Inositol 1,4,5-trisphosphate-independent calcium signalling by platelet-derived growth factor in the human SH-SY5Y neuroblastoma cell.

In adherent SH-SY5Y human neuroblastoma cells, activation of G-protein-coupled muscarinic M3 receptors evoked a biphasic elevation of both intracellular [Ca(2+)] ([Ca(2+)]i) and inositol-1,4,5-trisphosphate (D-Ins(1,4,5)P3) mass. In both cases, temporal profiles consisted of rapid transient elevations followed by a decline to a lower, yet sustained level. In contrast, platelet-derived growth factor (PDGF), a receptor tyrosine kinase agonist acting via PDGF receptor b chains in these cells, elicited a slow and transient elevation of [Ca(2+)]i that returned to basal levels within 5 to 10 min with no evidence of inositol phosphate generation. Full responses for either receptor type required intracellular and extracellular Ca(2+) and mobilization of a shared thapsigargin-sensitive intracellular Ca(2+) store. Strategies that affected the ability of D-Ins(1,4,5)P3 to interact with the Ins(1,4,5)P3-receptor demonstrated an Ins(1,4,5)P3-dependency of the muscarinic receptor-mediated elevation of [Ca(2+)]i but showed that PDGF-mediated elevations of [Ca(2+)]i are Ins(1,4,5)P3-independent in these cells.     

Carbachol-stimulated calcium entry in SH-SY5Y human neuroblastoma cells: which route?

M3 muscarinic receptors expressed on SH-SY5Y human neuroblastoma cells are linked to phosphoinositide turnover and rises in [Ca2+]i. The rise in [Ca2+]i is biphasic with the peak phase being due to release from an intracellular Ins(1,4,5)P3-sensitive site and the plateau phase being due to Ca2+ entry across the plasma membrane. Ca2+ entry does not appear to involve voltage sensitive Ca2+ channels, a pertussis toxin sensitive G-protein-operated Ca2+ channel or Ins(1,4,5)P3/Ins(1,3,4,5)P4-operated Ca2+ channel. We suggest that carbachol-stimulated Ca2+ entry in SH-SY5Y human neuroblastoma cells occurs via receptor operated Ca2+ channels and through capacitive refilling.     

Inositol 1,3,4,5-tetrakisphosphate-induced release of intracellular Ca2+ in SH-SY5Y neuroblastoma cells.

Inositol-polyphosphate-induced Ca2+ mobilization was investigated in saponin-permeabilized SH-SY5Y human neuroblastoma cells. Ins(1,4,5)P3 induced a dose-related release from intracellular Ca2+ stores with an EC50 (concn. giving half-maximal effect) of 0.1 microM and a maximal release of 70%. Ins(1,3,4)P3, DL-Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5 did not evoke Ca2+ mobilization in these cells when used at concentrations up to 10 microM. However, Ins(1,3,4,5)P4 was found to release Ca2+ in a dose-related manner, but the response was dependent on the source of Ins(1,3,4,5)P4 used. When commercially available D-Ins(1,3,4,5)P4 was used, the EC50 and maximal response values were 1 microM and 50% respectively, compared with values for chemically synthesized DL-Ins(1,3,4,5)P4 of 2 microM and 25%. The enhanced maximal response of commercial D-Ins(1,3,4,5)P4 was decreased by pretreatment with rat brain crude Ins(1,4,5)P3 3-kinase and was therefore concluded to be indicative of initial Ins(1,4,5)P3 contamination of the Ins(1,3,4,5)P4 preparation. When metabolism of DL-Ins(1,3,4,5)P4 (10 microM) in these cells at 25 degrees C was investigated by h.p.l.c., substantial amounts of Ins(1,4,5)P3 (0.2 microM) and Ins(1,3,4)P3 (0.8 microM) were found to be produced within 3 min. Analysis of DL-Ins(1,3,4,5)P4 incubation with cells at 4 degrees C, however, indicated that metabolism had been arrested ([3H]Ins(1,4,5)P3 detection limits were estimated to be approx. 0.01 microM). When chemically synthesized DL-Ins(1,3,4,5)P4 and incubation conditions of low temperature were used, the Ca2(+)-releasing properties of this compound were established to be 1 microM and 19% for the EC50 and maximal response values respectively. The results obtained strongly suggest that Ins(1,3,4,5)P4 alone has the ability to release intracellular Ca2+. However, in the presence of sub-maximal concentrations of Ins(1,4,5)P3, Ca2+ release appears to be synergistic with Ins(1,3,4,5)P4, but at supramaximal concentrations not even additive effects are observed.     

Interaction of synthetic D-6-deoxy-myo-inositol 1,4,5-trisphosphate with the Ca2(+)-releasing D-myo-inositol 1,4,5-trisphosphate receptor, and the metabolic enzymes 5-phosphatase and 3-kinase.

The ability of D-6-deoxy-myo-inositol 1,4,5-trisphosphate [6-deoxy-Ins(1,4,5)P3], a synthetic analogue of the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], to mobilise intracellular Ca2+ stores in permeabilised SH-SY5Y neuroblastoma cells was investigated. 6-Deoxy-Ins(1,4,5)P3 was a full agonist (EC50 = 6.4 microM), but was some 70-fold less potent than Ins (1,4,5)P3 (EC50 = 0.09 microM), indicating that the 6-hydroxyl group of Ins(1,4,5)P3 is important for receptor binding and stimulation of Ca2+ release, but is not an essential structural feature. 6-Deoxy-Ins(1,4,5)P3 was not a substrate for Ins (1,4,5)P3 5-phosphatase, but inhibited both the hydrolysis of 5-[32P]+ Ins (1,4,5)P3 (Ki 76 microM) and the phosphorylation of [3H]Ins(1,4,5)P3 (apparent Ki 5.7 microM). 6-Deoxy-Ins (1,4,5)P3 mobilized Ca2+ with different kinetics to Ins(1,4,5)P3, indicating that it is probably a substrate for Ins (1,4,5)P3 3-kinase.     

Muscarinic-receptor-mediated changes in intracellular Ca2+ and inositol 1,4,5-trisphosphate mass in a human neuroblastoma cell line, SH-SY5Y.

This study reports increased intracellular Ca2+ and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to muscarinic-cholinergic stimulation of human neuroblastoma (SH-SY5Y) cells. Carbachol stimulation leads to a rapid increase in intracellular Ca2+ and Ins(1,4,5)P3 mass, both reaching a peak at around 10 s and then declining to a new maintained phase significantly above basal. Dose-response analysis of peak and plateau phases of intracellular Ca2+ shows different agonist potencies for both phases, carbachol being more potent for the plateau phase. The plateau-phase intracellular Ca2+ was dependent on extracellular Ca2+, which is admitted to the cell through a non-voltage-sensitive Ni2(+)-blockable Ca2+ channel. Using a Mn2+ quench protocol, we have shown that Ca2+ entry occurs early during the discharge of the internal stores. The plateau phase (Ca2(+)-channel opening) is dependent on the continued presence of agonist, since addition of atropine closes the Ca2+ channel and intracellular Ca2+ declines rapidly back to basal. We also failed to detect a refilling transient when we added back Ca2+ after intracellular Ca2+ had reached a peak and then declined in Ca2(+)-free conditions. These data strongly suggest that muscarinic stimulation of SH-SY5Y cells leads to a rapid release of Ca2+ from an Ins(1,4,5)P3-sensitive internal store and a parallel early entry of Ca2+ across the plasma membrane.     

Ca2(+)-mobilising properties of synthetic fluoro-analogues of myo-inositol 1,4,5-trisphosphate and their interaction with myo-inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase

The ability of two fluoro-analogues of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) to mobilize intracellular Ca2+ stores in SH-SY5Y neuroblastoma cells has been investigated. DL-2-deoxy-2-fluoro-scyllo-Ins(1,4,5)P3 (2F-Ins(1,4,5)P3) and DL-2,2-difluoro-2-deoxy-myo-Ins(1,4,5)P3 (2,2-F2-Ins(1,4,5)P3) were full agonists (EC50s 0.77 and 0.41 microM respectively) and slightly less potent than D-Ins(1,4,5)P3 (EC50 0.13 microM), indicating that the axial 2-hydroxyl group of Ins(1,4,5)P3 is relatively unimportant in receptor binding and stimulation of Ca2+ release. Both analogues mobilized Ca2+ with broadly similar kinetics and were substrates for Ins(1,4,5)P3 3-kinase but, qualitatively, were slightly poorer than Ins(1,4,5)P3. 2F-Ins(1,4,5)P3 was a weak substrate for Ins(1,4,5)P3 5-phosphatase but 2,2-F2-Ins(1,4,5)P3 was apparently not hydrolysed by this enzyme, although it inhibited its activity potently (Ki = 26 microM).     

Sphingosine 1-phosphate induces Ca2+ transients and cytoskeletal rearrangement in C2C12 myoblastic cells

In many cell systems, sphingosine 1-phosphate (SPP) increases cytosolic Ca2+ concentration ([Ca2+]i) by acting as intracellular mediator and extracellular ligand. We recently demonstrated (Meacci E, Cencetti F, Formigli L, Squecco R, Donati C, Tiribilli B, Quercioli F, Zecchi-Orlandini S, Francini F, and Bruni P. Biochem J 362: 349-357, 2002) involvement of endothelial differentiation gene (Edg) receptors (Rs) specific for SPP in agonist-mediated Ca2+ response of a mouse skeletal myoblastic (C2C12) cell line. Here, we investigated the Ca2+ sources of SPP-mediated Ca2+ transients in C2C12 cells and the possible correlation of ion response to cytoskeletal rearrangement. Confocal fluorescence imaging of C2C12 cells preloaded with Ca2+ dye fluo 3 revealed that SPP elicited a transient Ca2+ increase propagating as a wave throughout the cell. This response required extracellular and intracellular Ca2+ pool mobilization. Indeed, it was significantly reduced by removal of external Ca2+, pretreatment with nifedipine (blocker of L-type plasma membrane Ca2+ channels), and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+ pathway inhibitors. Involvement of EdgRs was tested with suramin (specific inhibitor of Edg-3). Fluorescence associated with Ins(1,4,5)P3Rs and L-type Ca2+ channels was evident in C2C12 cells. SPP also induced C2C12 cell contraction. This event, however, was unrelated to [Ca2+]i increase, because the two phenomena were temporally shifted. We propose that SPP may promote C2C12 cell contraction through Ca2+-independent mechanisms.     

Inositol tetrakisphosphate as a frequency regulator in calcium oscillations in HeLa cells

Cellular signaling mediated by inositol (1,4,5)trisphosphate (Ins(1, 4,5)P(3)) results in oscillatory intracellular calcium (Ca(2+)) release. Because the amplitude of the Ca(2+) spikes is relatively invariant, the extent of the agonist-mediated effects must reside in their ability to regulate the oscillating frequency. Using electroporation techniques, we show that Ins(1,4,5)P(3), Ins(1,3,4, 5)P(4), and Ins(1,3,4,6)P(4) cause a rapid intracellular Ca(2+) release in resting HeLa cells and a transient increase in the frequency of ongoing Ca(2+) oscillations stimulated by histamine. Two poorly metabolizable analogs of Ins(1,4,5)P(3), Ins(2,4,5)P(3), and 2,3-dideoxy-Ins(1,4,5)P(3), gave a single Ca(2+) spike and failed to alter the frequency of ongoing oscillations. Complete inhibition of Ins(1,4,5)P(3) 3-kinase (IP3K) by either adriamycin or its specific antibody blocked Ca(2+) oscillations. Partial inhibition of IP3K causes a significant reduction in frequency. Taken together, our results indicate that Ins(1,3,4,5)P(4) is the frequency regulator in vivo, and IP3K, which phosphorylates Ins(1,4, 5)P(3) to Ins(1,3,4,5)P(4), plays a major regulatory role in intracellular Ca(2+) oscillations.     

Edg-6 as a putative sphingosine 1-phosphate receptor coupling to Ca(2+) signaling pathway

The endothelial differentiation gene-6 (Edg-6) was recently identified as an orphan G-protein-coupled receptor. Its predicted amino acid sequence is very close to Edg family of receptor proteins whose ligand is supposed to be lysophosphatidic acid (LPA) or lysosphingolipid such as sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC). Transfection of the Edg-6 into Chinese hamster ovary (CHO) cells and K562 cells resulted in the appearance of high-affinity [(3)H]S1P binding activity. Among lipids employed, S1P and, even though less potent, SPC, displaced the [(3)H]S1P binding, but LPA was inactive. In Edg-6-transfected CHO cells, an increase in cytosolic Ca(2+) concentration in response to S1P or SPC was clearly enhanced without change in the LPA-induced action as compared with the vector-transfected cells. The enhancement of the Ca(2+) response was associated with a significant accumulation of inositol phosphate, reflecting activation of phospholipase C. Similar enhancement of Ca(2+) response to S1P or SPC was also observed in Edg-6-expressing K562 cells. These lipid-induced actions in CHO cells and K562 cells expressing Edg-6 were markedly suppressed by pertussis toxin treatment. We conclude that Edg-6 is one of S1P or lysosphingolipid receptors that couple to phospholipase C-Ca(2+) system through pertussis toxin-sensitive G-proteins.     

Inositol 1,3,4,5-tetrakisphosphate increases the duration of the inositol 1,4,5-trisphosphate-mediated Ca2+ transient

The effect of Ins 1,3,4,5-P4 on the intracellular Ca2+ mobilization produced by Ins 1,4,5-P3 has been examined in permeabilized hepatocytes. Ins 1,3,4,5-P4 did not affect the magnitude of the Ins 1,4,5-P3-mediated Ca2+ release but did inhibit re-accumulation of the released Ca2+ back into intracellular stores. This effect was not mimicked by Ins 1,3,4-P3. In hepatocytes, the re-uptake phase of the response results from Ins 1,4,5-P3 hydrolysis. Measurements using labeled substrates indicate that Ins 1,3,4,5-P4 inhibits the hydrolysis of Ins 1,4,5-P3 and vice versa. Since the removal of the 5-phosphate on Ins 1,4,5-P3 and Ins 1,3,4,5-P4 is a common step in the disposal of both compounds, it is suggested that one of the biological effects of Ins 1,3,4,5-P4 may be to slow hydrolysis of Ins 1,4,5-P3 and thereby prolong the duration of a Ca2+ transient.     

Ca2+/calmodulin-dependent translocation of sphingosine kinase: role in plasma membrane relocation but not activation

Activation of sphingosine kinase (SPHK), thereby increasing cellular levels of sphingosine 1-phosphate (S1P), may be involved in a variety of intracellular responses including Ca(2+) signaling. This study uses mammalian SPHK1a, tagged with enhanced green fluorescent protein (eGFP), to examine whether translocation of this enzyme is linked with Ca(2+)-mobilizing responses. Real-time confocal imaging of SPHK1a-eGFP in human SH-SY5Y neuroblastoma cells visualized a relocation of the enzyme from the cytosol to the plasma membrane in response to Ca(2+)-mobilizing stimuli (muscarinic M(3)- or lysophosphatidic acid receptor activation, and thapsigargin-mediated store release). This redistribution was preceded by a transient increase in cytosolic SPHK1a-eGFP levels due to liberation of SPHK from localized higher intensity regions. Translocation was dependent on Ca(2+) mobilization from intracellular stores, and was prevented by pretreatment with the Ca(2+)/calmodulin inhibitor W-7, but not W-5 or KN-62. In functional studies, pretreatment with W-7 lowered basal and M(3)-receptor-mediated cellular S1P production. However, this pretreatment did not alter agonist-mediated Ca(2+) responses, and SPHK1a-eGFP activity itself appeared insensitive to Ca(2+)/calmodulin and W-7. These data suggest a role for Ca(2+)/calmodulin in controlling the subcellular distribution but not the activity of SPHK1a.     

Ca(2+)-induced Ca2+ release in insulin-secreting cells.

The sulphydryl reagent thimerosal (50 microM) released Ca2+ from a non-mitochondrial intracellular Ca2+ pool in a dose-dependent manner in permeabilized insulin-secreting RINm5F cells. This release was reversed after addition of the reducing agent dithiothreitol. Ca2+ was released from an Ins(1,4,5)P3-insensitive pool, since release was observed even after depletion of the Ins(1,4,5)P3-sensitive pool by a supramaximal dose of Ins(2,4,5)P3 or thapsigargin. The Ins(1,4,5)P3-sensitive pool remained essentially unaltered by thimerosal. Thimerosal-induced Ca2+ release was potentiated by caffeine. These findings suggest the existence of Ca(2+)-induced Ca2+ release also in insulin-secreting cells.     

Inhibition of inositol trisphosphate-stimulated calcium mobilization by calmodulin antagonists in rat liver epithelial cells

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), an intracellular second messenger produced from the hydrolysis of phosphatidylinositol 4,5-bisphosphate, interacts with cytoplasmic membrane structures to elicit the release of stored Ca2+. Ins(1,4,5)P3-induced Ca2+ mobilization is mediated through high affinity receptor binding sites; however, the biochemical mechanism coupling receptor occupation with Ca2+ channel opening has not been identified. In studies presented here, we examined the effects of naphthalenesulfonamide calmodulin antagonists, W7 and W13, and a new selective antagonist, CGS 9343B, on Ca2+ mobilization stimulated by Ins(1,4,5)P3 in neoplastic rat liver epithelial (261B) cells. Intact fura-2 loaded cells stimulated by thrombin, a physiological agent that causes phosphatidylinositol 4,5-bisphosphate hydrolysis and Ins (1,4,5)P3 release, responded with a rise in cytoplasmic free Ca2+ levels that was dose dependently inhibited by W7(Ki = 25 microM), W13 (Ki = 45 microM), and CGS 9343B (Ki = 110 microM). Intracellular Ca2+ release stimulated by the addition of Ins(1,4,5)P3 directly to electropermeabilized 261B cells was similarly inhibited by pretreatment with anti-calmodulin agents. W7 and CGS 9343B, which potently blocked Ca2+/calmodulin-dependent protein kinase, had no significant effect on protein kinase A or C in dose range required for complete inhibition of Ca2+ mobilization. Ca2+ release channels and Ca2+-ATPase pump activity were also unaffected by calmodulin antagonist treatment. These results indicate that calmodulin is tightly associated with the intracellular membrane mechanism coupling Ins(1,4,5)P3 receptors to Ca2+ release channels     

Lysophosphatidic acid inhibits Ca2+ signaling in response to epidermal growth factor receptor stimulation in human astrocytoma cells by a mechanism involving phospholipase C(gamma) and a G(alphai) protein

The effect of the lysophospholipid mediators lysophosphatidic acid (LPA) and sphingosine 1-phosphate and the polypeptide growth factor epidermal growth factor (EGF) on the human astrocytoma cell line 1321N1 was assessed. These agonists produced a rapid and transient increase of the intracellular Ca(2+) concentration. When LPA was perfused before addition of EGF, the EGF-dependent Ca(2+) transient was abrogated, whereas this was not observed when EGF preceded LPA addition. This inhibitory effect was not found for other EGF-mediated responses, e.g., activation of the mitogen-activated protein kinase cascade and cell proliferation, thus pointing to the existence of cross-talk between LPA and EGF for only a branch of EGF-induced responses. As 1321N1 cells expressed mRNA encoding the LPA receptors endothelial differentiation gene (Edg)-2, Edg-4, and Edg-7 and as sphingosine 1-phosphate did not interfere with LPA signaling, Edg-2, Edg-4, and/or Edg-7 could be considered as the LPA receptors mediating the aforementioned cross-talk. Attempts to address the biochemical mechanism involved in the cross-talk between the receptors were conducted by the immunoprecipitation approach using antibodies reacting with the EGF receptor (EGFR), phosphotyrosine, phospholipase Cgamma (PLCgamma)-1, and G(alphai) protein. LPA was found to induce coupling of PLCgamma-1 to the EGFR by a mechanism involving a G(alphai) protein, in the absence of tyrosine phosphorylation of both PLCgamma and the EGFR. These data show a cross-talk between LPA and EGF limited to a branch of EGFR-mediated signaling, which may be explained by a LPA-induced, G(alphai)-protein-mediated translocation of PLCgamma-1 to EGFR in the absence of detectable tyrosine phosphorylation of both proteins.     

Phosphatidylinositol 3-kinase regulates Ca2+ signaling in pancreatic acinar cells through inhibition of sarco(endo)plasmic reticulum Ca2+-ATPase

Calcium is a key mediator of hormone-induced enzyme secretion in pancreatic acinar cells. At the same time, abnormal Ca(2+) responses are associated with pancreatitis. We have recently shown that inhibition of phosphatidylinositol 3-kinase (PI3-kinase) by LY-294002 and wortmannin, as well as genetic deletion of PI3-kinase-gamma, regulates Ca(2+) responses and the Ca(2+)-sensitive trypsinogen activation in pancreatic acinar cells. The present study sought to determine the mechanisms of PI3-kinase involvement in Ca(2+) responses induced in these cells by CCK and carbachol. The PI3-kinase inhibitors inhibited both Ca(2+) influx and mobilization from intracellular stores induced by stimulation of acini with physiological and pathological concentrations of CCK, as well as with carbachol. PI3-kinase inhibition facilitated the decay of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) oscillations observed in individual acinar cells. The PI3-kinase inhibitors decreased neither CCK-induced inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] production nor Ins(1,4,5)P(3)-induced Ca(2+) mobilization, suggesting that the effect of PI3-kinase inhibition is not through Ins(1,4,5)P(3) or Ins(1,4,5)P(3) receptors. PI3-kinase inhibition did not affect Ca(2+) mobilization induced by thapsigargin, a specific inhibitor of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Moreover, SERCA blockade with thapsigargin abolished the effects of pharmacological and genetic PI3-kinase inhibition on [Ca(2+)](i) signals, suggesting SERCA as a downstream target of PI3-kinase. Both pharmacological PI3-kinase inhibition and genetic deletion of PI3-kinase-gamma increased the amount of Ca(2+) in intracellular stores during CCK stimulation. Finally, addition of the PI3-kinase product phosphatidylinositol 3,4,5-trisphosphate to permeabilized acini significantly attenuated Ca(2+) reloading into the endoplasmic reticulum. The results indicate that PI3-kinase regulates Ca(2+) signaling in pancreatic acinar cells through its inhibitory effect on SERCA.     

Synthetic D- and L-enantiomers of 2,2-difluoro-2-deoxy-myo-inositol 1,4,5-trisphosphate interact differently with myo-inositol 1,4,5-trisphosphate binding proteins: identification of a potent small molecule 3-kinase inhibitor.

The ability of two enantiomeric fluoro-analogues of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to mobilize intracellular Ca2+ stores in SH-SY5Y neuroblastoma cells has been investigated. (-)-D-2,2-difluoro-2-deoxy-myo-Ins(1,4,5)P3 [D-2,2-F2-Ins(1,4,5)P3] was a full agonist [EC50 0.21 microM] and slightly less potent than D-Ins(1,4,5)P3 [EC50 0.13 microM]. (+)-L-2,2-F2Ins(1,4,5)P3 was a very poor agonist, confirming the stereospecificity of the Ins(1,4,5)P3 receptor. D-2,2-F2-Ins(1,4,5)P3 mobilized Ca2+ with broadly similar kinetics to Ins(1,4,5)P3 and was a substrate for Ins(1,4,5)P3 3-kinase inhibiting Ins(1,4,5)P3 phosphorylation (apparent Ki = 10.2 microM) but was recognised less well than Ins(1,4,5)P3. L-2,2-F2-Ins(1,4,5)P3 was a potent competitive inhibitor of 3-kinase (Ki = 11.9 microM). Whereas D-2,2-F2-Ins(1,4,5)P3 was a good substrate for Ins(1,4,5)P3 5-phosphatase, L-2,2-F2Ins(1,4,5)P3 was a relatively potent inhibitor (Ki = 19.0 microM).     

Inositol 1,3,4,5-tetrakisphosphate stimulates calcium release from bovine adrenal microsomes by a mechanism independent of the inositol 1,4,5-trisphosphate receptor.

In bovine adrenal microsomes, Ins(1,4,5)P3 binds to a specific high-affinity receptor site (Kd = 11 nM) with low affinity for two other InsP3 isomers, Ins(1,3,4)P3 and Ins(2,4,5)P3. In the same subcellular fractions Ins(1,4,5)P3 was also the most potent stimulus of Ca2+ release of all the inositol phosphates tested. Of the many inositol phosphates recently identified in angiotensin-II-stimulated adrenal glomerulosa and other cells, Ins(1,3,4,5)P4 has been implicated as an additional second messenger that may act in conjunction with Ins(1,4,5)P3 to elicit Ca2+ mobilization. In the present study, an independent action of Ins(1,3,4,5)P4 was observed in bovine adrenal microsomes. Heparin, a sulphated polysaccharide which binds to Ins(1,4,5)P3 receptors in several tissues, inhibited both the binding of radiolabelled Ins(1,4,5)P3 and its Ca2(+)-releasing activity in adrenal microsomes. In contrast, heparin did not inhibit the mobilization of Ca2+ by Ins(1,3,4,5)P4, even at doses that abolished the Ins(1,4,5)P3 response. Such differential inhibition of the Ins(1,4,5)P3- and Ins(1,3,4,5)P4-induced Ca2+ responses by heparin indicates that Ins(1,3,4,5)P4 stimulates the release of Ca2+ from a discrete intracellular store, and exerts this action via a specific receptor site that is distinct from the Ins(1,4,5)P3 receptor.     

Auranofin dissociates chemotactic peptide-induced generation of inositol 1,4,5-trisphosphate from the subsequent mobilization of intracellular calcium in intact human neutrophils

Auranofin, an antiarthritic gold compound, modulates a number of chemotactic factor-induced inflammatory responses in human neutrophils. In order to unravel the mechanism involved, the present study investigated the effects of auranofin on early signal transduction events in these cells. Auranofin did not affect the chemotactic peptide (fMetLeuPhe)-induced formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), neither in the presence nor in the absence of extracellular calcium ions. In contrast, there was a progressive inhibition by auranofin on the fMet-Leu-Phe-induced mobilization of intracellular calcium. This demonstrates that auranofin can dissociate the generation of Ins(1,4,5)P3 from the subsequent release of intracellular calcium, perhaps by interfering with the intracellular binding of Ins(1,4,5)P3 to its receptor. In experiments performed in electro-permeabilized cells, however, a relatively high concentration of the drug failed to abolish the specific binding of Ins(1,4,5)P3. In addition, in the same system, auranofin also failed to abolish the Ins(1,4,5)P3-induced release of Ca2+. Consequently, auranofin-mediated dissociation of fMLP-induced Ins(1,4,5)P3 formation and intracellular calcium release can not be explained merely by an antagonistic effect of auranofin on the Ins(1,4,5)P3 receptor. Instead the interaction between auranofin and the plasma membrane seems to be an initial and important part of the mechanism by which this drug interferes with the transduction signalling system.     

Modulation of inositol(1,4,5)trisphosphate-sensitive calcium store content during continuous receptor activation and its effects on calcium entry.

Changes in intracellular Ca2+ concentration ([Ca2+]i) following the activation of muscarinic receptors with carbachol were studied in cells from the exocrine avian nasal gland that had been maintained in culture for 40-48 h. In these cells, the carbachol-induced sustained increase in [Ca2+]i could be further increased by the subsequent addition of thapsigargin. This increase was due to an additional release of intracellular Ca2+ and a corresponding further enhancement of Ca2+ entry. However, thapsigargin-sensitive and Ins(1,4,5)P3-sensitive stores appeared to be coincident and the initial carbachol stimulus was sufficient to completely empty these stores. It was concluded that the subsequent effect of thapsigargin was due to a partial refilling of the Ins(1,4,5)P3-sensitive stores despite the continued presence of agonist, an effect that was not the result of any decline in levels of cellular Ins(1,4,5)P3 or changes in the generation of Ins(1,3,4,5)P4, which were sustained throughout. Possible explanations for this refilling response include compartmentalization of intracellular Ins(1,4,5)P3, or a desensitization of the Ins(1,4,5)P3 receptor/Ca(2+)-release channel. Alternatively, the data are also compatible with a recently proposed kinetic separation of Ca2+ uptake and release sites. An important implication of this particular interpretation of our findings would be an apparent dependence of Ca2+ entry specifically on the status of the Ca(2+)-uptake component of the agonist-sensitive store, rather than the Ca(2+)-release component.     

Intercellular communication between follicular angiotensin receptors and Xenopus laevis oocytes: medication by an inositol 1,4,5- trisphosphate-dependent mechanism

In Xenopus laevis oocytes, activation of angiotensin II (AII) receptors on the surrounding follicular cells sends a signal through gap junctions to elevate cytoplasmic calcium concentration ([Ca2+]i) within the oocyte. The two major candidates for signal transfer through gap junctions into the oocyte during AII receptor stimulation are Ins(1,4,5)P3 and Ca2+. In [3H]inositol-injected follicular oocytes, AII stimulated two- to fourfold increases in phosphoinositide hydrolysis and production of inositol phosphates. Injection of the glycosaminoglycan, heparin, which selectively blocks Ins(1,4,5)P3 receptors, prevented both AII-stimulated and Ins(1,4,5)P3-induced Ca2+ mobilization in Xenopus follicular oocytes but did not affect mobilization of Ca2+ by ionomycin or GTP. These results indicate that the AII-regulated process of gap junction communication between follicular cells and the oocyte operates through an Ins(1,4,5)P3-dependent mechanism rather than through transfer of Ca2+ into the ooplasm and subsequent Ca(2+)-induced Ca2+ release.