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(Hypertension. 1996;28:1013-1017.)
© 1996 American Heart Association, Inc.

Articles

Chelerythrine Inhibits Na⁺-H⁺ Exchange in Platelets From Spontaneously Hypertensive Rats

Oscar A. Gende

Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Medicas, Universidad Nacional de La Plata (Argentina).

Correspondence to Oscar A. Gende, Centro de Investigaciones Cardiovasculares, Facultad de Ciencias Medicas, Universidad Nacional de La Plata, 60 Y 120, 1900 La Plata, Argentina. E-mail cicme@isis.unlp.edu.ar.

	Abstract

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Hypertension has been associated with increased activity of the Na⁺-H⁺ exchanger. To study the role played by protein kinase C in this process, we used chelerythrine, a potent and specific inhibitor of the kinase. After an acid load by ammonium chloride preincubation, platelets isolated from spontaneously hypertensive rats showed a faster and larger increase in intracellular pH than platelets from Wistar-Kyoto rats. The initial rate of intracellular pH recovery was 2.46±0.26 pH units per minute in spontaneously hypertensive rats and 1.74±0.19 in Wistar-Kyoto rats. For protein kinase C inhibition, platelets were incubated for 30 minutes with 10 µmol/L chelerythrine. This treatment induced a significant reduction in the recovery rate only in spontaneously hypertensive rat platelets, indicating that a pathway involving protein kinase C participates in the prestimulation of the exchanger in cells from this rat strain. Addition of chelerythrine reduced the baseline intracellular pH of platelets. No significant difference was found between the decrease of steady-state intracellular pH induced by chelerythrine in either rat strain. These findings indicate that this model of hypertension is characterized by increased Na⁺-H⁺ activity mediated by protein kinase C stimulation.

Key Words: hydrogen-ion concentration • blood platelets • ion exchange • sodium-hydrogen antiporter • protein kinases • chelerythrine • rats, inbred SHR

	Introduction

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Hypertension has been associated with abnormalities in a variety of ion transport mechanisms regulating H⁺, Ca²⁺, and Na⁺. Increased activity of the Na⁺-H⁺ exchanger has been described in SHR both in vivo¹ and in vitro,^{2 3} and this activity has been linked to the increased vascular tone and smooth muscle growth found in SHR. The increased Na⁺-H⁺ activity in hypertension might be caused by increased gene expression (transcriptional regulation) or increased function of the exchanger protein (posttranslational modification). Although controversial, most evidence suggests that transcriptional regulation does not account for the observed functional difference in Na⁺-H⁺ exchanger activity; therefore, the increased Na⁺-H⁺ antiport activity in hypertension would be due to an increased turnover rate at each site^{4 5} rather than to an increased number of transport sites.

Posttranslational processes such as phosphorylation are known to affect Na⁺-H⁺ antiport activity⁶ ; activation of the exchanger results from phosphorylation-induced conformational changes. It appears that phosphorylation shifts the pH range over which the pH_i sensor of the Na⁺-H⁺ antiport regulates ion transport.⁷ The Na⁺-H⁺ exchanger from quiescent vascular myocytes from SHR is more heavily phosphorylated than that of myocytes from WKY.⁸ These findings have led to the speculation that increases in Na⁺-H⁺ exchanger activity in hypertension may be caused by alterations in the balance between kinases and phosphatases that regulate the degree of phosphorylation of Na⁺-H⁺ antiport molecules. Several reports support the premise that PKC is involved in the maintenance of hypertension in SHR.^{9 10 11 12}

Phorbol esters, strong activators of PKC, increase Na⁺-H⁺ exchange in the renal tubules,¹³ smooth muscle,¹⁴ and platelets.¹⁵ If a marked degree of antiporter prestimulation were present in cells from hypertensive subjects before exposure to phorbol esters, the response to phorbol esters would become blunted because few sites remain available for phosphorylation. According to this speculation, it was found that the basal activity of the platelet Na⁺-H⁺ antiport is greater in hypertensive than in normotensive subjects, but this difference disappears when the platelets are treated with a phorbol ester.¹⁶ Phorbol esters also modify the kinetics of the Na⁺-H⁺ antiport in WKY but not in SHR vascular smooth muscle.¹⁴

The aim of these experiments was to compare the effect of PKC inhibition on the kinetics of pH_i recovery after an acid load in WKY and SHR. The inhibitory effect was more pronounced in hypertensive rats. Furthermore, the inhibitor was assayed in nonacidified platelets, and the responses to thrombin stimulation in the presence and absence of chelerythrine were compared.

	Methods

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Platelet Isolation
Washed platelets were obtained by a modification of the method of Kimura et al.¹⁷ Arterial blood was drained from the abdominal aorta of anesthetized rats and collected with one sixth volume of ACD (2.5% sodium citrate, 1.5% citric acid, 2.0% glucose, and 10 µmol/L prostaglandin E₁).

Platelet-rich plasma was obtained by centrifugation at 150g for 20 minutes and was centrifuged at 600g for 20 minutes to form a platelet pellet. The pellet was washed twice with a calcium-free buffer (mmol/L: NaCl 140, KCl 5, EGTA 0.5, aspirin 0.1, glucose 10, and HEPES 10 as well as 1% bovine serum albumin; pH 7.35). After resuspension in the same solution, but with EGTA omitted, the platelets were incubated for 15 minutes in 6 µmol/L 2',7-bis(2-carboxyethyl)-5,6-carboxyfluorescein acetoxy methyl ester (BCECF-AM) at 37°C.

The extracellular dye was washed in a calcium-free solution, and the pH_i of some platelets was measured after 5 to 10 minutes of stabilization in a HEPES-buffered solution (mmol/L: NaCl 140, KCl 5, CaCl₂ 1, MgSO₄ 1, glucose 10, and HEPES 10; pH 7.35). The stabilization time used allowed intracellular calcium to reach a steady-state concentration with a minimum of BCECF leakage.¹⁸ The effect of thrombin was evaluated after 8 minutes of calcium repletion. Other platelets were acidified in the calcium-free buffer by 30 minutes of exposure to 25 mmol/L NH₄Cl. The Na⁺-H⁺ exchanger was activated by addition of a 50-µL aliquot of acidified platelets into 2 mL of a prewarmed HEPES-buffered solution at pH 7.35.

Fluorescence Measurements
BCECF fluorescence was monitored in a spectrofluorimeter (SFM25, Kontron Instruments) with the use of excitation and emission wavelengths of 440/503 and 535 nm, respectively. Calibration of the fluorescence signals was carried out in a HEPES/high-K⁺ solution (130 mmol/L NaCl replaced with 130 mmol/L KCl) with 10 µmol/L nigericin, adjusted to defined pH values according to the method of Thomas et al.¹⁹ Autofluorescence (AutoF) was measured in platelets from the same batch that were not loaded with the dye, and the fluorescence ratio was calculated as follows: Fluorescence Ratio=(F₅₀₃-AutoF₅₀₃)/(F₄₄₀-AutoF₄₄₀).

For steady-state determinations, the ratio of successive measurements at both excitation wavelengths with an integration time of 2 seconds was calculated. Kinetic measurements were performed at 503 nm with integration times of 0.5 second in the recovery experiments and of 2 seconds for evaluation of the responses to thrombin or chelerythrine.

The initial rate of alkalinization (dpH_i/dt) during pH_i recovery from acid loads or after thrombin stimulation was obtained by least-squares fitting to the initial linear segment of the respective curves.

For estimation of the intrinsic buffer capacity (ß_i), the pH_i change was measured immediately on exposure of the cells to 10 mmol/L NH₄Cl. ß_i was defined as [NH₄⁺]_i/pH_i. The ammonium concentration was calculated with a pK_a value of 9.21.

Statistics
Results are expressed as mean±SE. Comparisons between groups were performed with the unpaired Student's t test. Paired comparisons were used for evaluation of the effects of drugs on platelets from the same rat. A value of P=.05 was considered to be the limit of statistical significance.

	Results

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Male WKY and SHR between 15 and 20 weeks of age were used. Systolic pressures obtained with the tail-cuff method were 118±3 and 168±3 mm Hg, respectively.

The buffer capacities of the washed platelets were 76±6 and 63±4 mmol/L per pH unit for WKY and SHR, respectively, at a pH_i of 6.52±0.03 and 40±2 and 32±4 for WKY and SHR, respectively, at a pH_i of 7.05±0.04. The pH_i indicated was the initial value of the spike produced by addition of 10 mmol/L NH₄Cl in the presence or absence of EIPA. Chelerythrine treatment did not modify buffer capacities.

Na⁺-H⁺ Exchanger Activity in Preacidified Platelets
Representative curves of the recovery from acidification in WKY and SHR platelets are plotted in Fig 1. In the presence of 10 µmol/L EIPA, a small increase in pH_i, at a similar rate in both groups, was observed. This could indicate processes that are independent of the exchanger, such as a minute BCECF leak. The initial pH_i of the preacidified platelets, estimated by the first values in the EIPA-inhibited runs, were similar (6.43±0.03 and 6.49±0.03 in SHR and WKY, respectively). After resuspension of control platelets in the HEPES-buffered solution, there was a short period during which fluorescence recording was affected by mixing artifacts. Thereafter, there was an almost linear increase that was adjusted with a regression line fitted to the values recorded during the first 8 seconds. This initial slope of pH_i recovery (dpH_i/dt) was faster in platelets from SHR (2.46±0.26 pH units per minute, n=6) than in platelets from WKY (1.74±0.19, n=9, P<.05, Fig 2). As shown above, no statistically significant difference in buffer capacities could be established between rat strains or between control and chelerythrine-treated platelets, so it was assumed that the differences in dpH_i/dt corresponded to changes in the fluxes of acid equivalents (J_H+). Preincubation during 30 minutes with 10 µmol/L chelerythrine significantly reduced the slope of recovery by 0.66±0.17 pH units per minute in SHR (paired differences, P<.05). Although a tendency toward a reduction appeared in WKY, chelerythrine did not induce a statistically significant inhibition in this rat strain (paired differences, 0.36±0.19 pH units per minute, P=NS); thus, the inhibition by chelerythrine is distinctly higher in SHR platelets. A similar conclusion was obtained when the initial slope of recovery in the inhibited platelets was expressed as a percentage of parallel control experiments (74±6% in SHR, P<.05; 83±9% in WKY, P=NS). In SHR, 1.6 minutes after recovery had begun, the pH_i of the platelets reached values of 7.13±0.06 in control experiments and 7.01±0.05 in platelets preincubated in chelerythrine (P<.05). On the other hand, the same pH_i (6.99±0.03) was reached in both cases with WKY platelets, suggesting that the PKC inhibitor eliminates the Na⁺-H⁺ exchanger activation associated with hypertension.²⁰ Thus, a treatment that presumably reduced the endogenous phosphorylation of the antiport in SHR by blocking a prestimulated kinase decreased the activity of the Na⁺-H⁺ antiport during recovery from an acid load.

View larger version (25K): [in this window] [in a new window]   Figure 1. Traces of representative experiments show pHi recovery after acid load. Washed rat platelets were preincubated 30 minutes at room temperature with 25 µmol/L NH4Cl in the presence or absence of 10 µmol/L chelerythrine (Cheler.). Platelets were diluted 20 times with a prewarmed HEPES-buffered solution at pH 7.35 in the presence or absence of 10 µmol/L EIPA. Platelets pretreated with chelerythrine showed inhibition by EIPA similar to that of control platelets; only one trace is shown for clarity. Note the faster and larger pHi rise in SHR traces. Inhibition produced by chelerythrine in SHR platelets was also greater. At the end of each trace, the spike produced by addition of 10 mmol/L NH4Cl is shown. This maneuver allowed estimation of buffer capacities.

View larger version (30K): [in this window] [in a new window]   Figure 2. Initial recovery rate after acid load in platelets from WKY (n=9) and SHR (n=6). Mean values ±SE are shown. Hatched bars indicate platelets preincubated for 30 minutes with 10 µmol/L chelerythrine. *Statistically significant difference, Student's t test for independent samples or for paired comparisons according to the case.

Effects of Chelerythrine on Basal pH_i
The pH_i values of the platelets maintained in a HEPES-buffered solution at pH 7.35 and 37°C were 7.09±0.02 in SHR and 7.04±0.02 in WKY (n=7 for both groups). Resting pH did not differ significantly between the two strains. Fig 3 shows the effect of chelerythrine (final concentration, 10 µmol/L) on these platelets. The addition of the PKC blocker induced a fall in pH_i that in 6.5 minutes amounted to 0.061±0.017 pH units in the case of SHR platelets. A similar acidification (0.072±0.18 pH units) was observed in WKY platelets.

View larger version (30K): [in this window] [in a new window]   Figure 3. Baseline pHi in platelets from WKY and SHR. Mean values ±SE are shown. Hatched bars indicate pHi reached 6.5 minutes after addition of 10 µmol/L chelerythrine. *P<.05 (n=7).

Effects of Chelerythrine on the Response to Thrombin
The response to 0.1 IU/mL thrombin was an initial drop, followed by an increase of pH_i that resulted in persistent alkalinization. The alkalinization was greater in WKY platelets (0.088±0.009 pH units versus 0.055±0.009 in SHR platelets, P<.05). When the rises of pH_i in response to thrombin were compared, the dpH_i/dt of the platelets was greater in WKY (0.173±0.020 pH units per minute) than in SHR (0.099±0.020, P<.05, Fig 4). Chelerythrine decreased the recovery rate in both groups of platelets to 0.054±0.008 pH units per minute in WKY platelets and 0.058±0.016 in SHR platelets (Fig 4).

View larger version (24K): [in this window] [in a new window]   Figure 4. Initial alkalinization rate after stimulation with thrombin (final concentration, 0.1 IU/mL) in platelets from WKY and SHR. Hatched bars indicate response obtained when thrombin was added 6.5 minutes after 10 µmol/L chelerythrine. *Statistically significant difference.

	Discussion

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A variety of hormones, neurotransmitters, and growth factors express their biological activities by stimulating phospholipase C–mediated hydrolysis of phosphoinositides. Two second messengers are generated—inositol triphosphate and diacylglycerol; diacylglycerol in turn activates PKC, a phospholipid-dependent protein kinase. PKC can also be stimulated by phorbol esters, which presumably bind to the same domain as diacylglycerol. PKC is a key element in signal transduction and cell regulation, eliciting a variety of cellular responses by phosphorylating target proteins on serine and threonine residues.

The PKC molecule contains two domains: a regulatory domain that interacts with calcium, phosphatidylserine, and diacylglycerol, and a catalytic domain with binding sites for ATP and protein substrates. One of the most straightforward approaches to the study of the role PKC plays in cellular processes is to inhibit the enzymatic activity in intact cells. For this purpose, permeable, potent, and selective compounds are required. Calphostin and sphingosine inhibit PKC, interacting with the regulatory moiety, and sanguivamicin, H-7, and staurosporine interact with the ATP binding site. The latter compound is the most potent PKC inhibitor, but its complete lack of specificity is a major problem when selective blockade of a PKC-dependent pathway is desired.²¹ In the present study, we used a potent and specific inhibitor, chelerythrine, which interacts with the catalytic domain of PKC competing with phosphate acceptors.²² It has been previously reported that chelerythrine inhibits calcium increase and platelet aggregation.²³ PKC activity has been shown to be high in platelets from SHR.²⁴ This activity seems to be related to a higher Na⁺-H⁺ antiport activity and phosphorylation even though it has been claimed that the Na⁺-H⁺ exchanger is not a direct substrate of PKC.

In this study, we measured Na⁺-H⁺ antiport activity after an acid load in normotensive and hypertensive rats. The velocity of pH_i recovery was 41% higher in SHR than in WKY. This finding is in agreement with previous data showing increased Na⁺-H⁺ transport activity in platelets,²⁵ lymphocytes,^{26 27} and myocardium²⁸ of SHR. An increased activity of the Na⁺-H⁺ exchanger in blood cells has also been detected in patients with essential hypertension.^{29 30} Whereas no differences in the recovery of platelet pH_i were detected in WKY platelets, the enhanced recovery rate of pH_i in SHR platelets was significantly reduced by the presence of the PKC inhibitor. These findings indicate a higher degree of prestimulation of the Na⁺-H⁺ antiport in hypertensive rats by this phosphorylating pathway.

Although in unstimulated cells at physiological extracellular pH the Na⁺-H⁺ exchanger will be nearly quiescent, some activity might be necessary to compensate metabolic acid loads and H⁺ leaks through the cell membrane. Therefore, PKC modulation could affect steady-state pH_i. Our experiments showed a pH_i decrease after 10 mmol/L chelerythrine was added to platelets maintained in a HEPES-buffered solution at pH 7.35. This is in agreement with the reduction of pH_i observed with another PKC inhibitor, staurosporine, in human platelets.³¹ A larger pH_i decrease in SHR platelets than in WKY platelets after inhibition of PKC with H-7 has been previously reported.³² This finding was interpreted as a demonstration of a higher basal activity of the exchanger in SHR. In the present study, chelerythrine treatment induced a reduction in baseline pH_i of similar magnitude in SHR and WKY despite the greater activity demonstrated in acidified platelets. Preliminary experiments suggested that other phenomena in addition to a possible modulation of the Na⁺-H⁺ exchanger were responsible for the chelerythrine-induced fall of pH_i: The pH_i fall was faster after addition of chelerythrine than when the exchanger was completely inhibited by EIPA. Furthermore, after blockade of the Na⁺-H⁺ antiport with EIPA, chelerythrine was still able to reduce pH_i (results not shown). This cumulative response indicates that chelerythrine lowers basal pH_i by a mechanism other than modulation of Na⁺-H⁺ exchanger activity.

Two parameters were used for characterization of thrombin responses: the initial velocity and the level of persistent alkalinization reached. Both were greater in WKY than in SHR. Although other factors could be involved in the different pH_i responses to thrombin, such as changes in calcium-calmodulin–stimulated pathways, it is tempting to use the same interpretation used for the phorbol experiments of Livne et al¹⁶ : Since cells from hypertensive animals have a greater basal phosphorylation, a smaller number of sites remains available for further stimulation, resulting in a lower response to phosphorylating agents. In contrast, it has been reported that platelets from hypertensive individuals show a significantly faster and larger pH_i rise in response to a saturating concentration of thrombin.²⁰

The interaction between the changes in intracellular calcium and pH_i during the responses of platelets to agonists is still controversial.³³ It has been reported that thrombin produces both a sustained alkalinization and an intracellular calcium peak. The former was antagonized with PKC blockers, with little effect on intracellular calcium concentration, suggesting that these changes are mediated by different mechanisms.³⁴ On the other hand, the involvement of intracellular calcium concentration in the activation of Na⁺-H⁺ exchange in some cells is supported by the inhibitory effect of calmodulin antagonists on the activation of the antiport.³⁵ The response of pH_i to thrombin in SHR could possibly be influenced by a different rise in intracellular calcium in platelets of hypertensive rats,¹⁸ a phenomenon that has been demonstrated in the response of human platelets to thrombin.³⁶

The evaluation of highly active inhibitors of PKC, such as chelerythrine, will help clarify the role of this enzyme in regulating pH_i, a determinant of smooth muscle tone and growth. Taking into account the similarities between platelets and smooth muscle cells, several attempts have been made since 1987³⁷ to correlate increases in platelet Na⁺-H⁺ exchanger activity with the degree of hypertension or to find a genetic link between these anomalies. It is well known that intracellular alkalinization leads to increased vascular wall tension.³⁸ Also, cell alkalinization has been related to an increased number of mitotic cycles in cell cultures. However, in physiological buffers, pH_i changes caused by Na⁺-H⁺ antiport hyperactivity became damped by an increased buffer capacity and by the existence of bicarbonate exchangers, and it is unlikely that cell alkalinization causes direct vascular changes.³⁹ A new clue to the biological significance of a PKC-stimulated increase in Na⁺-H⁺ exchanger activity in hypertension is given in a recent report.²⁸ Na⁺-H⁺ exchanger hyperactivity induces a simultaneous increase of Cl^--HCO₃^- exchange in physiological buffers. The decrease in intracellular H⁺ that is due to the first exchanger is compensated by an equivalent efflux of HCO₃^- leaving an uncompensated intracellular Na⁺_i increase that in turn would affect the intracellular calcium extrusion by the Na⁺-Ca²⁺ exchanger. We would speculate that this mechanism results in an indirect alteration of vascular wall tension.

	Selected Abbreviations and Acronyms

 

EIPA = ethyl isopropyl amiloride pHi = intracellular pH PKC = protein kinase C SHR = spontaneously hypertensive rat(s) WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This work was supported by grants from Universidad de la Plata and Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET) (Argentina). The author is grateful to Monica Rando for her technical assistance.

Received October 25, 1995; first decision November 30, 1995; first decision August 2, 1996;

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