Investigation of ion channels involved in cholinergic activity in mouse bronchial smooth muscles

Ritu, Ritu (2022) Investigation of ion channels involved in cholinergic activity in mouse bronchial smooth muscles. Doctoral thesis, Dundalk Institute of Technology.

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Abstract

In airway smooth muscle cells (ASMCs), multiple ion channels are expressed, however their involvement in cholinergic contractions have been controversial. In the present study, using isometric tension and live cell-Ca2+ imaging, the role of ion channels for instance TMEM16A, and voltage-dependent and independent Ca2+ channels were investigated in murine bronchial smooth muscle. CaCCinh- A01, benzbromarone and MONNA, three TMEM16A inhibitors inhibited sustained contractions rather than initial, suggesting involvement of TMEM16A on sustained part of cholinergic contractions. However, another TMEM16A inhibitor, Ani9 had little effect on cholinergic contractions. With the hypothesis that the other three inhibitors might be blocking L-type Ca2+ channels, their effects were tested after addition of nifedipine. Nifedipine (1 μM) itself reduced the effect of all of the concentrations of carbachol (CCh). However, TMEM16A inhibitors further reduced the responses when added in the presence of nifedipine. All three blockers increased intracellular Ca2+ concentration in calcium imaging experiments. With further investigation using 0 Ca2+ Hanks and caffeine, it was confirmed these blockers were causing Ca2+ release. Tetracaine (100 μM), ryanodine receptor blocker had an inhibitory effect on intracellular calcium increased by benzbromarone. Thus, confirming that TMEM16A blockers are causing Ca2+ release through ryanodine receptors. Store operated calcium entry (SOCE) is a major pathway in Ca2+ signalling which is activated upon depletion of Ca2+ from the SR during excitation-contraction coupling. Apart from store refilling channels and L-type Ca2+ channels, a role in cholinergic activity for Ca2+ release channels on the SR has also been reported. Ca2+ release from SR in the smooth muscle occurs through IP3Rs and RyRs (Kotlikoff & Wang, 1998). This project also investigated the involvement of calcium released activated calcium channel (CRAC; voltage independent Ca2+ channel) on cholinergic activity after inhibiting L-type Ca2+ channel (voltage 10 dependent Ca2+ channel). It was observed that blocking both the channels at the same time, completely inhibited CCh contractions of all concentrations. As similar effect was observed in Ca2+ signals induced by 0.3 μM CCh. It was also observed that after blocking CRAC channel using GSK-7975A, Ani9 has more of an inhibitory effect on cholinergic contractions. We have also found that in high KCl contractile activity in ASM, along with VDCC, Ca2+ release through RyR and CRAC channel are also actively involved. The key findings of the project was that TMEM16A has minor role in cholinergic activator induced contractile activity in healthy murine bronchial smooth muscle. SOCE and Ca2+ release through RyR are majorly involved in maintaining contractile activity in ASM.

Item Type:	Thesis (Doctoral)
Uncontrolled Keywords:	Airway smooth muscle, STIM/Orai, L-type Ca2+channels, TMEM16A, isometric tension, Ca2+ imaging
Subjects:	Science > Biology
Research Centres:	UNSPECIFIED
Depositing User:	Keith Thornbury
Date Deposited:	27 Feb 2025 12:07
Last Modified:	27 Feb 2025 12:07
URI:	https://eprints.dkit.ie/id/eprint/922

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