Brain Research 885 2000 231–239 www.elsevier.com locate bres
Research report
Activation of purinergic receptors by ATP inhibits secretion in bovine adrenal chromaffin cells
Amy B. Harkins , Aaron P. Fox
Department of Neurobiology , Pharmacology and Physiology, The University of Chicago, 947 E. 58th Street, Chicago, IL 60637, USA
Accepted 12 September 2000
Abstract
Autoinhibition is a common mechanism observed in neurons to regulate neurotransmission. Released neurotransmitter interacts with presynaptic autoreceptors to inhibit subsequent release. The requisite elements for autoinhibition are present in chromaffin cells: secretory
granules contain millimolar levels of ATP which is coreleased with catecholamines upon stimulation and the cells express purinergic receptors. We were interested to determine whether autoinhibition produced by ATP binding to purinergic receptors plays an important
role in catecholamine release from chromaffin cells. In these studies, short depolarizations were used to elicit transmitter release measured by membrane capacitance. We find that stimulation of chromaffin cells results in the release of endogenous ATP which may suppress
21
Ca channel currents and secretion. In the presence of a maximal concentration of ATP, both the amount of secretion and the maximal
rate of release are about half that observed in the absence of ATP. ATP-mediated inhibition of secretion was blocked by Reactive Blue-2
21
suggesting the involvement of P purinergic receptors. Prepulses to positive potentials that relieve the Ca
channel block largely relieve
2Y 21
the inhibition of secretion. Furthermore, when secretion is plotted as a function of Ca influx there is no apparent change in the
relationship between control cells and those stimulated in the presence of ATP and prepulses. These results suggest that ATP diminishes
21 21
secretion by inhibiting Ca influx into the cells. Our results indicate that feedback inhibition by ATP, mediated primarily by Ca
channels, may be an important regulator of catecholamine release in chromaffin cells.
2000 Elsevier Science B.V. All rights reserved.
Theme : Excitable membranes and synaptic transmission
Topic : Mechanisms of neurotransmitter release
21
Keywords : ATP; Purinergic receptor; Exocytosis; Ca
channels; Autoinhibition
21
1. Introduction Ca
channel current I by neurotransmitters which
Ca
activate a wide variety of G-protein-coupled receptors At most synapses, neuronal communication occurs by
[16,17,33]. Autoinhibition appears to involve activation of
21
Ca -dependent vesicular release of chemical neurotrans-
presynaptic receptors [34]. Inhibition is most likely me- mitters. Modulation of release can occur by a phenomenon
diated via a direct interaction of G-protein bg subunits
21
termed autoinhibition whereby a released neurotransmitter with N- and P Q-type Ca
channels [5,13,24,26], al- inhibits subsequent release of transmitter through an
though other inhibitory pathways are known to exist [25]. autoinhibitory feedback mechanism [19,41]. Neurotrans-
This inhibition of I is reversible. Inhibition is character-
Ca
mitters such
as noradrenaline,
g-aminobutyric acid
ized by slowing of activation kinetics, is voltage depen- GABA, serotonin, enkephalin and somatostatin reduced
dent, and is overcome by prepulses to strongly positive
21
Ca influx into cells and diminished release [15,35].
potentials [5,18,38].
21
Inhibition of Ca influx is due to a direct inhibition of the
Adrenal chromaffin cells provide an excellent model cell to study mechanisms of transmitter release. In chromaffin
cells, many exogenously applied neurotransmitters inhibit
Corresponding author. Tel.: 11-773-702-0020; fax: 11-773-702-
I through a number of receptors that include D dopa-
1216.
Ca 2
E-mail address : amydrugs.bsd.uchicago.edu A.B. Harkins.
mine receptors [6], GABA receptors [14], a-adrenergic
B
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 5 2 - 8
232 A
receptors [29], and opioid receptors [1,10,29,42]. Addition- mm in diameter positioned directly adjacent to the cell.
ally, exogenously applied ATP activates P purinergic
The outlet pipe was fed by six separate reservoirs equally
2Y
receptors which inhibit I via a G-protein-coupled path-
pressurized for similar flow rates |70 ml min and
Ca
way [1,10,12,21]. Chromaffin cell secretory granules con- controlled by valves operated and timed with the data
tain ATP at millimolar concentrations [45] leading to the acquisition software. The exchange time for switching
possibility that autoactivation of endogenous purinergic between two solutions was ,150 ms. The input rate of the
receptors regulates release. perfusion system was matched with the outflow rate of a
In this study, we sought to determine whether ATP peristaltic pump to maintain a constant bath volume in
could modulate secretion in chromaffin cells. Capacitance order to reduce artifactual capacitance changes.
measurements were employed to measure secretion in response to stimulation. We show that, as previously
2.2. Preconditioned media described [1,10,12,21], ATP significantly reduces I
Most
Ca.
importantly, ATP reduces release by .50. Stimulation of To determine whether chromaffin cells released suffi-
chromaffin cells which results in the release of endogenous cient ATP to activate autoinhibition, cells were plated at
ATP suppresses secretion in these cells. The inhibition high density |10-fold that of experimental dishes. Just
mediated by ATP was blocked by Reactive Blue-2 sug- prior to use, a dish of cells was incubated for 5 min in 1 ml
gesting the involvement of P receptors. Large prepulses,
of the TEA solution described above which induced
2Y
which relieve the inhibition of I , largely reverse the depolarization and thus large amounts of secretion. The
Ca
inhibition of secretion. Plots of secretion as a function of preconditioned solution was removed and immediately
21
Ca influx are not significantly different in the absence of
applied to voltage-clamped chromaffin cells. ATP or presence of ATP and prepulses. Our results
indicate that the primary mechanism by which ATP acts to 2.3. Capacitance recordings
inhibit secretion from chromaffin cells is by inhibiting I .
Ca
ATP autoinhibition may provide an important regulatory Capacitance measurements were made with the phase
mechanism for catecholamine release in chromaffin cells. tracking technique [27] in which a 60 mV peak to peak sine
wave was superimposed on a holding potential of 280 mV [36]. The combination of the sine wave and the holding
2. Materials and methods potential was chosen to provide a good signal to noise ratio
