Judith A. Creba

Changes in the Levels of Inositol Lipids and Phosphates during the Differentiation of HL60 Promyelocytic Cells towards Neutrophils or Monocytes

HL60 cells were adapted to grow in a serum-free medium containing 1 mg l-1 inositol, in which they differentiated normally towards neutrophils (in 0.9% by volume dimethylsulphoxide) and towards monocytes (in 10 nM phorbol myristate acetate). Cells that had been equilibrium-labelled with [2-3H] myo-inositol contained a complex pattern of inositol metabolites, several of which were at relatively high concentrations. These included InsP5 and InsP6, which were present at concentrations of about 25 μM and 60 μM, respectively. Striking and different changes occurred in the levels of some of the inositol polyphosphates as the cells differentiated towards either neutrophils or monocytes. Most notable were a large but gradual accumulation of Ins(1, 3, 4, 5, 6)P5 as HL60 cells decreased in size and acquired neutrophil characteristics, and much more rapid and sequential declines in InsP4, InsP5 and InsP6 as the cells started to take on monocyte character. There was a marked accumulation of free inositol and of phosphatidylinositol in the cells during neutrophil differentiation, probably caused at least in part by an increased rate of inositol uptake providing an increased intracellular inositol supply. The same accumulation of Ins( 1, 3, 4, 5, 6)P5 occurred during neutrophil differentiation, whether it was induced by dimethylsulphoxide or by a combination of retinoic acid and a T-lymphocyte cell line-derived differentiation factor. Ins(1, 4, 5)P3, a physiological intracellular mediator of Ca2+ release from membrane stores, did not change in concentration during these differentiation processes. These observations suggest that some of the more abundant cellular inositol polyphosphates play some important, but not yet understood, role either in the processes of haemopoietic differentiation or in the expression of differentiated cell character in myeloid cells.

Leukotriene D4 induced calcium changes in U937 cells may utilize mechanisms additional to inositol phosphate production that are pertussis toxin insensitive but are blocked by phorbol myristate acetate

DMSO differentiated U937 cells responded to 10(-6) M LTD4, LTB4 and FMLP with an increase in both InsP formation and [Ca2+]i. FMLP caused a greater rise in InsPs than either LTD4 or LTB4, which were equivalent. LTD4, however, caused a greater increase in [Ca2+]i than LTB4 (4-fold) or FMLP. The FMLP [Ca2+]i and InsP responses were abolished by pertussis toxin (100 ng/ml for 4 h) but were unaffected by PMA (10(-7) M for 3 min). In contrast, the LTD4 [Ca2+]i and InsP responses were reduced by only 50% by pertussis toxin, whilst PMA reduced the [Ca2+]i and InsP responses to LTD4 by 75 and 30%, respectively. These results suggest that mechanisms additional to InsP formation exist for mediating LTD4 evoked increases in [Ca2+]i.

Is Vasopressin-Stimulated Inositol Lipid Breakdown Intrinsic to the Mechanism of Ca2+-Mobilization at V1 Vasopressin Receptors?

Publisher Summary A role for Ca 2+ in the mechanism whereby vasopressin stimulates liver and smooth muscle is suggested by the observations that aortic contraction and hepatic glycogenolysis are diminished in Ca 2+ -free media and that the latter effect can be mimicked by the Ca 2+ ionophore A23187. This chapter describes that vasopressin-stimulated inositol lipid breakdown may have a role in the mechanism, whereby V 1 -receptor activation provokes Ca 2+ -mobilization in stimulated cells. There have been a number of suggestions as to how inositol lipid breakdown might be coupled to Ca 2+ -mobilization in stimulated cells. The chapter suggests that vasopressin might be a neurotransmitter in the central nervous system (CNS). It is interesting to note that V 1 -receptors have been identified in hippocampal neurons of the rat and latest studies have shown that vasopressin stimulates the accumulation of inositol phosphates in rat hippocampal slices and rat sympathetic ganglia. Therefore, it seems likely that information on the mechanism of action of V 1 -receptors in peripheral tissues may also help understand the actions of vasopressin in the CNS.

Metabolism of inositol 1- and 4-monophosphates in HL60 promyelocytic leukaemia cells

The metabolism of inositol 1- and 4-monophosphates in HL60 promyelocytic leukaemia cells was studied. LiCl, BeCl2 and NaF inhibited the hydrolysis of both monophosphates with half maximal inhibition occurring at 1.2 mM, 0.3 microM, 0.25 mM (Ins 1P) and 0.14 mM, 0.56 microM, 0.28 mM (Ins 4P) respectively. Lithium was an uncompetitive inhibitor with respect to both substrates. Ins 4P inhibited the hydrolysis of Ins 1P in a concentration dependent manner, suggesting that it acts as a competing substrate for the same enzyme. Half maximal inhibition occurred at 120 microM Ins 4P. The lithium sensitive activity responsible for the metabolism of both monophosphates was present in a soluble fraction made from the cells. Taken together these data suggest that Ins 1P and Ins 4P are hydrolysed by a single soluble enzyme activity which is sensitive to inhibition by lithium, beryllium and fluoride.