Restriction of calcium uptake in normal and sickle red cells by procaine hydrochloride and P-aminobenzoic acid.

A reduced capacity for calcium uptake by human red cells after procaine hydrochloride and p-aminobenzoic acid treatment has been measured by atomic absorption spectrophotometry and X-ray microprobe techniques. Cell-associated calcium was lower by 45% and 38% in deoxygenated sickle cells pre-exposed to 10 mM procaine HCl and PABA and incubated 2 hr. at 37°C in plasma, as compared with untreated cells. Prior exposure of washed red cells to 10 mM PABA reduced the intracellular calcium level by 39% after incubation for 45 min. at 37°C with 40 μM calcium ionophore A23187. Procaine HCl was more efficient than PABA in inhibiting calcium uptake at equimolar concentrations.     

Excitation-contraction coupling in cardiac Purkinje fibers. Effects of cardiotonic steroids on the intracellular [Ca2+] transient, membrane potential, and contraction

The [Ca2+]-activated photoprotein aequorin was used to measure [Ca2+] in canine cardiac Purkinje fibers during the positive inotropic and toxic effects of ouabain, strophanthidin, and acetylstrophanthidin. The positive inotropic effect of these substances was associated with increases in the two components of the aequorin signal, L1 and L2. On the average, strophanthidin at 10(-7) M produced steady, reversible increases in L1, L2, and peak twitch tension of 20, 91, and 240%, respectively. This corresponds to increases in the upper-limit spatial average [Ca2+] from 1.9 X 10(-6) M to 2.1 X 10(-6) M at L1 and from 1.4 X 10(-6) M to 1.8 X 10(-6) M at L2. Elevation of diastolic luminescence above the control level was not detected. At higher concentrations (5 X 10(-7) M), strophanthidin produced aftercontractions, diastolic depolarization, and transient depolarizations, all of which were associated with temporally similar changes in [Ca2+]. During these events, diastolic [Ca2+] rose from the normal level of approximately 3 X 10(-7) M up to 1-2 X 10(-6) M. The negative inotropic effect of 5 X 10(-7) M strophanthidin was not associated with a corresponding decrease in the [Ca2+] transient but was associated with a change in the relationship between [Ca2+] and tension. Assuming the Na+-lag mechanism of cardiotonic steroid action, we conclude the following: at low concentrations of drug, increased Ca2+ uptake by the sarcoplasmic reticulum prevents a detectable rise in cytoplasmic [Ca2+] during diastole, but this increased Ca2+ uptake results in increased release of Ca2+ during the action potential. At higher drug concentrations, observable [Ca2+] changes during diastole activate tension and membrane conductance changes.     

influence of Ca channel blockers on the positive inotropic effect of ionophore A23187 in the myocardium

Calcium ionophore A23187 (10(-5) M) increases the force of contraction of the frog atrium up to 27 + 4.8%. The calcium antagonists d-600 (5 X 10- M), Zn2+ (2 X 10(-5) M), Mn2+ (2 X 10(-4) M) decrease the force of contraction 50, 10 and 20%, respectively, and inhibit the positive inotropic effect of ionophore A23187. Inhibition of the ionophore effect by the blockers is determined by the ability of D-600, Zn2+ and Mn2+ to form complexes with the ionophore. Besides, the affinity of these blockers to the ionophore is higher than that of Ca2+. It is assumed that Zn2+, Mn2+ and D-600 possess higher affinity to ionophore A23187 as compared with myocardial Ca-channels. Fenigidin interacts with Ca-channels to a larger degree than with ionophore A23187.     

Modulation of Calcium Fluxes Across Synaptosomal Plasma Membrane by Local Anesthetics

We have studied the effects of local anesthetics (dibucaine, tetracaine, lidocaine, and procaine) on calcium fluxes through the plasma membrane of synaptosomes. All these local anesthetics inhibit the ATP-dependent calcium uptake by inverted plasma membrane vesicles at concentrations close to those that promote an effective blockade of the action potential. The values obtained for the K0.5 of inhibition of calcium uptake are the following: 23 microM (dibucaine), 0.44 mM (lidocaine), 1.5 mM (procaine), and 0.8 mM (tetracaine). There is a good correlation between these K0.5 values and the concentrations of the local anesthetics that inhibit the Ca2(+)-dependent Mg2(+)-ATPase of these membranes. In addition, except for procaine, these local anesthetics stimulate severalfold the Ca2+ outflow via the Na+/Ca2+ exchange in these membranes. This effect, however, is observed at concentrations slightly higher than those that effectively inhibit the ATP-dependent Ca2+ uptake, e.g., 80-700 microM dibucaine, 2-10 mM lidocaine, and 1-3 mM tetracaine. The results suggest that the Ca2+ buffering of neuronal cytosol is altered by these anesthetics at pharmacological concentrations.     

Head-to-head association in bovine spermatozoa induced by catecholamines

Noradrenaline, adrenaline, and isoproterenol induce head-to-head association in bovine spermatozoa in a Tris-HCl-buffered medium. Noradrenaline was the most and isoproterenol the least efficient. This effect was stimulated by Ca2+, Mg2+ and Mn2+, Ca2+ being more efficient than the other two ions. At 2 X 10(-6) M Ca2+, oxidation products of adrenaline dissociated spermatozoa associated by washing; at 2 X 10(-5) M Ca2+, the dissociating effect was transformed into association. The induction of association by adrenaline was blocked by both alpha- and beta-adrenergic blockers at low concentrations (2 X 10(-7) M). Both cAMP and dibutyryl substituted cAMP (db-cAMP) induced association in the Tris-buffered medium at 2 X 10(-6) M Ca2+. Further increase in association was brought about by increasing the Ca2+ concentration to 2 X 10(-5) M. Prolongation of the treatment with cAMP increased association. When combined with cAMP under the same conditions as used in the combination with adrenaline, L-propranolol did not inhibit association induced by cAMP. In an identical experiment, performed in Tyrode solution, L-propranolol inhibited association induced by cAMP. At 2 X 10(-5) M, theophylline, caffeine, and papaverine induced association in the presence of 2 X 10(-5) M Ca2+. The results are compatible with the hypothesis that catecholamines act via receptors and formation of cAMP.     

The divalent cation dependence of bovine brain calmodulin-dependent phosphatase

The divalent cation dependence of a calmodulin-stimulated phosphatase from bovine brain has been characterized kinetically using phosphorylated myelin basic protein and casein as substrates. At saturating concentrations of calmodulin, dephosphorylation of both myelin basic protein and casein was catalyzed 8- to 10-fold more rapidly at saturating concentrations of Mn2+ than at saturating concentrations of Ca2+. Half-maximal rates of dephosphorylation of both substrates occurred at either 15 microM Mn2+ or 1 microM Ca2+, and the Kact for each ion was not influenced appreciably by the presence of calmodulin. Half-maximal rates of dephosphorylation were observed at concentrations of calmodulin ranging from 3 X 10(-8) to 10(-6) M at saturating concentrations of divalent cations depending on the substrate used and the particular cation chosen. Trypsin treatment of the phosphatase activated the enzyme several-fold, eliminated its calmodulin dependence, but did not alter the Mn2+ concentration dependence of the activity. Ca2+ (10 microM) increased dephosphorylation rates without altering the Mn2+ concentration dependence of the phosphatase activity regardless of the presence of calmodulin. Mg2+ at millimolar concentrations did not alter the Ca2+ or Mn2+ concentration dependence of the activity. As measured without calmodulin, Ca2+ (90 microM) or Mn2+ (200 microM) produced nearly identical alterations of the far ultraviolet circular dichroic spectrum of the phosphatase.     

Metal ion binding properties of hen ovalbumin and S-ovalbumin: characterization of the metal ion binding site by 31P NMR and water proton relaxation rate enhancements

In this study, water proton relaxation rate (PRR) enhancements have been used to characterize the binding of metal ions to native ovalbumin, ovalbumin in which phosphate has been enzymatically cleaved from one or both of the two protein phosphoserines, and a heat-stabilized form of the protein (S-ovalbumin). With Scatchard plots constructed from water PRR enhancements, it was found that native ovalbumin and S-ovalbumin had one strong binding site for Mn2+ ion (KD approximately equal to 6.0 X 10(-4) M). Alkaline phosphatase treated ovalbumin, a protein having a single phosphoserine, had one Mn2+ binding site of slightly weaker affinity (KD approximately equal to 8.3 X 10(-4) M), while acid phosphatase treated ovalbumin, a dephosphorylated protein, had two much weaker Mn2+ ion binding sites (KD approximately equal to 1.3 X 10(-3) M). Competitive binding studies on the native protein suggested that Zn2+ ion competes with Mn2+ for the single strong-affinity site (KD approximately equal to 6.1 X 10(-3) M) while Mg2+ and Ca2+ do not. In a second set of experiments, the paramagnetic contribution to the 31P spin-lattice (T1P) and spin-spin (T2P) relaxation times at three separate magnetic field strengths was measured. Correlation times tau c characterizing Mn2+-31P dipolar relaxation were estimated from the ratios of T1P/T2P at a single field and from the ratios of spin-lattice relaxation rates at three different field strengths. The correlation times so obtained, ranging from about 0.7 to 7.7 ns at the three field strengths, were used in calculating distances from the bound Mn2+ ion to the phosphoserines of native ovalbumin, S-ovalbumin, and alkaline phosphatase treated ovalbumins. It was determined that the phosphate of phosphoserine-68 was 5.95 +/- 0.26 and 6.29 +/- 0.18 A from the Mn2+ in the native and alkaline phosphatase treated protein, respectively, and 6.99 +/- 0.30 A away from the Mn2+ in S-ovalbumin. The phosphate of phosphoserine-344 was determined to be 5.31 +/- 0.20 and 5.75 +/- 0.10 A from the Mn2+ ion in native ovalbumin and S-ovalbumin, respectively. The 13C nucleus of [1-13C]galactose enzymatically transferred to the nonreducing end of the ovalbumin oligosaccharide chain was not found to be significantly relaxed by Mn2+ bound to the protein, even at 1:1 stoichiometric ratio of metal:protein. Using this, we estimate the nonreducing terminal of the ovalbumin oligosaccharide to be at least 39 A from the metal ion binding site on the protein.     

Studies on phosphate transport in Escherichia coli. II. Effects of metabolic inhibitors and divalent cations.

1. Study has been made of the effects of a variety of metabolic inhibitors and divalent cations (Ni2+ and Mn2+), normally after 5 min exposure, on the biphasic uptake of inorganic phosphate (Pi) exhibited by phosphate-deprived cells of Escherichia coli, strains AB3311 (Reeves met-) and CBT302 (a (Ca2+ + Mg2+)-ATPase-deficient mutant). 2. In AB3311 cells cyanide (1-10 mM) produced comparable reductions in phosphate uptake to anaerobiosis, but in both instances significant uptake was maintained. Examination of intracellular Pi concentrations showed that, despite these inhibitions, Pi is still concentrated 130 times compared to 394 times under aerobic conditions. Arsenate (100 muM) and iodoacetate (100 muM pre-exposed 15 min) both abolished anaerobic-supported uptake. Under aerobic conditions the former eliminated primary uptake while the latter reduced both phases of uptake 60%. The uncouplers, dinitrophenol (100-1000 muM) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (50muM) produced very significant, but not complete inhibitions of both phases of uptake. Inhibitions by iodoacetate and dinitrophenol were additive while dithiothreitol protected against the effects of 50-250 mum CCCP. N,N'-Dicyclo-hexylcarbodiimide (DCCD), the potent inhibitor of membrane-bound (Ca2+ + Mg2+)-ATPase, at 10(-3) M caused significant inhibitions of aerobic- (approx. 60%) and anaerobic- (approx. 80%) supported uptakes thus suggesting some obligatory requirement for this ATPase. 3. CBT302 cells, like AB3311, supported Pi transport both aerobically and anaerobically. CCCP (50muM) reduced the primary uptake similarly to AB3311 cells, but the secondary uptake was less affected. DCCD (10(-5)-10(-3) M), as expected, showed no effects in contrast to AB3311 cells. 4. In AB3311 cells Ni2+ (10 mM) caused significant but different reductions of secondary (70%) and primary (33%) phases of phosphate uptake. Mn2+ (10 mM) showed a greater differential effect with the primary uptake being minimally affected and the secondary uptake being abolished (97%). Partial relief of these inhibitions by Mg2+ (10 mM), suggested that these ions compete with Mg2+ transport. High voltage electrophoresis studies showed that Ni2+ cause intensification in the labelling from 32Pi (i.e. during Pi uptake) of hexose phosphates and a reduction in the labelling of complex molecules left at the origin. With Mn2+, labelling of fructose 1,6-diphosphate was reduced, the triose phosphate area was intensified and an unknown area (X) was intensely labelled. When Mn2+ was combined with anaerobiosis, phosphate uptake though diminished in rate exceeded after 16 min the plateau level of uptake under aerobic conditions with Mn2+ present.     

Uptake of adrenaline by rat brain synaptosomes. Effects of several psychotropic drugs]

Comparative study of the uptake of 3H-epinephrine (3H-EN) and 3H-norepinephrine (3H-NE) into rat brain crude synaptosomes and effect of psychotropic drugs of different classes on this process showed that isolated nerve terminals had their own transport system for EN. The crude synaptosomal fraction had two transport system's for EN; high-specific active uptake with high affinity (KM = 3.7 + 0.21 microM) and low-affinity uptake (KM2 = 98.0 + 47.5 microM). En accumulation was saturable, stereo-specific and inhibited by ouabain (3 X 10(-3) M), protoveratrine A and B (10(-4) M), NaN3 (2 X 10(-3) M), 2,4-dinitrophenol (2 X 10(-3) M), p-chloromercuribenzoate (10(-4) M). Actinomycin D had no effect on the uptake of 3H-EN. 3H-HE was accumulated by two uptake system: 1-high affinity uptake system with KM values of 0.49 + 0.13 microM, 2-low affinity uptake system with KM values of 21.1 + 7.71 microM. Amphetamine, mesocarb, chlorpromazine, fluphenazine and haloperidol were equally effective inhibitors of 3H-EN and 2H-HE uptake. Imipramine, phenazepam, diazepam and carbamazepine (5 X 10(-5) M) had no effect on the uptake of 3H-NE. Imipramine, zimelidine, norzimelidine and viloxazine (5 X 10(-5) M) were more potent inhibitors of the 3H-EN uptake than that of 3H-NE.     

BAY K 8644, a 1,4-Dihydropyridine Ca2+ Channel Activator: Dissociation of Binding and Functional Effects in Brain Synaptosomes

K+-stimulated 45Ca2+ uptake into rat brain and guinea pig cerebral cortex synaptosomes was measured at 10 s and 90 s at K+ concentrations of 5-75 mM. Net increases in 45Ca2+ uptake were observed in rat and guinea pig brain synaptosomes. 45Ca2+ uptake under resting or depolarizing conditions was not increased by the 1,4-dihydropyridine BAY K 8644, which has been shown to activate Ca2+ channels in smooth and cardiac muscle. High-affinity [3H]nitrendipine binding in guinea pig synaptosomes (KD = 1.2 X 10(-10) M, Bmax = 0.56 pmol mg-1 protein) was competitively displaced with high affinity (IC50 2.3 X 10(-9) M) by BAY K 8644. Thus high-affinity Ca2+ channel antagonist and activator binding sites exist in synaptosome preparations, but their relationship to functional Ca2+ channels is not clear.     

Possible mechanisms of positive inotropic action of synthetic human calcitonin gene-related peptide in isolated rat atrium

The effect of synthetic human calcitonin gene-related peptide (hCGRP) on the isolated and electrically driven left atria of rats were investigated. The peptide at concentrations of 3 X 10(-9)-3 X 10(-7) M produced positive inotropic effects on the left atria in a dose-dependent manner. Verapamil (10(-5) M) and adenosine (10(-4) M) reduced the positive inotropic effect of hCGRP at concentrations of 3 X 10(-9) and 3 X 10(-8) M, but not at that of 3 X 10(-7) M. Ouabain (5 X 10(-5) M) inhibited the effect of hCGRP in concentrations of 3 X 10(-7) and 3 X 10(-8) M, but not in that of 3 X 10(-9) M. Simultaneous pretreatment with verapamil (10(-5) M) and ouabain (5 X 10(-5) M) suppressed the positive inotropy by hCGRP at all concentrations tested. On the other hand, tetrodotoxin (10(-6) M) potentiated only the positive inotropic effect of 3 X 10(-7) M hCGRP. Metoprolol (10(-7) M) and theophilline (10(-3) M) did not affect the inotropic effect of hCGRP. These results suggest that the positive inotropic effect of hCGRP is not mediated by beta-adrenoceptors but by two distinct mechanisms of action, which was inhibited by verapamil but not by ouabain (facilitation of Ca++ influx in lower concentrations of hCGRP) and which was blocked by ouabain but not by verapamil and potentiated by tetrodotoxin (inhibition of Na+/Ca++ exchange mechanism at higher concentrations of hCGRP).     

Cytoskeletal control of alanine transport across the rat and turtle small intestine

Procaine inhibited significantly (P less than 0.01) alanine accumulation in the rat intestinal strips in a concentration-dependent pattern, whereas it showed no effect on alanine uptake by the turtle intestinal cells. Colchicine and Vinca alkaloids at 5 X 10(-4) and 1.5 X 10(-6) M respectively caused a significant inhibition (P less than 0.01) of intracellular alanine concentration in the rat with no effect noticed in the turtle. Unidirectional influx of alanine across the brush border membrane of the rat was significantly (P less than 0.01) reduced in the presence of procaine, colchicine and vincristine in the preincubation medium. The same drugs did not show any effect on alanine influx into the turtle small intestine. Electron microscopy showed major structural alterations in the cytoskeletal organization of the turtle intestine in response to procaine, colchicine or vincristine treatment. It is proposed that microtubular system may participate in the overall transport mechanism of alanine across the small intestine.     

45Ca2+ uptake in rat brain neurons: absence of sensitivity to the Ca2+ channel ligands nitrendipine and Bay K 8644

K+-stimulated 45Ca2+ uptake into intact rat brain cells was biphasic, consisting of a fast first phase and a slow second phase; the latter was Na+ dependent. Cobalt and cadmium at 10(-4) and 10(-3) M produced 19-97% block of first phase 45Ca2+ uptake, but nitrendipine (to 10(-6) M) and Bay K 8644 (to 10(-6) M) were without effect on uptake and were similarly without effect in cells prepared in the presence of ATP, cAMP, Mg2+, and protease inhibitors. The second phase of K+-stimulated 45Ca2+ uptake was inhibited by 3,4-dichlorobenzamil (IC50, 29.6 microM). Depolarization-induced 45Ca2+ uptake into intact rat brain cells occurs by at least two different mechanisms. The first phase probably represents uptake through 1,4-dihydropyridine-insensitive Ca2+ channels, while the second phase is probably due to Na+-Ca2+ exchange.     

The effect of prostaglandins E1, E2, F1 alpha and F2 alpha on pig erythrocytes during haemolysis induced with aspirin and hypotonic NaCl solution

The effect of prostaglandins PGE1, PGE2, PGF1 alpha and PGF2 alpha was investigated on the haemolysis of pig erythrocytes induced with aspirin and hypotonic (0.119 M) NaCl solution. An inhibiting effect was observed of low concentrations (2 X 10(-5) M, 2 X 10(-4) M and 2 X 10(-3) M) of aspirin on haemolysis induced with hypotonic NaCl solution, while in a concentration of 2 X 10(-2) M aspirin itself caused haemolysis which amounted to 93% of the haemolysis induced with 0.041 M NaCl solution. No differences were observed in the degree of haemolysis inhibition in relation to the time of incubation of erythrocytes with aspirin. Aspirin concentrations from 0.035 M to 0.280 M caused slight haemolysis (9-15% of the haemolysis induced with water), the 0.560 M solution caused haemolysis corresponding to 85% of the water-induced haemolysis. None of the studied prostaglandins used in concentrations of 0.4 X 10(-3) M, 0.4 X 10(-4) M and 0.4 X 10(-5) M had any significant effect on aspirin-induced haemolysis. PGE1 and PGE2 in concentrations of 0.4 X 10(-3) M, 0.4 X 10(-4) M and 0.4 X 10(-5) M inhibited haemolysis induced with 0.119 M sodium chloride solution, and the degree of haemolysis inhibition was from 8% to 35%. Prostaglandins PGF1 alpha and PGF2 alpha in the same concentrations had no protective effect.     

Possible role of calmodulin in the control of histamine release from human basophil leukocytes

We investigated the possible role of calmodulin (CaM) in the control of histamine release from human basophil leukocytes using several CaM antagonists. Trifluoperazine (TFP) (10(-6)-2 X 10(-5) M), pimozide (10(-6)-1.5 X 10(-5) M), chlorpromazine (CPZ) (10(-5)-10(-4) M) and promethazine (PMZ) (2 X 10(-5)-10(-4) M) inhibited in vitro histamine secretion from human basophils induced by several immunological (antigen, anti-IgE, and formyl-L-methionyl-L-leucyl-L-phenylalanine: f-met peptide) and nonimmunological (Ca2+ ionophore A23187 and the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate: TPA) stimuli. Trifluoperazine sulfoxide (TFP-S) and chlorpromazine sulfoxide (CPZ-S), which have very low affinity to CaM, had practically no inhibitory effect on histamine release from human basophils. The inhibitory effect of TFP could be made irreversible by irradiating the cells with UV light. A sulfonamide derivative, the compound N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) (2.5 X 10(-5)-2 X 10(-4) M), which selectively binds to CaM, inhibited the release of histamine from basophils. In contrast, the chloride deficient analogue, W-5, which interacts only weakly with CaM, had practically no inhibiting effect. The IC50 for enzyme release by a series of eight CaM antagonists was closely correlated (r = 0.91; p less than 0.001) with the CaM specific binding, supporting the concept that these agents act by binding to CaM and thereby inhibiting histamine release. TFP and W-7 inhibited histamine release in the absence and in the presence of increasing concentrations of extracellular Ca2+. These results emphasize the possible role of CaM in the control of histamine secretion from human basophils.     

Modulation of phospholipase A2 activity associated with human sperm membranes by divalent cations and calcium antagonists

Phospholipase A2 activity in sonicates and acid extracts of ejaculated, washed human sperm was measured using [1-14C] oleate-labeled autoclaved E. coli and 1-[1-14C] stearoyl-2-acyl-3-sn- glycerophosphorylethanolamine as substrates. Phospholipase A was optimally active at pH 7.5, was calcium-dependent, and exclusively catalyzed the release of fatty acid from the 2-position of phospholipids. The activity was membrane-associated, and was solubilized by extraction with 0.18 N H2SO4. Acid extracts of human sperm had the highest specific activity (1709 nmols /h per mg), followed by mouse, rabbit and bull, which were 105, 36 and 1.7 nmols /h per mg, respectively. para-bromophenacyl bromide inhibited human sperm phospholipase A2 activity, but mepacrine was without effect. In the presence of 1.0 mM added CaCl2, phospholipase A2 activity was inhibited by Zn2+ and Mn2+; whereas Cu2+, Cd2+, Mg2+, or Sr2+ had no effect. Zn2+ stimulated activity at low concentrations (10(-6) to 10(-8) M), and inhibited activity in a dose-dependent manner at concentrations of 10(-5) M. The extent of stimulation by low concentrations of Zn2+ was dependent on Ca2+ concentration; at 10(-7) M, Zn2+ activity was stimulated 160% with 0.5 mM CaCl2, and only 120% with 1.0 mM CaCl2. At low concentrations (10(-5) to 10(-7) M), methoxyverapamil (D600) and trifluoperazine stimulated human sperm phospholipase A2 activity, and trifluoperazine but not D600 produced almost complete inhibition between 10(-5) and 10(-4) M of the drug. The significance of human sperm phospholipase A2 activity and its modulation by Ca2+, Zn2+ and Mn2+ in the sperm acrosome reaction is discussed.     

Change in the fructose 1,6-bisphosphatase activity in sea urchin eggs following fertilization.

The activity of fructose 1,6-bisphosphatase [EC 3.1.3.11] in sea urchin eggs decreased following fertilization. During the first 30 min after fertilization, the activity was considerably lower than that in unfertilized eggs, but by 30 min the activity was similar to that in unfertilized eggs. The enzyme activity in fertilized eggs, estimated in the presence of EGTA, was similar to that in unfertilized eggs. The activity in unfertilized eggs was reduced by Ca2+ at concentrations between 1 X 10(-5) M and 5 X 10(-3) M. Immediately after fertilization, the enzyme was insensitive to concentrations of Ca2+ lower than 2 X 10(-4) M, but the Ca2+ sensitivity of the enzyme recovered 30 min after fertilization. In the presence of Ca2+ at concentrations higher than 2 X 10(-4) M, the enzyme activity in unfertilized eggs was similar to that in fertilized eggs. Mg2+ restored the Ca2+-induced inhibition of fructose 1,6-bisphosphatase. 3-Phosphoglycerate and citrate hardly affected the enzyme activity, and AMP at concentrations above 10 mM inhibited it.     

A calmodulin antagonist inhibits histamine-stimulated acid production by isolated rat parietal cells

The role of calmodulin in the regulation of histamine-stimulated parietal cell function was studied in isolated rat parietal cells using [14C]aminopyrine uptake as a quantitative index of acid production. In enriched (77-87%) intact parietal cells the calmodulin antagonist naphthalene sulfonamide W 7 dose-dependently inhibited the response to 10(-4) M histamine (IC50: 2 X 10(-6) M). The mechanism of this inhibition was examined further with two other stimuli of H+-production: forskolin which directly activates the parietal cell adenylate cyclase without interacting at the histamine H2-receptor and dbcAMP which mimics the biological action of cAMP without preceding activation of adenylate cyclase. W 7 effectively inhibited the responses to 10(-4) M forskolin (IC50: 6 X 10(-7) M), 10(-3) M dbcAMP (IC50: 10(-6) M) and to 10(-2) M K+ (IC50: 3 X 10(-6) M). The action of W 7 followed non-competitive kinetics since the antagonist reduced the entire range of the concentration-response curves without shifting them rightwards towards higher concentrations of the respective stimulants. The effect of W 7 was reversed by washing the cells. ATP-induced [14C]aminopyrine uptake into digitonin-permeabilized oligomycin-inhibited parietal cells reflects H+-production independent of oxidative phosphorylation and was also inhibited by W 7 (IC50: 10(-5) M). Inhibition of K+-stimulated H+/K+-ATPase activity required even higher W 7-concentrations (IC50: 1.4 X 10(-4) M). Our data suggest that calmodulin might be involved in the intracellular mediation of the response to histamine. Between histamine-induced cAMP-generation and the H+-secreting tubulovesicular system W 7 seems to inhibit an intracellular step that finally activates the H+/K+-ATPase. Yet, direct inhibition of the ATPase requires W 7 concentrations of questionable specificity and is unlikely to be the mechanism behind the action of W 7 on the parietal cell response to histamine.     

The effect of ionic surface-active agents on macroconidial plasma membrane of Fusarium sulphureum.

A method was developed for direct assessment of changes in cytoplasmic volume and permeability of plasma membranes of intact cells to divalent cations. This technique, which ultilized an amphipathic spin label partitioning between intracellular aqueous and hydrophobic environments, allowed estimates of the proportion of cells in a homogenous population sustaining membrane damage associated with Ni+2 and water permeability and the rate at which such damage was induced. Several specific effects of cationic and anionic surfactants on the macroconidial plasma membranes of Fusarium sulphureum Schlect (isolate 1) were distinguished on the basis of detergent concentration and charge. The induction of water uptake by the cells was found to be an effect of dodecylguanidine acetate (Dodine), a cationic fungicide, at low concentration. At higher concentrations (greater than 5 X 10(-5) M) both the impermeability of the plasma membrane to divalent cations and the ability to accumulate actively L-phenylalanine were lost irreversibly and cell lysis occurred above 5 X 10(-4) M. Sodium dodecyl sulfate eliminated divalent cation impermeability more rapidly than Dodine but was less effective in inhibiting active transport function. An antagonistic effect between cationic and anionic detergents was observed.     

The effect of divalent metal cations on the inhibition of human coagulation factor Xa by plasma proteinase inhibitors

The inactivation of human coagulation factor Xa by the plasma proteinase inhibitors alpha 1-antitrypsin, antithrombin III and alpha 2-macroglobulin in purified systems was found to be accelerated by the divalent cations Ca2+, Mn2+ and Mg2+. The rate constant for the inhibition of factor Xa by antithrombin III rose from 2.62 X 10(4) M-1 X min-1 in the absence of divalent cations to a maximum of 6.40 X 10(4) M-1 X min-1 at 5 mM Ca2+, 8.10 X 10(4) M-1 X min-1 at 5 mM Mn2+, with a slight decrease in rate at higher cation concentrations. Mg2+ caused a gradual rise in rate constant to 5.65 X 10(4) M-1 X min-1 at 20 mM. The rate constant for the inhibition of factor Xa by alpha 1-antitrypsin in the absence of divalent cations was 5.80 X 10(3) M-1 X min-1. Ca2+ increased the rate to 1.50 X 10(4) M-1 X min-1 at 5 mM and Mn2+ to 2.40 X 10(4) M-1 X min-1 at 6 mM. The rate constant for these cations again decreased at higher concentrations. Mg2+ caused a gradual rise in rate constant to 1.08 X 10(4) M-1 X min-1 at 10 mM. The rate constant for the factor Xa-alpha 2-macroglobulin reaction was raised from 6.70 X 10(3) M-1 X min-1 in the absence of divalent cations to a maximum of 4.15 X 10(4) M-1 X min-1 at 4 mM Ca2+, with a decrease to 3.05 X 10(4) M-1 at 10 mM. These increases in reaction rate were correlated to the binding of divalent cations to factor Xa by studying changes in the intrinsic fluorescence and dimerization of factor Xa. The changes in fluorescence suggested a conformational change in factor Xa which may be responsible for the increased rate of reaction, whilst the decrease in rate constant at higher concentrations of Ca2+ and Mn2+ may be due to factor Xa dimerization.