Properties of the sarcoplasmic reticulum Ca2+-pump in coronary artery skinned smooth muscle

Pig coronary artery cultured smooth muscle cells were skinned using saponin. In the presence of an ATP-regenerating system and oxalate, the skinned cells showed an ATP-dependent azide insensitive Ca²⁺-uptake which increased linearly with time for >1 h. The Ca²⁺-uptake occurred with K_m values of 0.20±0.03 M for Ca²⁺ and 400±34 M for MgATP^2–. Thapsigargin and cyclopiazonic acid inhibited this uptake with IC₅₀ values of 0.13±0.02 and 0.56±0.04 M, respectively. These properties of SR Ca²⁺-pump are similar to those reported for membrane fractions isolated from fresh smooth muscle of coronary artery and other arteries. However, optimum pH of the uptake in the skinned cells (6.2) was lower than that reported previously using isolated membranes (6.4–6.8).Abbreviations SR sarcoplasmic reticulum - ER endoplasmic reticulum - PM plasma membrane - CPA cyclopiazonic acid - DTT dithiothreitol     

Erythrocyte membrane Ca2+-pumping ATPase of hypertensive humans: Reduced stimulation by calmodulin

The Ca²⁺-pumping ATPase of erythrocyte plasma membranes of hypertensive humans (HTN) show, in the absence ofcalmodulin, a low V_max comparable to that of the enzyme of the erythrocyte membranes of normotensive humans (NTN). Although the addition of calmodulin (1.5 gper ml) increased the maximum activity of the calcium pump of membranes of HTN and NTN individuals by at least 2-fold and 4-fold, respectively, the activator protein partially purifed from the erythrocytes of HTN individuals enhanced the activity of the enzyme in a fashion similar to that of the protein obtained from the haemolysate of NTN individuals. A determination of the dependence of the activity of the pump on concentration of ATP revealed that the K_m (ATP) of the enzyme of membranes of HTN individuals is 52% higher than that of the enzyme of membranes of NTN individuals, while the V_max (1.75±0.28 mol ATP mg protein^–1 h^–1) of the pump is 46% lower in the membranes of HTN humans than that of the enzyme of membranes of normal individuals (3.25 ±0.42 mol ATP mg protein^–1 h^–1) . It seems likely from these results that elevated erythrocyte Ca²⁺ concentration associated with essential hypertension may be due to a defective interaction between the Ca²⁺-pumping ATPase and the calmodulin Ca²⁺ complex,     

Effect of 2,6-Diisopropylphenol and Halogenated Anesthetics on Tetraphenylphosphonium Uptake by Rat Brain Synaptosomes: Determination of Membrane Potential

The effect of 2,6-diisopropylphenol (propofol) in comparison to that of the halogenated anesthetics enflurane, isoflurane, and halothane on tetrapenylphosphonium uptake by rat brain synaptosomes was studied. A direct method to separately measure the synaptosomal and the mitochondrial transmembrane potential by using the tetraphenylphosphonium cation (TPP⁺) was utilized. The latter is a lipophylic charged molecule which distributes between two compartments according to the transmembrane electrical potential in the presence or absence of 60 mM KCl as a synaptosomal membrane depolarizing agent. After previously reporting the damages induced by general anesthetics on isolated mitochondria, the aim of this paper was to study their possible action on the synaptosomal membrane potential and whether or not drugs concentrations damaging isolated mitochondria are also effective on synaptosomal mitochondria. The results indicated that, in the presence of glucose, mitochondria included in synaptosomes were able to maintain a transmembrane potential of 202 ± 8 mV (mean ± SD) while the synaptosomal membrane showed a potential of 78 ± 8 mV (mean ± SD). When anesthetic concentrations (0.6–1 mM propofol, 10–40 M enflurane, 30–50 M isoflurane, 8–15 M halothane) that impair mitochondrial energy metabolism were used, the synaptosomal transmembrane potential was maintained and, in addition, a slight increase of the TPP⁺ taken up was observed as the anesthetic concentration was increased.     

Opiates inhibit calmodulin activation of a high-affinity Ca2+-stimulated Mg2+-dependent ATPase in synaptic membranes

A high affinity Ca²⁺/Mg²⁺ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca²⁺ (1 M) believed to approximate the range of Ca²⁺ in the synaptosomal cytosol (0.1 to 5.0 M). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca²⁺-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca²⁺-stimulated ATP hydrolysis was characterized by a reduction inV _max for Ca²⁺. Levorphanol pretreatment reduced the Hill coefficient (H_N) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca²⁺ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca²⁺/Mg²⁺ ATPase in synaptic membranes. Levorphanol (10 M), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca²⁺-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca²⁺-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inV _max for both Ca²⁺ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca²⁺-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca²⁺/Mg²⁺ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca²⁺ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca²⁺ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca²⁺ following inhibition of Ca²⁺/Mg²⁺ ATPase may underlie some of the pharmacological effects of opiate drugs.     

Use of mitochondrial inhibitors to differentiate kinetic properties of the ATP-dependent Ca2+ uptake system in synaptic membranes

Synaptosomal membranes accumulate 3–6 times more Ca²⁺ in the presence of ATP (50–1000 M) than basal Ca²⁺ accumulation (-ATP). The location of this Ca²⁺ accumulation appears to reside on the cytosolic face of the synaptosome since lysed synaptosomes accumulate 4-times more Ca²⁺ than intact synaptosomes. The inclusion of mitochondrial inhibitors, oligomycin (0.7 g/ml), sodium azide (100 M) and dinitrophenol (100 M) differentiate mitochondrial from nonmitochondrial Ca²⁺ accumulation under conditions that are [Ca²⁺]- and ATP-dependent. In the presence of low concentrations of ATP (<150 M) and Ca _free ²⁺ (2.5 or 6.8 M), Ca²⁺ accumulation occurs as one process in both lysed synaptosomal membranes and purified synaptic plasma membranes in the presence and/or absence of MI. When ATP levels are increased (>200 M), the Ca²⁺ accumulation process remains independent of the presence of mitochondrial inhibitors when Ca _free ²⁺ =2.5 M. When Ca _free ²⁺ is increased to 6.8 M, mitochondrial inhibitors differentiate mitochondrial from nonmitochondrial accumulation. These studies suggest that optimal conditions for the measurement of Ca²⁺ accumulating mechanisms in synaptosomal membranes depend on both [Ca²⁺] and ATP. Use of these assay conditions provide evidence that ATP-dependent Ca²⁺ uptake may be a viable mechanism for the regulation of synaptosomal Ca²⁺ levels.     

Disulfiram lowers Ca2+, Mg2+-ATPase activity of rat brain synaptosomes

The chronic administration of disulfiram (DS) to rats resulted in significant decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase activity. In vitro studies indicated that DS (ID₅₀=20 M) produced a dose-dependent inhibition of Ca²⁺, Mg²⁺-ATPase. However, diethyldithio-carbamate, a metabolite of DS, failed to modify Ca²⁺, Mg²⁺-ATPase activity, implying that the decrease in ATPase activity in DS administered rats was due to the effect of parent compound. The DS-mediated inhibition (48%) of ATPase activity was comparable with a similar degree of inhibition (49%) achieved by treating the synaptosomal membranes with N-ethylmaleimide (ID₅₀=20 M) in vitro. Furthermore, the inhibition by DS was neither altered by washing the membranes with EGTA nor reversed by treatment with sulfhydryl reagents such as GSH or dithiothreitol. About 74% and 68% decrease of synaptosomal Ca²⁺, Mg²⁺-ATPase specific activity was observed when treated with DS (30 M) and EGTA (100 M) respectively. The remaining 25–30% of total activity is suggested to be of Mg²⁺-dependent ATPase activity. This indicates that both these drugs may act on a common target, calmodulin component that represents 70–75% of total Ca²⁺, Mg²⁺-ATPase activity. Therefore, DS-mediated modulation of synaptosomal Ca²⁺, Mg²⁺-ATPase activity could affect its function of maintaining intracellular Ca²⁺ concentration. This could contribute to the deleterious effects on CNS.     

Erythrocyte Membrane Digoxin-Sensitive (Na+–K+)-ATPase of Non-Insulin Dependent Diabetic Humans

Erythrocyte plasma membranes of non-insulin dependent diabetic humans (NIDDM) and healthy humans were prepared by hypotonic lysis. The specific activity of (Na⁺–K⁺)-ATPase of NIDDM membranes, both in the absence and presence of digoxin were lower than the specific activity of normal enzymes (83.6 percent and 74.0 percent of the normal enzyme respectively). Addition of digoxin decreased the activity of this enzyme (38.0 percent in NIDDM and 30.0 percent in normal enzyme).Although the affinity of the pump for ATP was similar in both membranes of NIDDM and normal humans (K_m for ATP=19.9±0.24M ATP and 20.0±0.21 M ATP respectively), the V_max of NIDDM membranes was more than 20 percent lower than that of the normal enzyme. The specific activity of Mg²⁺-dependent Ca²⁺-pumping ATPase (Ca²⁺–Mg²⁺)-ATPase) of NIDDM membrane was lower than 80 percent of the specific activity of the normal enzymes. While the affinity of the pump for ATP was lower in the membranes of NIDDM (K_m for ATP=50.0±4.3 M ATP) in comparison to normal membranes (K_m for ATP=63.1±38M ATP), the V_max of NIDDM membranes was similar to the normal enzyme. Altogether, these findings suggest that both the (Na⁺–K⁺)-ATPase and Ca²⁺-pumping ATPase of NIDDM membranes are less functional than the enzymes in normal erythrocytes.     

Parameters not influenced by vesamicol: Membrane potential,calcium uptake,and internal calcium concentration of synaptosomes

In our previous study vesamicol, an inhibitor of the acetylcholine transporter of the cholinergic vesicles, inhibited veratridine-evoked external Ca²⁺-dependent acetylcholine release from striatal slices but did not influence acetylcholine release observed in Ca²⁺-free medium (4). Here we examined if the effect of veratridine on membrane potential, Ca²⁺ uptake, and intracellular Ca²⁺ concentration of synaptosomes was altered by vesamicol in parallel with the inhibition of acetylcholine release. The depolarizing effect of 10 M veratridine (from 67±2.3 mV resting membrane potential to 50.7±2.5 mV) was not significantly influenced by vesamicol (1–20 M). Vesamicol (1–20 M) had no effect on either the overall curve of the veratridine-evoked⁴⁵Ca²⁺ uptake or the amount of Ca²⁺ taken up by synaptosomes. Veratridine caused a rise in intrasynaptosomal Ca²⁺ concentration as measured by Fura2 fluorescence, and the same increase both in characteristics and in magnitude was observed in the presence of vesamicol (20 M). The K⁺-evoked (40 mM) increase of Ca²⁺ uptake and of intracellular calcium concentration were also unaltered by vesamicol. In high concentration (50 M) vesamicol inhibited both the fall in membrane potential and the elevated Ca²⁺ uptake by veratridine, indicating a possible nonspecific effect on potential-dependent Na⁺ channels at this concentration. Vesamicol, in lower concentration (20 M) when neither of the above parameters was changed, completely prevented veratridine-evoked increase of [¹⁴C]acetylcholine release. This was observed only when vesamicol was present in the media throughout the experiment after loading the preparation with [¹⁴C]choline. The results suggest that vesamicol does not interfere with veratridine-induced changes in isolated nerve terminals other than with the release of acetylcholine, thus further supporting the involvement of a vesamicol-sensitive vesicular transmitter pool in Ca²⁺-dependent veratridine-elicited acetylcholine release.     

Met-enkephalin receptor-mediated increase of membrane fluidity modulates nitric oxide (NO) and cGMP production in rat brain synaptosomes

The association of [³H]-Met-enkephalin with synaptosomes isolated from rat brain cortex, when incubated for 30 min at 25°C follows a sigmoid path with a Hill coefficient h=1.25±0.04. Binding of Met-enkephalin into synaptosomes was saturable, with an apparent binding constant of 8.33±0.48 nM. At saturation, Met-enkephalin specific receptors corresponded to 65.5±7.2 nmol/mg synaptosomal protein. The Hill plot in combination with the biphasic nature of the curve to obtain the equilibrium constant, showed a moderate degree of positive cooperativity in the binding of Met-enkephalin into synaptosomes of at least one class of high affinity specific receptors. Met-enkephalin increased the lipid fluidity of synaptosomal membranes labelled with 1,6-diphenyl-1,3,5-hexatriene (DPH), as indicated by the steady-state fluorescence anisotropy [(r_o/r)–1]^–1. Arthenius-type plots of [(r_o/r)–1]^–1 indicated that the lipid separation of the synaptosomal membranes at 23.4±1.2°C was perturbed by Met-enkephalin such that the temperature was reduced to 15.8±0.8°C. Naloxone reversed the fluidizing effect of Met-enkephalin, consistent with the receptor-mediated modulation of membrane fluidity. Naloxone alone had no effect on membrane fluidity. NO release and cGMP production by NO-synthase (NOS) and soluble guanylate cyclase (sGC), both located in the soluble fraction of synaptosomes (synaptosol) were decreased by 82% and 80% respectively, after treatment of synaptosomes with Met-enkephalin (10^–10–10^–4 M). These effects were reversed by naloxone (10^–4 M) which alone was ineffective in changing NO and cGMP production. We propose that Met-enkephalin achieved these effects through receptor mediated perturbations of membrane lipid structure and that inhibition of the L-Arg/NO/cGMP pathway in the brain may result in the antinociceptive effects of Met-enkephalin.     

Regulatory effect of regucalcin on (Ca2+−Mg2+)-ATPase in rat liver plasma membranes: comparison with the activation by Mn2+ and Co2+

The effect of various metals and regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes of rat liver was investigated. Of various metals (Zn²⁺, Cu²⁺, Ni²⁺, Mn²⁺, Co²⁺ and Al³⁺; 100 M as a final concentration), Mn²⁺ and Co²⁺ increased markedly (Ca²⁺–Mg²⁺)-ATPase activity, while other metals had no effect. When Ca²⁺ was not added into enzyme reaction mixture, Mn²⁺ and Co²⁺ (25–100 M) did not significantly increase the enzyme activity, indicating that heavy metals act on Ca²⁺-stimulated phosphorylation of the enzyme. Meanwhile, regucalcin (0.25–1.0 M) caused a remarkable elevation of (Ca²⁺–Mg²⁺)-ATPase activity. This increase was not inhibited by the presence of 100 M vanadate, although the effects of Mn²⁺ and Co²⁺ (100 M) were inhibited by vanadate. Also, the inhibition of the Mn²⁺ and Co²⁺ effects by vanadate was not seen in the presence of regucalcin. Moreover, regucalcin (0.5 M) increased significantly the enzyme activity in the absence of Ca²⁺. This effect of regulcalcin was not altered by increasing concentrations of Ca²⁺ added, indicating that the regucalcin effect does not depend on Ca²⁺. The present results suggest that regucalcin activates directly (Ca²⁺–Mg²⁺)-ATPase in liver plasma membranes, and that the activation is not involved in the Ca²⁺-dependent phosphorylation of the enzyme.     

Electrogenic calcium transport in plasma membrane of rat pancreatic acinar cells

Summary ATP-dependent⁴⁵Ca²⁺ uptake was investigated in purified plasma membranes from rat pancreatic acinar cells. Plasma membranes were purified by four subsequent precipitations with MgCl₂ and characterized by marker enzyme distribution. When compared to the total homogenate, typical marker enzymes for the plasma membrane, (Na⁺,K⁺)-ATPase, basal adenylate cyclase and CCK-OP-stimulated adenylate cyclase were enriched by 43-fold, 44-fold, and 45-fold, respectively. The marker for the rough endoplasmic reticulum was decreased by fourfold compared to the total homogenate. Comparing plasma membranes with rough endoplasmic reticulum, Ca²⁺ uptake was maximal with 10 and 2 mol/liter free Ca²⁺, and half-maximal with 0.9 and 0.5 mol/liter free Ca²⁺. It was maximal at 3 and 0.2 mmol/liter free Mg²⁺ concentration, at an ATP concentration of 5 and 1 mmol/liter, respectively, and at pH 7 for both preparations. When Mg²⁺ was replaced by Mn²⁺ or Zn²⁺ ATP-dependent Ca²⁺ uptake was 63 and 11%, respectively, in plasma membranes; in rough endoplasmic reticulum only Mn²⁺ could replace Mg²⁺ for Ca²⁺ uptake by 20%. Other divalent cations such as Ba²⁺ and Sr²⁺ could not replace Mg²⁺ in Ca²⁺ uptake. Ca²⁺ uptake into plasma membranes was not enhanced by oxalate in contrast to Ca²⁺ uptake in rough endoplasmic reticulum which was stimulated by 7.3-fold. Both plasma membranes and rough endoplasmic reticulum showed cation and anion dependencies of Ca²⁺ uptake. The sequence was K⁺>Rb⁺>Na⁺>Li⁺>choline⁺ in plasma membranes and Rb⁺K⁺Na⁺>Li⁺>choline⁺ for rough endoplasmic reticulum. The anion sequence was Cl^–Br^–I^–>SCN^–>NO ₃ ^– >isethionate^– >cyclamate^–>gluconate^–>SO ₄ ^2– glutarate^– and Cl^–>Br>gluconate>SO ₄ ^2– >NO ₃ ^– >I^–>cyclamate^–SCN^–, respectively. Ca²⁺ uptake into plasma membranes appeared to be electrogenic since it was stimulated by an inside-negative K⁺ and SCN diffusion potential and inhibited by an inside-positive diffusion potential. Ca²⁺ uptake into rough endoplasmic reticulum was not affected by diffusion potentials. We assume that the Ca²⁺ transport mechanism in plasma membranes as characterized in this study represents the extrusion system for Ca²⁺ from the cell that might be involved in the regulation of the cytosolic Ca²⁺ level.     

Activatory effect of regucalcin on GTPase activity in rat liver plasma membranes

The effect of regucalcin, a regulatory protein of Ca²⁺ signaling, on guanosine-5-triphosphatase (GTPase) activity in isolated rat liver plasma membranes was investigated. GTPase activity was significantly increased by the addition of Ca²⁺ (25–100 M) in the enzyme reaction mixture. Such an increase was not seen by other metals (Mg, Co, Zn, Cu, Ni, and Mn) with 50 M. The activatory effect of calcium (50 M) was significantly decreased by calmodulin (2.5 and 5 g/ml), indicating that it does not depend on calmodulin. The presence of regucalcin (0.1–0.5 M) in the enzyme reaction mixture caused a significant increase in GTPase activity. This increase was not significantly enhanced by calcium (50 M). GTPase activity was significantly increased by dithiothreitol (DTT; 5 mM), a protecting reagent of thiol (SH)-groups, while it was decreased by N-ethylmaleimide (NEM; 5 mM), a modifying reagent of SH-groups. The effect of calcium or regucalcin in increasing GTPase activity was not seen in the presence of NEM. Also, the activatory effect of calcium or regucalcin on GTPase was not seen in the presence of vanadate, an inhibitor of protein phosphorylation, which could inhibit GTPase activity. Moreover, the effect of regucalcin was not seen in the presence of digitonin (0.01%), a solubilizing reagent of membranous lipids, while the effect of calcium was not inhibited by digitonin. The present study demonstrates that regucalcin has an activatory effect on GTPase activity independently of Ca²⁺ in rat liver plasma membranes.     

Hydrogen fluoride effects on plasma membrane composition,ATPase activity and cell structure in needles of eastern white pine (Pinus strobus) seedlings

Eastern white pine (Pinus strobus L.) seedlings were grown in controlled environment growth cabinets and fumigated with 0.4 and 1.6 g m^–3 hydrogen fluoride for 2–28 days. Plasma membranes were isolated from needles of treated and control seedlings and their chemical composition and ATPase activity examined to determine early effects of hydrogen fluoride action. In plants treated for 2 days with both fluoride levels, ratios of plasma membrane free sterols:phospholipids and sterols:proteins were drastically higher than ratios in control plants. Seedlings treated with hydrogen fluoride for 8 days contained plasma membranes with elevated phospholipid:protein and sterol:protein ratios and their plasma membrane ATPase activity was higher than that of control plants. Prolonged, 28-day hydrogen fluoride treatment with 1.6 g m^–3 level was the only treatment which produced a drastic inhibition of plasma membrane ATPase activity. During the initial stages of hydrogen fluoride treatment, treated cells did not show alterations of ultrastructure which were previously shown in cells of plants treated with soil applied sodium fluoride. The results of the present study indicate that the plasma membranes may be among the initial sites of hydrogen fluoride injury to plants as well as initial sites of defense reaction.     

Changes in lipid peroxidation, the redox system and ATPase activities in plasma membranes of rice seedling roots caused by lanthanum chloride

Highly purified plasma membranes were isolated by aqueous two-phase partitioning from rice (Oryza sativa) seedling roots. The effects of lanthanum chloride (LaCl₃) on the activities of lipid peroxidation, the redox system and H⁺-ATPase, Ca²⁺-ATPase of plasma membranes were studied. The lipid peroxidation of plasma membranes could be depressed by certain low concentrations of LaCl₃ and enhanced by high concentrations of LaCl₃, while the lipid peroxidation was also dependent on the plasma membrane protein and incubation time. The relative activity of O₂ uptake of plasma membranes was inhibited by all tested LaCl₃ concentrations. In contrast, the reduction rate of Fe(CN)₆ ^3– by plasma membranes was stimulated below 40 M of LaCl₃, but was reduced above 60 M of LaCl₃. The relative activities of both H⁺-ATPase and Ca²⁺-ATPase increased constantly from control to LaCl₃ of concentration 60 M where the activities of both enzymes were the maximum, but decreased remarkably at 80 M LaCl₃ concentrations various LaCl₃ were added to culture solutions. In the other measurement case in which various LaCl₃ concentrations were added directly to reaction medium and the plasma membrane vesicles only came from the control cultured rice seedling roots, the response of H⁺-ATPase activity to La³⁺ was similar to the response in culture solution. However, the La³⁺ concentration was only 20 M when the activity of H⁺-ATPase was the maximum. In contrast to the case of LaCl₃ addition to culture solution, Ca²⁺-ATPase activity was inhibited by all concentrations of La³⁺ which were added directly to the reaction medium. The above results revealed that REEs inhibited electron transfer from NADH to oxygen in plant plasma membranes, depressed the production of active oxygen radicals, and reduced the formation of lipid peroxides through plasma membrane lipid peroxidation. REEs ions also enhanced the H⁺ extrusion by both standard redox system and H⁺-ATPase in plasma membranes at certain concentrations. A possible role for the plant cell wall in REEs effects on plasma membranes was also suggested.     

Enhancement of plasma membrane (Ca2+-Mg2+)-ATPase activity in regenerating rat liver: Involvement of endogenous activating protein regucalcin

The alteration of (Ca²⁺-Mg²⁺)-ATPase activity in the plasma membranes of regenerating rat liver after a partial hepatectomy was investigated. Liver was surgically removed about two thirds of that of sham-operated rats. The reduced liver weight by partial hepatectomy was restored about 50% at 24 h after the surgery, and it was completely restored at 72 h. Regenerating liver significantly increased calcium content and plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity between 12–48 h after hepatectomy. Those increases were maximum at 24 h after the surgery. The regenerating liver-induced increase in hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was completely abolished by the presence of anti-regucalcin IgG (1.0–4.0 g/ml). The regenerating liver-induced increase in hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was clearly inhibited by N-ethylmaleimide (2.5 and 5.0 mM) addition into the enzyme reaction mixture. This NEM effect was also seen for the activatory effect with regucalcin (0.25 M) addition on the enzyme activity in the plasma membranes from normal rat liver. The endogenous regucalcin may play a cell physiological role in the activation of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase to maintain the intracellular calcium level in regenerating rat liver.     

Spermine Inhibits [3H]Glycine Binding at the NMDA Receptors from Plexiform Layers of Chick Retina

Saturable specific binding of glycine to synaptosomal membranes from plexiform layers of the retina has been described, which seems to correspond to the modulatory site on NMDA-receptors (26). Spermine inhibited specific [³H]glycine binding to membranes from synaptosomal fractions from the outer (P₁) and the inner (P₂) plexiform layers of 1–3 day-old chick retinas in a dose-dependent manner with an IC₅₀ = 35 M for the P₁ fraction and 32 M for the P₂ fraction. Kinetic experiments and non-linear regression analysis of [³H]glycine-specific binding showed a K_d ~ 100–150 nM in both fractions, and a higher B_max (4.11 ± 0.47 pmol/mg protein) for the inner plexiform layer compared to the outer plexiform layer (B_max = 2.76 ± 0.25 pmol/mg protein). Strychnine-insensitive [³H]glycine binding was inhibited by 100 M spermine, due to a reduction in B_max (P₁ = 0.84 ± 0.16 pmol/mg protein; P₂ = 0.81 ± 0.16 pmol/mg protein) without affecting the K_d. Association and dissociation constants in the absence and presence of 50 M spermine remained unchanged. Results demonstrate the presence of a single modulatory site for spermine on NMDA receptors, in both synaptic layers of the chick retina.     

Osmotic water permeability of small intestinal brush-border membranes

Summary A stopped-flow nephelometric technique was used to examine osmotic water flow across small intestinal brush-border membranes. Brush-border membrane vesicles (BBMV) were prepared from rat small intestine by calcium precipitation. Scattered 500 nm light intensity at 90° to incident was a linear function of the number of vesicles in suspension, and of the reciprocal of the suspending medium osmolality. When BBMV were mixed with hyperosmotic mannitol solutions there was a rapid increase in the intensity of scattered light that could be fit to a single exponential function. The rate constant for vesicle shrinking varied with temperature and the size of the imposed osmotic gradient. At 25°C and an initial osmotic gradient of 50 mOsm, the rate constant was 1.43±0.044 sec^–1. An Arrhenius plot of the temperature dependence of vesicle shrinking showed a break at about 25°C with an activation energy of 9.75±1.04 kcal/mole from 11 to 25°C and 17.2±0.55 kcal/mole from 25 to 37°C. The pore-forming antibiotic gramicidin increased the rate of osmotically driven water efflux and decreased the activation energy of the process to 4.51±0.25 kcal/mole. Gramicidin also increased the sodium permeability of these membranes as measured by the rate of vesicle reswelling in hyperosmotic NaSCN medium. Gramicidin had no effect on mannitol permeability. Assuming spherical vesicles of 0.1 m radius, an osmotic permeability coefficient of 1.2×10^–3 cm/sec can be estimated for the native brush-border membranes at 25°C. These fesults are consistent with the solubility-diffusion model for water flow across small intestinal BBMV but are inconsistent with the existence there of large aqueous pores.     

Erythrocyte membrane (Ca2++Mg2+)-ATPase in human protein-energy malnutrition

Calmodulin-free ghost membranes were prepared from erythrocytes of kwashiorkor children and from healthy children in the same age bracket. In the absence of calmodulin, the specific activity of Mg²⁺-dependent Ca²⁺-pumping ATPase (Ca²⁺+Mg²⁺-ATPase) of kwashiorkor membranes was more than 40 percent lower than the specific activity of the normal enzymes, whose maximum velocity was increased by at least four-fold by the modulator protein. In constrast, the maximum velocity of the enzymes of kwashiorkor membranes was enhanced by calmodulin by about 11/2 times the basal activity of the normal enzymes and by 2 times the basal activity of the kwashiorkor enzymes. The affinity of the pump for ATP was lower in the membranes of kwashiorkor children (K_m for ATP=30.6±2.8 M ATP) in comparison to normal membranes (K_m for ATP=21.7±2.0 M ATP). Similarly, calmodulin-affinity of the enzymes, was lower in kwashiorkor membranes than in the normal membranes irrespective of source of calmodulin. Calmodulin from haemolysates of kwashiorkor red cells activated the enzymes of normal and kwashiorkor membranes to the same degree as calmodulin partially purified from the haemolysate of healthy children. A determination of the dependence of the activity of the pump on calcium in the absence and presence of calmodulin reveals that the affinity of the kwashiorkor enzymes for Ca²⁺ is at least 70 percent lower than that of enzymes of normal membranes. Altogether, these findings suggest that the Ca²⁺-pumping ATPase of kwashiorkor membranes is less functional than the enzymes of healthy erythrocytes.     

Modulation of ATPase activities of human erythrocyte membranes by free fatty acids or phospholipase A2

Summary The artificial insertion of increasing amounts of unsaturated fatty acids into human erythrocyte membranes modulated ATPase activities in a biphasic manner, depending on the number and position of double bonds, their configuration, and the chain length. Uncharged long-chain fatty acid derivatives with double bonds and short-chain fatty acids were ineffective. Stearic acid stimulated Na⁺K⁺-ATPase only. Anionic and non-ionic detergents and -lysophosphatidylcholine failed to stimulate ATPase activities at low, and inhibited them at high concentrations.Mg²⁺-ATPase activity was maximally enhanced by a factor of 2 in the presence of monoenoic fatty acids; half-maximal stimulation was achieved at a molar ratio ofcis(trans)-configurated C18 acids/membrane phopholipid of 0.16 (0.26).Na⁺K⁺-ATPase activity was maximally augmented by 20% in the presence of monoenoic C18 fatty acids at 37°C. Half-maximal effects were attained at a molar ratio oleic (elaidic) acid/phospholipid of 0.032 (0.075). Concentrations of free fatty acids which inhibited ATPase activities at 37°C were most stimulatory at reduced temperatures. AT 10°C, oleic acid increased Na⁺K⁺-ATPase activity fivefold (molar ratio 0.22).Unsaturated fatty acids simulated the effect of calmodulin on Ca²⁺-ATPase of native erythrocyte membranes (i.e., increase ofV _max from 1.6 to 5 mol PO ₄ ^3– ·phospholipid^–1·hr^–1, decrease of K _Ca from 6 m to 1.4–1.8 m). Stearic acid decreasedK _Ca (2 m) only, probably due to an increase of negative surface charges.A stimulation of Mg²⁺-ATPase, Na⁺K⁺-ATPase, and Ca²⁺-ATPase could be achieved by incubation of the membranes with phospholipase A₂.An electrostatic segregation of free fatty acids by ATPases with ensuing alterations of surface charge densities and disordering of the hydrophobic environment of the enzymes provides an explanation of the results.     

Intracellular Calcium Changes in Neuronal Cells Induced by Alzheimer's ß-Amyloid Protein Are Blocked by Estradiol and Cholesterol

1. The elevation of intracellular Ca²⁺ levels ([Ca²⁺]_i) in immortalized hypothalamic neurons (GT1–7 cells) after exposure to Alzheimer's ß-amyloid protein (AßP[25–35]) was investigated using a multisite fluorometry system.2. The marked rise in [Ca²⁺]_i appeared afterexposure to 5–20-M AßP[25–35]. Analysis of the spatiotemporal patterns of [Ca²⁺]_i changes revealed that the magnitude and the latency of the response to AßP in each cell werehighly heterogeneous.3. The preadministration of 17ß-estradiol, 17-estradiol, phloretin and cholesterol, which influence the properties of membranes, such as membrane fluidity or membrane potential, significantly decreased the rise in [Ca²⁺]_i.4. These findings support the idea that disruption of calcium homeostasis by AßP channels may be the molecular basis of the neurotoxicity of AßP and of the pathogenesis of Alzheimer's disease. It is also suggested that membrane properties may play key roles in the expression of neurotoxicity.