ELSEVIER Biochimica et Biophysica Acta 1224 (1994) 454-458 BB Biochi~ic~a et Biophysica A~ta Rapid Report Autoregulation of endothelin-1 secretion by cultured human keratinocytes via the endothelin B receptor Joseph J. Yohn a,d,e,*, Christopher Smith a Troy Stevens c, Joseph G. Morelli a, Libby R. Shurnas a, Sandra J. Walchak b, Talia A. Hoffman b, Kim K. Kelley d, Antonio Escobedo-Morse d, Masashi Yanagisawa f, Madeleine A. Kane b,d,e, Martin R. Zamora h a Department of Dermatology, University of Colorado School of Medicine, 4200 East Ninth Avenue, Denver, CO 80262, USA Department of Medicine, University of Colorado School of Medicine, Denver, CO 80262, USA c Department of Anesthesia, University of Colorado School of Medicine, Denver, CO 80262, USA d Denver Department of Veterans Affairs Medical Center, Denver, CO 80262, USA e University of Colorado Cancer Center, Denver, CO 80262, USA f Howard Hughes Institute, Southwestern University Medical Center, Dallas, TX 75235, USA Received 8 September 1994 Abstract We investigated endothelin-1 (ET-1) receptor expression on normal human keratinocytes (HK). We show that HK express the ET B receptor isoform and respond to ET-1 with a 2.7-fold increase in intracellular free calcium. HK did not respond to ET-I with increased proliferation; however, 30 nM ET-1 caused a 51.7% decrease in ET-I accumulation in HK-conditioned medium. We propose that HK ET-1 receptors function in autocrine regulation of ET-1 secretion. Keywords: Keratinocyte; Endothelin-1; Receptor; Calcium; (Human) Human keratinocytes (HK) are a rich source of peptide growth factors that maintain epidermal home- ostasis via both autocrine and paracrine mechanisms. Of significant interest are the mechanisms orchestrat- ing the complex interplay between peptide growth fac- tor secretion, growth factor receptor expression and biological responses to growth factors. There is evi- dence that human keratinocytes regulate their own rate of proliferation via secreted epidermal growth factor (EGF) [1], transforming growth factor alpha and beta [2], insulin-like growth factor [3,4], and basic fi- broblast growth factor [3]. Furthermore, HK play a direct role in the control of human melanocyte growth, melanin synthesis and melanin distribution to HK [5]. The peptide growth factor, endothelin-1 (ET-1), is secreted by HK [6,7] and functions in the regulation of melanocyte proliferation and pigmentation [8]. In cul- tured HK, the rate of ET-1 secretion is high during the * Corresponding author. Fax: + 1 (303) 2708272. 0167-4889/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4889(94)00186-3 first six hours with a dramatic decrease during the following 18 hours [7]. From these data we hypoth- esized that HK express ET-1 receptors and that ET-1 acts to negatively regulate HK ET-1 secretion. Addi- tionally, ET-1 is a growth factor for a variety of cell types [9-11]. We therefore sought to determine if normal HK respond to ET-1 with increased prolifera- tion. Normal human keratinocytes and melanocytes were isolated from neonatal foreskins and maintained in cell culture as previously described [7]. HK received fresh keratinocyte serum-free medium (KSFM, Gibco Labo- ratories, Grand Island, NY) every 72 h and human melanocytes received melanocyte serum-free medium (Gibco) on the same schedule as HK. Experiments were performed using first passage cells. For [125I]ET-1 binding experiments, cells were seeded onto 24-well plates, 1.0" 105 cells per well, and cultured for 48 h. The medium was removed and the cells were gently washed with 4°C pH 7.4 incubation buffer (IB) consisting of 50 mM Tris base, 0.09 mM