Edward W. Westhead

Lipid Composition and Orientation in Secretory Vesiclesa

The exocytotic release of hormones and neurotransmitters from subcellular storage vesicles begins with the elevation of cytosolic calcium concentration and ends with the storage vesicle open to the cell exterior through fusion of the vesicle membrane with the plasma membrane. No one step in the intervening process is understood at the molecular level. The function, if not the intention, of this report will be to focus attention on how little we know about the role of the membrane lipids in the fusion of the two membranes. The fusion of two membranes requires an extreme reordering of the opposing lipid bilayers, very likely through intermediate formation on micelle-like structures. During reordering, lipids in the cytoplasmic monolayers which first make contact would form spherical inverted micelles with polar head groups in the centers of the spheres. Fatty acid chains would be in contact with neighboring spheres and with the fatty acid chains of the more distant monolayers of the two membranes. Evidence for the existence of such inverted micelles was developed through the study of artificial lipid bilayers. It was found that some phospholipids form only bilayers but that others are more likely to form the “hexagonal phase 11” composed of inverted micellar structures as proposed for the membrane fusion intermediate. Phosphatidyl ethanolamine and phosphatidyl serine alone tend to form the hexagonal phase rather than bilayers, and cholesterol also favors reordering of the bilayer.‘ The process of fusion is also expected to be favored by a high degree of membrane fluidity, which is determined largely by the content of unsaturated fatty acids. The steric irregularity of the chain which is caused by double bonds lowers van der Waals contacts and reduces cohesion of the lipid phase. The presence in a membrane of lysolecithin (or other lysolipids) will cause dislocations in the lipid array and add a relatively hydrophilic element to the lipid phase. Addition of lysolecithin to a suspension of cells or lipid vesicles can promote fusion between them? Free fatty acids too, if present in a membrane, should tend to induce rearrangement of membrane structure-after all, at physiological pH they are soap. Glycolipids or gangliosides on the outer surfaces of cells serve as receptors or surface recognition markers? Since the sialic acid portions of the carbohydrate have a high affinity for Ca” ion, gangliosides might play a role in exocytosis as membrane recognition sites or through their interaction with Cd+. The adrenal medulla has a ganglioside content almost as high as brain gray matter and much of that content is

Inhibition of Catecholamine Secretion from Bovine Chromaffin Cells by Adenine Nucleotides and Adenosine

Abstract: ATP, ADP, and adenosine have been found to inhibit acetylcholine‐stimulated secretion from isolated cells of bovine adrenal medulla (chromaffin cells). Maximal inhibition is −30% under the conditions studied; half‐maxmal inhibition occurs at nucleotide concentration in the micromolar range. Cells must be incubated with ATP for ‐90 s for maximal inhibition, but inhibition by adenosine occurs much faster, an observation suggesting the possibility that ATP and ADP exert their effect after being converted to adenosine. Experiments with cells preloaded with the fluorescent calcium chelator quin 2 indicate that external ATP can diminish the rise in cytosolic Ca2+ concentration that follows stimulation by acetylcholine.

Adenosine Receptors Activate Adenylate Cyclase and Enhance Secretion from Bovine Adrenal Chromaffin Cells in the Presence of Forskolin

Abstract: Cells of the adrenal medulla release not only catecholamines but also high concentrations of neuropeptides and nucleotides. Chromaffin cells, like many neuronal cells, have a diversity of receptors: adrenergic receptors, peptide receptors, histamine receptors, and dopamine receptors. We recently reported that these cells have nucleotide receptors that can mediate inhibition of the secretory response. The present studies show that adenosine, in the presence of enabling concentrations of forskolin, can potently enhance response to nicotinic stimulation. Neither adenosine nor forskolin alone produces a significant effect. A marked rise in intracellular cyclic AMP (cAMP) concentration is associated with the enhancement of secretion caused by forskolin plus adenosine. A phosphodiesterase inhibitor, Ro 20–1724, used together with forskolin produces significant increases in both cellular cAMP content and catecholamine secretion. However, the adenosine agonist 5′‐N‐ethylcarboxyadenosine elevates cellular cAMP content in the presence of forskolin without having any positive effect on secretion. This finding suggests that the rise in cAMP level may not be the sole cause of the increase in secretion by adenosine.

Okadaic acid, a protein phosphatase inhibitor, inhibits nerve growth factor-directed neurite outgrowth in PC12 cells

Abstract: The biochemical mechanisms involved in neurite outgrowth in response to nerve growth factor (NGF) have yet to be completely resolved. Several recent studies have demonstrated that protein kinase activity plays a critical role in neurite outgrowth. However, little information exists about the role of protein phosphatases in the process. In the present study, okadaic acid, a phosphatase inhibitor (specific for types 2A and 1) and tumor promoter, was used to investigate the role of protein phosphatases in neurite outgrowth in PC12 cells. PC12 cells cultured in the presence of 50 ng/ml of NGF started to extend neurites after 1 day. After 3 days, 20–25% of the cells had neurites. Okadaic acid inhibited the rate of neurite outgrowth elicited by NGF with an IC50, of sim7 nM. This inhibition was rapidly reversed after washout of okadaic acid. Okadaic acid also enhanced the neurite degeneration of NGF‐primed PC12 cells, indicating that continual phosphatase activity is required to maintain neurites. Taken together, these results reveal the presence of an okadaic acid‐sensitive pathway in neurite outgrowth and imply that protein phosphatase plays a positive role in regulating the neuritogenic effects of NGF.

Flow-injection analysis of catecholamine secretion from bovine adrenal medulla cells on microbeads

Bovine adrenal medullary cells have been cultured on microbeads which are placed in a low-volume flow system for measurements of stimulation-response parameters. Electronically controlled stream switching allows stimulation of cells with pulse lengths from 1 s to many minutes; pulses may be repeated indefinitely. Catecholamines secreted are detected by an electrochemical detector downstream from the cells. This flow-injection analysis technique provides a new level of sensitivity and precision for measurement of kinetic parameters of secretion. A manual injection valve allows stimulation by higher levels of stimulant in the presence of constant low levels of stimulant. Such experiments show interesting differences between the effects of K+ and acetylcholine on cells partially desensitized to acetylcholine.

Inhibition of yeast phosphoglycerate kinase by lanthanide—ATP complexes

The use of lanthanide cations as probes is assuming increasing importance in high resolution nuclear magnetic resonance structural studies of proteins [l] and nucleotides [2] , both with and without metal cofactor requirements. We describe accordingly in this report steady state kinetic investigations into the binding of lanthanide (Ln)-ATP to yeast phosphoglycerate kinase (PGK) (3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3.) in presence of the substrates Mg-ATP and 3-phosphoglycerate. These studies are intended as a complement to our n.m.r. investigations of this enzyme and together with earlier results [3] , will be necessary for the correct interpretation of lanthanide shifts and broadenings in the n.m.r. spectra of PGK-substrate complexes [4] . In this work La-ATP-was found to be an inhibitor competing with Mg-ATP’ ; similar results being obtained when Eu, Pr, and Yb were substituted for La. Direct binding determinations using Sephadex, and also an n.m.r. spectral titration, provided information about the number of binding sites for metalATP.

Two Distinct ATP Receptors Activate Calcium Entry and Internal Calcium Release in Bovine Chromaffin Cells

Abstract: ATP, an established neurotransmitter, causes elevation of cytosolic Ca2+ and catecholamine secretion when applied to chromaffin cells in the intact adrenal gland. The ATP‐induced rise in Ca2+ is due both to release from internal stores and to entry across the plasma membrane. The latter source of Ca2+ causes secretion; the primary role of Ca2+ released from internal stores remains undetermined. In this article, we have studied the nucleotide specificity for activating the two types of Ca2+ increases. The agonist potency order for the increase in fluorescence from fura‐2‐loaded chromaffin cells due to release of Ca2+ from internal stores is ATP = UTP > ADP > 2‐methylthio‐ATP, α,β‐methylene ATP, identifying the receptor as a P2U purinoceptor. The potency order for secretion is 2‐methylthio‐ATP > ATP > α,β‐methylene ATP, ADP, UTP, placing the receptor in the P2Y subtype. Thus, two distinct receptors are responsible for Ca2+ release and secretion. Agonists were more effective in the absence of extracellular Mg2+, suggesting that ATP uncomplexed with divalent cations binds preferentially to both receptors. The low response of both receptors to ADP distinguishes them from the ATP receptor on these cells that inhibits voltage‐dependent Ca2+ current and secretion.

ATP stimulated catecholamine secretion: Response in perfused adrenal glands and a subpopulation of cultured chromaffin cells

Extracellular ATP is shown to induce catecholamine secretion in bovine chromaffin cells. Our data indicate that cells in culture gradually increase their response to ATP, and we have separated freshly isolated cells on a density gradient and found that the lighter cells develop a much stronger response to ATP than do the heavier cells. To see if the ATP sensitivity is physiological, we have perfused intact adrenal glands. ATP induces a greater secretory response from glands than does acetylcholine without causing preferential secretion of norepinephrine or epinephrine. These data show that the response to ATP found in cultured cells is not an artifact of cell culture, and that ATP co-released with catecholamines from the storage vesicles may have a significant physiological role.

Accessibility of lysolecithin in catecholamine secretory vesicles to acyl coa: Lysolecithin acyl transferase

Abstract Lysolecithin (monoacylglycerophosphorylcholine) accounts for 13 to 20% of the lipid phosphorous of the bovine adrenal catecholamine secretory vesicles (chromaffin granules). We have incubated purified vesicles with [1-14C] oleyl coenzyme A and rat liver microsomes containing acyl coenzyme A: monoacylglycerophosphorylcholine acyl transferase to determine the accessibility of the granule membrane lysolecithin to another membrane. No acylation of lysolecithin occurs when the chromaffin granules are intact. The accessibility of the granule membrane lysolecithin increases markedly when the vesicles are broken.

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na+/Ca2+ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na+/Ca2+ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na+/Ca2+ exchanger of these cells. Cells treated with 1 mM dibutyryl cyclic AMP (dbcAMP), 1 µM phorbol 12,13‐dibutyrate, 1 µM okadaic acid, or 100 nM calyculin A showed lowered Na+/Ca2+ exchange activity and prolonged cytosolic Ca2+ transients caused by depolarization. A combination of 10 nM okadaic acid and 1 µM dbcAMP synergistically inhibited Na+/Ca2+ exchange activity. Conversely, 50 µM 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine, a protein kinase inhibitor, enhanced Na+/Ca2+ exchange activity. Moreover, we used cyclic AMP‐dependent protein kinase and calcium phospholipid‐dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na+/Ca2+ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na+/Ca2+ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.