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Effects of sodium azide on the Porichthys isolated luminous organs
Camp. f3io&m. fhysiol. Vol. 96C, No. I, pp. 105-109, 1990 Printed in Great Britain
EFFECTS
0
0306~4492/90 53.00 + 0.00 1990 Pergamon Press plc
OF SODIUM AZIDE ON THE PORICHTHYS ISOLATED LUMINOUS ORGANS J.
MALLEFET,*
J. F. F&ESand F. BACUET
Laboratoire de Physiologie GBnCrale et des Animaux Domestiques, Universite Catholique de Louvain, 5 place de la Croix du Sud, B-1348 Louvain-la-Neuve, Belgium. Telephone: (010) 473476 (Received 24 January 1990) Abstract-l.
Application
of sodium azide 10e3 M on isolated luminous organ of the epipelagic fish
Porichrhys always induced a light emission.
2. Although the parameters of azide luminescence were similar to those of the potassium cyanide induced luminescence, the metabolic mechanisms associated with this light emission appear to be different. 3. Effectively, neither glucose nor pyruvate showed inhibitory effects on the azide induced luminescence, although glucose totally suppressed KCN induced luminescence. 4. Further, contrary to the potassium cyanide luminescence, there is no important increase in the oxygen consumption during azide photogenesis. 5. Different hypotheses are discussed to explain azide induced luminescence.
INTRODUCTION Isolated photophores of the epipelagic fish Porichthys produce a large luminescence when exposed to potassium cyanide (KCN), suggesting that the light production is under the control of an energy dependent inhibitory mechanism (Baguet, 1975; Mallefet and Baguet, 1984; Rees and Baguet, 1989). As glucose but not pyruvate inhibits this luminescence, KCN is assumed to inhibit specifically the cellular respiration inducing the luminescence of the photocytes (Rees and Baguet, 1988; Mallefet and
Baguet, 1989a). In this new experimental series, we investigate the effects of sodium azide (NaN,), another respiratory chain inhibitor, on the isolated photophores of Porichthys.
The results show that, although the induction of luminescence is similar to that observed upon cyanide application, the oxidative phosphorylations should not be the exclusive target of sodium azide. MATERIALS AND Dissection
METHODS
qf the phosphores
Seven specimens of the midshipman fish Porichthys myriuster and notatus shipped by Pacific Bio-Marine Laboratories (Venice, California) were kept in aquaria provided with aerated and UV sterilized running seawater (18°C). After anaesthesia with quinaldine (0.037% v/v) in seawater (Allen and Sills, 1973), a strip of photophores from the mandibular, gular. branchial, ventral and pleural regions (using Greene’s terminology, 1899) was excised and immersed in saline. Experimental procedures
Two different installations were used to study the effects of sodium azide on isolated photophores: when solely the light emission was measured, we utilized the experimental installation previously described (Rees and Baguet, 1988). *Charge de Recherches FNRS.
When luminescence and oxygen consumption were simultaneously recorded, photophores were laid as in the experimental set-up described previously by Mallefet and Baguet (1988). Solutions
Potassium cyanide, sodium azide, glucose and pyruvate were purchased from Merck. They were dissolved in buffered Young’s saline (Young, 1933) containing (in mmol 1-r): NaCI, 150; KCI, 7.5; CaCl,, 3.5; MgCl,, 2.4; pH = 7.3 with Tris-HCI, 20, just prior to use. RESULTS 1. Luminescence
On application of 10m4M NaN,, isolated photophores never produced light. At the concentration of lo-) M, NaN, always induced the luminescence of the photophore (n = 38). The parameters of the light production are summarized in Table 1. Azide luminescence started 79.2 f 7.4 set after stimulation (LT) and reached a maximum value of 1554.4 f 275.8 Mq/sec (L,,,,,.) after 112 If: 7.7 set (TL,,,). Half-extinction time of luminescence (ER) was estimated at 263.6 f 22.5 sec. As azide and cyanide block the cellular respiration at the same level, the cytochrome aa, (Nicholls and Kimelberg, 1972), we have compared the light emission triggered by these two metabolic inhibitors (lO-3 M). The time courses of the luminescences induced by 10m3M KCN and NaN, were similar; neither the maximal light amplitude for KCN nor for NaN, were different, corresponding respectively to 4781 & 763 Mq/sec and 4824 rf: 697 Mq/sec (n = 6). As tt has been shown that glucose rather than pyruvate inhibits the KCN induced luminescence, we tested the effect of both substrates on the azide induced luminescence. Examination of Fig. 1 shows that neither glucose nor pyruvate significantly modified any parameters of the azide light production, although glucose totally suppressed the 105
J. MALLEFET et al.
106
Table I. Parameters of the light emission induced by the application of 10-j M NaN, on isolated photophores. Each value (mean k SEMI was calculated on 38 Dhotoohores isolated from 5 different fish LT (set)
NaN, IO-‘M
7X,,,
(set)
19.2k7.4 ll2.7f7.1
L,,,
(Mq/sec)
1554.4i275.8
ER (SC) 263.6i22.5
(n = 38)
luminescence triggered by KCN. These results suggest that the metabolic phenomenon in azide luminescence could be different from that involved in cyanide photogenesis. KCN
2. Oxygen consumption Figure 2 shows the time course evolution of the oxygen consumption and the light intensity of isolated photophores treated with azide 10m4M. One can observe that at a concentration that did not induce any light production, azide induced a slow decrease of the oxygen consumption (Fig. 2). After 30 min, the remaining level represented 69 If: 12% (p < 0.05; n = 6) of the previous resting level (0.137 f 0.02 nmO,/min). Simultaneous measurements of oxygen consumption and light production of isolated photophores treated with azide 10e3 M (Fig. 3) showed a significant increase in oxygen consumption above the resting level during the first 5 min following the application of azide (p < 0.05; n = 18). During the first 30sec, the oxygen consumption rate increased from 0.08 If: 0.01 nmO,/min, at rest, to 0.17 + 0.03 nmO,/min (p < 0.01). The oxygen uptake remained constant for 3 min, and decreased afterwards to 0.09 + 0.02 nmO,/min at 5 min, a value which is not significantly different from the previous resting level. Oxygen consumption remained at this steady level for the next 25 min. Light production started 139 f 41 set (LT) after azide application, i.e. long after the oxygen consumption increased to its maximal value, and reached a maximal intensity (L,,,) of 826 + 170 Mq/sec after 333 k 45 set (TL,,,). The half-extinction time (ER) was 625 f 66 sec. The time course of the mean oxygen consumption and the mean production of light in response to
=
CONTROLS
NaN3 m
GLUCOSE
m
PYRUVATE
Fig. 1. Effects of glucose and pyruvate on the maximal intensity of light responses of photophores treated with KCN or NaN,. Both metabolites (5.5 x 10-j M) were applied 10min prior to the application of the metabolic inhibitors (lo-‘M). Light intensities are expressed as a percentage of those measured on control photophores treated with the metabolic inhibitors in metabolite-free saline. n = 6 for each treatment (mean & SEM).
10m3M KCN (n = 8) are shown in Fig. 4. During the first minute following cyanide application, oxygen consumption showed an eight fold increase in its resting value, increasing from 0.08 k 0.01 nmO,/min to 0.64 f 0.19 nmO,/min after 60 set (p < 0.001). Subsequently, there was a slow decrease of the oxygen consumption rate, which remained higher than the previous resting rate (p < 0.05) during the following 25 min. Light emission started 118 &-28 set (LT) after cyanide application and reached a maximal value (L,,,) of 1128 rf: 297 Mq/sec after 260 + 121 set (7X,,,). Half-extinction rate (ER) was estimated at 352 f 17 sec. Since oxidative phenomena are very different during azide and cyanide luminescences, we calculated the average oxygen consumed above the resting rate level and the total amount of light produced during the 30 min of the experiments (Table 2). The results indicated that about ten times more oxygen is consumed during cyanide induced luminescence than
OXYGEN CONSUMPTION nmq/mln 0.7-
0.5-
0.5-
0.4-
0.3-
I ..__. ___,, . , . , . . . , AZIDE
Fig. 2. Time course
5 1O-4 M
of the oxygen
10
consumption
16
(0-n)
1 20
in response
I 25
to NaN,
I 20
’
TIME m,n
(10m4 M); mean k SE.
Azide luminescence of Porichthys photophores OXYGEN CONSUMPTION nmq/mln
107
LIGHT uwssc
30
Fig. 3. Time course of the oxygen consumption (II-_O)
and light production (o-0) NaN, (10-j M); mean f SE.
during azide light emission, although a similar amount of light is produced in both cases. DISCUSSION 1. The production
of light
Since azide and cyanide block the cellular respiration at the same level, the cytochrome aa3, (Nicholls and Kimelberg, 1972), and as NaN, and KCN both induce a similar luminescence of an isolated photophore, it is suggested that luminescence is triggered by the inhibition of cellular respiration, although the different effects of glucose on the light emission could imply that the effect of both metabolic inhibitors on the phenomenon associated to the luminescence is different. It has been suggested that isolated photophores are maintained in a non-luminous state by the activity of
TIME m1n
in response to
an energy consuming
inhibitory mechanism (Rees and Baguet, 1989). Since adenosine triphosphate is the major source of energy produced by glycolysis and oxidative phosphorylation, KCN should inhibit the supply of energy to the inhibitory mechanism and should induce the luminescence of the photophore. The application of glucose inhibits the KCN induced luminescence by stimulating the glycolytic pathway and the furniture of ATP. The lack of effect of glucose on the azide luminescence might be explained as a first hypothesis by a non-specific inhibitory effect of azide on glycolysis that suppresses any ATP supply to the inhibitory mechanism. Although glycolysis inhibition by azide has not yet been described, we cannot exclude such a non-specific effect of high concentration of azide. As a second hypothesis, azide should directly block the inhibitory mechanism; in this case, ATP supply by
OXYGEN CONSUMPTION
LIGHT nwsec
30
Fig. 4. Time course of the oxygen consumption (a-_O) and light production (o-0) KCN (IO-‘M); mean f. SE.
TIME mln
in response to
108
J. MALLEFET et al.
Table 2. Oxygen consumed above the resting level (Q02) and total amount of light produced (QL); mean + SE
NaN, IO-‘M(n
= 18)
PO2 (nm 0,) 0.58 + 0.43
KCN
=8)
7.19 f 1.78
Stimulation 10 ‘M(n
QL (109q) 957.8 f 386.2 703.6 f I I 1.8
glycolysis would be useless. The nature of the inhibitory mechanism is still unknown. As another possibility, azide could directly activate the luminous system, over-ruling the inhibitory mechanism control. This hypothesis is in opposition with earlier observations showing that sodium azide inhibited the light production of the Vargula isolated luminous system (Chase, 1942, 1948). However, this inhibition was observed at very low pH (5.4) and high azide concentrations (25-500 mM) i.e. in conditions which are very different from those we used (pH 7.3; 1 mM NaN,). In our experimental protocol, we cannot rule out the possibility of a direct activation of the Porichthys luminous system by sodium azide which is able to oxidize tryptophane (Land and Prutz, 1977), one of the constituents of the luciferin molecule, substrate of the light reaction (Kishi et al., 1966). Since azide is known to inhibit mitochondrial ATPase (Mitchell and Moyle, 1971), we suggest the azide luminescence might result from the inhibition of some ATPase activity controlling ATP hydrolysis necessary to the inhibitory mechanism. In this case, the production of ATP by glycolysis should be useless, and glucose should not prevent the azide induced luminescence. 2. Oxygen consumption Our results indicate a dual effect of sodium azide on the oxygen consumption: at a concentration of 10e4M that does not induce a luminescence, azide reduces the photophore respiration; when it induces a production of light (10-j M), a transitory increase of oxygen uptake occurs prior to the luminescence. KCN also induces a similar dual effect (Mallefet and Baguet, 1984). Assuming that both metabolic inhibitors block the cellular respiration, the question arises as to what is the origin of the enhancement of the oxygen consumption. Since the biochemical mechanism for luminescence in Porichthys photophores consists of a luciferasecatalyzed oxidation of luciferin by oxygen (Tsuji et al., 1971), the present result could suggest that the increase of oxygen uptake with light originated from the oxidation of luciferin by molecular oxygen. The number of oxygen molecules consumed, calculated for the production of one quantum of light, is 363 in azide and 6 131 in KCN; in both cases these values are much higher than the theoretical value (three molecules) calculated for the emission of one quantum of light in the oxidation of luciferin in vitro (Stone, 1968). Moreover, as sodium azide and KCN block the same site of the cellular respiration, the difference in the oxygen consumption associated with a light emission of similar intensity and kinetics is unexpected. In azide and cyanide luminescences, oxygen consumption increases prior to the production of light.
The same phenomenon was described for the light responses to adrenaline or noradrenaline (Mallefet and Baguet, 1989b). Although our results obviously demonstrate that mitochondrial respiration is not necessary to produce light, luminescence cannot be evoked in the absence of oxygen in saline (Mallefet and Baguet, 1987). Oxygen is thus necessary for the light reaction, but the oxygen consumption involved in the luciferin oxidation is so low that it cannot be detected by our technical approach. An isolated photophore seems to need a large amount of oxygen to trigger a light reaction that consumes a minute amount of oxygen. Nevertheless, this non-respiratory oxygen consumption can be very different in azide and cyanide induced luminescence. It is possible that the initial oxygen uptake might be due to an extra-mitochondrial respiration. Photophores seems to respond to specific stimuli like phagocytosis cells: in both cases a sharp burst of oxygen is consumed prior to the activity of the cells. Leukocytes and phagocytes consume oxygen to produce H20, through extra-mitochondrial single electron transfer reactions (Babior, 1978). Direct application of H,O, to isolated photophores triggers a bright production of light (Baguet, personal communication). Moreover a peroxide mechanism has been proposed for the luminescent reaction of the Vargula system (Tsuji et al., 1977), which is similar to the Porichthys photophore system (Cormier et al., 1967). In this case, as protons and oxygen are necessary to generate peroxides, the well known deprotonation activity of sodium azide (Oesterhelt & Tittor, 1989) should depress the enzymic reaction of peroxidation; this could explain the very low level of oxygen consumed prior to the generation of light by azide. Experiments are in progress to investigate the presence of an extra-mitochondrial respiration in Porichthys photophores.
REFERENCES Allen J. L. and Sills J. B. (1973) Preparation and properties of quinaldine sulphate, an improved fish anaesthetic. US Bur. Fish. and Wild., Invest in Fish Conrr. 47, 1-7. Babior B. M. (1978) Oxygen dependent microbial killing by phagocytes. New Eng. J. Med. 298, 659668. Baguet F. (1975) Excitation and control of isolated photophores of luminous fishes. Proc. Neurobiol. 5, 97-125. Chase A. M. (1942) The reaction of Cypridina luciferin with azide. J. cell. romp. Physiol. 19, 173-181. Chase A. M. (1948) The chemistry of Cypridina luciferin. Ann. NY Acad. Sci. 49, 353-375. Cormier M. J., Crane J. M. and Nakano Y. (1967) Evidence for the identity of the luminescent systems of Porichthys porosissimus (fish) and Cypridina hilgendorfi (crustacean). Biochem. biovhvs. Res. Commun. 29, 747-752. Greene C. W.‘(i899) The phosphorescent organs in the toadfish Porichthys notatus Girard. J. Morph. 15, 667-696. Kishi Y.. Goto T., Hirata Y., Shimomura 0. and Johnson F. H. (1966) Cypridino bioluminescence. I. Structure of Cypridina luciferin. Tetrahedron Lett. 29, 3427-3436. Land E. J. and Prutz W. A. (1977) Fast one-electron oxidation of tryptophan by azide radicals. Int. J. radiat. Biol. 32, 203-207. Mallefet J. and Baguet F. (1984) Oxygen consumption and luminescence of Porichfhys photophores stimulated by potassium cyanide. J. exp. Biol. 109, 341-352.
Azide luminescence of Porichthys photophores Mallefet J. and Baguet F. (1987) Effets d’inhibiteurs mitaboliques sur la consommation d’oxygene du photophore isnole du Poisson lumineux Porichrhys. J. physiol. Paris 82(3), 34A.
Mallefet J. and Baguet F. (1988) Effects of ouabain on isolated photophores of luminous fish. Comp. Biochem. Physiol. 89C, 159-163. Mallefet J. and Baguet F. (1989a) Oxygen consumption and luminescence of isolated photophores of Porichthys: effects of glucose. Arch. in!. Biochem. Physiol. 9’7(3), 38.
Mallefet J. and Baguet F. (1989b) Oxygen consumption and luminescence of isolated Porichthys photophores in response to adrenergic stimulation. Photochem. Photobiol. 50(2), 243-250.
Mitchell P. and Moyle J. (1971) Activation and inhibition of mitochondrial adenosine triphosphatase by various anions and other agents. Bioenergefics 2, l-l 1. Nicholls P. and Kimelberg H. K. (1972) Cytochrome oxidase in the mitochondrial membrane. In Biochemisfry and Biophysics of Mitochondrial Membranes, Azxone G. F., Carafoli E., Lehninger A. L., Quagliariello E. and Siliprandi N. teds), pp. 17-32. Academic Press, New York.
109
Oesterhelt D. and Tittor J. (1989) Two pumps, one principle: light-driven ion transport in Halobacteria. TIBS 14, 5761. Rees J. F. and Baguet F. (1988) Metabolic control of spontaneous glowing in isolated photophores of Porichthys. J. exp. Biol. 135, 289-299.
Rees J. F. and Baguet F. (1989) Metabolic control of luminescence in the luminous organs of the teleost Porichthys: effects of the metabolic inhibitors iodoacetic acid and potassium cyanide. J. exp. Biol. 143, 347-357. Stone H. (1968) The enzyme catalyzed oxidation of Cypridina luciferin. Biochem. Biophys. Res. Commun. 31,386-39 1. Tsuji F. I., De Luca M., Boyer P. D., Endo S. and Akutagawa M. (1977) Mechanism of the enzyme catalysed oxidation of Cypridina and firefly luciferin studied by means of “0, and HisO. Biochem. Biophys. Res. Commun. 74, 606-613.
Tsuji F. I., Haneda Y., Lynch R. V. and Sugiyaman N. (1971) Luminescence cross-reaction of Porichthvs luciferin .and theories on the origin of luciferin & some shallow-water fishes. Comp. Biochem. Physiol. 4OA, 163-179. Young J. Z. (1933) The preparation of isotonic solutions for use in experiments with fish. Publ. Sta. Zool. Nap. 12,424.
EFFECTS
0
0306~4492/90 53.00 + 0.00 1990 Pergamon Press plc
OF SODIUM AZIDE ON THE PORICHTHYS ISOLATED LUMINOUS ORGANS J.
MALLEFET,*
J. F. F&ESand F. BACUET
Laboratoire de Physiologie GBnCrale et des Animaux Domestiques, Universite Catholique de Louvain, 5 place de la Croix du Sud, B-1348 Louvain-la-Neuve, Belgium. Telephone: (010) 473476 (Received 24 January 1990) Abstract-l.
Application
of sodium azide 10e3 M on isolated luminous organ of the epipelagic fish
Porichrhys always induced a light emission.
2. Although the parameters of azide luminescence were similar to those of the potassium cyanide induced luminescence, the metabolic mechanisms associated with this light emission appear to be different. 3. Effectively, neither glucose nor pyruvate showed inhibitory effects on the azide induced luminescence, although glucose totally suppressed KCN induced luminescence. 4. Further, contrary to the potassium cyanide luminescence, there is no important increase in the oxygen consumption during azide photogenesis. 5. Different hypotheses are discussed to explain azide induced luminescence.
INTRODUCTION Isolated photophores of the epipelagic fish Porichthys produce a large luminescence when exposed to potassium cyanide (KCN), suggesting that the light production is under the control of an energy dependent inhibitory mechanism (Baguet, 1975; Mallefet and Baguet, 1984; Rees and Baguet, 1989). As glucose but not pyruvate inhibits this luminescence, KCN is assumed to inhibit specifically the cellular respiration inducing the luminescence of the photocytes (Rees and Baguet, 1988; Mallefet and
Baguet, 1989a). In this new experimental series, we investigate the effects of sodium azide (NaN,), another respiratory chain inhibitor, on the isolated photophores of Porichthys.
The results show that, although the induction of luminescence is similar to that observed upon cyanide application, the oxidative phosphorylations should not be the exclusive target of sodium azide. MATERIALS AND Dissection
METHODS
qf the phosphores
Seven specimens of the midshipman fish Porichthys myriuster and notatus shipped by Pacific Bio-Marine Laboratories (Venice, California) were kept in aquaria provided with aerated and UV sterilized running seawater (18°C). After anaesthesia with quinaldine (0.037% v/v) in seawater (Allen and Sills, 1973), a strip of photophores from the mandibular, gular. branchial, ventral and pleural regions (using Greene’s terminology, 1899) was excised and immersed in saline. Experimental procedures
Two different installations were used to study the effects of sodium azide on isolated photophores: when solely the light emission was measured, we utilized the experimental installation previously described (Rees and Baguet, 1988). *Charge de Recherches FNRS.
When luminescence and oxygen consumption were simultaneously recorded, photophores were laid as in the experimental set-up described previously by Mallefet and Baguet (1988). Solutions
Potassium cyanide, sodium azide, glucose and pyruvate were purchased from Merck. They were dissolved in buffered Young’s saline (Young, 1933) containing (in mmol 1-r): NaCI, 150; KCI, 7.5; CaCl,, 3.5; MgCl,, 2.4; pH = 7.3 with Tris-HCI, 20, just prior to use. RESULTS 1. Luminescence
On application of 10m4M NaN,, isolated photophores never produced light. At the concentration of lo-) M, NaN, always induced the luminescence of the photophore (n = 38). The parameters of the light production are summarized in Table 1. Azide luminescence started 79.2 f 7.4 set after stimulation (LT) and reached a maximum value of 1554.4 f 275.8 Mq/sec (L,,,,,.) after 112 If: 7.7 set (TL,,,). Half-extinction time of luminescence (ER) was estimated at 263.6 f 22.5 sec. As azide and cyanide block the cellular respiration at the same level, the cytochrome aa, (Nicholls and Kimelberg, 1972), we have compared the light emission triggered by these two metabolic inhibitors (lO-3 M). The time courses of the luminescences induced by 10m3M KCN and NaN, were similar; neither the maximal light amplitude for KCN nor for NaN, were different, corresponding respectively to 4781 & 763 Mq/sec and 4824 rf: 697 Mq/sec (n = 6). As tt has been shown that glucose rather than pyruvate inhibits the KCN induced luminescence, we tested the effect of both substrates on the azide induced luminescence. Examination of Fig. 1 shows that neither glucose nor pyruvate significantly modified any parameters of the azide light production, although glucose totally suppressed the 105
J. MALLEFET et al.
106
Table I. Parameters of the light emission induced by the application of 10-j M NaN, on isolated photophores. Each value (mean k SEMI was calculated on 38 Dhotoohores isolated from 5 different fish LT (set)
NaN, IO-‘M
7X,,,
(set)
19.2k7.4 ll2.7f7.1
L,,,
(Mq/sec)
1554.4i275.8
ER (SC) 263.6i22.5
(n = 38)
luminescence triggered by KCN. These results suggest that the metabolic phenomenon in azide luminescence could be different from that involved in cyanide photogenesis. KCN
2. Oxygen consumption Figure 2 shows the time course evolution of the oxygen consumption and the light intensity of isolated photophores treated with azide 10m4M. One can observe that at a concentration that did not induce any light production, azide induced a slow decrease of the oxygen consumption (Fig. 2). After 30 min, the remaining level represented 69 If: 12% (p < 0.05; n = 6) of the previous resting level (0.137 f 0.02 nmO,/min). Simultaneous measurements of oxygen consumption and light production of isolated photophores treated with azide 10e3 M (Fig. 3) showed a significant increase in oxygen consumption above the resting level during the first 5 min following the application of azide (p < 0.05; n = 18). During the first 30sec, the oxygen consumption rate increased from 0.08 If: 0.01 nmO,/min, at rest, to 0.17 + 0.03 nmO,/min (p < 0.01). The oxygen uptake remained constant for 3 min, and decreased afterwards to 0.09 + 0.02 nmO,/min at 5 min, a value which is not significantly different from the previous resting level. Oxygen consumption remained at this steady level for the next 25 min. Light production started 139 f 41 set (LT) after azide application, i.e. long after the oxygen consumption increased to its maximal value, and reached a maximal intensity (L,,,) of 826 + 170 Mq/sec after 333 k 45 set (TL,,,). The half-extinction time (ER) was 625 f 66 sec. The time course of the mean oxygen consumption and the mean production of light in response to
=
CONTROLS
NaN3 m
GLUCOSE
m
PYRUVATE
Fig. 1. Effects of glucose and pyruvate on the maximal intensity of light responses of photophores treated with KCN or NaN,. Both metabolites (5.5 x 10-j M) were applied 10min prior to the application of the metabolic inhibitors (lo-‘M). Light intensities are expressed as a percentage of those measured on control photophores treated with the metabolic inhibitors in metabolite-free saline. n = 6 for each treatment (mean & SEM).
10m3M KCN (n = 8) are shown in Fig. 4. During the first minute following cyanide application, oxygen consumption showed an eight fold increase in its resting value, increasing from 0.08 k 0.01 nmO,/min to 0.64 f 0.19 nmO,/min after 60 set (p < 0.001). Subsequently, there was a slow decrease of the oxygen consumption rate, which remained higher than the previous resting rate (p < 0.05) during the following 25 min. Light emission started 118 &-28 set (LT) after cyanide application and reached a maximal value (L,,,) of 1128 rf: 297 Mq/sec after 260 + 121 set (7X,,,). Half-extinction rate (ER) was estimated at 352 f 17 sec. Since oxidative phenomena are very different during azide and cyanide luminescences, we calculated the average oxygen consumed above the resting rate level and the total amount of light produced during the 30 min of the experiments (Table 2). The results indicated that about ten times more oxygen is consumed during cyanide induced luminescence than
OXYGEN CONSUMPTION nmq/mln 0.7-
0.5-
0.5-
0.4-
0.3-
I ..__. ___,, . , . , . . . , AZIDE
Fig. 2. Time course
5 1O-4 M
of the oxygen
10
consumption
16
(0-n)
1 20
in response
I 25
to NaN,
I 20
’
TIME m,n
(10m4 M); mean k SE.
Azide luminescence of Porichthys photophores OXYGEN CONSUMPTION nmq/mln
107
LIGHT uwssc
30
Fig. 3. Time course of the oxygen consumption (II-_O)
and light production (o-0) NaN, (10-j M); mean f SE.
during azide light emission, although a similar amount of light is produced in both cases. DISCUSSION 1. The production
of light
Since azide and cyanide block the cellular respiration at the same level, the cytochrome aa3, (Nicholls and Kimelberg, 1972), and as NaN, and KCN both induce a similar luminescence of an isolated photophore, it is suggested that luminescence is triggered by the inhibition of cellular respiration, although the different effects of glucose on the light emission could imply that the effect of both metabolic inhibitors on the phenomenon associated to the luminescence is different. It has been suggested that isolated photophores are maintained in a non-luminous state by the activity of
TIME m1n
in response to
an energy consuming
inhibitory mechanism (Rees and Baguet, 1989). Since adenosine triphosphate is the major source of energy produced by glycolysis and oxidative phosphorylation, KCN should inhibit the supply of energy to the inhibitory mechanism and should induce the luminescence of the photophore. The application of glucose inhibits the KCN induced luminescence by stimulating the glycolytic pathway and the furniture of ATP. The lack of effect of glucose on the azide luminescence might be explained as a first hypothesis by a non-specific inhibitory effect of azide on glycolysis that suppresses any ATP supply to the inhibitory mechanism. Although glycolysis inhibition by azide has not yet been described, we cannot exclude such a non-specific effect of high concentration of azide. As a second hypothesis, azide should directly block the inhibitory mechanism; in this case, ATP supply by
OXYGEN CONSUMPTION
LIGHT nwsec
30
Fig. 4. Time course of the oxygen consumption (a-_O) and light production (o-0) KCN (IO-‘M); mean f. SE.
TIME mln
in response to
108
J. MALLEFET et al.
Table 2. Oxygen consumed above the resting level (Q02) and total amount of light produced (QL); mean + SE
NaN, IO-‘M(n
= 18)
PO2 (nm 0,) 0.58 + 0.43
KCN
=8)
7.19 f 1.78
Stimulation 10 ‘M(n
QL (109q) 957.8 f 386.2 703.6 f I I 1.8
glycolysis would be useless. The nature of the inhibitory mechanism is still unknown. As another possibility, azide could directly activate the luminous system, over-ruling the inhibitory mechanism control. This hypothesis is in opposition with earlier observations showing that sodium azide inhibited the light production of the Vargula isolated luminous system (Chase, 1942, 1948). However, this inhibition was observed at very low pH (5.4) and high azide concentrations (25-500 mM) i.e. in conditions which are very different from those we used (pH 7.3; 1 mM NaN,). In our experimental protocol, we cannot rule out the possibility of a direct activation of the Porichthys luminous system by sodium azide which is able to oxidize tryptophane (Land and Prutz, 1977), one of the constituents of the luciferin molecule, substrate of the light reaction (Kishi et al., 1966). Since azide is known to inhibit mitochondrial ATPase (Mitchell and Moyle, 1971), we suggest the azide luminescence might result from the inhibition of some ATPase activity controlling ATP hydrolysis necessary to the inhibitory mechanism. In this case, the production of ATP by glycolysis should be useless, and glucose should not prevent the azide induced luminescence. 2. Oxygen consumption Our results indicate a dual effect of sodium azide on the oxygen consumption: at a concentration of 10e4M that does not induce a luminescence, azide reduces the photophore respiration; when it induces a production of light (10-j M), a transitory increase of oxygen uptake occurs prior to the luminescence. KCN also induces a similar dual effect (Mallefet and Baguet, 1984). Assuming that both metabolic inhibitors block the cellular respiration, the question arises as to what is the origin of the enhancement of the oxygen consumption. Since the biochemical mechanism for luminescence in Porichthys photophores consists of a luciferasecatalyzed oxidation of luciferin by oxygen (Tsuji et al., 1971), the present result could suggest that the increase of oxygen uptake with light originated from the oxidation of luciferin by molecular oxygen. The number of oxygen molecules consumed, calculated for the production of one quantum of light, is 363 in azide and 6 131 in KCN; in both cases these values are much higher than the theoretical value (three molecules) calculated for the emission of one quantum of light in the oxidation of luciferin in vitro (Stone, 1968). Moreover, as sodium azide and KCN block the same site of the cellular respiration, the difference in the oxygen consumption associated with a light emission of similar intensity and kinetics is unexpected. In azide and cyanide luminescences, oxygen consumption increases prior to the production of light.
The same phenomenon was described for the light responses to adrenaline or noradrenaline (Mallefet and Baguet, 1989b). Although our results obviously demonstrate that mitochondrial respiration is not necessary to produce light, luminescence cannot be evoked in the absence of oxygen in saline (Mallefet and Baguet, 1987). Oxygen is thus necessary for the light reaction, but the oxygen consumption involved in the luciferin oxidation is so low that it cannot be detected by our technical approach. An isolated photophore seems to need a large amount of oxygen to trigger a light reaction that consumes a minute amount of oxygen. Nevertheless, this non-respiratory oxygen consumption can be very different in azide and cyanide induced luminescence. It is possible that the initial oxygen uptake might be due to an extra-mitochondrial respiration. Photophores seems to respond to specific stimuli like phagocytosis cells: in both cases a sharp burst of oxygen is consumed prior to the activity of the cells. Leukocytes and phagocytes consume oxygen to produce H20, through extra-mitochondrial single electron transfer reactions (Babior, 1978). Direct application of H,O, to isolated photophores triggers a bright production of light (Baguet, personal communication). Moreover a peroxide mechanism has been proposed for the luminescent reaction of the Vargula system (Tsuji et al., 1977), which is similar to the Porichthys photophore system (Cormier et al., 1967). In this case, as protons and oxygen are necessary to generate peroxides, the well known deprotonation activity of sodium azide (Oesterhelt & Tittor, 1989) should depress the enzymic reaction of peroxidation; this could explain the very low level of oxygen consumed prior to the generation of light by azide. Experiments are in progress to investigate the presence of an extra-mitochondrial respiration in Porichthys photophores.
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