Specificity of Antibodies to Nitric Oxide Synthase Isoforms in Human, Guinea Pig, Rat, and Mouse Tissues

Coers, W; Timens, Wim; Kempinga, Claudia; Klok, Pieter; Moshage, Han

doi:10.1177/002215549804601207

Cited by 66 publications

(46 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Although no quantitative description about the density of nNOS-IR neurons in the hippocampus has been previously reported, there appears to be a greater number of nNOS-IR neurons in the present study than reported by others. The mouse monoclonal anti-nNOS antibody used has been shown to react specifically with the 150-kDa human nNOS of the recombinant protein or brain extract (Western blot), showing sufficient isoform specificity to undertake reliable immunohistochemical staining (30). We may have observed a greater density because previous studies used different fixation techniques (formalin-fixed tissue versus frozen sections) and detected nNOS expression using polyclonal antibodies or NADPH-diaphorase histochemistry.…”

Section: Discussionmentioning

confidence: 99%

Expression of neuronal nitric oxide synthase in the hippocampal formation in affective disorders

Oliveira

Guimarães

Deakin

2008

Braz J Med Biol Res

112

View full text Add to dashboard Cite

Hippocampal output is increased in affective disorders and is mediated by increased glutamatergic input via N-methyl-Daspartate (NMDA) receptor and moderated by antidepressant treatment. Activation of NMDA receptors by glutamate evokes the release of nitric oxide (NO) by the activation of neuronal nitric oxide synthase (nNOS). The human hippocampus contains a high density of NMDA receptors and nNOS-expressing neurons suggesting the existence of an NMDA-NO transduction pathway which can be involved in the pathogenesis of affective disorders. We tested the hypothesis that nNOS expression is increased in the human hippocampus from affectively ill patients. Immunocytochemistry was used to demonstrate nNOS-expressing neurons in sections obtained from the Stanley Consortium postmortem brain collection from patients with major depression (MD, N = 15), bipolar disorder (BD, N = 15), and schizophrenia (N = 15) and from controls (N = 15). nNOS-immunoreactive (nNOS-IR) and Nissl-stained neurons were counted in entorhinal cortex, hippocampal CA1, CA2, CA3, and CA4 subfields, and subiculum. The numbers of Nissl-stained neurons were very similar in different diagnostic groups and correlated significantly with the number of nNOS-IR neurons. Both the MD and the BD groups had greater number of nNOS-IR neurons/400 µm 2 in CA1 (mean ± SEM: MD = 9.2 ± 0.6 and BD = 8.4 ± 0.6) and subiculum (BD = 6.7 ± 0.4) when compared to control group (6.6 ± 0.5) and this was significantly more marked in samples from the right hemisphere. These changes were specific to affective disorders since no changes were seen in the schizophrenic group (6.7 ± 0.8). The results support the current view of the NMDA-NO pathway as a target for the pathophysiology of affective disorders and antidepressant drug development.

show abstract

Section: Discussionmentioning

confidence: 99%

Expression of neuronal nitric oxide synthase in the hippocampal formation in affective disorders

Oliveira

Guimarães

Deakin

2008

Braz J Med Biol Res

112

View full text Add to dashboard Cite

show abstract

“…One possible reason for this disagreement is the wide heterogeneity of the experimental models studied so far. Additional discrepancy may arise from the fact that the primary antibodies used to detect iNOS come from several sources, because the behavior of different antibodies directed against NOS isoforms can vary dramatically according to type (monoclonal versus polyclonal), species in which the antibody was raised, and tissue in which the antibody is tested (19).…”

mentioning

confidence: 99%

Evidence for the Existence of Two Distinct Functions for the Inducible NO Synthase in the Rat Kidney

Fujihara¹,

Mattar²,

Vieira³

et al. 2002

Journal of the American Society of Nephrology

View full text Add to dashboard Cite

Abstract. The functional role of the NO synthase (NOS) isoforms in the normal or diseased kidney is uncertain. This study examined the renal expression of the endothelial (eNOS), neuronal (nNOS), and inducible (iNOS) isoforms by both immunohistochemistry and Western blot analyses in shamoperated rats (S) and in rats subjected to 5/6 nephrectomy (Nx). Primary antibodies from two different sources were used to detect iNOS. Additional S and Nx rats were chronically treated with aminoguanidine (AG), a selective iNOS inhibitor. All three isoforms were clearly expressed in S kidney. Their renal abundance, evaluated by Western blot analysis, fell in Nx rats. With the use of anti-iNOS antibodies from two distinct sources, the immunohistochemical analysis showed the presence of what appeared to be two distinct iNOS fractions: a "tubular" fraction, present in S and with decreased intensity in Nx; and an "interstitial" fraction, observed only in inflamed areas of Nx rats. AG treatment greatly attenuated renal injury in Nx rats by a direct antiinflammatory effect, likely related to iNOS inhibition, rather than to amelioration of renal hemodynamics or to reduced protein glycation. These observations suggest that: (1) the functional role of the renal iNOS isoform may vary dramatically under different physiologic conditions; (2) caution should be taken in the interpretation of immunohistochemical iNOS data, because antibodies from different sources may detect different iNOS fractions; and (3) AG treatment may become useful in the treatment of human progressive nephropathies, even those not associated with diabetes or aging.

show abstract

“…Recent work 45,46 suggested that a novel mechanism involving iNOS ubiquitination and proteasomic proteolysis could also play a role in the regulation of iNOS activity. As previously proposed by Zhao et al 21 and Coers et al, 47 the small molecular weight bands revealed by Western blot could be considered as proteolytic degradation products. However, further work in this direction should be performed in order to confirm this hypothesis.…”

Section: Discussionmentioning

confidence: 52%

Post-transcriptional regulation of inducible nitric oxide synthase in chronic lymphocytic leukemia B cells in pro- and antiapoptotic culture conditions

et al. 2003

View full text Add to dashboard Cite

Functional inducible NOS (iNOS) may be involved in the prolonged lifespan of chronic lymphocytic leukemia cells (B-CLL), although the exact mechanisms implicated remain elusive as yet. In this work, we have examined iNOS expression in normal B lymphocytes and B-CLL cells in pro-and antiapoptotic conditions. Our results demonstrate: (1) The existence of a new splice variant characterized by a complete deletion of exon 14 (iNOS 13-16 14del ), which was preferentially detected in normal B lymphocytes and may represent an isoform that could play a role in the regulation of enzyme activity. (2) The existence of another alternatively spliced iNOS mRNA transcript involving a partial deletion of the flavodoxin region (iNOS 13-16 neg ) was correlated to a decreased B-CLL cell viability. The 9-b-D-arabinofuranosyl-2-fluoradenine or fludarabine (F-ara) treatment induced iNOS 13-16 neg transcript variants, whereas IL-4 enhanced both the transcription of variants, including these exons (iNOS 13-16 pos ), and the expression of a 122 kDa iNOS protein. These results suggest that in B-CLL, a regulation process involving nitric oxide ( K NO) levels could occur by a post-transcriptional mechanism mediated by soluble factors. Our results also provide an insight into a new complementary proapoptotic action of F-ara in B-CLL by the induction of particular iNOS splice variants, leading to the activation of a caspase-3-dependent apoptotic pathway.

show abstract

Specificity of Antibodies to Nitric Oxide Synthase Isoforms in Human, Guinea Pig, Rat, and Mouse Tissues

Cited by 66 publications

References 19 publications

Expression of neuronal nitric oxide synthase in the hippocampal formation in affective disorders

Expression of neuronal nitric oxide synthase in the hippocampal formation in affective disorders

Evidence for the Existence of Two Distinct Functions for the Inducible NO Synthase in the Rat Kidney

Post-transcriptional regulation of inducible nitric oxide synthase in chronic lymphocytic leukemia B cells in pro- and antiapoptotic culture conditions

Contact Info

Product

Resources

About