Philip A. Knauf

Changes in cytoskeletal actin content, F-actin distribution, and surface morphology during HL-60 cell volume regulation

Cell volume regulation occurs via the regulated fluxes of ions and solutes across the cell membrane in response to cell volume perturbations under anisotonic conditions. Our earlier studies in human promyelocytic leukemic HL‐60 cells showed that volume‐dependent changes in total cellular F‐actin content occur concomitantly as an inverse function of acute cell volume changes in anisotonic media (Hallows et al., 1991, Am. J. Physiol., 261:C1154–C1161). Although treatment with cytochalasin under anisotonic conditions significantly reduced total cellular F‐actin levels, cytochalasin did not significantly affect the ability of cells to undergo normal volume regulation responses, which suggested that these volume‐dependent changes in F‐actin content may not play a critical role in HL‐60 cell volume regulation. To examine more closely the possible role of the actin cytoskeleton in HL‐60 cell volume regulation, we quantitated changes in Triton‐insoluble cytoskeletal actin in the presence and absence of cytochalasin and also observed changes in F‐actin distribution and surface morphology during volume regulation. The quantity of cytoskeletal‐associated F‐actin, like total F‐actin, shifts inversely with initial cell volume changes in anisotonic media; however, subsequent changes in cytoskeletal actin levels during volume regulation are not significant. The soluble F‐actin pool in HL‐60 cells may thus be more susceptible to the physicochemical effects of shifts in cell volume than the insoluble (cytoskeletal) F‐actin pool. Twenty‐five micromolar dihydrocytochalasin B (DHB) treatment dramatically lowers cellular cytoskeletal actin levels by ∼75% under resting (isotonic) conditions, but there are no significant further changes in cytoskeletal actin as cells undergo anisotonic volume regulation in the presence of DHB. These results suggest that volume‐dependent changes in the absolute amounts of cytoskeletal‐associated F‐actin are not critical for HL‐60 cell volume regulation. However, because some portions of the actin cytoskeleton are resistant to cytochalasin disruption during volume regulation, a role for the cytoskeleton in the sensing and signaling of HL‐60 cell volume regulatory responses cannot be rigorously excluded. Particular F‐actin distribution patterns, as observed using confocal fluorescent microscopy, were correlated with particular phases of volume regulation. Also, comparison of cellular F‐actin distribution with surface morphology (observed by scanning electronic microscopy) of cells during volume regulation reveals a positive correlation between surface blebs and increased cortical F‐actin staining intensity.

Band 3 Mediated Transport

The band 3 protein is the first member, AE1, of the AE family of anion exchange proteins. The full-length 911 amino acid (Tanner et al. 1988; Lux et al. 1989) version of human AE1 (hAE1) is found primarily, though not exclusively (Papageorgiou et al. 2001), in red blood cells, where it mediates the very tightly coupled exchange of Cl- for HCO3. This permits HCO3 formed from CO2 in the systemic capillaries by the intracellular enzyme carbonic anhydrase to leave the cell in exchange for Cl-, thus effectively storing CO2 in the blood in the form of plasma HCO3, greatly increasing the CO2 carrying capacity of the blood (Gunn et al. 1989). In the lungs, the process is reversed to regenerate CO2 that is then exhaled. Although this function is important in facilitating the removal from the body of CO2 produced by tissue metabolism, it is not essential for life, as animals can survive despite nearly complete inhibition of this activity by disulphonic stil-benes (DS) (Swenson et al. 1993) or knockout of the functional gene for this protein (Peters et al. 1996).

Inhibition of Na+/H+ exchanger enhances low pH-induced L-selectin shedding and β2-integrin surface expression in human neutrophils

Ischemia-reperfusion injury is a common pathological occurrence causing tissue damage in heart attack and stroke. Entrapment of neutrophils in the vasculature during ischemic events has been implicated in this process. In this study, we examine the effects that lactacidosis and consequent reductions in intracellular pH (pH(i)) have on surface expression of adhesion molecules on neutrophils. When human neutrophils were exposed to pH 6 lactate, there was a marked decrease in surface L-selectin (CD62L) levels, and the decrease was significantly enhanced by inclusion of Na(+)/H(+) exchanger (NHE) inhibitor 5-(N,N-hexamethylene)amiloride (HMA). Similar effects were observed when pH(i) was reduced while maintaining normal extracellular pH, by using an NH(4)Cl prepulse followed by washes and incubation in pH 7.4 buffer containing NHE inhibitors [HMA, cariporide, or 5-(N,N-dimethyl)amiloride (DMA)]. The amount of L-selectin shedding induced by different concentrations of NH(4)Cl in the prepulse correlated with the level of intracellular acidification with an apparent pK of 6.3. In contrast, beta(2)-integrin (CD11b and CD18) was only slightly upregulated in the low-pH(i) condition and was enhanced by NHE inhibition to a much lesser extent. L-selectin shedding was prevented by treating human neutrophils with inhibitors of extracellular metalloproteases (RO-31-9790 and KD-IX-73-4) or with inhibitors of intracellular signaling via p38 MAP kinase (SB-203580 and SB-239063), implying a transmembrane effect of pH(i). Taken together, these data suggest that the ability of NHE inhibitors such as HMA to reduce ischemia-reperfusion injury may be related to the nearly complete removal of L-selectin from the neutrophil surface.

Hypertonicity induced apoptosis in HL-60 cells in the presence of intracellular potassium.

Cell shrinkage is a hallmark of apoptosis. Potassium efflux, which is involved in cell shrinkage, has been previously described as an essential event of apoptosis. This study was designed to address the importance of potassium efflux in hypertonicity (450 mOsm and 600 mOsm) induced apoptosis. We initiated apoptosis in HL-60 cells in hypertonic medium consisting of either high concentrations of NaCl, mannitol or KCl. Apoptotic activity was evaluated based on the DNA content of the cells, annexin-V staining and calcium content. Apoptosis was initiated in hypertonic conditions consisting of high intracellular K+. We demonstrate that apoptosis can occur in the presence of high intracellular potassium contrary to previous predictions.