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 Ca(2+) 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) 2beta in undifferentiated PC12 cells, but not CRFR1 or CRFR2alpha. KCl (40 mM) or Bay K8644 (1 microM), 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 [Ca(2+)](i) in tyrode's solution containing EGTA or Ca(2+)-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 microM), an L-type VGCC blocker. Urocortin 2 up-shifted the current-voltage curves. No frequency-dependence of urocortin 2 effects on I(Ba) was observed. The inhibitory effects of urocortin 2 on VGCC currents or [Ca(2+)](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.