calcimycin and obelin

Sendai virus induced fusion between rat polymorphonuclear leucocytes and human erythrocyte 'ghosts' containing the Ca-activated photoprotein obelin. This resulted in the production of more than 90% of the cell population as viable hybrid cells, containing active photoprotein. Substances entrapped originally within the 'ghosts' could be transferred to the hybrids as shown morphologically by fluorescence microscopy, and immunologically by the use of antibodies specific for each of the cell types. Resting free Ca2+ in the hybrids was estimated to be 0.1-0.3 microM. The relationship between intracellular Ca2+ within the hybrids and the production by the hybrids of oxygen radicals, as measured by luminol chemiluminescence, was investigated. The Ca ionophore A23187 stimulated both a rise in intracellular Ca2+ and oxygen radical production, the maximum rate of oxygen radical production being dependent upon the intracellular Ca2+ concentration. Complement activation at the cell surface, chemotactic peptide, and concanavilin A stimulated a rise in intracellular Ca2+, each with different characteristics: complement activation increased intracellular Ca2+ to 8 microM for at least 60 sec; chemotactic peptide raised it to approximately 0.6 microM for a prolonged period (at least 10 min) and concanavilin A stimulated a transient (t1/2 = approximately 80 sec) rise to approximately 0.6 microM. Un-opsinized particles, latex beads (diam. approximately equal to 1 micron), which stimulate oxygen radical production, did not produce a detectable rise in intracellular Ca2+. Intracellular EGTA (approximately, 2.5 mM) inhibited both the oxygen radical production and the rises in intracellular Ca2+ induced by chemotactic peptide and concanavilin A, and delayed both the rise in intracellular Ca2+ and the onset of oxygen radical production induced by complement. Intracellular EGTA had no effect on oxygen radical production stimulated by latex particles. An increase in intracellular Ca2+ triggered oxygen radical production by the hybrids in response to the following stimuli: concanavilin A, chemotactic peptide, complement and the Ca ionophore A23187. However, the extent and duration of the increase in cytoplasmic Ca2+ was different for each stimulus, and the apparent relationship between cytoplasmic Ca2+ and oxygen radical production was different for each stimulus. Un-opsinized particles stimulated oxygen radical production without requiring a rise in intracellular Ca2+ concentratio

Topics: Animals; Calcimycin; Calcium; Complement Activation; Egtazic Acid; Erythrocyte Membrane; Humans; Hybrid Cells; Luminescent Proteins; Neutrophils; Oxygen; Rats; Rats, Inbred Strains

1. Sealed pigeon erythrocyte 'ghosts' were prepared containing ATP and the Ca2+-activated photoprotein obelin to investigate the relationship cyclic AMP formation and internal free Ca2+. 2. The 'ghosts' were characterized by (a) morphology (optical and electron microscopy), (b) composition (haemoglobin, K+, Na+, Mg2+, ATP, obelin), (c) permeability to Ca2+, assessed by obelin luminescence and (d) hormone sensitivity (the effect of beta-adrenergic agonists and antagonists on cyclic AMP formation). 3. The effect of osmolarity at haemolysis and ATP at resealing on these parameters was investigated. 4. Sealed 'ghosts', containing approx. 2% of original haemoglobin, 150mM-K+, 0.5MM-ATP, 10(3)--10(4) obelin luminescence counts/10(6) 'ghosts', which were relatively impermeable to Ca2+ and in which cyclic AMP formation was stimulated by beta-adrenergic agonists over a concentration range similar to that for intact cells, could be prepared after haemolysis in 6mM-NaCl3mM-MgCl2/50mM-Tes, pH7, and resealing for 30min at 37 degrees C in the presence of ATP and 150mM-KCl. 5. The initial rate of adrenaline-stimulated cyclic AMP formation in these 'ghosts' was 30--50% of that in intact cells and was inhibited by the addition of extracellular Ca2+. Addition of Ca2+ to the 'ghosts' resulted in a stimulation of obelin luminescence, indicating an increase in internal free Ca2+ under these conditions. 6. The ionophore A23187 increased the rate of obelin luminescence in the 'ghosts' and also inhibited the adrenaline-stimulated increase in cyclic AMP. 7. The effect of ionophore A23187 on obelin luminescence and on cyclic AMP formation in the 'ghosts' was markedly decreased by sealing EGTA inside the 'ghosts'. 8. It was concluded that cyclic AMP formation inside sealed pigeon erythrocyte 'ghosts' could be inhibited by more than 50% by free Ca2+ concentrations in the range 1--10 micrometer.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Calcium; Columbidae; Cyclic AMP; Egtazic Acid; Erythrocyte Membrane; Erythrocytes; Luminescent Proteins; Sympathomimetics

1. Liposomes bearing different net surface charges have been prepared and their ability to entrap the Ca2+-activated photoprotein, obelin, has been studied 2. Negatively-charged liposomes, composed of egg-yolk lecithin, cholesterol and phosphatidylserine, consistently produced the most homogeneous populations of liposomes after sonication, as shown by electron microscopy after negative staining. These consisted of a large proportion of uni- and bilamellar vesicles within the size range of 20--50 nm, external diameter. 3. Sonicated negatively-charged liposomes had a mean aqueous obelin space of 6.8 +/- 0.8 microliter/mumol of phospholipid compared to a mean space for inulin [14C]carboxylic acid of 5.1 +/- 0.8 microliter/mumol of phospholipid. 4. Sonication reduced the Ca2+ permeability of the negatively-charged liposomes, as measured by the utilization of entrapped obelin. 5. Preparations of uncharged (no phosphatidylserine) and positively-charged (stearylamine instead of phosphatidylserine), sonicated liposomes contained a greater proportion of larger vesicles, which were more permeable to Ca2+ than sonicated, negatively-charged liposomes. 6. Obelin, trapped within sonicated, negatively-charged liposomes, responded to increases in the free Ca2+ concentration within the liposomes caused by the bivalent-cation ionophore A23187 at concentrations as low as 19 nM. 7. The effect of A23187 was inhibited by Mg2+ at a low concentration of Ca2+ (10 muM), but not at 1 mM Ca2+. 8. It was concluded that obelin could be trapped in the aqueous compartment of sonicated liposomes which remained relatively impermeable to Ca2+. Furthermore, trapped obelin could respond to changes in the free Ca2+ concentration within these liposomes.

Topics: Calcimycin; Calcium; Carboxylic Acids; Cholesterol; Insulin; Liposomes; Luminescent Measurements; Luminescent Proteins; Magnesium; Membranes, Artificial; Microscopy, Electron; Phosphatidylcholines; Phosphatidylserines; Polyethylene Glycols; Proteins; Sonication

Topics: Adipose Tissue; Animals; Calcimycin; Calcium; Carbonic Anhydrases; Cholesterol; In Vitro Techniques; Liposomes; Luminescent Measurements; Luminescent Proteins; Magnesium; Phospholipids; Proteins; Rats

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