The biological dynamics of lipoxygenase in rabbit red cells in the course of an experimental bleeding anaemia. Unexpected effects of the calcium ionophore A 23187.
The lipoxygenase activity of red cell lysates with linoleic acid as substrate, the concentration of immunologically detectable lipoxygenase protein as well as the metabolization of external [1-14C]arachidonic or -linoleic acid by intact cells were determined during bleeding anaemia and the recovery period of rabbits. All three criteria behaved in a parallel manner. Before bleeding no lipoxygenase was detectable. After the third day of strong bleeding high amounts and activities of lipoxygenase appeared in parallel to the appearance of megaloreticulocytes. As few as about a million of cells were sufficient to detect the utilization of [1-14C]polyenoic fatty acids via the lipoxygenase pathway in intact cells in the absence of ionophore A 23187. After discontinuation of strong bleeding the amount and activity of the lipoxygenase declined gradually but persisted for 2-3 months corresponding to the presumed life-span of red cells. Several types of evidence indicate the identity of the lipoxygenase of red cells during the recovery period with that of reticulocytes during strong bleeding: (1) comparable specific activity, (2) Western blot analysis, (3) identical cellular products from [1-14C]-linoleic and -arachidonic acid which were identified by means of HPLC analysis to be 13 S-hydroxyoctadecadienoic acid (13 S-HODE) and 15 S-hydroxyeicosatetraenoic acid (15 S-HETE), respectively. Use of large amounts of cells during the recovery period (5.10(9) cells) led to an apparent masking of the polyenoic fatty acid added. Haemolysis of the cells or addition of calcium and ionophore A 23187 abolished or reduced this masking. Both masking and influence of haemolysis or ionophore disappeared at sufficiently low concentrations of cells. Under these conditions the rate of the formation of lipoxygenase products in intact cells corresponded to the lipoxygenase activity measured in membrane-free cell lysates. In red cells during the recovery period but not during strong bleeding, calcium and ionophore A 23187 stimulated the secondary conversion of 15 S-HETE to more polar products after arachidonic acid had been exhausted. The implications of the results on the performance and the interpretation of the effects of the calcium ionophore A 23187 on cellular arachidonic acid metabolism are discussed.
Ludwig
P
P
Humboldt University, Medical School (Charité), Institute of Biochemistry, Berlin, GDR.
Höhne
M
M
Kühn
H
H
Schewe
T
T
Rapoport
S M
SM
D000740:Anemia / Q000097:blood / Q000201:enzymology*; D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000201:enzymology*; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis; D007120:Immunochemistry; D008041:Linoleic Acids / Q000378:metabolism; D008084:Lipoxygenase / Q000097:blood*; D011817:Rabbits
D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; D008041:Linoleic Acids; D016718:Arachidonic Acid; D000001:Calcimycin; D008084:Lipoxygenase; D002118:Calcium