Sulfation of minoxidil by multiple human cytosolic sulfotransferases

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Abstract

Minoxidil is an antihypertensive agent and hair growth promoter that is metabolized by sulfation to the active compound, minoxidil sulfate. Thermostable phenol sulfotransferase (TS PST or P-PST) was initially thought to catalyze the reaction, and the enzyme was designated minoxidil sulfotransferase (MNX-ST). Information about human ST activities toward minoxidil would be useful in developing the capacity to predict individual responses to minoxidil based on tissue levels of STs. Therefore, human STs were studied from platelet homogenates, partially purified platelets, scalp skin high speed supernatants and COS-1 cell cDNA expressed preparations using a radiochemical enzymatic assay with minoxidil as the substrate. Studies showed the presence of TS PST, TL (thermolabile) PST and MNX-ST activities in human scalp skin. Biochemical properties and correlation studies suggested that in addition to TS PST, the TL PST activity, another ST activity or both were involved in the reaction. Partially purified human platelet TL PST tested with minoxidil and dopamine showed identical thermal stabilities and similar responses to the inhibitors 2,6-dichloro-4-nitrophenol (DCNP) and NaCl. To characterize the activity of TL PST toward minoxidil, several biochemical properties of the enzyme expressed from a human liver cDNA clone were investigated. When assayed with minoxidil and dopamine, thermal stabilities of the expressed enzyme were identical and IC50 values for the inhibitors DCNP and NaCl were similar. It was also demonstrated that cDNA encoded human liver dehydroepiandrosterone sulfotransferase and estrogen sulfotransferase contributed to the sulfation of minoxidil. The results confirm that at least four human STs contribute to minoxidil sulfation. MNX-ST activity represents a combination of ST activities. The data indicate that multiple ST activities should be taken into account in attempts to predict the regulation of minoxidil sulfation and individual responses to minoxidil.

Introduction

Minoxidil (6-(1-piperidinyl)-2,4-pyrimidinediamine-3-oxide), an antihypertensive agent and hair growth promoter, is unique because it is activated by sulfate conjugation to the active metabolite minoxidil sulfate 1, 2, 3, 4, 5, 6, 7, 8. It was initially thought that thermostable phenol sulfotransferase (TS PST or P-PST) catalyzed the reaction (9, 10, Fig. 1). This activity was designated minoxidil sulfotransferase (MNX-ST). The active minoxidil sulfate is required for the hypotensive effect and for the stimulation of hair growth 3, 5, 8. Sulfotransferase activity toward minoxidil is localized in the monkey and rat hair follicle and skin 11, 12, 13but little is known about such activity in human skin.

Human platelet, liver and gut levels of PST activities show wide individual variations 14, 15, 16, 17, 18, 19. The platelet levels of TS PST have been particularly interesting because they are significantly correlated with the corresponding TS PST levels in the human liver, cerebral cortex and small intestinal mucosa 17, 18, 19. Thus, it should be feasible to use a test of the easily obtained blood platelet enzyme activity to reflect the potential drug metabolizing activity in other tissues of interest. Knowledge about human sulfotransferase activities toward minoxidil would be useful in developing the capacity to predict individual responses to minoxidil. Therefore, we carried out the studies reviewed in this manuscript to begin to test the hypothesis that a measure of the level of human blood platelet minoxidil sulfotransferase activity would predict an individual’s physiologic response to minoxidil. Additional questions addressed in the work determined whether there were sulfotransferase activities in human skin, whether these activities were the same as known platelet and liver sulfotransferase activities and whether any skin sulfotransferase activities were correlated with the relative platelet sulfotransferase activities. To approach these questions, human cytosolic sulfotransferases from platelet homogenates and partially purified platelet enzymes, scalp skin high speed supernatants and COS-1 cell cDNA expressed preparations were studied using a radiochemical enzymatic assay with minoxidil as the substrate 20, 21, 22, 23.

The emerging sulfotransferase classification and nomenclature eventually will ease the burden of identifying the enzymes being discussed 24, 25, 26. There are at least five characterized human cytosolic sulfotransferases; hTSPST1 (thermostable phenol sulfotransferase, SULT 1A1), hTSPST2 (SULT 1A2), hTLPST (thermolabile phenol sulfotransferase, SULT 1A3), hEST (estrogen sulfotransferase, SULT 1E1) and hDHEAST (dehydroepiandrosterone sulfotransferase, SULT 2A1). hTSPST1 is the major TS PST studied based on known substrate specificities and the use of the term TS PST will indicate hTSPST1. TL PST will be used for hTLPST, EST for hEST and DHEA ST for hDHEAST throughout this presentation.

Section snippets

Human scalp skin phenol sulfotransferases

Interest in the presence of phenol sulfotransferases in scalp skin has arisen due to the widespread use of topical minoxidil preparations for the treatment of androgenic alopecia 3, 11, 27. Detection and careful biochemical characterization of human scalp skin phenol sulfotransferases would be a necessary step toward determining whether a measurement of platelet PST activity would reflect or predict the scalp skin activity, and thus, the potential response of an individual to topical minoxidil.

Partially purified human platelet sulfotransferases

A confusing picture of the putative MNX-ST activity emerged. Thermal stability properties and sensitivities to DCNP were similar to TL PST activity behavior and there were significant positive correlations of MNX-ST activities with TL PST activities. Human platelet PST partially purified through DEAE anion exchange chromatography was used [20]to provide physically separated samples of TL PST and TS PST for testing with minoxidil as the substrate. When partially purified human platelet TL PST

Recombinant human liver sulfotransferases

The availability of cDNA clones of human cytosolic sulfotransferases provided the opportunity to test specific cDNA expressed sulfotransferases that were not contaminated with other similar enzymes 29, 30, 31, 32. COS-1 cells (an SV40 transformed simian cell line) were used for all expression studies. The cells were transfected by the calcium phosphate method [33]with the human liver cDNAs cloned into the EcoRI site of plasmid p91023(B) [34]. The cells were maintained and processed to high

Discussion

Minoxidil is widely used as a topical hair growth promoter and as an oral antihypertensive agent. Minoxidil sulfate is the active compound that leads to the therapeutic effect in both of these situations. Investigations of the sulfotransferases that catalyze minoxidil sulfation eventually may enhance the understanding of the regulation of the response to minoxidil treatment in alopecia and hypertension. Studies have shown that the phenol sulfotransferases TS PST and TL PST are present in human

Acknowledgements

We thank Gar Johnson, Ph.D., who, while with the Upjohn Company, contributed to these studies, and Christopher Huerter, M.D., for doing the scalp skin biopsies; Richard Weinshilboum, M.D. and Diane Otterness for the generous gifts of the human liver TL PST, DHEA ST and EST cDNAs; Christine Halgard for her assistance with these studies; and the Upjohn Company for providing the minoxidil and minoxidil sulfate. This work was supported by the VA Medical Research Service (RJA, DLC), Nebraska

References (47)

  • Y Yamazoe et al.

    Structural similarity and diversity of sulfotransferases

    Chem.-Biol. Interact.

    (1994)
  • V.H Price et al.

    Quantitative estimation of hair growth. I. Androgenetic alopecia in women: effect of minoxidil

    J. Invest. Dermatol.

    (1990)
  • T.C Wood et al.

    Human liver thermolabile phenol sulfotransferase: cDNA cloning, expression and characterization

    Biochem. Biophys. Res. Commun.

    (1994)
  • I.A Aksoy et al.

    Human liver estrogen sulfotransferase: identification by cDNA cloning and expression

    Biochem. Biophys. Res. Commun.

    (1994)
  • F.L Graham et al.

    A new technique for the assay of infectivity of human adenovirus 5 DNA

    Virology

    (1973)
  • M Bradford

    A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

    Anal. Biochem.

    (1976)
  • C.N Falany et al.

    Steroid sulfation by expressed human cytosolic sulfotransferases

    J. Steroid Biochem. Mol. Biol.

    (1994)
  • G.A Johnson et al.

    Minoxidil sulfate, a metabolite of minoxidil

    Drug Metab. Dispos.

    (1983)
  • W.A Pettinger

    Drug therapy: minoxidil and the treatment of severe hypertension

    N. Engl. J. Med.

    (1980)
  • J.M McCall et al.

    Pyrimidine and triazine 3-oxide sulfates: a new family of vasodilators

    J. Med. Chem.

    (1983)
  • R.N Earhart et al.

    Minoxidil-induced hypertrichosis: treatment with calcium thioglycolate depilatory

    South Med. J.

    (1977)
  • R.C Jacomb et al.

    The use of minoxidil in the treatment of severe essential hypertension: a report on 100 patients

    Clin. Sci. Mol. Med.

    (1976)
  • S.J Hirshey et al.

    Purification and characterization of rat liver minoxidil sulphotransferase

    Biochem. J.

    (1990)
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