A COMPARTMENTAL APPROACH TO THE MECHANISM OF CALCIFICATION IN HERMATYPIC CORALS

Ca2+ compartments, Ca2+ transport and the calcification process were s tudied by using Ca-45 as a tracer. The biological model used was clone s of Stylophora pistillata developed into microcolonies whose skeleton is entirely covered by tissues, thus avoiding direct radioisotope exc hange between the sea water and the skeleton. The study of Ca2+ compar tments was performed by measuring two complementary parameters: Ca2+ i nflux and Ca2+ efflux kinetics. Kinetic analysis of Ca-45 uptake revea led three exchangeable and one non-exchangeable Ca2+ compartments in t hese microcolonies. The first compartment was saturable with a short h alf-time (4 min), correlated to external Ca2+ concentration and insens itive to metabolic or ion transport inhibitors. This compartment (72.8 8 nmol Ca2+ mg(-1) protein) has been previously attributed to sea wate r present in the coelenteron. The second Ca2+ compartment (7.12 nmol C a2+ mg(-1) protein) was soluble in NaOH, saturable with a half-time of 20 min and displayed a combination of Michaelis-Menten kinetics and d iffusional entry. It was insensitive to a variety of inhibitors but it s loading was stimulated by Ca2+ channel inhibitors. On the basis of u ptake experiments, the existence of a third compartment with a rapid t urnover rate (about 2 min) and a very small size is predicted. It is s uggested that this compartment corresponds to the calicoblastic epithe lium. Ca2+ flux through this compartment was facilitated by voltage-de pendent Ca2+ channels (with L-type characteristics) and Ca2+-ATPase an d was coupled to an anion carrier. Transcellular Ca2+ movement was dep endent on the cytoskeleton. The rate of Ca2+ flux across this epitheli um was about 975 pmol mg(-1) protein min(-1). The fourth calcium compa rtment, corresponding to the skeleton, was soluble in HCl and non-exch angeable. After a short lag phase (about 2 min), the rate of Ca2+ depo sition was linear over a period of at least 5 h. The calcification rat e was 975 pmol mg(-1) protein h(-1) at an irradiance of 175 mu mol pho tons 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 a llowed identification of six compartments. The first two compartments were extracellular (bulk extracolonial water and coelenteron). The thi rd compartment may be part of the second Ca2+ compartment identified b y influx experiments. A fourth compartment was sensitive to the Ca2+ c hannel inhibitor D600 and appeared to be associated with the NaOH-solu ble (tissue) Ca2+ pool. Two compartments were identified during skelet al efflux, the first being small and due to either tissue carry-over o r a labile skeletal compartment, The second compartment corresponded t o bulk skeletal deposition. The various efflux protocols produced vary ing estimates of tissue Ca2+ levels and calcification rates and, thus, coral post-incubation processing has a profound impact on experimenta l interpretation.