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Purinergic cotransmission: parasympathetic and enteric nerves
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THE NEUROSCIENCES, Vol 8, 1996: pp 207–215
Purinergic cotransmission: parasympathetic and enteric nerves Charles H.V. Hoyle
The urinary bladder and the small intestine are presented as the principal models of purinergic cotransmission in the parasympathetic and enteric nervous systems, drawing upon evidence provided by functional, histochemical and ultrastructural studies. In the parasympathetic division ATP probably commonly transmits alongside acetylcholine, and in enteric nerves it is more likely to be transmitting alongside nitric oxide and VIP. Other organs, including some blood vessels and exocrine glands, in which there are hints that ATP might be involved as a parasympathetic cotransmitter are also given consideration.
cotransmitter, on which there is more information than there is on other regions such as the stomach or some of the sphincters (although interesting, these latter organs have not been discussed because of restrictions on space). Before a brief final summary and suggestions for future directions, other organs in which there are hints that ATP might be involved as a parasympathetic cotransmitter are given consideration.
Key words: acetylcholine / ATP / cotransmission / nitric oxide / vasoactive intestinal polypeptide
Anatomical considerations Parasympathetic ganglia lie very close to, or even embedded in, the organ that their postganglionic neurones innervate. Thus, in the vast majority of systems, it is difficult or impossible to denervate the target organ surgically. This is in marked contrast to the sympathetic system, in which the relatively long postganglionic nerves are more readily accessible for surgical intervention. Sensory neurones are also readily accessible in their dorsal roots. Pharmacologically, guanethidine and 6-hydroxydopamine are valuable tools in studying sympathetic activity since both these agents can destroy postganglionic sympathetic neurones. Capsaicin and resinifera toxin have proven useful for studying the sensory system as both these drugs can kill subpopulations of primary afferent neurones. For the parasympathetic system there are no comparable agents. Botulinum toxin and choline mustard derivatives have been used with limited success in parasympathetic studies, but these agents are dangerous to handle, and are not very specific, at best targetting cholinergic nerves rather than parasympathetic nerves.
©1996 Academic Press Ltd
IN THE PERIPHERAL nervous system ATP is better established as a cotransmitter in sympathetic, somatomotor and sensory nerves than it is in parasympathetic nerves. Although there are many examples where there is strong evidence that parasympathetic neuroeffector mechanisms utilize ATP as a neurotransmitter, the evidence for cotransmission tends to be rather circumstantial. The reasons for this lie in the anatomy of parasympathetic and enteric nervous systems, the lack of a particular feature of purinergic nerves that can be exploited histochemically, and in the lack of the right pharmacological tools. In this article the anatomical features that render organs innervated by the parasympathetic and enteric nervous systems difficult to denervate and the limited histochemical and pharmacological tools that are available are described. The urinary bladder is presented as the principal model of purinergic cotransmission in the parasympathetic nervous system, drawing upon evidence provided by functional, histochemical and ultrastructural studies. Then the small intestine is chosen as the model for ATP as an enteric
Histochemical localization of ATP
From the Department of Anatomy and Developmental Biology, University College London, Gower Street, London, WC1E 6BT, UK ©1996 Academic Press Ltd 1044-5765/96/040207 + 09 $18.00/0
At present there are no reliable histochemical stains for ATP, but a fluorescence method has been developed in which nerves that have taken up quinacrine 207
C. H. V. Hoyle can be visualized.1-3 Quinacrine has been demonstrated to form complexes with ATP, provided that the ratio of phosphate groups to quinacrine is high enough.4 The conjugation between quinacrine and ATP in forming the complex is strong enough to prevent ATP from diffusing down a concentration gradient.5,6 From studies on the guinea-pig taenia coli and urinary bladder it has been shown that following preloading of the tissue with [3H]-adenosine and [14C]-quinacrine, in sucrose density gradient homogenates the 3H and 14C peaks are correspondent. Since it has also been shown that [3H]-adenosine, when taken up by nerves, is rapidly phosphorylated with ATP being the preponderant product, these results imply that quinacrine is associated with adenine nucleotides. Just how specific quinacrine is for neurones that have a high ATP content is unknown:1 it will bind to DNA due to the high concentration of purine bases. Nevertheless quinacrine does bind compounds with transmitter-like properties, and when complexed it is released in a calcium-dependent fashion by depolarization.7,8 Quinacrine is taken up by platelets, which contain secretory granules that have a high concentration of ATP, but in platelets obtained from patients with storage pool deficiency, in which the levels of ATP are very low, there is little uptake of quinacrine.9,10 Organs innervated by postganglionic parasympathetic nerves, and regions of the gastrointestinal tract innervated by intrinsic enteric nerves that stain with quinacrine include: rat, guinea-pig and rabbit stomach and ileum myenteric plexus,1,3,7,11-14 rat colon,11 rat and mouse anococcygeus muscle,2,11 rabbit rectococcygeus,15 rabbit portal vein,15,16 guinea-pig and rabbit intrinsic cardiac ganglia.17 The uranaffin reaction may also be used to visualize neurones with high concentrations of ATP. In this reaction the divalent cation of uranium dioxide, UO22 + , complexes with the phosphate groups of ATP, and forms an electron-dense precipitate.18 In normal platelets and platelets depleted of serotonin by reserpine there is a strong reaction within storage granules, but in platelets from patients with storage pool deficiency there is a loss of reaction, as well as a loss of quinacrine staining.9,18 In the guinea-pig ileum, uranaffin reaction products can be seen in small vesicles with a diameter of 40–60 nm in nerve terminals in the myenteric plexus, submucous plexus, deep muscular plexus and circular muscle layer. Large granular vesicles, 80 nm in diameter, tend to remain unstained. Reactivity is unaffected by reserpine pretreatment or by surgical denervation of extrinsic
nerves, indicating that the nerves that contain the high levels of nucleotides are intrinsic and are not preganglionic.19,20
Urinary bladder The evidence in favour of both acetylcholine and ATP each being a neurotransmitter in the urinary bladder is strong, with functional and histological studies providing information (see refs 21-23 for reviews). In the absence of any direct evidence, the weight of the circumstantial evidence is indeed compelling that these two substances function as cotransmitters in this organ. The motor supply to the urinary bladder detrusor is provided by the parasympathetic nervous system, originating from the sacral or lower lumbar level, depending on species, of the spinal cord. The pelvic ganglia and intramural ganglia contain nerve cells that possess acetylcholinesterase activity, and in the smooth muscle of the detrusor there is an abundance of fibres that stain for acetylcholinesterase activity. Fibres that look like typical varicose autonomic nerve terminals are found in close association with the smooth muscle cells (see ref 24 for review). In the guinea-pig urinary bladder staining with quinacrine shows an extensive autonomic ground plexus, including intramural ganglia with varicose projections running to the smooth muscle.25-27 At an ultrastructural level autonomic nerve terminals in the bladder contain mixed transmitter-vesicle profiles,28-35 with cholinergic nerve terminals, i.e. those that contain mostly small clear vesicles, being the preponderant non-adrenergic type. There is no identifying vesicle morphology associated with purinergic nerves, and ATP has not been shown to be contained in the same vesicles as acetylcholine in postganglionic parasympathetic nerve terminals in the urinary bladder. Nor has the antithesis been shown, i.e. that ATP is not co-stored with acetylcholine in synaptic vesicles in the bladder. Certainly in other tissues, such as somatic nerve terminals innervating the torpedo ray electroplaque, the intravesicular acetylcholine:ATP ratio can be approximately 4:1, and the concentration of ATP may be as high as 120 mM.36 Based mostly on data obtained from somatic motor nerves, it has been stated that in all cholinergic vesicles where the analyses have been done, ATP is found at one fourth to one twelfth of the molar amount of acetylcholine.36 Unfortunately the list of tissues analysed does not include postganglionic 208
Parasympathetic and enteric cotransmission cholinergic excitatory junction potential (EJP) followed by a slow depolarization.65,70-73 Both the EJP and the slow depolarization may be associated with contractile events. The slow component is cholinergic, being abolished by atropine70-74 and represents a pharmacomechanical coupling between activation of the muscarinic receptors and the contractile apparatus, involving the inositol trisphosphate system. The EJPs, if large enough, will give rise to action potentials, and represent electromechanical coupling between the P2X-purinoceptor and the contractile apparatus. P2X-purinoceptors are ligand-gated ion channels, and in the bladder smooth muscle this channel is permeable to Na + and Ca2 + . 74-77 A feature of the P2-purinoceptor-mediated response is that it is only transient, whether evoked by nerve stimulation or applied ATP; there is some mechanism distal to receptor occupation that limits the duration of purinergic activity. Autoradiographic studies, utilizing tritiated α,β-methylene ATP as the radioligand have been carried out, and show a dense population of P2Xpurinoceptors in the smooth muscle of the urinary bladder detrusor of several species, including human.78,79 Furthermore, binding characteristics and ligand displacement profiles of the P2-purinoceptor have been determined in detrusor muscle membrane homogenates from rat, rabbit and human.79-82 All these are in line with it being a P2X-purinoceptor. Electrical stimulation of the postganglionic parasympathetic nerves in isolated preparations of guineapig urinary bladder detrusor muscle evokes release of ATP.25,26 That the release is prevented by the neurotoxin tetrodotoxin, but not affected by the sympatholytics guanethidine and 6-hydroxydopamine, indicates that the source of the ATP is parasympathetic nerves. ATP is not released from preganglionic parasympathetic nerve trunks in response to electrical stimulation,83,84 so in the isolated bladder preparations it almost certainly comes from the postganglionic nerve terminals. There is only one study that has directly addressed the question of whether or not ATP and acetylcholine are cotransmitters in the urinary bladder.54 In this work a partially purified botulinum toxin was utilized. Botulinum toxin is a parasympatholytic agent that destroys cholinergic but not purinergic nerves in the intestine.54 In the urinary bladder this toxin abolishes excitatory neuromuscular transmission, which implies that ATP is released from the same nerves that release acetylcholine.54 Another parasympatholytic agent is the choline mustard derivative AF64A. In the guineapig, sub-lethal doses of AF64A cause impairment of
parasympathetic nerves. Nevertheless it does not seem unreasonable to extrapolate such information. Responses to nerve stimulation and applied acetylcholine are potentiated by eserine (physostigmine), which inhibits acetylcholinesterase activity.37,38 Acetylcholine is released from postganglionic parasympathetic neurones in isolated preparations of the bladder.38 Moreover, choline is taken up by vesicular neurones, and is subsequently released as acetylcholine,39,40 and blockade of choline uptake by hemicholinium markedly reduces the release of acetylcholine.41 However, it seems to have become common practice to rely on atropine as an antagonist of muscarinic cholinoceptors to confirm or deny the role of acetylcholine in the transmission process. In many animals, including dogs, guinea-pigs, monkeys, rats, cats and humans stimulation of the preganglionic parasympathetic fibres either in the sacral ventral roots or in the pelvic nerve evokes contractile responses in the detrusor.42-53 In humans, baboons and rhesus monkeys contractions of the urinary bladder evoked by preganglionic parasympathetic nerve stimulation are abolished, or nearly so, by atropine.42-44 This indicates that in these old world primates and humans neuromuscular transmission is mediated only by acetylcholine acting on muscarinic receptors, and that cholinergic transmission is the predominant mechanism by which vesicular pressure is raised during voiding. In contrast, in the remaining species atropine causes at best only a partial inhibition of the motor response, indicating that in carnivores, rodents, lagomorphs, and new world primates there is a non-cholinergic component to postganglionic neuromuscular transmission in the bladder.22,23,44-46,48,52 Several studies have shown that the predominant or sole non-cholinergic neuromuscular transmitter in the urinary bladder detrusor, responsible for increasing intravesicular pressure or contraction is likely to be ATP (see refs 21-23). ATP mimics the contractions of the bladder due to stimulation of the postganglionic parasympathetic nerves in the presence of atropine.25,26,54-57 It acts on P2X-purinoceptors in the urinary bladder58 to evoke contractions, and these receptors can be blocked by antagonists such as photoactivated arylazidoaminopropionyl ATP (ANAPP3)59-63 or suramin,64 or following densensitization with α,β-methylene ATP.55,62,65-71 The important point here is that in all these cases the responses to ATP are blocked in parallel with the responses to stimulation of the non-cholinergic nerves. Electrophysiologically, stimulation of the postganglionic parasympathetic nerves evokes a non209
C. H. V. Hoyle synergistic interaction (see ref 91 for review) as found in the vas deferens where ATP and noradrenaline are sympathetic cotransmitters and noradrenaline markedly potentiates the postjunctional activity of ATP,92,93 but either facilitatory or inhibitory neuromodulatory interactions may be involved (see ref 91). In the urinary bladder acetylcholine potentiates ATP (unpublished observations).
cholinergic transmission in the urinary bladder, accompanied by an increase in the sensitivity of the P2X-purinoceptors, which is possibly indicative of an early stage of degeneration of the co-transmitting purinergic components.85 If we can accept it as fact that ATP and acetylcholine act as cotransmitters in the urinary bladder then we should also consider the functional significance. One would expect that in line with the natural themes and variations belonging to other combinations of cotransmitters there is a practical benefit to have ATP and acetylcholine as cotransmitters in the innervation of the urinary bladder as opposed to their being sole transmitters released from independent neuronal populations. In the bladder there is a marked temporal separation of the purinergic from the cholinergic excitation, whether evoked in vivo by stimulating a voiding cycle or in vitro by electrical field stimulation. The initial phase is transient and is purinergic, whereas the secondary sustained phase is cholinergic.51,86-89 The purinergic component is responsible for the initiation of voiding, whereas the cholinergic phase is responsible for the maintenance of voiding, the period of greatest vesicular pressure and during which most urine is expelled.48,86,87,89 In conscious rats, blocking the P2-purinoceptor results in increased bladder capacity and residual volume and decreased voiding pressure, but blocking the cholinoceptors results in decreased micturition pressure and micturition volume.90 The pattern of responses can be maintained by one type of nerve, i.e. one that uses ATP and acetylcholine as cotransmitters, because during prolonged activation there will still be only a transient rapid-onset initial phase mediated by ligandgated ion channels which is somehow self-limiting, and a sustained second phase, slower in onset because it is mediated by metabotropic receptors. Thus there is parsimony in using one population of neurones in this way because otherwise the activity of two populations of neurones would have to be co-ordinated. An aspect of the functional significance of cotransmission follows from the suggestion that the purinergic component is better developed in those animals that mark their territory by urination, and therefore need to urinate more frequently in short bursts (e.g. dogs, rats and New World primates) than it is in those animals that practice urinary continence until the need to empty the bladder arises (e.g. Old World primates and humans).23,44,52 Another aspect of the functional significance of cotransmission is that there should be an interaction of some sort between the cotransmitters.91 This is often manifest as a
Enteric nervous system The evidence that ATP acts as a neuromuscular transmitter in the enteric nervous system is now quite convincing, but whether or not it is a true cotransmitter with other substances has yet to be proven. Two other inhibitory substances are utilized widely throughout the gut, probably as cotransmitters, namely vasoactive intestinal polypeptide (VIP) and nitric oxide (NO).94 There is little evidence for the colocalization of ATP with these neurotransmitters, mostly because appropriate studies have not been attempted because of the lack of suitable tools. The quinacrine stain has been used in one investigation,11 but colocalization studies at the EM level with the uranaffin reaction have not been reported. Nevertheless there are many examples of regions of the gastrointestinal tract in which neuromuscular transmission is mediated by more than one transmitter, and generally, inhibitory transmission is carried out by members of the triumvirate, ATP, VIP and NO. It would be in keeping with the principles of autonomic transmission if indeed ATP were a true cotransmitter with these other substances. The evidence that ATP is a neurotransmitter in the gut has been reviewed extensively.21,95-97 The release of ATP, evoked by electrical stimulation of enteric nerves, has been measured.26,98,99 Pharmacological experiments have shown that responses to stimulation of the non-adrenergic, non-cholinergic nerves are blocked in parallel with the responses to applied ATP by P2-purinoceptor antagonists such as reactive blue 2100-103 or suramin,64,104,105 or the enzyme nucleotide pyrophosphatase.103,106 NO has only fairly recently been determined to be a neurotransmitter in the periphery, and it is now known to be synthesized in and released from enteric neurones. When its synthesis is inhibited so are the responses to neural stimulation, and application of authentic NO in aqueous solution or NO donor compounds mimic the responses of nerve stimulation and activate guanylate cyclase in the same way. This 210
Parasympathetic and enteric cotransmission evidence has been reviewed recently.107-110 VIP-containing nerves are well represented in the muscular coat throughout the gut, its release can be measured and in many instances exogenous VIP mimics responses to nerve stimulation. However selective antagonists of VIP-receptors have been slow in developing, and there is little evidence along the lines of pharmacological blockade of the receptor producing a parallel antagonism of responses to applied VIP and nerve stimulation.97,111 Histochemical localization of VIP with ATP has not been reported, but one study has attempted to colocalize ATP with NADPH-diaphorase, which can sometimes be utilized as a marker for nitric oxide synthase (NOS).11 In the myenteric plexus of the rat colon nearly all the quinacrinepositive nerves contain NADPH-diaphorase activity, but in the ileum and anococcygeus muscle only a subpopulation of quinacrine-positive nerves also contain NADPH-diaphorase activity.11 In the circular muscle of the guinea-pig ileum a single pulse of electrical field stimulation evokes a transient hyperpolarization that has a latency of onset of approximately 100 ms and a rise time of less than 500 ms, this is known as a fast inhibitory junction potential (IJP). This event is mimicked by local application of ATP and in parallel with the response to ATP it is abolished by apamin (a K + -channel blocker) or reactive blue 2 or following desensitization to α,β-methylene ATP.112,113 In the presence of apamin a short train of pulses of electrical field stimulation evokes a hyperpolarization that has a relatively long latency of onset, and a rise time approaching 1.5 s.113 This slow IJP is mimicked by VIP, and like the response to applied VIP it is antagnozied by the VIP-receptor antagonist VIP 10–28.113 Furthermore, when nitric oxide synthase is inhibited the slow IJP is attenuated while the fast IJP remains unaffected.112 Thus in this tissue ATP is the transmitter responsible for the fast IJP and both VIP and NO contribute to the slow IJP.112 It cannot be stated with certainty that these three substances are cotransmitters, but VIP is extensively colocalized with NOS in the nerves supplying the circular muscle,94,114 so these two are likely to be cotransmitters, but there is no evidence one way or the other as to whether or not ATP is released from this same population. Synaptosomal preparations of the ileal myenteric plexus contain a subpopulation of non-adrenergic vesicles that release ATP.115-117 Release of ATP is evoked by depolarizing stimuli, and by acetylcholine acting on nicotinic receptors.115,118 Since the release
of ATP from non-adrenergic vesicles is approximately 50% of that from the total pool of adrenergic and non-adrenergic vesicles, it follows that the nonadrenergic release comes from a substantial population of nerve terminals, from which it can be induced that ATP is costored with another transmitter substance. Physiologically the inhibitory transmission to the circular muscle is brought about via reflexes involved in descending inhibition. These reflexes prepare the bowel to receive the contents, and facilitate transit along the intestinal tract. It is not immediately clear what the advantage is of having a biphasic IJP or of having phases of fast and slow inhibitory transmission.
Other organs The rabbit portal vein has two muscle layers with the longitudinal muscle having a non-adrenergic, noncholinergic innervation that mediates relaxation independent of the endothelium. L-NAME partially inhibits this relaxation, an effect which is reversed by L-arginine. A combination of suramin and L-NAME abolishes neurogenic relaxation indicating that both ATP and NO are inhibitory transmitters in this vessel.119 This is possibly cotransmission from postganglionic parasympathetic nerves because terminal ganglia lie close to the rabbit portal vein and project into the muscle layers, and some of these ganglion cells stain with quinacrine.16,120 Salivary glands, in particular rat and mouse parotid glands, possess P2-purinoceptors that mediate intraacinar cell elevation of Ca2 + and amylase secretion.121-124 Following parasympathetic denervation, responses to ATP can be potentiated.124 This denervation supersensitivity is a hint that ATP is physiologically released from the parasympthatic nerve terminals, and since responses to stimulation of the parasympathetic nerves are markedly attenuated by atropine,122 it is possible that ATP is cotransmitting with acetylcholine in these glands. The nasal vasculature is under dual sympathetic and parasympathetic control125 with the parasympathetic element being responsible for non-cholinergic vasodilatation. Applied ATP also evokes dilatation,126 but rather than cotransmitting with acetylcholine it is more likely that the cotransmitter would be NO.127 211
C. H. V. Hoyle 10. McNicol A, Israels SJ, Robertson C, Gerrard JM (1994) The empty sack syndrome: a platelet storage pool deficiency associated with empty dense granules. Br J Haematol 86:574-582 11. Belai A, Burnstock G (1994) Evidence for coexistence of ATP and nitric oxide in non-adrenergic, non-cholinergic (NANC) inhibitory neurones in the rat ileum, colon and anococcygeus muscle. Cell Tissue Res 278:197-200 12. Crowe R, Burnstock G (1981) Comparative studies of quinacrine-positive neurones in the myenteric plexus of stomach and intestine of guinea-pig, rabbit and rat. Cell Tissue Res 221:93-107 13. Crowe R, Burnstock G (1981) Perinatal development of quinacrine-positive neurons in the rabbit gastrointestinal tract. J Auton Nerv Syst 4:217-230 14. Brizzi E, Calamai F, Staderini G, Viligiardi R (1983) The presence of purinergic, quinacrine-positive neurons in the rabbit stomach. Boll Soc Ital Biol Sper 59:962-964 15. Cocks T, Crowe R, Burnstock G (1979) Non-adrenergic, noncholinergic (purinergic?) inhibitory innervation of the rabbit rectococcygeus muscle. Eur J Pharmacol 54:261-271 16. Burnstock G, Crowe R, Wong HK (1979) Comparative pharmacological and histochemical evidence for purinergic inhibitory innervation of the portal vein of the rabbit, but not guinea-pig. Br J Pharmacol 65:377-388 17. Crowe R, Burnstock G (1982) Fluorescent histochemical localisation of quinacrine-positive neurones in the guinea-pig and rabbit atrium. Cardiovasc Res 16:384-390 18. Richards JG, Da Prada M (1977) Uranaffin reaction: a new cytochemical technique for the localization of adenine nucleotides in organelles storing biogenic amines. J Histochem Cytochem 25:1322-1336 19. Wilson AJ, Furness JB, Costa M (1979) A unique population of uranaffin-positive intrinsic nerve endings in the small intestine. Neurosci Lett 14:303-308 20. Wilson AJ, Furness JB, Costa M (1981) The fine structure of the submucous plexus of the guinea-pig ileum. II. Description and analysis of vesiculated nerve profiles. J Neurocytol 10:785-804 21. Hoyle CHV (1992) Transmission: purines, in Autonomic Neuroeffector Mechanisms (Burnstock G, Hoyle CHV, eds) pp 367-407. Harwood Academic Publishers, Chur 22. Hoyle CHV (1995) Non-adrenergic, non-cholinergic control of the urinary bladder. World J Urol 12:233-244 23. Hoyle CHV, Burnstock G (1993) Postganglionic efferent transmission in the bladder and urethra, in Nervous Control of the Urogenital System (Maggi CA, ed.) pp 349-381. Harwood Academic Publishers, Chur 24. Lincoln J, Burnstock G (1993) Autonomic innervation of the urinary bladder and urethra, in Nervous Control of the Urogenital Tract (Maggi CA, ed.) pp 33-68. Harwood Academic Publishers, Chur 25. Burnstock G, Cocks T, Crowe R, Kasakov L (1978) Purinergic innervation of the guinea-pig urinary bladder. Br J Pharmacol 63:125-138 26. Burnstock G, Cocks T, Kasakov L, Wong H (1978) Direct evidence for ATP release from non-adrenergic, non-cholinergic (‘purinergic’) nerves in the guinea-pig taenia coli and bladder. Eur J Pharmacol 49:145-149 27. Crowe R, Haven AJ, Burnstock G (1986) Intramural neurons of the guinea-pig urinary bladder: histochemical localization of putative neurotransmitters in cultures and newborn animals. J Auton Nerv Syst 15:319-339 28. Gilpin CJ, Dixon JS, Gilpin SA, Gosling JA (1983) The fine structure of autonomic neurons in the wall of the human urinary bladder. J Anat 137:705-713 29. Daniel EE, Cowan W, Daniel VP (1983) Structural bases for neural and myogenic control of human detrusor muscle. Can J Physiol Pharmacol 61:1247-1273
Summary and future directions Despite the title of this article there is no hard evidence for ATP acting as a cotransmitter in the parasympathetic and enteric nervous systems. However, based upon the received wisdom gained from studies of other divisions of the peripheral nervous system, where with appropriate tools it can be shown that ATP is indeed a cotransmitter, it is likely that cotransmission is the rule throughout the periphery rather than the exception. In postganglionic parasympathetic nerves ATP would commonly be transmitting alongside acetylcholine, and in enteric nerves it would be transmitting alongside NO and VIP. If the question of whether or not ATP is acting as a cotransmitter in the parasympathetic and enteric divisions were to be answered definitively, then new tools are needed. One’s wish-list would include a histochemical marker for purinergic nerves that is at least as good as the available immunocytochemical labels for any number of neuropeptides, a potent, specific competitive antagonist of the appropriate subtype of P2-purinoceptor, and a toxin that specifically destroys postganglionic parasympathetic nerves, and perhaps another toxin that selectively depletes synaptic vesicles of ATP.
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30. Hoyes AD, Barber P (1978) Effect of hydroxydopamine on the small dense-cored vesicles in the cholinergic nerve terminals of the rat bladder. J Anat 127:533-542 31. Hoyes AD, Barber P, Martin BG (1975) Comparative ultrastructure of the nerves innervating the muscle of the body of the bladder. Cell Tissue Res 164:133-144 32. Dixon JS, Gilpin CJ (1987) Presumptive sensory axons of the human urinary bladder: a fine structural study. J Anat 151:199-207 33. Gibbins IL (1982) Lack of correlation between ultrastructural and pharmacological types of non-adrenergic autonomic nerves. Cell Tissue Res 221:551-581 34. Feh´er E, Csanyi K, Vajda J (1980) Intrinsic innervation of the urinary bladder. Acta Anat Basel 106:335-344 35. McConnell J, Benson GS, Wood JG (1982) Autonomic innervation of the urogenital system: adrenergic and cholinergic elements. Brain Res Bull 9:679-694 36. Parsons SM, Prior C, Marshall IG (1993) Acetylcholine transport, storage, and release. Int Rev Neurobiol 35:279-390 37. Carpenter FG (1963) Excitation of the rat urinary bladder by coaxial electrodes and chemical agents. Am J Physiol 204:727-731 38. Carpenter FG, Rand SA (1965) Relation of acetylcholine release to responses of the rat urinary bladder. J Physiol 180:371-382 39. D’Agostino G, Chiari MC, Grana E (1989) Prejunctional effects of muscarinic agonists on 3H-acetylcholine release in the rat urinary bladder strip. Naunyn Schmiedeberg’s Arch Pharmacol 340:76-81 40. D’Agostino G, Kilbinger H, Chiari MC, Grana E (1986) Presynaptic inhibitory muscarinic receptors modulating acetylcholine release in the rat urinary bladder. J Pharmacol Exp Ther 239:522-528 41. Choo LK, Mitchelson F (1980) The effect of indomethacin and adenosine 5-triphosphate on the excitatory innervation of the rat urinary bladder. Can J Phys Pharmacol 58:1042-1048 42. Brindley GS, Craggs MD (1975) The effect of atropine in the urinary bladder of baboon and man. J Physiol 256:55P 43. Craggs MD, Stephenson JD (1985) Bladder electromyograms and function in monkeys after atropine. Br J Urol 57:341-345 44. Craggs MD, Rushton DN, Stephenson JD (1986) A putative non-cholinergic mechanism in urinary bladders of New but not Old World primates. J Urol 136:1348-1350 45. Edge ND (1955) A contribution to the innervation of the urinary bladder of the car. J Physiol 127:54-68 46. Elmer M (1974) Atropine sensitivity of the rat urinary bladder during nerve degeneration. Acta Physiol Scand 93:202-205 47. Carpenter FG (1981) Atropine and micturition responses by rats with intact and partially innervated bladder. Br J Pharmacol 73:837-842 48. Craggs MD, Stephenson JD (1982) The effects of parasympathetic blocking agents on bladder electrograms and function in conscious and anaesthetized cats. Neuropharmacol 21:695-703 49. Weetman DF, Turner N (1977) The effects of ATP-receptor blocking agents on the response to the guinea-pig isolated bladder preparation to hyoscine-resistant nerve stimulation. Arch Int Pharmacodyn Ther 228:10-14 50. Ursillo R, Clark B (1956) The action of atropine on the urinary bladder of the dog and on the isolated nerve-bladder strip preparation of the rabbit. J Pharmacol Exp Ther 118:338-347 51. Peterson JS, Noronha-Blob L (1989) Effects of selective cholinergic antagonists and α,β-methylene ATP on guinea-pig urinary bladder contractions in vivo following pelvic nerve stimulation. J Auton Pharmacol 9:303-313
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C. H. V. Hoyle 72. Creed KE, Ishikawa S, Ito Y (1983) Electrical and mechanical activity recorded from rabbit urinary bladder in response to nerve stimulation. J Physiol Lond 338:149-164 73. Brading AF, Mostwin JL (1989) Electrical and mechanical responses of guinea-pig bladder muscle to nerve stimulation. Br J Pharmacol 98:1083-1090 74. Brading AF, Inoue R (1991) Ion channels and excitatory transmission in the smooth muscle of the urinary bladder. Z Kardiol 80 Suppl 7:47-53 75. Brading AF (1992) Ion channels and control of contractile activity in urinary bladder smooth muscle. Jpn J Pharmacol 58 Suppl 2:120P-127P 76. Inoue R, Brading AF (1991) Human, pig and guinea-pig bladder smooth muscle cells generate similar inward currents in response to purinoceptor activation. Br J Pharmacol 103:1840-1841 77. Inoue R, Brading AF (1990) The properties of the ATPinduced depolarization and current in single cells isolated from the guinea-pig urinary bladder. Br J Pharmacol 100:619-625 78. Bo X, Burnstock G (1992) Species differences in characteristics and distribution of α,β-methylene ATP binding sites in urinary bladder and uretha of rat, guinea-pig and rabbit. Eur J Pharmacol 216:59-66 79. Bo X, Burnstock G (1995) Characterization and autoradiographic localization of [3H] α,β-methylene adenosine 5'-triphosphate binding sites in human urinary bladder. Br J Urol 76:297-302 80. Bo X, Burnstock G (1990) High- and low-affinity binding sites for α,β-methylene ATP in rat urinary bladder membranes. Br J Pharmacol 101:291-296 81. Levin RM, Jacoby R, Wein AJ (1983) High-affinity, divalent ion-specific binding of 3H-ATP to homogenate derived from rabbit urinary bladder. Comparison with divalent-ion ATPase activity. Mol Pharmacol 23:1-7 82. Ruggieri MR, Whitmore KE, Levin RM (1990) Bladder purinergic receptors. J Urol 144:176-181 83. Maire JC, Medilanski J, Straub RW (1982) Uptake of adenosine and release of adenine derivatives in mammalian nonmyelinated nerve fibres at rest and during activity. J Physiol Lond 323:589-602 84. Maire JC, Medilanski J, Straub RW (1984) Release of adenosine, inosine and hypoxanthine from rabbit non-myelinated nerve fibres at rest and during activity. J Physiol Lond 357:67-77 85. Hoyle CHV, Moss HE, Burnstock G (1986) Ethylcholine mustard aziridinium ion (AF64A) impairs cholinergic neuromuscular transmission in the guinea-pig ileum and urinary bladder, and cholinergic neuromodulation in the enteric nervous system of the guinea-pig distal colon. Gen Pharmacol 17:543-548 86. Chancellor MB, Kaplan SA, Blaivas JG (1992) The cholinergic and purinergic components of detrusor contractility in a whole rabbit bladder model. J Urol 148:906-909 87. Krell RD, McCoy JL, Ridley PT (1981) Pharmacological characterization of the excitatory innervation to the guineapig urinary bladder in vitro: evidence for both cholinergic and non-adrenergic, non-cholinergic neurotransmission. Br J Pharmacol 74:15-22 88. Levin RM, Ruggieri MR, Wein AJ (1986) Functional effects of the purinergic innervation of the rabbit urinary bladder. J Pharmacol Exp Ther 236:452-457 89. Maggi CA, Meli A, Santicioli P (1987) Neuroeffector mechanisms in the voiding cycle of the guinea-pig urinary bladder. J Auton Pharmacol 7:295-308 90. Igawa Y, Mattiasson A, Andersson KE (1993) Effects of GABAreceptor stimulation and blockade on micturition in normal rats and rats with bladder outflow obstruction. J Urol 150:537-542
91. Burnstock G (1990) Co-transmission: the fifth Heyman’s memorial lecture. Arch Int Pharmacodyn Ther 304:7-33 92. Kazic T, Milosavljevic D (1980) Interaction between adenosine triphosphate and noradrenaline in the isolated vas deferens of the guinea-pig. Br J Pharmacol 71:93-98 93. Huidobro Toro JP, Parada S (1988) Co-transmission in the rat vas deferens: postjunctional synergism of noradrenaline and adenosine 5'-triphosphate. Neurosci Lett 85:339-344 94. Keef KD, Shuttleworth CWR, Xue C, Bayguinov O, Publicover NG, Sanders KM (1994) Relationship between nitric oxide and vasoactive intestinal polypeptide in enteric inhibitory neurotransmission. Neuropharmacology 33:1303-1314 95. White TD (1988) Role of adenine compounds in autonomic neurotransmission. Pharmacol Ther 38:129-168 96. Hoyle CHV, Burnstock G (1991) ATP receptors and their physiological roles, in Adenosine in the Nervous System (Stone TW, ed.) pp 43-76. Academic Press, London 97. Hoyle CHV, Burnstock G (1989) Neuromuscular transmission in the gastrointestinal tract, in Handbook of Physiology, Section 6: The Gastrointestinal System, Vol 1 (Wood JD, ed.) pp 435-464. American Physiological Society, Bethesda 98. Su C, Bevan JA, Burnstock G (1971) [3H]adenosine triphosphate: release during stimulation of enteric nerves. Science 173:336-338 99. Rutherford A, Burnstock G (1978) Neuronal and nonneuronal components in the overflow of labelled adenyl compounds from guinea-poig taenia coli. Eur J Pharmacol 48:195-202 100. Crema A, Frigo GM, Lecchini S, Manzo L, Onori L, Tonini M (1983) Purine receptors in the guinea-pig internal and sphincter. Br J Pharmacol 78:599-603 101. Kerr DIB, Krantis A (1979) A new class of ATP antagonists. Proc Aust Physiol Pharmacol Soc 10:256 102. Manzini S, Hoyle CHV, Burnstock G (1986) An electrophysiological analysis of the effect of reactive blue 2, a putative P2-purinoceptor antagonist, on inhibitory junction potentials of rat caecum. Eur J Pharmacol 127:197-204 103. Manzini S, Maggi CA, Meli A (1985) Further evidence for involvement of adenosine-5'-triphosphate in non-adrenergic non-cholinergic relaxation of the isolated rat duodenum. Eur J Pharmacol 113:399-408 104. Den Hertog A, Van den Akker J, Nelemans A (1989) Suramin and the inhibitory junction potential in taenia caeci of the guinea-pig. Eur J Pharmacol 173:207-209 105. Den Hertog A, Nelemans A, Van den Akker J (1989) The inhibitory action of suramin on the P2-purinoceptor response in smooth muscle cells of guinea-pig taenia caeci. Eur J Pharmacol 166:531-534 106. Satchell DG (1981) Nucleotide pyrophosphatase antagonizes responses to adenosine 5'-triphosphate and non-adrenergic, non-cholinergic inhibitory nerve stimulation in the guinea-pig isolated taenia coli. Br J Pharmacol 74:319-321 107. Sneddon P, Graham A (1992) Role of nitric oxide in the autonomic innervation of smooth muscle. J Auton Pharmacol 12:445-456 108. Sanders KM, Ward SM, Thornbury KD, Dalziel HH, Westfall DP, Carl A (1992) Nitric oxide as a non-adrenergic, noncholinergic neurotransmitter in the gastrointestinal tract. Jpn J Pharmacol 58 Suppl 2:220P-225P 109. Lincoln J, Hoyle CHV, Burnstock G (1995) Transmission: nitric oxide, in Autonomic Neuroeffector Mechanisms (Burnstock G, Hoyle CHV, eds) pp 509-539. Harwood Academic, Reading 110. Sanders KM (1994) Nitric oxide as an inhibitory neurotransmitter in the gastrointestinal tract, in Nitric Oxide: Roles in Neuronal Communication and Neurotoxicity (Takagi H, Toda N, Hawkins RD, eds) pp 77-88. Japan Scientific Societies Press, Tokyo
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Parasympathetic and enteric cotransmission 111. Furness JB, Costa M (1989) Identification of transmitters of functionally defined enteric neurones, in Handbook of Physiology, Section 6: The Gastrointestinal System (Wood JD, ed.) pp 387-402. American Physiological Society, Bethesda, Maryland 112. He XD, Goyal RK (1993) Nitric oxide involvement in the peptide VIP-associated inhibitory junction potential in the guinea-pig ileum. J Physiol Lond 461:485-499 113. Crist JR, He XD, Goyal RK (1992) Both ATP and the peptide VIP are inhibitory neurotransmitters in guinea-pig ileum circular muscle. J Physiol Lond 447:119-131 114. Costa M, Furness JB, Pompolo S, Brookes SJH, Bornstein JC, Bredt DS, Snyder SH (1992) Projections and chemical coding of neurons with immunoreactivity for nitric oxide synthase in the guinea-pig small intestine. Neurosci Lett 148:121-125 115. White TD, Leslie RA (1982) Depolarization-induced release of adenosine 5'-triphosphate from isolated varicosities derived from the myenteric plexus of the guinea-pig small intestine. J Neurosci 2:206-215 116. Al-Humayyd M, White TD (1985) 5-Hydroxytryptamine releases adenosine 5'-triphosphate from nerve varicosities isolated from the myenteric plexus of the guinea-pig ileum. Br J Pharmacol 84:27-34 117. Hammond JR, MacDonald WF, White TD (1988) Evoked secretion of [3H]noradrenaline and ATP from nerve varicosities isolated from the myenteric plexus of the guinea pig ileum. Can J Physiol Pharmacol 66:369-375 118. White TD, Al-Humayyd M (1983) Acetylcholine releases ATP from varicosities isolated from guinea pig myenteric plexus. J Neurochem 40:1069-1075
119. Brizzolara AL, Crowe R, Burnstock G (1993) Evidence for the involvement of both ATP and nitric oxide in non-adrenergic, non-cholinergic inhibitory neurotransmission in the rabbit portal vein. Br J Pharmacol 109:606-608 120. Burnstock G, Crowe R, Kennedy C, Torok J (1984) Indirect evidence that purinergic modulation of perivascular adrenergic neurotransmission in the portal vein is a physiological process. Br J Pharmacol 82:359-368 121. Gallacher DV (1982) Are there purinergic receptors on parotid acinar cells? Nature 296:83-86 122. Gallacher DV, Petersen OH (1980) Electrophysiology of mouse parotid acina: effects of electrical field stimulation and ionophoresis of neurotransmitters. J Physiol 305:43-57 123. Sasaki T, Gallacher DV (1990) Extracellular ATP activates receptor-operated channels on mouse lacrimal acinar cells to promote calcium influx in the absence of phosphoinositide metabolism. FEBS Lett 264:130-134 124. McMillian MK, Soltoff SP, Cantley LC, Rudel RA, Talamo BR (1993) Two distinct cytosolic calcium responses to extracellular ATP in rat parotid acinar cells. Br J Pharmacol 108:451-461 125. Lung MA, Wang JCC (1989) Autonomic nervous control of nasal vasculature and airflow resistance in the anaesthetised dog. J Physiol 419:121-139 126. Lung MA, Wang JCC (1989) Dose-related effects of adenosine5'-triphosphate on canine nasal vascular and airway resistances. Br J Pharmacol 90:736P 127. Watanabe H, Tsuru H, Kawamoto H, Yajin K, Sasa M, Harada Y (1995) Nitroxidergic vasodilator nerve in the canine nasal mucosa. Life Sci 57:109-112
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THE NEUROSCIENCES, Vol 8, 1996: pp 207–215
Purinergic cotransmission: parasympathetic and enteric nerves Charles H.V. Hoyle
The urinary bladder and the small intestine are presented as the principal models of purinergic cotransmission in the parasympathetic and enteric nervous systems, drawing upon evidence provided by functional, histochemical and ultrastructural studies. In the parasympathetic division ATP probably commonly transmits alongside acetylcholine, and in enteric nerves it is more likely to be transmitting alongside nitric oxide and VIP. Other organs, including some blood vessels and exocrine glands, in which there are hints that ATP might be involved as a parasympathetic cotransmitter are also given consideration.
cotransmitter, on which there is more information than there is on other regions such as the stomach or some of the sphincters (although interesting, these latter organs have not been discussed because of restrictions on space). Before a brief final summary and suggestions for future directions, other organs in which there are hints that ATP might be involved as a parasympathetic cotransmitter are given consideration.
Key words: acetylcholine / ATP / cotransmission / nitric oxide / vasoactive intestinal polypeptide
Anatomical considerations Parasympathetic ganglia lie very close to, or even embedded in, the organ that their postganglionic neurones innervate. Thus, in the vast majority of systems, it is difficult or impossible to denervate the target organ surgically. This is in marked contrast to the sympathetic system, in which the relatively long postganglionic nerves are more readily accessible for surgical intervention. Sensory neurones are also readily accessible in their dorsal roots. Pharmacologically, guanethidine and 6-hydroxydopamine are valuable tools in studying sympathetic activity since both these agents can destroy postganglionic sympathetic neurones. Capsaicin and resinifera toxin have proven useful for studying the sensory system as both these drugs can kill subpopulations of primary afferent neurones. For the parasympathetic system there are no comparable agents. Botulinum toxin and choline mustard derivatives have been used with limited success in parasympathetic studies, but these agents are dangerous to handle, and are not very specific, at best targetting cholinergic nerves rather than parasympathetic nerves.
©1996 Academic Press Ltd
IN THE PERIPHERAL nervous system ATP is better established as a cotransmitter in sympathetic, somatomotor and sensory nerves than it is in parasympathetic nerves. Although there are many examples where there is strong evidence that parasympathetic neuroeffector mechanisms utilize ATP as a neurotransmitter, the evidence for cotransmission tends to be rather circumstantial. The reasons for this lie in the anatomy of parasympathetic and enteric nervous systems, the lack of a particular feature of purinergic nerves that can be exploited histochemically, and in the lack of the right pharmacological tools. In this article the anatomical features that render organs innervated by the parasympathetic and enteric nervous systems difficult to denervate and the limited histochemical and pharmacological tools that are available are described. The urinary bladder is presented as the principal model of purinergic cotransmission in the parasympathetic nervous system, drawing upon evidence provided by functional, histochemical and ultrastructural studies. Then the small intestine is chosen as the model for ATP as an enteric
Histochemical localization of ATP
From the Department of Anatomy and Developmental Biology, University College London, Gower Street, London, WC1E 6BT, UK ©1996 Academic Press Ltd 1044-5765/96/040207 + 09 $18.00/0
At present there are no reliable histochemical stains for ATP, but a fluorescence method has been developed in which nerves that have taken up quinacrine 207
C. H. V. Hoyle can be visualized.1-3 Quinacrine has been demonstrated to form complexes with ATP, provided that the ratio of phosphate groups to quinacrine is high enough.4 The conjugation between quinacrine and ATP in forming the complex is strong enough to prevent ATP from diffusing down a concentration gradient.5,6 From studies on the guinea-pig taenia coli and urinary bladder it has been shown that following preloading of the tissue with [3H]-adenosine and [14C]-quinacrine, in sucrose density gradient homogenates the 3H and 14C peaks are correspondent. Since it has also been shown that [3H]-adenosine, when taken up by nerves, is rapidly phosphorylated with ATP being the preponderant product, these results imply that quinacrine is associated with adenine nucleotides. Just how specific quinacrine is for neurones that have a high ATP content is unknown:1 it will bind to DNA due to the high concentration of purine bases. Nevertheless quinacrine does bind compounds with transmitter-like properties, and when complexed it is released in a calcium-dependent fashion by depolarization.7,8 Quinacrine is taken up by platelets, which contain secretory granules that have a high concentration of ATP, but in platelets obtained from patients with storage pool deficiency, in which the levels of ATP are very low, there is little uptake of quinacrine.9,10 Organs innervated by postganglionic parasympathetic nerves, and regions of the gastrointestinal tract innervated by intrinsic enteric nerves that stain with quinacrine include: rat, guinea-pig and rabbit stomach and ileum myenteric plexus,1,3,7,11-14 rat colon,11 rat and mouse anococcygeus muscle,2,11 rabbit rectococcygeus,15 rabbit portal vein,15,16 guinea-pig and rabbit intrinsic cardiac ganglia.17 The uranaffin reaction may also be used to visualize neurones with high concentrations of ATP. In this reaction the divalent cation of uranium dioxide, UO22 + , complexes with the phosphate groups of ATP, and forms an electron-dense precipitate.18 In normal platelets and platelets depleted of serotonin by reserpine there is a strong reaction within storage granules, but in platelets from patients with storage pool deficiency there is a loss of reaction, as well as a loss of quinacrine staining.9,18 In the guinea-pig ileum, uranaffin reaction products can be seen in small vesicles with a diameter of 40–60 nm in nerve terminals in the myenteric plexus, submucous plexus, deep muscular plexus and circular muscle layer. Large granular vesicles, 80 nm in diameter, tend to remain unstained. Reactivity is unaffected by reserpine pretreatment or by surgical denervation of extrinsic
nerves, indicating that the nerves that contain the high levels of nucleotides are intrinsic and are not preganglionic.19,20
Urinary bladder The evidence in favour of both acetylcholine and ATP each being a neurotransmitter in the urinary bladder is strong, with functional and histological studies providing information (see refs 21-23 for reviews). In the absence of any direct evidence, the weight of the circumstantial evidence is indeed compelling that these two substances function as cotransmitters in this organ. The motor supply to the urinary bladder detrusor is provided by the parasympathetic nervous system, originating from the sacral or lower lumbar level, depending on species, of the spinal cord. The pelvic ganglia and intramural ganglia contain nerve cells that possess acetylcholinesterase activity, and in the smooth muscle of the detrusor there is an abundance of fibres that stain for acetylcholinesterase activity. Fibres that look like typical varicose autonomic nerve terminals are found in close association with the smooth muscle cells (see ref 24 for review). In the guinea-pig urinary bladder staining with quinacrine shows an extensive autonomic ground plexus, including intramural ganglia with varicose projections running to the smooth muscle.25-27 At an ultrastructural level autonomic nerve terminals in the bladder contain mixed transmitter-vesicle profiles,28-35 with cholinergic nerve terminals, i.e. those that contain mostly small clear vesicles, being the preponderant non-adrenergic type. There is no identifying vesicle morphology associated with purinergic nerves, and ATP has not been shown to be contained in the same vesicles as acetylcholine in postganglionic parasympathetic nerve terminals in the urinary bladder. Nor has the antithesis been shown, i.e. that ATP is not co-stored with acetylcholine in synaptic vesicles in the bladder. Certainly in other tissues, such as somatic nerve terminals innervating the torpedo ray electroplaque, the intravesicular acetylcholine:ATP ratio can be approximately 4:1, and the concentration of ATP may be as high as 120 mM.36 Based mostly on data obtained from somatic motor nerves, it has been stated that in all cholinergic vesicles where the analyses have been done, ATP is found at one fourth to one twelfth of the molar amount of acetylcholine.36 Unfortunately the list of tissues analysed does not include postganglionic 208
Parasympathetic and enteric cotransmission cholinergic excitatory junction potential (EJP) followed by a slow depolarization.65,70-73 Both the EJP and the slow depolarization may be associated with contractile events. The slow component is cholinergic, being abolished by atropine70-74 and represents a pharmacomechanical coupling between activation of the muscarinic receptors and the contractile apparatus, involving the inositol trisphosphate system. The EJPs, if large enough, will give rise to action potentials, and represent electromechanical coupling between the P2X-purinoceptor and the contractile apparatus. P2X-purinoceptors are ligand-gated ion channels, and in the bladder smooth muscle this channel is permeable to Na + and Ca2 + . 74-77 A feature of the P2-purinoceptor-mediated response is that it is only transient, whether evoked by nerve stimulation or applied ATP; there is some mechanism distal to receptor occupation that limits the duration of purinergic activity. Autoradiographic studies, utilizing tritiated α,β-methylene ATP as the radioligand have been carried out, and show a dense population of P2Xpurinoceptors in the smooth muscle of the urinary bladder detrusor of several species, including human.78,79 Furthermore, binding characteristics and ligand displacement profiles of the P2-purinoceptor have been determined in detrusor muscle membrane homogenates from rat, rabbit and human.79-82 All these are in line with it being a P2X-purinoceptor. Electrical stimulation of the postganglionic parasympathetic nerves in isolated preparations of guineapig urinary bladder detrusor muscle evokes release of ATP.25,26 That the release is prevented by the neurotoxin tetrodotoxin, but not affected by the sympatholytics guanethidine and 6-hydroxydopamine, indicates that the source of the ATP is parasympathetic nerves. ATP is not released from preganglionic parasympathetic nerve trunks in response to electrical stimulation,83,84 so in the isolated bladder preparations it almost certainly comes from the postganglionic nerve terminals. There is only one study that has directly addressed the question of whether or not ATP and acetylcholine are cotransmitters in the urinary bladder.54 In this work a partially purified botulinum toxin was utilized. Botulinum toxin is a parasympatholytic agent that destroys cholinergic but not purinergic nerves in the intestine.54 In the urinary bladder this toxin abolishes excitatory neuromuscular transmission, which implies that ATP is released from the same nerves that release acetylcholine.54 Another parasympatholytic agent is the choline mustard derivative AF64A. In the guineapig, sub-lethal doses of AF64A cause impairment of
parasympathetic nerves. Nevertheless it does not seem unreasonable to extrapolate such information. Responses to nerve stimulation and applied acetylcholine are potentiated by eserine (physostigmine), which inhibits acetylcholinesterase activity.37,38 Acetylcholine is released from postganglionic parasympathetic neurones in isolated preparations of the bladder.38 Moreover, choline is taken up by vesicular neurones, and is subsequently released as acetylcholine,39,40 and blockade of choline uptake by hemicholinium markedly reduces the release of acetylcholine.41 However, it seems to have become common practice to rely on atropine as an antagonist of muscarinic cholinoceptors to confirm or deny the role of acetylcholine in the transmission process. In many animals, including dogs, guinea-pigs, monkeys, rats, cats and humans stimulation of the preganglionic parasympathetic fibres either in the sacral ventral roots or in the pelvic nerve evokes contractile responses in the detrusor.42-53 In humans, baboons and rhesus monkeys contractions of the urinary bladder evoked by preganglionic parasympathetic nerve stimulation are abolished, or nearly so, by atropine.42-44 This indicates that in these old world primates and humans neuromuscular transmission is mediated only by acetylcholine acting on muscarinic receptors, and that cholinergic transmission is the predominant mechanism by which vesicular pressure is raised during voiding. In contrast, in the remaining species atropine causes at best only a partial inhibition of the motor response, indicating that in carnivores, rodents, lagomorphs, and new world primates there is a non-cholinergic component to postganglionic neuromuscular transmission in the bladder.22,23,44-46,48,52 Several studies have shown that the predominant or sole non-cholinergic neuromuscular transmitter in the urinary bladder detrusor, responsible for increasing intravesicular pressure or contraction is likely to be ATP (see refs 21-23). ATP mimics the contractions of the bladder due to stimulation of the postganglionic parasympathetic nerves in the presence of atropine.25,26,54-57 It acts on P2X-purinoceptors in the urinary bladder58 to evoke contractions, and these receptors can be blocked by antagonists such as photoactivated arylazidoaminopropionyl ATP (ANAPP3)59-63 or suramin,64 or following densensitization with α,β-methylene ATP.55,62,65-71 The important point here is that in all these cases the responses to ATP are blocked in parallel with the responses to stimulation of the non-cholinergic nerves. Electrophysiologically, stimulation of the postganglionic parasympathetic nerves evokes a non209
C. H. V. Hoyle synergistic interaction (see ref 91 for review) as found in the vas deferens where ATP and noradrenaline are sympathetic cotransmitters and noradrenaline markedly potentiates the postjunctional activity of ATP,92,93 but either facilitatory or inhibitory neuromodulatory interactions may be involved (see ref 91). In the urinary bladder acetylcholine potentiates ATP (unpublished observations).
cholinergic transmission in the urinary bladder, accompanied by an increase in the sensitivity of the P2X-purinoceptors, which is possibly indicative of an early stage of degeneration of the co-transmitting purinergic components.85 If we can accept it as fact that ATP and acetylcholine act as cotransmitters in the urinary bladder then we should also consider the functional significance. One would expect that in line with the natural themes and variations belonging to other combinations of cotransmitters there is a practical benefit to have ATP and acetylcholine as cotransmitters in the innervation of the urinary bladder as opposed to their being sole transmitters released from independent neuronal populations. In the bladder there is a marked temporal separation of the purinergic from the cholinergic excitation, whether evoked in vivo by stimulating a voiding cycle or in vitro by electrical field stimulation. The initial phase is transient and is purinergic, whereas the secondary sustained phase is cholinergic.51,86-89 The purinergic component is responsible for the initiation of voiding, whereas the cholinergic phase is responsible for the maintenance of voiding, the period of greatest vesicular pressure and during which most urine is expelled.48,86,87,89 In conscious rats, blocking the P2-purinoceptor results in increased bladder capacity and residual volume and decreased voiding pressure, but blocking the cholinoceptors results in decreased micturition pressure and micturition volume.90 The pattern of responses can be maintained by one type of nerve, i.e. one that uses ATP and acetylcholine as cotransmitters, because during prolonged activation there will still be only a transient rapid-onset initial phase mediated by ligandgated ion channels which is somehow self-limiting, and a sustained second phase, slower in onset because it is mediated by metabotropic receptors. Thus there is parsimony in using one population of neurones in this way because otherwise the activity of two populations of neurones would have to be co-ordinated. An aspect of the functional significance of cotransmission follows from the suggestion that the purinergic component is better developed in those animals that mark their territory by urination, and therefore need to urinate more frequently in short bursts (e.g. dogs, rats and New World primates) than it is in those animals that practice urinary continence until the need to empty the bladder arises (e.g. Old World primates and humans).23,44,52 Another aspect of the functional significance of cotransmission is that there should be an interaction of some sort between the cotransmitters.91 This is often manifest as a
Enteric nervous system The evidence that ATP acts as a neuromuscular transmitter in the enteric nervous system is now quite convincing, but whether or not it is a true cotransmitter with other substances has yet to be proven. Two other inhibitory substances are utilized widely throughout the gut, probably as cotransmitters, namely vasoactive intestinal polypeptide (VIP) and nitric oxide (NO).94 There is little evidence for the colocalization of ATP with these neurotransmitters, mostly because appropriate studies have not been attempted because of the lack of suitable tools. The quinacrine stain has been used in one investigation,11 but colocalization studies at the EM level with the uranaffin reaction have not been reported. Nevertheless there are many examples of regions of the gastrointestinal tract in which neuromuscular transmission is mediated by more than one transmitter, and generally, inhibitory transmission is carried out by members of the triumvirate, ATP, VIP and NO. It would be in keeping with the principles of autonomic transmission if indeed ATP were a true cotransmitter with these other substances. The evidence that ATP is a neurotransmitter in the gut has been reviewed extensively.21,95-97 The release of ATP, evoked by electrical stimulation of enteric nerves, has been measured.26,98,99 Pharmacological experiments have shown that responses to stimulation of the non-adrenergic, non-cholinergic nerves are blocked in parallel with the responses to applied ATP by P2-purinoceptor antagonists such as reactive blue 2100-103 or suramin,64,104,105 or the enzyme nucleotide pyrophosphatase.103,106 NO has only fairly recently been determined to be a neurotransmitter in the periphery, and it is now known to be synthesized in and released from enteric neurones. When its synthesis is inhibited so are the responses to neural stimulation, and application of authentic NO in aqueous solution or NO donor compounds mimic the responses of nerve stimulation and activate guanylate cyclase in the same way. This 210
Parasympathetic and enteric cotransmission evidence has been reviewed recently.107-110 VIP-containing nerves are well represented in the muscular coat throughout the gut, its release can be measured and in many instances exogenous VIP mimics responses to nerve stimulation. However selective antagonists of VIP-receptors have been slow in developing, and there is little evidence along the lines of pharmacological blockade of the receptor producing a parallel antagonism of responses to applied VIP and nerve stimulation.97,111 Histochemical localization of VIP with ATP has not been reported, but one study has attempted to colocalize ATP with NADPH-diaphorase, which can sometimes be utilized as a marker for nitric oxide synthase (NOS).11 In the myenteric plexus of the rat colon nearly all the quinacrinepositive nerves contain NADPH-diaphorase activity, but in the ileum and anococcygeus muscle only a subpopulation of quinacrine-positive nerves also contain NADPH-diaphorase activity.11 In the circular muscle of the guinea-pig ileum a single pulse of electrical field stimulation evokes a transient hyperpolarization that has a latency of onset of approximately 100 ms and a rise time of less than 500 ms, this is known as a fast inhibitory junction potential (IJP). This event is mimicked by local application of ATP and in parallel with the response to ATP it is abolished by apamin (a K + -channel blocker) or reactive blue 2 or following desensitization to α,β-methylene ATP.112,113 In the presence of apamin a short train of pulses of electrical field stimulation evokes a hyperpolarization that has a relatively long latency of onset, and a rise time approaching 1.5 s.113 This slow IJP is mimicked by VIP, and like the response to applied VIP it is antagnozied by the VIP-receptor antagonist VIP 10–28.113 Furthermore, when nitric oxide synthase is inhibited the slow IJP is attenuated while the fast IJP remains unaffected.112 Thus in this tissue ATP is the transmitter responsible for the fast IJP and both VIP and NO contribute to the slow IJP.112 It cannot be stated with certainty that these three substances are cotransmitters, but VIP is extensively colocalized with NOS in the nerves supplying the circular muscle,94,114 so these two are likely to be cotransmitters, but there is no evidence one way or the other as to whether or not ATP is released from this same population. Synaptosomal preparations of the ileal myenteric plexus contain a subpopulation of non-adrenergic vesicles that release ATP.115-117 Release of ATP is evoked by depolarizing stimuli, and by acetylcholine acting on nicotinic receptors.115,118 Since the release
of ATP from non-adrenergic vesicles is approximately 50% of that from the total pool of adrenergic and non-adrenergic vesicles, it follows that the nonadrenergic release comes from a substantial population of nerve terminals, from which it can be induced that ATP is costored with another transmitter substance. Physiologically the inhibitory transmission to the circular muscle is brought about via reflexes involved in descending inhibition. These reflexes prepare the bowel to receive the contents, and facilitate transit along the intestinal tract. It is not immediately clear what the advantage is of having a biphasic IJP or of having phases of fast and slow inhibitory transmission.
Other organs The rabbit portal vein has two muscle layers with the longitudinal muscle having a non-adrenergic, noncholinergic innervation that mediates relaxation independent of the endothelium. L-NAME partially inhibits this relaxation, an effect which is reversed by L-arginine. A combination of suramin and L-NAME abolishes neurogenic relaxation indicating that both ATP and NO are inhibitory transmitters in this vessel.119 This is possibly cotransmission from postganglionic parasympathetic nerves because terminal ganglia lie close to the rabbit portal vein and project into the muscle layers, and some of these ganglion cells stain with quinacrine.16,120 Salivary glands, in particular rat and mouse parotid glands, possess P2-purinoceptors that mediate intraacinar cell elevation of Ca2 + and amylase secretion.121-124 Following parasympathetic denervation, responses to ATP can be potentiated.124 This denervation supersensitivity is a hint that ATP is physiologically released from the parasympthatic nerve terminals, and since responses to stimulation of the parasympathetic nerves are markedly attenuated by atropine,122 it is possible that ATP is cotransmitting with acetylcholine in these glands. The nasal vasculature is under dual sympathetic and parasympathetic control125 with the parasympathetic element being responsible for non-cholinergic vasodilatation. Applied ATP also evokes dilatation,126 but rather than cotransmitting with acetylcholine it is more likely that the cotransmitter would be NO.127 211
C. H. V. Hoyle 10. McNicol A, Israels SJ, Robertson C, Gerrard JM (1994) The empty sack syndrome: a platelet storage pool deficiency associated with empty dense granules. Br J Haematol 86:574-582 11. Belai A, Burnstock G (1994) Evidence for coexistence of ATP and nitric oxide in non-adrenergic, non-cholinergic (NANC) inhibitory neurones in the rat ileum, colon and anococcygeus muscle. Cell Tissue Res 278:197-200 12. Crowe R, Burnstock G (1981) Comparative studies of quinacrine-positive neurones in the myenteric plexus of stomach and intestine of guinea-pig, rabbit and rat. Cell Tissue Res 221:93-107 13. Crowe R, Burnstock G (1981) Perinatal development of quinacrine-positive neurons in the rabbit gastrointestinal tract. J Auton Nerv Syst 4:217-230 14. Brizzi E, Calamai F, Staderini G, Viligiardi R (1983) The presence of purinergic, quinacrine-positive neurons in the rabbit stomach. Boll Soc Ital Biol Sper 59:962-964 15. Cocks T, Crowe R, Burnstock G (1979) Non-adrenergic, noncholinergic (purinergic?) inhibitory innervation of the rabbit rectococcygeus muscle. Eur J Pharmacol 54:261-271 16. Burnstock G, Crowe R, Wong HK (1979) Comparative pharmacological and histochemical evidence for purinergic inhibitory innervation of the portal vein of the rabbit, but not guinea-pig. Br J Pharmacol 65:377-388 17. Crowe R, Burnstock G (1982) Fluorescent histochemical localisation of quinacrine-positive neurones in the guinea-pig and rabbit atrium. Cardiovasc Res 16:384-390 18. Richards JG, Da Prada M (1977) Uranaffin reaction: a new cytochemical technique for the localization of adenine nucleotides in organelles storing biogenic amines. J Histochem Cytochem 25:1322-1336 19. Wilson AJ, Furness JB, Costa M (1979) A unique population of uranaffin-positive intrinsic nerve endings in the small intestine. Neurosci Lett 14:303-308 20. Wilson AJ, Furness JB, Costa M (1981) The fine structure of the submucous plexus of the guinea-pig ileum. II. Description and analysis of vesiculated nerve profiles. J Neurocytol 10:785-804 21. Hoyle CHV (1992) Transmission: purines, in Autonomic Neuroeffector Mechanisms (Burnstock G, Hoyle CHV, eds) pp 367-407. Harwood Academic Publishers, Chur 22. Hoyle CHV (1995) Non-adrenergic, non-cholinergic control of the urinary bladder. World J Urol 12:233-244 23. Hoyle CHV, Burnstock G (1993) Postganglionic efferent transmission in the bladder and urethra, in Nervous Control of the Urogenital System (Maggi CA, ed.) pp 349-381. Harwood Academic Publishers, Chur 24. Lincoln J, Burnstock G (1993) Autonomic innervation of the urinary bladder and urethra, in Nervous Control of the Urogenital Tract (Maggi CA, ed.) pp 33-68. Harwood Academic Publishers, Chur 25. Burnstock G, Cocks T, Crowe R, Kasakov L (1978) Purinergic innervation of the guinea-pig urinary bladder. Br J Pharmacol 63:125-138 26. Burnstock G, Cocks T, Kasakov L, Wong H (1978) Direct evidence for ATP release from non-adrenergic, non-cholinergic (‘purinergic’) nerves in the guinea-pig taenia coli and bladder. Eur J Pharmacol 49:145-149 27. Crowe R, Haven AJ, Burnstock G (1986) Intramural neurons of the guinea-pig urinary bladder: histochemical localization of putative neurotransmitters in cultures and newborn animals. J Auton Nerv Syst 15:319-339 28. Gilpin CJ, Dixon JS, Gilpin SA, Gosling JA (1983) The fine structure of autonomic neurons in the wall of the human urinary bladder. J Anat 137:705-713 29. Daniel EE, Cowan W, Daniel VP (1983) Structural bases for neural and myogenic control of human detrusor muscle. Can J Physiol Pharmacol 61:1247-1273
Summary and future directions Despite the title of this article there is no hard evidence for ATP acting as a cotransmitter in the parasympathetic and enteric nervous systems. However, based upon the received wisdom gained from studies of other divisions of the peripheral nervous system, where with appropriate tools it can be shown that ATP is indeed a cotransmitter, it is likely that cotransmission is the rule throughout the periphery rather than the exception. In postganglionic parasympathetic nerves ATP would commonly be transmitting alongside acetylcholine, and in enteric nerves it would be transmitting alongside NO and VIP. If the question of whether or not ATP is acting as a cotransmitter in the parasympathetic and enteric divisions were to be answered definitively, then new tools are needed. One’s wish-list would include a histochemical marker for purinergic nerves that is at least as good as the available immunocytochemical labels for any number of neuropeptides, a potent, specific competitive antagonist of the appropriate subtype of P2-purinoceptor, and a toxin that specifically destroys postganglionic parasympathetic nerves, and perhaps another toxin that selectively depletes synaptic vesicles of ATP.
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