European Review for Medical and Pharmacological Sciences 2008; 12(Suppl 1): 69-80 Tachykinin receptors and gastrointestinal motility: focus on humans

A. LECCI, M. ALTAMURA, A. CAPRIATI, C.A. MAGGI

Clinical Research Department, Menarini Ricerche, Florence (Italy)

Abstract. – of the tachykinin (TK) thors observed that this extract had myotropic ac- family were first discovered in the gastrointesti- tivity on isolated intestinal segments and postu- nal tissue about 75 years ago and supposed to lated that SP (P means powder, the physical form be involved in gastrointestinal (GI) motility. This of the extract) was released in the intestine and hypothesis has been repeatedly proven, al- though the role of TKs on motility is modulatory was the mediator responsible for its movements. rather than pivotal. Furthermore, beyond the well This hypothesis was verified 50 years later when known excitatory role, it has been acknowledged it became evident that TKs were, together with that TKs can also inhibit GI motility. TKs act at 3 acetylcholine, the main excitatory transmitters to 1 receptors termed as TK NK1 (NK1r), NK2 (NK2r), the gastrointestinal (GI) smooth muscle . During and NK3 (NK3r) receptors. The view gained the course of these 50 years the TK family en- through intense preclinical research suggested that motor effects induced by the stimulation of larged. Erspamer discovered, characterized and sequenced from non-mammalian species pep- NK2r were prominently mediated by a direct ac- tion on smooth muscle, those produced by the tides with SP-like biological activity which were 2 stimulation of NK1r were due to both muscular named tachykinins , i.e, fast relaxants of vascular and neuronal effects, whereas the motor effects smooth muscle, which were later shown to have 3 induced by NK3r were exclusively mediated by sequence homology to SP . In the early 1980s, neuronal effects. Recent functional and anatomi- three independent groups discovered novel mam- cal findings in humans are challenging this con- cept since NK r have been found in several kinds malian TKs which were named 2 (NKA) and B (NKB), and soon after elongated of myenteric neurons and selective NK2r antago- nists can, in particular conditions, produce GI forms of NKA were described (- motor effects likely related to a neuronal site of kappa and -gamma). At the eve of the new mil- action. Furthermore, the evidence for a myotrop- lennium, novel mammalian TKs were identified: ic role of NK1r is scarce, and very few studies, if hemokinin-1 (HK-1) and its elongated forms en- any, have documented a functional role for NK3r. 4,5 The findings that an acute or a long lasting dokinin A and B (Table I). TKs must share the common amidated C-ter- blockade of NK2r does not alter normal GI func- tions and that these receptors can modulate vis- minal motif Phe-X-Gly-Leu-Met-NH2 (were X ceral sensitivity are good starting points for test- has to be a non-polar amino acid) to exert biolog- ing this class of in GI diseases charac- ical functions through TK receptors5-7 which terised by altered GI motility. have been termed NK1 (NK1r), NK2 (NK2r), and NK3 receptors (NK3r) (Table I). N-terminal SP Key Words: metabolites, such as SP1-5, also exert biological Clinical studies, , , . effects, although these effects are not mediated by TK receptors but could involve a site regulat- ing the expression of delta opioid receptors8. TKs are encoded by 3 genes termed TAC1 (SP, NKA, neuropeptide-gamma and -kappa), TAC3 Introduction (NKB), and TAC4 (HK-1, endokinin A, B, C, and D) and each of these genes produces multi- (SP), the most famous component ple mRNA isoforms. Thus, both the beta- and of the tachykinin (TK) family, was first gamma-TAC1 mRNAs produce both SP and extracted from the horse brain and intestine by NKA, whereas both alpha- and delta-TAC1 only von Euler and Gaddum in 1931. The same au- encode SP.

Corresponding Author: Alessandro Lecci, BS; e-mail: [email protected] 69 A. Lecci, M. Altamura, A. Capriati, C.A. Maggi

Table I. Amino acid sequence of tachykinins and tachykinin-related peptides and their receptor preference. In bold, the com- mon C-terminal sequence. Receptor preference has been assessed in functional experiments on Ca2+ mobilization or luciferase 5-7 assay (both responses are dependent on phospholipase C activation) in cells expressing human NK1 NK2 NK3 receptors .

Peptides Amino acid sequence Receptor preference

Substance P Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 NK1 > NK3 > NK2

Neurokinin A His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 NK2 > NK1 > NK3 Neuropeptide-gamma Asp-Ala-Gly-His-Gly-Gln-Ile-Ser-His-Lys-Arg-His-

Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 NK2 > NK1 > NK3

Neuropeptide-kappa Asp-Ala-Asp-Ser-Ser-Ile-Glu-Lys-Gln-Val-Ala- NK2 > NK1 > NK3 Leu-Leu-Lys-Ala-Leu-Tyr-Gly-His-Gly-Gln-Ile- Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-

Gly-Leu-Met-NH2

Neurokinin B Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2 NK3 > NK2 > NK1

Hemokinin-1 Thr-Gly-Lys-Ala-Ser-Gln-Phe-Phe-Gly-Leu-Met-NH2 NK1 > NK3 > NK2 Endokinin A Asp-Gly-Gly-Glu-Glu-Gln-Thr-Leu-Ser-Thr-Glu-Ala- Glu-Thr-Trp-Val-Ile-Val-Ala-Leu-Glu-Glu-Gly-Ala- Gly-Pro-Ser-Ile-Gln-Leu-Gln-Leu-Gln-Glu-Val-Lys-

Thr-Gly-Lys-Ala-Ser-Gln-Phe-Phe-Gly-Leu-Met-NH2 NK1 > NK3 > NK2 Endokinin B Asp-Gly-Gly-Glu-Glu-Gln-Thr-Leu-Ser-Thr-Glu-

Ala-Glu-Thr-Trp-Glu-Gly-Ala-Gly-Pro-Ser-Ile-Gln- NK1 > NK3 > NK2 Leu-Gln-Leu-Gln-Glu-Val-Lys-Thr-Gly-Lys-Ala-Ser-

Gln-Phe-Phe-Gly-Leu-Met-NH2

Endokinin C Lys-Lys-Ala-Tyr-Gln-Leu-Glu-His-Thr-Phe-Gln-Gly-Leu-Leu-NH2 Antagonist at NK1 Endokinin D Val-Gly-Ala-Tyr-Gln-Leu-Glu-His-Thr-Phe-Gln-Gly-Leu-Leu-NH2 Antagonist at NK1 Hemokinin-1* Arg-Ser-Arg-Thr-Arg-Gln-Phe-Tyr-Gly-Leu-Met-NH2 NK1 > NK2 > NK3

*Rat and mouse sequence.

TK receptors belong to class 1 (rhodopsin- of GI function such as immune-inflammatory like) seven transmembrane G-protein-coupled re- processes, tissue integrity, intestinal barrier func- ceptors. All tachykinin receptors can activate tion, carcinogenesis, blood supply, secretion, af- classical transduction mechanisms linked to ferent signaling and motor regulation. Although phospholipase C activation, and all natural TKs all these aspects can influence each other, this ar- having the common C-terminal sequence act as ticle is focussed on the role of TKs and their re- full agonists on these pathways, whereas their in- ceptors on GI motor regulation exerted at the pe- trinsic activity can vary when measured on other ripheral level with particular emphasis on evi- G-protein mediated pathways (e.g., increase of dence collected in humans. In this respect, it is cyclic adenosine monophosphate). TKs can also worth noting that TKs can also modulate GI exert biological effects through non G-protein motility by acting at sites located in the central mediated mechanisms (e.g., sustained activation nervous system, as it occurs for the anti-emetic of mitogen-activated kinases) but the agonist or- action produced by blood-brain barrier-penetrat- der of potency or intrinsic activity is unknown ing NK1r antagonists. for this transduction pathway9. Interestingly, en- dokinin C and D, which have been termed as TK-related peptides because both of them have a Expression of TKs and Leu residue replacing the common C-terminal Their Receptors in the GI Tract Met of TKs10 exert an antagonist effect through 11 NK1 receptors . In the GI tract the most abundant TK-encod- In the GI tract TKs and their receptors are lo- ing mRNAs are beta- and gamma-TAC1. TAC4 calized on many cell types, including neurons transcripts have also been detected, although and nerve fibres and exhibit a remarkable degree these are likely to be related to immune, rather of plasticity in response to environmental than motor function. A few papers have de- changes or diseases. As a matter of fact, there is scribed the expression of NKB and none that of evidence that TKs play a role in several aspects TAC3, in spite of the robust evidence (especially

70 Tachykinin receptors and gastrointestinal motility: focus on humans

in animals) for the expression of NK3r in the ly emerging from the cell body) where SP could gut12. This pattern of TK gene expression im- colocalize with either or calretinin; plies that SP and NKA are the most abundant iv) a small minority (0.5%) of type V neurons TKs in the GI tract and are always colocalized. (neurofilament-positive, small smooth cell body Thus, SP and NKA are expressed on nerve fibres with a single stem process from which very long from both extrinsic neurons and intrinsic neu- dendrites and one axon emerge). rons as well as in somata and varicosities of in- Likewise, Holzer and Holzer-Petsche13 pro- trinsic neurons. A scheme of TK expression in posed a scheme for the distribution of NK1r, the GI tract has been outlined on the basis of NK2r and NK3r based on animal studies. Accord- morphological evidence obtained in laboratory ing to this scheme, smooth muscle cells (both in animals13. According to this scheme, TKs are ex- the CM and LM) and enterocytes express both pressed in: (i) intrinsic primary afferent neurons NK1r and NK2r, whereas blood vessels and inter- (IPANs) which contain both choline acetyltrans- stitial cells of Cajal (ICC) seem to express NK1r ferase and calbindin and project to both circular only. Indeed, evidence for the expression of NK3r muscle (CM) and mucosa; (ii) secretomotor neu- in smooth muscle cells (functional evidence in rons having the same chemical coding as above; cultured cells, morphological evidence in colonic (iii) ascending myenteric interneurons which human specimens) and ICC (mRNA in cultured contain both choline acetyltransferase and calre- or freshly isolated cells) has been described but tinin; (iv) ascending (excitatory) motor neurons their functional meaning remains to be clari- 12,15 to the CM with long projections colocalizing fied . NK1r are particularly dense in ICC locat- choline acetyltransferase, neurofilament protein ed in the deep muscular plexus and some im- and ; (v) ascending (excitatory) motor munoreactivity has also been observed in ICC lo- neurons to the CM with short projections colo- cated close to the myenteric plexus, whereas in calizing choline acetyltransferase, gamma- the human antrum NK1r have been detected in in- aminobutyric acid and enkephalin; (vi) ascend- tramuscular ICC only16,17. ing (excitatory) motor neurons to the longitudi- NK1r and NK3r have been invariably localized nal muscle (LM) which contain both choline to cell bodies and nerve fibres of the GI nerve acetyltransferase and calretinin; (vii) nerve fi- plexuses. NK3r were found on vasomotor neu- bres of capsaicin-sensitive extrinsic primary af- rons in the submucous plexus (SMP) where they ferent neurons which have their cell bodies in play a physiological role in mucosal stroking- dorsal root ganglia and send collaterals to the and distension-induced vasodilatation18. Still in sympathetic prevertebral ganglia. Capsaicin-sen- the SMP, NK1r have been localised to IPANs sitive neurons have the peculiarity to release (those expressing TKs, calbindin and choline TKs at both peripheral and central varicosities, acetyltransferase), whereas secretomotor neurons so that they can influence motility through containing either vasoactive intestinal polypep- mechanisms involving spinal reflexes or through tide (VIP) or (NPY) express both 16,19-21 an axon reflex involving a local release of TKs NK1r and NK3r . Likewise, NK1r and NK3r upon stimulation. have been found on SMP neurons of the human In the human GI tract the expression of TKs gut, but the functional role and the neurochemi- (SP in particular) has been detected in the fol- cal characterization of these neurons remain un- lowing neurons classified according to morpho- known23. In the myenteric plexus (MP) of ro- 14 logical characteristics proposed by Brehmer . SP dents, both NK1r and NK3r are expressed on was found in: (i) a minority of type I stubby neu- IPANS (calbindin-positive), ascending interneu- rons (small cell body, one axon, short, non- rons and motor neurons (both calretinin-posi- branched lamellar dendrites) some of which have tive), descending interneurons and motor neurons been hypothesized to be ascending interneurons, (both positive for nitric oxide synthase, the latter whereas those expressing enkephalin and project- ones also containing VIP). Colocalization of ing to the LM and CM could be cholinergic mo- NK1r or NK3r with NPY has also been described tor neurons; (ii) type II neurons (multi-axonal, in the small intestine16,24. non dendritic) which project to the mucosa, ex- In rodents, NK2r were only described in nerve press calretinin and/or somatostatin and have terminals22 which in guinea-pigs were shown to been proposed to be intrinsic primary afferent belong to descending interneurons expressing ni- neurons; (iii) type III neurons (non-nitrergic, one tric oxide synthase (NOS) or -releasing 25 axon and several long, branched dendrites radial- peptide . NK2r were also detected in nerve fibres

71 A. Lecci, M. Altamura, A. Capriati, C.A. Maggi within the muscle layers and some of these fibres rons providing excitatory inputs to both CM and 17 expressed TKs . At variance with laboratory ani- LM, and NK1r and NK2r are located on both CM mals, in humans specific immunolabeling of and LM to mediate contractions due to the reflex

NK2r is also present on somata, and in the human activation of these neurons induced by mucosal colon NK2r are widely expressed on both neu- stimulation or by stretch. Indeed, SP is released ronal cell bodies and fibres of the MP23. The from the guinea-pig small intestine when peri- chemical coding of neurons expressing NK1r, stalsis is evoked by increasing intraluminal pres- NK2r and NK3r in the human gut is at present not sure through a capsaicin-resistant but partially known, except for the finding of some colocal- hexamethonium-sensitive mechanism27. Depolar- ization of NK2r and TKs in nerve fibres within isation-induced co-release of similar amounts of muscle layers, as previously observed in guinea- both SP and NKA from guinea-pig colon motor pigs26. neurons has also been demonstrated28. Likewise, similar amounts of SP and NKA were con- tained29 and released during the ascending excita- TK Receptors and GI Motility tory reflex in the human small intestine30. Whether TKs and acetylcholine are simultane- Given the extensive distribution of TKs and ously co-released is yet an unsolved issue. Func- their receptors in GI neurons and effector cells tional studies based on the effect of TK antago- respectively, it is no wonder that TKs can affect nists suggest that moderate intestinal distension GI motility by acting through multiple mecha- would induce the release of acetylcholine only, nisms and sites of action (even with opposite whereas more intense stimuli would be required consequences) within the GI wall. In this respect to induce the release of TKs in both humans and 30-32 preclinical models have highlighted that NK1r or animals . However, the difference between NK3r agonists can elicit either (or both) excitato- mild and intense stimulation in terms of release ry and inhibitory effects on GI motility, whereas of mediators could be quantitative rather than the NK2r agonists almost invariably produce ex- qualitative. citatory motor effects. However, the indiscrimi- In this context it should be pointed out that the nate access to receptors of exogenously adminis- efficiency of cholinergic mechanical coupling on tered agonists renders this approach physiologi- GI smooth muscle is much greater than that ob- cally unreliable, whereas studies with antagonists served with TKs33, and this implies that TK-me- allow to appreciate the role of discrete diated components of contractions are not evi- tachykininergic pathways in well-defined physio- dent unless muscarinic receptors are blocked. In- logical and pathological conditions. Overall pre- deed, this actually occurs in most of animal and clinical evidence indicates that TKs produce ex- human GI preparations when motility is elicited citatory effects by acting on smooth muscle through electrical stimuli or GI wall distension

(NK1r and NK2r), ICC (NK1r) and neurons since NK1r or NK2r antagonists have a very small (NK1r, NK2r, and NK3r), whereas TK-induced in- inhibitory effect (if any) on -sensitive hibitory effects are attributable to neuronal ef- contractions or peristalsis13. A notable exception fects (NK1r, NK2r, and NK3r) although other is represented by the colonic CM, where NK2r mechanisms cannot be excluded. antagonists decrease the amplitude of contrac- The involvement of TK receptors in three as- tions induced by electrical field stimulation pects of motility will be discussed: (i) active con- (EFS) even when muscarinic receptors are tractions elicited by the activation of excitatory viable34-36. Indeed, in all human segments exam- motor neurons that are part of the ascending exci- ined so far, i.e., esophagus37, ileum38, and colon39, tatory reflex involved in peristalsis; (ii) regulation NK1r and NK2r are expressed on both CM and of basal smooth muscle tone which is thought to LM. Despite the fact that selective NK1r and tune the threshold for both the activation of motor NK2r agonists evoke contractions, the inhibitory reflexes and visceral sensitivity; and (iii) direct effect of NK1r antagonists on non-adrenergic, activation of neuronal afferent pathways leading non-cholinergic (NANC) components of EFS-in- to an inhibition of reflex motility. duced contractions has been only observed in the human ileum and it was limited to short-lasting Active Contractions stimuli38,40. An inhibition of the cholinergic com- As pointed out in the previous paragraph, TKs ponent of EFS-induced twitches by an NK1r an- are colocalized with acetylcholine in motor neu- tagonist has also been described in the human

72 Tachykinin receptors and gastrointestinal motility: focus on humans colon LM, but this effect has been observed with CM35. As is shown in Figure 1, the sum of the in- antagonist concentrations which far exceed those hibitory effects produced by the muscarinic re- 41 specific for NK1r . The relative roles of TK re- ceptor and NK2r antagonists on their own is larg- ceptors in eliciting direct smooth muscle contrac- er than 100% at all frequencies, indicating that tions by exogenous application of selective ago- part of the cholinergic component is reduced by nists or by the release of endogenous TKs from NK2r antagonists and that part of the TK-mediat- ascending motor neurons in human GI segments ed component is inhibited by muscarinic receptor are summarized in Table II. antagonists. In the human colon, the largest component of In conclusion, in human GI segments, contrac-

NK2r-mediated contractions is due to a direct ef- tions due to the activation of motor neurons is me- fect on smooth muscle. A small atropine-sensitive diated by both acetylcholine and TKs acting 4-10 component has been described when NKA was through muscarinic and NK2r, respectively. In most 42 used as the contractile agent . However, this of intestinal segments the NK2r-mediated compo- finding was not replicated when the human colon nent is not evident unless transmission via mus- 43 CM was contracted by NK2r selective agonists . carinic receptors is blocked. In the human colon, a In this latter case, a small indomethacin-sensitive part of the cholinergic component is modulated by component has been detected, indicating that the NK2r, and selective antagonists are able to reduce stimulation of NK2r produces prostanoid re- part of the atropine-sensitive contractions. lease43.

The issue whether stimulation of NK2r can in- Spontaneous Motility duce the release of acetylcholine is not yet In addition to propulsive contractions outlined solved. Although NK2r have been localised to above, intestinal movements include two other nerve varicosities colocalizing TKs and acetyl- aspects: small amplitude rhythmic phasic con- 23 48 choline , no direct evidence for NK2r-induced tractions and smooth muscle tone . Rhythmic acetylcholine release has been provided yet. De- smooth muscle contractions and tone are thought spite this, several results (mostly preclinical) sug- to underlie the mixing of the luminal contents gest that such an interaction occurs. In both small and to regulate the threshold for the activation of intestine (guinea-pig) and colon (guinea-pig and motor and secretory reflexes but also for visceral rabbit) the inhibitory effect of sub-effective con- sensitivity, respectively. Rhythmic smooth mus- centrations of muscarinic antagonists on peristal- cle contractions developing spontaneously in vit- sis is enhanced by the addition of NK2r antago- ro and in vivo are believed to be largely myo- 44-46 nists . Furthermore, NK2r antagonists reduce a genic due to the propagation of slow waves gen- component of colonic hypermotility (giant con- erated by ICC pacemakers to the smooth muscle. tractions) elicited by intraluminal irritation, However, these rhythmic contractions are subject whereas the inhibitory effect of atropine is com- to neural regulation through both excitatory and plete, indicating that endogenous TKs act inhibitory inputs49. In the rat colon especially, the 47 through NK2r to enhance cholinergic motility . neural inhibitory modulation prevails since in the A similar conclusion can be drawn on the basis presence of tetrodotoxin or a NOS inhibitor the of the analysis of sequential addition of mus- amplitude of CM contractions is increased and carinic and NK2r antagonists or vice-versa on the effect of the combination of these drugs is not EFS-induced contractions in the human colon additive50,51. Nevertheless in NANC conditions,

Table II. Contractile effect induced by selective NK1r, NK2r, and NK3r agonists and inhibitory effect by selective NK1r, NK2r, NK3r antagonists (ant) on EFS- or NANC EFS-induced contractions. Legend: + = contraction; 0 = no effect; - = inhibition; n.a. = not assessed.

GI Segment NK1r agonist NK2r agonist NK3r agonist EFS+ NANC-EFS+

Oesophagus CM 0 + 0 n.a. NK2r ant - Oesophagus LM 0 + 0 n.a. 0

Ileum CM + + 0 0 NK1r ant - NK2r ant - Ileum LM + + 0 n.a. n.a.

Colon CM + + 0 NK2r ant - NK2r ant - Colon LM 0 + 0 n.a. n.a.

73 A. Lecci, M. Altamura, A. Capriati, C.A. Maggi

Normal Normal SR48968 Atropine ) Atropine + SR48968 2 SR48968 + Atropine Stress (mN/cm

Frequency (HZ) Frequency (HZ)

Human colon CM: inhibition produced by atropine or SR48968 expressed as % of inhibition produced by both antagonists

Hz Atropine SR48968

0.1 100% 100%

0.5 72% 83%

1.0 82% 85%

5.0 78% 47%

10.0 72% 50%

Figure 1. Effect of sequential addition of NK2r and muscarinic antagonists or muscarinic plus NK2r antagonists on frequen- cy-response curves on human colon CM (from 35 with permission of the publisher).

there is evidence that TKs play a modulatory role but not NK1r antagonists decreases resting tone on spontaneous, rhythmic, hexamethonium-resis- (up to 70% of the maximal effect of ) tant contractions since NK2r antagonists reduce in a concentration-dependent manner. The source spontaneous mechanical activity by about 50% from which TKs are released to mediate this ef- 50). A similar effect has been observed in the fect remains obscure since the relaxant effect of isolated human colon, where NK2r antagonists NK2r antagonists was resistant to apamin, NOS produce a concentration-dependent inhibition inhibitors, capsaicin pretreatment, indomethacin, (max 80%) of spontaneous phasic contractions52. tetrodotoxin, omega-conotoxin, hexamethonium, Interestingly, in this latter preparation neither nifedipine and to the removal of the mucosa54. No

NK1r antagonists nor atropine have any effect evidence for a relaxant effect by NK2r antagonists whereas tetrodotoxin substantially inhibits these has been provided in isolated human preparations. contractions, suggesting that TKs could be re- The selective NK2r antagonist nepadutant, howev- leased from tetrodotoxin-sensitive nerves other er, produced a significant increase of rectal com- than excitatory motor neurons52. pliance during ascending limit balloon distension A further effect of TKs which contributes to in volunteers who had previously received a glyc- spontaneous motility of GI smooth muscle con- erol enema12. Interestingly, glycerol enema has cerns the modulation of basal tension and intralu- been shown to induce a mild mucosal inflamma- minal pressure. In the mouse stomach neither tion and a reduction of rectal air nitric oxide55, 56 NK1r nor NK2r antagonists affect spontaneous suggesting that, as it occurs in animals , NK2r mechanical activity under normal conditions. antagonists could be able to reduce colonic CM

However, NK2r but not NK1r antagonists produce tone following NOS inhibition in humans, too. a concentration-dependent gastric relaxation in mdx dystrophic mice or in normal mice pretreated Neuronal Modulation of Motility with a NOS inhibitor53. Likewise, in isolated rat Beyond the relatively well characterized exci- small intestinal segments the application of NK2r tatory effects at the neuromuscular junction, TKs

74 Tachykinin receptors and gastrointestinal motility: focus on humans exert a role in the communica- to colonic distension without altering visceral no- tion amongst neurons in both MP and SMP. In ciception under normal conditions, whereas NK3r this context, both NK1r and NK3r contribute to antagonists produce a clear visceral hyposensitiv- slow excitatory postsynaptic potentials of MP ity in normal animals19,56. Interestingly, the effect

IPANs during the activation of ascending excita- of NK2r antagonists was associated with a specif- tory pathways while NK3r also participate in de- ic reversal of colonic distension-induced hyper- scending inhibitory circuitries19,57. Indeed, GI excitability and c-fos expression in spinal cord motor activity can be altered by TK agonists and dorsal horn neurons following colonic inflamma- antagonists also through an interaction with re- tion, whereas the responses elicited by electrical ceptors located on neuronal excitatory or in- stimulation of the pelvic nerve or exposure to so- hibitory motor pathways. In the preclinical set- matic noxious stimuli remained unaffected56. Al- ting, either the selective stimulation of NK3r or terations of the sensory transmission elicited by the concomitant stimulation of both NK1r and colonic distension have been described in NK1r 69 NK3r activates acetylcholine- and TK-expressing knock-out mice , and reversal of visceral hyper- 58,59 excitatory motor neurons , whereas a possible sensitivity by NK1r antagonists has been also re- 70 neuronal excitatory role of NK2r has been identi- ported but it is unclear if these effects are exert- fied in hexamethonium-resistant guinea-pig ed at the peripheral or central nervous system small intestine peristalsis60. On the other hand, level. the selective stimulation of NK1r by SP produces a nitric oxide-dependent inhibitory effect on on- going guinea-pig small intestine peristalsis61.

NK3r-mediated nitric oxide-dependent and -inde- Clinical Studies pendent inhibitory motor effects have been re- ported in guinea-pig small and large intestine The availability of potent and selective antago- preparations62,63. As a matter of fact, the selective nists at the human TK receptors could have theo- antagonism of NK1r, NK2r, or NK3r has been retically allowed a progress in the understanding shown to accelerate rabbit isolated colon peristal- about the role of these receptors in human GI sis64 or intestinal transit in a rat model of surgical diseases. Unfortunately, the progress made was 65 ileus . Unfortunately, no evidence has been pro- quite modest. , a selective NK1r antag- vided to indicate if such neuronal NK1r, NK2r, or onist, showed a promising effect in a pilot trial in NK3r-mediated excitatory or inhibitory mecha- IBS, suggesting a role of these receptors in the nisms operate in isolated human specimens, too. modulation of visceral sensitivity71. However,

Other neuronal targets through which TKs can blockade of NK1r had no effect on acid-induced potentially affect GI motility are fibres of extrin- hypersensitivity in the human esophagus72. At the sic primary afferent (DRG) neurons. The subset colonic level, a NK1r antagonist reduced both the of capsaicin-sensitive neurons is known to be a compliance and the volume threshold for dis- source of TKs but rarely a target for these pep- comfort under isobaric conditions but not when tides. Nevertheless, NK3r have been detected in distension was performed by the method of as- mouse nodose ganglion neurons projecting to the cending limits in healthy volunteers73. Interest- 66 viscera , and functional evidence for the expres- ingly, the stimulation of NK1r triggers a relax- sion of NK2r in capsaicin-sensitive pelvic DRG ation on precontracted human colon CM and neurons has been also provided67,68. Obviously an LM, but this response was impaired in specimens interference with reflex GI motility exerted taken from patients with inflammatory bowel through the activation of extrinsic sensory fibres diseases74. These patients suffer from diarrhea, is expected to occur in vivo rather than in vitro, and diarrhea has been reported as a common ad- although it should not be forgotten that cap- verse event in trials in which NK1r antagonists saicin-sensitive neurons are capable to release were tested as -antimigraine drugs75. mediators locally upon stimulation, which allows Overall, these results suggest that descending for a modulatory effect to occur even in vitro. motor neurons regulating colonic tone are toni-

A peripheral modulation of visceral sensitivity cally activated by NK1r and that blockade of this in preclinical models of colonic distension has mechanism could stimulate colonic motility. been clearly demonstrated by either NK2r or The blockade of NK3r by talnetant did not af- NK3r antagonists. In fact, NK2r antagonists revert fect colonic compliance nor sensory threshold inflammation- or stress-induced hypersensitivity recorded by the method of ascending limits or

75 A. Lecci, M. Altamura, A. Capriati, C.A. Maggi random phasic distensions in healthy nist on accommodation occurs during inflamma- volunteers76. The same antagonist had no effect tion only or under normal conditions, too. Ani- on IBS symptoms in 2 phase II studies where a mal studies indicate that NK2r antagonists do not wide range of doses (20-800 mg/day) was ad- affect GI compliance under normal conditions ministered to a consistent number of patients77. but do so following pharmacologically induced 53,56 The effect of NK2r antagonists in patients suf- or genetically determined NOS inhibition . fering from GI diseases has not yet been report- However, NK2r antagonists tend to decrease ed. The available evidence indicates that the in- compliance during guinea-pig small intestinal travenous infusion of NKA increases small in- peristalsis44 or in the rat colon during physiologi- testinal motility recorded by manometry in cal isovolumetric recordings47. healthy volunteers. The fasting pattern of the mi- The dose of nepadutant used in the above grating motor complex was disrupted after NKA mentioned clinical studies (8 mg intravenously) administration, since the total duration of phase allowed to block the GI effects of exogenously II motility (>2 contractions/min, without burst administered or endogenously released TKs act- organization) as well as the amplitude and fre- ing at NK2r, but the duration of this blockade was quency of contractions was increased, whereas limited to 2-3 hours78. Since the time frame of GI the duration of phase I motility (periods of rela- functions outlasts this period, a further study was tive quiescence with <2 contractions/min) was aimed at evaluating the effects of a longer lasting decreased. In contrast, phase III motility (bursts blockade of NK2r. Therefore, a larger dose of of propagating contractions with a frequency of nepadutant (16 mg intravenously every 12 h) was 11-12 contractions/min) was not altered by administered for 8 days in healthy male and fe-

NKA. Nepadutant, a selective NK2r antagonist, male subjects who had to fill in a questionnaire prevented the motor effects induced by NKA ad- reporting the bowel habits (number of bowel ministration in healthy volunteers without alter- movements and stool consistency according to ing the normal pattern of the migrating motor the Bristol Scale of Stool Form) of the day be- complex in volunteers treated with saline. Inter- fore. Nepadutant did not alter bowel habits as estingly, volunteers treated with NKA and place- compared to the baseline (the day before starting bo (n=10) experienced a series of gastrointestinal the treatment). Interestingly, in the group treated adverse events (n=10) including abdominal pain, with placebo, bowel movements significantly de- nausea, vomiting and borborygmi, whereas no creased during the first day of treatment and such adverse events were recorded in the group thereafter returned to the baseline value; no such receiving both NKA and nepadutant78. Overall decrease was observed in the group treated with 12 these results indicate that the blockade of NK2r nepadutant . We interpreted the drop of bowel does not affect physiological motility of the movements observed in the placebo group as small intestine or GI sensitivity, whereas the stress-related due to the expectation of receiving stimulation of these receptors increases motility 2 injections a day and to be confined to the clini- and alters GI sensitivity. cal unit for several days. Whatever the cause of The effect of nepadutant, at the same dose this drop, the fact that nepadutant can prevent an used as in the above mentioned study, was also inhibition of motility (whatever the cause of this investigated on sensory thresholds and compli- inhibition), provides a sort of proof of concept ance tested by rectal distension with a barostat by for the possibility to normalise bowel habits by the method of ascending limits. This study was blocking NK2r in intestinal (or other peripheral) carried out in healthy volunteers challenged with neurons. The presence of NK2r on varicosities of a glycerol enema. Nepadutant did not significant- guinea-pig myenteric neurons expressing either ly alter sensory thresholds, which were on the NOS or gastrin-releasing peptide25 provides an other hand only marginally decreased by glyc- anatomical substrate to explain a NK2r-mediated erol. However, a small but significant increase in inhibitory modulation of GI motility. rectal compliance (larger balloon volume at con- stant pressure) was recorded after nepadutant as compared to placebo12, suggesting that in the presence of mild inflammation, NK2r blockade Conclusions facilitates the accomodation of smooth muscle in response to radial stretch. A question arises TKs are important modulators of GI physiolo- whether this modulatory effect of a NK2r antago- gy, being able to affect motor, secretory and sen-

76 Tachykinin receptors and gastrointestinal motility: focus on humans

sory functions. Animal studies have provided ev- 7) KURTZ MM, WANG R, CLEMENTS MK, CASCIERI MA, AUSTIN CP, CUNNINGHAM BR, CHICCHI GG, LIU Q. idence that the stimulation of both NK1r and NK r located on effector cells (either ICC and Identification, localisation, and receptor charac- 2 terisation of novel mammalian substance P-like smooth muscle or smooth muscle only, respec- peptides. Gene 2002; 296: 205-212. tively) mediate the excitatory NANC component due to activation of motor neurons. An increase 8) GUAN JS, XU Z-Z, GAO H, HE S-Q, MA GQ, SUN T, WANG L-H, ZHANG Z-N, LENA I, KITCHEN I, ELDE R, of motility through stimulation of neuronal NK1r ZIMMER A, HE C, PEI G, BAO L, ZHANG X. Interaction or NK3r has also been shown. On the other hand, with vesicle luminal protachykinin regulates sur- in adequate preclinical models it is possible to face expression of delta-opioid receptors and opi- appreciate an inhibitory modulation of motility od analgesia. Cell 2005; 122: 619-631. exerted by neuronal NK1r, NK2r or NK3r. 9) LECCI A, MAGGI CA. Tachykinins and their recep- tors. Encyclopedic reference of molecular phar- In humans, NK2r play a pivotal role in motility by acting on either smooth muscle or neurons. macology. S. Offermanns and W. Rosenthal, eds. Springer, Heidelberg 2008. No NK3r-mediated motor effect has been yet de- scribed. The NANC excitatory input to both CM 10) PATACCHINI R, LECCI A, HOLZER P, M AGGI CA. Newly and LM is almost exclusively provided by NK r. discovered tachykinins raise new questions about 2 their periphearl roles and the tachykinin nomen- Neuronal NK1r or NK2r exert an inhibitory brake clature. Trends Pharmacol Sci 2004; 25: 1-3. on motility, although these receptors seem to op- erate in different contexts. Overall the results ob- 11) NAONO R, NAKAYAMA T, I KEDA T, M ATSUSHIMA O, NISHI- MORI T. Leucine at the carboxyl-terminal of en- tained in humans indicate that NK1r antagonists dokinins C and D contributes to elicitation of the could be useful in motility disorders character- antagonistic effect on substance P in rat pain pro- ized by constipation whereas NK2r antagonists cessing. Brain Res 2007; 1165: 71-80. could be tested in disorders characterized by ei- 12) LECCI A, CAPRIATI A, ALTAMURA M, MAGGI CA. ther diarrhea or constipation. Tachykinins and tachykinin receptors in the gut, 79 A recent paper has shown that administration with special reference to NK2 receptors in humans. Auton Neurosci Basic Clin 2006; 126-127: 232-249. of anti-emetic doses of aprepritant (a NK1r antag- onist) does not alter GI or colonic propulsion in 13) HOLZER P, H OLZER-PETSCHE U. Tachykinin receptors healthy volunteers. in the gut: physiological and pathological implica- tions. Curr Opin Pharmacol 2001; 1: 583-590.

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