In vitro stability of pancreatic zymogen granules: roles of pH and calcium

Abstract

Purified preparations of pancreatic zymogen granules have the peculiar property of lysing instantaneously at neutral pH, a property clearly irreconcilable with the cytoplasmic pH of the acinar cell. Two important factors known for regulating the stability of secretory granules are calcium and pH. Fluorescence microscopy of acinar cells in the presence of weak bases showed that zymogen granules have an acidic pH. In vivo, abolition of the delta pH by NH4Cl did not induce any lysis of the granules. In vitro, with purified granules, an acidic intragranular pH was measured. This delta pH was produced by a Donnan potential. The importance for granule stability of keeping the intragranular pH acidic has been confirmed in vitro by addition of K+ and nigericin to the suspension medium. These conditions produced alkalinization of the granule matrix and caused instantaneous solubilization of the granules. Concentrations of 15 mM total, and 10 mM free calcium were measured in purified granules. The importance of intragranular Ca2+ was evaluated by means of the ionophore A23187 which induced calcium efflux and granule lysis. The lysis induced by the calcium ionophore was in direct relation with the calcium efflux, since addition of Ca2+ to the medium, at concentrations corresponding to that measured in the granule, relieved the effect. The role of calcium-binding sites on the cytoplasmic surface of the granules was investigated with Ca2+, EGTA, and La3+. Calcium did not have any damaging effects; EGTA induced a slight lysis, while lanthanum yielded a strong and spontaneous lysis at micromolar concentrations. In addition to calcium-binding sites, La3+ would bind to specific sites on the granule that would be directly coupled to maintenance of its stability. These findings suggest that the intragranular acidic pH and calcium are both important for the in vitro stability of the zymogen granule and that purified granules have lost, in the course of purification, some cytoplasmic factors that in vivo, control the permeability of the membrane to protons, and chloride more particularly. Calcium-binding sites and other specific sites probed with La3+, presumably on proteins at the surface of the granule, are also believed to have key roles in preserving the integrity of the membrane and the resulting stability of the granule.

Parotid Secretory Granules: Crossroads of Secretory Pathways and Protein Storage

“…Lysis under these conditions is prevented by increased sucrose concentration of the medium, suggesting that the granules undergo osmotic lysis after calcium depletion (Sudhof, 1983). Similarly, exocrine pancreatic secretory granules require calcium for stability (Lebel et al, 1988). In this case, lysis occurred in the presence of A23187, but it did not require an external chelator, suggesting that granular calcium is more loosely bound in pancreatic exocrine granules than in adrenal chromaffin granules.…”

Calcium-dependent enzyme activation and vacuole formation in the apical granular region of pancreatic acinar cells

“…If the ER were empty because of excessive agonist stimulation or blockade of the ER Ca 2ϩ ATPase, refilling of the granular Ca 2ϩ store would not occur (30). Ca 2ϩ is important for the stability of the granules (31,32), and Ca 2ϩ ionophore-induced loss of Ca 2ϩ from ZGs causes granule lysis (31). The vast amount of Ca 2ϩ normally bound inside the granules (12) therefore plays an important role in making the proteins combine to osmotically and chemically inactive aggregates (33).…”

Secretory organelles of Paramecium cells (trichocysts) are not remarkably acidic compartments.

“…A0 is trapped by protonation, thus assuming on uv excitation green (less acidic) to orange (more acidic) fluorescence of increasing intensity (Anderson and Orci, 1988;Maxfield, 1985;Lee et al, 1982). Using A0 or related compounds, an acidic pH could be visualized within different intracellular compartments (Anderson and Orci, 1988;Mellman et al, 1986;Maxfield, 1985), including lysosomes (Allison and Young, 1969) and several secretory organelles, e.g., chromaffin granules (Salama et al, 1980), pancreatic zymogen granules (Lebel et al, 1988;DeLisle and Hbpfer, 1986), parafollicular secretory granules (Barasch et al, 1988), and granules in sea urchin eggs (Lee et al, 1982). However, some other secretory vesicles were found to be only slightly acidic (neurohypophysis: Saermark, 1989) or not acidic at all (parotis: Arvan and Castle, 1986;Arvan et al, 1984).…”