Neuropeptide B (NPB) and neuropeptide W (NPW) are neuropeptides, which constitute NPB/W signaling systems together with G-protein coupled receptors NPBWR1. The location and function of NPB/W signaling systems have been predominantly detected and mapped within the CNS, including their role in the modulation of inflammatory pain, neuroendocrine functions, and autonomic nervous systems. The aim of the study is to investigate the impact of diabetes on the neuropeptide B/W signaling system in different heart compartments and neurons which innervates it. In the RT-qPCR analysis, we observed the upregulation of mRNA for preproNPB in RV, for preproNPW in LA, and for NPBWR1 in DRG in diabetic rats. On the contrary, the expression of mRNA for NPBWR1 was downregulated in LV in diabetic rats. In the WB analysis, significant downregulation of NPBWR1 in LV (0.54-fold, p = 0.046) in diabetic rats was observed at the proteomic level. The presence of NPBWR1 was also confirmed in a dissected LCM section of cardiomyocytes and coronary arteries. The positive inotropic effect of NPW described on the diabetic cardiomyocytes in vitro could point to a possible therapeutic target for compensation of the contractile dysfunction in the diabetic heart. In conclusion, the NPB/W signaling system is involved in the regulation of heart functions and long-term diabetes leads to changes in the expression of individual members of this signaling system differently in each cardiac compartment, which is related to the different morphology and function of these cardiac chambers.

• Klíčová slova
• NPBWR1, RT-qPCR, ZDF rat, calcium transients, cardiomyocytes, contraction, laser capture microdissection, neuropeptide B, neuropeptide W, western blot,
• MeSH
• experimentální diabetes mellitus * genetika   MeSH
• krysa rodu Rattus MeSH
• messenger RNA genetika   MeSH
• proteomika MeSH
• receptory neuropeptidů * genetika   metabolismus   MeSH
• receptory spřažené s G-proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• messenger RNA MeSH
• receptory neuropeptidů * MeSH
• receptory spřažené s G-proteiny MeSH

The platform for precise proteomic profiling of targeted cell populations from heterogeneous tissue sections is developed. We demonstrate a seamless and systematic integration of LCM with an automated cap-IA for the handling of a very small-sized dissected tissues section from the kidney, liver and pancreatic Langerhans islet of rats. Our analysis reveals that the lowest LCM section area ≥ 0.125 mm2 with 10 µm thickness can be optimized for the detection of proteins through LCM-cap-IA integration. We detect signals ranging from a highly-abundant protein, β-actin, to a low-abundance protein, LC-3AB, using 0.125 mm2 LCM section from rat kidney, but, so far, a relatively large section is required for good quality of results. This integration is applicable for a highly-sensitive and accurate assessment of microdissected tissue sections to decipher hidden proteomic information of pure targeted cells. To validate this integration, PCK2 protein expression is studied within Langerhans islets of normal and diabetic rats. Our results show significant overexpression of PCK2 in Langerhans islets of rats with long-term diabetes.

• Klíčová slova
• Langerhans islets, PCK2, capillary Western blotting, laser capture microdissection, multiplexing,
• Publikační typ
• časopisecké články MeSH

Members of neuropeptide B/W signaling system have been predominantly detected and mapped within the CNS. In the rat, this system includes neuropeptide B (NPB), neuropeptide W (NPW) and their specific receptor NPBWR1. This signaling system has a wide spectrum of functions including a role in modulation of inflammatory pain and neuroendocrine functions. Expression of NPB, NPW and NPBWR1 in separate heart compartments, dorsal root ganglia (DRG) and stellate ganglia was proven by RT-qPCR, Western blot (WB) and immunofluorescence. Presence of mRNA for all tested genes was detected within all heart compartments and ganglia. The presence of proteins preproNPB, preproNPW and NPBWR1 was confirmed in all the chambers of heart by WB. Expression of preproNPW and preproNPB was proven in cardiac ganglionic cells obtained by laser capture microdissection. In immunofluorescence analysis, NPB immunoreactivity was detected in nerve fibers, some nerve cell bodies and smooth muscle within heart and both ganglia. NPW immunoreactivity was present in the nerve cell bodies and nerve fibers of heart ganglia. Weak nonhomogenous staining of cardiomyocytes was present within heart ventricles. NPBWR1 immunoreactivity was detected on cardiomyocytes and some nerve fibers. We confirmed the presence of NPB/W signaling system in heart, DRG and stellate ganglia by proteomic and genomic analyses.

• Klíčová slova
• NPBW receptor 1, RT-qPCR, Western blot, heart, neuropeptide B, neuropeptide W,
• MeSH
• exprese genu MeSH
• fluorescenční protilátková technika MeSH
• ganglion stellatum metabolismus   MeSH
• myokard metabolismus   MeSH
• neuropeptidy genetika   imunologie   metabolismus   MeSH
• potkani Zucker MeSH
• receptory neuropeptidů genetika   imunologie   metabolismus   MeSH
• receptory spřažené s G-proteiny genetika   imunologie   metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• signální transdukce MeSH
• spinální ganglia metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• neuropeptide B MeSH Prohlížeč
• neuropeptide W, rat MeSH Prohlížeč
• neuropeptidy MeSH
• Npbwr1 protein, rat MeSH Prohlížeč
• receptory neuropeptidů MeSH
• receptory spřažené s G-proteiny MeSH