Expression of urocortin 2 and its inhibitory effects on intracellular ca2+ via L-type voltage-gated calcium channels in rat pheochromocytoma (PC12) cells.
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.
Tao
Jin
J
Key Laboratory of Reproductive Medicine, Department of Pharmacology, Nanjing Medical University, Nanjing, PR China.
Zhang
Yuan
Y
Soong
Tuck Wah
TW
Li
Shengnan
S
D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000503:physiopathology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type / Q000187:drug effects* / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D003346:Corticotropin-Releasing Hormone / Q000235:genetics* / Q000494:pharmacology; D019610:Cytoprotection / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D005260:Female; D005434:Flow Cytometry; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018613:Microscopy, Confocal; D009433:Neural Inhibition / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009924:Organ Culture Techniques; D016716:PC12 Cells; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018019:Receptors, Corticotropin-Releasing Hormone / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics; D054832:Urocortins
C101531:CRF receptor type 2; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D012333:RNA, Messenger; D018019:Receptors, Corticotropin-Releasing Hormone; D054832:Urocortins; D003346:Corticotropin-Releasing Hormone; D002118:Calcium