cp-99994 and cyanine-dye-3

Previous immunohistochemical studies failed to reveal neurokinin (NK)(1) tachykinin receptors on intestinal muscle, despite convincing pharmacologic data indicating their presence. This study aimed to apply optimal immunohistochemical methods to reveal the receptors.. NK(1)-receptor immunoreactivity was examined by confocal microscopy in tissue incubated with or without 10(-7) mol/L substance P (SP), 10(-7) mol/L SP plus 10(-6) mol/L NK(1) receptor antagonist (CP99994), or with fluorescent cyanine 3.18 (Cy3) SP.. Without incubation, NK(1)-receptor immunoreactivity was strong on muscle of the rectum and distal colon and weak in proximal colon and small intestine. NK(1) receptor was located on the surface of muscle cells in all gut regions. Exposure to SP increased the intensity of immunoreactivity, and the receptor moved into the cytoplasm. Mobilization of the receptor by SP was blocked by the NK(1)-receptor antagonist CP99994. Cy3-SP was internalized by muscle cells and colocalized with the receptor. NK(1)-receptor immunoreactivity occurred on crypt epithelial cells in the small intestine and the base of glands in the proximal colon.. The NK(1) receptor occurs on the external muscle throughout the small and large intestines. SP binds and triggers NK(1)-receptor aggregation and internalization in the muscle.

Topics: Animals; Carbocyanines; Colon; Endocytosis; Epithelial Cells; Female; Fluorescent Dyes; Guinea Pigs; Immunohistochemistry; Intestinal Mucosa; Intestine, Small; Male; Muscle, Smooth; Nerve Fibers; Neurokinin-1 Receptor Antagonists; Piperidines; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Rectum; Substance P

The localisation of NK(3) tachykinin receptors in guinea-pig ileum was studied using the fluorescently labelled agonists, Cy3. 5-neurokinin A and Cy3.5-kassinin. Binding to nerve cell bodies in the myenteric and submucosal plexuses was visualised using confocal microscopy. Binding to NK(1) receptors was blocked by the NK(1) receptor antagonist, CP-99994. NK(3) receptors, demonstrated by binding in the presence of CP-99994, occurred in 72% of myenteric and 38% of submucosal neurons. Colocalisation with other markers was examined to deduce the classes of neurons with NK(3) receptors. In myenteric ganglia, NK(3) receptors were present on the following: 73% of calbindin-immunoreactive (IR) intrinsic primary afferent neurons, 63% of calretinin-IR excitatory motor neurons and ascending interneurons, 63% of nitric oxide synthase-IR inhibitory motor neurons and descending interneurons, 79% of strongly neuropeptide Y (NPY)-IR secretomotor neurons, 67% of weakly NPY-IR descending interneurons and motor neurons, and 46% of NK(1) receptor-IR neurons. In submucosal ganglia, NK(3) receptors were on 65% of calretinin-IR secretomotor/vasodilator neurons, 81% of NPY-IR cholinergic secretomotor neurons, 2% of vasoactive intestinal peptide-IR non-cholinergic secretomotor neurons and were completely absent from substance P-IR intrinsic primary afferent neurons. The results support physiological studies suggesting that NK(3) receptors mediate tachykinin transmission between myenteric sensory neurons and to interneurons and/or motor neurons in descending inhibitory and ascending excitatory pathways.

Topics: Animals; Carbocyanines; Cell Count; Female; Fluorescent Dyes; Guinea Pigs; Ileum; In Vitro Techniques; Kassinin; Male; Microscopy, Confocal; Myenteric Plexus; Neurokinin A; Neurons; Piperidines; Receptors, Neurokinin-3; Submucous Plexus