Scientific Contributions

Enalapril Induces Regression of Cardiac Hypertrophy and Normalization of pHi Regulatory Mechanisms

Irene L. Ennis, Bernardo V. Alvarez, María C. Camilión de Hurtado, Horacio E. Cingolani
https://doi.org/10.1161/01.HYP.31.4.961
Hypertension. 1998;31:961-967
Originally published April 1, 1998

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Abstract

Abstract—Intracellular pH is under strict control in myocardium; H+ are extruded from the cells by sodium-dependent mechanisms, mainly Na+/H+ exchanger and Na+/HCO3− symport, whereas Na+-independent Cl−/HCO3− exchanger extrudes bases on intracellular alkalinization. Hypertrophic myocardium from spontaneously hypertensive rats (SHR) exhibits increased Na+/H+ exchange activity that is accompanied by enhanced extrusion of bases through Na+-independent Cl−/HCO3− exchange. The present experiments were designed to investigate the effect of enalapril-induced regression of cardiac hypertrophy on the activity of these exchangers. Male SHR and normotensive Wistar-Kyoto rats (WKY) received enalapril maleate (20 mg/kg per day) in the drinking water for 5 weeks. Gender- and age-matched SHR and WKY were used as untreated controls. Enalapril treatment significantly reduced systolic blood pressure in SHR and completely regressed cardiac hypertrophy. Na+/H+ activity was estimated in terms of both steady pHi value in HEPES buffer and the rate of pHi recovery from CO2-induced acid load. Na+-independent Cl−/HCO3− activity was assessed by measuring the rate of pHi recovery from intracellular alkalinization produced by trimethylamine exposure. Regression of cardiac hypertrophy was accompanied by normalization of Na+/H+ and Na+-independent Cl−/HCO3− exchange activities. Inhibition of protein kinase C (PKC) activity with chelerythrine (10 mmol/L) or calphostin C (50 nmol/L) returned both exchange activities to normal values. These results show that angiotensin-converting enzyme inhibition normalizes the enhanced activity of both exchangers while regressing cardiac hypertrophy. Because normalization of exchange activities could be also achieved by PKC inhibition, the data would suggest that PKC-dependent mechanisms play a significant role in the increased ion exchange activities of hypertrophic myocardium and in their normalization by angiotensin-converting enzyme inhibition.

We have recently reported that NHE activity is increased in the hypertrophic myocardium of SHR.1 The blockade of angiotensin II production by ACE inhibitors is proven to be potent and effective for blood pressure reduction and cardiac hypertrophy regression.2 3

The enhancement of NHE activity has been described in various other cell types obtained both from hypertensive individuals and genetic animal models (for detailed review, see Reference 44 ), and it might result from an increased expression of exchanger protein units and/or an increased turnover rate of each unit. The question as to whether increased activity of NHE relates to increased turnover rate or increased mRNA and protein expression of the exchanger has been explored in different cell types, and it is still controversial.5 6 7 8 9 However, experiments in membranes from SHR cardiac tissue suggested a posttranslational processing mechanism involving phosphorylation as responsible for NHE hyperactivity.10 Activation of NHE by growth-promoting factors apparently involves an alkaline shift in the pHi dependency of NHE due to phosphorylation of the exchanger protein itself and/or of a putative regulatory protein.11 12 13

An increased activity of the AE in SHR myocardium has been reported by us.1 The parallel increase of AE activity induces net bicarbonate efflux and blunts the increase in myocardial pHi that would be induced by the NHE. It was therefore tempting to investigate whether enalapril-induced regression of cardiac hypertrophy had an influence on NHE and AE activities in SHR myocardium. In an attempt to further examine possible underlying mechanism(s), the effect of PKC inhibition was also studied.

Methods

Experiments were conducted in age-matched SHR and WKY male rats, which were originally derived from Charles River Breeding Farms, Wilmington, Mass. All animals were identically housed under controlled lighting and temperature conditions with free access to standard rat chow and tap water. All the experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (US Department of Health and Human Services). Beginning at 12 weeks of age, SBP was measured weekly in all animals by the standard tail-cuff method.14 By 16 weeks of age, SBP was significantly elevated in SHR (overall mean, 175±3 mm Hg; n=42) compared with WKY (118±2 mm Hg; n=27). By this time, rats from each breed were respectively divided at random into two groups. One group of each rat strain was treated with enalapril maleate (SHR-E and WKY-E) by inclusion of the drug in the drinking water. Concentration was adjusted every 2 days to match the BW/consumption ratio to ensure a dosage of 20 mg/kg per day. The second group of each strain served as untreated controls. Treatment lasted 5 weeks, and at the end of this period animals were deeply anesthetized with ether and their hearts were removed. From each heart, a papillary muscle was dissected free and mounted, as previously described,1 in an organ bath on the stage of an Olympus CK2 inverted microscope (Olympus Optical Co). Muscles were superfused with one of the following solutions: (1) HEPES- buffered solution containing (in mmol/L) 133.8 NaCl, 4.5 KCl, 1.35 CaCl2, 1.05 MgSO4, 11 glucose, and 25 HEPES (pH of the buffer solution was adjusted to 7.38±0.03 at 30°C with 3 N NaOH [total Na+ amounted to 148.8], and the solution was gassed with 100% O2) or (2) HCO3−/CO2-buffered solution containing (in mmol/L): 128.3 NaCl, 4.5 KCl, 1.35 CaCl2, 20.23 NaHCO3, 0.35 NaH2PO4, 1.05 MgSO4, and 11 glucose. The solution was equilibrated with CO2/O2 gas mixture to ensure a Pco2 value of 35 mm Hg at the chamber level, with the pH of CO2/HCO3−-buffered solutions being 7.37±0.01 at 30°C. Chelerythrine chloride (Research Biochemicals) was used at 10 mmol/L as specific inhibitor of PKC activity15 and calphostin C (Research Biochemicals) at 50 nmol/L.16 Atria and all adjacent connective tissue were removed, and the remaining tissue was blotted and weighed to determine HW. The free wall of the right ventricle was excised to determine LVW separately. HW and LVW were expressed as ratios to BW to determine the degree of hypertrophy.

Measurements of pHi

Measurements of pHi were made by epifluorescence as previously described.1 Briefly, muscles were loaded with BCECF-AM (Molecular Probes). BCECF fluorescence was excited at 450 and 495 nm, and the fluorescence emission was monitored after passage through a 535±5-nm filter every 20 seconds. To limit photobleaching, a neutral-density filter (1% transmittance) was placed in the excitation light path. At the end of each experiment, fluorescence emission was calibrated by the high K+-nigericin method.17 The calibration solution contained (in mmol/L) KCl 150.0, MgCl2 1.0, CaCl2 1.0, HEPES 5.0, nigericin 0.01, sodium cyanide 4.0, and 2,3-butanedione monoxime 20.0. Buffer pH was adjusted to four different values ranging from 7.5 to 6.5. Such a calibration gave a linear relation between buffer pH values and the fluorescence ratio (F495/F450) as previously reported.1 Values of autofluorescence at each wavelength were subtracted before the calculation of any F495/F450.

Assessment of NHE Activity

The activity of the antiporter was estimated in terms of both steady pHi values in the absence of external bicarbonate (HEPES buffer) and JH+ during the recovery from intracellular acidification. Acid loads were induced by switching from HEPES-buffered superfusate to CO2/HCO3− buffer. Recoveries of pHi after acid loads were analyzed by fitting the pHi versus time records to an exponential curve of the form ΔpHit=ΔpHi∞ (1−e−kΔt), where ΔpHit and ΔpHi∞ are the changes in pHi from the initial value at time t and after steady state has been reached, respectively, and k is the rate coefficient. The rate of change of pHi at any selected pHi value was obtained by calculating the derivative of the exponential fit at that selected pHi, and thus, intracellular buffer capacity×dpHi/dt represents JH+ (in mmol/L per minute) at that pHi. Intracellular buffer capacity was calculated as the sum of βi plus the buffering power due to intracellular CO2/HCO3−. The latter was considered to be 2.3×[HCO3−]i, assuming an open system for CO2 and that its solubility and pK value are the same at either side of the cell membrane. [HCO3−]i at any given pHi was calculated from the Henderson-Hasselbach equation to be [HCO3−]i=[HCO3−]o×10(pHi-pHo). βi was calculated as the ratio between Δ[HCO3−]i/ΔpHi observed when the superfusing solution was switched from HEPES to CO2/HCO3− buffer.18 Δ[HCO3−]i was considered to equal the value of [HCO3−]i immediately after CO2/HCO3− buffer introduction because in the absence of external CO2 the value of [HCO3−]i is very low, ≈50 μmol/L.19 The main problem for estimating βi is that acid extrusion during the loading period may blunt the acidosis, thus leading to overestimation of βi value (see Reference 1818 for details). Back-extrapolation of pHi recovery to a point where it intersected the line defining the maximum initial rate of acid loading was used to reduce errors in βi calculation, as previously shown.1 20 21

Assessment of AE Activity

The velocity of pHi recovery from imposed intracellular alkalinization was used to estimate AE activity. Exposure to TMACl (Sigma Chemical Co) has been previously demonstrated as a valid technique for investigating the activity of the AE based on the fact that no recovery from TMACl-induced intracellular alkalosis is detected in HCO3−-free solutions.22 23 Ten-minute pulses of different TMACl concentrations (10, 20, or 30 mmol/L) were applied without osmotic compensation and pHi values recorded during the first minute after peak alkalosis were fitted to a straight line to estimate the initial velocity of pHi recovery (dpHi/dti).23

Statistics

Data are expressed as mean±SEM. Statistical analysis of results was performed using either Student’s t test or ANOVA followed by Bonferroni’s test, as appropriate. Values of P≤.05 were considered significant.

Results

General characteristics for the various rat groups at the time of death are shown in Table 1⇓. SBP remained elevated at hypertensive levels in untreated SHR throughout the 5-week experimental period but significantly decreased in the SHR-E group. Enalapril treatment completely regressed cardiac hypertrophy as shown by HW/BW and LVW/BW ratios.

View this table:
Table 1.

General Characteristics of Rat Groups

Steady Myocardial pHi

Fig 1⇓ shows steady pHi values determined in papillary muscles superfused with HEPES-buffered medium. In the absence of bicarbonate, steady pHi is solely controlled by NHE activity. As a reflection of increased NHE activity in hypertrophic myocardium, steady pHi value was more alkaline in SHR-C than in WKY-C. These data confirm previous results from our laboratory1 and are probably the result of an alkaline shift of the antiporter “set point.” In the same study, we also showed that the inhibition of NHE activity with the amiloride derivative EIPA normalized myocardial pHi value in SHR.1 Enalapril treatment returned myocardial pHi of SHR to values not different from those found in myocardium of normotensive rats (Fig 1⇓). No significant changes in pHi values were detected after the treatment of normotensive rats with enalapril.

Figure 1.
Figure 1.

Myocardial steady pHi values in HEPES buffer. Note that after enalapril treatment, myocardial pHi in SHR was decreased to values similar to those observed in myocardium from normotensive rats. Enalapril did not change pHi significantly in WKY. Values in parentheses indicate the number of determinations. *P<.05 compared with every other group (ANOVA).

With HEPES used as extracellular buffer, NHE is the only mechanism regulating pHi therefore, the steady pHi values can be directly correlated to NHE activity. However, antihypertensive treatment could have changed the ability of cells to buffer protons. No significant difference between groups was detected when βi values were determined (Table 2⇓). Consequently, the reduction in steady myocardial pHi value in the SHR-E group can be interpreted as the result of normalization of NHE activity.

View this table:
Table 2.

Cellular Buffer Capacity

When hypertrophic myocardium of SHR-C was exposed to a specific inhibitor of PKC activity, chelerythrine, pHi value in HEPES buffer gradually decreased (Fig 2⇓). By contrast, no significant effect of PKC inhibition on myocardial pHi was observed in WKY-C. The different effect of PKC inhibition on SHR-C and WKY-C resulted in cancellation, after 25 minutes of drug administration, of the difference between pHi values in hypertrophic and control myocardium. The best fit of chelerythrine-induced decrease in pHi followed an exponential function that asymptotically approached a pHi value of 7.07±0.04 (n=6). Interestingly, this value was close to the steady pHi determined in WKY control-matched experiments (7.03±0.03, n=5) and not different from the overall mean value of steady pHi in WKY rats (7.12±0.03, n=20). The time constant of chelerythrine-induced pHi decay was found to be 12.5±2.01 minutes. Essentially the same results were obtained when a structurally and mechanistically different PKC inhibitor, calphostin C, was used. In these experiments, pHi in hypertrophic myocardium reached values not different from WKY-C (7.10±0.02, n=4) with a time constant of 16.1±4.1 minute after exposure to calphostin C. Therefore, the results suggest that the hyperactivity of NHE in hypertrophic myocardium is mediated by a PKC-dependent mechanism.

Figure 2.
Figure 2.

Effect of PKC activity inhibition on steady pHi of papillary muscles superfused with HEPES buffer. Specific inhibition of PKC by chelerythrine produced a significant decrease of pHi in SHR (n=6) without appreciable effect in WKY (n=5). *P<.05 (Student’s t test).

Fig 3⇓ shows steady pHi values in myocardium from control and enalapril-treated rats in CO2/HCO3− buffer. In the presence of the physiological buffer, no significant differences in pHi values were found between SHR-C and WKY-C or between enalapril-treated rats compared with their respective strain-matched untreated controls (ANOVA). We have previously reported the lack of pHi difference between normal and hypertrophic myocardium under bicarbonate.1 In the same article we also demonstrated that if anion exchangers were blocked by SITS, pHi value increased in hypertrophic myocardium bathed with bicarbonate buffer but not in WKY.1 The present results confirm therefore that NHE hyperactivity in hypertrophic myocardium is masked by a parallel enhancement of AE activity in the presence of bicarbonate.

Figure 3.
Figure 3.

Myocardial steady pHi values in CO2/HCO3− buffer. In the presence of the physiological buffer, no difference between resting pHi value in SHR compared with WKY was detected. Values in parentheses indicate the number of determinations.

Imposed Intracellular Acidification

When tissues are suddenly exposed to CO2/HCO3−-buffered media, CO2 easily permeates the cell membrane, causing rapid and transitory intracellular acidification. During the pHi recovery from CO2-induced acid load, at least 50% of proton extrusion is carried by NHE in cardiac muscle.24 25 The changes in pHi brought about on switching from HEPES to CO2/HCO3−-buffered superfusate were analyzed to appreciate the effect of enalapril treatment (Fig 4⇓). The initial fall in pHi was similar in all groups (Table 2⇑), a fact consistent with the lack of difference in βi values. However (and due to more alkaline steady pHi values in SHR superfused with HEPES buffer), pHi values of peak acidosis were higher in this group than in the remaining ones. For this reason, the rates of pHi recoveries (dpHi/dt) at a common pHi value of 6.98 were compared. Fig 5⇓ shows that in SHR the rate of myocardial pHi recovery was about three times faster than in any other group and that chronic treatment with enalapril decreased its value to normal.

Figure 4.
Figure 4.

Transient intracellular acidification induced on switching from HEPES- to CO2/HCO3−-buffered superfusate. Top, Data from untreated control SHR (n=6) and WKY (n=6); bottom, data from enalapril-treated SHR (n=10) and WKY (n=8). *P<.05 (ANOVA).

Figure 5.
Figure 5.

Rate of pHi recovery from CO2-induced intracellular acidification. Mean±SEM values of dpHi/dt calculated at a common pHi value of 6.98 are shown. Values in parentheses indicate the number of determinations. *Significant difference from any other group (ANOVA).

Acid-extruding mechanisms are modulated by pHi; therefore, values of JH+ during the recovery from CO2-induced intracellular acid load were estimated as a function of pHi in each experimental group. Fig 6⇓ shows that a rather linear relationship between JH+ and pHi was obtained in every case. However, for any given pHi value, JH+ values were larger in SHR compared with any other group, with the difference being larger for more acidic pHi values. The enhanced acid extrusion in SHR was reduced to values close to those seen in normotensive rats after enalapril-induced regression of cardiac hypertrophy. No significant difference was detected between WKY-C and SHR-E groups or between WKY-C and WKY-E. Thus, the enhancement of NHE activity detected after acid load in SHR was blunted by chronic treatment with enalapril. No significant difference was detected between the intercepts with the x axes among groups. Values of intercepts were 7.17±0.02, 7.14±0.02, 7.10±0.01, and 7.13±0.03 in SHR-C, SHR-E, WKY-C, and WKY-E, respectively (NS, ANOVA). The intercepts usually reflect the value of the “set point” for the NHE when the experiments are performed in the absence of bicarbonate. However, we should keep in mind that in our experiments the acid load was induced by introducing CO2/HCO3− buffer. The contribution of bicarbonate-dependent mechanisms to pHi regulation takes place along with the recovery in pHi.

Figure 6.
Figure 6.

JH+ as a function of pHi. JH+ was estimated to be equal to intracellular buffer capacity×dpHi/dt. Note that the enhanced acid extrusion in SHR was reduced close to values seen in normotensive rats after enalapril-induced regression of cardiac hypertrophy. *Significant difference from any other group (ANOVA).

Imposed Intracellular Alkalinization

To explore AE activity, and due to the anion exchanger’s sensitivity to increases in pHi, papillary muscles isolated from enalapril-treated and from untreated rat hearts were exposed to TMACl. TMACl-induced intracellular alkalinization has been previously demonstrated to be a valid technique for investigating the activity of the AE based on the fact that no pHi recovery is detected in HCO3−-free solutions.22 23 Fig 7⇓ (top) shows the results of representative experiments in which similar peak pHi values were attained during TMACl pulses carried out on papillary muscles obtained from SHR-C and SHR-E superfused with CO2/HCO3− buffer. The initial velocity of pHi recovery was used as indicative of AE activity, and it was estimated from the linear fits of pHi records during the first minute after peak TMACl-induced alkalinization.23 Using the same experimental approach, we have previously shown that recovery from alkaline loads was faster in SHR than in WKY and that lower pHi values were necessary to drive AE activity in hypertrophic myocardium.1 It can be appreciated now that enalapril treatment reduced the velocity of pHi recovery as well as it regressed cardiac hypertrophy (Fig 7⇓, top). Bars in Fig 7⇓ (bottom) depict the mean initial rate of pHi recovery in the overall experiments of both groups. A significant decrease in the rate of pHi recovery was observed after chronic treatment with enalapril. The initial rate of pHi recovery in SHR-E (0.019±.005 pH unit/min, n=10) was not different from the values measured in WKY-E and WKY-C (0.024±.01, n=8, and 0.021±.009, n=10, respectively).

Figure 7.
Figure 7.

Effect of enalapril treatment on pHi recovery from TMACl-induced intracellular alkalinization in SHR. Papillary muscles were superfused with CO2/HCO3−-buffered media. Top, Data from representative experiments carried out on myocardium from untreated control SHR (•) and enalapril-treated SHR (▴) exposed to 30 mmol/L TMACl. In both experiments, a similar peak pHi value was attained, but the rate of pHi recovery was reduced in SHR-E. Initial velocity of pHi recovery (dpHi/dti) was estimated by fitting the pHi values recorded during the first minute after peak alkalosis to a straight line. Bottom, Bars show mean±SEM values of dpHi/dti in the overall experiments (SHR-C, n=16; SHR-E, n=10). *P<.05 (Student’s t test).

The rate of pHi recovery from TMACl-induced intracellular alkalinization was also measured after the inhibition of PKC activity in hypertrophic myocardium. Two structurally different PKC inhibitors, chelerythrine and calphostin C, were used. Exposure to TMACl increased pHi to a similar value in both the absence and presence of PKC inhibitors. However, the rate of pHi recovery was significantly reduced by both PKC inhibitors (Fig 8⇓). After PKC inhibition with either chelerythrine or calphostin C, the rate of pHi recovery in hypertrophic myocardium was not different from the value measured in myocardium from normotensive rats at a comparable pHi value. The results therefore suggest that enhanced AE activity in hypertrophic myocardium from SHR is mediated by a PKC-dependent mechanism(s).

Figure 8.
Figure 8.

Effect of PKC inhibition on AE activity in SHR myocardium. Data are mean±SEM of the initial rate of pHi recovery after TMACl-imposed alkali load on papillary muscles from hypertrophic hearts (n=5) under control conditions (SHR-C) and after inhibition of PKC activity with either chelerythrine (SHR+Che, 10 mmol/L chelerythrine, n=5) or calphostin C (SHR+Cal, 50 nmol/L, n=4). For comparison, data from untreated normotensive rats (WKY-C, n=3) are shown. Similar values of peak intracellular alkalosis during TMACl pulses were attained in each group (7.45±.03 in SHR-C, 7.45±.03 in SHR+Che, 7.42±.07 in SHR+Cal, and 7.47±.01 in WKY-C). Notice that PKC inhibition significantly decreased the rate of pHi recovery of hypertrophic myocardium. *P<.05 compared with the other groups (ANOVA).

Discussion

Increased NHE activity is one of the most common phenotypic differences found in hypertension. NHE is a member of a multigene family, and four NHE isoforms (NHE-1 through NHE-4) have recently been cloned.11 The NHE-1 isoform is expressed in virtually all tissues and species; it controls cytosolic pH and may also participate in cell growth.26 Evidence of enhanced NHE activity in hypertension is provided by observations in skeletal muscle of SHR27 and of hypertensive patients28 ; in circulating blood cells such as platelets, leukocytes, erythrocytes5 ; and in immortalized lymphoblasts29 derived from individuals with essential hypertension. The vascular smooth muscle from SHR also seems to exhibit enhanced antiport activity.30 In addition, we have reported an increased NHE activity in hypertrophic myocardium of SHR.1 However, the data presented here and our previous results show that no difference in myocardial pHi between hypertrophic and normal myocardium can be detected in the presence of bicarbonate, despite enhanced NHE activity. This is due to the simultaneous hyperactivity of an acidifying (AE) and an alkalinizing (NHE) mechanism.

The question of whether antihypertensive therapy could affect the enhanced NHE activity was investigated before by Rosskopf et al31 in experiments conducted on platelets of hypertensive patients. These authors were unable to detect any normalization of NHE activity after 6 weeks of antihypertensive treatment with enalapril. They claimed that NHE hyperactivity was refractory to antihypertensive treatment and therefore appeared to be a relatively fixed parameter. Whether these contradictory results are a matter of difference in tissues, species, or the relative duration of treatments is not apparent at this time and requires further study. While this manuscript was in preparation, Sánchez et al32 reported that Na+/Li+ countertransport (a mode of operation of NHE) in erythrocytes from hypertensive individuals was normalized after 6 months of enalapril treatment.

The major finding of this study was that the enhancement of NHE and AE activities in hypertrophic myocardium of SHR normalized after PKC inhibition. The C terminal of the rat NHE-1 possesses a putative PKC phosphorylation site,33 and many agonists promote phosphoinositide hydrolysis generating inositol triphosphate and diacylglycerol, the latter then stimulating PKC activity. In connection with this, it was demonstrated that inhibitors of PKC were able to relax aortic tone in vitro and lower blood pressure of SHR in vivo.34 Recent experiments from our laboratory showed that PKC inhibition decreased NHE activity in platelets from SHR but not in WKY.35 These present and previous data are consistent with a “PKC syndrome” that was suggested to play a central pathogenic role in hypertension.36 Kimura et al37 and Aviv et al38 39 have also presented several lines of evidence supporting a connection between [Ca2+]i, PKC, and NHE in the increased peripheral vascular resistance, cardiovascular hypertrophy, salt sensitivity, and insulin resistance of established hypertension. Moreover, stimulation of cell hypertrophy and activation of PKC by stretching isolated cardiomyocytes have been described40 and seem to be linked to autocrine-paracrine secretion of angiotensin II and/or endothelin-1.41 42 Significantly, both of these agonists are well known to stimulate PKC activity in the myocardium.

The regulation of the AE has been less investigated than that of NHE, but evidence for PKC involvement in its regulation in Vero cells was presented by Ludt et al.43 The amino acid sequences of the cardiac-specific AE3 isoform has been recently examined in rats and compared with that of mice and humans.44 45 In all three species, potential consensus phosphorylation sites for protein kinases A and C were identified.

An increase in PKC activity would therefore explain the increased activity of both exchangers in hypertrophic myocardium. The normalization of their activities by enalapril treatment, as well as by PKC inhibition with chelerythrine and calphostin C, would suggest that the pharmacological intervention is mediated through a decrease in PKC activation. No measurements of PKC activity were performed in our study, but increases in PKC-β1,2 and PKC-ε isozymes were detected in left ventricular hypertrophy induced by aortic banding in the rat.46

We would like to emphasize that in the presence of the physiological bicarbonate buffer, the steady-state pHi value of the hypertrophic myocardium results from the interplay between the increased alkalinizing NHE activity and the enhanced acidifying activity of AE. Parallel hyperactivity of both exchangers will normalize pHi, but it will not prevent the increase in [Na+]i caused by NHE activity. Under these circumstances, an increase in [Ca2+]i would take place through the Na+/Ca2+ exchange. Increased [Ca2+]i is known to be an important signal for cellular growth47 and to activate conventional α, β, and γ PKC isoforms.48 49 However, novel PKC-δ has been also reported to be activated after Ca2+ infusion, and its activation was attributed to a Ca2+-dependent production of diacylglycerol through phospholipase C.50 In addition, by stimulating both acid loading and extrusion systems in parallel, the cell’s ability to recover from acid and alkaline loads is improved despite no significant change in pHi.51

Another possibility to be considered is a “primary” increase in [Ca2+]i, which could activate NHE through different mechanisms either directly37 52 or via PKC activation and/or a Ca2+-calmodulin complex.53 The increase in [Ca2+]i in hypertension as the result of different mechanisms has been reported by several authors.54 55 56

In summary, our data show that chronic treatment with enalapril induced normalization of enhanced NHE and AE activities while regressing cardiac hypertrophy in SHR and that its pharmacological effect was linked to a common PKC-related pathway. Whereas the link between PKC and NHE activity in hypertension is not new,5 34 35 36 37 38 39 the participation of the AE and its link with PKC in hypertension is novel and significant. Many questions related to the data presented herein need to be addressed. For example, which does enalapril treatment change first: hypertrophy, NHE activity, or AE activity? What is the temporal relationship between structural changes and exchanger activities? Is the increase in intracellular Ca2+ level found in hypertension the result or the cause of the hyperactivity of the exchangers? And finally, which PKC isoform is involved in determining the hyperactivity of the exchangers? All these questions should be addressed in future investigations.

Selected Abbreviations and Acronyms

βi=intrinsic buffer capacity
ACE=angiotensin-converting enzyme
AE=Na+-independent Cl−/HCO3− exchanger
BW=body weight
-C=control
-E=enalapril treated
HW=heart weight
JH+=net apparent acid equivalent efflux
LVW=left ventricular weight
NHE=Na+/H+ exchanger
PKC=protein kinase C
SBP=systolic blood pressure
SHR=spontaneously hypertensive rat(s)
TMACl=trimethylamine hydrochloride
WKY=Wistar-Kyoto rat(s)

Acknowledgments

Dr Ennis was the recipient of a predoctoral fellowship from La Plata University, and Dr Alvarez was the recipient of a predoctoral fellowship from Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina). Drs Camilión de Hurtado and Cingolani are established investigators of Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.

  • Received October 15, 1997.
  • Revision received November 10, 1997.
  • Accepted December 4, 1997.

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  • Enalapril Induces Regression of Cardiac Hypertrophy and Normalization of pHi Regulatory Mechanisms
    Irene L. Ennis, Bernardo V. Alvarez, María C. Camilión de Hurtado and Horacio E. Cingolani
    Hypertension. 1998;31:961-967, originally published April 1, 1998
    https://doi.org/10.1161/01.HYP.31.4.961
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