Coordination chemistry of the heme in cystathionine beta-synthase: formation of iron(II)-isonitrile complexes.

Interaction of rat and human cystathionine-beta-synthase (CBS) with various potential ligands has been studied by visible and EPR spectroscopy in order to explore the coordination chemistry of this atypical hemeprotein. Ferric CBS did not react with any classical hemeprotein ligands, such as various imidazole and pyridine derivatives, N(-)(3) and isonitriles RNC. Ferrous CBS also failed to bind these nitrogenous ligands or nitrosoalkanes. However, it reacts with various isonitriles RNC, leading to complexes characterized by a Soret peak at 433 +/- 2 nm. Binding of isonitriles to ferrous CBS is a relatively slow process; its rate markedly depends on the nature of R. It thus seems that the only exogenous ligands able to bind CBS iron are carbon-centered, very strong heme-Fe(II) ligands such as CNR, CO, and CN(-), presumably after dissociation of the CBS-iron(II)-cysteinate bond. Isonitriles appear as interesting tools for further studies on the topology of CBS active site.

Modulation of cholinergic airway reactivity and nitric oxide production by endogenous arginase activity.

Cholinergic airway constriction is functionally antagonized by agonist-induced constitutive nitric oxide synthase (cNOS)-derived nitric oxide (NO). Since cNOS and arginase, which hydrolyzes L-arginine to L-ornithine and urea, use L-arginine as a common substrate, competition between both enzymes for the substrate could be involved in the regulation of cholinergic airway reactivity. Using a perfused guinea-pig tracheal tube preparation, we investigated the modulation of methacholine-induced airway constriction by the recently developed, potent and specific arginase inhibitor N(Omega)-hydroxy-nor-L-arginine (nor-NOHA). Intraluminal (IL) administration of nor-NOHA caused a concentration-dependent inhibition of the maximal effect (E(max)) in response to IL methacholine, which was maximal in the presence of 5 microM nor-NOHA (E(max)=31.2+/-1.6% of extraluminal (EL) 40 mM KCl-induced constriction versus 51.6+/-2.1% in controls, P<0.001). In addition, the pEC(50) (-log(10) EC(50)) was slightly but significantly reduced in the presence of 5 microM nor-NOHA. The inhibition of E(max) by 5 microM nor-NOHA was concentration-dependently reversed by the NOS inhibitor N(Omega)-nitro-L-arginine methyl ester (L-NAME), reaching an E(max) of 89.4+/-7.7% in the presence of 0.5 mM L-NAME (P<0.01). A similar E(max) in the presence of 0.5 mM L-NAME was obtained in control preparations (85.2+/-9.7%, n.s.). In the presence of excess of exogenously applied L-arginine (5 mM), 5 microM nor-NOHA was ineffective (E(max)=33.1+/-5.8 versus 31.1+/-7.5% in controls, n.s.). The results indicate that endogenous arginase activity potentiates methacholine-induced airway constriction by inhibition of NO production, presumably by competition with cNOS for the common substrate, L-arginine. This finding may represent an important novel regulation mechanism of airway reactivity.

Inhibition of arginase in rat and rabbit alveolar macrophages by N omega-hydroxy-D,L-indospicine, effects on L-arginine utilization by nitric oxide synthase.

1. Alveolar macrophages (AM phi) exhibit arginase activity and may, in addition, express an inducible form of nitric oxide (NO) synthase (iNOS). Both pathways may compete for the substrate. L-arginine. The present study tested whether two recently described potent inhibitors of liver arginase (N omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine) might also inhibit arginase in AM phi and whether inhibition of arginase might affect L-arginine utilization by iNOS. 2. AM phi obtained by broncho-alveolar lavage of rat and rabbit isolated lungs were disseminated (2.5 or 3 x 10(6) cells per well) and allowed to adhere for 2 h. Thereafter, they were either used to study [3H]-L-arginine uptake (37 kBq, 0.1 microM, 2 min) or cultured for 20 h in the absence or presence of bacterial lipopolysaccharide (LPS). Cultured AM phi were incubated for 1 h with [3H]-L-arginine (37 kBq, 0.1 microM) and the accumulation of [3H]-L-citrulline (NOS activity) and [3H]-L-ornithine (arginase activity) was determined. 3. During 1 h incubation of rabbit AM phi with [3H]-L-arginine, no [3H]-L-citrulline, but significant amounts of [3H]-L-ornithine (150 d.p.m x 1000) were formed. N omega-hydroxy-D,L-indospicine and 4-hydroxyamidino-D,L-phenylalanine, present during incubation, concentration-dependently reduced [3H]-L-ornithine formation (IC50: 2 and 45 microM, respectively). 4. N omega-hydroxy-D,L-indospicine (up to 100 microM) had no effect on [3H]-L-arginine uptake into rabbit AM phi, whereas 4-hydroxyamidino-D,L-phenylalanine caused a concentration-dependent inhibition (IC50: 300 microM). 5. Rat AM phi, cultured in the absence of LPS, formed significant amounts of [3H]-L-citrulline and [3H]-L-ornithine (133 and 212 d.p.m x 1000, respectively) when incubated for 1 h with [3H]-L-arginine. When AM phi had been cultured in the presence of 0.1 or 1 microgram ml-1 LPS, the formation of [3H]-L-citrulline was enhanced by 37 +/- 8.3 and 99 +/- 12% and that of [3H]-L-ornithine reduced by 21 +/- 8.7 and 70 +/- 2.5%, respectively. 6. In rat AM phi, cultured in the absence or presence of LPS, N omega-hydroxy-D,L-indospicine (10 and 30 microM) greatly reduced formation of [3H]-L-ornithine (by 80-95%) and this was accompanied by increased formation of [3H]-L-citrulline. However, only 20-30% of the [3H]-L-arginine not metabolized to [3H]-L-ornithine after inhibition of arginase was metabolized to [3H]-L-citrulline, when the AM phi had been cultured in the absence of LPS (i.e. low level of iNOS). On the other hand, when the AM phi had been cultured in the presence of LPS (i.e. high level of iNOS), all the [3H]-L-arginine not metabolized by the inhibited arginase was metabolized to [3H]-L-citrulline. 7. In conclusion, N omega-hydroxy-D,L-indospicine is a potent and specific inhibitor of arginase in AM phi. In cells in which, in addition to arginase, iNOS is expressed, inhibition of arginase can cause a shift of L-arginine metabolism to the NOS pathway. However, the extent of this shift appears to depend in a complex manner on the level of iNOS.