Crosslinking by ligands to surface immunoglobulin triggers mobilization of intracellular 45Ca2+ in B lymphocytes.

Detailed studies of steady-state ion fluxes in murine lymphocytes were used to examine for possible ionic changes generated by surface Ig, the antigen receptor of B lymphocytes. When bound by ligands, surface Ig triggered the mobilization and release of 45Ca2+ from the cell interior by a transmembrane process requiring crosslinking of the bound receptors. This ionic event was unique for two reasons: (a) it did not occur when other common lymphocyte surface macromolecules were bound with rabbit anti-lymphocyte antibodies; and (b) it was not accompanied by a general perturbation of lymphocyte ionic properties such as a change in 42K+ fluxes nor did it depend on the presence of extracellular ions. Capping of surface Ig shares the same time sequence, dose response, requirement for crosslinking, and lack of dependence on extracellular ions. These correlations suggest that mobilization of intracellular Ca2+ may represent an early ionic signal for the contractile activation of lymphocytes that generates capping of surface Ig.

Transport of sodium, potassium, and calcium across rabbit polymorphonuclear leukocyte membranes. Effect of chemotactic factor.

The transport properties of the rabbit peritoneal polymorphonuclear leukocyte (PMN) plasma membrane to Na+, K+, and Ca2+ have been characterized. The use of a silicone oil centrifugation technique provided a rapid and reliable method for measuring ion fluxes in these cells. Na+ and K+ movements across PMN membranes were found to be rapid. The value for the unifirectional steady-state fluxes (in meq/liter cell X min) were of the order of 3.0 for Na+ and 7.4 for K+. Ouabian inhibited both K+ influx and Na+ efflux, the latter being also dependent on the presence of extracellular potassium. The rate constant (in min-1) for 45Ca influx was found to be .05 and that for 45Ca efflux .04. The synthetic chemotactic factor formyl-methionyl-leucyl-phenylalanine (FMLP) was found to affect the fluxes of Na+, K+, and Ca2+ at concentrations as low as 10(-10)M. FMLP induced a large and rapid increase in the permeability of the PMN plasma membrane to 22Na. Smaller and delayed enhancements of 42K influx and 22Na efflux were also noted. Some evidence that the latter findings are a consequence of the increased 22Na influx is presented. 45Ca influx and efflux were also stimulated by FMLP. In the presence of 0.25 mM extracellular calcium, FMLP induced an increase in the steady-state level of cell-associated 45Ca. In the presence of .01 mM extracellular calcium, however, a transient decrease in the steady-state level of cell-associated 45Ca was induced by FMLP. The curves relating the concentration of FMLP to its effects on cation fluxes are very similar to those found for its enhancement of migration.

Involvement of membrane calcium in the response of rabbit neutrophils to chemotactic factors as evidenced by the fluorescence of chlorotetracycline.

We have utilized the fluorescent chelate probe chlorotetracycline to investigate the possible involvement of membrane calcium in the response of rabbit peritoneal neutrophils to chemotactic factors. Two chemotactic factors, the small molecular weight fragment of the fifth component of complement C5a and the synthetic peptide formyl-methionyl-leucyl-phenylalanine (F-Met-Leu-Phe), were tested and found to decrease the fluorescence of cell-associated chlorotetracycline in a manner strongly suggesting stimulus-induced displacement of membrane calcium. The time-course, concentration dependence, and receptor specificity of the calcium redistribution induced by the stimuli are consistent with its early role in the initiation of the various neutrophil functions. F-Met-Leu-Phe and C5a appear to interact with the same pool of membrane calcium and to release it to the cytoplasmic side of the plasma membrane. Intracellular calcium then binds back to the membrane(s) from where it can be displaced by additional stimulation. The release of membrane calcium, experimentally defined here, appears to play a central role in the initiation of the various neutrophil functions.

Changes in ionic movements across rabbit polymorphonuclear leukocyte membranes during lysosomal enzyme release. Possible ionic basis for lysosomal enzyme release.

Changes in the movements of Na+, K+, and Ca+2 across rabbit neutrophils under conditions of lysosomal enzyme release have been studied. We have found that in the presence of cytochalasin B, the chemotactic factor formyl methionyl leucyl phenylalanine (FMLP) induces within 30 s large enhancements in the influxes of both 22Na+ and 45Ca+2 and an increase in the cellular pool of exchangeable calcium. The magnitude of the changes induced by cytochalasin B and FMLP exceeds that induced by FMLP or cytochalasin B alone, and cannot be explained on the basis of an additive effect of the two agents. However, these compounds either separately or together produce much smaller enhancements in 45Ca efflux. The divalent cation ionophore A23187 also produces a rapid and large increase in the influxes of both 22Na and 45Ca+2 in the presence and absence of cytochalasin B. We have also found an excellent correlation between calcium influx and lysosomal enzyme release. 42K influx is not significantly affected by any of these compounds. On the other hand, a large and rapid increase of 42K efflux is observed under conditions which give rise to lysosomal enzyme release. A flow diagram of the events that are thought to accompany the stimulation of polymorphonuclear leukocytes (PMNs) by chemotactic or degranulating stimuli is presented.

Activation of the rabbit polymorphonuclear leukocyte membrane "Na+, K+"-ATPase by chemotactic factor.

Addition of the synthetic chemotactic factor, formyl-methionyl-leucyl-phenylala-nine (F-Met-Leu-Phe) to medium containing magnesium, sodium, and potassium results in a doubling of the "Na+, K+"-ATPase activity of the plasma membrane fraction from polymophonuclear leukocytes (PMN). This activation is sensitive to ouabain inhibition and is dose dependent, maximal activity occuring at 10(-9)MF-Met-Leu-Phe. Equivalent activation was observed with the nonformylated derivative Met-Leu-Phe at 10(-9)M. The dipeptide, carbobenzoxy-methionylphenylalanine, which acts as an antagonist for F-Met-Leu-Phe, prevents the stimulation of the "Na+, K+"-ATPase by F-Met-Leu-Phe.

Is a rise in intracellular concentration of free calcium necessary or sufficient for stimulated cytoskeletal-associated actin?

The addition of the calcium ionophore A23187 to rabbit neutrophils increases the amount of actin associated with the cytoskeleton regardless of the presence or absence of calcium in the incubation medium. In the presence of extracellular calcium, the effect of A23187 is biphasic with respect to concentration. The action of the ionophore is rapid, transient, and is inhibited by pertussis toxin, hyperosmolarity, and quinacrine. On the other hand, the addition of pertussis toxin or hyperosmolarity has small if any, effect on the rise in intracellular calcium produced by A23187. While quinacrine does not affect the fMet-Leu-Phe-induced increase in cytoskeletal actin and the polyphosphoinositide turnover, its addition inhibits completely the stimulated increase in Ca-influx produced by the same stimulus. The results presented here suggest that a rise in the intracellular concentration of free calcium is neither necessary nor sufficient for the stimulated increase in cytoskeletal-associated actin. A possible relationship between the lipid remodeling stimulated by chemoattractants and the increased cytoskeletal actin is discussed.

Effects of chemotactic factors and other agents on the amounts of actin and a 65,000-mol-wt protein associated with the cytoskeleton of rabbit and human neutrophils.

Stimulation of rabbit neutrophils by the chemotactic factors fMet-Leu-Phe and leukotriene B4, by platelet activating factor, or by arachidonic acid produces a rapid and dose-dependent increase in the amounts of actin and of a 65,000-mol-wt protein associated with the cytoskeleton. Phorbol 12-myristate, 13-acetate, the calcium ionophore A23187 in the presence or absence of EGTA, and the fluorescent calcium chelator quin-2 also cause an increase in cytoskeletal actin. The stimulated increases in the cytoskeletal actin are not dependent on a rise in the intracellular concentration of free calcium and are not mediated by an increase in the intracellular pH or activation of protein kinase C. The increases in the cytoskeletal actin produced by fMet-Leu-Phe and leukotriene B4, but not by phorbol 12-myristate, 13-acetate, are inhibited by high osmolarity. The effect of hyperosmolarity requires a decrease in cell volume, is not mediated by an increase in basal intracellular concentration of free calcium, and is not prevented by pretreating the cells with amiloride. Preincubation of the cells with hyperosmotic solution also inhibits degranulation produced by all the stimuli tested. The inhibitory action of high osmolarity on the fMet-Leu-Phe and leukotriene B4 induced stimulation of cytoskeletal actin is discussed in terms of the possibility that the addition of high osmolarity, either directly or through activation of protein kinase C, causes receptor uncoupling.

The inhibition of neutrophil granule enzyme secretion and chemotaxis by pertussis toxin.

Pertussis toxin treatment of rabbit peritoneal neutrophils causes a concentration-dependent inhibition of granule enzyme secretion induced by formylmethionyl-leucyl-phenylalanine, C5a, and leukotriene B4. It also inhibits chemotaxis induced by formylmethionyl-leucyl-phenylalanine. The same toxin treatment, however, has no effect on granule enzyme secretion induced by the calcium ionophore A23187 or phorbol 12-myristate 13-acetate. Moreover, pertussis toxin treatment does not affect either the number or affinity of the formylpeptide receptors on the neutrophil nor does it have any effect on the unstimulated levels of cyclic AMP (cAMP) or the transient rise in cAMP induced by chemotactic factor stimulation in these cells. We hypothesize that pertussis toxin, as in other cells, interacts with a GTP binding regulatory protein identical with or analogous to either Ni or transducin which mediates the receptor-induced inhibition or activation of a target protein or proteins required in neutrophil activation. The nature of the target protein is unknown, but it is not the catalytic unit of adenylate cyclase. The target protein acts after binding of chemotactic factor to its receptor in the sequence that leads to the receptor-induced rise in intracellular Ca2+. It does not affect the responses elicited by the direct introduction of calcium into the cells or the activity of protein kinase C.

Chemotactic factor-induced release of membrane calcium in rabbit neutrophils.

The interaction of chemotactic factors (fMet-Leu-Phe and C5a) with rabbit neutrophils leads to rapid and specific release of membrane calcium, as evidenced by changes in the fluorescence of cell-associated chlorotetracycline. These two structurally different stimuli appear to interact with the same pool of membrane calcium.

Mono- and dihydroxyeicosatetraenoic acids alter calcium homeostasis in rabbit neutrophils.

Products of the lipoxygenation of arachidonic acid that express neutrophil chemotactic activity were examined in vitro for effects on the uptake of 45Ca by rabbit peritoneal neutrophils. At optimally chemotactic concentrations, 5- and 11-hydroperoxyeicosatetraenoic acid, 11- and 12L-hydroxyeicosatetraenoic acid, and leukotriene B4 enhanced 45Ca uptake within 1 min by a mean of 212-694% of the values for control neutrophils incubated in buffer alone, as compared with 75% for 5(S)-hydroxyeicosatetraenoic acid and no effect for 15-hydroperoxyeicosatetraenoic acid and 15-hydroxyeicosatetraenoic acid. The approximate rank of potency of the factors stimulating 45Ca uptake was similar to that observed for chemotaxis. Leukotriene B4, in addition to stimulating the rate of 45Ca uptake into rabbit neutrophils, also displaced intracellular calcium. The net result of the leukotriene B4-induced changes in calcium homeostasis ws to elevate transiently the intracellular level of exchangeable calcium. That neutrophil lipoxygenase metabolites of endogenous arachidonic acid rapidly enhance the influx of 45Ca supports a possible role for such metabolites in general, and leukotriene B4 in particular, in the regulation of the intracellular levels of free calcium in the neutrophils and possibly in other hormonally sensitive cells in which this cation is a second messenger.

Stimulated cytokineplasts from human polymorphonuclear leukocytes mobilize calcium and polymerize actin. Cytoplasts made in cytochalasin B retain a defect in actin polymerization.

Biologically active fragments from polymorphonuclear leukocytes (PMN) are simplified systems that can be used to elucidate specific pathways by which cell function is altered. In the current study we have found that cytokineplasts, which are motile fragments derived from the leading front (protopod, lamellipodium) of human PMN, rapidly increase their intracellular free calcium concentration when stimulated by chemotactic formyl peptide or by leukotriene B4, as measured by Quin-2 acetoxymethyl ester fluorescence. As in the parent cell, extracellular EGTA blunts this response only partially. Hence, cytokineplasts retain a mobilizable internal calcium pool, despite a general lack of intracellular organelles. In addition, formyl peptide more than doubles the amount of cytoskeleton-associated (polymerized) actin. In contrast, cytoplasts made by high-speed, discontinuous gradient centrifugation of cytochalasin B-treated leukocytes also increase their intracellular free calcium on stimulation, but cytoskeleton-associated actin increases by only approximately 14%. Thus, defective motile function in the latter cytoplast is associated with compromised effector function (actin polymerization).

A Ca2+-stimulated ATPase activity in rabbit neutrophil membranes.

An ATPase activity specifically stimulated by micromolar Ca2+ concentrations has been identified in association with rabbit neurophil membranes. These studies provide the basis of further characterization of the Ca2+-ATPase activity with regard to neutrophil function.

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions. Effect of local anesthetics, cholesterol and protein.

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 degrees C). Incorporation of cholesterol (30-50%) increased the microviscosity of lipid phases by 200-500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b5 did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since tha latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracain and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at 25 degrees C varied as follows: polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erythrocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol: phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important functional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.

Calmodulin antagonists modulate rabbit neutrophil degranulation, aggregation and stimulated oxygen consumption.

In an attempt to define further the sequence of events involved in the activation of rabbit peritoneal neutrophils, we have investigated the effects of trifluperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (compound W7), two compounds with known anti-calmodulin activity, on these cells' ability to respond to several stimuli. Neutrophil granule enzyme release, aggregation and oxygen consumption were monitored. These functions were elicited by the addition of one of the following stimuli to cell suspensions: the chemotactic factor f Met-Leu-Phe, arachidonic acid and the divalent cation ionophore A23187. Aggregation, oxygen consumption and granule enzyme release stimulated by the above three stimuli are all profoundly affected by calmodulin inhibitors. Low concentrations of trifluperazine slightly potentiates aggregation and oxygen consumption. At higher, but below lytic concentrations, compound W7 and trifluperazine significantly depress all three functions examined. In conjunction with previous results and by analogy to studies in other cell types, it therefore appears likely that calmodulin-stimulated events play central roles in the activation of rabbit neutrophils.

Demonstration of adenylate cyclase activity in human red blood cell ghosts.

The presence of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity was demonstrated in human erythrocyte ghosts and was found to be around 3 pmol adenosine 3',5'-monophosphate (cyclic AMP) - 2 h-1 - mg-1 protein. This enzymatic activity is strongly stimulated by NaF and 5'-guanylimidodiphosphate, is slightly stimulated by epinephrine, norepinephrine, isoproterenol, and prostaglandin E1 and is inhibited by calcium. The hormone stimulation is not potentiated by 5'-guanylylimidodiphosphate.

Phloretin is a potent inhibitor of rabbit neutrophil activation by chemotactic factors.

We have studied the effect of phloretin, a compound known to interfere with carrier mediated transport processes, on the functional responsiveness of rabbit neutrophils. Phloretin was found to inhibit neutrophil degranulation, aggregation and calcium uptake stimulated by the chemotactic peptide fMet-Leu-Phe. Part of these effects of phloretin may be accounted for by its interference with the binding of the synthetic peptide to its plasma membrane receptors. However, phloretin also inhibits the arachidonic acid and leukotriene B4 induced calcium uptake. These results imply that phloretin affects a component of membrane structure which is central to the activation sequences available to the neutrophils. The results of the present experiments demonstrate that phloretin can interfere with stimulus-response coupling in soluble mediator ('hormone') responsive cells.

Further studies of sodium transport in feline red cells.

The transport of radioactive sodium in high sodium cat red blood cells has been studied under various experimental conditions. It was found that iodoacetate (IAA) and iodoacetamide (IAM) inhibit Na influx by 50% whereas NaF has no effect. Reversible dyes, such as methylene blue (Mb), also inhibit this influx by 60%. Both IAA and Mb effects show a lag period of about 40 min. Cell starvation abolishes the volume-dependent Na influx which is generally observed in these cells. IAA reduces significantly the volume-dependent Na influx but does not inhibit it completely. 5 mM magnesium chloride produces a twofold increase in Na influx. On the other hand, MgCl(2) has no effect on Na transport in human red cells or on potassium or sulfate transport in cat red cells. The effect of MgCl(2) is quite rapid and does not interfere with the volume-dependent Na influx. This effect is abolished in starved cells. Reincubation of previously stored cells in buffered solutions containing glucose and MgCl(2) causes more than one order of magnitude increase in Na influx. These several observations are discussed in terms of the possibility of a link between Na transport and Na-Mg-activated ATPase.

Sulfate flux in high sodium cat red cells.

The transport of radioactive sulfate in cat red cells has been studied. The rate constant for (35)SO(4) inward movement under steady-state conditions is 0.24 +/- 0.02/hr. This movement was found to be sensitive to osmotic changes in cell volume and to the nature of anions in the incubation medium; it increases with increasing cell volume and decreases with decreasing cell volume. The anions SCN, NO(3), and I were found to inhibit the uptake of (35)SO(4). Furthermore, 1-fluoro-2,4-dinitrobenzene at a concentration of 1 mM inhibits (>90%) this uptake. The inward movement of erythritol-(14)C shows qualitatively the same dependence on cell volume as (35)SO(4), but it is insensitive to the nature of the anion present in the bathing medium. It was also found that the usually observed inhibition of radioactive Na uptake by SCN in cat red cells can be reversed when cell volume is increased.

Patterns of nonelectrolyte permeability in human red blood cell membrane.

The permeability of human red cell membrane to 90 different molecules has been measured. These solutes cover a wide spectrum of nonelectrolytes with varying chemical structure, chain length, lipid solubility, chemical reactive group, ability to form hydrogen bonds, and other properties. In general, the present study suggests that the permeability of red cell membrane to a large solute is determined by lipid solubility, its molecular size, and its hydrogen-bonding ability. The permeability coefficient increases with increasing lipid solubility and decreasing ability to form hydrogen bonds, whereas it decreases with increasing molecular size. In the case of small solutes, the predominant diffusion factor is steric hindrance augmented by lipid solubility. It is also found that replacement of a hydroxyl group by a carbonyl group or an ether linkage tends to increase permeability. On the other hand, replacement of a hydroxyl group by an amide group tends to decrease the permeability coefficient.

Sodium movement in high sodium feline red cells.

The transport of Na in the cat red cells has been studied under various experimental conditions. The unidirectional radioactive Na influx increased with increasing temperature until it reached a maximum value at 37 degrees C +/- 2 degrees C and then decreased with a further increase in temperature. Errors stated in this paper represent 1.0 standard errors of the mean. The apparent activation energy was calculated in the region between 25 and 37 degrees C and was found to be 4.9 +/- 0.5 kcal/mole. Copper at a concentration of 0.04 mM inhibited this influx by 65%. When cells were suspended in isosmotic KCl buffer, cell volume was found to decrease initially with time. This unusual behavior is discussed in terms of Na to K preference of the cell membrane. In cat red cells, Na influx was found to increase about 13-fold when cell volume was decreased from 1.16 normal to 0.87. This effect could not be reproduced when the medium osmolarity was changed only by the addition of urea, a permeating molecule. On the other hand, K influx was found to decrease from 0.24 +/- 0.03 mEq/liters RBC, hr at a relative cellular volume equal to 1.0 to 0.11 +/- 0.01 mEq/liters RBC, hr at a cell volume of 0.75. Na influx in human red cells did not show any significant dependence on cell volume. The properties of Na movement in the cat red cells are compared to those of human red cells.