thapsigargin and inositol-1-3-4-5-tetrakisphosphate

We have previously shown that addition of Ins(1,3,4,5)P4 to permeabilized L1210 cells increases the amount of Ca2+ mobilized by a submaximal concentration of Ins(2,4,5)P3, and we suggested that, in doing this, Ins(1,3,4,5)P4 is not working via an InsP3 receptor but indirectly via an InsP4 receptor [Loomis-Husselbee, Cullen, Dreikhausen, Irvine and Dawson (1996) Biochem. J. 314, 811-816]. Here we have investigated whether this effect might be mediated by GAP1(IP4BP), recently identified as a putative receptor for Ins(1,3, 4,5)P4. GAP1(IP4BP) is a protein that interacts with one or more monomeric G-proteins, so we sought evidence for involvement of monomeric G-proteins in the effects of Ins(1,3,4,5)P4 in permeabilized L1210 cells. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) enhanced the effect of Ins(1,3,4,5)P4 on Ins(2,4, 5)P3-stimulated Ca2+ mobilization, but had no effect on the action of Ins(2,4,5)P3 alone. A specific enhancement of only the action of Ins(1,3,4,5)P4 was also seen with GTP[S]-loaded R-Ras or Rap1a (two G-proteins known to interact with GAP1(IP4BP)), whereas H-Ras was inactive at similar concentrations. Guanosine 5'-[beta-thio]diphosphate (GDP[S]) did not alter the action of either Ins(2,4,5)P3 or Ins(1,3,4,5)P4. Finally, the addition of exogenous GAP1(IP4BP), purified from platelets, markedly enhanced the effect of Ins(1,3,4,5)P4, and again, the amount of Ca2+ mobilized by Ins(2,4,5)P3 alone was unaltered. We conclude that the increase in Ins(2,4,5)P3-stimulated Ca2+ mobilization by Ins(1,3,4, 5)P4 may be mediated by GAP1(IP4BP) or a closely related protein (such as GAP1(m)), and if so, the action of the GAP1 is not solely to regulate GTP loading of a G-protein, but rather it acts with a G-protein to cause its effect.

Topics: Animals; Calcium; GTP-Binding Proteins; GTPase-Activating Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Inositol Phosphates; Mice; Proteins; ras GTPase-Activating Proteins; ras Proteins; Receptors, Cytoplasmic and Nuclear; Thapsigargin; Thionucleotides; Tumor Cells, Cultured

To identify the Ca2+ influx pathway responsible for maintaining Ca2+ oscillations in hamster eggs, changes in intracellular Ca2+ concentration ([Ca2+]i) were recorded using the Fura-2 fluorescent imaging technique during iontophoretic injection of inositol phosphates under voltage clamp. Both inositol 1,4,5-trisphosphate (InsP3) and 1,3,4,5-tetrakisphosphate (InsP4) caused repetitive Ca2+ transients when injected continuously into eggs, although the latter was much less effective. These Ca2+ transients were inhibited by the monoclonal antibody 18A10 to the InsP3 receptor/Ca2+ channel. In Ca(2+)-free medium, InsP4-induced Ca2+ transients were absent or much less frequent than in normal medium. A small but persistent increase in [Ca2+]i during InsP4 injection was revealed when Ca2+ uptake into InsP3-sensitive Ca2+ stores was suppressed by thapsigargin. This Ca2+ rise is due to Ca2+ entry, but not Ca2+ release, because it was: (i) increased by raising the extracellular Ca2+ concentration and abolished in Ca(2+)-free medium; (ii) larger at more negative membrane potentials which provide greater electrical driving force for Ca2+ entry; and (iii) not affected by 18A10. A moderate dose of InsP3 did not cause substantial Ca2+ entry, as tested in thapsigargin- and 18A10-treated eggs. InsP4 facilitated the restoration of Ca2+ stores after Ca2+ releases induced by pulsatile InsP3 injections. Thus, we obtained evidence for a Ca2+ influx pathway activated by InsP4 which provides Ca2+ to refill InsP3-sensitive Ca2+ stores in intact cells.

Topics: Animals; Antibodies, Monoclonal; Biological Transport, Active; Calcium; Calcium-Transporting ATPases; Cricetinae; Enzyme Inhibitors; Female; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Mesocricetus; Ovum; Terpenes; Thapsigargin

Ins(1,4,5)P3 3-kinase is a key enzyme in the regulation of Ins(1,4,5)P3. Overexpression of Ins(1,4,5)P3 3-kinase inhibited agonist-evoked and Ins(1,3,4,5)P4-evoked Ca2+ entry in Xenopus oocytes, but did not inhibit Ca2+ entry evoked by thapsigargin or non-metabolizable Ins(1,4,5)P3 analogues. The data suggest that Ins(1,4,5)P3 alone plays the crucial role in the activation of capacitative Ca2+ entry by emptying intracellular stores.

Topics: Animals; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Female; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Intracellular Fluid; Oocytes; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Terpenes; Thapsigargin; Xenopus

The effects of inhibitors of the intracellular Ca2+ release mechanism on divalent cation fluxes were examined in acinar cells loaded with the Ca(2+)-sensitive, Mn(2+)-quenchable dye, fura-2. TMB-8 and dantrolene (DTL) dramatically inhibited the carbachol (CCh)-stimulated increase in [Ca2+]i and Mn2+ influx. These agents do not directly inhibit divalent cation entry since addition of TMB-8 or DTL after CCh stimulation did not block Mn2+ influx. TMB-8 did not influence the [Ca2+]i increase or the Mn2+ influx produced by thapsigargin. These results indicate that TMB-8 and DTL do not interfere with divalent cation influx by inhibiting a step distal to depletion of the intracellular Ca2+ pool. TMB-8 and DTL did not significantly influence the muscarinic-stimulated production of inositol trisphosphate (IP3) and inositol tetrakisphosphate (IP4), although TMB-8, but not DTL, did decrease the CCh-stimulated 1,4,5-IP3 levels approximately 55%. The above results directly demonstrate that the filling state of the intracellular Ca2+ store primarily regulates the Ca2+ entry mechanism in sublingual mucous acinar cells.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium-Transporting ATPases; Carbachol; Dantrolene; Fura-2; Gallic Acid; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Male; Manganese; Mucous Membrane; Rats; Rats, Wistar; Sublingual Gland; Terpenes; Thapsigargin

Injection of D-myo-inositol-1,4,5-trisphosphate (IP3) was found to induce a transient increase of intracellular Ca2+ concentration in cancerous mammary cells (MMT060562) and in normal mammary cells treated with epidermal growth factor. Responses to injection of either D-myo-inositol-1,4-bisphosphate (IP2) or D-myo-inositol-1,3,4,5-tetrakisphosphate (IP4) were small or absent. Furthermore, normal mammary cells cultivated with low-protein serum replacement alone or in the presence of differentiation-inducing hormones (insulin + cortisol + prolactin) were less sensitive to IP3. Thapsigargin induced a transient increase of Ca2+ due to the release of Ca2+ from an intracellular pool. There was no difference in the peak heights of the thapsigargin-induced Ca2+ increase when mammary cells were cultivated in the presence or absence of epidermal growth factor or insulin + cortisol + prolactin. These findings suggest that the releasable intracellular Ca2+ pool remained unchanged whereas sensitivity to IP3 increases during the proliferation stage. Mechanical stimulus of a mammary cell induces an increase of intracellular Ca2+ in the stimulated cell. A certain stimulating factor is released from the mechanically stimulated cell into the extracellular space, and it induces an increase of Ca2+ in surrounding cells. In contrast, the IP3-induced Ca2+ increase in both cancerous and epidermal growth factor-treated normal mammary cells did not spread to adjacent cells. Therefore, increase of Ca2+ is not sufficient to account for the release of stimulating substances from mammary cells in the mechanically-induced spreading response.

Topics: Animals; Caffeine; Calcium; Cell Division; Cells, Cultured; Drug Resistance; Epidermal Growth Factor; Epithelial Cells; Epithelium; Female; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Mammary Glands, Animal; Mammary Neoplasms, Experimental; Mice; Mice, Inbred ICR; Pregnancy; Ryanodine; Terpenes; Thapsigargin; Tumor Cells, Cultured

Carbachol and histamine both stimulate calcium-dependent chloride secretion in the colonic epithelial cell line, T84. However, pretreatment of cell monolayers with carbachol blocks subsequent chloride secretion induced by thapsigargin but not the calcium elevation stimulated by this agent, whereas histamine pretreatment blocks neither thapsigargin-induced chloride secretion nor calcium elevation. To examine whether inositol phosphate metabolism might account for this difference, we measured levels of radiolabeled inositol phosphates: Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4, Ins-(1,3,4,6)P4, Ins(3,4,5,6)P4, InsP5, and InsP6 after cell stimulation. Although both carbachol and histamine increase Ins (1,4,5)P3 at 5 s, there is a greater and more persistent increase in the levels of Ins(1,3,4)P3 and InsP4 at later time points after carbachol than histamine, which corresponded to the suppression of the chloride secretory response.

Topics: Calcium; Calcium-Transporting ATPases; Carbachol; Cell Line; Chlorides; Colon; Dose-Response Relationship, Drug; Epithelial Cells; Epithelium; Histamine; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Signal Transduction; Terpenes; Thapsigargin