Neuropeptide Receptor Ligands for the Treatment of Schizophrenia: Focus on Neurotensin and Tachykinins.

There is a wealth of evidence that various neuropeptides and their receptor ligands modulate schizophrenia- related behaviors in preclinical animal models, suggesting that neuropeptide systems may represent potential novel therapeutic targets for the treatment of schizophrenia. In particular, neurotensin and tachykinins have been the subject of significant research efforts, generating compelling preclinical data in the schizophrenia field. However, clinical studies with notably selective tachykinin NK3 receptor antagonists in schizophrenia have been disappointing, and they were unable to confirm the promising therapeutic potential from animal studies, thereby questioning the therapeutic utility of these compounds for this condition. This article reviews preclinical and clinical findings on ligands for neurotensin and tachykinin receptors in schizophrenia, and provides possible explanations for the failure so far to develop small-molecule neuropeptide ligands for the treatment of schizophrenia.

Targeting tachykinin receptors for the treatment of functional gastrointestinal disorders with a focus on irritable bowel syndrome.

BACKGROUND: Tachykinins (TKs) are a family of endogenous peptides widely expressed in the central and in the peripheral nervous systems as well as in the gastrointestinal (GI) tract. They act as full agonists at three different membrane receptors neurokinin (NK) 1, NK2, and NK3, which are G protein-coupled receptors and in the GI tract are expressed both on neurons and effector cells. PURPOSE: This article reviews the literature concerning the role of TKs in the GI tract function in physiological and pathological conditions and their potential relevance in the treatment of functional GI disorders with particular reference to irritable bowel syndrome (IBS). The efficacy of NK1 antagonists in chemotherapy-induced and postoperative nausea and vomiting is well established. While pharmacodynamic studies have reported conflicting and negative results concerning the effects of NK1 and of NK3 antagonists, respectively, on the GI tract function in humans, clinical studies applying the NK3 antagonist talnetant in IBS-D were negative. Pharmacodynamic studies applying NK2 antagonists have suggested a role for antagonism of NK2 receptors in modulation of GI chemical-induced altered motility and of stress-induced altered bowel habits. Clinical studies and in particular a recently completed Phase 2 study have reported that the NK2 antagonist ibodutant is effective and safe in treating symptoms of D-IBS, especially in females.

Natalisin, a tachykinin-like signaling system, regulates sexual activity and fecundity in insects.

An arthropod-specific peptidergic system, the neuropeptide designated here as natalisin and its receptor, was identified and investigated in three holometabolous insect species: Drosophila melanogaster, Tribolium castaneum, and Bombyx mori. In all three species, natalisin expression was observed in 3-4 pairs of the brain neurons: the anterior dorso-lateral interneurons, inferior contralateral interneurons, and small pars intercerebralis neurons. In B. mori, natalisin also was expressed in two additional pairs of contralateral interneurons in the subesophageal ganglion. Natalisin-RNAi and the activation or silencing of the neural activities in the natalisin-specific cells in D. melanogaster induced significant defects in the mating behaviors of both males and females. Knockdown of natalisin expression in T. castaneum resulted in significant reduction in the fecundity. The similarity of the natalisin C-terminal motifs to those of vertebrate tachykinins and of tachykinin-related peptides in arthropods led us to identify the natalisin receptor. A G protein-coupled receptor, previously known as tachykinin receptor 86C (also known as the neurokinin K receptor of D. melanogaster), now has been recognized as a bona fide natalisin receptor. Taken together, the taxonomic distribution pattern of the natalisin gene and the phylogeny of the receptor suggest that natalisin is an ancestral sibling of tachykinin that evolved only in the arthropod lineage.

Insight into molecular and functional diversity of tachykinins and their receptors.

Tachykinins (TKs) and their structurally related peptides constitute the largest peptide superfamily in the animal kingdom. TKs have been shown to play various physiological roles not only as major brain/gut peptides but also as endocrine/paracrine hormones in chordates and exocrine factors in amphibians. Recent studies have also revealed that the biological roles of TKs as brain/gut peptides and endocrine/paracrine factors are essentially conserved in protochordates, and that alternative splicing mechanism in mammalian TK genes were established during the evolution of vertebrates. Protostomes possess two structurally and functionally different peptides; invertebrate TKs (inv-TKs) serve as toxin-like compounds secreted from the salivary gland of several organisms, whereas TK-related peptides (TKRPs) are functional counterparts for chordate TKs. Additionally, a TKRP-like sequence was detected in a diploblastic organism. The dramatic difference in structural organizations between TKRP precursors and chordate TK precursors clearly indicates the distinct evolutionary processes of TKs and TKRPs. Despite high sequence homology, TK receptors manifest selective affinity to their endogenous ligands, while TKRPs exhibit redundant activity at their receptors. Moreover, in addition to nociceptive, inflammatory, and contractile effects as brain/gut peptides, a number of studies have revealed novel biological effects of TKs on the hypothalamus and genital organs, revealing the biological roles of TKs as pivotal regulators of reproduction. These findings shed light on complicated evolutionary lineages of both structures and functions of the TK/TKRP superfamily and their receptors. In this review, we present basic and latest knowledge of the TK/TKRP superfamily with various points of view.

Neuropeptide receptor ligands as drugs for psychiatric diseases: the end of the beginning?

The search for novel drugs for treating psychiatric disorders is driven by the growing medical need to improve on the effectiveness and side-effect profile of currently available therapies. Given the wealth of preclinical data supporting the role of neuropeptides in modulating behaviour, pharmaceutical companies have been attempting to target neuropeptide receptors for over two decades. However, clinical studies with synthetic neuropeptide ligands have been unable to confirm the promise predicted by studies in animal models. Here, we analyse preclinical and clinical results for neuropeptide receptor ligands that have been studied in clinical trials for psychiatric diseases, including agents that target the receptors for tachykinins, corticotropin-releasing factor, vasopressin and neurotensin, and suggest new ways to exploit the full potential of these candidate drugs.

The tachykinergic system as avenues for drug intervention.

This review discusses a family of receptors that interact with a family of small peptides that belong to the tachykinin family. The three subtypes of tachykinin receptors follow the following suggested nomenclature: NK1, NK2 and NK3. These receptors belong to the 7-transmembrane, G-protein coupled family. The NK receptor family has been implicated in a wide range of disorders, cancers, inflammation and neural-related diseases. The tachykinin NK receptors and their ligands have been implicated in solid and hematological tumors. Antagonists to NK receptors have been synthesized and some have been tested in preclinical and Phase II clinical studies. However, in order to be effective for treatment, it is imperative to understand the biology of NK receptor family as well as related molecules. One such molecule is HGFIN, also referred as nmb and osteoactivin. HGFIN is particularly relevant since it has been shown to interact with the high affinity ligand of NK1, substance P. This report discusses promising applications for targeting the NK receptors. The discussion proposes potential mechanisms that could occur when substance P interacts with HGFIN as well as the role of HGFIN when antagonists are used to block the NK receptors. This review is relevant for central and peripheral nervous system drug development and cancer targets and also discusses the indications for current patents.

[Vesical urothelium and new concepts]. / L'urotelio vescicale: nuovi concetti e possibili prospettive terapeutiche.

Vesical urothelium was long considered to simply be a protection barrier, which passively separates the urinary content from the underlying smooth muscle and the blood stream. Recent observations, though, have pointed out that vesical urothelium cells have clear active and sensory functions, in response to various physical and chemical stimuli. Among these characteristics are the expression of several neurotransmitters and receptors: Acetylcholine, Nitric Oxide, VIP, CGRP, NKA, SP and cholinergic, vanilloid, purinergic, and tachykinin receptors. Urothelium-produced neurotransmitters are likely supposed to act through a receptor stimulation of the afferent nerve fibers within the sub-urothelial spaces. Sub-urothelial myofibroblasts are considered to play a mediation role between urothelium-produced neurotransmitters and the underlying receptors. According to these observations, a pharmacologic modulation, directly affecting the urothelium, can be hypothesized.

Diverse roles of G-protein coupled receptors in the regulation of neurohypophyseal hormone secretion.

The magnocellular neurones in the supraoptic nucleus project to the neural lobe and release vasopressin and oxytocin into the peripheral circulation, where they act on the kidney to promote fluid retention or stimulate smooth muscles in the vasculature, uterus and mammary glands to support blood pressure, promote parturition or induce milk let-down, respectively. Hormone release is regulated by complex afferent pathways carrying information about plasma osmolality, blood pressure and volume, cervical stretch, and suckling. These afferent pathways utilise a broad array of neurotransmitters and peptides that activate both ligand-gated ion channels and G-protein coupled receptors (GPCRs). The ligand-gated ion channels induce rapid changes in membrane potential resulting in the generation of action potentials, initiation of exocytosis and the release of hormone into the periphery. By contrast, the GPCRs activate a host of diverse signalling cascades that modulate action potential firing and regulate other cellular functions required to support hormone release (e.g. hormone synthesis, processing, packaging and trafficking). The diversity of these actions is critical for integration of the distinct regulatory signals into a response appropriate for maintaining homeostasis. This review describes several diverse roles of GPCRs in magnocellular neurones, focusing primarily on adrenergic, purinergic and peptidergic (neurokinin and angiotensin) receptors.

Autocrine regulation of human sperm motility by tachykinins.

BACKGROUND: We examined the presence and function of tachykinins and the tachykinin-degrading enzymes neprilysin (NEP) and neprilysin-2 (NEP2) in human spermatozoa. METHODS: Freshly ejaculated semen was collected from forty-eight normozoospermic human donors. We analyzed the expression of substance P, neurokinin A, neurokinin B, hemokinin-1, NEP and NEP2 in sperm cells by reverse-transcriptase polymerase chain reaction (RT-PCR), western blot and immunocytochemistry assays and evaluated the effects of the neprilysin and neprilysin-2 inhibitor phosphoramidon on sperm motility in the absence and presence of tachykinin receptor-selective antagonists. Sperm motility was measured using WHO procedures or computer-assisted sperm analysis (CASA). RESULTS: The mRNAs of the genes that encode substance P/neurokinin A (TAC1), neurokinin B (TAC3), hemokinin-1 (TAC4), neprilysin (MME) and neprilysin-2 (MMEL1) were expressed in human sperm. Immunocytochemistry studies revealed that tachykinin and neprilysin proteins were present in spermatozoa and show specific and differential distributions. Phosphoramidon increased sperm progressive motility and its effects were reduced in the presence of the tachykinin receptor antagonists SR140333 (NK1 receptor-selective) and SR48968 (NK2 receptor-selective) but unmodified in the presence of SR142801 (NK3 receptor-selective). CONCLUSION: These data show that tachykinins are present in human spermatozoa and participate in the regulation of sperm motility. Tachykinin activity is regulated, at least in part, by neprilysins.

The mammalian tachykinin ligand-receptor system: an emerging target for central neurological disorders.

Our understanding of the complex signaling neurophysiology of the central nervous system has facilitated the exploration of potential novel receptor-ligand system targets for disorders of this most complex organ. In recent years, many relatively neglected receptor-ligand systems have been re-evaluated with respect to their ability to potently modulate discrete tracts in the central nervous system. One such system is the tachykinin (previously neurokinin) system. The multiple heptahelical G protein-coupled receptors and neuropeptide ligands that comprise this system may be significantly involved in more central nervous systems actions than previously thought, including sleep disorders, amyotrophic lateral sclerosis, Alzheimer's disease and Machado-Joseph disease. The development of our understanding of the role of the tachykinin receptor-ligand system in higher order central functions is likely to allow the creation of more specific and selective tachykinin-related neurotherapeutics.

Role of neurokinin receptors and ionic mechanisms within the respiratory network of the lamprey.

We have suggested that in the lamprey, a medullary region called the paratrigeminal respiratory group (pTRG), is essential for respiratory rhythm generation and could correspond to the pre-Bötzinger complex (pre-BötC), the hypothesized kernel of the inspiratory rhythm-generating network in mammals. The present study was performed on in vitro brainstem preparations of adult lampreys to investigate whether some functional characteristics of the respiratory network are retained throughout evolution and to get further insights into the recent debated hypotheses on respiratory rhythmogenesis in mammals, such as for instance the "group-pacemaker" hypothesis. Thus, we tried to ascertain the presence and role of neurokinins (NKs) and burst-generating ion currents, such as the persistent Na(+) current (I(NaP)) and the Ca(2+)-activated non-specific cation current (I(CAN)), described in the pre-Bötzinger complex. Respiratory activity was monitored as vagal motor output. Substance P (SP) as well as NK1, NK2 and NK3 receptor agonists (400-800 nM) applied to the bath induced marked increases in respiratory frequency. Microinjections (0.5-1 nl) of SP as well as the other NK receptor agonists (1 microM) into the pTRG increased the frequency and amplitude of vagal bursts. Riluzole (RIL) and flufenamic acid (FFA) were used to block I(NaP) and I(CAN), respectively. Bath application of either RIL or FFA (20-50 microM) depressed, but did not suppress respiratory activity. Coapplication of RIL and FFA at 50 microM abolished the respiratory rhythm that, however, was restarted by SP microinjected into the pTRG. The results show that NKs may have a modulatory role in the lamprey respiratory network through an action on the pTRG and that I(NaP) and I(CAN) may contribute to vagal burst generation. We suggest that the "group-pacemaker" hypothesis is tenable for the lamprey respiratory rhythm generation since respiratory activity is abolished by blocking both I(NaP) and I(CAN), but is restored by enhancing network excitability.

Identification of Eya3 and TAC1 as long-day signals in the sheep pituitary.

Seasonally breeding mammals such as sheep use photoperiod, encoded by the nocturnal secretion of the pineal hormone melatonin, as a critical cue to drive hormone rhythms and synchronize reproduction to the most optimal time of year. Melatonin acts directly on the pars tuberalis (PT) of the pituitary, regulating expression of thyrotropin, which then relays messages back to the hypothalamus to control reproductive circuits. In addition, a second local intrapituitary circuit controls seasonal prolactin (PRL) release via one or more currently uncharacterized low-molecular-weight peptides, termed "tuberalins," of PT origin. Studies in birds have identified the transcription factor Eya3 as the first molecular response activated by long photoperiod (LP). Using arrays and in situ hybridization studies, we demonstrate here that Eya3 is the strongest LP-activated gene in sheep, revealing a common photoperiodic molecular response in birds and mammals. We also demonstrate TAC1 (encoding the tachykinins substance P and neurokinin A) to be strongly activated by LP within the sheep PT. We show that these PRL secretagogues act on primary pituitary cells and thus are candidates for the elusive PT-expressed tuberalin seasonal hormone regulator.

Filicene obtained from Adiantum cuneatum interacts with the cholinergic, dopaminergic, glutamatergic, GABAergic, and tachykinergic systems to exert antinociceptive effect in mice.

In the present study, we describe the antinociceptive effect of filicene, a triterpene isolated from Adiantum cuneatum (Adiantaceae) leaves, in several models of pain in mice. When evaluated against acetic acid-induced abdominal constrictions, filicene (10, 30 and 60 mg/kg, i.p.) produced dose-related inhibition of the number of constrictions, being several times more potent [ID(50)=9.17 (6.27-13.18) mg/kg] than acetaminophen [ID(50)=18.8 (15.7-22.6) mg/kg], diclofenac [ID(50)=12.1(9.40-15.6) mg/kg] and acetylsalicylic acid [ID(50)=24.0(13.1-43.8) mg/kg] in the same doses as those used for the standard drugs. Filicene also produced dose-related inhibition of the pain caused by capsaicin and glutamate, with mean ID(50) values of 11.7 (8.51-16.0) mg/kg and <10 mg/kg, respectively. Its antinociceptive action was significantly reversed by atropine, haloperidol, GABA(A) and GABA(B) antagonists (bicuculline and phaclofen, respectively), but was not affected by L-arginine-nitric oxide, serotonin, adrenergic and the opioid systems. Together, these results indicate that the mechanisms involved in its action are not completely understood, but seem to involve interaction with the cholinergic, dopaminergic, glutamatergic, GABAergic and tachykinergic systems.

Central regulation of the cough reflex: therapeutic implications.

In many species including humans, antagonists of NMDA-type glutamate receptors such as dextromethorphan, when used at sufficient doses, have been found to be relatively safe and effective antitussives. Similarly, now in five different species (guinea pigs, rabbits, cats, dogs and pigs), neurokinin receptor antagonists have also proven to be safe and effective antitussive agents. Both of these classes of drugs act centrally to prevent cough. A brief review of what is known about the central encoding of cough is presented, as are the advantages of centrally acting antitussives. Also discussed are new insights into cough and NMDA receptor signaling that may lead to the development of more effective antitussive agents with limited side effects and broad application in treating cough associated with a variety of aetiologies.

Desflurane but not sevoflurane can increase lung resistance via tachykinin pathways.

BACKGROUND: Although there is evidence that the volatile anaesthetic desflurane directly relaxes preconstricted airway smooth muscle in vitro, the anaesthetic increases the lung resistance in vivo. The constrictive mechanisms of desflurane are, however, still unknown. This study was conducted to clarify the increasing mechanisms of desflurane on lung resistance by examining the vagal nerve reflexes in guinea pigs. METHODS: The effects of desflurane and sevoflurane on total lung resistance (R(L)) and dynamic lung compliance (C(Dyn)) were investigated in animals that were either untreated, pretreated with atropine or vagotomy, pretreated with the tachykinin receptor antagonists sendide or MEN-10376, or given chronic pretreatment with capsaicin. RESULTS: Desflurane biphasically and dose-dependently increased R(L) (by 180% and 230% at the first and second peaks, respectively, at 2 minimum alveolar concentration) concomitant with a decrease in C(Dyn). However, sevoflurane had little effect on either R(L) or C(Dyn). Although vagotomy partially inhibited the first peak of R(L) by 30%, neither atropine nor vagotomy had any effect on the other respiratory responses to desflurane. Antagonization of tachykinin receptors of airway smooth muscles completely diminished the increase in R(L) induced by desflurane. Desflurane also had little effect on respiratory parameters after the capsaicin pretreatment, in which tachykinin containing afferent C-fibres was desensitized. CONCLUSIONS: Desflurane but not sevoflurane increased R(L) concomitant with a decrease in C(Dyn) in guinea pigs. The increase in lung resistance by desflurane might be due to antidromic tachykinin release from afferent C-fibres but not acetylcholine release from parasympathetic efferent nerves.

Tachykinin receptor modulation of cyclooxygenase-2 expression in human polymorphonuclear leucocytes.

BACKGROUND AND PURPOSE: We investigated the ability of natural and synthetic selective NK receptors agonists and antagonists to modulate cyclooxygenase-2 (COX-2) expression in human polymorphonuclear leucocytes (PMNs). EXPERIMENTAL APPROACH: The presence of all three tachykinin in PMNs was assessed by Western blot and PCR techniques. Natural and synthetic ligands selective for the tachykinin receptors were used to modulate COX-2 protein (measured with Western blotting) and activity [as prostaglandin E(2) (PGE(2)) output]. Effects of substance P (SP) on phosphorylation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappaB) activation were studied to analyse the signalling pathway involved in COX-2 up-regulation mediated by SP. KEY RESULTS: Stimulation of NK receptors with the natural ligands SP, neurokinin A (NKA) and neurokinin B, in the pmol.L(-1)-micromol.L(-1) concentration range, modulated COX-2 expression and PGE(2) release in a concentration- and time-dependent manner. Experiments with synthetic selective agonists [Sar(9), Met(O(2))(11)]SP, [beta-Ala(8)] NKA(4-10), senktide or selective antagonists L703,606, SR48,968 or SR142801, confirmed that COX-2 up-regulation was mediated by NK receptors. We found that mainly p38, p42 and p46 MAPKs were phosphorylated by SP and SB202190, PD98059 and SP600125, which are selective inhibitors of these kinases, blocked SP-induced COX-2 expression. SP also induced nuclear translocation of NF-kappaB concentration-dependently, with a maximum effect at 1 nmol.L(-1). CONCLUSIONS AND IMPLICATIONS: Human PMNs possess functional NK(1), NK(2) and NK(3) receptors, which mediate the induction of COX-2 expression and NF-kappaB activation by SP.

Tweaking the gain on platelet regulation: the tachykinin connection.

Soluble factors such as ADP and thromboxane (TX) A(2) that are secreted or released by platelets at sites of tissue injury, mediate autocrine and paracrine regulation of platelet function, resulting in rapid localised thrombus formation. The suppression of platelet function, particularly through targeting such secondary regulatory mechanisms, that serve to 'fine-tune' the platelet response, has proven effective in the prevention of inappropriate platelet activation that results in thrombosis. The most commonly used anti-platelet approaches (ADP receptor antagonism or inhibition of TXA(2) synthesis), however, lack efficacy in many patients, suggesting the existence of additional uncharacterised mechanisms for the regulation of platelet function. Recent data, which form a focus of this review, have identified peripheral tachykinin peptide family members, such as substance P and the newly identified endokinins, as physiologically important positive feedback regulators of platelet function. The actions of tachykinins that are released from platelets during activation are mediated by the neurokinin-1 receptor. Initial analysis of the role of this receptor in platelet thrombus formation, and thrombosis in the mouse, indicate this to be a promising new target for the development of anti-thrombotic drugs.

Tachykinin receptors and gastrointestinal motility: focus on humans.

Peptides of the tachykinin (TK) family were first discovered in the gastrointestinal tissue about 75 years ago and supposed to be involved in gastrointestinal (GI) motility. This hypothesis has been repeatedly proven, although the role of TKs on motility is modulatory rather than pivotal. Furthermore, beyond the well known excitatory role, it has been acknowledged that TKs can also inhibit GI motility. TKs act at 3 receptors termed as TK NK1 (NK1r), NK2 (NK2r), and NK3 (NK3r) receptors. The view gained through intense preclinical research suggested that motor effects induced by the stimulation of NK2r were prominently mediated by a direct action on smooth muscle, those produced by the stimulation of NK1r were due to both muscular and neuronal effects, whereas the motor effects induced by NK3r were exclusively mediated by neuronal effects. Recent functional and anatomical findings in humans are challenging this concept since NK2r have been found in several kinds of myenteric neurons and selective NK2r antagonists can, in particular conditions, produce GI motor effects likely related to a neuronal site of action. Furthermore, the evidence for a myotropic role of NK1r is scarce, and very few studies, if any, have documented a functional role for NK3r. The findings that an acute or a long lasting blockade of NK2r does not alter normal GI functions and that these receptors can modulate visceral sensitivity are good starting points for testing this class of drugs in GI diseases characterised by altered GI motility.

Differential role of tachykinin NK3 receptors on cholinergic excitatory neurotransmission in the mouse stomach and small intestine.

BACKGROUND AND PURPOSE: Tachykinin NK(3) receptors are widely expressed in the mouse gastrointestinal tract but their functional role in enteric neuromuscular transmission remains unstudied in this species. We investigated the involvement of NK(3) receptors in cholinergic neurotransmission in the mouse stomach and small intestine. EXPERIMENTAL APPROACH: Muscle strips of the mouse gastric fundus and ileum were mounted in organ baths for tension recordings. Effects of NK(3) agonists and antagonists were studied on contractions to EFS of enteric nerves and to carbachol. KEY RESULTS: EFS induced frequency-dependent tetrodotoxin-sensitive contractions, which were abolished by atropine. The cholinergic contractions to EFS in the stomach were enhanced by the NK(3) antagonist SR142801, but not affected by the NK(3) agonist senktide or neurokinin B. The cholinergic contractions to EFS in the small intestine were not affected by SR142801, but dose-dependently inhibited by senktide and neurokinin B. This inhibitory effect was prevented by SR142801 but not by hexamethonium. SR142801, senktide or neurokinin B did not induce any response per se in the stomach and small intestine and did not affect contractions to carbachol. CONCLUSIONS AND IMPLICATIONS: NK(3) receptors modulate cholinergic neurotransmission differently in the mouse stomach and small intestine. Blockade of NK(3) receptors enhanced cholinergic transmission in the stomach but not in the intestine. Activation of NK(3) receptors inhibited cholinergic transmission in the small intestine but not in the stomach. This indicates a physiological role for NK(3) receptors in mouse stomach contractility and a pathophysiological role in mouse intestinal contractility.

A novel biological role of tachykinins as an up-regulator of oocyte growth: identification of an evolutionary origin of tachykininergic functions in the ovary of the ascidian, Ciona intestinalis.

Tachykinins (TKs) and their receptors have been shown to be expressed in the mammalian ovary. However, the biological roles of ovarian TKs have yet to be verified. Ci-TK-I and Ci-TK-R, characterized from the protochordate (ascidian), Ciona intestinalis, are prototypes of vertebrate TKs and their receptors. In the present study, we show a novel biological function of TKs as an inducible factor for oocyte growth using C. intestinalis as a model organism. Immunostaining demonstrated the specific expression of Ci-TK-R in test cells residing in oocytes at the vitellogenic stage. DNA microarray and real-time PCR revealed that Ci-TK-I induced gene expression of several proteases, including cathepsin D, chymotrypsin, and carboxy-peptidase B1, in the ovary. The enzymatic activities of these proteases in the ovary were also shown to be enhanced by Ci-TK-I. Of particular significance is that the treatment of Ciona oocytes with Ci-TK-I resulted in progression of growth from the vitellogenic stage to the post-vitellogenic stage. The Ci-TK-I-induced oocyte growth was blocked by a TK antagonist or by protease inhibitors. These results led to the conclusion that Ci-TK-I enhances growth of the vitellogenic oocytes via up-regulation of gene expression and enzymatic activities of the proteases. This is the first clarification of the biological roles of TKs in the ovary and the underlying essential molecular mechanism. Furthermore, considering the phylogenetic position of ascidians as basal chordates, we suggest that the novel TK-regulated oocyte growth is an "evolutionary origin" of the tachykininergic functions in the ovary.