Aging-related alterations in eNOS and nNOS responsiveness and smooth muscle reactivity of murine basilar arteries are modulated by apocynin and phosphorylation of myosin phosphatase targeting subunit-1

Abstract

Aging causes major alterations of all components of the neurovascular unit and compromises brain blood supply. Here, we tested how aging affects vascular reactivity in basilar arteries from young (<10 weeks; y-BA), old (>22 months; o-BA) and old (>22 months) heterozygous MYPT1-T-696A/+ knock-in mice. In isometrically mounted o-BA, media thickness was increased by ∼10% while the passive length tension relations were not altered. Endothelial denudation or pan-NOS inhibition (100 µmol/L L-NAME) increased the basal tone by 11% in y-BA and 23% in o-BA, while inhibition of nNOS (1 µmol/L L-NPA) induced ∼10% increase in both ages. eNOS expression was ∼2-fold higher in o-BA. In o-BA, U46619-induced force was augmented (pEC ∼6.9 vs. pEC ∼6.5) while responsiveness to DEA-NONOate, electrical field stimulation or nicotine was decreased. Basal phosphorylation of MLC-S19 and MYPT1-T-853 was higher in o-BA and was reversed by apocynin. Furthermore, permeabilized o-BA showed enhanced Ca-sensitivity. Old T-696A/+ BA displayed a reduced phosphorylation of MYPT1-T696 and MLC, a lower basal tone in response to L-NAME and a reduced eNOS expression. The results indicate that the vascular hypercontractility found in o-BA is mediated by inhibition of MLCP and is partially compensated by an upregulation of endothelial NO release.

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

“…L‐NAME had no effect on the concentration‐response relationship in FA (Figure A,C, Table ), but it induced a leftward shift in CA‐w ( P < .001), as well as a rise in basal (Figure B,D) and maximal U46619 elicited force (Figure B,D, c.f. Table ) as we reported before for BA . These results are in keeping with the idea that agonist‐induced tone and basal contractile activity in the absence of added agonists are blunted in cerebral arteries by constitutively released NO.…”

“…Next we tested the hypothesis that the observed differences in the expression of LZ + ‐MYPT1 splice variants and phosphorylation of MYPT1‐T696/T853 and the PKG sites (MYPT1‐S695/S668) are associated with differences in basal tone and the contractile response to stimulation with U46619. As we reported before for basilar arteries, the cumulative concentration‐response curve to U46619 was blunted and shifted to the right by nearly 1 order of magnitude in CA‐w compared to FA (pEC 50 7.7 ± 0.1 in FA, n = 5 vs 6.9 ± 0.06 in CA‐w, n = 6; P < .001, Figure A–D, Table ) with no difference in the Hill coefficients ( n H = 2.1 ± 0.1, n = 5 in FA vs 2.2 ± 0.4, n = 6 in CA‐w). After wash‐out of U46619 and pre‐incubation with 100 μmol/L L‐NAME in PSS for 20 minutes, a second cumulative concentration‐response relationship was obtained in the continued presence of L‐NAME.…”

“…In contrast, recent in vivo work using mouse models that target these phosphorylation sites indicated that phosphorylation of T696 is more important for force regulation than T853 . In line with this report, we recently observed that phosphorylation of T696 is critically involved in ageing‐associated intrinsic hypercontractility of basilar arteries (BA) that could be unmasked by L‐NAME …”

“…It is well established that cerebral blood vessels are under the tonic influence of NO released from the endothelium and non‐endothelial sources like perivascular nerves and astrocytes, which attenuates both basal and stimulated contractile activity . The major novel findings of our study are (i) that under conditions of constitutive release of NO, phosphorylation of the novel PKG site, MYPT1‐S668, which was proposed to activate PKG, was higher in cerebral than in femoral arteries, and (ii) that this was associated with a higher expression level of the LZ + ‐MYPT1 splice variants, which in turn was associated with a higher cGMP‐sensitivity of the contractile machinery; (iii) that the L‐NAME‐induced increase in basal tone in cerebral arteries was associated with an increase in inhibitory phosphorylations of MYPT1 and was attenuated in cerebral arteries from heterozygous knock‐in mice (MYPT1‐T696A/+) harbouring a non‐phosphorylatable alanine in place of T696; (iv) that cAMP relaxed permeabilized BA and FA to a similar degree indicating that cAMP‐induced relaxation does not depend on expression of the LZ + ‐MYPT1 isoform.…”

“…We have recently shown that the L‐NAME‐induced increase in basal tone of pre‐stretched basilar arteries was attenuated in 2‐year‐old heterozygous mice carrying a point mutation in the MYPT1 gene that replaced T696 by a non‐phosphorylatable alanine (MYPT1‐T696A/+) . Here, we tested whether this was also the case in cerebral arteries from young MYPT1‐T696A/+ mice.…”