Alkaline pH and internal calcium increase Na+ and K+ effluxes in LK sheep red blood cells in Cl--free solutions.

We examined the effects of pH, internal ionized Ca (Ca2+i), cellular ATP, external divalent cations and quinine on Cl-independent ouabain-resistant K+ efflux in volume-clamped sheep red blood cells (SRBCs) of normal high (HK) and low (LK) intracellular K+ phenotypes. In LK SRBCs the K+ efflux was higher at pH 9.0 (350%) than at pHs 7. 4 and 6.5, and was inhibited by external divalent cations, quinine, and cellular ATP depletion. The above findings suggest that the increased K+ efflux at alkaline pH is due to the opening of ion channels or specific transporters in the cell membrane. In addition, K+ efflux was activated (100%) when Ca2+i was increased (+A23187, +Ca2+o) into the microm range. However, in comparison to human red blood cells, the Ca2+i-induced increase in K+ efflux in LK SRBCs was fourfold smaller and insensitive to quinine and charybdotoxin. The Na+ efflux was also higher at pH 9.0 than at pH 7.4, and activated (about 40%) by increasing Ca2+i. In contrast, in HK SRBCs the K+ efflux at pH 9.0 was neither inhibited by quinine nor activated by Ca2+i. These studies suggest the presence in LK SRBCs, of at least two pathways for Cl--independent K+ and Na+ transport, of which one is unmasked by alkalinization, and the other by a rise in Ca2+i.

Modulation of K-Cl cotransport in volume-clamped low-K sheep erythrocytes by pH, magnesium, and ATP.

Cellular pH, ionized Mg (Mgi2+), and MgATP concentration of red blood cells, concomitantly with cell volume, change transiently during circulation. The action of these three effectors on Cl-dependent K efflux was examined in low-K sheep red blood cells with constant cell volume. Activation of K-Cl efflux by Mgi2+ extraction required ATP, suggesting that phosphorylation of a putative component occurred before Mgi2+ extraction. Conversely, Mg and ATP were synergistic inhibitors of K-Cl cotransport, since maximal inhibition was observed only in cells containing both ATP and > 300 microM Mgi2+. Both findings suggest dual roles for Mg and ATP. At 300-600 microM Mgi2+, lowering the pH from approximately 7.4 to approximately 6.5 stimulated K-Cl efflux only in fed cells, suggesting that protons oppose or release the inhibition by Mgi2+ and ATP. A direct effect of both protons and Mgi2+ on the cotransporter is suggested by their inhibition of K-Cl efflux in ATP-depleted cells. These findings are discussed in light of the current phosphorylation/dephosphorylation hypothesis.

Activation of bovine neutrophils by Pasteurella haemolytica leukotoxin is calcium dependent.

In this study, we used the fluorescent probe Fluo-3 to show that an increase in cytosolic free calcium, [Ca2+]i, occurred when suspensions of bovine neutrophils were incubated with sublethal concentrations of P. haemolytica leukotoxin. This increase in [Ca2+]i was dependent on the concentration of leukotoxin present in the medium and, at a given concentration of leukotoxin, dependent on the external calcium concentration. The calcium channel blocker verapamil and the beta-adrenergic antagonist propranolol inhibited leukotoxin-stimulated Ca2+ gain, as did a neutralizing antileukotoxin monoclonal antibody. As reported previously, incubation of bovine neutrophils with partially purified leukotoxin stimulated a vigorous luminol-dependent chemiluminescence response (LDCL). The present study shows that LDCL stimulation was dependent on the presence of extracellular calcium and was inhibited by the addition of verapamil and propranolol. These data indicate that bovine neutrophils exhibit a considerable increase in cytoplasmic free calcium when they are incubated with P. haemolytica leukotoxin in the presence of external calcium. They also provide evidence that an increased [Ca2+]i is required for functional activation of the bovine neutrophil oxidative burst by P. haemolytica leukotoxin.

Pasteurella haemolytica leukotoxin inhibits mitogen-induced bovine peripheral blood mononuclear cell proliferation in vitro.

In this study we demonstrate that partially purified Pasteurella haemolytica leukotoxin inhibits the proliferative response of bovine peripheral blood mononuclear cells (PBMC) to mitogens in vitro. Inhibition of PBMC proliferation did not appear to be due to cell death. Addition of a neutralizing anti-leukotoxin monoclonal antibody restored a normal proliferative response.

Activation of bovine neutrophils by partially purified Pasteurella haemolytica leukotoxin.

In this study we developed a new method for the partial purification of Pasteurella haemolytica leukotoxin by size-exclusion high-performance liquid chromatography. The partially purified leukotoxin had a molecular weight of 104,000, as estimated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reacted on an immunoblot with an antileukotoxin monoclonal antibody. As expected, high concentrations of the leukotoxin were inhibitory or lethal to bovine neutrophils. Incubation of bovine neutrophils with diluted leukotoxin, however, resulted in significant neutrophil activation that was comparable in magnitude to that obtained with standard activating agents such as opsonized zymosan or zymosan-activated serum. Dilute leukotoxin (1:128 to 1:8,192 dilutions) stimulated an oxidative burst (luminol-dependent chemiluminescence) by bovine neutrophils that was comparable in magnitude to that obtained with opsonized zymosan. Preincubation with leukotoxin did not significantly prime the neutrophils for an enhanced oxidative burst when they were then exposed to opsonized zymosan as a second stimulus. Dilute leukotoxin (1:100 to 1:1,000 dilutions) also stimulated cytoskeletal alterations in bovine neutrophils, as measured by a significant shape change response. Preferential release of secondary granule constituents (lactoferrin) occurred when neutrophils were incubated with 1:100 to 1:500 dilutions of leukotoxin. Significant release of primary granules, as measured by beta-glucosaminidase activity, was not observed except at low dilutions (1:20) of leukotoxin that resulted in significant release of cytosolic constituents (i.e., lactate dehydrogenase activity). The neutrophil-activating activity of the leukotoxin was heat labile, unaffected by polymyxin B, and abrogated by a leukotoxin-neutralizing monoclonal antibody. These data indicate that P. haemolytica leukotoxin, like the closely related Escherichia coli hemolysin, is a potent neutrophil-activating agent. Leukotoxin-stimulated release of neutrophil oxygen intermediates and granule constituents may contribute to the intense inflammation that characterizes bovine pulmonary pasteurellosis.