Characterisation of substance P-induced endocytosis of NK1 receptors on enteric neurons

Immunoreactivity for NK1 receptors is confined to specific nerve cell bodies in the guinea-pig ileum, including inhibitory motor neurons and secretomotor neurons. In the present work, endocytosis of NK1 receptors in these enteric neurons was studied following addition of substance P (SP) to isolated ileum. NK1 receptors were localised with antibodies against the C-terminus of this receptor. Some preparations were incubated with SP tagged with the fluorescent label, Cy3.18, so that the fate of SP bound to receptors could be followed. Preparations were analysed by confocal microcopy. In tissue that was incubated at 4° C in the absence of SP, most NK1 receptor immunoreactivity (IR) was confined to surface membranes of nerve cells. At 37° C in the presence of 10⁻⁷ M SP (plus 3×10⁻⁷M tetrodotoxin to prevent indirect activation via other neurons) the neuronal NK1 receptor was rapidly internalised. After 5 min, NK1 receptor IR was partially internalised, at 20 min NK1 receptor IR was throughout the cytoplasm and in perinuclear aggregates and at 30 min it was again at the cell surface. SP-induced NK1 receptor endocytosis was inhibited by the specific NK1 receptor antagonist, SR140333. Cy3-SP was colocalised with NK1 receptor IR and was internalised with the NK1 receptor. These results show that enteric neurons exhibit authentic NK1 receptors that are rapidly internalised when exposed to their preferred ligand.     

Localization of NK1 and NK3 receptors in guinea-pig brain

In this study the localizations of tachykinin neurokinin-1 (NK1) and neurokinin-3 (NK3) receptors in the guinea-pig brain are described. In agreement with studies in rat and human brain, the neurons that exhibited the most marked NK1 receptor immunoreactivity were found in the dorsomedial caudate putamen. NK1 receptors were also widely distributed in diencephalic structures and in the mid and hind brain. NK3 receptors were distributed in both superficial and deep layers of the cortex and many appeared to be located on cells with astrocyte-like morphology in the glia limitans. In several regions including the thalamus, hypothalamus, amygdala, periaqueductal gray, substantia nigra and area postrema, both NK1 and NK3 immunoreactivity were found. The present study revealed that tachykinin receptors are widely distributed in the guinea-pig central nervous system.     

Multiple opiate receptors in guinea-pig ileum

Actions of the prototypic μ-, κ-, and σ-opiate receptor agonists, morphine (M) ketocyclazocine (K) and SKF-10,047. (S), respectively, were examined and differentiated using the guinea-pig ileum preparation. S, like M and K, depressed the electrically stimulated ileum. Naloxone antagonized the depressant actions of the prototypic drugs with different potencies. PA₂ values of naloxone for M, K, and S, respectively, were 8.81, 7.58 and 7.74. Relative cross tolerance of each prototypic drug to normorphine, a comparison standard, was also examined in morphine-pretreated ilea and quantitatively estimated as follows: (1) the median effective dose of each drug and of the standard drug normorphine were determined in the nontolerant ileum (IC50_NT) and in ilea with varying degrees of tolerance IC50_T); (2) cross-tolerance ratios (IC50_T/IC50_NT) of each drug and of normorphine were calculated for the varying degrees of tolerance; (3) cross-tolerance ratios of each drug were plotted against those of normorphine, the data were fit by a least squares straight line, and the slope of the line determined as the Relative Cross Tolerance Index (RCTI). RCTI for M was 2.21. K and S, however, had lower RCTI's of 0.44 and 0.64 respectively. In the morphine-pretreated tolerant ilea, slopes of the dose response curves of the prototypic drugs were found to differ: while M and K possessed steep and constant slopes for ilea with different degrees of tolerance, the slopes for S became shallower as ilea became more tolerant to morphine. A maximum ceiling effect of less than 50% depression was obtained for S in the most highly tolerant ilea. The above observations are consistent with possible existence of the three types of hypothesized opiate receptors in the guinea-pig ileum.     

Properties of synaptic inputs from myenteric neurons innervating submucosal S neurons in guinea pig ileum

This study examined synaptic inputs from myenteric neurons innervating submucosal neurons. Intracellular recordings were obtained from submucosal S neurons in guinea pig ileal preparations in vitro, and synaptic inputs were recorded in response to electrical stimulation of exposed myenteric plexus. Most S neurons received synaptic inputs [>80% fast (f) excitatory postsynaptic potentials (EPSP), >30% slow (s) EPSPs] from the myenteric plexus. Synaptic potentials were recorded significant distances aboral (fEPSPs, 25 mm; sEPSPs, 10 mm) but not oral to the stimulating site. When preparations were studied in a double-chamber bath that chemically isolated the stimulating "myenteric chamber" from the recording side "submucosal chamber," all fEPSPs were blocked by hexamethonium in the submucosal chamber, but not by a combination of nicotinic, purinergic, and 5-hydroxytryptamine-3 receptor antagonists in the myenteric chamber. In 15% of cells, a stimulus train elicited prolonged bursts of fEPSPs (>30 s duration) that were blocked by hexamethonium. These findings suggest that most submucosal S neurons receive synaptic inputs from predominantly anally projecting myenteric neurons. These inputs are poised to coordinate intestinal motility and secretion.     

Neurochemical coding of myenteric neurons in the guinea-pig antrum

Electrophysiological studies of myenteric neurons in the guinea-pig antrum suggest that different neuroactive compounds are involved in synaptic transmission. It is not known what neurotransmitters and neuropeptides are present and to what extent they colocalize. Immunohistochemical stainings were performed on whole-mount preparations of the guinea-pig antrum. Immunoreactivity for neuron-specific enolase was used as a general marker and was set at 100%. There was no overlap between cholinergic and nitrergic neurons, resulting in two separate subpopulations. The presence of choline acetyltransferase immunoreactivity was used to identify the cholinergic subset, which accounted for 56% of the cells. Immunoreactivity for nitric oxide synthase, on the other hand, was displayed in 40.7% of the neurons. Substance-P immunoreactivity was present in 37.4% of the cells and vasoactive intestinal peptide and neuropeptide Y in 21.7% and 28.6%, respectively. Small subsets of neurons had immunoreactivity for serotonin (3.9%), calretinin (6.8%) and calbindin (0.5%). Colocalization studies revealed several subgroups of neurons, containing one or more of the screened markers. Though some similarity is found in the chemical coding of the antrum compared to that of the small intestine and the corpus, remarkable differences can be seen in the occurrence of some subpopulations. Cholinergic neurons are not as predominant as in other parts of the gut, serotonin presence is doubled and some vasointestinal-peptide-positive neurons express substance P. These differences might reflect the highly specialized function of the antrum; however, the exact role of these classes remains to be established.     

Identification of the populations of enteric neurons that have NK1 tachykinin receptors in the guinea-pig small intestine

Simultaneous immunofluorescence labelling was used to investigate the patterns of colocalisation of the NK1 tachykinin receptor with other neuronal markers, and hence determine the functional classes of neuron that bear the NK1 receptor in the guinea-pig ileum. In the myenteric plexus, 85% of NK1 receptor-immunoreactive (NK1r-IR) nerve cells had nitric oxide synthase (NOS) immunoreactivity and the remaining 15% were immunoreactive for choline acetyltransferase (ChAT). Of the latter group, about 50% were immunoreactive for both neuropeptide Y (NPY) and somatostatin (SOM), and had the morphologies of secretomotor neurons. Many of the remaining ChAT neurons were immunoreactive for calbindin or tachykinins (TK), but not both. These calbindin immunoreactive neurons had Dogiel type II morphology. No NK1r-IR nerve cells in the myenteric plexus had serotonin or calretinin immunoreactivity. In the submucosal ganglia, 84% of NK1r-IR nerve cells had neuropeptide Y immunoreactivity and 16% were immunoreactive for TK. It is concluded that NK1r-IR occurs in five classes of neuron; namely, in the majority of NOS-immunoreactive inhibitory motor neurons, in ChAT/TK-immunoreactive excitatory neurons to the circular muscle, in all ChAT/NPY/SOM-immunoreactive secretomotor neurons, in a small proportion of ChAT/calbindin myenteric neurons, and in about 50% of ChAT/TK submucosal neurons.     

Tetraethylammonium facilitates the stimulation-evoked loss of the enkephalins from the myenteric plexus of guinea-pig ileum

The effects of electrical field stimulation on the contents of [Met]enkephalin and [Leu]enkephalin were determined in myenteric plexus-longitudinal muscle preparations of the guinea-pig small intestine. Cycloheximide (0.1 mM) was present in all experiments to prevent de nouveau biosynthesis. The two enkephalins were separated by high performance liquid chromatography and assayed on the mouse vas deferens. Stimulation with submaximal pulses (50 mA, 0.5 ms) at a frequency of 10 Hz caused maximal losses of about 35% of [Met]enkephalin and [Leu]enkephalin after 3 h (108000 pulses). The plot of log (enkephalin content) against number of pulses was steeper during the first 30 min than during the later periods. Tetraethylammonium bromide (TEA, 10 mM) increased the [Met]enkephalin and [Leu]enkephalin contents of the non-stimulated preparations by about 50%. When the preparations were stimulated in the presence of TEA at 50 mA and 1 Hz, the plots of loss of enkephalins against number of pulses were linear until the maximum of about 50% was reached. Compared with stimulation in the absence of TEA, the rate constant was 8 times greater for [Leu]enkephalin and 20 times greater for [Met]enkephalin. The absolute losses per pulse were about 13 times greater for [Leu]enkephalin and 27 times greater for [Met]enkephalin than in the absence of TEA. In the presence of bacitracin and a mixture of dipeptides, the enzymatic degradation of the enkephalins was sufficiently suppressed to cause an overflow of 30-60% of the enkephalins lost from their stores into the perifusing Krebs solution. Until it is possible to determine the preformed precursors, which are present in large quantities, the kinetics relationship between these precursors and the enkephalins cannot be investigated. A similar dilemma exists for the relationship between "released' enkephalins and the losses from their stores.     

Stimulatory effect of trans-cinnamaldehyde on noradrenaline secretion in guinea-pig ileum myenteric nerve terminals

The effect of trans-cinnamaldehyde (CNMA) on the release of noradrenaline (NA) from nerve terminal was investigated using isolated ileal synaptosomes of guinea-pig. Release was determined as the amount of NA, quantified by h.p.l.c.-electrochemical detection, from samples incubated with CNMA minus that in parallel blanks treated with same volume of vehicle. CNMA stimulated the secretion of NA in a concentration-dependent manner from 5 microM to 50 microM, while the value of lactate dehydrogenase in the incubated medium was not influenced by CNMA. However, trans-cinnamic acid, cinnamoyl chloride and cinnamamide failed to produce similar effect. Specific action of CNMA can thus be considered. Guanethidine inhibited the release of NA by CNMA in a concentration- dependent manner. Saxitoxin attenuated the action of CNMA at concentrations sufficient to block sodium channels. The depolarizing effect of CNMA on the membrane potential was also illustrated by a concentration-dependent increase in the fluorescence of bisoxonol, a potential sensitive dye. The NA releasing action of CNMA was deleted by removal of calcium chloride from the bathing medium. This action of CNMA was also attenuated by Rp-cAMP at concentrations sufficient to inhibit the action of cyclic AMP. These findings suggest that CNMA can depolarize the membrane to result in a calcium-dependent and cyclic AMP-related release of NA from noradrenergic terminals.     

Neurons bearing NK3 tachykinin receptors in the guinea-pig ileum revealed by specific binding of fluorescently labelled agonists

The localisation of NK₃ 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 NK₁ receptors was blocked by the NK₁ receptor antagonist, CP-99994. NK₃ 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 NK₃ receptors. In myenteric ganglia, NK₃ 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 NK₁ receptor-IR neurons. In submucosal ganglia, NK₃ 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 NK₃ receptors mediate tachykinin transmission between myenteric sensory neurons and to interneurons and/or motor neurons in descending inhibitory and ascending excitatory pathways. Accepted: 22 June 1999     

Inputs from intrinsic primary afferent neurons to nitric oxide synthase-immunoreactive neurons in the myenteric plexus of guinea pig ileum

Nitric oxide synthase (NOS) immunoreactivity occurs in two groups of neurons in the guinea pig small intestine: descending interneurons that are also immunoreactive for choline acetyltransferase (ChAT), and inhibitory motor neurons that lack ChAT immunoreactivity. Interneurons that are involved in local reflexes would be expected to have inputs from intrinsic primary afferent (sensory) neurons, most of which are calbindin-immunoreactive. We examined this possibility using triple staining for NOS, ChAT and calbindin immunoreactivity and investigated the relationships between calbindin-immunoreactive varicosities and the cell bodies of NOS-immunoreactive neurons, using high-resolution confocal microscopy and electron microscopy. By confocal microscopy, we found that the cell bodies of ChAT/NOS interneurons received 84 +/- 23 (mean +/- SD) direct appositions from calbindin-immunoreactive varicosities and that the cell bodies of NOS-inhibitory motor neurons received 82 +/- 20 appositions. Electron-microscopic examination of the relations of 265-calbindin-immunoreactive varicosities, at distances within the resolution of the confocal microscope (300 nm), to 30 NOS-immunoreactive nerve cells indicated that 84% formed close contacts or synapses and 16% were separated from neurons by thin glial cell processes. Thus, each NOS-immunoreactive nerve cell receives about 70 synaptic inputs or close contacts from the calbindin-immunoreactive varicosities of intrinsic primary afferent neurons. It is concluded that there are monosynaptic reflex connections in which intrinsic primary afferent neurons synapse directly with motor neurons and di- or poly-synaptic reflexes in which ChAT- and NOS-immunoreactive neurons are interneurons, interposed between intrinsic primary afferent neurons and NOS-inhibitory neurons.     

Neurochemical identification of enteric neurons expressing P2X(3) receptors in the guinea-pig ileum

It was hypothesised that P2X(3) receptors, predominantly labelling spinal and cranial sensory ganglionic neurons, are also expressed in intrinsic sensory enteric neurons, although direct evidence is lacking. The aim of this study was to localise P2X(3) receptors in the enteric nervous system of the guinea-pig ileum, and to neurochemically identify the P2X(3)-expressing neurons. In the submucous plexus, cholinergic neurons expressing calretinin (CRT), were immunostained for P2X(3). These neurons made up about 12% of the submucous neurons. In the myenteric plexus, approximately 36% of the neurons expressed P2X(3). Half of the latter neurons were immunoreactive for CRT, whereas about 20% were immunoreactive for nitric oxide synthase (NOS). Based on earlier neurochemical analysis of enteric neurons in the guinea-pig, the myenteric neurons exhibiting P2X(3)/CRT immunoreactivity were identified as longitudinal muscle motor neurons, and those expressing P2X(3)/NOS immunoreactivity as short inhibitory circular muscle motor neurons. In both plexuses, no colocalisation was observed between P2X(3) and calbindin, a marker for intrinsic sensory neurons. Multiple staining with antisera raised against somatostatin, neuropeptide Y, substance P or neurofilament protein did not reveal any costaining. It can be concluded that in the guinea-pig ileum, intrinsic sensory neurons do not express P2X(3) receptors. However, this does not negate the possibility that extrinsic sensory nerves expressing P2X(3) are involved in a purinergic mechanosensory transduction pathway as demonstrated in other organs.     

Neurokinin 1 receptors mediate substance P-induced changes in ion transport in guinea-pig ileum.

Tachykinin receptors mediating substance P-induced secretion were examined in muscle-stripped segments of guinea-pig ileum set up in flux chambers. Changes in the short-circuit current (Isc) served as an index of active, electrogenic ion transport. Substance P evoked a transient increase in Isc which was concentration-dependent. The maximal change in Isc occurred at 1 microM concentration. [Sar9,Met(O2)11]-substance P, a neurokinin 1 (NK-1) receptor agonist, evoked a similar concentration-dependent increase in Isc. [Nle10]NKA(4-10) (1 microM) or [Pro7]NKB (1 microM), selective NK2 and NK3 agonists, respectively, had minimal effects on Isc. CP-96,345 (5 microM), a nonpeptide NK-1 antagonist, and the peptide NK-1 antagonist, GR82334 (1 microM), reduced the secretory response to substance P (50 nM) in the presence and absence of tetrodotoxin (0.2 microM). The NK2 antagonist, [Tyr5,D-Trp6,8,9,Arg10]NKA(4-10) MEN 10207 had no effect on the substance P response. Tetrodotoxin (0.2 microM) significantly reduced, but did not abolish the Isc response to substance P (1 microM) and [Sar9,Met(O2)11]substance P (1 microM). The substance P response was unaltered by 5 microM atropine and 50 microM mecamylamine. Piroxicam (10 microM) or pyrilamine (10 microM) or a combination of both had no effect on the tetrodotoxin-resistant substance P response. Electrical field stimulation evoked a biphasic increase in Isc which was significantly reduced by 0.2 microM tetrodotoxin. Atropine (5 microM) reduced the first peak of the biphasic response and mecamylamine (50 microM) had no effect. Similarly, 5 microM CP-96,345 and 1 microM GR82334 did not alter the EFS-induced change Isc. The results suggest that substance P-evoked secretory responses are independent of histamine or prostaglandins. Substance P responses are mediated by an NK-1 receptor type on enteric neurons and possibly epithelial cells.     

Storage and release of labelled acetylcholine in the myenteric plexus of the guinea-pig ileum.

Guinea-pig ileum myenteric plexus-longitudinal muscle preparation was superfused with [3H]choline for 15 min either without being stimulated or during field stimulation at 0.1 or 16 Hz; the preparation was then either removed immediately or after 75- or 135-min superfusion with hemicholinium-3 (HC-3) and the total acetylcholine (ACh) and [3H]ACh contents were determined. For measuring the release of [3H]ACh the preparation was stimulated for 60 min the second time at 0.1 or 16 HZ in the presence of hemicholinium. Exposure to [3H]choline without stimulation resulted in the formation of [3H]ACh stores which were maintained in the first 75 min but decreased therafter. Labelling during stimulation at 16 Hz produced the largest and best maintained [3H]ACh content. Following labelling during 0.1-Hz stimulation, more label could be released than following labelling in the absence of stimulation. Labelling during 16-Hz stimulation did not increase any further in fool of [3H]ACh accessible to release by 0.1-Hz stimulation, but caused a 2.5 times increase in the pool from which Hz stimulation released [3H]ACh. These results suggest that two populations of cholinergic neurons exist in the myenteric plexus, one activated only by high frequency stimulation, the other by both high and low frequency stimulation.     

Phenazine methosulfate induces a neurally-mediated contraction of the guinea-pig ileum

Phenazine methosulfate (PMS) and related phenazines are widely used in biochemistry and histochemistry and act as anti-bacterial agents, however, there is little information on their pharmacological actions. In the present paper the guinea-pig ileum was used as a model for studying the effects of PMS on nerve cells. PMS was found to contract intestinal muscle. This action appeared to be mediated by the activation of muscarinic receptors since it was blocked by atropine. Neostigmine potentiated the response to PMS. The nerve blocker tetrodotoxin prevented the effect of PMS and it is concluded that PMS causes the release of acetylcholine from nerve elements. The action of PMS on nerves is not mediated by nicotinic receptors. Receptors for serotonin, substance P or cholecystokinin also appear not to be involved. Of all the phenazines tested PMS was found to be the most potent and reversible.     

Multiple mechanisms contribute to myenteric plexus ablation induced by benzalkonium chloride in the guinea-pig ileum

Ablation of rat myenteric plexus with benzalkonium chloride has provided a model of intestinal aganglionosis, but the degenerative responses are not well understood. We examined the effects of this detergent on neurons and glia, including expression of c-Myc, c-Jun, JunB, and c-Fos, and on immunocytes in the guinea-pig ileum. Benzalkonium chloride (0.1%) or saline was applied to the serosal surface of distal ileum. Tissues were analyzed 2, 3, or 7 days later and compared with cyclosporine-treated and untreated animals. More than 90% of myenteric neurons were destroyed in ileal segments 3–7 days after benzalkonium-chloride treatment. Glia withdrew processes from around neurons after 2 days and were mostly gone after 3 days. Neuronal c-Myc began to disappear while c-Fos, c-Jun, and JunB were evident in some neuronal nuclei after 2 or 3 days. After 3 days, widespread apoptosis was evident in the myenteric plexus. Populations of T cells, B cells, and macrophage-like cells in untreated and saline-treated myenteric plexuses were substantially increased 3 and 7 days after benzalkonium-chloride treatment. Cyclosporine delayed significant neuronal loss. We conclude that a variety of degenerative mechanisms may be active in this model, including an immune response which may actively contribute to tissue destruction. Received: 13 September 1996 / Accepted: 20 January 1997     

Cholecystokinin-8 induces intracellular calcium signaling in cultured myenteric neurons from neonatal guinea pigs

The responsiveness of cultured myenteric neurons to cholecystokinin (CCK-8) was examined using fura-2-based digital microfluorimetric measurement of intracellular calcium ([Ca(2+)](i)). CCK-8 (10(-10)-10(-6)M) evoked concentration-dependent increases in percentage of neurons responding (8-52%) and delta[Ca(2+)](i) (76-169 nM). Gastrin (1 microM) also induced an increase in [Ca(2+)](i) in 29+/-6% of neurons (delta[Ca(2+)](i): 71+/-3 nM). L-364,718, an antagonist for the CCK-A receptor, blocked [Ca(2+)](i) response to CCK-8. Removal of extracellular calcium eliminated CCK-induced [Ca(2+)](i) increments, as did the addition of the calcium channel inhibitors nickel (1mM) and lanthanum (5mM). Nifedipine (1-50 microM) dose-dependently attenuated CCK-caused [Ca(2+)](i) responses. CCK evokes [Ca(2+)](i) signaling in myenteric neurons by the influx of extracellular calcium, likely through L-type calcium channels.     

Tachykinins mediate non-adrenergic,non-cholinergic excitatory neurotransmission to the hamster ileum via NK1 and NK2 receptors

The present study was designed to investigate Substance P (SP) and a related tachykinin, Neurokinin A (NKA), contributions to the excitatory neurotransmission to the circular smooth muscle of the hamster ileum. In the presence of atropine (0.5 microM), guanethidine (3 microM) and NG-nitro-L-arginine methyl ester (L-NAME) (200 microM), electrical field stimulation (EFS) evoked a non-adrenergic, non-cholinergic (NANC) excitatory junction potential (EJP) and contraction of circular smooth muscle. Applications of SP and NKA produced depolarizing and contractile responses in a concentration-dependent fashion. The EJP and contraction were almost abolished by the non-specific tachykininergic antagonist, spantide (3 microM). Application of SP antagonist, L-732,138, (1 microM) markedly inhibited EJP (82.5%) and contraction (68.9%) and completely blocked excitatory responses produced by exogenous application of SP. While application of NKA antagonist, SR48968 (1 microM) completely blocked the depolarising and contractile responses to NKA, it only slightly inhibited those to EFS (17.2% and 31.4% respectively).These results provide evidence that, in the circular muscle of hamster ileum, endogenous tachykinins are the main NANC excitatory neurotransmitters and their action is mediated by both NK1 and NK2 receptors.     

CCK-A receptor activation induces fos expression in myenteric neurons of rat small intestine

Cholecystokinin (CCK), a hormone secreted from endocrine cells of the small intestine, participates in the control of motility and secretion in the gastrointestinal tract, and in the control of food intake. At least some of the effects of CCK on intestinal function appear to be mediated via activation of intrinsic neurons in the myenteric plexus. However, the distribution of CCK-responsive enteric neurons within the rat small intestine is not known. Neither has the role of CCK-A receptors in the activation of rat myenteric neurons been investigated. Therefore, to determine the distribution of CCK-responsive neurons in the small intestinal myenteric plexus we utilized immunohistochemical detection of Fos, the protein product of the immediate early gene c-fos, to identify neurons that were activated by exogenous CCK. We also monitored Fos expression in the dorsal hindbrain, and examined CCK-induced Fos expression in the presence or absence of a receptor antagonist for the type-A CCK receptor. We found that CCK significantly increased Fos expression in the hindbrain and in myenteric neurons of the duodenum and jejunum, but not the ileum. Neuronal Fos responsiveness in both brain and myenteric neurons was mediated by CCK-A receptors, as CCK-induced Fos expression was eliminated in rats pretreated with a CCK-A receptor antagonist. We conclude that CCK activates small intestinal myenteric neurons, via CCK-A receptors. Activation of these intrinsic intestinal neurons may participate in reflexes and behaviors that are mediated by CCK.     

Cholecystokinin-8 increases Fos-like immunoreactivity in the brainstem and myenteric neurons of rats through CCK1 receptors

To examine the role of cholecystokinin1 receptor (CCK1) in the activation of brainstem and myenteric neurons by CCK, we compared the ability of exogenous CCK-8 to induce Fos-like immunoreactivity (Fos-LI) in these neurons in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, lacking CCK1 receptors, and Long-Evans Tokushima Otsuka (LETO) controls. Five groups (n=4 rats per group) of OLETF rats, and five LETO control groups, were injected intraperitoneally (IP) with 5, 10, 20, and 40 microg/kg CCK-8 or saline. Forty-micrometer brainstem sections containing the area postrema, nucleus of the solitary tract, and the dorsal motor nucleus of the vagus, and myenteric neurons of the duodenum, jejunum, and ileum underwent a diaminobenzidine reaction enhanced with nickel to reveal Fos-LI. CCK-8 did not increase Fos-LI in any of the tested neurons in the OLETF rats. CCK-8 increased Fos-LI in the brainstem of the LETO rats in a dose dependent manner. In the LETO rats only 40 microg/kg CCK-8 increased Fos-LI in the myenteric plexus of the jejunum. This study demonstrates that CCK-8 activates the brainstem and myenteric neurons through the CCK1 receptor.