Pharmacogenetics in critical care: association between CYP3A5 rs776746 A/G genotype and acetaminophen response in sepsis and septic shock.

BACKGROUND: Pharmacogenetics could represent a further resource to understand the interindividual heterogeneity of response of the host to sepsis and to provide a personalized approach to the critical care patient. METHODS: Secondary analysis of data from the prospective observational study NCT02750163, in 50 adult septic and septic shock patients treated with Acetaminophen (ACT) for pyrexia. We investigated the presence of two polymorphisms, located respectively in the genes UGT1A1 and CYP3A5, that encode for proteins related to the hepatic metabolism of ACT. The main dependent variables explored were plasmatic concentration of ACT, body temperature and hepatic parameters. RESULTS: 8% of the patients carried CYP3A5 rs776746 A/G genotypes and showed significantly higher plasma levels of ACT than GG wild type patients, and than patients with UGT1A1 rs8330 C/G genotypes. CONCLUSIONS: Identifying specific genotypes of response to ACT may be helpful to guide a more personalized titration of therapy in sepsis and septic shock. CYP3A5 might be a good biomarker for ACT metabolism; however further studies are needed to confirm this result. TRIAL REGISTRATION: NCT02750163.

Role of the mitochondrial sodium/calcium exchanger in neuronal physiology and in the pathogenesis of neurological diseases.

In neurons, as in other excitable cells, mitochondria extrude Ca(2+) ions from their matrix in exchange with cytosolic Na(+) ions. This exchange is mediated by a specific transporter located in the inner mitochondrial membrane, the mitochondrial Na(+)/Ca(2+) exchanger (NCX(mito)). The stoichiometry of NCX(mito)-operated Na(+)/Ca(2+) exchange has been the subject of a long controversy, but evidence of an electrogenic 3 Na(+)/1 Ca(2+) exchange is increasing. Although the molecular identity of NCX(mito) is still undetermined, data obtained in our laboratory suggest that besides the long-sought and as yet unfound mitochondrial-specific NCX, the three isoforms of plasmamembrane NCX can contribute to NCX(mito) in neurons and astrocytes. NCX(mito) has a role in controlling neuronal Ca(2+) homeostasis and neuronal bioenergetics. Indeed, by cycling the Ca(2+) ions captured by mitochondria back to the cytosol, NCX(mito) determines a shoulder in neuronal [Ca(2+)](c) responses to neurotransmitters and depolarizing stimuli which may then outlast stimulus duration. This persistent NCX(mito)-dependent Ca(2+) release has a role in post-tetanic potentiation, a form of short-term synaptic plasticity. By controlling [Ca(2+)](m) NCX(mito) regulates the activity of the Ca(2+)-sensitive enzymes pyruvate-, alpha-ketoglutarate- and isocitrate-dehydrogenases and affects the activity of the respiratory chain. Convincing experimental evidence suggests that supraphysiological activation of NCX(mito) contributes to neuronal cell death in the ischemic brain and, in epileptic neurons coping with seizure-induced ion overload, reduces the ability to reestablish normal ionic homeostasis. These data suggest that NCX(mito) could represent an important target for the development of new neurological drugs.

Up-regulation and increased activity of KV3.4 channels and their accessory subunit MinK-related peptide 2 induced by amyloid peptide are involved in apoptotic neuronal death.

The aim of the present study was to investigate whether K(V)3.4 channel subunits are involved in neuronal death induced by neurotoxic beta-amyloid peptides (Abeta). In particular, to test this hypothesis, three main questions were addressed: 1) whether the Abeta peptide can up-regulate both the transcription/translation and activity of K(V)3.4 channel subunit and its accessory subunit, MinK-related peptide 2 (MIRP2); 2) whether the increase in K(V)3.4 expression and activity can be mediated by the nuclear factor-kappaB (NF-kappaB) family of transcriptional factors; and 3) whether the specific inhibition of K(V)3.4 channel subunit reverts the Abeta peptide-induced neurodegeneration in hippocampal neurons and nerve growth factor (NGF)-differentiated PC-12 cells. We found that Abeta(1-42) treatment induced an increase in K(V)3.4 and MIRP2 transcripts and proteins, detected by reverse transcription-polymerase chain reaction and Western blot analysis, respectively, in NGF-differentiated PC-12 cells and hippocampal neurons. Patch-clamp experiments performed in whole-cell configuration revealed that the Abeta peptide caused an increase in I(A) current amplitude carried by K(V)3.4 channel subunits, as revealed by their specific blockade with blood depressing substance-I (BDS-I) in both hippocampal neurons and NGF-differentiated PC-12 cells. The inhibition of NF-kappaB nuclear translocation with the cell membrane-permeable peptide SN-50 prevented the increase in K(V)3.4 protein and transcript expression. In addition, the SN-50 peptide was able to block Abeta(1-42)-induced increase in K(V)3.4 K(+) currents and to prevent cell death caused by Abeta(1-42) exposure. Finally, BDS-I produced a similar neuroprotective effect by inhibiting the increase in K(V)3.4 expression. As a whole, our data indicate that K(V)3.4 channels could be a novel target for Alzheimer's disease pharmacological therapy.

In vitro evaluation of bio-functional performances of Ghimas titanium implants.

Titanium is the most widely used material for dental implants. The natural formation, in presence of oxygen, of different oxide films (passivation films) is correlated to titanium implant biocompatibility, resistance to corrosion and is responsible for implant bacteriostatic action. Surface roughness is another surface property of Ti-implants that, affecting implant-to-bone contact, improves integration. In the present study data concerning composition, surface roughness and biocompatibility of Ghimas implants and mini-implants undergoing sandblasting with Calcium Magnesium Carbonate (CaMg(CO3)2) are reported. AFM, SEM/EDX, XRD analyses and morpho-functional tests (MTT and ALP) were performed. Cell actin cytoskeletal modification (fluorescence phalloidin staining) was also observed with confocal laser microscopy (CLSM). Data related to surface geometry and chemical properties, associated with evidence of high purity of all the tested materials (XRD and EDX), highlighted the elevated biocompatibility of tested implants and mini-implants. CLSM investigation confirmed osteoblast features of an active cell behavior able to fit cell to chemico-mechanical stimuli present at the bone/implant interface and suggests an effective implant/alveolar bone integration in vivo.

A novel mutation in KCNQ2 associated with BFNC, drug resistant epilepsy, and mental retardation.

BACKGROUND: Benign familial neonatal convulsion (BFNC) is a rare autosomal dominant disorder caused by mutations in two genes, KCNQ2 and KCNQ3, encoding for potassium channel subunits underlying the M-current. This current limits neuronal hyperexcitability by causing spike-frequency adaptation. METHODS: The authors describe a BFNC family with four affected members: two of them exhibit BFNC only while the other two, in addition to BFNC, present either with a severe epileptic encephalopathy or with focal seizures and mental retardation. RESULTS: All affected members of this family carry a novel missense mutation in the KCNQ2 gene (K526N), disrupting the tri-dimensional conformation of a C-terminal region of the channel subunit involved in accessory protein binding. When heterologously expressed in CHO cells, potassium channels containing mutant subunits in homomeric or heteromeric configuration with wild-type KCNQ2 and KCNQ3 subunits exhibit an altered voltage-dependence of activation, without changes in intracellular trafficking and plasma membrane expression. CONCLUSION: The KCNQ2 K526N mutation may affect M-channel function by disrupting the complex biochemical signaling involving KCNQ2 C-terminus. Genetic rather than acquired factors may be involved in the pathophysiology of the phenotypic variability of the neurologic symptoms associated with BFNC in the described family.

OPSI (overwhelming postsplenectomy infection) syndrome: a case report.

Splenectomized patients are likely to suffer from severe infections, such as sepsis and meningitis. This syndrome is called overwhelming postsplenectomy infection (OPSI) in Europe and America. We present an adult case of OPSI syndrome, which occurred as respiratory insufficiency, and thrombocytopenia. The course is rapid, the clinical symptoms are serious, and the prognosis is very poor. Clinical examination showed cyanosis, mandibular hypertonia, psychomotor anxiety and purpura. Laboratory findings were thrombocytopenia, leukocytosis, hypoglycemia and altered coagulation parameters. A chest X-ray showed right pulmonary aspecific thickening. The autopsy findings occurred as Waterhouse-Friderichsen syndrome.

Granulocytic sarcoma (chloroma) in HIV patient: a report.

Isolated chloromas (granulocytic sarcomas) are rare tumours. Chloromas are masses composed of immature granulocytic cells. Granulocytic sarcoma occurs primarily in patients with acute myelogenous leukaemia, but can also arise in patients with other myeloproliferative disorders. We present an adult case of chloroma in HIV patient, which occurred as sudden death. Skin examination of right thigh showed dyschromia. Longitudinal incision of muscle revealed a "dark green" infiltration. Pathology showed in muscle fragments a infiltrate of granulocytes. The histologic sections of the excised tumour confirmed the cytologic diagnosis of chloroma.

[Intraoperative cholangiography during laparoscopic cholecystectomy: selective or routine?]. / Colangiografia intraoperatoria in corso di colecistectomia laparoscopica: selettiva o di routine?

Since its presentation by Mirizzi in 1931, the role of intraoperative cholangiography (ICHO) has been controversial and has become an argument even more disputed with the introduction of laparoscopic cholecystectomy (VLC) in 1988. The Authors reviewed their experience to determine the most appropriate use of ICHO during VLC on the basis of a retrospective analysis of cases of selective ICHO. From December 1991 to January 2001, 597 patients, 552 elective procedure and 45 emergency procedure, were reviewed. Of 552 patients 62 presented with at least one diagnostic criterion for symptomatic gallstone disease and were treated by means of ERCP completed with endoscopic sphincterectomy (ES) when a stone of the common bile duct was found, while the remaining 490 patients underwent VLC; a total of 10 ICHO were performed, two of which in the ERCP group and 8 in the VLC group. The 45 patients treated in emergency underwent VLC; in 43 cases ICHO was performed. Of all patients, there were 2 cases of common bile duct injuries (0.33%) and in both cases ICHO was not performed. A cholangiogram added 27 min to the average duration of surgery. On the basis of both the literature and Authors' experience, it can be stated that the routine use of ICHO is not useful to reduce bile duct injuries, while it significantly increases the cost of the surgical procedure due to the increase of average operative time. The use of ICHO seems to be effective to demonstrate clinically unsuspected choledocholitiasis, although, at present, the real clinical advantage deriving from the detection of these stones is not clear. The Authors conclude that further prospective, randomized studies are necessary to assess the precise role of ICHO with regard to VLC.

A novel KCNQ2 K+ channel mutation in benign neonatal convulsions and centrotemporal spikes.

Patients with benign familial neonatal convulsions (BFNC) may develop various epilepsies or epilepsy-associated EEG traits. A heterozygous 1-base pair deletion (2043DeltaT) in the KCNQ2 gene encoding for K+ channel subunits was found in a patient with BFNC who showed centrotemporal spikes at age 3 years. Electrophysiologic studies showed that mutant K+ channel subunits failed to give rise to functional homomeric channels or exert dominant-negative effects when expressed with KCNQ2/KCNQ3 subunits.

Inhibition of depolarization-induced [3H]noradrenaline release from SH-SY5Y human neuroblastoma cells by some second-generation H(1) receptor antagonists through blockade of store-operated Ca(2+) channels (SOCs).

In the present study, the effect of the blockade of membrane calcium channels activated by intracellular Ca(2+) store depletion on basal and depolarization-induced [3H]norepinephrine ([3H]NE) release from SH-SY5Y human neuroblastoma cells was examined. The second-generation H(1) receptor blockers astemizole, terfenadine, and loratadine, as well as the first-generation compound hydroxyzine, inhibited [3H]NE release induced by high extracellular K(+) concentration ([K(+)](e)) depolarization in a concentration-dependent manner (the IC(50)s were 2.3, 1.7, 4.8, and 9.4 microM, respectively). In contrast, the more hydrophilic second-generation H(1) receptor blocker cetirizine was completely ineffective (0.1-30 microM). The inhibition of high [K(+)](e)-induced [3H]NE release by H(1) receptor blockers seems to be related to their ability to inhibit Ca(2+) channels activated by Ca(i)(2+) store depletion (SOCs). In fact, astemizole, terfenadine, loratadine, and hydroxyzine, but not cetirizine, displayed a dose-dependent inhibitory action on the increase in intracellular Ca(2+) concentrations ([Ca(2+)](i)) obtained with extracellular Ca(2+) reintroduction after Ca(i)(2+) store depletion with thapsigargin (1 microM), an inhibitor of the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) pump. The rank order of potency for SOC inhibition by these compounds closely correlated with their inhibitory properties on depolarization-induced [3H]NE release from SH-SY5Y human neuroblastoma cells. Nimodipine (1 microM) plus omega-conotoxin (100 nM) did not interfere with the present model for SOC activation. In addition, the inhibition of depolarization-induced [3H]NE release does not seem to be attributable to the blockade of the K(+) currents carried by the K(+) channels encoded by the human Ether-a-Gogo Related Gene (I(HERG)) by these antihistamines. In fact, whole-cell voltage-clamp experiments revealed that the IC(50) for astemizole-induced hERG blockade is about 300-fold lower than that for the inhibition of high K(+)-induced [3H]NE release. Furthermore, current-clamp experiments in SH-SY5Y cells showed that concentrations of astemizole (3 microM) which were effective in preventing depolarization-induced [3H]NE release were unable to interfere with the cell membrane potential under depolarizing conditions (100 mM [K(+)](e)), suggesting that hERG K(+) channels do not contribute to membrane potential control during exposure to elevated [K(+)](e). Collectively, the results of the present study suggest that, in SH-SY5Y human neuroblastoma cells, the inhibition of SOCs by some second-generation antihistamines can prevent depolarization-induced neurotransmitter release.

Advanced glycation end product levels in eye lenses, aorta, and tail tendon in transplanted diabetic inbred Lewis rats.

BACKGROUND: Pancreatic islet transplantation in diabetes, by restoring euglycemia, should in time correct the abnormal accumulation of advanced glycation end products (AGEs) over target tissues, thus delaying the development of late diabetic complications. METHODS: Homologous islet transplantation was performed in inbred Lewis rats 15 days (TA), 4 months (TB), and 8 months (TC) after streptozotocin diabetes. Group TA was studied for 12 months and groups TB and TC were studied for 4 months after transplantation. Normal (N) and diabetic (D) rats formed the control groups. Metabolic control in the transplant (T) groups was evaluated by oral glucose tolerance test. Blood glucose, glycated hemoglobin, and body weight were determined in all groups. AGE levels were determined by spectrofluorometry in eye lens proteins and by ELISA in aortic and tail tendon collagen. RESULTS: T groups showed normal oral glucose tolerance tests and metabolic parameters. The latter were altered in all D groups (P<0.005 to P<0.0001 versus N and T groups). AGEs were increased in the D groups (P<0.05 to P<0.001) versus the N groups. AGEs in the TA and TB groups were not different from those of the N groups but were significantly reduced (P<0.05 to P<0.001) when compared with those of the D groups. In the TC group, eye lens AGEs were significantly elevated (P<0.001) or significantly reduced (P<0.01) when compared with those of the N or D groups, respectively. Aortic collagen AGEs were elevated (P<0.01) by comparison with those of the N groups and not statistically different from those of the D groups. Tail tendon collagen AGE levels lay between those of the N and D groups, without reaching a statistical significance. CONCLUSIONS: These results indicate that primary and early secondary (groups TA and TB) but not late secondary (group TC) islet transplantations are capable of blocking or reducing an abnormal accumulation of AGEs, thus confirming the importance of preventive transplantation therapies.

Plasma proteins containing damaged L-isoaspartyl residues are increased in uremia: implications for mechanism.

BACKGROUND: Several alterations of protein structure and function have been reported in uremia. Impairment of a transmethylation-dependent protein repair mechanism possibly related to a derangement in homocysteine metabolism is also present in this condition, causing erythrocyte membrane protein damage. Homocysteine may affect proteins via the accumulation of its parent compound S-adenosylhomocysteine (AdoHcy), a powerful in vivo methyltransferase inhibitor. However, since plasma homocysteine is mostly protein bound, a direct influence on protein structures cannot be ruled out. We measured the levels of L-isoaspartyl residues in plasma proteins of uremic patients on hemodialysis. These damaged residues are markers of molecular age, which accumulate when transmethylation-dependent protein repair is inhibited and/or protein instability is increased. METHODS: L-isoaspartyl residues in plasma proteins were quantitated using human recombinant protein carboxyl methyl transferase (PCMT). Plasma concentrations of homocysteine metabolites were also measured under different experimental conditions in hemodialysis patients. RESULTS: The concentration of damaged plasma proteins was increased almost twofold compared to control (controls 147.83 +/- 17.75, uremics 282.80 +/- 26.40 pmol of incorporated methyl groups/mg protein, P < 0.003). The major protein involved comigrated with serum albumin. Although hyperhomocysteinemia caused a redistribution of thiols bound to plasma proteins, this mechanism did not significantly contribute to the increase in isoaspartyl residues. The S-adenosylmethionine (AdoMet)/AdoHcy concentration ratio, an indicator of the flux of methyl group transfer, was altered. This ratio was partially corrected by folate treatment (0.385 +/- 0.046 vs. 0.682 +/- 0.115, P < 0.01), but protein L-isoaspartate content was not. CONCLUSIONS: Plasma protein damage, as determined by protein L-isoaspartyl content, is increased in uremia. This alteration is to be ascribed to an increased protein structural instability, rather than the effect of hyperhomocysteinemia.

Homocysteine and transmethylations in uremia.

Homocysteine is regarded as a cardiovascular risk factor in both the general population and chronic renal failure patients. Among the mechanisms for homocysteine toxicity, its interference with transmethylation reactions, through its precursor/derivative S-adenosylhomocysteine, plays a multifarious role. In uremia, inhibition of S-adenosylmethionine methyl transfer reactions has been reported by independent investigators, using multiple approaches. This has several possible consequences, which can ultimately affect the patient's relative state of health.

Retention in the endoplasmic reticulum as a mechanism of dominant-negative current suppression in human long QT syndrome.

Mutations in the cardiac potassium channel HERG (KCNH2) cause chromosome 7-linked long QT syndrome (LQT2) characterized by a prolonged QT interval, recurrent syncope and sudden cardiac death. Most mutations in HERG exhibit "loss of function" phenotypes with defective channels either inserted into the plasma membrane or retained in the endoplasmic reticulum. "Loss of function" mutations reduce I(Kr), the cardiac delayed rectifier current encoded by HERG, due to haploinsufficiency or suppression of wild-type function by a dominant-negative mechanism. One explanation for dominant-negative current suppression is that mutant subunits render tetrameric channel complexes non-conducting on co-assembly. In the present paper we describe an alternative mechanism for this phenomenon. We show (1) that the dominant-negative HERG mutation A561V is retained in the endoplasmic reticulum and (2) that wild-type channels are tagged for retention in the ER by co-assembly with trafficking deficient A561V subunits. Thus, in HERG A561V dominant-negative suppression of wild-type function is the result of an acquired trafficking defect.

Inhibition of HERG1 K(+) channels by the novel second-generation antihistamine mizolastine.

1. Ventricular arrhythmias are rare but life-threatening side effects of therapy with the second-generation H(1) receptor antagonists terfenadine and astemizole. Blockade of the K(+) channels encoded by the Human Ether-à-go-go-Related Gene 1 (HERG1) K(+) channels, which is the molecular basis of the cardiac repolarizing current I(Kr), by prolonging cardiac repolarization, has been recognized as the mechanism underlying the cardiac toxicity of these compounds. 2. In the present study, the potential blocking ability of the novel second-generation H(1) receptor antagonist mizolastine of the HERG1 K(+) channels heterologously expressed in Xenopus oocytes and in HEK 293 cells or constitutively present in SH-SY5Y human neuroblastoma cells has been examined and compared to that of astemizole. 3. Mizolastine blocked HERG1 K(+) channels expressed in Xenopus oocytes with an estimated IC(50) of 3.4 microM. Mizolastine blockade was characterized by a fast dissociation rate when compared to that of astemizole; when fitted to a monoexponential function, the time constants for drug dissociation from the K(+) channel were 72.4+/-11.9 s for 3 microM mizolastine, and 1361+/-306 s for 1 microM astemizole. 4. In human embryonic kidney 293 cells (HEK 293 cells) stably transfected with HERG1 cDNA, extracellular application of mizolastine exerted a dose-related inhibitory action on I(HERG1), with an IC(50) of 350+/-76 nM. Furthermore, mizolastine dose-dependently inhibited HERG1 K(+) channels constitutively expressed in SH-SY5Y human neuroblastoma clonal cells. 5. The results of the present study suggest that the novel second-generation H(1) receptor antagonist mizolastine, in concentrations higher than those achieved in vivo during standard therapy, is able to block in some degree both constitutively and heterologously expressed HERG1 K(+) channels, and confirm the heterogeneity of molecules belonging to this therapeutical class with respect to their HERG1-inhibitory action.

Sentinel lymph node identification in breast cancer: feasibility study.

The aim of the study was to evaluate in our institute the technique of sentinel node (SN) identification and biopsy in the surgical treatment of early breast cancer. Between June 1998 and November 1999 54 patients (age range, 31-75 years) where studied. Inclusion criteria were age less than 75 years, indication for conservative surgery, absence of palpable axillary nodes, Karnofksy index >70. Lymphoscintigraphy was performed 16-18 hours prior to surgery, following injection of 0.1-0.2 mL of 99mTc-Nanocoll: the administered activity was 3-4 MBq in group A (44 pts) and 7-8 MBq in group B (10 pts). The colloids were administered by transdermal supralesional injection in 49 patients with palpable nodules and by intraparenchymal ultrasound-guided injection in five patients with non-palpable nodules. Planar projections were performed starting from the 5th until the 80th min (or 180th in the event of late migration). In 10 patients further projections were acquired 14-18 h following tracer administration. All nodes identified by gamma probe (MR 100 Pol.Hi.Tech) were histologically evaluated by immunohistochemistry and standard histology. Scintigraphic visualization of the SN was obtained in 49 patients: in 38 of these patients there was only one SN while in 11 patients there were two or three SNs. The delayed scan made in 10 patients did not show any further nodes. In all patients given US-guided perilesional injections migration was late (after at least 60 min). Our study confirms the validity of the scintigraphic procedure, its safety for patients and health care workers, and the feasibility of interdisciplinary collaboration.

Do glia have heart? Expression and functional role for ether-a-go-go currents in hippocampal astrocytes.

Potassium homeostasis plays an important role in the control of neuronal excitability, and diminished buffering of extracellular K results in neuronal Hyperexcitability and abnormal synchronization. Astrocytes are the cellular elements primarily involved in this process. Potassium uptake into astrocytes occurs, at least in part, through voltage-dependent channels, but the exact mechanisms involved are not fully understood. Although most glial recordings reveal expression of inward rectifier currents (K(IR)), it is not clear how spatial buffering consisting of accumulation and release of potassium may be mediated by exclusively inward potassium fluxes. We hypothesized that a combination of inward and outward rectifiers cooperate in the process of spatial buffering. Given the pharmacological properties of potassium homeostasis (sensitivity to Cs(+)), members of the ether-a-go-go (ERG) channel family widely expressed in the nervous system could underlie part of the process. We used electrophysiological recordings and pharmacological manipulations to demonstrate the expression of ERG-type currents in cultured and in situ hippocampal astrocytes. Specific ERG blockers (dofetilide and E 4031) inhibited hyperpolarization- and depolarization-activated glial currents, and ERG blockade impaired clearance of extracellular potassium with little direct effect on hippocampal neuron excitability. Immunocytochemical analysis revealed ERG protein mostly confined to astrocytes; ERG immunoreactivity was absent in presynaptic and postsynaptic elements, but pronounced in glia surrounding the synaptic cleft. Oligodendroglia did not reveal ERG immunoreactivity. Intense immunoreactivity was also found in perivascular astrocytic end feet at the blood-brain barrier. cDNA amplification showed that cortical astrocytes selectively express HERG1, but not HERG2-3 genes. This study provides insight into a possible physiological role of hippocampal ERG channels and links activation of ERG to control of potassium homeostasis.

Modulation of the K(+) channels encoded by the human ether-a-gogo-related gene-1 (hERG1) by nitric oxide.

The inhibition of nitric oxide synthase by N-nitro-L-arginine methyl ester (0.03-3 mM) dose-dependently reduced nitric oxide (NO(*)) levels and enhanced the outward currents carried by human ether-a-gogo-related gene-1 (hERG1) K(+) channels expressed in Xenopus laevis oocytes, whereas the increase in NO(*) levels achieved by exposure to L-arginine (0.03-10 mM) inhibited these currents. Furthermore, four NO(*) donors belonging to such different chemical classes as sodium nitroprusside (1-1000 microM), 3-morpholino-sydnonimine (100-1000 microM), (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate (NOC-18; 1-300 microM), and S-nitroso N-acetylpenicillamine (1-300 microM) dose-dependently inhibited hERG1 outward K(+) currents. By contrast, the NO(*) donor NOC-18 (0.3 mM) did not affect other cloned K(+) channels such as rat neuroblastoma-glioma K(+) channel 2, rat delayed rectifier K(+) channel 1, bovine ether-a-gogo gene, rat ether-a-gogo-related gene-2, and rat ether-a-gogo-related gene-3. The inhibitory effect of NO(*) donors on hERG1 K(+) channels was prevented by the NO(*) scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide and hemoglobin. The membrane permeable analog of cGMP, 8-bromo-cGMP (1 mM), failed to reproduce the inhibitory action of NO(*) donors on hERG1 outward currents; furthermore, the specific inhibitor of the NO(*)-dependent guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (50 microM), neither interfered with outward hERG1 K(+) currents nor prevented their inhibition by 0.3 mM NOC-18. Both L-arginine (10 mM) and NOC-18 (0.3 mM) counteracted the stimulatory effect on hERG1 outward currents induced by the radical oxygen species-generating system FeSO(4) (25 microM)/ascorbic acid (50 microM; Fe/Asc). Finally, L-arginine (10 mM) and NOC-18 (0.3 mM) inhibited both basal and Fe/Asc (0.1 mM/0.2 mM)-stimulated lipid peroxidation in X. laevis oocytes. Collectively, the present results suggest that NO(*), both endogenously produced and pharmacologically delivered, may exert in a cGMP-independent way an inhibitory effect on hERG1 outward K(+) currents via an interaction with radical oxygen species either generated under resting conditions or triggered by Fe/Asc.

Homocysteine, a new cardiovascular risk factor, is also a powerful uremic toxin.

Homocystinuria, an inherited disease in which plasma levels of homocysteine are high, was discovered in the sixties and it soon became clear that the affected patients had striking features of generalized atherosclerosis. The most common causes of death were arterial and venous thrombosis, stroke, or myocardial infarction. Observations in this human model of hyperhomocysteinemia led to studies in the general population whose findings suggest - though not conclusively- that homocysteine is a cardiovascular risk factor. The same is true for patients with chronic renal failure who almost always have moderate to severe high blood homocysteine levels. Homocysteine accumulates in relation to the concentration of its precursor, S-adenosylhomocysteine, a powerful competitive transmethylation inhibitor. Inhibition of a methyltransferase required to repair damaged proteins has actually been detected in uremic patients' red blood cells. However, in view of the multiple, widespread metabolic roles of S-adenosylmethionine-dependent methyltransferases, in many organs and tissues including the vascular endothelium, hypomethylation is currently interpreted as one of homocysteine's most important mechanisms of action. Various biological compounds, including small molecules and nucleic acids, as well as proteins, which are involved in the pathophysiology of thrombosis and atherosclerosis, are all potential targets of hypomethylation. Epidemiological studies and experimental models tend to confirm that homocysteine is both a cardiovascular risk factor and a uremic toxin, acting through different mechanisms.