Characterization of Oligomer Formation of Surfactant Protein-D (SP-D) Using AF4-MALLS.

BACKGROUND: Surfactant protein-S (SP-D) is a naturally occurring lung protein with the potential to treat pulmonary infections. A recombinant surfactant protein-D (SP-D) has been produced and was previously found to exist in multiple oligomeric states. INTRODUCTION: Separation and characterization of interconverting oligomeric states of a protein can be difficult using chromatographic methods, so an alternative separation technique was employed for SPD to characterize the different association states that exist. METHODS: Samples of SP-D were analyzed using asymmetrical flow field-flow fractionation (AF4) using UV and multi-angle laser light scattering (MALLS) detection. The AF4 method appears to be able to separate species as small as the monomer up to the dodecamer (the dominant species) to much larger species with a molar mass greater than 5 MDa. RESULTS: Consistent elution of four distinct peaks was observed after repeated injections. The largest species observed under the last peak (labeled as Peak 4) were termed "unstructured multimers" and were resolved fairly well from the other species. The AF4-MALLS data suggest that only a small fraction of Peak 4 truly corresponds to high molar mass unstructured multimers. All other peaks demonstrated significant molar mass homogeneity consistent with AFM results. CONCLUSION: AF4-MALLS technology appears to be a powerful analytical approach to characterize the complex and dynamic interplay among different protein oligomeric species of SP-D in an aqueous solution.

Structure and activity of human surfactant protein D from different natural sources.

Surfactant protein D (SP-D) is a C-type lectin that participates in the innate immune defense of lungs. It binds pathogens through its carbohydrate recognition domain in a calcium-dependent manner. Human surfactant protein D (hSP-D) has been routinely obtained from bronchoalveolar lavage of patients suffering from pulmonary alveolar proteinosis (PAP) and from amniotic fluid (AF). As a consequence of the disease, hSP-D obtained from PAP is found in higher amounts and is mainly composed of higher order oligomeric forms. However, PAP-hSP-D has never been directly compared with nonpathological human protein in terms of structure and biological activity. Moreover, the quantitative distribution of the different hSP-D oligomeric forms in human protein obtained from a natural source has never been evaluated. In this work, we have determined the quantitative distribution of AF-hSP-D oligomers, characterized the sugars attached through the N-glycosylation site of the protein, and compared the activity of hSP-D from AF and PAP with respect to their ability to bind and agglutinate bacteria. We have found that fuzzy balls (40%) are the most abundant oligomeric form in AF-hSP-D, very closely followed by dodecamers (33%), with both together constituting 73% of the protein mass. The glycan attached to the N-glycosylation site was found to be composed of fucose, galactose, sialic acid, and N-acetylglucosamine. Finally, in the functional assays performed, hSP-D obtained from PAP showed higher potency, probably as a consequence of its higher proportion of large oligomers compared with hSP-D from AF.

Comparison of the effects of oestrogen deficiency and old age on primary bladder afferent activity and voiding behaviour in the ageing female rat.

OBJECTIVE: To determine if a decrease in oestrogen (E) levels increases activity in bladder afferent nerves, by investigating the effects of E loss on afferent nerve activity in ovariectomized (OVX) rats, and making comparisons with findings in ageing female rats. MATERIALS AND METHODS: Female Sprague-Dawley rats were divided into four groups: ageing; OVX+ E; OVX+ vehicle (V); and sham-operated (SHAM) + V. The E (250 µg/kg) or V (cottonseed oil) was injected s.c. once a week for 8 weeks. Frequency-volume recordings were performed with the rats in metabolic cages, and single Aδ- or C-fibre activities from the bladder were measured. After measurements, the blood serum was collected and the estradiol (E2) level was measured. RESULTS: The C-fibre activity of OVX + V rats was significantly lower than in the other groups. Aδ-fibre activity was unchanged. Despite low E2 levels, ageing rats showed high afferent activity and voiding frequency. CONCLUSION: Although a low E level may affect storage function, it is unlikely that it is the main cause of the high afferent activity and voiding frequency observed in the ageing female rat.

Cannabinor, a selective cannabinoid-2 receptor agonist, improves bladder emptying in rats with partial urethral obstruction.

PURPOSE: We studied the effects of chronic treatment with the novel selective cannabinoid 2 receptor agonist cannabinor (Procter & Gamble Pharmaceuticals, Cincinnati, Ohio) on bladder function in conscious rats with partial urethral obstruction and on the functional properties of isolated detrusor muscle. MATERIALS AND METHODS: A total of 24 female Sprague-Dawley® rats with surgically created partial urethral obstruction received daily intraperitoneal injections of 3 mg/kg cannabinor (12) or saline as controls (12) for 2 weeks. Cystometry was done, the rats were sacrificed and the bladders were prepared for in vitro studies. RESULTS: Mean ± SEM bladder weight was 0.97 ± 0.15 gm in controls and 0.53 ± 0.08 gm in cannabinor treated rats (p <0.05). There was no difference between the groups in the mean micturition interval, or mean baseline, threshold, flow or maximum pressure. In controls and cannabinor treated rats mean post-void residual volume was 0.28 ± 0.07 and 0.06 ± 0.02 ml, mean micturition compliance was 0.032 ± 0.006 and 0.069 ± 0.016 ml/cm H(2)O, and mean bladder wall force at the start of flow was 950 ± 280 and 1,647 ± 325 mN/gm, respectively (each p <0.05). Nonvoiding contractions were significantly less frequent in cannabinor treated rats than in controls. We noted no difference in carbachol (Sigma®) half maximum concentration between the groups but the carbachol maximum response in detrusor strips from cannabinor treated rats was significantly higher than that in control strips. CONCLUSIONS: In rats with partial urethral obstruction treated daily for 14 days with cannabinor bladder weight was lower, the ability to empty the bladder was preserved and nonvoiding contraction frequency was low compared to those in controls. Detrusor preparations from cannabinor treated rats showed a higher response to nerve stimulation than those from controls. Selective cannabinoid 2 receptor activation may be a novel principle to enable improved bladder function after partial urethral obstruction.

Urothelial beta-3 adrenergic receptors in the rat bladder.

AIMS: To investigate the distribution of beta-3 adrenergic receptors (ß(3)ARs) in the rat bladder and to examine the contribution of urothelial ß(3)ARs to agonist-induced suppression of bladder reflexes and relaxation of smooth muscle. METHODS: Bladder tissue was collected from 8- to 10-month old female SD rats. In some samples, the urothelium was surgically separated from the smooth muscle. The expression and localization of ßAR mRNA and ß(3)AR protein were determined using RT-PCR and immunohistochemistry. Contractile responses to the specific ß(3)AR agonists TAK-677 and BRL37344 were measured in bladder strips with or without the urothelium. The contribution of urothelial ß(3)ARs to the micturition reflex was assessed in continuous cystometry in urethane anesthetized rats using intravesical delivery of ß(3)AR agonists. RESULTS: RT-PCR detected mRNA of all ßARs in urothelium and smooth muscle. Immunostaining detected ß(3)ARs throughout the urothelium, in the smooth muscle, myofibroblast-like cells, and in the peripheral nerves. Ovariectomy did not change the distribution of ß(3)ARs in any bladder structure. Intravesical administration of TAK-677 and BRL37344 (1-5 × 10(-4) M) decreased voiding frequency and amplitude of bladder contractions. In bladder strips in vitro both ß(3)AR agonists (10(-12) to 10(-4) M) relaxed the smooth muscle in a concentration-dependent manner to the same extent in strips with and without the urothelium. CONCLUSIONS: In addition to their presence in bladder smooth muscle, ß(3)ARs are present in the urothelium where their activation may alter reflex voiding via release of factor(s) that act on non-myocyte structures including the afferent and/or efferent nerves to influence bladder contractility.

ß-Adrenergic receptor subtype expression in myocyte and non-myocyte cells in human female bladder.

ß(3)-Adrenergic receptor agonists are currently under clinical development for the treatment of overactive bladder, a condition that is prevalent in postmenopausal women. These agents purportedly relax bladder smooth muscle through a direct action at the myocyte ß(3)-receptor. The aim of this study was to examine the expression of the individual beta-adrenergic receptors in full thickness sections from ageing human female bladder. We obtained a series of rabbit polyclonal antibodies generated against each of the three ß-adrenergic receptors, and validated their receptor specificity in CHOK1 cells expressing each of the individual receptors. Immunostaining for ß(1), ß(2), and ß(3) were each more prominent in the urothelium than in the detrusor, with all receptors expressed in the same cell types, indicating co-expression of all three receptors throughout the urothelium in addition to the detrusor. Staining of all receptors was also observed in suburothelial myofibroblast-like cells, intramural ganglion cells, and in Schwann cells of intramural nerves. The ß(3)-receptor in the human urothelium appears to be functional, as two different selective ß(3)-receptor agonists, TAK677 and BRL37344, stimulate cAMP formation in URO tsa cells. Densitometry analysis indicates a persistent expression of all receptors throughout the bladder with increasing age, with the exception of the ß(2)-receptor in the urothelium of the trigone, which appears to decrease slightly in older women. These data indicate that ß(3)-receptor expression is maintained with age, but may function in concert with other ß-receptors. Activation of the myocyte receptor may be influenced by action on non-myocyte structures including the intramural ganglion cells and myofibroblasts.

Effects of cannabinor, a novel selective cannabinoid 2 receptor agonist, on bladder function in normal rats.

BACKGROUND: Cannabinoid (CB) receptors may be involved in the control of bladder function; the role of CB receptor subtypes in micturition has not been established. OBJECTIVES: Our aim was to evaluate the effects of cannabinor, a novel CB2 receptor agonist, on rat bladder function. DESIGN, SETTING, AND PARTICIPANTS: Sprague Dawley rats were used. Distribution of CB2 receptors in sensory and cholinergic nerves of the detrusor was studied. Selectivity of cannabinor for human and rat CB receptors was evaluated. Effects of cannabinor on rat detrusor and micturition were investigated. MEASUREMENTS: Immunohistochemistry, radioligand binding, tritium outflow assays, organ bath studies of isolated bladder tissue, and cystometry in awake rats were used. RESULTS AND LIMITATIONS: CB2 receptor immunoreactivity was expressed in the urothelium and in sensory and cholinergic bladder nerves. Cannabinor exhibited similar binding at human and rat CB2 receptors and a 321-fold functional selectivity for the CB2 receptor versus the CB1 receptor. Cannabinor had no effect on isolated detrusor muscle function. In vivo, cannabinor 3.0mg/kg increased micturition intervals and volumes by 52% (p<0.05) and 96% (p<0.01), respectively, and increased threshold and flow pressures by 73% (p<0.01) and 49% (p<0.001), respectively. Cannabinor 0.3 or 1.0mg/kg or vehicle did not affect urodynamic parameters. CONCLUSIONS: Considering that CB2 receptors are localized on sensory nerves and on the urothelium and that cannabinor had effects on "afferent" urodynamic parameters, peripheral CB2 receptors may be involved in sensory functions of rat micturition. Effects of cannabinor on cholinergic nerve activity in normal bladder tissue appear to be limited.

Effects of beta3-adrenergic receptor activation on rat urinary bladder hyperactivity induced by ovariectomy.

Voiding dysfunctions, including increased voiding frequency, urgency, or incontinence, are prevalent in the postmenopausal population. Beta(3)-adrenergic receptor (beta(3)AR) agonists, which relax bladder smooth muscle, are being developed to treat these conditions. We utilized the rat ovariectomy (OVX) model to investigate the effect of ovarian hormone depletion on bladder function and the potential for beta(3)AR agonists to treat bladder hyperactivity in this setting. OVX increased voiding frequency and decreased bladder capacity by approximately 25% in awake rats and induced irregular cystometrograms in urethane-anesthetized rats. Reverse transcription-polymerase chain reaction revealed three betaARs subtypes (beta(1,2,3)) in bladder tissue, and immunostaining indicated beta(3)AR localization in urothelium and detrusor. Receptor expression was not different in OVX and SHAM rats. The beta(3)AR agonist selectivity of BRL37344 [(+/-)-(R(*),R(*))-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium hydrate], TAK-677 [(3-((2R)-(((2R)-(3-chlorophenyl)-2-hydroxyethyl)amino)propyl)-1H-indol-7-yloxy)acetic acid], and FK175 [acetic acid, 2-[[(8S)-8-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-yl]oxy], ethyl ester, hydrochloride] was confirmed by examining the relative potency for elevation of cAMP in CHOK1 cells overexpressing the various rat betaARs. Intravenous injection of each of the beta(3)AR agonists (0.1-500 microg/kg) in anesthetized rats decreased voiding frequency, bladder pressure, and amplitude of bladder contractions. In bladder strips, beta(3)AR agonists (10(-12)-10(-4) M) decreased baseline tone and reduced spontaneous contractions. BRL37344 (5 mg/kg) and TAK-677 (5 mg/kg) injected intraperitoneally in awake rats decreased voiding frequency by 40 to 70%. These effects were not altered by OVX. The results indicate that OVX-induced bladder dysfunction, including decreased bladder capacity and increased voiding frequency, is not associated with changes in beta(3)AR expression or the bladder inhibitory effects of beta(3)AR agonists. This suggests that beta(3)AR agonists should prove effective for the treatment of overactive bladder symptoms in the postmenopausal population.

AMD3100 is a CXCR7 ligand with allosteric agonist properties.

The bicyclam AMD3100 is known as a small synthetic inhibitor of the CXCL12-binding chemokine receptor CXCR4. Here, we show that AMD3100 also binds to the alternative CXCL12 receptor CXCR7. CXCL12 or AMD3100 alone activate beta-arrestin recruitment to CXCR7, which we identify as a previously unreported signaling pathway of CXCR7. In addition, AMD3100 increases CXCL12 binding to CXCR7 and CXCL12-induced conformational rearrangements in the receptor dimer as measured by bioluminescence resonance energy transfer. Moreover, small but reproducible increases in the potency of CXCL12-induced arrestin recruitment to CXCR7 by AMD3100 are observed. Taken together, our data suggest that AMD3100 is an allosteric agonist of CXCR7. The finding that AMD3100 not only binds CXCR4, but also to CXCR7, with opposite effects on the two receptors, calls for caution in the use of the compound as a tool to dissect CXCL12 effects on the respective receptors in vitro and in vivo.

GPR30 contributes to estrogen-induced thymic atrophy.

The mechanisms by which prolonged estrogen exposures, such as estrogen therapy and pregnancy, reduce thymus weight, cellularity, and CD4 and CD8 phenotype expression, have not been well defined. In this study, the roles played by the membrane estrogen receptor, G protein-coupled receptor 30 (GPR30), and the intracellular estrogen receptors, estrogen receptor alpha (ERalpha) and beta (ERbeta), in 17beta-estradiol (E2)-induced thymic atrophy were distinguished by construction and the side-by-side comparison of GPR30-deficient mice with ERalpha and ERbeta gene-deficient mice. Our study shows that whereas ERalpha mediated exclusively the early developmental blockage of thymocytes, GPR30 was indispensable for thymocyte apoptosis that preferentially occurs in T cell receptor beta chain(-/low) double-positive thymocytes. Additionally, G1, a specific GPR30 agonist, induces thymic atrophy and thymocyte apoptosis, but not developmental blockage. Finally, E2 treatment attenuates the activation of nuclear factor-kappa B in CD25(-)CD4(-)CD8(-) double-negative thymocytes through an ERalpha-dependent yet ERbeta- and GPR30-independent pathway. Differential inhibition of nuclear factor-kappaB by ERalpha and GPR30 might underlie their disparate physiological effects on thymocytes. Our study distinguishes, for the first time, the respective contributions of nuclear and membrane E2 receptors in negative regulation of thymic development.

The Y2 receptor mediates increases in collateral-dependent blood flow in a model of peripheral arterial insufficiency.

We have utilized a rat model of peripheral artery disease (PAD) to examine whether the known angiogenic activity of the Y(2) receptor would translate into a meaningful increase in collateral blood flow. The maximal increase in collateral blood flow capacity of approximately 60% (p<0.001) was obtained with a 10microg/kgday (IA infusion, 14 days) of either PYY or PYY(3-36) and did not differ from that obtained with a maximally angiogenic dose of VEGF(165). Pharmacodynamic modeling based upon single dose pharmacokinetic plasma profiles of both agonists suggests that E(max) is reached when the Y(2) receptor is occupied by >or=50%. Furthermore, for PYY(3-36), occupancy of the Y(2) receptor is sufficient to promote a significant benefit in collateral blood flow.

Characterization of neuropilin-1 structural features that confer binding to semaphorin 3A and vascular endothelial growth factor 165.

Neuropilin-1 (Npn-1) is a receptor for both semaphorin 3A (Sema3A) and vascular endothelial growth factor 165 (VEGF(165)). To understand the role Npn-1 plays as a receptor for these structurally and functionally unrelated ligands, we set out to identify structural features of Npn-1 that confer binding to Sema3A or VEGF(165). We constructed Npn-1 variants containing deletions within the "a" and "b" domains of Npn-1. More than 16 variants were expressed in COS-1 cells and tested for alkaline phosphatase-Sema3A as well as alkaline phosphatase-VEGF(165) binding. Our results indicate that each of the two Npn-1 CUB domains and the amino-terminal coagulation factor V/VIII domain (CF V/VIII) are essential for Sema3A binding, but only the amino-terminal Npn-1 CF V/VIII domain is required for binding to VEGF(165). Guided by the structure of the bovine spermadhesin CUB domain, point mutants targeting defined surfaces of the Npn-1 a1 CUB domain were generated and tested for Sema3A and VEGF(165) binding. One Npn-1 variant, Npn-1(2ABC), exhibits complete loss of Sema3A binding while retaining normal VEGF(165) binding. Moreover, co-immunoprecipitation experiments show that Npn-1(2ABC) can form a signaling complex with the VEGF(165) signaling receptor KDR/VEGFR-2. These results establish the identity of contact sites between Npn-1 and its semaphorin ligands, and they provide a foundation for understanding how Npn-1 functions as a receptor for distinct classes of ligands in vivo.