Barbiturates Directly Inhibit the Calmodulin/Calcineurin Complex: a Novel Mechanism of Inhibition of Nuclear Factor of Activated T Cells

Abstract

Barbiturates are frequently used for the treatment of intracranial hypertension after brain injury but their application is associated with a profound increase in the infection rate. The mechanism of barbiturate-induced failure of protective immunity is still unknown. We provide evidence that nuclear factor of activated T cells (NFAT), an essential transcription factor in T cell activation, is a target of barbiturate-mediated immunosuppression in human T lymphocytes. Treatment of primary CD3 ϩ lymphocytes with barbiturates inhibited the PMA and ionomycin induced increase in DNA binding of NFAT, whereas the activity of other transcription factors, such as Oct-1, SP-1, or the cAMP response element-binding protein, remained unaffected. Moreover, barbiturates suppressed the expression of a luciferase reporter gene under control of NFAT (stably transfected Jurkat T cells), and of the cytokine genes interleukin-2 and interferon-␥ that contain functional binding motifs for NFAT within their regulatory promotor domains (human peripheral blood CD3 ϩ lymphocytes). Neither GABA receptor-initiated signaling nor direct interactions of barbiturates with nuclear proteins affected the activity of NFAT. In contrast, barbiturates suppressed the calcineurin-dependent dephosphorylation of NFAT in intact T cells and also inhibited the enzymatic activity of calcineurin in a cell-free system, excluding upstream regulation. Thus, our results demonstrate a novel mechanism of direct inhibition of the calcineurin/calmodulin complex that may explain some of the known immunosuppressive effects associated with barbiturate treatment.

Repression of T-Cell Function by Thionamides Is Mediated by Inhibition of the Activator Protein-1/Nuclear Factor of Activated T-Cells Pathway and Is Associated with a Common Structure

“…Fifteen hours before analysis of reporter gene expression, transfection reactions were pooled, equally redistributed at 10 5 cells into individual wells, and incubated with thionamides and 1 g/ml ionomycin plus 15 ng/ml PMA. Alternatively, the stably transfected Jurkat cell line C4-NFAT was used as described previously (Humar et al, 2004b). Luciferase reporter gene expression was measured in harvested cells that were lysed in 100 l of luciferase reporter lysis buffer (Promega, Madison, WI) using a Microlumat Plus LB 96P luminometer (Berthold Technologies, Bad Wildbad, Germany).…”

Inhibition of Activator Protein 1 by Barbiturates Is Mediated by Differential Effects on Mitogen-Activated Protein Kinases and the Small G Proteins Ras and Rac-1

“…These concentrations are within clinically relevant dose ranges and comparable with tissue concentrations in organs that play a central role in the development of nosocomial infections (Neuwelt et al, 1982;Schalen et al, 1992;Yasuda et al, 1993). Previous results demonstrate that 2-thiobarbiturates are more potent inhibitors of transcription factors such as NFAT or NF-B than their structurally identical oxy-analogs (Loop et al, 2002;Humar et al, 2004). In contrast to those results, AP-1 was affected similarly by both thiopental and pentobarbital or thiamylal and secobarbital, suggesting that C2 thiosubstitutions to the barbiturate backbone do not noticeable influence the potency of AP-1 inhibition.…”

“…Previously, we described that two transcription factors, NF-B and NFAT, were inhibited by thiobarbiturates but only marginally by their oxybarbiturate analogs (Loop et al, 2002;Humar et al, 2004). Surprisingly, at the same time, certain biological effects such as proliferation, cytokine production, or CD69 activation marker expression were repressed by all barbiturates, suggesting further mechanisms of barbiturate-mediated immunosuppression.…”

“…In previous studies, we demonstrated that two important regulators of immune function, the transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor-B (NF-B), are significantly inhibited by thiobarbiturates but are only marginally affected by their oxy-analogs (Loop et al, 2002;Humar et al, 2004). However, cellular immunomodulatory effects such as impaired proliferation, cytokine expression, and CD69 activation marker expression are exerted by both thiobarbiturates and their oxy-analogs, indicating additional, crucial immunosuppressive mechanisms.…”

Drug Repurposing by Simulating Flow Through Protein–Protein Interaction Networks

“…Based on drug actions, the five most prevalent drug groups were gamma‐aminobutyric acid (GABA) receptor activators (barbiturates), sympathomimetic drugs, vitamin D receptor (VDR) agonists, GnRH‐receptor agonists, and PPARG agonists. Barbiturates are yet not associated with psoriasis, but immunomodulatory effects of these substances have been suggested 13. Epinephrine is presumed to have a role in psoriasis pathogenesis, and, thus, the modulation of adrenergic signaling may have disease‐modifying effects 14, 15.…”