Regulation of the biosynthesis and processing of chromogranins in organotypic slices: influence of depolarization, forskolin and differentiating factors.

Slices from rat hippocampus in organotypic culture were used to study the biosynthesis regulation of chromogranins A and B and secretogranin II. Additionally, we investigated the proteolytic conversion of secretogranin II and the levels of prohormone convertases putatively involved. Forskolin treatment and depolarization with potassium plus BayK 8644 led to significant increases in secretogranin II mRNA in the principal cells of the hippocampus. Enhanced expression of secretogranin II was also reflected by a rise in peptide levels. Despite this induction of biosynthesis the extensive processing to secretoneurin normally observed in brain was maintained. Both forskolin and depolarization upregulated the prohormone convertase (PC)1, but not PC2, indicating that PC1 levels are critical for secretoneurin production under stimulating conditions. Results obtained for chromogranins A and B were less consistent. For chromogranin A mRNA, changes were restricted to granule cells; for chromogranin B, a response in granule cells was observed to depolarization but not to forskolin, and effects in pyramidal neurons were weak. Accordingly, we were unable to detect alterations in chromogranin A and B protein levels. Furthermore, we tested several neurotrophic growth factors and found that only basic fibroblast growth factor raised secretogranin II expression without affecting chromogranins A and B. The hippocampal slice preparation allowed well controlled treatment with identification of neuronal subpopulations and yielded data largely matching experiments in vivo and in cell culture. The pronounced regulation of secretogranin II and its effective processing underlines the importance of the resulting peptide secretoneurin as an active neuropeptide in the nervous system.

[Development and application of dynamic MR imaging in the evaluation of perfusion changes in rectal carcinoma during radiotherapy in clinical routine. Preliminary results]. / Entwicklung und Anwendung dynamischer MRT-Messungen zur Evaluierung von Perfusionsveränderungen bei Rektumkarzinomen unter Bestrahlung in der klinischen Routine. Erste Ergebnisse.

PURPOSE: This study was aimed at measuring microcirculatory parameters and contrast medium accumulation within the rectal carcinoma during fractionated radiotherapy in the clinical setting. MATERIAL AND METHODS: Perfusion data were observed in patients with rectal carcinoma (n = 8) who underwent a pre-operative combined chemo/radiotherapy. To acquire perfusion data, an ultrafast T1 mapping sequence was carried out on a 1.5-Tesla whole body imager to obtain T1 maps at intervals of 14 or 120 seconds. The overall measurement time was 40 minutes. The transaxial slice thickness (5 mm) was chosen in such a way that both arterial vessels and the tumor could be clearly identified. The gadolinium-DTPA (Gd-DTPA) concentration time curve was evaluated for arterial blood and tumor after intravenous constant rate infusion. The method allows a spatial resolution of 2 x 2 x 5 mm and a temporal resolution of 14 seconds. Patients underwent MR imaging before and at constant intervals during fractionated radiotherapy. RESULTS: Spatial and temporal resolution of dynamic T1 mapping was sufficient to reveal varying CM accumulation levels within the tumor and to identify the great arteries in the pelvis. In 6 patients Gd-DTPA concentration-time-curves were evaluated within the tumor during radiation. Pi index of Gd-DTPA versus radiation dose showed a significant increase in the first or second week of treatment, then either returned slowly to pretreatment level or a renewed increase was observed. The average Pi-value at the beginning was 0.16 (+/- 0.049), reaching highest level of 0.23 (+/- 0.058). In all groups the rise from the Pi-value to the Pi-maximum was statistically significant. The relative increase in perfusion ranged between 20 to 83%. CONCLUSION: The results show, that the ultrafast MR-technique described above provide a suitable tool for monitoring tumor microcirculation during therapeutic interventions and offers the potential for an individualized optimization of therapeutic procedures.

Ontogenic development of secretogranin II and of its processing to secretoneurin in rat brain.

The ontogenic development of secretogranin II was studied by immunochemistry and immunohistochemistry. Extracts of brains from various developmental stages were analyzed by a radioimmunoassay for secretoneurin, a peptide derived from secretogranin II. From gestational day 13 to adulthood the levels increased from 0.1 to 94 fmol/mg wet weight. Characterization of the immunoreactivity by molecular sieve chromatography revealed that throughout all developmental stages the proprotein secretogranin II was fully processed to the free peptide secretoneurin. In immunohistochemistry secretoneurin-IR was first detected at embryonic day 13. Between embryonic days 14 and 18 a strong increase in the number of secretoneurin immunopositive cells was observed in many brain areas, notably in the amygdala, hypothalamus, olfactory bulb and several brainstem nuclei. The pattern of staining during development is quite similar to that in the adult. The present paper demonstrates that secretoneurin immunoreactivity appears early in embryonic life. Processing of the proprotein secretogranin II starts when the protein is first synthesized apparently at about the same time when the prohormone convertase PC1 and PC2 can be demonstrated.

Processing of secretogranin II by prohormone convertases: importance of PC1 in generation of secretoneurin.

Secretoneurin is a recently characterized neuropeptide present in the primary amino acid sequence of secretogranin II. We investigated the proteolytic processing of secretogranin II by prohormone convertases in vivo in a cellular system using the vaccinia virus system. Both PC1 and PC2 can cleave the secretogranin II precursor at sites of pairs of basic amino acids to yield intermediate-sized fragments. Other convertases like PACE4, PC5 and furin were not active. For the formation of the free neuropeptide secretoneurin a different pattern was found. Only PC1 but none of the other convertases tested including PC2 were capable of generating secretoneurin. Our results demonstrate that the prohormone convertases PC1 and PC2 are involved in proteolytic processing of secretogranin II. The neuropeptide secretoneurin can only be generated by PC1 suggesting tissue-specific processing of secretogranin II in neurons expressing different subsets of the prohormone convertases.