The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. In order to observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.

A new fluorogenic substrate was developed for 3alpha-hydroxysteroid dehydrogenases (3alpha-HSD), including the human enzymes implicated in important physiological functions (androgen deactivation, neurosteroid activation). While ketone 5 is nonfluorescent, the corresponding alcohol exhibits high fluorescence with emission maximum at 510 nm, thus constituting a redox optical switch. This study began with a chemical concept of a ketone-alcohol optical switch which guided the synthesis of a focused array of compounds. Subsequently, seven compounds were selected (1-7) on the basis of their optical and chemical (stability) properties and were submitted to a screen against a panel of dehydrogenase enzymes. Probe 5 was found to be highly selective for bacterial, rat, and human 3alpha-HSD enzymes. The kinetic parameters were obtained for human 3alpha-HSD enzyme (type 2 isozyme, AKR 1C3; Km = 2.5 muM, kcat = 8.2 min-1). Remarkably, comparison to 5alpha-dihydrotestosterone (5alpha-DHT, Km = 26 muM, kcat = 0.25 min-1, Figure 4), a likely physiological substrate in prostate, revealed that synthetic probe 5 is in fact a far better substrate for this enzyme. Structure 5 represents an exciting lead for the development of a redox imaging probe.

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The current arsenal of tools and methods for the continuous monitoring and imaging of redox metabolic pathways in the context of intact cells is limited. Fluorogenic substrates allow for direct measurement of enzyme activity in situ; however, in contrast to proteases and exo-glycosidases, there are no simple guidelines for the design of selective probes for redox metabolic enzymes. Here, we introduce redox probe 1 and demonstrate its high selectivity in living cells for human hydroxysteroid dehydrogenases (HSDs) of the aldo-keto reductase (AKR) superfamily. AKR1C isoforms perform multiple functions among which the metabolism of potent steroid hormones is well documented. Moreover, expression of these enzymes is responsive to cellular stress and pathogenesis, including cancer. Our probe design is based on redox-sensitive optical switches, which couple a ketone-alcohol redox event to a profound change in fluorescence. The high selectivity of phenyl ketone 1 for AKR1C2 over the many endogenous reductases present in mammalian cells was established by a quantitative comparison of the metabolic rates between null control cells (COS-1) and AKR1C2-transfected cells. Phenyl ketone 1 is a cell-permeable fluorogenic probe that permits a direct, real-time, and operationally simple readout of AKR1C2 enzyme activity in intact mammalian cells. Furthermore, it was demonstrated that probe 1 enables the quantitative examination of physiological substrate 5␣-dihydrotestosterone (''dark substrate'') in situ by means of a two-substrate competitive assay. Similarly, inhibitor potency of physiological (ursodeoxycholate) and synthetic inhibitors (flufenamic acid, ibuprofen, and naproxen) was also readily evaluated.aldo-keto reductases ͉ fluorescent probes ͉ hydroxysteroid dehydrogenase ͉ metabolic reporters

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