Paracrine role of GABA in adrenal chromaffin cells

Abstract

The function of GABA in the adrenal medulla is still controversial. We will review experimental results in vivo and in vitro in adrenal chromaffin cells of various mammals to clarify what has been elucidated and what still remains to be settled.

Fig. 1

The presence of GABA signaling molecules in adrenal chromaffin cells. a Electrophoresis of PCR products for GAD67, GAD65, and β-actin. The PCR product of 280 bp for GAD67 was detected in cDNA samples of the rat brain (b) and adrenal medulla (m), but not in those of the adrenal cortex (c), whereas the PCR product of 338 bp for GAD65 was detected only in cDNA samples of the rat brain, but not of the adrenal medulla or adrenal cortex. Note that the 198 bp PCR products for β-actin were observed at similar levels in all three samples. s stands for standard ladder of DNAs. b Immunoblot analysis of fractionated bovine adrenal medulla for DβH, VGAT, synaptophysin (Syn), and early endosome antigen 1 (EEA1) with anti-DβH Ab (AB1538: Chemicon), anti-VGAT Ab (VGAT11-s: Alpha Diagnostic), anti-Syn Ab (sc-911: Santa Cruz), and anti-EEA1 Ab (sc-6414: Santa Cruz), respectively. Post-nuclear supernatants of bovine adrenal medulla were layered to the top of a discontinuous sucrose gradient from 10 to 65% in 5-point intervals, and ultracentrifuged at 5°C for 18 h. The same amount of proteins for each fraction was subjected to SDS-PAGE and immunoblotting. c Confocal fluorescence images of double immunostaining for VGAT and EEA1. Dissociated rat chromaffin cells were treated overnight with rabbit anti-VGAT Ab and goat anti-EEA1 Ab. Immunoreactivities to the former and the latter were visualized as FITC- and rhodamine-like fluorescence, respectively. The third image represents a merge of the first and the second image. The arrows indicate coincidence of both immunoreactivities. a Reproduced with modification from J Physiol (586: 4825–4842, 2008); b and c was from J Neurochem (114: 617–626, 2010)

Fig. 2

Secretion of catecholamine in response to nicotine, muscarine, and GABA in rat perfused adrenal medulla. Adrenal medulla was retrogradely perfused through the adrenal vein at a rate of 0.15 ml min⁻¹. The perfusate was collected for every minute and subjected to measurement of adrenaline and noradrenaline with HPLC. The amount of catecholamine was expressed as a fraction of that (Con) before application of nicotine (Nic), muscarine (Mus), or GABA, each at 30 μM. The data represent means ± SEM of six samples. One and two asterisks indicate statistical significance of P < 0.05 and P < 0.01 with reference to each control level before application, respectively

Fig. 3

Inhibitory effects of GABA on trans-synaptically evoked Ca²⁺ response in rat chromaffin cells. A Confocal images of fluo-4 fluorescence in rat adrenal medulla. The adrenal gland was retrogradely perfused with saline. The glands were subjected to a 40 min recurrent perfusion with 1 ml saline containing 10 μM fuo-4 AM and 0.2% Pluronic F 127. Then part of the adrenal cortex covering the medulla was removed by microcissors. The adrenal gland was placed between one pair of silver circles for electrical stimulation. Confocal images of FITC-like fluorescence were acquired every 5 s. B Relative values of change in fluorescence intensity are plotted against time. GABA at 30 μM was added to the perfusion solution during the period (interrupted line). Nerve fibers were electrically stimulated with 60 V pulses of 1.5 ms duration at 10 Hz for 30 s (bars). Fluorescence intensities in the areas (X and Y) indicated in Ac were measured and presented as filled (X) and open (Y) symbols, respectively. After correction for the decline due to photobleaching, an increase in fluorescence intensity to electrical stimulation and GABA was expressed as a fraction of the resting level. a, b, and c in A correspond to a, b and c in B. C Relative values of change in fluorescence intensity are plotted against time. Nerve fibers were electrically stimulated at 0.5, 1, and 5 Hz for 30 s in sequence before, during and after application of 30 μM GABA. D Summary of the overall Ca²⁺ responses to electrical stimulation with and without GABA application. The filled columns represent the maximum of the overall Ca²⁺ response to electrical stimulation and GABA. The open columns represent the maximum of the putative Ca²⁺ response to the electrical stimulation without GABA, a value which was estimated by averaging the maximum values of the electrically evoked Ca²⁺ responses before and after GABA application. The maximum of such Ca²⁺ responses with and without GABA application are expressed as a fraction of the Ca²⁺ response to electrical stimulation at each frequency before GABA application. * and ** represent statistical significance of P < 0.05 and P < 0.005, respectively. A, B, C, and D were reproduced from J Physiol (586:4825–4842, 2008)

Fig. 4

Measurement of intracellular [Cl⁻] in rat chromaffin cells. a Actual traces of whole-cell currents in response to 30 μM GABA at various holding potentials indicated next to the traces. The whole cell currents were recorded using a perforated patch clamp technique. Gramicidin was added as the perforating agent to the pipette solution at final concentrations of 4–40 μg ml⁻¹. b Peaks of GABA-induced currents are plotted against holding membrane potentials. c Summary of reversal potentials for GABA-induced currents under conditions where the pipette solution contained [Cl⁻] of 30 mM and 10 mM. The data represent means ± SEM of six and five experiments. a, b, and c were reproduced with modifications from J Physiol (586:4825–4842, 2008)

Fig. 5

Summary of paracrine role of GAGA in adrenal chromaffin cells. Each half of GABA_A receptors in rat chromaffin cells was estimated to contain either δ or γ2 subunit (Matsuoka et al. 2008)