Effects of new antiarrhythmic agent SS-68 on excitation conduction, electrical activity in Purkinje fibers and pulmonary veins: Assessment of safety and side effects risk.

The compound SS-68 has been selected among numerous new derivatives of indole and demonstrated antiarrhythmic effects in animal models. The present study concerns several aspects of SS-68 safety and efficacy as a potential antiarrhythmic drug. The first estimation of atrioventricular conduction in mammalian heart under SS-68 has been carried out; effects of SS-68 in Purkinje fibers and myocardium of pulmonary veins have been investigated. The drug weakly affects cardiac atrioventricular conduction: only high concentrations of SS-68 (≥10 µmol/L) significantly decrease this parameter. Also, the drug weakly affects Purkinje fibers automaticity, but effectively alters action potential waveform in Purkinje fibers in a concentration-dependent manner. SS-68 (0.1-100 µmol/L) failed to induce any early or delayed afterdepolarizations in Purkinje fibers both in basal conditions and under provocation of proarrhythmic activity by norepinephrine (NE). Moreover, 10 µmol/L SS-68 suppressed NE-induced extra-beats and rapid firing in Purkinje fibers. In pulmonary veins only high concentrations of SS-68 significantly increased action potential duration, while lower concentrations (0.1-1 µmol/L) were ineffective. Also, 0.1-100 µmol/L SS-68 was unable to elicit arrhythmogenic alternations of action potential waveform in pulmonary veins. In conclusion, SS-68 has no proarrhythmic effects, such as afterdepolarizations or abnormal automaticity in used experimental models.

Effects of a new antiarrhythmic drug SS-68 on electrical activity in working atrial and ventricular myocardium of mouse and their ionic mechanisms.

SS-68 is a derivative of indole, which demonstrated strong antiarrhythmic effects not associated with significant QT prolongation in dog models of atrial fibrillation. Therefore, SS-68 was proposed as a new antiarrhythmic drug and the present study is the first describing its effects on action potentials (APs) configuration and elucidating the ionic mechanisms of these effects. Sharp microelectrodes were used to record APs in isolated preparations of mouse atrial and ventricular myocardium. In both types of myocardium 10(-6) M SS-68 produced reduction of AP duration, 3 × 10(-6) M failed to alter AP waveform and 10(-5) - 3 × 10(-5) M prolonged APs. Sensitivity of main ionic currents to SS-68 was determined using whole-cell patch clamp. Transient potassium current Ito was slightly inhibited by SS-68 with IC50 = 1.43 × 10(-4) M. IKur was more sensitive with IC50 = 1.84 × 10(-5) M. Background inward rectifier showed very low sensitivity to SS-68 - only 10(-4) M SS-68 caused significant reduction of IK1. ICaL was significantly inhibited by 10(-6)M - 3 × 10(-5) M SS-68. The IC50 value for the ICaL was 1.84 × 10(-6) M. Thus, main ionic currents of mouse cardiomyocytes are inhibited by SS-68 in the following order of potency: ICaL > IKur > Ito > IK1. While lower concentration of SS-68 shorten APs via suppression of ICaL, higher concentrations inhibit K(+)-currents leading to APs prolongation.

Experimental allergic neuritis induced by sensitization with galactocerebroside.

Thirteen of 31 rabbits immunized repeatedly with bovine brain galactocerebroside developed experimental allergic neuritis, manifested by flaccid paresis and hypesthesia of four limbs, 2 to 11 months after the initial inoculation. Electrophysiological studies revealed multifocal conduction block of peripheral nerves. Perivenular demyelinative lesions associated with phagocytic mononuclear cells occurred in spinal ganglia, roots, and less frequently in distal nerves.

Toxic polyneuropathy produced by methyl N-butyl ketone.

A polyneuropathy affecting a large number of workers was recently observed at a plant producing plastic-coated and color-printed fabrics. Epidemiological data suggested strongly that methyl N-butyl ketone (MBK) was responsible for the outbreak. This hypothesis is now supported by the development of a peripheral neuropathy in chickens, rats, and cats exposed to MBK at atmospheric concentrations of 200 to 600 parts per million, 24 hours per day, 7 days per week. Although the animals were exposed continuously and the affected workers were exposed intermittently, the averages of the total number of hours of exposure for development of the peripheral neutropathy in the animals and workers were remarkably close.

Azathioprine-induced Agranulocytosis and Severe Alopecia After Kidney Transplantation Associated With a NUDT15 Polymorphism: A Case Report.

BACKGROUND: Azathioprine (AZA) is the drug recommended for the continuation of immunosuppressive treatment after renal transplant in women during pregnancy. CASE REPORT: A 37-year-old Japanese female developed agranulocytosis and severe alopecia after initiation of AZA (50 mg), used as an alternative to mycophenolate mofetil (MMF, 1000 mg) therapy in anticipation of a planned pregnancy. Within 4 days of the initiation of AZA therapy, the patient developed a high fever, leucopenia, and cranial alopecia. Genetic testing revealed a homozygous polymorphism of NUDT15 (rs116855232, NM_018283.3:c.415C>T: p.Arg139Cys), which has previously been identified as a risk factor for AZA-related complications in patients with inflammatory bowel disease. CONCLUSION: Genetic screening for NUDT15 could contribute to the prevention of serious adverse reactions to AZA and provide the opportunity for personalized medicine. Identification of a safe alternative to MMF during pregnancy after a renal transplant is a problem to be resolved in the future.

Caspase-dependent apoptosis by ectopic expression of E2F-4.

E2F is a family of transcription factors which regulates cell cycle and apoptosis of mammalian cells. E2F-1-3 localize in the nucleus, and preferentially bind pRb, while E2F-4 and 5 have no nuclear localization signal and preferentially bind p107/p130. E2F-6 suppresses the transcriptional activity of other E2F proteins. DP-1 and 2 are heterodimeric partners of each E2F protein. Using tetracycline-responsive promoters, here we compared the effects of ectopic expression of E2F-1, DP-1 and E2F-4 on cell cycle progression and apoptosis in Chinese hamster cell lines. We found that E2F-4, as well as DP-1 and E2F-1, induced growth arrest and caspase-dependent apoptosis. E2F-4 did not have a marked effect on cell cycle progression, while E2F-1 induced DNA synthesis of resting cells and DP-1 arrested cells in G1. Ectopic expression of E2F-4 did not activate E2F-dependent transcription. Our results suggest that expression of E2F-4 at elevated levels induces growth arrest and apoptosis of mammalian cells through a mechanism distinct from E2F-1 and DP-1.

cDNA cloning, sequence analysis and tissue distribution of a precursor for vasoactive intestinal contractor (VIC).

A full-length cDNA encoding preprovasoactive intestinal contractor (PPVIC) has been cloned. From the deduced 160 amino acid PPVIC, the mature VIC is predicted to be produced via a 37 residue intermediate, big VIC. The PPVIC also contains a VIC-like peptide of 16 amino acids structurally related to to the amino-terminal residues of VIC and flanked by pairs of dibasic amino acids, putative processing sites. RNA blot hybridization with PPVIC cDNA confirmed the PPVIC gene to be expressed in the small and large intestinal tract in a tissue specific manner.

Sequence and neuronal expression of mouse endothelin-1 cDNA.

We have isolated and sequenced a cDNA that encodes mouse endothelin-1 (ET-1). The putative protein contains 202 amino acids corresponds to the prepro-form of ET-1. Twenty-one amino acids sequence of the putative mature ET-1 was identical with that of rat, porcine, bovine, and human. In situ hybridization histochemistry indicate that ET-1 mRNA was expressed in several hypothalamic nuclei including the suprachiasmatic nucleus (SCN) in rodent brain.

Intracellular Ca release in skinned smooth muscle.

The release of internal Ca from saponin-treated skinned smooth muscle of guinea pig taenia caecum was studied. The amount of Ca released was estimated by the area under the contraction curve during treatment with 25 mM caffeine in the presence of 0.1 mM EGTA. The magnitude of the caffeine response in skinned muscle, after loading with 10(-6) M Ca for 3 min, was similar to that in the depolarized muscle in the presence of EGTA before treatment with saponin. This suggests that Ca in the skinned muscle was in a physiological range after loading. The release of Ca from the storage site could be facilitated by Ca itself when the skinned muscle was exposed to Ca above 3 x 10(-6) M. An increase in environmental MG concentration suppressed the Ca-induced Ca release mechanism. Sudden replacement of propionate with Cl in the bathing solution made it possible to release Ca from the storage site. This "depolarization"-induced Ca release occurred only immediately after the application of Cl; thereafter, the Ca release mechanism seemed to be inactivated by the prolonged presence of Cl. These results suggest that two mechanisms of Ca release operate in smooth muscle: (a) release induced by Ca itself, and (b) release by "depolarization".

Characteristics of Ca2+- and Mg2+-induced tension development in chemically skinned smooth muscle fibers.

Chemically skinned fibers from guinea pig taenia caecum were prepared by saponin treatment to study the smooth muscle contractile system in a state as close to the living state as posible. The skinned fibers showed tension development with an increase of Ca2+ in the solution, the threshold tension occurring as 5 X 10(-7) M Ca2+. The maximal tension induced with 10(-4) M Ca2+ was as large and rapid as the potassium-induced contracture in the intact fibers. The slope of the pCa tension curve was less steep than that of skeletal muscle fibers and shifted in the direction of lower pCa with an increase of MgATP. The presence of greater than 1 mM Mg2+ was required for Ca2+-induced contraction in the skinned fibers as well as for the activation of ATPase and superprecipitation in smooth muscle myosin B. Mg2+ above 2 mM caused a slow tension development by itself in the absence of Ca2+. Such a Mg2+-induced tension showed a linear relation to concentrations up to 8 mM in the presence of MgATP. Increase of MgATP concentration revealed a monophasic response without inhibition of Ca2+-induced tension development, unlike the biphasic response in striated muscle. When MgATP was removed from the relaxing solution, the tension developed slowly and slightly, even though the Mg2+ concentrations was fixed at 2 mM. These results suggest a substantial difference in the mode of actin-myosin interaction between smooth and skeletal muscle.

Cyclic AMP modulation of adrenoreceptor-mediated arterial smooth muscle contraction.

We examined the effects of cyclic AMP (cAMP) on the intracellular Ca2+ release in both the intact and skinned arterial smooth muscle. The amount of Ca2+ in the sarcoplasmic reticulum (SR) was estimated indirectly by caffeine-induced contraction of the skinned preparation and directly by caffeine-stimulated 45Ca efflux from the previously labeled skinned preparation. The norepinephrine-induced release contraction was markedly enhanced by dibutyryl cAMP (dbcAMP) and reduced by propranolol. The stimulatory effect of dbcAMP was best observed when the muscle was exposed to 10(-5) M dbcAMP and 2 X 10(-6) M norepinephrine was used to induce the release contraction. 10(-5) M cAMP had no effect on the Ca2+-induced contraction or on the pCa-tension relationship in the skinned preparation. This concentration of cAMP increased Ca2+ uptake into the SR of the skinned preparation when the Ca2+ in the SR was first depleted. 10(-5) M cAMP stimulated Ca2+-induced Ca2+ release from the SR after optimal Ca2+ accumulation by the SR. The results indicate that the stimulatory effect of cAMP on the norepinephrine-induced release contraction could be due to enhancement of the Ca2+-induced Ca2+ release from the SR in arterial smooth muscle.

Peripheral nerve changes induced by methyl n-butyl ketone and potentiation by methyl ethyl ketone.

A study of the sequential morphological changes in the peripheral nerve induced by experimental inhalation exposure of methyl n-butyl ketone (MBK) revealed that the earliest change was an increase in the number of neurofilaments in the large myelinated nerve fibers. This change occurred prior to axonal swelling or myelin thinning. As the duration of exposure lengthened the number of neurofilaments gradually increased and ultimately produced axonal swelling with secondary thinning of the myelin sheath. This appears to be the pathogenesis of the "giant axonal" neuropathy. Another change observed early in this neuropathy was the presence of inpouchings of the myelin sheath, which also increased in number in parallel to the duration of exposure. A careful study of the sequential changes in the entire motor unit did not show a predilection for early morphological changes at the axon terminal. Abnormalities at the neuromuscular junction occurred only after a full spectrum of changes were seen in the main nerve trunk, nerve roots and intramuscular nerves. An important observation was the marked potentiation of peripheral neurotoxicity observed when animals were exposed to MBK in combination with methyl ethyl ketone (MEK) at a ratio of 1:5, MBK:MEK. The latter solvent showed no neurotoxic effect alone. This might help explain a recent outbreak of a polyneuropathy affecting many workers. One further observation was that the sural nerve of a patient with prolonged exposure to MBK showed changes similar to those induced experimentally.

Peripheral nerve demyelination induced by intraneural injection of experimental allergic encephalomyelitis serum.

Intraneural injection of sera from rabbits with experimental allergic encephalomyelitis, induced by sensitization with bovine brain white matter in complete Freund's adjuvant, produced focal primary demyelinative lesions in rat sciatic nerves. Demyelinating activity was removed by prior incubation of antisera with central (CNS) and peripheral nervous system (PNS) myelin but not with liver or kidney, and was heat-labile and complement-dependent. Recipient animals developed a sensorimotor disturbance of their toes and ankles on the side injected with antiserum. Twenty minutes after antiserum injection, Schwann cells showed focal cytoplasmic outpouching and their external mesaxons opened. Between 1 and 8 hours after injection vacuolation, splitting and vesiculation of myelin became increasingly prominent at Schmidt-Lanterman clefts and paranodal regions, with concomitant degenerative changes in Schwann cell cytoplasm. Polymorphonuclear cell infiltration and endoneurial edema were apparent at this time. Substantial demyelination occurred before the appearance of phagocytic cells. Between 8 hours and 3 days many nerve fibers were surrounded and attacked by invading macrophages. Axons became demyelinated progressively over several internodes by macrophage phagocytosis. Early signs of remyelination were observed by 5 days. These findings suggest that antibodies directed against antigens common to both CNS and PNS myelin can produce in vivo peripheral nerve demyelination.

Differential activities of two distinct endothelin family peptides on ileum and coronary artery.

A synthesized mouse vasoactive intestinal contractor peptide, which belongs to a novel member of the endothelin family, induced a prolonged contraction in mouse ileum as well as porcine coronary artery in vitro. Studies comparing the effects of vasoactive intestinal contractor and endothelin on different tissues revealed that the maximum ileum contraction of vasoactive intestinal contractor was much higher than that of endothelin in both guinea pig and mouse systems, but that the vasoconstriction activity of vasoactive intestinal contractor was weaker than that of endothelin in porcine artery. These results show that vasoactive intestinal contractor might be a novel gastrointestinal hormone.

Effects of vasoactive intestinal contractor (VIC) and endothelin on intracellular calcium level in neuroblastoma NG108-15 cells.

Effects on [Ca2+]i levels of endothelin-l (ET) and vasoactive intestinal contractor peptide (VIC), which is a novel member of the endothelin family, were examined in fura 2-loaded neuroblastoma NG108-15 cells. VIC was found to be a very effective stimulus for intracellular Ca2+ mobilization and to be more potent than ET. Intracellular calcium response to sequential addition of two stimulants exhibited the homologous desensitization of either ET or VIC, but no heterologous desensitization between ET and VIC. This indicates evidence suggesting that these two peptides act through distinct receptors.

Congenital myopathy with type II muscle fiber hypoplasia.

We examined a 14-month-old boy with severe muscular hypotonia and weakness. Loss of tendon reflexes were noted at age 2 months. Right-sided heart failure and dyspnea during sleep developed at age 4 months. Muscle biopsy revealed selective hypoplasia of type II fibers and normal type I fibers. Intercostal and diaphragmatic muscles showed similar changes at necropsy and probably accented the respiratory failure.

Failure to transfer multiple sclerosis into severe combined immunodeficiency mice by mononuclear cells from CSF of patients.

To confirm the reported transfer of multiple sclerosis (MS) by CSF cells, we injected CSF cells from six MS patients in the exacerbation stage into the cisterna magna of 18 severe combined immunodeficiency mice. No clinical neurologic abnormalities or light- or electron-microscopic pathologic changes were present in any transferred mice, and the reported results could not be reproduced.

Axonal lesions in acute experimental demyelination: a sequential teased nerve fiber study.

The relationship between axonal degeneration and primary demyelination was studied in isolated rat sciatic nerve fibers previously exposed to antiserum from rabbits with either experimental allergic neuritis or experimental allergic encephalomyelitis, or immunized with antigalactocerebroside antiserum. Continuous demyelination over one to eight or more internodes was seen in association with phagocytic cells or, later, with increased numbers of Schwann cells. Paranodal demyelination was prominent proximal ahd distal to the zone of continuous demyelination. Axonal degeneration affected 5 to 15% of myelinated fibers exposed to antiserum and was not related to the length of demyelination must proximal to the axonal changes. At times, there were seven or eight consecutive demyelinated internodes with no distal axonal degeneration; in contrast, one demyelinated internode was often associated with axonal degeneration beginning just distally. The inflammatory reaction could account for axonal degeneration in antiserum-mediated demyelination.

Familial neuropathy with dementia, retinitis pigmentosa, and dysautonomia.

We studied a 59-year-old woman with dementia, retinitis pigmentosa, sensorimotor neuropathy, and attacks of vomiting associated with blood pressure lability and loss of consciousness. Abnormalities included CT evidence of cerebral atrophy, low IQ, slow central and peripheral nerve conduction velocities, axonal degeneration in sural nerve biopsy, and elevated levels of catecholamines and slow waves in EEG during attacks. Her sister, two brothers, and daughter also had progressive muscle weakness, visual disturbance, and similar vomiting attacks. The hereditary nervous system disorder does not fit any previously described condition.

Non-Menkes-type copper deficiency with regression, lactic acidosis, and granulocytopenia.

A 2-year-old girl with granulocytopenia developed fever followed by truncal ataxia and progressive neurologic regression. CT demonstrated symmetric low-density areas in the cerebral white matter. Sural nerve biopsy revealed axonal degeneration. Blood lactate levels were high, and serum levels of copper and ceruloplasmin and urinary excretion of copper were low. Cultured skin fibroblasts showed normal copper uptake. Treatment with oral copper administration normalized serum copper and ceruloplasmin levels, blood lactate levels, and granulocyte count. However, copper levels in the CSF were still low, and the patient showed no clinical improvement. We speculated that copper transport across the intestinal wall and across the blood-brain barrier was impaired.