Inhibition of myosin light chain phosphorylation decreases rat mesenteric lymphatic contractile activity

Abstract

Muscular lymphatics use both phasic and tonic contractions to transport lymph for conducting their vital functions. The molecular mechanisms regulating lymphatic muscle contractions are not well understood. Based on the well-established finding that the phosphorylation of myosin light chain 20 (MLC(20)) plays an essential role in blood vessel smooth muscle contraction, we investigated if phosphorylated MLC(20) (pMLC(20)) would modulate the tonic and/or phasic contractions of lymphatic muscle. The effects of ML-7, a MLC kinase inhibitor (1-10 microM), were tested on the contractile parameters of isolated and cannulated rat mesenteric lymphatics during their responses to the known modulators, pressure (1-5 cm H(2)O) and substance P (SP; 10(-7) M). Immunohistochemical and Western blot analyses of pMLC(20) were also performed on isolated lymphatics. The results showed that 1) increasing pressure decreased both the lymphatic tonic contraction strength and pMLC(20)-to-MLC(20) ratio; 2) SP increased both the tonic contraction strength and phosphorylation of MLC(20); 3) ML-7 decreased both the lymphatic tonic contraction strength and pMLC(20)-to-MLC(20) ratio; and 4) the increase in lymphatic phasic contraction frequency in response to increasing pressure was diminished by ML-7; however, the phasic contraction amplitude was not significantly altered by ML-7 either in the absence or presence of SP. These data provide the first evidence that tonic contraction strength and phasic contraction amplitude of the lymphatics can be differentially regulated, whereby the increase in MLC(20) phosphorylation produces an activation in the tonic contraction without significant changes in the phasic contraction amplitude. Thus, tonic contraction of rat mesenteric lymphatics appears to be MLC kinase dependent.

Fig. 1.

Increases in transmural pressure and ML-7 decrease the tonic contraction of the lymphatics. The tonic index was calculated as described in materials and methods. APSS, albumin-enriched physiological salt solution. Values are means ± SE for 5 experiments. ^×P < 0.05 vs. 1 cmH₂O of pressure; *P < 0.05 vs. control (APSS); #P < 0.05 vs. 10⁻⁶ M ML-7.

Fig. 2.

Increases in transmural pressure decrease myosin light chain 20 (MLC₂₀) phosphorylation in pressurized rat mesenteric lymphatics. A: representative blot for the MLC₂₀ reaction on lymphatic proteins isolated from lymphatics at different pressures. P-1, P-3, and P-7 indicate pressures of 1, 3, and 7 cmH₂O, respectively. B: representative blot for phosphorylated MLC₂₀ (pMLC₂₀). C: quantitative analyses of the pMLC₂₀-to-MLC₂₀ ratio. Signal intensities for MLC₂₀ and pMLC₂₀ from three different blots were used for the quantitative analyses. **P < 0.005 between the values at P-1 to P-3 and ***P < 0.001 between the values at P-1 to P-7.

Fig. 3.

ML-7 decreases the phasic contraction frequency of the lymphatics. A: ML-7 decreases the contraction frequency [in counts/min (cpm)] of the lymphatics. B: ML-7 decreases the substance P (SP)-stimulated contraction frequency. Values are means ± SE for 5 experiments. *P < 0.05 vs. control; +P < 0.05 vs. SP; ^×P < 0.05 vs. 1 cmH₂O.

Fig. 4.

ML-7 does not change the phasic contraction amplitude of the lymphatics. Effects of ML-7 on the phasic contraction amplitude were calculated as described in materials and methods. Values are means ± SE for 5 experiments.

Fig. 5.

ML-7 decreases SP-induced tonic contraction of the lymphatics. Values are means ± SE for 5 experiments. *P < 0.05 vs. control; +P < 0.05 vs. SP.

Fig. 6.

ML-7 does not significantly change the phasic contraction amplitude of the lymphatics in the presence of SP. Values are means ± SE for 5 experiments. *P < 0.05 vs. control.

Fig. 7.

Immunohistochemical staining analysis for pMLC₂₀ in the lymphatics. pMLC₂₀ in the isolated rat mesenteric lymphatics with the corresponding treatments described earlier were subjected to immunohistochemical detection. A: APSS-treated lymphatic vessel; B: 10⁻⁷ M SP-treated vessel; C: 10⁻⁵ M ML-7-treated vessel; D: 10⁻⁵ M ML-7 + 10⁻⁷ M SP-treated lymphatic vessel.

Fig. 8.

Quantitative analyses of pMLC₂₀ to MLC₂₀ in lymphatics. A and B: representative antibody reaction blots for the relative levels of MLC₂₀ (A) and pMLC₂₀ (B) in protein samples from ML-7 (10⁻⁵ M)-treated or untreated lymphatic vessels. C: signal intensities for MLC₂₀ and pMLC₂₀ from three different blots were used for the quantitative analysis. **P ≤ 0.001, significant difference between untreated and ML-7-treated samples. D: MLC₂₀ phosphoprotein analysis by urea-glycerol gel. Un, mono, and di represent the unphosphorylated, monophosphorylated, and diphosphorylated forms of MLC₂₀. Top, representative antibody reaction blot for the relative levels of MLC₂₀ and pMLC₂₀ in samples treated with either SP (10⁻⁷ M) or ML-7 (10⁻⁵ M) after urea-glycerol gel electrophoresis. LMC, lymphatic muscle cells. Bottom, quantitative analysis for the percentage of pMLC₂₀, which was calculated as described in materials and methods from three different blots. *P ≤ 0.05, significant difference between untreated (control) and SP- or ML-7-treated samples.