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Abstract

Previous studies have shown that hormonal activation of the Cl⁻ conductance in pancreatic zymogen granules (ZG) is closely related to enzyme secretion from acinar cells. We have now examined the role of guanine nucleotides in stimulated and unstimulated protein secretion from isolated digitonin‐permeabilized pancreatic acini and in the Cl⁻ conductance of isolated ZG. Protein secretion from permeabilized isolated acini, measured at 0.1 mM Ca²⁺, increased with increasing cholecystokinin octapeptide (CCK‐8) concentrations and decreased at high CCK‐8 concentrations. The maximum secretion, approximately twice the control level, was reached at 1 nM CCK‐8. The CCK analog, CCK JMV‐180, which supposedly acts as an agonist on high‐affinity CCK receptors and as an antagonist on low‐affinity CCK receptors, stimulated maximum enzyme secretion at a CCK JMV‐180 concentration of 0.1 μM and no decrease in secretion was observed at higher CCK JMV‐180 concentrations. 0.1 mM guanosine 5′‐[γ‐thio]triphosphate (GTP [S]) also increased the protein release by approximately twice that of the control and shifted the CCK‐8 concentration causing maximum stimulation from 1 nM to 0.01 nM. GTP[S] concentrations greater than 0.1 mM inhibited protein release evoked by an optimal concentration of 1 nM CCK‐8. 0.1 mM GTP[S] had no pronounced effect on the protein secretion stimulated by low concentrations of CCK JMV‐180, but inhibited protein secretion evoked by CCK JMV‐180 concentrations greater than 0.1 μM. This indicates that guanosine‐nucleotide‐binding proteins [G protein(s) coupling to CCK receptors also mediate both CCK‐induced increases and CCK‐induced decreases of enzyme secretion at low and high CCK concentration, respectively.

ZG were prepared on a Percoll gradient from CCK‐8‐stimulated or CCK‐JMV‐180‐stimulated and unstimulated acini. Their Cl⁻ conductances were estimated in the absence of Ca2+ and in the presence of 1 mM EGTA from the rate of decrease in absorbance following addition of the K⁺ ionophore valinomycin as a measure of ZG osmotic lysis. The Cl⁻ conductance in ZG from CCK‐8‐stimulated and CCK‐JMV‐180‐stimulated acini was maximally activated at 1 pM and 10 nM respectively. At higher agonist concentrations, Cl⁻ conductance was decreased. Direct addition of 10 μM GTP[S] to isolated ZG from unstimulated acini increased the rate of lysis by approximately 40% of the control value. This effect was approximately additive to that of CCK‐8 or of CCK JMV‐180 prestimulation. In the presence of Ca²⁺ concentrations greater than 1 μM, the ZG lysis rate was increased by approximately 30% of the control value and this effect was additive to the effect of CCK‐8 prestimulation, but not additive to GTP[S]‐stimulated ZG lysis.

We conclude from our data that G proteins in the plasma membrane Gp and in the ZG membrane GE are involved in stimulated enzyme secretion. In the presence of GTP, activated GE couples to a Cl⁻ channel in the ZG membrane, which leads to an increase in Cl⁻ conductance, an influx of Cl⁻, cations and H₂O into the ZG and flushing of stored enzymes over the ZG membrane which is fused to the luminal cell membrane. Inhibition of enzyme secretion by supramaximal CCK concentrations is reflected by a decrease in Cl⁻ conductance. The mechanism of this inhibition is not known. It probably involves messengers or metabolites generated at the plasma membrane by CCK‐induced signal transduction, rather than an inhibitory G protein in the ZG membrane.