Differential Bile Acid Detection in Refractory GERD Patient Saliva Using a Simple and Sensitive Liquid Chromatography Tandem Mass Spectrometry Approach.

GOALS: The aim was to measure bile acids in human saliva using a sensitive ultraperformance liquid chromatography tandem mass spectrometry analysis method to distinguish quantitative differences in refractory gastroesophageal reflux disease (GERD) patients as compared with proton pump inhibitor (PPI) controlled GERD patients and healthy volunteers. STUDY: Human saliva samples were analyzed from 2 separate studies. The first a meal-controlled pilot, in which premeal and postmeal saliva samples were analyzed from 20 healthy subjects and 20 patients with GERD symptoms controlled by PPIs. In a subsequent exploratory study, saliva was collected from 34 patients with continuing GERD symptoms despite PPI treatment (refractory GERD), 30 healthy subjects, and 30 PPI-controlled GERD patients at ≥4 hours postmeal. RESULTS: In the meal-controlled pilot study, both healthy subjects and patients with PPI-controlled GERD, had total saliva bile acid increase for the first hour after consumption of a meal and returned to baseline levels 4 hours later. There was no difference in bile acid levels between the 2 groups. In the exploratory study, the saliva from patients with refractory GERD had statistically significant higher levels of total bile acid concentration compared with those of healthy volunteers and patients with PPI-controlled GERD (P=0.0181). CONCLUSIONS: Bile acids can be detected and accurately quantitated in human saliva using a sensitive ultraperformance liquid chromatography tandem mass spectrometry assay. Increases above threshold could indicate an underlying disease.This method could potentially be used to evaluate biliary reflux as an underlying pathophysiology of refractory GERD.

Guanylate cyclase 2C agonism corrects CFTR mutants.

Cystic fibrosis (CF) is a genetic disorder in which epithelium-generated fluid flow from the lung, intestine, and pancreas is impaired due to mutations disrupting CF transmembrane conductance regulator (CFTR) channel function. CF manifestations of the pancreas and lung are present in the vast majority of CF patients, and 15% of CF infants are born with obstructed gut or meconium ileus. However, constipation is a significantly underreported outcome of CF disease, affecting 47% of the CF patients, and management becomes critical in the wake of increasing life span of CF patients. In this study, we unraveled a potentially novel molecular role of a membrane-bound cyclic guanosine monophosphate-synthesizing (cGMP-synthesizing) intestinal enzyme, guanylate cyclase 2C (GCC) that could be targeted to ameliorate CF-associated intestinal fluid deficit. We demonstrated that GCC agonism results in functional rescue of murine F508del/F508del and R117H/R117H Cftr and CFTR mutants in CF patient-derived intestinal spheres. GCC coexpression and activation facilitated processing and ER exit of F508del CFTR and presented a potentially novel rescue modality in the intestine, similar to the CF corrector VX-809. Our findings identify GCC as a biological CFTR corrector and potentiator in the intestine.

Linaclotide activates guanylate cyclase-C/cGMP/protein kinase-II-dependent trafficking of CFTR in the intestine.

The transmembrane receptor guanylyl cyclase-C (GC-C), expressed on enterocytes along the intestine, is the molecular target of the GC-C agonist peptide linaclotide, an FDA-approved drug for treatment of adult patients with Irritable Bowel Syndrome with Constipation and Chronic Idiopathic Constipation. Polarized human colonic intestinal cells (T84, CaCo-2BBe) rat and human intestinal tissues were employed to examine cellular signaling and cystic fibrosis transmembrane conductance regulator (CFTR)-trafficking pathways activated by linaclotide using confocal microscopy, in vivo surface biotinylation, and protein kinase-II (PKG-II) activity assays. Expression and activity of GC-C/cGMP pathway components were determined by PCR, western blot, and cGMP assays. Fluid secretion as a marker of CFTR cell surface translocation was determined using in vivo rat intestinal loops. Linaclotide treatment (30 min) induced robust fluid secretion and translocation of CFTR from subapical compartments to the cell surface in rat intestinal loops. Similarly, linaclotide treatment (30 min) of T84 and CaCo-2BBe cells increased cell surface CFTR levels. Linaclotide-induced activation of the GC-C/cGMP/PKGII signaling pathway resulted in elevated intracellular cGMP and pVASPser239 phosphorylation. Inhibition or silencing of PKGII significantly attenuated linaclotide-induced CFTR trafficking to the apical membrane. Inhibition of protein kinase-A (PKA) also attenuated linaclotide-induced CFTR cell surface trafficking, implying cGMP-dependent cross-activation of PKA pathway. Together, these findings support linaclotide-induced activation of the GC-C/cGMP/PKG-II/CFTR pathway as the major pathway of linaclotide-mediated intestinal fluid secretion, and that linaclotide-dependent CFTR activation and recruitment/trafficking of CFTR from subapical vesicles to the cell surface is an important step in this process.

The Soluble Guanylate Cyclase Stimulator IWP-953 Increases Conventional Outflow Facility in Mouse Eyes.

PURPOSE: The nitric oxide (NO)-cyclic guanosine-3',5'-monophosphate (cGMP) pathway regulates aqueous humor outflow and therefore, intraocular pressure. We investigated the pharmacologic effects of the soluble guanylate cyclase (sGC) stimulator IWP-953 on primary human trabecular meshwork (HTM) cells and conventional outflow facility in mouse eyes. METHODS: Cyclic GMP levels were determined in vitro in HEK-293 cells and four HTM cell strains (HTM120/HTM123: predominantly myofibroblast-like phenotype, HTM130/HTM141: predominantly endothelial-like phenotype), and in HTM cell culture supernatants. Conventional outflow facility was measured following intracameral injection of IWP-953 or DETA-NO using a computerized pressure-controlled perfusion system in enucleated mouse eyes ex vivo. RESULTS: IWP-953 markedly stimulated cGMP production in HEK-293 cells in the presence and absence of DETA-NO (half maximal effective concentrations: 17 nM, 9.5 µM). Similarly, IWP-953 stimulated cGMP production in myofibroblast-like HTM120 and HTM123 cells, an effect that was greatly amplified by the presence of DETA-NO. In contrast, IWP-953 stimulation of cGMP production in endothelial-like HTM130 and HTM141 cells was observed, but was markedly less prominent than in HTM120 and HTM123 cells. Notably, cGMP was found in all HTM culture supernatants, following IWP-953/DETA-NO stimulation. In paired enucleated mouse eyes, IWP-953 at 10, 30, 60, and 100 µM concentration-dependently increased outflow facility. This effect (89.5%) was maximal at 100 µM (P = 0.002) and in magnitude comparable to DETA-NO at 100 µM (97.5% increase, P = 0.030). CONCLUSIONS: These data indicate that IWP-953, via modulation of the sGC-cGMP pathway, increases aqueous outflow facility in mouse eyes, suggesting therapeutic potential for sGC stimulators as novel ocular hypotensive drugs.

MRP4 Modulation of the Guanylate Cyclase-C/cGMP Pathway: Effects on Linaclotide-Induced Electrolyte Secretion and cGMP Efflux.

MRP4 mediates the efflux of cGMP and cAMP and acts as an important regulator of these secondary messengers, thereby affecting signaling events mediated by cGMP and cAMP. Immunofluorescence staining showed high MRP4 expression localized predominantly in the apical membrane of rat colonic epithelium. In vitro studies were performed using a rat colonic mucosal layer mounted in an Ussing chamber. Linaclotide activation of the guanylate cyclase-C (GC-C)/cGMP pathway induced a concentration-dependent increase in transepithelial ion current [short-circuit current (Isc)] across rat colonic mucosa (EC50: 9.2 nM). Pretreatment of colonic mucosa with the specific MRP4 inhibitor MK571 potentiated linaclotide-induced electrolyte secretion and augmented linaclotide-stimulated intracellular cGMP accumulation. Notably, pretreatment with the phosphodiesterase 5 inhibitor sildenafil increased basal Isc, but had no amplifying effect on linaclotide-induced Isc. MRP4 inhibition selectively affected the activation phase, but not the deactivation phase, of linaclotide. In contrast, incubation with a GC-C/Fc chimera binding to linaclotide abrogated linaclotide-induced Isc, returning to baseline. Furthermore, linaclotide activation of GC-C induced cGMP secretion from the apical and basolateral membranes of colonic epithelium. MRP4 inhibition blocked cGMP efflux from the apical membrane, but not the basolateral membrane. These data reveal a novel, previously unrecognized mechanism that functionally couples GC-C-induced luminal electrolyte transport and cGMP secretion to spatially restricted, compartmentalized regulation by MRP4 at the apical membrane of intestinal epithelium. These findings have important implications for gastrointestinal disorders with symptoms associated with dysregulated fluid homeostasis, such as irritable bowel syndrome with constipation, chronic idiopathic constipation, and secretory diarrhea.

Gastrointestinal pain: unraveling a novel endogenous pathway through uroguanylin/guanylate cyclase-C/cGMP activation.

The natural hormone uroguanylin regulates intestinal fluid homeostasis and bowel function through activation of guanylate cyclase-C (GC-C), resulting in increased intracellular cyclic guanosine-3',5'-monophosphate (cGMP). We report the effects of uroguanylin-mediated activation of the GC-C/cGMP pathway in vitro on extracellular cGMP transport and in vivo in rat models of inflammation- and stress-induced visceral hypersensitivity. In vitro exposure of intestinal Caco-2 cells to uroguanylin stimulated bidirectional, active extracellular transport of cGMP into luminal and basolateral spaces. cGMP transport was significantly and concentration dependently decreased by probenecid, an inhibitor of cGMP efflux pumps. In ex vivo Ussing chamber assays, uroguanylin stimulated cGMP secretion from the basolateral side of rat colonic epithelium into the submucosal space. In a rat model of trinitrobenzene sulfonic acid (TNBS)-induced visceral hypersensitivity, orally administered uroguanylin increased colonic thresholds required to elicit abdominal contractions in response to colorectal distension (CRD). Oral administration of cGMP mimicked the antihyperalgesic effects of uroguanylin, significantly decreasing TNBS- and restraint stress-induced visceromotor response to graded CRD in rats. The antihyperalgesic effects of cGMP were not associated with increased colonic spasmolytic activity, but were linked to significantly decreased firing rates of TNBS-sensitized colonic afferents in rats in response to mechanical stimuli. In conclusion, these data suggest that the continuous activation of the GC-C/cGMP pathway along the intestinal tract by the endogenous hormones guanylin and uroguanylin results in significant reduction of gastrointestinal pain. Extracellular cGMP produced on activation of GC-C is the primary mediator in this process via modulation of sensory afferent activity.

Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.

Linaclotide, a potent guanylate cyclase C agonist, is a therapeutic peptide approved in the United States for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation. We present for the first time the metabolism, degradation, and disposition of linaclotide in animals and humans. We examined the metabolic stability of linaclotide in conditions that mimic the gastrointestinal tract and characterized the metabolite MM-419447 (CCEYCCNPACTGC), which contributes to the pharmacologic effects of linaclotide. Systemic exposure to these active peptides is low in rats and humans, and the low systemic and portal vein concentrations of linaclotide and MM-419447 observed in the rat confirmed both peptides are minimally absorbed after oral administration. Linaclotide is stable in the acidic environment of the stomach and is converted to MM-419447 in the small intestine. The disulfide bonds of both peptides are reduced in the small intestine, where they are subsequently proteolyzed and degraded. After oral administration of linaclotide, <1% of the dose was excreted as active peptide in rat feces and a mean of 3-5% in human feces; in both cases MM-419447 was the predominant peptide recovered. MM-419447 exhibits high-affinity binding in vitro to T84 cells, resulting in a significant, concentration-dependent accumulation of intracellular cyclic guanosine-3',5'-monophosphate (cGMP). In rat models of gastrointestinal function, orally dosed MM-419447 significantly increased fluid secretion into small intestinal loops, increased intraluminal cGMP, and caused a dose-dependent acceleration in gastrointestinal transit. These results demonstrate the importance of the active metabolite in contributing to linaclotide's pharmacology.

Linaclotide, through activation of guanylate cyclase C, acts locally in the gastrointestinal tract to elicit enhanced intestinal secretion and transit.

Linaclotide is a first-in-class, orally administered 14-amino acid peptide that is in development for the treatment of irritable bowel syndrome with constipation and chronic constipation. We have characterized the solution structure of linaclotide, the in vitro binding and agonist activity to guanylate cyclase C receptors, the stability of linaclotide under conditions mimicking the gastric environment, oral bioavailability, and the pharmacodynamic effects in rat models of gastrointestinal transit and intestinal secretion. Nuclear magnetic resonance spectroscopy analysis determined that the molecular structure of linaclotide is stabilized by three intramolecular disulfide bridges. Linaclotide exhibited high affinity and pH-independent binding (K(i): 1.23-1.64 nM) to guanylate cyclase C receptors on human colon carcinoma T84 cells and concomitantly, linaclotide binding resulted in a significant, concentration-dependent accumulation of intracellular cyclic guanosine-3', 5'-monophosphate (cGMP) (EC50:99 nM). Linaclotide was stable after 3 h incubation in simulated gastric fluid (pH 1) and similarly, was completely resistant to hydrolysis by pepsin. Pharmacokinetic analysis of linaclotide showed very low oral bioavailability (0.1%). Orally administered linaclotide elicited a significant, dose-dependent increase in gastrointestinal transit rates in rats at doses of ≥5 µg/kg. Exposure of surgically ligated small intestinal loops to linaclotide induced a significant increase in fluid secretion, accompanied by a significant increase in intraluminal cGMP levels. These results suggest that the guanylate cyclase C agonist linaclotide elicits potent pharmacological responses locally in the gastrointestinal tract, and that orally administered guanylate cyclase C agonists may be capable of improving bowel habits in patients suffering from irritable bowel syndrome with constipation and chronic constipation.

Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract.

AIMS: Linaclotide is an orally administered 14-amino acid peptide being developed for the treatment of constipation-predominant irritable bowel syndrome (IBS-C) and chronic constipation. We determined the stability of linaclotide in the intestine, measured the oral bioavailability, and investigated whether the pharmacodynamic effects elicited in rodent models of gastrointestinal function are mechanistically linked to the activation of intestinal guanylate cyclase C (GC-C). MAIN METHODS: Linaclotide binding to intestinal mucosal membranes was assessed in competitive binding assays. Stability and oral bioavailability of linaclotide were measured in small intestinal fluid and serum, respectively, and models of gastrointestinal function were conducted using wild type (wt) and GC-C null mice. KEY FINDINGS: Linaclotide inhibited in vitro [(125)I]-STa binding to intestinal mucosal membranes from wt mice in a concentration-dependent manner. In contrast, [(125)I]-STa binding to these membranes from GC-C null mice was significantly decreased. After incubation in vitro in jejunal fluid for 30 min, linaclotide was completely degraded. Pharmacokinetic analysis showed very low oral bioavailability (0.10%). In intestinal secretion and transit models, linaclotide exhibited significant pharmacological effects in wt, but not in GC-C null mice: induction of increased fluid secretion into surgically ligated jejunal loops was accompanied by the secretion of elevated levels of cyclic guanosine-3',5'-monophosphate and accelerated gastrointestinal transit. SIGNIFICANCE: Linaclotide is a potent and selective GC-C agonist that elicits pharmacological effects locally in the gastrointestinal tract. This pharmacological profile suggests that orally administered linaclotide may be capable of improving the abdominal symptoms and bowel habits of patients suffering from IBS-C and chronic constipation.

The natriuretic peptide uroguanylin elicits physiologic actions through 2 distinct topoisomers.

The peptide uroguanylin regulates electrolyte transport in the intestine and kidney. Human uroguanylin has 2 conformations that can be stably isolated because of their slow interconversion rate. The A isomer potently activates the guanylate cyclase C receptor found primarily in the intestine. The B isomer, by contrast, is a very weak agonist of this receptor, leading to a widely held assumption that it is physiologically irrelevant. We show here, however, that human uroguanylin B has potent natriuretic activity in the kidney. Interestingly, uroguanylin A and B both induce saluretic responses, but the activity profiles for the 2 peptides differ markedly. The uroguanylin B dose-response curve is sigmoidal with a threshold dose of approximately 10 nmol/kg of body weight, whereas uroguanylin A has a comparable threshold but a bell-shaped dose-response curve. In addition, our study indicates a unique interplay between the A and B isoforms, such that the A form at high concentrations antagonizes the natriuretic action of the B form. These data show that the kidney contains a uroguanylin receptor of which the pharmacological profile does not match that of the well-defined intestinal uroguanylin receptor (guanylate cyclase C), an observation consistent with previous studies showing that the kidney of the guanylate cyclase C knockout mouse remains responsive to uroguanylin. The results presented here also support the unconventional notion that distinct conformations of a single endocrine peptide can elicit different responses in different tissues.

Identification of potential cell-surface proteins in Candida albicans and investigation of the role of a putative cell-surface glycosidase in adhesion and virulence.

Cell-surface proteins are attractive targets for the development of novel antifungals as they are more accessible to drugs than are intracellular targets. By using a computational biology approach, we identified 180 potential cell-surface proteins in Candida albicans, including the known cell-surface adhesin Als1 and other cell-surface antigens, such as Pra1 and Csa1. Six proteins (named Csf1-6 for cell-surface factors) were selected for further biological characterization. First, we verified that the selected CSF genes are expressed in the yeast and/or hyphal form and then we investigated the effect of the loss of each CSF gene on cell-wall integrity, filamentation, adhesion to mammalian cells and virulence. As a result, we identified Csf4, a putative glycosidase with an apparent orthologue in Saccharomyces cerevisiae (Utr2), as an important factor for cell-wall integrity and maintenance. Interestingly, deletion of CSF4 also resulted in a defect in filamentation, a reduction in adherence to mammalian cells in an in vitro adhesion assay, and a prolongation of survival in an immunocompetent mouse model of disseminated candidiasis. A delay in colonization of key organs (e.g. kidney) was also observed, which is consistent with a reduction in virulence of the csf4-deletion strain. These data indicate a key role for extracellular glycosidases in fungal pathogenesis and represent a new site for therapeutic intervention to cure and prevent fungal disease.

Systematic discovery of new genes in the Saccharomyces cerevisiae genome.

We used genome-wide comparative analysis of predicted protein sequences to identify many novel small genes, named smORFs for small open reading frames, within the budding yeast genome. Further analysis of 117 of these new genes showed that 84 are transcribed. We extended our analysis of one smORF conserved from yeast to human. This investigation provides an updated and comprehensive annotation of the yeast genome, validates additional concepts in the study of genomes in silico, and increases the expected numbers of coding sequences in a genome with the corresponding impact on future functional genomics and proteomics studies.

The use of direct cDNA selection to rapidly and effectively identify genes in the fungus Aspergillus fumigatus.

Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. To rapidly identify genes in this fungus, including potential targets for chemotherapy, diagnostics, and vaccine development, we constructed cDNA libraries. We began with non-normalized libraries, then to improve this approach we constructed a normalized cDNA library using direct cDNA selection. Normalization resulted in a reduction of the frequency of clones with highly expressed genes and an enrichment of underrepresented cDNAs. Expressed sequence tags generated from both the original and the normalized libraries were compared with the genomes of Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans, indicating that a large proportion of A. fumigatus genes do not have orthologs in these fungal species. This method allowed the expeditious identification of genes in a fungal pathogen. The same approach can be applied to other human or plant pathogens to rapidly identify genes without the need for genomic sequence information.