thapsigargin and inositol-1-4-5-trisphosphate-1-(2-nitrophenyl)ethyl-ester

By voltage clamp technique and intracellular calcium measurements, we recorded in prawn oocytes simultaneous [Ca2+]i and ionic current changes stimulated by external Mg2+. The [Ca2+]i response consists of an oscillation period followed by a second state of sustained [Ca2+]i level. The oscillation period successively comprises a first [Ca2+]i peak, a series of [Ca2+]i transients, and a [Ca2+]i oscillatory plateau respectively concurrent with an initial transient outward K+Ca current, an inward Na+Ca current, and a final K+Ca outward current. By using inhibitor (heparin) or sensitizers (thimerosal or caffeine) of calcium release ER channels, and caged InsP3, we established that InsP3 is the sole second messenger releasing Ca2+ from intracellular stores. By sequential substitutions and reapplications of external Ca2+, and using econazole (50 microM), a Ca2+ influx inhibitor, we documented Ca2+ influx during the [Ca2+]i oscillatory plateau. The intracellular Ca2+ store was depleted with thapsigargin (75-350 nM) in Ca2+-free ASW. Reapplication of external Ca2+ evoked a rise in [Ca2+]i, indicating a store-dependent capacitative Ca2+ influx, correlated with a K+Ca outward current increase. No measurable Ca2+ release-activated Ca2+ current (Icrac) could be detected, but was indirectly demonstrated using the sensitivity of the K+Ca channels to [Ca2+]i. We propose that the involvement of external Ca2+, in the physiological [Ca2+]i response of prawn oocytes to external Mg2+, consists of a store-dependent capacitative Ca2+ influx.

Topics: Animals; Caffeine; Calcium; Calcium Channels; Cell Membrane; Decapoda; Female; Heparin; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Magnesium; Microinjections; Oocytes; Patch-Clamp Techniques; Photolysis; Potassium Channels; Thapsigargin; Thimerosal

Depletion of intracellular calcium stores activates the plasma membrane capacitative calcium entry pathway in many cell types. The nature of the signal that couples the depletion of the intracellular calcium stores to the activation of the plasma membrane calcium influx pathway is as yet unknown. It has recently been suggested that a highly diffusible calcium influx factor is involved in the activation of capacitative calcium entry, and that its action is potentiated by the protein phosphatase inhibitor okadaic acid. Depletion of intracellular calcium stores in a localised region of a Xenopus oocyte was found to evoke capacitative calcium entry exclusively colocalised across the stimulated area of the plasma membrane, arguing against the involvement of a highly diffusible calcium influx factor. Equally, no evidence could be found for the presence of a soluble calcium influx factor in the bulk cytosol of Xenopus oocytes. The potentiation of capacitative calcium entry by okadaic acid resembled that mediated by the activation of protein kinase C, thus suggesting that okadaic acid activity may not necessarily be related to the action of a putative calcium influx factor.

Topics: Animals; Calcium; Calcium-Transporting ATPases; Cell Extracts; Cytoplasm; Diffusion; Electric Conductivity; Female; Injections; Inositol 1,4,5-Trisphosphate; Okadaic Acid; Oocytes; Protein Kinase C; Thapsigargin; Tissue Distribution; Xenopus laevis