MSH(4–10) peptide increases postnatal metabolic activity of EDL muscle following early prenatal administration

Pepndes, Vol 11, pp 177-179 ©Pergamon Press plc, 1990 Pnnted m the U S A

0196-9781/90 $3 00 + 00

BRIEF COMMUNICATION

ACTH/MSH(4-10) Peptide Increases Postnatal Metabolic Activity of EDL Muscle Following Early Prenatal Administration K E N N E T H J. R O S E A N D F L E U R L. S T R A N D 1

Department of Biology, Washington Square, New York University, New York, N Y 10003 R e c e i v e d 21 April 1989

ROSE, K J. AND F L STRAND. ACTH/MSH(4-10)peptlde increasespostnatal metabohc acttwty of EDL musclefollowmg early prenatal admmistratton PEPTIDES 11(1) 177-179, 1990 --ACTH/MSH(4-10) (10 ~g/kg/b 1 d, IP), admmtstered to pregnant Sprague-Dawley rats dunng gestatlonal days (GD) 3 to 12, s~gmficantly increased the metabohc actlwty of extensor dtgttorum longus (EDL) muscle at postnatal day 14 ACTH/MSH peptlde, administered from day of birth to postnatal day 13, had no effect on EDL muscle metabohc actlwty using the 2,3,5-tnphenyltetrazohum chloride indicator. By postnatal day 30, no differences were seen between the early prenatally treated group and sahne controls These results confLrm our previous electrophyslologlcal studtes that showed that early prenatal ACTH/MSH(4-10) administration accelerates EDL muscle maturation Muscle

Metabohsm

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dunng postnatal days (DPN) 1 (day of birth) to 13; 2) those that received ACTH/MSH(4-10) (10 ixg/kg/2 × day, IP) during G D 3 GD12 followed by ACTH/MSH(4-10) (10 ixg/kg/day, SC) dunng DPN 1 to 13; 3) those that received the saline vehicle dunng GD3-GD12; pups received ACTH/MSH(4-10) (10 p.g/kg/day, SC) dunng DPN 1 to 13; and 4) those that received the sahne vehicle during GD3 to GD12; pups received the saline vehicle during DPN 1 to 13. Testing occurred on DPN 14 and DPN 30. The dose of peptlde used (10 Ixg/kg) had been found to be most effective in elicitlng posmve postnatal motor responses (1, 19, 20) which is in agreement with the work on regeneration from our and other laboratories (4, 11, 19, 20). At the day of experimentation, pups were anaesthetized wlth 40 mg/kg sodmm pentobarbital (IP; Butler Co.) before surgery. Whole EDL muscles were qmckly removed from the anaesthetized rats and immediately placed m beakers (10 ml) containing 95% ethyl alcohol precooled with dry ice, and left m this frozen state for no more than 20 minutes. The muscles were then incubated m a fresh solution of 1% 2,3,5-tnphenyltetrazolium chloride (TC1) m 0.05 M phosphate buffer (pH 7 3) at 36°C (water bath) for one hour. TC1 was used as an indicator to measure the extent and rate of

PREVIOUS electrophysxologlcal results in our laboratory have indicated that ACTH/MSH(4-10) peptlde, the noncortlcotropic fragment of adrenocorticotropic hormone (12), admimstered early m gestation, accelerates the maturat~onal rate of developing extensor dzgztorum longus (EDL) muscle (18). In this communication, we report on the effects this peptlde fragment of ACTH/ MSH has on the metabolic activity of developmg EDL muscle METHOD Pregnant Sprague-Dawley rats [ordered 2 days pregnant on dehvery (Taconic Farms)], housed separately and maintained on a 12-hr hght/12-hr dark cycle at a room temperature of 22°C, were supphed with rat chow and water ad lib dunng the period of peptide admimstratlon. On the day of birth (day 1) each htter was culled to 8 with approximately equal numbers of males and females. The pups were assigned randomly to htters within treatment groups for experimentation. Dams (N = 4 for each group) were divided into four groups' 1) those that received ACTH/MSH(4-10) (Org 0163; Organon Pharmaceutical Co.) [10 t~g/kg/2xday (1000 hr and 1600 hr), IP] during gestational days GD3-GD12; pups recexved saline vehicle

1Requests for repnnts should be addressed to Dr Fleur L Strand, 1009 Main Budding, Washington Square, New York Umversxty, New York, NY 10003

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FIG 1 EDL metabohc acUvlty with ACTH/MSH(4-10) Absorbance/ EDL dry weight values of EDL muscles from 14-day-old rat pups treated with ACTH/MSH(4-10) (10 p~g/kg) dunng an early prenatal period (GD3-GD12) (pre-ACTH) or dunng postnatal days 1-13 (post-ACTH) Peptlde adrmmstratxon either dunng GD3-GD12 (*p<0.05) or dunng GD3~3D12 and DPN 1-13 (pre-post-ACTH) (**p<0 001) showed slgmflcantly greater values vs. sahne controls or vs pups treated with peptxde dunng DPN 1-13 (post-ACTH) (N= 10 for each group ) (ANOVA, ad hoc Student's t-test ) cellular (mitochondnal) oxidative metabohsm in muscle. Muscle glycolytic and oxidative enzyme activities change dunng early muscle development (2, 3, 13, 17, 24). The greater the extent of muscle maturation, the higher the oxidative metabolism in muscle (2, 23, 24). Thus, the more mature the muscle the greater will be the conversion of TC1 to its reduced product, formazan (22). Dunng the postlncubation period, the solutions were decanted, leaving only the EDL muscles in the test tubes. The formazan red dye was extracted with the addition of 2 ml of acetone for 10 min at room temperature. The muscles were removed and dried on filter paper (Whatman L t d ; 1 qualitative, 15.0 cm) at room temperature (10 min) and weighed. The acetone-formazan dye solution was analyzed m a Spectrotonic 20 spectrophotometer (435 nm setting), and the absorbance/EDL dry weight values were recorded. Control muscles were incubated in solutions without TCl or with 1% NaCN. RESULTS

Absorbance/EDL dry weight values of experimental groups treated dunng GD3-GD12, or during both GD3--GD12 and DPN 1-13 were significantly higher than either saline controls or the group treated with peptide fragment during DPN 1-13 when viewed two weeks postnatal. Those animals that were treated with ACTH/MSH(4-10) throughout gestation and also postnatally did not differ significantly from the group that was exposed to the peptide only during gestation. Nor did the values from the postnatally peptide-treated group differ significantly from control (Fig. 1). By 30 DPN, absorbance/EDL dry weight values of the group treated with ACTH/MSH(4-10) during GD3-GD12 did not differ from control values (Fig. 2). DISCUSSION

The present study demonstrates that early gestatlonal administration of ACTH/MSH(4-10) to pregnant dams increases the metabolic activity of developing EDL of the resultant pups at 14 days postnatal. The fact that this increased metabolic activity also occurs when the ACTH peptide fragment is administered during early gestation as well as during DPN 1-13, yet does n o t occur when ACTH/MSH(4-10) is administered exclusively during DPN 1-13, indicates that this increased metabolic activity is triggered by the peptide only during early prenatal life (GD3-GD 12). While

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FIG. 2. EDL metabohc activity 30 DPN Absorbance/EDL dry weight values of EDL muscles from 30-day-old (DPN) rat offspnng treated with ACTH/MSH(4-10) (10 p,g/kg) or sahne vehicle dunng GD3-GD12 There were no significant differences (Student's t-test) m the two groups at this age (N = 6 for each group )

this effect is clearly seen at two weeks of age, it is no longer evident when the animals reach one month of age. It has been well established that during early development, fast-twitch muscles, like the EDL, initially show a slow-twitch phenotype (9). This slow-twitch phenotype reflects the immaturity of the EDL muscle's sarcoplasmic retlculum (24) and the composition of the different regulatory proteins involved in its contraction (13, 16, 23), as well as the state of its glycolytic, and oxidative and citric acid enzyme activities (2, 3, 13, 17, 24). That EDL muscle of early prenatally ACTH/MSH(4-10)-treated rats (GD3-GD12) has been shown to present a faster isometric twitch at two weeks postnatal vs. controls is an indication of accelerated muscle maturation (9,18). The higher metabohc activity (Fig. 1), coupled with the faster isometric twitch durations seen previously with day 14 ACTH/ MSH-treated animals (18), leads us to believe that the greater metabolic activity IS indicative of advanced EDL muscle maturation. This higher activity may signify accelerated oxidative enzyme changes required to reach the enzymatic situation in adult muscle. Moreover, the fact that the differential increase in metabolic rate seen in peptide-treated pups at two weeks of age d i s a p p e a r s by one month of age (Fig. 2) may signify that the EDL muscle has already reached its fast-twitch, low metabolic steadystate activity indicative of a fully developed and mature muscle (9, 13, 23, 24) How this peptide fragment of ACTH/MSH works in this regard is not known. In this experiment, the peptide fragment was administered before ACTH, ot-MSH, or vasopressin are detected in the fetal pituitary gland (5, 8, 21) and before CRF (6, 7, 10) or the CRF receptor (14) is detetcted in the fetal hypothalamus Further complicating the situation is the evidence of ACTH(139)'s difficulty in penetrating the placental barrier (15). If the 4-10 fragment of ACTH/MSH is unable to pass through the placental barrier--and this has yet to be convincingly demonstrated--it is possible that the 4-10 ACTH/MSH molecule may act as a trophic agent on placental or maternal systems unknown to us at this time. Still, it is clear that these results reinforce our electrophysiological data indicating that the early prenatal administration of this noncortlcotropic fragment of ACTH/MSH accelerates muscle metabolic activity and possibly accelerates muscle maturation as well ACKNOWLEDGEMENTS This research was funded by The Council for Tobacco Research We thank Organon for its generous glft of Org 0163

ACTH/MSH(4-10) AFFECTS EDL MUSCLE

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REFERENCES 1 Acker, G R , Berran, J., Strand, F L. ACTH neuromodulaUon of the developing motor system and neonatal learning m the rat Peptldes 6(Suppl. 2)41-49, 1985 2 Bass, A., Lusch, G , Pette, D Postnatal dffferentlataon of the enzyme actwlty pattern of energy supply metabohsm m slow (red) and fast (white) muscles of chicken Eur J Blochem. 13:288-292, 1970. 3 Beatty, C. H.; Basmger, G M , Bocek, R M Dffferentmtlon of red and white fibers m muscle from fetal, neonatal, and infant rhesus monkeys. J. Hlstochem Cytochem 15"93-103, 1967. 4 Bljlsma, W. A.; Jennekms, F G. I , Schotman, P , Glspen, W. H. Effects of cortlcotropln (ACTH) on recovery of sensonmotor funcUon m the rat: structure-acUvlty study Eur J Pharmacol. 76'73-79, 1981. 5 Brayon, A , Shoemster, W J , Bloom, F F , Manns, A., Gudlemln, R. Pennatal development of the endorphm- and enkephahn-containlng systems m the rat brain Brain Res 179'93-101; 1979 6 Bugnon, C ; Fellmann, D , Gouget, A., Cardot, J. Ontogeny of the cort~cohbenn neuroglandular system m rat brain. Nature 298 159161, 1982 7. Bugnon, C., Fellmann, D , Gouget, A , Bresson, J L , Clavequln, M. C., Hadjlyassemls, M ; Cardot, J Cortacohbenn neurons cytophysmlogy, phylogeny and ontogeny J Steroid B~ochem 20:183195, 1984 8. Chatelam, A , Dupouy, J P , Dubots, M P Ontogenesls of ceils producing polypeptlde hormones (ACTH, MSH, LPH, GH, prolactln) m the fetal hypophysls of the rat mfluence of the hypothalamus Cell Tissue Res 196'409-427, 1979 9. Close, R Dynamic properties of fast and slow skeletal muscles of the rat dunng development J Physiol. 173 74-95; 1964 10 Dalkoku, S. Development of the hypothalamlc-hypophyseal axis m rats. In. Yoshlmura, F , Gorbman, A., eds. Pars dlstahs of the pituitary gland--structure, function and regulatmn. Amsterdam: Elsevier, 1986 21-26 11 de Konmg, P ; Brakee, J H , Glspen, W. H Methods for producing a reproducible crush m the sclattc nerve of the rat and the rapid and precise testing of return of sensory function: beneficial effects of melanocortms J Neurol Scl 74 237-246, 1986. 12. de Wled, D Effects of pepttde hormones on behawor. In: Ganong, W. F., Martin, L , eds Frontiers in neuroendocnnology. New York

Oxford Umv Press, 1969:97-140. 13 Gutmann, E , Mehchna, J., Syrovy, I Developmental changes in contractaon tame, myosin properties and fiber pattern of fast and slow skeletal muscles Physiol. Bohemoskov 23 19-27, 1974 14. Insel, T R , Battaglia, G., Falrbanks, D. W., de Souza, E B. The ontogeny of brain receptors for cortlcotropm-releasmg factor and the development of their functional assooatlon with adenylate cyclase J Neuroscl. 8 4151--4158, 1988 15 Mdkovlc, K , Mllkovlc, S. Reactiveness of fetal pltuaary to stressful sUmuh Does maternal ACTH cross the placenta9 Proc. Soc. Exp Biol. Med. 107:47-49, 1961 16 Montarras, D , Flszman, M. Y , Gros, F Changes m tropomyosm dunng development of chick embryomc skeletal muscles m v z v o and dunng differentiation of chick muscle cells m v t t r o J Biol. Chem 257 545-548; 1982 17 Nystrom, B. Succlnate dehydrogenase in developing cat leg muscles Nature 212 954-955; 1966 18 Rose, K. J , Strand, F. L Mammahan neuromuscular development accelerated w~th early but slowed w~th late gestatlonal administration of ACTH peptlde Synapse 2 2013-204, 1988 19 Saint-Come, C., Acker, G R , Strand, F. L. Peptlde influences on the development and regeneraUon of motor performance PepUdes 3 439--449, 1982 20 Saint-Come, C., Acker, G R , Strand, F. L Development and regeneration of motor systems under the influence of ACTH peptldes. Psychoneuroendocnnology 10 445-459, 1985. 21. Smdmg, C , Robinson, A. G., Self, S M Neurohypophyseal peptldes in the developing rat fetus. Brain Res 195 177-186; 1980 22. Weiss, L , Greep, R. O. Histology New York' McGraw-Hill Book Co, 1977 105-106 23. Whalen, R. G ; Sell, S M , Butler-Browne, G S., Schwartz, K., Bouveret, P , Plnset-Harstrom, I. Three myosin heavy chain ~sozymes appear sequentmlly in rat muscle development Nature 292 805-809, 1981 24. Zubrzycka-Gaarn, E , Sarzala, M G Sarcoplasmlc retlculum and sarcolemma dunng development. In: Pette, D , ed. Plasticity of muscle Berhn de Gruyter, 1980 35-52