The involvement of phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and MYPT1 isoform expression in NO/cGMP mediated differential vasoregulation of cerebral arteries compared to systemic arteries

Abstract

Aim: Constitutive release of NO blunts intrinsic and stimulated contractile activity in cerebral arteries (CA). Here, we explored whether phosphorylation and expression levels of the PKG-sensitive, leucine zipper positive (LZ⁺ ) splice variants of the regulatory subunit of myosin phosphatase (MYPT1) are involved and whether its expression is associated with higher cGMP sensitivity.

Methods: Vascular contractility was investigated by wire myography. Phosphorylation of MYPT1 was determined by Western blotting.

Results: Constitutive phosphorylation of MYPT1-T696 and T853 was lower and that of S695 and S668 was higher in cerebral arteries from the circulus arteriosus (CA-w) than in femoral arteries (FA), while total MYPT1 expression was not different. In CA-w but not in FA, L-NAME lowered phosphorylation of S695/S668 and increased phosphorylation of T696/T853 and of MLC₂₀ -S19, plus basal tone. The increase in basal tone was attenuated in CA-w and basilar arteries (BA) from heterozygous MYPT1-T696A/+ mice. Compared to FA, expression of the LZ⁺ -isoform was ~2-fold higher in CA-w coincident with a higher sensitivity to DEA-NONOate, cinaciguat and Y27632 in BA and 8-Br-cGMP (1 μmol/L) in pre-constricted (pCa 6.1) α-toxin permeabilized CAs. In contrast, 6-Bnz-cAMP (10 μmol/L) relaxed BA and FA similarly by ~80%.

Conclusion: Our results indicate that (i) regulation of the intrinsic contractile activity in CA involves phosphorylation of MYPT1 at T696 and S695/S668, (ii) the higher NO/cGMP/PKG sensitivity of CAs can be ascribed to the higher expression level of the LZ⁺ -MYPT1 isoform and (iii) relaxation by cAMP/PKA pathway is less dependent on the expression level of the LZ⁺ splice variants of MYPT1.

Keywords: MYPT1 alternative splicing; NO/cGMP/PKG and cAMP/PKA sensitivity; myosin light-chain phosphatase; vascular tone.