Increased Ca++ uptake by erythrocytes infected with malaria parasites: Evidence for exported proteins and novel inhibitors

Malaria parasites export many proteins into their host erythrocytes and increase membrane permeability to diverse solutes. Although most solutes use a broad‐selectivity channel known as the plasmodial surface anion channel, increased Ca⁺⁺ uptake is mediated by a distinct, poorly characterised mechanism that appears to be essential for the intracellular parasite. Here, we examined infected cell Ca⁺⁺ uptake with a kinetic fluorescence assay and the virulent human pathogen, Plasmodium falciparum. Cell surface labelling with N‐hydroxysulfosuccinimide esters revealed differing effects on transport into infected and uninfected cells, indicating that Ca⁺⁺ uptake at the infected cell surface is mediated by new or altered proteins at the host membrane. Conditional knockdown of PTEX, a translocon for export of parasite proteins into the host cell, significantly reduced infected cell Ca⁺⁺ permeability, suggesting involvement of parasite‐encoded proteins trafficked to the host membrane. A high‐throughput chemical screen identified the first Ca⁺⁺ transport inhibitors active against Plasmodium‐infected cells. These novel chemical scaffolds inhibit both uptake and parasite growth; improved in vitro potency at reduced free [Ca⁺⁺] is consistent with parasite killing specifically via action on one or more Ca⁺⁺ transporters. These inhibitors should provide mechanistic insights into malaria parasite Ca⁺⁺ transport and may be starting points for new antimalarial drugs.     

Muscle Contraction during Hyperpolarizing Currents in the Crab

Isolated muscle fibers from the motor legs of the crab Trichodactilus dilocarcinus were submitted to strong hyperpolarizing currents of varied intensities which produced tension during the current pulse. Threshold for tension was obtained with intensities of about 0.2 x 10^–5 A, changing E_m to ca. –150 mV (starting from a resting potential ofca. –80 mV). At the closure of the anodic square pulse, a second phase of tension usually appeared superimposed upon the one obtained during hyperpolarization. The first phase of tension increased with the increase of Ca⁺⁺ concentration in the bath. Sr⁺⁺ produced the same type of mechanical output as Ca⁺⁺. When added to the normal Ca⁺⁺ concentration, Ba⁺⁺ and Mn⁺⁺ in low concentrations (up to 21.5 mM) also increased the tension of this phase, but at higher concentrations they blocked both phases while Mg⁺⁺ did not alter the tension. Of all the divalent cations employed, only Sr⁺⁺ is capable of developing tension as a substitute for Ca⁺⁺ in the external media. Procaine administered in a dosage (5 x 10^–3 W/V)which would suppress the contracture due to caffeine (10 mM), did not modify the tension developed during the hyperpolarization. The preceding data indicate that the Ca⁺⁺ required for tension during hyperpolarization comes from sites which would differ from those usually postulated for tension due to depolarization in the muscle fibers of other crustaceans (American crayfish). Furthermore, the external source of Ca⁺⁺ appears to be one mainly implicated in the induction of tension due to inward current pulses.     

Junctional Membrane Uncoupling : Permeability transformations at a cell membrane junction

The permeability of the membrane surfaces where cells are in contact (junctional membranes) in Chironomus salivary glands depends on Ca⁺⁺ and Mg⁺⁺. When the concentration of these ions at the junctional membranes is raised sufficiently, these normally highly permeable membranes seal off; their permeability falls one to three orders, as they approach the nonjunctional membranes in conductance. This permeability transformation is achieved in three ways: (a) by iontophoresis of Ca⁺⁺ into the cell; (b) by entry of Ca⁺⁺ and/or Mg⁺⁺ from the extracellular fluid into the cell through leaks in the cell surface membrane (e.g., injury); or (c) by entry of these ions through leaks arising, probably primarily in the perijunctional insulation, due to trypsin digestion, anisotonicity, alkalinity, or chelation. Ca⁺⁺ and Mg⁺⁺ appear to have three roles in the junctional coupling processes: (a) in the permeability of the junctional membranes; (b) in the permeability of the perijunctional insulation; and (c) a role long known— in the mechanical stability of the cell junction. The two latter roles may well be closely interdependent, but the first is clearly independent of the others.     

Effects of managanese salts on the AIDS-related pathogen,Cryptosporidium parvum in vitro and in vivo

The authors examined the effects of manganese salts on the interaction of the AIDS-related pathogen,Cryptosporidium parvum, with human ileoadenocarcinoma (HCT-8) cells in vitro. Manganese (Mn) inhibited binding ofC. parvum sporozoite membrane antigens to intact, fixed HCT-8 cells in a dose-dependent fashion, whereas Ca⁺⁺, Mg⁺⁺, and Zn⁺⁺ salts had no effect. Manganese was also found to affect sporozoite penetration of live HCT-8 cells, which resulted in a dose-dependent inhibition of parasite development. However, the levels of Mn⁺⁺ needed in the live cell assays was approx 10-fold greater than in the fixed-cell assays. This inhibition of parasite development was not reversible when Ca⁺⁺ or Mg⁺⁺ were used as competitors. Oral supplementation of suckling mice infected withC. parvum with MnSO₄ resulted in significant reductions and, in some cases, elimination of intestinally derived oocysts.     

Microvillar Ca++ signaling: A new view of an old problem

Proceeding from the recent finding that the main components of the Ca⁺⁺ signal pathway are located in small membrane protrusions on the surface of differentiated cells, called microvilli, a novel concept of cellular Ca⁺⁺ signaling was developed. The main features of this concept can be summarized as follows: Microvilli are formed on the cell surface of differentiating or resting cells from exocytic membrane domains, growing out from the cell surface by elongation of an internal bundle of actin filaments. The microvillar tip membranes contain all functional important proteins synthesized such as ion channels and transporters for energy-providing substrates and structural components, which are, in rapidly growing undifferentiated cells, distributed over the whole cell surface by lateral diffusion. The microvillar shaft structure, a bundle of actin filaments, forms a dense cytoskeletal matrix tightly covered by the microvillar lipid membrane and represents an effective diffusion barrier separating the microvillar tip compartment (entrance compartment) from the cytoplasm. This diffusion barrier prevents the passage of low molecular components such as Ca⁺⁺ glucose and other relevant substrates from the entrance compartment into the cytoplasm. The effectiveness of the actin-based diffusion barrier is modulated by various signal pathways and effectors, most importantly, by the actin-depolymerizing/reorganizing activity of the phospholipase C (PLC)-coupled Ca⁺⁺ signaling. Moreover, the microvillar bundle of actin filaments plays a dual role in Ca⁺⁺ signaling. It combines the function of a diffusion barrier, preventing Ca⁺⁺ influx into the resting cell, with that of a high-affinity, ATP-dependent, and IP₃-sensitive Ca⁺⁺ store. Activation of Ca⁺⁺ signaling via PLC-coupled receptors simultaneously empties Ca⁺⁺ stores and activates the influx of external Ca⁺⁺. The presented concept of Ca⁺⁺ signaling is compatible with all established data on Ca⁺⁺ signaling. Properties of Ca⁺⁺ signaling, that could not be reconciled with the basic principles of the current hypothesis, are intrinsic properties of the new concept. Quantal Ca⁺⁺ release, Ca⁺⁺-induced Ca⁺⁺ release (CICR), the coupling phenomen between the filling state of the Ca⁺⁺ store and the activity of the Ca⁺⁺ influx pathway, as well as the various yet unexplained complex kinetics of Ca⁺⁺ uptake and release can be explained on a common mechanistic basis. J. Cell. Physiol. 180:19–34, 1999. © 1999 Wiley-Liss, Inc.     

Role of external potassium in the calcium-induced potassium efflux from human red blood cell ghosts

Summary The exposure of red cell ghosts to external Ca⁺⁺ and K⁺ leads to a rapid net K⁺ efflux. Preincubation of the ghosts for various lengths of time in the absence of K⁺ in the external medium prior to a challenge with maximally effective concentrations of Ca⁺⁺ and K⁺ renders the ghosts unresponsive to that challenge with a half-time of about 7–10 min. Preincubation at a range of K⁺ concentrations for a fixed length of time (60 min) prior to the challenge revealed that K⁺ concentrations of about 500 m or more suffice to maintain the K⁺ channel in a maximally responsive state for at least 60 min. These K⁺ concentrations are considerably lower than the K⁺ concentrations required to make the responsive channel respond with a maximal rate of K⁺ efflux. Thus, external K⁺ is not only necessary to induce the permeability change but also to maintain the transport system in a functional state.The presence of Mg⁺⁺ or ethylenediamine-tetraacetic acid (EDTA) in the K⁺-free preincubation media preserves the responsiveness to a challenge with Ca⁺⁺ plus K⁺. In contrast to external K⁺, the presence of external Ca⁺⁺ does not reduce but rather enhances the loss of responsiveness. An excess of EDTA prevents the effects of Ca⁺⁺ while washes with EDTA after exposure to Ca⁺⁺ do not reverse them.In red cell ghosts that contain Ca⁺⁺ buffers, the transition from a responsive to a nonresponsive state incubation in the absence of external K⁺ is enhanced. The effects of incubation in the presence of Ca⁺⁺ in K⁺-free media are reversed; external Ca⁺⁺ now reduces the rate at which the responsiveness is lost. The loss of responsiveness after incubation in K⁺-free media prior to a challenge with external K⁺ and internal Ca⁺⁺ does also take place when K⁺-efflux from red cell ghosts is measured by means of⁴²K⁺ into media that have the same K⁺ concentrations as the ghost interior. This confirms that the effects of K⁺-free incubation are due to the modification of the K⁺-selective channel rather than to an inhibition of diffusive Cl^–-efflux.Abbreviation used in text TRIS Tris (hydroxymethyl) aminomethan This paper is dedicated to the memory of Walther Wilbrandt.     

Calcium regulation of skeletal myogenesis. I. Cell content critical to myotube formation

Summary Primary cultures of embryonic chick pectoral skeletal muscle were used to study calcium regulation of myoblast fusion to form multinucleated myotubes. Using atomic absorption spectrometry to measure total cellular calcium and the⁴⁵Ca-exchange method to determine free cellular Ca⁺⁺, our data suggest that only the free cellular calcium changes significantly during development under conditions permissive for myotube formation (0.9 mM external Ca⁺⁺). Increases in calcium uptake occurred before and toward the end of the period of fusion with the amount approximating 2 to 4 pmol per cell in mass cultures. If the medium [Ca⁺⁺] is decreased to 0.04 mM, as determined with a calcium electrode, a fusion-block is produced and free cell Ca⁺⁺ decreased 5- to 10-fold. Removal of the fusion-block by increasing medium [Ca⁺⁺] results in a release of the fusion-block and an increase in cellular Ca⁺⁺ to approximately 1 pmol per cell during fusion, and higher thereafter. Cation ionophore A23187 produced transient increases in cellular calcium and stimulated myoblast fusion and the final extent of myotube formation only when added at the onset of culture. Results suggest that transient increased calcium uptake alone is insufficient for fusion because critical cellular content in conjunction with permissive amounts of medium [Ca⁺⁺] must exist. The latter suggests further that cell surface Ca⁺⁺ was also critical.     

Lanthanum in heart cell culture. Effect on calcium exchange correlated with its localization

Correlation of the localization of La⁺⁺⁺ with its effects on Ca⁺⁺ exchange in cultured rat heart cells is examined with the use of a recently developed technique. 75% of cellular Ca⁺⁺ is exchangeable and is completely accounted for by two kinetically defined phases. The rapidly exchangeable phase has a t ½ = 1.15 min and accounts for 1 1 mmoles Ca⁺⁺/kg wet cells or 43% of the exchangeable Ca⁺⁺ (cells perfused with [Ca⁺⁺]_o = 1 mM) Phase 2 has a t ½ = 19.2 min and accounts for 1.5 mmoles Ca⁺⁺/kg wet cells or 57% of the exchangeable Ca⁺⁺. 0.5 mM [La⁺⁺⁺]_o displaces 0 52 mmoles Ca⁺⁺/kg wet cells—all from phase 1—and almost completely abolishes subsequent Ca⁺⁺ influx and efflux The presence of La⁺⁺⁺ in the washout converts the washout pattern to a single phase system with a t ½ = 124 min. The effects upon Ca⁺⁺ exchange are coincident with abolition of contractile tension but regenerative depolarization of the tissue is maintained Electron microscope localization of the La⁺⁺⁺ places it exclusively in the external lamina or basement membrane of the cells. The study indicates that negatively charged sites in the basement membrane play a crucial role in the E-C coupling process in heart muscle     

Junctional membrane permeability

Summary Substitution of extracellular Na⁺ by Li⁺ causes depression of junctional membrane permeability inChironomus salivary gland cells; within 3 hr, permeability falls to so low a level that neither fluorescein nor the smaller inorganic ions any longer traverse the junctional membrane in detectable amounts (uncoupling). The effect is Li-specific: if choline⁺ is the Na⁺ substitute, coupling is unchanged. The Li-produced uncoupling is not reversed by restitution of Na⁺. Long-term exposure (>1 hr) of the cells to Ca, Mg-free medium leads also to uncoupling. This uncoupling is fully reversible by early restitution of Ca⁺⁺ or Mg⁺⁺. Coupling is maintained in the presence of either Ca⁺⁺ or Mg⁺⁺, so long as the total divalent concentration is about 12mm. The uncoupling in Ca, Mg-free medium ensues regardless of whether the main monovalent cation is Na, Li or choline.The uncouplings are accompanied by cell depolarization. Repolarization of the cells by inward current causes restoration of coupling; the junctional conductance rises again to its normal level. The effect was shown for Li-produced uncoupling, for uncoupling by prolonged absence of external Ca⁺⁺ and Mg⁺⁺, and for uncoupling produced by dinitrophenol. In all cases, the recoupling has the same features: (1) it develops rapidly upon application of the polarizing current; (2) it is cumulative; (3) it is transient, but outlasts the current; and (4) it appears not to depend on the particular ions carrying the current from the electrodes to the cell. The recoupling is due to repolarization of nonjunctional cell membrane; recoupling can be produced at zero net currernt through the junctional membrane. Recoupling takes place also as a result of chemically produced repolarization; restoration of theK gradients in uncoupled cells causes partial recoupling during the repolarization phase.An explanation of the results on coupling is proposed in terms of known mechanisms of regulation of Ca⁺⁺ flux in cells. The uncouplings are explained by actions raising the Ca⁺⁺ level in the cytoplasmic environment of the junctional membranes; the recoupling is explained by actions lowering this Ca⁺⁺ level.     

Calcium transport and cellular distribution in quiescent and serum-stimulated primary cultures of bone cells and skin fibroblasts

Primary cultures of bone cells and skin fibroblasts were examined for their Ca⁺⁺ content, intracellular distribution and Ca⁺⁺ fluxes. Kinetic analysis of ⁴⁵Ca⁺⁺ efflux curves indicated the presence of three exchangeable Ca⁺⁺ compartments which turned over at different rates: a “very fast turnover” (S₁), a “fast turnover” (S₂), and a “slow turnover” Ca⁺⁺ pool (S₃). S₁ was taken to represent extracellular membrane-bound Ca⁺⁺, S₂ represented cytosolic Ca⁺⁺, and S₃ was taken to represent Ca⁺⁺ sequestered in some intracellular organelles, probably the mitochondria. Bone cells contained about twice the amount of Ca⁺⁺ as compared with cultured fibroblasts. Most of this extra Ca⁺⁺ was localized in the “slow turnover” intracellular Ca⁺⁺ pool (S₃). Serum activation caused the following changes in the amount, distribution, and fluxes of Ca⁺⁺: (1) In both types of cells serum caused an increase in the amount of Ca⁺⁺ in the “very fast turnover” Ca⁺⁺ pool, and an increase in the rate constant of ⁴⁵Ca⁺⁺ efflux from this pool, indicating a decrease in the strength of Ca⁺⁺ binding to ligands on cell membranes. (2) In fibroblasts, serum activation also caused a marked decrease in the content of Ca⁺⁺ in the “slow turnover” Ca⁺⁺ pool (S₃), an increase in the rates of Ca⁺⁺ efflux from the cells to the medium, and from S₃ to S₂, as well as a decrease in the rate of influx into S₃. (3) In bone cells the amount of Ca⁺⁺ in S₃ remained high in “serum activated” cells, the rate of efflux from S₃ to S₂ increased, and the rate of influx into S₃ also increased. The rate of efflux from the cells to the medium did not change. The results suggest specific properties of bone cells with regard to cell Ca⁺⁺ presumably connected with their differentiation. Following serum activation we investigated the time course of changes in the amount of exchangeable Ca⁺⁺ in bone cells and fibroblasts, in parallel with measurements of ³H-thymidine incorporation and cell numbers. Serum activation caused a rapid decrease in the content of cell Ca⁺⁺ which was followed by a biphasic increase lasting until cell division.     

A Possible Role for Ligatin and the Phosphoglycoproteins It Binds in Calcium-Dependent Retinal Cell Adhesion

Ligatin is a filamentous plasma membrane protein that serves as a baseplate for the attachment of peripheral glycoproteins to the external cell surface. Ligatin can be released from intact, embryonic chick neural retinal cells by treatment with 20 mM Ca⁺⁺ without adversely affecting their viability. α-Glucose-1-phos phate is also effective in removing ligatin-associated glycoproteins from intact cells. After either of these treatments, the retinal cells seem not to exhibit Ca⁺⁺ -dependent adhesion for one another. It is thus suggested that ligatin in neural retina may serve as a baseplate for the attachment to the cell surface of glycoproteins active in Ca⁺⁺-dependent adhesion. The finding that Ca⁺⁺ serves to protect Ca⁺⁺-dependent adhesion molecules from digestion by trypsin is discussed in relation to steric constraints on trypsin's accessibility to these adhesion molecules because of their possible binding to arrayed ligatin filaments.     

Factors influencing the degradation of photoreceptor membrane in the crayfish,Procambarus clarkii

Summary Light-induced degradation of photoreceptor membrane in the crayfish was studied by quantitative light and electron microscopy. The production of lysosomal organelles within the photoreceptor cells was enhanced by presenting the light stimulus intermittently (i.e., flicker) or by doubling its intensity. The enhancement was seen primarily as an increase in the number and size of multivesicular bodies. As these stimulus conditions are likely to facilitate intracellular Ca⁺⁺ fluxes, the results are compatibl with recent speculations that Ca⁺⁺ ions may regulate membrane degradation. To test the possibility that Ca⁺⁺ acts as a signal coupling receptor stimulation with membrane loss, retinas were incubated in the dark with the ionophore A23187 in the presence or absence of external Ca⁺⁺. The results demonstrate that A23187 produces a Ca⁺⁺-dependent increase in lysosomal organelles, predominantly multivesicular bodies. These data are consistent with a role for intracellular Ca⁺⁺ in the degradative process; however, the exact locus of the effect is unclear.Supported by a grant (BNS 8004587) from the National Science Foundation to G.S.H. The authors gratefully acknowledge the helpful discussions and expert technical assistance of Thomas R. Tokarski     

Active Uptake of Ca++ and Ca++-Activated Mg++ ATPase in Red Cell Membrane Fragments

Isolated human red blood cell membrane fragments (RBCMF) were found to take up Ca⁺⁺ in the presence of ATP.¹ This ATP-dependent Ca⁺⁺ uptake by RBCMF appears to be the manifestation of an active Ca⁺⁺ transport mechanism in the red cell membrane reported previously (Schatzmann, 1966; Lee and Shin, 1969). The influences of altering experimental conditions on Ca⁺⁺-stimulated Mg⁺⁺ ATPase (Ca⁺⁺ ATPase) and Ca⁺⁺ uptake of RBCMF were studied. It was found that pretreatment of RBCMF at 50°C abolished both Ca⁺⁺ ATPase and Ca⁺⁺ uptake. Pretreatment of RBCMF with phospholipases A and C decreased both Ca⁺⁺ ATPase and Ca⁺⁺ uptake, whereas pretreatment with phospholipase D did not significantly alter either Ca⁺⁺ ATPase or Ca⁺⁺ uptake. Both Ca⁺⁺ ATPase and Ca⁺⁺ uptake had ATP specificity, similar optimum pH's, and optimum incubation temperatures. From these results, it was concluded that Ca⁺⁺ uptake is intimately linked to Ca⁺⁺ ATPase.     

The Initiation of Spike Potential in Barnacle Muscle Fibers under Low Intracellular Ca++

Electrical properties of the muscle fiber membrane were studied in the barnacle, Balanus nubilus Darw. by using intracellular electrode techniques. A depolarization of the membrane does not usually produce an all-or-none spike potential in the normal muscle fiber even though a mechanical response is elicited. The intracellular injection of Ca⁺⁺-binding agents (K₂SO₄ and K salt of EDTA solution, K₃ citrate solution, etc.) renders the fiber capable of initiating all-or-none spikes. The overshoot of such a spike potential increases with increasing external Ca concentration, the increment for a tenfold increase in Ca concentration being about 29 mv. The threshold membrane potential for the spike and also for the K conductance increase shifts to more positive membrane potentials with increasing [Ca⁺⁺]_out. The removal of Na ions from the external medium does not change the configuration of the spike potential. In the absence of Ca⁺⁺ in the external medium, the spike potential is restored by Ba⁺⁺ and Sr⁺⁺ but not by Mg⁺⁺. The overshoot of the spike potential increases with increasing [Ba⁺⁺]_out or [Sr⁺⁺]_out. The Ca influx through the membrane of the fiber treated with K₂SO₄ and EDTA was examined with Ca⁴⁵. The influx was 14 pmol per sec. per cm² for the resting membrane and 35 to 85 pmol per cm² for one spike. From these results it is concluded that the spike potential of the barnacle muscle fiber results from the permeability increase of the membrane to Ca⁺⁺ (Ba⁺⁺ or Sr⁺⁺).     

Muscle Calcium Metabolic Effects of Hypokinesia in Physically Healthy Subjects

The incompleteness of electrolyte deposition during hypokinesia (HK; diminished movement) is the defining factor of electrolyte metabolic changes, yet the effect of prolonged HK upon electrolyte deposition is poorly understood. The objective of this investigation was to determine the effect of muscle calcium (Ca⁺⁺) changes upon Ca⁺⁺ losses during prolonged HK. Studies were conducted on 20 physically healthy male volunteers during a pre-experimental period of 30 days and an experimental period of 364 days. Subjects were equally divided in two groups: control subjects (CS) and experimental subjects (ES). The CS group ran average distances of 9.2?±?1.2 km day^?l, and the ES group walked average distances of 2.3?±?0.2 km day^?l. Muscle Ca⁺⁺ contents, plasma Ca⁺⁺ concentrations, and Ca⁺⁺ losses in urine and feces were measured in the experimental and control groups of subjects. The muscle Ca⁺⁺ contents decreased (p?<?0.05), and plasma Ca⁺⁺ levels and Ca⁺⁺ losses in the urine and feces increased (p?<?0.05) in the ES group compared with their pre-experimental levels and the values in their respective CS group. Muscle Ca⁺⁺ contents and plasma Ca⁺⁺ levels and urinary and fecal Ca⁺⁺ losses did not change in the CS group compared to their pre-experimental levels. It is concluded that prolonged HK increase plasma Ca⁺⁺ concentrations and Ca⁺⁺ losses in Ca⁺⁺ deficient muscle indicating decreased Ca⁺⁺ deposition.     

Calcium and facilitation at two classes of crustacean neuromuscular synapses

The closer muscle of the crab, Chionoecetes, has at least two classes of excitatory neuromuscular synapses. In one class of synapses an action potential depolarizing the synaptic region releases much more transmitter if it has been preceded recently by another action potential. The other class of synapses shows this property, called facilitation, to a far lesser extent. Immediately after one conditioning stimulus the level of facilitation is similar in both classes. The rate of the ensuing decay of the facilitation is the critical factor differentiating the two classes of synapses. The relationship between external Ca⁺⁺ concentration and transmitter release is similar for both classes of synapses. The slope of a double logarithmic plot of this relationship varies from 3.1 between 5 and 10 mM Ca⁺⁺ to 0.9 between 30 and 40 mM Ca⁺⁺. Facilitation does not significantly change when tested in external Ca⁺⁺ concentrations ranging from 7 to 30 mM. The extracellularly recorded nerve terminal action potential does not increase in amplitude during facilitation. The results suggest that the mechanism of synaptic facilitation is similar for both classes of synapses and occurs after the stage in transmitter release involving Ca⁺⁺.     

Bile acid uptake and calcium flux in brush border membrane vesicles

Our laboratory has recently reported that intestinal bile acid malabsorption in cystic fibrosis (CF) is a primary mucosal cell defect. Others have suggested that elevated intracellular Ca⁺⁺ levels in other cell types in CF may represent a common primary dysfunction in Ca⁺⁺ efflux in these cells. We examined the possibility that intestinal bile acid absorption and Ca⁺⁺ efflux in mucosal cells may be linked physiologically. Brush border membrane vesicles (BBMV) prepared from guinea pig ileum served as the experimental model to test this hypothesis. Ca⁺⁺ (2.5×10^?3M) present in the incubation medium did not alter the uptake of taurocholic acid (TCA) by BBMV. Also, TCA uptake into BBMV preloaded with Ca⁺⁺ was not significantly different from that in BBMV not previously loaded with Ca⁺⁺. Furthermore, with TCA present in the incubation medium, Ca⁺⁺ efflux from preloaded BBMV was not altered. These data suggest that ileal TCA uptake, as measured by BBMV, is not dependent upon either intra- or extravesicular Ca⁺⁺. Also, Ca⁺⁺ efflux from BBMV is unaffected by TCA uptake. Although separate lines of evidence suggest that intestinal bile acid malabsorption and reduced plasma membrane Ca⁺⁺ flux are primary defects in CF, we conclude that in the normal intestine these functions are independent physiological processes.     

Action of insulin and cell calcium: Effect of ionophore A23187

Summary We have measured the effects of the carboxylic Ca⁺⁺ ionophore A23187 on muscle tension, resting potential and 3-O-methylglucose efflux. The ionophore produces an increase in tension that is dependent on external Ca⁺⁺ concentration since (a) the contracture was blocked by removing external Ca⁺⁺ and (b) its size was increased by raising outside Ca⁺⁺. Neither resting potential nor resting and insulin-stimulated sugar efflux were modified by the ionophore. These data imply that the action of insulin is not mediated by increasing cytoplasmic [Ca⁺⁺]. Additional support for this conclusion was obtained by testing the effects of caffeine on sugar efflux. This agent, which releases Ca⁺⁺ from the reticulum, did not increase resting sugar efflux and inhibited the insulin-stimulated efflux. Incubation in solutions containing butyrated derivatives of cyclic AMP or cyclic GMP plus theophylline did not modify the effects of insulin on sugar efflux. Evidence suggesting that our experimental conditions increased the cytoplasmic cyclic AMP activity was obtained.     

Ion metabolism in a halophilic blue-green alga,Aphanothece halophytica

The intracellular ion content of the halophilic blue-green alga, Aphanothece halophytica was studied as a function of age, external sodium and external potassium concentration. Intracellular Na⁺ was found to be about 0.38 millimoles/g dry mass. Intracellular K⁺ concentrations were as high as 1 M and varied directly with external salinity. Intracellular Ca⁺⁺ and Mg⁺⁺ were in the range previously reported for fresh water blue-green algae despite their extremely high extracellular concentrations. Average cell size is consistent at room temperature with two exceptions. When the outside K⁺ is lower than 6.5 mM the cells tend to be smaller with less intracellular K⁺ and high Ca⁺⁺. In stationary phase cultures the cells are larger with high intracellular Mg⁺⁺ and low K⁺.     

Muscle: A Three Phase System : The partition of divalent ions across the membrane

The partition of sulfate, Ca⁺⁺, and Mg⁺⁺ across the membrane of the sartorius muscle has been studied, and the effect of various concentrations of these ions in the Ringer solution on the cellular level of Na⁺, K⁺, and Cl^- has been determined. The level of the three divalent ions in toad plasma and muscle in vivo has been assayed. Muscle was found to contain an almost undetectable amount of inorganic sulfate. Increases in the external level of these ions brought about increases in intracellular content, calculated from the found extracellular space as determined with radioiodinated serum albumin or inulin. Less of the cell water is available to sulfate than to Cl^-, and the Mg⁺⁺ space is less than the Na⁺ space. An amount of muscle water similar to that found for Li⁺ and I^- appears to be available to these divalent ions. Sulfate efflux from the cell was extremely rapid, and it was not found possible to differentiate kinetically between intra- and extracellular material. These results are consistent with the theory of a three phase system, assuming the muscle to consist of an extracellular phase and two intracellular phases. Mg⁺⁺ and Ca⁺⁺ are adsorbed onto the ordered phase, and increments in cellular content found on raising the external level are assumed to occur in the free intracellular phase.