Imidazole-induced elevations of intracellular calcium in HL-60 cells: effect of inhibition of phospholipase C by the steroidal maleimide U73122

Abstract

A variety of agents, including imidazoles, such as miconazole, elicit elevations of calcium in human leukemic HL-60 cells through a stimulation of influx of calcium and/or a release of intracellular calcium. Release of intracellular calcium by such agents may involve stimulation of IP 3 formation. The steroidal maleimide U73122, a potent phospholipase C (PLC) inhibitor, at 3-10 mM blocked elevations of calcium elicited in HL-60 cells by ATP. At a 10 mM concentration, U73122 either blocked or altered elevations of calcium elicited by imidazoles, such as miconazole, SKF 96365, and clotrimazole that are often used to block capacitative calcium entry, and by calmidazolium, a quaternary imidazole that can trigger massive influx of calcium in HL-60 cells. Elevations of calcium elicited by thapsigargin were not affected by 3 mM U73122, but were reduced by higher concentrations. Remarkably, U73122 at 10 mM did not block, but instead slowed and potentiated the elevation of calcium elicited by higher concentrations of imidazoles, an effect that may involve inhibition of Ca 21 -ATPases of the endoplasmic reticulum. The steroidal succinimide U73343 had minimal effects on calcium elevations in HL-60 cells. The results indicate that U73122 requires cautious use in assessing the role of PLC in controlling calcium levels in HL-60 cells, in particular when used with imidazoles, such as SKF 96365, which are used to block capacitative calcium entry. The effects of U73122 on cell calcium are reviewed. In toto, the results suggest that many lipophilic agents may affect intracellular calcium by activating PLC through unknown mechanisms. Drug Dev. Res.

Further studies on the interaction of loperamide with capacitative calcium entry in Leukemic HL‐60 cells

“…The blockade of CCE by various agents is also poorly defined with respect to mechanism. The imidazoles, in particular, have other effects on calcium release [Harper et al, 1999;Daly and Camerini-Otero, 2006]. A striking example of an agent with diverse effects is 2-aminoethoxydiphenyl borane (2-APB), an agent first considered a selective antagonist at IP 3 -receptors [Ma et al, 2000], but now known to have many effects [see Hu et al, 2004 and references therein].…”

“…Intracellular calcium concentrations and intracellular pH levels were calculated from the fluorescent excitation ratios of the appropriate probes as described [Grynkiewicz et al, 1985;Rink et al, 1982]. Representative experiments (n 5 3-4) are de- Daly and Camerini-Otero, 2006]. Results are expressed as the excitation ratio, i.e., the ratio of emission intensities from the two excitation wavelengths.…”

“…Loperamide also could augment elevations of intracellular calcium elicited in HL-60 cells by miconazole, clotrimazole, and SKF 96365 [Harper and Daly, 1999], perhaps by reversing the well-known but poorly defined blockade of CCE by these imidazoles. In addition to blocking CCE, such imidazoles appeared to activate phospholipase C in HL-60 cells and thereby trigger an IP 3 -dependent elevation of intracellular calcium [Harper and Daly, 1999;Daly and Camerini-Otero, 2006]. All the above studies were conducted with differentiated HL-60 cells.…”