The natural product noformycin is an inhibitor of inducible-nitric oxide synthase

Inducible-Nitric oxide synthase (iNOS, EC 1.14.13.39) catalyzes the formation of nitric oxide (NO) and L-citrulline from L-Arg. NADPH and dioxygen. The natural product, (-)-noformycin was found to be a potent, competitive inhibitor of recombinant human iNOS with respect to L-Arg with a Ki = 1.3 +/- 0.3 microM. The reversible binding of noformycin caused a high spin type I spectral perturbation of the iNOS heme group with a Kd = 1.5 +/- 0.2 microM. These results demonstrate that natural products may be a useful source for inhibitors of NO-biosynthesis.     

Comparative functioning of dihydro- and tetrahydropterins in supporting electron transfer, catalysis, and subunit dimerization in inducible nitric oxide synthase

The nitric oxide synthases (NOS) are the only heme-containing enzymes that require tetrahydrobiopterin (BH4) as a cofactor. Previous studies indicate that only the fully reduced (i.e., tetrahydro) form of BH4 can support NO synthesis. Here, we characterize pterin-free inducible NOS (iNOS) and iNOS reconstituted with eight different tetrahydro- or dihydropterins to elucidate how changes in pterin side-chain structure and ring oxidation state regulate iNOS. Seven different enzyme properties that are important for catalysis and are thought to involve pterin were studied. Only two properties were found to depend on pterin oxidation state (i.e., they required fully reduced tetrahydropterins) and were independent of side chain structure: NO synthesis and the ability to increase heme-dependent NADPH oxidation in response to substrates. In contrast, five properties were exclusively dependent on pterin side-chain structure or stereochemistry and were independent of pterin oxidation state: pterin binding affinity, and its ability to shift the heme iron to its high-spin state, stabilize the ferrous heme iron coordination structure, support heme iron reduction, and promote iNOS subunit assembly into a dimer. These results clarify how structural versus redox properties of the pterin impact on its multifaceted role in iNOS function. In addition, the data reveal that during NO synthesis all pterin-dependent steps up to and including heme iron reduction can take place independent of the pterin ring oxidation state, indicating that the requirement for fully reduced pterin occurs at a point in catalysis beyond heme iron reduction.     

Oxidation of NG-hydroxy-L-arginine by nitric oxide synthase: evidence for the involvement of the heme in catalysis

The involvement of the protoporphyrin IX heme iron of macrophage nitric oxide synthase (NOS) in the oxidation of NG-hydroxy-L-arginine (L-NHA) to nitric oxide (NO) and citrulline was investigated by carbon monoxide (CO) inhibition studies and binding difference spectroscopy. A CO:oxygen mixture (80:20) was found to inhibit the reaction by 33% with L-NHA as a substrate compared to 57% with L-arginine. Spectral perturbations were observed upon the addition of L-NHA to oxidized NOS, producing a type I binding difference spectrum with a maximum at 384 nm and minimum at 420 nm. In addition, L-NHA was incapable of reducing anaerobic oxidized NOS in the absence of NADPH. These studies support the involvement of the heme in the oxidation of L-NHA to NO and citrulline, indicating that the heme functions in both of the currently characterized oxidative steps of the NOS reaction.     

Fast cytochrome bo from Escherichia coli binds two molecules of nitric oxide at CuB

The reaction of nitric oxide (NO) with fast cytochrome bo from Escherichia coli has been studied by electronic absorption, MCD, and EPR spectroscopy. Titration of the enzyme with NO showed the formation of two distinct species, consistent with NO binding stoichiometries of 1:1 and 2:1 with observed dissociation constants at pH 7.5 of approximately 2.3 x 10(-)6 and 3.3 x 10(-)5 M. Monitoring the titration by EPR spectroscopy revealed that the broad EPR signals at g approximately 7.3, 3.7, and 2.8 due to magnetic interaction between high-spin heme o (S = 5/2) and CuBII (S = 1/2) are lost. A high-spin heme o signal at g = 6.0 appears as the 1:1 complex is formed but is lost again on formation of the 2:1 complex, which is EPR silent. The absorption spectrum shows that heme o remains in the high-spin FeIII state throughout the titration. These results are consistent with the binding of up to two NO molecules at CuBII. This has been confirmed by studies with the Cl- adduct of fast cytochrome bo. MCD evidence shows that heme o remains ligated by histidine and water. Addition of excess NO to the Cl- adduct leads to the appearance of a high-spin FeIII heme EPR signal. Hence chloride ion binds to CuB, blocking the binding of a second NO molecule. These results suggest a mechanism for the reduction of NO to nitrous oxide by cytochrome bo and cytochrome c oxidase in which the binding of two cis NO molecules at CuB permits the formation of an N-N bond and the abstraction of oxygen by the heme group.     

Crystal structure of tyrosine hydroxylase with bound cofactor analogue and iron at 2.3 A resolution: self-hydroxylation of Phe300 and the pterin-binding site

TyrOH is a non-heme iron enzyme which uses molecular oxygen to hydroxylate tyrosine to form L-dihydroxyphenylalanine (L-DOPA), and tetrahydrobiopterin to form 4a-hydroxybiopterin, in the rate-limiting step of the catecholamine biosynthetic pathway. The 2.3 A crystal structure of the catalytic and tetramerization domains of rat tyrosine hydroxylase (TyrOH) in the presence of the cofactor analogue 7,8-dihydrobiopterin and iron shows the mode of pterin binding and the proximity of its hydroxylated 4a carbon to the required iron. The pterin binds on one face of the large active-site cleft, forming an aromatic pi-stacking interaction with Phe300. This phenylalanine residue of TyrOH is found to be hydroxylated in the meta position, most likely through an autocatalytic process, and to consequently form a hydrogen bond to the main-chain carbonyl of Gln310 which anchors Phe300 in the active site. The bound pterin forms hydrogen bonds from N-8 to the main-chain carbonyl of Leu295, from O-4 to Tyr371 and Glu376, from the C-1' OH to the main-chain amides of Leu294 and Leu295, and from the C-2' hydroxyl to an iron-coordinating water. The part of the pterin closest to the iron is the O-4 carbonyl oxygen at a distance of 3.6 A. The iron is 5.6 A from the pterin 4a carbon which is hydroxylated in the enzymatic reaction. No structural changes are observed between the pterin bound and the nonliganded enzyme. On the basis of these structures, molecular oxygen could bind in a bridging position optimally between the pterin C-4a and iron atom prior to substrate hydroxylation. This structure represents the first report of close interactions between pterin and iron in an enzyme active site.     

Urokinase and the intestinal mucosa: evidence for a role in epithelial cell turnover

The enzyme nitric oxide synthase catalyzes the oxidation of the amino acid L-arginine to L-citrulline and nitric oxide in an NADPH-dependent reaction. Nitric oxide plays a critical role in signal transduction pathways in the cardiovascular and nervous systems and is a key component of the cytostatic/cytotoxic function of the immune system. Characterization of nitric oxide synthase substrates and cofactors has outlined the broad details of the overall reaction and suggested possibilities for chemical steps in the reaction; however, the molecular details of the reaction mechanism are still poorly understood. Recent evidence suggests a role for the reduced bound pterin in the first step of the reaction--the hydroxylation of L-arginine.     

Electron transfer and catalytic activity of nitric oxide synthases. Chimeric constructs of the neuronal, inducible, and endothelial isoforms

The nitric oxide synthases (NOS) are single polypeptides that encode a heme domain, a calmodulin binding motif, and a flavoprotein domain with sequence similarity to P450 reductase. Despite this basic structural similarity, the three major NOS isoforms differ significantly in their rates of .NO synthesis, cytochrome c reduction, and NADPH utilization and in the Ca2+ dependence of these rates. To assign the origin of these differences to specific protein domains, we constructed chimeras in which the reductase domains of endothelial and inducible NOS, respectively, were replaced by the reductase domain of neuronal NOS. The results with the chimeric proteins confirm the modular organization of the NOS polypeptide chain and demonstrate that (a) similar residues establish the necessary contacts between the reductase and heme domains in the three NOS isoforms, (b) the maximal rate of .NO synthesis is determined by the maximum intrinsic ability of the reductase domain to deliver electrons to the heme domain, (c) the Ca2+ independence of inducible NOS requires interactions of calmodulin with both the calmodulin binding motif and the flavoprotein domain, and (d) the effects of tetrahydrobiopterin and L-arginine on electron transfer rates are mediated exclusively by heme domain interactions.     

In vivo ESR-CT imaging of the liver in mice receiving subcutaneous injection of nitric oxide-bound iron complex

We have cloned the human liver inducible isoform of nitric oxide synthase (NOS) into an Escherichia coli expression vector and have expressed and purified the enzyme. The protein has been expressed with and without a polyhistidine tail. In both cases, expression of functional protein requires coexpression with calmodulin and inclusion of tetrahydrobiopterin (H4B) in the purification buffers. Unlike the constitutive isoforms of NOS, this isoform is unstable in the absence of L-arginine (L-Arg) and H4B toward loss of the heme group and the formation of a low-spin species spectroscopically distinct from that of the cofactor-bound protein. The enzyme purified in the presence of both L-Arg and H4B is highly active, with a Vmax of approximately 800 nmol NO min(-1) mg(-1) and a Km for L-Arg of 22 microM. The cytochrome c reductase activity is 38,000 nmol x min(-1) mg(-1). Similar values are obtained for the enzyme with and without the polyhistidine tail. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid does not inhibit the activity of the protein, nor is the activity of the enzyme increased by the addition of exogenous calmodulin and/or Ca2+. These findings contrast with an earlier report, based on experiments with extracts of COS-1 cells expressing the recombinant enzyme, that the enzyme responds to changes in the Ca2+ concentration. The human hepatic isoform is similar in its properties to the inducible NOS isoform purified from macrophages.     

High concentration of L-arginine suppresses nitric oxide synthase activity and produces reactive oxygen species in NB9 human neuroblastoma cells

Hereditary argininemia manifests as neurological disturbance and mental retardation, features not observed in other amino acidemias. The cytotoxic effect of a high concentration of L-arginine (L-Arg) was investigated using NB9 human neuroblastoma cells (NB9), which express neuronal nitric oxide synthase (nNOS). When the concentration of L-Arg in the medium increased from 50 microM to 2 mM after incubation for 48 hr, the intracellular concentration of L-Arg increased from 68.0 +/- 1 pmol/10(6) cells to 1310.0 +/- 5 pmol/10(6) cells and that of L-citrulline (L-Cit) from undetectable levels to 47.1 +/- 0.2 pmol/10(6) cells (mean +/- SD of three independent analyses). This increase in intracellular L-Arg levels caused a decrease in NOS activity by approximately 71%. Flow cytometric analysis showed that reactive oxygen species (ROS) are produced in NB9 exposed to 2 mM L-Arg. The production of ROS was abolished by a NOS inhibitor, NG-nitro-L arginine-methylester. Production of ROS was also observed when NB9 were treated with L-Cit for 48 hr. To investigate the effect of L-Cit on the activity of NOS, a kinetic study on nNOS was conducted using cellular extracts from NB9. The apparent Km value of nNOS for L-Arg was 8.4 microM, with a Vmax value of 8.2 pmol/min/mg protein. L-Cit competitively inhibited NOS activity, as indicated by an apparent Ki value of 65 nM. These results suggest that L-Cit formed by nNOS in L-Arg-loaded neuronal cells inhibits NOS activity and nNOS in these L-Arg-loaded cells functions as a NADPH oxidase to produce ROS, which may cause neurotoxicity in argininemia.     

Riluzole interacts with voltage-activated sodium and potassium currents in cultured rat cortical neurons

Inhaled nitric oxide is a selective pulmonary vasodilator used for the treatment of pulmonary hypertension. The potential adverse effects of inhaled nitric oxide are unknown and represent the focus of the present studies. Whereas inhalation of nitric oxide (10 to 100 ppm, 5 h) by Balb/c mice had no effect on the number or type of cells recovered from the lung, a dose-related increase in bronchoalveolar lavage protein was observed, suggesting that nitric oxide induces alveolar epithelial injury. To determine if this was associated with altered alveolar macrophage activity, we quantified production of reactive oxygen and nitrogen intermediates by these cells. Interferon-gamma, alone or in combination with lipopolysaccharide (LPS), induced expression of inducible nitric oxide synthase (iNOS) protein and nitric oxide production by alveolar macrophages. Cells from mice exposed to 20 to 100 ppm nitric oxide produced significantly more nitric oxide and expressed greater quantities of iNOS than cells from control animals. Superoxide anion production and peroxynitrite generation by alveolar macrophages were also increased after exposure of mice to nitric oxide. This was correlated with increased antinitrotyrosine antibody binding to macrophages in histologic sections. Taken together, these data demonstrate that inhaled nitric oxide primes lung macrophages to release reactive oxygen and nitrogen intermediates. Increased production of these mediators by macrophages following inhalation of nitric oxide may contribute to tissue injury.     

Effects of various imidazole ligands on heme conformation in endothelial nitric oxide synthase

We have evaluated the influence of a series of substituted imidazoles on the heme structure of endothelial nitric oxide synthase (eNOS). Optical, MCD, and EPR spectra reveal widely differing effects on heme spin state and geometry. 1-Substituted imidazoles always yield low-spin heme complexes, but the size of the 2- and 4-substituent influences their structural effects on the heme. Methyl substituents lead to low-spin complexes while the bulky phenyl group yields high-spin complexes. The only exception to this behavior is provided by 2-aminoimidazole. Although this compound has three functional groups which can serve as an axial ligand to the heme, its binding to eNOS leads to a pure high-spin complex. This result can only be interpreted as due to a direct binding of 2-aminoimidazole to the guanidine binding subdomain of L-arginine. MCD spectra also imply that an O-ligand is present in the low-spin resting eNOS, while EPR data reveal the presence of two low-spin heme complexes in resting eNOS and its imidazole complexes. EPR also distinguishes four different high-spin forms of eNOS generated by different imidazole analogues. This series of ligands promises to be useful in probing the subtle structural difference among the active sites of three NOS isozymes and in developing selective inhibitors to these important enzymes.     

Substrate binding and calmodulin binding to endothelial nitric oxide synthase coregulate its enzymatic activity

Endothelial nitric oxide synthase (NOS) is a constitutively expressed flavin-containing heme protein that catalyzes the formation of NO from L-arginine, NADPH, and molecular oxygen. We purified bovine endothelial NOS from transfected embryonic kidney cells by conventional chromatographic techniques and characterized the activity of the detergent-solubilized enzyme. Endothelial NOS displays a much lower specific activity of NO synthesis (143 +/- 11 nmol NO/min/mg enzyme) than the constitutive neuronal NOS or inducible NOS isoforms. Like the neuronal isoform, endothelial NOS requires binding of Ca2+/calmodulin to achieve Vmax NO synthase activity; however, we observed a basal level of NO synthesis even when Ca2+/calmodulin was omitted and 0.5 mM EDTA was present in the assay solution. Moreover, endothelial NOS demonstrates a high-affinity bonding interaction with calmodulin such that the enzyme as purified has a NO synthase activity at about 80% of Vmax. We also observed a more than twofold increase in NADPH consumption by endothelial NOS when it was coupled to arginine oxygenation as opposed to when oxygen is activated in the absence of substrate. Substrate binding was also shown to stimulate heme reduction in the absence of added calmodulin. Thus, the enzymatic synthesis of NO from L-arginine by endothelial NOS appears to be partially regulated by binding of both calmodulin and substrate. These findings for endothelial NOS represent a significant departure from the enzymatic properties of the other constitutive NOS isoform, neuronal NOS, and we interpret this result in terms of the physiological implications.     

Heme oxygenase: the physiological role of one of its metabolites, carbon monoxide and interactions with zinc protoporphyrin, cobalt protoporphyrin and other metalloporphyrins

In 1991, we postulated that carbon monoxide, which is formed endogenously from heme catabolism catalyzed by heme oxygenase and shares some of the chemical and biological properties of nitric oxide, may play a role similar to that of nitric oxide as a widespread signal transduction mechanism for the regulation of cell function and communication. We review the experimental evidence that tests this postulate. Carbon monoxide appears to be involved in the neurophysiological phenomenon of long-term potentiation, which appears to play a key role in memory and learning. Zinc protoporphyrin, an inhibitor of heme oxygenase, prevents induction of long-term potentiation. Zinc protoporphyrin is an endogenous substance, the levels of which are increased in iron deficiency states and in lead poisoning, and by inhibiting heme oxygenase may modulate long-term potentiation and memory. It has been shown that, when cobalt protoporphyrin is injected into the medial nuclei of the rat hypothalamus, weight loss occurs. These nuclei contain heme oxygenase, and we postulate that weight loss is due to cobalt protoporphyrin induction of heme oxygenase and increased formation of carbon monoxide, which serves as a signal transduction mechanism in the medial hypothalamus to suppress appetite.     

Characterization of C415 mutants of neuronal nitric oxide synthase

Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. C415H and C415A mutants of the neuronal isoform of NOS (nNOS) were expressed in a baculovirus system and purified to homogeneity for spectral analysis and activity measurements. UV-visible spectra of each mutant lacked an observable Soret peak, suggesting that neither mutant contained heme. When reduced in the presence of CO, however, a small Soret centered at 417 nm could be detected for the C415H mutant, further supporting the assignment of C415 as the axial ligand to the heme. In addition to a deficiency in bound heme, neither mutant had any detectable bound tetrahydrobiopterin, as compared to wild-type enzyme, which had a ratio of 0.84 mol of bound pteridine:1 mol of nNOS 160 kDa subunit. The C415H mutant contained bound FAD and FMN at levels of 1.0 +/- 0.1 and 0.9 +/- 0.1 mol/mol of nNOS subunit, respectively. UV-visible spectra of both nNOS mutants retained the distinctive absorbance due to tightly associated oxidized flavin prosthetic groups. Further, the spectra suggested the presence of a neutral flavin semiquinone. Ferricyanide oxidation of the C415A mutant yielded a spectrum that was essentially that of oxidized flavin. Ferricyanide titration showed that the C415A mutant contained approximately 1 reducing equiv. Circular dichroism spectra suggested that each mutant was folded properly, in that both spectra were found to be essentially identical to the spectrum of wild-type nNOS. Neither mutant could synthesize nitric oxide, and neither mutant had the ability to oxidize NADPH unless an exogenous electron acceptor was added. The rate of cytochrome c reduction by each mutant was found to be slightly less, but very similar to the rate (approximately 20 mumol mg-1 min-1) observed with wild-type nNOS. In all cases, the rate of cytochrome c reduction increased approximately 15-fold with the addition of calmodulin. Overall, these spectral and activity data suggest that C415 is the axial heme ligand and that a point mutation at C415 prevents binding of heme and tetrahydrobiopterin without interfering with the global folding or the reductase function of nNOS.     

Further evidence that nitric oxide modifies acute and chronic morphine actions in mice

The effects of the nitric oxide (NO) synthase inhibitor, N(omega)-nitro-L-arginine (L-NNA, 2.5-10 microg i.c.v.), and the NO synthesis precursor, L-arginine (L-Arg, 2.5-10 microg i.c.v.), on morphine-induced analgesia, and on morphine-induced tolerance and dependence were examined in mice. Administration of L-NNA diminished the morphine-induced analgesia. L-Arg pretreatment increased the analgesic effect of morphine. Repeated pretreatment (three times, at 24-h intervals) with L-NNA diminished both acute and chronic tolerance to morphine, whereas both the acute and the chronic morphine-induced tolerance increased after the repeated (three times, at 24-h intervals) administration of L-Arg. Neither L-NNA nor L-Arg affected the signs of morphine dependence, as assessed by naloxone (1 mg/kg, s.c.)-precipitated withdrawal. Our data suggest that increased NO synthesis potentiates morphine analgesia and enhances the development of morphine tolerance in mice.     

An unusual effect of application of the amino acid L-arginine on cat visual cortical cells

Iontophoretic application of L-arginine (L-Arg) resulted in a profound decrease in visually elicited and spontaneous activity in 22 of 77 (29%) cells in area 17 of the anaesthetized/paralysed cat. Duration was long, and cells did not recover pre-application activity levels, indicating permanent decline. This effect was obtained without change in the extracellularly recorded wave-form, demonstrating that this did not result from depolarization block. In the remaining 55 cells, application of L-Arg alone, at levels capable of eliciting inhibition as described above, was without effect. In 29 cells, L-Arg application was able to reverse the effect of inhibition of nitric oxide (NO) production. Populations of cells showing the depressive effect described above and those affected by NO modulation levels were mutually exclusive.     

Generation of nitric oxide by a nitrite reductase activity of xanthine oxidase: a potential pathway for nitric oxide formation in the absence of nitric oxide synthase activity

The effects of substrate, L-Arg and cofactors, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (H4B) and calmodulin (CaM), on chiral discrimination by rat neuronal nitric oxide synthase (nNOS) for binding the enantiomers of 1-(1-naphthyl)ethylamine (ligand I), 1-cyclohexylethylamine (ligand II), and 1-(4-pyridyl)ethanol (ligand III) were studied under anaerobic conditions by optical absorption spectroscopy. The ratio of the dissociation constant (Kd) values for the S- and R-enantiomers of ligand I (S/R) was 30, while the S/R ratio for ligand II and the R/S ratio for ligand III were 1.8 and < 0.14, respectively, in the presence of 0.15 microM H4B. However, in the presence of 1 mM L-Arg, the S/R ratio of the Kd values for ligand I was decreased down to 5.9. In the presence of both 1 mM L-Arg and 0.1 mM H4B, the S/R ratios for ligands I and II and the R/S ratio for ligand III were enormously increased up to 29, > 80, and 60, respectively. These and other spectral observations strongly suggest that strict chiral recognition at the active site of nNOS during catalysis is exhibited only in the presence of the active effector.     

Comparison of the effects of various clinically applied mistletoe preparations on peripheral blood leukocytes

The nitric oxide synthases (NOS), although unrelated to the cytochromes P450 in terms of sequence, exhibit spectroscopic and catalytic properties strongly reminiscent of those of the P450 system. One important difference is the requirement of the NOS enzymes for tetrahydrobiopterin. The biopterin cofactor is shown by chemical studies to bind close to pyrrole ring D of the prosthetic heme group, a position confirmed recently for inducible NOS and endothelial NOS by crystal structures. The only plausible role so far for the tetrahydrobiopterin is as a transient electron donor for the activation of molecular oxygen. NADPH-derived electrons are provided to the heme by the NOS flavin domain, but the biopterin may be required to provide an electron at a faster rate than that supported by the flavin groups. Chimeras in which the reductase domains of the isoforms have been exchanged indicate that the overall rate of catalytic turnover is directly governed by the ability of the flavin domain to deliver electrons. Electron transfer from the flavin to the heme domain, and within the flavin and heme domains, is thus a critical determinant of the catalytic turnover of NOS.     

Egg culture: the foundation

Bacterial lipopolysaccharide and a diverse array of other immunostimulants and cytokines suppress the metabolism of endogenous and exogenous substances by reducing the activity of hepatic cytochrome P-450 mixed function oxidase system. Although this effect of immunostimulants was first described almost 40 years ago, the mechanism is obscure. Immunostimulants are now known to cause nitric oxide overproduction by cells via induction of nitric oxide synthase. The highly reactive NO radical binds to prosthetic groups such as heme or iron-sulfur clusters leading to either activation or (more often) inhibition of iron-containing enzymes. It has been known for years that NO also binds to the heme moiety of cytochrome P-450 (CYP) with high affinity. However it was only recently demonstrated that binding of NO to CYPs also inhibits their enzymatic activity. This applies to both exogenously derived as well as endogenously synthesized NO. Suppression of CYP-dependent metabolism, which is a major problem of inflammatory liver diseases, can be significantly reversed by inhibition of NO synthesis in vivo under experimental conditions. The present paper reviews the findings implicating NO as a major factor mediating the suppression of CYP expression caused by endotoxins and immunostimulants in general. NO-mediated suppression of the metabolism of endogenous and exogenous substances under inflammatory conditions may contribute to the clinical manifestations and may be an important consideration for rational drug therapy in these conditions.     

Rediscovering the science of the history of life

The effects induced by L-arginine (L-Arg) on the short-circuit current and potential difference of Pleurodema thaul skin were investigated. L-Arg, but not D-Arg significantly increased the short-circuit current and potential difference when applied to the serosal surface. The effects of L-Arg were antagonized by amiloride, NG-nitro-methyl-L-arginine (L-NAME) and by methylene blue. Carbachol and acetylcholine induced significant increases of both electrical parameters of the toad skin. These effects of the muscarinic cholinergic drugs were potentiated by a low concentration of L-Arg and antagonized by L-NAME or methylene blue. Carbachol and acetylcholine induced significant increases of both electrical parameters of the toad skin. These effects of the muscarinic cholinergic drugs were potentiated by a low concentration of L-Arg and antagonized by L-NAME or methylene blue. Addition of dibutyryl cyclic guanosyl monophosphate (db cGMP) or dibutyryl cyclic adenosine monophosphate (db cAMP) increased short-circuit current and potential difference. The effects of db cGMP, but not those of db cAMP were antagonized by L-NAME. The consecutive application of db cGMP and db cAMP induced additive effects. These results suggest that L-Arg increases transport in toad skin presumably acting through the formation of nitric oxide, which then stimulates cytoplasmic guanylate cyclase and leads to increased Na+ and K+ transport. The effects of L-Arg and carbachol were antagonized by acute application of morphine; however, a rebound response was observed when carbachol or noradrenaline were given after prolonged exposure of the skin to morphine, which suggests an adaptive response of the skin involving both cGMP and cAMP. Responses to both nucleotides were unchanged by morphine.