237
Biochimica et Biophysica Acta, 436 (1976) 237--241
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA R e p o r t BBA 7 1 2 5 4
I N T E R A C T I O N OF MORPHINE WITH C H O L E S T E R O L MO N O L A Y E RS
J. P A B L O H U I D O B R O - T O R O * , M I T Z Y C A N E S S A * * and S I G M U N D F I S C H E R * * *
Department of Physiology and Biophysics, School of Medicine, University of Chile, Santiago (Chile) (Received F e b r u a r y 19th, 1976)
It appears that a first step in t he pharmacological response to a narcotic drug involves a stereochemical interaction of t he opiate to a m e m b r a n e component. The binding of narcotic drugs to a receptor substance present primarily in the synaptic m e m b r a n e fraction from brains of different animal species, as first described by Pert and Snyder [ 1 ] , has aroused m uch interest in the molecular mechanism o f action of the opiates. To study the effects of m o r p h in e on a model m e m b r a n e system, we investigated the interaction of mo r p h in e with cholesterol monolayers. This m e t h o d o l o g y seemed appropriate since drugs t hat are t h o u g h t t o p r o d u c e their pharmacological effect through modification of m e m br a ne c o m p o n e n t s have also been observed to affect artificial lipid membranes (for reviews see refs. 2 and 3). Surface tension was measured by a Cahn recording electrobalance (model RG) with surface tension a t t a c h m e n t and an X-Y recorder (Servogor). T h r o u g h o u t the experiments the t e m p e r a t u r e was maintained at 19 +- 1°C. Cholesterol monolayers were f or m ed in a 70 ml teflon bath by adding sequentially 1-t~l aliquots of freshly prepared cholesterol over a subphase of 150 mM NaC1, with a 50 pl Hamilton microsyringe. The m o n o l a y e r was c o m p leted in ab o ut 2 min. Morphine was studied under two different experimental conditions. It was added as the base, dissolved in a mixture of c h l o r o f o r m / m e t h a n o l (2:1) to the interphase, or as the hydrochl ori de dissolved in twice-distilled water (pH 5.5) to t he subphase. In all cases, the subphase was gently stirred with a magnetic bar after the addition of morphine, and th en the cholesterol m o n o l a y e r was formed. Control experiments were p e r f o r m e d adding the same volume of solvent alone to the interphase or subphase.
*To w h o m c o r r e s p o n d e n c e s h o u l d b e addressed at D e p a r t m e n t o f P h a r m a c o l o g y , U n i v e r s i t y of California, San Francisco, Calif. 94143, U.S.A. **Present address: L a b o r a t o r y o f R e n a l B i o p h y s i c s , M a s s a c h u s e t t s General Hospital and Department o f P h y s i o l o g y , Harvard M e d i c a l S c h o o l , B o s t o n , Mass. 02114, U.S.A. ***Present address: D e p a r t m e n t o f B i o l o g y , M a s s a c h u s e t t s I n s t i t u t e o f T e c h n o l o g y , Mass. 02139, U.S.A.
238 All the experimental data were processed by an 1130 IBM computer connected to the X-Y recorder output. The computer program utilized regression analysis to construct area vs. pressure curves from the combined results of 3--4 experiments. Thus, each experimental determination of the area per molecule of cholesterol represents the mean value obtained from at least 3--4 monolayers performed under identical conditions. The area per molecule of cholesterol (A 2/mol.) was calculated from the area vs. pressure curves at a constant pressure of 10 dynes/cm, assuming a molecular weight of 386 for cholesterol. The variability of the A 2/mol. for cholesterol in different determinations was less than 6%. In 8 control experiments, the values obtained for cholesterol in a 150 mM NaC1 subphase were between 46--52 A 2/mol., with a mean of 48 A 2/mol. Analytical grade salts and solvents were from Merck (Darmstadt, G.F.R.); cholesterol and Tris base from Sigma (St. Louis, Mo.); morphine base and hydrochloride were from Merck (Darmstadt, G.F.R.). Distilled water was deionized and re-distilled through an all-glass Corning apparatus. Surface tension values of 72.75 dynes/cm at 20°C verified the water purity. Fresh cholesterol solutions were prepared every 2--3 days in a mixture of chlorof o r m / m e t h a n o l (2:1). Salt solutions of the subphase were prepared daffy and buffered to the appropriate pH using Tris. HC1, 100 mM. Morphine base, when applied to the interphase of a 150 mM NaC1 solution (pH 7.4), strikingly increased the area per molecule of the cholesterol monolayers. As shown in Fig. 1, the effect followed a steep biphasic doseeffect relation. The maximal effect was observed at a final concentration of
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Biochimica et Biophysica Acta, 436 (1976) 237--241
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA R e p o r t BBA 7 1 2 5 4
I N T E R A C T I O N OF MORPHINE WITH C H O L E S T E R O L MO N O L A Y E RS
J. P A B L O H U I D O B R O - T O R O * , M I T Z Y C A N E S S A * * and S I G M U N D F I S C H E R * * *
Department of Physiology and Biophysics, School of Medicine, University of Chile, Santiago (Chile) (Received F e b r u a r y 19th, 1976)
It appears that a first step in t he pharmacological response to a narcotic drug involves a stereochemical interaction of t he opiate to a m e m b r a n e component. The binding of narcotic drugs to a receptor substance present primarily in the synaptic m e m b r a n e fraction from brains of different animal species, as first described by Pert and Snyder [ 1 ] , has aroused m uch interest in the molecular mechanism o f action of the opiates. To study the effects of m o r p h in e on a model m e m b r a n e system, we investigated the interaction of mo r p h in e with cholesterol monolayers. This m e t h o d o l o g y seemed appropriate since drugs t hat are t h o u g h t t o p r o d u c e their pharmacological effect through modification of m e m br a ne c o m p o n e n t s have also been observed to affect artificial lipid membranes (for reviews see refs. 2 and 3). Surface tension was measured by a Cahn recording electrobalance (model RG) with surface tension a t t a c h m e n t and an X-Y recorder (Servogor). T h r o u g h o u t the experiments the t e m p e r a t u r e was maintained at 19 +- 1°C. Cholesterol monolayers were f or m ed in a 70 ml teflon bath by adding sequentially 1-t~l aliquots of freshly prepared cholesterol over a subphase of 150 mM NaC1, with a 50 pl Hamilton microsyringe. The m o n o l a y e r was c o m p leted in ab o ut 2 min. Morphine was studied under two different experimental conditions. It was added as the base, dissolved in a mixture of c h l o r o f o r m / m e t h a n o l (2:1) to the interphase, or as the hydrochl ori de dissolved in twice-distilled water (pH 5.5) to t he subphase. In all cases, the subphase was gently stirred with a magnetic bar after the addition of morphine, and th en the cholesterol m o n o l a y e r was formed. Control experiments were p e r f o r m e d adding the same volume of solvent alone to the interphase or subphase.
*To w h o m c o r r e s p o n d e n c e s h o u l d b e addressed at D e p a r t m e n t o f P h a r m a c o l o g y , U n i v e r s i t y of California, San Francisco, Calif. 94143, U.S.A. **Present address: L a b o r a t o r y o f R e n a l B i o p h y s i c s , M a s s a c h u s e t t s General Hospital and Department o f P h y s i o l o g y , Harvard M e d i c a l S c h o o l , B o s t o n , Mass. 02114, U.S.A. ***Present address: D e p a r t m e n t o f B i o l o g y , M a s s a c h u s e t t s I n s t i t u t e o f T e c h n o l o g y , Mass. 02139, U.S.A.
238 All the experimental data were processed by an 1130 IBM computer connected to the X-Y recorder output. The computer program utilized regression analysis to construct area vs. pressure curves from the combined results of 3--4 experiments. Thus, each experimental determination of the area per molecule of cholesterol represents the mean value obtained from at least 3--4 monolayers performed under identical conditions. The area per molecule of cholesterol (A 2/mol.) was calculated from the area vs. pressure curves at a constant pressure of 10 dynes/cm, assuming a molecular weight of 386 for cholesterol. The variability of the A 2/mol. for cholesterol in different determinations was less than 6%. In 8 control experiments, the values obtained for cholesterol in a 150 mM NaC1 subphase were between 46--52 A 2/mol., with a mean of 48 A 2/mol. Analytical grade salts and solvents were from Merck (Darmstadt, G.F.R.); cholesterol and Tris base from Sigma (St. Louis, Mo.); morphine base and hydrochloride were from Merck (Darmstadt, G.F.R.). Distilled water was deionized and re-distilled through an all-glass Corning apparatus. Surface tension values of 72.75 dynes/cm at 20°C verified the water purity. Fresh cholesterol solutions were prepared every 2--3 days in a mixture of chlorof o r m / m e t h a n o l (2:1). Salt solutions of the subphase were prepared daffy and buffered to the appropriate pH using Tris. HC1, 100 mM. Morphine base, when applied to the interphase of a 150 mM NaC1 solution (pH 7.4), strikingly increased the area per molecule of the cholesterol monolayers. As shown in Fig. 1, the effect followed a steep biphasic doseeffect relation. The maximal effect was observed at a final concentration of
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