Immunohistochemical evidence from co-localization and denervation studies for four types of substance P-containing nervous structures in the rat superior cervical ganglion.

Four types of substance P-immunoreactive structures have been distinguished in the rat superior cervical ganglion by double-immunofluorescence microscopy: (1) A major population of mainly varicose fibres enmeshed singly-scattered neuronal perikarya, some of which contained vasoactive intestinal polypeptide-immunoreactivity. These substance P-immunoreactive fibres did not contain colocalized calcitonin gene-related peptide (CGRP) and were absent after transection of the cervical sympathetic trunk. (2) A rather small substance P-immunoreactive fibre population with colocalized calcitonin gene-related peptide-immunoreactivity was distributed in a patchy manner and disappeared after cutting the postganglionic branches. (3) Most of the intraganglionic small intensely fluorescent (SIF) cell clusters were intensely substance P-immunoreactive. SIF cells were not visibly changed in number and fluorescence intensity by either surgical procedure. (4) Immunoreactivity was not visible in principal ganglionic neurons of control ganglia, but occurred in cell bodies after pre- as well as after postganglionic nerve transection. Some of the substance P-immunolabelled perikarya in addition revealed immunostaining to antisera against the catecholamine-synthesizin enzyme tyrosine hydroxylase or against the neuropeptides leu-enkephalin and vasoactive intestinal polypeptide, respectively. The results strongly suggest that, in addition to a substance P-containing preganglionic input (1), and a supply by substance P-containing sensory axon collaterals (2), the superior cervical ganglion of the rat gives origin to a paraganglionic (3) and a postganglionic (4) substance P-immunoreactive intrinsic system, the latter becoming visible only after disconnection of the sympathetic pathway.

Nitric oxide synthase in guinea pig lower airway innervation.

The inhibitory non-adrenergic non-cholinergic (i-NANC) innervation of the guinea pig airways was suggested to be mediated, at least partially, by nitric oxide (NO). The enzyme catalyzing the generation of NO and citrulline from L-arginine, nitric oxide synthase (NOS), was found to be identical with neuronal nicotinamide-adenine dinucleotide hydrogen phosphate (NADPH)-diaphorase. In the present study, we report the distribution of NOS in guinea pig lower airways and in vagal sensory and sympathetic ganglia as revealed by NOS immunohistochemistry and NADPH-diaphorase histochemistry. The distribution of NOS was identical using either technique and displayed a similar distribution pattern in all parts of the lower airways. Yet, the number of NOS-containing fibres was increasing from cervical trachea towards principal bronchi and decreasing to complete absence in bronchioli. Innervation with NOS-containing nerve fibres was densest in the smooth muscle layer and in the lamina propria of the mucosa. Single fibres were found in the respiratory epithelium. Labelling was absent from nerve fibres innervating the submucosal glands. Perivascular fibre networks enmeshed tracheal arteries, pulmonary arteries and veins. A substantial number of NOS-immunoreactive and NADPH-diaphorase-positive neurons was observed in vagal sensory ganglia, whereas such neurons were rather sparse in sympathetic ganglia. Tracheal and peribronchial ganglia of the airways were devoid of labelling. These findings suggest that extrinsic rather than intrinsic (tracheal and peribronchial) neurons are the source of NO release from guinea pig airway nerve fibres after electrical field stimulation. These extrinsic nerve fibres may originate from both sympathetic and vagal sensory ganglia.

Nitric oxide synthase in cardiac nerve fibers and neurons of rat and guinea pig heart.

Participation of nitric oxide (NO) in the autonomic innervation of rat and guinea pig hearts was investigated by applying the NADPH diaphorase technique and immunohistochemistry with NO synthase antiserum. We present evidence that NO synthase is localized in cardiac ganglion cells and nerve fibers innervating the sinuatrial and atrioventricular nodes, the myocardium, local neurons, coronary arteries, and pulmonary vessels, suggesting an involvement of NO in neurogenic heart rate regulation, myocardial cell function, neuronal transmission in cardiac ganglia, and coronary as well as pulmonary vasodilation.

Nitric oxide synthase in VIP-containing vasodilator nerve fibres in the guinea-pig.

We investigated the possibility of nitric oxide (NO), a powerful vasodilator agent, being synthesized by perivascular nerve fibres. Immunoreactivity and catalytic activity of the NO synthesizing enzyme, NO synthase (NOS), were demonstrated in perivascular nerve fibres of blood vessels receiving autonomic vasodilator innervation, but not of those innervated exclusively by vasoconstrictor nerve fibres. Double-labelling techniques allowed identification of NOS-containing nerve fibres as belonging to the vasoactive intestinal peptide (VIP)/acetylcholine-containing class whereas noradrenergic and substance P-containing perivascular fibres were devoid of NOS. We suggest that, in addition to its endothelial source, NO is a neuronal co-mediator of VIP/cholinergic vasodilation.

Immunohistochemistry of the guinea-pig trachea using an anti-idiotypic antibody recognizing substance P receptors.

The airways receive a dense innervation from sensory neurons containing substance P (SP). An anti-SP anti-idiotypic antibody (anti-Id ab) recognizing SP receptors was previously characterized pharmacologically and proved to be useful in immunohistochemistry of the central nervous system. This antibody was used to localize SP binding sites in the guinea-pig trachea by immunohistochemistry. Immunolabelling was considered as specific when it could be prevented by a) preabsorption of the anti-Id ab with a C-terminal specific monoclonal anti-SP antibody, and b) preincubation of the tissue sections with either of the tachykinins, substance P and neurokinin A, in the presence of the inhibitor of neutral endopeptidase, phosphoramidon, and addition of these compounds into the antibody incubation medium. Moreover, immunofluorescence was absent when the acetone-fixed of fresh frozen sections were exposed to the detergent Tween 20 prior to immunohistochemistry, which points to a membrane localization of the detected tissue antigen, as expected for SP receptors. Compared with previous reports on autoradiographic localization of SP receptors in the guinea-pig trachea, the present immunohistochemical approach proved to be superior in enabling discrimination of labelled elements: Trachealis muscle, cylindrical epithelial cells and some roundish, singly lying cells in the epithelium and subepithelial lamina propria displayed specific immunofluorescence. These morphological findings match well with the known pharmacological actions of SP on the guinea-pig trachea.