Abstract 5878: Protein Kinase C-Dependent Calcium Sensitization of Permeabilized Rat Lymphatic Muscle
The contractile activity of the muscle cells lining the walls of collecting lymphatics is responsible for generating and regulating flow within the lymphatic system. Activation of protein kinase C (PKC) signaling contributes to the regulation of smooth muscle contraction by enhancing sensitivity of the contractile apparatus to Ca2+. It is currently unknown whether PKC signaling contributes to the regulation of lymphatic muscle contraction. We hypothesized that activation of PKC signaling would increase the sensitivity of the lymphatic myofilament to Ca2+. To test this hypothesis, we determined the effects of PKC activation with phorbol esters [phorbol-12-myristate-13-acetate (PMA); phorbol dibutyrate (PDBu)] on the contractile behavior of α-toxin permeabilized rat mesenteric lymphatics. Addition of PMA or PDBu induced slowly developing, sustained contractions of lymphatic muscle under Ca2+-clamped conditions (at pCa 6.5, 0.06±0.01 mN/mm, control vs. 0.63±0.05 mN/mm, PMA; 0.63±0.05 mN/mm, PDBu; n=6; p<0.05). Furthermore, the Ca2+-force relationship of lymphatic muscle was significantly left-shifted in the presence of phorbol ester, indicating greater myofilament Ca2+ sensitivity (pCa50: 6.05±0.10, control vs. 6.70±0.03, PMA; 6.60±0.04, PDBu; n=6; p<0.05). Phorbol ester treatment was also associated with slowed relaxation kinetics of lymphatic muscle (T½ relaxation for control, 0.28±0.02 min vs. PMA or PDBu, 1.60±0.13 min; n=6; p<0.05). To determine whether changes in phosphorylation states of the regulatory proteins could be one of the mechanisms that are activated during the steady state contraction induced by PKC signaling, western blots or immunofluorescence were conducted. There were no significant increases either in the phosphorylation of CPI-17 at thr38 or caldesmon at ser789 or ser19 on MLC20. Thus, these data demonstrate that activation of PKC signaling results in a significant increase in lymphatic myofilament Ca2+ sensitivity. The sustained contraction induced by phorbol ester is not associated with increased phosphorylation of MLC20, CPI-17 or caldesmon, indicating that PKC-dependent Ca2+ sensitization of lymphatic muscle may involve different mechanisms than those observed in other types of smooth muscle.