pineal organ and retina

2 downloads 47 Views 433KB Size Report
(Esox lucius, L.) pineal organ and retina: effects of temperature. Received: 11 September 1995/Received after revision: 19 November 1995/Accepted: 17 ...
Pflügers Arch – Eur J Physiol (1996) 342 : 386 –393

© Springer-Verlag 1996

O R I G I NA L A RT I C L E

Jack Falcón · Valérie Bolliet · Jean Pierre Collin

Partial characterization of serotonin N - acetyltransferases from northern pike (Esox lucius, L.) pineal organ and retina: effects of temperature

Received: 11 September 1995 /Received after revision: 19 November 1995 /Accepted: 17 January 1996

Abstract In vertebrates, the nocturnal rise in pineal organ and retinal melatonin synthesis results from the increase in the activity of the serotonin N-acetyltransferase (NAT), a cAMP-dependent enzyme. In the fish pineal organ in culture, light and temperature act in a similar manner on cAMP content and NAT activity. It is not known whether the effects of temperature are mediated through cAMP or through modifications of NAT kinetics. The present study was designed : (1) to find out whether NAT activity from pineal organ homogenates is similar to NAT activity from pineal organs in culture, with regard to variations in temperature, and (2) to compare NAT activity from the pineal organ and the retina. Pineal organ and retinal NAT activity increased linearly with protein concentrations. Higher activities were obtained with 0.2 mol/ l of phosphate buffer, pH 6. Higher molarity or a higher pH induced a decrease in retinal and pineal organ NAT activity: retinal NAT was more sensitive than pineal organ NAT to changes in molarity, whereas the opposite held true as far as pH was concerned. Pineal organ and retinal NAT obeyed the Michaelis-Menten equation with respect to increasing concentrations of acetylcoenzyme A. With increasing concentrations of tryptamine: (1) pineal organ NAT activity increased in a manner suggesting positive co-operativity, (2) retinal NAT displayed, after an initial increase, inhibition by substrate. The kinetics of the reactions were temperature dependent. Maximal activities were reached at 18/20 °C in the pineal organ and at 37 °C in the retina.

J. Falcón (*) · V. Bolliet · J.P. Collin Laboratoire de Neurobiologie et Neuroendocrinologie Cellulaires, URA CNRS 1869, Université de Poitiers, 40 avenue du Recteur Pineau, F-86022 Poitiers-Cedex, France V. Bolliet Département de Biologie, Université de Montréal, CP 6128, Succ. A, Montréal, Québec, Canada H3C 3J7

The present study is the first to describe the optimum conditions for the assay of NAT activity in homogenates from the retina of fish and from the pineal organ of poikilotherms, and also the first to compare some characteristics of NAT activity from these two analogous organs. Our results suggest that the effects of temperature on melatonin production are mediated, at least in part, through modifications of NAT kinetics. Future studies will aim to clarify whether the activities measured in the pineal organ and retinal homogenates reflect the presence of one or of several enzymes. Key words Serotonin N-acetyltransferase · Melatonin · Photoperiod · Temperature · Pineal organ · Retina · Fish

Introduction Melatonin, a 5-methoxyindole produced by the pineal organ and the retina of vertebrates, is an internal “Zeitgeber” involved in the timing and control of a number of rhythmic functions and behaviours (refs. in [23, 38]). In all species investigated so far, melatonin biosynthesis and release are lower during day-time than during night-time [6]. These oscillations result from daily variations in the activity of the acetyl-coenzyme A (acetyl-coA) arylalkylamine N-acetyltransferase (NAT; EC 2.3.1.87) which converts serotonin to Nacetylserotonin [25]. The activity of hydroxyindole-Omethyltransferase (HIOMT; 2.1.1.4), which catalyses methylation of N-acetylserotonin to produce melatonin, does not display significant variations throughout a 24-h light/ dark (L / D) cycle [25]. In poikilotherms, light acting on pineal organ and retinal photoreceptors inhibits NAT activity from these cells (refs. in [11]). Thus, under a 24-h L / D cycle, the rhythmic pattern of melatonin secretion reflects the

387

rhythmic activity of NAT. In addition to light, evidence has been accumulating to indicate that temperature is also involved in the control of melatonin production. It has been suggested that the amplitude, duration and phase of the melatonin rhythm in the pineal organ and in the serum of ectotherms result from the interaction of photoperiod and temperature cycles [12, 13, 22, 29, 34]. Thus, the pineal organ might function as a transducer of both photic and thermal information [12, 21, 29, 37]. It is known that the pineal organ, by means of its neurohormonal output, may influence thermoregulated functions and behaviours in ectotherms and endotherms as well [16, 20, 21, 29, 33, 38]. Most of our knowledge on the effects of temperature on melatonin secretion refers to in vivo studies of a few amphibians and reptiles [8, 13, 22, 32, 34]. In vitro studies showing a direct effect of temperature on the pineal organ or retinal melatonin secretion are scarce. In the frog (Rana perezi): (1) previous acclimatation influences occular NAT activity and melatonin secretion in culture, and (2) biochemical characterization of NAT indicated thermal sensitivity of the enzyme [1, 2, 30]. In the pineal organ, it has been observed that the circadian oscillators which drive the melatonin rhythm in the anole, pike and white sucker, are damped at low temperatures [4, 17, 39]. Temperature cycles can influence the amplitude of the pineal organ melatonin secretion rhythm in the pike and white sucker maintained either under L / D or under D/D cycles [12, 37]. Finally, it has been shown that temperature can affect cAMP formation and NAT activity in a similar manner, either in trout or in pike pineal organs in culture [9, 11, 26, 27]. Because NAT is a cAMP-dependent enzyme, it may be possible that the effects of temperature on NAT activity, observed in cultured fish pineal organ, result from a modulation of cAMP metabolism. Alternatively they might be due to a direct effect on enzyme kinetics. To our knowledge, very few studies deal with the characterization of NAT in vertebrates, and none in fish. The present investigation was initiated to determine optimum conditions for quantitative measurement of NAT activity in pineal organ and retinal homogenates from northern pike, with special attention to the effects of temperature.

Materials and methods Animals Pike (Esox lucius L., Teleost) of both sexes, with body weights ranging from 500 g to 2000 g, originated from ponds at Poitou-Charentes (France). They were obtained between December and March. In the laboratory, they were maintained for no more than 24 h in oxygenated recycled filtered pond water under conditions resembling their natural winter habitat with respect to temperature (+7 ± 3 °C) and illumination. Animals were killed by decapitation in the middle of the dark period. The pineal organs and the retinas were quickly removed

under dim red light and frozen in liquid nitrogen. Usually, darkadapted retinas were easily detached from the pigmented epithelium. Samples were kept at [80 °C for no more than a week before the NAT assay was run. Because pike are wild animals, which cannot be bred in hatcheries, their availability depended totally on the fisheries. This explains why (1) we could not choose the sex, age size and season for the experiments, and (2) why we could not repeat each experiment more than twice.

NAT activity assay Unless otherwise specified, individual or pooled organs were sonicated in 0.25 mol / l phosphate buffer (pH 6.8). An aliquot of the homogenate was kept for protein determination, using the colorimetric method of Bradford [5], with BSA as a standard. Homogenates were then centrifuged for 30 s at 11 000 rpm (8000 g), and + 4 °C. Supernatants were kept on ice (no more than 5 min) until the NAT assay. The enzymatic assay was based on the method described by Sugden et al. [25] with slight modifications [10]. In a typical assay a 50-µl homogenate aliquot (containing 150 / 250 µg pineal organ proteins or 650 / 750 retinal proteins) was added to 50 µl of a solution containing tryptamine (Sigma, France), acetyl-CoA (Boehringer Manheim, France) (final concentrations were, respectively, 10 and 0.5 mmol / l) and [3H]acetyl-CoA (Amersham, France; final specific activity 148 GBq mmol). Special conditions are described below. After 20 min, the reaction was stopped by the addition of 1 ml of chloroform, at + 4 °C. Acetylated products were extracted into the chloroform by vigorous shaking. After removal of the aqueous phase, the chloroform was washed once with phosphate buffer. The organic phase was evaporated to dryness in a counting scintillation vial, and 10 ml of scintillant (Optiphase, LKB, France) was added. Radioactivity was counted using a Wallak rack b scintillation counter. Each assay was run in duplicate, and each experiment was repeated twice.

Experimental design Effects of protein concentration Pineal organ and retinal homogenates were sequentially diluted in phosphate buffer (0.25 mol /l, pH 6.8), and incubated for 20 min at +37 °C.

Effects of duration of incubation The effects of different durations of incubation (5 – 60 min) were tested at + 4, + 25 and + 37 °C.

Effects of different temperatures of incubation Homogenates were incubated for 20 min at temperatures ranging from + 4 to + 56 °C.

Effects of buffer molarity Phosphate buffer solutions (pH 6.8) of different molarities (0.1, 0.2, 0.25 and 0.3 mol /l) were made to homogenize tissues and to prepare incubation solutions. Assays were run for 20 min at + 20 °C.

388 Effects of buffer pH Different tissue extracts and incubation solutions were prepared in phosphate buffer (0.25 mol /l) of pH ranging between 6 and 8. Assays were run for 20 min at +20 °C.

Effects of substrate and co-substrate concentrations Assays were run in phosphate buffer (0.25 mol/l, pH 6.8) for 20 min at +20 °C. When the tryptamine concentration was varied (0.125 to 10 mmol/l) acetyl-CoA was maintained constant at 0.5 mmol / l. When the acetyl-CoA concentration was varied (0.125 to 2 mmol /l) tryptamine was maintained constant at 10 mmol/l.

Analysis of the data Data were analysed using the GraphPAD iterating curve-fitting programme (GraphPAD, ISI software). Graphs were drawn using the Sigmaplot curve-fitting programme (Jandel Scientific). For the time-course experiments, data were fitted to a linear regression (+4 °C) or to a rectangular hyperbola equation (+ 25 and +37 °C): V=

Vmax × t t + t½

(1)

(V = velocity, t = time) To determine the apparent Km and Vmax, data were fitted to the Michaelis-Menten equation: V=

Vmax × [S] [S] + Km

(2)

(Km = Michaelis constant), ([S] = substrate concentration). Linearization was achieved by plotting S/V against S (refs, in [15, 40]). According to Henderson [15], this type of plot is the safest to use, when compared to the 1/V against 1/S, or V against V/S plots. When linearization was not perfectly acheived by plotting S/V against S, we used the Hill equation: V=

Vmax × [S]h [S]h + Km

(3)

(h = Hill coefficient), which allows the detection of possible co-operative phenomena [15, 28]. In some cases, the model from Haldane was followed in order to determine a possible inhibition by substrate (refs. in [28]): V=

Vmax 1+Km / [S] + [S]/ Ki

(4)

Ki = inhibition constant Values are expressed as the mean ± SE of two independent experiments.

Results Effects of protein concentration The relationship between pineal organ NAT activity (expressed in pmol/ h of [3H]acetyl-tryptamine formed) and homogenate protein concentration, ranging from 0 to 1250 µg was linear (Fig. 1), leading to an average activity of 0.46 ± 0.03 pmol· h-1 ·µg-1 of proteins (r = 0.99).

Fig. 1 N-Acetyltransferase (NAT) activity as a function of protein concentration. Pineal and retinal homogenates were sequentially diluted in phosphate buffer (0.25 mol /l, pH 6.8) and incubated for 20 min at +37 °C in the presence of tryptamine (10 mmol /l), acetylCoA (0.5 mmol /l) and [3H]acetyl-CoA (148 GBq / mmol). Each point corresponds to the mean of duplicate determinations from one representative experiment

Retinal NAT activity increased linearly with protein concentrations up to 1000 µg (r = 0.99) giving an average activity of 6.23 ± 0.53 pmol ·h[1 · µg[1 of proteins. Over 1000 µg, the relationship displayed a downward curve (Fig. 1). Effects of duration of incubation At + 4 °C, pineal organ and retinal NAT activity increased linearly with increasing incubation times (Fig. 2). Activity was, respectively, 0.46 ± 0.11 and 1.8 ± 0.23 pmol· h[1 · µg[1. At 25 and 37 °C, data obtained both from the pineal organ and the retina fitted a rectangular hyperbola equation (r = 0.95; Fig. 2). 1. In the pineal organ the relationship “activity as a function of time” was linear for the first 20 /30 min of incubation at 25 °C, and for the first 10 / 15 min at 37 °C (Fig. 2). Thereafter it tended to reach a plateau both at 25 °C (Vmax = 1.19 ± 0.17 pmol /µg; t1/2 = 36.63 ± 8.5 min) and at 37 °C (Vmax = 0.35 ± 0.2 pmol/ µg; t1/2 = 12.28 ± 9 min).

389

Fig. 3 N-Acetyltransferase (NAT) activity as a function of temperature. Pineal and retinal homogenates were incubated for 20 min at the temperatures indicated, in the presence of tryptamine (10 mmol / l), acetyl-CoA (0.5 mmol / l) and [3H]acetyl-CoA (148 GBq / mmol), all in phosphate buffer (0.25 mol / l, pH 6.8). Each point corresponds to the mean of duplicate determinations from one representative experiment

homogenates, NAT activity increased progressively from + 4 °C to + 37 °C, and then decreased abruptly. Effects of buffer molarity and pH Fig. 2 N-Acetyltransferase (NAT) activity as a function of time. Pineal and retinal homogenates were incubated for the times and at the temperatures indicated, in the presence of tryptamine (10 mmol/l), acetyl-CoA (0.5 mmol/l) and [3H]acetyl-CoA (148 GBq/mmol), all in phosphate buffer (0.25 mol/l, pH 6.8). Each point corresponds to the mean of duplicate determinations from one representative experiment

2. In the retina linearity was observed for an incubation period of at least 40 min at 25 °C and 20 min at 37 °C, after which the rate of product formation started to decline (Fig. 2). When fitted to a rectangular hyperbolic function, the calculated Vmax was 3.3 ± 0.8 pmol/µg (t1/2 = 98.7 ± 34.1 min) at 25 °C, and 1.56 ± 0.1 pmol /µg (t1/2 = 11 ± 2.4 min) at 37 °C. Effects of different temperatures of incubation Under the general conditions described in Materials and methods, NAT activity was assayed for 20 min at different temperatures ranging from + 4 °C to + 56 °C (Fig. 3). In the pineal organ homogenates, NAT activity increased with increasing temperatures, to reach optimal values between + 17 °C and + 20 °C, after which it decreased progressively. In the retinal

Both pineal organ and retinal NAT activity were two to four times higher with 0.2 mol/ l than with 0.1 or 0.3 mol /l of phosphate buffer pH 6.8 (Fig. 4). Fig. 4 N-Acetyltransferase (NAT) activity as a function of buffer molarity. Pineal and retinal homogenates were prepared in phosphate buffer (pH 6.8) at the molarity indicated, and incubated for 20 min at + 20 °C. Tryptamine (10 mmol /l), acetyl-CoA (0.5 mmol / l) and [3H]acetyl-CoA (148 GBq / mmol) were also prepared in pH 6.8 buffer at the corresponding molarity. Each point corresponds to the mean of duplicate determinations from one representative experiment

390

Fig. 5 N-Acetyltransferase (NAT) activity as a function of buffer pH. Pineal and retinal homogenates were prepared in phosphate buffer (0.25 mol/l) at the pH indicated, and incubated for 20 min at +20 °C. Tryptamine (10 mmol/l), acetyl-CoA (0.5 mmol /l) and [3H]acetyl-CoA (148 GBq/mmol) were also prepared in 0.25 mol / l buffer at the corresponding pH. Each point corresponds to the mean of duplicate determinations from one representative experiment

At a fixed molarity (0.25 mol / l), increasing pH from 6 to 7.5 induced a slight but progressive decrease in pineal organ NAT activity. This decrease was more pronounced at pH 8 (a 50 % reduction was observed vs the activity measured at pH 6; Fig. 5). Retinal NAT activity was also maximal at pH 6, but increasing pH to 6.5 resulted in a 60 % reduction in enzyme activity (Fig. 5). With higher pH values, retinal NAT activity continued to decrease and represented, at pH 8, less than 10% of the activity measured at pH 6. Effects of different acetyl-CoA concentrations NAT activity, assayed at + 25 °C for 20 min, increased with increasing acetyl-CoA concentrations to reach a plateau (Fig. 6). The experimental data fitted adequately the Michaelis-Menten equation both in the pineal organ (Vmax = 1.06 ± 0.15 pmol· h[1 · µg[1, Km = 0.34 ± 0.13 mmol/ l) and in the retina (Vmax = 0.99 ± 0.07 pmol· h[1 · µg[1, Km = 0.1 ± 0.03 mmol /l; Fig. 6). These values were in good agreement with those obtained after transformation of the data by plotting S/V against S (insets in Fig. 6), or 1 /V against 1 /S (data not shown).

Fig. 6 N-Acetyltransferase (NAT) activity as a function of acetylCoA concentration. Pineal and retinal homogenates were incubated for 20 min at +20 °C, in the presence of tryptamine (10 mmol /l), [3H]acetyl-CoA (148 GBq /mmol), and acetyl-CoA at the concentrations indicated, all in phosphate buffer (0.25 mol / l, pH 6.8). Insets correspond to the S/V against S plot. Each point corresponds to the mean of duplicate determinations from one representative experiment. Pineal : Vmax = 1.06 ± 0.15 pmol · h[1 ·µg[1 and Km = 0.34 ± 0.13 mmol /l; retina : Vmax = 0.99 ± 0.07 pmol · h[1 · µg[1 and Km= 0.1 ± 0.03 mmol /l

found, in agreement with the values obtained after S/V was plotted against S (Fig. 7). Observation of the S/V against S plot might suggest a possible departure from linearity (upper inset in Fig. 7), which was still more pronounced with the 1 /V against 1 /S double reciprocal plot (data not shown). Thus, the Hill equation was applied. A Hill coefficient of 0.73 ± 0.14 was found, and the corresponding Vmax and Km were, respectively, 7.34 ± 2.72 pmol· h[1 · µg[1 and 7.48 ± 2.85 mmol/l. Retina

Effects of different tryptamine concentrations Pineal organ When the experimental data were fitted to the Michaelis-Menten equation, a Vmax of 5.1 ± 0.45 pmol·h[1 ·µg[1, and a Km of 5.96 ± 1.29 mmol / l were

NAT activity as a function of tryptamine concentration did not follow the Michaelis-Menten equation, but the model from Haldane (Fig. 7). The parameters, estimated by computer calculation, were Vmax = 1.43 ± 0.17 pmol· h[1 · µg[1, Km = 0.12 ± 0.08 mmol/l and Ki = 25.6 ± 1 mmol /l.

391

Fig. 7 N-Acetyltransferase (NAT) activity as a function of tryptamine concentration. Pineal and retinal homogenates were incubated for 20 min at +20 °C, in the presence of acetyl-CoA (0.5 mmol /l), [3H]acetyl-CoA (148 GBq/mmol), and tryptamine at the concentrations indicated, all in phosphate buffer (0.25 mol /l, pH 6.8). Insets correspond to the S/V against S plot. Each point corresponds to the mean of duplicate determinations from one representative experiment. Pineal : Vmax = 7.34 ± 2.72 pmol· h[1 · µg[1 and Km = 7.48 ± 2.85 mmol /l; retina: Vmax = 1.43 ± 0.17 pmol · h[1 · µg[1. Km = 0.12 ± 0.08 mmol /land Ki = 25.6 ± 1 mmol / l

Discussion Our results indicate that there was a direct proportionality between pineal organ NAT activity and protein concentration (up to 1250 µg) in the homogenates, suggesting that the initial velocity of the reaction was proportional to the amount of enzyme present [28]. This also held true for the retinal homogenates as long as the protein concentration did not exceed 1000 µg. For higher concentrations, a very slight downward curving was observed. According to Tipton [28], three possible explanations may account for a downward curvature of this type : (1) the detection method may become rate-limiting at high enzyme concentrations, (2) the reaction is reaching equilibrium within the assay time used, (3) a dissociable inhibitor is being formed which accumulates in noticeable amounts. The latter possibility might well be retained in view of the effects

of substrate concentration on retinal NAT activity (see below). In this experimental series, retinal NAT activity appeared higher than pineal organ NAT activity. These variations probably result from the assay conditions (e.g. temperature, buffer molarity and pH). Indeed, when phosphate buffer was 0.1 or 0.2 mol/l, pineal organ and retinal NAT activity were similar. In contrast, in 0.25 mol/ l phosphate buffer the enzymatic activity remained rather high in the retina, but was more drastically affected in the pineal organ. Initially, our experiments used 0.25 mol/ l phosphate buffer, a choice dictated by previous experiments in the chick, indicating that NAT activity improved at this molarity [14]. In this respect, NAT activity from pike and chicken pineal organs differ from that of the rat pineal organ, which is insensitive to buffer molarity [14]. The molarity-dependent differences in pike retinal and pineal organ NAT activity might reflect different properties of the respective enzymes. This hypothesis is supported by the observation that pineal organ NAT activity was less sensitive to increasing pH when compared to retinal NAT. Both exhibited optimal activity at pH 6. It might be interesting to investigate whether these pH-dependent differences are still observed at other times of the year. Indeed, results obtained in the chinook salmon pineal organ indicated that sensitivity of HIOMT to pH varied with season [3]. Maximal NAT activity is obtained at pH 6.8 in the retina and in the pineal organ of mammals [18, 19, 31], at pH 7.4 in the Harderian gland of the syrian hamster, and at pH 6.4 /6.5 in the retina of the frog ([1] and refs. therein). In the frog retina, the enzyme activity was not detected at pH below 6.1 ([1] and refs. therein). Altogether, this points to species-dependent variations in NAT activity from fish to mammals. With respect to the acetyl donor, pike retinal and pineal organ NAT behaved similarly, obeying the Michaelis-Menten relationship. The apparent of Km values for acetyl-CoA, observed in the pike pineal organ and retina, were within the range of those previously reported for mammals [18, 24, 35]. However, in the pike, affinity for the acetyl donor appeared higher for retinal than for pineal organ NAT. Retinal and pineal organ NAT behaved differently with regard to variations in tryptamine concentration. Data from the pineal organ NAT followed the Hill equation, suggesting the existence of some co-operative phenomena [28]. The apparent Km obtained for tryptamine (7.4 mmol / l) was within the range of those values previously obtained for the mammalian NAT (0.1 to 4.5 mmol /l) [7, 18, 19, 24, 35]. In the pike retina, NAT activity failed to obey the Michaelis-Menten equation. Indeed, after an initial rapid increase in acetyl-tryptamine formation, an inhibition in activity was observed with acceptor-amine concentrations over 0.3 mmol/ l. It is unlikely that inhibition resulted from a contaminant present in the substrate [28]. If this were

392

to have been the case, one would have expected to see inhibition also with the pineal organ NAT. The observed relationship between velocity and substrate concentration may reflect inhibition by a high substrate concentration in the pike retina. A similar situation has been described for HIOMT activity in the pineal organ of the chinook salmon [3] as well as for ocular NAT activity in Rana perezi [2]. In the frog, inhibition of NAT activity by substrate was observed with serotonin but not with tryptamine, and only at high (+ 25 °C) temperatures. It is not known why tryptamine inhibited NAT activity in the eye of the pike but not in that of the frog. Both in the pineal organ and retina, the reaction was linear with the time of incubation. After a while, the length of which depended on the temperature, linearity was progressively lost. Several explanations may account for the departure from linearity (substrate depletion, equilibrium, product inhibition, enzyme instability, etc. [28]). Whatever it may be, here again some differences appeared between pike pineal organ and retinal NAT assayed under identical conditions. As described for the frog retina, there was a positive thermal modulation of ocular NAT activity in the pike, the activity increasing with temperature from +4 to + 37 °C. Among other explanations, the decrease observed at temperatures over + 37 °C might reflect modifications in the structure (tertiary or quaternary ?) of the enzyme, or degradation. In contrast, pineal organ NAT activity increased with temperature to peak between +17 and + 21 °C. For warmer temperatures a progressive decline was observed. It is noteworthy that the profile of the curve obtained with pineal organ homogenates could be superimposed on the profiles obtained after measurement of cAMP accumulation and NAT activity in pike pineal organs cultured at different temperatures [9, 11, 27]. Thus, although NAT is a cAMP-dependent enzyme (see Introduction), our results suggest that the response of NAT to varying temperatures mainly reflects, in organ culture, thermosensitivity of the enzyme and not a regulatory mechanism involving cAMP. These results are in good agreement with the observation that, under constant darkness, temperature cycles (13 / 17 °C or 10/20 °C) can synchronize melatonin production, which peaks with the warm temperature [12]. The observation that in other species (white sucker, anole) melatonin peaks with the low temperature [29, 37] might reflect different adaptative mechanisms at the level of the enzyme. For example, NAT activity of trout pineal organs cultured at different temperatures exhibited variations similar to those described above for the pike, but with peak activities between 12 °C and 17 °C [9, 11, 26]. Whatever they may be, at the cellular level the mechanisms involved in the control of melatonin production by temperature are too complex to be explained by an effect of the external stimulus on NAT kinetics. Indeed, in pike pineal organs cultured under

a L / D cycle at 10 °C, a 20 °C temperature pulse enhanced melatonin production when applied during the night, but inhibited it when applied during the preceding light phase [12]. The question also remains as to what is the functional significance of the differences herein evidenced between pineal organ and retinal NAT, with regard to the effects of temperature as well as of substrate concentration, pH and molarity. They reflect either the presence of a different NAT in each organ, or of more than one enzyme in the retina. Interestingly, it has been recently shown that the chicken pineal organ contains an arylamine NAT (E.C. 2.3.1.5, not regulated by light) together with an arylalkylamine NAT inhibited by light (E.C. 2.3.1.87 [36]). Activity of the former, but not of the latter, was preserved at + 37 °C. However, both enzymes displayed high specificity for different substrates. In conclusion, the present study provides the first information available, (1) describing the optimal conditions for the assay of NAT activity in the retina of fish and the pineal organ of cold-blooded vertebrates, and (2) comparing some biochemical characteristics of NAT activity from two analogous organs. The best protocol assay for pineal organ NAT activity was a 20min incubation, at 20 °C, in 0.2 mol /l, pH 6 phosphate buffer in the presence of 0.5 / 1 mmol/ l acetyl-CoA and 40 mmol / l tryptamine; similar conditions held true for the retinal homogenates except for temperature (37 °C) and tryptamine concentration (1 mmol/ l). Our study also allows us to conclude that the effects of temperature on the rhythmic melatonin production by the pineal organ are mediated, at least in part, through modifications of NAT kinetics. Future studies will aim to clarify whether the activity measured in the pineal organ and retinal homogenates reflects the presence of one or of several enzymes. Acknowledgements The study was supported by the University of Poitiers, Foundation Langlois (Dinard), CNRS, MEN (AS no, 91–1522). We express our thanks to F. Chevalier and J. Dérisson for their technical assistance.

References 1. Alonso-Gómez AL, Alonso-Bedate M, Delgado MJ (1992) Thermal sensitivity and effect of temperature acclimatation on ocular serotonin N-acetyltransferase activity in Rana perezi. Neurosci Lett 142 : 187–190 2. Alonso-Gómez AL, Alonso-Bedate M, Delgado MJ (1993) The inhibition by indoleamines (tryptamine and serotonin) of ocular serotonin N-acetyltransferase from Rana perezi is temperature-dependent. Neurosci Lett 155 : 33–36 3. Birks EK, Ewing RD (1981) Characterization of hydroxyindole-O-methyltransferase (HIOMT) from the pineal gland of the chinook salmon (Oncorhynchus tshawytscha). Gen Comp Endocrinol 43 : 269–276 4. Bolliet V, Bégay V, Ravault JP, Ali MA, Collin JP, Falcón J (1994) Multiple circadian oscillators in the photosensitive pike pineal organ. A study using organ and cell culture. J Pineal Res 16 : 77–84

393 5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 : 248–254 6. Collin JP, Voisin P, Falcón J, Faure JP, Brisson P, Defaye JR (1989) Pineal transducers in the course of evolution : molecular organization, rhythmic metabolic activity and role. Arch Histol Cytol 52:441–449 7. Deguchi T (1975) Characteristics of serotonin-acetyl coenzyme A N-acetyltransferase in pineal gland of the rat. J Neurochem 24:1083–1085 8. Delgado MJ, Vivien-Roels B (1989) Effect of environmental temperature and photoperiod on melatonin levels in the pineal, lateral eye and plasma of the frog, Rana perezi: importance of ocular melatonin. Gen Comp Endocrinol 75:46–53 9. Falcón J, Collin JP (1989) Photoreceptors in the pineal of lower vertebrates: functional aspects. Experientia 45: 909–913 10. Falcón J, Brun Marmillon J, Claustrat B, Collin JP (1989) Regulation of melatonin secretion in a photoreceptive pineal organ: an in vitro study in the pike. J Neurosci 9 : 1943–1950 11. Falcón J, Thibault C, Bégay V, Zachmann A, Collin JP (1992) Regulation of the rhythmic melatonin secretion by fish pineal photoreceptor cells. In: Ali MA (ed) Rhythms in fishes. NATOASI Ser A, vol 236. Plenum, New York, pp 167–198 12. Falcón J, Bolliet V, Ravault JP, Chesneau D, Ali MA, Collin JP (1994) Rhythmic secretion of melatonin by the superfused pike pineal organ: thermo- and photoperiod interaction. Neuroendocrinology 60:535–543 13. Firth BT, Kennaway DJ (1987) Melatonin content of the pineal, parietal eye and plasma of the lizard, Trachydosaurus rugosus: effects of constant and fluctuating temperature. Brain Res 404:313–318 14. Hamm E, Menaker M (1981) Pineal and retinal serotonin Nacetyltransferase activity: modulation by phosphate. J Neurochem 37:1567–1572 15. Henderson PJF (1992) Statistical analysis of enzyme kinetic data. In: Eisenthal R, Danson MJ (eds) Enzyme assays, a practical approach. IRL, Oxford, pp 277–335 16. Kavaliers M (1982) Effects of pineal shielding on the thermoregulatory behavior of the white sucker Catostomus commersoni. Physiol Zool 55:155–161 17. Menaker M, Wisner S (1983) Temperature-compensated circadian clock in the pineal of Anolis. Proc Natl Acad Sci USA 80:6119–6121 18. Namboodiri MA, Dubbels R, Klein DC (1987) Arylalkylamine N-acetyltransferase from mammalian pineal gland. Methods Enzymol 142:583–590 19. Olcese J, Moller M (1989) Characterization of serotonin Nacetyltransferase in the retina of the mongolian gerbil, Meriones unguiculates. Neurosci Lett 102:235–240 20. Ralph CL (1992) Pineal bodies and thermoregulation. In : Reiter RJ (ed) The pineal gland. Raven, New York, pp 193–219 21. Ralph CL, Firth BT, Gern WA, Owens DW (1979) The pineal complex and thermoregulation. Biol Rev 54:41–72 22. Rawding RS, Hutchison VH (1992) Influence of temperature and photoperiod on plasma melatonin in the mudpuppy, Necturus maculosus. Gen Comp Endocrinol 88:364–374 23. Reiter RJ (1991) Pineal melatonin: cell biology of its synthesis and of its physiological interactions. Endocr Rev 12 : 151–180 24. Reiter RJ, King TS, Steinlechner S, Steger RW, Richardson BA (1990) Tryptophan administration inhibits nocturnal Nacetyltransferase activity and melatonin content in the rat pineal gland. Evidence that serotonin modulates melatonin produc-

25.

26.

27.

28. 29. 30.

31. 32.

33.

34.

35.

36. 37.

38.

39.

40.

tion via a receptor-mediated mechanism. Neuroendocrinology 52 : 291–296 Sugden D, Namboodiri MAA, Weller JL, Klein DC (1983) Melatonin synthesizing enzymes : serotonin N-acetyltransferase and hydroxyindole-O-methyltransferase. In : Parvez S, Nagatsu T, Nagatsu I, Parvez H (eds) Methods in biogenic amine research. Elsevier, Amsterdam, pp 567–572 Thibault C, Falcón J, Greenhouse SS, Lowery CA, Gern WA, Collin JP (1993) Regulation of melatonin production by pineal photoreceptor cells : role of cyclic nucleotides in the trout (Oncorhynchus mykiss). J Neurochem 61 : 332–339 Thibault C, Collin JP, Falcón J (1993) Intrapineal circadian oscillator(s), cyclic nucleotides and melatonin production in the pike pineal photoreceptor cells. In : Touitou Y, Arendt J, Pévet P (eds) Melatonin and the pineal gland. From basic science to clinical application, vol 1017. Elsevier, Amsterdam, pp 11–18 Tipton KF (1992) Principles of enzyme assay and kinetic studies. In : Eisenthal R, Danson MJ (eds) Enzyme assays, a practical approach. IRL, Oxford, pp 1–58 Underwood H (1989) The pineal and melatonin : regulators of circadian function in lower vertebrates. Experientia 45 : 914–922 Valenciano AI, Alonso-Gómez AL, Alonso-Bedate M, Delgado MJ (1994) Serotonin N-acetyltransferase activity as a target for temperature in the regulation of melatonin production by the frog retina. Pflügers Arch 429 : 153–159 Vanecek J, Illnerova H (1980) Some characteristics of the night N-acetyltransferase in the rat pineal gland. J Neurochem 35 : 1455–1457 Vivien-Roels B, Arendt J (1983) How does the indolamine production of the pineal gland respond to variations of the environment in a non mammalian vertebrate, Testudohermanni Gmelin. Psychoneuroendocrinology 8 : 327–332 Vivien-Roels B, Pévet P (1983) The pineal gland and the synchronization of reproductive cycles with variations of the environmental climatic conditions, with special reference to temperature. Pineal Res Rev 1 : 91–143 Vivien-Roels B, Pévet P, Claustrat B (1988) Pineal and circulating melatonin rhythms in the box turtle, Terrapene carolina triunguis: effect of photoperiod, light pulses, and environmental temperature. Gen Comp Endocrinol 69 : 163–173 Voisin P, Namboodiri MAA, Weller JL, Klein DC (1984) Arylamine N-acetyltransferase and arylalkylamine N-acetyltransferase in the mammalian pineal gland. J Biol Chem 259 : 10913–10918 Wolfe MS, Lee NR, Zatz M (1995) Properties of clock-controlled and constitutive N-acetyltransferases from chick pineal cells. Brain Res 669 : 100–106 Zachmann A, Knijff SCM, Bolliet V, Ali MA (1991) Effects of temperature cycles and photoperiod on rhythmic melatonin secretion from the pineal organ of the white sucker (Catostomus commersoni) in vitro. Neuroendocr Lett 13 : 325–330 Zachmann A, Ali MA, Falcón J (1992) Melatonin rhythms in the pineal organ of fishes and its effects. An overview. In : Ali MA (ed) Rhythms in fishes. NATO-ASI Ser A, vol 236. Plenum, New York, pp 149–166 Zachmann A, Falcón J, Knijff SCM, Bolliet V, Ali MA (1992) Effects of photoperiod and temperature on rhythmic melatonin secretion from the pineal organ of the white sucker (Catostomus commersoni) in vitro. Gen Comp Endocrinol 86 : 26–33 Zivin JA, Waud DR (1982) How to analyse binding, enzyme and uptake data : the simplest case, a single phase. Life Sci 30 : 1407–1422