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Active neurokinin (NK) receptors were visualized with 5-nm colloidal gold-protein-substance P (GPSP) and -senktide (GPSenk) complexes on vascular tissues. Electron micrographs of pig coronary strips incubated with GPSP showed gold particles either bound to the plasmalemma or inside intracytoplasmic vesicles of endothelial cells. Preincubation with SP or the NK1 receptor antagonist L-703606 prevented GPSP marking. No gold particles were seen after incubation with GPSenk. On coronary strips in vitro, which had been precontracted with U46619, GPSP induced relaxations similar to those produced by equimolar concentrations of SP, both relaxations being inhibited by L-703606. Analogous to senktide, GPSenk was totally inactive on arterial strips. Incubation of rat portal veins with GPSenk showed gold particles bound to the plasmalemma or inside intracytoplasmic vesicles of smooth muscle cells. Preincubation with senktide or the NK3 receptor antagonist R-820 prevented GPSenk marking. On portal veins in vitro GPSenk induced contractions similar to those induced by equimolar concentrations of senktide or NKB; these effects were inhibited by R-820. Our results show that colloidal gold-protein complexes present biological activity and selectivity similar to those of their respective native ligand and detect the presence of active receptors; in addition, they suggest the presence of NK-receptor-mediated endocytosis in endothelial cells of coronary artery and in smooth muscle cells of the portal vein.

We describe a rapid method for the preparation and binding site labeling of cryostat sections for use in light microscopy. Instead of using antibodies to bind to specific sites, substance P, delta-sleep-inducing peptide, oxytocin, and dopamine were covalently attached to BSA and then the BSA-ligand complex was adsorbed on 5-nm colloidal gold particles. Bioassays carried out on isolated organs indicated that the physiological activity of the ligand GPL complex was maintained. Most of the technical steps included use of an ordinary microwave oven (MWO), with tissues exposed for less than 1 min in any given step. Cryostat sections of unfixed rat brain were pre-incubated for 50 sec in the MWO in a Tris-buffered solution (pH 7.4) containing 1.5% BSA, then further incubated for 50 sec in the MWO in Tris-buffered solution containing 1% gelatin and the diluted colloidal gold suspension. After washing, the preparations were postfixed for 30 sec in the MWO in 5% formaldehyde solution, pH 7.4. Finally, the cell-bound gold particles were enlarged by a silver-enhancing process and counterstained. Preparations observed at high magnification provided excellent resolution of the cell binding sites. Positive and negative controls performed by addition of BSA-conjugated ligands to the pre-incubation and incubation medium, and displacement of the markers by an excess of unbound ligand in the pre-incubation or the incubation medium, showed the specificity of the tissue labeling.

Substance P-like immunoreactivity was found in Hydra attenuata mainly but not exclusively in the nerve and interstitial cells, localized in the cytoplasm and on the cell surface membranes.

The distribution of immunoreactive substance P (sP)-containing structures in the newt brain and spinal cord was explored with an indirect immunofluorescence method. Five sP-positive elements were detected: perikarya, dots, fibers, pericellular appositions, and pipe-shaped structures. Perikarya were seen at the levels of the spinal ganglia, spinal cord, raphe nucleus, interpeduncular nucleus, mesencephalon, preoptic area, infundibulum, dorsocaudal part of the ventral hypothalamus, habenula, and corpus striatum. Pericellular terminals were observed in periventricular areas, known to be rich in catecholaminergic cells; pipe-shaped structures were observed from the corpus striatum to diencephalon, and in mesencephalon. The olfactory nerve and nuclei were devoid of sP-positive elements. Six sP-immunofluorescent pathways were detected. One of them is composed of axons with huge varicosities and extends from the lateral spinal cord area to the mesencephalon. This pathway has not been described as yet in other animals and could be peculiar to the newt.