Peptides,Vol. 12, pp. 529-533. ©PergamonPress plc, 1991. Printedin the U.S.A.

0196-9781/91 $3.00 + .00

Pineal Vasoactive Intestinal Peptide Is Reduced During the Proestrous Stage of the Rat Estrous Cycle F. M O U J I R , M. J. L O R E N Z O , * R. A L O N S O , 1 C. S A N T A N A , L. C A C I C E D O * A N D F. S A N C H E Z - F R A N C O *

Laboratory of Neuroendocrinology, Department of Physiology University of La Laguna School of Medicine, 38320 Tenerife, Spain *Department of Endocrinology, Hospital "Ramon y Cajal," 28034 Madrid, Spain Received 16 April 1990

MOUJIR, F., M. J. LORENZO, R. ALONSO, C. SANTANA, L. CACICEDO AND F. SANCHEZ-FRANCO. Pinealvasoactive intestinal peptide is reduced during the proestrous stage of the rat estrous cycle. PEPTIDES 12(3) 529-533, 1991.--Immunoreactive levels of vasoactive intestinal peptide (IR-VIP) in the rat pineal gland were examined during the estrous cycle. IR-VIP was shown to be identical to the synthetic porcine material by its similarity in competitive binding studies, and by both gel filtration and high pressure liquid chromatography. Pineal IR-VIP decreased at early proestrus (0300 h, dark), partially as a function of the rise in serum estradiol levels. Although the functional role of VIP in the pineal remains to be clearly elucidated, these results suggest that it might be involved in the reproductive function of the female rat, and that estrogens could partially modulate its pineal concentration. Pineal gland

VIP

Estrous cycle

Estrogens

EVEN though mammalian pineal rhythms are mainly controlled by the light-dark cycle through a well-known noradrenergic mechanism (1, 4, 9), experimental evidence suggests that circulating levels of reproductive hormones may influence, at least in part, pineal indole metabolism (6,22). In the female rat, it has been shown that changes in gonadal or pituitary hormones occurring during the estrous cycle are able to induce parallel variations in pineal biosynthetic activity and secretion (3,13). However, even though these relationships between serum hormone levels and pineal indoleamine metabolism are well documented, little is known about the effects of reproductive hormones on other pineal components. It has been indicated that VIP can be a second transmitter candidate in the regulation of mammalian pineal function. Nerve fibers containing VIP have been identified in the pineal gland of several species (19,28) and immunoreactive levels of VIP have shown circadian changes in the male rat pineal (15). In addition, VIP receptors are abundant in pineal tissue (14), and VIP stimulates cAMP and cGMP accumulation as well as N-acetyltransferase (NAT) activity in the pineal gland (12, 16, 25, 30). On the other hand, it was recently suggested that the pineal is under dual receptor regulation, one system being activated by beta-adrenergic agonists and the other by VIP (7). In the present work, we

have examined the immunoreactive levels of VIP in the pineal gland of female rats, in order to determine whether it could be influenced by the hormonal variations during the estrous cycle. METHOD

Animals Female Sprague-Dawley rats, caged in groups of three to five, were maintained in a temperature (23 __.2°C)-controlled room under a 12:12 light-dark cycle (50-80 p,W/cm2; lights on, 9 a.m.; lights off, 9 p.m.), with ad lib access to rat chow and water. Vaginal smears were performed dally between 1200 and 1400 hours, and only rats showing at least three consecutive 4-day cycles were used for the experiments. Smears were identified as proestrus, estrus, diestrus-1 or diestrus-2. Rats were killed by decapitation at 0300 (dark) or 1800 hours (light) and their pineals quickly removed, immersed in liquid nitrogen and stored frozen ( - 8 0 ° C ) until assayed. Blood was collected from the trunk and serum stored frozen for hormone assays.

Tissue Extracts Individual pineals were thawed at room temperature and

1Requests for reprints should be addressed to Dr. R. Alonso.

529

530 homogenized by sonication in 600 Ixl of 0.1 M HC1. After boiling for five minutes, the homogenates were centrifuged at 3000 × g for 30 minutes and the supernatants kept frozen at -20°C until analyzed for radioimmunoassayable VIP (5,17).

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Chromatographic Characterization IR-VIP from pooled rat pineal glands was fractionated by gel filtration through a (1 x40 cm) column of Sephadex G-50 fine (Pharmacia Fine Chemicals, Upsala, Sweden). Twenty pineal glands were homogenized by sonication in 1 ml of 0.1 M HCI. After centrifugation at 3000 x g for 30 minutes, the supernatants were applied on the column and fractions of 1 ml eluted with 0.1 M acetic acid containing 0.25% BSA, at 4°C with constant gravity perfusion, and assayed by RIA. To achieve further characterization, IR-VIP from rat pineals was also analyzed by HPLC. Reversed phase analysis was carried out on a ix-Bondapak C-18 column (Waters Associates, Milford, MA). Pineal extracts were prepared as described above and, after centrifugation at 3000 x g, 500 ixl aliquots from the supernatants were injected into the HPLC system. The column was eluted with 0-100% acetonitrile in 0.1% trifluoroacetic acid for 30 rain at a flow rate of 1 ml/min; 2 rnl fractions were collected, lyophilized, and then reconstituted in 500 Ixl of the RIA buffer and assayed for IR-VIP.

Hormone Assays Serum luteinizing hormone (LH) levels were determined by RIA according to previously validated procedures (2), and resuits are expressed in terms of NIAMDD rat LHRP-2. Serum estradiol concentrations were measured by RIA (Diagnostic Products Co., Los Angeles, CA).

Peptides Porcine VIP(1-28), PHM-27, PHI, pancreatic polypeptide, insulin, gastrin, glucagon, somatostatin, AVP, LH-RH, 13-endorphin and gastric inhibitory peptide were supplied by Bachem

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VIP antiserum was raised by immunizing rabbits with synthetic porcine VIP coupled to bovine serum albumin with carbodimide (Sigma Chemical Co., St. Louis, MO) (17). Specificity of the antiserum was tested against PHM-27, PHI, pancreatic polypeptide, insulin, gastrin, glucagon, somatostatin, argininevasopressin (AVP), gonadotropin-releasing hormone (LH-RH), 13-endorphin, secretin, pentagastrin and gastrin inhibitory peptide, and was shown to be highly specific. VIP was iodinated by the chloramine-T method (11). The iodination mixture was fractionated in a Sephadex G-50 fine column by elution with 0.1 M acetic acid in 0.25% bovine serum albumin (BSA). The standard diluent used was 0.05 M phosphate buffer, pH 7.5, containing 0.1% BSA, 0.25 M EDTA, and 0.1% sodium azide. Samples of unknown or standard solutions of porcine VIP(1-28) were incubated at 4°C with 100 ix1 of VIP antibody (final dilution 1/14,000) in standard diluent containing 100 I~1 of normal rabbit serum (Diagnostic Biochem. Inc., Canada). After 24 h, 100 lxl of ~2~I-VIP were added and the incubation continued for another 48 h. To separate bound from free complexes, 100 ILl of anti-gamma-globulin serum were added and, after 24 h, the mixture was centrifuged at 3000 × g for 60 min at 4°C. The assay sensitivity was 19-39 pg/ml, and the intra- and interassay variation coefficients 4-6 and 10-15%, respectively.

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Inc. (Torrance, CA). Porcine secretin was obtained from Kabi Vitrum (Stockholm, Sweden), and pentagastrin from Imperial Chemical Industries PLC (Cheshire, England).

Statistics Data were analyzed by two-way ANOVA followed by pairwise test to compare pairs of means (27). Statistical differences between estrous phases were judged to be significant when associated with a probability of 5% or less. To evaluate possible relationships between pineal VIP content and plasma estradiol levels, linear regression analysis was performed. RESULTS

As shown in Fig. 1, serial dilutions of pineal extracts resulted in binding inhibition curves which paralleled those of synthetic VIP. The specificity of the antiserum was tested against pentagastrin, secretin, PHM-27, PHI, pancreatic polypeptide, insulin, gastrin, glucagon, somatostatin, AVP, LH-RH, 13-endorphin, and gastric inhibitory peptide. No cross-reactivity was shown in concentrations up to 1 ~g/ml. After fractionation of pineal extracts on a Sephadex G-50 column, IR-VIP was apparently present in four molecular forms (Fig. 2). Peak II showed an elution profile identical to that of synthetic VIP(1-28), whereas component I emerged earlier, and components III and IV later. Component II accounted for 62% of the total immunoreactivity in the extracts, while components I, III, and IV each accounted for 12.6, 17.2, and 7.8%, respectively. The analysis of rat pineal extracts by HPLC is shown in Fig. 3. Only one component was shown at a retention time of 20 min, which corresponds to that of synthetic VIP(1-28), Figure 4 shows the variation of immunoreactive VIP in the pineal gland of female rats killed at different hours during the estrous cycle. Serum estradiol and LH levels displayed typical estrous-related variations. Estradiol concentrations were elevated from late diestrus-2 to late proestrus, as compared with other phases, F(7,58)= 20.22, p