Loss of sarcolemmal nNOS is common in acquired and inherited neuromuscular disorders.

OBJECTIVE: Neuronal nitric oxide synthase (nNOS), normally expressed at the sarcolemmal membrane, is known to be mislocalized to the sarcoplasm in several forms of muscular dystrophy. Our objectives were to characterize further the range of patients manifesting aberrant nNOS sarcolemmal immunolocalization and to study nNOS localization in animal models of nondystrophic myopathy. METHODS: We carried out a retrospective cross-sectional study. We performed immunofluorescent staining for nNOS on biopsy specimens from 161 patients with acquired and nondystrophin inherited neuromuscular conditions. The localization of sarcolemmal nNOS correlated with mobility and functional status. Muscle specimens from mouse models of steroid-induced and starvation-related atrophy were studied for qualitative and quantitative nNOS expression. RESULTS: Sarcolemmal nNOS staining was abnormal in 42% of patients with inherited myopathic conditions, 25% with acquired myopathic conditions, 57% with neurogenic conditions, and 93% with hypotonia. Interestingly, we found significant associations between mobility status or muscle function and sarcolemmal nNOS expression. Furthermore, mouse models of catabolic stress also demonstrated mislocalization of sarcolemmal nNOS. CONCLUSION: Our analyses indicate that nNOS mislocalization is observed in a broad range of nondystrophic neuromuscular conditions associated with impaired mobility status and catabolic stress. Our findings suggest that the assessment of sarcolemmal localization of nNOS represents an important tool for the evaluation of muscle biopsies of patients with a variety of inherited and acquired forms of neuromuscular disorders.

Differential expression of calcium channel subtypes in the bovine adrenal medulla.

This study aimed at determining the distribution and expression levels of different subtypes of Ca(2+) channels in the bovine adrenal medulla, and whether individual subtypes were more abundant in chromaffin cells exhibiting an adrenergic or a noradrenergic phenotype. In situ hybridization using riboprobes specific for the pore-forming Ca(2+) channel alpha(1D) (L-type channel), alpha(1B) (N-type channel), and alpha(1A) (P/Q-type channel) subunits of bovine chromaffin cells showed a broad distribution of the three transcripts in adrenal medulla tissue. However, a tissue-specific expression pattern of individual subunits was found; whereas alpha(1B) mRNA was homogeneously distributed throughout the medulla, alpha(1D) and alpha(1A) transcripts were present at higher densities in the internal medullary area, far away from the adrenal cortex. These results were corroborated by comparative analysis of the alpha(1B), alpha(1D), and alpha(1A) products amplified by RT-PCR from total RNA extracted from small pieces of tissue dissected out from external or internal medullary areas. Interestingly, immunohistochemical experiments performed in adrenal gland sections, using antidopamine-beta-hydroxylase and anti-phenylethanolamine-N-methyltransferase antibodies, indicated a higher density of noradrenergic over adrenergic chromaffin cells in the internal medullary region. These results provide direct evidence in favor of a heterogeneous distribution of Ca(2+) channel subtypes in the adrenal medulla, in agreement with previous functional data showing that blockade of the high K+ -elicited responses by dihydropyridines was greater in noradrenergic than in adrenergic chromaffin cells. These differences may be relevant for the differential release regulation of each catecholamine under physiological and pathophysiological conditions.

Intracellular glutathione deficiency is associated with enhanced nuclear factor-kappaB activation in older non-insulin dependent diabetic patients.

Diabetes mellitus may be associated with intracellular glutathione (GSH) deficiency. Since in vivo studies have shown that plasma intracellular GSH plays a key role in regulating the activation of nuclear factor kappaB (NF-kappaB), we have investigated the relationship between intracellular thiols (GSH, homocysteine, cysteine and cysteinyglycine) and NF-kappaB activity in the peripheral blood mononuclear cells (PBMC) of 63 elderly non-insulin dependent diabetes mellitus (NIDDM) patients (28 microalbuminurics and 35 normoalbuminurics) and 30 healthy age- and sex-matched subjects. In addition, we have measured plasma concentrations of these thiol compounds, serum concentrations of interleukin-6 (IL-6) and vascular cell adhesion molecule-1 (sVCAM-1), that are partly dependent on the NF-kappaB activation, as well as the serum levels of thiobarbituric acid reacting substances (TBARS), as index of lipid peroxidation. Diabetic patients with microalbuminuria (MAB) and normoalbuminuria had NF-kappaB activity 2.1- and 1.5-fold greater, respectively, than the control group. As compared to normoalbuminuric patients, patients with MAB had significantly higher levels of glycemia, plasma homocysteine, and serum concentrations of TBARS, IL-6 and sVCAM-1 (in all cases, p < 0.01), and significantly lower GSH content in the PBMC (p < 0.05). The intracellular GSH in PBMC correlated with NF-kappaB activation (r = -0.82; p < 0.0001), serum TBARS (r = -0.60; p < 0.001), and with fasting glycemia (r = -0.56; p < 0.001) in patients with MAB, whereas a weaker association between GSH levels in PBMC and NF-kappaB activation (r = -0.504, p < 0.001) was seen in patients without MAB. These results suggest that the decrease of intracellular GSH content in elderly NIDDM patients with MAB is strongly associated with enhanced NF-kappaB activation, which could contribute to the development of increased glomerular capillary permeability and its rapid progression.