Wolfgang Meyerhof

Expression patterns of rat somatostatin receptor genes in pre- and postnatal brain and pituitary.

Abstract: The relative abundances of mRNAs encoding four different somatostatin receptors were examined using PCR techniques during postnatal development of the rat brain and hypophysis. In most tissues, somatostatin receptor 1 and 4 mRNAs are more abundant than those encoding somatostatin receptor 2 and 3. Transcript levels of somatostatin receptor subtype 4 are relatively high in the cortex, hippocampus. and striatum, those of subtype 1 in the cortex and brainstem, and those of subtype 3 in the cerebellum. In situ hybridization revealed the presence of significant amounts of somatostatin receptor 1 mRNA. as early as prenatal day 14, in the trigeminal ganglion and in the neuroepithelial layers surrounding the lateral, third, and fourth ventricles. In the developing cortex a morphological change in the sites of somatostatin receptor 1 gene expression occurs; mRNA is present superficially in the cortex at prenatal stages, appears in all layers shortly after birth, and in adult rats is restricted to the deep cortical layers. In the cerebellum, somatostatin receptor 1 mRNA levels are highest around birth, declining thereafter. In contrast, cerebellar somatostatin receptor 3 transcripts are absent at birth, become detectable around postnatal day 7, and reach a maximal level during maturation.

Somatostatin receptor imaging in somatotroph and non-functioning pituitary adenomas: correlation with hormonal and visual responses to octreotide.

A multicentre study was undertaken to determine the value of somatostatin receptor (sst) scintigraphy in predicting hormonal and visual responses to octreotide treatment in GH‐secreting and non‐functioning pituitary adenomas.

Cloning, functional expression and mRNA distribution of an inwardly rectifying potassium channel protein

In GH3/B6 cells at least two different inward K+ currents are observed that are regulated by thyrotropin‐releasing hormone and somatostatin, respectively. Using a polymerase chain reaction based approach a cDNA was isolated and functionally expressed in human embryonic kidney cells that encodes an inward rectifier K+ channel, rIRK3, with a predicted molecular mass of 49.7 kDa. Corresponding transcripts of 2.6 kb have been detected in rat brain, pituitary and GH3/B6 cells. In situ hybridization revealed that rIRK3 mRNA is distributed throughout the brain and occurs predominantly in the piriform cortex, indusium griseum, supraoptic nucleus, facial nucleus and cerebellar Purkinje cells.

Sequence analysis of the promoter region of the rat somatostatin receptor subtype 1 gene

Somatostatin receptor (SSTR) subtype genes are differentially expressed in brain and various peripheral tissues. RNA blotting and semiquantitative PCR analyses have revealed low levels of SSTR1 mRNA in the gastrointestinal tract and relatively high levels in GH3 anterior pituitary cells. As a first step in the investigation of the regulation of SSTR1 gene expression, we isolated a genomic fragment that contains the promoter region and determined the transcriptional initiation site. The SSTR1 gene lacks introns and TATA and CAAT motifs, but possesses several consensus recognition sequences for the transcription factors GCF and AP‐2. The presence, also, of two Pit‐1 binding sites could explain the high SSTR1 mRNA levels in GH3 cells.

Molecular cloning of a bovine MSH receptor which is highly expressed in the testis

A novel G protein‐coupled receptor (BDF3) was isolated from bovine genomic DNA by a combined approach of polymerase chain reaction (PCR) and hybridization techniques. The predicted amino acid sequence is 317 amino acids in length and displays 80% homology to the human α‐MSH receptor MC1. Stably transfected into CHO‐K1 cells, BDF3 mediates an increase of intracellular cAMP‐levels following incubation with NLE‐α‐MSH, a potent α‐MSH analog. The stimulation with ACTH1–10 is only moderate and γ‐MSH is ineffective. Northern blot analysis of bovine tissues revealed that the BDF3 gene is higly expressed in the testis as a single 2.3 kb mRNA species, suggesting an involvement of the BDF3 receptor in spermatogenesis.

Characterization of N- and C-terminal deletion mutants of the rat serotonin HT2 receptor in Xenopus laevis oocytes

Deletion mutants of the serotonin HT2 receptor have been constructed in which the N- and C-terminal sequences have been gradually removed. The mutant constructs were assayed for their biological activity by electrophysiological measurements in Xenopus oocytes previously injected with the respective in vitro synthesized cRNAs. No significant loss of biological activity was observed when either the extracellular N-terminal sequence, including all potential glycosylation sites, up to the beginning of the first transmembrane domain or the C-terminal sequence up to cystein residue 397, which is conserved in most G-protein coupled receptor known so far, was deleted from the serotonin HT2 receptor constructs.

A Rat Pituitary Tumour K+ Channel Expressed in Frog Oocytes Induces a Transient K+ Current Indistinguishable from that Recorded in Native Cells

A voltage‐gated K+ channel protein has been cloned from a cDNA library derived from poly(A)+ RNA of the rat pituitary tumour cell line GH3/B6 by the polymerase chain reaction technique. The clone referred to as RGHK9 encodes a protein sequence very similar to a recently cloned K+ channel protein from rat brain and heart, with deviations in a few amino‐acid positions. In situ hybridization experiments show that RGHK9 mRNA is also present in the anterior pituitary as well as in other brain regions and that it is particularly abundant in the hippocampus. After injection of cRNA transcribed from the RGHK9 cDNA clone into Xenopus oocytes, the expressed protein induces a transient K+ current. Except for the activation kinetics the properties of this current are indistinguishable from that of the native transient K+ current measured in GH3/B6 cells, e.g. both K+ currents are blocked by 4‐aminopyridine and show the same voltage dependence and slope of steady state activation and inactivation as well as identical time constants of, and slow recovery from, inactivation. Taken together, these data show that the outward‐rectifying voltage‐gated K+ channel protein encoded by the RGHK9 cDNA correlates well in its functional properties with that of a very similar, if not identical, K+ channel present in GH3/B6 cells.

Identification of G protein‐coupled receptors by RNase H‐mediated hybrid depletion using Xenopus laevis oocytes as expression system

A method has been developed for rapidly identifying putative G protein‐coupled receptors isolated initially as small cDNA fragments, following reverse transcription and polymerase chain reaction (PCR) amplification of mRNA. The method is based upon the use of synthetic oligonucleotides deduced from the sequence of the amplified receptor fragments, to direct a RNase H‐mediated specific degradation of hybrids formed between the oligonucleotides and the corresponding receptor‐encoding mRNA. Loss of an agonist‐dependent receptor response in the Xenopus laevis oocyte expression system identifies the amplified receptor fragment. Taking in vitro synthesised serotonin HT2‐receptor (SR)‐encoding mRNA as a model, it was shown that following incubation with RNase H and SR antisense oligonucleotides, injection of this message no longer caused the acquisition of agonist‐dependent membrane currents in voltage‐clamped oocytes. In contrast, when corresponding sense oligonucleotides were used, the serotonin‐evoked membrane responses in oocytes were acquired as normal. The method should allow the identification of receptors which can functionally be expressed and measured in Xenopus oocytes.

Chapter 13 The elucidation of neuropeptide receptors and their subtypes through the application of molecular biology

Publisher Summary Neuropeptides are found widely distributed throughout the animal kingdom where they play a key role, in both the central nervous system and peripheral endocrine system, in the regulation of physiology and behavior. The cloning of cDNAs for a variety of neuropeptide receptors has revealed that these are all the members of the G-protein-coupled receptor family. This chapter focuses on those neuropeptide receptors that are predicted to contain seven membrane-spanning domains and whose sequences have been elucidated by the application of recombinant DNA techniques. The chapter discusses the salient features of neuropeptide receptors and specific examples are used to illustrate particular points. The paucity of specific neuropeptide agonists and antagonists has prevented the affinity purification of many neuropeptide receptors and restricted studies on the possible existence of receptor subtypes. For somatostatin, three receptor cDNAs, which presumably represent subtypes, have been cloned from the mouse, rat, and human. When these cDNAs are expressed individually in cultured mammalian cells, one of the receptor types that is inserted into the membrane binds somatostatin-14 with a higher affinity than that for somatostatin-28, whereas the second receptor type prefers somatostatin-28 over somatostatin-14 and the third type displays no selectivity.

Expression of Histamine H1-Receptors in Xenopus Oocytes Injected with Messenger Ribonucleic Acid from Bovine Adrenal Medulla: Pertussis Toxin Insensitive Activation of Membrane Chloride Currents

Histamine H2‐receptors have been identified in Xenopus oocytes previously microinjected with poly(A) + ribonucleic acid from bovine adrenal glands. Bath application of histamine to ribonucleic acid‐primed oocytes evoked concentration‐dependent, oscillating membrane currents under voltage‐clamp conditions. H1‐receptor specific antagonists clemastine, doxepin, pyrilamine, promethacine, diphenylhydramine, dephenylpyraline and chlorpheniramine, but not H2‐receptor antagonists, cimetidine and ranitidine, inhibited histamine‐induced responses. Membrane currents evoked by bath‐applied histamine were insensitive to pertussis toxin, carried by chloride ions and dependent on intracellular but not extracellular calcium.