Molecular mechanisms supporting a paracrine role of GABA in rat adrenal medullary cells

Abstract

GABA is known to produce membrane depolarization and secretion in adrenal medullary (AM) cells in various species. However, whether the GABAergic system is intrinsic or extrinsic or both in the adrenal medulla and the role that GABA plays are controversial. Therefore, these issues were addressed by combining a biochemical and functional analysis. Glutamic acid decarboxylase (GAD), a GABA synthesizing enzyme, and vesicular GABA transporter (VGAT) were expressed in rat AM cells at the mRNA and protein levels, and the adrenal medulla had no nerve fibre-like structures immunoreactive to an anti-GAD Ab. The double staining for VGAT and chromogranin A indicates that GABA was stored in chromaffin granules. The α1, α3, β2/3, γ2 and δ subunits of GABA A receptors were identified in AM cells at the mRNA and protein levels. Pharmacological properties of GABA-induced Cl − currents, immunoprecipitation experiments and immunocytochemistry indicated the expression of not only γ2-, but also δ-containing GABA A receptors, which have higher affinities for GABA and neurosteroids. Expression of GATs, which are involved in the clearance of GABA at GABAergic synapses, were conspicuously suppressed in the adrenal medulla, compared with expression levels of GABA A receptors. Increases in Ca 2+ signal in AM cells evoked trans-synaptically by nerve stimulation were suppressed during the response to GABA, and this suppression was attributed to the shunt effect of the GABA-induced increase in conductance. Overall Ca 2+ responses to electrical stimulation and GABA in AM cells were larger or smaller than those to electrical stimulation alone, depending on the frequency of stimulation. The results indicate that GABA functions as a paracrine in rat AM cells and this function may be supported by the suppression of GAT expression and the expression of not only γ2-, but also δ-GABA A receptors.

Ca2+ Imaging in Perfused Adrenal Medullae

“…Figure 5 shows the results of the Ca 2+ imaging experiments in rat adrenal medulla. In contrast to the fluorescence images in guinea-pig adrenal medulla, the 30 µM GABA-sensitive area in the rat adrenal medulla was much smaller than the 30 µM nicotine-sensitive area, being about 40% of the latter [11]. In Fig.…”

“…4B, a relative change in f luorescence intensity in each frame is plotted against time. Application of 1.5 ms square pulses with 50 V to the adrenal gland for 30 s induced an increase in f luorescence in a frequency-dependent manner [11]. Administration of nicotine or GABA resulted in an increase in fluorescence intensity, which decayed due to desensitization of receptors in the presence of the agonist.…”

“…Administration of nicotine or GABA resulted in an increase in fluorescence intensity, which decayed due to desensitization of receptors in the presence of the agonist. The Ca 2+ signal in response to electrical stimulation during application of nicotine and GABA also diminished markedly, probably because of occlusion of nicotinic ACh receptors with nicotine [13] and a shunt effect by activation of GABA A receptors [11], respectively. However, saturation of Ca 2+ signal can not be excluded.…”

“…Comparison of f luorescence images in response to nicotine and GABA in rat and guinea-pig adrenal medullae clearly indicates that about 40% and almost all of AM cells in the former and the latter responded to GABA. This GABA-induced increase in Ca 2+ signal may be ascribable to GAGA A receptor-mediated depolarization and the subsequent activation of voltage-dependent Ca 2+ channels [11,12]. It would be worth investigating what molecular mechanism is involved in this difference in expression of GABA A receptors between rat and guinea-pig AM cells.…”

“…To accomplish this goal, we have developed a special technique to obtain Ca 2+ image from adrenal medulla which are perfused retrogradely through the adrenal vein [10]. With this method, the properties of Ca 2+ store sites [10], the effects of γ-aminobutyric acid (GABA) on neuronally evoked increase in excitability [11,12], and the role of muscarinic receptors in neurotransmission in rat AM cells [13] have been investigated in the past decade. Because of the technical difficulty, however, this method is not common.…”