Download Influence of Synthetic Somatostatin upon Growth Hormone Release

Influence of Synthetic Somatostatin upon Growth Hormone
Release from Perifused Rat Pituitaries
MAX E. STACHURA
Division of Endocrinology and Metabolism, Michael Reese Hospital and Medical Center, Chicago,
Illinois 60616; and the Pritzker School of Medicine, University of Chicago, Chicago, Illinois
60637
ABSTRACT. Basal release of pre-labeled, stored tritiated rat growth hormone ([3H]rGH) from perifused
rat pituitary explants is a constant fraction of
pituitary [3H]rGH content.
Synthetic somatostatin (SRIF) inhibits the release
of pre-labeled, stored [3H]rGH in a dose-dependent
fashion. Prolonged exposure to SRIF results in an immediate and continuous inhibition of [3H]rGH release. In the in vitro perifusion system, the maximal
inhibition, which is achieved with 25 nM SRIF,
results in a rate of [3H]rGH release which is 30 to 40
per cent of basal release.
Pulses of SRIF produce inhibition of [3H]rGH release followed by rebound release after withdrawal
of SRIF. When the time of exposure to SRIF is
held constant, both the SRIF-induced inhibition and
the rebound release of [3H]rGH are dose-dependent.
A similar progressive response is seen when a
constant SRIF concentration is pulsed for variable
periods of time. There is no net inhibition of [3H]rGH
release by pulses of SRIF. (Endocrinology 99: 678,
1976)
OMATOSTATIN (SRIF) is a tetrade- pituitary glands were removed, cut into 1 mm3
capeptide of hypothalamic origin fragments, and incubated in a metabolic shaker
which is capable of inhibiting the in vivo for 3 h at 37 C, in 2 ml of Krebs-Ringer bicaror in vitro secretion of immunoreactive bonate (KRB) containing glucose (1.5 mg/ml),
serum albumin (10 mg/ml), and 20 fxCi
growth hormone (GH). Both the natural (cy- bovine
[3H]leucine (SA 31.7 Ci/mmol, New England Nuclized) and the synthetic linear forms are
clear) under a 95% O -5% CO2 atmosphere,
active (1-5). A transient period of increased at pH 7.2-7.3. Under 2 these conditions,
GH
GH release following the withdrawal of synthesis has been shown to be linear for 8 h
SRIF has also been noted in vivo and in vitro (14). The prelabeled explants were then washed,
following SRIF alone or after SRIF paired weighed, and transferred to a perifusion system
with stimulators of GH release (6-12).
(15) through which KRB without radioactive
This report describes investigations of the amino acids was perifused at a rate of 0.2
SRIF-induced inhibition and rebound re- ml/min, during which no further synthesis of
lease of pre-labeled [3H]rGH in an in vitro [3H]rGH occurs (14). After an equilibration periperifusion system. The studies were under- fusion period of 90 min during which the release
taken in an attempt to examine the role of of prelabeled rGH could be expected to attain
SRIF as a physiologic regulator of growth a constant rate (15), experimental manipulations
hormone release. The perifusion system was were begun. Effluent fractions were collected at
min intervals, and perifusion buffer reservoirs
selected because it permits analysis of the 5could
be changed instantaneously without
time course of a response and because manipulation of the tissue explant or alteration of
small responses are not obscured by the bulk the flow rate (15).
of ongoing basal release (13).
Somatostatin (SRIF) was added to the peri-
S
Materials and Methods
Pituitary perifusion
Male Holtzman rats weighing from 200-300 g
were sacrificed by decapitation. The anterior
Received December 29, 1975.
fusion buffer in several concentrations and for
variable periods of time, as described in Results. At the end of the experiment, perifused
pituitary explants were homogenized at 4 C in
1.0 ml O.OIN NaOH with a 1.0 ml saline wash,
neutralized with I N HC1, and immediately centrifuged at 12,000 x g for 10 min at 4 C. The
supernatant solution and the perifusion fractions
were stored at - 2 0 C.
678
679
SRIF-INHIBITED rGH RELEASE
FIG. 1. Basal release of immunoprecipitable rGH prelabeled
with [3H]leucine by perifused
pituitary explants. Release is expressed as [3H]rGH DPM per
collection interval •*• pituitary
[3H]rGH DPM at the onset of the
collection interval x 100. The
rate of release is initially rapid,
due to explant manipulation during transfer to the perifusion
chamber, and stabilizes by 60
min. Once equilibrated, the release rate is not altered by the
mechanics of changing the perifusion medium (arrows). Subsequent figures omit the stabilization period.
Growth hormone
T T
immunoprecipitation
Tritium incorporated in rGH was measured
by a. double-antibody immunoprecipitation technique, using rGH specific antiserum from a
rhesus monkey immunized with iodination grade
rGH (16,17). This antiserum exhibited sufficient
capacity to bind all of the rGH present in the
aliquot of pituitary extract or perifusion medium
with which it was reacted. The rGH-monkey
antibody complex was precipitated with excess
goat anti-monkey gamma globulin. Normal monkey serum substituted for monkey rGH antiserum
in a parallel immunoprecipitation for each fraction. The radioactivity in the washed precipitates
was counted by liquid scintillation using an
external standard-based quench correction.
Calculation of [3H]rGH release
Total [3H]rGH is the sum of the total [3H]rGH
released into the perifusion buffer during the
SRIF (lOnM)
O.2-
0.1FIG. 2. Inhibition of release of
prelabeled [3H]rGH from perifused pituitary by continuous
SRIF. The rate of [3H]rGH release is immediately inhibited
by SRIF. Within 20 min, stable
inhibition is achieved by each
concentration of SRIF (10, 25,
and 50 nM), and the rate of
[3H]rGH release remains constant during continuous exposure to SRIF. Maximal inhibition is produced by 25 nM SRIF.
T
I
SRIF
0.2-
(25nM)
S 0.1-
SRIF ( 5 0 n M )
O.2"
O.I-
90
150
210
MINUTES
STACHURA
680
Endo • 1976
Vol 99 • No 3
FIG. 3. Release of prelabeled
[3H]rGH from perifused pituitary pulsed with SRIF. [3H]rGH
release is immediately inhibited
by SRIF. Removal of SRIF from
the perifusion medium is followed by rebound release of
pre-labeled [3H]rGH.
I2O
I8O
MINUTES
experiment and the total [3H]rGH remaining
in the pituitary explant at the end of the experiment. The rate of [3H]rGH release is expressed
as a per cent of the [3H]rGH remaining in
the pituitary explant at the onset of the particular
collection interval, the latter calculated by subtracting [3H]rGH already released from total
[3H]rGH.
Results
3
Basal release of stored [ H]rGH
The pre-incubation of pooled rat adenohypophysial fragments (20 mg) with [ 3 Hlieucine established a homogeneous pool of
stored [3H]rGH (18). After equilibration in
the perifusion system, the rate of stored
[3H]rGH release was constant when expressed as a per cent of the [3H]rGH remaining in the pituitary explant at the onset of the
individual collection interval (first order
function) (15). This release rate was not altered
by the mechanics of changing the perifusion
medium (Fig. 1) and in different experiments ranged between 0.08 and 0.12 per
cent per min (15).
Effect of continuous SRIF upon release of
stored [3H]rGH
SRIF in final concentrations of 10, 25,
or 50 nM was added to the perifusion
medium for 120 min after the equilibration
of pre-labeled explants had occurred. The
addition of SRIF resulted in an immediate
decrease in the rate of stored [3H]rGH release (Fig. 2). The maximal release inhibition by each SRIF dose was achieved
within 20 min, and this new rate of [3H]rGH
release remained constant for the remainder
of the experiment. The SRIF inhibition of
stored [3H]rGH release was never complete.
FIG. 4. Dose responsive inhibition and rebound release of prelabeled [3H]rGH by 20 min
pulses of SRIF. The magnitude
of inhibition of [3H]rGH release
by SRIF, and of rebound release
after SRIF in a single experiment is compared. Displacement from basal release rate is
calculated by planimetry, the
inhibitory effect of 25 nM SRIF
being defined as —1.0.
min
min
O.25
2.5
25
SOMATOSTATIN CONCENTRATION (nM)
681
SRIF-INHIBITED rGH RELEASE
SRIF
SRIF
O.2-
i...
rw
6O
24O
ISO
ISO
3OO
MINUTES
FIG. 5. Inhibition and rebound release of prelabeled [3H]rGH by variable periods of exposure to 25 nM SRIF.
Inhibition of [3H]rGH release is seen during 10, 20, or 40 min of exposure to SRIF. The rebound
release of [3H]rGH is barely detectable after 10 min of SRIF, but increases progressively following 20
and 40 min of SRIF inhibition.
The maximally inhibited rate of [3H]rGH
release over several experiments averaged
30 to 40% of the basal release rate in the
presence of either 25 or 50 nM SRIF.
Effect of pulses of SRIF upon release of
stored [3H]rGH
total rebound release of [3H]rGH exceeded
the inhibition so that there was net release.
Rebound release which was equal to, or
slightly less than, the inhibition was observed in other experiments. The use of
50 nM SRIF did not increase the inhibition
or rebound stimulation of stored [3H]rGH
release beyond that seen with 25 nM SRIF.
Variable SRIF concentration-fixed exposure
time. Prelabeled, equilibrated rat pituitary Fixed SRIF concentration-variable exexplants pulsed with SRIF exhibited a char- posure time. The correlation between SRIF
acteristic biphasic response (Fig. 3). The inhibited release of stored [3H]rGH and the
exposure to SRIF again produced an im- rebound release after SRIF was further
mediate inhibition of stored [3H]rGH re- investigated by exposing perifused pituitary
lease. The removal of SRIF resulted in an explants to 25 nM SRIF for different time
immediate increase in the rate of [3H]rGH periods (Fig. 5). While exposure to 25 nM
release. The rate of release rose transiently SRIF for 10 min (or 5 min in other experiabove the previously determined basal rate ments) invariably produced inhibition of
and then returned to the basal rate. Both [3H]rGH release, clear rebound release was
the inhibition of stored [3H]iGH release by seen only irregularly. Longer exposure to
SRIF and the rebound release which fol- SRIF (Fig. 5; 20 and 40 min) resulted in
lowed the withdrawal of SRIF were dose- more pronounced inhibition of [3H]rGH
responsive. The maximal inhibition of re- release and was associated with clear release and the maximal rebound release were bound release after withdrawal of the inachieved with 25 nM SRIF. The results of hibitor. In this experiment, the rebound rea single experiment in which 20 min pulses lease after 20 min of SRIF totalled less than
of 0.25, 2.5, and 25 nM SRIF were applied the inhibition, while the rebound release
sequentially are summarized in Fig. 4. after 40 min of SRIF exceeded the inhibiThe total inhibition and rebound release tion.
(displacement from the basal release rate)
were calculated. Maximal inhibition (25 nM
Discussion
SRIF) was defined as -1.0, and the other
responses were expressed proportionately.
Rat adenohypophysial explants mainIn this experiment, for each SRIF dose the tained in an in vitro perifusion system re-
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Endo • 1976
Vol 99 • No 3
3
3
spond to stimulators and inhibitors of GH the release of [ H]rGH (3,6,7,9). The [ H]release (6,9,13,15). Because perifusion per- rGH release which continues in the presmits analysis of the time course of the re- ence of maximal SRIF inhibition (approxisponse to any stimulus, it offers at least mately one-third the basal release rate)
two advantages over static incubation sys- could represent only non-physiologic retems: a) the response to a given agent can be lease secondary to cell death and autolysis.
fingerprinted to show the latency of the ini- If such were the case, however, one might
tial and maximal effects, the persistence of not expect it to continue as a first-order
the effect after removal of the stimulus, and function.
any post-stimulatory phenomena, and b) a
Characteristically, explants exposed to 20
minimal response will not be lost within the min pulses of SRIF exhibited inhibition of
bulk of the ongoing basal release, while [3H]rGH release during SRIF exposure,
oscillating responses will not cancel.
and rebound release immediately after SRIF
As shown in Fig. 1, after preincubation in exposure (Fig. 3). Both the inhibition and the
the presence of [3H]leucine, the [3H]rGH re- rebound release of [3H]rGH were doselease in the perifusion system is atfirstrapid, responsive (Fig. 4). Although in this experiprobably due to explant manipulation dur- ment (Fig. 4) rebound release regularly
ing transfer, and, within an hour, achieves exceeded inhibition, over several experia stable rate of release. The basal release ments, SRIF-induced inhibition of [3H]rGH
of pre-labeled [3H]rGH is a constant frac- release was approximately equal to the intion of the total [3H]rGH in the pituitary creased release observed during the reexplant at the onset of the collection interval, bound phase. A correlation of the inhibition
suggesting a close relationship between hor- and rebound release of [3H]rGH was also
mone synthesis, storage, and release (14,15). seen when a single dose of SRIF was pulsed
Although any in vitro release of GH could for varying periods of time (Fig. 5).
be due either to cell death or physiologic
Protein secretion is thought to be an
mechanisms, the contribution of cell death is energy-dependent process (19), and these
minimized in this system since the cells data contribute additional evidence that the
releasing [3H]rGH in perifusion must have basal secretion of GH by pituitary explants
been sufficiently viable to synthesize and in vitro is not passive, but rather an active
store hormone during the preincubation process during which GH is randomly reperiod. Stable rGH release rates in peri- leased from a homogeneous storage pool
fusion are not observed using radioimmuno- (15,20-22). SRIF is capable of partially
assay, where the viability of all the cells blocking this release. The accumulation of
contributing to the release process cannot be releasable GH behind the block is not alassessed (18).
tered by SRIF, however, since removal of
When equilibrated, pre-labeled pituitary the SRIF inhibition is followed by a period
explants are exposed to continuous SRIF of increased release. This rebound, or catch(Fig. 2), there is an immediate inhibition up release, approximately equals the amount
of [3H]rGH release. Whichever SRIF dose of GH which would have been secreted
is used, a maximally inhibited release rate is if basal release had been allowed to proceed
established by 20 min, and this rate of [3H]- without interruption.
rGH release remains constant for the duration
After stalk section in the living animal,
of SRIF exposure. The degree of inhibition of plasma GH falls to undetectable levels.
release is related to the concentration of SRIF Therefore, it is difficult to suggest that SRIF
in the perifusate, with 50 nM SRIF produc- exerts tonic inhibition upon GH release
ing no more inhibition than does 25 nM (analogous to PIF-prolactin), producing
SRIF. It is of interest that no dose of spikes of increased GH release by dereSRIF tested was able totally to inhibit pression. According to the data presented,
SRIF-INHIBITED rGH RELEASE
while this mechanism alone could produce
the pulsatile GH release which is observed
in vivo, no net alteration in total GH release
would result.
8.
Acknowledgments
9.
The excellent technical assistance of Rosemary Fitzer
is acknowledged with appreciation. These studies
were supported by the Michael Reese Medical Research Institute and by a grant from the Human
Growth Foundation. Synthetic somatostatin was kindly
supplied by Dr. R. Reese of Wyeth Laboratories.
10.
11.
12.
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