Ca2+ compartments, Ca2+ transport and the calcification process were studied by using 45Ca as a tracer. The biological model used was clones of Stylophora pistillata developed into microcolonies whose skeleton is entirely covered by tissues, thus avoiding direct radioisotope exchange between the sea water and the skeleton. The study of Ca2+ compartments was performed by measuring two complementary parameters: Ca2+ influx and Ca2+ efflux kinetics. Kinetic analysis of 45Ca uptake revealed three exchangeable and one non-exchangeable Ca2+ compartments in these microcolonies. The first compartment was saturable with a short half-time (4 min), correlated to external Ca2+ concentration and insensitive to metabolic or ion transport inhibitors. This compartment (72.88 nmol Ca2+ mg-1 protein) has been previously attributed to sea water present in the coelenteron. The second Ca2+ compartment (7.12 nmol Ca2+ mg-1 protein) was soluble in NaOH, saturable with a half-time of 20 min and displayed a combination of Michaelis-Menten kinetics and diffusional entry. It was insensitive to a variety of inhibitors but its loading was stimulated by Ca2+ channel inhibitors. On the basis of uptake experiments, the existence of a third compartment with a rapid turnover rate (about 2 min) and a very small size is predicted. It is suggested that this compartment corresponds to the calicoblastic epithelium. Ca2+ flux through this compartment was facilitated by voltage-dependent Ca2+ channels (with L-type characteristics) and Ca2+-ATPase and was coupled to an anion carrier. Transcellular Ca2+ movement was dependent on the cytoskeleton. The rate of Ca2+ flux across this epithelium was about 975 pmol mg-1 protein min-1. The fourth calcium compartment, corresponding to the skeleton, was soluble in HCl and non-exchangeable. After a short lag phase (about 2 min), the rate of Ca2+ deposition was linear over a period of at least 5 h. The calcification rate was 975 pmol mg-1 protein h-1 at an irradiance of 175 µmol photons m-2 s-1. It followed Michaelis-Menten kinetics and saturated at levels (9 mmol l-1) close to the Ca2+ concentration of sea water. Wash-out (efflux) experiments employing several different protocols allowed identification of six compartments. The first two compartments were extracellular (bulk extracolonial water and coelenteron). The third compartment may be part of the second Ca2+ compartment identified by influx experiments. A fourth compartment was sensitive to the Ca2+ channel inhibitor D600 and appeared to be associated with the NaOH-soluble (tissue) Ca2+ pool. Two compartments were identified during skeletal efflux, the first being small and due to either tissue carry-over or a labile skeletal compartment. The second compartment corresponded to bulk skeletal deposition. The various efflux protocols produced varying estimates of tissue Ca2+ levels and calcification rates and, thus, coral post-incubation processing has a profound impact on experimental interpretation.
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