Addition of docosahexaenoic acid, but not arachidonic acid, activates glutathione and thioredoxin antioxidant systems in murine hippocampal HT22 cells: potential implications in neuroprotection.

Docosahexaenoic acid (DHA, 22:6n-3) is a major constituent of nerve cell membrane phospholipids. Besides a role in membrane architecture, DHA is a pleiotropic molecule involved in multiple facets of neuronal biology and also in neuroprotection. We show here that supplementation with DHA (but not arachidonic acid) to mouse hippocampal HT22 cells modulates the expression of genes encoding for antioxidant proteins associated with thioredoxin/peroxiredoxin and glutathione/glutaredoxin systems. Thus, within the thioredoxin system, DHA increased Txn1-2, Trxrd1-2, Prdx3, and Srxn1 gene expression. Paralleling these changes, DHA increased thioredoxin reductase activity, the main enzyme involved in thioredoxin regeneration. For the glutathione system, the most important change triggered by DHA was the upregulation of Gpx4 gene, encoding for the nuclear, cytosolic and mitochondrial isoforms of phospholipid-hydroperoxide glutathione peroxidase (PH-GPx/GPx4, the main enzyme protecting cell membranes against lipid peroxidation), which was followed by a significant increase in total glutathione peroxidase and GPx4 activities. Noticeably, DHA also upregulated a new Gpx4 splicing variant that retained part of the first intronic region. Finally, we demonstrate that DHA treatment, under the same time course, protects HT22 cells from the oxitoxic exposure to amyloid beta (Aß25-35 ) peptide. Altogether, our data pinpoint to a role of DHA as Indirect Antioxidant that modulates neuronal defences in neuroprotection. DHA improves the antioxidant capacity of cultured hippocampal HT22 cells. We propose that DHA supplementation induces the remodelling of membrane phospholipids, and also triggers a transcriptional program to increase the expression of members of the glutathione and thioredoxin systems. We postulate that this transcriptional effect is mediated by a signal arising from non-enzymatic oxidation of DHA.

Polyamines transduce the nongenomic, androgen-induced calcium sensitization in intestinal smooth muscle.

Androgens regulate body development and differentiation through a variety of genotropic mechanisms, mostly in reproductive organs. In recent years a different scenario for sex hormone actions has emerged: the intestinal muscle. Thus, although estrogens relax intestinal muscle, androgens are powerful inducers of mechanical potentiation. This effect of androgens was intriguing because it is observed at physiological concentrations, is mediated by nongenomic mechanisms, and involves a phenomenon of calcium sensitization of contractile machinery by stimulating phosphorylation of 20 kDa myosin light chain by Rho-associated kinase. Here we have deciphered the molecular mechanisms underlying calcium sensitization and mechanical potentiation by androgens in male intestinal muscle as well as its tight relationship to polyamine metabolism. Thus, androgens stimulate polyamine synthesis, and the inhibition of polyamine synthesis abolishes androgen-induced calcium sensitization and 20 kDa myosin light chain phosphorylation. We demonstrate that the first molecular step in the induction of calcium sensitization is a nonconventional activation of the adaptor protein RhoA, triggered by a transglutaminase-catalyzed polyamination of RhoA, which is then targeted to the membrane to activate Rho-associated kinase. Altogether, these results demonstrate that the physiological levels of androgens, through the modulation of polyamine metabolism and posttanslational modification of RhoA, activate a new signal transduction pathway in the intestinal smooth muscle to induce calcium sensitization. Furthermore, apart from being one of the few physiologically relevant nongenomic effects of androgens, these results might underlie the well-known gender differences in intestinal transits, thus expanding the nature's inventory of sex hormones effects.

Androgens induce nongenomic stimulation of colonic contractile activity through induction of calcium sensitization and phosphorylation of LC20 and CPI-17.

We show that androgens, testosterone and 5alpha-dihydrotestosterone (DHT), acutely (approximately 40 min) provoke the mechanical potentiation of spontaneous and agonist-induced contractile activity in mouse colonic longitudinal smooth muscle. The results using flutamide, finasteride, cycloheximide, and actinomycin D indicate that androgen-induced potentiation is dependent on androgen receptors, requires reduction of testosterone to DHT, and occurs independently of transcriptional and translational events. Using permeabilized colonic smooth muscle preparations, we could demonstrate that mechanical potentiation is entirely due to calcium sensitization of contractile machinery. In addition, DHT (10 nm) increased phosphorylation of both 20-kDa myosin light chain (LC(20)) [regulatory myosin light chain, (MLC)] and CPI-17 (an endogenous inhibitor of MLC phosphatase). Paralleling these findings, inhibition of Rho-associated Rho kinase (ROK) and/or protein kinase C (PKC) with, respectively, Y27632 and chelerythrine, prevented LC(20) phosphorylation and abolished calcium sensitization. In addition, inhibition of ROK prevents CPI-17 phosphorylation, indicating that ROK is located upstream PKC-mediated CPI-17 modulation in the signalling cascade. Additionally, androgens induce a rapid activation of RhoA and its translocation to the plasma membrane to activate ROK. The results demonstrate that androgens induce sensitization of colonic smooth muscle to calcium through activation of ROK, which in turn, activates PKC to induce CPI-17 phosphorylation. Activation of this pathway induces a potent steady stimulation of LC(20) by inhibiting MLC phosphatase and displacing the equilibrium of the regulatory subunit towards its phosphorylated state. This is the first demonstration that colonic smooth muscle is a physiological target for androgen hormones, and that androgens modulate force generation of smooth muscle contractile machinery through nongenomic calcium sensitization pathways.

Androgens are powerful non-genomic inducers of calcium sensitization in visceral smooth muscle.

Androgens are recognized as genotropic inducers of a number of physiological functions mainly associated with the development of sexual characteristics. However, as in the case of estrogens, the number of studies evidencing androgen actions in non-reproductive tissues has steadily grown over the past years. Here, we show that androgens acutely ( approximately 30min) alter the frequency spectrum of peristaltic activity of intestinal smooth muscle and augment the amplitude agonist-induced contractile activity. Maximal stimulation occurred at physiological concentrations of androgens with EC(50) values in the picomolar range. Androgen-induced potentiation was prevented by preincubation with androgen receptor (AR) antagonists but unaffected by cycloheximide plus actinomycin D, indicating that potentiation was mediated by ARs via a non-genomic mechanism. The effects of androgens were mimicked by polyamines and were completely blocked by inhibitors of polyamine synthesis. Using ionomycin-permeabilized intestinal smooth muscle preparations, we demonstrate that androgens exert their effects by inducing a mechanism of sensitization to calcium and not by altering intracellular calcium homeostasis. Correspondingly, the potentiation of mechanical activity induced by androgens was accompanied by an increase in the phosphorylation of the regulatory myosin light chain (LC(20)) within the same time-course than calcium sensitization and mechanical potentiation. The pursuit of potential signalling pathways linking androgen receptor activation with calcium sensitization revealed that mechanical potentiation of intestinal muscle by androgens involve activation of the Rho pathway, whose downstream effector, Rho-associated kinase (ROCK), is eventually responsible for displacement of the phosphorylation/dephosphorylation state of LC(20) towards its phosphorylated form.

Androgens differentially potentiate mouse intestinal smooth muscle by nongenomic activation of polyamine synthesis and Rho kinase activation.

We demonstrate that testosterone and its active metabolite 5alpha-dihydrotestosterone acutely (approximately 30 min) potentiate mouse ileal, but not duodenal, muscle activity. Androgens augment the amplitude of spontaneous peak-to-peak oscillations, alter the spontaneous activity frequency spectrum, and increase the amplitude of calcium-induced and carbachol-induced contractions. Concentration-dependence analyses revealed that maximal potentiation (449-910%) occurred at physiological concentrations of androgens (100 pM to 10 nM) with EC50 values in the picomolar range (8-20 pM). Western blot analyses using an antiandrogen receptor (anti-AR) antibody revealed the presence of two different AR proteins migrating at 87 and 110 kDa in ileal, but not duodenal, extracts. Androgen-induced potentiation was prevented by preincubation with AR antagonists flutamide or cyproterone acetate but was unaffected by pretreatment with cycloheximide plus actinomycin D, indicating that potentiation was mediated by ARs via a novel nongenomic mechanism. Androgen effects were mimicked by polyamines putrescine and spermine and were blocked by the ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase inhibitors alpha-difluoromethylornithine and berenil, respectively. Accordingly, androgens increase alpha-difluoromethylornithine-sensitive ornithine-decarboxylase- mediated L-ornithine decarboxylation in ileal tissues within the same time course as isometric potentiation. Both putrescine and dihydrotestosterone induced Ca2+ sensitization of ionomycin-permeabilized ileal smooth muscle. Finally, inhibition of the Rho kinase (ROK) pathway with the specific inhibitor Y27632 completely prevented androgen-induced potentiation. In agreement, androgens elicited ROK-induced Ser19 phosphorylation of myosin light chain 2 in ileal muscle. These data indicate that androgens potentiate ileal contractile activity by an AR-dependent nongenomic mechanism involving intracellular polyamine signaling and Ca2+ sensitization via ROK activation.