Ca2+ transport by the sarcoplasmic reticulum Ca2+-ATPase in sea cucumber (Ludwigothurea grisea) muscle

In muscle cells, the excitation-contraction cycle is triggered by an increase in the concentration of free cytoplasmic Ca2+. The Ca2+-ATPase present in the membrane of the sarcoplasmic reticulum (SR) pumps Ca2+ from the cytosol into this intracellular compartment, thus promoting muscle relaxation, The microsomal fraction derived from the longitudinal smooth muscle of the body wall from the sea cucumber Ludwigothurea grisea retains a membrane-bound Ca2+-ATPase that is able to transport Ca2+ mediated by ATP hydrolysis. Immunological analyses reveal that monoclonal antibodies against sarco-endoplasmic reticulum Ca2+-ATPase (SERCA1 and SERCA2a) cross-react with a 110 kDa band, indicating that the sea cucumber Ca2+-ATPase is a SERCA-type ATPase, Like the mammalian Ca2+-ATPase isoforms so far described, the enzyme also shows a high affinity for Ca2+ and ATP, has an optimum pH of approximately 7.0 and is sensitive to thapsigargin and cyclopiazonic acid, specific inhibitors of the SERCA pumps. However, unlike the mammalian SERCA isoforms, concentrations of ATP above 2 mmol l(-1) inhibit Ca2+ transport, but not ATP hydrolysis, in sea cucumber vesicles, suggesting that high ATP concentrations uncouple the Ca2+-ATPase. Another unique feature observed with the sea cucumber Ca2+-ATPase is the high dependence of maximal activity on K+ or Na+. Similar activation promoted by these cations was observed with various mammalian Ca2+-ATPase preparations when they were incubated in the presence of low concentrations of sulphated polysaccharides, In control experiments, K+ and Na+ have almost no effect on Ca2+ transport, but in the presence of heparin or fucosylated chondroitin sulphate, the activity of the different mammalian Ca2+-ATPases is inhibited and they are activated by either K+ or Na+ in a manner similar to the native sea cucumber ATPase, These results led us to investigate the possible occurrence of a highly sulphated polysaccharide on vesicles from the SR of sea cucumber smooth muscle that could act as an 'endogenous' Ca2+-ATPase inhibitor, In fact, SR vesicles derived from the sea cucumber, but not from rabbit muscle, contain a highly sulphated polysaccharide. After extraction and purification of these polysaccharide molecules, their effect was tested on vesicles obtained from rabbit muscle. This compound inhibited Ca2+ uptake in rabbit SR vesicles. at concentrations lower than heparin, and restored the dependence on monovalent cations, These results strongly suggest that the sea cucumber Ca2+-ATPase is activated by monovalent cations because of the presence of endogenous sulphated polysaccharides.