Increased degradation of MYPT1 contributes to the development of tolerance to nitric oxide in porcine pulmonary artery

Abstract

Myosin phosphatase target subunit 1 (MYPT1) is the regulatory subunit of myosin light chain phosphatase (MLCP). It plays a critical role in vasodilatation induced by cGMP-elevating agents such as nitric oxide (NO). The present study was performed to determine the role of MYPT1 in the development of tolerance of the pulmonary artery to NO. Incubation of isolated porcine pulmonary arteries for 24 or 48 h with DETA NONOate (DETA NO) significantly reduced protein levels of MYPT1 and the leucine zipper-positive (LZ+) isoform of MYPT1 but not that of PP1cdelta. The extent of reduction in total MYPT1 protein level was comparable to that of MYPT1 (LZ+). The decrease in MYPT1 protein caused by 48-h DETA NO incubation was prevented by ODQ, an inhibitor of guanylyl cyclase, and by inhibitors of proteasomes (MG-132 and lactacystin) but was not affected by the inhibitor of protein synthesis, cycloheximide. A reduction in MYPT1 protein was also obtained with 8-bromo-cGMP, but this was prevented by Rp-8-bromo-PET-cGMP [inhibitor of cGMP-dependent protein kinase (PKG)]. Incubation for 48 h with DETA NO also reduced dephosphorylation of myosin light chain and relaxation of the artery in response to DETA NO, which was prevented by MG-132. These results suggest that the reduction in MYPT1 protein contributes to the development of tolerance of pulmonary arteries to NO. This may result from increased degradation of MYPT1 after prolonged PKG activation.

Fig. 1.

Protein levels of total myosin phosphatase target subunit 1 (MYPT1) and the leucine zipper-positive (LZ+) isoform of MYPT1 [MYPT1 (LZ+)] of porcine pulmonary arteries were reduced to a similar extent following incubation with DETA NONOate (DETA NO; 10⁻⁴ M) for 24 and 48 h (A) and incubation for 48 h with different concentrations of the nitric oxide (NO) donor (B). Incubation with DETA NO (10⁻⁴ M) for 48 h had no effect on the expression of PP1cδ protein (C). Top: Western blots; bottom, summaries of densitometric scanning of MYPT1 or PP1cδ protein normalized to actin. Data are means ± SE; n = 4–6 for each group. *P < 0.05, significantly different from values at 0 h (A) or basal values (B).

Fig. 2.

Reduction in protein levels of MYPT1 of porcine pulmonary arteries caused by incubation with DETA NO (10⁻⁴ M) was prevented when 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; 3 × 10⁻⁵ M) was presented in the incubation medium. Top, Western blots; bottom, summaries of densitometric scanning of MYPT1 protein normalized to actin. Data are means ± SE; n = 4–6 for each group. *P < 0.05, significantly different from control.

Fig. 3.

Protein levels of MYPT1 in porcine pulmonary arteries were reduced by 48-h incubation with 8-bromo-cGMP (8-Br-cGMP; 10⁻⁴ M) but not by 8-Br-cAMP (10⁻⁴ M; A). The effect of 8-Br-cGMP was inhibited by Rp-Br-PET-cGMPS (PKG-I; 3 × 10⁻⁵ M; B). Top, Western blots; bottom, summaries of densitometric scanning of MYPT1 protein normalized to actin. Data are means ± SE; n = 4–5 for each group. *P < 0.05, significantly different from control.

Fig. 4.

Reduction of MYPT1 protein in porcine pulmonary arteries following 48-h incubation with DETA NO (10⁻⁴ M; A) or 8-Br-cGMP (10⁻⁴ M; B) was not affected by cycloheximide (CHX; 10⁻⁴ M) but was prevented by MG-132 (10⁻⁵ M). Incubation with DETA NO (10⁻⁴ M) or MG-132 (10⁻⁵ M) for 48 h had no effect on the expression of PP1cδ protein (C). Top, Western blots; bottom, summaries of densitometric scanning of MYPT1 or PP1cδ protein normalized to actin. Data are means ± SE; n = 4–6 for each group. * P < 0.05, significantly different from basal values.

Fig. 5.

Reduction of MYPT1 protein in porcine pulmonary arteries following 48-h incubation with DETA NO (10⁻⁴ M) was prevented by lactacystin (10⁻⁶ M). Top, Western blots; bottom, summaries of densitometric scanning of MYPT1 protein normalized to actin. Data are means ± SE; n = 4 for each group. *P < 0.05, significantly different from solvent group.

Fig. 6.

Myosin light chain (MLC) phosphorylation [judged as the ratio of phosphorylated MLC to total MLC (MLC-p/MLC)] in porcine pulmonary arteries evoked by U46619 (3 × 10⁻⁷ M) was inhibited by DETA NO (10⁻⁴ M) in arteries preincubated for 48 h with solvent but not with DETA NO (10⁻⁵ M). The effect of preincubation with DETA NO on MLC phosphorylation was prevented by MG-132 (3 × 10⁻⁶ M). The MLC-p/MLC values at basal and after U46619 were determined using tissues taken before and 10 min after administration of the thromboxane A₂ mimetic, respectively. The MLC-p/MLC values for U46619 plus DETA NO were obtained from tissues treated with U46619 for 10 min followed by DETA NO (10⁻⁴ M) for 5 min. Top, Western blots (lane 1, basal value; lane 2, U46619; lane 3, U46619 plus DETA NO); bottom, summaries of densitometric scanning of MLC-p and MLC proteins expressed as MLC-p/MLC. Data are means ± SE; n = 4 for each group. *P < 0.05, significant difference between basal and U46619-treated arteries. †P < 0.05, significant difference between vessels treated with U46619 and those treated with U46619 plus DETA NO.

Fig. 7.

A: acute responses in porcine pulmonary arteries induced by DETA NO in the presence of solvent, ODQ (3 × 10⁻⁵ M), and PKG-I (3 × 10⁻⁵ M). The vessels were preconstricted with U46619 (3 × 10⁻⁷ M) to similar tension levels. B and C: effects of 48-h incubation with DETA NO on relaxation of porcine pulmonary arteries to DETA NO and 8-Br-cGMP, respectively. After 48-h incubation, the vessels were repeatedly rinsed and preconstricted with U46619 (3 × 10⁻⁷ M) to similar tension levels before the effect of DETA NO or 8-Br-cGMP was tested. Data are means ± SE; n = 5–6 for each group. *P < 0.05, significantly different from control. †P < 0.05, significantly different from vessels treated with PKG-I (A) or from vessels-treated with DETA NO at 10⁻⁵ M (B).

Fig. 8.

Relaxation of porcine pulmonary artery to DETA NO after 48-h incubation with this nitrovasodilator (10⁻⁴ M) in the presence and absence of MG-132 (3 × 10⁻⁶ M). After incubation, the vessels were repeatedly rinsed and preconstricted with U46619 (3 × 10⁻⁷ M) to similar tension levels before the effect of DETA NO was tested. Data are means ± SE; n = 4–6 for each group. *P < 0.05, significant difference between vessels treated with DETA NO and those treated with solvent or MG-132. †P < 0.05, significant difference between vessels treated with DETA NO and those treated with MG-132 plus DETA NO.