Concurrent analysis of intracellular glutathione content and gap junctional intercellular communication

Abstract

The potential for performing dual analysis of intracellular glutathione levels and assessment of gap junctional intercellular communication with thiol-specific fluorescent probes in anchored cells was evaluated. Gap junction-mediated diffusion of monochlorobimane and 5-chloromethylfluorescein diacetate following intracellular loading and conjugation with glutathione was compared with 5-carboxyfluorescein diacetate (which is routinely used in laser cytometry to monitor intercellular communication) by means of fluorescence recovery after photobleaching using a variety of communication-competent and communication-incompetent cells. The rate of diffusion of fluorescence among communication-competent cells was inversely proportional to the size of the fluorescent probe employed. The thiol-specific probes were also employed to monitor depletion and synthesis of glutathione following treatments to inhibit glutathione synthesis or consume glutathione by adduct formation. Analysis of gap junctional intercellular communication following glutathione depletion revealed a direct correlation between glutathione levels and intercellular communication. These studies support the utility of the thiol-specific probes to monitor the respective role of cellular glutathione and intercellular communication in the mechanisms of cellular injury. 0 1993 Wiley-Liss, Inc.

Activity and expression of the multidrug resistance proteins P-glycoprotein, MRP1, MRP2, MRP3 and MRP5 in de novo and relapsed acute myeloid leukemia

“…Although a colocalization of both proteins has been reported, 51 a coregulation of MDR1 and MRP2 has not been described before. These findings suggest that interactions might exist between the different MDR transporter proteins.…”

“…As described previously, MRP1 and MRP2 show the same substrate specificities 49,50 and both have been found to transport CF. 45,51 With regard to MRP3, which shows 58% amino acid identity with MRP1, it has been demonstrated that MRP3, in contrast to MRP1 does not transport GSH. 21 CF, however, is transported independently of GSH, 51 which makes it a potential candidate to be pumped by MRP3.…”

Kinetic analysis of glutathione in anchored cells with monochlorobimane

“…As shown, the mBCI-GSH conjugation during the first 8 min initially follows a first-order kinetic reaction. However, the conjugation reaction will not reach a plateau due to the second-order kinetic reaction caused by a number of possible additional reactions [e.g., conjugation of mBCl to nonprotein thiols (21), new synthesis of GSH (3,17,34), feedback inhibition by mBC1-GSH on GST activity (9), slow leakage of the mBC1-GSII conjugate from cells (32)]. Curve-fitting regression analysis was performed using scans that follow the first-order kinetic reaction to minimize the confounding factors referenced above ( i c , where the correlation coefficient = 1 ).…”

“…Because laser cytometry with the ACAS 570 can be performed on anchored cells, there are unique opportunities to examine a variety of responses by individual cells over time (see, e.g., 3,28). The potential for diffusion of intracellular GSH between individual contacting ROSE-199 and ras-R199 was examined.…”

“…Analysis of the conjugation of mBCl with GSH has been used to evaluate GST activity in cells (10,ll). In addition to flow cytometry, mBCl has been used with digital imaging systems on anchored cells to evaluate GSH status (3,4,8), GSH synthesis (3,17), and GST activity on a single-cell basis and/or to perform multiple measurements on individual cells (3).…”