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Steroidogenic properties of isolated turkey adrenocortical cells
DOMESTIC ANIMAL ENDOCRINOLOGY
Vol 6(2) 121-131, 1989
STEROIDOGENIC PROPERTIES OF ISOLATED TURKEY ADRENOCORTICAL CELLS J.F. Kocsis and R.V. Carsia Department of Anatomy Un,vers,ty of Medic,ne and Dentistry of New Jersey School of Osteopathic Med,cine Plscataway, NJ 08854 Received September 15, 1988
ABSTRACT Some functional properties of highly enriched turkey poult adrenocortlcal cellswere characterized Cellswere incubated with various mammalian and avian A C T H analogues, 8-Br-cAMP, and 25.hydroxycholesterol for 2 hr Corticosterone production and, where appropriate, cyclic A M P (cAMP) production were measured by radloimmunoassay H u m a n ACI~.(I-24) was the most efficaciousand potent A C T H analogue for stimulating corticosterone and cAMP production, whereas turkey ACTH-(I-39) was among the least efficaciousand least potent analogues Maximal corticosterone production Induced by 8.Br-cAMP and supported by 25.hydroxycholesterol was 67-109% greater than that induced by A C T H analogues The data suggest that intracellularconcentrations of cAMPdependent factors and steroidogenlc enzymes exceed those which are accessible to ACTH-aCtlvated cellular processes In addition, there were sex.dependent contrasts in some functionalparameters, despite the immature statusof the birds Basal corticosterone production of female ceils was 19% greater than that of male cells, albeit maximal A C T H analogue-induced corticosterone production was not differentbetween male and female cells In contrast, maximal 8-Br-cAMP-Induced and 25-hydroxycholesterol-supported cortlcosterone production of male ceilswere, respectavely,72 and 45% greater than that of female,ceils,thus suggesting greater Intracellularconcentrations of protein kanase A-dependent factorsand steroidogenic enzymes in male cellscompared to female ceils However, maximal ACTH-induced cAMP production of female cells was 21% greater than those of male cells,thus sugl~estinga compensatory mechanism In female ceils In addition, there were sex.dependent ddferences in sensitivityto A C T H as indicated by corticosterone and cAMP responses to A C T H analogues sensitivityof male cells was 1 2-3 2 times that of female cells A sex-dependent difference in ACTH-cell interaction, possibly including A C T H receptors, is implicated since there were no sex diflrerencesin cellularsensitivitiesto 8-Br-cAMP and 25-hydroxycholesterol These data indicate that the turkey adrenal gland is amenable for the preparation of isolated adrenocortical cells that have functional integrity Thus, turkey adrenocortlCal cells expand the in vitro repertoire for elucidating the regulatory mechanisms of avian adrenocortical function INTRODUCTION Studies in vitro using isolated adrenocortical ceils have been important in understanding adrenocortical physiology, as well as the cellular mechanisms underlying alterations in adrenocortical function with various physiological states in m a m m a l s In recent years, similar techniques of cell isolation have been a p p h e d to the avian adrenal gland with considerable success To date the most widely studied species has been the domestic fowl (GaUus &allus domestlcus) For example, the steroidogenlc (I-6) and cyclic A M P (2-4,6) responses of isolated domestic fowl adrenocortical cells have been described and A C T H receptors in intact cell suspensions have been partially characterized (7) Furthermore, changes in domestic fowl adrenocortical cell function after hypophysectomy (8) and orchiectomy (9), during the perihatch period (I0)
Copyright@ 1989 by DOMENDO, INC
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KOCSIS AND CARSIA
and with maturation (10,11) have been described, as well as alterations in cellular function accompanying genetic differences m stress response (12) and alterations in response to dietary protein deprivation (5,7) In addition to studies with domestic fowl adrenocorucal cells, other recent work has characterized the steroidogemc properties of isolated adrenocorucal cells from the Japanese quail (Coturnix coturnix japomca) and the impact of selecuon for high serum corticosterone response to stress on these cells (13) Our understanding of avian adrenocorucal f u n c u o n has been enhanced by studms using the domestic turkey (Meleagris gallopavo) Experiments tn vtvo have described turkey a d r e n o c o m c a l response to exogenous ACTH and a variety of stressors (14-22) Undoubtedly, our current knowledge of avian adrenocortical function at the cellular level w o ul d be enhanced by m form auon from studies with turkey adrenal tissue and adrenocortlcal cells However, to our knowledge, no studms have assessed turkey adrenocortical function in vitro Accordingly, in the present study, we evaluated the corucosterone and cychc AMP (cAMP) responses of highly enriched adrenocortmal cell populations isolated from the adrenal glands of male and female poults MATERIALS AND METHODS A n i m a l s . Male and female turkeys (1 day old) (Nicholas Turkey Breeding Farms, Sonoma, CA) were housed in deep litter brooder pens under controlled conditions (12-hr light 12-hr dark photocycle) and had free access to commercml diet and water At 9 weeks of age, birds were collected together into plastic carrying crates and transported to a necropsy room where they were randomly kMled by decapitation and exsangumated In each experiment, 4-10 birds of each sex were used Adrenal glands were quickly removed, trimmed free of connective tissue, and then diced into small pieces (about 2 mm 3) for cell lsolauon The number of birds used in each experiment reflects the amount of adrenal tissue required to achieve equal adrenocortmal cell concentrations b etween sexes and to provide a sufficient volume of cell suspension to test cellular responses to steroidogemc agents A d r e n o c o r t l c a l cell i s o l a t i o n a n d i n c u b a t i o n . The standard medium for cell lsolauon and incubation was Krebs-Rmger HEPES (N-2-hydroxyethylplperazme-N'-2-ethane sulfomc acid) buffer (24 2 mM HEPES, 118 5 mM NaCI, 4 75 mM KCI, 2 54 mM CaC12, 1 2 mM KH2PO4, 1 2 mM MgSO4, 11 1 mM glucose, pH 7 5) Adrenocortmal cells were isolated by techmques described previously for the isolation of domestic fowl (Gallus gallus domesticus) adrenocortmal cells (5,7,11) In brief, dined tissue was subjected to a collagenase solution (0 24%, 127 U/mg, Cooper Biomedical, Malvern, PA) and mechanical agitation until c o m p l e t e l y dissociated Adrenocortmal cells were isolated from the resultant adrenal cell population by Percoll (40%, Pharmacla Fine Chemicals, Ptscataway, NJ) continuous density gradient centrlfugauon Cells were washed free of Percoll, and then resuspended in incubation medium [standard medium containing 0 25% bovine serum albumin (Fraction V) and 0 5 mM 1.methyl-3-1sobutylxanthme (Sigma Chemical C o , St Louis, MO)] Routinely, 85-89% of the cell populations were comprised of adrenocortical cells as adjudged by light microscopy The light microscopic ldenuficauon of avian adrenocorucal cells has been verified by electron microscopy (1) Cell suspensions were adjusted to a c onc e nt rauon of 2 X 105 cells/ml The ymld of male and female adrenocortmal cells was 35,738 - 2,911 and 24,012 + 622 cells/mg adrenal tissue, respectively (means +_ SEM from 3 experiments)
TURKEY ADRENOCORTICAL CELLS
123
Ahquots of cell suspensions were incubated with various steroldogenlc agents Purified synthetic ACTH peptides used were human (h) ACTH-(1-24) (cortrosyn, Organon, I n c , West Orange, NJ), hACTH-(1-39) (Peninsula Laboratories, Belmont, CA) ostrich (os) ACTH-(I-39), and turkey (tu) ACTH-(I-39) Avian ACTH peptides were synthetized by the solid state method, were purified to homogenetty as determined by high-performance hquld chromatography (HPLC) and had chromatograms that were identical to those of the respective highly purified natural hormones (23) (gift from Dr C H Li, Laboratory of Molecular Endocrinology, University of California, San Francisco) In addmon, 8-bromocyclic AMP (8-Br-cAMP) (Sigma), and the sterotdogemc precursor, 25-hydroxycholesterol (Steraloids, I n c , Wilton, NH) were used Prior to the addition of these agents to the incubation medium, ACTH peptldes were dissolved m a solution of 0 9% NaC1 containing 0 I% bovine serum albumin (pH 3 0), whereas 8-Br.cAMP and 25-hydroxycholesterol were made up m the standard medium Imtial dilutions of 25-hydroxycholesterol were carried out m absolute ethanol, however, the final concentration of ethanol in the incubations (0 4%) did not affect corticosterotdogenesis The final incubation volume (90% cell suspension, 10%, a solution containing one of the agents) was 300 ~tl Incubations were carried out at 40 C m a metabolic shaking water bath for 2 hr Routinely, 8793% of the cells were viable after a 2-hr incubation as indicated by trypan blue dye exclusion (24) Incubations were then frozen until radioimmunoassay for cortmosterone and cAMP R a d i o l m m u n o a s s a y f o r c o r t i c o s t e r o n e a n d cAMP. Corticosterone, the predominant corticosteroid released by the adult avian adrenal gland (25), was measured by a modification of the radloimmunoassay procedure of Roy, Jr et al (26) The modifications were the direct radlolmmunoassay of appropriately diluted ahquots of the cell incubations and the use of a commercially available, specific antibody (ICN ImmunoBlologlcals, Lisle, IL) Incubation medium was used to dilute cell incubations prior to radiolmmunoassay As httle as 0 1 ng cortlcosterone/ml cell Incubation could be detected Crossreactlvittes with two corucosteroids of the steroldogemc pathway leading to cortlCOsterone, progesterone and 11-deoxycortmosterone, were 17% and 27%, respectively In each radiolmmunoassay, ahquots of pooled cell incubations from other experzments were included as quality control references Radlotmmunoassay of these references showed tntraassay and interassay coefficients of variation of 3 8 and 5 3%, respectively Cellular cAMP production was measured by a highly sensitive and specific, succmylated cAMP radloimmunoassay (ICN ImmunoBtologmals) As little as 1 pmole cAMP/ml cell incubation could be detected Quality control references showed lntraassay and lnterassay coefficients of variation of 4 1 and 8 7%, respectively A n a l y s i s o f d a t a . The data of corticosterone and cAMP production by isolated adrenocorttcal cells were statistically analyzed using a four-parameter logistic equation model that is available as a computer program (ALLFIT, Biomedical Computing Technology Information Center, Vanderbilt Medical Center, Nashville, TN) This program allowed for the simultaneous analysis of a family of dose-response curves (27) Values for half-maximal steroldogenic concentration of an agent (EDso) and values for maximal production were calculated and statistically analyzed using this program In a statistical analysis run, parameters to be compared were set equal to one another This decreased the number of parameters in the analysis, but increased the extra sum of squares
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KOCSIS AND CARSIA
of residuals due to the constraint An F ratio test (contained within the program) of the constrained fit to the unconstrained fit was performed Values hypothesized to be equal were deem e d significantly different w hen P < 0 05 The average within- and between- exper i m ent coefficmnts of variation of cell mcubauons (calculated from basal corticosterone values after 2 hr of incubation) were 5 7 and 9 4%, respectively Other cellular response data were stausucally analyzed by analysis of variance Means were separated by the Newman-Keuls test and were deem e d significantly different w hen P < 0 05 (28) RESULTS
Corticosterone production induced by ACTH analogues. Figure 1 shows corttcosterone production by isolated turkey adrenocorucal cells in response to various concentrations of ACTH analogues ACTH analogues stimulated cortlcosterone production over about two log orders of concent rauon Human analogues were 48% more efficacious than avian analogues for sumulatlng cortlcosterone p r o d u c u o n [859 8 _ 72 6 and 580 8 _ 51 3 ng cortmosterone/ ml, respectively (means _ SEM), pool ed data of maximal ACTH-mduced corticosterone production by male and female cells] In addition to differences in efficacy, analogues exhibited different potencies as indicated by the different half-maximal steroidogemc concentrations (EDso values) (Table 1) Human ACTH-(1-24) was the most potent analogue, whereas tuACTH-(1-39) was one of the least potent analogues, the average pot ency of hACTH-(1-24) was 11 times that of tuACTH-(1-39) A stausucal l y significant --//, FEMALE 120C
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Fig 1 ACTH a n a l o g u e - m d u c e d cortlcosterone p r o d u c t i o n by isolated turkey adrenocortlcal cells Cells (2 X 10 5 c e l l s / m l ) w e r e incubated w i t h various concentrations of analogues for 2 hr. Each s y m b o l r e p r e s e n t s the m e a n o f corticosterone values from nine cell incubations (3 cell mcuDatlons from each o f 3 e x p e r i m e n t s ) SEM are not s h o w n
TURKEY ADRENOCORTICAL CELLS
125
TABLE 1 I-IALF-MAXtMAL STeROtDOGm~aCCONCENTgATION(EDs0) OF STERO1DOGENICAGENTFOR CORTICOSTERONE PRODUCTIONBY ISOIATm) ~
ADmmOCOgnO.L Ct~LLS
Source of Cells ACTH analogues hACTH-(1-24) hACTH-(1-39) osACTH-(1-39) tuACTH-(1-39) 8Br-cAMP 2 5-hydroxycholesterol
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(2 49 + 0 08) X 101~M '
(3 51 (1 20 _+ 0 06) X 1 0 ' ° i (1 99 (2 08 -+ 0 12) X 10t°M (3 18 (2 43 -+ 0 21) X 104M (2 42 (4 1 0 (3 76 + 0 2 0 ) X 10"6M Note- Values are the means _+ SEMS calculated from the data shown in ALLFIT computer program
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*Values significantly different from corresponding values for male cells (P < 0 05)
(P<0 05) order of potency was as follows hACTH-(1-24) > osACTH-(1-39) >[hACTH-(1-39) ---- tuACTH-(1-39)] cAMP p r o d u c t i o n i n d u c e d by ACTH a n a l o g u e s . Fig 2 shows cAMP production by isolated turkey adrenocortlcal cells in response to various concentrations of ACTH analogues The pattern of analogue efficacy and potency for cAMP production was somewhat different than that for cortlcosterone producuon Human ACTH-(I-24) was the most efficacious analogue, however, the efficacy of hACTH-(I-39) was not different from that of the avian analogues Concentrations of ACTH analogue required to half-maximally stimulate cAMP production were about 400 times those required to half-maximally stimulate corttcosterone production (cf Tables 1 and 2) Nevertheless, some aspects of the analogue potency data for cAMP production were consistent with those for corttcosterone production Here again, human ACTH-(I-24) was the most 900 800 70£
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a d r e n o c o r t i c a l cells C e l l s (2 c e u s / m U were incubated With various concentrations of analogues for 2 hr Each symbol represents the mean of cAMP values from nine cell incubations (3 cell incubations from each of 3 experiments) SEM are not shown tu-
126
KOCSIS AND CARSIA
CYCLIC
TABLE 2 HALF-MAXIMALSTEROIDOGENICCONCENTRATION(EDso) OF ACTH ANALOGUEFOR AMP PRODUCTIONBY ISOLATEDTURKEYADRENOCORTICALCELLS Source of Cells Male Female ACTH analogues hACTH-(1-24) (4 61 -+ 0 82) X 109M (1 42 + 0 28) X lOaM. hACTH-(1-39) (2 67 -+ 0 39) X 10aM (4 48 _+ 0 67) X 10aM. osACTH-(1-39) (2 88 _+ 0 42) X 10aM (5 O1 _+ 0 86) X 10aM• tuACTH-(1-39) (3 97 -+ 0 41) X 10aM (1 07 -+ 0 21) X 107M' Note. Values are the means + SEMScalculated from the data shown m Figure 2 by the ALLFIT computer program "Values significantly different from corresponding values for male cells (P < 0 05) p o t e n t a n a l o g u e , w h e r e a s t u A C T H - ( I - 3 9 ) w a s t h e least p o t e n t , w i t h t h e p o t e n c y of hACTH-(I-24) b e i n g 9 times that of tuACTH-(I-39) However, the order of a n a l o g u e p o t e n c y for cAMP p r o d u c t i o n w a s d i f f e r e n t f r o m t h a t for c o r u c o s t e r o n e p r o d u c t i o n h A C T H - ( I - 2 4 ) > [hACTH-(I-39) ---- o s A C T H - ( I - 3 9 ) ] > tuACTH-(I39)
Corticosterone p r o d u c t i o n i n d u c e d by 8-Br-cAMP and supported by 25-hydroxycholesterol. Figures 3 and 4 show cellular c o r u c o s t e r o n e prod u c u o n s t i m u l a t e d b y 8-Br.cAMP a n d s u p p o r t e d b y 2 5 - h y d r o x y c h o l e s t e r o l m t h e a b s e n c e o f ACTH, r e s p e c t i v e l y T h e a v e r a g e m a x i m a l 8 - B r - c A M P - m d u c e d and 25-hydroxycholesterol-supported cortmosterone production values were, r e s p e c t i v e l y , 109 a n d 67% g r e a t e r t h a n t h e a v e r a g e m a x i m a l ACTH a n a l o g u e i n d u c e d c o r t l C O s t e r o n e p r o d u c u o n ( T a b l e 3) S e x d i f f e r e n c e s i n adrenocortical cell f u n c t i o n . D e s p i t e t h e I m m a t u r e p h y s l o l o g m a l status of the poults, there w e r e some clear sexual differences tn a d r e n o c o r t m a l c e l l r e s p o n s e to s t e r o l d o g e m c a g e n t s a n d to t h e p r e c u r s o r , 25h y d r o x y c h o l e s t e r o l Basal c o r u c o s t e r o n e p r o d u c u o n o f f e m a l e c e l l s w a s 19% --/f/L j
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Fig 3 Corticosterone productxon by isolated turkey adrenocortlcal cells sumulated with 8-BrcAMP Cells (2 X 105 cells/ml) were incubated with various concentrauons of 8-Br-cAMP for 2 hr Each symbol represents the mean of corticosterone values from rune cell incubations (3 lncubauons from each of 3 experiments) SEM are represented by bars when they are larger than the symbols
127
TURKEY ADRENOCORTICAL CELLS -7///i
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Fig 4 Conversion of 25-hydroxycholesterol to corticosterone by isolated turkey adrenocortical cells Cells (2 X 105 cells/ml) were incubated with various concentrations of 25-hydroxycholesterol in the absence of ACTH for 2 hr Each symbol represents the mean of corticosterone values from nine cell incubations (3 cell incubations from each of 3 experiments) SEM are represented by bars w h e n they are larger than the symbols
greater than that of male cells (Table 3) However, maximal ACTH analoguereduced corticosterone production of male and female cells was not different In contrast, maximal 8-Br.cAMP-mduced and 25-hydroxycholesterol-supported cortmosterone production values of female cells were, respectively, 42 and 31% less than those for male cells On the other hand, although basal cAMP production by male and female cells was not different, maximal ACTH analogueinduced cAMP production of female cells was 21% greater than that of male cells (Table 4) In addition to sex differences in cAMP and cortlcosterone production, there were also sex differences in ACTH analogue potency, or put another way, cellular sensitivity to ACTH analogues (Tables 1 and 2) Sex differences in cellular sensitivity to ACTH analogues varied with the ACTH analogue but were stguificant (P < 0 05) for corticosterone and cAMP production, the range of sensitivity of male cells to ACTH analogues was, respectively, 1 2-2 1 times and 1.6-3 2 times that of female cells TABLZ 3 BASAL"AND MAXIMALb CORTICOSTERO
~lqE
PRODUCTION BY ISOLATED~
21kDRENOCORTICAL
CELLS Additions to Source of Cells Cell Incubations Male Female Diluent 537 ± 24 6 3 8 :t: 13" ACTH analogues 788 8 ± 69 4 729 4 ± 54 3 8-Br-cAMP 2021 7 ± 9 4 4 11776 ± 348' 25-hydroxycholesterol 1513 3 ± 41 8 1040 2 ± 27 5" Note:. Values (ng/ml) are the means ± SEMS calculated from the data shown in Figures 1,3 and 4 by the ALLFIT computer program •Basal corticosterone production after 2-hr incubation with agent diluent bMaximal corticoster?ne production after 2-hr incubation with ACTH analogues (pooled), or with 8-Br-cAMi', or with 25-hydroxycholesterol "Values significantly different from corresponding values for male cells (P < 0 05)
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KOCSIS AND CAFISIA
TABLE4 BASAL"ANDMAXIMALb CYCHC A M P PRODUCTION BY ISOLATED TURKL~ ADRENOCORTICAL CELLS Additions to Source of Cells Cell Incubations Male Female Diluent 24 _+ 0 1 24 _+ 0 2 ACTH analo~ues 538 3 -+ 26 4 653 0 _+ 42 3' Note. Values (pmoles/ml) are the means _+ SEMScalculated from the data shown in Figures 2 •Basal cAMP production after 2-hr incubation with ACTH diluent bMaxlmal cAMP production after 2-hr incubation with ACTH analogues (pooled) "Value significantly different from corresponding value for male cells (P < 0 05) DISCUSSION Both m a m m a l i a n and avian ACTH a n a l o g u e s s t i m u l a t e d c o r u c o s t e r o n e and cAMP p r o d u c t i o n b y isolated t u r k e y a d r e n o c o r u c a l cells (Figures 1 and 2) S o m e w h a t p u z z l i n g is the o b s e r v a t i o n that tuACTH-(1-39) was the least p o t e n t a n a l o g u e (Tables 1 and 2) H o w e v e r , similar results w e r e o b t a i n e d w i t h isolated c h i c k e n a d r e n o c o r t m a l cells (5) F u r t h e r m o r e , o u r results of differences m p o t e n c i e s b e t w e e n avian and m a m m a l i a n ACTH a n a l o g u e s are consistent w i t h the results o f w o r k w i t h isolated rabbit and rat fat cells ( 2 3 ) , albeit differences in p o t e n c i e s w o u l d b e e x p e c t e d w i t h m a m m a l i a n cells It s h o u l d b e p o i n t e d that the o b s e r v e d differences in p o t e n c i e s b e t w e e n avian and m a m m a l i a n ACTH a n a l o g u e s in the p r e s e n t s t u d y and in o t h e r w o r k ( 5 , 2 3 ) w e r e not d u e to differences in specific activities b e t w e e n analogues, b e c a u s e the analogues u s e d w e r e synthetic, highly purified p e p u d e s that w e r e identical to the res p e c u v e natural p e p t l d e s as a d j u d g e d b y HPLC ( 2 3 ) In a d d i t i o n to the l o w p o t e n c y o f tuACTH-(1-39), b o t h tuACTH-(I-39) and osACTH-(I-39) w e r e the least efficacious for s t i m u l a t i n g c o r t l c o s t e r o n e prod u c u o n (Figure 1 and data u n d e r RESULTS) The data suggest that, wittx avian a d r e n o c o r t m a l cells, d o m a i n s w i t h i n tuACTH-(I-39) act to m o d u l a t e steroldogenesis, that is, b o t h s t i m u l a t e a n d d a m p e n s t e r o i d o g e n e s i s Presumably, the " d a m p e n i n g " d o m a i n ( s ) resides b e y o n d a m i n o acid 24, since hACTH-(I-24) had the greatest efficacy and p o t e n c y for s t i m u l a t i n g c o r t m o s t e r o n e and cAMP p r o d u c t i o n b y t u r k e y a d r e n o c o r t m a l cells (Figures 1 and 2, Tables 1 and 2), a n d c o r t m o s t e r o n e p r o d u c t i o n b y c h i c k e n a d r e n o c o r t m a l cells (5) Indeed, m o s t o f the n o n c o n s e r v a u v e a m i n o acid variation b e t w e e n the p e p u d e s o c c u r s after a m i n o acid 24 ( 2 3 ) Perhaps the p o s t u l a t e d d a m p e n i n g effect o f the native p e p u d e serves to p r e v e n t d e l e t e r i o u s o v e r s t i m u l a t l o n of the avian adr e n o c o r t l c a l cell d u r i n g p e r s i s t e n t stress in vtvo The cAMP analogue, 8-Br-cAMP, a s t e r o i d o g e m c a g e n t that bypasses p l a s m a m e m b r a n e events in c o r t l c o s t e r o l d o g e n e s i s , s t i m u l a t e d c o r t l c o s t e r o n e p r o d u c tion in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r (Figure 3) In addition, the readily c o n v e r t i b l e s t e r o i d o g e m c p r e c u r s o r , 2 5 - h y d r o x y c h o l e s t e r o l ( 2 9 ) i n c r e a s e d cort t c o s t e r o n e p r o d u c t i o n m the a b s e n c e o f ACTH (Figure 4) H o w e v e r , the average maxamal cortlCOsterone p r o d u c t i o n s t i m u l a t e d b y 8-Br-cAMP and s u p p o r t e d b y 2 5 - h y d r o x y c h o l e s t e r o l was, respectively, 109 and 67% greater than that stimu l a t e d b y ACTH a n a l o g u e s (Table 3) T h e data suggest that the i n t r a c e l l u l a r c o n c e n t r a t i o n s o f p r o t e i n kinase A - d e p e n d e n t factors a n d s t e r o l d o g e n i c e n z y m e s e x c e e d t h o s e w h i c h are a c c e s s i b l e to ACTH-acuvated c e l l u l a r p r o c e s s e s Similar results w i t h 25-hydroxych01esterol (5,9,10,13) and the p r e c u r s o r p r e g n e n o l o n e ( 1 , 8 , 1 1 , 1 2 ) have b e e n o b t a i n e d f r o m e x p e r i m e n t s w i t h c h i c k e n ( 1 , 5 , 8 - 1 2 ) and J a p a n e s e quail ( 1 3 ) a d r e n o c o r t m a l cells H o w e v e r , the o b s e r v a t i o n that the average m a x i m a l c o r t m o s t e r o n e p r o d u c t i o n i n d u c e d b y 8.Br-cAMP was 109% g r e a t e r t h a n that i n d u c e d b y ACTH a n a l o g u e s m i g h t b e a u n i q u e feature
TURKEY ADRENOCORTICAL CELLS
129
of turkey adrenocortmal cells In experiments with chicken and Japanese quail adrenocortical cells, the magnitude of maximal cortlcosterone production stimulated by ACTH and 8-Br-cAMP was not different (1,5,8-13) Sex steroids can influence adrenocortlcal function in the domestic fowl (9,30) However, their involvement in turkey adrenocortical function is unknown In the present study, presumably plasma sex steroid levels were very low in the sexually immature, 9-week-old poults Thus, we were perplexed to find clear sex differences in adrenocortlcal cell function On an equal cell concentration basis, maximal 8-Br-cAMP-induced and 25-hydroxycholesterolsupported cormcosterone production of male cells were, respectively, 72 and 45% greater than those of female cells, albeit, maximal ACTH analogue-induced corticosterone production of male and female cells was not different (Table 3) The data suggest a greater intracellular concentration of protein kanase Adependent factors and steroldogenic enzymes of male cells compared to female cells In contrast, maximal ACTH analogue-induced cAMP production of female cells was 21% greater than that of male cells (Table 4) Possibly, this observation indicates a compensatory mechanism of female cells However, the physiological significance of these differences is unclear since presumably the lntracellular concentrations of cAMP-dependent factors and steroidogenic enzymes exceed those that are accessible to ACTH-actwated cellular processes Even more puzzling than the aforementioned observations are the differences in sensitivity to ACTH analogues between male and female cells, as indicated by the different analogue EDso values (Tables 1 and 2) Depending on the analogue used and the parameter measured (i e , corucosterone or cAMP production), sensitivity of male cells to ACTH was 1 2-3 2 times that of female cells This sex difference in cellular sensitivity to ACTH appears to be due to a difference in steroidogenlc steps prior to the formation of cAMP, possibly including the ACTH receptor, since 8-Br-cAMP and 25-hydroxycholesterol EDs0 values for corticosterone production were not different between male and female cells (Table 1) Obviously, recent techniques of characterizing domesuc fowl adrenocortmal cell ACTH receptors (7) need to be applied to turkey adrenocortmal cells to test this postulate. Data presented here indicate that the turkey adrenal gland is amenable for the preparation of isolated adrenocortical cells that are responsive to ACTH and steroidogemc agents which bypass plasma membrane events (e g , 8-BrcAMP), and readily convert steroidogenic precursors (e g , 25-hydroxycholesterol) to the predominant end product, corticosterone In addition, recently we have demonstrated that both turkey and domestic fowl adrenocortical cells are responsive to prostaglandms (31) Thus, isolated turkey adrenocortlcal cells expand the in vitro repertoire for understanding the regulation of avian adrenocortlcal function in terms of both basic cellular mechanisms and comparative adrenocortical function ACKNOWLEDGMENTS AND FOOTNOTES This work was funded by USDA grant 85-CRCR-l-1846 We thank Helen Weber and Leslie A Schwarz for expert technical assistance Please address all correspondence to Rocco V Carsla, Ph D , Department of Anatomy, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, 675 Hoes Lane, Piscataway, NJ 08854-5635
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KOCSIS AND CARSIA REFERENCES
1 Carsia RV, Scanes CG, Malamed S Isolated adrenocortmal cells of the domesuc fowl (Gallus domesttcus) Steroidogemc and ultrastructural properties J Steroid Blochem 22 273-279, 1985 2 Rosenberg J, Pines M, Hurwitz S Relationship between endogenous cyclic AMP production and steroid hormone secretion in chick adrenal cells Comp Blochem Physlol 84B 71-75, 1986 3 Carsia RV, Scanes CG, Malamed S Polyhormonal regulation of avian and mammalian cortmosteroidogenesis in vitro Comp Blochem Physiol 88A 131-140, 1987 4 Rosenberg J, Pines M, Hurwitz S Response of adrenal cells to parathyroid hormone stimulatxon J Endocrmol 112 431-437, 1987 5 Carsia RV, Weber H, Lauteno, TJ Protein malnutrition in the domestic fowl induces alterations in adrenocortical function Endocrinology 122 673-680, 1988 6 Rosenberg J, Panes M, Hurwltz S Stimulation of chick adrenal steroldogenesis by avian parathyroid hormone J Endocrmol 116 91-95, 1988 7 Carsla RV, Weber H Protein malnutrition in the domestic fowl induces alterations in adrenocortmal cell adrenocorticotropm receptors Endocrinology 122 681-688, 1988 8 Carsla RV, Weber H, King DB, Scanes CG Adrenocortmal cell function m the hypophysectomized domestic fowl Effects of growth hormone and 3,5,3'-trllodothyronme replacement Endocrinology 117 928-933, 1985 9 Carsla RV, Reisch NM, Fennell MJ, Weber H Adrenocortical function of the domestic fowl Effects of orchlectomy and androgen replacement Proc Soc Exp Blol Med 185 223-232, 1987 10 Carsia RV, Morro ME, Rosen HD, Weber H Ontogemc corticosteroidogenesis of the domestic fowl Response of isolated adrenocortical cells Proc Soc Exp Blol Med 184 436-445, 1987 11 Carsia RV, Scanes CG, Malamed S Loss of sensitivity to ACTH of adrenocortical cells isolated from maturing domestic fowl Proc Soc Exp Blol Med 179 279-282, 1986 12 Carsia RV, Weber H Genetm-dependent alterations in adrenal stress response and adrenocortical cell function of the domestic fowl (Gallus domesticus) Proc Soc Exp Blol Med 183 99-105, 1986 13 Carsia RV, Weber H, Satterlee DG Steroidogenlc properties of isolated adrenocortical cells from Japanese quail selected for high serum cortlcosterone response to immobilization Domestic Animal Endocrmol (in press) 14 Brown KI Enwronmentally imposed stress In Enwronmental Control m Poultry Production, Carter TC (ed) Oliver and Boyd, Edmburg and London, p101-107, 1967 15 Brown KI, Nestor KE Some physiological responses of turkeys selected for high and low adrenal response to cold stress Poultry Sci 52 1948-1954, 1973 16 E1 Halawanl ME, Waibel PE, Appel JR, Good AL Effects of temperature stress on catecholammes and corticosterone of male turkeys J Physiol 224 384-388, 1973 17 Brown KI, Nestor KE Implications of selection for high and low adrenal response to stress Poultry Sci 53 1297-1306, 1974 18 S~mensen E, Olson LD, Ryan MP Determination of cortmosterone concentrauons in plasma of turkeys usmg radioimmunoassay Poultry Sci 57 1701-1704, 1978 19 McCorkle FM, Edens FW, Simmons DG Adrenal secretory involvement in Alcahgenes faecahs mfectton m turkey poults Poultry Sci 61 1510, 1982 (abstract) 20 Daws GS, Slopes TD Adrenal cortical response of tom poults Poultry Sci 64 21892194, 1985 21 Davis GS, Slopes TD Plasma cortmosterone response of turkeys to adrenocortlCOtropic hormone Age, dose and route of administration effects Poultry Sci 66 17271732, 1987 22 Davis GS, Slopes TD The ontogeny of diurnal rhythmicity in plasma corticosterone of poults and the influence of this rhythm on the plasma cortlcosterone (B) response to ACTH or cold Poultry Sci 66 (Suppl 1) 89, 1987 (abstract) 23 Yamashlro D, Ho CL, Ll CH Adrenocortmotropm 57 Synthesis and biological activity of ostrich and turkey hormones Int J Pepude Protein Res 23 42-46, 1984
TURKEY ADRENOCORTICAL CELLS 24 25 26 27 28 29 30 31
131
Liotta A, Krleger DT A sensluve bioassay for the determmauon of human plasma ACTH levels J Clin Endocrinol Metab 40 268-277, 1975 Harvey S, Scanes CG, Brown KI Adrenals In Avian Physiology, Sturlae, PD (ed) Sprmger-Verlag, New York, p479-493, 1986 Roy K, Jr, Garza R, Marouhs G, Abraham GE Radlolmmunoassay of plasma corucosterone Anal Lett 7 109-118, 1974 De Lean A, Munson, PJ, Rodbard D Simultaneous analysis of famd~es of slgmo~dal curves Apphcauon to bloassay, radlohgand assay, and physiological dose-response curves Amer J Physiol 235 E97-E102, 1978 Snedecor GW, Cochran WG Stausucal Methods, Iowa State Umverslty Press, Ames, IA, 1980 Falke HE, Degenhart HI, Abeln GJA, Vlsser HKA Effects of 25-hydroxycholesterol and ammoglutetham~de m isolated rat adrenal cells A model for congemtal hpotd adrenal hyperplasla; Mol Cell Endocrinol 4 107-114, 1976 Nagra CL, Sauers AK, Wlttma~er HN Effect of testosterone, progestagens and metoplrone on adrenal acuvlty m cockerels Gen Comp Endocrmol 5 69-73, 1965 Kocsls, JF, Carsla RV Effect of prostaglandms on awan cortlcostermdogenesls in vitro Poultry SCl 67(Suppl 1) 105, 1988 (abstract)
Vol 6(2) 121-131, 1989
STEROIDOGENIC PROPERTIES OF ISOLATED TURKEY ADRENOCORTICAL CELLS J.F. Kocsis and R.V. Carsia Department of Anatomy Un,vers,ty of Medic,ne and Dentistry of New Jersey School of Osteopathic Med,cine Plscataway, NJ 08854 Received September 15, 1988
ABSTRACT Some functional properties of highly enriched turkey poult adrenocortlcal cellswere characterized Cellswere incubated with various mammalian and avian A C T H analogues, 8-Br-cAMP, and 25.hydroxycholesterol for 2 hr Corticosterone production and, where appropriate, cyclic A M P (cAMP) production were measured by radloimmunoassay H u m a n ACI~.(I-24) was the most efficaciousand potent A C T H analogue for stimulating corticosterone and cAMP production, whereas turkey ACTH-(I-39) was among the least efficaciousand least potent analogues Maximal corticosterone production Induced by 8.Br-cAMP and supported by 25.hydroxycholesterol was 67-109% greater than that induced by A C T H analogues The data suggest that intracellularconcentrations of cAMPdependent factors and steroidogenlc enzymes exceed those which are accessible to ACTH-aCtlvated cellular processes In addition, there were sex.dependent contrasts in some functionalparameters, despite the immature statusof the birds Basal corticosterone production of female ceils was 19% greater than that of male cells, albeit maximal A C T H analogue-induced corticosterone production was not differentbetween male and female cells In contrast, maximal 8-Br-cAMP-Induced and 25-hydroxycholesterol-supported cortlcosterone production of male ceilswere, respectavely,72 and 45% greater than that of female,ceils,thus suggesting greater Intracellularconcentrations of protein kanase A-dependent factorsand steroidogenic enzymes in male cellscompared to female ceils However, maximal ACTH-induced cAMP production of female cells was 21% greater than those of male cells,thus sugl~estinga compensatory mechanism In female ceils In addition, there were sex.dependent ddferences in sensitivityto A C T H as indicated by corticosterone and cAMP responses to A C T H analogues sensitivityof male cells was 1 2-3 2 times that of female cells A sex-dependent difference in ACTH-cell interaction, possibly including A C T H receptors, is implicated since there were no sex diflrerencesin cellularsensitivitiesto 8-Br-cAMP and 25-hydroxycholesterol These data indicate that the turkey adrenal gland is amenable for the preparation of isolated adrenocortical cells that have functional integrity Thus, turkey adrenocortlCal cells expand the in vitro repertoire for elucidating the regulatory mechanisms of avian adrenocortical function INTRODUCTION Studies in vitro using isolated adrenocortical ceils have been important in understanding adrenocortical physiology, as well as the cellular mechanisms underlying alterations in adrenocortical function with various physiological states in m a m m a l s In recent years, similar techniques of cell isolation have been a p p h e d to the avian adrenal gland with considerable success To date the most widely studied species has been the domestic fowl (GaUus &allus domestlcus) For example, the steroidogenlc (I-6) and cyclic A M P (2-4,6) responses of isolated domestic fowl adrenocortical cells have been described and A C T H receptors in intact cell suspensions have been partially characterized (7) Furthermore, changes in domestic fowl adrenocortical cell function after hypophysectomy (8) and orchiectomy (9), during the perihatch period (I0)
Copyright@ 1989 by DOMENDO, INC
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and with maturation (10,11) have been described, as well as alterations in cellular function accompanying genetic differences m stress response (12) and alterations in response to dietary protein deprivation (5,7) In addition to studies with domestic fowl adrenocorucal cells, other recent work has characterized the steroidogemc properties of isolated adrenocorucal cells from the Japanese quail (Coturnix coturnix japomca) and the impact of selecuon for high serum corticosterone response to stress on these cells (13) Our understanding of avian adrenocorucal f u n c u o n has been enhanced by studms using the domestic turkey (Meleagris gallopavo) Experiments tn vtvo have described turkey a d r e n o c o m c a l response to exogenous ACTH and a variety of stressors (14-22) Undoubtedly, our current knowledge of avian adrenocortical function at the cellular level w o ul d be enhanced by m form auon from studies with turkey adrenal tissue and adrenocortlcal cells However, to our knowledge, no studms have assessed turkey adrenocortical function in vitro Accordingly, in the present study, we evaluated the corucosterone and cychc AMP (cAMP) responses of highly enriched adrenocortmal cell populations isolated from the adrenal glands of male and female poults MATERIALS AND METHODS A n i m a l s . Male and female turkeys (1 day old) (Nicholas Turkey Breeding Farms, Sonoma, CA) were housed in deep litter brooder pens under controlled conditions (12-hr light 12-hr dark photocycle) and had free access to commercml diet and water At 9 weeks of age, birds were collected together into plastic carrying crates and transported to a necropsy room where they were randomly kMled by decapitation and exsangumated In each experiment, 4-10 birds of each sex were used Adrenal glands were quickly removed, trimmed free of connective tissue, and then diced into small pieces (about 2 mm 3) for cell lsolauon The number of birds used in each experiment reflects the amount of adrenal tissue required to achieve equal adrenocortmal cell concentrations b etween sexes and to provide a sufficient volume of cell suspension to test cellular responses to steroidogemc agents A d r e n o c o r t l c a l cell i s o l a t i o n a n d i n c u b a t i o n . The standard medium for cell lsolauon and incubation was Krebs-Rmger HEPES (N-2-hydroxyethylplperazme-N'-2-ethane sulfomc acid) buffer (24 2 mM HEPES, 118 5 mM NaCI, 4 75 mM KCI, 2 54 mM CaC12, 1 2 mM KH2PO4, 1 2 mM MgSO4, 11 1 mM glucose, pH 7 5) Adrenocortmal cells were isolated by techmques described previously for the isolation of domestic fowl (Gallus gallus domesticus) adrenocortmal cells (5,7,11) In brief, dined tissue was subjected to a collagenase solution (0 24%, 127 U/mg, Cooper Biomedical, Malvern, PA) and mechanical agitation until c o m p l e t e l y dissociated Adrenocortmal cells were isolated from the resultant adrenal cell population by Percoll (40%, Pharmacla Fine Chemicals, Ptscataway, NJ) continuous density gradient centrlfugauon Cells were washed free of Percoll, and then resuspended in incubation medium [standard medium containing 0 25% bovine serum albumin (Fraction V) and 0 5 mM 1.methyl-3-1sobutylxanthme (Sigma Chemical C o , St Louis, MO)] Routinely, 85-89% of the cell populations were comprised of adrenocortical cells as adjudged by light microscopy The light microscopic ldenuficauon of avian adrenocorucal cells has been verified by electron microscopy (1) Cell suspensions were adjusted to a c onc e nt rauon of 2 X 105 cells/ml The ymld of male and female adrenocortmal cells was 35,738 - 2,911 and 24,012 + 622 cells/mg adrenal tissue, respectively (means +_ SEM from 3 experiments)
TURKEY ADRENOCORTICAL CELLS
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Ahquots of cell suspensions were incubated with various steroldogenlc agents Purified synthetic ACTH peptides used were human (h) ACTH-(1-24) (cortrosyn, Organon, I n c , West Orange, NJ), hACTH-(1-39) (Peninsula Laboratories, Belmont, CA) ostrich (os) ACTH-(I-39), and turkey (tu) ACTH-(I-39) Avian ACTH peptides were synthetized by the solid state method, were purified to homogenetty as determined by high-performance hquld chromatography (HPLC) and had chromatograms that were identical to those of the respective highly purified natural hormones (23) (gift from Dr C H Li, Laboratory of Molecular Endocrinology, University of California, San Francisco) In addmon, 8-bromocyclic AMP (8-Br-cAMP) (Sigma), and the sterotdogemc precursor, 25-hydroxycholesterol (Steraloids, I n c , Wilton, NH) were used Prior to the addition of these agents to the incubation medium, ACTH peptldes were dissolved m a solution of 0 9% NaC1 containing 0 I% bovine serum albumin (pH 3 0), whereas 8-Br.cAMP and 25-hydroxycholesterol were made up m the standard medium Imtial dilutions of 25-hydroxycholesterol were carried out m absolute ethanol, however, the final concentration of ethanol in the incubations (0 4%) did not affect corticosterotdogenesis The final incubation volume (90% cell suspension, 10%, a solution containing one of the agents) was 300 ~tl Incubations were carried out at 40 C m a metabolic shaking water bath for 2 hr Routinely, 8793% of the cells were viable after a 2-hr incubation as indicated by trypan blue dye exclusion (24) Incubations were then frozen until radioimmunoassay for cortmosterone and cAMP R a d i o l m m u n o a s s a y f o r c o r t i c o s t e r o n e a n d cAMP. Corticosterone, the predominant corticosteroid released by the adult avian adrenal gland (25), was measured by a modification of the radloimmunoassay procedure of Roy, Jr et al (26) The modifications were the direct radlolmmunoassay of appropriately diluted ahquots of the cell incubations and the use of a commercially available, specific antibody (ICN ImmunoBlologlcals, Lisle, IL) Incubation medium was used to dilute cell incubations prior to radiolmmunoassay As httle as 0 1 ng cortlcosterone/ml cell Incubation could be detected Crossreactlvittes with two corucosteroids of the steroldogemc pathway leading to cortlCOsterone, progesterone and 11-deoxycortmosterone, were 17% and 27%, respectively In each radiolmmunoassay, ahquots of pooled cell incubations from other experzments were included as quality control references Radlotmmunoassay of these references showed tntraassay and interassay coefficients of variation of 3 8 and 5 3%, respectively Cellular cAMP production was measured by a highly sensitive and specific, succmylated cAMP radloimmunoassay (ICN ImmunoBtologmals) As little as 1 pmole cAMP/ml cell incubation could be detected Quality control references showed lntraassay and lnterassay coefficients of variation of 4 1 and 8 7%, respectively A n a l y s i s o f d a t a . The data of corticosterone and cAMP production by isolated adrenocorttcal cells were statistically analyzed using a four-parameter logistic equation model that is available as a computer program (ALLFIT, Biomedical Computing Technology Information Center, Vanderbilt Medical Center, Nashville, TN) This program allowed for the simultaneous analysis of a family of dose-response curves (27) Values for half-maximal steroldogenic concentration of an agent (EDso) and values for maximal production were calculated and statistically analyzed using this program In a statistical analysis run, parameters to be compared were set equal to one another This decreased the number of parameters in the analysis, but increased the extra sum of squares
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of residuals due to the constraint An F ratio test (contained within the program) of the constrained fit to the unconstrained fit was performed Values hypothesized to be equal were deem e d significantly different w hen P < 0 05 The average within- and between- exper i m ent coefficmnts of variation of cell mcubauons (calculated from basal corticosterone values after 2 hr of incubation) were 5 7 and 9 4%, respectively Other cellular response data were stausucally analyzed by analysis of variance Means were separated by the Newman-Keuls test and were deem e d significantly different w hen P < 0 05 (28) RESULTS
Corticosterone production induced by ACTH analogues. Figure 1 shows corttcosterone production by isolated turkey adrenocorucal cells in response to various concentrations of ACTH analogues ACTH analogues stimulated cortlcosterone production over about two log orders of concent rauon Human analogues were 48% more efficacious than avian analogues for sumulatlng cortlcosterone p r o d u c u o n [859 8 _ 72 6 and 580 8 _ 51 3 ng cortmosterone/ ml, respectively (means _ SEM), pool ed data of maximal ACTH-mduced corticosterone production by male and female cells] In addition to differences in efficacy, analogues exhibited different potencies as indicated by the different half-maximal steroidogemc concentrations (EDso values) (Table 1) Human ACTH-(1-24) was the most potent analogue, whereas tuACTH-(1-39) was one of the least potent analogues, the average pot ency of hACTH-(1-24) was 11 times that of tuACTH-(1-39) A stausucal l y significant --//, FEMALE 120C
o h A C T H - ( 1 - 24) z~ h A C T H - ( 1 - 3 9 ) v o s A C T H - (1 - 39}
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Fig 1 ACTH a n a l o g u e - m d u c e d cortlcosterone p r o d u c t i o n by isolated turkey adrenocortlcal cells Cells (2 X 10 5 c e l l s / m l ) w e r e incubated w i t h various concentrations of analogues for 2 hr. Each s y m b o l r e p r e s e n t s the m e a n o f corticosterone values from nine cell incubations (3 cell mcuDatlons from each o f 3 e x p e r i m e n t s ) SEM are not s h o w n
TURKEY ADRENOCORTICAL CELLS
125
TABLE 1 I-IALF-MAXtMAL STeROtDOGm~aCCONCENTgATION(EDs0) OF STERO1DOGENICAGENTFOR CORTICOSTERONE PRODUCTIONBY ISOIATm) ~
ADmmOCOgnO.L Ct~LLS
Source of Cells ACTH analogues hACTH-(1-24) hACTH-(1-39) osACTH-(1-39) tuACTH-(1-39) 8Br-cAMP 2 5-hydroxycholesterol
Male
Female
(2 10 -+ 0 08) X 10i'M (1 68 + 0 07) X 10t°M
(2 49 + 0 08) X 101~M '
(3 51 (1 20 _+ 0 06) X 1 0 ' ° i (1 99 (2 08 -+ 0 12) X 10t°M (3 18 (2 43 -+ 0 21) X 104M (2 42 (4 1 0 (3 76 + 0 2 0 ) X 10"6M Note- Values are the means _+ SEMS calculated from the data shown in ALLFIT computer program
-+ 0 + 0 + 0 _+ 0
13) X 10tOM" 10) × 10'°M ' 16) X 10'°M' 13) X 104M - 0 26) x 10"6M Figures 3 and 4 by the
*Values significantly different from corresponding values for male cells (P < 0 05)
(P<0 05) order of potency was as follows hACTH-(1-24) > osACTH-(1-39) >[hACTH-(1-39) ---- tuACTH-(1-39)] cAMP p r o d u c t i o n i n d u c e d by ACTH a n a l o g u e s . Fig 2 shows cAMP production by isolated turkey adrenocortlcal cells in response to various concentrations of ACTH analogues The pattern of analogue efficacy and potency for cAMP production was somewhat different than that for cortlcosterone producuon Human ACTH-(I-24) was the most efficacious analogue, however, the efficacy of hACTH-(I-39) was not different from that of the avian analogues Concentrations of ACTH analogue required to half-maximally stimulate cAMP production were about 400 times those required to half-maximally stimulate corttcosterone production (cf Tables 1 and 2) Nevertheless, some aspects of the analogue potency data for cAMP production were consistent with those for corttcosterone production Here again, human ACTH-(I-24) was the most 900 800 70£
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126
KOCSIS AND CARSIA
CYCLIC
TABLE 2 HALF-MAXIMALSTEROIDOGENICCONCENTRATION(EDso) OF ACTH ANALOGUEFOR AMP PRODUCTIONBY ISOLATEDTURKEYADRENOCORTICALCELLS Source of Cells Male Female ACTH analogues hACTH-(1-24) (4 61 -+ 0 82) X 109M (1 42 + 0 28) X lOaM. hACTH-(1-39) (2 67 -+ 0 39) X 10aM (4 48 _+ 0 67) X 10aM. osACTH-(1-39) (2 88 _+ 0 42) X 10aM (5 O1 _+ 0 86) X 10aM• tuACTH-(1-39) (3 97 -+ 0 41) X 10aM (1 07 -+ 0 21) X 107M' Note. Values are the means + SEMScalculated from the data shown m Figure 2 by the ALLFIT computer program "Values significantly different from corresponding values for male cells (P < 0 05) p o t e n t a n a l o g u e , w h e r e a s t u A C T H - ( I - 3 9 ) w a s t h e least p o t e n t , w i t h t h e p o t e n c y of hACTH-(I-24) b e i n g 9 times that of tuACTH-(I-39) However, the order of a n a l o g u e p o t e n c y for cAMP p r o d u c t i o n w a s d i f f e r e n t f r o m t h a t for c o r u c o s t e r o n e p r o d u c t i o n h A C T H - ( I - 2 4 ) > [hACTH-(I-39) ---- o s A C T H - ( I - 3 9 ) ] > tuACTH-(I39)
Corticosterone p r o d u c t i o n i n d u c e d by 8-Br-cAMP and supported by 25-hydroxycholesterol. Figures 3 and 4 show cellular c o r u c o s t e r o n e prod u c u o n s t i m u l a t e d b y 8-Br.cAMP a n d s u p p o r t e d b y 2 5 - h y d r o x y c h o l e s t e r o l m t h e a b s e n c e o f ACTH, r e s p e c t i v e l y T h e a v e r a g e m a x i m a l 8 - B r - c A M P - m d u c e d and 25-hydroxycholesterol-supported cortmosterone production values were, r e s p e c t i v e l y , 109 a n d 67% g r e a t e r t h a n t h e a v e r a g e m a x i m a l ACTH a n a l o g u e i n d u c e d c o r t l C O s t e r o n e p r o d u c u o n ( T a b l e 3) S e x d i f f e r e n c e s i n adrenocortical cell f u n c t i o n . D e s p i t e t h e I m m a t u r e p h y s l o l o g m a l status of the poults, there w e r e some clear sexual differences tn a d r e n o c o r t m a l c e l l r e s p o n s e to s t e r o l d o g e m c a g e n t s a n d to t h e p r e c u r s o r , 25h y d r o x y c h o l e s t e r o l Basal c o r u c o s t e r o n e p r o d u c u o n o f f e m a l e c e l l s w a s 19% --/f/L j
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127
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greater than that of male cells (Table 3) However, maximal ACTH analoguereduced corticosterone production of male and female cells was not different In contrast, maximal 8-Br.cAMP-mduced and 25-hydroxycholesterol-supported cortmosterone production values of female cells were, respectively, 42 and 31% less than those for male cells On the other hand, although basal cAMP production by male and female cells was not different, maximal ACTH analogueinduced cAMP production of female cells was 21% greater than that of male cells (Table 4) In addition to sex differences in cAMP and cortlcosterone production, there were also sex differences in ACTH analogue potency, or put another way, cellular sensitivity to ACTH analogues (Tables 1 and 2) Sex differences in cellular sensitivity to ACTH analogues varied with the ACTH analogue but were stguificant (P < 0 05) for corticosterone and cAMP production, the range of sensitivity of male cells to ACTH analogues was, respectively, 1 2-2 1 times and 1.6-3 2 times that of female cells TABLZ 3 BASAL"AND MAXIMALb CORTICOSTERO
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PRODUCTION BY ISOLATED~
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CELLS Additions to Source of Cells Cell Incubations Male Female Diluent 537 ± 24 6 3 8 :t: 13" ACTH analogues 788 8 ± 69 4 729 4 ± 54 3 8-Br-cAMP 2021 7 ± 9 4 4 11776 ± 348' 25-hydroxycholesterol 1513 3 ± 41 8 1040 2 ± 27 5" Note:. Values (ng/ml) are the means ± SEMS calculated from the data shown in Figures 1,3 and 4 by the ALLFIT computer program •Basal corticosterone production after 2-hr incubation with agent diluent bMaximal corticoster?ne production after 2-hr incubation with ACTH analogues (pooled), or with 8-Br-cAMi', or with 25-hydroxycholesterol "Values significantly different from corresponding values for male cells (P < 0 05)
128
KOCSIS AND CAFISIA
TABLE4 BASAL"ANDMAXIMALb CYCHC A M P PRODUCTION BY ISOLATED TURKL~ ADRENOCORTICAL CELLS Additions to Source of Cells Cell Incubations Male Female Diluent 24 _+ 0 1 24 _+ 0 2 ACTH analo~ues 538 3 -+ 26 4 653 0 _+ 42 3' Note. Values (pmoles/ml) are the means _+ SEMScalculated from the data shown in Figures 2 •Basal cAMP production after 2-hr incubation with ACTH diluent bMaxlmal cAMP production after 2-hr incubation with ACTH analogues (pooled) "Value significantly different from corresponding value for male cells (P < 0 05) DISCUSSION Both m a m m a l i a n and avian ACTH a n a l o g u e s s t i m u l a t e d c o r u c o s t e r o n e and cAMP p r o d u c t i o n b y isolated t u r k e y a d r e n o c o r u c a l cells (Figures 1 and 2) S o m e w h a t p u z z l i n g is the o b s e r v a t i o n that tuACTH-(1-39) was the least p o t e n t a n a l o g u e (Tables 1 and 2) H o w e v e r , similar results w e r e o b t a i n e d w i t h isolated c h i c k e n a d r e n o c o r t m a l cells (5) F u r t h e r m o r e , o u r results of differences m p o t e n c i e s b e t w e e n avian and m a m m a l i a n ACTH a n a l o g u e s are consistent w i t h the results o f w o r k w i t h isolated rabbit and rat fat cells ( 2 3 ) , albeit differences in p o t e n c i e s w o u l d b e e x p e c t e d w i t h m a m m a l i a n cells It s h o u l d b e p o i n t e d that the o b s e r v e d differences in p o t e n c i e s b e t w e e n avian and m a m m a l i a n ACTH a n a l o g u e s in the p r e s e n t s t u d y and in o t h e r w o r k ( 5 , 2 3 ) w e r e not d u e to differences in specific activities b e t w e e n analogues, b e c a u s e the analogues u s e d w e r e synthetic, highly purified p e p u d e s that w e r e identical to the res p e c u v e natural p e p t l d e s as a d j u d g e d b y HPLC ( 2 3 ) In a d d i t i o n to the l o w p o t e n c y o f tuACTH-(1-39), b o t h tuACTH-(I-39) and osACTH-(I-39) w e r e the least efficacious for s t i m u l a t i n g c o r t l c o s t e r o n e prod u c u o n (Figure 1 and data u n d e r RESULTS) The data suggest that, wittx avian a d r e n o c o r t m a l cells, d o m a i n s w i t h i n tuACTH-(I-39) act to m o d u l a t e steroldogenesis, that is, b o t h s t i m u l a t e a n d d a m p e n s t e r o i d o g e n e s i s Presumably, the " d a m p e n i n g " d o m a i n ( s ) resides b e y o n d a m i n o acid 24, since hACTH-(I-24) had the greatest efficacy and p o t e n c y for s t i m u l a t i n g c o r t m o s t e r o n e and cAMP p r o d u c t i o n b y t u r k e y a d r e n o c o r t m a l cells (Figures 1 and 2, Tables 1 and 2), a n d c o r t m o s t e r o n e p r o d u c t i o n b y c h i c k e n a d r e n o c o r t m a l cells (5) Indeed, m o s t o f the n o n c o n s e r v a u v e a m i n o acid variation b e t w e e n the p e p u d e s o c c u r s after a m i n o acid 24 ( 2 3 ) Perhaps the p o s t u l a t e d d a m p e n i n g effect o f the native p e p u d e serves to p r e v e n t d e l e t e r i o u s o v e r s t i m u l a t l o n of the avian adr e n o c o r t l c a l cell d u r i n g p e r s i s t e n t stress in vtvo The cAMP analogue, 8-Br-cAMP, a s t e r o i d o g e m c a g e n t that bypasses p l a s m a m e m b r a n e events in c o r t l c o s t e r o l d o g e n e s i s , s t i m u l a t e d c o r t l c o s t e r o n e p r o d u c tion in a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r (Figure 3) In addition, the readily c o n v e r t i b l e s t e r o i d o g e m c p r e c u r s o r , 2 5 - h y d r o x y c h o l e s t e r o l ( 2 9 ) i n c r e a s e d cort t c o s t e r o n e p r o d u c t i o n m the a b s e n c e o f ACTH (Figure 4) H o w e v e r , the average maxamal cortlCOsterone p r o d u c t i o n s t i m u l a t e d b y 8-Br-cAMP and s u p p o r t e d b y 2 5 - h y d r o x y c h o l e s t e r o l was, respectively, 109 and 67% greater than that stimu l a t e d b y ACTH a n a l o g u e s (Table 3) T h e data suggest that the i n t r a c e l l u l a r c o n c e n t r a t i o n s o f p r o t e i n kinase A - d e p e n d e n t factors a n d s t e r o l d o g e n i c e n z y m e s e x c e e d t h o s e w h i c h are a c c e s s i b l e to ACTH-acuvated c e l l u l a r p r o c e s s e s Similar results w i t h 25-hydroxych01esterol (5,9,10,13) and the p r e c u r s o r p r e g n e n o l o n e ( 1 , 8 , 1 1 , 1 2 ) have b e e n o b t a i n e d f r o m e x p e r i m e n t s w i t h c h i c k e n ( 1 , 5 , 8 - 1 2 ) and J a p a n e s e quail ( 1 3 ) a d r e n o c o r t m a l cells H o w e v e r , the o b s e r v a t i o n that the average m a x i m a l c o r t m o s t e r o n e p r o d u c t i o n i n d u c e d b y 8.Br-cAMP was 109% g r e a t e r t h a n that i n d u c e d b y ACTH a n a l o g u e s m i g h t b e a u n i q u e feature
TURKEY ADRENOCORTICAL CELLS
129
of turkey adrenocortmal cells In experiments with chicken and Japanese quail adrenocortical cells, the magnitude of maximal cortlcosterone production stimulated by ACTH and 8-Br-cAMP was not different (1,5,8-13) Sex steroids can influence adrenocortlcal function in the domestic fowl (9,30) However, their involvement in turkey adrenocortical function is unknown In the present study, presumably plasma sex steroid levels were very low in the sexually immature, 9-week-old poults Thus, we were perplexed to find clear sex differences in adrenocortlcal cell function On an equal cell concentration basis, maximal 8-Br-cAMP-induced and 25-hydroxycholesterolsupported cormcosterone production of male cells were, respectively, 72 and 45% greater than those of female cells, albeit, maximal ACTH analogue-induced corticosterone production of male and female cells was not different (Table 3) The data suggest a greater intracellular concentration of protein kanase Adependent factors and steroldogenic enzymes of male cells compared to female cells In contrast, maximal ACTH analogue-induced cAMP production of female cells was 21% greater than that of male cells (Table 4) Possibly, this observation indicates a compensatory mechanism of female cells However, the physiological significance of these differences is unclear since presumably the lntracellular concentrations of cAMP-dependent factors and steroidogenic enzymes exceed those that are accessible to ACTH-actwated cellular processes Even more puzzling than the aforementioned observations are the differences in sensitivity to ACTH analogues between male and female cells, as indicated by the different analogue EDso values (Tables 1 and 2) Depending on the analogue used and the parameter measured (i e , corucosterone or cAMP production), sensitivity of male cells to ACTH was 1 2-3 2 times that of female cells This sex difference in cellular sensitivity to ACTH appears to be due to a difference in steroidogenlc steps prior to the formation of cAMP, possibly including the ACTH receptor, since 8-Br-cAMP and 25-hydroxycholesterol EDs0 values for corticosterone production were not different between male and female cells (Table 1) Obviously, recent techniques of characterizing domesuc fowl adrenocortmal cell ACTH receptors (7) need to be applied to turkey adrenocortmal cells to test this postulate. Data presented here indicate that the turkey adrenal gland is amenable for the preparation of isolated adrenocortical cells that are responsive to ACTH and steroidogemc agents which bypass plasma membrane events (e g , 8-BrcAMP), and readily convert steroidogenic precursors (e g , 25-hydroxycholesterol) to the predominant end product, corticosterone In addition, recently we have demonstrated that both turkey and domestic fowl adrenocortical cells are responsive to prostaglandms (31) Thus, isolated turkey adrenocortlcal cells expand the in vitro repertoire for understanding the regulation of avian adrenocortlcal function in terms of both basic cellular mechanisms and comparative adrenocortical function ACKNOWLEDGMENTS AND FOOTNOTES This work was funded by USDA grant 85-CRCR-l-1846 We thank Helen Weber and Leslie A Schwarz for expert technical assistance Please address all correspondence to Rocco V Carsla, Ph D , Department of Anatomy, University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, 675 Hoes Lane, Piscataway, NJ 08854-5635
130
KOCSIS AND CARSIA REFERENCES
1 Carsia RV, Scanes CG, Malamed S Isolated adrenocortmal cells of the domesuc fowl (Gallus domesttcus) Steroidogemc and ultrastructural properties J Steroid Blochem 22 273-279, 1985 2 Rosenberg J, Pines M, Hurwitz S Relationship between endogenous cyclic AMP production and steroid hormone secretion in chick adrenal cells Comp Blochem Physlol 84B 71-75, 1986 3 Carsia RV, Scanes CG, Malamed S Polyhormonal regulation of avian and mammalian cortmosteroidogenesis in vitro Comp Blochem Physiol 88A 131-140, 1987 4 Rosenberg J, Pines M, Hurwitz S Response of adrenal cells to parathyroid hormone stimulatxon J Endocrmol 112 431-437, 1987 5 Carsia RV, Weber H, Lauteno, TJ Protein malnutrition in the domestic fowl induces alterations in adrenocortical function Endocrinology 122 673-680, 1988 6 Rosenberg J, Panes M, Hurwltz S Stimulation of chick adrenal steroldogenesis by avian parathyroid hormone J Endocrmol 116 91-95, 1988 7 Carsla RV, Weber H Protein malnutrition in the domestic fowl induces alterations in adrenocortmal cell adrenocorticotropm receptors Endocrinology 122 681-688, 1988 8 Carsla RV, Weber H, King DB, Scanes CG Adrenocortmal cell function m the hypophysectomized domestic fowl Effects of growth hormone and 3,5,3'-trllodothyronme replacement Endocrinology 117 928-933, 1985 9 Carsla RV, Reisch NM, Fennell MJ, Weber H Adrenocortical function of the domestic fowl Effects of orchlectomy and androgen replacement Proc Soc Exp Blol Med 185 223-232, 1987 10 Carsia RV, Morro ME, Rosen HD, Weber H Ontogemc corticosteroidogenesis of the domestic fowl Response of isolated adrenocortical cells Proc Soc Exp Blol Med 184 436-445, 1987 11 Carsia RV, Scanes CG, Malamed S Loss of sensitivity to ACTH of adrenocortical cells isolated from maturing domestic fowl Proc Soc Exp Blol Med 179 279-282, 1986 12 Carsia RV, Weber H Genetm-dependent alterations in adrenal stress response and adrenocortical cell function of the domestic fowl (Gallus domesticus) Proc Soc Exp Blol Med 183 99-105, 1986 13 Carsia RV, Weber H, Satterlee DG Steroidogenlc properties of isolated adrenocortical cells from Japanese quail selected for high serum cortlcosterone response to immobilization Domestic Animal Endocrmol (in press) 14 Brown KI Enwronmentally imposed stress In Enwronmental Control m Poultry Production, Carter TC (ed) Oliver and Boyd, Edmburg and London, p101-107, 1967 15 Brown KI, Nestor KE Some physiological responses of turkeys selected for high and low adrenal response to cold stress Poultry Sci 52 1948-1954, 1973 16 E1 Halawanl ME, Waibel PE, Appel JR, Good AL Effects of temperature stress on catecholammes and corticosterone of male turkeys J Physiol 224 384-388, 1973 17 Brown KI, Nestor KE Implications of selection for high and low adrenal response to stress Poultry Sci 53 1297-1306, 1974 18 S~mensen E, Olson LD, Ryan MP Determination of cortmosterone concentrauons in plasma of turkeys usmg radioimmunoassay Poultry Sci 57 1701-1704, 1978 19 McCorkle FM, Edens FW, Simmons DG Adrenal secretory involvement in Alcahgenes faecahs mfectton m turkey poults Poultry Sci 61 1510, 1982 (abstract) 20 Daws GS, Slopes TD Adrenal cortical response of tom poults Poultry Sci 64 21892194, 1985 21 Davis GS, Slopes TD Plasma cortmosterone response of turkeys to adrenocortlCOtropic hormone Age, dose and route of administration effects Poultry Sci 66 17271732, 1987 22 Davis GS, Slopes TD The ontogeny of diurnal rhythmicity in plasma corticosterone of poults and the influence of this rhythm on the plasma cortlcosterone (B) response to ACTH or cold Poultry Sci 66 (Suppl 1) 89, 1987 (abstract) 23 Yamashlro D, Ho CL, Ll CH Adrenocortmotropm 57 Synthesis and biological activity of ostrich and turkey hormones Int J Pepude Protein Res 23 42-46, 1984
TURKEY ADRENOCORTICAL CELLS 24 25 26 27 28 29 30 31
131
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