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

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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. a. Disappearance rate of plasma (Fig. lb). beta-MSH. The calculated half-life was 5.1 min. b. Concentration of beta-MSH in CSF Immunoreactive beta-MSH was after intravenous injection of beta-MSH or saline, c. CSF to plasma ratios of undetectable in all preinjection sam­ beta-MSH. Data expressed as in Fig. 1. ND = not detectable. ples of both plasma and CSF. At 2 min

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post-injection, the mean plasma con­ min after the injection of beta-LPH, The final question addressed by centration was 26±4nM. This is an the majority of the IR-LPH, -MSH, this study concerns the nature of the order of magnitude less than the 2 and -endorphin eluted with beta-LPH IR-LPH found in the CSF. In order to min concentrations of either beta- (Fig. 4 a, b, c), but there was also a determine if this was beta-LPH, endorphin or beta-LPH, which were clear peak of IR-endorphin eluting gamma-LPH or LPHI-M, we fraction­ injected at the same dose. Between 2 near standard beta-endorphin (Fig. ated the pooled 30 and 45 min CSF and 30 min beta-MSH disappeared 4c). The calculated "gamma-LPH" samples on Sephadex G-50 as with an apparent half-time of 5.1 min peak in tubes 16-18 (shaded area) is described for the plasma samples. (Fig. 2a). The maximum concentra­ approximately equal to the beta- IR-MSH elutes as two peaks in both tion of beta-MSH in CSF (0.72 ±0.35 endorphin peak (tubes 19-21, Fig. the 30 and 45 min CSF samples (Fig. nM) was observed at 2 min. The level 3c). At 15 min, the IR-N-fragment, 5a, c). The first peak coincides with had declined slightly at 15 and 30 min -MSH and -endorphin all appeared in beta-LPH and the second may rep­ and was only 0.10 ±0.03 nM at 45 min two peaks (Fig, 4d, e, f)- In each case resent gamma-LPH. IR-endorphin (Fig. 2b). Beta-MSH remained unde­ the first peak coincides with that of elutes principally as beta-LPH (Fig. tectable in saline injected rabbit CSF. standard beta-LPH and is taken to be 5b, d). The identity and significance The CSF to plasma ratios for beta- the intact molecule. In addition, the of the second peak of IR-endorphin MSH were variable and reached a second peak of IR-endorphin elutes seen at 30 min is unknown. No free maximum of 1.48+1.75 at 30 min with standard endorphin (Fig. 4f) beta-MSH or beta-endorphin were (Fig. 2c). This high value is largely while the second peak of both found. attributable to one animal in which a ratio of 3.48 was calculated. The preinjection concentration of plasma beta-LPH averaged 0.12±0.03 nM. Beta-LPH was unde­ tectable in two of four of the prein­ jection CSF samples and was only 0.08 and 0.14 nM in the other two samples. The apparent half-life of beta-LPH in the circulation was 13.7 min, considerably longer than that of beta-endorphin or beta-MSH (Fig. 3a). The concentration of beta-LPH in CSF as well as the CSF to plasma ratio, continued to increase for the duration of the experiments. The 0 (5 30 maximum concentration (5.5 + 0.7 TIME (min) nM) and the maximum ratio (0.08 0.03) were at 45 min (Fig. 3b, Figure 3—The fate of exogenous beta-LPH in vivo. a. Disappearance rate of plasma c). The controls for the beta- beta-LPH. The calculated half-life was 13.7 min. b. Concentration of beta-LPH in endorphin (Fig. lb) and beta-MSH CSF after intravenous injection of beta-LPH. c. CSF to plasma ratios of beta-LPH. (Fig. 2b) experiments also apply to Data expressed as in Fig. 1. beta-LPH. Any increase in the con­ trol levels of beta-LPH would have N-fragment and beta-MSH can be DISCUSSION been detected with either the ant- accounted for as "gamma-LPH", There is growing awareness that endorphin or anti-MSH serum. The calculated as above (Fig. 4 d, c). By peptides related to ACTH and beta- pooled plasma samples for each time 30 and 45 min, an increasing fraction LPH can have profound effects on brain period were fractionated on of the IR-N-fragment and -MSH can functions. The heptapeptide sequence Sephadex G-50 and each fraction was be accounted for as "gamma-LPH" that is common to ACTH, beta- assayed for immunoreactive (IR) rather than beta-LPH (Fig. 4 g-1). In and gamma-LPH and beta-MSH N-fragment, beta-MSH and beta- fact, at 45 min there is essentially no has been shown to influence extinc­ endorphin. We also calculated the remaining beta-LPH as is evidenced tion of conditioned active, condi­ approximate concentration of by the paucity of IR-endorphin in tioned passive and appetitive "gamma-LPH" in each fraction by fractions 14-16 (Fig. 4 1). responses (Kastin et al., 1973), re­ subtracting the concentration of IR- action time Kastin et al., 1973), endorphin from the concentration of Since no IR-MSH was detected at visual memory (Sandman et al., IR-N-fragment (i.e. if a tube contains the elution volume of beta-MSH, we 1972) and attention and stimulus a molar excess of N-fragment over conclude that beta-LPH was not processing (Sandman et al., 1977). beta-endorphin, we tentatively as­ transformed peripherally into beta- In addition, the endorphins sume that to represent gamme-LPH). MSH. This conclusion is supported and enkephalins which are believed The results are shown in Fig. 4. At 2 by our failure to find any IR-N frag­ ment the size of beta-LPHIOS. to derive from beta-LPH are capable

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of inducing analgesia, catatonia ment and reached the highest level of beta-LPH is cleaved to beta- (akinesia), sedation and excessive the three peptides tested. The endorphin in the circulation, we grooming (Belluzzi et al., 1976; physiological significance of this is failed to detect beta-endorphin in the Buscheretal., 1976; Graf etal., 1976; uncertain since intact beta-LPH is CSF of beta-LPH injected rabbits. Gispen et al., 1976; Motomatsu et al., not known to have central actions. A This is undoubtedly due to the sen­ 1976; Bloom et al., 1976; Jacquet and second peak of IR-MSH was also sitivity of the method. Since only a Marks, 1976; Tseng et al., 1977; found in the CSF of beta-LPH treated very small volume of CSF was avail­ Bradbury et al., 1977). Our results rabbits. We tentatively conclude that able for fractionation, we were un­ show that beta-endorphin, beta- this represents "gamma-LPH" that able to detect the anticipated trace MSH and beta-LPH can pass from has entered the CSF from the circula­ quantities of beta-endorphin. The the circulation to the CSF in the tion, but cannot exclude the possibil­ generation of beta-endorphin from rabbit. The concentration of beta- ity that it is a cleavage product gener­ circulating beta-LPH is of uncertain LPH in the CSF continued to in­ ated by the CSF or brain. Although significance. It does not seem likely crease for the duration of the experi­ we have shown that circulating that, in physiological situations, a concentration of beta-endorphin suf­ ficient to influence behavior would N-FRAGMENT 0-MSH Q-ENDORPHIN be attained. 15 r 2min Beta-MSH levels in the CSF were highest at 2 min post-injection. The 12 - i 0 • E decline that followed is likely a con­ a sequence of degradation or uptake by 9 - the brain rather than return to the circulation, since the blood levels 6 " , remained higher (except in one sam­ ple) than CSF levels. Brain tissue is 3 known to inactivate beta-MSH en- • i± i_ zymatically (Long et al., 1961). Since beta-MSH can influence be­ 15 -15 min CjB r~i 0 2 • • havior (Ferrari et al., 1963) and 12 d neuronal activity (Krivoy et al., 1 e 1977), our finding that it does p_ass O 9 m r from the circulation to the CSF may be of physiological importance. 6 Smith and Shuster (1976) found high Ir 1 levels of immunoreactive beta-MSH UJ 3 in human CSF, a finding that further o J liy-: i J i.. supports the possibility that this pep­ I tide may be involved in the regulation O 9 -30min 1 Q • D 0 of central nervous system activity. o 6 1 Physiological studies have yielded V 9 conflicting results concerning the 3 transfer of endorphins and enkepha­

i / / . .l\^ i lins from the circulation to the CSF (Pert et al., 1976; Plotnikoff et al., 6 r45min • ra 1976; Tseng et al., 1976). Our de­ • @ ; monstration that in certain species circulating beta-endorphin has ac­ J^l A^TV. cess to the CSF supports the possi­ 0 10 20 30 0 10 20 30 0 10 20 30 bility that pituitary beta-endorphin is FRACTION NUMBER (Iml) a functioning ligand for brain opiate receptors. Previously, Kastin et al. CZI O-LPH ZZ2 0-ENDORPHIN WM 0-MSH (1976b) showed that enkephalin cros­ sed the blood-brain barrier of rats, a finding supported by our results with endorphin and the work of Plotnikoff Figure 4—Immunoreactive N-fragment (a, d, g, j), beta-MSH (b, e, h, k) and beta-endorphin (c, f, i, I) in plasma fractionated on Sephadex G-50. Plasma was et al. (1976). collected 2, 15, 30 and 45 min after intravenous injection of 20 nmoles of beta-LPH. Our results show that the three The shaded area indicates the calculated concentration of gamma-LPH (see text). peptides tested can pass from the The blocks indicate the elution positions of 125I-labelled beta-LPH, beta-endorphin, circulation to the CSF. Further and beta-MSH. a, b, c, 2 min; d, e, f, 15 min; g, h, i, 30 min; j, k, 1, 45 min. studies are needed to determine the

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point or points of entry into the CSF. likely that the active N-terminus of physiological actions on the central Oliver et al. (1977) have provided beta-endorphin is removed. We nervous system. evidence that pituitary hormones are found no evidence for the in vivo transported by retrograde blood flow cleavage of beta- or gamma-LPH to from the pituitary toward the beta-MSH. It appears the circulating ACKNOWLEDGMENTS hypothalamus. It is possible that this beta-LPH may serve as a source of This work was supported by a Medical vascular system delivers high con­ beta-endorphin but not beta-MSH. Research Council of Canada Program Grant centrations of pituitary hormones to This report provides the first direct and the Edwards Foundation. We thank Mrs. the CSF. Marie-Claude Guerinot for the cheerful and evidence that beta-LPH, beta-MSH skilled technical assistance and Mrs. D. Marcil Accurate determinations of disap­ and beta-endorphin can pass from the for typing the manuscript. pearance rates require more rapid circulation to the CSF and that cir­ and frequent sampling than was used culating beta-LPH can be cleaved to in this study. Because our first sam­ beta-endorphin. Our findings provide REFERENCES ples were not taken until 2 min after additional support for the notion that AUSTEN, B. M. and SMYTH, D. G. (1977), the injection, we undoubtedly missed these pituitary peptides may have The NHMerminus of C-fragment is resis­ the early, rapid phase of the disap­ pearance curves. This is especially apparent in the case of beta-MSH. ts O-LPH Nonetheless, our results do show B3 0-ENDORPHIN that beta-MSH and beta-endorphin are rapidly cleared from the circula­ • 0-MSH tion. Kastin et al. (1976a, b) found the half-time disappearance of alpha- 0-MSH 0-ENDORPHIN MSH and enkephalin to be 1.5 minor 0.6 r i i vm less for the early component of the 30mln disappearance curve. In addition, for enkephalin the second component of the curve gave a half-time of 4.8 min, a value quite close to those for 0.4 beta-MSH and beta-endorphin. The long apparent half-time for the disappearance of beta-LPH is proba­ -. 0.2 h bly related to the specificity of the E antiserum used. The antiserum rec­ c ognizes the N-terminal portion of beta-LPH and gamma-LPH. By gel filtration and RIA we have shown that the catabolism of beta-LPH be­ gins at the C-terminus (Fig. 4). 2 Within 2 min after administration, beta-LPH is partially cleaved to pep­ u.b 45 min c tides tentatively identified as beta- o endorphin and gamma-LPH by o molecular weight and immunological tzn w& W7A characterization. These techniques 0.4 - are obviously not adequate to deter­ mine the exact position of the cleav­ age. However, in view of the known lability of the peptide bond between 0.2 arginine (position 60) and tyrosine (position 61) of beta-LPH to trypsin and trypsin-like enzymes (Bradbury et al., 1976; Seidah et al., 1977) it i k i ±J- _L seems likely that cleavage occurs at 10 20 30 10 20 30 this point. Furthermore, the FRACTION NUMBER (Iml) N-terminal tyrosine of beta- endorphin is quite resistant to aminopeptidases and to brain en­ Figure 5—Immunoreactive beta-MSH (a, c) and beta-endorphin (b, d) in CSF fraction­ ated on Sephadex G-50. CSF was collected 30 and 45 min after intravenous injection zymes (Austen and Smyth, 1977; 125 Marks et al., 1977). Thus, it is un­ of beta-LPH. The elution positions of l-labelled beta-LPH, beta-endorphin, and beta-MSH are indicated by the blocks, a, b 30 min; c, d. 45 min.

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