responses in submucosal guinea pig ileum. NK1 receptor distribution in whole mount preparations of the submucosa was examined using a rabbit polyclonal ...
Characterization of neurokinin-1 receptors in the submucosal plexus of guinea pig ileum B. A. MOORE,l S. VANNER,l N. W. BUNNETT,” AND K. A. SHARKEY3 lGastrointestinal Disease Research Unit, Queen’s University, Hotel Dieu Hospital, Kingston, Ontario K7L 5G2; and 3Neuroscience Research Group, University of Calgary, Alberta, Canada T2N 4Nl; and “Departments of Surgery and Physiology, University of California, San Francisco, California 94143-0660 Moore, B. A., S. Vanner, N. W. Bunnett, and K. A. Sharkey. Characterization of neurokinin-1 receptors in the submucosal plexus of guinea pig ileum. Am. J. PhysioZ. 273 (Gastrointest. Liver Physiol. 36): G670-G678, 1997. -This study combined immunohistochemical double-labeling techniques with functional studies to characterize the neurokinin-I (NKI) receptors mediating neuronal and vasodilator responses in submucosal guinea pig ileum. NK1 receptor distribution in whole mount preparations of the submucosa was examined using a rabbit polyclonal antibody directed against the COOH terminus of the rat NKi receptor. Results showed that 97% of neuropeptide Y immunoreactive submucosal neurons colocalized NKi receptor immunoreactivity, whereas vasoactive intestinal polypeptide immunoreactive neurons were not NKi immunoreactive. Intracellular recordings were made using neurobiotin-filled electrodes to enable reidentification of recorded neurons for immunohistochemical study. The selective NK1 agonists [Sarg,Met(02)11]substance P (SP) and septide depolarized S-type submucosal neurons. Of these neurons, 36% were NK1 immunoreactive and 64% were not. NK1 immunoreactivity was not observed on submucosal arterioles, but superfusion of [Sarg,Met(O#l]SP and septide dilated preconstricted submucosal arterioles. Agonist-evoked responses in both neurons and blood vessels were blocked by the selective NKi antagonist CP-99994. These findings suggest that NK1 receptors are found on submucosal neurons and arterioles and that electrophysiological and immunohistochemical techniques may identify conformational variants of the receptor.
SUBSTANCE P (SP) and related tachykinins are important mediators of regulatory pathways in the gastrointestinal (GI) tract. SP immunoreactivity is found in a subpopulation of submucosal and myenteric neurons as well as in extrinsic primary sensory afferent nerves projecting within the intestinal wall (7). Functional studies have shown that SP modulates ion secretion (lZ), mucosal blood flow (27), motility (6), and local immune function (24). Although these studies have helped to clarify the functional importance of tachykinins as neurotransmitters in the GI tract, the properties of the receptors that mediate their actions on effector systems are less clear. Three distinct receptor subtypes are currently known to mediate tachykinin-evoked responses: NK1, NK2, and NK3. Their distribution varies within the GI tract (23), but in the submucosa of the intestine the actions of NK1 receptors appear to play a prominent role. In the guinea pig ileum, SP, which has high affinity at the NK1 receptor, has been shown to evoke neurally mediated
ion secretion and to dilate submucosal arterioles (27). In addition, selective NK1 agonists depolarize neurons of the submucosal plexus, and these neurons have the morphological and electrophysiological properties of secretomotor neurons (25, 28). Results from these functional studies are supported by autoradiography analyses that revealed specific binding sites for 1251Bolton-Hunter SP on neurons of the submucosal plexus and on large, branching submucosal arterioles (3). These findings imply that SP-evoked alterations in fluid secretion and mucosal blood flow are mediated by a homogeneous distribution of NK1 receptors on these effector systems. Growing evidence suggests, however, that NK1 receptor distribution is more complex. This originates in part from several functional and ligand-binding studies that demonstrate significant differences in the expected pharmacological profiles of NKI receptors, which could not be attributed to interspecies differences (16, 23). For example, a “septide-sensitive” receptor has been proposed based on observations that the NK1 receptorselective agonist septide was less effective than other NK1-selective ligands at displacing labeled SP derivatives from NK1 sites within the same host species (4,X, 20,30). In addition, compared with other NK1 agonists, responses to septide were more potently inhibited by members of the nonpeptide class of NK1 antagonists (14, 15,20,30) and had somewhat increased activity at certain NKI sites relative to others (14, 30). Such findings were interpreted as evidence that pharmacologically distinct subtypes of the NK1 receptor mediate smooth muscle contraction in the guinea pig ileum and trachea. In view of the important role NK1 receptors play in regulating intestinal blood flow and secretion, it was of interest to further characterize NK1 receptors in the submucosal plexus. In a recent immunohistochemical study conducted in the guinea pig ileum, NK1 receptor immunoreactivity was found only on a single subpopulation of submucosal secretomotor neurons (21). This may suggest that NK1 receptor-mediated events are confined to a single neuronal effector system. However, an alternative explanation may be that a conformational variant of the NK1 receptor, which is not recognized by the antibody, may exist on the remaining neuronal systems. Distinguishing between these two possibilities is important in view of the significance attributed to NK1 receptor-mediated events in the intestine in both physiological and pathophysiological states (16,24).
G670
the American
substance
P; tachykinin
receptors;
secretion;
0193-1857/97
blood flow
$5.00
Copyright
o 1997
Physiological
Society
SUBMUCOSAL
NKI
Consequently, the aim of this study was to further examine the properties and distribution of NK1 receptors in the guinea pig ileal submucosal plexus by combining morphological and functional techniques in the examination of single neuronal cells and effector systems. By using the intracellular tracer neurobiotin, it was possible to correlate electrophysiological responses to selective NK1 agonists with the immunohistochemical staining profiles of specific peptide and NK1 receptor antibodies within a single cell. In addition, by using a videomicroscopic system to monitor changes in blood vessel diameter, this same approach could be applied to a separate effector system in the same tissue. METHODS
Adult Hartley guinea pigs (100-200 g) of either sex were obtained from Charles River Laboratories. Experiments were performed according to the guidelines of the Canadian Council of Animal Care. Animals were rendered unconscious by a single blow to the head and immediately killed by carotid and cervical transection. The abdomen was opened, and segments of ileum were collected -10 cm proximal to the ileocecal junction. Sections of submucosa were dissected by removal of the mucosa and muscle layers, as described previously (27). Immunohistochemistry. The distribution of a rabbit polyclonal antisera directed against the rat NK1 receptor [1:500; raised against the COOH-terminal region of the receptor (amino acids 393-407)] (10) was examined in whole mount preparations of guinea pig submucosa. Tissues were fixed by immersion in Zamboni’s fixative for 24 h at 4°C and then processed using indirect immunofluorescence double labeling, as previously described (5). NKi receptor immunoreactivity was colocalized with neuropeptide Y (NPY, 1500) or vasoactive intestinal polypeptide (VIP; 1:500), and both primary antibodies were simultaneously visualized with appropriate secondary antibodies [sheep anti-rabbit conjugated to CY3, goat anti-rat conjugated to fluorescein isothiocyanate (FITC) or goat anti-mouse conjugated to FITC]. To assess the specificity of the NKi labeling, the primary antibody was preabsorbed with the original hapten (10 nmol/ml). Colocalization of the NKi receptor antibody and neurobiotin was determined in submucosal preparations in which previous intracellular recordings had been made from single submucosal neurons using neurobiotin-filled electrodes. Tissues were incubated for 48 h at 4°C in a mixture of streptavidin conjugated to FITC (1:200) and the primary anti-NKi. The NKi antibody was subsequently visualized with goat anti-rabbit CY3. In all cases, preparations were viewed using a Zeiss Axioplan fluorescence microscope and photographed using Kodak Tmax 400 film. EEectrophysioEogy. Submucosal preparations for electrophysiology studies were pinned serosal side up in the organ bath and perfused with Krebs physiological saline solution containing (in mM) 126 NaCl, 2.5 NaH2P04, 1.2 MgC12, 2.5 CaC12, 5 KCl, 25 NaHC03, and 11 glucose, which was continuously gassed with 95% 02-5% CO2 at 35-36°C. Stable impalements of submucosal neurons that lasted for hours were accomplished by standard electrophysiological techniques, using glass microelectrodes with tip resistances of 90-120 Ma filled with 1 M KC1 containing 2% neurobiotin. Changes in membrane potential were recorded with an Axoclamp 2A amplifier and displayed on a Gould TA240 chart recorder or digitized at 5-10 kHz using a Digidata 1200A acquisitions board and Axoscope software (Axon Instru-
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ments). Fiber tracts and adjacent ganglia were stimulated (20 Hz, 0.7 ms, 200- to 500-ms train duration) with the use of a bipolar platinum electrode placed one or two interconnecting ganglionic nodes from the recording site. Neurobiotin-filled neurons were identified with streptavidin conjugated with FITC. Videomicroscopy. Submucosal preparations were dissected and superfused as described above for electrophysiological experiments. The outside diameter of submucosal arterioles was monitored using a computer-assisted videomicroscopy system (Diamtrak), as previously described (18). Briefly, this system uses an Imaging Technology PC VisionPlus framegrabber board in an IBM PC/AT computer to digitize television images of the arteriole and converts this result to an analog signal, which is stored on a computer-assisted data acquisition system (Axotape; Axon Instruments). Sampling rate is lo-20 Hz; resolution of the system is
