The T-type calcium channel Ca V 3.2 regulates bladder afferent responses to mechanical stimuli.

ABSTRACT: The bladder wall is innervated by a complex network of afferent nerves that detect bladder stretch during filling. Sensory signals, generated in response to distension, are relayed to the spinal cord and brain to evoke physiological and painful sensations and regulate urine storage and voiding. Hyperexcitability of these sensory pathways is a key component in the development of chronic bladder hypersensitivity disorders including interstitial cystitis/bladder pain syndrome and overactive bladder syndrome. Despite this, the full array of ion channels that regulate bladder afferent responses to mechanical stimuli have yet to be determined. Here, we investigated the role of low-voltage-activated T-type calcium (Ca V 3) channels in regulating bladder afferent responses to distension. Using single-cell reverse-transcription polymerase chain reaction and immunofluorescence, we revealed ubiquitous expression of Ca V 3.2, but not Ca V 3.1 or Ca V 3.3, in individual bladder-innervating dorsal root ganglia neurons. Pharmacological inhibition of Ca V 3.2 with TTA-A2 and ABT-639, selective blockers of T-type calcium channels, dose-dependently attenuated ex-vivo bladder afferent responses to distension in the absence of changes to muscle compliance. Further evaluation revealed that Ca V 3.2 blockers significantly inhibited both low- and high-threshold afferents, decreasing peak responses to distension, and delayed activation thresholds, thereby attenuating bladder afferent responses to both physiological and noxious distension. Nocifensive visceromotor responses to noxious bladder distension in vivo were also significantly reduced by inhibition of Ca V 3 with TTA-A2. Together, these data provide evidence of a major role for Ca V 3.2 in regulating bladder afferent responses to bladder distension and nociceptive signalling to the spinal cord.

Disengaging spinal afferent nerve communication with the brain in live mice.

Our understanding of how abdominal organs (like the gut) communicate with the brain, via sensory nerves, has been limited by a lack of techniques to selectively activate or inhibit populations of spinal primary afferent neurons within dorsal root ganglia (DRG), of live animals. We report a survival surgery technique in mice, where select DRG are surgically removed (unilaterally or bilaterally), without interfering with other sensory or motor nerves. Using this approach, pain responses evoked by rectal distension were abolished by bilateral lumbosacral L5-S1 DRG removal, but not thoracolumbar T13-L1 DRG removal. However, animals lacking T13-L1 or L5-S1 DRG both showed reduced pain sensitivity to distal colonic distension. Removal of DRG led to selective loss of peripheral CGRP-expressing spinal afferent axons innervating visceral organs, arising from discrete spinal segments. This method thus allows spinal segment-specific determination of sensory pathway functions in conscious, free-to-move animals, without genetic modification.

TGR5 agonists induce peripheral and central hypersensitivity to bladder distension.

The mechanisms underlying chronic bladder conditions such as interstitial cystitis/bladder pain syndrome (IC/BPS) and overactive bladder syndrome (OAB) are incompletely understood. However, targeting specific receptors mediating neuronal sensitivity to specific stimuli is an emerging treatment strategy. Recently, irritant-sensing receptors including the bile acid receptor TGR5, have been identified within the viscera and are thought to play a key role in neuronal hypersensitivity. Here, in mice, we identify mRNA expression of TGR5 (Gpbar1) in all layers of the bladder as well as in the lumbosacral dorsal root ganglia (DRG) and in isolated bladder-innervating DRG neurons. In bladder-innervating DRG neurons Gpbar1 mRNA was 100% co-expressed with Trpv1 and 30% co-expressed with Trpa1. In vitro live-cell calcium imaging of bladder-innervating DRG neurons showed direct activation of a sub-population of bladder-innervating DRG neurons with the synthetic TGR5 agonist CCDC, which was diminished in Trpv1-/- but not Trpa1-/- DRG neurons. CCDC also activated a small percentage of non-neuronal cells. Using an ex vivo mouse bladder afferent recording preparation we show intravesical application of endogenous (5α-pregnan-3ß-ol-20-one sulphate, Pg5α) and synthetic (CCDC) TGR5 agonists enhanced afferent mechanosensitivity to bladder distension. Correspondingly, in vivo intravesical administration of CCDC increased the number of spinal dorsal horn neurons that were activated by bladder distension. The enhanced mechanosensitivity induced by CCDC ex vivo and in vivo was absent using Gpbar1-/- mice. Together, these results indicate a role for the TGR5 receptor in mediating bladder afferent hypersensitivity to distension and thus may be important to the symptoms associated with IC/BPS and OAB.

Activation of MrgprA3 and MrgprC11 on Bladder-Innervating Afferents Induces Peripheral and Central Hypersensitivity to Bladder Distension.

Understanding the sensory mechanisms innervating the bladder is paramount to developing efficacious treatments for chronic bladder hypersensitivity conditions. The contribution of Mas-gene-related G protein-coupled receptors (Mrgpr) to bladder signaling is currently unknown. Using male and female mice, we show with single-cell RT-PCR that subpopulations of DRG neurons innervating the mouse bladder express MrgprA3 (14%) and MrgprC11 (38%), either individually or in combination, with high levels of coexpression with Trpv1 (81%-89%). Calcium imaging studies demonstrated MrgprA3 and MrgprC11 agonists (chloroquine, BAM8-22, and neuropeptide FF) activated subpopulations of bladder-innervating DRG neurons, showing functional evidence of coexpression between MrgprA3, MrgprC11, and TRPV1. In ex vivo bladder-nerve preparations, chloroquine, BAM8-22, and neuropeptide FF all evoked mechanical hypersensitivity in subpopulations (20%-41%) of bladder afferents. These effects were absent in recordings from Mrgpr-clusterΔ-/- mice. In vitro whole-cell patch-clamp recordings showed that application of an MrgprA3/C11 agonist mixture induced neuronal hyperexcitability in 44% of bladder-innervating DRG neurons. Finally, in vivo instillation of an MrgprA3/C11 agonist mixture into the bladder of WT mice induced a significant activation of dorsal horn neurons within the lumbosacral spinal cord, as quantified by pERK immunoreactivity. This MrgprA3/C11 agonist-induced activation was particularly apparent within the superficial dorsal horn and the sacral parasympathetic nuclei of WT, but not Mrgpr-clusterΔ-/- mice. This study demonstrates, for the first time, functional expression of MrgprA3 and MrgprC11 in bladder afferents. Activation of these receptors triggers hypersensitivity to distension, a critically valuable factor for therapeutic target development.SIGNIFICANCE STATEMENT Determining how bladder afferents become sensitized is the first step in finding effective treatments for common urological disorders such as overactive bladder and interstitial cystitis/bladder pain syndrome. Here we show that two of the key receptors, MrgprA3 and MrgprC11, that mediate itch from the skin are also expressed on afferents innervating the bladder. Activation of these receptors results in sensitization of bladder afferents, resulting in sensory signals being sent into the spinal cord that prematurely indicate bladder fullness. Targeting bladder afferents expressing MrgprA3 or MrgprC11 and preventing their sensitization may provide a novel approach for treating overactive bladder and interstitial cystitis/bladder pain syndrome.

A spider-venom peptide with multitarget activity on sodium and calcium channels alleviates chronic visceral pain in a model of irritable bowel syndrome.

ABSTRACT: Chronic pain is a serious debilitating condition that affects ∼20% of the world's population. Currently available drugs fail to produce effective pain relief in many patients and have dose-limiting side effects. Several voltage-gated sodium (NaV) and calcium (CaV) channels are implicated in the etiology of chronic pain, particularly NaV1.1, NaV1.3, NaV1.7-NaV1.9, CaV2.2, and CaV3.2. Numerous NaV and CaV modulators have been described, but with few exceptions, they display poor potency and/or selectivity for pain-related channel subtypes. Here, we report the discovery and characterization of 2 novel tarantula-venom peptides (Tap1a and Tap2a) isolated from Theraphosa apophysis venom that modulate the activity of both NaV and CaV3 channels. Tap1a and Tap2a inhibited on-target NaV and CaV3 channels at nanomolar to micromolar concentrations and displayed moderate off-target selectivity for NaV1.6 and weak affinity for NaV1.4 and NaV1.5. The most potent inhibitor, Tap1a, nearly ablated neuronal mechanosensitivity in afferent fibers innervating the colon and the bladder, with in vivo intracolonic administration reversing colonic mechanical hypersensitivity in a mouse model of irritable bowel syndrome. These findings suggest that targeting a specific combination of NaV and CaV3 subtypes provides a novel route for treatment of chronic visceral pain.

Histamine induces peripheral and central hypersensitivity to bladder distension via the histamine H1 receptor and TRPV1.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a common chronic pelvic disorder with sensory symptoms of urinary urgency, frequency, and pain, indicating a key role for hypersensitivity of bladder-innervating sensory neurons. The inflammatory mast cell mediator histamine has long been implicated in IC/BPS, yet the direct interactions between histamine and bladder afferents remain unclear. In the present study, we show, using a mouse ex vivo bladder afferent preparation, that intravesical histamine enhanced the mechanosensitivity of subpopulations of afferents to bladder distension. Histamine also recruited "silent afferents" that were previously unresponsive to bladder distension. Furthermore, in vivo intravesical histamine enhanced activation of dorsal horn neurons within the lumbosacral spinal cord, indicating increased afferent signaling in the central nervous system. Quantitative RT-PCR revealed significant expression of histamine receptor subtypes (Hrh1-Hrh3) in mouse lumbosacral dorsal root ganglia (DRG), bladder detrusor smooth muscle, mucosa, and isolated urothelial cells. In DRG, Hrh1 was the most abundantly expressed. Acute histamine exposure evoked Ca2+ influx in select populations of DRG neurons but did not elicit calcium transients in isolated primary urothelial cells. Histamine-induced mechanical hypersensitivity ex vivo was abolished in the presence of the histamine H1 receptor antagonist pyrilamine and was not present in preparations from mice lacking transient receptor potential vanilloid 1 (TRPV1). Together, these results indicate that histamine enhances the sensitivity of bladder afferents to distension via interactions with histamine H1 receptor and TRPV1. This hypersensitivity translates to increased sensory input and activation in the spinal cord, which may underlie the symptoms of bladder hypersensitivity and pain experienced in IC/BPS.

Linaclotide treatment reduces endometriosis-associated vaginal hyperalgesia and mechanical allodynia through viscerovisceral cross-talk.

Endometriosis, an estrogen-dependent chronic inflammatory disease, is the most common cause of chronic pelvic pain. Here, we investigated the effects of linaclotide, a Food and Drug Administration-approved treatment for IBS-C, in a rat model of endometriosis. Eight weeks after endometrium transplantation into the intestinal mesentery, rats developed endometrial lesions as well as vaginal hyperalgesia to distension and decreased mechanical hind paw withdrawal thresholds. Daily oral administration of linaclotide, a peripherally restricted guanylate cyclase-C (GC-C) agonist peptide acting locally within the gastrointestinal tract, increased pain thresholds to vaginal distension and mechanical hind paw withdrawal thresholds relative to vehicle treatment. Furthermore, using a cross-over design, administering linaclotide to rats previously administered vehicle resulted in increased hind paw withdrawal thresholds, whereas replacing linaclotide with vehicle treatment decreased hind paw withdrawal thresholds. Retrograde tracing of sensory afferent nerves from the ileum, colon, and vagina revealed that central terminals of these afferents lie in close apposition to one another within the dorsal horn of the spinal cord. We also identified dichotomizing dual-labelled ileal/colon innervating afferents as well as colon/vaginal dual-labelled neurons and a rare population of triple traced ileal/colon/vaginal neurons within thoracolumbar DRG. These observations provide potential sources of cross-organ interaction at the level of the DRG and spinal cord. GC-C expression is absent in the vagina and endometrial cysts suggesting that the actions of linaclotide are shared through nerve pathways between these organs. In summary, linaclotide may offer a novel therapeutic option not only for treatment of chronic endometriosis-associated pain, but also for concurrent treatment of comorbid chronic pelvic pain syndromes.

Chronic linaclotide treatment reduces colitis-induced neuroplasticity and reverses persistent bladder dysfunction.

Irritable bowel syndrome (IBS) patients suffer from chronic abdominal pain and extraintestinal comorbidities, including overactive bladder (OAB) and interstitial cystitis/painful bladder syndrome (IC-PBS). Mechanistic understanding of the cause and time course of these comorbid symptoms is lacking, as are clinical treatments. Here, we report that colitis triggers hypersensitivity of colonic afferents, neuroplasticity of spinal cord circuits, and chronic abdominal pain, which persists after inflammation. Subsequently, and in the absence of bladder pathology, colonic hypersensitivity induces persistent hypersensitivity of bladder afferent pathways, resulting in bladder-voiding dysfunction, indicative of OAB/IC-PBS. Daily administration of linaclotide, a guanylate cyclase-C (GC-C) agonist that is restricted to and acts within the gastrointestinal tract, reverses colonic afferent hypersensitivity, reverses neuroplasticity-induced alterations in spinal circuitry, and alleviates chronic abdominal pain in mice. Intriguingly, daily linaclotide administration also reverses persistent bladder afferent hypersensitivity to mechanical and chemical stimuli and restores normal bladder voiding. Linaclotide itself does not inhibit bladder afferents, rather normalization of bladder function by daily linaclotide treatment occurs via indirect inhibition of bladder afferents via reduced nociceptive signaling from the colon. These data support the concepts that cross-organ sensitization underlies the development and maintenance of visceral comorbidities, while pharmaceutical treatments that inhibit colonic afferents may also improve urological symptoms through common sensory pathways.

Structure-Activity Studies Reveal the Molecular Basis for GABAB-Receptor Mediated Inhibition of High Voltage-Activated Calcium Channels by α-Conotoxin Vc1.1.

α-Conotoxins are disulfide-bonded peptides from cone snail venoms and are characterized by their affinity for nicotinic acetylcholine receptors (nAChR). Several α-conotoxins with distinct selectivity for nAChR subtypes have been identified as potent analgesics in animal models of chronic pain. However, a number of α-conotoxins have been shown to inhibit N-type calcium channel currents in rodent dissociated dorsal root ganglion (DRG) neurons via activation of G protein-coupled GABAB receptors (GABABR). Therefore, it is unclear whether activation of GABABR or inhibition of α9α10 nAChRs is the analgesic mechanism. To investigate the mechanisms by which α-conotoxins provide analgesia, we synthesized a suite of Vc1.1 analogues where all residues, except the conserved cysteines, in Vc1.1 were individually replaced by alanine (A), lysine (K), and aspartic acid (D). Our results show that the amino acids in the first loop play an important role in binding of the peptide to the receptor, whereas those in the second loop play an important role for the selectivity of the peptide for the GABABR over α9α10 nAChRs. We designed a cVc1.1 analogue that is >8000-fold selective for GABABR-mediated inhibition of high voltage-activated (HVA) calcium channels over α9α10 nAChRs and show that it is analgesic in a mouse model of chronic visceral hypersensitivity (CVH). cVc1.1[D11A,E14A] caused dose-dependent inhibition of colonic nociceptors with greater efficacy in ex vivo CVH colonic nociceptors relative to healthy colonic nociceptors. These findings suggest that selectively targeting GABABR-mediated HVA calcium channel inhibition by α-conotoxins could be effective for the treatment of chronic visceral pain.

Structure-Activity Studies of Cysteine-Rich α-Conotoxins that Inhibit High-Voltage-Activated Calcium Channels via GABA(B) Receptor Activation Reveal a Minimal Functional Motif.

α-Conotoxins are disulfide-rich peptides that target nicotinic acetylcholine receptors. Recently we identified several α-conotoxins that also modulate voltage-gated calcium channels by acting as G protein-coupled GABA(B) receptor (GABA(B)R) agonists. These α-conotoxins are promising drug leads for the treatment of chronic pain. To elucidate the diversity of α-conotoxins that act through this mechanism, we synthesized and characterized a set of peptides with homology to α-conotoxins known to inhibit high voltage-activated calcium channels via GABA(B)R activation. Remarkably, all disulfide isomers of the active α-conotoxins Pu1.2 and Pn1.2, and the previously studied Vc1.1 showed similar levels of biological activity. Structure determination by NMR spectroscopy helped us identify a simplified biologically active eight residue peptide motif containing a single disulfide bond that is an excellent lead molecule for developing a new generation of analgesic peptide drugs.

Conopeptide-Derived κ-Opioid Agonists (Conorphins): Potent, Selective, and Metabolic Stable Dynorphin A Mimetics with Antinociceptive Properties.

Opioid receptor screening of a conopeptide library led to a novel selective κ-opioid agonist peptide (conorphin T). Intensive medicinal chemistry, guided by potency, selectivity, and stability assays generated a pharmacophore model supporting rational design of highly potent and selective κ-opioid receptor (KOR) agonists (conorphins) with exceptional plasma stability. Conorphins are defined by a hydrophobic benzoprolyl moiety, a double arginine sequence, a spacer amino acid followed by a hydrophobic residue and a C-terminal vicinal disulfide moiety. The pharmacophore model was supported by computational docking studies, revealing receptor-ligand interactions similar to KOR agonist dynorphin A (1-8). A conorphin agonist inhibited colonic nociceptors in a mouse tissue model of chronic visceral hypersensitivity, suggesting the potential of KOR agonists for the treatment of chronic abdominal pain. This new conorphine KOR agonist class and pharmacophore model provide opportunities for future rational drug development and probes for exploring the role of the κ-opioid receptor.

Linaclotide inhibits colonic nociceptors and relieves abdominal pain via guanylate cyclase-C and extracellular cyclic guanosine 3',5'-monophosphate.

BACKGROUND & AIMS: Linaclotide is a minimally absorbed agonist of guanylate cyclase-C (GUCY2C or GC-C) that reduces symptoms associated with irritable bowel syndrome with constipation (IBS-C). Little is known about the mechanism by which linaclotide reduces abdominal pain in patients with IBS-C. METHODS: We determined the effects of linaclotide on colonic sensory afferents in healthy mice and those with chronic visceral hypersensitivity. We assessed pain transmission by measuring activation of dorsal horn neurons in the spinal cord in response to noxious colorectal distention. Levels of Gucy2c messenger RNA were measured in tissues from mice using quantitative reverse transcription polymerase chain reaction and in situ hybridization. We used human intestinal cell lines to measure release of cyclic guanosine-3',5'-monophosphate (cGMP) by linaclotide. We performed a post-hoc analysis of data from a phase III, double-blind, parallel-group study in which 805 patients with IBS-C were randomly assigned to groups given an oral placebo or 290 µg linaclotide once daily for 26 weeks. We quantified changes in IBS-C symptoms, including abdominal pain. RESULTS: In mice, linaclotide inhibited colonic nociceptors with greater efficacy during chronic visceral hypersensitivity. Intra-colonic administration of linaclotide reduced signaling of noxious colorectal distention to the spinal cord. The colonic mucosa, but not neurons, was found to express linaclotide's target, GC-C. The downstream effector of GC-C, cGMP, was released after administration of linaclotide and also inhibited nociceptors. The effects of linaclotide were lost in Gucy2c(-/-) mice and prevented by inhibiting cGMP transporters or removing the mucosa. During 26 weeks of linaclotide administration, a significantly greater percentage of patients (70%) had at least a 30% reduction in abdominal pain compared with patients given placebo (50%). CONCLUSIONS: We have identified an analgesic mechanism of linaclotide: it activates GC-C expressed on mucosal epithelial cells, resulting in the production and release of cGMP. This extracellular cGMP acts on and inhibits nociceptors, thereby reducing nociception. We also found that linaclotide reduces chronic abdominal pain in patients with IBS-C.

Ahogamiento incompleto, valoración de la injuria cerebral al ingreso: terapia intensiva pediátrica / Nearly drowning, valuation of the cerebral injury when entering the hospital: intensive pediatric therapy

Se realizó una investigación descriptiva retrospectiva que incluyó a todos los pacientes que fueron ingresados en el Servicio de Terapia Intensiva del Hospital Eliseo Noel Caamaño, en el período comprendido desde junio de 1982 hasta junio de 2004, con el diagnóstico de Ahogamiento Incompleto, con el propósito de determinar el estado neurológico y su relación con diferentes variables que pueden haber influido en la intensidad del daño, así como la sobrevivencia final de los mismos. Los resultados obtenidos nos permitieron conocer el manejo del paciente pediátrico que ha sufrido este tipo de accidentes, así como su estado a la llegada al Servicio. El 68 por ciento de los pacientes tuvieron una afección de moderada a severa según las escalas de Conn y Glasgow, además existió una correlación de hasta un 97 por ciento entre dichos métodos de evaluación. Dentro de las variables que influenciaron en el estado de los niños al ingreso se destacó un tiempo de inmersión mayor de 5 minutos para los clasificados como severos, 70 por ciento de la muestra, fallecieron el 7 por ciento de todos los pacientes estudiados, todos ellos clasificados como severos.

We carried out a descriptive retrospective study including all the patients that were entered in the Service of Intensive Therapy of the hospital Eliseo Noel Caamaño, in the period from June 1982 to June 2004, with the diagnosis of nearly drowning, with the purpose of determining the neurological state and its relation with different variables that might have influence in the intensity of the damage, as well as in the final surviving of the patients. The obtained results allowed us knowing the managing of the paediatrics patient that have suffered this kind of accidents, as well as their state at the arrival to the service; 68 percent of the patients had a moderated to severe affection according to the Conn and Glasgow scales; moreover, there was a correlation of up to 97 percent between these evaluation methods. Among the variables that influenced in the state of the children at the entering, there was an immersion time of more than 5 minutes for those classified as severe, 70 percent of the sample; 7 percent of all the studied patients died, all of them classified as severe.

Ion movements in cell death: from protection to execution.

Cell death is preceded by severe disruption of inorganic ion homeostasis. Seconds to minutes after an injury, calcium, protons, sodium, potassium and chloride are exchanged between the cell and its environment. Simultaneously, ions are shifted between membrane compartments inside the cell, whereby mitochondria and endoplasmic reticulum play a crucial role. Depending of the type and severity of injury, two mutually exclusive metastable states can be reached, which predict the final outcome. Cells characterized by large increases in cytosolic [Ca2+], [Na+] and [Mg2+] swell and die by necrosis; alternatively, cells characterized by high [H+] and low [K+], with normal [Na+] and normal to moderate [Ca2+] increases die by apoptosis. The levels of these ions represent central determinants in signaling events leading to cell death. Their movements are explained mechanistically by specific modulation of membrane transport proteins including channels, pumps and carriers.

Ion movements in cell death: from protection to exection

Cell death is preceded by severe disruption of inorganic ion homeostasis. Seconds to minutes after an injury, calcium, protons, sodium, potassium and chloride are exchanged between the cell and its environment. Simultaneously, ions are shifted between membrane compartments inside the cell, whereby mitochondria and endoplasmic reticulum play a crucial role. Depending of the type and severity of injury, two mutually exclusive metastable states can be reached, which predict the final outcome. Cells characterized by large increases in cytosolic [Ca2+], [Na+] and [Mg2+] swell and die by necrosis; alternatively, cells characterized by high [H+] and low [K+], with normal [Na+] and normal to moderate [Ca2+] increases die by apoptosis. The levels of these ions represent central determinants in signaling events leading to cell death. Their movements are explained mechanistically by specific modulation of membrane transport proteins including channels, pumps and carriers