Donald J. Wolff

Pharmacological characterization of guanidinoethyldisulphide (GED), a novel inhibitor of nitric oxide synthase with selectivity towards the inducible isoform.

1 . Guanidines, amidines, S‐alkylisothioureas, and recently, mercaptoalkylguanidines have been described as inhibitors of the generation of nitric oxide (NO) from L‐arginine by NO synthases (NOS). We have recently demonstrated that guanidinoethyldisulphide (GED), formed from the dimerisation of mercaptoethylguanidine (MEG), is a novel inhibitor of nitric oxide synthases. Here we describe the pharmacological properties of GED on purified NOS isoforms, various cultured cell types, vascular ring preparations, and in endotoxin shock. 2 . GED potently inhibited NOS activity of purified inducible NOS (iNOS), endothelial NOS (ecNOS), and brain NOS (bNOS) enzymes with Ki values of 4.3, 18 and 25 μm, respectively. Thus, GED has a 4 fold selectivity for iNOS over ecNOS at the enzyme level. The inhibitory effect of GED on ecNOS and iNOS was competitive vs. L‐arginine and non‐competitive vs. tetrahydrobiopterin. 3 . Murine J774 macrophages, rat aortic smooth muscle cells, murine lung epithelial cells, and human intestinal DLD‐1 cells were stimulated with appropriate mixtures of pro‐inflammatory cytokines or bacterial lipopolysaccharide to express iNOS. In these cells, GED potently inhibited nitrite formation (EC50 values: 11,9, 1 and 30 μm, respectively). This suggests that uptake of GED may be cell type‐ and species‐ dependent. The inhibitory effect of GED on nitrite production was independent of whether GED was given together with immunostimulation or 6 h afterwards, indicating that GED does not interfere with the process of iNOS induction. 4 . GED caused relaxations in the precontracted vascular ring preparations (EC50: 20 μm). Part of this relaxation was endothelium‐dependent, but was not blocked by methylene blue (100 μm), an inhibitor of soluble guanylyl cyclase. In precontracted rings, GED enhanced the acetylcholine‐induced, endothelium‐dependent relaxations at 10 μm and caused a slight inhibition of the relaxations at 100 μm. The vascular studies demonstrate that the inhibitory potency of GED on ecNOS in the ring preparations is considerably lower than its potency against iNOS in the cultured cells. These data suggest that the selectivity of GED towards iNOS may lie, in part, at the enzyme level, as well as differential uptake by cells expressing the various isoforms of NOS. 5 . In a rat model of endotoxin shock in vivo, administration of GED, at 3 mg kg−1 bolus followed by 10 mg kg−1 h−1 infusion, starting at 90 min after bacterial lipopolysaccharide (LPS, 15 mg kg−1, i.v.), prevented the delayed fall in mean arterial blood pressure, prevented the development of the vascular hyporeactivity to noradrenaline of the thoracic aorta ex vivo and protected against the impairment of the endothelium‐dependent relaxations associated with this model of endotoxaemia. The same bolus and infusion of the inhibitor did not alter blood pressure or ex vivo vascular reactivity in normal animals over 90 min. 6 . Administration of GED (10 mg kg−1, i.p.) given at 2 h after LPS (120 mg kg−1, i.p.) and every 6 h thereafter caused a significant improvement in the survival rate in a lethal model of endotoxin shock in mice between 12 and 42 h. 7 . In conclusion, we found that GED is a competitive inhibitor of iNOS activity. Its selectivity towards iNOS may lie both at the enzyme level and at the level of cell uptake. GED has beneficial effects in models of endotoxin shock that are driven by iNOS. GED or its derivatives may be useful tools in the experimental therapy of inflammatory conditions associated with NO overproduction due to iNOS expression.

Pharmacological modulation of nitric oxide synthesis by mechanism-based inactivators and related inhibitors

Nitric oxide synthase (NOS) (EC 1.14.13.39) is a homodimeric cytochrome P450 monooxygenase analog that generates nitric oxide (NO) from the amino acid L-arginine. Enzymatically produced NO acts as an intracellular messenger in neuronal networks, blood pressure regulatory mechanisms, and immune responses. Isoform-selective pharmacological modulation of NO synthesis has emerged as a new therapeutic strategy for the treatment of diverse clinical conditions associated with NO overproduction. Mechanism-based inactivators (MBIs) represent a class of NOS mechanistic inhibitors that require catalytic turnover to produce irreversible inactivation of the ability of NOS to generate NO. Diverse isoform-selective NOS MBIs have been characterized with respect to their kinetic parameters and chemical mechanisms of inactivation. In studies with isolated and purified NOS isoforms, MBIs produce irreversible inactivation of NOS enzymatic activities. The inactivation process is associated with covalent modification of the NOS active site and proceeds either through heme destruction, its structural alteration, or covalent modification of the NOS protein chain. The behavior of NOS MBIs in intact cells is different from their behavior observed with the isolated NOS isoforms. In cytokine-induced RAW 264.7 macrophages, treatment with MBIs produces a complete loss of cellular NOS synthetic competence and inducible NOS activity. However, following drug removal, cells can recover at least partially in the absence of protein synthesis. In GH3 cells containing the neuronal NOS isoform, calcium transients are too low and abbreviated to allow significant NOS inactivation; hence, the cellular effects of MBIs on the neuronal isoform are almost completely and immediately reversible.

Inactivation of nitric oxide synthases and cellular nitric oxide formation by N6-iminoethyl-l-lysine and N5-iminoethyl-l-ornithine

The kinetics of inactivation of affinity-purified nitric oxide synthase isoforms by N6-iminoethyl-L-lysine (NIL) and N5-iminoethyl-L-ornithine (NIO) has been examined. Each of the agents produced a time and concentration dependent first order inactivation of the nitric oxide synthase isoforms that required exposure of the NO synthase to drug under conditions that supported catalysis, consistent with the proposal that these agents act as alternate substrate, mechanism-based inactivators. As measured at 100 microM arginine, NIL and NIO were equally efficient as inactivators of the cytokine-inducible nitric oxide synthase exhibiting apparent second order inactivation rate constants of 31.5 and 32.0 mM(-1) min(-1) respectively. By contrast, NIL and NIO were less efficient as inactivators of the constitutive neuronal nitric oxide synthase isoform exhibiting apparent second order inactivation rate constants of 0.79 and 8.4 mM(-1) min(-1) respectively. As measured at 100 microM extracellular arginine, NIL and NIO produced a time and concentration dependent inactivation of the NO synthetic capability of cytokine-induced murine macrophage RAW 264.7 cells exhibiting apparent second order inactivation rate constants of 3.1 and 1.8 mM(-1) min(-1). The inactivated RAW cell NO synthetic capability was restored to 30% of its pretreatment value over a 3-h period despite the presence of cycloheximide.

Inhibition of nitric oxide synthase with pyrazole-1-carboxamidine and related compounds

Guanidines, amidines, S-alkylisothioureas, and other compounds containing the amidine function (-C(=NH)NH2) have been described as inhibitors of the generation of nitric oxide (NO) by NO synthase (NOS). Here we report on the inhibition of the activity of NOS isoforms by compounds in which the amidine function is attached to a nitrogen of 1,2-diazo heterocycles to form N-carboxamidines and related compounds. 1H-Pyrazole-1-carboxamidine HCl (PCA) inhibited the activity of purified inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) isoforms to a similar extent (IC50 = 0.2 microM). 3-Methyl-PCA and 4-methyl-PCA showed reduced potencies, but a preference for iNOS [IC50 = 5 and 2.4 microM, respectively; cf. N(G)-methyl-L-arginine (NMA) IC50 = 6 microM]. Inhibition of purified iNOS by PCAs could be reversed completely by excess L-arginine, while their inhibition of NO production by stimulated RAW macrophages could be reversed by transfer to a drug-free medium. This suggests a competitive mode of inhibition. PCA caused potent concentration-dependent inhibition of the acetylcholine-induced, endothelium-dependent relaxations of precontracted rat thoracic aorta (IC50 = 30 microM). 4-Methyl-PCA inhibited the relaxations only at > or = 300 microM. In contrast, 4-methyl-PCA was more effective than both PCA and NMA in restoring the ex vivo contractility of aortic rings taken from lipopolysaccharide-treated rats. PCA and NMA, but not 4-methyl-PCA, caused marked increases in mean arterial pressure when administered i.v. to anesthetized rats. In conclusion, PCA and related compounds caused potent inhibition of NOS. Substitution of the pyrazole ring reduced potency, but improved selectivity towards iNOS as exemplified by 4-methyl-PCA.

Calmodulin-Dependent Phosphatases of PC12, GH3, and C6 Cells: Physical, Kinetic, and Immunochemical Properties

Calmodulin‐dependent phosphoprotein phosphatase (CaMDP) activity has been found in each of three cultured cell lines: rat pheochromocytoma (PC 12), glioma (C6), and pituitary adenoma (GH3) cells. These CaMDP activities bind to immobilized calmodulin in the presence of Ca2+ and are eluted by EGTA. Sucrose density centrifugation revealed that the phosphatase activities exhibited sedimentation coefficients of 4.37, 4.23, and 4.59 for proteins derived from C6, GH3, and PC 12 cells, respectively. The Stokes radii measured for the PC 12 and C6 activities were 41.8 and 40.0 A, respectively. The estimated molecular weights calculated for the enzymes from these data are 79, 100 and 72, 200. The phosphatase activities required the presence of divalent cations such as Ca2+ or Mn2+ for expression of activity, which was optimal only in the presence of calmodulin. The apparent Km for phosphorylated myelin basic protein substrate was 8 μM. Affinity‐purified antibodies to the B subunit of bovine brain CaMDP were found by immunoblot (Western blot) to cross‐react with a single protein among proteins extracted from PC 12, C6, and GH3 cells that had been resolved by two‐dimensional electrophoresis. In each case, the cross‐reacting protein exhibited an Mr of 16,000 and an isoelectric point of 4.7, values virtually identical to those reported previously for the B subunit of bovine brain CaMDP (sometimes called calcineurin). This cross‐reacting protein was found among cellular proteins eluted from immobilized calmodulin by EGTA. Immuno‐cytochemical localization of the cross‐reacting protein in undifferentiated PC 12 cells or in cells differentiated in response to nerve growth factor revealed its presence diffusely throughout the cytoplasm. These experiments support the contention that each of these cell lines contains a calmodulin‐regulated phosphatase homologous physically and kinetically, and immunologically related to bovine brain CaMDP.

Diffusivity of 125I-labelled macromolecules through collagen: mechanism of diffusion and effect of adsorption

Diffusion of angiotensin II, albumin and aldolase was studied through collagen membranes with swelling ratios between 4 and 15. The diffusion coefficient was measured from the time-lag for the onset of steady-state flux through the membrane. Binding of macromolecules to collagen was evaluated from the results of sorption studies conducted as a function of macromolecular concentration. Results presented indicate that the diffusion of macromolecules through collagen membrane is slowed by electrostatic and hydrogen bonding between individual macromolecular chains and collagen. The extent of adsorption is increased as the molecular weight of the diffusant increases. Diffusion of water soluble macromolecules through collagen occurs rapidly, suggesting that diffusion occurs through water filled channels as opposed to between collagen molecules. The results of these studies are useful in understanding diffusion through connective tissues and in the design of drug delivery systems based on collagen.

The antithyroid agent 6-n-propyl-2-thiouracil is a mechanism-based inactivator of the neuronal nitric oxide synthase isoform.

6-n-Propyl-2-thiouracil (6-PTU), the antithyroid agent, produces a time-, concentration-, and turnover-dependent inactivation of the NO synthetic capability of the neuronal nitric oxide synthase isoform irreversible by either arginine or (6R)-5,6,7,8-tetrahydro-L-biopterin. By contrast 6-PTU produces an inhibition of the cytokine-inducible and endothelial nitric oxide synthases fully reversible by arginine. The inactivation of neuronal nitric oxide synthase by 6-PTU follows first order kinetics, and is inhibited competitively by both arginine and (6R)-5,6,7,8-tetrahydro-L-biopterin, but is not accompanied by either a loss of heme-CO binding, heme fluorescence, or disassembly of dimeric structure. 2-Thiouracil behaves qualitatively identically to 6-PTU. Turnover-dependent inactivation of neuronal nitric oxide synthase by [2-14C]-2-thiouracil is accompanied by incorporation of radioactivity into the polypeptide chain. Ca2+-dependent NO formation by GH3 pituitary cells is inhibited by 6-PTU in a manner enhanced by depletion of either extracellular arginine or intracellular (6R)-5,6,7,8-tetrahydro-L-biopterin. These observations establish that 6-PTU is an alternate substrate, mechanism-based inactivator of the neuronal nitric oxide synthase isoform with the ability to suppress cellular NO formation.