NO synthase II in mouse skeletal muscle is associated with caveolin 3.

The inducible-type NO synthase (NOS II; iNOS) is constitutively expressed in slow-twitch skeletal muscle fibres of guinea-pigs [Gath, Closs, Gödtel-Armbrust, Schmitt, Nakane, Wessler and Förstermann (1996) FASEB J. 10, 1614-1620]. Here we studied the expression of NOS II in skeletal muscle of wild-type and NOS II-deficient mice and investigated the molecular basis for the membrane association of this NOS in muscle. A basal expression of NOS II mRNA and protein was detected in skeletal muscle from untreated wild-type mice; expression increased when mice were treated with bacterial lipopolysaccharide (LPS). No NOS II was found in any tissue of untreated or LPS-treated NOS II-deficient mice. Immunoprecipitation experiments were performed with homogenates of gastrocnemius muscle from untreated or LPS-treated wild-type mice. A NOS II-specific antibody precipitated caveolin 3 in all homogenates investigated, the effect being most pronounced in skeletal muscle from LPS-treated animals. Conversely, an antibody against caveolin 3 co-precipitated NOS II in muscle homogenates. Similarly, a weak co-precipitation of NOS II and caveolin 3 was seen in homogenates of untreated murine C2C12 myotubes; co-precipitation was markedly enhanced in cells stimulated with LPS/interferon gamma. The association of NOS II with caveolin 3 might have implications for the regulation of contraction of, and/or glucose uptake by, slow-twitch muscle fibres.

Analysis of NO synthase expression in neuronal, astroglial and fibroblast-like derivatives differentiating from PCC7-Mz1 embryonic carcinoma cells.

We studied the expression of the NO synthase isoforms in an in vitro model of neural development using RT-PCR, Western blot and immunohistochemistry. Murine PCC7-Mz1 cells (Jostock et al., Eur. J. Cell Biol. 76, 63-76, 1998) differentiate in the presence of all-trans retinoic acid and dibutyryl cAMP along the neural pathway into neuron-like, fibroblast-like and astroglia-like cells. Undifferentiated cells showed immunofluorescent staining for neuronal-type NOS I and endothelial-type NOS III. This expression pattern was retained in those cells differentiating into neurofilament- and tau protein-positive neuronal cells. Thymocyte alloantigen (Thy1.2/CD 90.2)-positive fibroblasts, appearing around day 3, and glial fibrillary acidic protein (GFAP)-positive astroglial cells, appearing after day 6 of differentiation, stained negative for any NOS isoform. Starting at day 6 of differentiation, expression of inducible-type NOS II could be stimulated with cytokines in a subset of cells, which may represent activated astrocytes. NOS II was always undetectable in non-induced cultures. These data indicate that the ability of stem cells to express NOS I and NOS III is only retained when the cells differentiate along the neuronal lineage, while a small subpopulation of cells acquires the ability to express NOS II in response to cytokines.

Increased expression of constitutive nitric oxide synthase III, but not inducible nitric oxide synthase II, in human heart failure.

OBJECTIVES: The purpose of the present study was to examine the expression of the endothelial-type nitric oxide synthase (NOS III) and the inducible-type NOS (NOS II) in human myocardium and their regulation in heart failure from patients with different etiologies. BACKGROUND: In heart failure, plasma levels of nitrates were found to be elevated. However, data on myocardial NOS expression in heart failure are conflicting. METHODS: Using RNase protection analysis and Western blotting, the expression of NOS III and NOS II was investigated in ventricular myocardium from nonfailing (NF) hearts (n=5) and from failing hearts of patients with idiopathic dilated cardiomyopathy (dCMP, n=14), ischemic cardiomyopathy (iCMP, n=9) or postmyocarditis cardiomyopathy (mCMP, n=7). Furthermore, immunohistochemical studies were performed to localize NOS III and NOS II within the ventricular myocardium. RESULTS: In failing human hearts, NOS III mRNA levels were increased to 180% in dCMP, 200% in iCMP and to 210% in mCMP as compared to NF hearts. Similarly, in Western blots (using constitutively expressed beta-tubulin as a reference) NOS III protein expression was increased about twofold in failing compared to NF hearts. Immunohistochemical studies with a selective antibody to NOS III showed no obvious differences in the staining of the endothelium of cardiac blood vessels from NF and failing human hearts. However, NOS III-immunoreactivity in cardiomyocytes was significantly more intense in failing compared to NF hearts. Low expression of NOS II mRNA was detected in only 2 of 30 failing human hearts and was not found in NF hearts. Inducible-type NOS protein was undetectable in either group. CONCLUSIONS: We conclude that the increased NOS III expression in the ventricular myocardium of failing human hearts may contribute to the contractile dysfunction observed in heart failure and/or may play a role in morphologic alterations such as hypertrophy and apoptosis of cardiomyocytes.

System response of the sinoatrial node during vagal stimulation.

The SA node response to modulations in canine vagal tone was investigated by means of the heart rate variability power spectrum. A new algorithm that was developed for accurate power spectrum estimation of short R-R segments is described. The performance of the algorithm was assessed for ECG recordings obtained from a controlled experiment, in which a frequency modulated pulse train was applied to the vagal nerve after vagal transaction and blockade of the sympathetic system. The power spectrum calculated for 20-25 heartbeats showed conspicuous spectral peaks in accordance with the different modulating frequencies between 0.1 and 0.3 Hz. The presence of spurious peaks was negligible even when the analysed signal segment consisted of only 20-25 beats. These results imply that for a certain range of modulating frequencies the sinoatrial node responds linearly to fluctuations in the parasympathetic tone. System identification methods that include fitting a linear model to the heart rate variability signal and analysis of the residuals were used for confirming the hypothesis of linearity. For higher frequencies of the modulating signal, usually above 0.3 Hz, the system was found to deviate from linearity.

Prediction of epileptic seizures from two-channel EEG.

Multivariate spectral estimation based on parametric modelling has been applied to epileptic surface EEG in order to detect EEG changes that occur prior to the clinical outbreak of the seizure. A better time/frequency resolution has been achieved using residual energy ratios (Dickinson's method). Prediction of oncoming seizures was based on detection of increased preictal synchronisation by calculation of coherence and pole trajectories. The method has been tested on simulated EEG data and on real EEG data from patients with primary generalised epilepsy. Prediction times of 1-6 s have been found in several seizures from five patients.

Expressional downregulation of neuronal-type NO synthase I in guinea pig skeletal muscle in response to bacterial lipopolysaccharide.

We have investigated the expression of neuronal-type NO synthase I (NOS I) and inducible-type NOS II in guinea pig skeletal muscle (diaphragm). Expression of NOS I mRNA and protein was highest in muscle of specific pathogen-free animals, lower in normally bred animals, and lowest in lipopolysaccharide (LPS)-treated animals. NOS II mRNA and protein levels were highest in muscle of LPS-treated animals. Elevated NOS activity in muscle from LPS-treated animals was less susceptible to the NOS I-selective inhibitor N(G)-nitro-L-arginine. Expressional downregulation of NOS I in sepsis may have implications for contractile function of skeletal muscle.

Characterization of nitric oxide synthase isoforms expressed in different structures of the guinea pig cochlea.

Nitric oxide synthase (NOS) activity and NADPH diaphorase staining has previously been reported in mammalian cochlea. Here we demonstrate immunoreactivity for neuronal-type NOS I and endothelial-type NOS III in the cochlea of the guinea pig. NOS I immunoreactivity was seen in inner and outer hair cells, and spiral ganglion cells. Staining for NOS I was also shown in basal and intermediate cells of the stria vascularis, spiral ligament cells, and the media of vessels near the modiolus. An antibody to NOS III stained primarily vascular endothelial cells. Some NOS III immunoreactivity was also detected in spiral ganglion cells. An antibody to the inducible-type NOS II did not stain any structure of the guinea pig cochlea, suggesting that this isoform is not expressed under physiological conditions. Nitric oxide produced by NOS I and/or NOS III may act as a neuromodulator in the organ of Corti and could also play a role as a regulator of cochlear blood flow.

Identification of the NO synthase isoforms expressed in human neutrophil granulocytes, megakaryocytes and platelets.

Using Western blot and fluorescent immunocytochemistry, NOS III (or ecNOS) and NOS II (or iNOS), but no NOS I (or ncNOS), were identified in preparations of human platelets. Reverse-transcription polymerase chain reactions (RT-PCR) demonstrated NOS III mRNA, but no NOS II mRNA (which is short-lived) and no NOS I mRNA in platelets. Immunofluorescent staining of human bone marrow smears showed the presence of NOS III, but not NOS I in megakaryocytes. A subpopulation of megakaryocytes also expressed NOS II. In preparations of human neutrophils, immunocytochemistry demonstrated NOS I in all cells, whereas no NOS III was detected. The few NOS II positive cells were characterized as contaminating eosinophils. Similarly, in RT-PCR, transcripts for NOS I and NOS II, but not for NOS III, were identified. Thus, the constitutive NOS isoform in megakaryocytes and platelets is NOS III, whereas neutrophils express NOS I. Some megakaryocytes and eosinophils also express NOS II.

Activation of soluble guanylyl cyclase by YC-1 in aortic smooth muscle but not in ventricular myocardium from rat.

1. The effects of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole), an activator of soluble guanylyl cyclase, on tension, levels of cyclic GMP and cyclic AMP, and cardiac L-type Ca2+-current (I[Ca(L)]) were investigated in aortic smooth muscle and ventricular heart muscle from rat. 2. YC-1 (0.1-30 microM) induced a concentration-dependent relaxation in aortic rings precontracted with phenylephrine (3 microM). The relaxant effects of YC-1 were reversed by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (30 microM; ODQ), potentiated by zaprinast (10 microM) and antagonized by Rp-8-Br-cGMPS (100 microM). 3. In ventricular heart muscle strips, YC-1 (30 microM) exhibited no effects on force of contraction (Fc) in the absence or presence of either zaprinast (10 microM) or 3-isobutyl-1-methylxanthine (30 microM). Fc was slightly increased by YC-1 (30 microM) in the presence of isoprenaline (100 nM), but this effect was not influenced by ODQ (30 microM). 4. Cardiac I[Ca(L)] was not significantly affected by YC-1 (30 microM), either in the absence or presence of isoprenaline (30 nM). 5. In aortic rings, cyclic GMP levels were increased almost 3 fold by YC-1 (30 microM); this effect was abolished by ODQ (30 microM). In isolated ventricular cardiomyocytes, cyclic GMP levels were not affected by YC-1 (30 microM) but almost doubled by activation of particular guanylyl cyclase with atriopeptin II (100 nM). 6. YC-1 (30 microM) did not increase cyclic AMP levels either in aortic rings or in ventricular cardiomyocytes. In contrast, isoprenaline (3 microM) increased cyclic AMP levels about two fold in both tissues. In cardiomyocytes, the effect of isoprenaline (3 microM) was slightly enhanced by YC-1 (30 microM). 7. It is concluded that relaxation of smooth muscle preparations by YC-1 is mediated mainly by activation of soluble guanylyl cyclase and subsequent increase in cyclic GMP levels. The failure of YC-1 to affect cardiac Fc, levels of cyclic GMP, and I[Ca(L)] suggests that soluble guanylyl cyclase is not influenced by YC-1 in rat heart muscle or only barely present in this tissue.

Inducible NO synthase II and neuronal NO synthase I are constitutively expressed in different structures of guinea pig skeletal muscle: implications for contractile function.

The expression of NOS isoforms was studied in guinea pig skeletal muscle at the mRNA and protein level, and the effect of NO on contractile response was examined. Ribonuclease protection analyses demonstrated NOS I and NOS II mRNAs in diaphragm and gastrocnemius muscle. In Western blots, NOS I and NOS II immunoreactivities were found in the particulate but not the soluble fraction of skeletal muscle. NOS activity was found almost exclusively in the particulate fraction. About 50% of this activity was Ca2+ independent. In immunohistochemistry, the anti-NOS I antibody stained distinct membrane regions of muscle fibers. The most intense staining was seen in neuromuscular endplates identified by labeling with alpha-bungarotoxin. The anti-NOS II antibody labeled muscle fibers that contained alkali-labile myosin ATPase (type I fibers). NOS II was located to intracellular structures and was also seen in "specific pathogen-free" animals. Pretreatment of guinea pigs with bacterial lipopolysaccharide (LPS) markedly intensified NOS II staining. Significant NOS III immunoreactivity was detected only in vascular endothelium. In functional experiments, tetanic muscle contractions were induced in diaphragm and gastrocnemius muscle by electrical stimulation of the innervating nerves. Pretreatment of guinea pigs with LPS or addition of S-nitroso-N-acetyl-D,L-penicillamine to the organ bath markedly decreased tetanic contractions. N(G)-nitro-L-arginine, on the other hand, increased contractile force and reversed the effect of LPS. Our data indicate that NOS II and NOS I are expressed in different structures of skeletal muscle and are involved in the regulation of contractile response.

Conjugation of anti-dihydrodiol epoxides of benzo[a]pyrene, chrysene, benzo[c]phenanthrene and dibenz[a,h]anthracene with glutathione catalyzed by cytosol and by the Mu-class glutathione transferase HTP II from rat liver.

The (+/-)-anti-dihydrodiol epoxides (DE) of benzo[a]pyrene (BP), chrysene (Chr), benzo[c]phenanthrene (BcPh) and dibenz[a,h]anthracene (DBA) were incubated in the presence of glutathione (GSH) with hepatic cytosol from untreated and Aroclor 1254 pretreated rats and with the Mu-class glutathione transferase (GST) HTP II from rat liver. The diastereoisomeric GSH conjugates formed were separated, identified and quantified by HPLC employing synthetic reference compounds. All (+/-)-anti-dihydrodiol epoxides investigated in this study were proven to be substrates of the cytosolic GSTs. The highly mutagenic and carcinogenic (+)-anti-DE with R,S,S,R absolute configuration was preferentially conjugated in the case of BP and Chr. Aroclor 1254 pretreatment increased the turnover 2-3-fold and changed the enantioselectivity. The previously purified GST HTP II exhibited a high degree of enantioselectivity (> or = 95%) towards the R,S,S,R-configurated enantiomer in the case of the bay-region (+/-)-anti-BPDE, (+/-)-anti-ChrDE and (+/-)-anti-DBADE, whereas in the case of fjord-region (+/-)-anti-BcPhDE both enantiomers were good substrates. The contribution of HTP II to the enzymatic activity of the cytosolic GST pool was estimated to be in the range of 11-32%. In agreement with previous results, the observed enantioselectivity of the purified enzyme seems to be of minor significance considering the total GST pool in the liver.

On time delay estimation of epileptic EEG.

The present study deals with comparative evaluation of various methods for time delay estimations applied to multichannel seizure EEG. The different methods included block algorithms, both in time and frequency domains (such as General Crosscorrelation, FFT, AR), and a new method for time delay estimation based on adaptive least-squares filtering. The various time delay estimators were tested on simulated signals and on real multichannel EEG recorded from rats having generalized seizures with focal onset. The adaptive least-squares filtering (the lattice-ladder type) has been found as the most efficient for time delay estimation.

Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions.

Three isozymes of nitric oxide (NO) synthase (EC 1.14.13.39) have been identified and the cDNAs for these enzymes isolated. In humans, isozymes I (in neuronal and epithelial cells), II (in cytokine-induced cells), and III (in endothelial cells) are encoded for by three different genes located on chromosomes 12, 17, and 7, respectively. The deduced amino acid sequences of the human isozymes show less than 59% identity. Across species, amino acid sequences for each isoform are well conserved (> 90% for isoforms I and III, > 80% for isoform II). All isoforms use L-arginine and molecular oxygen as substrates and require the cofactors NADPH, 6(R)-5,6,7,8-tetrahydrobiopterin, flavin adenine dinucleotide, and flavin mononucleotide. They all bind calmodulin and contain heme. Isoform I is constitutively present in central and peripheral neuronal cells and certain epithelial cells. Its activity is regulated by Ca2+ and calmodulin. Its functions include long-term regulation of synaptic transmission in the central nervous system, central regulation of blood pressure, smooth muscle relaxation, and vasodilation via peripheral nitrergic nerves. It has also been implicated in neuronal death in cerebrovascular stroke. Expression of isoform II of NO synthase can be induced with lipopolysaccharide and cytokines in a multitude of different cells. Based on sequencing data there is no evidence for more than one inducible isozyme at this time. NO synthase II is not regulated by Ca2+; it produces large amounts of NO that has cytostatic effects on parasitic target cells by inhibiting iron-containing enzymes and causing DNA fragmentation. Induced NO synthase II is involved in the pathophysiology of autoimmune diseases and septic shock. Isoform III of NO synthase has been found mostly in endothelial cells. It is constitutively expressed, but expression can be enhanced, eg, by shear stress. Its activity is regulated by Ca2+ and calmodulin. NO from endothelial cells keeps blood vessels dilated, prevents the adhesion of platelets and white cells, and probably inhibits vascular smooth muscle proliferation.

Different enzyme kinetics during the glutathione conjugation of the four stereoisomers of the fjord-region diolepoxides of benzo[c]phenanthrene by the mu-class rat liver glutathione S-transferase HTP II.

The enzyme-catalysed conjugation of each of the four stereoisomers of trans-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]phenanthrene (B[c]PhDE) with glutathione (GSH) by HTP II, a novel isolated mu-class GSH transferase from the liver of untreated rat, was studied. All four stereoisomers were substrates for GSH transferase HTP II. The enzymatic reaction shows three different types of enzyme kinetics: substrate inhibition for (-)-anti-B[c]PhDE with (R,S,S,R)-absolute configuration, allosteric behavior using (+)-anti-B[c]PhDE with (S,R,R,S)-absolute configuration and Henri-Michaelis-Menten kinetics with both the (-)-syn- and (+)-syn-enantiomers, with (S,R,S,R)- and (R,S,R,S)-absolute configuration, respectively. When the concentration of these diolepoxides was varied (using 2 mM GSH), the apparent Vmax values were 1975 nmol/min x mg for (-)-anti-B[c]PhDE and about 60 nmol/min x mg for both (-)-syn- and (+)-syn-B[c]PhDE, with the corresponding Km values of 1.05 and 0.20 mM. The reaction of (+)-anti-B[c]PhDE determined by applying the Hill equation had an estimated Vmax value of 930 nmol/min x mg. On varying the concentration of GSH, linear Lineweaver-Burk plots were obtained. No competitive effect could be observed using a mixture of (-)-anti- and (+)-anti-enantiomers, indicating that their binding sites are different and independent. It was also shown, that the binding sites of (+)-anti- and both syn-enantiomers were different and independent of each other, while there was a small effect on the binding of the syn-enantiomers caused by (-)-anti-B[c]PhDE. All products of the reaction between GSH and the dihydrodiol epoxides of benzo[c]phenanthrene could be resolved by HPLC and were identified and quantitated using the corresponding synthetic GSH conjugates.

Effects of sodium butyrate on DNA content, glutathione S-transferase activities, cell morphology and growth characteristics of rat liver nonparenchymal epithelial cells in vitro.

The effects of sodium butyrate, which has been shown to act as a differentiation promoting agent in several different tumor cell lines, were studied in a rat liver nonparenchymal epithelial cell line. Exposure of these cells to 3.75 mM butyrate resulted in an inhibition of cell proliferation and, at the same time, an increase in cell diameter (2- to 6-fold) and size of the nuclei (approximately 2-fold) after 3 days in culture. Binucleated cells arose, comprising approximately 12% of the cells investigated, and the number of cells with an abnormal set of chromosomes was increased. Intercellular communication, measured by dye transfer of Lucifer Yellow, was unchanged. From the various xenobiotic metabolizing enzyme activities measured, only those of glutathione S-transferases were significantly altered (increases of 4- to 9-fold) by butyrate treatment. These increases were mainly due to the predominant rise in the pi class isoenzyme which is a well-known tumour marker in rat hepatocarcinogenesis. Thus, our results cannot be interpreted as being either due to promotion of differentiation or due to transformation. The state and type of cell under study has to be considered and investigations of further differentiation parameters are needed to obtain a deeper insight into the biological activity and the underlying mechanisms of cell state modifying agents like butyrate.

On the tracking of rapid dynamic changes in seizure EEG.

Estimation of autospectra and coherence and phase spectra of seizure EEG, using the FFT technique, will cause "smearing" of the rapid dynamic changes which occur during the seizure. This is inherent to FFT spectral estimation, due to the averaging process which is necessary in order to get consistent spectral estimates. A different approach suggested in the present study is to carry out multivariate autoregressive modeling of the multichannel seizure EEG, combined with adaptive segmentation. In order to obtain good estimates in cases of short record length, the vectorial AR modeling was based on residual energy ratios. The method has been tested on multichannel seizure EEG recordings from rats with focal epilepsy, caused by intracerebral administration of Kainic acid, and in depth EEG recordings in patients with temporal lobe epilepsy.