Stimulus-dependent changes in calcium metabolism in rabbit neutrophils

We have found that the changes in calcium metabolism in rabbit neutrophils produced by the chemotactic synthetic peptide f-Met-Leu-Phe are not sensitive to the calcium chelator EGTA. The present results demonstrate unambiguously that the previously described chemotactic factor induced changes in 45Ca fluxes in rabbit neutrophils do indeed reflect intracellular events. The pool of calcium mobilized by f-Met-Leu-Phe and increase in cell associated 45Ca upon stimulation are both insensitive to the presence of EGTA.     

Molecular properties of the red cell calcium pump: I. Effects of calmodulin,proteolytic digestion and drugs on the kinetics of active calcium uptake in inside-out red cell membrane vesicles

In calmodulin-stripped inside-out human red cell membrane vesicles /IOV/ ATP + Mg²⁺-dependent active calcium uptake is stimulated by the addition of calmodulin. Calmodulin increases the maximum calcium transport rate /V_max/, decreases K_Ca, and does not affect K_ATP of calcium uptake. The action of both membrane bound and external calmodulin is competitively inhibited by phenothiazines. Drugs reacting with SH groups of proteins reversibly inhibit calcium pumping by decreasing V_max and not affecting K_Ca and K_ATP. The relative magnitude of calmodulin stimulation of calcium transport is unaltered by SH reagents.Mild proteolytic digestion of IOVs stimulates active calcium uptake and mimics the effects of calmodulin on the kinetic parameters — that is converts the system to a “high calcium-affinity” state. Proteolysis eliminates calcium-dependent calmodulin binding to IOV membranes and any further stimulation of calcium uptake by calmodulin. Based on these results the presence of a calmodulin-binding regulatory subunit of the red cell calcium pump at the internal membrane surface is postulated.     

Inhibition by sodium of A23187-mediated calcium translocation

Sodium inhibits in a dose-related fashion the translocation of calcium from an aqueous milieu into an organic phase containing the divalent-cation ionophore A23187. This inhibitory effect is reproduced by other monovalent cations, modulated by the nature of the anion in the sodium halide, and inversely related to the absolute amount of calcium translocated. The inhibitory effect cannot be attributed to a change in osmolarity or ionic strength, to sequestration of the ionophoretic molecule at the interface between the aqueous and organic phases, or to translocation of sodium or chloride. These findings indicate that sodium may directly affect the handling of calcium by ionophoretic systems specifically mediating the transport of divalent cations.     

Effects of ionophore A23187 on calcium flux and amylase release in isolated mouse pancreatic acini

The divalent cation ionophore A23187 has been used extensively to demonstrate the importance of Ca²⁺ in the control of pancreatic enzyme secretion. The relative importance, however, of the ability of the ionophore to facilitate Ca²⁺ movement across plasma and intracellular membranes in the stimulation of amylase release is not clear. We therefore studied these relationships in isolated pancreatic acini, a preparation in which it is possible to precisely measure both ⁴⁵Ca²⁺ fluxes, Ca²⁺ content and amylase release. A23187 increased the initial rates of both ⁴⁵Ca²⁺ uptake and washout. In addition, the content of both exchangeable ⁴⁵Ca²⁺ and total Ca²⁺ were reduced. These results indicated, therefore, that A23187 increases Ca²⁺ fluxes across both plasma and intracellular membranes. Consistent with this observation, the initial stimulation of amylase release by A23187 was independent of extracellular Ca²⁺. In the absence of extracellular Ca²⁺, however, A23187 caused a rapid fall in acinar Ca²⁺ and subsequent amylase release was abolished. Depletion of intracellular Ca²⁺ by the ionophore also blocked the subsequent stimulation by cholecystokinin (CCK). The results indicate certain similarities in the actions of A23187 and CCK on pancreatic acini; both the agonists have striking effects on intracellular Ca²⁺ which in turn mediates their actions.     

Molecular properties of the red cell calcium pump: II. Effects of calmodulin,proteolytic digestion and drugs on the calcium-induced membrane phosphorylation by ATP in inside-out red cell membrane vesicles

In inside-out human red cell membrane vesicles /IOV/, in the absence of Mg²⁺, the only calcium-induced labelling by γ³²P-ATP occurs in a 140–150 000 molecular weight protein fraction, representing the hydroxylamine-sensitive phosphorylated intermediate /EP/ of the calcium pump. In the presence of Mg²⁺ calcium-induced phosphorylation is accelerated but several other membrane proteins are also phosphorylated through protein kinase action forming hydroxylamine-insensitive bonds. Addition of calmodulin accelerates EP formation both in the absence and presence of Mg²⁺.Treatment of the membrane with SH-group reagents significantly reduces EP formation. Mild trypsin digestion of IOVs, stimulating active calcium transport, eliminates calmodulin action and decreases the steady-state level of EP. In trypsin-digested IOVs the molecular weight of the ³²P-labelled EP is shifted to lower values /110–120 000/ We suggest that trypsin digestion cleaves off a 20–40 000 molecular weight calmodulin-binding regulatory subunit of the calcium pump molecule.     

Evidence for calcium mediation of gonadotropin releasing hormone action in the pituitary

Despite the relatively long time since the isolation, characterization, and complete chemical synthesis of the gonadotropin releasing hormone (GnRH), very little information has become available which has elucidated the manner by which this hormone evokes gonadotropin release from the pituitary. Recently, a line of evidence has developed which suggests that calcium (Ca²⁺) may play a central role in GnRH stimulation of gonadotropin release from cultured rat pituitary cells.     

Perturbation of the phagocytic response in P388D1 cultured macrophages by agents altering cell calcium

Phagocytosis in adherent P388D₁ (D₁) cells was monitored utilizing formalin treated $\text{Listeria}$$\text{monocytogenes}$ ($\text{Lm}$) previously labeled with ¹²⁵iododeoxyuridine. The dependence of this phagocytic process on calcium was studied by using several agents which alter calcium metabolism. The calcium antagonist ruthenium red (RR) produced a dose and time dependent stimulation (60–70%) of $\text{Lm}$ phagocytosis by D₁ cells. Utilizing another calcium antagonist, D-600, a prolonged inhibition (4 hours) of phagocytosis (40%) was observed. The addition of the cation ionophore A23187 produced a transient stimulatory increase (38% at 2 hours) in the phagocytic response. The concomitant addition of RR and D-600 did not alter the phagocytosis of $\text{Lm}$ by D₁ cells as compared to control cells. However, this complete drug/drug antagonism was not seen with the combinations of A23187 and D-600 or RR and A23187. The addition of A23187 and D-600 resulted in a time dependent inhibition of phagocytosis which did not become maximal until 3 to 4 hours. A23187 and RR produced a time independent stimulation of phagocytosis which was significantly less than that which was observed for RR alone, but was of longer duration than the response produced by A23187 alone. The use of these calcium probes in the P388D₁ macrophage model suggests a role for calcium in the phagocytic process.     

The calcium hypothesis of cystic fibrosis

Data have been presented which suggests that various CF cell types show evidence of alterations in calcium homeostasis. The significance of these observations and the exact nature of the putative calcium defect in CF remains to be elucidated. It must also be determined whether this possible defect is primary, or is secondary or tertiary to some more basic lesion. The data reviewed suggests that altered calcium homeostasis may play some focal role in the aetiology or the pathogenesis of CF.     

Calcium binding capacity and calcium sensitive protein content in denervated frog muscles

Total and ionic calcium content, calcium binding capacity of sarcoplasmic proteins and calcium insensitive proteins were examined in atrophying leg muscles of frog after 1-5 months period of denervation. Different muscles showed different levels of atrophy and the total calcium content varied with reference to the type of muscle. Ionic calcium levels doubled in the gastrocnemius muscle after three months denervation. Calcium binding capacity of proteins and calcium insensitive proteins decreased rapidly up to four months after denervation in the gastrocnemius muscle. However no significant changes in the levels of calcium binding capacity and calcium insensitive proteins were found with reference to the type of muscle. Since total calcium content remains constant and wet muscle mass (expressed as atrophy) decreased markedly, an apparent increase in calcium concentration occurs in each muscle on denervation.     

Vitamin D-dependent calcium binding protein in the chick brain

A radioimmunoassay for chick CaBP has been used to measure the distribution of the protein in the chick central nervous system. The cerebellum contained 7.4 μg/mg protein, six times more than the medulla, and eleven times more than the cerebral cortex. Most of the CaBP was found in the supernate, after ultracentrifugation, with only trace amounts in the secretosome, microsome and other fractions. Slices of cerebellum from 19 and 21 days $\text{in}$$\text{ovo}$ chicks were maintained in organ culture. Treatment with kainic acid resulted in a loss of CaBP from the slices while metabolites of vitamin D were without effect.     

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 induced calcium transients in human red cells

A transient increase of cellular calcium was induced by addition of the divalent cation ionophore A23187 to human red cells in the absence or presence of drugs. The peak height of the calcium transient was increased about five times at pH 6.9 and up to eighteen times at pH 7.4 by trifluoperazine (0.30 mM), and two to three times at pH 6.9 by compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). The time-dependent changes of cellular calcium were analysed by the aid of a pump-leak model based partly on the calcium dependent parameters obtained from calcium ATPase experiments, partly on the A23187 induced calcium fluxes determined in experiments with ATP depleted cells. The transient increase of cellular calcium induced within few minutes after the addition of ionophore A23187 could be explained satisfactorily by the model both in the absence and presence of the four drugs, whereas the final level of cellular calcium in the drug experiments was more difficult to predict from the pump-leak model. Comparison of experimental and model calcium transients suggested that trifluoperazine and TMB-8 affected both pump and leak, whereas compound 48/80, probably due to low membrane-permeability, mainly affected the leak and verapamil affected the pump only.     

Evidence for the mobilization of cellular calcium by prostacyclin in cat adrenocortical cells: The effect of TMB-8

Stimulation of steroid production by isolated cat adrenocortical cells is partially dependent upon the presence of extracellular Ca²⁺ when elicited by prostacyclin (PGI₂) and completely dependent upon extracellular Ca²⁺ when elicited by corticotropin. TMB-8, an intracellular Ca²⁺ antagonist, completely blocked PGI₂-evoked steroid output in the absence of external Ca²⁺; this inhibition was partially reversed by the addition of Ca²⁺. The increase in secretion caused by corticotropin or PGI₂ in the presence of Ca²⁺ was also reduced in a dose-dependent manner by TMB-8. The steroidogenic action of pregnenolone, which is induced by a Ca²⁺ independent mechanism, was not blocked by TMB-8, either in the presence or absence of Ca²⁺. Corticotropin significantly potentiated the Ca²⁺-independent aspect of PGI₂ action. These studies provide evidence for an internal, PGI₂-sensitive Ca²⁺ store in cat adrenocortical cells.     

Effect of trifluoperazine, compound 48/80, TMB-8 and verapamil on ionophore A23187 mediated calcium uptake in ATP depleted human red cells

The A23187 induced calcium uptake in ATP depleted cells was determined at pH 6.9 in the presence of trifluoperazine (TFP, 0.30 mM), compound 48/80 (0.89 mg/ml), 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8, 2.13 mM) and verapamil (1.81 mM). Apart from verapamil the drugs all increased the maximum rate of ionophore-mediated calcium flux by 50-60 per cent. After the ionophore addition some time elapsed before the calcium flux attained the maximum value, and this time dependence could be interpreted as a slow uptake of A23187 into the membrane: five seconds after the addition of A23187 half of the added ionophore was able to transport calcium through the membrane. The effect of pH on the ionophore-mediated calcium uptake was determined in the absence and presence of TFP. At pH 7.4 the maximum rate of calcium flux in the absence of TFP was two to three times higher than that at pH 6.9 and TFP increased the uptake rate by 98 per cent.     

In situ accumulation of calcium by organelles of squid axoplasm

Existing morphological and physiological evidence indicates that axoplasm of squid axons sequesters calcium by both mitochondrial and non-mitochondrial buffers. The present work demonstrates that essentially all of the non-mitochondrial component is located in organelles. Extruded axoplasm was loaded with varying amounts of calcium by mixing with small volumes of solutions containing pH buffered ⁴⁵Ca. Ethyleneglycol-bis(β-amino-ethyl ether)N,N′-tetraacetic acid (EGTA) or diethylenetriamine pentaacetic acid (DTPA) was used to stabilize the free calcium. The axoplasm was then sucked up in a polyethylene tube and centrifuged at 100,000 g for 2–3 hours to produce a loose pellet comprising 10–20% of the axoplasm volume. After centrifugation, the tube was frozen, sliced into segments, and counted by liquid scintillation. No significant pellet accumulation of exogenous calcium occurred at physiological concentrations of free calcium (ca. 50 nM); however, a threshold for accumulation existed at 150–200 nM. Essentially complete pellet sequestration of the exogenous load occurred at a free calcium concentration above 1 μM. About half of the pellet buffering capacity was sensitive to carbonyl cyanide, p-trifluoromethoxy phenylhydrazone (FCCP). Variation of exogenous load between 0.1 – 3 mmole/kg axoplasm did not affect the buffering capacity of either the FCCP sensitive or insensitive components when the free calcium concentration was above threshold.     

The effect of Ca on virus-cell fusion and permeability changes

Sendai virus-mediated permeability changes in Lettre cells or red blood cells are affected by extracellular Ca2+ in the following way: the lag period to onset of permeability changes is lengthened and the subsequent extent of leakage is reduced. Ca2+ neither stimulates nor inhibits fusion of the viral envelope to the plasma membrane of Lettre cells or red blood cells. It is concluded that Ca2+ protects cells against virally-induced permeability changes in a manner not involving membrane fusion.     

Alterations in calcium metabolism in phorbol ester-treated mouse peritoneal macrophages

Phorbol 12-myristate 13-acetate (PMA)-treated macrophages exhibited a two-fold increase in the rate of 45Ca++ efflux and over a three-fold increase in the size of the exchangeable calcium pool, resulting in almost a seven-fold increase in the slow phase of calcium efflux. The calcium antagonist 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) by itself did not affect calcium efflux in macrophages; but abolished the PMA-induced increase in the rate of calcium efflux. The divalent cationphore A23187 increased the rate constant of the fast phase of calcium efflux two-fold when applied alone or when applied with PMA. These effects might be linked to ionophore enhancement and TMB-8 inhibition of PMA-induced macrophage chemotaxis and spreading (previously reported in Cell Calcium 3:503-514 and Cancer Research 43:3385-3391). No change in calcium efflux was observed if cells were exposed to PMA only during the efflux experiment suggesting that a prolonged exposure to PMA is required to elicit changes in calcium flux. Increased 45Ca++ remained in treated cells at each time point perhaps reflecting the PMA-induced increase in exchangeable calcium.     

Antagonism of calmodulin and phosphodiesterase by nifedipine and related calcium entry blockers

Nifedipine, a 1,4-dihydropyridine Ca2+ entry blocker, partially inhibits calmodulin-activated and, to a lesser extent, basal (non-activated) cyclic AMP phosphodiesterase activity at 10-440 microM. The inhibition of calmodulin-activated phosphodieserase does not parallel Ca2+ entry blockade, since analogs of nifedipine, which are 500-fold less potent than nifedipine as Ca2+ entry blockers (Bolger et al. (1982) Biochemical and Biophysical Research Communications 104, 1604-1609), are equal in potency to nifedipine as calmodulin-activated phosphodiesterase inhibitors. Furthermore, the inhibition of calmodulin-activated phosphodiesterase by nifedipine is about 500-fold less potent than its inhibition of Ca2+ entry blockade. It is suggested that the low affinity interaction of nifedipine and related 1,4-dihydropyridines with calmodulin and phosphodiesterase is also of low specificity and therefore is unlikely to contribute to the cardiac and vascular muscle relaxant actions of these drugs at normal pharmacological concentrations.     

The role of calcium ions and the thioredoxin system in regulation of spinach chloroplast fructosebisphosphatase

Illumination of isolated type A spinach chloroplasts causes a rapid increase in their activity of fructosebisphosphatase, as assayed at physiological substrate and Mg²⁺ concentrations. Activation is accelerated by addition of dihydroxyacetone phosphate to the chloroplasts and decreased by inorganic phosphate concentrations greater than those optimal for CO₂ fixation. At all times, measured fructosebisphosphatase activity was greater than was necessary to account for the observed rates of CO₂ fixation. Activation of purified fructosebisphosphatase $\text{in vitro}$ by dithiothreitol or reduced thioredoxin is extremely slow, but is greatly accelerated in the presence of physiological concentrations of Mg²⁺ and fructosebisphosphate if Ca²⁺ ions are present. Increased concentrations of fructosebisphosphate greatly increase the rate and extent of activation whereas in the absence of fructosebisphosphate Ca²⁺ ions have no effect. Neither inorganic phosphate nor dihydroxyacetone phosphate significantly affect the rate of activation. Ca²⁺ ions strongly inhibit the activity of the activated form of fructosebisphosphatase. It is proposed that free Ca²⁺ ions within chloroplasts are involved in preventing fructosebisphosphatase from functioning in the dark, and that free and/or bound Ca²⁺ facilitates the rapid reductive activation of this enzyme when the light is turned on again.     

The low affinity binding sites of the vitamin D-dependent calcium-binding protein and their functional role in calcium transport

Calcium- and other divalent cation-binding properties of the vitamin D-dependent calcium-binding protein isolated from the duodenal mucosa of chicks were studied using the flow dialysis technique and ⁴⁵Ca. It was found that the calcium-binding protein along with the high affinity binding sites has approximately 40 low affinity binding sites with K_a of about 1000 M^?1. The low affinity sites possess of certain specificity towards binding of Ca. The affinity of the calcium-bindin protein for other divalent cations depends on the ionic radius. It is suggested that the low affinity binding sites of the calcium-binding protein take part in calcium transport organization across the intestinal epithelium.     

Effects of manganese and other divalent cations on calcium uptake and catecholamine secretion by primary cultures of bovine adrenal medulla cells

Primary cultures of bovine adrenal medullary chromaffin cells were used to examine the effect of replacing divalent cations in the extracellular media on secretion. When calcium was replaced by manganese, nicotine-stimulated secretion was delayed in onset for 3 to 5 minutes, but continued for approximately 60 minutes. In contrast, calcium-supported secretion began immediately on stimulation and plateaued by 10 minutes. ⁵⁴Mn²⁺ uptake occurred on stimulation but at a lower rate than ⁴⁵Ca²⁺ uptake. There was no delay of ⁵⁴Mn²⁺ uptake upon stimulation and ⁵⁴Mn²⁺ uptake was considerably prolonged compared to ⁴⁵Ca²⁺ uptake. Replacement of calcium with strontium gave results similar to those with calcium, and, in addition, strontium was able to bring about secretion by itself in a manner similar to barium. Inhibition experiments showed that the potency for inhibiting calcium uptake was Cd²⁺>Mn²⁺>Ca²⁺>Sr²⁺.