Rho-kinase inhibition attenuates calcium-induced contraction in β-escin but not Triton X-100 permeabilized rabbit femoral artery

Abstract

K+-depolarization (KCl) of smooth muscle has long been known to cause Ca2+-dependent contraction, but only recently has this G protein-coupled receptor (GPCR)-independent stimulus been associated with rhoA kinase (ROCK)-dependent myosin light chain (MLC) phosphatase inhibition and Ca2+ sensitization. This study examined effects of ROCK inhibition on the concentration-response curves (CRCs) generated in femoral artery by incrementally adding increasing concentrations of KCl to intact tissues, and Ca2+ to tissues permeabilized with Triton X-100, β-escin and α-toxin. For a comparison, tissue responses were assessed also in the presence of protein kinase C (PKC) and MLC kinase inhibition. The ROCK inhibitor H-1152 induced a strong concentration-dependent inhibition of a KCl CRC. A relatively low GF-109203X concentration (1 μM) sufficient to inhibit conventional PKC isotypes also inhibited the KCl CRC but did not affect the maximum tension. ROCK inhibitors had no effect on the Ca2+ CRC induced in Triton X-100 or α-toxin permeabilized tissues, but depressed the maximum contraction induced in β-escin permeabilized tissue. GF-109203X at 1 μM depressed the maximum Ca2+-dependent contraction induced in α-toxin permeabilized tissue and had no effect on the Ca2+ CRC induced in Triton X-100 permeabilized tissue. The MLC kinase inhibitor wortmannin (1 μM) strongly depression the Ca2+ CRCs in tissues permeabilized with Triton X-100, α-toxin and β-escin. H-1152 inhibited contractions induced by a single exposure to a submaximum [Ca2+] (pCa 6) in both rabbit and mouse femoral arteries. These data indicate that β-escin permeabilized muscle preserves GPCR-independent, Ca2+- and ROCK-dependent, Ca2+ sensitization.

Size‐dependent heterogeneity of contractile Ca2+ sensitization in rat arterial smooth muscle

“…At 3 min, phosphorylation of MLC but not CPI‐17 decreased to about 60% of the control at 30 s. MYPT1 phosphorylation at ROCK‐specific Thr853 (Murányi et al 2005) was already high (76 ± 7%) at rest and only slightly increased with time after PE stimulation (Supplemental Fig. S8 D ), suggesting an existence of constitutively active ROCK(s) at rest (Clelland et al 2011).…”

The AMP-Dependent Protein Kinase (AMPK) Activator A-769662 Causes Arterial Relaxation by Reducing Cytosolic Free Calcium Independently of an Increase in AMPK Phosphorylation

“…Thus, if A-769662 can cause relaxation by inhibition of ROCK and PKC, then A-769662 would be expected to suppress PE-induced contractions. Likewise, A-769662 would be expected to inhibit Ca 2+ -activated contraction of β-escin-permeabilized VSM because ROCK and PKC inhibitors attenuate this response ( Clelland et al, 2011 ). However, a PE-induced contraction of EA was entirely resistant to A-769662.…”

“…Thus, when considering MA, no data ( Figures 3D,T , n.d.) was obtained for Ang II and VP CRCs, and mouse MA was used to assess the effect of A-769662 on a U-46619 CRC ( Figure 3P ). Permeabilized tissues were activated by addition of Ca 2+ and GTPγS as described previously ( Clelland et al, 2011 ). Data were reported as active force normalized to F 0 (F/F 0 ), and thus, an F/F 0 value of 1 was equal to the maximum KPSS-induced contraction achieved during the initial determination of L 0 .…”

“…Rabbit MA was chemically permeabilized with β-escin as described previously ( Clelland et al, 2011 ) with minor modifications. In short, each artery ring at L 0 was incubated at 30°C in a Ca 2+ -free “relaxing solution” (in mM: 74.1 potassium methanesulfonate, 4.0 magnesium methanesulfonate, 4.0 Na2ATP, 4.0 EGTA, 5.0 creatine phosphate, 30.0 PIPES, adjusted to pH 7.1 with 1 N KOH and ionic strength 180 with additional 0.5 M potassium methanesulfonate).…”

Pregnancy-associated adaptations in [Ca2+]i-dependent and Ca2+sensitization mechanisms of venous contraction: implications in pregnancy-related venous disorders

“…Studies have also shown that Y27632 and other ROCK inhibitors inhibit KCl-induced contraction, Ca 2ϩ influx, and MLC phosphorylation in rat aorta and mesenteric artery (25,97), rat tail artery (58), rabbit femoral artery (13), and porcine airway smooth muscle (34). ROCK is a family of Ca 2ϩ -dependent and Ca 2ϩ -independent isoforms (46,79,91 ] i -dependent contraction mechanisms are likely underestimated, and that, in the absence of these rescue Ca 2ϩ -sensitization pathways, the veins could be even more prone to dilation during pregnancy.…”