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Influence of bacterial toxins and forskolin upon vasopressin-induced inositol phosphate accumulation in WRK 1 cells.

ABSTRACT: The accumulation of inositol phosphates in WRK 1 cells, stimulated with a range of vasopressin concentrations, was diminished by prior exposure to cholera toxin or forskolin, whilst that observed in the presence of maximal concentrations of the hormone was enhanced in pertussis-toxin-treated cells. In the presence of [32P]NAD+, both cholera toxin and pertussis toxin provoked the labelling of peptides with approximate Mrs of 45,000 and 41,000 respectively in the membranes of WRK 1 cells. Exposure to cholera toxin or forskolin for 15-18 h enhanced cyclic AMP accumulation in these cells. The concentrations of these agents which provoked half-maximal cyclic AMP accumulation were similar to those required to diminish receptor-mediated inositol phosphate accumulation by 50%. In contrast, half-maximal ADP-ribosylation of the 45,000Mr peptide needed 100-fold greater concentrations of the toxin than were effective in provoking half-maximal inhibition of inositol phosphate accumulation. Cholera toxin or forskolin also reduced the maximal specific binding, to intact WRK 1 cells, of both [3H][Arg8]vasopressin and the V1a antagonist [3H][beta-mercapto-beta,beta-cyclopentamethylenepropionic acid,O-methyl-Tyr2, Arg8]vasopressin. The kinetics for the loss of this binding capacity following cholera-toxin treatment were very similar to those describing the diminution of vasopressin-stimulated inositol phosphate accumulation in the same cells.

SUBMITTER: Guillon G

PROVIDER: S-EPMC1138729 | biostudies-other | 1989 Jun

REPOSITORIES: biostudies-other

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Project description:1. Temporal changes in the levels of many inositol phosphates, whose structural characterization is presented in the preceding paper [Wong, Barker, Morris, Craxton, Kirk & Michell (1991) Biochem. J. 286, 459-468], have been monitored in vasopressin-stimulated WRK-1 cells. 2. Upon stimulation, Ins(1,4,5)P3 accumulated within 1 s, consistent with its role as a rapidly acting second messenger produced by receptor activation of phosphoinositidase C. Ins(1,4)P2 and Ins(1,3,4,5)P4, both of which are immediate products of Ins(1,4,5)P3 metabolism, also accumulated quickly. Ins4P, Ins(1,3,4)P3, Ins(3,4)P2, Ins(1,3)P2, Ins1P and Ins3P, which are intermediates in the metabolism of Ins(1,4)P2 and Ins(1,3,4,5)P4 to inositol, accumulated after seconds or within a few minutes, and in a temporal sequence consistent with their known metabolic interrelationships. 3. The stimulated accumulation of Ins(1,3,4,6)P4 was delayed, as expected if it is formed by phosphorylation of Ins(1,3,4)P3. 4. Ins(3,4,5,6)P4 accumulated 2-3-fold in a few minutes, and mainly before Ins(1,3,4,6)P4. 5. Using a [3H]-/[14C]-inositol double-labelling protocol, we obtained evidence that all of the compounds that accumulated upon stimulation, except Ins(3,4,5,6)P4, originated from lipid-derived Ins(1,4,5)P3, but that the newly formed Ins(3,4,5,6)P4 came from a different source. 6. There were no consistent changes in the levels of Ins(1,3,4,5,6)P5 and InsP6 during stimulation. 7. Alongside the gradual accumulation of Ins(1:2-cyclic,4,5)P3 during stimulation [Wong, Barker, Shears, Kirk & Michell (1988) Biochem. J. 252, 1-5], there was an accumulation of Ins(1:2-cyclic,4)P2 and Ins(1:2-cyclic)P, probably as either minor side products of phosphoinositidase C action or metabolites of Ins(1:2-cyclic,4,5)P3. 8. When Li+ was present during stimulation, it redirected the dephosphorylation pathways downstream of Ins(1,4,5)P3 in the manner expected from its inhibition of inositol monophosphatase and Ins(1,4)P2/Ins(1,3,4)P3 1-phosphatase: there were marked increases in the accumulation of Ins(1,4)P2 and Ins(1,3,4)P3 and of monophosphates. Moreover, Li+ shifted the Ins1P/Ins3P balance in favour of Ins1P, thus demonstrating redirection of the metabolism of the accumulated Ins(1,3,4)P3 towards Ins(1,3)P2 rather than Ins(3,4)P2.

Project description:The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of Archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol. 188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression in Escherichia coli of the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3'-phosphate-1'-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus, Pyrococcus furiosus, Thermococcus kodakaraensis, Aquifex aeolicus, and Rubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP and l-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol and l-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP and l-myo-inositol-1-P; the DIPP synthase domain used only l-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted in Aeropyrum pernix, Thermotoga maritima, and Hyperthermus butylicus. Additionally, using l-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3'-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.

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Influence of bacterial toxins and forskolin upon vasopressin-induced inositol phosphate accumulation in WRK 1 cells.

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