100 uM ATP. Also, 100 uM UTP or UDP induced robust responses of 205 18% and 221 17%, respectively, while at 1 mM they generated increases of 216 37% and 183 23%, respectively. The results strongly suggested that P2Y receptors were able to activate a proliferative response in TIC. The possibility of regulatory cross talk between P2Y2 and LH receptors was also e amined. First, it Sorafenib B-Raf was shown that TIC responded to a 2 IU hCG stimulus by increasing CREB phosphorylation, and this response reached a ma imum in 10 min. This observation demon strated that the LH receptor efficiently activated the cAMP pathway in TIC cultures. It is well established that the cAMP PKA CREB pathway partici pates in the canonical signal transduction cascade for regulation of androgen biosynthesis through the LH receptor.

With this rationale, cultures of TIC were incubated in 0, 10, or 100 uM of UTP for 10 min, then 2 IU hCG were added for 10 min more, cell lysates were obtained, and CREB phosphorylation was evaluated by Western blot as an indicator of LH receptor activity. It was found that UTP, either 10 or 100 uM, completely blocked CREB phosphorylation, strongly suggesting that P2Y2 receptor stimulation inhibited the cAMP pathway activated by LH. This raises the possibility that the puri nergic system participates in regulating the physiological actions promoted by LH, for e ample, the steroid hor mones synthesis pathway. Discussion It has been recognized that neurotransmitters might play distinct, regulatory roles in ovarian physiology.

for e am ple, it has been proposed that, in addition to the regula tory actions of gonadotropins, the activity of sympathetic fibers that innervate the ovary controls different aspects of ovarian function, such as steroidogenesis, folliculogen esis, and ovulation ]. most of these stud ies have e amined the role of the catecholaminergic system and specifically, of norepinephrine, but there is also a great deal of important evidence for partic ipation of an ovarian cholinergic system. Although knowledge about the purinergic system in the ovary is scarce, it is well established that ATP and norepi nephrine are co released at similar concentrations from sympathetic terminals in many cell systems, and release of ATP by the oocyte has already been docu mented in other species.

Thus, the study in the ovary of the molecular components e pressed and cellu Anacetrapib lar mechanisms activated by the purinergic system will be of importance to understand the possible role of different ATP in ovarian physiology and pathology. Here, we present clear evidence of functional e pression of UTP sensitive P2Y receptors in TIC cultures, suggesting a role for these receptors in ovarian physiology. RT PCR and Western blot studies indicated that cul tured TIC e press P2Y2 and P2Y6 receptors. In func tional e periments, UTP and UDP, specific agonists for P2Y2 and P2Y6, respectively, induced robust Ca2 signals in normal Krebs or in Ca2 free solution, which indicated that the nucleotides