Wolf-Eberhard Siems

Gene expression and activity of specific opioid-degrading enzymes in different brain regions of the AA and ANA lines of rats

There is increasing evidence that alcoholism runs in families suggesting that genetic factors may play a role. In support of this hypothesis, the alcohol-preferring (AA) and the alcohol-avoiding (ANA) rat lines have been developed through selective outbreeding. Numerous studies indicate that the endogenous opioid system may be involved in controlling ethanol consumption. Changes in opioid peptides and opioid receptors have been described after ethanol intake. But, the influence of ethanol on peptidolytic degradation of opioid peptides has been largely ignored, although the peptidase-mediated metabolism of neuropeptides is known as an important regulatory site of peptidergic transmission. Neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE) degrade neuropeptides, including enkephalin and are expressed in the brain. Furthermore, a good correspondence between the regional distribution of NEP and opioid receptors in rat brain has already been reported pointing to a possible role of NEP in regulating opioid peptides. For both enzymes studied, the gene expression pattern was found to be in good agreement with the corresponding enzyme activities in the brain regions investigated, showing the highest levels for both specific mRNAs and enzyme activities in the striatum. Differences in both measured parameters were detected in distinct brain regions of AA and ANA rats. Furthermore, in some brain regions discrepancies between ACE and NEP mRNA levels and the corresponding enzyme activities were observed. For example, in olfactory bulb and striatum such discrepancies were found for both enzymes studied. In tegmentum/colliculi a higher NEP gene expression in AA rats was associated with a higher NEP enzyme activity compared to the amounts found in ANA rats.

Expression and characterization of the substance P (NK1) receptor in the rat pituitary and AtT20 mouse pituitary tumor cells

Although substance P is known to take part in the regulation of the anterior pituitary, no conclusive evidence for the expression of the tachykinin NK1 receptor has been found yet in the pituitary or pituitary derived cells. With the reverse transcription-polymerase chain reaction (RT-PCR) method we could detect the low abundant transcripts of the NK1 receptor in the rat pituitary and in the AtT20 cell line (clone D16v). Furthermore, the functional expression of the NK1 receptor in AtT20 cells was confirmed by activation of the phosphatidylinositol-calcium second messenger system when the cells were treated with substance P. In addition, binding studies also indicated the functional expression of this receptor in AtT20 cells. Thus we provide the first evidence that the NK1 receptor is expressed in AtT20 cells and the rat pituitary.

Elements of the kallikrein–kinin system are present in rat seminiferous epithelium

Peptide hormones are involved in the paracrine regulation of several physiological processes. A possible function of the kallikrein-kinin system (KKS) in mammalian reproduction has been discussed. To evaluate its putative role in spermatogenesis, we searched for components of the KKS (kallikrein, kininases, kinin receptor) in the rat testis. Specific immunostaining demonstrated that the kininogenase tissue kallikrein was present in round and elongated spermatids. Leydig cells, Sertoli cells, peritubular cells, spermatogonia and spermatocytes were not stained. Bradykinin in the supernatant of Sertoli cell cultures was effectively degraded. The resulting metabolites were analysed by high-performance liquid chromatography (HPLC). Specific protease inhibition in the degrading experiments confirmed the occurrence of several metalloproteases on Sertoli cell membranes, including neutral metalloendopeptidases (NEP 24.11 and NEP 24.15), kininase type II (angiotensin converting enzyme, ACE), and kininase type I (metallocarboxypeptidase). Northern blots hybridized with a bradykinin B2 receptor probe showed the presence of B2 receptor mRNA in testis homogenate and Sertoli cell extract. All components of the kallikrein-kinin system are present within the seminiferous epithelium of the rat. Therefore, this paracrine peptide system may play a role in the regulation of Sertoli cell function or in the Sertoli cell-germ cell crosstalk.

Expression and Location of the Bradykinin B2 Receptor in Rat Testis

Abstract To investigate the possible role of the local tissue kallikrein-kinin system in spermatogenesis, we analyzed gene expression and cellular distribution of the bradykinin subtype-2 receptor (B2 receptor) in the rat testis. Reverse transcription-polymerase chain reaction revealed B2 receptor expression in testis and primary cultures of Sertoli cells and peritubular cells isolated from immature and mature rats. In situ hybridization of the B2-receptor mRNA showed intense labeling of cells on the base of the seminiferous tubule, whereas the autoradiographic signals gradually decreased toward the lumen. Immune histochemistry using testicular sections of pubertal and adult rats showed specific staining for the B2-receptor protein in cells of the adluminal compartment of the seminiferous tubules, especially on pachytene spermatocytes and spermatids. This immunostaining varied with the stages of the seminiferous cycle. The receptor protein was also observed on peritubular cells of pubertal rats. In conclusion, we demonstrated a stage-specific expression of the bradykinin B2 receptor in different cells of the seminiferous tubules of the rat testis. The results point to a possible function of the tissue kallikrein-kinin system in the local regulation of spermatogenesis.

Degradation of bradykinin in semen of ram and boar

The pattern of bradykinin (BK; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9)-inact iva ting peptidases in semen of boar and ram was investigated. The degradation of BK in semen was completely abolished by the metalloprotease inhibitors EDTA and o-phenanthroline. Inhibitors of angiotensin-converting enzyme (ACE; EC 3.4.15.1) and phosphoramidon, an inhibitor of neutral metalloendopeptidase (NEP; EC 3.4.24.11), were only partially effective in preventing BK degradation in semen. An additive effect was seen with simultaneous inhibition of both enzymes, resulting in complete abolition of BK degradation. HPLC analysis demonstrated that exogenous BK in semen is cleaved at Gly4-Phe5, Phe5-Ser6 and Pro7-Phe8. These results indicate that NEP and ACE are the main peptidases responsible for rapid BK inactivation in semen. The involvement of other peptidases known to be responsible for BK cleavage in other tissues and body fluids, namely carboxypeptidase N (EC 3.4.12.7), post proline cleaving enzyme (EC 3.4.21.26) and aminopeptidase P (EC 3.4.11.9) was excluded. NEP and ACE were shown to be localized mainly in seminal plasma and to a lesser extent on sperm cells.