The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells

Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16-160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M(-1) cm(-1)). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca(2+) rapidly released Ca(2+) (rate of 20,000 s(-1)) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of approximately 0.6 GM and two-photon photolysis induced localized Ca(2+)-induced Ca(2+) release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.     

Bichromophoric fluorescent photolabile protecting group for alcohols and carboxylic acids

Novel bichromophoric fluorescent photolabile protecting group, (5-dansyloxy-3-hydroxynaphthalen-2-yl)methyl (DNS-NQMP), allows for the independent photochemical release and fluorescent imaging of caged substrates. Irradiation of DNS-NQMP-caged alcohols and carboxylic acids with 300 or 350 nm light results in fast (k(release) ~ 10(5) s(-1)), efficient (Φ = 0.2), and quantitative release of the substrates. This uncaging chemistry is compatible with aqueous media and DNS-NQMP-protected hydroxy compounds are hydrolytically stable at neutral pH. Upon excitation with 400 nm light, caged compounds show intense green emission (λ(max) = 559 nm) with 21% fluorescence quantum yield. Fluorescent readout conducted using 400 nm or longer wavelengths does not cause substrate release. The DNS-NQMP chromophore retains its fluorescent properties after photo-uncaging reaction.     

Photo-control of nitric oxide synthase activity using a caged isoform specific inhibitor

Nitric oxide (NO) plays a critical role in a number of physiological processes and is produced in mammalian cells by nitric oxide synthase (NOS) isozymes. Because of the diverse functions of NO, pharmaceutical interventions which seek to abrogate adverse effects of excess NOS activity must not interfere with the normal regulation of NO levels in the body. A method has been developed for the control of NOS enzyme activity using the localized photochemical release of a caged isoform-specific NOS inhibitor. The caged form of an iNOS inhibitor has been synthesized and tested for photosensitivity and potency. UV and multiphoton uncaging were verified using a hemoglobin-based assay. IC(50) values were determined for the inhibitor (70+/-11 nM), the caged inhibitor (1098+/-172 nM), the UV uncaged inhibitor (67+/-26 nM) and the multiphoton uncaged inhibitor (73+/-11 nM). UV irradiation of the caged inhibitor resulted in a 86% reduction in iNOS activity after 5 min. Multiphoton uncaging had an apparent first order time constant of 0.007+/-0.001 min(-1). A therapeutic range exists, with molar excess of inhibitor to enzyme from 3- to 7-fold, over which the full dynamic range of the inhibition can be exploited.     

Alignment and calibration of a focal neurotransmitter uncaging system

Photolysis of caged compounds is a powerful tool for studying subcellular physiological functions. Here we describe protocols for the alignment and calibration of a focal uncaging system. We also report procedures for convenient quantitative calibration of uncaging. Using these methods, we can achieve submicron lateral resolution of photolysis and probe biological function in spines, the smallest signaling compartments of neurons. Initially, the entire alignment procedure takes 4-6 h to perform; periodic fine-tuning of the system takes 1-2 h.     

Flash photolysis of caged nitric oxide inhibits proximal tubular fluid reabsorption in free-flow nephron

A nonobstructing optical method was developed to measure proximal tubular fluid reabsorption in rat nephron at 0.25 Hz. The effects of uncaging luminal nitric oxide (NO) on proximal tubular reabsorption were investigated with this method. Proximal fluid reabsorption rate was calculated as the difference of tubular flow measured simultaneously at two locations (0.8-1.8 mm apart) along a convoluted proximal tubule. Tubular flow was estimated on the basis of the propagating velocity of fluorescent dextran pulses in the lumen. Changes in local tubular flow induced by intratubular perfusion were detected simultaneously along the proximal tubule, indicating that local tubular flow can be monitored in multiple sites along a tubule. The estimated tubular reabsorption rate was 5.52 +/- 0.38 nl.min(-1).mm(-1) (n = 20). Flash photolysis of luminal caged NO (potassium nitrosylpentachlororuthenate) was induced with a 30-Hz UV nitrogen-pulsed laser. Release of NO from caged NO into the proximal tubule was confirmed by monitoring intracellular NO concentration using a cell-permeant NO-sensitive fluorescent dye (DAF-FM). Emission of DAF-FM was proportional to the number of laser pulses used for uncaging. Photolysis of luminal caged NO induced a dose-dependent inhibition of proximal tubular reabsorption without activating tubuloglomerular feedback, whereas uncaging of intracellular cGMP in the proximal tubule decreased tubular flow. Coupling of this novel method to measure reabsorption with photolysis of caged signaling molecules provides a new paradigm to study tubular reabsorption with ambient tubular flow.     

A protecting group for carboxylic acids that can be photolyzed by visible light

We report on a photolabile protecting (caging) group that is new for carboxylic acids. Unlike previously used caging groups for carboxylic acids, it can be photolyzed rapidly and efficiently in the visible wavelength region. The caging group 7-N,N-diethyl aminocoumarin (DECM) was used to cage the gamma-carboxyl group of glutamic acid, which is also a neurotransmitter. The caged compound has a major absorption band with a maximum at 390 nm (epsilon(390) = 13651 M(-)(1) cm(-)(1)). Experiments are performed at 400 nm (epsilon(400) = 12232 M(-)(1) cm(-)(1)) and longer wavelengths. DECM-caged glutamate is water soluble and stable at pH 7.4 and 22 degrees C. It photolyzes rapidly in aqueous solution to release glutamic acid within 3 micros with a quantum yield of 0.11 +/- 0.008 in the visible region. In whole-cell current-recording experiments, using HEK-293 cells expressing glutamate receptors and visible light for photolysis, DECM-caged glutamate and its photolytic byproducts were found to be biologically inert. Neurotransmitter receptors that are activated by various carboxyl-group-containing compounds play a central role in signal transmission between approximately 10(12) neurons of the nervous system. Caged neurotransmitters have become an essential tool in transient kinetic investigations of the mechanism of action of neurotransmitter receptors. Previously uncaging the compounds suitable for transient kinetic investigations required ultraviolet light and expensive lasers, and, therefore, special precautions. The availability of caged neurotransmitters suitable for transient kinetic investigations that can be photolyzed by visible light allows the use of simple-to-use, readily available inexpensive light sources, thereby opening up this important field to an increasing number of investigators.     

Masked red-emitting carbopyronine dyes with photosensitive 2-diazo-1-indanone caging group

Caged near-IR emitting fluorescent dyes are in high demand in optical microscopy but up to now were unavailable. We discovered that the combination of a carbopyronine dye core and a photosensitive 2-diazo-1-indanone residue leads to masked near-IR emitting fluorescent dyes. Illumination of these caged dyes with either UV or visible light (λ < 420 nm) efficiently generates fluorescent compounds with absorption and emission at 635 nm and 660 nm, respectively. A high-yielding synthetic route with attractive possibilities for further dye design is described in detail. Good photostability, high contrast, and a large fluorescence quantum yield after uncaging are the most important features of the new compounds for non-invasive imaging in high-resolution optical microscopy. For use in immunolabelling the caged dyes were decorated with a (hydrophilic) linker and an (activated) carboxyl group.     

Photolysis of caged phosphatidic acid induces flagellar excision in Chlamydomonas

Phosphatidic (PtdOH) acid formation is recognized as an important step in numerous signaling pathways in both plants and mammals. To study the role of this lipid in signaling pathways, it is of major interest to be able to increase the amount of this lipid directly. Therefore, "caged" PtdOH was synthesized, which releases the biologically active PtdOH upon exposure to UV. Analysis of the product revealed that two 2-nitrophenylethyl (NPE) caging groups were coupled to the phosphate headgroup of PtdOH. To measure the quantum efficiency of uncaging, a fluorimetric assay, based on the notion that the NPE cage is an efficient quencher of pyrene fluorescence, was developed. Consequently, after NPE-caged PtdOH and (N-pyrene)-PtdEtn had been mixed in DOPC vesicles, the extent of photolysis of caged PtdOH can be quantified by monitoring the increase in pyrene fluorescence. Using this assay, a quantum yield of 9.6% was determined for the uncaging reaction. The swimming green alga Chlamydomonas moewusii deflagellates upon addition of PtdOH. This response was used to study the release of PtdOH in vivo. Algae incubated with caged PtdOH only arrested swimming after exposure to UV, indicative of PtdOH release. This effect was not observed in the absence of the caged compound or when a control caged compound (caged acetic acid) was added. Fluorescein diacetate staining was used to show that the cells remained viable after UV exposure. The anticipated effect of PtdOH release is confirmed by phase contrast images of UV-exposed algae showing excision of flagella. Together, these results show that caged PtdOH can be used to efficiently increase PtdOH levels, demonstrating that it is a promising precursor for studying PtdOH-dependent signaling.     

Rapid neurotransmitter uncaging in spatially defined patterns

Light-sensitive 'caged' molecules provide a means of rapidly and noninvasively manipulating biochemical signals with submicron spatial resolution. Here we describe a new optical system for rapid uncaging in arbitrary patterns to emulate complex neural activity. This system uses TeO(2) acousto-optical deflectors to steer an ultraviolet beam rapidly and can uncage at over 20,000 locations per second. The uncaging beam is projected into the focal plane of a two-photon microscope, allowing us to combine patterned uncaging with imaging and electrophysiology. By photolyzing caged neurotransmitter in brain slices we can generate precise, complex activity patterns for dendritic integration. The method can also be used to activate many presynaptic neurons at once. Patterned uncaging opens new vistas in the study of signal integration and plasticity in neuronal circuits and other biological systems.     

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.     

Design and synthesis of photolabile caged cytokinin

Cytokinins are phytohormones that regulate diverse developmental processes throughout the life of a plant. trans-Zeatin, kinetin, benzyladenine and dihydrozeatin are adenine-type cytokinins that are perceived by the AHK cytokinin receptors. Endogenous cytokinin levels are critical for regulating plant development. To manipulate intracellular cytokinin levels, caged cytokinins were designed on the basis of the crystal structure of the AHK4 cytokinin receptor. The caged cytokinin was photolyzed to release the cytokinin molecule inside the cells and induce cytokinin-responsive gene expression. The uncaging of intracellular caged cytokinins demonstrated that cytokinin-induced root growth inhibition can be manipulated with photo-irradiation. This caged cytokinin system could be a powerful tool for cytokinin biology.     

Cell membrane permeable esters of D-myo-inositol 1,4,5-trisphosphate

d-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3, or IP3) is a ubiquitous second messenger that regulates cytosolic Ca2+ activities ([Ca2+]i). To study this signaling branch in intact cells, we have synthesized a caged and cell permeable derivative of IP3, ci-IP3/PM, from myo-inositol in 9 steps. Ci-IP3/PM is a homologue of cm-IP3/PM, a caged and cell permeable IP3 ester developed earlier. In ci-IP3/PM, 2- and 3-hydroxyl groups of myo-inositiol are protected by an isopropylidene group; whereas in cm-IP3/PM, a methoxymethylene is used. Ci-IP3/PM can be loaded into cells non-invasively to high concentrations without activating IP3 receptors (IP3Rs). UV uncaging of loaded ci-IP3 released i-IP3, a potent agonist of IP3Rs, and evoked Ca2+ release from internal stores. Interestingly, elevations of [Ca2+]i by i-IP3 lasted longer than [Ca2+]i transients by m-IP3, the uncaging product of cm-IP3. To understand this difference, we measured the metabolic stability of i-IP3 and m-IP3. Like natural IP3 which is known to be rapidly metabolized in cells, m-IP3 could only be detected within several seconds after uncaging cm-IP3. In contrast, i-IP3 was metabolized at a much slower rate. By exploiting different metabolic rates of m-IP3 and i-IP3, we developed two procedures for activating IP3Rs in cells without UV uncaging. The first method involves photolyzing ci-IP3/PM in vitro to generate i-IP3/PM. Successive additions of low micromolar i-IP3/PM to NIH 3T3 cells caused graded Ca2+ releases, confirming that "quantal Ca2+ release" occurs in fully intact cells with normal ATP supplies and undisrupted endoplasmic reticulum. The second technique utilizes two photon uncaging. After locally illuminating cells loaded with cm-IP3 with femtosecond-pulsed near-infrared light (730 nm), we observed a burst of Ca2+ activity in the uncaging area. This local Ca2+ rise rapidly propagated across cells and could be repeated many times in different sub-cellular locations to produce artificial Ca2+ oscillations of defined amplitudes and frequencies. The complementary advantages of these IP3 prodrugs should provide new approaches for studying IP3-Ca2+ signaling in intact cell populations with high spatiotemporal resolutions.     

Preparation and photophysical properties of a caged kynurenine

We have prepared l-kyurenine 4-hydroxyphenacyl ester, a caged derivative of L-kynurenine. N(α)-tBOC-L-tryptophan was reacted with 4-hydroxyphenacyl bromide in DMF with K(2)CO(3) as the base to give the N(α)-tBOC 4-hydroxyphenacyl ester. The ester was then treated with O(3) in MeOH at -20°C, followed by trifluoroacetic acid in CH(2)Cl(2), then aqueous HCl to obtain the caged kynurenine as the dihydrochloride salt. The caged kynurenine is stable as a dry solid in the dark at -78°C, but in aqueous solutions in phosphate buffer at pH 7-8 hydrolyzes rapidly (t(1/2) ～5 min). Solutions in Tris at pH 7 are more stable (t(1/2) >30 min), and solutions in 1mM HCl are stable for several hours. As expected, the ester is cleaved in microseconds with laser pulses at 355 nm. The caged kynurenine may be useful for preparation of substrate complexes for crystallography or in biological studies on kynurenine.     

A Cell Permeable NPE Caged ADP-Ribose for Studying TRPM2

Transient potential receptor melastatin-2 (TRPM2) is a non-selective Ca²⁺-permeable cation channel of the TRPM channel subfamily and is mainly activated by intracellular adenosine diphosphate ribose (ADPR). Here we synthesized a 1-(2-nitrophenyl)ethyl caged ADPR (NPE-ADPR) and found that uncaging of NPE-ADPR efficiently stimulated Ca²⁺, Mg²⁺, and Zn²⁺ influx in a concentration-dependent manner in intact human Jurkat T-lymphocytes. The cation influx was inhibited by inhibitors or knockdown of TRPM2. Likewise, uncaging of NPE-ADPR markedly induced cation entry in HEK 293 cells that overexpress TRPM2. As expected, high temperature increased the ability of the photolyzed NPE-ADPR to induce cation entry, whereas acidic pH inhibited. Moreover, the absence of extracellular Ca²⁺ significantly inhibited Mg²⁺ and Zn²⁺ influx after uncaging NPE-ADPR. On the other hand, the absence of extracellular Na⁺ or Mg²⁺ had no effect on photolyzed NPE-ADPR induced Ca²⁺ entry. Taken together, our results indicated that NPE-ADPR is a cell permeable ADPR analogue that is useful for studying TRPM2-mediated cation entry in intact cells.     

Photocontrol of nitric oxide production in cell culture using a caged isoform selective inhibitor

Over the past decade, multiphoton microscopy has progressed from a photonic novelty to a technique whose application is currently experiencing exponential growth in the biological sciences. A novel application of this technology with significant therapeutic potential is the control of drug activity by multiphoton photolysis of caged therapeutics. As an initial case study, the potent isoform selective inhibitor N-(3-(aminomethyl)benzyl) acetamidine (1400W) of inducible nitric oxide synthase (iNOS) has been conjugated to a caging molecule 6-bromo-7-hydroxy-4-hydroxyquinoline-2-ylmethyl acetyl ester (Bhc). Here we present the first report of a bulk therapeutic effect, inhibition of nitric oxide production, in mammalian cell culture by multiphoton photolysis of a caged drug, Bhc-1400W. Mouse macrophage RAW 264.7 cells induced with bacterial lipopolysaccharides to express iNOS were used to assess the therapeutic value of the conjugated inhibitor. Both 1400W and Bhc-1400W are stable in metabolically active cells and an optimal time interval for the photorelease of the inhibitor was determined. The ratios of the IC(50) values of Bhc-1400W over 1400W calculated in the presence of iNOS enzyme and in RAW 264.7 cell culture are 19 and 100, respectively, indicating that a broad therapeutic range exists in cell culture. Multiphoton uncaging protocols and therapeutic doses of inhibitors were not cytotoxic. Photocontrol of LPS induced nitric oxide production was achieved in mammalian cell culture using a single laser focal volume. This technology has the potential to control active drug concentrations in vivo, a lack of which is one of the main problems currently associated with systemic drug administration.     

Kinetics and mechanism of DNA repair. Preparation, purification and some properties of caged dideoxynucleoside triphosphates.

Caged dideoxyribosylthymine triphosphate, dideoxyadenosine triphosphate and arabinosylcytosine triphosphate were prepared in high yield by reaction with 1-(2-nitrophenyl)diazoethane at pH 4 and room temperature for 24 h. Synthesis of caged alpha-32P-labelled dideoxyadenosine triphosphate (approx. 5000 Ci/mmol) in 85% yield was achieved by a modification of the method used for the synthesis of the unlabelled compounds. ATP was shown to be an excellent buffer in the synthesis of alpha-32P-labelled material, and in caged form to be an effective carrier in h.p.l.c. purification. Preparative h.p.l.c. was used to achieve purification of unlabelled caged compounds to greater than 98% purity and 32P-labelled material to 97% purity. Photolysis of unlabelled and 32P-labelled caged compounds by using XeF-excimer laser irradiation at 351 nm was characterized by using difference spectrophotometry and h.p.l.c. analysis. The stability of caged dideoxyadenosine [a-32P]triphosphate in the presence of cultured mammalian cells was evaluated; the adenosine derivative is essentially stable for 1 h.     

PKCalpha: a versatile key for decoding the cellular calcium toolkit

Conventional protein kinases C (cPKCs) play an essential role in signal transduction and are believed to integrate both global Ca(2+) transients and diacylglycerol signals. We provide evidence that PKCalpha is a ubiquitous readout sensor for the cellular Ca(2+) toolkit, including highly restricted elementary Ca(2+) release. Threshold stimulations of cells with Ca(2+)-mobilizing agonists resulted in PKCalpha translocation events with limited spatial spreads (<4 microm) comprising two groups of lifetimes; brief events (400-1,500 ms) exclusively mediated by Ca(2+)-C2 domain membrane interactions and long-lasting events (>4 s) resulting from longer DAG-C1a domain-mediated membrane interactions. Although upon uncaging NP-EGTA, which is a caged Ca(2+) compound, WT-PKCalpha displayed rapid membrane translocations within <250 ms, PKCalpha constructs with C2 domains mutated in their Ca(2+)-binding region lacked any Ca(2+)-dependent translocation. Flash photolysis of diazo-2, a photosensitive caged Ca(2+) buffer, revealed a biphasic membrane dissociation (slow and fast period) of WT-PKCalpha. The slow phase was absent in cells expressing PKCalpha-constructs containing mutated C1a-domains with largely reduced DAG binding. Thus, two groups of PKCalpha membrane interactions coexist; C2- and C1a-mediated interactions with different lifetimes but rapid interconversion. We conclude that PKCalpha can readout very fast and, spatially and temporally, very complex cellular Ca(2+) signals. Therefore, cPKCs are important transducers for the ubiquitous cellular Ca(2+) signaling toolkit.     

Localized Ca2+ uncaging reveals polarized distribution of Ca2+-sensitive Ca2+ release sites: mechanism of unidirectional Ca2+ waves

Ca2+-induced Ca2+ release (CICR) plays an important role in the generation of cytosolic Ca2+ signals in many cell types. However, it is inherently difficult to distinguish experimentally between the contributions of messenger-induced Ca2+ release and CICR. We have directly tested the CICR sensitivity of different regions of intact pancreatic acinar cells using local uncaging of caged Ca2+. In the apical region, local uncaging of Ca2+ was able to trigger a CICR wave, which propagated toward the base. CICR could not be triggered in the basal region, despite the known presence of ryanodine receptors. The triggering of CICR from the apical region was inhibited by a pharmacological block of ryanodine or inositol trisphosphate receptors, indicating that global signals require coordinated Ca2+ release. Subthreshold agonist stimulation increased the probability of triggering CICR by apical uncaging, and uncaging-induced CICR could activate long-lasting Ca2+ oscillations. However, with subthreshold stimulation, CICR could still not be initiated in the basal region. CICR is the major process responsible for global Ca2+ transients, and intracellular variations in sensitivity to CICR predetermine the activation pattern of Ca2+ waves.     

Kinetics of local helix formation in poly-L-glutamic acid studied by time-resolved photoacoustics: neutralization reactions of carboxylates in aqueous solutions and their relevance to the problem of protein folding

Photoactivatable caged protons have been used to trigger proton transfer reactions in aqueous solutions of acetate, glutamate, and poly-L-glutamic acid, and the volumetric and enthalpic changes have been detected and characterized by means of time-resolved photoacoustics. Neutralization of carboxylates in aqueous solutions invariably results in an expansion of the solution due to the disappearance of two charges and is accompanied by little enthalpic change. The reactions occur with thermally activated, apparent bimolecular rates on the order of 10(10) M(-1)s(-1). In the case of aqueous solutions of poly-L-glutamic acid at pH around the pK(a) of the coil-to-helix transition, diffusional binding of a proton by carboxylates is followed by a sequential reaction with rate 1.06 (+/- 0.05) x 10(7)s(-1). This step is not thermally activated in the temperature range we have investigated and is likely related to local formation of hydrogen bonds near the protonation site. This structural event may constitute a rate-limiting step in helix propagation.     

Simultaneous capacitance and amperometric measurements of exocytosis: a comparison.

We measured the exocytotic response induced by flash photolysis of caged compounds in isolated mast cells and chromaffin cells. Vesicle fusion was measured by monitoring the cell membrane capacitance. The release of vesicular contents was followed by amperometry. In response to a GTP gamma S stimulus we found that the time integral of the amperometric current could be superimposed on the capacitance trace. This shows that the integrated amperometric signal provides an alternative method of measuring the extent and kinetics of the secretory response. Very different results were obtained when photolysis of caged Ca2+ (DM-nitrophen) was used to stimulate secretion. In mast cells, there was an immediate, graded increase in membrane capacitance that was followed by step increases (indicative of granule fusion). During the initial phase of the capacitance increases, no release of oxidizable secretory products was detected. In chromaffin cells we also observed a considerable delay between increases in capacitance, triggered by uncaging Ca2+, and the release of oxidizable secretory products. Here we demonstrate that there can be large increases in the membrane capacitance of a secretory cell, triggered by flash photolysis of DM-nitrophen, which indicate events that are not due to the fusion of granules containing oxidizable substances. These results show that increases in capacitance that are not resolved as steps cannot be readily interpreted as secretory events unless they are confirmed independently.