LE JOURNAL CANAD1EN DES SCIENCES NEUROLOGIQUES Lipotropin, Melanotropin and Endorphin: In Vivo Catabolism and Entry into Cerebrospinal Fluid P. D. PEZALLA, M. LIS, N. G. SEIDAH AND M. CHRETIEN SUMMARY: Anesthetized rabbits were INTRODUCTION (Rudman et al., 1974). These findings given intravenous injections of either Beta-lipotropin (beta-LPH) is a suggest, albeit weakly, that the pep­ beta-lipotropin (beta-LPH), beta- peptide of 91 amino acids that was tide might cross the blood-brain bar­ melanotropin (beta-MSH) or beta- first isolated from ovine pituitary rier. In the case of beta-endorphin, endorphin. The postinjection concentra­ glands (Li et al., 1965). Although there are physiological studies both tions of these peptides in plasma and cerebrospinal fluid (CSF) were measured beta-LPH has a number of physiologi­ supporting and negating the possibil­ by radioimmunoassay (RIA). The plasma cal actions including the stimulation ity that beta-endorphin crosses the disappearance half-times were 13.7 min of lipolysis and melanophore disper­ blood-brain barrier. The study of for beta-LPH, 5.1 min for beta-MSH, and sion, it is believed to function princi­ Tseng et al. (1976) supports this pos­ 4.8 min for beta-endorphin. Circulating pally as a prohormone for beta- sibility since they observed analgesia beta-LPH is cleaved to peptides tenta­ melanotropin (beta-MSH) and beta- in mice following intravenous injec­ tively identified as gamma-LPH and endorphin. Beta-MSH, which com­ tion of beta-endorphin. However, beta-endorphin. Each of these peptides prises the sequence 41-58 of beta- Pert et al. (1976) were unable to elicit appeared in the CSF within 2 min postin­ LPH, is considerably more potent central effects in rats by intravenous jection. The maximum CSF to plasma than beta-LPH in either lipolytic or injection of the smaller, proteolytic ratios were 0.08 for beta-LPH, 1.48 for enzyme resistant analogue beta-MSH, and 0.23 for beta-endorphin. melanophore stimulating assays (Chretien, 1973). In addition, the ac­ (D-Ala2)-Met-enkepa!inamide and RESUME: Les lupins anesthesies tive heptapeptide core of beta-MSH surmised that it did not cross the recurent des injections intraveineuses, exhibits both behavioral and elec- blood-brain barrier. soil de beta-lipotropine (beta-LPH), de troencephalographic actions in the The goals of this study were (a) to beta-melanotropine (beta-MSH) ou de rat and man (Kastin et al., 1976c). determine if beta-LPH, beta-MSH beta-endorphine. Les concentrations de The second peptide for which beta- and beta-endorphin could cross the ces peptides dans le plasma et le liquide LPH is believed to be the prohor­ blood-brain barrier of the rabbit, (b) cephalorachidien (LCR) a la suite de ces mone is beta-endorphin. This injections furent mesurees par des essais to estimate their half-lives in the radioimmunologiques (RIA). Les demi- peptide corresponds to the sequence circulation and (c) to determine if the temps plasmatiques de disparition furent 61-91 of beta-LPH and, like beta- prohormone, beta-LPH, is cleaved in de 13.7 min pour la beta-LPH, 5.1 min MSH, has both central and vivo to one or both of its constituent pour la beta-MSH, et 4.8 min pour la peripheral actions. Beta-endorphin is hormones. beta-endorphine. La beta-LPH cir- produced in the pars intermedia culante est degrade e et les produite de (LaBella et al., 1976; Queen et al, MATERIALS AND METHODS degradation initials sont tentativement 1976; LaBella et al., 1977; Crine et Animals: Male New Zealand white identifies comme etant le al., 1977) and is well known for rabbits (2-3 kg) were anesthetized by beta-endorphine et la gamma-LPH. Chacun de ces peptides apparait dans le its potent opiate-like actions on the intraperitoneal injection of 5-7 g of LCR dans les 2 minutes qui suivent central nervous system and on peri­ urethane in 10-14 ml of 0.9% NaCI. I'injection. Le rapport maximum de con­ pheral neuromuscular transmission After anesthesia had been induced, centration dans le LCR par rapport aux (reviewed by Goldstein, 1976; Gold­ blood and CSF samples were taken les taux plasmatiques etait de 0.08 pour la stein and Cox, 1977; Scherrer et al., simultaneously. Blood (0.5-1 ml) was beta-LPH, 1.48 pour la beta-MSH et 0.23 1977). allowed to drop from a small cut in a pour la beta-endorphine. An important question that has not marginal ear vein into a tube contain­ been directly addressed is whether ing EDTA. The blood was im­ beta-MSH and beta-endorphin can mediately cooled on ice, centrifuged cross the blood-brain barrier to reach and the plasma stored at —20°C. CSF their putative site of action, the cen­ (0.1-0.2 ml) was taken from the cis- From the Clinical Research Institute of Montreal, tral nervous system. Beta-MSH has terna magna with a 23-gauge 3*/2 inch Affiliated to the Universite de Montreal and the Hotel-Dieu de Montreal. been found in CSF (Smith and Shus- spinal needle inserted percutane- ter, 1976) and peripherally adminis­ ously. The needle was equipped with Reprint requests to: Dr. P. D. Pezalla, Clinical Research Institute of Montreal, 110 avenue des Pins tered beta-MSH can increase pro­ an occluding stylet that was kept in ouest, Montreal, Quebec H2W IR7, Canada. tein synthesis in certain brain regions place between samplings. The CSF Vol. 5, No. 2 MAY 1978 - 183 Downloaded from https://www.cambridge.org/core. IP address: 170.106.33.22, on 29 Sep 2021 at 21:36:32, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0317167100024537 THE CANADIAN JOURNAL OF NEUROLOGICAL SCIENCES was frozen in a dry ice-ethanol bath previously described (Chretien et al., as tracer and standard. Under these immediately after withdrawal. CSF 1976; Li et al., 1965; Pezaila et al., in conditions, beta-LPH and gamma- samples were judged to be free of press). The peptides for injection as LPH cross react 46% and 79% while contamination by blood if they were well as for RIA iodination and stan­ beta-MSH does not cross react. The clear and colorless. Injections of the dardization were purified to anti-beta-endorphin serum cross test substances were made 15 min homogeneity as determined by elec­ reacts 58% with beat-LPH and not after the first sample was taken. trophoresis and amino acid analysis. at all with gamma-LPH or beta- Twenty nmoles of either beta-LPH, RIA: The details of our procedure MSH. More complete characteriza­ beta-MSH or beta-endorphin in 0.5 for RIA have been described (Pezaila tions of the antisera can be found ml of 0.9% NaCl were injected into et al., In Press). Antisera against in Guillemin et al (1977) and the marginal vein of the ear not used beta-MSH and beta-LPH were raised Pezaila et al (in press). for blood sampling. The injections in this laboratory. Antiserum against Chromatography: Gel filtration took about 1 min. Four rabbits re­ beta-endorphin was generously sup­ chromatography was done at 4°C on 1 ceived each peptide and an additional plied by Dr. Roger Guillemin. The x 42 cm columns of Sephadex G-50 four received saline alone. Each rab­ anti-beta-MSH serum cross reacts superfine (Pharmacia). The columns bit was used only once. with both beta- and gamma-LPH were equilibrated and eluted with pH Peptides: Ovine beta-LPH, (47% and 31% respectively on a 7.6 phosphate buffered saline con­ beta-LPH 1-47 and beta-endorphin and molar basis). The anti-beta-LPH taining 25 mM EDTA and 1% (w/v) porcine beta-MSH were purified as serum was used with beta-LPH 1-47 bovine serum albumin (Pezaila et al., in press). One ml fractions were col­ lected and stored frozen. The col­ umns were calibrated with 125I-labelled beta-LPH, beta-MSH and beta-endorphin. RESULTS Concentrations of immunoreactive beta-endorphin in the preinjection samples of plasma and CSF were near or below the limits of detectabil- ity. For those animals which had measurable beta-endorphin in both CSF and plasma, the concentration ratios were between 0.43 and 2.70. No 0 15 30 TIME (mfti) apparent correlation between the levels in the two fluids was seen, Figure I—The fate of exogenous beta-endorphin in vivo. a. Disappearance rate of possibly because of the error in plasma beta-endorphin. The dashed line is the least squares regression from which a measuring such low concentrations. half-life of 4.8 min was calculated, b. Concentration of beta-endorphin in CSF after intravenous injection of beta-endorphin or beta-MSH. c. CSF to plasma ratios of The mean half-time of disappearance beta-endorphin. Data expresses as mean ± standard error of the mean. from the plasma was 4.8 min (Fig. la). t> Beta-endorphin appears in the 100 10 - CSF within 2 min after injection. The 50 5 . concentration (0.5 nM) is nearly ten 0-MSH wjEcrto times the preinjection level. The z 2 c 1 concentration is maximal and does I / L_ J not change significantly between 15 ^ T T 05 - and 45 min (Fig. lb). The stability of beta-endorphin in CSF as opposed to plasma is responsible for the con­ 0.1 \[ tinued rise in the CSF to plasma 0.5 0.05 SdllNE concentration ratio. The maximum INJECTED ratio (0.23 ± 0.06) was seen at 45 min NO NO (Fig. lc). Beta-endorphin in the CSF ' 1 0 15 30 30 45 of control (beta-MSH injected) rab­ TIME (min) bits remained low or undetectable Figure 2—The fate of exogenous beta-MSH in vivo.