Mokori, E.B.
(2016)
The role of pannexin channels in small arteries.
PhD thesis, University of Nottingham.
Abstract
Pannexins (Panx) are membrane bound channels which allow the exchange of molecules, particularly ATP, between the cytoplasm and the extracellular space. Panx channels have been shown to be expressed in the vasculature, and there is evidence suggesting that they are involved in the regulation of vasoconstriction in small arteries. In mice thoracodorsal resistance arteries Panx1 channels are co localized with α1D-adrenoceptors on smooth muscle cells, and have been reported to open after α1D-adrenoceptor stimulation to release ATP which is then responsible for causing vasoconstriction by activating P2 purinoceptors (Billaud et al., 2011). This thesis examined the expression and the role of Panx channels in rat mesenteric and cerebral arteries.
Western blotting was used to probe for the presence of pannexin proteins in rat mesenteric and cerebral arteries. The functional role of pannexin channels in the mesenteric and cerebral arterial vasoconstriction was studied using pressure myography. The effects of the pannexin channel inhibitors, mefloquine, probenecid, and carbenoxolone were tested against sympathetic nerve mediated vasoconstriction in 2nd order mesenteric arteries. The effects of purinoceptor antagonists, suramin, and NF449 as well as apyrase an ectonucleotidase that degrades ATP and αβ-MeATP which desensitises P2X1 purinoceptors and mefloquine, probenecid and carbenoxolone were examined against responses to noradrenaline (NA) and potassium chloride (KCl) in pressurized 2nd order mesenteric arteries. The effects of increasing concentrations of pannexin channel inhibitors, mefloquine, probenecid, and carbenoxolone, and purinoceptor antagonists, suramin, and NF449 were also examined against the myogenic tone of 3rd order mesenteric arteries and cerebral arteries. Panx1 siRNAs were used to try to knock down the Panx1 protein in isolated mesenteric arterial smooth muscle cells and mesenteric arteries.
Panx1 and Panx2 proteins were detected in rat mesenteric and cerebral arteries, but no Panx3 protein was detected. The activation of the sympathetic nerves in the 2nd order mesenteric arteries resulted in a frequency-dependent vasoconstriction, which was reduced in the presence of all the pannexin channel inhibitors (mefloquine, probenecid and carbenoxolone). Mefloquine caused a significant difference in the vessel diameter, the vessel diameter was 91.00 ± 36.19 µm in the absence of mefloquine and 1.00 ± 0.70 µm in its presence (Student’s t test, p>0.05). Probenecid caused a significant change in the vessel diameter, at 10Hz the vessel diameter was 60.75 ± 4.59 µm in the absence of probenecid and 26.25 ± 2.83 µm in the presence of probenecid (Student’s t test, p>0.05). Similarly NA and KCl caused a concentration dependent contraction of the 2nd order mesenteric arteries. All the agents tested against the NA mediated responses, pannexin channel inhibitors, purinoceptor antagonists (suramin, NF449), and αβ-MeATP except apyrase resulted in the reduction in the contraction. The concentration that caused a 20% reduction in the vessel diameter in the absence of carbenoxolone was -6.82 ± 0.09 M and it was significantly different in the presence of carbenoxolone, it was -6.22 ± 0.16 M (student’s t test, p>0.05). The concentration that cause a 20% reduction in the vessel diameter, in the absence of suramin was -6.72 ± 0.18 M and it was significantly different in its presence -6.00 ± 0.15 M (Student’s t test, p>0.05). However, mefloquine and probenecid also inhibited the contraction produced by raising extracellular KCl.
The myogenic tone of 3rd order mesenteric arteries was reduced by both pannexin channel inhibitors and purinoceptor antagonists. With the exception of mefloquine, none of the agents had any effect on the myogenic tone of cerebral arteries.
The attempt to knock down the Panx1 protein in isolated mesenteric arterial smooth muscle cells and mesenteric arteries was unsuccessful, despite demonstrating that the delivery systems worked.
In conclusion, Panx1 and Panx2 proteins are expressed in both mesenteric and cerebral arteries. Some of the observations in this study provide evidence that support the role for pannexin channels in sympathetic nerve responses and those to exogenous NA, these include the inhibitory effects seen with the P2 purinoceptor antagonists, suramin, and NF449 as well as αβ-MeATP on exogenous NA. This effect was also mimicked by the pannexin channel inhibitors, mefloquine, probenecid and carbenoxolone which also inhibited the nerve mediated contractions. Thus the data is consistent with the hypothesis that transduction of responses to NA involves the opening of pannexin channels and the release of ATP as an intercellular messenger, to enable synchronized vasoconstriction of the vascular smooth muscle cells. However, some caution needs to be applied since mefloquine and probenecid also reduced the response to raised extracellular KCl indicating that they may not be selective.
In the case of the myogenic tone major differences in the effects of the pannexin channel inhibitors were seen between the cerebral and mesenteric arteries showing that probenecid and carbenoxolone were acting selectively, however mefloquine was non-selective. Thus the effects obtained with probenecid and carbenoxolone coupled with those obtained with P2 purinoceptor antagonists, suramin and NF449 on the myogenic tone of mesenteric arteries suggests a role for pannexin channels as the stress/pressure sensor in mesenteric arteries.
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