The Chemotactic Factors Induced Movement of Calcium and Sodium across Rabbit Neutrophil Membranes: Effect of Desensitization to Cytochalasin B P. H. NACCACHE, H. J. SHOWELL, E. L. BECKER AND R. I. S H A A F I ' Departments of Physiology and Pathology, Uniuersity of Connecticut Health Center, Farmington, Connecticut 06032
ABSTRACT The preincubation of rabbit neutrophils with the chemotactic factor F-Met-Leu-Phe and the subsequent addition of cytochalasin B has previously been shown to induce a time, concentration and calcium dependent loss of secretory responsiveness in neutrophils. This has been termed desensitization. The results reported here first confirm that lysosomal enzyme release from neutrophils will still occur in the absence of extracellular calcium. In addition, a time dependent decrease in the magnitude of the cytochalasin B induced influxes of 45Caand 22Nawas found upon preincubation with F-Met-Leu-Phe. In the presence of extracellular Ca2+,this decrease in ionic responsiveness reaches a maximum by five minutes preincubation with F-Met-Leu-Phe. In the absence of added extracellular Ca2+an initial and rapid ( < 1minute) loss of ionic responsiveness is followed by partial recovery as the length of the preincubation with the chemotactic factor is increased from one to five minutes. These changes in ionic responses correspond exactly to the changes in secretory behavior of the neutrophils. Desensitization can thus be explained on the same ionic basis as that underlying the secretory response of t h e neutrophils. In addition, these results provide information about the sequence of events involved in the cytochalasin B and chemotactic factor induced release of lysosomal enzymes in neutrophils. Rabbit polymorphonuclear leukocytes, or neutrophils, in suspension, will selectively secrete lysosomal constituents in the extracellular medium if exposed simultaneously to any of a variety of natural or synthetic chemotactic factors and cytochalasin B or to the calcium ionophore A23187 in t h e presence of extracellular calcium (Goldstein et al., '73; Showell e t al., '76, '77). F-Met-Leu-Pheinduces among other things an influx of Na+ and Ca2+(Naccache et al., '77a,b; Petroski et al., '79). The latter flux can be correlated with the changes in levels of exchangeable calcium and can thus serve as an index of such intracellular events. The changes in Na+ and Caz+movements stimulated by F-Met-Leu-Pheare greatly increased in the presence of cytochalasin B (Naccache et al., '77b). The extent of lysosomal enzyme release stimulated by either F-Met-Leu-Phe or the calcium ionophore A23187 correlates quite precisely with the increase in exchangeJ. CELL. PHYSIOL. (1979) 100; 239-250.
able calcium. From the available data on neutrophils and the behavior of other secretory cells (Douglas, '68; Becker and Henson, '73; Gallin and Rosenthal, '74; Goldstein, '76; Boucek and Snyderman, '76; Naccache et al., '77a,b; Gallin et al., '78) we have postulated t h a t lysosomal enzyme release in neutrophils is mediated by an increase in the intracellular level of free Ca2+.Moreover, we have also postulated t h a t the sodium influx response to F-Met-Leu-Phe is involved in signal transmission and possibly also in the regulation of the level of intracellular free Ca2+. Recently it has been found that increasing t h e time interval between the addition of FMet-Leu-Phe, or C5,, and cytochalasin B causes a rapid loss of functional responsiveness in rabbit and human neutrophils (Henson Received Dec. 7, '78. Accepted Feb. 25, '79. ' Please address all correspondence to: Dr. R. 1. Sha'afi, Department of Physiology, University of Chnecticut Health Center, Farmington, Connecticut 06032.
239
240
P. H. NACC!ACHE, H. J. SHOWELL, E. L. BECKER A N D R. I. SHA'AFI
quired number of cells were transferred into the necessary number of thermally equilibrated flasks and allowed to equilibrate for three to five additional minutes. F-Met-LeuPhe was then added to the vessels at the appropriate time. After the cells had been preincubated with the chemotactic factor for the desired length of time ( 0 , l or 5 minutes), cytochalasin B premixed with 22Naor 45Ca was added to all flasks. The time a t which cytochalasin B and labelled ions were added was taken as zero time. Aliquots were then sampled a t the desired intervals, layered above a silicone oil layer and centrifuged for one minute in an Eppendorf microcentrifuge. The cell pellets were analyzed for radioactivity either directly in the case of *"a or after digestion in 5% nitric acid and 0.1% Triton X-100 when 45Ca was used. The terms influx is used only to denote inward moveMATERIALS AND METHODS ment. General procedure Cytochalasin B was used throughout a t 5 Neutrophils from white albino rabbits were pg/ml and was diluted prior to use in the collected as described previously (Becker and appropriate buffer from a 5 Fg/ml stock in Showell, '72) from the peritoneum of rabbits dimethyl sulfoxide. No effects of dimethyl 12 to 14 hours after the intraperitoneal injec- sulfoxide could be detected in these experition of 400 ml of sterile 0.1% glycogen in ments. isotonic saline. The cells were washed twice Lysosomal enzyme release and suspended in Hanks' balanced salt solution containing either 0.5 mM Ca2+or when no Lysosomal enzyme release was measured as M. The com- previously described in the presence of 1.7 mM calcium was added less than position of the Hank's balanced salt solution Ca2+(Showell et al., '79). This is the calcium was otherwise as previously reported (Showell concentration routinely used in our laboratory et al., '76) with 1 mg/ml glucose and 1 mg/ml for the study of lysosomal enzyme release. Decrystalline bovine serum albumin added. creasing the concentration of extracellular The experiments were carried out at a cell calcium to 0.5 mM, the concentration required concentration of lo7 celldm1 and a t 37°C. Cell for 45Caflux studies (Naccache et al., '77a,b), viability and non-specific release were as- does not modify in any significant way the sesessed from the amount of lactate dehydroge- cretory behavior of the neutrophils (Showell nase recovered in the supernates from the cell et al., '77) or its response to desensitization. suspension. In all of the experiments to be re- The time interval between the addition of Fported, lactate dehydrogenase release was Met-Leu-Phe and cytochalasin B was varied negligible (
ABSTRACT The preincubation of rabbit neutrophils with the chemotactic factor F-Met-Leu-Phe and the subsequent addition of cytochalasin B has previously been shown to induce a time, concentration and calcium dependent loss of secretory responsiveness in neutrophils. This has been termed desensitization. The results reported here first confirm that lysosomal enzyme release from neutrophils will still occur in the absence of extracellular calcium. In addition, a time dependent decrease in the magnitude of the cytochalasin B induced influxes of 45Caand 22Nawas found upon preincubation with F-Met-Leu-Phe. In the presence of extracellular Ca2+,this decrease in ionic responsiveness reaches a maximum by five minutes preincubation with F-Met-Leu-Phe. In the absence of added extracellular Ca2+an initial and rapid ( < 1minute) loss of ionic responsiveness is followed by partial recovery as the length of the preincubation with the chemotactic factor is increased from one to five minutes. These changes in ionic responses correspond exactly to the changes in secretory behavior of the neutrophils. Desensitization can thus be explained on the same ionic basis as that underlying the secretory response of t h e neutrophils. In addition, these results provide information about the sequence of events involved in the cytochalasin B and chemotactic factor induced release of lysosomal enzymes in neutrophils. Rabbit polymorphonuclear leukocytes, or neutrophils, in suspension, will selectively secrete lysosomal constituents in the extracellular medium if exposed simultaneously to any of a variety of natural or synthetic chemotactic factors and cytochalasin B or to the calcium ionophore A23187 in t h e presence of extracellular calcium (Goldstein et al., '73; Showell e t al., '76, '77). F-Met-Leu-Pheinduces among other things an influx of Na+ and Ca2+(Naccache et al., '77a,b; Petroski et al., '79). The latter flux can be correlated with the changes in levels of exchangeable calcium and can thus serve as an index of such intracellular events. The changes in Na+ and Caz+movements stimulated by F-Met-Leu-Pheare greatly increased in the presence of cytochalasin B (Naccache et al., '77b). The extent of lysosomal enzyme release stimulated by either F-Met-Leu-Phe or the calcium ionophore A23187 correlates quite precisely with the increase in exchangeJ. CELL. PHYSIOL. (1979) 100; 239-250.
able calcium. From the available data on neutrophils and the behavior of other secretory cells (Douglas, '68; Becker and Henson, '73; Gallin and Rosenthal, '74; Goldstein, '76; Boucek and Snyderman, '76; Naccache et al., '77a,b; Gallin et al., '78) we have postulated t h a t lysosomal enzyme release in neutrophils is mediated by an increase in the intracellular level of free Ca2+.Moreover, we have also postulated t h a t the sodium influx response to F-Met-Leu-Phe is involved in signal transmission and possibly also in the regulation of the level of intracellular free Ca2+. Recently it has been found that increasing t h e time interval between the addition of FMet-Leu-Phe, or C5,, and cytochalasin B causes a rapid loss of functional responsiveness in rabbit and human neutrophils (Henson Received Dec. 7, '78. Accepted Feb. 25, '79. ' Please address all correspondence to: Dr. R. 1. Sha'afi, Department of Physiology, University of Chnecticut Health Center, Farmington, Connecticut 06032.
239
240
P. H. NACC!ACHE, H. J. SHOWELL, E. L. BECKER A N D R. I. SHA'AFI
quired number of cells were transferred into the necessary number of thermally equilibrated flasks and allowed to equilibrate for three to five additional minutes. F-Met-LeuPhe was then added to the vessels at the appropriate time. After the cells had been preincubated with the chemotactic factor for the desired length of time ( 0 , l or 5 minutes), cytochalasin B premixed with 22Naor 45Ca was added to all flasks. The time a t which cytochalasin B and labelled ions were added was taken as zero time. Aliquots were then sampled a t the desired intervals, layered above a silicone oil layer and centrifuged for one minute in an Eppendorf microcentrifuge. The cell pellets were analyzed for radioactivity either directly in the case of *"a or after digestion in 5% nitric acid and 0.1% Triton X-100 when 45Ca was used. The terms influx is used only to denote inward moveMATERIALS AND METHODS ment. General procedure Cytochalasin B was used throughout a t 5 Neutrophils from white albino rabbits were pg/ml and was diluted prior to use in the collected as described previously (Becker and appropriate buffer from a 5 Fg/ml stock in Showell, '72) from the peritoneum of rabbits dimethyl sulfoxide. No effects of dimethyl 12 to 14 hours after the intraperitoneal injec- sulfoxide could be detected in these experition of 400 ml of sterile 0.1% glycogen in ments. isotonic saline. The cells were washed twice Lysosomal enzyme release and suspended in Hanks' balanced salt solution containing either 0.5 mM Ca2+or when no Lysosomal enzyme release was measured as M. The com- previously described in the presence of 1.7 mM calcium was added less than position of the Hank's balanced salt solution Ca2+(Showell et al., '79). This is the calcium was otherwise as previously reported (Showell concentration routinely used in our laboratory et al., '76) with 1 mg/ml glucose and 1 mg/ml for the study of lysosomal enzyme release. Decrystalline bovine serum albumin added. creasing the concentration of extracellular The experiments were carried out at a cell calcium to 0.5 mM, the concentration required concentration of lo7 celldm1 and a t 37°C. Cell for 45Caflux studies (Naccache et al., '77a,b), viability and non-specific release were as- does not modify in any significant way the sesessed from the amount of lactate dehydroge- cretory behavior of the neutrophils (Showell nase recovered in the supernates from the cell et al., '77) or its response to desensitization. suspension. In all of the experiments to be re- The time interval between the addition of Fported, lactate dehydrogenase release was Met-Leu-Phe and cytochalasin B was varied negligible (
