Cardiovascular Research

Cardiovascular Research 1998 38(3):703-710; doi:10.1016/S0008-6363(98)00039-X
© 1998 by European Society of Cardiology

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Copyright © 1998, European Society of Cardiology

Phenylephrine-induced stimulation of Na⁺/Ca²⁺ exchange in rat ventricular myocytes

Milan Stengl, Kanigula Mubagwa, Edward Carmeliet and Willem Flameng^*

Centre for Experimental Surgery and Anaesthesiology, Katholieke Universiteit Leuven, Provisorium 1, Minderbroederstraat 17, B-3000 Leuven, Belgium

^* Corresponding author. Tel.: +32 (16) 337298; Fax: +32 (16) 337855.

Received 17 September 1997; accepted 16 January 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: The effect of an -adrenergic agonist, phenylephrine, on the Na⁺/Ca²⁺ exchange current in rat ventricular myocytes was investigated. Methods: The Na⁺/Ca²⁺ exchange current was measured at room temperature in rat ventricular myocytes as the whole-cell current induced by addition of extracellular Na⁺ and Ca²⁺, while blocking Na⁺ current by setting the holding potential at –30 mV, K⁺ currents by intracellular Cs⁺, TEA⁺ and by extracellular Ba²⁺, Ca²⁺ current by nifedipine and Na⁺ pump current by ouabain or by 0 extracellular K⁺. Results: Under these experimental conditions, application of external Na⁺ and Ca²⁺ induced a current which was further increased by phenylephrine. Phenylephrine (80 µM) increased the current by up to 31.0±5.4% of control at all membrane potentials tested both below and above the reversal potential. The reversal potential (+21.0±3.2 mV), which corresponded with the theoretical reversal potential for the Na⁺/Ca²⁺ exchange current under our ionic conditions (+21.3 mV), was not changed by phenylephrine (+23.2±4.1 mV). Applying phenylephrine in the absence of Na⁺/Ca²⁺ exchange (0 Na_e⁺, 0 Ca_e²⁺) did not change the current. The effect was resistant to propranolol, a β-adrenergic blocker, but prevented by prazosin, an -receptor antagonist, by neomycin, an inhibitor of phospholipase C, and by chelerythrine, a selective inhibitor of protein kinase C. Phorbol 12-myristate 13-acetate failed to stimulate the current. The effect remained similar under conditions of high (HEPES_i=5 mM) and low (HEPES_i=0.5 mM) intracellular pH buffering. Conclusion: Our data indicate that phenylephrine stimulates the Na⁺/Ca²⁺ exchange, both in the forward and the reverse modes, probably via a protein kinase C-dependent pathway.

KEYWORDS Experimental; Heart; Electrophysiology; Pharmacology; Adrenergic agonist; Membrane current; Myocyte; Na/Ca exchanger; Protein kinase; Rat

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
-Adrenergic receptors affect the inotropic state of the mammalian myocardium. The effect is complex: a sustained positive inotropic effect is usually preceded by a transient decrease in contraction amplitude [1]; under certain conditions, a sustained negative inotropic effect can be obtained [2, 3]. Different mechanisms have been suggested to explain the positive effect: a reduction of K⁺ currents and subsequent prolongation of the action potential with an increased Ca²⁺ influx through Ca²⁺ channels [4], a stimulation of phosphatidylinositol turnover [1]and/or an increased myofibrillar sensitivity, either directly by an action on contractile proteins or indirectly by inducing intracellular alkalinization [5].

The Na⁺/Ca²⁺ exchanger which catalyses the electrogenic exchange of 3 Na⁺ for 1 Ca²⁺ across the plasma membrane plays a major role in the excitation–contraction coupling (ECC) of the heart. The Na⁺/Ca²⁺ exchange is particularly important for relaxation, by maintaining a balance between Ca²⁺ entry via the Ca²⁺ channels and Ca²⁺ extrusion from the cell via the exchanger [6]. On the other hand, the Na⁺/Ca²⁺ exchanger may be involved in contraction, when Ca²⁺ entering the cell via the reverse mode contributes to sarcoplasmic reticular (SR) Ca²⁺ loading and/or to triggering SR Ca²⁺ release [7]. However, despite the importance of the exchanger for ECC and Ca²⁺ homeostasis, regulation of the exchanger is still unclear. It has been shown that the exchanger is regulated by the phospholipid environment within the sarcolemma [8, 9], by cytoplasmic ATP [10], by protons [11]and by insulin [12]. Recently, Ballard and Schaffer [13]demonstrated that Gq-linked agonists (phenylephrine, endothelin I and angiotensin II) stimulate Na⁺-dependent ⁴⁵Ca²⁺-uptake in rat heart sarcolemmal vesicles. The objective of the present study was to investigate the effect of -adrenergic stimulation by phenylephrine on the current carried by the Na⁺/Ca²⁺ exchange in isolated rat cardiomyocytes and to investigate the possible signalling pathways involved. Part of the results was presented to the Biophysical Society [14].

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Cell preparation and voltage clamp
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). The technique for cell isolation and voltage clamping are the same as those described previously [15]. In brief, cells were dissociated from Langendorff-perfused rat hearts by a protocol which consisted of a constant-flow perfusion (8–10 ml/min) with: (1) 5 min of Ca²⁺-free Tyrode solution; (2) 3 min of Ca²⁺-free Tyrode solution, containing collagenase (Type A, Boehringer Mannheim; 0.22 mg/ml) and protease (Type XIV, Sigma; 0.12 mg/ml); (3) 7–11 min of Ca²⁺-free, collagenase-containing Tyrode; and (4) 10 min of 0.18-mM Ca²⁺ solution. The isolated myocytes were stored in normal Tyrode solution at room temperature (22–25°C).

The cell suspension was placed into a small chamber (0.4 ml) on the stage of an inverted microscope (Diaphot 200, Nikon, Japan). They were constantly perfused with the external solution at a flow rate of 0.7 ml/min. Voltage-clamp was performed in the whole-cell configuration of the patch-clamp method using an Axopatch 200A amplifier (Axon Instruments, USA) and heat-polished borosilicate glass pipettes (horizontal puller; Zeitz Instrumente, Germany) with resistances of 2–6 M when filled with pipette solution. Voltage-clamp signals were low-pass filtered (5 kHz 4-pole Bessel), digitized by an A/D converter (Labmaster DMA, Scientific solutions) at 1 kHz and stored in an IBM-AT personal computer using the pClamp software (Axon Instruments). Cell capacitance was measured by integrating the area of the capacitive transient elicited by a 10-mV depolarizing step from the holding potential –80 mV and digitized at 100 kHz. Mean value was 231±17 pF (n=46). Either square or ramp clamp pulses were employed. The ramp pulse contained three phases: an initial 90-mV depolarizing phase from the holding potential of –30 mV, a second hyperpolarization of 180 mV and then a third phase returning to the holding potential at a speed of 90–120 mV/s. The current–voltage relationship was measured during the second hyperpolarizing part. All experiments were performed at room temperature (22–25°C).

2.2 Solutions
The solutions and protocol for measuring the Na⁺/Ca²⁺ exchange current were similar to those used by Kimura et al. [16]. The normal Tyrode solution contained (in mM): NaCl 135, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.9, Na₂HPO₄ 0.33, HEPES 10, glucose 10; pH adjusted to 7.4 with NaOH. The solution to activate the Na⁺/Ca²⁺ exchange had the same composition as the normal Tyrode except for CaCl₂ which was at 1 mM and for pH which was titrated (to 7.4) with Tris. In the Na⁺- and Ca²⁺-free external solution used to suppress Na⁺/Ca²⁺ exchange, NaCl was replaced with N-methyl-D-glucamine (NMDG, 135 mM) or LiCl (135 mM), CaCl₂ was omitted without substitution and pH titrated to 7.4 with HCl (or Tris). In both external solutions, BaCl₂ (1 mM) and CsCl (2 mM) were added to block K⁺ currents, ouabain (100 µM) or 0 K_e⁺ to block Na⁺-pump current, nifedipine (10–50 µM) to block Ca²⁺ current. The internal solution contained (in mM): Cs-glutamate 50, TEA-Cl 25, MgCl₂ 1, Na₂ATP 5, Na₂GTP 0.1, HEPES 5, EGTA 42, CaCl₂ 36, pH titrated with CsOH to 7.2. To prevent any change of the Ca²⁺ concentration caused by Ca²⁺ either entering the cell from extracellular space or released from the sarcoplasmic reticulum, 42 mM EGTA was included in the pipette solution to increase the buffering capacity for Ca²⁺. The free Ca²⁺ concentration was calculated using the software Cabuf (G. Droogmans, University of Leuven) which is based on Fabiato and Fabiato [17]. The stability constants for EGTA were according to Owen [18]. Free Ca²⁺ concentration was 1 µM (pCa 6.0).

2.3 Drugs
Phenylephrine (Sigma) was prepared as stock solution (20 mM) in distilled water. Stock solutions of phorbol 12-myristate 13-acetate (Sigma; 1 mM), prazosin (Sigma; 5 mM) and chelerythrine (Calbiochem; 10 mM) were made in dimethylsulphoxide and diluted to the desired concentration. Neomycin sulphate, propranolol and ouabain were from Sigma. Other chemicals were from Sigma or Merck.

2.4 Data presentation and statistics
Data are presented as mean±s.e.m. When appropriate, a paired t-test using the software Origin (Microcal Software, USA) was performed to compare currents in the presence and absence of phenylephrine and/or other drugs in the same cell.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The Na⁺/Ca²⁺ exchange current in general was measured as the current activated by the addition of extracellular Na⁺ and Ca²⁺ or, in another series of experiments, by the addition of extracellular Na⁺ alone while most other membrane currents were suppressed [16]. In the absence of exchanger current, the current–voltage relationship was very flat, suggesting that most membrane conductances were blocked. Fig. 1A shows pooled data from 7 cells in which the external solution was changed from 0 Na⁺, 0 Ca²⁺ Tyrode to 135 mM Na⁺, 1 mM Ca²⁺ containing Tyrode. The application of external Na⁺, Ca²⁺ induced an increase in membrane conductance. The Na⁺,Ca²⁺ induced current illustrated in Fig. 1B reversed at +21.0±3.2 mV (n=7), i.e. near the theoretical reversal potential for Na⁺/Ca²⁺ exchange current under our ionic conditions (E_rev=3 E_Na–2 E_Ca=3x65.1–2x87=+21.3 (mV)). The effect of adding Na⁺ and Ca²⁺ was reversible (Fig. 1B, inset). Under our experimental conditions with high intracellular Ca²⁺, the Na⁺/Ca²⁺ exchange current can also be activated by increasing external Na⁺ alone. Fig. 1C shows the change of the membrane current, when Ca²⁺-free, 135 mM Na⁺-containing Tyrode was applied. Under such conditions the current was shifted inwardly at all membrane potentials tested (i.e. from –120 to +60 mV) and no reversal potential could be obtained. Moreover, the shift was induced only by Na⁺, but not by Li⁺, indicating that the current is really due to the Na⁺/Ca²⁺ exchanger and not to a non-specific leak. These results are similar to those of Kimura et al. [16]obtained in guinea pigs. The development of the Na⁺/Ca²⁺ exchange current was rather slow. In all cells of the series, the time-to-peak current was 310±23 s (n=46). While in most cells (n=31) the current remained stable, in 6, the current continued to increase by 19±4% of the initial apparent peak value; in 9 cells, an overshoot was seen and the current showed a secondary decrease of 23±3%. Steady-state level for the whole group was obtained after 361±20 s (n=46). The time for return to the basal level upon wash-out of Na_e⁺ and Ca_e²⁺ was much faster and similar in all three subgroups (68±7 s, n=46).

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Isolation of the Na+/Ca2+ exchange current. K+ channels blocked by intracellular Cs+, TEA+ and extracellular Ba2+,Ca2+ channels blocked by nifedipine (50 µM), Na+ channels blocked by a holding potential of –30 mV, Na+ pump blocked by ouabain (100 µM) or 0 extracellular K+. A: current–voltage relationships in 0 Na+, 0 Ca2+ (open symbols) and 135 mM Na+, 1 mM Ca2+ (filled symbols) external solutions, n=7. Inset: voltage-clamp protocol. Current measured during the second, hyperpolarizing part. B: Na+/Ca2+ exchange current obtained by subtracting the current in 0 Na+, 0 Ca2+ external solution from the current in Na+, Ca2+ external solution, n=7. Inset: current traces before, during and after extracellular application of Na+ and Ca2+ in one cell. C: current–voltage relationships in the Na+-, Ca2+-free (Na+ replaced either with Li+ or with NMDG+) and Ca2+-free, Na+-containing external solutions in one cell. Inset: time course of the holding current at –30 mV in the same cell. Similar results were obtained in 3 cells.

 
Addition of phenylephrine (80 µM) resulted in an increase of the external Na⁺,Ca²⁺-dependent current. Fig. 2A shows the time course of changes of the holding current at –30 mV upon adding Na⁺ and Ca²⁺ in the external solution, with and without phenylephrine. The application of external Na⁺,Ca²⁺ induced an inward current. After reaching the plateau, phenylephrine was applied and induced a further increase of the holding current. In 22 experiments, phenylephrine consistently caused stimulation of the external Na⁺,Ca²⁺-dependent current by up to 31.0±5.4% of control (taken as 100%). From these 22 cells, 1 cell did not respond to phenylephrine, in two cells, the current was increased by up to 80 and 100% and in the other cells, the stimulation varied between 10 and 50% of control. The effect was partially reversible; in 4 experiments, the current went from 30.0±5.9% above control in the presence of phenylephrine to 10.9±5.5% above control after 5 min of phenylephrine wash-out. In one cell, the effect did not reverse upon wash-out of phenylephrine. To exclude the possibility, that phenylephrine stimulates a different current which is not blocked under our experimental conditions (without affecting Na⁺/Ca²⁺ exchanger), phenylephrine was applied under conditions where no Na⁺/Ca²⁺ exchange current is present (Fig. 2B). In 4 experiments, Na⁺ was replaced with Li⁺ and Ca²⁺ omitted from extracellular solution. Li⁺ was used as the Na⁺ substituent in these experiments, because it is known to effectively substitute for Na⁺ as charge carrier in most membrane currents [19, 20], but not to substitute for Na⁺ in the Na⁺/Ca²⁺ exchange. Application of phenylephrine under such conditions failed to change the holding current. To investigate the voltage dependence of the phenylephrine-induced stimulation of Na⁺/Ca²⁺ exchange current, ramp voltage-clamp commands in the range from –120 to +60 mV were used. Fig. 3 demonstrates that phenylephrine stimulates the current at all membrane potentials tested, both above and below the reversal potential. Moreover, the reversal potential itself was not influenced by phenylephrine (+21.0±3.2 mV in the absence and +23.2±4.1 mV in the presence of phenylephrine, P>0.05, n=7). The effect seems to be rather voltage independent. Fig. 3C shows that the ratio of the current in the presence of phenylephrine over the control current (taken as 100%) was constant (32% above control) at different membrane potentials.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Effect of phenylephrine on membrane current in the presence or in the absence of Na+/Ca2+ exchange current. A: effect of phenylephrine in the presence of Na+/Ca2+ exchange current. Time course of changes in the holding current at –30 mV. The current upon addition of extracellular Na+ and Ca2+ was further increased in the presence of phenylephrine (80 µM) by up to 30% of control. B: lack of effect of phenylephrine in the absence of Na+/Ca2+ exchange current. Time course of holding current at –30 mV in the presence and absence of phenylephrine (PE, 80 µM). To remove Na+/Ca2+ exchange current, extracellular NaCl was replaced with LiCl and CaCl2 was omitted.

 

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Effect of phenylephrine on current–voltage relationship of the Na+/Ca2+ exchange current. A: current–voltage relationships in the absence of extracellular Na+ and Ca2+, in the presence of extracellular Na+ and Ca2+ and in the presence of extracellular Na+ and Ca2+ after addition of phenylephrine (PE, 80 µM), n=7. B: Na+/Ca2+ exchange currents in the absence and presence of phenylephrine (80 µM) obtained by subtracting the current in the absence of extracellular Na+ and Ca2+ from the current in the presence of them and absence or presence of phenylephrine, n=7. The Na+/Ca2+ exchange current was stimulated at all membrane potentials tested, both below and above the reversal potential, and the reversal potential was not changed. Inset: current–voltage relationships in the absence of extracellular Na+ and Ca2+, in the presence of them and in the presence of them after addition of phenylephrine in one cell. C: membrane potential dependence of the phenylephrine effect. Data points were calculated as ratios of the Na+/Ca2+ exchange current in the presence of phenylephrine (80 µM) and of the Na+/Ca2+ exchange current in the absence of it taken as 100%, n=7. Unreliable ratios near the reversal potential were omitted.

 
The effect of phenylephrine was resistant to β-adrenergic blocker propranolol (3 µM). In 5 experiments, the Na⁺/Ca²⁺ exchange current was stimulated by up to 36.79±11.44% in the presence of propranolol, indicating that the phenylephrine-induced stimulation of the Na⁺/Ca²⁺ exchange current is mediated via the -adrenergic pathway. This conclusion was further supported by experiments where prazosin (1 µM) prevented the stimulatory effect of phenylephrine; (at –30 mV the current was –52.9±7.9 pA in the absence and –54.8±9.9 pA in the presence of phenylephrine added on top of prazosin, P>0.05, n=5).

Fig. 4 summarizes the results of the experiments performed with the aim to investigate possible cellular signalling mechanisms underlying the phenylephrine effect. The first hypothesis was that a mild intracellular alkalinization could be responsible for the observed effect. Cytoplasmic protons are known to inhibit the Na⁺/Ca²⁺ exchange [11], hence a mild alkalinization induced by phenylephrine [5]can cause a stimulation of the Na⁺/Ca²⁺ exchange current. If this hypothesis were true, the phenylephrine-induced alkalinization and subsequent stimulation of the Na⁺/Ca²⁺ exchange current should be more pronounced under conditions with low intracellular buffering. We therefore tested the phenylephrine effect under conditions where the intracellular pH buffering was low (HEPES_i=0.5 mM). However, the magnitude of the phenylephrine effect was similar with 0.5 mM or 5 mM HEPES (25.3±4.9% and 31.0±5.4% stimulation). We next looked at a possible involvement of the phospholipase C and protein kinase C. Neomycin (200 µM), an inhibitor of phospholipase C [21], prevented the effect of phenylephrine in 4 experiments (–47.9±12.1 pA in the absence and –47.8±13.1 pA in the presence of phenylephrine at V_m –30 mV). The selective inhibitor of the protein kinase C, chelerythrine (25 µM) also prevented the effect of phenylephrine in 4 experiments (–40.5±5.3 pA without and –42.3±5.3 pA with phenylephrine at V_m –30 mV). Since a protein kinase C antagonist inhibited the stimulation of the Na⁺/Ca²⁺ exchange current, a protein kinase C activator, phorbol 12-myristate 13-acetate was next examined. In 6 experiments, phorbol 12-myristate 13-acetate, however, failed to stimulate the Na⁺/Ca²⁺ exchange current (–55.9±15.2 pA in the absence and –55.5±15.7 pA in the presence of phorbol 12-myristate 13-acetate at V_m –30 mV).

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Possible mechanisms underlying the phenylephrine effect. Values represent ratios of the Na+/Ca2+ exchange current in the presence of phenylephrine and/or other pharmacological intervention at –30 mV and of the control Na+/Ca2+ exchange current obtained as the extracellular Na+- and Ca2+-dependent change of the current at –30 mV and taken as 100%. Figures above the error bars indicate numbers of experiments. *Significantly different from control, P<0.01.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
The present study was performed to determine a direct effect of -adrenergic receptor activation on the Na⁺/Ca²⁺ exchanger. The Na⁺/Ca²⁺ exchange current was measured according to Kimura et al. [16]with a small difference in the composition of experimental solutions to allow the determination of the reversal potential of the current. We are confident that the measured extracellular Na⁺- and Ca²⁺-dependent current is the Na⁺/Ca²⁺ exchange current, because its reversal potential was nearly identical to the theoretical reversal potential (+21.3 mV) for the Na⁺/Ca²⁺ exchange current under our ionic conditions. The steady value of the reversal potential, moreover, suggests that the intracellular clamping of sodium and calcium was quite effective, at least in the bulk solution. We have to admit, however, that in the subsarcolemmal ‘fuzzy’ space, which is invisible to macroscopic electrodes, the effectiveness of dialysis is probably incomplete and therefore the local Na⁺ (and/or Ca²⁺) concentration may be significantly different depending on the Na⁺/Ca²⁺ exchange activity (under conditions when other transporters and channels are blocked). Since no ion has its equilibrium potential close to this level, the only other candidate (beside the Na⁺/Ca²⁺ exchange current) is a non-selective cation current activated by intracellular Ca²⁺ [22]. An increase in intracellular Ca²⁺ could occur secondary to application of external Na⁺ and Ca²⁺. However, this is not probable under our experimental conditions: the concentration of EGTA in the pipette solution was very high, Ca²⁺ channels were blocked and the membrane potential was most of the time held at potentials negative to the reversal potential of the Na⁺/Ca²⁺ exchange current. Moreover, an application of Ca²⁺-free, Na⁺-containing Tyrode which cannot increase intracellular Ca²⁺, but only result in a decrease via activation of the Na⁺/Ca²⁺ exchange, still induced a current. This current was inward-going at all membrane potentials tested (from –120 to +60 mV), which is fully consistent with Na⁺/Ca²⁺ exchange current. The possibility of a non-specific leak current carried by small cations, induced by replacing NMDG⁺ with Na⁺, was excluded. The experiment in Fig. 1C shows that only application of Na⁺ was capable of inducing the current, while the application of another small monovalent cation (Li⁺) was ineffective. The change in leak current upon addition of Na_e⁺ and Ca_e²⁺, moreover, should only result in an inward current (only external permeant cations are added while intracellular ions remain unchanged). This was not the case, since the difference current showed a clear reversal (Fig. 1B). It is possible, however, that a small part of the external Na⁺- and Ca²⁺-dependent current is carried by this leak current (less than 10% according to [11]) which would result in a small underestimation of the stimulation of the exchange current. The development of the exchanger current was quite slow. These kinetics are partly due to the rate of perfusion which was 0.7 ml/min with a bath volume 0.4 ml. It should also be mentioned that the K_0.5 for Na_e⁺ of the exchanger is rather elevated and amounts to 87.5±10.7 mM [16], meaning that a complete exchange of the solution around the cell is required before the current can attain its steady-state. Local subsarcolemmal changes in Na_i⁺ and Ca_i²⁺ furthermore may occur and be responsible for the slower secondary phases.

Phenylephrine increased the membrane current when extracellular Na⁺ and Ca²⁺ were present, but not in their absence. Therefore, it is quite safe to conclude that phenylephrine stimulates the Na⁺/Ca²⁺ exchange current. Similarly, Ballard and Schaffer [13]reported stimulation by up to 111% of control of the Na⁺-dependent ⁴⁵Ca²⁺ uptake by phenylephrine (100 µM) in cardiac sarcolemmal vesicles. The quantitative difference with our results is probably related to the use of different preparations and experimental approaches.

The exchange current was stimulated both above and below the reversal potential, indicating that both forward and reverse modes of the exchange are stimulated. Since the effect was resistant to β-adrenergic blocker, propranolol, and prevented by ₁-adrenergic antagonist, prazosin, it should be mediated via the ₁-adrenergic pathway. However, the exact underlying pathway remains unclear. The possibility that intracellular alkalinization secondary to activation of the Na⁺/H⁺ exchanger [5]was responsible for the effect on the Na⁺/Ca²⁺ exchanger was excluded on the basis that the magnitude of the effect was not affected by the intracellular H⁺ buffering capacity. Moreover, activation of the Na⁺/H⁺ exchanger should lead to a change of intracellular Na⁺ which would result in a change of the reversal potential of the Na⁺/Ca²⁺ exchange current and this was not the case.

The membrane phospholipid environment influences the exchanger activity. The hydrolysis of phosphatidylinositol by specific phospholipase C has been reported to stimulate exchange measured as Na⁺-dependent ⁴⁵Ca²⁺ uptake in cardiac sarcolemmal vesicles [9]. Since phenylephrine activates phospholipase C, which hydrolyses preferentially phosphatidylinositol 4,5-bisphosphate, but also phosphatidylinositol [1, 23], this hydrolysis can hypothetically be responsible for the stimulation of the exchanger after application of phenylephrine. In support of this hypothesis, neomycin, a blocker of phospholipase C [21], prevented phenylephrine induced stimulation of the Na⁺/Ca²⁺ exchange current in our experiments. Phospholipid hydrolysis could activate the exchanger either directly, if phosphatidylinositol itself or a modulatory factor anchored by it, inhibit the exchanger, or indirectly via products of the hydrolysis. With regard to the direct effect of hydrolysis, negatively charged phospholipids, such as phosphatidylinositol or phosphatidylserine have been shown to stimulate the Na⁺/Ca²⁺ exchange in canine cardiac sarcolemmal vesicles [8]or in cardiac giant membrane patches [10]and recently Hilgemann and Ball [24]demonstrated stimulation of the exchange by phosphatidylinositol 4,5-bisphosphate in cardiac giant patches. Therefore, the second possibility, indirect activation by hydrolysis products, appears to be more appropriate. It is well established that the -receptor mediated activation of phospholipase C results in formation of diacylglycerol and subsequently activation of protein kinase C. Stimulation of the Na⁺-dependent ⁴⁵Ca²⁺ uptake by protein kinase C-dependent phosphorylation has been suggested in aortic smooth muscle [25], in rat neonatal cardiomyocytes and CCL39 cells stably overexpressing canine cardiac Na⁺/Ca²⁺ exchanger [26]and a kinase-phosphatase modulation of the Na⁺/Ca²⁺ exchange fluxes has been shown in squid axons [27]. Consistent with these findings, chelerythrine, a selective antagonist of protein kinase C, prevented the effect of phenylephrine in our experiments. In contrast, phorbol 12-myristate 13-acetate, a protein kinase C activator, failed to stimulate the Na⁺/Ca²⁺ exchange current. Similar results were obtained by Ballard and Schaffer [13]in cardiac sarcolemmal vesicles, when the phenylephrine-induced stimulation of Na⁺-dependent ⁴⁵Ca²⁺ uptake was significantly attenuated by chelerythrine, but phorbol 12-myristate 13-acetate required high levels of ATP to stimulate the exchanger. However, a stimulation of the exchanger by phorbol 12-myristate 13-acetate was reported in aortic smooth muscle [25]and in rat neonatal cardiomyocytes [26]. These conflicting results are probably related to different protein kinase C isoforms activated by phorbol esters and by phenylephrine. Phorbol esters have a lower affinity for Ca²⁺-independent isoforms; on the other hand, the endogenous activator, diacylglycerol, has similar affinity for all isoforms [28]suggesting that perhaps a Ca²⁺-independent isoform of protein kinase C is involved in the -adrenergic regulation of the Na⁺/Ca²⁺ exchange. Another possible pathway consistent with our results is a mediation of the phenylephrine effect via phospholipase D. Phenylephrine activates phospholipase D in cardiac myocytes [29]. The mechanism of its activation is unclear, but it can be mediated either via Gq protein or via protein kinase C [30]. Phospholipase D catalyses hydrolysis of phosphatidylcholine to choline and phosphatidic acid [30], which can stimulate Na⁺/Ca²⁺ exchange either directly [31]or via the activation of protein kinase C isoform [32]. This isoform is insensitive to phorbol esters as well as to diacylglycerol [33, 34]. Moreover, phosphatidic acid is further metabolized to diacylglycerol, activator of protein kinase C. This pathway should be inhibited by neomycin which blocks phospholipase D [35]and by chelerythrine (if protein kinase C is involved). However, if protein kinase C isoform involved were , phorbol esters failure to activate the pathway will be fully consistent with our hypothesis.

What is the physiological meaning of the phenylephrine effect? To answer this question is not simple, because phenylephrine does not influence only Na⁺/Ca²⁺ exchange, but many other membrane transporters and channels [36, 37]. Stimulation of the reverse mode of the exchange can contribute to the positive inotropic effect of -adrenergic agonists by an increase of the sarcoplasmic reticular Ca²⁺ loading and/or by an increase of trigger Ca²⁺ [7]. On the other hand, stimulation of the forward mode of the exchange can represent a feedback mechanism which should prevent damage of the cell and ensure proper Ca²⁺ homeostasis when Ca²⁺ transients and/or myofibrillar Ca²⁺ sensitivity are increased. Stimulation of the forward mode could also be responsible for the transient and sustained negative inotropic effect observed under certain experimental conditions.

Time for primary review 22 days.

	Acknowledgements

 
We thank Mr. Peter Matejovic for the preparation of cells.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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