The Role of Cl-cotransporters in the Excitation Produced by GABAA Receptors in Juvenile Bovine Adrenal Chromaffin Cells

GABA is the primary inhibitory neurotransmitter in 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 four to five month old animals are excited by GABA. Activation of GABAA receptors depolarizes the cells, opens voltage-dependent Ca channels, elevates [Ca]i and promotes the release of catecholamines. Blockade of voltage-dependent Ca channels prevents the elevation of [Ca]i by GABA. The extrapolated anion reversal potential in these cells is ~-28 mV indicating a resting intracellular anion concentration of ~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 [Ca]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 [Ca]i that were comparable to those produced by cholinergic receptors alone. Our data showing the selective expression of chloride cotransporters 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.