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Involvement of ANO1 currents in pacemaking of PDGFRα-positive specialised smooth muscle cells in rat caudal epididymis

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Abstract

The epididymal duct exhibits spontaneous phasic contractions (SPCs) to store and transport sperm. Here, we explored molecular identification of pacemaker cells driving SPCs in the caudal epididymal duct and also investigated properties of pacemaker currents underlying SPCs focusing on ANO1 Ca2+-activated Cl− channels (CaCCs). Immunohistochemistry was performed to visualise the distribution of platelet-derived growth factor receptor α (PDGFRα)- or ANO1-positive cells in the rat caudal epididymal duct. Perforated whole-cell patch clamp technique was applied to enzymatically isolated epididymal cells, while SPCs were recorded with video edge-tracking technique. Immunohistochemistry revealed the distribution of α-smooth muscle actin (α-SMA)-positive cells co-expressing both PDGFRα and ANO1 in the innermost smooth muscle layer. Approximately one-third of isolated epididymis cells exhibited spontaneous transient inward currents (STICs) at the holding potential −60 mV. The reversal potential for STICs was close to the calculated chloride equivalent potential depending on intracellular Cl− concentrations. Ani9 (3 µM), the ANO1 specific inhibitor, decreased both amplitude and frequency of STICs, while cyclopiazonic acid (CPA, 30 µM), a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, abolished STICs. Ani9 (3 or 10 µM) reduced the frequency of SPCs without changing their amplitude. Thus, PDGFRα+, ANO1+ specialised smooth muscle cells (SMCs) appear to function as pacemaker cells to electrically drive epididymal SPCs by generating ANO1-dependnet STICs. STICs arising from spontaneous Ca2+ release from intracellular Ca2+ store and subsequent opening of ANO1 result in depolarisations that spread into adjacent SMCs where L-type voltage-dependent Ca2+ channels are activated to develop SPCs.

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The data supporting the findings of present study are available from the corresponding author upon reasonable request.

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Acknowledgements

The authors wish to thank Dr. Richard Lang (Monash University) for his critical reading of the manuscript.

Funding

The present study was partly supported by Grant-in-Aid for Scientific Research (C) from Japan Society for Promotion of the Science to R.M. (No. 19K08426) and H.H. (No. 23K08767).

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Authors and Affiliations

  1. Department of Cell Physiology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

    Wataru Kudo, Retsu Mitsui & Hikaru Hashitani

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  1. Wataru Kudo
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  2. Retsu Mitsui
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  3. Hikaru Hashitani
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Correspondence to Retsu Mitsui.

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The experimental protocols used in the present study were approved by the animal experimentation ethics committee at Nagoya City University Graduate School of Medical Sciences (No. 21–002).

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Kudo, W., Mitsui, R. & Hashitani, H. Involvement of ANO1 currents in pacemaking of PDGFRα-positive specialised smooth muscle cells in rat caudal epididymis. Cell Tissue Res (2024). https://doi.org/10.1007/s00441-024-03890-x

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