Ruth A. Senter

Relationship Between Ca2+ Uptake and Catecholamine Secretion in Primary Dissociated Cultures of Adrenal Medulla

Abstract: Carbachol or elevated K+ stimulated 45Ca2+ uptake into chromaffin cells two‐ to fourfold. The uptake was stimulated by cholinergic drugs with nicotinic activity, but not by those with only muscarinic activity. Ca2+ uptake and catecholamine secretion induced by the mixed nicotinic‐muscarinic agonist carbachol were inhibited by the nicotinic antagonist mecamylamine, but not by the muscarinic antagonist atropine. Significant Ca2+ uptake occurred within 15 s of stimulation by carbachol or elevated K+ at a time before catecholamine secretion was readily detected. At later times the time course of secretion induced by carbachol or elevated K+ was similar to that of Ca2+ uptake. There was a close correlation between Ca2+ uptake and catecholamine secretion at various concentrations of Ca2+. The concentration dependencies for inhibition of both processes by Mg2+ or Cd2+ were similar. Ca2+ uptake saturated with increasing Ca2+ concentrations, with an apparent Km for both carbachol‐induced and elevated K+‐induced Ca2+ uptake of approximately 2 mM. The Ca2+ dependency, however, was different for the two stimuli. The studies provide strong support for the notion that Ca2+ entry and a presumed increase in cytosolic Ca2+ concentration respectively initiates and maintains secretion. They also provide evidence for the existence of saturable, intracellular, Ca2+‐ dependent processes associated with catecholamine secretion. Ca2+ entry may, in addition, enhance nicotinic receptor desensitization and may cause inactivation of voltage‐sensitive Ca2+ channels.

Effects of Osmolality and Ionic Strength on Secretion from Adrenal Chromaffin Cells Permeabilized with Digitonin

Hyperosmotic solutions inhibit exocytosis of catecholamine from adrenal chromaffin cells at a step after Ca2+ entry into the cells. The possibility that the inhibition resulted from an inability of shrunken secretory granules to undergo exocytosis was investigated in cells with plasma membranes permeabilized by digitonin. The osmoticants and salts used in this study rapidly equilibrated across the plasma membrane and bathed the intracellular organelles. When sucrose was the osmoticant, secretion was not significantly inhibited unless the osmolality was raised above 1,000 mOs. When the osmolality was raised with the tetrasaccharide stachyose or a low‐molecular‐weight maltodextrin fraction (average size a tetrasaccharide), one‐half maximal inhibition occurred at 900–1,000 mOs. Prior treatment of permeabilized cells with Ca2+ in hyperosmotic solution did not result in enhanced secretion when cells were restored to normal osmolality. Increased concentrations of potassium glutamate or sodium isethionate were more potent than carbohydrate in inhibiting secretion. Half‐maximal inhibition occurred at 600–700 mOs or when the ionic strength was approximately doubled. The inhibition by elevated potasium glutamate also occurred when the osmolality was kept constant with sucrose. Increasing the ionic strength did not alter the Ca2+ sensitivity of the secretory response. Reducing the ionic strength by substituting sucrose for salt reduced the Ca2+ concentration required for half‐maximal stimulated secretion from approximately 1.2 μM. Chromaffin granules, the secretory granules, are known to shrink in hyperosmotic solution. The experiments indicate that shrunken chromaffin granules can undergo exocytosis and suggest that in intact cells elevated ionic strength rather than chromaffin granule shrinkage contributes to the inhibition of secretion by hyperosmotic solutions. The experiments place limits on the possible osmotic mechanisms that could be involved in exocytosis.

Energetic Determinants of Tyrosine Phosphorylation of Focal Adhesion Proteins during Hypoxia/Reoxygenation of Kidney Proximal Tubules

Anaerobic mitochondrial metabolism of alpha-ketoglutarate and aspartate or alpha-ketoglutarate and malate can prevent and reverse severe mitochondrial dysfunction during reoxygenation after 60 minutes of hypoxia in kidney proximal tubules.(34) The present studies demonstrate that, during hypoxia, paxillin, focal adhesion kinase, and p130(cas) migrated faster by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their phosphotyrosine (pY) content decreased to approximately 5% of that in oxygenated tubules without changes in total protein, and the normally basal immunostaining of beta1 and alpha6 integrin subunits, pY, and paxillin was lost or markedly decreased. During reoxygenation without supplemental substrates, recovery of pY and basal localization of the focal adhesion proteins was poor. alpha-Ketoglutarate and aspartate, which maintained slightly higher levels of ATP during hypoxia, also maintained 2.5-fold higher levels of pY during this period, and promoted full recovery of pY content and basal localization of focal adhesion proteins during subsequent reoxygenation. Similarly complete recovery was made possible by provision of alpha-ketoglutarate and aspartate or alpha-ketoglutarate and malate only during reoxygenation. These data emphasize the importance of very low energy thresholds for maintaining the integrity of key structural and biochemical components required for cellular survival and reaffirm the value of approaches aimed at conserving or generating energy in cells injured by hypoxia or ischemia.

Plasma Membrane and Chromaffin Granule Characteristics in Digitonin-Treated Chromaffin Cells

Abstract: Digitonin permeabilizes the plasma membranes of bovine chromaffin cells to Ca2+, ATP, and proteins and allows micromolar Ca2+ in the medium to stimulate directly catecholamine secretion. In the present study the effects of digitonin (20 μM) on the plasma membrane and on intracellular chromaffin granules were further characterized. Cells with surface membrane labeled with [3H]galactosyl moieties retained label during incubation with digitonin. The inability of digitonin‐treated cells to shrink in hyperosmotic solutions of various compositions indicated that tetrasaccharides and smaller molecules freely entered the cells. ATP stimulated [3H]norepinephrine uptake into digitonin‐treated chromaffin cells fivefold. The stimulated [3H]norepinephrine uptake was inhibited by 1 μM reserpine, 30 mM NH4+, or 1 μM carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP). The data indicate that [3H]norepinephrine was taken up into the intracellular storage granules by the ATP‐induced H+ electrochemical gradient across the granule membrane. Reduction of the medium osmolality from 310 mOs to 100 mOs was required to release approximately 50% of the catecholamine from chromaffin granules within digitonin‐treated chromaffin cells which indicates a similar osmotic stability to that in intact cells. Chromaffin granules in vitro lost catecholamine when the digitonin concentration was 3 μM or greater. Catecholamine released into the medium by micromolar Ca2+ from digitonin‐treated chromaffin cells that had subsequently been washed free of digitonin could not be pelleted in the centrifuge and was not accompanied by release of membrane‐bound dopamine‐β‐hydroxylase. The studies demonstrate that 20 μM of digitonin caused profound changes in the chromaffin cell plasma membrane permeability but had little effect on intracellular chromaffin granule stability and function. It is likely that the intracellular chromaffin granules were not directly exposed to significant concentrations of digitonin. Furthermore, the data indicate that during catecholamine release induced by micromolar Ca2+, the granule membrane was retained by the cells and that catecholamine release did not result from release of intact granules into the extracellular medium.

Regulated exocytotic fusion I : chromaffin cells and PC12 cells

Publisher Summary The focus of this chapter is the use of the detergent digitonin in the study of secretion from primary cultures of bovine chromaffin cells and the continuous cell line PC12 (rat adrenal pheochromocytoma), both of which store and secrete catecholamine. Digitonin gives reproducible results, is easy to use, and permits exogenous proteins to enter the cells. High-voltage discharges caused dielectric breakdown of the plasma membrane of suspended chromaffin cells and platelets, thus rendering the membranes permeable to small salts and nonelectrolytes. The studies demonstrated the central role of Ca 2+ in stimulating exocytosis and the necessity of ATP as a cofactor. Subsequently, a variety of other techniques were developed to permeabilize selectively the plasma membrane, many of which were suitable for use with monolayer cells. This chapter compares some of the methods of permeabilization that are commonly used. The preparation and maintenance of adrenal medullary chromaffin cells in vitro are reviewed.

Effects of trypsin on secretion stimulated by micromolar Ca2+ and phorbol ester in digitonin-permeabilized adrenal chromaffin cells.

Summary1.Catecholamine secretion from digitonin-treated chromaffin cells is stimulated directly by micromolar Ca2+ in the medium. The permeabilized cells are leaky to proteins.2.In this study trypsin (30–50µg/ml) added to cells after digitonin treatment completely inhibited subsequent Ca2+-dependent catecholamine secretion. The same concentrations of trypsin did not inhibit secretion from permeabilized cells if trypsin was present only prior to cell permeabilization.3.The data indicate that trypsin entered digitonin-treated chromaffin cells which were capable of undergoing secretion and that an intracellular, trypsinsensitive protein is involved in secretion. Chymotrypsin was less potent but had effects similar to those of trypsin.4.The enhancement of Ca2+-dependent secretion from permeabilized chromaffin cells induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by trypsin added simultaneously with Ca2+ to permeabilized cells at concentrations (3–10µg/ml) which had little or no effect on Ca2+-dependent secretion from cells untreated with TPA. Ca2+-dependent secretion in TPA-treated cells was reduced by trypsin only to the level that would have occurred in cells not treated with TPA. Trypsin reduced the large TPA-induced increment of membrane-bound protein kinase C.5.The data indicate that Ca2+-dependent secretion in the absence of TPA does not require aTPA-like effect of Ca2+ to activate protein kinase C. Protein kinase C activation by TPA probably enhances Ca2+-dependent secretion by modulating the normal Ca2+-dependent pathway or by activating another Ca2+-dependent pathway which functions in parallel to the normal pathway.

Investigation by transient transfection of the effects on regulated exocytosis of Rab3a.

Publisher Summary Techniques have been developed to render the plasma membrane of secretory cells permeable to ions and proteins while still maintaining the secretory response to micromolar Ca 2+ . These techniques have successfully determined the role of various soluble factors in exocytosis and have allowed a careful analysis of steps in the secretory pathway. A key part of the technique is the transfection of an effector plasmid of interest together with a reporter plasmid encoding human growth hormone (GH). GH is targeted to the regulated secretory pathway. Adrenal chromaffin cells contain secretory granules (chromaffin granules) that contain catecholamine, ATP, opiate peptides, and a variety of proteins, including chromogranins and opiate peptide precursors. The expressed GH is stored in chromaffin granules and serves as a reporter for the secretory pathway in the transfected cells. The measurement of catecholamine secretion is predominantly a measure of secretion from nontransfected cells as they represent the vast majority of the cells on the dish.

Mitochondrial dysfunction induced by pancreatic and crotalic (Crotalus durissus terrificus) phospholipases A2 on rabbit proximal tubules suspensions.

In the present study we show that phospholipases A2 isolated from porcine pancreas (PP-PLA2) and Crotalus durissus terrificus snake venom (SV-PLA2) induced dose-dependent increases of LDH release from rabbit proximal tubules in suspension. Both porcine and crotalic PLA(2)s induced increases in non-esterified fatty acid (NEFA) levels (microg of NEFA/mg of tubule protein). It was observed that the NEFA levels in the pellets were higher than in the supernatant for both PLA2, and were dose-dependent for the crotalic PLA2 group. Furthermore, snake venom PLA2 induced a decrease in mitochondrial membrane potential (DeltaPsi(m)) assessed by both JC-1 uptake and safranin O uptake. Porcine PLA2 produced no effects on JC-1 uptake with the highest concentrations and an unexpected increase in the group treated with the lowest concentration. In contrast, the safranin O method revealed decreases of energization with both phospholipases, so it had higher sensitivity to the presence of the increased NEFA levels. Addition of delipidated bovine serum albumin (dBSA) completely reversed the effects induced by phospholipases on DeltaPsi(m) measured with safranin O. Incubation with pancreatic and crotalic phospholipases A2 produced no changes on cell ATP levels. We conclude that the treatment of proximal tubule suspensions with porcine or crotalic phospholipases disturbed membrane integrity as well as mitochondrial function. Specific early NEFA-mediated mitochondrial effects of the phospholipases used in the present study are indicated by the benefit provided by dBSA.