First localisation of somatostatin sst4 receptor protein in selected human brain areas: an immunohistochemical study

doi:10.1016/S0169-328X(00)00186-8

Molecular Brain Research

Volume 82, Issues 1–2, 20 October 2000, Pages 114-125

https://doi.org/10.1016/S0169-328X(00)00186-8 Get rights and content

Abstract

Somatostatin is known to have diverse neurophysiological effects in the mammalian CNS. To date, genes for five different receptors, termed sst_1–5, have been isolated. Recently several reports have been published on the localisation of the individual receptor protein in the rat CNS, but their localisation in the human CNS remains largely unknown. Until now little information about the function of the sst₄ receptor is available, and there is a lack of receptor specific agonists and antagonists. Here, we report for the first time the immunohistochemical localisation of the sst₄ receptor in selected human brain areas using an anti-peptide antibody raised against a carboxy-terminal portion of the receptor protein. Strong receptor immunoreactivity was found in several brain regions, including the hippocampal formation, the cerebellar cortex and the medulla. Further immunohistochemical labelling was observed in the cerebral cortex, the red nucleus and the globus pallidus. Somatodendritic as well as axonal staining was observed. Specific signals were entirely absent following antibody pre-adsorption with the synthetic peptide. The results are in good agreement with the previously published immunohistochemical localisation of the sst₄ receptor in the rat brain. This is the first immunohistochemical study of the localisation of the sst₄ receptor in the human brain, and implicates this receptor in the function of higher centres of the human nervous system.

Introduction

Somatostatin (somatotrophin-release inhibiting factor, SRIF) was initially isolated as a tetradecapeptide that reduced the release of growth hormone from the pituitary [4]. In addition to its neuroendocrine role, somatostatin has diverse neurophysiological effects (for recent reviews see Refs. [6], [8], [28], [32]).

The cellular physiological processes modulated by somatostatin are mediated through a family of somatostatin receptors of which five receptors have been cloned, termed sst_1–5 [16]. In mice and rats, the sst₂ receptor exists in two splice variants, sst_2(a) and a C-terminal truncated receptor isoform sst_2(b) [39], [41]. The somatostatin receptors are G-protein coupled seven transmembrane receptors, which interact with a wide range of downstream signalling targets (for review see Ref. [21], [32]). Based upon pharmacological studies and sequence analysis the receptors have been divided into two subgroups: the SRIF₁ group consists of sst₂, sst₃ and sst₅ and the SRIF₂ group consists of sst₁ and sst₄.

The distribution of somatostatin receptor mRNAs in the adult human brain has been investigated by in situ hybridisation studies [24], [26], [38]. These showed the mRNAs of the sst_1–4 receptors to have distinct but overlapping distribution patterns [24], [26], [38]. The expression of sst₅ receptor mRNA was detected in the cerebellum and in the pituitary at a low level only [38]. Overall, these findings in the human brain are similar to the expression patterns of the somatostatin receptors in the adult rat brain [11], [18], [22], [23], [34].

Recently, a number of investigators have developed specific antibodies raised against the somatostatin receptors. Using these tools, the cellular localisation of the sst_1–5 receptors have been mapped in the adult rodent brain [7], [9], [12], [13], [14], [29], [30], [33], [37]. To date, only the localisation of the sst_2(a) receptor has been investigated in the human brain [27]. In this study we report for the first time the cellular localisation of the sst₄ receptor in selected regions of the human brain. Parts of these results have been reported in abstract form at the British Pharmacological Society January 2000.

Section snippets

Human tissue

Human brain tissue was obtained from the New Zealand Neurological Foundation Human Brain Bank at the University of Auckland using ethical consent procedures approved by the University of Auckland Human Subjects Ethics Committee. Each case was devoid of previous history of neurological illness. The post mortem delay for each case was between 8 and 20 h (see Table 1).

Chemicals

All chemicals used where obtained from Sigma (Poole, UK) unless otherwise stated.

Anti-sst₄ antibody preparation and purification

The generation off the sst₄ selective antibody has

Specificity of the antibody

In immunohistochemical control experiments using preabsorbed antibody a complete loss of sst₄ receptor-like immunoreactivity in all brain regions examined was observed, confirming the specificity of the sst₄ receptor antibody labelling (Fig. 2, Fig. 4, Fig. 5). Further controls were carried out using the pre-immune serum and no staining was detected using these sera (data not shown).

Distribution of sst₄ receptor immunoreactivity in selected human brain regions

The affinity-purified sst₄ receptor antibody was used to localise the sst₄ receptor immunoreactivity in selected

Discussion

The sst₄ receptor antibody used in our immunohistochemical analysis has previously been shown to be specific. The specificity was demonstrated by Western blots and immunocytochemistry, using host cell lines [33]. Pre-absorption experiments carried out on all the human brain regions examined here, showed that the specific signal was abolished after pre-incubating the affinity-purified antibody with the C-terminal sst₄ receptor antigen.

Common problems associated with the use of human post mortem

Acknowledgements

Inger-Sofie Selmer is grateful for the financial support from the Cambridge Overseas Research Trust.

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Research reportFirst localisation of somatostatin sst4 receptor protein in selected human brain areas: an immunohistochemical study

Abstract

Introduction

Section snippets

Human tissue

Chemicals

Anti-sst4 antibody preparation and purification

Specificity of the antibody

Distribution of sst4 receptor immunoreactivity in selected human brain regions

Discussion

Acknowledgements

J. Biol. Chem.

Neuroscience

Neuroscience

Neurosci. Lett.

Neuroscience

Neuroscience

Trends Pharmacol. Sci.

Brain Res.

Neuroscience

Neuroscience

Biochem. Biophys. Res. Commun.

Mol. Brain Res.

Prog. Neurobiol.

Neuroscience

J. Biol. Chem.

Regul. Pept.

Neuroscience

Biochem. Biophys. Res. Commun.

FEBS Lett.

Brain Res.

Research report
First localisation of somatostatin sst₄ receptor protein in selected human brain areas: an immunohistochemical study

Anti-sst₄ antibody preparation and purification

Distribution of sst₄ receptor immunoreactivity in selected human brain regions