Drug effects on neuropeptides and their receptors: Big hopes but moderate success in the treatment of chronic pain.

Neuropeptides, including tachykinins, CGRP, and somatostatin, are localized in a peptidergic subgroup of nociceptive primary afferent neurons. Tachykinins and CGRP are pronociceptive, somatostatin is an antinociceptive mediator. Intensive drug research has been performed to develop tachykinin and CGRP antagonists, and somatostatin agonists as analgesics. CGRP receptor antagonists are efficacious and well-tolerated drugs in migraine. Monoclonal antibodies against CGRP or its receptor are used for the prophylactic treatment of migraine. Tachykinin NK1 receptor antagonists failed as analgesics but are used for chemotherapy-induced nausea and vomiting. New, orally active somatostatin 4 receptor agonists are promising drug candidates for treating various pain conditions.

A vagal-brainstem interoceptive circuit for cough-like defensive behaviors in mice.

Coughing is a respiratory behavior that plays a crucial role in protecting the respiratory system. Here we show that the nucleus of the solitary tract (NTS) in mice contains heterogenous neuronal populations that differentially control breathing. Within these subtypes, activation of tachykinin 1 (Tac1)-expressing neurons triggers specific respiratory behaviors that, as revealed by our detailed characterization, are cough-like behaviors. Chemogenetic silencing or genetic ablation of Tac1 neurons inhibits cough-like behaviors induced by tussive challenges. These Tac1 neurons receive synaptic inputs from the bronchopulmonary chemosensory and mechanosensory neurons in the vagal ganglion and coordinate medullary regions to control distinct aspects of cough-like defensive behaviors. We propose that these Tac1 neurons in the NTS are a key component of the airway-vagal-brain neural circuit that controls cough-like defensive behaviors in mice and that they coordinate the downstream modular circuits to elicit the sequential motor pattern of forceful expiratory responses.

Two C-terminal isoforms of Aplysia tachykinin-related peptide receptors exhibit phosphorylation-dependent and phosphorylation-independent desensitization mechanisms.

Diversity, a hallmark of G protein-coupled receptor (GPCR) signaling, partly stems from alternative splicing of a single gene generating more than one isoform for a receptor. Additionally, receptor responses to ligands can be attenuated by desensitization upon prolonged or repeated ligand exposure. Both phenomena have been demonstrated and exemplified by the deuterostome tachykinin signaling system, although the role of phosphorylation in desensitization remains a subject of debate. Here, we describe the signaling system for tachykinin-related peptides (TKRPs) in a protostome, mollusk Aplysia. We cloned the Aplysia TKRP precursor, which encodes three TKRPs (apTKRP-1, apTKRP-2a, and apTKRP-2b) containing the FXGXR-amide motif. In situ hybridization and immunohistochemistry showed predominant expression of TKRP mRNA and peptide in the cerebral ganglia. TKRPs and their posttranslational modifications were observed in extracts of central nervous system ganglia using mass spectrometry. We identified two Aplysia TKRP receptors (apTKRPRs), named apTKRPR-A and apTKRPR-B. These receptors are two isoforms generated through alternative splicing of the same gene and differ only in their intracellular C termini. Structure-activity relationship analysis of apTKRP-2b revealed that both C-terminal amidation and conserved residues of the ligand are critical for receptor activation. C-terminal truncates and mutants of apTKRPRs suggested that there is a C-terminal phosphorylation-independent desensitization for both receptors. Moreover, apTKRPR-B also exhibits phosphorylation-dependent desensitization through the phosphorylation of C-terminal Ser/Thr residues. This comprehensive characterization of the Aplysia TKRP signaling system underscores the evolutionary conservation of the TKRP and TK signaling systems, while highlighting the intricacies of receptor regulation through alternative splicing and differential desensitization mechanisms.

Estrogen promotes gonadotropin-releasing hormone expression by regulating tachykinin 3 and prodynorphin systems in chicken.

The "KNDy neurons" located in the hypothalamic arcuate nucleus (ARC) of mammals are known to co-express kisspeptin, neurokinin B (NKB), and dynorphin (DYN), and have been identified as key mediators of the feedback regulation of steroid hormones on gonadotropin-releasing hormone (GnRH). However, in birds, the genes encoding kisspeptin and its receptor GPR54 are genomic lost, leaving unclear mechanisms for feedback regulation of GnRH by steroid hormones. Here, the genes tachykinin 3 (TAC3) and prodynorphin (PDYN) encoding chicken NKB and DYN neuropeptides were successfully cloned. Temporal expression profiling indicated that TAC3, PDYN and their receptor genes (TACR3, OPRK1) were mainly expressed in the hypothalamus, with significantly higher expression at 30W than at 15W. Furthermore, overexpression or interference of TAC3 and PDYN can regulate the GnRH mRNA expression. In addition, in vivo and in vitro assays showed that estrogen (E2) could promote the mRNA expression of TAC3, PDYN, and GnRH, as well as the secretion of GnRH/LH. Mechanistically, E2 could dimerize the nuclear estrogen receptor 1 (ESR1) to regulate the expression of TAC3 and PDYN, which promoted the mRNA and protein expression of GnRH gene as well as the secretion of GnRH. In conclusion, these results revealed that E2 could regulate the GnRH expression through TAC3 and PDYN systems, providing novel insights for reproductive regulation in chickens.

Substance P in the medial amygdala regulates aggressive behaviors in male mice.

Behavioral and clinical studies have revealed a critical role of substance P (SP) in aggression; however, the neural circuit mechanisms underlying SP and aggression remain elusive. Here, we show that tachykinin-expressing neurons in the medial amygdala (MeATac1 neurons) are activated during aggressive behaviors in male mice. We identified MeATac1 neurons as a key mediator of aggression and found that MeATac1→ventrolateral part of the ventromedial hypothalamic nucleus (VMHvl) projections are critical to the regulation of aggression. Moreover, SP/neurokinin-1 receptor (NK-1R) signaling in the VMHvl modulates aggressive behaviors in male mice. SP/NK-1R signaling regulates aggression by influencing glutamate transmission in neurons in the VMHvl. In summary, these findings place SP as a key node in aggression circuits.

Tachykinins Play a Major Role in Micro and Macrovascular Complications in Type 2 Diabetes Patients.

Diabetes Mellitus is a metabolic disorder, which is characterized by an increase in blood glucose levels. The defects in the secretion or action of insulin are the major cause of diabetes. Increase in the blood glucose level exerts a negative effect on the normal functions of the body organs and this leads to the dysfunctions of cells and tissue and causes vascular complications in diabetic patients. Several studies indicate that neuropeptides are released from the neurosensory cells which are mainly known as tachykinins which provoke major vascular complications in diabetic patients. Tachykinins are known as pro-inflammatory peptides which increase vascular complications and vascular permeability. The duration and severity of diabetes disease increase the risk of vascular complication in patients. The aim of this review is to elaborate the role of tachykinins in microvascular and macrovascular complications in diabetic patients. The study concluded that tachykinins increase micro and macrovascular complications in diabetic patients.

A class of secreted mammalian peptides with potential to expand cell-cell communication.

Peptide hormones and neuropeptides are signaling molecules that control diverse aspects of mammalian homeostasis and physiology. Here we provide evidence for the endogenous presence of a sequence diverse class of blood-borne peptides that we call "capped peptides." Capped peptides are fragments of secreted proteins and defined by the presence of two post-translational modifications - N-terminal pyroglutamylation and C-terminal amidation - which function as chemical "caps" of the intervening sequence. Capped peptides share many regulatory characteristics in common with that of other signaling peptides, including dynamic physiologic regulation. One capped peptide, CAP-TAC1, is a tachykinin neuropeptide-like molecule and a nanomolar agonist of mammalian tachykinin receptors. A second capped peptide, CAP-GDF15, is a 12-mer peptide cleaved from the prepropeptide region of full-length GDF15 that, like the canonical GDF15 hormone, also reduces food intake and body weight. Capped peptides are a potentially large class of signaling molecules with potential to broadly regulate cell-cell communication in mammalian physiology.

Deciphering specificity and cross-reactivity in tachykinin NK1 and NK2 receptors.

The tachykinin receptors neurokinin 1 (NK1R) and neurokinin 2 (NK2R) are G protein-coupled receptors that bind preferentially to the natural peptide ligands substance P and neurokinin A, respectively, and have been targets for drug development. Despite sharing a common C-terminal sequence of Phe-X-Gly-Leu-Met-NH2 that helps direct biological function, the peptide ligands exhibit some degree of cross-reactivity toward each other's non-natural receptor. Here, we investigate the detailed structure-activity relationships of the ligand-bound receptor complexes that underlie both potent activation by the natural ligand and cross-reactivity. We find that the specificity and cross-reactivity of the peptide ligands can be explained by the interactions between the amino acids preceding the FxGLM consensus motif of the bound peptide ligand and two regions of the receptor: the ß-hairpin of the extracellular loop 2 (ECL2) and a N-terminal segment leading into transmembrane helix 1. Positively charged sidechains of the ECL2 (R177 of NK1R and K180 of NK2R) are seen to play a vital role in the interaction. The N-terminal positions 1 to 3 of the peptide ligand are entirely dispensable. Mutated and chimeric receptor and ligand constructs neatly swap around ligand specificity as expected, validating the structure-activity hypotheses presented. These findings will help in developing improved agonists or antagonists for NK1R and NK2R.

Sequences analysis and pituitary actions of tachykinins in Chinese sturgeon (Acipenser sinensis).

Tachykinins belong to a large, evolutionarily conserved family of brain/gut peptides that are involved in a variety of physiological functions in mammals, such as reproductive regulation. However, little information was available about tachykinins in ancient fish lineage. In the present study, we firstly identified three tachykinin genes (named tac1, tac3 and tac4) and three neurokinin receptors (named nk1r, nk2r and nk3r) from Chinese sturgeon brain and pituitary. Sequence analysis showed that tac1 encoded substance P (SP) and neurokinin A (NKA), tac3 encoded neurokinin B (NKB) and NKB-related peptide (NKBRP), and tac4 encoded hemokin 1 (HK-1) and hemokin 2 (HK-2), respectively. The luciferase reporter assay results showed that NK1R preferentially selected asSP, NK2R preferentially selected asNKA, and NK3R preferentially selected asNKB. Tissue expression analysis showed that the three tac genes were highly detected in the telencephalon and hypothalamus, whereas nkr genes were widely expressed in peripheral tissues. Spatio-temporal expression analysis showed that all three tac genes were highly expressed in unknown sex individuals. Intraperitoneal injection experiments showed that both asSP and asNKB could stimulate luteinizing hormone (LH) release in Chinese sturgeon serum. At the transcriptional level, asSP and asNKB could significantly reduce pituitary follicle-stimulating hormone beta (fshß) mRNA expression, but induce pituitary growth hormone (gh) mRNA expression. In addition, estradiol (E2) could stimulate tac3 mRNA expression in hypothalamus. Taken together, this study provided information on the tachykinin family in Chinese sturgeon and demonstrates that asNKB and asSP could be involved in reproductive and growth regulation in pituitary.

Tachykinins and the potential causal factors for post-COVID-19 condition.

The most prevalent symptoms of post-COVID-19 condition are pulmonary dysfunction, fatigue and muscle weakness, anxiety, anosmia, dysgeusia, headaches, difficulty in concentrating, sexual dysfunction, and digestive disturbances. Hence, neurological dysfunction and autonomic impairments predominate in post-COVID-19 condition. Tachykinins including the most studied substance P are neuropeptides expressed throughout the nervous and immune systems, and contribute to many physiopathological processes in the nervous, immune, gastrointestinal, respiratory, urogenital, and dermal systems and participate in inflammation, nociception, and cell proliferation. Substance P is a key molecule in neuroimmune crosstalk; immune cells near the peripheral nerve endings can send signals to the brain with cytokines, which highlights the important role of tachykinins in neuroimmune communication. We reviewed the evidence that relates the symptoms of post-COVID-19 condition to the functions of tachykinins and propose a putative pathogenic mechanism. The antagonism of tachykinins receptors can be a potential treatment target.

Drosophila Tachykininergic Neurons Modulate the Activity of Two Groups of Receptor-Expressing Neurons to Regulate Aggressive Tone.

Neuropeptides influence animal behaviors through complex molecular and cellular mechanisms, the physiological and behavioral effects of which are difficult to predict solely from synaptic connectivity. Many neuropeptides can activate multiple receptors, whose ligand affinity and downstream signaling cascades are often different from one another. Although we know that the diverse pharmacological characteristics of neuropeptide receptors form the basis of unique neuromodulatory effects on distinct downstream cells, it remains unclear exactly how different receptors shape the downstream activity patterns triggered by a single neuronal neuropeptide source. Here, we uncovered two separate downstream targets that are differentially modulated by tachykinin, an aggression-promoting neuropeptide in Drosophila Tachykinin from a single male-specific neuronal type recruits two separate downstream groups of neurons. One downstream group, synaptically connected to the tachykinergic neurons, expresses the receptor TkR86C and is necessary for aggression. Here, tachykinin supports cholinergic excitatory synaptic transmission between the tachykinergic and TkR86C downstream neurons. The other downstream group expresses the TkR99D receptor and is recruited primarily when tachykinin is overexpressed in the source neurons. Differential activity patterns in the two groups of downstream neurons correlate with levels of male aggression triggered by the tachykininergic neurons. These findings highlight how the amount of neuropeptide released from a small number of neurons can reshape the activity patterns of multiple downstream neuronal populations. Our results lay the foundation for further investigations into the neurophysiological mechanism by which a neuropeptide controls complex behaviors.SIGNIFICANCE STATEMENT Neuropeptides control a variety of innate behaviors, including social behaviors, in both animals and humans. Unlike fast-acting neurotransmitters, neuropeptides can elicit distinct physiological responses in different downstream neurons. How such diverse physiological effects coordinate complex social interactions remains unknown. This study uncovers the first in vivo example of a neuropeptisde from a single neuronal source eliciting distinct physiological responses in multiple downstream neurons that express different neuropeptide receptors. Understanding the unique motif of neuropeptidergic modulation, which may not be easily predicted from a synaptic connectivity map, can help elucidate how neuropeptides orchestrate complex behaviors by modulating multiple target neurons simultaneously.

Development of Neuropeptide Hemokinin-1 Analogues with Antimicrobial and Wound-Healing Activity.

Wound healing is a complex process that can be delayed in some pathological conditions, such as infection and diabetes. Following skin injury, the neuropeptide substance P (SP) is released from peripheral neurons to promote wound healing by multiple mechanisms. Human hemokinin-1 (hHK-1) has been identified as an SP-like tachykinin peptide. Surprisingly, hHK-1 shares similar structural features with antimicrobial peptides (AMPs), but it does not display efficient antimicrobial activity. Therefore, a series of hHK-1 analogues were designed and synthesized. Among these analogues, AH-4 was found to display the greatest antimicrobial activity against a broad spectrum of bacteria. Furthermore, AH-4 rapidly killed bacteria by membrane disruption, similar to most AMPs. More importantly, AH-4 showed favorable healing activity in all tested mouse full-thickness excisional wound models. Overall, this study suggests that the neuropeptide hHK-1 can be used as a desirable template for developing promising therapeutics with multiple functions for wound healing.

Activation of Parabrachial Tachykinin 1 Neurons Counteracts Some Behaviors Mediated by Parabrachial Calcitonin Gene-related Peptide Neurons.

Many threats activate parabrachial neurons expressing calcitonin gene-related peptide (CGRPPBN) which transmit alarm signals to forebrain regions. Most CGRPPBN neurons also express tachykinin 1 (Tac1), but there are also Tac1-expressing neurons in the PBN that do not express CGRP (Tac1+;CGRP- neurons). Chemogenetic or optogenetic activation of all Tac1PBN neurons in mice elicited many physiological/behavioral responses resembling the activation of CGRPPBN neurons, e.g., anorexia, jumping on a hot plate, avoidance of photostimulation; however, two key responses opposed activation of CGRPPBN neurons. Activating Tac1PBN neurons did not produce conditioned taste aversion and it elicited dynamic escape behaviors rather than freezing. Activating Tac1+;CGRP- neurons, using an intersectional genetic targeting approach, resembles activating all Tac1PBN neurons. These results reveal that activation of Tac1+;CGRP- neurons can suppress some functions attributed to the CGRPPBN neurons, which provides a mechanism to bias behavioral responses to threats.

Kobayashi Award 2021: Neuropeptides, receptors, and follicle development in the ascidian, Ciona intestinalis Type A: New clues to the evolution of chordate neuropeptidergic systems from biological niches.

Ciona intestinalis Type A (Ciona robusta) is a cosmopolitan species belonging to the phylum Urochordata, invertebrate chordates that are phylogenetically the most closely related to the vertebrates. Therefore, this species is of interest for investigation of the evolution and comparative physiology of endocrine, neuroendocrine, and nervous systems in chordates. Our group has identified>30 Ciona neuropeptides (80% of all identified ascidian neuropeptides) primarily using peptidomic approaches combined with reference to genome sequences. These neuropeptides are classified into two groups: homologs or prototypes of vertebrate neuropeptides and novel (Ciona-specific) neuropeptides. We have also identified the cognate receptors for these peptides. In particular, we elucidated multiple receptors for Ciona-specific neuropeptides by a combination of a novel machine learning system and experimental validation of the specific interaction of the predicted neuropeptide-receptor pairs, and verified unprecedented phylogenies of receptors for neuropeptides. Moreover, several neuropeptides were found to play major roles in the regulation of ovarian follicle development. Ciona tachykinin facilitates the growth of vitellogenic follicles via up-regulation of the enzymatic activities of proteases. Ciona vasopressin stimulates oocyte maturation and ovulation via up-regulation of maturation-promoting factor- and matrix metalloproteinase-directed collagen degradation, respectively. Ciona cholecystokinin also triggers ovulation via up-regulation of receptor tyrosine kinase signaling and the subsequent activation of matrix metalloproteinase. These studies revealed that the neuropeptidergic system plays major roles in ovarian follicle growth, maturation, and ovulation in Ciona, thus paving the way for investigation of the biological roles for neuropeptides in the endocrine, neuroendocrine, nervous systems of Ciona, and studies of the evolutionary processes of various neuropeptidergic systems in chordates.

Functional homology of tachykinin signalling: The influence of human substance P on the immune system of the mealworm beetle, Tenebrio molitor L.

Tachykinin-related peptides (TRPs) are one of the most prominent families of neuropeptides in the animal kingdom. Insect TRPs display strong structural and functional homology to vertebrate tachykinins (TKs). To study functional homologies between these two neuropeptide families, the influence of human substance P (SP, one of the essential vertebrate TKs) on the immune system of the mealworm beetle, Tenebrio molitor L., was analysed. Human SP influences the phagocytic abilities of T. molitor haemocytes. Peptide injection leads to an increase in the number of haemocytes participating in the phagocytosis of latex beads. In contrast, incubation of haemocytes from non-injected beetles in a solution of physiological saline and SP causes a decrease in phagocytic activity. Treatment with human SP also led to increased adhesion of haemocytes, but no changes in the arrangement of the F-actin cytoskeleton were observed. Interestingly, 6 h after human SP injection, increased DNA integrity in T. molitor haemocytes was reported. The opposite effects were observed 24 h after SP injection. Human SP caused the upregulation of humoral immune responses, such as phenoloxidase (PO) activity in the T. molitor haemolymph, and the downregulation of immune-related genes encoding coleoptericin A, tenecin 3 and Toll receptor. However, genes encoding attacin 2 and cecropin were upregulated. Despite these differences, the antimicrobial activity of T. molitor haemolymph was significantly lower in beetles injected with SP than in control beetles. Moreover, an analysis of the direct influence of SP on lysozyme activity was performed. Our results suggest that SP at a concentration of 10-6 M can directly inhibit lysozyme activity. However, an opposite effect was reported after the application of SP at a concentration of 10-4 M. The presented results suggest structural and functional homology between TK signalling in vertebrates and insects. Primarily, this was visible in the context of the humoral response and general antimicrobial activity of T. molitor haemolymph. However, some of the results related to haemocyte function may also indicate the importance of the TK and TRP sequences for evoking immunological effects.

Female Infertility Is Associated with an Altered Expression Profile of Different Members of the Tachykinin Family in Human Granulosa Cells.

Neurokinin B (NKB) and its cognate receptor, NK3R, play a key role in the regulation of reproduction. NKB belongs to the family of tachykinins, which also includes substance P and neurokinin A, both encoded by the by the gene TAC1, and hemokinin-1, encoded by the TAC4 gene. In addition to NK3R, tachykinin effects are mediated by NK1R and NK2R, encoded by the genes TACR1 and TACR2, respectively. The role of these other tachykinins and receptors in the regulation of women infertility is mainly unknown. We have analyzed the expression profile of TAC1, TAC4, TACR1, and TACR2 in mural granulosa and cumulus cells from women presenting different infertility etiologies, including polycystic ovarian syndrome, advanced maternal age, low ovarian response, and endometriosis. We also studied the expression of MME, the gene encoding neprilysin, the most important enzyme involved in tachykinin degradation. Our data show that TAC1, TAC4, TACR1, TACR2, and MME expression is dysregulated in a different manner depending on the etiology of women infertility. The abnormal expression of these tachykinins and their receptors might be involved in the decreased fertility of these patients, offering a new insight regarding the diagnosis and treatment of women infertility.

Regional differences of tachykinin effects on smooth muscle and pacemaker potentials of the stomach, duodenum, ileum and colon of an emetic model, the house musk shrews.

BACKGROUND AND AIMS: The contractile effects of tachykinins on the gastrointestinal tract are well-known, but how they modulate slow-waves, particularly in species capable of emesis, remains largely unknown. We aimed to elucidate the effects of tachykinins on myoelectric and contractile activity of isolated gastrointestinal tissues of the Suncus murinus. METHODS: The effects of substance P (SP), neurokinin (NK)A, NKB and selective NK1 (CP122,721, CP99,994), NK2 (SR48,968, GR159,897) and NK3 (SB218,795, SB222,200) receptor antagonists on isolated stomach, duodenum, ileum and colon segments were studied. Mechanical contractile activity was recorded using isometric force displacement transducers. Electrical pacemaker activity was recorded using a microelectrode array. RESULTS: Compared with NKA, SP induced larger contractions in stomach tissue and smaller contractions in intestinal segments, where oscillation magnitudes increased in intestinal segments, but not the stomach. CP122,721 and GR159,897 inhibited electrical field stimulation-induced contractions of the stomach, ileum and colon. NKB and NK3 had minor effects on contractile activity. The inhibitory potencies of SP and NKA on the peristaltic frequency of the colon and ileum, respectively, were correlated with those on electrical pacemaker frequency. SP, NKA and NKB inhibited pacemaker activity of the duodenum and ileum, but increased that of the stomach and colon. SP elicited a dose-dependent contradictive pacemaker frequency response in the colon. CONCLUSION: This study revealed distinct effects of tachykinins on the mechanical and electrical properties of the stomach and colon vs. the proximal intestine, providing a unique aspect on neuromuscular correlation in terms of the effects of tachykinin on peristaltic and pacemaker activity in gastrointestinal-related symptoms.

Efficacy and Safety of Drugs for Gastroparesis: Systematic Review and Network Meta-analysis.

BACKGROUND & AIMS: Although there have been multiple drugs tested in gastroparesis, their relative efficacy and safety are unknown. We evaluated this in a network meta-analysis of randomized controlled trials (RCTs). METHODS: We searched the literature to September 7, 2022. We judged the efficacy of drugs based on global symptoms of gastroparesis; individual symptoms, including nausea, vomiting, abdominal pain, bloating, or fullness; and safety according to total adverse events and adverse events leading to withdrawal. We extracted data as intention-to-treat analyses, assuming dropouts to be treatment failures and reporting pooled relative risks (RRs) of not improving with 95% confidence intervals (CIs), ranking drugs according to P-score. RESULTS: We identified 29 RCTs (3772 patients). Based on global symptoms, clebopride ranked first for efficacy (RR, 0.30; 95% CI, 0.16-0.57; P-score = .99) followed by domperidone (RR, 0.68; 95% CI, 0.48-0.98; P-score = .76). No other drug was superior to placebo. Only 2 drug classes were efficacious: in rank order, oral dopamine antagonists (RR, 0.58; 95% CI, 0.44-0.77; P-score = .96) and tachykinin-1 antagonists (RR, 0.69; 95% CI, 0.52-0.93; P-score = .83). For individual symptoms, oral metoclopramide ranked first for nausea (RR 0.46; 95% CI, 0.21-1.00; P-score = .95), fullness (RR 0.67; 95% CI, 0.35-1.28; P-score = .86), and bloating (RR 0.53; 95% CI, 0.30-0.93; P-score = .97), based on only 1 small trial. Only prucalopride was more likely to be associated with adverse events than placebo. CONCLUSIONS: In a network meta-analysis, oral dopamine antagonists and tachykinin-1 antagonists were more efficacious than placebo for gastroparesis, but confidence in the evidence was low to moderate for most comparisons. There is an unmet need for efficacious therapies for gastroparesis.

Multiple tachykinins and their receptors characterized in the gastropod mollusk Pacific abalone: Expression, signaling cascades, and potential role in regulating lipid metabolism.

Tachykinin (TK) families, including the first neuropeptide substance P, have been intensively explored in bilaterians. Knowledge of signaling of TK receptors (TKRs) has enabled the comprehension of diverse physiological processes. However, TK signaling systems are largely unknown in Lophotrochozoa. This study identified two TK precursors and two TKR isoforms in the Pacific abalone Haliotis discus hannai (Hdh), and characterized Hdh-TK signaling. Hdh-TK peptides harbored protostomian TK-specific FXGXRamide or unique YXGXRamide motifs at the C-termini. A phylogenetic analysis showed that lophotrochozoan TKRs, including Hdh-TKRs, form a monophyletic group distinct from arthropod TKRs and natalisin receptor groups. Although reporter assays demonstrated that all examined Hdh-TK peptides activate intracellular cAMP accumulation and Ca2+ mobilization in Hdh-TKR-expressing mammalian cells, Hdh-TK peptides with N-terminal aromatic residues and C-terminal FXGXRamide motifs were more active than shorter or less aromatic Hdh-TK peptides with a C-terminal YXGXRamide. In addition, we showed that ligand-stimulated Hdh-TKRs mediate ERK1/2 phosphorylation in HEK293 cells and that ERK1/2 phosphorylation is inhibited by PKA and PKC inhibitors. In three-dimensional in silico Hdh-TKR binding modeling, higher docking scores of Hdh-TK peptides were consistent with the lower EC50 values in the reporter assays. The transcripts for Hdh-TK precursors and Hdh-TKR were highly expressed in the neural ganglia, with lower expression levels in peripheral tissues. When abalone were starved for 3 weeks, Hdh-TK1 transcript levels, but not Hdh-TK2, were increased in the cerebral ganglia (CG), intestine, and hepatopancreas, contrasting with the decreased lipid content and transcript levels of sterol regulatory element-binding protein (SREBP). At 24 h post-injection in vivo, the lower dose of Hdh-TK1 mixture increased SREBP transcript levels in the CG and hepatopancreas and accumulative food consumption of abalone. Higher doses of Hdh-TK1 and Hdh-TK2 mixtures decreased the SREBP levels in the CG. When Hdh-TK2-specific siRNA was injected into abalone, intestinal SREBP levels were significantly increased, whereas administration of both Hdh-TK1 and Hdh-TK2 siRNA led to decreased SREBP expression in the CG. Collectively, our results demonstrate the first TK signaling system in gastropod mollusks and suggest a possible role for TK peptides in regulating lipid metabolism in the neural and peripheral tissues of abalone.

BmGr4 responds to sucrose and glucose and expresses in tachykinin-related peptide-secreting enteroendocrine cells.

The regulatory hormones known as tachykinin-related peptides (TRPs) are identified as brain-gut peptides in insects. Dietary components from mulberry leaves, including glucose, induce secretion of TRPs from Bombyx mori midgut. However, the sensory molecules that recognize these compounds are still unknown. Here, we identified the gustatory receptor, BmGr4, as a sucrose and glucose receptor using Ca2+ imaging. Immunostaining revealed BmGr4 expression not only in the midgut, but also in the brain. In addition, BmGr4 expression was found to co-localize with TRP-expressing cells in both midgut enteroendocrine cells (EECs) and brain neurosecretory cells (NSCs). Furthermore, dietary nutrients after food intake result in an increase of TRP-level in hemolymph of silkworm larvae. These results provide significant circumstantial evidence for the involvement of the sucrose and glucose receptor, BmGr4, in the elicitation of TRP secretion in midgut EECs and brain NSCs.