Is myosin phosphatase regulated in vivo by inhibitor-1? Evidence from inhibitor-1 knockout mice

Abstract

1. The Ca(2+) sensitivity of smooth muscle contractility is modulated via regulation of phosphatase activity. Protein phosphatase inhibitor-1 (I-1) is the classic type-1 phosphatase inhibitor, but its presence and role in cAMP-dependent protein kinase (PKA) modulation of smooth muscle is unclear. To address the relevance of I-1 in vivo, we investigated smooth muscle function in a mouse model lacking the I-1 protein (I-1((-/-)) mice). 2. Significant amounts of I-1 protein were detected in the wild-type (WT) mouse aorta and could be phosphorylated by PKA, as indicated by (32)P-labelled aortic extracts from WT mice. 3. Despite the significant presence of I-1 in WT aorta, phenylephrine and KCl concentration- isometric force relations in the presence or absence of the PKA pathway activator isoproterenol (isoprenaline) were unchanged compared to I-1((-/-)) aorta. cGMP-dependent protein kinase (PKG) relaxation pathways were also not different. Consistent with these findings, dephosphorylation rates of the 20 kDa myosin light chains (MLC(20)), measured in aortic extracts, were nearly identical between WT and I-1((-/-)) mice. 4. In the portal vein, I-1 protein ablation was associated with a significant (P < 0.05) rightward shift in the EC(50) of isoproterenol relaxation (EC(50) = 10.4 +/- 1.4 nM) compared to the WT value (EC(50) = 3.5 +/- 0.2 nM). Contraction in response to acetylcholine as well as Ca(2+) sensitivity were similar between WT and I-1((-/-)) aorta. 5. Despite the prevalence of I-1 and its activation by PKA in the aorta, I-1 does not appear to play a significant role in contractile or relaxant responses to any pharmacomechanical or electromechanical agonists used. I-1 may play a role as a fine-tuning mechanism involved in regulating portal vein responsiveness to beta-adrenergic agonists.

Figure 1. Protein phosphatase inhibitor-1 is present and phosphorylated in mouse aorta

A, homogenates for both wild-type (WT) and I-1^(−/−) aorta (100 μg) were subjected to Western blot analysis. Homogenates (10 μg) from WT brain (lane 1) and I-1^(−/−) brain (lane 2) were run as positive and negative controls, respectively. An immunoreactive band at ∼29 kDa in both WT aorta and brain (lanes 1 and 3) was absent in the I-1^(−/−) tissues (lanes 2 and 4). B, extracts phosphorylated by PKA in the presence of [γ-³²P]ATP were subjected to SDS-PAGE and autoradiography. WT extracts contained a signal at ∼29 kDa, which was absent in the I-1^(−/−) extracts, consistent with the phosphorylation of I-1 under these conditions.

Figure 2. Phenylephrine concentration-isometric force relations

Receptor-mediated responses to phenylephrine (PE) were generated in WT (•) and I-1^(−/−) (□) denuded aortas. A, force/area responses to phenylephrine were not different between the two groups (P > 0.05). B, percentage maximal force was unchanged and pretreatment with isoproterenol (dotted lines) resulted in a rightward shift in both WT (▿) and I-1^(−/−) (▵) curves to a similar extent (P > 0.05, n = 4 for all groups). Values represent means ±s.e.m.

Figure 3. KCl concentration-isometric force relations

Responsiveness to KCl was assessed in WT (•, n = 4) and I-1^(−/−) (□, n = 4) denuded aortas, in parallel. A, KCl did not affect the absolute force per area generated in either group (P > 0.05). B, percentage maximal response was also not different between the two groups. Pretreatment with isoproterenol (dotted lines) resulted in a rightward shift of the concentration curves in a similar manner in WT (▿) and I-1^(−/−) (▵) aorta (P > 0.05). Values represent means ±s.e.m.

Figure 4. PKA-mediated (forskolin) and PKG-mediated (SNP) concentration-relaxation relations

Denuded aortas from WT (•, n = 4) and I-1^(−/−) (□, n = 4) mice were contracted with a submaximal concentration of PE and subsequently relaxed with increasing concentrations of forskolin (A) or sodium nitroprusside (B0. Relaxation in response to either agonist, expressed as a percentage of PE-induced contraction, was nearly identical between the two groups (P > 0.05). Values represent means ±s.e.m.

Figure 5. MLC₂₀ dephosphorylation rates

³²P-labelled MLC₂₀ dephosphorylation rates were measured in aortic extracts from WT (•, n = 5) and I-1^(−/−) (□, n = 5) mice treated with 50 μm forskolin. Type-2A (2 nm okadaic acid) and type-2B (50 nm deltamethrin) phosphatase inhibitors were included to maintain the phosphorylation status of I-1 during the assay. Values represent means ±s.e.m. of duplicate experiments.

Figure 6. Concentration-tension-time integral relations in response to isoproterenol in WT and I-1^(−/−) portal vein

Spontaneous mechanical activity in response to isoproterenol was measured in WT (•, n = 10) and I-1^(−/−) (□, n = 10) portal veins. The concentration of isoproterenol resulting in 50 % relaxation was significantly rightward shifted (P < 0.05) in I-1^(−/−) portal veins, compared to the WT. Values represent means ±s.e.m.

Figure 7. Percentage maximal force or tension-time integral from portal vein in response to increased calcium or ACh concentrations

Portal vein contraction in response to calcium (A) or ACh (B) was measured in WT (•, n = 5) and I-1^(−/−) (□, n = 5) mice. Portal vein contractility in WT and I-1^(−/−) mice was similar at every calcium concentration (P > 0.05). Pretreatment with isoproterenol (dotted lines), to fully activate I-1, resulted in a parallel rightward shift in the calcium dose-response curve in both the WT (▿) and I-1^(−/−) (▵) portal veins. Contraction in response to ACh, expressed as a percentage of basal values, increased in a comparable manner in both WT (•, n = 5) and I-1^(−/−) (□, n = 5) portal vein (P > 0.05). Values represent means ±s.e.m.