Type IV collagen stimulates an increase in intracellular calcium. Potential role in tumor cell motility.

Type IV collagen (Coll IV), a component of the extracellular matrix, stimulates motility in the A2058 human melanoma cell line, a response that is inhibited by pertussis toxin (PT). Fibronectin (FN)-induced chemotaxis in this cell line is not affected by PT. To understand the mechanism of cellular signaling, single cell intracellular Ca2+ responses to Coll IV and FN were studied using Fura-2 and digital imaging fluorescence microscopy. Coll IV, at a dose that stimulates motility (100 micrograms/ml, 185 nM), induces a significant rise in cytosolic free Ca2+ concentration ([Ca2+]i) within 100 s. This response is not inhibited by PT. Treatment of the cells with FN 30 micrograms/ml (70 nM), a dose that stimulates near-maximal chemotaxis, does not increase [Ca2+]i appreciably. Removal of extracellular Ca2+ fails to inhibit the Coll IV-stimulated rise in Ca2+ in all cells. Depletion of extracellular Ca2+ and pretreatment of cells with Ca2+ channel blockers only partially inhibits Coll IV-induced motility. Depletion of intracellular Ca2+ inhibits both chemotaxis and the Coll IV-induced increase in intracellular Ca2+. Coll IV does not stimulate membrane phosphoinositide hydrolysis. We conclude that Coll IV treatment induces an inositol 1,4,5-trisphosphate-independent release of intracellular Ca2+ stores which appears to play a necessary role in the chemotactic response of A2058 cells but is not mediated by a PT-sensitive G-protein. This response is not seen in cells exposed to FN, suggesting different intracellular signaling mechanisms for stimulated motility between these two extracellular matrix molecules.     

Inositol trisphosphate levels in cells expressing wild-type and mutant polyomavirus middle T antigens: evidence for activation of phospholipase C via activation of pp60c-src.

The transforming protein of polyomavirus, middle T (mT), forms a complex with two cellular enzymes: the protein tyrosine kinase pp60c-src and a phosphatidylinositol (PtdIns) 3-kinase. A mutant virus, Py1178T, encodes an mT protein which associates with and activates pp60c-src to the same extent as the wild type but fails to associate with PtdIns 3-kinase. To investigate relationships between activation of pp60c-src, association of PtdIns 3-kinase, and cellular levels of the second messenger inositol 1,4,5-trisphosphate (InsP3), we examined the effects of wild-type and mutant mT proteins on inositol metabolism in rat and mouse fibroblasts. Expression of either wild-type or 1178T mT caused a 300 to 500% increase in the InsP3 level. Cells transformed by Rous sarcoma virus also showed similar increases in InsP3 levels. Mutant mT proteins which failed to activate pp60c-src (NG59 and 1387T) had no effect on InsP3 levels. Pulse-chase experiments with [3H]inositol showed that the turnover of phosphoinositides was increased in cells transformed by either wild-type polyomavirus or Py1178T as compared with the normal parent cell line. The turnover of inositol phosphates was unchanged upon transformation. These data indicate that cells expressing either wild-type or mutant 1178T mT or pp60v-src exhibit elevated levels of InsP3 because of activation of phospholipase C. This activation appears to depend, directly or indirectly, upon activation of pp60src protein kinase activity. Activation of pp60c-src and elevation of InsP3 content are not sufficient for full transformation. Full transformation also requires the association of mT-pp60c-src complexes with PtdIns 3-kinase.     

Phosphatidylinositol 4,5-bisphosphate hydrolysis in human sperm stimulated with follicular fluid or progesterone is dependent upon Ca2+ influx.

Hydrolysis of the phospholipid phosphatidylinositol 4,5-bisphosphate is thought to be intimately involved in agonist-induced changes in intracellular Ca2+ levels. Recently we have shown that human preovulatory follicular fluid, which induces exocytosis in human sperm, can stimulate a rapid, transient increase in sperm cytosolic [Ca2+] [Thomas & Meizel (1988) Gamete Res. 20, 397-411]. We report here that both a Sephadex G-75 column fraction, derived from follicular fluid, and progesterone (a component of both the G-75 fraction and whole follicular fluid) stimulate rapid hydrolysis of PtdIns(4,5)P2 and PtdIns4P in human sperm. We also report that progesterone stimulates a rapid influx of Ca2+ in human sperm. Human spermatozoa were labelled for 24 h with myo-[3H]inositol and then treated with either the G-75 fraction or progesterone. A 30-65% loss of label was detected in PtdIns(4,5)P2 and PtdIns4P within 15 s of stimulus addition; no changes were observed in PtdIns during 2 min of treatment. The loss of label from both lipids was accompanied by an increase in water-soluble inositol phosphates. Production of both InsP3 and InsP2 was seen within 10 s; however, InsP3 was rapidly removed and had reached control levels by 1 min. Similarly, formation of InsP2 reached a peak by 30 s and then began a decline accompanied by a corresponding increase in InsP. No increases in InsP4 were seen in sperm treated in this fashion. Stimulated hydrolysis of the phosphoinositides and release of inositol phosphates were both blocked by the Ca2+ antagonist La3+. Likewise, the progesterone-induced increase in intracellular Ca2+ was inhibited by La3+, and phosphoinositide hydrolysis stimulated by this hormone was dependent upon the presence of extracellular Ca2+.     

Production of inositol pentakisphosphate in a human T lymphocyte cell line

The human T lymphocyte cell line, Jurkat, produced five distinct water soluble, inositol-containing compounds following a period of labeling with 3H-myo-inositol and several hours of incubation in non-radioactive complete medium. The less polar four peaks had been previously shown to be inositol phosphates, InsP through InsP4. Here, we demonstrate that the prominent fifth, very polar, peak was inositol pentakisphosphate, InsP5. The pattern of incorporation of 3H-myo-inositol into InsP5 differed from that of incorporation into other inositol phosphates. InsP5, unlike the second messengers, InsP3 and InsP4, was not increased by perturbation of the T cell receptor/T3 complex.     

Epidermal growth factor (EGF) and hormones stimulate phosphoinositide hydrolysis and increase EGF receptor protein synthesis and mRNA levels in rat liver epithelial cells. Evidence for protein kinase C-dependent and -independent pathways

Epidermal growth factor (EGF) stimulated the rapid accumulation of inositol trisphosphate in WB cells, a continuous line of rat hepatic epithelial cells. Since we previously had shown that EGF stimulates EGF receptor synthesis in these cells, we tested whether hormones that stimulate PtdIns(4,5)P2 hydrolysis would increase EGF receptor protein synthesis and mRNA levels. Epinephrine, angiotensin II, and [Arg8]vasopressin activate phospholipase C in WB cells as evidenced by the accumulation of the inositol phosphates, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. A 3-4-h treatment with each hormone also increased the rate of EGF receptor protein synthesis by 3-6-fold as assessed by immunoprecipitation of EGF receptor from [35S]methionine-labeled cells. Northern blot analyses of WB cell EGF receptor mRNA levels revealed that agents linked to the phosphoinositide signaling system increased receptor mRNA content within 1-2 h. A maximal increase of 3-7-fold was observed after a 3-h exposure to EGF and hormones. The phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), which activates protein kinase C also stimulated EGF receptor synthesis. Pretreatment of WB cells for 18 h with high concentrations of TPA "down-regulated" protein kinase C and blocked TPA-directed EGF receptor mRNA synthesis. In contrast, the effect of EGF on EGF receptor mRNA levels was not significantly decreased by TPA pretreatment. Epinephrine-induced increases in EGF receptor mRNA were reduced from 4- to 2-fold. Similarly, 18 h TPA pretreatment abolished the effect of TPA on EGF receptor protein synthesis but did not affect EGF-dependent EGF receptor protein synthesis. The 18-h TPA pretreatment diminished by 30-50% the induction of receptor protein synthesis by epinephrine or angiotensin II. We conclude that in WB cells EGF receptor synthesis can be regulated by EGF and other hormones that stimulate PtdIns(4,5)P2 hydrolysis. In these cells, EGF receptor synthesis appears to be regulated by several mechanism: one pathway is dependent upon EGF receptor activation and can operate independently of protein kinase C activation; another pathway is correlated with PtdIns(4,5)P2 hydrolysis and is dependent, at least in part, upon protein kinase C activation.     

Carbachol causes rapid phosphodiesteratic cleavage of phosphatidylinositol 4,5-bisphosphate and accumulation of inositol phosphates in rabbit iris smooth muscle; prazosin inhibits noradrenaline- and ionophore A23187-stimulated accumulation of inositol phosphates.

Rabbit iris smooth muscle was prelabelled with myo-[3H]inositol for 90 min and the effect of carbachol on the accumulation of inositol phosphates from phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol (PtdIns) was monitored with anion-exchange chromatography. Carbachol stimulated the accumulation of inositol phosphates and this was blocked by atropine, a muscarinic antagonist, and it was unaffected by 2-deoxyglucose. The data presented demonstrate that, in the iris, carbachol (50 microM) stimulates the rapid breakdown of PtdIns(4,5)P2 into [3H]inositol trisphosphate (InsP3) and diacylglycerol, measured as phosphatidate, and that the accumulation of InsP3 precedes that of [3H]inositol bisphosphate (InsP2) and [3H]inositol phosphate (InsP). This conclusion is based on the following findings. Time course experiments with myo-[3H]inositol revealed that carbachol increased the accumulation of InsP3 by 12% in 15s and by 23% in 30s; in contrast, a significant increase in InsP release was not observed until about 2 min. Time-course experiments with 32P revealed a 10% loss of radioactivity from PtdIns(4,5)P2 and a corresponding 10% increase in phosphatidate labelling by carbachol in 15s; in contrast a significant increase in PtdIns labelling occurred in 5 min. Dose-response studies revealed that 5 microM-carbachol significantly increased (16%) the accumulation of InsP3 whereas a significant increase in accumulation of InsP2 and InsP was observed only at agonist concentrations greater than 10 microM. Studies on the involvement of Ca2+ in the agonist-stimulated breakdown of PtdIns(4,5)P2 in the iris revealed the following. Marked stimulation (58-78%) of inositol phosphates accumulation by carbachol in 10 min was observed in the absence of extracellular Ca2+. Like the stimulatory effect of noradrenaline, the ionophore A23187-stimulated accumulation of InsP3 was inhibited by prazosin, an alpha 1-adrenergic blocker, thus suggesting that the ionophore stimulation of PtdIns(4,5)P2 breakdown we reported previously [Akhtar & Abdel-Latif (1978) J. Pharmacol. Exp. Ther. 204, 655-688; Akhtar & Abdel-Latif (1980) Biochem. J. 192, 783-791] was secondary to the release of noradrenaline by the ionophore. The carbachol-stimulated accumulation of inositol phosphates was inhibited by EGTA (0.25 mM) and this inhibition was reversed by excess Ca2+ (1.5 mM), suggesting that EGTA treatment of the tissue chelates extracellular Ca2+ required for polyphosphoinositide phosphodiesterase activity. K+ depolarization, which causes influx of extracellular Ca2+ in smooth muscle, did not change the level of InsP3.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pertussis toxin inhibits stimulated motility independently of the adenylate cyclase pathway in human melanoma cells

The human melanoma cell line, A2058, has previously been shown to respond to an autocrine motility factor (AMF). We have studied biochemical pathways that may be involved in the generation of such a motile response. Pertussis toxin (PT) caused a profound, rapid decrease in stimulated motility that was both dose and time-dependent. Preincubation of cells for 2 hr with as little as 1 ng/ml of PT significantly inhibited motility. A concentration of PT (0.5 microgram/ml) that completely eliminated migration after a 30 min. preincubation had a markedly reduced effect when added 1 hr after the start of the assay. In contrast, agents which selectively modulate or have a role in the adenylate cyclase pathway, e.g., cholera toxin, forskolin, the cAMP analogue 8-bromoadenosine 3':5'-cyclic monophosphate and the cyclase inhibitor 2',5'-dideoxyadenosine, all had negligible effect upon motility. These data are consistent with the presence of a receptor coupled to a PT sensitive G protein initiating motility independently of the adenylate cyclase system.     

Phosphatidylinositol metabolism in cells transformed by polyomavirus middle T antigen.

Associated with the middle T antigen of polyomavirus is a novel phosphatidylinositol (PtdIns) kinase activity which phosphorylates PtdIns at the D-3 position of the inositol ring. We have undertaken an analysis of myo-[3H]inositol-containing compounds in a panel of NIH 3T3 cell lines stably transfected with transforming and nontransforming middle T antigen mutants. All cell lines from which PtdIns 3-kinase activity coprecipitated with middle T antigen exhibited modestly elevated levels of PtdIns(3)P and compounds with predicted PtdIns(3,4)P2 and PtdIns(3,4,5)P3 structures. Complex formation between middle T antigen and PtdIns 3-kinase correlated not with an increase in total inositol phosphate levels but rather with elevated levels of InsP2 and InsP4. A specific increase in the level of an InsP2 species which comigrated in high-pressure liquid chromatography analysis with Ins(3,4)P2 was observed. These results suggest that association of the polyomavirus middle T antigen with PtdIns 3-kinase activates a distinct inositol metabolic pathway.     

Accumulation of the inositol phosphates in thrombin-stimulated, washed rabbit platelets in the presence of lithium.

Experiments with washed rabbit platelets demonstrate that stimulation with a low concentration of thrombin (0.1 unit/ml), that causes maximal aggregation and partial release of amine granule contents, also causes increased accumulation of [3H]inositol-labelled inositol trisphosphate (InsP3) in the presence of 20 mM-Li+. This concentration of Li+ was found to inhibit the degradation of inositol phosphates by phosphomonoesterases. This result indicates that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is degraded early after platelet stimulation with thrombin, although in a previous study we had found no decrease in amount. In the absence of Li+, the labelling of inositol bisphosphate (InsP2) increased more rapidly than that of InsP3, consistent with rapid degradation of InsP3 by phosphomonoesterase. After 30s the increase in InsP2 was augmented by Li+. This increase in InsP2 could have been due to increased degradation of phosphatidylinositol 4-phosphate or inhibition of breakdown of InsP2 to InsP with a lesser inhibition of breakdown of InsP3 to InsP2. The effect on InsP3 and InsP2 of stimulation of the platelets with 1.0 unit of thrombin/ml was comparable with the effect of the lower concentration of thrombin. Inositol phosphate (InsP) labelling did not increase in response to 0.1 unit of thrombin/ml, but increased when the platelets were stimulated with 1.0 unit of thrombin/ml. Whether the increase in InsP was due to increased degradation of phosphatidylinositol or a greater rate of breakdown of InsP2 to InsP than InsP to inositol cannot be determined in these experiments. These results indicate that degradation of PtdIns(4,5)P2 is an early event in platelet activation by thrombin and that formation of inositol phosphates and 1,2-diacylglycerol rather than a decrease in PtdIns(4,5)P2 may be the important change.     

Detection of inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase by thioinosinic acid and azathioprine by a new colorimetric assay.

The role of insulin in modulating phosphoinositide breakdown and accumulation of inositol phosphates was investigated in isolated rat pancreatic islets by using GPAIS (guinea-pig anti-insulin antiserum) that neutralizes effects of insulin in the medium. At either 3.0 mM- or 16.7 mM-glucose or 3.0 mM-glucose plus 10 microM-arecaidine propargyl ester (muscarinic receptor agonist), GPAIS (but not control serum) was able to increase InsP2 and InsP3, but not InsP, in myo-[3H] inositol-prelabelled islets. The effect of GPAIS on 3H incorporation into InsP3 was dose-dependent, with a half-maximal effect at a concentration able to bind 4004 +/- 163 microunits of insulin. A specific mass assay of the biologically relevant isomer Ins (1,4,5)P3 revealed a huge increase (greater than 3-folf). Formation of PtdIns, PtdInsP and PtdInsP2 was not affected by GPAIS. This is indirect evidence for an effect of insulin on inositide metabolism, and therefore endogenously released insulin may have led to an underestimation in earlier studies of effects of insulinotropic substances on inositol phosphate accumulation.     

Stimulation, by vasopressin and other agonists, of inositol-lipid breakdown and inositol phosphate accumulation in WRK 1 cells.

WRK 1 cells were labelled to equilibrium with 2-myo-[3H]inositol and stimulated with vasopressin. Within 3 s of hormone stimulation there was a marked accumulation of 3H-labelled InsP2 and InsP3 (inositol bis- and tris-phosphate), but not of InsP (inositol monophosphate). There was an associated, and rapid, depletion of 3H-labelled PtdInsP and PtdInsP2 (phosphatidylinositol mono- and bis-phosphates), but not of PtdIns (phosphatidylinositol), in these cells. Some 4% of the radioactivity in the total inositol lipid pool of WRK 1 cells was recovered in InsP2 and InsP3 after 10 s stimulation with the hormone. The selectivity of the vasopressin receptors of WRK 1 cells for a variety of vasopressin agonists and antagonists revealed these to be of the V1a subtype. There was no receptor reserve for vasopressin-stimulated inositol phosphate accumulation in WRK 1 cells. The accumulation of inositol phosphates was enhanced in the presence of Li+ions. Half-maximal accumulation of InsP, InsP2 and InsP3 in vasopressin-stimulated cells was observed with 0.9, 3.0 and 3.6 mM-Li+ respectively. Bradykinin and 5-hydroxytryptamine also provoked inositol phosphate accumulation in WRK 1 cells. The effects of sub-optimal concentrations of bradykinin and vasopressin upon inositol phosphate accumulation were additive, but those of optimal concentrations of the hormones were not.     

Thyrotropin-releasing hormone and GTP activate inositol trisphosphate formation in membranes isolated from rat pituitary cells

Stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by a phospholipase C to produce inositol trisphosphate (InsP3) and 1,2-diacylglycerol appears to be the initial step in signal transduction for a number of cell-surface interacting stimuli, including thyrotropin-releasing hormone (TRH). In suspensions of membranes isolated from rat pituitary (GH3) cells that were prelabeled to isotopic steady state with [3H]inositol and incubated with ATP, [3H] PtdIns(4,5)P2, and [3H]phosphatidylinositol 4-phosphate, the polyphosphoinositides, and [3H]InsP3 and [3H]inositol bisphosphate, the inositol polyphosphates, accumulated. TRH and GTP stimulated the accumulation of [3H]inositol polyphosphates in time- and concentration-dependent manners; half-maximal effects occurred with 10-30 nM TRH and with 3 microM GTP. A nonhydrolyzable analog of GTP also stimulated [3H] inositol polyphosphate accumulation. Moreover, when TRH and GTP were added together their effects were more than additive. Fixing the free Ca2+ concentration in the incubation buffer at 20 nM, a value below that present in the cytoplasm in vivo did not inhibit stimulation by TRH and GTP of [3H]inositol polyphosphate accumulation. ATP was necessary for basal and stimulated accumulation of [3H]inositol polyphosphates, and a nonhydrolyzable analog of ATP could not substitute for ATP. These data demonstrate that TRH and GTP act synergistically to stimulate the accumulation of InsP3 in suspensions of pituitary membranes and that ATP, most likely acting as substrate for polyphosphoinositide synthesis, was necessary for this effect. These findings suggest that a guanine nucleotide-binding regulatory protein is involved in coupling the TRH receptor to a phospholipase C that hydrolyzes PtdIns(4,5)P2.     

Calcium mobilization in permeabilized fibroblasts: effects of inositol trisphosphate, orthovanadate, mitogens, phorbol ester, and guanosine triphosphate

Utilizing a digitonin-permeabilized cell system, we have studied the release of calcium from a non-mitochondrial intracellular compartment in cultured human fibroblasts (HSWP cells). Addition of 1 mM MgATP to a monolayer of permeabilized cells in a cytosolic media buffered to 150 nM Ca with EGTA rapidly stimulates 45Ca uptake, and the subsequent addition of the putative intracellular messenger inositol trisphosphate (InsP3) induces rapid release of 85% (+/- 6% n = 6) of the 45Ca taken up in response to ATP. Mitogenic peptides (bradykinin, vasopressin, epidermal growth factor [EGF], and insulin) and orthovanadate, which are effective in mobilizing intracellular Ca in intact cells, have little or no effect when added alone to permeabilized cells. However, in the presence of GTP these agents stimulate accumulation of inositol phosphates and release Ca from the InsP3-sensitive pool. These data suggest that a GTP binding protein is involved in receptor mediated activation of phospholipase C, which leads to release of inositol phosphates. The GTP-dependent release of InsP3 and the mobilization of 45Ca from the intracellular compartment are inhibited by pretreatment of cells, prior to permeabilization, with the protein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). TPA pretreatment does not affect the InsP3 stimulated Ca release. These results suggest that protein kinase C is involved in down-regulation or inhibition of phospholipase C, or the GTP binding protein responsible for relaying the mitogenic signal from the cell surface receptor to the phospholipase C activity.     

Phorbol ester inhibits phosphoinositide hydrolysis and calcium mobilization in cultured astrocytoma cells

In cultured human 1321N1 astrocytoma cells, muscarinic receptor stimulation leads to phosphoinositide hydrolysis, formation of inositol phosphates, and mobilization of intracellular Ca2+. Treatment of these cells with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) completely blocks the carbachol-stimulated formation of [3H]inositol mono-, bis-, and trisphosphate ( [3H]InsP, [3H]InsP2, and [3H]InsP3). The concentrations of PMA that give half-maximal and 100% inhibition of carbachol-induced [3H]InsP formation are 3 nM and 0.5 microM, respectively. Inactive phorbol esters (4 alpha-phorbol 12,13-didecanoate and 4 beta-phorbol), at 1 microM, do not inhibit carbachol-stimulated [3H]InsP formation. The KD of the muscarinic receptor for [3H]N-methyl scopolamine is unchanged by PMA treatment, while the IC50 for carbachol is modestly increased. PMA treatment also abolishes carbachol-induced 45Ca2+ efflux from 1321N1 cells. The concomitant loss of InsP3 formation and Ca2+ mobilization is strong evidence in support of a causal relationship between these two responses. In addition, our finding that PMA blocks hormone-stimulated phosphoinositide turnover suggests that there may be feedback regulation of phosphoinositide metabolism through the Ca2+- and phospholipid-dependent protein kinase.     

Cross-linking membrane IgM induces production of inositol trisphosphate and inositol tetrakisphosphate in WEHI-231 B lymphoma cells

The addition of anti-IgM to the immature B lymphoma cell line WEHI-231 resulted in breakdown of phosphatidylinositol 4,5-bisphosphate, generating diacylglycerol and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). These reactions have recently been demonstrated in mature resting B cells stimulated with anti-IgM, as well. In addition to Ins(1,4,5)P3, inositol tetrakisphosphate (InsP4) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) were rapidly generated in WEHI-231 cells upon stimulation of the antigen receptor with anti-IgM. These two inositol polyphosphates are probably generated from Ins(1,4,5)P3 by phosphorylation to yield InsP4 and removal of the 5-phosphate from InsP4 to yield Ins(1,3,4)P3. It is possible that these inositol polyphosphates play a second messenger role in mediating the biologic effects of antigen-receptor signaling. It had previously been shown that anti-IgM also causes an increase in cytoplasmic free calcium. Therefore, the relationship between Ca2+ elevation and phosphoinositide breakdown was investigated. Although elevation of cytoplasmic Ca2+ with ionophores can trigger phosphoinositide breakdown, this required levels of Ca2+ well beyond those normally seen in response to anti-IgM. Thus, the Ca2+ elevation seen in response to anti-IgM cannot be the event controlling phosphoinositide breakdown. WEHI-231 cells have been shown to have a calcium storage compartment that releases Ca2+ in the presence of Ins(1,4,5)P3; therefore, it is likely that anti-IgM stimulates phosphoinositide breakdown as a primary event and this leads to the elevation of cytoplasmic Ca2+.     

Control of cellular phosphatidylinositol 4,5-bisphosphate levels by adhesion signals and rho GTPases in NIH 3T3 fibroblasts involvement of both phosphatidylinositol-4-phosphate 5-kinase and phospholipase C.

The involvement of small GTPases of the Rho family in the control of phosphoinositide metabolism by adhesion signals was examined in NIH 3T3 fibroblasts. Abrogation of adhesion signals by detachment of cells from their substratum resulted in a time-dependent decrease in the cellular level of PtdIns(4,5)P2 by approximately 50%. This effect could be mimicked by treatment of adherent cells with Clostridium difficile toxin B and toxin B-1470, which inhibit specific subsets of Rho and Ras GTPases. Detachment of cells that had been pretreated with the clostridial toxins did not cause a further reduction in PtdIns(4,5)P2 levels, suggesting that the target GTPases are integrated into the control of phosphoinositide levels by adhesion signals. The reduction in PtdIns(4,5)P2 levels could be attributed to reduced activity of the major PtdIns(4, 5)P2-producing enzyme, PtdIns4P 5-kinase. Unexpectedly, both cell detachment and toxin treatment resulted in a twofold to threefold increase in inositol phosphate production in intact cells. In lysates of these cells, in vitro phospholipase C activity was found to be elevated by 30-50%. The effects of cell detachment and toxin treatment on inositol phosphate formation could be mimicked by expression of dominant-negative N17 Rac1. Taken together, these data suggest that adhesion-controlled small GTPases of the Rho family are involved in the regulation of the cellular PtdIns(4,5)P2 levels in NIH 3T3 fibroblasts, by controlling the activities of both PtdIns4P 5-kinase and phospholipase C.     

Long-term phorbol ester treatment down-regulates protein kinase C and sensitizes the phosphoinositide signaling pathway to hormone and growth factor stimulation. Evidence for a role of protein kinase C in agonist-induced desensitization

Exposure of a nontransformed, continuous line of epithelial cells derived from rat liver (WB cells) to epidermal growth factor, angiotensin II, [Arg8]vasopressin, and epinephrine resulted in rapid accumulation of the inositol phosphates (InsP) InsP1, InsP2, and InsP3. Although short-term (5-60 min) pretreatment of WB cells with the phorbol ester 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly attenuated InsP accumulation in response to all agonists, the inhibitory effects on the InsP response were lost after 2 h incubation with PMA; and, with extended (6-24 h) preincubation, a time-dependent potentiation of the InsP response to angiotensin II, epidermal growth factor and [Arg8]vasopressin was observed. The InsP response during a 15-min challenge with angiotensin II in cells pretreated for 18 h with 600 nM and 10 microM PMA was increased by 2-3-fold and 4-6-fold, respectively. Long-term (18 h) treatment with 600 nM and 10 microM PMA caused a similar 90-100% loss of measurable Ca2+/phospholipid-dependent enzyme (protein kinase C) activity in cytosolic and soluble particulate fractions. The effects of long-term PMA pretreatment do not represent a general enhancement of hormone responsiveness since the InsP response to epinephrine was not affected. In control cells, the InsP response to angiotensin II and epinephrine desensitized very rapidly. Long-term pretreatment with PMA greatly reduced the contribution of agonist-induced desensitization to the angiotensin II response; in contrast, the extent of desensitization occurring during incubation of WB cells with epinephrine was unaltered by long-term treatment with PMA suggesting that an additional mechanism may be involved in alpha 1-adrenergic receptor desensitization. No PMA-induced change in resting levels of [3H]phosphoinositides or the metabolism of exogenous [3H]inositol 1,4,5-trisphosphate by WB homogenates occurred. Stimulation of InsP formation in intact cells by NaF and activation of phospholipase C by GTP gamma S in membranes both were unaltered by short-term or long-term PMA pretreatment. These data are consistent with the idea that following long-term treatment of WB cells with PMA, the occurrence of agonist-induced desensitization of receptors linked to the phosphoinositide/Ca2+ signaling system is reduced, apparently at least in part due to the loss of contribution of a negative feedback regulatory role of protein kinase C.     

Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase

To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.     

Down-regulation of protein kinase C potentiates angiotensin II-stimulated polyphosphoinositide hydrolysis in vascular smooth-muscle cells.

In smooth-muscle cells (SMC) isolated from rat aorta, angiotensin II stimulates a phospholipase C with subsequent formation of inositol trisphosphate (InsP3). Short-term (10 min) pretreatment of SMC with 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) decreases the angiotensin II-induced InsP3 formation. However, this inhibition is not observed after incubating the cells for 2 h with TPA. Longer-term pretreatments even lead to an enhanced generation of InsP3. This increased response to angiotensin II occurs without a significant change in the receptor number or Kd value of angiotensin II binding to the cells. The biologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect on angiotensin II-stimulated InsP3 generation, irrespective of the time of preincubation. In parallel with this potentiation of angiotensin II-induced generation of InsP3 by TPA, a down-regulation of protein kinase C activity is observed. A 24 h pretreatment of SMC with TPA decreases protein kinase C activity to less than 10% of that of control cells. Longer-term pretreatment also increases the angiotensin II-induced release of Ca2+ and delays the decay of the transient Ca2+ increase. All these data suggest that protein kinase C exerts a negative feedback control on angiotensin II-stimulated polyphosphoinositide turnover, and that protein kinase C is an important factor in limiting the production of InsP3 in stimulated cells.     

Effects of homo- and heterodimeric isoforms of PDGF on signalling events in rat renal mesangial cells.

Platelet-derived growth factor (PDGF) exists in three dimeric isoforms, AA, BB and AB. Mesangial cells exclusively bound the BB homodimer and responded only to the BB isoform in terms of DNA synthesis and phosphoinositide hydrolysis. PDGF-BB stimulated a dose-dependent formation of inositol trisphosphate (InsP3). Neither pertussis toxin nor short-term (10 min) treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited the PDGF-BB-evoked production of InsP3. In contrast, the response to PDGF-BB was attenuated in cells in which protein kinase C has been down-regulated by long-term (24 h) treatment with TPA. In parallel to the generation of InsP3, there was a biphasic increase in 1,2-diacylglycerol (DAG). The second peak of DAG generation was associated with a concomitant 2-fold increase in choline formation. In addition, PDGF-BB stimulated the accumulation of phosphatidylpropanol, produced by phospholipase D phosphatidyl transferase activity, when 1-propanol was added to mesangial cells. Stimulation of mesangial cells with PDGF-BB caused a dose-dependent formation of prostaglandin E2. Furthermore, mesangial cells secreted PDGF-AA into the culture supernatant.