L-myo-inositol 1,4,5,6-tetrakisphosphate is present in both mammalian and avian cells.

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.

The haemopoietic growth factors interleukin 3 and colony stimulating factor-1 stimulate proliferation but do not induce inositol lipid breakdown in murine bone-marrow-derived macrophages.

The haemopoietic growth factors interleukin 3 (IL-3) and colony stimulating factor-1 (CSF-1) stimulate the survival and proliferation of murine normal bone-marrow-derived macrophages. To establish whether these growth factors elicit their effects via the hydrolysis of phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] to form the second messengers inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] and diacylglycerol, macrophages were labelled with tracer quantities of [3H]inositol. Treatment of these cells with either IL-3 or CSF-1 did not alter the levels of PtdIns(4,5)P2 or Ins(1,4,5)P3.

Phorbol esters activate protein kinase C and glucose transport and can replace the requirement for growth factor in interleukin-3-dependent multipotent stem cells.

Interleukin 3 (IL-3) promotes the survival, proliferation and development of progenitor cells from several distinct haemopoietic lineages and can also stimulate the self-renewal of stem cells. We have explored the mode of action of this growth factor in promoting survival and proliferation, using a multipotent haemopoietic stem cell line FDC-Mix 1. In the absence of IL-3 these cells died within 16-48 h.

Characterisation of monoclonal antibodies which specifically recognise the human erythrocyte glucose transport protein.

Two monoclonal antibodies (mabs) of subclass IgG1 have been raised against the human erythrocyte glucose transport protein. The mabs bound to the purified glucose transporter in both its membrane-bound and detergent-solubilised forms. However, they exhibited little or no binding to the detergent-solubilised nucleoside transport protein, which is present as a minor contaminant in the glucose transport protein preparation.

Insulin inhibits the cholera-toxin-catalysed ribosylation of a Mr-25000 protein in rat liver plasma membranes.

A method is described for preparing a plasma-membrane fraction from hepatocytes by a rapid, gentle, Percoll fractionation procedure. Cholera toxin elicited the ribosylation of a number of proteins in these membranes, including the components of the stimulatory guanine nucleotide regulatory protein, Ns. Insulin, however, inhibited the ability of cholera toxin to ribosylate a protein of Mr 25 000.

Inhibitors of cholera toxin-induced adenosine diphosphate ribosylation of membrane-associated proteins block stem cell differentiation.

Two potent inhibitors of mono-adenosine diphosphate (ADP) ribosylation have recently been described and characterized, named p-methoxylbenzylaminodecamethylene guanidine sulfate (MBAMG) and benzylaminododecylguanine hydrochloride (BADGH). We have used these agents to investigate the role of ADP ribosylation in hematopoiesis using long-term marrow cultures. The addition of MBAMG (10(-6) mol/L) or BADGH (5 X 10(-4) mol/L) led to both an inhibition of mature cell production and the development of colony-stimulating factor (CSF-1)-responsive GM-CFC, but had no effect upon spleen colony-forming units (CFU-S) or on progenitor cells which respond to the multilineage stimulating factor present in WEHI-3B cell-conditioned medium.

Stimulation of hexose uptake by haemopoietic cell growth factor occurs in WEHI-3B myelomonocytic leukaemia cells: a possible mechanism for loss of growth control.

WEHI-3B myelomonocytic leukaemia cells secrete a haemopoietic cell growth factor (HCGF) which facilitates the proliferation and development of multipotential stem cells and committed progenitor cells. Several cloned, nonleukaemic cell lines (FDC-P cells) are absolutely dependent on HCGF and die in the absence of it. In these cell lines, factor dependence is associated with the ability of HCGF to increase glucose uptake, thereby controlling glycolytic flux and intracellular ATP levels.

The role of haemopoietic cell growth factor (interleukin 3) in the development of haemopoietic cells.

Haemopoietic cell development in vivo occurs in restricted sites in association with stromal cells. Haemopoiesis in vitro can be induced in the absence of stromal cells, provided the haemopoietic cells are supplied with appropriate growth stimulatory molecules. Evidence indicates that the same, or functionally similar, growth factors are normally supplied in vivo by the surrounding stromal cells and that the control of haemopoietic cell proliferation and development is regulated locally and is mediated by cell-cell interactions.

The role of stromal cells and growth factors in haemopoiesis and modulation of their effects by the src oncogene.

In the haemopoietic system the mature blood cells have only a finite lifetime. For example, a circulating granulocyte in the peripheral blood has an approximate half-life of 7 h (Cartwright, Athens & Wintrobe, 1964; Dancey, Dubelbeiss, Harker & Finch, 1976) whilst the lifetime of an erythrocyte is approximately 120 days (Wickramasinghe & Weatherall, 1982). This constant 'death' of mature functional haemopoietic cells means that new blood cells must replace those that are removed.

Dimethylnitrosamine inhibits the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes and decreases plasma membrane fluidity.

The effect of the hepatocarcinogen dimethylnitrosamine on rat liver plasma membrane adenylate cyclase activity and lipid fluidity was assessed. Glucagon-stimulated adenylate cyclase activity exhibited a complex response to increasing concentrations of dimethylnitrosamine, whereas fluoride-stimulated adenylate cyclase activity was progressively inhibited. Maximal inhibitory effects were observed at a concentration of 15 mM in both cases.

The phorbol ester, TPA inhibits glucagon-stimulated adenylate cyclase activity.

The ability of glucagon (10 nM) to increase hepatocyte intracellular cyclic AMP concentrations was reduced markedly by the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate). The half-maximal inhibitory effect occurred at 0.14 ng/ml TPA.

Haemopoietic cell growth factor mediates cell survival via its action on glucose transport.

A number of haematopoietic precursor cell lines have been established which exhibit an absolute dependence on haematopoietic cell growth factor (HCGF) which is secreted by WEHI-3 myelomonocytic leukaemia cells. In the presence of HCGF, ATP levels are maintained in these factor-dependent cells (FDC-P cells); in the absence of HCGF, intracellular ATP levels undergo a steady depletion. The cell death that follows this ATP depletion can be prevented by supplying exogenous ATP suggesting that HCGF maintains these cells via its effects on energy metabolism.

Membrane-mediated responses to 12-O-tetradecanoylphorbol-13-acetate in human skin fibroblasts.

The effects on primary human skin fibroblasts of the structurally unrelated tumour promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and teleocidin B were compared with those of epidermal growth factor and the non-promoting derivative 4-O-methyl TPA by means of two-dimensional gel electrophoresis. Both TPA and teleocidin B caused a marked increase in the synthesis of two polypeptides of molecular weights 44 kilodaltons (P44) and 55 kilodaltons (P55). P55 was complexed in cell lysates by antiactin antibody and was shown to be a component of the cytoskeleton.

The lipid fluidity of rat liver membrane subfractions.

1. The lipid fluidity of three major rat liver plasma-membrane subfractions, as well as Golgi apparatus and endocytic fractions, was assessed with a fatty acid spin probe by using e.s.

5'-Nucleotidase is activated upon cholesterol-depletion of liver plasma membranes.

The cholesterol content of rat liver plasma membranes was manipulated using either cholesterol-free or cholesterol-enriched liposomes. Removal of cholesterol from the membranes led to a marked increase in 5'-nucleotidase activity. However, increase in cholesterol content failed to exert any significant effect on 5'-nucleotidase activity.

Effect of haematopoietic cell growth factor on intracellular ATP levels.

Growth and development of haematopoietic cells in vitro require the presence of specific regulatory molecules. Some of these molecular species appear to have a broad specificity, being able to promote the proliferation and differentiation of multipotential cells, as well as megakaryocytic, erythroid and granulocytic-progenitor cells. Such factors are present in medium conditioned by the growth of lectin-stimulated mouse spleen cells or WEHI-3 myelomonocytic leukaemia cells (WEHI-CM).

Adenylate cyclase is inhibited upon depletion of plasma-membrane cholesterol.

A procedure has been developed that allows for the depletion of rat liver plasma membrane cholesterol by incubation with liposomes at 4 degrees C. Upon cholesterol depletion, adenylate cyclase activity was inhibited and the membranes became more rigid, as determined by the flexibility of an incorporated fatty acid spin probe. Decreasing the cholesterol/phospholipid molar ratio elicited a pronounced drop in the net fold-stimulation of adenylate cyclase activity by glucagon.

Forskolin and ethanol both perturb the structure of liver plasma membranes and activate adenylate cyclase activity.

Both forskolin and ethanol elicit the activation of basal and ligand-stimulated adenylate cyclase activities in rat liver plasma membranes. Ethanol is most potent at activating the fluoride- and glucagon-stimulated activities whilst having little effect on basal activity. In contrast forskolin exerts its greatest effect on basal activity.

Mechanism of glucagon activation of adenylate cyclase in the presence of Mn2+.

For a variety of ligand states, adenylate cyclase activity in the presence of Mn2+ was greater than with Mg2+. Trypsin treatment of intact hepatocytes, under conditions which destroy cell surface glucagon receptors, led to a first order loss of glucagon-stimulated adenylate cyclase activity in isolated membranes assayed in the presence of Mn2+ whether or not GTP (100 microM) was present in the assays. Arrhenius plots of basal activity exhibited a break at around 22 degrees C, those with NaF were linear and those with glucagon +/- GTP (100 microM) were biphasic with a break at around 28 degrees C.

Perturbations of liver plasma membranes induced by Ca2+ are detected using a fatty acid spin label and adenylate cyclase as membrane probes.

Ca2+ decreased the lipid fluidity of rat liver plasma membranes labeled with 5-nitroxide stearate, I(12,3), as indicated by the order parameter (S). These effects form a reversible, saturable process with an association constant of 1 x 10(3) M-1. Arrhenius-type plots of S indicated that the lipid phase separation, present in the external leaflet of native membranes between 28 and 19 degrees C, is perturbed by mM Ca2+ such that the high temperature onset is elevated to 32-34 degrees C.

Elevated membrane cholesterol concentrations inhibit glucagon-stimulated adenylate cyclase.

A method was devised which increases the cholesterol concentration of rat liver plasma membranes by exchange from cholesterol-rich liposomes at low temperature (4 degrees C). When the cholesterol concentration of liver plasma membranes is increased, there is an increase in lipid order as detected by a decrease in mobility of an incorporated fatty acid spin probe. This is accompanied by an inhibition of adenylate cyclase activity.

Thermotropic lipid phase separations in human platelet and rat liver plasma membranes.

Electron spin resonance (ESR) studies were conducted on human platelet plasma membranes using 5-nitroxide stearate, I(12,3). The polarity-corrected order parameter S and polarity-uncorrected order parameters S(T parallel) and S(T perpendicular) were independent of probe concentration at low I(12.3)/membrane protein ratios.