The cyclic-ADP–ribose signaling pathway in human myometrium Eduardo N. Chini, * Claudia C.S. Chini, Hosana Barata da Silva, and Weronika Zielinska Department of Anesthesiology and Department of Internal Medicine, Mayo Clinic and Foundation, Rochester, MN 55905, USA Received 7 June 2002, and in revised form 15 August 2002 Abstract Human myometrial contraction plays a fundamental role in labor. Dysfunction of uterine contraction is an important cause of failure in progression of labor. The mechanisms of control of uterine contractions are not completely understood. It appears that intracellular Ca 2þ mobilization may play an important role during uterine contraction. Several mechanisms of intracellular Ca 2þ mobilization have been described. However, in human uterus only the inositol 1,4,5-trisphosphate-induced Ca 2þ release has been extensively studied to date. In view of the identification of the presence of functional ryanodine channels in myometrium, we ex- plored the role of the endogenous regulator of the ryanodine channel cyclic-ADP–ribose in human myometrial Ca 2þ regulation. Cyclic-ADP–ribose (cADPR) is a naturally occurring nucleotide implicated in the regulation of the gating properties of the ry- anodine channel, in fact cADPR may be a second messenger that activates the ryanodine receptor. Here we explore the components of the cADPR system in human myometrium. We found that human myometrium contains all the components of the cADPR pathway including (1) cADPR-activated microsomal Ca 2þ release and (2) enzymes responsible for synthesis and degradation of cADPR and, furthermore, that intracellular levels of cADPR were detected in human myometrial tissue. These data indicate that the cADPR system is present and operational in human myometrial tissue. Further research is warranted to determine the role of this new signaling molecule in uterine contraction. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: cADPR; IP 3 ; Endoplasmic reticulum; Ryanodine channel; NAADP Release of Ca 2þ from intracellular stores is a wide- spread component in several signaling pathways [7,9,12,13,15,17]. It is well known that inositol 1,4,5- trisphosphate (IP 3 ) 1 triggers Ca 2þ release from intra- cellular stores [12,13,15,17] however, cells possess other intracellular Ca 2þ releasing systems [4–7,9,12,13,15,17], including the so-called Ca 2þ -induced Ca 2þ release sys- tem, mediated by the ryanodine receptor/channel (RyR) [9]. Recently it was found that the endogenous nucleo- tide cyclic-ADP–ribose (cADPR) is a potent activator of the RyR [7,9,12,13,15,17]. Biosynthesis of cADPR from NAD is catalyzed by ADP–ribosyl cyclase, and cADPR is hydrolyzed by the cADPR hydrolyzed to ADP–ribose (ADPR) [7,9,12,13,15,17]. Recently, cADPR has been proposed to be the second messenger in several intra- cellular signaling pathways [7,9,12,13,15,17]. The presence of functional RyR in uterus from several species has been described before [1,3,18,21,22]. In addi- tion,itappearsthattheexpressionoftheRyRsubtypesin the myometrium changes during different stages of preg- nancy [1]; however, the mechanism of regulation of the RyR-mediated Ca 2þ release in myometrium is not well defined. We have previously shown that rat uterus can catalyze synthesis and degradation of cADPR [8]. Fur- thermore expression of the ADP–ribosyl cyclase is con- trolled by 17b-estradiol [8,11]. These data taken together raisethepossibilitythatcADPRmayplayaroleinuterine contractionviaactivationoftheRyR.However,nodirect evidence for the presence of functional cADPR-induced Ca 2þ releaseinuterushasbeenpresentedtodate.Herewe study the cADPR-induced Ca 2þ -releasing system in hu- man myometrium. We found for the first time that all components of the cADPR system are present in human Archives of Biochemistry and Biophysics 407 (2002) 152–159 www.academicpress.com ABB * Corresponding author. Fax: 1-507-284-8566. E-mail address: Chini.eduardo@mayo.edu (E.N. Chini). 1 Abbreviations used: cADPR, cyclic-ADP–ribose; RyR, ryanodine channel; NAADP, nicotinic acid adenine dinucleotide phosphate; IP 3 , inasitol 1,4,5-triphosphate; TCA, tricholoroacetic acid; HM, human myometrium; DTT, dithiothreitol; FMN, flavin mononucleotide. 0003-9861/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0003-9861(02)00486-1