Bidirectional control of cytosolic free calcium by thyrotropin-releasing hormone in pituitary cells

It is now established that a key step in the action of calcium-mobilizing agonists is stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to 1,2-diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). The latter substance acts as a second messenger, controlling the release of calcium from intracellular stores (see ref. 3 for review). The bifurcating nature of the signalling system is exemplified by the fact that the other product of PtdIns(4,5)P2 hydrolysis, 1,2-diacylglycerol, can alter cellular function by activating protein kinase C, the cellular target for several tumour-promoting agents such as the phorbol esters. In various tissues, including GH3 pituitary tumour cells, a synergistic interaction between calcium ions and protein kinase C underlies agonist-induced changes in cell activity. The data presented here suggest that when GH3 cells are stimulated by thyrotropin-releasing hormone (TRH), an agonist inducing PtdIns(4,5)P2 hydrolysis, the two limbs of the inositol lipid signalling system interact to control free cytosolic calcium levels [( Ca2+]i). At low levels of TRH receptor occupancy, [Ca2+]i increases rapidly, then declines relatively slowly. As receptor occupancy increases, the calcium signal becomes more short-lived due to the appearance of a second, inhibitory, component. This latter component, which is enhanced when [Ca2+]i is elevated by high potassium depolarization, is mimicked by active phorbol esters and by bacterial phospholipase C. It seems likely that protein kinase C subserves a negative feedback role in agonist-induced calcium mobilization.     

Inositol trisphosphate, a novel second messenger in cellular signal transduction

There has recently been rapid progress in understanding receptors that generate intracellular signals from inositol lipids. One of these lipids, phosphatidylinositol 4,5-bisphosphate, is hydrolysed to diacylglycerol and inositol trisphosphate as part of a signal transduction mechanism for controlling a variety of cellular processes including secretion, metabolism, phototransduction and cell proliferation. Diacylglycerol operates within the plane of the membrane to activate protein kinase C, whereas inositol trisphosphate is released into the cytoplasm to function as a second messenger for mobilizing intracellular calcium.     

Purified inositol 1,4,5-trisphosphate receptor mediates calcium flux in reconstituted lipid vesicles

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), a second messenger molecule involved in actions of neurotransmitters, hormones and growth factors, releases calcium from vesicular non-mitochondrial intracellular stores. An Ins(1,4,5)P3 binding protein, purified from brain membranes, has been shown to be phosphorylated by cyclic-AMP-dependent protein kinase and localized by immunohistochemical techniques to intracellular particles associated with the endoplasmic reticulum. Although the specificity of the Ins(1,4,5)P3 binding protein for inositol phosphates and the high affinity of the protein for Ins(1,4,5)P3 indicate that it is a physiological Ins(1,4,5)P3 receptor mediating calcium release, direct evidence for this has been difficult to obtain. Also, it is unclear whether a single protein mediates both the recognition of Ins(1,4,5)P3 and calcium transport or whether these two functions involve two or more distinct proteins. In the present study we report reconstitution of the purified Ins(1,4,5)P3 binding protein into lipid vesicles. We show that Ins(1,4,5)P3 and other inositol phosphates stimulate calcium flux in the reconstituted vesicles with potencies and specificities that match the calcium releasing actions of Ins(1,4,5)P3. These results indicate that the purified Ins(1,4,5)P3 binding protein is a physiological receptor responsible for calcium release.     

Occurrence and extracellular actions of inositol pentakis- and hexakisphosphate in mammalian brain

Although inositol 1,3,4,5,6-pentakisphosphate (InsP5) and hexakisphosphate (InsP6) have been recognized for some time as naturally-occurring metabolites of inositol, their occurrence in mammalian cell types, including one of neural origin, has only recently been documented. This is of interest because of the recognized second messenger role of inositol 1,4,5-trisphosphate (InsP3) in intracellular signalling; coupling surface stimuli to cytoplasmic calcium discharge. The metabolism, existence in normal mature tissues, and possible functional roles of these inositol polyphosphates are unknown. Here we report evidence that InsP5 and InsP6 are synthesized in intact brain after labelling with [3H]inositol in vivo. We also show that local infusion of InsP5 and InsP6 into a discrete brain stem nucleus implicated in cardiovascular regulation, results in dose-dependent changes in heart rate and blood pressure.     

Early steps of lymphocyte activation bypassed by synergy between calcium ionophores and phorbol ester

Although it has been proposed that the activation of T lymphocytes is mediated by an early rise in cytosolic calcium concentration, it has not been possible to mimic antigen- or mitogen-induced mouse lymphocyte activation by calcium ionophores that bypass receptor-mediated processes. There is now evidence from other systems that the rise in cytosolic calcium which follows receptor triggering is preceded by the breakdown of phosphatidylinositol bisphosphate into 1,2-diacylglycerol and inositol trisphosphate. The latter is known to cause release of calcium from intracellular stores. The cellular target for the former is now widely accepted to be protein kinase C. Therefore, ligand-induced cellular response follows a rise in cytosolic calcium concentration and protein kinase C activation. Here we confirm that the calcium ionophores A23187 and ionomycin do not activate mouse T lymphocytes. However, either one in combination with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which is structurally related to 1,2-diacylglycerol, induces in lymphoid cell populations the expression of receptors for interleukin-2 (IL-2), the secretion of IL-2 and cell proliferation as measured by 3H-thymidine uptake. The growth-promoting effect of IL-2 on an exogenous IL-2-dependent clone could not be substituted for by ionomycin either alone or with TPA. Thus, the combination of calcium ionophores and TPA bypasses the requirement for antigen- or lectin-induced signal at the onset of lymphocyte activation.     

Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400

Cloning and expression of functional P400 protein from cerebellar Purkinje neurons shows that this protein is a receptor for inositol 1,4,5-trisphosphate, a second messenger that mediates the release of intracellular calcium.     

Activation of Ca2+ entry into acinar cells by a non-phosphorylatable inositol trisphosphate.

In many cell types, receptor activation of phosphoinositidase C results in an initial release of intracellular Ca2+ stores followed by sustained Ca2+ entry across the plasma membrane. Inositol 1,4,5-trisphosphate is the mediator of the initial Ca2+ release, although its role in the mechanism underlying Ca2+ entry remains controversial. We have now used two techniques to introduce inositol phosphates into mouse lacrimal acinar cells and measure their effects on Ca2+ entry: microinjection into cells loaded with Fura-2, a fluorescent dye which allows the measurement of intracellular free calcium concentration by microspectrofluorimetry, and perfusion of patch clamp pipettes in the whole-cell configuration while monitoring the activity of Ca(2+)-activated K+ channels as an indicator of intracellular Ca2+. We report here that inositol 1,4,5-trisphosphate serves as a signal that is both necessary and sufficient for receptor activation of Ca2+ entry across the plasma membrane in these cells.     

棉籽饼综合加工的研究——联产肌醇、蛋白饲料和磷酸氢钙

探讨了以农副产品棉籽饼为原料，联联肌醇，蛋白饲料和磷酸氢钙的可行性，加工路线和重要的工艺条件。     

Alpha-haemolysin of uropathogenic E. coli induces Ca2+ oscillations in renal epithelial cells

Pyelonephritis is one of the most common febrile diseases in children. If not treated appropriately, it causes irreversible renal damage and accounts for a large proportion of end stage renal failures. Renal scarring can occur in the absence of inflammatory cells, indicating that bacteria may have a direct signalling effect on renal cells. Intracellular calcium ([Ca2+]i) oscillations can protect cells from the cytotoxic effects of prolonged increases in intracellular calcium. However, no pathophysiologically relevant protein that induces such oscillations has been identified. Here we show that infection by uropathogenic Escherichia coli induces a constant, low-frequency oscillatory [Ca2+]i response in target primary rat renal epithelial cells induced by the secreted RTX (repeats-in-toxin) toxin alpha-haemolysin. The response depends on calcium influx through L-type calcium channels as well as from internal stores gated by inositol triphosphate. Internal calcium oscillations induced by alpha-haemolysin in a renal epithelial cell line stimulated production of cytokines interleukin (IL)-6 and IL-8. Our findings indicate a novel role for alpha-haemolysin in pyelonephritis: as an inducer of an oscillating second messenger response in target cells, which fine-tunes gene expression during the inflammatory response.     

The colony stimulating factor-1 receptor associates with and activates phosphatidylinositol-3 kinase

Colony stimulating factor-1 (CSF-1) is a lineage-specific growth factor required for proliferation and survival of mononuclear phagocytes and their precursors. The CSF-1 receptor belongs to a family of ligand-activated protein-tyrosine kinases. Activation of the platelet-derived growth factor receptor, but not the CSF-1 receptor, leads to an increase in phospholipase C activity and a subsequent elevation in intracellular calcium. Recent studies have shown that a novel phosphoinositol (PtdIns) kinase, termed PtdIns-3 kinase, is stimulated by the platelet-derived growth factor receptor and certain oncogenes in the protein-tyrosine kinase family. PtdIns-3 kinase phosphorylates the D-3 hydroxyl position of the inositol ring of PtdIns, and its products do not participate in the generation of the second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Here we report that addition of CSF-1 is followed by activation of PtdIns-3 kinase in a macrophage cell line (P388 D1), which contains CSF-1 receptors, and in BALB/c fibroblasts made to express the human CSF-1 receptor. Furthermore, we show that activation of the CSF-1 receptor results in the accumulation in intact cells of polyphosphoinositides phosphorylated at the D-3 position of the inositol ring. Thus activation of the CSF-1 receptor stimulates PtdIns-3 kinase activity, indicating a novel pathway for CSF-1 receptor-mediated signal transduction.     

Depletion of intracellular calcium stores activates a calcium current in mast cells.

In many cell types, receptor-mediated Ca2+ release from internal stores is followed by Ca2+ influx across the plasma membrane. The sustained entry of Ca2+ is thought to result partly from the depletion of intracellular Ca2+ pools. Most investigations have characterized Ca2+ influx indirectly by measuring Ca(2+)-activated currents or using Fura-2 quenching by Mn2+, which in some cells enters the cells by the same influx pathway. But only a few studies have investigated this Ca2+ entry pathway more directly. We have combined patch-clamp and Fura-2 measurements to monitor membrane currents in mast cells under conditions where intracellular Ca2+ stores were emptied by either inositol 1,4,5-trisphosphate, ionomycin, or excess of the Ca2+ chelator EGTA. The depletion of Ca2+ pools by these independent mechanisms commonly induced activation of a sustained calcium inward current that was highly selective for Ca2+ ions over Ba2+, Sr2+ and Mn2+. This Ca2+ current, which we term ICRAC (calcium release-activated calcium), is not voltage-activated and shows a characteristic inward rectification. It may be the mechanism by which electrically nonexcitable cells maintain raised intracellular Ca2+ concentrations and replenish their empty Ca2+ stores after receptor stimulation.     

Regulation of intracellular calcium by a signalling complex of IRAG, IP3 receptor and cGMP kinase Ibeta

Calcium release from the endoplasmic reticulum controls a number of cellular processes, including proliferation and contraction of smooth muscle and other cells. Calcium release from inositol 1,4,5-trisphosphate (IP3)-sensitive stores is negatively regulated by binding of calmodulin to the IP3 receptor (IP3R) and the NO/cGMP/cGMP kinase I (cGKI) signalling pathway. Activation of cGKI decreases IP3-stimulated elevations in intracellular calcium, induces smooth muscle relaxation and contributes to the antiproliferative and pro-apoptotic effects of NO/cGMP. Here we show that, in microsomal smooth muscle membranes, cGKIbeta phosphorylated the IP3R and cGKIbeta, and a protein of relative molecular mass 125,000 which we now identify as the IP3R-associated cGMP kinase substrate (IRAG). These proteins were co-immunoprecipitated by antibodies directed against cGKI, IP3R or IRAG. IRAG was found in many tissues including aorta, trachea and uterus, and was localized perinuclearly after heterologous expression in COS-7 cells. Bradykinin-stimulated calcium release was not affected by the expression of either IRAG or cGKIbeta, which we tested in the absence and presence of cGMP. However, calcium release was inhibited after co-expression of IRAG and cGKIbeta in the presence of cGMP. These results identify IRAG as an essential NO/cGKI-dependent regulator of IP3-induced calcium release.     

Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca(2+)-permeable channel.

Receptor-mediated increases in the cytosolic free calcium ion concentration in most mammalian cells result from mobilization of Ca2+ from intracellular stores as well as transmembrane Ca2+ influx. Inositol 1,4,5-trisphosphate (InsP3) releases calcium from intracellular stores by opening a Ca(2+)-permeable channel in the endoplasmic reticulum. But the mechanism and regulation of Ca2+ entry into nonexcitable cells has remained elusive because the entry pathway has not been defined. Here we characterize a novel inositol 1,3,4,5-tetrakisphosphate (InsP4) and Ca(2+)-sensitive Ca(2+)-permeable channel in endothelial cells. We find that InsP4, which induces Ca2+ influx into acinar cells, enhances the activity of the Ca(2+)-permeable channel when exposed to the intracellular surface of endothelial cell inside-out patches. Our results suggest a molecular mechanism which is likely to be important for receptor-mediated Ca2+ entry.     

Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum.

Release of calcium from intracellular stores occurs by two pathways, an inositol 1,4,5-trisphosphate (InsP3)-gated channel and a calcium-gated channel (ryanodine receptor). Using specific antibodies, both receptors were found in Purkinje cells of cerebellum. We have now compared the functional properties of the channels corresponding to the two receptors by incorporating endoplasmic reticulum vesicles from canine cerebellum into planar bilayers. InsP3-gated channels were observed most frequently. Another channel type was activated by adenine nucleotides or caffeine, inhibited by ruthenium red, and modified by ryanodine, characteristics of the ryanodine receptor/channel6. The open probability of both channel types displayed a bell-shaped curve for dependence on calcium. For the InsP3-gated channel, the maximum probability of opening occurred at 0.2 microM free calcium, with sharp decreases on either side of the maximum. Maximum activity for the ryanodine receptor/channel was maintained between 1 and 100 microM calcium. Thus, within the physiological range of cytoplasmic calcium, the InsP3-gated channel itself allows positive feedback and then negative feedback for calcium release, whereas the ryanodine receptor/channel behaves solely as a calcium-activated channel. The existence in the same cell of two channels with different responses to calcium and different ligand sensitivities provides a basis for complex patterns of intracellular calcium regulation.     

Translational control of InsP3-induced chromatin condensation during the early cell cycles of sea urchin embryos

The cycles of DNA synthesis and chromatin condensation in dividing cells are controlled by signals from the cytoplasm. Changes in the concentration of free calcium (Cai) in the cytoplasm control a variety of cellular functions and it has thus been suggested that observed variations in Cai during the cell cycle may be the cytoplasmic signal that co-ordinates nuclear and cytoplasmic division. We show here that increases in Cai induced by the calcium-releasing second messenger inositol 1,4,5-triphosphate (Ins(1,4,5)P3), or by calcium buffers, cause premature chromatin condensation and breakdown of the nuclear envelope in sea urchin (Lytechinus pictus) early embryos. Both natural and induced chromatin condensation are prevented by calcium chelators. The nucleus becomes sensitive to the Cai signal 45 min after fertilization, but remains insensitive if protein synthesis is prevented. Our experiments demonstrate that Cai regulates the behaviour of the nucleus during the cell cycle, suggest that Ins(1,4,5)P3 is a cell cycle messenger and indicate that there is an interaction between the protein and ionic signals that control the state of chromatin during the cell cycle.     

Activation of two signal-transduction systems in hepatocytes by glucagon

The ability of glucagon to stimulate glycogen breakdown in liver played a key part in the classic identification of cyclic AMP and hormonally stimulated adenylate cyclase. But several observations indicate that glucagon can exert effects independent of elevating intracellular cAMP concentrations. These effects are probably mediated by an elevation of the intracellular concentration of free Ca2+ although the mechanism by which this occurs is unknown. We show here that glucagon, at the low concentrations found physiologically, causes both a breakdown of inositol phospholipids and the production of inositol phosphates. Indeed, we show that the glucagon analogue, (1-N-alpha-trinitrophenylhistidine,12-homoarginine)glucagon (TH-glucagon), which does not activate adenylate cyclase or cause any increase in cAMP in hepatocytes yet can fully stimulate glycogenolysis, gluconeogenesis and urea synthesis, stimulates the production of inositol phosphates. This stimulation of inositol phospholipid metabolism by low concentrations of glucagon provides a mechanism whereby glucagon can exert cAMP-independent actions on target cells. We suggest that hepatocytes possess two distinct receptors for glucagon, a GR-1 receptor coupled to stimulate inositol phospholipid breakdown and a GR-2 receptor coupled to stimulate adenylate cyclase activity.     

Molecular cloning and complete amino-acid sequence of form-I phosphoinositide-specific phospholipase C

We report the molecular cloning and sequence of a phosphoinositide-specific phospholipase C (PI-PLC), an enzyme that is of particular interest because of its central role in cell signal transduction. The signals in question are those delivered by hormones to their cell-surface receptors that activate PI-PLC by means of a guanine nucleotide binding protein. Activation of the enzyme leads to the hydrolysis of phosphatidylinositol 4,5-bisphosphate to two second messengers, 1,2-diacylglycerol and inositol 1,4,5-trisphosphate, the second of which ultimately mobilizes internal pools of calcium. There are at least five PI-PLC isoenzymes, whose differences in structure and function are unknown. We have focused on isoenzyme I, which we have recently purified and characterized from guinea pig uterus. We have now determined the sequence of a full length complementary DNA of this isoenzyme from the rat. Although the sequence has little similarity with the only other sequenced PI-PLC isoenzyme, it has a surprising degree of similarity to thioredoxins, protein co-factors in thiol-dependent redox reactions.     

不同维生素组合对南美白对虾生长性能的影响

选取我国海水养殖种类中的南美白对虾为研究对象,采用均匀设计法,在相同基础日粮中,分别添加不同配比的维生素组合,其中有VE（X1）、烟酸（X2）、肌醇（X3）、泛酸钙（X4）、VB6（X5）,经过45 d的试验,以相对体重和相对体长为指标,建立了多元二次多项式回归模型,并利用SAS软件在计算机上拟合,用求极值的方法给出南美白对虾不同维生素组合的最佳配方.     

InsP4 facilitates store-operated calcium influx by inhibition of InsP3 5-phosphatase

Receptor-mediated generation of inositol 1,4,5-trisphosphate (InsP3) initiates Ca2+ release from intracellular stores and the subsequent activation of store-operated calcium influx. InsP3 is metabolized within seconds by 5-phosphatase and 3-kinase, yielding Ins(1,4)P2 and inositol 1,3,4,5-tetrakisphosphate (InsP4), respectively. Some studies have suggested that InsP4 controls Ca2+ influx in combination with InsP3 (refs 3 and 4), but another study did not find the same result. Some of the apparent conflicts between these previous studies have been resolved; however, the physiological function of InsP4 remains elusive. Here we have investigated the function of InsP4 in Ca2+ influx in the mast cell line RBL-2H3, and we show that InsP4 inhibits InsP3 metabolism through InsP3 5-phosphatase, thereby facilitating the activation of the store-operated Ca2+ current I(CRAC) (ref. 9). Physiologically, this mechanism opens a discriminatory time window for coincidence detection that enables selective facilitation of Ca2+ influx by appropriately timed low-level receptor stimulation. At higher concentrations, InsP4 acts as an inhibitor of InsP3 receptors, enabling InsP4 to act as a potent bi-modal regulator of cellular sensitivity to InsP3, which provides both facilitatory and inhibitory feedback on Ca2+ signalling.