The role of arterial smooth muscle in vasorelaxation.

The concept of endothelium-derived relaxing factor (EDRF) implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium in response to vasorelaxants such as acetylcholine (ACh) acts on the underlying vascular smooth muscle cells (VSMC) inducing vascular relaxation. The EDRF concept was derived from experiments on denuded blood vessel strips and, in frames of this concept, VSMC were regarded as passive recipients of NO from endothelial cells. However, it was later found that VSMC express NOS by themselves, but the principal question remained unanswered, is the NO generation by VSMC physiologically relevant? We hypothesized that the destruction of the vascular wall anatomical integrity by rubbing off the endothelial layer might increase vascular superoxides that, in turn, reduced the NO bioactivity as a relaxing factor. To test our hypothesis, we examined ACh-induced vasorelaxation under protection against oxidative stress and found that superoxide scavengers restored vasodilatory responses to ACh in endothelium-deprived blood vessels. These findings imply that VSMC can release NO in amounts sufficient to account for the vasorelaxatory response and challenge the concept of the obligatory role of endothelial cells in the relaxation of arterial smooth muscle.

Nitric oxide synthase is up-regulated in muscle fibers in muscular dystrophy.

Nitric oxide (NO) mediates fundamental physiological actions on skeletal muscle. The neuronal NO synthase isoform (NOS1) was reported to be located exclusively in the sarcolemma. Its loss from the sarcolemma was associated with development of Duchenne muscular dystrophy (DMD). However, new studies evidence that all three NOS isoforms-NOS1, NOS2, and NOS3-are co-expressed in the sarcoplasm both in normal and in DMD skeletal muscles. To address this controversy, we assayed NOS expression in DMD myofibers in situ cytophotometrically and found NOS expression in DMD myofibers up-regulated. These results support the hypothesis that NO deficiency with consequent muscle degeneration in DMD results from NO scavenging by superoxides rather than from reduced NOS expression.

Nitric oxide synthase in muscular dystrophies: a re-evaluation.

Duchenne and Becker muscular dystrophies (DMD and BMD) are associated with decreased total nitric oxide (NO). However, mechanisms leading to NO deficiency with consequent muscle-cell degeneration remain unknown. To address this issue, we examined skeletal muscles of DMD and BMD patients for co-expression of NO synthase (NOS) with nitrotyrosine and transcription factor CREB, as well as with enzymes engaged in NO signaling. Employing immunocytochemical labeling, Western blotting and RT-PCR, we found that, in contrast to the most commonly accepted view, neuronal NOS was not restricted to the sarcolemma and that muscles of DMD and BMD patients retained all three NOS isoforms with an up-regulation of the inducible NOS isoform, CREB and nitrotyrosine. We suggest that enhanced nitrotyrosine immunostaining in muscle fibers as well as in the vasculature of DMD and BMD specimens reflects massive oxidative stress, resulting in withdrawal of NO from its regular physiological course via the scavenging actions of superoxides.

Compartmentalization of NO signaling cascade in skeletal muscles.

Skeletal muscle functions regulated by NO are now firmly established. However, the literature on the compartmentalization of NO signaling in myocytes is highly controversial. To address this issue, we examined localization of enzymes engaged in L-arginine-NO-cGMP signaling in the rat quadriceps muscle. Employing immunocytochemical labeling complemented with tyramide signal amplification and electron microscopy, we found NO synthase expressed not only in the sarcolemma, but also along contractile fibers, in the sarcoplasmic reticulum and mitochondria. The expression pattern of NO synthase in myocytes showed striking parallels with the enzymes engaged in L-arginine-NO-cGMP signaling (arginase, phosphodiesterase, and soluble guanylyl cyclase). Our findings are indicative of an autocrine fashion of NO signaling in skeletal muscles at both cellular and subcellular levels, and challenge the notion that the NO generation is restricted to the sarcolemma.

An in situ evidence for autocrine function of NO in the vasculature.

The concept of endothelium derived relaxing factor (EDRF) implies that nitric oxide (NO) generated by NO synthase in the endothelium diffuses to the underlying vascular smooth muscle cells (VSMC) modulating thereby vascular tone. VSMC were regarded as passive recipients of NO from endothelial cells. However, this paradigm of a paracrine function of NO became currently subject to considerable debate. To address this issue, we examined the localization of enzymes engaged in l-arginine-NO-cGMP signaling in the rat blood vessels. Employing multiple immunocytochemical labeling complemented with signal amplification, electron microscopy, Western blotting, and RT-PCR, we found that NO synthase was differentially expressed in blood vessels depending on the blood vessel type. Moreover, the expression pattern of NO synthase in VSMC showed striking parallels with arginase and soluble guanylyl cyclase. Our findings challenge the commonly accepted view that the expression of NO synthase is restricted to vascular endothelial cells and lends further support to an alternative mechanism, by which constitutive local NOS expression in VSMC may modulate vascular functions in an endothelium-independent manner. Moreover, the co-expression of enzymes engaged in l-arginine-NO-cGMP signaling (NO synthase, arginase, and soluble guanylyl cyclase) in VSMC is indicative of an autocrine fashion of NO signaling in the vasculature in addition to the paracrine role of NO generated in the endothelium.

Cellular control of nitric oxide synthase expression and activity in rat cardiomyocytes.

Potential ortho- and pathophysiological roles for nitric oxide synthases (NOS) in cardiac functions have been and are continuing to be described. However, cellular signaling mechanisms controlling nitric oxide (NO) production in the heart remain obscure. The aim of this study was to investigate signaling mechanisms involved in regulation of NOS expression and NO generation in cardiomyocytes. Using immunocytochemical methods in conjunction with western blotting, we have found that cultured neonatal rat cardiomyocytes express constitutively all three NOS isoforms targeted predominantly to the particulate component of cardiomyocytes - mitochondria and along contractile fibers, as well as along plasma membrane including T-tubules. Biochemical assay of NO generation has shown that exposure of cultured neonatal rat cardiac cells to isoproterenol (beta-adrenergic stimulation), iloprost [stable prostaglandin I(2) (PGI(2)) analogue], as well as inflammatory cytokines and dibutyryl adenosine-3',5'-monophosphate (db-cAMP), resulted in a marked up-regulation of NOS expression by cardiomyocytes. In db-cAMP-stimulated cells, inhibition of protein kinase A (PKA) and protein kinase C (PKC) reduced immunolabeling of NOS and concomitantly lowered NO production. Taken together, these data point to an involvement of beta-adrenergic mechanisms, cytokine and PGI(2) receptors, adenylyl cyclase, PKA, and PKC in the control of NO generation and expression of NOS in rat cardiomyocytes.

Vascular smooth muscle and nitric oxide synthase.

The concept of endothelium-derived relaxing factor (EDRF) put forward in 1980 by Furchgott and Zawadzki implies that nitric oxide (NO) produced by NO synthase (NOS) in the endothelium diffuses to the underlying vascular smooth muscle, where it modulates vascular tone as well as vascular smooth muscle cell (VSMC) proliferation by increasing cGMP formation with subsequent activation of cGMP-dependent protein kinase. According to this concept, VSMC do not express NOS by themselves. This attractive, simple scheme is now under considerable debate. To address this issue, we designed this study with the use of a novel supersensitive immunocytochemical technique of signal amplification with tyramide and electron microscopic immunogold labeling complemented with Western blotting, as in our recent studies demonstrating NOS in the myocardial and skeletal muscles. We provide the first evidence that, in contrast to the currently accepted view, VSMC in various blood vessels express all three NOS isoforms depending on the blood vessel type. These findings suggest an alternative mechanism by which local NOS expression may modulate vascular functions in an endothelium-independent manner.