Propionate-induced relaxation in rat mesenteric arteries: a role for endothelium-derived hyperpolarising factor

Abstract

Short chain fatty acids, including propionate, are generated in the caecum and large intestine, and when absorbed may elicit localised increases in intestinal blood flow. We sought to assess the mechanism by which propionate caused vasorelaxation. Propionate-mediated relaxation of noradrenaline-preconstricted rat mesenteric small arteries (RMSAs, i.d. 200-300 microm) was studied using small vessel myography. Propionate (1-30 mM) produced a concentration-dependent relaxation. Relaxation induced by 10 mM propionate (the approximate EC50) was almost abolished by endothelial denudation, although a marked relaxation to a very high concentration of propionate (50 mM) persisted in the absence of the endothelium. In endothelium-intact RMSAs, relaxation to 10 mM propionate was almost abolished by elevating [K+]o to 25 mM, but was unaffected by 100 microM N(omega)-nitro-L-arginine methyl ester (L-NAME) (68 +/- 4 vs. 66 +/- 3% in controls, n = 35), or by 1 microM indomethacin (60 +/- 4 vs. 61 +/- 7 % in controls, n = 15). In the presence of L-NAME, relaxation to 10 mM propionate was significantly and markedly (i.e. > 50 %) inhibited by 50 microM Ba2+ and by the combination of 100 nM charybdotoxin and 100 nM apamin. A similar effect on propionate-mediated relaxation was also exerted by 100 microM ouabain, and by the combination of 50 microM barium with ouabain. Relaxation was also significantly and markedly inhibited by pre-treatment of RMSAs with 100 nM thapsigargin or 10 microM cyclopiazonic acid (CPA). The results demonstrate that 10 mM propionate relaxes RMSAs via endothelium-derived hyperpolarising factor (EDHF). The observation that relaxation by propionate is inhibited by thapsigargin and CPA suggests that this action of propionate involves the release of endothelial cell Ca2+ stores.

Figure 1. Responses to 10 mm sodium propionate compared to time control contractions

A, examples of tension measurements in a single artery showing time control and effect of 10 mm propionate. The arrow indicates where the bath solution was changed and the dotted line represents basal tone. B, mean (± s.e.m.) contraction amplitude measured at 1 min intervals and normalised to amplitude at 5 min (n = 41). For each time point following the solution change, values ‘a’ and ‘b’ were calculated and, using the formula a/(a + b), propionate-induced relaxation was corrected for spontaneous time-related changes in contraction amplitude, as shown in C.

Figure 2. Endothelium and concentration dependence of propionate-induced relaxation

A-C, mean (± s.e.m.) normalised and corrected contraction amplitude plotted at 1 min intervals, showing the effects of endothelium removal (A, n = 6), 100 μm l-NAME (B, n = 35) or 25 mm K⁺ (C, n = 5) in the bath solution on responses to 10 mm propionate. D, concentration-dependent relaxation induced by propionate. Points show the mean of the percentage relaxation measured at 12, 13 and 14 min for 0.1 (n = 6), 0.3 (n = 7), 1 (n = 13), 3 (n = 13), 10 (n = 41) and 30 mm propionate (n = 10). * Significant relaxation (P < 0.05).

Figure 3. Combined effect of charybdotoxin and apamin on propionate-induced relaxation

A, mean (± s.e.m.) normalised contraction amplitude plotted at 1 min intervals, showing the effects of 100 nm charybdotoxin (ChTx) and 100 nm apamin (Ap) on responses to 10 mm propionate and paired time controls (n = 9). B, propionate-induced relaxations after correction for time controls, before and after treatment with ChTx and Ap.

Figure 4. Effect of Ba²⁺ or ouabain on propionate-induced relaxation

A and C, mean (± s.e.m.) normalised contraction amplitude plotted at 1 min intervals, showing the effects of 50 μm Ba²⁺ (A, n = 12) or 100 μm ouabain (C, n = 6) on responses to 10 mm propionate and paired time controls. B and D, propionate-induced relaxations after correction for time controls, showing the effects of Ba²⁺ or ouabain, respectively.

Figure 5. Combined effects of Ba²⁺ and ouabain on propionate-induced relaxation

A and C, mean (± s.e.m.) normalised contraction amplitude plotted at 1 min intervals, showing the effects of 50 μm Ba²⁺ and 1 μm ouabain (A, n = 8) or 50 μm Ba²⁺ and 100 μm ouabain (C, n = 10) on responses to 10 mm propionate and paired time controls. B and D, propionate-induced relaxations after correction for time controls, showing the effects of Ba²⁺ together with 1 or 100 μm ouabain, respectively.

Figure 6. Effect of combining Ba²⁺ with charybdotoxin and apamin on propionate-induced relaxation

A, mean (± s.e.m.) normalised contraction amplitude plotted at 1 min intervals, showing the effects of combining 50 μm Ba²⁺ with 100 nm charybdotoxin (ChTx) and 100 nm apamin (Ap) on responses to 10 mm propionate and paired time controls (n = 10). B, propionate-induced relaxations after correction for time controls, before and after treatment with Ba²⁺ plus ChTx and Ap.

Figure 7. Effect of iberiotoxin on propionate-induced relaxation

A, mean (± s.e.m.) normalised contraction amplitude plotted at 1 min intervals, showing the effects of 100 nm iberiotoxin (IbTx) on responses to 10 mm propionate and paired time controls (n = 13). B, propionate-induced relaxations after correction for time controls, before and after treatment with IbTx.

Figure 8. Example of effect of thapsigargin on propionate-induced relaxation

Trace showing acetylcholine (Ach, a, b, e), time control (c, f) and propionate responses (d, g) in the absence and presence of 100 μm l-NAME, and the combination of l-NAME with 100 nm thapsigargin. The arrows show the points at which solutions were exchanged (and addition of 10 mm propionate). The dotted lines represent basal tone.

Figure 9. Effect of thapsigargin and cyclopiazonic acid

Mean (± s.e.m.) normalised contraction amplitude plotted at 1 min intervals, showing the effects of 100 nm thapsigargin (thaps, A) or cyclopiazonic acid (CPA, C) on responses to 10 mm propionate and paired time controls (n = 8), and propionate-induced relaxations after correction for time controls, before and after treatment with thapsigargin (B) or CPA (D).