Purinergic cotransmission: sympathetic nerves

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THE NEUROSCIENCES, Vol 8, 1996: pp 201–205

Purinergic cotransmission: sympathetic nerves Peter Sneddon, Gerald J. McLaren and Charles Kennedy

Studies of the corelease of sympathetic neurotransmitters

During the past decade it has become clear that cotransmission is the rule rather than the exception in the autonomic nervous system. The role of ATP as a cotransmitter has been most extensively investigated in sympathetic nerves innervating smooth muscle preparations such as isolated vas deferens and arteries. This article describes how the role of ATP as a sympathetic cotransmitter has been established by a combination of various experimental methods including classical organ bath pharmacology, electrophysiology and a variety of biochemical methods for measuring neurotransmitter release.

Experiments in which the overflow of putative cotransmitters into the perfusate is measured by biochemical analysis have been put forward to support the cotransmitter hypothesis in a variety of tissues. In the late 1970’s experiments examining the release of radiolabelled NA and adenosine provided evidence for ATP as a cotransmitter in sympathetic nerves. Exogenously added 3H-adenosine was presumed to be taken up by nerves and converted to 3H-ATP, which was subsequently released upon nerve stimulation. However, this type of study was criticised on the basis that the released purine might come from other pools loaded by the addition of exogenous adenosine, such as smooth muscle cells. More recent studies measuring the release of endogenous ATP, using detection by HPLC or luciferin-luciferase assays, seem to confirm that only some of the measured ATP is being released from the nerve, but there are still widely disparate estimates of how much of the measured ATP is from neuronal or non-neuronal sources. One recent study in guinea-pig vas deferens estimated that only about 16% of the nerve-evoked overflow of ATP actually comes from the sympathetic nerves, the majority probably originating from smooth muscle cells when they are stimulated by NA acting on α1-adrenoceptors.4 In contrast, other recent studies measuring the release of endogenous ATP and NA produced by sympathetic nerve stimulation in guinea-pig vas deferens suggest that almost all the ATP measured comes from the nerves.5 The latter study also indicates that the two transmitters are released with different time courses and can be differentially modulated by prejunctional receptors. This supports the view that there are different sub-populations of vesicles with different proportions of the two transmitters. It has also been shown that stimulation of the sympathetic nerves to the guinea-pig vas deferens coreleases neuropeptide Y (NPY) with NA and ATP into the perfusate, all of which can be measured simultaneously using various biochemical techniques. It has been proposed that NA and ATP packaged in small synaptic vesicles are released at low stimulation

Key words: ATP / cotransmission / neurotransmission / purinergic / sympathetic ©1996 Academic Press Ltd

IN THE 1930’s the idea that noradrenaline (NA) was the sole neurotransmitter in sympathetic nerves was firmly established by the pioneering studies performed chiefly by Sir Henry Dale and his colleagues.1 By the early 1970’s evidence had been accumulated to support the proposal that the endogenous purine, adenosine 5'-triphosphate (ATP) was also an autonomic neurotransmitter. The evidence supporting what Burnstock called purinergic nerves was presented in his landmark review of this topic in 1972.2 The idea of cotransmission was given its first real impetus by the review by Burnstock3 in 1976 which posed the question ‘Do some nerve cells release more than one transmitter?’ Now cotransmission involving NA, ATP and other agents is regarded as the rule rather than the exception in sympathetic nerves. The early evidence for cotransmission emerged particularly from studies on the isolated vas deferens and arteries of various species, which are considered in detail below.

From the Department of Physiology & Pharmacology, University of Strathclyde, Royal College, 204 George Street, Glasgow, G1 1XW, UK ©1996 Academic Press Ltd 1044-5765/96/040201 + 05 $18.00/0

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P. Sneddon et al nyl-ATP (ANAPP3) which was shown to inhibit the initial phase of the sympathetic contraction, and the contraction to exogenous ATP, but had no effect on the second phase of the neurogenic response or the contractions produced by NA or other agonists.7 This result was soon confirmed by using the stable ATP analogue α,β-methyleneATP to produce selective desensitization of P2X-purinoceptors in the vas deferens8 and more recently by the use of selective P2Xpurinoceptor antagonists such as PPADS.9 An important feature of the function of the vas deferens is that the smooth muscle cells in different regions have different relative sensitivities to ATP and NA. Segments of vas deferens taken from the prostatic region of the rat, rabbit or guinea-pig vas deferens are approximately 10 times more sensitive than epididymal segments to exogenous ATP (or the stable analogue α,β-methyleneATP) whilst segments from the epididymal region are at least 10 times more sensitive than prostatic segments to application of exogenous α-adrenoceptor agonists such as NA.10 Obviously this implies that even if the sympathetic nerves release constant amounts of NA and ATP throughout a train of pulses, the purinergic and noradrenergic contribution to the neurogenic response will vary from one region of the organ to another.

rates, whilst NPY, which only occurs in large vesicles, is released during periods of higher frequency bursts of nerve activity.

Functional significance of ATP as a sympathetic cotransmitter in the vas deferens In rat and mouse vas deferens a single stimulus of sympathetic nerves produces a biphasic contraction of the smooth muscle. The initial peak is thought to be mediated by ATP and the second component by NA. The most compelling arguments in favour of this interpretation have come from pharmacological investigations using selective antagonists. The initial phase of the response is selectively reduced by agents which inhibit the action of ATP on P2X-purinoceptors, whilst the second component is selectively reduced by α1-adrenoceptor antagonists. See review by von K¨ugelgen and Starke.6 In guinea-pig and rabbit vas deferens a single stimulus of the sympathetic nerves produces little or no mechanical response, and therefore trains of pulses, usually from 2–32 Hz applied for 10–30 s, are used to investigate the neurogenic response. In these tissues the response to trains of pulses is biphasic, with an initial peak occurring after 3–4 s, which then subsides before a second phase reaches a plateau after about 10 s. Again, antagonist studies have provided convincing evidence that the initial phasic component of this sympathetic response is mediated by ATP whilst the secondary component is mediated by NA (see Figure 1). The first clear pharmacological evidence for ATP as a cotransmitter came from studies using the photo-affinity label, arylazidoaminopropio-

Electrophysiological evidence for ATP as a cotransmitter in vas deferens In vas deferens, ATP and NA activate separate transduction mechanisms to produce contraction. Activation of P2X-purinoceptors by neuronally released ATP produces rapid depolarization of the muscle, in the form of excitatory junction potentials (e.j.p.s) a train of which will summate to produce action potentials, which results in an influx of calcium through voltage dependent channels, leading to the initial, rapid contraction. Noradrenaline, on the other hand, does not appear to contribute significantly to nerve-mediated membrane depolarization, and α1-adrenoceptor stimulation mediates the slow phase of the neurogenic contraction mainly by generation of the second messenger inositol trisphosphate (IP3) which is able to release calcium stored in the sarcoplasmic reticulum. When e.j.p.s were first recorded in vas deferens in the early 1960’s it was assumed that they were mediated by NA. This review prevailed for about 20 years until selective antagonists of ATP became available. We first showed that e.j.p.s

Rabbit vas deferens

2g

Control

Prazosin

30 sec

Prazosin +ANAPP3

Figure 1. Trains of pulses at 16 Hz for 30 seconds produced a biphasic contraction in rabbit isolated vas deferens (control panel on left). The initial, phasic component was selectively reduced by the P2-purinoceptor antagonist ANAPP3, whereas the secondary, tonic contraction was selectively blocked by the α1-adrenoceptor antagonist prazosin.

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Purinergic cotransmission in sympathetic nerves in guinea-pig vas deferens were almost abolished by the P2-purinoceptor antagonist ANAPP3, but were not reduced by selective concentrations of α-adrenoceptor antagonists.11 We also found that in reserpinized animals, in which almost all neuronal NA was depleted, the magnitude of fully facilitated e.j.p.s was no less than in controls.12 The main action of cocaine, which inhibits NA inactivation, was actually to depress the magnitude of e.j.p.s probably by increasing feedback inhibition of ATP release by NA.12 Subsequent electrophysiological studies using P2-purinoceptor desensitization by α,β-methyleneATP and the P2-purinoceptor antagonist suramin13 have supported the proposal that e.j.p.s are mediated by ATP (Figure 2). These studies led us to propose the model for sympathetic neurotransmission which is depicted in Figure 3. Many of the features of this model seem to apply to a variety of autonomically innervated smooth muscles, particularly arteries as described below.

Sympathetic nerve varicosity

Smooth muscle

NPY NA NA ATP

NPY NPY

NA ATP

Postjunctional potentiation

α1 NA ATP P2X

e.j.p.

Potential-independent Ca++ mobilisation Tonic contraction

Phasic contraction Action potentials

Figure 3. Model of cotransmission in sympathetic nerves involving ATP, NA and NPY as cotransmitters. There may be sub-populations of vesicles within the nerve terminal varicosities which could allow for different ratios of release of ATP:NA:NPY at different stimulation durations and intensities. Each transmitter produces a different effect on the smooth muscle cells, ATP mediating rapid depolarization (e.j.p.s) and phasic contraction, whilst NA produces a slow, maintained contraction mainly due to release of internal Ca2 + stores. NPY at the concentrations achieved during nerve stimulation probably has a predominantly prejunctional inhibitory action, but may also act postjunctionally to enhance the action of both ATP and NA.

Cotransmission in arteries Sympathetic innervation of many arteries involves

ATP, NA and various peptides. In arteries the electrical response of the muscle to the cotransmitters is more complex than in vas deferens, and varies considerably from one tissue to another. In rat tail artery for example, each stimulus of the sympathetic nerves produces a rapid e.j.p. similar in magnitude, time-course and pharmacological profile to those in the vas deferens. The e.j.p.s are abolished by suramin, but during a train of pulses a slow depolarization develops, which is inhibited by adrenoceptor antagonists such as phentolamine (ref 14, Figure 4). Since

10 mV Control 2s

Phentolamine

Suramin 10 mins

10 mV 10 s

20 mins Control 30 mins

Suramin

Suramin and phentolamine

Figure 4. In rat tail artery each stimulus of the sympathetic nerves at 1 Hz produces an e.j.p. of about 8–10 mV and a slow depolarization as the train of pulses progresses (left panel). Addition of the P2-purinoceptor antagonist suramin (10-4 M) abolished e.j.p.s within 30 minutes, but did not reduce the slow depolarization, which was abolished by subsequent addition of the α-adrenoceptor antagonist phentolamine (10-6 M). From ref 14.

Figure 2. In the guinea-pig vas deferens each stimulus of the sympathetic nerves at 0.5 Hz (d) produced e.j.p.s. of about 6 mV (top panel). Addition of the α-adrenoceptor antagonist phentolamine (10-6 M) slightly enhanced e.j.p. magnitude, whereas the P2-purinoceptor antagonist suramin (10-4 M) abolished e.j.p.s within 30 minutes.

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P. Sneddon et al and the relative amounts of cotransmitters released also changes with the frequency and duration of stimulation. Again we shall use as an example the sympathetic nerves in the rodent vas deferens and arteries, which have been extensively studied to determine the role of the three putative cotransmitters, ATP, NA and NPY, in modulation of transmitter release. NA acts on prejunctional α2-adrenoceptors to modulate its own release and the release of ATP, and NPY. The important role of NA-mediated autoinhibition of transmitter release can be demonstrated by the use of α2-adrenoceptor antagonists such as yohimbine, which greatly enhance both phases of the sympathetic contractile response, and also enhance the excitatory junction potentials, which are purely purinergic. There is also clear evidence that stimulation of prejunctional purinoceptors can inhibit transmitter release and therefore inhibit both phases of the neurogenic contraction and reduce e.j.p. magnitude. These prejunctional purinoceptors are stimulated by adenosine and its analogues, and blocked by P1-purinoceptor antagonists such as 8-sulphonylphenyltheophylline, and are therefore classified as P1-purinoceptors. These receptors could respond to adenosine coming from the breakdown of neuronally released ATP, or adenosine from other sources, such as the effector smooth muscle. It has also been proposed that in vas deferens and some arteries ATP per se can inhibit transmitter release by acting on prejunctional P2-purinoceptors21 or possibly on a novel class of purinoceptors designated P3-purinoceptors.22 The release of transmitters from the vas deferens is also inhibited by NPY, probably acting at the NPY2 subclass of receptors. Since NPY is thought to be released only during higher frequency bursts of nerve stimulation, this negative feedback mechanism may complement the α2-adrenoceptor mediated autoinhibition mechanism which can be demonstrated at stimulation frequencies as low as 0.5 Hz. Therefore, during stimulation with a prolonged train of pulses, the regulation of transmitter release will change with the concentrations of the various transmitters. Circulating hormones and autocoids can also modulate cotransmitter release from sympathetic nerves, such as angiotensin II, prostaglandin E2, bradykinin, histamine, atrial naturetic factor and acetylcholine via M1-receptors. (For detailed discussion see review by Bartfai et al, 23.) It should also be borne in mind that the cotransmitters which a particular nerve utilizes may not be fixed,

the combination of the purinoceptor antagonist and the adrenoceptor antagonist abolishes the electrical response to nerve stimulation, it seems that NPY does not contribute directly to membrane depolarization, although it may enhance the action of the other transmitters, as discussed below. The contribution which ATP and NA make to sympathetic vasoconstriction varies enormously from one artery to another.6 In dog mesenteric artery the response to sympathetic nerve stimulation looks similar to that found in vas deferens. ATP contributes mainly to the initial, rapid, phasic contraction, whilst NA mediates the slower, secondary, tonic component of the vasoconstriction. In rat mesenteric arteries and rabbit ileocolonic arteries there is also a biphasic response with purinergic and adrenergic phases. In some arteries, such as rat tail artery, ATP makes little contribution to sympathetic vasoconstriction,15 whereas in rabbit saphenous artery16 and small jejunal arteries17 the sympathetic vasoconstriction is largely mediated by ATP. The role of NPY in sympathetic vasoconstriction is less clear. In many cases it has little or no constrictor effect on its own, but potentiates the action of ATP and NA.18 In some human arteries it has been found that vasoactive peptides are released from sympathetic nerves (NPY with NA), parasympathetic nerves (vasoactive intestinal polypeptide (VIP) with histidine isoleucine and acetylcholine) and sensory nerves (CGRP with tachykinins). The peptides appear to serve as cotransmitters in the sympathetic and parasympathetic nerves only at high stimulation frequencies, but are released from sensory nerves during low frequency stimulation.

Prejunctional modulation of the release of cotransmitters from sympathetic nerves The idea that a neurotransmitter can act not only postjunctionally on the effector cell, but also prejunctionally on autoreceptors to regulate its own release, was first established in sympathetic nerves. NA was shown to promote its own release via prejunctional β-adrenoceptors and, at higher concentrations, inhibit its own release via prejunctional α-adrenoceptors. The fact that several cotransmitter substances are released from some sympathetic nerves suggests that each transmitter could differentially modulate release.20 Prejunctional control of transmitter release is known to be dependent upon stimulation frequency, 204

Purinergic cotransmission in sympathetic nerves 11. Sneddon P, Westfall DP, Fedan JS (1982) Cotransmitters in the motor nerves of the guinea-pig vas deferens: Electrophysiological evidence. Science 218:693-695 12. Sneddon P, Westfall DP (1984). Pharmacological evidence that adenosine triphosphate and noradrenaline are co-transmitters in the guinea-pig vas deferens. J Physiol 347:561-580 13. Sneddon P (1992) Suramin inhibits excitatory junction potentials in guinea-pig vas deferens. Br J Pharmacol 107:101-103 14. McLaren GJ, Kennedy C, Sneddon P (1995) The effects of suramin on purinergic and noradrenergic neurotransmission in the rat isolated tail artery. Eur J Pharmacol 277:57-61 15. Bao JX (1993) Sympathetic neuromuscular transmission in rat tail artery: A study based on electrochemical, electrophysiological and mechanical recording. Acta Physiol Scand Sup 148, 610:1-58 16. MacDonald A, Daly CJ, Bulloch JM, McGrath JC (1992) Contributions of alpha1-adrenoceptors, alpha 2-adrenoceptors and P2X-purinoceptors to neurotransmission in several rabbit isolated blood vessels: Role of neuronal uptake and autofeedback. Br J Pharmacol 105:347-354 17. Evans RJ, Cunnane TC (1992) Relative contributions of ATP and noradrenaline to the nerve evoked contraction of the rabbit jejunal artery. Dependence on stimulation parameters. Naunyn-Schmied Arch Pharmacol 345:424-430 18. Westfall TC, Yang C-L, CurfmanFalvey M (1995) NeuropeptideY-ATP interactions at the vascular sympathetic neuroeffector junction. J Cardiol Pharmacol 26:682-687 19. Lundberg JM, Franco-Cereceda A, Hemsen A, Lacroix JS, Pernow J (1990) Pharmacology of noradrenaline and neuropeptide tyrosine (NPY) — mediated sympathetic cotransmission. Fundam Clin Pharmacol 4:373-391 20. Von Kugelgen ¨ I, Kurz K, Bultmann R, Driessen B, Starke K (1994) Presynaptic modulation of the release of the cotransmitters noradrenaline and ATP. Fund Clin Pharmacol 8:207-213 21. Kurz K, Von Kugelgen ¨ I, Starke K (1993) Prejunctional modulation of noradrenaline release in mouse and rat vas deferens: Contribution of P1- and P2-purinoreceptors. Br J Pharmacol 110:1465-1472 22. Todorov LD, Bjur RA, Westfall DP (1994) Inhibitory and facilitatory effects of purines on transmitter release from sympathetic nerves. J Pharmacol Exp Ther 268:985-989 23. Bartfai T, Iverfeldt K, Fisone G (1988). Regulation of the release of co-existing neurotransmitters. Annu Rev Pharmacol Toxicol 28:285-310 24. Burnstock G (1990) Changes in expression of autonomic nerves in ageing and disease. J Auton Nerv Syst 30:S25-S34

but may vary during the development of the animal, perhaps as a result of ageing or disease.24

Acknowledgements This work was supported by grants from Astra plc and the MRC.

References 1. Dale HH (1935) Pharmacology and nerve endings. Proc Roy Soc Med 28:319-322 2. Burnstock G (1972) Purinergic nerves. Pharmacol Rev 24:509-581 3. Burnstock G (1976) Do some nerve cells release more than transmitter? Neuroscience 1:239-248 4. Von Kugelgen ¨ I, Starke K (1994) Corelease of noradrenaline and ATP by brief pulse trains in guinea-pig vas deferens. Naunyn-Schmied Arch Pharmacol 350:123-129 5. Todorov LD, Bjur RA, Westfall DP (1994) Temporal dissociation of the release of the sympathetic co-transmitters ATP and noradrenaline. Clin Exp Pharmacol Physiol 21:931-932 6. Von Kugelgen ¨ I, Starke KS (1991) Noradrenaline and ATP cotransmission in the sympathetic nervous system. Trend Pharmacol Sci 12:319-324 7. Fedan JS, Hogaboom GK, Westfall DP, O’Donnell (1982) Comparison of the effects of arylazido aminopropionyl ATP (ANAPP3.) an ATP antagonist of responses of the smooth muscle of the guinea-pig vas deferens to ATP and related nucleotides. Eur J Pharmacol 85:227-290 8. Meldrum LA, Burnstock G (1984) Evidence that ATP acts as a cotransmitter with noradrenaline in the sympathetic nerves supplying the guinea-pig vas deferens. Eur J Pharmacol 92:161-163 9. McLaren GJ, Lambrecht G, Mutschler E, B¨aumert HG, Sneddon P, Kennedy C (1994) Investigation of the actions of PPADS, a novel P2X-purinoceptor antagonist, in the guinea-pig isolated vas deferens. Br J Pharmacol 111:913-917 10. Sneddon P, Machaly M (1992) Regional variation in purinergic and adrenergic responses in isolated vas deferens of rat, rabbit and guinea-pig. J Auton Pharmacol 12:421-428

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