- Email: [email protected]
Differential effects of somatostatin on adenylate cyclase as functional correlate for different brain somatostatin receptor subpopulations
Neuroscience Letters, 104 (1989) 13 18 Elsevier Scientific Publishers Ireland Ltd.
13
NSL 06295
Differential effects of somatostatin on adenylate cyclase as functional correlate for different brain somatostatin receptor subpopulations R. Markstein l, K.A. St6ckli l'* and J.C. Reubi 2 ISandoz Preclinical Research, Basel (Switzerland) and 2Sandoz Research Institute, Bern (Switzerland) (Received 12 September 1988; Revised version received 11 May 1989; Accepted 12 May 1989) Key words." Somatostatin; Adenylate cyclase; Somatostatin receptor In homogenates of rat hippocampus and striatum, but not substantia nigra, somatostatin (SRIF) inhibits forskolin-activated adenylate cyclase in nanomolar concentrations. However, SRIF can also stimulate adenylate cyclase in micromolar concentrations in homogenates of rat hippocampus and substantia nigra. The SRIF octapeptide SMS 201-995 solely inhibits the forskolin-activated adenylate cyclase in the 3 preparations. These results suggest that the SRlF-specific stimulation of adenylate cyclase may be a functional correlate for the brain-specific SRIF receptor subpopulation, whereas the SRIF and SMS 201-995 inhibition of stimulated adenylate cyclase correlate with the SRIF receptor subpopulation present in brain and non-neuronal tissues.
The tetradecapeptide somatostatin (SRIF) is widely distributed throughout the central nervous system [8], where it is thought to function as a neurotransmitter or neuromodulator [12]. Its mechanism of action is not yet completely elucidated. However, there is convincing evidence suggesting that its effects are mediated by specific SRIF receptors located in various brain areas such as cortex, limbic system and basal ganglia [14, 15]. Interestingly, it has been proposed on the basis of pharmacological studies that at least 2 different SRIF receptor subtypes exist in brain tissue, which can be differentiated by reduced-size SRIF analogs such as SMS 201-995 [1]. One type of SRIF receptors has high affinity for SMS 201-995, is present in most brain areas including cortex, hippocampus or striatum, as well as in non-neuronal, peripheral tissues, and has been named SSI or SSA [10, 13, 17]. The other receptor type has only low affinity for the SMS 201-995, is preferentially located in neuronal tissue, often in the same areas as SSI receptors, and is named SS2 or SSB receptor [13, 17]. Since the post-receptor mechanism of action of SRIF includes, at least in part, effects on adenylate cyclase both in brain and in non-neuronal tissue such as pituitary Correspondence: R. Markstein, Sandoz Preclinical Research, 4002 Basel, Switzerland. *Present address: Max Planck Institute, Martinsried, F.R.G. 0304-3940/89,$ 03.50 (~'~1989 Elsevier Scientific Publishers Ireland Ltd.
14 [2, 3, 5-7, 9, I I], we investigated in this study how the different S R I F receptor subpopulations in rat h i p p o c a m p u s , striatum a n d s u b s t a n t i a nigra are related to the adenylate cyclase system. The d e t e r m i n a t i o n of adenylate cyclase activity was performed as tbllows: male rats (Sandoz O F A strain) weighing 150 200g were pretreated twice with reserpine (2.5 mg/kg, s.c.) 18 a n d 24 h before sacrifice by decapitation. A n i m a l s were pretreated with reserpine with the aim of depleting g r a n u l a r stores of d o p a m i n e a n d serotonin, in order to avoid e n d o g e n o u s m o n o a m i n e s interacting with the action of somatostatin on the adenylate cyclase. The brains were rapidly removed a n d striatum, hippoc a m p u s a n d s u b s t a n t i a nigra dissected. The tissues were homogenized in 15 volumes (w/v) of ice-cold 2 m M Tris-acetate buffer (pH 7.4) c o n t a i n i n g 2 m M ethylenglycol-
Fig. I. Visualization of SRIF receptors in the rat brain, a ~: autoradiograms showing SRIF receptors labelled with ~251-[Leu~,D-Trpz2, Tyr25]SRIF-28as radioligand, a: total binding (SS~ and SS2 receptors). b: non-specificbinding in presence of 0.1 /~M SRIF. c: binding in presence of 0.1 ttM SMS 201-995; only SS, receptors remain visualized, d, e: autoradiograms showing SRIF receptors labelled with 12511204-0901 as radioligand, d: total binding showing SSI sites, c: non-specificbinding in presence of 0.1 I~M 204-090. I = cortex, 2 = hippocampus, 3 = substantia nigra, 4 = central grey. The right-left asymmetry in receptor densities in some areas is due to the use of cryostat sections slightly non-parallel to the coronal plane. Exposure time: I week. Bar - 1 mm.
15
bis-(2-aminoethylether)-N,N-tetraacetic acid (EGTA). Two ml aliquots were quickly frozen by immersing the tubes into a mixture of dry ice and acetone and stored at - 7 0 ° C until use. Test compounds either alone or in combination, were incubated in 400/11 of a mixture containing 120 mM Tris-acetate (pH 7.4), 2 mM MgC12, 1.12 mM EGTA, 0.25 mM 3 isobutyl-l-methylxanthine, 0.15% bovine serum albumine, and 100/zl tissue homogenate (diluted to a final protein content of 107.5/tg/ml) during 3 min at 37°C. In some experiments MgCI2 concentration was increased to 4 mM. Bacitracin (10/zM) was included in all assays where SRIF or SMS 201-995 were present in order to prevent degradation by proteases. The enzyme reaction was initiated by adding 100/tl of a solution containing 2.5 mM ATP and 500/zM GTP (pH 7.4) and terminated 4 min later by heating the mixture to 95°C for 4 rain. Supernatant samples (50/zl) were acetylated according to Frandsen and Krishna [4] and cyclic AMP content measured by radioimmunoassay as described by Steiner et al. [16]. Pro-
TABLE I EFFECT OF SRIF AND SMS 201-995 ON CYCLIC AMP FORMATION IN HOMOGENATES OF VARIOUS RAT BRAIN REGIONS Homogenates were incubated in a standard assay buffer (Mg 2+ concentration 4 mM) containing bacitracin (10/~M). Various concentrations of drugs were added as indicated. Cyclic A M P content was measured 3 min after addition of ATP/GTP solution. Values are changes of cyclic A M P content (pmol/mg protein per rain) versus control values. Control values were: hippocampus 245 ± 3.7; substantia nigra 310.7__+3.9; striatum 222.9±2.8. All values are mean ± S.E.M. All data are means of at least 3 independent assays which were performed in triplicates. 5-HT, serotonin; DA, dopamine.
Dose (,uM)
0.31 0.63 1.25 2.5 5.0 10.0
Hippocampus
Substantia nigra
Striatum
SRIF
SMS 201-995
SRIF
SRIF
21.1± 0.1"* 47.7± 4.7** 49.6± 4,5*** 31.9± 1.1"** 21.1± 2.1"* 3.2± 3.1
0.1± 1.0 4.1± 4.0 0.2± 1.1 6.8± 4.3 3.4± 3.1 0.2± 0.7
1.25/zM 5-HT 63.8 ± 2.8** 125 ItM DA
7.2± 1.1 15.6± 4.4* 30.6± 7.8** 12.2± 0.5* 2.8± 0.5 0 ± 0.5
120.3+ 44***
SMS 201-995 1.1± 1.1 2.8± 2.9 7.2± 4.4 2.2± 2.2 4.4+ 2.2 2.8± 2.2
--32.5± 3.8*** --40.1± 6,7** --48.6± 4.9*** -36.1± 7.1"* --18.3± 8.2 0 ± 2.6
SMS 201-995 -25 ± 9.0* -19.4± 2.6* -- 4.0± 2.2 0 ± 0.4 -- 2.4± 3.8 - 0.1± 1.6
179.8+ 2.0***
Statistical significance: *2P< 0.05; **P<0.01; ***2P<0.001 (Student's two tailed t-test).
16
tein content was determined by the biuret method. All determinations were performed in triplicate. Data was expressed as mean values i S.E.M. and significance levels for the comparison between mean values were calculated according to Student’s r-test for unpaired SRIF either
receptors the SRIF-28
““I-[Tyr3]SMS
observation.
were visualised analog
201-995
in IO ,Lim cryostat
“‘I- [ Leus , D-Trp’?,
(‘z~1-204-090)
sections
Tyrz5]SRIF-28
as radioligands,
of the rat brain
using
or the octapeptide
as described
in detail
else-
where [l4, 151. Fig. I. is an autoradiographic representation of the 2 SRIF receptor populations found in the rat cortex, hippocampus and substantia nigra. using 2 different SRIF radioligands, as described extensively for the human brain [14]. It can be seen that in the rat cortex and hippocampus both SS, and SSz receptors are present (Fig. la,d). whereas in the substantia nigra only SS2 receptors are found (Fig. la,c) which are not labelled with the octapeptide SRIF radioligand “51-204-090 (Fig. Id). In the striatum (not shown) SS, sites predominate [I 31. Table I shows that in homogenates of hippocampus and substantia nigra SRIF (in the presence of2 mM Mg’ +) significantly stimulated adenylate cyclase in the micromolar concentration range. The doses response curves were biphasic in both tissue preparations with a maximum effect occurring at 1.25 ,YM and amounting to 120% and I IO % of basal activity in homogenates of hippocampus and substantia nigra, respectively. The ECs,,-value was 0.3 /IM in both tissue preparations. Under the same experimental conditions SMS 201-995 failed to stimulate adenylate cyclase in both tissue preparations. In homogenates of striatum, SRIF and SMS 201-995 by itself had no stimulating effect but reduced basal activity of adenylate cyclase by 22% at I .X jtM and by 9% at 0.63 /IM. respectively. Table II shows that forskolin markedly stimulated adenylate cyclase activity in all 3 tissue preparations. In homogenates of hippocampus and substantia nigra 5 /IM forskolin (in the presence of 4 mM Mg’ ’ ) enhanced basal activity approximately -I-fold and in homogenates of striatum approximately ?I)-fold. SRIF and SMS 201-995 inhibited the effect of 5 PM forskolin dose-dependently in homogenates of hippocampus and striatum in the nanomolar concentration range. The maximal inhibitory effect of SRIF occurred at I and IO ,uM and amounted to 13.2~ in the hippocampal and to 39.5% in the striatal homogenate. SMS 201-995. although less potent than SRIF. produced the same maximal inhibition of the forskolin effect in the hippocampus. The inhibitory effect of SMS 201-995 in the striatal homogenate was clearly smaller than that of SRIF. In homogenates of the substantia nigra SMS 20 l-995 in a concentration range of 0.001 IO ,uM significantly inhibited the effect of forskolin whereas SRIF was inactive. The present data. therefore. show that SRIF can produce a differential, opposite effect on adenylate cyclase in different brain regions. In homogenates of hippocampus, SRIF both inhibits stimulated adenylate cyclase activity in the nanomolar range and stimulates basal adenylate cyclase in the micromolar range. In the substantia nigra. only a stimulation of basal adenylate cyclase is seen whereas in the striatum only inhibition of stimulated cyclasc is observed in presence of SRIF. A correlation may be drawn between these SRIF effects and the various SRIF receptor popda-
17 TABLE 1I EFFECT OF SRIF AND SMS 201-995 ON FORSKOLIN-STIMULATED CYCLIC AMP FORMATION IN HOMOGENATES OF VARIOUS BRAIN AREAS Homogenates were incubated in a standard assay buffer (Mg 2+ concentration 2 mM) containing bacitracin (10 ItM) and forskolin (5/tM). Various concentrations of drugs were added as indicated. Cyclic AMP content was measured 3 min after addition of ATP/GTP solution. Values represent cyclic AMP content (pmol/mg protein per min) expressed as percentage of the increase by 5 ltM forskolin. The increase of cyclic AMP content by forskolin (5 :tM) was (basal value in parenthesis): hippocampus 520.9+3.2 (68.8+ 1.5); substantia nigra 719+ 10.2 (100.4+ 1.2); striatum 4081 +26.9 (200.9+3.8). All values are mean +_ S.E.M.,n=4. % of Forskolin effect Dose
Hippocampus
Substantia nigra
Striatum
SRIF
SMS 201-995
SRIF
SMS 201-995
SRIF
SMS 201-995
93.9+ 1.3"* 89.0+ 2.8** 79.6+ 0.7*** 76.8+ 1.1"**
99.1 + 0.2 94.5+ 1.4' 92.2+ 0.6*** 78.6+ 1.1"**
96.7+ 3.1 92.5+ 3.3
72.4+ 2.6***
87.4+ 3.0** 83.8+ 0.9*** 83.7+ 1.3*** 81.0+ 3.7** 82.7-t3.1"* 90.4+ 0.7***
72.8+ 4.9** 65.3+ 4.3***
74.0+ 1.1"**
95.5+ 4.4 95.3+ 3.7 99.7+ 3.6 102.7__+ 5.9 112.1+ 2.5 103.8+ 0.3
60.5+ 4.7*** 79.3+ 5.4** 90.7+ 3.0*
89.6+ 2.1"* 89.5__ 2.8* 86.6+ 4.4*
~M)
0.001 0.01 0.1 1.0 5.0 10.0
Statistical significance: *2P< 0.05; **2P<0.01; ***2P< 0.001 (Student's two tailed t-test)
tions, since we know that the hippocampus has both SS1 and S S 2 receptors, whereas the substantia nigra has mainly SS2 and the striatum SSI receptors [10, 13, 14, 17]. Moreover, SMS 201-995, devoid of stimulating effects on adenylate cyclase in homogenates of rat hippocampus and substantia nigra has only a low affinity to the SS2 sites [13]. These results therefore suggest that SS2 sites (i.e. in the substantia nigra) mediate a stimulation of adenylate cyclase, whereas SSI sites mediate inhibitory effects. Inhibitory effects of SRIF adenylate cyclase activity have been described earlier in non-neuronal tissues considered SRIF target tissues [5-7, 9, 11]. However, SRIFspecific stimulatory effects on this enzyme have to our knowledge not yet been reported. Its physiological significance as well as its cellular localisation (gliai vs neuronal) remain to be determined. 1 Bauer, W., Briner, U., Doepfner, W., Haller, R., Huguenin, R., Petcher, T.P. and Pless, J., SMS 201-995: a very potent and selective octapeptide analogue of somatostatin with prolonged action, Life Sci., 31 (1982) 1133-1140.
18 2 Schneiweiss, H.. Bertrand, Ph., Epelbaum, J., Kordon, C., Glowinski, J., Premont, J. and Enjalbert, A., Somatostatin receptors on cortical neurones and adenohypophysis: comparison between specific binding and adenylate cyclase inhibition, Eur. J. Pharmacol. 138 (1987) 249 255. 3 Schneiweiss, H., Glowinski. J. and Pr6mont, J., Modulation of monoamines of somatostatin-sensitive adenylate cyclasc on neuronal and glial cells from the mouse brain in primary cultures, .I. Neurochem., 44(1985) 1825 1831. 4 Frandsen, E.K. and Krishna, G., A simple ultrasensitive method for the assay of cyclic AMP and cyclic GMP in tissues, Life Sci., 18 (1976) 529 542. 5 tlarwood, J.P., Grewe, (7, and Aguilera, G., Actions of growth hormone-releasing l:actor and somatostatin on adenylate cyclase and growth hormone release in rat anterior pituitary, Mol. Cell. Endocrinol., 37 (19841 277 284. 6 lfcisler, S. and Reisine, T., Forskolin stimulates adenylate cyclase activity, cyclic A M P accumulation and adrenocorticolropin secretion from mouse anterior pituitary tumor cells, J. Neurochem., 42 (1984) 1659 1666. 7 Heisler, S. and Srikant, C,B., Somatostatin-14 and somatostatin-28 pretreatment down-regulate somatostatm-14 receptors and have biphasic effects on forskolin-stimulated cyclic adenosine, 3",5"-monophosphate synthesis and adrenocorticotropin secretion in mouse anterior pituitary tumor cells, Endocrinology, 117 (1985) 217 225. 8 .lohansson. O., tt6kfelt, T. and Else. R., lmmunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the rat, Neuroscience, 13 (1984) 265 339. 9 Koch, B.D. and Schonbrunn. A., The somatostatin receptor is directly coupled to adenylate cyclase in GH4C~ pituitary cell membranes, Endocrinology, 114 (1984) 1784 1790. 10 Maurer, R. and Reubi, J.C., Brain somatostatin receptor subpopulation visualized by autoradiography, Brain Res., 333 (1985) 178 181. I I Ra~, K.P., Gomm, J.J., Law, G.J., Sigournay, C. and Wallis, M., Dopamine and somatostatin inhibit forskolin-stimulated prolactin and growth hormone secretion but not stimulated cyclic AMP levels in sheep anterior pituitary cell cultures, Mol. Cell. Endocrinol., 45 (1986) 175 182. 12 Reichlin, S., Somatostatin. In D.T. Krieger, M.J. Brownstein and J.B. Martin (Eds.), Brain Peptides, Wiley. New York, 1983, pp. 711 752. 13 Reubi. J.C., Evidence for two somatostatin-14 receptor types in rat brain cortex, Neurosci. Lett., 49 (1984) 259 263. 14 Reubi, J.C., Probst, A., Cort6s, R. and Palacios, J.M., Distinct topographical locatisation of two somatostatin receptor subpopulations in the human cortex, Brain Res., 406 (1987) 391 396. 15 Reubi, J.C. and Maurer, R., Autoradiographic mapping o f somatostatin receptors in the rat central nervous system and pituitary, Neuroscience, 15 (1985) 1183~-1193. 16 Steiner, A.L., Kipnis, D.M., Utiger, R. and Parker, C., Radioimmunoassay for the measurement of adenosine 3'-5'-cyclic phosphate, Proc. Natl. Acad. Sci. U.S.A., 64 (1969) 367 373. 17 Tran, V.T., Beal, M.F. and Martin, J.B., Two types of somatostatin receptors differentiated by cyclic somatostatin analogs, Science, 228 (1985) 492~.495.
13
NSL 06295
Differential effects of somatostatin on adenylate cyclase as functional correlate for different brain somatostatin receptor subpopulations R. Markstein l, K.A. St6ckli l'* and J.C. Reubi 2 ISandoz Preclinical Research, Basel (Switzerland) and 2Sandoz Research Institute, Bern (Switzerland) (Received 12 September 1988; Revised version received 11 May 1989; Accepted 12 May 1989) Key words." Somatostatin; Adenylate cyclase; Somatostatin receptor In homogenates of rat hippocampus and striatum, but not substantia nigra, somatostatin (SRIF) inhibits forskolin-activated adenylate cyclase in nanomolar concentrations. However, SRIF can also stimulate adenylate cyclase in micromolar concentrations in homogenates of rat hippocampus and substantia nigra. The SRIF octapeptide SMS 201-995 solely inhibits the forskolin-activated adenylate cyclase in the 3 preparations. These results suggest that the SRlF-specific stimulation of adenylate cyclase may be a functional correlate for the brain-specific SRIF receptor subpopulation, whereas the SRIF and SMS 201-995 inhibition of stimulated adenylate cyclase correlate with the SRIF receptor subpopulation present in brain and non-neuronal tissues.
The tetradecapeptide somatostatin (SRIF) is widely distributed throughout the central nervous system [8], where it is thought to function as a neurotransmitter or neuromodulator [12]. Its mechanism of action is not yet completely elucidated. However, there is convincing evidence suggesting that its effects are mediated by specific SRIF receptors located in various brain areas such as cortex, limbic system and basal ganglia [14, 15]. Interestingly, it has been proposed on the basis of pharmacological studies that at least 2 different SRIF receptor subtypes exist in brain tissue, which can be differentiated by reduced-size SRIF analogs such as SMS 201-995 [1]. One type of SRIF receptors has high affinity for SMS 201-995, is present in most brain areas including cortex, hippocampus or striatum, as well as in non-neuronal, peripheral tissues, and has been named SSI or SSA [10, 13, 17]. The other receptor type has only low affinity for the SMS 201-995, is preferentially located in neuronal tissue, often in the same areas as SSI receptors, and is named SS2 or SSB receptor [13, 17]. Since the post-receptor mechanism of action of SRIF includes, at least in part, effects on adenylate cyclase both in brain and in non-neuronal tissue such as pituitary Correspondence: R. Markstein, Sandoz Preclinical Research, 4002 Basel, Switzerland. *Present address: Max Planck Institute, Martinsried, F.R.G. 0304-3940/89,$ 03.50 (~'~1989 Elsevier Scientific Publishers Ireland Ltd.
14 [2, 3, 5-7, 9, I I], we investigated in this study how the different S R I F receptor subpopulations in rat h i p p o c a m p u s , striatum a n d s u b s t a n t i a nigra are related to the adenylate cyclase system. The d e t e r m i n a t i o n of adenylate cyclase activity was performed as tbllows: male rats (Sandoz O F A strain) weighing 150 200g were pretreated twice with reserpine (2.5 mg/kg, s.c.) 18 a n d 24 h before sacrifice by decapitation. A n i m a l s were pretreated with reserpine with the aim of depleting g r a n u l a r stores of d o p a m i n e a n d serotonin, in order to avoid e n d o g e n o u s m o n o a m i n e s interacting with the action of somatostatin on the adenylate cyclase. The brains were rapidly removed a n d striatum, hippoc a m p u s a n d s u b s t a n t i a nigra dissected. The tissues were homogenized in 15 volumes (w/v) of ice-cold 2 m M Tris-acetate buffer (pH 7.4) c o n t a i n i n g 2 m M ethylenglycol-
Fig. I. Visualization of SRIF receptors in the rat brain, a ~: autoradiograms showing SRIF receptors labelled with ~251-[Leu~,D-Trpz2, Tyr25]SRIF-28as radioligand, a: total binding (SS~ and SS2 receptors). b: non-specificbinding in presence of 0.1 /~M SRIF. c: binding in presence of 0.1 ttM SMS 201-995; only SS, receptors remain visualized, d, e: autoradiograms showing SRIF receptors labelled with 12511204-0901 as radioligand, d: total binding showing SSI sites, c: non-specificbinding in presence of 0.1 I~M 204-090. I = cortex, 2 = hippocampus, 3 = substantia nigra, 4 = central grey. The right-left asymmetry in receptor densities in some areas is due to the use of cryostat sections slightly non-parallel to the coronal plane. Exposure time: I week. Bar - 1 mm.
15
bis-(2-aminoethylether)-N,N-tetraacetic acid (EGTA). Two ml aliquots were quickly frozen by immersing the tubes into a mixture of dry ice and acetone and stored at - 7 0 ° C until use. Test compounds either alone or in combination, were incubated in 400/11 of a mixture containing 120 mM Tris-acetate (pH 7.4), 2 mM MgC12, 1.12 mM EGTA, 0.25 mM 3 isobutyl-l-methylxanthine, 0.15% bovine serum albumine, and 100/zl tissue homogenate (diluted to a final protein content of 107.5/tg/ml) during 3 min at 37°C. In some experiments MgCI2 concentration was increased to 4 mM. Bacitracin (10/zM) was included in all assays where SRIF or SMS 201-995 were present in order to prevent degradation by proteases. The enzyme reaction was initiated by adding 100/tl of a solution containing 2.5 mM ATP and 500/zM GTP (pH 7.4) and terminated 4 min later by heating the mixture to 95°C for 4 rain. Supernatant samples (50/zl) were acetylated according to Frandsen and Krishna [4] and cyclic AMP content measured by radioimmunoassay as described by Steiner et al. [16]. Pro-
TABLE I EFFECT OF SRIF AND SMS 201-995 ON CYCLIC AMP FORMATION IN HOMOGENATES OF VARIOUS RAT BRAIN REGIONS Homogenates were incubated in a standard assay buffer (Mg 2+ concentration 4 mM) containing bacitracin (10/~M). Various concentrations of drugs were added as indicated. Cyclic A M P content was measured 3 min after addition of ATP/GTP solution. Values are changes of cyclic A M P content (pmol/mg protein per rain) versus control values. Control values were: hippocampus 245 ± 3.7; substantia nigra 310.7__+3.9; striatum 222.9±2.8. All values are mean ± S.E.M. All data are means of at least 3 independent assays which were performed in triplicates. 5-HT, serotonin; DA, dopamine.
Dose (,uM)
0.31 0.63 1.25 2.5 5.0 10.0
Hippocampus
Substantia nigra
Striatum
SRIF
SMS 201-995
SRIF
SRIF
21.1± 0.1"* 47.7± 4.7** 49.6± 4,5*** 31.9± 1.1"** 21.1± 2.1"* 3.2± 3.1
0.1± 1.0 4.1± 4.0 0.2± 1.1 6.8± 4.3 3.4± 3.1 0.2± 0.7
1.25/zM 5-HT 63.8 ± 2.8** 125 ItM DA
7.2± 1.1 15.6± 4.4* 30.6± 7.8** 12.2± 0.5* 2.8± 0.5 0 ± 0.5
120.3+ 44***
SMS 201-995 1.1± 1.1 2.8± 2.9 7.2± 4.4 2.2± 2.2 4.4+ 2.2 2.8± 2.2
--32.5± 3.8*** --40.1± 6,7** --48.6± 4.9*** -36.1± 7.1"* --18.3± 8.2 0 ± 2.6
SMS 201-995 -25 ± 9.0* -19.4± 2.6* -- 4.0± 2.2 0 ± 0.4 -- 2.4± 3.8 - 0.1± 1.6
179.8+ 2.0***
Statistical significance: *2P< 0.05; **P<0.01; ***2P<0.001 (Student's two tailed t-test).
16
tein content was determined by the biuret method. All determinations were performed in triplicate. Data was expressed as mean values i S.E.M. and significance levels for the comparison between mean values were calculated according to Student’s r-test for unpaired SRIF either
receptors the SRIF-28
““I-[Tyr3]SMS
observation.
were visualised analog
201-995
in IO ,Lim cryostat
“‘I- [ Leus , D-Trp’?,
(‘z~1-204-090)
sections
Tyrz5]SRIF-28
as radioligands,
of the rat brain
using
or the octapeptide
as described
in detail
else-
where [l4, 151. Fig. I. is an autoradiographic representation of the 2 SRIF receptor populations found in the rat cortex, hippocampus and substantia nigra. using 2 different SRIF radioligands, as described extensively for the human brain [14]. It can be seen that in the rat cortex and hippocampus both SS, and SSz receptors are present (Fig. la,d). whereas in the substantia nigra only SS2 receptors are found (Fig. la,c) which are not labelled with the octapeptide SRIF radioligand “51-204-090 (Fig. Id). In the striatum (not shown) SS, sites predominate [I 31. Table I shows that in homogenates of hippocampus and substantia nigra SRIF (in the presence of2 mM Mg’ +) significantly stimulated adenylate cyclase in the micromolar concentration range. The doses response curves were biphasic in both tissue preparations with a maximum effect occurring at 1.25 ,YM and amounting to 120% and I IO % of basal activity in homogenates of hippocampus and substantia nigra, respectively. The ECs,,-value was 0.3 /IM in both tissue preparations. Under the same experimental conditions SMS 201-995 failed to stimulate adenylate cyclase in both tissue preparations. In homogenates of striatum, SRIF and SMS 201-995 by itself had no stimulating effect but reduced basal activity of adenylate cyclase by 22% at I .X jtM and by 9% at 0.63 /IM. respectively. Table II shows that forskolin markedly stimulated adenylate cyclase activity in all 3 tissue preparations. In homogenates of hippocampus and substantia nigra 5 /IM forskolin (in the presence of 4 mM Mg’ ’ ) enhanced basal activity approximately -I-fold and in homogenates of striatum approximately ?I)-fold. SRIF and SMS 201-995 inhibited the effect of 5 PM forskolin dose-dependently in homogenates of hippocampus and striatum in the nanomolar concentration range. The maximal inhibitory effect of SRIF occurred at I and IO ,uM and amounted to 13.2~ in the hippocampal and to 39.5% in the striatal homogenate. SMS 201-995. although less potent than SRIF. produced the same maximal inhibition of the forskolin effect in the hippocampus. The inhibitory effect of SMS 201-995 in the striatal homogenate was clearly smaller than that of SRIF. In homogenates of the substantia nigra SMS 20 l-995 in a concentration range of 0.001 IO ,uM significantly inhibited the effect of forskolin whereas SRIF was inactive. The present data. therefore. show that SRIF can produce a differential, opposite effect on adenylate cyclase in different brain regions. In homogenates of hippocampus, SRIF both inhibits stimulated adenylate cyclase activity in the nanomolar range and stimulates basal adenylate cyclase in the micromolar range. In the substantia nigra. only a stimulation of basal adenylate cyclase is seen whereas in the striatum only inhibition of stimulated cyclasc is observed in presence of SRIF. A correlation may be drawn between these SRIF effects and the various SRIF receptor popda-
17 TABLE 1I EFFECT OF SRIF AND SMS 201-995 ON FORSKOLIN-STIMULATED CYCLIC AMP FORMATION IN HOMOGENATES OF VARIOUS BRAIN AREAS Homogenates were incubated in a standard assay buffer (Mg 2+ concentration 2 mM) containing bacitracin (10 ItM) and forskolin (5/tM). Various concentrations of drugs were added as indicated. Cyclic AMP content was measured 3 min after addition of ATP/GTP solution. Values represent cyclic AMP content (pmol/mg protein per min) expressed as percentage of the increase by 5 ltM forskolin. The increase of cyclic AMP content by forskolin (5 :tM) was (basal value in parenthesis): hippocampus 520.9+3.2 (68.8+ 1.5); substantia nigra 719+ 10.2 (100.4+ 1.2); striatum 4081 +26.9 (200.9+3.8). All values are mean +_ S.E.M.,n=4. % of Forskolin effect Dose
Hippocampus
Substantia nigra
Striatum
SRIF
SMS 201-995
SRIF
SMS 201-995
SRIF
SMS 201-995
93.9+ 1.3"* 89.0+ 2.8** 79.6+ 0.7*** 76.8+ 1.1"**
99.1 + 0.2 94.5+ 1.4' 92.2+ 0.6*** 78.6+ 1.1"**
96.7+ 3.1 92.5+ 3.3
72.4+ 2.6***
87.4+ 3.0** 83.8+ 0.9*** 83.7+ 1.3*** 81.0+ 3.7** 82.7-t3.1"* 90.4+ 0.7***
72.8+ 4.9** 65.3+ 4.3***
74.0+ 1.1"**
95.5+ 4.4 95.3+ 3.7 99.7+ 3.6 102.7__+ 5.9 112.1+ 2.5 103.8+ 0.3
60.5+ 4.7*** 79.3+ 5.4** 90.7+ 3.0*
89.6+ 2.1"* 89.5__ 2.8* 86.6+ 4.4*
~M)
0.001 0.01 0.1 1.0 5.0 10.0
Statistical significance: *2P< 0.05; **2P<0.01; ***2P< 0.001 (Student's two tailed t-test)
tions, since we know that the hippocampus has both SS1 and S S 2 receptors, whereas the substantia nigra has mainly SS2 and the striatum SSI receptors [10, 13, 14, 17]. Moreover, SMS 201-995, devoid of stimulating effects on adenylate cyclase in homogenates of rat hippocampus and substantia nigra has only a low affinity to the SS2 sites [13]. These results therefore suggest that SS2 sites (i.e. in the substantia nigra) mediate a stimulation of adenylate cyclase, whereas SSI sites mediate inhibitory effects. Inhibitory effects of SRIF adenylate cyclase activity have been described earlier in non-neuronal tissues considered SRIF target tissues [5-7, 9, 11]. However, SRIFspecific stimulatory effects on this enzyme have to our knowledge not yet been reported. Its physiological significance as well as its cellular localisation (gliai vs neuronal) remain to be determined. 1 Bauer, W., Briner, U., Doepfner, W., Haller, R., Huguenin, R., Petcher, T.P. and Pless, J., SMS 201-995: a very potent and selective octapeptide analogue of somatostatin with prolonged action, Life Sci., 31 (1982) 1133-1140.
18 2 Schneiweiss, H.. Bertrand, Ph., Epelbaum, J., Kordon, C., Glowinski, J., Premont, J. and Enjalbert, A., Somatostatin receptors on cortical neurones and adenohypophysis: comparison between specific binding and adenylate cyclase inhibition, Eur. J. Pharmacol. 138 (1987) 249 255. 3 Schneiweiss, H., Glowinski. J. and Pr6mont, J., Modulation of monoamines of somatostatin-sensitive adenylate cyclasc on neuronal and glial cells from the mouse brain in primary cultures, .I. Neurochem., 44(1985) 1825 1831. 4 Frandsen, E.K. and Krishna, G., A simple ultrasensitive method for the assay of cyclic AMP and cyclic GMP in tissues, Life Sci., 18 (1976) 529 542. 5 tlarwood, J.P., Grewe, (7, and Aguilera, G., Actions of growth hormone-releasing l:actor and somatostatin on adenylate cyclase and growth hormone release in rat anterior pituitary, Mol. Cell. Endocrinol., 37 (19841 277 284. 6 lfcisler, S. and Reisine, T., Forskolin stimulates adenylate cyclase activity, cyclic A M P accumulation and adrenocorticolropin secretion from mouse anterior pituitary tumor cells, J. Neurochem., 42 (1984) 1659 1666. 7 Heisler, S. and Srikant, C,B., Somatostatin-14 and somatostatin-28 pretreatment down-regulate somatostatm-14 receptors and have biphasic effects on forskolin-stimulated cyclic adenosine, 3",5"-monophosphate synthesis and adrenocorticotropin secretion in mouse anterior pituitary tumor cells, Endocrinology, 117 (1985) 217 225. 8 .lohansson. O., tt6kfelt, T. and Else. R., lmmunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the rat, Neuroscience, 13 (1984) 265 339. 9 Koch, B.D. and Schonbrunn. A., The somatostatin receptor is directly coupled to adenylate cyclase in GH4C~ pituitary cell membranes, Endocrinology, 114 (1984) 1784 1790. 10 Maurer, R. and Reubi, J.C., Brain somatostatin receptor subpopulation visualized by autoradiography, Brain Res., 333 (1985) 178 181. I I Ra~, K.P., Gomm, J.J., Law, G.J., Sigournay, C. and Wallis, M., Dopamine and somatostatin inhibit forskolin-stimulated prolactin and growth hormone secretion but not stimulated cyclic AMP levels in sheep anterior pituitary cell cultures, Mol. Cell. Endocrinol., 45 (1986) 175 182. 12 Reichlin, S., Somatostatin. In D.T. Krieger, M.J. Brownstein and J.B. Martin (Eds.), Brain Peptides, Wiley. New York, 1983, pp. 711 752. 13 Reubi. J.C., Evidence for two somatostatin-14 receptor types in rat brain cortex, Neurosci. Lett., 49 (1984) 259 263. 14 Reubi, J.C., Probst, A., Cort6s, R. and Palacios, J.M., Distinct topographical locatisation of two somatostatin receptor subpopulations in the human cortex, Brain Res., 406 (1987) 391 396. 15 Reubi, J.C. and Maurer, R., Autoradiographic mapping o f somatostatin receptors in the rat central nervous system and pituitary, Neuroscience, 15 (1985) 1183~-1193. 16 Steiner, A.L., Kipnis, D.M., Utiger, R. and Parker, C., Radioimmunoassay for the measurement of adenosine 3'-5'-cyclic phosphate, Proc. Natl. Acad. Sci. U.S.A., 64 (1969) 367 373. 17 Tran, V.T., Beal, M.F. and Martin, J.B., Two types of somatostatin receptors differentiated by cyclic somatostatin analogs, Science, 228 (1985) 492~.495.