Population fluctuations of light-attracted chrysomelid beetles in relation to supra-annual environmental changes in a Bornean rainforest.

In Southeast Asian tropical rainforests, two events, severe droughts associated with the El Niño-Southern Oscillation and general flowering, a type of community-wide mass flowering, occur at irregular, supra-annual intervals. The relationship between these two supra-annual events and patterns of insect population fluctuations has yet to be clearly elucidated. Leaf beetles (Chrysomelidae) are major herbivores and flower-visitors of canopy trees, affecting their growth and reproduction and, in turn, affected by tree phenology; but their population fluctuations in the Southeast Asian tropics have not been extensively investigated. We examined population fluctuation patterns of the 34 most dominant chrysomelid species in relation to the two supra-annual events by conducting monthly light-trapping over seven years in a lowland dipterocarp forest in Borneo. Our results showed large community variation in population fluctuation patterns and a supra-annual (between-year) variation in abundance for most of the dominant chrysomelids that was significantly larger than the annual (within-year) variation. Specifically, in response to a severe drought in 1998, chrysomelid species exhibited different population responses. These results show that population fluctuations of individual species, rather than the entire assemblage, must be analyzed to determine the effects of changes in environmental conditions on the structure of insect assemblages in the tropics, especially in regions where supra-annual environmental changes are relatively more important than seasonal changes.

relationship between the binary kernels of visually evoked potentials and the visual responses on the magnocellular and parvocellular pathways.

OBJECTIVES: To investigate the nonlinear characteristics of visual evoked potentials (VEPs), and their correlation with the visual responses on parvocellular and magnocellular pathways. First and second-order kernels of the VEPs elicited by several checkerboard patterns were estimated, and their relations to the visual pathway responses were investigated. METHODS: VEPs elicited by checkerboard pattern (0.5, 1.0, 2.0 and 4.0 c/d) alternating based on pseudorandom binary sequence were measured, and their binary kernels were calculated. First and second-order binary kernels were compared with amplitudes of the steady-state VEPs (S-VEPs) to pattern reversal stimulation with a constant temporal frequency (4, 8, 12, 16, and 32 Hz). RESULTS: Positive peak latencies at 150 ms (P150) of second-order first and second slices were correlated with S-VEP amplitude for higher temporal frequencies, indicating that the first and second slices reflect the response of the magnocellular. However, for second and third slices, their amplitudes were partially correlated with 4-16 Hz S-VEP, and this indicated that the second slice contains both magno- and parvocellular pathway responses. P150 latencies of third slices were correlated with S-VEP for lower temporal frequencies, indicating that third slice reflects the response of the parvocellular pathway. CONCLUSIONS: The lower slices of second-order binary kernels reflect the response of the magnocellular pathway and the higher slices reflect those on the parvocellular pathway in the human visual system of VEPs.

Lysophosphatidic acid positively regulates the fluid flow-induced local Ca(2+) influx in bovine aortic endothelial cells.

Using real-time confocal microscopy, we have demonstrated that lysophosphatidic acid (LPA), a bioactive phospholipid existing in plasma, positively regulates fluid flow-induced [Ca(2+)](i) response in fluo 4-loaded, cultured, bovine aortic endothelial cells. The initial increase in [Ca(2+)](i) was localized to a circular area with a diameter of <4 microm and spread concentrically, resulting in a mean global increase in [Ca(2+)](i). The local increase often occurred in a stepwise manner or repetitively during constant flow. The percentage of cells that responded and the averaged level of increase in [Ca(2+)](i) were dependent on both the concentration of LPA (0.1 to 10 micromol/L) and the flow rate (25 to 250 mm/s). The response was inhibited by removing extracellular Ca(2+) or by the application of Gd(3+), an inhibitor of mechanosensitive (MS) channels, but not by thapsigargin, an inhibitor of the endoplasmic reticular Ca(2+)-ATPASE: It was also inhibited by 8-bromo-cGMP, and the inhibition was completely reversed by KT5823, an inhibitor of protein kinase G (PKG). These results suggest that the [Ca(2+)](i) response arises from Ca(2+) influx through Gd(3+)-sensitive MS channels, which are negatively regulated by the activation of PKG. The spatiotemporal properties of the [Ca(2+)](i) response were completely different from those of a Ca(2+) wave induced by ATP, a Ca(2+)-mobilizing agonist. Therefore, we called the phenomenon Ca(2+) spots. We conclude that LPA positively regulates fluid flow-induced local and oscillatory [Ca(2+)](i) increase, ie, the Ca(2+) spots, in endothelial cells via the activation of elementary Ca(2+) influx through PKG-regulating MS channels. This indicates an important role for LPA as an endogenous factor in fluid flow-induced endothelial function.

Alternations of diacylglycerol kinase in streptozotocin-induced diabetic rats.

Dysfunction of organs has been reported in diabetic rats, suggesting an association with changes in intracellular signal transduction pathways including phosphatidylinositol (PI) turnover. Diacylglycerol (DG) kinase catalyses the phosphorylation of DG, which is considered to play a major physiological role in the metabolism of the intracellular messenger DG. However, no relation between DG kinase activity and any disease in mammalian tissue has been reported to date. In the present study, we investigated whether the changes in DG kinase activity are related to diabetes. Basal resting level of DG kinase activity changed in tissue isolated from diabetic rats. Decreases in resting activity detected in aorta and kidney and agonist-induced responses differed between these tissues. Submaximal increases in basal activity also were detected in vas deferens and hepatocytes. These changes in DG kinase activity resemble the functional changes associated with complications of diabetes, suggesting that changes in PI turnover followed by DG kinase activity are a key element in the complications. It is the first study about the changes in DG kinase activity in mammalian disease.

Lysophosphatidic acid sensitises Ca2+ influx through mechanosensitive ion channels in cultured lens epithelial cells.

We investigated the effect of lysophosphatidic acid (LPA), a bioactive phospholipid, on the response in cytosolic free Ca2+ concentration ([Ca2+]i) to mechanical stress in cultured bovine lens epithelial cells. Spritzing of bath solution onto cells as mechanical stress caused marked increase in [Ca2+]i in the presence of LPA and this increase was concentration-dependent (1-10 microM), whereas neither addition of LPA alone nor the mechanical stress in the absence of LPA affected [Ca2+]i. The mechanical stress-induced increase in [Ca2+]i in the presence of LPA was inhibited by removing extracellular Ca2+ or by addition of Gd3+, a blocker of mechanosensitive cation channels, but not by nicardipine, thapsigargin, an inhibitor of endoplasmic reticulum-ATPase pump, or U73122, a phospholipase C inhibitor. These results show that LPA sensitises Ca2+ influx through cation-selective mechanosensitive channels, but does not sensitise Ca2+ release from intracellular stores, triggered by changes in mechanical stress. On the other hand, phosphatidic acid had less of a sensitising effect than LPA, and neither lysophosphatidylcholine nor chlorpromazine had any effect. Also Ca2+ mobilising agonists, ATP, histamine and carbachol, did not sensitise Ca2+ response to the mechanical stress. These results show that LPA sensitises mechanoreceptor-linked response in lens epithelial cells, suggesting that it plays a role in the development of cataracts due to increases in [Ca2+]i induced by mechanical stress.

Hepatocyte growth factor-induced calcium waves in hepatocytes as revealed with rapid scanning confocal microscopy.

Cytosolic Ca2+ transients induced by hepatocyte growth factor (HGF) were imaged in primary cultured rat hepatocytes using newly developed rapid scanning confocal microscopes and indo-1. HGF (40 ng/ml) increased cytosolic free Ca2+ concentration ([Ca2+]i) in about 60% of hepatocytes, in 45% of which the increases were oscillatory. In each of the oscillatory hepatocytes, the repetitive increases in [Ca2+]i originated from a specific same region adjacent to the cell membrane and propagated across the cell like waves. Phenylephrine (10 microM) also induced Ca2+ waves. The locus where HGF-induced Ca2+ waves and phenylephrine-induced Ca2+ waves were originated was the same, and there was a correlation in the peak height between HGF-induced Ca2+ waves and phenylephrine-induced Ca2+ waves in each cell, although the mechanisms of inositol 1,4,5-trisphosphate (ins(1,4,5)P3) formation induced by HGF should be different from those by phenylephrine. On the other hand, there was no correlation between sensitivity of each cell to HGF and that to phenylephrine which were measured as latent periods prior to Ca2+ rises after an addition of the agonists. These results suggested the following: the spatial patterns of Ca2+ waves were decided by a common mechanism, probably not the propagation of ins(1,4,5)P3 but the distribution of ins(1,4,5)P3-sensitive Ca2+ pools; sensitivities of each cell to the agonists did not mainly depend on the common mechanism.

Effect of DFP on loading of fura 2/AM and quin 2/AM into single smooth muscle cells prepared from guinea pig taenia coli.

The effect of diisopropyl fluorophosphate (DFP), a potent cholinesterase (ChE) inhibitor, on loading quin 2 acetoxymethyl ester (quin 2/AM) and fura 2/AM into smooth muscle cells isolated from guinea pig taenia coli was investigated spectrofluorometrically. The presence of DFP during the loading permitted the incorporation of quin 2 into the cells, so that it became possible to measure intracellular Ca2+ concentrations using the ester of this dye. Also, DFP significantly enhanced the incorporation of fura 2 into the cells. These results indicate that loading of quin 2/AM and fura 2/AM into the smooth muscle cells may depend on the suppression of ChE or various serine protease activities outside cells.

[3H]9-Methyl-7-bromoeudistomin D, a caffeine-like powerful Ca2+ releaser, binds to caffeine-binding sites distinct from the ryanodine receptors in brain microsomes.

[3H]9-Methyl-7-bromoeudistomin D ([3H]MBED), the most powerful Ca2+ releaser from sarcoplasmic reticulum, specifically bound to the brain microsomes. Caffeine competitively inhibited [3H]MBED binding. [3H]MBED binding was markedly blocked by procaine, whereas that was enhanced by adenosine-5'-(beta,gamma-methylene)triphosphate. The Bmax value was 170 times more than that of [3H]ryanodine binding. The profile of sucrose-density gradient centrifugation of solubilized microsomes indicated that [3H]MBED binding protein was different from [3H]ryanodine binding protein. These results suggest that there are MBED/caffeine-binding sites in brain that are distinct from the ryanodine receptor and that MBED becomes an essential molecular probe for characterizing caffeine-binding protein in the central nervous system.

Evaluation of neonatal intracranial hemorrhage by computerized tomography.

Computerized tomographic (CT) brain scan was performed on 28 infants with unexplained cardiorespiratory and neurologic deterioration and bloody lumbar cerebrospinal spinal fluid. Fourteen of 20 with intraventricular hemorrhage (IVH) died; the six infants with lesser degrees of IVH survived. Significant subarachnoid hemorrhage (SAH) was demonstrable in three infants and three had negative scans despite bloody CSF. We have found that CT scans provide useful information about the size and extent of neonatal IVH and distinguished it from SAH. It also confirms the diagnosis of post-hemorrhagic hydrocephalus in these infants. Continued use of the CT scan will help us to understand the natural history and the effects of neonatal intracranial hemorrhage among the survivors of intensive care.

Visual temporal frequency characteristics determined by pseudorandom stimuli.

PURPOSE: To investigate whether a rapid and practical determination of the temporal frequency characteristic (TFC) of the visual system can be obtained by using the visually evoked potentials (VEPs) elicited by pseudorandom binary sequence (PRBS) stimulation. METHODS: VEPs were recorded from eight volunteers. For the conventional steady state VEPs (S-VEP), the eye was stimulated with five stimulus frequencies. To acquire the PRBS-VEPs, the eye was stimulated with a PRBS stimulus for 40 seconds. The TFC for the S-VEP was calculated from the root mean squared amplitude for each frequency using Fourier transform. For the PRBS stimulus, a cross-correlation function between PRBS (x[t]) and PRBS-VEP (y[t]) was calculated to obtain the TFC. RESULTS: The TFCs obtained by the PRBS and S-VEP methods were highly correlated (P < 0.05), and the TFC curves resembled those in the literature. Most important, the data necessary to determine the TFCs using the PRBS stimulus could be obtained in 4 minutes, whereas that for the S-VEP required 60 minutes for the two eyes. CONCLUSIONS: The high correlation between the TFCs obtained by the two methods indicated that the PRBS technique gives a good measure of the TFC of the human visual system. The significantly shorter time required for this method demonstrated that it is a practical method for determining the linear (and nonlinear) property of the visual system and that it may be useful in clinical applications.

Reduction by NG-nitro-L-arginine of H2O2-induced endothelial cell injury.

1. The effects of three analogues of NG-nitro-L-arginine (L-NOARG) and NG-monomethyl-L-arginine (L-NMMA), inhibitors of nitric oxide (NO) synthase, on hydrogen peroxide (H2O2)-induced endothelial cell injury were studied. 2. Endothelial cell injury was assessed by measuring the release of intracellular lactate dehydrogenase (LDH) and 51Cr. 3. Addition of H2O2 (250-1,000 microM) to endothelial cells induced the release of LDH dose-dependently. The release of LDH was reduced by pretreatment with NG-nitro-L-arginine methyl ester (L-NAME, 10(-4)-4 x 10(-3) M), L-NOARG (10(-4)-4 x 10(-3) M) and NG-nitro-L-arginine benzyl ester (L-NABE, 10(-4)-4 x 10(-3) M), inhibitors of NO synthase. 4. L-NOARG analogues also reduced H2O2-induced 51Cr release from endothelial cells, while L-NMMA had no effect. 5. The protective effect of L-NAME was not reversed by addition of L-arginine (L-Arg, 1-10 mM). 6. Both L-NAME and L-NMMA completely inhibited L-Arg metabolism to L-citrulline coupled with NO synthesis. 7. These findings suggest that L-NOARG analogues but not L-NMMA reduced H2O2-induced endothelial cell injury, and that these effects may not be related to inhibition of NO production.

The mode of inotropic action of ciguatoxin on guinea-pig cardiac muscle.

1. Ciguatoxin (CTX) caused a dose-dependent increase in the contractile force of the guinea-pig isolated left atria at concentrations ranging from 0.1 to 10 ng ml-1 with the ED50 value of 0.5 ng ml-1. 2. In the atria, tetrodotoxin (5 x 10(-7) M) inhibited markedly the inotropic action of CTX. The inotropic effect of CTX at low concentrations was abolished by practolol (10(-5) M) and reserpine (2 mg kg-1 daily, for 3 days), whereas that of CTX at high concentrations was partially inhibited by both drugs. 3. In single atrial cells, CTX (3 ng ml-1) produced a marked increase in the amplitude of longitudinal contractions. 4. CTX (3 ng ml-1) caused marked prolongation in the falling phase of action potentials of atrial strips without affecting the maximum rate of rise of action potentials and membrane resting potentials. The effect of CTX on action potentials was abolished by tetrodotoxin (10(-6) M). 5. The whole-cell patch-clamp experiments on myocytes revealed that CTX (20 ng ml-1) shifted the current-voltage curve of Na inward currents by 40 mV in the negative direction. CTX caused a small sustained Na inward current even at resting membrane potentials. 6. These results suggest that the inotropic action of lower concentrations of CTX is primarily due to an indirect action via noradrenaline release, whereas that of higher concentrations is caused not only by an indirect action but also by a direct action on voltage-dependent Na channels of cardiac muscle. It is also suggested that CTX activates cardiac muscle Na channels by modifying the voltage-dependence of channel activation to increase Na inward currents, thus producing cardiotonic actions.

Activation of diacylglycerol kinase by carbachol in guinea pig taenia coli.

Changes in diacylglycerol kinase (DG kinase) activity in carbachol (CCh)-stimulated guinea pig taenia coli were investigated. In a mixed micellar assay system, added 1,2-dioctanoyl-sn-glycerol (diC8) and endogenous DG were competitively bound to common DG kinase isozymes from guinea pig taenia coli and phosphorylated, suggesting that diC8 is useful as a probe of agonist effects on DG kinase activity. In phosphorus-32 ([32P]Pi)- and diC8-prelabeled guinea pig taenia coli, diC8 was phosphorylated by DG kinase to [32P]dioctanoyl-phosphatidic acid ([32P]diC8-PA). CCh increased the accumulation of both [32P]diC8-PA and endogenous [32P]phosphatidic acid ([32P]PA) in a time- and dose-dependent manner (0.1-100 microM CCh). CCh-induced increases in [32P]diC8-PA and [32P]PA were inhibited by 1 microM atropine and 3 microM DG kinase inhibitor (R59022). These findings indicated the activation of DG kinase by muscarinic receptor stimulation in guinea pig taenia coli. Therefore, DG kinase activation may play an important role in CCh-induced PA formation. CCh-induced [32P]diC8-PA and [32P]PA accumulation was dependent on intracellular calcium concentrations. However, a KCl-induced increase in intracellular calcium, without receptor stimulation, was ineffective. Moreover, treatment with phorbol ester also increased accumulation of both PA species in KCl-treated tissues. These findings suggest that muscarinic receptor mediated activation of DG kinase may require both an increase in intracellular calcium and PKC activation in guinea pig taenia coli.

Receptor-mediated diacylglycerol kinase translocation dependent on both transient increase in the intracellular calcium concentration and modification by protein kinase C.

Diacylglycerol kinase (DG kinase) is activated by various stimuli in many types of cells. We reported earlier that carbachol (CCh) induced DG kinase translocation from the cytosolic fraction to the membrane fraction in guinea pig taenia coli (Biochem. Pharmacol., 50: 591-599, 1995). In this study, the regulation mechanisms of DG kinase translocation are reported, based on the following findings: 1) CCh sustained an increase in DG kinase in the membrane fraction and a decrease in the cytosolic fraction; 2) blocking calcium influx by removing extracellular calcium did not affect the CCh-induced sustained DG kinase translocation; 3) exposing purified protein kinase C (PKC) to DG kinase increased DG kinase affinity to octylglycoside micelles only with the enzyme extracted from the cytosolic fraction; and 4) CCh-induced DG kinase translocation was reversed by removing CCh, and the serine/threonine phosphatase inhibitor, okadaic acid, blocked the reversal of the translocation. These results suggest that CCh-induced DG kinase translocation is promoted by both a transient increase in intracellular calcium, which may be released from the intracellular store, and by DG kinase phosphorylation by PKC.

Protein kinase C is involved in translocation of diacyglycerol kinase induced by carbachol in guinea pig taenia coli.

The regulatory mechanisms of diacylglycerol (DG) kinase activity were studied in guinea pig taenia coli. In an octylglycoside mixed micellar assay system, DG kinase activities were distributed in both membrane and cytosolic fractions. Treatment of the tissue with carbachol (CCh) increased the activity in the membrane fraction and decreased the cytosolic fraction without affecting total DG kinase activity. The Km value of DG kinase in the membrane fraction was unchanged by treatment with CCh, although Vmax was increased. These findings suggest that DG kinase may be translocated from the cytosol to the membrane by CCh-stimulation. Increase in DG content by treatment of tissue with a cell-permeable species of DG, dioctanoyl-sn-glycerol, did not induce DG kinase translocation. Each treatment with protein kinase C (PKC) inhibitor and PKC-desensitization blocked CCh-induced DG kinase translocation; and phorbol ester induced the translocation only in intracellular calcium-accumulated tissues. Considering these results, CCh-induced DG kinase activation appears to involve DG kinase translocation from the cytosol to the membrane in association with both PKC and intracellular calcium concentration rather than cellular DG content.

Reduction in the numbers of muscarinic receptors by an endogenous protein.

The soluble fraction from the ileal longitudinal muscle of guinea pigs was examined for the presence of an endogenous modulator of muscarinic receptors. In the presence of the soluble fraction, the binding of [3H]quinuclidinyl benzilate to the membranes from the tissue was inhibited in a concentration-dependent manner. The inhibitory activity in the soluble fraction was heat stable, but was inactivated by trypsin treatment. Several protease inhibitors had no effect on the inhibitory activity. These results suggest the existence of an endogenous protein that inhibits the binding of the muscarinic ligand to the receptor. Ultrafiltration demonstrated that the protein factor had a molecular mass of more than 10,000 Da. Saturation binding and dissociation kinetic experiments indicate neither a competitive nor allosteric mode of inhibitory action and suggest that an irreversible block or internalization of muscarinic receptors is induced by the endogenous protein.

Stimulation of nitric oxide synthase during oxidative endothelial cell injury.

The purpose of this study was to determine changes in nitric oxide synthase (NOS) activity during the process of lethal oxidative cell injury following H2O2 treatment of endothelial cells. NOS activity was determined by measuring the conversion of [3H]arginine ([3H]Arg) to [3H]citrulline ([3H]Cit). Cell death was assessed by measuring the release of intracellular lactate dehydrogenase (LDH). Moreover, cell death and changes in cytosolic free Ca2+ (Ca(i)2+) were measured simultaneously using a confocal laser scanning system, and propidium iodide and fluo-3 as fluorescent indicators, respectively. Treatment with H2O2 (125-1000 microM) concentration dependently increased L-Cit formation from L-Arg, and a peak was obtained at 90 min after the addition of 500 or 1000 microM H2O2. The H2O2-induced increase in L-Cit formation was blocked completely by N(G)-nitro-L-arginine (L-NNA) or N(G)-methyl-L-arginine (L-NMA), both inhibitors of NOS. LDH release from endothelial cells was evoked from 120 min after the addition of H2O2 (125-1000 microM) in a concentration-dependent manner. Moreover, H2O2 increased Ca(i)2+ before cell death, and addition of Ca2+ chelator inhibited both the increase in L-Cit formation and LDH release by H2O2. The H2O2-induced LDH release was reduced by L-NNA, but not by L-NMA. These results suggest that H2O2 treatment of endothelial cells increases Ca(i)2+ before cell death, and stimulates NOS activity. The activation of NOS may be involved in oxidative endothelial cell death.

Effect of tributyltin chloride on the release of calcium ion from intracellular calcium stores in rat hepatocytes.

The effects of tri-n-butyltin chloride (TBT), an environmental pollutant, on the release of Ca(2+) from intracellular stores were investigated in isolated rat hepatocytes. Isolated hepatocytes permeabilized with digitonin were suspended in solution, and the concentration of extracellular Ca(2+) was measured, using a fluorescent Ca(2+) dye, fura-2. In the solution containing permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min, the extracellular Ca(2+) concentration was high, but the inositol 1,4,5-trisphosphate (InsP(3))-induced increase in Ca(2+) concentration was suppressed, suggesting that the extracellular release of Ca(2+) in response to TBT treatment was from intracellular stores. Images of the Ca(2+) concentration in the intracellular stores of primary cultured hepatocytes loaded with fura-2 were obtained after digitonin-permeabilization, using digitalized fluorescence microscopy. The permeabilized hepatocytes that had been preincubated with 4.0 microM TBT for 30 min had a very low fura-2 fluorescence ratio (340/380 nm), suggesting that stored Ca(2+) was released. When the hepatocytes were treated with 4.0 microM TBT after digitonin-permeabilization, the decrease in the fura-2 fluorescence ratio was very small. However, when the permeabilized hepatocytes were incubated with 4.0 microM TBT and 2.0 microM NADPH, the decrease was enhanced, raising the possibility that TBT might be metabolized to the active form(s), thus releasing Ca(2+) from intracellular stores. When the hepatocytes were preincubated with 0.1 microM TBT for 30 min and then were permeabilized, the fura-2 fluorescence ratio was almost the same as that in the control permeabilized hepatocytes. However, the InsP(3)-induced decrease in the fluorescence ratio was suppressed significantly in the permeabilized hepatocytes. These results suggest that TBT released Ca(2+) from the intracellular stores at high concentrations, and suppressed the InsP(3)-induced Ca(2+) release at non-toxic low concentrations. It is probable that the latter effect was responsible for the previously reported suppression of Ca(2+) response induced by hormonal stimulations (Kawanish et al., Toxicol Appl Pharmacol 1999;155:54-61).

Extracranial optic nerve decompression for traumatic optic neuropathy.

We examined 14 patients with acute, unilateral optic nerve injury after blunt head trauma. In each patient the optic canal was decompressed through an ipsilateral external ethmoidectomy. The patients also received treatment with dexamethasone during the perioperative period. There was no morbidity or mortality. Eleven of the 14 patients improved, including 3 of the 5 who could not perceive light preoperatively. Transethmoid-sphenoid optic canal decompression is a safe and effective treatment for indirect optic nerve trauma.

Inhibitory effects of catecholamines and anti-oxidants on the fluorescence reaction of 4,5-diaminofluorescein, DAF-2, a novel indicator of nitric oxide.

4,5-Diaminofluorescein (DAF-2) is a newly developed indicator of nitric oxide (NO). Two amino groups of DAF-2 are oxidized by NO. We investigated the effects of reducers on the NO-induced oxidation of DAF-2. NOC-5 (0.1-10 microM), a NO-donor, concentration-dependently elicited fluorescence with 10 microM DAF-2. The rate of the fluorescence reaction was dependent on the width of the excitation band path. The presence of catecholamines (1 microM), but not tyrosine or phenylephrine, attenuated the fluorescence induced by NOC-5. Ascorbate and other reducers like dithiothreitol, 2-mercaptoethanol, or glutathione (all 1 mM) abolished the fluorescence. These results suggest that reducers attenuate the NO-induced fluorescence of DAF-2 mainly through an anti-oxidative action.