Members of both major families of intracellular Ca 2+ channels, ryanodine and inositol 1,4,5-trisphosphate (IP 3 ) receptors, are stimulated by substantial increases in cytosolic free Ca 2+ concentration ([Ca 2+ ] c ). They thereby mediate Ca 2+ -induced Ca 2+ release (CICR), which allows amplification and regenerative propagation of intracellular Ca 2+ signals. In permeabilized hepatocytes, increasing [Ca 2+ ] c to 10 μ M stimulated release of 30±1% of the intracellular stores within 60s; the EC 50 occurred with a free [Ca 2+ ] of 170±29nM. This CICR was abolished at 2°C. The same fraction of the stores was released by CICR before and after depletion of the IP 3 -sensitive stores, and CICR was not blocked by antagonists of IP 3 receptors. Ryanodine, Ruthenium Red and tetracaine affected neither the Ca 2+ content of the stores nor the CICR response. Sr 2+ and Ba 2+ (EC 50 = 166nM and 28 μ M respectively) mimicked the effects of increased [Ca 2+ ] on the intracellular stores, but Ni 2+ blocked the passive leak of Ca 2+ without blocking CICR. In rapid superfusion experiments, maximal concentrations of IP 3 or Ca 2+ stimulated Ca 2+ release within 80ms. The response to IP 3 was complete within 2s, but CICR continued for tens of seconds despite a slow [half-time ( t 1/2 ) = 3.54±0.07s] partial inactivation. CICR reversed rapidly ( t 1/2 = 529±17ms) and completely when the [Ca 2+ ] was reduced. We conclude that hepatocytes express a novel temperature-sensitive, ATP-independent CICR mechanism that is reversibly activated by modest increases in [Ca 2+ ], and does not require IP 3 or ryanodine receptors or reversal of the sarcoplasmic/endoplasmic-reticulum Ca 2+ -ATPase. This mechanism may both regulate the Ca 2+ content of the intracellular stores of unstimulated cells and allow even small intracellular Ca 2+ signals to be amplified by CICR.

The functional properties of the only inositol trisphosphate (IP 3 ) receptor subtype expressed in Drosophila were examined in permeabilized S2 cells. The IP 3 receptors of S2 cells bound (1,4,5)IP 3 with high affinity ( K d = 8.5±1.1nM), mediated positively co-operative Ca 2+ release from a thapsigargin-sensitive Ca 2+ store (EC 50 = 75±4nM, Hill coefficient = 2.1±0.2), and they were recognized by an antiserum to a peptide conserved in all IP 3 receptor subtypes in the same way as mammalian IP 3 receptors. As with mammalian IP 3 receptors, (2,4,5)IP 3 (EC 50 = 2.3±0.3 μ M) and (4,5)IP 2 (EC 50 approx. 10 μ M) were approx. 20- and 100-fold less potent than (1,4,5)IP 3 . Adenophostin A, which is typically approx. 10-fold more potent than IP 3 at mammalian IP 3 receptors, was 46-fold more potent than IP 3 in S2 cells (EC 50 = 1.67±0.07nM). Responses to submaximal concentrations of IP 3 were quantal and IP 3 -evoked Ca 2+ release was biphasically regulated by cytosolic Ca 2+ . Using rapid superfusion to examine the kinetics of IP 3 -evoked Ca 2+ release from S2 cells, we established that IP 3 (10 μ M) maximally activated Drosophila IP 3 receptors within 400ms. The activity of the receptors then slowly decayed ( t 1/2 = 2.03±0.07s) to a stable state which had 47±1% of the activity of the maximally active state. We conclude that the single subtype of IP 3 receptor expressed in Drosophila has similar functional properties to mammalian IP 3 receptors and that analyses of IP 3 receptor function in this genetically tractable organism are therefore likely to contribute to understanding the roles of mammalian IP 3 receptors.

Adenophostin A, the most potent known agonist of inositol 1,4,5-trisphosphate (Ins P 3 ) receptors, stimulated 45 Ca 2+ release from the intracellular stores of permeabilized hepatocytes. The concentration of adenophostin A causing the half-maximal effect (EC 50 ) was 7.1±0.5nM, whereas the EC 50 for Ins P 3 was 177±26nM; both responses were positively co-operative. In rapid superfusion analyses of 45 Ca 2+ release from the intracellular stores of immobilized hepatocytes, maximal concentrations of adenophostin A or Ins P 3 evoked indistinguishable patterns of Ca 2+ release. The Ca 2+ release evoked by both agonists peaked at the same maximal rate after about 375ms and the activity of the receptors then decayed to a stable, partially (60%) inactivated state with a half-time ( t 1/2 ) of 318±29ms for adenophostin A and 321±22ms for Ins P 3 . Dissociation rates were measured by recording rates of Ins P 3 -receptor channel closure after rapid removal of agonist. The rate of adenophostin A dissociation ( t 1/2 , 840±195ms) was only 2-fold slower than that of Ins P 3 ( t 1/2 , 436±48ms). We conclude that slow dissociation of adenophostin A from Ins P 3 receptors does not underlie either its high-affinity binding or the reported differences in the Ca 2+ signals evoked by Ins P 3 and adenophostin A in intact cells.