Brain somatostatin receptor subpopulation visualized by autoradiography

Brain somatostatin receptor subpopulation visualized by autoradiography

178 Ei,,cvic~ BRE 20753 Brain somatostatin receptor subpopulation visualized by autoradiography R. MAURER andJ. C. REUBI Preclinical Research, Sando...

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178 Ei,,cvic~

BRE 20753

Brain somatostatin receptor subpopulation visualized by autoradiography R. MAURER andJ. C. REUBI Preclinical Research, Sandoz Ltd., CH 4002 Basel (Switzerland)

(Accepted December 4th, 1984) Key words: somatostatin - - subpopulation - - receptor .... rat brain - - autoradiography - - SMS 201-995 analogs

[12SI-Tyr11]somatostatin-14as well as iodinated D-Tyr~and Tyr-3derivatives of the cyclicoctapeptide somatostatin analog, SMS 20t-995 (H-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-ol) have been used as radioligands for somatostatin receptor autoradiography in rat brain. Although the cyclic octapeptide ligands label the majority of the regions labeled with [lZSI-Tyr~L]somatostatin,in the cortex and hippocampus only a subpoputation of somatostatin receptors is labeled. Cyclic octapeptide ligands have improved resolution due to their very low non-specific binding.

N u m e r o u s studies suggest that somatostatin (SS) fulfills most of the conditions necessary for a neurotransmitter or n e u r o m o d u l a t o r role in the CNS 4. One of these n e u r o t r a n s m i t t e r criteria is the presence of a high density of specific, high-affinity SS receptors in selected brain regions. Such receptors have been described using both SS-14 and SS-28 radiotigands in in vitro h o m o g e n a t e binding studies2, s,w and more recently using autoradiographical techniques3,12. Although pharmacological characterization of these receptors revealed that CNS SS receptors have a different affinity for selected SS analogs than peripheral, i.e. pituitary or pancreatic SS receptors6,11, there has been no conclusive evidence for the presence of multiple SS receptors in these various organs. Recently, however, we could d e m o n s t r a t e that, in a [azsI-Ty61]SS r e c e p t o r assay, a stable cyclic octapeptide analog of SS 1, SMS 201-995 ( H - D - P h e C } s - P h e - D - T r p - L y s - T h r - C ~ s - T h r - o l ) only binds to a part of the rat cortical SS receptors but to all pituitary and pancreatic SS receptorsS. The same is true for a Tyr3-derivative of SMS 201-9959. F u r t h e r m o r e , the iodinated c o m p o u n d seems to label only one subpopulation of cortical and h i p p o c a m p a l SS receptors 9 in vitro. The aim of the present study is to visualize these SS receptors in the rat CNS with the D-TyrO-and Tyr3-derivative of SMS 201-995, and to compare the

autoradiographic pattern with that from receptors labeled with [125I-Tyrll]SS. T y r m S S has been iodinated and purified according to Reubi et al.7, whereas D-Tyr 1- and Tyr3-deriva tives of SMS 201-995 have been iodinated with the chloramine T m e t h o d using molar ratios of peptide : chloramine T:1251-Na of 1 : 4.6 : 0.6. The reaction was terminated with 100 ul of 10% BSA. The iodinated peptide was separated from the free iodine using a Sep-pack (Waters Associates) column, tt was purified by H P L C on C-18 Waters column using an isocratic mixture of t r i e t h y l - a m m o n i u m - f o r m a t e acetonitrile (pH 3). Two peaks were obtained: only the c o m p o u n d eluted in the first peak showed high affinity to cortical membranes. The fractions corresponding to this peak, which coeluted with the respective m o n o - i o d i n a t e d peptide, were freeze-dried and diluted in 10 vols. 0.1 M H O A c containing 1% BSA. Aliquots were stored at - 4 °C. The H P L C purification of the radioligand allows the assumption that its specific activity is near the theoretically calculated one. All 3 iodinated compounds were extensively characterized using binding assays~.'L Frozen brains from male rats ( O F A strain, 200-250 g) were cut on a cryostat (Leitz 1720), the coronal or sagittal sections (10 u m ) m o u n t e d on precleaned microscope slides and stored at - 2 0 °C for at

Correspondence: R. Maurer, Sandoz Ltd., 386-355. Preclinical Research, CH 4002, Basel, Switzerland.

0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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A

B

C

Fig. 1. Subpopulation of [125I-Tyrll]somatostatin-14 in rat brain sections. Coronal sections incubated with the ligand alone (A) or in the presence of 1ruM unlabeled SS (B) or 1~M Tyr3-SMS 201-995 (C). Photos from autoradiograms on [3H]LKB films exposed for 1 week are shown; arrows indicate hippocampus and habenuta: reference bar 1 ram. least 3 days to improve adhesion of the tissue to the slide. For autoradiography sections were preincubated in Tris-HC1 buffer (50 mM, pH 7.4) containing CaCI 2 (2 mM) and KC1 (5 mM) for 10 min at ambient temperature and then washed twice for 2 min in the same buffer without additional salts added. Incuba-

tion was carried out for 40 min at ambient temperature in Tris-HC1 buffer (17~ mM, pH 7.4) containing bovine serum albumin (1%), bacitracin (40 gg/ml) and MgCI 2 (10 mM) to inhibit endogenous proteases in the presence of iodinated ligands (0.16 x I0 n dpm/ml, ca. 80 pM). Non-specific binding was determined by admixing unlabeled Tyr11-SS at a concentration of 1 ,uM. Incubated sections were washed twice for 5 min in cold incubation buffer containing only 0.25% BSA, prolonged washing times with either ligand did not change the amount of specifically or non-specifically bound tracer. After a brief dip in distilled water to remove excess salts, the sections were dried quickly, apposed to [3H]LKB films and exposed for 1 week in X-ray cassettes. The lower cortical layers, the CA1 region of the hippocampus and the habenula are highly enriched in [125I-Tyrll]SS receptor sites (Fig. 1A), the dentate gyrus has an intermediate density, whereas the hypothalamic region is almost devoid of receptor sites. Coincubation with 1 k~M of unlabeled somatostatin eliminates the specific binding in the described regions completely (Fig. 1B), although the remaining non-specific background is considerably higher than the film background. In the presence of 1/~M Tyr)SMS 201-995, however, the ligand is only partially displaced in the most densely labeled regions, particularly in the hippocampus (Fig. 1C). In an additional study 3 somatostatin ligands [125ITyrll]SS, [125I-D-Tyrl] and [125I-Tyr3]SMS 201-995 were compared under identical experimental conditions. Three major differences are obvious. Firstly the ratio of total to non-specific binding is much greater using either the D-Tyr l- or Tyr3-SMS 201-995 analog than with the TyrlI-SS (Fig. 2). Whereas the remaining non-specific binding using [1251-Tyrlt]SS is still clearly visible (Fig. 2D), the nonspecific binding using [125I-Tyr3] (Fig. 2E) or [125I-D-TyrqSMS 201-995 (Fig. 2F) is almost as low as the background of the [3H]LKB film. Secondly the labeling of the dentate gyrus is much greater with the somatostatin analogs than with [125I-Tyrll]SS, and thirdly the resolution, particularly in areas with intermediate to low receptor densities (i.e. hypothalamus), using the SMS 201-995 analogs, is highly superior to the [1251Tyrll]SS. This study shows the autoradiographic distribution of SS receptors in rat brain using 3 different radioli-

180

Fig. 2. Autoradiograms obtained with 3 different iodinated somatostatin analogs. Identical incubation of rat brain co,onal sections with the ligands Tyrla-SS (A, D), Tyr3-SMS201-995 (B, E) and o-Tyrl-SMS 201-995 (C, F). Non-specificbinding (D, E, t:.)~.as done in the presence of 1BM unlabeled SS. Sections were exposed for 1 week with [3H]LKB films; arrows point to dentate gyrus ;andhypothalamic area; reference bar 1 mm.

gands, an SS-14 analog and two octapeptide derivatives of SS. All tracers appear to reveal a similar regional distribution of SS-receptors, cortex and hippocamigus as well as h a b e n u l a and amygdala being heavily labeled, whereas hypothalamus and striatum

are moderately labeled. This distribution is in agreement with recent autoradiographic data by others 3,12 using SS-14 and SS-28 analog tracers. However, it appears that the use of the stable octapeptide tracers considerably improves the autoradiographic

181 sensitivity of the [Tyr11]SS tracer, mainly because of

has been performed, these data suggest that the high-

their very low non-specific binding9. The stability of the octapeptides 1 furthermore explains the higher

est densities of the SS receptor population without affinity for the octapeptides are located in cortex and

n u m b e r of receptors visualized with these tracers, as has already been discussed by others 2,9.

hippocampus, confirming recent data obtained in homogenate binding studiesS. 9.

These data secondly demonstrate that the octapeptide tracers only visualize one subpopulation of

other in vitro binding studies8.9 demonstrate that

brain SS receptors, since only incomplete displacement of the [125I-Tyr11]SS tracers occurs in the presence of excess amounts of SMS 201-995 in some regions, whereas a complete displacement of the [1251Tyr3]SMS 201-995 tracer is obtained with the octa-

The present autoradiographic data together with these new octapeptide radioligands are valuable tools to visualize one CNS SS receptor subpopulation, and, due to their greatly improved stability, represent an improvement over SS-14 and SS-28 analog ligands.

peptide analog. Although no quantitative evaluation

1 Bauer, W., Briner, U., Doepfner, W., Hailer, R., Huguenin, R., Marbach, P., Petcher, T. J. and Pless, J., SMS 201-995: a very potent and selective octapeptide analogue of somatostatin with prolonged action, Life Sci., 31 (1982) 1133-1140. 2 Czernik, A. J. and Petrack, B., Somatostatin receptor binding in rat cerebral cortex, J. biol. Chem., 258 (1983) 5525-5530. 3 Leroux, P. and Pelletier, G., Radioautographic localization of somatostatin-14 and somatostatin-28 binding sites in the rat brain, Peptides, 5 (1984) 503-506. 4 Reichlin, S., Somatostatin. In D. T. Krieger, M. J. Brownstein and J. B. Martin (Eds.), Brain Peptides, Wiley, New York, 1983, pp. 711-752. 5 Reubi, J. C., Perrin, M. H., Rivier, J. E. and Vale, W., High affinity binding sites for a somatostatin-28 analog in rat brain, Life Sci., 28 (1981) 2191-2198. 6 Reubi, J. C., Rivier, J., Perrin, M., Brown, M. and Vale, W., Specific high affinity binding sites for somatostatin-28 on pancreatic cells: differences with brain somatostatin receptors, Endocrinology,, 110 (1982) 1049-1051.

7 Reubi, J. C., Perrin, M., Rivier, J. and Vale, W., High affinity binding sites for somatostatin to rat pituitary, Biochem. biophys. Res. Commun., 105 (1982) 1538-1545. 8 Reubi, J. C., Evidence for two somatostatin-14 receptor types in rat brain cortex, Neurosci. Lett., 49 (1984) 259-263. 9 Reubi, J. C., Novel selective radioligand for one subpopulation of rat cortex somatostatin receptors, Brit. J. Pharmacol., 83 (1984) Suppl., 23P. 10 Srikant, C. B. and Patel, Y. C., Somatostatin receptors: identification and characterization in rat brain membranes. Proc. nat. Acad. Sci. U.S.A., 78 (1981) 3930-3934. 11 Srikant, C. B. and Patel, Y. C., Receptor binding of somatostatin-28 is tissue specific, Nature (Lond.), 294 (1981) 259-269. 12 Tran, V. T., Uhl, G. R., Perry, D. C., Manning, D. C., Vale, W., Perrin, M. H., Rivier, J. E., Martin, J. B. and Snyder, S. H., Autoradiographic localization of somatostatin receptors in rat brain, Europ. J. Pharmacol., 101 (1984) 307-309.