The objective was to characterize ATP-dependent Ca uptake into inside out basolateral membrane vesicles prepared from three crayfish tissues that routinely transport Ca during stages of the crustacean molting cycle. The initial uptake rate of 45Ca (free Ca 5 uM) into vesicles in the presence or absence of ATP (5 mM) was determined by rapid filtration and found to be linear for 10 mins. ATP independent accumulation was typically 20% of ATP dependent uptake. ATP dependent uptake was abolished by pretreatment with either vanadate or the ionophore A23187- Calcium efflux was observed when A23187 was added to actively loading vesicles. Ca affinity was equivalent in liver and gill (Km 0.27 uM) but was higher in kidney (0.11 uM). Maximal uptake was 10.13 pmol/mg/min in liver and was 5-6 fold higher in gill and kidney. An ATP titration curve indicated a Km of 0.01 mM in liver and gill and 0.024 mM in kidney. EGTA treatment of liver and kidney vesicles decreased Ca uptake by 50%. Uptake was restorable by calmodulin. However in gill Ca uptake was unaffected by EGTA treatment and calmodulin decreased uptake. Q10 was 1.43 in liver, 2.06 in gill and 1-64 in kidney. Addition of Na (5 mM) caused a 30% increase in Ca uptake that was inhibitable by preincubation with ouabain indicating that the Na pump generates a Na gradient favorable for Ca accumulation via the Na/Ca exchanger. Kinetics have been related to the role of crayfish liver, gill and kidney in Ca homeostasis and compared with other known Ca transporting epithelia.
|Original language||English (US)|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology