| Abstract: | The uptake of Quin-2 by human polymorphonuclear (PMN) leukocytes permitted accurate fluorimetric quantification of the cytosolic concentration of intracellular calcium ( Ca+2]in), without altering the expression of the two subsets of leukotriene B4 (LTB4) receptors, as assessed by the binding of 3H]LTB4. Chemotactic concentrations of LTB4 elicited a rapid increase in Ca+2]in, which reached a peak within 0.6 to 1 min and then decayed back to baseline levels by 6 to 10 min. The maximal increase and the half-maximal increase in Ca+2]in were achieved by LTB4 at mean concentrations of 5 X 10(-10) M and 2 X 10(-10) M, respectively, where the binding of LTB4 to high-affinity receptors predominates. A rank order of potency of LTB4 greater than 5(S),12(S)-6-trans-LTB4 greater than 12(S)-LTB4 was established for the elicitation of increases in Ca+2]in, which reflects the binding of the isomers to low-affinity receptors. PMN leukocytes were preincubated with 10(-8) M LTB4 to induce chemotactic deactivation, which eliminates the expression of high-affinity receptors without altering the expression of the low-affinity receptors for LTB4. LTB4 elicited an increase in Ca+2]in in the deactivated PMN leukocytes with an EC50 of 3 X 10(-8) M, which is similar to the Kd for LTB4 binding to the low-affinity receptors. Two lines of cultured human leukemic cells, IM-9 and HL-60, did not bind LTB4 specifically and did not show any change in Ca+2]in upon the addition of 3 X 10(-8) M LTB4. The HL-60 human promyelocytic leukemia cell line was induced to differentiate in 1% dimethyl sulfoxide to leukocytes with more mature myelocytic characteristics. Differentiated HL-60 cells expressed an average of 54,000 low-affinity receptors for LTB4 per cell with an average dissociation constant of 7.3 X 10(-8) M and concurrently developed the capacity to respond to LTB4 with an increase in Ca+2]in. The binding of LTB4 to either high-affinity or low-affinity receptors appears to be sufficient to initiate an increase in Ca+2]in in human PMN leukocytes and differentiated HL-60 cells. The specificity of LTB4 receptors in transducing maximum increases in Ca+2]in is determined by the subset of receptors that predominate as a result of the concentration of LTB4 and the state of the responding cells. |
