- Email: [email protected]
Effect of testosterone on renal and hepatic L -gulonolactonase activities in male, female and pseudohermaphroditic rats
Lüe Sciences Vol . 10, Part II, pp. 175-180, 1971 . Printed in Great Britain
Pergamon Press
EFFECT OF TFSTOSTERONE ON RENAL AND HEPATIC IrGUIONOIACTONASE ACTIVITI~ IN MALE
FBMALE
AND PSEUDOHERMAPiIIaDDITIC RATS l Steven H. Groasman 2~ Bernard Axelrod~
C. Wayne Hardie
Department of Biochemistry Purdue University Lafayette~ Indiana 47907 and Department of Medicine Pennsylvania State University The Milton S. Hershey Medical Centers Hershey P~enneylvania 17033
(Received 23 October 1970; in final form 24 December 1970) L-GUIANOLACTONASE (3 .1.1 .19) catalyzes the reversible lactonization of Ir qulonic acid (1) .
The enzyme has been purified from rat liver and its role in
Irascorbic acid biosynthesis established (2) .
Irc~lonolactonase also exists is
the rat kidneys where its function is not clear.
It has been observed that
kidneys from adult males exhibit approximately ten times as much activity as kidneys from females (3) .
This difference in activity can be dimittiahed~ but
not eliminated, by administration of testosterone to female rate
(4) .
Stanley and C~mbreok have described a hereditable form of peeudohermarphroditism in the rat.
Affected animals are characterised by inguinal testes
a female phenotype and a male genotype (5) .
The recent observation that aev~
eral tissues of these animals were resiatam to physiologic doses of teetosterone suggested that the lack of masculine differentiation was due to an end-organ insensitivity to androgens (6) .
Since Irqulonolactonase provides
an index of androgen action on the kidney it was pertinent to examine th
1Journal Paper No . 4220 Purdue University Lafayette, Indiana 47907 .
Agricultural 8xparimant Station
2Predoctoral trainees PHS Training Grant No . GM 1195 General Medical Science.
175
National Institute for
178
L-gulonolacto~ae Activity
Vol. 10, No . 3
effect of testosterone on the activity of this enzyme in the pseudohermaphroditic rat.
7Y~is is the subject of the presem report . Materials a~ Methods
o-(~ulonolactone ~s purchased from Sigma Chemical OompanY"
IrC,tiloao
lectors vas prepared by the redaction of sodium ~glucuronate with sodium borohydride.
The psocaduro followed vas similar to that described for the
praparatioa of L-galactonolaotone from ~galaoturonic acid (7)~ except that 2matho~grethanol was omitted and the product vas recryetallized from absolute ethanol. Moraal and pseudoharnaphroditic rate
(250-300 gm) rare obtained from the
University of Ok1aAo~on Medical Centers Departments of Anatomy and Physiology. Testosterone ~anthata (0.06-60 .0 n4) rae administered daily for 10 days in 0.1 or 0.3 ml of sesame oil.
Animals rare decapitated
rare stored in dry ice for 48 hours.
a~ the kidneys and livers
Prior to assay tissues rare thared and
homgenized in five times their reight in rater (4 C) using a 40 ml capacity ground-glass mrtar and a rotor-driven ground-glass pestle
(Pyrex
No . 7727) .
Aliquots rare imwediately assayed. Assays were performed titrimetrically at 28 C~ pH 6.4~ using a Type TTTlb Radiometer (Copenhagen)
and Type SHRc Titrigraph (Copenhagen) .
wee equipped with a abtor driven syringe~
The instrument
Type SHOla (Oopenhagen), which de
livered s+~+~.:_+~ base as a function of the hydrolysis of D-qulonolactone . usual assay mixture contained 8.0 ml oP 11 .2 umoles/1m1 D-qulonolactone~ of 0.1 N MnCl~ and 0.2 ml of saturated NaCl .
The
1.0 ml
The pH vas adjusted quickly to
6.4~ and noa-enzymatic hydrolysis was recorded for 3 minutes.
When D-giilono-
lectors was the substrates 0.O1 ml of liver homogenate or 0.5 ml of kidneyy homogenate was addeds and the rate of enzymatic plus norrenzymatic hydrolysis recorded for 5 minutes.
A similar procedure was followed when irqulonolactone
vas the substrates except that L5 ml of kidney homogenate was used for the assay.
All activities are in umolee/min/g fresh reight of tissue.
L-gulonolactoimee Activity
Vol . 10, No . 3
177
Results Renal irqulonolactonase has a broad substrate specificity catalysing the hydrolysis of D and L-galactonolactone and ~ and irgulonolactone (8) .
The
enzyme hydro]yzes D-qulono]actone about six time faster than Irqulonolactoae~ and hence D-gulonolactone was used in the routine assay.
In order to be sure
that we were measuring irgulonolactonaae activity when using ~gulonolactoae as a substrates we compared certain preparations with both D and L-qulonolactone as substrates .
The remits are shown in Table L
The similarity in
ratios indicates the validity of measuring irgulonolactoaase activity by D~ quloaolactone hydrolysis . TAHLE 1 Ratio of ~gulonolactonase to L-qulorwlnctonase Activities of Males Females and Pseudohe=aaphroditic Rat Renal Preparations Sex
Testosterone Bnanthate* mg/day
pseudo**
none 60. 0
pseudo** female fe®nle
female male
~qulonolactone/ L-qulonolactone 6.00
none 0. 06
6.10 5,77 6. 30
none
5.59
60. 0
6. 72
*Given for 10 days **Male pseudohermnphrodite Renal i.-gulonolactonase activities in control and testosterone treated rats are given in Fiq. 1.
L-qulonolncronase from nozual male kidneys was ten
times as active as that of normal females.
In the kidneys of pseudoherma-
phroditic males* however* this enzyme activity ms essemially the sam as that in females.
Testosterone enanthate treatmem (0.6 mg/day) of female rats en-
hanced renal Ir-gulonolactonase approximately 5 fold and higher àoses produced no further increment.
A similar increase of activity in pseudohermaphroditic
Vol. 10, No. 3
L -gulonolactonase Activity
178
rate was achieved when a 100 fold greater dose of androgen was given (60 This large dose of testosterone enamhate resulted in renal Irqulorn-
ag/day) .
lactonase activities in fmoale and pseudohermaphroditic rats which were respectively only 38 and 29t of that of normal males. The results of similar studies on rat liver are given in Fiq. 2.
In the
rat, L-gulonolactonase activity is much higher in the liver than in the kidney but does not vary between the sexes or during testosterone treatment .
~ s.o ~ â ~o ô ~ s.o
81ALE
W
~ ~ 6.0 ~ 4.0 ! ~ 3"o ~ t.0
FE8IALE P8E000HER~AAPHRODITE . r~
J
o ~os as s so 0 os s so 0 TE8T08TERONE ENANTHATE 81ß/DAY Fig. 1 Bffect of testosterone enanthate given daily for 10 days on renal L-qulonolactonaae activities in mnle~ female and pseudohermaphroditic rats . Brackets indicate range of values .
PSEUDOHER8IAPHRODITE
o.os as s so
TESTOSTERONE
81ALE
0 0~ s so 0 81ß/DAY ENANTHATE
Fiq. 2 Sffect of testosterone ananthate given daily for 10 days on hepatic Irqulonolaotonase activities in mnle~ female and pseudoharmaphroditic rats . Brackets indicate range of values .
Yol . 10, No. 3
L-gulonolactonaee Acüvity
179
Discussion Previous studies of the Stanley-Gumbreck male peeudohermnphroditic rat have demonstrated that genitalia
livery preputial glands and hypothalemis are
insensitive to testosterone administered at 2-3 times the replaceoent dose for a castrated male (6~9) .
Therefore the effect o! testosterone enamhate on
androgen inducible renal irgulonolactonase activity in the present study vas of considerable interest .
Even though for a given Boas of an~~ogen there vas a
greater increase in enzyme activity in the female it vas significant that there vas a detectable effect of this androgen in ps~dohermnphroditic rats as well. These observations indicate these animals have a relative rather than an absolute resistance to testosterone .
Similar etudieé
of the preputial gland of
the pseudohermaphroditic rat indicate that these organs will grow in response to 100 folB higher level of testosterone tkan is required in the normal rat (10) . Evidence from several laboratories suggest that the action of testosterone in the cell may be mediated by dihydxotestosterone.
Pbilovinq testosterorons-3H
administration dihydrotestosterone- 3A is retained by prostatic nuclei long alter tritium is cleared from the blrood (11) .
The specificity of the nuclei for di-
hydroteatoaterone suggests that this steroid might ba the intranuclear effeotor of testosterone action .
The ability of dihydrotestoaterons rather than testo-
sterone to enhance cytidine incorporation into "nucleic acid" of isolated pa~ostatic nuclei supports this interpretation (12) .
We have recently proposed
that the androgen insensitivity in pseudohermaphroditic rate is due to the inability of nuclei to retain dihydrotestosterone (13) .
The present stud sug~
gents this postulate could be substantiated if nuclear 8lhydroteatosterone retention could be increased by administration of large Bows of testosterone . Acknowledgements The authors viah to thank Drs. J. E. Allison L G. Gumbreck and A. J. Stanley.
IInivnraity of Oklahoma Medical Center for supplying the rata .
180
L-gulonolactonaee Activity
vol. 10, No . s
References 1.
C.
BDHLITZ and A, L . LâHNINGBN Biochim. Biophys . Acta 47, 288 (1961) .
2.
C.
BUHLITZ and A. L .
3.
R HJGEL and H. A7~LROD~ unpublished observations .
4.
I, L. A2JiA1C~ 8ffect of Androgens on the Renal Gulonolactonase of the Rat, M . S . Thesis Purdué University (1962j .
5.
A. J. STANLB}( and L. G. Q1M~2HCIC~ (1964) .
6.
C. w, Hi4RDIIi~ L. HOLI4CIC and G. SCEIIiBIDER~ Science 167, 1136 (1970) .
7,
N. L. FRUSH and H. S. ISHFT"T " in Methods in Carbohydrate Chemistry, 8" 127 R. L. whistler and M . L, wolfram (e~)i Academic Presa~ New York (1962) .
H.
S . B. GROSSMAti and H, AXBLROD~ unpublished observations .
9.
T . VANB1hPERTTUT,A~ C, w, HARDIN~ J . E, ALUSON L. G . (T8lHRHC1C and A. J. STANLEY~ &idocrinology 89, 611 (1970) .
~a Biochim. eiophys . Acta 32, 290 (1959) .
ras of the Pt~docrine Society, p . 40
10.
R, J. SFH:RII~ô and C. w, HARDIN~ Pr (1970) .
1L
N. BRUCf~VSlO! and J . D. 1PIhSON~ J . Hiol. Chem. 243, 2012 (1968) .
12 .
N. g6S srrstur .aur~ G . J. CI~DBR, and C. A . VILLSFy Biochem , Biophy , Res, ( .bam. 37, 976 (1969) .
13 .
L, BULLOCK and C . w, HilRDINr J, Clin.
ras of the Phdocrine Society, p . 50
Bt~docr. 31, 113 (1970) .
Pergamon Press
EFFECT OF TFSTOSTERONE ON RENAL AND HEPATIC IrGUIONOIACTONASE ACTIVITI~ IN MALE
FBMALE
AND PSEUDOHERMAPiIIaDDITIC RATS l Steven H. Groasman 2~ Bernard Axelrod~
C. Wayne Hardie
Department of Biochemistry Purdue University Lafayette~ Indiana 47907 and Department of Medicine Pennsylvania State University The Milton S. Hershey Medical Centers Hershey P~enneylvania 17033
(Received 23 October 1970; in final form 24 December 1970) L-GUIANOLACTONASE (3 .1.1 .19) catalyzes the reversible lactonization of Ir qulonic acid (1) .
The enzyme has been purified from rat liver and its role in
Irascorbic acid biosynthesis established (2) .
Irc~lonolactonase also exists is
the rat kidneys where its function is not clear.
It has been observed that
kidneys from adult males exhibit approximately ten times as much activity as kidneys from females (3) .
This difference in activity can be dimittiahed~ but
not eliminated, by administration of testosterone to female rate
(4) .
Stanley and C~mbreok have described a hereditable form of peeudohermarphroditism in the rat.
Affected animals are characterised by inguinal testes
a female phenotype and a male genotype (5) .
The recent observation that aev~
eral tissues of these animals were resiatam to physiologic doses of teetosterone suggested that the lack of masculine differentiation was due to an end-organ insensitivity to androgens (6) .
Since Irqulonolactonase provides
an index of androgen action on the kidney it was pertinent to examine th
1Journal Paper No . 4220 Purdue University Lafayette, Indiana 47907 .
Agricultural 8xparimant Station
2Predoctoral trainees PHS Training Grant No . GM 1195 General Medical Science.
175
National Institute for
178
L-gulonolacto~ae Activity
Vol. 10, No . 3
effect of testosterone on the activity of this enzyme in the pseudohermaphroditic rat.
7Y~is is the subject of the presem report . Materials a~ Methods
o-(~ulonolactone ~s purchased from Sigma Chemical OompanY"
IrC,tiloao
lectors vas prepared by the redaction of sodium ~glucuronate with sodium borohydride.
The psocaduro followed vas similar to that described for the
praparatioa of L-galactonolaotone from ~galaoturonic acid (7)~ except that 2matho~grethanol was omitted and the product vas recryetallized from absolute ethanol. Moraal and pseudoharnaphroditic rate
(250-300 gm) rare obtained from the
University of Ok1aAo~on Medical Centers Departments of Anatomy and Physiology. Testosterone ~anthata (0.06-60 .0 n4) rae administered daily for 10 days in 0.1 or 0.3 ml of sesame oil.
Animals rare decapitated
rare stored in dry ice for 48 hours.
a~ the kidneys and livers
Prior to assay tissues rare thared and
homgenized in five times their reight in rater (4 C) using a 40 ml capacity ground-glass mrtar and a rotor-driven ground-glass pestle
(Pyrex
No . 7727) .
Aliquots rare imwediately assayed. Assays were performed titrimetrically at 28 C~ pH 6.4~ using a Type TTTlb Radiometer (Copenhagen)
and Type SHRc Titrigraph (Copenhagen) .
wee equipped with a abtor driven syringe~
The instrument
Type SHOla (Oopenhagen), which de
livered s+~+~.:_+~ base as a function of the hydrolysis of D-qulonolactone . usual assay mixture contained 8.0 ml oP 11 .2 umoles/1m1 D-qulonolactone~ of 0.1 N MnCl~ and 0.2 ml of saturated NaCl .
The
1.0 ml
The pH vas adjusted quickly to
6.4~ and noa-enzymatic hydrolysis was recorded for 3 minutes.
When D-giilono-
lectors was the substrates 0.O1 ml of liver homogenate or 0.5 ml of kidneyy homogenate was addeds and the rate of enzymatic plus norrenzymatic hydrolysis recorded for 5 minutes.
A similar procedure was followed when irqulonolactone
vas the substrates except that L5 ml of kidney homogenate was used for the assay.
All activities are in umolee/min/g fresh reight of tissue.
L-gulonolactoimee Activity
Vol . 10, No . 3
177
Results Renal irqulonolactonase has a broad substrate specificity catalysing the hydrolysis of D and L-galactonolactone and ~ and irgulonolactone (8) .
The
enzyme hydro]yzes D-qulono]actone about six time faster than Irqulonolactoae~ and hence D-gulonolactone was used in the routine assay.
In order to be sure
that we were measuring irgulonolactonaae activity when using ~gulonolactoae as a substrates we compared certain preparations with both D and L-qulonolactone as substrates .
The remits are shown in Table L
The similarity in
ratios indicates the validity of measuring irgulonolactoaase activity by D~ quloaolactone hydrolysis . TAHLE 1 Ratio of ~gulonolactonase to L-qulorwlnctonase Activities of Males Females and Pseudohe=aaphroditic Rat Renal Preparations Sex
Testosterone Bnanthate* mg/day
pseudo**
none 60. 0
pseudo** female fe®nle
female male
~qulonolactone/ L-qulonolactone 6.00
none 0. 06
6.10 5,77 6. 30
none
5.59
60. 0
6. 72
*Given for 10 days **Male pseudohermnphrodite Renal i.-gulonolactonase activities in control and testosterone treated rats are given in Fiq. 1.
L-qulonolncronase from nozual male kidneys was ten
times as active as that of normal females.
In the kidneys of pseudoherma-
phroditic males* however* this enzyme activity ms essemially the sam as that in females.
Testosterone enanthate treatmem (0.6 mg/day) of female rats en-
hanced renal Ir-gulonolactonase approximately 5 fold and higher àoses produced no further increment.
A similar increase of activity in pseudohermaphroditic
Vol. 10, No. 3
L -gulonolactonase Activity
178
rate was achieved when a 100 fold greater dose of androgen was given (60 This large dose of testosterone enamhate resulted in renal Irqulorn-
ag/day) .
lactonase activities in fmoale and pseudohermaphroditic rats which were respectively only 38 and 29t of that of normal males. The results of similar studies on rat liver are given in Fiq. 2.
In the
rat, L-gulonolactonase activity is much higher in the liver than in the kidney but does not vary between the sexes or during testosterone treatment .
~ s.o ~ â ~o ô ~ s.o
81ALE
W
~ ~ 6.0 ~ 4.0 ! ~ 3"o ~ t.0
FE8IALE P8E000HER~AAPHRODITE . r~
J
o ~os as s so 0 os s so 0 TE8T08TERONE ENANTHATE 81ß/DAY Fig. 1 Bffect of testosterone enanthate given daily for 10 days on renal L-qulonolactonaae activities in mnle~ female and pseudohermaphroditic rats . Brackets indicate range of values .
PSEUDOHER8IAPHRODITE
o.os as s so
TESTOSTERONE
81ALE
0 0~ s so 0 81ß/DAY ENANTHATE
Fiq. 2 Sffect of testosterone ananthate given daily for 10 days on hepatic Irqulonolaotonase activities in mnle~ female and pseudoharmaphroditic rats . Brackets indicate range of values .
Yol . 10, No. 3
L-gulonolactonaee Acüvity
179
Discussion Previous studies of the Stanley-Gumbreck male peeudohermnphroditic rat have demonstrated that genitalia
livery preputial glands and hypothalemis are
insensitive to testosterone administered at 2-3 times the replaceoent dose for a castrated male (6~9) .
Therefore the effect o! testosterone enamhate on
androgen inducible renal irgulonolactonase activity in the present study vas of considerable interest .
Even though for a given Boas of an~~ogen there vas a
greater increase in enzyme activity in the female it vas significant that there vas a detectable effect of this androgen in ps~dohermnphroditic rats as well. These observations indicate these animals have a relative rather than an absolute resistance to testosterone .
Similar etudieé
of the preputial gland of
the pseudohermaphroditic rat indicate that these organs will grow in response to 100 folB higher level of testosterone tkan is required in the normal rat (10) . Evidence from several laboratories suggest that the action of testosterone in the cell may be mediated by dihydxotestosterone.
Pbilovinq testosterorons-3H
administration dihydrotestosterone- 3A is retained by prostatic nuclei long alter tritium is cleared from the blrood (11) .
The specificity of the nuclei for di-
hydroteatoaterone suggests that this steroid might ba the intranuclear effeotor of testosterone action .
The ability of dihydrotestoaterons rather than testo-
sterone to enhance cytidine incorporation into "nucleic acid" of isolated pa~ostatic nuclei supports this interpretation (12) .
We have recently proposed
that the androgen insensitivity in pseudohermaphroditic rate is due to the inability of nuclei to retain dihydrotestosterone (13) .
The present stud sug~
gents this postulate could be substantiated if nuclear 8lhydroteatosterone retention could be increased by administration of large Bows of testosterone . Acknowledgements The authors viah to thank Drs. J. E. Allison L G. Gumbreck and A. J. Stanley.
IInivnraity of Oklahoma Medical Center for supplying the rata .
180
L-gulonolactonaee Activity
vol. 10, No . s
References 1.
C.
BDHLITZ and A, L . LâHNINGBN Biochim. Biophys . Acta 47, 288 (1961) .
2.
C.
BUHLITZ and A. L .
3.
R HJGEL and H. A7~LROD~ unpublished observations .
4.
I, L. A2JiA1C~ 8ffect of Androgens on the Renal Gulonolactonase of the Rat, M . S . Thesis Purdué University (1962j .
5.
A. J. STANLB}( and L. G. Q1M~2HCIC~ (1964) .
6.
C. w, Hi4RDIIi~ L. HOLI4CIC and G. SCEIIiBIDER~ Science 167, 1136 (1970) .
7,
N. L. FRUSH and H. S. ISHFT"T " in Methods in Carbohydrate Chemistry, 8" 127 R. L. whistler and M . L, wolfram (e~)i Academic Presa~ New York (1962) .
H.
S . B. GROSSMAti and H, AXBLROD~ unpublished observations .
9.
T . VANB1hPERTTUT,A~ C, w, HARDIN~ J . E, ALUSON L. G . (T8lHRHC1C and A. J. STANLEY~ &idocrinology 89, 611 (1970) .
~a Biochim. eiophys . Acta 32, 290 (1959) .
ras of the Pt~docrine Society, p . 40
10.
R, J. SFH:RII~ô and C. w, HARDIN~ Pr (1970) .
1L
N. BRUCf~VSlO! and J . D. 1PIhSON~ J . Hiol. Chem. 243, 2012 (1968) .
12 .
N. g6S srrstur .aur~ G . J. CI~DBR, and C. A . VILLSFy Biochem , Biophy , Res, ( .bam. 37, 976 (1969) .
13 .
L, BULLOCK and C . w, HilRDINr J, Clin.
ras of the Phdocrine Society, p . 50
Bt~docr. 31, 113 (1970) .