INHIBITIOX OF CHOLESTEROLGENESIS
March 1970 TABLL I Average .\.I concentration required t o depolarize t o 45 mV
Compound
Acetylcholine ilcetylthiolcholine ilcet ylthionocholine
5 5 5
x x x
10-6 10-5
10-8
Equipotent .1! ratio
1 17 1.7
enzyme formed during the hydroly-i. of either . h C h or AcSCh. Electric eel acetylcholinebtera>e 1va.s used for these studies. The K , of the thiono e+ter was found to be M, compared with a Kmof 1 X for hcCh 6 X and 0.6 X for AcSCh. The I*,,, of the hydrolysis of acetylthionocholine was significantly lower than that of AcCh or AcSCh. Experimental Section Acetylthionocholine Bromide.--?;a (200 mg) was dissolved in 5.0 g of 2-dimethylaminoethanol, fullowed by the addition of a solution of 8.0 g of ethyl thionoacetateI8 in 100 ml of toluene. A slow stream of NZ was passed into the mixture which was heated at 97-98' for 2 hr. The reaction mixtiire was concentrated in vacuo in a 50" bat,h. The rehidue was acidified with 5 ml of concentrated HC1 in 70 ml of ice-cold H?O and the mixture was filtered. The filtrate was extracted with 200 ml of Et?O to remove unreacted ethyl thionoacetate. The aqueous layer was shaken with 35 ml of ice-cold saturated Na2C03, followed by extraction with 40-ml portions of Et,O. The organic extracts xere washed with H 2 0 and dried 0IplPO1). Addition of 2.0 ml of MeBr to t,he Et,O led, after refrigeration, t o the formation
215
of 2.4 g of light yellow crystals. The product was recrystallized from 2: 1 LLIe2CO-EtOH; mp 151-152';19 uv Amax (EtOH) 237 mp (e, 8440). A n a l . (C7HloBrKOS) C, 34.71; H, 6.65; S,13.23. Found: C, 34.81; H, 6.68; S, 12.82. Acetylthionothiolcholine Bromide.-This compound was obtained in 20% yield by the reaction of 6.0 g of 2-dimethylaminoethanethiol and 8.0 g of ethyl thionoacetate in toluene followed by quaternization with MeBr. After three recrystallizations from EtOH the product melted a t 169"; uv (EtOH) Amax 298 mp (emax 7830). 9nal. (C?HleBrNS2)C, H, S. Depolarizing Activity.-The depolarizing activity of acetylthionocholine was measured in the isolated single cell electroplax preparation, iising cell. from the electrir organ of E. electricus.20,21 Eserine was added to prevent hydrolysis by acetylcholinesterase present in the tissue. Hydrolysis by Acetylcholinesterase.-Highly purified electric eel acetylcholinesterase was used for these studies. Enzyme assays were carried out titrimetrically, using a Radiometer autotitrator. A constant p H of 7.5 =t0.02 was maintained during enzymatic hydrolysis by the automatic addition of 0.01 NaOH to neutralize the acetic acid produced by substrate hydrolysis. Initial rates were used, the rate being constant for a t least 2 min.
Acknowledgments.-We are indebted to A h . Eva Bartels of Columbia University for the electroplax assays and to Professor David Sachmansohn of Columbia University for a gift of the acetylcholinesterase used in these studies. This n-orli was supported by grants from the National Science Foundation (GB6833) and the Xational Institute for Seurological Diseases (XB-07835). (19) T h e melting point was determined with a Gallenkamp melting point apparatus a n d has been corrected. (20) E. Schoeffeniels, Biochim. Biophys. A c t a , 26, 585 (1957). (21) H. E.Higman and E. Bartels, ibid., 64, 543 (1062).
(18) U. Schmidt, E. Heymann, and 1;. Iiairitzke, C h r m . Be?.. 96, 1178 (1963). -
In Vitro Inhibition of Cholesterolgenesis by Various Thyroid Hormone Analogs C. D. ESKELSOS, C. R. CAZEE,IT,AXTHONY, J. C. TOWSE,,4ND B. R.
\yALSKE
Radioisotope SerL I C P , T'eterans Administration Hospital, Tztcson, Arzzona 85713 Received July 17, 1969 Nineteen thyroid hormone analog5 were tested in an in vitro cholesterolgenic liver homogenate rystem obtained from rats. "C-.\cetate was uhed as substrate, DMSO was used as a solvent for adding the thyroid hormone analogs to the system. Of thwe compoiinds tested, L-triiodothyronine, n- thyroxine, DI,-triiodoth\.ronine, at 1.0 x 10-4 M inhibited cholesterolgenesis from I4C-acetate substrate. and ~~-3,5-diiodo-3'-phenylthyronine No effect was elicited by Ta 01' T, when mevalonate was substrate. These st,iidies indicated that the 3'-I or a bulky 3'-Ph associated Kith either D- o r L-thyronine is necessary for inhibition of in vitro cholesterolgenesis. The D isomer is more active than the .I iwmer in this system. The need for higher than physiological levels of the thyroid hormones (triiodothyroniiLe or thyroxine) in this system is in part a resiilt of the nonspecific binding of the hormones to inert protein3 iu the homogenate system. When enoiigh hormone is present to saturate the binding sites on the inert protein3 rhe remaining hormone binds to active proteins contained in the microsomes resulting in an inhibition of chole.terolgenesis.
The alteration of blood chole-terol concentrations associated with thyroid function i y well kno~vn. The hypocholesterolemia associated with thyroxine administration is attributed in part to ;in increased conversion of cholesterol into bile acid> which overrides the increased cholesterolgene-i- cau-ed by thyroxine.' In thyroidectomized animal- a decreaye in the level of P-hydroxy-P-methylglutaryl-coenzyme -4reductase (HAIG-reductase) occurs wherea; the adminibtration of thyroxine increases the level- of HJIG-reductase re(1) D. Kritehevsky, Metabolism, 9, 984 (1964).
sulting in increased ch~lesterolgenesis.~~~ I n addition to the studies above, a new parameter was recently reported in which the addition of L-triiodothyronine (T3) and L-thyroxine (T4) to an in vitro cholesterolgenic rat liver homogenate from euthyroid rats resulted in decreased ch~lesterolgenesis.~The studies presented here are an extension of those studies and indicate that a structural specificity similar to that of TS and Td is (2) W. Gruder, I. Nolte, and 0. Wieland, Eur. J . Biochem., 4, 273 (1968). (3) F. 4.Gries, F. Matschinsky, and 0. Wieland, Biochim. Biophys. Acta. 66, 615 (1962). (4) C. D. Eskelson, Life Sci., 467 (1968).
3500
217
INHIBITION OF CHOLESTEROLGENESIS
March 1970
t
TABLE 1 THEEFFECT OF VARIOUSTHYROID AKALOGS O N in VifroCHOLESTEROLGENESW
/
Group No. 1
2 3 4
5 6 1 2 3
/,
,’-
e’
I
I
,
I
4
5 6
, 1 2 3
whereas the percentage binding to the protein fraction decreases from 44% to 8% a t the respective T3 concentrations. I n this system most of the T? binding sites on the soluble proteins are saturated a t 1 X lop3 JI Td, since the total T, bound to the proteins drops off significantly at this hormone concentration. The data for T, binding in Figure 4 are generally similar to those for the T, binding curve
