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ANALOGS OF GROWTH HORMONE-RELEASING HORMONE INDUCE
RELEASE OF GROWTH HORMONE IN THE BOVINE 1'2
R. S c a r b o r o u g h , J. G u l y a s 3, A. V. Schally 3 a n d J. J. Reeves 4
Washington State University 5
Pullman 99164-6332
ABSTRACT
Biological potencies of three 29 amino acid growth hormone-releasing hormone analogs
(GHRH[1-29]) were determined in the bovine and compared to synthetic human GHRH (44 amino
acids; hGHRH[1-44]NH 2) for their ability to increase serum growth hormone (GH)concentrations. Four prepubertal Holstein heifers (179 • 10 kg) received hGHRH(1-44)NH 2 or analogs
(D-Ala 2 , Nle 27, Agrn 29 GHRH[1-29], [JG-73]; D-N-MeAIa 2, Nle 27, A g l n 29 GHRH[1-29], [JG-75];
and desamino-Tyr I , D-AIa2 , Nle aT, Agm 29 GHRH[1-29], [JG-77]) at the following doses: O, 6.25,
25, 100 and 400 #g/animal. All treatment-dose combinations were administered to each heifer with
at least a 1-d interval between treatments. Sixteen blood samples were collected via jugular cannulas
20 min before and up to 6 h after treatment injection. There was a linear dose-dependent GH
release in response to hGHRH(1-44)NH~ and the three analogs. Growth hormone peak amplitudes
for the three analogs were similar to those observed after administration of the hGHRH(1-44)NH 2
(P > .05). However, when total area under the GH response curves for each treatment was averaged
over all the doses, JG-73 stimulated greater GH release than hGHRH(1-44)NH 2 (P < .05). Heifers
injected with the 401Y#g dose of hGHRH(1-44)NH 2 or the three analogs showed a primary release
of GH followed by a secondary release 1 h later. At all other doses, only a primary GH release was
observed. In conclusion, JG-73, a GHRH analog, was found to be 4.65 times more potent on a
weight basis and 3.3 times more potent on a molar basis, whereas JG-75 and JG-77 were as potent
as synthetic hGHRH(1-44)NH 2 in stimulating the release of GH in heifers.
(Key Words: Growth Hormone Releasing Hormone Analogs.)
Introduction
Manipulation of serum growth hormone
(GH) c o n c e n t r a t i o n s to e n h a n c e p r o d u c t i o n
traits in d o m e s t i c livestock has r e c e n t l y received
c o n s i d e r a b l e a t t e n t i o n . E x o g e n o u s p i t u i t a r y or
r e c o m b i n a n t G H has b e e n used in t h e b o v i n e t o
i m p r o v e N r e t e n t i o n (Moseley et al., 1982),
t Scientific paper no. 7847. College of Agric. and
Home Econ. Res. Center, Washington State Univ.,
Pullman 99164. This research was supported by W. R.
Grace & Co., Columbia, MD.
2Appreciation is expressed to the National Hormone and Pituitary Program, Baltimore, MD for
supplying oGH antibody and to R. G. Eggert, American
Cyanamid Co., for the recombinant bGH used for
iodination and standards.
3 Endocrine, Polypeptide and Cancer Inst., Veterans
Admin. Medical Center, Tulane Univ. School of Medicine, New Orleans, LA 70146.
4Reprint requests: Dr. J. J. Reeves, Dept. Anita.
Sci., Washington State Univ., Pullman 99164-6332.
s Dept. of Anim. Sci.
Received September 18, 1987.
Accepted January 15, 1988.
w e i g h t gains (Grings et al., 1 9 8 7 a ) a n d m i l k
p r o d u c t i o n ( B a u m a n et al., 1985). A viable
a l t e r n a t i v e to t h e a d m i n i s t r a t i o n o f G H is t o
increase e n d o g e n o u s release o f G H b y adm i n i s t e r i n g t h e n a t u r a l 4 4 a m i n o acid g r o w t h
h o r m o n e - r e l e a s i n g h o r m o n e ( G H R H [ 1-44] NH2 )
or its analogs ( P l o u z e k a n d T r e n k l e , 1 9 8 6 ;
Moseley et al., 1986).
N a t u r a l G H R H a n d its s y n t h e t i c replicates
h a v e b e e n s h o w n to s t i m u l a t e t h e release o f G H
in t h e b o v i n e ( P l o u z e k et al., 1 9 8 3 ; M o s e l e y et
al., 1984). Full biological a c t i v i t y was s h o w n to
b e p r e s e n t in a 29 a m i n o acid f r a g m e n t ( G H R H [1-29]; Ling et al., 1 9 8 4 ; H o d a t e et al., 1986).
A f u r t h e r r e m o v a l of C - t e r m i n a l a m i n o acids
r e s u l t e d in a m a r k e d d e c r e a s e in GH-releasing
activity (Ling et al., 1 9 8 4 ; H o d a t e et al., 1986).
M o d i f i c a t i o n s a n d s u b s t i t u t i o n s w i t h Di s o m e r a m i n o acids in G H R H ( 1 - 2 9 ) have b e e n
made that alter the duration and potency of the
GH-releasing activity ( B a r r o n et al., 1985;
H e i m a n et al., 1 9 8 5 ; P l o u z e k a n d Trenkle,
1986). However, little is k n o w n a b o u t t h e
GH-releasing p o t e n c i e s
of these peptides
c o m p a r e d w i t h n a t u r a l G H R H ( 1 - 4 4 ) N H 2 in
1386
J. Anim. Sci. 1988. 6 6 : 1 3 8 6 - 1 3 9 2
GROWTH HORMONE, RELEASING HORMONE, ANALOGS
domestic animals. The objective of this study
was to evaluate the biological potencies of three
new analogs (D-AIa2, Nle 27, Agm 29 GHRH~
[1-29], [JG-73]; D-N-MeAla 2, Nle 27, Agm 29
GHRH[1-29], [JG-75]; and desamino-Tyr 1,
D-Ala z, Nle 27, Agm 29 GHRH[1-29], [JG-77])
and to compare them to hGHRH(1-44)NH2 for
the ability to increase serum concentrations of
GH in the bovine.
Materials and Methods
Four prepubertal Holstein heifers (179 + 10
kg) were maintained indoors in individual pens
and were exposed to a 15-h light:9-h d a r k
photoperiod. Animals were accustomed to
being handled prior to the study to decrease
stress at sampling times. A complete pelleted
diet was fed once daily at 1500. At least 12 h
before treatments, heifers were fitted with
indwelling jugular cannulas. The analogs JG-73,
JG-75 and JG-77 and synthetic hGHRH(1-44)NH2 were dissolved in 1 ml physiological
saline for all doses and stored at - 7 0 ~
The analogs and hGHRH(1-44)NH2 were
synthesized by solid-phase methods and repurified by high performance liquid chromatography. Amino acid substitutions and modifications in the hGHRH analogs were as follows.
Methionine 27 was replaced with norleucine
(Nle), and arginine 29 was decarboxylated to
give agmatine (Agm) in all three analogs. The
D-isomer of alanine replaced L-alanine 2 in
JG-73 and JG-77. Analog JG-77 lacked the
amino group of the Tyr 1 residue (desaminoTyr 1). A methyl group was attached to the N
of the D-isomer of alanine 2 (D-N-MeAIa2) in
JG-75.
Heifers received i.v. injections of hGHRH(1-44)NH2 or analogs at the foUowing doses: 0,
6.25, 25, 100 and 400 /Jg/animal. All treatment-dose combinations were administered to
each heifer with at least a 1-d interval between
treatments. Sampling began at 0800 on each
treatment day. Btood samples were collected
via jugular cannulas at - 2 0 , - 1 0 , 0 (injection
time), 5, 10, 15, 20, 25, 30, 45, 60, 120, 180,
240, 300 and 360 min. All blood samples were
allowed to clot at 4~ for 24 h and were
centrifuged. Serum was decanted and stored at
180-A, American Cyanamid Co., Wayne, NJ.
STylan 200, Elanco Products, Div. of Eli Lilly and
Co., Indianapolis, IN.
1387
-20~
for subsequent determination of GH
concentrations by a heterologous double
antibody radioimmunoassay (Grings et al.,
1988) using recombinant bovine GH 6 as the
radioiodinated antigen and standard.
Mean GH peak amplitude and area under the
GH response curve for 6 h after GHRH treatment were determined for each heifer at each
treatment-dose combination. Growth hormone
peak amplitude was reported as the maximum
concentration of GH achieved between 5 and
60 min postinjection. The area under the GH
response curves, which is a measure of the total
amount of GH released over time minus metabolic clearance rate, was estimated using
trapezoidal summation for the entire 6-h period
(Thomas, 1953).
Each trait was analyzed as a completely
random design (Steel and Torrie, 1980) with
heifer, day, close and treatment as class variables
and the residual used as the error term. The
interactions between treatments and doses also
were analyzed. Comparisons among treatment
means and dose means were made by Duncan's
multiple range test (Steel and Torrie, 1980).
Orthogonal contrasts were used to test for
differences in slopes of the best fit lines of the
analogs compared to the hGHRH(1-44)NH2.
Treatment variance was subdivided into linear,
quadratic and cubic components using the F
test to determine significant responses. Potencies
of analogs based on total area under the GH
response curves were calculated according to a
four-point assay method as described by
Pugsley (1946).
Throughout the study, heifer #3 displayed
raised body temperature (40~
and was
treated with an antibiotic 7 i.m. It was evident
from the serum GH data that either therapeutic
administration of antibiotic, the illness, or a
combination of both interfered with GH
release. Therefore, all data collected from this
heifer were excluded from statistical analyses.
Results
A rapid increase in plasma GH was elicited in
response to hGHRH(1-44)NH2 and the analogs,
but not to saline. There was a linear dosedependent response in GH release as measured
by the area under the GH response curve for all
treatments (Figure 1). Quadratic and cubic
effects were not significant. The slopes of the
best fit lines were not different when each
analog was compared to hGHRH(1-44)NH2 (P
1388
SCARBOROUGH ET AL.
O
IJLi
JG-73
8000
>
JG-75
O'E
:3
tu=>,
az m
~
JO
GHRH 1 - 4 4
4000'
JG-77
I1:
,<
O,
A
|
H
I
SALINE 6.25
2'5
1(~0
400
DOSE GHRH OR GHRH-ANALOG (I.Ig)
Figure 1. Dose response curves to growth hormone-releasing hormone (GHRH) (1-44) NH 2 (r = .95, o--o),
JG-73 (r = .92, o--o), JG-75 (r = .97, o - - o ) and JG-77 injection (r = .97, m--m) on area under GH response
curves for 6 h postinjection. Best fit lines were determined by linear regression. The doses are on a log scale, and
each data point represents the mean of three observations. The standard errors for the least square means were
319, 389, 1,564 and 1,578 for the doses of 6.25, 25, 100 and 400/ag, respectively.
> .05). Correlation coefficients of the best fit
lines ranged from .92 to .97. No evidence for a
diminished response at the highest dose utilized
was seen when using area under the curve.
No differences (P > .05) were found among
the three analogs and hGHRH(1-44)NH2 when
total area under the GH response curves was
compared at the doses of 6.25, 100 and 400 pg.
The analog JG-77 was the only analog to
stimulate greater total area under the GH
response curves (P < .05) than hGHRH(1-44)NH2 at the 25-pg dose. When treatments
were averaged over the doses of 6.25, 25, 100
and 400 pg, total area under the GH response
curves was greater for JG-73 than hGHRH(1-44)NH2 (P < .05). The analog JG-73 was
calculated to stimulate 4.65 times greater total
area under the GH response curves compared
with hGHRH(1-44)NH2. No significant day
effect or treatment-dose interaction was found
( P > .10).
Greater total area under the GH response
curves stimulated by 400 pg of hGHRH(1-44)-
NH2 and the analogs was the result of both a
primary GH release occurring 5 to 25 min after
treatment injection and a secondary release
between 60 to 180 min after treatment injection. At the lower doses, only a primary GH
release was observed (Figure 2).
Comparison of GH peak amplitudes at 6.25,
100 and 400 pg indicated no differences (P >
.05) between the three analogs and hGHRH(1-44)NH2 (Figure 3). At the 25-pg dose, JG-77
stimulated greater (P < .05) GH peak amplitudes than hGHRH(1-44)NH2. When GH peak
amplitudes for each treatment were averaged
over all four doses, no differences (P > .05)
were found between the three analogs and
hGHRH(1-44)NH2. No day effect or treatmentdose interaction was found for GH peak amplitudes (P > .05).
Discussion
This study demonstrated that the three
GHRH analogs tested and hGHRH(1-44)NH2
GROWTH HORMONE, RELEASING HORMONE, ANALOGS
1389
.0 i 'o
40
20-
g
-r
6
'~
4.
6
TIME
6
2
~
(HOURS)
Figure 2. Mean serum growth hormone (GH) profiles of heifers (n = 3) at 6.25 ( o - - o ) , 25 ( o - - o ) , 100
( o - - e ) and 400 ( i - - a ) within each growth hormone-releasing hormone (GHRH) treatment after i.v. injection
of JG-73, JG-75, JG-77 and synthetic human GHRH (hGHRH [1-441NH 2) at time 0.
stimulated
a release of GH in heifers.
Considerable animal variation was noted. Large
variation in individual responsiveness to G H R H
also has been observed in rats (Wehrenberg et
al., 1982), chickens (Leung and Taylor, 1983),
h u m a n s ( T h o r n e r et al., 1983), pigs (Kraft et
al., 1985), sheep (Kensinger et al., 1987) and
cows (McCutcheon et al., 1984; Enright et al.,
1986). The cause of this variation is n o t clear,
but a relationship between the different re9 sponses and the a m o u n t of somatostatin in the
peripheral circulation at the time of G H R H
t r e a t m e n t has been postulated to be involved
(Kraft et al., 1985; Enright et al., 1986; Kensinger et al., 1987).
A s the doses of h G H R H ( 1 - 4 4 ) N H 2 or
analogs were increased, the total area u n d e r the
GH response curves was enlarged. However,
a m p l i t u d e o f the GH peak after t r e a t m e n t did
n o t increase in a d o s e - d e p e n d e n t manner. These
results suggest that with doses o f 100 and 400
,.., 100~
75-
O
~)
50-
25"
O
~;
0
6.25
25
100
40(
DOSE (pg)
Figure 3. Mean growth hormone (GH) peak amplitudes (maximum concentration of GH achieved
between 5 and 60 min postinjection) for JG-73 (white
bars), JG-75 (black bars), JG-77 (striped bars) and
synthetic human growth hormone-releasing hormone
(hGHRH [1-441NH 2) (shaded bars)9 *P < .05 compared with hGHRH at that dose. There were three
observations at each dose level.
1390
SCARBOROUGH ET AL.
/ag, the GH peak amplitude may have been near
maximal, but the duration of the stimulation
increased as measured by total area under the
GH response curves. The greater total area
under the GH response curves stimulated by
400/ag hGHRH(1-44)NHz and the analogs was
the result of a biphasic release of GH. Biphasic
types of response to GHRH have been reported
previously in humans (Vance et al., 1984) and
cows (Moseley et al., 1984; Petitclerc et al.,
1985; A1-Raheem et al., 1986).
In vitro studies with rat pituitary fragments
have demonstrated that GH stored in the rat
pituitary can be divided into at least two
functional compartments (Stachura and Tyler,
1986, 1987). These two compartments were
described as a relatively small pool of previously
synthesized hormone that responds quickly to
GHRH but is rapidly exhaustible, and a larger
pool that responds more slowly and continuously to long-term stimulation. The biphasic
response of GH measured in the serum after
administration of the high dose of hGHRH or
analogs therefore may represent mobilization
and release of stored GH from these two
compartments.
The results of this study indicate that an
analog (JG-73) with D-AIa 2, Nle 27 and Agm 29
substitutions in hGHRH(1-29) exhibited a
significant increase in potency compared with
hGHRH(1-44)NH2. Other studies using analogs
substituted with D-Ala s in position 2 of
hGHRH(1-29) also have demonstrated significant increases in potency (Barron et al., 1985;
Hodate et al., 1986; Karashima et al., 1987).
The increased GH-releasing potency of JG-73 in
the heifers is probably linked with increased
affinity of the receptor to this peptide. Data
that lend support to this suggestion were
collected by Seifert et al. (1985) from a radioreceptor assay using rat anterior pituitary cell
primary cell cultures. In their study, the labeled
GHRH analogs had higher relative binding
affinities than human pancreatic growth hormone-releasing factor (hpGRF[1-40]). Due to
the differences in molecular weight of the
GHRH analogs and the native hormone, the
potency estimates on a weight basis, as done in
this study, would overestimate the potency
calculated on a molar basis by approximately
30%.
Another possibility for the increased GHreleasing activity of JG-73 may be greater
biological stability of the analog. Rate of
degradation of this peptide likely is reduced
due to the D-isomer substitution. The decarboxylation of arginine 29 to produce agmatine
also may play a role in conferring resistance to
proteolytic degradation from the C-terminus. Similar results of increased biological
activity of analogs substituted with D-isomer
amino acids have been obtained using several
other peptides. The substitutions of D-amino
acids in LHRH, enkephalin and glucagon
analogs were suggested to have stabilized the
B-bend conformation centered around the
changed residue, thus increasing biological
activity (Monahan et al., 1973; Coy et al.,
1976; Sueiras-Diaz et al., 1984). Substitutions
in JG-73 may act to stabilize the conformation
of GHRH(1-29) in a similar manner.
The analogs used in this study may possibly
have greater potency when administered s.c., as
was recently demonstrated by Karashima et al.
(1987) using a similar GHRH analog. Differences
between i.v. and s.c. potencies of GHRH
analogs also have been described by Rafferty et
al. (1985) and for an analog of somatostatin by
Bauer et al. (1982). The analogs may be more
resistant to degradation in s.c. tissue than in
blood (Karashima et al., 1987). Further studies
are being conducted in our laboratory with the
administration of the JG-73 GHRH analog in
the form of an s.c. injection to determine its
efficacy in increasing milk production.
Because JG-73 was 4.65 times more potent
on a weight basis and 3.3 times more potent on
a molar basis, and JG-75 and JG-77 appeared as
p o t e n t as hGHRH(1-44)NH2, all three offer
potential applications in the induction of GH
secretion in domestic livestock for promotion
of growth and lactation.
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