Journal articles

In A7r5 vascular smooth muscle cells, Arg(8)-vasopressin (AVP) stimulates phospholipase C leading to activation of two distinct Ca(2+) entry pathways. The capacitative Ca(2+) entry (CCE) pathway is activated by depletion of Ca(2+) stores, is permeable to Mn(2+), Ba(2+) and Ca(2+), and is selectively blocked by Gd(3+)(1 microM). A7r5 cells also express a non-capacitative Ca(2+) entry (NCCE) pathway, which is activated by arachidonic acid that is released by the sequential activities of phospholipase C and diacylglycerol lipase.

Members of both major families of intracellular Ca(2+) channels, ryanodine and inositol 1,4,5-trisphosphate (IP3) 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 microM stimulated release of 30+/-1% of the intracellular stores within 60 s; the EC(50) occurred with a free [Ca(2+)] of 170+/-29 nM. This CICR was abolished at 2 degrees C.

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+/-1.1 nM), mediated positively co-operative Ca(2+) release from a thapsigargin-sensitive Ca(2+) store (EC(50)=75+/-4 nM, 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.

Recent results indicate that 'regulators of G-protein signalling' may contribute to the generation of receptor-specific patterns of cytosolic Ca2+ oscillations by associating with specific receptors, accelerating G-protein inactivation and responding to changes in cytosolic Ca2+.

Adenophostin A is the most potent known agonist of inositol 1,4,5-trisphosphate (InsP(3)) receptors. Ca(2+) release from permeabilized hepatocytes was 9.9 +/- 1.6-fold more sensitive to adenophostin A (EC(50), 14.7 +/- 2.4 nM) than to InsP(3) (145 +/- 10 nM), consistent with the greater affinity of adenophostin A for hepatic InsP(3) receptors (K(d) = 0.48 +/- 0.06 and 3.09 +/- 0.33 nM, respectively).

Synthetic analogues of inositol trisphosphate (IP(3)), all of which included structures equivalent to the 4,5-bisphosphate of (1,4,5)IP(3), were used to probe the recognition properties of rat full-length type 1, 2 and 3 IP(3) receptors expressed in insect Spodoptera frugiperda 9 cells. Using equilibrium competition binding with [(3)H](1,4,5)IP(3) in Ca(2+)-free cytosol-like medium, the relative affinities of the receptor subtypes for (1,4,5)IP(3) were type 3 (K(d)=11+/-2 nM)>type 2 (K(d)=17+/-2 nM)>type 1 (K(d)=24+/-4 nM).

Adenophostin A, the most potent known agonist of inositol 1,4, 5-trisphosphate (InsP(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+/-0.5 nM, whereas the EC(50) for InsP(3) was 177+/-26 nM; 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 InsP(3) evoked indistinguishable patterns of Ca(2+) release.

In cells expressing different receptors linked to Ins(1,4,5)P(3) formation, maximal stimulation of any one of them often releases all the Ins(1,4,5)P(3)-sensitive Ca(2+) stores, suggesting that Ins(1,4, 5)P(3) is used similarly by many receptors. In single HEK-293 cells, ATP and carbamylcholine (CCh) stimulated Ca(2+) release from intracellular stores via a pathway that was entirely dependent on Ins(1,4,5)P(3).

In HEK 293 cells stably expressing type 1 parathyroid (PTH) receptors, PTH stimulated release of intracellular Ca(2+) stores in only 27% of cells, whereas 96% of cells responded to carbachol. However, in almost all cells PTH potentiated the response to carbachol by about 3-fold. Responses to carbachol did not desensitize, but only the first challenge in Ca(2+)-free medium caused an increase in [Ca(2+)](i), indicating that the carbachol-sensitive Ca(2+) stores had been emptied.