Membrane fluidity and myotonia: effects of cholesterol and desmosterol on erythrocyte membrane fluidity in rats with 20,25-diazacholesterol-induced myotonia.
Bioscience Reports 4, 115-120 (1984) Printed in Great Britain
115
Membrane f l u i d i t y and myotonia: e f f e c t s
of c h o l e s t e r o l
and d e s m o s t e r o l on e r y t h r o c y t e m e m b r a n e f l u i d i t y in rats w i t h 2 0 , 2 5 - d i a z a c h o l e s t e r o l - i n d u c e d
myotonia
and on phospholipid l i p o s o m e s
3. ASHRAF*, 3. 5. FEIX**, and D. A. BUTTERFIELD% Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, U.S.A. (Received 9 December 1983)
Previous spin-label and electromyographic experiments with rats fed 20,25-diazacholesterol, an inhibitor of the biosynthetic conversion of desmostero[ to cholesterol, demonstrated an increased e r y t h r o c y t e membrane fluidity and myotonia, a prolonged muscle contraction upon stimulation. The current studies with rats showed normal erythrocyte fluidity in animals fed 20,25-diazacholesterol but maintained on a high-cholesterol diet and no myotonia. Studies of model membrane systems composed of p h o s p h o l i p i d v e s i c l e s c o n t a i n i n g desmosterol, cholesterol, or both demonstrated that desmosterol increased membrane lipid fluidity relative to c h o l e s t e r o l , s u g g e s t i n g t h a t in 20,25-diazacholesterol-induced myotonia, in which desmosterot accounts for g5% of the plasma sterol, the increased membrane fluidity previously observed in erythrocytes and sarcolemma m this animal model of human congenital myotonia may be due to desmosterol. Myotonia, a prolonged muscle contraction upon stimulation resulting from r e p e t i t i v e membrane depolarizations, occurs congenitally in humans (1) and animals (2-5) and can be induced in both by various chemicals (6). One of the chemicals by which myotonia can be induced in animals and man is 20,25-diazacholesterol (20,25-DC) (6-g), a potent reversible inhibitor of A2r the enzyme which catalyses the reduction of 2/~-dehydrocholesterol (desmosterol) to cholesterol. In rats made myotonic with 20,25-DC, desmostero] accumulates to g5% of the plasma sterol level (g). In both human congenital myotonia (CM) and 20,25-DC-induced myotonia a decreased chloride conductance and increased membrane resistance is observed, suggesting that the physiological basis of myotonia may be the same *Present address: Pituitary Hormones and Antisera Center, Harbor/UCLA Medical Center, Torrance, CA 90509, U.S.A. **Present address: National Biomedical ESR Laboratory, Medical College of Wisconsin, Milwaukee, WI 53226, U.S.A. %To whom correspondence should be addressed. ~1984
The Biochemical
Society
116
ASHRAF ET AL.
(9,10). In addition, a curare-resistant repetitive membrane depolarization is reported in myotonic rats (11), suggesting that similar to CM (9), the defect responsible is located in the muscle surface membrane. Spin-labeling studies in our laboratory of membrane lipids and proteins in e r y t h r o c y t e s obtained from rats made myotonic by t r e a t m e n t w i t h 20,25-DC suggested an increased membrane lipid fluidity with no changes in the physical state of membrane proteins (12). Similar spin-labeling results were also found in erythrocyte membranes from patients with CM (13), suggesting that both myotonic conditions might be assoc[afed w i t h diffuse membrane defects. Recently, others using fluorescence spectroscopy have also observed a more fluid lipid environment of both erythrocytes and sarcolemma from rats made myotonic with 20,25-DC (1#). The c u r r e n t e x p e r i m e n t s were performed to further study the possible correlation of increased desmosterol levels in the membrane and increased lipid fluidity with myotonia in this model of human CM. The results support the role of cholesterol replacement by desmosterol in membrane fluidity alterations in 20,25-De-induced myotonia. M a t e r i a l s and M e t h o d s
T h e s p i n - l a b e l u s e d was 2 - ( 3 - c a r b o x y p r o p y l ) - 2 - t r i d e c y l #,#-dimethyl-3-oxazolidinyloxyl (5-nitroxide stearic acid or 5-NS) and was o b t a i n e d from Syva. Desmosterol (2#-dehydrocholesterol) and p h o s p h a t i d y l c h o l i n e (egg) were o b t a i n e d from Applied S c i e n c e Laboratories and Supelco Inc. These compounds were sufficiently pure to use without further t r e a t m e n t (as indicated by thin-layer chromatograms furnished by the suppliers). Cholesterol used was obtained from Sigma Chemical Company and was recrystallized twice from methanol. 20,25-DC was kindly provided by G. D. Searle Co. All other reagents were of the highest purity available. Male Wistar rats weighing 230 grams were either Ied a diet of n o r m a l P u r i n a l a b o r a t o r y chow (1% cholesterol according to the m a n u l a c t u r e r ) or l a b o r a t o r y chow c o n t a i n i n g an additional 5% c h o l e s t e r o l by w e i g h t . Each group of rats were dosed daily by esophageal cannula with 30 mg of 20,25-DC per kg weight in one ml of distilled water. A third group of rats was fed normal Purina l a b o r a t o r y chow and dosed with one ml oI d i s t i l l e d water by esophageal cannula. Two rats from each group were studied by e!ectromyography at 2 and #89 weeks after initiation of the 20,25-DC t r e a t m e n t . EMG data were obtained and responses evaluated as previously described (12). After 589 weeks from the initiation of the experiment rats were s a c r i f i c e d by decapitation and the blood collected into heparinized syringes. Washed intact erythrocytes were prepared and spin-labeled with 5-NS as previously reported (12). Histological examination of the left gastrocnemius muscle immediately obtained after the blood was c o l l e c t e d from the s a c r i f i c e d rat was performed as described (12). Phospholipid liposomes were prepared according to the procedure described by Butler and Smith (15). Magnetic-resonance spectra were recorded on a Varian E-109 or Magnion-Ventron MVR-9X ESR spectrometer. M o d u l a t i o n and power broadening of spectral lines were avoided.
DESMOSTEROL AND MEMBRANE FLUIDITY
I17
T
HEIGHT m
i
I
89
..L
Fig. I. Half-width at half-height of the M I = +i low-field line of 5-NS in intact erythrocytes. Spectrometer conditons: scan range (40 G), modulation frequency and amplitude (I00 kHz and 0.32 G, respectively), and power incident on the resonance cavity (16 mW).
Results
M y o t o n i a was d e m o n s t r a t e d by electromyography in rats given 20,25-DC (12); however, myotonia could not be observed in 20,25DC-treated rats maintained on a high-cholesterol diet, in agreement with the results of others (16) (data not shown). In addition, no histological or histochemical alterations in the gastrocnemius muscle of each group of rats were detected, as also previously observed (12). L i p i d - s p e c i f i c , amphipathic spin labels like 5-NS used in these experiments are thought to be oriented in the lipid bilayer with its polar, charged carboxylic acid group near the phospholipid head group and its long alkyl chain on the average parallel to the chains of the lipids (17). Rapid anisotropic motion about the long axis of the probe with a t t e n d a n t rapid m o t i o n of t h e spin-label moiety through a restricted angle around its point of a t t a c h m e n t is though to occur. The resulting ESR s p e c t r u m is sensitive to this angular motion and p r o v i d e s a m e a s u r e of the lipid m e m b r a n e f l u i d i t y near the paramagnetic center of the spin label, via the order parameter (17) or half-width at half-height of the low-field line (18) (Fig. 1). An i n c r e a s e d membrane lipid fluidity in erythrocytes #ore rats made myotonic by 20,25-DC was previously observed in our laboratory (12). However, no alteration in the order parameter of 5-NS in e r y t h r o c y t e m e m b r a n e s from 20,25-DC-treated animals on a highcholesterol diet was observed in the present study compared to control animals (Table 1), suggesting no alteration in lipid fluidity in these rats on a cholesterol-supplemented regimen. Table i. Difference in the order parameters (S) of 5-NS in erythrocyte membranes* obtained from rats given H20 [control (C)] or 20,25-DC and with or without a high-cholesterol diet (HCD)**
Mean • S.E.M.
Sc-S20,25-DC 0.026•
N
9
P
