216
BIOCHEMICAL SOCIETY TRANSACTIONS
Jones, G. R. N. (1975)Med. Hypotheses 1, 118-127 Jones, G.R. N. (1976~)Med. Hypotheses 2, 50-54 Jones, G. R.N. (19766)Annual Report, Inst. Basic Med. Sci., University ofLondon in the press Jones, G. R. N. (1977)Biochem. SOC.Trans. 5, 213-214 Klingenberg, M. & Slenczka, W. (1959)Biochem. Z . 331,486-517 Nauts, H.C.,Swift, W. E. & Coley, B. L. (1946) Cancer Res. 6,205-216 Shapiro, C . J. (1940)Am..I. Hyg. 31B, 114-126 Shear, M.J. & Andervont, H. B. (1936)Proc. SOC.Exp. Biol. Med. 34,323-325 Thorne, R. F. W. & Bygrave, F. L. (1974)Biochem. J. 144, 551-558 Weinbach, E. C. & Garbus, J. (1966)J. Biol. Chem. 241, 3708-3713
The Mechanism of the Stimulation of Pyruvate Transport into Rat Liver Mitochondria by Glucagon ANDREW P. HALESTRAP Department of Biochemistry, University of Bristol, Bristol BS8 1T D , U.K.
Mitochondria isolated from the livers of rats previously treated with glucagon show an enhanced rate of pyruvate metabolism (Adam & Haynes, 1969). This effect has also been demonstrated with isolated liver cells (Garrison & Haynes, 1975). The suggestion was made that this effect on pyruvate metabolism was at the level of pyruvate transport into the mitochondrion (Adam & Haynes, 1969). However, I have shown previously that the pyruvate transporter is not activated under these conditions (Halestrap, 1975). In contrast, Titheradge & Coore (1976) have reported that activation of pyruvate transport does occur. In the present paper I demonstrate that mitochondria from glucagon-pretreated rats do show an enhanced rate of pyruvate transport under conditions of metabolism, but that this is not caused by an increase in the V,,,. activity of the pyruvate transporter. Rather it is produced by an increase in the mitochondrial OH- ion concentration, which is rate-limiting for pyruvate transport. Female Wistar rats (250-3OOg) were treated with glucagon (Wpglkg, intraperitoneally) as described by Adam & Haynes (1969). Liver mitochondria were prepared as described previously (Halestrap, 1975) and initial rates of pyruvate transport and pyruvate metabolism were measured as described in the legends to the Figures. Mitochondria] matrix volumes and uptake of [14C]methylaminewere measured under the same conditions as for pyruvate metabolism, but with additions of 3Hz0(1 pCi/ml) and [U-'4C]sucrose (0.1 pCi/ml), or [6,6'-3H]sucrose (1pCi/ml) and [14C]methylamine (0.5m ~ 0.1 , pCi/rnl) as required. Mitochondria were separated from the incubation medium and analysed for I4C, 3H and protein as described previously (Halestrap, 1975). Treatment of rats with glucagon consistently allowed the isolation of liver mitochondria which metabolized pyruvate more rapidly than mitochondria from control rats. In 16 separate experiments the rate of pyruvate metabolism at 37°C was increased from 15.56k2.17 to 32.4:f:4.76nmol/min per mg of mitochondrial protein. As shown in Fig. 1, however, no change in the rate of pyruvate transport at 6°C was detected after glucagon treatment of rats, despite large changes in the rate of pyruvate metabolism. This is in contrast with the findings of Titheradge & Coore (1976), who, however, measured pyruvate transport under conditions where pyruvate is accumulated by means of a respiration-induced pH gradient, whereas in the experiments reported here pH gradients were induced artificially. To test whether this difference in technique might explain the difference in results, it seemed necessary to establish whether pyruvate transport was rate-limiting in mitochondrial metabolism of pyruvate. To do this, use was made of the specific non-competitive inhibitor of pyruvate transport, a-cyano-4-hydroxycinnamate (Halestrap, 1975; Halestrap & Denton, 1975). By adding increasing concentrations of this inhibitor and measuring the rate of pyruvate metabolism it was possible to show that pyruvate transport does limit metabolism under these conditions. Further, the maximal rate of pyruvate transport and the K , for inhibition by a-cyano-4-hydroxycinnamatecan be calculated from a Dixon plot (Fig. 2). 1977
217
566th MEETING, CAMBRIDGE
I 0
I
I
I
I
1
3
I
4
Time (min)
Fig. 1. Initial rates of pyruvate transport into mitochondriafrom control and glucagontreated animals Pyruvate uptake was measured at 6°C by the inhibitor-stop technique described previously (Halestrap, 1975). Uptake was initiated by the addition of mitochondria at pH 7.5 and 0°C (final concentration 5mg of protein/ml) into medium 0.25~-sucrose-lOm~3-(N-morpholino)propanesulphonate 1m~-EGTA, pH 6.8 containing 10,uuM-rotenone, S,u~-antirnycin,0.5 mwpyruvate (0.1 PCilrnl) and [6,6'-3H]sucrose (1PCilml)} at 6°C and terminated by addition of 2m~-a-cyano-4-hydroxycinnamate.The initial rates of pyruvate transport calculated by non-linear least-squares regression were 2.01 kO.19 and 2.08 kO.13 nmol/min per mg of protein for mitochondria from control (0)and glucagon-treated (m) rats respectively. The rates of metabolism of pyruvate by these mitochondria at 37"C, measured as described in Fig. 2, were 8.1 and 14.6nmol/min per mg of protein respectively (results are the mean of two estimatesagreeing within 5 %). This experiment is representative of six similar experiments. The plot of Fig. 2, which is representative of several such experiments, shows that not only does glucagon pretreatment of rats increase the rate of pyruvate transport, but that it also decreases the K, for a-cyano4hydroxycinnamate by an equivalent amount. The rate of pyruvate transport into the mitochondria is limited by the matrix concentration of exchangeable ions (Halestrap, 1975; Paradies & Papa, 1976; Titheradge & Coore, 1976) and the normal exchangeable ion is OH-. a-Cyanocinnamate and its derivatives inhibit pyruvate transport by attacking a thiol group on the inside of the inner mitochondrial membrane (Halestrap, 1976; A. P. Halestrap, unpublished work). Since these inhibitors cross the mitochondrial membrane in exchange for an OH- ion (Halestrap, 1975), their Kifor inhibition of pyruvate transport into intact mitochondria will depend on the mitochondrial pH gradient. Thus the increase in pyruvate transport and the decrease in the Kifor a-cyano4hydroxycinnamatecaused by glucagon is consistent with an increase in mitochondrial pH. Measurement of this by an adaptation of the method of Nicholls (1974) showed that under metabolizing conditions in the presence of b i c a r b nate, the matrix was more acid than the medium and accumulation of r4C]methylamine VOl. 5
218
BIOCHEMICAL SOCIETY TRANSACTIONS
-
7
0.18-
h
!I 2
0.16-
0
100
200
300
400
500
[a-Cyano-4-hydroxycinnamate](PM)
Fig. 2. Dixonplot of the inhibition ofpyruoate metabolism by a-cyano4hydroxycinnamate in mitochondria from control and glucagon-treated rats Pyruvate metabolism was measured by the disappearance of enzymically assayable pyruvate from the medium. Mitochondria (approx. 3mg of protein) were incubated at 37°C in medium containing 250m~-sucrose, 10mM-Tris/HCl, 1mM-EGTA, 2 m ~ potassium phosphate, 10m - K H C 03, 3.5 mM-sodium pyruvate and a-cyano-4-hydroxycinnamate as indicated. Uptake of pyruvate, which was linear with time for at least 20min, was terminated after lOmin by the addition of 0.1 ml of 20% (w/v) HC104/ml of medium, and the pyruvate remaining in the medium assayed enzymically (Hohorst, 1963) after centrifugation. The maximal rate of pyruvate metabolism and Kl for a-cyanoChydroxycinnamate were calculated by a non-linear least-squares regression of the data. was 14.23+0.32nmol/min per mg of proFor mitochondria from control rats ( 0 )V,,,,,. tein and the Kiwas 3 2 4 f 2 7 b ~ ,and for mitochondria from glucagon-treated rats).( the values were 36.48+ 0.33 and 135f6 respectively. This experiment is representative of five similar experiments.
could be. used to estimate the mitochondria1 pH. From measurements of the matrix volume and the uptake of [14C]methylamineit was shown that control mitochondria concentrated methylamine 17.62f 1.I-fold, whereas glucagon mitochondria showed a concentration of 11.45+0.59-fold (results are the mean+_s.E.M.of 60 observations on 15 different mitochondrial preparations). Thus glucagon appears to allow the maintenance of a less acid matrix pH, which in turn would stimulate pyruvate transport. The presence of an acid matrix in mitochondria metabolizing pyruvate under these conditions was not 1977
566th MEETING, CAMBRIDGE
219
expected. However, it was confirmed by measuring the accumulation of enzymically assayable ammonia. The very high K , for a-cyano-4-hydroxycinnamatemeasured under these conditions is also consistent with an acid matrix. The value of 6 p reported ~ previously (Halestrap, 1975) was determined under conditions where the mitochondrial matrix was more alkaline than the medium. Addition of valinomycin to mitochondria metabolizing pyruvate caused the matrix to become alkaline, and pyruvate metabolism was increased considerably. The results in the present paper are consistent with an action of glucagon on the mitochondrial pH gradient. Consistent with this finding is the observation that glucagon treatment of the perfused liver may increase the mitochondrial content of anionic metabolites (Parrilla et al., 1975) and that glucagon causes a stimulation of the phosphorylation of the mitochondrial membrane (Zahlten et al., 1972). During the course of this work I was a Beit Memorial Research Fellow. Adam, P. A. J. & Haynes, R. C. (1969) J. Biol. Chem. 244,6444-6450 Garrison, J. C. & Haynes, R. C. (1975) J. Biol. Chem. 250,2769-2777 Halestrap, A. P. (1975) Biochem. J. 148,85-96 Halestrap, A. P. (1976) Biochem. J. 156, 181-183 Halestrap, A. P. & Denton, R. M. (1975) Biochem. J . 148,97-106 Hohorst, H. J. (1963) in Methods of Enzymafic Analysis (Bergmeyer, H.-U., ed.), pp. 266-270, Verlag Chemie, Berlin Nicholls, D. G. (1974) Eur. J. Biochem. 50,305-315 Paradies, G. & Papa, S. (1976) FEBSLPtt. 62,318-321 Parrilla, R., Jimenez, I. & Ayuso-Parrilla, M. S. (1975) Eur. J. Biochem. 56, 375-383 Titheradge, M. A. & Coore, H. G. (1976) FEBS Left. 63,45-50 Zahlten, R. N., Hochberg, A. A., Stratman,F. W. & Lardy, H.A. (1972) Proc. Nufl. Acad. Sci. U.S.A. 69,800-804
Mechanism of Action of Somatostatin:Growth-Hormone Release, [45Ca]CalciumIon Efflux and Cyclic Nucleotide Metabolism of Bovine Anterior-Pituitary Slices in the Presence of Prostaglandin E2 and 1-Methyl-3-isobutylxanthine R. J. BICKNELL,* P. W. YOUNG,* J. G. SCHOFIELD* and JANET ALBANOt *Department of Biochemistry, and ?Department of Medicine, Medical School, University of Bristol, University Walk, Bristol BS8 1 TD, U.K.
Several lines of evidence indicate that the increase in growth-hormone release observed in vitro in the presence of prostaglandins and methylxanthines, dibutyryl cyclic AMP and
hypothalamic extracts presumed to contain the growth-hormone-releasing factor, is due to an increase in the intracellular concentration of cyclic AMP. Thus the ability of prostaglandins and hypothalamic extracts to stimulate growth-hormone release is enhanced in the presence of methylxanthine phosphodiesterase inhibitors, and the rise in cyclic AMP concentration apparently precedes the release of growth hormone (Steiner et al., 1970; Schofield & McPherson, 1974). Some additional evidence indicates that a redistribution of intracellular Caz+ mediates this increased growth-hormone release. Thus the increase in hormone release, but not cyclic AMP concentration, is inhibited by although verapamil (an inhibitor of Caz+entry into cells) does depletion of tissue Caz+, not inhibit the hormone release @to et al., 1974). It has been suggested that somatostatin, a peptide isolated from sheep hypothalami and capable of inhibiting growth-hormone secretion, acts by modifying the cyclic nucleotide response in the pituitary. In support of this suggestion, it has been shown in rat pituitaries that somatostatin decreases the rise in cyclic AMP in the presence of prostaglandins and theophylline (Borgeat et al., 1974), and that somatostatin increases the concentration of cyclic GMP (Kaneko et al., 1974). We here report that somatostatin, VOl. 5
BIOCHEMICAL SOCIETY TRANSACTIONS
Jones, G. R. N. (1975)Med. Hypotheses 1, 118-127 Jones, G.R. N. (1976~)Med. Hypotheses 2, 50-54 Jones, G. R.N. (19766)Annual Report, Inst. Basic Med. Sci., University ofLondon in the press Jones, G. R. N. (1977)Biochem. SOC.Trans. 5, 213-214 Klingenberg, M. & Slenczka, W. (1959)Biochem. Z . 331,486-517 Nauts, H.C.,Swift, W. E. & Coley, B. L. (1946) Cancer Res. 6,205-216 Shapiro, C . J. (1940)Am..I. Hyg. 31B, 114-126 Shear, M.J. & Andervont, H. B. (1936)Proc. SOC.Exp. Biol. Med. 34,323-325 Thorne, R. F. W. & Bygrave, F. L. (1974)Biochem. J. 144, 551-558 Weinbach, E. C. & Garbus, J. (1966)J. Biol. Chem. 241, 3708-3713
The Mechanism of the Stimulation of Pyruvate Transport into Rat Liver Mitochondria by Glucagon ANDREW P. HALESTRAP Department of Biochemistry, University of Bristol, Bristol BS8 1T D , U.K.
Mitochondria isolated from the livers of rats previously treated with glucagon show an enhanced rate of pyruvate metabolism (Adam & Haynes, 1969). This effect has also been demonstrated with isolated liver cells (Garrison & Haynes, 1975). The suggestion was made that this effect on pyruvate metabolism was at the level of pyruvate transport into the mitochondrion (Adam & Haynes, 1969). However, I have shown previously that the pyruvate transporter is not activated under these conditions (Halestrap, 1975). In contrast, Titheradge & Coore (1976) have reported that activation of pyruvate transport does occur. In the present paper I demonstrate that mitochondria from glucagon-pretreated rats do show an enhanced rate of pyruvate transport under conditions of metabolism, but that this is not caused by an increase in the V,,,. activity of the pyruvate transporter. Rather it is produced by an increase in the mitochondrial OH- ion concentration, which is rate-limiting for pyruvate transport. Female Wistar rats (250-3OOg) were treated with glucagon (Wpglkg, intraperitoneally) as described by Adam & Haynes (1969). Liver mitochondria were prepared as described previously (Halestrap, 1975) and initial rates of pyruvate transport and pyruvate metabolism were measured as described in the legends to the Figures. Mitochondria] matrix volumes and uptake of [14C]methylaminewere measured under the same conditions as for pyruvate metabolism, but with additions of 3Hz0(1 pCi/ml) and [U-'4C]sucrose (0.1 pCi/ml), or [6,6'-3H]sucrose (1pCi/ml) and [14C]methylamine (0.5m ~ 0.1 , pCi/rnl) as required. Mitochondria were separated from the incubation medium and analysed for I4C, 3H and protein as described previously (Halestrap, 1975). Treatment of rats with glucagon consistently allowed the isolation of liver mitochondria which metabolized pyruvate more rapidly than mitochondria from control rats. In 16 separate experiments the rate of pyruvate metabolism at 37°C was increased from 15.56k2.17 to 32.4:f:4.76nmol/min per mg of mitochondrial protein. As shown in Fig. 1, however, no change in the rate of pyruvate transport at 6°C was detected after glucagon treatment of rats, despite large changes in the rate of pyruvate metabolism. This is in contrast with the findings of Titheradge & Coore (1976), who, however, measured pyruvate transport under conditions where pyruvate is accumulated by means of a respiration-induced pH gradient, whereas in the experiments reported here pH gradients were induced artificially. To test whether this difference in technique might explain the difference in results, it seemed necessary to establish whether pyruvate transport was rate-limiting in mitochondrial metabolism of pyruvate. To do this, use was made of the specific non-competitive inhibitor of pyruvate transport, a-cyano-4-hydroxycinnamate (Halestrap, 1975; Halestrap & Denton, 1975). By adding increasing concentrations of this inhibitor and measuring the rate of pyruvate metabolism it was possible to show that pyruvate transport does limit metabolism under these conditions. Further, the maximal rate of pyruvate transport and the K , for inhibition by a-cyano-4-hydroxycinnamatecan be calculated from a Dixon plot (Fig. 2). 1977
217
566th MEETING, CAMBRIDGE
I 0
I
I
I
I
1
3
I
4
Time (min)
Fig. 1. Initial rates of pyruvate transport into mitochondriafrom control and glucagontreated animals Pyruvate uptake was measured at 6°C by the inhibitor-stop technique described previously (Halestrap, 1975). Uptake was initiated by the addition of mitochondria at pH 7.5 and 0°C (final concentration 5mg of protein/ml) into medium 0.25~-sucrose-lOm~3-(N-morpholino)propanesulphonate 1m~-EGTA, pH 6.8 containing 10,uuM-rotenone, S,u~-antirnycin,0.5 mwpyruvate (0.1 PCilrnl) and [6,6'-3H]sucrose (1PCilml)} at 6°C and terminated by addition of 2m~-a-cyano-4-hydroxycinnamate.The initial rates of pyruvate transport calculated by non-linear least-squares regression were 2.01 kO.19 and 2.08 kO.13 nmol/min per mg of protein for mitochondria from control (0)and glucagon-treated (m) rats respectively. The rates of metabolism of pyruvate by these mitochondria at 37"C, measured as described in Fig. 2, were 8.1 and 14.6nmol/min per mg of protein respectively (results are the mean of two estimatesagreeing within 5 %). This experiment is representative of six similar experiments. The plot of Fig. 2, which is representative of several such experiments, shows that not only does glucagon pretreatment of rats increase the rate of pyruvate transport, but that it also decreases the K, for a-cyano4hydroxycinnamate by an equivalent amount. The rate of pyruvate transport into the mitochondria is limited by the matrix concentration of exchangeable ions (Halestrap, 1975; Paradies & Papa, 1976; Titheradge & Coore, 1976) and the normal exchangeable ion is OH-. a-Cyanocinnamate and its derivatives inhibit pyruvate transport by attacking a thiol group on the inside of the inner mitochondrial membrane (Halestrap, 1976; A. P. Halestrap, unpublished work). Since these inhibitors cross the mitochondrial membrane in exchange for an OH- ion (Halestrap, 1975), their Kifor inhibition of pyruvate transport into intact mitochondria will depend on the mitochondrial pH gradient. Thus the increase in pyruvate transport and the decrease in the Kifor a-cyano4hydroxycinnamatecaused by glucagon is consistent with an increase in mitochondrial pH. Measurement of this by an adaptation of the method of Nicholls (1974) showed that under metabolizing conditions in the presence of b i c a r b nate, the matrix was more acid than the medium and accumulation of r4C]methylamine VOl. 5
218
BIOCHEMICAL SOCIETY TRANSACTIONS
-
7
0.18-
h
!I 2
0.16-
0
100
200
300
400
500
[a-Cyano-4-hydroxycinnamate](PM)
Fig. 2. Dixonplot of the inhibition ofpyruoate metabolism by a-cyano4hydroxycinnamate in mitochondria from control and glucagon-treated rats Pyruvate metabolism was measured by the disappearance of enzymically assayable pyruvate from the medium. Mitochondria (approx. 3mg of protein) were incubated at 37°C in medium containing 250m~-sucrose, 10mM-Tris/HCl, 1mM-EGTA, 2 m ~ potassium phosphate, 10m - K H C 03, 3.5 mM-sodium pyruvate and a-cyano-4-hydroxycinnamate as indicated. Uptake of pyruvate, which was linear with time for at least 20min, was terminated after lOmin by the addition of 0.1 ml of 20% (w/v) HC104/ml of medium, and the pyruvate remaining in the medium assayed enzymically (Hohorst, 1963) after centrifugation. The maximal rate of pyruvate metabolism and Kl for a-cyanoChydroxycinnamate were calculated by a non-linear least-squares regression of the data. was 14.23+0.32nmol/min per mg of proFor mitochondria from control rats ( 0 )V,,,,,. tein and the Kiwas 3 2 4 f 2 7 b ~ ,and for mitochondria from glucagon-treated rats).( the values were 36.48+ 0.33 and 135f6 respectively. This experiment is representative of five similar experiments.
could be. used to estimate the mitochondria1 pH. From measurements of the matrix volume and the uptake of [14C]methylamineit was shown that control mitochondria concentrated methylamine 17.62f 1.I-fold, whereas glucagon mitochondria showed a concentration of 11.45+0.59-fold (results are the mean+_s.E.M.of 60 observations on 15 different mitochondrial preparations). Thus glucagon appears to allow the maintenance of a less acid matrix pH, which in turn would stimulate pyruvate transport. The presence of an acid matrix in mitochondria metabolizing pyruvate under these conditions was not 1977
566th MEETING, CAMBRIDGE
219
expected. However, it was confirmed by measuring the accumulation of enzymically assayable ammonia. The very high K , for a-cyano-4-hydroxycinnamatemeasured under these conditions is also consistent with an acid matrix. The value of 6 p reported ~ previously (Halestrap, 1975) was determined under conditions where the mitochondrial matrix was more alkaline than the medium. Addition of valinomycin to mitochondria metabolizing pyruvate caused the matrix to become alkaline, and pyruvate metabolism was increased considerably. The results in the present paper are consistent with an action of glucagon on the mitochondrial pH gradient. Consistent with this finding is the observation that glucagon treatment of the perfused liver may increase the mitochondrial content of anionic metabolites (Parrilla et al., 1975) and that glucagon causes a stimulation of the phosphorylation of the mitochondrial membrane (Zahlten et al., 1972). During the course of this work I was a Beit Memorial Research Fellow. Adam, P. A. J. & Haynes, R. C. (1969) J. Biol. Chem. 244,6444-6450 Garrison, J. C. & Haynes, R. C. (1975) J. Biol. Chem. 250,2769-2777 Halestrap, A. P. (1975) Biochem. J. 148,85-96 Halestrap, A. P. (1976) Biochem. J. 156, 181-183 Halestrap, A. P. & Denton, R. M. (1975) Biochem. J . 148,97-106 Hohorst, H. J. (1963) in Methods of Enzymafic Analysis (Bergmeyer, H.-U., ed.), pp. 266-270, Verlag Chemie, Berlin Nicholls, D. G. (1974) Eur. J. Biochem. 50,305-315 Paradies, G. & Papa, S. (1976) FEBSLPtt. 62,318-321 Parrilla, R., Jimenez, I. & Ayuso-Parrilla, M. S. (1975) Eur. J. Biochem. 56, 375-383 Titheradge, M. A. & Coore, H. G. (1976) FEBS Left. 63,45-50 Zahlten, R. N., Hochberg, A. A., Stratman,F. W. & Lardy, H.A. (1972) Proc. Nufl. Acad. Sci. U.S.A. 69,800-804
Mechanism of Action of Somatostatin:Growth-Hormone Release, [45Ca]CalciumIon Efflux and Cyclic Nucleotide Metabolism of Bovine Anterior-Pituitary Slices in the Presence of Prostaglandin E2 and 1-Methyl-3-isobutylxanthine R. J. BICKNELL,* P. W. YOUNG,* J. G. SCHOFIELD* and JANET ALBANOt *Department of Biochemistry, and ?Department of Medicine, Medical School, University of Bristol, University Walk, Bristol BS8 1 TD, U.K.
Several lines of evidence indicate that the increase in growth-hormone release observed in vitro in the presence of prostaglandins and methylxanthines, dibutyryl cyclic AMP and
hypothalamic extracts presumed to contain the growth-hormone-releasing factor, is due to an increase in the intracellular concentration of cyclic AMP. Thus the ability of prostaglandins and hypothalamic extracts to stimulate growth-hormone release is enhanced in the presence of methylxanthine phosphodiesterase inhibitors, and the rise in cyclic AMP concentration apparently precedes the release of growth hormone (Steiner et al., 1970; Schofield & McPherson, 1974). Some additional evidence indicates that a redistribution of intracellular Caz+ mediates this increased growth-hormone release. Thus the increase in hormone release, but not cyclic AMP concentration, is inhibited by although verapamil (an inhibitor of Caz+entry into cells) does depletion of tissue Caz+, not inhibit the hormone release @to et al., 1974). It has been suggested that somatostatin, a peptide isolated from sheep hypothalami and capable of inhibiting growth-hormone secretion, acts by modifying the cyclic nucleotide response in the pituitary. In support of this suggestion, it has been shown in rat pituitaries that somatostatin decreases the rise in cyclic AMP in the presence of prostaglandins and theophylline (Borgeat et al., 1974), and that somatostatin increases the concentration of cyclic GMP (Kaneko et al., 1974). We here report that somatostatin, VOl. 5
