Mulugu V. Brahmajothi

In Situ Hybridization Reveals Extensive Diversity of K+ Channel mRNA in Isolated Ferret Cardiac Myocytes

The molecular basis of K+ currents that generate repolarization in the heart is uncertain. In part, this reflects the similar functional properties different K+ channel clones display when heterologously expressed, in addition to the molecular diversity of the voltage-gated K+ channel family. To determine the identity, regional distribution, and cellular distribution of voltage-sensitive K+ channel mRNA subunits expressed in ferret heart, we used fluorescent labeled oligonucleotide probes to perform in situ hybridization studies on enzymatically isolated myocytes from the sinoatrial (SA) node, right and left atria, right and left ventricles, and interatrial and interventricular septa. The most widely distributed K+ channel transcripts in the ferret heart were Kv1.5 (present in 69.3% to 85.6% of myocytes tested, depending on the anatomic region from which myocytes were isolated) and Kv1.4 (46.1% to 93.7%), followed by kv1.2, Kv2.1, and Kv4.2. Surprisingly, many myocytes contain transcripts for Kv1.3, Kv2.2, Kv4.1, Kv5.1, and members of the Kv3 family. Kv1.1, Kv1.6, and Kv6.1, which were rarely expressed in working myocytes, were more commonly expressed in SA nodal cells. IRK was expressed in ventricular (84.3% to 92.8%) and atrial (52.4% to 64.0%) cells but was nearly absent (6.6%) in SA nodal cells; minK was most frequently expressed in SA nodal cells (33.7%) as opposed to working myocytes (10.3% to 29.3%). Two gene products implicated in long-QT syndrome, ERG and KvLQT1, were common in all anatomic regions (41.1% to 58.2% and 52.1% to 71.8%, respectively). These results show that the diversity of K+ channel mRNA in heart is greater than previously suspected and that the molecular basis of K+ channels may vary from cell to cell within distinct regions of the heart and also between major anatomic regions.

Regional Localization of ERG, the Channel Protein Responsible for the Rapid Component of the Delayed Rectifier, K+ Current in the Ferret Heart

Repolarization of the cardiac action potential varies widely throughout the heart. This could be due to the differential distribution of ion channels responsible for repolarization, especially the K+ channels. We have therefore studied the cardiac localization of ERG, a channel protein known to play an important role in generation of the rapid component of the delayed rectifier K+ current (IKr), an important determinant of the repolarization waveform, Cryosections of the ferret atrium and ventricle were prepared to determine the localization of ERG by fluorescence in situ hybridization (FISH) and immunofluorescence. We found that in the ferret, ERG transcript and protein expression was most abundant in the epicardial cell layers throughout most of the ventricle, except at the base. In the atrium, we found that ERG is most abundant in the medial right atrium, especially in the trabeculae and the crista terminalis of the right atrial appendage. It also is present in areas within the sinoatrial node. In all regions studied, FISH and immunofluorescence showed concordant localization patterns. These data suggest that repolarization mediated by IKr is not uniform throughout the ferret heart and provide a molecular explanation for heterogeneity in action potential repolarization throughout the mammalian heart.

Heterogeneous Basal Expression of Nitric Oxide Synthase and Superoxide Dismutase Isoforms in Mammalian Heart Implications for Mechanisms Governing Indirect and Direct Nitric Oxide–Related Effects

The basal expression patterns of NO synthase (NOS; endothelial [eNOS], neuronal [nNOS], and cytokine-inducible [iNOS]) and superoxide dismutase (SOD; extracellular membrane bound [ECSOD], MnSOD, and CuZnSOD) isoforms in ferret heart (tissue sections and isolated myocytes) were determined by immunofluorescent localization. We demonstrate the following for the first time in the mammalian heart: (1) heterogeneous expression patterns of the 3 NOS and 3 SOD isoforms among different tissue and myocyte types; (2) colocalization of eNOS and ECSOD at both the tissue and myocyte levels; (3) a significant gradient of eNOS and ECSOD expression across the left ventricular (LV) wall, with both enzymes being highly expressed and colocalized in LV epicardial myocytes but markedly reduced in LV endocardial myocytes; and (4) specific subcellular localization patterns of eNOS and the 3 SOD isoforms. In particular, eNOS and ECSOD are demonstrated (electron and confocal microscopy) to be specifically localized to the sarcolemma of ventricular myocytes. Similar heterogeneous eNOS and ECSOD expression patterns were also obtained in human LV tissue sections, underscoring the general importance of these novel findings. Our data suggest a strong functional correlation between the activities of sarcolemmally localized myocyte eNOS and ECSOD in governing NO*/O(2-) interactions and suggest that NO-related modulatory effects on cardiac myocyte protein and/or ion channel function may be significantly more complex than is presently believed.

Heterogeneity in K+ Channel Transcript Expression Detected in Isolated Ferret Cardiac Myocytes

The molecular basis of the potassium ion (K+) channels that generate repolarization in heart tissue remains uncertain, in part because of the molecular diversity of the voltage‐gated K+ channel family. In our investigation, we used fluorescent labeled oligonucleotide probes to perform in situ hybridization studies on enzymatically isolated myocytes to determine the identity, regional distribution, and cellular distribution of voltage‐gated K+ channel, a‐subunit mRNA expressed in ferret heart. The regions studied were from the sinoatrial node (SA), right and left atrium, right and left ventricle, and interatrial and interventricular septa, Kv1.5 and Kv1.4 were the most widely distributed K+ channel transcripts in the ferret heart (present in approximately 70%–86% and approximately 46%–95% of tested myocytes, respectively), followed byKv1.2, Kv2.1, and Kv4.2. In addition, many myocytes contain transcripts for Kv1.3, Kv2.2, Kv4.1, Kv5.1, and members of the Kv3 family, Kv1.1, Kv1.6, and Kv6.1 were rarely expressed in working myocytes, but were more commonly expressed in SA nodal cells. Two other transcripts whose genes have been implicated in the long QT syndrome, erg and KvLQT1, were common in all regions (approximately 41%–58% and 52%–72%, respectively). These results show that both the diversity and heterogeneity of K+ channel mRNA in heart tissue is greater than previously suspected

Hyperoxia impairs postnatal alveolar epithelial development via NADPH oxidase in newborn mice

Hyperoxia disrupts postnatal lung development in part through inducing inflammation. To determine the contribution of leukocyte-derived reactive oxygen species, we exposed newborn wild-type and NADPH oxidase p47(phox) subunit null (p47(phox-/-)) mice to air or acute hyperoxia (95% O(2)) for up to 11 days. Hyperoxia-induced pulmonary neutrophil influx was similar in wild-type and p47(-/-) mice at postnatal days (P) 7 and 11. Macrophages were decreased in wild-type hyperoxia-exposed mice compared with p47(phox-/-) mice at P11. Hyperoxia impaired type II alveolar epithelial cell and bronchiolar epithelial cell proliferation, but depression of type II cell proliferation was significantly less in p47(-/-) mice at P3 and P7, when inflammation was minimal. We found reciprocal results for the expression of the cell cycle inhibitor p21(cip/waf) in type II cells, which was induced in 95% O(2)-exposed wild-type mice, but significantly less in p47(phox-/-) littermates at P7. Despite partial preservation of type II cell proliferation, deletion of p47(phox) did not prevent the major adverse effects of hyperoxia on alveolar development estimated by morphometry at P11, but hyperoxia impairment of elastin deposition at alveolar septal crests was significantly worse in wild-type vs. p47(phox-/-) mice at P11. Since we found that p47(phox) is expressed in a subset of alveolar epithelial cells, its deletion may protect postnatal type II alveolar epithelial proliferation from hyperoxia through effects on epithelial as well as phagocyte-generated superoxide.

Intra-amniotic LPS amplifies hyperoxia-induced airway hyperreactivity in neonatal rats

Background:We previously showed that intra-amniotic lipopolysaccharide (LPS) amplifies alveolar hypoplasia induced by postnatal hyperoxia. We determined whether the priming effect of intra-amniotic LPS amplifies hyperoxia-induced airway hyperreactivity (AHR).Methods:LPS or normal saline was injected into the amniotic cavities of pregnant rats at the 20th day of gestation. After birth, rat pups were exposed to 60% O2 or air for 14 d. On postnatal day 14, rat pups underwent forced oscillometry, which included a challenge with nebulized methacholine, and the lungs were harvested for morphological studies.Results:Hyperoxia significantly increased airway reactivity and decreased compliance. Intra-amniotic LPS further increased hyperoxia-induced AHR but did not further impair respiratory system compliance. Hyperoxia-induced changes in lung parenchymal and small airway morphology were not further altered by intra-amniotic LPS. However, combined exposure to intra-amniotic LPS and hyperoxia increased the proportion of degranulating mast cells in the hilar airways.Conclusion:Intra-amniotic LPS amplified postnatal hyperoxia-induced AHR. This was associated with increased airway mast cell degranulation, which has previously been linked with hyperoxia-induced AHR. There were no morphologic changes of parenchyma or airways that would account for the LPS augmentation of hyperoxia-induced AHR.

NOS2 REGULATION OF LPS-INDUCED AIRWAY INFLAMMATION VIA S-NITROSYLATION OF NF-κB p65

Inducible nitric oxide synthase (NOS2) expression is increased in the airway epithelium in acute inflammatory disorders although the physiological impact remains unclear. We have previously shown that NOS2 inhibits NF-κB (p50-p65) activation in respiratory epithelial cells by inducing S-nitrosylation of the p65 monomer (SNO-p65). In addition, we have demonstrated that mouse lung SNO-p65 levels are acutely depleted in a lipopolysaccharide (LPS) model of lung injury and that augmenting SNO-p65 levels before LPS treatment results in decreased airway epithelial NF-κB activation, airway inflammation, and lung injury. We now show that aerosolized LPS induces NOS2 expression in the respiratory epithelium concomitant with an increase in lung SNO-p65 levels and a decrease in airway NF-κB activity. Genetic deletion of NOS2 results in an absence of SNO-p65 formation, persistent NF-κB activity in the respiratory epithelium, and prolonged airway inflammation. These results indicate that a primary function of LPS-induced NOS2 expression in the respiratory epithelium is to modulate the inflammatory response through deactivation of NF-κB via S-nitrosylation of p65, thereby counteracting the initial stimulus-coupled denitrosylation.

Transport rather than diffusion-dependent route for nitric oxide gas activity in alveolar epithelium

The pathway by which inhaled NO gas enters pulmonary alveolar epithelial cells has not been directly tested. Although the expected mechanism is diffusion, another route is the formation of S-nitroso-L-cysteine, which then enters the cell through the L-type amino acid transporter (LAT). To determine if NO gas also enters alveolar epithelium this way, we exposed alveolar epithelial-rat type I, type II, L2, R3/1, and human A549-cells to NO gas at the air liquid interface in the presence of L- and D-cysteine+/-LAT competitors. NO gas exposure concentration dependently increased intracellular NO and S-nitrosothiol levels in the presence of L- but not D-cysteine, which was inhibited by LAT competitors, and was inversely proportional to diffusion distance. The effect of L-cysteine on NO uptake was also concentration dependent. Without preincubation with L-cysteine, NO uptake was significantly reduced. We found similar effects using ethyl nitrite gas in place of NO. Exposure to either gas induced activation of soluble guanylyl cylase in a parallel manner, consistent with LAT dependence. We conclude that NO gas uptake by alveolar epithelium achieves NO-based signaling predominantly by forming extracellular S-nitroso-L-cysteine that is taken up through LAT, rather than by diffusion. Augmenting extracellular S-nitroso-L-cysteine formation may augment pharmacological actions of inhaled NO gas.

Hyperoxia Inhibits Nitric Oxide Treatment Effects in Alveolar Epithelial Cells via Effects on L-Type Amino Acid Transporter-1

AIMS The aims of this study were to determine hyperoxia effects on S-nitrosothiol (SNO) accumulation and L-type amino acid transporter 1 (LAT1) expression/function in alveolar epithelium and to determine whether hyperoxia impairs exogenous nitric oxide (NO) treatment effects in alveolar epithelium through effects on LAT1 expression and/or function. RESULTS SNO accumulation in vitro and in vivo after NO treatment was dependent on the LAT1 system transport. Hyperoxia (60% or 90%) impaired NO effects on SNO accumulation and soluble guanylyl cyclase activation in proportion to the magnitude of hyperoxia and the duration of exposure, up to 12 h, in type I-like (R3/1) and type II-like (L2) rat and human (A549) alveolar epithelial cells. LAT function, determined by sodium-independent (3)H-leucine uptake, was impaired in a parallel manner. Hyperoxia impaired LAT1 expression in alveolar epithelial cells, determined by immunoblots and immunofluorescence, and in newborn rats exposed to 60% O2 for 4 days, determined by immunohistochemistry. INNOVATION Despite significant preclinical evidence, inhaled NO has shown disappointing limitations in clinical applications. Our studies suggest an important explanation: oxidative stress, a common feature of diseases in which therapeutic NO would be considered, impairs LAT1 expression and function, blocking a major route for inhaled NO (iNO) action, that is, the uptake of S-nitrosocysteine via LAT1. CONCLUSIONS SNO uptake after NO treatment is dependent on LAT1. Hyperoxia impairs SNO uptake and NO effects during NO exposure and impairs LAT system function and LAT1 expression. Effects on SNO formation and transport must be considered for rational optimization of NO-based therapeutics.

S-nitrosothiol transport via PEPT2 mediates biological effects of nitric oxide gas exposure in macrophages.

The pharmacological effects of nitric oxide (NO) administered as a gas are dependent on the conversion to S-nitrosocysteine, and as such are largely mediated by the L-type amino-acid transporters (LATs) in several cell types. The dipeptide transporter PEPT2 has been proposed as a second route for S-nitrosothiol (SNO) transport, but this has never been demonstrated. Because NO governs important immune functions in alveolar macrophages, we exposed rat alveolar macrophages (primary and NR8383 cells) to NO gas at the air-liquid interface ± LPS stimulation in the presence of PEPT2 substrate Cys-Gly (or the LAT substrate L-Cys) ± transporter competitors. We found that SNO uptake and NO-dependent actions, such as the activation of soluble guanylyl cyclase (sGC), the augmentation of sGC-dependent filamentous actin (F-actin) polymerization, phagocytosis, and the inhibition of NF-κB activation, were significantly augmented by the addition of Cys-Gly in a manner dependent on PEPT2 transport. We found parallel (and greater) effects that were dependent on LAT transport. The contribution of cystine/cysteine shuttling via system x cystine transporter (xCT) to SNO uptake was relatively minor. The observed effects were unaffected by NO synthase inhibition. The NO gas treatment of alveolar macrophages increased SNO uptake, the activation of sGC, F-actin polymerization, and phagocytosis, and inhibited NF-κB activation, in a manner dependent on SNO transport via PEPT2, as well as via LAT.