Calcium ions and 1-palmitoyl carnitine reduce erythrocyte electrophoretic mobility: Test of a surface charge hypothesis

J Mol Cell Cardio120,481-492 (1988)

Calcium Ions and l-Palmitoyl Carnitine Reduce Erythrocyte Electrophoretic Mobility: Test of a Surface Charge Hypothesis

Janos M~sz~ros, Lynnea Villanova and Achilles J. Pappano Department of Pharmacology, The University of Connecticut Health Center, Farmington, C T 06032, USA (Received 12 October 1987, accepted in revisedform 28January 1988) j. Ms

L. VILLANOVAANDA. J. PAPPANO.Calcium Ions and 1-Pahnitoyl Carnitine Reduce Erythrocyte Electrophoretic Mobility: Test of a Surface Charge Hypothesis. Journal of Molecularand Cellular Cardiology (1988), 20, 481 492. In ventricular muscle, l-palmitoyl carnitine (1-PC) acted like calcium ions to shift the voltage-dependent inactivation of excitatory ion currents to less negative potentials [20]. We proposed that I-PC affected ion current kinetics by reducing surface negative charge. This hypothesis was tested in cell electrophoresis experiments where the electrophoretic mobility (EPM) of erythrocytes was measured in the absence and presence of test ligands. Calcium (0.18 to 3.6 raM) or I-PC (10 -v to 10-6 M) reduced erythrocyte EPM in a concentration-dependent manner; the maximum reduction of EPM by either ligand was ~ 40%. In the presence of calcium, I-PC produced a smaller decrement of EPM as expected from an occlusive interaction, Treatment oferythrocytes to remove sialic acids not only predictably reduced EPM but also diminished the ability of I-PC and calcium to do so. These results indicate that the surface negative charge ofsialic acid carboxyl groups is art important determinant both of erythrocyte EPM and of erythrocyte interaction with either 1-PC or calcium. The findings are consistent with the surface charge hypothesis for I-PC action. We propose that I-PC is not a neutral molecule at the cell surface but is able to neutralize surface negative charge by electrostatic interaction between the sialic acid carboxyl groups and the 1-PC quaternary ammonium moiety on the one hand and between the I-PC carboxyl group and counterions near the membrane surface. KEY WORDS:Electrophoretic mobility; Erythrocyte; Gouy-Chapman theory; Neuraminidase; l-Palmitoyl carnitine; Sialic acid; Surface charge; Zeta potential.

Introduction Ischemia-induced arrhythmias have been related, a t least i n part, to the a c t i o n of accum u l a t e d lipid m e t a b o l i t e s (acylcarnitines, lysophosphatidylcholine) on cardiac plasma m e m b r a n e s (reviewed in [9, 22, 24]. W e h a v e r e p o r t e d t h a t 1-palmitoyl c a r n i t i n e (/-PC) modified the kinetics of e x c i t a t o r y ionic currents carried b y N a + (isa) a n d C a 2+ (/ca) in the a v i a n v e n t r i c l e [20]. W e p r o p o s e d t h a t 1-PC, like elevated [ C a 2+] r e d u c e d the surface n e g a t i v e c h a r g e in the m e m b r a n e n e a r N a + a n d C a 2+ c h a n n e l s [20, 29] because v o l t a g e - d e p e n d e n t shifts for i n a c t i v a t i o n of iN~ a n d / c a b y 1-PC were q u a l i t a t i v e l y a n d q u a n t i tatively similar to those p r o d u c e d b y e l e v a t i o n of the e x t r a c e l l u l a r C a z + c o n c e n t r a t i o n ( [ C a 2 + ] 0 ) . 1-Palmitoyl c a r n i t i n e d i d n o t c h a n g e the resting p o t e n t i a l of a v i a n v e n t r i c u -

lar cells w h e n it p r o d u c e d a positive shift in the v o l t a g e - d e p e n d e n t i n a c t i v a t i o n of iNa a n d i c , . I n c a n i n e c a r d i a c P u r k i n j e fibers, 1-PC d e p o l a r i z e d the resting m e m b r a n e and r e d u c e d iNa [10], b u t the r e d u c t i o n of iN~ was g r e a t e r t h a n t h a t expected b y d e p o l a r i z a t i o n alone. L y s o p h o s p h a t i d y l c h o l i n e , w h i c h has electrophysiological effects like 1-PC, r e d u c e d iNa in P u r k i n j e fibers at low c o n c e n t r a t i o n s (10 #~t) t h a t h a d n o effect o n the resting p o t e n t i a l [3]. I n n e r v e a n d c a r d i a c m e m b r a n e s , a surface n e g a t i v e c h a r g e exists whose properties c a n be expressed in terms of the G o u y - C h a p m a n t h e o r y for a diffuse d o u b l e layer [19, 26, 27]. A c c o r d i n g to this theory, diffusible c o u n t e r ions (cations) are a t t r a c t e d to the n e g a t i v e l y c h a r g e d cell surface w h e r e they either b i n d or screen, r e d u c e the n e g a t i v e surface p o t e n t i a l

This work was supported by USPHS Program Project Grant HL-33026. This paper was handled by Dr Schwartz. 0022-2828/88/060481 + 12 $03.00/0

9 1988 Academic Press Limited

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and thereby modify the gating of ionic currents by voltage-dependent sensors that are components of ion channels. The diffuse double layer concept has also been applied to the estimation of surface potentials and surface charge density of phospholipid vesicles (reviewed in [-26]) and of cells including erythrocytes (reviewed in [-34]). In the case of erythrocytes, the surface potential and negative charge density can be estimated from measurements of the velocity of cell movement in an applied electric field (reviewed in [1, 4, 16, 30, 34]). The advantages and limits of erythrocyte electrophoretic mobility (EPM) measurements for the evaluation of the biophysical properties of the cell surface and the changes produced by chemical probes have been addressed by many investigators (reviewed in [1, 4, 16, 30, 34]). Because of its special advantages, we selected the erythrocyte model system to test in a more direct fashion, the surface charge hypothesis for the actions of 1-PC. According to the surface charge hypothesis, the 1-PC would be expected to reduce EPM of erythrocytes. Additionally, this effect should be shared by Ca 2+. This report describes an evaluation of the surface charge hypothesis for 1-PC action.

Materials and Methods

Experiments were carried out on washed human erythrocytes obtained by finger puncture. The EPM of blood cells has been reported to be independent of the blood collection method, the age, sex and race of the donor and of the blood group [3/]. Blood was drawn up in a blood dilution pipette and added directly into 7.5 ml physiological saline solution (1500-fold dilution) composed of 145 mM NaC1 and 0.3 mM N a H C O a ; the pH was 7.2 -t- 0.1. The cell suspension was centrifuged for l0 min at 30x g, the supernatant removed and the cells were resuspended in an equal volume of fresh physiological solution. This washing procedure was performed four times. Unless noted otherwise, the cell suspension was incubated with the appropriate concentration of ligand at 25~ for at least 10 rain and then it was transferred directly into the electrophoresis tube and the mobility

of the cells was determined as described below. The microscopic electrophoresis apparatus (Grant Instrument, Cambridge, England) used in our experiments was essentially the same as described by others [30]. The electrophoresis tube consist of a thin horizontal capillary (15.7 cm in length, and 0.24 cm in diameter) with an optical flat in center and two upright cylindrical chambers for insertion of the platinum electrodes at each end. The electrophoresis tube was immersed in a water bath and fixed by a holder in horizontal position. A standard microscope tube (160 mm) was attached horizontally to a holder and adjusted to the optical flat of the chamber. An adjustable zero dial test indicator was also linked mechanically to the fine adjustment of the microscope so as to give an accurate and rapid reading of the objective setting. The objective is waterproofed as it inserts through the wall of water bath using a rubber grommet. The source of illumination was mounted behind the waterbath, coaxial with the objective and at right angles to the optical flat of the chamber. Before starting an experiment, the tube was washed in situ with 1 M NaC1 solution and rinsed with distilled water and then with the physiological saline solution. The tube was filled with the cell suspension and the electrodes were inserted in the end chambers, forcing out excess liquid. The microscope was focused on the stationary layer [30] where net water flow due to electroosmosis is zero. The electrodes were connected to a GRASS SM5E stimulator, in DC mode. The applied field strength ranged between 3 and 4 V/cm. The eyepiece of the microscope contains an ocular micrometer to measure the distance covered by the cell in the electric field. Cell movement was timed with a stopwatch and the EPM (always given as mean _+ S.D.) was calculated from the field strength, time, and distance, and was expressed in #m/s/V/cm. Solutions with ionic strength less than 0.145 were prepared with sorbitol [18]. Either HC1 or N a O H was added to the physiological saline solution (I = 0.145) to obtain the desired pH in those experiments directed at an evaluation of the effect o f p H on EPM. When saline solutions (I = 0.145) were prepared with 1.0 #M 1-PC and 1.8 mM Ca, we did not detect the formation of crystalline

P a l m i t o y l C a r n i t i n e a n d Cell S u r f a c e C h a r g e

precipitates with microscope observation. However, we cannot exclude the possibility that a calcium salt o f 1-PC was produced when the two substances were mixed. T h e electrophoresis tube contained a 5 ml suspension of erythrocytes at a concentration of about 2.45 x 107 erythrocytes/ml. At a stromal protein content of 5.7 x 10 - l ~ mg/ hemoglobin-free ghost [35], the total membrane protein content of the suspension is approximately 1.4 x 10 -2 m g and the total membrane lipid content amounts to 1.1 x 10 -2 mg. We studied the effect of Ca and 1-PC on the E P M of erythrocytes treated with neuraminidase which liberates neuraminic acids from the m e m b r a n e of erythrocytes [8, 12]. Neuraminidase (Type V I I I ; E.C. No. 3.2.1.18; purified from Clostridium perfringens) was obtained from Sigma (St. Louis, MO~. The erythrocytes (1500 x diluted) were incubated with 0.002, 0.02, and 0.2 U / m l neuraminidase (diluted from a stock solution of 1 U / m l in distilled water) in normal ionic strength solution (NaC1 0.145 M) at 25~ for 30 rain. T h e reaction was stopped by placing the test tube containing the mixture in ice for 5 rain. T h e erythrocytes were sedimented by centrifugation (3000 g for 10 min), the supernatant removed and replaced by the same a m o u n t (5 ml) of fresh physiological saline solution. This suspension of erythrocytes was then used in E P M experiments. Measurements of surface charge density (a in esu/cm 2) were initially done with the "flat plate" model [7, 12] with the following equation : a = (1 + h:Ri) -1

x { (NDkT/2000u) [ZCr (e- zoe~/kT _ 1) nt- s

2"eUkT -- 1)]} 1/2

(1)

where ~c = the reciprocal of the Debye length (cm-lo), Ri = counterion radius (for N a = 2.67 A), N = Avogadros's number (6.023 x 1023), D is the dielectric constant o f water (78 at 25~ k = Boltzman constant (1.38 x 10-a6), T = a b s o l u t e temperature (~ C c and Ca are the concentrations of cation and anion, respectively, Z~ and Z~ are the corresponding valences, e = electronic charge (4.8 x 10 - l ~ electrostatic units) and = zeta potential ( = 12.9 x E P M according

483

to [11]). Alternatively, and as indicated by others [1, 12], a was estimated from the following equation : a = EPMt/tc

(2)

where E P M = electrophoretic mobility in pm/s/V/cm and t / = viscosity of the test solution in poise ( q H 2 0 = 8.9 x 10 -3 at 25~ Viscosities were obtained with an Ostwald viscometer and were standardized against distilled water at 25~ T h e determination of ~: depends upon the ionic strength (/5 and is given by ~c = 3.27 x 107x/1 at 25~ When equation (2) was used, no correction for finite ion size was included [12]. Equation (1) provides a correction for the finite size of counterions. Equations (1] and (2) are accurate for large smooth particles, that is, when the radius of curvature Ca) is large and 1ca > 100 [4]. Because the radius of curvature of the erythrocyte surface is not known, the assumption that ~ca > 100 is suspect. I f the assumption is not correct, then either equation will underestimate a by ~ 50% [4]. In our experiments, the changes of E P M and a are emphasized rather than the absolute values. We obtained values of ~ and a that are very m u c h like those reported by others (e.g., [12, 18]. T h e erythrocytes used in these experiments were analyzed morphologically by a light microscope. Washed erythrocytes, suspended in physiological saline solution, were placed on a covered, glass microscope slide and examined with a Zeiss differential interference contrast microscope fitted with a photomicrographic camera. At 0.6 and 1.0/~M, 1-PC caused some erythrocytes to undergo crenation. Crenated cells, which could also be detected with the light microscope used for E P M measurements, were excluded from E P M determinations. T h e hemolytic effect of 1-PC was studied by a spectrophotometric method [5, 6]. T h e normal physiological saline solution was used as a 0% hemolyzed blank, and the 100% hemolyzed blank was prepared by adding 5 /zl of blood to 7.5 ml of distilled water. T o analyze the effect of palmitoyl carnitine, 5 #1 of blood was added to 7.5 ml physiological solution containing the desired concentration of the drug. T h e samples were incubated at 25~ for 15 rain, centrifuged, the supernatant was removed and placed in a quartz cuvette.

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TABLE 1. Effect of[Ca2+]0 and of 1-PC on EPM of human erythrocytes Condition

EPM (#m/s/V/cm) a

Control [Ca2+]0 0.18 mM 0.36 mM 1.8 mM 3.6 mM

1.10 +_ 0.02 1.02 0.97 0.82 0.74

__ 0.02 ___0.03 + 0.04 _ 0.03

Condition Control I-PC 1 X 10 .7 3 X 10 -v 6 x 10 -v 1 X 10 -6

EPM (/~m/s/V/cm) a 1.14 +_ 0.04 M M M M

0.98 0.83 0.74 0.69

__ 0.03 __+0.03 _+ 0.03 +_ 0.02

a I = 0.145; p H = 7.3. M e a s u r e m e n t s are means ___S.D. of five experiments.

A b s o r b a n c e was read at a wavelength of 415 n m with a s p e c t r o p h o t o m e t e r (Gilford 250). T h e extent of hemolysis was expressed as the percent of m a x i m u m o b t a i n e d with distilled water. Results

Extracellular Ca 2 + ([Ca 2 +] o) and E P M : effects

of concentration, ionic strength and pH I n isotonic saline solution with an ionic strength (/) of 0.145, erythrocytes m i g r a t e d t o w a r d the a n o d e with an electrophoretic m o b i l i t y ( E P M ) of ~ 1.10/~m/s/V/cm ( T a b l e 1). This E P M is characteristic u n d e r these conditions (reviewed in [30]). A d d i t i o n of CaC1 z to the isotonic saline solution r e d u c e d E P M in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r (Table 1). T h e reduction of E P M b y C a 2+ was not due to the small increase of I because raising I by a d d i t i o n of an equivalent concentration of NaC1 h a d no effect on E P M . It is well-known t h a t E P M increases as I is r e d u c e d [15, 18]. W h e n ionic strength was r e d u c e d by replacing NaC1 with sorbitol, E P M increased non-linearly (Fig. 1). A t I = 0.0145, E P M was 2.74__+ 0.05 # m / s / V / cm. This value is quite similar to t h a t r e p o r t e d b y others [15, 18] as is the n o n - l i n e a r relationship between E P M a n d I. A d d i t i o n of CaC1 z (3.6 mM) r e d u c e d E P M at all I in the r a n g e tested (0.0073 to 0.145) with g r e a t e r reductions observed at lower I (Fig. 1). As an a d d i t i o n a l check of e x p e r i m e n t a l conditions, we ascertained the effect of p H on E P M . As shown in F i g u r e 2, E P M decreased slightly from 1.13 +_ 0.03 p m / s / V / c m at p H 11 to 0.98__+ 0.04 / t m / s / V / c m at p H 4. A t p H < 4, E P M decreased more r a p i d l y to reach a value of 0.35 __+0.04 # m / s / V / c m at p H 2.5. E r y t h r o c y t e fragility was m a r k e d l y

increased at p H < 3 a n d hemolysis occurred within 1 to 2 m i n at p H 2. T h e r e d u c t i o n of E P M at p H < 4 is g r e a t e r t h a n that r e p o r t e d by H e a r d a n d S e a m a n [18] b u t is c o m p a r a b l e to the results r e p o r t e d by others [12, 15, 32].

l-Palmitoyl carnitine (1-PC) and E P M : effects of concentration, ionic strength and pH T h e E P M of h u m a n erythrocytes was r e d u c e d by 1-PC in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r ( T a b l e 1). T h e reductions of E P M by 1-PC, (I = 0.145), in the c o n c e n t r a t i o n range from 10 - 7 t o 1 0 - 6 M were c o m p a r a b l e to those p r o d u c e d by a d d i t i o n s of C a z + from 0.18 to 3.6 mM (see T a b l e 1). T h e similarity between 1-PC a n d C a 2+ actions on E P M was also -4.5 -4.0

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F I G U R E 1. R e l a t i o n s h i p between erythrocyte E P M and ionic strength in the absence, ( 0 ) and presence ( O ) of 3.6 mM CaC12 . pH, 7.2; t e m p e r a t u r e 25~ Lines are d r a w n by eye; m e a s u r e m e n t s are m e a n 3-S.D. for this F i g u r e a n d for Figures 2, 3, 4, 6.

Palmitoyl Carnitine and Cell Surface Charge

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FIGURE 2. The effect of pH on EPM in the absence (O) and presence (O) of 3.6 mMCaCI2 . Ionic strength, 0.145; temperature, 25~ Lines are drawn by eye ; arrows indicate pH at which EPM is half the maximum value. revealed in experiments where I a n d p H were varied. T h e inverse relationship between E P M a n d / w a s r e t a i n e d in the presence of 10 - 6 M 1-PC, the highest c o n c e n t r a t i o n tested (Fig. 3). As with Ca 2 +, the reduction of E P M by 1-PC was greater at lower L M o r e o v e r , the ability of -4.5 -g

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1-PC to reduce E P M was r a t h e r constant when the bulk p H of the suspending m e d i u m ( I = 0.145 M) varied between 4 a n d 11 (Fig. 4). A t bulk p H < 4, the m a g n i t u d e of the reduction of E P M by 1-PC diminished. These results are q u a l i t a t i v e l y similar to those seen with 3.6 m u [ C a 2 + ] o . T h e changes of E P M by 1-PC a n d by [ C a 2 + ] 0 in saline solution of n o r m a l I are illustrated in Figure 5. Comparable reductions of h u m a n e r y t h r o c y t e E P M required a b o u t ,,~10-3-fold less 1-PC t h a n [ C a 2 + ] o which a p p r o x i m a t e the physiologic range. I n the presence of 0.36 a n d 1.8 m g [ C a 2 + ] 0 , the r e d u c t i o n of E P M by 1-PC was diminished b o t h in m a g n i t u d e a n d sensitivity (Fig. 5). T h e fact t h a t concentrations of 1PC > 1 0 - 6 ~ tended to hemolyze erythrocytes p r e c l u d e d a more q u a n t i t a t i v e analysis of the interaction between this a m p h i p h i l e and the d i v a l e n t cation. Therefore, the occlusive interaction between 1-PC a n d [ C a 2 + ] 0 , while consistent with a similar action to reduce surface negative charge, c a n n o t be defined as to its n a t u r e (i.e., competitive, non-competitive, uncompetitive).

I

0.020.040.060.080.10 0.12 0.14 1.16 Ionic strength

FIGURE 3. Relationship between EPM and ionic strength for human erythrocytes in the absence (0) and presence (O) of 10 6M 1-PC. pH, 7.2; temperature, 25~

Neuraminidase treatment Because sialic acid is an i m p o r t a n t determin a n t of the negative charge at the e r y t h r o c y t e surface ]-8, 12], we treated cells with increas-

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(0.58 __+0.04 #m/s/V/cm) because raising the enzyme concentration 10-fold did not change EPM significantly (0.52 + 0.03 #m/s/V/cm) from that at 0.1 Unit. The threshold concentration of neuraminidase to reduce EPM was betweert 0.O l and 0.1 U. The small reduction of EPM by 1-PC in erythrocytes treated with 0.1 and 110 total units ofneuraminidase could arise from interaction with negatively charged membrane phospholipids,

ing concentrations of neuraminidase (see Methods) to evaluate the effect of sialic acid removal on EPM and on the changes in this variable caused by 1-PC and [Ca 2 +]0. As shown in Figure 6, treatment with neuraminidase alone significantly reduced EPM at a total of 0.1 and 1.0 U of the enzyme which corresponds to 0.02 and 0.2 U/ml incubation medium. The effect of neuraminidase on EPM was about maximal at 0.1 U

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[Ligond] (m) F I G U R E 5. Change of EPM (negative sign indicates decrease) by [Ca 2+ ] alone ([~) and by l-PC alone (O) and in the presence of 0.36 mM [Ca2+]0 (/X)or of 1.8 mM [CaZ+]0 ( 0 ) .

Palmitoyl Carnitine and Cell Surface Charge

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FIGURE 6. Reduction of EPM as a function of the total amount of enzyme (abscissa; 1.0 U corresponds to 0.2 U/ml). Erythrocytes treated with neuraminidase alone (0) displayed maximum reduction of EPM at 0.i U . The effect of I-PC (10-6 M) on EPM of neuraminidase-treated cells is indicated by (9 "C" indicates control measurements in the absence of any treatment (O) and in the presenceof 10-6 MI-PC (O). I n the absence of n e u r a m i n i d a s e treatment, 1-PC (10 - 6 M) reduced E P M by 0.43/2m/s/V/ cm (Fig. 6). T h e decrement of E P M by 1-PC was progressively reduced to 0.40, 0.19 a n d 0.17 after t r e a t m e n t with 0.01, 0.1 a n d 1.0 U neuraminidase, respectively. T h e ability of [Ca2+]0 to reduce E P M was also diminished by t r e a t m e n t with the same concentrations of neuraminidase. For example, 3.6 mM [ C a 2 + ] , reduced E P M by 0.46 _+ 0.03 # m / s / V / c m in the absence a n d by 0 . 1 4 - t - 0 . 0 6 in the presence of 1.0 U of neuraminidase, respectively (n = 4). Q u a n t i t a t i v e differences between 1-PC a n d [ Caz +]o included not only the concentrations needed to reduce E P M b u t also the rate at which E P M diminished as a function of time. As shown in Figure 7, the reduction of E P M b y 3.6 mM [CaZ+]0 occurred with a tl/2 of "" < 0 . 5 m i n (n = 3) and the m a x i m u m effect was attained within 3 to 4 min. T h e time required to make the measurements after mixing a n d addition to the electrophoresis a p p a r a t u s precluded a more definitive estimation of the initial rate of reaction with 3.6 mM [ Ca2 +]0- By contrast, the onset of 1-PC action was slower a n d thereby more readily resolved. I n the case of 10 . 6 M 1-PC, the ta/2 was

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Time (minutes]

FIGURE 7. Time course for change of EPM by 10-6 M 1-PC (O) and by 3.6 mM Ca2+ (O). Ordinate: indicates reduction, n = 3. 1.5 rain a n d a quasi-steady effect was reached by 10 to 12 rain (Fig. 7). T h e times required for Ca / + a n d 1-PC to reduce E P M to steadystate values are c o m p a r a b l e to those observed for the actions of these ligands on the m a x i m u m rate of rise (l)max) of the N a + c u r r e n t in h e a r t cells [20].

Electrophoretic mobility : tests of l-palmitic acid, and l-carnitine Experiments were carried out to determine if the constituents of 1-PC, palmitic acid a n d carnitine, affected E P M . Each of these compounds was applied to erythrocytes suspended in physiological saline solution (I = 0.145 M; pH=7.3). Neither of these c o m p o u n d s change E P M significantly. I n experiments with carnitine (10 - 4 M), E P M (#m/s/V/cm) was 1.05 _ 0.05 in the absence a n d 1 . 0 3 _ 0.04 in the presence of carnitine ( n = 3). With palmitic acid (10 - 4 M), the E P M was 1.04 • 0.05 in the absence a n d 1.02 __+0.05 in the presence of palmitic acid (n = 2).

Hemolysis and erythrocyte shape I n the c o n c e n t r a t i o n range 10 - 7 to 10 - 6 M, 1-PC had no significant hemolytic effect (Fig. 8). T h e extent of hemolysis for control a n d treated samples was < 1% up to 10 - 6 M 1-PC. Significant hemolysis was detected at

488

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I O0

IO-r~l)

FIGURE 8. Hemolysisof erythrocytesby I-PC. Ordinate: hemolysisas percent of maximum; abscissa: I-PC concentration on logarithmicscale. Experimentswith 1-PCwere done in the absence (O) of added CaCl2 (0.145 ~t NaC1; pH, 7.2; temperature, 25~ and in the presenceof either 0.36 mMCaC12(O) or 1.8 mMCaC12(A). 3 x 10-6M I-PC and complete hemolysis occurred at 10 .5 ~ 1-PC (Fig. 8). Addition of 0.36 mM and 1.8 mM CaC12 to the physiological saline solution provided slight protection against the hemolytic effect of 1-PC (Fig: 8). Estimates of the concentration of 1-PC that produced 50% of maximum hemolysis were 3 #M (no Ca2+)' 4 tim (0.36 m~t Ca 2+) and 5.2 #M (1.8 mM Ca2+). Changes in e r y t h r o c y t e shape were obtained from photomicrographs as shown in Figure 9. There was no detectable difference in erythrocyte morphology between untreated cells [Fig. 8(a)] and those treated with 10-TM [Fig. 8(b)] and 3 x 10-7M [Fig. 9(c)] 1-PC, respectively. In the presence of 6 x 10 -7 M 1-PC (Fig. 9(d)], about 5% of the erythrocytes were crenated. The percentage of crenated cells increased to about 16% in 10 -6 M 1-PC (Fig. 9(e)]. It should be noted that crenated cells were not included in the estimates of EPM. Discussion

The negatively charged surface of the erythrocyte, in the presence of solutions with altered ionic strength or pH, displayed the changes in electrophoretic mobility described by others (reviewed in [34]). The surface charge system

may be viewed as an equilibrium between ions bound at the surface and ions in solution [1, 4, 16, 26"]. As ionic strength was reduced from the normally used value of 0.145, EPM increased in a non-linear fashion [15]. The intensity of the local electric field at a given distance from the surface is increased as the ionic strength decreases as predicted by the G o u y - C h a p m a n theory. We were unable to measure a reversal of charge at very acidic pH. Nevertheless, our results indicated that at bulk [ H + ] 0 ranging from 10 -4 to 10 -11 M, there was only a slight change of EPM as reported by others [12, 18]. The importance of sialic acid (pK ~ 2.6) as an ionogenic moiety at the erythrocyte surface has been emphasized [8, 12] and the ionogenic role of membrane phospholipids in the erythrocyte is viewed as subsidiary [16, 34]. Our results are in accordance with this view (vide infra). The EPM of human erythrocytes decreased as the [Ca2+]0 increased over the range 0.18 to 3.6 m~a. This result is consistent with the hypothesis that Ca 2+, either by screening or binding surface negative charge, diminishes the ( potential and thereby reduces the EPM of the erythrocyte. The observation agrees with that ofPape et al. [28] who studied erythrocytes of Amphiuma means. The EPM of other cell types, including embryonic chick epider-

Palmitoyl Carnitine and Cell Surface Charge

489

FIGURE 9. Photomicrographs of erythrocytes in the absence (a) and presence of 1-PC at 0.1 /~M (b), 0.3 ,u~ (c), 0.6 #M (d) and 1.0 ,uM (e). Calibration bar (#m) in (a) applies to all panels. The erythrocytes were suspended in 0.145 MNaC1, at pH 7.2. mal, cardiac, hepatic and neural retinal cells [7] and toad bladder epithelium [25] has also been reported to diminish in the presence of Ca 2+. The effect of [Ca2+]0 on erythrocyte E P M is consonant with its well-known action to reduce negative surface charge in electrically excitable cells (reviewed in [19, 27] and to interact with phosphate and carboxyl groups, the latter in sialic acid [21]. This similarity strengthens the usefulness of E P M measurements as an indicator of surface charge properties. 1-Palmitoyl carnitine reduced E P M , albeit at concentrations about three orders of magnitude less than those of [Ca2+]0 . Both 1-PC and Ca 2+, at maximally effective concentrations, reduced E P M by the same extent. Estimates of a obtained from equations (1) and (2) are given in Table 2. I n 0.145 M NaC1, a

was comparable to the values reported by others (reviewed in [30]). Because 1-PC and Ca z+ reduced E P M without changing viscosity appreciably ( < 2 % ) , it is apparent from inspection of equation (2) that a has been diminished. I f correction for ion size is included in equation (2) [12], the estimated values for 6 ( x l 0 3 e s u / c m 2) are: 2.78

TABLE 2. Estimated changes of surface charge density (r from equations (1) and (2) o-(esu/cm 2) Condition

Equation (1)

Equation (2)

0.145 ~ NaC1 +l-PC (1 /~M) + C a 2+ (3.6 mM)

3.18 x 103 2.57 x 103 2.72 x 103

3.69 x 103 2.42 x 103 2.61 x 103

490

J.M+szAros

(control), 1.81 (1-PC at 1 pg) and 1.93 (Ca at 3.6 raM). Despite the quantitative fluctuations in cr obtained from the different equations, the evidence is consistent with the hypothesis that 1-PC and Ca 2 + reduce a and thereby are able to impede EPM. It is assumed that 1-PC is incorporated into the erythrocyte by insertion of the fatty acyl chain into the hydrophobic environment of the lipid bilayer. From values of erythrocyte membrane protein and lipid contents (see Methods) a bulk concentratration of 1 0 - 6 N 1-PC and a total of 2.45 x 1 0 7 erythrocytes in a 5 ml sample one obtains ratios of 0.36 #mol 1-PC/mg membrane protein and 0.46 #mol l-PC/mg membrane lipid. However, we do not know the extent of 1-PC incorporation into the erythrocyte membrane. We have previously proposed that 1-PC acted like Ca z+ to reduce surface negative charge at or near voltage- and time- dependent Na and Ca channels in heart cell membranes [20]. The present results provide additional evidence for this hypothesis. In general, two mechanisms may be proposed for the reduction of a by 1-PC. First, 1-PC is incorporated into the membrane by the fatty acyl chain. (At concentrations > 1 0 -6 M, 1-PC hemolyzed erythrocytes, a result in accordance with membrane disruption by incorporated amphiphile [6].) This mechanism arises from the bilayer couple hypothesis (reviewed in [33]). In this model, anionic and neutral amphiphiles incorporated preferentially into the outer membrane leaflet and caused crenation. At low concentrations, the amphiphile-induced membrane expansion would produce microvesicles (400 to 600 nm). Accordingly, the average distance between surface charges would increase and this would diminish a. While our EPM measurements did not include crenated cells, microvesicles could not have been detected with the light microscope. The application of this mechanism to the action of 1-PC on EPM assumes that the molecule is neutral. This supposition is probably satisfied in bulk solution where the C O O - groups of 1-PC can neutralize the positive change of the quaternary amine. Secondly, 1-PC could neutralize electrostatically some of the negative charge on carboxyl groups of sialic acid. This model, like the first one, also assumes that the fatty acyl chain has been incorporated into the erythro-

cyte membrane as has been reported for cardiac sarcolemmal membranes [13]. Additionally, this model assumes that 1-PC is not a zwitterion at the cell surface. There are two reasons for suspecting that 1-PC is not a neutral molecule at the ionogenic surface of the erythrocyte. First, others have shown that a neutral polymer (dextran) adsorbed at the cell surface expanded the diffuse double layer, increased the ~ potential and thereby increased EPM [31]. If 1-PC were neutral at the ionogenic surface, EPM should also have increased. Second, the G o u y - C h a p m a n model posits an ordered array of mobile counterions at the region of fixed membrane charge (reviewed in [26]. According to this theory, the electrostatic potential at the cell surface dissipates with increasing distance from the plane of fixed membrane charges. (The average distance for the position of counterions from the charged surface is given by the Debye length. At 25~ the Debye length (~c)-1 is given by the relation: = 3.27 x 10 v (/) 1/2 where I is ionic strength. At I = 0.145, ~c = 1.25 x 106 cm 1 and the Debye length is 8 A. The ~ potential will decrease to e-1 of its m a x i m u m value within 8 A of the ionogenic surface [1, 9, 26].) Because of the surface negative charge, cationic counterions will be attracted and their concentration will increase greatly as the fixed negative charge surface is approached [4]. A univalent electrolyte whose bulk concentration is 10 - l M will have a cation concentration at the surface of 1 M while the diffusible anion concentration will be practically zero [26]. In other words, the surface negative charge imposes a redistribution of diffusable ions such that mobile cations will have a greater concentration and mobile anions a lesser concentration than in bulk solution as the fixed negative charge surface is approached. In light of these considerations, it may be proposed that the orientation of the quaternary amine and C O O - groups in 1-PC is highly ordered at the erythrocyte surface so as to have the quaternary amine moiety nearer the fixed surface negative charge and the C O O - group positioned at some distance away. In the case of 1-PC, the separation between quaternary amine and C O O - moieties is approximately 5 A, an estimate that could place the quaternary amine group at the plane of surface negative charge and the

Palmitoyl Carnitine and Cell Surface Charge C O 0 - within one D e b y e length of the field a n d therefore subject to electrostatic repulsion. Several aspects of our experiments can be i n t e r p r e t e d as consistent with either the bilayer couple m e c h a n i s m or the electrostatic mechanism. First, in solutions of r e d u c e d /, the E P M increased a n d the reduction of E P M by 1-PC b e c a m e greater. E i t h e r m e c h a n i s m is consistent with the latter observation. W h e n I is reduced, the ~ potential becomes m o r e n e g a t i v e a n d at constant a, the D e b y e length increases (to ,-~25 ,~ in 0.0145 M NaC1). A c c o r d i n g to the electrostatic mechanism, the ability of surface negative charge to induce a dipole in a zwitterion should be increased because the D e b y e length is ~ 25 A while the distance between q u a t e r n a r y a m i n e a n d C O O - moieties in I-PC should r e m a i n at ,-~ 5 A. N e i t h e r m e c h a n i s m excludes the possibility that there is some m o d u l a t i o n by the COOg r o u p of the q u a t e r n a r y aminei n d u c e d reduction in surface negative charge. I n c a r d i a c s a r c o l e m m a l p r e p a r a t i o n s , 1-PCi n d u c e d increases in m e m b r a n e fluidity were not due to d i s p l a c e m e n t of endogenous Ca 2 + ['13]. I t was p r o p o s e d that long chain acyl carnitine w o u l d increase C a b i n d i n g at the m e m b r a n e surface by electrostatic interaction between Ca 2 + a n d the C O O - moiety in 1-PC [13]. (Low concentrations of 1-PC increased the b i n d i n g of Ca z+ to sarcoplasmic retic u l u m m e m b r a n e s from h e a r t a n d skeletal muscle [2].) W i t h this provision, the a m p h i phile would also be expected to a t t r a c t N a + and t h e r e b y e n h a n c e its ability to reduce a. Second, the ability of 1-PC to reduce E P M was diminished in the presence of C a 2 +. Provided that Ca 2+ does not i n t e r a c t with C O O - in 1-PC to reduce its concentration, this p h e n o m e n o n can be a t t r i b u t e d to the well-known ability of Ca 2 + to reduce a a n d t h e r e b y diminish the surface negative charge available for i n t e r a c t i o n with 1-PC. A l t e r n a -

491

tively, Ca 2+ could p r e v e n t i n c o r p o r a t i o n of 1-PC into the bilayer. T h e fact t h a t the extent of E P M r e d u c t i o n b y 3.6 mM Ca 2+ a n d 1 /tM 1-PC was q u a n t i t i v e l y the same in 145 mM NaC1 a n d increased p r o p o r t i o n a t e l y in 14.5 mM NaC1 is consistent with the view that C a / + a n d 1-PC are i n t e r a c t i n g with the same fraction of surface negative charge. T h i r d , t r e a t m e n t of cells with n e u r a m i n i d a s e not only r e d u c e d E P M as r e p o r t e d b y others [17, 31, 33] b u t also blocked the ability of C a 2+ a n d 1-PC to reduce E P M . Assuming t h a t the effect of n e u r a m i n i d a s e is due to r e m o v a l of sialic acid residues from the m e m b r a n e surface, this observation is consistent with the concept t h a t the negative charges on C O O g r o u p of sialic acid are the site of interaction with Ca 2 + a n d 1-PC. T h e r e is one i m p o r t a n t aspect in which the two mechanisms differ. As m e n t i o n e d previously, there is reason to suspect t h a t 1-PC is not n e u t r a l at the cell surface because if it were, 1-PC should have increased E P M like n e u t r a l d e x t r a n [31] r a t h e r t h a n decrease it. W h i l e this observation argues against the b i l a y e r couple mechanism, it does not constitute p r o o f of the electrostatic mechanism. S u m m a r i l y , we have observed t h a t I-PC, like Ca z+, reduces E P M of h u m a n erythrocytes in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r . Because t r e a t m e n t with n e u r a m i n i d a s e diminished the changes of E P M caused by either 1-PC or C a 2 +, the C O O - moiety ofsialic acid is required for interaction with these ligands. These results are consistent with our previously a d v a n c e d hypothesis that 1-PC interacts with surface negative charge at excitatory ion channels in the h e a r t in a m a n n e r analogous to that of Ca 2. T h e reduction of E P M by 1-PC or Ca z+, when e v a l u a t e d by equations derived from the G o u y - C h a p m a n m o d e l of the diffuse d o u b l e layer, would of necessity be associated with a reduction of surface negative charge density.

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