Calcium Ionophore X-537A Research Articles

Prolonged hormone secretion from neuroendocrine cells of Aplysia is independent of extracellular calcium.

The role of Ca2+ from extracellular and intracellular sources in stimulating neurosecretion was investigated in four experiments using neuroendocrine bag cells of the marine mollusk Aplysia. (i) Bag cells were treated with either an extracellular calcium chelator (BAPTA) or Co(2+)-substitution within 30 s after onset of an electrical afterdischarge to prevent influx of Ca2+ from extracellular fluid. These treatments shortened the duration of the afterdischarge, but did not significantly affect the overall pattern or total amount of egg laying hormone (ELH) secretion, suggesting that extracellular Ca2+ is not required for maintenance of ELH release. (ii) Substitution of Ba2+ for Ca2+ has previously been shown to support bag cell afterdischarges that trigger transient elevations in intracellular Ca2+. We showed that this treatment also stimulates ELH secretion, suggesting that Ca2+ release from intracellular stores can stimulate ELH secretion. (iii) To raise intracellular Ca2+ levels in the absence of an afterdischarge, the calcium ionophore X537A was used to transport Ca2+ across plasma and organelle membranes. When this treatment was combined with extracellular calcium chelators so that the only source of Ca2+ was from intracellular compartments, ELH secretion was stimulated. Taken together, these findings are consistent with the hypothesis that release of Ca2+ from intracellular stores is sufficient to stimulate ELH secretion.

Annexin I and involucrin are cross-linked by particulate transglutaminase into the cornified cell envelope of squamous cell carcinoma Y1

The squamous cell carcinoma line, SqCC/Y1, like natural squamous epithelia, forms a cornified cell envelope during differentiation which can be directly correlated with an increase in particulate transglutaminase activity. When transglutaminase is activated in these cells by calcium ionophore X-537A, annexin I and involucrin become incorporated into the cornified cell envelope and cannot be extracted with solutions containing sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. This effect is specific for annexin I; thus, the amounts of annexins II and IV that were extractable from cells by SDS and beta-mercaptoethanol did not change following treatment with ionophore X-537A. Annexin I could be cross-linked in vitro to itself and to other endogenous proteins by transglutaminase extracted from the particulate fraction of SqCC/Y1 cells. Immunofluorescence studies showed that cross-linked annexin I and involucrin form an envelope-like structure in SqCC/Y1 cells during differentiation that cannot be extracted by EGTA and Triton X-100. The amount of staining of this envelope structure corresponded directly to the particulate transglutaminase activity of these cells. Annexin I monoclonal and polyclonal antibodies were shown to bind to purified cornified cell envelopes from SqCC/Y1. These studies suggest that particulate transglutaminase regulates a function of annexin I during the differentiation of SqCC/Y1 cells by covalently cross-linking this protein into the cornified cell envelope.

Proteoglycan inhibition of calcium phosphate precipitation in liposomal suspensions.

The major proteoglycan in cartilage (aggrecan) is a complex macromolecule with numerous chondroitin sulphate, keratan sulphate, and oligosaccharide substituents. It has been proposed that this macromolecule has an important role in regulating mineralization in this tissue, a process which is initiated by the deposition of apatite in matrix vesicles. We have used a liposome-centred endogenous precipitation method as a model for matrix vesicle mineralization to study the effect of the rat chondrosarcoma aggrecan and its chondroitin sulphate and core protein components on apatite formation from solution. Precipitation was initiated by encapsulating buffered (pH 7.4) 50 mmol/l KH2PO4 solutions in the aqueous centres of 7:2:1 phosphatidylcholine:dicetylphosphate:cholesterol liposomes, adding 2.25-2.65 mmol/l Ca2+ and 1.5 mmol/l total inorganic phosphate (PO4) to the suspending medium (pH 7.4, 22 degrees C), then making the intervening lipid membranes permeable to the Ca2+ ions with the calcium ionophore X-537A. Aggrecan (0.5%) in the suspending medium had no effect on intraliposomal precipitation, but severely reduced (approximately 70% reduction at 24 h) its subsequent spread into the medium. The chondroitin sulphate and core protein were similarly inhibitory. The degree to which aggrecan and its constituent parts inhibited precipitation correlated with their capacity to bind Ca2+ ions. These findings suggest that functional groups in aggrecan blocked apatite growth by linking via Ca2+ bridges to growth sites on the crystal surfaces. Similar Ca-mediated interactions may well have a critical regulatory role in cartilage mineralization.

Release of endogenous brain epinephrine by the calcium ionophores X537A and A23187

The Ca 2+ ionophores X537A and A23187 produced dose-dependent release of endogenous epinephrine (Epi) as well as norepinephrine (NE) and dopamine (DA) from chopped rat hypothalamus and brainstem. The X537A-induced release of these catecholamines (CAs) was found to be Ca 2+-independent, whereas the A23187-induced release was Ca 2+-dependent. X537A and A23187 were approximately equipotent in causing the release of the hypothalamic Epi, NE and DA, but X537A was much more effective than A23187 in causing the maximal release. X537A, but not A23187, reduced the total endogenous CA content and increased the total 3,4-dihydroxyphenylacetic acid (DOPAC) content in the chopped hypothalamus. Ca 2+-independent release of Epi, NE and DA in the chopped hypothalamus was also observed with indirectly acting sympathomimetic amines (e.g. tyramine and amphetamine). Tyramine and amphetamine did not affect the total endogenous CA contents nor the total DOPAC content. These results suggest that X537A caused release of endogenous brain Epi, NE and DA by transporting thebiogenic amines across vesicular or intracellular storage sites. However, A23187 caused release of these CAs by exocytosis via transport of Ca 2+ into the neurons.

Depolarization of motor nerve terminals by pyrethroids in susceptible and kdr-resistant house flies

When applied at concentrations of one n M or higher to a house fly larval neuromuscular preparation, deltamethrin (DM) and fenvalerate (FV) greatly increased miniature excitatory postsynaptic potential (mepsp) rate and blocked neuromuscular transmission. The DM-induced mepsp discharge was abolished by tetrodotoxin (TTX), removal of Ca 2+ from the saline, or by application of hyperpolarizing stimuli to the nerve, indicating that it was due to depolarization of the presynaptic terminals. Also, in the presence of TTX, K + depolarization increased mepsp rate at the same external K + concentration before and after DM treatment, confirming that DM released transmitter by depolarizing the nerve terminals rather than by altering the voltage dependence of transmitter release. The potassium channel blocker tetraethylammonium (TEA) increased mepsp rate somewhat, while aconitine (20 μ M), which keeps sodium channels open, increased mepsp rate consistently. Pretreatment of nerves with a subthreshold dose of TEA greatly increased the mepsp rate-increasing activity of DM and aconitine, while a subthreshold level of aconitine did not synergize DM. These observations suggest that DM, like aconitine, depolarized nerves by modifying the sodium channels. Knockdown resistant ( kdr) larvae were resistant to the depolarizing action of DM and aconitine but not to that of TEA, indicating that the kdr gene produced a modified sodium channel which was less sensitive to the action of pyrethroids and aconitine. During sustained transmitter release by DM, evoked release gradually declined, resulting in a condition called early block in which spontaneous release was high and release could be evoked by electrotonic depolarization of the nerve terminals, but not by a nerve action potential. Early block was probably due to conduction block in the nerve terminals. Early block eventually gave way to late block, characterized by the decline of spontaneous release to subnormal levels and complete failure of evoked release. After late block, the calcium ionophore X-537A could not release transmitter, suggesting that late block was due to depletion of available transmitter. DM did not have a direct effect upon extrasynaptic muscle membrane. However, after late block, muscles were left insensitive to the putative transmitters glutamate and aspartate when these were bath or iontophoretically applied. A low rate of mepsps persisted after late block, indicating that the muscles were still sensitive to the natural transmitters.

The sarcoplasmic reticulum-glycogenolytic complex in mammalian fast twitch skeletal muscle. Proposed in vitro counterpart of the contraction-activated glycogenolytic pool.

Evidence is presented that the sarcoplasmic reticulum (SR)-glycogenolytic complex isolated from fast twitch skeletal muscle is a highly specific, functionally defined compartment for phosphorylase regulation. The addition of ATP alone results in prompt phosphorylase activation which demonstrates calcium dependence similar to the calcium-magnesium ATPase that catalyzes SR calcium transport suggesting that these two calcium-requiring -ystems might interact within the complex. Lowering extravesicular calcium concentration by transport of calcium into the SR lumen resulted in inactivation of phosphorylase a. This effect could be prevented by the addition of the calcium ionophore X537A which inhibits SR calcium sequestration or a calcium EGTA buffer which maintains free calcium. It was mimicked by EGTA addition. Since exogenous phosphorylase b and phosphorylase a were not activated or inactivated, respectively, by the endogenous activating enzymes or phosphatase in the SR-glycogenolytic complex, these regulatory enzymes may be compartmented. In addition, endogenous phosphorylase could be uncoupled from its activating enzymes by amylase treatment. These results suggest that the SR-glycogenolytic complex in fast twitch skeletal muscle is a compartmented system for phosphorylase activation controlled by SR calcium flux, a feature in contrast to the cardiac complex (Entman, M.L., Kaniike, K., Goldstein, M.A., Nelson, T.E., Bornet, E.P., Futch, T.W., and Schwartz, A. (1976) J. Biol. Chem. 251, 3140-3146). We suggest that the complex is the in vitro counterpart of the well documented rapid burst of glycogenolysis which ensures with the onset of contraction.

An improved procedure for the isolation of plasma membranes from rat mesenteric arteries

A plasma membrane enriched fraction has been prepared from rat mesenteric arteries by an improved procedure which eliminated the contamination by cells other than smooth muscle; i.e. those derived from mesenteries and fat tissues. Morphological and enzymatic characterization of the plasma fraction revealed only minimal contamination by other intracellular membrane fragments in the smooth muscle cell. The plasma membrane was concentrated approximately 8-fold from the post nuclear supernatant fraction as suggested by the enrichment of 5′-nucleotidase, alkaline phosphatase and ATP-dependent calcium accumulation activities. Calcium accumulation by various membrane fractions in the presence of ATP was closely associated with plasma membrane marker enzyme activities, and was not affected by azide and oxalate. The ATP-dependent calcium accumulation by plasma membrane enriched fraction was not affected by the sodium ionophore, monensin, but was substantially inhibited by calcium ionophores X537A and A23187. This suggests that the functional properties of the plasma membrane of vascular smooth muscle include calcium extrusion across the cellular membrane via an energy-dependent transport process and that the plasma membrane plays the major role in regulating the intracellular calcium in vascular smooth muscle.

Collagenase Isolation and <sup>45</sup>Ca Efflux Studies of Human Islets of Langerhans

22 human pancreases were removed soon after circulatory arrest from donors aged 15--63 years. Part of the pancreas was used for isolation of islets by the collagenase technique. The mean yield +/- SEM, expressed as the number of islets isolated from 4 g pancreas was 79 +/- 15. The yield varied considerably even when the pancreas was removed immediately after circulatory arrest and the histology was normal, and there was no correlation with the age of the donors. The islet content of insulin (ng/microgram dry islet) was 8.5 +/- 1.2 (mean +/- SEM). Isolated islets were loaded with 45Ca in the presence of 20 mM glucose and placed in a perfusion apparatus for further studies of the 45Ca washout. The decrease of washout of radioactivity in Ca2+-deficient medium offers support for the existence of a Ca2+-Ca2+ exchange process. When added in a concentration of 20 mM, glucose tended to stimulate 45Ca efflux in perfusion medium of ordinary ionic composition but inhibited this process when the medium was deficient in Ca2+. Exposure to the calcium ionophore X-537A resulted in immediate stimulation of of 45Ca efflux from human islets as previously observed for islets from rats and mice. This suggests that Ca2+ has a direct regulatory role for insulin release also in humans.

Use of calcium ionophores to determine the effects of intracellular calcium on the action potential of canine cardiac Purkinje fibers.

The effects of calcium ionophores X-537A, A23187, and PR-47 on the action potential of canine cardiac Purkinje fibers were studied using standard microelectrode techniques. The ionophores hyperpolarize the membrane potential, eliminate the notch on the action potential plateau, decrease action potential duration, decrease plateau amplitude and duration, decrease spontaneous diastolic depolarization and excitability, and decrease the rate effect on action potential duration. These effects were shown to be independent of catecholamines, membrane-bound calcium, and the patency of the slow inward current channel. The effects of the ionophores depended on the presence of calcium in the external solution. A plausible interpretation of the data is that an ionophore-mediated increase in intracellular calcium shifts the voltage dependence of transmembrane potassium currents to more negative levels.

The release of endogenous amino acids from rat visual cortex by calcium ions in the presence of the calcium ionophores X537A and A23187

Journal of NeurochemistryVolume 28, Issue 2 p. 461-463 The release of endogenous amino acids from rat visual cortex by calcium ions in the presence of the calcium ionophores X537A and A23187 G. G. S. COLLINS, G. G. S. COLLINS Department of Pharmacology, The School of Pharmacy, 29/39 Brunswick Square, London WC1N 1AXSearch for more papers by this author G. G. S. COLLINS, G. G. S. COLLINS Department of Pharmacology, The School of Pharmacy, 29/39 Brunswick Square, London WC1N 1AXSearch for more papers by this author First published: February 1977 https://doi.org/10.1111/j.1471-4159.1977.tb07773.xCitations: 11Read the full textAboutPDF 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 Citing Literature Volume28, Issue2February 1977Pages 461-463 RelatedInformation

The electrophysiological effects of ionophore X-537A on cardiac purkinje fibres.

Using classical microelectrode techniques in canine cardiac Purkinje fibres, calcium ionophore X-537A was shown to shorten the action potential, hyperpolarizing the membrane and lowering the plateau, suggesting that intracellular calcium controls membrane permeability to potassium in this preparation.

Inhibition of the noradrenergic induction of pineal N-acetyltransferase by dibutyryl cyclic guanosine monophosphate and by ionophore X-537A

Utilizing rat pineals in vitro, it can be demonstrated that dibutyryl cyclic GMP and the calcium ionophore X-537A are both able to reversibly inhibit the elevation of pineal N-acetyltransferase induced by the β-agonists norepinephrine or isoproterenol. These results are discussed in terms of the known hyposensitivy of the pineal gland to overstimulation by β-agonists.

Modifying actions of calcium ionophores on insulin release

β-Cell-rich pancreatic islets were microdissected from noinbred ob/ob-mice and exposed to the calcium ionophores X-537A and A-23187. X-537A differed from A-23187 in being a potent insulin secretagogue at non-stimulating glucose concentrations. Both ionophores inhibited the stimulation of insulin release obtained after adding 20 mM glucose to the incubation medium. The latter observation is consistent with the idea of a reduced β-cell function when the Ca 2+ in the functionally important intracellular pool(s) exceeds a certain concentration. The ionophore inhibition of the glucose-stimulated insulin release may at least in part result from decreased formation of cyclic AMP, since X-537A proved to be as effective as L-epinephrine in reducing the islet content of this nucleotide in the presence of a phosphodiesterase inhibitor. The secretagogic action of X-537A at a low glucose concentration persisted when different ions were omitted from the incubation medium and was actually considerably enhanced in the absence of extracellular Ca 2+. The insulin-releasing action of X-537A was neither influenced by 3- O-methylglucose nor by drugs blocking the α or β-adrenergic receptor sites. Exposure of the pancreatic β-cells to metabolic inhibitors in concentrations which significantly reduced the secretory response to glucose, potentiated stimulation of insulin release by X-537A, suggesting that this effect may in part be accounted for by intracellular dissolution of secretory granules.

The mechanism of calcium ionophore-induced secretion from the rat neurohypophysis.

THE release of neurohypophyseal hormones, whether evoked by electrical stimulation or by a high potassium concentration, is associated with an uptake of calcium from the extracellular space1–4. It has therefore been proposed that calcium entry plays a key role in stimulus-secretion coupling in the neurohypophysis5–7. Recently, calcium ionophores have been described which increase calcium flux across the cell membrane and induce secretion in a number of secretory systems8–12. In contrast, we report that although the calcium ionophore X-537A (Lasalocid, Hofmann-LaRoche) promotes secretion from the rat neurohypophysis, there is no corresponding increase in calcium uptake; furthermore, ionophore-induced secretion persists in the absence of external calcium ions. The most logical explanation for these results is that, rather than increasing the influx of extra-cellular calcium, the ionophore actually releases calcium from intracellular binding sites, probably mitochondria; and that it is the mobilisation of this internal calcium which, inter alia, promotes secretion.

Calcium ionophore X-537A increases spontaneous and phasic quantal release of acetylcholine at frog neuromuscular junction.

TRANSMITTER release from nerve endings is believed to be triggered by the influx of Ca2+, which is thought to enter the terminal as a result of an increase in conductance produced by the action potential1. At the neuromuscular junction the evidence for the role of Ca2+ is largely indirect, based on changes in endplate potential (e.p.p.) amplitude following variations in [Ca2+]0. Miledi2 showed that microinjection of Ca2+ into the presynaptic terminal at the synapse of the squid giant axon causes a brief period of enhanced quantal release. Ca2+ also affects the rate of spontaneous quantal release at the neuromuscular junction; miniature end-plate potential (m.e.p.p.) frequencies are very low in solutions with reduced Ca2+ (ref. 3–5) The mode of action of Ca2+ within the terminal remains largely a matter of speculation. Now a new tool has become available: an ionophore, X-537A, that transfers both univalent and divalent cations across lipid bilayers and cell membranes6–10. Using X-537A it may be possible to change intracellular Ca2+ levels while observing changes in quantal release. The ionophore might also be used to transfer other divalent metal ions into the terminal, to see how effective they are compared with Ca2+.