Jackie D. Wood, PhD, Department of Physiology and Cell Biology, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210–1218, USA. Tel.: (614) 292 5449; fax: (614) 292 4888; e-mail: email@example.com
Metabotropic mechanisms of excitatory signalling in enteric neurones underlie both slow synaptic transmission and paracrine transmission from enteric non-neuronal cells. The type of neurone in which signalling occurs determines the characteristics of synaptic- and paracrine-mediated slow excitatory responses. Slow excitatory responses in neurones with AH-type electrophysiological behaviour and multipolar Dogiel type II morphology are characterized by membrane depolarization associated with closure of Ca2+-gated K+ channels that is reflected by increased neuronal input resistance. Slow excitatory responses in neurones with S-type electrophysiological behaviour and uniaxonal morphology are characterized by membrane depolarization associated with opening of cationic channels and decreased neuronal input resistance. Postreceptor signalling that involves activation of adenylate cyclase, stimulation of cAMP formation and activation protein kinase A generates excitatory responses characterized by increased neuronal input resistance in AH neurones. Postreceptor signalling that involves activation of phospholipase C, release of IP3 and diacylglycerol and activation of protein kinase C and calmodulin kinases generates excitatory responses characterized by decreased neuronal input resistance in S neurones. Slow excitatory responses that are characterized by increased neuronal input resistance are a property of AH-type neurones that function as interneurones in the neural networks of the ENS. Slow excitatory responses that are characterized by decreased neuronal input resistance are a property of S-type neurones that function either as interneurones or as musculomotor and secretomotor neurones in the neural networks of the ENS.
Neurocrine, endocrine and paracrine are the primary forms of chemical signalling to the neuronal elements of the enteric nervous system (ENS). Neurocrine signalling refers to chemical transmission from neurone to neurone (i.e. synaptic transmission) or from neurones to muscles, glands and blood vessels (i.e. neuroeffector transmission). Endocrine signals are hormones transported into the vicinity of the neurones by the blood. Paracrine signals are chemical substances that reach the neurones by diffusion in the extracellular milieu following release by non-neuronal neighbouring cells in proximity to the neurones.
The fundamental mechanisms for neurocrine transmission in the ENS are the same as elsewhere in the nervous system. Neurotransmitters are released by Ca2+-triggered exocytosis from stores localized in vesicles at axonal terminals or axonal varicosities. Release is triggered by the depolarization phase of action potentials when they arrive at the release site and open voltage-activated Ca2+ channels. Once released, enteric neurotransmitters bind to their specific postsynaptic receptors to evoke ionotropic or metabotropic synaptic events. When the receptors are coupled directly to the ionic channel, they are classified as ionotropic. They are metabotropic receptors when their effects to open or close ionic channels are mediated indirectly by guanosine triphosphate (GTP) binding proteins and the induction of cytoplasmic second messengers (e.g. cyclic adenosine monophosphate, inositol trisphosphate and diacylglycerol).
Excitatory postsynaptic potentials (EPSPs), inhibitory postsynaptic potentials (IPSPs) and presynaptic inhibition and facilitation are the principal synaptic events in the ENS of the guinea-pig small and large intestine, which serves as the primary model for the cellular neurophysiology of enteric neurones. Any enteric neurone might express mechanisms for both slow and fast forms of synaptic neurotransmission. Fast synaptic potentials have durations in the millisecond range; slow synaptic potentials last for several seconds, minutes or hours. Fast synaptic potentials are usually EPSPs and are mediated by ionotropic receptors. Slow synaptic events in the ENS may be either EPSPs or IPSPs and are mediated by metabotropic receptors.
Enterochromaffin cells, lymphocytes, macrophages, polymorphonuclear leucocytes and mast cells are sources of paracrine signals to the ENS. Signalling from enteric mast cells and serotonin-containing enterochromaffin cells to the ENS has been the focus of multiple studies.1–6 The actions of most paracrine signal substances in the ENS are mediated by metabotropic receptors. Paracrine signal substances mimic slow synaptic excitation when applied to ENS neurones in vitro or when released in vivo during degranulation of enteric mast cells in antigen-sensitized animal models.,1,2,6
Slow synaptic excitation
Focal electrical stimulation applied experimentally to interganglionic fibre tracts or to the surfaces of ganglia in the myenteric or submucosal plexus evokes slow EPSPs in ENS neurones with AH-type electrophysiological behaviour and multipolar–Dogiel type II morphology and neurones with S type electrophysiological behaviour and uniaxonal morphology. Neurones with slow EPSPs are found in the small and large intestine and gastric antrum, but not the gastric corpus, gallbladder or pancreas.7–14 Slow EPSPs appear to be restricted to the enteric microcircuits of specialized digestive compartments where propulsive motility is a significant function.
Slowly activating membrane depolarization continuing for several seconds to minutes, and sometimes hours, after termination of release of the neurotransmitter from the presynaptic terminal underlies the slow EPSP (Fig. 1). Enhanced excitability reflected by prolonged trains of action potentials is the hallmark of the event. Enhanced excitability during the slow EPSP is apparent experimentally as repetitive spike discharge during depolarizing current pulses in both AH- and S-type neurones and as anodal break excitation at the offset of hyperpolarizing current pulses in AH neurones. AH neurones, that will not discharge at all or will fire only a single spike at the beginning of a depolarizing current pulse in the inactivated state and which show no anodal break excitation, will fire repetitively in response to depolarizing pulses and discharge at the offset hyperpolarizing pulses when the slow EPSP is in effect. When activated by either slow synaptic or paracrine inputs, electrophysiological behaviour of AH neurones is very similar to S neurones and may be confused as such, if the AH neurones happen to be in an activated state due to ongoing release of the transmitter or a paracrine mediator (e.g. inflammatory/immune mediators). The characteristic postspike hyperpolarization in AH neurones is suppressed or abolished during slow EPSPs. Suppression of the after-hyperpolarization is part of the mechanism that permits repetitive spike discharge at increased frequencies during the enhanced state of excitability.
Slow EPSPs differ for neurones with AH-type electrophysiological behaviour and multipolar Dogiel type II morphology and neurones with S-type electrophysiological behaviour and uniaxonal morphology (Fig. 2). Stimulus-evoked slow EPSPs and responses to paracrine mediators in AH neurones are generally associated with an increase in neuronal input resistance that reflects closure of ionic channels and decreased membrane ionic conductance. In S neurones, a large proportion of which are musculomotor or secretomotor neurones, stimulus-evoked slow EPSPs and responses to paracrine mediators are associated with a decrease in neuronal input resistance that reflects opening of ionic channels and increased membrane ionic conductance.
Ionic mechanisms in ah neurones
Conductance changes in several kinds of ionic channels underlie the slow EPSPs in AH neurones. The depolarizing phase occurs when Ca2+ channels that are normally open at rest are closed.15–17 Closure of the Ca2+ channels lowers intraneuronal Ca2+ which, in turn, leads to closure of Ca2+-activated K+ channels. Closure of the K+ channels accounts for the increased input resistance seen in the neurone during the depolarization phase of the slow EPSP. Changes in input resistance are often biphasic, with an increase followed by a decrease as the depolarization progresses. Elevated Cl– conductance accounts for the second phase of decreased input resistance. Bertrand & Galligan18 estimated that 90% of the conductance change during slow EPSPs and the EPSP-like responses to an NK-3 agonist was accounted for by closure of Ca2+-activated K+ channels and the remaining 10% by increased Cl– conductance. Chloride channels also contribute to the conductance changes associated with histamine-evoked slow EPSP-like depolarizing responses.19
Inward current in N-type Ca2+ channels during the rising phase of the action potential, which in the resting state would inject Ca2+ and lead to postspike increase in Ca2+-activated K+ conductance, is suppressed during slow excitatory responses in AH neurones.20,21 This accounts for suppression of postspike hyperpolarization (i.e. the AH) during the response. Enhanced excitability, seen as repetitive spike discharge and anodal break excitation during stimulus-evoked EPSPs and during exposure to paracrine mediators, is related to suppression of A-type and delayed rectifier K+ currents.22
Ionic mechanisms in s neurones
The ionic mechanism for the depolarization phase of slow EPSPs in S-type neurones is opening of cationic conductance channels and is opposite to the mechanism in AH-type neurones. Elevated Na+ and Ca2+ conductance accounts for most of the depolarization phase and decreased input resistance seen during the EPSP and paracrine-evoked excitatory responses in S neurones.23,24
Mediators of slow epsps
Several messenger substances found in neurones, endocrine or immune cells mimic slow EPSPs when applied experimentally to enteric neurones. Receptors for more than one of the messenger substances may be present on the same neurone. Table 1 is a partial listing of the substances together with receptor subtypes.
Substance P, 5-hydroxytryptamine (5-HT), adenosine triphosphate (ATP) and acetylcholine fulfil criteria for function as a neurotransmitter for metabotropic slow EPSPs in enteric neurones. Other substances are implicated mainly by their presence in enteric neurones and/or by mimicry of the slow EPSP when applied experimentally. Receptors for these substances, as well as combinations of receptors for some of the substances listed in Table 1, can be colocalized on the same enteric neuronal cell body.
Histamine, interleukin-1β and mast cell proteases are examples of slow EPSP mimetics of paracrine origin. Histamine and interleukin-1β are released from intestinal mast cells to become neuromodulatory signals that are decoded by the enteric microcircuits. The evidence for 5-HT is as complete as for any known neurotransmitter, including synthesis, storage and release from enteric neurones and the activity of reuptake transporters for termination of action.25 Nevertheless, the strongest evidence is that agents such as N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, renzapride and anti-idiotypic antibodies to 5-HT block both the slow EPSP-like actions of 5-HT and the slow EPSP in the same AH neurones.25,26 Aside from its role as a putative enteric neurotransmitter, 5-HT also has a paracrine signalling function. A large fraction of the body's 5-HT is stored in enterochromaffin cells interspersed in the intestinal epithelial lining. Mechanical stimulation (e.g. shearing forces) at the mucosal surface or noxious stimulation (e.g. stimulant laxatives like sennosides) releases the 5-HT, which may then reach receptors on AH neuronal projections in the mucosa, other enteric neural elements and spinal and vagal sensory afferents.3,4,25,27
Bornstein et al.28 reviewed five criteria for transmitter function that were fulfilled by substance P: (i) pharmacological evidence suggests the intramural release of substance P within intestinal segments; (ii) the K+ conductance decrease produced by substance P and the slow EPSP is the same; (iii) chymotrypsin, which digests substance P, reduces both the response to the peptide and the slow EPSP; (iv) the widespread occurrence of slow EPSPs implies that terminals of the responsible axons synapse with most cell bodies in the ganglion, as is the case for the multipolar (AH-Dogiel type II) neurones that contain substance P; and (v) slow EPSPs were evoked in myenteric neurones with ganglia that contained substance P, but no immunocytochemically demonstrable 5-HT containing fibres. Slow EPSPs can be initiated by substance P released from AH neurones or from collateral projections of spinal afferents inside the intestinal wall.29
Neurokinin-3 receptors are implicated as mediators of the action of substance P.28,30 The slow EPSP-like action of 5-HT is related to the 5-HP1P serotonergic receptor, so named by Gershon and his coworkers.31 Slow EPSP mimetic action of acetylcholine is mediated by the M1 muscarinic receptor subtype.32,33 The action of histamine occurs at the H2 receptor subtype and protease-activated receptors (PARs) PAR-1, -2 and -4 mediate the slow EPSP-like responses evoked by mast cell proteases.23,34,35
Motilin is a slow EPSP mimetic of particular interest because it is implicated as an endocrine messenger in the initiation of the intestinal migrating motor complex in the interdigestive period.36 Additional interest emerges from findings that macrolide antibiotics (e.g. erythromycin) might act at motilin receptors to facilitate gastric emptying in disorders such as diabetic gastroparesis.37 Slow EPSP-like actions of motilin are prominent in AH neurones of the gastric antrum.38
Metabotropic signal transduction
Slow activation and the prolonged time course of slow EPSPs are clues to the mechanism of signal transduction. A 30-millisec experimental ‘puff’ of a slow EPSP mimetic from a micropressure ejection pipette or electrical stimulation of synaptic inputs for the slow EPSP evokes changes in excitability that last for several minutes.7,39 This happens in experiments where the exposure is limited to the 30-millisec duration of the ‘puff’. The slow EPSP mimetics act transiently at localized receptor sites on the somal membrane, while closure of Ca2+-activated K+ channels in AH neurones and opening of cation channels in S neurones occurs globally. Global activation in the whole cell suggests involvement of intraneuronal second messenger systems that connect localized surface receptors (i.e. metabotropic receptors) to biochemical processes in the neuronal interior and ultimately to globally distributed ion channels. Receptor occupancy by messenger substances in these cases stimulate intraneuronal synthesis or release of a second messenger, which in turn initiates the neurochemical reactions and molecular conformational changes responsible for transformation of the somal membrane from hypo- to hyperexcitability.
Metabotropic transduction in ah neurones
Several lines of evidence point to receptor mediated activation of adenylate cyclase, elevation of intraneuronal cyclic adenosine 3′, 5′- phosphate (cAMP) and activation of protein kinase A (PKA) as steps in the signal transduction process for the slow EPSP in AH neurones. Forskolin, a substance that directly activates adenylate cyclase, is a useful tool for the study of signal transduction in AH neurones. Application of forskolin elevates cAMP in enteric ganglia40 and mimics the slow EPSP in AH neurones, but has no effect on excitability in S-Type neurones.41 Similarly, other treatments that elevate cAMP, such as intraneuronal injection of cAMP, application of membrane permeant analogues of cAMP and treatment with phosphodiesterase inhibitors each evoke slow EPSP-like behaviour in AH neurones.42
Treatment of guinea-pig myenteric ganglia with, 5-HT,43,44 or histamine45 stimulates cAMP formation. Stimulation of cAMP by 5-HT is mediated by 5-HT1p receptors and histamine action is mediated by the H2 receptor subtype. Most of the evidence supports the hypothesis that cAMP is an intracellular second messenger in the process of signal transduction in AH neurones. Occupancy of receptors for the slow EPSP leads to activation of adenylate cyclase, which in turn leads to synthesis of cAMP, phosphorylation of protein kinases, and/or membrane channel proteins27,46(Fig. 3) and eventually to the dramatic changes in neuronal excitability and suppression of postspike hyperpolarizing potentials that occur during the slow EPSP in AH neurones.
G proteins couple receptors for multiple signal substances to adenylate cyclase and/or phospholipase C to initiate postreceptor cascades of reactions that culminate in the slow EPSP in AH neurones. Bertrand & Galligan47 reported that G protein-coupling for the slow EPSP-like action of the NK-3 agonist senktide did not involve a pertussis toxin-sensitive G protein in AH neurones. On the other hand, Pan et al.48 offered evidence that pertussis toxin-sensitive Gαo coupled the 5-HT1P receptor to the signalling cascade for the slow EPSP-like responses to 5-HT. There is little doubt that activation of adenylate cyclase, stimulation of formation of cAMP and activation of PKA are steps in the signal transduction cascade for the increased-resistance slow EPSP in AH neurones. On the other hand, the evidence of Bertrand & Galligan47 and Pan et al.48,49 suggests that G protein-coupling to phosphatidylcholine phospholipase C (PC-PLC), formation of diacylglycerol and activation of protein kinase C (PKC) might be components of the transduction mechanism. If it is indeed operational, the PKC transduction pathway undoubtedly depends on PC-PLC rather than phosphatidylinositol-specific PLC (PI-PLC), because liberation of inositol-1,4,-5 trisphosphate by PI-PLC would be expected to release Ca2+ from intraneuronal stores, which in turn would open Ca2+-gated K+ channels, decrease input resistance and oppose depolarization of the membrane potential. Pan et al.48 postulated for the cascade mediated by Gαo? PC-PLC that activation of PKC phosphorylates adenylate cyclase and thereby stimulates formation of cAMP and activation of PKA. In this scenario, the receptors do not couple directly to adenylate cyclase through Gαs protein as in other cell types.
Adenosine a1 receptors
Tonic release of endogenous adenosine is a property of the guinea-pig intestinal preparations that are used commonly in electrophysiological studies of the ENS.50 Whether released spontaneously or applied experimentally, adenosine acts to suppress initiation of the increased resistance slow EPSP in AH neurones and to abort the responses once they are evoked.50–53 Suppression by adenosine of an EPSP in progress is evidence that an action of adenosine is at postsynaptic receptors. Adenosine or selective adenosine A1 receptor agonists suppress slow EPSPs and the slow EPSP-like actions of forskolin or histamine, but do not suppress slow EPSP-like responses to 5-HT, calcitonin gene-releated peptide or substance P in AH neurones.52 Neither slow EPSP-like responses to intraneuronal injection of cAMP nor application of membrane permeant analogs of cAMP is affected by adenosine. Lack of suppression of effects of elevation of intraneuronal cAMP suggests that the action of adenosine is direct inhibition of adenylate cyclase. Blockade of the inhibitory action of adenosine by preincubation with pertussis toxin suggests that Gαi protein links the inhibitory A1 adenosine receptors to adenylate cyclase in AH neurones.54
The ionic mechanisms responsible for the increased-resistance slow EPSPs in AH neurones and for the slow EPSP-like actions of the large number of substances that mimic the EPSPs appear to be the same. This raises the question of how 5-HT, substance P and calcitonin gene-related peptide evoke the same kind of response as forskolin, vasoactive intestinal peptide, cholecystokinin, pituitary adenylate cyclase-activating peptide and histamine and yet, unlike the latter, are insensitive to the inhibitory action of adenosine A1 receptor agonists in the same neurones. One possibility is that the transduction mechanism for 5-HT, calcitonin gene-related peptide and substance P might not involve the PKA pathway. Findings of Pan et al.,48 that inhibition of PKA suppressed the slow EPSP-like responses to 5-HT, and reports that 5-HT stimulate production of cAMP in myenteric ganglia,43,44 argues against the hypothesis that the PKA pathway is not involved. The general conclusion is that an unequivocal explanation for the differential action of adenosine A1 receptor activation on responses to slow EPSP mimetics is presently unavailable.
Suppression of resting CA2+ influx
Changes that result from blocking resting influx of Ca2+ are another interesting aspect of slow EPSP-like events in AH neurones. Treatments that block Ca2+ channels evoke responses that mimic all aspects of slow EPSPs in AH-, but not S-type neurones.15 Exposure to solutions with elevated Mg2+ and depleted Ca2+ or to multivalent cations that impede transmembrane influx of Ca2+ and suppress Ca2+-dependent processes (e.g. Mn2+, Cd2+ or La2+) results in slowly activating membrane depolarization associated with increased input resistance, suppression of postspike hyperpolarizing potentials and enhanced membrane excitability reflected by repetitive spike discharge during injection of depolarizing current pulses and anodal-break excitation at the offset of hyperpolarizing current pulses. These effects of blockade of Ca2+ entry to mimic increased-resistance slow EPSPs and the actions of slow EPSP mimetics suggest that part of the downstream effects of activation of the variety of metabotropic receptors that lead to the EPSPs or EPSP-like responses is suppression of resting Ca2+ influx, reduction of free intraneuronal Ca2+ and closure of Ca2+-gated K+ channels.
Metabotropic transduction in s-type neurones
Characteristics of slow EPSPs in enteric neurones with S-type electrophysiological behaviour and uniaxonal morphology differ from the slow EPSPs in AH neurones. The EPSPs in both types of neurones are characterized by slowly activating depolarization of the membrane potential. Slow EPSPs in S-type neurones are distinguished from those in AH neurones by association of decreased input resistance (i.e. increased ionic conductance) with the depolarizing responses (Fig. 1B). When a putative signal substance evokes a response in an AH neurone, the depolarizing response is associated with an increase in input resistance and a reversal potential near the K+ equilibrium potential of −90 mV (Figs 1C, 2B). Exposure of S-type neurones to the same signal substance evokes depolarizing responses associated with decreased input resistance and reversal potentials in the range of −20–0 mV (Fig. 2A).23
The evidence available suggests that signal transduction for the decreased-resistance slow EPSPs in S neurones involves a phospholipase C/Ca2+–calmodulin second messenger mechanism (Fig. 3).23,24,55–59 Receptors for the slow EPSP on S neurones are G protein-coupled to activation of PI-PLC. PI-PLC catalyses the formation of inositol-1,4,-5 trisphosphate (IP3) and diacylglycerol. Once released, IP3 acts as a second messenger to release Ca2+ from intraneuronal membrane stores. Binding of the released Ca2+ to calmodulin activates calmodulin kinases II and IV (CaMKII-IV). Diacylglycerol, together with Ca2+, activates PKC. PKC phosphorylates cation conductance channels that open when phosphorylated to increase cationic conductance and thereby depolarize the membrane potential. Opening of the cationic conductance channels accounts for the decreased input resistance observed while recording with microelectrodes during the depolarization phase of the EPSP (Fig. 1B). Termination of the EPSP results from activation of the intraneuronal phosphatase, calcineurin, which catalyses dephosphorylation of the cationic channels.58,59
Pharmacological evidence, obtained for decreased-resistance slow EPSPs in S-type neurones and for mimetics of the EPSPs, supports the hypothesis that the signal transduction cascade for the decreased-resistance slow EPSP is G protein coupling of multiple kinds of receptors to PI-PLC. Purinergic P2Y1 receptors mediate one kind of decreased-resistance slow EPSP in S neurones and exposure to ATP mimics the slow EPSP.24 Application of trypsin, which is a PARs agonist, also evokes depolarizing responses that mimic decreased-resistance slow EPSPs.23,35 Inhibition of PI-PLC with U73122 or blockade of intraneuronal IP3 by 2-APB suppresses both the stimulus-evoked slow EPSPs and the slow EPSP-like actions of ATP and the PARS agonist trypsin.23,24 The calmodulin inhibitor W-7 abolishes stimulus-evoked slow EPSPs in S-type uniaxonal neurones in both the myenteric and submucosal plexuses. Inhibition of CaMKIIα with KN-62 also suppresses the amplitude of the depolarizing response of slow EPSPs associated with decreased input resistance in S neurones.58,59 Immunoreactivity (IR) for CaMKIIα is expressed exclusively in uniaxonal enteric neurones in the guinea-pig. Most of the submucosal neurones that express CaMKIIα-IR in the guinea-pig, rat and human also express VIP-IR.59