Hermann Lübbert

Differential regulation of three sodium channel messenger RNAs in the rat central nervous system during development.

The levels of the mRNAs encoding sodium channels I, II and III in various regions of the developing rat central nervous system (from embryonal day 10 to postnatal day 90) have been examined by blot hybridization analysis with specific probes. The three sodium channel mRNAs exhibit different temporal and regional expression patterns. The expression of sodium channel I mRNA rises after a lag phase to adult levels during the second and third postnatal weeks with stronger increases in caudal regions of the brain and in spinal cord. Sodium channel II mRNA increases steadily until the first postnatal week, keeping high adult levels in rostral regions of the brain or reaching low adult levels after the second postnatal week in most caudal regions of the brain and in spinal cord; cerebellum shows low levels during the first two postnatal weeks but high adult levels. In all regions, sodium channel III mRNA attains maximum levels around birth and decreases during the first and second postnatal weeks to reach variable low adult levels. These results suggest that sodium channel III is expressed predominantly at fetal and early postnatal stages and sodium channel I predominantly at late postnatal stages, whereas sodium channel II is expressed throughout the developmental stages studied with greater regional variability.

Expression of serotonin receptor mRNAs in blood vessels

Using RT‐PCR we distinguished mRNAs for all known G‐protein coupled serotonin receptors expressed in various rat and porcine blood vessels. Nearly all vessels expressed 5HT1 dβ, 5‐HT2A, 5‐HT2B, 5‐HT4, and 5‐Ht7 receptor mRNA to different extents. New splice variants of the porcine 5‐HT4 receptor were observed. Similar PCR assays were performed with endothelial and smooth muscle cells from human pulmonary artery, aorta, and with endothelial cells from human coronary artery and umbilical vein. All endothelial cells expressed 5‐HT1dβ, 5‐HT2b, and 5‐HT4 receptor mRNA, whereas in smooth muscle cells 5‐HT1dβ, 5‐HT2A, 5‐HT7, and in some experiments 5‐HT2B receptor mRNA were found. A model for the regulation of vascular tone by different 5‐HT receptors is proposed.

The distribution and cellular localization of the serotonin 1C receptor mRNA in the rodent brain examined by in situ hybridization histochemistry. Comparison with receptor binding distribution

The regional distribution and cellular localization of mRNA coding for the serotonin 1C receptor were investigated in tissue sections of mouse and rat brain by in situ hybridization histochemistry. Several 32P-labelled riboprobes derived from mouse genomic clones were used. The serotonin 1C receptor binding sites were visualized autoradiographically and quantified using [3H]mesulergine as ligand, in the presence of spiperone to block serotonin 1C receptors. Strong hybridization signal was observed in the choroid plexus of all brain ventricles. High levels of hybridization were also seen in the anterior olfactory nucleus, pyriform cortex, amygdala, some thalamic nuclei, especially the lateral habenula, the CA3 area of the hippocampal formation, the cingulate cortex, some components of the basal ganglia and associated areas, particularly the nucleus subthalamicus and the substantia nigra. The midbrain and brainstem showed moderate levels of hybridization. The distribution of the serotonin 1C receptor mRNA corresponded well to that of the serotonin 1C receptors. The highest levels of serotonin 1C receptor binding were observed in the choroid plexus. In addition, significant levels of the serotonin 1C receptor binding were seen in the anterior olfactory nucleus, pyriform cortex, nucleus accumbens, ventral aspects of the striatum, paratenial and paracentral thalamic nuclei, amygdaloid body and substantia nigra pars reticulata. The cingulate and retrosplenial cortices as well as the caudal aspects of the hippocampus (CA3) were also labelled. Binding in brainstem and medulla was low and homogeneously distributed. No significant binding was seen in the habenular and subthalamic nuclei. Similar findings were obtained in rat brain. These results demonstrate that, in addition to their enrichment in the choroid plexus, the serotonin 1C receptor mRNA and binding sites are heterogeneously distributed in the rodent brain and thus could be involved in the regulation of many different brain functions. The combination of in situ hybridization histochemistry with receptor autoradiography opens the possibility of examining the regulation of the serotonin 1C receptor synthesis after pharmacological or physiological alterations.

Molecular pharmacology of somatostatin receptors

The neuropeptide somatostatin (SRIF) is widely expressed in the brain and in the periphery in two main forms, SRIF-14 and SRIF-28. Similarly, the presence of SRIF receptors throughout the whole body has been reported. SRIF produces a variety of effects including modulation of hormone release (e.g. GH, glucagon, insulin), of neurotransmitter release (e.g. acetylcholine, dopamine, 5-HT), and its own release is modulated by many neurotransmitters. SRIF affects cognitive and behavioural processes, the endocrine system, the gastrointestinal tract and the cardiovascular system and also has tumor growth inhibiting effects. Initially, two classes of SRIF receptors have been proposed on the basis of biochemical and functional studies. However, the recent cloning of five putative SRIF receptor subtypes which belong to the G-protein coupled receptor superfamily suggests that SRIF mediates its various effects via a whole family of receptors. Here we review, in this new context, the molecular pharmacology of the SRIF receptor subtypes present in the brain and in the periphery, and address the question of nomenclature of SRIF receptors.

Cloning and characterization of MUPP1, a novel PDZ domain protein.

Using the yeast two‐hybrid system we isolated a cDNA clone encoding a novel protein interacting with the C‐terminal domain of the 5‐HT2C receptor. The protein, named MUPP1 (multi‐PDZ‐domain protein), contains thirteen PDZ domains and no obvious catalytic domain; it is related to hINADL and a putative C. elegans polypeptide referred to as C52A11.4 containing six or ten PDZ domains, respectively. Domains highly similar to those of MUPP1 are arrayed in the same order in all three proteins. The MUPP1 gene is localized on human chromosome 9p24‐p22. Transcripts encoding MUPP1 are abundant in the brain as well as in several peripheral organs.

Cloning and functional characterization of the rat stomach fundus serotonin receptor.

A DNA segment homologous to the third exons of the serotonin 1C and 2 receptor genes was isolated from a mouse genomic library. The positions of the introns flanking these exons were conserved in the three genes. To examine whether the new fragment was part of an active gene, we used a quantitative PCR protocol to analyse rat RNAs from different tissues and ages. The gene was expressed in stomach fundus at an abundance of 1 × 10(5) mRNA molecules. This tissue contracts in response to serotonin via a receptor that has previously resisted classification. We constructed a cDNA library from rat stomach fundus and isolated clones containing 2020 bp inserts with open reading frames of 465 amino acids comprising seven putative membrane‐spanning regions. The protein was transiently expressed in COS cells and binding of serotonergic ligands to the membranes was analysed. The pharmacological profile resembled that described for the serotonin‐stimulated contraction of the stomach fundus. After expression of this receptor in Xenopus oocytes, the application of serotonin triggered the typical chloride current which presumably results from the activation of phospholipase C. The coupling to this response system was less efficient than that of the 5‐HT1C or 5‐HT2 receptors.

The mouse MPTP model: gene expression changes in dopaminergic neurons

Parkinsons disease (PD) is a common neurodegenerative disorder, characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Although valuable animal models have been developed, our knowledge of the aetiology and pathogenic factors implicated in PD is still insufficient to develop causal therapeutic strategies aimed at halting its progression. The neurotoxicity induced by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) is one of the most valuable models for analysing pathological aspects of PD. In this paper we studied the gene expression patterns underlying the pathogenesis of MPTP‐induced neurodegeneration. We treated young and old C57BL/6 mice with different schedules of MPTP to induce degenerative processes that vary in intensity and time‐course. During the first week after intoxication we used nonradioactive in situ‐hybridization to investigate the expression patterns of genes associated with (i) dopamine metabolism and signalling; (ii) familial forms of PD; (iii) protein folding and (iv) energy metabolism. MPTP injections induced different severities of neuronal injury depending on the age of the animals and the schedule of administration as well as a significant degeneration in the striatum. In situ hybridization showed that MPTP intoxication initiated a number of gene expression changes that (i) were restricted to the neurons of the substantia nigra pars compacta; (ii) were correlated in intensity and number of changes with the age of the animals and the severity of histopathological disturbances; (iii) displayed in each a significant down‐regulation by the end of one week after the last MPTP injection, but (iv) varied within one MPTP regimen in expression levels during the observation period. The subacute injection of MPTP into one‐year‐old mice induced the most severe changes in gene expression. All genes investigated were affected. However, α‐synuclein was the only gene that was exclusively up‐regulated in MPTP‐treated animals displaying cell death.

Cloning and functional characterization of the human 5-HT2B serotonin receptor

Recently, we have reported the cloning of the rat 5‐HT2B receptor cDNA. This receptor is particularly interesting since it may be involved in diseases such as migraine. Here, we describe the isolation of a human 5‐HT2B receptor clone from a cDNA library derived from SH‐SY5Y cells. Although the receptor sequence was only 80% homologous to the rat sequence, the exon‐intron distribution was conserved between the two species. In the human body, the receptor mRNA was detected in most peripheral organs. Only low expression levels were found in the brain. After expression in HEK 293 cells, activation of the receptor stimulated the production of phosphatidylinositol. The pharmacology of this functional response correlated well with that of the rodent receptor.

Expression and regulation of human and rat phosphodiesterase type IV isogenes

Type IV phosphodiesterases (PDE IV) specifically hydrolyze cAMP and are inhibited by rolipram. RT‐PCR was applied to analyze the expression patterns of mRNAs for four cloned human and rat phosphodiesterase type IV isogenes (PDE IV‐A, ‐B, ‐C and ‐D). Although these patterns were mostly coincident for the human and rat PDE IV genes, some differences were found between the two species. PDE IV‐A expression was detectable in human blood but not in rat blood, suggesting a species‐specific difference in the expression of this PDE IV isogene. PDE IV‐C was neither detected in human or rat blood nor in different cell populations of the human immune system. It is further demonstrated that the PDE IV isogene expression is differentially regulated by cAMP in different cell types.

Activation of Meningeal 5-HT2B Receptors: An Early Step in the Generation of Migraine Headache?

Several pharmaceuticals are frequently dispensed to prevent or reduce the occurrence of migraine attacks. The prophylactic effect of these drugs has been suggested to be caused through blockade of serotonin (5‐HT) receptors of type 5‐HT2B or 5‐HT2C. To elucidate which of these receptors is involved, we first used radioligand binding assays to determine the pharmacological profile of the human and rat 5‐HT2B receptor. Furthermore, the potency of drugs used in migraine prophylaxis to stimulate or inhibit 5‐HT2B or 5‐HT2C receptor‐mediated phosphatidyl inositol hydrolysis was measured. All these drugs were found to block both human receptors. Correlation of the receptor affinities with the potencies used in migraine prophylaxis showed significant correlations, which were better for the 5‐HT2B (P= 0.001) than for the 5‐HT2C receptor (P= 0.005). Migraine headache is thought to be transmitted by the trigeminal nerve from the meninges and their blood vessels. Using the reverse transcription–polymerase chain reaction, the expression patterns of all cloned G‐protein‐coupled serotonin receptors were analysed in various human meningeal tissues. All tissues expressed 5‐HT1Dβ, 5‐HT2A, 5‐HT2B, 5‐HT4 and 5‐HT7 mRNAs. Only trace amounts of 5‐HT2C receptor mRNA were found. With organ bath experiments we showed that the 5‐HT2B receptor stimulated the relaxation of the pig cerebral artery via the release of nitric oxide. Our data support the hypothesis that 5‐HT2B receptors located on endothelial cells of meningeal blood vessels trigger migraine headache through the formation of nitric oxide.