Disparate efficacy of tobramycin on Ca(2+)-, Mg(2+)-, and HEPES-treated Pseudomonas aeruginosa biofilms.

Mucoid exopolysaccharide (MEP) obtained from Pseudomonas aeruginosa 579 was suspended in 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) pH 7.2 containing 0.1-10.0 mM of CaCl2.2H2O or MgCl2.4H2O. MEP treated with HEPES or < 5.0 mM of the Ca2+ or Mg2+ salts remained soluble and bound tobramycin in an equilibrium dialysis bioassay. MEP treated with 5.0 or 10.0 mM of the Ca2+ or Mg2+ salts did not bind tobramycin. Five and 10 mM Ca(2+)-treated MEP precipitated but Mg(2+)-treated MEP did not. Pseudomonas aeruginosa 579 biofilms formed using a defined growth medium having < 1 mM Ca2+ or Mg2+ were treated for 1 h with 10 mM HEPES +/- 5.0 mM CaCl2.2H2O or MgCl2.4H2O, prior to an 8-h exposure to HEPES, or the defined growth medium, +/- 125 micrograms/mL of tobramycin. The tobramycin kill kinetics for the HEPES-, Mg(2+)-, and Ca(2+)-treated biofilms were similar and gradual from T = 0-6 h. The viability of the HEPES- and Mg(2+)-treated populations declined sharply (from 6 to 8 h). Bacteria dispersed from the MEP in control biofilms at 0 and 8 h did not grow in the presence of 7.81 micrograms/mL of tobramycin. Thus, binding of tobramycin of P. aeruginosa 579 MEP may not be as influential to the impediment of tobramycin diffusion as is the steric hindrance imposed by the Ca2+ condensation of the polymer.     

Cyclic AMP-dependent regulation of ornithine decarboxylase activity in Chinese hamster ovary cells maintained with a salts/glucose medium.

Ornithine decarboxylase activity (ODC) increased about 7 fold 6--8 h following 10mM asparagine (ASN) addition to confluent cultures that had been previously serum deprived and then placed in a salts/glucose medium. Optimal concentrations of dibutyryl cAMP (dB cAMP) when incubated with the ASN caused up to a 50 fold increase in the activity of this enzyme after 7--8 h. The enhancement of ODC activity by ASN and dB cAMP was not sensitive to continuous (0--7 h) treatment with actinomycin D but similar treatment with cycloheximide depressed enzyme activity 40--60%. The synergistic stimulation of ODC activity by dB cAMP added with ASN was dose dependent and the dB cAMP stimulation of ODC activity displayed an absolute requirement for ASN when cells were maintained in the salts/glucose medium. The addition of dB cAMP always further enhanced ODC activity above the levels produced by addition of various levels of ASN (1 to 40mM) to the salts/glucose medium. Other agents which elevated cAMP levels such as 1-methyl-3-isobutylxanthine (IBMX) also enhanced ODC activity when administered with ASN. Additionally, treatment with sodium butyrate at concentrations ranging from 0.001mM to 5.0mM did not elevate ODC activity above the activity obtained with ASN alone. Addition of dB cAMP at various times after placing cells in salts/glucose medium with ASN further stimulated ODC activity only when added during the first 3-4 h. These results demonstrate the involvement of cAMP in the ASN mediated stimulation of ODC activity using cells maintained in a salts/glucose medium.     

Hormone-specific combinations of isoforms of adenylyl cyclase and phosphodiesterase in the rat liver

Since many isoforms of adenylyl cyclase and adenosine 3', 5'-monophosphate (cAMP) phosphodiesterase have been cloned, it is likely that receptors of each hormone have a specific combination of these isoforms. Types I, III and VIII adenylyl cyclases are reported to be stimulated by Ca(2+)-calmodulin, type I phosphodiesterase by Ca(2+)-calmodulin, but types IV and VII (cAMP-specific) phosphodiesterases by Co2+. In the present study, we examined different effects of Ca2+ and Co2+ on hormone-induced cAMP response in the isolated perfused rat liver.The removal of Ca2+ from the perfusion medium (0 mM CaCl(2 ) + 0.5 mM EGTA) did not affect glucagon (0.1 nM)-responsive cAMP but reduced secretin (1 nM)-, vasoactive intestinal polypeptide (VIP, 1-10 nM)- and forskolin (1 microM)-responsive cAMP considerably. The addition of 1 mM CoCl2 reduced glucagon- and secretin-responsive cAMP considerably, forskolin-responsive cAMP partly, did not affect 1 nM VIP-responsive cAMP, but enhanced 10 nM VIP-responsive cAMP. Forskolin- and VIP-responsive cAMP was greater in the combination (0 mM CaCl(2) + 0.5 mM EGTA + 3 mM CoCl2) than in the Ca(2+)-free perfusion alone.These results suggest that secretin, VIP1 and VIP2 receptors are linked to Ca(2+)-calmodulin-sensitive adenylyl cyclase; glucagon receptor to Ca(2+)-calmodulin-insensitive adenylyl cyclase; VIP1 receptor to Ca(2+)-calmodulin-dependent phosphodiesterase; glucagon, secretin and VIP2 receptors to cAMP-specific phosphodiesterase, respectively, in the rat liver.     

Change in the activity of calcium ions during illumination of a suspension of visual cell outer segment fragments]

Changes in the activity of calcium ions in the medium containing outer fragments suspension of bovine eye retina rods have been studied by the method of calcium-selective electrodes. Illumination of the suspension increases calcium ion activity in the incubation medium. Photoinduced yield of calcium ions depends on Ca+2 concentration: it equals 0.11+/-0.015 M Ca2+/1m rodopsin in the medium containing 0.1 mM CaCl2 and 0.046+/-0.002Ca2+/1M rodopsin in the medium containing 0.05 mM CaCl2. In the medium containing more than 10(-4) M CaCl2 both an increase and a decrease of Ca2+ ions have been observed.     

Polyamines and Ca2+ Mediate Hyperosmolal Opening of the Blood-Brain Barrier: In Vitro Studies in Isolated Rat Cerebral Capillaries

We recently presented evidence that the reversible opening of the blood-brain barrier (BBB) by the infusion of 1.6 M mannitol into the rat internal carotid artery is mediated by a rapid stimulation of ornithine decarboxylase (ODC) activity and putrescine synthesis in cerebral capillaries. We have now investigated this hypothesis further, using isolated rat cerebral capillaries as an in vitro model of the BBB. The ODC activity of cerebral capillary preparations was enriched up to 15-fold over that of the cerebral homogenate. Hyperosmolal mannitol in physiological buffer evoked a rapid (less than 15 s), concentration- and time-dependent increase in capillary ODC activity and an accumulation of putrescine and spermidine which was blocked by the specific ODC inhibitor, alpha-difluoromethylornithine (DFMO, 10 mM). Mannitol (1 M), as well as 2 M urea, evoked a two- to fivefold increase in the temperature-sensitive influx of 45Ca2+ and uptake of horseradish peroxidase (HRP) and 2-deoxy-D-[1-3H]glucose (DG), but not alpha-[1-14C]aminoisobutyrate, during a 2-min incubation. DFMO (10 mM) abolished 1 M mannitol-mediated stimulation of 45Ca2+ influx and uptake of HRP and DG, whereas 1 mM putrescine replenished capillary polyamines and reversed the DFMO effects. Mannitol (1 M)-induced stimulation of ODC activity and membrane transport processes was Ca2+-dependent and verapamil- and nisoldipine-sensitive. Phorbol myristate acetate (PMA, 10 nM), a protein kinase C activator, also evoked a two- to threefold stimulation of 45Ca2+ transport and HRP and DG uptake. This PMA effect was abolished by DFMO, suggesting involvement of rapid, ODC-controlled polyamine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of ions in activation of human lymphocytes

Phytohemagglutinin (PHA)-stimulated lymphocytes were cultured in media containing varying levels of K+, Mg2+, Ca2+. Cell activation was monitored by measuring nuclear diameter and by evaluating the area of nucleolus which reacted with silver nitrate. Decreasing extracellular K+ from normal levels (5.0 mM) to 14% (0.7 mM) and decreasing extracellular Mg2+ from normal levels (1.0 mM) to 14% (0.14 mM) did not affect nuclear diameter or silver nitrate reactivity of PHA-stimulated lymphocytes. Chelation of extracellular Ca2+ with EGTA during the first 24 h after PHA stimulation completely inhibited the increases in silver reactivity and nuclear diameter associated with stimulation. Chelation of extracellular Ca2+ 48 h after PHA stimulation did not inhibit lymphocyte stimulation. Inhibitory effects of EGTA were completely reversed if CaCl2 was added to the medium within 24 h of PHA stimulation. By 48 h the effects were irreversible.     

Insulin action in isolated fat cells. I. Effects of divalent cations on the stimulation by insulin of glucose uptake.

The effects of divalent cations, in particular Ca2+ and Mg2+, on glucose uptake by rat isolated fat cells in the presence and absence of insulin have been studied. EDTA (disodium salt) was used to deplete the bovine serum albumin present in the incubation medium of endogenous divalent cations prior to incubation with the cells, but was not present in the incubation medium during the incubation of the cells. The removal of Ca2+ and Mg2+ from the incubation medium did not affect the basal glucose uptake, but abolished the ability of insulin to stimulate glucose uptake by the cells. Addition of 25 microM MgCl2 or CaCl2 to the incubation medium restored a significant insulin stimulation, and this stimulation was maximal when 0.1 mM MgCl2 or CaCl2 had been added. SrCl2 and BaCl2 were also effective in restoring the insulin stimulation, but did not substitute fully for Ca2+ and Mg2+ in the incubation medium. Possible explanation for these observations are discussed.     

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 effects of alpha- and beta-adrenergic agents, Ca2+ and insulin on 2-deoxyglucose uptake and phosphorylation in perfused rat heart

Insulin (0.1 microM) and 1 microM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent Vmax without altering the Km. Isoproterenol (10 microM), 50 microM methoxamine and 10 mM CaCl2 also increased uptake. Lowering of the perfusate Ca2+ concentration from 1.27 to 0.1 mM Ca2+, addition of the Ca2+ channel blocker nifedipine (1 microM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 microM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 microM insulin was only partly inhibited by Ca2+ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca2+, respectively. Maximal concentrations of insulin (0.1 microM) and epinephrine (1 microM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 microM but maximal concentrations of epinephrine (e.g., 1 microM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca2+ in signal reversal was also studied. Removal of 1 microM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca2+ concentration was lowered to 0.1 mM or nifedipine (1 microM) was added. Similarly, removal of 0.1 microM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca2+ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca2+ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca2+-dependent increase in 2-deoxyglucose transport from either alpha or beta receptors. Insulin has both a Ca2+-dependent and a Ca2+-independent component. Reversal studies suggest an additional role for Ca2+ in maintaining the activated transport state when activated by either epinephrine or insulin.     

Azaspiracids modulate intracellular pH levels in human lymphocytes

The azaspiracids (AZAs) are a group of marine toxins implicated in several intoxications whose mechanism of action is unknown. These phycotoxins include the five compounds shown in : AZA-1 (1), AZA-2 (2), AZA-3 (3), AZA-4 (4), and AZA-5 (5). The aim of this work was to study the effects of the five naturally occurring azaspiracids (AZA-1 to -5, Fig. 1) and four synthetic analogues (6-9, Fig. 2) on intracellular pH, and the influence of Ca2+ upon this effect. The AZAs (1-5) were found to modulate cytosolic Ca2+ levels in human lymphocytes, while some of them, but not all, had effects on the intracellular pH. AZA-1 (1) and AZA-2 (2) did not modify intracellular pH in a Ca2+-containing or a Ca2+-free medium. AZA-3 (3) increased intracellular pH by 0.16 units in the presence of extracellular Ca2+, an effect that was blocked when a 1 mM solution of Ni2+ was added. In a Ca2+-free medium, the increase in pH induced by AZA-3 (3) was reduced to 0.08 pH units. AZA-4 (4) inhibited the basal pH increase even in the presence of a 1 mM solution of Ni2+. In a Ca2+-free medium, the inhibition caused by AZA-4 (4) was small, but when Ca2+ was added back to the medium, the pH basal increase was again significantly inhibited. The alkalinization was also inhibited when AZA-4 (4) was added simultaneously, 10 min before or 10 min after thapsigargin (Tg), and also when the Ca2+-influx induced by Tg was inhibited by Ni2+. AZA-5 (5), on the other hand, did not modulate the intracellular pH profile in either a Ca2+-containing or a Ca2+-free medium. Finally, we investigated four synthetic analogues (6-9, Fig. 2) whose structures were based on the four originally proposed structures of azaspiracid-1, with an opened E-ring. Compound 6 induced a small cytosolic Ca2+ increase, but did not modify intracellular pH in saline solution. In a Ca2+-free medium, compound 6 blocked the pH fall when Ca2+ was added back to the medium. Compound 7 also did not modify intracellular pH in saline solutions, however it significantly blocked basal pH increases in a Ca2+-free medium. Compound 8 did not alter intracellular pH, however compound 9 induced a small acidification when Ca2+ was present in the extracellular medium. These results point to a structure-activity relationship in AZAs pH effect that affects the modulation and the coupling of intracellular pH and Ca2+.     

Calcium and contractions of isolated smooth muscle cells from rat myometrium

Smooth muscle cells were isolated from estrogenized rat myometrium by collagenase digestion. Electron microscopic examination and measurement of cell lengths by image-splitting micrometry were carried out after fixation with acrolein. Mean lengths of cells before and after isolation were 81.7 and 66.9 micron, respectively. Responses of cells were compared with contractions of isolated strips recorded isometrically. Effects of carbachol and KCl were examined in 2 mM Ca, 2 mM Ca + 4 mM EGTA, and 2 mM Ca + 10(-8) M nitrendipine solution. Carbachol and KCl produced concentration-dependent shortening of isolated cells maximal at 30 s after addition. The concentrations of carbachol required to produce shortenings were about 100-fold less than those required to produce isometric contractions; but no major difference was observed in the concentration dependence of cell shortening and isometric contraction produced by potassium-induced depolarization. In 2 mM Ca solution, there was a phasic response, followed by a tonic response such that more than 50% of maximum cell shortening was maintained for 10 min. However, in 2 mM Ca + 4 mM EGTA or 10(-8) M nitrendipine, the tonic contraction was abolished and cells rapidly relaxed after 30 s. If carbachol was added to cells after varying times in the EGTA-containing solution, the ability to initiate a contraction declined exponentially with a half-time of 160 s. Effects of depolarization by KCl were examined in 2 mM Ca plus nitrendipine and 2 mM Ca + 4 mM EGTA solution. Shortening occurred in 2 mM Ca solution by depolarization but not if nitrendipine was added. Though shortening was not observed in 2 mM Ca + 4 mM EGTA solution by KCl, subsequent addition of carbachol induced shortening. These results suggested that there was an intracellular Ca store site from which Ca was released by carbachol and which was not affected by depolarization in the absence of external Ca. No evidence was obtained that the contraction persists in Ca2+-free medium in isolated cells, which is in agreement with previous findings in small muscle strips in which only a similar transient response was obtained.     

Degranulation of rough endoplasmic reticulum in M phase and adenosine-triphosphate-treated interphase cells is reversible.

In the preferential harvesting of rounded mitotic (M phase) cells of human Chang liver monolayer cultures by mechanical agitation in Ca(2+)-free phosphate-buffered saline, degranulation of endoplasmic reticulum (ER) was observed. Mitotic cells are known to have a series of Ca2+ transients and, without being subjected to Ca(2+)-free washings, did not have degranulated ER. Quiescent cells incubated with 0.7 mM adenosine 5'-triphosphate (ATP) in Ca(2+)-free HEPES-buffered saline produced very similar ER degranulations. Confocal argon laser imaging of fluo-3-loaded cells showed a Ca2+ transient peaking at 2 min after ATP treatment. In the absence of extracellular Ca2+, transients of Ca2+ elevation in the cytosol would exit the cell in a down-gradient, draining the ER Ca2+ stores. Substituting ATP with 1 microM brominated A23187 calcium ionophore in the incubation that contained 1-100 mM CaCl2, respectively, did not produce ER degranulation, thereby excluding raised cytosolic Ca2+ per se as the cause of ER degranulation. In fact, incubation with 0.7 mM ATP in the presence of 1-5 mM CaCl2 failed to produce ER degranulation. ER degranulated cells, from treatment with ATP without extracellular Ca2+ as well as from Ca(2+)-free washings at M phase, could be rescued by subsequent incubation in growth medium that contains Ca2+ whereupon the rounded cells re-flatten (a round-to-flat change) and have well-defined rough ER. It therefore seems possible for Ca2+ depletion, or at least a reduction, to be causally related to ER degranulation. If that were the case, ER granularity would appear to be a facultative rather than a constitutive state.     

Caffeine effects on buccal muscles of Aplysia

1. Caffeine (35-70 mM) elicited contractions of Aplysia buccal muscle El. In a Ca2+-free medium, in which ACh-elicited contractions rapidly fail, caffeine elicited contractions of approximately the same size as in normal medium. 2. 5-HT (10(-8) M and 10(-7) M) did not enhance caffeine-elicited contractions. 3. Lower concentrations (1-10 mM) of caffeine inhibited ACh-elicited contractions. Caffeine (7 mM) reduced the contraction by 80%. 4. Caffeine (7 mM) reduced ACh-elicited depolarization by 60%. 5. Caffeine (7 mM) increased 45Ca2+ influx into Aplysia buccal muscle I5. The stimulation of influx of 45Ca2+ by 10(-3) M ACh was non-additive with the stimulation caused by caffeine, and 7 mM caffeine reduced the influx caused by 10(-3) M ACh.     

Two thrombin-activated Ca2+ channels in human platelets.

The regulation of extracellular Ca2+ entry into fura-2-loaded human platelets was examined following stimulation with thrombin. In the presence of external Ca2+, stimulation of platelets with thrombin resulted in a rapid increase, followed by a plateau, in intracellular Ca2+ concentration ([Ca2+]i). Pretreatment with wortmannin, a specific inhibitor of myosin light chain kinase, suppressed only the plateau phase and had no effect on the initial rapid increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thrombin induced a transient and relatively small increase in [Ca2+]i caused by Ca2+ release from internal stores. When Ca2+ was added subsequently to the Ca(2+)-free medium within 10 min after thrombin activation, a marked increase in [Ca2+]i was seen, reflecting thrombin-stimulated external Ca2+ entry. With the Ca(2+)-free medium, wortmannin did not affect either the Ca2+ mobilization from the internal stores or the rapid external Ca2+ entry at early time points (within 5 s) after thrombin stimulation, whereas it significantly inhibited Ca2+ entry when Ca2+ was added later (at 3 min). Wortmannin inhibition of this late Ca2+ entry and that of 20-kDa myosin light chain phosphorylation after thrombin stimulation were dose- and preincubation time-dependent and correlated well with each other. These results suggest that two different channels are responsible for Ca2+ entry in human platelets at the early and late phases of thrombin stimulation and that the channel responsible for the late phase of Ca2+ entry may be activated by a mechanism involving myosin light chain kinase.     

Calcium dependence of human sperm fertilizing ability

The Ca2+ dependency of human-sperm fertilizing ability was investigated using a modified Tyrode's medium either containing 2.4 mM CaCl2 (CA medium) or with the CaCl2 replaced by SrCl2 and 0.1 mM EGTA added to chelate any residual Ca2+ ions (SREG-medium). Ten washed sperm populations incubated in either medium for 0, 6, and 22 hours showed the same occurrence of acrosome reactions (by fluorescent lectin labelling and triple stain). A further 3-hour incubation after washing into fresh CA medium resulted in only a slight increase in acrosome reactions in both media. Eight sperm populations preincubated overnight in CA and SREG media were coincubated for 1 hour with previously salt-stored human zonae pellucidae also in the same media. Significantly more motile spermatozoa were bound to more of the zonae in CA medium (53.9% vs. 27.6% of zonae with 13.8 vs. 4.3 sperm/bound zona). In three hamster egg penetration test (HEPT) experiments, sperm populations preincubated overnight in either CA or SREG media were coincubated with hamster oocytes prepared in the same media. Only 2.1% of oocytes (1.0 polyspermy) were penetrated in SREG medium, cf., 30.9% of oocytes (1.3 polyspermy) in CA medium. These results demonstrate that while Sr2+ ions can substitute fully for Ca2+ in the capacitation and acrosome reaction of human spermatozoa, sperm-zona, and sperm-oolemma interaction seem to involve some more Ca2+-specific process(es). Furthermore, the increased HEPT fertilizing ability of human spermatozoa using overnight preincubation in SREG medium and CA medium for the test cannot be explained on the basis of differential kinetics of capacitation or the acrosome reaction.     

Sickle-cell membrane-bound (Ca2+ + Mg2+)-ATPase: activation by 3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-6-butyric acid, a novel antisickling agent

The effects of 3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-6-butyric acid (DBA), an antisickling agent, on the rates of Ca2+-dependent ATP hydrolysis by the human red cell (Ca2+ + Mg2+)-ATPase, have been studied in membranes (normal and sickle-cell) stripped of endogenous calmodulin. The activity of the enzyme is increased by DBA in a manner which is dependent on both the concentrations of DBA and Ca2+. At 37 degrees C, the normal red cell (Ca2+ + Mg2+)-ATPase activity is stimulated maximally by 133% in the presence of 1 mM DBA and 0.2 mM CaCl2, while the sickle-cell enzyme is stimulated maximally by 81% in the presence of 0.5 mM DBA and 0.2 mM CaCl2. The stimulation of the enzyme in both systems is antagonized by increasing the CaCl2 concentration in the medium to 0.5 mM, in contrast to the well established mode of activation by the modulator protein, calmodulin. This suggests that the two effectors, DBA and calmodulin, probably act by different mechanisms. From our present observations, we suggest that the antisickling effect of DBA may be connected with the mobilization of calcium within red cells.     

Studies on the mechanism of Ca2+ stimulation of plasminogen activator synthesis/release by Swiss 3T3 cells.

Stimulation of postconfluent Swiss 3T3 cells in serum-free medium with 4.3 mM Ca2+ results in marked increases in both released and cell-associated plasminogen activator (PA). Increased release of PA commenced approximately 10 to 12 hours post-stimulation and continued to increase steadily until 48 hours at which time the stimulates cells (4.3 mM Ca2+) released approximately 14 times more PA than control cells (1.8 mM Ca2+). Sr2+, like Ca2+, also stimulates PA synthesis/release either in the presence or in the absence of 1.8 mM Ca2+ whereas an excess of Mg2+ inhibits Ca2+ stimulation. Supranormal [Pi] in the medium stimulates PA synthesis/release in the presence of 1.8 mM mM Ca2+. Further, optimal stimulation by 4.3 mM Ca2+ requires a normal level of Pi (1.0 mM). Elevation of medium [Ca2+] or [Pi] results in an enhanced uptake of Ca2+. The facts that cycloheximide treatment completely abolishes the Ca2+ stimulatory effect and that an increase in cell associated PA precedes release indicate that PA release is coupled to synthesis of new PA. Ca2+ stimulation of PA synthesis/release also requires continuous energy production and RNA as well as protein synthesis. A hypothesis is proposed to explain the relationship between stimulation of PA production and its enhanced release from cells stimulated by elevated [Ca2+] or [Pi] in the media. The possibility that PA release may be an example of the phenomenon of membrane shedding as opposed to secretion is discussed.     

Induction of mouse epidermal ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate: dependence on calcium availability

The role of calcium in epidermal ornithine decarboxylase (ODC) induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) was determined in adult mouse skin pieces incubated in serum-free minimal essential medium (MEM). Addition of TPA to skin pieces incubated in serum-free MEM, which contains 1.82 mM Ca2+ and 0.83 mM Mg2+, resulted in about a 200-fold increase in epidermal ODC activity at about 8 h after TPA treatment. TPA failed to induce epidermal ODC in skin pieces incubated in calcium-free medium. Similarly, chelation of extracellular calcium by ethyleneglycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented ODC induction by TPA, which could be resumed upon calcium restoration in the medium. Furthermore, calcium ionophore A23187, which facilitates efflux of Ca2+ across cellular membranes, induced ODC activity in incubated skin pieces. Epidermal ODC activity increased by TPA appears to be the result of an increase in both the amount of ODC protein and the level of hybridizable ODC messenger. Inhibition of the induction of ODC activity by EGTA was the result of the inhibition of the amount of active ODC protein and the level of ODC mRNA.     

Flocculation and adsorption of enzymes during growth of a moderate halophile, Micrococcus varians var. Halophilus.

Flocculation of a moderate halophile, Micrococcus varians ATCC 2197, occurred during growth in complex medium containing 3 M NaCl and a concentration of MgSO4 and KH2PO4 greater than 40 and 14 mM, respectively. Extracellular nuclease activity was absent in the flocculated cultures. Repeated washing of flocs by Mg2+-free Tris buffer containing 3 M NaCl, lowering of pH value of floc suspension below 6.3, or addition of ethylenediaminetetraacetic acid resulted in complete dissociation of the flocs and release of Mg2+ ions as well as nuclease and amylase. Inhibition of extracellular enzyme production accompanied by flocculation appeared to be the result of adsorption of enzyme proteins to surfaces of the flocs, but not of inhibition of biosynthesis. Floc formation could also occur in media containing 18 mM CaCl2 and 3.0 mM KH2PO4, but the Ca flocs were not deflocculated by washing with Ca2+-free buffer, suggesting that the affinity of Ca2+ for cell envelopes was stronger than that of Mg2+. It was also observed that most halophilic Planococcus and Micrococcus flocculated in the presence of MgSO4 and phosphate but halophilic Pseudomonas, Acinetobacter, and Bacillus did not.