Novel dual ‘small’ vesicle model of ATP- and noradrenaline-mediated sympathetic neuromuscular transmission
Introduction
That chromaffin cells secrete catecholamines and ATP in the same (∼4/1) molar ratio in which they occur in the average vesicle (‘chromaffin granule’) is generally accepted Stjärne, 1989, Todorov et al., 1996. Whether or not this also applies to sympathetic nerves is debated, in part because the transmitter storing vesicles and their content of noradrenaline and ATP are less well known.
What is known is that these vesicles are not homogeneous. According to size and electron density of the core they belong to three categories: ‘large dense core vesicles’ (LDCVs; external diameter 80–120 nm), and two classes of ‘small vesicles’ (SVs; external diameter about 50 nm), some with a dense core (‘small dense core vesicles’, SDCVs) but the majority without (here termed ‘small clear vesicles’, SCVs) Bauerfeind et al., 1995, Gabella, 1992, Klein and Lagercrantz, 1981, Luff et al., 1987, Stjärne, 1989. The relative proportions of these vesicle types vary with the species. In small laboratory animals, e.g. mouse, rat or guinea-pig, LDCVs make up ≤5% of all transmitter vesicles. They are probably of little importance for ‘fast’ sympathetic neuromuscular transmission (especially at low frequency), because they are few, triggered to release mainly at high frequency and then discharge their content ‘extrasynaptically’ Klein and Lagercrantz, 1981, Stjärne, 1989. In these species SVs are therefore probably the main sources of the noradrenaline and ATP which mediate sympathetic neuromuscular transmission.
In the present work the guinea-pig isolated vas deferens was used to find out if action potentials in sympathetic nerve terminals always release ATP and noradrenaline in parallel, by exocytosis of the contents of the same SVs. A conclusive answer requires methods which make it possible to directly measure exocytosis of single ‘quanta’ of endogenous ATP and noradrenaline from a single SV. This is not technically possible as yet; what may be achieved at present, at best, is to decide whether or not ATP and noradrenaline are always secreted in parallel from the whole varicosity population.
Not even that is simple. No current method measures directly and quantitatively the exocytosis per se of endogenous ATP or noradrenaline; indirect approaches have to be used. In the present work the action potential-evoked EJC, i.e. the extracellularly recorded, ATP-induced, P2x-receptor-mediated excitatory junction current in smooth muscle (Brock and Cunnane, 1988), and the fractional secretion of tritium (ΔT) in preparations preincubated with [3H]noradrenaline (Alberts et al., 1981), were used for that purpose. Parallel variations in these parameters under all experimental conditions would support, but not conclusively prove, that ATP and noradrenaline were secreted from the same SV. Non-parallel variations might exclude that, provided that the two parameters always linearly reflect per pulse secretion of endogenous ATP and noradrenaline. One of the questions discussed is to what extent they do that.
The main finding was that the facilitation, depression and α2-autoinhibition of EJCs and ΔT were largely parallel during electrical field stimulation with trains of 100–300 pulses at ≤2 Hz, but increasingly non-parallel at higher frequencies. These observations are not easily explained in terms of exocytosis of ATP and noradrenaline from a single class of SVs. The paper proposes therefore a working hypothesis in which nerve impulses at low and high frequencies release the ATP and noradrenaline driving sympathetic neuromuscular transmission from two different classes of SVs.
Section snippets
Materials and methods
Male guinea-pigs (250–350 g) were stunned and bled to death and the vasa deferentia dissected out. The isolated organ was used to analyse electrophysiologically, radiometrically and mechanically the exocytosis of ATP and noradrenaline and the twitch contraction. The experiments were mostly (except when stated otherwise in the text) performed under either maximal or minimal α2-autoinhibition. The first was achieved by adding 3 μM cocaine and 30 μM corticosterone to the medium to block neuronal
Nerve terminal spike and EJC at 0.1–40 Hz
At all stimulation frequencies (0.1–40 Hz) each pulse triggered a positive-going, monophasic (as TTX was present in the recording electrode) nerve terminal spike (NTS), followed by a positive-going EJC (Fig. 1, Fig. 2). The presence of 0.1 μM prazosin and 50 μM nifedipine in the bath had no effect on the NTS or EJC (Blakeley et al., 1981). Increases in the stimulation frequency, or drug additions to the perfusion medium (3 μM cocaine, with or without 30 μM corticosterone, or 10 μM PBA, or 10 μM
Main findings and questions, overall plan
The main question in this paper is if action potentials in the sympathetic nerves in the guinea-pig vas deferens release noradrenaline and ATP in parallel, by exocytosis of the contents of the same ‘small’ vesicles (SVs). The approach was to find out if the parameters used to monitor the per pulse secretion of noradrenaline (ΔT/p) and ATP (EJC/p) varied in parallel under all experimental conditions. The main findings were that facilitation and α2-autoinhibition of ΔT/p and EJC/p were strikingly
Conclusions
The main question asked in this paper is if sympathetic nerves always release the two co-transmitters, ATP and noradrenaline, in fixed combinations from a single set of SVs, or are capable of releasing them in varying proportions because they are stored in and secreted from different SVs. The results strongly support the latter alternative. A tentative working hypothesis is proposed according to which (i) these nerves possess two classes of SVs, a small pool containing ‘big’ noradrenaline and
Acknowledgements
This work was supported by the Swedish Medical Research Council (project K97-14X-03027-28B) and Karolinska Institutets Fonder.
References (56)
Autonomic neuromuscular transmission at a varicosity
Prog. Neurobiol.
(1996)Noradrenaline and ATP as cotransmitters in sympathetic nerves
Neurochem. Int.
(1990)- et al.
Origin of adenosine released from rat vas deferens by nerve stimulation
Eur. J. Pharmacol.
(1982) - et al.
Prostaglandin E2 selectively affects purinergic transmission in guinea-pig vas deferens
Neuropharmacology
(1991) - et al.
SV2A and SV2B function as redundant Ca2+ regulators in neurotransmitter release
Neuron
(1999) - et al.
Uptake of adenosine triphosphate by isolated adrenal chromaffin granules: a carrier-mediated transport
Neuroscience
(1977) - et al.
Sympathetic transmitter release in rat tail artery and mouse vas deferens: facilitation and depression during high frequency stimulation
Neurosci. Lett.
(1993) - et al.
Is the neuronal ATP release from guinea-pig vas deferens subject to α2-adrenoceptor-mediated modulation?
Neuroscience
(1992) - et al.
Neuronal synthesis, storage and release of ATP
Semin. Neurosci.
(1996) - et al.
Relative pre- and postjunctional roles of noradrenaline and adenosine 5′-triphosphate as neurotransmitters of the sympathetic nerves of guinea-pig and mouse vas deferens
Neuroscience
(1985)
Geometry, kinetics and plasticity of release and clearance of ATP and noradrenaline as sympathetic cotransmitters: roles for the neurogenic contraction
Prog. Neurobiol.
P2x-receptors bring new structure to ligand-gated ion channels
Trends Neurosci.
Indirect evidence for separate vesicular neuronal origins of norepinephrine and ATP in the rabbit vas deferens
Eur. J. Pharmacol.
Modulation of neural ATP release through presynaptic receptors
Semin. Neurosci.
Site(s) and ionic basis of α-autoinhibition and facilitation of [3H]-noradrenaline secretion in guinea-pig vas deferens
J. Physiol.
Neurosecretory vesicles can be hybrids of synaptic vesicles and secretory granules
Proc. Natl. Acad. Sci. USA
An electropharmacological analysis of the effects of some drugs on sympathetic neuromuscular transmission in the vas deferens of the guinea-pig
J. Auton. Pharmacol.
Effects of nifedipine on electrical and mechanical responses of rat and guinea-pig vas deferens
Nature
Calcium in sympathetic varicosities of mouse vas deferens during facilitation, augmentation and autoinhibition
J. Physiol.
Electrical activity at the sympathetic neuroeffector junction in the guinea-pig vas deferens
J. Physiol.
Local application of drugs to sympathetic nerve terminals: an electrophysiological analysis of the role of prejunctional α-adrenoceptors in the guinea-pig vas deferens
Br. J. Pharmacol.
Inhibition of purinergic transmission by prostaglandin E1 and E2 in the guinea-pig vas deferens: an electrophysiological study
Br. J. Pharmacol.
Effects of Ca2+ concentration and Ca2+ channel blockers on noradrenaline release and purinergic neuroeffector transmission in rat tail artery
Br. J. Pharmacol.
β-Adrenoceptor mediated facilitation of noradrenaline and adenosine 5′-triphosphate release from sympathetic nerves supplying the rat tail artery
Br. J. Pharmacol.
α2-Adrenoceptor-mediated autoinhibition of sympathetic transmitter release in guinea-pig vas deferens studied by intracellular and focal extracellular recording of junction potentials and currents
Naunyn-Schmiedeberg’s Arch. Pharmacol.
Real-time measurement of transmitter release from single synaptic vesicles
Nature
Neural ATP release and its α2-adrenoceptor-mediated modulation in guinea-pig vas deferens
Naunyn-Schmiedeberg’s Arch. Pharmacol.
The fade of the purinergic neurogenic contraction of the guinea-pig vas deferens: analysis of possible mechanisms
Naunyn-Schmiedeberg’s Arch. Pharmacol.
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