scholarly journals Specific detection of the endogenous transient receptor potential (TRP)-1 protein in liver and airway smooth muscle cells using immunoprecipitation and Western-blot analysis

2002 ◽  
Vol 364 (3) ◽  
pp. 641-648 ◽  
Author(s):  
Hwei Ling ONG ◽  
Jinglong CHEN ◽  
Tim CHATAWAY ◽  
Helen BRERETON ◽  
Lei ZHANG ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Western Blot ◽  
Airway Smooth Muscle ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Airway Smooth Muscle Cells ◽  
Transient Receptor

Although there are numerous reports of the presence of mRNA encoding the transient receptor potential (TRP)-1 protein in animal cells and of the detection of the heterologously expressed TRP-1 protein by Western-blot analysis, it has proved difficult to unequivocally detect endogenous TRP-1 proteins. A combination of immunoprecipitation and Western-blot techniques, employing a polyclonal antibody and a monoclonal antibody respectively, was developed. Using this technique, a band of approx. 80kDa was detected in extracts of H4-IIE rat liver hepatoma cell line and guinea-pig airway smooth muscle (ASM) cells transfected with human TRPC-1 cDNA. In extracts of untransfected H4-IIE cells, ASM cells, rat brain and guinea-pig brain, a band of approx. 92kDa was detected. Reverse transcriptase PCR experiments detected cDNA encoding both the α- and β-isoforms of TRP-1 in H4-IIE cells. Treatment of protein extracts with peptide N-glycosidase F indicated that the 92kDa band represents an N-glycosylated protein. Western blots conducted with a commercial polyclonal anti-(TRP-1) antibody (Alm) detected a band of 120kDa in extracts of H4-IIE cells and guinea-pig ASM cells. A combination of immunoprecipitation and Western-blotting techniques with the Alm antibody did not detect any bands at 92kDa or 120kDa in extracts of H4-IIE and ASM cells. It is concluded that (a) the 92-kDa band detected in untransfected H4-IIE and ASM cells corresponds to the N-glycosylated β-isoform of endogenous TRP-1, (b) the combined immunoprecipitation and Western-blot approach, employing two different antibodies, provides a reliable and specific procedure for detecting endogenous TRP-1 proteins, and (c) that caution is required in developing and utilizing anti-(TRP-1) antibodies.

scholarly journals Role of transient receptor potential vanilloid 1 in the modulation of airway smooth muscle tone and calcium handling

2017 ◽  
Vol 312 (6) ◽  
pp. L812-L821 ◽  
Author(s):  
Gene T. Yocum ◽  
Jun Chen ◽  
Christine H. Choi ◽  
Elizabeth A. Townsend ◽  
Yi Zhang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Airway Smooth Muscle ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Nerve Fibers ◽  
Calcium Oscillations ◽  
Transient Receptor Potential Vanilloid ◽  
Organ Bath ◽  
Transient Receptor

Asthma is a common disorder characterized, in part, by airway smooth muscle (ASM) hyperresponsiveness. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel expressed on airway nerve fibers that modulates afferent signals, resulting in cough, and potentially bronchoconstriction. In the present study, the TRPV1 transcript was detected by RT-PCR in primary cultured human ASM cells, and the TRPV1 protein was detected in ASM of human trachea by immunohistochemistry. Proximity ligation assays suggest that TRPV1 is expressed in the sarcoplasmic reticulum membrane of human ASM cells in close association with sarco/endoplasmic reticulum Ca2+-ATPase-2. In guinea pig tracheal ring organ bath experiments, the TRPV1 agonist capsaicin led to ASM contraction, but this contraction was significantly attenuated by the sodium channel inhibitor bupivacaine ( n = 4, P < 0.05) and the neurokinin-2 receptor antagonist GR-159897 ( n = 4, P < 0.05), suggesting that this contraction is neutrally mediated. However, pretreatment of guinea pig and human ASM in organ bath experiments with the TRPV1 antagonist capsazepine inhibited the maintenance phase of an acetylcholine-induced contraction ( n = 4, P < 0.01 for both species). Similarly, capsazepine inhibited methacholine-induced contraction of peripheral airways in mouse precision-cut lung slice (PCLS) experiments ( n = 4–5, P < 0.05). Although capsazepine did not inhibit store-operated calcium entry in mouse ASM cells in PCLS ( n = 4–7, P = nonsignificant), it did inhibit calcium oscillations ( n = 3, P < 0.001). These studies suggest that TRPV1 is expressed on ASM, including the SR, but that ASM TRPV1 activation does not play a significant role in initiation of ASM contraction. However, capsazepine does inhibit maintenance of contraction, likely by inhibiting calcium oscillations.

20-HETE inotropic effects involve the activation of a nonselective cationic current in airway smooth muscle

2003 ◽  
Vol 285 (3) ◽  
pp. L560-L568 ◽  
Author(s):  
Martin Cloutier ◽  
Shirley Campbell ◽  
Nuria Basora ◽  
Sonia Proteau ◽  
Marcel D. Payet ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Guinea Pig ◽  
Airway Smooth Muscle ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Isometric Tension ◽  
Transient Receptor Potential Channels ◽  
Inotropic Effects ◽  
Cationic Current ◽  
Transient Receptor

20-Hydroxyeicosatetraenoic acid (20-HETE) controls several mechanisms such as vasoactivity, mitogenicity, and ion transport in various tissues. Our goal was to quantify the effects of 20-HETE on the electrophysiological properties of airway smooth muscle (ASM). Isometric tension measurements, performed on guinea pig ASM, showed that 20-HETE induced a dose-dependent inotropic effect with an EC50 value of 1.5 μM. This inotropic response was insensitive to GF-109203X, a PKC inhibitor. The sustained contraction, requiring Ca2+ entry, was partially blocked by either 100 μM Gd3+ or 1 μM nifedipine, revealing the involvement of noncapacitative Ca2+ entry and L-type Ca2+ channels, respectively. Microelectrode measurements showed that 3 μM 20-HETE depolarized the membrane potential in guinea pig ASM by 13 ± 2mV( n = 7), as did 30 μM 1-oleoyl-2-acetyl- sn-glycerol. Depolarizing effects were also observed in the absence of epithelium. Patch-clamp recordings demonstrated that 1 μM 20-HETE activated a nonselective cationic inward current that may be supported by the activation of transient receptor potential channels. The presence of canonical transient receptor potential mRNA was confirmed by RT-PCR in guinea pig ASM cells.

scholarly journals Xiangsha Liujunzi Decoction Alleviates Symptoms in Rats With Functional Dyspepsia Through EGC-Derived NGF

2021 ◽  
Author(s):  
juanjuan li ◽  
Lin Xu ◽  
Lin Lv ◽  
Enjin Zeng ◽  
Zedan Zhang ◽  
...  
Keyword(s):  
Food Intake ◽  
Western Blot ◽  
Functional Dyspepsia ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Transient Receptor Potential ◽  
Receptor Potential ◽  
Transient Receptor Potential Vanilloid ◽  
Expression Levels ◽  
Enteric Glial Cells

Abstract Background: Xiangsha Liujunzi (XSLJZ) decoction is a traditional chinese prescription that shows to be effective in functional dyspepsia (FD). However, the underlying mechanism remains to be elucidated. Nerve growth factor (NGF) secreted from enteric glial cells (EGCs) is associated with visceral hypersensitivity (VH), which has been wildly accepted as the main pathological mechanism of FD. But it is not clear whether and how EGC-derived NGF contributes to VH in FD. The present study aimed to evaluate the effects of XSLJZ decoction on FD and investigate whether XSLJZ decoction relieves VH by inhibiting EGC-derived NGF and TrkA-Akt-TRPV1 axis.Methods: FD rat models were established using iodoacetamide and modified multiple platform method. The validation of FD model and effects of XSLJZD decoction on FD were evaluated in terms of weight, food intake, hematoxylin-eosin staining and Von Frey test. The expression levels of glial fibrillary acidic protein (GFAP), NGF and transient receptor potential vanilloid receptor 1 (TRPV1) were detected by immunohistochemistry and western blot analysis. The expression of TrkA and Akt was determined by western blot analysis. The co-localization of GFAP and NGF was observed by double-label immunofluorescence.Results: XSLJZ decoction increased the weight, food intake and pain threshold of FD rats. The expression levels of GFAP and NGF, together with NGF-positive EGCs were increased in FD rats, but decreased following XSLJZ decoction treatment. Furthermore, XSLJZ decoction downregulated the over-expression of TrkA, Akt and TRPV1 in FD rats.Conclusions: XSLJZ decoction can effectively improve the symptoms of FD and alleviate VH in FD, which may be mediated via EGC-derived NGF and TrkA-Akt-TRPV1 axis.

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