Different temperature dependence of carrier-mediated (cytoplasmic) and stimulus-evoked (exocytotic) release of transmitter: a simple method to separate the two types of release

Abstract

The temperature dependence of transmitter release associated with axonal conduction, evoked by ligand-gated mechanism and by reversed operation of plasma membrane transporter was studied in superfused slice preparation. When the temperature was reduced from 37–17°C the release of [³H]noradrenaline ([³H]NA) and [³H]dopamine ([³H]DA) in response to field stimulation was significantly enhanced in slice preparations of the hippocampus and main olfactory bulbs. The release of [³H]dopamine evoked by a ligand-gated mechanism (nicotine receptor stimulation) was potentiated at low temperature (12°C). In contrast, when transmitter release was evoked by ouabain, a drug inhibiting Na⁺\K⁺-activated ATPase and thereby increasing [Na⁺]_i the release of [³H]GABA was enhanced. This release was very sensitive to cooling (Q₁₀ = 3.5 between 37°C and 27°C), indicating that the release was induced by a reversed operation of the transporter. The excessive release of [³H]NA from the hippocampal slice in response to oxygen and glucose deprivation (simulation of ischemia) was also inhibited in a temperature-dependent manner. Because at low temperatures (17–12°C) only one type of release mechanism (exocytosis) is operational and carrier-mediated uptake and release is inhibited, this temperature condition provides a method to study the mode of action of different drugs (e.g. amphetamine) and the effect(s) of certain conditions (e.g. ischemia) on the mechanisms of transmitter release, specifically whether they exert their transmitter releasing effect through an exocytotic process or through the reversed operation of plasma membrane transporter.

This finding also suggests that it would be important to re-examine mechanistic conclusions based on results from electrophysiological, neurochemical and pharmacological studies that have been carried out at room temperature (∼20°C). In particular because transmitter release associated with the action potential, diffusion, receptor kinetics, active transport in both directions (uptake and release) and the probability of transmitter release are all temperature dependent, it would seem important to carry out experiments involving these processes at physiological temperature (37°C).

Introduction

Over the past 10 years we have seen an exponential increase in our understanding of the diversity and mechanism of transmitter release (cf. Vizi, 1979; Clements, 1996; Wu and Saggau, 1997; Langley and Grant, 1997; Zimmermann, 1997). It is generally accepted that, when the action potential invades the nerve terminal, Ca² flows into the terminal through voltage-gated Ca² channels, which in turn triggers neurotransmitter release by stimulating vesicle exocytosis. Another way that Ca² influx results in exocytotic release is through the mediation of ligand-gated ion channels. It is also known that there is a cellular transport mechanism that has the dual capacity to clear the extracellular space of transmitter and to transport the transmitter in either direction across the cell membrane (cf. Trendelenburg, 1991). But if the transporter operates in a reverse mode, due to excessive accumulation of [Na⁺]_i, the release of transmitter is [Ca²]_o-independent and the transmitter originates from the cytoplasm. This transporter located in the basal membrane is different from the vesicular transport system (cf. Henry et al., 1998).

The inward transport of transmitter depends on the presence of [Na⁺]_o (Iversen and Kravitz, 1966) and Cl⁻ (Sanchez-Armass and Orrego, 1977) and its capacity is temperature dependent; at low temperatures it fails to operate completely (Kirpekar and Wakade, 1968; Lindmar and Loffelholz, 1972). Because the action potential is still present at relatively low temperatures (Frankenhaeuser and Moore, 1963;Katz and Miledi, 1965; Paintal, 1967), we tested the hypothesis by measuring the effect of varying the temperature on the release of transmitters (noradrenaline and dopamine) evoked by electrical stimulation and by applying nicotine, a nicotinic acetylcholine receptor (nAChR) agonist, i.e. to test the release evoked by stimulation of ligand-gated ion channels.

We also attempted to study the temperature dependence of [³H]GABA release evoked by sodium pump inhibition and of [³H]noradrenaline release induced by ischemic condition, i.e. under a condition when the transporter operates in a reverse mode. Our aim was to separate the [Ca²]_o-independent carrier-mediated (cytoplasmic) and stimulus-evoked, [Ca²]_o-dependent (exocytotic) release of transmitter.