RESPONSES OF WHOLE-ANIMAL AND ISOLATED HEARTS OF GROUND SQUIRRELS, CITELLUS LATERAT,IS TO MELATONIN

7/29/2019 RESPONSES OF WHOLE-ANIMAL AND ISOLATED HEARTS OF GROUND SQUIRRELS, CITELLUS LATERAT,IS TO MELATONIN

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Palmer, Dennis L., and M. L. Riedesel. L976. Responses ofwhole-animal and isolated heart of ground squirrels' Cilgfryelateralis, to melatonin. C_omp. Biochem. Physiol. 53C: 69-72.Canp. Bio.heDt. Physirt.. 1976, Vol. 53C. t1t1. 69 to 12. Pergamott Press Prited in Gredt Btitaitl

RESPONSES OF WHOLE-ANIMAL AND ISOLATED HEARTSOF GROUND SQUIRRELS, CITELLUS LATERAT,ISTO MELATONIN1DrNNrs L. Parwn aNl M. L. RrprpselDepartment of Biology, University of New Mexico, Albuquerque, 87131, U.S.A.

(Receir:ed 14 August 1975)Abstract 1. The pineal hormone, melatonin, has been demonstrated to influence hibernation in thegronnd squilrel, C. lateralis. Daily subcutaneous iniections of a commercial preparation of melatoninresulted in an increase in the incidence and duration of hibernation.2. Isolated hearts from hibernating and non-hibernating ground squirrels had a depressed durationof electrical aotivity when melatonin was present in the bathing solution.

INTRODUCTIONIN pnrpanarroN for the hibernating state, the mam-malian hibernator becomes increasingly obese duringthe late summer months with the deposition of bothbrown and white fat. There is also atrophy ol theovaries and testes before hibernation (Lyman & Chat-fleld, 1955). The pineal hormone, melatonin (5-meth-oxy N-acetyltryptamine), has been demonstrated tostimulate the growth of brown adipose tissrre withinthe Djungarian hamster (Heldmaier & Hoffman,1914).It also has been described to produce antigona-dal effects within rodents (Wurtman et al., 1963).Spafford & Pengelley (1971) reported an influenceof serotonin, the metabolic precursor to melatonin,on hibernating golden-mantied ground squirrels,Citellus lateralis. Their results demonstrate that whole-brain serotonin levels decrease during the entranceinto hibelnation and that inhibition of serotonin syn-thesis disrupted hibernation.The metabolic conversion of serotonin into mela-tonin is a light dependent mechanism with the highestactivity of the melatonin forming enzyme coming indarkness (Ax1erod, 1974). Within C. lateralis, Pengel-1ey (1969) observed that light does play a role in theperiod of hibernation. In view of these reports, thepurpose of this study was to determine if melatoninhad an influence on hibernation within the golden-mantled ground squirrel.Our results from administering a commercial prep-aration of melatonin into C. lateralis indicate thatmelatonin does have an influence on hibernation.These results in turn posed the question of what wasthe physiological role of melatonin within the groundsquirrel.A dramatic slowing ol the heart rate is one of thefirst physiological changes identified with the onsetof hibernation (Dawe & Morrison, 1955). It isbelieved that the hibernating heart is devoid of in-fluences of vagal tone (Dawe & Landau, 1960) butthat the slow rate in hibernation is due primarily

I Taken in part from a thesis submitted in partial fu1fiil-ment of the M.S. degree. Universitl' of New Mexico.

to a reduction in the number of impulses sent outfrom the pacemaker (Dawe & Morrison, 1955). Thisinfers that the decrease in rate is attributable to bio-chemical effects on cardiac tissue or pacemaker.Therefore, the objective of the second portion of thisstudy was to determine if melatonin had a direct effecton the heart.

MATERIALS AND METHODS:The golden-mantled ground squirrels, C. Iateralis, werctrapped in the area of Fenton Lake, New Mexico. Animalswere caged individually with food and water ad lib. Thebody weights of the animals selected for the study rangedfrom 200 Io 324 g. The hibernaculum temperature was5 + 2"C with a daily 10 L and 14 D photoperiod. Animalswere provided with burlap as a nesting material.Melatonin was obtained from Sigma Biochemicals, St.Louis, Missouri and Nutritional Biochemicals, Cleveland,Ohio. Each commercial preparation of melatonin wasassayed for its relative biological effectiveness by observingthe degree of compensatory ovarian hypertrophy withinlab mice.Crystalline melatonin was stored frozen until the soiu-tions were to be prepared. Fresh solutions were preparedweekly and refrigerated, 5"C. Melatonin, either 12'5 or 25 0mg, was dissolved in 0'3 ml absolute ethyl alcohol anddiluted to l0 m1 with 0'9?4 NaCl.The study of the response of whole animals to mela-tonin consisted of two continuous phases which involvedfour groups of nine animals per group. In the first phasethe animals of groups A and B were administered injec-tions of physiological saline. The experimental animals ofgroups C and D were given injections equalling 100 and

500 pg of melatonin per day respectively. The second phasewas a reversal ofthe first phase in which the ground squir-rels of groups A and B were administered dosages of 100and 500 pg of melatonin per day respectively and thoseanimals of groups D and C received injections of physio-logical saline. The ground squirrels were observed twicedaily (7 00 a.m. & 5 00 p.m.) at which time skin tempera-ture measurements were made with a copper constantanthermocouple probe and a Leeds Northrup Potenti-ometer. Each animal was administered two subcutaneousinjections daily iust after the observations for hibernation.Each phase continued for 10 days.69


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70 DrNNrs L. Pelwn aNo M. L. Rrror:srSkin temperature was the criterion for determiningwhether a ground squirrel was hibernating or active. Ani-mals with skin temperature below 10"C were consideredto be hibernating. Hibernating animals disturbed regularlyrequire several days to become habituated to regular hand-ling and injections (Pengelley & Fisher, 1967). Once habi-tllated most animals do not arouse following injections,however, bouts of hibernation less than 24 consecutivehours were not included in the statistical analvsis. The dataon the frequency and duration ol the hibernation periodswere submitted to statistical analysis in accordance withmethods discussed by Simpson et al. (1960).The experimental animals in the study of isolated heartsconsisted of two groups: summer-active and winter-hiber-nating ground squirrels. The animals were sacrificed bydecapitation and the heart was immediately excised andbathed in two consecutive washes of cotd safine prep-aration (7 + 2'C) for 3 min. The heart was then placedinto a final saline bath with two graphite eleclrodesarranged to make contact with the surface of the heart,one on an auricle and one on a ventricle. Experimentalhearts had melatonin added to the fina1 saline bath(46 x l0 4 M). Electrical activity was recorded con-tinuously on a Narco DMP 44 Physiograph. Totalnumber of electrical pulses and the length of time the iso-lated hearts continued to show activity were recorded.After t hr of having no activity the hearts were consideredto be terminated and no attempt was made to mechani-cal11 stimulate them.

SULTSResponse of ground squirrels to cold and melatoninIn the first phase, two groups of ground squirrelswere administered physiological saline injectionswhile the other two groups received 100 and 500 pgof melatonin respectively as a daily dosage. In thesecond phase the dosage given to the groups wasreversed, i.e. the two groups of ground squirrels whichhad initially received the saline injections were givenmelatonin injections and those two groups which hadbeen administered the melatonin in the first phasewere given the saline injections. The two phases werecontinuous with the duration of each phase being 10days.The incidence of hibernation bouts (Table 1) in-creased in all groups when melatonin was injecteddaily. Whereas there was no difference in the meanduration of hibernation (Table 2), each animal hadTable l. Average number ofhibernations observed per day

Table 2. Duration of hibernation period (24 hr). p valuesrepresent differences in the length of the hibernation periodfor individual animals in the two phases6roup SaI ineInj ections Me1 atoninInj ect ions

3.70L0.84112.500 .291t2.940.2215

2.440.1515

4.130,71113.250.44

113.600.3315

0.01

0.02

0.01

0. 001.930 .361.5

6raup Sa1 ineInj ections Melatoninlnl ectlons

1. xs.E.Na significant increase in the duration of hibernationwhen injected with melatonin. The results of this ex-periment indicate that melatonin did have an in-fluence on hibernation within the golden-mantledground squirrel, C. lateralis.Response of isolated hearts to melatonin.From the two groups of experimental animals, i.e.summer-active and winter-hibernating ground squir-rels, isolated hearts bathed in a standard saline solu-tion containing melatonin (4.6 x 10-aM) alwaysresulted in a lower total number of pulses recordedand a shorter duration of electricai activity than thecontrol isolated hearts (Figs. i & 2). Within the sum-mer-active animals the mean values for the totalnumber of pulses recorded were 576 for the controlanimals and 253 for the experimental ground squir-rels. Totai duration of organized activity ranged from122 to 188 min for the controls and 80 88 min forthe isolated hearts bathed in the saline preparationcontaining melatonin. Winter-hibernating groundsquirrels ranged from 738 to 1335 in the total number

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6.90.7096.30.77

9

+.al0.s090.37

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0-70I5.50.699

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0.001

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HIBERNATINGJANUARY 196N:4 e btqalls

Fig. 1. Total number ofpulses recorded for isolated hearts


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Responses of whole-animal and isolated hearts of ground squirrels 7l

Fig. 2. Duration of pulses recorded for isolated heartsof pulses recorded for the controls and 370 538 forthe experimental animals. The mean values for theduration of organized activity were 197 min for thecontrol ground squirrels and 96 for the experimentalgroups.

DISCUSSIONThe pineal has been reported to participate in theregulation of seasonal cycles (Reiter, 1973). In mam-mals, melatonin is produced in the pineai gland andthe activity of hydroxyindole-O-methyltansferase(HIOMT), the enzyme responsible for the last stepin melatonin formation, has been reported to be lightdependent with high activity in darkness and 1ow ac-tivity in light (Wurtman et al., 1968). Environmentailighting messages reach the pineal via the retina of

the mammalian eye (Axlerod,1974). Pengelley (1969)noted light does play a role in the period of hiberna-tion in C. lateralis as evidenced by experiments in-volving enucleation.Hoffman and his colleagues (1965) reported thatin the golden hamster there is an increase of brownadipose tissue (BAT) weight following exposure toshort photoperiods. There was a drastic increase ofBAT mass in hamsters treated with melatonin whichindicates regulation of the growth of BAT involvesthe pineal and increased BAT is not simply a conse-quence ol cold exposure (Heldmaier & Hoffman,1974\.These previous reports along with the results pre-sented in this study support the concept that mela-tonin may be involved in the onset and maintenanceof hibernation. The physiological mechanisms bywhich this pineal indoleamine could influence hiber-nation are unknown.Our results from the isolated heart experiment indi-cate the heart may be an important melatonin targettissue in the mammalian hibernator. Because manycompounds which prolong the effects of hypnotics arevasoactive agents, Barchas et al., (1967) speculatedthat melatonin would alter the inotropic and chrono-tropic actions ol isolated hearts. Their resuits demon-

strated this not to be the case for isolated guineapig and rat hearts periused with melatonin at a con-centration of 10 a M. Studies conducted to datewithin our laboratory on perfused ground squirrelhearts confirm their report of no response. Theabsence ola response to melatonin by periused heartswhereas non-perfused hearts do respond, presumablyindicates melatonin is most effective in a deterioratingheart.

When the data obtained for two groups were com-pared both the duration of electrical activity and thetotal number ol' pulses recorded were higher withinthe winter-hibernating animals than in the summer-active animals. These data support the hypothesis(Dawe & Landau, 1960) that the mammalian hiber-nating heart has undergone some physiological transi-tion(s) which enables it to have a longer survival timethan the heart from the active animal.It is interesting to speculate as to the relationshipbetween melatonin and the hibernation "trigger" de-scribed by Dawe & Spurrier (1974). The action olmelatonin in this seasonal hibernator, C- lateralis, isvery different from the action of the blood borne trig-ger. Both compounds change the activity of isolatedhearts but the trigger increased whereas melatonindecreased the number of spontaneous heartbeats.Melatonin may either be responsible for changing thereceptiveness of target tissue to the trigger or asso-ciated with the seasonal changes in the amount oftrigger which is present in the blood. Most certainlythere needs to be additional information before it ispossible to relate these two systems within anyreasonable doubt.The data collected in these whole-animal and iso-lated heart experiments support the hypothesis thatmelatonin has a role in the preparation for and main-tenance of hibernation within the golden-mantledground squirrel, C. lateralis.

REFERENCESAxr.rnoo J. (1974) The Pineal Gland: A NeurochemicalTransducer. Science. N.I 184. 1341-1348.Brrncnas J., D.q.Cosrn F. & SpEcron S. (1967) Acute phar-macology of melatonin. Nature, Lond. 214,919 920.Dl,ws A. R.& LlNor,u B. R. (1960) The hibernating mam-malian heart. Am. Heart J. 59, 78-89.D,qwE A. R. & MonnrsoN P. R. (1955) Characteristics ofthe hibernating hearl. Ant. Heart J. 49,361*389.D,q.wr A. R. & Spunnnn W. A. (1974) Summer hibernationof infant (six-week old) l3Jined ground squirrels.Citellus tridecemlineatus. Cryobiology. l1(1), 33 43.HsroMAlen G. & HoFpnr.tr K. (1974) Melatonin stimulatesgrowth of brown adipose tissue. l{atare, Lond. 247,t1/ 11

RESPONSES OF WHOLE-ANIMAL AND ISOLATED HEARTS OF GROUND SQUIRRELS, CITELLUS LATERAT,IS TO MELATONIN

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