BRAIN RESEARCH

E L S E V I E R Brain Research 731 (1996) 254-257

Short communication

Pyroglutamyl peptidase I levels and their left-right distribution in the rat retina and hypothalamus are influenced by light-dark conditions

Begofia Sfinchez a Francisco Alba b, Juan de Dios Luna c Jos~ Manuel Martfnez d Isabel Prieto d Manuel Ramfrez d,,

a Department of Physiology, University of the Basque Country, Leioa, Vizcaya, Spain b Department of Biochemistry and Molecular Biology, University of Granada, Granada, Spain

c Department of Biostatistics, University of Granada, Granada, Spain d Unit of Physiology, University ofJadn, Bldg 5, E-23071 Jadn, Spain

Accepted 18 June 1996

Abstract

To evaluate the effects of light and darkness on pyroglutamyl peptidase I activity (pGluPI) and its left-right distribution, pGluPI was measured bilaterally in the retina and hypothalamus under selected light-dark schedules. Rats under a 12 h light-dark cycle were divided into four experimental groups. After the end of the 12 h dark period, the animals were kept two additional hours in darkness (group 1), or light (group 2). After the end of the 12 h light period, the animals were kept two additional hours in darkness (group 3), or light (group 4). Experiments were done in light or darkness depending on the 2 h period. In the retina, a previous 12 h light period led to higher values of enzyme activity than dark periods. Left-right predominance, however, depended on the previous 2 h period: the light period led to left predominance, whereas fight predominance was found after the 2 h dark period. In the hypothalamus, a left predominance was found only in group 3. These results demonstrate that environmental light conditions influence pGluPI activity in the rat retina and hypothalamus.

Keywords: Pyroglutamyl peptidase I; Asymmetry; Retina; Hypothalamus; Light; Darkness; Rat

Pyroglutamyl peptidase I (pGluPI; EC 3.4.19.3) re- moves pyroglutamyl N-terminal residues from peptides and arylamide derivatives in a highly selective manner [4]. We previously reported significant changes for pGluPI activity in the retina and anterior hypothalamus, together with an asymmetrical distribution of this activity at se- lected time points during 12 h light and 12 h dark periods [5]. Because the retina and hypothalamus are functionally connected by the retinohypothalamic tract, it is plausible that environmental light influences pGluPI activity. To test this hypothesis, we studied the effect of light and dark conditions on pGluPI and on its left-fight distribution in the retina and anterior hypothalamus, pGluPI activity was measured in the left and right sides of the retina and in the anterior hypothalamus under selected light-dark schedules. To analyze the activity of pGluPI we used pyroglutamyl- [3-naphthylamide (pGluNNap) as the substrate [1,4].

* Corresponding author. Fax: (34) (53) 212-141; e-mail: msanchez @piturda.ujaen.es

Thirty-two male Sprague-Dawley rats weighing 200- 250 g were used in this study. The animals were given food and water ad lib and were housed under a controlled temperature (25°C). Four experimental groups were used with a regular 12-12 h light-dark cycle (groups 1 and 2, light on from 7.00 to 19.00 h; groups 3 and 4, light on from 19.00 to 7.00 h). Previous to the experiments, rats were adjusted to their corresponding cycle for at least 2 weeks. Experiments were performed under the following schedules (Fig. 1). Group 1: animals kept two additional hours in darkness at the end of the 12 h dark period; the experiment was done under dark conditions (n = 5). Group 2: two hours in light after the end of the 12 h dark period, the experiment was done under light conditions (n = 9). Group 3: two hours in darkness after the end of the 12 h light period, the experiment was done under dark condi- tions (n = 9). Group 4: two additional hours in light at the end of the 12 h light period; the experiment was done under light conditions (n = 9).

The brains were perfused with saline transcardially under equithensin anesthesia (2 m l / k g body weight),

0006-8993/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S0006-8993(96)00753-6

B. Sdnchez et al. / Brain Research 731 (1996)254-257 255

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t Fig. 1. Experimental groups used to evaluate the effects of light and darkness on pGluPI activity. The animals were previously maintained for at least 2 weeks under a 12 h light-12 h dark cycle (groups 1 and 2, light on from 7.00 to 19.00 h; groups 3 and 4, light on from 19.00 to 7.00 h). Group 1: Animals kept two additional hours in darkness at the end of the 12 h dark period; the experiment was done under dark conditions. Group 2: Two hours in light after the end of the 12 h dark period; the experiment was done under light conditions. Group 3: Two hours in darkness after the end of the 12 h light period; the experiment was done under dark conditions. Group 4: Two additional hours in light at the end of the 12 h light period; the experiment was done under light conditions.

quickly removed (less than 60 s) and cooled in dry ice. The experiments were done in winter (northern hemi- sphere). In dark conditions, the animals were perfused under a dim red light and additional light was used only after the eyes were removed. Before the brain was re- moved, both eyes were taken out and their corresponding retinas quickly dissected and cooled in dry ice. The left and right anterior hypothalamus were dissected according to the stereotaxic atlas of K~Snig and Klippel [3]. The

anterior hypothalamic area was considered that between the stereotaxic planes A6360 and A5150, anterior to the interauricular line. Tissue samples were homogenized in 10 volumes of 10 mmol/1 HC1-Tris buffer (pH 7.4) and ultracentrifuged (100,000 × g, 30 min, 4°C) to obtain the soluble fraction. The resulting supernatants were used to detect enzymatic activity and protein content, assayed in triplicate.

pGluPI activity was measured in a fluorometric assay with pGluNNap as the substrate, according to the modified method of Schwabe and McDonald [6]: 10 ~1 of each supernatant was incubated during 120 min at 37°C with 1 ml of the substrate solution (60 mol/1 of pGluNNap, 0.5 mmol/1 of dithiothreitol, 1 mmol/1 of EDTA in 50 mmol/1 of phosphate buffer, pH 7.4). The reaction was stopped by adding 1 ml 0.1 tool/1 of acetate buffer (pH 4.2). The quantity of -naphthylamine released as a result of enzymatic activity was determined fluorometrically at an emission wavelength of 412 nm with an excitation wave- length of 345 nm. Proteins were quantified in triplicate by the method of Bradford [2]. Specific pGluPI activity was expressed as pmol of pGluNNap hydrolysed per min per mg of protein. The results of the fluorogenic assay were linear with respect to time of hydrolysis and protein con- tent.

A four-factor design was used to analyze the data. The first factor was long period (12 h), with two levels (light and darkness). The second factor was short period (2 h), with two levels (light and darkness). The third factor was laterality, with two levels (left and right). These were fixed effect factors, and were crossed between themselves. The fourth factor was the animal, and was considered a random effect factor. The fourth factor was nested in the long

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Fig. 2. Bars represent the percentage differences between the left and right sides of retina, for each of the rats studied, in the four different groups programmed. H, higher value; L, lower value.

256 B. Sdnchez et al. /Brain Research 731 (1996) 254-257

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Fig. 3. Bars represent the percentage differences between the left and fight sides of anterior hypothalamus, for each of the rats studied, in the four different groups programmed. H, higher value; L, lower value.

period vs. the short period crossing, and involved two measurements: one from the left side and the other from the right. The data were analyzed with an ANOVA table. If interactions (from highest to lowest) were significant, pairwise comparisons were made using the Bonferroni method. If main effects were found with no significant interactions, differences between the levels were obtained directly from the ANOVA table. All the left-right compar- isons were made for paired data.

The percentage of differences between the left and right values of retina and anterior hypothalamus, for each ani- mal, in the four groups studied are shown in Figs. 2 and 3.

In the retina, statistical analysis demonstrated a signifi- cant difference between long periods (P = 0.037): a previ- ous 12 h light period (long period, groups 3 and 4) led to higher values of enzyme activity than long dark periods (groups 1 and 2). In addition, a significant interaction was found between side and short period (P = 0.001): in the right side. a previous 2 h dark period (short period, groups 1 and 3) led to higher values of activity than short light periods (groups 2 and 4) (P < 0.001). However, in the left side, a previous short light or dark period did not lead to differences in enzymatic activity. On the other hand, left- right predominance depended on the previous short period (2 h): a short light period led to left predominance (groups 2 and 4, P < 0.01, paired data) (90% and 100% of left predominance respectively), whereas a tendency to fight predominance was found after the short dark period (groups 1 and 3) (80% and 67% of right predominance respec- tively).

In the hypothalamus, previous exposure to light or darkness did not influence enzymatic activities, whereas left predominance was found only in group 3 (P < 0.001,

paired data) (89% of left predominance), which was condi- tioned by the interaction between the previous long light period and the subsequent short dark period.

The present results show the existence of laterality which depends on light (left predominance) or dark (right predominance) conditions. These observations support our previous findings [5] of left predominance of retinal en- zyme activity in the light period of a 12-12 h light-dark cycle, and right predominance in the dark period. In hypothalamus, we found only left predominance in the light and dark period [5]. In the present study we demon- strated conclusively that an external factor, environmental light conditions, influences endogenous neurochemical change in the levels of pGluPI activity in the retina and hypothalamus. Specific environmental conditions, such as programmed rhythms of light and darkness, can determine the existence and the side (left or right predominance) of laterality of pGluPI activity in the same locations.

Putative endogenous substrates of pGluPI (such as thy- rotropin releasing hormone or gonadotropin releasing hor- mone) may play a functional role in the retina, apart from their well-known role in the hypothalamus. Since this enzymatic activity may play an important role in the hydrolysis of susceptible substrates, changes in pGluPI levels under different environmental conditions may reflect modifications in the function of the enzyme's substrates.

Acknowledgements

i Special thanks are due to Mrs. Raquel Villares for

valuable technical assistance in laboratory work. We also

B. Sdnchez et al. / Brain Research 731 (1996) 254-257 257

thank Karen Shashok for improving the English style of the manuscript.

References

[1] Bauer, K., Degradation and biological inactivation of thyrotropin-re- leasing hormone (TRH): regulation of the membrane-bound TRH-de- grading enzyme from rat anterior pituitary by estrogens and thyroid hormones, Biochimie, 70 (1988) 69-74.

[2] Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248-254.

[3] KiSnig, J.F.R. and Klippel, R.A., The Rat Brain. A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Krieger Hunting- ton, New York, 1967, p.p 24-95.

[4] McDonald, J.K. and Barret, J.A, Mammalian Proteases, Academic Press, London, Vol. 2, 1986, pp. 23-100.

[5] Ramfrez, M., Sanchez, B., Arechaga, G., Garcia, S., Lardelli, P., Venzon, D. and De Gandarias, LM., Diurnal variation and left-right distribution of pyroglutamyl peptidase I activity in the rat brain and retina, Acta Endocrinol., 125 (1991) 570-573.

[6] Schwabe, C. and McDonald, J.K., Demonstration of a pyroglutamyl residue at the N terminus of the B-chain of porcine relaxin, Biochem. Biophys. Res. Commun., 74 (1977) 1501-1504.