Stimulation of neutrophils by prenylcysteine analogs: Ca2+ release and influx

Abstract

Farnesylthiosalicylic acid (FTS), a synthetic analog of the terminal prenylcysteine present in signaling proteins induces generation of superoxide ions, phospholipase C-driven hydrolysis of inositol lipids and calcium elevation in human neutrophils and DMSO-differentiated HL60 cells. These effects were ascribed to an interaction of the analog with elements responsible for recognition of specific prenylated proteins. The present study demonstrated that in addition to the release of intracellular calcium stores, FTS enhanced entry of Ca(2+) and Mn(2+) from the medium. The biphasic dependence of the influx on the concentration of FTS, as well as its insensitivity to inhibition by PMA and La(3+) suggest that the influx pathway activated by FTS is distinct from the previously described store-operated calcium channels of neutrophils. Consistent with the participation of a cellular membrane component in the interaction, FTS enhanced (45)Ca uptake in neutrophils and neutrophil cell membranes, but not in multilamellar vesicles. To establish specificity of the farnesyl moiety of FTS (C(15)), effects of three other analogs, geranylthiosalicylate, GTS (C(10)), geranylgeranylthiosalicylate, GGTS (C(20)), as well as the carboxymethyl ester FTS-Me on calcium homeostasis and superoxide production were investigated. GGTS dose-dependently elevated [Ca(2+)](i), induced quenching of the 360 nm Fura-2-calcium fluorescence by Mn(2+) and stimulated superoxide release, while GTS and FTS-Me were inactive. These results defined specific structural requirements for the functional interaction of prenylcysteine analogs with myeloid cells.

Studies on G-protein α·βγ heterotrimer formation reveal a putative S-prenyl-binding site in the α subunit

“…Thus half-maximal and maximal inhibition were observed at ≈ 40 and 80 µM FTS, respectively, for both experimental conditions. Note that these concentrations are lower than the critical micelle concentration of FTS, which has been determined to be ≈ 125 µM [37].…”

Above and beyond C5a Receptor Targeting by Staphylococcal Leucotoxins: Retrograde Transport of Panton–Valentine Leucocidin and γ-Hemolysin

“…In the present study, maintaining human neutrophils in RPMI-10% FBS medium improved cell survival and supported vigorous intracellular Ca 2+ responses. Accordingly, estimates of [Ca 2+ ] i were obtained in resting and activated neutrophils based on the Fura-2 fluorescent signal ( Figure 2 ) [ 56 , 72 ]. Additionally, the neutrophil responses were better estimated in cells challenged by leucotoxins under various pharmacological conditions ( Figure 2 ).…”

In vivo, trans‐synaptic tract‐tracing utilizing manganese‐enhanced magnetic resonance imaging (MEMRI)

“…Manganese ion (Mn 2þ ) has long been used in biological research in conjunction with fluorescent calcium (Ca 2þ ) indicator dyes such as fura-2 to monitor Ca 2þ influx as Mn 2þ is known to enter cells via L-type voltage gated calcium Ca 2þ channels. [1][2][3][4][5][6] In such studies the binding of Mn 2þ to fluorescent molecules results in the quenching of the associated fluorescence. [1][2][3][4][5][6] The rate of these quenching phenomena can be measured and is proportional to the rate of Ca 2þ influx only.…”

“…[1][2][3][4][5][6] In such studies the binding of Mn 2þ to fluorescent molecules results in the quenching of the associated fluorescence. [1][2][3][4][5][6] The rate of these quenching phenomena can be measured and is proportional to the rate of Ca 2þ influx only. This measurable unidirectional flux is owing to the fact that, after Mn 2þ enters the cell, it is not processed in a similar fashion to Ca 2þ by plasma membrane extrusion or by sarcoplasmic reticulum uptake via Ca 2þ pumps.…”

“…This measurable unidirectional flux is owing to the fact that, after Mn 2þ enters the cell, it is not processed in a similar fashion to Ca 2þ by plasma membrane extrusion or by sarcoplasmic reticulum uptake via Ca 2þ pumps. [1][2][3][4][5][6] Although Mn 2þ has proven to be quite useful in the realm of monitoring Ca 2þ influx in biological research, it is important to note that this ion is also a neurotoxin at high concentrations. 7,8 For example, Mn 2þ is released in welding fumes and individuals exposed to high levels of inhaled Mn 2þ exhibit enhanced accumulation within the basal ganglia and eventually can develop manganism (manganese poisoning).…”