Aggregation of marine sponge cells induced by Ca pulses, Ca ionophores, and phorbol esters proceeds in the absence of external Ca

Cells of the sponge Microciona prolifera dissociated in Ca,Mg-free sea water reaggregate upon addition of Ca, an observation classically attributed to the requirement for Ca of a species-specific aggregation factor. We now report that, unexpectedly, extracellular Ca is not required during aggregation; brief Ca pulses (1-3 sec) terminated by excess EDTA suffice to prepare the cells for aggregation by Ca ionophores (e.g. A23187). We also show that phorbol myristate acetate (PMA) promotes aggregation of pulse-prepared cells. Since PMA and A23187 act synergistically in Microciona, the "twin signal" hypothesis, signalling by Ca and protein kinase C in parallel, is validated in this primitive animal.     

The contribution of the calcium-dependent interaction of aggregation factor molecules to recognition: A system providing additional specificity forces?

Cells from the sponge Microciona prolifera display on their surfaces large but defined proteoglycan complexes (Microciona aggregation factor = MAF) that mediate species-specific cell aggregation by a process requiring high calcium ion concentrations. An analysis of MAF-MAF interactions based on binding studies of MAF to glutaraldehyde-fixed sponge cells and MAF-derivatized beads demonstrates that the requirement for high calcium concentrations can be overcome by extremely small amounts of certain polycations such as polybrene, polylysine, or histones. For measurements of the affinity of these substances to MAF, a method was adopted that partitions ¹²⁵I-labeled MAF between dextran and polyethyleneglycol in an aqueous two-phase polymer system depending on the net charge of the complex formed. Since only polymers of positive charges affect binding and partitioning at low concentrations, large areas of interaction similar to those found in glycosaminoglycans are proposed for MAF. Through a multitude of appropriately spaced interaction sites, the rather weak selectivity of single charged sites could in such a system still provide strong enough specificities to explain species-specific cell sorting. The biological significance of naturally occurring polycations as well as extracellular calcium includes their role in cell recognition, sorting out as well as the ordered and continual streaming movements of groups of cells seen in the mesohyl of live sponges.     

Involvement of carbohydrates as multiple low affinity interaction sites in the self-association of the aggregation factor from the marine sponge Microciona prolifera

Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular weight aggregation factor (MAF) and is based on two functional properties, a Ca2+-independent cell binding activity and a Ca2+-dependent factor-factor self-interaction. Monoclonal antibodies were prepared against purified MAF, and one clone was characterized which selectively inhibited the MAF-MAF association activity. Binding of the blocking antibody (Block 1) to MAF demonstrated that this epitope structure was present in 1100 copies per one MAF molecule of Mr = 2 X 10(7). Such blocking antibodies precipitated a small molecular weight protein-free glycan fraction prepared from MAF by Pronase digestion, thus indicating that the highly repetitive epitope is located in the carbohydrate portion of the molecule. Since the inhibitory activity of the Block 1 antibody could only be achieved when most of the sites were occupied by Fab fragments of this antibody, the self-association of MAF seemed to be based on the polyvalency of the carbohydrate determinants. The affinity of the protein-free glycans was very low as shown by the fact that they did not display any measurable self or MAT binding activity in their monomeric form. After cross-linking them with glutaraldehyde into polymers of the size of MAF, however, the self-interaction could be reconstituted. Thus, the MAF-MAF association activity, which is needed for cell aggregation in sponges, seems to be based on multiple low affinity carbohydrate-carbohydrate interactions, which is different from most interactions mediated by adhesion molecules characterized so far.     

Involvement of a highly polyvalent glycan in the cell-binding of the aggregation factor from the marine sponge Microciona prolifera

A proteoglycan-like aggregation factor from the marine sponge Microciona prolifera (MAF) mediates cell-cell recognition via a cell-binding and a self-association domain. After repetitive and prolonged treatment of MAF with glycopeptide-N-glycosidase (PNGase) the specific binding of MAF to homotypic cells was decreased by 72%. Polyacrylamide gel electrophoresis and gel filtration analysis of such PNGase digests showed that: 1) the enzyme released a single glycan type of Mr = 6 X 10(3) (G-6) from MAF, 2) 1 mole of MAF contains at least 830 moles of N-linked chains of G-6 glycan. The correlation between the loss of the binding activity of MAF and the extent of the release of the repetitive G-6 polysaccharide strongly suggests its involvement in MAF-cell association via highly polyvalent interactions.     

Effect of caffeine on azide-sensitive Mg2+, ATP-dependent increase of tetracycline fluorescent response in modeling of Ca ions accumulation in mitochondria

The influence of caffeine on the Mg2+, ATP-dependent Ca(2+)-uptake was investigated in the experiments, conducted on mitochondria isolated from myometrium of nonpregnant estrogenized rats. NaN3-sensitive CTC fluorescence increasing (lambda f = = 520 nm) was used as a test for active Ca2+ transport. Kinetics of NaN3-sensitive Mg2+, ATP-dependent component of CTC fluorescence change fits to the pattern of the first-order reaction either in the absence or in the presence of caffeine (20 mM). Caffeine (0-20 mM) inhibited both the stationary level (settled on the 2-3d min. of incubation) and the initial rate V0, and rate constant k of CTC fluorescence change. Magnitude of the apparent inhibition constant I0.5 for caffeine is 10.41 +/- 1.81 mM, inhibition process has weak positive cooperativity--the value of apparent Hill coefficient for caffeine is equal to 1.2 +/- 0.3. Data obtained suggest that caffeine inhibits both stationary Ca2+ capacity of mitochondria and the rate of NaN3-sensitive Mg2+, ATP-dependent Ca(2+)-accumulation in case of myometrium. These data could be useful for further investigation of molecular and membrane mechanisms of caffeine action on the intracellular Ca2+ homeostasis in uterus smooth muscle and its contractive activity.     

Use of chlortetracycline fluorescence for the detection of Ca storing intracellular vesicles in normal human erythrocytes

The uptake of chlortetracycline (CTC) and the nature of the fluorescence of CTC was studied in intact human erythrocytes from apparently healthy donors. The uptake of CTC at 22 degrees C proceeded with a t1/2 of about 3 min, and after 15 min a stable equilibrium was achieved with an intracellular accumulation by a factor of 5-6 relative to the medium concentration. The accumulation did not change in the range of CTC concentrations tested (20-500 microM). The Ca specificity of the CTC fluorescence spectrum was confirmed by Ca depletion of red cells using A23187 in the presence of EGTA and 0.2 mM Mg. This procedure decreased the total intracellular calcium content by about 70% and reduced the fluorescence intensity to one-fourth. Fluorescence microscopy of red cells incubated with 100 microM CTC at 22 degrees C showed that the fluorescence originated mainly from the red cell membrane. In addition, in about 15% of erythrocytes one or more fluorescent dots (diameter greater than 0.2 less than 1 microns) were detected. The fluorescence of the dots and membranes was related to calcium, as evidenced by the reduction of their intensity in Ca depleted cells. The number of erythrocytes with fluorescent dots and the frequency of the dots per cell was largely unaffected by lowering the incubation temperature to 0 degrees C, indicating that the dots most probably do not represent endocytotic artifacts induced by CTC. The number of dots was increased in erythrocytes preincubated with primaquine, demonstrating that CTC fluorescence can be applied to monitor the appearance of intracellular Ca storing vesicles. It is concluded that in (at least) 15% of erythrocytes obtained from apparently healthy donors intracellular vesicles containing Ca can be detected by CTC fluorescence microscopy.     

Chlorotetracycline fluorescence is a quantitative measure of the free internal Ca2+ concentration achieved by active transport. In situ calibration and application to bovine cardiac sarcolemmal vesicles

Chlorotetracycline (CTC) fluorescence is shown to be a competent and quantitative measure of the free internal calcium concentration, [Ca2+]i, obtained by ATP supported active uptake by bovine cardiac sarcolemmal (SL) vesicles. The fluorescence response of CTC to [Ca2+]i is calibrated by pre-equilibrating the vesicles with known Ca2+ concentrations and then diluting into a Ca2+-free medium containing CTC. The experiments show that CTC comes into equilibrium with the internal Ca2+ more rapidly than the latter can passively leak from the vesicles. The amplitude of the fluorescence increase is proportional to the Ca2+ concentration with which the vesicles are pre-equilibrated. This constitutes a calibration procedure for the use of CTC fluorescence as a quantitative measure of the free internal Ca2+ concentrations achieved in active transport. This method is applied to the determination of the average free Ca2+ concentrations achieved in ATP-energized uptake with sarcolemmal vesicles. Under optimal conditions an initial rate of 13 mM/min (37 nmol/mg/min) is observed. Uptake reaches a maximum corresponding to 70 mM (179 nmol/mg). Half-maximal values are obtained after 5 min of reaction. The mechanism of the CTC response to free internal Ca2+ concentration is discussed and is compared with measurements of vesicle-associated 45Ca2+.     

Influence of divalent cations upon complement-mediated enzyme release from human polymorphonuclear leukocytes.

The complement component, C5a provokes the selective release of granule-associated enzymes from the intact, viable cytochalasin B-treated human polymorphonuclear leukocytes (PMN) in the absence of phagocytosis or cellular adherence to surfaces. Consquently, in this experimental system the influence of divalent cations on these two processes can be disregarded and their effects on enzymes secretion can be studied directly. Cytochalasin B-treated PMN exposed to C5a in calcium and magnesium-free media consistently secreted significant amounts of the granule-associated enzymes, beta-glucuronidase and lysozyme. The basal secretory response was not diminished if cells were preincubated with 5.0 mM EDTA, nor was it influenced if 1.0 mm or 2.0 mM EDTA were present in the reaction mixtures. The addition of calcium (up to 1.5 to 2.0 mM) produced a concentration-dependent enhancement of beta-glucuronidase release, whereas increasing amounts of calcium (above 2.0 mM) inhibited secretion of this enzyme. Lysozyme release was similarly enhanced by the addition of calcium, but inhibition with high concentrations was not observed. Calcium per se, in the absence of C5a, provoked only the release of lysozyme from these cells. The effects of calcium upon enzyme release were not associated with alterations in the state of assembly of cytoplasmic microtubules. These findings provide another example of the role of calcium in "stimulus-secretion coupling" and provide evidence that exocytosis of various granules in human PMN is regulated by independent mechanisms involving calcium.     

Reconstitution of high cell binding affinity of a marine sponge aggregation factor by cross-linking of small low affinity fragments into a large polyvalent polymer

Species-specific reaggregation of cells from the marine sponge Microciona prolifera is mediated by a proteoglycan-like aggregation factor (MAF) of Mr = 2 X 10(7) which has two functional domains, a cell binding domain and an aggregation factor interaction domain. After extensive trypsin digestion, over 60% of the MAF mass was converted into a glycopeptide fragment of Mr = 10,000 (T-10) which is therefore a representative part of the major portion, but not of the entire MAF molecule. The T-10 fragment has a similar amino acid and carbohydrate composition as the intact MAF and displays species-specific binding. Although T-10 also inhibited MAF association with homotypic cells, its apparent affinity is 3 X 10(6) M-1, i.e. 13,000 times lower than that of native MAF. Reconstitution of binding affinity in the same order of magnitude as native MAF (Ka = 10(10) M-1) was obtained by cross-linking the glycopeptide fragment into polymers of the approximate size of MAF (Mr greater than 1.5 X 10(7) using diepoxybutane and glutaraldehyde, or periodate oxidation and glutaraldehyde. The apparent association constants of intermediate polymers with Mr = 1 X 10(5), 6 X 10(5), 9 X 10(5), 2 X 10(6) and above 1.5 X 10(7) increased proportionally to their size and were in line with association constants of MAF degradation fragments. Since the binding affinity of the T-10 glycopeptide fragment could be reconstituted by cross-linking, and since this fragment accounts for over 60% of MAF, we propose that the specificity and high affinity of the MAF-cell association is based on a highly polyvalent interaction of low affinity cell-binding sites. Such a polyvalency of the cell binding domain is advantageous for efficient cell-cell interactions and thus differs from most known interaction molecules and receptors characterized.     

Dual adhesion systems of chick myoblasts

Cultured chick myoblasts (Mb) were resuspended by incubation with 100 micrograms/ml trypsin/2.5 mM CaCl2 (to yield TC-Mb), or with 5 micrograms/ml trypsin/2.5 mM EDTA (to yield LTE-Mb). As measured in a particle counter, TC-Mb aggregation was Ca2+ dependent, whereas LTE-Mb aggregated equally well in the presence of CaCl2 or EDTA. Cells subjected to the same treatments in sequence, like cells dissociated directly with 100 micrograms/ml trypsin/2.5 mM EDTA, did not aggregate significantly in the presence or absence of Ca2+. Adhesive specificity was assessed by mixing unlabeled cells with cells labeled with a fluorescent dye and then analyzing the distribution of fluorescent and nonfluorescent cells in aggregates. No adhesive specificity was seen in controls (i.e., TC-Mb aggregated randomly with TC-Mb, or LTE-Mb with LTE-Mb), but TC-Mb and LTE-Mb did not cross-adhere. These results indicate the existence of two independent, noncomplementing, adhesion systems, and suggest that the differential treatments preserve or activate one system while destroying the other. Myoblasts dissociated with 2.5 mM EDTA in the absence of exogenous trypsin (E-Mb) have both adhesion systems active on their surfaces, as do Mb grown in Ca2+-free medium and then dissociated with 0.7 mM EDTA (Knudsen, K. A., and Horwitz, A. F., Dev. Biol. 58, 328-338, 1977). Although aggregation of E-Mb is largely Ca2+ independent and that of Knudsen/Horwitz-Mb is largely Ca2+ dependent, they adhere well to each other and to LTE-Mb while segregating from TC-Mb. Fibroblasts also have dual adhesion systems, one Ca2+ dependent and the other Ca2+ independent, but TC-Fb do not cross-adhere to TC-Mb (nor E-Fb to E-Mb). Cell type-specific adhesive selectivity may thus contribute to the selectivity of myocyte fusion.     

The effect of divalent cations on aggregation of Dictyostelium discoideum.

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20--40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl2 accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 X 10(-5) M progesterone considerably delays it. These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg2+ or the ionophore and greatly decreased accumulation in the case of the steroid. Treatment with EDTA and Mg2+ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn2+ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg2+ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca2+ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca2+. These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

Thrombin-induced calcium movements in platelet activation

The thrombin-induced Ca2+ fluxes and their coupling to platelet aggregation of the human platelet were studied using quin2 as a measure of the cytoplasmic Ca2+ concentration [( Ca2+]cyt) and chlorotetracycline (CTC) as a measure of internally sequestered Ca2+. Evidence is given that the CTC fluorescence change is proportional to the free internal Ca2+ concentration in the dense tubular lumen. The intracellular quin2 concentration was 1 mM and analysis showed that it did not perturb the processes reported herein. The value of [Ca2+]cyt at rest and during thrombin activation was analyzed in terms of Ca2+ influx, Ca2+ release, Ca2+ sequestration, and Ca2+ extrusion. Influx was distinguished from internal release by removing extracellular Ca2+ 1 min before thrombin activation. In the presence of 2 mM external Ca2+, the thrombin-induced Ca2+ influx accounts for most of the increase in [Ca2+]cyt (over 80%). Thrombin-induced Ca2+ influx and release have somewhat different EC50 values (0.17 U/ml vs. 0.35 U/ml). The contribution of influx can be inhibited by verapamil, bepridil and Cd2+ (IC50 values of 19 microM, 2 microM and 50 microM). The influx results were analyzed in terms of a thrombin-activated channel. Indomethacin pretreatment experiments suggest that activation of the arachidonic pathway accounts for approx. 50% of the influx-related [Ca2+]cyt elevation. Elevation of [Ca2+]cyt by intracellular release is not inhibited by verapamil or Cd2+ but is inhibited by bepridil with a high IC50 (25 microM). It is only 15-20% inhibited by indomethacin and is thus not dependent on thromboxane A2 formation. The release reaction does not require Ca2+ influx. The rate of thrombin-activated platelet aggregation is shown to have an approximately fourth-power dependence on [Ca2+]cyt with an apparent Km of 0.4 microM. Comparisons of aggregation rates of the partially thrombin-activated vs. fully thrombin-activated, partially verapamil-inhibited conditions suggest that this dependence on [Ca2+]cyt is the major determinant of the aggregation behavior. Analysis shows that calcium influx is the major pathway for elevating [Ca2+]cyt by thrombin when physiological concentrations of external Ca2+ are present.     

Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. I. Use of chlorotetracycline as fluorescent probe

Stimulus-secretion coupling in pancreatic exocrine cells was studied using dissociated acini, prepared from mouse pancreas, and chlorotetracycline (CTC), a fluorescent probe which forms highly fluorescent complexes with Ca2+ and Mg2+ ions bound to membranes. Acini, preloaded by incubation with CTC (100 microM), displayed a fluorescence having spectral properties like that of CTC complexed to calcium (excitation and emission maxima at 398 and 527 nm, respectively). Stimulation with either bethanechol or caerulein resulted in a rapid loss of fluorescence intensity and an increase in outflux of CTC from the acini. After 5 min of stimulation, acini fluorescence had been reduced by 40% and appeared to be that of CTC complexed to Mg2+ (excitation and emission maxima at 393 and 521 nm, respectively). The fluorescence loss induced by bethanechol was blocked by atropine and was seen at all agonist concentrations that elicited amylase release. Maximal fluorescence loss, however, required a bethanechol concentration three times greater than that needed for maximal amylase release. In contrast, acini preloaded with ANS or oxytetracycline, probes that are relatively insensitive to membrane-bound divalent cations, displayed no secretagogue-induced fluorescence changes. These results are consistent with the hypothesis that CTC is able to probe some set of intracellular membranes which release calcium during secretory stimulation and that this release results in dissociation of Ca(2+)-complexed CTC.     

Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. II. Subcellular localization of the fluorescent probe chlorotetracycline

Subcellular distribution of the divalent cation-sensitive probe chlorotetracycline (CTC) was observed by fluorescence microscopy in isolated pancreatic acinar cells, dissociated hepatocytes, rod photoreceptors, and erythrocytes. In each cell type, areas containing membranes fluoresced intensely while areas containing no membranes (nuclei and zymogen granules) were not fluorescent. Cell compartments packed with rough endoplasmic reticulum or Golgi vesicles (acinar cells) or plasma membrane-derived membranes (rod outer segments) exhibited a uniform fluorescence. In contrast, cell compartments having large numbers of mitochondria (hepatocytes and the rod inner segment) exhibited a punctate fluorescence. Punctate fluorescence was prominent in the perinuclear and peri-granular areas of isolated acinar cells during CTC efflux, suggesting that under these conditions mitochondrial fluorescence may account for a large portion of acinar cell fluorescence. Fluorometry of dissociated pancreatic acini, preloaded with CTC, showed that application of the mitochondrial inhibitors antimycin A, NaCN, rotenone, or C1CCP, or of the divalent cation ionophore A23187 (all agents known to release mitochondrial calcium) rapidly decreased the fluorescence of acini. In the case of mitochondrial inhibitors, this response could be elicited before but not following the loss of CTC fluorescence induced by bethanechol stimulation. Removal of extracellular Ca2+ and Mg2+ or addition of EDTA also decreased fluorescence but did not prevent secretagogues or mitochondrial inhibitors from eliciting a further response. These data suggest that bethanechol acts to decrease CTC fluorescence at the same intracellular site as do mitochondrial inhibitors. This could be due to release of calcium from either mitochondria or another organelle that requires ATP to sequester calcium.     

Influence of thyroid status on Ca2+ mobilization and amylase secretion in rat pancreatic acini

Thyroid hormones influence Ca2+ homeostasis in both skeletal and cardiac muscle. Since secretory cells, like muscle cells, store and use Ca2+ in stimulus-response coupling, we have studied the effects of thyroid status on Ca2+ mobilization and secretion in a model secretory tissue, the pancreatic acinar cell. Hyperthyroidism was induced by rats by daily, subcutaneous injections of triiodothyronine for 8 days and hypothyroidism by adding 6-n-propyl-2-thiouracil to the drinking water for 14 days. Pancreatic acini were prepared by collagenase digestion of pancreatic tissue from hyper- and hypo-thyroid animals and from euthyroid controls. Ca2(+)-mobilization was assessed using Quin-2 fluorescence and secretion by assaying amylase release. The data indicate that the amount of Ca2+ mobilized by the muscarinic agonist carbachol or by cholecystokinin octapeptide increases with increasing thyroid hormone concentrations. Only in hypothyroidism was this change in Ca2+ homeostasis reflected by a parallel change in amylase secretion. This implies the existence of some compensatory mechanism which stabilizes secretory rate in the face of stimulus-evoked increases in intracellular Ca2+ concentration.     

Effect of low doses of ethanol on platelet function in long-life abstainers and moderate-wine drinkers

In vitro, high concentrations of ethanol (EtOH) reduce platelet aggregation. Less is known about the effect of low EtOH doses on platelet function in a selected human population of long-life abstainers and low moderate-wine drinkers to avoid rebound effect of EtOH on platelet aggregation. Results of our experiments suggest that moderate-wine drinkers have higher levels of high density lipoprotein (HDL) than long-life abstainers while fibrinogen levels are unchanged. Furthermore, platelets obtained from these individuals do not differ in their response when stimulated by agonists such as AA and collagen. The effect of in vitro exposure of low doses of EtOH has been studied in PRP and in washed platelets. EtOH (0.1-10 mM) inhibits platelet aggregation induced by collagen at its ED50 while is ineffective when aggregation was triggered by U-46619 and by 1 microM adenosine diphosphate (ADP). 5-10 mM EtOH partially reduces the second wave of aggregation induced by 3 microM ADP. 0.1-10 mM EtOH dose-dependently lowers the aggregation induced by AA at its ED50 but it is less effective at ED75 of AA. The antiaggregating effect of EtOH on aggregation induced by AA is unchanged by inhibitor of nitric oxide synthase. In addition, 10 mM EtOH reduces thromboxane (Tx) formation. In washed platelets, 1-10 mM EtOH partially inhibits platelet aggregation induced by thrombin. In washed resting platelets, 10 mM EtOH does not change the resting [Ca++]i while significantly reduces the increase in [Ca++]i triggered by AA. The results of ex vivo experiments have demonstrated that wine increases the HDL. However, this observation may or may not influence the response of platelets to agonists. Results of our studies demonstrate that low doses of alcohol reduces platelet function.     

The species-specific cell-binding site of the aggregation factor from the sponge Microciona prolifera is a highly repetitive novel glycan containing glucuronic acid, fucose, and mannose

Species-specific adhesion of dissociated cells from the marine sponge Microciona prolifera is mediated by a Mr = 2 x 10(7) proteoglycan-like aggregation factor (MAF) via two highly polyvalent functional domains, a cell-binding and a self-interaction domain. Glycopeptide N-glycosidase F release of a major glycan of Mr = 6.3 gamma 10(3) (G-6) from the MAF protein core resulted in the loss of cell binding activity, indicating a role of this polysaccharide molecule in MAF-cell association. The G-6 glycan was isolated and purified after complete Pronase digestion of MAF using gel electrophoresis, gel filtration, and ion exchange chromatography. Quantification of the amount of carbohydrate recovered in G-6 showed that one MAF molecule has about 950 repeats of this glycan. In its monomeric state G-6 did not display any measurable binding to cells (K alpha less than or equal to 10(3) M-1). Intermolecular cross-linking of the G-6 glycan with glutaraldehyde resulted, however, in the concomitant recovery of polyvalency (about 2200 repeats of G-6 per polymer of Mr greater than or equal to 1.5 x 10(7) and species-specific high cell binding affinity (K alpha = 1.6 x 10(9) M-1) but not of the MAF-MAF self-interaction activity. Thus, the G-6 glycan is the multiple low affinity cell-binding site involved in cell-cell recognition and adhesion of sponge cells. The G-6 glycan has 7 glucuronic acids, 3 fucoses, 2 mannoses, 5 galactoses, 14 N-acetylglucosamines, 2 sulfates, and 1 asparagine. Such a unique chemical composition indicates a new type of structure which includes features of glycosaminolycans and N-linked polysaccharides.     

Na(+)-dependent Ca(2+) transport modulates the secretory response to the Fcepsilon receptor stimulus of mast cells

Immunological stimulation of rat mucosal-type mast cells (RBL-2H3 line) by clustering of their Fcepsilon receptors (FcepsilonRI) causes a rapid and transient increase in free cytoplasmic Ca(2+) ion concentration ([Ca(2+)](i)) because of its release from intracellular stores. This is followed by a sustained elevated [Ca(2+)](i), which is attained by Ca(2+) influx. Because an FcepsilonRI-induced increase in the membrane permeability for Na(+) ions has also been observed, and secretion is at least partially inhibited by lowering of extracellular sodium ion concentrations ([Na(+)](o)), the operation of a Na(+)/Ca(2+) exchanger has been considered. We found significant coupling between the Ca(2+) and Na(+) ion gradients across plasma membranes of RBL-2H3 cells, which we investigated employing (23)Na-NMR, (45)Ca(2+), (85)Sr(2+), and the Ca(2+)-sensitive fluorescent probe indo-1. The reduction in extracellular Ca(2+) concentrations ([Ca(2+)](o)) provoked a [Na(+)](i) increase, and a decrease in [Na(+)](o) results in a Ca(2+) influx as well as an increase in [Ca(2+)](i). Mediator secretion assays, monitoring the released beta-hexosaminidase activity, showed in the presence of extracellular sodium a sigmoidal dependence on [Ca(2+)](o). However, the secretion was not affected by varying [Ca(2+)](o) as [Na(+)](o) was lowered to 0.4 mM, while it was almost completely inhibited at [Na(+)](o) = 136 mM and [Ca(2+)](o) < 0.05 mM. Increasing [Na(+)](o) caused the secretion to reach a minimum at [Na(+)](o) = 20 mM, followed by a steady increase to its maximum value at 136 mM. A parallel [Na(+)](o) dependence of the Ca(2+) fluxes was observed: Antigen stimulation at [Na(+)](o) = 136 mM caused a pronounced Ca(2+) influx. At [Na(+)](o) = 17 mM only a slight Ca(2+) efflux was detected, whereas at [Na(+)](o) = 0.4 mM no Ca(2+) transport across the cell membrane could be observed. Our results clearly indicate that the [Na(+)](o) dependence of the secretory response to FcepsilonRI stimulation is due to its influence on the [Ca(2+)](i), which is mediated by a Na(+)-dependent Ca(2+) transport.     

Salinity affects intracellular calcium in corn root protoplasts

Previous work with the fluorescent Ca probe chlorotetracycline (CTC) showed that salinity displaces Ca from membranes of root cells. Using a variety of indirect approaches, we studied whether salinity displaces Ca from the cell surface or from internal membranes of corn (Zea mays L. cv Pioneer 3377) root protoplasts. Preloading the cells with supplemental Ca counteracted subsequent NaCl effects on CTC fluorescence. CTC quenching by exogenous EGTA was not competitive with CTC quenching by NaCl. The Ca channel reagent (+)-202-791 had significant interactions with the effect of NaCl on CTC fluorescence. The effect of NaCl on CTC fluorescence was attenuated by pretreatment with Li, but was restored by inositol. Salinity increased Na influx, decreased Ca influx, and increased Ca efflux from the cells. Fluorescence anisotropy indicated that NaCl decreased the fluidity of the external face of the plasmalemma but increased the fluidity of cell membranes in general. Our results suggest that salinity displaces Ca associated with intracellular membranes through activation of the phosphoinositide system and depletion of intracellular Ca pools.     

Effects of platelet-derived growth factor on Ca2+ in 3T3 cells

The effect of platelet-derived growth factor (PDGF) on cellular Ca2+ was examined in BALB/c-3T3 cells. PDGF induced: A decrease in cell 45Ca2+ content. An apparent increased rate of efflux of preloaded 45Ca2+. A decrease in residual intracellular 45Ca2+ remaining after rapid efflux. When added after the rapid phase of efflux of 45Ca2+ had occurred, an immediate decrease in post-efflux residual intracellular 45Ca2+. All of the observed changes in 45Ca2+ induced by PDGF are consistent with a rapid release of Ca2+ from an intracellular Ca2+ pool that has the slowest efflux and is relatively inaccessible to extracellular EDTA. When incubated with chlortetracycline (CTC), a fluorescent Ca2+ probe, 3T3 cell mitochondria became intensely fluorescent. Addition of PDGF resulted in a rapid decrease in CTC fluorescence intensity in both adherent and suspended 3T3 cells. The effects of PDGF on 3T3 cell Ca2+ stores and CTC fluorescence intensity were identical with the effects of the Ca2+ ionophore A23187 and of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone. Serum, which contains PDGF, also altered intracellular Ca2+ stores, but platelet-poor plasma, which does not contain PDGF, had no effect. EGF, insulin, and tetradecanoyl phorbol acetate (TPA), other factors which stimulate 3T3 cell growth, did not alter 3T3 cell Ca2+ stores. Release of Ca2+ from intracellular sequestration sites may be a mechanism by which PDGF stimulates cell growth.