Expression of urocortin 2 and its inhibitory effects on intracellular Ca2+ via L-type voltage-gated calcium channels in rat pheochromocytoma (PC12) cells

Abstract

Urocortin 2, a new member of the corticotrophin-releasing factor (CRF) neuropeptide family, was reported to be widely expressed in the central nervous system and peripheral tissues. Here, we detected urocortin 2 mRNA in PC12 cells using reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, we observed its effects on intracellular Ca2+ concentration ([Ca 2+]i) using confocal microscopy and flow cytometry and on voltage-gated calcium channel (VGCC) currents using whole-cell patch clamp. Our results showed that urocortin 2 mRNA was coexpressed with CRF, and CRF receptor (CRFR) 2β in undifferentiated PC12 cells, but not CRFR1 or CRFR2α. KCl (40 mM) or Bay K8644 (1 μM), an L-type VGCC activator, increased [Ca 2+]i. Pretreatment of the cells with urocortin 2 significantly diminished the effect of Bay K8644 or KCl. Urocortin 2 showed no influence on [Ca2+]i in tyrode's solution containing EGTA or Ca2+-free tyrode's solution. It reversibly inhibited the VGCC currents in a concentration-dependent manner, but had no apparent effects on the cells treated with nifedipine (1 μM), an L-type VGCC blocker. Urocortin 2 up-shifted the current-voltage curves. No frequency-dependence of urocortin 2 effects on IBa was observed. The inhibitory effects of urocortin 2 on VGCC currents or [Ca2+]i were not affected by astressin 2B, an antagonist of CRFR2. As calcium overload play a key role in some neuronal degenerative diseases such as Alzheimer's and Parkinson's diseases, our results suggest that urocortin 2 may be a potentially interesting agent for the treatment of these diseases. © 2006 Nature Publishing Group All rights reserved.