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Coexpression of somatostatin receptors SSTR-3 and SSTR-4 mRNAS in the rat brain
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25
APRIL SUPPLEMENT
P5/7 Coexpression of Somatostatin Receptors SSTR-3 and SSTR-4 mRNAS in the Rat Brain J. P&z and D. Hoyer Preclinical Research, 360/614, SANDOZ Pharma Ltd. CH-4002 Basel, Switzerland In situ hybridization histochemistry was used to analyze the distribution of the messenger RNA @RNA) of the somatostatin receptor subtypes SSTR3 (Yasuda et al. J. Biol. Chem. 1992; 267: 20422) and SSTR-4 (Rohrer et al. Proc. Natl. Acad. Sci. 1993; 90: 4196) in rat brain. Coexpression of both receptor mRNAs was assessed by the simultaneous use of digoxigenin- and isotopic-labeled probes. SSTR-3 mRNA displayed a homogeneous distribution in the cerebral cortex and was mainly expressed in the olfactory bulb, pyramidal cells of the hippocampus, granular cell layer of the dentate gyms, motor and sensory metencephalic nuclei, and the granular and Purkinje cell layers of the cerebellum. SSTR-4 mRNA expression was restricted to the telencephalon and diencephalon, and the highest hybridization signals were found in layers IV and VI of the cerebral cortex, pyramidal cell layer of the hippocampus, especially in the CA1 and CA2 areas, anterior olfactory nuclei and amygdala. Our results show that somatostatin receptors (SSTR-3 and SSTR-4) display different, although partially overlapping, distributions in the rat brain. Interestingly a significant proportion of neurons coexpress both receptors in the hippocampus, cortex and amygdala. It remains to be seen whether SSTR-3 and SSTR-4 receptors coexpressed in the same cell have similar or different functions and/or localizations (e.g. somatodendritic autoreceptors, postsynaptic receptors).
P5/8 Localization and Regulation of Secretoneurin in the Rat Sympathetic Nervous System L. Kfimaschewski, K. Bermdorf, R. Kirchamair*, R. Fischer-Colbrie* and Ch. Heym Institute of Anatomy and Cell Biology, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany and *Institute of Pharmacology, Peter-Mayr-Str. la, A-6020 Innsbruck, Austria Participation of a novel neuropeptide, secretoneurin (SN), in the rat sympathetic pathway to head and neck has been studied by double-labeling imrnunofluorescence, imnnmoelectromnicroscopy, retrograde tracing and radioimmunoassay. SN-immunoreactive (ir) neurons were detected in the intermediate zone of spinal cord levels C8-T5 where the majority of preganglionic neurons projecting to the superior cervical ganglion (SCG) are located. More than
90% of retrogradely labeled preganglionic neurons were apposed by SN-ir nerve endings which often additionally contained substance P or leu-enkephalin. Numerous SNir nerve terminals were also detected in the SCG which disappeared after transection of the cervical sympathetic trunc. Some SN-ir varicosities stained positive for leuenkephalin. SN-IR was localized in large dense-core vesicles in nerve endings and in paraganglionic (small intensely fluorescent) cells which were strongly SN-ir. The majority of postganglionic neurons exhibited a faint SN-signal. At day 6 after transection of postganglionic axons the level of SN-IR (86 f 18 fmol/mg, n = 6) decreased by about 50% compared to the contralateral SCG (157 f 17 finol/mg, n = 6). This study indicates an involvement of SN in modulation of sympathetic outflow, possibly by fimctional interaction with substance P and leu-enkephalin. Furthermore, synthesis of SN may depend on the presence of targetderived factors since the level of SN decreased markedly in response to axotomy of the postganglionic neuron.
P5/9 Secretoneurin and Chromogranin A in Human and Bovine Cerebrospinal Fluid R. Kirchmair, J. Marksteiner*, A. Saria*, R. Fischer-Colbrie and H. Winkler Department of Pharmacology and *Department of Psychiatry, University of Innsbruck, Austria Secretoneurin is a recently discovered neuropeptide, produced by endoproteolytic processing of its precursor secretogranin II. It is present in a variety of brain regions but also in peripheral, capsaicin sensitive afferent Cfibers. In rat brain secretoneurin releases dopamin from striatal slices and in the periphery this peptide is a potent chemoattractor for monocytes. We investigated levels and molecular forms of secretoneurin and chromogranin A, a related protein to secretogranin II, in human and bovine cerebrospinal fluid (CSF) and brain by HPLC and radioimmunoassay (RIA). In CSF and brain samples the majority of secretoneurinimmunoreactivity eluted at the position of the standard peptide in gel-filtration and reversed-phase I-IPLC. This indicates that the precursor secretogranin II is nearly completely processed to the free peptide. For chromogranin A a different elution pattern was seen: most of the chromogranin A immunoreactivity was present as intermediate breakdown products. Small amounts of the precursor chromogranin A and of a peptide, i.e. GE-25 (bovine chromogranin A 367-391) were also detectable. The levels of secretoneurin and chromogranin A were 1503.7 f 90.7 and 2192.9 f 142.6 fmol/ml respectively. These concentrations are comparable to those of other chromogranins, i.e. chromogranin B and 7B2, but lo- to lOO-fold higher than classical neuropeptides like NPY or substance P.
25
APRIL SUPPLEMENT
P5/7 Coexpression of Somatostatin Receptors SSTR-3 and SSTR-4 mRNAS in the Rat Brain J. P&z and D. Hoyer Preclinical Research, 360/614, SANDOZ Pharma Ltd. CH-4002 Basel, Switzerland In situ hybridization histochemistry was used to analyze the distribution of the messenger RNA @RNA) of the somatostatin receptor subtypes SSTR3 (Yasuda et al. J. Biol. Chem. 1992; 267: 20422) and SSTR-4 (Rohrer et al. Proc. Natl. Acad. Sci. 1993; 90: 4196) in rat brain. Coexpression of both receptor mRNAs was assessed by the simultaneous use of digoxigenin- and isotopic-labeled probes. SSTR-3 mRNA displayed a homogeneous distribution in the cerebral cortex and was mainly expressed in the olfactory bulb, pyramidal cells of the hippocampus, granular cell layer of the dentate gyms, motor and sensory metencephalic nuclei, and the granular and Purkinje cell layers of the cerebellum. SSTR-4 mRNA expression was restricted to the telencephalon and diencephalon, and the highest hybridization signals were found in layers IV and VI of the cerebral cortex, pyramidal cell layer of the hippocampus, especially in the CA1 and CA2 areas, anterior olfactory nuclei and amygdala. Our results show that somatostatin receptors (SSTR-3 and SSTR-4) display different, although partially overlapping, distributions in the rat brain. Interestingly a significant proportion of neurons coexpress both receptors in the hippocampus, cortex and amygdala. It remains to be seen whether SSTR-3 and SSTR-4 receptors coexpressed in the same cell have similar or different functions and/or localizations (e.g. somatodendritic autoreceptors, postsynaptic receptors).
P5/8 Localization and Regulation of Secretoneurin in the Rat Sympathetic Nervous System L. Kfimaschewski, K. Bermdorf, R. Kirchamair*, R. Fischer-Colbrie* and Ch. Heym Institute of Anatomy and Cell Biology, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany and *Institute of Pharmacology, Peter-Mayr-Str. la, A-6020 Innsbruck, Austria Participation of a novel neuropeptide, secretoneurin (SN), in the rat sympathetic pathway to head and neck has been studied by double-labeling imrnunofluorescence, imnnmoelectromnicroscopy, retrograde tracing and radioimmunoassay. SN-immunoreactive (ir) neurons were detected in the intermediate zone of spinal cord levels C8-T5 where the majority of preganglionic neurons projecting to the superior cervical ganglion (SCG) are located. More than
90% of retrogradely labeled preganglionic neurons were apposed by SN-ir nerve endings which often additionally contained substance P or leu-enkephalin. Numerous SNir nerve terminals were also detected in the SCG which disappeared after transection of the cervical sympathetic trunc. Some SN-ir varicosities stained positive for leuenkephalin. SN-IR was localized in large dense-core vesicles in nerve endings and in paraganglionic (small intensely fluorescent) cells which were strongly SN-ir. The majority of postganglionic neurons exhibited a faint SN-signal. At day 6 after transection of postganglionic axons the level of SN-IR (86 f 18 fmol/mg, n = 6) decreased by about 50% compared to the contralateral SCG (157 f 17 finol/mg, n = 6). This study indicates an involvement of SN in modulation of sympathetic outflow, possibly by fimctional interaction with substance P and leu-enkephalin. Furthermore, synthesis of SN may depend on the presence of targetderived factors since the level of SN decreased markedly in response to axotomy of the postganglionic neuron.
P5/9 Secretoneurin and Chromogranin A in Human and Bovine Cerebrospinal Fluid R. Kirchmair, J. Marksteiner*, A. Saria*, R. Fischer-Colbrie and H. Winkler Department of Pharmacology and *Department of Psychiatry, University of Innsbruck, Austria Secretoneurin is a recently discovered neuropeptide, produced by endoproteolytic processing of its precursor secretogranin II. It is present in a variety of brain regions but also in peripheral, capsaicin sensitive afferent Cfibers. In rat brain secretoneurin releases dopamin from striatal slices and in the periphery this peptide is a potent chemoattractor for monocytes. We investigated levels and molecular forms of secretoneurin and chromogranin A, a related protein to secretogranin II, in human and bovine cerebrospinal fluid (CSF) and brain by HPLC and radioimmunoassay (RIA). In CSF and brain samples the majority of secretoneurinimmunoreactivity eluted at the position of the standard peptide in gel-filtration and reversed-phase I-IPLC. This indicates that the precursor secretogranin II is nearly completely processed to the free peptide. For chromogranin A a different elution pattern was seen: most of the chromogranin A immunoreactivity was present as intermediate breakdown products. Small amounts of the precursor chromogranin A and of a peptide, i.e. GE-25 (bovine chromogranin A 367-391) were also detectable. The levels of secretoneurin and chromogranin A were 1503.7 f 90.7 and 2192.9 f 142.6 fmol/ml respectively. These concentrations are comparable to those of other chromogranins, i.e. chromogranin B and 7B2, but lo- to lOO-fold higher than classical neuropeptides like NPY or substance P.