No abstract

Radioligand binding studies were performed in membranes of rat cerebellum using [125I]-[Tyr3]octreotide ([125I]204-090) to characterize the nature of cerebellar somatostatin receptors. Saturation experiments suggest the presence of a single class of binding sites with high affinity, pKd = 9.53 +/- 0.11, but low receptor density, Bmax = 12.7 +/- 1.0 fmol/mg protein. The pharmacological profile of [125I]204-090 sites in cerebellar membranes was established using a range of ligands known to interact with SSTR-2 (now called sst2) and other somatostatin (SRIF) receptors. SRIF analogues such as octreotide (SMS 201-995), seglitide (MK 678) and somatuline (BIM 23014) displayed very high affinity for cerebellar [125I]204-090 binding sites. The data were compared to results obtained using the same ligand in rat cerebral cortex membranes known to represent sst2 binding. The pharmacological characteristics of the cerebellar sites were in close correlation with those of the cerebral cortex (r = 0.976, n = 19, p < 0.001) and CHO-cells expressing human recombinant sst2 receptor (r = 0.977, n = 19, p < 0.001). By contrast, there was very little correlation between cerebellar binding and published affinities for rat sst5 receptors (r = 0.465), for which octreotide has also high affinity. In vitro autoradiographic studies performed in cerebellar slices using [125I]204-090 demonstrated the presence of binding sites in the molecular layer of the rat cerebellum. In situ hybridization studies using sst2 receptor mRNA selective oligoprobes confirmed the presence of sst2 receptor mRNA in the rat cerebellum. Together, the present data demonstrate the presence of a low density of SRIF receptors in the molecular layer of the adult rat cerebellum which are best characterized as sst2. This is the first pharmacological characterization and localization of sst2 receptors in the adult rat cerebellum.

The pharmacological profile and localization of somatostatin (SRIF) receptors were determined in rat, monkey and human cerebellum. In rat cerebellar cortex, low sst1/sst4, intermediate sst2 and very high sst3 receptor mRNA levels were found. sst1 mRNA was also expressed in the deep cerebellar nuclei. [125I]Tyr3-octreotide binding sites in cerebellar membranes correlated with recombinant sst2, but not with sst5 or sst3 receptors and were found in the molecular layer of the cerebellum. [125I]CGP 23996 (in Na(+)-buffer) binding in rat cerebellum correlated with sst1 or sst4, but not with sst2, sst3 or sst5 receptor binding. Similar data were obtained in rhesus monkey cerebellum. mRNAs for all five receptors were found in the granule cell layer of the human cerebellum and/or in the dentate nucleus. [125I]Tyr3-octreotide binding was strong in the molecular layer and correlated with that of recombinant sst2 receptors, but not with sst3 or sst5 receptors. [125I]CGP 23996 (in Mg(++)-buffer) binding was heterogeneous (about 75%, to sst2 and 25% to sst1 and/or sst4 receptors). The molecular and granular layers were equally and the dentate nucleus strongly labeled. Thus, SRIF receptors of the sst2, sst1 and/or sst4 subtype are presnt in the rat, monkey and human cerebellum. In the latter two species, the sst2 type appears to be predominant. Surprisingly, the high expression of sst3 receptor mRNA is not supported by radioligand binding data in any of the species studied. The reason for this discrepancy remains to be elucidated.

The distribution of somatostatin (SRIF) receptor sites was studied by in vitro receptor autoradiography in the human brain and pituitary using the SRIF1 (sst2) receptor selective [125I]Tyr3-octreotide, the non-subtype selective [125I]LTT-SRIF-28 ([Leu8,D-Trp22, 125I-Tyr25]SRIF-28) and the SRIF2-receptor selective [125I]CGP 23996 (c[Asu- Lys-Asn-Phe-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) in buffer containing 120 mM Na+. SRIF receptor autoradiography was compared with mRNA expression of somatostatin receptors sst1-5 as studied by in situ hybridisation in human brain. High levels of [125I]LTT-SRIF-28 and [125I]Tyr3-octreotide recognition sites were found in the deep layers of cerebral cortex and molecular layer of cerebellum of the human brain. The hypothalamus, choroid plexus, most areas of the brainstem and dentate nucleus were associated with low levels of binding. In contrast to [125I]LTT-SRIF-28 and [125I]Tyr3-octreotide, no difference was observed for [125I]CGP 23996 labelling in the various layers of cerebral cortex. The choroid plexus, substantia nigra and molecular layer of the cerebellum presented high densities of [125I]CGP 23996 binding sites whereas no binding was observed in the hypothalamus and locus coeruleus using this radioligand. Both lobes of the human pituitary displayed low levels of [125I]LTT-SRIF-28 and [125I]Tyr3-octreotide binding. By contrast, the anterior lobe of the pituitary displayed very high levels of [125I]CGP 23996 labelled sites whereas intermediate levels were found in the posterior lobe. There was a partial overlap between sst2 receptor mRNA and [125I]Tyr3-octreotide binding, although the distribution of the binding sites was much wider than that of receptor mRNA. The same observation was made for sst1 and/or sst4 receptor mRNA and [125I]CGP 23996 labelled sites. The present data show that SRIF1 and SRIF2 receptors are present in the human brain with different distributions, especially in the cerebral cortex and the pituitary. The very similar distribution of sites labelled with [125I]LTT-SRIF-28 and [125I]Tyr3-octreotide suggests (i) that sst2 receptors are predominant within the SRIF1 family in the human brain and (ii) that [125I]LTT-SRIF-28 under the conditions used in the present study, does not significnatly label SRIF2 sites.

To characterize the nature and distribution of somatostatin (SRIF) receptors, radioligand binding studies and in vitro receptor autoradiography were performed in Rhesus monkey brain using either [125I]LTT-SRIF-28 ([Leu8, D-Trp22, 125I-Tyr25]SRIF-28) alone or in the presence of 3 nM seglitide (to block sst2 sites), [125I]Tyr3-octreotide or [125I]CGP 23996 (c[Asu-Lys-Asn-Phe-Trp-Lys-Thr-Tyr-Thr-Ser]) in buffer containing either 120 mM Na+ or 5 mM Mg2+. [125I]Tyr3 -octreotide labelled an apparently homogeneous population of sites in cerebral and cerebellar cortex (Bmax = 27.3 +/- 2.8 fmol/mg protein and 52.6 +/- 8.6 fmol/mg protein, PKd = 9.46 +/- 0.03 and] 9.93 +/- 0.03, respectively). The pharmacological profile of these sites correlated highly significantly with that of human recombinant sst2 receptors (r = 0.996), but not or much less with that of human recombinant sst3 and sst5 receptors (r = 0.12 and 0.45, respectively). [125I]CGP 23996 (in Na(+)-buffer) also labelled an apparently homogeneous population of sites in Rhesus monkey cerebral cortex membranes (Bmax = 3.1 +/- 0.3 fmol/mg protein, pKd = 10.57 +/- 0.08), the pharmacological profile of which was highly significantly correlated with the profiles of human recombinant sst1 and sst4 receptors (r = 0.98 and 0.96, respectively). Using receptor autoradiography, high levels of [125I]LTT-SRIF-28 and [125I]Tyr3 -octreotide recognition sites were found in basal ganglia, molecular and granular layers of the cerebellum and layers III, V and VI of entorhinal cortex. In these regions, the addition of 3 nM seglitide produced a marked decrease of [125I]LTT-SRIF-28 binding. Low levels of [125I]LTT-SRIF-28 binding were observed in subiculum, pituitary and choroid plexus. By contrast, [125I]CGP 23996 labelling in the presence of Mg2+ as well as Na+ ions was highest in pituitary and choroid plexus. However, [125I]CGP 23996 binding was diversely affected by these ionic conditions in several regions of hippocampus and cerebral cortex. Displacement of [125I]CGP 23996 (in Mg(2+)-buffer) with seglitide in the molecular layer of the cerebellum, deep layers of the entorhinal cortex, layers I, II and V of the insular cortex and frontal pole yielded complex competition curves suggesting the presence of two populations of SRIF receptors. By contrast, [125I]CGP 23996 binding (in Mg(2+)-buffer) in the choroid plexus, hilus of the dentate gyrus and stratum oriens and radiatum of the CA3 field of hippocampus was not affected by seglitide up to 10 microM, suggesting only sst1 and/or sst4 sites which have a negligible affinity for seglitide to be present in these structures. Taken together, these results suggest that [125I]CGP 23996 (in the presence of Na+) labels exclusively SRIF-2 receptors (sst1 and/or sst4), whereas in the presence of Mg2+ ions, [125I]CGP 23996 labels both SRIF-2 and SRIF-1 receptors (sst2, sst3 and sst5). The present study also demonstrates the presence and differential distribution of sst2 and sst1/sst4 receptors in the Rhesus monkey brain.