Cotransmission in the autonomic nervous system

Handbook of Clinical Neurology, Vol. 117 (3rd series) Autonomic Nervous System R.M. Buijs and D.F. Swaab, Editors © 2013 Elsevier B.V. All rights reserved

Chapter 3

Cotransmission in the autonomic nervous system GEOFFREY BURNSTOCK* Autonomic Neuroscience Centre, University College Medical School, London, UK and Department of Pharmacology, University of Melbourne, Australia

EARLY STUDIES For many years, understanding of neurotransmission incorporated the concept that one neuron releases only a single transmitter, termed “Dale’s Principle” by Eccles (1957). This idea arose from a widely adopted misinterpretation of Dale’s suggestion in 1935 that the same neurotransmitter was stored in and released from all terminals of a single sensory neuron, a suggestion which did not specifically preclude the possibility that more than one transmitter may be associated with the same neuron (Dale, 1935). Early hints that nerves might release more than one transmitter began in the 1950s with evidence for the involvement of both noradrenaline/norepinephrine (NA) and acetylcholine (ACh) in sympathetic transmission. Koelle identified acetylcholinesterase in some adrenergic neurons in 1955 (Koelle, 1955), while Burn and Rand introduced the concept of a “cholinergic” link in adrenergic transmission (Burn and Rand, 1959). Another line of evidence, provided by Hillarp, concerned the coexistence of adenosine 50 -triphosphate (ATP) with catecholamines, first in adrenal chromaffin cells (Hillarp umann, et al., 1955) and later in sympathetic nerves (Sch€ 1958). Ultrastructural studies of the enteric nervous system suggested that there were several different cotransmitters in nerves; at least nine distinguishable types of axon profiles showing different combinations of vesicle types were described in the guinea pig myenteric plexus (Cook and Burnstock, 1976). Inconsistencies in the single transmitter hypothesis provided by these and other studies, including those concerned with invertebrate neurotransmission (Brownstein et al., 1974; Cottrell, 1976), were rationalized in an article by Burnstock in 1976 with the provocative title: “Do some nerve cells release more than one transmitter?” Later, it was widely accepted that

“cotransmission” is an integral feature of neurotransmission (see Cuello, 1982; Osborne, 1983; Burnstock, 1990a, 2004, 2009b; Kupfermann, 1991; Lundberg, 1996). A role for ATP as a cotransmitter in sympathetic, parasympathetic, sensory-motor, and enteric nonadrenergic, noncholinergic (NANC) inhibitory nerves was supported by research from Burnstock and colleagues (see Burnstock 1983, 1990b, 2007, 2009b), while H€okfelt and colleagues focused on the colocalization, vesicular storage, and release of peptides from both peripheral and central nerves (see H€okfelt et al., 1977, 1986). Evidence for ATP being a cotransmitter with established neurotransmitters in the central nervous system (CNS) (including brainstem and hypothalamus, which regulate autonomic activities) as well as in the periphery has been reported (see Table 3.1). Furness and Costa introduced the concept of “chemical coding” to describe the combination of potential neurotransmitters found in enteric nerves and this concept has since been applied to other nerve types, in both peripheral and central nervous systems (Furness et al., 1989). Colocalized substances are not necessarily cotransmitters; they can (especially peptides) act as pre- and/or postjunctional neuromodulators of the release and actions of the principal cotransmitters. The proportions of cotransmitters vary considerably between species and organs, and show plasticity of expression during development and in pathological conditions. In general, classical transmitters are contained in small synaptic vesicles, whereas peptides are stored in large granular (dense-cored) vesicles (LGVs), although small molecule transmitters are sometimes stored together with peptides in LGVs. Pharmacological studies of pre- and postjunctional neuromodulation provide evidence, which is complementary to the concept of cotransmission. For example,

*Correspondence to: Geoffrey Burnstock, Autonomic Neuroscience Centre, University College Medical School, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK. Tel: þ44-(0)20-7830-2948, Fax: þ44-(0)20-7830-2949, E-mail: [email protected]

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G. BURNSTOCK

Table 3.1 Cotransmitters in the peripheral and central nervous systems Cotransmitters Peripheral nervous system Sympathetic nerves Parasympathetic nerves Sensory-motor NANC enteric nerves Motor nerves (in early development)

ATP + NA + NPY ATP+ACh+VIP ATP+CGRP+SP ATP+NO+VIP ATP+ACh

Central nervous system Cortex, caudate nucleus Hypothalamus, locus ceruleus Hypothalamus, dorsal horn, retina Mesolimbic system Hippocampus, dorsal horn

ATP+ACh ATP+NA ATP+GABA ATP+DA ATP+glutamate

Compiled from Burnstock, 2007. ACh, acetylcholine; ATP, adenosine 50 -triphosphate; CGRP, calcitonin gene-related peptide; DA, dopamine; GABA, g-amino butyric acid; NA, noradrenaline; NO, nitric oxide; NPY, neuropeptide Y; SP, substance P; VIP, vasoactive intestinal peptide.

parallel presynaptic modulation of transmitter overflow supports the concept of closely associated corelease, while postjunctional synergism between colocalized transmitters provides justification of cotransmission in terms of transmitter economy.

SYMPATHETIC NERVES There is compelling evidence that under certain conditions in vitro, single sympathetic neurons may release NA, ACh, or a mixture of these two transmitter substances (Le Douarin et al., 1975; Furshpan et al., 1976; Patterson et al., 1976). It seems likely that this represents a true reflection of events that occur in vivo during perinatal development (Hill and Hendry, 1977). It appears that a population of sympathetic nerve cells are present at birth that have the potential to synthesize both NA and ACh. These multipotential cells require nerve growth factor (NGF) to survive and they respond to NGF with an increased production of both choline acetyltransferase and tyrosine hydroxylase, enzymes that are involved in the synthesis of ACh and NA, respectively. Under the influence of conditioning factors, most of the cells appear to differentiate into either cholinergic or adrenergic neurons soon after birth. However, it is possible that some sympathetic neurons, supplying some organs in some animals, retain the ability to produce and release both ACh and NA (see Burn and Rand, 1965; Burnstock, 1978). In late pregnancy, sympathetic innervation of guinea pig uterine blood vessels exhibits a remarkable

switch from adrenergic vasoconstrictor to cholinergic vasodilator control (Bell, 1968). There is substantial evidence to show that NA and ATP are cotransmitters in sympathetic nerves, having differentially important roles as transmitters and neuromodulators depending on the tissue, the species, and on the parameters of stimulation (see Burnstock and Sneddon, 1985; Burnstock, 1990b). The first indication that ATP might be released from sympathetic nerves was the demonstration that stimulation of sympathetic nerves led to release of tritium from taenia coli preincubated in [3H]adenosine (which is taken up and converted to [3H]ATP) (Su et al., 1971). Later, Langer and Pinto (1976) suggested that the substantial residual NANC response of the cat nictitating membrane observed after depletion of NA by reserpine was due to the release of ATP remaining in the sympathetic nerves. Most of the early studies of sympathetic cotransmision, involving NA and ATP, were made by Dave Westfall and colleagues on the vas deferens, a tissue with a high density of sympathetic nerves (Westfall et al., 1978; Fedan et al., 1981). Subsequently, numerous studies demonstrated that cotransmission of NA and ATP also occurs in other visceral organs and many different blood vessels in a variety of species (see Burnstock, 1990a). In retrospect, there was a good indication that the excitatory junction potentials (EJPs) recorded in the guinea pig vas deferens when the electrophysiology of sympathetic nervesmooth muscle transmission was first described by Burnstock and Holman (1960) (Fig. 3.1A) were due to ATP released as a cotransmitter from sympathetic nerves, rather than to NA. It was puzzling at the time that adrenoceptor antagonists failed to block the EJPs, although guanethidine, a drug that prevents the release of sympathetic transmitters, was effective. It was over 20 years later that NANC EJPs were shown to be blocked by desensitization of the ATP (P2) receptors by a,bmethylene ATP (a,b-meATP) (Fig. 3.1B) and were mimicked by ATP (Sneddon and Burnstock, 1984b; Sneddon and Westfall, 1984) (Fig. 3.1C). Later, it was shown that reserpine, which depletes NA in the sympathetic nerves, did not affect the twitch contractions of the vas deferens in response to nerve stimulation or release of ATP (Kirkpatrick and Burnstock, 1987). After destruction of sympathetic nerves with 6-hydroxydopamine, purinergic nerve-mediated responses were abolished, establishing that the ATP was released from sympathetic nerves and not from separate “purinergic” nerves. ATP is costored with NA in small and large vesicles. Differential prejunctional modulation of the release of NA and ATP by various agents has been shown in the vas deferens, perhaps indicating that NA and ATP are stored in different vesicles (Ellis and Burnstock, 1990). ATP has been shown to be a cotransmitter with NA in

COTRANSMISSION IN THE AUTONOMIC NERVOUS SYSTEM

Fig. 3.1. Electrical activity in smooth muscle cells of the guinea pig vas deferens during stimulation of sympathetic nerves. (A) Sucrose gap recording of the activity of smooth muscle cells of the vas deferens in response to nerve stimulation (white dots). The upper trace records the tension, the lower trace electrical activity. Note both facilitation and summation of successive excitatory junction potentials (EJPs) and that at a critical depolarization threshold an action potential is initiated which results in contraction. (Reproduced from Burnstock and Costa, 1975, with permission from Springer.) (B) The effect of a,b-methylene (a,b-meATP) ATP on EJPs recorded from the guinea pig vas deferens (intracellular recording). The control responses to stimulation of the motor nerves at 0.5 Hz are shown on the left. After at least 10 minutes in the continuous presence of a,b-meATP, EJPs were recorded using the same stimulation parameters. The EJPs are clearly reduced in magnitude in the presence of a,b-meATP (3 108 M and 3  107 M). Notice also that in control cells several large spontaneous EJPs were seen, whereas after a,b-meATP no spontaneous EJPs were recorded. The EJPs were virtually abolished with a,b-meATP (3 106 M). (Reproduced from Sneddon and Burnstock, 1984b, with permission from Elsevier.) (C) The effect of NA, ATP and nerve stimulation (EJP) on guinea pig vas deferens recorded using microelectrodes (From Burnstock and Verkhratsky, 2012, with permission from Springer.)

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sympathetic nerves supplying the human vas deferens (Banks et al., 2006). Cotransmission of NA and ATP in perivascular sympathetic nerves supplying the aorta, portal vein, and saphenous, pulmonary, tail, ear, basilar, hepatic, coronary, and mesenteric arteries of different mammalian species has been described (e.g., see Sneddon and Burnstock, 1984a; Kennedy and Burnstock, 1985; Ramme et al., 1987; Warland and Burnstock, 1987; Burnstock, 1990b, 1995, 2009c). Electrophysiological studies have shown that in a number of vessels the electrical response to stimulation of perivascular sympathetic nerves is biphasic; an initial fast, transient depolarization or EJP of the vascular smooth muscle is followed by a slow, prolonged depolarization. The EJP and slow depolarization are mimicked by the effects of ATP and NA, respectively. Considerable variation exists in the proportions of NA and ATP utilized by sympathetic nerves. For example, in guinea pig submucosal arterioles both vasoconstriction and EJPs, evoked in response to electrical stimulation of sympathetic nerves, are mediated exclusively by ATP, with NA assuming the role of a neuromodulator, by acting through prejunctional a2-adrenoceptors to depress transmitter release (Evans and Surprenant, 1992). At the other extreme, in rat renal arteries the purinergic component is relatively small. It has also been noted that the purinergic component is optimal with short bursts of low frequency stimulation, whereas longer durations of higher frequency favor adrenergic transmission. Neuropeptide Y (NPY) has been found to be present in LGV in most sympathetic nerves. The release of NPY, as well as NA and ATP, in response to electrical stimulation of sympathetic nerve terminals is prevented by guanethidine. The major role of NPY in the vasculature, and in the vas deferens, appears to be that of a pre- and/ or postjunctional modulator of sympathetic transmission, since it has little direct postjunctional action or causes contraction only at high concentrations (see Fig. 3.2A). Direct vasoconstrictor actions of NPY have, however, been demonstrated in some vessels. At the prejunctional level, NPY has potent inhibitory effects, reducing the release of NA and ATP from sympathetic nerves. Postjunctionally, NPY generally acts to enhance the actions of sympathetic nerve stimulation, NA and ATP. Although 5-hydroxytryptamine (5-HT) immunofluorescent nerves have been localized in a number of vessels, for the most part 5-HT is not synthesized and stored in separate nerves, but is taken up, stored in, and released as a “false transmitter” from sympathetic nerves (Jackowski et al., 1989). Enkephalins have been shown to coexist with NA in cell bodies and fibers of some postganglionic sympathetic neurons (Schultzberg

26

G. BURNSTOCK

Fig. 3.2. (A) Schematic of sympathetic cotransmission. ATP (adenosine 50 -triphosphate) and NA (noradrenaline) released from small granular vesicles (SGV) act on P2X and a1 receptors on smooth muscle, respectively. ATP acting on inotropic P2X receptors evokes excitatory junction potentials (EJPs), increase in intracellular calcium ([Ca2þ]i) and fast contraction; while occupation of metabotropic a1 adrenoceptors leads to production of inositol triphosphate (IP3), increase in [Ca2þ]i and slow contraction. Neuropeptide Y (NPY) stored in large granular vesicles (LGV) acts after release both as a prejunctional inhibitory modulator of release of ATP and NA and as a postjunctional modulatory potentiator of the actions of ATP and NA. Soluble nucleotidases are released from nerve varicosities, and are also present as ectonucleotidases. (Reproduced from Burnstock, 2009e, with permission from Elsevier.) (B) A classic transmitter (ACh) coexists with vasoactive intestinal polypeptide (VIP) in parasympathetic nerves supplying the cat salivary gland. ACh and VIP are stored in separate vesicles; they can be released differentially at different stimulation frequencies to act on acinar cells and glandular blood vessels. ACh is released during low frequency stimulation to increase salivary secretion from acinar cells and to elicit some minor dilatation of blood vessels in the gland. At high stimulation frequencies, VIP is released to produce marked dilatation of the blood vessels in the gland and to act as a neuromodulator, postjunctionally on the acinar gland to enhance the actions of ACh, and prejunctionally on the nerve varicosities to enhance the release of ACh. ACh also has an inhibitory action on the release of VIP. (Reproduced from Burnstock, 1983, with permission from Elsevier.) (C). Diagram showing the basis of the “axon reflex” in the skin leading to vasodilatation and inflammation. It is suggested that calcitonin generelated peptide (CGRP), substance P (SP), and ATP are released during antidromic activation of sensory collatorals. (Adapted from Burnstock, 1977, with permission of the Nature Publishing Group.)

COTRANSMISSION IN THE AUTONOMIC NERVOUS SYSTEM et al., 1979). The functional significance of sympathetic coexistence of opioids is likely to be related to their prejunctional inhibitory effects on sympathetic transmission.

PARASYMPATHETIC NERVES Evidence was presented for seasonal changes in release of ACh, 5-HT, histamine, and a peptide from vagus nerves supplying the frog stomach (Singh, 1964). The evidence for cotransmission of ACh and vasoactive intestinal polypeptide (VIP) in certain postganglionic parasympathetic neurons comes from pharmacological studies performed by Lundberg (1981) on cat salivary glands (see Fig. 3.2B). ACh and VIP are released from the same parasympathetic nerve terminals in response to transmural nerve stimulation. During low-frequency stimulation, ACh is released to cause an increase in salivary secretion from acinar cells and also to elicit some minor dilatation of blood vessels in the gland. VIP is preferentially released at high frequencies to cause marked vasodilatation of blood vessels and, while it has no direct effect on acinar cells, it acts as a neuromodulator to enhance both the postjunctional effect of ACh on acinar cell secretion and the release of ACh from nerve varicosities via prejunctional receptors. Vasodilator nerves to the uterine arteries in the guinea pig contain immunoreactivity to VIP, which coexists with dynorphin, NPY, and somatostatin (Morris et al., 1987). NPY-like immunoreactivity has been reported in some of the choline acetyltransferase-/VIP-containing neurons of the parasympathetic ciliary, sphenopalatine, otic, and pterygopalatine ganglia with targets including the iris and cerebral vessels (Leblanc and Landis, 1988). Autonomic control of penile erection, involving relaxation of the smooth muscle of the corpus cavernosum as well as dilatation of other penile vascular beds, has traditionally been attributed to the vasodilator effects of ACh and VIP released from parasympathetic neurons. Nitric oxide (NO) released from nerves arising from neurons in the pelvic ganglia, have been claimed to play a role in smooth muscle relaxation leading to penile erection (Burnett et al., 1992). NO synthase (NOS)-containing fibers, shown by lesion studies to arise from parasympathetic cell bodies in the sphenopalatine ganglia, have been localized in the adventitia of cerebral arteries and many of these also contain VIP (Bredt and Snyder, 1992). A functional role for perivascular neuronal NO in cerebral arteries has been identified in studies showing that stimulation of adventitial nerve fibers causes vascular relaxation, which is attenuated by inhibitors of NOS (Toda et al., 1990). Parasympathetic nerves supplying the urinary bladder utilize ACh and ATP as cotransmitters (Burnstock et al., 1972, 1978; Burnstock, 2001b), in variable proportions in

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different species, and by analogy with sympathetic nerves, ATP again acts through P2X ionotropic receptors to produce EJPs and fast contraction, while the slow component of the response is mediated by a metabotropic receptor, in this case muscarinic. There is also evidence for parasympathetic, purinergic cotransmission to resistance vessels in the heart and airways.

SENSORY-MOTOR NERVES The neuropeptides substance P (SP) and calcitonin generelated peptide (CGRP) are the principal transmitters of primary afferent nerves and have been shown to coexist in the same terminals (Gibbins et al., 1985; Rubino and Burnstock, 1996). Furthermore, with the use of colloidal gold particles of different sizes, they have been shown to coexist in the same large granular vesicles (Gulbenkian et al., 1986). The motor (efferent) function of sensory nerves has been demonstrated in rat mesenteric arteries where evidence exists for a role for CGRP as the mediator of vasodilatation following release from sensory motor nerves. In contrast, SP is not coreleased with CGRP by electrical stimulation and SP has little or no vasodilator action on rat mesenteric arteries. While it is possible that SP released from nerves supplying the microvasculature could produce vasodilatation via SP receptors on endothelial cells, it is most unlikely to reach the endothelium without degradation in larger blood vessels. Other peptide and nonpeptide substances, including neurokinin A, somatostatin, VIP, and ATP, have been described in capsaicin-sensitive sensory neurons (Maggi and Meli, 1988). Unmyelinated sensory neurons containing cholecystokinin (CCK)/CGRP/dynorphin/SP have been shown to project to cutaneous arterioles in guinea pig skin (Gibbins et al., 1987). Neurons from the same ganglia which contain CCK/CGRP/SP innervate arterioles of skeletal muscle, CGRP/dynorphin/SP nerve fibers mostly supply the pelvic viscera, and CGRP/SP fibers run mainly to the heart, large arteries, and veins. There is also evidence for a sensory role for ATP and it has been proposed that ATP may coexist in sensory nerve terminals with SP and CGRP, and with glutamate in primary afferent neurons (Holton, 1959; Burnstock, 2009d) (Fig. 3.2C).

INTRINSIC ENTERIC AND CARDIAC NEURONS Intrinsic neurons exist in most of the major organs of the body. Many of these are part of the parasympathetic nervous system, but certainly in the gut, and perhaps also in the heart and airways, some of these intrinsic neurons are derived from neural crest tissue, which differs from that which forms the sympathetic and parasympathetic systems, and appear to participate in local reflex actions independent of the CNS.

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G. BURNSTOCK

The enteric nervous system contains several hundred million neurons located in the myenteric plexuses between muscle coats and the submucous plexus. The chemical coding of these nerves has been examined in detail (see Furness, 2006). A subpopulation of these intramural enteric nerves provides NANC inhibitory innervation of the gastrointestinal smooth muscle. It seems likely that three major cotransmitters are released from these nerves. ATP produces fast inhibitory junction potentials, NO also produces inhibitory junction potentials but with a slower time course, while VIP produces slow tonic relaxations (see Burnstock, 2001a, 2008). The proportions of these three transmitters varies considerably in different regions of the gut and in different species; for example, in some sphincters the NANC inhibitory nerves utilize largely VIP, in others largely NO, while in nonsphincteric regions of the intestine ATP is more prominent. In recent papers, evidence has suggested that ACh and ATP are fast excitatory cotransmitters to myenteric neurons and that there may be colocalization of ACh, ATP, and serotonin in enteric Dogiel type I/S neurons. Detailed studies have allowed a very complete mapping of the complex neuronal markers and projections of enteric neurons. Several peptidergic substances, including NPY, VIP, enkephalin, somatostatin, peptide histidine isoleucine, galanin, SP, and CGRP, have been identified in enteric neurons, often coexisting (up to five peptides in the same neuron) with the neurotransmitters NA, ACh, 5-HT, NO, and ATP (see Table 3.2) (Furness and Costa, 1987). Studies of intrinsic cardiac neurons in culture have shown that subpopulations of intrinsic nerves in the atrial and intra-atrial septum contain and/or release cotransmitters, including ATP, NO, NPY, ACh, and 5-HT (Hassall and Burnstock, 1986; Burnstock et al., 1987). Many of these nerves project to the coronary microvasculature and produce potent vasomotor actions (Saffrey et al., 1992). NO and ATP have been shown to be the mediators of NANC vasodilatation of the rabbit portal vein.

and parasympathetic cotransmission, release of ATP is favored at low frequency stimulation, whereas NA and ACh are released at higher frequencies (Bradley et al., 2003; Ventura et al., 2003). There are instances where more than one fast cotransmitter is released (e.g., glutamate and ATP) together with one or more peptides.

PHYSIOLOGICAL SIGNIFICANCE OF COTRANSMISSION

Synergism

In general, autonomic cotransmission offers more diverse physiological control by mechanisms other than the all-or-none control by messages coming from the CNS that was the dominant view for many years (see Burnstock, 2004, 2009a) (Fig. 3.3).

Cotransmitters with different firing patterns Although single presynaptic action potentials release small molecule neurotransmitters, trains of impulses are needed to release neuropeptides. For sympathetic

Different cotransmitters act on different postjunctional cells Neurons using multiple transmitters may project to two or more targets. For example, ACh released at low frequency stimulation from parasympathetic nerves supplying salivary glands acts on acinus cells to produce secretion and a minor dilatation of vessels, whereas, at higher frequency stimulation, its cotransmitter VIP causes powerful vasodilatation of vessels in the glands and postjunctional enhancement of ACh-induced saliva secretion (Lundberg, 1996).

Neuromodulation A cotransmitter can feed back on presynaptic receptors that increase or decrease its own release and/or that of its cotransmitter(s) (see Vizi, 1979). For example, ATP released as a cotransmitter with glutamate from primary afferent fibers in lamina II of the spinal cord can act on prejunctional P2X3 receptors to facilitate the release of its cotransmitter, glutamate, whereas adenosine resulting from ectoenzymatic breakdown of ATP acts on presynaptic P1 receptors to inhibit glutamate release. Both NA and ATP can prejunctionally modulate sympathetic transmission, NA via prejunctional a2-adrenoceptors and ATP via P1 receptors following breakdown to adenosine or directly via P2X and P2Y receptors (see Burnstock, 2007). Modulation of cotransmitter release and presynaptic action by other local agents also occurs and might provide another level of synaptic flexibility.

There is an increasing number of reports of the synergistic actions of cotransmitters. ATP and NA released from sympathetic nerves have synergistic actions on smooth muscle of vas deferens and blood vessels, and ATP released with ACh from motor neurons facilitates the nicotinic actions of ACh at the skeletal neuromuscular junction. The mechanisms underlying cotransmitter synergism are not well understood. However, it has been suggested that postjunctional synergism between the responses of vas deferens to NA and ATP is caused by the ability of NA to potentiate the contractile responses to ATP by sensitizing smooth muscle cells

COTRANSMISSION IN THE AUTONOMIC NERVOUS SYSTEM

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Table 3.2 Types of neurons in the enteric nervous system{ {

Myenteric neurons Excitatory circular muscle motor neurons Inhibitory circular muscle motor neurons

Excitatory longitudinal muscle motor neurons Inhibitory longitudinal muscle motor neurons Ascending interneurons (local reflex) Descending interneurons (local reflex) Descending interneurons (secretomotor reflex) Descending interneurons (migrating myoelectric complex) Myenteric intrinsic primary afferent (primary sensory) neurons Intestinofugal neurons *Motor neurons to gut endocrine cells

Submucosal neurons Noncholinergic secretomotor/ vasodilator neurons Cholinergic secretomotor/ vasodilator neurons

Proportion

Chemical coding

Function/comments

12%

To all regions, primary transmitter ACh, cotransmitter TK Several cotransmitters with varying prominence: NO, ATP, VIP, PACAP

25%

Short: ChAT/TK/ENK/GABA Long: ChAT/TK/ENK/NFP Short: NOS/VIP/PACAP/ENK/ NPY/GABA Long: NOS/VIP/PACAP/ Dynorphin/BN/NFP ChAT/Calretinin/TK

2%

NOS/VIP/GABA

5% 5%

ChAT/Calretinin/TK ChAT/NOS/VIP  BN  NPY

2%

ChAT/5-HT

4%

ChAT/SOM

26% <1% N/A

ChAT/Calbindin/TK/NK3 receptor ChAT/BN/VIP/CCK/ENK N/A

45%

VIP/GAL

15%

ChAT/Calretinin/Dynorphin

16%

Primary transmitter ACh, cotransmitter TK Several cotransmitters with varying prominence: NO, ATP, VIP, PACAP Primary transmitter ACh Primary transmitter ACh, ATP may be a cotransmitter Primary transmitter ACh, 5-HT (at NK3 receptors) Primary transmitter ACh Primary transmitter TK Primary transmitter ACh For example, myenteric neurons innervating gastrin cells. Neurons of this type may be in submucosal ganglia Primary transmitter VIP. A small proportion of these have cell bodies in myenteric ganglia Primary transmitter ACh

{ This table lists the neuron types that are found in the guinea-pig small intestine, some of their defining characteristics, and percentages of occurrence in each of the ganglionated plexuses. Also listed are three types of motor neuron that are found in other parts of the tubular digestive tract, marked by an asterisk*. ACh, acetylcholine; ATP, adenosine 50 -triphosphate; BN, bombesin; CCK, cholecystokinin; CGRP, calcitonin generelated peptide; ChAT, choline acetyltransferase; DA, dopamine; ENK, enkephalin; GABA, g-amino butyric acid; GAL, galanin; 5-HT, 5-hydroxytryptamine; NA, noradrenaline; NFP, neurofilament protein; NK, neurokinin; NOS, nitric oxide synthase; NPY, neuropeptide Y; PACAP, pituitary adenylyl cyclase-activating peptide; SOM, somatostatin; SP, substance P; TK, tachykinin; VIP, vasoactive intestinal peptide. { Reproduced from Furness, 2000, with permission from Elsevier.

to Ca2þ via an inhibitory action on myosin light chain phosphatase, an action mediated by protein kinase C (Smith and Burnstock, 2004).

Negative cross-talk Coapplication of nicotinic and P2X receptor agonists produces less than the additive responses predicted by independent receptor activation. Inhibitory interactions between 5-HT3 and P2X receptors have been described in submucosal and myenteric neurons (Barajas-Lo´pez et al., 2002; Boue´-Grabot et al., 2003).

Trophic factors Some costored and coreleased substances can act as long-term (trophic) factors, as well as fast neurotransmitters. For example, ATP can act on P2 receptors, or P1 (adenosine) receptors after ectoenzymatic breakdown, to promote vascular cell proliferation, motility, differentiation or death (see Burnstock, 2002; Burnstock and Verkhratsky, 2010). NPY released from sympathetic nerves has cardiovascular trophic effects in end-stage renal disease. There is growing evidence that neuropeptide trophic factors are synthesized, stored and released from nerve terminals together with fast neurotransmitters.

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G. BURNSTOCK Fast and slow cotransmitters

C1

C2

low stimulation frequency

C1

C2

R1

R2

high stimulation frequency

R1

A

Fast cotransmitters

R2

B Presynaptic neuromodulation of cotransmitter release

Cotransmitters act on different postjunctional cells

C1

C2

+ C1 + – – C2

Excitatory and inhibitory cotransmitters Although cotransmitters generally have similar actions on postjunctional cells, there are some examples of cotransmitters having opposite actions. For example, in the mammalian uterus, one or other cotransmitter dominates depending on the hormonal and/or tonic status of the postjunctional muscle cells. Brain-derived neurotrophic factor (BDNF) increases the release of ACh and reduces NA release from sympathetic nerves to cause a rapid shift from excitatory to inhibitory transmission (Yang et al., 2002; Landis, 2002).

False cotransmitters R1

R2

C

D Synergism

C1

C2

R1

R2

F

Cotransmitters and trophic factors

C1

R1

R2

Excitatory and inhibitory cotransmitters

C2

C1

For example, it has been known for some time that sympathetic nerves take up 5-HT, which can then be released as a “false transmitter,” rather than a genuine “cotransmitter.” A “false transmitter” is a substance actively taken up and subsequently released by a neuron that does not synthesize it.

Coexisting peptides acting as neuromodulators

– –

+ +

E

R2

Negative cross-talk

C2

C1

R1

C2

In general, most neuropeptides act as neuromodulators rather than neurotransmitters (Milner and Burnstock, 1994). For example, NPY released from sympathetic nerves acts as a prejunctional inhibitor of release of ATP and NA and a postjunctional potentiator of the actions of ATP and NA (Ellis and Burnstock, 1990).

– high tone R2

G

R1 neurotransmission

+ R1

R2 trophic events

H

False cotransmitters

C1 C2

low tone

Coexisting peptide acts as a neuromodulator

C3 C1 C2 – –

C3

uptake of C3

R1

I

R2

+

R3

J

R1

+ R2

Fig. 3.3. Spectrum of signaling variations offered by cotransmission (black arrows ¼ neurotransmission; gray arrows ¼ pre- or postjunctional neuromodulation). (A) Fast transmission is usually produced by small molecules (C1) released at low frequency nerve stimulation acting on ionotropic receptors (R1), whereas slow transmission is usually produced by release of peptides (C2) or other molecules at high frequency stimulation acting on G protein-coupled receptors (R2). (B) Cotransmitters C1 and C2 can both be fast messengers acting via ionotropic receptors (R1 and R2). (C) Cotransmitters C1 and C2 act on receptors (R1 and R2) localized on different postjunctional cells. (D) Cotransmitters C1 and C2 not only act

postjunctionally via R1 and R2 receptors but can also act as prejunctional modulators to either inhibit () or enhance (þ) the release of C1 and/or C2. (E) Cotransmitters C1 and C2 act synergistically to enhance the combined responses produced via R1 and R2 receptors. (F) Cotransmitters C1 and C2 act to inhibit the responses evoked via R1 and/or R2 receptors. (G) Cotransmitter C1 evokes neurotransmission via R1 receptors, while C2 evokes long-term (trophic) responses of postjunctional cells via R2 receptors. (H) Cotransmitter C1 produces excitation via R1 receptors when the postjunctional smooth muscle target has low tone, with C2 having little influence; however, when the smooth muscle tone is high, the dominant response might be relaxation produced by C2 via R2 receptors. (I) Substance C3 is taken up by nerve terminals, rather than being synthesized and stored as is true for the cotransmitters C1 and C2. C3 can then be released on nerve stimulation to act on postjunctional R3 receptors. In these circumstances, C3 would be known as a “false transmitter.” (J) A coexisting substance C3 (often a peptide) can be synthesized and stored in a nerve, but not act directly via a postjunctional receptor to produce changes in postjunctional cell activity. It could, however, act as a prejunctional inhibitor () of the release of the cotransmitters C1 and C2, or as a postjunctional enhancer (þ) of the responses mediated by R1 and R2. (Reproduced from Burnstock, 2004, with permission from Elsevier.)

COTRANSMISSION IN THE AUTONOMIC NERVOUS SYSTEM 31 outflow obstruction, there is a substantial increase in COTRANSMITTER PLASTICITY ATP as the cotransmitter with ACh in parasympathetic Neurons possess the genetic potential to produce many nerves supplying the human bladder (Palea et al., 1993; neurotransmitters. The particular combination and quanSmith and Chapple, 1994; O’Reilly et al., 2001; tity that results is partly preprogrammed and partly deterAndersson and Hedlund, 2002), and there is increase mined by “trophic” factors that trigger the expression or in ATP as a cotransmitter with NA in sympathetic nerves suppression of the appropriate genetic machinery. A numin spontaneous hypertensive rats (Vidal et al., 1986). ber of studies have demonstrated plasticity of expression of autonomic nerves during development and aging, folCONCLUDING COMMENTS lowing trauma or surgery, after chronic exposure to drugs and in disease (Burnstock 1990c, 2006; Burnstock and A compelling body of evidence is now available to supMilner, 1999). NA and NPY show different expression port the cotransmitter hypothesis, which implies more sophisticated local control mechanisms than were envisin cerebrovascular nerves during development. Whereas aged in earlier times. However, a number of issues still there is a decrease in expression of vasoconstrictor cereneed to be resolved, including: brovascular (NA and 5-HT) in aging rats, there is an increase in vasodilator neurotransmitters (VIP and CGRP) 1. resolution of the different roles of coexisting sub(Mione et al., 1988). BDNF was claimed to switch sympastances (including neurotransmitter, pre- and postthetic neurotransmission to the heart from an adrenergic junctional neuromodulator and/or trophic roles) excitation to cholinergic inhibition. under different physiological conditions and patIn patients with bladder areflexia following lower motor terns of discharge in the nerves spinal lesion there is increased innervation by VIP, NPY, 2. identification of different transmitter substances in and NA-containing nerve fibers to the striated muscle of vesicular storage sites in nerve terminals at the ultrathe intrinsic external urethral sphincter, which may indicate structural level, perhaps utilizing colloidal gold para regulatory mechanism via the intrinsic ganglia and/or ticle techniques the somatic nervous system to help overcome this type 3. studies of the molecular mechanisms that are conof bladder dysfunction. Surgical ganglionectomy leads cerned with the regulation of transmitter and recepto increased SP levels in the iris and ciliary body, increased tor expression during development (including old CGRP in pial vessels, and increased expression of NPY in age) and during the compensatory changes that parasympathetic neurons supplying cerebral vessels occur in nerves in the adult, which remain following (Mione et al., 1990). Long-term guanethidine sympathectrauma, surgery and disease. tomy of both neonate and adult rats results in a drastic increase in the innervation of tissues by the sensory neuroIn view of the growing number of examples of the plaspeptide, CGRP, probably due to increased availability to ticity of the nervous system in the adult in aging and disnerve growth factor for which sensory and sympathetic ease, neuropathologists should consider increases in neurons compete (Aberdeen et al., 1992). After extrinsic expression of transmitters and density of nerves as well denervation of human respiratory tract by heart–lung as the more traditional approach of looking for degrees transplantation, the intrinsic parasympathetic neurons that of damage in loss of nerves. In terms of drug developpersist express an NA-synthesizing enzyme and NPY, subment, since the expression of cotransmitters and receptors stances normally found in sympathetic nerves. changes so markedly with age, sex, and disease states, we During the course of experimentally induced diabetes cannot assume that drugs tested on young healthy male there are marked changes in the expression of neurovolunteers will be appropriate for, say, a man of 80; thus transmitters/neuromodulators in nerves supplying the the age, sex, and pathological history of individual patients bowel. While there are degenerative changes in VIP will need to be taken into account in prescribing therapeuand NA-containing nerves early on in the development tic strategies. Another possibility, although clearly not of the disease, the expression of 5-HT, SP, and CGRP popular, is that the use of drug cocktails designed to match in nerve fibers changes at different times during the the coding of neurons projecting to a damaged area may progression of the disease. Reduction in the expression be a more efficient strategy than using a single compound of VIP and 5-HT, but not NPY and NA, has been with widespread actions and then trying to find a dosage or demonstrated in perivascular nerves supplying the cereprocedure to avoid unwanted side-effects. bral blood vessels of 8-week streptozotocin-induced diaIt has been particularly difficult to establish cotransbetic rats (Lagnado et al., 1987). Changes in chemical mitter roles for the many peptides found in nerves, parcoding of myenteric neurons in ulcerative colitis have ticularly in the enteric nervous system, where up to five been reported, with a shift from cholinergic to more neuropeptides are found in some neurons, partly SP-mediated cotransmission. In interstitial cystitis and because specific receptors and physiological roles have

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not been established for some of these and partly because of the lack of selective antagonists. It will be important to distinguish between neuromodulator, neurotransmitter and neurotrophic roles for released peptides or indeed for as yet unrecognized roles. It has become clear that ATP is a primitive signaling molecule that has been retained as a cotransmitter in every nerve type in both the peripheral and central nervous systems, although the relative role of ATP varies considerably in different species and pathophysiological conditions. ATP appears to become a more prominent cotransmitter in stress and inflammatory conditions. Most nerves contain and release ATP as a fast cotransmitter together with classical transmitters such as ACh, NA, dopamine, glutamate, GABA, and one or more peptides. Now that cotransmission is recognized as a universal mechanism, it is recommended that the terms “adrenergic,” “cholinergic,” “peptidergic,” “purinergic,” “aminergic,” and “nitrergic” should not be used when nerves are described, although adrenergic, cholinergic, peptidergic, purinergic, aminergic, or nitrergic transmission is still meaningful.

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