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

2002 ◽  
Vol 283 (6) ◽  
pp. R1303-R1313 ◽  
Author(s):  
Steven M. Simasko ◽  
Jason Wiens ◽  
Adrienne Karpiel ◽  
Mihai Covasa ◽  
Robert C. Ritter
Keyword(s):  
Cytosolic Calcium ◽  
Threshold Concentration ◽  
Extracellular Calcium ◽  
Calcium Stores ◽  
Afferent Neurons ◽  
Sensory Stimuli ◽  
Adult Rat ◽  
Nodose Ganglia ◽  
Vagal Afferent ◽  
High Concentrations

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.

scholarly journals Cooperative Activation of Cultured Vagal Afferent Neurons by Leptin and Cholecystokinin

Endocrinology ◽  
2004 ◽  
Vol 145 (8) ◽  
pp. 3652-3657 ◽  
Author(s):  
J. H. Peters ◽  
A. B. Karpiel ◽  
R. C. Ritter ◽  
S. M. Simasko
Keyword(s):  
Primary Cultures ◽  
Cytosolic Calcium ◽  
Extracellular Calcium ◽  
Vagal Afferents ◽  
Afferent Neurons ◽  
Adult Rat ◽  
Nodose Ganglia ◽  
Vagal Afferent ◽  
Acute Changes ◽  
And Control

Abstract To test the hypothesis that leptin can directly activate vagal afferent neurons, we used fluorescence imaging to detect acute changes in cytosolic calcium after leptin application to primary cultures of vagal afferent neurons dissociated from adult rat nodose ganglia. We found that approximately 40% of vagal afferent neurons exposed to leptin (40 ng/ml) responded with rapid and reversible increases in cytosolic calcium. These responses were dependent upon extracellular calcium. As previously reported, about 35% of vagal afferents increase cytosolic calcium in response to the gut-peptide cholecystokinin (CCK). A majority (74%) of neurons that responded to CCK also exhibited increases in cytosolic calcium in response to leptin. In addition, synergistic increases in cytosolic calcium were observed when leptin and CCK were applied in combination. These results demonstrate that leptin acts directly on vagal afferent neurons to trigger acute influxes of extracellular calcium. Our results also suggest cooperation between leptin and CCK in the activation of some vagal afferent neurons. Acute activation of vagal afferents by leptin alone and in combination with CCK may contribute to modulation of visceral reflexes and control of food intake.

Leptin and CCK modulate complementary background conductances to depolarize cultured nodose neurons

2006 ◽  
Vol 290 (2) ◽  
pp. C427-C432 ◽  
Author(s):  
J. H. Peters ◽  
R. C. Ritter ◽  
S. M. Simasko
Keyword(s):  
Food Intake ◽  
Outward Current ◽  
Vagal Afferents ◽  
Afferent Neurons ◽  
Inward Current ◽  
Adult Rat ◽  
Nodose Ganglia ◽  
Sodium Dependent ◽  
Ionic Mechanisms ◽  
Vagal Afferent

We have previously reported that intraceliac infusion of leptin induces a reduction of meal size that depends on intact vagal afferents. This effect of leptin is enhanced in the presence of cholecystokinin (CCK). The mechanisms by which leptin and CCK activate vagal afferent neurons are not known. In the present study, we have begun to address this question by using patch-clamp electrophysiological techniques to examine the mechanisms by which leptin and CCK activate cultured vagal afferents from adult rat nodose ganglia. We found that leptin depolarized 41 (60%) of 68 neurons. The magnitude of membrane depolarization was dependent on leptin concentration and occurred in both capsaicin-sensitive and capsaicin-insensitive neurons. We also found that a majority (16 of 22; 73%) of nodose neurons activated by leptin were also sensitive to CCK. CCK-induced depolarization was primarily associated with the increase of an inward current (11 of 12), whereas leptin induced multiple changes in background conductances through a decrease in an outward current (7 of 13), an increase in an inward current (3 of 13), or both (3 of 13). However, further isolation of background currents by recording in solutions that contained only sodium or only potassium revealed that both leptin and CCK were capable of increasing a sodium-dependent conductance or inhibiting a potassium-dependent conductance. Our results support the hypothesis that vagal afferents are a point of convergence and integration of leptin and CCK signaling for control of food intake and suggest multiple ionic mechanisms by which leptin and CCK activate vagal afferent neurons.

scholarly journals Morphologically Identified Cutaneous Afferent DRG Neurons Express Three Different Potassium Currents in Varying Proportions

1998 ◽  
Vol 79 (4) ◽  
pp. 1814-1824 ◽  
Author(s):  
Brian Everill ◽  
Marco A. Rizzo ◽  
Jeffery D. Kocsis
Keyword(s):  
Outward Current ◽  
Potassium Currents ◽  
Entire Population ◽  
Channel Blockers ◽  
Relative Distribution ◽  
Afferent Neurons ◽  
Drg Neurons ◽  
Adult Rat ◽  
High Concentrations ◽  
The Right

Everill, Brian, Marco A. Rizzo, and Jeffery D. Kocsis. Morphologically identified cutaneous afferent DRG neurons express three different potassium currents in varying proportions. J. Neurophysiol. 79: 1814–1824, 1998. Outward K+ currents were recorded using a whole cell patch-clamp configuration, from acutely dissociated adult rat cutaneous afferent dorsal root ganglion (DRG) neurons (L4 and L5) identified by retrograde labeling with Fluoro-gold. Recordings were obtained 16–24 h after dissociation from cells between 39 and 49 mm in diameter with minimal processes. These cells represent medium-sized DRG neurons relative to the entire population, but are large cutaneous afferent neurons giving rise to myelinated axons. Voltage-activated K+ currents were recorded routinely during 300-ms depolarizing test pulses increasing in 10-mV steps from −40 to +50 mV; the currents were preceded by a 500-ms conditioning prepulse of either −120 or −40 mV. Coexpression of at least three components of K+ current was revealed. Separation of these components was achieved on the basis of sensitivities to the K+ channel blockers, 4-aminopyridine (4-AP) and dendrotoxin (DTx), and by the current responses to variation in conditioning voltage. Changing extracellular K+ concentration from 3 to 40 mM resulted in a shift to the right of the I-V curve commensurate with K+ being the principal charge carrier. Presentation of 100 mM 4-AP revealed a rapidly activating K+ current sensitive to low concentrations of 4-AP. High concentrations of 4-AP (6 mM) extinguished all inactivating current, leaving almost pure sustained current ( I K). On the basis of the relative distribution of K+ currents neurons could be separated into three distinct categories: fast inactivating current ( I A), slow inactivating current ( I D), and sustained current ( I K); only I A and I K; and slow inactivating current and I K. However, I K was always the dominant outward current component. These results indicate that considerable variation in K+ currents is present not only in the entire population of DRG neurons, as previously reported, but even within a restricted size and functional group (large cutaneous afferent neurons).

scholarly journals Role of Transient Receptor Potential Channels in Cholecystokinin-Induced Activation of Cultured Vagal Afferent Neurons

Endocrinology ◽  
2010 ◽  
Vol 151 (11) ◽  
pp. 5237-5246 ◽  
Author(s):  
Huan Zhao ◽  
Steven M. Simasko
Keyword(s):  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Cytosolic Calcium ◽  
Membrane Depolarization ◽  
Induced Response ◽  
Trpc Channels ◽  
Afferent Neurons ◽  
Trpv Channels ◽  
Vagal Afferent ◽  
Transient Receptor

Cholecystokinin (CCK), an endogenous brain-gut peptide, is released after food intake and promotes the process of satiation via activation of the vagus nerve. In vitro, CCK increases cytosolic calcium concentrations and produces membrane depolarization in a subpopulation of vagal afferent neurons. However, the specific mechanisms and ionic conductances that mediate these effects remain unclear. In this study we used calcium imaging, electrophysiological measurements, and single cell PCR analysis on cultured vagal afferent neurons to address this issue directly. A cocktail of blockers of voltage-dependent calcium channels (VDCC) failed to block CCK-induced calcium responses. In addition, SKF96365, a compound that blocks both VDCC and the C family of transient receptor potential (TRP) channels, also failed to prevent responses to CCK. Together these results suggest that CCK-induced calcium influx is not subsequent to the membrane depolarization. Ruthenium red, an inhibitor of the TRPV family and TRPA1, blocked both depolarizing responses to CCK and CCK-induced calcium increases, but had no effect on the KCl-induced calcium response. Selective block of TRPV1 and TRPA1 channels with SB366791 and HC030031, respectively, had minor effects on the CCK-induced response. Application of 2-aminoethoxydiphenyl borate, an activator of select TRPV channels but a blocker of several TRPC channels, either had no effect or enhanced the responses to CCK. Further, results from PCR experiments revealed a significant clustering of TRPV2-5 in neurons expressing CCK1 receptors. These observations demonstrate that CCK-induced increases in cytosolic calcium and membrane depolarization of vagal afferent neurons are likely mediated by TRPV channels, excluding TRPV1.

II. Integration of afferent signaling from the viscera by the nodose ganglia

2003 ◽  
Vol 284 (1) ◽  
pp. G8-G14 ◽  
Author(s):  
Kirsteen N. Browning ◽  
David Mendelowitz
Keyword(s):  
Sensory Neurons ◽  
Tissue Injury ◽  
Large Body ◽  
Sensory Function ◽  
Afferent Neurons ◽  
Nodose Ganglia ◽  
Vagal Afferent ◽  
Afferent Signaling ◽  
Recent Demonstration ◽  
Stimulation Of

To understand vago-vagal reflexes, one must have an appreciation of the events surrounding the encoding, integration, and central transfer of peripheral sensations by vagal afferent neurons. A large body of work has shown that vagal afferent neurons have nonuniform properties and that distinct subpopulations of neurons exist within the nodose ganglia. These sensory neurons display a considerable degree of plasticity; electrophysiological, pharmacological, and neurochemical properties have all been shown to alter after peripheral tissue injury. The validity of claims of selective recordings from populations of neurons activated by peripheral stimuli may be diminished, however, by the recent demonstration that stimulation of a subpopulation of nodose neurons can enhance the activity of unstimulated neuronal neighbors. To better understand the neurophysiological processes occurring after vagal afferent stimulation, it is essential that the electrophysiological, pharmacological, and neurochemical properties of nodose neurons are correlated with their sensory function or, at the very least, with their specific innervation target.

Oxygen increases ductus arteriosus smooth muscle cytosolic calcium via release of calcium from inositol triphosphate-sensitive stores

2005 ◽  
Vol 288 (5) ◽  
pp. L917-L923 ◽  
Author(s):  
Maggie Keck ◽  
Ernesto Resnik ◽  
Bradley Linden ◽  
Franklin Anderson ◽  
David J. Sukovich ◽  
...  
Keyword(s):  
Intracellular Calcium ◽  
Oxygen Tension ◽  
Ductus Arteriosus ◽  
Cytosolic Calcium ◽  
Extracellular Calcium ◽  
Inositol Triphosphate ◽  
Calcium Stores ◽  
Low Oxygen ◽  
Low Oxygen Tension ◽  
Pharmacological Blockade

In utero, blood shunts away from the lungs via the ductus arteriosus (DA) and the foramen ovale. After birth, the DA closes concomitant with increased oxygen tension. The present experimental series tests the hypothesis that oxygen directly increases DA smooth muscle cell (SMC) cytosolic calcium ([Ca2+]i) through inactivation of a K+ channel, membrane depolarization, and entry of extracellular calcium. To test the hypothesis, DA SMC were isolated from late-gestation fetal lambs and grown to subconfluence in primary culture in low oxygen tension (25 Torr). DA SMC were loaded with the calcium-sensitive fluorophore fura-2 under low oxygen tension conditions and studied using microfluorimetry while oxygen tension was acutely increased (120 Torr). An acute increase in oxygen tension progressively increased DA SMC [Ca2+]i by 11.7 ± 1.4% over 40 min. The effect of acute normoxia on DA SMC [Ca2+]i was mimicked by pharmacological blockade of the voltage-sensitive K+ channel. Neither removal of extracellular calcium nor voltage-operated calcium channel blockade prevented the initial increase in DA SMC [Ca2+]i. Manganese quenching experiments demonstrated that acute normoxia initially decreases the rate of extracellular calcium entry. Pharmacological blockade of inositol triphosphate-sensitive, but not ryanodine-sensitive, intracellular calcium stores prevented the oxygen-induced increase in [Ca2+]i. Endothelin increased [Ca2+]i in acutely normoxic, but not hypoxic, DA SMC. Thus acute normoxia 1) increases DA SMC [Ca2+]i via release of calcium from intracellular calcium stores, and subsequent entry of extracellular calcium, and 2) potentiates the effect of contractile agonists. Prolonged patency of the DA may result from disordered intracellular calcium homeostasis.

scholarly journals Diet-induced obesity leads to the development of leptin resistance in vagal afferent neurons

2011 ◽  
Vol 301 (1) ◽  
pp. E187-E195 ◽  
Author(s):  
Guillaume de Lartigue ◽  
Claire Barbier de la Serre ◽  
Elvis Espero ◽  
Jennifer Lee ◽  
Helen E. Raybould
Keyword(s):  
High Fat Diet ◽  
Leptin Resistance ◽  
Afferent Neurons ◽  
High Fat ◽  
Leptin Signaling ◽  
Protein Levels ◽  
Diet Induced Obesity ◽  
Nodose Ganglia ◽  
Vagal Afferent

Ingestion of high-fat, high-calorie diets is associated with hyperphagia, increased body fat, and obesity. The mechanisms responsible are currently unclear; however, altered leptin signaling may be an important factor. Vagal afferent neurons (VAN) integrate signals from the gut in response to ingestion of nutrients and express leptin receptors. Therefore, we tested the hypothesis that leptin resistance occurs in VAN in response to a high-fat diet. Sprague-Dawley rats, which exhibit a bimodal distribution of body weight gain, were used after ingestion of a high-fat diet for 8 wk. Body weight, food intake, and plasma leptin levels were measured. Leptin signaling was determined by immunohistochemical localization of phosphorylated STAT3 (pSTAT3) in cultured VAN and by quantifaction of pSTAT3 protein levels by Western blot analysis in nodose ganglia and arcuate nucleus in vivo. To determine the mechanism of leptin resistance in nodose ganglia, cultured VAN were stimulated with leptin alone or with lipopolysaccharide (LPS) and SOCS-3 expression measured. SOCS-3 protein levels in VAN were measured by Western blot following leptin administration in vivo. Leptin resulted in appearance of pSTAT3 in VAN of low-fat-fed rats and rats resistant to diet-induced obesity but not diet-induced obese (DIO) rats. However, leptin signaling was normal in arcuate neurons. SOCS-3 expression was increased in VAN of DIO rats. In cultured VAN, LPS increased SOCS-3 expression and inhibited leptin-induced pSTAT3 in vivo. We conclude that VAN of diet-induced obese rats become leptin resistant; LPS and SOCS-3 may play a role in the development of leptin resistance.

scholarly journals Expression of transient receptor potential channels and two-pore potassium channels in subtypes of vagal afferent neurons in rat

2010 ◽  
Vol 298 (2) ◽  
pp. G212-G221 ◽  
Author(s):  
Huan Zhao ◽  
Leslie K. Sprunger ◽  
Steven M. Simasko
Keyword(s):  
Single Cell ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Transient Receptor Potential Channels ◽  
Vagal Afferents ◽  
Afferent Neurons ◽  
Single Cell Pcr ◽  
Nodose Ganglia ◽  
Vagal Afferent ◽  
Transient Receptor

Vagal afferent neurons relay important information regarding the control of the gastrointestinal system. However, the ionic mechanisms that underlie vagal activation induced by sensory inputs are not completely understood. We postulate that transient receptor potential (TRP) channels and/or two-pore potassium (K2p) channels are targets for activating vagal afferents. In this study we explored the distribution of these channels in vagal afferents by quantitative PCR after a capsaicin treatment to eliminate capsaicin-sensitive neurons, and by single-cell PCR measurements in vagal afferent neurons cultured after retrograde labeling from the stomach or duodenum. We found that TRPC1/3/5/6, TRPV1-4, TRPM8, TRPA1, TWIK2, TRAAK, TREK1, and TASK1/2 were all present in rat nodose ganglia. Both lesion results and single-cell PCR results suggested that TRPA1 and TRPC1 were preferentially expressed in neurons that were either capsaicin sensitive or TRPV1 positive. Expression of TRPM8 varied dynamically after various manipulations, which perhaps explains the disparate results obtained by different investigators. Last, we also examined ion channel distribution with the A-type CCK receptor (CCK-RA) and found there was a significant preference for neurons that express TRAAK to also express CCK-RA, especially in gut-innervating neurons. These findings, combined with findings from prior studies, demonstrated that background conductances such as TRPC1, TRPA1, and TRAAK are indeed differentially distributed in the nodose ganglia, and not only do they segregate with specific markers, but the degree of overlap is also dependent on the innervation target.

scholarly journals Effect of Extracellular Calcium on Excitability of Guinea Pig Airway Vagal Afferent Nerves

2003 ◽  
Vol 89 (3) ◽  
pp. 1196-1204 ◽  
Author(s):  
Bradley J. Undem ◽  
Eun Joo Oh ◽  
Eric Lancaster ◽  
Daniel Weinreich
Keyword(s):  
Guinea Pig ◽  
Action Potential ◽  
Extracellular Calcium ◽  
C Fibers ◽  
Afferent Nerves ◽  
Nodose Ganglia ◽  
Cation Conductance ◽  
Afferent Fibers ◽  
Low Threshold ◽  
Vagal Afferent

The effect of reducing extracellular calcium concentration ([Ca2+]o) on vagal afferent excitability was analyzed in a guinea pig isolated vagally innervated trachea-bronchus preparation. Afferent fibers were characterized as either having low-threshold, rapidly adapting mechanosensors (Aδ fibers) or nociceptive-like phenotypes (Aδ and C fibers). The nociceptors were derived from neurons within the jugular ganglia, whereas the low-threshold mechanosensors were derived from neurons within the nodose ganglia. Reducing [Ca2+]o did not affect the excitability of the low-threshold mechanosensors in the airway. By contrast, reducing [Ca2+]o selectively increased the excitability of airway nociceptors as manifested by a substantive increase in action potential discharge in response to mechanical stimulation, and in a subset of fibers, by overtly evoking action potential discharge. This increase in the excitability of nociceptors was not mimicked by a combination of ω-conotoxin and nifedipine or tetraethylammonium. Whole cell patch recordings from airway-labeled and unlabeled neurons in the vagal jugular ganglia support the hypothesis that [Ca2+]o inhibits a nonselective cation conductance in vagal nociceptors that may serve to regulate excitability of the nerve terminals within the airways.

scholarly journals Ghrelin receptors in rat and human nodose ganglia: putative role in regulating CB-1 and MCH receptor abundance

2006 ◽  
Vol 290 (6) ◽  
pp. G1289-G1297 ◽  
Author(s):  
Galina Burdyga ◽  
Andrea Varro ◽  
Rod Dimaline ◽  
David G. Thompson ◽  
Graham J. Dockray
Keyword(s):  
Receptor Expression ◽  
Afferent Neurons ◽  
Growth Hormone Secretagogue Receptor ◽  
Growth Hormone Secretagogue ◽  
Ghrelin Receptor ◽  
Nodose Ganglia ◽  
Melanin Concentrating Hormone ◽  
Ghrelin Receptors ◽  
Vagal Afferent ◽  
Immunohistochemical Studies

Intact vagal afferent neurons are required for the satiety effects of the intestinal hormone cholecystokinin (CCK) and the orexigenic effects of the gastric regulatory peptide ghrelin. In this study, we examined the localization of ghrelin receptors in nodose ganglia and their function in regulating the expression of other orexigenic receptors, notably cannabinoid (CB)-1 and melanin-concentrating hormone (MCH)-1 receptors. With the use of RT-PCR, transcripts corresponding to both functional [growth hormone secretagogue receptor (GHS-R)1a] and truncated forms (GHS-R1b) of the ghrelin receptor were detected in rat nodose ganglia. There was no difference in expression between rats fed ad libitum or fasted for up to 48 h. Immunohistochemical studies using antibodies directed at GHS-R1a revealed expression in over 75% of neurons also expressing CCK-1 receptors in the mid- and caudal regions of the ganglion. There was also expression in human nodose ganglia. In fasted rats in which CB-1 and MCH-1 receptor expression was increased, administration of ghrelin prevented the downregulation by refeeding. We conclude that the actions of CCK and ghrelin are mediated by a common population of vagal afferent neurons. Ghrelin may act to limit the action of CCK in depressing expression of CB-1 and MCH-1 receptors and other receptors.

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