Heiko Mühl

Amplification of nitric oxide synthase expression by nitric oxide in interleukin 1 beta-stimulated rat mesangial cells.

Nitric oxide (NO) plays an important role in immunological reactions as a host defense mechanism against tumor cells and invasive microorganisms, but it may also damage healthy tissue. The excessive formation of NO in IL-1 beta-stimulated renal mesangial cells not only alters glomerular filtration, but it may also cause tissue injury and thus contribute to the pathogenesis of certain forms of glomerulonephritis. We report here that, although NO alone has no evident effect on NO synthase expression, it potently augments IL-1 beta-stimulated NO synthase expression in mesangial cells. NO donors such as sodium nitroprusside and S-nitroso-N-acetyl-D,L-penicillamine markedly increase IL-1 beta-induced NO synthase mRNA and protein levels as well as enzyme activity. Nuclear run-on experiments suggest that NO acts to increase IL-1 beta-induced NO synthase gene expression at the transcriptional level. Furthermore, inhibition of NO synthesis by different pharmacological approaches reduces IL-1 beta-induced NO synthase expression, thus suggesting that NO functions in a positive feedback loop that speeds up and strengthens its own biosynthesis. We suggest that this potent amplification mechanism forms the basis for the excessive formation of NO in acute and chronic inflammatory diseases.

Expression of nitric oxide synthase in rat glomerular mesangial cells mediated by cyclic AMP.

1 Treatment of rat mesangial cells with interleukin 1β (IL‐1β) or tumour necrosis factor α (TNFα) has been shown to induce a macrophage‐type of nitric oxide (NO) synthase. Here we report that adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) is another mediator that triggers induction of NO synthase in mesangial cells. 2 Incubation of mesangial cells with the β‐adrenoceptor agonist, salbutamol, forskolin or cholera toxin, which all activate adenylate cyclase and increase intracellular cyclic AMP concentration, increased nitrite formation in a dose‐dependent manner. Likewise, the addition of the membrane‐permeable cyclic AMP analogue, N6, 0–2′‐dibutyryladenosine 3′,5′‐phosphate (Bt2cyclic AMP) or the phosphodiesterase inhibitor, 3‐isobutyl‐1‐methylxanthine enhanced NO synthase activity in a dose‐dependent manner. 3 There was a lag period of about 8 h before a significantly enhanced secretion of nitrite could be detected upon exposure of cells to forskolin and for maximal stimulation, forskolin had to be present during the whole incubation period. 4 Treatment of mesangial cells with actinomycin D, cycloheximide or dexamethasone completely suppressed forskolin‐stimulated NO‐synthase activity, thus demonstrating that transcription and protein synthesis are necessary for nitrite formation. 5 Bt2 cyclic AMP, the most potent inducer of nitrite production, increased NO synthase mRNA levels in mesangial cells in a time− and dose‐dependent fashion. Dexamethasone completely inhibited the increase of NO synthase mRNA in response to Bt2 cyclic AMP. 6 Combination of Bt2 cyclic AMP and IL‐1β or TNFα revealed a strong synergy in terms of nitrite formation. Time‐course studies indicated that cyclic AMP needed to be increased during the whole period of IL‐1β stimulation for maximal nitrite production. 7 These observations suggest that cyclic AMP controls NO synthase expression in mesangial cells. Furthermore, the signalling cascades triggered by IL‐1β and TNFα synergize with the cyclic AMP pathway to stimulate NO synthase activity.

THERAPEUTIC STRATEGIES FOR THE INHIBITION OF INDUCIBLE NITRIC OXIDE SYNTHASE—POTENTIAL FOR A NOVEL CLASS OF ANTI-INFLAMMATORY AGENTS

In recent years, NO, a gas previously considered a potentially toxic chemical, has become established as a diffusible universal messenger mediating cell—cell communication throughout the body. In mammals, NO is a recognized mediator of blood vessel relaxation that helps to maintain blood pressure. In the central nervous system NO acts as a non‐conventional neurotransmitter and participates in the establishment of long‐term plasticity required for memory formation. In addition, NO is responsible for some parts of the host response to sepsis and inflammation and contributes to certain disease states. A number of strategies have emerged with regard to a pharmacological control of pathological NO overproductions. This review will discuss these novel therapeutic approaches that may provide new means for clinical medicine.

Cyclosporin derivatives inhibit interleukin 1β induction of nitric oxide synthase in renal mesangial cells

Treatment of mesangial cells with recombinant human interleukin 1 beta dose dependently increased nitrite formation due to the induction of a macrophage-type of nitric oxide (NO) synthase. Addition of cyclosporin A, cyclosporin G or cyclosporin H dose dependently inhibited interleukin 1 beta-induced nitrite generation. Half-maximal inhibition was observed at concentrations of 0.9 microM, 2.0 microM and 3.8 microM of cyclosporin A, cyclosporin G and cyclosporin H, respectively. Time-course studies indicated that cyclosporin A could be added up to 6 h after the interleukin 1 beta stimulus and still caused maximal inhibition of nitrite production. Furthermore, interleukin 1 beta increased NO synthase mRNA levels in mesangial cells and this effect was potently suppressed by all three cyclosporin derivatives. As cyclosporin H has no immunosuppressive activity, these data indicate that the inhibitory effect of the cyclosporin derivatives on NO synthase expression is not related to the immunosuppressive action of the drugs. This suggestion is further substantiated by the observation that the potent immunosuppressants rapamycin and FK506 did not alter interleukin 1 beta-induced NO synthase mRNA levels or nitrite generation in mesangial cells. In summary, these data demonstrate that cyclosporin derivatives potently modulate the L-arginine-NO pathway in renal mesangial cells.

Transforming growth factors type-β and dexamethasone attenuate group II phospholipase A2 gene expression by interleukin-1 and forskolin in rat mesangial cells

Treatment of rat mesangial cells with interleukin‐1β(IL‐1β) and forskolin induced, in a synergistic fashion,the expression of group II phospholipase A2 (PLA2) mRNA, with subsequent increased synthesis and secretion of PLA2. In contrast, interleukin‐6 did not increase PLA2 mRNA levels of PLA2 activity. Transforming growth factor (TGF) β1, TGFβ2 and TGFβ3 equipotently attenuated the IL‐1β‐ and forskolin‐induced elevation of PLA2 mRNA, as well as PLA2 synthesis and secretion. The glucocorticoid dexamethasone only partially suppressed the IL‐1β‐ and forskolin‐induced elevation of PLA2 mRNA, but totally inhibited PLA2 synthesis and secretion.

Apoptosis is triggered by the cyclic AMP signalling pathway in renal mesangial cells.

Glomerular mesangial cells are regarded as specialized smooth muscle cells located within the renal glomeruli and fulfilling important functions in glomerular physiology and pathophysiology. Here, we report that activation of the cyclic AMP signalling pathway by dibutyryl cyclic AMP, forskolin, or the β 2‐adrenergic receptor agonist salbutamol results in induction of apoptosis in mesangial cells. Activation of the apoptotic programme results in DNA fragmentation which is visible for most forms of apoptosis and is paralleled by enrichment of cytosolic DNA/histone complexes, an increasing number of cellular 3′‐OH‐fragmented DNA ends and typical nuclear chromatin condensation. Induction of apoptosis was found to be dependent on translation and independent of nitric oxide synthase activity.

Cytokine regulation of group II phospholipase A2 expression in glomerular mesangial cells.

SummaryPhospholipase A2 (PLA2) is believed to play an essential role in inflammatory processes by releasing arachidonic acid from membrane phospholipids for synthesis of important lipid mediators, such as prostaglandins, leukotrienes and platelet activating factor.We have used glomerular mesangial cells as a model system to study the regulation of PLA2 under inflammatory conditions. Potent pro-inflammatory cytokines, such as interleukin 1 (IL-1) and tumour necrosis factor α (TNFα), as well as agents that increase cellular cAMP levels have been found to increase Group II PLA2 gene expression in a time- and dose-dependent manner.In all cases cytokine-induced synthesis of PLA2 occurred in parallel with cytokine-stimulated prostaglandin (PG) E2 synthesis. Three important classes of compounds that potently antagonise the stimulatory effect of IL-1, TNFα and cAMP on Group II PLA2 expression in mesangial cells have been identified, namely, glucocorticoids, transforming growth factors (TGF) type-β and platelet-derived growth factor (PDGF). Those agents may act sequentially to protect the kidney from damage resulting from cytokine-stimulated mediator release and the subsequent inflammatory reactions.