Conformational Stability of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Dictates Its Substrate Selectivity

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP₅) to inositol hexakisphosphate (IP₆). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP₅ in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in k_cat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP₄) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.     

KCS1 deletion in Saccharomyces cerevisiae leads to a defect in translocation of autophagic proteins and reduces autophagosome formation

Inositol phosphates are implicated in the regulation of autophagy; however, the exact role of each inositol phosphate species is unclear. In this study, we systematically analyzed the highly conserved inositol polyphosphate synthesis pathway in S. cerevisiae for its role in regulating autophagy. Using yeast mutants that harbored a deletion in each of the genes within the inositol polyphosphate synthesis pathway, we found that deletion of KCS1, and to a lesser degree IPK2, led to a defect in autophagy. KCS1 encodes an inositol hexakisphosphate/heptakisposphate kinase that synthesizes 5-IP 7 and IP 8; and IPK2 encodes an inositol polyphosphate multikinase required for synthesis of IP 4 and IP 5. We characterized the kcs1Δ mutant strain in detail. The kcs1Δ yeast exhibited reduced autophagic flux, which might be caused by both the reduction in autophagosome number and autophagosome size as observed under nitrogen starvation. The autophagy defect in kcs1Δ strain was associated with mislocalization of the phagophore assembly site (PAS) and a defect in Atg18 release from the vacuole membrane under nitrogen deprivation conditions. Interestingly, formation of autophagosome-like vesicles was commonly observed to originate from the plasma membrane in the kcs1Δ strain. Our results indicate that lack of KCS1 interferes with proper localization of the PAS, leads to reduction of autophagosome formation, and causes the formation of autophagosome-like structure in abnormal subcellular locations.     

Roles for inositol polyphosphate kinases in the regulation of nuclear processes and developmental biology

Our laboratory studies the biology and enzyme regulation of inositol signal transduction pathways, which are activated in response to a wide range of stimuli. As a six-carbon cyclitol, inositol and its numerous phosphorylated derivatives efficiently generate combinatorial ensembles of signaling molecules. Through the cloning and characterization of inositol polyphosphate kinases (IPK), novel roles for inositol tetrakisphosphate (IP₄), inositol pentakisphosphate (IP₅), and inositol hexakisphosphate (IP₆) and inositol pyrophosphates (PP-IPs), have been identified. Studies have linked the IPKs and their inositide products to the regulation of nuclear processes including gene expression, chromatin remodeling, mRNA export, DNA repair and telomere maintenance. Analysis of IPK knockout animals has revealed a role for production of IPs in regulation of embryogenesis and organism development.The discoveries of the IPK proteins and their connection to nuclear signaling have generated significant interest in the field. Furthermore, they have provided interesting clues into the evolution of inositide-signaling pathways. Ipk2/IPMK and IPS/IP6K family members are conserved from yeast to man. In contrast, the IP₃ 3-kinase (ITPK) branch is observed in selected metazoans and not in plant or fungi. This may imply that Ipk2 and IPS activities evolved first among the group. The promiscuity of the Ipk2 protein further supports this notion and may provide the cell with a means to generate many IP species in a genetically economical fashion. Studies of yeast inositide signaling reveal that these simple eukaryotes do not have an IP₃ receptor in their genome and do not utilize diacylglycerol to activate protein kinase C. Thus, it appears that the canonical “text book” aspects of inositide-signaling pathways are not conserved throughout eukaryotic evolution. In light of the conservation of Ipk2/IPMK, Ipk1 and IPS/IP6K pathways from yeast to man it is interesting to speculate that a primordial role of phospholipase C-induced, IPK-dependent inositide signaling was to regulate nuclear processes. As calcium and PKC signaling evolved in metazoans, these may have greatly enhanced signaling capabilities. Recent studies demonstrating an essential role for IP₅, IP₆ and possibly PP-IP production in metazoan development highlight the importance of IPK signaling in cellular responses in metazoans. With these thoughts in mind, we eagerly await future studies aimed at further elucidating how these signaling codes participate in developmental processes and the control of gene expression, mRNA export, and DNA metabolism.     

Expression pattern of inositol phosphate-related enzymes in rice (Oryza sativa L.): implications for the phytic acid biosynthetic pathway

Phytic acid, myo-inositol-hexakisphosphate (InsP(6)), is a storage form of phosphorus in plants. Despite many physiological investigations of phytic acid accumulation and storage, little is known at the molecular level about its biosynthetic pathway in plants. Recent work has suggested two pathways. One is an inositol lipid-independent pathway that occurs through the sequential phosphorylation of 1D-myo-inositol 3-phosphate (Ins(3)P). The second is a phospholipase C (PLC)-mediated pathway, in which inositol 1,4,5-tris-phosphate (Ins(1,4,5)P(3)) is sequentially phosphorylated to InsP(6). We identified 12 genes from rice (Oryza sativa L.) that code for the enzymes that may be involved in the metabolism of inositol phosphates. These enzymes include 1D-myo-inositol 3-phosphate synthase (MIPS), inositol monophosphatase (IMP), inositol 1,4,5-tris-phosphate kinase/inositol polyphosphate kinase (IPK2), inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1), and inositol 1,3,4-triskisphosphate 5/6-kinase (ITP5/6K). The quantification of absolute amounts of mRNA by real-time RT-PCR revealed the unique expression patterns of these genes. Outstanding up-regulation of the four genes, a MIPS, an IPK1, and two ITP5/6Ks in embryos, suggested that they play a significant role in phytic acid biosynthesis and that the lipid-independent pathway was mainly active in developing seeds. On the other hand, the up-regulation of a MIPS, an IMP, an IPK2, and an ITP5/6K in anthers suggested that a PLC-mediated pathway was active in addition to a lipid-independent pathway in the anthers.     

Enhancement by sphingosine 1‐phosphate in vasopressin‐induced phosphoinositide hydrolysis in aortic smooth‐muscle cells: Involvement of p38 MAP kinase

We previously reported that sphingosine 1‐phosphate (S‐1‐P), a sphingomyelin metabolite, activates p44/p42 mitogen‐activated protein (MAP) kinase and p38 MAP kinase in aortic smooth‐muscle A10 cells. In the present study, we investigated the effect of sphingomyelin metabolites on phospholipase C‐catalyzing phosphoinositide hydrolysis induced by arginine vasopressin (AVP) in A10 cells. C₂‐ceramide and sphingosine had little effect on inositol phosphate (IP) formation stimulated by AVP. S‐1‐P, which alone slightly stimulated the IPs formation, dose‐dependently amplified the AVP‐induced formation of IPs. Tumor necrosis factor‐α enhanced the AVP‐induced formation of IPs. However, S‐1‐P did not enhance the formation of IPs by NaF, a heterotrimeric GTP‐binding protein activator. Pertussis toxin inhibited the effect of S‐1‐P. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, had little effect on the enhancement by S‐1‐P. SB203580, an inhibitor of p38 MAP kinase, suppressed the effect of S‐1‐P on the formation of IPs by AVP. SB203580 inhibited the AVP‐induced phosphorylation of p38 MAP kinase. Pertussis toxin suppressed the phosphorylation of p38 MAP kinase by S‐1‐P. These results indicate that S‐1‐P amplifies AVP‐induced phosphoinositide hydrolysis by phospholipase C through p38 MAP kinase in vascular smooth‐muscle cells. J. Cell. Biochem. 80:46–52, 2000. © 2000 Wiley‐Liss, Inc.     

A miniaturized column chromatography method for measuring receptor-mediated inositol phosphate accumulation

Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP(3)), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen assays, offer higher throughput. However, these techniques rely on measurement of IP(3) itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP(3). The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP(3) and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.     

A role for rat inositol polyphosphate kinases rIPK2 and rIPK1 in inositol pentakisphosphate and inositol hexakisphosphate production in rat-1 cells

Over 30 inositol polyphosphates are known to exist in mammalian cells; however, the majority of them have uncharacterized functions. In this study we investigated the molecular basis of synthesis of highly phosphorylated inositol polyphosphates (such as inositol tetrakisphosphate, inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6)) in rat cells. We report that heterologous expression of rat inositol polyphosphate kinases rIPK2, a dual specificity inositol trisphosphate/inositol tetrakisphosphate kinase, and rIPK1, an IP5 2-kinase, were sufficient to recapitulate IP6 synthesis from inositol 1,4,5-trisphosphate in mutant yeast cells. Overexpression of rIPK2 in Rat-1 cells increased inositol 1,3,4,5,6-pentakisphosphate (I(1,3,4,5,6)P5) levels about 2-3-fold compared with control. Likewise in Rat-1 cells, overexpression of rIPK1 was capable of completely converting I(1,3,4,5,6)P5 to IP6. Simultaneous overexpression of both rIPK2 and rIPK1 in Rat-1 cells increased both IP5 and IP6 levels. To reduce IPK2 activity in Rat-1 cells, we introduced vector-based short interference RNA against rIPK2. Cells harboring the short interference RNA had a 90% reduction of mRNA levels and a 75% decrease of I(1,3,4,5,6)P5. These data confirm the involvement of IPK2 and IPK1 in the conversion of inositol 1,4,5-trisphosphate to IP6 in rat cells. Furthermore these data suggest that rIPK2 and rIPK1 act as key determining steps in production of IP5 and IP6, respectively. The ability to modulate the intracellular inositol polyphosphate levels by altering IPK2 and IPK1 expression in rat cells will provide powerful tools to study the roles of I(1,3,4,5,6)P5 and IP6 in cell signaling.     

Regulation of nuclear processes by inositol polyphosphates

Inositide signaling pathways represent a multifaceted ensemble of cellular switches capable of regulating a number of processes, for example, intracellular calcium release, membrane trafficking, chemotaxis, ion channel activity and several nuclear functions. Over 30 inositide messengers are found in eukaryotic cells that may be grouped into two classes: (1) inositol lipids, phosphatidylinositols or phosphoinositides (PIPs) and (2) water-soluble inositol polyphosphates (IPs). This review will focus on inositol polyphosphate kinases (IPK) and inositol pyrophosphate synthases (IPS) responsible for the cellular production of IP(4), IP(5) IP(6) and PP-IPs. Of interest, IPK and IPS proteins localize, in part, within the nucleus and their activities are necessary for proper regulation of gene expression, mRNA export, DNA repair and telomere maintenance. The breadth of nuclear processes regulated and the evolutionary conservation of the genes involved in their synthesis have sparked renewed interest in inositide messengers derived from sequential phosphorylation of inositol 1,4,5-trisphosphate.     

A multi-well filtration assay for quantitation of inositol phosphates in biological samples

The quantitation of inositol phosphates (IPs), mediators of certain signal transduction processes, typically involves laborious and time consuming conventional ion-exchange chromatography procedures. We have developed a high throughput microtiter plate-based IP assay that utilizes vacuum rather than gravitational flow and has significant advantages over existing methods. The response of recombinant HEK-293 cells expressing human LHRH receptor cDNA to LHRH agonists was used as a model system to develop the assay conditions. Cell lysates containing labeled IPs were applied in 96-well plates fitted with filtration discs containing regenerated Dowex AGI-X8 resin. Specifically bound inositol phosphates were eluted with 1 M ammonium formate in 0.1 M formic acid directly into a fresh 96-well plate and an aliquot of the eluate from each well is transferred into a 96-well plate and counted. The results were comparable to those obtained with the conventional column method and the variation among replicates was significantly improved. This assay facilitates rapid quantitation of inositol phosphates from a large number of samples with relative ease and reduced generation of radioactive waste.     

Mutagenesis of isopentenyl phosphate kinase to enhance geranyl phosphate kinase activity

Isopentenyl phosphate kinase (IPK) catalyzes the ATP-dependent phosphorylation of isopentenyl phosphate (IP) to form isopentenyl diphosphate (IPP) during biosynthesis of isoprenoid metabolites in Archaea. The structure of IPK from the archeaon Thermoplasma acidophilum (THA) was recently reported and guided the reconstruction of the IP binding site to accommodate the longer chain isoprenoid monophosphates geranyl phosphate (GP) and farnesyl phosphate (FP). We created four mutants of THA IPK with different combinations of alanine substitutions for Tyr70, Val73, Val130, and Ile140, amino acids with bulky side chains that limited the size of the side chain of the isoprenoid phosphate substrate that could be accommodated in the active site. The mutants had substantially increased GP kinase activity, with 20-200-fold increases in k(cat)(GP) and 30-130-fold increases in k(cat)(GP)/K(M)(GP) relative to those of wild-type THA IPK. The mutations also resulted in a 10(6)-fold decrease in k(cat)(IP)/K(M)(IP) compared to that of wild-type IPK. No significant change in the kinetic parameters for the cosubstrate ATP was observed, signifying that binding between the nucleotide binding site and the IP binding site was not cooperative. The shift in substrate selectivity from IP to GP, and to a lesser extent, FP, in the mutants could act as a starting point for the creation of more efficient GP or FP kinases whose products could be exploited for the chemoenzymatic synthesis of radiolabeled isoprenoid diphosphates.     

Regulation of telomere length by fatty acid elongase 3 in yeast. Involvement of inositol phosphate metabolism and Ku70/80 function

In this study, we investigated the roles of very long-chain fatty acid (VLCFA) synthesis by fatty acid elongase 3 (ELO3) in the regulation of telomere length and life span in the yeast Saccharomyces cerevisiae. Loss of VLCFA synthesis via deletion of ELO3 reduced telomere length, and reconstitution of the expression of wild type ELO3, and not by its mutant with decreased catalytic activity, rescued telomere attrition. Further experiments revealed that alterations of phytoceramide seem to be dispensable for telomere shortening in response to loss of ELO3. Interestingly, telomere shortening in elo3Delta cells was almost completely prevented by deletion of IPK2 or KCS1, which are involved in the generation of inositol phosphates (IP4, IP5, and inositol pyrophosphates). Deletion of IPK1, which generates IP6, however, did not affect regulation of telomere length. Further data also suggested that elo3Delta cells exhibit accelerated chronologic aging, and reduced replicative life span compared with wild type cells, and deletion of KCS1 helped recover these biological defects. Importantly, to determine downstream mechanisms, epistasis experiments were performed, and data indicated that ELO3 and YKU70/80 share a common pathway for the regulation of telomere length. More specifically, chromatin immunoprecipitation assays revealed that the telomere binding and protective function of YKu80p in vivo was reduced in elo3Delta cells, whereas its non-homologues end-joining function was not altered. Deletion of KCS1 in elo3Delta cells recovered the telomere binding and protective function of Ku, consistent with the role of KCS1 mutation in the rescue of telomere length attrition. Thus, these findings provide initial evidence of a possible link between Elo3-dependent VLCFA synthesis, and IP metabolism by KCS1 and IPK2 in the regulation of telomeres, which play important physiological roles in the control of senescence and aging, via a mechanism involving alterations of the telomere-binding/protection function of Ku.     

The Streptomyces-produced antibiotic fosfomycin is a promiscuous substrate for archaeal isopentenyl phosphate kinase

Isopentenyl phosphate kinase (IPK) catalyzes the phosphorylation of isopentenyl phosphate to form the isoprenoid precursor isopentenyl diphosphate in the archaeal mevalonate pathway. This enzyme is highly homologous to fosfomycin kinase (FomA), an antibiotic resistance enzyme found in a few strains of Streptomyces and Pseudomonas whose mode of action is inactivation by phosphorylation. Superposition of Thermoplasma acidophilum (THA) IPK and FomA structures aligns their respective substrates and catalytic residues, including H50 and K14 in THA IPK and H58 and K18 in Streptomyces wedmorensis FomA. These residues are conserved only in the IPK and FomA members of the phosphate subdivision of the amino acid kinase family. We measured the fosfomycin kinase activity of THA IPK [K(m) = 15.1 ± 1.0 mM, and k(cat) = (4.0 ± 0.1) × 10?2 s?1], resulting in a catalytic efficiency (k(cat)/K(m) = 2.6 M?1 s?1) that is 5 orders of magnitude lower than that of the native reaction. Fosfomycin is a competitive inhibitor of IPK (K(i) = 3.6 ± 0.2 mM). Molecular dynamics simulation of the IPK·fosfomycin·MgATP complex identified two binding poses for fosfomycin in the IP binding site, one of which results in a complex analogous to the native IPK·IP·ATP complex that engages H50 and the lysine triangle formed by K5, K14, and K205. The other binding pose leads to a dead-end complex that engages K204 near the IP binding site to bind fosfomycin. Our findings suggest a mechanism for acquisition of FomA-based antibiotic resistance in fosfomycin-producing organisms.     

Thrombin and IgE antigen induce formation of inositol phosphates by mouse E-mast cells

Stimulation of murine chondroitin sulfate E containing mast cells (E-MC) in vitro either by thrombin or immunologically resulted in a rapid formation of inositol phosphates (IPs). Increase in all of the three IPs (IP1, IP2 and IP3) could be detected 20 s after stimulation. The depletion of Ca2+ from the medium resulted in more than 80% reduction in beta-hexosaminidase release from either thrombin or IgE antigen stimulated cells. However, both thrombin and IgE antigen increased the formation of IP3 under these conditions independent of the presence of extracellular Ca2+.     

Putative role of inositol phospholipid metabolism in neurons

Inositol phospholipids play a crucial role in the intracellular signal transduction in most cell types. Activation of an enzyme called phospholipase C or PIP2-phosphodiesterase (PIP2-PDE) leads to the production of two second messenger molecules, diacylglycerol (DG) and inositol 1,4,5-triphosphate (IP3). DG activates a kinase called protein kinase C, whereas IP3 mediates the release of Ca2+ from intracellular storage sites. The measurement of IP3 and its degradation products, inositol diphosphate (IP2) and inositol monophosphate (IP1) provides a way of assessing the extent to which this complex system has been activated. In the central nervous system (CNS) most of the studies on the neurotransmitter stimulated formation of inositol phosphates (IPs) have been performed on brain slices, a mixture of mainly neurons and glial cells. The recent development of pure neuronal cultures provides a means of determining which of these responses were of neuronal origin. The purpose of this review is to summarize the results obtained in neurons in primary culture together with a brief appraisal of the possible function of this second messenger system in neurons.     

Anion-exchange high-performance liquid chromatographic analysis of inositol phosphates

The analysis of inositol phosphates by anion-exchange HPLC is described. The method employs a citrate buffer gradient to resolve several inositol phosphates including inositol 1-phosphate, inositol 1,4-bisphosphate (IP2), and inositol 1,4,5-trisphosphate (IP3), as well as some of the isomers of these compounds. Since the buffer system does not contain any phosphate, we can use a phosphate assay to examine the chromatographic behavior of phosphate-containing compounds. The method shows good resolution and recovery (greater than 95% for IP2 and IP3). Total analysis time, including reequilibration, is about 90 min. In addition, an isocratic system that can rapidly (less than 10 min) measure IP3 is described. The HPLC system was used to characterize inositol phosphate turnover in thrombin-stimulated platelets and formylmethionyl-leucyl-phenylalanine-stimulated HL-60 cells.     

Regulation of casein kinase-2 (CK2) activity by inositol phosphates

Casein kinase 2 (CK2) was one of the first protein kinases to be discovered and has been suggested to be responsible for as much as one-fifth of the eukaryotic phosphoproteome. Despite being responsible for the phosphorylation of a vast array of proteins central to numerous dynamic cellular processes, the activity of CK2 appears to be unregulated. In the current study, we identified a protein kinase activity in rat liver supernatant that is up-regulated by inositol 1,3,4,5-tetrakisphosphate (IP4) and inositol hexakisphosphate (IP6). The substrate for the inositol phosphate-regulated protein kinase was identified as a phosphatidylcholine transfer protein-like protein. Using the phosphorylation of this substrate in an assay, we purified the inositol phosphate-regulated protein kinase and determined it to be CK2. Bacterially expressed recombinant CK2, however, showed very high basal activity and was only modestly activated by IP6 and not regulated by IP. We found that an endogenous component present in rat liver supernatant was able to inhibit both recombinant and liver-purified CK2 basal activity. Under these conditions, recombinant CK2 catalytic activity could be increased substantially by IP4, inositol 1,3,4,5,6-pentakisphosphate (IP5), and IP6. We concluded that, contrary to the previously held view, CK2 can exist in a state of low constitutive activity allowing for its regulation by inositol phosphates. The ability of the higher inositol phosphates to directly stimulate CK2 catalytic activity provides the first evidence that these signaling molecules can operate via a direct control of protein phosphorylation.     

Luteinizing hormone stimulates the formation of inositol trisphosphate and cyclic AMP in rat granulosa cells. Evidence for phospholipase C generated second messengers in the action of luteinizing hormone.

The following studies were conducted to determine whether luteinizing hormone (LH), a hormone which increases cellular levels of cyclic AMP, also provokes increases in 'second messengers' derived from inositol lipid metabolism (i.e. inositol phosphates and diacylglycerol). Rat granulosa cells isolated from mature Graafian follicles were prelabelled for 3 h with myo-[2-3H]inositol. LH provoked rapid (5 min) and sustained (up to 60 min) increases in the levels of inositol mono-, bis, and trisphosphates (IP, IP2 and IP3, respectively). Time course studies revealed that IP3 was formed more rapidly than IP2 and IP following LH treatment. The response to LH was concentration-dependent with maximal increases at LH concentrations of 1 microgram/ml. LiCl (2-40 mM) enhanced the LH-provoked accumulation of all [3H]inositol phosphates, presumably by inhibiting the action of inositol phosphate phosphatases. The effectiveness of LH, however, was dependent on the concentration of lithium employed; maximal increases in IP were observed at 10 mM-LiCl, whereas maximal increases in IP2 and IP3 were observed at 20 mM- and 40 mM-LiCl, respectively. The stimulatory effects of LH on inositol phosphate and progesterone accumulation were also compared with changes in cyclic nucleotide levels. LH rapidly increased levels of inositol phosphates, progesterone and cyclic AMP, but transiently reduced levels of cyclic GMP. These results demonstrate that LH increases both cyclic AMP and inositol trisphosphate (and presumably diacylglycerol) in rat granulosa cells. Our findings suggest that two messenger systems exist to mediate the action of LH in granulosa cells.     

Enhancement by sphingosine 1-phosphate in vasopressin-induced phosphoinositide hydrolysis in aortic smooth-muscle cells: involvement of p38 MAP kinase

We previously reported that sphingosine 1-phosphate (S-1-P), a sphingomyelin metabolite, activates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in aortic smooth-muscle A10 cells. In the present study, we investigated the effect of sphingomyelin metabolites on phospholipase C-catalyzing phosphoinositide hydrolysis induced by arginine vasopressin (AVP) in A10 cells. C(2)-ceramide and sphingosine had little effect on inositol phosphate (IP) formation stimulated by AVP. S-1-P, which alone slightly stimulated the IPs formation, dose-dependently amplified the AVP-induced formation of IPs. Tumor necrosis factor-alpha enhanced the AVP-induced formation of IPs. However, S-1-P did not enhance the formation of IPs by NaF, a heterotrimeric GTP-binding protein activator. Pertussis toxin inhibited the effect of S-1-P. PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase, had little effect on the enhancement by S-1-P. SB203580, an inhibitor of p38 MAP kinase, suppressed the effect of S-1-P on the formation of IPs by AVP. SB203580 inhibited the AVP-induced phosphorylation of p38 MAP kinase. Pertussis toxin suppressed the phosphorylation of p38 MAP kinase by S-1-P. These results indicate that S-1-P amplifies AVP-induced phosphoinositide hydrolysis by phospholipase C through p38 MAP kinase in vascular smooth-muscle cells.     

Agonist-induced production of inositol phosphates in human airway smooth muscle cells in culture.

Smooth muscle cells (SMC) from human bronchi were isolated by elastase treatment, subcultured, and characterized by their positive reaction with a monoclonal antibody against alpha-smooth muscle actin (alpha SMA). In each cell line tested, at least 95% of the cells were positively stained. The functional properties of these cells were examined by measuring the metabolism of inositol phosphates (IPs). For that purpose, cells were incubated for 3 days before reaching confluency in the presence of myo-[3H]inositol in order to label the phosphoinositide pool, and the various [3H]IPs were separated by HPLC on a SAX column with a phosphate gradient. IP1 isomers were separated in three peaks; IP2, IP3, IP4, IP5 and IP6 (phytic acid) were each eluted as single peaks. The identity of the [3H]peaks was verified with corresponding [3H]IP standards. The accumulation of [3H]IPs was measured by incubating cells up to 30 min in the presence of 10 mM LiCl, with or without a bronchoconstrictor agent (carbachol, histamine, PGF2 alpha). Histamine, 10(-4) M, elicited a four times larger IP accumulation than carbachol, 10(-4) M, and than PGF2 alpha, 5 10(-5) M. Dose-response curves were established for histamine and carbachol in the range 10(-7)-10(-4) M. At 10(-7) M, carbachol was more effective than histamine in stimulating the IP metabolism. Atropine blocked the response to carbachol, and diphenhydramine inhibited the effect of histamine, indicating the specificity of the response to the agonists. These results indicate that cultured human bronchial SMC are a suitable preparation for studying physiological aspects of membrane transduction in the airways.