Electrically Evoked Release of [³H]Noradrenaline from Mouse Cultured Sympathetic Neurons

Abstract

Cultured neurons from the thoracolumbar sympathetic chain of newborn mice are known to possess release-inhibiting ␣ 2 -autoreceptors. The present study was carried out in a search for release-modulating heteroreceptors on these neurons. Primary cultures were preincubated with [ 3 H]noradrenaline and then superfused and stimulated by single pulses, trains of 8 pulses at 100 Hz, or trains of 36 pulses at 3 Hz. The cholinergic agonist carbachol reduced the evoked overflow of tritium. Experiments with antagonists indicated that the inhibition was mediated by M 2 muscarinic receptors. The cannabinoid agonist WIN 55,212-2 reduced the evoked overflow of tritium through CB 1 receptors. Prostaglandin E 2 , sulprostone, and somatostatin also caused presynaptic inhibition. The inhibitory effects of carbachol, WIN 55,212-2, prostaglandin E 2 , and somatostatin were abolished (at the highest concentration of WIN 55,212-2 almost abolished) by pretreatment of the cultures with pertussis toxin (250 ng/ml). Several drugs, including the ␤ 2 -adrenoceptor agonist salbutamol, opioid receptor agonists, neuropeptide Y, angiotensin II, and bradykinin, failed to change the evoked overflow of tritium. These results demonstrate a distinct pattern of presynaptic inhibitory heteroreceptors, all coupled to pertussis toxin-sensitive G proteins. The lack of operation of several presynaptic receptors known to exist in adult mice in situ may be due to the age of the (newborn) donor animals or to the culture conditions. Key Words: Mouse sympathetic neuron cultures-Noradrenaline release -Presynaptic receptors-Muscarinic receptors-Cannabinoid receptors-Prostanoid receptors-Somatostatin receptors. J. Neurochem. 75, 2087Neurochem. 75, -2094Neurochem. 75, (2000.As in intact tissues, various receptors modulate the action potential-evoked release of noradrenaline in cultures of postganglionic sympathetic neurons from, for example, chicken and rats (for review, see Boehm and Huck, 1997). Neurons of mice, however, were not studied in this respect until we recently prepared primary cultures of thoracolumbar sympathetic ganglion cells from this species. The cells possessed release-inhibiting ␣ 2A/D -autoreceptors, but not release-modulating P2, A 1 , or A 2A receptors, which are alternative autoreceptors due to the cotransmitter role of ATP (Trendelenburg et al., 1999a, b). We have now carried out a search for releasemodulating heteroreceptors on these neurons. Most heteroreceptors ever observed were included (see Fuder and Muscholl, 1995;Powis and Bunn, 1995). The study concentrated on receptors modifying action potentialevoked release; receptors that, when activated, elicit exocytotic release of noradrenaline will be described in a subsequent report. Neuronal action potentials were triggered by electrical stimulation, and release of previously incorporated [ 3 H]noradrenaline was measured. MATERIALS AND METHODS Culturing and [ 3 H]noradrenaline releaseThese techniques were modified from our previous work (Trendelenburg et al., 1999a). Thora...

A study of presynaptic α 2 -autoreceptors in α 2A/D -, α 2B - and α 2C -adrenoceptor-deficient mice

“…A likely reason is the greater duration of a train of 120 pulses at 3 Hz (40 s) than of 4 POPs at 1 Hz (4 s), leaving time for the development of a greater degree of negative feedback. A second possible reason is the lower sensitivity of high frequency pulse trains (50 Hz in the POPs) than of low frequency pulse trains to presynaptic modulation (see for example Göbel et al 2000). More pertinent for the aim of the present study than these questions is the observation that not only α 2A/D deletion but also α 2C deletion decreased the releaseenhancing effect of α 2 antagonists, i.e.…”

Stimulation frequency-noradrenaline release relationships examined in α2A-, α2B- and α2C-adrenoceptor-deficient mice

“…The curves, therefore, became tilted at the right-hand extreme when α 2 -autoinhibition was removed. The general resistance of high-frequency-induced transmitter release to presynaptic modulation is well known (p. 5 of Starke 1977;Göbel et al 2000). In the present case, the 100-Hz trains (0.3 s vas deferens, 0.5 s brain cortex) may moreover have been too short for the development of α 2 -autoinhibition (Story et al 1981).…”

Postnatal development of presynaptic receptors that modulate noradrenaline release in mice

“…For example, activation of α 2D -adrenoceptors inhibited the release of noradrenaline from the cultured sympathetic neurons but presynaptic non-α 2A/Dadrenoceptors seemed to be absent (Trendelenburg et al 2001a), in accord with the operation of α 2D -and non-operation of non-α 2A/D -autoreceptors in atria and vasa deferentia from P1 mice. Similarly, the three classical opioid receptors were not detected in the neuron cultures (Göbel et al 2000) and also did not operate, or operated weakly, in P1 atria and vasa deferentia. Moreover, lack of modulation of release in the cultures through AT 1 and bradykinin B 2 receptors (Göbel et al 2000) mirrors the relatively minor modulation through the two receptors in P1 atria and vasa deferentia.…”

“…Similarly, the three classical opioid receptors were not detected in the neuron cultures (Göbel et al 2000) and also did not operate, or operated weakly, in P1 atria and vasa deferentia. Moreover, lack of modulation of release in the cultures through AT 1 and bradykinin B 2 receptors (Göbel et al 2000) mirrors the relatively minor modulation through the two receptors in P1 atria and vasa deferentia. Possibly the program for the synthesis or transport to the terminals of some presynaptic receptors (e.g.…”