Ping-Sheng Hu

4-aminopyridine-induced increase in basal and stimulation-evoked [3H]-NA release in slices from rat hippocampus : Ca2+ sensitivity and presynaptic control

1 We have examined the mechanisms by which the K+‐channel blocker 4‐aminopyridine (4‐AP) can dose‐dependently increase both basal [3H]‐noradrenaline ([3H]‐NA) release and the [3H]‐NA release evoked by electrical stimulation, but not the release of [3H]‐acetylcholine ([3H]‐ACh), from slices of rat hippocampus. 2 Both the electrically evoked and the 4‐AP‐induced release were blocked by tetrodotoxin (TTX) (3 μm). The Ca2+‐dependence of the 4‐AP‐induced release (EC50 0.15 mm) was, however, different from that of the electrically evoked [3H]‐NA release (EC50 0.76 mm). 3 The 4‐AP‐induced release could be inhibited by CdCl2(10 μm) and ω‐conotoxin (30 nm), but not by nifedipine (1 μm). 4 Transmitter release evoked by 100 μm 4‐AP could be blocked by the α2‐adrenoceptor agonist, UK 14,304 (0.1 μm) and by the A1‐receptor agonist R‐N6‐phenylisopropyl adenosine (R‐PIA, 1 μm) and increased by the α2‐adrenoceptor antagonist, yohimbine (1 μm), both in 0.25 and 1.3 mm Ca2+‐containing medium. By contrast, the effect of α2‐adrenoceptor agonists and antagonists on transmitter release evoked by electrical stimulation was markedly reduced in the presence of 4‐AP (100 μm). 5 The results suggest that 4‐AP can depolarize some nerve endings in the central nervous system, leading to transmitter release that is dependent on nerve impulses and Ca2+. Furthermore, the fact that α2‐receptors and adenosine A1 receptor agonists can influence the release of NA evoked by 4‐AP suggests that these drugs may have actions that are independent of blockade of aminopyridine‐sensitive K+‐channels.

Comparison of the effects of four dendrotoxin peptides, 4-aminopyridine and tetraethylammonium on the electrically evoked [3H]noradrenaline release from rat hippocampus

We have examined the effects of four dendrotoxin (DaTX) peptides, alpha-, beta-, gamma- and delta-DaTX, separated from the venom of the green mamba (Dendroaspis angusticeps), on field stimulation-evoked [3H]noradrenaline (NA) release from rat hippocampus and compared their effects with those of two other inhibitors of K+ channels, 4-aminopyridine (4-AP) and tetraethylammonium (TEA). 4-AP (10-300 microM) and TEA (0.1-5 mM) facilitated the evoked [3H]NA release in a concentration-dependent manner. The evoked [3H]NA release was reduced to about half by alpha 2-adrenoceptor stimulation (UK 14,304; 100 nM) and this reduction was antagonized by 4-AP (10-100 microM), whereas TEA even at 5 mM was a poor inhibitor of alpha 2-effects. alpha-DaTX (10-200 nM) mimicked 4-AP in increasing the electrically evoked [3H]NA release and diminishing the inhibitory effects of UK 14,304 in a concentration-dependent manner. delta-DaTX did not itself alter the electrically evoked [3H]NA release, but at 200 nM, it reduced the effects of alpha 2-receptor stimulation. beta- and gamma-DaTX (up to 200 nM) had no significant effects. 4-AP, 3,4-diaminopyridine (3,4-DAP), TEA and the four dendrotoxins displaced the binding of [3H]p-aminoclonidine ([3H]PAC) from alpha 2-receptors. The IC50 values were 6.6 x 10(-4), 1.42 x 10(-3), 5.6 x 10(-2) for 4-AP, 3,4-DAP and TEA, respectively, and 3.19 x 10(-6) M for alpha-DaTX. Thus, their potency as inhibitors of alpha 2-receptors is apparently too low to account alone for the antagonism by K+ channel inhibitors of alpha 2-effects on NA release.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of an intracellular calcium chelator on the regulation of electrically evoked [3H]-noradrenaline release from rat hippocampal slices.

1 The electrically (3 Hz, 5 min) evoked [3H]‐noradrenaline ([3H]‐NA) release from rat hippocampal slices was reduced by prior treatment of the slices with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′N′‐tetraacetomethylester (BAPTA/AM) in a concentration‐(10 to 500 μm) dependent manner (40% at 30 μm). Reduction of medium calcium from 1.3 to 0.5 mm caused a larger (70%) decrease. BAPTA free acid (100 mm), a non‐permeable Ca2+‐chelator had no significant effect. 2 Basal [3H]‐noradrenaline release was reduced by BAPTA/AM in a concentration‐dependent manner (50% at 30 μm), but reduction of external Ca2+ from 1.3 to 0.5 mm did not alter basal release. 3 About 10% of total [3H]‐NA in the slices was released at 3 Hz stimulation in 1.3 mm Ca2+ buffer. Addition of the α2‐adrenoceptor antagonist, idazoxan (1 μm), increased electrically evoked [3H]‐NA release to 26% but stimulated release was not altered by the adenosine A1‐receptor antagonist, 8‐cyclopentyl theophylline (8‐CPT) (1 μm). 4 Evoked release was reduced by the α2‐receptor agonist, UK 14,304, in a concentration‐dependent manner in the presence of 8‐CPT (1 μm). The magnitude of this effect was not altered by the treatment of slices with 30 μm BAPTA/AM. 5 The adenosine A1 receptor agonist, N6‐cyclohexyl adenosine (CHA) (1 μm) inhibited electrically evoked [3H]‐NA release by about 40% in the presence of idazoxan (1 μm). The effect of CHA was not significantly altered by treatment of slices with BAPTA/AM. 6 The N‐type Ca2+ ‐channel antagonist, ω‐conotoxin (30 nm), inhibited electrically evoked [3H]‐NA release by 30–50% and this was not altered by treatment of the slices with BAPTA/AM. 7 The present results show that spontaneous [3H]‐NA release is affected by reduction of intracellular Ca2+, but not by reduction of extracellular Ca2+ or by the presynaptic agonists or ω‐conotoxin. By contrast, electrically evoked release was affected more strongly by alterations of extracellular Ca2+ than by buffering intracellular Ca2+. The reduction of electrically evoked [3H]‐NA release by agonists at the adenosine A1‐receptor and α2‐adrenoceptor is probably mediated through the control of Ca2+ entry via membrane ion channels or at a low affinity Ca2+‐site governing evoked release.

Postendocytotic traffic of the galanin R1 receptor: a lysosomal signal motif on the cytoplasmic terminus.

The neuropeptide galanin R1 receptor (GalR1) was tagged at its C terminus with EGFP (GalR1–EGFP) to study receptor localization and trafficking. In PC12 and HEK293 cells, functional GalR1–EGFP was expressed on the plasma membrane and internalized into cytoplasmic vesicles after galanin stimulation. The internalization was blocked by 0.4 M sucrose and by silencing of clathrin with siRNA methodology. Internalized GalR1–EGFP and LysoTracker, a lysosomal marker, overlapped in intracellular vesicles after prolonged galanin stimulation. This colocalization was strongly reduced after site-directed mutagenesis of the motif YXXØ on the C terminus of GalR1 (where Ø is a bulky hydrophobic residue and X any amino acid). Taken together, these data suggest that GalR1 is internalized via the clathrin-dependent, endocytic pathway and then, to a large extent, delivered to lysosomes for degradation through the lysosome-targeting signal YXXØ.

Pharmacological Implications of A Multiplicity of Adenosine Actions in the CNS

Abstract Adenosine (50 nM - 50 μM) in brain extracellular space acts on two major classes of receptors present on virtually every cell. Specificity of action may be achieved by altering brain adenosine levels and by using partial agonists and/or drugs that affect more than one biochemical target.

Mechanisms of Adenosine Action

As clearly shown during this symposium adenosine may play an important modulatory role in several cells and tissues (see figure 1). Most of the effects of adenosine are mediated via cell surface receptors which can be grouped into two major categories A1 and A2. The pharmacological characterization of these two receptor types has been dealt with extensively elsewhere — the aim of this paper is instead to briefly discuss some aspects of the signalling from these receptors.

Mechanisms of Inhibition of Transmitter Release by Adenosine Analogs

ABSTRACT Adenosine receptors similar to A1 receptors mediate inhibition of the release of several neurotransmitters in the rat hippocampus. A G-protein, that is probably not a pertussis toxin substrate, is involved. Inhibition of cyclic AMP formation is usually not an important mechanism, neither is an interaction with protein kinase C. Adenosine effects are different from autoreceptor effects in that they are less sensitive to 4-AP and are similar to the effects of ω-conotoxin.