Molecular and Cellular Biology
1
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 <0.0001 MNS vs MHS (U/mg) and MLCS Ca2+ATP 14316*_315 147.5_+9.1 170.7:h5.6 0.016 MNS vs MHS ase (mmol and MLCS /mK/min) , I
2 VITAMIN B-6 DEFICIENCY IN GROWING RATS CONSTITUTES IN VIVO MODEL OF CELLULAR CALCIUM STORE DEPLETION. E. Karczmarewicz. H. Natyaszczyk, J.Marowska, L. Bielecka N. Jarocewicz, E. Czarnowska, ~.Przybyls~i.,RD. ReynoldsJ.,RS. Lorenc, Dept Biochem & Expt Ned, Child's Health Centre, Warsaw; .Dept Physiol, Ned Academy, Warsaw; • -Dept Human Nutr & Dietetics, Univ Illinois at Chicago, Chicago.
An experimental model o£ vitamin B-6 deficiency in growing rats have been developed. 4-5 fold decrease in pycidoxal 5"-phosphate (PLP) of isolated enterocytes, p r o x i m a l kidney tubules and platelets was observed, which induced profound changes in cellular calcium homeostasis but not in basic m o r p h o l o g i c a l and functional c e l l features. Compared to control rats, extraceltular Ca decreased by 25Z, total intracellular calcium by 50Z, ionized cytosol Ca by 40-50Z, and t o t a l e x c h a n g e a b l e Ca by n e a r l y TOZ. ATP-dependent Ca u p t a k e i n t o non-mitochondrial pool decreased by 3 0 Z and the concentration o£ i n o s i t o l 1 , 4 , 5 - t r i p h o s p h a t e - i n d u c e d h a l f - m a x i m a l Ca r e l e a s e i n c r e a s e d J - f o l d , s u g g e s t i n g d i m i n i s h e d i n o s i t o l i.~,5 - tciphosphate ccceptuc sensitivity. Vitamin D-6 deficiency also decreased the A2318T-mediated increase i n mitochondrial respiration by 50X, probably resulting from the pathological behaviour of the mitochondrial Ca translocator. These changes did not affect jejunal and kidney calcium transport, because of the greater paracellular Ca movement compensating for the lower transcellulac Ca transport. Our hypothesis of cellular Ca store depletion in vitamin B-6 deficiency was supported by blood pressure normalization in g-6 deficient spontaneously hypertensive rats (SHR), apparently being a consequence o£ decreased Ca in vascular smoth muscle cells.
© 1995 by Elsevier Science Inc. 393
Bone Vol. 16, No. 3 March 1995:391 410
3 Ca SOLUBILITY, INTESTINAL SOJOURN TIME AND PARACELLULAR PERMEABILITY CODETERMINE PASSIVE Ca ABSORPTION. D.Pansu t C.Duflos t C.Bellatont L.Escoffier r F.Bronner. EPHE & INSERM U 45, Lyon France, Universdity of Connecticut Health Center. Farmington, CT, USA.
6 wk old male rats were placed on 3 high Ca diets, 1.5 % Ca (3a), 3.0 % Ca (5a) with most Ca in carbonate form, and 3.0 % Ca (5b) with most in gluconate form. 2 wk later 4-day balance experiments were done and net Ca absorption determined. The luminal contents of 12 cm intestinal segments were then analyzed for pH, transit time, soluble and insoluble Ca. The pH increased from <6.6 in the duodenum to >8.0 in the ileum in all 3 diet groups. Intestinal transit time was 3h in diet groups 3a and 5a, lh in the rats fed the diarrhea-inducing gluconate diet. In the duodenum of all 3 groups the soluble Ca averaged 25% of total. Distally soluble Ca averaged 2.6%, 2% and 8.7% in groups 3a, 5a and 5b respectively. When the net Ca absorption values of the diet groups were divided by the total soluble Ca in their intestines, 58/1.28, 100/2.12, 180/4.54, the resulting turnover of soluble luminal Ca averaged 44.d -I This means soluble Ca moves out of the lumen at the average rate of 3.1% .min -I and is instantly replaced by Ca that goes into solution. Clearance calculations indicate that Ca movement through the paracellular space is much slower than if the ion were to diffuse down its chemical gradient through water. Thus intestinal sojourn time, Ca solubility and membrane permeability to Ca determine how much Ca is absorbed passively, i.e. paracellular ly, a route which in rats on high Ca intakes accounts for 80-90 % of the total absorbed.
4 CELLULAR AND PARACELLULAR Ca ++ TRANSPORT IN CACO-2 CELL MONOLAYERS: EFFECT OF VITAMIN D. M, v. Chiravath. L. Gaidzik. J. Graf. H. S. Cross. M.
p0terlik, Department of General and Experimental Pathology, University of Vienna Medical School, Austria
Previous work from our laboratory shows that in intestinal epithelial cells, expression of genomic effects of vitamin D at the cellular level apparently depends on their degree of differentiation (Acta Endocrinol. 124, 679, 1991 ). Thus, human colon adenocarcinoma-derived Caco-2 cells, though expressing vitamin receptors (VDR) in appropriate numbers, still might have acquired defects in VDR- mediated gene expression, viz. calbindin-mediated Ca ++ transport, during neoplastic transformation. The present study was therefore conducted to dissect the effects of vitamin D on different pathways of transepithelial Ca ++ transfer in Caco-2 cell monolayers.
Caco-2 cells grown on Transwell filter supports were exposed to 10 nM 1,25(OH)2D3 from confluency on. Transepithelial electric resistance (TEER) as a measure of junc t iona l pe rmeab i l i t y was mon i to red pr ior to determinat ion of [14C]mannitol transport and 45Ca++ fluxes.
The gradual rise of TEER during., post-confluent differentiation (to 2000 (+60 SEM) ~.cm z on day 10 past confluency) was most notably attenuated by 1,25(OH)2D3 to 850 (+150 SEM) fLcm 2. At the same time the hormone had no significant influence on [14C]mannitol transport, but increased transepithelial 45Ca++ transfer in parallel with its positive effect on junctional permeabil i ty. In addition, 1,25(OH)2D3 increased cellular 45Ca++ uptake from the mucosal solution in post-confluent Caco-2 cells (to 160 Yo of vitamin D-free control).
From the combined data we conclude that, in accordance with our previous results obtained in organ- cultured embryonic chick intestine (Mol. Ceil. Endocrinol. 53: 53, 1987), 1,25(OH)2D3 increases paracellular ionic permeability and thereby could facilitate paracellular Ca ++ transport as a major route of transepithelial Ca ++ transfer in Caco-2 cell monolayers.
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