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Co-storage and co-secretion of somatostatin and catecholamine in bovine adrenal medulla
Neuroscience Letters, 52 (1984) 43-47
43
Elsevier Scientific Publishers Ireland Ltd. NSL 03021
C O - S T O R A G E A N D CO-SECRETION OF S O M A T O S T A T I N A N D C A T E C H O L A M I N E IN B O V I N E A D R E N A L M E D U L L A
H A R U H 1 K O SAITO 1'*, SHIRO SAITO l, TAKESHI O H U C H I 2, M O T O O O K A 2, TOSHIAKI SANO 3 and EIJI HOSOI 1
Departments of 1Internal Medicine, 2Pharmacologyand 3Pathology, University of Tokushima School of Medicine, 3-18 Kuramoto-cho, Tokushima 770 (Japan) (Received July 7th, 1984; Revised version received August 31st, 1984; Accepted September 5th, 1984)
Key words: somatostatin - catecholamine - co-storage - co-secretion - bovine adrenal medulla chromaffin vesicle
Co-storage and co-secretion of somatostatin (SRIF) and catecholamine (CA) were demonstrated using bovine adrenal medulla. In the experiment of retrograde perfusion system of isolated adrenal gland, the basal concentration o f immunoreactive (IR)-SRIF was 20 p g / 4 m l / m i n , but a significant increase of the value (142 pg/4 ml/min) was observed at 1-2 min after a brief infusion of acetylcholine (10 mM) and the marked release continued for over 6 min. Similar result was obtained for CA release. On gel-filtration chromatography of a soluble lysate of chromaffin vesicles prepared by differential and discontinuous sucrose density centrifugation, IR-SRIF was separated into three components with molecular sizes corresponding to prepro-SRIF (76.8070 of the total immunoreactivity), SRIF28 (14.1 070) and SRIFt4 (9.1 07o).
Recent studies have shown that immunoreactive (IR)-enkephalins and IRcalcitonin are present in catecholamine (CA) storage granules in cells of normal adrenal medulla and p h e o c h r o m o c y t o m a , respectively [5, 8, 10, 14, 16]. F r o m immunochemical and ultrastructural studies on a somatostatin (SRIF)-secreting adrenal pheochromocytoma, we suggested that SRIF and C A are present in the same tumor cells [13]. Corder et al. [2] reported simultaneous release of IR-SRIF and CA from perfused cat adrenal gland. However, the subcellular storage site and the characteristics of SRIF-like peptide in normal adrenal gland have been unclear. Here we report that a SRIF~4-1ike substance and components corresponding to precursor molecules are present in bovine adrenal chromaffin vesicles. The left adrenal gland was removed from oxen as soon after slaughter as possible and taken on ice to the laboratory, where it was freed from surrounding fat. Many incisions were then made through the cortex, without damaging the medulla and a glass cannula was inserted into the adrenal vein. The gland was perfused in a *Author for correspondence at: First Department of Internal Medicine, University of T o k u s h i m a School of Medicine, 3-18 Kuramoto-cho, T o k u s h i m a 770, Japan. 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
44
retrograde manner with a solution of 20 mM Tris-HCl buffer (pH 7.4) containing 154 mM NaCl, 5.6 mM KC1, 2.2 mM CaC12 and 10 mM glucose saturated with 100% Oa and maintained at 37°C. The gland was perfused at a rate of 4.0 ml/min for 60 min, when the spontaneous release of CA had reached a fairly constant rate. The perfusate was collected at 1-min intervals in ice-cold vessels containing 2 N HCIO4 to give a final concentration of about 0.4 N. The medulla was stimulated by injecting 1 ml of 10 m M acetylcholine (ACh) into the perfusion fluid immediately before it entered the gland. The concentration of CA was measured fluorimetrically
131. Chromaffin vesicles of bovine adrenal medulla were isolated by differential and discontinuous sucrose density centrifugation [15]. Purified vesicles were lyzed by suspending them in ice-cold 10 mM Tris maleate buffer, pH 6.0, and the preparation was then centrifuged at 105,000 g for 20 min. An aliquot of the supernatant was applied to a column (1.6 x 85 cm) of Sephadex G-50 (fine) to analyse the molecular heterogeneity of IR-SRIF.
100
160
g140
om
100
60
¢.-
8O
20c, ""
20 0
0
I
2 3 4 5 6 7 8 9 I0 11 Fraction Number (4ml/fraction.min)
12
Fig. 1. Effect of ACh on the release of IR-SRIF and CA from isolated perfused bovine adrenal gland. The arrow indicates the time of addition of 1 ml of 10 mM ACh to the perfusion medium. Open and shaded bars represent concentrations of IR-SR1F and CA, respectively. The gland was perfused at a rate of 4.0 ml/min, and the perfusate was collected at intervals of 1 rain. Then 3.5 ml of each fraction was lyophilized, and the residue was dissolved in 0.7 ml of 0.01 M phosphate bufer (pH 7.4) containing 0.14 M NaCI, 0.05 M EDTA, 0.01% NaN3 and 0.1070 gelatin and subjected to SRIF radioimmunoassay (RIA). Duplicate samples of 0.2 ml were used for RIA of SRIF with an additional tube containing 0.2 ml of sample but no SRIF antibody to evaluate the non-specific interference in the RIA system. The dotted line indicates the limit of sensitivity of the SRIF assay. The concentration of CA was measured fluorimetrically.
45 A n t i - S R I F s e r u m was raised a g a i n s t synthetic S R I F t e t r a d e c a p e p t i d e [1] (SRIF14; P r o t e i n R e s e a r c h F o u n d a t i o n ) a n d was c o n f i r m e d to be h i g h l y specific for the r e g i o n o f the disulfide b r i d g e a n d T r p s o f SRIFI4. Tyrt-SRIF14 was r a d i o i o d i n a t e d b y a m o d i f i c a t i o n o f the l a c t o p e r o x i d a s e m e t h o d [6] a n d was p u r i f i e d on c a r b o x y m e t h y l - c e l l u l o s e ( C M 23; W h a t m a n ) . T h e p r o c e d u r e for r a d i o i m m u n o a s s a y o f S R I F was as r e p o r t e d in detail p r e v i o u s l y [12]. T h e b i n d i n g o f [125I]Tyrl-SRIF14 to a n t i s e r u m was i n h i b i t e d d o s e - d e p e n d e n t l y b y u n l a b e l e d h o r m o n e in the range o f 5 to 1000 p g / t u b e . T h e i n t r a a s s a y coefficients o f v a r i a t i o n in three m e a s u r e m e n t s each at three c o n c e n t r a t i o n s (12.5, 25.0 a n d 50.0 p g / t u b e ) were 12.00/0, 7 . 8 % a n d 6 . 1 % , respectively. T h e i n t e r a s s a y coefficients o f v a r i a t i o n at the s a m e c o n c e n t r a tions were 13.4%, 7 . 2 % a n d 8 . 3 % , respectively. The c r o s s - r e a c t i v i t y o f S R I F oct a c o s a p e p t i d e [9] (SRIF28; P e n i n s u l a ) with anti-SRIF14 a n t i b o d y was 109.2 +_4.5O7o ( m e a n ___ S . D . ) o n a m o l a r basis. T h e levels o f I R - S R I F a n d C A in the p e r f u s a t e b e f o r e a n d a f t e r b r i e f s t i m u l a t i o n with 10 m M A C h are s h o w n in Fig. 1. T h e c o n c e n t r a t i o n o f I R - S R I F was initially b e l o w 5 p g / m l (20 p g / f r a c t i o n ) , a n d increased significantly to 35.5 p g / m l (142 p g / f r a c t i o n ) at 1-2 m i n after the b e g i n n i n g o f A C h infusion. T h e n , a l t h o u g h the c o n c e n t r a t i o n in the p e r f u s a t e g r a d u a l l y decreased, the release c o n t i n u e d for over 6 min. S i m i l a r results were o b t a i n e d for C A release (Fig. 1).
~o
Cytochrome c 1 SRIF28 SRIFlZl
Vt
E
3~ .E
E 0 i
0
~"
2222~222~2~
60
80
100
120
140
160
180
260
Effluent (ml) Fig. 2. Elution profile of IR-SRIF in a lysate of chromaffin vesicles. The material was applied to a column (1.6 × 85 cm) of Sephadex G-50 (fine) and eluted with 0.2 N acetic acid at a flow rate of 15 ml/h at 4°C and fractions of 3 ml were collected. For accurate determination of the IR-SRIF concentration, the standards used were also diluted with the elution buffer used for chromatography. The column was calibrated with Blue Dextran (Sigma; mol.wt. 2,000,000), cytochrome c (Sigma; mol.wt. 12,300), synthetic SRIF28 (Peninsula; mol.wt. 3150), synthetic SRIFI4 (Protein Research Foundation; mol.wt. 1650) and Na1251 (New England Nuclear).
4(~ The inhibition curve of IR-SRIF in a lysate of chromaffin vesicles was parallel with that of synthetic SRIFH, suggesting that the SRIF-like material is immunologically indistinguishable from synthetic SRIF14. However, on gel-filtration chromatography IR-SRIF separated into three components: the first peak was eluted in about the position of cytochrome c (mol.wt. 12,300), and the second and third peaks were eluted in the same positions as synthetic SRIF28 (mol.wt. 3150) and SRIF~4 (mol.wt. 1650), respectively. The amounts of IR-SRIF in the respective peaks were calculated to be 76.8, 14.1 and 9.1% of the total immunoreactivity on a molar basis (Fig. 2). These findings indicate that (1) SRIF produced in the adrenal chromaffin cells is stored, at least in part, in some CA storage vesicles and released into the blood circulation with CA by exocytotic secretion, and (2) SRIF in the vesicles is heterogeneous in size, the major component corresponding in size to prepro-SRIF [4]. Recently, in in vitro experiments using isolated retrogradely perfused cat adrenal gland, Corder et al. [2] reported that ACh (5.5 × 10- 5 M), nicotin (2.2 × 10 5 M) and K + (5.5 raM) all evoked the release of noradrenaline, adrenaline and the four neuropeptide-like substances; IR-SRIF, -neurotensin, -Met-enkephalin and -Leuenkephalin. In addition, IR-enkephalins (Met-enkephalin, Leu-enkephalin, Metenkephalin-Arg6-Phe 7 and Met-enkephalin-Arg~-GlyV-Leu8) and IR-calcitonin have been demonstated in CA storage vesicles [8, 10, 16]. However, it is unknown whether these various peptides including SRIF are present in the same cell type of CA. Another problem is the physiological function of SRIF in the adrenal medulla. In in vitro studies on cultured or freshly dispersed adrenal medullary cells, Role et al. [11] and Mizobe et al. [7] found that SRIF (10-3-10 -6 M) reduced nicotinic stimulation of CA release, suggesting that SRIF exerted inhibitory control of CA secretion. However, in the present study, the maximum concentration of IR-SRIF in the perfusate after ACh stimulation was at most 2 . 2 x 10 ~E M (35.5 pg/ml) which is less than the minimum concentration required for biological effects (1 x 10 9 M) [1]. From the view points described above, we suspect that the number of SRIF-producing cells is small in the adrenal medulla, a n d / o r that SRIF acts not only via the microcirculation in the gland but also via a paracrine mechanism. Further studies are required on these points. This work was supported by a Grant-in Aid for Somatostatin Research from the Clinical Pathology Research Foundation of Japan, and by a Grant-in-Aid for Cancer Research (No. 56-6) from the Ministry of Health and Welfare of Japan. l Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J. and Guillemin, R., Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone, Science, 179 (1973) 77-79. 2 Corder, R., Mason, D.[~..I., Perrett, D., Lowry, P.J., Clement-Jones, V., Linton, E.A., Besser, G.M. and Rees, L.H., Simultaneous release of neurotensin, somatostatin, enkephalins and catecholamines from perfused cat adrenal glands, Neuropeptide, 3 (1982) 9-17.
47 3 Crout, J.R., Catecholamines in urine. In D. Seligson (Ed.), Standard Methods of Clinical Chemistry, Vol. 3, Academic Press, 1961, pp. 62-80. 4 Goodman, R.H., Jacobs, J.W., Chin, W.W., Lund, P.K., Dee, P.C. and Habener, J.F., Nucleotide sequence of a cloned structural gene coding for a precursor of pancreatic somatostatin, Proc. Natl. Acad. Sci. USA, 77 (1980) 5869-5873. 5 Livett, B.G., Dean, D.M., Whelau, L.G., Udenfriend, S. and Rossier, J., Co-release of enkephalin and catecholamines from cultured adrenal chromaffin cells, Nature (Lond.), 289 (1981) 317-319. 6 Miyachi, Y., Vaitukaitis, J.L., Nieschlag, E. and Lipsett, M.B., Enzymatic radioiodination of gonadotropins, J. Clin. Endocrinol. Metab., 34 (1972) 23-28.' 7 Mizobe, F., Kozousek, V., Dean, D.M. and Livett, R.G., Pharmacological characterization of adrenal-paraneurons: substance P and somatostatin as inhibitory modulators of the nicotinic response, Brain Res., 178 (1979) 555-566. 8 O'Connor, D.T., Frigon, R.P. and Deftos, L.J., lmmunoreactive calcitonin in catecholamine storage vesicles of human pheochromocytoma, J. Clin. Endocrinol., Metab., 56 (1983) 582-585. 9 Pradayrol, L., J6rnvall, H., Mutt, V. and Ribet, A., N-terminally extended somatostatin: the primary structure of somatostatin-28, FEBS Lett., 109 (1980) 55-58. 10 Roisin, M.P., Artola, A., Henry, J.P. and Rossier, J., Enkephalins are associated with adrenergic granules in bovine adrenal medulla, Neuroscience, 10 (1983) 83-88. 11 Role, L.W., Leeman, S.E. and Perlman, R.L., Somatostatin and substance P inhibit catecholamine secretion from isolated cells of guinea-pig adrenal medulla, Neuroscience, 6 (1981) 1813-1821. 12 Saito, H., Radioimmunoassay of plasma somatostatin: methods and levels in normal and pathological states, Ligand Rev., 2 (1980) 17-22. 13 Sano, T., Saito, H., lnaba, H., Hizawa, K., Saito, S., Yamanoi, A., Mizunuma, Y., Matsumura, M., Yuasa, M. and Hiraishi, K., Immunoreactive somatostatin and vasoactive intestinal polypeptide in adrenal pheochromocytoma. An immunochemical and ultrastructural study, Cancer, 52 (1983) 282-289. 14 Schulzberg, M., H6kfelt, T., Lundberg, J.M., Terenius, L., Elfvin, L.G. and Elde, R., Enkephalinlike immunoreactivity in nerve terminals in sympathetic ganglia and adrenal medulla and in adrenal medullary gland cells, Acta Physiol. Scand., 103 (1978) 475-477. 15 Smith, A.D. and Winkler, H., Purification and properties of an acidic protein from chromaffin granules of bovine adrenal medulla, Biochem. J., 103 (1967) 483-492. 16 Viveros, O.H., Diliberto, Jr., E.J., Hazum, E. and Chang, K.J., Opiate-like materials in the adrenal medulla: evidence for storage and secretion with catecholamines, Mol. Pharmacol., 16 (1979) 1101-1108.
43
Elsevier Scientific Publishers Ireland Ltd. NSL 03021
C O - S T O R A G E A N D CO-SECRETION OF S O M A T O S T A T I N A N D C A T E C H O L A M I N E IN B O V I N E A D R E N A L M E D U L L A
H A R U H 1 K O SAITO 1'*, SHIRO SAITO l, TAKESHI O H U C H I 2, M O T O O O K A 2, TOSHIAKI SANO 3 and EIJI HOSOI 1
Departments of 1Internal Medicine, 2Pharmacologyand 3Pathology, University of Tokushima School of Medicine, 3-18 Kuramoto-cho, Tokushima 770 (Japan) (Received July 7th, 1984; Revised version received August 31st, 1984; Accepted September 5th, 1984)
Key words: somatostatin - catecholamine - co-storage - co-secretion - bovine adrenal medulla chromaffin vesicle
Co-storage and co-secretion of somatostatin (SRIF) and catecholamine (CA) were demonstrated using bovine adrenal medulla. In the experiment of retrograde perfusion system of isolated adrenal gland, the basal concentration o f immunoreactive (IR)-SRIF was 20 p g / 4 m l / m i n , but a significant increase of the value (142 pg/4 ml/min) was observed at 1-2 min after a brief infusion of acetylcholine (10 mM) and the marked release continued for over 6 min. Similar result was obtained for CA release. On gel-filtration chromatography of a soluble lysate of chromaffin vesicles prepared by differential and discontinuous sucrose density centrifugation, IR-SRIF was separated into three components with molecular sizes corresponding to prepro-SRIF (76.8070 of the total immunoreactivity), SRIF28 (14.1 070) and SRIFt4 (9.1 07o).
Recent studies have shown that immunoreactive (IR)-enkephalins and IRcalcitonin are present in catecholamine (CA) storage granules in cells of normal adrenal medulla and p h e o c h r o m o c y t o m a , respectively [5, 8, 10, 14, 16]. F r o m immunochemical and ultrastructural studies on a somatostatin (SRIF)-secreting adrenal pheochromocytoma, we suggested that SRIF and C A are present in the same tumor cells [13]. Corder et al. [2] reported simultaneous release of IR-SRIF and CA from perfused cat adrenal gland. However, the subcellular storage site and the characteristics of SRIF-like peptide in normal adrenal gland have been unclear. Here we report that a SRIF~4-1ike substance and components corresponding to precursor molecules are present in bovine adrenal chromaffin vesicles. The left adrenal gland was removed from oxen as soon after slaughter as possible and taken on ice to the laboratory, where it was freed from surrounding fat. Many incisions were then made through the cortex, without damaging the medulla and a glass cannula was inserted into the adrenal vein. The gland was perfused in a *Author for correspondence at: First Department of Internal Medicine, University of T o k u s h i m a School of Medicine, 3-18 Kuramoto-cho, T o k u s h i m a 770, Japan. 0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.
44
retrograde manner with a solution of 20 mM Tris-HCl buffer (pH 7.4) containing 154 mM NaCl, 5.6 mM KC1, 2.2 mM CaC12 and 10 mM glucose saturated with 100% Oa and maintained at 37°C. The gland was perfused at a rate of 4.0 ml/min for 60 min, when the spontaneous release of CA had reached a fairly constant rate. The perfusate was collected at 1-min intervals in ice-cold vessels containing 2 N HCIO4 to give a final concentration of about 0.4 N. The medulla was stimulated by injecting 1 ml of 10 m M acetylcholine (ACh) into the perfusion fluid immediately before it entered the gland. The concentration of CA was measured fluorimetrically
131. Chromaffin vesicles of bovine adrenal medulla were isolated by differential and discontinuous sucrose density centrifugation [15]. Purified vesicles were lyzed by suspending them in ice-cold 10 mM Tris maleate buffer, pH 6.0, and the preparation was then centrifuged at 105,000 g for 20 min. An aliquot of the supernatant was applied to a column (1.6 x 85 cm) of Sephadex G-50 (fine) to analyse the molecular heterogeneity of IR-SRIF.
100
160
g140
om
100
60
¢.-
8O
20c, ""
20 0
0
I
2 3 4 5 6 7 8 9 I0 11 Fraction Number (4ml/fraction.min)
12
Fig. 1. Effect of ACh on the release of IR-SRIF and CA from isolated perfused bovine adrenal gland. The arrow indicates the time of addition of 1 ml of 10 mM ACh to the perfusion medium. Open and shaded bars represent concentrations of IR-SR1F and CA, respectively. The gland was perfused at a rate of 4.0 ml/min, and the perfusate was collected at intervals of 1 rain. Then 3.5 ml of each fraction was lyophilized, and the residue was dissolved in 0.7 ml of 0.01 M phosphate bufer (pH 7.4) containing 0.14 M NaCI, 0.05 M EDTA, 0.01% NaN3 and 0.1070 gelatin and subjected to SRIF radioimmunoassay (RIA). Duplicate samples of 0.2 ml were used for RIA of SRIF with an additional tube containing 0.2 ml of sample but no SRIF antibody to evaluate the non-specific interference in the RIA system. The dotted line indicates the limit of sensitivity of the SRIF assay. The concentration of CA was measured fluorimetrically.
45 A n t i - S R I F s e r u m was raised a g a i n s t synthetic S R I F t e t r a d e c a p e p t i d e [1] (SRIF14; P r o t e i n R e s e a r c h F o u n d a t i o n ) a n d was c o n f i r m e d to be h i g h l y specific for the r e g i o n o f the disulfide b r i d g e a n d T r p s o f SRIFI4. Tyrt-SRIF14 was r a d i o i o d i n a t e d b y a m o d i f i c a t i o n o f the l a c t o p e r o x i d a s e m e t h o d [6] a n d was p u r i f i e d on c a r b o x y m e t h y l - c e l l u l o s e ( C M 23; W h a t m a n ) . T h e p r o c e d u r e for r a d i o i m m u n o a s s a y o f S R I F was as r e p o r t e d in detail p r e v i o u s l y [12]. T h e b i n d i n g o f [125I]Tyrl-SRIF14 to a n t i s e r u m was i n h i b i t e d d o s e - d e p e n d e n t l y b y u n l a b e l e d h o r m o n e in the range o f 5 to 1000 p g / t u b e . T h e i n t r a a s s a y coefficients o f v a r i a t i o n in three m e a s u r e m e n t s each at three c o n c e n t r a t i o n s (12.5, 25.0 a n d 50.0 p g / t u b e ) were 12.00/0, 7 . 8 % a n d 6 . 1 % , respectively. T h e i n t e r a s s a y coefficients o f v a r i a t i o n at the s a m e c o n c e n t r a tions were 13.4%, 7 . 2 % a n d 8 . 3 % , respectively. The c r o s s - r e a c t i v i t y o f S R I F oct a c o s a p e p t i d e [9] (SRIF28; P e n i n s u l a ) with anti-SRIF14 a n t i b o d y was 109.2 +_4.5O7o ( m e a n ___ S . D . ) o n a m o l a r basis. T h e levels o f I R - S R I F a n d C A in the p e r f u s a t e b e f o r e a n d a f t e r b r i e f s t i m u l a t i o n with 10 m M A C h are s h o w n in Fig. 1. T h e c o n c e n t r a t i o n o f I R - S R I F was initially b e l o w 5 p g / m l (20 p g / f r a c t i o n ) , a n d increased significantly to 35.5 p g / m l (142 p g / f r a c t i o n ) at 1-2 m i n after the b e g i n n i n g o f A C h infusion. T h e n , a l t h o u g h the c o n c e n t r a t i o n in the p e r f u s a t e g r a d u a l l y decreased, the release c o n t i n u e d for over 6 min. S i m i l a r results were o b t a i n e d for C A release (Fig. 1).
~o
Cytochrome c 1 SRIF28 SRIFlZl
Vt
E
3~ .E
E 0 i
0
~"
2222~222~2~
60
80
100
120
140
160
180
260
Effluent (ml) Fig. 2. Elution profile of IR-SRIF in a lysate of chromaffin vesicles. The material was applied to a column (1.6 × 85 cm) of Sephadex G-50 (fine) and eluted with 0.2 N acetic acid at a flow rate of 15 ml/h at 4°C and fractions of 3 ml were collected. For accurate determination of the IR-SRIF concentration, the standards used were also diluted with the elution buffer used for chromatography. The column was calibrated with Blue Dextran (Sigma; mol.wt. 2,000,000), cytochrome c (Sigma; mol.wt. 12,300), synthetic SRIF28 (Peninsula; mol.wt. 3150), synthetic SRIFI4 (Protein Research Foundation; mol.wt. 1650) and Na1251 (New England Nuclear).
4(~ The inhibition curve of IR-SRIF in a lysate of chromaffin vesicles was parallel with that of synthetic SRIFH, suggesting that the SRIF-like material is immunologically indistinguishable from synthetic SRIF14. However, on gel-filtration chromatography IR-SRIF separated into three components: the first peak was eluted in about the position of cytochrome c (mol.wt. 12,300), and the second and third peaks were eluted in the same positions as synthetic SRIF28 (mol.wt. 3150) and SRIF~4 (mol.wt. 1650), respectively. The amounts of IR-SRIF in the respective peaks were calculated to be 76.8, 14.1 and 9.1% of the total immunoreactivity on a molar basis (Fig. 2). These findings indicate that (1) SRIF produced in the adrenal chromaffin cells is stored, at least in part, in some CA storage vesicles and released into the blood circulation with CA by exocytotic secretion, and (2) SRIF in the vesicles is heterogeneous in size, the major component corresponding in size to prepro-SRIF [4]. Recently, in in vitro experiments using isolated retrogradely perfused cat adrenal gland, Corder et al. [2] reported that ACh (5.5 × 10- 5 M), nicotin (2.2 × 10 5 M) and K + (5.5 raM) all evoked the release of noradrenaline, adrenaline and the four neuropeptide-like substances; IR-SRIF, -neurotensin, -Met-enkephalin and -Leuenkephalin. In addition, IR-enkephalins (Met-enkephalin, Leu-enkephalin, Metenkephalin-Arg6-Phe 7 and Met-enkephalin-Arg~-GlyV-Leu8) and IR-calcitonin have been demonstated in CA storage vesicles [8, 10, 16]. However, it is unknown whether these various peptides including SRIF are present in the same cell type of CA. Another problem is the physiological function of SRIF in the adrenal medulla. In in vitro studies on cultured or freshly dispersed adrenal medullary cells, Role et al. [11] and Mizobe et al. [7] found that SRIF (10-3-10 -6 M) reduced nicotinic stimulation of CA release, suggesting that SRIF exerted inhibitory control of CA secretion. However, in the present study, the maximum concentration of IR-SRIF in the perfusate after ACh stimulation was at most 2 . 2 x 10 ~E M (35.5 pg/ml) which is less than the minimum concentration required for biological effects (1 x 10 9 M) [1]. From the view points described above, we suspect that the number of SRIF-producing cells is small in the adrenal medulla, a n d / o r that SRIF acts not only via the microcirculation in the gland but also via a paracrine mechanism. Further studies are required on these points. This work was supported by a Grant-in Aid for Somatostatin Research from the Clinical Pathology Research Foundation of Japan, and by a Grant-in-Aid for Cancer Research (No. 56-6) from the Ministry of Health and Welfare of Japan. l Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J. and Guillemin, R., Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone, Science, 179 (1973) 77-79. 2 Corder, R., Mason, D.[~..I., Perrett, D., Lowry, P.J., Clement-Jones, V., Linton, E.A., Besser, G.M. and Rees, L.H., Simultaneous release of neurotensin, somatostatin, enkephalins and catecholamines from perfused cat adrenal glands, Neuropeptide, 3 (1982) 9-17.
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