Habib, Alaa
(2017)
Role of pannexins in vasculature.
PhD thesis, University of Nottingham.
Abstract
Pannexins are newly discovered proteins that were first discovered by Panchin in 2000. The pannexin family has three isomers, i.e. pannexin-1, pannexin-2, and pannexin-3. In 2011, Billaud et al suggested that pannexin1 channels contribute to the spread of vasoconstriction after activation of α1D-adrenoceptors present on the surface of vascular smooth muscle cell (VSMCs) of thoracodorsal resistance arteries (TDA) isolated from mice. Phenylephrine acting upon the α1D-adrenoceptors activated pannexin1 channels present in the cell to release ATP, which in turn activated P2Y receptors on neighbouring cells to produce a co-ordinated contractile response. This aim of this work was to further investigate the role of pannexin in the regulation of contractile responses in the vasculature. To this end, the present study examined the presence and function of pannexins in the porcine splenic artery (PSA) and the rat aorta (RA), in which α1A- and α1D-adrenoceptors are present respectively. The role of pannexin channels and ATP (via activation of P2 purinoceptors) in the response to exogenous NA-induced contractile responses in the PSA and the RA were investigated, as were responses to sympathetic nerve activation in the PSA. The involvement of pannexin channels was studied using several pannexin inhibitors, i.e. mefloquine (a non-selective pannexin inhibitor), probenecid (a selective pannexin1 inhibitor at low concentrations), carbenoxolone (a selective pannexin1 inhibitor) and Brilliant Blue FCF (a selective pannexin1 inhibitor). Additionally, the involvement of ATP in NA-induced responses was examined using P2 purinoceptor antagonists (PPADS, suramin and NF449). Further experiments examined the role of pannexins in contributing to endothelium-dependent responses in a large vessel i.e. the porcine coronary artery (PCA).
The results showed that both pannexin1 and pannexin2 are present in the PSA and the RA. In the PSA, mefloquine and probenecid reduced the responses to both NA-induced contractions and the frequency-dependent response curves generated to sympathetic nerve stimulation, whereas carbenoxolone, suramin and PPADS had no effect on responses to either exogenous NA or those caused by activating the sympathetic nerves. In the RA, mefloquine and probenecid reduced the response to NA-induced contractions, whereas BB-FCF had no effect. Purinoceptor antagonists (suramin, PPADs and NF449) had no effect on responses mediated by either α1A–adrenoceptors in the PSA or α1D–adrenoceptors in the RA, arguing against the role of ATP (via activation of P2 receptors) in mediating NA-induced responses in either the PSA or the RA. Conflicting results were obtained, in some cases, upon the use of three different pannexin inhibitors. The most likely reason for this is that mefloquine demonstrated non-selective inhibitory actions on contractile responses since it was also shown to inhibit responses to KCl, 5-HT, U46619 (the thromboxane mimetic), and responses to re-addition of calcium to depolarised preparations, suggesting that it acts to block L-type Ca2+ currents. Both mefloquine and probenecid demonstrated non-selective inhibitory effects when used at relatively high concentrations. Therefore, mefloquine and probenecid should only be used in low concentrations as pannexin1 inhibitors. It has been suggested that pannexin proteins may be involved in mediating endothelium-derived hyperpolarizing factor (EDHF) responses (Gaynullina, Shestopalov et al 2015). Bradykinin (BK) was shown to induce relaxation in PCA and to a lesser extent in the PSA after inhibition of NO-synthase and cyclooxygenase. The evidence for the involvement of pannexin in mediating an EDHF response was limited in both the PSA and the PCA, since neither carbenoxolone nor probenecid had any effect. While mefloquine reduced EDH-mediated responses to bradykinin in the PCA, the questions about its selectivity make this observation difficult to interpret.
The present work therefore provided some evidence for the involvement of pannexin channels in conducting responses to NA-induced α1-adrenoceptor-mediated vasoconstriction in blood vessels in PSA and the RA, although great care must be taken in interpreting this data on the basis of a lack of selectivity of the pharmacological agents currently available as pannexin inhibitors. In addition, there was no evidence that activation of α1-adrenoreceptors causes the release of ATP from inside cells to act as an intercellular messenger, leading to P2 receptor-mediated contractions.
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