Lowering pH in blood platelets dissociates myosin phosphorylation from shape change and myosin association with the cytoskeleton.
Platelet shape change induced by ADP is relatively independent of external pH over the range 6-7. If the chloride ion in the buffer is replaced by weak acids, however, shape change is rapidly and reversibly inhibited as a function of lowered pH (92% at pH 6.0). This inhibition is correlated with lowered internal pH caused by the weak acids, as measured by the 5,5-dimethyloxazolidine 2,4-dione technique. Shape change was 50% inhibited at internal pH 6.4 when 50 mM NaCl was replaced by propionate (PR). When platelets were stimulated with ADP 10-20 s after addition of PR to a final pH of 6 (PR6), both myosin light chain (MLC) phosphorylation and myosin and actin association with the cytoskeleton were reduced in correlation with the inhibition of shape change. But when ADP was added 30 s after PR6, the MLC phosphorylation was essentially the same in PR or in chloride, although shape change and myosin and actin association with the cytoskeleton remained inhibited. This was shown to be due mainly to endogenous phosphorylation of MLC. On return to neutral pH, platelets in PR immediately changed shape and myosin and actin became associated with the cytoskeleton. Two-dimensional tryptic peptides of MLC showed two major spots after PR6 treatment, indicating that both the MLC kinase site and the protein kinase C sites were phosphorylated. The results show that increased internal pH is not required for shape change, although it may affect the rate. In PR6, as after phorbol esters, MLC phosphorylation can be uncoupled from shape change. The association of myosin and actin with the cytoskeleton is closely correlated with shape change, suggesting that shape change requires the active interaction of these contractile proteins.
Nachmias
V T
VT
Department of Anatomy, School of Medicine, University of Pennsylvania, Philadelphia 19104.
Yoshida
K
K
Glennon
M C
MC
D001792:Blood Platelets / Q000648:ultrastructure*; D002264:Carboxylic Acids; D003599:Cytoskeleton / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009218:Myosins / Q000502:physiology*; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000502:physiology; D010766:Phosphorylation; D013997:Time Factors
D002264:Carboxylic Acids; D010750:Phosphoproteins; D009218:Myosins