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The accumulation of unfolded proteins within the Endoplasmic Reticulum (ER) activates a signal transduction pathway termed the unfolded protein response (UPR), which attempts to restore ER homeostasis. If this cannot be done, UPR signalling ultimately induces apoptosis. Ca(2+) depletion in the ER is a potent inducer of ER stress. Despite the ubiquity of Ca(2+) as an intracellular messenger, the precise mechanism(s) by which Ca(2+) release affects the UPR remains unknown. Tethering a genetically encoded Ca(2+) indicator (GCamP6) to the ER membrane revealed novel Ca(2+) signalling events initiated by Ca(2+) microdomains in human astrocytes under ER stress, induced by tunicamycin (Tm), an N-glycosylation inhibitor, as well as in a cell model deficient in all three inositol triphosphate receptor isoforms. Pharmacological and molecular studies indicate that these local events are mediated by translocons and that the Ca(2+) microdomains impact (PKR)-like-ER kinase (PERK), an UPR sensor, activation. These findings reveal the existence of a Ca(2+) signal mechanism by which stressor-mediated Ca(2+) release regulates ER stress.

GM2-gangliosidosis, a subgroup of lysosomal storage disorders, is caused by deficiency of hexosaminidase activity, and comprises the closely related Tay-Sachs and Sandhoff diseases. The enzyme deficiency prevents normal metabolization of ganglioside GM2, usually resulting in progressive neurodegenerative disease. The molecular mechanisms whereby GM2 accumulation in neurons triggers neurodegeneration remain unclear. In vitro experiments, using microsomes from Sandhoff mouse model brain, showed that increase of GM2 content negatively modulates sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (Pelled et al., 2003). Furthermore, Ca2+ depletion in endoplasmic reticulum (ER) triggers Unfolded Protein Response (UPR), which tends to restore homeostasis in the ER; however, if cellular damage persists, an apoptotic response is initiated. We found that ER GM2 accumulation in cultured neurons induces luminal Ca2+ depletion, which in turn activates PERK (protein kinase RNA [PKR]-like ER kinase), one of three UPR sensors. PERK signaling displayed biphasic activation; i.e., early upregulation of cytoprotective calcineurin (CN) and, under prolonged ER stress, enhanced expression of pro-apoptotic transcription factor C/EBP homologous protein (CHOP). Moreover, GM2 accumulation in neuronal cells induced neurite atrophy and apoptosis. Both processes were effectively modulated by treatment with the selective PERK inhibitor GSK2606414, by CN knockdown, and by CHOP knockdown. Overall, our findings demonstrate the essential role of PERK signaling pathway contributing to neurodegeneration in a model of GM2-gangliosidosis.

The accumulation of unfolded proteins within the endoplasmic reticulum (ER), caused by any stress condition, triggers the unfolded protein response (UPR) through the activation of specialized sensors. UPR attempts first to restore homeostasis; but if damage persists the signaling induces apoptosis. There is increasing evidence that sustained and unresolved ER stress contributes to many pathological conditions including neurodegenerative diseases. Because the UPR controls cell fate by switching between cytoprotective and apoptotic processes, it is essential to understand the events defining this transition, as well as the elements involved in its modulation. Recently, we demonstrated that abnormal GM2 ganglioside accumulation causes depletion of ER Ca2+ content, which in turn activates PERK (PKR-like-ER kinase), one of the UPR sensors. Furthermore, PERK signaling participates in the neurite atrophy and apoptosis induced by GM2 accumulation. In this respect, we have established an experimental system that allows us to molecularly modulate the expression of downstream PERK components and thus change vulnerability of neurons to undergo neuritic atrophy. We performed knockdown of calcineurin (cytoprotective) and CHOP (pro-apoptotic) expression in rat cortical neuronal cultures. Cells were infected with lentivirus-delivered specific shRNA and then treated with GM2 at different times, fixed and immunostained with anti-MAP2 (microtube-associated protein 2) antibody. Later, cell images were recorded using a fluorescence microscope and total neurite outgrowth was evaluated by using the public domain image processing software ImageJ. The inhibition of expression of those PERK signaling components clearly made it possible to either accelerate or delay the neuritic atrophy induced by ER stress. This approach might be used in cell system models of ER stress to evaluate the vulnerability of neurons to neurite atrophy.

The accumulation of unfolded proteins within the Endoplasmic Reticulum (ER) activates a signal transduction pathway termed theunfoldedprotein response (UPR), which attempts to restore ER homeostasis. If homeostasis cannot be restored, UPR signalling ultimately induces apoptosis. Ca2+depletion in the ER is a potent inducer of ER stress. Despite the ubiquity of Ca2+as intracellular messenger, the precise mechanism (s) by which Ca2+release affects the UPR remains unknown. Use of a genetically encoded Ca2+indicator (GCamP6) that is tethered to the ER membrane, uncovered novel Ca2+signalling events initiated by Ca2+microdomains in human astrocytes under ER stress, as well as in a cell model deficient in all three IP3Receptor isoforms. Pharmacological and molecular studies indicate that these local events are mediated by translocons. Together, these data reveal the existence of a previously unrecognized mechanism by which stressor-mediated Ca2+release regulates ER stress.

Cyclophilin D (CyPD), a mitochondrial matrix protein, has been widely studied for its role in mitochondrial-mediated cell death. Unexpectedly, we previously discovered that overexpression of CyPD in a stable cell line, increased mitochondrial membrane potentials and enhanced cell survival under conditions of oxidative stress. Here, we investigated the underlying mechanisms responsible for these findings. Spectrophotometric measurements in isolated mitochondria revealed that overexpression of CyPD in HEK293 cells increased respiratory chain activity, but only for Complex III (CIII). Acute treatment of mitochondria with the immumosupressant cyclosporine A did not affect CIII activity. Expression levels of the CIII subunits cytochrome b and Rieske-FeS were elevated in HEK293 cells overexpressing CyPD. However, CIII activity was still significantly higher compared to control mitochondria, even when normalized by protein expression. Blue native gel electrophoresis and Western blot assays revealed a molecular interaction of CyPD with CIII and increased levels of supercomplexes in mitochondrial protein extracts. Radiolabeled protein synthesis in mitochondria showed that CIII assembly and formation of supercomplexes containing CIII were significantly faster when CyPD was overexpressed. Taken together, these data indicate that CyPD regulates mitochondrial metabolism, and likely cell survival, by promoting more efficient electrons flow through the respiratory chain via increased supercomplex formation. Fil: Etzler, Julie C.. University of Texas Health Science Center at San Antonio; Estados Unidos Fil: Bollo, Mariana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Holstein, Deborah. University of Texas Health Science Center at San Antonio; Estados Unidos Fil: Deng, Janice Jianhong. University of Texas Health Science Center at San Antonio; Estados Unidos Fil: Perez, Viviana. State University of Oregon; Estados Unidos Fil: Lin, Da-ting. University of Texas Health Science Center at San Antonio; Estados Unidos Fil: Richardson, Arlan. Oklahoma State University; Estados Unidos Fil: Bai, Yidong. University of Texas Health Science Center at San Antonio; Estados Unidos Fil: Lechleiter, James D.. University of Texas Health Science Center at San Antonio; Estados Unidos

The Ca2 +-dependent phosphatase, calcineurin (CN) is thought to play a detrimental role in damaged neurons; however, its role in astrocytes is unclear. In cultured astrocytes, CNβ expression increased after treatment with a sarco/endoplasmic reticulum Ca2 +-ATPase inhibitor, thapsigargin, and with oxygen and glucose deprivation, an in vitro model of ischemia. Similarly, CNβ was induced in astrocytes in vivo in two different mouse models of brain injury - photothrombotic stroke and traumatic brain injury (TBI). Immunoprecipitation and chemical activation dimerization methods pointed to physical interaction of CNβ with the unfolded protein response (UPR) sensor, protein kinase RNA-like endoplasmic reticulum kinase (PERK). In accordance, induction of CNβ resulted in oligomerization and activation of PERK. Strikingly, the presence of a phosphatase inhibitor did not interfere with CNβ-mediated activation of PERK, suggesting a hitherto undiscovered non-enzymatic role for CNβ. Importantly, the cytoprotective function of CNβ was PERK-dependent both in vitro and in vivo. Loss of CNβ in vivo resulted in a significant increase in cerebral damage, and correlated with a decrease in astrocyte size, PERK activity and glial fibrillary acidic protein (GFAP) expression. Taken together, these data reveal a critical role for the CNβ-PERK axis in not only prolonging astrocyte cell survival but also in modulating astrogliosis after brain injury. Fil: Chen, Yanan. University of Texas Health Science Center at San Antonio; Estados Unidos Fil: Holstein, Deborah M.. University of Texas Health Science Center at San Antonio; Estados Unidos Fil: Aime, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Bollo, Mariana Ines. University of Texas Health Science Center at San Antonio; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Lechleiter, James D.. University of Texas Health Science Center at San Antonio; Estados Unidos

Here we identify a cytosolic factor essential for MgATP up-regulation of the squid nerve Na(+)/Ca(2+) exchanger. Mass spectroscopy and Western blot analysis established that this factor is a member of the lipocalin super family of lipid binding proteins of 132 amino acids in length. We named it Regulatory protein of the squid nerve sodium calcium exchanger (ReP1-NCXSQ). ReP-1-NCXSQ was cloned, over expressed and purified. Far-UV circular dichroism and infrared spectra suggest a majority of beta-strand in the secondary structure. Moreover, the predicted tertiary structure indicates ten beta-sheets and two short alpha-helices characteristic of most lipid binding proteins. Functional experiments showed that in order to be active ReP1-NCXSQ must become phosphorylated in the presence of MgATP by a kinase that is Staurosporin insensitive. Even more, the phosphorylated ReP1-NCXSQ is able to stimulate the exchanger in the absence of ATP. In addition to the identification of a new member of the lipid binding protein family, this work shows, for the first time, the requirement of a lipid binding protein for metabolic regulation of an ion transporting system.

We studied the effect of Na + extracellular on Ca 2+ mobilization from intracellular store evoked by carbachol in Trypanosoma cruzi . We report that slow component of Ca 2+ signaling evoked by agonist is dependent on extracellular Na + but not on Ins P 3 increase. Moreover, this Ca 2+ signaling progressively increased when pH of the medium changed from 7.0 to 7.8. In addition, we found that it was regulated by PKC. The agonist was also able to induce the alkalinization of the acidic compartment, and both Ca 2+ signaling and alkalinization were inhibited by the EIPA-inhibitor of the Na + /H + exchanger. These results demonstrated the alkalinization of acidic vacuoles and PKC are involved in the triggering of the epimastigote Ca 2+ signaling.

The synthetic peptide carryingresidues 1–40 of chicken a D -globin, which promotes differentiation in Trypanosoma cruzi epimastigote, stimulated PPtdIns-k, DAG-k, and PA-k activities in a dose-dependent manner. A biphasic behavior only for PPtdIns-k and DAG-k was demonstrated by changes in [ 32 P]PPtdIns and PtdOH levels, the earlier phase peakingat 3 min with a return to basal levels by 6 min and then a second phase with a sustained increase in time. This behavior was not observed for PA-k; the DGPP levels peaked at 6 min and were sustained in time. PMA pretreatment only abolished the first peak of PPtdIns-k, DAG-k activities, and InsPs/InsP3 levels. There was also a transient elevation in intracellular calcium concentration, but this variation was modified only 50% by PMA. The results suggest that peptide 1–40 induces activation of the inositol cycle through lipid kinase activation in a biphasic manner. In this response, the early increase of enzymatic activities would be regulated by PKC and the InsP3 may only be responsible, in part, for the calcium signaling. 2002 Elsevier Science (USA). All rights reserved.

This work provides evidence that nicotine (1 × 10 −5 M) can cause changes in the intracellular calcium concentration of Trypanosoma cruzi epimastigotes, which can be blocked by α-bungarotoxin but not by atropine. Moreover, parasite membranes also bind such nicotinic acetylcholine receptor antagonist as well as agonists such as carbamylcholine (IC 50 : 7.6 × 10 −7 M) and nicotine (IC 50 : 1 × 10 −7 M). Results suggest that there is a molecular species in the surface of the parasite able to bind nicotinic ligands; therefore, nicotine interaction could lead to the activation of the mechanisms involved in intracellular calcium concentration increase in the parasite.

The endoplasmic reticulum (ER) is a Ca2+ storing organelle that plays a critical role in the synthesis, folding and post-translational modifications of many proteins. The ER enters into a condition of stress when the load of newly synthesized proteins exceeds its folding and processing capacity. This activates a signal transduction pathway called the unfolded protein response (UPR) that attempts to restore homeostasis. The precise role of ER Ca2+ in the initiation of the UPR has not been defined. Specifically, it has not been established whether ER Ca2+ dysregulation is a cause or consequence of ER stress. Here, we report that partial depletion of ER Ca2+ stores induces a significant induction of the UPR, and leads to the retention of a normally secreted protein Carboxypeptidase Y. Moreover, inhibition of protein glycosylation by tunicamycin rapidly induced an ER Ca2+ leak into the cytosol. However, blockade of the translocon with emetine inhibited the tunicamycin-induced Ca2+ release. Furthermore, emetine treatment blocked elF2α phosphorylation and reduced expression of the chaperone BiP. These findings suggest that Ca2+ may be both a cause and a consequence of ER protein misfolding. Thus, it appears that ER Ca2+ leak is a significant co-factor for the initiation of the UPR. Fil: Paredes, R. Madelaine. University of Texas; Estados Unidos Fil: Bollo, Mariana Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina Fil: Holstein, Deborah. University of Texas; Estados Unidos Fil: Lechleiter, James D.. University of Texas; Estados Unidos

In squid nerves, MgATP modulation of the Na(+)/Ca(2+) exchanger requires the presence of a cytosolic protein which becomes phosphorylated during the process. This factor has been recently identified. Mass spectroscopy and Western blot analysis established that it is a member of the lipocalin superfamily of lipid-binding proteins (LBP or FABP) of 132 amino acids. We called it regulatory protein of squid nerve sodium/calcium exchanger (ReP1-NCXSQ, access to GenBank EU981897).ReP1-NCXSQ was cloned, expressed, and purified. Circular dichroism, far-UV, and infrared spectroscopy suggest a secondary structure, predominantly of beta-sheets. The tertiary structure prediction provides ten beta-sheets and two alpha-helices, characteristic of most of LPB. Functional experiments showed that, to be active, ReP1-NCXSQ must be phosphorylated by MgATP, through the action of a kinase present in the plasma membrane. Moreover, PO4-ReP1-NCXSQ can stimulate the exchanger in the absence of ATP. An additional crucial observation was that, in proteoliposomes containing only the purified Na(+)/Ca(2+) exchanger, PO4-ReP1-NCXSQ promotes activation; therefore, this upregulation has no other requirement than a lipid membrane and the incorporated exchanger protein.Recently, we solved the crystal structure of ReP1-NCXSQ which was as predicted: a "barrel" consisting of ten beta-sheets and two alpha-helices. Inside the barrel is the fatty acid coordinated by hydrogen bonds with Arg126 and Tyr128. Point mutations showed that neither Tyr20Ala, Arg58Val, Ser99Ala, nor Arg126Val is necessary for protein phosphorylation or activity. On the other hand, Tyr128 is essential for activity but not for phosphorylation. We can conclude that (1) for the first time, a role of an LBP is demonstrated in the metabolic regulation of an ion exchanger; (2) phosphorylation of this LBP can be separated from the activation capacity; and (3) Tyr128, a candidate to coordinate lipid binding inside the barrel, is essential for activity.

In squid nerves, MgATP modulation of the Na+/Ca2+ exchanger requires the presence of a cytosolic protein which becomes phosphorylated during the process. This factor has been recently identified. Mass spectroscopy and Western blot analysis established that it is a member of the lipocalin superfamily of lipid-binding proteins (LBP or FABP) of 132 amino acids. We called it regulatory protein of squid nerve sodium/calcium exchanger (ReP1-NCXSQ, access to GenBank EU981897).

Trypanosoma cruzi undergoes differentiation in the rectum of triatomine, where increased osmolarity is caused mainly by elevated content of NaCl from urine. Early biochemical events in response to high osmolarity in this parasite have not been totally elucidated. In order to clarify the relationship between these events and developmental stages of T. cruzi, epimastigotes were subjected to hyperosmotic stress, which caused activation of Na(+)/H(+) exchanger from acidic vacuoles and accumulation of inositol trisphosphate (InsP(3)). Suppression of InsP(3) levels was observed in presence of intracellular Ca(2+) chelator or pre-treatment with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), which also inhibited the alkalinization of acidic vacuoles via a Na(+)/H(+) exchanger and the consequent increase in cytosolic calcium. These effects were activated and inhibited by PMA and Chelerythrine respectively, suggesting regulation by protein kinase C. The T. cruzi Na(+)/H(+) exchanger, TcNHE1, has 11 transmembrane domains and is localized in acidic vacuoles of epimastigotes. The analyzed biochemical changes were correlated with morphological changes, including an increase in the size of acidocalcisomes and subsequent differentiation to an intermediate form. Both processes were delayed when TcNHE1 was inhibited by EIPA, suggesting that these early biochemical events allow the parasite to adapt to conditions faced in the rectum of the insect vector.

The protein ReP1-NCXSQ was isolated from the cytosol of squid nerves and has been shown to be required for MgATP stimulation of the squid nerve Na(+)/Ca(2+) exchanger NCXSQ1. In order to determine its mode of action and the corresponding biologically active ligand, sequence analysis, crystal structures and mass-spectrometric studies of this protein and its Tyr128Phe mutant are reported. Sequence analysis suggests that it belongs to the CRABP family in the FABP superfamily. The X-ray structure at 1.28 A resolution shows the FABP beta-barrel fold, with a fatty acid inside the barrel that makes a relatively short hydrogen bond to Tyr128 and shows a double bond between C9 and C10 but that is disordered beyond C12. Mass-spectrometric studies identified this fatty acid as palmitoleic acid, confirming the double bond between C9 and C10 and establishing a length of 16 C atoms in the aliphatic chain. This acid was caught inside during the culture in Escherichia coli and therefore is not necessarily linked to the biological activity. The Tyr128Phe mutant was unable to activate the Na(+)/Ca(2+) exchanger and the corresponding crystal structure showed that without the hydrogen bond to Tyr128 the palmitoleic acid inside the barrel becomes disordered. Native mass-spectrometric analysis confirmed a lower occupancy of the fatty acid in the Tyr128Phe mutant. The correlation between (i) the lack of activity of the Tyr128Phe mutant, (ii) the lower occupancy/disorder of the bound palmitoleic acid and (iii) the mass-spectrometric studies of ReP1-NCXSQ suggests that the transport of a fatty acid is involved in regulation of the NCXSQ1 exchanger, providing a novel insight into the mechanism of its regulation. In order to identify the biologically active ligand, additional high-resolution mass-spectrometric studies of the ligands bound to ReP1-NCXSQ were performed after incubation with squid nerve vesicles both with and without MgATP. These studies clearly identified palmitic acid as the fatty acid involved in regulation of the Na(+)/Ca(2+) exchanger from squid nerve.

The fatty acid-binding protein ReP1 is a soluble protein that shows the common beta-barrel motif with two alpha-helix in the portal region. Its isoelectric point of 5.85 leads to net charge of −1 at neutral pH. Previous evidence of its interaction with anionic lipids [1], makes it an attractive model to test the hypothesis that the binding and orientation of soluble proteins within lipid membranes are driven by the interaction of its macrodipole with the interphasial electric field [2].The interaction of ReP1 with lipid membranes of anionic and zwitterionic phospholipids was studied by multiple-run molecular dynamics, Potential of Mean Force calculations and filtration assays. ReP1 has a macrodipole of 310 Debyes pointing towards the portal region. We found that it interacted selectively with anionic interphases, aligning its macrodipole in the configuration of lowest energy within the membrane electric field. Additional evidence of this orientation was achieved experimentally by FRET measurements. This orientation led to the portal region in contact with the membrane. The strength and range of the interaction and the preference in spacial configuration was attenuated by the presence of salt. A global loss of compactness was seen by MD and FT-IR spectroscopy.A similar behaviour has been described for L-BABP [2], a protein with identical tertiary structure.These electrostatic-like features of the interaction suggest that the interphasial electric field could be the driving force for the binding and orientation, and may be involved on the ligand delivery mechanism in this family of proteins.[1] Berberian G., Bollo M., Montich G., Roberts G., DeGiorgis JA., DiPolo R., Beauge L., Biochimica et Biophysica Acta, 1788, 1255-1262 (2009).[2] Villarreal MA., Perduca M., Monaco HL., Montich GG., Biochimica et Biophysica Acta, 1778, 1390-1397 (2008).

This work shows, for the first time, a properly metabolically regulated squid nerve Na(+)/Ca(2+) exchanger (NCXSQ1) heterologous expressed in Saccharomyces cerevisiae. The exchanger was fused to the enhanced green fluorescence protein (eGFP) on its C-terminus and had two tags, a Strep-tag II and 6 histidines, added to the N-terminal region (ST-6HB-NCXSQ1-eGFP). The eGFP fluorescence signal co-localized with that of the plasma membrane marker FM1-43 in whole cells that displayed an uptake of Ca(2+) with the expected characteristics of the reverse Na(+)/Ca(2+) exchange mode. Similar to squid nerve membrane vesicles, inside-out yeast plasma membrane vesicles (ISOV) showed a Ca(2+)(i) regulation of the forward mode that was modulated by previously phosphorylated regulatory cytosolic protein (ReP1-NCXSQ). On the other hand, a close association between NCXSQ1 and ReP1-NCXSQ, estimated by co-immunoprecipitation, was independent of ReP1-NCXSQ phosphorylation. An additional crucial observation was that in proteoliposomes containing only the ST-6HB-NCXSQ1-eGFP protein, Na(+)/Ca(2+) exchange was stimulated by phosphorylated ReP1-NCXSQ; i.e., this up-regulation needs no other requirement besides the lipid membrane and the exchanger protein. Finally, this work provides a potential approach to obtain enough purified NCXSQ1 for structural and biochemical studies which have been delayed due to the lack of sufficient material.

Stimulation of epimastigote forms of Trypanosma cruzi with carbachol resulted in a long-lasting response. The earlier phase for inositol phosphates was rapid and transient, peaking at 1 min with a return to basal levels by 6 min. In a second phase, these metabolite levels reached maximal values at 10-12 min, with a later declination to basal values at about 20 min. The inositol phosphate response was quenched by parasite treatment with atropine. The elevation in intracellular free calcium ([Ca(2+)]i) was transient, reaching the resting level at 87+/-8 s (n=48) of agonist addition. Myo-inositol 1,4,5-triphosphate (InsP(3)) production and [Ca(2+)]i mobilisation were carbachol dose-dependent. The maximally effective concentrations of agonist ranged between 1x10(-6) and 1x10(-5) M. The increase in carbachol concentration resulted in an evident attenuation of [Ca(2+)]i mobilisation and in [3H]InsP(3) levels. Pretreatment of the cells with 10 microM U73122, a phospholipase C (PLC) inhibitor, showed that both InsP(3) peaks triggered by carbachol were completely abolished, whereas there was not substantial change on the maximum elevation in [Ca(2+)]i. The first peak of InsP(3) and InsP(s) was completely abolished when the cells were incubated with phorbol 12-myristate 13-acetate ester (TPA) for 30 min before carbachol stimulation. A biphasic behaviour for PtdIns 4-kinase activity was demonstrated by changes in [32P]PtdInsP levels. The time-course of PtdIns4P 5-kinase activity showed a rapid, significant and transient decrease of [32P]PtdInsP(2) from 0 time to the third min. At the end of this time the polyphosphoinositide began to return towards control levels but, interestingly, after 5-6 min of stimulation there was a subsequent more important increase over control levels which peaked at 10 min. There was also a detectable increment of DAG at 1 min with a maximum at 3 min, this level remaining elevated until at least 10 min. Subsequently, these levels returned to the base line or even below it.