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(Circulation Research. 1996;79:705-715.)
© 1996 American Heart Association, Inc.

Articles

Thrombin Activates the Sarcolemmal Na⁺-H⁺ Exchanger

Evidence for a Receptor-Mediated Mechanism Involving Protein Kinase C

Masahiro Yasutake, Robert S. Haworth, Anna King, Metin Avkiran

Cardiovascular Research, The Rayne Institute, St. Thomas' Hospital, London, UK.

Correspondence to Dr Metin Avkiran, Cardiovascular Research, The Rayne Institute, St. Thomas' Hospital, London SE1 7EH, UK. E-mail m.avkiran@umds.ac.uk.

	Abstract

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Thrombin can activate the plasma membrane Na⁺-H⁺ exchanger in a variety of noncardiac cells. We have studied (1) the effect of thrombin on the activity of the sarcolemmal Na⁺-H⁺ exchanger in freshly isolated quiescent ventricular myocytes from the adult rat heart and (2) the signaling mechanism(s) underlying any effect. Reverse-transcription polymerase chain reaction analysis revealed thrombin receptor mRNA expression in a myocyte-enriched cell preparation. As an index of Na⁺-H⁺ exchanger activity, acid efflux rates (J_Hs) were determined in single myocytes (n=4 to 11 per group) loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1 after two consecutive intracellular acid pulses (induced by transient exposure to 20 mmol/L NH₄Cl) in bicarbonate-free medium. At a pH_i of 6.9, J_H did not change significantly during the second pulse relative to the first in control cells. However, when the second pulse occurred in the presence of 0.2, 1, or 5 U/mL thrombin, J_H increased by 30%, 62% (P<.05), and 87% (P<.05), respectively. A hexameric thrombin receptor–activating peptide (SFLLRN) mimicked the effect of thrombin and increased J_H by 73% (P<.05) at 25 µmol/L. In contrast, an inactive control peptide (FLLRN) was without effect at 25 µmol/L. In cells pretreated with 100 nmol/L GF109203X or 5 µmol/L chelerythrine (protein kinase C inhibitors), neither 5 U/mL thrombin nor 25 µmol/L SFLLRN produced a significant increase in J_H. In the presence of 10 µmol/L HOE-694 (a Na⁺-H⁺ exchanger inhibitor), pH_i did not recover after an acid load, even during exposure to 5 U/mL thrombin or 25 µmol/L SFLLRN, confirming that the Na⁺-H⁺ exchanger was the primary acid efflux mechanism under the conditions used. Neither 5 U/mL thrombin nor 25 µmol/L SFLLRN affected resting pH_i and Ca²⁺ or background acid loading. We conclude that (1) adult rat ventricular myocytes express a functional thrombin receptor, whose stimulation results in increased activity of the sarcolemmal Na⁺-H⁺ exchanger, and (2) this effect appears to occur through a protein kinase C–mediated mechanism.

Key Words: thrombin receptor • protein kinase C • Na⁺-H⁺ exchanger • heart • myocyte

	Introduction

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The sarcolemmal Na⁺-H⁺ exchanger is a major acid extrusion system in cardiac myocytes and plays an important role in restoration of pH_i following an acid load.¹ Recent evidence suggests that the exchanger may also play an important role in determining the severity of the unfavorable sequelae of myocardial ischemia and reperfusion, such as arrhythmias, contractile dysfunction, and infarction.^{1 2} In cardiac myocytes, as in other cell types, the activity of the Na⁺-H⁺ exchanger is regulated not only by pH_i but also by a number of extracellular stimuli (such as exposure to adrenergic agonists, endothelin, angiotensin II, and adenosine triphosphate) through receptor-mediated mechanisms (for recent review, see Puceat and Vassort³ ).

Thrombin is a multifunctional protease, which, in addition to its established role in blood coagulation and thrombus formation, induces a variety of cellular responses through the recently cloned thrombin receptor (for review, see Coughlin⁴ ). The receptor can be activated not only by thrombin but also by synthetic peptides (thrombin receptor–activating peptides), which mimic the "tethered ligand" domain of the receptor that is revealed after cleavage by thrombin.⁴ A number of cell types within the cardiovascular system, including platelets,⁵ endothelial cells,⁵ and vascular smooth muscle cells,⁶ have been shown to express mRNA coding for the cloned thrombin receptor. Indeed, exposure to thrombin can produce a range of physiological effects in these cell types; these include activation of the plasma membrane Na⁺-H⁺ exchanger, which occurs (at least in part) through a PKC-mediated pathway.^{7 8 9} With respect to cardiac myocytes, Steinberg et al¹⁰ have shown that thrombin can alter phosphoinositide metabolism and cytosolic Ca²⁺ in cultured neonatal rat ventricular myocytes. In a similar model, Glembotski et al¹¹ have demonstrated recently that the cloned thrombin receptor is expressed and may mediate a hypertrophic response after exposure to thrombin. However, there is a paucity of data regarding the effects of thrombin in adult cardiac myocytes and the role of the cloned thrombin receptor in this cell type, particularly with regard to regulation of sarcolemmal Na⁺-H⁺ exchanger activity.

The primary objectives of the present study were to use freshly isolated adult rat ventricular myocytes to (1) obtain molecular evidence for expression of the cloned thrombin receptor in this cell type, (2) determine the effects of thrombin and a thrombin receptor–activating peptide on sarcolemmal Na⁺-H⁺ exchanger activity, and (3) delineate the roles of intracellular Ca²⁺ and PKC in mediating any regulation of exchanger activity via the thrombin receptor. Our results provide molecular and physiological evidence that adult rat ventricular myocytes express a functional thrombin receptor, whose stimulation results in increased activity of the sarcolemmal Na⁺-H⁺ exchanger. This effect occurs in the absence of a detectable increase in [Ca²⁺]_i and appears to involve a PKC-mediated mechanism.

	Materials and Methods

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The present investigation was performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act of 1986, published by Her Majesty's Stationery Office, London, UK.

Isolation of Ventricular Myocytes
Ventricular myocytes were isolated from the hearts of adult male Wistar rats (200 to 250 g body weight) using a collagenase-based enzymatic digestion technique, which has been described previously.¹² In brief, rats were anesthetized by inhalation of diethyl ether, and hearts were excised and perfused (37°C) in the Langendorff mode for four sequential periods, as follows: (1) with Tyrode's solution (mmol/L: NaCl 137, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.5, HEPES 10, and glucose 10, adjusted to pH 7.4 at 34°C with NaOH) for 5 minutes, (2) with nominally Ca²⁺-free Tyrode's solution (mmol/L: NaCl 135, KCl 5.4, NaH₂PO₄ 0.33, MgCl₂ 1.0, HEPES 10, and glucose 10, adjusted to pH 7.2 at 34°C with NaOH) for 5.5 minutes, (3) with nominally Ca²⁺-free Tyrode's solution containing collagenase (Worthington type 1, 200 U/mL) for 10 minutes, and (4) with storage buffer (mmol/L: KOH 78, KCl 30, KH₂PO₄ 30, MgSO₄ 3, EGTA 0.5, HEPES 10, glutamic acid 50, taurine 20, and glucose 10, adjusted to pH 7.2 at 34°C with KOH) for 5 minutes. All solutions were gassed with 100% O₂. After the perfusion procedure, the ventricles were removed and chopped into several pieces in storage buffer. The tissue fragments were then gently agitated to facilitate cell dispersion, which commonly resulted in a myocyte yield of >80% rod-shaped cells. The cell suspension was maintained in storage buffer at 25°C for at least 1 hour before use in the microepifluorescence studies. When required for RNA extraction, myocyte-enriched preparations were obtained by centrifugation (25g for 5 minutes) of the cell suspension, followed by gravity sedimentation for 15 minutes.

Extraction of RNA From Myocyte-Enriched Preparations
Myocyte-enriched preparations (2x10⁶ cells per heart) were pelleted by centrifugation at 160g for 5 minutes. The supernatant was discarded, and total RNA was prepared from the cell pellet using Trisolv reagent (Biotecx Laboratories Inc) according to the manufacturer's instructions. mRNA was subsequently prepared from total RNA using the Invitrogen FastTrack mRNA isolation kit (R & D Systems Europe Ltd) as recommended by the manufacturer.

Reverse-Transcription PCR
mRNA (1 µg) from myocyte-enriched preparations was reverse-transcribed in a volume of 20 µL, using the Invitrogen cDNA Cycle kit and oligo dT primer (R & D Systems Europe Ltd). Samples were extracted with phenol/chloroform, and the cDNA was precipitated with ethanol at -70°C for 20 minutes. The cDNA was dissolved in 20 µL RNase-free water before PCR. Aliquots (5 µL) were subjected to 35 cycles of PCR (1 minute at 94°C, 1 minute at 55°C, and 1 minute at 72°C) using primers (100 pmol) specific for the rat thrombin receptor (5'-TGTGAACTGATTATGTCAATT-3' and 5'-TAACCAGGAAAAGGATATG-3'), as described by Glembotski et al.¹¹ As a negative control, reverse transcriptase was omitted from the cDNA synthesis step. As a positive control, plasmid (1 pg) containing subcloned rat thrombin receptor cDNA (see Glembotski et al¹¹ ) was also subjected to PCR amplification. PCR products were incubated for 60 minutes in the presence or absence of the restriction enzyme EcoRV before separation on 5% acrylamide gels. Based on the known sequence of the rat thrombin receptor cDNA,¹¹ the intact PCR product obtained using these primers should be 946 bp, while cleavage at the EcoRV recognition site should result in two products of 585 and 361 bp. To further confirm the identity of the intact PCR product obtained from myocyte cDNA, this was additionally subjected to sequence analysis in both directions, using the primers described above and an ABI 373 automated sequencing system (Molecular Medicine Unit, King's College, London, UK).

Determination of Sarcolemmal Na⁺-H⁺ Exchanger Activity
Measurement of pH_i
pH_i was monitored in single ventricular myocytes using the pH-sensitive fluorescent dye C-SNARF-1, as described previously.^{13 14} Briefly, aliquots of cells were loaded with C-SNARF-1 within 6 hours of their isolation by incubating them in a 4 µmol/L solution of the acetoxymethyl ester (Calbiochem) for 10 minutes at room temperature. Cells loaded with C-SNARF-1 were then allowed to settle on a glass coverslip at the bottom of a 100-µL chamber, mounted on the stage of an inverted microscope (Nikon Diaphot), and viewed using a x40 oil immersion objective with a numerical aperture of 1.3. After adherence to the coverslip, cells were superfused (3.5 mL/min) with Tyrode's solution at 34°C. Cells were excited with light at 540 nm, and the resulting fluorescence emission intensity from a selected area of a single myocyte was measured simultaneously at 580 nm (I₅₈₀) and 640 nm (I₆₄₀), using a dual-emission photometer system (model D104C, Photon Technology International Inc), which contained an adjustable aperture and two multialkali photomultiplier tubes (type R928, Hamamatsu Photonics UK Ltd). Background fluorescence was measured using an identical aperture and subtracted from the signal. After current-voltage conversion, the acquired signals were digitized at 1.7 Hz and stored on computer hard disk, using pClamp software (Axon Instruments).

Calibration of Fluorescence Signal
The emission intensity ratio (I₅₈₀/I₆₄₀) was calculated and converted to a pH_i scale, using in situ calibration data obtained by exposing cells loaded with C-SNARF-1 to nigericin-containing calibration solutions of pH 5.8 to 8.0.¹⁵ The calibration solutions consisted of 140 mmol/L KCl, 1 mmol/L MgCl₂, and 10 µmol/L nigericin and were buffered with 10 mmol/L of one of the following zwitterionic buffers: MES at pH 5.8, 6.2, and 6.4; PIPES at pH 6.6, 6.8, and 7.0; and HEPES at pH 7.2, 7.4, 7.7, and 8.0. All calibration solutions were adjusted to the correct pH with NaOH. Fig 1A shows a recording from a typical calibration experiment in a single cell. The data acquired from each such calibration experiment were normalized by dividing all I₅₈₀/I₆₄₀ ratios by the ratio obtained in that cell at a pH_i of 7.0¹⁶ ; thus, the normalized I₅₈₀/I₆₄₀ ratio at pH_i 7.0 was always 1. The calibration curve shown in Fig 1B was obtained by a nonlinear least squares fit of the normalized data from seven cells to the equation given below, with the curve constrained to pass through the point having the coordinates (I₅₈₀/I₆₄₀)/(I₅₈₀/I₆₄₀)_pH7=1.0, pH 7.0.¹⁶

(E1)

The best-fit values for pK and a were 7.12 and -1.38, respectively.

View larger version (16K): [in this window] [in a new window]   Figure 1. A, Representative recording of the C-SNARF-1 fluorescence emission intensity ratio (I580/I640) from a single cell during exposure to nigericin-containing calibration solutions of varying pH (numbers indicate pH). In this trace, the I580/I640 ratio was averaged over 5-second intervals for clarity. B, Calibration curve constructed using normalized I580/I640 ratio data from seven experiments (see text for details).

Estimation of ß_i
ß_i was estimated by stepwise removal of extracellular NH₄Cl, as described in detail previously.^{17 18} Twelve cells from three different hearts were exposed to Tyrode's solution containing 20 mmol/L NH₄Cl for 1 to 2 minutes, followed by a stepwise reduction of extracellular NH₄⁺ to 10, 5, 2.5, 1, and 0 mmol/L. At each step, calculated changes in [NH₄⁺]_i and measured changes in pH_i were used to estimate ß_i, from the equation ß_i=[NH₄⁺]_i/pH_i. These experiments were carried out in the presence of 30 µmol/L HOE-694 in order to prevent acid extrusion via the Na⁺-H⁺ exchanger and 5 mmol/L Ba²⁺ to reduce NH₄⁺ efflux through K⁺ channels.¹⁸ By plotting ß_i as a function of pH_i from 51 determinations and subsequent linear least squares regression analysis, the following equation was obtained:

(E2)

Equation 2 was used in all subsequent experiments to estimate ß_i at different pH_i values.

Calculation of Sarcolemmal Acid Efflux
Since all experiments were carried out in the nominal absence of bicarbonate, J_H was used as a direct index of sarcolemmal Na⁺-H⁺ exchanger activity.¹⁸ J_H was estimated during recovery from an intracellular acidosis using the equation J_H=ß_i·dpH_i/dt (where dpH_i/dt is the rate of recovery of pH_i), as previously described.¹⁸ No corrections were made for cell volume–to–surface area ratio, since all experiments were carried out in the same cell type (ie, adult rat ventricular myocytes) in a randomized manner, with contemporary controls. Intracellular acidosis was induced by the washout (6 minutes) of 20 mmol/L NH₄Cl after its transient (2- to 3-minute) application. The pH_i trace during the recovery phase was fitted to a single exponential function, as previously described.¹⁹ At pH_i intervals of 0.05 during this phase, J_H values were calculated from ß_i (estimated as described above) and dpH_i/dt (calculated as the differential derivative of the exponential fit), thus enabling the construction of pH_i-versus-J_H relationships. A rightward shift of this curve (signifying a greater J_H at a given pH_i) was taken to indicate an increased Na⁺-H⁺ exchanger activity.

Estimation of Intracellular Ca²⁺
[Ca²⁺]_i was estimated using the Ca²⁺-sensitive fluorescent dye indo 1 (Cambridge BioScience), as described previously.²⁰ Aliquots of cells were loaded with indo 1, by incubating them for 20 minutes at room temperature in a 10 µmol/L solution of the acetoxymethyl ester. As in the studies with pH_i measurement, cells were superfused (3.5 mL/min) with Tyrode's solution at 34°C. The microepifluorescence approach used was also similar to that described above for C-SNARF-1, except that only a portion of a selected cell was illuminated (at 360 nm), and the fluorescence emission intensity was measured simultaneously at 405 and 485 nm, through the use of appropriate optical filters. The emission intensity ratio (I₄₀₅/I₄₈₅) was used as an index of [Ca²⁺]_i, without calibration.²⁰

Solutions
All chemicals were purchased from Sigma Chemical Co, unless stated otherwise. The thrombin receptor–activating peptide SFLLRN and the inactive control peptide FLLRN were gifts from Glaxo (Greenford, UK). The Na⁺-H⁺ exchanger inhibitor HOE-694 was a gift from Hoechst (Frankfurt, Germany).

All experiments were performed in HEPES-buffered Tyrode's solution. In Na⁺-free Tyrode's solution, NaCl was replaced with 137 mmol/L choline chloride, and pH was adjusted to 7.4 with KOH. NH₄Cl (20 mmol/L) was added directly to Tyrode's solution without osmotic compensation. Stock solutions of thrombin (T-6759, Sigma) and HOE-694 were prepared in deionized water; SFLLRN and FLLRN were dissolved directly in Tyrode's solution. The PKC inhibitors GF109203X (Calbiochem) and chelerythrine were dissolved in dimethyl sulfoxide and subsequently diluted with Tyrode's solution to obtain appropriate stock solutions. All agents were added to superfusion solutions, at the appropriate concentrations, shortly before the beginning of experiments.

Experimental Protocols
Experiments were performed using the protocols described below, unless stated otherwise. Basal pH_i was noted after 5 to 10 minutes of superfusion with normal Tyrode's solution (pH 7.4). To study the effects of thrombin and SFLLRN on resting pH_i, cells were then exposed to these agents for a further 5 minutes. To study the effects of these stimuli on sarcolemmal Na⁺-H⁺ exchanger activity, cells were subjected to intracellular acidosis by consecutive transient exposures to NH₄Cl. After the initial 6-minute period of NH₄Cl washout (first acid pulse), cells were superfused with normal Tyrode's solution for an additional 6 minutes to allow further recovery of pH_i before the second transient exposure to NH₄Cl (second acid pulse). In control cells, both acid pulses occurred under identical conditions. When studying the effects of thrombin, SFLLRN, or FLLRN, these agents were present throughout the second pulse (ie, during exposure to and washout of NH₄Cl). When studying the effects of thrombin and SFLLRN in the presence of GF109203X or chelerythrine, these agents were included in all solutions from 6 minutes before the second pulse to the end of the experiment. J_H-versus-pH_i curves were constructed using data obtained during the pH_i recovery phases following both acid pulses. This double-pulse protocol was necessitated by the intercell variability in J_H even at identical pH_i values (eg, see Figs 4, 5, and 7) and enabled paired data analysis. At the end of every experiment, the cell under study was exposed to nigericin-containing calibration solution at pH 7.0, thus enabling normalization of the I₅₈₀/I₆₄₀ ratios. Equation 1 and the fitted values for pK and the constant a were then used to calculate pH_i from the normalized I₅₈₀/I₆₄₀ values.

View larger version (37K): [in this window] [in a new window]   Figure 4. JH values at an identical pHi of 6.9 after consecutive acid pulses in control cells (C, n=11) and in cells that were exposed to thrombin (0.2 U/mL, n=7; 1 U/mL, n=6; and 5 U/mL, n=5) throughout the second pulse. Open columns indicate the first pulse; hatched columns, the second pulse. *P<.05.

View larger version (38K): [in this window] [in a new window]   Figure 5. JH values at an identical pHi of 6.9 after consecutive acid pulses in control cells (C, n=11) and in cells that were exposed to SFLLRN (1 µmol/L, n=4; 5 µmol/L, n=4; and 25 µmol/L, n=8) throughout the second pulse. Open columns indicate the first pulse; hatched columns, the second pulse. *P<.05.

View larger version (30K): [in this window] [in a new window]   Figure 7. JH values at an identical pHi of 6.9 after consecutive acid pulses in control cells (C) and in cells that were exposed to 5 U/mL of thrombin (Th) or 25 µmol/L of SFLLRN (SFLL) throughout the second pulse, in the absence of PKC inhibitor (left) or in the presence of either 100 nmol/L of GF109203X (GF, middle) or 5 µmol/L of chelerythrine (CH, right). Open columns indicate the first pulse; hatched columns, the second pulse. Respective group sizes for C, Th, and SFLL were as follows: n=6, 5, and 5 in the absence of PKC inhibitor; n=5, 5, and 5 in the presence of GF; and n=4, 4, and 4 in the presence of CH. *P<.05.

To study the effects of thrombin and SFLLRN on [Ca²⁺]_i, each cell was consecutively exposed to (1) field stimulation at 0.5 Hz (to enable the recording of Ca²⁺ transients), (2) Tyrode's solution with or without 5 U/mL of thrombin or 25 µmol/L of SFLLRN, and (3) Tyrode's solution with 10 mmol/L of caffeine (at an increased superfusion rate of 12 to 15 mL/min), with the indo 1 emission intensity ratio (I₄₀₅/I₄₈₅) monitored throughout.

Statistical Analysis
Experiments within each study subsection were carried out in a randomized manner, with contemporary controls. Gaussian-distributed variables were expressed as mean±SEM. To assess changes in J_H within groups (ie, between first and second acid pulses), a paired t test was used. For intergroup comparisons, data were subjected to one-way ANOVA. If a difference among mean values was established, further analysis was carried out using either Dunnett's test (to compare every group with the control group) or Tukey's test (to compare every group with every other). A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thrombin Receptor Expression in Ventricular Myocytes
Fig 2 illustrates the results of reverse-transcription PCR analysis of mRNA from myocyte-enriched preparations. No signal was obtained in the absence of reverse transcriptase (lane 2), confirming the absence of contamination. The intact PCR product (lane 3) was 946 bp in size, as expected from the known thrombin receptor cDNA sequence, and matched exactly that obtained by PCR amplification of thrombin receptor cDNA (lane 6). Cleavage of the PCR products by EcoRV gave two bands of the sizes 585 and 361 bp (lanes 4 and 7, respectively), as expected from the rat thrombin receptor cDNA sequence, regardless of the source of the template cDNA. These results suggest that mRNA coding for the cloned thrombin receptor was expressed in this cell preparation. The identity of the intact reverse-transcription PCR product was further confirmed by sequence analysis (data not shown), which revealed it to be homologous with the published rat thrombin receptor cDNA sequence.¹¹

View larger version (68K): [in this window] [in a new window]   Figure 2. Reverse-transcription PCR analysis of thrombin receptor mRNA expression. Acrylamide gel electrophoresis was used to analyze the products from PCR amplification of either cDNA obtained by reverse transcription of mRNA from myocyte-enriched preparations (lanes 3 and 4) or rat thrombin receptor cDNA (lanes 6 and 7). Lanes 3 and 6 show intact PCR products, and lanes 4 and 7 show products obtained after exposure to EcoRV. Lane 2 shows negative control (no reverse transcriptase), and lanes 1 and 5 show pGEM DNA markers (Promega) of the sizes (from the top) 2645, 1605, 1198, 676, 517, 460, 396, 350, 222, and 179 bp. The sizes of the PCR products were 946 bp without exposure to EcoRV and 585 and 361 bp after exposure to EcoRV, as predicted from the rat thrombin receptor cDNA sequence.

Effects of Thrombin Receptor Stimulation on Resting pH_i
Neither thrombin (5 U/mL) nor SFLLRN (25 µmol/L) produced a significant change in resting pH_i (n=3 per group). In control cells and those exposed to thrombin or SFLLRN, basal pH_i was 7.27±0.06, 7.31±0.08, and 7.24±0.07 (P=NS), respectively. After 3 minutes of exposure to vehicle, thrombin, or SFLLRN, pH_i remained at similar values, measuring 7.23±0.07, 7.25±0.09, and 7.20±0.06 (P=NS), respectively.

Regulation of Sarcolemmal Na⁺-H⁺ Exchanger Activity via the Thrombin Receptor
The Table shows basal and minimal pH_i values in the various study groups, obtained just before and immediately after the first and second acid pulses. Within each study subsection, there was no significant difference between groups in basal or minimal pH_i at either time point. The overall mean value for basal pH_i before exposure to any acid load or pharmacological intervention, taking into account data from all cells included in this part of the study, was 7.25±0.01 (n=93). This value is comparable to basal pH_i values previously reported for rat ventricular myocytes maintained in HEPES-buffered medium.^{21 22 23}

View this table: [in this window] [in a new window]   Table 1. Mean Values for Basal pHi (Measured Just Before NH4Cl Application) and Minimal pHi (Measured Immediately After NH4Cl Washout) During the First and Second Acid Pulses

Fig 3 shows representative recordings of pH_i during two consecutive acid pulses in single myocytes as well as the pH_i-versus-J_H relationships constructed using data from several such experiments. In control experiments (Fig 3A), the profiles of pH_i recovery from intracellular acidosis were similar after both pulses; consequently, the acid efflux curves were superimposed. Thus, at any given pH_i, J_H values were similar at both time points during the experimental protocol, showing that temporal changes in Na⁺-H⁺ exchanger activity do not occur in the absence of agonist stimulation.

View larger version (24K): [in this window] [in a new window]   Figure 3. Representative pHi recordings (top) and pHi-vs-JH curves (bottom) obtained during two consecutive acid pulses in control cells (A, n=11) and cells in which 5 U/mL thrombin was present throughout the second pulse (B, n=5). Thrombin accelerated recovery from acidosis and resulted in a rightward shift in the pHi-vs-JH relationship.

When cells were exposed to 5 U/mL of thrombin during the second acid pulse, pH_i recovery from intracellular acidosis was accelerated (Fig 3B). This effect was reflected by a rightward shift of the pH_i-versus-J_H curve such that over the pH_i range of 6.80 to 7.10, J_H was significantly greater in the presence of thrombin. In order to investigate the dose dependence of the effects of thrombin, such experiments were performed with three concentrations of thrombin, ranging from 0.2 to 5 U/mL. Fig 4 shows J_H at an identical pH_i of 6.9 after both the first acid pulse (in the absence of thrombin) and the second acid pulse (in the presence of thrombin). Thrombin increased J_H in a dose-dependent manner, with statistically significant increases of 62% and 87% at 1 and 5 U/mL, respectively.

The effect of the synthetic thrombin receptor–activating peptide SFLLRN was also examined to determine whether this could mimic the Na⁺-H⁺ exchanger stimulatory effect of thrombin, thereby supporting an involvement of the cloned receptor. As with thrombin, SFLLRN produced a dose-dependent rightward shift of the pH_i-versus-J_H curve. Consequently, J_H at pH_i 6.9 was increased in a dose-dependent manner, resulting in a statistically significant increase of 73% at 25 µmol/L (Fig 5). Indeed, with this concentration, J_H was increased significantly throughout the pH_i range of 6.70 to 7.05. In contrast, 25 µmol/L of the inactive control peptide FLLRN did not produce any significant increase in J_H throughout this pH_i range (data not shown).

Role of Intracellular Ca²⁺
The intracellular Ca²⁺ study was carried out to determine whether thrombin and SFLLRN (at the concentrations shown to increase sarcolemmal Na⁺-H⁺ exchanger activity) could also increase [Ca²⁺]_i within an appropriate time frame, thereby implicating a role for Ca²⁺ in the relevant signaling mechanism(s). Fig 6 shows a representative recording of the indo 1 emission intensity ratio from a single cell, during consecutive exposure to field stimulation, 5 U/mL of thrombin, and 10 mmol/L of caffeine. Although predictable changes in the signal were observed in response to both field stimulation and exposure to caffeine (at the beginning and end of the protocol), there was no detectable change in the signal during 3 minutes of exposure to thrombin. Similar observations were made during exposure to 25 µmol/L of SFLLRN or Tyrode's solution alone (n=3 per group).

View larger version (9K): [in this window] [in a new window]   Figure 6. Recording of the indo 1 fluorescence emission intensity ratio (I405/I485) from a single cell during consecutive exposure to field stimulation (left), 5 U/mL of thrombin (middle), and 10 mmol/L of caffeine (right). In the middle and right panels, arrows indicate the start of superfusion with solution containing thrombin or caffeine. The recording is representative of three experiments following an identical protocol.

Role of PKC
The PKC inhibitor study was carried out to determine the role of PKC in thrombin receptor–mediated stimulation of sarcolemmal Na⁺-H⁺ exchanger activity. To this end, the effects of thrombin and SFLLRN on the pH_i-versus-J_H relationship were reexamined in the presence of one of two PKC inhibitors, GF109203X²⁴ or chelerythrine,²⁵ at concentrations shown in our preliminary studies²⁶ to be sufficient to inhibit phorbol ester–induced stimulation of sarcolemmal Na⁺-H⁺ exchanger activity. The results are summarized in Fig 7. GF109203X (100 nmol/L) and chelerythrine (5 µmol/L) had no significant effect on J_H in control cells. Consistent with our earlier observations, in the absence of the PKC inhibitors, both 5 U/mL of thrombin and 25 µmol/L of SFLLRN produced significant increases in J_H of 74% and 81%, respectively. However, in the presence of either GF109203X or chelerythrine, thrombin and SFLLRN were no longer able to produce a significant increase in J_H.

Effect of Na⁺-H⁺ Exchanger Inhibition
The Na⁺-H⁺ exchanger inhibitor study was carried out to confirm that the accelerated recovery of pH_i following intracellular acidosis (and hence the rightward shift of the pH_i-versus-J_H relationship) in the presence of thrombin or SFLLRN was mediated exclusively through activation of the sarcolemmal Na⁺-H⁺ exchanger. Cells (n=3 per group) were once again subjected to consecutive acid pulses, this time both in the presence of 5 U/mL of thrombin or 25 µmol/L of SFLLRN and with 10 µmol/L of HOE-694 (a novel Na⁺-H⁺ exchanger inhibitor²⁷ ) also present during the second pulse. As illustrated by the representative recordings in Figs 8A and 9A, a rapid recovery of pH_i was observed after the first acid pulse in both cases. However, there was little recovery of pH_i after the second pulse, in the presence of HOE-694, despite the continued presence of thrombin or SFLLRN. These observations confirm that in the nominally bicarbonate-free conditions used, the sarcolemmal Na⁺-H⁺ exchanger is the primary mechanism of pH_i recovery from acidosis and that activation of other acid equivalent efflux mechanisms is unlikely to play a significant role in mediating the actions of thrombin and SFLLRN.

View larger version (24K): [in this window] [in a new window]   Figure 8. A, Representative pHi recording (n=3) from a single myocyte during two consecutive acid pulses in the presence of thrombin (5 U/mL). During the second pulse, HOE-694 (10 µmol/L) was also present. B, Representative pHi recording from a single myocyte (n=3) during an acid pulse in the absence of extracellular Na+. Thrombin (5 U/mL) was applied for 3.5 minutes, as indicated, after the induction of intracellular acidosis.

Role of Background Acid Loading
The acid-loading experiment was carried out, using a protocol that has been described previously,²⁸ to confirm that the accelerated recovery of pH_i following intracellular acidosis in the presence of thrombin or SFLLRN was not due to an inhibition of background acid-loading mechanisms (eg, metabolic acid production) under low pH_i conditions. Cells (n=3 per group) were subjected to an acid pulse in the nominal absence of extracellular Na⁺ (to block Na⁺-dependent acid efflux mechanisms), and 5 U/mL of thrombin or 25 µmol/L of SFLLRN was applied after the induction of intracellular acidosis. As illustrated by the representative recordings in Figs 8B and 9B, under these conditions, background acid loading was not observed, and neither thrombin nor SFLLRN had any significant effect on pH_i.

View larger version (16K): [in this window] [in a new window]   Figure 9. A, Representative pHi recording (n=3) from a single myocyte during two consecutive acid pulses in the presence of SFLLRN (25 µmol/L). During the second pulse, HOE-694 (10 µmol/L) was also present. B, Representative pHi recording from a single myocyte (n=3) during an acid pulse in the absence of extracellular Na+. SFLLRN (25 µmol/L) was applied for 3.5 minutes, as indicated, after the induction of intracellular acidosis.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study provides evidence, for the first time, that thrombin is capable of activating the sarcolemmal Na⁺-H⁺ exchanger in isolated adult rat ventricular myocytes. This effect of thrombin is mimicked by the synthetic thrombin receptor–activating peptide SFLLRN, thus indicating the involvement of a receptor-mediated mechanism. The Na⁺-H⁺ exchanger stimulatory actions of both thrombin and SFLLRN are abolished by selective inhibitors of PKC, suggesting an important role for this enzyme in the intracellular signaling mechanisms downstream from thrombin receptor activation.

Assessment of Na⁺-H⁺ Exchanger Activity
In the present study, an agonist-induced rightward shift of the pH_i-versus-J_H relationship (in the absence of bicarbonate-dependent pH_i regulatory mechanisms) has been taken to indicate activation of the Na⁺-H⁺ exchanger. Similar approaches have been used in various cell types to study changes in plasma membrane Na⁺-H⁺ exchanger activity (eg, in response to pharmacological manipulation¹⁶ or oncogenic transformation²⁹ ). Of particular relevance to the present study, shifts in the pH_i-versus-J_H relationship have been used previously in isolated ventricular myocytes as indicators of altered sarcolemmal Na⁺-H⁺ exchanger activity in response to extracellular agonists.^{18 22} Recently, Wu et al²¹ have suggested that even in nominally bicarbonate-free medium, bicarbonate-dependent pH_i regulatory mechanisms may be operative, thereby complicating the interpretation of data with regard to Na⁺-H⁺ exchanger activity. Nevertheless, under the conditions used in the present study, mechanisms other than sarcolemmal Na⁺-H⁺ exchange are unlikely to have contributed significantly to acid equivalent efflux, since pH_i recovery from acidosis was blocked completely in the presence of the Na⁺-H⁺ exchanger inhibitor HOE-694. Furthermore, thrombin and SFLLRN did not appear to alter background acid loading within the relevant pH_i range. Thus, it is reasonable to assume that any rightward shift of the pH_i-versus-J_H relationship in response to thrombin receptor activation, by either thrombin or SFLLRN, was indeed a reflection of increased sarcolemmal Na⁺-H⁺ exchanger activity.

Actions of Thrombin on Cardiac Myocytes
The role of thrombin in modulating cardiac myocyte function has received little attention to date. Steinberg et al¹⁰ were the first to study the effects of thrombin on mammalian myocytes, by showing that it could modulate phosphoinositide metabolism and cytosolic Ca²⁺ in cultured neonatal rat ventricular cells. Subsequently, the same group showed that thrombin-induced phosphoinositide hydrolysis in these cultured cells could be enhanced by hypoxia.³⁰ Recently, Glembotski et al¹¹ have demonstrated that the thrombin receptor is expressed by cultured neonatal rat ventricular myocytes, thereby providing a mechanistic basis for the cellular effects of thrombin in this preparation. With regard to adult mammalian cardiac myocytes, there is evidence that thrombin can activate the L-type Ca²⁺ channel in guinea pig cells³¹ and induce lysophosphatidylcholine accumulation in rabbit cells,³² with the latter effect mimicked by a thrombin receptor–activating peptide.³² However, the present study is the first to provide molecular as well as physiological evidence for thrombin receptor expression in adult ventricular myocytes.

It is distinctly possible that contaminating cells of nonmyocyte origin (eg, fibroblasts, endothelial cells, or smooth muscle cells) may have contributed to the thrombin receptor mRNA expression detected in our myocyte-enriched cell preparation by reverse-transcription PCR analysis. However, the stimulatory effects of thrombin and SFLLRN on sarcolemmal Na⁺-H⁺ exchanger activity (monitored in individual myocytes) are strongly suggestive of functional thrombin receptor expression by the myocyte fraction. It may be argued that such effects on cardiac myocytes could have a paracrine basis, through the thrombin receptor–mediated release (from contaminating nonmyocyte cells) of an unknown Na⁺-H⁺ exchanger stimulatory factor. However, this is unlikely for several reasons. First, all visible cells that adhered to the coverslip at the bottom of the cell chamber had the morphological characteristics of cardiac myocytes. Second, cell density in the chamber was very low, ensuring considerable dilution of any paracrine factor that is released from contaminating nonmyocyte cells. Finally, the cells in the chamber were continuously superfused at 3.5 mL/min (equivalent to a complete change of the chamber volume every 1.7 seconds); thus, any released paracrine factor would be rapidly removed. Nevertheless, definitive confirmation of thrombin receptor expression by cardiac myocytes awaits analysis by immunocytochemistry and/or in situ hybridization.

Regulation of Sarcolemmal Na⁺-H⁺ Exchanger Activity via the Thrombin Receptor
Thrombin has been shown to increase the activity of the plasma membrane Na⁺-H⁺ exchanger in other cell types of the cardiovascular system, such as platelets,⁷ endothelial cells,⁸ and vascular smooth muscle cells.⁹ Our observations show that thrombin increases the activity of the sarcolemmal Na⁺-H⁺ exchanger also in adult rat ventricular myocytes and that this effect occurs in a dose-dependent manner. Furthermore, by demonstrating that the Na⁺-H⁺ exchanger stimulatory effect of thrombin can be mimicked by SFLLRN, our study strongly suggests the involvement of the cloned thrombin receptor.⁴ It is important to note that thrombin receptor stimulation increased J_H (ie, acid efflux via the Na⁺-H⁺ exchanger) throughout a pH_i range of 6.70 to 7.10. Despite this, however, thrombin receptor stimulation did not alter resting pH_i over a 5-minute period. This is probably because the actual J_H achieved at pH_i values approaching basal pH_i (mean value of 7.25) was very low (<1 mmol/L/min), even in the presence of thrombin or SFLLRN.

Signaling Mechanisms Downstream From Receptor Stimulation
The thrombin receptor is a member of the seven-transmembrane-domain receptor family, and its stimulation has been linked with G protein–mediated activation of phospholipase C and, subsequently, PKC.³³ Indeed, in several cell types, thrombin-induced activation of the Na⁺-H⁺ exchanger has been shown to be mediated, at least in part, via PKC activation.^{7 9} Our demonstration that the thrombin receptor–mediated stimulation of sarcolemmal Na⁺-H⁺ exchanger activity is abolished by both GF109203X and chelerythrine, two potent and selective inhibitors of PKC,^{24 25} suggests a key role for this enzyme in the relevant intracellular signaling pathway in rat ventricular myocytes. This is consistent with previous studies in this cell type that have shown that agents capable of activating PKC (eg, phorbol esters,^{22 26} ₁-adrenergic agonists,^{22 26} and endothelin²³ ) are potent activators of the sarcolemmal Na⁺-H⁺ exchanger.

Which of the isoforms of PKC are involved in thrombin receptor–mediated stimulation of sarcolemmal Na⁺-H⁺ exchanger activity cannot be deduced on the basis of the present study. The lack of a rapid effect on [Ca²⁺]_i by thrombin and SFLLRN (at concentrations sufficient to increase sarcolemmal Na⁺-H⁺ exchanger activity), taken together with the marked inhibitory effects of GF109203X and chelerythrine, might suggest a role for Ca²⁺-independent isoforms of PKC in thrombin receptor–mediated stimulation of exchanger activity. In this regard, the Ca²⁺-independent novel PKC isoforms and have been detected as the most abundant isoforms of this enzyme in adult rat ventricular myocardium (for recent review, see Sugden and Bogoyevitch³⁴ ) and have been shown to be readily activated by exposure to a number of extracellular stimuli.^{35 36} However, it should be noted that regardless of the identity of the isoform(s) involved, PKC is unlikely to regulate sarcolemmal Na⁺-H⁺ exchanger activity by direct phosphorylation of the exchanger.³⁷

It is important to note also that the lack of effect of thrombin receptor stimulation on [Ca²⁺]_i in our quiescent myocytes is contrary to observations made in platelets⁷ and vascular smooth muscle cells.⁹ This apparent discrepancy is likely to have arisen because of the insignificant role played by phosphoinositide hydrolysis products in inducing Ca²⁺ release from intracellular stores in mammalian cardiac myocytes,^{38 39} relative to other cell types.⁴⁰ The dominant mechanism of Ca²⁺ release from the sarcoplasmic reticulum in cardiac myocytes is widely accepted to be Ca²⁺-induced release via ryanodine receptors,^{39 41} which itself might be subject to some modulation by phosphoinositide hydrolysis products.^{38 39} Therefore, the possibility cannot be discounted that thrombin receptor stimulation may affect [Ca²⁺]_i regulation in nonquiescent myocardium. Indeed, recent evidence⁴² suggests that in spontaneously contracting or electrically driven neonatal rat ventricular myocytes, thrombin receptor activation by a high concentration (300 µmol/L) of SFLLRN can increase both systolic and diastolic [Ca²⁺]_i.

Physiological/Pathophysiological Relevance
Glembotski et al¹¹ have shown that in cultured neonatal rat ventricular myocytes, activation of the thrombin receptor induces the phenotypic and morphological characteristics of cellular hypertrophy. Although other extracellular agonists that can induce a hypertrophic response in this preparation (eg, phenylephrine⁴³ and endothelin⁴⁴ ) also share the ability to activate the sarcolemmal Na⁺-H⁺ exchanger,³ it is currently unclear whether this action plays a role in the initiation of the hypertrophic response.

As noted earlier, the sarcolemmal Na⁺-H⁺ exchanger has been implicated as a key determinant of the severity of ischemia/reperfusion-induced cardiac dysfunction, including arrhythmias.^{1 2} Therefore, it is possible that thrombin-induced activation of the sarcolemmal Na⁺-H⁺ exchanger could modulate the outcome of ischemia and reperfusion. In this regard, we have shown that although reperfusion-induced arrhythmias are suppressed by interventions designed to inhibit the Na⁺-H⁺ exchanger,^{45 46} they are exacerbated by stimuli that can activate the exchanger.⁴⁷ Of particular relevance to the present study, Goldstein et al⁴⁸ have shown that the incidence of malignant ventricular arrhythmias during acute ischemia is greater after thrombotic coronary occlusion than after nonthrombotic balloon occlusion, implicating an arrhythmogenic role for factors (such as thrombin) that are associated with thrombus formation. More recently, the same group suggested that during myocardial ischemia, activation of the thrombin receptor may contribute to arrhythmogenesis by inducing an increase in intracellular Na⁺,⁴⁹ an observation that is consistent with Na⁺-H⁺ exchanger activation. An arrhythmogenic role for thrombin during ischemia/reperfusion, possibly via Na⁺-H⁺ exchanger activation, could have clinical significance, since intracoronary thrombosis is the commonest cause of acute ischemia in patients with coronary artery disease.⁵⁰

Potential Limitations of Study
Although the concentrations of thrombin (1 and 5 U/mL) that we have shown to significantly activate the sarcolemmal Na⁺-H⁺ exchanger in freshly isolated adult rat ventricular myocytes are within the range proposed to occur in the vicinity of intracoronary thrombi,⁴⁸ they are nevertheless higher than those previously shown to elicit cellular responses in cultured neonatal ventricular myocytes from the same species.^{10 11} In contrast, the concentration of SFLLRN that significantly increased sarcolemmal Na⁺-H⁺ exchanger activity in the present study was comparable to that shown recently to induce physiological responses in cultured neonatal rat ventricular myocytes.⁵¹ This may be a reflection of partial receptor proteolysis during our collagenase-based cell isolation procedure, since it is known that the cleaved thrombin receptor becomes desensitized to further activation by thrombin.⁵² Indeed, we have preliminary evidence (data not shown) that in myocytes isolated using a combination of protease and collagenase (rather than collagenase alone), the Na⁺-H⁺ exchanger is no longer activatable by thrombin. It would be of interest to determine whether in these cells SFLLRN retains the ability to increase the exchanger's activity. It would also be of value to determine whether the maximal responses elicited by thrombin versus SFLLRN in our cells, in terms of stimulation of sarcolemmal Na⁺-H⁺ exchanger activity, are comparable.

The sequence of the thrombin receptor–activating peptide SFLLRN is based on the human thrombin receptor sequence and differs by one amino acid from the corresponding sequence of the rat thrombin receptor (SFFLRN). Therefore, it may be argued that this could have led to an underestimation of the effects of SFLLRN in rat ventricular myocytes. Contrary to this, however, it has been shown recently⁵³ that the thrombin receptors of rat vascular smooth muscle cells can be activated with comparable potency by synthetic receptor–activating peptides based on either the human or the rat thrombin receptor sequence.

It should be noted that the findings of the present study do not preclude an involvement of changes in [Ca²⁺]_i in the signaling pathway(s) underlying thrombin receptor–mediated stimulation of sarcolemmal Na⁺-H⁺ exchanger activity. In this regard, in the present study, the effects of thrombin and SFLLRN on [Ca²⁺]_i were examined only under conditions in which pH_i was unaffected. To ascertain the potential role of changes in [Ca²⁺]_i, it would be necessary to determine whether thrombin and SFLLRN modulate [Ca²⁺]_i under conditions in which they also increase Na⁺-H⁺ exchanger activity (ie, in the presence of acute intracellular acidosis).

Concluding Comments
In conclusion, the present study indicates that adult rat ventricular myocytes express a functional thrombin receptor, whose stimulation leads to increased activity of the sarcolemmal Na⁺-H⁺ exchanger. PKC appears to play a key role in the intracellular signaling mechanisms(s) downstream from thrombin receptor stimulation, although direct phosphorylation of the Na⁺-H⁺ exchanger by this enzyme is unlikely to be involved. The physiological and/or pathophysiological significance of thrombin receptor–mediated regulation of sarcolemmal Na⁺-H⁺ exchanger activity remains to be determined.

	Selected Abbreviations and Acronyms

 

ßi = intracellular intrinsic buffering power C-SNARF-1 = carboxy-seminaphthorhodafluor-1 HOE-694 = 3-methylsulfonyl-4-piperidinobenzoyl guanidine I405, I485, I580, I640 = fluorescence emission intensity at 405, 485, 580, and 640 nm JH = rate of acid efflux PCR = polymerase chain reaction PKC = protein kinase C

	Acknowledgments

 
This study was supported in part by the British Heart Foundation, the St. Thomas' Hospital Heart Research Trust, and The David and Frederick Barclay Foundation. Dr Yasutake was an International Research Fellow from the Nippon Medical School, Tokyo, Japan. Dr Avkiran is the holder of a British Heart Foundation (Basic Science) Senior Lectureship Award. The authors thank C.J. McGill for technical assistance, Drs W.A. Coetzee and G. Brooks for many valuable discussions, C.E. Fuller (Glaxo, Greenford, UK) for the gift of the peptides SFLLRN and FLLRN, Dr W. Scholz, Dr H.J. Lang, and Prof B.A. Scholkens (Hoechst AG, Frankfurt, Germany) for the gift of HOE-694, and Dr C.C. Glembotski (San Diego State University, San Diego, Calif) for the gift of the rat thrombin receptor clone. The helpful advice of Dr W.F. Boron (Yale University, New Haven, Conn) on the assessment of Na⁺-H⁺ exchanger activity and the assistance of Drs K.T. MacLeod and C.M.N. Terracciano (National Heart and Lung Institute, London, UK) in carrying out the studies with indo 1 are gratefully acknowledged.

Received December 27, 1995; accepted July 12, 1996.

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