Fernand Baguet

Antioxidative properties of natural coelenterazine and synthetic methyl coelenterazine in rat hepatocytes subjected to tert-butyl hydroperoxide-induced oxidative stress

Coelenterazine (CLZn; 3, 7-dihydro-2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)-8-benzylimidazolo++ +[1 ,2-a]pyrazin-3-one), the substrate for bioluminescence reactions in many marine animals, is endowed with high antioxidant properties. This work investigated the antioxidative properties of CLZn in primary cultures of rat hepatocytes subjected to the oxidant tert-butyl hydroperoxide (t-BHP). Micromolar concentrations of CLZn increased survival and decreased lipid peroxidation in rat hepatocytes subjected for 6 hr to 2.5 x 10(-4) M t-BHP. However, the extent of protection was limited by a strong toxicity of CLZn (IC(50) = 6.9 x 10(-5) M). The presence of t-BHP increased the cellular toxicity of CLZn. Methyl coelenterazine (CLZm, 3, 7-dihydro-2-methyl-6-(p-hydroxyphenyl)-8 benzylimidazolo[1, 2-a]pyrazin-3-one), a synthetic analogue of CLZn, demonstrated excellent antioxidant properties, even at very low (3 x 10(-6) M) concentrations and was not toxic throughout most of its effective concentration range. CLZm proved far more effective than reference antioxidants such as Trolox C(R), alpha-tocopherol, BHT, and probucol. The assay of thiobarbituric reactive substances (TBARS) associated with cells and in the culture medium indicated that 10(-5) M CLZm provided a total protection against t-BHP-induced lipid peroxidation. This coelenterazine analogue could be used as a model compound for investigating the action mechanism of imidazolopyrazinones in mammalian hepatocytes.

Kinetics of light emission and oxygen consumption by bioluminescent bacteria.

Oxygen plays a key role in bacterial bioluminescence. The simultaneous and continuous kinetics of oxygen consumption and light emission during a complete exhaustion of the exogenous oxygen present in a closed system has been investigated. The kinetics are performed with Vibrio fischeri, V. harveyi, and Photobacterium phosphoreum incubated on respiratory substrates chosen for their different reducing power. The general patterns of the luminescence time courses are different among species but not among substrates. During steady-state conditions, substrates, which are less reduced than glycerol, have, paradoxally, a better luminescence efficiency. Oxygen consumption by luciferase has been evaluated to be ≈17% of the total respiration. Luciferase is a regulatory enzyme presenting a positive cooperative effect with oxygen and its affinity for this final electron acceptor is about 4–5 times higher than the one of cytochrome oxidase. The apparent Michaelis constant for luciferase has been evaluated to be in the range of 20 to 65 nM O2. When O2 concentrations are as low as 10 nM, luminescence can still be detected; this means that above this concentration, strict anaerobiosis does not exist. By n-butyl malonate titration, it was clearly shown that electrons enter the luciferase pathway only when the cytochrome pathway is saturated. It is suggested that, in bioluminescent bacteria, luciferase acts as a free-energy dissipating valve when anabolic processes (biomass production) are impaired.

The control mechanism of relaxation in molluscan catch-muscle (ABRM)

Summary1.The relaxing effects of 5-Hydroxtryptamine (5-HT) have been tested on glycerol-extracted and chemically treated fibres (EDTA and Triton X-100 solutions) of ABRM in a low Ca2+ solution (10−9 M) on a high Ca2+ solution (10−6 M): at two ionic strengths (0.06 and 0.28) and two pHs (6.5 and 7.0). The resistance to stretch of the two muscle preparations has been studied in the low Ca2+ medium i.e. without contractile activity in presence and in absence of 5-HT. The presence of 5-HT reduces significantly the resistance to stretch of chemically treated fibres in conditions of ionic strength 0.28 and pH 7.0, but has no effect on glycerol extracted fibres.2.After the maximal tension has been developed in a high Ca2+ solution (10−6 M) the rate of relaxation of chemically treated fibres, induced by a low Ca2+ solution (10−9M) at ionic strength 0.28 and pH 7.0 is increased by the addition of 5-HT. No effect is observed in glycerol extracted fibres.3.Chemically treated fibres in catch-state induced at ionic strength 0.28 and pH 7.0, relaxe after addition of 5-HT to the low Ca2+ solution. No relaxing effect is observed when the catch-state is induced at ionic strength 0.06 and pH 6.5.4.Cyclic AMP (c-AMP) induces the same relaxing effects as 5-HT on chemically treated fibres in the same conditions of ionic strength, pH and Ca2+ concentration.5.The results are in disagreement with the hypothesis that the catch-state in ABRM is due to a lowering of the intracellular ionic strength and pH; they suggest that the intracellular physiological conditions are rather near ionic strength 0.28 and pH 7.0 even in catch-state. We suppose that the relaxing effect of 5-HT, is not be due to a decrease of the intracellular free Ca2+ level but rather to an increase of the rate of Ca2+ release from the contractile proteins, and that it is mediated through c-AMP and intracellular relaxing mediator.

The catch-state in glycerol extracted fibres from a lamellibranch smooth muscle (ABRM).

Summary1.The influence of four ionic strengths (0.06 to 0.28) and two pHs (7.0 and 6.5) was investigated on the contractile properties of glycerol extracted ABRM bundles in presence of different free Ca2+ concentrations (10−6 to 10−4 M).2.The isometric force is maximal at Ca2+ 10−6 M for pH 7.0 and ionic strength 0.28; at Ca2+ 10−4 M for the other conditions studied. During the plateau in presence of Ca2+ 10−6 M, a quick release applied at any time, is followed by a large redevelopment of tension, except at ionic strength 0.29 and pH 7.0: in this case, tension redevelops only during the first 5 min afterwards no tension redevelops. The disappearance of the active state is completely reversible and depends on the ionic strength and the presence of Ca2+.3.In a relaxing solution (Ca2+ 10−9 M), tension disappears at a rate which is maximal at pH 7.0, ionic strength 0.28 and minimum at pH 6.5, ionic strength 0.06. In this last condition, no tension redevelops after a quick release applied during the relaxation.4.Those results suggest that the catch-state is spontaneously induced after the activation of the contractile mechanism, in presence of Ca2+ ions which concentration depends on ionic strength and pH.

The stimulation of isolated photophores (Argyropelecus) by epinephrine and norepinephrine

Abstract 1. 1. Photophores isolated from the ventral region of the bathypelagic fish Argyropelecus hemigymnus respond to epinephrine and norepinephrine by a long-lasting luminescence: the maximal light emission occurring at 10 −6 M for epinephrine and 10 −5 M for norepinephrine, is estimated at 8 × 10 6 quanta/sec per photophore. 2. 2. The LD 50 value of the light response calculated from the dose-response curves to both neuromediators, is 2 × 10 −7 M for epinephrine and 2 × 10 −6 M for norepinephrine. 3. 3. The light response to epinephrine is depressed by propranol and phentolamine 10 −5 M, and the maximum is shifted to concentration higher than 10 −6 M. Phentolamine completely blocks the light evoked by norepinephrine at a concentration lower than 10 −5 M and shifts the light maximum to 10 −3 M. 4. 4. Photophores show a greenish fluorescence when excited with 365 nm u.v. light; the fluorescence intensity, is largely decreased after the light emission.

Bioluminescence of bathypelagic fish from the strait of messina

Abstract 1. Photophores isolated from bathypelagic fish emit bright flashes (6 × 10 7 to 6 × 10 9 quanta/sec) lasting a few msec when they are electrically stimulated. In Argyropelecus, Diaphus and Ichthyococcus . the flashes are short, fatigable and have short latency. In Chauliodus and Stomias the flashes are multimodal, and have a long latency. 2. Adrenaline, nor-adrenaline, serotonine and acetylcholine are without effect on the isolated photophores, and do not influence the electrical stimulation. 3. Photophores of Argyropelecus have a low amount of ATP, less than 0·2 μmole/g, and larger amount of ADP (about 1 μmole/g) and AMP (0·5 μmole/g). No inosine monophosphate was detected, nor inosine nor adenosine. 4. Oral and caudal extremities of the gut harbour photomicrobes, which appears to be normal hosts of bathypelagic fish.

Nitric oxide in control of luminescence from hatchetfish (Argyropelecus hemigymnus) photophores.

SUMMARY Nitric oxide synthase-like immunoreactivity (NOS-LI IR) was detected by immunohistochemistry in ventral light organs of the mesopelagic fish, Argyropelecus hemigymnus. Strong NOS-LI IR was present in nerve fibres and in other cells central for production or modulation of light: immunoreactive fibres surrounded the photophores, and were also present in the filter area. Filter cells, particularly in the outer layers, showed strong IR throughout the cytoplasm. Pharmacological studies suggested that nitric oxide (NO) modulates adrenaline-stimulated light emission, and that the modulation is correlated to the ability of the light organ to respond to adrenaline. Adrenaline is known to produce two different types of light response in isolated photophores from Argyropelecus: a slow, long-lasting, high intensity response, or a fast and weak response of short duration. Incubation of photophores in the NO donors sodium nitroprusside or S-nitroso-N-acetylpenicillamine prior to adrenaline stimulation reduced the intensity of the strong and long-lasting type of response, but had little or even a potentiating effect on the weakly responding photophores. Hydroxylamine, which is converted to NO if catalase activity is present in the tissue, reduced the duration and the intensity of the adrenaline response in all tested organs. The NOS-inhibitor l-thiocitrulline potentiated the adrenaline response in the weakly responding organs; the weaker the adrenaline effect, the stronger the potentiation caused by l-thiocitrulline. The strongly responding organs were instead inhibited by l-thiocitrulline. The results suggest that NO has an important role in the control of light emission from Argyropelecus hemigymnus photophores. The cGMP analogue dibutyryl cGMP, the guanylate cyclase inhibitor ODQ and the phosphodiesterase inhibitor pentoxiphylline had no effect, indicating that the NO effect does not involve cGMP.

Detection of Coelenterazine and Related Luciferase Activity in the Tissues of the Luminous Fish, Vinciguerria-attenuata

Abstract 1. 1. We attempted to identify the components of the light-emitting system of the bioluminescent fish, Vinciguerria attenuata (Photichthyidae). 2. 2. Methanol extracts of the digestive tract and body tissues were analysed by HPLC. Assays for the detection of coelenterazine or Vargula luciferin were performed on the collected fractions. 3. 3. The corresponcling luciferase activities were also assayed in the solid residues. 4. 4. No cross-reactions with Vargula luciferase and luciferin were detected. 5. 5. Coelenterazine was detected in the tissues of the fish. The eoelenterazine content of the digestive tract represented as much as 95% of the total coelenterazine content of the fish. Luciferase activity, specific for eoelenterazine, was absent from the digestive tract, but present in the rest of the body. 6. 6. These results suggest that the luminescent system in Vinciguerria photophores is of the coelenterate type. Coelenterazine distribution in the tissues of the fish suggests that it might be obtained from the diet.

The muscular membrane and calcium activation of the contractile system of a lamellibranch smooth muscle (ABRM).

Summary1.Thin bundles of fresh ABRM treated by EDTA solution or Triton X-100 0.1%, can be brought like glycerol-extracted fibres through contraction-relaxation cycles by changing the free Ca2+ level of the bathing medium from 10−6 M to 10−9 M. The kinetics of isometric force development and relaxation has been studied in the two conditions i.e. ionic strength 0.06, pH 6.5 and ionic strength 0.28, pH 7.0, which are known to induce the catch-state in glycerol extracted fibres. When the low Ca2+ solution (10−9 M) is substituted for the high Ca2+ solution (10−6 M) immediately after the maximal force has been developed, relaxation occurs at a higher rate at ionic strength 0.28 and pH 7.0 than at ionic strength 0.06 and pH 6.5. In this last condition, no tension redevelops after a quick release applied during the slow relaxation.2.During the plateau, in presence of Ca2+ 10−6 M, a quick release applied at any time is followed by a large redevelopment of tension at ionic strength 0.06 and pH 6.5. At ionic strength 0.28 and pH 7.0, the tension redevelops only during the 10 first min, and not more afterwards. After that time the Ca2+ solution level can be decreased from 10−6 to 10−9 M without any change in the time course of the tension maintained.3.These results suggest that in EDTA and Triton X-100 treated fibres of ABRM, the catch-state is spontaneously induced after the activation of the contractile mechanism under the same conditions of ionic strength and pH as in glycerol extracted fibres. However, in the EDTA and Triton treated fibres, the presence of a high free Ca2+ level is not necessary to maintain the tension in the catch-state induced at ionic strength 0.28 and pH 7.0.

Bioluminescence and luminescent fish in the Strait of Messina from the mesoscaph “Forel”

Light irradiance was measured at 430, 470 and 500 nm aboard the mesoscaph “Forel” in the Strait of Messina from the surface to 550 m depth in May 1979. The underwater light regime is partly due to the downwelling residual sunlight and partly to bioluminescence. An intense bioluminescence is localized at about 450 m at midday and moves upwards in the evening to reach an area extending from 100 m depth to the surface late in the evening. Two types of luminescence were observed: one associated with luminescent organisms and another diffuse, probably due to bacteria. Three types of luminescent fish were recognized, namely Argyropelecus hemigymnus, myctophids and Cyclothone braueri, and their time and space distribution were studied. While myctophids were encountered from the surface (21:00 hrs) to 550 m depth (16:00 hrs), A. hemigymnus were only observed between 180 m (19:45 hrs) and 500 m (12:15 hrs), and C. braueri between 330 m (16:00 hrs) and 500 m (19:00 hrs). The results do not show a significant relation between the absolute ambient light intensity and the time or the depth where the fish were observed.