N- and P/Q-type Ca2+ channels in adrenal chromaffin cells.

Ca2+ is the most ubiquitous second messenger found in all cells. Alterations in [Ca2+]i contribute to a wide variety of cellular responses including neurotransmitter release, muscle contraction, synaptogenesis and gene expression. Voltage-dependent Ca2+ channels, found in all excitable cells (Hille 1992), mediate the entry of Ca2+ into cells following depolarization. Ca2+ channels are composed of a large pore-forming subunit, called the alpha1 subunit, and several accessory subunits. Ten different alpha1 subunit genes have been identified and classified into three families, Ca(v1-3) (Dunlap et al. 1995, Catterall 2000). Each alpha1 gene produces a unique Ca2+ channel. Although chromaffin cells express several different types of Ca2+ channels, this review will focus on the Cav(2.1) and Cav(2.2) channels, also known as P/Q- and N-type respectively (Nowycky et al. 1985, Llinas et al. 1989b, Wheeler et al. 1994). These channels exhibit physiological and pharmacological properties similar to their neuronal counterparts. N-, P/Q and to a lesser extent R-type Ca2+ channels are known to regulate neurotransmitter release (Hirning et al. 1988, Horne & Kemp 1991, Uchitel et al. 1992, Luebke et al. 1993, Takahashi & Momiyama 1993, Turner et al. 1993, Regehr & Mintz 1994, Wheeler et al. 1994, Wu & Saggau 1994, Waterman 1996, Wright & Angus 1996, Reid et al. 1997). N- and P/Q-type Ca2+ channels are abundant in nerve terminals where they colocalize with synaptic vesicles. Similarly, these channels play a role in neurotransmitter release in chromaffin cells (Garcia et al. 2006). N- and P/Q-type channels are subject to many forms of regulation (Ikeda & Dunlap 1999). This review pays particular attention to the regulation of N- and P/Q-type channels by heterotrimeric G-proteins, interaction with SNARE proteins, and channel inactivation in the context of stimulus-secretion coupling in adrenal chromaffin cells.

Etomidate elevates intracellular calcium levels and promotes catecholamine secretion in bovine chromaffin cells.

Etomidate, an intravenous imidazole general anaesthetic, is thought to produce anaesthesia by modulating or activating ionotropic Cl(-)-permeable GABA(A) receptors. Chromaffin cells are known to express functional GABA(A) receptors with properties similar to their neuronal counterparts. We have shown that activation of the GABA(A) receptors, with specific GABA(A) agonists, leads to cellular excitation. Our goal was to determine whether etomidate mimicked this response and to explore the functional consequences of this activation. Imaging experiments with the Ca(2+)-indicator dye fura-2 were used to assay [Ca(2+)](i). Bovine adrenal chromaffin cells were superfused with a variety of GABA(A)-selective drugs to determine their effects on [Ca(2+)](i). Amperometric measurements were used to assay catecholamine release in real-time. We show that bovine adrenal chromaffin cells were excited by etomidate at clinically relevant concentrations. Etomidate directly activated GABA(A) receptors found in chromaffin cells thereby elevating [Ca(2+)](i). The effects of etomidate were mimicked by the specific GABA(A) agonist muscimol and blocked by the specific antagonist bicuculline. Our data show that low concentrations of etomidate modulated GABA(A) receptor activation by muscimol. Blockade of voltage-dependent Ca(2+) channels prevented the elevation of [Ca(2+)](i) by GABA. Application of etomidate directly to the chromaffin cells elicited robust catecholamine secretion from these cells. The data indicate that clinically relevant concentrations of etomidate can directly activate GABA(A) receptors, which, due to the positive anion equilibrium potential, depolarizes chromaffin cells. This depolarization activates voltage-dependent Ca(2+) channels thereby stimulating catecholamine release. Our data suggest that circulating catecholamine levels may be elevated after etomidate application.

Role of Cl- co-transporters in the excitation produced by GABAA receptors in juvenile bovine adrenal chromaffin cells.

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.