L-NAME inhibits Mg(2+)-induced rat aortic relaxation in the absence of endothelium

Abstract

1 L-NG-nitro-arginine methyl ester (L-NAME; 100 microM), a nitric oxide synthase (NOS) inhibitor, reversed the relaxation induced by 3 microM acetylcholine (ACh) and 2-10 mM Mg2+ in endothelium-intact (+E) rat aortic rings precontracted with 1 microM phenylephrine (PE). In PE-precontracted endothelium-denuded (-E) rat aorta, 3 microM ACh did not, but Mg2+ caused relaxation which was reversed by L-NAME, but not by D-NAME. 2 The concentration response profiles of L-NAME in reversing the equipotent relaxation induced by 5 mM Mg2+ and 0.2 microM ACh were not significantly different. 3 L-NAME (100 microM) also reversed Mg(2+)-relaxation of -E aorta pre-contracted with 20 mM KCl or 10 microM prostaglandin F2alpha (PGF2alpha). L-NG-monomethyl-arginine (L-NMMA; 100 microM) was also effective in reversing the Mg(2+)-relaxation. 4 Addition of 0.2 mM Ni2+, like Mg2+, caused relaxation of PE-pre-contracted -E aorta, which was subsequently reversed by 100 microM L-NAME. 5 Reversal of the Mg(2+)-relaxation by 100 microM L-NAME in PE-precontracted -E aorta persisted following pre-incubation with 1 microM dexamethasone or 300 microM aminoguanidine (to inhibit the inducible form of NOS, iNOS). 6 Pretreatment of either +E or -E aortic rings with 100 microM L-NAME caused elevation of contractile responses to Ca2+ in the presence of 1 microM PE. 7 Our results suggest that L-NAME exerts a direct action on, as yet, unidentified vascular smooth muscle plasma membrane protein(s), thus affecting its reactivity to divalent cations leading to the reversal of relaxation. Such an effect of L-NAME is unrelated to the inhibition of endothelial NOS or the inducible NOS.

Figure 1

(A) Typical tracings (left) and data summary (right) of the effects of L-NAME on aortic contraction, Top tracings: In endothelium-intact (+E) aortic rings precontracted with 1 μM PE, 100 μM L-NAME reversed the relaxation induced by 3 μM ACh or cumulatively added 10 mM Mg²⁺; Middle tracings: Pre-incubation with 100 μM L-NAME, inhibited the relaxation in response to 3 μM ACh or 10 mM Mg²⁺. Bottom tracings: In endothelium-denuded (−E) rings precontracted with 1 μM PE, 3 μM ACh had no effect but 10 mM Mg²⁺ (added cumulatively) induced concentration-dependent relaxation, which was reversed by 100 μM L-NAME. (B) The data summary shows that 100 μM L-NAME inhibits the concentration-dependent relaxation induced by added Mg²⁺ (2–10 mM) in +E and −E rat aortic rings. D-NAME (100 μM) did not affect the relaxation induced by 2, 4 and 10 mM Mg²⁺ in −E or +E aortic rings (only the data for +E rings are shown for clarity). Values are expressed as mean±s.e.mean; n=6–8. ^*P<0.001, comparison between corresponding values, in the presence and absence of L-NAME.

Figure 2

The concentration response profiles of the reversal effect of L-NAME on the relaxation by 5 mM Mg²⁺ and 0.2 μM ACh, which are equipotent in inhibit1ing the contraction to 1 μM PE by about 50% in the presence of L-NAME. Data are expressed as mean±s.e.mean in four separate experiments. ^*P<0.05. significantly different from the corresponding control value in the absence of L-NAME.

Figure 3

In endothelium-denuded aortic rings contracted with added 20 mM KCl or 10 μM PGF_2α, 100 μM L-NAME reversed the relaxation induced by 2 mM Mg²⁺. The time controls for KCl or PGF_2α-induced contraction remained sustained for the entire duration of the experiments (not shown). +L-NAME values are significantly different from −L-NAME values in 4–6 separate experiments (^*P<0.05; as mean±s.e.mean).

Figure 4

Top tracing, as a positive control, shows that 100 μM L-NMMA completely reversed the relaxation induced by 3 μM ACh of endothelium-intact (+E) rings precontracted with 1 μM PE. Lower tracing shows that in endothelium-denuded (−E) rings precontracted with 20 mM KCl, L-NMMA also reversed the relaxation induced by 2 mM Mg²⁺. The bar graph shows that similar effect of L-NMMA on Mg²⁺ (2 mM)-induced relaxation was also observed in rings precontracted with 1 μM PE. +L-NMMA values are significantly different from −L-NMMA values (^*P<0.05; mean±s.e.mean; n=3–4).

Figure 5

The effect of 100 μM L-NAME on the relaxation induced by 0.2 mM Ni²⁺ of endothelium-denuded aortic rings precontracted with 1 μM PE. +L-NAME values are significantly different from −L-NAME values (^*P<0.001; mean±s.e.mean; n=5).

Figure 6

Effect of L-NAME on the contractile responses to added Ca²⁺ in the presence of PE in endothelium-intact (+E) and -denuded (−E) aortic rings. Rings were incubated in nominally Ca²⁺-free medium with (+) or without (−) 100 μM L-NAME for 1 h prior to the addition of 1 μM PE, aliquots of Ca²⁺ were subsequently added in cumulative manner to initiate contractions. ^*P<0.05 (mean±s.e.mean; n=6) compared to values in the absence of L-NAME in +E rings. ^**P<0.05 (mean±s.e.mean; n=4) compared to values in the absence of L-NAME in −E rings.

Figure 7

Preincubation with 300 μM aminoguanidine (AG) for 20 min or with 1 μM dexamethasone (DXM) for 5 h did not modify the resting tension or the relaxation induced by 4 mM Mg²⁺ in endothelium-denuded rings precontracted with 1 μM PE. Subsequent addition of 100 μM L-NAME still exerted significant reversal effect on Mg²⁺-induced relaxation in both cases (^*P<0.01; mean±s.e.mean; n=3–4).