Richard J.H. Wojcikiewicz

Desensitization of cell signalling mediated by phosphoinositidase C

The waning of responses to cell-surface receptor activation during persistent stimulation with agonists (desensitization) is a feature common to many forms of transmembrane signalling. However, information is scarce regarding the regulatory processes that modulate the extensive group of receptors linked via phosphoinositidase C to the production of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. This situation is now beginning to change. Recent data indicate (1) that very rapid desensitization, possibly associated with receptor phosphorylation, regulates receptors linked to phosphoinositidase C, (2) that different receptors are desensitized to varying extents, (3) that receptor internalization can mediate desensitization at later times and (4) that signalling can be regulated at additional sites downstream of phosphoinositidase C. As Richard Wojcikiewicz and colleagues discuss here, these diverse regulatory events provide the means by which the breakdown of phosphoinositides and cellular responsiveness to their products are controlled during cell stimulation.

Muscarinic Receptor‐Mediated Inositol 1,4,5‐Trisphosphate Formation in SH‐SY5Y Neuroblastoma Cells Is Regulated Acutely by Cytosolic Ca2+ and by Rapid Desensitization

Abstract: Stimulation of muscarinic receptors expressed in SH‐SY5Y human neuroblastoma cells resulted in a complex profile of inositol 1,4,5‐trisphosphate (InsP3) accumulation, with a dramatic increase (six‐ to eightfold) over the first 10 s (the “peak” phase) and subsequently, from ∼60 s onward, maintained at a lower but sustained level (the “plateau” phase). Chelation of extracellular Ca2+ with EGTA or inhibition of Ca2+ channels with Ni2+ showed that the plateau phase was dependent upon Ca2+ entry. Furthermore, use of thapsigargin and EGTA to discharge and sequester Ca2+ from intracellular stores revealed that Ca2+ from this source was capable of supporting the peak phase of the InsP3 response. Carbachol‐stimulated phosphoinositidase C activity in permeabilized SH‐SY5Y cells was also shown to be highly dependent on free Ca2+ concentration (20–100 nM) and suggests that under normal conditions, InsP3 formation is enhanced by increases in cytosolic free Ca2+ concentration that accompany muscarinic receptor activation. Measurement of carbachol‐stimulated total inositol phosphate accumulation in the presence of Li+ indicated that the initial rate of phosphoinositide hydrolysis (from 0 to 30 s) was about fivefold greater than that from 30 to 300 s. This rapid but partial desensitization of receptor‐mediated phosphoinositide hydrolysis provides strong evidence for the mechanism underlying the changes in InsP3 accumulation over this time. Because very similar data were obtained in Chinese hamster ovary cells transfected with human m3 receptor cDNA, we suggest that although increases in cytosolic free Ca2+ concentration amplify InsP3 formation during stimulation of m3 muscarinic receptors, the primary factor that governs the profile of InsP3 accumulation is rapid, but partial, desensitization. Such desensitization does not appear to be mediated by changes in cytosolic Ca2+ or protein kinase C activity.

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 (EC30 = 6.4 μM), but was some 70‐fold less potent than Ins (1,4,5)P3 (EC30 = 0.09 μM), indicating that the 6‐hydroxyl group of Ins(1,4,5)P3 is most 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‐[22P] + Ins (1,4,5)P3 (K 1 76 μM) and the phosphorylation of [3H]Ins(1,4,5)P3 (apparent K 1 5.7 μM) 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.

Regulation of Muscarinic Agonist‐Induced Activation of Phosphoinositidase C in Electrically Permeabilized SH‐SY5Y Human Neuroblastoma Cells by Guanine Nucleotides

myo‐[3H]Inositol‐labelled SH‐SY5Y cells were permeabilized with electrical discharges. 3H‐Inositol phosphate formation in cells shown to be fully permeable was stimulated by the muscarinic agonist carbachol, by guanosine 5′‐(γ‐thio)triphosphate [GTP(S)], and by guanosine 5′‐(β‐thio)diphosphate (GppNHp). Synergism was observed on coincubation of these GTP analogues with carbachol. GTP was also stimulatory and guanosine 5′‐(β‐thio)diphosphate was inhibitory in the presence of agonist. Atropine blocked the effects of carbachol. Stimulation by GTP(S) (0.1 mM) occurred after a 1‐2‐min lag, whereas Ca2+ (0.5 mM), carbachol (1 mM), and carbachol plus GTP(S) stimulated without delay. The effects of carbachol plus GTP(S) but not those of Ca2+ were inhibited by spermine (4 mM). Accumulation of 3H‐inositol phosphates was enhanced by Li+ (4 mM) only in intact cells. In intact or permeabilized cells, the “partial” agonist arecoline was maximally 40–50% as efficacious as carbachol. In permeabilized cells, the maximal effects of carbachol and arecoline were enhanced 2.8‐ and 5.3‐fold, respectively, by 0.1 mM GTP(S), but only the EC50 for carbachol was substantially reduced. The binding affinity of carbachol but not that of arecoline in permeabilized cells was significantly reduced by 0.1 mM GppNHp. These data indicate that a guanine nucleotide‐binding regulatory protein is involved in coupling muscarinic receptors to phosphoinositidase C in SH‐SY5Y cells and that the activity of this protein influences the relationship between receptor occupation and phosphoinositide response.

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 μM respectively) and slightly less potent than D‐Ins(1,4,5)P3 (EC50 0.13 μM), 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 μM).

Inositol 1,4,5-Trisphosphate Receptor Immunoreactivity in SH-SY5Y Human Neuroblastoma Cells Is Reduced by Chronic Muscarinic Receptor Activation

Inositol 1,4,5‐trisphosphate (InsP3) receptor immunoreactivity in SH‐SY5 Y human neuroblastoma cells was monitored with a monoclonal antibody raised against the mouse cerebellar InsP3 receptor. Recognition of a protein corresponding to the InsP3 receptor (molecular mass, ∼275 kDa) was inhibited markedly following exposure of cells to 0.1 mM carbachol. This effect was half‐maximal and maximal at ∼2 and ∼6 h, respectively; was blocked by atropine; but was not mimicked by thapsigargin, K+, or phorbol 12‐myristate 13‐acetate. However, the decrease in immunoreactivity following exposure of cells to carbachol for 5 h was blocked if the extracellular Ca2+ concentration was reduced from 1.3 mM to 200 nM. This manipulation also reduced markedly carbachol‐induced increases in InsP3 concentration at 5 h. These data indicate that chronic muscarinic stimulation of phosphoinositide hydrolysis reduces InsP3 receptor concentration in SH‐SY5 Y cells, perhaps via a mechanism that involves prolonged elevation of InsP3 levels.

Muscarinic receptors, phosphoinositide metabolism and intracellular calcium in neuronal cells.

1. We have utilised SH-SY5Y human neuroblastoma cells and primary cultures of rat neonatal cerebellar granule cells, both expressing M3 muscarinic receptors, to examine agonist driven polyphosphoinositide hydrolysis and alterations in intracellular calcium. 2. Stimulation of SH-SY5Y cells leads to a biphasic increase in intracellular calcium, the initial peak being due to the release of calcium from an intracellular store and the second maintained phase being due to calcium entry across the plasma membrane. The channel involved does not appear to be voltage sensitive, to involve a pertussis toxin sensitive G protein, or be opened by inositol polyphosphates. 3. Muscarinic receptor stimulation also leads to increased inositol polyphosphate formation in SH-SY5Y cells. Ins(1,4,5)P3 mass formation was biphasic in profile whereas Ins(1,3,4,5)P4 mass formation was slower and monophasic in profile. These data are consistent with substantial activity of 5-phosphatase (dephosphorylating Ins(1,4,5)P3 to Ins(1,4)P2) and 3-kinase (phosphorylating Ins(1,4,5)P3 to Ins(1,3,4,5)P4) in SH-SY5Y cells. 4. In order to better understand the role of Ins(1,4,5)P3 and its metabolites in calcium homeostasis we have examined the ability of a variety of natural and synthetic analogues to release intracellular sequestered calcium. The Ins(1,4,5)P3 calcium mobilizing receptor displays a remarkable degree of stereo- and positional selectivity with the most potent agonist to date being Ins(1,4,5)P3 (EC50 = 0.09 microM). 5. As an alternative to the continuous SH-SY5Y neuroblastoma (tumour derived) cell line we have used the primary cultured cerebellar granule cell. These cells also display a biphasic increase in Ins(1,4,5)P3 mass and a subsequent release of intracellular stored calcium. In our hands carbachol appears to increase calcium influx, a response which is only visible in the absence of magnesium.

Phosphoinositide hydrolysis in permeabilized SH-SY5Y human neuroblastoma cells is inhibited by mastoparan

The effects of mastoparan on phospholipase C‐catalysed phosphoinositide hydrolysis were examined in [3H]inositol‐labelled human neuroblastoma SH‐SY5Y cells. [3H]Inositol phosphate formation in intact cells was not altered by 20 μM mastoparan. In contrast, [3H]inositol phosphate formation in electrically permeabilized cells stimulated with guanosine 5′[γ‐thio]triphosphate and/or carbachol was inhibited by mastoparan with half‐maximal effects at approx. 3 μM. The peptide was much less effective in inhibiting stimulatory effects of Ca2+. Similar but less potent inhibitory effects were observed with the cations, neomycin and spermine, indicating that direct interaction of mastoparan with polyphosphoinositides might account for its inhibitory effects on inositol phosphate formation.

Aluminium Inhibits Muscarinic Agonist‐Induced Inositol 1,4,5‐Trisphosphate Production and Calcium Mobilization in Permeabilized SH‐SY5Y Human Neuroblastoma Cells

Abstract: The effects of aluminium (as Al3+) on carbachol‐induced inositol 1,4,5‐trisphosphate (lnsP3) production arid Ca2+ mobilisation were assessed in electropermeabilised human SH‐SY5Y neuroblastoma cells. Al3+ had no effect on lnsP3‐induced Ca2+ release but appreciably reduced carbachol‐induced Ca2+ release (lC50 of ∼90 μM). Aβ3+ also inhibited lnsP3 production (lC60 of ∼15 μM). Dimethyl hydroxypyridin‐4‐one, a potent Al3+ chelator (K5= 31), at 100 μM was able to abort and reverse the effects of Al3+ on both Ca2+ release and lnsP3 production. These data suggest that, in permeabilised cells, the effect of Al3+ on the phosphoinositide‐mediated signalling pathway is at the level of phosphatidylinositol 4,5‐bisphosphate hydrolysis. This may reflect interference with receptor‐G protein‐phospholipase C coupling or an interaction with phosphatidylinositol 4,5‐bisphosphate.

Inositol 1,4,5-Trisphosphate Receptor Down-Regulation

Publisher Summary This chapter discusses down regulation of inositol 1, 4, 5-trisphosphate receptor in human neuroblastoma cell line (SH-SY5Y). These cells express muscarinic receptors—predominantly the M3 subtype—that when activated stimulate phosphoinositide hydrolysis, and they also possess substantial IP 3 -sensitive Ca 2+ stores. The essential features of the procedure are permeabilizing the cell plasma membrane, loading of intracellular stores with 45 Ca 2+ , and following experimental incubations and 45 Ca 2+ mobilization, effectively separating released 45 Ca 2+ from that remaining in intracellular stores. During the measurement of IP 3 -induced Ca 2+ mobilization it is very important to monitor and control [Ca 2+ free ] in buffers A and B used to prepare and incubate the permeabilized cells, as both the potency and efficacy of IP 3 are influenced by changes in [Ca 2+ free ]. The procedure relies on the use of [ 32 P] IP 3 to identify IP 3 binding sites in crude SH-SY5Y cell membrane preparations. Thus, chronic activation of muscarinic receptors suppresses IP 3 receptor immunoreactivity in SH-SY5Y cells, providing direct evidence that IP 3 receptor concentration can be modified by cell-surface receptor activation.