Angiotensin II and Angiotensin-(1-7) Effects on Free Cytosolic Sodium, Intracellular pH, and the Na+-H+ Antiporter in Vascular Smooth Muscle
Abstract The aim of the present study was to define the effects of angiotensin II (Ang II) and Ang-(1-7) on free cytosolic Na+ (Na+i), intracellular pH (pHi), and the Na+-H+ antiporter in cultured vascular smooth muscle cells from rat aorta. Cells were loaded with either BCECF-AM or SBFI-AM for measurement of pHi and Na+i, respectively. Ang II (10−6 mol/L) caused a rapid rise in Na+i followed by a progressive increase that peaked at about 10 minutes (from 11±1.5 to 16±1.5 mmol/L, P<.001), whereas Ang-(1-7) (10−6 mol/L) did not affect Na+i significantly (from 11.5±1.1 to 11.8±0.07 mmol/L). The effect of Ang II on Na+i was concentration dependent (ΔNa+i, 5.1±0.9, 3.8±0.6, 1.6±0.6, and 0.14±0.18 mmol/L with decreasing concentrations of 10−6, 10−7, 10−8, and 10−9 mol/L, respectively). Ang II caused a brief acidification followed by an increase in pHi (from 7.34±0.03 to 7.43±0.03 after 10 minutes, P<.005), and Ang-(1-7) had no significant effect on pHi (from 7.23±0.03 to 7.23±0.03). To investigate whether pHi and Na+i changes induced by Ang II were due to cell Na+ entry via stimulation of the Na+-H+ antiporter, we pretreated cells with EIPA (25 μmol/L) or ouabain (2.0 mmol/L). Ang II in the presence of ouabain caused a greater increase than that seen with ouabain alone (ΔNa+i, 13±1.5 versus 6.3±1.2 mmol/L, P<.0025). EIPA by itself decreased Na+i and pHi. After EIPA, Ang II failed to increase both Na+i and pHi, demonstrating that the Na+-H+ antiporter is responsible for the rises in Na+i and pHi during stimulation with Ang II. To further characterize the mechanism of Ang II action, we exposed cells to an Ang II type 1 receptor antagonist (L-158,809, 10−6 mol/L) or two different type 2 receptor antagonists (PD 123177 and CGP 421112A, 10−6 mol/L). L-158,809 completely blocked the rise in pHi caused by Ang II, whereas PD 123177 and CGP 421112A did not. We conclude that Ang II increases both Na+i and pHi, and both effects are mediated by stimulation of the Na+-H+ antiporter. Ang-(1-7), by contrast, has no significant effect on Na+i, pHi, or the Na+-H+ antiporter. Stimulation of this antiporter by Ang II is exerted through the type 1 receptor.
Ang II has a vast array of effects on ion transport in vascular and nonvascular tissues.1 In vascular smooth muscle Ang II increases vascular tone and elicits a hyperplastic2 and hypertrophic3 response. Since the Na+-H+ antiporter is stimulated by many growth factors,4 it seems plausible that stimulation of this transporter may be involved in the growth-promoting actions of Ang II. Ang-(1-7) is a biologically active fragment of the renin-angiotensin system generated from Ang I or Ang II.5 Unlike Ang II, this peptide does not cause vasoconstriction or mitogenesis but has a vasodilator action.6 Whether this peptide affects the Na+-H+ antiporter, to our knowledge, has not been studied.
The use of a fluorescence-sensitive Na+ indicator, SBFI, has emerged as a noninvasive method to measure Na+i.7 8 9 Our studies in cultured aortic smooth muscle cells have shown the suitability of SBFI for assessing Na+-H+ exchange activity by continuous Na+i monitoring during recovery from cell acidification.8 We reasoned that direct monitoring of Na+i could also be used to evaluate Na+-H+ exchange activity in the steady state and after stimulation with Ang II, particularly if used in conjunction with concurrent pHi measurements.
Smith and Brock10 first provided indirect evidence suggestive of Na+ entry via Na+-H+ exchange stimulated by Ang II. These authors used 86Rb uptake as an index of Na+,K+-ATPase activity and found that in the presence of ouabain Ang II stimulated Na+ uptake by cultured aortic smooth muscle cells. The stimulation of Na+ uptake with Ang II was insensitive to tetrodotoxin, a blocker of Na+ channels, and furosemide, an Na+-K+-Cl− cotransport inhibitor, but was sensitive to amiloride. This suggested that the peptide stimulates Na+ entry via the Na+-H+ antiporter. Studies by Vallega et al11 and Berk et al12 later showed that Ang II increases the Vmax of the Na+-H+ antiporter after activation by cell acidification.
We wanted to characterize the action of Ang II on Na+i and H+ (pHi), reasoning that this approach would unravel whether activation of the Na+-H+ antiporter, which is much less active under steady-state conditions than during cell acidification, is responsible for changes in these intracellular cations during Ang II stimulation. We also studied which type of Ang II receptor is responsible for the stimulation of the Na+-H+ antiporter by this agonist.
VSMCs from the thoracic aorta of male Sprague-Dawley rats were isolated by collagenase and elastase digestion as previously described.13 Cells were seeded onto 9×35-mm coverslips (Wheaton Glass) resting in a 60-mm tissue culture dish. The cells were grown in Dulbecco’s modified Eagle’s medium supplemented with Ham’s nutrient F-12 (Sigma Chemical Co), 10% fetal calf serum (Hazleton Biologics Inc), penicillin (100 U/mL), streptomycin (100 μg/mL), and Fungizone (250 μg/mL) at 37°C in a humidified atmosphere of 5% CO2. Cells were fed twice weekly. Twenty-four hours before measurements, cells on the coverslips were placed in new culture dishes and made quiescent by feeding of serum-free medium, and the cells remaining adherent to the original culture dish were passaged onto fresh coverslips for future use. The subcultures grew to confluence in 5 to 7 days. Experiments were performed on confluent subcultures between passages 3 and 7.
On the day of the study cells were loaded with 1.5 μmol/L BCECF-AM for 30 minutes at 37°C as previously described.14 After dye loading, the coverslips were washed three times with assay buffer and allowed to sit at room temperature before the experiment was begun. The basic assay solution had the following composition (mmol/L): NaCl 133.8, KCl 4.7, CaCl2 1.25, MgCl2 1.25, Na2HPO4 0.97, NaH2PO4 0.23, glucose 3.0, and HEPES 5.0, pH 7.4.
At the start of the experiment, a coverslip was placed in a customized holder and inserted into a suction cuvette resting in a water-jacketed cuvette holder. The temperature was maintained constant at 37°C. The coverslips were then superfused at a rate of 2 mL/min with prewarmed assay buffer using a syringe pump, and the effluent was constantly removed with a peristaltic pump.13 14 When switching from one experimental solution to another, we increased the superfusion rate to 8 mL/min for 30 seconds to enhance the rate of exchange. At this rate, a greater than 95% exchange of solutions occurs within 20 seconds. The contribution of external BCECF to the total fluorescence signal due to dye leakage was found to be negligible at the superfusion rate used, as demonstrated by the lack of fluorescence in the effluent.
BCECF fluorescence was monitored continuously, alternating between the desired excitation wavelengths (500 and 440 nm), with an emission wavelength of 520 nm. The excitation and emission slits were set at 5 and 10 nm, respectively. For calculation of pHi, the 500/440 BCECF ratio was calibrated with the use of nigericin (6 μg/mL) in 120 mmol/L potassium buffer. The pH of the superfusate was adjusted in a stepwise fashion between 6.4 and 7.8 by progressive addition of NaOH.
The sodium-sensitive fluorescent probe SBFI was used for measurement of Na+i as described previously.8 On the day of the study VSMCs were loaded for 2.5 to 3 hours at room temperature with freshly prepared loading solution containing SBFI-AM (10 μmol/L) (Molecular Probes) and pluronic (0.1%).15 16 Probenecid (10 μmol/L) was added to the loading solution to minimize SBFI leakage. This agent has been used previously to prevent fura 2 secretion and sequestration.8 17 After loading, the coverslips were washed three times with the assay buffer and inserted into the suction cuvette and superfused for 10 to 15 minutes until a stable fluorescence signal was obtained. SBFI fluorescence was continuously measured at an emission wavelength of 519 nm and alternating excitation wavelengths of 348 and 383 nm. Data were obtained every 15 seconds with a DMX 1000 spectrofluorometer (SLM Instruments). The 348/383 excitation ratio was calibrated at pH 7.4 by exposing the cells to different concentrations of Na+ (between 0 and 60 mmol/L) and maintaining the osmotic pressure by adjusting the K+ concentration (Fig 1⇓). For equilibration of intracellular and extracellular sodium, gramicidin (10 μmol/L) and monensin (5 μmol/L) were added.15 Sodium concentrations of the calibration buffer were verified by flame photometry.
After a stable baseline of Na+i was recorded, cells were exposed to Ang II at various concentrations (see “Results”). Because the rise in Na+i during Ang II represents the balance of Na+ entry into and Na+ extrusion from the cells, additional experiments were performed after inhibition of Na+ extrusion via Na+,K+-ATPase using ouabain (2 mmol/L) for 3 minutes before superfusion with Ang II.
In other experiments EIPA was used before Na+i measurement to ensure that the Na+-H+ exchanger was inhibited before exposure to Ang II. Because of EIPA-related autofluorescence at the wavelengths used to monitor SBFI fluorescence, cells could not be continuously perfused with EIPA. To obviate this technical problem, cells were preincubated for 2 minutes with 25 μmol/L EIPA and then immediately inserted into the cuvette and superfused for 10 minutes with the desired experimental solution. There was no difference between the SBFI curves generated from cells preincubated with EIPA (25 μmol/L) or control cells (Fig 1⇑). Preincubation of cells with EIPA for 2 minutes lowered the baseline Na+i value compared with controls, indicating effective blockade of the Na+-H+ antiporter. According to our previous work,8 EIPA preincubation with this approach blocks the pHi recovery from acid loading, further indicating effective blockade of the Na+-H+ antiporter (see “Results”).
Analysis of the time course of Na+i or pHi changes was done by one-way ANOVA. Two-way ANOVA was used to seek differences between experimental groups over time. Analysis of differences between two experimental groups at a certain end point (ie, 30 seconds, 10 minutes, etc) was done with Student’s t test (unpaired analysis). Differences were considered significant at a value of P<.05. All data are reported as mean±SE.
SBFI-AM, BCECF-AM, and pluronic were purchased from Molecular Probes, Inc. EIPA was a gift from Dr Michael Ganz. Ang-(1-7), L-158,809, CGP 421112A, and PD 123155 were kindly provided by Dr Mahesh Khosla and Dr Carlos Ferrario. All other chemicals were of analytic grade and were purchased from Sigma.
Effect of Ang II and Ang-(1-7) on Na+i
After a stable baseline was recorded, addition of Ang II (10−6 mol/L) to the superfusate caused a rapid and progressive increase in Na+i (from 11.6 to 13.1 mmol/L at 30 seconds). By 10 minutes Na+i had increased (from 11.6±1.5 to 16.5±1.5 mmol/L, P<.001) and then stabilized, reflecting a new steady state in which Na+ exit, presumably via increased Na+,K+-ATPase activity, matched Na+ entry (Fig 2⇓). In time control studies Na+i remained stable (from 11.2±2.2 to 10.3±2.1 mmol/L at 10 minutes).
The action of Ang II on Na+i was concentration dependent, as shown by a rise of Na+i above baseline of 5.1±0.9, 3.8±0.6, 1.6±0.6, and 0.14±0.18 mmol/L at 10 minutes with decreasing Ang II concentrations of 10−6, 10−7, 10−8, and 10−9 mol/L, respectively (Fig 3⇓).
Ang-(1-7) (10−6 mol/L) did not have a significant effect on Na+i at any time during the entire perfusion (from 11.5±1.1 mmol/L at baseline to 11.8±0.7 after 30 seconds and 10.6±0.9 at 10 minutes) (Fig 2⇑).
Effect of Ang II on Na+i in the Presence of Ouabain
Because an increase in Na+ entry would be expected to be accompanied by increases in Na+ extrusion via stimulation of the Na+ pump (Na+,K+-ATPase), we examined the effect of Ang II on Na+i after prior addition of ouabain (2 mmol/L). Ouabain by itself caused a significant increase in Na+i (Fig 4⇓). In the presence of ouabain Ang II caused an increase in Na+i that was greater than the increase seen with ouabain alone (ΔNa+i, 13.1±1.5 and 6.3±1.2 mmol/L at 10 minutes, respectively; P<.0025) (Fig 4⇓).
Effect of Ang II and Ang-(1-7) on pHi
Changes in pHi in response to Ang II measured in parallel with Na+i experiments are shown in Fig 5⇓. As previously shown by us18 and others19 Ang II caused an initial and transient fall of pHi, with a peak response at about 1 minute (from 7.34±0.03 to 7.29±0.03, n=13, P<.025). Thereafter, pHi rose progressively, with a peak response at about 10 minutes (from 7.34±0.03 to 7.43±0.03, P<.005), and remained stable thereafter.
The effect of Ang II on pHi, like that on Na+i, was concentration dependent, as shown by an increase in pHi of 0.09±0.01, 0.06±0.03, 0.04±0.02, and 0.003±0.006 pH units with decreasing Ang II concentrations of 10−6, 10−7, 10−8, and 10−9 mol/L, respectively (Fig 6⇓). The rise in pHi occurred in parallel with the Na+i increase, suggesting H+ efflux coupled to Na+ influx.
Ang-(1-7) (10−6 mol/L) did not have a significant effect on pHi at either 1 minute (from 7.23±0.03 to 7.23±0.04) or after 10 minutes (from 7.23±0.03 to 7.22±0.02) of superfusion (Fig 5⇑).
Effect of Pretreatment With EIPA on Na+i and pHi
If Ang II–induced cytosolic alkalinization and Na+i increase were both due to the stimulation of Na+-H+ antiporter, then an inhibitor of this transporter should prevent the effect on both pHi and Na+i. This possibility was examined by the addition of 25 μmol/L EIPA, a specific blocker of the Na+-H+ exchanger.
Pretreatment with EIPA for 2 minutes caused a marked decrease in Na+i and pHi, probably reflecting a new steady state of activity of the Na+-H+ antiporter. After EIPA pretreatment Ang II failed to increase Na+i (Fig 7⇓) and pHi (Fig 8⇓). This shows that activation of the Na+-H+ exchanger is the pathway of cell Na+ influx and H+ efflux that is stimulated by this agonist.
Effect of AT1 and AT2 Receptor Antagonists on pHi and Na+i
We also studied the effect of Ang II on pHi using L-158,809, a potent antagonist of the AT1 receptor.20 In the presence of L-158,809, Ang II failed to increase both pHi (Fig 9⇓) and Na+i (Fig 10⇓). By contrast, two different AT2 receptor antagonists, CGP 421112A (10−6 mol/L) and PD 123177 (10−6 mol/L), did not prevent the effect of Ang II on either pHi (Fig 9⇓) or Na+i (data not shown).
Previous studies in vascular smooth muscle have shown that Ang II has profound effects on ion transport.1 This agonist, which has both vasoconstrictor and mitogenic properties, increases Ca2+i and has a biphasic effect on pHi.18 19 The effect of Ang II on Na+i has received less attention largely because the free cytoplasmic concentration of this ion has been difficult to measure until recently. Johnson et al,7 using individual rat aortic smooth muscle cells, showed that Ang II induces an elevation of Na+i, but they did not define the mechanism underlying this action. We now demonstrate that the rise in Na+i elicited by Ang II in cultured VSMCs is sustained and due to the activation of the Na+-H+ antiporter. The rise in Na+i was temporarily associated with the increase in pHi, suggesting H+ efflux coupled with Na+ entry (Figs 2⇑ and 5⇑). Moreover, EIPA, a specific inhibitor of the Na+-H+ antiporter, obliterated the increases in both Na+i and pHi (Figs 7⇑ and 8⇑). Taken together, these findings show that Ang II increases Na+i and decreases H+i (ie, increases pHi) by stimulating the Na+-H+ antiporter. This action is probably due to stimulation of the type 1 Na+-H+ exchanger isoform, which is abundantly present in cultured aortic VSMCs.8 Other Na+-H+ antiporter isoforms, types 2, 3, and 4, are not present in VSMCs.20
By using selective and specific antagonists of Ang II receptors,20 we were able to demonstrate that the effects of Ang II on pHi, Na+i, and Na+-H+ exchange are mediated by stimulation of the AT1 receptor (Figs 9⇑ and 10⇑). Numerous studies have shown that Ang II receptors on aortic smooth muscle are of the AT1 subtype, which is responsible for the hypertrophic response to Ang II.21 Unlike the AT1 receptor, the exact physiological function of the AT2 receptor is yet to be demonstrated. AT2 receptors are highly represented embryonically in tissues such as immature aorta, adrenal glands, and fetal kidney.22 23 After birth these receptors decrease dramatically.24 Ang II might act through this receptor as a differentiation/growth factor during nephron development.24 There is also evidence that AT2 receptors reappear in high concentrations after wound healing and myocardial ischemia and may inhibit tissue growth. However, all the hemodynamic effects of Ang II, including vasoconstriction and mitogenic actions, are mediated through the AT1 receptor.25 Our data show that stimulation of the Na+-H+ antiporter by Ang II is mediated via activation of the AT1 receptor, whereas the AT2 receptor plays no role in this stimulation.
The Na+-H+ antiporter contributes noticeably to sodium influx as reflected by the marked fall in Na+i seen after a few minutes of preincubation with EIPA, a specific inhibitor of this antiporter (Fig 7⇑). The marked fall in Na+i associated with EIPA (from about 10 to 5 mmol/L) was not an artifact related to interference of EIPA with SBFI fluorescence because EIPA was not added to the superfusate. Brief preincubation with EIPA did not alter the SBFI calibration procedure (see “Methods” and Fig 1⇑). Owing to the exquisite sensitivity of the type 1 isoform of the Na+-H+ antiporter, brief preincubation with EIPA appears to provide EIPA binding in a concentration sufficient to ensure inhibition of the Na+-H+ antiporter during subsequent superfusion for Na+i measurements.
The possible effect of Ang II–induced Na+i changes on vascular cell growth deserves comment. Sodium influx via the Na+-H+ antiporter may influence the ability of vascular smooth muscle to synthesize protein late in the G1 phase of the mitotic cell cycle.26 It is thus possible that the development of vascular hyperplasia in hypertensive states involves cell Na+ entry via activation of the Na+-H+ antiporter.26 Ang II could cause vascular hyperplasia by this mechanism in Ang II–dependent forms of hypertension, such as malignant and renovascular hypertension. It is also worthy of mention that Ang II has been shown to induce vascular hypertrophy in association with rapid increases in Na+,K+-ATPase activity.2 When Na+ exit via the plasma membrane Na+,K+-ATPase was blocked with ouabain, Na+i increased, and this action was greatly potentiated by the addition of Ang II (Fig 4⇑). This suggests that the reported stimulatory effect of Ang II on Na+,K+-ATPase activity is likely secondary to the enhancement of Na+ entry via activation of the Na+-H+ antiporter.
When smooth muscle or glomerular mesangial cells are exposed to agonists that increase Ca2+i, such as Ang II or vasopressin, a brief acidification followed by a marked alkalinization occurs when HCO3−/CO2 is absent from the medium.19 27 Initial cell acidification occurs whether or not HCO3−/CO2 is present in the medium.27 We have shown that cell acidification requires an increase in Ca2+i and is caused by H+ entry coupled to Ca2+ efflux.18 Presumably, the Ca2+-ATPase in the plasma membrane, acting as a Ca2+-H+ exchanger, is responsible for this initial acidification.18 The delayed cell alkalinization induced by Ang II, when cells are studied in media lacking HCO3−/CO2, is blocked by amiloride and its derivatives, indicating that Ang II-induced increase in pHi and Na+i is exerted via activation of the Na+-H+ antiporter.28 When the medium contains HCO3−/CO2, however, the fall in pHi is not followed by a subsequent cell alkalinization because the rise in pHi is opposed and compensated by the concurrent activation of the Na+-independent Cl−/HCO3− exchanger, a process that leads to cell HCO3− exit and thus acidifies the cell.18 28
We conducted the present studies under conditions in which the media lacked HCO3−/CO2 to be able to examine Na+i and pHi changes related to the Na+-H+ antiporter independently of changes that could be related to activation of the Na+-dependent Cl−/HCO3− exchanger. Under this condition, Ang II increases Na+i solely by activation of the Na+-H+ antiporter, causing it to increase markedly (ie, about 50% above baseline) (Fig 2⇑). This sustained increase is in contrast to the rapid recovery in Ca2+i.18 Activation of the Na+-H+ antiporter does not appear to require the initial increase in Ca2+i that Ang II and other agonists produce as a result of activation of phospholipase C and subsequent mobilization of Ca2+ from the sarcoplasmic reticulum by inositol triphosphate.29 This conclusion is based on our finding that when the initial Ca2+i spike is prevented by preincubation with 1,2-bis(2-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid (BAPTA), Ang II still produces an increase in pHi.29 Moreover, BAPTA prevents the initial decrease in pHi, indicating that cell acidification is not a mechanism whereby Ang II increases the Na+-H+ antiporter.29
In contrast to the profound ionic effects of Ang II just outlined, Ang-(1-7) (10−6 mol/L) did not exert any discernible effects on either Na+i or pHi and therefore lacks any stimulatory or inhibitory action on the Na+-H+ antiporter in VSMCs. Ang-(1-7) appears to exert a vasodilator action while lacking mitogenic activity.6 Previous studies with intact blood vessels have shown that Ang-(1-7) causes an initial increase in systemic blood pressure of short duration followed by a decline to values below baseline.6 Osei et al30 suggested that the vasodilator effect could be mediated by the release of endothelium-derived relaxing factor and the vasoconstrictor effect via activation of the AT1 receptor subtype. Andreatta–Van Leyen et al31 showed that in proximal tubular cells Ang-(1-7) increased phospholipase activity and inhibited 22Na+ flux. They suggested that this peptide could have a potential role in the regulation of electrolyte transport in the kidney. It has also been suggested that in proximal tubular cells the stimulatory effect of Ang-(1-7) on fluid absorption is concentration dependent, with lower concentrations being stimulatory and higher concentrations inhibitory.32 However, in cultured VSMCs decreasing concentrations of Ang-(1-7) (10−6 to 10−8 mol/L) had no effect on either pHi or Na+i (data not shown).
In summary, in cultured VSMCs the Na+-H+ antiporter is a major pathway of Na+ influx and H+ efflux during steady-state conditions and after Ang II stimulation. The effect of Ang II on Na+i and pHi is secondary to stimulation of the Na+-H+ antiporter via activation of the AT1 receptor. Ang-(1-7), unlike Ang II, has no effect on Na+i, pHi, or the Na+-H+ antiporter.
Selected Abbreviations and Acronyms
|Ang I, II||=||angiotensin I, II|
|AT1, AT2||=||angiotensin II type 1, type 2|
|Ca2+i||=||free cytosolic calcium|
|Na+i||=||free cytosolic Na+|
|SBFI||=||sodium-binding benzofuran isophthalate|
|VSMC||=||vascular smooth muscle cell|
This work was supported by a Veterans Administration Merit Review grant (D. Batlle). G. Flores was supported by grants from the Instituto Mexicano del Seguro Social, Hospital de Especialidades del Centro Medico Nacional and the National Kidney Foundation (Illinois chapter). Dr Batlle is a member of the Feinberg Cardiovascular Institute at Northwestern University Medical School. We thank Drs Makesh Khosla and Carlos Ferrario for providing us with Ang-(1-7) and the AT1 and AT2 antagonists used in this study.
- Received June 14, 1995.
- Revision received July 24, 1995.
- Accepted August 8, 1995.
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