Calmodulin and respiratory burst dependence of the leukotriene generation in eosinophils

PROSTAGLANDINS

CALMODULIN GENERATION

AND

RESPIRATORY

CHAVAILLAZ

P.A.,

Department

of Biochemistry,

BURST

DEPENDENCE

OF THE LEUKOTRIENE

IN EOSINOPHILS ZILTENER

H.J.

and JOERGA.

University,

CH-1700

Fribourg,

Switzerland

Horse eosinophils (25~10~ cells/ml) incubated with the ionophore A23187 (10 pg/ml) generate leukotrienes (LT) which can be separated by ion-pair reverse phase HPLC on a 3 urn Spherisorb ODS 2 column (250x4.6 mm) into at least 11 compounds. Four of them exhibited the biological activity of slow-reacting substance and were identified as LTC,,, ll-trans LTC,,, LTD, and ll-trans LTDk. The other compounds showed LTB, spectra. Since ionophore induces a respiratory burst with an increase (s 10x) of hydrogen peroxide production to about 30 ? 8 nmol per 25~10~ cell, we investigated the influence of this metabolic pathway on the leukotriene formation. Z-deoxyglucose (56 mM), an inhibitor of the burst, suppressed the ionophore-induced formation of leukotrienes completely. Chlorpromazine (100 uM) and phenothiazine (25 nM), known calmodulin antagonists, almost suppressed the leukotriene formation and H20,generation. Because the phospholipase activity in leukocytes is very probably calmodulin dependent, eosinophils were incubated with 50 ng arachidonic acid per ml cell suspension in the presence or absence of chlorpromazine or phenothiazine. While in the absence of these calmodulin antagonists the leukotriene formation was normal, their presence inhibited the transformation of external arachidonic acid into leukotrienes, an indication that either the calmodulin dependent respiratory burst is necessary for the leukotriene formation or the lipoxygenase is calmodulin dependent.

98

SUPPLEMENT TO VOL. 27