W Gil Wier
Department of Physiology, University of Maryland, Baltimore, MD, USA

https://doi.org/10.36866/pn.53.16

The sympathetic nervous system regulates blood pressure in part by controlling the diameter of muscular resistance arteries. Nevertheless, the mechanisms controlling release of the ‘triad’ of sympathetic neuro­transmitters (NA, ATP and neuro­peptide Y, or NPY) are largely unknown. And, perhaps surprisingly to those not in the field, even the post-junctional effects of two of these, ATP and NPY, are still obscure. Recently, however, a new ‘window’ on sympathetic neuromuscular transmission has been opened, through the combination of an old experimental technique, electrical stimulation of sympathetically innervated whole tissues (arteries or vas deferens), with simultaneous high resolution confocal imaging of pre-and post­junctional [Ca2+] transients.

Sympathetic neuromuscular transmission

Sympathetic motor neurons are good examples of the new principle of ‘multiplicity of neurotransmitters’ (see Stjarne, 1999), by which the old ‘ergic’ classification of neurons (Dale’s Principle) has been rendered obsolete. At the varicosities present along the terminals of these nerves (Fig. 1) not one, but multiple neuro­transmitters are ‘co-released’: nor­adrenaline (NA), ATP, and NPY. In small arteries and vas deferens, it is likely that NA and ATP are not always released in parallel. Details of co-transmitter release are sketchy at best, but Stjarne (2001) has advanced the provocative hypothesis that NA and ATP are present in two types of small synaptic vesicles; a ‘big’ quantum type containing a relatively much larger amount of ATP compared to NA than the other, ‘small’ quantum type. It is certainly true, in small arteries, that the effects of ATP predominate at lower frequencies of nerve activity, while NA predominates at higher frequencies. After release, ATP binds primarily to ionotropic (ion-channel) purinergic receptors (probably homomeric P2X1) on smooth muscle cells, activating inward current, including entry of Ca2+, and produces the excitatory junction potential (EJP) (see Ralevic & Burnstock, 2003 for review of purinergic signalling). On mouse mesenteric arteries at least, other purinergic receptors of the metabotropic type, such as P2Y6, may also be present (Vial & Evans, 2002), but these will be activated only by pyrimidine nucleotides (released from endothelial cells or platelets). On smooth muscle, NA binds mainly to α1-adrenoceptors and activates the well-known signaling cascade involving production of 1,4,5-InsP3, activation of receptor activated channels (ROCs), activation of protein kinase C and other events (see Wier & Morgan, 2003, for review). These events culminate in increases in intracellular [Ca2+], activation of Ca-calmodulin dependent myosin light-chain kinase (MLCK), as well as modulation of myosin light-chain phosphatase (MLCP) activity. As with all synapses, the spatio-temporal dynamics of transmitter action at sympathetic varicosities are influenced by diffusion, uptake and enzymatic degradation of transmitter, and receptor desensitization. Through pre-synaptic α2­adrenoceptors, NA inhibits its own release, as does ATP though pre­synaptic P1 (adenosine) and P2Y receptors.

Observing sympathetic neuromuscular transmission

Classically, the release of ATP has been detected electrically, as excitatory junctional currents (EJCs) recorded with loose-patch electrodes. These electrodes are ~ 12 µm in diameter and thus may encompass several sympathetic varicosities. With this technique, it can also be difficult to distinguish small EJCs arising beneath the electrode from large EJCs arising one cell layer deeper in the wall (Bennett et al. 2001). Perhaps surprisingly for this relatively mature field, a new technique has emerged recently for observing purinergic sympathetic neuromuscular transmission, with temporal and spatial resolution sufficient to resolve events at single sympathetic varicosities. As so often since the early 1990s, the combination of fluorescent Ca2+ indicators with confocal microscopy has provided the new observations. Cunnane and his colleagues in Oxford (Brain et al. 2002) were investigating pre-synaptic Ca2+ signaling in mouse vas deferens and found, unexpectedly, their fluorescent Ca2+ indicator (dextran­linked, low affinity Oregon Green) also in a subset of smooth muscle cells. By whatever means the indicator might have gotten there, it revealed localized smooth muscle Ca2+ transients, adjacent to sympathetic varicosities, after nerve stimulation. The Ca2+ transients were termed ‘neuroeffector Ca2+ transients’ (NCTs). At about the same time, we (Lamont & Wier, 2002) were asking the question, ‘Does NA elicit propagating Ca2+ waves in SMC during neurogenic contractions of small arteries, just as it does (Zang et al. 2001) during bath-application of NA?’ Unexpectedly, we observed brief, spatially localized Ca2+ transients that occurred in the vicinity of nerve fibres, before the adrenergic Ca2+ waves began. These localized Ca2+ transients were completely resistant to blockade of α1-adrenoceptors, L-type Ca2+ channels or ryanodine receptors. They were abolished, however, by the purinergic receptor blocker suramin. Thus, they certainly represented Ca2+ that had entered the cell through post-junctional purinergic receptors (probably P2X1) activated by neurally released ATP. As a manifestation of the excitatory junction current (EJC) we termed them ‘junctional Ca2+ transients’ or ‘jCaTs’. It seems clear that NCTs and jCaTs represent very similar phenomena but in vas deferens and arteries, respectively.

Opening the window

Synaptic physiologists can expect to use jCaTs to examine function of an individual sympathetic varicosity at an unprecedented level of detail. For example, the ability to monitor pre­and post-junctional activity for long periods at a single varicosity should provide the best estimates yet of the probability of transmitter release at a single varicosity. It may also provide new information on quantal content, by carefully observing amplitude distributions of jCaTs at single varicosities, and reveal differences amongst individual varicosities.

Students of smooth muscle contraction can also expect to benefit. Briefly, an initial transient component of the neurogenic contractions of small arteries appears to be activated by jCaTs (Fig. 1A), while a larger, maintained component of the contraction appears to be associated with asynchronous propagating Ca2+ waves (Fig. 1B), similar to those elicited by exogeneous α1­adrenoceptor agonists, such as phenylephrine (PE) (Lamont et al. 2003).

Clearly, bath-application of ATP (in particular), as typically done in the past, does not mimic the events involved in contractile activation by sympathetic nerves. Most importantly, the pattern of ‘tonic’ sympathetic nerve activity to small arteries consists of intermittent bursts of action potentials. Given the frequency dependence of ATP and NA release (and/or their effects) this should provide a spatio-temporal pattern of activation within the artery wall that is very markedly different than that achieved with simple bath­application of NA or ATP. Furthermore, the sympathetic co­transmitters act synergistically. The most recent data indicate that: ‘All three co-transmitters contribute significantly to vascular responses and their contribution varies markedly with impulse numbers’ (Bradley et al. 2003). Optical indicators of sympathetic neuromuscular transmission, such as jCaTs and NCTs now provide another view into this complex picture.

References

Bennett MR, Farnell L, Gibson WG, Lin YQ & Blair DH (2001). Quantal and non-quantal current and potential fields around individual sympathetic varicosities on release of ATP. Biophys J 80, 1311-1328.

Bradley E, Law A, Bell D & Johnson C (2003). Effects of varying impulse number on cotransmitter contributions to sympathetic vasoconstriction in rat tail artery. Am J Physiol Heart Circ Physiol 284, H2007-H2014.

Brain KL, Jackson VM, Trout SJ & Cunnane TC (2002). Intermittent ATP release from nerve terminals elicits focal smooth muscle Ca2+ transients in mouse vas deferens. J Physiol 549, 849-862.

Lamont C, Vainorius E & Wier WG (2003) Purinergic and adrenergic Ca2+ transients during neurogenic contractions of rat mesenteric small arteries. J Physiol 549, 801-808.

Lamont C & Wier WG (2002). Evoked and spontaneous purinergic junctional Ca2+ transients in rat small arteries. Circ Res 91, 454-456.

Ralevic V & Burnstock G (2003). Involvement of purinergic signaling in cardiovascular diseases. Drug News Perspect 16, 133-140.

Stjarne L (1999). Catecholaminergic neurotransmission: flagship of all neurobiology. Acta Physiol Scand 166, 251-259.

Stjarne L (2001). Novel dual ‘small’ vesicle model of ATP- and noradrenaline-mediated sympathetic neuromuscular transmission. Autonomic Neuroscience: Basic and Clinical 87, 16-36.

Vial C & Evans RJ (2002). P2X1 Receptor-deficient mice establish the native P2X receptor and a P2Y6-like receptor in arteries. Mol Pharmacol 62, 1438-1445.

Wier WG & Morgan KG (2003). α1-Adrenergic signaling mechanisms in contraction of resistance arteries. Reviews of Physiol Biochem Pharmacol (Published On-Line, 20 July 2003). DOI: 10.1007/s10254-003-0019-8

Zang W-J, Balke CW & Wier WG (2001). Graded α1-adrenoceptor activation of arteries involves recruitment of smooth muscle cells to produce ‘all or none’ Ca2+ signals. Cell Calcium 29, 327-334.