Human Eccrine Sweat Contains Tissue Kallikrein and Kininase II Toshihiko Hibino, Toshiyuki Takemura, and Kenzo Sato Marshall Dermatology Research Laboratories, Department of Dermatology, Universiry of Iowa Co llege of Medicine, Iowa Ciry, Iowa, U.S.A. We attempted to determine the level of sweat kallikrein kallikrein is the glandular type. Purified sweat and salivary (kininogenase) and to purify and characterize it using sweat kallikrein showed similar Mr and responses to inhibitors and collected over a white petrolatum barrier. Thermally induced antibodies. Using immunohistochemistry, kallikrein activity eccrine sweat obtained from 24 healthy subjects showed kal­ was localized in luminal ductal cells and in the peripheral rirn likrein activity of 24.4 ng kinins generated/1 mg of sweat of secretory coil segments, presumably the outer membrane protein when heated plasma was used as the substrate and of the myoepithelium. We also observed kininase activity in 16.1 ng kinin when purified low molecular weight bovine sweat at Mr 160,000, which was inhibited by ethylenedia­ kininogen was used as the substrate. Sweat was sequentially mine tetraacetic acid, captopril, and angiotensin converting purified by Sephacryl S-200, diethyaminoethyl Sephacel, enzyme inhibitor peptide, indicating that it is kininase II (or and fast flow liquid chromatography Mono Q chromatogra­ angiotensin converting enzyme). Sweat also contains abun­ phy. Sweat kallikrein had aMrof 40,000 and was inhibited by dant non-kallikrein hydrolases for S-2266 and S-2302. The aprotinin but not by soybean trypsin inhibitor. The peptide demonstration of glandular kallikrein, its tissue localization, generated by sweat kallikrein was identified as lys-bradykinin and the presence of kininase II in sweat provide the basis for using reverse phase high-performance liquid chromatogra­ future studies on the physiologic role of the kallikrein/kinin phy and by its amino acid sequence. Anti-human urinary system in the eccrine sweat gland. Key words: ki,tinoge1lase/ kallikrein immunoglobulin G neutralized the sweat kal­ sweat gland/protease. ] Invest Dermatol1 02:214 - 220, 1994 likrein activity completely, indicating that the sweat he eccrine sweat gland shares a number of features the presence of a kallikrein/kinin system in the eccrine sweat gland with other exocrine glands, especially with regard to is hard to dismiss. Fox and Hilton [16] first reported in 1958 that the mechanisms involved in the secretion of fluids, eccrine sweat and peri glandular dermal fluid contained a substance electrolytes, and nonelectrolytes [1 ,2]. Both the sali­ that contracted the rat uterus when such fluids were preincubated vary gland and saliva contain high concentrations of with pseudo-globulin. They interpreted the finding as indicating Tglandular kallikrein, a serine protease [3]. Kallikreins (or kinino­ that bradykinin was produced from pseudo-globulin by kallikrein in genases) are also present in a variety of other tissues and body fluids sweat or dermal fluid. Frewin et al [17] also observed weak kinin­ [4-8]. It has been postulated that glandular kallikrein serves many generating activity in sweat samples from some, but not all, subjects functions including the regulation of salivary glandular blood flow during exercise. Fraki et al [18] showed benzoyl-L-arginine ethyl through kinin generation [9], proteolytic processing of various ester (BAEE) - hydrolyzing, aprotinin-sensitive proteinase in growth factors and polypeptide hormones [10], and the regulation human sauna sweat, which suggests that sweat may contain kal­ of membrane transport also via kinin generation [11-13]. likrein-like enzymes. During the course of the present study, May­ Thermoregulatory eccrine sweating is associated with active re­ field et al [19] also observed that human sweat contains immunore_ flex vasodilation (ARV) [14]. In patients with hereditary anhidrotic active glandular kallikrein with kinin-generating activity by using ectodermal dysplasia where sweat glands are congenitally absent, radioimmunoassays for kallikrein and for generated kinin. Never­ ARV does not occur during heat exposure despite the fact that their theless, the kallikrein activity was absent or near the detection limit sympathetic innervation to cutaneous blood vessels appears to be of the ~ssay ~ystem. in some unconcentrated sweat samples. Further­ normal [15]. Whether or not the ARV during heat-induced sweat­ more, 111 their studies, sweat samples were not collected over a White ing is due to bradykinin produced by the sweat gland and the lack of petrolatum barrier and the sodium concentration in some of their ARV in anhidrotic ectodermal patients is due to the absence oflocal sweat samples was abnormally high [19]. Thus the possibility of bradykinin production are unknown. Nevertheless, evidence for epidermal contamination and evaporative water loss in some of their sweat samples cannot be entirely ruled out. The goals of the present study have been 1) to confirm the pres­ ence of kallikrein activity in clean sweat, 2) to determine whether Manuscript received March 19, 1993; accepted for publication September hydrolysis of synthetic substrates reflects the kallikrein activity in 2,1993. Reprint requests to: Dr. Kenzo Sato, Department of Dermatology, Uni­ sweat, 3) to determine that sweat kallikrein is of the glandular (or versiry ofIowa College of Medicine, 271 Medical Laboratories, Iowa Ciry, tissue) type, but not the plasma type, 4) to study whether or not IA 52242-1181. kinin-degrading enzyme(s) (i.e., kininase II) is also present in sweat, Abbreviations: ARV, active reflex vasodilation; HMW, high molecular and 5) to study the localization of kallikrein in the sweat gland using weight; HP, heated plasma; LMW, low molecular weight. immunohistochemistry . 0022-202X/94/S07.00 Copyright © 1994 by The Sociery for Investigative Dermatology, Inc. 214 VOL. 102, NO.2 FEBRUARY 1994 KALLIKREIN IN SWEAT 215 I: Q) 60 ~ 0 (TOc) ~ A n=24 ~52.5 ~ :!i ...I~ t 45 c 40 Ea;... _!:?C5 0 0 co. .210 -Q) 20 B -Sll)u~ 0 ~ o~ ... D..e Y=0.68X + 0.68 m::::" R=0.80, P<0.001 ~E .~~ 0 .- c .~~ n=5 ~~ 0 10 20 30 40 50 60 70 Kinin production from H.P. 4 B n=14 0 5-2266 A 5-2302 .... 1: 3 0 ft:l~:i! A ~ ~ LI--.----r---r--~--_,,_--r_--,_--_. ~S 0 r I OA 15 20 0 30 40 50 min 2 AO AO 0 80 II) • 0 C GI 0 ~ .. OA .. o fromH.P. ...ra i n=24 0 • from LMW K-gon - II 60 o iii .. • .Q ~ :::l~ .. f o (1).5 uCD 40 .:'0 0 CD-- • • .co. 0 10 20 30 40 50 60 70 0 o 0 ~ AI 0 Cra Kinin production from H.P. 20 0 -->-CD OJ. 0 II)~ 4 t • all) 0 5-2266 • • ~ • C n=14 0 rn~ A 5-2302 0 200 400 600 800 1000 Ui.E .?;os 3 0 Total sweat volume (mI/50minlm2) f:?o A "tie ~s 0 Figure 1. Kininogenase and kininase activity in thermally induced sweat 2 ~ 0 A from the back. The solid line in A is the temperature protocol used through­ 0 0 out the present sauna experiments. The mean sweat rate was derived from 0 ~ ~ ~ [20]. B) Time course of kallikrein (solid symbols) and kininase (open symbols) 0 A activity in sweat, expressed as ng kinin production from LMW kininogen or .. ng bradykinin degradation per ml of sweat/h, respectively. C) Kinin pro­ ~ A duction versus total sweat output (m1j50 min/m2 on the back) in 24 men, ages 18-30. tI, number of subjects. Open circles, kinin production from HP 0 and solid triallgles from bovine LMW kininogen. Kinin production was ex­ 0 10 20 30 40 50 60 pressed on a per-sweat protein basis (which is similar to that expressed on the Kinin production from LMW K-gen sweat volume basis because sweat protein content is similar in different sweat (ng/h/mg sweat protein) samples; not shown). Approximately 24.4 ± 15.4 ng of kinin was generated by 1 mg of sweat protein for the HP (used as substrate) and 16.1 ± 13.6 ng for bovine LMW kininogen (used as substrate), which correspond to 0.18 ng Figure 2. Kininogenase activity as determined using natural subrates versus and 0.16 ng of kinins generated by 1 ml of original sweat, respectively. synthetic substrates. A) Comparison of kinin generation from bovine LMW When EDTA and 1,10 phenanthroline were not included in the assay mix­ kininogen and human HP. B) Comparison of kinin generation from HP and ture for kinin, detection of kinin was dramatically decreased (data not hydrolysis of S-2266 (widely used substrate for glandular kallikrein) and shown), consistent with the observation that kininase is also present in 5-2302 (substrate for plasma kallikrein). C} Comparison of kinin generation human eccrine sweat (see B) . from bovine LMW kininogen and hydrolysis ofS-2266 and S-2302. Sweat samples from each subject were pooled and concentrated. The correlation between the hydrolysis ofS-2266 and that ofS-2302 is R = 0.90, P < 0.001, Y = 0.95X -0.12, not shown. MATERIALS AND METHODS Materials Trizma base, phenylmethyl sulfonyl fluoride (PMSF), diiso­ propylfluorophosphatc (DFP), l,lO-phenanthroline, trypsin (type III), pa­ ular weight (LMW) kininogen was purchased from Seikagaku Kogyou, pain (type III), benzoyl-arginine-p-nitroanilide (BANA), aprotinin, soybean Tokyo, Japan. Purified bovine pancreatic kallikrein was a gift of Showa trypsin inhibitor (SBTI), bradykinin, Iys-bradykinin (kallidin), angiotensin Denko Co. Ltd., Tokyo, Japan. StrAvigen was purchased from Biogenex converting enzyme (ACE) inhibitor (Gln-Trp-Pro-Arg-Pro-Gln-I1e-Pro­ Laboratories, San Ramon, CA. Rabbit antiserum to human urinary ka.Ilikrein Pro), and angiotensin I were obtained from Sigma Chemicals, St. Louis, was obtained from Japan Chemical Research, Kobe,Japan. Norit A was the MO. Ethylenediamine tetraacetic acid (EDTA) was purchased from MCB product of Fisher Scientific, Fair Lawn, NJ.