Chemiosmotic Properties of Isolated Secretory Granules from Parotid Glands of Normal and Isoproterenol Treated Rats

Peter Raymond Arvan, Yale University.

This is an Open Access Thesis


Secretory granule fractions were prepared isoosmotically from the parotid glands of normal rats and from rats that had received repeated injections of isoproterenol. These granules, purified extensively, are stable after isolation, making it possible to study biophysical parameters which may relate to their role in intracellular transport and secretion.Normal parotid granules have an internal pH of (TURN)6.8 and a low ionic permeability with respect to internal buffering capacity. By contrast, granules from the treated rats have an internal pH ranging from (TURN)7.7 in (0.3 M) sucrose media to (TURN)7.1 in (100 mM) KCl media; suggesting a large ionic permeability with respect to buffering capacity. These intragranular pH values are the highest reported for any secretory vesicle type. The major content protein within control granules is (alpha)-amylase (pI (LESSTHEQ) 7), while the major species in granules from the treated rats are the proline-rich proteins (pI > 10). Intragranular pH values probably differ because of these compositional differences.Normal parotid granules have a low H('+) permeability and cannot utilize ATP either to acidify their interior or to create an inside-positive membrane potential. ATP-stimulated parotid granule lysis (cited by others as evidence implicating a granule H('+)-ATPase) is mimicked entirely by non-hydrolyzable ATP analogs, and thus most likely does not depend on ATP hydrolysis.Parotid granules isolated from isoproterenol-treated rats, however, exhibit a measurable ATP-dependent acidification which is abolished by proton ionophores. The granules are also able to utilize ATP in creating a more inside-positive membrane potential. These effects depend on ATP hydrolysis.The underlying mechanism which permits the expression of electrogenic H('+)-translocating ATPase activity in these granules (but fails to do so in the case of normal parotid granules) has not yet been found. An hypothesis is presented from which a strategy in approaching this question may be planned.