Effects of Catecholamines and Purines on Luminescence in the Brittlestar Amphipholis Squamata (Echinodermata)

Amphipholis squamata is a polychromatic luminescent ophiuroid. Six coloration varieties can be distinguished (Binaux and Bocquet, 1971; Deheyn et al., 1997), and the luminescent capacity varies from one variety to another (De Bremaeker et al., 1996; Deheyn et al., 1997). Two abundant and easily distinguishable varieties (brown and black) were examined in this study (the brown variety will be termed clear in comparison with the black variety). The isolated arms of Amphipholis squamata (only the arms are luminescent) emit monophasic light in response to KCl depolarization (Mallefet et al., 1992). Pharmacological studies have shown that acetylcholine (ACh), applied to isolated arms, induces a series of light flashes mediated by muscarinic receptors (De Bremaeker et al., 1993, 1996). The intensity of luminescence induced by ACh, even at a concentration of 10⁻³ mol l⁻¹, is lower than that obtained in response to applications of KCl (De Bremaeker et al., 1996). To explain the low intensity of ACh-induced luminescence compared with that of the KCl-induced light response, it has been postulated (De Bremaeker et al., 1996) that KCl might induce the release of substances that could act in synergy with endogenous ACh to modulate the luminescence.

A recent study has shown that amino acids and echinoderm neuropeptides have modulatory effects on the luminescence of A. squamata (De Bremaeker et al., 1999). However, these results only partly explained the difference between ACh- and KCl-induced luminescence. This suggests that other modulators could be involved in the control of luminescence in A. squamata. Catecholamines and purines have been implicated in, respectively, triggering and modulating luminescence in various species (Anctil et al., 1989; Awad and Anctil, 1993; Rees et al., 1991; Mallefet et al., 1993). Hence, these two groups of mediators are potential candidates for the control of luminescence in the ophiuroid A. squamata. Catecholamines and purines are present in the tissues of echinoderms and have been shown to be pharmacologically active.

Noradrenaline and dopamine have been found in the nervous system of various echinoderms (for a review, see Pentreath and Cobb, 1972; Fänge, 1969; Welsh, 1966, 1972; Bachmann and Goldschmid, 1978) and have also been been implicated in tube foot movement in Asterias rubens (Cottrell and Pentreath, 1970) and in the process of tissue differentiation during arm tip regeneration in Asterina gibbosa (Huet and Franquinet, 1981). 5-Hydroxytryptamine has been detected histochemically in the nervous system of larval echinoderms (Bisgrove and Burke, 1986; Nakajima, 1988) and in the tube feet of adult specimens of a holothurian and a starfish (Dolder, 1975), in the radial nerve cord of an ophiuroid (Ghyoot and Cobb, 1994) and in a feather star (Candia-Carnevali et al., 1989). Purines have been shown to be pharmacologically active in tissues of several echinoderm species (Hoyle and Greenberg, 1988; Shingyoji and Yamaguchi, 1995).

In the present study, we consider the effects of catecholamines (dopamine, adrenaline, noradrenaline and its derivatives), 5-hydroxytryptamine (5-HT) and purines (adenosine, ATP and their derivatives) on ACh-induced luminescence in isolated arms and dissociated photocytes from clear and black Amphipholis squamata.

Specimens of Amphipholis squamata (Delle Chiaje) collected at Langrune-sur-Mer (Normandy, France) were transported to the Laboratory of Animal Physiology at the Catholic University of Louvain and maintained in closed-circuit marine aquaria (32 ‰ salinity, 12 °C).

The arms are the only luminescent body parts of A. squamata (Brehm and Morin, 1977), and the five arms from a single specimen produce light of the same intensity provided that they are of the same length (Mallefet et al., 1992). Specimens were anaesthetized by immersion (3 min) in artificial sea water (ASW) containing 3.5 % (w/w) MgCl₂. The arms were removed and placed in small chambers containing 200 μl of ASW of the following composition (in mmol l⁻¹): 400.4 NaCl, 9.6 KCl, 52.3 MgCl₂, 9.9 CaCl₂, 27.7 Na₂SO₄, 20 Tris-HCl, pH 8.3. One of the five arms was used as a control, i.e. stimulated by ACh only, and the others were stimulated by application of exogenous acetylcholine after 10 min of treatment with catecholamines or their derivatives (adrenaline, noradrenaline, dopamine, isoproterenol, octopamine, propranolol, alprenolol, benextramine, phentolamine), with 5-HT or with purines and their derivatives [adenosine, N⁶-cyclohexyladenosine (CHA), 5′-N-ethylcarboxamidoadenosine (NECA), 8-cyclopentyl-1,3-dimethylxanthine (CPT), aminophylline, adenosine-5′-triphosphate (ATP), β,γ-methylene adenosine-5′-triphosphate (Me-ATP), 2-chloroadenosine triphosphate tetrasodium (Cl-ATP), suramin hexasodium and Reactive Blue (RB)]. All chemicals were obtained from Sigma Chemical Co. except adrenaline and noradrenaline, which were purchased from Fluka, and Reactive Blue 2, suramin hexasodium and CPT, which were from Research Biochemicals International.

The method used is based on that described by Mallefet et al. (1991) as adapted and developed by Deheyn (1998). Cell dissociations were performed using 80 specimens. First, the brittlestars were anaesthetized as above. Next, the arms were removed from the discs, cut into small fragments and incubated in 3 ml of pronase (Sigma; 0.5 % in ASW). Tissue digestion took place at 28 °C for 35 min under mild mechanical agitation. Arm fragments were left to settle before the supernatant was removed. The pellet was then rinsed in ASW, resuspended and subjected to further digestion with pronase. This procedure was repeated twice more. Each of the collected supernatants was centrifuged (10 min, 536 g, at room temperature 18 °C). The pellets were resuspended in 1 ml of ASW before being combined and kept at 4 °C. At the end of the dissociation process, 3 ml of cell suspension in ASW was obtained.

The photocytes were then separated from other cell types using a continuous Percoll density gradient. The cell suspension was added to the Percoll solution (Pharmacia, 63 % in ASW, 1050 mosmol l⁻¹) and centrifuged for 30 min at 50 160 g (Beckman L8-70 ultracentrifuge, rotor SW27). The fraction of Percoll solution enriched in photocytes was then collected with a Pasteur pipette, diluted (1:10) in ASW and centrifuged (15 min, 744 g, room temperature). The resulting pellet, i.e. the photocyte concentrate, was resuspended in 2 ml of ASW and kept at 4 °C. This protocol provides a photocyte suspension, the purity of which was verified in a former study using fluorescence microscopy and transmission electron microscopy. Photocytes make up 80 % of the total number of cells, and muscle cells make up 20 %; no remaining nervous elements were detected on the photocytes or in the cell suspension (Deheyn, 1998). Samples (50 μl) of this photocyte suspension were used for the experiments. Injection of drug solutions was controlled manually to avoid mechanical stimulation of luminescence during the assays. Injections of corresponding volumes of ASW served as controls before the assays. These controls indicated that luminescence due to mechanical excitability was negligible.

Light emission was monitored using either a PM 270 D photomultiplier connected via an IL500 amplifier (International Light, USA) to a chart recorder (Servogor S, Germany) or a 1250 Bioorbit luminometer (Bioorbit, Finland) connected to a computer.

The amplitude of light emission (L_max) was the parameter used to characterize light emission by A. squamata and was expressed in megaquanta s⁻¹ (Mq s⁻¹). Results are expressed as a percentage of the corresponding values measured in control arms or in dissociated cells stimulated with acetylcholine without any pretreatment.

Statistical analyses were performed using analysis of variance (ANOVA) or Student’s t-test; each mean value is expressed with its standard error (mean ± S.E.M.) and the number of responses observed (n) in the number of preparations stimulated (N).

Dose–response curves (10⁻⁶ mol l⁻¹ to 10⁻³ mol l⁻¹) for adrenaline, noradrenaline, dopamine and 5-HT were obtained for both clear and black specimens. Except for dopamine on black specimens (which had no significant effect), all the catecholamines tested and 5-HT had a dose-dependent inhibitory effect on ACh-induced luminescence (Table 1), and the number of arms responding (n) was reduced in nearly all cases. This inhibitory effect was not significant at 10⁻⁶ mol l⁻¹. Since the catecholamines had an inhibitory effect on the luminescence of isolated arms, we attempted to define the receptor subtypes involved in the light response. Specific adrenergic agonists (octopamine, isoproterenol) and antagonists (propranolol, alprenolol, phentolamine, benextramine) were tested.

In the first series of experiments, the adrenergic agonists octopamine, which binds selectively to α-adrenoceptors, and isoproterenol, which binds selectively to β-adrenoceptors, were tested at 10⁻⁴ mol l⁻¹ (10 min treatment before ACh stimulation) on isolated arms from eight clear specimens and seven black specimens. The results from these experiments were compared with those from arms treated with noradrenaline and ACh control arms. For both types of specimen, the treatments with α- and β-adrenergic agonists had an inhibitory effect on ACh-induced luminescence and reduced the number of arms responding. The isolated arms of clear specimens treated with noradrenaline, octopamine and isoproterenol showed, respectively, an inhibition of ACh-induced luminescence of 85±4.8 % (P<0.01; n/N=6/8), 97±2.6 % (P<0.01; n/N=2/8) and 95±3.5 % (P<0.01; n/N=2/8).

For the black specimens, there was 68±8.2 % (P<0.01; n/N=7/7) inhibition with noradrenaline, 60±12.4 % (P<0.01; n/N=6/7) with octopamine and 75±6.8 % (P<0.01; n/N=6/7) with isoproterenol.

In another series of experiments, the adrenergic antagonists phentolamine and benextramine, which selectively block α-adrenoceptors, and the adrenergic antagonists propranolol and alprenolol, which selectively block β-adrenoceptors, were tested at 10⁻⁴ mol l⁻¹ (10 min pretreatment, followed by 10 min of noradrenaline treatment before ACh stimulation) on isolated arms from nine clear specimens and eight black specimens. The results showed that, instead of antagonizing the effects of noradrenaline, the α- and β-adrenergic antagonists enhanced the inhibitory action of noradrenaline non-selectively. All the antagonists tested reduced ACh-induced luminescence by more than 92 % (P<0.01) compared with the control (results not shown).

To determine whether the inhibitory effects of catecholamines occur directly on the photocytes or indirectly via nerve endings acting on the photocytes, the catecholamine adrenaline was tested on dissociated photocytes. No significant differences were observed between the samples treated for 5 min with adrenaline (5×10⁻⁴ mol l⁻¹) before ACh stimulation and the controls stimulated by ACh (10⁻³ mol l⁻¹) only (results not shown).

Dose–response curves (10⁻⁶ mol l⁻¹ to 10⁻³ mol l⁻¹) to adenosine and ATP were obtained for clear and black specimens. The purines triggered light responses in the absence of ACh stimulation and, in most cases, potentiated ACh-induced luminescence (Table 2).

Fourteen clear and eight black specimens were used to study the dose-dependent effects of adenosine on ACh-induced luminescence according to the same experimental procedures described above.

Table 2 shows that an application of adenosine to isolated arms of both clear and black specimens triggered luminescence. The pattern of light emission was similar to that of the control. In clear specimens, 10⁻³ mol l⁻¹ adenosine induced luminescence in seven isolated arms out of fourteen. The mean maximal light production (L_max) recorded for adenosine represented 112±54 % of the ACh control response. In black specimens, adenosine (10⁻⁵ mol l⁻¹) induced luminescence in five isolated arms out of eight. At 10⁻⁴ and 10⁻³ mol l⁻¹ adenosine, all the stimulated arms emitted light. The mean maximal light production for adenosine represented 3±1.5 % (10⁻⁵ mol l⁻¹), 11±6.2 % (10⁻⁴ mol l⁻¹) and 28±12.3 % (10⁻³ mol l⁻¹) of the ACh-induced control response.

After treatment with 10⁻⁴ mol l⁻¹ and 10⁻³ mol l⁻¹ adenosine, ACh-induced luminescence for the black specimens was enhanced by, respectively, 247±87 % (P<0.05) and 362±101 % (P<0.01) of the control value. The clear specimen showed no significant difference from the control.

To evaluate the specificity of the adenosine receptors (P₁-purinoceptors) involved in light emission, we tested the effects of adenosine agonists (N⁶-cyclohexyladenosine, CHA; 5′-N-ethylcarboxamidoadenosine, NECA) and antagonists (8-cyclopentyl-1,3-dimethylxanthine, CPT; aminophylline).

Fig. 1A shows the results with the adenosine agonists CHA, which selectively activates the A₁ receptors, and NECA, which actives A₁ and A₂ receptors. Tests were conducted at a concentration of 10⁻⁴ mol l⁻¹ on isolated arms from nine clear specimens and seven black specimens. The results were compared with arms stimulated with adenosine and the ACh control arms. In clear specimens, adenosine, CHA and NECA triggered luminescence, which represented, respectively, 300±108 %, 41±9 % and 308±112 % of the control value. In black specimens, adenosine-stimulated luminescence represented 153±41 %, CHA-stimulated luminescence represented only 8±3 % and NECA-stimulated luminescence represented 204±28 % of the control luminescence.

Fig. 1B shows the results with the adenosine antagonist CPT, which selectively blocks the A₁ receptors, and the adenosine antagonist aminophylline, which blocks A₁ and A₂ receptors. Tests were performed at 10⁻⁴ mol l⁻¹ on isolated arms from thirteen clear specimens and eight black specimens. For the clear specimens, CPT and aminophylline reduced the luminescence induced by adenosine by 47±16 % (P<0.05) and 91±2 % (P<0.01) respectively. Similar results were obtained for black specimens, (Fig. 1B) in which CPT induced a 35±13 % (P<0.05) reduction of light emission and aminophylline a 96±1.5 % reduction (P<0.01).

To test the reversibility of these purinergic antagonists, the arms treated with CPT and aminophylline were washed for 1 h with sea water. After washing, the effect were 100 % reversible.

Eight clear and seven specimens were used to study the effects of ATP (10⁻⁶ to 10⁻³ mol l⁻¹) on ACh-induced luminescence.

Table 2 shows that the application of ATP at all concentrations to isolated arms from clear specimens failed to induce light emission, but that ACh-induced light emission was enhanced by 314 % (P<0.01) of the control value by 10⁻³ mol l⁻¹ ATP. Application of ATP to isolated arms from black specimens triggered luminescence at 10⁻⁴ mol l⁻¹ (n/N=3/7) and 10⁻³ mol l⁻¹ (n/N=7/7). The luminescence represented respectively 1.3 % (10⁻⁴ mol l⁻¹) and 16 % (10⁻³ mol l⁻¹) of the ACh control response. ACh-induced luminescence was also enhanced at 10⁻⁴ mol l⁻¹ and 10⁻³ mol l⁻¹ ATP and represented, respectively, 222 % and 244 % (P<0.05) of the control response.

To evaluate the specificity of the ATP receptors (P₂ purinoceptors) involved in light emission, we tested the effects of ATP agonists (2-chloroadenosine triphosphate tetrasodium, Cl-ATP; β,γ-methylene adenosine-5′-triphosphate, Me-ATP) and antagonists (suramin; Reactive Blue 2, RB).

Fig. 2A shows the results with the ATP agonists, Cl-ATP, which selectively activates P_2y purinoceptors, and Me-ATP, which selectively activates P_2x purinoceptors. Tests were conducted at 10⁻⁴ mol l⁻¹ on isolated arms from ten clear specimens and six black specimens and compared with the arms stimulated with ATP and the ACh control arms. For the clear specimens, ATP, Cl-ATP and Me-ATP triggered luminescence which represented, respectively, 48±17 %, 67±31 % and 7.6±0.8 % of the ACh response. For the black specimens, ATP-induced luminescence represented 96±8 % of the control value, Cl-ATP-induced luminescence represented 83±6 % and Me-ATP-induced luminescence represented only 5±3 %.

Fig. 2B illustrates the results with the ATP antagonist suramin, which blocks P_2x and P_2y purinoceptors, and the ATP antagonist RB, which selectively blocks P_2y purinoceptors. Tests were performed at 10⁻⁴ mol l⁻¹ on isolated arms from eight clear specimens and seven black specimens. For the clear specimens, suramin and RB significantly reduced the luminescence induced by ATP by 81±8 % (P<0.05) and 93±7 % (P<0.01), respectively. We obtained a similar result for the black specimens, in which suramin induced a 75±13 % (P<0.05) reduction in light emission and RB totally inhibited luminescence.

To test the reversibility of these purinergic antagonists, arms treated with suramin and 10⁻⁴ mol l⁻¹ RB were washed for 1 h with sea water. After washing, reversibility was 100 % for suramin and 61 % for RB in the presence of ATP.

To determine whether the purines trigger luminescence by acting directly on the photocytes via purinergic receptors or indirectly via nerve endings, adenosine and its agonist NECA and ATP and its agonist Cl-ATP were tested on dissociated photocytes.

In the first series of experiments, samples of dissociated photocytes (N=6) were stimulated by application of ACh, adenosine or NECA (all 10⁻⁴ mol l⁻¹). All three drugs triggered luminescence in photocytes. Maximal levels of light production induced by ACh and adenosine were not significantly different, while NECA induced a higher level of luminescence (P<0.05) (Fig. 3).

In another series of experiments, samples of dissociated photocytes (N=6) were stimulated by application with ACh, ATP or Cl-ATP (all 10⁻⁴ mol l⁻¹). All three drugs triggered luminescence of photocytes. Maximal levels of light production induced by ACh, ATP and Cl-ATP were not significantly different (Fig. 3).

Previous studies on clear and black specimens of Amphipholis squamata have shown a difference in the intensity of light emission and pharmacological differences between the two (De Bremaeker et al., 1996; Deheyn et al., 1997). In the present study, we also observed a difference in the intensity of light emission (of two orders of magnitude), but our results are presented as percentages, not as absolute values. Some pharmacological differences were also observed: in both types of specimen, light production was inhibited by adrenaline, noradrenaline and 5-HT, with inhibition being more marked in clear specimens. Dopamine strongly inhibited ACh-induced luminescence in clear specimens, but had no effect in black specimens. Adenosine induced luminescence in isolated arms of black specimens at 10⁻⁵ mol l⁻¹, but 10⁻³ mol l⁻¹ adenosine was required to induce luminescence in clear specimens. In addition, ATP induced luminescence in isolated arms of black specimens at 10⁻⁴ mol l⁻¹, but failed to trigger luminescence at all concentrations in clear specimens. Differences between clear and black specimens may be either at the receptor level, i.e. the extrinsic control mechanism, or at the level of the regulation of light production inside the photocyte, i.e. the intrinsic control mechanism.

Previous studies have reported immunohistochemical and pharmacological evidence for the presence and involvement of adrenergic substances in the control of bioluminescence in invertebrates: catecholamines induced luminescence in the sea pansy Renilla koellikeri (Anctil et al., 1984; Awad and Anctil, 1993); adrenaline and 5-HT showed excitatory effects in the scale-worm Harmothoe (Miron et al., 1987; Anctil et al., 1989).

In the present study, all the catecholamines tested (adrenaline, noradrenaline, dopamine) and 5-HT had a dose-dependent (10⁻⁵ mol l⁻¹ to 10⁻³ mol l⁻¹) inhibitory effect on ACh-induced luminescence. This result suggests the involvement of adrenergic receptors in the control of luminescence in Amphipholis squamata. We then attempted to define the receptor subtypes involved in the light response. The α-adrenergic agonist octopamine and the β-adrenergic agonist isoproterenol were tested. Both inhibited ACh-induced luminescence. The experiments with selective α- and β-adrenergic antagonists did not confirm the presence of both α- and β-subtypes of adrenoceptor; all the antagonists had agonistic effects and strongly inhibited ACh-induced luminescence. As in other studies on invertebrates (Hanai and Kitajima, 1984; Awad and Anctil, 1993), the adrenergic receptors implicated seem to differ from those of vertebrates. The experiments with dissociated photocytes showed that catecholamines have no effect if applied directly onto the photocytes. This result could indicate that the inhibitory effect of catecholamines on luminescence is indirect, via nerve endings, and not on photocytes.

Our study shows that luminescence in the ophiuroid is triggered by ACh and inhibited by catecholamines. This antagonistic effect between cholinergic and catecholaminergic systems is consistent with observations in other echinoderms, such as the starfish Asterias forbesi (Anderson, 1954) and holothurians (Wyman and Lutz, 1930; Prosser and Judson, 1952; Von Euler et al., 1952).

The extracellular actions of purine compounds are mediated by P₁ purinoceptors activated by adenosine and AMP and P₂ purinoceptors activated by ADP and ATP (Burnstock, 1978). P₁ purinoceptors can be divided into A₁, A₂ and A₃ subclasses (Fredholm et al., 1994), and P₂ purinoceptors can be divided into P_2x, P_2y, P_2z, P_2t or P_2u subclasses (Burnstock and Kennedy, 1985; Gordon, 1986; O’Connor et al., 1991).

There are many reports of the effects of purine compounds on invertebrates, including echinoderms (for a review, see Burnstock, 1996). Purines have been shown to be pharmacologically active on several muscle tissue preparations isolated from starfish (Hoyle and Greenberg, 1988; Knight et al., 1990), sea urchins (Hoyle and Greenberg, 1988; Shingyoji and Yamaguchi, 1995) and holothurians (Hoyle and Greenberg, 1988). Although the presence of P₁- and P₂-like purinoceptors was suggested, attempts to define the receptor subtypes failed (Hoyle and Greenberg, 1988; Knight et al., 1990).

In the present study, the purine compounds adenosine (10⁻⁵ mol l⁻¹ to 10⁻³ mol l⁻¹) and ATP (10⁻⁴ mol l⁻¹ and 10⁻³ mol l⁻¹) triggered luminescence in isolated arms of the ophiuroid Amphipholis squamata, with adenosine being more potent. These results suggest the presence of both P₁- and P₂-like purinoceptors. Selective purinoceptor agonists and antagonists were used to define the receptor subtypes involved in the light response.

Experiments with the selective P₁ purinoceptor agonists CHA (A₁ agonist) and NECA (A₁ and A₂ agonist) indicated the presence of both A₁- and A₂-like receptors, with a predominance of the latter type. The results with the P₁ purinoceptor antagonists CPT (A₁ antagonist) and aminophylline (A₁ and A₂ antagonist) confirmed these results. Experiments with the selective P₂ purinoceptor agonists Me-ATP (P_2x agonist) and Cl-ATP (P_2y agonist) demonstrated the presence of both P_2x- and P_2y-like receptors, with a predominance of the latter type. The results with the P₂ purinoceptor antagonists RB (P_2y antagonist) and suramin (P_2x and P_2y antagonist) supported this view. This is the first report of the presence of P₁- and P₂-like purinoceptors subtypes in echinoderms.

Adenosine and ATP and their respective analogues, NECA and Cl-ATP, were able to trigger luminescence in the dissociated photocyte preparations. This result suggests the presence of purinoceptors on the photocyte membrane.

Our results also showed that adenosine and ATP had a potentiating effect on ACh-induced luminescence. The potentiation induced by adenosine represented 2.5 times (10⁻⁴ mol l⁻¹) and 3.6 times (10⁻³ mol l⁻¹) the ACh control response, while for ATP it represented 3.1 times the control response (10⁻³ mol l⁻¹). The potentiation was obtained both when the isolated arms were treated for 10 min with purines and when ACh and the purines were applied together. Numerous studies have shown ATP to be co-stored and co-released with ACh (Silinsky, 1975; Meunier et al., 1975; Stadler and Fuldner, 1981; Kolb and Wakelam, 1983; Hume and Honig, 1986). Purines have also been shown to potentiate the effects of ACh (Akasu and Koketsu, 1985; Cox and Walker, 1987; Fu, 1994; Schrattenholz et al., 1994; Nakaoka and Yamashita, 1995). There are fewer reports on the stimulating effects of adenosine. However, muscular excitatory actions have been demonstrated in Actinia equina, a sea anemone (Hoyle et al., 1989), in Psammechinus miliaris, a sea urchin (Gustafson, 1991), and in Helix, a snail (Cox and Walker, 1987). This result is not the first report of a modulatory effect of adenosine or ATP on light emission; such modulation has already been observed in isolated photophores of the teleost Porichthys notatus (Rees et al., 1991; Mallefet et al., 1993).

The concentrations used, 10⁻⁴ mol l⁻¹ and 10⁻³ mol l⁻¹, for catecholamines and purines to be effective may seem high, but a low sensitivity to putative neuromediators is a feature that has previously been observed for isolated luminescent organs from invertebrates (Nicolas et al., 1978; Nathanson, 1979; Anctil, 1981).

All our pharmacological results were obtained using drugs designed to characterize mammalian receptors. We cannot rule out the possibility that ophiuroid receptors are somewhat different from those encountered in mammalian tissues. Therefore, we must be careful in extrapolating pharmacological results from mammalian to invertebrate tissue.

In summary, our results suggest that, in addition to the cholinergic system involved in the control of luminescence in Amphipholis squamata, purines play a synergistic role through P₁- and P₂-like purinoceptors and catecholamines play a neuromodulatory inhibitory role.

We thank I. Cogneau for technical assistance, D. Deheyn and E. Lamarque for field assistance and Professor J. Avoine for providing facilities at the Marine Laboratory of Luc-sur-Mer (France).. This work is a contribution of the CIBIM (centre interuniversitaire de biologie marine) and was supported financially by grants FRFC 6.231.85 and FDS/UCL. J.M. is research associate of the National Fund for Scientific Research (FNRS, Belgium).