A PKC wave follows the calcium wave after activation of Xenopus eggs

Calcium waves are well-known hallmarks of egg activation that trigger resumption of the cell cycle and development of the embryo. These waves rapidly and efficiently assure that activation signals are transmitted to all regions of the egg. Although the mechanism by which the calcium wave propagates across an egg as large as that of Xenopus is not known, two models prevail. One model is a wave of calcium-induced calcium release (CICR) and the other is propagation by inositol-induced calcium release (IICR). IICR requires a wave of phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, generating two second messengers, IP3, which then releases calcium and DAG, which activates protein kinase C (PKC). We show here that a wave of PKC-green fluorescent protein travels across the egg immediately following, and at the same velocity as, the calcium wave. This is the first example of a PKC wave in a vertebrate egg and supports the IICR model of wave propagation.     

The substrate specificity of phosphoinositide phospholipase C in rat heart sarcolemma

In rat cardiac sarcolemmal membranes a phosphoinositide-specific phospholipase C (PLC) was found to be present. The enzyme hydrolysed exogenous [³H-]phosphatidylinositol 4,5-biphosphate ([³H-]PtdIns(4,5)P ₂) in an optimized assay mixture containing 15 leg SL protein, 100 mM NaCl, 1 mM free Ca²⁺,14 mM Na-cholate and 20 AM [³H-]PtdIns (4,5)P ₂ (400–500 dpm/gm-l) in 30 mM HEPES-Tris buffer (pH 7.0). The average specific activity was 9.14±0.55 nmol-mg^–1·2.5 min^–1. The addition of Mg²⁺ to the assay mixture did not change PLC activity but increased the relative amounts of dephosphorylated inositol products. In the absence of Na⁺ and at a low Ca²⁺ concentration (0.3 M), Mg²⁺ also enhanced the intraSL levels of PtdIns4P and PtdIns, and, moreover, inhibited PLC activity (IC₅₀0.07 mM). PtdIns4P seemd to be a good substrate for the rat SL PLC (23.07 ± 1.57 nmol·mg^–1·2.5 min^–1) whereas PtdIns was hydrolysed at a very low rate (0.36 ± 0.08 nmol·mg^–1·2.5 min^–1). Unlike PtdIns(4,5)P ₂, PLC-dependent PtdIns4P and PtdIns hydrolysis was not inhibited by Ca²⁺ concentrations over 1 mM. The possibility of distinct isozymes being responsible for the different hydrolytic activities is discussed. (Mol Cell Biochem116: 27–31, 1992).Abbreviations DAG sn-1,2-diacylglycerol - EGTA ethyleneglycol-O,O-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - Ins(1,4,5)P ₃ inositol 1,4,5-trisphosphate - InsP inositol monophosphate (unidentified isomer) - InsP ₂ inositol bisphosphate (unidentified isomer) - InsP ₃ inositol trisphosphate (unidentified isomer) - InsP _x any inositol phosphate - PLC phospholipase C - PtdIns phosphatidylinositol - PtdIns(4,5)P ₂ phosphatidylinositol 4,5-bisphosphate - PtdIns4P phosphatidylinositol 4-monophosphate - SL sarcolemma     

Enhanced phosphoinositide metabolism in colorectal carcinoma cells derived from familial adenomatous polyposis patients

The production of the second messenger molecules diacylglycerol and inositol 1,4,5-trisphosphate is mediated by activated phosphatidylinositol-specific phospholipase C (PLC) enzymes. We report the enhancement of the phosphoinositide metabolism pathway in KMS-4 and KMS-8 cells, both of which are human colorectal carcinoma cell lines derived from familial adenomatous polyposis patients. In these cells, the cellular contents of diacylglycerol and inositol 1,4,5-trisphosphate were constitutively increased and the PLC activity in vitro was significantly high, as compared with those in normal colon cells or in other sporadic colorectal carcinoma cells. Northern and Western analyses showed the high expression levels of both PLC-γ1 and PLC-δ1 in KMS-4 and KMS-8 cells. Moreover, we detected the enhancement of protein–tyrosine kinase activity and tyrosine phosphorylation of PLC-γ1 in these KMS cells. These results suggest the involvement of activated phosphoinositide signaling pathways in the colorectal tumorigenesis of familial adenomatous polyposis. © 1994 Wiley-Liss, Inc.     

alpha Latrotoxin of black widow spider venom binds to a specific receptor coupled to phosphoinositide breakdown in PC12 cells

alpha Latrotoxin of black widow spider is known to bind with high affinity to surface sites of rat pheochromocytoma (PC12) cells, thereby causing depolarization, calcium influx and massive neurotransmitter release. We show here that the toxin causes the accumulation of inositol phosphates, the products of phosphoinositide breakdown. Inositol 1,4,5, trisphosphate was predominantly accumulated shortly after toxin application. Phosphoinositide breakdown appears to be a direct consequence of toxin binding because high K+ and ionophores (which induce depolarization, calcium influx and transmitter release by different mechanisms) were without such effect. Phosphoinositide breakdown is known as an event coupled to the activation of receptors of various hormones and transmitters. We suggest therefore that the alpha latrotoxin binding site is a receptor coupled across the membrane to the phosphoinositide hydrolysing system.     

Progesterone triggers rapid transmembrane calcium influx and/or calcium mobilization from endoplasmic reticulum,via a pertussis-insensitive G-protein in granulosa cells in relation to luteinization process

We investigated the early effects (5–60 s) of progesterone (1 pM–0.1 μM) on cytosolic free calcium concentration ([Ca²⁺]_i) and inositol 1,4,5-trisphosphate (InsP₃) formation in nonluteinized and in vitro luteinized porcine granulosa cells (pGCs). Progesterone increased [Ca²⁺]_i and InsP₃ formation within 5 s in both cell types. Progesterone induced calcium mobilization from the endoplasmic reticulum via the activation of a phospholipase C linked to a pertussis-insensitive G-protein. This process was controlled by protein kinases C and A. In contrast, only nonluteinized pGCs showed a Ca²⁺ influx via dihydropyridine-insensitive calcium channel. In both cell types, the nuclear progesterone receptor antagonist RU-38486 did not inhibit the progesterone-induced increase in [Ca²⁺]_i; progesterone immobilized on bovine serum albumin, which did not enter the cell, increased [Ca²⁺]_i within 5 s and was a full agonist, but less potent than the free progesterone; pertussis toxin did not inhibit progesterone effect on InsP₃. In conclusion, progesterone may interact with membrane unconventional receptors that belong to the class of membrane receptors coupled to a phospholipase C via a pertussis toxin-insensitive G-protein. The source of the Ca²⁺ for the progesterone-induced increase in [Ca²⁺]_i also depends on the stage of cell luteinization. © 1996 Wiley-Liss, Inc.     

Odorant receptors directly activate phospholipase C/inositol-1,4,5-trisphosphate coupled to calcium influx in Odora cells

Mechanisms by which odorants activate signaling pathways in addition to cAMP are hard to evaluate in heterogeneous mixtures of primary olfactory neurons. We used single cell calcium imaging to analyze the response to odorant through odorant receptor (OR) U131 in the olfactory epithelial cell line Odora (Murrell and Hunter 1999), a model system with endogenous olfactory signaling pathways. Because adenylyl cyclase levels are low, agents activating cAMP formation do not elevate calcium, thus unmasking independent signaling mediated by OR via phospholipase C (PLC), inositol-1,4,5-trisphosphate (IP(3)), and its receptor. Unexpectedly, we found that extracellular calcium is required for odor-induced calcium elevation without the release of intracellular calcium, even though the latter pathway is intact and can be stimulated by ATP. Relevant signaling components of the PLC pathway and G protein isoforms are identified by western blot in Odora cells as well as in olfactory sensory neurons (OSNs), where they are localized to the ciliary zone or cell bodies and axons of OSNs by immunohistochemistry. Biotinylation studies establish that IP(3) receptors type 2 and 3 are at the cell surface in Odora cells. Thus, individual ORs are capable of elevating calcium through pathways not directly mediated by cAMP and this may provide another avenue for odorant signaling in the olfactory system.     

A rapid attenuation of muscarinic agonist stimulated phosphoinositide hydrolysis precedes receptor sequestration in human SH-SY-5Y neuroblastoma cells

Agonist occupancy of muscarinic cholinergic receptors in human SH-SY-5Y neuroblastoma cells elicited two kinetically distinct phases of phosphoinositide hydrolysis when monitored by either an increased mass of inositol 1,4,5-trisphosphate, or the accumulation of a total inositol phosphate fraction. Within 5s of the addition of the muscarinic agonist, oxotremorine-M, the phosphoinositide pool was hydrolyzed at a maximal rate of 9.5%/min. This initial phase of phosphoinositide hydrolysis was short-lived (t_1/2=14s) and after 60s of agonist exposure, the rate of inositol lipid breakdown had declined to a steady state level of 3.4%/min which was then maintained for at least 5–10 min. This rapid, but partial, attenuation of muscarinic receptor stimulated phosphoinositide hydrolysis occurred prior to the agonist-induced internalization of muscarinic receptors.Abbreviations I(1,4,5)P₃ inositol 1,4,5-trisphosphate - IP total inositol phosphate fraction - IPL total inositol lipid fraction - mAChR muscarinic acetylcholine receptor - NMS N-methylscopolamine - Oxo-M oxotremorine-M - PI phosphatidylinositol - PIP phosphatidylinositol 4-phosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PPI phosphoinositide - QNB quinuclidinyl benzilate Special issue dedicated to Dr. Bernard W. Agranoff     

Reduction of epidermal growth factor receptor affinity by heterologous ligands: evidence for a mechanism involving the breakdown of phosphoinositides and the activation of protein kinase C

The tetradecapeptide bombesin converts epidermal growth factor (EGF) receptors on Swiss 3T3 cells from a high affinity state (KD = 9.8 X 10(-11)M) to a lower affinity state (KD = 1.8 X 10(-9)M). This conversion occurs when the cells are incubated with bombesin at 37 degrees C but not when incubated at 4 degrees C. Previously, a number of other (chemically unrelated) cell growth-promoting peptides and polypeptides have been shown to induce a similar indirect, temperature-dependent reduction of EGF receptor affinity. We have now demonstrated that hormones and growth factors which cross-regulate EGF receptor affinity in Swiss 3T3 cells have a common ability to stimulate the breakdown of phosphoinositides in these cells. We propose that the reduction of EGF receptor affinity is a consequence of the activation of protein kinase C by the diacylglycerol generated by this breakdown. In support of this proposal we have found that exogenously added diacylglycerol reduces the affinity of the Swiss 3T3 cell EGF receptor.     

Agonist-dependent phosphorylation of the inositol 1,4,5-trisphosphate receptor: A possible mechanism for agonist-specific calcium oscillations in pancreatic acinar cells.

The properties of inositol 1,4,5-trisphosphate (IP3)-dependent intracellular calcium oscillations in pancreatic acinar cells depend crucially on the agonist used to stimulate them. Acetylcholine or carbachol (CCh) cause high-frequency (10-12-s period) calcium oscillations that are superimposed on a raised baseline, while cholecystokinin (CCK) causes long-period (>100-s period) baseline spiking. We show that physiological concentrations of CCK induce rapid phosphorylation of the IP3 receptor, which is not true of physiological concentrations of CCh. Based on this and other experimental data, we construct a mathematical model of agonist-specific intracellular calcium oscillations in pancreatic acinar cells. Model simulations agree with previous experimental work on the rates of activation and inactivation of the IP3 receptor by calcium (DuFour, J.-F., I.M. Arias, and T.J. Turner. 1997. J. Biol. Chem. 272:2675-2681), and reproduce both short-period, raised baseline oscillations, and long-period baseline spiking. The steady state open probability curve of the model IP3 receptor is an increasing function of calcium concentration, as found for type-III IP3 receptors by Hagar et al. (Hagar, R.E., A.D. Burgstahler, M.H. Nathanson, and B.E. Ehrlich. 1998. Nature. 396:81-84). We use the model to predict the effect of the removal of external calcium, and this prediction is confirmed experimentally. We also predict that, for type-III IP3 receptors, the steady state open probability curve will shift to lower calcium concentrations as the background IP3 concentration increases. We conclude that the differences between CCh- and CCK-induced calcium oscillations in pancreatic acinar cells can be explained by two principal mechanisms: (a) CCK causes more phosphorylation of the IP3 receptor than does CCh, and the phosphorylated receptor cannot pass calcium current; and (b) the rate of calcium ATPase pumping and the rate of calcium influx from the outside the cell are greater in the presence of CCh than in the presence of CCK.     

Protein kinase C modulator effects on parathyroid hormone-induced intracellular calcium and morphologic changes in UMR 106-H5 osteoblastic cells

The effects of parathyroid hormone (PTH) on 1,4,5-inositol triphosphate (1,4,5-IP₃) and intracellular free calcium (Ca_i²⁺) in osteoblasts are variable, whereas adenylate cyclase activity is consistently stimulated. Cyclic AMP is considered a mediator in the contractile effects of PTH on osteoblasts, but the regulation and role of Ca_i²⁺ remains unclear. Recent studies indicate that protein kinase C (PKC) inhibits PTH-stimulated Ca_i²⁺ increases in osteoblastic cells. Therefore, the objectives of this study were to determine the effects of PKC modulators and PTH on UMR 106-H5 rat osteoblastic cell morphology, and the relationship between cell shape and PTH-induced Ca_i²⁺ changes. In suspended cells loaded with the calcium indicator dye fura-2, pretreatment with PKC inhibitors calphostin C (100 nM × 1 h) and H-7 (30 μM × 18 h) potentiated the effects of 1 μg/ml bPTH(1–84) on Ca_i²⁺ (83% increase over basal) by 1.4- and 1.65-fold, respectively. In comparison, PTH (10 ng-1 μg/ml) was without significant effect on adherent cell Ca_i²⁺ as measured by single-cell image analysis, although another in vitro bone resorbing agent, thrombin (10 U/ml), produced an acute 3-fold increase in the ratio (R) of emission (∼ λ510 nm) detected and optimized at λ348/374 nm (i.e., Ca-bound dye/free dye) in control cells. Phase-contrast microscopy revealed PKC inhibitor-treated cells changed from a spread configuration to a stellate form with retracting processes or cell rounding and a collapse of actin stress fibers. Within 1 h of PTH addition, PKC inhibitor-treated cells continually became extended/respread up to 3 h with an associated increase in actin stress fibers that was preceded by an acute 1.6-fold Ca_i²⁺ increase. In contrast, control or PKC activator-treated cells (phorbol 12,13-dibutyrate or 12-O-tetradecanoylphorbol-13-acetate; TPA) contracted/retracted within 5 min in response to PTH. A role for Ca_i²⁺ in PTH-induced cell spreading was further indicated by a contractile response to PTH when PKC-inhibitor-treated cells were loaded with the intracellular calcium chelator dimethyl BAPTA (3 μM × 30 min). PTH-induced Ca_i²⁺ increases in adherent PKC inhibitor-treated cells were also associated with a 1.8-fold 1,4,5-IP₃ increase as measured by mass assay. The data suggest PKC contributes to UMR 106-H5 cell morphology and selectively regulates signal pathways activated by PTH to promote either cell contraction (cAMP) or extension (1,4,5-IP₃/Ca_i²⁺). J. Cell. Biochem. 65:276–285. © 1997 Wiley-Liss, Inc.     

Regional Activities of Phospholipase C after Experimental Brain Injury in the Rat

Regional activities of phosphoinositide-specific phospholipase C (PLC) were measured after lateral fluid percussion (FP) brain injury in rats. The activity of PLC on phosphatidylinositol 4,5-bisphosphate (PIP₂) in the rat cortex required calcium, and at 45 M concentration it increased PLC activity by about ten-fold. The activity of PLC was significantly increased in the cytosol fraction in the injured (left) cortex (IC) at 5 min, 30 min and 120 min after brain injury. However, in the same site, increases were observed in the membrane fraction only at 5 min after brain injury. In both the contralateral (right) cortex (CC) and ipsilateral hippocampus (IH), the activity of PLC was increased in the cytosol only at 5 min after brain injury. These results suggest that increased activity of PLC may contribute to increases in levels of cellular diacylglycerol and inositol trisphosphate in the IC (the greatest site of injury), and to a smaller extent in the IH and CC, after lateral FP brain injury. It is likely that this increased PLC activity is caused by alteration in either the levels or activities of one or more of its isozymes (PLC, PLC, and PLC) after FP brain injury.     

Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes

Microsomes were prepared from cultured neonatal rat cardiomyocytes. Incubation of microsomes in buffer containing 5µM CaCl₂, 5 mM cholate and 100 nM [3H-]Phosphatidylinosito14,5-bisphosphate (PtdIns(4,5) P₂) resulted in the formation of [³H-]InsP ₃. GTP-gamma-S (125 µM) stimulated the production of [³H-]InsP ₃. Microsomes prepared from phorbol ester-treated (100 nM phorbol 12-myristate 13-acetate, PMA) cardiomyocytes showed decreased activities of basal as well as GTP-gamma-S-stimulated [³H-]Ptdlns(4,5)P ₂ hydrolysis. In the microsomes a 15 kD protein was demonstrated to be the major substrate phosphorylated by intrinsic protein kinase C, which was activated by 0.5 mM Ca²⁺. Addition of phorbol ester (100 nM PMA) enhanced the ³²P-incorporation into the 15 kD protein. Protein kinase C, purified from rat brain, in the presence of Ca²⁺, diglyceride, and phosphatidylserine did not change the phosphorylation pattern any further. In conclusion, it was shown that phorbol ester pretreatment of neonatal rat cardiomyocytes reduces microsomel GTP-gamma-S-stimulated Ptdlns(4,5)P ₂-specific phospholipase C activity, as estimated with exogenous substrate, and that in cardiomyocyte microsomes phorbol ester activates protein kinase C-induced 15 kD protein phosphorylation. The results indicate that phorbol ester may down-regulate -adrenoceptor mediated Ptdlns(4,5)P ₂ hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.List of abbreviations ATP Adenosine 5-Trphosphate - CSU Catalytic Subunit of cyclic AMP-dependent protein kinase - DG Diacylglycerol - DMSO Dimethylsulfoxide - DTT DL-dithiothreitol - EDTA Ethylenedinitrilotetraacetic Acid - EGTA Ethyleneglycol-0,0-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - GTP-gamma-S Guanosine 5-O-(3-thiotriphosphate) - HPTLC High Performance Thin Layer Chromatography - InsP ₃ Inositol monophosphate - InsP ₂ Inositol bisphosphate - InsP ₃ Inositol trisphosphate - MES 2-Morpholinoethanesulfonic acid - MOPS 3-[N-Morpholino]Propanesulfonic acid - PAGE Polyacrylamide-gel Electrophoresis - PKC Protein Kinase C - PLase C Phospholipase C - PMA Phorbol 12-Myristate 13-Acetate - PMSF Phenylmethylsulfonyl Fluoride - PtdSer Phosphatidylserine - PtdIns Phosphatidyl inositol - PT Pertussis Toxin - Ptdlns(4)P Phosphatidylinositol 4-monophosphate - Ptdlns (4,5)PZ-Phosphatidylinositol4,5-bisphosphate - SDS-Sodium Dodecyl Sulfate Tris-Tris(hydroxymethyl) aminomethane     

The role of calcium on protein secretion of the albumen gland in Helisoma duryi (Gastropoda)

Abstract. The albumen gland of the freshwater pulmonate snail Helisoma duryi produces and secretes the perivitelline fluid, which coats fertilized eggs and provides nutrients to the developing embryos. It is known that perivitelline fluid secretion is stimulated by dopamine through the activation of a dopamine D₁‐like receptor, which in turn stimulates cAMP production leading to the secretion of perivitelline fluid. This paper examines the glandular release of perivitelline fluid and provides evidence for the role of Ca²⁺ in the regulated secretion of perivitelline fluid based on protein secretion experiments and inositol 1,4,5‐trisphosphate assays. Dopamine‐stimulated protein secretion by the albumen gland is reduced in Ca²⁺‐free medium or in the presence of plasma membrane Ca²⁺ channel blockers, although the Ca²⁺ channel subtype involved is unclear. In addition, dopamine‐stimulated protein secretion does not directly involve phospholipase C‐generated signaling pathways and Ca²⁺ release from intracellular stores. Sarcoplasmic/endoplasmic reticulum Ca²⁺‐ATPase inhibitors had little effect on protein secretion when applied alone; however, they potentiated dopamine‐stimulated protein secretion. Dantrolene, an inhibitor of ryanodine receptors, 8‐(N,N‐diethylamino)‐octyl‐3,4,5‐trimethoxybenzoate hydrochloride, a nonspecific inhibitor of intracellular Ca²⁺ channels, and 2‐aminoethyldiphenylborate, an inhibitor of inositol 1,4,5‐trisphosphate receptors, did not suppress protein secretion, suggesting Ca²⁺ release from internal stores does not directly regulate protein secretion. Thus, the influx of Ca²⁺ from the extracellular space appears to be the major pathway mediating protein secretion by the albumen gland. The results are discussed with respect to the role of Ca²⁺ in controlling exocytosis of proteins from the albumen gland secretory cells.     

Phosphatidylinositol 4,5-bisphosphate: Light-mediated breakdown in the vertebrate retina

Isolated frog ($\text{Rana}\text{Pipiens}$) retinas were labeled in the dark with either [³²P]PO₄-orthophosphate or $\text{myo}$-[2-³H]inositol for 2.5–4 hrs. After washing the retinas with cold buffer, they were exposed to brief flashes of light (5 secs or 15 secs) and their rod outer segments isolated. Upon separation of labeled phospholipids, a specific decrease in label in phosphatidylinositol 4,5-bisphosphate was observed, whereas there was no significant effect on the labeling of phosphatidylinositol 4-phosphate, phosphatidylinositol, or phosphatidic acid. These results are indicative of a light-activated phosphatidylinositol 4,5-bisphosphate-specific phospholipase C in frog rod outer segments.     

Lithium treatment of affective disorders: effects of lithium on the inositol phospholipid and cyclic AMP signalling pathways

The effects of lithium (Li⁺) on the adenylyl cyclase and inositol phospholipid receptor signalling pathways were compared directly in noradrenergic and carbachol stimulated rat brain cortical tissue slices. Li⁺ was a comparatively weak inhibitor of noradrenaline-stimulated cyclic AMP accumulation with an IC₅₀ of approx. 20 mM. By contrast, half-maximal effects of Li⁺ on inositol monophosphate (InsP) accumulation in [³H]inositol labelled tissue slices occurred at about 1 mM. A similar IC₅₀ for Li⁺ of about 1 mM was also obtained for noradrenaline-stimulated accumulation of CMP-phosphatidate (CMPPA), a sensitive indicator of intracellular inositol depletion, in tissue slices that had been prelabelled with [³H]cytidine. The effect of myo-inositol (inositol) depletion on the prolonged activity of phosphoinositidase C (PIC) was examined in carbachol-stimulated corticol slices using a novel mass assay fro InsP. Exposure to a maximal dose of carbachol for 30 min in the presence of 5 mM Li⁺ caused a 10-fold increase in the level of radioactivity associated with the InsP fraction, but only a 2-fold increase in InsP mass. During prolonged incubations in the presence of both carbachol and Li⁺ the accumulation of InsP mass was enhanced if 30 mM inositol was included in the medium. The results are comptable with the inositol depletion hypothesis of Li⁺ action but do not support the concept that adenylyl cyclase or guanine nucleotide dependent proteins represent therapeutically relevant targets of this drug.     

白介素-2对心肌细胞[Ca~(2 )]_i的作用及其信号转导途径

为研究白介素 2 (interleukin 2 ,IL 2 )对心肌细胞内钙浓度 ([Ca2 ]i)的影响及其信号转导途径 ,实验采用酶解法分离成年大鼠心室肌细胞 ,以Fura 2 /AM为钙探针 ,用细胞内双波长钙荧光系统检测细胞 [Ca2 ]i 的变化。结果发现 :(1)IL 2 (0 5～ 2 0 0U/ml)浓度依赖性地降低单个心室肌细胞内钙瞬态 ,IL 2 (2 0 0U/ml)对咖啡因诱导的肌浆网内储钙的释放无影响 ;(2 )纳洛酮 (naloxone ,Nal) (10 -8mol/L)和nor binaltorphimine (nor BNI,10 -8mol/L)可阻断IL 2对心肌细胞钙瞬态的作用 ,而纳曲吲哚 (naltrindole ,NTI) (10 -6mol/L)不能阻断此作用 ;(3)κ阿片受体激动剂U5 0 488H (10 -6mol/L)降低心肌细胞钙瞬态 ,nor BNI (10 -8mol/L)可阻断此作用 ;(4 ) 5mg/L百日咳毒素 (PTX)预处理可取消IL 2降低心肌细胞钙瞬态的作用 ,而酪氨酸激酶抑制剂genistein (10 -4 mol/L)不能取消IL 2的作用 ;(5 )U7312 2预处理可阻断IL 2的作用。研究结果表明 ,IL 2降低心肌细胞钙瞬态的作用 ,是通过心肌细胞上κ阿片受体介导的 ,其下游途径包括PTX敏感的G蛋白和磷脂酶C。     

Aluminium impacts elements of the phosphoinositide signalling pathway in neuroblastoma cells

Inositol phosphate formation was examined in aluminium-treated murine neuroblastoma cells labelled with [³H]-myoinositol. Employing fluoride-stimulated intact cells, aluminium (0.2M to 1 mM) reduced inositol phosphate formation in a dose-dependent manner. In digitonin-permeabilized cells, stimulated with nonhydrolyzable GTP[S], inositol phosphate formation was also inhibited by increasing aluminium doses; the IC₅₀ value was about 20M aluminium, while the inositol phosphate level was reduced 2.5 to 3 fold by 50M aluminium. The inhibitory effect of aluminium (50M) could not be reversed by increasing GTP[S] concentrations up to 500M. Prechelation of aluminium to citrate or EGTA completely abolished the aluminium-triggered inhibition of fluoride-stimulated inositol phosphate formation in intact cells, but had little effect on the inhibition of permeabilized cells stimulated with GTP[S]. In neuroblastoma cells phosphoinositide hydrolysis could be evoked either through a pathway involving the Mg²⁺/guanine nucleotide binding (G_p) protein, or via a pathway operative in the presence of high intracellular Ca²⁺ concentrations. In the Mg²⁺/G_p protein-mediated pathway, formation of inositol triphosphate, IP₃, inositol diphosphate, IP₂, and inositol monophosphate, IP, was apparently inhibited by aluminium in an interdependent manner. As to the Ca²⁺-mediated pathway, aluminium application mainly diminished the release of IP₃. Following interiorization, aluminium thus acts upon elements critical for phosphoinositide-associated signal transduction. An aluminium target apparently resides on the G_p protein. Phosphatidylinositol-4,5-diphosphate-specific phospholipase C probably harbours a second aluminium target.     

Bradykinin effects on phospholipid metabolism and its relation to arachidonic acid turnovers in neuroblastoma × glioma hybrid cells (NG 108-15)

In neuroblastoma × glioma hybrid cells (NG 108-15) labelled with [³²P]-trisodium phosphate, [³H]-inositol and [¹⁴C]-arachidonic acid, bradykinin stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) while it had no effect on the release of [¹⁴C]-arachidonic acid (AA). The effect on PIP, was time- and dose-dependent with a maximal effect on [³H]-inositol- and [³²P]-labelled cells after 10–30 s of stimulation with 10⁻⁶ M bradykinin. However, the hydrolysis of [¹⁴C]-AA labelled PIP₂ was delayed compared to the effect on [³H]- and [¹⁴C]-PIP₂ and was not detectable until after 60 s of stimulation. Bradykinin stimulation resulted in an increased formation of [³H]-inositol phosphates (IP) and [³²P]- and [¹⁴P]- and [¹⁴C]-phosphatidic acid (PA) but the time course for PA formation did not allow the time-course for PIP₂ hydrolysis. A reduced labelling of [²³P]- and [¹⁴C]-phosphatidylcholine was also found in stimulated cells suggesting that PA may derive from other sources than PIP₂. In conclusion, our results indicate that bradykinin activates phospholipase C, but not phospholipase A₂, in NG 108-15 cells.     

Enzymes of phospholipid metabolism in rat pancreatic islets: subcellular distribution and the effect of glucose and calcium

The effect of glucose and calcium on the activities of the phosphatidylinositol cycle enzymes, CDP-diglyceride inositol transferase, diacylglycerokinase, and lysophosphatidylcholine 2-acyltransferase in rat pancreatic islets was studied. Calcium inhibited the activity of CDP-diglyceride inositol transferase but had no effect on lysophosphatidylcholine 2-acyltransferase and diacylglycerokinase activities. Upon preincubation of islets in a concentration of glucose known to stimulate insulin release, the activity of lysophosphatidylcholine 2-acyltransferase, but not that of diacylglycerokinase or the CDP-diglyceride inositol transferase, was stimulated. Subcellular fractionation of pancreatic islets showed that secretory granule membranes were enriched in CDP-diglyceride inositol transferase, whereas lysophosphatidylcholine 2-acyltransferase activity was highest in the microsomal membranes. The activation of 2-acyltransferase by incubating islets in insulinotropic glucose, and the calcium sensitivity of CDP-diglyceride inositol transferase, suggest that these enzymes may have roles in regulation of insulin secretion.