Michael J. O. Wakelam

Development of a novel, Ins(1,4,5)P3-specific binding assay. Its use to determine the intracellular concentration of Ins(1,4,5)P3 in unstimulated and vasopressin-stimulated rat hepatocytes.

The binding of [3H]Ins(1,4,5)P3 to bovine adrenocortical microsomes has been shown to be rapid, reversible and saturable. The microsomal preparation contained a single population of high affinity sites (KD = 6.82+/-2.3 nM, Bmax = 370+/-38 fmol/mg protein). The binding site was shown to exhibit positional specificity with respect to inositol trisphosphate binding, i.e. Ins(2,4,5)P3 was able to compete with [3H]Ins(1,4,5)P3 whereas Ins(1,3,4)P3 was not. Ins(1,3,4,5)P4 showed a similar affinity for the receptor as Ins(2,4,5)P3 whereas the other inositol phosphates tested, ATP, GTP and 2,3-DPG, were poor competitors. [3H]Ins(1,4,5)P3-binding was independent of free Ca2+ concentrations. The adrenocortical microsomal preparation has been incorporated into an assay which has been used to determine the basal and vasopressin-stimulated content of neutralised acid extracts of rat hepatocytes. Intracellular concentrations of Ins(1,4,5)P3 were calculated to be 0.22+/-0.15 microM basal and 2.53+/-1.8 microM at peak stimulation. This assay provides a simple, specific and quantitative method for the measurement of Ins(1,4,5)P3 concentrations in the picomolar range.

Widespread distribution of Gqα/Gllα detected immunologically by an antipeptide antiserum directed against the predicted C-terminal decapeptide

Antisera were raised to a synthetic peptide which represents the predicted C‐terminal decapeptide of the α subunit of the G‐proteins Gq and Gll. Competitive ELISA indicated that antiserum CQ2 displayed strong reactivity against this peptide. Antiserum CQ2 identified an apparently single polypeptide of 42 kDa which was expressed widely. The mobility of this polypeptide in SDS‐PAGE was not modified by pretreatment of cells with pertussis toxin, indicating that it was not a substrate for this toxin. Furthermore, the levels and mobility of this polypeptide were unaltered by treatment of cells with cholera toxin, defining that it was not related to Gsα.

Vasopressin‐stimulated [3H]‐inositol phosphate and [3H]‐phosphatidylbutanol accumulation in A10 vascular smooth muscle cells

1 The characteristics of vasopressin‐stimulated phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) and phosphatidylcholine (PtdCh) hydrolysis were examined in A10 vascular smooth muscle cells (VSMC), by assessing the formation of [3H]‐inositol phosphates ([3H]‐IP) and the accumulation of the phospholipase D (PLD) specific product, [3H]‐phosphatidylbutanol ([3H]‐PtdBuOH). 2 Vasopressin ([Arg8]‐VP) and a number of related analogues stimulated the accumulation of [3H]‐IP and [3H]‐PtdBuOH with similar EC50 values, generating the same rank order of potency for each response (Arg8‐VP = vasotocin = Lys8‐VP ≫ oxytocin). 3 Inhibition of vasopressin‐stimulated [3H]‐IP and [3H]‐PtdBuOH accumulation by the V1a receptor antagonists, Des‐Gly9[β‐mercapto‐β,β,‐cyclopentamethylene propionyl, O‐Et‐Tyr2,Val4,Arg8]‐vasopressin generated similar IC50 values suggesting that both these responses are mediated through the activation of a single V1a receptor subtype. 4 The onset of vasopressin‐stimulated inositol‐1,4,5‐trisphosphate (Ins(1,4,5)P3) mass formation proceeded [3H]‐PtdBuOH accumulation indicating that PtdCh hydrolysis was activated subsequent to PtdIns(4,5)P2 breakdown. 5 The protein kinase C (PKC) activator, tetradecanoylphorbol acetate (TPA) also stimulated [3H]‐PtdBuOH accumulation. Preincubation with the PKC inhibitor Ro‐31–8220 abolished both TPA‐ and vasopressin‐stimulated [3H]‐PtdBuOH, suggesting that the intermediate activation of protein kinase C is involved in the regulation of PLD by vasopressin. 6 Pretreatment of the A10 VSMC with Ro‐31–8220 (100 μm) also potentiated vasopressin‐stimulated Ins(1,4,5)P3 mass formation. Therefore stimulation of PKC may have opposing roles in the regulation of agonist activation of PLC and PLD. 7 Preincubation of the cells with EGTA, verapamil, or the receptor‐operated calcium channel antagonist, SK&F 96365, reduced vasopressin‐stimulated [3H]‐PtdBuOH accumulation by approximately 30%, suggesting that influx of calcium has a significant role to play in the regulation of vasopressin‐stimulated PLD activity.

Mass measurement of inositol phosphates

This review summarises the methods available for the mass measurement of inositol phosphates, i.e., use of radioactive inositol lipid precursors, optical techniques, gas chromatography, mass spectrometry, nuclear magnetic resonance, fast atom bombardment and assays specific for Ins(1,4,5)P3. Examples of the use of each method, its sensitivity, advantages and drawbacks are given.

L6 skeletal muscle cells have functional V1-vasopressin receptors coupled to stimulated inositol phospholipid metabolism

The effects of vasopressin and related peptides upon the rat skeletal muscle cell line, L6, have been examined. No effects upon cellular cyclic AMP levels were found indicating that L6 cells possess no functional V2‐vasopressin receptors. Vasopressin and its analogues did, however, stimulate the rapid and dose‐dependent accumulation of inositol phosphates. This effect and the rank order of potency of vasopressin analogues demonstrate the presence of functional V1‐vasopressin receptors upon L6 cells. These results suggest that the L6 line may be a useful model for vasopressin effects upon skeletal muscle metabolism.

Insulin and platelet-derived growth factor acutely stimulate glucose transport in 3T3-L1 fibroblasts independently of protein kinase C

Insulin and platelet-derived growth factor (PDGF) are mitogenic for murine 3T3-L1 fibroblasts. Both these mitogens acutely stimulate glucose transport by 2-4-fold in these cells, evident within minutes of agonist exposure. The tumour promoter and protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) also stimulates glucose transport by 2-3-fold over a similar time frame, suggesting that protein kinase C may be involved in the mitogenic action of insulin and PDGF in this cell line. In an attempt to address this, we have measured intracellular sn-1,2-diacylglycerol (DAG) levels in response to insulin, PDGF and PMA. We show that PDGF and PMA induce a rapid elevation in intracellular diacylglycerol levels, but insulin was without effect. In addition, we have shown that PMA and PDGF, but not insulin, stimulate protein kinase C activity. However, depletion of protein kinase C by overnight exposure to PMA, abolished PMA-stimulated glucose transport but had no effect on insulin- and PDGF-stimulated glucose transport, suggesting that the stimulation of glucose transport by these mitogens does not involve protein kinase C. The use of the selective protein kinase C inhibitor, Roche 31-8220, which inhibited PMA-stimulated glucose transport, but was without effect on insulin- and PDGF-stimulated glucose transport further supports this conclusion. Taken together, these data argue against a role for protein kinase C in the stimulation of glucose transport in 3T3-L1 fibroblasts caused by acute exposure to insulin or PDGF.

v-Src Induces elevated levels of diglyceride by stimulation of phosphatidylcholine hydrolysis

When Rat-1 cells bearing the ts LA29 mutant of Rous sarcoma virus (Rat1 LA29) are shifted from restrictive to permissive temperature, the pp60v-Src tyrosine kinase is activated and there is an increase in the cellular level of sn1,2-diacylglycerol (DRG) within 30 min which is not accompanied by increased inositol phospholipid hydrolysis. Temperature shift also increases the hydrolysis of phosphatidylcholine (PC), as determined by an increase in the generation of water soluble choline metabolites. Transphosphatidylation studies have shown that this occurs at least in part via a phospholipase D (PLD) catalysed pathway.

Mechanical and biochemical responses to endothelin-1 and endothelin-3 in bovine bronchial smooth muscle.

1 In this study, mechanical responses to endothelin‐1 and endothelin‐3 were examined in bovine bronchial smooth muscle. In addition, the involvement of phosphatidylinositol 4,5‐bisphosphate hydrolysis (PIP2) in the responses to these peptides was assessed by measurement of inositol (1,4,5) trisphosphate (I(1,4,5)P3) production using a specific mass assay. 2 ET‐1 evoked contractions of bovine bronchi which were concentration‐dependent and initiated at between 10−9m and 10−8m. ET‐1‐evoked responses were unaffected by slight elevation of tone with potassium chloride (3 × 10−2m), methacholine (10−6m) or U46619 (10−7m). 3 Contractions to ET‐1 were not altered by pre‐incubation with atropine (10−5m), indomethacin (10−5m), nifedipine (10−5m), phosphoramidon (3.67 × 10−5m) or by removal of the epithelium. 4 ET‐3 evoked small contractions which were not concentration‐dependent. In the presence of phosphoramidon (3.67 × 10−5m) however, concentration‐dependent contractions were obtained to ET‐3 which were unaffected by atropine (10−5m) or by removal of the epithelium, but were significantly attenuated by indomethacin (10−5m). Nifedipine (10−5m) virtually abolished this response. 5 Both ET‐1 and ET‐3 (in the presence of phosphoramidon)‐evoked contractions were significantly enhanced by the presence of the phorbol ester phorbol 12,13‐dibutyrate (10−8m). Neither ET‐1‐, nor ET‐3‐mediated responses were antagonized by the protein kinase C (PKC) inhibitor, Ro 31–8220 (3 × 10−9–3 × 10−8m). 6 ET‐1 (3 × 10−7m) evoked a biphasic rise in levels of I(1,4,5)P3 which was unaltered by pre‐incubation with atropine, whilst ET‐3 (10−10–3 × 10−7m) failed to alter levels of I(1,4,5)P3 at any time point examined, even in the presence of phosphoramidon (3.67 × 10−5m). 7 These results suggest that, in bovine bronchial smooth muscle, ET‐1 does not evoke contraction via cyclo‐oxygenase metabolites, does not evoke release of the neurotransmitter substance acetylcholine, or require calcium influx via dihydropyridine‐sensitive channels. ET‐1 evokes I(1,4,5)P3 production, but stimulation of protein kinase C may not be critical for the associated contraction. In contrast, ET‐3‐evoked contractions are partly mediated by cyclo‐oxygenase metabolites. ET‐3 does not stimulate PIP2 hydrolysis, nor activate PKC, but may, either directly or as a requirement of intermediates released in response to ET‐3, rely upon extracellular calcium.

Inhibition by glucocorticoid and staurosporine of IL-4-dependent CD23 production in B lymphocytes is reversed on engaging CD40.

IL‐4 synergizes with signals delivered through CD40 both for the induction of CD23/FcRH expression and for IgE synthesis. Moreover, engagement of CD40 on the B cell surface by MoAb overcomes the ability of intcrferons. transforming growth factor‐beta, or anti‐CD 19 to inhibit IL‐4‐dependent change. We now report that occupancy of CD40 relieves potent suppression of IL‐4‐induced CD23 production by glucocorticoid or the relatively broad‐acting kinase inhibitor staurosporine. Interruption of the IL‐4 signal was observed with concentrations of staurosporine considered to be selective for protein kinase C (PKC) inhibition (IC50= 10 nm) but not with genistein or tyrphostins. effective inhibitors of tyrosine kinase activity. On ligation of CD40, staurosporine no longer inhibited the IL‐4 signal: at concentrations of between I and 20 nM. staurosporine actually increased by as much as 100% the rate of CD23 production stimulated on simultaneous activation through CD40 and 1L‐4R. Such augmentation was not observed when the more specific PKC inhibitor RO‐31‐8220 was used; indeed. CD40 engagement was unable to overcome the ability of this inhibitor to block IL‐4‐promoted CD23 induction (IC50= 10 μm). Occupancy of CD40 did, however, thwart completely the usual ability of prednisolone to inhibit the IL‐4 signal leading to CD23 induction. Activation through CD40 left inhibition of phorbol ester‐induced CD23 expression by staurosporine, RO‐31‐8220, or glucocorticoid unchecked. These findings further highlight the intimate level of cross‐talk existing between CD40 and IL‐4R on resting B lymphocytes to promote CD23 expression, a phenotypic change which preludes IgE synthesis.

Inhibition of the amplified bombesin‐stimulated inositol phosphate response in N‐ras transformed cells by high density culturing

The bombesin‐stimulated inositol phosphate response is only increased in cells transformed by the overexpression of N‐ras when they are cultured under sub‐confluent conditions. The inhibition of the amplified bombesin stimulation can be partially reversed by either, incubation of the cells for 5 h with 1 mM suramin, or by a 30 min preincubation of cells in excess buffer. The inhibitory effects are probably caused by the effects of autocrines and may explain some of the different published observations concerning the effects of ras gene products upon inositol phospholipid metabolism.