Calcium-mobilizing receptors, polyphosphoinositides, generation of second messengers and contraction in the mammalian iris smooth muscle: historical perspectives and current status

It is well established now that activation of Ca2+ -mobilizing receptors results in the phosphodiesteratic breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), instead of phosphatidylinositol (PI), into myoinositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG). There is also accumulating experimental evidence which indicates that IP3 and DG may function as second messengers, the former to mobilize Ca2+ from intracellular sites and the latter to activate protein kinase C (PKC). In this review, I have recounted our early studies, which began in 1975 with the original observation that activation of muscarinic cholinergic and adrenergic receptors in the rabbit iris smooth muscle leads to the breakdown of PIP2, instead of PI, and culminated in 1979 in the discovery that the stimulated hydrolysis of PIP2 results in the release of IP3 and DG and that this PIP2 breakdown is involved in the mechanism of smooth muscle contraction. In addition, I have summarized more recent work on the effects of carbachol, norepinephrine, substance P, the platelet-activating factor, prostaglandins, and isoproterenol on PIP2 hydrolysis, IP3 accumulation, DG formation, myosin light chain (MLC) phosphorylation, cyclic AMP production, arachidonic acid release (AA) and muscle contraction in the iris sphincter muscle. These studies suggest: (a) that the IP3-Ca2+ signalling system, through the Ca2+ -dependent MLC phosphorylation pathway, is probably the primary determinant of the phasic component of the contractile response; (b) that the DG-PKC pathway may not be directly involved in the tonic component of muscle contraction, but may play a role in the regulation of IP3 generation; (c) that there are biochemical and functional interactions between the IP3-Ca2+ and the cAMP second messenger systems, cAMP may act as regulator of muscle responses to agonists that exert their action through the IP3-Ca2+ system; and (d) that enhanced PIP2 turnover is involved in desensitization and sensitization of alpha 1-adrenergic- and muscarinic cholinergic-mediated contractions of the dilator and sphincter muscles of the iris, respectively. The contractile response is a typical Ca2+ -dependent process, which makes smooth muscle an ideal tissue to investigate the second messenger functions of IP3 and DG and their interactions with the cAMP system.     

Effect of staurosporine on fMet-Leu-Phe-stimulated human neutrophils: dissociated release of inositol 1,4,5-trisphosphate, diacylglycerol and intracellular calcium.

Staurosporine, a microbial alkaloid, enhances inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) production rapidly and dose-dependently in fMet-Leu-Phe (FMLP)-stimulated human neutrophils showing maximal effects at 1 microM concentration. The IP3 increase was specific for staurosporine as three other putative protein kinase C (PKC) inhibitors, H7, sphingosine and palmitoylcarnitine were unable to enhance the IP3 generation in FMLP-stimulated human neutrophils. Staurosporine, at concentrations 0.3-1.0 microM, did not affect the initial mobilization of FMLP-induced intracellular Ca2+ (Ca2+i), although a sustained elevation of cytosolic Ca2+ level was observed within 5 min. This effect could not be suppressed, even by 1 microM phorbol-myristate 12,13-acetate (PMA). Whereas lower concentrations of staurosporine (less than or equal to 100 nM) were unable to affect FMLP-induced IP3 production, DG accumulation and Ca2+i, the PMA-inhibited initial Ca2+i signal and IP3 formation triggered by FMLP were almost completely restored. At higher concentrations (greater than or equal to 300 nM) staurosporine reversed the inhibitory effect of other protein kinases, distinct from the PMA-inducible one, which may be responsible for the phosphatidyl inositol 4,5-bisphosphate (PIP2) breakdown, thus causing accumulation of IP3 and DG and an elevation of C2+i level. Whereas IP3 declined to basal level within 5 min, the DG level remained elevated during the same period. This phenomenon is attributed to phospholipase D (PLD) stimulation by staurosporine, which augments the DG synthesis, in part through PA degradation via phosphatidic acid (PA) phosphohydrolase.     

The inhibitory effect of PGE2 on T cell activation is not associated with inhibition of PKC translocation

We have recently reported on the effect of PGE2 on T cell activation and suggested that their immunosuppressive effect may involve the PKC activation pathway. In the present study, we further investigated the potential interference of PGE2 with PHA induced signaling in T lymphocytes. We demonstrate that the PHA mediated increase in IP3, the putative mobilizer of intracellular Ca2+, is slightly affected following cell incubation with PGE2. Treatment of cell culture with the tumor promoter TPA abrogates the suppressive activity of PGE2 whereas exogenous diacylglycerol (1,2-diolein) has only a marginal effect. This suggests that PGE2 affect PKC activity at sites distal to IP3 and DG generation. We also demonstrate that the PGE2 suppressive effect on T lymphocyte activation is not related to an inhibition of PKC translocation.     

Alzheimer's presenilin-1 mutation potentiates inositol 1,4,5-trisphosphate-mediated calcium signaling in Xenopus oocytes

Perturbations in intracellular Ca2+ signaling may represent one mechanism underlying Alzheimer's disease (AD). The presenilin-1 gene (PS1), associated with the majority of early onset familial AD cases, has been implicated in this signaling pathway. Here we used the Xenopus oocyte expression system to investigate in greater detail the role of PS1 in intracellular Ca2+ signaling pathways. Treatment of cells expressing wild-type PS1 with a cell surface receptor agonist to stimulate the phosphoinositide second messenger pathway evoked Ca2+-activated Cl- currents that were significantly potentiated relative to controls. To determine which elements of the signal transduction pathway are responsible for the potentiation, we used photolysis of caged inositol 1,4,5-trisphosphate (IP3) and fluorescent Ca2+ imaging to demonstrate that PS1 potentiates IP3-mediated release of Ca2+ from internal stores. We show that an AD-linked mutation produces a potentiation in Ca2+ signaling that is significantly greater than that observed for wild-type PS1 and that cannot be attributed to differences in protein expression levels. Our findings support a role for PS1 in modulating IP3-mediated Ca2+ liberation and suggest that one pathophysiological mechanism by which PS1 mutations contribute to AD neurodegeneration may involve perturbations of this function.     

Dishevelled activates Ca2+ flux,PKC, and CamKII in vertebrate embryos

Wnt ligands and Frizzled (Fz) receptors have been shown to activate multiple intracellular signaling pathways. Activation of the Wnt-beta-catenin pathway has been described in greatest detail, but it has been reported that Wnts and Fzs also activate vertebrate planar cell polarity (PCP) and Wnt-Ca2+ pathways. Although the intracellular protein Dishevelled (Dsh) plays a dual role in both the Wnt-beta-catenin and the PCP pathways, its potential involvement in the Wnt-Ca2+ pathway has not been investigated. Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII). Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development. These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.     

Ca2+- and protein kinase C-dependent signaling pathway for nuclear factor-kappaB activation, inducible nitric-oxide synthase expression, and tumor necrosis factor-alpha production in lipopolysaccharide-stimulated rat peritoneal macrophages

Lipopolysaccharide (LPS)-activated macrophages are pivotal in innate immunity. With LPS treatment, extracellular signals are transduced into macrophages via Toll-like receptor 4 and induce inflammatory mediator production by activating signaling pathways, including the nuclear factor-kappaB (NF-kappaB) pathway and the mitogen-activated protein kinase (MAPK) pathway. However, the mechanisms by which the intracellular free Ca2+ concentration ([Ca2+]i) increases and protein kinase C (PKC) is activated remain unclear. Therefore, we investigated the signaling pathway for Ca2+- and PKC-dependent NF-kappaB activation, inducible nitric-oxide synthase expression, and tumor necrosis factor-alpha (TNF-alpha) production in LPS-stimulated rat peritoneal macrophages. The results demonstrated that the LPS-induced transient [Ca2+]i increase is due to Ca2+ release and influx. Extracellular and intracellular Ca2+ chelators inhibited phosphorylation of PKCalpha and PKCbeta. A PKCbeta-specific and a general PKC inhibitor blunted phosphorylation of serine in mitogen-activated/extracellular signal-regulated kinase kinase kinase (MEKK) 1. Moreover, a MEKK inhibitor reduced activation of inhibitorykappaB kinase and NF-kappaB. Upstream of the [Ca2+]i increase, a protein-tyrosine kinase inhibitor reduced phosphorylation of phospholipase C (PLC) gamma. Furthermore, a PLC inhibitor eliminated the transient [Ca2+]i increase and decreased the amount of activated PKC. Therefore, these results revealed the following roles of Ca2+ and PKC in the signaling pathway for NF-kappaB activation in LPS-stimulated macrophages. After LPS treatment, protein-tyrosine kinase mediates PLCgamma1/2 phosphorylation, which is followed by a [Ca2+]i increase. Several PKCs are activated, and PKCbeta regulates phosphorylation of serine in MEKK1. Moreover, MEKKs regulate inhibitory kappaB kinase activation. Sequentially, NF-kappaB is activated, and inducible nitric-oxide synthase and tumor necrosis factor-alpha production is promoted.     

Transactivation of hsp70-1/2 in geldanamycin-treated human non-small cell lung cancer H460 cells: involvement of intracellular calcium and protein kinase C

Geldanamycin is an antitumor drug that binds HSP90 and induces a wide range of heat shock proteins, including HSP70s. In this study we report that the induction of HSP70s is dose-dependent in geldanamycin-treated human non-small cell lung cancer H460 cells. Analysis of the induction of HSP70s specific isoform using LC-ESI-MS/MS analysis and Northern blotting showed that HSP70-1/2 are the major inducible forms under geldanamycin treatment. Transactivation of hsp70-1/2 was determined by electrophoretic mobility-shift assay using heat shock element (HSE) as a probe. The signaling pathway mediators involved in hsp70-1/2 transactivation were screened by the kinase inhibitor scanning technique. Pretreatment with serine/threonine protein kinase inhibitors H7 or H8 blocked geldanamycin-induced HSP70-1/2, whereas protein kinase A inhibitor HA1004, protein kinase G inhibitor KT5823, and myosin light chain kinase inhibitor ML-7 had no effect. Furthermore, the protein kinase C (PKC)-specific inhibitor Ro-31-8425 and the Ca2+-dependent PKC inhibitor G?-6976 diminished geldanamycin-induced HSP70-1/2, suggesting an involvement of the PKC in the process. In addition, geldanamycin treatment causes a transient increase of intracellular Ca2+. Chelating intracellular Ca2+ with BAPTA-AM or depletion of intracellular Ca2+ store with A23187 or thapsigargin significantly decreased geldanamycin-transactivated HSP70-1/2 expression. Taken together, our results demonstrate that geldanamycin-induced specific HSP70-1/2 isoforms expression in H460 cells through signaling pathway mediated by Ca2+ and PKC.     

Calcium signal transduction from caveolae

Caveolae are specialized membrane microdomains that are found on the plasma membrane of most cells. Recent studies indicate that a variety of signaling molecules are highly organized in caveolae, where their interactions initiate specific signaling cascades. Molecules enriched in this membrane include G protein-coupled receptors, heterotrimeric GTP binding proteins, IP3 receptor-like protein, Ca2+ ATPase, eNOS, and several PKC isoforms. Direct measurements of calcium changes in endothelial cells suggest that caveolae may be sites that regulate intracellular Ca2+ concentration and Ca2+ dependent signal transduction. This review will focus on the role of caveolae in controlling the spatial and temporal pattern of intracellular Ca2+ signaling.     

Thrombin-induced translocation of protein kinase C in human megakaryoblastic leukemia cells (MEG-01)

Our previous immunocytochemical study showed that Ca2+ ionophore-induced translocation of protein kinase C (PKC) in human megakaryoblastic leukemia cells (MEG-01) was potentiated by a synthetic diacylglycerol (T. Ito, T. Tanaka, T. Yoshida, K. Onoda, H. Ohta, M. Hagiwara, Y. Itoh, M. Ogura, H. Saito, and H. Hidaka, 1988, J. Cell Biol. 107, 929). In the present study, we analyzed the roles of the intracellular Ca2+ levels ([Ca2+]i) and diacylglycerol (DG) levels in thrombin-induced translocation of PKC using MEG-01 cells. When the cells were treated with thrombin (0.5 U/ml), PKC was translocated from the cytosol to the plasma membrane after 15 s, and the maximal membrane association was observed after 90 s. The [Ca2+]i of the cells rapidly increased (15 s) and reached a maximum level at 60 s which was sustained for a total of 600 s after thrombin addition. The increase in DG was biphasic with the first phase occurring in the first 15 s and the increase during the second phase lasting less than 600 s. The experiments without extracellular Ca2+ indicated that Ca2+ efflux accompanied by DG in the first phase was sufficient to initiate the membrane association of the PKC and that the large Ca2+ influx enhanced the binding. PKC returned to the cytosol within 600 s despite high levels of both [Ca2+]i and DG. We found that a relatively selective PKC inhibitor, H-7, enhanced thrombin-induced translocation of PKC without modulating [Ca2+]i or DG levels. These results indicate that certain protein phosphorylation events, potentially those mediated by PKC, may be responsible for, at least in part, inhibiting membrane association and further activation of the enzyme.     

Measurement of intracellular IP3 during Ca2+ oscillations in mouse eggs with GFP-based FRET probe

Intracellular Ca2+ oscillations in fertilized mammalian eggs, the key signal that stimulates egg activation and early embryonic development, are regulated by inositol 1,4,5-trisphosphate (IP3) signaling pathway. We investigated temporal changes in intracellular IP3 concentration ([IP3]i) in mouse eggs, using a fluorescent probe based on fluorescence resonance energy transfer between two green fluorescent protein variants, during Ca2+ oscillations induced by fertilization or expression of phospholipase Czeta (PLCzeta), an egg-activating sperm factor candidate. Fluorescence measurements suggested the elevation of [IP3]i in fertilized eggs, and the enhancement of PLCzeta-mediated IP3 production by cytoplasmic Ca2+ was observed during Ca2+ oscillations or in response to CaCl2 microinjection. The results supported the view that PLCzeta is the sperm factor to stimulate IP3 pathway, and suggested that high Ca2+ sensitivity of PLCzeta activity and positive feedback from released Ca2+ are important for triggering and maintaining Ca2+ oscillations.     

Amplification of Ca2+ signaling by diacylglycerol-mediated inositol 1,4,5-trisphosphate production

Stimulation of various cell surface receptors leads to the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through phospholipase C (PLC) activation, and the IP3 and DAG in turn trigger Ca2+ release through IP3 receptors and protein kinase C activation, respectively. The amount of IP(3) produced is particularly critical to determining the spatio-temporally coordinated Ca(2+)-signaling patterns. In this paper, we report a novel signal cross-talk between DAG and the IP3-mediated Ca(2+)-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of protein kinase C activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was completely abolished by depletion of Ca2+ stores or inhibition of PLC and IP3 receptors, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IP3-induced Ca2+ release. Furthermore, intracellular accumulation of endogenous DAG by a DAG-lipase inhibitor greatly increased the number of cells responding to agonist stimulation at low doses. These results suggest a novel physiological function of DAG, i.e. amplification of Ca2+ signaling by enhancing IP3 production via its positive feedback effect on PLC activity.     

IL-4 promotes anti-Ig-mediated protein kinase C translocation and reverses phorbol ester-mediated protein kinase C down-regulation in murine B cells

Co-stimulation of B lymphocytes with IL-4 plus nonmitogenic concentrations of anti-Ig antibodies, or protein kinase C (PKC) activators, drives resting B cells into DNA synthesis. Although cross-linking of the sIg receptors provokes the generation of the intracellular second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, the molecular mechanism utilized by IL-4R in murine B cells has not, as yet, been defined. In human B cells IL-4 has been shown to induce a transient rise in IP3 followed by a sustained elevation of cAMP. However, in murine B cells, IL-4 does not induce the release of IP3, Ca2+ mobilization, PKC translocation, or indeed modify signaling via the phosphoinositide pathway induced by ligation of sIg receptors. We now present evidence that, in murine B cells, IL-4 synergizes with nonmitogenic concentrations of anti-Ig to provoke translocation of PKC from the cytosol to membranes. In addition, the lymphokine up-regulates PKC levels and activity and prevents phorbol ester-induced PKC down-regulation in B cells. We therefore propose that (unknown) signals generated via IL-4R potentiate and/or prolong sIg-induced PKC activation. These observations may therefore provide a biochemical basis for explaining how IL-4 and anti-Ig synergize to induce B cell activation.     

Signal transduction by the polymeric immunoglobulin receptor suggests a role in regulation of receptor transcytosis

Many membrane traffic events that were previously thought to be constitutive recently have been found to be regulated by a variety of intracellular signaling pathways. The polymeric immunoglobulin receptor (pIgR) transcytoses dimeric IgA (dIgA) from the basolateral to the apical surface of polarized epithelial cells. Transcytosis is stimulated by binding of dIgA to the pIgR, indicating that the pIgR can transduce a signal to the cytoplasmic machinery responsible for membrane traffic. We report that dIgA binding to the pIgR causes activation of protein kinase C (PKC) and release of inositol 1,4,5- trisphosphate (IP3). The IP3 causes an elevation of intracellular Ca. Artificially activating PKC with phorbol myristate acetate or poisoning the calcium pump with thapsigargin stimulates transcytosis of pIgR, while the intracellular Ca chelator BAPTA-AM inhibits transcytosis. Our data suggest that ligand-induced signaling by the pIgR may regulate membrane traffic via well-known second messenger pathways involving PKC, IP3, and Ca. This may be a model of a general means by which membrane traffic is regulated by receptor-ligand interaction and signaling pathways.     

The variety of cytosolic calcium responses and possible roles of PLC and PKC

A model of ligand-induced intracellular calcium (Ca2+) responses incorporating phospholipase C (PLC) and protein kinase C (PKC) is developed for the purpose of understanding the mechanisms underlying the observed temporal patterns of intracellular calcium (Ca(i)2+) under sustained agonist stimulation. Some studies have suggested that inhibition of ligand receptors and PLC by PKC could generate sinusoidal Ca2+ oscillations, while PKC-independent Ca2+-induced Ca2+ release (CICR) via IP(3)-gated Ca2+ channels on the endoplasmic reticulum (ER) is believed to be responsible for baseline spiking. However, some evidence also indicates that baseline spiking can be observed under high-PKC activity, or under low-PKC activity with low agonist stimulus, as well. Insight into the basis of these observations regarding the role of PKC in Ca(i)2+ response patterns can be gained by developing and analyzing a mathematical model of Ca(i)2+ responses. We do this herein and find that (1) interaction of CICR and the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump is enough to generate both types of Ca(i)2+ oscillations, (2) there exist four possible Ca(i)2+ response patterns under sustained agonist stimulus: a sub-threshold response (SR), baseline spiking, sinusoidal oscillations (SO) and transient with plateau, and (3) the IP(3) concentration, which is controlled by the strength of the interaction between PKC and PLC, can be used to predict the Ca(i)2+ response patterns. From this analysis we conclude that the different patterns of Ca(i)2+ oscillations can be understood as a generic consequence of the interactions between CICR via the IP(3)-gated Ca(2+) channels in response to changes in the level of IP(3), and re-uptake into the ER/SR via the SERCA pump. PKC, in conjunction with PLC, can act as a switch between different Ca(i)2+ response patterns by modulating the cytosolic IP(3) level, which determines the Ca(i)2+ patterns.     

Differential inositol 1,4,5-trisphosphate receptor signaling in a neuronal cell line

Differential intracellular distribution of the three pharmacologically and biophysically distinct types of IP3Rs can lead to different subcellular Ca2+ transients each coupled to discrete intracellular functions. Here, we report the functional localization of differentially distributed IP3 receptor types in the commonly-used hippocampal cell line HT22. The distinct subcellular localization and Ca2+ signaling properties of these receptors determine the potential role of specific IP3 receptor types in cellular function. By utilizing immunochemistry, we conclude that HT22 cells express all three IP3 receptors with types 1 and 3 being expressed predominantly in the endoplasmic reticulum and perinuclear regions and type 2 being expressed predominantly in the nuclear envelope. Optical imaging studies using the Ca2+-sensitive indicator dye fluo-3 show that nuclear IP3 responses have greater amplitude and faster kinetics than cytosolic IP3 responses corresponding to the biophysical characteristics of the differentially distributed receptor types. These results support the hypothesis that differentially distributed IP3R isotypes mediate distinct cellular functions through differential, organelle-specific Ca2+ signaling.     

Acetylcholine increases Ca2+ influx by activation of CaMKII in mouse oocytes

IP3-induced Ca2+ release is the primary mechanism that is responsible for acetylcholine (ACh)-induced Ca2+ oscillation. However, other mechanisms remain to explain intracellular Ca2+ elevation. We here report that ACh induces Ca2+ influx via T-type Ca2+ channel by activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the ACh-induced Ca2+ influx facilitates the generation of Ca2+ oscillation in the mouse ovulated oocytes (oocytes(MII)). ACh increased Ca2+ current by 50+/-21%, and produced Ca2+ oscillation. However, the currents and Ca2+ peaks were reduced in Ca2+ -free extracellular medium. ACh failed to activate Ca2+ current and to produce Ca2+ oscillation in oocytes pretreated with KN-93, a CaMKII inhibitor. KN-92, an inactive analogue of KN93, and PKC modulators could not prevent the effect of ACh. These results show that ACh increases T-type Ca2+ current by activation of CaMKII, independent of the PKC pathway, in the mouse oocytes.     

A novel follicle-stimulating hormone-induced G alpha h/phospholipase C-delta1 signaling pathway mediating rat sertoli cell Ca2+-influx

FSH is known to activate Gs/cAMP signaling pathway in Sertoli cells (SCs) to support spermatogenesis. However, the molecular mechanism of FSH-induced Gs/cAMP-independent Ca2+-influx in SCs is not clear. In this study, FSH indeed induced an immediate and dose-dependent intracellular Ca2+-elevation in rat SCs. In the presence of EDTA (2.5 mm) or in the absence of extracellular Ca2+, the FSH-induced intracellular Ca2+-elevation was abolished. The confocal microscopic observation of Ca2+ image revealed that the SC cellular Ca2+ level was gradually increased after 50 sec of FSH treatment. Dantrolene, a blocker of intracellular Ca2+ release, did not affect this FSH-induced intracellular Ca2+ elevation. The pretreatment of rat SCs with phosphatidylinositol-phospholipase C (PLC)-specific inhibitor, U73122 (3 and 10 microm), inhibited the FSH-induced Ca2+-influx in a dose-dependent manner, but treatment with Gs-specific inhibitor, NF449 (0.1 and 0.3 microm), did not. On the other hand, the activation of G alpha h was immediately induced by FSH in the rat SCs within 5 sec of treatment. The translocation of PLC-delta1 from cytosol to cell membrane and the formation of G alpha h /PLC-delta1 complexes occurred within 5 and 10 sec, respectively, of FSH exposure. The intracellular inositol 1,4,5-triphosphate (IP3) production was also detected after 30 sec of FSH treatment. The synthetic peptide of PLC-delta1 (TIPWNSLKQGYRHVHLL), not Gs inhibitor, predominantly inhibited the FSH-induced PLC-delta1 translocation, formation of G alpha h /PLC-delta1 complex, intracellular IP3 production, and Ca2+ influx. In contrast, the peptide did not interfere with FSH-induced intracellular cAMP accumulation. In conclusion, the FSH-induced immediate Ca2+ influx is unambiguously mediated by an alternative G alpha h /PLC-delta1/IP3 pathway that is distinct from the Gs/cAMP pathway in rat SCs.     

The Pro335 --> Leu polymorphism of type 3 inositol 1,4,5-trisphosphate receptor found in mouse inbred lines results in functional change

Inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel involved in various cellular signaling. Type 3 IP3R (IP3R3) retains ligand-gated Ca2+ channel properties differing from other subtypes in terms of IP3-binding affinity and regulation of its channel activity by effector molecules. In this study, we found the natural Pro335 --> Leu polymorphism of mouse IP3R3 between BALB/c and C57BL/6J. We investigated the functional differences between Pro335IP3R3 and Leu335IP3R3 with purified receptors reconstituted into proteoliposomes as well as with soluble ligand binding domains. Pro335IP3R3 exhibited significantly higher IP3-binding affinity and IP3-induced Ca2+ release than those of Leu335IP3R3 in both forms of the receptor. Moreover, the polymorphic change caused differences in the effect of external Ca2+ on IP3-induced Ca2+ release. The Pro335 --> Leu substitution alters the conformation of soluble ligand binding domain as revealed by intrinsic fluorescence and circular dichroism spectra with or without Ca2+. The results indicate that the polymorphism of IP3R3 causes changes in receptor function, presumably affecting intracellular Ca2+ signaling.     

Phorbol esters and dioctanoylglycerol block anti-IgM-stimulated phosphoinositide hydrolysis in the murine B cell lymphoma WEHI-231

Cross-linking of membrane IgM (mIgM) on both normal resting B cells and on the murine B cell lymphoma WEHI-231 activates the phosphoinositide signal transduction pathway. The initial event in this pathway is the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2), which results in the generation of two second-messengers: inositol trisphosphate (InsP3), which can cause the release of Ca2+ from intracellular stores, and diacylglycerol (DG), which activates protein kinase C. In examining the effects of exogenous activation of protein kinase C on WEHI-231 cells, we found that phorbol esters blocked some of the biologic effects of anti-IgM on WEHI-231 cells. The mechanism of this effect was investigated. Phorbol ester treatment of WEHI-231 cells blocked the ability of anti-IgM to stimulate production of inositol phosphates and accumulation of phosphatidic acid, the phosphorylated product of DG. Phorbol esters also blocked the ability of anti-IgM to cause an increase in intracellular Ca2+. Thus, it is clear that phorbol esters block anti-IgM-stimulated PtdInsP2 hydrolysis in WEHI-231 cells. In addition, a synthetic DG, dioctanoylglycerol (diC8), also blocked anti-IgM-stimulated inositol phosphate production and the anti-IgM-stimulated rise in cytoplasmic Ca2+. The ability of phorbol esters and diC8 to block mIgM-mediated signaling may reflect a feedback inhibition mechanism by which activated protein kinase C limits the magnitude and duration of receptor signaling.