Does melatonin have a time-dependent effect on brain and gill ionic metabolism in a teleost, <i>Anabas testudineus</i> (Bloch)?

Does melatonin have a time-dependent effect on brain and gill ionic metabolism in a teleost, Anabas testudineus (Bloch)?

L Divya, A S Vijayasree, P Sreejith, R S Beyo, M Smita & O V Oommen*

Department of Zoology, University of Kerala, Kariavattom, Thiruvananthapuram 695 581, India Received 1 September 2005; revised 1 May 2006

Exogenous administration of 0.20, 0.40 and 0.60 μg/g body weight melatonin over a 24 hr cycle caused an inhibition of Na⁺, K⁺ ATPase activity in both brain and gills of A. testudineus. However, Ca²⁺ ATPase activity in the brain was significantly inhibited by the highest dose, and that in the gill at all the doses of melatonin. Evening injection of melatonin had an inhibitory effect on both brain and gill Na⁺ K⁺ and Ca²⁺ ATPase activity. Melatonin treatment in the morning for 12 hrs did not have an effect on brain Na⁺, K⁺ ATPase, while Ca²⁺ ATPase was inhibited. Similar treatment stimulated Na⁺, K⁺ and Ca²⁺ ATPase activity in the gills. Sodium, potassium and calcium ions in the gill were significantly reduced in the evening treated group while no change was observed in the morning melatonin injected group. The results suggest that melatonin elicits a time - dependent effect on the enzymes and ionic content in the brain and gills of A. testudineus.

Keywords: Brain, Ca²⁺ ATPase, Gills, Melatonin, Na⁺, K⁺ ATPase, Teleost

The pineal organ of ectotherms is a part of the circadian pacemaker system transducing photothermal environmental inputs into a neurochemical signal¹. In the vertebrate endocrine network, the pineal hormone melatonin has emerged as a multifunctional hormone and its wide distribution² emphasizes a key role as an intracellular neuroendocrine regulator and co-ordi- nator of many complex and interrelated biological processes. The sodium-potassium activated adenosine triphosphatase (Na⁺, K⁺ATPase) and calcium (Ca²⁺) ATPase are P-type membrane pumps present mainly on cell membrane regulating transportation of ions and are widely used as indices of osmoregulation in fish³. Ca²⁺ channels and Ca²⁺ ATPase in the chloride cells regulate the influx of Ca²⁺ ion in a freshwater fish. Although Na⁺, K⁺ ATPase and Ca²⁺ATPase are different enzymes, their mechanism of action may have several features in common⁴. Abundant evidences are there to show that activity of these enzymes is regulated by a number of hormones^3,5. Sunny et al.⁶ have reported a rapid action of glucocorticoids on branchial ATPase activity.

Recently, short-term in vivo administration of insulin and alloxan was shown to influence Na⁺, K⁺ ATPase and Ca²⁺ATPase activity in the brain and gills of A.testudineus⁷. However, any functional relationship

between the time-dependent action of melatonin and ionic metabolism in teleosts is yet a topic of speculation. Hence, the present study evaluates the time and dose-dependent response of melatonin on total brain and gill Na⁺, K⁺ ATPase, Ca²⁺ATPase and inorganic ions like Na⁺, K⁺ and Ca²⁺ in the gills of a freshwater teleost, Anabas testudineus (Bloch).

Materials and Methods

Adult freshwater teleost, Anabas testudineus were obtained locally and kept in large aerated storage tanks. They were acclimated for 2 to 3 weeks at 26^о±3^oC and 12:12 hr L:D natural photoperiod. Two weeks prior to the experiment, adult healthy fish (body weight 50±5g) were transferred to aerated aquarium tanks maintained under conditions identical to those of stock tanks. The fish were fed ad libitum with 40% protein feed prepared in the laboratory⁸. The components were rice bran, tapioca, fish meal, groundnut oil cake with adequate amount of vitamins.

Melatonin and other chemicals used were of analytical grade and purchased from SRL, Mumbai, India.

Experiments were performed independently to assess the effect of melatonin on the activity of brain and branchial Na⁺, K⁺ ATPase and Ca²⁺ ATPase and branchial Na⁺, K⁺ and Ca²⁺ ionic contents.

Acclimatized fish were divided into four groups of 12 each. Group I served as control and received 0.1ml of 0.9 % saline as intraperitoneal injection. The fish in groups II, III and IV received 0.20, 0.40, 0.60μg/g

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*Correspondent author

Phone: + 91- 471 2418906 (off); 471 2598940 (res) Fax: + 91-471 2307158

E-mail: oommen@bigfoot.com

body weight melatonin, respectively in 0.1 ml vehicle between 0600 and 0630 hrs in a 24 hr cycle.

In another experiment, the time-dependent effect of melatonin was assessed by dividing the fish into 4 batches, each containing 12 individuals. Group I and III served as control fish and received 0.1ml saline.

Group II and IV fish received a dose of 0.40 μg/g body weight melatonin. Group I and II were injected between 0600 and 0630 hrs and were sacrificed after 12 hour. Both group III and IV were injected between 1800 and 1830 hrs. All the injections were intraperitoneal.

Fish were sacrificed by spinal concussion. The brain and gills were excised and kept separately in SEI (75 mM sucrose, 20 mM EDTA, 100 mM imidazole) buffer. Activity of Na⁺, K⁺ ATPase (EC 3.6.1.3) and Ca²⁺ ATPase (EC 3.6.3.8) was determined. The homogenate prepared to measure the ATPase activity was similar to that described by Zaugg⁹. The preparation of samples was carried out at 4^oC throughout. Gill and brain (100 mg each) was homogenized in 2ml sucrose buffer and centrifuged at 1000 g for 10 min. The supernatant was used to measure the specific activity of ATPase¹⁰. Experimental and control cocktails were prepared separately. The experimental cocktail of Na⁺K⁺ATPase contained (mM) 30 Tris, 10 Sodium Azide, 1 EDTA, 3 MgCl2, 120 NaCl, 10 KCl and 3 ATP. The control cocktail contained each of these reagents along with 3mM ouabain. The pH of all the cocktails was adjusted to 7.4. The specific activity of Ca²⁺ATPase was assayed employing similar conditions of Na⁺K⁺ATPase as described above. The Ca²⁺ATPase experimental cocktail contained 100 mM CaCl₂ instead of 120 mM NaCl and 10 mM KCl. The tubes were shaken well and incubated at room temperature for 15 min, after which the reaction was terminated by adding 1ml of 10% TCA. Adenosine triphosphate (3mM) was also added to the control.

The tubes were kept on ice bath for 40 min and they were centrifuged at 2500 g for 10 min thereafter. The clear supernatant was collected for the determination of inorganic phosphate¹¹. Ammonium molybdate was added to this and kept for 10 min, followed by ANSA (1-amino-2-naphthol-4-sulphonic acid). Absorbance was measured after 10 min with a Shimadzu UV-1609 spectrophotometer at 640 nm. ATPase activity was calculated as the difference in ouabain-sensitive and ouabain-insensitive ATP hydrolysis. The phosphate liberated was expressed as nanomoles/min/mg protein. The protein content of original brain and gill

homogenate was measured using modified Biuret assay¹².

Na⁺, K⁺ and Ca²⁺ in gills were determined by APHA method¹³. Three to five gill lamellae from each fish were excised and washed in double distilled water and allowed to air dry for 30 min. The tissue was then kept in hot air oven for 2-3 hr. Subsequently, it was powdered with the help of motar and pestle.

Powdered gill tissue (100 mg) was weighed and transferred to a conical flask. It was digested with 5ml of concentrated HNO3 and HClO3 in 4:1 ratio for 3-4 hr on a hotplate with constant stirring at regular intervals. When the acid mixture was completely evaporated, a white precipitate was formed. The residue was cooled and dissolved in 20 ml distilled water. Na⁺, K⁺ and Ca²⁺ ion contents were determined in a Systronics 128 flame photometer using five known standards and the values were expressed as μg/g body weight.

Statistical analysis was done with the help of a SPSS package. Data were analyzed by analysis of variance (ANOVA). Groups that were not significantly different in Duncan's multiple range test¹⁴ were considered homogeneous. Significant difference among the means was considered at a level of P < 0.05.

Results

Administration of employed dose of melatonin had diverse effects on gill and brain Na⁺, K⁺ and Ca²⁺

ATPase in Anabas testudineus. Injections of 0.20 or 0.40 or 0.60 μg/g body weight melatonin inhibited both brain and gill Na⁺, K⁺ ATPase activity. Ca²⁺

ATPase activity in the brain was significantly inhibited in response to the highest dose of melatonin (Fig. 1a). However, branchial Ca²⁺ ATPase was significantly decreased at each dose of melatonin (Fig. 1b).

Control groups of fish that received vehicle injections at two different time periods (0600 or 1800 hrs) showed significant differences, activity of Na⁺, K⁺ and Ca²⁺ ATPase in both brain and gills was higher in evening than the morning group.

Administration of 0.40 μg/g body weight melatonin in the morning for 12 hr did not have any effect on Na⁺, K⁺ ATPase of the brain while an evening injection of the same resulted in a significant decrease, compared to the corresponding control fish. Likewise, in both morning and evening treated groups, activity of Ca²⁺

ATPase was significantly inhibited compared to their respective control groups (Fig. 2a).

Na⁺, K⁺ ATPase activity in gills was significantly stimulated following treatment of melatonin in the morning, while it was inhibited in the evening treated group. Ca²⁺ATPase activity also followed a similar trend, on comparison with their respective control groups of fish (Fig. 2b).

Branchial ionic content

A significant difference (P<0.05) was noticed in the concentration of Na⁺, K⁺ and Ca²⁺ ions between the two control groups of fish injected with physiological saline in the morning and evening. Both Na⁺ and K⁺ increased in evening control group while Ca²⁺ was reduced (P<0.05), compared to morning control group. There was no significant change in the concentrations of Na⁺, K⁺ and Ca²⁺ in the morning melatonin treated fish while a significant decrease (P<0.05) was noticed in the evening treated fish compared to the corresponding control groups (Table 1).

Discussion

The present study provides evidence for a time- dependent effect of melatonin on ATPases in A.testudineus. A single injection of melatonin at

various doses caused a differential organ-related influence on Na⁺, K⁺ and Ca²⁺ ATPase activity. The significant inhibition of both Na⁺, K⁺ and Ca²⁺

ATPase in response to different doses of melatonin treatment apparently contributed to a decline in Na⁺, K⁺ and Ca²⁺ pump activity and ionic content in the fish.

The data of the present study reveal an inhibitory action of melatonin on total brain and gill Na⁺, K⁺ ATPase activity. A hypoglycemic effect of melatonin

Fig: 1 — Dose - dependent effects of melatonin on Na⁺, K⁺and Ca²⁺ ATPases in (a) brain and (b) gills. [Each histogram represents mean ± SE of 5 animals; groups with different letters are significantly different (P<0.05)]

Table 1 — Effect of morning and evening injections of melatonin on branchial ionic content

[Values presented as µg/g/wt are mean ± SE from 5 animals]

Gills Control (morning

injected)

Melatonin

(0.40 μg/gbw) Control (evening injected)

Melatonin (0.40 μg/

g bw) Na⁺ 4.08±0.16^c 4.40±0.13^ac 4.64±0.17^a 3.45±0.16^b K⁺ 0.12±0.03^c 0.04±0.007^c 2.51±0.16^a 1.76±0.11^b Ca²⁺ 34.68±0.71^a 35.32±0.50^a 28.40±0.89^b 24.008±0.59^c The significant difference between the groups was analysed by one-way analysis of variance, mean values of groups with different superscript letters in a given row are significantly different (P<0.05) as determined by Duncan’s multiple range test.

Fig. 2 — Time-dependent response of melatonin on Na⁺, K⁺and Ca²⁺ ATPases in (a) brain and (b) gills. (Other details as in Fig. 1)

is reported in goldfish where a decrease in plasma glucose and an increase in liver glycogen were observed after melatonin injection¹⁵. Melatonin is known to elevate gluconeogenesis in liver and changes in plasma glucose level, which in turn, affects hepatic melatonin binding¹⁶. Sodium (Na⁺) coupled glucose co-transport was reported in the mucosa of the pigmented conjunctiva of rabbit¹⁷. The decreased activity of Na⁺/K⁺ ATPase by melatonin may account for an inhibition of glucose co-transport and its utilization for energy requirement. This may have contributed to an inhibitory action on the neurons in the brain of A.testudineus. The ion gradients formed by the enzyme are necessary for Na⁺ coupled transport of nutrients into cells for osmotic balance, cell volume regulation and restoration of membrane potential in excitable tissues¹⁸. A direct consequence of Na⁺, K⁺ ATPase inhibition is an increase in the intracellular Na⁺ and depletion of K⁺ concentration. An increase in intracellular Na⁺ in turn raises intracellular concentration of Ca²⁺ by stimulating Na⁺/Ca²⁺ exchanges. The variation in ionic concentration at different time periods also suggests an involvement of melatonin in Na⁺/Ca²⁺

pump dynamics and the transport of ions. In basolateral membrane of some Ca²⁺ transporting epithelia, ATP dependent Ca²⁺ transport as well as Na⁺/Ca²⁺ exchanges activity has been demonstrated¹⁹. The decrease in Ca²⁺ ATPase in the present study may support the necessity of providing sufficient amount of intracellular Ca²⁺ required for the biochemical events taking place within these tissues after melatonin injection. Blocking of Ca²⁺ voltage dependent channels is known to suppress melatonin synthesis in trout pineal cells²⁰.

Melatonin treatment in the morning did not have an effect on Na⁺, K⁺, ATPase in the brain. The early morning injection probably coupled to the endogenous evening melatonin synthesis. As melatonin is chronically available, there is a possibility that it binds all available melatonin receptors, probably making the animal refractory to the action of additional melatonin. Melatonin administration in the evening produced a significant decrease in brain and gill Na⁺, K⁺, ATPase. The present results support the earlier report that melatonin administered in the afternoon advanced the circadian sensitive phase coincident with endogenous melatonin peak²¹. This strengthens the hypothesis that the coincidence of melatonin injection during the sensitivity phase may be a factor responsible for the

appearance of a physiological effect²¹. The data further showed a decrease in brain Ca²⁺ATPase in both morning and evening treated groups. Evening melatonin treatment also showed a similar influence on the gills. Certain forms of adenylate cyclase, the cAMP-synthesizing enzyme are regulated by calcium/calmodulin²². Melatonin is highly lipid soluble; hence it can readily enter cells and may form a complex with calmodulin in the cytosol. Melatonin alone has no effect on a number of signal transduction pathways including the accumulation of cAMP²³, the mobilization of calcium or the activities of phosphatases C^24,25. The major effect of melatonin appears to be the inhibition of activated second messenger pathway as melatonin dose dependently inhibits forskolin-stimulated cAMP production²⁵. Both Na⁺, K⁺ and Ca²⁺ ATPases in the gills were stimulated in the morning treated group. Studies on 2-[¹²⁵I] iodomelatonin binding in the kidneys and gills of freshwater rainbow trout and seawater flounder²⁶ suggest the possibility of an action of melatonin at these sites. Melatonin administration produced a 2^о-3^oC decline in temperature in mice²⁷. It has also been shown that circulating levels of melatonin increased significantly in homing pigeons after a flight so as to reduce flight-induced hyperthemia²⁸. Gill Na⁺, K⁺, ATPase activity was elevated compared to that in kidney suggesting that rainbow trout may rely more on branchial uptake of ions at higher temperature than in kidney²⁹. Activity of Na⁺, K⁺, ATPase in trout and goldfish fell sharply with the increase in acclimation temperature. This phenomenon was believed to be the result of temperature related changes in membrane lipid composition rather than alterations in type or amount of enzyme³⁰. The present study therefore, suggests a time-dependent activity of melatonin and its regulatory role on the ionic content (Na⁺, K⁺ and Ca²⁺) and enzyme activity in the brain and gills of Anabas testudineus. Na⁺, K⁺, ATPase involvement via Protein kinase C activation has been suggested as a mechanism of action by which melatonin increases water transport in MDCK cells³¹. The response of melatonin in our study reflects variations in ion exchanges across the two tissues studied and suggests an involvement of melatonin in osmotic adaptation mechanisms. This study throws light on yet another physiological function of melatonin. The variety of melatonin sites of action in cells may explain the enormous variability of melatonin effects in organisms. The relationship between melatonin and

systems controlling fluid balance is more complex.

The precise mechanism of action of melatonin in teleosts remains to be understood.

Acknowledgement

We thank DST, New Delhi for the FIST programme.

References

1 Firth B T & Kennaway D J, Thermoperiod and photoperiod interact to affect the phase of the plasma melatonin rhythm in the lizard, Tiliqua rogosa, Neurosci Lett, 106 (1989) 125.

2 Kvetnoy I M, Extra pineal melatonin location and function within diffuse neuroendocrine system, Histochemistry, 31 (1999) 1.

3 Peter M C S, Lock R A C & Wendelaar Bonga S E, Evidence for an osmoregulatory role of thyroid hormones in the freshwater Mozambique Tilapia Oreochromis mossambicus, Gen Comp Endocrinol, 120 (2000) 157.

4 Garrahan P J & Rega A F, Comparison between plasma membrane Ca²⁺ ATPase and Na⁺, K⁺, ATPase: Short review, Braz J Med Biol, 21 (1998) 1261.

5 Leena S & Oommen O V, Hormonal control on enzymes of osmoregulation in a teleost Anabas testudineus (Bloch): an in vivo and in vitro study, Endocrine Res, 26 (2000) 169.

6 Sunny F, Jacob A & Oommen O V, Genomic effect of glucocorticoids on enzymes of intermediary metabolism in Oreochromis mossambicus, Endocrine Res, 29 (2003) 119.

7 Vijayasree A S, Divya L, Sreejith P, Cyril J, Smita M &

Oommen O V, Insulin regulates ionic metabolism in a freshwater teleost, Anabas testudineus (Bloch), Indian J Exp Biol, 43 (2005) 702.

8 Hardy R, Fish feed formulation, FAO/ UNDP training course in fish feed technology, University of Washington, Seattle (1980) 233.

9 Zaugg W S, A simplified preparation for ATP determination in gill tissue, Can J Fish Aqua Sci, 39 (1982) 215.

10 Brungberg J A & Hahn N S, The role of ouabain sensitive adenosine triphosphatase in the stimulatory effect of thyrotropin on the iodine pump of rat thyroid, Endocrinology, 79 (1966) 801.

11 Fiske C H & Subbarow Y, The colorimetric estimation of phosphorus, J Biol Chem, 66 (1925) 375.

12 Alexander J B & Ingram G A, A Comparison of five of the methods commonly used to measure protein concentration in fish sera, J Fish Biol, 16 (1980) 115.

13 Greenberg A D, Clescus L S & Easton A D, APHA standard methods for the examination of water and wastewater, prepared and published by American Public Health association, American water works association, water environmental Federation, 18 (1992).

14 Duncan D B, Multiple range and multiple F test, Biometrics, 11, (1955) 1.

15 Delahunty G G, Bauer M & Prack de Vlaming V I, Effects of pinealectomy and melatonin treatment on liver and

plasma metabolites in gold fish, Carassius auratus, Gen Comp Endocrinol, 35 (1978) 99.

16 Poon A M S, Nehoy E H Y & Pang S F, Modulation of blood glucose by melatonin: A direct action on melatonin receptors in mouse hepatocytes, Biol Neurosignals, 10 (2001) 6.

17 Horbie Y, Hosoya K, Kim K J & Lee V H, Kinetic evidence for Na⁺, glucose co-transport in the pigmented rabbit conjunctiva, Curr Eye Res, 16 (1997) 1050.

18 Gloor S M, Relevance of Na⁺, K⁺, ATPase to local extracellular potassium homeostasis and modulation of synaptic transmission, FEBS Lett, 412 (1997) 1.

19 Taylor A, The role of sodium – calcium exchange in sodium transporting epithelia, in Sodium – calcium exchange, edited by T J Allen, D Noble & H Reuter (Oxford, Oxford University Press) 1989, 298.

20 Begay V, Bois P, Collin J P, Lenfant J & Falcon J, Calcium and melatonin production in dissociated trout pineal photoreceptor cells in culture. Cell Calcium, 16 (1994) 37.

21 Reiter R J, The melatonin message: Duration versus coincidence hypothesis, Life Sci, 40 (1987) 111.

22 Tang W J, Krupinski J & Gilman A G, Expression and characterization of calmodulin–activated (Type1) adenylyl cyclase, J Biol Chem, 266 (1991) 8595.

23 Morgan P J, Hastings M H, Barett P, Lawson W &

Davidson G, Intracellular signalling in the ovine pars tuberalis: An investigation using aluminium fluoride and melatonin, J Mol Endocrinol, 7 (1991) 137.

24 Mc Nulty S, Morgan P J, Thompson M G, Davidson L W &

Hastings M H, Phospholipases and melatonin signal transduction in ovine pars tuberalis, Mol Cell Endocrinol, 99 (1994) 73.

25 Morgan P J, King T P & Lawson W, Ultra structure of melatonin responsive cells in the ovine pars tuberalis, Cell Tissue Res, 263 (1991) 529.

26 Kulczykowska E, Warne J M & Balment R J, Day – night specific binding of 2 – [¹²⁵I] Iodomelatonin and melatonin content in gill, small intestine and kidney of three fish species. J Comp Physiol B, 130 (2005) 827.

27 Arutyunyan G S, Mashovskii M D & Roshchina L F, Pharmacological properties of melatonin, Fed Proc, 23 (1964) T1330.

28 George J C, Thermoregulatory and metabolic responses to cold exposure in birds, in Frontiers in environmental and metabolic endocrinology, edited by S K Maitra, (University of Burdwan, India) 1997, 135.

29 Houston A H & Mearow K M, Branchial and renal (Na⁺, K⁺) ATPase and carbonic anhydrase activities in a eurythermal freshwater teleost, Carassius auratus, J Comp Biochem Physiol, 71 (1982) 175.

30 Hazel J R & Prosser C L, Molecular mechanism of temperature compensation in poikilotherms, Physiol Rev, 54 (1974) 620.

31 King-Benitez G, Melatonin as a cytoskeletal modulator:

implications for cell physiology and disease, J Pineal Res, 40 (2006) 1.