Pharmaco/ogicalResearchCommunications, VoL 1~ No. Z 1984

PROTECTIVE EFFECT OF EPOMEDIOL ON CALCIUM RELEASE INDUCED BY PALMITOYL

CoA IN THE SARCOPLASMIC RETICULUM.

A. Bindoli, L. Cavallini and N. Siliprandi

Centre for the Study of Mitochondrial Physiology CNR and Institute of

Biological Chemistry of the University of Padova - Via Marzolo 3 - Padova

Receivedmfinal ~rm 13February1984

SUMMARY

Epomedioi, used at 1 mM concentration, does not modify the calcium move-

ments in the sarcoplasmic reticulum vesicles. Nevertheless it reduces by

about 35% the calcium release induced by 30 ~M palmitoyl CoA. This effect

can be attributed to the stabilizing action of epomediol on the sarcopla-

smic reticulum membrane.

647

INTRODUCTION

The sarcoplasmic reticulum (SR) is a membrane system present in the skele-

tal and cardiac muscle, attending to the regulation of the contraction

and relaxation of the muscl( ~ fiber.

The Caiion release by the SR vesicles and the subsequent reuptake contri-

bute, in a manner which seems to be determinant, to the oscillations of

the cytoplasmic concentration of the Ca-ions representing the signal for

the contraction and relaxation of the muscular fiber-cells.

The SR membrane system is an intracellular ramification of the plasma

membrane, with several chemico-physical and physiological characteristics

typical of the plasma membrane, but it is more suitable to investigate

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648 Pharmacolog~alResea~hCommun~at/ons, Vof f~ No. Z f984

some functional activities such as the Ca-ion accumulation and release.

For this reason skeletal muscle SR was chosen as biological material to

test "in vitro" the activity of epomediol (l,7,7-trimethyl-8-oxabicycle

2-2-2 ottan-2,6-diol) a drug with hepatokinetic activity (Campana" e

coll. 1981, Sabbatini e coll. 1982).

The action of palmitoyl CoA on calcium release from the SR was considered

an appropriate experimental condition to test the above activity. When

this metabolite exceeds a critical concentration in the tissue, it indu-

ces a Ca-ion release, the mechanism of which has not yet been completely

explained as well as its function. The high concentrations that the va-

rious acyl CoA can reach in certain pathological conditions such as ano-

xia (Shug, Shrago, Bittar, Folts and Koke, 1975; Whitemer, Idell-Wanger,

Rovetto and Neely, 1978) and the consequent functional and structural

lesions, allow the "in vitro" reproduction of lesions equal or similar to

the lesions which can occur "in vivo".

The present study was aimed at investigating the epomediol activity on

the Ca-ion release induced by palmitoyl CoA in the sarcoplasmic vesicles.

MATERIAL AND METHODS

Sarcoplasmic reticulum vesicles were isolated from rat skeletal muscle

following the method of Kawakita et al. (1980). The final pellet, suspen-

ded in 0.3 M saccharose, 0.i M KCI, I0 mM imidazole at pH 6.8, was frozen

*) CLESIDREN (R) - Camillo Corvi S.p.A.

PharmacologicalResea~hCommunications, VoL 1~ No. Z 1984

at -80°C. Proteins were measured following the method of Lowry et al.

(1951) using bovine serum albumin as standard.

Calcium movements (uptake and release) in the vesicles were measured with

the specific indicator arsenazo III (Scarpa, 1979) using 0.3 mg of SR

proteins/ml in a medium constituted by 0.i M KCI, 20 mM imidazole, 5 mM

phosphate, 50 ~M arsenazo III, 5 mM MgCI2, 5 mM ATP at pH 7.0 and 25°C.

The absorbance variations at 660-740 nm were measured using an AMINCO

double wavelenght spectrophotometer.

649

RESULTS AND DISCUSSION

As shown in Figure IA, the addition of 1 mM Epomediol to a SR suspension

before the addition of Ca-ions, does not modify the rate of Ca-ions accu-

mulation; similarly, when epomediol is added to the suspension after the

accumulation of all the Ca-ions, no release is observed. This result indi-

cates that neither the calcium pump activity, nor the SR membrane proper-

ties are modified by epomediol.

Figure IB evidences that the addition of 30 ~M palmitoyl CoA to the SR

vesicles previously loaded with Ca-ions, causes a release of these ions

at a rate of 90.2 nmoles/min/mg protein. This effect of palmitoyl CoA can

be due to an activation of a protein channel facilitating calcium release

or, more generally, to an increase of the SR membranes permeability due

to the interaction of the palmitoyl CoA molecule which is amphipatic and

has a negative charge (Cavallini, Siliprandi, Valente, Bindoli, 1982}.

6 5 0 Pharmacological Research Communications, VoL 16, No. 7, 1984

FIGURE 1

I CaCI. 2 + I~POMEDIOL _+ EPOHEDIOL

\

CaCI. 2

t CaCL 2

f 9a~// t

PALHITOYL Co A

T O.D. 0.1

L3.~ MIN.

t EPOMEDIOL 1

PALMITOYL Co A

Effect of epomediol on calcium movements in the sarcop!asmic reticulum.

The experimental conditions are described in "Materials and Methods". The

opticsl density decrease indicates calcium entry in the sarcoplasmic

reticulum vesicles, whilst the increase indicates calcium release. I00 ~M

CaCI2, 1 mM Epomediol and 30 ~M pm]mitcyl CoA were added at the times

indicated by the arrows.

When 1 mM Epomediol is added to the suspension before palmitoyl CoA (fi-

gure 1C) a decrease is noted in the rate of Ca-ion release (67.3 nmoles/

min/mg protein) as well as a decrease of the total concentration of rele-

ased Ca-ions.

PharmacologicalResearchCommunications, VoL I~ No. Z 1984

The results evidence that epomediol, at the used experimental conditions,

is able to protect the functional integrity of the membranes from the

action of a metabolite, palmitoyl CoA, which is potentially dangerous,

both specifically (at low concentrations) and because of its detergent

characteristics (at high concentration). Although the concentrations of

epomediol used in the present experiment is probably higher that those

attained "in vivo" upon oral administration of the drugtthe results seem

all the same significant in elucidating the stabilizing action of the

drug on SR membranes. In fact a direct competition between epomediol and

palmitoyl CoA (the compound used in these experiments for the induction

of membrane alterations) may be ruled out both for the reason that no

interactio~ occurs between the two compounds and for the high improbabili-

ty that epomediol could interfere in the membrane with the same sites of

palmitoyl CoA.

In conclusion, we can state that epomediol may act as "stabilizing agent"

of the functional integrity of the biomembranes since it appears to nor-

malize the SR membrane permeability when altered by the action of the

various acyl CoA. Considering the structure analogy of the SR membrane

with the plasma membrane, it can be reasonably supposed that epomediol

exerts a similar action also on the plasma membrane.

651

BIBLIOGRAPHY

Campana G., Petrali G.R., Guzzaloni A., Capretti G.(198]) Giorn. Ital.

Ric. Clin. Ter. 2, 115

Cavallini L., Siliprandi N., Valente M. and Bindoli A. (1982) 28 ° National

Congress of the Italian Society of Biochemistry, Firenze, p. 194-195

652 Pharmacological Research Communications, Vo/. !6, No. 7, !984

Kawakita H.~ Yasuoka K. and Kaziro Y. (1980) J. Biochem. 87, 609

Lowry O.H., Rosebrough N.J., Farr A.L. and Randall R.J. (1951) J. Biol.

Chem. 193, 265

Sabbatini F., D'Argenio G., Sollazzo R., Piai G. (1982) Rass. Intern.

Clin. Ter. 62, 1OO9

Scarpa A. (1979) Methods in Enzymology 56, 301

Shug A.L., Shrago E., Bittar N., Folts J.D. and Koke J.R. (1975) Am.

J. Physiol. 228, 689

Whitemer J.T., Idell-Wanger JoA., Rovetto H.J. and Neely J.R. (1978)

J. Biol. Chem. 253, 4305