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L-myo-inositol 1,4,5,6-tetrakisphosphate is present in both mammalian and avian cells
L Stephens1, P T Hawkins, N Carter
1Smith Kline & French Research Ltd., Welwyn, Herts., U.K.
Abstract:
When myo-[3H]inositol-prelabelled primary-cultured murine bone-marrow-derived macrophages were challenged with platelet-activating factor (PAF; 200 ng/ml), there was a rapid (2.5-fold at 10 s) rise in the intracellular concentration of D-myo-[3H]inositol 1,4,5-trisphosphate, followed by a rise in myo-[3H]inositol tetrakisphosphate. myo-[3H]Inositol tetrakisphosphate fractions were isolated by high-performance anion-exchange chromatography from myo-[3H]inositol-prelabelled chick erythrocytes and primary-cultured macrophages. In both cases [3H]iditol and [3H]inositol were the only significant products (greater than 90% of recovered radioactivity) after oxidation to completion with periodic acid, reduction with NaBH4 and dephosphorylation with alkaline phosphatase. The presence of [3H]inositol after this procedure is consistent with the occurrence of [3H]inositol 1,3,4,5-tetrakisphosphate in the cell extracts, whereas [3H]iditol could only be derived from D- or L-inositol 1,4,5,6-tetrakisphosphate. When [3H]inositol tetrakisphosphate fractions obtained from (A) unstimulated macrophages, (B) macrophages that had been stimulated with PAF for 40s or (C) chick erythrocytes were subjected to the above procedure, radioactivity was recovered in these polyols in the following proportions: A, 60-90% in iditol, with 10-40% in inositol; B, total radioactivity increased by a factor of 9.8, 94% being recovered in inositol and 8% in iditol; C, 70-80% in iditol and 20-30% in inositol. [3H]Iditol derived from myo-[3H]inositol tetrakisphosphate fractions from macrophages and chick erythrocytes was oxidized to sorbose by L-iditol dehydrogenase (L-iditol:NAD+2-oxidoreductase, 1.1.1.14) at the same rate as authentic L-iditol. D-[14C]Iditol, derived from D-myo-inositol 1,4,5-trisphosphate, was not oxidized by L-iditol dehydrogenase. This result indicates that the [3H]iditol was derived from L-myo-inositol inositol 1,4,5,6-tetrakisphosphate. The data are consistent with rapid PAF-sensitive synthesis of D-myo-[3H]inositol 1,3,4,5-tetrakisphosphate in macrophages, and demonstrate that L-myo-inositol 1,4,5,6-tetrakisphosphate is synthesized in both mammalian and avian cells. The levels of L-myo-[3H]inositol 1,4,5,6-tetrakisphosphate in primary-cultured macrophages are not acutely sensitive to PAF.
Insights
Platelet-activating factor (PAF) rapidly increases D-myo-inositol 1,3,4,5-tetrakisphosphate in macrophages. Both mammalian and avian cells synthesize L-myo-inositol 1,4,5,6-tetrakisphosphate, but its levels are not acutely affected by PAF.
Area of Science:
- Biochemistry
- Cell Biology
- Signal Transduction
Background:
- Platelet-activating factor (PAF) is a potent lipid mediator involved in inflammation and allergic responses.
- Inositol phosphates are crucial second messengers in intracellular signaling pathways.
- Understanding the synthesis and regulation of inositol phosphates is vital for deciphering cellular responses.
Purpose of the Study:
- To investigate the synthesis of inositol tetrakisphosphates in response to PAF stimulation in macrophages.
- To identify and quantify specific inositol tetrakisphosphate isomers produced in primary-cultured murine macrophages and chick erythrocytes.
- To elucidate the role of L-myo-inositol 1,4,5,6-tetrakisphosphate in cellular signaling.
Main Methods:
- Primary-cultured murine bone-marrow-derived macrophages and chick erythrocytes were prelabelled with myo-[3H]inositol.
- Cells were stimulated with PAF, and intracellular inositol phosphates were extracted and analyzed.
- High-performance anion-exchange chromatography was used to isolate myo-[3H]inositol tetrakisphosphate fractions.
- Enzymatic and chemical degradation methods were employed to identify the structures of inositol tetrakisphosphates.
Main Results:
- PAF stimulation led to a rapid increase in D-myo-[3H]inositol 1,4,5-trisphosphate and subsequent rise in myo-[3H]inositol tetrakisphosphates in macrophages.
- Analysis revealed the presence of both D-myo-inositol 1,3,4,5-tetrakisphosphate and L-myo-inositol 1,4,5,6-tetrakisphosphate in macrophages and chick erythrocytes.
- PAF-sensitive synthesis of D-myo-[3H]inositol 1,3,4,5-tetrakisphosphate was observed, while L-myo-[3H]inositol 1,4,5,6-tetrakisphosphate levels remained largely unaffected by acute PAF challenge.
- L-myo-inositol 1,4,5,6-tetrakisphosphate was identified as a common inositol tetrakisphosphate in both mammalian and avian cells.
Conclusions:
- The study demonstrates the rapid synthesis of D-myo-inositol 1,3,4,5-tetrakisphosphate in response to PAF in macrophages.
- L-myo-inositol 1,4,5,6-tetrakisphosphate is a conserved inositol phosphate synthesized in both mammalian and avian cells.
- The findings contribute to understanding the complex landscape of inositol phosphate signaling pathways and their regulation.