Vagally mediated, nonparacrine effects of cholecystokinin-8s on rat pancreatic exocrine secretion

Cholecystokinin (CCK) has been proposed to act in a vagally dependent manner to increase pancreatic exocrine secretion via actions exclusively at peripheral vagal afferent fibers. Recent evidence, however, suggests the CCK-8s may also affect brain stem structures directly. We used an in vivo preparation with the aims of 1) investigating whether the actions of intraduodenal casein perfusion to increase pancreatic protein secretion also involved direct actions of CCK at the level of the brain stem and, if so, 2) determining whether, in the absence of vagal afferent inputs, CCK-8s applied to the dorsal vagal complex (DVC) can also modulate pancreatic exocrine secretion (PES). Sprague-Dawley rats (250-400 g) were anesthetized and the common bile-pancreatic duct was cannulated to collect PES. Both vagal deafferentation and pretreatment with the CCK-A antagonist lorglumide on the floor of the fourth ventricle decreased the casein-induced increase in PES output. CCK-8s microinjection (450 pmol) in the DVC significantly increased PES; the increase was larger when CCK-8s was injected in the left side of the DVC. Protein secretion returned to baseline levels within 30 min. Microinjection of CCK-8s increased PES (although to a lower extent) also in rats that underwent complete vagal deafferentation. These data indicate that, as well as activating peripheral vagal afferents, CCK-8s increases pancreatic exocrine secretion via an action in the DVC. Our data suggest that the CCK-8s-induced increases in PES are due mainly to a paracrine effect of CCK; however, a relevant portion of the effects of CCK is due also to an effect of the peptide on brain stem vagal circuits.     

Vanilloid, purinergic, and CCK receptors activate glutamate release on single neurons of the nucleus tractus solitarius centralis

Baroreceptor inputs to nucleus of the tractus solitarius medialis (mNTS) neurons can be differentiated, among other features, by their response to vanilloid or purinergic agonists, active only on C- or A-fibers, respectively. A major aim of this study was to examine whether neurons of NTS centralis (cNTS), a subnucleus dominated by esophageal inputs, exhibit a similar dichotomy. Since it has been suggested that cholecystokinin (CCK), exerts its gastrointestinal (GI)-related effects via paracrine activation of vagal afferent C-fibers, we tested whether CCK-sensitive fibers impinging upon cNTS neurons are responsive to vanilloid but not purinergic agonists. Using whole cell patch-clamp recordings from cNTS, we recorded miniature excitatory postsynaptic currents (mEPSCs) to test the effects of the vanilloid agonist capsaicin, the purinergic agonist α,β-methylene-ATP (α,β-Met-ATP), and/or CCK-octapeptide (CCK-8s). α,β-Met-ATP, capsaicin; and CCK-8s increased EPSC frequency in 37, 71, and 46% of cNTS neurons, respectively. Approximately 30% of cNTS neurons were responsive to both CCK-8s and α,β-Met-ATP, to CCK-8s and capsaicin, or to α,β-Met-ATP and capsaicin, while 32% of neurons were responsive to all three agonists. All neurons responding to either α,β-Met-ATP or CCK-8s were also responsive to capsaicin. Perivagal capsaicin, which is supposed to induce a selective degeneration of C-fibers, decreased the number of cNTS neurons responding to capsaicin or CCK-8s but not those responding to α,β-Met-ATP. In summary, GI inputs to cNTS neurons cannot be distinguished on the basis of their selective responses to α,β-Met-ATP or capsaicin. Our data also indicate that CCK-8s increases glutamate release from purinergic and vanilloid responsive fibers impinging on cNTS neurons.     

Cholecystokinin-8s excites identified rat pancreatic-projecting vagal motoneurons

It is known that cholecystokinin (CCK) acts in a paracrine fashion to increase pancreatic exocrine secretion via vagal circuits. Recent evidence, however, suggests that CCK-8s actions are not restricted to afferent vagal fibers, but also affect brain stem structures directly. Within the brain stem, preganglionic neurons of the dorsal motor nucleus of the vagus (DMV) send efferent fibers to subdiaphragmatic viscera, including the pancreas. Our aims were to investigate whether DMV neurons responded to exogenously applied CCK-8s and, if so, the mechanism of action. Using whole cell patch-clamp recordings we show that perfusion with CCK-8s induced a concentration-dependent excitation in approximately 60% of identified pancreas-projecting DMV neurons. The depolarization was significantly reduced by tetrodotoxin, suggesting both direct (on the DMV membrane) and indirect (on local synaptic circuits) effects. Indeed, CCK-8s increased the frequency of miniature excitatory currents onto DMV neurons. The CCK-A antagonist, lorglumide, prevented the CCK-8s-mediated excitation whereas the CCK-B preferring agonist, CCK-nonsulfated, had no effect, suggesting the involvement of CCK-A receptors only. In voltage clamp, the CCK-8s-induced inward current reversed at -106 +/- 3 mV and the input resistance increased by 150 +/- 15%, suggesting an effect mediated by the closure of a potassium conductance. Indeed, CCK-8s reduced both the amplitude and the time constant of decay of a calcium-dependent potassium conductance. When tested with pancreatic polypeptide (which reduces pancreatic exocrine secretion), cells that responded to CCK-8s with an excitation were, instead, inhibited by pancreatic polypeptide. These data indicate that CCK-8s may control pancreas-exocrine secretion also via an effect on pancreas-projecting DMV neurons.     

Differential mechanism and site of action of CCK on the pancreatic secretion and growth in rats

Recent studies demonstrated that cholecystokinin (CCK) at physiological levels stimulates pancreatic enzyme secretion via a capsaicin-sensitive afferent vagal pathway. This study examined whether chemical ablation of afferent vagal fibers influences pancreatic growth and secretion in rats. Bilateral subdiaphragmatic vagal trunks were exposed, and capsaicin solution was applied. Pancreatic wet weight and pancreatic secretion and growth in response to endogenous and exogenous CCK were examined 7 days after capsaicin treatment. Perivagal application of capsaicin increased plasma CCK levels and significantly increased pancreatic wet weight compared with those in the control rats. Oral administration of CCK-1 receptor antagonist loxiglumide prevented the increase in pancreatic wet weight after capsaicin treatment. In addition, continuous intraduodenal infusion of trypsin prevented the increase in plasma CCK levels and pancreatic wet weight after capsaicin treatment. There were no significant differences in the expression levels of CCK-1 receptor mRNA and protein in the pancreas in capsaicin-treated and control rats. Intraduodenal administration of camostat or intravenous infusion of CCK-8 stimulated pancreatic secretion in control rats but not in capsaicin-treated rats. In contrast, repeated oral administrations of camostat or intraperitoneal injections of CCK-8 significantly increased pancreatic wet weight in both capsaicin-treated and control rats. Present results suggest that perivagal application of capsaicin stimulates pancreatic growth via an increase in endogenous CCK and that exogenous and endogenous CCK stimulate pancreatic growth not via vagal afferent fibers but directly in rats.     

Anti-inflammatory effects of leptin and cholecystokinin on acetic acid-induced colitis in rats: role of capsaicin-sensitive vagal afferent fibers

Leptin and cholecystokinin (CCK) have a synergistic interaction in the suppression of food intake, and afford similar gastroprotective activity. The present study was designed to investigate the putative protective effects of CCK and leptin on acute colonic inflammation. Leptin or CCK-8s was injected to rats intraperitoneally immediately before and 6 h after the induction of colitis with acetic acid. CCK-A receptor antagonist (L-364,718) or CCK-B receptor antagonist (L-365,260) was injected intraperitoneally 15 min before leptin or CCK treatments. In a group of rats, vagal afferent fibers were denervated by topical application of capsaicin on the cervical vagi. Rats were decapitated at 24 h, and the distal 8 cm of the colon were removed for macroscopic scoring, determination of tissue wet weight index (WWI), histologic assessment and tissue myeloperoxidase (MPO) activity. All inflammation parameters were increased by acetic acid-induced colitis compared to control group. Leptin or CCK-8s treatment reduced these parameters in a similar manner, while co-administration of leptin and CCK was found to be more effective in reducing the macroscopic score and WWI. CCK-8s-induced reduction in the score and WWI was prevented by CCK-A, but not by CCK-B receptor antagonist, whereas neither antagonist altered the inhibitory effect of leptin on colitis-induced injury. On the other hand, perivagal capsaicin prevented the protective effects of both CCK-8s and leptin on colitis. Our results indicate that leptin and CCK have anti-inflammatory effects on acetic acid-induced colitis in rats, which appear to be mediated by capsaicin-sensitive vagal afferent fibers involving the reduction in colonic neutrophil infiltration.     

Inhibitory effects on intake of cholecystokinin-8 and cholecystokinin-33 in rats with hepatic proper or common hepatic branch vagal innervation

The relative potencies of cholecystokinin (CCK)-8 and CCK-33 for decreasing meal size depend on the route of administration. Inhibitory potencies are equal after intraperitoneal administration, but CCK-33 is significantly more potent after intraportal administration. This suggests that CCK-33 is a more effective stimulant of hepatic afferent vagal nerves than is CCK-8. To investigate this possibility, we administered both peptides intraperitoneally in rats with abdominal vagotomies that spared only the hepatic proper vagal nerves (H) and in rats with abdominal vagotomies that spared the common hepatic branch that contains the fibers of the hepatic proper and gastroduodenal nerves (HGD). The vagal afferent innervation in H and HGD rats was verified with a wheat germ agglutinin-horseradish tracer strategy. Intraperitoneal administration of CCK-33 decreased 30-min intake of 10% sucrose in H rats as much as in sham rats, but CCK-8 decreased intake significantly less in H rats than in sham rats. The larger inhibitory effect of CCK-33 than of CCK-8 in H rats is consistent with the hypothesis that CCK-33 is a more effective stimulant of the hepatic proper vagal afferent nerves than CCK-8. In contrast to the results in H rats, the inhibitory potencies of both peptides were significantly and equivalently reduced in HGD rats compared with sham rats. This suggests that there is an inhibitory interaction between the stimulation of the gastroduodenal and hepatic proper afferent fibers by CCK-33.     

D-glucose modulates synaptic transmission from the central terminals of vagal afferent fibers

Experimental evidence suggests that glucose modulates gastric functions via vagally mediated effects. It is unclear whether glucose affects only peripheral vagal nerve activity or whether glucose also modulates vagal circuitry at the level of the brain stem. This study used whole cell patch-clamp recordings from neurons of the nucleus of the tractus solitarius (NTS) to assess whether acute variations in glucose modulates vagal brain stem neurocircuitry. Increasing D-glucose concentration induced a postsynaptic response in 40% of neurons; neither the response type (inward vs. outward current) nor response magnitude was altered in the presence of tetrodotoxin suggesting direct effects on the NTS neuronal membrane. In contrast, reducing d-glucose concentration induced a postsynaptic response (inward or outward current) in 54% of NTS neurons; tetrodotoxin abolished these responses, suggesting indirect sites of action. The frequency, but not amplitude, of spontaneous and miniature excitatory postsynaptic currents (EPSCs) was correlated with d-glucose concentration in 79% of neurons tested (n = 48). Prior surgical afferent rhizotomy abolished the ability of D-glucose to modulate spontaneous EPSC frequency, suggesting presynaptic actions at vagal afferent nerve terminals to modulate glutamatergic synaptic transmission. In experiments in which EPSCs were evoked via electrical stimulation of the tractus solitarius, EPSC amplitude correlated with D-glucose concentration. These effects were not mimicked by L-glucose, suggesting the involvement of glucose metabolism, not uptake, in the nerve terminal. These data suggest that the synaptic connections between vagal afferent nerve terminals and NTS neurons are a strong candidate for consideration as one of the sites where glucose-evoked changes in vagovagal reflexes occurs.     

Comparative pharmacology of cholecystokinin induced activation of cultured vagal afferent neurons from rats and mice

Cholecystokinin (CCK) facilitates the process of satiation via activation of vagal afferent neurons innervating the upper gastrointestinal tract. Recent findings indicate CCK acts on these neurons via a ruthenium red (RuR) sensitive pathway that involves members of the vanilloid (V) subfamily of transient receptor potential (TRP) channels. To further test this mechanism, the mouse provides an ideal model in which genetic tools could be applied. However, whether CCK acts by similar mechanism(s) in mice has not been determined. In the present study we explored the actions of CCK on nodose neurons isolated from Sprague Dawley (SD) rat and two strains of mice; C57BL/6 and BalbC using fluorescence-based calcium imaging. With minor exceptions nodose neurons isolated from all species/strains behaved similarly. They all respond to brief depolarization with a large calcium transient. A significant subset of neurons responded to capsaicin (CAP), a TRPV1 agonist, although neurons from C57BL/6 were 10-fold more sensitive to CAP than SD rats or BalbC mice, and a significantly smaller fraction of neurons from BalbC mice responded to CAP. CCK-8 dose-dependently activated a subpopulation of neurons with similar dose dependency, percent responders, and overlap between CCK and CAP responsiveness. In all species/strains CCK-8 induced activation was significantly attenuated (but not completely blocked) by pretreatment with the TRPV channel blocker RuR. Surprisingly, the CCK analogue JMV-180, which is reported to have pure antagonistic properties in rat but mixed agonist/antagonist properties in mice, behaved as a pure antagonist to CCK in both rat and mouse neurons. The pure antagonistic action of JMV-180 in this in vitro preparation suggests that prior reported differential effects of JMV-180 on satiation in rats versus mouse must be mediated by a site other than vagal afferent activation.     

Cholecystokinin increases cytosolic calcium in a subpopulation of cultured vagal afferent neurons

Imaging fluorescent measurements with fura 2 were used to examine cytosolic calcium signals induced by sulfated CCK octapeptide (CCK-8) in dissociated vagal afferent neurons from adult rat nodose ganglia. We found that 40% (184/465) of the neurons responded to CCK-8 with a transient increase in cytosolic calcium. The threshold concentration of CCK-8 for inducing the response varied from 0.01 to 100 nM. In most neurons (13/16) the response was eliminated by removing extracellular calcium. Depleting intracellular calcium stores with thapsigargin slightly augmented the response. Most neurons were unresponsive to nonsulfated CCK-8. The response was eliminated by the CCK-A receptor antagonist lorglumide. Low concentrations of JMV-180 had no effect; however, high concentrations of JMV-180 reduced responses to CCK-8. These results demonstrate that CCK acts at the low-affinity site of the CCK-A receptor to trigger the entry of extracellular calcium into vagal afferent neurons. Increased cytosolic calcium may participate in acute activation of vagal afferent neurons, or it may initiate long-term changes, which modulate future neuronal responses to sensory stimuli.     

In vitro analysis of the effects of cholecystokinin on rat brain stem motoneurons

Using whole cell patch clamp in thin brain stem slices, we tested the effects of cholecystokinin (CCK) on identified gastric-projecting neurons of the rat dorsal motor nucleus of the vagus (DMV). Perfusion with the sulfated form of CCK octapeptide (CCK8s, 30 pM-300 nM, EC50 approximately 4 nM) induced a concentration-dependent inward current in 35 and 41% of corpus- and antrum/pylorus-projecting DMV neurons, respectively. Conversely, none of the fundus-projecting DMV neurons responded to perfusion with CCK8s. The CCK8s-induced inward current was accompanied by a 65 +/- 17% increase in membrane input resistance and reversed at 90 +/- 4 mV, indicating that the excitatory effects of CCK8s were mediated by the closure of a potassium conductance. Pretreatment with the synaptic blocker TTX (0.3-1 microM) reduced the CCK8s-induced current, suggesting that a portion of the CCK8s-induced current was mediated indirectly via an action on presynaptic neurons apposing the DMV membrane. Pretreatment with the selective CCK-A receptor antagonist lorglumide (0.3-3 microM) attenuated the CCK8s-induced inward current in a concentration-dependent manner, with a maximum inhibition of 69 +/- 12% obtained with 3 microM lorglumide. Conversely, pretreatment with the selective CCK-B antagonist triglumide did not attenuate the CCK8s-induced inward current; pretreatment with triglumide (3 microM) and lorglumide (1 microM) attenuated the CCK8s-induced current to the same extent as pretreatment with lorglumide alone. Immunohistochemical experiments showed that CCK-A receptors were localized on the membrane of 34, 65, and 60% of fundus-, corpus-, and antrum/pylorus-projecting DMV neurons, respectively. Our data indicate that CCK-A receptors are present on a subpopulation of gastric-projecting neurons and that their activation leads to excitation of the DMV membrane.     

Low-affinity CCK-A receptors are coexpressed with leptin receptors in rat nodose ganglia: implications for leptin as a regulator of short-term satiety

The paradigm for the control of feeding behavior has changed significantly. Research has shown that leptin, in the presence of CCK, may mediate the control of short-term food intake. This interaction between CCK and leptin occurs at the vagus nerve. In the present study, we aimed to characterize the interaction between CCK and leptin in the vagal primary afferent neurons. Single neuronal discharges of vagal primary afferent neurons innervating the gastrointestinal tract were recorded from rat nodose ganglia. Three groups of nodose ganglia neurons were identified: group 1 responded to CCK-8 but not leptin; group 2 responded to leptin but not CCK-8; group 3 responded to high-dose CCK-8 and leptin. In fact, the neurons in group 3 showed CCK-8 and leptin potentiation, and they responded to gastric distention. To identify the CCK-A receptor (CCKAR) affinity states that colocalize with the leptin receptor OB-Rb, we used CCK-JMV-180, a high-affinity CCKAR agonist and low-affinity CCKAR antagonist. As expected, immunohistochemical studies showed that CCK-8 administration significantly potentiated the increase in the number of c-Fos-positive neurons stimulated by leptin in vagal nodose ganglia. Administration of CCK-JMV-180 eliminated the synergistic interaction between CCK-8 and leptin. We conclude that both low- and high-affinity CCKAR are expressed in nodose ganglia. Many nodose neurons bearing low-affinity CCKAR express OB-Rb. These neurons also respond to mechanical distention. An interaction between CCKAR and OB-Rb in these neurons likely facilitates leptin mediation of short-term satiety.     

Mechanisms of action of CCK to activate central vagal afferent terminals

Cholecystokinin [CCK] is a peptide released as a hormone by the proximal gut in response to the presence of peptones and fatty acid in the gut. Considerable evidence suggests that CCK inhibits feeding behavior and gastric function by acting as a paracrine modulator of vagal afferents in the periphery, especially in the duodenum. CCK is also widely distributed throughout the mammalian brain and appears to function as a neurotransmitter and neuromodulator. More recent studies have suggested that CCK may act directly within the CNS to activate central vagal afferent terminal inputs to the solitary nucleus. We have developed an in vitro calcium imaging method that reveals, for the first time, the direct effects of this peptide on vagal terminals in the solitary nucleus. In vitro imaging reveals that CCK provokes increases in intracellular calcium in vagal afferent terminals as a consequence of a complex interaction between protein kinase A [PKA] and phospholipase C [PLC] transduction mechanisms that open L-type calcium channels and causes endoplasmic reticular [ER] calcium release. The subsequent activation of PKC may be responsible for initiating calcium spiking which is dependent on a TTX-sensitive mechanism. Thus, imaging of the isolated but spatially intact hindbrain slice has allowed a more complete appreciation of the interdependent transduction mechanisms used by CCK to excite identified central vagal afferent fibers and varicosities.     

Effect of CCK pretreatment on the CCK sensitivity of rat polymodal gastric vagal afferent in vitro

To prevent the blood-borne interference and reflex actions via neighboring organs and the central nervous system, the study was conducted in an in vitro isolated stomach-gastric vagus nerve preparation obtained from overnight-fasted, urethan-anesthetized rats. Afferent unit action potentials were recorded from the gastric branch of the vagus nerve. The left gastric artery was catheterized for intra-arterial injection. In vitro we found that 1) 55/70 gastric vagal afferents (GVAs) were polymodal, responding to CCK-8 and mechanical stimuli, 13 were mechanoreceptive, and 2 were CCK-responsive; 2) sequential or randomized intra-arterial injections of CCK-8 (0.1-200 pmol) dose-dependently increased firing rate and reached the peak rate at 100 pmol; 3) the action was suppressed by CCK-A (Devazepide) but not by CCK-B (L-365,260) receptor antagonist; 4) neither antagonist blocked the mechanosensitivity of GVA fibers. These results are consistent with corresponding in vivo well-documented findings. Histological data indicate that the layered structure of the stomach wall was preserved in vitro for 6-8 h. Based on these results, it seems reasonable to use the in vitro preparation for conducting a study that is usually difficult to be performed in vivo. For instance, because there was no blood supply in vitro, the composition of the interstitial fluid, i.e., the ambient nerve terminals, can be better controlled and influenced by intra-arterial injection of a defined solution. Here we report that acutely changing the ambient CCK level by a conditioning stimulus (a preceding intra-arterial injection of increasing doses of CCK-8) reduced the CCK sensitivity of GVA terminals to a subsequent test stimulus (a constant dose of CCK-8 intra-arterial injection).     

Canine vagus nerve stores cholecystokinin-58 and -8 but releases only cholecystokinin-8 upon electrical vagal stimulation

Cholecystokinin-58 has been shown to be the major form of cholecystokinin (CCK) released to the circulation upon lumenal stimulation of the small intestine in humans and dogs. In anesthetized dogs, electrical vagal stimulation evokes pancreatic exocrine secretion that is in part mediated through the release of CCK. We studied the molecular form of CCK stored in canine vagus nerves and that released into circulation upon electrical vagal stimulation. Gel filtration and radioimmunoassay of the water and acid extracts of canine vagus nerves indicated CCK-8 (35%) and CCK-58 (65%) as the major molecular forms in the vagus nerve. Both forms of CCK isolated from the vagal extracts were equally bioactive as the standard CCK-8 and CCK-58, respectively, in stimulation of amylase release from isolated rat pancreatic acini. Analysis of plasma collected after electrical vagal stimulation indicated that CCK-8 is the only form released into the circulation. The release of CCK-8 upon electrical vagal stimulation was not affected by application of lidocaine to the upper small intestinal mucosa, suggesting that it was released from vagal nerve terminals.     

Targeted disruption of the murine CCK1 receptor gene reduces intestinal lipid-induced feedback inhibition of gastric function

Cholecystokinin (CCK), acting at CCK1 receptors (CCK1Rs) on intestinal vagal afferent terminals, has been implicated in the control of gastrointestinal function and food intake. Using CCK1R(-/-) mice, we tested the hypothesis that lipid-induced activation of the vagal afferent pathway and intestinal feedback of gastric function is CCK1R dependent. In anesthetized CCK1R(+/+) ("wild type") mice, meal-stimulated gastric acid secretion was inhibited by intestinal lipid infusion; this was abolished in CCK1R(-/-) mice. Gastric emptying of whole egg, measured by nuclear scintigraphy in awake mice, was significantly faster in CCK1R(-/-) than CCK1R(+/+) mice. Gastric emptying of chow was significantly slowed in response to administration of CCK-8 (22 pmol) in CCK1R(+/+) but not CCK1R(-/-) mice. Activation of the vagal afferent pathway was measured by immunohistochemical localization of Fos protein in the nucleus of the solitary tract (NTS; a region where vagal afferents terminate). CCK-8 (22 pmol ip) increased neuronal Fos expression in the NTS of fasted CCK1R(+/+) mice; CCK-induced Fos expression was reduced by 97% in CCK1R(-/-) compared with CCK1R(+/+) mice. Intralipid (0.2 ml of 20% Intralipid and 0.04 g lipid), but not saline, gavage increased Fos expression in the NTS of fasted CCK1R(+/+) mice; lipid-induced Fos expression was decreased by 47% in CCK1R(-/-) compared with CCK1R(+/+)mice. We conclude that intestinal lipid activates the vagal afferent pathway, decreases gastric acid secretion, and delays gastric emptying via a CCK1R-dependent mechanism. Thus, despite a relatively normal phenotype, intestinal feedback in response to lipid is severely impaired in these mice.     

Gastric emptying in response to IAPP and CCK in rats with subdiaphragmatic afferent vagotomy

In the subdiaphragmatic vagal deafferentation procedure (SDA), the afferent fibers of the vagus are surgically severed unilaterally where they enter the brain stem. The technique includes a subdiaphragmal truncal vagotomy performed on the contralateral side. This procedure has been used to study the control of food intake, but it has not been used previously to investigate the role of vagal afferent fibers in the control of gastric emptying (GE). The current experiment studied the effect of SDA on the inhibition of GE by islet amyloid polypeptide (IAPP or amylin) and cholecystokinin (CCK) in awake, unrestrained rats with gastric cannulas. The experimental group underwent subdiaphragmatic vagal deafferentation; the control group had sham operations. All rats received 20-min intravenous infusions of IAPP (1, 3, 9, 27, and 81 pmol/kg/min), CCK (3, 30 and 90 pmol/kg/min), and normal saline in random order. Gastric emptying of saline was measured by the phenol red method during the last 5 min of each infusion period. CCK dose-dependently inhibited gastric emptying in both the control and SDA animals. The inhibition of GE by CCK was significantly attenuated by SDA (p<0.01). IAPP also inhibited gastric emptying dose-dependently, but the difference between the SDA and control groups was not significant. The current experiment, which used a different methodology than previous studies, provides support for the hypothesis that the inhibition of gastric emptying by CCK, but not by IAPP, is mediated partly by afferent vagal fibers.     

Characterization of CCK(A) receptor affinity states and Ca(2+) signal transduction in vagal nodose ganglia

CCK(A) receptors are present on vagal afferent fibers. The objectives of this study were to identify the presence of high- and low-affinity CCK(A) receptors on nodose ganglia and to characterize the intracellular calcium signal transduction activated by CCK. Stimulation of acutely isolated nodose ganglion cells from rats with 1 nM CCK-8 primarily evoked a Ca(2+) transient followed by a sustained Ca(2+) plateau (45% of cells responded), whereas 10 pM CCK-8 evoked Ca(2+) oscillations (37% of cells responded). CCK-OPE, a high-affinity agonist and low-affinity antagonist of CCK(A) receptors, primarily elicited Ca(2+) oscillations (29% of cells responded). CCK-OPE inhibited the Ca(2+) transient induced by 1 nM CCK-8 but not by carbachol and high K(+). This result suggests the presence of high- and low-affinity states of CCK(A) receptors on nodose ganglia. We further demonstrated that nicardipine (10 microM) but not omega-conotoxins GVIA and MVIIC (10-100 nM) abolished Ca(2+) signaling induced by CCK-8, indicating that an L-type voltage-dependent Ca(2+) channel and not an N- or Q-type Ca(2+) channel is coupled to CCK(A) receptors. In a separate study, we showed that the G protein activator NaF (10 mM) elicited a Ca(2+) transient and inhibited CCK-8-evoked Ca(2+) signaling, indicative of G protein(s) involvement in CCK(A) receptor activity. The G(q) protein antagonist Gp antagonist-2A (10 microM) also abolished the action of CCK-8. This study indicates that CCK(A) receptors exist in both high- and low-affinity states in the nodose ganglia. Activation of high-affinity CCK(A) receptors elicits Ca(2+) oscillations, whereas stimulation of low-affinity CCK(A) receptors evokes a sustained Ca(2+) plateau. These Ca(2+)-signaling modes are mediated through the L-type Ca(2+) channel and involve the participation of G(q) protein.     

CCK enhances response to gastric distension by acting on capsaicin-insensitive vagal afferents

Capsaicin treatment destroys vagal afferent C fibers and markedly attenuates reduction of food intake and induction of hindbrain Fos expression by CCK. However, both anatomical and electrophysiological data indicate that some gastric vagal afferents are not destroyed by capsaicin. Because CCK enhances behavioral and electrophysiological responses to gastric distension in rats and people, we hypothesized that CCK might enhance the vagal afferent response to gastric distension via an action on capsaicin-insensitive vagal afferents. To test this hypothesis, we quantified expression of Fos-like immunoreactivity (Fos) in the dorsal vagal complex (DVC) of capsaicin-treated (Cap) and control rats (Veh), following gastric balloon distension alone and in combination with CCK injection. In Veh rats, intraperitoneal CCK significantly increased DVC Fos, especially in nucleus of the solitary tract (NTS), whereas in Cap rats, CCK did not significantly increase DVC Fos. In contrast to CCK, gastric distension did significantly increase Fos expression in the NTS of both Veh and Cap rats, although distension-induced Fos was attenuated in Cap rats. When CCK was administered during gastric distension, it significantly enhanced NTS Fos expression in response to distension in Cap rats. Furthermore, CCK's enhancement of distension-induced Fos in Cap rats was reversed by the selective CCK-A receptor antagonist lorglumide. We conclude that CCK directly activates capsaicin-sensitive C-type vagal afferents. However, in capsaicin-resistant A-type afferents, CCK's principal action may be facilitation of responses to gastric distension.     

Glucose increases synaptic transmission from vagal afferent central nerve terminals via modulation of 5-HT3 receptors

Acute hyperglycemia has profound effects on vagally mediated gastrointestinal functions. We have reported recently that the release of glutamate from the central terminals of vagal afferent neurons is correlated directly with the extracellular glucose concentration. The present study was designed to test the hypothesis that 5-HT(3) receptors present on vagal afferent nerve terminals are involved in this glucose-dependent modulation of glutamatergic synaptic transmission. Whole-cell patch-clamp recordings were made from neurons of the nucleus tractus solitarius (NTS) in thin rat brainstem slices. Spontaneous and evoked glutamate release was decreased in a concentration-dependent manner by the 5-HT(3) receptor selective antagonist, ondansetron. Alterations in the extracellular glucose concentration induced parallel shifts in the ondansetron-mediated inhibition of glutamate release. The changes in excitatory synaptic transmission induced by extracellular glucose concentration were mimicked by the serotonin uptake inhibitor, fenfluramine. These data suggest that glucose alters excitatory synaptic transmission within the rat brainstem via actions on tonically active 5-HT(3) receptors, and the number of 5-HT(3) receptors on vagal afferent nerve terminals is positively correlated with the extracellular glucose concentration. These data indicate that the 5-HT(3) receptors present on synaptic connections between vagal afferent nerve terminals and NTS neurons are a strong candidate for consideration as one of the sites where glucose acts to modulate vagovagal reflexes.