ALBERT

Notes: In two field experiments acetylene (C2H2) reduction by white clover in mixed swards was compared to N2-fixed measured by 15N dilution. In both experiments, samples for C2H2 reduction were 7.5 cm diameter turves taken from plots within which microplots of 24 cm diameter were delimited and to which 15N was applied as ammonium sulphate (15NH4)2SO4). C2H2 reduction was assayed every 6–7 d. The rate of C2H2 reduction per unit length of stolon was applied to the estimated stolon length within the appropriate microplot at the time of assay, and the amount of C2H2 that would have been reduced within the microplot was estimated by integration.In experiment 1, turves taken from grass/clover swards to which 0, 1·5, 3·0, 4·5 or 6·0 g N m−2 had been applied were incubated in sealed chambers (10% C2H2, 90% air). The mean ratio of C2H2 reduced to N2 fixed during 5 weeks was 0.74:1. Application of N fertilizer lowered the proportion of assimilated N derived from N2 fixation from 95% in unfertilized swards to 83% in those receiving 6 g N m−2 (60 kg N ha−1). In experiment 2, clover roots and stolons from plots that previously had been grazed were dissected from turves and incubated in a stream of C2H2 and air (i.e. the open system). The maximum rate of ethylene (C2H4) produced during the first 12 min was taken as a measure of true nitrogenase activity. The relationship between C2H2 reduced and N2 fixed was significant (r=0.80**). The mole ratio was 0.55:1 for the 6 weeks duration of the experiment, the low ratio possibly being due to disturbance of the nodules adversely affecting acetylene reduction. Mole ratios from both experiments were well short of the theoretical 4·3:1. Using the open system does not, therefore, overcome the shortcomings of the acetylene reduction technique for measuring N2 fixation of white clover in mixed swards.

Notes: We have previously reported that platelet-activating factor (PAF) elevates cytosolic free calcium concentration ([Ca2+]i) in fura-2-loaded glomerular mesangial cells. To confirm that this increase in [Ca2+]i is a result of receptor-mediated activation of phospholipase C, we investigated hydrolysis of phosphaphatidylinositol-4,5-bisphosphate (Ptdlns-4,5-P2) in PAF-treated mesangial cells. PAF (10-7 M) stimulated a rapid and transient formation of inositol trisphosphate. In concomitant experiments, PAF stimulated a biphasic accumulation of 3H-arachidonatelabeled 1,2-diacylglycerol (DAG). The secondary elevation in DAG was coincident with a rise in 3H-phosphorylcholine (PC) and 3H-phosphorylethanolamine (PE) suggesting that PAF stimulates delayed phospholipase activities which hydrolyze alternate phospholipids besides the polyphosphoinositides. This PAF-stimulated elevation in 3H-water soluble phosphorylbases was seen at 5 min but not at 15 sec suggesting that the initial rise in DAG as well as the initial elevation in [Ca2+]i are due primarily to Ptdlns-4,5-P2 hydrolysis. PAF also stimulated PGE2 as well as 3H-arachidonic acid and 3H-lyso phosphatidylcholine (PtdCho) formation. We suggest that arachidonate released specifically from PtdCho via phospholipase A2 is a source of this PAF-elevated PGE2. It has been postulated that anti-inflammatory prostaglandins may antagonize the contractile and proinflamatory effects of PAF via activation of adenylate cyclase. Surprisingly, exogenous PAF reduced basal and receptor-mediated cAMP concentration indicating that PAF-stimulated transmembrane signaling pathways may oppose receptor-mediated activation of adenylyl cyclase. We have taken advantage of the different sensitivities of phospholipases A2 and C(s) to PMA, EGTA, and pertussis toxin to dissociate phospholipase A2 and C activities. Acute PMA-treatment enhanced PAF-stimulated PGE2 formation, reduced PAF-induced elevations in [Ca2+]i and had no effect upon PAF-stimulated 3H-PE. We have also demonstrated that phospholipase A2, but not Ptdlns-specific phospholipase C, was sensitive to external calcium concentration. The role of a GTP-binding protein to couple PAF-receptors to the Ptdlns-specific phospholipase C was confirmed as GTPγS synergistically elevated PAF-stimulated inositol phosphate formation. We also demonstrated that pertussis toxin ADP-ribosylates a single protein of an apparent 42 kD mass and that PAF pretreatment reduced subsequent ADP-ribosylation in a time-dependent manner. However, pertussis toxin had no effect upon phospholipase C-generated water soluble phosphorylbases or inositol phosphates. In contrast, PAF-stimulated phospholipase A2 and PAF-inhibited adenylyl cyclase activities were sensitive to pertussis toxin. These results suggest that a pertussis toxin-sensitive GTP binding protein(s) may couple PAF receptors to both phospholipase A2 and adenylyl cyclase which is distinct from a pertussis toxin-insensitive GTP binding protein that links PAF receptors to phospholipase C(s). Thus, we conclude that PAF activates rat mesangial cells through multiple signaling pathways.