Elsevier

Autonomic Neuroscience

Volume 87, Issue 1, 20 February 2001, Pages 16-36
Autonomic Neuroscience

Novel dual ‘small’ vesicle model of ATP- and noradrenaline-mediated sympathetic neuromuscular transmission

https://doi.org/10.1016/S1566-0702(00)00246-0Get rights and content

Abstract

The main question asked was if sympathetic nerves in guinea-pig vas deferens release the co-transmitters ATP and noradrenaline from the same, or different vesicles, i.e. in fixed combinations or independently. The extracellularly recorded excitatory junction current (EJC) and the fractional increase in overflow of tritium (ΔT) were used to monitor the per pulse secretion of ATP and [3H]NA, respectively, during electrical stimulation with 1–3000 pulses at 0.1–40 Hz. The frequency- and train length-dependence and α2-adrenoceptor-mediated autoinhibition of these parameters, and of the ATP-mediated twitch contraction, were compared first in the presence of cocaine (to block noradrenaline reuptake), then after brief exposure to phenoxybenzamine (PBA, to irreversibly ‘destroy’ α2-autoreceptors). Parallel variations of EJC/p(ulse) and ΔT/p(ulse) under all conditions would support, non-parallel variations argue against exocytosis of ATP and noradrenaline from the same vesicles. The main findings were that facilitation and α2-autoinhibition of EJC/p and ΔT/p were remarkably similar during stimulation at 2 Hz but increasingly dissimilar at higher frequencies. ΔT/p remained strongly facilitated and tightly controlled by activation of α2-autoreceptors at 10–40 Hz, but both the facilitation and the sensitivity to α2-autoinhibition of EJC/p were inversely related to frequency. At 40 Hz EJCs were ‘small’, minimally facilitated and totally unaffected by cocaine or PBA, i.e. insensitive to α2-autoinhibition. Nevertheless, activation of α2-receptors during the 40 Hz train strongly restricted the ‘post-tetanic augmentation’ (PTA) of the first EJC 10 s after the tetanus. Comparison between the frequency dependence of EJCs and the twitch contraction in the presence of cocaine or after PBA treatment indicates that it is the ‘summed EJC per second’, i.e. the ATP-driven current injection per unit time into smooth muscle, which triggers the twitch. The working hypothesis is proposed that these nerves use two classes of ‘small vesicles’ (SVs) to store and release either ‘big’ or ‘small’ ATP and noradrenaline ‘quanta’, and that differences in properties (Ca2+ affinity, capacity) of Ca2+ receptors in the SV membranes enable the nerves to selectively secrete ‘big quanta’ at low frequency and ‘small quanta’ during trains at high frequency.

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)

  • L Stjärne et al.

    Geometry, kinetics and plasticity of release and clearance of ATP and noradrenaline as sympathetic cotransmitters: roles for the neurogenic contraction

    Prog. Neurobiol.

    (1995)
  • A Surprenant et al.

    P2x-receptors bring new structure to ligand-gated ion channels

    Trends Neurosci.

    (1995)
  • G.J Trachte et al.

    Indirect evidence for separate vesicular neuronal origins of norepinephrine and ATP in the rabbit vas deferens

    Eur. J. Pharmacol.

    (1989)
  • I von Kügelgen

    Modulation of neural ATP release through presynaptic receptors

    Semin. Neurosci.

    (1996)
  • P Alberts et al.

    Site(s) and ionic basis of α-autoinhibition and facilitation of [3H]-noradrenaline secretion in guinea-pig vas deferens

    J. Physiol.

    (1981)
  • R Bauerfeind et al.

    Neurosecretory vesicles can be hybrids of synaptic vesicles and secretory granules

    Proc. Natl. Acad. Sci. USA

    (1995)
  • A.G.H Blakeley et al.

    An electropharmacological analysis of the effects of some drugs on sympathetic neuromuscular transmission in the vas deferens of the guinea-pig

    J. Auton. Pharmacol.

    (1981)
  • A.G.H Blakeley et al.

    Effects of nifedipine on electrical and mechanical responses of rat and guinea-pig vas deferens

    Nature

    (1981)
  • K.L Brain et al.

    Calcium in sympathetic varicosities of mouse vas deferens during facilitation, augmentation and autoinhibition

    J. Physiol.

    (1997)
  • J.A Brock et al.

    Electrical activity at the sympathetic neuroeffector junction in the guinea-pig vas deferens

    J. Physiol.

    (1988)
  • J.A Brock et al.

    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.

    (1991)
  • J.A Brock et al.

    Inhibition of purinergic transmission by prostaglandin E1 and E2 in the guinea-pig vas deferens: an electrophysiological study

    Br. J. Pharmacol.

    (1996)
  • J.A Brock et al.

    Effects of Ca2+ concentration and Ca2+ channel blockers on noradrenaline release and purinergic neuroeffector transmission in rat tail artery

    Br. J. Pharmacol.

    (1999)
  • J.A Brock et al.

    β-Adrenoceptor mediated facilitation of noradrenaline and adenosine 5′-triphosphate release from sympathetic nerves supplying the rat tail artery

    Br. J. Pharmacol.

    (1997)
  • J.A Brock et al.

    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.

    (1990)
  • D Bruns et al.

    Real-time measurement of transmitter release from single synaptic vesicles

    Nature

    (1995)
  • B Driessen et al.

    Neural ATP release and its α2-adrenoceptor-mediated modulation in guinea-pig vas deferens

    Naunyn-Schmiedeberg’s Arch. Pharmacol.

    (1993)
  • B Driessen et al.

    The fade of the purinergic neurogenic contraction of the guinea-pig vas deferens: analysis of possible mechanisms

    Naunyn-Schmiedeberg’s Arch. Pharmacol.

    (1994)
  • Cited by (29)

    • Purinergic modulation of glutamate transmission: An expanding role in stress-linked neuropathology

      2018, Neuroscience and Biobehavioral Reviews
      Citation Excerpt :

      Both ATP and adenosine can act on their respective receptors to induce synaptic currents in neurons; however the manner in which these two ligands enter the synaptic cleft is quite different (discussed below). ATP can be packaged into synaptic vesicles by vesicular nucleotide transporter (VNUT or SLC17A9) (Sawada et al., 2008; Larsson et al., 2012), and is typically released alongside other neurotransmitters, including glutamate (Pankratov et al., 2003, 2002; Pankratov et al., 1998), acetylcholine (ACh) (Reigada et al., 2003; Morel and Meunier, 1981), noradrenaline (NA) (Stjarne, 2001; Sesti et al., 2002), and γ-aminobutyric acid (GABA) (Jo and Schlichter, 1999; Jo and Role, 2002). These neurotransmitters have all been shown to be meaningfully modulated by purinergic signalling, however the present review will focus primarily on the interactions of purines and glutamate.

    • Imaging sympathetic neurogenic Ca<sup>2 +</sup> signaling in blood vessels

      2017, Autonomic Neuroscience: Basic and Clinical
      Citation Excerpt :

      In sympathetic varicosities in the walls of arteries, the amount and timing of the release of NE, ATP and NPY are dependent on the duration and frequency of the nerve action potentials (Todorov et al., 1999, Bradley et al., 2003). The reasons for this remain obscure, but a plausible hypothesis has been advanced and supported by Stjarne (2001). Briefly, he hypothesized that NE and ATP are stored in both ‘big’ or ‘small’ vesicles within sympathetic varicosities and that mechanisms exist to enable the selective secretion of ‘big’ quanta at low frequencies and ‘small’ quanta at high frequencies of action potential arrival in the nerve terminal.

    • The purinergic neurotransmitter revisited: A single substance or multiple players?

      2014, Pharmacology and Therapeutics
      Citation Excerpt :

      Therefore, the temporal dissociation of ATP and NE release described in the guinea-pig vas deferens might not be universal for the sympathetic nervous system. For example, analysis of excitatory junction currents (EJCs) to measure the release of ATP and differential pulse amperometry to measure the release of NE suggested that ATP and NE were likely released in parallel in the mouse vas deferens (Stjarne et al., 1994; Msghina et al., 1998), whereas in the guinea-pig vas deferens it was proposed that the release of ATP and NE may originate from different classes of small vesicles with different Ca2+ sensitivities at low and high frequencies of stimulation (Stjarne, 2001). Furthermore, analysis of electrical and mechanical responses to electrical stimulation has demonstrated that in mouse vas deferens N-, P- and Q-type voltage-dependent calcium channels are involved similarly in the release of ATP and NE (Waterman, 1997), whereas ATP and NE overflow studies in the guinea-pig vas deferens suggest differential modulation of ATP and NE release by N-type and P-type Ca2+ channels (Westfall et al., 1996).

    • Vas deferens neuro-effector junction: From kymographic tracings to structural biology principles

      2014, Autonomic Neuroscience: Basic and Clinical
      Citation Excerpt :

      A variety of microscopic techniques have consistently visualized two types of synaptic vesicles in the sympathetic nerve endings of this tissue as well as many sympathetic organs: the small and the large dense cored synaptic vesicles (Neuman et al., 1984). It is not at all clear whether each transmitter is separately stored in the small vesicles and therefore each transmitter is individually released, or both transmitters are co-stored in common vesicles that contain ATP and NE (Stjärne, 2001). Notwithstanding, not all varicosities discharge at the same time with each nerve stimuli nor secrete the same proportion of co-transmitters (Westfall et al., 2002).

    View all citing articles on Scopus
    View full text