Ionophore X537A Research Articles

Environmental pro-oxidants induce altered envelope protein profiles in human keratinocytes.

Cornified envelopes (CEs) of human epidermis ordinarily consist of transglutaminase-mediated cross-linked proteins and are essential for skin barrier function. However, in addition to enzyme-mediated isopeptide bonding, protein cross-linking could also arise from oxidative damage. Our group recently demonstrated abnormal incorporation of cellular proteins into CEs by pro-oxidants in woodsmoke. In this study, we focused on 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), mesquite liquid smoke (MLS), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to further understand the mechanisms through which environmental pro-oxidants induce CE formation and alter the CE proteome. CEs induced by the ionophore X537A were used for comparison. Similar to X537A, DMNQ- and MLS-induced CE formation was associated with membrane permeabilization. However, since DMNQ is non-adduct forming, its CEs were similar in protein profile to those from X537A. By contrast, MLS, rich in reactive carbonyls that can form protein adducts, caused a dramatic change in the CE proteome. TCDD-CEs were found to contain many CE precursors, such as small proline-rich proteins and late cornified envelope proteins, encoded by the epidermal differentiation complex. Since expression of these proteins is mediated by the aryl hydrocarbon receptor (AhR), and its well-known downstream protein, CYP1A1, was exclusively present in the TCDD group, we suggest that TCDD alters the CE proteome through persistent AhR activation. This study demonstrates the potential of environmental pro-oxidants to alter the epidermal CE proteome and indicates that the cellular redox state has an important role in CE formation.

Role of Presynaptic Purinoceptors and Cyclic AMP on the Noradrenaline Release in Cat Cerebral Arteries

Field electrical stimulation (ES), K+ (50 mM) or ionophore X-537A (0.01 mM) induced tritium release from cat cerebral arteries preincubated with [3H]noradrenaline (NA). Adenosine and AMP (0.5 mM) did not modify tritium release caused by ionophore X-537A, but these agents and ATP (0.5 mM) significantly reduced that elicited by ES and K+; this reduction was antagonized by 1-methyl-3-isobutylxanthine (MIX; 0.05 mM). Inosine (0.5 mM) and the agonist of purinergic A2-receptors, 5'N-ethyl-carboxamide adenosine (NECA; 0.5 mM) had no effect, but the agonist of purinergic A2-receptors L-N6-phenylisopropyl adenosine (L-PIA; 0.1 mM) diminished tritium efflux caused by ES and K+. The adenosine inhibition of ES-induced radioactivity release was not affected by indomethacin (0.05 mM). MIX (0.05 mM) increased tritium release evoked by ES and K+. Agents that increase intracellular cyclic (c)AMP levels, such as dibutyryl cAMP (0.5 mM), the phosphodiesterase inhibitor Ro 20-1724 (0.1 mM), and the activators of adenylate cyclase, forskolin (0.005 mM) and NaF (2 mM) reduced tritium secretion elicited by ES and K+. However, the intracellular increase of cyclic GMP (cGMP) caused by 8-Br-cGMP did not affect this secretion. Dipyridamole (0.05 mM) and the adenosine deaminase inhibitor erythro-9-2-hydroxy-3 nonyl adenosine (EHNA; 0.1 mM) also produced inhibition of tritium secretion elicited by ES and K+. Dipyridamole reduced both the uptake of [3H]NA and [3H]adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Functional evidence for an extracellular calcium receptor mechanism triggering tyrosine kinase activation associated with mouse keratinocyte differentiation.

Calcium-induced keratinocyte differentiation is associated with tyrosine phosphorylation of a p62 protein which associates with the ras-GTPase activating protein (GAP). We have examined the nature of the calcium signal triggering p62 phosphorylation. EGTA, a specific chelator of calcium, was able to completely block calcium-induced p62 phosphorylation, even after using conditioned medium from calcium-treated keratinocytes. Preventing calcium-induced cell-cell contacts by anti-cadherin antibodies did not inhibit tyrosine phosphorylation. Slight increases in extracellular calcium concentrations (0.15 or 0.30 mM) were already sufficient to induce p62 phosphorylation. Other divalent cations, such as magnesium, zinc, nickel, and cobalt, but not the trivalent cation lanthanum, induced p62 phosphorylation to a similar extent as calcium. There was no close correlation between the ability of the various ions to induce p62 phosphorylation and increase free intracellular calcium. Similarly, treatment of primary keratinocytes with the calcium ionophores A23187 or X537A did not induce p62 phosphorylation, although it increased free intracellular calcium levels. Finally, blockers of potassium uptake, which is induced by calcium, did not inhibit p62 phosphorylation. Thus, in keratinocyte differentiation, calcium is likely to provide the primary signal for p62 tyrosine phosphorylation and may act directly at the cell membrane through a "cationic receptor mechanism" analogous to that described in other cell types.

Facilitation and delayed release at about 0 degree C at the frog neuromuscular junction: effects of calcium chelators, calcium transport inhibitors, and okadaic acid.

1. We studied two-pulse facilitation and delayed release at 0 degree C, because at low temperature facilitation is enhanced and extended whereas delayed release is increased. Our major goal was to test, by a number of approaches, the residual Ca2+ hypothesis of facilitation and delayed release. 2. As we increased the interval between pulses from 30 to 100-200 ms facilitation declined steeply. As we lengthened the interval further facilitation declined more slowly. In our entire series facilitation was still seen at 700 ms, in some preparations facilitation was apparent at 2 s. 3. We measured delayed release in preparations in which excitation-contraction was uncoupled. The decline in the rate of delayed release following the endplate potential (EPP) is similar to the decay of facilitation, both at 0 and 22 degrees C. 4. When we replaced the Ca2+ in the Ringer by Sr2+, facilitation persisted for a longer time, there was significant facilitation 2 s after an EPP. Delayed release also continued longer; the time courses for the decline of facilitation and delayed release were very similar. 5. We measured delayed release after EPPs triggered by electrotonic depolarization in isotonic CaCl2 solution or in Ringer in which the Na+ was replaced by methylamine (these solutions also contained 3,4-diaminopyridine). The time course of delayed release was very similar to that in Ringer. 6. We found that delayed release also facilitated, in the sense that the number of delayed releases, and the rate at which they were released, increased markedly after a second or third closely spaced EPP. The facilitation of delayed release and of EPPs were quantitatively similar. 7. We soaked preparations for 2 h in 200 microM bis-(aminophenoxy) ethane-tetraacetic acid (BAPTA/AM), a cell permeable Ca2+ chelator. In about one-half of these preparations facilitation was clearly diminished, judging from the EPPs evoked by a series of four to five stimuli at 30-ms intervals. The summed results from those preparations in which facilitation was reduced at 30 ms showed that it was also reduced at longer intervals. There was a comparable shortening in delayed release. Facilitation was significantly reduced when we pretreated with ethylene glycol bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (EGTA/AM), another cell-permeable Ca2+ chelator. 8. It has been reported that in BAPTA loaded preparations facilitation during trains of EPPs transiently reappears after exposure to the ionophore X-537A, which presumably elevates intracellular [Ca2+].(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Membrane-mediated precipitation of calcium phosphate in model liposomes with matrix vesicle-like lipid composition

The present study examined calcium phosphate precipitation in aqueous suspensions of artificial liposomes which closely resembled matrix vesicles (MV) in membrane lipid composition. At 22 °C, the liposomes per se did not initiate precipitation in the suspending medium for up to 120 h when the latter was made supersaturated with respect to hydroxyapatite (2.25 mM Ca 2+, 1.5 mM PO 4, 240 mosmol, pH 7.4). Likewise, the suspending medium remained stable for up to 72 h when precipitation was induced within the aqueous interiors of the liposomes by encapsulating pH 7.4-buffered 50 mM PO 4 solutions in the interior spaces and making the enclosing membranes permeable to external solution Ca 2+ ions with the ionophore X-537A. However, extraliposomal precipitation readily occurred under these latter conditions when phosphatidylserine (PS) and sphingomyelin (Sph) were deleted from the MV-like lipid formulation used to prepare the liposomes. These results suggest that lipidic membrane constituents such as PS and Sph may have a controlling influence on MV-mediated calcification in vivo by affecting the release of intravesicularly formed mineral crystals into the extracellular matrix space where they can subsequently grow and proliferate.

Secretory organelles of Paramecium cells (trichocysts) are not remarkably acidic compartments.

Acridine orange (AO) trapping in conjunction with fluorescence microscopy was applied to Paramecium cells. Trichocysts were not labeled when analyzed with an image intensification system (as opposed to a lysosomal population). Only with increasing intensity of ultraviolet light (UV) did trichocysts (and to some extent the cytosol) exhibit orange fluorescence, both effects being paralleled by increasing cell damage. Therefore, in comparison with the reported cytosolic pH (6.8), trichocysts cannot be considered as essentially acidic compartments. This is supported by experiments in vitro, using isolated cortex fragments or isolated fractions of membrane-bounded trichocysts (greater than or equal to 90% non-leaky). Again, during UV illumination orange fluorescence was observed even in the absence of ATP and Mg2+. Furthermore, this AO fluorescence and the condensation state of trichocyst contents were not affected by NH3 or by any of the widely differing ion- and H(+)-exchange inhibitors or ionophores tested. Decondensation of trichocyst contents occurred only when Ca2+ ionophore A23187 or X537A was incorporated into trichocyst membranes and when Ca2+ was then added. In this case all trichocysts partially decondensed within their intact membranes. We conclude that AO might be trapped in trichocysts by the abundant acidic secretory components during observation with UV light, rather than by acidic luminal pH.

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.

Purinergic and cyclic AMP modulation of noradrenaline release in cat femoral arteries

1. 1. Adenosine, AMP, ATP (5 × 10 −4 M), 5′- N-ethylcarboxamide adenosine (NECA) and N6- l-phenylisopropyl adenosine ( l-PIA) (10 −4 M) decreased tritium release elicited by electrical stimulation (ES) or 50 mM K + in cat femoral arteries preincubated with [ 3H]noradrenaline (NA). 2. 2. This effect was antagonized by 1-methyl-3-isobutylxanthine (MIX, 5 × 10 −5 M). 3. 3. The release induced by ionophore X-537A (10 −5 M) was unaffected by adenosine and AMP. 4. 4. The increase of intracellular cAMP levels caused by dibutyryl cAMP (5 × 10 −4 M), Ro-20 1724 (10 −4 M), forskolin (5 × 10 −6 M), NaF (2 × 10 −3 M) reduced, but MIX (5 × 10 −5 M) increased tritiu release elicited by ES and K +. 5. 5. Dipyridamole (5 × 10 −5 M) and erythro-9-2-hydroxy-3 nonyl adenosine (EHNA) (10 −4 M) also reduced tritium release. 6. 6. Dipyridamole decreased both the uptake of [ 3H]NA and [ 3H]adenosine. 7. 7. These data indicate: (a) the existence of A 1 and A 2 subtypes of purinoceptors situated presynaptically, which modulates NA release, (b) the intracellular increase of cAMP negatively modulates this secretion, and (c) these arteries possess an active system for incorporating and degrading adenosine.

Non-genomic mechanism of action of Δ-4 and 5-reduced androgens and progestins on the contractility of the isolated rat myometrium

Effective concentrations 50 of androgens, i.e. testosterone, androsterone, androstanediol, 5β-dihydrotestosterone and progestins: progesterone, pregnanolone, pregnanedione, epipregnanolone, allopregnanolone and allopregnanedione were assayed on the tonic contractions of the isolated rat myometrium induced by calcium in high-potassium calcium-free depolarizant solutions. Steroids showed their relaxant effect by fadding the sustained contraction induced by calcium in a depolarized state. Also, the addition of the calcium ionophores A-23187 and X-537A reversed the steroid relaxant effect by increasing sharply the tonic contraction. The possibility of steroid-induced relaxation through release of noradrenaline or histamine was discarded by blocking their specific receptors. From the results it is concluded that Δ-4 and 5-reduced androgens and progestins produce relaxation by a myogenic mechanism acting on the smooth muscle cell, most likely by directly blocking the calcium channels they causing modulation of: the contraction-relaxation cycle.

Characteristics of Histamine Release from Rat Mast Cells Induced by a Bracken Toxin, Braxin A1

The effect of braxin A1, a new bracken glucoside, on histamine release from isolated rat peritoneal mast cells was studied. Braxin A1 caused the release of histamine in a dose-dependent manner; the release was slow and increased gradually with time, finally reaching a maximum release of 100%. The action of braxin A1 depended on the incubation temperature in the range from 4°C to 49°C, while it was almost abolished at 0°C. The action of braxin A1 was unaffected by removing calcium or any inorganic ions from the incubation medium and by the addition of 2,4-dinitrophenol or theophylline. The mast cells exposed to braxin A1 were vitally stained with trypan blue and swelled greatly. The cell swelling was characterized by the protrusion of swollen cytoplasmic granules. The present results for braxin A1 were similar to those for the ionophore X537A except for the extracellular inorganic ion dependency, but they were different from those observed with compound 48/80. These results suggest that braxin A1 releases histamine from mast cells without both exocytosis and membrane lysis, but with a cytotoxic action on cytoplasmic membranes by a different mode of action from that of X537A.

Effects of Na + and Ca 2+ gradients on intracellular free Ca 2+ in voltage-clamped Aplysia neurons

Selected neurons of the abdominal ganglion of Aplysia californica were voltage-clamped and intracellular free Ca ([Ca 2+] i) and Na ([Na +] i) concentrations were monitored with ion selective microelectrodes. Reducing [Na +] o from 500 mM (normal seawater, NSW) to 5 mM resulted in a decrease of the potential measured by the Ca electrode ( V Ca). Increasing [Ca 2+] o from 10 to 50 mM increased [Ca 2+] i two-fold, keeping [Ca 2+] o at 50 mM and decreasing [Na +] o to 5 mM still led to a decrease in V Ca. With 100 mM [Ca 2+] o, which also increased [Ca 2+] i, decreasing [Na +] o increased V Ca in two of the eight cells tested. This indicates that in normal or moderately high resting [Ca 2+] i, Ca 2+ extrusion by Na/Ca exchange (forward mode) is not essential for [Ca 2+] i buffering. [Na +] i was12.9 ± 3.6mM (S.E.M., n = 7) in NSW; reducing [Na +] o to 5 mM decreased [Na +] i to2.0 ± 1.1mM (S.E.M.). Keeping [Na +] o at 5 mM and increasing [Ca 2+] o from 10 to 20 mM further decreased [Na +] i to about 1.0 mM, evidence of Na/Ca exchange operating in the reverse mode. Attempts to increase [Ca 2+] i by bath application of the Ca ionophores A23187, X537A, ionomycin or ETH 1001 resulted in no measurable change of the resting [Ca 2+] i. Application of Ouabain caused an apparent increase in [Ca 2+] i in two of the six cells tested. In cells injected with the metallochromic indicator arsenazo III (AIII), the rate of the falling phase of the AIII absorbance increase, following a voltage-clamp pulse, was significantly slower in 5 mM [Na +] o. This indicates that in its forward mode Na/Ca exchange is active in clearing large submembrane increases in [Ca 2+] i.

An ultrastructural study of calcium phosphate formation in multilamellar liposome suspensions.

Calcium phosphate precipitation can be induced within liposomes containing buffered inorganic phosphate by the ionophore-mediated loading of calcium ions. Negative staining, positive staining for thin sectioning, and freeze-fracture electron microscopy were used to characterize these synthetic vesicles and to evaluate the liposome-mineral interactions resulting from apatite formation. Suspensions of phosphate (0-50 mM KH2PO4)-encapsulated liposomes were prepared from mixtures of phosphatidylcholine, dicetyl phosphate, and cholesterol in the molar ratios of 7:2:1. Precipitation reactions were initiated by first suspending the liposomes in a buffered solution containing calcium (1.3-2.2 mM Ca(NO3)2) and then adding the cationic ionophore X-537A. All experiments were carried out at 22 degrees C, pH 7.4, and 240 mosm. Transmission electron microscopical analysis showed that the liposome preparation consisted of multilamellar, multicompartmental vesicular structures. The liposomes were typically heterogeneous with respect to both the size and number of phospholipid bilayers surrounding the aqueous cores. In Ca-loaded liposomes, discrete clusters of apatite mineral were present within the lumen, and in close proximity to the inner lipid membranes. These nascent crystallites eventually penetrated the lipid envelope to provide a focus for external precipitation events. Crystalline apatite phases were not observed when the incubation conditions prevented intraliposomal precipitation. The de novo calcification of these liposomes had many features in common with the sequence of mineral deposition occurring in matrix vesicle-mediated calcification. These results reinforce the conclusions of earlier chemical and kinetic studies and further support the use of this system as an experimental model for examining the membrane-mineral interactions associated with tissue mineralization.

Dihydropyridine Modulation of the Chromaffin Cell Secretory Response

Prolonged perfusion of cat adrenal glands with Krebs-bicarbonate solutions containing nicotine, muscarine, or excess K rapidly increased the rate of catecholamine output proportional to the concentrations of secretagogue used. The secretory responses to nicotine or high K reached a peak and declined to almost basal rates of secretion after about 10 min of stimulation. The dihydropyridine Ca channel agonist Bay K 8644 potentiated markedly the secretory responses to 1 microM nicotine and to 17.7 mM K but not to higher concentrations of these secretagogues. The muscarinic response did not decrease with time and was modestly potentiated by Bay K 8644. Similar curves were obtained with 17.7 mM K plus Bay K 8644 and with 59 mM K alone. CGP28392, another agonist, was about 10 times less potent than Bay K 8644 in potentiating the secretory responses to 17.7 mM K. Bay K 8644 also potentiated the release of [3H]noradrenaline evoked by stimulation of cultured bovine adrenal chromaffin cells with 17.7 mM K or 2 microM nicotine but not with higher concentrations of K or nicotine. Dihydropyridine Ca channel antagonists reversed the effects of Bay K 8644 with the following order of potency: niludipine greater than nifedipine = nimodipine greater than nitrendipine. The secretory rates from intact chromaffin cells treated with the Ca ionophores X537A or A23187, or those evoked by Ca-EGTA buffers from digitonin-permeabilized cells, were not affected by Bay K 8644. These results are compatible with the following conclusions: Bay K 8644 selectively potentiates catecholamine secretory responses mediated through the activation of voltage-sensitive Ca channels; during nicotine or high-K stimulation, Ca gains access to the cell interior through a common permeability pathway, the Ca channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of the calcium ionophores A23187 and X537A on calcitonin secretion from the isolated perfused porcine thyroid

The present study examined the influence of the calcium ionophores A23187 and X537A on the calcitonin (CT) secretory process. The isolated perfused porcine thyroid was used to evaluate ionophore effects on CT secretion and thyroid slices were used to measure 45Ca uptake. Both A23187 and X537A enhanced the rate of CT release from the perfused thyroid. A23187 at a concentration of 19 μM (10 μg/ml) produced a maximal increase in CT secretion of 325% above control levels. X537A at a concentration of 16 μM (10 μg/ml) produced a peak rise in CT release of approximately 2000% over control levels. The CT secretory response to A23187 was found to be completely calcium dependent; however, the secretory response to X537A was partially, but not completely dependent upon the presence of perfusate calcium. The results demonstrate that these calcium ionophores are very potent CT secretagogues which vary considerably in their calcium dependency.

Culture and Characterization of Rat Middle-ear Epithelium

This study was performed to design a method for the culture of rat middle-ear epithelium and to apply the method to investigate the characteristics of this epithelium. Culture of explants of middle-ear epithelium in the presence of the epidermal growth factor was successful, whereas serial cultivation required 3T3 feeder cells in addition to the epidermal growth factor. Cultured middle-ear epithelium was studied by phase-contrast microscopy, transmission and scanning electron microscopy, and combined light and scanning electron microscopy (LM/SEM). These techniques showed similarity between the cultured and the natural middle-ear epithelium. Explants and outgrowths showed both flat polygonal and ciliated epithelial cells. In serial cultivation, however, only the first of these cell types was observed. Frequently, a single primary cilium was found on the cell surface. Transmission electron microscopy showed cross-linked envelopes whose formation was promoted by ionophore X537A. Cytokeratin was demonstrated by immunoblotting, immunofluorescence, and immunoperoxidase methods, using an anti-cytokeratin monoclonal antibody. The model described here permits study of the differentiation of middle-ear epithelium in vitro and may be of future value for the study of chronic middle-ear diseases.

Gadolinium ions inhibit exocytotic vasopressin release from the rat neurohypophysis.

Single rat neurointermediate lobes (n.i.l.s) were fixed by their stalks to a platinum wire clip electrode and incubated in oxygenated Krebs-HEPES medium. Vasopressin release int the medium was determined by radioimmunoassay. Vasopressin secretion was increased by different stimuli and the effects of gadolinium (Gd3+) were tested. Electrical stimulation (15 Hz, three times 1 min with 1 min intervals) increased vasopressin release in a calcium-dependent manner. Gd3+ (10 microM to 3 mM) inhibited the evoked release of vasopressin in a concentration-dependent fashion; at 3 mM the inhibition was 98%. The inhibitory effect of Gd3+ up to 300 microM was antagonized by increasing the calcium concentration in the medium up to 6 mM. The effects of 1 and 3 mM-Gd3+ were unaffected by increasing the calcium concentration. Exposure of n.i.l.s to depolarizing concentrations of potassium (high K+, 60 mM, 30 min) increased the vasopressin release more than 33-fold. The elevated vasopressin release remained constant during six consecutive 5 min periods. In the initial 5 min period 300 microM-Gd3+ reduced the evoked vasopressin release by 80% but during the last 5 min period only by 30%. At 3 mM-Gd3+ vasopressin release was completely blocked during the whole time of incubation with high K+. Vasopressin release induced by exposure of n.i.l.s to cold (4 degrees C, 20 min) was completely inhibited by 3 mM-Gd3+, but reduced by only 25% in the presence of 300 microM-Gd3+. Vasopressin release induced by incubation of n.i.l.s with the ionophore X-537A (lasalocid) (10 microM, 30 min) was reduced by 90% in the presence of 300 microM-Gd3+ and completely prevented by 3 mM-Gd3+. 300 microM-Gd3+, added to the incubation medium, had no significant effect on the vasopressin release from crude synaptosomal preparations evoked by high K+. However, when 300 microM-Gd3+ was already present during the tissue homogenization, the evoked vasopressin release from the synaptosomes was completely blocked. It is concluded that Gd3+ inhibits exocytotic vasopressin release at two different sites. First, Gd3+ may block voltage-regulated calcium channels. Secondly, Gd3+ may inhibit the exocytotic release mechanism by an intracellular site of action. It is speculated that contractile proteins may be the intracellular target for Gd3+.

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.

Liposome-mediated calcium phosphate formation in metastable solutions.

The present study examined the effect of anionic liposomes on precipitate formation in supersaturated calcium phosphate solutions. The liposomes were prepared by dispersing 7:2:1 molar mixtures of phosphatidylcholine, dicetyl phosphate, and cholesterol in buffered aqueous solutions containing 0 or 50 mM inorganic phosphate (PI). Unencapsulated PI was removed by gel filtration. The liposomes were then suspended in reaction solutions containing 2.25 mM Ca2+ and either 0, 1.0, or 1.5 mM PI. All experiments were carried out at 22 degrees C, pH 7.4, and 240 mOsm. External solution Ca2+ and PI losses were found to be appreciable only when the membranes of liposomes containing entrapped PI were made permeable to Ca2+ with the addition to the suspension of the ionophore X-537A. The Ca2+ losses, moreover, were up to 3 times as great (1.5 vs 0.5 mM) when accompanied by external PI losses than in PI-free external solutions where Ca2+ alone was involved. Previous studies showed that in this latter situation, decline in external Ca2+ concentration was the result of precipitate formation in the aqueous interiors of the liposomes. The present findings suggest that when the external solution phase was metastable, the apparent coupling of large additional Ca2+ losses with intraliposomal precipitation was due to secondary precipitation brought about by the seeding action of intraliposomal crystals penetrating into the external solution. The results may explain in part the origin of extravesicular mineral deposits in matrix vesicle calcification.