Further characterisation of particulate neuronal nitric oxide synthase in rat small intestine.
Neuronal NO-synthase (nNOS) was investigated in rat longitudinal muscle/myenteric plexus (LM/MP) tissue at the cellular and subcellular level. Using preparations and double immune staining and light and electron microscopy, we concluded that, in these preparations, nNOS is only present in neuronal cells. However, in spite of numerous attempts to morphologically identify the NOS-containing subcellular structure, no firm conclusions were possible. Consequently, the problem was approached by biochemical methods including gradient centrifugation followed by analysis of the fractions. Using a protocol involving gentle homogenisation of the tissue, we found that about 10% of the nNOS immune reactivity was particle-bound confirming previous results (Biochem. Pharmacol. 60 (2000) 145). However, applying a different protocol including strong homogenisation, we now demonstrated that about 50% of the immune reactive nNOS was sedimentable. The results suggested that particulate nNOS is associated with one single subcellular structure, which is different from the plasma membrane, rough and smooth endoplasmic reticulum, mitochondria and lysosomes. The equilibrium sedimentation characteristics of the nNOS containing particles corresponded partly to those containing vasoactive intestinal polypeptide (VIP) or synaptobrevin. Application of non-equilibrium centrifugation conditions, however, demonstrated that almost no co-localisation occurred. We conclude that, in the LM/MP tissue, nNOS is about 50% particle-bound in a subcellular structure, which is different from the VIP-containing particle and from synaptobrevin-containing exocytotic particles.
Van Geldre
Lieve A
LA
Heymans Institute of Pharmacology, Ghent University, De Pintelaan 185, B-9000 Ghent, Belgium.
Fraeyman
Norbert H
NH
Peeters
Theo L
TL
Timmermans
Jean-Pierre
JP
Lefebvre
Romain A
RA
D000818:Animals; D002451:Cell Compartmentation / Q000502:physiology; D002478:Cells, Cultured; D007150:Immunohistochemistry; D007421:Intestine, Small / Q000201:enzymology* / Q000294:innervation* / Q000648:ultrastructure; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D009130:Muscle, Smooth / Q000201:enzymology* / Q000294:innervation* / Q000648:ultrastructure; D009197:Myenteric Plexus / Q000201:enzymology* / Q000648:ultrastructure; D009474:Neurons / Q000201:enzymology / Q000648:ultrastructure; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D015388:Organelles / Q000201:enzymology / Q000648:ultrastructure; D050683:R-SNARE Proteins; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions / Q000201:enzymology; D022161:Transport Vesicles / Q000378:metabolism / Q000648:ultrastructure; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D008565:Membrane Proteins; D050683:R-SNARE Proteins; D014660:Vasoactive Intestinal Peptide; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat