Differential vasodilation of human placental and myometrial arteries related to myofilament Ca2+-desensitization and the expression of Hsp20 but not MYPT1

Abstract

Endothelial-dependent regulation of vascular tone occurs in part via protein kinase G1α-mediated changes in smooth muscle myofilament sensitivity to Ca(2+). Tissue-specific differences in PKG-dependent relaxation have been attributed to altered expression of myofilament-associated proteins that are substrates for PKG binding. These include the alternative splicing of the myosin targeting subunit (MYPT1) of myosin light chain phosphatase to yield leucine zipper positive (LZ(+)) and negative (LZ(-)) isovariants, with the former being required for PKG-mediated relaxation, and/or altered expressions of telokin, vasodilator-stimulated phosphoprotein (VASP) or heat shock protein Hsp20. During human pregnancy the uterine and placental circulations remain distinct entities and, as such, their mechanisms of vascular tone regulation may differ. Indeed, the sensitivity of myometrial arteries to endothelial-dependent agonists has been suggested to be greater than that of placental arteries. We tested the hypothesis that this was related to tissue-specific changes in PKG-mediated myofilament Ca(2+)-desensitization and/or the expressions of PKG-interacting myofilament-associated proteins. Permeabilized human placental and myometrial arteries were constricted with maximal activating Ca(2+) (pCa 4.5), or sub-maximal Ca(2+) (pCa 6.7) and the thrombane mimetic U46619, and exposed to 8-Br-cGMP. In each case, relaxation was significantly greater in myometrial arteries (e.g. relaxation in pCa 4.5 to 8-Br-cGMP was 49 ± 9.7%, n = 7) than placental arteries (relaxation of 23 ± 6.6%, n = 6, P < 0.05). MYPT1 protein levels, or MYPT1 LZ(+)/LZ(-) mRNA ratios, were similar for both artery types. Of other proteins examined, only Hsp20 expression was significantly elevated in myometrial arteries than placental arteries. These results demonstrate that the reduced human placental artery relaxation to PKG stimulation lies partly at the level of myofilament (de)activation and may be related to a lower expression of Hsp20 than in myometrial arteries.

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

“…). Differential expression of HSP20, but not of the MYPT1 splice variants, was associated with observed different cGMP responsiveness in human placental vs uterine vessels, to our knowledge the only report of MYPT1 splice variants, related to cGMP sensitivity in human arteries. The PKG target and activator of MLCP, telokin, is unlikely involved since it is not expressed in CA (Figure ).…”

Altered Expression of Human Smooth Muscle Myosin Phosphatase Targeting (MYPT) Isovariants with Pregnancy and Labor

“…We have previously reported PPP1R12ALZ+ and LZ- mRNA expression in human uterine and placental blood vessel tissues [14]. The PPP1R12ALZ- nucleotide and amino acid sequences generated are consistent with recently predicted PPP1R12ALZ- human sequences obtained from automated computational analysis using an NCBI eukaryotic gene prediction tool [http://www.ncbi.nlm.nih.gov/nuccore: PPP1R12ALZ- transcript variant X3: XM_011538376.1/ transcript variant X8: XM_005268887.2] and other bioinformatic analyses [15].…”

“…In addition, a reduction in PPP1R12ALZ+ and PPP1R12CLZ+ isovariants with labor could confer reduced tissue sensitivity to PKG-mediated relaxatory influences. Of note, differing levels of PKG-induced Ca 2+ desensitization in uterine and placental vasculature have been associated with changes in the expression of PPP1R12ALZ+ and other contractile associated proteins such as hsp20 and VASP[14]. However other workers have suggested that only small proportions of NO mediated relaxation in myometrium are actually mediated via PKG and cGMP related pathways, underlining the need for future investigations to explore alternative relaxatory pathways [18].…”

“…Micro-dissected myometrial tissues were homogenized and RNA extracted using a Qiagen RNeasy fibrous kit as previously described in [14]. Briefly, 30mg of tissue was homogenized in lysis buffer using a mechanical homogenizer (Minilys, Bertin Technologies).…”

Estrogenic vascular effects are diminished by chronological aging

“…Arteries were cut into 5–10 mm segments and immediately snap frozen with 2-methylbutane (isopentane) cooled by liquid nitrogen and stored at −80 °C for further analysis of protein expression. Frozen samples were homogenized in cell lysis buffer (62.5 mM Tris – HCl, 2% SDS, 10% Sucrose) with 2% (vol/vol) protease inhibitor and 0.5% (vol/vol) phosphatase inhibitor at 5 µl per mg of tissue (minimum of 60 µl) and prepared for western blotting as previously described 77 . Thirty μg of protein was resolved by SDS-PAGE, transferred to a PVDF membrane for western blotting with ERα (Vector Labs #E-613 mouse monoclonal 1:200) or ERβ (Abcam #288 mouse monoclonal 1:500) and viewed chemiluminescently following incubation with HRP-goat anti-mouse-IgG (1:2000 Dakocytomation #PO447).…”