Divergence of growth hormone actions on hepatic drug metabolism in the presence and absence of the pituitary gland

Pergamoa Press

Life Sciences, Vol. 17, pp .779-786 Priatad in the II .S .A .

DIVEBGEIiCE OF GBO'iTrH HOBl~äB ACTIONS ON ~ATIC DBDG ~TAHOLISë IIi THL PBESEBCE A1® 118SEBCE OF THB PITUITABT CLADm Bobart E. xrasrr, John W. Greinet sad Hward D. Colby Daparts~sat of Physiology aad Biop~ysics üest Virginia üaiveraity ësdical Ceatar ëorgaatavs, üsat Virgiaia 26506 (ASOaiwd is Baal loan August 4, 1975) $ussaary Ssparisrnts were carried oat to cas~pare the effects of groreh horsoae oa hepatic drug oxidation in aorul and hypophywctosdsed rats . Adsiaistratioa of grarth horsnaa to nosaal sale rats located hepatic sicrosasal cytochrase P-iS0 content sad dacrwed the rata of athyLorphiae a-dsathyLtioa and aailias hydroxylation . Those effects care folly sanifutad in orchiactasisad or adreaalactcaisad ss11u , szcludin~ a dapsa deaca upon endopaoas steroids . Grovth horsoae ~ withoat affect oa hepatic drag sataboliss or cytochra~e P-4S0 contest is norsal fsula rate . Ia coatrat to its action is aa1uL with intact pituitary glands, adainistration of grarth horaona to hypophysactoaiisad rats of aithsr su iacrwad the rata of ethylaorphias rtabolisa. Phrthersore, in both oleo and fasalas, aniliaa hydraagLtion sad adcrosaeial cytoehrasr P-~50 contest ware uaaffacted by growth horawna is the absence of the pituitary gland. ProLctia adsinistration did not affect hepatic ozidative utaboliss or cytochrau P-450 levels is hypophysectosdxad or is norul rata of either saz. The taints ladi eata that the nature of growth horsone actions on hepatic drag oxidation is pituitary-dependent and probably iatartvined with the effects of other horsonea . Furtharswre, the direct physiological affects of growth horsaae on hepatic si:ad function ozidasa susl~to depend upon the substrate asployad . äaay hor~oaas have baea shove to iaflussca the activity of hepatic drnS-aetaboli :ia4 ensyaes (1,2) . The role of Soaadal horaoaas, particularly tutostarons, in the regulation of hepatic sired-fsmction oaidasas is rats hu base rather ezteasivaly iavutigatad (3-S) . Various substrata, including hazobarbital, athyLorphine sad testosterone, are setaboliaed far scars rapidly by sale rate thaw fouler . In addition, hepatic oxidation of such caspounda decrwes attar orchiectosy and iacrauu after testosterone adstiaiatratioa to leula or csatratad sale (1,3,6,9-12) . Observations such u thue have lead sort invutigators to aa~sa that taatostarone aorta a direct affect oa thn liver to iacrusa sized fimctioa ozidase activity . Havwar, ve have recently noted that the action of tutostarone to iacrwe hepatic athy]aorphiae setaboliss is not dasoastrabla is hypophy779

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aactastiised rats of either saz (13), sugguting radiation by (a) pituitary factor(s) . The nature of androgen dependence on the pituitary gland as wll as the identity of pituitary factors contributing to the effects of testosterone on hepatic drug rstabolisr are presently unknorn. Among the pituitary non-tropic hormones whose secretion is inflaencad by testosterone and which affects drug rstabolisr (14-19) is growth hormone. Ths present studies wre carried out to further asasins the role of the pituitary gland is the regulation of hepatic drug mstabolisn and to consider if grarth horrona sight contribute to the affects of testosterone oa hepatic rstabolisr . The results indicate that the effects of grarth hormone on microsoral oziiativa rtabolism, like those of testosterone, era pituitary-dependent and vary with the specific substrata saploysd . Msthoda !lain and fsrale Sprague-Darley rats, 60-75 days old, (obtained fran 2lvio-äillar Laboratories, Pittsburgh, PA) rare raintaiasd under standardised conditions of light (0600-1800) and tasparaturs (22.0 + 1.0 C) on a diet of Purina i.aboratory Chav and wtsr ad lib. Orchiectoâi.as, adrsaslectcsiea or parapharyngsal hypophyssctas~ias, as indicated, were psrfoasd on rats approsisstsly 60 days of age and horsonal treatment initiated 24-72 hours later . Hypophysactosiu wre psrforasd by the breeder. Cosrpletsnass of hypophysacto~r ws datirsiasd by vLual inspection at autopsy a~ by a significant decline is adrenal Wight. Growth bosons (äIH-GS-B17) sad prolactia (1fI8-P-B3) ware adrinistarad subeutaasoasly is 0 .9x salsas at a dose of 250 vg/100 g body Wight trice daily. All anisals wre sacrificed by decapitation bntwm 9:fl0 and 10 :00 a.r . seven days after beg ~r{rte horsonal trsatssnt. Livers wre gaickly meowed and hasagsaiaad is 1 .1SZ potusius chloride . Hosogeaates (0 .5 g/al) wrs centrifuged at 9000 g for 20 sdantss is a Sorvall refrigerated centrifuge . Aliquots of ehs sapernataat wre rwo~sd for sas7ss assays or centrifuged at 103,000 g for 60 riautss in a àsai~an preparative nltracsatrifuge . äicrosasal. pellets wre rasaspeadad is 1 .15x potassi~ss chloride containing 0.05 ë trig-HC1 buffer (p$ 7 .4) at a concenentratioa of 3-4 erg protein per rl . All stapa is the preparation of aicrososisa wrs perfosasd rith the tissera kept at 0-4° . Ethylaorphina derathylation and aniline hydrozylation rers assayed a. the asotimt of forraldshyda (20) or pua-asiaophaaol (7) found reapecCivaly by O .S rl liver 9000 g supsrnataat (200 rg/ml) incubated rith glucose-6 phosphate (9 .0 usolss), liADP (2 .08 uosolu), l1g304 (24.2 yaolss), tria -HCl buffer (0 .02 K; pH 7.4) sad either sthylaorphine-HC1 (10 broies) or aniline (S unies) is a final voler of 3.0 al . Sssicarba:ide-HC1 (23 pmolu) sawed as a trapping agnat for forraldahyde produced frac sthylsorphins. Iacubatioaa wre carried oat is a Dubnoff ëstabolic Incubator at 37° C for 1S riantes in air. All sasplss wre rand against appropriate tissus blanks and standards. Cytochroaa P-430 was asasured a. described by Osera sad Sato (21) sad sicrosasial protein detersiasd by the rethod of Lawry at al (22) . Results Adsinistration o! growth hormone to norral male or fasala rats did not affect body or liver weights (Tabls 1) . Hepatic microsanal protein concmtratioas wre also unaffected by growth horroas. As previously noted by Wilson (23), traatmeat of vale rats with growth horrona produced a decrease in both hepatic microsaml cytochroma P-450 concentration and the rates of

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TABLE 1 Effscta of Growth Horawae Administration to Horsul. Male wad Fa~ala Bata oa Hepatic Ozidatlve Metabolisms

Horsal Male Rata Control Grwth Hormone

Normal Faule Rata ~ Control Grarth Horaana

Sody Weight (g)

282 .2±5 .0

275 .0±4 .6

~ 203 .8±4 .2

Livsr Aeight (g)

12 .8±0 .6

12 .1±0 .3

~

211 .3±5 8

7_7±0.5

8.5±0 .3

i Microsamal Proteia (mg/g liver)

33 .4±1 .6

33 .1±0 .6

~

37 .2±1 .1

36 .3±1 .3

Cytochrase P-450 (nmolea/~ prot)

0.77±0 .04

0.57±0.02a

~

0.43±0.03

0.43±0 .01

496 .6±29 .3

212 .ß±24.2b

20 .4±0 .7

12 .4t1 .4b

51 .2±2 .6

43 .4±1 .S a 2 .6±0 .2

1~thTisorphine Metabolism (nmolaa/nia/g liver) (nnoluhia/P-450)

~ 157.1±11 .8

148.8±5 .0

10 .1±0 .4

9 .6±0 .4

~

36 .4±2 .5

35 .5±3 .0

~

2.4±0 .1

2 .3±0 .1

Aniliaa Mstabo],(r~mola/min/g liver) (nmolas/ain/P-450)

2 .2± 0.1

Values asprusad as mua ± 88 ; 5-10 animals par group a

p<0 .05

(vs control group of name sa:)

b

p<0 .01

(va control group of acne se>J

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ethylcorphiae and sanies metabolism (Tabla 1) . The fractional decline is aniline hydrozylaee activity was similar to the fall in cytochrome P-450 contant. The decrease is athylmorphina setabolism, hwavar, vas indapendmt of the mode of e:presaion and apparent vhathar ezpresaed per unit tissue vaight or per unit cytochros~e P-450. Whoa administered to normal female rate, growth hormone affected neither hepatic cytochrom P-450 contort nor the rates of ethylmorphine and aailinn metabolisc (Table 1) . Growth hormone adciaiatration to hypophyeectamized male or female rata increased body vaighta but did not significantly affect liver weights (Table 2) . In contrast to its actions is the presence of the pituitary gland, growth hoxcoae, then adminiatarad to hypophysectociznd male rats, increased the rate of hepatic ethylmorphina setaboliec (Table 2) . Growth horcona =~+  += tratioa to hypophysactamizad female rats also significantly increased athylsorphina satabolias (Table 2), although to a smaller eztnnt . Neither aicroaasal protaia nor cytochroma P-450 contort vas affected by grwth horsône is hypophysectomizad male or female rats . The rata of aailiaa hydroacylatioa aLo vas not affected by growth hormone adciaistration to hypophysactaaizad rats of aithar aaz. The affecta of grwth horsona rien in normal ca].s rats ware aLo raifastsd is adreaalactaad.sad or orchiactomisad sn+,.=~_ (Tabla 3) . Both orchiactaay and adraaalactaay produced dacreasaa is hepatic cytochrome P-450 contant sad in the rata of ethylmorphina cetaboliss but did not affect dcrosocal protein concentration. Grwth hormone administration to adrenal- . ectaaisad or orchiactomised = +~~= further lowered the rata of ethylmorphiae desathylation and the concentration of cytochrome P-450 in hepatic cicroaomsa (Table 3) . Prolactin and growth horsona overlap sasevhat is structura and prolaotin aorta varions aosatotrophic affects is 8any vartabrata apaciss (24) . Haravar, adsiaiatration of prolactia to either normal or hypophyesctamisad rats of sithar saz had no affect on hepatic athylmorphins or sanies rtaboliem or oa dcrosomal cytochrome P-450 contant . Dissuasion Wilson and carorkara haws auggaatad, that 8r~h horsons servo ss an sadogaaous aodulator of the hepatic microaoaal si=ed function ozidaaa systas is sal~ rats (17-19, 23, 25) . They fonad that admiaiatration of growth horsoae to cals rate with intact pituitary glands prodncad a dacrsaaa is hepatic cytochrome P-450 contort and decreased the rates of catabolism of various substrates iaclndiag athy]aorphina sad =~+~+~~ (23,25) . WiLoa aLo rsported that growth hotmona had no affect on drug aetabolisc fatale rata (26) . Our data comfits each of thoes observations . Howwar, the rasps of a:psricents is which growth horsoaa is admiaistarad to ~+++_~= with intact pituitary glands are difficult to iatarpret. Lhralnation of chaagas is hepatic function under such circisstaaces wst take into account ~mcoatrollad changer in eadogsaow growth horaow secretion. In addition, the sffacta of growth horcoae administration on aacretiaa by other endocrine glands vhosa hotsooal products say also affect drug cataboliam cost ba canaidarad. Hacansa of thaw cosplicatiom , the direct physiological actions of growth horcona on the liver say be obscured vhea the horcona is givsn to anisals with intact pitüitariaa .

in

Tha divergence of nffacte of grarth hormone in the presaace sad abasnce of the pituitary gland is clearly illwtrated by the prisant etndiss. None of the effects of growth bosons treatmaat seen in normal rats wre cani-

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783

TABLE 2 Effects of Grwth Hormone Adsinistration to Hypophysectosi :ed Male and Female Bats on Hepatic Ozidative Metabolism Hypophysactasisad Males rovtb Control Sorsona

Body Weight (g) Liver Weight (g)

252 .0±7 .8

283 .5±8 .2a

8 .5±0 .3

9 .2±0 .5

ëicrososal Protein (~/g liver)

29 .T±1 .3

31 .7±1 .2

Cytochrome P-450 (r~soLs/aE prot)

0 .57±0 .07

0 .59±0 .06

Eth~lsorphiae Metabolism (nsolu/sin/g liver) (nsolu/:xtn/P-450)

~_

~ Hvnoyhvsactasised Feinalu Gr°vth ; Control ~ Hormone i { iv { i {

202 .5±6 .6

~

6 .1±0 .5

6 .9±0 .3

{ i

27 .0±1 .0

27 .7±1 .5

0 .54±0 .04

0 .53±0 .07

{ ~ { i

220 .1±22 .8

411 .8±69 .Oe

13 .9±1 .6

-

233 .3±4 .8s

' 91 .3±8 .0

22 .3±2 .3 a

~ i i

15 .4±1 .4

18 .1±1 .7

{

12 .2±0 .8

13 .8±1 .1

1 .1±0 .1

1 .3±0 .1

~

0 .8±0 .1

0 .9±0 .1

6 .3±0 .4

128 .5±13 .Oa 8 .8+0 .ß a

Aniline Metabolism (nsolu/sin/g liver) (nmoles/sin/P-45o)

i

Values e~rused as scan + SE ; 5-10 anima]r per group a

p<0 .05

(vs control group of sate sez)

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TABLB 3 Effecu of Grarth Horsons ate++++~tration to Orchiactos~isad and Adrenalactaed.sad !lala Rats on Hepatic Eth~laarphiaa Kataboliaa

Group

Microeaaal Protein (~B 8 liwir

tochrar P-450 (n~olae sag prot)

Sth~laorphina ëatabolisa n~o aa g li~ar)

~t1 Nasal Male (8)

37 .1 + 0 .6

0 .77 + 0 .02

819 .6 ± 40 .2

Orchiactaaizad (8)

33 .8 + 1 .1

0 .61 + 0 .04a

352 .8 ± 43 .8a

33 .5 + 0 .8 -

0 .51 + _ 0 .02a~b

175 .0 + _ 20 .8a+b

Norrl Male (69

35 .4 + 0 .4

0 .91 + 0 .04

578 .1 + 20 .8

Adranalactoaised (6)

38 .5 + 0 .6

0 .67 + 0 .06a

391 .0 + 33 .1a

39 .1 + 1 .9 -

0 .48 + _ O .OSa'b

243 .4 + _ 19 .3a~b

Orchiectaaaised Grautb Harmes (8) Ss~t 2

Adrenalactosized Grautb Horaona (6)

Values a:preasad as ran _+ SS ; amber of aaiaala par group indicaud in paraathaau . a

p<0 .05

(vs nord Talaa)

b

p<0 .05

(va orchiactoaisad or adraaalactastised rlaa)

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Growth Hormone and Drug Nstabolism

fated is hypophysectamiaad animals . For eLmple, aailiae hydrazylation sad cytochrame P-450 levels ware decreased by growth hormone administration to normal male rats but unaffected in the absence of the pituitary gland . Furthermore, the rate of ethylmorphine metaboliw, although lasted by growth hormone is normal males and unaffected is normal females, vas markedly increased after grwth hormone administration to hypophyeectamind males sad fsoales . These obsarvatiaos iadicata a far more compla: role for grarth hormone is the regulation of hepatic mind fuactioa o:idaaa than previously suspected . üei sad WiLon (18) previously reported that hamobarbital ozidatloa vas decreased by growth hormone traatmsat in both normal and hypophysactasd .zed maL rats, but apparently did sot datesmiae affects on cytochrose P-450 content or on the metabolism of other substrata in the absence of the pituitary gland . Oa the basis of his observatiass, iülaoa (18,26) coaclnded that grarth hormone actions to lorar microsamal drag matabo 7 ; a vase independent of pituitary factors . Hw~ever, oar scraps clearly demcastrate that the pituitary gland doe iniluenea growth hormone effects an drag metabo lism sad that its specific roL varies with the sabatrate employed . Thus, in delineating the actions of growth hormone, care mast ba takes to distiagaish between the effects sees is the pruence sad absence of the pit~dtary gland . The former may be maaifated is response to large chaaga in the rata of wcration of grarth hormone . The latter, hosavar, probably rspreseat the direct physiological actions of growth ho:moae as hepatic aind fmctian ozidasa . The mschaaism of interaction batuaen grarth hormone sad the pituitary gland to affect drag metabo liw is presently sm]oDOSn . Specificity of growth hormone action is indicated by the failure of prolactia to mrt similar affects in normal or hypophysactamised aaimaL . Effects of growth hormone sees is normal male rats were aLo manifested is orchieetasdsed or adrenalectcmind animals, eloeluding a dependence on sadogeaous steroids . Farther studies are ear needed to evaluate the interactions of growth horaoas dth still other hormones, particularly throe of pituitary origin. The stndiu presented here ware originally ~mdertskea to further e:amiaa the reLtianship of the pituitary gland to aadrogeaic effects oa hepatic drag metabolism . üe raceatly reported that the affects of testos terone to eahaace athylmorphine metabolism in rata ors desionstrabls only is the presence of the pituitary (13) . Intesating~y, the affects of tutosterone is the presence of the pituitary gland are similar to those of grarth hormone is the absence of the pituitary . Furthermore, tutosterone ha bean shorn to pramota grarth hormone secretion (hi-16) . Thus observations snggut that aadrogeaic sad growth hormone affects an hepatic drug ozidatioa may be iatarrelated . In ary event, the results praeated clearly demonstrate a pituiury-dapendeaca for growth hormone effects on hepatic drug metabolism sad further emphasis! the probabia importance of hormonal interactions in tiu regulation of hepatic mind function osidasa . Acknarladg.aats The technical usistance of Manses Pope la gratefully acknowledged . Growth hormone sad prolactia warn provided by the Mtaitary IIormone Distribution Program of AIAMD, NIH . Thus iavaatigatioas were supported by NSF grant GH-41215 .

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Beferaacas J.B . Gillatta, D.C . Daviu aad H.A . Sasasn, Aaa. Bav. Pharmscol. 12 57-84 (1972) . 2. A.H. Connay, Pbar~acol. Bsv. 19 317-366 (1967) . 3. G.P . Quina, J . Asialrod aad B.B . Hrodie, Hiochaa. Pharaacol. 1 152-159 (1958) . 4. D.B . Daviu, P.L . Gigoa aad J.B . Gillatta, Lifs Sci . 85-91 (1969) . 5. J.B . Schaaimn, I . Frsy, H. Aarr aad B.ü . ßstabrooic, liolac . Pharssticol . 3 516-525 (1967) . 6. B. Lato wwdL. Oaada, Hiochu . Phaascol . 19 1649-1660 (1970) . 7. B. Yato aad J.B. GilLtte, J. Pharsacol . ßsp. Thsr . 150 279-284 (1965) . 8. P.L . Gigoa, T.ß . Graw aad J.B . Gillette, Biochs . Hiopips . Bu . Caasam . 31 SS8-562 (1969) . 9 . B.ß . Hsaiiclc, N.G . Zaapaglioaa, B. Stripp and J.B . Gillette, Biochs. ~+~~~ol . 22 293-310 (1973) . 10 . B. Yato and J.Y. Gillatta, J . Phas>lacol . ßsp. There. 150 285-291 (1965) . 11 . B. Aato, R. Oaada and 11. Sasjiaa, Jap. J. Phartlacol. 20 194-209 (1970) . 12 . 3. ßl Dafravy ßl ëury aad G.J . ëaaa ring, Drug lfatab.Msp . 2 279284 (1974) . 13 . B.ß . Sras+ar, J.W . Graiaar aad H.D . Colby, Diocha . Pitas9acol . (1975) (In prass) . 14 . L.G . ëartin, J .W. Clark and T.B . Coaaer, J. Clin . ßndocriaol. liatab . 28 425-428 (1968) . 1S . S. Illig and A. Pradar, J. Clia . ßadocriaol . ëatab. 615-61 8 (1970) . 16 . [.J . Catt, Tha Lancet 1 933-939 (1970) . 17 . J.T . WiLon, 1latnra 22S 861-863 (1970) . 18 . ß. Wai aad J.T . WiLon, J. phar ~~ ol . Esp . Thar . 177 227-233 (1971) . 19 . J.T . üilson and L.A . Frohafa, J. Pharsacol. ßsp . Thsr . 189 255-270 (1974) . 20 . T . äuh, Biochaa. J. 5S 416-421 (1953) . 21 . T. Ooura aad B. Sato, J. Biol . Chaa. 239 2370-2378 (1964) . wad B.J . Baadall, J . Hiol . 22 . O .H. Lavry, ä.J . Bosabroagh, A .L . Farr Chat . 193 265-275 (1951) . 23 . J .T . gilron, Hiochat. Pharwacol. 2Z 1717-1728 (1973) . 24 . C.S . gicoll, in Haadbook of PhVSiôlo~ IV, part 2: 253-292, edited by S.B. Gaigar, TJilliau aadWiltias : Halti~ora (1975) . 25 . J.T . Wilsoa, P. Soc. Esp . !t . 143 978-983 (1973) . 26 . J.T. i111son, Fad. Procad . (abst) 30 281 (1971) . 1.