Inositol phospholipids and cell surface receptor function.

Isolated pancreatic acini were prepared by a new method from mouse and rat pancreases by digestion with purified collagenase and chymotrypsin followed by mechanical shearing. Acini were structurally similar to those of the intact pancreas, having a normal luminal structure but with the basal acinar cell membranes exposed to the incubation medium. Amylase release in response to both cholinergic analogues and the cholecystokinin analogues caerulein and pentagastrin was comparable to that of the intact pancreas, but was much greater than previously reported for isolated acinar cells. Cholinergic-stimulated release was inhibited by atropine with a Ki value of 1.4 nM which is comparable to other muscarinic receptors. All agonists tested, when added at supramaximal concentrations, produced a submaximal release of amylase even though ATP levels and the release of slowly exchanging 45Ca2+ were normal or increased. Acini releasing amylase submaximally after being exposed to supramaximal concentrations of carbachol failed to respond to a maximal amount of caerulein or to the Ca2+ ionophore A23187. It is concluded that the decreased response (desensitization) is a postreceptor phenomenon and possibly mediated by Ca2+ itself.

https://www.physiology.org/doi/pdf/10.1152/ajpendo.1978.235.5.E517

Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini.

SUMMARY
1After an outline description of pancreatic structure and function, and a more detailed account of acinar cell morphology, this review traces the pathway of amino acids as they are taken up by the acinar cell, incorporated into digestive hydrolases, transported through the cell and finally discharged from the cell, and considers the mechanisms by which these steps are controlled. At all stages comparisons are made with other secretory cells.
2The use of radioautography and cell-fractionation techniques in determining this pathway in the pancreas are described. The route and kinetics of the process in pancreas are compared with those in other cells.
3Amino-acid entry is by an active mechanism. However the intracellular pool of accumulated amino acids may not be used directly in protein synthesis. Selection of amino acids for incorporation into proteins may occur whilst they are associated with carrier systems within the plasma membrane. There is no convincing evidence that amino-acid entry can be influenced by the pancreatic secretagogues, cholecystokin-pancreazymin (CCK-PZ) or acetylcholine.
4Secretory proteins are synthesized on ribosomes bound to the endoplasmic reticulum (ER) and the nascent proteins vectorially transferred across the ER membrane into the ER cisternae. All messenger RNA molecules which are templates for secretory proteins appear to possess an initial sequence of codons whose translation produces a ‘signal’ sequence of amino acids. This signal sequence somehow triggers attachment of the ribosomes to the ER, thereby automatically determining that the final translation product is destined for the ER cisternae.
5The effects of CCK-PZ and acetylcholine on pancreatic protein synthesis are controversial. Whereas stimulation can be observed in vivo, this has not been convincingly demonstrated in vitro. I conclude that while CCK-PZ and acetylcholine may accelerate protein synthesis, the physiological significance of this effect remains to be clarified. Long-term stimulation can modify pancreatic enzyme synthesis and this, together with other factors, may be the means of dietary adaptation by the gland.
6Newly synthesized proteins travel from the ER cisternae via the peripheral Golgi components to the Golgi cisternae. Transport from ER to Golgi cisternae may occur by a vesicle shuttle service or by direct tubular connexions. Although sustained stimulation with CCK-PZ analogues can accelerate this intracellular transport step, pancreatic secretagogues have not yet been shown to accelerate transport under physiological conditions.
7The Golgi complex has a number of functions including: glycosylation and, where appropriate, sulphation of glycoprotein and mucopolysaccharide components of the zymogen granules (ZG) and granule membranes; sequestration of divalent cations which bind to secretory proteins; the formation of condensing vacuoles (CV) from the inner Golgi cisternae.
8Aggregation of proteins occurs passively within CV so as to form osmotically inert complexes, thereby reducing internal osmotic activity and causing water to diffuse out. This condensation imparts a gel-like consistency to the mature ZG so formed.
9Discharge of ZG occurs by a process of exocytosis involving fusion of the ZG membrane with the apical plasma membrane, release of the ZG contents, and retrieval of the ZG membrane from the plasma membrane by endocytotic mechanisms. The mechanisms responsible for migration of ZG towards the cell apex and for exocytosis remain unknown but may involve the participation of microtubules and/or microfilaments. Although there is a small, basal discharge of ZG at all times, stimulation with CCK-PZ or acetylcholine greatly accelerates the process.
10The basic tenet of the secretory mechanism summarized above is that, following synthesis, secretory proteins are confined within an intracellular organelle at all times. This ‘segregation’ hypothesis has been challenged by the ‘equilibrium’ hypothesis in which secretory proteins are suggested to move across cellular membranes and are therefore at equilibrium within the various compartments of the cell. While many of the observations on which the equilibrium hypothesis are based are tenuous, some others cannot readily be explained by the segregation model. Proponents of the equilibrium hypothesis therefore suggest that preferential release of individual hydrolases from ZG occurs, followed by their separate transport across the apical cell membrane. The claims of this alternative model are discussed.
11In the final section are discussed the intracellular mechanisms by which CCK-PZ and acetylcholine act on the acinar cell to cause discharge. The overall membrane perturbations brought about by CCK-PZ and acetylcholine appear to be the same and include cell depolarization, and perhaps increased phospholipid turnover. Both events may be related to an altered membrane permeability to cations. CCK-PZ, but not acetylcholine, will activate adenylate cyclase, but cyclic AMP does not appear to be involved in regulating enzyme discharge. Instead, Ca2+ is the major intracellular second messenger. However, rather than increase Ca2+ uptake into the cell, CCK-PZ and acetylcholine appear to raise the intracellular Ca2+ concentration by causing release of Ca2+ from intracellular stores. The mechanism by which they do this, and the role of Ca2+ in the discharge process remain unknown.

Synthesis, intracellular transport and discharge of exportable proteins in the pancreatic acinar cell and other cells

1. The role of extracellular and intracellular Ca(2+) in pancreatic enzyme secretion has been assessed by correlating the exchange of (45)Ca with amylase secretion in the isolated uncinate pancreas of baby rats.2. The rate coefficient of (45)Ca efflux from pre-loaded glands declined continually (indicating that (45)Ca is retained in several different pools) and probably reflects changes in the concentration of cytoplasmic free (45)Ca, which is determined by the rate at which (45)Ca is released from intracellular organelles into the cytoplasm.3. The rate coefficient of (45)Ca release was not influenced by extracellular Ca(2+) or Mg(2+) concentrations.4. Cholecystokinin-pancreozymin (CCK-PZ) and acetylcholine accelerated the release of both (45)Ca and amylase in a dose-dependent fashion, even when extracellular Ca(2+) was reduced to 0.1 mM, but did not affect the initial rate of (45)Ca uptake by the tissue.5. In Ca(2+)-free media (containing 0.5 mM-EGTA) basal amylase secretion slowly declined and stimulated secretion was virtually abolished, but the accelerated release of (45)Ca was maintained.6. These observations indicate that natural stimuli of pancreatic enzyme secretion alter (45)Ca distribution in the cell by a process which is independent of extracellular Ca(2+) and which is associated with amylase secretion provided that the plasma membrane has not been depleted of Ca(2+).7. Secretin, glucagon and insulin did not influence (45)Ca release. Secretin slightly increased amylase secretion, but this may have been a washout effect.8. Replacement of extracellular Na(+) by Li(+) increased the release of (45)Ca and amylase, but only in the presence of extracellular Ca(2+). Li(+)-substitution also increased (45)Ca uptake. Thus, under special conditions, secretion may be stimulated when increased amounts of Ca(2+) are made available from extracellular sources.9. Hyperosmolarity (known to increase (45)Ca release in muscle) also accelerated (45)Ca release and amylase secretion.10. 2,4-Dinitrophenol markedly accelerated (45)Ca efflux but did not stimulate amylase secretion, indicating that a rise in cytoplasmic Ca(2+) will not initiate secretion if energy metabolism is impaired.11. CCK-PZ slightly increased the rate coefficient of (42)K release, indicating a changed membrane permeability.12. The stimulatory effects of CCK-PZ and acetylcholine were suppressed during Na(+)-substitution by Li(+), suggesting that the Na(+) concentration gradient across the membrane is important in secretion.13. It is concluded that the primary action of CCK-PZ and acetylcholine may be to increase the influx of Na(+) into the cell by changing membrane permeability. This in turn is responsible for the release of Ca(2+) from intracellular stores (probably endoplasmic reticulum), leading to a rise in Ca(2+) concentration close to the structures involved in enzyme secretion. Secretion then follows provided that ATP is available and the plasma membrane is not depleted of Ca(2+).

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1973.sp010379

L. Rochus

Papers

Role of cyclic 3′,5t́-AMP in the action of physiological secretagogues on the metabolism of rat pancreas in vitro