thapsigargin and Adenoma

The mechanism behind Ca(2)(+) entry into the parathyroid cells has been widely debated, and the molecular identities of the responsible ion channels have not been established yet. In this study, we show that the parathyroid cells lack voltage-operated Ca(2)(+) channels. Passive store depletion by thapsigargin, on the other hand, induces a large non-voltage-activated non-selective cation current. The increase in intracellular Ca(2)(+) caused by thapsigargin is attenuated by 2-aminoethoxydiphenyl borate, a blocker of store-operated Ca(2)(+) entry (SOCE). Candidate molecules for non-voltage-operated Ca(2)(+) signaling were investigated. These included members of the transient receptor potential canonical (TRPC) ion channel family, as well as Ca(2)(+) release-activated Ca(2)(+) modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1) that are key proteins in the SOCE pathway. Using RT-PCR screening, quantitative real-time PCR, and western blot, we showed expression of TRPC1, TRPC4, and TRPC6; Orai1; and STIM1 genes and proteins in normal and adenomatous human parathyroid tissues. Furthermore, co-immunoprecipitation experiments demonstrated a ternary complex of TRPC1-Orai1-STIM1, supporting a physical interaction between these molecules in human parathyroid.

Topics: Adenoma; Boron Compounds; Calcium; Calcium Channel Blockers; Calcium Channels; Cells, Cultured; Enzyme Inhibitors; Humans; Membrane Proteins; Neoplasm Proteins; ORAI1 Protein; Parathyroid Glands; Parathyroid Neoplasms; Stromal Interaction Molecule 1; Thapsigargin; TRPC Cation Channels

The response of the L-type Ca2+ current (ICa,L) in pituitary GH3 cells to variations in the action potential (AP) waveform was examined using the whole-cell configuration of the patch-clamp technique. ICa,L evoked during an AP waveform exhibited an early and a late component. The early component occurred on the rising phase of the AP; the late component coincided with the falling phase. Prolonging the falling phase of the AP increased the Ca2+ charge carried by ICa,L, although the amplitude of the late ICa,L was reduced. Prolonging the peak voltage of the AP waveform, however, increased the amplitude of the late component. ICa,L inactivated during a train of AP waveforms. When Ba2+ was used as the charge carrier, current inactivation during a train of APs decreased. Likewise, ICa,L evoked by the AP templates with irregular bursting pattern was inactivated. When the repetitive firing of APs with depolarizing potentials was replayed to cells, Ca2+ entry was not only spread over the entire AP, but also occurred during the interspike voltage trajectory. After application of thyrotropin releasing hormone (TRH; 10 microM), ICa,L in response to rectangular pulses was increased and the current/voltage relation shifted slightly to more negative values. TRH (10 microM), thapsigargin (10 microM) or cyclopiazonic acid (30 microM) enhanced the late component of the AP-evoked ICa,L. TRH also attenuated the inactivation of ICa,L during a train of APs. These results indicate that in pituitary GH3 cells, the time course and kinetics of ICa,L during the AP waveforms is distinct from that evoked by rectangular voltage clamp. Changes in the shape and firing pattern of APs in GH3 cells can modulate Ca2+ influx through L-type Ca2+ channels. Ca2+ release from internal stores may affect the magnitude of AP-evoked ICa,L in these cells.

Topics: Action Potentials; Adenoma; Animals; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Dantrolene; Enzyme Inhibitors; Indoles; Ionomycin; Ionophores; Muscle Relaxants, Central; Nifedipine; Patch-Clamp Techniques; Pituitary Neoplasms; Rats; Thapsigargin; Thyrotropin; Tumor Cells, Cultured