PKC activation increases Ca²⁺sensitivity of permeabilized lymphatic muscle via myosin light chain 20 phosphorylation-dependent and -independent mechanisms

Abstract

The contractile activity of muscle cells lining the walls of collecting lymphatics is responsible for generating and regulating flow within the lymphatic system. Activation of PKC signaling contributes to the regulation of smooth muscle contraction by enhancing sensitivity of the contractile apparatus to Ca(2+). It is currently unknown whether PKC signaling contributes to the regulation of lymphatic muscle contraction. We hypothesized that the activation of PKC signaling would increase the sensitivity of the lymphatic myofilament to Ca(2+). To test this hypothesis, we determined the effects of PKC activation with phorbol esters [PMA or phorbol dibutyrate (PDBu)] on the contractile behavior of α-toxin-permeabilized rat mesenteric and cervical lymphatics or the thoracic duct. The addition of PMA or PDBu induced a significant increase in the contractile force of submaximally activated α-toxin-permeabilized lymphatic muscle independent of a change in intracellular Ca(2+) concentration, and the Ca(2+)-force relationship of lymphatic muscle was significantly left shifted, indicating greater myofilament Ca(2+) sensitivity. Phorbol esters increased the maximal rate of force development, whereas the rate of relaxation was reduced. Western blot and immunohistochemistry data indicated that the initial rapid increase in tension development after stimulation by PDBu was associated with myosin light chain (MLC)20 phosphorylation; however, the later, steady-state Ca(2+) sensitization of permeabilized lymphatic muscle was not associated with increased phosphorylation of MLC20 at Ser(19), 17-kDa C-kinase-potentiated protein phosphatase-1 inhibitor at Thr(38), or caldesmon at Ser(789). Thus, these data indicate that PKC-dependent Ca(2+) sensitization of lymphatic muscle may involve MLC20 phosphorylation-dependent and -independent mechanism(s).

Hypoxia-induced force increase (HIFI) is a novel mechanism underlying the strengthening of labor contractions, produced by hypoxic stresses

“…The increased biosynthesis of prostaglandins, and possibly Ca 2+ sensitization also resulting from these changes, culminates in increased uterine contractions. Dougherty et al (33) have recently shown that PKC activation increases Ca 2+ sensitivity of permeabilized lymphatic muscle via myosin light chain 20 phosphorylation-dependent and -independent mechanisms, and this may contribute to the sensitization in myometrium. It is also likely that actin dependent mechanisms of Ca 2+ sensitization are evoked (34,35).…”

Mechanobiological oscillators control lymph flow

“…(ii) The lymphatic muscle contractile force increases with cytosolic Ca 2+ concentration (with saturating kinetics); the maximum possible force decreases with the radius (to reproduce the known length-force relationship) (22)(23)(24)(25)(26). (iii) Ca 2+ accumulates in the cytosol at a constant, slow rate (i.e., influx through T-type channels) (23,27,28) and is depleted via ion pumps (23,29).…”

Mechanical forces and lymphatic transport

“…Rho Kinase (ROCK), which phosphorylates the MLC phosphatase (MLCP) targeting subunit MYPT-1, leading to inactivation of MLCP, also promotes tonic constriction of lymphatics (Hosaka et al, 2003; Kurtz et al, 2014b; Souza-Smith et al, 2013). There is also evidence that PKC increases Ca 2+ -sensitivity by activating CPI-17-mediated inhibition of MLCP to increase tone (Dougherty et al, 2008; Dougherty et al, 2014). New evidence also suggests that lymphatic tone can be sustained by slow-cycling latch bridges (Dougherty et al, 2014), as seen in vascular smooth muscle (Dillon et al, 1981).…”

“…There is also evidence that PKC increases Ca 2+ -sensitivity by activating CPI-17-mediated inhibition of MLCP to increase tone (Dougherty et al, 2008; Dougherty et al, 2014). New evidence also suggests that lymphatic tone can be sustained by slow-cycling latch bridges (Dougherty et al, 2014), as seen in vascular smooth muscle (Dillon et al, 1981). …”