Regulation of noradrenaline release by presynaptic receptor systems.

This lecture summarizes the knowledge in a field dubbed autopharmacology: the secretion by a body cell of a substance prompted by an internal stimulus. The majority of the work reported on here was with the adrenal chromaffin cell. Topics covered describing and proving the release of secretion into the chromaffin cells extracellular environment are means of intracellular recording from chromaffin cells and the effect of acetylcholine on membrane potential and consequences of exposing the cell to acetylcholine (altered membrane chemistry increased membrane permeability entry of sodium and calcium ions and depolarization). Also postulated are 2 possible mechanisms of catecholamine secretion (both involve release directly from chromaffin granules to the cell exterior through frank rupture of the adherent membranes of granule and cell i.e. exocytosis or through some greatly increased permeability at the adhesion site). The lecture then focuses on calcium and stumulus secretion coupling in other systems including the exocrine glands neurones and neurosecretory cells the mast cell and polymorphonuclear leukocytes. In a concluding discussion on the role of calcium in secretory mechanisms the presence of prostaglandin in effluent from adrenal glands exposed to acetylcholine is mentioned as evidence of the secretory mechanism being a chemical reaction.

Stimulus-secretion coupling: the concept and clues from chromaffin and other cells.

Gonadotropin binding and stimulation of cyclic adenosine 3': 5' monophosphate and testosterone production in isolated Leydig cells

Addition of ruthenium red to mitochondria isolated from brain, adrenal cortex, parotid gland and skeletal muscle inhibits the further uptake of Ca2+ by these mitochondria but induces little or no net Ca2+ efflux; the further addition of Na+, however, induces rapid efflux of Ca2+. The velocity of the Na+-induced efflux of Ca2+ from these mitochondria exhibits a sigmoidal dependence on the [Na+]. Addition of Na+ to mitochondria exhibiting the most active Na+-dependent efflux of Ca2+ (brain and adrenal cortex) also releases Ca2+ in the absence of ruthenium red and, under these conditions, the mitochondria become uncoupled. It is concluded that the efflux of Ca2+ from these mitochondria occurs via a Na+-dependent pathway, possibly a Na+-Ca2+ antiporter, that is distinct from the ruthenium-red-sensitive carrier that catalyses energy-linked Ca2+ influx. The possible role of the Na+-dependent efflux process in the distribution of Ca2+ between the mitochondria and the cytosol is discussed. In contrast, mitochondria from liver, kidney, lung, uterus muscle and ileum muscle exhibit no Na+-dependent efflux of Ca2+.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1978.tb11993.x

The Interrelations between the Transport of Sodium and Calcium in Mitochondria of Various Mammalian Tissues

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

Studies were carried out on cat adrenal glands perfused in situ to determine the effects of magnesium (Mg2+) and the local anesthetic, tetracaine, on catecholamine secretion and calcium movement. The presence of tetracaine (3 x 10-4 M) or Mg2+(5 x 10-3 M) profoundly inhibited (> 90%) the rate of catecholamine release when calcium (Ca2+) was subsequently added to Ca2+-free Locke's solution or to Ca2+-free Locke's solution containing 56 mM KCl. Inhibition by tetracaine was detectable at concentrations as low as 3 x 10-6 M. The inhibitory effects of tetracaine and Mg2+ were reversed by raising the Ca2+ concentration in the perfusion fluid. Tetracaine, at concentrations as low as 1.5 x 10-6 M, depressed the secretory response to acetylcholine (ACh). When tetracaine and magnesium were employed at concentrations which produced nearly equivalent inhibition of the ACh response, excess Ca2+(8 and 16 mM) was more effective in reversing the Mg2+-induced depression. Both tetracaine (3 x 10-4 M) and Mg2+(5 x 10-3 M) markedly depressed calcium exchange between the gland and the perfusate. Tetracaine also decreased Ca45 uptake resulting from ACh stimulation. It is concluded that tetracaine and Mg2+ inhibit secretion by interfering with Ca2+ entry into the medullary chromaffin cells, presumably by an action at the cell membrane.

Inhibition of catecholamine secretion and calcium exchange in perfused cat adrenal glands by tetracaine and magnesium

1. Isolated cat adrenal glands were perfused with Locke solution, and the corticosteroid outputs in response to adrenocorticotrophin (ACTH) were studied.

2. Steroid outputs varied with the ACTH concentration, as well as with the duration of exposure to a given ACTH concentration.

3. Omission of calcium from the perfusion medium markedly depressed ACTH-evoked steroid release. The steroid output was directly related to the extracellular calcium concentration up to 0·5 mM.

4. During a constant exposure to ACTH, steroid output was maintained for at least 2-3 hr, provided that calcium was present in the perfusion medium.

5. Strontium, but not barium or magnesium, replaced calcium in maintaining the secretory response to ACTH.

6. Magnesium depressed ACTH-evoked secretion in the presence of calcium, and this depression of secretion was antagonized by increasing the calcium concentration.

7. Prolonged perfusion with sodium-free or potassium-free solutions did not markedly inhibit steroid output in response to ACTH. Excess potassium (56 mM) did not produce a consistent or marked increase in spontaneous steroid output and did not affect the response to ACTH.

8. The steroid content of adrenal glands perfused with Locke solution and exposed to ACTH was about 10% of the amount which was secreted. By contrast, adrenal glands perfused with calcium-free media and exposed to ACTH contained much higher amounts of steroid, despite the negligible amount secreted.

9. These data suggest that calcium plays a critical role in the mechanism of corticosteroid secretion from the adrenal cortex.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1970.sp009178

On the mode of action of ACTH on the isolated perfused adrenal gland

1. Cyclic adenosine 3′,5′-monophosphate (cyclic AMP) levels and catecholamine release were measured in cat adrenal glands perfused in situ with Locke solution.



2. Cyclic AMP was present in the medulla in an amount which represented approximately one fifth of that present in the cortex.



3. Perfusion with acetylcholine (ACh) or nicotine increased cyclic AMP both in the intact adrenal and in the perfusate. The time course of the changes in tissue cyclic AMP during stimulation was out of phase with the time course of catecholamine release. Maximal increases in cyclic AMP were not manifest until after 8 min of exposure to the secretogogue, whereas maximal rates of secretion occurred during the first minute.



4. Theophylline (0·5 m M) increased basal and stimulated adrenal cyclic AMP levels, but did not potentiate the secretory response to ACh or nicotine.



5. Perfusion with cyclic AMP or its dibutyryl derivative (0·2–4 m M) failed to effect a consistent or significant increase in the rate of catecholamine release and was unable to potentiate the secretory response to a submaximal concentration of ACh or calcium.



6. The results suggest that, unlike calcium, cyclic AMP is not a direct mediator of medullary secretion.

Analysis of the role of cyclic adenosine 3',5'-monophosphate in catecholamine release.

Isolated cat adrenal glands were perfused in situ with Locke’s solution. The addition of synthetic adrenocorticotropin (0.4-400 µunits/ml) to the perfusion solution for 40 min caused a sustained rise in both tissue adenosine cyclic 3',5'-phosphate levels and corticosteroid release. After a 5-min exposure to ACTH, tissue cyclic AMP levels fell to control values within 30 min, while corticosteroid release was still near maximum. Theophylline increased both basal and ACTH-stimulated tissue cyclic AMP levels but did not further enhance corticosteroid release. Perfusion with calcium-free or potassium-free Locke's solution increased tissue cyclic AMP levels 3-6 fold but did not augment steroid output. Under these ion-deficient conditions, ACTH produced only a very small additional increment in tissue cyclic AMP concentrations but caused a marked increase in steroid release. The addition of cyclic AMP or its dibutyryl derivative to the perfusion solution for up to 30 min did not increase corticoid output. Cyclic AMP was detected in the adrenal perfusate, and its rate of release was augmented by ACTH and by calcium deprivation. These observations are discussed in light of the concept that an increase in tissue cyclic AMP is not sufficient to trigger steroid release, but that a redistribution of cell calcium produced by ACTH is also required.

ACKNOWLEDGMENT The authors wish to thank Mrs. Marcia Feinberg for her indispensable and enthusiastic assistance.

The role of adrenocorticotropin and calcium in adenosine cyclic 3', 5'-phosphate production and steroid release from the isolated, perfused cat adrenal gland.

1. Experiments were carried out to study the effects of adrenocorticotrophin (ACTH) on calcium distribution in the isolated cat adrenal gland perfused with Locke solution.

2. The addition of ACTH to Locke solution caused a small, but significant, increase in the radiocalcium (45Ca) space and content of the adrenal cortex.

3. The average 45Ca washout curve obtained after exposure to ACTH was significantly displaced above the average washout curve for the control glands during the first 20 min of washout. During the time that the two curves were significantly different, maximum corticosteroid secretion was attained.

4. The rate of 45Ca efflux was slowed after ACTH was added to the perfusion medium.

5. The total calcium content of the cortex was not altered by ACTH.

6. Perfusion with calcium-free Locke caused a rapid decrease in the calcium content of the cortex to about 20% of the control value. Dinitrophenol was required to deplete completely the cortical calcium content during perfusion with the calcium-deprived medium.

7. The results are consistent with the suggestion that a translocation of calcium occurs during stimulation of the cortex by ACTH. The source of the calcium ions needed to support the secretory response to ACTH is probably not the extracellular fluid; but ACTH may shift calcium from a rapidly exchanging to a more slowly exchanging cellular pool.

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/jphysiol.1971.sp009402

R. P. Rubin

Papers

Inhibition of catecholamine secretion and calcium exchange in perfused cat adrenal glands by tetracaine and magnesium

On the mode of action of ACTH on the isolated perfused adrenal gland

Analysis of the role of cyclic adenosine 3',5'-monophosphate in catecholamine release.

The role of adrenocorticotropin and calcium in adenosine cyclic 3', 5'-phosphate production and steroid release from the isolated, perfused cat adrenal gland.

The effect of ACTH on calcium distribution in the perfused cat adrenal gland.