Specific binding of [3H]ouabain to heart is inhibited by long-chain fatty acids and alcohols

3ourd of Molecular and Cellular Cardiology (1980) 12, 847-856

Specific

Binding of [3H]0 ua b ain to Heart is Inhibited Long-chain Fatty Acids and Alcohols*

RYUNG

SOON (SONG) KIM?

AND

FRANK

by

S. LABELLA:

Department of Pharmacolog and Thrapeutics, Fact& of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada, R3E 0 W3 (Received 19 November 1979, accepted in revised farm 20 December 1979) R. S. KIM and F. S. LABELLA. Specific Binding of [3H]Ouabain to Heart is Inhibited by Long-chain Fatty Acids and Alcohols. 3ournal of Molecular and Cellular Cardiologv (1980) 12, 847-856. Long-chain fatty acids and alcohols inhibit specific binding of [sH]ouabain to a particulate fraction from dog heart. The magnitude of inhibition increases with chain length, reaching a maximum with lauric acid, myristoleic acid and decanol in a series of saturated fatty acids, unsaturated fatty acids, and alcohols, respectively, followed by a decline with longer chain members of homologous series. Unsaturated fatty acids and alcohols are more inhibitory than their corresponding saturated congeners; in general, inhibitory potency is greater in cis than tram forms and increases with higher degree of unsaturation. Methyl esters of both saturated and unsaturated fatty acids tested were inactive at 10d3 M or lower concentrations. The inhibition of ouabain binding by fatty acids and alcohols was specific and appears to be competitive. This study extends our earlier findings on the inhibitory activity of fatty acid fractions from bovine liver. KEY

WORDS:

Ouabain;

Specific

binding;

Heart;

Fatty

acids; Fatty

alcohols;

Liver

lipids.

1e Introduction In recent studies in our laboratory [14, 151, lipid fractions from bovine liver were found to inhibit specific binding of [3H]ouabain to dog heart particulate fraction rich in (Na+ + K+)-ATPase. In addition to an apparent potent neutral lipid [.2.5] inhibition of ouabain binding was shown to be due to the presence of longchain fatty acids (FA) and their methyl esters in the acidic fraction of lipid extract. In order to investigate the contribution of free FA to the observed inhibitory activity, a series of authentic saturated and unsaturated FA, their methyl esters, and saturated and unsaturated fatty alcohols§ were examined in the [3H]ouabain radioreceptor assay (RRA). Correspondence to: F. S. LaBella. * Supported by the Manitoba Heart Fbundation and the Richardson Foundation. 7 Dr Kim is a Postdoctoral Fellow of the Canadian Heart Foundation. $’ Dr LaBella is an MRC Career Investigator. 4 (Fatty) alcohols in this paper refer to straight-chain primary alcohols with chain length of c, to c,,. 0022-2828/80/090847+ 10 ros.oojo 0 1980 Academic Press Inc. (London) Limited

848

R. S. KIM

AND

2. Materials

F. S. LABELLA

and Methods

[3H]Ouabain RRA on dog heart particulate fraction was carried out as described previously [S, 181, and additional details are described in another paper [15]. Free FA and alcohols were obtained from Applied Science Laboratories, College Park, Pennsylvania, and solutions were freshly prepared prior to assay.

3. Results Effect offree fat9

acids and alcohols on [3H]ouabain

binding

Inhibition of [3H]ouabain binding by FA and alcohols was dependent not only on concentration but on chain length. Figure 1 shows the relationships between chain length of FA (a) and alcohols (b) and inhibition of binding. Among saturated acids, lauric acid (C,,) was most active, followed by Crs, Ci4, C,, acids in decreasing order. Other shorter (Cr5) acids had weak or no inhibitory activity at the highest concentration (1 mM) tested. Among the unsaturated acids maximum activity was obtained with myristoleic acid. Unsaturated FA were more potent inhibitors of [3H]ouabain binding than the corresponding saturated FA of identical chain length (C,, to C,,). This is illus(C 14:0), palmitic trated by comparing the effects of myristic (C,,:,) and stearic (C,,:,) acids with myristoleic (C&t), palmitoleic (C,,:,) and oleic acids (C&r) (Table 1). The degree of unsaturation also affected binding potency; unsaturated FA with two or more double bonds were more active than those with a single double bond in members of chain length up to C,,. Thus, linolenic (C,,:,) and linoleic (C,s:J acids were approximately equipotent, but both were more potent than oleic (C&t) acid. The cis form of unsaturated acids was consistently more potent than its corresponding tram form. All methyl esters tested, of both saturated and unsaturated FA, were inactive at 1O-3 M. The results are summarized in Table 1. Similar structure-activity relationships are observed with alcohols [Figure 1 (b)]. It is noted that the chain length activity curve for alcohols is shifted to the left of that for acids; thus, maximum inhibition of ouabain binding by the alcohol series occurred at C,, (decanol), compared with C,, for the fatty acid series. Unsaturated alcohols were more potent in inhibiting ouabain binding than the activity increased with degree of corresponding saturated congeners; inhibitory unsaturation as was observed for the unsaturated fatty acids. C&alcohols were slightly more potent than the corresponding tram forms. Concentration-displacement curves of ouabain (IC,, : 23 nM), lauric acid acid (IC,, : 0.27 mM), octanol (IC,, : 1 mM), and (I&l : 0.4 mM), myristoleic inactive stearic acid are shown in Figure 2. There is reasonable parallelism between active FA/alcohols and ouabain curves.

FATTY

ACIDS/ALCOHOLS

AND

OUABAIN

RECEPTOR

849

(a)

P I E

.-

0

I

1

6

12 Acid

d

1

I

I6

24

chain length

i (b)

I

0

6

I

I

16 12 Alcohol chain lrngth

I

24

FIGURE 1. Relationship between specific binding of [sH]ouabain (96 bound) and chain length of (a) free fatty acids and (b) alcohols. e---e, saturated fatty acids/alcohols. O--0, unsaturated fatty acids/alcohols. Only cis form with single dokble bond is shown. The total particulate fraction from 12.5 mg dog heart was incubated for 60 min at 37°C in 50 nm Tris-HCl buffer (pH 7.4) containing 150 nm NaCl, 1 mm EDTA, 1.25 rn~ MgCl,, 1.25 rn~ ATP (freshly added) and 9 nM [3H]ouabain (11 n/pmol) in a total volume of 1 ml. All test solutions were freshly prepared prior to use and added in 10 ~1 ethanol to give final concentrations of 10-s M. Ouabain standard and control test tubes had the same amount of ethanol. Specific binding of [SH]ouabain, represents the difference in bound counts in the presence and absence of expressed as s& control, 1.25 rn~ ATP. Details of assay procedures are described in the accompanying article of this journal. The mean values of two separate duplicate experiments, which did not vary more than 5%, are plotted in the graph.

850

R. S. KIM

AND

F. S. LABELLA

The characteristics of inhibition of ouabain binding by FA and alcohols were studied. Figure 3 shows saturation curves for specific binding of [3H]ouabain in the absence of competing substances (control), in the presence of octanol (2 mM), myristoleic acid (0.5 mM) and excess unlabelled ouabain (nonspecific binding). Double reciprocal plots of the above are shown in Figure 4. The result indicates that FA and alcohols inhibit specific binding of ouabain in a competitive manner.

TABLE

1. Comparison of [sH]ouabain alcohols (b) *

receptor

binding

Abbreviation

(a) Fatty

acids and fatty

activity

of fatty

Specific binding of [sH]ouabain (%)**

acid esters

Saturated Caproic acid Caprylic Capric Laurie N-Tridecanoic Myristic Pentadecanoic Palmitic Stearic Arachidic Behemic

G:ll

98 92 88 16 24 34 75 85 90 98 95

Gl(C) %:1(c) GM) Gxlw %:1(t) G7:z(c) Gs:z(t) G?:3w %3:1(c) Gz:l(c) C 24:1(c)

9 32 35 56 77 25 44 23 60 85 98

MC,,:, MC,,:, MCI,:, MG,:m

102 102 102 99

C 8%

Unsaturated Myristoleic Palmitoleic Palmitelaidic Oleic Elaidic Linoleic Linolelaidic Linolenic cis-5-Eicosaenoic Erucic Nervonic

Methyl esters Methyl Methyl Methyl Methyl

myristate palmitate stearate palmitoleate

acids

(a) and

FATTY

TABLE

ACIDS/ALCOHOLS

AND

OUABAIN

851

RECEPTOR

I-continued Abbreviation

Specific binding of [3H]ouabain (%)**

(b) Alcohols Saturated 1-Hexanol I-Octanol 1-Decanol 1-Dodecanol 1-Tetradecanol 1-Hexadecanol I-Octadecanol 1-Eicosanol I -Docosanol I-Tetracosanol

96 56 13 55 87 94 96 97 100 93

Unsaturated

c16:1w Clfd)

Palmitoleyl Palmitelaidyl Oleyl Elaidyl Linoleyl Linolelaidyl Linolenyl Eicosenyl Erucyl Nervonyl

88 87 93 99 44 57 48 92 95 94

C 18:1(c) Gsx(t) Gs:z(c) Gs:z(t) &x3(C) C 20:1(c) Gmw GdC)

(c) = cis; (t) = trans. * All test solutions were freshly prepared prior to use and added in 10 ~1 ethanol to give final concentrations of 10m3M. Ouabain standard and control test tubes had the same amount of ethanol. Specific binding of [3H]ouabain, expressed as o/0 control, represents the difference in bound counts in the prksence and absence of 1.25 XIIM ATP. ** Mean value of two separate duplicate experiments.

4. Discussion The

results

of the present

study

indicate

that

specific

binding

of [3H]ouabain

to

dog heart is inhibited by long-chain fatty acids (FA) and alcohols; inhibition is dependent on concentration and chain length. The maximum inhibiting activity in the saturated acid series occurs with lauric acid (C,,) and slightly less with tridecanoic (C,,) and myristic acid (C,,). The shorter or longer acids were weaker or inactive. The degree

of unsaturation,

as well

affects

inhibitory

potency;

for

example,

852

R.

S. KIM

AND

F.

S. LABELLA

myristoleic and palmitoleic acids were more active than their corresponding saturated congeners, myristic and palmitic acids. Among C,, unsaturated acids, linolenic and linoleic acids were much more potent than oleic acid. This general Log concentration -8 I

of ouabain (~1

-7 I

-6

I

Log concentration

I

of liplds (u)

stearic FIGURE 2. The effects of myristoleic acid (RIYR), 1auric acid (LAU), octanol (OCT) on pH]ouabain binding. Varying amount of lipids were incubated conditions to those described in Figure 1.

[‘HI

acid (STE) and in the identical

ouobain (nu)

FIGURE 3. Saturation curves of specific binding of [3H]ouabain. The indicated concentrations of [sH]ouabain were incubated’for 60 rnin at 37°C under identical conditions to those described in Figure 1. The specific binding, shown as solid lines, represents the difference in bound counts in the presence and absence of excess (1OV M) unlabelled ouabain, assayed in the absence of cornpeting substances (0) and in the presence of 2 rn~ octanol (v), or 0.5 rn~ myristoleic acid (a). Non-specific binding, assayed in the presence of excess unlabelled ouabain, is shown as dashed lines.

FATTY

0

0

ACIDS/ALCOHOLS

0.1 l/[3H]

AND

0.2 ouabain

OUABAIN

0.3

RECEPTOR

853

0.4

(nu)

FIGURE 4. Double reciprocal (Lineweaver-Burk) plots of the data from Figure 3. The reciprocal values of specific binding in the absence of competing substances (0, control), and in the presence of 2 mu octanol (v) or 0.5 no myristoleic acid (m) are plotted against the reciprocal values of [3H]ouabain.

pattern was also observed with alcohols, although the maximum activity was observed at decanol (C,,) rather than C,, as for the acid series. Investigations on the effects of chain length and unsaturation of FA in other biological systems [I, 2, 3, 10, 21, 2.51 report similar results. For example, anesthetic potency of a homologous series of organic compounds increases with increasing length of the hydrocarbon chain up to a point, and potency is abruptly reduced or abolished in longer members. Brink and Posternak [6] found that the straight chain primary alcohols showed a steady increase in anesthetic potency up to Cllr but tridecanql was nonanesthetic. Similar observations were made point occurs at decane. It was with alkanes [24] where the anesthetic “cut-off” suggested that the longer alkanes at high concentration do not exist in true solution (molecular dispersion) but are present as aggregates in suspension. There appears to be an optimum lipid solubility, therefore, for any particular set of anesthetic congeners [7]. However, there are indications that structural rather than solubility considerations must also be invoked for anesthetic action. Similar conclusions can be made from the present study of FA and alcohols with respect to inhibitory activity on [3H]ouabain binding. Short-chain FA (C, to C,,) induced narcosis in vivo and inhibited (Na+ + K+)ATPase in vitro; the two phenomena were shown to be related [9]. With increasing chain length, a close parallelism existed between enhanced enzyme inhibition and increased narcotic potency. It was suggested that both phenomena result from an ordered array of FA molecules along the membrane in association with membrane lipids, thereby interfering with movements of critical inorganic ions. These

854

R. S. KIM

AND

F. S. LABELLA

studies suggest that inhibition of (Na+ + K+)-ATPase may contribute to the arrhythmogenic action of local, high levels of FA which disturb ionic balance. A similar explanation was given for the inhibitory effect of palmityl-carnitine and palmityl-CoA on partially purified bovine heart (Na+ + K+)-ATPase [26]. The Lineweaver-Burk analysis indicates that inhibition of ouabain binding by FA and alcohols is of a competitive nature. It has been demonstrated that certain long-chain FA at low concentrations inhibited (Na+ + K+)-ATPase of rat brain microsomes [4] and guinea-pig heart microsomes [19]. As we observed for ouabain binding, Ahmed and Thomas [4] reported that the magnitude of inhibition of (Naf + K+)-ATPase increases with increasing chain length, reaching a maximum with myristate and declining with higher members. Unsaturated FA were more inhibitory than their corresponding saturated congeners. The inhibition was specific, reversible and competitive with K +. Attachment of FA to the enzyme appeared to be close to the K+ site but overlapped the Na+ site. It was suggested that the effects of FA on this enzyme are mediated by induction of conformational changes [ 101. Because (Naf + K+)-ATPase appears to be one important ouabain receptor [18], FA may normally be concerned with the regulation of ion transport. These lipid substances may represent just one type of endogenous factor influencing ouabain-receptors. In the present study, inhibition of ouabain binding by some FA and alcohols were obtained at concentrations less than 1 mM, in the range of values reported for normal human plasma [5, II]. Free fatty acids (FA) have been implicated as metabolic control agents in mammalian tissues [20, 21, 251. Toxic effects of high levels of Free FA have been ascribed to a non-specific detergent action on biomembranes, inhibition of enzymes, uncoupling of oxidative phosphorylation or stimulation of mitochondrial ATPase [13, 221. There is growing evidence of cause-effect relationships between elevated plasma free FA and cardiovascular disease states such as myocardial ischemia [23], cardiac arrhythmias [17], thrombosis, [ 121 and atherosclerosis [16]. Thus, studies on homologous series of alkanes, straight-chain alcohols and FA generally indicate that maximum biological effects are obtained with chain length of C,, to C,, for anesthetic/nerve block effects, inhibition of (Na+ + K+)ATPase, [3H]ouabain binding activity, and several metabolic activities. Furthermore, these lipid substances may subserve a variety of regulatory functions in the normal physiology of the individual, and may be responsible for a number of pathological conditions. REFERENCES 1.

K. & SCHOLEFIELD, P. G. Effects of fatty acids on the [32P]-adenosine triexchange reaction in rat liver mitochondria. Nature 186, 1046-1047 (1960). AHMED, K. & SCHOLEFIELD, P. G. Studies of fatty-acid oxidation: The effects of fatty acids on the phosphate metabolism of slice and mitochondrial preparation of rat liver. Biochemical Journal 81, 3745 (1961). AHMED,

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5. 6. 7.

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OUABAIN

RECEPTOR

855

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R. S. KIM AND F. S. LABELLA SCHWARTZ, A., WOOD, J.M., ALLEN, J. C., BORNET, E.P., ENTMAN, M.L., GOLDSTEIN, M. A., SORDAHL, L. A. & SUZUKI, M. Biochemical and morphological correlates of cardiac &hernia. The American Journal of Cardiology 32, 46-61 (1973). SEEMAN, P. The membrane actions of anesthetics and tranquilizers. Pharmacological Reviews 24, 583-655 (1972). TSUTSUMI, E. & TAKENAKA, F. Inhibition of pyruvate kinase by free fatty acids in rat heart muscle. Biochimica et biophysics acta 171, 355-357 (1969). WOOD, J. M., BUSH, B., PITTS, B. J. R. & SCHWARTZ, A. Inhibition of bovine heart Nat, K+-ATPase by palmityl carnitine and palmityl-CoA. Biochemical and Biophysical Research Communications 74, 677-684 (1977).