Synergism between diacylglycerols and calcium ionophore in the induction of human B cell proliferation mimics the inositol lipid polyphosphate breakdown signals induced by crosslinking surface immunoglobulin

Resting human tonsillar B cells were stimulated to divide by heat killed Staphylococcus aureus Cowan strain 1 which was shown to induce hydrolysis of phosphatidylinositol 4, 5-bisphosphate known to give rise to diacylglycerol and an increase in cytosolic free calcium. Addition of the diacylglycerols, 1-oleoyl-2 acetyl glycerol or sn-1, 2-dioctanoylglycerol, together with the calcium ionophore ionomycin to B cell cultures induced marked cell proliferation whereas these agents were ineffective when used alone. Both diacylglycerols were shown to compete with [3H] phorbol 12,13 dibutyrate in binding to protein kinase C. These data support the hypothesis that synergism between cytosolic calcium and endogenous diacylglycerol, which activates protein kinase C, is involved in signal transduction in the proliferation of human B cells.     

Metabolism of D-myo-inositol 1,3,4,5-tetrakisphosphate by rat liver, including the synthesis of a novel isomer of myo-inositol tetrakisphosphate.

1. We have studied the metabolism of Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) by rat liver homogenates incubated in a medium resembling intracellular ionic strength and pH. 2. Ins(1,3,4,5)P4 was dephosphorylated to a single inositol trisphosphate product, Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate), the identity of which was confirmed by periodate degradation, followed by reduction and dephosphorylation to yield altritol. 3. The major InsP2 (inositol bisphosphate) product was inositol 3,4-bisphosphate [Shears, Storey, Morris, Cubitt, Parry, Michell & Kirk (1987) Biochem. J. 242, 393-402]. Small quantities of a second InsP2 product was also detected in some experiments, but its isomeric configuration was not identified. 4. The Ins(1,3,4,5)P4 5-phosphatase activity was primarily associated with plasma membranes. 5. ATP (5 mM) decreased the membrane-associated Ins(1,4,5)P3 5-phosphatase and Ins(1,3,4,5)P4 5-phosphatase activities by 40-50%. This inhibition was imitated by AMP, adenosine 5'-[beta gamma-imido]triphosphate, adenosine 5'-[gamma-thio]triphosphate or PPi, but not by adenosine or Pi. A decrease in [ATP] from 7 to 3 mM halved the inhibition of Ins(1,3,4,5)P4 5-phosphatase activity, but the extent of inhibition was not further decreased unless [ATP] less than 0.1 mM. 6. Ins(1,3,4,5)P4 5-phosphatase was insensitive to 50 mM-Li+, but was inhibited by 5 mM-2,3-bisphosphoglycerate. 7. The Ins(1,3,4,5)P4 5-phosphatase activity was unchanged by cyclic AMP, GTP, guanosine 5'-[beta gamma-imido]triphosphate or guanosine 5'-[gamma-thio]triphosphate, or by increasing [Ca2+] from 0.1 to 1 microM. 8. Ins(1,3,4)P3 was phosphorylated in an ATP-dependent manner to an isomer of InsP4 that was partially separable on h.p.l.c. from Ins(1,3,4,5)P4. The novel InsP4 appears to be Ins(1,3,4,6)P4. Its metabolic fate and function are not known.     

The pathway of myo-inositol 1,3,4-trisphosphate dephosphorylation in liver.

We studied the dephosphorylation pathway for Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) by liver homogenates and soluble and particulate subfractions incubated in media resembling physiological ionic strength and pH. Ins(1,3,4)P3 was dephosphorylated to two InsP2 (inositol bisphosphate) isomers, one of which is Ins(3,4)P2 [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147]. The second InsP2 is the 1,3 isomer. Ins(3,4)P2 is dephosphorylated to inositol 3-phosphate by an enzyme activity located in both soluble and particulate fractions. The phosphatase(s) that attacks Ins(1,3)P2 was largely soluble, but we have not determined which phosphate(s) is removed. When the initial substrate concentration was 1 nM, the rate of dephosphorylation of Ins(1,4)P2 greater than Ins(1,3)P2 greater than Ins(3,4)P2. None of these bisphosphates was phosphorylated when incubated with liver homogenates and 5 mM-ATP, but their rates of dephosphorylation were then decreased.     

Multiple metabolic pools of phosphoinositides and phosphatidate in human erythrocytes incubated in a medium that permits rapid transmembrane exchange of phosphate.

1. A Hepes-based medium has been devised which allows rapid Pi exchange across the plasma membrane of the human erythrocyte. This allows the metabolically labile phosphate pools of human erythrocytes to come to equilibrium with [32P]Pi in the medium after only 5 h in vitro. 2. After 5-7 h incubation with [32P]Pi in this medium, only three phospholipids, phosphatidic acid (PtdOH), phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) are radioactively labelled. The concentrations of PtdIns4P and PtdIns4,5P2 remain constant throughout the incubation, so this labelling process is a reflection of the steady-state turnover of their monoester phosphate groups. 3. During such incubations, the specific radioactivities of the monoesterified phosphates of PtdIns4, PtdIns4,5P2 and PtdOH come to a steady value after 5 h that is only 25-30% of the specific radioactivity of the gamma-phosphate of ATP at that time. We suggest that this is a consequence of metabolic heterogeneity. This heterogeneity is not a result of the heterogeneous age distribution of the erythrocytes in human blood. Thus it appears that there is metabolic compartmentation of these lipids within cells, such that within a time-scale of a few hours only 25-30% of these three lipids are actively metabolized. 4. The phosphoinositidase C of intact human erythrocytes, when activated by Ca2+-ionophore treatment, only hydrolyses 50% of the total PtdIns4,5P2 and 50% of 32P-labelled PtdIns4,5P2 present in the cells: this enzyme does not discriminate between the metabolically active and inactive compartments of lipids in the erythrocyte membrane. Hence at least four metabolic pools of PtdIns4P and PtdIns4,5P2 are distinguishable in the human erythrocyte plasma membrane. 5. The mechanisms by which multiple non-mixing metabolic pools of PtdOH, PtdIns4P and PtdIns4,5P2 are sustained over many hours in the plasma membranes of intact erythrocytes are unknown, although some possible explanations are considered.     

Inositol phosphates in receptor-mediated cell signaling: metabolic origins and interrelationships

We have investigated the metabolic interrelationships of the major inositol phosphates in vasopressin-stimulated WRK 1 mammary tumor cells which were labeled to equilibrium with [14C]inositol and briefly, just prior to stimulation, with [3H]inositol. A comparison of the 3H/14C ratios of these compounds with those of the cellular inositol lipids suggests that most of the known inositol mono-, bis-, tris-, and tetrakis-phosphates are derived from precursors with turnover rates similar to those of these lipids. However, Ins(3,4,5,6)P4 (which is the major inositol tetrakisphosphate to accumulate in stimulated WRK 1 cells), Ins(1,3,4,5,6)P5, and InsP6 had 3H/14C ratios of 0 in this experiment, indicating that they must have a different metabolic origin.     

Partial purification and some properties of rat brain inositol 1,4,5-trisphosphate 3-kinase.

An enzyme which catalyses the ATP-dependent phosphorylation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was purified approx. 180-fold from rat brain cytosol by (NH4)2SO4 precipitation, chromatography through hydroxyapatite, anion-exchange fast protein liquid chromatography and gel-filtration chromatography. Gel filtration on Sepharose 4B CL gives an Mr of 200 x 10(3) for the native enzyme. The inositol tetrakisphosphate (InsP4) produced by the enzyme has the chromatographic, chemical and metabolic properties of Ins(1,3,4,5)P4. Ins(1,4,5)P3 3-kinase displays simple Michaelis-Menten kinetics for both its substrates, having Km values of 460 microM and 0.44 microM for ATP and Ins(1,4,5)P3 respectively. When many of the inositol phosphates known to occur in cells were tested, only Ins(1,4,5)P3 was a substrate for the enzyme; the 2,4,5-trisphosphate was not phosphorylated. Inositol 4,5-bisphosphate and glycerophosphoinositol 4,5-bisphosphate were phosphorylated much more slowly than Ins(1,4,5)P3. CTP, GTP and adenosine 5'-[gamma-thio]triphosphate were unable to substitute for ATP. When assayed under conditions of first-order kinetics, Ins(1,4,5)P3 kinase activity decreased by about 40% as the [Ca2+] was increased over the physiologically relevant range. This effect was insensitive to the presence of calmodulin and appeared to be the result of an increase in the Km of the enzyme for Ins(1,4,5)P3. Preincubation with ATP and the purified catalytic subunit of cyclic AMP-dependent protein kinase did not affect the rate of phosphorylation of Ins(1,4,5)P3 when the enzyme was assayed at saturating concentrations of Ins(1,4,5)P3 or at concentrations close to its Km for this substrate.     

Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

Inositol lipid-mediated signalling in response to endothelin and ATP in the mammalian testis

Molecular and Cellular Biochemistry - The testis is a complex organ in which local control is achieved by signalling between its constituent cells. Herein we describe the responses of cultured rat...