9 Springer-Verlag 1993. Lack of effect of oxytocin on the numbers of "synaptic" ribbons, cyclic guanosine monophosphate and serotonin N-acetyltransferase.
Cell Tissue Res (1993) 274:337 342
Cell&Tissue Research 9 Springer-Verlag 1993
Lack of effect of oxytocin on the numbers of "synaptic" ribbons, cyclic guanosine monophosphate and serotonin N-acetyltransferase activity in organ-cultured pineals of three strains of rats Stefan Reuss, Eva Mattern, Rainer Spessert, Randolf Riemann, Achim Weber, Lutz Vollrath Department of Anatomy,Johannes Gutenberg University,Saarstrasse 19 21, D-55099 Mainz, Germany Received: 2 February 1993/Accepted: 22 March 1993
Abstract. In addition to the stimulating influence of the sympathetic system on the function of the mammalian pineal gland, neuropeptides such as neuropeptide Y, vasoactive intestinal polypeptide and arginine-vasopressin (AVP) are thought to function as modulators. Since AVP has been shown to influence pineal melatonin synthesis, the aim of the present study was to investigate the possible effects of the second hypothalamic nonapeptide oxytocin (OT), which likewise has been detected in the pineal gland. We therefore studied "synaptic" ribbon (SR) numbers, N-acetyltransferase (NAT) activity and the intracellular concentration of cyclic guanosine monophosphate (cGMP) following in vitro incubation of rat pineals in media containing OT (10 5 M), noradrenaline (NA, 10 .5 M) or both NA and OT. Pineal glands were derived from rats of three different strains (Sprague-Dawley, Long-Evans and the AVP-deficient strain Brattleboro). Neither morphological nor biochemical analyses showed a difference between control and OT-incubated organs in any of the strains tested. In Brattleboro rats, but not in the other strains, noradrenaline slightly increased the number of SR which was not observed when NA and OT were combined. The addition of NA resulted in distinct augmentation of NAT activity and cGMP content, which were not affected by additional OT application. These results suggest that oxytocin is not crucially involved in the regulation of pineal gland function. Key words: Pineal gland - Oxytocin Arginine vasopressin - cGMP Paraventricular nucleus - Circadian rhythm - Rat (Brattleboro, Long Evans, Sprague Dawley)
Introduction The mammalian pineal gland is connected to the optic system via the retino-hypothalamic pathway (Moore and Correspondence to:
S. Reuss
Lenn 1972), hypothalamus, spinal cord (Saper et al. 1976), superior cervical ganglia (SCG) and postganglionic sympathetic fibers innervating the pineal gland (Kappers 1960; Reuss and Moore 1989). A circadian rhythm, originating in the suprachiasmatic nuclei (cf. Moore 1983), is mediated to the pineal and found in several biochemical, physiological and morphological parameters (cf. Sugden 1989; Reuss 1987; Vollrath 1981). These rhythms, e.g., of melatonin synthesis, are characterized by low daytime and high nighttime levels and are induced by the release of noradrenaline from sympathetic terminals. In addition, there is increasing evidence that other neurotransmitters or hormones may influence pineal function. For example, muscarinic and nicotinic cholinoceptors have been found in the gland (Taylor et al. 1980; Reuss et al. 1992). Furthermore, neuropeptides such as vasoactive intestinal polypeptide (Yuwiler 1983) or neuropeptide Y (Reuss and Schr6der 1987; Vacas etal. 1987; Olcese 1991) have been shown to alter pineal melatonin synthesis in rats. Of further interest are the nonapeptides arginine vasopressin (AVP) and oxytocin (OT), which are synthesized in hypothalamic nuclei and act as peptidergic transmitters or as hormones via cerebrospinal fluid or blood circulation. These substances have been detected immunocytochemically in intrapineal nerve fibers in rat (Buijs and P~vet 1980), hedgehog (Nfirnberger and Korf 1981) and monkey (Ronnekleiv 1988). Further, these substances have been identified by radioimmunoassay (RIA) in the rat pineal (Dogterom et al. 1980; Liu et al. 1991). Despite the detection of both substances in the mammalian pineal, their functions remain obscure. The modulation of rat pineal melatonin metabolism has been demonstrated for AVP (Schr6der etal. 1988; Simonneaux et al. 1990; Stehle et al. 1991). Recently, a modulating effect of OT, which has similar hypothalamic sites of production and differs from AVP by only two amino acids, on melatonin secretion from perifused rat pineal glands has been demonstrated (Simonneaux et al. 1990). In order to investigate further the role of OT for the
338 r e g u l a t i o n o f p i n e a l f u n c t i o n , we s t u d i e d the effects o f i n c u b a t i o n in o x y t o c i n - c o n t a i n i n g m e d i a o n selected pineal m o r p h o l o g i c a l ( n u m b e r o f " s y n a p t i c " r i b b o n s ) a n d metabolic parameters (serotonin N-acetyltransferase and g u a n y l a t e cyclase activities). A s e x p e r i m e n t a l a n i m a l s , we selected three different r a t strains, i.e., the a l b i n o S p r a g u e - D a w l e y r a t (SD) as o n e o f the m o s t w i d e l y u s e d a n i m a l m o d e l s , the p i g m e n t e d (" h o o d e d " ) L o n g - E v a n s r a t ( L E ) as well as a n L E m u t a n t , the B r a t t l e b o r o r a t ( h e r e d i t a r y d i a b e t e s i n s i p i d u s d u e to v a s o p r e s s i n deficiency).
Materials and methods
ture medium was changed daily. After two days, drugs were added to the medium and the organs were incubated for 10 or 60 min, respectively. Glands were then sonicated in 0.1 ml 0.05 M TRISHC1 buffer (pH 8.0), 1 mM theophylline, 0.1 mM isobutylmethylxanthine and 0.05 ml 0.9 M sodium acetate buffer (pH 4.0). An aliquot was taken for protein determination, the remainder heated at 90~ for 3 min, centrifuged and frozen in liquid N2. Cyclic GMP was determined following acetylation (Harper and Brooker I975) by means of a radioimmunoassay kit (Amersham Buchler) with [~2~I] cyciic GMP as tracer.
Protein determination Protein concentrations of all pineal samples were determined according to Lowry et al. (1951) with bovine serum albumin as a standard.
First experimental series (determination o f N A T and "synaptic " ribbon numbers) Statistics For these studies (conducted in December), 24 Sprague-Dawley (SD) rats, 24 Long-Evans (LE) rats and 24 Brattleboro (BB) (di/di) rats were used. The animals, aged 10-14 weeks (180-220 g b wt), were obtained from the Zentralinstitut fiir Versuchstierzucht, Hanhover, Germany (LE, SD) or stem from our own breeding colony (BB), the original stock of which was kindly provided by Professor Dr. D. Richter, Universit~itsklinik Eppendorf, Hamburg, Germany. All animals were housed under a constant light-dark regimen (LD 12:12; lights on at 05.00 h) with food and water ad tibitum. Between 15:00 h and 16:00 h, the rats were decapitated, the pineal glands quickly removed and placed three each in culture dishes on plastic meshes under sterile conditions. Dishes contained 1 ml BGJb medium, Fitton Jackson modified (Gibco; 0.125 mg/ml CaCO3, 0.1 mg/ml ascorbic acid, 2 mM glutamate, 100 U/0.1 mg penicillin/streptomycin). The organs were incubated for 6 h under an atmosphere of 95% 02 and 5% COz at 37~ C. Each pharmacological treatment involved 2 culture dishes, i.e., 6 pineal glands. The glands were incubated either in medium alone or in medium containing either oxytocin (OT, 10 5 M), noradrenaline (NA, t0 -s M) or a combination of OT and NA. At the end of the incubation period, pineals were removed, cut in halves and fixed for electron microscopy or processed for biochemical analysis.
Electron microscopy. Tissue specimens were fixed by immersion according to Karnovsky (1965) and processed for routine electron microscopy as described (Reuss et al. 1989). Examination of the sections was performed in such a way that the investigator was unaware from which group of animals the material was taken. For quantitative assessment of SR, pineal tissue covering 5 adjacent grid holes was carefully scanned at x 20000 magnification, and the total number of SR was counted. SR numbers are calculated per 21 125 gm 2 corresponding to 1 unit area (UA). Furthermore, the structures were analysed with respect to their intracellular location (i.e., near to or distant from the cell membrane).
Biochemical analysis. At the end of the experiments, pineal tissues were immediately frozen in liquid nitrogen. Within 24 h, they were homogenised in an ice-cold tryptamine solution (2.1 mM tryptamine in 50 mM phosphate buffer, pH 6.5). The activity of pineal N-acetyltransferase (EC 2.3.1.5) was determined from glands of all experimental series (Deguchi and Axelrod 1972).
Second experimental series ( c G M P assay) For this study (conducted in May), pineal glands from 30 male SD rats were obtained at 10 : 00-11 : 00 h, placed in plastic dishes and preincubated for 48 h under conditions described above. Cul-
The Wilcoxon-Mann-Whitney U-test and the Kruskal-Wallis Htest were used for statistical evaluation of differences in number of SR among animal groups. The Spearman-Rank correlation coefficient was computed to test correlation between parameters. Statistical treatment of the NAT and cGMP data involved an analysis of variance followed by Duncan's multiple range comparison of means.
Results and discussion
" Synaptic " ribbons In p i n e a l tissue f r o m each r a t strain, " s y n a p t i c " b o d i e s were f o u n d (Fig. 1). In all cases, r i b b o n s clearly o u t n u m b e r e d i n t e r m e d i a t e s t r u c t u r e s a n d spherules. T h e n u m b e r o f S R in c o n t r o l s were in the n o r m a l range, i.e., 15-20 p e r u n i t a r e a at d a y t i m e . W i t h one e x c e p t i o n , the a d d i t i o n o f N A , O T o r N A + O T to the i n c u b a t i o n m e d i u m d i d n o t result in a n y significant differences in the n u m b e r o f p i n e a l SR. In the case o f N A i n c u b a t i o n o f B r a t t l e b o r o r a t pineals, n u m b e r s o f S R were a u g m e n t e d a p p r o x i m a t e l y t w o f o l d w h e n c o m p a r e d to c o n t r o l s ( P < 0.02; Fig. 2). This effect was n o t o b s e r v e d w h e n BB r a t p i n e a l s were i n c u b a t e d in m e d i u m e n r i c h e d w i t h b o t h N A a n d OT.
N-acetyltransferase activity (Fig. 3) Levels o f p i n e a l N A T a c t i v i t y f o l l o w i n g i n c u b a t i o n in c o n t r o l m e d i u m were b e l o w 5 n m o l / m g p r o t e i n / h supp o r t i n g earlier r e p o r t s (Stehle et al. 1991). I n c u b a t i o n with n o r a d r e n a l i n e a l o n e i n c r e a s e d p i n e a l N A T activity significantly in each strain. This i n c r e a s e was l o w e r in BB a n d L E strains t h a n in the S D rats. In p i n e a l s i n c u b a t e d in O T - c o n t a i n i n g m e d i u m , no significant differences in N A T a c t i v i t y levels were o b served w h e n c o m p a r e d to c o n t r o l s . S i m u l t a n e o u s i n c u b a t i o n in b o t h N A a n d O T h a d b a s i c a l l y similar effects w h e n c o m p a r e d to the i n c u b a tion in N A alone.
339
Fig. 1 A, B. "Synaptic" ribbons (arrows) in pinealocytes of Sprague-Dawley rats following 6 h of incubation in OT-enriched medium. A Two ribbons in a pinealocyte process. Note the enlarged end of a SR. B Ribbons and ribbon fields located at the cytoplasmic membrane. Bar: 300 nm
Numbers
of " s y n a p t i c " r i b b o n s
40 30 20 I0 0
r m
;~ Brattleboro
o~ ~
Long-Evans
NA;0T ~
Sprague-Dawley
Cyclic guanosine monophosphate (Fig. 4) Pineal c G M P levels were n o t altered following incubation in OT-enriched medium. The incubation in N A enriched m e d i u m a u g m e n t e d c G M P levels after 10 rain, with a similar effect when N A and O T were combined. The present results reveal that, u n d e r the experimental conditions tested, o x y t o c i n does n o t exert any effect
Fig. 2. Numbers of "synaptic" ribbons in pinealocytes of Brattleboro, LongEvans and Sprague-Dawley rats following a 6 h-incubation in either medium alone (control), medium enriched with noradrenaline (NA, 10- s M), oxytocin (OT, 10 -5 M) or combination of NA+ OT. Columns represent mean + SEM (n = 5). Asterisk indicates statistical significance (P
