nNOS Splice Variants Research Articles

Expression of neuronal NO synthase α- and β-isoforms in skeletal muscle of mice.

Knowledge of the primary structure of neuronal NO synthase (nNOS) in skeletal muscle is still conflicting and needs further clarification. To elucidate the expression patterns of nNOS isoforms at both mRNA and protein level, systematic reverse transcription (RT)-PCR and epitope mapping by qualitative immunoblot analysis on skeletal muscle of C57/BL6 mice were performed. The ability of the nNOS isoforms to form aggregates was characterized by native low-temperature polyacrylamide electrophoresis (LT-PAGE). The molecular analysis was focused on the rectus femoris (RF) muscle, a skeletal muscle with a nearly balanced ratio of nNOS α- and β-isoforms. RT-PCR amplificates from RF muscles showed exclusive exon-1d mRNA expression, either with or without exon-μ. Epitope mapping demonstrated the simultaneous expression of the nNOS splice variants α/μ, α/non-μ, β/μ and β/non-μ. Furthermore, immunoblotting suggests that the transition between nNOS α- and β-isoforms lies within exon-3. In LT-PAGE, three protein nNOS associated aggregates were detected in homogenates of RF muscle and tibialis anterior muscle: a 320 kDa band containing nNOS α-isoforms, while 250 and 300 kDa bands consist of nNOS β-isoforms that form homodimers or heterodimers with non-nNOS proteins.

Heat shock protein 90α increases superoxide generation from neuronal nitric oxide synthases

Neuronal nitric oxide synthase (nNOS) generates superoxide, particularly at sub-optimal l-arginine (l-Arg) substrate concentrations. Heat shock protein 90 (Hsp90) was reported to inhibit superoxide generation from nNOS protein. However, commercially available Hsp90 product from bovine brain tissues with unspecified Hsp90α and Hsp90β contents and an undefined Hsp90 protein oligomeric state was utilized. These two Hsp90s can have opposite effect on superoxide production by NOS. Importantly, emerging evidence indicates that nNOS splice variants are involved in different biological functions by functioning distinctly in redox signaling. In the present work, purified recombinant human Hsp90α, in its native dimeric state, was used in electron paramagnetic resonance (EPR) spin trapping experiments to study the effects of Hsp90α on superoxide generation from nNOS splice variants nNOSμ and nNOSα. Human Hsp90α was found to significantly increase superoxide generation from nNOSμ and nNOSα proteins under l-Arg-depleted conditions and Hsp90α influenced superoxide production by nNOSμ and nNOSα at varying degrees. Imidazole suppressed the spin adduct signal, indicating that superoxide was produced at the heme site of nNOS in the presence of Hsp90α, whereas l-Arg repletion diminished superoxide production by the nNOS-Hsp90α. Moreover, NADPH consumption rate values exhibited a similar trend/difference as a function of Hsp90α and l-Arg. Together, these EPR spin trapping and NADPH oxidation kinetics results demonstrated noticeable Hsp90α-induced increases in superoxide production by nNOS and a distinguishable effect of Hsp90α on nNOSμ and nNOSα proteins.

NNOS splice variants differentially regulate myofilament function but are dispensable for intracellular calcium and force transients in cardiac papillary muscles.

Cardiac muscle expresses three neuronal nitric oxide synthase (nNOS) splice variants: nNOSα, nNOSμ and nNOSβ. The functions of these nNOS splice variants in cardiac muscle, particularly myofilament-associated nNOSβ are unclear. To decipher cardiac nNOS splice variant function we investigated myofilament function and intracellular calcium and force transients in demembranated and intact papillary muscles from two lines of nNOS knockout mice. The first line (KN1) lacks nNOSα and nNOSμ. The second line (KN2) lacks active nNOSα, nNOSμ and nNOSβ. Demembranated KN1 papillary muscles exhibited reduced myofilament ATPase activity (-35%) and specific force (-10%) relative to controls. Demembranated KN2 muscles exhibited a smaller decrease in myofilament ATPase activity (-21%), but a greater reduction in specific force (-26%) relative to controls. Myofilament calcium sensitivity in demembranated KN1 and KN2 papillary muscles was similar to controls. Thus, papillary muscle-expressed nNOS splice variants are necessary for control levels of myofilament ATPase activity and force generation, but dispensable for myofilament calcium sensitivity. The greater reduction in myofilament ATPase relative to specific force in KN1, but not KN2 muscle, reduced the energy cost of muscle contraction, suggesting that nNOSβ increased the energetic efficiency of contraction in the absence of nNOSμ and nNOSα. Analyses of intact KN1 and KN2 papillary muscles showed that both intracellular calcium transients and their evoked force transients were similar to controls at stimulation frequencies between 1 and 3 Hz. Therefore, nNOS was dispensable for baseline excitation-contraction coupling. In summary, these data suggest that nNOS splice variants differentially regulate myofilament function, but not baseline calcium handling in papillary muscles. More importantly, they suggest that nNOSβ is a novel modulator of myofilament function, and ultimately the energetic efficiency of cardiac papillary muscle contraction.

Superoxide generation from nNOS splice variants and its potential involvement in redox signal regulation.

We previously demonstrated different spacial expression profiles of the neuronal nitric oxide (NO) synthase (nNOS) splice variants nNOS-µ and nNOS-α in the brain; however, their exact functions are not fully understood. Here, we used electron paramagnetic resonance to compare the electron-uncoupling reactions of recombinant nNOS-µ and nNOS-α that generate reactive oxygen species (ROS), in this case superoxide. nNOS-µ generated 44% of the amount of superoxide that nNOS-α generated. We also evaluated the ROS production in HEK293 cells stably expressing nNOS-α and nNOS-µ by investigating these electron-uncoupling reactions as induced by calcium ionophore A23187. A23187 treatment induced greater ROS production in HEK293 cells expressing nNOS-α than those expressing nNOS-µ. Also, immunocytochemical analysis revealed that A23187-treated cells expressing nNOS-α produced more 8-nitroguanosine 3',5'-cyclic monophosphate, a second messenger in NO/ROS redox signaling, than did the cells expressing nNOS-µ. Molecular evolutionary analysis revealed that the ratio of nonsynonymous sites to synonymous sites for the nNOS-µ-specific region was higher than that for the complete gene, indicating that this region has fewer functional constraints than does the complete gene. These observations shed light on the physiological relevance of the nNOS-µ variant and may improve understanding of nNOS-dependent NO/ROS redox signaling and its pathophysiological consequences in neuronal systems.

PS 07-10 EFFECT OF CONSTITUTIVE NITRIC OXIDE SYNTHASE PROFILING IN REGULATION OF HYPERTENSIVE ATRIAL MYOCARDIUM BY FREE FATTY ACIDS

Objective: Constitutive nitric oxide synthases (eNOS and nNOS) in heart, have been implicated to regulate myocardial Ca2+ handling, electrical activity and the contractility of cardiac myocytes. Recently, we have shown that nNOSa and/or nNOSμ are functionally expressed in the cytosol of left ventricular (LV) myocytes from rat hearts and nNOSa/μ are up-regulated in hypertension (HTN) whereas eNOS is decreased. In addition to, we revealed for the first time that nNOSß is expressed in the myofilament fraction of LV myocytes, whose expression was unaffected in HTN. In hypertensive atrial myocardium, eNOS and nNOS profiling are not demonstrated systematically. Design and Method: 1. Left atrium (LA) from sham Sprague-Dawley rats (12 weeks old, male) and HTN rats were used. HTN was induced by infusing Ang II subcutaneously using osmotic minipumps (Alzet model 2004, infusion rate 125 ng/min/kg) for 4 weeks. 2. Immunoblotting: cytosol and myofilament fractions of LA homogenates were separated by centrifugation at 8000 rpm for10 min 10times at 4°C. Specific antibodies targeting anti-nNOS, anti-eNOS, anti-GAPDH were used and secondary antibody were followed relevant primary antibodies. Results: Our result showed that nNOSa/μ were expressed in cytosol fractions of LA and the expression was not different between sham and HTN. Interestingly, none of the nNOS splice variants (including nNOSa/μ and nNOSß) was detected in myofilament fraction of LA from either group. eNOS protein expression was not different between groups and among different fractions of LA. Incubation of LA with PA (100 mM, 1 hr) failed to affect total protein levels of eNOS or nNOS in cytosol fractions of LA from sham or HTN rats. Conclusions: Expression profiles of eNOS and nNOS splice variants in LA are not different between sham and HTN. Furthermore, acute application of PA (1hr) did not affect the protein levels or the translocation of eNOS or nNOS splice variants in atrial myocardium.

Novel Splice Variant of nNOS in Cerebrovascular Endothelial Cells Contributes to Superoxide and Peroxynitrite Generation

Experimental stroke in animals with gene deletions of endothelial (eNOS) and neuronal (nNOS) nitric oxide synthase isoforms have shown opposite effects on infarct injury. nNOS has been identified recently in endothelial cells, however, its functional significance is unclear. Our objective was to identify nNOS in the brain microvascular endothelial cells (MECs) and characterize its functional role. Primary MECs from humans (hMECs), rats (rMECs), and mice (mMECs) along with cultured primary rat cortical neurons were used in the studies. In addition, rat brain microvessels were isolated by homogenization of cortices and separation by filtration followed by gradient centrifugation. Immunohistochemistry identified von Willebrand factor (endothelial marker), eNOS, and nNOS but stained negative for GFAP (glial marker) and Neu1 (neuronal marker) in rMECs and microvessels. Western blot analysis using antibodies targeting N‐terminal domain of nNOS revealed immunoband of a potential nNOS splice variant at ~130 kD in rMECs as opposed to 160 kD in rat cerebral cortex and cultured neurons. PCR experiments identified the nNOS mRNA in all MECs and rat microvessels. 3′ Rapid Amplification of cDNA Ends method using nNOS specific exon 2 primers revealed a prominent truncated nNOS band in rMECs compared to full length nNOS cDNA (RefSeq NP_434686) found in the brain cortex. Furthermore, siRNA targeting rat nNOS in rMECs was able to knockdown nNOS mRNA. We utilized electron spin resonance spectrometry to measure superoxide (1‐Hydroxy‐3‐methoxycarbonyl‐2,2,5,5‐tetramethyl‐pyrrolidine), peroxynitrite (1‐Hydroxy‐3‐carboxy‐2,2,5,5‐tetramethylpyrrolidine), and NO (colloid Fe(DETC)2). siRNA knockdown or pharmacological inhibition (N‐ω‐Propyl‐L‐arginine and ARL‐17477) of nNOS reduced superoxide levels but increased NO levels in rMECs compared to untreated cells. Inhibition of nNOS also decreased peroxynitrite levels in rMECs. In contrast, eNOS inhibitor (L‐N5‐(1‐Iminoethyl)ornithine) increased superoxide levels but reduced NO levels. Similarly, inhibition of nNOS in neurons increased ROS levels and decreased NO levels. Moreover, co‐treatment with tetrahydrobiopterin, a co‐factor for NOS, reduced the superoxide levels in hMECs. Furthermore, treatment of rMECs with angiotensin II (ATII) and high glucose levels increased superoxide levels. Superoxide generation in MECs at baseline and following the treatment with ATII was sensitive to inhibition by gp‐91‐ds‐tat peptide (NADPH oxidase inhibitor) or ARL‐17477 with combined inhibition greater than individual agent administered alone. Thus, we identified a constitutively active nNOS splice variant in MECs that is distinct from the nNOS isoform expressed in neurons and eNOS and makes significant contribution to superoxide and peroxynitrite generation. In addition, endothelial nNOS contributes to the angiotensin II‐ as well as high glucose‐induced superoxide generation that is comparable to but independent of NADPH oxidase.Support or Funding InformationSupport: PVK: Louisiana BoRSF‐RCS (LEQSF(2014‐17)‐RD‐A‐11) and AHA Scientist Development Grant (14SDG20490359); IR: AHA Postdoctoral Fellowship Grant (15POST23040005); and DWB: NIH grants (HL‐077731and HL093554).

Commentary: A Hypothesis for Examining Skeletal Muscle Biopsy-Derived Sarcolemmal nNOSμ as Surrogate for Enteric nNOSα Function

Commentary: A Hypothesis for Examining Skeletal Muscle Biopsy-Derived Sarcolemmal nNOSμ as Surrogate for Enteric nNOSα Function

Identification of a novel splice variant of neuronal nitric oxide synthase, nNOSβ, in myofilament fraction of murine cardiomyocytes

Splice variant forms of neuronal nitric oxide synthase (nNOS or NOS1), nNOSα and nNOSμ, are well established to be functionally expressed in discrete compartments in cardiomyocytes (e.g. sarcoplasmic reticulum, SR, caveolae in plasma membrane or mitochondria). So far, whether nNOS is expressed in myofilament fraction of cardiomyocytes and the splice variant form of nNOS are unknown. Immunoblotting results using two nNOS specific antibodies (BD Transduction Laboratories aa 1095–1289 and Santa Cruz Biotechnology aa 2–300) clearly demonstrated that nNOS was abundantly expressed in myofilament-enriched fraction of cardiomyocytes. Whilst the molecular weight of nNOS in membrane/cytosol fractions was ∼165 kDa, nNOS in myofilament was below 140 kDa, suggesting that the predominant splice variant of nNOS in myofilament is nNOSβ. RT-PCR results confirmed the expressions of both nNOSα and nNOSβ mRNAs in rat cardiomyocytes. Similarly, immunoprecipitation experiments using myofilament lysates of cardiomyocytes identified nNOS with low molecular weight (M.W. ∼140 kDa), confirming nNOSβ. Intriguingly, all three splice variants of nNOS were undetectable in the lysates of cardiomyocytes (including myofilament fractions) from nNOS−/− mice (which lacks nNOSα/μ). Furthermore, nNOSβ expression in myofilament of cardiomyocytes was not different in hypertensive rats compared to the level expressed in sham. iTRAQ-based quantitative proteomics analysis revealed that nNOS regulates phosphorylations of ∼20 proteins in cardiac myofilaments. Collectively, we provide direct evidence that different splice variants of nNOS are expressed in myofilament and membrane/cytosol fractions of cardiomyocytes. Discrete expressions of various splice variants in different compartments of cardiomyocytes suggest diverse roles nNOS play in healthy and diseased heart.

The ontogeny of Mauthner cells in the brain of Labeo rohita as revealed by NADPH-d and nNOS immunohistochemistry

Nitric oxide (NO) is well demonstrated to act as a neuronal messenger in neurotransmission in vertebrate animals. We are for the first time reporting nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and neuronal nitric oxide synthase (nNOS) in the Mauthner cells (MC) of the carp, Labeo rohita brain during post-embryonic development. The NADPH-d and nNOS-positive MC were seen intensely labeled in all young stages on either side of the ventricle in the hindbrain. In adult, these cells were moderately stained with NADPH-d but not with nNOS. Further, the morphometeric analysis of the staining intensities showed the decrease in the optical densities of the MC as the development progresses. The gradual increase in cell diameter was evident from hatchling to adult (18-22μm to 98-106μm), while the nuclear diameter remained nearly unchanged (10-12μm to 18-20μm). In fishes, MC are known to be involved in the control of startle response. The NADPH-d enzymatic activity seen in the MC of early development may play an important role to activate the startle response to fast escape from the predators and unfavorable environmental conditions to survive during early phase of life. As nNOS is absent in the adult MC, we assume that the adult MC express a different splice variant of nNOS which is detected by NADPH-d, but not by the currently used antibody. Hence, the presence of nNOS (enzyme required for the synthesis of NO) in the MC suggests the involvement of NO in neurotransmission to generate the startle response rapidly. Furthermore, we also suggest that NO may be involved in other aspects such as pathway finding, neuronal cell migration, signaling and refinement during the development of the brain of L. rohita.

Salt-sensitive splice variant of nNOS expressed in the macula densa cells

Neuronal nitric oxide synthase (nNOS), which is abundantly expressed in the macula densa cells, attenuates tubuloglomerular feedback (TGF). We hypothesize that splice variants of nNOS are expressed in the macula densa, and nNOS-beta is a salt-sensitive isoform that modulates TGF. Sprague-Dawley rats received a low-, normal-, or high-salt diet for 10 days and levels of the nNOS-alpha, nNOS-beta, and nNOS-gamma were measured in the macula densa cells isolated with laser capture microdissection. Three splice variants of nNOS, alpha-, beta-, and gamma-mRNAs, were detected in the macula densa cells. After 10 days of high-salt intake, nNOS-alpha decreased markedly, whereas nNOS-beta increased two- to threefold in the macula densa measured with real-time PCR and in the renal cortex measured with Western blot. NO production in the macula densa was measured in the perfused thick ascending limb with an intact macula densa plaque with a fluorescent dye DAF-FM. When the tubular perfusate was switched from 10 to 80 mM NaCl, a maneuver to induce TGF, NO production by the macula densa was increased by 38 +/- 3% in normal-salt rats and 52 +/- 6% (P < 0.05) in the high-salt group. We found 1) macula densa cells express nNOS-alpha, nNOS-beta, and nNOS-gamma, 2) a high-salt diet enhances nNOS-beta, and 3) TGF-induced NO generation from macula densa is enhanced in high-salt diet possibly from nNOS-beta. In conclusion, we found that the splice variants of nNOS expressed in macula densa cells were alpha-, beta-, and gamma-isoforms and propose that enhanced level of nNOS-beta during high-salt intake may contribute to macula densa NO production and help attenuate TGF.

Golgi and sarcolemmal neuronal NOS differentially regulate contraction-induced fatigue and vasoconstriction in exercising mouse skeletal muscle

Signaling via the neuronal NOS (nNOS) splice variant nNOSmu is essential for skeletal muscle health and is commonly reduced in neuromuscular disease. nNOSmu is thought to be the predominant source of NO in skeletal muscle. Here we demonstrate the existence of what we believe to be a novel signaling pathway, mediated by the nNOS splice variant nNOSbeta, localized at the Golgi complex in mouse skeletal muscle cells. In contrast to muscles lacking nNOSmu alone, muscles missing both nNOSmu and nNOSbeta were severely myopathic, exhibiting structural defects in the microtubule cytoskeleton, Golgi complex, and mitochondria. Skeletal muscles lacking both nNOSmu and nNOSbeta were smaller in mass, intrinsically weak, highly susceptible to fatigue, and exhibited marked postexercise weakness. Our data indicate that nNOSbeta is a critical regulator of the structural and functional integrity of skeletal muscle and demonstrate the existence of 2 functionally distinct nNOS microdomains in skeletal muscle, created by the differential targeting of nNOSmu to the sarcolemma and nNOSbeta to the Golgi. We have previously shown that sarcolemmal nNOSmu matches the blood supply to the metabolic demands of active muscle. We now demonstrate that nNOSbeta simultaneously modulates the ability of skeletal muscle to maintain force production during and after exercise. We conclude therefore that nNOS splice variants are critical regulators of skeletal muscle exercise performance.

Nitric oxide regulates skeletal muscle performance by compartmentalization of nNOS splice variants

Regulation of the subcellular localization of nitric oxide synthases is important for conferring specificity to intracrine nitric oxide signaling. We report that two splice variants of nNOS, nNOSμ and nNOSβ, are differentially targeted to the sarcolemma and the Golgi complex in skeletal muscle, respectively. Sarcolemmal nNOSμ and Golgi nNOSβ?exhibit independent and overlapping functions in modulating the muscle phenotype. First, nNOSμ and NOSβ?differentially regulate skeletal muscle mass through sex‐specific mechanisms. Second, nNOSμ‐deficient skeletal muscles display increased susceptibility to contraction‐induced fatigue, independently of structural defects. However, muscles lacking both nNOSμ and nNOSβ display markedly exacerbated fatigue associated with specific force deficits, an aberrant microtubule cytoskeleton, fewer fatigue‐resistant type IIa fibers and greater numbers of fatigable type IIb fibers. Thus, Golgi nNOSβ appears to modulate susceptibility to contraction‐induced fatigue through alterations in muscle structural integrity and shifts in fiber composition. These data suggest that one function of alternative splicing of the NOS1 gene is the creation of isozyme‐specific signaling compartments. We conclude that sarcolemmal nNOSμ and Golgi nNOSβ? are critical regulators of skeletal muscle performance and that the spatial confinement of nNOS isozymes plays an important role in imparting specificity to NO signaling.

Expression of neuronal nitric oxide synthase splice variants in atherosclerotic plaques of apoE knockout mice

Objective We previously reported that deletion of brain type neuronal nitric oxide synthase-α (nNOS-α) accelerates atherosclerosis in apolipoproteinE (apoE) knockout (ko) mice. The regulation of nNOS expression is complex, involving the generation of mRNA splice variants. The current study investigates occurrence and distribution of nNOS variants in atherosclerotic lesions of apoE ko and apoE/nNOS-α double ko (dko) animals. Methods Mice were fed a high fat diet for 20 weeks. Immunohistochemistry and western blot analysis were performed using antibodies detecting the carboxy terminal-, or amino terminal-residue of the nNOS protein. Confocal microscopy and in situ hybridization were used to identify the compartment of cellular expression. Results In situ hybridization revealed the presence of nNOS-α and -γ mRNA variants in apoE ko plaques, while only nNOS-γ was detectable in apoE/nNOS dko plaques. Consistent with mRNA expression nNOS-α protein can be detected in the neointima of apoE ko, but not apoE/nNOS dko animals. In contrast, the carboxy terminal antibody stained the neointima and media in apoE ko vessels and showed residual nNOS immunoreactivity in apoE/nNOS dko lesions. Confocal microscopy showed predominant nNOS expression in vascular smooth muscle cells, while colocalization with macrophages was less pronounced. Conclusions Our study shows that nNOS-α and -γ splice variants are expressed in atherosclerotic plaques of apoE ko mice. nNOS variants colocalized with markers for vascular smooth muscle cells and macrophages but not for endothelial cells. Since nNOS-α is atheroprotective, other nNOS splice variants which differ in enzyme kinetic and subcellular localization may also influence plaque formation.

Splice variants of neuronal nitric oxide synthase are present in the rat kidney

Decreased renal cortical neuronal NO synthase (nNOS) abundance/activity correlates with progression of chronic kidney disease (CKD) in a number of animal models. Western blotting with both N-terminal and C-terminal antibodies, immunoprecipitation, proteomics, RT-PCR and in situ hybridization were used to identify nNOS splice variants in the rat kidney. We have identified two nNOS proteins and transcripts in the rat kidney; nNOSalpha (approximately 160 kDa) and nNOSbeta (approximately 140 kDa), a catalytically active exon-2 deletion variant, lacking both the PDZ and protein inhibitor of nNOS (PIN) domains. We also report that nNOSbeta protein abundance is increased in the kidney at 11 weeks following 5/6th nephrectomy (5/6NX)-induced CKD while nNOSalpha protein abundance is diminished. The transcript data parallel the protein data in 5/6NX. By in situ hybridization, there is abundant nNOSalpha mRNA widely distributed throughout the normal kidney cortex, with very sparse nNOSbeta mRNA confined to a few proximal tubules. In a second injury model (6 weeks after 5/6 renal mass reduction by combined right kidney ablation and infarction of approximately 2/3 of the left kidney; 5/6 A/I), nNOSalpha mRNA almost disappears from the kidney cortex while nNOSbeta mRNA abundance increases in tubules and tubulo-interstitium. The renal cortical nNOSbeta protein is present in low abundance in the normal kidney and increases with injury, in an inverse pattern of change with the nNOSalpha.

A Novel nNOS Signaling Pathway Regulates Skeletal Muscle Size, Strength and Fatigue Resistance

Nitric oxide (NO) is a highly versatile signaling molecule which participates in a broad array of pathways in the cardiovascular, immune and nervous systems. We present further evidence for this physiological diversity by identifying a novel nNOS signaling pathway in skeletal muscle. NO is synthesized by neuronal nitric oxide synthase mu isoenzyme (nNOSμ) in skeletal muscle. nNOSμ is found associated with the dystrophin‐associated glycoprotein complex and in a soluble pool. During investigation of the soluble nNOS pool, we identified a splice variant of nNOS called nNOSβ localized to the Golgi complex. Soluble guanylyl cyclase and protein kinase G also localized to the Golgi suggesting that a classical NO–cGMP signaling pathway functioned at the Golgi complex. Loss of nNOSβ activity resulted in reduced muscle mass with muscles exhibiting lower force‐generating capacity, even after correcting for decreased muscle size. Furthermore, the absence of nNOS decreased the ability of muscle to resist exercise‐induced fatigue. In summary, we have identified a novel nNOS‐signaling pathway that regulates skeletal muscle size and strength and provided the first evidence that differential targeting of nNOS isozymes facilitates the creation of spatially and functionally distinct nNOS signaling pathways by localized synthesis of an otherwise directionless free radical gas. Furthermore, these results make nNOSβ of potential relevance to myopathies and dystrophies where preventing the loss of muscle bulk and strength is of clinical importance.

Atheroprotective Effects of Neuronal Nitric Oxide Synthase in Apolipoprotein E Knockout Mice

All 3 isoforms of the nitric oxide synthase (NOS) are expressed in atherosclerotic lesions. To test whether neuronal NOS (nNOS) deficiency affects atherosclerosis, we studied apoE/nNOSalpha double knockout (DKO) and apolipoprotein E (apoE) knockout (KO) control mice. Lesion area was significantly increased in male DKO (66%) mice after 14 weeks and in female DKO animals (31%) after 24 weeks of "western" diet. Moreover, mean arterial blood pressure was significantly reduced in female DKO animals. Immunohistochemistry revealed nNOS expression in the neointima of KO mice. In DKO animals, residual nNOS staining was caused by the presence of nNOS splice variants. Whereas nNOSalpha was present in vessels of KO and absent in DKO animals, nNOSgamma was expressed in KO and DKO mice. nNOSalpha protects against atherosclerosis as nNOSalpha deletion leads to an increase in plaque formation in apoE/nNOSalpha DKO mice. Female DKO mice showed a significant reduction in mean arterial blood pressure. Additionally, we found expression of nNOS splice variants in vessels of apoE KO mice. Our data highlights nNOSalpha overexpression as a potential therapeutic strategy and naturally occurring splice variants that lack exon 2 of the nNOS gene as a potential risk factor for vascular disease.

Alternatively spliced neuronal nitric oxide synthase mediates penile erection

A key role for nitric oxide (NO) in penile erection is well established, but the relative roles of the neuronal NO synthase (nNOS) versus endothelial forms of NOS are not clear. nNOS- and endothelial NOS-deficient mice maintain erectile function and reproductive capacity, questioning the importance of NO. Alternatively, residual NO produced by shorter transcripts in the nNOS(-/-) animals might suffice for normal physiologic function. We show that the beta splice variant of nNOS elicits normal erection despite a decrease in stimulus-response characteristics and a 5-fold increased sensitivity to the NOS inhibitor, l-NAME. Residual nNOSbeta generates only 10% of the normal NO level in vitro but produces citrulline and diaphorase staining reflecting in vivo NOS activity in pelvic ganglion nerves that is comparable to WT animals. Thus, alternatively spliced forms of nNOS are major mediators of penile erection and so may be targets for therapeutic intervention.

Myenteric neurons and interstitial cells of Cajal of mouse colon express several nitric oxide synthase isoforms

Information on equipment and subcellular distribution of nitric oxide synthase (NOS) isoforms in myenteric neurons and pacemaker cells (ICC) might help to identify nitric oxide (NO) pathway(s) acting on gastrointestinal motility. In sections of mouse colon labelled with neuronal (n)NOS, endothelial (e)NOS and inducible (i)NOS antibodies, all myenteric neurons co-expressed eNOS and iNOS and a subpopulation of them co-expressed nNOS. ICC co-expressed nNOS and eNOS. In the neurons, nNOS-labeling was intracytoplasmatic, in the ICC at cell periphery. In both cell types, eNOS-labeling was on intracytoplasmatic granules, likely mitochondria. In conclusion, myenteric neurons and ICC co-express several NOS isoforms with specific subcellular distribution. Different nNOS splice variants are presumably present: intracytoplasmatic nNOSβ and nNOSα producing neurogenic NO, plasma membrane-bound nNOSα producing ICCgenic NO. eNOS might be implicated in mitochondrial respiration and, in ICC, also in pacemaker activity. Neurons express iNOS also in basal condition.

Differential distribution and transcriptional control of human nnos splice variants in the gastrointestinal tract

Differential distribution and transcriptional control of human nnos splice variants in the gastrointestinal tract

Isolation and Characterization of a Novel, Human Neuronal Nitric Oxide Synthase cDNA

A splice variant of nNOS has recently been identified in both rat and mouse which contains an in-frame insertion of 34 conserved amino acids between the N-terminal oxygenase and the C-terminal reductase domains. In the present study we report the isolation and characterization of a similar, but not identical (76% amino acid identity), human variant (nNOSμ) which arises from the splicing in of an additional exon between exons 16 and 17 of the human nNOS gene. Furthermore, we describe two additional splice variants which, if translated, would give rise to truncated forms of nNOS lacking the C-terminal reductase domain. These additional variants and nNOSμ have a distinct and more restricted expression pattern compared to nNOS. Further studies are required to elucidate the possible physiological roles of these novel human nNOS splice variants in NO signaling.