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Somatostatin secretion from the rat neurohypophysis and stalk median eminence (SME) in vitro: Calcium-dependent release by high potassium and electrical stimulation
Somatostatin Secretion From the Rat Neurohypophysis and Stalk Median Eminence (SME) In Vitro: Calcium-Dependent Release by High Potassium and Electrical Stimulation Yogesh
W
ITHIN
C. Patel. H. H. Zingg, and J. J. Dreifuss
THE
CENTRAL NERVOUS SYSTEM, immunoreactive so(SRIF) has been localized to fibers in the external zone of the median eminence, neuronal cell bodies in the anterior hypothalamus, axons in the posterior pituitary lobe, and spinal ganglia and fibers in the spinal cord.’ This extensive brain distribution, the synaptosomal localization, and the effects of SRIF on behavior and on the spontaneous electrical activity of single neuron units in many parts of the brain suggest that the peptide may serve as a central neurotransmitter or a modulator of neuronal function.” Although these observations imply an important role for SRIF in brain function, little is currently known about the factors controlling the secretion of SRIF from nerve cells. Since the neurohypophysis and stalk median eminence (SME) region of the hypothalamus contain a high concentration of SRIF and can be readily isolated, we have used them as models for studying the release of the peptide from nerve terminals in vitro.” matostatin
MATERIALS
AND
METHODS
killed by decapitation. The SME (approximately 0.5 mg wet weight) was removed by gross dissection; the posterior pituitary lobes were isolated and separated from adhering intermediate lobe tissues. SMEs and neural lobes in pools of five were incubated in I ml bicarbonate-buffered Locke’s solution at 37” C under an atmosphere of 95% 09-5% CO, with constant shaking. SRIF was found to be stable for at least 60 min under these conditions with more than 90% recovery of synthetic SRIF from medium previously in contact with five neural lobes or SMEs for 30 min. Following a 30-min preincubation period, the medium was replaced by fresh Locke’s solution or a modified Locke’s solution at 30-min intervals. For electrical stimulation studies, neural lobes or SME fragments were placed between two silver-plate electrodes and current (40 mA, 20 Hz, 2-msec duration impulses) was passed through the medium for the first IO min of the 30-min incubation period. Media and acetic acid extracts of neural lobes or SME at the end of each experiment were assayed for SRIF by a specific radioimmunoassay.’ The protein content of the extracts was determined by the Male
Sprague-Dawley
rats were
method of Lowry et al.”
RESULTS AND DISCUSSION
A high concentration of immunoreactive SRIF was found in posterior pituitary extracts; the amount of 4.9 * 0.4 pg/pg protein (mean * SE, n = 20) was comparable to that of other SRIF-rich neural tissues (e.g., cerebral cortex, brainstem, spinal cord) but approximately 66 times lower than the SME concentration, 324 f From the Medical Research Centre. Prince Henry’s Hospital. Melbourne. Australia; and the Instilure of Histology and Embryology and the Department of Physiology. Geneva Medical School, Geneva. Switzerland. Supported by the NH and MRC of Australia and the Swiss NSF. Address reprint requests to Dr. Yogesh C. Parel. Medical Research Centre. Prince Henry’s Hospital. Melbourne, Australia. ~9 1978 by Grune & Stratton, Inc. 00260495/78/2713-0022$01.00/0 Merabolism, Vol. 27. No. 9, Suppl. 1 (September),
1978
1243
1244
PATEL,
Table 1. Stimulation
ZINGG.
AND
DREIFUSS
of SRIF Release From Neural Lobe and SME by High K+, low Na+ with and without Ca2+
lncubatlon
Condltaons’
Normal Locke’s with Low
Na+
Low
Na+.
Normal Low Low ‘Normal
with
Locke’s
and glucose
no Cat+
Na+.
high
10. The
K+ concentration priate Mn’+
Na+
was
(in mM):
concentration to 56
mM
When
Na+
1.0
32
1.6
28.8
*
9.6t
172.0
5.2
*
2.7
36.3
f
12.8
3.8
f
1 9
30.3
f
14.0
*
1.5$
f
9.ot
160;
K+) where
Ca*+
was
K+ 5.6; medium
indicated.
omitted
from
was
medium
Mg’+
lowered was (no
1 .O: Cl-
to 12 mM maintained
Ca’+),
7.3
f
5 4
zt 24.0’
34.5 Ca *+ 2.2;
lsoosmolarity the
3 f
mm)
f
of the incubation
(high
(pg/SME/30
f
2.6
contamed
mm)
9.6 57.0
K+. no Ca2+
of choline
mM)
Ca2+
lobe/30
6.4
Ca2+
solution
raised
amounts (2.2
with
Na+,
Locke’s
Ca2+
K+ with
SME SRIF Release
(pg/neural
Ca2+
high
Neural Lobe SRIF Release
160, (low
HCOJNa+)
by adding
an equivalent
12:
and the appro-
amount
of
added.
tp
<
0.01
compared
to incubation
in normal
$p
<
0.01
compared
to incubation
in medium
Locke’s
solution.
containing
high
K+ with
Ca2+
27 pg/pg protein (n = 16). The total content of SRIF per neural lobe was 743 =t 91 pg (n = 20) compared to 36 * 2.7 ng/SME (n = 16). As shown in Table I and Fig. I, SRIF was secreted at a low basal rate from both tissues; the amount released was 5.9-26 pg/neural lobe/30 min or 11.5-32.3 pg/SME/30 min, representing 0.8%-3.5% and 0.03%~~0.09% respectively, of total content. High extracellular potassium is a potent stimulus for neurohypophysial hormone secretion and this response can be potentiated by a lowering of the extracellular Na+ concentration.” The release of SRIF (Table I) from both tissues was also markedly increased following high-potassium (56 mM), low-sodium (12 mM) stimulation to 57 f 9.6 pg/neural lobe and 172 _t 24 pg/SME, representing 7.8% and OS%, respectively, of total content per 30 min. The stimulatory effect of high potassium and low sodium was abolished when Ca ‘+ in the medium was replaced by Mn2+, an antagonist of Ca’)+ in secretory processes (Table 1). In response to electrical stimulation (Fig. 1) there was a significant three-fold increase in SRIF release from neural lobes (10% of total content) and a six-fold increase from SME (0.2% of content). This response was also Ca’+ dependent and failed to occur in CaZ+-free medium. It is concluded that the mechanism of SRIF release from the posterior pituitary and SME shares two important characteristics with the neurosecretory process: stimulation of release in response to membrane depolarization, and dependence of
-s
s
B&SAL
s
s
Fig. 1. Effect of electrical stimulation(s) release from neural lobes or SMEs.
on SRIF
SECRETION
FROM RAT NEUROHYPOPHYSIS
AND
SME
this process on extracellular Ca2+. The fractional is much smaller than from the posterior pituitary. in the neurohypophysis, the bulk of SME SRIF The isolated SME and neurohypophysis should studies of SRIF secretion from nerves.
1245
release of SRIF from the SME This suggests that, unlike SRIF exists in a nonreleasable form. provide a useful tool for further
REFERENCES I. Hokfelt T, Effendic S, Hellerstriim C, et al: Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special reference to the A-l cells of the pancreatic islets and to the hypothalamus. Acta Endocrinol [Suppl] (Kbh)80:5541, 1975 2. Epelbaum J, Brazeau P, Tsang D, et al: Subcellular distribution of radioimmunoassayable somatostatin in rat brain. Brain Res 126:309314, 1977 3. Pate1 YC, Zingg HH, Dreifuss JJ: Calciumdependent somatostatin secretion from rat neurohypophysis in vitro. Nature 267:852-853, 1977
4. Pate1 YC, Rao K, Reichlin S; Somatostatin in human cerebrospinal fluid. N Engl J Med 296~524533, 1977
5. Lowry OH, Rosebrough NJ, Ferrell AL, et al: Protein measurement with Folinphenol reagent. J Biol Chem 193:2655271, 1951
6. Dreifuss JJ, Grau JD, Bianchi RE: Antagonism between Ca and Na ions at neurohypophyseal nerve terminals. Experientia 27:129551296, 1971
W
ITHIN
C. Patel. H. H. Zingg, and J. J. Dreifuss
THE
CENTRAL NERVOUS SYSTEM, immunoreactive so(SRIF) has been localized to fibers in the external zone of the median eminence, neuronal cell bodies in the anterior hypothalamus, axons in the posterior pituitary lobe, and spinal ganglia and fibers in the spinal cord.’ This extensive brain distribution, the synaptosomal localization, and the effects of SRIF on behavior and on the spontaneous electrical activity of single neuron units in many parts of the brain suggest that the peptide may serve as a central neurotransmitter or a modulator of neuronal function.” Although these observations imply an important role for SRIF in brain function, little is currently known about the factors controlling the secretion of SRIF from nerve cells. Since the neurohypophysis and stalk median eminence (SME) region of the hypothalamus contain a high concentration of SRIF and can be readily isolated, we have used them as models for studying the release of the peptide from nerve terminals in vitro.” matostatin
MATERIALS
AND
METHODS
killed by decapitation. The SME (approximately 0.5 mg wet weight) was removed by gross dissection; the posterior pituitary lobes were isolated and separated from adhering intermediate lobe tissues. SMEs and neural lobes in pools of five were incubated in I ml bicarbonate-buffered Locke’s solution at 37” C under an atmosphere of 95% 09-5% CO, with constant shaking. SRIF was found to be stable for at least 60 min under these conditions with more than 90% recovery of synthetic SRIF from medium previously in contact with five neural lobes or SMEs for 30 min. Following a 30-min preincubation period, the medium was replaced by fresh Locke’s solution or a modified Locke’s solution at 30-min intervals. For electrical stimulation studies, neural lobes or SME fragments were placed between two silver-plate electrodes and current (40 mA, 20 Hz, 2-msec duration impulses) was passed through the medium for the first IO min of the 30-min incubation period. Media and acetic acid extracts of neural lobes or SME at the end of each experiment were assayed for SRIF by a specific radioimmunoassay.’ The protein content of the extracts was determined by the Male
Sprague-Dawley
rats were
method of Lowry et al.”
RESULTS AND DISCUSSION
A high concentration of immunoreactive SRIF was found in posterior pituitary extracts; the amount of 4.9 * 0.4 pg/pg protein (mean * SE, n = 20) was comparable to that of other SRIF-rich neural tissues (e.g., cerebral cortex, brainstem, spinal cord) but approximately 66 times lower than the SME concentration, 324 f From the Medical Research Centre. Prince Henry’s Hospital. Melbourne. Australia; and the Instilure of Histology and Embryology and the Department of Physiology. Geneva Medical School, Geneva. Switzerland. Supported by the NH and MRC of Australia and the Swiss NSF. Address reprint requests to Dr. Yogesh C. Parel. Medical Research Centre. Prince Henry’s Hospital. Melbourne, Australia. ~9 1978 by Grune & Stratton, Inc. 00260495/78/2713-0022$01.00/0 Merabolism, Vol. 27. No. 9, Suppl. 1 (September),
1978
1243
1244
PATEL,
Table 1. Stimulation
ZINGG.
AND
DREIFUSS
of SRIF Release From Neural Lobe and SME by High K+, low Na+ with and without Ca2+
lncubatlon
Condltaons’
Normal Locke’s with Low
Na+
Low
Na+.
Normal Low Low ‘Normal
with
Locke’s
and glucose
no Cat+
Na+.
high
10. The
K+ concentration priate Mn’+
Na+
was
(in mM):
concentration to 56
mM
When
Na+
1.0
32
1.6
28.8
*
9.6t
172.0
5.2
*
2.7
36.3
f
12.8
3.8
f
1 9
30.3
f
14.0
*
1.5$
f
9.ot
160;
K+) where
Ca*+
was
K+ 5.6; medium
indicated.
omitted
from
was
medium
Mg’+
lowered was (no
1 .O: Cl-
to 12 mM maintained
Ca’+),
7.3
f
5 4
zt 24.0’
34.5 Ca *+ 2.2;
lsoosmolarity the
3 f
mm)
f
of the incubation
(high
(pg/SME/30
f
2.6
contamed
mm)
9.6 57.0
K+. no Ca2+
of choline
mM)
Ca2+
lobe/30
6.4
Ca2+
solution
raised
amounts (2.2
with
Na+,
Locke’s
Ca2+
K+ with
SME SRIF Release
(pg/neural
Ca2+
high
Neural Lobe SRIF Release
160, (low
HCOJNa+)
by adding
an equivalent
12:
and the appro-
amount
of
added.
tp
<
0.01
compared
to incubation
in normal
$p
<
0.01
compared
to incubation
in medium
Locke’s
solution.
containing
high
K+ with
Ca2+
27 pg/pg protein (n = 16). The total content of SRIF per neural lobe was 743 =t 91 pg (n = 20) compared to 36 * 2.7 ng/SME (n = 16). As shown in Table I and Fig. I, SRIF was secreted at a low basal rate from both tissues; the amount released was 5.9-26 pg/neural lobe/30 min or 11.5-32.3 pg/SME/30 min, representing 0.8%-3.5% and 0.03%~~0.09% respectively, of total content. High extracellular potassium is a potent stimulus for neurohypophysial hormone secretion and this response can be potentiated by a lowering of the extracellular Na+ concentration.” The release of SRIF (Table I) from both tissues was also markedly increased following high-potassium (56 mM), low-sodium (12 mM) stimulation to 57 f 9.6 pg/neural lobe and 172 _t 24 pg/SME, representing 7.8% and OS%, respectively, of total content per 30 min. The stimulatory effect of high potassium and low sodium was abolished when Ca ‘+ in the medium was replaced by Mn2+, an antagonist of Ca’)+ in secretory processes (Table 1). In response to electrical stimulation (Fig. 1) there was a significant three-fold increase in SRIF release from neural lobes (10% of total content) and a six-fold increase from SME (0.2% of content). This response was also Ca’+ dependent and failed to occur in CaZ+-free medium. It is concluded that the mechanism of SRIF release from the posterior pituitary and SME shares two important characteristics with the neurosecretory process: stimulation of release in response to membrane depolarization, and dependence of
-s
s
B&SAL
s
s
Fig. 1. Effect of electrical stimulation(s) release from neural lobes or SMEs.
on SRIF
SECRETION
FROM RAT NEUROHYPOPHYSIS
AND
SME
this process on extracellular Ca2+. The fractional is much smaller than from the posterior pituitary. in the neurohypophysis, the bulk of SME SRIF The isolated SME and neurohypophysis should studies of SRIF secretion from nerves.
1245
release of SRIF from the SME This suggests that, unlike SRIF exists in a nonreleasable form. provide a useful tool for further
REFERENCES I. Hokfelt T, Effendic S, Hellerstriim C, et al: Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special reference to the A-l cells of the pancreatic islets and to the hypothalamus. Acta Endocrinol [Suppl] (Kbh)80:5541, 1975 2. Epelbaum J, Brazeau P, Tsang D, et al: Subcellular distribution of radioimmunoassayable somatostatin in rat brain. Brain Res 126:309314, 1977 3. Pate1 YC, Zingg HH, Dreifuss JJ: Calciumdependent somatostatin secretion from rat neurohypophysis in vitro. Nature 267:852-853, 1977
4. Pate1 YC, Rao K, Reichlin S; Somatostatin in human cerebrospinal fluid. N Engl J Med 296~524533, 1977
5. Lowry OH, Rosebrough NJ, Ferrell AL, et al: Protein measurement with Folinphenol reagent. J Biol Chem 193:2655271, 1951
6. Dreifuss JJ, Grau JD, Bianchi RE: Antagonism between Ca and Na ions at neurohypophyseal nerve terminals. Experientia 27:129551296, 1971