Expression of nicotinic acetylcholine receptor subunit mRNA in mouse bladder afferent neurons.

Nicotinic acetylcholine receptors (nAChR) influence bladder afferent activity and reflex sensitivity, and have been suggested as potential targets for treating detrusor overactivity. Mechanisms may include indirect effects, e.g. involving the urothelium, and direct action on nAChR expressed by afferent neurons. Here we determined the nAChR repertoire of bladder afferent neurons by retrograde neuronal tracing and laser-assisted microdissection/reverse transcriptase polymerase chain reaction (RT-PCR), and quantified retrogradely labelled nAChRα3-subunit-expressing neurons by immunohistochemistry in nAChR α3ß4α5 cluster enhanced green fluorescent protein (eGFP) reporter mice. Bladder afferents distinctly expressed mRNAs encoding for nAChR-subunits α3, α6, α7, ß2-4, and weakly α4. Based upon known combinatorial patterns of subunits, this predicts the expression of at least three basically different subunits of nAChR - α3(∗), α6(∗) and α7(∗) - and of additional combinations with ß-subunits and α5. Bladder afferents were of all sizes, and their majority (69%; n=1367) were eGFP-nAChRα3 positive. Immunofluorescence revealed immunoreactivities to neurofilament 68 (NF68), transient receptor potential cation channel vanilloid 1 (TRPV1), substance P (SP) and calcitonin gene-related peptide (CGRP) in eGFP-nAChRα3-positive and -negative neurons. For each antigen, all possible combinations of colocalisation with eGFP-nAChRα3 were observed, with eGFP-nAChRα3-positive bladder neurons without additional immunoreactivity being most numerous, followed by triple-labelled neurons. In conclusion, more than one population of bladder afferent neurons expresses nAChR, indicating that peripheral nicotinic initiation and modulation of bladder reflexes might result, in addition to indirect effects, from the direct activation of sensory terminals. The expression of multiple nAChR subunits offers the potential of selectively addressing functional aspects and/or sensory neuron subpopulations.

Muscarinic acetylcholine receptor subtypes expressed by mouse bladder afferent neurons.

Cell bodies of afferent neurons located in lumbosacral dorsal root ganglia (DRG) provide Adelta- and C-fibres to the urinary bladder, reporting bladder wall tension, volume and noxious stimuli. Recent studies suggested an involvement of muscarinic acetylcholine receptors (mAChRs) not only in detrusor contractility but also in modulating afferent function, and this has been linked to the beneficial effects of muscarinic antagonists in the treatment of overactive bladder. Here, we aimed to determine the inventory of mAChR subtypes expressed by bladder afferent neurons in the mouse. Bladder afferent neurons were identified by retrograde neuronal tracing using Fast Blue (FB) or 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorhydrate (DiI) injection into the detrusor muscle. DRG L6-S1 were recognized as the major location of bladder afferent perikarya with an additional smaller peak at L1/L2. Retrogradely labelled bladder afferents located in DRG L4-S2 were subjected to immunohistochemistry or to laser-assisted microdissection with subsequent RT-PCR to study expression of mAChRs subtypes M1R-M5R. Immunolabelling for mAChR subtype M2R, validated on DRG from M2R gene-deficient mice, demonstrated this subtype on 35% of FB-labelled bladder afferents. RT-PCR demonstrated expression of subtypes M2R, M3R and M4R, but not of M1R and M5R, in pooled samples (30 section profiles each) of laser microdissected DiI-labelled bladder afferent cell bodies. In conclusion, bladder afferent neurons express different subtypes of mAChRs (M2R, M3R and M4R). Thus, processing of sensory information from the bladder appears to be under direct cholinergic control.

Loss of serotonin oxidation as a component of the altered substrate specificity in the Y444F mutant of recombinant human liver MAO A.

To investigate the roles of tyrosyl residues located near the covalent 8alpha-S-cysteinyl FAD in monoamine oxidase A (MAO A) and to test the suggestion that MAO A and plant polyamine oxidase may have structural homology, tyrosyl to phenylalanyl mutants of MAO A at positions 377, 402, 407, 410, 419, and 444 were constructed and expressed in Saccharomyces cerevisiae. All mutant enzymes were expressed and exhibited lower specific activities as compared to WT MAO A using kynuramine as substrate. The lowest specific activities in this assay are exhibited by the Y407F and Y444F mutant enzymes. On purification and further characterization, these two mutants were found to each contain covalent FAD. Both mutant enzymes are irreversibly inhibited by the MAO A inhibitor clorgyline and exhibit binding stoichiometries of 0.54 (Y407F) and 0.95 (Y444F) as compared to 1.05 for WT MAO A. Y444F MAO A oxidizes kynuramine with a k(cat) <2% of WT enzyme and is greater than 100-fold slower in catalyzing the oxidation of phenylethylamine or of serotonin. In contrast, Y444F MAO A oxidizes p-CF(3)-benzylamine at a rate 25% that of WT enzyme. Steady state and reductive half-reaction stopped-flow data using a series of para-substituted benzylamine analogues show Y444F MAO A exhibits quantitative structure activity relationships (QSAR) properties on analogue binding and rates of substrate oxidation very similar to that exhibited by the WT enzyme (Miller and Edmondson (1999) Biochemistry 38, 13670): log K(d) = -(0.37 +/- ()()0.07)V(W)(x0.1) - 4.5 +/- 0.1; log k(red) = +(2.43 +/- 0.19)sigma + 0.17 +/- 0.05. The Y444F MAO A mutant also exhibits similar QSAR properties on the binding of phenylalkyl side chain amine analogues as WT enzyme: log K(i) = (4.37 +/- 0.51)E(S) + 1.21 +/- 0.77. These data show that mutation of Y444F in MAO A results in a mutant that has lost its ability to efficiently oxidize serotonin (its physiological substrate) but, however, exhibits unaltered quantitative structure-activity parameters in the binding and rate of benzylamine analogues. The mechanism of C-H abstraction is therefore unaltered. The suggestion that polyamine oxidase and monoamine oxidase may have structural homology appears to be valid as regards Y444 in MAO A and Y439 in plant polyamine oxidase.

Structure-activity relations in the oxidation of phenethylamine analogues by recombinant human liver monoamine oxidase A.

The interaction of recombinant human liver monoamine oxidase A (MAO A) with a series of phenethylamine substrate analogues has been investigated by steady-state and stopped-flow kinetic techniques. Substrate analogues with para substituents exhibit large deuterium kinetic isotope effect on k(cat), on k(cat)/K(m), and on the limiting rate of enzyme reduction in reductive half-reaction experiments. These kinetic isotope effect values range from 5 to 10 with the exception of tyramine, which exhibited smaller steady-state isotope effects (2.3-3.5) than that observed on the rate of flavin reduction (6.9). The stopped-flow data show that imine release from the reduced enzyme is slower than the rate of catalytic turnover. Phenethylamine oxidation by MAO A can be described as the C-H bond cleavage step being rate limiting in catalysis and with oxygen reacting with the reduced enzyme-imine complex. In the case of tyramine, the product release from the oxidized enzyme-imine complex contributes to the rate limitation in catalysis. The binding affinities of a series of para-substituted phenethylamine analogues to MAO A show an increase in affinity of the deprotonated amine with increasing van der Waals volume of the substituent. The limiting rate of enzyme reduction decreases with increasing van der Waals volume of the substituent in a linear manner with no observable electronic contribution as observed previously with benzylamine reduction of MAO A [Miller, J. R., and Edmondson, D. E. (1999) Biochemistry 38, 13670-13683]. Examination of side chain analogues of phenethylamine show 3-phenylpropylamine to be oxidized 2.5-fold more slowly and bound 75-fold more tightly than phenethylamine. 4-Phenylbutylamine is not a substrate for MAO A but is a good competitive inhibitor with a K(i) value of 31 +/- 5 microM. Analysis of the effect of alkyl side chain alterations on binding affinities of a series of arylalkylamine analogues taken from this study and from the literature show a linear correlation with the Taft steric value (E(s)) of the side chain. These results suggest that the binding site for the aryl ring is identical for phenethylamine and for benzylamine analogues and that steric interactions of the alkyl side chain with the enzyme strongly contribute to the binding affinities of a series of reversible inhibitors of MAO A.

Influence of FAD structure on its binding and activity with the C406A mutant of recombinant human liver monoamine oxidase A.

The FAD binding site of human liver monoamine oxidase A (MAO A) has been investigated by mutagenesis of the amino acid site of covalent FAD attachment (Cys-406) to an alanyl residue. Expression of the C406A mutant in Saccharomyces cerevisiae results in the formation of an active enzyme, as found previously with the rat liver enzyme. The activity of this mutant enzyme is labile to solubilization, thus requiring all experiments to be done with membrane preparations. C406A MAO A was expressed in a rib 5(-) strain of S. cerevisiae in the presence of 16 different riboflavin analogues. Inactive apoC406A MAO A is formed by induction of the enzyme in the absence of riboflavin. FAD but not FMN or riboflavin restores catalytic activity with an apparent K(d) of 62 +/- 5 nm. The results from both in vivo and in vitro reconstitution experiments show increased activity levels (up to approximately 7-fold higher) with those analogues exhibiting higher oxidation-reduction potentials than normal flavin and decreased activity levels with analogues exhibiting lower potentials. Analogues with substituents on the pyrimidine ring bind to C406A MAO A more weakly than normal FAD, suggesting specific interactions with the N(3) and N(1) positions. Analogues with substituents in the 7 and 8 positions bind to C406A MAO A with affinities comparable with that of normal FAD. These results are discussed in regard to functional significance of 8alpha-covalent binding of flavins to proteins.