Journal of Dental Research, Vol 76, 744-753, Copyright © 1997 by International & American Associations for Dental Research Online Journals
Effects of calcium and calmodulin on the binding of rat parotid secretion granules to the plasma membrane
D. T. Watkins and S. J. Cooperstein
Department of Anatomy, University of Connecticut Health Center, Farmington 06030, USA.
Since numerous studies suggest that Ca++ and calmodulin may modulate the
fusion of secretion granules to the plasma membrane which takes place in
exocytosis, we have examined the role of calcium and calmodulin in the
binding of isolated parotid secretion granules to plasma membrane vesicles.
125I-labeled inside-out plasma membrane vesicles were incubated with
secretion granules, the mixture was layered over 20% sucrose, the gradient
was centrifuged, and the amount of 125I in the granule pellet was
determined. Addition of Ca++ (20 nM to 10 microM) produced a
concentration-dependent increase in the binding of 125I-labeled plasma
membrane vesicles to the secretion granules, reaching a maximum value at 10
microM free Ca++; half-maximal binding occurred at 400 nM. Neither
right-side-out parotid plasma membrane vesicles nor inside-out pancreatic
islet plasma membrane vesicles bound to granules in the presence of 1
microM Ca++. Calmodulin produced a concentration-dependent increase in
binding above that of Ca++ alone, and this effect was inhibited by the
calmodulin antagonists, trifluoperazine and calmidazolium. Incubation of
secretion granules with octadecylrhodamine B (R18)-loaded inside-out plasma
membrane vesicles and 2 microM Ca++ caused de-quenching of fluorescence,
indicating that the lipids in the granule membrane and the plasma membrane
had intermixed. Added calmodulin increased the fluorescence two-fold above
that with Ca++ alone. These results suggest that Ca++ and calmodulin may
play a role in parotid gland exocytosis by modulating the interaction
between the secretion granules and plasma membrane.
