Calcium Signaling System Research Articles

The regulation of leukotriene D 4-induced calcium influx in human epithelial cells involves protein tyrosine phosphorylation

Leukotriene D 4 (LTD 4) has been found to induce calcium signalling in the intestinal epithelial cell line Int 407, and this action involves the activation of both different GTP-binding proteins (G-proteins) and phospholipase C of the γ-subtype (PLC-γ). With this knowledge as the incentive, we investigated the possible regulatory role of protein tyrosine kinase activities in the calcium signalling system of the LTD 4 receptor. The tyrosine kinase inhibitors genistein and herbimycin A both reduced the LTD 4-induced calcium signal by 70% when Int 407 cells were stimulated in the presence of extracellular calcium, but had no effect on the signal when the cells were stimulated in a calcium-free medium. In accordance with these findings, pretreatment with a tyrosine kinase inhibitor also blocked thapsigargin-induced cellular influx of calcium. These inhibitors had no effect on the intracellular mobilisation of calcium, which was supported by the findings that LTD 4 was able to induce an increase in the tyrosine phosphorylation of PLC-γ even when one of the tyrosine kinase inhibitors was present. Of possible interest regarding the effect of genistein on LTD 4-induced calcium influx is that two major tyrosine phosphorylated protein bands were detected in immunoprecipitates obtained with PLC-γ antibodies from LTD4-stimulated cells. These proteins, which associate with PLC-γ have estimated molecular weights of 84 and 97 kD. Preincubation with genistein completely abolished the LTD4-induced increase in tyrosine phosphorylation of the major 97 kD band, whereas the 84 kD protein band, like the PLC-γ band, still exhibited an increased phosphorylation of tyrosine residues in response to LTD 4. Neither this effect nor any of the other effects of genistein were induced when cells were preincubated with daidzein, an inactive analogue of genistein. The present results suggest that LTD 4-induced calcium signalling in epithelial cells involves not only tyrosine phosphorylation of PLC-γ, but also a tyrosine kinase-dependent step which occurs downstream of PLC-γ activation and is directly implicated in the regulation of agonist-mediated calcium influx.

Effects of pharmacological modulation of intracellular signalling systems on retinal pigment epithelial cell attachment to extracellular matrix proteins.

Complication of retinal detachment by proliferative vitreoretinopathy (PVR) is common. In the contraction of intraocular collagen matrices which occurs in PVR cell proliferation, migration and adhesion seem to be more important than any inherent cellular contractility. The aim of this study was to investigate the pharmacological inhibition of adhesion of retinal pigment epithelial cells to extracellular matrices. The adherence of human RPE lines to a range of ten substrates was assessed to determine their preferred substrates for attachment. The effect of pharmacological inhibitors and stimulators of protein kinase C, cyclic AMP and calcium/calmodulin intracellular signal transduction systems on attachment to substrates was investigated. RPE cells showed a clear substrate preference for fibronectin, and slight preference for collagen type I. Modulation of the protein kinase C and cAMP pathways had relatively minor effects upon RPE attachment. Increasing intracellular calcium concentration reduced RPE attachment to 12% of control, whilst reducing intracellular calcium had a less marked, although significant effect. Down-regulation of calmodulin reduced attachment to 17% of control. The drug tamoxifen, and the experimental calmodulin antagonist J8, produced significant inhibition of attachment even when cells had been allowed to adhere for 24 h prior to exposure to these agents. The adhesion of RPE to extracellular matrices may be markedly affected by drugs which modulate the intracellular calcium and calmodulin signalling systems. Calmodulin antagonists warrant further investigation as possible pharmacological inhibitors of PVR.

P-520 - The difference in calcium signaling systems of bradykinin and histamine in human gingival fibroblasts.

P-520 - The difference in calcium signaling systems of bradykinin and histamine in human gingival fibroblasts.

Effects of Surgical Sympathetic Denervation on myo-Inositol Trisphosphate Production and Contraction in the Dilator and Sphincter Smooth Muscles of the Rabbit Iris: Evidence for Interaction Between the Cyclic AMP and Calcium Signaling Systems

The effects of norepinephrine (NE), carbachol (CCh), NaF, 3-isobutyl-1-methylxanthine (IBMX), and high K+ concentration (80 mM) depolarization on inositol trisphosphate (IP3) accumulation, cyclic AMP (cAMP) formation, and contraction were investigated in the dilator and sphincter smooth muscles of the sympathetically denervated as well as the normal rabbit eye. (a) In the denervated dilator muscle, NE-stimulated IP3 production and contraction are enhanced. (b) In the sphincter muscle of rabbits that have undergone sympathetic denervation. CCh-stimulated IP3 production and contraction are attenuated. (c) The increase in tension by a maximal effective dose of NaF (209 mM) in the dilator was 12.5 and 18 mg of tension/mg wet weight in normal and denervated tissue, respectively, and in the sphincter was 33.8 and 15.2 mg of tension/mg wet weight in normal and denervated tissue, respectively. NaF had no effect on cAMP formation. (d) Addition of NE had no effect on cAMP formation in both the normal and denervated dilator, whereas basal and IBMX-induced cAMP formation increased. in the denervated sphincter over that of the normal tissue by 15 and 60%, respectively. (e) Isoproterenol (5 microM) increased cAMP formation in the normal and denervated sphincter by 47 and 91%, respectively. (f) Whereas CCh inhibits cAMP formation in the normal sphincter, it lost its inhibitory effect in the sphincter with denervation. (g) IBMX (0.1 mM) attenuated the CCh-stimulated IP3 production and contraction of the sphincter by approximately 30% of their respective controls. (h) High K+ concentration depolarization attenuated contraction in both dilator and sphincter muscles with denervation. These observations suggest that an increase in the level of cAMP in the iris sphincter due to sympathetic denervation could lead to inhibition of phospholipase C (or other target sites, such as phosphorylation of the muscarinic receptor, Gp protein itself, myosin light chain kinase, or the IP3 receptor), IP3 production, and contraction. In conclusion, we suggest that the supersensitivity and subsensitivity observed after surgical sympathetic denervation of the iris dilator and sphincter muscles, respectively, are caused by alterations in the efficiency of coupling, probably through the Gp proteins, between their respective receptors and the breakdown of polyphosphoinositides by phospholipase C. In addition, we propose that the sympathetic nervous system can regulate, through alterations in cAMP levels, the muscarinic stimulation of IP3 accumulation and contraction in the iris sphincter. These findings add further support to the hypothesis that there are reciprocal interactions between the cAMP and IP3-Ca2+ signaling systems and the contractile response in the iris smooth muscle.

Widespread synchronous [Ca2+]i oscillations due to bursting electrical activity in single pancreatic islets

Pancreatic beta cells, tightly organized in the islet of Langerhans, secrete insulin in response to glucose in a calcium-dependent manner. The calcium input required for this secretory activity is thought to be provided by an oscillatory electrical activity occurring in the form of "bursts" of calcium action potentials. The previous observation that islet intracellular free Ca2+ levels undergo spontaneous oscillations in the presence of glucose, together with the fact that islet cells are coupled through gap junctions, hinted at a highly effective co-ordination between individual islet cells. Through the use of simultaneous recordings of intracellular calcium and membrane potential it is now reported that the islet calcium waves are synchronized with the beta cell bursting electrical activity. This observation suggests that each calcium wave is due to Ca2+ entering the cells during a depolarized phase of electrical activity. Moreover, fura-2 fluorescence image analysis indicates that calcium oscillations occur synchronously across the whole islet tissue. The maximal phase shift between oscillations occurring in different islet cells is estimated as 2 s. This highly co-ordinated oscillatory calcium signalling system may underlie pulsatile insulin secretion and the islet behaviour as a secretory "syncytium". Since increasing glucose concentration lengthens calcium wave and burst duration without significantly affecting wave amplitude, we further propose that it is the fractional time at an enhanced Ca2+ level, rather than its amplitude, that encodes for the primary response of insulin-secreting cells to fuel secretagogues.

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

Calcium signaling systems in nonexcitable cells involve activation of Ca2+ entry across the plasma membrane and release from intracellular stores as well as activation of Ca2+ pumps and inhibition of passive Ca2+ pathways to ensure exact regulation of free cytosolic Ca2+ concentration [( Ca2+]i). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca2+ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) both stimulated Ca2+ entry and release while bradykinin appeared only to release Ca2+ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca2+]i response to these agonists was examined by four methods. Low concentrations of TPA (2 x 10(-10) M) had no effect on Ca2+ release due to EGF, TGR-alpha or bradykinin but resulted in a rapid return of [Ca2+]i to baseline levels for EGF or TGF-alpha. Addition of the PKC inhibitor staurosporine (1 and 10 nM) completely inhibited the action of TPA on EGF-induced [Ca2+]i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 microM TPA produced the converse effect, namely prolonged Ca2+ entry following stimulation with EGF or TGF-alpha. To show that one effect of TPA was on Ca2+ entry, fura-2 loaded cells were suspended in Mn2+ rather than Ca2+ buffers. Addition of EGF or TGF-alpha resulted in Ca2+ release and Mn2+ entry. TPA but not the inactive phorbol ester, 4-alpha-phorbol-12,13-didecanoate, inhibited the Mn2+ influx. Thus, PKC is able to regulate Ca2+ entry due to EGF or TGF-alpha in this cell type. A431 cells treated with higher concentrations of TPA (5 x 10(-8) M) inhibited not only Ca2+ entry but also Ca2+ release due to EGF/TGF-alpha but had no effect on bradykinin-mediated Ca2+ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF-alpha gave a single transient of [Ca2+]i, showing a common pool of Ca2+ for these agonists. In contrast, sequential addition of EGF (or TGF-alpha) and bradykinin resulted in two [Ca2+]i transients equal in size to those obtained with a single agonist.(ABSTRACT TRUNCATED AT 400 WORDS)

Inositol Trisphosphate, Calcium, Lithium, and Cell Signaling

Inositol lipids play a major role in cell signaling by functioning as precursors of second messengers. Of the three common inositol-containing lipids found in the plasma membrane, phosphatidylinositol (4,5)-bisphosphate is hydrolyzed to give diacylglycerol, which stimulates protein kinase C, and inositol 1,4,5-trisphosphate, which diffuses into the cell to release intracellular calcium. Inositol 1,4,5-trisphosphate is metabolized to give free inositol by two separate pathways. Lithium inhibits the final dephosphorylation step of both pathways, thus reducing the supply of the free inositol required to maintain the lipid precursors used for signaling. An inositol-depletion hypothesis may explain both the teratogenic effects of lithium and its therapeutic action in controlling manic-depressive illness. One of the metabolic pathways generates inositol tetrakisphosphate, which may also play a messenger role by expanding the size of the inositol 1,4,5-trisphosphate-sensitive pool of calcium. Calcium imaging of single cells has begun to reveal that this inositol 1,4,5-trisphosphate/calcium signaling system is organized in complex patterns, which include localization of calcium signals to discrete regions of cells and the generation of both calcium waves and calcium oscillations.

Palmitate dependence of insulin secretion, "de novo" phospholipid synthesis and 45Ca2+-turnover in glucose stimulated rat islets.

Palmitate ability to modify D-[U-14C]glucose incorporation into different lipids ("de novo" synthesis), as well as sugar-stimulation of insulin release and 45Ca2+-fluxes, was investigated in islets of fed and 48-h starved rats. The fatty-acid induced dose-dependent, correlative increments of insulin secretion, 45Ca2+-influx and the "de novo" synthesis of each phospholipid fraction analysed at 20 mmol/l (but not 3 mmol/l) glucose. Omission of calcium reduced drastically (p less than 0.001) insulin release and the "de novo" synthesis of neutral glycerolipids, leaving unaltered that of acidic phospholipids (phosphatidate and phosphoinositides). The increased synthesis of the latter is therefore not the consequence of stimulated secretion. It could initiate or contribute to maintain an increased turnover of islet phosphoinositides, thus generating some mediators of the calcium signalling system (inositol phosphates). Starvation led to a drastic reduction (p less than 0.001) of both insulin secretion, "de novo" synthesis of each lipid fraction, and 45Ca2+-influx in response to glucose and palmitate. The presence of a fatty-acid oxidation inhibitor (2-bromostearate or 2-tetradecylglycidate) prevented the effect of starvation on 45Ca2+-influx, as it has been shown to do on insulin secretion and palmitate incorporation into islet lipids. It is finally suggested that palmitate might amplify the insulin secretory response of islets to glucose, through the stimulation of the "de novo" synthesis of phosphoinositides and the subsequent generation of inositol phosphates, which would contribute to accelerated calcium turnover.