Comp. Biochem. Physiol. Vol. 72B, No. 4, pp. 547 to 549, 1982 0305-0491/82/080547-03503.00/0 Printed in Great Britain © 1982 Pergamon Press Ltd

BINDING OF H O M O L O G O U S VERSUS HETEROLOGOUS LOW DENSITY LIPOPROTEINS

BY DIFFERENT TISSUES OF THE RAT

WALTER K. K. Ho and ALEXANDRA M. LEUNG

Department of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

(Received 4 January 1982)

Abstract--1. The ability of five different tissues of the rat to bind homologous and heterologous (human) low density lipoprotein (LDL) was compared.

2. Human LDL was as effective as rat LDL in displacing rat [125I]LDL from the plasma membrane fraction of rat skeletal muscle.

3. Membranes from rat liver appeared to have no affinity for homologous LDL, while human LDL was slightly more effective in displacing labeled rat LDL from this tissue.

4. A similar pattern of displacement was observed in membranes derived from rat heart. 5. In contrast, membranes from both the aorta and adrenal glands of the rat displayed a significantly

greater affinity for binding LDL of homologous origin. 6. These results suggest that lipoprotein receptors analogous to those described for cultured human

cells may exist in the rat and that individual tissues exhibit distinctly different affinities for binding LDL of both homologous and human origin.

INTRODUCTION

The binding and internalization of serum low density lipoproteins (LDL) by specific receptors on the cell surface is well established (Brown et al., 1981). This mechanism is regarded as a major pathway by which the cell obtains extracellular cholesterol for metab- olism. A number of cell types derived from both human and other animal species have been shown to contain such receptors. Cells derived from pig aorta (Weinstein et al., 1976) and the adrenal cortex of the mouse (Faust et al., 1977) and the cow (Kovanen et al., 1979) show no obvious distinction in the binding of human or their homologous lipoproteins. These results suggest that human lipoproteins may possess a determinant, possibly either apolipoprotein B (apo B) or apolipoprotein E (apo E), which is commonly recognized by LDL receptors of different tissues and different species.

In contrast to the above trend, Drevon et al. (1981) reported recently that rat skin fibroblasts appear to possess LDL receptors which recognize rat LDL with much greater affinity than human LDL whereas the converse is observed in human fibroblasts. These results suggest that there may be species as well as tissue differences in the recognition of homologous versus heterologous lipoproteins. It is possible that LDL receptors having varied affinities for human LDL may exist in different species and/or different tissues of the same species. The aim of our study is to evaluate this question by comparing the ability of dif- ferent tissues of the rat to bind heterologous (human) versus homologous (rat) low density lipoproteins.

MATERIALS AND METHODS

Serum lipoproteins were isolated by sequential ultracen- trifugal flotation essentially as described by Hatch & Lees

(1968). The low density lipoproteins of human and rat serum were isolated between d 1.006 and d 1.063 g/ml. Pur- ity of the lipoprotein fractions was checked by agarose electrophoresis. A chloramine T method was used to iodin- ate rat LDL (Greenwood et al., 1963) and the specific ac- tivity of the final product was 350 cpm/ng. Protein concen- tration was determined by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as a standard.

Rats (Sprague-Dawley) were anaesthetized by intraperi- toneal injection of a solution of pentobarbital (40 mg/ml, 1 ml injected per kg body weight). The various tissues ot interest were removed and placed into a Krebs-Ringer bicarbonate (KRB) solution on ice. Known quantities o! each tissue were finely minced and suspended in KRB at an approximate concentration of 1 g tissue/10 mi. The sus- pensions were homogenized on ice with a polytron hom- ogenizer, then centrifuged at 600 g for 10 min in a refriger- ated centrifuge. The supernatant was spun at 4000g, 20 min, and the resulting pellet was washed twice at the same speed with KRB. Each tissue pellet, constituting the crude plasma membrane fraction, was suspended in an appropriate volume of KRB with 4% (w/v) BSA. Binding assays were performed in a final volume of 1 ml, composed of 0.8ml membrane suspension, 0.1 ml rat [IzsI]LDL (10 ~g/ml), and either 0.1 ml unlabeled human or rat LDL. Membrane suspensions were incubated for 4 hr at room temperature. After incubation, 500#1 of the mixture was layered onto an equal volume of KRB containing 4% BSA, 0.25 M sucrose in a microfuge tube. The tubes were spun for 3 min in an Eppendorf microfuge, after which the pel- lets were washed twice with 1 ml aliquots of KRB contain- ing 4% BSA followed by a final wash with KRB. Mem- brane pellets were assayed for gamma activity, then solu- bilized in 1 M NaOH for determination of protein concen- tration.

RESULTS

In an effort tO ascertain whether human LDL and rat LDL possess similar binding affinities for the membrane fractions of rat tissues, a competitive bind-

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LDL /.Lg Iml Fig. 1. Binding of human and rat LDL to membrane frac- tions of rat adrenal glands and aorta. Plasma membrane fractions were prepared as described in the text and incu- bated with the indicated concentrations of human LDL or rat LDL together with 10 #g/ml of rat [125I]LDL. Specific binding was assayed as described in the text. Values are presented as the mean _+ SD of four determinations.

Human LDL (-O-O-q; rat LDL (Q--O-).

ing study was performed using iodinated rat LDL and unlabeled human or rat LDL in various concen- trations. Because of the undefined and heterogeneous nature of rat LDL (Chapman, 1980), the density frac- tion (i.e. 1.006 < d < 1.063 g/ml) corresponding to human LDL was used in these studies. Although we have designated this fraction "LDL" for convenience, it must be emphasized that some very low density (VLDL) and high density (HDL~) lipoproteins were also present. Membrane fractions derived from various rat tissues were incubated in KRB/4% BSA (to minimize non-specific association), along with rat [12SI]LDL and the concentration of unlabeled human LDL or rat LDL indicated in Figs 1 and 2. The results demonstrate that all tissues tested possess some affinity for either human or rat LDL, but that each tissue is strikingly different from any other with respect to its binding affinity.

Human LDL was somewhat less effective than rat LDL in displacing iodinated rat LDL from skeletal muscle membranes (Fig. 2, lower panel). Unlabeled rat LDL reduced the amount of rat [a25I]LDL bound by 27%, whereas human LDL reduced the amount by 18.7% at the highest concentration tested. In contrast, in the adrenal glands (Fig. l, lower panel), human LDL was much less effective than rat LDL and essen- tially no displacement of rat [125I]LDL was detected. Rat LDL, however, was very effective in displacing

homologous iodinated LDL from adrenal gland membrane. At the highest concentration tested, un- labeled rat LDL reduced the amount of rat [125I]LDL bound by about 59%.

The pattern of displacement was very similar in the aorta (Fig. 1, upper panel). Human LDL displayed some ability to displace rat [125I]LDL, reducing the amount originally bound in the absence of unlabeled LDL by approx. 32%. However, unlabeled rat LDL was extremely effective in displacing iodinated rat LDL from aorta membranes. At the highest concen- tration, the displacement of labeled rat LDL was reduced by approx 88%.

In both the liver and heart membranes, the pattern of binding affinity was reversed, with human LDL displaying a greater capacity for displacing rat [125I]LDL than unlabeled rat LDL. Membranes from rat liver did not appear to have any affinity for homologous LDL (Fig. 2, upper panel). Human LDL was somewhat more effective, reducing the amount of rat [~2SI]LDL originally bound by approx 15% at the highest concentration tested. Membranes derived from rat heart once again displayed no significant af- finity for LDL of homologous origin, whereas human LDL was just slightly more effective in displacing iodinated rat LDL (Fig. 2, middle panel). At the high. est concentration tested, human LDL reduced the amount of rat LDL originally bound by approx 7%.

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Fig. 2. Binding of human and rat LDL to membrane frac- tions of rat skeletal muscle, heart and liver. Plasma mem- brane fractions of rat tissues were prepared as described in the text and incubated with the indicated concentrations of either human LDL or rat LDL, along with 10 pg/ml of rat [1251]LDL. The amount of specifically bound human (-O--O-) and rat (-0-0--) lipoprotein was determined as described in the test. Values are presented as the

mean + SD of four determinations.

LDL binding by different rat tissues 549

DISCUSSION

The present study was undertaken to evaluate whether different tissues of the rat have distinct affini- ties for binding homologous (rat) versus heterologous (human) LDL. Our results indicate that there is a definite difference amongst the five tissues tested. Both the adrenal gland and the aorta seem to have sites which bind rat LDL with high specificity. Human LDL even at very high concentration was comparatively ineffective in displacing rat LDL. On the other hand, in the liver and heart homogenates human LDL was slightly more efficient than rat LDL in displacing bound iodinated rat LDU However, the maximum amount of radiolabeled rat LDL which could be displaced by human LDL in these tissues was several orders of magnitude lower than the corre- sponding amount displaceable by rat LDL in the aorta and adrenal gland membranes. Thus, unlabeled rat LDL reduced the amount of rat [~25I]LDL bound to the adrenal gland and aorta membranes by about 59 and 88% respectively, whereas an equivalent con- centration of unlabeled human LDL displaced the amount of iodinated rat LDL bound to liver and heart membranes by only about 15 and 7~o, respect- ively. The skeletal muscle homogenate showed no dis- tinctive differences in the binding of human or rat LDL. In this tissue the amount of labeled rat LDL which could be displaced by the highest concentration of either rat or human LDL was nearly the same.

In the foregoing discussion, it is apparent that dif- ferent tissues of the rat may possess different affinities for binding LDL of different origin. The preference in the binding of rat LDL in the adrenal and aorta sug- gests that these two organs may have specific recep- tors which can interact primarily with the homolo- gous lipoprotein. The nature and function of these receptors are unclear, nevertheless, it is not unreason- able to speculate that they may be the counterpart of the LDL receptor in the human system. On the other hand, both the liver and the heart muscle showed a higher affinity for human rather than rat LDL. This preference for binding of heterologous LDL indicates that the sites of binding in these two tissues are prob- ably not highly specific. In fact, most of the binding may be due to non-specific adsorption. In this respect, Kovanen et al. (1979) have noticed that over 50% of the LDL bound onto bovine liver homogenate is of a non-specific nature.

Apolipoprotein B (apo B) and apolipoprotein E (apo E) have been shown to be the primary com- ponents recognized by LDL receptors in cells of a number of species including man (Brown et al., 1981; Mahley et al., 1979). Chemical analysis of the protein components in the rat 1.006 < d < 1.063 g/ml frac- tion indicate that both of these apolipoproteins may exist (Chapman, 1980). On the other hand, in the LDL (1.006 < d < 1.063) fraction of human serum, apo B is the major component while apo E exists probably only in minute quantity if at all (Osborne &

Brewer, 1977). Along this line of reasoning, the higher affinity of rat LDL for receptors in the adrenal and aortic homogenate may be due to the preference of the rat LDL receptors in these tissues to bind apo E. In support of this contention, Drevon et al. (1981) have observed that rat HDL, which contains no apo B (Chapman, 1980), was very effective in regulating the two enzymes, viz., HMGCoA reductase and ACAT, associated with the LDL-pathway. In another study, Kovanen et al. (1981) have reported that mem- branes derived from canine liver possess high affinity binding sites which recognize lipoproteins containing apo E with higher affinity than those containing only apo B. These results together with ours suggest strongly that if a counterpart of the LDL-pathway exists in the adrenal gland and aorta of the rat, the primary apolipoprotein involved in receptor recog- nition may be apo E rather than apo B.

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