Abstract
Hypertensive rats from the Milan strain show a significant decrease in calpastatin activity as compared with normotensive control animals. Calpastatin deficiency is age-related and highly relevant in kidney, heart, and erythrocytes and of minor entity in brain tissue. In normotensives the changes during aging in the levels of calpastatin activity and mRNA are consistent with an increase of calpastatin protein. In hypertensive rats such a relationship during aging is not observed, because a progressive accumulation of mRNA is accompanied by a lower amount of calpastatin protein as compared with control rats. Together with the low level of calpastatin in kidney of hypertensive rats, a progressive accumulation of an active 15-kDa calpastatin fragment, previously shown to represent a typical product of calpain-mediated calpastatin degradation, is also observed. Evidence for such intracellular proteolysis by Ca(2+)-activated calpain is provided by the normalization of the calpastatin level, up to that of control animals, in hypertensive rats treated with drugs known to reduce both blood pressure and intracellular Ca(2+) influx. Further evidence is provided by the disappearance, in these conditions, of the 15-kDa calpastatin fragment. These data allow the conclusion that calpastatin degradation is a relevant part of the overall mechanism for regulating calpain activity.
Highlights
Calpain activation is a multistep process involving transient conformational changes as well as irreversible limited autoproteolysis
Together with the low level of calpastatin in kidney of hypertensive rats, a progressive accumulation of an active 15-kDa calpastatin fragment, previously shown to represent a typical product of calpain-mediated calpastatin degradation, is observed. Evidence for such intracellular proteolysis by Ca2؉-activated calpain is provided by the normalization of the calpastatin level, up to that of control animals, in hypertensive rats treated with drugs known to reduce both blood pressure and intracellular Ca2؉ influx
The preferential interaction of calpastatin with the autoproteolyzed calpain form explains why intracellular calpain can become active even in the presence of an excess of calpastatin as we have observed in human erythrocytes enriched with calcium [11]
Summary
Materials—Leupeptin, phenylmethylsulfonyl fluoride, and Ca2ϩ ionophore A23187 were purchased from Sigma. The absorbed proteins were eluted with a linear gradient of NaCl from 0 to 0.35 M, and the amount of calpastatin activity was measured in the eluted fractions using human erythrocyte calpain and human acid denatured globin as substrate [28] in the presence of 1 mM Ca2ϩ as described previously [26]. This enzyme has been selected because it is sensitive to all calpastatin forms regardless of their sources. Systolic blood pressure was measured during treatment by the tail-cuff method using the plethysmographic system based on techniques originally described by Byrom and Wilson [38]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
