Role of Cl^–Co-Transporters in the Excitation Produced by GABA_AReceptors in Juvenile Bovine Adrenal Chromaffin Cells

Abstract

GABA is the primary inhibitory neurotransmitter in the adult mammalian brain. However, in neonatal animals, activation of Cl(-)-permeable GABA receptors is excitatory and appears to depend on the expression of a Na(+)-K(+)-2Cl- cotransporter (NKCC) that elevates intracellular Cl- levels, leading to a depolarized Cl- equilibrium potential (ECl). The change from excitation to inhibition appears to involve the expression of the K+/Cl- co-transporter, KCC2, which lowers intracellular Cl- levels resulting in a hyperpolarized ECl. In this study, we show that bovine chromaffin cells from 4- to 5-mo-old animals are excited by GABA. Activation of GABAA receptors depolarizes the cells, opens voltage-dependent Ca2+ channels, elevates [Ca2+]i, and promotes the release of catecholamines. Blockade of voltage-dependent Ca2+ channels prevents the elevation of [Ca2+]i by GABA. The extrapolated anion reversal potential in these cells is approximately -28 mV, indicating a resting intracellular anion concentration of approximately 50 mM. Expression of KCC2 protein was not detected in the juvenile chromaffin cells. In contrast, clear expression of NKCC1 was observed. Blockade of NKCC1 should reduce the intracellular Cl- concentration and hyperpolarize ECl. Bumetanide, an NKCC1 blocker, reduced the elevation of [Ca2+]i by GABA. In some cells, activation of GABAA receptors inhibits responses to excitatory neurotransmitters, even though GABA itself is depolarizing. Co-activation of cholinergic and GABAA receptors in chromaffin cells produced elevations in [Ca2+]i that were comparable to those produced by cholinergic receptors alone. Our data showing the selective expression of chloride co-transporters and the resulting strongly depolarized anion reversal potential may help explain how activation of GABAA receptors causes sufficient excitation to elicit catecholamine release from chromaffin cells.

Regulation of large dense-core vesicle volume and neurotransmitter content mediated by adaptor protein 3

“…For immunoblotting, we separated proteins by SDS͞PAGE and transferred them to Immobilon-P membranes (Millipore) using standard procedures (53). A monoclonal antibody against ␤3B (610892; BD Biosciences, Pharmingen) was used at a dilution of 1:500, followed by an anti-mouse IgG-horseradish peroxidase-coupled secondary antibody (Jackson ImmunoResearch) used at a dilution of 1:100,000.…”

Investigating the stress attenuating potential of furosemide in immobilization and electric foot-shock stress models in mice

“…However, it is the first report suggesting the antistress effects of furosemide in acute immobilization and electric foot-shock models of experimental stress in mice. Studies have documented the widespread presence of NKCC1 in different stress-responsive brain regions, including the hypothalamus, pituitary, hippocampus, amygdala, and fornix Wang et al 2002;Nugent et al 2012;Xie et al 2003;Zemkova et al 2008). Furosemide is a nonselective blocker of NKCC and inhibits both NKCC1 and NKCC2.…”

“…However, NKCC1 is widely distributed in various types of tissues, including the stomach, heart, skeletal muscle, lungs, brain, and kidney (Russell 2000;Pedersen et al 2006). There is extensive distribution of NKCC1 on the components of the HPA axis including the hypothalamus (Nugent et al 2012), anterior pituitary (Zemkova et al 2008), and adrenal gland (Xie et al 2003). Furthermore, these channels are also widely distributed in other stress-responsive regions also including the hippocampus, amygdala, and fornix Wang et al 2002).…”

Cytosolic organelles shape calcium signals and exo–endocytotic responses of chromaffin cells

“…GABA A receptor agonists also cause cell depolarisation, an elevation of [Ca 2+ ] c , likely due to opening of VDCCs and the release of catecholamines [46][47][48]. Furthermore, GABA enhances the [Ca 2+ ] c elevation elicited by low-frequency electrical field stimulation of perfused rat adrenals [49].…”