Inverse expression of prostaglandin E2-related enzymes highlights differences between diverticulitis and inflammatory bowel disease.

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.

Correlation between cystometric volumes, ATP release, and pH in women with overactive bladder versus controls.

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).

Hemokinin-1 stimulates prostaglandin E2 production in human colon through activation of cyclooxygenase-2 and inhibition of 15-hydroxyprostaglandin dehydrogenase.

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.

Immunocytochemical characterisation of cultures of human bladder mucosal cells.

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.

Does adenosine triphosphate released into voided urodynamic fluid contribute to urgency signaling in women with bladder dysfunction?

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.

Hemokinin-1 and substance P stimulate production of inflammatory cytokines and chemokines in human colonic mucosa via both NK1 and NK2 tachykinin receptors.

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).

Cyclooxygenase-dependent alterations in substance P-mediated contractility and tachykinin NK1 receptor expression in the colonic circular muscle of patients with slow transit constipation.

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.

Comparison of receptor binding characteristics of commonly used muscarinic antagonists in human bladder detrusor and mucosa.

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.

Tachykinin NK2 receptor and functional mechanisms in human colon: changes with indomethacin and in diverticular disease and ulcerative colitis.

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.

Age-related changes of P2X(1) receptor mRNA in the bladder detrusor from men with and without bladder outlet obstruction.

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.

Muscarinic receptor subtypes in human bladder detrusor and mucosa, studied by radioligand binding and quantitative competitive RT-PCR: changes in ageing.

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.

Quantitative structure-activity analyses of bufokinin and other tachykinins at bufokinin (bNK1) receptors of the small intestine of the cane toad, Bufo marinus.

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.

Tachykinin peptides and receptors: putting amphibians into perspective.

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.

Structure-activity studies of bufokinin, substance P and their C-terminal fragments at bufokinin receptors in the small intestine of the cane toad, Bufo marinus.

Bufokinin is a substance P-related tachykinin peptide with potent spasmogenic actions, isolated from the intestine of the cane toad, Bufo marinus. Bufokinin acts via a tachykinin receptor with similarities to the mammalian NK(1) receptor. In this structure-activity study of bufokinin, substance P (SP) and their C-terminal fragments, we have used isolated segments and homogenates of toad small intestine to compare the contractile potencies and abilities to compete for the binding of [125I]-Bolton-Hunter bufokinin. In general, potency was very similar in both studies (r=0.956) and was primarily related to peptide length, with the natural undecapeptide tachykinins bufokinin - ranakinin>SP- cod SP -trout SP being most potent. The weakest peptides were [Pro(9)]SP, BUF(7-11) and SP(7-11). Bufokinin fragments (BUF) were approximately equipotent to the corresponding SP fragments, with only BUF(5-11) showing unexpectedly low binding affinity. Data obtained with SP, bufokinin and fragments were subjected to quantitative structure--activity (QSAR) analysis which demonstrated that molecular connectivity and shape descriptors yielded significant regression equations (r approximately 0.90). The predictive capacity of the equations was confirmed using ranakinin, trout SP and cod SP, but not using the synthetic analogs [Pro(9)]SP and [Sar(9)]SP. The study suggests that the full undecapeptide sequence of bufokinin is required for optimal activity, with high potency conferred by Lys(1), Pro(2), Gly(9) and probably Tyr(8). The finding that receptor-ligand interactions were correlated with the shape descriptor 2kappa(alpha) and favored by basic and rigid residues at position 1-3 is consistent with an important role of conformation at the N-terminus of bufokinin.

Structure-activity relationship of neurokinin A(4-10) at the human tachykinin NK(2) receptor: the effect of amino acid substitutions on receptor affinity and function.

A structure-activity study of the neurokinin A (NKA) fragment NKA(4-10) was performed to investigate the importance of amino acid residues for receptor efficacy, potency and affinity at the NK(2) receptor in human colon circular muscle. Fourteen analogs of NKA(4-10) were produced with substitutions at positions 4, 5, 7, 9 and/or 10 of NKA. Their potencies were determined by in vitro contractile responses and affinities by radioligand binding using [125I]NKA. Functional potency was enhanced 8-fold by single amino acid substitutions with Lys(5) and MeLeu(9) but not significantly altered by substitutions Glu(4), Arg(5), His(5) and Nle(10). The multiply-substituted analogs [MeLeu(9),Nle(10)]NKA(4-10), [Lys(5),MeLeu(9),Nle(10)]NKA(4-10) and [Lys(5),(Tyr(7)),MeLeu(9),Nle(10)]NKA(4-10) displayed 6-9-fold increase in potency. Although [Arg(5),Nle(10)]NKA(4-10) was similar in potency to NKA(4-10), it was the only analog to show significantly reduced efficacy. All analogs were able to compete fully for [125I]NKA binding. [Lys(5),MeLeu(9)]NKA(4-10), [MeLeu(9),Nle(10)]NKA(4-10), [Lys(5),Nle(10)]NKA(4-10) and analogs containing single substitutions with Glu(4), Arg(5), Lys(5) and MeLeu(9) displayed significantly higher affinity, whereas those with Nle(10) and [Glu(4),Nle(10)] substitutions showed significantly lower affinity than NKA(4-10). There was a positive correlation (r=0.63) between binding affinity and functional potency, which was markedly improved (r=0.95) by removal of three analogs: [Lys(5),MeLeu(9),Nle(10)]NKA(4-10), [Lys(5),Tyr(7),MeLeu(9),Nle(10)]NKA(4-10) and [Lys(5),Tyr(I(2))(7),MeLeu(9),Nle(10)]NKA(4-10). These exhibited similar binding affinities to that of NKA(4-10) but were more potent in functional studies, possibly indicating a different mechanism of receptor interaction. In conclusion, substitution of Ser(5) with Lys, and/or N-methylation of Leu(9), were the most effective changes to increase functional and binding potency of NKA(4-10) at the human colon NK(2) receptor.

The tachykinin NK-2 receptor antagonist SR48968 does not block noncholinergic contractions in unstable human bladder.

Concentration-response curves to acetylcholine, and responses to electrical field stimulation (EFS) were compared in detrusor muscle strips, from control patients and those with idiopathic detrusor instability (IDI). Responses were similar in both groups. However, atropine abolished responses to EFS in 80% of control but only 33% of IDI patients (P>0.05), with the residual atropine-resistant response in most IDI patients abolished by tetrodotoxin. The post-atropine residual response was unaffected by the tachykinin NK-2 receptor antagonist SR48968. Despite the known existence of NK-2 receptors in the human detrusor, there was no evidence for tachykinin contribution to EFS-induced contractions.

Muscarinic receptor subtypes in the human colon: lack of evidence for atypical subtypes.

Characteristics of muscarinic receptors were investigated in circular muscle from normal human colon. In saturation studies (n=18), binding of [3H]quinuclidinyl benzylate (QNB) was of high affinity (K(d) 87.3 pM) and capacity (B(max) 362+/-27 fmol/mg protein), with no differences between ascending and sigmoid colon. Kinetic studies gave a K(d) of 55 pM. Methoctramine and darifenacin displayed biphasic binding profiles, the high affinity components being compatible with a population of approximately 80+/-5% M(2) and 13+/-2% M(3) muscarinic receptors, respectively. Pirenzepine, mamba toxin 1 and mamba toxin 3 were very weak competitors, indicating negligible expression of muscarinic M(1) and M(4) receptors. Six other subtype-preferring antagonists exhibited K(i) values typical of those reported at cloned human muscarinic M(2) receptors. In the presence of methoctramine, pre-treatment with alkylating agent 4-diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP mustard) inhibited [3H]quinuclidinyl benzylate binding to 26% of sites. Following alkylation of muscarinic M(3) receptors, darifenacin bound to a single low affinity site, indicating binding to muscarinic M(2) receptors.

Enhancement of neurokinin A-induced smooth muscle contraction in human urinary bladder by mucosal removal and phosphoramidon: relationship to peptidase inhibition.

Neurokinin A (NKA) is potent in contracting the human detrusor muscle. Here, we have investigated whether these contractile responses are influenced by the presence of the mucosa, by the peptidase inhibitor phosphoramidon or by possible modulators, prostaglandins and nitric oxide. Contractile responses to neurokinin A were unaffected by indomethacin or N-omega-nitro-L-arginine, but were significantly reduced in strips containing mucosa. Phosphoramidon, an inhibitor of neutral endopeptidase 24.11 (neprilysin, CD10), was ineffective at 10 microM, but at 100 microM, significant increase in the maximum response was achieved by neurokinin A in detrusor strips with and without mucosa. In immunohistochemical studies, neutral endopeptidase immunoreactivity occurred in peripheral nerve trunks in the detrusor and in a fibrous meshwork in the subepithelial lamina propria. Our data indicate that neutral endopeptidase is present in bladder mucosa and detrusor, and support the concept that this metalloprotease and/or related enzymes are important in regulating the actions of tachykinins.

Characterization of receptors for two Xenopus gastrointestinal tachykinin peptides in their species of origin.

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.

Gene expression of muscarinic, tachykinin, and purinergic receptors in porcine bladder: comparison with cultured cells.

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.