Membrane potential changes associated with pinocytosis of serum lipoproteins in L cells

CopyrIght @ 1981 by Academic Pres\. Inc. All rights of reproduction in any form reserved 0014-48?7W11120?71-08$o?.oo10

Experimental

MEMBRANE

Cell Research 136 (1981) 271-278

POTENTIAL

PINOCYTOSIS

CHANGES

OF SERUM

ASSOCIATED

LIPOPROTEINS

WITH

IN L CELLS

WAKOH TSUCHIYA,‘, * YASUNOBU OKADA,’ JURI YANO,’ ATSUSHI MURAI,’ TADAO MIYAHARA” and TOMOJI TANAKA:’ ‘Department

of Physiology,

2Department oj’Obstetrics und Gynecology, and “Department Faculty oj’Medicine, Kyoto University, Kyoto 606. Japan

of Geriatrics.

SUMMARY L cells exhibit spontaneous oscillations of membrane potential in accord with fluctuations of the cytoplasmic Ca’+ concentration. Upon addition of low-density lipoprotein (LDL), L cells show a prolonged hyperpolarization which is followed by an increase in the frequency of membrane potential oscillations. These membrane potential changes induced by LDL were inhibited by Ca2+-channel blockers. LDL-induced membrane potential changes were accompanied by a vigorous pinocytosis which was coupled with the formation of ring-like ridge structures on the cell surface. These electrical and morphological changes were also induced by high-density lipoprotein (HDL) but not by very-low-density lipoprotein (VLDL). These results suggest that the application of LDL or HDL to the membrane surface elicits a rapid influx of Ca2+into the cytosol, resulting in membrane hyperpolarization. A rise in cytoplasmic Ca2+ may be implicated in the primary factor for the pinocytic process.

Cholesterol is known to be essential for cell survival and growth. Unlike other cell lines that synthesize cholesterol, the L cell is incapable of producing cholesterol [1] and uses exogenous cholesterol in both lowdensity lipoprotein (LDL) and high-density lipoprotein (HDL) [2]. It has been firmly established, in a variety of cells including fibroblasts, that LDL binds to the receptor on the cell surface, is rapidly internalized, and is delivered to the lysosome, in which degradation into amino acids and free cholesterol occurs [3]. HDL is also taken up in some cells [4, 51, but the mechanism of internalization is virtually unknown. Fibroblastic L cells, which are known to have the LDL receptor [6], show oscillation of the membrane potential composed of repeated hyperpolarizing responses [7]. The oscillation is apparently dependent

upon intracellular metabolism, since it can be suppressed by metabolic inhibitors or by low temperature [7]. The ionic mechanism responsible for the oscillation has been extensively studied [8-121. In brief, the hyperpolarizing response results from an increase in the intracellular Ca*+ concentration, which, in turn, increases the K+ permeability. Although the physiological significance of the membrane potential oscillation has not been established, we have previously suggested that the Ca*+ inflow inducing hyperpolarizing response is related to membrane motility [13] and phagocytic activity

[111. We report here that LDL induces a rapid membrane hyperpolarization which is fol* Present address: Department of Dermatology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.

272

Tsuchiya et al.

Table 1. Membrane potential profile in the 3rd phase of LDL effect

Control LDL” with oscillation without oscillation

Resting level (mV)

Hyperpolarized level (mV)

Frequency of oscillation (cyclelmin)

- 12.7k2.6 (67)

-40.5+ 9.1 (67)

4.95 1.4 (67)

-13.1&3.4(95) -

-35.OklO.l (95) -38.3+ 7.5 (29)

9.2k2.6 (95) -

u Membrane potentials were measured 5-30 min after exposure to LDL (IO mglml). All values are presented as means + SD. Numbers in parentheses indicate no. of cells observed.

lowed by changes in the pattern of membrane potential oscillation and that these electrical changes are accompanied by a chain of mbrphological alterations leading to pinocytic vesicle formation. A preliminary account of some of these data has been published in abstract form [ 141. MATERIALS

AND METHODS

Cell culture The cell culture techniques employed were identical with those described in a previous paper [15]. A monolayer of giant L cells obtained by X-ray irradiation (1500-2000 R) was subjected to both electrophysiological and morphological studies.

Isolation

of lipoproteins

The method originally reported by Have1 et al. [16] was used with some modification. Lipoproteins were *

1 m,n

.

.

.

I

.

mv

Other proteins

~-20

-0

Fig. I. Serum-induced hyperpolarization. Arrow and dots indicate time of addition of heat-inactivated bovine serum (final concentration: 0.2%) and that of application of outward current (0.3 nA) to monitor membrane resistance, respectively.

E.up Cd

Res 136 (IYXI)

isolated from the freshly collected serum of fasting human donors by a sequential ultra-centrifugal floatation in NaCI-NaBr solution, containing 0.05 % EDTA (pH 7.1) at IO’C, using a Hitachi RP65TA rotor. Very-low-density lipoprotein (VLDL) was initially isolated by centrifugation at d=l.O06 (g/ml) for I6 h at 40000 rpm. To obtain LDL, the solvent density was then raised to 1.063 by adding the NaCl-NaBr solution, followed by centrifugation of the mixture for 24 h at 40000 rpm. The NaCl-NaBr solution was subsequently added to LDL-free serum to raise the density to 1.125 and then to 1.210. For each density the serum was centrifuged for 36 h at 40000 rpm to obtain HDL, and HDL,. The concentrations of lipoproteins were estimated from their cholesterol contents. Each fraction was dialysed against 0.9% NaCl or a Tris-buffered saline (TBS) for 2 days at 5°C and then diluted in TBS or concentrated, using Minicon (Amicon Corp., USA), to desired levels. In some experiments, VLDL and LDL were isolated in an EDTA-free solution, and each fraction was washed twice by reflotation at their respective densities, since the contamination of a small amount of the chelator is known to affect the membrane properties of the cells [lo]. No contamination of albumin was detected when each fraction was examined against rabbit anti-human albumin serum. The lipoproteins were freshly prepared before the experiments in order to prevent protein aggregation and added directly to the medium or applied to the cell surface through a micropipette.

Human P-lipoprotein obtained as Cohn’s fraction III-O and P-globulin (fraction III) were purchased from ICN pharmaceuticals Inc. (Cleveland). o-Globulin (fraction IV), fibrinogen (fraction I) and albumin (fraction V) were purchased from Miles Laboratories Inc. (USA)). y-Globulin was the product of AB Kabi (Stockholm). All these proteins were solubilized in TBS at desired concentrations and used for the experiments.

Electrophysiology The electrophysiological equipment and techniques used were the same as those described in previous papers [8, IO]. 3 M KCI-filled microelectrodes with

LDL-induced membrane potential changes

01

273

imln I

Fig.

2. LDL-induced changes in membrane potential profile. Arrows and dots indicate time of addition of LDL and that of application of outward current (0.3 nA) to monitor membrane resistance, respectively.

Since the latency period before a response was dependent on the distance between the site of LDL addition and the cell surface, it seems to be due to the diffusion of LDL.

a resistance of 10-20 Ma were used. Their tip potentials were less than 5 mV.

interference or scanning electron microscope photographs (SEMs), cells cultured on coverslips were used and observed under Nikon Optiphoto NT microscope or Shimadzu SEM (ASM-SX). The techniques of fixation and drying for SEM studies have been described previously [ll, 151.

Morphological studies Studies with a phase contrast microscope (Tiyoda T-2) were made on the cells cultured in plastic dishes during or after the electrophysiological studies. To take

Media and drugs Fischer’s medium (Nissui Co.) supplemented with 10% heat-inactivated bovine serum was used for the cell culture. A bathing solution used in electrophysiological and morphological experiments was TBS which was composed of 143 mM NaCI, 4.2 mM KCI, 0.9 mM CaCI,, 0.5 mM MgCI, and 20 mM mannitol, and buffered to pH 7.3-tO.l with 10 mM Tris-HCI. In order to change the K+, Na+ or Cl- concentration, TBS was modified by the equimolar replacement with Na+, Tris+ or SOi- (and mannitol for adjustment of osmolarity), respectively. Verapamil was a gift from Eisai Co. (Tokyo). Ethanol was used as a vehicle for the drug. Nifedipine was a gift from Bayer Yakuhin Ltd (Osaka), and was dissolved in dimethyl sulfoxide (DMSO). The addition of ethanol or DMSO, at least in the doses used, did not affect the electrical and morphological properties of L cells.

RESULTS Serum- and P-lipoprotein-induced / membrane potential changes A”, I” In a control solution (TBS), almost all the Fig. S. Suppression of LDL effect by a Ca2+-free giant L cells impaled showed hyperpolarizmedium and Ca2+-channel blockers. Thick arrows: time of LDL addition (10 mglml). Dots: time of ap- ing oscillation (about 4-5 cycle/min) of the plication of small outward current (0.3 nA) to monimembrane potential up to about -40 mV tor membrane resistance. Asterisks: time of application of large inward current (10 nA) to stimulate the from the resting level (about - 13 mV) and membrane. Fine arrows: time of impalement or withresponded with a hyperpolarizing response drawal of microelectrode. (a) In a Ca*+-free medium containing 2 mM EGTA; (b) in the presence of 0.4 (about -40 mV) to electrical stimulus mM verapamil; (c) in the presence of 50 PM nifedi(k 10 nA), as reported previously [7]. pine. Exp Cell Rus 136 c/98/)

274

Tsuchiya et al.

a) YLDLW!$~l~ VLWLlrrJlml) 1. rn” 1 . a-

b)

p,(Lmglml) h

Fig. 4. Effects of(u) VLDL; (b) HDL, on the membrane potential profile. Arrows: time of addition of lipoproteins. Dofs: time of application of small outward current (0.3 nA) to monitor membrane resist-

ante. Asferisks: time of application of large outward current (10 nA) to stimulate the membrane. The delay in response after addition of lipoproteins is due to diffusion of lipoproteins.

Upon exposure to bovine serum during the spontaneous potential oscillation, the cell membrane was always rapidly hyperpolarized, causing the oscillation to cease (fig. 1). The magnitude of the peak hyperpolarization (-46.1k10.4 (S.D.) mV,n=31) was usually larger than that of the peak hyperpolarization of the oscillation (table 1). To determine which component of the serum affects the membrane, we examined the effects of serum proteins, since other serum constituents (amino acids, sugars and fatty acids) did not elicit the hyperpolarizing response. Of all the protein fractions tested, a-globulin (Cohn’s fraction IV), P-globulin (fraction III) and /3-lipoprotein (fraction III-O) always produced strong hyperpolarizing responses at a concentration of 5 mg/ml. Albumin (fraction V) could induce a similar but weak hyperpolarization in about 50% of the cells examined at a concentration of 10 mg/ml. Fibrinogen (fraction I) and y-globulin (fraction II) were ineffective.

brane hyperpolarization, the effect of freshly isolated human LDL was examined. As shown in fig. 2a, an addition of LDL (10 mg/ml) elicited a rapid hyperpolarization which was associated with a decrease in membrane resistance (1st phase of the response). This rapid response was followed by a slowly depolarizing phase which gradually approached the resting level and remained at a certain level of hyperpolarization (2nd phase of the response). After 2-5 min of LDL addition, about 80% of the cells examined resumed oscillation (3rd phase of the response) which was higher in frequency but smaller in amplitude than the control oscillation (table 1). An increase in the dose of added LDL increased the hyperpolarization and frequency of the oscillation (fig. 2 b). The LDL-induced changes in the membrane potential were reversible within 20-30 min after replacement of the medium by LDL-free TBS.

LDL-induced changes in membrane potential Since P-lipoprotein was found to be the most powerful fraction that induced memExp Cell Res 136 (1981)

Ionic mechanism The ionic mechanism underlying the initial hyperpolarization (i.e. 1st phase) was evaluated by changing the external ion concentrations. The P-lipoprotein (or LDL)induced hyperpolarization was dependent

LDL-induced

membtwne potential

changes

275

Fig. 5. LDL-induced changes observed under light microscopy. Phase micrograph of (u) control giant L cell; (b) giant L cell incubated with 10 mglml LDL for 5 min; (c) giant L cell incubated with 10 mg/ml LDL for 25 min. (d) Interference micrograph of

giant L cell incubated with 10 mg/ml P-lipoprotein for 70 min. Note the accumulated pinocytic vesicles (long UWWS) over the nucleus and many lysosomelike granules (d-m? arvo~~~s) around the nucleus. Bars, (u-c) 50 pm; (d) 10pm.

upon extracellular K+ concentration but not upon the external Na+ and Cl-. A lo-fold increase in the extracellular K+ concentration resulted in depolarization of the peak level of P-lipoprotein (or LDL)-induced hyperpolarization by 26.4 mV. The polarity of a P-lipoprotein-induced response was reversed when the external K+ concentration was increased over 150 mM which is a value close to an intracellular KC concentration [7]. In a Ca*+-free 2 mM EGTA medium, the LDL-induced hyperpolarizing response was greatly suppressed, and the spontaneous as well as electrically-induced hyperpolarizing responses were also inhibited completely (fig. 3~). Moreover, in

the presence of a calcium channel inhibitor, verapamil (0.4 mM) or nifedipine (50 PM), the LDL-induced hyperpolarizing response was completely inhibited even in the presence of 0.9 mM Caz+ (fig. 3b, c). Since intracellular Ca”+ injection was able to produce hyperpolarization in the presence of these doses of calcium channel inhibitors, this inhibition cannot be attributed to non-specific effects of these drugs. These data suggest that the ionic mechanism involved in the LDL-induced hyperpolarizing response is basically the same as that of the spontaneous hyperpolarizing oscillation [lo, 111; i.e., the Ca2+ influx through CaZ+ channel results from exposure to LDL, and

276

Tsuchiya et al.

Fig. 6. LDL-induced changes observed under SEM. (u) SEM of a giant L cell incubated with 10 mg/ml /3-lipoprotein for 5 min; (b) higher magnification from (a). Note that the majority of microvilli are seen in

the region around the circled ridge structure. (Compare with SEM of a control giant L ceil: fig. 4 in the preceding paper [ 151.)Bars: (a) 50 pm; (b) 10 pm.

the hyperpolarization is induced by stimulation of Ca2+-activated K+ conductance.

black wavy circle under the phase contrast microscope (fig. 5 b). The ring-like structure initially appeared along the cell periphery and then surged together with the vesicles toward the central region around the nucleus. The ring-like structure was seen as a circled ridge covered with dense microvilli under a scanning electron microscope (SEM) (fig. 6). In a small population of the cells, increased formation of membrane ruffles preceded formation of pinocytic vesicles. However, such a rufflemediated process was not so dominant as ridge-mediated pinocytosis. Subsequently, these vesicles moved centripetally (fig. 5~) and accumulated around the cell nucleus (fig. 5d). The same phenomena were observed when HDL was applied to the cells, whereas VLDL did not induce these morphological changes. In a Ca*+-free 2 mM EGTA solution, those morphological changes induced by LDL or HDL were considerably suppressed, although the inhibition was not complete. These results are in good accordance with the electrical changes (fig. 3a) and may suggest a close relationship between the two phenomena.

Effect of VLDL and HDL

As shown in fig. 4a, application of VLDL (total 5 mglml) failed to affect the spontaneous oscillation, whereas addition of HDLz (5 mg/ml) and HDL, (4 mg/ml, fig. 4b) produced a potential profile similar to that by LDL. The mean maximum levels of the 1st phase of responses induced by HDL, and HDL, were -39.6k11.5 (S.D.) mV (n=14), and -39.2k11.6 (S.D.) mV (n= 12), respectively. These data suggest that not only LDL, but also HDL (but not VLDL) interacts with the membrane of the L cell to induce the ionic permeability change. Morphological

observations

In the giant L cells, addition of LDL (or P-lipoprotein) resulted in pinocytosis that could be observed under the light microscope (fig. 5 b-d). The pinocytic vesicles were prominently formed within 2-3 min after LDL addition along the ring-like surface structure which was observed as a Exp Cell RPS 136 (1981)

LDL-induced membrane potential changes DISCUSSION The present study provides the first demonstration that LDL as well as HDL triggers rapid changes in ionic permeability across the cell membrane and thus induces pronounced changes in membrane potential profiles. It is suggested that similar effects observed with whole serum are due to those of LDL and HDL. This idea was supported by the fact that the same electrical response was induced by application of CP and pglobulins which contain HDL and LDL, respectively, or by that of commercially available P-lipoprotein which is expected to contain native LDL in addition to denatured LDL. Goldstein et al. [6] have reported that L cells have a specific receptor for LDL, which mediates pinocytosis of LDL [3]. However, the primary involvement of LDL receptor in LDL-induced hyperpolarizing response is unlikely for the following reasons. (1) The concentration of LDL which is necessary to induce the electrical changes was much higher than that for the receptormediated LDL pathway [3]. (2) HDL also produced the same effect. (3) VLDL, which is considered to share the common binding site for LDL [3], did not induce any changes. (4) Half the cells tested responded to albumin as well. Therefore, there is a possibility of involvement of rather nonspecific binding sites for lipoproteins, as suggested in rat liver cells [ 171. The present study strongly suggests that the hyperpolarizing responses are closely related to pinocytosis of several serum proteins (especially LDL and HDL) in L cells. Since deprivation of external Ca’+ or application of Ca”+-channel inhibitors suppressed both LDL-induced hyperpolarization and the subsequent pinocytosis, it can be conceived that Ca2+ influx leading to the

277

hyperpolarizing response plays an important role in the pinocytic activity. This agrees with the previous. suggestion for a close relationship between Ca*+ and pinocytosis [18-201. In addition, we have provided the data which suggest that membrane potential oscillation and the hyperpolarizing responses in L cells are closely related to the phagocytic activity [ 1l] and to membrane movement [13]. Thus, it may be hypothesized that membrane potential oscillation and hyperpolarizing response are associated with membrane-mobile cell activities in the L cell. The authors are grateful to Professor M. Kuno for kindly reading the manuscript. Thanks are also due to Dr T. Yada for discussion and to Mr T. Araki, Shimadzu Co., for technical assistance with SEM. This work was supported by Grant no. 557020 from the Ministry of Education, Science and Culture of Japan.

REFERENCES 1. Rothblat, G H, Bums, C H, Conner, R L & Landrev. J R. Science 169(1970) 880. 2. Bates, S R & Rothblat; G H, Biochim biophys acta 360 (1974) 38. 3. Goldstein, J L & Brown, M S, Ann rev biochem 46 (1977) 897. 4. Rechless, J P D, Weinstein, D B & Steinberg, D. Biochim biophys acta 529 (1978) 475. 5. Ose, L, Ose, T, Norum, K R & Berg, T, Biochim biophys acta 574 (1979) 521. 6. Goldstein, J L, Ho, Y K, Basu, S K & Brown, M S, Proc natl acad sci US 76 (1979) 333. 7. Okada, Y, Doida, Y, Roy, G, Tsuchiya, W, Inouye, K & Inouye, A, J membrane biol 35 (1977) 319. 8. Okada, Y, Roy, G, Tsuchiya, W, Doida, Y & Inouye, A, J membrane biol 35 (1977) 337. 9. Roy, G & Okada, Y, J membrane biol 38 (1978) 347. 10. Okada. Y. Rsuchiva. W & Inouve. A. J membrane biol 47’(1979) 357.. 11. Okada, Y, Tsuchiya, W, Yada, T, Yano, J & Yawo, H, J physiol 313 (1981) 101. 12. Nelson, P G & Henkart, M P, J exp biol81 (1979) 49. 13. Tsuchiya, W, Okada, Y & Inouye, A, 6th Int biophys congr, abstr, p. 223, Kyoto (1978). 14. Tsuchiya, W, Okada, Y, Yawo, H, Yada, T & Yano, J, J physiol sot Japan 41 (1979) 297 (abstr). 15. Tsuchiya, W, Okada, Y, Yano, J, Inouye, A, Sasaki, S & Doida, Y, Exp cell res 133 (1981) 83. 16. Havel, R J, Eder, H A & Bragdon, J H, J clin invest 34 (1955) 1345.

2’78 Tsuchiya et al. 17. Ose, L, Rsken, I, Norum, K R&Berg, T, Exp cell res 130 (1980) 127. 18. Josefsson, J 0, Holmer, N G & Hansson, S E, Acta physiol Stand 94 (1975) 278. 19. F’rusch, R D & Hannafin, J A, J gen physiol 74 (1979) 523.

E-ul, Cc// Rrs 136 (1981~

20. Duncan, R & Lloyd, J B, Biochim biophys acta 544 (1978) 647. Received April 14, 1981 Revised version received June 16, 1981 Accepted June 22, 1981

Printed

in Sweden