Pannexin inhibitors decrease responses to sympathetic nerve stimulation in porcine splenic arteries

A Habib, V Ralevic, WR Dunn. university of Nottingham, Nottingham, UK

Pannexins are a newly discovered family of proteins with sequence homology to innexins (2). The pannexin family consists of Panx1, Panx2 and Panx3. These proteins form channels in cell membranes to allow ATP to pass through to act as an intercellular messenger. In mouse thoracodorsal resistance arteries it has been shown that Panx1 channels are opened following activation of α-1D adrenoceptors to release ATP, which in turn activates purinergic receptors to cause vasoconstriction (1). This project examined the presence and function of pannexins in the porcine splenic artery (PSA).

PSA were dissected from pig spleens obtained from a local abattoir. The presence of Panx1, Panx2 and Panx3 proteins was studied by Western blotting. Segments of PSA were prepared for isometric tension recording in oxygenated Krebs solution warmed to 37oC. Concentration-response curves (CRC) to noradrenaline (NA) were generated in the absence and presence of the Panx channel inhibitors mefloquine (2x10-5 M) and probenecid (5x10-4 M). Some tissue was prepared to allow electrical field stimulation (2-20 Hz) to be applied to the PSA to assess sympathetic nerve-mediated responses. Responses to sympathetic nerve stimulation are expressed as a % of the contractile response to 10-5 M NA. Data were analysed by two-way ANOVA followed by a Bonferroni post-hoc test. P<0.05 was considered significant.

Using Western blotting it was shown that Panx1 and Panx2, (50 kDa for Panx1 and 100kDa for Panx2), but not Panx3, proteins were present in the PSA. Inhibitors of pannexin channels, mefloquine and probenecid, had no effect on responses to exogenous NA in the PSA (P>0.05). The log EC50 value for NA was -5.7 ± 0.2 in the control CRC compared to -5.7 ± 0.1 in the presence of mefloquine (n=8), and -5.6 ± 0.09 for control versus -5.5 ± 0.12 in the presence of probenecid (n=6). However, mefloquine and probenecid both significantly decreased the frequency-dependent response curves generated to nerve stimulation. For example, a frequency of 20 Hz (applied for 10 seconds, 0.5 ms,20 V) produced a contractile response of 55.4 ± 4.5% (n=9) in control arteries, and this was reduced to 27.6 ± 7.5% (n=9) (P < 0.001) in the presence of mefloquine. Probenecid also reduced the response at 20Hz, from 82.2 ± 15.4% to 14.9 ± 4.0% (P < 0.0001).

Panx1 and Panx2 proteins are present in the PSA. While inhibitors of pannexin channels had no effect on responses to exogenous NA in the PSA, both mefloquine and probenecid decreased responses to sympathetic nerve stimulation. These data could indicate either, that pannexins are located prejunctionally and modify sympathetic neurotransmitter release, or that the transduction of a nerve-mediated response, following discrete neurotransmitter release, requires pannexin channel activity. By contrast exogenous NA application does not require pannexin involvement, in the PSA.

References:

(1) Billaud, M et al.(2011). Circ Res 109(1): 80-85.

(2) Dahl G. and R. W. Keane. (2012). Brain Res 1487: 150-159.