Control of neurosecretion and synthesis of neurotransmitters appears to be regulated through second messengers that change the phosphorylation state of critical enzymes and proteins. Relatively little is known about the role of protein kinases in these processes in neuronal cells and even less is known about the role of protein phosphatases. We have examined the phosphatase activities of the bovine adrenal medulla using chromatographic separation, substrate specificity and inhibitor sensitivity. When fractionated, using an HPLC ion exchange DEAE column, four distinct peaks (peaks I,II,III and IV) of phosphatase activity were observed in the supernatant of homogenized bovine adrenal medulla. These phosphatases have distinctly different specific activities toward different substrates. Peak IV which contains most of the activity toward phosphocasein, showed preferential dephosphorylation of the α subunit of phosphorylase kinase relative to the β subunit and was strongly inhibited by okadaic acid, attributes of the type 2A phosphatase. The apparent molecular weight of phosphatase peak IV is also comparable to the heterotrimeric form of the known protein phosphatase type 2A of mammalian cells.

Annals of the New York Academy of SciencesVolume 779, Issue 1 p. 395-396 Agents That Promote Protein Phosphorylation Increase Catecholamine Secretion and Inhibit the Activity of the Na+-Ca2+ Exchanger in Bovine Chromaffin Cells L. F. LIN, L. F. LIN Program in Molecular and Cellular Biology University of Massachusetts Amherst, Massachusetts 01003Search for more papers by this authorL-S. KAO, L-S. KAO Institute for Biomedical Science Academia Sinica TaiwanSearch for more papers by this authorE. W. WESTHEAD, Corresponding Author E. W. WESTHEAD Program in Molecular and Cellular Biology University of Massachusetts Amherst, Massachusetts 01003Corresponding author.Search for more papers by this author L. F. LIN, L. F. LIN Program in Molecular and Cellular Biology University of Massachusetts Amherst, Massachusetts 01003Search for more papers by this authorL-S. KAO, L-S. KAO Institute for Biomedical Science Academia Sinica TaiwanSearch for more papers by this authorE. W. WESTHEAD, Corresponding Author E. W. WESTHEAD Program in Molecular and Cellular Biology University of Massachusetts Amherst, Massachusetts 01003Corresponding author.Search for more papers by this author First published: April 1996 https://doi.org/10.1111/j.1749-6632.1996.tb44811.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume779, Issue1Sodium–Calcium Exchange: Proceedings of the Third International ConferenceApril 1996Pages 395-396 RelatedInformation

Three phosphodiesterase (PDE) isoenzymes were separated by Mono Q h.p.l.c. column chromatography from the soluble fraction of a homogenate of pig aortic smooth muscle cells. The first peak of PDE activity was stimulated by calmodulin in the presence of calcium. The second broad peak contained at least two activities, which were sensitive to inhibition by CI-930 or rolipram respectively. The distribution of total cellular enzyme activity in different subcellular fractions was also determined. The majority (78%) of the total activity was present in the cytosolic fraction, 18% of activity was in a membrane-bound form and 4% of activity was associated with the cytoskeleton. Rolipram-sensitive PDE was present predominantly in the cytosolic fraction, whereas cyclic GMP-inhibited, CI-930-sensitive PDE was evenly distributed between the cytosolic and particulate fractions. All of the calmodulin-dependent PDE activity was found in the soluble fraction. CI-930 and rolipram enhanced, by 2-fold and 3-4-fold respectively, the adenosine-stimulated rise in cellular cyclic AMP level. The increase in cyclic AMP levels due to CI-930 or rolipram was dose-dependent. Removal of adenosine once cyclic AMP had risen resulted in a rapid fall in cyclic AMP levels even in the presence of rolipram and CI-930. M&B 22,948, the calmodulin-dependent PDE inhibitor, caused less than a 25% increase of the adenosine-stimulated cyclic AMP levels by itself, but it contributed substantially to controlling the cyclic AMP levels after the removal of adenosine when used together with CI-930 and rolipram. These phenomena suggested that all three PDE isoenzymes participated in modulating cellular cyclic AMP levels after adenosine stimulation, and that differential importance of the individual isoenzymes depends on cellular cyclic AMP levels.

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.

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.

Abstract: Desensitization or habituation to repeated or prolonged stimulation is a common property of secretory cells. Phosphorylation of receptors mediates some desensitization processes, but the relationship of phosphorylation to desensitization at postreceptor sites is not well understood. We have tested the effect of protein phosphorylation on desensitization in bovine chromaffin cells. To increase protein phosphorylation, we have used the protein phosphatase inhibitor okadaic acid at 12.5 nM, 100 pA4 8‐bromo‐cyclic AMP to activate protein kinase A, and 10 nM phorbol 12,13‐dibutyrate to activate protein kinase C . During repeated 6‐s stimulation at 5‐min intervals, catecholamine secretion from control cells decreases. Cells exposed to 8‐bromo‐cyclic AMP or okadaic acid alone show slightly decreased rates of desensitization. In cells pretreated with phorbol 12,13‐dibutyrate, desensitization is blocked. Okadaic acid‐treated cells stimulated in the presence of 8‐bromo‐cyclic AMP show potentiation of secretion with repeated stimulation. The protein kinase inhibitor 1 ‐(5‐iso‐quinolinylsulfonyl)‐2‐methylpiperazine (H7) increases the desensitization rate. Because these phenomena are observed during secretion evoked with elevated Kf as well as by a nicotinic agonist, the effect of phosphorylation is at a postreceptor site. In contrast to desensitization to the repeated stimulations, desensitization to prolonged stimulation with high K+ is not altered by the above protocols in chromaffin Cells.

Abstract: We reported earlier that adenine nucleotides and adenosine inhibit acetylcholine‐induced catecholamine secretion from bovine adrenal medulla chromaffin cells. In this article, we used an adenosine analogue, N6‐L‐phenyliso‐propyladenosine (PIA), to study the mechanism underlying inhibition of catecholamine secretion by adenosine. PIA inhibits secretion induced by a nicotinic agonist, 1,1‐di‐methyl‐4‐phenylpiperazinium, or by elevated external K+. The half‐maximal effect on 1,1‐dimethyl‐4‐phenylpiperazinium‐induced secretion occurred at ∼5 ± 10‐‐5M. The inhibition is immediate and reversible. Fura‐2 measurements of cytosolic free Ca2+ indicate that PIA inhibits Ca2+ elevation caused by stimulation; measurements of 45Ca2+ influx show that PIA inhibits uptake of Ca2+. PIA does not inhibit calcium‐evoked secretion from digitonin‐permeabilized cells, nor does PIA cause any significant change in the dependence of catecholamine secretion on calcium concentration. These data suggest that inhibition by PIA occurs at the level of the voltage‐sensitive calcium channel.

Annals of the New York Academy of SciencesVolume 632, Issue 1 p. 241-248 Effects of Substance P on Secretion of Catecholamines from Populations of Bovine Chromaffin Cells and on Calcium Transients in Individual Cells J. A. KENT-BRAUN, J. A. KENT-BRAUN Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003Search for more papers by this authorL. K. LYFORD, L. K. LYFORD Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003Search for more papers by this authorD. J. GROSS, D. J. GROSS Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003Search for more papers by this authorE. W. WESTHEAD, Corresponding Author E. W. WESTHEADTo whom all correspondence should be addressed.Search for more papers by this author J. A. KENT-BRAUN, J. A. KENT-BRAUN Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003Search for more papers by this authorL. K. LYFORD, L. K. LYFORD Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003Search for more papers by this authorD. J. GROSS, D. J. GROSS Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003Search for more papers by this authorE. W. WESTHEAD, Corresponding Author E. W. WESTHEADTo whom all correspondence should be addressed.Search for more papers by this author First published: September 1991 https://doi.org/10.1111/j.1749-6632.1991.tb33112.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Volume632, Issue1Substance P and Related Peptides: Cellular and Molecular PhysiologySeptember 1991Pages 241-248 RelatedInformation

Hormone secretion from chromaffin cells is evoked by calcium influx through voltage-dependent channels in the plasma membrane. Previous studies have shown that ATP, cosecreted with catecholamines from chromaffin granules, can modulate the secretion resulting from depolarization by nicotinic agonists. The immediate effect of ATP is to enhance secretion; more prolonged exposure to the nucleotide results in inhibition. These receptor-mediated actions of ATP involve the activation of at least two separate classes of GTP-binding protein. Results from electrophysiological experiments reported here demonstrate that the modulatory actions of ATP can, in large part, be explained by the effects of the nucleotide on inward calcium current. ATP shows a rapid enhancement and a slower, persistent inhibition of the depolarization-induced inward current.

Cells of the adrenal medulla (chromaitin cells) cosecrete catecholamines and nucleotides, chiefly ATP. To investigate possible effects of ATP on secretion, it is important to minimize the unquantifiable effects of secreted ATP. We accomplish this by studying the effects of exogenous ATP on cultured cells in a constant stream of buffer that quickly dilutes and washes away secreted compounds. Cells are cultured on fibrinectincoated quartz plates that become part of a 2 0 4 chamber through which buffer flows constantly. Stimulants are injected into the stream via an HPLC-type sample loop. The chamber is put into a fluorometer so that cytosolic Ca + transients can be monitored in fura-2-loaded cells. Secreted catecholamines are detected downstream electrochemically to measure secretion simultaneously with Ca2 ' transients. ATP alone ( 5 100 pM) will stimulate secretion. ATP also rapidly increases inositol triphosphate turnover and causes a rise in CAMP that plateaus in 15 min during continuous ATP perfusion. Cytosolic C a 2 ' is elevated by extracellular ATP both in the presence and absence of extracellular Ca2*. Most of the rise is from intracellular stores, but secretion requires extracellular CaZ I . UTP elevates cytosolic Ca2 + but does not stimulate secretion in the presence or absence of extracellular Ca" . When cells are stimulated for 5-10 sec by a nicotinic agonist, 1,l-dimethyl-4phenylpiperazinium chloride (DMPP), or by elevated K +, secretion is enhanced 30-5070 by simultaneous addition of ATP. Enhancement of secretion by ATP is associated with an elevated cytosolic Ca2 + transient. Surprisingly, enhancement of secretion appears not to be simply additive secretion expected from ATP stimulation. Pretreatment of cells with cholera toxin (Ctx) has little or no effect on ATP-stimulated secretion but completely blocks the enhancing effect of ATP added with DMPP or K + . If cells are exposed to ATP prior to stimulation by DMPP or K I , the enhancing effect gives way to an inhibitory effect that reaches a plateau at 30% inhibition within 3 min. The inhibitory, but not the enhancing effect of ATP, is mimicked by ADP, which is marginally more potent than ATP. Inhibition by ATP is seen instantly and increases over the same time course as the ATP effect. Although Ctx pretreatment of cells does not alter the inhibitory effects of ATP and ADP, pretreatment with pertussis toxin (Ptx) eliminates the inhibitory effects

A fundamental process in neurosecretion is desensitization, or a declining response to a stimulus. The response of chromaffin cells to continuous nicotinic stimulation, secretion of catecholamines, desensitizes within a few minutes. The neuropeptide substance P (SP) has been reported to prevent desensitization in culture dish experiments and to enhance desensitization in patch clamp studies. In the present study, these contradictory responses have been demonstrated and the apparent contradictions resolved. We have measured catecholamine secretion by on-line electrochemical detection in a constant-pressure flow system. Isolated chromaffin cells cultured on quartz plates were stimulated with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in the presence and absence of SP. SP inhibited secretion and increase the rate of desensitization compared with stimulation by DMPP alone. However, when the cells were stimulated a second time with DMPP alone immediately after 5-min stimulation with SP + DMPP, the rate of desensitization was markedly lower than the control. Removal of SP after a desensitizing stimulation with SP + DMPP caused a slow secondary release of catecholamine in response to the continued stimulation with DMPP. The kinetic analysis of the secretory response shows that the primary response to SP is enhanced desensitization, but that upon removal of SP the response to DMPP desensitizes less rapidly. We suggest that SP protects some receptors from nicotinic desensitization while holding them in an inactive state, and that upon removal of SP these receptors can slowly respond to DMPP.

Bovine adrenal medullary cells, cultured on quartz plates, were superfused with buffer to which pulses of stimulant were added. Cytosolic Ca2+ was measured by the fura-2 fluorescence method and the simultaneously released catecholamine was measured electrochemically. When stimulant concentrations were adjusted to given equivalent elevations of cytosolic Ca2+, secretion depended entirely on whether Ca2+ came from internal stores or from the extracellular medium. Calcium from internal stores did not support secretion under these conditions. This nonequivalence of the two sources of cytosolic Ca2+ points to important differences in the physiological roles of the two sources of calcium. Dimethylphenylpiperazinium (a cholinergic agonist) and elevated K+ increased cytosolic Ca2+ and caused secretion only in the presence of external Ca2+. Bradykinin, muscarine, and ATP elevated cytosolic Ca2+ in the presence and absence of extracellular Ca2+ but caused secretion only in the presence of extracellular Ca2+. UTP, which in the absence of extracellular Ca2+ elevated cytosolic Ca2+ as effectively as ATP, did not cause detectable secretion under any circumstance. Because of the high Ca2+-buffering capacity of the cytosol, we expected that Ca2+ gradients, perhaps quite steep, would be produced by a pulse of Ca2+ entering the cytosol. Fura-2 fluorescence measures only the average free cytosolic Ca2+. Our data show that Ca2+ entering across the plasma membrane was much more effective at triggering exocytosis than was Ca2+ released from internal stores, suggesting that the two sources of Ca2+ are effectively compartmentalized, probably by concentration gradients in the cytosol.

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.

The major Mn2+-activated phosphoprotein phosphatase of the human erythrocyte has been purified to homogeneity from the cell hemolysate. It is sensitive to both inhibitors 1 and 2 of rabbit skeletal muscle, preferentially dephosphorylates the beta subunit of the phosphorylase kinase, and dephosphorylates a broad range of substrates including phosphorylase a, p-nitro-phenyl phosphate, phosphocasein, the regulatory subunit of cyclic AMP-dependent protein kinase, and both spectrin (Km = 10 microM) and pyruvate kinase (Km = 18 microM) purified from the human erythrocyte. The purified enzyme is stimulated by Mn2+ and to a lesser extent by higher concentrations of Mg2+. The purification procedure was selected to avoid any change in molecular weight, hence subunit composition, between the crude and purified enzyme. Maintenance of the original structure is demonstrated by non-denaturing gel electrophoresis and gel filtration chromatography. Gel filtration of the purified holoenzyme shows a single active component with a Stokes radius of 58 A at a molecular weight position of 180,000. Sedimentation velocity in a glycerol gradient gives a value of 6.1 for S20, w. Together these data indicate a molecular weight of about 135,000. Two bands of equal intensity appear on sodium dodecyl sulfate-gel electrophoresis at molecular weights of 61,700 and 36,300, suggesting a subunit composition of two 36,000 and one 62,000 subunits. The 36-kDa catalytic subunit can be isolated by freezing and thawing the holoenzyme or by hydrophobic chromatography of the holoenzyme. The catalytic subunit shows unchanged substrate and inhibitor specificity but altered metal ion activation.

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.

Bovine adrenal medullary cells have been cultured on microbeads which are placed in a lowvolume 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.

The activation of yeast enolase by cobaltous ion in 0.1 M KCl is characterized by an activation constant of 1 microM and an inhibition constant of 18 microM. Measurements of binding of Co2+ to the apoenzyme show that a maximum of four Co2+ ions are bound per dimer in the presence or absence of substrate although binding is far tighter in the presence of substrate. Ultraviolet spectral titrations show evidence for a conformational change due exclusively to the binding of the first two ions of Co2+. Both visible and EPR spectra confirm that the environment of the first pair of cobalt ions ("conformational sites") is markedly different from that of the second pair in the "catalytic" sites. Cobalt at the conformational site appears to be a tetragonally distorted octahedral complex while the second pair of metal ions appears to be in a more regular tetrahedral symmetry. Addition of either Mg2+ or substrate to the enzyme with only one pair of cobalt ions per dimer causes striking changes in the metal ion environment. The conformational metal sites appear sufficiently shielded from solvent to be inaccessible to oxidation by H2O2, in contrast to the second pair of cobaltous ions whose ready oxidation by H2O2 inactivates the enzyme. Comparison of kinetic and binding data suggests that only one site of the dimeric enzyme can be active, since activity requires more than two metals bound per dimer and inactivation results from the binding of the fourth ion per dimer.

This chapter discusses how modification of pyruvate kinase activity can control oxygen delivery from the erythrocyte. Hemoglobins of a variety of animals fall into two classes: low-02-affinity hemoglobins insensitive to P2GA and high-O2-affinity, P2GA-sensitive hemoglobins. How the levels of 2,3-P2GA in the red cell are regulated is an important question that has not yet been fully answered. The discovery that the human red cell contains phosphoglycolate, a powerful activator of the phosphatase may hint at a previously unsuspected mechanism for control of 2, 3-P2GA concentration. The best established correlation with 2,3-P2GA concentration in red cells is the activity of the enzyme pyruvate kinase. Red cell pyruvate kinase activity can be affected by a variety of factors. The enzyme is an allosteric enzyme showing cooperative homotropic interactions with its substrate phosphoenolpyruvate (PEP) but not with adenosine diphosphate (ADP). The enzyme is activated by fructose bisphosphate (FBP) that appears to stabilize the PEP-binding conformation, decreasing the Kmfor PEP and changing the PEP saturation curve to a rectangular hyperbola. Activation is not specific to FBP because mannose bisphosphate and glucose bisphosphate show the same activating effects although at high affector concentrations. Erythrocyte pyruvate kinase, in the absence of FBP, is very sensitive to oxidation. Oxidation raises the Kmfor PEP but not for ADP and causes increased sigmoidicity of PEP saturation. No evidence has been obtained that oxidation of pyruvate kinase in vivo is a reversible control mechanism but it has been suggested that abnormal pyruvate kinase found in some genetic diseases may be an oxidized form of a normal enzyme rather than a mutant enzyme. The chapter presents the role of phosphorylation in the red cell as a determinant of the oxygen delivery capacity of that cell. Cyclic adenosine monophosphate (cAMP) added externally is clearly capable of stimulating phosphorylation of pyruvate kinase and decreasing its affinity for PEP. This necessarily causes an increase in the concentration of 2,3-P2GA and a decreased affinity of hemoglobin for oxygen.

Binding of actin to chromaffin granules was confirmed and shown to be salt dependent and eliminated by prior trypsin treatment of the granules. However, purified granules bind less actin than do crude granules. A mitochondria-enriched fraction was found to bind substantially more actin per mg protein than did the secretory vesicle fraction. Binding of actin by the secretory vesicles therefore is not a good indication that actin plays an active role in exocytosis.