MEDICIÓN

The use of agonists to define receptor heterogeneity presents certain problems, particularly if the agonist is not entirely selective and the suspected receptor subtypes exist together as a mixed population in the same tissue. However, whilst recognising the iimitations of this approach (see further discussion in Chapter 7), the present data suggest a mixed population of somatostatin- receptors may exist in severai rat brain areas and are in agreement with the original observations of Reubi (1984) and Tran et ai. (1985). The case for receptor heterogeneity was most striking in the GA1 region of the hippocampus where oniy 25-35% of the total receptor population (representing the SSa component as defined by Tran et a!., 1985 or the SSi as described by Reubi, 1984 ) had a high affinity for octreotide and seglitide.

In the cortex and dentate gyrus the proportionality appears to be somewhat different with the majority of the sites (80-90% in the cortex, 95% in the dentate) being the high affinity or SSa subtype. Cortical somatostatin binding sites have been the subject of several previous studies, but there is disagreement as to the relative proportion of high and iow affinity sites. Thus, Tran et al. (1985) found approximately 50% of sites to be of the iow affinity SSb subclass. The present

results compare more favourably with those of Krantic et al. (1990) who found approximately 86% of p ^ l] Tyr^^ SSI 4 binding to deep layers of occipital cortex to be of the high affinity SSaor SSi subclass. It is not surprising that differences in

SSa-'SSb binding site proportionality have been quoted for the cortex, not oniy

because of its iaminar structure but because the reiative area occupied by each lamina varies between corticai regions. The situation is further compounded by data in the literature generated from conventional homogenate binding assays

which do not possess the same degree of resolution associated with the autoradiographic approach.

By comparing the degree of displacement of [i2S|]Tyi^-D-Trp^-SSI 4 binding by octreotide independently in the superficial and deep corticai layers, Krantic et al.

(1990) have suggested that the superficial layers generally show a higher proportion of SS2 receptors whilst the deep layers contain more of the SSi subtype. Reubi et al. (1987) have also shown a differential distribution of somatostatin receptors within the various laminae of the human cortex. These authors have shown that the density of SSi receptors, defined using the selective radioligand, p25|]$Ms 204-090, is particularly prominent in human cortical laminae V and Vi, whèreas SS2 receptors are preferentially located in lamina IV with lower but still reasonable levels in laminae I, V and VI. Further experiments will be required to confirm these findings and it will be interesting to determine whether somatostatin receptor subtypes are preferentially associated with the different types of synapse, i.e. symmetrical vs. asymmetrical, both of which are known to be associated with somatostatin neurones in the cortex (Hendry etal. 1984).

The concept of somatostatin-receptor heterogeneity is not one of universal acceptance. Moyse et al (1989) have shown octreotide (IpM) to fully displace ail [i25|]jyr0.p_Trp8-ss14 binding in both frontal cortex and hippocampal CA1 regions and with monophasic displacement curves. These authors suggest that the lack of topographical differences in octreotide binding sites found in their experiments argues in favour of different affinity states rather than different receptors. This might occur, for example, if the receptor showed regional variations in the extent of GTP coupling which may effect the affinity of one agonist to a greater extent than another. Clearly, even in the case of the present experiments this explanation remains a possibility and will always represent a credible alternative argument when attempting to define receptor differences with agonists. However, whilst unequivocal evidence demonstrating fundamental differences in receptors remains elusive there is, equally, no published evidence to support regional variations in GTP coupling to somatostatin receptors. Undoubtedly, the use of potent, selective and competitive somatostatin antagonists would overcome many

the cerebral cortex. The present data show somatuiine to be the most potent of the short-chain analogues in displacing p25|j Tyr^^-SS14 binding from the pituitary and the weakest, by far, in the hippocampus and cortex (Table 4.2). Surprisingly, somatuiine was also extremely potent at displacing radiolabelled somatostatin binding in the LC. However, when compared to other competing ligands, the rank order of potency was different to that seen in the pituitary. This suggests that the binding sites in these two tissues may be different but the magnitude of the differences were not large enough to support a definitive conclusion in this regard. However, recent autoradiographic studies published by Martin et al. (1991) appear to support this contention since another peptide analogue of somatostatin, CGP23-996, bound with high affinity to anterior pituitary but showed no affinity whatsoever for the site in the LG.

Taken together, these data appear to indicate that, contrary to previous suggestions, neither the site in the LC (Gagne at a/., 1990) nor that in the pituitary (Tran et a/., 1985) can be the same as the high affinity (SSa or SSi) site in the

cortex.

The region of SSI 4 which comprises the pharmacophore involves residues Phe^- Trp®-Lys®-Thr^° (Veber et a/., 1979). This region is essentially conserved in all short chain analogues with only minor modifications around Trp®-Lys®, the latter being a fundamental requirement for activity (see Chapter 1, Section 1.6.2). Thus, seglitide, octreotide and somatuiine differ more significantly in substitutions around this region. As is evident from the present experiments, the effects of such substitutions in the case of seglitide and octreotide do not appear to have resulted in any obvious differences in the binding sites recognised by these peptides. The more profound differences in affinity seen with somatuiine can be attributed to a single modification at a site distal to the central sequence, in this instance at the C-terminal (Heiman et a/., 1987). Whilst this offers hope for the development of other somatostatin-receptor-selective ligands, it is also recognised that certain amino acid substitutions can also influence the selectivity of the resulting peptide. Consequently, several short-chain somatostatin analogues, including octreotide and somatuiine (Maurer et a/., 1982; Walker et a/., 1987), have been shown to have high affinity for opiate p-receptors. Such an effect is unlikely to explain any of the present findings, since the potent opiate p-receptor antagonist, naloxone, failed to displace ^^1] Tyrii-SS14 binding and does not antagonise the electrophysiological effects of short-chain somatostatin analogues in the LC (see Chapter 5).