obelin and Hemolysis

Using polymorphonuclear leucocyte-erythrocyte ghost hybrids entrapping the calcium-activated photoprotein obelin, we have demonstrated that sublytic amounts of the complement membrane attack complex induce a rapid but transient increase in intracellular free calcium ion concentration ([Ca2+]i). This increase in [Ca2+]i occurs prior to, and is required for, rapid removal of membrane attack complexes from the cell surface. The increase in [Ca2+]i is not only due to increased influx from outside the cell, but also results from mobilization of intracellular stores. The possible mechanism of mobilization of calcium, and the importance of an increase in [Ca2+]i as a mediator of recovery processes in nucleated cells, are discussed.

Topics: Calcium; Complement Membrane Attack Complex; Complement System Proteins; Erythrocyte Membrane; Hemolysis; Humans; Hybridization, Genetic; Luminescent Proteins; Neutrophils

1. The effect of rabbit anti-(pigeon erythrocyte) antibodies plus human complement on the concentration of intracellular free Ca2+ in sealed pigeon erythrocyte 'ghosts' was investigated with the photoprotein obelin. 2. The addition of human serum, as a source of complement, to 'ghosts' coated with antibody caused a rapid increase in intracellular free Ca2+ after a lag of 20-40 s, as detected by an increase in obelin luminescence. 3. The increase in obelin luminescence could not be explained by release of obelin into the medium. It was also Ca2+-dependent in that extracellular EGTA abolished the effect and intracellular EGTA inhibited it and required the complete terminal complex (C56789). No effect was seen with C5678. 4. The concentration of intracellular free Ca2+ before addition of complement was approx. 0.3 microM. This increased to a maximum of 5-30 microM after complement addition and then remained constant for at least 1-2 min. 5. Antibody plus complement induced a rapid increase in 42K+ efflux and an inhibition of cyclic AMP formation. 6. When partially purified complement components (C5b-9) were used in 'reactive lysis' it was possible to inhibit the release of macromolecules from pigeon erythrocyte 'ghosts' by extracellular EGTA. 7. It was concluded that the increase in intracellular free Ca2+ concentration caused by anti-cell antibody plus complement occurred before cell lysis and may be involved in the mechanism of complement-induced cell injury.

Topics: Animals; Antibodies; Body Fluids; Calcium; Columbidae; Complement C9; Complement System Proteins; Cyclic AMP; Erythrocyte Membrane; Hemolysis; Intracellular Fluid; Luminescent Measurements; Luminescent Proteins; Potassium

1. Obelin, the Ca(2+)-activated luminescent protein from the hydroid Obelia geniculata, was sealed inside pigeon erythrocyte ;ghosts' in order to investigate effects on their permeability of different methods of preparation and of the bivalent cation ionophore A23187. 2. Changes in free Ca(2+) within the ;ghosts' were studied by following the rate of luminescence of obelin. The possibility that the obelin might have been released from the ;ghosts' during an experiment was investigated by studying the release of inulin and pyruvate kinase from the ;ghosts'. Less than 10% of the inulin or pyruvate kinase sealed within the ;ghosts' was released under any of the experimental conditions. 3. Triton X-100 (0.1-10%, v/v) made the ;ghosts' highly permeable to Ca(2+). In the presence of 1mm-Ca(2+) and Triton, 95-100% of the obelin was utilized within 10-20s. 4. A time-course of resealing ;ghosts' at 37 degrees C showed that over a period of 90min, the ;ghosts' became gradually less permeable to Ca(2+). ;Ghosts' which remained at 0 degrees C retained only a small concentration of obelin and ATP, and were highly permeable to Ca(2+). 5. Erythrocyte ;ghosts' resealed for 30min at 20 degrees C rather than 37 degrees C were more permeable to Ca(2+), as shown by the fact that 92% of the obelin in the ;ghosts' was utilized during the first 60s after the addition of 1mm-Ca(2+), as opposed to 44% for ;ghosts' resealed at 37 degrees C. 6. Haemolysis at pH6.0 rather than 7.0 resulted in ;ghosts' which were highly permeable to Ca(2+) after resealing for 60min at 37 degrees C. Of the obelin in the ;ghosts', produced by haemolysis at pH6.0, 90% was utilized in the first 60s after the addition of 1mm-Ca(2+) compared with 23% for ;ghosts' produced at pH7.0. 7. The bivalent cation ionophore A23187 increased the permeability of the ;ghosts' to Ca(2+). Maximum effects of the ionophore (16mug/ml) were obtained by preincubating the ;ghosts' with the ionophore A23187 (16mug/ml) in the presence of a low concentration of Mg(2+) and in the absence of Ca(2+).

Topics: Adenosine Triphosphate; Animals; Calcimycin; Calcium; Cell Membrane Permeability; Columbidae; Erythrocytes; Hemoglobins; Hemolysis; Hydrogen-Ion Concentration; In Vitro Techniques; Inulin; Ionophores; Luminescent Measurements; Luminescent Proteins; Polyethylene Glycols; Proteins; Pyruvate Kinase; Temperature; Time Factors