title: Delayed autoregulation of the Ca2+ signals resulting from capacitative Ca2+ entry in bovine pulmonary artery endothelial cells.
abstract: 1. In calf pulmonary artery endothelial (CPAE) cells loaded with fura-2, the effects of ATP on Ca2+ entry were mediated entirely by the ability of P2U purinoceptors to stimulate InsP3 formation, empty intracellular Ca2+ stores and thereby activate capacitative Ca2+ entry. 2. Restoration of extracellular Ca2+ to cells with empty intracellular stores evoked transient increases in cytosolic [Ca2+] ([Ca2+]i) which then declined to an elevated plateau. These overshoots in [Ca2+]i were not a consequence of store refilling nor of desensitization of the capacitative pathway. Similar responses were recorded from cells in which Ca2+ uptake into mitochondria had been inhibited by microinjection of Ruthenium Red. The amplitudes of the capacitative Ca2+ signals decreased at lower extracellular [Ca2+], but [Ca2+]i invariably overshot before slowly declining to an elevated plateau. Even modest increases in [Ca2+]i therefore caused a delayed attenuation of the Ca2+ signal evoked by capacitative Ca2+ entry. 3. Modest pre-elevation of [Ca2+]i inhibited the ability of subsequent capacitative Ca2+ entry to further increase [Ca2+]i. The onset of the inhibition was slow (half-time (t1/2), approximately 100 s) and more tightly correlated with the preceding peak [Ca2+]i than with the [Ca2+]i immediately preceding Ca2+ entry. Recovery was also slow and complete only after [Ca2+]i had returned to its basal level for 320 +/- 3 s. 4. In thapsigargin-treated cells loaded with mag-fura-2, the peak [Ca2+]i that followed restoration of extracellular Ca2+ was accompanied by an abrupt approximately 2.5-fold decrease in the rate of Mn2+ entry, which then continued indefinitely at the reduced rate, demonstrating a rapid partial inactivation of the capacitative pathway. 5. The half-time for Ca2+ removal from the cytosol was significantly slower during the rising (t 1/2 = 22 +/- 2.5 s) than during the falling (t 1/2 = 7.1 +/- 0.7 s) phase of the Ca2+ overshoot evoked by addition of extracellular Ca2+ to thapsigargin-treated cells. 6. We conclude that an increase in [Ca2+]i rapidly inhibits the capacitative pathway and more slowly activates mechanisms that remove Ca2+ from the cytosol. Reversal of either or both of these regulatory mechanisms can occur only a considerable time after [Ca2+]i has been completely restored to its resting level. These mechanisms are likely to protect cells from excessive increases in [Ca2+]i and contribute to oscillatory changes in [Ca2+]i.
authors.affiliation: Department of Pharmacology, University of Cambridge, UK.
authors.forename: L
authors.initials: L
authors.lastname: Madge
authors.forename: I C
authors.initials: IC
authors.lastname: Marshall
authors.forename: C W
authors.initials: CW
authors.lastname: Taylor
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D002138:Calibration; D002417:Cattle; D002460:Cell Line; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2; D006207:Half-Life; D006706:Homeostasis / Q000502:physiology; D008345:Manganese / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D015220:Calcium Channels; D005456:Fluorescent Dyes; D008345:Manganese; D000255:Adenosine Triphosphate; D002118:Calcium; D016257:Fura-2