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European Journal of Pharmacology, 194 (1991) 107-110 © 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 0014299991002085 EJP 20784

Short communication

Effect of palmitoylcamifine on the passive electrical properties of isolated guinea pig ventricular myocytes J~nos M~sz~ros Department of Pharmacology, University of Connecticut Health Center, Farmington, CT 06032, U.S.A. Received 28 December 1990, accepted 2 January 1991

The effects of palmitoylcarnitine (PC) on the passive electrical properties of isolated guinea pig ventricular myocytes were studied using conventional microelectrode techniques. The amphiphile depolarized the cell membrane, and at low concentration (10 -6 M), increased the input resistance (Rin) from 12.4 to 17.9 M~2 and the time constant (rm) from 1.38 to 2.20 ms. A higher concentration of PC (10 -5 M) decreased Rin to 8.5 M$2 and ~m to 1.14 ms. No significant change was observed in the membrane capacitance and in the capacitative membrane area. The results suggest that PC incorporates into the cell membrane and in low concentration, it increases the membrane resistance, while its high concentration increases the membrane permeability probably by causing serious damages in the membrane structure. Myocytes (isolated); Palmitoylcamitine; Electrical properties (passive)

1. Introduction

The role of the toxic products of lipid metabolism, such as palmitoylcamitine (PC) and lysophosphatidylcholine, in the generation of electrical derangements and arrhythmias in ischaemic myocardium is well established (Katz and Messineo, 1981). These amphiphiles are released from the diseased heart cells and incorporated into the healthy cell membrane and alter its fluidity and molecular dynamics (Fink and Gross, 1984; Katz and Messineo, 1981). It has also been demonstrated that PC decreases the amplitude and maximum rate of rise of the action potential in avian ventricular muscle (Inoue and Pappano, 1983). Since PC, like elevated extracellular Ca 2+, shifted the steady state inactivation of excitatory inward currents (INa and Ica ) to more positive potentials, Inoue and Pappano (1983) proposed the hypothesis that PC reduced the surface negative charge density near voltage-dependent Na + and Ca 2+ channels. This hypothesis was verified by the results that PC reduced erythrocyte electrophoretic mobility, which directly indicated a reduction in the surface charge density (M6sz~tros et al., 1988). More recently, we also demonstrated that PC induced arrhythmogenic transient depolarizations by causing an intracellular Ca 2+ overload in single ventricular myocytes of the guinea pig (M6szhros and Pappano, 1990).

Correspondence to: J. Mrsz~ros, Department of Physiology and Biophysics, University of Cincinnati, Cincinnati, OH 45267, U.S.A.

Since all the above results suggest the incorporation of PC into the cell membrane, it seemed reasonable to examine whether PC had any effect on the passive electrical properties of the membrane. The aim of the present work was, therefore, to investigate the effect of PC on the membrane resistance, capacitance, time constant and capacitative membrane area in single ventricular myocytes of the guinea pig.

2. Materials and methods

The experiments were carried out on single ventricular myocytes isolated enzymatically from guinea pig heart by the method of Isenberg and Kl~zkner (1982) with modifications as previously described by Rardon and Pappano (1986). Cells were isolated by gentle mechanical agitation as soon as the collagenase-treated heart was removed from the Langendorff perfusion apparatus, and the cells were kept in recovery medium for, at least, 1 h at 4 ° C temperature before beginning the experiments. An aliquot of the cell suspension was placed in a small (0.5 ml volume) chamber mounted on the stage of an inverted microscope (400 × magnification with Hoffman contrast optics). After a settling period of 8-10 min, the cells were perfused with Tyrode solution at a rate of 2-4 ml/min. The composition of the Tyrode solution was (mM): NaC1 137.0, KC1 5.4, MgC12 1.0, CaC12 1.8, N a H C O 3 6.0, NaH2PO 4 1.0, HEPES 5.0, glucose 5.5. The solution was aerated with

108 O 2 gas, the p H was 7.2 and the temperature was maintained at 35 ° C. PC was dissolved in this solution just before starting the experiments. Incubation time was 30 min with each concentration used. Cells showing a rod shape and clear striations without any blebs on the surface were used for the experiments. Membrane potential was recorded with glass microelectrodes filled with 160 m M KC1 solution (Isenberg and Klt~ckner, 1982). Microelectrodes with tip resistance of 40-80 MI2 were mounted on a Leitz micromanipulator and connected to the input of a WPI KS700 dual-microprobe system that permits simultaneous voltage measurement and current injection. Before impaling a cell, the voltage drop across the microelectrode tip and the tip capacitance were minimized electronically. Passive membrane parameters, such as input resistance ( R i n ) , time constant ('rm), specific membrane resistance (Rm), capacitance (Cm) and the capacitative membrane area (Ac) were estimated by applying small hyperpolarizing current pulses near the resting potential at a basal rate of 0.2 Hz, and measuring the voltage responses to them (Isenberg and Kl~Sckner, 1982). A detailed description of the procedure is given in Results. The assumptions and limitations of this method are presented elsewhere (Rardon and Pappano, 1986). PC used in this study was purchased from Sigma Chemical Comp. (St. Louis, MO, USA). Data are presented as means + S.E.M. The statistical significance of difference from the control was estimated using Student's t-test. A P value less than 0.05 was considered significant.

amplitude) near the restintg potential to avoid activation of voltage-gated conductances and recorded voltage responses. The procedure is shown in fig. 1. It has been verified that during a small hyperpolarizing pulse, the myocyte is isopotential (Isenberg and Kl5ckner, 1982; Rardon and Pappano, 1986), and the input resistance ( R i n ) c a n be calculated by Ohm's law from the steady state voltage deflection produced by 0.15-0.35 nA current pulses (fig. 1). The control value of R i n in seven myocytes averaged 12.4 MI2. The zm, the time required to achieve 63% of the steady state voltage deflection, was found 1.38 ms. From the experimentally obtained Rin and ~'m we calculated data for the R m and C m and Ac, because the time constant is interpreted as R m times specific membrane capacitance (Cm; generally considered 1 / ~ F / c m 2, as a constant), and R m equals to Ac times Ri,, and C m equals to C-m multiplied by A~ (Isenberg and Kli3ckner, 1982; Rardon and Pappano, 1986). For these parameters the following values were obtained under control conditions: R m w a s 1.38 kI2. cm 2, A c was 1.13 × ] 0 - 4 cm 2 and C m w a s 113.6 pF. In addition, we also characterized the cell with a morpho-

A

B

. . . . . . "-~ - F --~ O

C

-s

~ F I 5mV,

I



Voltage

-4

-3

(mV)

3. Results Since, in a former study (M~szhros and Pappano, 1990), we have observed that PC exerted a concentration-dependent dual effect on the repolarization process in single ventricular myocytes, insofar as 10 -6 M was the most effective concentration in prolonging the action potential duration (APD), while 10-5 M made the A P D shorter, we focused attention on the effects of these two concentrations in the present study. Recordings were taken after a 30 min incubation period when the changes in the parameters reached a steady state under the effect of each amphiphile concentration. We studied each concentration on a different cell. When superfused with Tyrode solution and stimulated by intracellular current pulses, the control resting potential of the myocytes averaged - 8 4 . 9 mV. The value of APDs0 was 255.4 ms, and the threshold current needed to evoke an action potential was 1.22 nA. To estimate passive electrical parameters of the myocyte m e m b r a n e we applied small intracellular hyperpolarizing current pulses (0.2 Hz; 50 ms duration; m a x i m u m 0.4 nA

0.25nA

• Control I0-6 molll O PC

~/-c

25ms

~ -2

/

/

-I

o

-0.I "~ ~.=

e3~ 10.4

Fig. 1. Concentration-dependent effects of PC on the passive electrical properties of the myocyte membrane. In each panel symbols are: (O) control; (©) PC 10 -6 M; (zx) PC 10 -5 M. Original recordings show that 10 - 6 M PC increases the voltage response to a constant hyperpolarizing current pulse applied intracellularly through the same microelectrode by which the voltage was recorded (A). PC at 10 -5 M decreases the voltage response to the current pulse (B). Graph: changes in the membrane potential (abscissa) in response to the small hyperpolarizing current pulses (ordinate) applied near the resting potential. The straight lines were fitted to the data points by linear regression analysis. The 0 mV corresponds to -84.9 mV (control), -82.1 mV(PC 10 - 6 M) and -78.4 mV (PC 10-5 M).

109 TABLE 1 Concentration-dependent effect of PC on the passive electrical parameters of single ventricular myocytes isolated from guinea pig heart. M e a n s + S.E.M. n = 7. Vm: resting membrane potential; Rin: input resistance; ~m: m e m b r a n e t i m e constant; Rm: specific membrane resistance; Ac: capacitative membrane area; Cm: membrane capacitance. Treatment

Vm (mV)

R in (MI2)

'rm (ms)

Rm (kl2- cm 2)

Ac ( × 10 - 4 cm 2)

Cm (pF)

Control PC 10 -6 M PC 10 -5 M

- 84.9 + 0.88 -82.1 4-0.46 a - 78.4+0.51 b

12.4 + 0.87 17.9+0.52 b 8.5 4-0.74 b

1.38 4- 0.10 2.204-0.15 b 1.14+0.30 a

1.38 + 0.10 2.204-0.15 b 1.14+0.12 b

1.13 4- 0.07 1.23+0.10 1.34+0.09

113.6 4- 7.7 123.5+10.1 134.04-12.4

a p < 0.05; b p < 0.002.

logical surface area (As) calculated from its geometry. The dimensions of the cells averaged 124 + 9 /~m in length, 23 + 2/~m in width and 5/~m in diameter (taken as a constant). The value of A s was 6.9 × 10 -5 cm 2, which is somewhat lower than the value of 1.13 × 10 -4 cm 2 obtained for the capacitative membrane area calculated from the electrophysiological data. The discrepancy may have resulted from the existence of significant infoldings of the cell membrane. The effects of low (10 -6 M) and high (10 -5 M) concentrations of PC on the above parameters are summarized in table 1, and fig. 1 depicts original recordings. The amphiphile depolarized the cell membrane gradually at each concentration, but 10-6 and 10-5 M evoked opposite effects on the passive electrical parameters. PC at 10 -6 M increased Rin by about 45%, and *m by about 60%, and R m by about 60%. Raising the concentration to 10 -5 M was associated with a decrease in Rin by 32%, in ~'m by 18%, and in R m by 18%. No significant changes were observed in A c and in C m under the influence of PC.

4. Discussion

The main conclusion of the results presented here is that the changes in the action potential configuration observed under PC action (Inoue and Pappano, 1983; M6szhros and Pappano, 1990) is associated with similar changes in the passive electrical parameters of the myocyte membrane, namely, in the membrane resistance and time constant. The finding that 10 -6 M PC increases the membrane resistance (both Rin and Rm) suggests that the amphiphile by binding to the cell membrane blocks potassium channels and consequently, reduces not only the resting potassium permeability which results in depolarization but also the K ÷ conductance associated with the excitation which leads to the prolongation of APD. These findings are in good agreement with those obtained with lysophosphatidylcholine, an amphiphile sharing some of the action of PC (Fink and Gross, 1984), which also prolonged APD and depolarized the cell membrane and decreased the resting potassium conductance (Kiyosue and Arita, 1986),

and consequently increased membrane resistance (Arnsdorf and Sawicki, 1981). The hypothesis that the positively charged PC molecule incorporates into the cell membrane (Fink and Gross, 1984) and reduces surface negative charge density near voltage-dependent ion channels ( I n o u e and Pappano, 1983; M&zhros et al., 1988) also offers an explanation for PC action. An elevated R m can explain the increased Tm, since this parameter has been shown to depend o n R m and C m (Rardon and Pappano, 1986), and in our experiments, only R m was altered significantly by PC. In this study, we have experienced that a higher concentration of PC (10 -5 M) exerted opposite effects on the passive membrane parameters. After 30 min incubation with 10 -5 M PC, R i n and "rm were significantly decreased. In this case, a different explanation for the mechanism underlying this apparently toxic effect of the high concentration of PC must be considered. If we accept the incorporation hypothesis for PC action (Fink and Gross, 1984; Katz and Messineo, 1981) we can assume that PC applied in high concentration incorporates into the cell membrane in a higher concentration and increases its fluidity and eventually makes the sarcolemma 'leaky' and more permeable for ions. This proposition seems to be confirmed by the finding that PC at concentrations higher than 10 -5 M always damaged the isolated myocytes. A similar phenomenon was observed by Busselen et al. (1988), and in our laboratory with erythrocytes (M6szhros et al., 1988). The alterations in the membrane fluidity and molecular dynamics induced by PC cause the membrane resistance to decrease which, consequently, reduces the time constant, because of the above detailed correlations. On the other hand, a shorter Tm observed under the effect of high concentration of PC can also be caused by an elevated intracellular Ca 2+ concentration (Rardon and Pappano, 1986). This latter also looks a likely mechanism, because it has been demonstrated that PC causes an intracellular Ca 2+ overload (M6szhros and Pappano, 1990). In summary, it can be concluded that low concentration of PC incorporates into the cell membrane, thus increasing its volume and, consequently, exerting a membrane stabilizing effect, which decreases the potas-

110 s i u m p e r m e a b i l i t y . W h e r e a s , t h e lytic e f f e c t o f h i g h a m p h i p h i l e c o n c e n t r a t i o n s r e p r e s e n t s d e t e r g e n t - l i k e action, w h i c h d a m a g e s t h e m e m b r a n e s t r u c t u r e , a n d c a u s e s i n t r a c e l l u l a r C a 2÷ o v e r l o a d a n d r e d u c e s R m a n d ~'m"

Acknowledgements The author thanks Dr. Achilles J. Pappano for providing expert advices and stimulating discussions. This work was supported by National Heart, Lung, and Blood Institute Program Project Grant HL-33026.

References Arnsdorf, M.F. and G.J. Sawicki, 1981, The effects of lysophosphatidylcholine, a toxic metabolite of ischemia, on the components of cardiac excitability in sheep Purkinje fibers, Circ. Res. 49, 16. Busselen, P., D. Sercu and F. Verdonck, 1988, Exogenous palmitoyl carnitine and membrane damage in rat hearts, J. Mol. Cell. Cardiol. 20, 905.

Fink, K.L. and R.W. Gross, 1984, Modulation of canine myocardial sarcolemmal membrane fluidity by amphiphilic compounds, Circ. Res. 55, 585. Inoue, D. and A.J. Pappano, 1983, 1-Palmitoylcarnitine and calcium ions act similarly on excitatory ionic currents in avian ventricular muscle, Circ. Res. 52, 625. Isenberg, G. and U. K16ckner, 1982, Calcium tolerant ventricular myocytes prepared by preincubation in a 'KB medium', Pfliigers Arch. European J. Physiol. 395, 6. Katz, A.M. and F.C. Messineo, 1981, Lipid-membrane interactions and the pathogenesis of ischemic damage in the myocardium, Circ. Res. 48, 1. Kiyosue, T. and M. Arita, 1986, Effects of lysophosphatidylcholine on resting potassium conductance of isolated guinea pig ventricular ceils, Pfliigers Arch. European J. Physiol. 406, 296. M6szhros, J. and A.J. Pappano, 1990, Electrophysiological effects of 1-palmitoylcarnitine in single ventricular myocytes, Am. J. Physiol. 258, H931. M6sz/~ros, J., L. Villanova and A.J. Pappano, 1988, Calcium ions and 1-palmitoylcarnitine reduce erythrocyte electrophoretic mobility: Test of a surface charge hypothesis, J. Mol. Cell. Cardiol. 20, 481. Rardon, D.P. and A.J. Pappano, 1986, Carbachol inhibits electrophysiological effects of cyclic AMP in ventricular myocytes, Am. J. Physiol. 251, H601.

Effect of palmitoylcarnitine on the passive electrical properties of isolated guinea pig ventricular myocytes.

The effects of palmitoylcarnitine (PC) on the passive electrical properties of isolated guinea pig ventricular myocytes were studied using conventiona...
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