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

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. Incubation of the permeabilized fibres with M1301-633 (a fragment containing amino acid residues 1-633) or M13044-633 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, M1301-633; 163 nM, M13044-633). Pre-incubation for 1-3 h was needed for these long constructs. M1301-374, M130304-511 and M130297-374, i.e. relatively short constructs compared with M1301-633 and M13044-633, 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. Deletion of residues 304-374 from the most potent construct, M1301-374, abolished the Ca2+-sensitizing effect. 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. M1301-374 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. The levels of myosin light chain phosphorylation (22.4 %) and force (34.5 %) obtained with 300 nM Ca2+ were increased by 3 μM M1301-374 to 35.7 and 92.2 %, respectively. However, M1301-374 had no effect on the phosphorylation-force relationship. 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.