AD-A273 107 USAARL Report No. 93-30

Effect of Photoperiodon Blood Cholinesterase Activity

and Melatonin Concentrations in Adult Cats

By

Alfred T. TownsendAlbert W. Kirby

Carolyn D. PopeGeorge Evans

Sensory Research Division

August 1993

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Effect of Photoperiod on Blood Cholinesterase Activity and Melatonin Concentrations inAdult Cats

12. PERSONAL AUTHOR(S)A.T. Townsend, A.W. Kirby, C.D. Pope, and G. Evans

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FROM TO 1993 August 1016. SUPPLEMENTARY NOTATION

17. COSATI CODES 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number)

FIELD GROUP SUB-GROUP Cholinesterases, Circadian rhythms, flelatonin,06 01 Photoperiod06 04

19. ABSTRACT (Continue on reverse if necessary and identify by block number)

We studied the effects of photoperiod on plasma melatonin (MEL) concentrations, butyryl-

cholinesterase (BUChE), and blood acetylcholinesterase (AChE) activity in 7 adult cats. Cats

were exposed to a 14:10 light-dark (LD) cycle with lights on from 0630 to 2030 for 3 monthsbefore blood sampling. Blood samples were obtained every 4 hours for up to 52 hours byrepeated venipuncture of the jugular vein in three animals, while four animals had indwellingcatheters implanted prior to sampling. All animals were handled repeatedly for several weeksprior to the study to minimize stress. MEL concentrations were measured in plasma by radio- 0immunoassay. Plasma BUChE activity and whole blood AChE activity were determined from thesame blood samples. In all of the animals, mean MEL concentrations were higher during thedark period of the LD cycle, while plasma BUChE and blood AChE activity had no observablechange in response to the LD cycle. These findings indicate that MEL secretion, but not

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19. Abstract (Continued)BUChE and AChE activity in cats, is responsive to changes in photoperiod. Under controlledconditions, secretion of MEL may be used as a control marker for physiological rhythmicity incats when studying the effects of photoperiod on other compounds.

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Table of contents

Introduction ................................................. I S

Materials and methods ........................................ 3

Results ...................................................... 3

Discussion ................................................... 6

Acknowledgments .............................................. 6

References ................................................... 7

List of figures

1. The effect of photoperiod on blood acetylcholin-esterase activity in cats determined at 4-hourintervals. Data are represented as the mean +SEM of seven animals assayed in duplicate at each •time period ............................................. 3

2. The effect of photoperiod on plasma butyrylcho-linesterase activity in cats determined at 4-hourintervals. Data are represented as the mean +SEM of seven animals assayed in duplicate at eachtime period ............................................. 4

3. The effect of photoperiod on plasma melatonin con-centrations in cats determined at 4-hour intervals.Data are represented as the mean + SEM of sevenanimals assayed in duplicate at each time period ........ 5

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Introduction

Activity of synaptically released acetylcholine (ACh) isterminated by the degradative enzyme acetylcholinesterase (AChE),or true cholinesterase. There is a second class ofcholinesterase found in living systems, pseudocholinesterase(butyrylcholinesterase, BUChE) which preferentially hydrolyzeshigher choline esters. Some of our previous studies withcholinesterase inhibitors relate decrements in the visual evokedresponse (VER) to inhibition of AChE or BUChE (Harding, Wiley,and Kirby, 1983; Harding, Kirby and Wiley, 1985; Kirby, HardinganO Wiley, 1987). Initial decrements in the VER appear to berelated to blood activity of cholinesterases. In addition toAChE inhibition, other neurochemical alterations have beenreported after exposure of animals to organophosphate (OP) 6(Kirby, Harding, and Wiley, 1987).

It is essential to the interpretation of results fromstudies involving cholinesterase inhibitors to know if acircadian variation in cholinesterase activity is present, and ifso, how much of the observed results can be explained on thebasis of that rhythm. Establishing baseline enzyme activity at apeak or base of a circadian period could result in a significantdifference in the calculated percent inhibition.

Circadian variations in AChE have been described in manyspecies, including the mouse brain (Lewandowski, 1983), the ratbrain (Schiebeler and Mayerbach, 1974), the cockroach nervoussystem (Vijayalakshmi, Mohan, and Babu, 1977), the centralnervous system of the grasshopper (Vijayalakshmi and Babu, 1978),and it has been suggested in human brain enzymes (Perry, et al.,1977). Experimental conditions and amount of demonstratedrhythmicity vary widely between species and laboratories. Weknow of no previous studies investigating circadian variations inAChE and BUChE in the cat.

To lend validity to changes in BUChE and AChE activitieswhich might be linked to the LD cycle, plasma levels of melatonin(MEL) also were determined. Leyva et al. (1984) showed that MELconcentration in cat blood is responsive to the dark phase of theLD cycle. Melatonin plasma levels were shown to increase in thedark period due to an increase in the activation of the enzyme N-acetyltransferase (Rudeen, Reiter, and Vaughan, 1975; Reiter,1987). In this study, we report the effects of the LD cycle onAChE and BUChE activity and melatonin secretion in cats.

Materials and methods

Five adult male and two noncycling female cats (mixed breed,3.1-4.9 Kg) were used in this study and were maintained in rooms

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at 21 0C under a 14:10 LD cycle (600 lux) for approximately 3months before the experiment. Animals were free of disease andreceived food (Purina high protein fortified chow) and water adlibitum. Blood samples were obtained every 4 hr for 52 hours byrepeated venipuncture of the jugular vein in 3 animals, while 4animals had indwelling catheters surgically implanted in thejugular vein prior to sampling. All animals were monitored byhematocrit to ascertain if the amount of blood withdrawnaltered total blood volume. In an effort to minimize stressduring sample collection, animals were placed in a canvas bagseveral times a day for several weeks prior to the study.

AChE and BUChE activity were determined according to themethod of Ellman et al. (1961). AChE activity is expressed asmoles of acetylthiocholine iodide hydrolyzed/min/rbc x 1016.BUChE activity is expressed as moles of butyrylthiocholine iodidehydrolyzed/Al plasma/min.

All immunoreagents and procedures employed for thedetermination of MEL in this study were supplied by StockgrandLTD, Department of Biochemistry, University of Surrey, GuildfordSurrey, UK. Briefly, 500 Al of sample or standard was added tothe assay tubes in duplicates. Two hundred Al of antiserum(G/S/704-8483) were added and the tubes were vortexed andincubated at room temperature for 30 minutes. One hundred Al of3H-melatonin then were added, vortexed and incubated at 40C for18 hours in a cold room. The antibody-bound melatonin wasseparated from the free fraction by vortexing and subsequentincubation with dextran coated charcoal for 15 minutes at 40C.The resulting mixture was centrifuged at 1500 x g at 40C for 15minutes. An aliquot of the supernatant was removed and placedinto vials containing 6 ml of scintillant. The vials then wereshaken at room temperature for 1 hour and counted by liquidscintillation spectrometry. Repeated analysis of this RIA over a3-month period revealed a limit of detection of 2.2-5 pg/tube.Parallelism existed between standard curves prepared using pooledstripped human plasma and pooled stripped feline plasma. Intraand interassay coefficients of variation were 7.6 and 4.1percent, respectively. Specificity was demonstrated through theuse of a high performance liquid chromatography (HPLC) methodutilizing electrochemical detection (Medford and Barchas, 1980).Concentrations of MEL (1-3 ng) were injected through the HPLCsystem, and peaks were identified by retention. MEL wasquantified by peak height.

Differences between light and dark concentrations of MEL orBUChE and AChE activity values in the blood of individual animalswere assessed using one-way analyses of variance and covariancewith repeated measures. The minimum acceptable level ofsignificance in all cases was p < 0.05.

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Results

Figure 1 illustrates the effect of the LO cycle on bloodAChE activity in male and female cats. When comparing animal Sgender, AChE activity did not differ significantly; therefore,results from male and female cats were combined. AChE activitydid not show any significant elevation or decrement in responseto photoperiod. These findings demonstrate that blood AChEactivity in cats does not change significantly during a 14:10 LDcycle. 0

Time of day 0

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Figure 1. The effect of photoperiod on blood acetylcholines-terase activity in cats determined at 4-hour inter-vals. Data are represented as the mean + SEM! of sevenanimals assayed in duplicate at each time period.

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Figure 2 illustrates the effect of the LD cycle on plasmaBUChE activity in male and female cats. As with AChE activity,BUChE activity did not differ between animals of different sex.In addition, BUChE activity did not show any significantelevation or decrement in response to the LD cycle. Thesefindings demonstrate that plasma BUChE activity in cats does notchange significantly during a 14:10 LD cycle.

Time of day

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Fiyure 2. The effect of photoperiod on plasma butyrylcholines-terase activity in cats determined at 4-hour inter-vals. Data are represented as the mean + SEM of sevenanimals assayed in duplicate at each time period.

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Figure 3 illustrates the effect of the LD cycle on plasmaMEL concentrations in adult cats. In all of the animals studied,mean MEL concentrations were higher during the dark period of thephotoper 4 od regime. This pattern and magnitude of change appearsto be consistent in animals regardless of their gender. Inagreement with an earlier report (Leyva, Addiego, andStabenfeldt, 1984) these findings indicate that MELconcentrationg are sensitive to the LD cycle and are elevated inthe dark.

Time of Day

0 0 0 0 0 0 0150-

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Figure 3. The effect of photoperiod on plasma melatonin con-centrations in cats determined at 4-hour intervals.Data are represented as the mean + SEM of sevenanimals assayed in duplicate at each time period.

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Discussion

Results of this study ,*owed that blood AChE and plasma 0BUChE activity in the cat did not vary significantly in responseto the LD cycle. Elsmore (1981) reported no significantdifferences in blood cholinesterase activity as a function oftime of day in rats. Mabood, Newman, and Nimmo, (1978)showedthat variations in enzyme activity, detected in humanerythrocytes, did not appear to be circadian or semicircadian in 9nature. Quay et al. (1971) suggested that results obtained fromprevious studies in rat brain have led to the conclusion thatAChE activities are more closely related to factors other than 24hour or photoperiod rhythmicity. Our finding that bloodcholinesterase activity in cats was not linked to photoperiodagree with these reports on other species. 9

This study also confirmed results from an earlier report(Leyva, Addiego, and Stabenfeldt, 1984) which linked changes inMEL concentrations to the LD cycle. High MEL concentrations wereobserved in the dark phase of the LD cycle. The rise in plasmaMEL concentrations during the dark phase of the LD cycle and the 5fall of the concentrations during the light indicates thesecretion pattern of MEL is related strongly to photoperiod. Itis suggested that the secretion pattern of MEL during a 14:10 LDcycle may be used as a marker for circadian rhymicity whenstudying the effects of photoperiod on other compounds.

Acknowledcgments

We thank Dr. Teresa Jernigan, School of Veterinary Medicine,Auburn University, Auburn Alabama, for supplying a generousamount of pooled feline plasma used for the validation of themelatonin RIA. Many thanks to SGT Lovell of Walter ReedInstitute of Research, Washington, D.C., for surgicallyimplanting the catheters used for sampling. We thank R. Espejo,B. Lancue, J.B. Lopez, and T. Tapia for their excellent technicalassistance. The experiments reported here were conductedaccording to the Guide for Care and Use of Laboratory Animals,National Research Council, DHEW Publication # (NIH) 85-23.

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Refere.ices

Ellman, G. L., Courtney ,K. D., Andres, V. Jr., and Featherstone,R. M. 1961. A new and rapid colorimetric determination ofacetyicholinesterase activity. Biochemical pharmacology.7:88-95.

Elsmore, T. F. 1981. Circadian susceptibility to somanpoisoning. Fundamental applied toxicology. 1:238-241.

Harding, T. H., Kirby, A. W., Wiley, R. W., 1985. The effects ofdiisopropylfluorophosphate on spatial frequency responsivityin the cat visual system. Brain research. 325:357-361.

Harding, T. H., Wiley, R. W., and Kirby A. W., 1983. A 0cholinergic-sensitive channel in the cat visual system tunedto low spatial frequencies. Science. 221:1076-1078.

Kirby, A. W., Harding, T. H., and Wiley, R. W., 1987. Recoveryof the visual evoked response in the cat followingadministration of diisopropylfluorophosphate, an irreversible Icholinesterase inhibitor. Life sciences. 41:2669-2677.

Lewandowski, M. 1983. Circadian changes of acetylcholinesteraseactivity in the reticular formation of the mouse brain underthe influence of different light conditions, with reference tolocomotor activity. Folia biolo i. 31:53-64. 6

Leyva, H., Addiego, L., and Stabenfeldt, G. 1984. The effectof different photoperiods on plasma concentrations ofmelatonin, prolactin, and cortisol in the domestic cat.Endocrinology. 115:1729-1736.

Mabood, S. F., Newman, P. F. 7., Nimmo, I. A. 1978. Circadian

rhythms in the activity of acetylcholinesterase of humanerythrocytes incubated in vitro. Biochemical societytransactions. 572nd meeting. V6:305-308.

Mefford, I. N., and Barchas, J. D. 1980. Determination oftryptophan and metabolites in rat brain and pineal tissue byreversed phase high-performance liquid chromatography withelectrochemical detection. Journal of chromatography.181:187.

Perry, E. K., Perry, R. H., Taylor M. J., and Tomlinson, B. E. 01977. Circadian variation in human brain enzymes. Lancet.VI(8014):753-754.

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Quay, W. B., Bennett, E. L., Morimoto, H., and Hebert, M. 1971.Evidence for the absence of 24 hour rhythms in cholinesteraseactivities of brain regions. Comparative general pharma- 0cology. 2:402-410.

Reiter, R. J. 1987. The melatonin message: duration versuscoincidence hypotheses. Life sciences. 40:2119-2131.

Rudeen, P. K, Reiter, R. J., and Vaughan, M. K. 1975. Pinealserotonin-n-acetyltransferase activity in four mammalianspecies. Neuroscience letters. 1:225.

Schiebeler, H., and Mayerbach, H. V. 1974. Circadian variationsof acetylcholine esterase (E.C.3.1.1.7) in rat brains.International iournal of chronobiology. 2:281-289. 0

Townsend, A. T., Adams, D. K., Lopez, J. B., and Kirby, A. W.1991. Effect of diisopropylfluorophosphate on muscarinic andgamma-aminobutyric receptors in visual cortex of cats. Lifesciences. 49:1053-1060.

Vijayalakshmi, S., and Babu, K. S. 1978. Diurnal rhythmicity ofcholinesterase activity in central nervous system of thegrasshopper Poecilocerus pictus. Indian Journal ofexperimental biology. 16(12):1305-7.

Vijayalakshmi, S., Mohan, P. M., and Babu, K. S. 1977. Circadian 0rhythmicity in the nervous system of the cockroach,Periplaneta americana. Journal of insect physiology. 23:195-202.

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CA Av Med COL C. Fred TynerHQ DAAC U.S. Army Medical ResearchMiddle Wallop & Development CommandStockbridge, Hants S020 8DY UK SGRD-ZB

Fort Detrick, Frederick, MD 21702-5012Dr. Christine SchlichtingBehavioral Sciences Department DirectorBox 900, NAVUBASE NLON Directorate of Combat DevelopmentsGroton, CT 06349-5900 ATZQ-CD

Building 515Brazilian Army Liaison Office Fort Rucker, AL 36362Building 602Fort Rucker, AL 36362

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