Abstract
The nature of the mechanism underlying store-mediated Ca(2+) entry has been investigated in human platelets through a combination of cytoskeletal modifications. Inhibition of actin polymerization by cytochalasin D or latrunculin A had a biphasic time-dependent effect on Ca(2+) entry, showing an initial potentiation followed by inhibition of Ca(2+) entry. Moreover, addition of these agents after induction of store-mediated Ca(2+) entry inhibited the Ca(2+) influx mechanism. Jasplakinolide, which reorganizes actin filaments into a tight cortical layer adjacent to the plasma membrane, prevented activation of store-mediated Ca(2+) entry but did not modify this process after its activation. In addition, jasplakinolide prevented cytochalasin D-induced inhibition of store-mediated Ca(2+) entry. Calyculin A, an inhibitor of protein serine/threonine phosphatases 1 and 2 which activates translocation of existing F-actin to the cell periphery without inducing actin polymerization, also prevented activation of store-mediated Ca(2+) entry. Finally, inhibition of vesicular transport with brefeldin A inhibited activation of store-mediated Ca(2+) entry but did not alter this mechanism once initiated. These data suggest that store-mediated Ca(2+) entry in platelets may be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, which shows close parallels to the events mediating secretion.
Highlights
In many cell types, including human platelets, depletion of the intracellular Ca2ϩ stores induces entry of Ca2ϩ across the plasma membrane (PM)1 [1]
We report here that modification of the actin cytoskeleton by stabilizing actin filaments prevents store-mediated Ca2ϩ entry (SMCE) without affecting chemical coupling mediated by InsP3; this modification has no effect once Ca2ϩ entry has been activated by depletion of the stores
Pretreatment of human platelets with 10 M cytochalasin D (Cyt D), a widely utilized membrane-permeant inhibitor of actin polymerization which binds to the barbed end of actin filaments [26], prevents TG-evoked actin filament formation in a time-dependent manner, reaching complete inhibition after 40 min of treatment (Table I)
Summary
(Received for publication, October 18, 1999, and in revised form, December 8, 1999). Juan A. In support of this hypothesis, small GTP-binding proteins, which modulate actin reorganization and vesicular trafficking, have been shown to be important for store-mediated Ca2ϩ entry (SMCE) in different cell types (e.g. 12, 13) including platelets [14]. We report here that modification of the actin cytoskeleton by stabilizing actin filaments prevents SMCE without affecting chemical coupling mediated by InsP3; this modification has no effect once Ca2ϩ entry has been activated by depletion of the stores These results suggest that under these conditions actin filaments act as a physical barrier that prevents a close interaction between the ER and the PM. May need mechanical support, is more likely than the incorporation of channels into the PM by vesicle fusion
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
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
