Is myosin phosphatase regulated in vivo by inhibitor-1? Evidence from inhibitor-1 knockout mice.
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.
Carr
A N
AN
Department of Pharmacology and Cell Biophysics, University of Cincinnati, Cincinnati, OH 45267, USA.
Sutliff
R L
RL
Weber
C S
CS
Allen
P B
PB
Greengard
P
P
de Lanerolle
P
P
Kranias
E G
EG
Paul
R J
RJ
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins; D005576:Colforsin / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D009599:Nitroprusside / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011169:Portal Vein / Q000201:enzymology; D011189:Potassium Chloride / Q000494:pharmacology; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology; D014665:Vasodilator Agents / Q000494:pharmacology
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D016601:RNA-Binding Proteins; D011943:Receptors, Adrenergic, beta; D014662:Vasoconstrictor Agents; D014665:Vasodilator Agents; C021042:protein phosphatase inhibitor-1; D009599:Nitroprusside; D005576:Colforsin; D010656:Phenylephrine; D011189:Potassium Chloride; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D000109:Acetylcholine; D002118:Calcium