Characterization of CCKA receptor affinity states and Ca2+ signal transduction in vagal nodose ganglia

Tim O. Lankisch, Yasuhiro Tsunoda, Yuanxu Lu, and Chung Owyang

Gastroenterology Research Unit, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CCKA receptors are present on vagal afferent fibers. The objectives of this study were to identify the presence of high- and low-affinity CCKA 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 Ca2+ transient followed by a sustained Ca2+ plateau (45% of cells responded), whereas 10 pM CCK-8 evoked Ca2+ oscillations (37% of cells responded). CCK-OPE, a high-affinity agonist and low-affinity antagonist of CCKA receptors, primarily elicited Ca2+ oscillations (29% of cells responded). CCK-OPE inhibited the Ca2+ 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 CCKA receptors on nodose ganglia. We further demonstrated that nicardipine (10 µM) but not omega -conotoxins GVIA and MVIIC (10-100 nM) abolished Ca2+ signaling induced by CCK-8, indicating that an L-type voltage-dependent Ca2+ channel and not an N- or Q-type Ca2+ channel is coupled to CCKA receptors. In a separate study, we showed that the G protein activator NaF (10 mM) elicited a Ca2+ transient and inhibited CCK-8-evoked Ca2+ signaling, indicative of G protein(s) involvement in CCKA receptor activity. The Gq protein antagonist Gp antagonist-2A (10 µM) also abolished the action of CCK-8. This study indicates that CCKA receptors exist in both high- and low-affinity states in the nodose ganglia. Activation of high-affinity CCKA receptors elicits Ca2+ oscillations, whereas stimulation of low-affinity CCKA receptors evokes a sustained Ca2+ plateau. These Ca2+-signaling modes are mediated through the L-type Ca2+ channel and involve the participation of Gq protein.

cholecystokinin; cholecystokinin A receptors; Ca2+ signaling


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CCK, WHICH BELONGS TO THE brain-gut peptide family, regulates gastrointestinal functions such as pancreatic enzyme secretion, gallbladder contraction, gastrointestinal motility, and cell growth (6, 7, 8a, 12-14). In the central nervous system (CNS), CCK may regulate anxiety, behavior, and learning/memory processes (11). These actions are mediated through two different CCK receptors, CCAA and CCKB, which have been cloned and characterized in different tissues. CCKA and CCKB receptors each consist of seven hydrophobic transmembrane domains homologous to other members of the G protein-coupled receptor superfamily (8, 23). CCKA and CCKB receptors are distributed throughout the gastrointestinal tract, and they are localized in several regions of the CNS. In addition, CCK receptors have been detected in the rat vagus nerve (12, 25, 28). These receptors are transported from the nodose ganglion to the peripheral nerve ending.

The existence of two different affinity sites of the CCKA receptor was first demonstrated in pancreatic acini (10). The high affinity of the CCKA receptor elicits Ca2+ oscillations and amylase secretion, whereas the low-affinity state evokes a large Ca2+ transient and may mediate both stimulation and inhibition of enzyme secretion, depending on experimental conditions. These different affinity states of the CCKA receptor may also exist and function in the autonomic nervous system. In vivo studies in rats have shown that high-affinity CCKA receptors on the vagus nerve mediate CCK-8-stimulated pancreatic enzyme secretion (9). Conversely, low-affinity vagal CCKA receptors appear to mediate satiety (24). Evidence of high- and low-affinity CCKA receptors on the vagus nerve has not been directly demonstrated in vitro, and the mode of calcium signal transduction activated by CCK has yet to be determined. The objectives of this study were to identify the presence of high- and low-affinity CCKA receptors on vagal nodose ganglia by performing intracellular Ca2+ studies using various CCKA-receptor agonists and antagonists and to characterize the intracellular Ca2+ signaling activated by CCK.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Chemicals were purchased from the following sources: CCK-8, collagenase type IA, trypsin, soybean trypsin inhibitor (SBTI), nicardipine, and NaF from Sigma (St. Louis, MO); CCK-OPE from Research Plus (Bayonne, NJ); Hanks' balanced salt solution (HBSS) from Life Technologies (Grand Island, NY); fura 2-AM, omega -conotoxin GVIA, and omega -conotoxin MVIIC from Calbiochem (San Diego, CA); Gp antagonist-2A from Biomol (Plymouth Meeting, PA); and Cell-Tak from Collaborative Biomedical Products (Bedford, MA). All chemicals were dissolved in either dimethyl sulfoxide, ethanol, acetic acid, or distilled water, the final concentrations being 0.01-0.1%, which did not affect cell responses.

Methods. Acutely isolated nodose ganglion neurons were obtained from 4-wk-old male Sprague-Dawley rats and prepared by collagenase digestion described previously (6). Rats were anesthetized with an intramuscular injection of xylazine and ketamine; then nodose ganglia were removed from connective tissues and minced into fine pieces. In each experiment, three rats were used to obtain isolated nodose ganglion cells. The tissue fragments were incubated with 3 ml HBSS containing 1 mg/ml collagenase (type I-A), 0.5 mg/ml trypsin (type 1), and 1 mg/ml BSA for 30 min at 37°C. After incubation, the ganglia fragments were gently triturated six times to disperse the cells under dissecting microscope, and the solution was diluted with 3 ml HBSS followed by centrifugation at 900 revolutions/min (rpm). The neurons were then incubated in 3 ml HBSS containing 1.5 ml SBTI (type II-S) and 1 mg/ml BSA for 8 min at 37°C. The incubation was stopped by centrifugation at 900 rpm, and the nodose ganglion cells were resuspended in a physiological salt solution (PSS) and washed twice followed by centrifugation (900 rpm). The PSS contained 0.1% BSA and (in mM) 137 NaCl, 4.7 KCl, 0.56 MgCl2, 1.28 CaCl2, 1 NaH2PO4, 10 HEPES, Eagle's minimum essential amino acid neutralized with NaOH, 2 L-glutamine, and 5.5 D-glucose. The PSS was adjusted to pH 7.4 and equilibrated with 100% O2. The intracellular Ca2+ concentration ([Ca2+]i) measurements were performed as previously described (20). In brief, isolated nodose ganglion cells were incubated with 2 µM fura 2-AM in 3 ml PSS for 30 min at 37°C. All experiments were done using a dual-excitation wavelength (340/380 nm emitted at 505 nm) modular fluorometer system (SPEX) coupled to a Nikon Diaphot inverted microscope magnification (×40). Isolated nodose ganglion cells were attached to a glass coverslip that was coated with the natural adhesive Cell-Tak. These coverslips were mounted on a closed chamber and superfused with PSS from a reservoir (1 ml/min). A fluoroscence ratio was converted to [Ca2+]i according to in vitro calibration with an external standard and 50 µM fura 2-AM potassium salt (5). All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Every effort was made to minimize animal suffering and reduce the number of animals used. Statisitical analysis was performed by the Student's t-test. Results were expressed as means ± SE. P < 0.05 was considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CCK-8 and the high-affinity CCKA receptor agonist CCK-OPE induce Ca2+ signaling in individual nodose ganglion cells. To investigate whether CCK analogs stimulate Ca2+ signaling in nodose ganglia, we measured [Ca2+]i in fura 2-AM-loaded single cells. Mean basal [Ca2+]i in individual nodose ganglion cells was 67 ± 3 nM (n = 48). Some nodose ganglion cells demonstrated Ca2+ oscillation in the resting state (~15% occurrence); these cells were not used for [Ca2+]i measurement. On stimulation with 1 nM CCK-8, an initial transient followed by a sustained increase in [Ca2+]i to 182 nM was observed in 21 of 47 cells (Fig. 1A and Table 1). In contrast, 10 pM CCK-8 elicited repetitive Ca2+ oscillations in 15 of 41 cells. Ca2+ oscillations occurred with a frequency of 2.5 ± 0.4 cycles/5 min and an amplitude (peak-basal) of 113 ± 16 nM (Fig. 1B). The threshold of CCK-8 concentration to evoke Ca2+ oscillations was 1 pM, with two of nine cells responding. Application of the high-affinity CCKA receptor agonist CCK-OPE (100 nM) to nodose ganglion cells elicited repetitive Ca2+ oscillations in 12 of 41 cells (Fig. 1C). Ca2+ oscillations occurred with a frequency of 2.1 ± 0.3 cycles/5 min and an amplitude of 86 ± 11 nM. The threshold CCK-OPE concentration to evoke Ca2+ spiking was 10 nM, with two of nine cells responding. No response was observed when lower concentrations of CCK-8 (0.5 pM) or CCK-OPE (5 nM) were used (9 of 9 cells). These data indicate that stimulation of low-affinity CCKA receptors evokes an initial transient followed by a sustained increase in [Ca2+]i, whereas activation of high-affinity CCKA receptors elicits Ca2+ oscillations. Administration of the CCKA receptor antagonist L-364,718 (10-6 M) but not the CCKB receptor antagonist L-365,260 (10-6M) inhibited calcium oscillations evoked by CCK-8 (1 nM and 10 pM; 57 ± 6 mM vs. 171 ± 7 nM, P < 0.05, and 63 ± 10 vs. 185 ± 10 nM, P < 0.05; n = 6) or CCK-OPE (100 nM), indicating that these CCK analogs were acting via CCKA receptors. All calculations and statistical analyses, including the Ca2+ spike amplitude and frequency, were made with cells responding to CCK analogs.


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Fig. 1.   Effects of CCK-8 and CCK-OPE on Ca2+ signaling in fura 2-AM-loaded individual nodose ganglion cells from 4-wk-old rats. A: 1 nM CCK-8 induced a sustained intracellular Ca2+ concentration ([Ca2+]i) plateau. B: application of 10 pM CCK-8 stimulated Ca2+ oscillations. C: 100 nM CCK-OPE elicited Ca2+ oscillations. Each panel is representative of 21 (A), 15 (B), and 12 (C) separate determinations.


                              
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Table 1.   Amplitude and frequency of Ca2+ signaling induced by high and low concentrations of CCK-8 and CCK-OPE in individual nodose ganglion cells

High- and low-affinity CCKA receptors exist in nodose ganglion cells. As shown in Fig. 2A, a low concentration (10 pM) of CCK-8 induced Ca2+ oscillations (n = 8). When the concentration of CCK-8 was increased to 1 nM, a transient increase in [Ca2+]i occurred, followed by a prolonged but smaller elevation of [Ca2+]i without oscillations (n = 8). CCK-OPE as well as JMV-180 act as high-affinity agonists and as low-affinity antagonists of the CCKA receptor (4). CCK-OPE (100 nM) evoked Ca2+ oscillations and blocked Ca2+ signalings elicited by 1 nM CCK-8 (n = 11, P < 0.05; Fig. 3). However, subsequent application of 10 µM carbachol evoked a transient increase in [Ca2+]i even in the presence of CCK-OPE (n = 5; Fig. 2B; Fig. 3), suggesting that CCK-OPE acts as a specific antagonist of the low-affinity CCKA receptor. Similarly, CCK-8 (1 nM) evoked a Ca2+ transient, which was inhibited by application of CCK-OPE (n = 11, P < 0.05; Fig. 2C). In separate experiments, we showed that CCK-OPE (100 nM) inhibited Ca2+ transients induced by 10 nM CCK-8 (Fig. 3), but it did not affect Ca2+ transient stimulated by 50 mM K+ (n = 4; Fig. 4). This indicates that the inhibitory action of CCK-OPE on CCK-8-induced Ca2+ signalings is agonist specific. These results suggest that high- and low-affinity CCKA receptors are present in nodose ganglion cells.


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Fig. 2.   Effects of CCK-8 and CCK-OPE on Ca2+ signaling in nodose ganglion cells. A: 10 pM CCK-8 elicited Ca2+ oscillations, whereas 1 nM CCK-8 evoked a Ca2+ transient in the same cell. B: 100 nM CCK-OPE elicited Ca2+ oscillations but inhibited the Ca2+ transient in response to 1 nM CCK-8. C: 1 nM CCK-8 induced a Ca2+ transient, which was inhibited by 100 nM CCK-OPE. Each panel is representative of 8 (A) and 10 (B and C) separate determinations.



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Fig. 3.   Effects of CCK-OPE on the amplitude of Ca2+ signaling induced by high concentration of CCK-8 in nodose ganglion cells. CCK-8 induced an initial transient followed by a sustained increase in [Ca2+] (n = 8). CCK-OPE 100 nM inhibited Ca2+ transients induced by 1 nM of CCK-8 (P < 0.01; n = 10), but it did not affect Ca2+ spikes evoked by 100 µM carbachol (n = 5). **P < 0.01.



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Fig. 4.   Representative tracings to demonstrate the effects of CCK-OPE, CCK-8, and K+ on Ca2+ signaling in the nodose ganglia. CCK-OPE (100 nM) stimulated a Ca2+ transient but inhibited Ca2+ signaling evoked by CCK-8 (10 nM). CCK-OPE did not inhibit Ca2+ transient elicited by K+ (50 mM; n = 4). When K+ was increased from 5 to 50 mM in physiological saline, concentration of Na+ in medium was reduced to 90 mM.

Elimination of extracellular Ca2+ and an L-type Ca2+ channel blocker inhibit Ca2+ signaling in response to CCK-8. We next examined whether extracellular Ca2+ was required for Ca2+ signaling induced by CCK-8 and CCK-OPE. Elimination of extracellular calcium abolished the Ca2+ plateau induced by 1 nM CCK-8 and the Ca2+ oscillations evoked by 10 pM CCK-8 in seven of seven cells (P < 0.01; Fig. 5, A and B). These changes were reversible with the reintroduction of extracellular Ca2+ (1.28 mM). CCK-OPE (100 nM) also failed to elicit Ca2+ signaling in the absence of extracellular Ca2+ in three of three cells (data not shown). These results indicate that CCK analogs elicit Ca2+ signaling using an extracellular Ca2+ source. We further characterized the type of Ca2+ channel coupled to the CCKA receptor in nodose ganglion cells. The L-type Ca2+ channel blocker nicardipine (10 µM) abolished 1 nM CCK-8-stimulated Ca2+ signaling in all eight cells tested (P < 0.01; Figs. 6 and 7A). Nicardipine also abolished Ca2+ oscillations elicited by 10 pM CCK-8 in all six cells tested. In contrast, neither the N-type Ca2+ channel blocker omega -conotoxin GVIA (10-100 nM) nor the Q type channel blocker omega -conotoxin MVIIC (10 nM) inhibited Ca2+ signaling evoked by 1 nM CCK-8 in the 13 cells that responded to CCK-8 (Figs. 6 and 7, B and C). Our results showed that the voltage-dependent Ca2+ channel coupled to the vagal CCKA receptor in the nodose ganglia is an L-type channel.


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Fig. 5.   Representative tracings to demonstrate the effect of elimination of extracellular Ca2+ on CCK-8-induced Ca2+ signaling. Extracellular Ca2+ ([Ca2+]o) was removed from medium, and 1 mM EGTA was added 1 min before administration of CCK-8. Neither high nor low doses of CCK-8 induced Ca2+ signaling in the absence of [Ca2+]o. These changes were reversible with the reintroduction of extracellular Ca2+ (1.28 mM, no lines indicated). A and B are representative tracings of 7 separate determinations.



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Fig. 6.   Effects of elimination of [Ca2+]0 and various Ca2+-channel blockers on Ca2+ signaling induced by CCK-8. Elimination of extracellular Ca2+ abolished Ca2+ transient and plateau induced by 1 nM CCK-8 (P < 0.01; n = 7). Similarly, the L-type Ca2+-channel blocker nicardipine (10 µM) also abolished Ca2+ signaling evoked by 1 nM CCK-8 (P < 0.01; n = 8). In contrast, neither the N-type Ca2+-channel blocker omega -conotoxin GVIA (100 nM) nor the Q-type channel blocker omega -conotoxin MVIIC (10 nM) had any effect on Ca2+ signaling evoked by 1 nM CCK-8 (n = 13). Delta , Change in. **P < 0.01.



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Fig. 7.   Representative tracings to demonostrate the effects of Ca2+-channel blockers on Ca2+ signaling induced by CCK-8. A: preincubation of cells with the L-type Ca2+-channel blocker nicardipine (10 µM) for 8 min inhibited the 1 nM CCK-8-mediated Ca2+ transient. Data represent 8 separate determinations. B and C: the N-type channel blocker omega -conotoxin GVIA (10-100 nM) and the Q-type channel blocker omega -conotoxin MVIIC (10 nM) did not affect CCK-8-induced Ca2+ signaling. B and C represent 13 separate experiments.

G protein(s) is involved in mediating Ca2+ signaling evoked by CCK-8. It is well accepted that activation of G protein(s) will inactivate receptors coupled to G protein(s) (16). As shown in Fig. 8A, the G protein activator NaF (10 mM) evoked a Ca2+ transient and abolished the Ca2+ signaling evoked by CCK-8 (1 nM) in five of five cells [CCK-8: 115 ± 10.5 nM (n = 21) vs. CCK-8 + NaF: 28.5 ± 9.7 nM (n = 5); P < 0.05 by unpaired t-test]. This suggests that CCKA receptors in the nodose ganglia are coupled to G protein(s). To further confirm this observation and characterize the type of G protein coupled to CCKA receptors in the nodose ganglion cells, we used the Gq protein antagonist Gp antagonist-2A (19). Administration of Gp antagonist-2A (10 µM) caused a marked reduction of the Ca2+ signaling stimulated by 1 nM CCK-8 in nine of nine cells (Fig. 8B), suggesting that Gq protein is involved in CCKA receptor activity [CCK-8: 115 ± 10.5 nM (n = 21) vs. CCK-8 + Gp antagonist-2A: 49.6 ± 11.5 nM (n = 9); P < 0.05 by unpaired t-tests].


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Fig. 8.   Representative tracings to demonstrate the effects of NaF, a G protein activator, and Gp antagonist-2A, a Gq protein inhibitor, on Ca2+ signaling elicited by CCK-8. A: NaF (10 mM) evoked a Ca2+ transient and inhibited Ca2+ spiking elicited by CCK-8 (1 nM; n = 5). B: Gp antagonist-2A (10 µM) abolished the Ca2+ signaling stimulated by CCK-8 (1 nM; n = 9).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study showed that, in rat vagal nodose ganglia, 1) 30-45% of nodose ganglion cells responded to CCK stimulation; 2) high doses of CCK-8 elicited a sustained Ca2+ plateau, whereas low doses caused Ca2+ oscillations; 3) CCK-OPE, a high-affinity CCKA receptor agonist, elicited Ca2+ oscillations and inhibited the CCK-induced Ca2+ signaling, indicating the presence of high- and low-affinity CCKA receptors; 4) extracellular Ca2+ is required for Ca2+ signaling elicited by CCK; 5) the voltage-dependent Ca2+ channel coupled to the CCKA receptor is L type; and 6) CCKA receptors in the nodose ganglia are coupled to Gq protein.

The presence of CCKA receptors on the neurons of the nodose ganglion has been demonstrated by receptor autoradiography (25, 28, 29). Recent functional studies have demonstrated that postprandial satiety appears to be mediated by vagal low-affinity CCKA receptors (24), whereas high-affinity CCKA receptors mediate pancreatic enzyme secretion (9). Using [Ca2+]i recording systems in single nodose ganglion cells, we demonstrated that 37% of cells (48 of 129 cells) responded to CCK analogs. In a variety of cell systems, submaximal concentrations of Ca2+-mobilizing agonists elicit Ca2+ oscillations, whereas supramaximal doses evoke an initial large Ca2+ transient followed by a sustained Ca2+ plateau (17). This phenomenon is also found in nodose ganglion cells. The Ca2+ response to CCK-8 was blocked by CCKA receptor antagonist L364,718 and not by CCKB receptor antagonist L365,260, indicating mediation by CCKA receptor. The main objective of this study was to identify the presence of high- and low-affinity CCKA receptors on nodose ganglia. CCK-OPE is a synthetic decapeptide that acts as an agonist to high-affinity CCKA receptors and as an antagonist to low-affinity CCKA receptors (4). We showed that CCK-OPE elicited Ca2+ oscillations. It also blocked the Ca2+ signaling stimulated by high doses of CCK-8 in individual nodose ganglion cells. This finding indicates the presence of high- and low-affinity CCKA receptors in single nodose ganglion cells.

In pancreatic acini, intracellular Ca2+ is used for the Ca2+ oscillations or the large initial Ca2+ transient stimulated by CCK analogs (17, 26), whereas in nodose ganglion cells, the Ca2+ source was strictly extracellular. We further showed that the L-type Ca2+-channel blocker nicardipine abolished the Ca2+ signaling induced by CCK. L-type voltage-dependent Ca2+ channels are concentrated on neuronal cell bodies and at the base of the dendrites in the brain, spinal cord, and retinal neurons, whereas N-type voltage-dependent Ca2+ channels are clustered on the presynaptic plasma membrane of neural fibers mediating neuromuscular functions (15). Thus it is not surprising that the voltage-dependent Ca2+ channel coupled to the CCKA receptors found on the cell bodies of the nodose ganglia is type L and not N or Q. However, our finding requires confirmation by patch-clamp studies.

CCKA receptors are Gq protein-coupled heptahelical receptors. They are coupled to well-characterized intracellular effectors and second-messenger systems and mediate Ca2+ signal transduction in a variety of cell types (22). For example, it has been shown that the low-affinity CCKA receptor in pancreatic acinar cells is coupled to the conventional Galpha q/11/PLC-beta 1 pathway, resulting in production of 1,4,5-inositol trisphosphate (to release Ca2+ and activate calmodulin) and diacylglycerol (to activate protein kinase C) (26). These low-affinity CCKA receptors also appear to be coupled to the nonreceptor protein tyrosine kinase pathway that mediates extracellular Ca2-dependent pancreatic exocytosis (21). In contrast, high-affinity CCKA receptors are coupled to the Gbeta 8 subunit of the Gq protein, which is linked to the cPLA2 pathway, resulting in production of arachidonic acid, which enhances intracellular Ca2+ oscillations (18, 19). It remains to be determined whether high- and low-affinity CCKA receptors in the nodose ganglia use intracellular pathways similar to those used in pancreatic acini. However, the source of Ca2+ used for Ca2+ oscillations or the large Ca2+ transient is completely different in these two cell systems.

To investigate whether the CCKA receptors in the nodose ganglia are coupled to G protein(s), we examined the effect of NaF, a G protein activator. It is well recognized that activation of G protein(s) will inhibit binding to receptors coupled to G protein(s) (20). In this study, we showed that NaF elicited a Ca2+ transient and inhibited the Ca2+ signaling evoked by CCK-8. Because NaF abolished the action of CCK-8, it suggests that the CCKA receptor in the nodose ganglia is coupled to a G protein. This is likely to be Gq, because CCKA receptors are in general linked to Galpha q/11 in a variety of tissues, including pancreatic acinar cells and neurons (26). To exclude coupling of CCKA receptors to Gi, we showed that pertusis toxin treatment did not affect Ca2+ signaling evoked by CCK-8 in the nodose ganglia (unpublished data). In other neuronal systems, such as the substantia nigra dopaminergic neurons, Galpha q/11 mediates CCK-8 activiation of the cationic conductance (27). In the thalamic reticular neurons and hippocampal neurons, CCK-8 increases neuronal excitability by suppressing the resting K+ conductance by an as yet to be determined mechanism (1). Our study indicates that the Gq protein coupled to the CCKA receptor in rat nodose ganglia is involved in the opening of the L-type Ca2+ channels.

In conclusion, this study indicates that CCKA receptors exist in both high- and low-affinity states in the nodose ganglia. Activation of high-affinity CCKA receptors elicits Ca2+ oscillations, whereas stimulation of low-affinity CCKA receptors evokes a Ca2+ transient followed by a small sustained Ca2+ plateau. These Ca2+-signaling modes are mediated through L-type Ca2+ channels and involve the participation of Gq proteins.


    ACKNOWLEDGEMENTS

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-32830 and 5P 30-DK-34933.


    FOOTNOTES

Address for reprint requests and other correspondence: C. Owyang, 3912 Taubman Center, Box 0362, Univ. of Michigan Health System, Ann Arbor, MI 48109 (E-mail: cowyang{at}umich.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpgi.00313.2001

Received 18 July 2001; accepted in final form 15 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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