cellular and paracellular ca++ transport in caco-2 cell monolayers: effect of vitamin d
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Molecular and Cellular Biology
EFFECT OF CALCIUM-ACTIVATED NEUTRAL PROTEINASE (CALPAIN) ON RAT ERYTHROCYTE PLASMA MEMBRANE CALCIUM PUMP. Laura Soldati. Giusenoe Vezzoli. Sergio Salardi*, Barry 1~, Barber*, Tiziana Azzani and Giuseppe Bianctli, Department of Sciences and Biomedical Technologies, University of Milan: and *Prassis Research Institute, Settimo Milanese. Italy,
Calpain I, the only form present in human and rat erythrocytes, is a cytoplasmic calcium-dependent proteinase, consisting of two subunits: both of them also have a Ca 2+ binding domain, which has a marked similarity to Ca 2+ binding proteins, such as calmodulin (CAM). Erythrocytes contain also a protein termed calpastatin that specifically inhibits calpain I. A large number of proteins have been reported to be proteolysed by calpain I, but in most cases it is neither known if these proteins are really substrates for caipain in intact cells, nor if calpain plays a regulatory rather than a degradative role on these substrates. The caip,'fin domain structure suggested that the plasma membrane Ca2+ATPase could be a target for proteolytic action of calpain I and it was reported that calpain produces on Ca2+ATPase purified from human erythrocytes a 124 KDa fragment in absence of CaM. In order to assess the influence of calpain/calpastatin system on plasma membrane Ca 2+ ATPase activity we utilized a recently selected animal model that has an imbalance of erythrocyte calpain:calpastatin ratio: Milan low calpastatin rat strain (MLCS), derived by crosses between Milan hypertensive strain (MHS) and Milan normotensive strain (MNS). MLCS rats have extremely low calpastatin values, as parental MHS, and normal blood pressure values, as parental MNS. Calpaln values are similar in all three strains. We found that erythrocytes from mrs with less inhibited calpain, MLCS and MHS, had decreased calcium pump activity in comparison with MNS (results are summarized in the following table). Moreover, in erythrocytes from MLCS and MIlS we found the appearance of 124 KDa fragments, while in erythrccytes from MNS they were absent and it was present only the 136 KDa Ca2+ATPase native form.. Our data show that the rat erythrocyte calcium pump is a target for calpain and that its activity is decreased by proteinase action.
MLCS ] MHS MNS F P I ealpain 0.60&0,11 I 0.80-10.23 0.82-+0.13 0.53 n.s. (U/mR) I calpastatin 1.66:h0.48 0.91i-0.31 8,17i-0.22