinson, Franklin Lakes, NJ, USA). The fluorescence intensity was measured before the unique stimulus and at different time points (t = 1, 3, 9 and 15 min) after the unique stimulus with HCl or NaOH. HCl was used to decrease the pH of Hanks solution containing endothelial cells from 7.4 to 7.0 (final concentration of HCl≈6 mM) and from 7.4 to 6.5 (final concentration of HCl≈10 mM). NaOH was used to increase the pH of Hanks solution containing endothelial cells from 7.4 to 8.0 (final concentration of NaOH≈15 mM) and from 7.4 to 8.5 (final concentration of NaOH≈27 mM); Hanks solution, at pH 7.4, served as control. The fluorescence intensity before the stimulus was designated F₀, and the fluorescence intensity after the stimulus for each time point was designated F_t. In this way, the difference in fluorescence intensity (ΔF = F_t - F₀) was obtained for each time point.

Before starting the pH_i measurement with SNARF-1, we evaluated the specificity of this dye for acid and alkali stimuli using nigericin and NH₄Cl at different emitted fluorescence wavelengths (580 and 640 nm), respectively.

5. Aorta pH_i and [NO]_i measurement by confocal microscopy

The fresh aorta cross sections (100 µm thick) were placed on a poly-L-lysine-coated slide. The tissue was loaded with 10 µM SNARF-1 or 5 µM DAF-FM DA and maintained in a humidified 37°C incubator gassed with 5% CO₂ for 30 or 20 min, respectively. The pH_i and [NO]_i were assessed using a confocal scanning laser microscope (Leica TCS SP2, Leica Microsystems CMS GmbH, Mannheim, Baden-Württemberg, Germany). The fluorescence was excited with the 488 nm line of an argon ion laser for both dyes, and the emitted fluorescence was measured at 580 nm for SNARF-1 in the acidification experiments, 640 nm for SNARF-1 in the alkalization experiments, and 515 nm for DAF-FM DA. Time-course software was used to capture cross sections images at intervals of 2 s (xyt) in Live Data Mode acquisition at 512×512 pixels at 700 Hz. Aorta cross sections were stimulated at 3^rd and 9^th min with Hanks acidified or alkalinized solution and cross sections images were captured during 15 min. From 0 to 3^rd min the cross sections were immersed in Hanks solution (pH 7.4), from 3^rd to 9^th min the cross sections were immersed in Hanks acidified (pH 7.0) or alkalinized (pH 8.0) solution, and from 9^th to 15^th min the cross sections were immersed in Hanks acidified (pH 6.5) or alkalinized (pH 8.5) solution. We used HCl or NaOH to change the pH of the Hanks solution; Hanks solution, at pH 7.4, served as the control. Using the Leica Microsystem LAS AF software (Leica Microsystems CMS GmbH, Mannheim, Baden-Württemberg, Germany), the fluorescence intensity was measured in the endothelial and the smooth muscle layers, separately. The fluorescence intensity before the stimulus (immediately before the 3^rd min) was designated F, and the fluorescence intensity at 6^th min after the stimulus for each pH value was designated F_pH. In this way, the difference in fluorescence intensity (ΔF = F_pH - F) was obtained for each pH value.

6. Statistical analysis

The data are expressed as means ± SEM. The statistical analysis was performed using two-way repeated-measures ANOVA or one-way ANOVA and Bonferroni post-test (Prism 4.0, GraphPad Software, San Diego, CA, USA). p values lower than 0.05 were considered statistically significant.

Results

1. Effect of extracellular alkalinization on pH_i in isolated endothelial cells

NaOH-induced extracellular alkalinization increased the SNARF-1 ΔF (pH 8.5 compared with pH 7.4), showing that extreme extracellular alkalinization increased the pH_i while less severe pH_o augmentation did not change the pH_i in freshly isolated endothelial cells (Figure 1).

Figure 1. Effect of extracellular alkalinization on pH_i in isolated endothelial cells from rat aorta.

Cells were loaded with SNARF-1 (10 µM) and analyzed by flow cytometry. NaOH was used to increase pH_o from 7.4 to 8.0 and from 7.4 to 8.5; Hanks solution pH 7.4 served as control. Fluorescence intensity was measured before the NaOH stimulus (F₀) and at different time points (t = 1, 3, 9 and 15 min) after this stimulus (F_t). Results are reported as ΔF = F_t - F₀. All values are means ± SEM (n = 7). Two-way ANOVA, Bonferroni's post-test, * p<0.05 versus control.

2. Effect of extracellular alkalinization on pH_i and [NO]_i in aorta cross sections

Confirming the result above, the pH_o 8.5 increased the SNARF-1 ΔF in the endothelial layer of fresh aorta cross sections while the pH_o 8.0 did not result in a change (Figures 2 A and 2 C). Furthermore, the result was the same in the muscular layer: the pH_o 8.5 increased the SNARF-1 ΔF while the pH_o 8.0 did not change it (Figures 2 B and 2 C).

Figure 2. Effect of extracellular alkalinization on pH_i of rat aorta cross sections assessed using a confocal scanning laser microscope.

A) Fluorescence intensity for endothelial layer. B) Fluorescence intensity for muscular layer. C) Representative confocal photomicrograph of one aorta cross section. Aorta cross sections were loaded with SNARF-1 (10 µM) and analyzed by confocal microscopy. Aorta cross sections were stimulated at 3^rd and 9^th min with Hanks alkalinized solution pH 8.0 and 8.5, respectively. NaOH was used to change the pH of Hanks solution; Hanks solution pH 7.4 served as control. Fluorescence intensity was measured before the stimulus (F) and at 6^th min after the stimulus for each pH_o value (F_pH). Results are reported as ΔF = F_pH – F. All values are means ± SEM (n = 7). One-way ANOVA, Bonferroni's post-test, ^# p<0.001 versus control, * p<0.05 versus control.

In the endothelial layer of freshly obtained aorta cross sections, the pH_o 8.0 reduced while the pH_o 8.5 increased the DAF-FM DA ΔF (Figures 3 A and 3 C). However, in the muscular layer, both pH_o values increased the DAF-FM DA ΔF (Figures 3 B and 3 C). These data indicate that, at pH_o 8.0, a reduction in the endothelial [NO]_i was observed simultaneously with an increase in muscular [NO]_i, and that, at pH_o 8.5, the [NO]_i increased in both vessel layers.

Figure 3. Effect of extracellular alkalinization on [NO]_i of rat aorta cross sections assessed using a confocal scanning laser microscope.

A) Fluorescence intensity for endothelial layer. B) Fluorescence intensity for muscular layer. C) Representative confocal photomicrograph of one aorta cross section.. Aorta cross sections were loaded with DAF-FM DA (5 µM) and analyzed by confocal microscopy. Aorta cross sections were stimulated at 3^rd and 9^th min with Hanks alkalinized solution pH 8.0 and 8.5, respectively. NaOH was used to change the pH of Hanks solution; Hanks solution pH 7.4 served as control. Fluorescence intensity was measured before the stimulus (F) and at 6^th min after the stimulus for each pH_o value (F_pH). Results are reported as ΔF = F_pH – F. All values are means ± SEM (n = 7). One-way ANOVA, Bonferroni's post-test, ^# p<0.001 versus control, * p<0.05 versus control.

3. Effect of extracellular acidification on pH_i in isolated endothelial cells

HCl-induced extracellular acidification had no effect on the SNARF-1 fluorescence, showing that extracellular acidification did not change the pH_i in freshly isolated endothelial cells (Figure 4).

Figure 4. Effect of extracellular acidification on pH_i in isolated endothelial cells from rat aorta.

Cells were loaded with SNARF-1 (10 µM) and analyzed by flow cytometry. HCl was used to reduce pH_o from 7.4 to 7.0 and from 7.4 to 6.5; Hanks solution pH 7.4 served as control. Fluorescence intensity was measured before the HCl stimulus (F₀) and at different time points (t = 1, 3, 9 and 15 min) after this stimulus (F_t). Results are reported as ΔF = F_t - F₀. All values are means ± SEM (n = 7). Two-way ANOVA, Bonferroni's post-test.

4. Effect of extracellular acidification on pH_i and [NO]_i in aorta cross sections

Confirming the result above, the SNARF-1 fluorescence in the endothelial layer of freshly obtained aorta cross sections did not change when the pH_o was reduced to 7.0 or 6.5 (Figures 5 A and 5 C). However, in the muscular layer, the pH_o 7.0 increased the SNARF-1 ΔF while the pH_o 6.5 resulted in no change in the SNARF-1 ΔF, showing that the pH_o 7.0 acidified the smooth muscle cells pH_i which returned to the basal levels with extreme extracellular acidification (Figures 5 B and 5 C).

Figure 5. Effect of extracellular acidification on pH_i of rat aorta cross sections assessed using a confocal scanning laser microscope.

A) Fluorescence intensity for endothelial layer. B) Fluorescence intensity for muscular layer. C) Representative confocal photomicrograph of one aorta cross section. Aorta cross sections were loaded with SNARF-1 (10 µM) and analyzed by confocal microscopy. Aorta cross sections were stimulated at 3^rd and 9^th min with Hanks acidified solution pH 7.0 and 6.5, respectively. HCl was used to change the pH of Hanks solution; Hanks solution pH 7.4 served as control. Fluorescence intensity was measured before the stimulus (F) and at 6^th min after the stimulus for each pH_o value (F_pH). Results are reported as ΔF = F_pH – F. All values are means ± SEM (n = 7). One-way ANOVA, Bonferroni's post-test, * p<0.01 versus control.

In the endothelial layer of fresh aorta cross sections, the pH_o 7.0 reduced the DAF-FM DA ΔF while the pH_o 6.5 increased it without statistical significance (Figures 6 A and 6 C). However, in the muscular layer, the DAF-FM DA ΔF was increased with both pH_o values (Figures 6 B and 6 C). These data demonstrate that, at pH_o 7.0, the [NO]_i is reduced in the endothelial layer at the same time in which it is increased in the muscular layer. Moreover, at pH_o 6.5, the [NO]_i is increased in both vessel layers.

Figure 6. Effect of extracellular acidification on [NO]_i of rat aorta cross sections assessed using a confocal scanning laser microscope.

A) Fluorescence intensity for endothelial layer. B) Fluorescence intensity for muscular layer. C) Representative confocal photomicrograph of one aorta cross section. Aorta cross sections were loaded with DAF-FM DA (5 µM) and analyzed by confocal microscopy. Aorta cross sections were stimulated at 3^rd and 9^th min with Hanks acidified solution pH 7.0 and 6.5, respectively. HCl was used to change the pH of Hanks solution; Hanks solution pH 7.4 served as control. Fluorescence intensity was measured before the stimulus (F) and at 6^th min after the stimulus for each pH_o value (F_pH). Results are reported as ΔF = F_pH – F. All values are means ± SEM (n = 7). One-way ANOVA, Bonferroni's post-test, * p<0.05 versus control, ^# p<0.001 versus control.

Discussion

The main findings of the present study are: a) extracellular alkalinization increased the pH_i in the endothelium and vascular smooth muscle; b) extracellular acidification did not change the endothelial pH_i but reduced the smooth muscle pH_i; c) severe extracellular alkalinization (pH_o 8.5) and acidification (pH_o 6.5) increased the endothelial [NO]_i; and d) both extracellular alkalinization and acidification increased the vascular smooth muscle [NO]_i.

In the present investigation, we selected aorta because it is the most important conductance artery. In addition, we wanted to clarify previous results obtained with this artery. Another methodological concern that deserves consideration is the use of freshly isolated endothelial cells and fresh aorta cross sections. Studies using freshly isolated cells should be distinguished from those using cultured cells or cell lines, as these cells may no longer express an in vivo phenotype [2], [8], [9]. Another important methodological issue is the pH range. We decided to study the pH_o from 6.5 to 8.5 because the literature shows that, in this pH_o range, the cells are viable [8], [9], [10] and we confirmed this literature data in our previous studies [4], [5].

It is known that both pH_o and pH_i can alter vascular tone and that they can influence each other. The main factor responsible for this mutual interaction between pH_o and pH_i is the high H⁺ permeability of arterial vascular smooth muscle. An alternative route would be the flux of weak acids across the membrane. This acid flux can occur either by passive diffusion of the protonated form or via a transporter mechanism. Thus, it is believed that changes in the pH_o can induce changes in the pH_i in the same direction [2]. However, here we have demonstrated that not all changes in the pH_o induce changes in the pH_i, especially in the endothelium. The extracellular acidification did not produce any change in the endothelial pH_i of isolated cells or cells covering the aorta internal surface. Concerning extracellular alkalinization, a great change in the pH_o was necessary to induce an increase in the pH_i in both isolated endothelial cells and endothelium-intact rings. This suggests that the endothelium, the largest functional organ responsible for regulating vascular tone, coagulation, inflammation and permeability [11], probably has efficient mechanisms of controlling the pH_i to avoid changes in enzymatic function and signal transduction, which could impair the important endothelial control of body homeostasis or result in cell death. Moreover, these control mechanisms seem to be more efficient for acidification than for alkalinization. In contrast to the endothelial findings, the extracellular acidification reduced the vascular smooth muscle pH_i. Actually, at pH_o 7.0, the pH_i was decreased, corroborating previous data in rat mesenteric arteries [12], [13], cerebral arteries [14] and portal vein [15], but the pH_i returned to basal levels when the pH_o was reduced to 6.5, probably due to slow recovery to the initial pH_i as acid equivalents are removed from the fiber by the pH_i regulating mechanism [16]. The vascular smooth muscle exhibited the same behavior as the endothelium when the pH_o was increased. Oly extreme extracellular alkalinization increased the pH_i, which is different from the findings of earlier studies showing that the pH_o 7.9 was sufficient to increase the smooth muscle pH_i [12], [13], [15]. These variations between endothelial and vascular smooth muscle cells and between distinct vessels can be due to different proton permeabilities or different mechanisms of controlling the pH_i [2].

Although the mechanisms by which the pH influences vascular tone remain inconclusive, some evidences suggest the involvement of NO [17], [18], [19], [20], prostacyclin [20], [21], potassium channels [17], [22], [23] and calcium flux [14], [22], [23]. We have previously demonstrated that both extracellular alkalinization and acidification induce vasodilation, and although the mechanisms responsible for this relaxation are not the same in each acid-base disorder, the NO is a common pathway [4], [5]. Here, we have confirmed that changes in acid-base balance did increase the [NO]_i in freshly obtained aorta cross sections. However, this [NO]_i increase was not necessarily dependent on pH_i changes, it could have been due to pH_o changes. This observation came from our results showing that, in some cases, even without changing the endothelial or muscular pH_i, the [NO]_i was altered. Moreover, the pH_o changes can alter cellular permeability and then activate different signaling pathways [24], [25].

In the present study, we showed that mild extracellular alkalinization (pH_o 8.0) and acidification (pH_o 7.0) reduced the [NO]_i in the endothelial layer and increased the [NO]_i in the smooth muscle layer, while severe extracellular alkalinization (pH_o 8.5) and acidification (pH_o 6.5) increased the [NO]_i in the entire vascular wall. The reduction in endothelial [NO]_i can be explained by the fast diffusion of this gas to the muscular layer. This event was clearly observed during the confocal experiments, when it was possible to see that the increase in fluorescence intensity in the endothelial layer coincided with the addition of the base or acid, and that, immediately after this addition, the fluorescence intensity in the endothelial layer decreased simultaneously with its increase in the muscular layer. This is confirmed by our previous results showing that the [NO]_i in isolated endothelial cells was raised not only at pH_o 8.5 and 6.5, but also at pH_o 8.0 and 7.0 [4], [5]. Moreover, it was demonstrated that NaOH-induced extracellular alkalinization (pH_o 8.0) stimulated the endothelial nitric oxide synthase (eNOS) activity in cultured human pulmonary arterial endothelial cells [8]. Conversely, in cultured human umbilical vein endothelial cells and freshly isolated porcine aortic endothelial cells, the extracellular alkalinization-induced intracellular alkalinization diminished the eNOS activity [26]. Regarding acidosis, it has been shown that the pH reduction predisposes to an increase in the [NO]_i. In pig cerebellum, the acidification favored the NO synthesis, probably due to an increase in the enzymatic activity [27]. It has also been suggested that, in an acidic milieu, the nitrite can be non-enzymatically reduced to NO [28] and that the NO is more stable in this milieu as it is protected from degradation [19], [29]. However, there are some reports describing that acidification decreases the eNOS activity [8], [26]. Discrepancies between our results (obtained from both extracellular alkalinization and acidification) and this literature data may be attributed to the use of different animal species, different alkali or acid stimulus [2], [24] or different phenotypes (cultured versus fresh cells) [2], [8], [9] and the presence or absence of pH_i change when the pH_o is altered.

An important consideration about [NO]_i is that although both extracellular alkalinization and acidification raised the [NO]_i in the muscular layer, the NO synthesis was restricted to the endothelium during the augmentation of the pH_o. On the other hand, during the acidification, the NO was synthesized by the endothelium and smooth muscle. These inferences are based on the fact that the extracellular alkalinization promoted an endothelium-dependent relaxation, which was exclusively mediated by NO [4], while the extracellular acidification-induced relaxation was partially reduced after inhibition of the NO synthesis in endothelium-denuded rings [5]. Another intriguing detail is the endothelial pH_i change induced by extracellular alkalinization compared with the lack of change in the endothelial pH_i when the pH_o was reduced. Based on this observation, would be valid to speculate that this difference is responsible for the exclusive role of NO in the alkalinization-induced relaxation while the vasodilation elicited by extracellular acidification needs factors other than NO?

Conclusions

In summary, we have demonstrated that not all changes in pH_o are reflected in pH_i alterations and that both acid-base disorders induce NO synthesis in the endothelium and/or vascular smooth muscle.

Funding Statement

The authors are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FAEPA/HCFMRP/USP) for financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.