NH2-terminal fragments of the 130 kDa subunit of myosin phosphatase increase the Ca2+ sensitivity of porcine renal artery

1. The effects of the NH2-terminal fragments of M130, a 130 kDa regulatory subunit of smooth muscle myosin phosphatase, on contraction and myosin light chain phosphorylation were investigated in Triton X-100-permeabilized porcine renal artery. 2. Incubation of the permeabilized fibres with M130(1-633) (a fragment containing amino acid residues 1-633) or M130(44-633) enhanced the Ca2+-induced contraction and shifted the [Ca2+](i)-force relationship to the left (EC50 of Ca2+: 330 nM, control, without fragment; 145 nM, M130(1-633); 163 nM, M130(44-633)). Pre-incubation for 1-3 h was needed for these long constructs. 3. M130(1-374), M130(304-511) and M130(297-374), i.e. relatively short constructs compared with M130(1-633) and M1303(44-633), also induced leftward shifts of the [Ca2+](i)-force relationship (EC50 of Ca2+: 65 nM, 72 nM and 180 nM, respectively). However, these required no pre-incubation. 4. Deletion of residues 304-374 from the most potent construct, M130(1-374), abolished the Ca2+ sensitizing effect. 5. Wortmannin inhibited the enhancement of contraction induced by M130 fragments when added before contraction was initiated and partially inhibited the effects when added after steady-state contraction. 6. M130(1-374) slowed the rate of relaxation in Ca2+-free medium. The time for 50% relaxation with this fragment was 510 +/- 51 s, compared with 274 +/- 14 s for control. 7. The levels of myosin light chain phosphorylation (22.4%) and force (34.5%) obtained with 300 nM Ca2+ were increased by 3 mu M M130(1-374) to 35.7 and 92.2%, respectively. However, M130(1-374) bad no effect on the phosphorylation-force relationship. 8. In conclusion, the NH2-terminal M130 fragments containing residues 304-374 inhibited myosin phosphatase, increased myosin light chain phosphorylation and increased the Ca2+ sensitivity of the contractile apparatus in permeabilized porcine renal artery.