Inhibition of NOS II prevents cardiac dysfunction in myocardial infarction and congestive heart failure

Strong expression of the inducible form of nitric oxide synthase (NOS II) has been shown in the myocardium of patients with myocardial infarction (MI). We hypothesized that NOS II plays an important role in the development of MI and subsequent heart failure and that inhibition of NOS II may beneficially alter the course of the disease. Long-term administration (2 mo) of the selective NOS II inhibitor S-methylisothiourea (SMT) to rats with MI significantly improved cardiac function. A significant drop in mortality, lung water content, infarct size, and cardiomyocyte hypertrophy was also associated with the use of SMT. Plasma concentration of nitrite and nitrate was also reduced by SMT. Short-term administration of SMT (first 2 wk only) significantly reduced infarct size; however, it did not improve cardiac dysfunction measured 2 mo after MI. These findings demonstrate that induction of NOS II during MI exerts negative effects on cardiac function and structure. Long-term administration of a selective NOS II inhibitor may prove to be beneficial in the treatment of MI and congestive heart failure.     

Effects of sex differences on constitutive nitric oxide synthase expression and activity in response to pressure overload in rats

Clinical studies have documented sex differences in left ventricular (LV) hypertrophy patterns, but the mechanisms are so far poorly defined. This study aimed to determine whether 1) severe pressure overload altered expression and/or activity of cardiac constitutive nitric oxide synthase (NOS1 and NOS3) and 2) these changes were modulated according to sex. Analyses were performed 0.4-20 wk after thoracic aortic constriction (TAC) in male and female Wistar rats. Male rats with TAC exhibited early signs of cardiac dysfunction, as shown by echocardiographic and LV end-diastolic pressure measurements, whereas females with TAC exhibited higher LV hypertrophy (+96% vs. males at 20 wk; P < 0.05). After TAC, cardiac NOS1 expression was rapidly induced (0.4 wk) and stable afterward in males (P < 0.05 vs. sham groups), whereas it was delayed in females. Accordingly, specific NOS1 activity was increased by 2 wk in male rats with TAC (+122%; P < 0.001 vs. sham groups) and only by 20 wk in females (+220%; P < 0.001 vs. sham groups). NOS1 activity was correlated with NOS1 level. Regarding cardiac NOS3, expression was unaffected by TAC, and the decrease in activity observed at early and late times in male and female rats with TAC, respectively, is shown to be related to NOS3 allosteric regulator caveolin-1 level. The data demonstrated a unique sex-dependent regulation of the constitutive NOSs in response to TAC in rats; such a difference might play a role in the sex-dependent adaptability of the heart in response to pressure overload.     

Ruminal muscle of sheep is innervated by non-polarized pathways of cholinergic and nitrergic myenteric neurones

The motility patterns of the reticulorumen evoke mainly mixing of the ingesta. So far unknown, intrinsic neural circuits of the enteric nervous system are involved in the control of these motility patterns. The aim of the study was to characterize neurochemically sheep ruminal myenteric neurones, in particular the neural pathways innervating the ruminal muscle layers. Cell bodies within the myenteric plexus projecting to the longitudinal or circular muscle layer were retrogradely labelled by direct application of the fluorescent tracer 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) onto the circular or longitudinal muscle. The neurochemical code of myenteric neurones was identified by their immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). According to their neurochemical code, ruminal myenteric neurones were divided into three populations: ChAT/SP (68% of all myenteric neurones), NOS/VIP (26% of all myenteric neurones) and ChAT/- (5% of all myenteric neurones). Application of DiI onto the circular or longitudinal muscle revealed on average 64 or 44 labelled cell bodies in the myenteric plexus, respectively. DiI-labelled neurones expressed the code ChAT/SP or NOS/VIP. In the pathways to circular or longitudinal muscle, ChAT/SP-positive neurones outnumbered NOS/VIP-immunoreactive neurones by 5:1 and 2:1. Pathways to the circular or longitudinal muscle did not exhibit any pronounced polarized innervation patterns. This study demonstrated specific projections of myenteric neurones to the ruminal muscle. Neurones expressing the code ChAT/SP might function as excitatory muscle motor neurones, whereas NOS/VIP neurones are likely to act as inhibitory muscle motor neurones.     

Enkephalin-immunoreactive subpopulations in the myenteric plexus of the guinea-pig fundus project primarily to the muscle and not to the mucosa

Enkephalin (ENK) immunoreactivity was localised in different neuronal subpopulations of the myenteric plexus in the guinea-pig gastric fundus using immunohistochemistry for neurone-specific enolase (NSE), ENK, choline acetyltransferase (ChAT), substance P (SP), neuropeptide Y (NPY), calretinin (CALRET), and somatostatin (SOM). NADPH-diaphorase staining was used to label nitric oxide synthase (NOS)-containing neurones. ENK was observed in 44% of the myenteric neurones. The major ENK-positive subpopulations were ChAT/ENK (35% of ENK-positive neurones), ChAT/SP/ENK (26%), NOS/NPY/ENK (22%) and ChAT/SP/ENK/CALRET (9%). The projection pathways of these ENK-positive subpopulations to the circular muscle and the mucosa were determined using retrograde labelling with DiI in organ culture followed by immunohistochemistry. Of myenteric neurones retrogradely labelled from the mucosa and the circular muscle, 13% and 48% exhibited ENK immunoreactivity, respectively. Three major ENK-positive subpopulations innervating the mucosa or circular muscle were identified: ascending ChAT/SP/ENK (7% of all mucosa neurones; 24% of all circular muscle neurones), ascending ChAT/ENK (4%; 15%) and descending NOS/NPY/ENK (1%; 8%) neurones. Only very few CALRET- or SOM-positive neurones projected to the mucosa or circular muscle. ChAT/SP/ENK and ChAT/ENK neurones might function as ascending excitatory muscle motor neurones, whereas NOS/NPY/ENK neurones are most likely descending inhibitory muscle motor neurones. The relatively few ENK-positive mucosa neurones do not favour a major involvement of ENK-positive myenteric neurones in the control of gastric mucosa activity.     

Different subpopulations of cholinergic and nitrergic myenteric neurones project to mucosa and circular muscle of the guinea-pig gastric fundus

Since the stomach lacks a well-developed ganglionated submucous plexus, the somata of enteric neurones innervating the muscle or the mucosa have to be localised within the myenteric plexus. The aim of this study was to determine the projection pathways and the neurochemical coding of myenteric neurones innervating these different targets in the gastric fundus. Myenteric cell bodies projecting to the mucosa or the circular muscle were retrogradely labelled by mucosa or muscle application of the fluorescent tracer DiI and subsequently characterised by their immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and/or neuropeptide Y (NPY). On average 143±91 and 89±49 myenteric neurones were labelled from the mucosa and the circular muscle, respectively. DiI-labelled neurones were either ChAT- or NOS-positive. DiI-labelled ChAT-positive neurones were mainly ascending and outnumbered NOS-positive neurones, which were mainly descending (79.3±6.2% vs 20.7±6.2% for mucosa neurones; 69.3±11.1% vs 30.7±11.1% for muscle neurones). Three ChAT-positive subpopulations (ChAT/–, ChAT/SP, ChAT/NPY) and two NOS-positive subpopulations (NOS/–, NOS/NPY) were found. ChAT/SP neurones projected mainly to the circular muscle (36.1±11.9% of the cholinergic muscle neurones; mucosa projection: 8.0±2.1%), whereas ChAT/NPY neurones projected mainly to the mucosa (38.1±9.2% of the cholinergic mucosa neurones; muscle projection: 5.7±2.4%). NOS/– cells projected predominantly to the muscle. This study demonstrates polarised pathways in the myenteric plexus consisting of ascending ChAT and descending NOS cells that innervate the circular muscle and the mucosa of the gastric fundus. The ChAT/SP neurones might function as circular muscle motor neurones, whereas ChAT/NPY neurones might represent secretomotor neurones.     

Cardiomyocyte-restricted restoration of nitric oxide synthase 3 attenuates left ventricular remodeling after chronic pressure overload

Although nitric oxide synthase (NOS)3 is implicated as an important modulator of left ventricular (LV) remodeling, its role in the cardiac response to chronic pressure overload is controversial. We examined whether selective restoration of NOS3 to the hearts of NOS3-deficient mice would modulate the LV remodeling response to transverse aortic constriction (TAC). LV structure and function were compared at baseline and after TAC in NOS3-deficient (NOS3(-/-)) mice and NOS3(-/-) mice carrying a transgene directing NOS3 expression specifically in cardiomyocytes (NOS3(-/-TG) mice). At baseline, echocardiographic assessment of LV dimensions and function, invasive hemodynamic measurements, LV mass, and myocyte width did not differ between the two genotypes. Four weeks after TAC, echocardiographic and hemodynamic indexes of LV systolic function indicated that contractile performance was better preserved in NOS3(-/-TG) mice than in NOS3(-/-) mice. Echocardiographic LV wall thickness and cardiomyocyte width were greater in NOS3(-/-) mice than in NOS3(-/-TG) mice. TAC-induced cardiac fibrosis did not differ between these genotypes. TAC increased cardiac superoxide generation in NOS3(-/-TG) but not NOS3(-/-) mice. The ratio of NOS3 dimers to monomers did not differ before and after TAC in NOS3(-/-TG) mice. Restoration of NOS3 to the heart of NOS3-deficient mice attenuates LV hypertrophy and dysfunction after TAC, suggesting that NOS3 protects against the adverse LV remodeling induced by prolonged pressure overload.     

一氧化氮在血管紧张素Ⅱ诱导心肌细胞肥大中的作用

在培养新生大鼠心肌细胞上,探讨一氧化氮（ＮＯ）在血管紧张素Ⅱ诱导的心肌细胞以大中的作用。结果显示,血管紧张素Ⅱ可使心肌细胞蛋白质含量显著增加,心肌细胞一氧化氮合酶（ＮＯＳ）活性和培养液ＮＯ浓度明显降低。血管紧张素Ⅱ可明显降低心肌细胞ｅＮＯＳｍＲＮＡ水平。Ｓａｒａｌｓｉｎ和百日咳毒素（ＰＴＸ）可抑制血管紧张素Ⅱ诱导的蛋白质含量增加、心肌细胞ＮＯＳ活性减弱和培养液ＮＯ浓度降低。硝普钠提高心肌细胞培养     

Nitric oxide-compromised hypertension: facts and enigmas

NO concentration in the femoral artery and femoral vein of anesthetized dogs was found to be 154.2+/-5.6 nM and 90.0+/-12 nM, respectively. Inhibition of NO synthase (NOS) slightly decreased the basal NO concentration in femoral artery from 154.2+/-5.6 to 137.2+/-3.3 nM. Acetylcholine-induced increase in NO concentration was slightly but still significantly attenuated, suggesting that very probably L-NAME did not inhibit all sources of nitric oxide (NO). Local NOS inhibition in the posterior hypothalamus dose-dependently increased systemic blood pressure (BP) in rats. Short-term general NOS inhibition in anesthetized dogs increased diastolic BP but not systolic BP. The heart rate after one-hour down-fluctuation returned to initial values. Proteosynthesis in the myocardium and both branches of the left coronary artery increased, but this was not supported by polyamines, since the activity of ornithine decarboxylase declined. Long-term general NOS inhibition elicited a sustained BP increase, a decrease in heart rate, cardiac hypertrophy and an increase in wall thickness of the coronary and carotid artery. The results indicate that NO deficiency itself plays a role in proteosynthesis and cardiac hypertrophy, in spite of relatively small increase in diastolic blood pressure and no change in systolic blood pressure, at least after an acute L-NAME administration. The hypotension response to acetylcholine and bradykinin studied in anesthetized NO-compromised rats, was unexpectedly enhanced. The elucidation of this paradoxical phenomenon will require further experiments.     

Reduced neuronal nitric oxide synthase expression contributes to cardiac oxidative stress and nitroso-redox imbalance in ob/ob mice.

Disruption of leptin signaling in the heart may contribute to obesity-related cardiac disease, as leptin deficient (oblob) mice display cardiac hypertrophy, increased cardiac apoptosis and reduced survival. Since leptin maintains a tonic level of neuronal nitric oxide synthase (NOS1) expression in the brain, we hypothesized that leptin deficiency would decrease NOS1 cardiac expression, in turn activating xanthine oxidoreductase (XOR) and creating nitroso-redox imbalance. We studied 2- to 6-month-old oblob (n=26) and C57Bl/6 controls (n=27). Cardiac NOS1 protein abundance (P<0.01) and mRNA expression (P=0.03) were reduced in oblob (n=10 and 6, respectively), while NOS3 protein abundance and mRNA expression were unaltered. Importantly, cardiac NOS1 protein abundance was restored towards normal in oblob mice after leptin treatment (n=3; P<0.05 vs leptin untreated oblob mice). NO metabolite (nitrite and nitrate) production within the myocardium was also reduced in oblob mice (n=5; P=0.02). Furthermore, oxidative stress was increased in oblob mice as GSH/GSSG ratio was decreased (n=4; P=0.02). Whereas XOR activity measured by Amplex Red fluorescence was increased (n=8; P=0.04), XOR and NADPH oxidase subunits protein abundance were not changed in oblob mice (n=6). Leptin deficiency did not disrupt NOS1 subcellular localization, as NOS1 co-localized with ryanodine receptor but not with caveolin-3. In conclusion, leptin deficiency is linked to decreased cardiac expression of NOS1 and NO production, with a concomitant increase in XOR activity and oxidative stress, resulting in nitroso-redox imbalance. These data offer novel insights into potential mechanisms of myocardial dysfunction in obesity.     

Neuronal subpopulations in autonomic ganglia associated with the chicken ureter: an immunohistochemical study

The neurochemical coding of neurones located in ganglia of the nerve trunk accompanying the chicken ureter was analysed and quantified using NADPH-diaphorase reactivity and immunohistochemistry against tyrosine hydroxylase (TH), nitric oxide synthase (NOS), calbindin (CAL), vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and calcitonin gene-related peptide (CGRP) in untreated or colchicine-treated preparation. Almost all neurones were either positive for TH (38%) or for SOM (60%). Only 4% of the neurones were both TH- and SOM-positive and 3% of the neurones exhibited neither TH nor SOM immunoreactivity. The relative numbers of NPY-, NOS-, CAL- and VIP-positive neurones were 57%, 28%, 14% and 7%, respectively. No SP- or CGRP-positive neurones were observed. All NADPH-diaphorase-positive neurones expressed NOS immunoreactivity. Only in some TH-positive neurones was NPY and/or NOS found. Four major subpopulations were found in the ureteric ganglia. The SOM-positive neurones were subdivided into SOM/NPY/NOS- (28% of all neurones), SOM/NPY- (18%) and SOM/CAL/NPY-positive neurones (14%). A subpopulation of these peptid- ergic neurones also contained VIP. About 35% of the neurones contained TH only. Neurones of all subpopulations (72% of the neurones), except most of the CAL-positive neurones, were encircled by dense plexus of varicose SP/CGRP-positive, presumably sensory nerve fibres. Dense plexus of VIP-positive fibres were observed around 89% of the neurones. The chemical coding of the neuronal subpopulations identified in the ganglia accompanying the chicken ureter resembled that observed in the ganglia of Remak’s nerve but was remarkably different from that of the autonomic neurones described in mammalian species.     

Complementary distribution of NADPH-diaphorase and l-arginine in the snail nervous system

Since the interneuronal messenger nitric oxide (NO) can not be stored in neurones, the regulation of the NO-producing enzyme nitric oxide synthase (NOS) is crucial. Neuronal NOS metabolises L-arginine to nitric oxide (NO) and L-citrulline in a Ca(2+)-dependent manner. Thus, availability of L-arginine to NOS may modulate NO production. In this study, we examined the cellular distribution of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, L-arginine and L-citrulline. Using NADPH-diaphorase histochemistry to visualise putative NO-producing cells and immunocytochemistry to localise L-arginine, we showed that the distribution of L-arginine-immunoreactive neurones correlates well with those of NADPH-diaphorase-positive neurones in cerebral ganglia of the pulmonate Helix pomatia. However, substrate and enzyme were visualised in separate but adjacent neurones. We further examined whether NADPH-diaphorase-labelled cells contain the L-citrulline. Following elevation of intracellular Ca(2+) by the Ca(2+) ionophore, ionomycin, or by a high-K(+) solution, the number of L-citrulline-immunoreactive neurones in mesocerebrum and pedal lobe increased up to tenfold. Preincubation of ganglia with the NOS inhibitor N(G)-nitro-L-arginine prevented ionomycin or high-K(+) solution-induced L-citrulline synthesis. Most L-citrulline-immunoreactive neurones contain NADPH-diaphorase activity. In conclusion, these experiments indicate a complementary distribution of NOS and L-arginine and suggest an unknown signalling pathway between neurones to maintain L-arginine and NO homeostasis.     

Sex Differences in the Beneficial Cardiac Effects of Chronic Treatment with Atrial Natriuretic Peptide In Spontaneously Hypertensive Rats

Introduction

The aim of this study was to investigate both the effects of chronic treatment with atrial natriuretic peptide (ANP) on systolic blood pressure (SBP), cardiac nitric oxide (NO) system, oxidative stress, hypertrophy, fibrosis and apoptosis in spontaneously hypertensive rats (SHR), and sex-related differences in the response to the treatment.

Methods

10 week-old male and female SHR were infused with ANP (100 ng/hr/rat) or saline (NaCl 0.9%) for 14 days (subcutaneous osmotic pumps). SBP was recorded and nitrites and nitrates excretion (NOx) were determined. After treatment, NO synthase (NOS) activity, eNOS expression, thiobarbituric acid-reactive substances (TBARS) and glutathione concentration were determined in left ventricle, as well as the activity of glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD). Morphological studies in left ventricle were performed in slices stained with hematoxylin-eosin or Sirius red to identify collagen as a fibrosis indicator; immunohistochemistry was employed for identification of transforming growth factor beta; and apoptosis was evaluated by Tunel assay.

Results

Female SHR showed lower SBP, higher NO-system activity and less oxidative stress, fibrosis and hypertrophy in left ventricle, as well as higher cardiac NOS activity, eNOS protein content and NOx excretion than male SHR. Although ANP treatment lowered blood pressure and increased NOS activity and eNOS expression in both sexes, cardiac NOS response to ANP was more marked in females. In left ventricle, ANP reduced TBARS and increased glutathione concentration and activity of CAT and SOD enzymes in both sexes, as well as GPx activity in males. ANP decreased fibrosis and apoptosis in hearts from male and female SHR but females showed less end-organ damage in heart. Chronic ANP treatment would ameliorate hypertension and end-organ damage in heart by reducing oxidative stress, increasing NO-system activity, and diminishing fibrosis and hypertrophy.     

Alteration of gene expression during progression of hypertension-induced cardiac dysfunction in rats

Hypertension induced by high-salt diet in Dahl salt-sensitive rats leads to compensatory cardiac hypertrophy by approximately 11 wk, cardiac dysfunction at approximately 17 wk, and death from cardiac dysfunction at approximately 21 wk. It is unclear what molecular hallmarks distinguish the compensatory hypertrophy from the decompensated cardiac dysfunction phase. Here we compared the gene expression in rat cardiac tissue from the compensatory hypertrophic phase (11 wk, n = 6) with the cardiac dysfunction phase (17 wk, n = 6) and with age-matched normotensive controls. Messenger RNA levels of 93 genes, selected based on predicted association with cardiac dysfunction, were measured by quantitative real-time PCR. In the hypertrophic phase, the expression of three genes, atrial natriuretic peptide (ANP; P = 0.0089), brain natriuretic peptide (P = 0.0012), and endothelin-1 precursor (P = 0.028), significantly increased, whereas there was decreased expression of 24 other genes including SOD2 (P = 0.0148), sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (P = 0.0002), and ryanodine receptor 2 (P = 0.0319). In the subsequent heart cardiac dysfunction phase, the expression of an additional 20 genes including inducible nitric oxide synthase (NOS; P = 0.0135), angiotensin I-converting enzyme (P = 0.0082), and IL-1beta (P < 0.0001) increased, whereas the expression of seven genes decreased compared with those of age-matched controls. Furthermore, the expression of 22 genes, including prepro-endothelin-1, ANP, angiotensin I-converting enzyme, beta(1)-adrenergic receptor, SOD2, and endothelial NOS, significantly changed in the cardiac dysfunction phase compared with the compensatory hypertrophic phase. Finally, principal component analysis successfully segregated animals with decompensatory cardiac dysfunction from controls, as well as from animals at the compensated hypertrophy phase, suggesting that we have identified molecular markers for each stage of the disease.     

Changes in short‐chain acyl‐coA dehydrogenase during rat cardiac development and stress

This study was designed to investigate the expression of short‐chain acyl‐CoA dehydrogenase (SCAD), a key enzyme of fatty acid β‐oxidation, during rat heart development and the difference of SCAD between pathological and physiological cardiac hypertrophy. The expression of SCAD was lowest in the foetal and neonatal heart, which had time‐dependent increase during normal heart development. In contrast, a significant decrease in SCAD expression was observed in different ages of spontaneously hypertensive rats (SHR). On the other hand, swim‐trained rats developed physiological cardiac hypertrophy, whereas SHR developed pathological cardiac hypertrophy. The two kinds of cardiac hypertrophy exhibited divergent SCAD changes in myocardial fatty acids utilization. In addition, the expression of SCAD was significantly decreased in pathological cardiomyocyte hypertrophy, however, increased in physiological cardiomyocyte hypertrophy. SCAD siRNA treatment triggered the pathological cardiomyocyte hypertrophy, which showed that the down‐regulation of SCAD expression may play an important role in pathological cardiac hypertrophy. The changes in peroxisome proliferator‐activated receptor α (PPARα) was accordant with that of SCAD. Moreover, the specific PPARα ligand fenofibrate treatment increased the expression of SCAD and inhibited pathological cardiac hypertrophy. Therefore, we speculate that the down‐regulated expression of SCAD in pathological cardiac hypertrophy may be responsible for ‘the recapitulation of foetal energy metabolism’. The deactivation of PPARα may result in the decrease in SCAD expression in pathological cardiac hypertrophy. Changes in SCAD are different in pathological and physiological cardiac hypertrophy, which may be used as the molecular markers of pathological and physiological cardiac hypertrophy.     

Regulation of the mammalian heart function by nitric oxide

The mammalian heart expresses all three isoforms of nitric oxide synthases (NOS) in diverse cell types of the myocardium. Despite their apparent promiscuity, the NOS isoforms support specific signaling because of their subcellular compartmentation with colocalized effectors and limited diffusibility of NO in muscle cells. eNOS and nNOS sustain normal EC coupling and contribute to the early and late phases of the Frank-Starling mechanism of the heart. They also attenuate the beta1-/beta2-adrenergic increase in inotropy and chronotropy, and reinforce the pre- and post-synaptic vagal control of cardiac contraction. By doing so, the NOS protect the heart against excessive stimulation by catecholamines, just as an "endogenous beta-blocker". In the ischemic and failing myocardium, induced iNOS further reinforces this effect, as does eNOS coupled to overexpressed beta3-adrenoceptors. nNOS expression also increases in the aging and infarcted heart, but its role (compensatory or deleterious) is less clear. In addition to their direct regulation of contractility, the NOS modulate oxygen consumption, substrate utilization, sensitivity to apoptosis, hypertrophy and regenerative potential, all of which illustrate the pleiotropic effects of this radical on the cardiac cell biology.     

Genistein prevents isoproterenol-induced cardiac hypertrophy in rats

Genistein, an isoflavone and a rich constituent of soy, possesses important regulatory effects on nitric oxide (NO) synthesis and oxidative stress. Transient and low release of NO by endothelial nitric oxide synthase (eNOS) has been shown to be beneficial, while high and sustained release by inducible nitric oxide synthase (iNOS) may be detrimental in pathological cardiac hypertrophy. The present study was designed to evaluate whether genistein could prevent isoproterenol-induced cardiac hypertrophy in male Wistar rats (150-200 g, 10-12 weeks old) rats. Isoproterenol (5 mg·(kg body weight)(-1)) was injected subcutaneously once daily for 14 days to induced cardiac hypertrophy. Genistein (0.1 and 0.2 mg·kg(-1), subcutaneous injection once daily) was administered along with isoproterenol. Heart tissue was studied for myocyte size and fibrosis. Myocardial thiobarbituric acid reactive substances (TBARS), glutathione (GSH), superoxide dismutase (SOD), catalase levels, and 1-OH proline (collagen content) were also estimated. Genistein significantly prevented any isoproterenol-induced increase in heart weight to body weight ratio, left ventricular mass (echocardiographic), myocardial 1-OH proline, fibrosis, myocyte size and myocardial oxidative stress. These beneficial effects of genistein were blocked by a nonselective NOS inhibitor (L-NAME), but not by a selective iNOS inhibitor (aminoguanidine). Thus, the present study suggests that the salutary effects of genistein on isoproterenol-induced cardiac hypertrophy may be mediated through inhibition of iNOS and potentiation of eNOS activities.     

Transient expression of NOS-II during development of the murine enteric nervous system

In the enteric nervous system, nitric oxide (NO) is regarded as an important messenger for the non-adrenergic and non-cholinergic neurotransmission. Synthesized mainly by the constitutive nitric oxide synthase (NOS) isoforms NOS I and NOS III, this molecule exerts prejunctional inhibitory effects in the submucosal plexus as well as relaxation of enteric smooth muscles. In order to elucidate the role for NO during enteric development, we looked for the expression of all three NOS-isoforms in the enteric nervous system during mouse development from E8 to E20 using immunohistochemistry. Starting around midgestation, a transient expression of the NOS-II isoform during the very early development of enteric neurones was detected in parallel to that of HNK-1 exclusively in the myenteric plexus. Similar to findings for other neuronal systems, NOS-I and NOS III isoforms could be traced starting significantly later to increase toward the end of embryonic development when NOS II immunoreactivity faded and a strong expression of the vasointestinal peptide could be detected. In contrast to the NOSII expression, the constitutive isoforms can also be detected in the submucosal plexus. Altogether, these findings suggest NOS-II to be exclusively involved during early steps of enteric nervous system development. Absence of downstream signalling elements, such as sGC and cGMP both in neurons and in enteric muscle until the end of the second third of gestation, may indicate different effects executed by NO during development, expressed by Ca²⁺ -dependent and Ca²⁺ -independent NOS isoforms.     

LKB1IP promotes pathological cardiac hypertrophy by targeting PTEN/Akt signalling pathway

Pathological cardiac hypertrophy represents a leading cause of morbidity and mortality worldwide. Liver kinase B1 interacting protein 1 (LKB1IP) was identified as the binding protein of tumour suppressor LKB1. However, the role of LKB1IP in the development of pathological cardiac hypertrophy has not been explored. The aim of this study was to investigate the function of LKB1IP in cardiac hypertrophy in response to hypertrophic stimuli. We investigated the cardiac level of LKB1IP in samples from patients with heart failure and mice with cardiac hypertrophy induced by isoproterenol (ISO) or transverse aortic constriction (TAC). LKB1IP knockout mice were generated and challenged with ISO injection or TAC surgery. Cardiac function, hypertrophy and fibrosis were then examined. LKB1IP expression was significantly up-regulated on hypertrophic stimuli in both human and mouse cardiac samples. LKB1IP knockout markedly protected mouse hearts against ISO- or TAC-induced cardiac hypertrophy and fibrosis. LKB1IP overexpression aggravated ISO-induced cardiomyocyte hypertrophy, and its inhibition attenuated hypertrophy in vitro. Mechanistically, LKB1IP activated Akt signalling by directly targeting PTEN and then inhibiting its phosphatase activity. In conclusion, LKB1IP may be a potential target for pathological cardiac hypertrophy.