Futile cycling of a sulfate conjugate by isolated hepatocytes

• Published in: Molecular pharmacology Vol. 39; no. 3; pp. 414 - 420
• Main Authors: KAUFFMAN, F. C, WHITTAKER, M, ANUNDI, I, THURMAN, R. G
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Pharmacology and Experimental Therapeutics 01.03.1991
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Arylsulfatases - metabolism; Biological and medical sciences; Biological Transport - drug effects; Cell-Free System; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Glucuronates - metabolism; Hydrolases; Hymecromone - metabolism; In Vitro Techniques; Liver - metabolism; Microsomes, Liver - metabolism; Pregnenolone - pharmacology; Rats; Rats, Inbred Strains; Sulfates - metabolism; Sulfotransferases - metabolism; Rat; Enzyme; Rodentia; Glucuroconjugate; Futile cycle; Isolated organ; Sulfatation; Vertebrata; Mammalia; Enzymatic activity; Hepatocyte; Arylsulfatase; Conjugation
• ISSN: 0026-895X; 1521-0111

The sulfate conjugate of the model compound 4-methylumbelliferone was taken up and hydrolyzed considerably more rapidly by isolated hepatocytes than was the glucuronide conjugate. Using intact hepatocytes or homogenates of hepatocytes, compounds were identified that either inhibited 4-methylumbelliferyl sulfate hydrolysis via arylsulfatase or impaired its uptake into cells. For example, sodium sulfate inhibited hydrolysis of 4-methylumbelliferyl sulfate by intact hepatocytes (half-maximal inhibition, 0.1 mM) but not by homogenates, suggesting a selective action on organic sulfate uptake at the plasma membrane. In contrast, cholesterol sulfate inhibited hydrolysis of 4-methylumbelliferyl sulfate by homogenates but not by hepatocytes, consistent with the hypothesis that cholesterol sulfate does not readily enter intact cells. Compounds that inhibited hydrolysis of 4-methylumbelliferyl sulfate by both isolated hepatocytes and microsomes include sodium sulfite (half-maximal inhibition, 0.1 mM), pregnenolone sulfate (half-maximal inhibition, 1 microM), and estrone sulfate (half-maximal inhibition, 10 microM). To test whether production of sulfate conjugates could be modified by agents affecting arylsulfatase in intact hepatocytes, we examined the effects of pregnenolone sulfate on the production of 4-methylumbelliferyl sulfate from 4-methylumbelliferone. Addition of pregnenolone sulfate (100 microM) to intact cells increased rates of 4-methylumbelliferone sulfate production and decreased the fraction of 4-methylumbelliferone converted into the glucuronide. Hydrolysis of 4-methylumbelliferyl sulfate by isolated microsomes was inhibited in a dose-dependent manner by adenosine 3'-phosphate 5'-phosphosulfate (PAPS) when cytosol, a source of sulfotransferase was present. Furthermore, addition of low concentrations of PAPS (0.5 microM) to a reconstituted system of microsomes and cytosol impaired the formation of fluorescent product from 4-methylumbelliferyl sulfate until PAPS was consumed, indicating that futile cycling via arylsulfatase and sulfotransferase occurred. Subsequent futile cycling of free 4-methylumbelliferone and 4-methylumbelliferyl sulfate occurred upon repeated additions of PAPS and was prevented by sodium sulfite, an inhibitor of arylsulfatase. These results argue strongly that sulfate conjugate production within hepatocytes is regulated by futile cycling via sulfotransferase and arylsulfatase. Thus, drugs and endogenous substances that affect arylsulfatase may have marked effects on sulfate conjugate production by the liver.