Chronic nitric oxide synthase inhibition prevents new coronary capillary generation.

L-NAME-induced hypertension has been shown to produce concentric (eutrophic) remodeling of the heart despite an enhanced afterload. We postulated that nitric oxide synthase inhibition could limit coronary capillary growth to explain the nature of remodeling. To test our hypothesis, we aimed at determining the effect of endogenous and exogenous nitric oxide on coronary neovascularization. Aortic and coronary rings from normotensive animals were incubated in a three-dimensional type I collagen matrix in the presence of L-NAME or the nitric oxide donor SNAP. L-NAME inhibited, while SNAP stimulated, neovascularization from aortic and coronary rings after 12 days of in vitro incubation. In arterial rings harvested from rats treated with L-NAME for 14 days and in which no further in vitro treatment was added, only coronary rings showed a reduction in new capillary generation. While confirming that chronic L-NAME-treated rats develop concentric remodeling, the evaluation of capillary density did not reveal any difference as compared with the controls in 3 areas of the myocardium. In conclusion, chronic inhibition of nitric oxide synthesis in vivo produces a long-lasting reduction in the capacity of coronary arteries to generate new capillaries in vitro. Thus, our results lend support to the hypothesis that an inhibition of new capillary formation could prevent the development of compensatory ventricular hypertrophy, in favor of concentric remodeling.

Comparison of toxic effects of nitric oxide and peroxynitrite on Uronema marinum (Ciliata: Scuticociliatida).

To discover the effects of nitric oxide (NO) and peroxynitrite on Uronema marinum (a ciliate responsible for systemic scuticociliatosis in cultured olive flounder Paralichthys olivaceus), the dose-dependent inhibitory effect of NO donors, S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine (SIN-1) on the proliferation and survival of U. marinum was investigated. The inhibitory effects of exogenous superoxide dismutase (SOD) and catalase on the toxicity of SIN-1 were also investigated. After 24 h of incubation in the presence of 0.2 mM SNAP, the number of ciliates was not statistically different from that of the controls, whereas incubation in the presence of 0.5 mM SNAP reduced the number of parasites significantly to 59.1% of controls. Concentrations of SNAP higher than 0.5 mM resulted in greater reductions in the number of ciliates, but levels of generated NO far exceeded physiological ranges. The number of viable ciliates incubated for 24 h with 0.2 mM SIN-1 was reduced significantly to 25.0%, and all ciliates were killed by incubation in concentrations above 0.5 mM SIN-1. Although SOD decreased the toxic effect of SIN-1 on U. marinum, protection was not complete and did not improve after increasing the SOD concentration from 50 to 400 U ml(-1). Addition of catalase ranging from 500 to 10000 U ml(-1) completely protected U. marinum from SIN-1 toxicity. Ciliates exposed to catalase alone or catalase plus SIN-1 showed significantly higher and dose-dependent proliferation rates compared to controls. Addition of haemoglobin, ranging from 0.5 to 2.0 mg ml(-1), also protected U. marinum from SIN-1 toxicity, and increased the proliferation rate dose-dependently. In conclusion, resistance of U. marinum to oxidative and nitrative stress may allow this pathogen to withstand the NO- and oxygen-radical-dependent killing mechanisms of phagocytic cells.

Modulation of D-penicillamine-induced autoimmunity in the Brown Norway rat using pharmacological agents that interfere with arachidonic acid metabolism or synthesis of inducible nitric oxide synthase.

The D-penicillamine-induced autoimmune syndrome observed in Brown Norway (BN) rats is similar to an idiosyncratic reaction seen in some patients. We have previously shown that pretreatment of BN rats with aminoguanidine, an inducible nitric oxide synthase (iNOS) inhibitor, and misoprostol, a prostaglandin E (PGE) analog, completely prevented the development of D-penicillamine-induced autoimmunity. In an effort to further understand the role of arachidonic acid metabolism and iNOS in the pathogenesis of D-penicillamine-induced autoimmunity we had 3 objectives: (1) to test whether aminoguanidine and misoprostol could reverse D-penicillamine-induced autoimmunity; (2) whether BN rats that had previously developed D-penicillamine-induced autoimmunity could be protected on re-challenge with drug by pretreatment with aminoguanidine and misoprostol and (3) whether non-steroidal anti-inflammatory drugs, which inhibit PGE synthesis, would potentiate D-penicillamine-induced autoimmunity. We found that neither aminoguanidine nor misoprostol had any significant effect on the speed of recovery from D-penicillamine-induced autoimmunity. Prevention of disease on re-challenge after a 4 week recovery was less effective than on initial treatment with 7/8 animals pretreated with aminoguanidine getting sick again, while only 5/13 animals pretreated with misoprostol became ill. The effect of aminoguanidine was not significantly different from control (16/17) but that of misoprostol was (P=0.002). A single dose of the non-selective cyclooxygenase (COX) inhibitor, ketoprofen, decreased the time to onset of D-penicillamine-induced autoimmunity and continuous treatment significantly increased the incidence (P=0.024). Diclofenac, which is more selective, did not have a significant effect, and one dose of the selective inhibitor, rofecoxib, actually appeared to lower the incidence of D-penicillamine-induced autoimmunity (P=0.001). In this animal model of drug-induced autoimmunity, non-selective COX inhibitors appear to increase the incidence of disease. However, once the reaction occurs, prostaglandins are not effective for treatment and are only partially protective in an already sensitized animal.

Reversible inhibition of the human xenobiotic-metabolizing enzyme arylamine N-acetyltransferase 1 by S-nitrosothiols.

Human arylamine N-acetyltransferase 1 (NAT1) is a polymorphic phase II xenobiotic-metabolizing enzyme which catalyzes the biotransformation of primary aromatic amines, hydrazine drugs, and carcinogens. Structural and functional studies have shown that the NAT1 and factor XIII transglutaminase catalytic pockets are structurally related with the existence of a conserved catalytic triad (Cys-His-Asp). In addition, it has been reported that factor XIII transglutaminase activity could be regulated by nitric oxide (NO), in particular S-nitrosothiols (RSNO). We thus tested whether NAT1 could be a target of S-nitrosothiols. We show here that human NAT1 is reversibly inactivated by S-nitrosothiols such as SNAP (S-nitroso-N-acetyl-DL-penicillamine). A second-order rate constant for the inactivation of NAT1 by SNAP was determined (k(inact)=270M(-1)min(-1)) and shown to be in the same range of values reported for other enzymes. The inhibition of NAT1 by S-nitrosothiols was reversed by dithiothreitol and reduced glutathione, but not by ascorbate. As reported for some reactive cysteine-containing enzymes, our results suggest that inactivation of NAT1 by S-nitrosothiols is due to direct attack of the highly reactive cysteine residue in the enzyme active site on the sulfur of S-nitrosothiols to form a mixed disulfide between these NO-derived oxidants and NAT1. Finally, our findings suggest that, in addition to the polymorphic-dependent variation of NAT1 activity, NO-derived oxidants, in particular S-nitrosothiols, could also regulate NAT1 activity.

Expression of prostaglandin endoperoxide H synthase-2 induced by nitric oxide in conditionally immortalized murine colonic epithelial cells.

Increased expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) has been implicated in pathological conditions such as inflammatory bowel diseases and colon cancer. Recently, it has been demonstrated that inducible nitric oxide synthase (NOS II) expression and nitric oxide (NO) production are up-regulated in these diseases as well. However, the apparent link between PGHS-2 and NOS II has not been thoroughly investigated in nontransformed and nontumorigenic colonic epithelial cells. In the present study, we examined the concomitant expression of PGHS-2 and NOS II as well as the production of prostaglandin E2 (PGE2) and NO in conditionally immortalized mouse colonic epithelial cells, namely YAMC (Apc(+/+)). We found that the induction of PGHS-2 and generation of PGE2 in these cells by IFN-gamma and lipopolysaccharide (LPS) were greatly reduced by two selective NOS II inhibitors, L-NIL and SMT. To ascertain the effect of NO on PGHS-2 overexpression, we tested NO-releasing compounds, NOR-1 and SNAP, and found that they caused PGHS-2 expression and PGE2 production. This effect was abolished by hemoglobin, a NO scavenger. Using electrophoretic mobility shift assays, we found that both NOR-1 and SNAP caused beta-catenin/LEF-1 DNA complex formation. Super-shift by anti-beta-catenin antibody confirmed the presence of beta-catenin in the complex. Cell fractionation studies indicated that NO donors caused an increase in free soluble cytoplasmic beta-catenin. This is further corroborated by the immunocytochemistry data showing the redistribution of beta-catenin from the predominantly membrane localization into the cytoplasm and nucleus after treatment with NO donors. To further explore the possible connection between PGHS-2 expression and beta-catenin/LEF-1 DNA complex formation, we studied IMCE (Apc(Min/+)) cells, a sister cell line of YAMC with similar genetic background but differing in Apc genotype and, consequently, their beta-catenin levels. We found that IMCE cells, in comparison with YAMC cells, had markedly higher beta-catenin/LEF-1 DNA complex formation under both resting conditions as well as after induction with NO. In parallel fashion, IMCE cells expressed significantly higher levels of PGHS-2 mRNA and protein, and generated more PGE2. Overall, this study suggests that NO may be involved in PGHS-2 overexpression in conditionally immortalized mouse colonic epithelial cells. Although the molecular mechanism of the link is still under investigation, this effect of NO appears directly or indirectly to be a result of the increase in free soluble beta-catenin and the formation of nuclear beta-catenin/LEF-1 DNA complex.

Nitric oxide synthase inhibitors enhance 5-HT2 receptor-mediated behavior, the head-twitch response in mice.

The purpose of this study was to characterize behavioral interactions between nitric oxide synthase (NOS) inhibitors and serotonergic 5-HT2 receptors. In the present study, NOS inhibitors, N(G)-nitro-L-arginine, N(G)-nitro-L-arginine methylester, N(G)-monomethyl-L-arginine, 7-nitroindazole, trifluoperazine and NO scavenger, methylene blue markedly enhanced 5-hydroxytryptamine (5-HT)-induced selective serotonergic behavior, the head twitch response (HTR), in mice. However NO generators, sodium nitroprusside, 3-morpholinosydnonimine and S-nitroso-N-acetylpenicillamine as well as NO precursor, L-arginine markedly inhibited 5-HT induced HTR in mice. In the previous study, it was demonstrated that the N-methyl-D-aspartate (NMDA) receptor antagonists markedly enhanced 5-HT-induced selective serotonergic behavior, HTR, whereas NMDA itself inhibited 5-HT-induced HTR in mice. In the present study, it was demonstrated that the inhibition by a NMDA receptor agonist, NMDA of 5-HT-induced HTR was reversed by the treatment with NOS inhibitors, N(G)-nitro-L-arginine and N(G)-nitro-L-arginine methylester. The suppressive action by a NO generator, S-nitroso-N-acetylpenicillamine of 5-HT-induced HTR was also reversed by the treatment with NMDA receptor antagonists, MK-801 and dextromethorphan. These results have shown that the NO system is located down stream of NMDA receptors involved in modulation of 5-HT2-mediated HTR. Therefore, the enhanced effects of NOS inhibitors on 5-HT-induced HTR support experimental evidence for the NO/5-HT2 as well as NMDA/5-HT2 receptor interactions indicating that NO plays an important role in the glutamatergic modulation of the serotonergic function at the 5-HT2 receptor.

Chronic hyperammonemia in rats impairs activation of soluble guanylate cyclase in neurons and in lymphocytes: a putative peripheral marker for neurological alterations.

Chronic hyperammonemia impairs the glutamate-nitric oxide-cGMP pathway in rat brain in vivo. The aims of this work were to assess whether hyperammonemia impairs modulation of soluble guanylate cyclase, and to look for a peripheral marker for impairment of this pathway in brain. We activated the pathway at different steps using glutamate, SNAP, or YC-1. In control neurons these compounds increased cGMP by 7.4-, 9.7- and 7.2-fold, respectively. In ammonia-treated neurons formation of cGMP induced by glutamate, SNAP, and YC-1 was reduced by 50%, 56%, and 52%, respectively, indicating that hyperammonemia impairs activation of guanylate cyclase. This enzyme is also present in lymphocytes. Activation of guanylate cyclase by SNAP or YC-1 was impaired in lymphocytes from hyperammonemic rats. These results suggest that determination of the activation of soluble guanylate cyclase in lymphocytes could serve as a peripheral marker for impairment of the neuronal glutamate-nitric oxide-cGMP pathway in brain.

Synergism of nitric oxide and iron in killing the transformed murine oligodendrocyte cell line N20.1.

Nitric oxide (NO) produced in inflammatory lesions may play a major role in the destruction of oligodendrocytes in multiple sclerosis and experimental allergic encephalomyelitis. The transformed murine oligodendroglial line N20.1 is much more resistant than primary oligodendrocytes to killing by the NO generator S-nitroso-N-acetyl-DL-penicillamine (SNAP). This observation prompted investigation of the mechanisms leading to cell death in the N20.1 cells and comparison of SNAP with another NO donor, sodium nitroprusside (SNP). We observed that N20.1 cells were 30 times more sensitive to SNP than to SNAP. The specific NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) protected against SNP only, not against SNAP. However, dithiothreitol protected against both SNAP and SNP, indicating that S-nitrosylation of cysteines plays a major role in the cytotoxicity of both NO donors. We did not observe any formation of peroxynitrite or increase of Ca2+ concentration with either SNAP or SNP, thus excluding their involvement in the mechanisms leading to N20.1 cell death. Based on two observations, (a) potentiation of the cytotoxic effect of SNP when coincubated with ferricyanide or ferrocyanide, but not sodium cyanide, and (b) protection by deferoxamine, an iron cyanide chelator, we conclude that the greater sensitivity of N20.1 cells to SNP compared with SNAP is due to synergism between NO released and the iron cyanide portion of SNP, with the cyanide accounting for very little of the cytotoxicity. Finally, SNP but not SNAP induces some apoptosis, as shown by DNA laddering and protection by a caspase-3 inhibitor. These results suggest that low levels of NO in combination with increased iron content lead to apoptotic cell death rather than the necrotic cell death seen with higher levels of NO generated by SNAP.

The protective role of thiols against nitric oxide-mediated cytotoxicity in murine macrophage J774 cells.

Nitric oxide (NO) plays an important role in the cytotoxic activity of macrophages towards tumour cells and microbial pathogens. We investigated whether alteration of intracellular thiol levels modulates the cytotoxic effects of different NO donors and lipopolysaccharide-induced NO in the murine macrophage cell lin J774A.1. The NO-releasing compound S-nitroso-N-acetylpenicillamine caused a significant concentration-dependent loss of viability of the macrophages only under glucose-limiting conditions. The cytotoxic effect of S-nitroso-N-acetylpenicillamine was prevented by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO). Depletion of total glutathione before exposure to S-nitroso-N-acetylpenicillamine further decrease cell viability while pretreatment with N-acetylcysteine was protective. Comparing equimolar concentrations of various NO donors including S-nitrosoglutathione, S-nitrosocysteine and 3-morpholino-sydnonimine hydrochloride, cytotoxicity appeared to be related to the relative stability of the test compound. Both the order of stability and the order of potency for cell killing was S-nitrosoglutathione > S-nitroso-N-acetylpenicillamine > S-nitrosocysteine = 3-morpholino-sydnonimine hydrochloride. Stimulation of the macrophages with lipopolysaccharide and interferon-gamma resulted in dose-dependent cell injury and NO production. Glutathione depletion prior to stimulation considerably decreased macrophage viability as well as the NO production. In contrast to the protective effect on S-nitroso-N-acetylpenicillamine-mediated injury, pretreatment with N-acetylcysteine did not influence the lipopolysaccharide-mediated cytotoxicity. These results demonstrate that (a) reduction in the availability of glucose and intracellular glutathione renders the cells more vulnerable to the cytotoxic effects of NO donors, (b) in this model of cytotoxicity, long-lived NO donors were more cytotoxic than short-lived NO donors, (c) the differential effects of N-acetylcysteine on S-nitroso-N-acetylpenicillamine-induced and bacterial lipopolysaccharide-mediated cytotoxicity support the existence of other toxic species different from NO or NO-related compounds with a potent cytotoxic activity in immunostimulated macrophages, and (d) other non-protein thiols like N-acetylcysteine may substitute for glutathione as a major component of the cellular antioxidant defense system.

Effects of nitric oxide (NO) and NO donors on the membrane conductance of circular smooth muscle cells of the guinea-pig proximal colon.

1. The membrane conductance changes underlying the membrane hyperpolarizations induced by nitric oxide (NO), S-nitroso-L-cysteine (NC) and sodium nitroprusside (SNP) were investigated in the circular smooth muscle cells of the guinea-pig proximal colon, by use of standard intracellular microelectrode recording techniques. 2. NO (1%), NC (2.5-25 microM) and SNP (1-1000 microM) induced membrane hyperpolarization in a concentration-dependent manner, the hyperpolarizations to NO and NC developing more rapidly than those to SNP. The slower-developing responses to SNP were mimicked by the membrane permeable analogue of guanosine 3':5' cyclic-monophosphate (cyclic GMP), 8-bromo-cyclic GMP (500 microM), and by isoprenaline (10 microM). 3. The hyperpolarizations to NC and SNP were reduced in a low Ca2+ (0.25 mM) saline and upon the addition of haemoglobin (20 microM), but were not effected by NG-nitro-L-arginine (L-NOARG) (100 microM) or omega-conotoxin GVIA (100 nM). the hyperpolarizations to SNP were also significantly reduced by methylene blue (50 microM). 4. Apamin (250 nM) depolarized the membrane potential approximately 10 mV and reduced the initial transient component of the hyperpolarization to NO (1%) and NC (25 microM), but had no effects on the hyperpolarizations to SNP and cyclic GMP. Tetraethylammonium (TEA) (5-15 mM), had little effect on the membrane responses to NO(1%), NC(2.5-25 microM), SNP(100(-1000) microM) or cyclic GMP(500 microM). However, TEA (5-15 mM) reduced the membrane hyperpolarizations to SNP (10 microM) and isoprenaline (10 microM) in a concentration-dependent manner. The hyperpolarization to isoprenaline (10 microM) remaining in the presence of 15 mM TEA was blocked by ouabain (10 microM). 5. The amplitude of electronic potentials (1 s duration) elicited during NO donor hyperpolarizations were little changed or only slightly reduced (5-25%). However, the amplitude of the electrotonic potentials elicited during maintained electrically-induced hyperpolarizations of similar amplitude were significantly increased (30-150%), suggesting that the non-linear membrane properties of the proximal colon partially mask an increase in membrane conductance elicited during the NO donor hyperpolarizations. 6. Membrane hyperpolarization in the presence of an NO donor, 8-bromo-cyclic GMP, isoprenaline, or upon application of a maintained hyperpolarizing electrical current, often evoked oscillations of the membrane potential. These oscillations were prevented by Cs+ (1 mM). 7. These results indicate that NO and NC hyperpolarize the circular muscle of the proximal colon by activating at least two TEA-resistant membrane K+ conductances, one of which is sensitive to apamin blockade. The K+ conductance increases activated by SNP or 8-bromo-cyclic GMP were little effected by apamin, perhaps suggesting a common mechanism. In contrast, the hyperpolarization to isoprenaline appears to involve the activation of TEA-sensitive Ca2(+)-activated K+ ('BK') channels, as well as a Na:K ATPase. Finally, the 'background' membrane conductance of the circular muscle cells of the proximal colon decreased upon membrane hyperpolarization to reveal oscillations of the membrane potential which may well represent 'pacemaker' or 'slow wave' activity.

Inhibition of purified (Na+,K+)-ATPase activity from porcine cerebral cortex by NO generating drugs.

We tested the effects of several nitric oxide (NO) generating compounds on the activity of sodium-potassium adenosine 5'-triphosphatase [(Na+,K+)-ATPase] purified from porcine cerebral cortex. Sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine (SIN-1) and (d1)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR 3) inhibited the (Na+,K+)-ATPase activity dose dependently. Superoxide dismutase, a NO scavenger, and sulfhydryl (SH) compounds, reduced-form glutathione (rGSH) and dithiothreitol (DTT), prevented the inhibitory action of SNAP, SIN-1 and NOR 3 but not of SNP, when applied simultaneously with NO generating compounds, and this enzyme inhibition could be reactivated by the incubation with these SH compounds but not with SOD. The inhibitory action by SNP was magnified by simultaneous application of DTT. These results suggest that NO generating compounds, SNAP, SIN-1 and NOR 3 but not SNP, may release NO or NO-derived products and may inhibit (Na+,K+)-ATPase activity by interacting with a SH group at the active site of the enzyme.

Differential effects of hydroxocobalamin on relaxations induced by nitrosothiols in rat aorta and anococcygeus muscle.

In aortic rings, hydroxocobalamin (30 microM) reduced the relaxant actions of S-nitrosocysteine (0.1-3 microM), S-nitrosoglutathione (0.1-3 microM) and S-nitroso-N-acetylpenicillamine (SNAP, 0.01-3 microM), but did not affect the relaxant action of S-nitroso-coenzyme A (0.1-3 microM). In anococcygeus muscles, hydroxocobalamin (30 microM) had little effect on relaxations produced by nitrosocysteine (0.1-3 microM) and SNAP (0.01-1 microM), and enhanced those produced by nitrosoglutathione (0.1-3 microM) and nitroso-coenzyme A (0.1-3 microM). Since hydroxocobalamin is thought to act like haemoglobin by sequestering NO, some of the effects of hydroxocobalamin were compared with those of haemoglobin. Haemoglobin (10 microM) inhibited relaxations of aortic rings produced by nitrosocysteine and nitrosoglutathione and relaxations of anococcygeus muscles produced by nitrosocysteine, nitrosoglutathione and SNAP. Thus the effects of hydroxocobalamin on nitrosothiol-induced relaxations differ between the rat aorta and anococcygeus muscle, and depend on the exact nature of the nitrosothiol; however, the effects of haemoglobin did not differ qualitatively between the two tissues. Since hydroxocobalamin reduced relaxations of rat anococcygeus muscles elicited by NO, but not those elicited by nitrergic nerve stimulation or nitrosothiols, the nitrergic transmitter more closely resembles a nitrosothiol than free NO. Of those tested, the best correspondence was with nitrosocysteine; however, there were some differences between it and the transmitter.

Studies on the metabolism of D-penicillamine and its interaction with probenecid in cystinuria and rheumatoid arthritis.

Four patients with recurrent cystine stones and 5 with rheumatoid arthritis (RA) were studied. After a single dose of D-penicillamine to cystinuric patients, cystine excretion decreased considerably. Cysteine-penicillamine mixed disulfide (CSSP) and penicillamine disulfide (PSSP) metabolites appeared within 1-2 h (CSSP/PSSP approximately equal to 4.8-8.6). In RA, cystine excretion remained negligible (CSSP/PSSP approximately equal to 1.4-2.9). With daily D-penicillamine in RA (CSSP/PSSP ratios were usually greater than 7 in those with favorable clinical response. CSSP/PSSP ratios may help to predict prognosis and adjust penicillamine dosage. Coadministration of probenecid is contraindicated in hyperuricemic cystinuric patients because of increased cystine and decreased CSSP and PSSP excretion.

Reduction by copper supplementation of teratogenic effects of D-penicillamine.

Previous experiments from this laboratory demonstrated that oral administration of D-penicillamine (DPA) throughout gestation produced significant teratogenic effects and low copper concentrations in both maternal and fetal tissues in a dose-related manner. To test the hypothesis that the teratogenicity of DPA was due to induced copper deficiency, Sprague-Dawley rats were fed throughout gestation complete, purified diets containing 5 (control), 50, or 100 micrograms/g (supplemental) copper. One-half of the rats in each group received DPA at 0.83% of the diet. On day 21 of gestation, fetuses were removed and examined for gross malformations. Selected maternal and fetal tissues were analyzed for copper, zinc, iron and manganese. Animals fed the drug and supplemental copper had a very low incidence of fetal resorption (4%) and malformation (4%) compared to a high frequency of resorption (23%) and malformation (21%) in drug-fed nonsupplemented animals. There were no consistent differences in tissue iron, zinc or manganese levels among the groups. Tissue copper concentrations were lowest in the drug-fed, nonsupplemented group; those of the group fed DPA and supplemental copper were higher, but did not reach control values. Thus, there was a correlation between tissue copper levels and the frequency of malformations, supporting our hypothesis that DPA teratogenicity is due at least in part to induced copper deficiency.

Penicillamine nephropathy and iron.

7 of 16 patients with rheumatoid arthritis in whom penicillamine glomerulonephritis had developed had been taking oral iron, usually without the knowledge of their hospital clinician, while the dose of penicillamine was being gradually increased to an effective level. In 4 patients glomerulonephritis had appeared after the patients had stopped iron, with proteinuria developing with 2-5 months of discontinuation. Chelation of penicillamine by iron in the gut reduces its absorption, and in these 4 patients toxicity only became apparent after iron was stopped and there was a sudden increase in penicillamine absorption.

Effect of different doses of D-penicillamine and combined administration of D-penicillamine and methylprednisolone on collagen, glycosaminoglycans, DNA and RNA of granulation tissue, skin, bone and aorta in rats.

D-penicillamine (D-pen) in doses of 20, 100 and 500 mg/kg/day or D-pen 100 mg/kg/day plus methylprednisolone (MP) 2.0 mg/kg/day was administered daily for 42 days to rats implanted with viscose-cellulose sponges. Operated, pairfed rats served as controls. D-pen increased the DNA content of granulation tissue, but had no effect on the amount of tissue produced. In contrast, high dose D-pen reduced the content of DNA and collagen in skin. A dose related inhibition of collagen crosslink formation occurred in all tissues, particularly in skin, as indicated by increased proportions of extractable collagen with increased alpha/beta chain ratio and aldehyde content. Moreover, low doses of D-pen increased the hydroxyproline/proline ratio of acid soluble skin collagen, presumably due to solubilization of type III collagen as demonstrated by SDS-polyacrylamide gel electrophoresis in the presence of 3.6 M urea. These changes were associated with increased skin fragility and edema plus excess elastin deposition in the aorta after high dose D-pen treatment. Low dose D-pen stimulated the 35S-sulphate uptake into the sulphated glycosaminoglycans (GAGs) of granulation tissue without altering their relative amounts, whereas high dose D-pen reduced the concentration of chondroitin-4/6-sulphate in skin. MP antagonized the solubilizing effect of D-pen on collagen, probably by inhibition of the collagen synthesis. In addition, MP inhibited the cell proliferation and GAG metabolism. Food restriction reduced the DNA content of granulation tissue. The inhibitory effect of D-pen on the formation of granuloma collagen crosslinks in the presence of unaltered rate of collagen biosynthesis may diminish the amount of fibrotic tissue due to increased degradability of crosslink deficient collagen. Simultaneous administration of MP may facilitate this effect by inhibiting the biosynthesis of collagen. However, long-term D-pen treatment seems to increase the susceptibility of normal tissues to mechanical injury.

The inhibitory effect of D-penicillamine on human lymphocyte cultures stimulated by phytohaemagglutinin, the antagonistic action of L-cysteine and synergistic inhibition by copper sulphate.

The inhibitory effects of D-penicillamine (D-Pen) on lymphocyte activation by PHA are found to be dose-dependent, showing significant effects above a concentration of 50 microgram/ml. The cells show a diminished sensitivity to D-Pen added after 24 hours in culture, although the dose-response relationship is maintained. L-cysteine is usually found to have an insignificant effect on PHA stimulation, although enhancement and inhibition are also sometimes observed. The supplementation of culture medium with L-cysteine abolishes the inhibitory effect of D-Pen, whilst copper sulphate synergistically inhibits PHA-induced transformation. The balance between the availability of naturally occurring thiols, copper ions and administered D-Pen may be critical in determining the eventual effect of the drug on the lymphoid system. Lymphocyte suppressive soluble factors appear in the sera of patients with rheumatoid arthritis and are modified by treatment with D-Pen. Although our experiments suggest possible modes of action of the drug on the immune system as a therapeutic agent as well as a potent inducer of autoimmunity in its own right, further work is required to delineate its precise mechanism of action.

Effect of organic mercurials and sulfhydryl compounds on the urease activity of Proteus: inhibition by urine and ascorbic acid.

Meralluride, mercaptomerin, ethacrynic acid, and penicillamine inhibited urease activity of Proteus mirabilis. The activity of the organic mercurials and ethacrynic acid was markedly inhibited by human and dog urine. Antiurease activity could not be detected in the urine of a human and a dog given meralluride by injection. Urine from patients receiving penicillamine also failed to inhibit urease activity. Ascorbic acid inhibited, whereas dehydroascorbic acid enhanced, the activity of the mercurials, but neither agent altered the inhibitory effect of urine. The lethal effect of meralluride against Proteus occurred at the same concentration at which urease activity was inhibited, but penicillamine inhibited the enzymatic activity without affecting viability of the organism. The data suggest that these sulfhydryl-reactive compounds will not be useful against Proteus infections of the urinary tract.

17 alpha estradiol: separation of estrogen effect on collagen from other clinical and biochemical effects in man.

In rodents the effect of 17alpha estradiol upon collagen is identical to that of 17beta estradiol, but the 17alpha estradiol effect upon uterine lining is 1/1000 that of 17beta estradiol. Both steroids reverse the effect of D-penicillamine on rodent skin collagen. Five human beings with the skin collagen changes associated with D-penicillamine were treated with 17alpha estradiol for three to six weeks. 17alpha estradiol caused no detectable changes in blood pressure, breast development, menstrual periods, serum liver enzymes, serum proteins, plasma growth hormone, insulin, serum clotting factors, serum triglycerides, serum copper or serum ceruloplasma. In contrast, 17alpha estradiol increased skin prolyl hydroxylase activity, increased soluble collagen content in the skin and increased urinary hydroxyproline excretion. These studies with 17alpha estradiol, point out a specificity difference between the various sites of estrogen action in human beings.