Lipid associated calcium ionophores in islet cell plasma membrane following glucose stimulation

The effect of glucose exposure on lipid associated calcium ionophoretic activity was measured in cultured neonatal rat pancreatic islet cells using two model systems. The first measured the ability of a lipid extract of islet cells to facilitate calcium transfer from an aqueous to organic phase and thus detected lipids which transfer calcium in the manner of authentic ionophores or which chelate the ion. In this system glucose stimulation was followed by an increase in total cell ionophoretic activity and a decrease in the activity associated with the plasma membrane. The second system measured the transfer of calcium across an artificial phospholipid membrane and detected authentic ionophoretic activity. In this model an increase in total ionophoretic activity was again seen following glucose but there was no change in the ionophoretic activity of a plasma membrane extract. The results indicate that the lipid modifications which accompany glucose-induced insulin release may alter cellular calcium stores by decreasing lipid bound calcium at the plasma membrane and increasing the capacity for calcium ionophoresis at intracellular sites.     

Calcium antagonists and islet function: VII. Effect of calcium deprivation

Summary The role of extracellular Ca²⁺ in the regulation of islet function is investigated. Decreasing extracellular Ca²⁺ concentrations cause a dose-related inhibition of glucose-induced insulin release. Whereas the efflux of⁴⁵Ca from perifused islets is transiently increased on exposure to Ca²⁺-deprived media, it is unaffected by a partial lowering of the extracellular Ca²⁺ concentration. Under the latter condition, therefore, the observed reduction in the size of the islets' exchangeable calcium pool(s) appears to be due to reduced Ca²⁺ entry. The proper effect of glucose on Ca handling by the islets is apparently not affected by a lowering in the extracellular Ca²⁺ concentration. Nevertheless, in islets exposed to glucose and incubated in Ca²⁺-deprived media, glucose uptake and oxidation and lactate output are decreased, whereas the islet ATP level is increased, as if extracellular Ca²⁺ shortage were to affect not only the cellular pool of Ca regulating insulin release, but also energy-consuming processes possibly located at the cell membrane.     

(Ca2+ + Mg2+)-ATPase of density-separated human red cells. Effects of calcium and a soluble cytoplasmic activator (Calmodulin)

The effect of calcium and a soluble cytoplasmic activator on (Ca²⁺ + Mg²⁺)-ATPase of density-separated human red cells was investigated. At all calcium concentrations tested, dense (old) lysed cells and their isolated membranes displayed lower activities as compared to the light (young) cells and their membranes. Isolated membranes from all density red cell fractions showed two distinct (Ca²⁺ + Mg²⁺)-ATPase activities; one at low calcium and another at moderate calcium concentrations. At high calcium concentration, (Ca²⁺ + Mg²⁺)-ATPase activity of isolated membranes was low in all cell fractions. In contrast to the isolated membranes, lysed cells from all density fractions had a maximum (Ca²⁺ + Mg²⁺)-ATPase activity only at a low concentration of calcium, while moderate and high calcium concentrations produced low activity. Upon isolation of membranes, a substantial loss of (Ca²⁺ + Mg²⁺)-ATPase activity took place from all density cell fractions. Upon membrane isolation, the relative loss of (Ca²⁺ + Mg²⁺)-ATPase activity at low Ca²⁺ concentration was greater in older cells. The extent of stimulation of (Ca²⁺ + Mg²⁺)-ATPase by the activator at low calcium concentration was 3–4-fold greater in older cell membranes than in the young ones.These data suggest that the lower (Ca²⁺ + Mg²⁺)-ATPase activity in old cells could be accounted for by a selective loss of (Ca²⁺ + Mg²⁺)-ATPase activity at low Ca²⁺ concentration presumably due to reduced affinity of old cell membranes to activator protein.     

Calcium-binding properties and ATPase activities of rat liver plasma membranes

Plasma membranes from rat liver purified according to the procedure of Neville bind calcium ions by a concentration-dependent, saturable process with at least two classes of binding sites. The higher affinity sites bind 45 nmol calcium/mg membrane protein with a K_D of 3 µM. Adrenalectomy increases the number of the higher affinity sites and the corresponding K_D. Plasma membranes exhibit a (Na⁺-K⁺)-independent-Mg²⁺-ATPase activity which is not activated by calcium between 0.1 µM and 10 mM CaCl₂. Calcium can, with less efficiency, substitute for magnesium as a cofactor for the (Na⁺-K⁺)-independent ATPase. Both Mg²⁺- and Ca²⁺-ATPase activities are identical with respect to pH dependence, nucleotide specificity and sensitivity to inhibitors. But when calcium is substituted for magnesium, there is no detectable membrane phosphorylation from [γ-³²P] ATP as it is found in the presence of magnesium. The existence of high affinity binding sites for calcium in liver plasma membranes is compatible with a regulatory role of this ion in membrane enzymic mechanisms or in hormone actions. Plasma membranes obtained by the procedure of Neville are devoid of any Ca²⁺-activated-Mg²⁺-ATPase activity indicating the absence of the classical energy-dependent calcium ion transport. These results would suggest that the overall calcium-extruding activity of the liver cell is mediated by a mechanism involving no direct ATP hydrolysis at the membrane level.     

The stimulus secretion coupling of glucose-induced insulin release.

In isolated rat pancreatic islets, exogenous l-lactate causes a dose-related enhancement of glucose-induced insulin release and shifts the sigmoidal curve relating insulin output to ambient glucose concentrations to the left. l-Lactate also enhances α-ketoisocaproate-induced insulin release and glucose-induced proinsulin biosynthesis. l-Lactate rapidly accumulates in the islet cells, is converted to pyruvate and CO₂, and raises the intracellular concentration of both ATP and NAD(P)H. On a molar basis, the insulinotropic capacity of nutrients ranges as follows d-glucose ? l-lactate > pyruvate = d/l-lactate > d-lactate and does not correlate with their respective oxidation rates. However, when allowance is made for the intracellular interconversion of these exogenous nutrients, for their reciprocal influence upon oxidation rates, and for their sparing action on the utilization of endogenous fuels, a close correlation is found between the aptitude of glucose, l-lactate, and pyruvate to generate reducing equivalents and to stimulate insulin release. It is proposed that the concentration of NAD(P)H in islet cells affects the ionophoretic fluxes of cations (K⁺, Ca²⁺) across membrane systems and, hence, regulates the net uptake of Ca²⁺ and subsequent release of insulin. The effect of l-lactate upon Ca²⁺ handling is sufficiently rapid to account for the immediate secretory response to this nutrient.     

Lysophospholipid-mediated alterations in the calcium transport systems of skeletal and cardiac muscle sarcoplasmic reticulum

Summary The effects of various lysophospholipids on the calcium transport activity of sarcoplasmic reticulum (SR) from rabbit skeletal and canine cardiac muscles were examined. The lipids decreased calcium transport activity in both membrane types; the effectiveness being in the order lysoPC > lsyoPS, lysoPG > lysoPE. The maximum inhibition induced by lysoPC, lysoPG and lysoPS was greater than 85% of the normal Ca²⁺-transport rate. In cardiac SR lysoPE had a maximal inhibition of about 50%. Half maximal inhibition of calcium transport by lysoPC was achieved at 110 nmoles lysoPC/mg SR. At this concentration of lysoPC, the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺-uptake activities were inhibited to the same extent (about 60%) in skeletal sarcoplasmic reticulum, while in cardiac sarcoplasmic reticulum, there was less than 20% inhibition of the Ca²⁺ + Mg²⁺-ATPase activity. Studies with EGTA-induced passive calcium efflux showed that up to 200 nmoles lysoPC/mg SR did not alter calcium permeability significantly in cardiac sarcoplasmic reticulum. In skeletal muscle membranes the lysophospholipid mediated decrease in calcium uptake correlated well with the increase in passive calcium efflux due to lysophosphatidylcholine. The difference in the lysophospholipid-induced effects on the sarcoplasmic reticulum from the two muscle types probably reflects variations in protein and other membrane components related to the respective calcium transport systems.     

Calcium content and binding in synaptosomal subfractions during chronic morphine treatment

Chronic exposure to morphine in mice produced an increase in Ca²⁺ content of synaptosomes, synaptic plasma membranes (SPM), and synaptic vesicles. Ca²⁺ binding capacity was significantly reduced in tolerant SPM fractions. Naloxone significantly reversed the increased calcium content and reduced binding capacity of SPM when administered to 72-h-treated mice. Scatchard analysis of binding curves reveals three distinct classes of Ca²⁺ binding sites. During tolerance, the high- and low-affinity sites exhibit a reduced capacity to bind calcium, which may be reversed by in vivo and in vitro administration of naloxone. The increase in SPM and synaptic vesicle calcium content may reflect adaptive changes in the cell membrane during tolerance development, which may contribute to changes in neurotransmitter and second messenger function.     

Proton countertransport by the reconstituted erythrocyte Ca2+-translocating ATPase: Evidence using ionophoretic compounds

Summary Human erythrocyte Ca²⁺-translocating ATPase was solubilized from calmodulin-depleted membranes using the detergent Triton X-100, and subsequently purified by calmodulin-affinity chromatography. The purified enzyme was reconstituted in artificial phospholipid vesicles using a cholate-dialysis method and various phospholipids. The reconstituted enzyme was able to translocate Ca²⁺ inside the vesicles, both in the absence and in the presence of the Ca²⁺-chelating agent, oxalate, inside the vesicles. The tightness of coupling between ATP hydrolysis and cation translocation was investigated by the use of different ionophoretic compounds. The efficiency of Ca²⁺ translocation was measured by the ability of the ionophores to stimulate ATP hydrolytic activity of the reconstituted enzyme. It was found that the maximum stimulation of the ATP hydrolytic activity was induced by the electroneutral Ca²⁺/2H⁺ ionophore A23187 (9 to 10-fold). A Ca²⁺ ionophore unable to translocate H⁺, CYCLEX-2E, was less efficient in stimulating the activity of the reconstituted enzyme (two- to threefold). However, the combined addition of CYCLEX-2E plus protonophores further increased the ATP hydrolytic activity (around fourfold), whereas, the protonophores did not further stimulate ATP hydrolysis in the presence of A23187. Furthermore, in the absence of Ca²⁺ ionophore, the electroneutral K⁺(Na⁺)/H⁺ ionophoretic exchanger, nigericin, or the electroneutral Na⁺(K⁺)/H⁺ ionophoretic exchanger, monensin, stimulated the rate of ATP hydrolysis in the reconstituted enzyme two- or threefold, respectively. These results suggest that the Ca²⁺-ATPase not only translocates Ca²⁺ but also H⁺ in the opposite direction.     

Changes in islet plasma membrane calcium-ATPase activity and isoform expression induced by insulin resistance

We studied the effect of insulin resistance (IR) induced by administration of a fructose-rich diet (FRD) to normal Wistar rats for 21 days, upon islet plasma membrane calcium ATPases (PMCAs) and insulin secretion. FRD rats showed significantly higher triglyceride and insulin levels, insulin:glucose ratio and HOMA-IR index than controls. FRD islets released significantly more insulin in response to glucose and showed (a) marked changes in PMCA isoform protein content (decreased PMCA 2 and increased PMCA 3), (b) a decrease in total PMCAs activity, and (c) higher levels of cytosolic calcium [Ca²⁺]_i. The lower PMCAs activity with the resultant increase in [Ca²⁺]_i would favor the compensatory greater release of insulin necessary to cope with the IR state present in FRD rats and to maintain normal glucose homeostasis. Thus, changes in PMCAs activity and isoform expression play a modulatory role upon insulin secretion during long-term adaptation to an increased hormone demand.     

Enhanced glucose-induced insulin release and endogenous Ca2+ ionophoretic activity in neonatal rat islets following islet-activating protein

Islet-Activating Protein, purified from the culture medium of Bordetella pertussis, enhanced glucose-induced insulin secretion from cultured neonatal rat islets in a calcium dependent manner. This effect was accompanied by an increase in lipid associated Ca2+ ionophoretic activity, as measured by passage of Ca2+ through multilamellar planar membranes containing islet lipid extracts. These findings suggest that the action of IAP in the neonatal islet may be mediated by enhanced entry of extracellular calcium following an effect on membrane lipid composition.     

The Relationship between Membrane Potential and Calcium Dynamics in Glucose-Stimulated Beta Cell Syncytium in Acute Mouse Pancreas Tissue Slices

Oscillatory electrical activity is regarded as a hallmark of the pancreatic beta cell glucose-dependent excitability pattern. Electrophysiologically recorded membrane potential oscillations in beta cells are associated with in-phase oscillatory cytosolic calcium activity ([Ca²⁺]_i) measured with fluorescent probes. Recent high spatial and temporal resolution confocal imaging revealed that glucose stimulation of beta cells in intact islets within acute tissue slices produces a [Ca²⁺]_i change with initial transient phase followed by a plateau phase with highly synchronized [Ca²⁺]_i oscillations. Here, we aimed to correlate the plateau [Ca²⁺]_i oscillations with the oscillations of membrane potential using patch-clamp and for the first time high resolution voltage-sensitive dye based confocal imaging. Our results demonstrated that the glucose-evoked membrane potential oscillations spread over the islet in a wave-like manner, their durations and wave velocities being comparable to the ones for [Ca²⁺]_i oscillations and waves. High temporal resolution simultaneous records of membrane potential and [Ca²⁺]_i confirmed tight but nevertheless limited coupling of the two processes, with membrane depolarization preceding the [Ca²⁺]_i increase. The potassium channel blocker tetraethylammonium increased the velocity at which oscillations advanced over the islet by several-fold while, at the same time, emphasized differences in kinetics of the membrane potential and the [Ca²⁺]_i. The combination of both imaging techniques provides a powerful tool that will help us attain deeper knowledge of the beta cell network.     

Activatory effect of regucalcin on hepatic plasma membrane (Ca2+-Mg2+)-ATPase is impaired by liver injury with carbon tetrachloride administration in rats

The alteration of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver of rats administered orally carbon tetrachloride (CCl₄) solution was investigated. Rats received a single oral administration of CCl₄ (10, 25 and 50%, 1.0 ml/100 g body weight), and 3 or 24 h later they were sacrificed. CCl₄ administration caused a remarkable elevation of liver calcium content and a corresponding increase in liver plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity, indicating that the increased Ca²⁺ pump activity is partly involved in calcium accumulation in liver cells. Moreover, the participation in regucalcin, which is an intracellular activating factor on the enzyme, was examined by using anti-regucalcin IgG. The plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity increased by CCl₄ administration was not entirely inhibited by the presence of anti-regucalcin IgG (1.0 and 2.5 ug/ml) in the enzyme reaction mixture. However, the effect of regucalcin (0.25–1.0 uM) to activate (Ca²⁺-Mg²⁺)-ATPase in the liver plasma membranes of normal rats was not revealed in the liver plasma membranes obtained from CCl₄-administered rats. Also, the effect of regucalcin was not seen when the plasma membranes were washed with 1.0 mM EGTA, indicating that the disappearance of regucalcin effect is not dependent on calcium binding to the plasma membranes due to liver calcium accumulation. Now, the presence of dithiothreitol (5 mM) or heparin (20 ug/ml) caused a remarkable elevation of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver obtained from CCl₄-administered rats. Thus, the regucalcin effect differed from that of dithiothreitol or heparin. The present study suggests that the impairment of regucalcin effect on Ca²⁺ pump activity in liver plasma membranes is partly contribute to hepatic calcium accumulation induced by liver injury with CCl₄ administration.     

Impact of Ca2+ on membrane catalyzed IAPP amyloid formation and IAPP induced vesicle leakage

Human islet amyloid polypeptide (hIAPP, also known as amylin) is a 37 amino acid pancreatic polypeptide hormone that plays a role in regulating glucose levels, but forms pancreatic amyloid in type-2 diabetes. The process of amyloid formation by hIAPP contributes to β-cell death in the disease. Multiple mechanisms of hIAPP induced toxicity of β-cells have been proposed including disruption of cellular membranes. However, the nature of hIAPP membrane interactions and the effect of ions and other molecules on hIAPP membrane interactions are not fully understood. Many studies have used model membranes with a high content of anionic lipids, often POPS, however the concentration of anionic lipids in the β-cell plasma membrane is low. Here we study the concentration dependent effect of Ca²⁺ (0 to 50 mM) on hIAPP membrane interactions using large unilamellar vesicles (LUVs) with anionic lipid content ranging from 0 to 50 mol%. We find that Ca²⁺ does not effectively inhibit hIAPP amyloid formation and hIAPP induced membrane leakage from binary LUVs with a low percentage of POPS, but has a greater effect on LUVs with a high percentage of POPS. Mg²⁺ had very similar effects, and the effects of Ca²⁺ and Mg²⁺ can be largely rationalized by the neutralization of POPS charge. The implications for hIAPP-membrane interactions are discussed.     

Small subpopulations of β-cells do not drive islet oscillatory [Ca2+] dynamics via gap junction communication

The islets of Langerhans exist as multicellular networks that regulate blood glucose levels. The majority of cells in the islet are excitable, insulin-producing β-cells that are electrically coupled via gap junction channels. β-cells are known to display heterogeneous functionality. However, due to gap junction coupling, β-cells show coordinated [Ca²⁺] oscillations when stimulated with glucose, and global quiescence when unstimulated. Small subpopulations of highly functional β-cells have been suggested to control [Ca²⁺] dynamics across the islet. When these populations were targeted by optogenetic silencing or photoablation, [Ca²⁺] dynamics across the islet were largely disrupted. In this study, we investigated the theoretical basis of these experiments and how small populations can disproportionality control islet [Ca²⁺] dynamics. Using a multicellular islet model, we generated normal, skewed or bimodal distributions of β-cell heterogeneity. We examined how islet [Ca²⁺] dynamics were disrupted when cells were targeted via hyperpolarization or populations were removed; to mimic optogenetic silencing or photoablation, respectively. Targeted cell populations were chosen based on characteristics linked to functional subpopulation, including metabolic rate of glucose oxidation or [Ca²⁺] oscillation frequency. Islets were susceptible to marked suppression of [Ca²⁺] when ~10% of cells with high metabolic activity were hyperpolarized; where hyperpolarizing cells with normal metabolic activity had little effect. However, when highly metabolic cells were removed from the model, [Ca²⁺] oscillations remained. Similarly, when ~10% of cells with either the highest frequency or earliest elevations in [Ca²⁺] were removed from the islet, the [Ca²⁺] oscillation frequency remained largely unchanged. Overall, these results indicate small populations of β-cells with either increased metabolic activity or increased frequency are unable to disproportionately control islet-wide [Ca²⁺] via gap junction coupling. Therefore, we need to reconsider the physiological basis for such small β-cell populations or the mechanism by which they may be acting to control normal islet function.     

Role of voltage-dependent ionic currents in coupling glucose stimulation to insulin secretion in canine pancreatic islet B-Cells

Summary Glucose-induced electrical activity in canine pancreatic islet B cells is distinct from that in rodent islets, though both display Ca²⁺-dependent insulin secretion. Rodent islet B cells undergo regular bursts of Ca²⁺-dependent action potentials, while canine islet B cells generate isolated Na⁺-dependent action potentials which often give way to a plateau depolarization. Here we present evidence to reconcile the species difference in electrical activity with the similarity of Ca²⁺ dependence of secretion. (i) In canine B cells increasing glucose concentrations produce membrane depolarization and increasing frequency of Na_o-dependent action potentials until a background membrane potential (-40mV) is reached where Na⁺ currents are inactivated. (ii) Voltage-dependent Ca²⁺ currents are present which are activated over the voltage excursion of the action potential (–50 to +20 mV) and inactivate slowly, (over seconds) in the range of the plateau depolarization (–40 to –25 mV). Hence, they are available to contribute to both phases of depolarization. (iii) Tetrodotoxin (TTX) reduces by half an early transient phase of glucosestimulated insulin secretion but not a subsequent prolonged plateau phase. The transient phase of secretion often corresponds well in time to the period of initial high frequency action potential activity. These latter results suggest that in canine B cells voltagedependent Na⁺ and Ca²⁺ currents mediate biphasic glucose-induced insulin secretion. The early train of Na⁺-dependent action potentials, by transiently activating Ca²⁺ channels and allowing pulsatile Ca²⁺ entry, may promote an early transient phase of insulin secretion. The subsequent sustained plateau depolarization, by allowing sustained Ca²⁺ entry, may permit steady insulin release.     

Calcium-Lipid Interactions Observed with Isotope-Edited Infrared Spectroscopy

Calcium ions bind to lipid membranes containing anionic lipids; however, characterizing the specific ion-lipid interactions in multicomponent membranes has remained challenging because it requires nonperturbative lipid-specific probes. Here, using a combination of isotope-edited infrared spectroscopy and molecular dynamics simulations, we characterize the effects of a physiologically relevant (2 mM) Ca²⁺ concentration on zwitterionic phosphatidylcholine and anionic phosphatidylserine lipids in mixed lipid membranes. We show that Ca²⁺ alters hydrogen bonding between water and lipid headgroups by forming a coordination complex involving the lipid headgroups and water. These interactions distort interfacial water orientations and prevent hydrogen bonding with lipid ester carbonyls. We demonstrate, experimentally, that these effects are more pronounced for the anionic phosphatidylserine lipids than for zwitterionic phosphatidylcholine lipids in the same membrane.     

25-Hydroxysterols increase the permeability of liposomes to Ca2+ and other cations

25-Hydroxycholesterol and 25-hydroxy vitamin D-3 increased the permeability of liposomes to Ca²⁺ measured by the arsenazo III encapsulation technique. This effect was sensitive to the lipid composition of the membrane, with changes that decreased the motional freedom of phospholipid acyl chains decreasing Ca²⁺ permeability. The greatest permeability was observed with the zwitter-ionic phospholipids, phosphatidylcholine and phosphatidylethanolamine, whereas the acidic phospholipids, phosphatidylinositol and phosphatidylserine, depressed Ca²⁺ permeability. The effect was not specific for Ca²⁺. Other divalent cations were translocated in the order Mn²⁺ > Mg²⁺  Ca²⁺ ? Sr²⁺  Ba²⁺. The permeability of liposomes to the monovalent cation, Na⁺, was also substantially increased. The effect did not appear to be due to ionophoretic properties of the sterols, and it is suggested that perturbation of the membranes by the polar 25-hydroxyl group may play a role in increasing membrane permeability.     

High-resolution structures of annexin A5 in a two-dimensional array

Annexins are soluble cytosolic proteins that bind to cell membranes. Annexin A5 self-assembles into a two-dimensional (2D) array and prevents cell rupture by attaching to damaged membranes. However, this process is not fully understood at the molecular level. In this study, we determined the crystal structures of annexin A5 with and without calcium (Ca²⁺) and confirmed the Ca²⁺-dependent outward motion of a tryptophan residue. Strikingly, the two structures exhibited the same crystal packing and 2D arrangement into a p3 lattice, which agrees well with the results of low-resolution structural imaging. High-resolution structures indicated that a three-fold interaction near the tryptophan residue is important for mediating the formation of the p3 lattice. A hypothesis on the promotion of p3 lattice formation by phosphatidyl serine (PS) is also suggested. This study provides molecular insight into how annexins modulate the physical properties of cell membranes as a function of Ca²⁺ concentration and the phospholipid composition of the membrane.     

Calcium Binding to Plasma Membranes from Normal and SV40 Transformed Hamster Lymphocytes

Abstract

The calcium binding properties of isolated plasma membranes from normal and SV40 transformed hamster lymphocytes were compared over the Ca²⁺ concentration range of 10^?5M to 5 × 10^?3M and at physiological ionic strength. At all Ca²⁺ concentrations, normal membranes bound more Ca²⁺ than tumor membranes; at blood Ca²⁺ levels (1–2 mM) plasma membranes of normal cells bind twice as much as membranes from tumor cells. Normal plasma membranes demonstrated positive cooperative Ca²⁺ binding whereas tumor membranes displayed non-interacting Ca²⁺-binding sites. Ca²⁺ binding to both membranes was insensitive to Mg²⁺ (0.1 to 2.5 mM). A pH shift from 7 to 6 resulted in a 70% decrease of normal membrane-bound Ca²⁺ compared to a 40% decrease observed with tumor membranes. Extracellular surface Ca²⁺ binding to intact cells was also studied after a 72-hour equilibration of cells with ⁴⁵Ca²⁺ and with ethylene-glycol-bis-(β-amino-ethyl ether) N, N′-tetraacetate chelation as marker for surface Ca²⁺. Tumor cell surface Ca²⁺ binding was only 10% of that observed with quiescent lymphocytes. Normal lymphocytes stimulated to divide with phytohemagglutinin also showed a decreased level of surface Ca²⁺ (50%). However, plasma membranes isolated from non-dividing and phytohemagglutinin-stimulated lymphocytes exhibited equivalent Ca²⁺ binding.     

The effect of membrane stabilizers on phytochrome-controlled anthocyanin biosynthesis in Brassica oleracea

The promotion of anthocyanin synthesis in red-cabbage seedlings by 5 min exposure to R light is inhibited by subsequent application of CaCl₂. The stimulation of dark synthesis of anthocyanin by n-PrOH and by kinetin is also reduced by Ca²⁺ and by cholesterol, both of which are well known to stabilize cell membranes. By contrast, EDTA, which chelates Ca²⁺, promotes dark synthesis of anthocyanin. Assay of native Ca²⁺ extractable from seedlings immersed in EDTA demonstrates that R light exposure promotes a highly significant increase in extractable Ca²⁺. It is suggested that the molecular configuration of the phytochrome molecule affects the ability of a membrane to bind Ca²⁺ and that this in turn affects the permeability to substrates which are required for anthocyanin biosynthesis.