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Similar to Expression of the non-neuronal cholinergic system in human knee synovial tissue from patients with rheumatoid arthritis and osteoarthritis
Similar to Expression of the non-neuronal cholinergic system in human knee synovial tissue from patients with rheumatoid arthritis and osteoarthritis (20)
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Expression of the non-neuronal cholinergic system in human knee synovial tissue from patients with rheumatoid arthritis and osteoarthritis
- 2. Acetylcholine (ACh) 7. local signaling molecule 8. Cellular proliferation Introduction and Background Importance and role in the body: 1. Neurotransmitter 2. Activation of skeletal muscles 3. Direct effect on vascular tone 4. Enhancement of alertness 5. Sustaining attention 6. Learning and memory
- 3. Neuronal and Non-neuronal CS Function Neuronal CS Non-Neuronal CS SYNTHESIS Nerve terminals whole cell STORAGE vesicles cytosol RELEASE Exocytosis on demand Transporter mediator continuously ACTION last short last long ELIMINATION rapid slow Acting as Acting as
- 4. Neuronal-type Muscle-type I II III IV α9, α10 α7 , α8 1 2 3 α1, β1, δ, γ, εα2, α3, α4, α 6 β2, β4 β3, α5 ACh Receptors Muscarinic Nicotinic DECREASE amount of: I. TNFα II. IL-6 III. Other pro-inflammatory cytokines
- 5. Transporters Muscarinic Receptors Enzymes Choline AcetylTransferase Muscarinic acetylcholine receptors 1-5 Organic cation transporters 1-3 Carnitine AcetylTransferase AcetylcholinEsterase Butyrylcholinesterase high-affinity choline transporter Vesicular Acetylcholine Transporter A hit about the Terms
- 6. NM OC+ OC+ OCT3 OC+ OC+ OCT2 OC+ OC+ OCT1 CarAT Carnitine Acetyl Carnitine Acetyl-CoA CoA Krebs cycle Mitochondrial Matrix BChE: Butyrylcholinesterase
- 7. Methods and Materials Patients and Samples synovial tissue (each n=5) 30 to 73 years old RA and OA patients Knee joint replacement surgery frozen in liquid nitrogen
- 8. Reverse Transcription Polymerase Chain Reaction (Qualitative) RNA of synovial tissue & (+ )controls was isolated Lipid Tissue Mini Kit RNA was reverse ‐ transcribed Quantitect® Kit (Qiagen) Amplifying cDNAs AmpliTaq Gold polymerase RT-PCR products were separated electrophoresis RT-PCR
- 9. qPCR Real-time Polymerase Chain Reaction (Quantitative) cDNA + Quantifast SYBR Green PCR Mastermix + primers denaturation step of 5 min at 95 °C 40 cycles of 10 s at 95 °C and 30 s at 60 °C Verifying specificity and identity by ↑ tempt. from 60 °C to 95 °C in steps of 0.5 °C every 10 s GOI expression was normalized to the housekeeping gene (regulatory gene) βMG Followed by Indirect immunofluorescence on OA and RA synovial tissue Thermal Cycling separation of the NA double chain allows the binding of the primers with the DNA template
- 10. ACh synthesizing and degrading enzymes and transportersResult s
- 13. ACh Receptors
- 15. box and whisker diagram Reminder interquartile range (IQR) Or middle 50%
- 16. OCT3 M3R nAChRα7 Real-time Polymerase Chain Reaction box and whisker diagram
- 17. Discussion Expressed in OA α2 α3 α4 α5 α6 α7 α9 α10 ✗ Expressed in RA ✗ α3 ✗ α5 α6 α7 α9 α10 β4 Not able to build a functional receptor β-subunits only α-homopentameric & α-heteropentameric nAChRs
- 18. , IL-1, IL-6 Fibroblast-like synoviocytes (FLS) &
- 19. non-neuronal CS components DIFFERENTLY expressed between RA and OA α2 α4 β4 M3 M4 M5 OCT1&3 Peripheral sites of anti nociceptive action Pain regulation Choline deficiency Cellular apoptosis Specific Both
- 20. Any Questions ?
Editor's Notes
- controls were performed: (a) samples processed without reverse transcription (−RT) were run to control for contamination with genomic DNA, (b) RT-PCR runs without template (H2O), and (c) primers for β2-microglobulin (βMG) were used as a positive control for efficiency of RNA isolation and cDNA synthesis. (d) Several human tissues and the neuroblastoma cell line SH-SY5Y were used as positive control for quality of the genespecific primers.
- Boxplot (with an interquartile range) and a probability density function (pdf) of a Normal N(0,σ2) Population
- It could be speculated that in the synovial tissue of OA patients the expressed nAChR subunits α2–α6 might act as non-channel effectors as these subunits could also be detected on protein level in the OA synovium despite the lack of β-subunit.