Signaling and Signal Transduction in Endocrine Tissues
(W741) THE IMPORTANCE AND REGULATION OF TrpC ION CHANNELS IN MYOMETRIAL SMOOTH MUSCLE.
Kim, Yoon-Sun1, Romero, Alejandro2, Ku, Chun-Ying1, Sanborn, Barbara1, 1 Colorado State University, Fort Collins, CO2 Duke University, Durham, NC
ABSTRACT- Contraction of myometrium is regulated by intracellular Ca2+ ([Ca2+]i). Increases in [Ca2+]i can be caused by release of Ca2+ from intracellular stores or by influx of extracellular Ca2+ through voltage-dependent Ca2+ channels (VDCC) and signal-regulated Ca2+ entry (SRCE) mechanisms. Myometrial cells exhibit SRCE in response to G-protein-coupled receptor stimulation, store-depletion and diacylglycerol. One of our goals is to determine the relative contributions of these two entry pathways to the change in [Ca2+]i and to contractile activity in myometrium. In order to do this, we assessed the relative specificity of inhibitors for these entry pathways. We used two immortalized human myometrial cell lines: PHM1 cells, which exhibit SRCE but do not respond to KCl depolarization and lack appreciable VDCC, and PHM2 cells, which exhibit SRCE but also respond to KCl with an increase in [Ca2+]i. In PMH1 cells, SKF96365, a putative SRCE blocker, inhibited oxytocin-stimulated and store-depletion SRCE, while verapamil, a VDCC blocker, had little effect on SRCE below 1uM. In PHM2 cells, 100 nM verapamil had little effect on store-depletion SRCE but completely inhibited KCl-stimulated Ca2+ entry. Conversely, 500nM SKF96365 inhibited store-deletion SRCE, but not KCl-stimulated Ca2+ entry. Myometrium expresses a number of TrpC channels implicated in SRCE, including TrpC3 and TrpC6. These proteins are activated by diacylglycerols (OAG); OAG stimulates cell-specific patterns of [Ca2+]i oscillations in PHM1 cells that require extracellular Ca2+ and are PKC-independent. In PMH1 cells, SKF96365 inhibited OAG-stimulated Ca2+ entry, while verapamil had no effect. Therefore, SKF96365 appeared quite specific for SCRE in myometrium. In estrogen-primed rat uterine strips, SKF96365 did not inhibit KCl-stimulated contraction but decreased frequency and amplitude of contractions induced by oxytocin. In contrast, the specific VDCC inhibitor nifedipine inhibited KCl-stimulated contraction but only weakly affected the amplitude of oxytocin-stimulated contractions at high concentrations. These data support the specificity of SKF96365 for SRCE and provide evidence that SRCE contributes to modulation of contractile properties in myometrium. Supported by HD38978 and R25DK067017.
KEY WORDS: myometrium, calcium, contraction, TrpCs