Arachidonic acid metabolites follow the preferential course of cyclooxygenase pathway for the basal tone in the internal anal sphincter

Abstract

Present studies determined the roles of the cyclooxygenase (COX) versus the lipoxygenase (LO) pathways in the metabolic pathway of arachidonic acid (AA) in the internal anal sphincter (IAS) tone. Studies were performed in the rat IAS versus the nontonic rectal smooth muscle (RSM). Indomethacin, the dual COX inhibitor, but not nordihydroguaiaretic acid (NDGA), the LO inhibitor, produced a precipitous decrease in the IAS tone. However, when added in the background of indomethacin, NDGA caused significant reversal of the IAS tone. These inhibitors had no significant effect on the RSM. To follow the significance of COX versus LO pathways, we examined the effects of AA and its metabolites. In the IAS, AA caused an increase in the IAS tone (Emax=38.8+/-3.0% and pEC50=3.4+/-0.1). In the RSM, AA was significantly less efficacious and potent (Emax=11.3+/-3.5% and pEC50=2.2+/-0.3). The AA metabolites (via COXs) PGF2alpha and U-46619 (a stable analog of thromboxane A2) produced increases in the IAS tone and force in the RSM. Conversely, AA metabolites (via 5-LO) lipoxin A4, 5-HETE, and leukotriene C4 decreased the IAS tone. Finally, the contractile effects of AA in the IAS were selectively attenuated by the COX-1 but not the COX-2 inhibitor. Collectively, the specific effects of AA and the COX inhibitor, the Western blot and RT-PCR analyses showing specifically higher levels of COX-1, suggest a preferential role of the COX (specifically COX-1) pathway versus the LO in the regulation of the IAS tone.

Fig. 1.

Cumulative concentration-response curves for arachidonic acid (AA), showing significant increase in the internal anal sphincter (IAS) tone (A) and rectal smooth muscle (RSM) force (B; *P < 0.05). AA is more efficacious and potent in producing increase in the IAS tone versus force in the RSM (**P < 0.05). Indomethacin causes significant attenuation of AA effects both in the IAS and RSM (***P < 0.05). #P < 0.05. Data represent means ± SE of 4 independent determinations. NDGA, nordihydroguaiaretic acid.

Fig. 2.

Cumulative concentration-response curves for the dual cyclooxygenases (COXs) inhibitor indomethacin, the selective inhibitor of 5-lipoxygenase (5-LO) NDGA, and vehicle in the IAS of rats. Indomethacin produces a significant decrease in the IAS basal tone (*P < 0.05). NDGA and vehicle do not produce any significant effect (P > 0.05). Data represent means ± SE of 4–9 independent determinations.

Fig. 3.

Time course of the effect of indomethacin or NDGA (both at 100 μM) and the combination of indomethacin plus NDGA on the basal IAS tone. Data show significant (*P < 0.05; n = 4) sustained decrease in the IAS tone with indomethacin alone, whereas NDGA has no significant effect (P > 0.05; n = 4). NDGA administered 5 min following pretreatment with indomethacin causes significant reversal of the suppressed basal tone (**P < 0.05; n = 4).

Fig. 4.

Concentration-response curves of the COX products thromboxane A₂ (TxA₂; represented here by its synthetic analog U-46619), prostaglandin F_2α (PGF_2α), and leukotrienes B₄ and D₄ (LTB₄ and LTD₄, respectively) in the IAS (A) and RSM (B). Note that only PGF_2α and TxA₂ produce significant increase in the IAS basal tone (*P < 0.05; n = 4). In addition, data show significantly higher potency and efficacy of the metabolites in the case of IAS versus RSM (**P < 0.05; n = 4).

Fig. 5.

Concentration-response curves with leukotriene C₄ (LTC₄), lipoxin A₄ (LXA₄), and 5-HETE, the major products of AA metabolism via the 5-LO pathway. These agents produce concentration-dependent and significant (*P < 0.05; n = 4) decreases in the IAS basal tone.

Fig. 6.

Western blots (WB) analysis of IAS and RSM protein extracts for COXs and 5-LO. The integrated optical density was analyzed, and the percent relative density was calculated relative to that of α-actin. Columns show means ± SE of 4 independent determinations. Note that IAS expresses significantly (P < 0.05) higher levels of COX-1 than the RSM. Data show no significant differences in the levels of 5-LO and COX-2 between the IAS and the RSM (P > 0.05). Unpaired Student's t-test: *P < 0.05.

Fig. 7.

RT-PCR for 5-LO, COX-1, and COX-2 using total RNA from the IAS and the RSM. Total RNA was subjected to RT at 37°C for 2 h and amplification in a thermocycler. The RT-PCR products were separated in an ethidium bromide-containing 1.5% agarose gel by electrophoresis and visualized on a UV transluminator, and the relative density of each band was analyzed. The percent relative density of COX and 5-LO bands was calculated as the ratio of β-actin. Data show significantly higher expression of COX-1 and COX-2 in the IAS versus RSM (*P < 0.05; n = 4). However, there were no significant differences in the levels of 5-LO in the IAS versus RSM (P > 0.05; n = 4). Unpaired Student's t-test: *P < 0.05.

Fig. 8.

Effect of COX-1 (SC-560; A) and COX-2 (rofecoxib; B) selective inhibitors on the increase in the IAS tone by AA. As shown, SC-560 causes significant and concentration-dependent rightward shift in the AA concentration-response curves (*P < 0.05; n = 4; A). Rofecoxib, on the other hand, has no significant effect (P > 0.05; n = 4; B). These data suggest significant contribution of COX-1 versus COX-2 in AA metabolic pathway in the IAS.