Arachidonic acid and calcium metabolism in rnelittin stimulated neutrophils

Melittin, the predominant fraction of bee venom proteins, was studied in an experimental model of human neutrophil granulocytes to reveal its influence on eicosanoid release, metabolism and receptor function in relation to intracellular calcium metabolism. Melittin (2 μmol/l) was as potent as the calcium ionophore A23187 (10 μmol/l) for activation of 5-lipoxygenase, releasing arachidonate only from phosphatidyl-choline and phosphatidyl-ethanolamine of cellular membranes, as judged from the decreases in radioactivity by 15.4% and 30.5%, respectively. The mechanism responsible for the release of arachidonate from cellular membranes is closely coupled to cellular calcium metabolism, and melittin was found to promote calcium entry through receptor gated calcium channels, probably due to an activation of phospholipase A2. Furthermore, a down-regulation of leukotriene B4 receptors was seen. The maximal number of binding sites per cell was reduced from a median of 1520 to 950 with melittin (1 μmol/l). The study has revealed some factors important for the inflammatory mechanisms mediated by melittin.


Introduction
Melittin is a polypeptide toxin which constitutes more than 50% of bee venom proteins. It has been claimed to release endogenous arachidonic acid (AA) from cultured cells and to increase formation of prostanoids through activation of membrane bound enzymes. [2][3][4] Further, melittin was found to be able to stimulate exogenous non-incorporated AA metabolism in human polymorphonuclear neutrophils (PMN), and recent research has dealt with the interaction between melittin and cellular membranes. 6 The aims of the present work were: (1) to assess if melittin was a stimulator of endogenous AA metabolism in purified human PMNs; (2) to compare its potency with that of the calcium ionophore A23187; (3) to reveal where in the phospholipid pool AA was mobilized by melittin challenge; (4) to evaluate its influence on cellular calcium metabolism; and finally (5) to investigate its possible action on surface leukotriene B 4 receptors.
In separate receptor studies duplicate suspensions of PMNs (107/ml) were incubated with radioactive 3H-LTB4 (specific activity 6.3-8.5 x 103 GBq/ mmol, Amersham International, UK), 0.1 nmol/1-2.5 nmol/1 at 4C for 60 min. Following incubation, the ceils were rapidly centrifuged through a precooled oil phase. 9 The tips of the tubes were cut off, and cell bound radioactivity was determined in a tracer analytical scintillation counter with an automatic quench correction. Nonspecific binding was determined by adding a 1000-fold excess of non-radioactive LTB 4 (Paesel Gmbh, Germany). 1 In specific experiments, 1/tmol/1 melittin was added to the cell suspensions at 37C for 5 min. The cells were then rapidly cooled to 4C and binding experiments were done. For estimation of the dissociation constant (Kd) and receptor number per cell (Brnax) a Scatchard plot was applied. 11
The substrate for formation of most of the AA following melittin stimulation was mobilized from phosphatidyl-choline (PC) and phosphatidyl-ethanolamine (PE), owing to the relative decreases in median radioactivity by 15.4% (p < 0.01), and 30.5% (p < 0.01), respectively (Fig. 2). No significant changes were found for phosphatidylinositol (PI) or phosphatidyl-serine (PS). Calcium mobilization" Addition of melittin to PMNs caused a dose-related increase in intracellular Ca 2+ (Ca2+)i concentrations (Fig. 3). This rise was   Ca 2+ rise shows a sustained phase which is due to influx of Ca 2 + across the plasma membrane, and the effect is sustained as long as melittin is present in the medium. Validation of these results is supported by the experiments performed in a cae+-free medium (Fig. 5). When extracellular Ga g+ was removed, the melittin-induced Ca2+-influx was inhibited (Fig. 5). However, in a Cai+-free medium, LTB4 and fMLP still result in a transient rise in free (Ca2+)i (data not shown).
However, in a cae+-free medium, if melittin is added before LTB4, the increase in cytosolic free calcium elicited by LTB 4 is completely inhibited (Fig, 5). To obtain a rise in cytosolic free Ca2+, a higher concentration (50-fold) of LTB4 is needed.    (1/imol/1), respectively. However, melittin significantly reduced the maximal number of LTB 4 binding sites per cell (Bmax) from 1520 to 950 under identical experimental conditions. The viability of the PMNs after challenge with melittin or A23187 in the concentration ranges used was more than 97% as assessed by the trypan blue exclusion technique.

Discussion
The data demonstrate that the bee venom polypeptide, melittin, is a potent stimulator of endogenous AA metabolism to mono-and di-hydroxy products, including LTB4, in human PMNs which are of importance for inflammatory reactions. 12'13 Its potency regarding phospholipases stimulation in human PMNs is about 2/3 that of A23187, which is assumed to produce a maximal stimulation of phospholipases and synthesis of leukotrienes in response to calcium influx. However, regarding 5-1ipoxygenase stimulation, an enzyme which has been shown to exhibit an absolute requirement of calcium ions, 14  relative distribution of the eicosanoids LTB 4 and 5-HETE. The characteristic responses of the PMNs to LTB4, resemble those of the primary stimulus, fMLP, in the ability to promote a rapid accumulation of inositol trisphosphate (IP3) and calcium mobilization through the phospholipase C system. 1s-iv Further, melittin stimulates cyclooxygenase, as evaluated from the production of HHT, with a similar potency as found for A23187.
Stimulation of PLA2 as well as PLC, which are essential for AA metabolism and which have been demonstrated in PMN membranes, 18 appears to involve cellular calium. 19 Increase in (Ca2+)i could be effected either by PLC mediated IP3 accumulation, or by Ca 2 influx through receptor operated (voltage independent) Ca 2+ channels. If calcium dependent phospholipases are regulators of AA release, then agents that stimulate P LA2 might be expected to increase calcium availability. Melittin has earlier been described to stimulate phospholipase A 2 in human leukocytes in the presence of exogenous AA, and an ability to alter membrane permeability to calcium has been further suggested, The calcium antagonists, verapamil and nifedipine, at concentrations known to block classical voltage dependent calcium channels, had no effect on the Ca + influx induced by melittin, possibly due to receptor operated Ca 2+ influx. 9 This is contrary to apamin, another toxin from bee venom, which affects the calcium channel function. 6 The possibility that melittin interferes with calcium influx via secondary messengers (i.e. IP3) is out of question, since there was no increase in (gag+)i in calcium-free media. Further, melittin only released AA from PC and PE and not via the classical way, which involves PI, PE, PC as the three sources of AA. 19 It has been described earlier, that AA is mobilized from PC, PE, and PI using the calcium ionophore A23187. 8 The present investigation has demonstrated that melittin increases the plasma permeability to calcium, and that cytosolic free Ca 2+ is closely coupled to melittin induced activation of PLA2.
Further, melittin affects PMN surface LTB 4 receptors, as it significantly reduces the maximal number of LTB 4 binding sites per cell (Bmax). One possible explanation is that melittin generates LTB 4 from PMNs. 5 LTB 4 is then exported to the extracellular medium where it subsequently acts as a receptor agonist on the PMN surfaces with a resulting down-regulation of LTB 4 receptors.
Another explanation is that activators of protein kinase C (PKC), an important component of the signal transduction pathway in human PMNs, cause cells to become unresponsive to LTB4, but not to fMLP. 2 However, in this respect melittin has earlier been found to be a PKC inhibitor. 21 Therefore melittin may act indirectly as an activator of PKC through AA release. 22 The latter (AA) is known to induce diacylglycerol (DAG) generation, which then activates PKC. 23 Since the production of AA is high following addition of melittin, we believe that bee venom via DAG is an indirect PKC-activator. This could explain the deactivation and the decrease in the expression of LTB4 receptors found in the present study, as explained above. 2'23 In conclusion, melittin is a potent challenger of 5-1ipoxygenase AA metabolism in human PMNs. Its potency regarding phospholipases stimulation is about 2/3 that of A23187 which is assumed to produce a maximal stimulation in response to calcium influx. Further, melittin mobilizes AA from PC and PE, whereas P1 and PS seem to be unaffected, and melittin promotes Ca 2+ entry through receptor gated Ca2+-channels, probably due to a direct activation of phospholipase A 2.
Receptor studies indicate that melittin further affects the total number of LTB4-receptors, either by a down-regulatory mechanism or via the PKC system. The present data provide evidence for the complicated mechanism of melittin, and its sensitivity to arachidonate metabolism, cellular eicosanoid receptors and intracellular calcium suggests that these factors may play a role for the inflammation mediated by melittin.