HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 18/12/1468 04-1828fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by FUENTEALBA, J. Articles by GARCÍA, A. G. Search for Related Content PubMed PubMed Citation Articles by FUENTEALBA, J. Articles by GARCÍA, A. G.

A choline-evoked [Ca²⁺]_c signal causes catecholamine release and hyperpolarization of chromaffin cells

JORGE FUENTEALBA^*, ROMÁN OLIVARES^*, EVA ALÉS^*, LAURA TAPIA^*, JONATHAN ROJO^*, GLORIA ARROYO^*, MARCOS ALDEA^*, MANUEL CRIADO, LUIS GANDÍA^* and ANTONIO G. GARCÍA^*,,1

Instituto Teófilo Hernando y Departamento de Farmacología y Terapéutica;
Servicio de Farmacología Clínica e Instituto de Gerontología del Hospital de la Princesa; Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, Spain; and
Instituto de Neurociencias, Universidad Miguel Hernández-CSIC, Alicante, Spain

¹Correspondence: Instituto Teófilo Hernando, Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, C/Arzobispo Morcillo 4, 28029, Madrid, Spain. E-mail: agg{at}uam.es

The simplistic view of choline as a mere precursor and hydrolytic product of acetylcholine (ACh), the physiological neurotransmitter at central and peripheral cholinergic synapses, is changing. This change is due to the discovery of various activities of choline (i.e., muscarinic) effects on brain neurons, secretion of catecholamines from bovine adrenal medullary chromaffin cells, inward currents in neurons, or an increase in the cytosolic concentration of calcium ([Ca²⁺]_c) in sympathetic neurons. In oocytes expressing different subtypes of neuronal nicotinic receptors for ACh (nAChR, choline does not activate 4ß2 or 3ß2 receptors; it is, however, a partial agonist of several heteromeric receptors, including the 3ß4 subtype, and behaves as a full agonist at homomeric 7 receptors.

SPECIFIC AIMS

In this study we chose a different approach to study the choline effects in chromaffin cells, using choline as a selective agonist for 7 receptors and ACh as a nonselective agonist for 7 and 3ß4 receptors, as well as high [Ca²⁺] solutions.

We compared secretory responses, confocal [Ca²⁺]_c signals, and changes of membrane potential elicited by choline and ACh in bovine chromaffin cells as well as the inward currents elicited by ACh and choline in high Ca²⁺ solutions in oocytes expressing 7 and 3ß4 nAChRs.

PRINCIPAL FINDINGS

1. The secretory responses to increasing concentrations of choline and ACh in a high Ca²⁺ solution
The strict concentration dependence of ACh responses was not observed with choline.

2. Different Ca²⁺-dependent catecholamine release responses from chromaffin cells stimulated with choline, ACh, and K⁺
The choline secretory responses exhibited a stronger dependence of the [Ca²⁺]_e (Fig. 1 ); however, at the lower ACh concentration used here (0.03 mM) no Ca²⁺ dependence was observed, and a more desensitizing response to choline than to ACh was observed in the secretory kinetics at different [Ca²⁺]_e studied.

View larger version (17K): [in this window] [in a new window]   Figure 1. Time course of the secretory responses to choline and ACh at different [Ca2+]e. 0.03ACh, 0.1Ach, and 10Cho refer to the mM concentrations of choline and ACh of the stimulating solutions at the mM concentrations of Ca2+ shown on top of each panel (1Ca2+ indicates a solution containing 1 mM Ca2+, and so on). These curves were plotted with data obtained through normalization of the secretory responses, as % of the first spike area, in each individual experiment from experiments using protocols of Fig. 2A ; they are means ± SE of the number of experiments shown in parentheses from least at 6 different batches of cells. Data of ACh were significantly different at P < 0.001 compared with those of choline for all [Ca2+]e tested (Student’s t test).

3. Effects of mecamylamine and atropine on the secretory responses elicited by choline and ACh in high extracellular Ca²⁺
The nAChR blocker mecamylamine (10 µM) reduced the secretory response to 10 mM choline (in Krebs-HEPES with 20 mM Ca²⁺) by >90%, and those to 0.03 mM ACh by >70%. In contrast, 30 nM of the muscarinic receptor blocker atropine did not affect the choline responses, but reduced by 80% those of ACh.

4. Cytosolic Ca²⁺ increments elicited by choline and ACh, measured by confocal imaging in chromaffin cells
The [Ca²⁺]_c increments elicited by choline were similar to those elicited by ACh, showing no statistical differences.

5. The effects of choline and ACh on the membrane potential of chromaffin cells
In contrast to ACh, 10 mM choline (in Krebs-HEPES with 20 mM Ca²⁺) produced little or no depolarization, followed by a large transient hyperpolarization (Fig. 2 ).

View larger version (14K): [in this window] [in a new window]   Figure 2. Original traces of the changes in membrane potential of chromaffin cells elicited by choline and ACh measured with the patch-clamp technique under the whole-cell configuration and current-clamp mode. A) A typical trace of the changes in membrane potential most often seen when applying 5 s pulses of 10Cho/20Ca2+. B) Changes in membrane potential when applying a 5 s pulse of 0.1ACh/20Ca2+ solution. C) Changes in membrane potential generated by 10Cho/2Ca2+. D) Changes in membrane potential generated by 0.1ACh/2Ca2+.

6. Effects of choline on the inward currents through 7 and 3ß4 nicotinic receptors expressed in oocytes
Choline (10 mM) generated a small current in 3ß4-injected oocytes, 4.2% of the inward current elicited by ACh (0.03 mM); in 7-injected oocytes choline generated a full inward current, 118% of the current elicited by ACh.

CONCLUSIONS

Our results are compatible with the following pattern of chromaffin cell activation. It is most plausible that under physiological conditions, ACh activates the abundant low Ca²⁺ permeability 3ß4 receptors; then Na⁺ entering through them causes depolarization of chromaffin cells, leading to firing of action potentials, the recruitment of voltage-dependent Ca²⁺ channels, massive Ca²⁺ entry, and fast exocytosis. In this frame, Ca²⁺ entry through the scarce but highly Ca²⁺-permeable and strategically located 7 nAChRs might cause the quick activation of closely localized SK channels to hyperpolarize the chromaffin cell and terminate a secretory cycle. Whether 7 receptors are activated by ACh or by choline present at synaptic and/or extrasynaptic sites is not known. Our results, however, suggest that 1) 7 nAChRs have a role in controlling Ca²⁺-dependent K⁺ channels and the electrical activity of chromaffin cells, during ACh stimulation; and 2) choline might have a functional role in regulating cholinergic synaptic activity by acting on 7 nAChRs (Fig. 3 ).

View larger version (22K): [in this window] [in a new window]   Figure 3. Scheme showing the possible role of choline and 7 nAChRs in modulating synaptic cholinergic neurotransmission at the splachnic nerve-chromaffin cell synapse. 1) Endogenously released ACh activates abundant synaptic 3ß4 nAChRs, causing Na+ entry, depolarization, and firing of action potentials that recruit voltage-dependent Ca2+ channels (VDCC) to allow Ca2+ entry and exocytosis of catecholamine. 2) Synaptic acetylcholinesterase (AChase) rapidly hydrolyzes ACh, and choline accumulates intra- and/or extrasynaptically. 3) Choline, as a full selective agonist, activates 7 nAChRs that are scarce but highly localized near SK and/or BK channels. Ca2+ entering through 7 receptors activates these channels very efficiently; thus, the chromaffin cell is hyperpolarized and the firing of action potentials and exocytotic release of catecholamine stops. In this way, choline and the nAChR might play a physiological role in controlling the electrical activity of the chromaffin cell in a Ca2+-dependent manner and, hence, the release of catecholamine.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1828fje; doi: 10.1096/fj.04-1828fje

This article has been cited by other articles:

				B. Lendvai and E. S. Vizi Nonsynaptic Chemical Transmission Through Nicotinic Acetylcholine Receptors Physiol Rev, April 1, 2008; 88(2): 333 - 349. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 18/12/1468 04-1828fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by FUENTEALBA, J. Articles by GARCÍA, A. G. Search for Related Content PubMed PubMed Citation Articles by FUENTEALBA, J. Articles by GARCÍA, A. G.