Bombesin-like peptide recruits disinhibitory cortical circuits and enhances fear memories.

Disinhibitory neurons throughout the mammalian cortex are powerful enhancers of circuit excitability and plasticity. The differential expression of neuropeptide receptors in disinhibitory, inhibitory, and excitatory neurons suggests that each circuit motif may be controlled by distinct neuropeptidergic systems. Here, we reveal that a bombesin-like neuropeptide, gastrin-releasing peptide (GRP), recruits disinhibitory cortical microcircuits through selective targeting and activation of vasoactive intestinal peptide (VIP)-expressing cells. Using a genetically encoded GRP sensor, optogenetic anterograde stimulation, and trans-synaptic tracing, we reveal that GRP regulates VIP cells most likely via extrasynaptic diffusion from several local and long-range sources. In vivo photometry and CRISPR-Cas9-mediated knockout of the GRP receptor (GRPR) in auditory cortex indicate that VIP cells are strongly recruited by novel sounds and aversive shocks, and GRP-GRPR signaling enhances auditory fear memories. Our data establish peptidergic recruitment of selective disinhibitory cortical microcircuits as a mechanism to regulate fear memories.

A neuropeptide code for itch.

Itch is one of the most primal sensations, being both ubiquitous and important for the well-being of animals. For more than a century, a desire to understand how itch is encoded by the nervous system has prompted the advancement of many theories. Within the past 15 years, our understanding of the molecular and neural mechanisms of itch has undergone a major transformation, and this remarkable progress continues today without any sign of abating. Here I describe accumulating evidence that indicates that itch is distinguished from pain through the actions of itch-specific neuropeptides that relay itch information to the spinal cord. According to this model, classical neurotransmitters transmit, inhibit and modulate itch information in a context-, space- and time-dependent manner but do not encode itch specificity. Gastrin-releasing peptide (GRP) is proposed to be a key itch-specific neuropeptide, with spinal neurons expressing GRP receptor (GRPR) functioning as a key part of a convergent circuit for the conveyance of peripheral itch information to the brain.

Structures of human gastrin-releasing peptide receptors bound to antagonist and agonist for cancer and itch therapy.

Gastrin releasing peptide receptor (GRPR), a member of the bombesin (BBN) G protein-coupled receptors, is aberrantly overexpressed in several malignant tumors, including those of the breast, prostate, pancreas, lung, and central nervous system. Additionally, it also mediates non-histaminergic itch and pathological itch conditions in mice. Thus, GRPR could be an attractive target for cancer and itch therapy. Here, we report the inactive state crystal structure of human GRPR in complex with the non-peptide antagonist PD176252, as well as two active state cryo-electron microscopy (cryo-EM) structures of GRPR bound to the endogenous peptide agonist gastrin-releasing peptide and the synthetic BBN analog [D-Phe6, ß-Ala11, Phe13, Nle14] Bn (6-14), in complex with Gq heterotrimers. These structures revealed the molecular mechanisms for the ligand binding, receptor activation, and Gq proteins signaling of GRPR, which are expected to accelerate the structure-based design of GRPR antagonists and agonists for the treatments of cancer and pruritus.

Single-cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin-driven circadian network.

Circadian rhythms in mammals are governed by the hypothalamic suprachiasmatic nucleus (SCN), in which 20,000 clock cells are connected together into a powerful time-keeping network. In the absence of network-level cellular interactions, the SCN fails as a clock. The topology and specific roles of its distinct cell populations (nodes) that direct network functions are, however, not understood. To characterise its component cells and network structure, we conducted single-cell sequencing of SCN organotypic slices and identified eleven distinct neuronal sub-populations across circadian day and night. We defined neuropeptidergic signalling axes between these nodes, and built neuropeptide-specific network topologies. This revealed their temporal plasticity, being up-regulated in circadian day. Through intersectional genetics and real-time imaging, we interrogated the contribution of the Prok2-ProkR2 neuropeptidergic axis to network-wide time-keeping. We showed that Prok2-ProkR2 signalling acts as a key regulator of SCN period and rhythmicity and contributes to defining the network-level properties that underpin robust circadian co-ordination. These results highlight the diverse and distinct contributions of neuropeptide-modulated communication of temporal information across the SCN.

GRP/GRPR enhances alcohol-associated liver injury through the IRF1-mediated Caspase-1 inflammasome and NOX2-dependent ROS pathway.

BACKGROUND AND AIMS: The common characteristics of alcohol-associated liver injury (ALI) include abnormal liver function, infiltration of inflammatory cells, and generation of oxidative stress. The gastrin-releasing peptide receptor (GRPR) is activated by its neuropeptide ligand, gastrin-releasing peptide (GRP). GRP/GRPR appears to induce the production of cytokines in immune cells and promotes neutrophil migration. However, the effects of GRP/GRPR in ALI are unknown. APPROACH AND RESULTS: We found high GRPR expression in the liver of patients with alcohol-associated steatohepatitis and increased pro-GRP levels in peripheral blood mononuclear cells of these patients compared with that of the control. Increased expression of GRP may be associated with histone H3 lysine 27 acetylation induced by alcohol, which promotes the expression of GRP and then GRPR binding. Grpr-/- and Grprflox/floxLysMCre mice alleviated ethanol-induced liver injury with relieved steatosis, lower serum alanine aminotransferase, aspartate aminotransferase, triglycerides, malondialdehyde, and superoxide dismutase levels, reduced neutrophil influx, and decreased expression and release of inflammatory cytokines and chemokines. Conversely, the overexpression of GRPR showed opposite effects. The pro-inflammatory and oxidative stress roles of GRPR might be dependent on IRF1-mediated Caspase-1 inflammasome and NOX2-dependent reactive oxygen species pathway, respectively. In addition, we verified the therapeutic and preventive effects of RH-1402, a novel GRPR antagonist, for ALI. CONCLUSIONS: A knockout or antagonist of GRPR during excess alcohol intake could have anti-inflammatory and antioxidative roles, as well as provide a platform for histone modification-based therapy for ALI.

Descending dopaminergic pathway facilitates itch signal processing via activating spinal GRPR+ neurons.

A11 dopaminergic neurons regulate somatosensory transduction by projecting from the diencephalon to the spinal cord, but the function of this descending projection in itch remained elusive. Here, we report that dopaminergic projection neurons from the A11 nucleus to the spinal dorsal horn (dopaminergicA11-SDH ) are activated by pruritogens. Inhibition of these neurons alleviates itch-induced scratching behaviors. Furthermore, chemogenetic inhibition of spinal dopamine receptor D1-expressing (DRD1+ ) neurons decreases acute or chronic itch-induced scratching. Mechanistically, spinal DRD1+ neurons are excitatory and mostly co-localize with gastrin-releasing peptide (GRP), an endogenous neuropeptide for itch. In addition, DRD1+ neurons form synapses with GRP receptor-expressing (GRPR+ ) neurons and activate these neurons via AMPA receptor (AMPAR). Finally, spontaneous itch and enhanced acute itch induced by activating spinal DRD1+ neurons are relieved by antagonists against AMPAR and GRPR. Thus, the descending dopaminergic pathway facilitates spinal itch transmission via activating DRD1+ neurons and releasing glutamate and GRP, which directly augments GRPR signaling. Interruption of this descending pathway may be used to treat chronic itch.

Gastrin-releasing peptide regulates fear learning under stressed conditions via activation of the amygdalostriatal transition area.

The amygdala, a critical brain region responsible for emotional behavior, is crucially involved in the regulation of the effects of stress on emotional behavior. In the mammalian forebrain, gastrin-releasing peptide (GRP), a 27-amino-acid mammalian neuropeptide, which is a homolog of the 14-amino-acid amidated amphibian peptide bombesin, is highly expressed in the amygdala. The levels of GRP are markedly increased in the amygdala after acute stress; therefore, it is known as a stress-activated modulator. To determine the role of GRP in emotional behavior under stress, we conducted some behavioral and biochemical experiments with GRP-knockout (KO) mice. GRP-KO mice exhibited a longer freezing response than wild-type (WT) littermates in both contextual and auditory fear (also known as threat) conditioning tests only when they were subjected to acute restraint stress 20 min before the conditioning. To identify the critical neural circuits associated with the regulation of emotional memory by GRP, we conducted Arc/Arg3.1-reporter mapping in the amygdala with an Arc-Venus reporter transgenic mouse line. In the amygdalostriatal transition area (AST) and the lateral side of the basal nuclei, fear conditioning after restraint stress increased neuronal activity significantly in WT mice, and GRP KO was found to negate this potentiation only in the AST. These results indicate that the GRP-activated neurons in the AST are likely to suppress excessive fear expression through the regulation of downstream circuits related to fear learning following acute stress.

Chronic corticosterone exposure evokes itch hypersensitivity and sexual dysfunction in male rats: Relationship between the two distinct gastrin-releasing peptide systems in the spinal cord.

In today's society, people are subjected to many social stressors, and excessive chronic stress causes functional disruption of the neuroendocrine system and many diseases. Although the exacerbation of atopic dermatitis with symptoms of itching and erectile dysfunction is induced by chronic stress, the details of the mechanisms are unknown. Here, we examined the effects of chronic stress on itch sensation and male sexual function at the behavioral and molecular levels, focusing on two distinct gastrin-releasing peptide (GRP) systems that independently regulate itch transmission, i.e., the somatosensory GRP system, and male sexual function, i.e., the lumbosacral autonomic GRP system, in the spinal cord. In a rat model of chronic stress induced by chronic corticosterone (CORT) administration, we observed increased plasma CORT concentrations, decreased body weight, and increased anxiety-like behavior, similar to that observed in humans. Chronic CORT exposure induced hypersensitivity to itch and increased the Grp mRNA level in the spinal somatosensory system, but there was no change in pain or tactile sensitivity. Antagonists of the somatosensory GRP receptor, an itch-specific mediator, suppressed itch hypersensitivity induced by chronic CORT exposure. In contrast, chronic CORT exposure decreased male sexual behavior, ejaculated semen volume, vesicular gland weight, and plasma testosterone levels. However, there were no effects on the expression of Grp mRNA or protein in the lumbosacral autonomic GRP system, which regulates male sexual function. In summary, chronic stress model rats showed itch hypersensitivity and impaired sexual function in males, and the involvement of the spinal GRP systems was apparent in itch hypersensitivity.

Analgesic effect of gastrin-releasing peptide in the dorsal horn.

Itch and pain are both unpleasant, but they are discrete sensations. Both of these sensations are transmitted by C-fibers and processed in laminae I-II of the dorsal horn. To examine whether pruriception modulates pain, we first confirmed the activation of cells in the itch-related circuits that were positive for gastrin-releasing peptide (GRP) and GRP receptor (GRPR) using a paw formalin injection model. This pain model with typical biphasic pain behavior increased c-Fos but did not affect the expressions of GRP and GRPR mRNAs in the dorsal horn. Using c-Fos expression as a marker for activated cells, we confirmed that formalin injection increased the number of cells double-labeled for c-Fos and GRP or GRPR in the dorsal horn. The emergence of these neurons indicates the activation of itch-related circuits by acute pain signals. The effect of an antagonist for a GRPR was examined in the paw formalin injection model. Intrathecal chronic antagonization of spinal GRPR enhanced the onset of phase II of paw formalin injection-induced pain behavior. Exogenous intrathecal GRP infusion to the paw-formalin injection model not only showed significant reduction of pain behavior but also increased c-Fos in the inhibitory neurons in the dorsal horn. The anti-nociceptive effect of spinal GRP infusion was observed in the peripheral inflammation model (complete Freund's adjuvant injection model). In this study we suggest that painful stimuli activated itch-related neuronal circuits and uncovered the spinal activation of the itch-induced analgesic effect on acute and established inflammatory pain.

Constitutively active BRS3 is a genuinely orphan GPCR in placental mammals.

G protein-coupled receptors (GPCRs) play an important role in physiology and disease and represent the most productive drug targets. Orphan GPCRs, with their endogenous ligands unknown, were considered a source of drug targets and consequently attract great interest to identify their endogenous cognate ligands for deorphanization. However, a contrary view to the ubiquitous existence of endogenous ligands for every GPCR is that there might be a significant overlooked fraction of orphan GPCRs that function constitutively in a ligand-independent manner only. Here, we investigated the evolution of the bombesin receptor-ligand family in vertebrates in which one member-bombesin receptor subtype-3 (BRS3)-is a potential orphan GPCR. With analysis of 17 vertebrate BRS3 structures and 10 vertebrate BRS3 functional data, our results demonstrated that nonplacental vertebrate BRS3 still connects to the original ligands-neuromedin B (NMB) and gastrin-releasing peptide (GRP)-because of adaptive evolution, with significantly changed protein structure, especially in three altered key residues (Q127R, P205S, and R294H) originally involved in ligand binding/activation, whereas the placental mammalian BRS3 lost the binding affinity to NMB/GRP and constitutively activates Gs/Gq/G12 signaling in a ligand-independent manner. Moreover, the N terminus of placental mammalian BRS3 underwent positive selection, exhibiting significant structural differences compared to nonplacental vertebrate BRS3, and this domain plays an important role in constitutive activity of placental mammalian BRS3. In conclusion, constitutively active BRS3 is a genuinely orphan GPCR in placental mammals, including human. To our knowledge, this study identified the first example that might represent a new group of genuinely orphan GPCRs that will never be deorphanized by the discovery of a natural ligand and provided new perspectives in addition to the current ligand-driven GPCR deorphanization.

The peptidergic control circuit for sighing.

Sighs are long, deep breaths expressing sadness, relief or exhaustion. Sighs also occur spontaneously every few minutes to reinflate alveoli, and sighing increases under hypoxia, stress, and certain psychiatric conditions. Here we use molecular, genetic, and pharmacologic approaches to identify a peptidergic sigh control circuit in murine brain. Small neural subpopulations in a key breathing control centre, the retrotrapezoid nucleus/parafacial respiratory group (RTN/pFRG), express bombesin-like neuropeptide genes neuromedin B (Nmb) or gastrin-releasing peptide (Grp). These project to the preBötzinger Complex (preBötC), the respiratory rhythm generator, which expresses NMB and GRP receptors in overlapping subsets of ~200 neurons. Introducing either neuropeptide into preBötC or onto preBötC slices, induced sighing or in vitro sigh activity, whereas elimination or inhibition of either receptor reduced basal sighing, and inhibition of both abolished it. Ablating receptor-expressing neurons eliminated basal and hypoxia-induced sighing, but left breathing otherwise intact initially. We propose that these overlapping peptidergic pathways comprise the core of a sigh control circuit that integrates physiological and perhaps emotional input to transform normal breaths into sighs.

Dorsal Horn Gastrin-Releasing Peptide Expressing Neurons Transmit Spinal Itch But Not Pain Signals.

Gastrin-releasing peptide (GRP) is a spinal itch transmitter expressed by a small population of dorsal horn interneurons (GRP neurons). The contribution of these neurons to spinal itch relay is still only incompletely understood, and their potential contribution to pain-related behaviors remains controversial. Here, we have addressed this question in a series of experiments performed in GRP::cre and GRP::eGFP transgenic male mice. We combined behavioral tests with neuronal circuit tracing, morphology, chemogenetics, optogenetics, and electrophysiology to obtain a more comprehensive picture. We found that GRP neurons form a rather homogeneous population of central cell-like excitatory neurons located in lamina II of the superficial dorsal horn. Multicolor high-resolution confocal microscopy and optogenetic experiments demonstrated that GRP neurons receive direct input from MrgprA3-positive pruritoceptors. Anterograde HSV-based neuronal tracing initiated from GRP neurons revealed ascending polysynaptic projections to distinct areas and nuclei in the brainstem, midbrain, thalamus, and the somatosensory cortex. Spinally restricted ablation of GRP neurons reduced itch-related behaviors to different pruritogens, whereas their chemogenetic excitation elicited itch-like behaviors and facilitated responses to several pruritogens. By contrast, responses to painful stimuli remained unaltered. These data confirm a critical role of dorsal horn GRP neurons in spinal itch transmission but do not support a role in pain.SIGNIFICANCE STATEMENT Dorsal horn gastrin-releasing peptide neurons serve a well-established function in the spinal transmission of pruritic (itch) signals. A potential role in the transmission of nociceptive (pain) signals has remained controversial. Our results provide further support for a critical role of dorsal horn gastrin-releasing peptide neurons in itch circuits, but we failed to find evidence supporting a role in pain.

Immediate Release of Gastrin-Releasing Peptide Mediates Delayed Radiation-Induced Pulmonary Fibrosis.

Radiation-induced pulmonary fibrosis (RTPF) is a progressive, serious condition in many subjects treated for thoracic malignancies or after accidental nuclear exposure. No biomarker exists for identifying the irradiated subjects most susceptible to pulmonary fibrosis (PF). Previously, we determined that gastrin-releasing peptide (GRP) was elevated within days after birth in newborns exposed to hyperoxia who later developed chronic lung disease. The goal of the current study was to test whether radiation (RT) exposure triggers GRP release in mice and whether this contributes to RTPF in vivo. We determined urine GRP levels and lung GRP immunostaining in mice 0 to 24 after post-thoracic RT (15 Gy). Urine GRP levels were significantly elevated between 24 hours post-RT; GRP-blocking monoclonal antibody 2A11, given minutes post-RT, abrogated urine GRP levels by 6 to 12 hours and also altered phosphoprotein signaling pathways at 24 hours post-RT. Strong extracellular GRP immunostaining was observed in lung at 6 hours post-RT. Mice given one dose of GRP monoclonal antibody 2A11 24 hours post-RT had significantly reduced myofibroblast accumulation and collagen deposition 15 weeks later, indicating protection against lung fibrosis. Therefore, elevation of urine GRP could be predictive of RTPF development. In addition, transient GRP blockade could mitigate PF in normal lung after therapeutic or accidental RT exposure.

Gastrin-releasing peptide induces fibrotic response in MRC5s and proliferation in A549s.

Idiopathic pulmonary fibrosis (IPF) is a complex lung disease, whose build-up scar tissue is induced by several molecules. Gastrin-releasing peptide (GRP) is released from pulmonary neuroendocrine cells, alveolar macrophages, and some nerve endings in the lung. A possible role of GRP in IPF is unclear. We aimed to investigate the fibrotic response to GRP, at the cellular level in MRC5 and A549 cell lines. The proliferative and fibrotic effects of GRP on these cells were evaluated by using BrdU, immunoblotting, immunofluorescence and qRT-PCR for molecules associated with myofibroblast differentiation, TGF-ß and Wnt signalling. All doses of GRP increased the amount of BrdU incorporation in A549 cells. In contrast, the amount of BrdU increased in MRC5 cells in the first 24 h, though progressively decreased by 72 h. GRP did not stimulate epithelial-mesenchymal transition in A549 cells, rather, it stimulated the differentiation of MRC5 cells into myofibroblasts. Furthermore, GRP induced gene and protein expressions of p-Smad2/3 and Smad4, and reduced the levels of Smad7 in MRC5 cells. In addition, GRP decreased Wnt5a protein levels and stimulated ß-catenin activation by increasing Wnt4, Wnt7a and ß-catenin protein levels. GRP caused myofibroblast differentiation by inducing TGF-ßand Wnt pathways via paracrine and autocrine signalling in MRC5 cells. In conclusion, GRP may lead to pulmonary fibrosis due to its proliferative and fibrotic effects on lung fibroblasts. The abrogation of GRP-mediated signal activation might be considered as a treatment modality for fibrotic lung diseases. Video Abstract.

Role of reactive astrocytes in the spinal dorsal horn under chronic itch conditions.

Astrocytes are the most abundant glial cells in the central nervous system (CNS), including the spinal cord. Neuronal damage induces astrocytes to become reactive and contribute to various CNS pathologies. Recent studies have demonstrated that astrocytes in the spinal dorsal horn (SDH) become reactive in a transcription factor signal transducer and activator of transcription 3-dependent manner without neuronal damage under chronic itch conditions, causing release of the factor lipocalin-2, leading to induction of sensitization of gastrin releasing peptide-induced chemical itch signaling in the SDH. In this review, we describe recent advances in our understanding of SDH neuronal pathways for itch transmission, the mechanisms of SDH astrocytic activation and its contribution to abnormal itch processing and discuss the role of reactive astrocytes in the SDH in abnormal sensory processing under chronic itch conditions.

[Neuropathic pruritus]. / Neuropathischer Pruritus.

In the past 10 years specific pathways for pruritus have been characterized on a cellular and molecular level but their exact role in the pathophysiology of neuropathic pruritus remains unclear. This also applies to the question which of the competing theories for pruritus, e.g. specificity, temporal/spatial pattern or intensity, would best apply. While experimental trials on mice have mostly confirmed the theory of specificity, the results on humans indicate a role of spatial and temporal patterns. The skin innervation is greatly reduced by the neuropathy and could provide a "spatial contrast pattern" and the axotomy could induce a de novo expression of gastrin-releasing peptide (GRP) in primarily afferent nociceptors and thus modulate spinal pruritus processing. In addition, the overlap of pruritus and pain in neuropathy patients complicates the direct translation from animal experiments and requires collaboration at the clinical level between pain medicine and dermatology.

Spinal IL-33/ST2 signaling mediates chronic itch in mice through the astrocytic JAK2-STAT3 cascade.

Interleukin-33 (IL-33) and its receptor ST2 contribute to spinal glial activation and chronic pain. A recent study showed that peripheral IL-33 plays a pivotal role in the pathogenesis of chronic itch induced by poison ivy. However, how IL-33/ST2 signaling in the spinal cord potentially mediates chronic itch remains elusive. Here, we determined that St2-/- substantially reduced scratching behaviors in 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis (ACD) as well as acetone and diethylether followed by water-induced dry skin in mice. Intrathecal administration of the neutralizing anti-ST2 or anti-IL-33 antibody remarkably decreased the scratching response in DNFB-induced ACD mice. Expression of spinal IL-33 and ST2 significantly increased in ACD mice, as evidenced by increased mRNA and protein levels. Immunofluorescence and in situ hybridization demonstrated that increased expression of spinal IL-33 was predominant in oligodendrocytes and astrocytes, whereas ST2 was mainly expressed in astrocytes. Further studies showed that in ACD mice, the activation of astrocytes and increased phosphorylation of signal transducer and activator of transcription 3 (STAT3) were markedly attenuated by St2-/- . Intrathecal injection of Janus Kinase 2 Inhibitor AG490 significantly alleviated scratching behaviors in ACD mice. rIL-33 pretreatment exacerbated gastrin-releasing peptide (GRP)-evoked scratching behaviors. This increased gastrin-releasing peptide receptor (GRPR) expression was abolished by St2-/- . Tnf-α upregulation was suppressed by St2-/- . Our results indicate that the spinal IL-33/ST2 signaling pathway contributes to chronic itch via astrocytic JAK2-STAT3 cascade activation, promoting TNF-α release to regulate the GRP/GRPR signaling-related itch response. Thus, these findings provide a potential therapeutic option for treating chronic pruritus.

Independent prognostic genes and mechanism investigation for colon cancer.

PROPOSE: We aimed to explore the potential molecular mechanism and independent prognostic genes for colon cancer (CC). METHODS: Microarray datasets GSE17536 and GSE39582 were downloaded from Gene Expression Omnibus. Meanwhile, the whole CC-related dataset were downloaded from The Cancer Genome Atlas (TCGA) database. Differentially expressed mRNA (DEMs) were identified between cancer tissue samples and para-carcinoma tissue samples in TCGA dataset, followed by the KEGG pathway and GO function analyses. Furthermore, the clinical prognostic analysis including overall survival (OS) and disease-free survival (DFS) were performed in all three datasets. RESULTS: A total of 633 up- and 321 down-regulated mRNAs were revealed in TCGA dataset. The up-regulated mRNAs were mainly assembled in functions including extracellular matrix and pathways including Wnt signaling. The down-regulated mRNAs were mainly assembled in functions like Digestion and pathways like Drug metabolism. Furthermore, up-regulation of UL16-binding protein 2 (ULBP2) was associated with OS in CC patients. A total of 12 DEMs including Surfactant Associated 2 (SFTA2) were potential DFS prognostic genes in CC patients. Meanwhile, the GRP and Transmembrane Protein 37 (TMEM37) were two outstanding independent DFS prognostic genes in CC. CONCLUSIONS: ULBP2 might be a potential novel OS prognostic biomarker in CC, while GRP and TMEM37 could be served as the independent DFS prognostic genes in CC. Furthermore, functions including extracellular matrix and digestion, as well as pathways including Wnt signaling and drug metabolism might play important roles in the process of CC.

The preterm infant stomach actively degrades milk proteins with increasing breakdown across digestion time.

AIM: This study investigated the effect of time post-ingestion on gastric digestion and gastric hormones after feeding preterm infants unfortified and fortified human milk. METHODS: Human milk and infant gastric samples were collected from 14 preterm (23-32 weeks birth gestational age) mother-infant pairs within 7-98 days postnatal age. Gastric samples were collected one, two and three hours after beginning of feeding. Samples were analysed for pH, proteolysis, general protease activity and the concentrations of pepsin, gastrin and gastrin-releasing peptide (GRP). One-way ANOVA with repeated measures followed by Tukey's multiple comparisons test was used. RESULTS: Gastric pH was significantly decreased after each hour in the preterm infant stomach from one to three hours postprandial. Proteolysis increased significantly from human milk to gastric contents at one, two and three hours postprandial (by 62, 131% and 181%, p < 0.05). General protease activity increased significantly by 58% from human milk to the gastric contents at two hours postprandial. GRP was present in human milk, whereas gastrin was produced in the infant stomach. CONCLUSION: Although preterm infants may digest human milk proteins to a lesser extent than term infants, we demonstrated that the preterm infant stomach actively degrades milk proteins with increasing breakdown over digestion time.

A sexually dimorphic peptidergic system in the lower spinal cord controlling penile function in non-human primates.

STUDY DESIGN: Experimental animal study. OBJECTIVES: Although a population of gastrin-releasing peptide (GRP) neurons in the lumbar spinal cord has an important role in erection and ejaculation in rats, little information exists on this GRP system in primates. To identify the male-specific GRP system in the primate spinal cord, we studied the lumbosacral cord in macaque monkeys as a non-human primate model. SETTING: University laboratory in Japan. METHODS: To determine the gene sequence of GRP precursors, the rhesus macaque monkey genomic sequence data were searched, followed by phylogenetic analysis. Subsequently, immunocytochemical analysis for GRP was performed in the monkey spinal cord. RESULTS: We have used bioinformatics to identify the ortholog gene for GRP precursor in macaque monkeys. Phylogenetic analysis suggested that primate prepro-GRP is separated from that of other mammalian species and clustered to an independent branch as primates. Immunocytochemistry for GRP further demonstrated that male-dominant sexual dimorphism was found in the spinal GRP system in monkeys as in rodents. CONCLUSION: We have demonstrated in macaque monkeys that the GRP system in the lower spinal cord shows male-specific dimorphism and may have an important role in penile functions not only in rodents but also in primates. SPONSORSHIP: Tissues of Nihonzaru (Japanese macaque monkeys) were provided in part by National Institutes of Natural Sciences (NINS) through the National Bio-Resource Project (NBRP) of the MEXT, Japan. This work was supported in part by KAKENHI from the Japan Society for the Promotion of Science (JSPS) (to KT; 15KK0343, 15J40220 and HS; 15K15202, 15KK0257, 15H05724).