Korean J Physiol Pharmacol.  2024 Jan;28(1):49-57. 10.4196/kjpp.2024.28.1.49.

Differential expression of the enzymes regulating myosin light chain phosphorylation are responsible for the slower relaxation of pulmonary artery than mesenteric artery in rats

• Oh SB
• Cho S
• Kim HJ
• Kim SJ

• KMID: 2550288
• DOI: http://doi.org/10.4196/kjpp.2024.28.1.49

Abstract

While arterial tone is generally determined by the phosphorylation of Ser¹⁹ in myosin light chain (p-MLC2), Thr¹⁸ /Ser¹⁹ diphosphorylation of MLC2 (ppMLC2) has been suggested to hinder the relaxation of smooth muscle. In a dual-wire myography of rodent pulmonary artery (PA) and mesenteric artery (MA), we noticed significantly slower relaxation in PA than in MA after 80 mM KCl-induced condition (80K-contraction). Thus, we investigated the MLC2 phosphorylation and the expression levels of its regulatory enzymes; soluble guanylate cyclase (sGC), Rho-A dependent kinase (ROCK) and myosin light chain phosphatase target regulatory subunit (MYPT1). Immunoblotting showed higher sGC-α and ROCK2 in PA than MA, while sGC-β and MYPT1 levels were higher in MA than in PA. Interestingly, the level of ppMLC2 was higher in PA than in MA without stimulation. In the 80K-contraction state, the levels of p-MLC2 and pp-MLC2 were commonly increased. Treatment with the ROCK inhibitor (Y27632, 10 µM) reversed the higher pp-MLC2 in PA. In the myography study, pharmacological inhibition of sGC (ODQ, 10 µM) slowed relaxation during washout, which was more pronounced in PA than in MA. The simultaneous treatment of Y27632 and ODQ reversed the impaired relaxation in PA and MA. Although treatment of PA with Y27632 alone could increase the rate of relaxation, it was still slower than that of MA without Y27632 treatment. Taken together, we suggest that the higher ROCK and lower MYPT in PA would have induced the higher level of MLC2 phosphorylation, which is responsible for the characteristic slow relaxation in PA.

Keyword

Muscle, smooth, vascular; Myosin light chains; Phosphorylation; Pulmonary artery; Relaxation; Rho-associated kinases

Figure

• 

  Fig. 1 Comparison of mechanical characteristics and length-tension property between PAs and MAs. (A) Representative images of isolated PA and MA mounted for myography. (B) Summary of the length and inner diameter (I.D.) of PAs (n = 9, N = 3) and MAs (n = 8, N = 3). (C) Representative traces showing the active and passive tension recording procedure. Step-like increase of I.D. is indicated by upward arrow with ‘S’. After confirming a quasi-steady state passive tension, 80 mM KCl (80K) was applied to induce an active tension increase, which was reversed by washout with PSS. (D, E) The relationship between the calculated inner circumferential length (I.C.) and tension (both active and passive) of PAs (n = 9, N = 4) and MAs (n = 8, N = 4). Data are presented as means ± SD. PA, pulmonary artery; MA, mesenteric artery; PSS, physiological salt solution.

• 

  Fig. 2 Properties of the K+-induced contraction and the relaxation by washout in PAs and MAs. (A) Representative original traces showing contractions induced by cumulative increase of [K+]ext in PA and MA. A standard contraction by 80K was commonly induced at the initial phase of each experiment. Note that MA showed more prominent spontaneous relaxation under the raised [K+]ext. (B) Representative original trace of 80K-contraction and the summary of peak amplitude of active tension increase in PAs (n = 16, N = 5) and MAs (n = 10, N = 4). Note the marked difference of the relaxation speed by washout with PSS. (C) The summary of peak amplitudes from PAs and MAs. (D) Summary of the traces of relaxation phase normalized to the peak amplitude of the 80K-contraction (Norm.T/T80K). (E) The 50% and 75% relaxation time to baseline (t1/2 and t3/4, in sec) was measured in each vessel and summarized for the comparison. Data are presented as means ± SD. PA, pulmonary artery; MA, mesenteric artery; PSS, physiological salt solution. **p < 0.01, ***p < 0.001, using two-tailed Student’s t-test.

• 

  Fig. 3 Expression of MLC2, p-MLC2, pp-MLC2 and cGMP-dependent signaling molecules in PAs and MAs. (A) Western blot analysis of mono- and di-phosphorylated states of MLC2 in PAs and MAs. (B) Summary of total MLC2 expression normalized to β-actin and the levels of p-MLC2 (S19-p) and pp-MLC2 (T18/S19-pp) normalized to the expression of MLC2. (C) Representative immunoblot analysis of sGCα, sGCβ, PKG, ROCK1, ROCK2 and MYPT1 in PAs and Mas. (D) Summary of the protein levels normalized to β-actin. (E) Western blot analysis of phosphorylated states of MLC2 induced by 80K and the effect of pretreatment with Y27632 (10 µM) in PAs and MAs. (F) Summary of normalized levels of p-MLC2 (left panel) and pp-MLC2 (right panel). Data are means ± SD. PA, pulmonary artery; MA, mesenteric artery; PSS, physiological salt solution. *p < 0.05, **p < 0.01, using two-tailed Student’s t-test and one-way ANOVA test.

• 

  Fig. 4 Pharmacological vasodilatory response in PAs and MAs treated with sGC inhibitor and its recovery by ROCK inhibitor. (A) Representative traces of 80K-contraction and response to washout with or without pretreatment with ODQ in the PA (left panel) and MA (right panel). (B) The relaxation times (t1/2 and t3/4, in sec) were measured in each vessel and summarized for the comparison (PA; n = 6, N = 3, MA; n = 6, N = 3). (C) Representative traces of 80K-contraction treated with ODQ and response to washout with or without pretreatment with Y27632 in the PA (left panel) and MA (right panel). (D) The relaxation time to baseline (t1/2 and t3/4, in sec) was measured in each vessel and summarized for the comparison (PA; n = 5, N = 3, MA; n = 5, N = 3). (E) Representative traces of 80K-contraction and response to washout with or without pretreatment with Y27632 in the PA. (F) The summary of t1/2 and t3/4 (sec) for PAs and further comparison with MAs (PA; n = 5, N = 2, MA; n = 9, N = 4). Data are means ± SD. PA, pulmonary artery; MA, mesenteric artery; PSS, physiological salt solution. *p < 0.05, **p < 0.01, ***p < 0.001, using two-tailed Student’s t-test and one-way ANOVA test.

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  Fig. 5 A schematic diagram of the regulatory pathways for MLC phosphorylation in smooth muscle comparing the relative expressions between PA and MA. Higher level of ROCK2 with lower levels of sGC and MYPT1 in PA than MA would lead to the higher level of basal MLC diphosphorylation and slower relaxation property of PA than MA. PA, pulmonary artery; MA, mesenteric artery.

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