ABSTRACT
There is increasing focus on the involvement of tachykinins in immune and inflammatory responses. Hemokinin-1 (HK-1) is a recently identified tachykinin that originates primarily from immune cells, and has structural similarities to substance P (SP), found mainly in neurons. However, there are species differences in HK-1, and the role of HK-1 in humans, particularly the intestine, has received minimal attention. The aim of this study was to investigate the inflammatory role of human HK-1 in the human colon. The effects of HK-1 and SP were compared on the production of multiple inflammatory cytokines and chemokines from human colonic mucosal explants. Data generated by Procarta multiplex assay and QuantiGene assay demonstrated that 4 h incubation with HK-1 (0.1 µM) significantly stimulated transcript expression and release of MCP-1, MIP-1α and ß, RANTES, TNF-α, IL-1ß and IL-6 from the mucosa. SP (0.1 µM) had comparable actions, but had no effect on MCP-1 or RANTES. These effects were inhibited separately by tachykinin NK1 and NK2 receptor antagonists SR140333 and SR48968 (both 0.1 µM), suggesting that these responses were mediated by both NK1 and NK2 receptors. In conclusion, these data support a novel inflammatory role for HK-1 in human colon, signaling via NK1 and NK2 receptors (and possibly other tachykinin-preferring receptors) to regulate the release of a broad spectrum of proinflammatory mediators. The study suggests that along with SP, HK-1 is also a proinflammatory mediator, likely involved in colonic inflammation, including inflammatory bowel disease (IBD).
Subject(s)
Inflammation/metabolism , Intestinal Mucosa/metabolism , Receptors, Neurokinin-1/metabolism , Receptors, Neurokinin-2/metabolism , Substance P/metabolism , Tachykinins/metabolism , Adult , Aged , Aged, 80 and over , Chemokines/metabolism , Cytokines/metabolism , Female , Gene Expression , Humans , Inflammation Mediators/metabolism , Intestinal Mucosa/drug effects , Male , Middle Aged , Substance P/administration & dosage , Tachykinins/administration & dosageABSTRACT
BACKGROUND: Prostaglandin E2 (PGE2) is the dominant prostaglandin in the colon and is associated with colonic inflammation. PGE2 levels are regulated not only by cyclooxygenases (COX-1 and COX-2) but also by 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the major PGE2-degrading enzyme. Information about the involvement of 15-PGDH in colonic inflammation is sparse. AIM: We thus aimed to determine the gene expression and immunoreactivity (IR) of COX-1, COX-2, and 15-PGDH in colonic mucosa from patients with diverse inflammatory disorders: ulcerative colitis (UC), Crohn's disease (CD), and acute diverticular disease (DD). METHODS: RNA from human colonic mucosa was extracted and assessed for gene expression by real-time PCR. Intact colon sections were processed for immunohistochemistry with immunostaining of the mucosal areas quantified using ImageJ. RESULTS: In colonic mucosa of both UC and CD, COX-2 mRNA and COX-2-IR were significantly increased, whereas 15-PGDH mRNA and 15-PGDH-IR were significantly reduced. In macroscopically undamaged acute DD mucosa, the opposite findings were seen: for both gene expression and immunoreactivity, there was a significant downregulation of COX-2 and upregulation of 15-PGDH. COX-1 mRNA and COX-1-IR remained unchanged in all diseases. CONCLUSIONS: Our study for the first time demonstrated differential expression of the PGE2-related enzymes COX-2 and 15-PGDH in colonic mucosa from UC, CD, and acute DD. The reduction of 15-PGDH in IBD provides an additional mechanism for PGE2 increase in IBD. With respect to DD, alterations of PGE2-related enzymes suggest that a low PGE2 level may precede the onset of inflammation, thus providing new insight into the pathogenesis of DD.
Subject(s)
Colitis, Ulcerative/enzymology , Colon/enzymology , Crohn Disease/enzymology , Cyclooxygenase 1/analysis , Cyclooxygenase 2/analysis , Dinoprostone/metabolism , Diverticulosis, Colonic/enzymology , Hydroxyprostaglandin Dehydrogenases/analysis , Intestinal Mucosa/enzymology , Adult , Aged , Aged, 80 and over , Case-Control Studies , Colitis, Ulcerative/genetics , Crohn Disease/genetics , Cyclooxygenase 1/genetics , Cyclooxygenase 2/genetics , Diverticulosis, Colonic/genetics , Female , Gene Expression Regulation, Enzymologic , Humans , Hydroxyprostaglandin Dehydrogenases/genetics , Male , Middle Aged , RNA, Messenger/analysisABSTRACT
Urothelial cells, myofibroblasts, and smooth muscle cells are important cell types contributing to bladder function. Multiple receptors including muscarinic (M3/M5), tachykinin (NK1/NK2), and purinergic (P2X1/P2Y6) receptors are involved in bladder motor and sensory actions. Using female pig bladder, our aim was to differentiate between various cell types in bladder by genetic markers. We compared the molecular expression pattern between the fresh tissue layers and their cultured cell counterparts. We also examined responses to agonists for these receptors in cultured cells. Urothelial, suburothelial (myofibroblasts), and smooth muscle cells isolated from pig bladder were cultured (10-14 days) and identified by marker antibodies. Gene (mRNA) expression level was demonstrated by real-time PCR. The receptor expression pattern was very similar between suburothelium and detrusor, and higher than urothelium. The gene expression of all receptors decreased in culture compared with the fresh tissue, although the reduction in cultured urothelial cells appeared less significant compared to suburothelial and detrusor cells. Cultured myofibroblasts and detrusor cells did not contract in response to the agonists acetylcholine, neurokinin A, and ß,γ-MeATP, up to concentrations of 0.1 and 1 mM. The significant reduction of M3, NK2, and P2X1 receptors under culture conditions may be associated with the unresponsiveness of cultured suburothelial and detrusor cells to their respective agonists. These results suggest that under culture conditions, bladder cells lose the receptors that are involved in contraction, as this function is no longer required. The study provides further evidence that cultured cells do not necessarily mimic the actions exerted by intact tissues.
ABSTRACT
AIMS: In the bladder, ATP is an important signaling molecule, which is released by bladder stretch and acid. We hypothesized that ATP might play a unique role in patients with OAB, characterized by low bladder volumes at first desire to void (FDV) and maximal cystometric capacity (MCC) and symptoms of frequency/urgency [mild bladder pain syndrome (BPS)]. Our aim was to investigate the correlation between ATP release and urodynamic parameters, as well as urine pH, in OAB patients. METHODS: Routine cystometry was performed in a consecutive series of 249 women. The voided urodynamic fluid (VUF) was stored at -20°C and ATP measured using bioluminescence. Catheter urine was collected for pH measurement. Correlations between two factors were tested by linear regression analysis. RESULTS: Subjects with urinary tract infection, voiding dysfunction, and detrusor overactivity (DO) were excluded. For OAB patients (n = 25), there was an inverse correlation between ATP concentration in VUF and FDV (r(2) = 0.25; P = 0.01) but not MCC. This was not seen in controls (n = 69). In OAB, but not controls, there was a significant reverse correlation (r(2) = 0.16; P = 0.047) between ATP in VUF and urine pH. Urine pH was not significantly correlated with MCC in either group. CONCLUSIONS: In OAB patients, ATP is an important factor for initial perception of need to urinate (as indicated by FDV). This is similar to our previous findings in patients with DO, suggesting that ATP may mediate initial afferent sensation in patients with bladder dysfunctions characterized by urgency. ATP release was also strongly affected by urine pH, in patients with OAB (at FDV).
Subject(s)
Adenosine Triphosphate/urine , Urinary Bladder, Overactive/diagnosis , Urinary Bladder/metabolism , Urinary Bladder/physiopathology , Urodynamics , Adult , Aged , Aged, 80 and over , Biomarkers/urine , Case-Control Studies , Female , Humans , Hydrogen-Ion Concentration , Linear Models , Luminescent Measurements , Middle Aged , Predictive Value of Tests , Urinary Bladder, Overactive/physiopathology , Urinary Bladder, Overactive/urine , Urinary Catheterization , UrinationABSTRACT
Stretch-evoked ATP release from the bladder mucosa is a key event in signaling bladder fullness. Our aim was to examine whether acid and capsaicin can also release ATP and to determine the receptors involved, using agonists and antagonists at TRPV1 and acid-sensing ion channels (ASICs). Strips of porcine bladder mucosa were exposed to acid, capsaicin or stretch. Strip tension was monitored. Bath fluid was collected for ATP measurement. Gene expression of ASICs and TRPV1 in porcine bladders was quantified using quantitative real-time PCR (qRT-PCR). Stretch stimulus (150% of original length) repeatedly and significantly increased ATP release to approximately 45 times basal release. Acid (pH 6.5, 6.0, 5.6) contracted mucosal strips and also increased ATP release up to 30-fold, without evidence of desensitization. Amiloride (0.3 µM) reduced the acid-evoked ATP release by approximately 70%, while capsazepine (10 µM) reduced acid-evoked ATP release at pH 6.0 and pH 5.6 (by 68% and 61%, respectively). Capsaicin (0.1-10 µM) was ineffective in causing ATP release, and also failed to contract porcine mucosal or detrusor strips. Gene expression for ASIC1, ASIC2, ASIC3 and TRPV1 was seen in the lateral wall, dome, trigone and neck of both detrusor and mucosa. In conclusion, stretch and acid induce ATP release in the porcine bladder mucosa, but capsaicin is ineffective. The pig bladder is a well-known model for the human bladder, however these data suggest that it should be used with caution, particularly for TRPV1 related studies.
Subject(s)
Adenosine Triphosphate/metabolism , Mucous Membrane/metabolism , Nerve Tissue Proteins/metabolism , Sodium Channels/metabolism , TRPV Cation Channels/metabolism , Urinary Bladder/metabolism , Acid Sensing Ion Channels , Animals , Capsaicin/pharmacology , Female , Gene Expression Regulation/drug effects , Hydrogen-Ion Concentration , In Vitro Techniques , Male , Membrane Transport Modulators/pharmacology , Mucous Membrane/drug effects , Muscle Contraction/drug effects , Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , Nerve Tissue Proteins/agonists , Nerve Tissue Proteins/antagonists & inhibitors , Physical Stimulation , Protein Isoforms/agonists , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Sodium Channel Agonists , Sus scrofa , TRPV Cation Channels/agonists , TRPV Cation Channels/antagonists & inhibitors , Urinary Bladder/drug effectsABSTRACT
Hemokinin-1 (HK-1) is a newly identified tachykinin, originating from the immune system rather than neurons, and may participate in the immune and inflammatory response. In colonic mucosa of patients with inflammatory bowel disease (IBD), up-regulation of the TAC4 gene encoding HK-1 and increased production of prostaglandin E2 (PGE2) occur. Our aim was to examine the mechanistic link between human HK-1 and PGE2 production in normal human colon. Exogenous HK-1 (0.1 µM) for 4 h evoked an increased PGE2 release from colonic mucosal and muscle explants by 10- and 3.5-fold, respectively, compared with unstimulated time controls. The HK-1-stimulated PGE2 release was inhibited by the tachykinin receptor antagonists (S)1-2-[3-(3,4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidin-3-yl]ethyl-4-phenyl-l azonia-bicyclo[2.2.2]octane (SR140333) [neurokinin-1 (NK1)] and N-[(2S)-4-(4-acetamido-4-phenylpiperidin-1-yl)-2-(3,4-dichlorophenyl)butyl]-N-methylbenzamide (SR48968) [neurokinin-2 (NK2)] and was also inhibited by the cyclooxygenase (COX)-2 inhibitor N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide) (NS-398) but not by the COX-1 inhibitor 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC-560). A parallel study with substance P showed similar results. Molecular studies with HK-1-treated explants demonstrated a stimulatory effect on COX-2 expression at both transcription and protein levels. It is noteworthy that this was coupled with HK-1-induced down-regulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) mRNA and protein expression. Immunoreactivity for 15-PGDH occurred on inflammatory cells, epithelial cells, platelets, and ganglia. This finding provides an additional mechanism for HK-1-evoked PGE2 increase, in which HK-1 may interfere with the downstream metabolism of PGE2 by suppressing 15-PGDH expression. In conclusion, our results uncover a novel inflammatory role for HK-1, which signals via NK1 and NK2 receptors to regulate PGE2 release from human colonic tissue, and may further explain a pathological role for HK-1 in IBD when abnormal levels of PGE2 occur.
Subject(s)
Colon/metabolism , Cyclooxygenase 2/metabolism , Dinoprostone/biosynthesis , Enzyme Inhibitors/pharmacology , Hydroxyprostaglandin Dehydrogenases/antagonists & inhibitors , Tachykinins/pharmacology , Adult , Aged , Blotting, Western , Colitis/physiopathology , Colon/drug effects , Colon/enzymology , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme-Linked Immunosorbent Assay , Female , Gene Expression Regulation, Enzymologic/drug effects , Humans , Immunohistochemistry , In Vitro Techniques , Intestinal Mucosa/drug effects , Intestinal Mucosa/metabolism , Male , Middle Aged , Muscle, Smooth/drug effects , Muscle, Smooth/metabolism , Real-Time Polymerase Chain Reaction , Receptors, Neurokinin-1/drug effects , Receptors, Neurokinin-1/physiology , Receptors, Neurokinin-2/drug effects , Receptors, Neurokinin-2/physiology , Stimulation, Chemical , Tachykinins/antagonists & inhibitorsABSTRACT
ATP is released from the bladder mucosa in response to stretch, but the cell types responsible are unclear. Our aim was to isolate and characterize individual populations of urothelial, myofibroblast, and detrusor muscle cells in culture, and to examine agonist-stimulated ATP release. Using female pig bladders, urothelial cells were isolated from bladder mucosa following trypsin-digestion of the luminal surface. The underlying myofibroblast layer was dissected, minced, digested, and cultured until confluent (10-14 days). A similar protocol was used for muscle cells. Cultures were used for immunocytochemical staining and/or ATP release investigations. In urothelial cultures, immunoreactivity was present for the cytokeratin marker AE1/AE3 but not the contractile protein α-smooth muscle actin (α-SMA) or the cytoskeletal filament vimentin. Neither myofibroblast nor muscle cell cultures stained for AE1/AE3. Myofibroblast cultures partially stained for α-SMA, whereas muscle cultures were 100% stained. Both myofibroblast and muscle stained for vimentin, however, they were morphologically distinct. Ultrastructural studies verified that the suburothelial layer of pig bladder contained abundant myofibroblasts, characterized by high densities of rough endoplasmic reticulum. Baseline ATP release was higher in urothelial and myofibroblast cultures, compared with muscle. ATP release was significantly stimulated by stretch in all three cell populations. Only urothelial cells released ATP in response to acid, and only muscle cells were stimulated by capsaicin. Tachykinins had no effect on ATP release. In conclusion, we have established a method for culture of three cell populations from porcine bladder, a well-known human bladder model, and shown that these are distinct morphologically, immunologically, and pharmacologically.
ABSTRACT
BACKGROUND: The functional role of the bladder urothelium has been the focus of much recent research. The bladder mucosa contains two significant cell types: urothelial cells that line the bladder lumen and suburothelial interstitial cells or myofibroblasts. The aims of this study were to culture these cell populations from human bladder biopsies and to perform immunocytochemical characterisation. METHODS: Primary cell cultures were established from human bladder biopsies (n = 10). Individual populations of urothelial and myofibroblast-like cells were isolated using magnetic activated cell separation (MACS). Cells were slow growing, needing 3 to 5 weeks to attain confluence. RESULTS: Cytokeratin 20 positive cells (umbrella cells) were isolated at primary culture and also from patients' bladder washings but these did not proliferate. In primary culture, proliferating cells demonstrated positive immunocytochemical staining to cytokeratin markers (AE1/AE3 and A0575) as well fibroblasts (5B5) and smooth muscle (αSMA) markers. An unexpected finding was that populations of presumptive urothelial and myofibroblast-like cells, isolated using the MACS beads, stained for similar markers. In contrast, staining for cytokeratins and fibroblast or smooth muscle markers was not co-localised in full thickness bladder sections. CONCLUSIONS: Our results suggest that, in culture, bladder mucosal cells may undergo differentiation into a myoepithelial cell phenotype indicating that urothelial cells have the capacity to respond to environmental changes. This may be important pathologically but also suggests that studies of the physiological function of these cells in culture may not give a reliable indicator of human physiology.
Subject(s)
Keratin-20/metabolism , Mucous Membrane/cytology , Mucous Membrane/metabolism , Urinary Bladder/cytology , Urinary Bladder/metabolism , Aged , Cells, Cultured , Female , Humans , Male , Tissue DistributionABSTRACT
The brain-gut peptide neurotensin has complex effects on gastrointestinal smooth muscle. Our objective was to elucidate the mechanisms underlying neurotensin contractions in human colon. Discrete concentration response curves to neurotensin were obtained in strips of circular muscle and taenia coli from "normal" ascending and sigmoid colon segments, in the presence and absence of various pharmacological inhibitors. Potency of neurotensin in all regions was similar (pD(2) ~7). Atropine and the selective muscarinic receptor antagonists, methoctramine and darifenacin, had no effect on neurotensin contractions. In ascending colon circular muscle, responses were enhanced by indomethacin (indicating inhibitory prostaglandin mechanisms) and by tetrodotoxin (TTX), hexamethonium and L-NAME, suggesting nicotinic and enteric inhibitory neurotransmission, with involvement of nitric oxide. In sigmoid circular muscle, neurotensin responses were also enhanced by TTX and hexamethonium, but were attenuated in the presence of mepyramine, MEN10627 and CP99994, suggesting inhibitory neuronal mechanisms and involvement of histamine and tachykinins, respectively; L-NAME and the GABA(B) receptor antagonist, CGP36742, were without effect. The transcripts of NTS1 and NTS3 receptors, but not NTS2 receptors, were detected in sigmoid colon circular muscle and taenia coli. No age and gender differences in NTS1 mRNA expression were found. In conclusion, neurotensin contracts circular muscle strips from ascending and sigmoid regions of the human colon via direct (muscle) and indirect (neuronal/non-neuronal mechanisms). The enteric mediators influenced by neurotensin are regionally specific. In taenia coli strips from both ascending and sigmoid colon, neurotensin contractions were unchanged in the presence of inhibitors, suggesting direct actions only.
Subject(s)
Colon, Ascending/metabolism , Colon, Sigmoid/metabolism , Neurotensin/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Adult , Aged , Female , Humans , Male , Middle Aged , Muscle Contraction/drug effects , RNA, Messenger/metabolism , Receptors, Neurotensin/metabolismABSTRACT
PURPOSE: Adenosine triphosphate released from urothelium during stretch stimulates afferent nerves and conveys information on bladder fullness. We measured adenosine triphosphate released during cystometric bladder filling in women with idiopathic detrusor overactivity and stress incontinence (controls), and assessed whether the level of released adenosine triphosphate is related to cystometric parameters. MATERIALS AND METHODS: Routine cystometry was done in 51 controls and 48 women with detrusor overactivity who were 28 to 87 years old. Voided urodynamic fluid was collected and stored at -30 C. Adenosine triphosphate was measured by a bioluminescence assay. RESULTS: Adenosine triphosphate levels were similar in voided urodynamic fluid of controls and patients with detrusor overactivity (p = 0.79). A significant inverse correlation was seen between adenosine triphosphate and maximal cystometric capacity in controls (p = 0.013), and between voided volume and adenosine triphosphate in controls (p = 0.015) and detrusor overactivity cases (p = 0.019). A significant correlation between first desire to void and adenosine triphosphate was also noted in detrusor overactivity cases (p = 0.033) but not in controls (p = 0.58). No correlation was seen between adenosine triphosphate and detrusor pressure during filling or voiding. CONCLUSIONS: Adenosine triphosphate measurement in voided urodynamic fluid is a novel approach to understanding signals that may contribute to the urgency sensation (a sudden compelling desire to pass urine). The inverse correlation between adenosine triphosphate in voided urodynamic fluid and first desire to void suggests that adenosine triphosphate has a role in modulating the early filling sensation in patients with detrusor overactivity.
Subject(s)
Adenosine Triphosphate/physiology , Adenosine Triphosphate/urine , Urinary Bladder, Overactive/physiopathology , Urinary Bladder, Overactive/urine , Urinary Incontinence, Stress/physiopathology , Urinary Incontinence, Stress/urine , Adult , Aged , Aged, 80 and over , Female , Humans , Middle Aged , UrodynamicsABSTRACT
Tachykinins are important neurotransmitters regulating intestinal motility. Slow transit constipation (STC) represents an extreme colonic dysmotility with unknown etiology that predominantly affects women. We examined whether the tachykinin system is involved in the pathogenesis of STC. Isolated sigmoid colon circular muscle from female STC and control patients was studied using functional and quantitative reverse transcriptase-polymerase chain reaction methods. A possible alteration of neurotransmission was investigated by electrical field stimulation (EFS) and ganglionic stimulation by dimethylphenylpiperazinium (DMPP). Substance P (SP)-mediated contractions in circular muscle strips were significantly diminished in STC compared with age-matched control (P < 0.001). In contrast, contractile responses to neurokinin A, the selective tachykinin NK(2) receptor agonist, [Lys(5),MeLeu(9),Nle(10)]NKA(4-10), and acetylcholine were unaltered in STC. The reduced responses to SP in STC were fully restored by indomethacin, partially reversed by tetrodotoxin (TTX), but unaffected by atropine or hexamethonium. The restoration by indomethacin was blocked by the NK(1) receptor antagonist CP99994 [(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine] and TTX. In STC colonic muscle, there was a significant increase of NK(1) receptor mRNA expression, but no difference in NK(2) mRNA level. DMPP generated biphasic responses, relaxation at lower and contraction at higher concentrations. Although the responses to DMPP were similar in STC and control, an altered contractile pattern in response to EFS was observed in STC circular muscle. In conclusion, we postulate that the diminished contractile response to SP in STC is due to an increased release of inhibitory prostaglandins through activation of up-regulated NK(1) receptors. Our results also indicate some malfunction of the enteric nervous system in STC.
Subject(s)
Colon, Sigmoid/metabolism , Constipation/metabolism , Muscle, Smooth/metabolism , Prostaglandin-Endoperoxide Synthases/physiology , Receptors, Neurokinin-1/biosynthesis , Substance P/pharmacology , Acetylcholine/pharmacology , Adult , Aged , Aging/physiology , Atropine/pharmacology , Colon, Sigmoid/enzymology , Constipation/physiopathology , Dimethylphenylpiperazinium Iodide/pharmacology , Electric Stimulation , Enteric Nervous System/drug effects , Female , Ganglionic Stimulants/pharmacology , Gastrointestinal Transit/physiology , Humans , In Vitro Techniques , Kinetics , Middle Aged , Muscarinic Agonists/pharmacology , Muscle Contraction/physiology , Muscle, Smooth/enzymology , Neurokinin A/pharmacology , Nicotinic Agonists/pharmacology , Receptors, Neurokinin-1/genetics , Receptors, Nicotinic/drug effects , Reverse Transcriptase Polymerase Chain Reaction , Substance P/antagonists & inhibitors , Tetrodotoxin/pharmacology , Young AdultABSTRACT
Recent studies have described muscarinic receptors on the mucosa and the detrusor of the human urinary bladder. Muscarinic receptor antagonists are effective in the treatment of overactive bladder (OAB), but their site(s) of action and actual therapeutic target are unclear. Our aim was to compare, in human bladder mucosa and detrusor, the radioligand binding characteristics of newer, clinically effective agents: darifenacin, its hydroxylated metabolite UK-148,993, fesoterodine, solifenacin, tolterodine, and trospium. Specimens were collected from asymptomatic patients (50-72 years old) undergoing open bladder surgery. Radioligand binding studies with the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) were performed separately on detrusor and mucosal membranes. All antagonists displayed high affinity when competing for [3H]QNB binding in both detrusor and mucosa. Inhibition constants were also obtained for all antagonists against individual muscarinic receptor subtypes expressed in Chinese hamster ovary cells. Here, fesoterodine showed anomalous binding results, suggesting that some conversion to its metabolite had occurred. Global nonlinear regression analysis of bladder binding data with five antagonists demonstrated 82% low-affinity sites in mucosa and 78% low-affinity sites in detrusor, probably representing M(2)/M(4) receptors. There was an excellent correlation (r(2) = 0.99) of low-affinity global estimates between detrusor and mucosa, whereas the corresponding high-affinity estimates ( approximately 20% of sites) were dissimilar. In conclusion, commonly used and clinically effective muscarinic receptor antagonists bind to receptors located on the bladder mucosa and the detrusor, providing support for the hypothesis that muscarinic receptors in the mucosa may represent an important site of action for these agents in OAB.
Subject(s)
Mucous Membrane/metabolism , Muscarinic Antagonists/pharmacology , Receptors, Muscarinic/metabolism , Urinary Bladder, Overactive/metabolism , Urinary Bladder/metabolism , Aged , Benzhydryl Compounds/pharmacology , Benzofurans/pharmacology , Cresols/pharmacology , Cystectomy , Female , Humans , Male , Middle Aged , Phenylpropanolamine/pharmacology , Prostatectomy , Pyrrolidines/pharmacology , Quinuclidines/pharmacology , Quinuclidinyl Benzilate/metabolism , Radioligand Assay , Solifenacin Succinate , Tetrahydroisoquinolines/pharmacology , Tolterodine TartrateABSTRACT
Neurokinin A (NKA) is an important spasmogen in human colon. We examined inflammatory disease-related changes in the tachykinin NK(2) receptor system in human sigmoid colon circular muscle, using functional, radioligand binding, and quantitative reverse transcription-polymerase chain reaction methods. In circular muscle strips, indomethacin enhanced contractile responses to NKA (p < 0.01) and to the NK(2) receptor-selective agonist [Lys(5),MeLeu(9),Nle(10)]-NKA(4-10) (p < 0.05) in both normal and acute diverticular disease (DD) specimens, indicating NK(2) receptor-mediated release of relaxant prostanoids. Contractile responses to both tachykinins were reduced in strips from DD (p < 0.001) and ulcerative colitis (UC) (p < 0.05) specimens. Responses to acetylcholine were no different in other strips from the same disease patients, demonstrating that the change in responsiveness to tachykinins in disease is specifically mediated by the NK(2) receptor. In membranes from UC specimens, receptor affinity for (125)I-NKA (median K(D) 0.91 nM, n = 16) was lower (p < 0.01) than that in age-matched control specimens (K(D) 0.55 nM, n = 40), whereas K(D) (0.65 nM, n = 28) in DD was no different from control. No disease-related changes in receptor number (B(max)) were found (mean, 2.0-2.5 fmol/mg of wet weight tissue), suggesting that the reduced contractile responses in disease are not due to a loss of receptor number. Different mechanisms may account for the reduced contractility in DD compared with UC. A gender-related difference in receptor density was seen in controls, with B(max) lower in females (1.77 fmol/mg, n = 15) than in males (2.60 fmol/mg, n = 25, p = 0.01). In contrast, no gender-related differences were seen in NK(2) receptor mRNA in control colonic muscle, indicating that the gender difference is a post-translational event.
Subject(s)
Colitis, Ulcerative/physiopathology , Diverticulum, Colon/physiopathology , Indomethacin/pharmacology , Receptors, Neurokinin-2/physiology , Adult , Aged , Aged, 80 and over , Colitis, Ulcerative/metabolism , Colon/drug effects , Colon/metabolism , Colon/physiopathology , Diverticulum, Colon/metabolism , Female , Humans , In Vitro Techniques , Male , Middle Aged , Muscle Contraction/drug effects , Neurokinin A/analogs & derivatives , Neurokinin A/pharmacology , Peptide Fragments/pharmacology , RNA, Messenger/metabolism , Receptors, Neurokinin-2/agonists , Receptors, Neurokinin-2/geneticsABSTRACT
The urinary bladder purinergic system is reported to change with age and with bladder dysfunction. Here, we examined the expression of purinergic P2X(1) receptors in detrusor and mucosa (urothelium+lamina propria) from male control bladder and investigated age-related P2X(1) receptor mRNA expression in control and obstructed detrusor. Biopsy specimens were obtained at cystoscopy from control patients (n=46, age range 30-86years) and patients diagnosed with outlet obstruction (n=29, 46-88years). Calponin expression (measured by RT-PCR) was similar in control and obstructed detrusor and did not change with age. Quantitative competitive RT-PCR was used to measure P2X(1) receptor and GAPDH mRNA in control and obstructed detrusor. P2X(1) receptor mRNA expression was 9-fold (p<0.0001) higher in the detrusor than in the mucosa. Expression of mRNA for the internal control GAPDH remained stable with age and across control and obstructed detrusor. No difference in P2X(1) receptor expression was observed between control and obstructed detrusor (p=0.35). However, an age-related decrease in P2X(1) mRNA expression was observed in control (n=27; p=0.0054; Spearman coefficient r=-0.520) but not obstructed detrusor (n=19; p=0.093; r=-0.396). Downregulation of P2X(1) mRNA expression might occur as a result of an increased component of neural ATP release in the aging bladder.
Subject(s)
Aging/genetics , RNA, Messenger/genetics , Receptors, Purinergic P2/genetics , Urinary Bladder Neck Obstruction/genetics , Urinary Bladder/physiology , Aged , Aged, 80 and over , Biopsy , Humans , Male , Middle Aged , Receptors, Purinergic P2X , Reference Values , Reverse Transcriptase Polymerase Chain Reaction , Urinary Bladder/innervation , Urinary Bladder/pathology , Urinary Bladder/physiopathology , Urinary Bladder Neck Obstruction/pathology , Urinary Bladder Neck Obstruction/physiopathologyABSTRACT
OBJECTIVE: To examine the expression of muscarinic M2 and M3 receptors in human bladder detrusor and mucosa, from controls and patients with idiopathic detrusor overactivity (IDO), as antimuscarinic agents are the primary pharmacological treatment for IDO. PATIENTS AND METHODS: Biopsies from the bladder body were collected at cystoscopy from 20 women with urodynamically confirmed refractory IDO (age range 25-86 years); biopsies were also collected from 30 asymptomatic female controls (age range 32-87 years). Samples were collected into RNA extraction medium and dissected into mucosa (urothelium plus lamina propria) and detrusor. RNA was extracted and the expression of M2 and M3 receptor mRNA determined by quantitative competitive reverse transcription-polymerase chain reaction. Results were normalized to beta-actin expression in the same sample. RESULTS: Expression of M3 receptor mRNA, in mucosa of IDO patients (median 0.057 pg M3/100 ng total RNA; interquartile range 0.03-0.13, 12 samples), was four times (P = 0.039, Mann-Whitney) lower than from the control (median 0.22 pg M3/100 ng total RNA; 0.13-0.51, 11 samples). The expression of muscarinic M3 receptor mRNA was higher (14-35 times) in detrusor (control median 3.17; 26 samples) than in mucosa and did not change in IDO (median 2.03; 14 samples). M2 expression was not significantly different with region or with IDO. CONCLUSIONS: These data show that M3 muscarinic receptor mRNA expression was significantly less in mucosa from IDO patients than from age-matched controls. The role of mucosal M3 receptors is unknown at present and elucidation of this role might provide a greater understanding of the aetiology of IDO.
Subject(s)
Muscarinic Antagonists/pharmacology , Receptor, Muscarinic M2/metabolism , Receptor, Muscarinic M3/metabolism , Urinary Bladder, Overactive/metabolism , Actins/metabolism , Adult , Aged , Aged, 80 and over , Biopsy/methods , Case-Control Studies , Female , Humans , Middle Aged , Mucous Membrane/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Urinary Bladder/metabolism , Urinary Bladder/pathology , Urinary Bladder, Overactive/drug therapy , Urinary Bladder, Overactive/etiology , Urinary Bladder, Overactive/pathologyABSTRACT
AIM: Treatments targeting vanilloid receptor TRPV1 are effective in some bladder disorders. Our aim was to determine the expression profiles of TRPV1 in regions of human bladder and test the hypothesis that there would be an upregulation of TRPV1 in mucosa of patients with bladder hypersensitivity but not idiopathic detrusor overactivity (IDO). MATERIALS AND METHODS: Women with sensory urgency (SU), interstitial cystitis (IC), and IDO were investigated by videourodynamics and cystoscopy. Control biopsies were used for comparison. Biopsies were dissected into mucosa and muscle, and evaluated for TRPV1 mRNA expression using quantitative competitive RT-PCR (QC-RT-PCR). RESULTS: TRPV1 mRNA from SU trigonal mucosa was significantly higher than control trigonal mucosa or SU bladder body mucosa. In contrast, in IDO patients, there was no difference between trigonal mucosa and body mucosa. In IC biopsies, RNA quality was substandard and unable to be used for analysis. The most striking finding was that TRPV1 mRNA expressed in SU trigonal mucosa was significantly inversely correlated with the bladder volume at first sensation of filling during cystometry. No such relationship was seen for IDO trigonal mucosa. No difference was seen in bladder body mucosa from any disease groups compared with age-matched control. CONCLUSIONS: The symptoms of SU were associated with the increased expression of TRPV1 mRNA in the trigonal mucosa. No upregulation or regional differences of TRPV1 mRNA were seen in IDO patients. TRPV1 may play a role in SU and premature first bladder sensation on filling.
Subject(s)
TRPV Cation Channels/genetics , TRPV Cation Channels/metabolism , Urinary Bladder, Overactive/physiopathology , Urinary Bladder/physiology , Urodynamics/genetics , Adult , Aged , Aged, 80 and over , Biopsy , Female , Gene Expression/physiology , Humans , Middle Aged , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sensation , Up-Regulation/physiology , Urinary Bladder/innervation , Urinary Bladder/pathology , Urinary Bladder, Overactive/pathology , Urothelium/physiologyABSTRACT
The tachykinins form one of the largest peptide families in nature. In this review, we describe the comparative features of the tachykinin peptides and their receptors, focusing particularly on amphibians. We also summarize our systematic studies of the localization, characteristics, and actions of bufokinin, a toad substance P-related peptide, in its species of origin. In addition, we discuss the establishment of multiple isoforms of the NK1-like receptor in the toad, and their structure, pharmacology and tissue distributions. We conclude that tachykinin peptides and receptors are well conserved in terms of their structures, physiological functions and coupling mechanisms during tetrapod evolution.
Subject(s)
Amphibians/metabolism , Peptide Fragments , Receptors, Tachykinin , Tachykinins , Amino Acid Sequence , Animals , Humans , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/metabolism , Intercellular Signaling Peptides and Proteins/physiology , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Peptide Fragments/physiology , Phylogeny , Receptors, Tachykinin/chemistry , Receptors, Tachykinin/metabolism , Receptors, Tachykinin/physiology , Sequence Alignment , Tachykinins/chemistry , Tachykinins/metabolism , Tachykinins/physiologyABSTRACT
1. We investigated muscarinic receptors in the detrusor and mucosa of the human bladder body. Radioligand-binding studies with [(3)H]QNB were conducted using specimens collected from patients (36-77 years) with normal bladder function, undergoing surgery. For RT-PCR, biopsies of normal bladder were obtained from patients (30-88 years) undergoing check cystoscopy. 2. Binding of [(3)H]QNB in detrusor (n=20) was of high affinity (K(D) 77.1 (55.2-99.0) pM) and capacity (B(max) 181+/-7 fmol mg protein(-1)). Similar values were obtained in mucosa (n=6) (K(D) 100.5 (41.2-159.9) pM; B(max) 145+/-9 fmol mg protein(-1)). 3. Competition-binding experiments in detrusor membranes with muscarinic receptor antagonists including trospium, darifenacin, 4-DAMP, methoctramine, AQ-RA 741, AF-DX 116 and pirenzepine indicated a receptor population of 71% M(2), 22% M(3) and 7% M(1). In the mucosa, 75% of sites were M(2) receptors, with 25% M(3)/M(5). 4. Using RT-PCR, expression of M(1), M(2), M(3) and M(5) mRNA was demonstrated in both detrusor and mucosa. 5. The presence of a high density of mainly M(2) muscarinic receptors in the mucosa appears to be a novel finding and raises the question of their physiological significance and the source of their endogenous ligand. 6. There was a negative correlation of receptor number (B(max)) with age in detrusor muscle from male patients (P=0.02). Quantitative competitive RT-PCR demonstrated a selective age-related decrease in mRNA for muscarinic M(3) but not M(2) receptors, in both male (P<0.0001) and female (P=0.019) detrusor. These findings correspond with reports of decreased detrusor contractility with ageing.
Subject(s)
Aging/metabolism , Protein Isoforms/classification , Radioligand Assay/methods , Receptors, Muscarinic/classification , Receptors, Muscarinic/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Urinary Bladder/metabolism , Adult , Aged , Dose-Response Relationship, Drug , Female , Humans , Male , Middle Aged , Mucous Membrane/metabolism , Protein Binding/physiology , Protein Isoforms/metabolism , Quinuclidinyl Benzilate/metabolismABSTRACT
The toad tachykinin, bufokinin (Lys-Pro-Arg-Pro-Asp-Gln-Phe-Tyr-Gly-Leu-Met amide; BUF), acts via tachykinin NK1-like receptors to contract the intestine of the cane toad, Bufo marinus. In this structure-activity study, we used isolated segments of toad small intestine and performed binding studies with [125I] Bolton-Hunter BUF in intestinal membranes to compare the contribution of individual amino acid residues to the potencies of 18 naturally occurring tachykinins and 13 BUF analogs. Potencies were similar (r=0.94) in functional and binding studies, with BUF and ranakinin being most potent. Ranatachykinin A, physalaemin, hylambatin and cod, trout and mammalian SPs exhibited 10-60% of the potency of BUF. The Ala-substituted BUF analogs were 11-60% as potent as BUF in functional studies, with [Ala2]-BUF and [Ala4]-BUF the least efficacious, indicating the importance of both proline residues. QSAR equations were developed using 12 connectivity, shape and steric parameters for each of the 7 hypervariable amino acid residues in these peptides. For the binding data, the optimal regression equation explained 81% of the variance, and indicated the importance of the steric function at [Pro2] and simple connectivity functions at [Gln6] and [Tyr8]. The optimal functional regression equation (80% of variance) confirmed the importance of connectivity functions at [Gln6] and [Tyr8], as well as the shape of residues [Lys1] and [Pro4]. The potencies of most full-length peptides were well predicted using the leave-one-out procedure, as were the potencies of a series of model Ala-substituted BUFs, thus emphasising the potential utility of these equations in the design of new ligands interacting with tachykinin receptors.
Subject(s)
Intercellular Signaling Peptides and Proteins/chemistry , Intestinal Mucosa/metabolism , Intestine, Small/physiology , Quantitative Structure-Activity Relationship , Receptors, Neurokinin-1/genetics , Tachykinins/chemistry , Amino Acid Substitution , Animals , Bufo marinus , Dose-Response Relationship, Drug , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Intercellular Signaling Peptides and Proteins/pharmacology , Intestinal Mucosa/chemistry , Intestinal Mucosa/drug effects , Intestine, Small/drug effects , Intestine, Small/metabolism , Receptors, Neurokinin-1/agonists , Receptors, Neurokinin-1/metabolism , Tachykinins/genetics , Tachykinins/metabolism , Tachykinins/pharmacologyABSTRACT
Two tachykinin peptides, bufokinin and Xenopus neurokinin A (X-NKA) were recently isolated from Xenopus laevis. In this study we investigated the tachykinin receptors in the Xenopus gastrointestinal tract. In functional studies using stomach circular muscle strips, all peptides had similar potencies (EC50 values 1-7 nM). The rank order of potency to contract the intestine was physalaemin (EC50 1 nM)> or =bufokinin (EC50 3 nM)>substance P (SP)> or =cod SP>NKA>>X-NKA (EC50 1,900 nM). No maximum response could be obtained for [Sar9,Met(O2)11]SP, eledoisin and kassinin. In stomach strips, the mammalian tachykinin receptor antagonists RP 67580 (NK1) and MEN 10376 (NK2) had agonistic effects but did not antagonize bufokinin or X-NKA. In intestinal strips, RP 67580 (1 microM) reduced the maximal response to X-NKA but not bufokinin, while MEN 10376 was ineffective. [125I]BH-bufokinin bound with high affinity to a single class of sites, of KD 213+/-35 (stomach) and 172+/-9.3 pM (intestine). Specific binding of [125I]BH-bufokinin was displaced by bufokinin> or =SP>NKA> or =eledoisin approximately kassinin>X-NKA, indicating binding to a tachykinin NK1-like receptor. Selective tachykinin receptor antagonists were weak or ineffective. Other iodinated tachykinins ([125I]NKA and [125I]BH-eledoisin) displayed biphasic competition profiles, with the majority of sites preferring bufokinin rather than X-NKA. In conclusion, there is evidence for two different tachykinin receptors in Xenopus gastrointestinal tract. Both receptors may exist in stomach, whereas the bufokinin-preferring NK1-like receptor predominates in longitudinal muscle of the small intestine. Antagonists appear to interact differently with amphibian receptors, compared with mammalian receptors.