High-Content Imaging of Immunofluorescently Labeled TRPV1-Positive Sensory Neurons

Isensee, Jörg; Hucho, Tim

doi:10.1007/978-1-4939-9446-5_8

Cited by 8 publications

(10 citation statements)

References 28 publications

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“…This allows monitoring of the activity of endogenous PKA-II in primary sensory neurons expressing the neuronal marker ubiquitin C-terminal hydrolase L1 (UCHL1, formerly PGP9.5; Isensee et al, 2018 ) using an automated HCS microscopy approach ( Fig. 1 A ; Isensee et al, 2014a ; Isensee and Hucho, 2019 ; Isensee et al, 2018 ; Isensee et al, 2017a ; Isensee et al, 2017b ; Isensee et al, 2014b ).…”

Section: Resultsmentioning

confidence: 99%

“…This antibody allows quantifying endogenous PKA-II activity in nociceptors. Previously, we used this method in combination with high-content screening (HCS) microscopy–based quantification to study G protein–coupled receptor (GPCR)–mediated pro- and anti-nociceptive signaling in nociceptors ( Isensee et al, 2014a ; Isensee and Hucho, 2019 ; Isensee et al, 2018 ; Isensee et al, 2017a ; Isensee et al, 2017b ; Isensee et al, 2014b ). Using this approach, we found that Ca V 1.2 mediates depolarization-induced PKA-II activity in nociceptors and identified a feed-forward mechanism of cAMP-independent regulation of Ca V 1.2 by PKA-II.…”

Section: Introductionmentioning

confidence: 99%

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Depolarization induces nociceptor sensitization by CaV1.2-mediated PKA-II activation

Isensee

Cann

Despang

et al. 2021

Journal of Cell Biology

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Depolarization drives neuronal plasticity. However, whether depolarization drives sensitization of peripheral nociceptive neurons remains elusive. By high-content screening (HCS) microscopy, we revealed that depolarization of cultured sensory neurons rapidly activates protein kinase A type II (PKA-II) in nociceptors by calcium influx through CaV1.2 channels. This effect was modulated by calpains but insensitive to inhibitors of cAMP formation, including opioids. In turn, PKA-II phosphorylated Ser1928 in the distal C terminus of CaV1.2, thereby increasing channel gating, whereas dephosphorylation of Ser1928 involved the phosphatase calcineurin. Patch-clamp and behavioral experiments confirmed that depolarization leads to calcium- and PKA-dependent sensitization of calcium currents ex vivo and local peripheral hyperalgesia in the skin in vivo. Our data suggest a local activity-driven feed-forward mechanism that selectively translates strong depolarization into further activity and thereby facilitates hypersensitivity of nociceptor terminals by a mechanism inaccessible to opioids.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Depolarization induces nociceptor sensitization by CaV1.2-mediated PKA-II activation

Isensee

Cann

Despang

et al. 2021

Journal of Cell Biology

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“…Pharmacological investigations have approved TRP channels as therapeutic targets [16] . It is worthwhile to mention that, the TRPV6 is also found in a variety of mammalian tissues such as placenta, pancreas, and prostate suggesting its further physiological functions [17] .…”

Section: Introductionmentioning

confidence: 99%

“…[15] Pharmacological investigations have approved TRP channels as therapeutic targets. [16] It is worthwhile to mention that, the TRPV6 is also found in a variety of mammalian tissues such as placenta, pancreas, and prostate suggesting its further physiological functions. [17] Furthermore, it has been shown that the TRPV6 gene expressed abnormally in human malignancies such as prostate, breast, ovary, thyroid, and colon cancers.…”

Section: Introductionmentioning

confidence: 99%

One‐Pot Synthesis of Hantzsch 1,4‐Dihydropyridines by a Series of Iron Oxide Nanoparticles: Putative Synthetic TRPV6 Calcium Channel Blockers

Jafari-Chermahini

Tavakol

2021

ChemistrySelect

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Various spinel iron oxide nanoparticles (MFe2O4, M: Ca, Co, Cu, Fe, Hg, Mg, Mn, Ni, Sn and Zn) have been prepared using a single step coprecipitation method. Nanoparticles were characterized using XRD, FESEM, FT‐IR and UV‐Vis diffuse‐reflectance spectroscopy. The catalytic activities of the synthesized nanoparticles in Hantzsch reaction have been studied. HgFe2O4 NPs exhibits the highest catalytic activity than others. The structural properties of synthesized 1,4‐dihydropyridine were characterized by mass spectrometry, FT‐IR and 1H‐ and 13C‐NMR. Finally, the in silico molecular simulations for screening of blocking ability of synthesized 1,4‐dihydropyridine against various TRPV6 proteins were performed. These studies revealed that examined 1,4‐dihydropyridines are promising TRPV6 calcium channel blockers. As a result, molecular modeling confirmed that synthesized 1,4‐DHPs are docked on the pore region of the examined TRPV6 channels.

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“…Over the last years, an assay based on a high content screening (HCS) microscopy approach has been developed allowing the analysis of primary sensory neurons from dissociated dorsal root ganglia (DRG) [23–27]. Fluorescence intensities of labeled antibodies binding to different targets are quantified in tens of thousands of cells on a single‐cell level.…”

Section: Introductionmentioning

confidence: 99%

Scorpion toxin MeuNaTxα‐1 sensitizes primary nociceptors by selective modulation of voltage‐gated sodium channels

et al. 2020

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Nociceptors are specialized sensory neurons that initiate the perception of pain. We applied high content screening microscopy to screen animal venoms for toxins, which activate pain enhancing signal transduction in sensory neurons. We identified the scorpion α‐toxin MeuNaTxα‐1 from Mesobuthus eupeus, which selectively affected tetrodotoxin‐sensitive voltage‐gated sodium channels, in particular NaV1.2, and thereby depolarized the nociceptors, leading to calcium influx, subsequent PKA‐II activation, and thermal hyperalgesia.

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High-Content Imaging of Immunofluorescently Labeled TRPV1-Positive Sensory Neurons

Cited by 8 publications

References 28 publications

Depolarization induces nociceptor sensitization by CaV1.2-mediated PKA-II activation

Depolarization induces nociceptor sensitization by CaV1.2-mediated PKA-II activation

One‐Pot Synthesis of Hantzsch 1,4‐Dihydropyridines by a Series of Iron Oxide Nanoparticles: Putative Synthetic TRPV6 Calcium Channel Blockers

Scorpion toxin MeuNaTxα‐1 sensitizes primary nociceptors by selective modulation of voltage‐gated sodium channels

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