Ultradian rhythm of pontomesencephalic opiate receptor: Modification by chloramphenicol

EXPERIMENTAL

NEUROLOGY

85,209-2 15 ( 1984)

RESEARCH Ultradian

NOTE

Rhythm of Pontomesencephalic Opiate Receptor: Modification by Chloramphenicol

Departamento de Neurociencias, Centro de Investigaciones en Fisiologia Celular, Universidad National Authoma de Mkxico, Apartado Postal 70-600, 04510 Mkxico, D. F.. Mixico Received September 6. 1983; revision received February 13, 1984 Pontomesencephalic receptor binding was measured in rata at 4-h intervals throughout a 24-h day. Two hours prior to each interval the animals were treated with either saline, thiamphenicol, or chloramphenicol. The results showed the existence of an ultradian rhythm of receptor binding. Such rhythm was abolished by chlommphenicol, but not by thiamphenicol. These results suggest that the number of available binding sites changes during a 24-h day, and that such availability is modified by chloramphenicol.

A number of studies have demonstrated that both the metabolism of neurotransmitters (15, 16, 2 1, 29) as well as receptor binding (9, 10, 15, 26) undergo circadian and in some cases ultradian changes (14). In relation to binding some studies showed that such cyclic variations reflected changes in the number of binding sites rather than receptor affinity. Recently it was shown also that antidepressants, be they tricyclic or MAO inhibitors, had a tendency to phase delay the peak number of binding sites of several receptor types, as well as change the amplitude of their circadian variation (10, 26, 27). Antidepressants share with chloramphenicol (CAP), a protein synthesis inhibitor, the effect of producing a specific and selective decrease of REM Abbreviations: CAP-chloramphenicol, TAP-thiamphenicol. r We thank the collaboration of Dr. Jorge Arauz and Juan Lopez in some aspects of this study. We also are grateful to Aurelio Jimenez Huesca for the statistical analysis. This study was partially subsidized by grant PCCBBNA-000800 from CONACYT to A.M.L.C. 209 00144886184 $3.00 CopyrigJtt 0 1984 by Academic Press, Inc. All rights of reproduction in any form mewed

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sleep (4, 20). Chloramphenicol has in addition been shown to decrease the discharge rate of medial reticular neurons as cats pass from slow-wave sleep into REM sleep (5). We determined the effect of CAP on opiate receptor by measuring [‘Hlnaloxone binding. Ninety male Wistar rats were maintained 1 month (March 1983) on a strict 12: 12 light-dark cycle. They were decapitated at 4-h intervals (beginning at 08:OO h) throughout 24 h. The 90 animals were divided into three groups of 30 each according to pretreatment prior to decapitation, thus yielding five rats per time interval in each group. All pretreatments were administered i.p. 2 h prior to decapitation and consisted of administration of saline, CAP (150 mg/kg), or thiamphenicol (TAP, 150 mg/kg) a structural analog of CAP, without effects on sleep (5) and on DNA synthesis (28). Alter decapitation the rat brains were removed and the pontomesencephalic region dissected and placed on dry ice. The entire procedure lasted less than 2 min. The brain stems were stored at -40°C in a Revco freezer until they were analyzed. For preparing membranes, tissue was thawed and homogenized in a glass/ glass homogenizer in 20 vol (w/v) TRIS-HCl buffer, pH 7.4,0.05 Mwithout NaCl and kept on ice for 15 min to allow osmotic disruption. The homogenate was centrifuged 20 min at 4°C at 45,000g. The supematant was discarded and the pellet was resuspended in the same volume of buffer and recentrifuged. Each pellet was washed three times and the membrane pellets were kept frozen at -40°C until assayed. Freezing to 3 months did not alter the specific binding values in our conditions. Binding of [3H]naloxone was measured following basically the procedure described by Bardo et al. (3). Each pellet was homogenized in a glass/glass homogenizer, in TRIS-HCl buffer, pH 7.1, 0.05 M, to give a concentration close to 1 mg protein/ml. The buffer was 100 mM in NaCl, because it has been demonstrated that sodium decreases opiate receptor affinity for agonists, whereas affinity for antagonist is increased or unchanged (22, 24). Samples of 0.94 ml were placed in polycarbonate tubes and incubated 120 min at 0°C with increasing concentrations of [3H]naloxone (1, 2, 4, and 8 nM) in the presence or absence of 1.0 pit4 cold naloxone. All samples were assayed in duplicate or triplicate and the final incubation volume was 1 ml. After incubation, the samples were filtered under vacuum pressure in a Millipore manifold and washed twice with 10 ml cold buffer. Filters were counted for radioactivity after the addition of 10 ml Tritosol(8). Before counting, samples were kept 12 h in the dark to eliminate chemoluminescence. Protein was measured by the method of Lowry et al. (13). Previous to the assay, Whatman GF/B filters were swirled in distilled water and then in isoamyl alcohol saturated with water at room temperature; when on the filtering unit, the filters were washed twice with 10 ml buffer. Under this condition, nonspecific binding of [‘Hlnaloxone to the filters was eliminated. Specific binding was obtained by subtracting binding values in the presence of excess (1 PM) cold naloxone

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from those obtained in the absence of the cold compound. All values were corrected for quenching and counting efficiency. Ultradian variations in [3H]naloxone binding were observed in membranes obtained from the pontomesencephalic formation of animals treated either with saline or with TAP. Two maxima were observed, at 1230 h (87 fmol/mg protein) and at 24:00 h (97 fmol/mg protein) whereas minima were observed at 800 and 20:00 h. (61 fmol/mg protein). These values were obtained using a saturating concentration of [3H]naloxone (8 nM) as is seen from the B/F graphs in Fig. 2. Examination of kinetic variables through double reciprocal (not shown) and Scatchard analysis, revealed a single component of the binding system, with a high affinity KB in the nanomolar range. As seen in Scatchard plots in Fig. 2, the KB values at all selected times remained practically unchanged (from 1.8 nM to 2.6 nM). The kinetic analysis in Fig. 2 also indicates that the highly significant increases in binding observed at 12:00 and 24:00 h were due to changes in the number of receptors (B,,) rather than in the affinity of the binding to those receptors. In membranes from animals treated with CAP, the ultradian rhythm was altered, as no increase in receptor binding was observed at the times in which the controls (treated with NaCl) presented maxima (12:00 and 24:00 h). This contrasted with the animals treated with TAP, the structural analogue of CAP with no effects on sleep, in which variations of receptor binding were almost identical to those of controls (Fig. 1). This study clearly shows that pontomesencephalic receptor sites change

a

12

TIME

1620

24

4 8

OF

DAY

(hrs)

12

16

FIG. 1. Effect of chloramphenicol (CAP) on the rhythm of [3H]naloxone specific binding to pontomesencephalic membranes of the rat. Specific binding at 8 nM [3H]naloxone concentration was measured at 4-h intervals during a 24-h period. Effect of CAP (0) and thiamphenicol (TAP) (A) on the values for control (0) animals are shown. Data are the mean * SE of three or four different experiments in triplicate. Shaded areas indicate the dark phase of the lightdark cycle. Points without standard errors were repeated for accuracy.

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44 1b 2

4

6

[%I-NALOXONE]

8

2

nM

Sb

4

6

8

nM

Kb -2.8

2b

1

K, -2.5

nM

2b

. PO

PO . 0 I?.

4 h 40

(tmolbe

Imp

protbln)

l

NoCl

o CAP h TAP

[‘H-NAL~X~NE]

nM

19.0

900

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throughout a 24-h day. There appears to be differences between forebrain sites as to the periodicity of these changes, because in our study the brain stem presented an ultradian rhythm, whereas the forebrain was reported to show mainly a circadian rhythm (15). This, however, might not be a true difference, for the discrepancy is only at the 12:00-h value which has not been measured in brain by other authors (15, 27). The mean specific binding of [3H]naloxone to membranes from the pontomesencephalic region throughout the 24-h period (6.28 pmol/g wet weight tissue) was found to be about three times higher than that reported by Naber et al. ( 15) in membranes obtained from rat forebrain ( 1.87 pmol/g wet weight) although the KB was in the same range (2.5 r&f). As the mean difference between the lowest and the highest points ( 1.46 times) was the same in both regions and the KB values corresponded, the higher specific binding values obtained in our preparation could indicate a higher capacity of pontomesencephalic membranes for binding opiates. Another fact which could explain the discrepancy could be that previous studies were conducted using low specific activity [3H]naloxone (24.5 Ci/mmol), whereas the specific activity of naloxone used in our study was 48.6 Ci/mmol. As we conducted assays under conditions in which the resolution level of determination was highly increased (3), our values might be more accurate than those reported for other regions. One-way analysis of variance (ANOVA) revealed significant changes with time of day in controls treated with NaCl (I; = 9.00, P < 0.00 1). In the CAPtreated group, however, differences with time of day were nonsignificant. Two-way ANOVA comparing NaCl with CAP as well as NaCl with TAP with time, showed no changes in mean number of binding sites in all three treatments. However, NaCl and CAP treatments showed significant changes with time (F = 2.77, P < 0.05) as well as with the interaction between time and experimental conditions (F = 3.47, P < 0.05). Using the same analysis (two-way ANOVA), treatments with NaCl and TAP showed parallel changes with time (F = 9.55, P < 0.0001). FIG. 2. Saturation curves and Scatchard analysis of [3H]naloxone specific binding to opiate receptors. Saturation curves at six different times were determined in membranes from the rat pontomesencephalic region. AU points are the mean + SE of five animals assayed in triplicate. O-control with NaC1, O-treated with CAP, A-treated with TAP. Scatchard analysis of control and CAP-treated animals is shown at the top of each saturation curve. Plots were drawn as described (25), and KB values were calculated as the negative reciprocal of the slope of each plot. The density of receptors sites (B-) was calculated from the total concentration of receptor sites by correcting for the protein concentration: l , control (NaCl); 0, CAP. Statistical analysis: Nail group one-way ANOVA, F = 9.00, P < 0.001; CAP group one-way ANOVA = NS. Two-way ANOVA comparing NaCl and CAP showed changes with time, F = 2.77, P < 0.05; time and experimental condition interaction, F = 3.47, P < 0.05. Two-way ANOVA comparing NaCl and TAP showed changes with time, F = 9.55, P < 0.001; time and experimental condition interaction = NS.

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The results of this study clearly show that although the total number of opiate binding sites does not change during 24 h, their availability does change at different times of the day. Chloramphenicol appears to modify this &radian availability. It has been demonstrated that the number of neurotransmitter synaptic receptors can vary under different physiologic conditions. Such is the case for GABA receptors which were decreased in number in epileptic foci induced by aluminium (1) and increased under certain stress conditions (23). Glutamate receptors in the hippocampus were highly increased after repetitive stimulation, and this fact has been related to long-term potentiation phenomena (2). The fact that naloxone receptors seem to be concentrated in the pontomesencephalic formation and undergo rhythmic changes could suggest the participation of opiates in the modulation of neurotransmission at this level. It is also interesting to note that CAP, which has been studied mostly as a mitochondrial protein synthesis inhibitor (6, 7, 12), has potent actions on synaptic receptors (18). This effect of CAP on receptors may underlie its actions on sleep. It was shown by several authors that CAP inhibits REM sleep in rats ( 11, 19) and cats (4, 17), whereas TAP does not. Because CAP’s effect on sleep seems to be mediated through decrease of the firing frequency of medial reticular neurons (5), it is conceivable that such effect in turn could be mediated by a decrease in available binding sites for putative neurotransmitters at precise time periods, as shown in this study for opiate receptors. REFERENCES 1. BAKAY, R. A. E., AND A. B. HARRIS. 1981. Neurotransmitter, receptor and biochemical changes in monkey cortical epileptic foci. Brain Rex 206: 398-403. 2. BAUDRY, M., M. OLIVER, R. CREAGER, A. NIERASZKO, AND G. LYNCH. 1980. Increase in 8hnamate receptors following repetitive electrical stimulation in bippocampal slices. Life

Sci. 27: 325-330. 3. BARDO, M. T., R. K. BHATNAGAR, AND G. F. GEBHART. 1982. An improved filtration procedure for measuring opiate receptors in small regions of rat brain. J. Neurochem. 39: 1751-1754. 4. DRUCKER-COLON, R., J. ZAMORA, J. BERNAL-PEDRA~A, AND B. 80s~. 1979. Modification of REM sleep and associated phasic activities by protein synthesis inhibitors. Exp. Neural. 63: 458-467. 5. DRUCKER-COLON, R., S. S. BOWERSOX, AND D. J. MCGINTY. 1982. Sleep and medial reticular unit responses to protein synthesis inhibitors: effects of chloramphenicol and thiamphenicol. Brain Rex 252: 117-I 27. 6. FREEMAN, K. B. 1970. Inhibition of mitochondrial and bacterial protein synthesis by chloramphenicol. Can. J. B&hem. 48: 479-485. 7. FIRKIN, F. C., AND A. W. LINNAE. 1968. Differential effects of chloramphenicol on the growth and respiration of mammalian cells. B&hem. Biophys. Rex Commun. 32: 398. 8. PRICKE’, V. ( 1975). Trimsok a new scintillation cocktail based on Triton X- 100. And. Biochem. 63: 555-558. 9. KAFKA, M. 8, A. WIRZ-JUSTICE, D. NABER, AND T. A. WEHR. 198 1. Circadian acetylcholine

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receptor rhythm in rat brain and its modification by imipramine. Neuropharmacology 20: 421-425.

10. KAFKA, M. S., A. WIRZ-JUSTICE, AND D. NABER. 1981. Circadian and seasonal rhythms in (Y- and @drenetgic receptors in the rat brain. Brain Rex 207: 109-l 19. 11. KITAHAMA, K., AND J. L. VALATAX. 1975. Effect du cbIoramphenico1 et du thiamphenicol sur le sommeil de la souris. C. R. Sot. Biol. (Paris) 169: 1522-1525. 12. KROON, A. M., AND H. DE VRIES. 1969. The effect of chloramphenicol on the biogenesis of mitochondria of rat liver in vivo. FEBS Len. 3: 208-210. 13. LOWRY, 0. H., W. J. ROSEBROUGH, A. L. FARR, AND R. S. RANDALL. 195 I. Protein measurements with the Folin phenol reagent. J. Biol. Chern. 193: 265-275. 14. NABER, D., A. WIRZ-JUSTICE, M. S. KAFKA, AND T. A. WEHR. 1980. Dopamine receptor binding in rat striatum: &radian rhythm and its modification by chronic imipramine. Psychopharmacology 68: l-5. 15. NABER, D., A. WIRZ-JUSTICE, AND M. S. KAFKA. 1981. Circadian rhythm in rat brain opiate receptor. Neurosci. Lett. 21: 45-50. 16. PERLOW, M. J., M. H. EBERT, E. K. GORDON, M. G. ZIEGLER, R. C. LAKE, AND T. N. CHASE. 1978. The circadian variation of catecholamine metabolism in the subhuman primate. Brain Res. 139: 101-l 13. 17. PETITJEAN, F. 1977. Antibiotiques et sommeleis. Effet suppreseur du chloramphenicol sur le sommeil paradoxal chez le chat. Absence d’effet du thiamphenicol. These de Medicine, Universite de Lyon. 18. RAMIREZ, G. 1973. Synaptic plasma membrane protein synthesis: selective inhibition by chloramphenicol in vivo. Biochem. Biophys. Res. Commun. 50: 452-458. 19. ROJAS-RAMIREZ,J. A., E. AGUILARJIMENEZ, A. POSADAS-ANDREW&J. BERNAL-PEDRAZA, AND R. DRUCKER-COLIN. 1977. The effects of various protein synthesis inhibitors on the sleep-wake cycle of rats. Psychopharmacology 53: 147-150. 20. SCHERSCHLICHT,R., J. POLC, J. SCHNEEBERGER,M. STEINER, AND W. HAEFELY. 1982. Selective suppression of rapid eye movement sleep (REMS) in cats by typical and atypical antidepressants. Pages 359-364 in E. COSTA AND G. RACAGNI, Eds., Typical and Atypical Antidepressants: Molecular Mechanisms. Raven Press, New York. 21. SCHAVING, L. E., W. H. HARRISON, P. GORDON, AND J. E. PAULY. 1978. Daily fluctuations in biogenic amines of the rat brain. Am. J. Physiol. 214: 166-173. 22. SIMON, E. J. 1976. The opiate receptors. Neurochem. Rex 1: 3-28. 23. SKERRIT, J. H., P. TRISDIKOON, AND G. JOHNSTON. 1981. Increased GABA binding in mouse following acute swim stress. Brain Res. 206: 387-403. 24. SNYDER,S. H., G. W. PASTERNAK, AND C. B. PERT. 1975. Opiate receptors mechanisms. Pages 329-360 in L. L. IVERSEN, S. D. IVERSEN, AND S. H. SNYDEREds., Handbook of Psychopharmacology. Plenum, New York. 25. WEITAND, G. A., AND P. B. MOLINOFF. 1981. Quantitative analysis of drug-receptor interactions. I. Determination of kinetic and equilibrium properties. Life Sci. 29: 3 13-330. 26. WIRZ-JUSTICE, A., M. S. KAFKA, D. NABER, AND T. A. WEHR. 1980. Circadian rhythms in rat alpha- and beta-adrenergic receptors are modified by chronic imipramine. Life Sci. 27: 341-347.

WIRZ-JUSTICE, A., M. S. KAFKA, D. NABER, I. CAMPBELL, P. MARANGOS, L. TAMARKIN, AND T. A. WEHR. 1982. Clorgyline delays the phase-position ofcircadian neurotransmitter receptor rhythms. Brain Res. 241: 115-122. 28. YUNIS, A. A., D. R. MANYAN, AND G. K. ARIMURA. 1973. Comparative effect of chloramphenicol and thiamphenicol on DNA and mitochondrial protein synthesisin mammalian ceils. Lab. C/in. Med. 81: 7 13-7 18. 29. ZIEGLER, M. G., C. R. LAKE, J. H. WOOD, AND M. H. EBERT. 1976. Circadian rhythm in cerebrospinal 8uid noradrenaline of man and monkey. Nature (London) 261: 656-658. 27.