Lisa D. Baier

p38 mitogen-activated protein kinase down-regulates nitric oxide and up-regulates prostaglandin E2 biosynthesis stimulated by interleukin-1beta.

The inflammatory cytokine interleukin 1β (IL-1β) induces both cyclooxygenase-2 (Cox-2) and the inducible nitric-oxide synthase (iNOS) with increases in the release of prostaglandins (PGs) and nitric oxide (NO) from glomerular mesangial cells. However, the intracellular signaling mechanisms by which IL-1β induces iNOS and Cox-2 expression is obscure. Our current studies demonstrate that IL-1β produces a rapid increase in p38 mitogen-activated protein kinase (MAPK) phosphorylation and activation. Serum starvation and SC68376, a drug which selectively inhibits p38 MAPK in mesangial cells, were used to investigate whether p38 MAPK contributes to the signaling mechanism of IL-1β induction of NO and PG synthesis. Serum starvation and SC68376 selectively inhibited IL-1β-induced activation of p38 MAPK. Both SC68376 and serum starvation enhanced NO biosynthesis by increasing iNOS mRNA expression, protein expression, and nitrite production. In contrast, both SC68376 and serum starvation suppressed PG release by inhibiting Cox-2 mRNA, protein expression, and PGE2 synthesis. These data demonstrate that IL-1β phosphorylates and activates p38 MAPK in mesangial cells. The activation of p38 MAPK may provide a crucial signaling mechanism, which mediates the up-regulation of PG synthesis and the down-regulation of NO biosynthesis induced by IL-1β.

Nitric oxide amplifies interleukin 1-induced cyclooxygenase-2 expression in rat mesangial cells.

Interleukin 1 and nitric oxide (NO) from infiltrating macrophages and activated mesangial cells may act in concert to sustain and promote glomerular damage. To evaluate if such synergy occurs, we evaluated the effect if IL-1 beta and NO on the formation of prostaglandin (PG)E2 and cyclooxygenase (COX) expression. The NO donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, alone did not increase basal PGE2 formation. However, these compounds amplified IL-1 beta-induced PGE2 production. Similarly, sodium nitroprusside and S-nitroso-N-acetylpenicillamine by themselves did not induce mRNA and protein for COX-2, the inducible isoform of COX; however, they both potentiated IL-1 beta-induced mRNA and protein expression of COX-2. The stimulatory effect of NO is likely to be mediated by cGMP since (a) an inhibitor of the soluble guanylate cyclase, methylene blue, reversed the stimulatory effect of NO donors on COX-2 mRNA expression; (b) the membrane-permeable cGMP analogue, 8-Br-cGMP, mimicked the stimulatory effect of NO donors on COX-2-mRNA expression; and (c) atrial natriuretic peptide, which increases cellular cGMP by activating the membrane-bound guanylate cyclase, also amplified IL-1 beta-induced COX-2 mRNA expression. These data indicate a novel interaction between NO and COX pathways.

Antioxidants Inhibit Interleukin-1-induced Cyclooxygenase and Nitric-oxide Synthase Expression in Rat Mesangial Cells EVIDENCE FOR POST-TRANSCRIPTIONAL REGULATION

Glomerular mesangial cells produce reactive oxygen intermediates when stimulated by interleukin-1 (IL-1) or tumor necrosis factor. Recent observations suggest that reactive oxygen intermediates may play a role in IL-1 and tumor necrosis factor signaling and may up-regulate gene expression. We therefore evaluated the effects of antioxidants on IL-1β-induced cyclooxygenase-2 (Cox-2) and inducible nitric-oxide synthase (iNOS) expression in rat mesangial cells. The oxidant scavenger, pyrrolidine dithiocarbamate (PDTC), inhibited iNOS expression at the transcriptional level, since PDTC abolished iNOS mRNA accumulation. In contrast, PDTC inhibited Cox-2 expression at the post-transcriptional level, since PDTC did not affect IL-1β-induced Cox-2 mRNA levels but inhibited Cox-2 protein expression and prostaglandin E production. Another antioxidant, rotenone, which inhibits reactive oxygen intermediate production by inhibiting the mitochondrial electron transport system, did not inhibit IL-1β-induced iNOS and Cox-2 mRNA expression but inhibited iNOS and Cox-2 protein expression, suggesting a post-transcriptional target for the inhibition of iNOS and Cox-2 expression induced by IL-1β. These results suggest that not only transcriptional regulation but also post-transcriptional mechanisms are involved in redox-sensitive inhibition of cytokine induced Cox-2 and iNOS expression. These results suggest a novel approach for intervention in cytokine-mediated inflammatory processes.

Barbiturate tolerance: effects on GABA-operated chloride channel function.

Male ICR mice were fed powdered laboratory chow containing phenobarbital for 7 days to induce tolerance. Mice were sacrificed and brains assayed for changes in GABA-mediated chloride flux into brain membrane vesicles (microsacs). Concentration-dependent stimulation of chloride flux by GABA alone was not affected by the development of tolerance to phenobarbital. Phenobarbital potentiation of GABA-mediated chloride flux was significantly attenuated in the membranes prepared from phenobarbital-tolerant mice compared with those from pair-fed control mice. Similarly, stimulation of GABA-mediated flux by the benzodiazepine, flunitrazepam was also depressed in membranes from tolerant mice. However, the ability of ethanol and the benzodiazepine inverse agonist FG-7142 to modulate GABA-gated chloride flux was not affected by the development of phenobarbital tolerance. No significant changes in saturation [3H]diazepam binding parameters were observed. These findings suggest that there is a degree of cross-tolerance between phenobarbital and benzodiazepine agonist at the level of the GABA-operated chloride channel. Furthermore, although some reports have demonstrated behavioral cross-tolerance between ethanol and barbiturates, the present data suggest different mechanisms of tolerance development for these intoxicants at the level of the GABAA receptor chloride channel complex.

IGF-I and insulin amplify IL-1β-induced nitric oxide and prostaglandin biosynthesis

The inflammatory cytokine interleukin-1β (IL-1β) induces both cyclooxygenase-2 (Cox-2) and the inducible nitric oxide synthase (iNOS) with concomitant release of PGs and nitric oxide (NO) by glomerular mesangial cells. In our current studies, we determine whether insulin and IGF-I are involved in the signal transduction mechanisms resulting in IL-1β-induced NO and PGE2biosynthesis in renal mesangial cells. We demonstrate that both insulin and IGF-I increase IL-1β-induced Cox-2 and iNOS protein expression, which in turn enhance PGE2 and NO production. Our data also indicate that both insulin and IGF-I enhance IL-1β-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation and SAPK activation. These findings implicate the possible role of the MAPK pathway in mediating the effects of insulin and IGF-I on the upregulation of cytokine-stimulated NO and PG biosynthesis. Together, our results indicate that IGF-I and insulin may function to modulate the renal inflammatory process.

Effects of lorazepam tolerance and withdrawal on GABAA receptor operated chloride channels in mice selected for differences in ethanol withdrawal severity

Withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) mice were treated with 5 mg/kg lorazepam for 7 days via implanted osmotic mini pumps. Following chronic drug treatment, brains were assayed for GABA-mediated chloride flux (GABA-Cl-). Under control (drug naive) conditions, brain membranes prepared from WSP and WSR lines did not differ in flunitrazepam or ethanol stimulation of GABA-mediated 36Cl- uptake, but the WSP lines were more sensitive to inhibition of 36Cl- flux by the inverse agonist, FG-7142. Membranes from lorazepam tolerant WSP and WSR mice were resistant to flunitrazepam- and ethanol-stimulation of GABA-Cl-. Withdrawal from chronic treatment, by an acute injection with the benzodiazepine antagonist RO15-1788, returned flunitrazepam stimulation of GABA-Cl- to near control levels in WSR membranes but not in WSP membranes and restored ethanol modulation of the channel to control levels in both lines. Inhibition of chloride flux by the benzodiazepine partial inverse agonist, FG-7142 was greater in membranes from WSP mice compared with WSR mice. Tolerance to lorazepam increased sensitivity of the WSR membranes to FG-7142 without altering the response in the WSP line. Again, withdrawal restored the Cl- flux response to FG-7142 back to near control levels. Lorazepam tolerance lowered [3H]-flunitrazepam binding affinity slightly only in the WSR strain with no change in binding number. Withdrawal from chronic lorazepam treatment produced no significant change in binding affinity or number. The initial genotypic differences in benzodiazepine inverse agonist sensitivity, may be related to the selection for withdrawal seizure severity. Chronic administration of lorazepam reduces the coupling between the benzodiazepine agonist site and the chloride channel and concomitantly increases coupling between the channel and the inverse agonist site, while withdrawal resets the receptor coupling back to control response levels. However, for the WSP line, this drug environment dependent shift in channel coupling bias appears to be deficient compared with the WSR line.