Sickle erythrocytes are known to have an increased adhesion

Sickle erythrocytes are known to have an increased adhesion to endothelial droperidol synthesis (Hebbel et al., 1980, Hebbel et al., 1981a, Hebbel et al., 1981b, Wautier et al., 1985, Mohandas and Evans, 1987). Understanding the events associated with vaso-occlusion requires a detailed knowledge of the interactions between circulating sickle cells and endothelial cells. Does the Gárdos channel contribute to this phenomenon? Evidence exists that CD-1 mouse erythrocytes express endothelin-1 (ET-1) receptors and that ET-1 activates the Gárdos channels in these cells (Rivera, Rotter, & Brugnara, 1999). A subsequent study showed that ET-1, platelet activation factor (PAF), “regulated on activation normal T cells expressed and secreted” (RANTES) and interleukin-10 (IL-10) all increase the Vmax and lower the Ca2+-activation constant of the Gárdos channel (Rivera, Jarolim, & Brugnara, 2002). It is postulated that there may be a functional coupling between the ET-1 receptor on erythrocyte membranes and the GC protein in local environments (such as capillaries) where ET-1 concentrations may be sufficiently high to bring about Gárdos channel activation. A recent study also reveals an increased level of activated monocytes (defined by production of intracellular cytokines) in SSA patients, leading to increased adhesion of such cells to platelets (Wun, Cordoba, Rangaswami, Cheung, & Paglieroni, 2002). It is feasible that these contribute to Gárdos channel-mediated dehydration of nearby erythrocytes. Nitric oxide (NO) plays an important role as a vasodilator in vascular capillaries, and its presence has been shown to reduce the extent of sickle cell-endothelium binding (Space, Lane, Pickett, & Weil, 2000). However, the exact mechanism remains unknown. NO is produced in vivo from arginine and O2, and arginine depletion has been noted in sickle cell patients (Morris, Kuypers, Larkin, Vichinsky, & Styles, 2000). Romero, Suzuka, Nagel, and Fabry (2002) have shown that supplementing the diets of transgenic sickle cell mice with arginine results in decreased red cell density and reduces the Vmax (but not the Ca2+ affinity constant) of their Gárdos channels. It is postulated that the presence of arginine may reduce endothelin levels, thereby lowering Gárdos channel activity. Clinical studies have shown successful reduction in the severity of symptoms of SSA (Benjamin et al., 1986, Orringer et al., 1986). Recent work has targeted the obligatory chloride conductance to alleviate the Gárdos effect in sickle cells. The poorly cation selective Psickle (which can be activated by oxygenation–deoxygenation cycling of sickle cells) can be inhibited up to 35% (for Na+ and K+ fluxes) by dipyridamole (Joiner et al., 2001a). Interestingly, dipyridamole also inhibits K+ efflux via the Gárdos channel by inhibiting the Cl− conductance (separate from its effects on Psickle). Thus, this drug appears to have multiple modes of action in reducing erythrocyte dehydration. Some recently synthesized reversible inhibitors of anion exchange, called NS1652 and NS3623, also show promise as high affinity inhibitors of Cl− conductance in normal and sickle cells (Bennekou et al., 2001; Bennekou, Pedersen, Moller, & Christophersen, 2000).
Functional studies of the gárdos channel In our own work, we have made extensive use of NMR spectroscopy to study various metabolic and membrane transport processes (Kuchel et al., 1984; Kuchel, Bulliman, Chapman, Kirk, & Potts, 1987; Kuchel, 1989; Kuchel, Kirk, & King, 1994). Therefore, we reasoned that the alternative perspective it offers on the function of membrane proteins, as they operate in situ in whole cells, is worthy of discussion here. Here, the erythrocyte presents an example of a relatively simple cell in which NMR can be used to study ion transport. This field of application of NMR was given a significant boost by Gupta & Gupta (1982) when they showed that the paramagnetic complex dysprosium tripolyphosphate, DyPPP serves as a ‘shift reagent’ thus resolving intra- and extra-cellular NMR spectral peaks. This technique has since been developed, and extended to several other nuclei (Adam, Koretsky, & Weiner, 1987; Boulanger & Vinay, 1989; Boulanger, Vinay, & Boulanger, 1987; Boulanger et al., 1992; Brophy, Hayer, & Riddell, 1983; Ogino et al., 1985; Pettegrew, Woessner, Minshew, & Glonek, 1984; Pike et al., 1985). Most shift reagents are lanthanide metal-based membrane-impermeable compounds (such as DyPPP and dytriethylene tetraamine hexaacetate, DyTTHA) that induce a chemical shift of the resonance frequency of nuclei in their immediate surroundings. For example, erythrocytes can be suspended in a medium containing 10mM DyPPP and the transport of Na+ or K+ can be followed over time by using the integral of the corresponding peak in the NMR spectrum. Due to the relatively low sensitivity of the nucleus, is often used as a K+ congener. When cells are pre-loaded with RbCl, the signal behaves in a similar manner to that expected for K+ (Helpern, Welch, & Halvorson, 1989). A simple example of such an experiment is as follows: by introducing the divalent-cation ionophore A23187 to a suspension of erythrocytes, with RbCl (in the presence of CaCl2), it is possible to follow the time-dependent change in the intensity of the corresponding peaks in the NMR spectrum (see Fig. 3).