Prog Med Phys.  2014 Sep;25(3):157-166. 10.14316/pmp.2014.25.3.157.

Effect of ATP on Calcium Channel Modulation in Rat Adrenal Chromaffin Cells

• Kim KA
• Goo YS

• KMID: 1910548
• DOI: http://doi.org/10.14316/pmp.2014.25.3.157

Abstract

ATP in quantity co-stored with neurotransmitters in the secretory vesicles of neurons, by being co-released with the neurotransmitters, takes an important role to modulate the stimulus-secretion response of neurotransmitters. Here, in this study, the modulatory effect of ATP was studied in Ca2+ channels of cultured rat adrenal chromaffin cells to investigate the physiological role of ATP in neurons. The Ca2+ channel current was recorded in a whole-cell patch clamp configuration, which was modulated by ATP. In 10 mM Ba2+ bath solution, ATP treatment (0.1 mM) decreased the Ba2+ current by an average of 36+/-6% (n=8), showing a dose-dependency within the range of 10(-4)~10(-1) mM. The current was recovered by ATP washout, demonstrating its reversible pattern. This current blockade effect of ATP was disinhibited by a large prepulse up to +80 mV, since the Ba2+ current increment was larger when treated with ATP (37+/-5%, n=11) compared to the control (25+/-3%, n=12, without ATP). The Ba2+ current was recorded with GTPgammaS, the non-hydrolyzable GTP analogue, to determine if the blocking effect of ATP was mediated by G-protein. The Ba2+ current decreased down to 45% of control with GTPgammaS. With a large prepulse (+80 mV), the current increment was 34+/-4% (n=19), which 25+/-3% (n=12) under control condition (without GTPgammaS). The Ba2+ current waveform was well fitted to a single-exponential curve for the control, while a double-exponential curve best fitted the current signal with ATP or GTPgammaS. In other words, a slow activation component appeared with ATP or GTPgammaS, which suggested that both ATP and GTPgammaS caused slower activation of Ca2+ channels via the same mechanism. The results suggest that ATP may block the Ca2+ channels by G-protein and this Ca2+ channel blocking effect of ATP is important in autocrine (or paracrine) inhibition of adrenaline secretion in chromaffin cell.

Keyword

Adrenal chromaffin cell; Ca2+ channel; ATP; G-protein; Autocrine inhibition

MeSH Terms

Adenosine Triphosphate*
Animals
Baths
Calcium Channels*
Chromaffin Cells*
Epinephrine
GTP-Binding Proteins
Guanosine 5'-O-(3-Thiotriphosphate)
Guanosine Triphosphate
Neurons
Neurotransmitter Agents
Rats*
Secretory Vesicles
Adenosine Triphosphate
Calcium Channels
Epinephrine
GTP-Binding Proteins
Guanosine 5'-O-(3-Thiotriphosphate)
Guanosine Triphosphate
Neurotransmitter Agents

Figure

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  Fig. 1. Peak current (IBa)-voltage (Vm) relationship recorded in adrenal chromaffin cell held at −80 mV activated by 150 ms depolarizing pulses applied in 10 mV steps. (a) Activation of Ba2＋ currents. Upper and lower traces are voltage and current traces, respectively. (b) Voltage-current relationship of Ba2＋ current from 25 cells.

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  Fig. 2. Effect of ATP on Barium current (IBa). (a) Upper trace is voltage protocol. Lower traces are control Ba2＋ current trace (left), current trace when the external solution is switched to ATP-containing solution (middle), and current trace after wash-out of ATP (right), respectively. (b) Effect of ATP on Ba2＋ current. Peak amplitude of control Ba2＋ current was 281.37±53.53 pA while that with ATP was 168.05±28.6 pA, with wash-out of ATP was 243.45±70.7 pA (mean±S.E.M., n=8). ∗p-value＜0.05. (c) Left: Current traces show Ba2＋ currents recorded and concentration of ATP in one cell. Right: Time course of inhibitory effects of ATP on IBa. IBa was activated by 50 ms depolarization pulses from －80 mV to 0 mV applied at a frequency of 0.1 Hz. As indicated by the bars, ATP (0.1, 1, 10, 100 μM) was added to or removed (W/O, washed out) from the external solution. Concentration was successively changed from 0.1 μM to 100 μM. (d) Left: Current traces of control, and with and after washout of ATP. Curve fitting was performed and activation time constant (τ) was calculated in each trace. Right: Normalized current: normalization of raw current with respect to the final point. Upper trace (●) is current during ATP, middle (□), current after washout of ATP, and lower trace (■), control current.

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  Fig. 3. Effect of GTPγS (50 μM) on Ba2＋ current. (a) Effect of GTPγS on gating kinetics of Ca2＋ channel. Current trace of control condition vs intracellular GTPγS. Curve fitting was performed and activation time constant was calculated for each trace as shown. (b) Averaged results are shown (mean±S.E.M.) with the number of cells given in parentheses. Peak amplitude of IBa was 261.65±38.84 pA (n=11) while that with GTPγS was 118.78±17.59 pA (n=19). ∗p-value＜0.05.

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  Fig. 4. Prepulse induced facilitation recorded in different dialyzing conditions. (a) Left: Ba2＋ currents were recorded without (●) and with (■) prepulse of 20 ms to ＋80 mV from Vh=−80 mV(upper trace) in control condition. Right: Records were obtained using the same pulse protocol but with extracellular ATP. Notice the pronounced inactivation on the facilitated currents that slowly relaxes to its control level. (b) Left: Ba2＋ currents were recorded without (●) and with (■) prepulse of 30 ms to ＋80 mV from Vh=−80 mV(upper trace) in the application of intracellular GTPγS. Right: Records were obtained using same pulse protocol but with the application of intracellular GTPγS and additional extracellular ATP. There was no additional facilitation increment with GTPγS and ATP in comparison with GTPγS alone. (c) Prepulse induced facilitation in control condition was 25±3% (n=12) increase while that with extracellular ATP, with intracellular GTPγS, with intracellular GTPγS and extracellular ATP was 37±5% (n=11) increase, was 34±4% (n=19) increase, and 35±4% (n=8) increase, respectively. There was significant difference between control group and ATP-and or GTPγS- treated group. ∗p-value＜0.05.

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  Fig. 5. Schematic diagram of chromaffin cell stimulus-secretion coupling. The splanchnic nerve that innervates chromaffin cell release acetylcholine (Ach) that activates the nicotinic acetylcholine receptor (nAChR) that gates Na＋, causing the membrane to depolarize (ΔV) sufficiently to trigger the action potentials to activate voltage-operated Ca2＋ channels (VOCC). Entry of external Ca2＋ increases the intracellular Ca2＋ concentration ([Ca2＋ i]) that triggers exocytosis and the release of chromaffin granule contents that contain adrenaline and ATP. The ATP may feedback to activate receptors that use the G protein to inhibit the VOCC. (Broken arrow represents the possibility).

Reference

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