Ncurocndocrinology 15: 99 105 (1974) L-Dopa as a Stimulus for LH Release in the Proestrous Rat: Blockade of its Action by Pentobarbital Anesthesia1 J.H. W edig 3 and V.L. G ay 3 Interdepartmcm Toxicology Program and Reproductive Endocrinology Program, Department of Pathology, University of Michigan, Ann Arbor, Mich. Abstract. Intra-arterial injcctions(8 75mg/kg)of l-3,4 . dihydroxyKey Words phenylalanine (t.-dopa) at I p.m. on the afternoon of proestrus proL-dopa duced a rapid increase in scrum LH in unanesthetized female rats. LH release Maximum responses were observed following doses of 25 mg/kg alPentobarbital though the smallest (2.7 mg/kg) and largest (225 mg/kg) doses were Proestrous rat ineffective in stimulating LH release. Pentobarbital anesthesia (40mg/kg, i.p.) at 12.30 p.m. on proestrous prevented the responses to L-dopa. The 25 mg/kg dose of t.-dopa, which produced maximum effects in the unanesthetized proestrous rat, caused no detectable LH release in unanesthetized dicstrous rats. These results support the con cept that dopaminergic mechanisms are involved in the pre-ovulatory release of LH and indicate that two of the conditions which prevent spontaneous pre-ovulatory LH release (pentobarbital anesthesia or a lack of circulating estrogens) may similarly inhibit L-dopamediated LH release. In rats, the incidence of ovulation is markedly reduced following treatments which alter brain catecholamine levels [R ubinstein and S awyer , 1970; K ordon and G lowinski, 1969; Coppola el al., 1966; Barraclough and S awyer , 1957], Conversely, ovulation may be Received: July 6th, 1973: revised MS accepted: December 13th, 1973. Downloaded by: Kings's College London 18.104.22.168 - 10/25/2017 8:31:25 PM 1 Supported by USPHS grants ES-0016 and HD-05318. 2 Recipient of an NIH Toxicology Training Fellowship. Present address: Environ mental Hygiene and Toxicology Dept., Olin Research Center, 275 Winchester Ave., New Haven, Connecticut. 3 Career Development Awardee of the National Institute of Child Health and Human Development. Present address: Department of Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania. I(K) W edig/CJay induced or reinstated by treatments which increase the availability of endogenous or exogenous catecholamines [ R u b in s t e in and S a w y e r , 1970; M e y er so n and S a w y e r , 1968; R a z ia n o el u L, 1971], Apparently the catecholamines do not produce these effects by a direct action on the pituitary gland [S c h n e id e r and M c C a n n , 1969] but, rather, initiate a sequence of events involving the release of both LRH [K a m b e r i et al., 1969; S c h n e id e r and M c C a n n , 1970b] and LH [S c h n e id e r and M c C a n n , 1970a], The physiological importance of the LH-releasing action of dopamine (DA) or one precursor of DA, l-3,4, dihydroxyphenylalanine (L-dopa), has been suggested by the observation that the release of LH induced by these compounds, like the spontaneous pre-ovulatory LH release of the cyclic female rat, is considerably reduced in the absence of circulating estrogens [ R a z ia n o et al., 1971; S c h n e id e r and M c C a n n , 1970a], We have sought to extend this correlation by demonstrating that pentobarbital anesthesia, a potent blocker of the spontaneous pre-ovulatory LH release [C r a m e r and B a r r a c l o u g h , 1971; C r a m e r et al., 1970], also prevents L-dopa-induced LH release in the proestrous rat. Mature female Holtzmann rats (240 260 g) were housed in air-conditioned quarters. All animals were exposed to the lighting schedule of lights on 5 a.m. to 7 p.m. for a period of 10 days after which vaginal smears were obtained daily at 9 a.m. for an additional 2 weeks. The responses of rats with regular 4- or 5-day cycles were assessed separately and were combined only after it was determined that responses were similar. At 9 a.m. on the morning of functional proestrous (as predicted by vaginal cytology) each rat was anes thetized with ether, laparotomized to confirm uterine 'ballooning' and cannulated via the carotid artery [G ay et at., 1969], Approximately 4 h after these surgical procedures each unanesthetized rat received 100 units of heparin via the cannula. Blood samples of 50 //I were obtained at 20-min intervals prior to and following injections of L-dopa. The cannula was flushed with 40 /d of warm physiological saline immediately after each 50 /d blood sample had been obtained and after the injection of 0.5 n HCI or L-dopa (Calbiochem, lot no. 979028). Each blood sample was added directly to an assay tube containing 475 /d of phosphate-buffered saline (PBS) with I % egg albumin (Sigma Client. Co.) and assayed for LH by radioimmunoassay [N iswender cl al. 1968] as described previously [G ay et at., 1969]. Serum LH concentrations have been expressed in terms of B160, a partially purified rat anterior pituitary gland extract, 1 mg of which is equivalent to 0.17 mg of NIH-LH-Si or 5 mg of NIAMD-LH-RP-I, as determined by bioassay. A total of 42 proestrous rats (6/group) were injected via the carotid cannula with 100 //I of 0.5 n HCI containing varying concentrations of L-dopa or, in the case of the control group, with an equal volume of HCI alone. Doses of L-dopa were 2.7, 8, 25. 75 or Downloaded by: Kings's College London 22.214.171.124 - 10/25/2017 8:31:25 PM Materials and Methods L-Dopa and Pentobarbital on LH 101 225 mg/kg and were injected over a 2.5-min interval beginning at approximately I p.m. One group of proestrous rats was injected with pentobarbital (40 mg/kg, i.p.) 30 min prior to the injection of L-dopa (25 mg/kg). An additional 6 rats were cannulated on the morning of diestrous and injected with L-dopa (25 mg/kg) at I p.m. On the morning following the injection each rat was anesthetized with ether. The ovaries and oviducts were examined and, in those animals in which they were present, tubal ova were counted. Results No increase in serum LH concentration was observed during the 40-min interval following injection of acid into unanesthetized proestrous rats. However, L-dopa (25 mg/kg) caused a significant increase in circulating LH in such animals (table I), producing serum LH levels comparable to those measured in castrated and intact proestrous rats [G ay and M idgley, 1969; G ay et at., 1970]. The 8 and 75 mg/kg doses of L-dopa caused only a moderate increase in serum LH concentration, and the smallest (2.7 mg/kg ) and largest (225 mg/kg) doses of L-dopa were totally ineffective in inducing LH release. Large doses of L-dopa which failed to stimulate LH release did not prevent subsequent spontaneous LH release as indicated by the occurrence of ovulation in such rats. In proestrous rats pentobarbital anesthesia abolished the increase in serum LH following L-dopa (25 mg/kg) injections, abolished the spontaneous pre-ovulatory LH release, and prevented ovulation. Unanesthetized diestrous rats failed to respond to the dose of L-dopa (25 mg/kg) which was most effective in stimulating LH release in unanesthetized proestrous animals. In proestrous rats anesthetized with ether, 3rd ventricle injections of DA, L-dopa or epinephrine are effective stimuli for LH release or ovulation [S c h n e id e r and M c C a n n , 1970a; R a z ia n o et al., 1971], Systemic (i.p.) injections of L-dopa also produce ovulation in unanesthetized immature rats in which PMS-induced ovulation has been prevented by treatment with «-methyl-/?-tyrosine [K o r d o n and G l o w in s k i , 1969], Under these experimental conditions, 3rd ventricular injections of norepinephrine and serotonin, or systemic injections of a precursor of norepinephrine (DOPS) were ineffective Downloaded by: Kings's College London 126.96.36.199 - 10/25/2017 8:31:25 PM Discussion Table 1. Plasma LH concentrations and incidence of ovulation in anesthetized and untreated cyclic female rats following systemic injections of various doses o f L-dopa Type of rat L-dopa. mg/kg1 Plasma LH relative to time of day and time of injection, ng B160/mI 12.45 p.m. LOO p.m. 1.40 p.m. -15 min 0 min -f40 min Proestrous + HCI 0 ND1*3 ND ND 4.00 p.m. + 180 min Number Number Number Tubal ova, with with ovumean ± elevated elevated lating SE serum serum LH at LH at 1.40 p.m. 4.00 p.m. 72.6 ± 18.0 0/6 5/6 6 14.1 ±0.9 2.7 10.8 ± 1.1 11.0 ±1.6 13.4 ± 2.6 88.8 ±15.6 0/6 2/6 6 12.1 ±0.9 8 12.6 ± 1.5 ND 52.8 ±19.5 117.0 ±37.7 3/6 4/6 6 13.0 ± 1.9 Proestrous 25 I2.0± 1.7 11.0 ±2.1 158.6 ±65.1 265.2 ±70.5 6/6 6/6 6 14.1 ±1.5 Proestrous 75 14.4 ± 11.0 12.8 ±6.9 25.2 ± 8.7 89.0 ±20.8 1/6 3/6 6 13.1 ±1.7 Proestrous 225 10.5 ± 3.5 10.1 ±4.4 16.0 ± 6.4 100.0±44.1 0/6 2/6 6 13.0 ± 1.6 Proestrous 25 + pentobarbital-' ND ND ND 17.8 ± 7.2 0/6 0/6 0 0 Diestrous4 ND ND ND 0/6 0/6 0 0 25 ND Wedig/G ay Proestrous Proestrous Downloaded by: Kings's College London 188.8.131.52 - 10/25/2017 8:31:25 PM 1 L-dopa dissolved in 100 /d o f 0.5 n HCI injected at 1.00 p.m .: animals receiving no L-dopa were injected with 100 /d o f 0.5 n HCI. - ND = LH not detectable in quantity of blood (50 /d) used in each assay tube. In untreated proestrous rats, at times other than the critical period, serum LH normally ranges from 5 to 10 ng/ml. 3 Pentobarbital (40 mg/kg, i.p.) given at 12.30 p.m. 4 Diestrous = day before functional proestrous and pre-ovulatory LH release. 103 in stimulating LH release. By contrast, R u b in s t e in and S a w y e r  found epinephrine to be the most effective stimulus for ovulation in the pentobarbital-anesthetized proestrous rat. a condition which, in light of the results presented here, may be attributed to the inhibitory action of pentobarbital on the LH-reieasing action of DA. Although R a z ia n o et al.  also used pentobarbital-blocked proestrous rats, they administered DA 105 min prior to the anesthesia and the results presented here, as well as the data of S c h n e id e r and M c C a n n [1970b], suggest that DA-induced LH release would have occurred during the interval between injections. Attempts have been made to correlate dopaminergic neurons with the areas of the brain thought to control LH release and it is now clear that biogenic amines are not uniformly distributed in the brain but are segregated into distinct tracts and nuclei. The tuberoinfundibular tract which originates in the arcuate nucleus of the hypothalamus and terminates in the median eminence region just above the pituitary may be dopaminergic, as indicated by histochemical fluorescence studies of the cellular localization of catecholamines in these neurons [C a r l s so n et at., 1962], This tuberoinfundibular tract has been implicated as the final pathway in the release of LH [F u x e el al., 1969], However, the most direct evidence suggesting that DA can function as a neural trigger (transmitter) for the ovulatory mechanism is provided by injections of DA into the 3rd ventricle. In our experiments we chose to inject L-dopa systemically rather than to infuse DA into the 3rd ventricle. t.-dopa, which crosses the blood brain barrier, is the immediate precursor of DA. Unanesthe tized proestrous rats responded to the systemic injections of L-dopa with a rapid increase in serum LH concentrations. 25 mg/kg of L-dopa was found to be the optimal dose: it induced a prompt release of LH in 100% of the unanesthetized proestrous rats. Because L-dopa was capable of inducing LH release in our unanestheti zed proestrous rats we tested the ability of pentobarbital anesthesia to block this response and found that this anesthesia blocked the L-dopa-induced increase in serum LH. Although it is not clear why the larger doses of L-dopa failed to induce LH release in our rats, it is evident that L-dopa may be converted to norepinephrine within the animal and may act at various sites within the brain. K a m b er i et al.  have reported a similar diminished effect of higher doses of DA injected into the 3rd ventricle of male rats. Downloaded by: Kings's College London 184.108.40.206 - 10/25/2017 8:31:25 PM u-Dopa and Pentobarbital on LH I(M W edig/G ay In both PMS-treated immature and mature cyclic rats spontaneous pre-ovulatory LH release is prevented by systemic injections of an antiserum which neutralizes endogenous estrogens [Ferin et a!., 1969a, b]. This antiserum is also effective in preventing ovulation induced by injections of L-dopa into the 3rd ventricle of anesthetized proestrous rats [R aziano et al., 1971]. Schneider and McC ann [1970a] have also reported that the diestrous rat is relatively insensitive to injections of DA (4 /ig) into the 3rd ventricle. These data, combined with our observation that the unanesthetized diestrous rat failed to respond to the 25 mg/kg dose of i.-dopa, suggest that the mechanisms of L-dopa-induced LH release are similar to those which cause LH release in the proestrous rat. Spontaneous LH release is dependent upon the presence of serum estrogen and on the activities of the unanesthetized brain, and it is now clear that similar conditions are required if L-dopa injections arc to produce optimal release of LH. References Barraclough , C.A. and S awyer, C.H.: Blockade of the release of pituitary ovulating Downloaded by: Kings's College London 220.127.116.11 - 10/25/2017 8:31:25 PM hormone in the rat by chlorpromazinc and rescrpinc: possible mechanisms of action. Endocrinology 61: 341 351 (1957). C arlsson, A .: F alck , B., and H illarp , N.A.: Cellular localization of brain monoamines. Acta physiol, scand. 56, suppl. 196 (1962). 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