Immunological detection of nitric oxide synthase(s) in human tissues using heterologous antibodies suggesting different isoforms

Abstract

Nitric oxide (NO) is generated from L-arginine by NO synthases. Localization of the brain enzyme has been carried out in the rat; however, despite data suggesting that NO is a major regulator of vascular and neural functions in man, there is no information about the localization of NO synthase in human tissues. Rabbit antisera to NO synthase purified from rat brain (antisera A and B) were raised, tested by Western blotting, affinity purification and enzyme immunoprecipitation assay, and used to investigate the distribution of the enzyme in a variety of human tissues by immunohistochemistry. Antisera to two synthetic peptides from cloned neural NO synthase were used to aid specificity testing. Anti-sera A and B reacted with a approximately 160-kDa protein in Western blots of human brain extracts, gave immunostaining of nerves, and precipitated enzyme activity from rat brain homogenates. Antiserum B to NO synthase also reacted with proteins of M(r) between 125 and 140 kDa in extracts of well-vascularised tissues, and immunostained vascular endothelium; the neural and vascular immunoreactivity persisted after affinity purification of antiserum B with the approximately 160 kDa protein. Endothelial staining with antiserum B was seen in respiratory tract, liver, skin and umbilicus; syncytial trophoblasts stained in the placenta. Neural staining with antiserum A and B was seen in the myenteric and submucous plexus, and in nerve fibres in smooth muscle of the gut and in many areas of the central nervous system, particularly cortex, hippocampus, hypothalamus, cerebellum, brain stem and spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide synthase activity in human breast cancer

“…Two antibodies were used for NO synthase detection: (1) a monoclonal antibody to inducible NO synthase from murine macrophages (ANTI-macNOS; Transduction Laboratories, Lexington, KY, USA) which immunoreacts with human inducible NO synthase (I Charles, unpublished observation) and (2) polyclonal antiserum to NO synthase raised in rabbits using purified rat brain NO synthase (Spnmgall et al, 1992). At the dilution used this antiserum has proven reactivity with endothelial and neuronal constitutive NO synthase isoenzymes across several species including humans (Springall et al, 1992;Brave et al, 1993;Terenghi et al, 1993). At a lower dilution (1:100) in Western blots this antiserum also cross-reacts with inducible NO synthase (Thomsen et al, 1994 'One benign cystic change, one stromal fibrosis and three fibroadenomas.…”

“…consistent with the presence of constitutive NO synthase was immunoreactive with the polyclonal antiserum to NO synthase in these tumours. This antiserum, which immunoreacts with endothelial and neuronal constitutive NO synthases (Springall et al, 1992;Brave et al, 1993;Terenghi et al, 1993). labelled vascular endothelial cells and myoepithelial cells in addition to macrophages.…”

Nitric oxide synthase

“…The macrophage enzyme was able to compete weakly with the binding of the brain enzyme to its own antibody, but 10 times more inducible protein was required. We propose that conformational epitopes are responsible for the cross-reaction, suggesting that the overall tertiary structures of the two enzymes share a degree of similarity.Nitric oxide (NO) synthase has been the subject of intensive study, focusing on the mode of action Stuehr, 1992;Bredt et al, 1992), distribution in different tissues, organs and species (Salter et al, 1991 ;Springall et al, 1992;Bredt et al, 1990), as well as comparisons between the enzymes at the molecular level (Bredt et al …”

“…Nitric oxide (NO) synthase has been the subject of intensive study, focusing on the mode of action Stuehr, 1992;Bredt et al, 1992), distribution in different tissues, organs and species (Salter et al, 1991 ;Springall et al, 1992;Bredt et al, 1990), as well as comparisons between the enzymes at the molecular level (Bredt et al, 1991;Lyons et al, 1992;Xie et al, 1992;Janssens et al, 1992;Lowenstein et al, 1992;Sessa et al, 1992).…”

“…We have made antibodies against the rat brain constitutive enzyme and used them to identify specific cross-reactivity in human tissues by immunohistochemical techniques (Springall et al, 1992;Terenghi et al, 1993).…”