Depletion of the Ca2+ stores of A7r5 cells stimulated Ca2+, though not Sr2+, entry. Vasopressin (AVP) or platelet‐derived growth factor (PDGF) stimulated Sr2+ entry. The cells therefore express a capacitative pathway activated by empty stores and a non‐capacitative pathway stimulated by receptors; only the former is permeable to Mn2+ and only the latter to Sr2+. Neither empty stores nor inositol 1,4,5‐trisphosphate (InsP3) binding to its receptors are required for activation of the non‐capacitative pathway, because microinjection of cells with heparin prevented PDGF‐evoked Ca2+ mobilization but not Sr2+ entry. Low concentrations of Gd3+ irreversibly blocked capacitative Ca2+ entry without affecting AVP‐evoked Sr2+ entry. After inhibition of the capacitative pathway with Gd3+, AVP evoked a substantial increase in cytosolic [Ca2+], confirming that the non‐capacitative pathway can evoke a significant increase in cytosolic [Ca2+]. Arachidonic acid mimicked the effect of AVP on Sr2+ entry without stimulating Mn2+ entry; the Sr2+ entry was inhibited by 100 μM Gd3+, but not by 1 μM Gd3+ which completely inhibited capacitative Ca2+ entry. The effects of arachidonic acid did not require its metabolism. AVP‐evoked Sr2+ entry was unaffected by isotetrandrine, an inhibitor of G protein‐coupled phospholipase A2. U73122, an inhibitor of phosphoinositidase C, inhibited AVP‐evoked formation of inositol phosphates and Sr2+ entry. The effects of phorbol esters and Ro31‐8220 (a protein kinase C inhibitor) established that protein kinase C did not mediate the effects of AVP on the non‐capacitative pathway. An inhibitor of diacylglycerol lipase, RHC‐80267, inhibited AVP‐evoked Sr2+ entry without affecting capacitative Ca2+ entry or release of Ca2+ stores. Selective inhibition of capacitative Ca2+ entry with Gd3+ revealed that the non‐capacitative pathway is the major route for the Ca2+ entry evoked by low AVP concentrations. We conclude that in A7r5 cells, the Ca2+ entry evoked by low concentrations of AVP is mediated largely by a non‐capacitative pathway directly regulated by arachidonic acid produced by the sequential activities of phosphoinositidase C and diacylglycerol lipase.

We investigated the putative roles of phospholipase C, polyphosphoinositides, and inositol 1,4,5-trisphosphate (IP 3 ) in capacitative calcium entry and calcium releaseactivated calcium current (I crac ) in lacrimal acinar cells, rat basophilic leukemia cells, and DT40 B-lymphocytes. Inhibition of phospholipase C with U73122 blocked calcium entry and I crac activation whether in response to a phospholipase C-coupled agonist or to calcium store depletion with thapsigargin. Run-down of cellular polyphosphoinositides by concentrations of wortmannin that block phosphatidylinositol 4-kinase completely blocked calcium entry and I crac . The membrane-permeant IP 3 receptor inhibitor, 2-aminoethoxydiphenyl borane, blocked both capacitative calcium entry and I crac . However, it is likely that 2-aminoethoxydiphenyl borane does not inhibit through an action on the IP 3 receptor because the drug was equally effective in wildtype DT40 B-cells and in DT40 B-cells whose genes for all three IP 3 receptors had been disrupted. Intracellular application of another potent IP 3 receptor antagonist, heparin, failed to inhibit activation of I crac . Finally, the inhibition of I crac activation by U73122 or wortmannin was not reversed or prevented by direct intracellular application of IP 3 . These findings indicate a requirement for phospholipase C and for polyphosphoinositides for activation of capacitative calcium entry. However, the results call into question the previously suggested roles of IP 3 and IP 3 receptor in this mechanism, at least in these particular cell types.

Sazetidine-A has been recently proposed to be a "silent desensitizer" of ␣4␤2 nicotinic acetylcholine receptors (nAChRs), implying that it desensitizes ␣4␤2 nAChRs without first activating them. This unusual pharmacological property of sazetidine-A makes it, potentially, an excellent research tool to distinguish between the role of activation and desensitization of ␣4␤2 nAChRs in mediating the central nervous system effects of nicotine itself, as well as those of new nicotinic drugs. We were surprised to find that sazetidine-A potently and efficaciously stimulated nAChR-mediated dopamine release from rat striatal slices, which is mediated by ␣4␤2* and ␣6␤2* subtypes of nAChR. The agonist effects on native striatal nAChRs prompted us to re-examine the effects of sazetidine-A on recombinant ␣4␤2 nAChRs in more detail. We expressed the two alternative stoichiometries of ␣4␤2 nAChR in Xenopus laevis oocytes and investigated the agonist properties of sazetidine-A on both ␣4(2)␤2(3)and ␣4(3)␤2(2) nAChRs. We found that sazetidine-A potently activated both stoichiometries of ␣4␤2 nAChR: it was a full agonist on ␣4(2)␤2(3) nAChRs, whereas it had an efficacy of only 6% on ␣4(3)␤2(2) nAChRs. In contrast to what has been published before, we therefore conclude that sazetidine-A is an agonist of native and recombinant ␣4␤2 nAChRs but shows differential efficacy on ␣4␤2 nAChRs subtypes.

No abstract