- Email: [email protected]
Physiological characteristics of platelet serotonin in rats
Maxwell Pergamon Macmillan
Life Sciences, Vol. 45, pp. 485-492 Printed in the U.S.A.
PHYSIOLOGICAL
Branimir
Jernej,
CHARACTERISTICS OF PLATELET SEROTONIN IN RATS
Lipa &in-Sain*
and Sergije
Kveder”
Departments of Experimental Biology and Medicine and of *Organic Chemistry and Biochemistry,“Ruder Bogkovi6t’ Institute, Yu-41001 Zagreb, Yugoslavia, P.O.Box 1016 (Received in final form May 31,
1989)
Summary Physiological
characteristics
of platelet
serotonin
(5HT)
levels
in rats of Wistar origin were investigated by use of a recently developed method. By comparison of populations of male and female rats (N=281) similar unimodal frequency distributions, with a tendency to higher values in females (1.61 vs. 1.70 ug 5HT/mg platelet protein; pc O.Ol), were found. For a group of 55 animals, monitored twice for this parameter within a week interval, a remarkable intraindividual constancy in time, the mean difference between two determinations being 5.5%, was shown. No age-dependence could be demonstrated for platelet serotonin concentrations in 5- to 30week-old rats, nor were there significant circadian or seasonal oscillations. The
physiology of platelet serotonin (5_hydroxytryptamine, 5HT) has so far mostly been the subject of clinical investigations, while considerably less data have been derived from experimeqtal work on laboratory animals. A recently developed method for quantitative determination of platelet serotonin levels (PSL) in rats (1) has made it possible to monitor changes of this parameter in individual animals through prolonged periods of time. Having in mind forthcoming (patho)phy siological investigations of platelet 5HT in rats, we first wanted to become familiar with the basic physiological characteristics of this biological variable, to be able to recognize their possible influences on the results of further experimental treatments.
There is disagreement in the literature concerning sex and age dependence ofPSL. Some reports indicate tendencies to higher, or even significantly higher values of this parameter in females (2,3,4,5), but the absence of sex differences has also been claimed (6,7,8). In a similar manner, a decrease of circulating serotonin with age has been stated in most reports (2,7,9,10,11) while, in other studies, the absence of age dependence (3,5,12) or even tendencies towards an increase of PSL during ageing (8,13) have been claimed. Individual stability of PSL in humans has been reported by several investigators (2,3,6,7,13), but the range of intraindividual variability has been stated only in few reports (3,7). With the exception of our previous observations obtained on a small group of rats (l), we found only one paper adressed to this problem in experimental animals (14). Periodic oscillations of central and peripheral serotonin-related parameters have been reported in the literature. In humans, data supporting a circadian
rhythm in circulating or platelet serotonin (3,15,16) can be contrasted to results which failed to confirm the existence of such rhythmic oscillations (2,6).
0024-3205189 $3.00 +.OO Copyright (c) 1989 Maxwell Pergamon Macmillan plc
486
Platelet Serotonin in Rats
Vol. 45, No. 6, 1989
Seasonal variation of platelet serotonin in man has also been described (3). As to experimental animals, circadian variation of whole-blood serotonin in rabbits (17) and of serum serotonin in rats (18,19) has been demonstrated, but we are not aware of any reports on seasonal variation in laboratory animals. In summary, the literature data are inconsistent and, at least for rats, incomplete. Here we present results, all obtained by use of the same methodology, on sex and age dependence, intraindividual oscillation in time and on circadian and seasonal variations of platelet serotonin levels in rats. Methods Animals. Zgr:Wistar rats of either sex from the breeding facilities of the "R. BoZkovTC" Institute, Zagreb, were used. Animals were housed 3-5 per cage with commercial rat chow and water ad libitum. The animal quarter was maintained at a temperature of 22 t 2"C, with natural light-dark cycle, and usually entered during the morning f% cleaning and supplementing fresh food and water. Sex-de endence studies were performed on 140 males (135-240 g) and 141 females rom which no blood samples had been taken before. Whenever pos* sible, samples of males and females were processed simultaneously. Age-dependence studies were performed on three groups of male animals (N=lO) of different age: prepubertal (5-6 weeks old; 65-80 g), young postpubertal, commonly used in experimental work (12-15 weeks old; 180-250 g) and middle-aged animals (24-30 weeks old; 420-490 g). Individual stability of PSL in rats was investigated on 55 males (190-280 g) by determining this parameter twice in a week interval. The measurements were compared by plotting the results of the first estimation (x-axis) against the second one (y-axis) and calculating the correlation between these two sets of values. Periodic oscillations of PSL were investigated with respect to circadian rhythm and seasonal variation. Circadian variation was studied for males (210-260 g), at the end of March (light/dark intervals approximately 12h/12 h). In the first experiment, 6 groups (N=12) were controlled for PSL at the times indicated in Fig.4. In the second experiment, performed one week later, 3 additional groups (N=12) were used for repeating determinations at 10 and 13 h, and for extension of the night part of the experiment until 02 h. For every group, blood sampling was performed within 30 minutes. Care was taken to minimize disturbance of the animals during the withdrawal of cages. During the dark period, exposing the animals to light was prevented by inducing narcosis in the dark room and transferring them to the laboratory after loss of consciousness. Information on seasonal variation of PSL was obtained by evaluation of data accumulated through a period of more than two years. Althogether, 29 groups of males and 13 groups of females, each consisting of 5-30 animals, were analyzed. Determination of platelet 5HT content. The method for determination of platelet serotonin content in rats has been described in detail elsewhere (1). Shortly, under light aether narcosis, 1 mL of blood was withdrawn, by jugular venipuncture, into a plastic syringe preloaded with ACD anticoagulant. Platelet-rich plasma (PRP) was prepared by centrifuging the sample in the same syringe. The supernatant PRP was quantitatively transferred to a plastic tube and platelets were pelleted by recentrifugation. After homogenizing the pellets by ultrasonication, proteins were precipitated with ZnSO /NaOH and serotonin measured in the clear supernatant by the orthophthaldialdehyde-fluorometric method. The PRP sample obtained from 1 mL of blood by use of this method possesses well-defined characteristics and is termed standardized PRP (sPRP) sample (1). Platelet serotonin content is expressed either per sPRP sample (reflecting platelet serotonin levels in the circulation) or per mg of platelet protein (reflecting se-
Platelet
vol. 45, No. 6, 1989
Serotonin
in Rats
487
rotonin concentration in platelets). Statistics. Student's t-test, regression analysis and the method of differences were used. Variability was expressed in terms of standard deviations (SD) and coefficients of variation (CV). The level of confidence was 99% or above. Results and Discussion Influence of sex. The frequency distributions of PSL in rats of either sex are presented in tig.1. The histograms are of similar unimodal shape and the distribution resembles that found in humans (20). The mean value of PSL in female rats (1.70 t 0.19 ug 5HT/mg protein, N=141) was somewhat higher than in males (1.61 t 0.17 iig 5HT/mg protein, N=140). That small difference (5.6 %), although reaching statistical significance in such a large sample (p < O.Ol), can, in a smaller sample, easily be overshadowed by interindividual variability. Indeed, our previous studies on smaller groups of animals failed to demonstrate significant differences of PSL between sexes (21). It is possible that such differences in sample size could also explain the discrepancies in the literature data on humans, some of which support tendencies toward higher platelet serotonin values in females (2,3,5). while other claim no influence of sex on this parameter (6,7,8).
40
n = 1.61 SD = 0.17
35
N = 140
i E
30
’L
25
1.2
1.4 1.6 1.8
2.0 2.2
n = 1.70
SD= 0.19 N = 1'11
1.2 1.4 w
1.6 1.8 2.0 2.2
5-Hl/~g platelet
protein
Fig. 1 Frequency distributions of PSL in male and female rats of Zgr:Wistar strain The influence of sex has also been investigated with regard to some other serotonin-related parameters. There appear to3be no differences between males and females in either platelet 5HT uptake or .H-imipramine binding, while higher activity of platelet MAO in females has been noted (reviewed in ref. 8). Concerning the platelet MAO activity, a deviation from normal distribution in humans, both males and females, has also been shown (22).
488
Platelet Serotonin in Rats
vol. 45, No. 6. 1989
We conclude that the observed sex differences in PSL, although statistically significant when large populations are compared, are likely too small to be of much biological relevance. Therefore, they could be rather considered only as a tendency toward higher values in females. Influence of age. Fig. 2 shows that the values of PSL do not vary significantly with age, at least from the prepubertal up to the middle age. The means were 1.55 t 0.14 ug 5HT/mg protein in prepubertal animals (N=lO), 1.60 t 0.15 ug 5HT/ /mg protein in young postpubertal animals (N=lO) and 1.54 + 0.08 ug 5HT/mg protein in middle-aged animals (N=lO). Serotonin concentrations in the brain of humans and rodents remain stable during ageing (for a review see ref. 12). Literature data on alterations of the level of serotonin in the circulation with ageing mostly refer to humans and are inconsistent. The results, showing a decrease, an increase, or absence of changes, for this parameter with ageing (see Introduction) are not easily comparable because of different sampling intervals and different compartments of circulating serotonin (whole blood or platelets) investigated. For experimental animals, we did not find data as to the influence of age on platelet 5HT except for one paper reporting no changes of rat PSL with ageing (4). The results of our investigations are in accordance with this finding, at least for rats up to 30 weeks of age. Data for PSL in prepubertal animals are given here for the first time. The similar PSL values in pre- and postpubertal rats argue against an important role of gonadal hormones in the control of PSL in males, but their influence on platelet 5HT was studied in rats of both sexes more extensively (21) and will be discussed in a subsequent paper.
1.80
8
: A _*_ :
1.60
-T 0
1.40
i
0
h
-_-
8 8
1.20
S-6
12-15
24-30 weeks
Fig. 2 A comparative representation of individual PSL values in three groups of rats of different age. Group means are indicated by horizontal lines.
Intraindividual oscillations. We have previously demonstrated intraindividual stability of PSL in a small group of rats (1). Here, the study is extended to a large sample, enabling quantitative estimation of intraindividual variability. Individual stability of PSL or whole-blood serotonin in man, when supported by quantitative data, has been stated in the literature in terms of CV (3,6,7) or SD (11). In our case, when a larger group of animals was sampled twice, we found it more convenient to operate in terms of correlation between repeated samples, similarly to Brammer et al (14). The data presented on the scatter-digram (Fig. 3) indicate a high positive correlation (r = 0.858, p < 0.001) between two determinations of PSL in the same animal. Practically all points are within the limits of + 10 % from the line of ideal congruence (y = x). The mean of the differences between two determinations in the same animal amounted to 5.5 % (0 - 13 %). The method of differences confirmed the absence of significant variations between these two determinations (p > 0.2). A similar range of intraindividual variability has been shown for whole-blood serotonin
Vol.
45,
No.
6,
Platelet
1989
Serotonin
in Rats
489
in monkeys (23). The mean interindividual variation (calculated as the arithmetic mean of the differences of individual values from the group mean) in the same sample of 55 rats amounted to 10.2 % (0 - 26 %). Therefore, the intraindividual oscillations of PSL amounted to only half of the interindividual variation in the investigated population. This ratio is very similar to that found in man (3), although both intra- and interindividual variations are much higher in humans. The observed intraindividual variations of PSL of 5.5 % did not significantly exceed the estimated error of the analytical method used, which amounted to approximately 4 % (1). Thus, part of the observed differences could be attributed to this error. The described stability of PSL in the individual rat is in accordance with our recent preliminary observations indicating a genetic background of this biological parameter (24). 2
2.2
z P e
2.0
2 UI s
Fig. 3 Correlation between the values obtained in two independent determinations of platelet serotonin (N = 55) performed within the week interval in the same individual. The t 10 % confidence limits are shown in the shaded area.
la8
E ;; 1.6 { L 1.4 B
r = O.&a P < 0.001 Y = 0.81x l 0.31
i 1.2 v)
N = 55
1.2
1.4 first
1.6
1.8
deter~lnation
2.0
2.2
2.4
(w cj~~,~ prOtej
Periodic oscillations v a r i a t i o n. The results presented in Fig. 4 show Circadian that there are no significant circadian variations of PSL in rats. The suspected decrease between 10 and 13 h, obtained in the first experiment, was not confirmed in the second one. The group size (N = 12) was chosen such as to assure the required accuracy of sample means. Because of the heterogeneity of methodological approaches, it is difficult to compare the literature data on circadian variation of circulating serotonin. In humans, diurnal oscillations of 5HT content in whole blood (2,16), blood serum (15) or platelets (3,6,13,25) have been reported, but the data are not consistent. This is also true for experimental animals: An early study on rabbits, which were kept under usual housing conditions, showed a higher level of blood serotonin in the morning with lowering towards the evening (17). In rats, maintained in strict isolation under a fixed light-dark cycle, circadian variations in serum serotonin levels were observed, but there are pronounced inconsistencies as to the time-course and supposed source of the described oscillations. While, according to Scheving et al. (IB), the peak serum serotonin level coincided with the last period of the light span and the beginning of the dark span, Ho et al. (19) showed a similar peak 10 - 13 hours after feeding regardless of light-dark phasing. The later authors concluded that the feeding schedule is a
Platelet Serotonin in Rats
490
vol. 45, No. 6, 1989
stronger entrainer than the light phasing, but in their own hands, withholding of food had no effect on the observed rhythm. In both papers, the source of the described rhythmical oscillations remained unexplained; they were tentatively related to the rhythm of platelet numbers in the circulation (18,19), diurnal variations in clotting time (18) or the rhythm of blood tryptophan (18). A simple precursor-product relationship influencing the blood serotonin levels, was, however, considered unlikely (19). Such a reasoning is supported by our results which showed resistance of PSL to tryptophan loading (21). Variations in platelet number or the blood clotting process can indeed influence the serum serotonin level, but they do not affect the concentration of serotonin in platelets. In the case of serum, results can, moreover, be affected by the low reproducibility of serotonin release from platelets in the course of the coagulation process (20,26), especially in the light of the mentioned diurnal oscillation of the clotting process (18,27). Our data clearly show that there are neither significant oscillations of 5HT in platelets, nor fluctuations of the number of platelets in the circulation. Namely, any variation of platelet number would be directly reflected in the serotonin content of the sPRP sample. Actually, circadian stability of platelet counts has recently been shown for man (28). Also, the circadian constancy of PSL found here, by itself provides an argument against potential methodological artifacts. Namely, every artificial variation at any time would tend to support, rather than to disprove, the existence of 5HT oscillations. The observed absence of diurnal rhythm of PSL in rats may, however, be related to the experimental conditions under which our investigations were performed. The presented results are, therefore, not necessarily inconsistent with earlier observations in rats maintained under restricted experimental conditions (18, 19). For example, diurnal variations of platelet counts in such conditions would result in changes of serum 5HT without affecting PSL
07
10
13
16
19
22
02
time of daY
Fig. 4 Circadian variation of platelet serotonin content in sPRP samples. Presentation of individual values with indicated group means and standard deviation. N=12. o=first experiment, A=second experiment. C i r c a n n u a 1 (s e a s o n a 1) v a r i a t i o n. A random fluctuation of the mean values of PSL in 42 groups of animals of either sex (N = 412), determined at various times of the year, are summarized in Fig. 5. No indication of a systemic shift of values can be noticed in any season. An absence of
Vol.
45,
No.
6,
1989
Platelet
Serotonin
in Rats
491
marked differences between sexes can also be observed. Practically all values are within the limits of 1.50 and 1.80 ug 5HT/mg platelet protein with the average CV amounting to 9 %. Among the literature data on periodic variations of central and peripheral serotonergic parameters, reports on seasonal variation of circulating serotonin levels are scant for humans and practically non-existent for rats. In humans, seasonal variations in platelet 5HT uptake have been observed by most authors (8), while there are positive and negative reports (29,30) concerning platelet imipramine binding, which appear to be related to a serotonin transporter at the plasma membrane. Regarding the rat there is, to our knowledge, only one paper reporting higher serum serotonin levels in autumn than in summer (18). Since serum serotonin content also depends on variables other than PSL, as discussed above, it can not be easily compared with our results which disprove significant seasonal variation of PSL in rats.
Uu-ll---‘l----
I
II
111
IV
V
VI
VII
VIII
IX
X
Xl
XII
monthof the year Fig. 5 Circanual variations of PSL in rats. Each symbol represents a mean value of a group of animals. N=5-30. o=males, r=females. Conclusions The physiological characteristics of platelet serotonin in rats, summarized here for the first time, may offer useful information to investigators researching this parameter in vivo. The platelet serotomel in rats is an individually stable biological parameter neither subject to periodic oscillations, nor markedly influenced by sex and age. This stability should be regarded in terms of a dynamic equilibrium between intraplatelet and free plasma serotonin (31), with PSL being controlled by the platelet serotonin uptake mechanism (14).
Acknowledgements We are grateful to dr Sonja Iskric, dr fivan OeanoviC and dr Volker Magnus for critical discussions and to Vlado VraneSa and Ivanka Fresl for skillful1 technical assistance. References 1. B. JERNEJ, L. CICIN-SAIN and S. ISKRIC, Life Sci. 43 1663-1670 (1988). 2. G.W. ASHCROFT, T.B.B. CRAWFORD, J.K. BINNS and E.JJacDOUGALL, Clin. Chim.
492
Platelet
Serotonin
in Rats
vol.
45,
No. 6,
1989
Acta 9 364-369 (1964). 3. A. WIEZ-JUSTICE, M. LICHTSTEINER and H. FEER, J. Neural Transm. 41 7-15 (1977). 4. J. WEIL-FUGAZZA, F. GOOEFROY and M. STUPFEL, Mech. Ageing Dev. -13 199204 (1980). P. GUICHENEY, Method. Find. Exp. Pharmacol. 10 253-258 (1988). 2: I.K. GENEFKE and P. MANDEL, Clin. Chim. Acta 131-138 (1968). 7. R.D. SHUTTLEWORTH and J.R. O'BRIEN, Blood 57 g5-509 (1981). a. A. WIRZ-JUSTICE, Experientia 44 145-152 (lv88). 9. E.R. RITVO, A. YUWILER, E. GEKER, E.M. ORNITZ, K. SAEGER and S. PLOTKIN, Arch. Gen. Psychiat. 23 566-572 (1970). N. CRAWFORD, Clin. Chiifi. Acta 18 297 (1967). 1:: E.R. RITVO, A. YUWILER, E. GELER, S. PLOTKIN, A. MASON and K. SAEGER, Biochem. M&d. 5 90-96 (1971). D.G. MORGAN, PFog. Neuro-Psychopharmacol. Biol. Psychiat. 11 153-157 (1987). 1:: S.M. STAHL, R.D. CIARANELLO and P.A. BERGER, Serotonin in Biological Psychiatry, B.T. Ho, J.C. Schoolar and E. Usdin (eds.) pp. 183-198, Raven =New York (1982). 14. G.L. BRAMMER, M.I. McGUIRE and M.J. RALEIGH, Life Sci. 41 1539-1546 (1987). 15. R. FOGARI and L. CORRADI, Folia Endocrinol. 27 463-469 n974). 16. I. SAUERBIER and H. von MAYERSBACH, Horm. Me?%. Res. 8 157-158 (1976). 17. W. NAKAMURA, K. SHIBATA, H. MOMA, M. NE1 and H. ETO, CTR. Sot. Biol. -158 1416-1429 (1964). 18. L.E. SCHEVING, J.D. DUNN, J.E. PAULY and W.H. HARRISON, Am. J. Physiol. 222 252-255 (1972). HO, C.L. CHIK and G.M. BROWN, Life Sci. 37 1619-1626 (1985). 19. m. R.M. HARDISTY and R.S. STACEY, 3. Physiol. 13Tr711-720 (1955). ::: L. CICIN-SAIN. Ph.D. Thesis, University Of Zagreb (1988). 22. D.L. MURPHY, C. WRIGHT, M. BuCHSBAUM, A. NICHOLS, J.L. COSTA and R.J. WYATT, Biochem. Med. 16 254-265 (1976). 23. M.J. RALEIGH, M-1. McmIRE, G.L. BRAMMER and A. YUWILER, Arch. Gen. Psychiat. 41 405-410 (1984 . 8. JERNm and L. tICIN- IAIN, Iugosl. Physiol. Pharmacol. Acta (in press). ::: J. ORTIZ, F. ARTIGAS and E. GELPI, Life Sci. 43 983-990 (1988) 26. N. CRAWFORD and B.I. RUDD, Clin. Chim. Acta 7714-122 (1962). 27. L.E. SCHEWING and J.E. PAULY, Anat Record -157 657-666 (1967). 88 28. I. MODAI, R. MALMGREN, M. ASBERG and H. BEVING, Psychopharmacology 493-495 (1986). 29. A.M. GALZIN, H. LOO, D. SECHTER and S.Z. LANGER, Biol. Psychiat. -21 876-882 (1986). WARSH, H.C. STANCER, E. PERSAD and C.K. VINI, Psychiat. 30. P.M. WITHAKER, J.J. Res. 11 127-141 (1984). 340 77-90 (1983). 31. E.E. mIM and J.H. WYLLIE, J. Physiol. -
Life Sciences, Vol. 45, pp. 485-492 Printed in the U.S.A.
PHYSIOLOGICAL
Branimir
Jernej,
CHARACTERISTICS OF PLATELET SEROTONIN IN RATS
Lipa &in-Sain*
and Sergije
Kveder”
Departments of Experimental Biology and Medicine and of *Organic Chemistry and Biochemistry,“Ruder Bogkovi6t’ Institute, Yu-41001 Zagreb, Yugoslavia, P.O.Box 1016 (Received in final form May 31,
1989)
Summary Physiological
characteristics
of platelet
serotonin
(5HT)
levels
in rats of Wistar origin were investigated by use of a recently developed method. By comparison of populations of male and female rats (N=281) similar unimodal frequency distributions, with a tendency to higher values in females (1.61 vs. 1.70 ug 5HT/mg platelet protein; pc O.Ol), were found. For a group of 55 animals, monitored twice for this parameter within a week interval, a remarkable intraindividual constancy in time, the mean difference between two determinations being 5.5%, was shown. No age-dependence could be demonstrated for platelet serotonin concentrations in 5- to 30week-old rats, nor were there significant circadian or seasonal oscillations. The
physiology of platelet serotonin (5_hydroxytryptamine, 5HT) has so far mostly been the subject of clinical investigations, while considerably less data have been derived from experimeqtal work on laboratory animals. A recently developed method for quantitative determination of platelet serotonin levels (PSL) in rats (1) has made it possible to monitor changes of this parameter in individual animals through prolonged periods of time. Having in mind forthcoming (patho)phy siological investigations of platelet 5HT in rats, we first wanted to become familiar with the basic physiological characteristics of this biological variable, to be able to recognize their possible influences on the results of further experimental treatments.
There is disagreement in the literature concerning sex and age dependence ofPSL. Some reports indicate tendencies to higher, or even significantly higher values of this parameter in females (2,3,4,5), but the absence of sex differences has also been claimed (6,7,8). In a similar manner, a decrease of circulating serotonin with age has been stated in most reports (2,7,9,10,11) while, in other studies, the absence of age dependence (3,5,12) or even tendencies towards an increase of PSL during ageing (8,13) have been claimed. Individual stability of PSL in humans has been reported by several investigators (2,3,6,7,13), but the range of intraindividual variability has been stated only in few reports (3,7). With the exception of our previous observations obtained on a small group of rats (l), we found only one paper adressed to this problem in experimental animals (14). Periodic oscillations of central and peripheral serotonin-related parameters have been reported in the literature. In humans, data supporting a circadian
rhythm in circulating or platelet serotonin (3,15,16) can be contrasted to results which failed to confirm the existence of such rhythmic oscillations (2,6).
0024-3205189 $3.00 +.OO Copyright (c) 1989 Maxwell Pergamon Macmillan plc
486
Platelet Serotonin in Rats
Vol. 45, No. 6, 1989
Seasonal variation of platelet serotonin in man has also been described (3). As to experimental animals, circadian variation of whole-blood serotonin in rabbits (17) and of serum serotonin in rats (18,19) has been demonstrated, but we are not aware of any reports on seasonal variation in laboratory animals. In summary, the literature data are inconsistent and, at least for rats, incomplete. Here we present results, all obtained by use of the same methodology, on sex and age dependence, intraindividual oscillation in time and on circadian and seasonal variations of platelet serotonin levels in rats. Methods Animals. Zgr:Wistar rats of either sex from the breeding facilities of the "R. BoZkovTC" Institute, Zagreb, were used. Animals were housed 3-5 per cage with commercial rat chow and water ad libitum. The animal quarter was maintained at a temperature of 22 t 2"C, with natural light-dark cycle, and usually entered during the morning f% cleaning and supplementing fresh food and water. Sex-de endence studies were performed on 140 males (135-240 g) and 141 females rom which no blood samples had been taken before. Whenever pos* sible, samples of males and females were processed simultaneously. Age-dependence studies were performed on three groups of male animals (N=lO) of different age: prepubertal (5-6 weeks old; 65-80 g), young postpubertal, commonly used in experimental work (12-15 weeks old; 180-250 g) and middle-aged animals (24-30 weeks old; 420-490 g). Individual stability of PSL in rats was investigated on 55 males (190-280 g) by determining this parameter twice in a week interval. The measurements were compared by plotting the results of the first estimation (x-axis) against the second one (y-axis) and calculating the correlation between these two sets of values. Periodic oscillations of PSL were investigated with respect to circadian rhythm and seasonal variation. Circadian variation was studied for males (210-260 g), at the end of March (light/dark intervals approximately 12h/12 h). In the first experiment, 6 groups (N=12) were controlled for PSL at the times indicated in Fig.4. In the second experiment, performed one week later, 3 additional groups (N=12) were used for repeating determinations at 10 and 13 h, and for extension of the night part of the experiment until 02 h. For every group, blood sampling was performed within 30 minutes. Care was taken to minimize disturbance of the animals during the withdrawal of cages. During the dark period, exposing the animals to light was prevented by inducing narcosis in the dark room and transferring them to the laboratory after loss of consciousness. Information on seasonal variation of PSL was obtained by evaluation of data accumulated through a period of more than two years. Althogether, 29 groups of males and 13 groups of females, each consisting of 5-30 animals, were analyzed. Determination of platelet 5HT content. The method for determination of platelet serotonin content in rats has been described in detail elsewhere (1). Shortly, under light aether narcosis, 1 mL of blood was withdrawn, by jugular venipuncture, into a plastic syringe preloaded with ACD anticoagulant. Platelet-rich plasma (PRP) was prepared by centrifuging the sample in the same syringe. The supernatant PRP was quantitatively transferred to a plastic tube and platelets were pelleted by recentrifugation. After homogenizing the pellets by ultrasonication, proteins were precipitated with ZnSO /NaOH and serotonin measured in the clear supernatant by the orthophthaldialdehyde-fluorometric method. The PRP sample obtained from 1 mL of blood by use of this method possesses well-defined characteristics and is termed standardized PRP (sPRP) sample (1). Platelet serotonin content is expressed either per sPRP sample (reflecting platelet serotonin levels in the circulation) or per mg of platelet protein (reflecting se-
Platelet
vol. 45, No. 6, 1989
Serotonin
in Rats
487
rotonin concentration in platelets). Statistics. Student's t-test, regression analysis and the method of differences were used. Variability was expressed in terms of standard deviations (SD) and coefficients of variation (CV). The level of confidence was 99% or above. Results and Discussion Influence of sex. The frequency distributions of PSL in rats of either sex are presented in tig.1. The histograms are of similar unimodal shape and the distribution resembles that found in humans (20). The mean value of PSL in female rats (1.70 t 0.19 ug 5HT/mg protein, N=141) was somewhat higher than in males (1.61 t 0.17 iig 5HT/mg protein, N=140). That small difference (5.6 %), although reaching statistical significance in such a large sample (p < O.Ol), can, in a smaller sample, easily be overshadowed by interindividual variability. Indeed, our previous studies on smaller groups of animals failed to demonstrate significant differences of PSL between sexes (21). It is possible that such differences in sample size could also explain the discrepancies in the literature data on humans, some of which support tendencies toward higher platelet serotonin values in females (2,3,5). while other claim no influence of sex on this parameter (6,7,8).
40
n = 1.61 SD = 0.17
35
N = 140
i E
30
’L
25
1.2
1.4 1.6 1.8
2.0 2.2
n = 1.70
SD= 0.19 N = 1'11
1.2 1.4 w
1.6 1.8 2.0 2.2
5-Hl/~g platelet
protein
Fig. 1 Frequency distributions of PSL in male and female rats of Zgr:Wistar strain The influence of sex has also been investigated with regard to some other serotonin-related parameters. There appear to3be no differences between males and females in either platelet 5HT uptake or .H-imipramine binding, while higher activity of platelet MAO in females has been noted (reviewed in ref. 8). Concerning the platelet MAO activity, a deviation from normal distribution in humans, both males and females, has also been shown (22).
488
Platelet Serotonin in Rats
vol. 45, No. 6. 1989
We conclude that the observed sex differences in PSL, although statistically significant when large populations are compared, are likely too small to be of much biological relevance. Therefore, they could be rather considered only as a tendency toward higher values in females. Influence of age. Fig. 2 shows that the values of PSL do not vary significantly with age, at least from the prepubertal up to the middle age. The means were 1.55 t 0.14 ug 5HT/mg protein in prepubertal animals (N=lO), 1.60 t 0.15 ug 5HT/ /mg protein in young postpubertal animals (N=lO) and 1.54 + 0.08 ug 5HT/mg protein in middle-aged animals (N=lO). Serotonin concentrations in the brain of humans and rodents remain stable during ageing (for a review see ref. 12). Literature data on alterations of the level of serotonin in the circulation with ageing mostly refer to humans and are inconsistent. The results, showing a decrease, an increase, or absence of changes, for this parameter with ageing (see Introduction) are not easily comparable because of different sampling intervals and different compartments of circulating serotonin (whole blood or platelets) investigated. For experimental animals, we did not find data as to the influence of age on platelet 5HT except for one paper reporting no changes of rat PSL with ageing (4). The results of our investigations are in accordance with this finding, at least for rats up to 30 weeks of age. Data for PSL in prepubertal animals are given here for the first time. The similar PSL values in pre- and postpubertal rats argue against an important role of gonadal hormones in the control of PSL in males, but their influence on platelet 5HT was studied in rats of both sexes more extensively (21) and will be discussed in a subsequent paper.
1.80
8
: A _*_ :
1.60
-T 0
1.40
i
0
h
-_-
8 8
1.20
S-6
12-15
24-30 weeks
Fig. 2 A comparative representation of individual PSL values in three groups of rats of different age. Group means are indicated by horizontal lines.
Intraindividual oscillations. We have previously demonstrated intraindividual stability of PSL in a small group of rats (1). Here, the study is extended to a large sample, enabling quantitative estimation of intraindividual variability. Individual stability of PSL or whole-blood serotonin in man, when supported by quantitative data, has been stated in the literature in terms of CV (3,6,7) or SD (11). In our case, when a larger group of animals was sampled twice, we found it more convenient to operate in terms of correlation between repeated samples, similarly to Brammer et al (14). The data presented on the scatter-digram (Fig. 3) indicate a high positive correlation (r = 0.858, p < 0.001) between two determinations of PSL in the same animal. Practically all points are within the limits of + 10 % from the line of ideal congruence (y = x). The mean of the differences between two determinations in the same animal amounted to 5.5 % (0 - 13 %). The method of differences confirmed the absence of significant variations between these two determinations (p > 0.2). A similar range of intraindividual variability has been shown for whole-blood serotonin
Vol.
45,
No.
6,
Platelet
1989
Serotonin
in Rats
489
in monkeys (23). The mean interindividual variation (calculated as the arithmetic mean of the differences of individual values from the group mean) in the same sample of 55 rats amounted to 10.2 % (0 - 26 %). Therefore, the intraindividual oscillations of PSL amounted to only half of the interindividual variation in the investigated population. This ratio is very similar to that found in man (3), although both intra- and interindividual variations are much higher in humans. The observed intraindividual variations of PSL of 5.5 % did not significantly exceed the estimated error of the analytical method used, which amounted to approximately 4 % (1). Thus, part of the observed differences could be attributed to this error. The described stability of PSL in the individual rat is in accordance with our recent preliminary observations indicating a genetic background of this biological parameter (24). 2
2.2
z P e
2.0
2 UI s
Fig. 3 Correlation between the values obtained in two independent determinations of platelet serotonin (N = 55) performed within the week interval in the same individual. The t 10 % confidence limits are shown in the shaded area.
la8
E ;; 1.6 { L 1.4 B
r = O.&a P < 0.001 Y = 0.81x l 0.31
i 1.2 v)
N = 55
1.2
1.4 first
1.6
1.8
deter~lnation
2.0
2.2
2.4
(w cj~~,~ prOtej
Periodic oscillations v a r i a t i o n. The results presented in Fig. 4 show Circadian that there are no significant circadian variations of PSL in rats. The suspected decrease between 10 and 13 h, obtained in the first experiment, was not confirmed in the second one. The group size (N = 12) was chosen such as to assure the required accuracy of sample means. Because of the heterogeneity of methodological approaches, it is difficult to compare the literature data on circadian variation of circulating serotonin. In humans, diurnal oscillations of 5HT content in whole blood (2,16), blood serum (15) or platelets (3,6,13,25) have been reported, but the data are not consistent. This is also true for experimental animals: An early study on rabbits, which were kept under usual housing conditions, showed a higher level of blood serotonin in the morning with lowering towards the evening (17). In rats, maintained in strict isolation under a fixed light-dark cycle, circadian variations in serum serotonin levels were observed, but there are pronounced inconsistencies as to the time-course and supposed source of the described oscillations. While, according to Scheving et al. (IB), the peak serum serotonin level coincided with the last period of the light span and the beginning of the dark span, Ho et al. (19) showed a similar peak 10 - 13 hours after feeding regardless of light-dark phasing. The later authors concluded that the feeding schedule is a
Platelet Serotonin in Rats
490
vol. 45, No. 6, 1989
stronger entrainer than the light phasing, but in their own hands, withholding of food had no effect on the observed rhythm. In both papers, the source of the described rhythmical oscillations remained unexplained; they were tentatively related to the rhythm of platelet numbers in the circulation (18,19), diurnal variations in clotting time (18) or the rhythm of blood tryptophan (18). A simple precursor-product relationship influencing the blood serotonin levels, was, however, considered unlikely (19). Such a reasoning is supported by our results which showed resistance of PSL to tryptophan loading (21). Variations in platelet number or the blood clotting process can indeed influence the serum serotonin level, but they do not affect the concentration of serotonin in platelets. In the case of serum, results can, moreover, be affected by the low reproducibility of serotonin release from platelets in the course of the coagulation process (20,26), especially in the light of the mentioned diurnal oscillation of the clotting process (18,27). Our data clearly show that there are neither significant oscillations of 5HT in platelets, nor fluctuations of the number of platelets in the circulation. Namely, any variation of platelet number would be directly reflected in the serotonin content of the sPRP sample. Actually, circadian stability of platelet counts has recently been shown for man (28). Also, the circadian constancy of PSL found here, by itself provides an argument against potential methodological artifacts. Namely, every artificial variation at any time would tend to support, rather than to disprove, the existence of 5HT oscillations. The observed absence of diurnal rhythm of PSL in rats may, however, be related to the experimental conditions under which our investigations were performed. The presented results are, therefore, not necessarily inconsistent with earlier observations in rats maintained under restricted experimental conditions (18, 19). For example, diurnal variations of platelet counts in such conditions would result in changes of serum 5HT without affecting PSL
07
10
13
16
19
22
02
time of daY
Fig. 4 Circadian variation of platelet serotonin content in sPRP samples. Presentation of individual values with indicated group means and standard deviation. N=12. o=first experiment, A=second experiment. C i r c a n n u a 1 (s e a s o n a 1) v a r i a t i o n. A random fluctuation of the mean values of PSL in 42 groups of animals of either sex (N = 412), determined at various times of the year, are summarized in Fig. 5. No indication of a systemic shift of values can be noticed in any season. An absence of
Vol.
45,
No.
6,
1989
Platelet
Serotonin
in Rats
491
marked differences between sexes can also be observed. Practically all values are within the limits of 1.50 and 1.80 ug 5HT/mg platelet protein with the average CV amounting to 9 %. Among the literature data on periodic variations of central and peripheral serotonergic parameters, reports on seasonal variation of circulating serotonin levels are scant for humans and practically non-existent for rats. In humans, seasonal variations in platelet 5HT uptake have been observed by most authors (8), while there are positive and negative reports (29,30) concerning platelet imipramine binding, which appear to be related to a serotonin transporter at the plasma membrane. Regarding the rat there is, to our knowledge, only one paper reporting higher serum serotonin levels in autumn than in summer (18). Since serum serotonin content also depends on variables other than PSL, as discussed above, it can not be easily compared with our results which disprove significant seasonal variation of PSL in rats.
Uu-ll---‘l----
I
II
111
IV
V
VI
VII
VIII
IX
X
Xl
XII
monthof the year Fig. 5 Circanual variations of PSL in rats. Each symbol represents a mean value of a group of animals. N=5-30. o=males, r=females. Conclusions The physiological characteristics of platelet serotonin in rats, summarized here for the first time, may offer useful information to investigators researching this parameter in vivo. The platelet serotomel in rats is an individually stable biological parameter neither subject to periodic oscillations, nor markedly influenced by sex and age. This stability should be regarded in terms of a dynamic equilibrium between intraplatelet and free plasma serotonin (31), with PSL being controlled by the platelet serotonin uptake mechanism (14).
Acknowledgements We are grateful to dr Sonja Iskric, dr fivan OeanoviC and dr Volker Magnus for critical discussions and to Vlado VraneSa and Ivanka Fresl for skillful1 technical assistance. References 1. B. JERNEJ, L. CICIN-SAIN and S. ISKRIC, Life Sci. 43 1663-1670 (1988). 2. G.W. ASHCROFT, T.B.B. CRAWFORD, J.K. BINNS and E.JJacDOUGALL, Clin. Chim.
492
Platelet
Serotonin
in Rats
vol.
45,
No. 6,
1989
Acta 9 364-369 (1964). 3. A. WIEZ-JUSTICE, M. LICHTSTEINER and H. FEER, J. Neural Transm. 41 7-15 (1977). 4. J. WEIL-FUGAZZA, F. GOOEFROY and M. STUPFEL, Mech. Ageing Dev. -13 199204 (1980). P. GUICHENEY, Method. Find. Exp. Pharmacol. 10 253-258 (1988). 2: I.K. GENEFKE and P. MANDEL, Clin. Chim. Acta 131-138 (1968). 7. R.D. SHUTTLEWORTH and J.R. O'BRIEN, Blood 57 g5-509 (1981). a. A. WIRZ-JUSTICE, Experientia 44 145-152 (lv88). 9. E.R. RITVO, A. YUWILER, E. GEKER, E.M. ORNITZ, K. SAEGER and S. PLOTKIN, Arch. Gen. Psychiat. 23 566-572 (1970). N. CRAWFORD, Clin. Chiifi. Acta 18 297 (1967). 1:: E.R. RITVO, A. YUWILER, E. GELER, S. PLOTKIN, A. MASON and K. SAEGER, Biochem. M&d. 5 90-96 (1971). D.G. MORGAN, PFog. Neuro-Psychopharmacol. Biol. Psychiat. 11 153-157 (1987). 1:: S.M. STAHL, R.D. CIARANELLO and P.A. BERGER, Serotonin in Biological Psychiatry, B.T. Ho, J.C. Schoolar and E. Usdin (eds.) pp. 183-198, Raven =New York (1982). 14. G.L. BRAMMER, M.I. McGUIRE and M.J. RALEIGH, Life Sci. 41 1539-1546 (1987). 15. R. FOGARI and L. CORRADI, Folia Endocrinol. 27 463-469 n974). 16. I. SAUERBIER and H. von MAYERSBACH, Horm. Me?%. Res. 8 157-158 (1976). 17. W. NAKAMURA, K. SHIBATA, H. MOMA, M. NE1 and H. ETO, CTR. Sot. Biol. -158 1416-1429 (1964). 18. L.E. SCHEVING, J.D. DUNN, J.E. PAULY and W.H. HARRISON, Am. J. Physiol. 222 252-255 (1972). HO, C.L. CHIK and G.M. BROWN, Life Sci. 37 1619-1626 (1985). 19. m. R.M. HARDISTY and R.S. STACEY, 3. Physiol. 13Tr711-720 (1955). ::: L. CICIN-SAIN. Ph.D. Thesis, University Of Zagreb (1988). 22. D.L. MURPHY, C. WRIGHT, M. BuCHSBAUM, A. NICHOLS, J.L. COSTA and R.J. WYATT, Biochem. Med. 16 254-265 (1976). 23. M.J. RALEIGH, M-1. McmIRE, G.L. BRAMMER and A. YUWILER, Arch. Gen. Psychiat. 41 405-410 (1984 . 8. JERNm and L. tICIN- IAIN, Iugosl. Physiol. Pharmacol. Acta (in press). ::: J. ORTIZ, F. ARTIGAS and E. GELPI, Life Sci. 43 983-990 (1988) 26. N. CRAWFORD and B.I. RUDD, Clin. Chim. Acta 7714-122 (1962). 27. L.E. SCHEWING and J.E. PAULY, Anat Record -157 657-666 (1967). 88 28. I. MODAI, R. MALMGREN, M. ASBERG and H. BEVING, Psychopharmacology 493-495 (1986). 29. A.M. GALZIN, H. LOO, D. SECHTER and S.Z. LANGER, Biol. Psychiat. -21 876-882 (1986). WARSH, H.C. STANCER, E. PERSAD and C.K. VINI, Psychiat. 30. P.M. WITHAKER, J.J. Res. 11 127-141 (1984). 340 77-90 (1983). 31. E.E. mIM and J.H. WYLLIE, J. Physiol. -