In vivo monitoring of norepinephrine and hydroxyl free radical generation by ferrous iron in the myocardium with a microdialysis technique

1. We examined in vivo monitoring of norepinephrine and hydroxyl radical generation in rat myocardium with a microdialysis technique. For this purpose, we designed the microdialysis probe holding system which includes loose fixation of the tube and synchronization of the movement of the heart and the probe. 2. The hydroxyl free radical (.OH) reacts with salicylate and generates 2,3- and 2,5-dihydroxybenzoic acid (DHBA) which can be measured electrochemically in picomole quantity by high performance liquid chromatography (HPLC). 3. After probe implantation, norepinephrine concentration of dialysate decreased over the first 150 min and then reached an almost steady level. A positive linear correlation between the ferrous iron and .OH formation trapped as 2,3-DHBA (R2 = 0.960) and 2,5-DHBA (R2 = 0.982) was observed using the microdialysis technique. 4. The present results indicate that non-enzymatic oxidation in the extracellular fluid may play a key role in hydroxyl radical generation by ferrous iron.     

Oxidative inactivation of gastric peroxidase by site-specific generation of hydroxyl radical and its role in stress-induced gastric ulceration

We have shown earlier that restraint-cold stress-induced gastric ulceration in rats is caused by metal ion-dependent generation of hydroxyl radical (OH.) and oxidative inactivation of the gastric peroxidase (GPO), an important H2O2 scavenging enzyme. To study the mechanism of the oxidative damage of GPO, the purified enzyme was exposed to an OH. generating system containing Cu2+, ascorbate, and H2O2. Kinetic studies indicate that the enzyme is inactivated in a time-dependent process showing saturation with respect to Cu2+ concentration. The enzyme specifically requires Cu2+ and is not inactivated by the same concentration of Fe2+, Mn2+, or Zn2+. Sensitivity to catalase indicates the critical role of H2O2 in the inactivation. Inactivation is insensitive to superoxide dismutase, suggesting no role of superoxide. The rate of inactivation is not increased in D2O excluding the involvement of singlet oxygen in the process. However, OH. scavengers such as benzoate or mannitol cannot prevent inactivation. The results indicate a plausible generation of OH. within the enzyme molecule as the cause of inactivation. Fragmentation of peptide linkage or intramolecular crosslinking, gross change of tertiary structure, or change in intrinsic tryptophan fluorescence which occurs in "global" oxidation are not evident. Inactivation is dependent on pH and from a plot of K(obs) of inactivation against pH, the controlling role of an ionizable group of the enzyme having a pka of 7.8 could be suggested, deprotonation of which favors inactivation. Amino acid analysis shows a specific loss of two lysine residues in the inactivated enzyme. Competitive kinetic studies indicate that pyridoxal phosphate, a specific modifier of the lysine residue, prevents inactivation by competing with Cu2+ for binding at the GPO. A Cu2+ binding motif consisting at least of two lysine residues exists in GPO, which specifically binds Cu2+ and generates OH.. The radical oxidizes the lysine residues and perturbs the heme environment to cause inactivation. We suggest that oxidative damage of GPO is mediated by site-specific generation of OH. and not by the OH. generated in the bulk phase.     

A novel melatonin metabolite, cyclic 3-hydroxymelatonin: a biomarker of in vivo hydroxyl radical generation

In the current study, we characterized a urinary melatonin metabolite which could provide a safe and effective method to monitor generation of HO* in humans. Using mass spectrometry (MS), proton nuclear magnetic resonance (1H NMR), COSY 1H NMR analysis, and calculations on the relative thermodynamic stability, a novel melatonin metabolite was identified as cyclic 3-hydroxymelatonin (3-OHM). 3-OHM is the product of the reaction of melatonin with HO* which was generated in two different cell-free in vitro systems. Interestingly, this same metabolite, 3-OHM, was also identified in the urine of both rats and humans. A proposed reaction pathway suggests that 3-OHM is the footprint product that results when a melatonin molecule scavenges two HO*. When rats were challenged with ionizing radiation which results in HO* generation, urinary 3-OHM increased dramatically compared to that of controls. These results strongly indicate that the quantity of 3-OHM produced is associated with in vivo HO* generation. Since melatonin exists in virtually all animal species and has a wide intracellular distribution and 3-OHM is readily detected noninvasively in urine, we suggest that 3-OHM is a valuable biomarker that can be used to monitor in vivo HO* levels in humans and other species. The measurement of urinary 3-OHM as a biomarker of HO* generation could provide clinical benefits in the diagnosis and treatment of diseases.     

Copper-binding amyloid precursor protein undergoes a site-specific fragmentation in the reduction of hydrogen peroxide

The extracellular domain of transmembrane Abeta amyloid precursor protein (APP) has a Cu(II) reducing activity upon Cu(II) binding associated with the formation of a new disulfide bridge. The complete assignment of the disulfide bond revealed the involvement of cysteines 144 and 158 around copper-binding histidine residues. The vulnerability of APP-Cu(I) complexes to reactive oxygen species was elaborated as a site-specific and random fragmentation of APP in a time-dependent manner and at low concentrations of H2O2. Analysis of the specific reaction revealed the generation of C-terminal polypeptides, containing the Abeta domain. APP catalyzed the reduction of H2O2 and oxidation of Cu(I) to Cu(II) in a "peroxidative" reaction in vitro. The resulting bound copper-hydroxyl radical intermediate [APP-Cu(II)(.OH)] then likely participated in a Fenton type of reaction with radical formation as a prerequisite for protein degradation. Evidence from two observations suggests that the reaction takes place in two phases. Bathocuproine, a trapping agent for Cu(I), abolished the initial fragmentation, and chelation of Cu(II) by DTPA (diethylenetriaminepentaacetic acid) interrupted the reaction cascade induced by H2O2 at later stages. Consequently, the results suggest that a cytotoxic gain-of-function of APP-Cu(I) complexes might result in a perturbation of free radical homeostasis. What significance such a perturbation may have for the pathogenesis of Alzheimer's disease remains to be determined.     

Detection of a tryptophan radical as an intermediate species in the reaction of horseradish peroxidase mutant (Phe-221 --> Trp) and hydrogen peroxide

The crucial reaction intermediate in the reaction of peroxidase with hydrogen peroxide (H2O2), compound I, contains a porphyrin pi-cation radical in horseradish peroxidase (HRP), which catalyzes oxidation of small organic and inorganic compounds, whereas cytochrome c peroxidase (CcP) has a radical center on the tryptophan residue (Trp-191) and oxidizes the redox partner, cytochrome c. To investigate the roles of the amino acid residue near the heme active center in discriminating the function of the peroxidases in these two enzymes, we prepared a CcP-like HRP mutant, F221W (Phe-221 --> Trp). Although the rapid spectral scanning and stopped-flow experiments confirmed that the F221W mutant reacts with H2O2 to form the porphyrin pi-cation radical at the same rate as for the wild-type enzyme, the characteristic spectral features of the porphyrin pi-cation radical disappeared rapidly, and were converted to the compound II-type spectrum. The EPR spectrum of the resultant species produced by reduction of the porphyrin pi-cation radical, however, was quite different from that of compound II in HRP, showing typical signals from a Trp radical as found for CcP. The sequential radical formation from the porphyrin ring to the Trp residue implies that the proximal Trp is a key residue in the process of the radical transfer from the porphyrin ring, which differentiates the function of peroxidases.     

Elevated "hydroxyl radical" generation in vivo in an animal model of amyotrophic lateral sclerosis

Mutations in the enzyme copper/zinc superoxide dismutase-1 (SOD1) are associated with familial amyotrophic lateral sclerosis (FALS). The means by which the mutations cause FALS appears to be due to an adverse property of the mutant SOD1 protein that may involve increased generation of free radicals. We used in vivo microdialysis to measure the conversion of 4-hydroxybenzoic acid to 3,4-dihydroxybenzoic acid (3,4-DHBA) as a measure of "hydroxyl radical-like" production in transgenic amyotrophic lateral sclerosis (ALS) mice with the G93A mutation as well as littermate controls. The conversion of 4-hydroxybenzoic acid to 3,4-DHBA was significantly increased in the striatum of transgenic ALS mice at baseline but not in mice overexpressing wild-type human SOD1. Following administration of 3-nitropropionic acid 3,4-DHBA generation was significantly increased as compared with baseline, and the increase in the transgenic ALS mice was significantly greater than those in controls, whereas the increase in mice overexpressing wild-type human SOD1 was significantly attenuated. The present results provide in vivo evidence that expression of mutations in SOD1 can lead to increased generation of "hydroxyl radical-like" activity, which further implicates oxidative damage in the pathogenesis of ALS.     

Oxidant stress with hydrogen peroxide attenuates calcium paradox injury: role of protein kinase C and ATP-sensitive potassium channel

Available literature on the use of pharmacologic agents for the treatment of sleep-disordered breathing was reviewed by evidenced-based methodology. Evidence tables were created and studies were graded according to study design and the number of subjects included. Scores for each group of studies evaluating each pharmacologic agent were established so that the quality of research for different drugs could be compared. The use of various ventilatory stimulants, psychotropic drugs, and antihypertensive agents were reviewed. The most objective data are available on theophylline and opioid antagonist/nicotine groups. Although more controlled studies would be helpful, relatively clear-cut indications for the use of ventilatory stimulants exist for hypercapnic obesity-hypoventilation patients (medroxyprogesterone), myxedema (thyroid replacement), central apnea (acetazolamide), and periodic breathing in congestive heart failure (theophylline). Few randomized, well-controlled trials have been published that evaluate pharmacologic agents in the treatment of classic OSA. To date, no one agent stands out as being useful for OSA. Future research will need to characterize subjects so that various subsets of patients can be tried on one or on a combination of various pharmacologic agents.     

Augmentation of human neutrophil and alveolar macrophage LTB4 production by N-acetylcysteine: role of hydrogen peroxide

1. The actions of N-acetylcysteine (NAC) on hydrogen peroxide (H2O2) and leukotriene B4 (LTB4) production by human resting and stimulated peripheral blood neutrophils and alveolar macrophages were investigated. 2. At a concentration of 100 microM, NAC significantly (P < 0.01) suppressed the accumulation of H2O2 in the incubation medium of resting and opsonized zymosan (OZ; 0.5 mg ml[-1])- or N-formylmethionyl-leucyl-phenylalanine (fMLP; 1 microM)-stimulated neutrophils and of resting and OZ-stimulated macrophages. At concentrations of 10 microM and above, NAC augmented significantly the level of LTB4 in the supernatants of OZ- and fMLP-stimulated neutrophils (P < 0.01 and P < 0.05, respectively) and OZ-stimulated macrophages (P < 0.05 at 10 microM, P < 0.01 at 100 microM NAC). 3. NAC (100 microM) caused a significant (P < 0.01) reduction in the quantity of measurable H2O2 when incubated with exogenous H2O2 concentrations equivalent to those released from OZ-stimulated neutrophils and macrophages. At no concentration did NAC affect quantitites of measurable LTB4 when incubated with exogenous LTB4. 4. Superoxide dismutase (SOD), which catalyzes the conversion of superoxide anion to H2O2 had no significant effect on LTB4 production by human neutrophils. In contrast, catalase, which catalyzes the conversion of H2O2 to H2O and O2, caused a pronounced, statistically significant (P < 0.01) increase in the levels of LTB4 measured in the supernatants of OZ- and fMLP-stimulated neutrophils. 5. H2O2 (12.5 microM and 25 microM, concentrations equivalent to those measured in the supernatants of activated neutrophils and alveolar macrophages, respectively) caused a small (13%) decrease in the quantity of measurable LTB4 (P = 0.051 and P < 0.05 at 12.5 microM and 25 microM, respectively) that was inhibited by NAC (100 microM) but not by catalase (400 u ml[-1]). 6. In conclusion, the anti-oxidant drug, NAC, increases LTB4 production by human neutrophils and alveolar macrophages, probably through the elimination of cell-derived H2O2. LTB4 undergoes a H2O2-dependent oxidation that is inhibited by NAC but this is unlikely to account fully for the increased levels of LTB4, suggesting that NAC may increase LTB4 production by blocking the H2O2-dependent inhibition of a synthetic enzyme, such as 5-lipoxygenase.     

Transformation in vitro of a nontumorigenic rat urothelial cell line by hydrogen peroxide

Chronic infection/inflammation of the urinary tract is a significant risk factor for the development of bladder cancer. The present study examined the hypothesis that hydrogen peroxide (H202) and cytokines released during inflammation are involved in the enhancement of bladder carcinogenesis. Using growth in soft agar and tumorigenicity in athymic nude mice as indices of transformation, we examined the effect of H202 and cytokines on the enhancement of N-methyl-N-nitrosourea (MNU)-initiated transformation of MYP3 cells, an anchorage-dependent nontumorigenic rat bladder epithelial cell line. MYP3 cells pretreated with or without MNU were exposed to H202 (0.001 to 0.1 mM) daily for 1 week in monolayer culture and were then tested for growth in soft agar. A marked increase in colony numbers was observed in the cells that were MNU-initiated and exposed to H202 (P < 0.01). Furthermore, H202 exposure alone at 0.01 mM or 0.1 mM caused colony formation in soft agar. The transformants induced by MNU plus H202 or H202 alone formed high-grade transitional cell carcinomas when injected into nude mice. The growth of these transformants was stimulated by several cytokines (interleukin 1alpha, interleukin 6, and tumor necrosis factor-alpha) better than the parental cells both on a plastic surface and in soft agar. Our results indicate that H202 causes genetic change(s) to induce tumorigenic conversion in urothelial cells and that the transformants are stimulated to grow because of their selective response to several cytokines. We suggest that these mechanisms may be involved in the in vivo carcinogenesis associated with chronic urinary tract infection.     

Evidence of hydroxyl free radical generation by calcium overload in rat myocardium

Although calcium (Ca2+) is important in cardiac dysfunction and has also been reported as a source of oxidative toxicity, the connection between Ca2+ overload and oxygen free radicals in the myocardium is not clear. We have investigated whether Ca2+ overload generates hydroxyl free radicals in rat ventricle. HPLC with electrochemical detection was used to measure the levels of 2,3- and 2,5-dihydroxybenzoic acid (DHBA) formed when the hydroxyl free radical reacts with salicylate. Ringer's solution containing salicylic acid (0.5 nmol microL-1 min-1) was infused through a microdialysis probe in the region of the left anterior descending coronary artery of the rat ventricle. A positive linear correlation was obtained between Ca2+ and hydroxyl free radical formation trapped as 2,3-DHBA (r2 = 0.976) and 2,5-DHBA (r2 = 0.982) in the myocardial dialysate. The administration of ouabain (1 mg kg-1, i.v.), a Ca2+ elevator, into the femoral vein significantly increased the level of 2,3- and 2,5-DHBA. These results indicate that Ca2+ overload generates hydroxyl free radicals in rat heart.     

In vivo analysis of hydrogen peroxide and lipid radicals in the striatum of rats under long-term administration of a neuroleptic

It has been hypothesized that free radicals play a causative role in tardive dyskinesia, which is an inveterate movement disorder caused by chronic administration of neuroleptics. To verify this hypothesis, rats were reared while being regularly treated with water containing a neuroleptic, haloperidol (HPD), for 1 year (HPD group). The changes in the striatal hydrogen peroxide content of the rats in the HPD and control groups were measured by using a Pt-disk microelectrode while the animals were in a freely moving state following intraperitoneal administration of HPD (HPD challenge). We also performed electron spin resonance (ESR) detection of lipid radicals in the striatum before the HPD challenge. HPD challenge led to significant elevation of the intrastriatal hydrogen peroxide in all animals, but the elevation in the HPD group was smaller than that in the control group. However, in the HPD group, marked ESR signals of intrastriatal lipid radicals were observed. We think that these results support the hypothesis on the role of free radicals in tardive dyskinesia.     

Myelin basic protein is a zinc-binding protein in brain: possible role in myelin compaction

The zinc-binding proteins (ZnBPs) in porcine brain were characterized by the radioactive zinc-blot technique. Three ZnBPs of molecular weights about 53 kDa, 42 kDa, and 21 kDa were identified. The 53 kDa and 42 kDa ZnBPs were found in all subcellular fractions while the 21 kDa ZnBP was mainly associated with particulate fractions. This 21 kDa ZnBP was identified by internal protein sequence data as the myelin basic protein. Further characterization of its electrophoretic properties and cyanogen bromide cleavage pattern with the authentic protein confirmed its identity. The zinc binding properties of myelin basic protein are metal specific, concentration dependent and pH dependent. The zinc binding property is conferred by the histidine residues since modification of these residues by diethyl-pyrocarbonate would abolish this activity. Furthermore, zinc ion was found to potentiate myelin basic protein-induced phospholipid vesicle aggregation. It is likely that zinc plays an important role in myelin compaction by interacting with myelin basic protein.     

Rat pachytene spermatocytes down-regulate a polo-like kinase and up-regulate a thiol-specific antioxidant protein, whereas sertoli cells down-regulate a phosphodiesterase and up-regulate an oxidative stress protein after exposure to methoxyethanol and methoxyacetic acid

2-Methoxyethanol (ME) and its metabolite, methoxyacetic acid (MAA), produce testicular lesions characterized by pachytene spermatocyte degeneration. To understand the molecular basis of this action on meiotic prophase cells, mRNA differential display was used to identify gene expression changes in control and treated cells. When pachytene spermatocytes were cultured with 5 mM ME or 5 mM MAA for 24 h, two complementary DNAs (cDNAs), of 557 nucleotides (clone 5) and 388 nucleotides (clone 6), were up-regulated; and a cDNA of 648 nucleotides (clone 1) was down-regulated. The altered expression pattern shown by differential display was confirmed by Northern blotting. Sequence analyses indicate that clones 1 and 6 have 83% and 79% homology at the nucleotide level to a polo-like kinase and a thiol-specific antioxidant, respectively. Clone 5 shows no homology to any known gene in the database. Messenger RNAs (mRNAs) encoding the thiol-specific antioxidant and clone 5 are up-regulated within 30 min of the addition of MAA, whereas the polo-like kinase mRNA decreased to undetectable levels after 6 h. Changes in Sertoli cell gene expression were also detected when Sertoli cells were cultured with 5 mM ME or MAA for 24 h. Two cDNAs, of 367 nucleotides (clone 2) and 676 nucleotides (clone 3), were up-regulated; and a cDNA of 538 nucleotides (clone 4) was down-regulated. Homology searches revealed that clones 3 and 4 have 90 and 91% homology at the nucleotide level to an oxidative stress protein and a phosphodiesterase (PDE), respectively. Northern blotting confirmed the differential display expression pattern for the PDE and oxidative stress protein. mRNAs for the latter were induced within 30 min, and PDE mRNAs were down-regulated within one h, after the addition of MAA. To determine whether the changes in gene expression seen with cells in culture also occur in vivo, rats were given a single oral dose of 250 mg/kg ME or MAA. After 24 h, total testis RNAs from control and treated rats were purified and hybridized. The expression patterns seen in vivo for the differentially expressed cDNAs were identical to those seen in vitro. We conclude that, although pachytene spermatocytes seem to be selectively affected by ME and MAA, changes in gene expression are also detected in Sertoli cells, suggesting that the action(s) of ME or MAA on pachytene spermatocytes could be mediated through Sertoli cells.     

Biotinylation in vivo as a sensitive indicator of protein secretion and membrane protein insertion

Escherichia coli biotin ligase is a cytoplasmic protein which specifically biotinylates the biotin-accepting domains from a variety of organisms. This in vivo biotinylation can be used as a sensitive signal to study protein secretion and membrane protein insertion. When the biotin-accepting domain from the 1.3S subunit of Propionibacterium shermanii transcarboxylase (PSBT) is translationally fused to the periplasmic proteins alkaline phosphatase and maltose-binding protein, there is little or no biotinylation of PSBT in wild-type E. coli. Inhibition of SecA with sodium azide and mutations in SecB, SecD, and SecF, all of which slow down protein secretion, result in biotinylation of PSBT. When PSBT is fused to the E. coli inner membrane protein MalF, it acts as a topological marker: fusions to cytoplasmic domains of MalF are biotinylated, and fusions to periplasmic domains are generally not biotinylated. If SecA is inhibited by sodium azide or if the SecE in the cell is depleted, then the insertion of the MalF second periplasmic domain is slowed down enough that PSBT fusions in this part of the protein become biotinylated. Compared with other protein fusions that have been used to study protein translocation, PSBT fusions have the advantage that they can be used to study the rate of the insertion process.     

Glutathione peroxidase degrades intracellular hydrogen peroxide and thereby inhibits intracellular protein iodination in thyroid epithelium

Protein iodination in the thyroid is largely confined to the surface of the epithelium. Intracellular iodine binding is insignificant. We have tested our hypothesis that the key mechanism in the control of intracellular iodination is the control of the intracellular availability of H2O2. The sites of iodination were identified by locating bound radioiodine in electron microscopic autoradiographs, produced from porcine thyroid epithelium grown on filter in Transwell bicameral culture chambers. Autoradiographs obtained after standard incubations with 125I for 15 min to 3 h were all characterized by concentrations of autoradiographic grains along the external surface of the plasma membrane and very few grains over the cytoplasm. The presence of 10 microM H2O2 in the incubation medium resulted in a drastically changed labeling pattern now showing a dissemination of grains over the entire cytoplasm. Epithelia with elevated GSH peroxidase activity produced autoradiographs showing the same restriction of grains to the cell surface as controls; this pattern was the same in the absence and presence of H2O2 (up to 10 microM). Cultures with subnormal GSH peroxidase activity presented cytoplasmic labeling both in the absence and presence of H2O2. In conclusion, iodine binding in filter-cultured thyroid epithelium under normal conditions is an extracellular process located at the cell surface. When H2O2 is available intracellularly, iodination takes place in the cytoplasm, evidently catalyzed by intracellular thyroperoxidase. Normally, this iodination is prevented by cytosolic GSH peroxidase that effectively degrades H2O2 and thus controls intracellular iodination. The observations should be applicable to the thyroid in vivo.     

Possible role of glutathione in resistance to heavy metals and hydrogen peroxide in a radioresistant Chinese hamster V79 cell strain

To understand the cellular and biochemical nature of radioresistance in the strain M5 derived from Chinese hamster V79 cells, the sensitivity of the resistant cells towards CdCl2, Zn(Ac)2, and H2O2 by the colony forming ability has been tested. D0 values for these compounds in Chinese hamster V79 cells were 5.4 microM, 27.8 microM and 4.3 micrograms/ml respectively while for M5 cells these were 8.3 microM, 142.9 microM and 11.9 micrograms/ml respectively. The resistance to heavy metals as well as the oxidative damage could be reversed by the inhibition of glutathione synthesis by the drug buthionine sulfoximine (BSO). These set of data indicate that the cellular antioxidant glutathione plays an important role in the observed oxidant-resistant phenotype as well as heavy metal resistance in M5 cells.     

Hydrogen peroxide activation of multiple mitogen-activated protein kinases in an oligodendrocyte cell line: role of extracellular signal-regulated kinase in hydrogen peroxide-induced cell death

Oxidative stress is known to induce cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. In this study, we have examined the activation of mitogen-activated protein kinase (MAPK) cascades in relation to oxidant-induced cell death in an oligodendrocyte cell line, central glia-4 (CG4). Exposure of CG4 cells to hydrogen peroxide (H2O2) resulted in an increased tyrosine phosphorylation of several protein species, including the abundantly expressed platelet-derived growth factor (PDGF) receptor and the activation of the three MAPK subgroups, i.e., extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK). Dose-response studies showed differential sensitivities of PDGF receptor phosphorylation (>1 mM) and ERK/p38 MAPK (>0.5 mM) and JNK (>0.1 mM) activation by H2O2. The activation of ERK was inhibited by PD98059, a specific inhibitor of the upstream kinase, MAPK or ERK kinase (MEK). H2O2 also activated MAPK-activated protein kinase-2, and this activation was blocked by SB203580, a specific inhibitor of p38 MAPK. The oxidant-induced cell death was indicated by morphological changes, decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, and DNA fragmentation. These effects were suppressed dose-dependently by the MEK inhibitor PD98059. The results demonstrate that H2O2 induces the activation of multiple MAPKs in oligodendrocyte progenitors and that the activation of ERK is associated with oxidant-mediated cytotoxicity.     

The importance of sodium pyruvate in assessing damage produced by hydrogen peroxide

OBJECTIVES: The external striated urethral sphincter (rhabdosphincter) is a tubular muscle sleeve that extends from the prostato-membranous urethra and perineal membrane to the bladder neck. The male rhabdosphincter neuroanatomy remains unclear, and a better understanding of its innervation may provide insight into potential modifications of radical pelvic surgery to improve urinary continence. METHODS: Fresh cadaveric dissections of 12 male hemipelves were undertaken to investigate the neuroanatomy of the urinary rhabdosphincter. RESULTS: Neuroanatomic courses of the nerve supply to the rhabdosphincter revealed that, in the perineum, the perineal nerve (a terminal branch of the pudendal nerve) provided branches directly to the bulbospongiosus muscle and the urinary rhabdosphincter. In the pelvis, the course of the pelvic nerve was as follows: (1) arising from the inferior hypogastric plexus, it had a weblike course beneath the muscle fascia of the levator ani muscle; (2) traveling posterolateral to the rectum, it gave many branches that perforated into the lateral rectum; and (3) at the level of the prostatic apex, still beneath the levator ani muscle fascia (superior fascia), it sent multiple direct branches to the inferolateral aspect of urinary rhabdosphincter. The pudendal nerve traversed the pelvis in the pudendal canal, and, before leaving the pelvis to enter the perineum, it gave an intrapelvic branch that courses with the pelvic nerve to innervate the rhabdosphincter. CONCLUSIONS: Our understanding of the neuroanatomy of what may be the continence nerves has been improved by fresh cadaveric dissection. The rhabdosphincter receives nerve fibers from the pelvic nerve and dual innervation from an intrapelvic branch and a perineal branch of the pudendal nerve. Better understanding of these anatomic findings may have potential surgical significance with respect to improvement in postoperative urinary continence.