Neuromedin B receptor stimulation of Cav3.2 T-type Ca2+ channels in primary sensory neurons mediates peripheral pain hypersensitivity.

Background: Neuromedin B (Nmb) is implicated in the regulation of nociception of sensory neurons. However, the underlying cellular and molecular mechanisms remain unknown. Methods: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of Nmb on the sensory neuronal excitability and peripheral pain sensitivity mediated by Cav3.2 T-type channels. Results: Nmb reversibly and concentration-dependently increased T-type channel currents (IT) in small-sized trigeminal ganglion (TG) neurons through the activation of neuromedin B receptor (NmbR). This NmbR-mediated IT response was Gq protein-coupled, but independent of protein kinase C activity. Either intracellular application of the QEHA peptide or shRNA-mediated knockdown of Gß abolished the NmbR-induced IT response. Inhibition of protein kinase A (PKA) or AMP-activated protein kinase (AMPK) completely abolished the Nmb-induced IT response. Analysis of phospho-AMPK (p-AMPK) revealed that Nmb significantly activated AMPK, while AMPK inhibition prevented the Nmb-induced increase in PKA activity. In a heterologous expression system, activation of NmbR significantly enhanced the Cav3.2 channel currents, while the Cav3.1 and Cav3.3 channel currents remained unaffected. Nmb induced TG neuronal hyperexcitability and concomitantly induced mechanical and thermal hypersensitivity, both of which were attenuated by T-type channel blockade. Moreover, blockade of NmbR signalling prevented mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain, and this effect was attenuated by siRNA knockdown of Cav3.2. Conclusions: Our study reveals a novel mechanism by which NmbR stimulates Cav3.2 channels through a Gßγ-dependent AMPK/PKA pathway. In mouse models, this mechanism appears to drive the hyperexcitability of TG neurons and induce pain hypersensitivity.

Central opioid receptors mediate morphine-induced itch and chronic itch via disinhibition.

Opioids such as morphine are mainstay treatments for clinical pain conditions. Itch is a common side effect of opioids, particularly as a result of epidural or intrathecal administration. Recent progress has advanced our understanding of itch circuits in the spinal cord. However, the mechanisms underlying opioid-induced itch are not fully understood, although an interaction between µ-opioid receptor (MOR) and gastrin-releasing peptide receptor (GRPR) in spinal GRPR-expressing neurons has been implicated. In this study we investigated the cellular mechanisms of intrathecal opioid-induced itch by conditional deletion of MOR-encoding Oprm1 in distinct populations of interneurons and sensory neurons. We found that intrathecal injection of the MOR agonists morphine or DAMGO elicited dose-dependent scratching as well as licking and biting, but this pruritus was totally abolished in mice with a specific Oprm1 deletion in Vgat+ neurons [Oprm1-Vgat (Slc32a1)]. Loss of MOR in somatostatin+ interneurons and TRPV1+ sensory neurons did not affect morphine-induced itch but impaired morphine-induced antinociception. In situ hybridization revealed Oprm1 expression in 30% of inhibitory and 20% of excitatory interneurons in the spinal dorsal horn. Whole-cell recordings from spinal cord slices showed that DAMGO induced outward currents in 9 of 19 Vgat+ interneurons examined. Morphine also inhibited action potentials in Vgat+ interneurons. Furthermore, morphine suppressed evoked inhibitory postsynaptic currents in postsynaptic Vgat- excitatory neurons, suggesting a mechanism of disinhibition by MOR agonists. Notably, morphine-elicited itch was suppressed by intrathecal administration of NPY and abolished by spinal ablation of GRPR+ neurons with intrathecal injection of bombesin-saporin, whereas intrathecal GRP-induced itch response remained intact in mice lacking Oprm1-Vgat. Intrathecal bombesin-saporin treatment reduced the number of GRPR+ neurons by 97% in the lumber spinal cord and 91% in the cervical spinal cord, without changing the number of Oprm1+ neurons. Additionally, chronic itch from DNFB-induced allergic contact dermatitis was decreased by Oprm1-Vgat deletion. Finally, naloxone, but not peripherally restricted naloxone methiodide, inhibited chronic itch in the DNFB model and the CTCL model, indicating a contribution of central MOR signalling to chronic itch. Our findings demonstrate that intrathecal morphine elicits itch via acting on MOR on spinal inhibitory interneurons, leading to disinhibition of the spinal itch circuit. Our data also provide mechanistic insights into the current treatment of chronic itch with opioid receptor antagonist such as naloxone.

Imaging the Distribution of Gastrin-Releasing Peptide Receptors in Cancer.

Targeting tumor-expressed receptors using selective molecules for diagnostic, therapeutic, or both diagnostic and therapeutic (theragnostic) purposes is a promising approach in oncologic applications. Such approaches have increased significantly over the past decade. Peptides such as gastrin-releasing peptide receptors targeting radiopharmaceuticals are small molecules with fast blood clearance and urinary excretion. They demonstrate good tissue diffusion, low immunogenicity, and highly selective binding to their target cell-surface receptors. They are also easily produced. Gastrin-releasing peptide receptors, part of the bombesin family, are overexpressed in many tumors, including breast and prostate cancer, and therefore represent an attractive target for future development.

Recent insights into biological functions of mammalian bombesin-like peptides and their receptors.

PURPOSE OF REVIEW: The current review highlights recent advances in physiological and pharmacological researches in biology of mammalian bombesin-like peptides (BLPs). RECENT FINDINGS: BLPs and their receptors were found to have regulatory roles in many biological processes in central nervous system. Two BLPs, neuromedin B and gastrin-releasing peptide (GRP), and their receptors are required for regulation of basal and induced sighing activity in rodents. This is the first study demonstrating central pathways involved in regulation of sighing activity. GRP receptor (GRPR) expressing neurons are excitatory glutamatergic interneurons located in the dorsal lamina without projections outside the spinal cord and mediate itch signals via vesicular glutamate transporter 2. Those neurons receive itch signals and make synapses with the parabrachial nucleus projecting spinal neurons to transmit itch signals to parabrachial nucleus. GRP expressing interneurons function in a proposed 'leaky gate model' to interpret the mechanism of both pain and itch transmission. In addition to recent advances of biology in nervous system, BLPs and their receptors were found to play potential regulatory roles in innate and adaptive immune responses and tissue development. SUMMARY: Several important biological roles of BLPs and their receptors in nervous system were identified. Together with researches regarding central roles of BLPs, studies revealing the regulatory roles of BLPs and their receptors in immunology and tissue development provide us with novel insights into understanding of the biology of BLPs and their receptors.

Molecular and neural basis of contagious itch behavior in mice.

Socially contagious itch is ubiquitous in human society, but whether it exists in rodents is unclear. Using a behavioral paradigm that does not entail prior training or reward, we found that mice scratched after observing a conspecific scratching. Molecular mapping showed increased neuronal activity in the suprachiasmatic nucleus (SCN) of the hypothalamus of mice that displayed contagious scratching. Ablation of gastrin-releasing peptide receptor (GRPR) or GRPR neurons in the SCN abolished contagious scratching behavior, which was recapitulated by chemogenetic inhibition of SCN GRP neurons. Activation of SCN GRP/GRPR neurons evoked scratching behavior. These data demonstrate that GRP-GRPR signaling is necessary and sufficient for transmitting contagious itch information in the SCN. The findings may have implications for our understanding of neural circuits that control socially contagious behaviors.

A Stress-Related Peptide Bombesin Centrally Induces Frequent Urination through Brain Bombesin Receptor Types 1 and 2 in the Rat.

Stress exacerbates symptoms of bladder dysfunction including overactive bladder and bladder pain syndrome, but the underlying mechanisms are unknown. Bombesin-like peptides and bombesin receptor types 1 and 2 (BB1 and BB2, respectively) in the brain have been implicated in the mediation/integration of stress responses. In this study, we examined effects of centrally administered bombesin on micturition, focusing on their dependence on 1) the sympathoadrenomedullary system (a representative mechanism activated by stress exposure) and 2) brain BB receptors in urethane-anesthetized (1.0-1.2 g/kg, i.p.) male rats. Intracerebroventricularly administered bombesin significantly shortened intercontraction intervals (ICI) at both doses (0.1 and 1 nmol/animal) without affecting maximal voiding pressure. Bombesin at 1 nmol induced significant increments of plasma noradrenaline and adrenaline levels, which were both abolished by acute bilateral adrenalectomy. On the other hand, adrenalectomy showed no effects on the bombesin-induced shortening of ICI. Much lower doses of bombesin (0.01 and 0.03 nmol/animal, i.c.v.) dose-dependently shortened ICI. Pretreatment with either a BB1 receptor antagonist (BIM-23127; d-Nal-cyclo[Cys-Tyr-d-Trp-Orn-Val-Cys]-Nal-NH2; 3 nmol/animal, i.c.v.) or a BB2 receptor antagonist (BEA; H-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt; 3 nmol/animal, i.c.v.), respectively, suppressed the BB (0.03 nmol/animal, i.c.v.)-induced shortening of ICI, whereas each antagonist by itself (1 and 3 nmol/animal, i.c.v.) had no significant effects on ICI. Bombesin (0.03 nmol/animal, i.c.v.) significantly reduced voided volume per micturition and bladder capacity without affecting postvoid residual volume or voiding efficiency. These results suggest that brain bombesin and BB receptors are involved in facilitation of the rat micturition reflex to induce bladder overactivity, which is independent of the sympathoadrenomedullary outflow modulation.

Spinal Functions of B-Type Natriuretic Peptide, Gastrin-Releasing Peptide, and Their Cognate Receptors for Regulating Itch in Mice.

B-type natriuretic peptide (BNP)-natriuretic peptide receptor A (NPRA) and gastrin-releasing peptide (GRP)-GRP receptor (GRPR) systems contribute to spinal processing of itch. However, pharmacological and anatomic evidence of these two spinal ligand-receptor systems are still not clear. The aim of this study was to determine the spinal functions of BNP-NPRA and GRP-GRPR systems for regulating scratching activities in mice by using pharmacological and immunohistochemical approaches. Our results showed that intrathecal administration of BNP (0.3-3 nmol) dose dependently elicited scratching responses, which could be blocked by the NPRA antagonist (Arg6,ß-cyclohexyl-Ala8,D-Tic16,Arg17,Cys18)-atrial natriuretic factor(6-18) amide (A71915). However, A71915 had no effect on intrathecal GRP-induced scratching. In contrast, pretreatment with a GRPR antagonist (D-Tpi6,Leu13ψ(CH2-NH)-Leu14)bombesin(6-14) (RC-3095) inhibited BNP-induced scratching. Immunostaining revealed that NPRA proteins colocalize with GRP, but not GRPR, in the superficial area of dorsal horn, whereas BNP proteins do not colocalize with either GRP or GRPR in the dorsal horn. Intradermal administration of ligands including endothelin-1, U-46619, bovine adrenal medulla 8-22, and Ser-Leu-Ile-Gly-Arg-Leu-NH2 (SLIGRL) increased scratching bouts at different levels of magnitude. Pretreatment with intrathecal A71915 did not affect scratching responses elicited by all four pruritogens, whereas pretreatment with RC-3095 only inhibited SLIGRL-induced scratching. Interestingly, immunostaining showed that RC-3095, but not A71915, inhibited SLIGRL-elicited c-Fos activation in the spinal dorsal horn, which was in line with behavioral outcomes. These findings demonstrate that: 1) BNP-NPRA system may function upstream of the GRP-GRPR system to regulate itch in the mouse spinal cord, and 2) both NPRA and GRPR antagonists may have antipruritic efficacy against centrally, but not peripherally, elicited itch.

Bombesin-like peptides and their receptors: recent findings in pharmacology and physiology.

PURPOSE OF REVIEW: To highlight the research progress of roles of bombesin-like peptides and their receptors in pharmacology and physiology. RECENT FINDINGS: Several new bombesin-derived radioactive or nonradioactive compounds were designed for the diagnosis and therapy of tumors that are overexpressing bombesin receptors. Both gastrin-releasing peptide receptor and neuromedin B receptor activation were shown to induce membrane depolarization and excite neurons in brain. Bombesin receptor subtype-3 was found to be downregulated in the muscle cells and myocytes from obese and type 2 diabetes patients, and its relevant cell signaling events in glucose homeostasis were also investigated. The molecular events triggered by bombesin receptors activation in different types of malignancies is being explored recently and new clues were provided for a better understanding of the biological roles of abnormal expression of bombesin receptors in tumors. Novel cross-talk between gastrin-releasing peptide receptor cell signaling and Sonic hedgehog pathways was identified in small-cell lung carcinoma. SUMMARY: Increasing evidence shows bombesin-like peptides and their receptors play important roles in both physiological state and diseases. More specific and safe tumor targeting Bombesin derivatives are being developed for tumor diagnosis and therapy.

Cross talk between the bombesin neuropeptide receptor and Sonic hedgehog pathways in small cell lung carcinoma.

Small cell lung carcinoma (SCLC) often features the upregulation of the Sonic hedgehog (Shh) pathway leading to activation of Gli transcription factors. SCLC cells secrete bombesin (BBS)-like neuropeptides that act as autocrine growth factors. Here, we show that SCLC tumor samples feature co-expression of Shh and BBS-cognate receptor (gastrin-releasing peptide receptor (GRPR)). We also demonstrate that BBS activates Gli in SCLC cells, which is crucial for BBS-mediated SCLC proliferation, because cyclopamine, an inhibitor of the Shh pathway, hampered the BBS-mediated effects. BBS binding to GRPR stimulated Gli through its downstream Gαq and Gα12/13 GTPases, and consistently, other Gαq and Gα13 coupled receptors (such as muscarinic receptor, m1, and thrombin receptor, PAR-1) and constitutively active GαqQL and Gα12/13QL mutants stimulated Gli. By using cells null for Gαq and Gα12/13, we demonstrate that these G proteins are strictly necessary for Gli activation by BBS. Moreover, by using constitutively active Rho small G-protein (Rho QL) as well as its inhibitor, C3 toxin, we show that Rho mediates G-protein-coupled receptor (GPCR)-, Gαq- and Gα12/13-dependent Gli stimulation. At the molecular level, BBS caused a significant increase in Shh gene transcription and protein secretion that was dependent on BBS-induced GPCR/Gαq-12/13/Rho mediated activation of nuclear factor κB (NFκB), which can stimulate a NF-κB response element in the Shh gene promoter. Our data identify a novel molecular network acting in SCLC linking autocrine BBS and Shh circuitries and suggest Shh inhibitors as novel therapeutic strategies against this aggressive cancer type.

Excitatory effects of bombesin receptors in urinary tract of normal and diabetic rats in vivo.

AIMS: Bombesin receptors (BB receptors) and bombesin related peptides are expressed in the lower urinary tract of rodents. Here we investigated whether in vivo activation of BB receptors can contract the urinary bladder and facilitate micturition in sham rats and in a diabetic rat model of voiding dysfunction. MATERIAL AND METHODS: In vivo cystometry experiments were performed in adult female Sprague-Dawley rats under urethane anesthesia. Diabetes was induced by streptozotocin (STZ; 65mg/kg, i.p.) injection. Experiments were performed 9 and 20weeks post STZ-treatment. Drugs included neuromedin B (NMB; BB1 receptor preferring agonist), and gastrin-releasing peptide (GRP; BB2 receptor preferring agonist). KEY FINDINGS: NMB and GRP (0.01-100µg/kg in sham rats; 0.1-300µg/kg in STZ-treated rats, i.v.) increased micturition frequency, bladder contraction amplitude and area under the curve dose dependently in both sham and STZ-treated rats. In addition, NMB (3, 10µg/kg i.v.) triggered voiding in >80% of STZ-treated rats when the bladder was filled to a sub-threshold voiding volume. NMB and GRP increased mean arterial pressure and heart rate at the highest doses, 100 and 300µg/kg. SIGNIFICANCE: Activation of bombesin receptors facilitated neurogenic bladder contractions in vivo. Single applications of agonists enhanced or triggered voiding in sham rats as well as in the STZ-treated rat model of diabetic voiding dysfunction. These results suggest that BB receptors may be targeted for drug development for conditions associated with poor detrusor contraction such as an underactive bladder condition.

Gastrin-releasing peptide receptor signaling in the integration of stress and memory.

Neuropeptides act as signaling molecules that regulate a range of aspects of brain function. Gastrin-releasing peptide (GRP) is a 27-amino acid mammalian neuropeptide, homolog of the amphibian peptide bombesin. GRP acts by binding to the GRP receptor (GRPR, also called BB2), a member of the G-protein coupled receptor (GPCR) superfamily. GRP produced by neurons in the central nervous system (CNS) plays a role in synaptic transmission by activating GRPRs located on postsynaptic membranes, influencing several aspects of brain function. Here we review the role of GRP/GRPR as a system mediating both stress responses and the formation and expression of memories for fearful events. GRPR signaling might integrate the processing of stress and fear with synaptic plasticity and memory, serving as an important component of the set of neurobiological systems underlying the enhancement of memory storage by aversive information.

Human BRS-3 receptor: functions/role in cell signaling pathways and glucose metabolism in obese or diabetic myocytes.

Several studies showed that the orphan Bombesin Receptor Subtype-3 (BRS-3) - member of the bombesin receptor family - has an important role in glucose homeostasis (v.g.: BRS-3-KO mice developed mild obesity, and decreased levels of BRS-3 mRNA/protein have been described in muscle from obese (OB) and type 2 diabetic (T2D) patients). In this work, to gain insight into BRS-3 receptor cell signaling pathways, and its implication on glucose metabolism, primary cultured myocytes from normal subjects, OB or T2D patients were tested using high affinity ligand - [d-Tyr(6),ß-Ala(11),Phe(13),Nle(14)]bombesin6-14. In muscle cells from all metabolic conditions, the compound significantly increased not only MAPKs, p90RSK1, PKB and p70s6K phosphorylation levels, but also PI3K activity; moreover, it produced a dose-response stimulation of glycogen synthase a activity and glycogen synthesis. Myocytes from OB and T2D patients were more sensitive to the ligand than normal, and T2D cells even more than obese myocytes. These results widen the knowledge of human BRS-3 cell signaling pathways induced by a BRS-3 agonist, described its insulin-mimetic effects on glucose metabolism, showed the role of BRS-3 receptor in glucose homeostasis, and also propose the employing of BRS-3/ligand system, as participant in the obese and diabetic therapies.

Chronic itch development in sensory neurons requires BRAF signaling pathways.

Chronic itch, or pruritus, is associated with a wide range of skin abnormalities. The mechanisms responsible for chronic itch induction and persistence remain unclear. We developed a mouse model in which a constitutively active form of the serine/threonine kinase BRAF was expressed in neurons gated by the sodium channel Nav1.8 (BRAF(Nav1.8) mice). We found that constitutive BRAF pathway activation in BRAF(Nav1.8) mice results in ectopic and enhanced expression of a cohort of itch-sensing genes, including gastrin-releasing peptide (GRP) and MAS-related GPCR member A3 (MRGPRA3), in nociceptors expressing transient receptor potential vanilloid 1 (TRPV1). BRAF(Nav1.8) mice showed de novo neuronal responsiveness to pruritogens, enhanced pruriceptor excitability, and heightened evoked and spontaneous scratching behavior. GRP receptor expression was increased in the spinal cord, indicating augmented coding capacity for itch subsequent to amplified pruriceptive inputs. Enhanced GRP expression and sustained ERK phosphorylation were observed in sensory neurons of mice with allergic contact dermatitis­ or dry skin­elicited itch; however, spinal ERK activation was not required for maintaining central sensitization of itch. Inhibition of either BRAF or GRP signaling attenuated itch sensation in chronic itch mouse models. These data uncover RAF/MEK/ERK signaling as a key regulator that confers a subset of nociceptors with pruriceptive properties to initiate and maintain long-lasting itch sensation.

Integrin ß1 is critical for gastrin-releasing peptide receptor-mediated neuroblastoma cell migration and invasion.

BACKGROUND: Gastrin-releasing peptide (GRP) and its receptor, GRP-R, are critically involved in neuroblastoma tumorigenesis; however, the molecular mechanisms and signaling pathways that are responsible for GRP/GRP-R-induced cell migration and invasion remain unclear. In this study, we sought to determine the cell signals involved in GRP/GRP-R-mediated neuroblastoma cell migration and invasion. METHODS: Human neuroblastoma cell lines SK-N-SH, LAN-1, and IMR-32 were used for our study. Transwell migration and invasion assays were performed after GRP (10(-7) M) stimulation. The cDNA GEArray Microarray kit was used to determine GRP-R-induced gene expression changes. Protein and membrane expression of integrin subunits were confirmed by Western blotting and flow cytometry analysis. siRNA transfection was performed using Lipofectamine 2000. For scratch assay, a confluent monolayer of cells in 6-well plates were wounded with micropipette tip and observed microscopically at 24 to 72 h. RESULTS: GRP increased neuroblastoma cell migration and expressions of MMP-2 whereas the TIMP-1 level decreased. GRP-R overexpression stimulated SK-N-SH cell migration and upregulated integrin α2, α3, and ß1 protein as well as mRNA expression. Targeted silencing of integrin ß1 inhibited cell migration. CONCLUSION: GRP/GRP-R signaling contributes to neuroblastoma cell migration and invasion. Moreover, the integrin ß1 subunit critically regulates GRP-R-mediated neuroblastoma cell migration and invasion.

miR-335 and miR-363 regulation of neuroblastoma tumorigenesis and metastasis.

BACKGROUND: microRNA (miRNA) functions broadly as post-transcriptional regulators of gene expression, and disproportionate miRNAs can result in dysregulation of oncogenes in cancer cells. We have previously shown that gastrin-releasing peptide receptor (GRP-R) signaling regulates tumorigenicity of neuroblastoma cells. Herein, we sought to characterize miRNA profile in GRP-R silenced neuroblastoma cells, and to determine the role of miRNAs on tumorigenicity and metastatic potential. METHODS: Human neuroblastoma cell lines, BE(2)-C and SK-N-SH, were used for our study. Stably transfected GRP-R silenced cells were assessed for miRNA profiles. Cells were transfected with miR-335, miR-363, or miR-CON, a nontargeting control, and in vitro assays were performed. In vivo functions of miR-335 and miR-363 were also assessed in a spleen-liver metastasis murine model. RESULTS: GRP-R silencing significantly increased expression of miR-335 and miR-363 in BE(2)-C cells. Overexpression of miR-335 and miR-363 decreased tumorigenicity as measured by clonogenicity, anchorage-independent growth, and metastasis determined by cell invasion assay and liver metastasis in vivo. CONCLUSION: We report, for the first time, that GRP-R-mediated tumorigenicity and increased metastatic potential in neuroblastoma are regulated, in part, by miR-335 and miR-363. A better understanding of the anti-tumor functions of miRNAs could provide valuable insights to discerning molecular mechanisms responsible for neuroblastoma metastasis.

Gastrin-releasing peptide acts via postsynaptic BB2 receptors to modulate inward rectifier K+ and TRPV1-like conductances in rat paraventricular thalamic neurons.

Gastrin-releasing peptide (GRP) is a bombesin-like peptide with a widespread distribution in mammalian CNS, where it has a role in food intake, circadian rhythm generation, fear memory, itch sensation and sexual behaviour. While it has been established that GRP predominantly excites neurons, details of the membrane mechanism involved in this action remain largely undefined. We used perforated patch clamp recording in acute brain slice preparations to investigate GRP-affected receptors and ionic conductances in neurons of the rat paraventricular thalamic nucleus (PVT). PVT is a component of the midline and intralaminar thalamus that participates in arousal, motivational drives and stress responses, and exhibits a prominence of GRP-like immunoreactive fibres. Exposure of PVT neurons to low nanomolar concentrations of GRP induced sustained TTX-resistant membrane depolarizations that could trigger rhythmic burst discharges or tonic firing. Membrane current analyses in voltage clamp revealed an underlying postsynaptic bombesin type 2 receptor-mediated inward current that resulted from the simultaneous suppression of a Ba(2+)-sensitive inward rectifier K(+) conductance and activation of a non-selective cation conductance with biophysical and pharmacological properties reminiscent of transient receptor potential vanilloid (TRPV) 1. A role for a TRPV1-like conductance was further implied by a significant suppressant influence of a TRPV1 antagonist on GRP-induced membrane depolarization and rhythmic burst or tonic firing. The results provide a detailed picture of the receptor and ionic conductances that are involved in GRP's excitatory action in midline thalamus.

Activation of gastrin-releasing peptide receptors in the lumbosacral spinal cord is required for ejaculation in male rats.

INTRODUCTION: Ejaculation is a complex reflex mediated by a spinal ejaculation generator located in the lumbosacral spinal cord and consisting of a population of lumbar spinothalamic (LSt) neurons. LSt neurons and their intraspinal axonal projections contain several neuropeptides, including gastrin-releasing peptide (GRP). AIM: To test the hypothesis that GRP is critically involved in mediating ejaculation by acting in autonomic and motor areas of the lumbosacral spinal cord, utilizing a physiological paradigm to investigate ejaculatory reflexes in isolation of supraspinal inputs. METHODS: Dual immunohistochemistry for GRP and galanin was performed to investigate co-expression of GRP in LSt cells of control male rats. Next, anesthetized, spinalized male rats received intrathecal infusions of either GRP antagonist RC-3095 (0, 10, or 20 nmol/10 µL) or GRP (0, 0.2, 0.5 nmol/10 µL). Ejaculatory reflexes were induced by electrical stimulation of the dorsal penile nerve (DPN) which reliably triggers rhythmic increases in seminal vesicle pressure (SVP) and contractions of the bulbocavernosus muscle (BCM), indicative of the emission and expulsion phases of ejaculation, respectively. MAIN OUTCOME MEASURES: GRP in LSt cells was expressed as percentages of co-expression. SVP and electromyographic recording (EMG) of BCM activity following drug treatment and DPN stimulation were recorded and analyzed for numbers of SVP increases, BCM events and bursts. RESULTS: GRP was exclusively expressed in LSt cells and axons. Intrathecal infusion of RC-3095, but not saline, blocked SVP increases and BCM bursting induced by DPN stimulation. Intrathecal infusions of GRP, but not saline, triggered SVP increases and BCM bursting in 43-66% of animals and facilitated SVP increases and BCM bursting induced by subthreshold DPN stimulation in all animals. CONCLUSION: These data support a critical role for GRP for control of the emission and expulsion phases of ejaculation in male rats by acting in LSt target areas in the lumbosacral spinal cord.

Neuromedin B and its receptor influence the activity of myometrial primary cells in vitro through regulation of Il6 expression via the Rela/p65 pathway in mice.

The neuromedin B receptor (Nmbr) is an important physiological regulator of spontaneous activities and stress responses through different cascades as well as its autocrine and paracrine effects. Previous studies have revealed that neuromedin B (Nmb) and its receptor signal via the Rela (also known as p65)/Il6 pathway in a mouse model of pregnancy. This study investigated the mechanism of Nmbr signaling via the Rela/p65-Il6 pathway and regulation of the concentration of intracellular free calcium ([Ca(2+)](i)) during the onset of labor in primary mouse myometrial cell cultures isolated from mice in term labor. Data demonstrated Nmbr agonist-mediated upregulation of the DNA binding activity of Rela/p65, Il6 expression, and [Ca(2+)](i) in a concentration-dependent manner. Furthermore, a significant correlation was observed between DNA binding activity of Rela/p65 and Il6 expression. Moreover, this up-regulation was blocked by Nmbr and Rela/p65 knockdown, achieved by RNA interference (RNAi) technology. No significant differences were identified in the inhibition of Il6 expression as a result of Nmbr or Rela/p65 knockdown. However, significant differences were observed between the [Ca(2+)](i) in Rela/p65-specific group and that in the Nmbr-specific small interfering RNA (siRNA)-treated groups. These data demonstrated that the Nmb/Nmbr interaction in pregnant myometrial primary cells in vitro predominantly influenced uterine activity through regulation of Il6 expression via the Rela/p65 pathway, although the effects of Nmbr on [Ca(2+)](i) involved several pathways that remain to be elucidated.

A selective human bombesin receptor subtype-3 peptide agonist mediates CREB phosphorylation and transactivation.

The native ligand for the G protein-coupled bombesin receptor subtype-3 (BRS-3) has currently not been identified. Studies in mice showed robust BRS-3 expression in the hypothalamic satiety centers, and genetic receptor inactivation resulted in obesity, diabetes, and hypertension. BRS-3 was also detected in normal human pancreatic islet cells suggesting a critical role of BRS-3 in regulating energy metabolism and satiety via central and peripheral mechanisms of action. The cyclic AMP response element binding protein (CREB) is a main regulator of pancreatic ß-cell gene expression required for glucose homeostasis and islet cell survival, and hypothalamic regulation of satiety. Therefore, in this study we examined whether agonist-dependent hBRS-3 stimulation mediates CREB activation. A selective hBRS-3 peptide agonist and two non-selective hBRS-3 peptide agonists were used to activate ectopically expressed hBRS-3. Stimulation with hBRS-3 peptide agonists resulted in transient calcium mobilization, whereby the selective peptide agonist acted exclusively via hBRS-3 but not through the gastrin-releasing peptide receptor (GRP-R). A selective high-affinity GRP-R antagonist did not inhibit hBRS-3-mediated calcium signals. We also found time-dependent CREB phosphorylation in response to the selective hBRS-3 activation, which was abrogated by pretreatment with protein kinase A and protein kinase C inhibitors. Human BRS-3 agonists also stimulated CREB transactivation and resulted in modest increases of CRE-dependent gene transcription. These changes were significantly reduced after pretreatment with inhibitors of PKA, PKC, and MEK-1. Thus, our results suggest that hBRS-3 agonist-dependent signaling mediates CREB phosphorylation and transactivation through partially PKA, PKC, and MEK-1 pathways.