Rapid Increase In Cytosolic Ca2 Research Articles

Oomycete effector AVRblb2 targets cyclic nucleotide-gated channels through calcium sensors to suppress pattern-triggered immunity.

Transient and rapid increase in cytosolic Ca2+ plays a crucial role in plant-pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Cyclic nucleotide-gated channels (CNGCs) have been implicated in mediating this Ca2+ influx; however, their regulatory mechanisms remain poorly understood. Here, we have found that AVRblb2 requires the calmodulin (CaM) and calmodulin-like (CML) proteins as co-factors to interact with the NbCNGCs, resulting in the formation of AVRblb2-CaM/CML-NbCNGCs complex. Furthermore, CaM and CML are dissociated from NbCNGC18 during PTI response to increase Ca2+ influx; however, Avrblb2 inhibits calcium channel activation by disrupting the release of CaM and CML from NbCNGC18. Following recognition of PAMP, NbCNGC18 forms active heteromeric channels with other NbCNGCs, which may give selectivity of CNGC complex against diverse signals for fine-tuning of cytosolic Ca2+ level to mediate appropriate responses. Silencing of multiple NbCNGCs compromised the function of AVRblb2 on the pathogenicity of Phytophthora infestans, confirming that AVRblb2 contributes to pathogen virulence by targeting CNGCs. Our findings provide new insights into the regulation of CNGCs in PTI and the role of pathogen effectors in manipulating host cell physiology to promote infection.

The architecture and function of cardiac dyads.

Cardiac excitation-contraction (EC) coupling, which links plasma membrane depolarization to activation of cardiomyocyte contraction, occurs at dyads, the nanoscopic microdomains formed by apposition of transverse (T)-tubules and junctional sarcoplasmic reticulum (jSR). In a dyadic junction, EC coupling occurs through Ca2+-induced Ca2+ release. Membrane depolarization opens voltage-gated L-type Ca2+ channels (LTCCs) in the T-tubule. The resulting influx of extracellular Ca2+ into the dyadic cleft opens Ca2+ release channels known as ryanodine receptors (RYRs) in the jSR, leading to the rapid increase in cytosolic Ca2+ that triggers sarcomere contraction. The efficacy of LTCC-RYR communication greatly affects a myriad of downstream intracellular signaling events, and it is controlled by many factors, including T-tubule and jSR structure, spatial distribution of ion channels, and regulatory proteins that closely regulate the activities of channels within dyads. Alterations in dyad architecture and/or channel activity are seen in many types of heart disease. This review will focus on the current knowledge regarding cardiac dyad structure and function, their alterations in heart failure, and new approaches to study the composition and function of dyads.

Targeting the Activating Mutations of NOTCH1 in T-Cell Lymphoblastic Leukemia with a New SERCA Inhibitor CAD204520

Given its oncogenic role in human cancers, Notch1 signaling has garnered increased attention as a therapeutic target. Several Notch modulators, including γ-secretase inhibitors, have shown therapeutic efficacy in preclinical tumor models. However, despite this promise, few of these candidates have shown clinical benefit in patients, in part due to tissue-dependent on-target toxicities from the repression of both mutant and wild type Notch proteins. The discovery of the P-type ATPase Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) as a modulator of oncogenic NOTCH1 suggested a new approach to treat T-cell Acute Lymphoblastic Leukemia (T-ALL). In fact, thapsigargin mediated SERCA inhibition had a stronger effect on the most common type of activating NOTCH1 mutants compared to wild type. However, the consequence of thapsigargin binding to SERCA is a rapid increase in cytosolic Ca2+ and a depletion of Ca2+ stored in the endoplasmic reticulum (ER). This shift of Ca2+ efflux might cause cardiac toxicity in human, suggesting the need to identify inhibitors with better drug-like properties and reduced off-target toxicity. From a small molecule screening of 191,000 P-type ATPase modulators CAD204520 displayed ~25 and ~79-fold greater selectivity toward human SERCA compared to Na+/K+ and H+-ATPase respectively and emerged as a candidate for the development of novel anti-NOTCH1 agents. In a series of in vitro studies, we showed that CAD204520 impairs the proliferation of a panel of lymphoid malignancies carrying activating mutations in the NOTCH1 heterodimerization domain (HD, such as T-ALL) and/or deletions in the degradation domain (PEST, such as mantle cell lymphoma (MCL)). Importantly, T-ALL lymphoblasts from patients carrying NOTCH1 mutations were more sensitive to CAD204520 suppression compared with normal lymphocytes or ALL wild type NOTCH1. Similar to thapsigargin, CAD204520 causes a defect in NOTCH1 trafficking that results in an accumulation of full-length NOTCH1 and in a depletion of the activated form: ICN1. Consequently, the NOTCH1 targets, MYC and DTX1, were repressed as measured by qRT-PCR. In contrast, we observed no effect at protein level in T-ALL wild type NOTCH1 suggesting that clinically relevant NOTCH1 mutations including PEST deletions are more sensitive to CAD204250 treatment than wild type proteins. The next question is whether CAD204520 activity has limitations in vivo due to Ca2+ shifts. Thus we analysed cardiac mechanics in isolated rat cardiomyocytes and showed that compared to thapsigargin, CAD204520 minimally alters myocardial performance (~25%) suggesting that the heart will likely tolerate CAD204520 modulation in vivo. This effect may be caused to differences between Ca2+ mediated ER stress upon thapsigargin or CAD204520 treatment. To test this hypothesis, we treated T-ALL cell lines at equimolar doses of CAD204520 and thapsigargin. We showed that, despite a similar effect on NOTCH1 protein levels, CAD204520 does not induce the unfolded protein response (UPR) pathway as measured by the phosphorylation of eIF2α or the induction of the ER chaperone BIP. This result suggests that CAD204520 retains ATPase activity, responsible for Notch inhibition, decoupled from the Ca2+ transport activity responsible for the off-target effects. A tempting hypothesis to explain these differences is that thapsigargin and CAD204520 inhibit SERCA in distinct pockets. Consistently, docking poses showed that CAD204520 binds SERCA in the trans-membrane helices M1, M3 and M4 while thapsigargin in the M3, M5 and M7 groove. Consequently, thapsigargin and CAD204520 co-treatment resulted in synergistic anti-proliferative effect in T-ALL. To test CAD204520 in vivo we studied its effect in CD1 mice and established a maximum tolerated dose. Mice exposed to CAD204520 for 21 days at 30 mg/Kg BID showed no decrease in body weight and, importantly no effect on cardiac hemodynamic. Finally, to assess the in vivo efficacy of CAD204520 we xenografted SKW-3/KE-37 T-ALL cell line in L-2Rγ null mice and demonstrated that drug's treatment reduced circulating and tissue infiltrating human leukemia T-ALL cells without causing heart-related off target effects. In conclusion, this study supports the development of tolerated SERCA inhibitors for Notch dependent cancers and extends its application to cases with mutations in the PEST degradation domain such as MCL or chronic lymphocytic leukemia. Disclosures Lund Winter: CaDo Biotechnology IvS: Employment. Stegmaier:Novartis: Research Funding; Rigel Pharmaceuticals: Consultancy. Dalby-Brown:CaDo Biotechnology IvS: Employment.

G-protein-coupled receptor participates in 20-hydroxyecdysone signaling on the plasma membrane

BackgroundAnimal steroid hormones are conventionally known to initiate signaling via a genomic pathway by binding to the nuclear receptors. The mechanism by which 20E initiates signaling via a nongenomic pathway is unclear.ResultsWe illustrate that 20E triggered the nongenomic pathway through a plasma membrane G-protein-coupled receptor (named ErGPCR) in the lepidopteran insect Helicoverpa armigera. The transcript of ErGPCR was increased at the larval molting stage and metamorphic molting stage by 20E regulation. Knockdown of ErGPCR via RNA interference in vivo blocked larval–pupal transition and suppressed 20E-induced gene expression. ErGPCR overexpression in the H. armigera epidermal cell line increased the 20E-induced gene expression. Through ErGPCR, 20E modulated Calponin nuclear translocation and phosphorylation, and induced a rapid increase in cytosolic Ca2+ levels. The inhibitors of T-type voltage-gated calcium channels and canonical transient receptor potential calcium channels repressed the 20E-induced Ca2+ increase. Truncation of the N-terminal extracellular region of ErGPCR inhibited its localization on the plasma membrane and 20E-induced gene expression. ErGPCR was not detected to bind with the steroid hormone analog [3H]Pon A.ConclusionThese results suggest that ErGPCR participates in 20E signaling on the plasma membrane.

Characterization of signaling pathways coupled to melatonin receptors in gastrointestinal smooth muscle

Melatonin, a close derivative of serotonin, is involved in physiological regulation of circadian rhythms. In the gastrointestinal (GI) system, melatonin exhibits endocrine, paracrine and autocrine actions and is implicated in the regulation of GI motility. However, it is not known whether melatonin can also act directly on GI smooth muscle cells. The aim of the present study was to determine the expression of melatonin receptors in smooth muscle and identify their signaling pathways. MT1, but not MT2 receptors are expressed in freshly dispersed and cultured gastric smooth muscle cells. Melatonin selectively activated Gq and stimulated phosphoinositide (PI) hydrolysis in freshly dispersed and cultured muscle cells. PI hydrolysis was blocked by the expression of Gq, but not Gi minigene in cultured muscle cells. Melatonin also caused rapid increase in cytosolic Ca2+ as determined by epifluorescence microscopy in fura-2 loaded single smooth muscle cells, and induced rapid contraction. Melatonin-induced PI hydrolysis and contraction were blocked by a non-selective MT1/MT2 antagonist luzindole (1μM), but not by a selective MT2 antagonist 4P-PDOT (100nM), and by the PLC inhibitor U73122. MT2 selective agonist IIK7 (100nM) had no effect on PI hydrolysis and contraction. We conclude that rabbit gastric smooth muscle cells express melatonin MT1 receptors coupled to Gq. Activation of these receptors causes stimulation of PI hydrolysis and increase in cytosolic Ca2+, and elicits muscle contraction.

Effect of dexamethasone on voltage-gated K + channels in Jurkat T-lymphocytes

The voltage-gated K+ channel Kv1.3 is an important regulator of lymphocyte function. Activation of lymphocytes is accompanied by stimulation, whereas CD95-induced apoptosis by inhibition, of Kv1.3. The channel serves to maintain cell membrane potential, a prerequisite for signalling through the Ca2+ release-activated Ca2+ channel I(CRAC). As glucocorticoids are known to regulate lymphocyte function, the present study addressed the effect of dexamethasone on voltage-gated K+ channels in Jurkat T-lymphocytes. In whole-cell patch-clamp experiments current families evoked by 200-ms potential steps every 15 s from -70 mV to values from -120 to +100 mV revealed the functional expression of voltage-gated K+ channels. Pre-treatment of Jurkat T-lymphocytes for 2-3 h with 1 microM dexamethasone led to a significant decrease of voltage-gated K+ currents. Fura-2-fluorescence measurements showed that the readdition of Ca2+ to Ca(2+)-depleted cells led to a rapid increase of cytosolic Ca2+ activity. This increase of Ca2+ activity was blunted by both the K+ channel blocker margatoxin (10 nM) and 24 h pre-treatment with dexamethasone (1 microM). In conclusion, dexamethasone inhibits voltage-gated K+ channels in Jurkat T-lymphocytes, an effect impeding Ca2+ entry through I(CRAC).

Transgenic aequorin monitors cytosolic calcium transients in soybean cells challenged with β-glucan or chitin elicitors

Transgenic soybean (Glycine max L.) cells expressing aequorin were used to monitor changes in cytosolic Ca2+ concentrations in response to treatment with fungal elicitors. After an apparent lag phase of about 60 s, both chitin fragments and β-glucan elicitors caused a rapid increase in cytosolic Ca2+ concentration, which peaked within 2–2.5 min of treatment. The Ca2+ concentration then decreased and reached the basal level after about 5 min in the case of the treatment with chitin fragments, while a second rise in the Ca2+ concentration with a maximum occurring after about 7–8 min was observed in the case of β-glucan treatment. Calibration of the signals showed that the elicitors enhanced the cytosolic Ca2+ concentration from resting concentrations as low as 0.1 lM to highest levels of about 2 lM. Dose-response experiments showed that the concentration of elicitors giving a Ca2+ response at the 50% level was 0.4 nM for the chitin fragment and 28 lM and 72 lM, respectively, for a synthetic hepta-β-glucoside and a fungal β-glucan fraction. The β-glucan- or N,N′,N′′,N′′′-tetraacetyl chitotetratose (CH4)-induced Ca2+ signals were inhibited by both the Ca2+ chelator 1,2-bis-(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and by the Ca2+-channel inhibitor La3+. Neomycin, whose target in plant cells has not yet been clearly identified, reduced predominantly the expression of the second peak of the biphasic Ca2+ curve following β-glucan treatment. Bacterial cyclic β-glucans known to suppress β-glucan-induced phytoalexin production were also found to function as a suppressor for the Ca2+ response that was elicited by the fungal β-glucans. The results clearly show that the increase in the cytosolic Ca2+ concentration is an early and rapid event in the elicitor-sensing mechanism of soybean cells, and is probably connected with the subsequent activation of defence responses.

Stimulation of phospholipase D via alpha1-adrenergic receptors in Madin-Darby canine kidney cells is independent of PKCalpha and -epsilon activation.

We have demonstrated previously that protein kinase Calpha (PKCalpha) plays a key role in regulating phospholipase D (PLD) activation by nucleotides and the phorbol ester phorbol-12-myristate-13-acetate in Madin-Darby canine kidney (MDCK-D1) cells. In the current work, we investigated PLD activation in MDCK-D1 cells triggered by the adrenergic receptor agonist epinephrine and its mechanism of activation. Epinephrine, acting through the alpha1-adrenergic receptor subtype, promoted transient translocation of PKCalpha and more prolonged translocation of PKCepsilon to the membrane fraction, indicating activation of these two isoforms. In addition, epinephrine promoted activation of PLD, as shown by a sustained accumulation of phosphatidylethanol. All of these events were blocked by pretreatment of cells with the alpha1-adrenergic antagonist prazosin. D609, an inhibitor of phosphatidylcholine hydrolysis, blocked translocation of PKCalpha and PKCepsilon but did not inhibit PLD activation. Unlike results with PMA, or with the P2 purinergic receptor agonist ATP, epinephrine-stimulated PLD activity was not inhibited in MDCK-D1 cells in which PKCalpha expression is attenuated by an antisense cDNA construct or in cells in which PKC activity was inhibited by 1 microM GF 109203X. However, PLD activation by epinephrine was abolished by concomitant incubation of cells with the calcium chelator EGTA. These data, together with previous results, are consistent with the hypothesis that in MDCK-D1 cells, epinephrine acting on alpha1-adrenergic receptors, promotes a rapid increase in cytosolic Ca2+ that promotes activation of PLD through an as-yet poorly defined mechanism. The data demonstrate that different types of G protein-linked receptors that activate PLD can mediate this activation in either a PKC activation-dependent or -independent manner within a single cell type.

Macrophage Inflammatory Protein-1α Mediated Growth Inhibition in a Haemopoietic Stem Cell Line is Associated with Inositol 1,4,5 Trisphosphate Generation

Macrophage Inflammatory Protein-1 alpha (MIP-1 alpha) can inhibit the proliferation of multipotent haemopoietic cells. Using the FDCP-Mix A4 multipotent stem cell line, MIP-1 alpha was shown to inhibit 1L-3 stimulated cell cycling (assessed using the [3H]-thymidine "suicide" assay). Furthermore, MIP-1 alpha can inhibit 1L-3-stimulated [3H]-thymidine incorporation in FDCP-Mix cells, with half maximal inhibition observed at 3 ng/ml MIP-1 alpha. Prostaglandin E2, but not MIP-1 alpha was able to elevate cyclic AMP levels in FDCP-Mix A4 cells although both agents can cause growth inhibition. However, MIP-1 alpha addition resulted in a pertussis-toxin-insensitive increase in the level of the second messenger inositol 1,4,5 triphosphate (Ins 1,4,5P3). This response was both rapid (maximal at 5 seconds) and transient. A half maximal effect was observed at 5 ng/ml MIP-1 alpha and the dose dependency correlated with that for MIP-1 alpha mediated growth inhibition. A rapid increase in cytosolic Ca2+ levels was also observed in response to MIP-1 alpha. Inositol lipid hydrolysis and an increase in cytosolic Ca2+ (signals normally associated with proliferation) may therefore be implicated in growth inhibitory mechanisms in multipotent cells.

Carbachol-Induced Potassium Release in Rat Parotid Acini: Comparison of the Roles of Cytosolic Ca2+ and Protein Kinase C

Carbachol (CCh) stimulated K+ release from rat parotid acini. Treatment with the intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) or the intracellular Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) strongly suppressed the CCh-induced K+ release. Combined addition of the Ca2+ ionophore ionomycin and the microsomal Ca(2+)-ATPase inhibitor thapsigargin caused a rapid increase in cytosolic Ca2+ concentration ([Ca2+]i) and resulted in a marked release of K+. In the absence of extracellular Ca2+, CCh or a combination of ionomycin and thapsigargin caused a transient release of K+ which correlated well with the transient change in [Ca2+]i. On the other hand, phorbol 12-myristate 13-acetate (PMA) did not potentiate the CCh-induced K+ release, although the CCh-induced amylase release was significantly enhanced in the presence of PMA. Staurosporine, a protein kinase C-inhibitor, did not inhibit the CCh-induced K+ release, which was in contrast with its inhibitory effect on amylase release. These results suggest that the K+ release from rat parotid acini induced by CCh stimulation is mediated by a rapid increase in [Ca2+]i but is not associated with activation of protein kinase C. This signal pathway is different from that for amylase release where activation of protein kinase C plays an important role.

Cytotoxic and genotoxic effects of styrene-7,8-oxide in neuroadrenergic Pc 12 cells.

Exposure of Pc 12 cells to styrene-7,8-oxide (SO) (0.5-1 mM) caused a rapid increase in cytosolic Ca2+, depletion of intracellular glutathione and ATP, DNA damage and loss of cell viability. Lower SO concentrations (less than or equal to 100 microM), did not cause loss of cell viability or affect cell growth rate. However, at 30 and 100 microM, SO stimulated the formation of alkali-sensitive, DNA single-strand breaks (SSB). DNA SSB were fully repaired when cells exposed to 30 microM SO were subsequently incubated for 3 h in fresh medium, whereas DNA repair was only partial after exposure to 100 microM SO. When cells exposed to 30 or 100 microM SO were incubated with the inhibitors of repair synthesis 1-beta-D-arabinofuranosyl-cytosine (AraC) and hydroxyurea (HU), SSB accumulated, indicating the involvement of the excision-repair system in the removal of DNA lesions. A SO adduct with guanine at the N7 position was detected in the DNA extracted from treated cells. SO did not induce the formation of double-strand breaks, interstrand cross-links, or DNA-protein cross-links. Although cells exposed to 30 or 100 microM SO underwent normal cell division, latent DNA damage was retained for up to 14 subsequent replicative cycles. In addition, SO-treated cells partially lost their normal ability to differentiate in response to nerve growth factor (NGF) stimulation. NGF failed to induce differentiation in cells that had replicated for 20 generations after exposure to 100 microM SO. Spontaneous differentiation stimulated by high-density culture was also inhibited in SO-treated cells. These results indicate that non-lethal concentrations of SO can cause modifications that compromise the ability of Pc 12 cells to respond to NGF and differentiate.

Effects of prostaglandins, cAMP, and changes in cytosolic calcium on platelet aggregation induced by a thromboxane A2 mimic (U46619).

The effects of U46619, a thromboxane mimic, on cytosolic Ca2+ concentration and platelet aggregation were determined in human platelets. Cytosolic Ca2+ concentration was determined by Quin-2 fluorescence and platelet aggregation quantitated with an aggregometer. Addition of U46619 (1 x 10(-7) M) to the platelet suspension produced a rapid increase in cytosolic Ca2+ and platelet aggregation. Pretreatment of platelets with EGTA (3 x 10(-3) M), verapamil (5 x 10(-4) M), a calcium entry blocker, or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (1 x 10(-3) M), an inhibitor of intracellular Ca2+ release, either blunted or markedly delayed the rate, but not the magnitude, of increase in cytosolic Ca2+ and prevented platelet aggregation by U46619. Pretreatment of platelets with prostaglandin I2 (PGI2) (5 x 10(-8) M), PGD2 (5 x 10(-8) M), PGE1 (5 x 10(-8) M), PGF2 alpha (1 x 10(-5) M), dibutyryl cAMP (5 x 10(-3) M), or forskolin (1 x 10(-6) M) prevented both the increase in cytosolic Ca2+ and the associated platelet aggregation induced by U46619. These data suggest that U46619 may induce platelet aggregation through an increase in cytosolic Ca2+, and that both Ca2+ entry and its release from intracellular storage sites probably contribute to the increase in cytosolic Ca2+. Furthermore, the rate of the increase in cytosolic Ca2+ concentration, as well as the magnitude of the increase, appear to be critical for platelet aggregation induced by U46619. Our data are consistent with the hypothesis that PGs inhibit U46619-induced platelet aggregation by preventing the increase in cytosolic Ca2+, and that these effects may be mediated via an increase in cAMP, since they were induced by PGs and cAMP.

Evidence that muscarinic cholinergic receptors selectively interact with either the cyclic AMP or the inositol phosphate second-messenger response systems.

The relative capacities of muscarinic cholinergic receptor (MR) and bradykinin (BK)-receptor activation to increase phosphoinositide hydrolysis and to increase cytosolic Ca2+ were compared in NG108-15 neuroblastoma x glioma and 1321N1 human astrocytoma cells. In 1321N1 cells, the muscarinic cholinergic agonist carbachol and BK each stimulated a concentration-dependent accumulation of inositol phosphates (K0.5 approximately 10 microM and approximately 10 nM respectively) and a rapid increase in cytosolic Ca2+ as determined by quin2 fluorescence. In NG108-15 cells, BK alone stimulated a pertussis-toxin-insensitive accumulation of inositol phosphates (K0.5 approximately 10 nM) under conditions in which pertussis toxin completely inhibited MR-mediated inhibition of adenylate cyclase. BK also stimulated a rapid increase in cytosolic Ca2+ in NG108-15 cells. In contrast, no MR-mediated increase in phosphoinositide hydrolysis or change in cytosolic Ca2+ concentration was observed in NG108-15 cells. These results support the idea that MR selectively interact with either the cyclic AMP or the inositol phosphate second-messenger systems.

Role of guanine nucleotide regulatory protein in polyphosphoinositide degradation and activation of phagocytic leukocytes by chemoattractants.

Leukocyte activation by chemoattractants provides an important model to study the biochemical mechanisms of stimulus-response coupling in these cells. Well-defined chemotactic factors induce readily quantifiable responses in phagocytic leukocytes. These include directed migration and the production and release of toxic substances including oxygen radicals and lysosomal enzymes. The development of radiolabeled synthetic oligopeptides with potent chemotactic activity allowed the demonstration of chemoattractant receptors on polymorphonuclear leukocytes (PMNs) as well as macrophages. In membrane preparations from these cells, these receptors exist in high- and low-affinity states which are regulated by guanosine di- and triphosphates. This suggested that chemoattractant receptors interact with guanine nucleotide regulatory proteins (N or G proteins). Although chemoattractants elicit a rapid but transient increase in intracellular cAMP levels, they neither stimulate nor inhibit membrane-bound adenylate cyclase, suggesting a novel role for N proteins in certain receptor-transduction mechanisms. Stimulation of phagocytes by chemoattractants is also associated with a rapid increase in cytosolic Ca2+ concentrations ([ Ca2+]i) which appears to result from the production of inositol 1,4,5-triphosphate (IP3) as a consequence of the diesteric cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2). Treatment of phagocytes with pertussis toxin (PT), which ADP-ribosylates and thereby inactivates certain N proteins, abolishes the cells' responsiveness to chemoattractants. More direct evidence for a role of a PT-sensitive N protein in leukocyte activation was provided by the demonstration that chemoattractants stimulate the hydrolysis of PIP2 in PMN membranes only in the presence of GTP. This receptor-mediated hydrolysis of PIP2 is not observed in plasma membranes prepared from PT-treated PMNs. Therefore, these studies suggest that occupancy of chemoattractant receptors activates a PT-sensitive N protein. The activated N protein shifts the Ca2+ requirement for phospholipase C activity from supraphysiological levels to ambient cytosolic Ca2+ concentrations. Cleavage of PIP2 results in the formation of the second messenger molecules, IP3 and 1,2-diacylglycerol, which can initiate cellular activation. These messengers also seem to activate responses which feed back to attenuate receptor stimulation of phospholipase.