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In single bovine aortic endothelial (BAE) cells pre-loaded with Fura-2, Ca2+transients in a Ca2+-free medium have been revealed, which evidently reflects Ca2+release from intracellular stores. In cells with different levels of resting basal cytoplasmic Ca2+([Ca2+]ifrom about 50 to 110 nM, a biphasic dependence of the Ca2+transients on resting [Ca2+]iwas shown and spontaneous Ca2+oscillations were observed. At a [Ca2+]ilevel over 110 nM, a pronounced rise in Ca2+transients occurred and only single transients were observed. Ryanodine (10 μM) produced a transient [Ca2+]ielevation, suggesting the presence of ryanodine receptors in intracellular store membranes. The results imply that both inositol 1,4,5-trisphosphate-sensitive Ca2+release (IICR) and Ca2+-sensitive Ca2+release (CICR) take place in BAE cells. Only IICR seems to be sufficient for generating baseline Ca2+oscillations in BAE cells, whereas the ATP-induced (5–100 μM) Ca2+response involves the CICR set in motion by an oscillatory IICR of high frequency. The completion of both the spontaneous and ATP-induced Ca2+transients was associated with a [Ca2+]idecrease to a level below the initial resting [Ca2+]i(undershoot). Its depth biphasically depended on the resting [Ca2+]ifrom 50 to 110 nM, suggesting that the lack of a Ca2+leak from inositol 1,4,5-trisphosphate-sensitive stores is responsible for the undershoot in this range. The Ca2+leak is concluded to play a key role in the initiation and termination of regenerative IICR both in spontaneous oscillations and in ATP-induced transients.

In many cells, activation of receptors coupled to PIP2turnover results in Ca2+release from the intracellular stores accompanied by Ca2+influx across the PM. It is not well established yet whether Ca2+influx is activated by IP3or by an unknown signal generated upon Ca2+store depletion. We report here a single‐channel study of low‐conductance IP3‐activated channels of very high selectivity for Ca2+in the PM of A431 carcinoma cells. The channels are strongly potential dependent and sensitive to [Ca2+]iwithin the physiological range. The data obtained argues for IP3acting directly on plasma membrane Ca2+channels.

Iminis a plasma membrane-located, Ca2+-selective channel that is activated by store depletion and regulated by inositol 1,4,5-trisphosphate (IP3). In the present work we examined the coupling between Iminand IP3receptors in excised plasma membrane patches from A431 cells. Iminwas recorded in cell-attached mode and the patches were excised into medium containing IP3. In about 50% of experiments excision caused the loss of activation of Iminby IP3.In the remaining patches activation of Iminby IP3was lost upon extensive washes of the patch surface. The ability of IP3to activate Iminwas restored by treating the patches with rat cerebellar microsomes reach in IP3receptors but not by control forebrain microsomes. The re-activated Iminhad the same kinetic properties as Iminwhen it is activated by Ca2+-mobilizing agonists in intact cells and by IP3in excised plasma membrane patches and it was inhibited by the Icracinhibitor SKF95365. We propose that Iminis a form of Icracand is gated by IP3receptors.

The patch-clamp technique was used to study mechanisms of ATP-induced Ca 2+influx in rat peritoneal macrophages. The experiments on whole-cell and patch membranes have shown that extracellular ATP activates channels permeable to di- and monovalent inorganic cations. Ratios of unitary channel conductances in 105 mM Ca 2+, Sr 2+, Mn 2+, Ba 2+and normal sodium solutions were 1.0, 0.95, 0.75, 0.55 and 0.85, respectively. The channels could open in the presence of non-hydrolyzable GTP analogues in artificial intracellular solution. The data are consistent with the hypothesis that a GTP-binding protein is involved in receptor-to-channel coupling.

Ca 2+-permeable channels in human carcinoma A431 cells were studied using the patch clamp technique. We have found two types of Ca 2+-penneable channels which are activated by inositol 1,4,5-trisphosphate (IP 3) applied to the intracellular side of the plasma membrane. Unitary conductances of these channels are 3.7 and 13 pS (105 mM Ca 2+in recording pipette, 30–33°C). From the extracellular side of the membrane the channels are activated by EGF. It is assumed that extracellular agonists open both channel types by stimulating the release of IP 3from the membrane.