550 Honn. Metab. Res. 11 '1979) 550-554
Effect of Pentobarbital and Urethane on the Release of Hypothalamic Somatostatin and Pituitary Growth Hormone H. Saito. T. Ogawa. K. Ishimaru. I. Oshima and S. Saito Department of Laboratory Medicine. Tokushima University. School of Medicine. Tokushima. Japan
Hypothalamic somatostatin release was investigated in the rat to elucidate the mechanism of anesthetic action on growth hormone (GH) release from the pituitary. Intraperitoneal injection of sodium pentobarbital (5 mg/lOO gm B.W.) significantly elevated serum GH levels and increased hypothalamic somatostatin concentration from basal values of 0.98 ± 0.04 to 1.21 ± 0.06 ng/mg wet wt. In contrast, urethane (\ 50 mg/! 00 gm B.W., IP) administration lowered serum GH levels and hypothalamic somatostatin concentration (0.64 ± 0.04 ng/mg wet wt.). However, the mean concentration of pancreatic somatostatin showed no change in either case. In rats receiving passive immunization with 0.5 ml rabbit antiserum to somatostatin (SRIF-AS), serum GH levels were significantly increased (67.5 ± 12.3 ng/ml) and did not differ from those in the group treated with normal rabbit serum (NRS) plus pentobarbital (101.3 ± 18.5 ng/ml). However, serum GH levels in rats injected with SRIF-AS plus pentobarbital were increased to lligher values than in rats given SRIF-AS alone. When urethane was administered to rats after passive immunization with SRIF-AS, urethane-induced suppression of serum GH levels was markedIy inhibited (5.5 ± 2.0 vs. 33.5 ± 7.5 ng/ml). These results suggest a possibility that the changes in serum GH levels observed with pentobarbital or urethane administration may be induced at least in one part by somatostatin released from the hypothalamus. Key-Words: Hypothlliamus - Sorruztostatin - Growth Hor· mone - Pentobarbital - Urethllne
Introduction Somatostatin has been isolated from the ovine hypothalamus and characterized by Brazeau, Vale, Burgus, Ling, Butcher, Rivier and Guillemin (1973) on the basis of its ability to inhibit the secretion of growth hormone (GH). Subsequently, they also reported that the synthetic somatostatin inhibited the release of GH induced by the administration of sodium pentobarbital (Brazeau, Rivier, Vale and Guillemin 1974). On the other hand, it has been known that pentobarbital or urethane affect serum GH levels. The mechanism of this has not been known but Collu, Fraschini, Visconti and Martini (1972) suggested that the central nervous system, which was involved in the control of GH secretion, may be influenced by these kinds of anesthetics. There· fore, the present study was undertaken to clarify Received: 28 Febr. 1979 0018-5043/79
1032-0550
Accepted: 6 March 1979 S 03.00
©
the mechanism of pentobarbital or urethane action on GH release by measuring the concentrations of hypothalamic somatostatin and serum GH levels with or without pretreatment of the antiserum to somatostatin (SRlF -AS).
Material and Methods Experimental design Adult male Wistar rats weighing about 300 gm were used after overnight 12 hr fast. Experiment I: The animals were divided into 3 groups of 6 to 8 rats each. Group I rats were injected intraperitonea1ly with sodium pentobarbital (5 mg/I 00 gm B.W.), and group 2 rats received urethane (150 mg/IOO gm B.W., IP). The animals of group land group 2 were killed at 60 and 30 min after each injection, respectively. Group 3 rats were administered 0.1 ml physiological saline intraperitonea1ly and used as the con trol. Experiment 2: The animals were divided into 6 groups, consisting of 5 rats each. As the pretreatment, group I to group 3 rats were injected with 0.5 ml nonnal rabbit serum (NRS) and groups 4 to group 6 rats with 0.5 ml SRIF-AS. Thirty minutes later group 2 and group 5 rats were given pentobarbital (5 mg/100 gm B.W., IP), and group 3 and group 6 rats were treated with urethane (150 mg/100 gm B.W., IP) at 60 min after the first injection. All the animals were then killed at 90 min after the injection of NRS or SRIF-AS. In the experiment land 2, the blood sam pies were collected from the trunk by decapitation, and the hypothalamus and pancreas were removed immediately and kept frozen at --20 oe until extraction. Each experiment was carried out between 900h and 1200 h.
Antiserum to sorruztostatin (SRIF-AS) Anti-somatostatin serum used in this study was prepared according to the method of Arimura, Sato, Coy and SchlllIy (1975) and proved to be highly specific for somatostatin as reported previously (S. Saito, H. Saito. Ogawa, Ishimaru, Oshirruz, K. Saito, Kakuta and HiTaishi 1978). Radioiodination of somatostain Tyr'-somatostatin (Pro tein Research Foundation. Minoo. Osaka, Japan) was radioiodinated with ' 25 1 (New England Nuclear, Boston, U.S.A.) using a modification of the lactoperoxidase method (Miyachi, Vaitukatitis, Nieschlag and Lipsett 1972) and 125 1_ Tyr '-somatostatin was separated on carboxymethyl cellulose column as described previously (S. Saito et al. 1978).
Extraction of sorruztostatin {rom tissues The organs removed were cut into small pieces and homogenized with 10 volumes of 2N acetic acid using the Uni-
1979 Georg Thieme Publ ishers
Downloaded by: University of British Columbia. Copyrighted material.
Summary
551
Effect of Anesthetics on GH and SRIF Levels
Radioimmunoassay o[ somatostatin
curves parallel to that obtained with synthetic somatostatin, the active materials present in the extracts seemed to be immunologically indistinguishable from somatostatin. These materials were considered to have similar molecular size as somatostatin, because both were eluted in the same portion of effluent fractions as synthetic somatostatin.
For the radioimmunoassay, 100 111 sampies (standard or tissue extract), 100 111 anti-somatostatin serum (3,OOO-fold dilution) Ef[ect of anesthetics on serum GH levels and hypoand 400 111 0.01 M phosphate buffer solution (pH 7.4) c o n t a m - . . . . ing 0.14 M NaCI, 0.05 M EDTA, 0.1 % NaN 3 • 0.1 % gelatin (Difco, thalamlc and pancreatlc somatostatm concentratlOns U.S.A.) and 1 mM gabexate mesilate (Ono pharmaceutical Co., As shown in Figure 2 a significant increment in Osaka, Japan) were added to glass tubes and mixed. All deterGH b' d f b al al f minations were performed in duplicate. All tubes were incubat- serum was 0 serve rom as v ues 0 ed at 4°C for 48hr,and then 1001l11251-Tyrl-somatostatin 28.6 ± 2.4 to 99.8 ± 21.2 ng/ml (mean ± SE, P < (approx. 10,000 cpm) was added to each tube and mixed. After 0.0 I) at 60 min after the intraperitoneal injection incubation at 4°C for 48 hr, 100 111 NRS (IOO-fold dilution) of pentobarbital (5 mg/100 gm B.W.). Urethane a~d 100 111 goat antl-rabblt -y-globuhn antiserum ~~ O-fold (150 mg/100 gm BW., IP) administration depressed dilutIOn) were added. The tubes were mcubated turther for . . 24 hr at 4 °C and centrifuged at 3,000 rpm at 4 °C for 30 the serum GH levels conslstently after 30 mm; min. The supernatant solutions were decanted and precipimean serum GH value was 8.5 ± 1.4 ng/ml tates eounted in an automatie gamma counter with approx. (P < 0.01 vs.basal values). 50% counting efficieney (Aloca Co., Tokyo, Japan). ConOn the other hand, hypothalamic somatostatin concentrations of immunoreactive somatostatin in the unknown centrations in the group of pentobarbital were sigsampies were determined by comparison to the standard curve. nificantly increased from 0.98 ± 0.04 to 1.21 ± 0.06
Radioimmunoassay o[ serum GH Serum GH was determined by the dextran-coated charcoal method using the rat radioimmunoassay kit supplied by the NIAMDD, NIH, Bethesda, Maryland, U.S.A. Purified rat GH (NIAMDD-Rat GH-I-3) was used for radioiodination with 125 1 and all results were expressed in ng/ml in terms of the reference standard (NIAMDD-Rat GH-RP-I). Statistical significance between groups was analysed according to Student's t-test.
Results
Inhibition curves o[ tissue extracts in somatostatin radioimmunoassay The inhibition curves of the extracts obtained from the hypothalamus and pancreas are shown in Figure 1. Since two preparations tested produced inhibition
ng/mg wct wt. CP < 0.01), while in the group of urethane, hypothalamic somatostatin concentrations were remarkably decreased to 0.64 ± 0.04 ng/mg wet wt. CP < 0.01). There was a significant correlation between serum GH levels and hypothalamic somatostatin concentrations (r = 0.6033, P < 0.0 I) as depicted in Figure 3. However pancreatic somatostatin concentrations in the three groups did not differ as shown in Figure 2 (saline group, 0.28 ± 0.02; pentobarbital group, 0.27 ± 0.01; urethane group, 0.26 ± 0.03 ng/mg wet wt.). The data shown in Figures 2 and 3 suggest a possibility that changes in serum GH levels in rats treated with pentobarbital or urethane are probably related to somatostatin release from the hypothalamus.
3
2
o ,e
, 5
10
50
100
Somatostat in ( pg I tube) Fig. (ll
. -
200
0.1
0.5
5
10
T Issue extract ( mg wet wt.)
Inhibition curves in the somatostatin radioimmunoassay with tissue extracts obtained from the hypothalamus 'J) and pancreas (C' ,.)
20
Downloaded by: University of British Columbia. Copyrighted material.
versal Homogenizer®, Nihon Seiki Co., Tokyo, Japan. The homogenate was heated in boiling water bath for 10min, quickly cooled to 4 °C and centrifuged at 15,000 x g for 30 min at 4 °C after adjusting to pH 7.0. The supernatant was Iyophilized and dissolved in the assay buffer be fore radioimmunoassay.
552
H. Saito, T. Ogawa, K. Ishimaru, I. Oshima. S. Saito Hypothalamic SR I F I ngl mg wet wt. I
PB
C
U
PB
C
U
1.2
On the other hand, urethane-induced suppression of serum GH levels was markedly recovered by the pretreatment with SRIF-AS (from 5.5 ± 2.0 to 33.5 ± 7.5 ng/mI, P < 0.01), but the ex tent of decrease in hypothalamic somatostatin concentrations did not significantly differ between both groups. However, the animals who received SRIF-AS prior to urethane administration had levels of serum GH and hypothalamic somatostatin that did not differ from those in rats with SRIF-AS alone (Figure 5).
.
0
'" E
00
.
1.0 c: '"
~
•
0.8 VI
.~
E 0.6 "' (101.3 ± 18.5 ng/mI). However, the further eleva:>: 0.2 tion of circulating GH levels were found in rats which received SRIF-AS prior to pentobarbital ado ministration as compared with the rats given with NRS SR IF-AS NRS SRIF-AS SRIF-AS alone (P < 0.05). The hypothalamic somaPB PB tostatin concentrations were similar in both groups, Fig. 4 E1'fect of anti-somatostatin serum on the levels of but these levels were significantly lower than those in serum GH and hypothalamic somatostatin in intact and the group injected with NRS plus pentobarbital (P pentobarbital treated rats. Each column represents the < 0.005). [n the graup treated with SRIF-AS plus mean ± SE of 5 animals. N.S. = not significant ",-
-
Downloaded by: University of British Columbia. Copyrighted material.
Serum GH ( nglmll
553
Effeet of Anestheties on GH and SRIF Levels
Discussion
As to the mechanism of pentobarbital-induced GH secretion in the rat, there have been so me arguments: Howard and Martin (1972) first reported a possibility of its direct effect on GH release from the pituitary in in vitro experiment. However, some investigators suggested that the mechanism through which pentobarbital elicit pituitary GH release was probably mediated via the central nervous system (Martin 1973/74; Brown and Vale 1975). On the other hand, possibilities that urethane anesthesia interferes with GH secretion in the central nervous system either by activating the inhibitory system or by suppressing the stimulatory system have been reported (Collu et al. 1972), but still remains to be confirmed.
..--N.S.~ r-
80
P< 0.01-.
r--
P
Effect of Pentobarbital and Urethane on the Release of Hypothalamic Somatostatin and Pituitary Growth Hormone H. Saito. T. Ogawa. K. Ishimaru. I. Oshima and S. Saito Department of Laboratory Medicine. Tokushima University. School of Medicine. Tokushima. Japan
Hypothalamic somatostatin release was investigated in the rat to elucidate the mechanism of anesthetic action on growth hormone (GH) release from the pituitary. Intraperitoneal injection of sodium pentobarbital (5 mg/lOO gm B.W.) significantly elevated serum GH levels and increased hypothalamic somatostatin concentration from basal values of 0.98 ± 0.04 to 1.21 ± 0.06 ng/mg wet wt. In contrast, urethane (\ 50 mg/! 00 gm B.W., IP) administration lowered serum GH levels and hypothalamic somatostatin concentration (0.64 ± 0.04 ng/mg wet wt.). However, the mean concentration of pancreatic somatostatin showed no change in either case. In rats receiving passive immunization with 0.5 ml rabbit antiserum to somatostatin (SRIF-AS), serum GH levels were significantly increased (67.5 ± 12.3 ng/ml) and did not differ from those in the group treated with normal rabbit serum (NRS) plus pentobarbital (101.3 ± 18.5 ng/ml). However, serum GH levels in rats injected with SRIF-AS plus pentobarbital were increased to lligher values than in rats given SRIF-AS alone. When urethane was administered to rats after passive immunization with SRIF-AS, urethane-induced suppression of serum GH levels was markedIy inhibited (5.5 ± 2.0 vs. 33.5 ± 7.5 ng/ml). These results suggest a possibility that the changes in serum GH levels observed with pentobarbital or urethane administration may be induced at least in one part by somatostatin released from the hypothalamus. Key-Words: Hypothlliamus - Sorruztostatin - Growth Hor· mone - Pentobarbital - Urethllne
Introduction Somatostatin has been isolated from the ovine hypothalamus and characterized by Brazeau, Vale, Burgus, Ling, Butcher, Rivier and Guillemin (1973) on the basis of its ability to inhibit the secretion of growth hormone (GH). Subsequently, they also reported that the synthetic somatostatin inhibited the release of GH induced by the administration of sodium pentobarbital (Brazeau, Rivier, Vale and Guillemin 1974). On the other hand, it has been known that pentobarbital or urethane affect serum GH levels. The mechanism of this has not been known but Collu, Fraschini, Visconti and Martini (1972) suggested that the central nervous system, which was involved in the control of GH secretion, may be influenced by these kinds of anesthetics. There· fore, the present study was undertaken to clarify Received: 28 Febr. 1979 0018-5043/79
1032-0550
Accepted: 6 March 1979 S 03.00
©
the mechanism of pentobarbital or urethane action on GH release by measuring the concentrations of hypothalamic somatostatin and serum GH levels with or without pretreatment of the antiserum to somatostatin (SRlF -AS).
Material and Methods Experimental design Adult male Wistar rats weighing about 300 gm were used after overnight 12 hr fast. Experiment I: The animals were divided into 3 groups of 6 to 8 rats each. Group I rats were injected intraperitonea1ly with sodium pentobarbital (5 mg/I 00 gm B.W.), and group 2 rats received urethane (150 mg/IOO gm B.W., IP). The animals of group land group 2 were killed at 60 and 30 min after each injection, respectively. Group 3 rats were administered 0.1 ml physiological saline intraperitonea1ly and used as the con trol. Experiment 2: The animals were divided into 6 groups, consisting of 5 rats each. As the pretreatment, group I to group 3 rats were injected with 0.5 ml nonnal rabbit serum (NRS) and groups 4 to group 6 rats with 0.5 ml SRIF-AS. Thirty minutes later group 2 and group 5 rats were given pentobarbital (5 mg/100 gm B.W., IP), and group 3 and group 6 rats were treated with urethane (150 mg/100 gm B.W., IP) at 60 min after the first injection. All the animals were then killed at 90 min after the injection of NRS or SRIF-AS. In the experiment land 2, the blood sam pies were collected from the trunk by decapitation, and the hypothalamus and pancreas were removed immediately and kept frozen at --20 oe until extraction. Each experiment was carried out between 900h and 1200 h.
Antiserum to sorruztostatin (SRIF-AS) Anti-somatostatin serum used in this study was prepared according to the method of Arimura, Sato, Coy and SchlllIy (1975) and proved to be highly specific for somatostatin as reported previously (S. Saito, H. Saito. Ogawa, Ishimaru, Oshirruz, K. Saito, Kakuta and HiTaishi 1978). Radioiodination of somatostain Tyr'-somatostatin (Pro tein Research Foundation. Minoo. Osaka, Japan) was radioiodinated with ' 25 1 (New England Nuclear, Boston, U.S.A.) using a modification of the lactoperoxidase method (Miyachi, Vaitukatitis, Nieschlag and Lipsett 1972) and 125 1_ Tyr '-somatostatin was separated on carboxymethyl cellulose column as described previously (S. Saito et al. 1978).
Extraction of sorruztostatin {rom tissues The organs removed were cut into small pieces and homogenized with 10 volumes of 2N acetic acid using the Uni-
1979 Georg Thieme Publ ishers
Downloaded by: University of British Columbia. Copyrighted material.
Summary
551
Effect of Anesthetics on GH and SRIF Levels
Radioimmunoassay o[ somatostatin
curves parallel to that obtained with synthetic somatostatin, the active materials present in the extracts seemed to be immunologically indistinguishable from somatostatin. These materials were considered to have similar molecular size as somatostatin, because both were eluted in the same portion of effluent fractions as synthetic somatostatin.
For the radioimmunoassay, 100 111 sampies (standard or tissue extract), 100 111 anti-somatostatin serum (3,OOO-fold dilution) Ef[ect of anesthetics on serum GH levels and hypoand 400 111 0.01 M phosphate buffer solution (pH 7.4) c o n t a m - . . . . ing 0.14 M NaCI, 0.05 M EDTA, 0.1 % NaN 3 • 0.1 % gelatin (Difco, thalamlc and pancreatlc somatostatm concentratlOns U.S.A.) and 1 mM gabexate mesilate (Ono pharmaceutical Co., As shown in Figure 2 a significant increment in Osaka, Japan) were added to glass tubes and mixed. All deterGH b' d f b al al f minations were performed in duplicate. All tubes were incubat- serum was 0 serve rom as v ues 0 ed at 4°C for 48hr,and then 1001l11251-Tyrl-somatostatin 28.6 ± 2.4 to 99.8 ± 21.2 ng/ml (mean ± SE, P < (approx. 10,000 cpm) was added to each tube and mixed. After 0.0 I) at 60 min after the intraperitoneal injection incubation at 4°C for 48 hr, 100 111 NRS (IOO-fold dilution) of pentobarbital (5 mg/100 gm B.W.). Urethane a~d 100 111 goat antl-rabblt -y-globuhn antiserum ~~ O-fold (150 mg/100 gm BW., IP) administration depressed dilutIOn) were added. The tubes were mcubated turther for . . 24 hr at 4 °C and centrifuged at 3,000 rpm at 4 °C for 30 the serum GH levels conslstently after 30 mm; min. The supernatant solutions were decanted and precipimean serum GH value was 8.5 ± 1.4 ng/ml tates eounted in an automatie gamma counter with approx. (P < 0.01 vs.basal values). 50% counting efficieney (Aloca Co., Tokyo, Japan). ConOn the other hand, hypothalamic somatostatin concentrations of immunoreactive somatostatin in the unknown centrations in the group of pentobarbital were sigsampies were determined by comparison to the standard curve. nificantly increased from 0.98 ± 0.04 to 1.21 ± 0.06
Radioimmunoassay o[ serum GH Serum GH was determined by the dextran-coated charcoal method using the rat radioimmunoassay kit supplied by the NIAMDD, NIH, Bethesda, Maryland, U.S.A. Purified rat GH (NIAMDD-Rat GH-I-3) was used for radioiodination with 125 1 and all results were expressed in ng/ml in terms of the reference standard (NIAMDD-Rat GH-RP-I). Statistical significance between groups was analysed according to Student's t-test.
Results
Inhibition curves o[ tissue extracts in somatostatin radioimmunoassay The inhibition curves of the extracts obtained from the hypothalamus and pancreas are shown in Figure 1. Since two preparations tested produced inhibition
ng/mg wct wt. CP < 0.01), while in the group of urethane, hypothalamic somatostatin concentrations were remarkably decreased to 0.64 ± 0.04 ng/mg wet wt. CP < 0.01). There was a significant correlation between serum GH levels and hypothalamic somatostatin concentrations (r = 0.6033, P < 0.0 I) as depicted in Figure 3. However pancreatic somatostatin concentrations in the three groups did not differ as shown in Figure 2 (saline group, 0.28 ± 0.02; pentobarbital group, 0.27 ± 0.01; urethane group, 0.26 ± 0.03 ng/mg wet wt.). The data shown in Figures 2 and 3 suggest a possibility that changes in serum GH levels in rats treated with pentobarbital or urethane are probably related to somatostatin release from the hypothalamus.
3
2
o ,e
, 5
10
50
100
Somatostat in ( pg I tube) Fig. (ll
. -
200
0.1
0.5
5
10
T Issue extract ( mg wet wt.)
Inhibition curves in the somatostatin radioimmunoassay with tissue extracts obtained from the hypothalamus 'J) and pancreas (C' ,.)
20
Downloaded by: University of British Columbia. Copyrighted material.
versal Homogenizer®, Nihon Seiki Co., Tokyo, Japan. The homogenate was heated in boiling water bath for 10min, quickly cooled to 4 °C and centrifuged at 15,000 x g for 30 min at 4 °C after adjusting to pH 7.0. The supernatant was Iyophilized and dissolved in the assay buffer be fore radioimmunoassay.
552
H. Saito, T. Ogawa, K. Ishimaru, I. Oshima. S. Saito Hypothalamic SR I F I ngl mg wet wt. I
PB
C
U
PB
C
U
1.2
On the other hand, urethane-induced suppression of serum GH levels was markedly recovered by the pretreatment with SRIF-AS (from 5.5 ± 2.0 to 33.5 ± 7.5 ng/mI, P < 0.01), but the ex tent of decrease in hypothalamic somatostatin concentrations did not significantly differ between both groups. However, the animals who received SRIF-AS prior to urethane administration had levels of serum GH and hypothalamic somatostatin that did not differ from those in rats with SRIF-AS alone (Figure 5).
.
0
'" E
00
.
1.0 c: '"
~
•
0.8 VI
.~
E 0.6 "' (101.3 ± 18.5 ng/mI). However, the further eleva:>: 0.2 tion of circulating GH levels were found in rats which received SRIF-AS prior to pentobarbital ado ministration as compared with the rats given with NRS SR IF-AS NRS SRIF-AS SRIF-AS alone (P < 0.05). The hypothalamic somaPB PB tostatin concentrations were similar in both groups, Fig. 4 E1'fect of anti-somatostatin serum on the levels of but these levels were significantly lower than those in serum GH and hypothalamic somatostatin in intact and the group injected with NRS plus pentobarbital (P pentobarbital treated rats. Each column represents the < 0.005). [n the graup treated with SRIF-AS plus mean ± SE of 5 animals. N.S. = not significant ",-
-
Downloaded by: University of British Columbia. Copyrighted material.
Serum GH ( nglmll
553
Effeet of Anestheties on GH and SRIF Levels
Discussion
As to the mechanism of pentobarbital-induced GH secretion in the rat, there have been so me arguments: Howard and Martin (1972) first reported a possibility of its direct effect on GH release from the pituitary in in vitro experiment. However, some investigators suggested that the mechanism through which pentobarbital elicit pituitary GH release was probably mediated via the central nervous system (Martin 1973/74; Brown and Vale 1975). On the other hand, possibilities that urethane anesthesia interferes with GH secretion in the central nervous system either by activating the inhibitory system or by suppressing the stimulatory system have been reported (Collu et al. 1972), but still remains to be confirmed.
..--N.S.~ r-
80
P< 0.01-.
r--
P
