Effect of calmodulin antagonists on Ca2+ uptake by boar spermatozoa

Calcium uptake by washed boar sperm suspensions is markedly stimulated by the calmodulin antagonists trifluoperazine and calmidazolium. Both ⁴⁵Ca²⁺ uptake and net Ca²⁺ uptake are increased by these drugs. Drug stimulated Ca²⁺ uptake is blocked by verapamil (1 mM), by ruthenium red (25 μM) and by carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Calmodulin antagonists do not slow ATP-dependent Ca²⁺ extrusion from plasma membrane vesicles, and they do not inhibit plasma membrane Ca²⁺-ATPase. It is proposed that calmodulin is involved in the control of Ca²⁺ entry in boar spermatozoa. Most entering Ca²⁺ in uncapacitated spermatozoa is sequestered by mitochondria or rapidly extruded by plasma membrane pumps. In contrast to the uptake mechanism, ATP-dependent Ca²⁺ extrusion does not appear to be regulated by calmodulin.     

Spontaneous inactivation of the Ca2+-sensitive K+ channels of human red cells at high intracellular Ca2+ activity

Ionophore A23187-mediated Ca²⁺-induced oscillations in the conductance of the Ca²⁺-sensitive K⁺ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K⁺ activity were followed with a K⁺-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular ⁴⁵Ca content. An increased cellular ⁴⁵Ca content at the successive minima of the oscillations where the K⁺ channels are closed indicates that the activation of the channels might be a $\text{(}\text{dCa}{}^{\mathrm{2+}}\text{/}\text{d}\text{t)-}\text{sensitive}$ process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K⁺ channels at intracellular ionized calcium levels of about 10 μM and a concurrent membrane potential of about ?65 mV was observed. At a membrane potential of about ?70 mV and an intracellular concentration of about 2·10^?4M no inactivation of K⁺ channels took place. Inactivation of the K⁺ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular ⁴⁵Ca content support the notion that a necessary component of the oscillatory system is a Ca²⁺ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium.     

The rate of Ca2+ translocation by sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase measured with intravesicular arsenazo III

Release of Ca²⁺ from the (Ca²⁺ + Mg²⁺)-ATPase into the interior of intact sarcoplasmic reticulum vesicles was measured using arsenazo III, a metallochromic indicator of Ca²⁺. Arsenazo III was placed inside the sarcoplasmic reticulum vesicles by making the vesicles transiently leaky with an osmotic gradient in the presence of arsenazo III. External arsenazo III was then removed by centrifugation. Addition of ATP to the (Ca²⁺ + Mg²⁺)-ATPase in the presence of Ca²⁺ causes the rapid phosphorylation of the enzyme at which time the bound Ca²⁺ becomes inaccessible to external EGTA. The release of Ca²⁺ from the (Ca²⁺ + Mg²⁺)-ATPase to the interior of the vesicle measured with intravesicular arsenazo III was much slower indicating that there is an occluded from the Ca²⁺-binding site which precedes the release of Ca²⁺ into the vesicle. The rate of Ca²⁺ accumulation by sarcoplasmic reticulum vesicles is increased by K⁺ (5–100 mM) and ATP (50–1000 μM) but the initial rate of Ca²⁺ translocation measured after the simultaneous addition of ATP and EGTA to vesicles that were preincubated in Ca²⁺ was not influenced by these concentrations of K⁺ and ATP.     

Isolation of sarcoplasmic reticulum by zonal centrifugation and purification of Ca2+-pump and Ca2+-binding proteins

A procedure for the isolation of highly purified sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been described using sucrose gradient centrifugation in zonal rotors. The yield of our purest fraction was 300 mg of sarcoplasmic reticulum protein using 1 kg muscle. The sarcoplasmic reticulum vesicles were relatively simple in composition. The Ca²⁺-pump protein accounted for most (approx. two-thirds) of the sarcoplasmic reticulum protein. Two other protein components, a Ca²⁺-binding protein and a M₅₅ protein (approx. 55 000 daltons) each accounted for about 5–10% of the protein. Enrichment in the level of phosphoenzyme by the Ca²⁺-pump protein was regarded as an important index of the purification of sarcoplasmic reticulum vesicles. The sarcoplasmic reticulum vesicles were capable of forming 6.4 nmoles of ³²P-labelled phosphoenzyme per mg protein and had a high capacity of energized Ca²⁺ uptake. The Ca²⁺-dependent formation of phosphoenzyme has been used to estimate the sarcoplasmic reticulum protein content in rabbit skeletal muscle and found to be about 2.5% of the total muscle protein.The Ca²⁺-pump and Ca²⁺-binding proteins were isolated with a purity of 90% or more by treating the purified sarcoplasmic reticulum vesicles with bile acids in the presence of salt. The solubilized Ca²⁺-pump protein reaggregated during dialysis together with phospholipid to form membranous vesicles which were capable of forming approx. 9 nmoles ³²P-labelled phosphoenzyme per mg protein. The Ca²⁺-binding protein was water soluble and contained a high percentage of acidic amino acids (35% of total residues).Ca²⁺ binding by sarcoplasmic reticulum vesicles and by the Ca²⁺-pump and Ca²⁺-binding proteins was studied by equilibrium dialysis. Sarcoplasmic reticulum vesicles and Ca²⁺-pump protein contained nonspecific high-affinity Ca²⁺ binding sites with a capacity of 90–100 and 55–70 nmoles Ca²⁺ per mg protein, respectively. Both of them specifically bound 10–15 nmoles Ca²⁺ per mg protein. The binding constants for nonspecific and specific Ca²⁺ binding by both preparations were approx. 1 μM^?1. The Ca²⁺-binding protein nonspecifically bound 900–1000 nmoles Ca²⁺ per mg protein with a binding constant of about 0.25 μM^?1.     

Localization of (Ca2+ + Mg2+)-ATPase,Ca2+ pump and other ATPase activities in cardiac sarcolemma

N-Ethylmaleimide was employed as a surface label for sarcolemmal proteins after demonstrating that it does not penetrate to the intracellular space at concentrations below 1·10^?4 M. The sarcolemmal markers, ouabain-sensitive (Na⁺ + K⁺)-ATPase and Na⁺/Ca²⁺-exchange activities, were inhibited in N-ethylmaleimide perfused hearts. Intracellular activities such as creatine phosphokinase, glutamate-oxaloacetate transaminase and the internal phosphatase site of the Na⁺ pump (K⁺-p-nitrophosphatase) were not affected. Almost 20% of the (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump were inhibited indicating the localization of a portion of this activity in the sarcolemma. Sarcolemma purified by a recent method (Morcos, N.C. and Drummond, G.I. (1980) Biochim. Biophys. Acta 598, 27–39) from N-ethylmaleimide-perfused hearts showed loss of approx. 85% of its (Ca²⁺ + Mg²⁺-ATPase and Ca²⁺ pump compared to control hearts. (Ca²⁺ + Mg²⁺)-ATPase and Ca²⁺ pump activities showed two classes of sensitivity to vanadate ion inhibition. The high vanadate affinity class ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for inhibition approx. 1.5 μM) may be localized in the sarcolemma and represented approx. 20% of the total inhibitable activity in agreement with estimates from N-ethylmaleimide studies. Sucrose density fractionation indicated that only a small portion of Mg²⁺-ATPase and Ca²⁺-ATPase may be associated with the sarcolemma. The major portion of these activities seems to be associated with high density particles.     

Steady-state kinetics of immobilized enzymes at very low substrate concentrations studied using the asarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

A method for determining initial velocities of enzymatic reactions at very low substrate concentrations is presented. It is based on teh continuous perfusiion of substrate-containing media through the enzyme, previously deposited as a thin layer on a solid support. An analytical rationalization of the dependence of the enzymatic activity upon the substrate supply and the flow rate was developed (substrate supply (μmol/min) = flow rate (ml/min) × inflowing substrate concentration (μmol/ml). This paper shows that a straight line should be expected from a double-reciprocal plot of the velocity of the enzymatic reaction and flow rate. The reciprocal of the ordinate at the origin is the strict initial velocity for a given, constant, and very low substrate concentration, since substrate consumption and product accumulation tend to zero. Results obtained with two different sarcoplasmic reticulum (Ca²⁺ + Mg²⁺)-ATPase preparations agree with the theoretical predictions. The method enabled the use of ATP concentrations in the range of 10^?8 M: it required neither an ATP-regerating system nor the dilution of the enzyme protein, and it presented no limitations for the reaction time. Both ATPase preparations showed two apparent K_m values for the substrate in the submicromolar and micromolar ranges: 0.25–12.0 μM for the purified ATPase, and 0.17–1.65 μM for the microsomal ATPase.     

Ca2+-dependent K+ transport in the Ehrlich ascites tumor cell

The possible presence and properties of the Ca²⁺-dependent K⁺ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187+Ca²⁺, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K⁺ (balanced by the uptake of Na⁺) and a stimulation of both the influx and the efflux of ⁸⁶Rb. These effects were antagonized by quinine, a known inhibitor of the Ca²⁺-dependent K⁺ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca²⁺-dependent K⁺ channel whose characteristics are similar to those described in other cell systems.     

Ca2+ and Mn2+ mediated binding of the glycoprotein peroxidase to membranes of Pharbitis cotyledons

Ca²⁺ and Mn²⁺ promote the binding of the basic isoperoxidase to a crude membrane preparation in extracts from Pharbitis cotyledons. The Ca²⁺- or Mn²⁺-induced binding is resistant to high ionic strength and can be saturated by increasing the divalent ion or the isoperoxidase concentrations. Treatments in vitro with glucosaminidase or in vivo with tunicamycin show that the carbohydrate part of the isoperoxidase is necessary for the binding. The amino sugar galactosamine inhibits the binding at rather high concentrations. Pharbitis basic isoperoxidase can be bound to zucchini squash microsomes in the presence of Ca²⁺ and conversely.     

Inhibition by Sr2+ of specific mitochondrial Ca2+-efflux pathways

The effect of Sr²⁺ on the set point for external Ca²⁺ was studied in rat heart and liver mitochondria with the aid of a Ca²⁺-sensitive electrode. In respiring mitochondria the set point is determined by the rates of Ca²⁺ influx on the Ca²⁺ uniporter and efflux by various mechanisms. We studied the Ca²⁺-Na⁺ exchange pathway in heart mitochondria and the Δψ-modulated efflux pathway in liver mitochondria. Prior accumulation of Sr²⁺ was found to shift the set points towards lower external Ca²⁺ both in heart mitochondria under conditions of Ca²⁺-Na⁺ exchange and in liver mitochondria under conditions that should promote opening of the Δψ-modulated pathway. The effect on the set point was found to be due to inhibition of Ca²⁺ efflux by Sr²⁺ taken up by the mitochondria, while Sr²⁺ efflux was too slow to be measurable.     

Effects of glucagon and Na+ on the control of extramitochondrial free Ca2+ concentration by mitochondria from liver and heart

Rat liver mitochondria maintain the extramitochondrial free Ca²⁺ concentration at pCa²⁺ of about 6.15. Glucagon pre-treatment of the rats does not alter this value. Rat heart mitochondria maintain free Ca²⁺ at a pCa²⁺ value of about 6.7, addition of 5mM Na⁺ changes this to a value of about 5.85.     

Electroneutral H+-K+ exchange in liver mitochondria Regulation by membrane potential

The paper analyzes the factors affecting the H⁺-K⁺ exchange catalyzed by rat liver mitochondria depleted of endogenous Mg²⁺ by treatment with the ionophore A23187. The exchange has been monitored as the rate of K⁺ efflux following addition of A23187 in low-K⁺ media. (1) The H⁺-K⁺ exchange is abolished by uncouplers and respiratory inhibitors. The inhibition is not related to the depression of ΔpH, whereas a dependence is found on the magnitude of the transmembrane electrical potential, Δψ. Maximal rate of K⁺ efflux is observed at 180–190 mV, whereas K⁺ efflux is inhibited below 140–150 mV. (2) Activation of H⁺-K⁺ exchange leads to depression of ΔpH but not of Δψ. Respiration is only slightly stimulated by the onset of H⁺-K⁺ exchange in the absence of valinomycin. These findings indicate that the exchange is electroneutral, and that the Δψ control presumably involves conformational changes of the carrier. (3) Incubation in hypotonic media at pH 7.4 or in isotonic media at alkaline pH results in a marked activation of the rate of H⁺-K⁺ exchange, while leaving unaffected the level of Mg²⁺ depletion. This type of activation results in partial ‘uncoupling’ from the Δψ control, suggesting that membrane stretching and alkaline pH induce conformational changes on the exchange carrier equivalent to those induced by high Δψ. (4) The available evidence suggests that the activity of the H⁺-K⁺ exchanger is modulated by the electrical field across the inner mitochondrial membrane.     

H+ uptake by chromatophores from Rhodopseudomonas spheroides; the relation between rapid H+ uptake and the H+ pump

1. H⁺ uptake induced by repeated flash excitation approached the full extent of H⁺ uptake induced by continuous light. At low repetition rates, the H⁺ uptake was seen to consist of repeated occurrences of rapid H⁺ uptake.2. The effects of ionophores and uncoupling agents on H⁺ uptake induced by continuous light could be adequately accounted for in terms of their effects on the flash induced changes. It is concluded that the reaction disclosed by rapid H⁺ uptake is an integral part of the process observed on continuous illumination, and therefore, in view of the association between rapid H⁺ uptake and the reduction of a hydrogen-carrying secondary acceptor, that the electron transport system is an integral part of the mechanism of the H⁺ pump.3. When the frequency of repetition of the flashes was increased, the full extent of H⁺ uptake or of the carotenoid change was seen only after the first few flashes. Thereafter, the extent decreased, and depended on the dark time between flashes. The full extent of the change could be restored even at high frequencies if uncoupling agents or valinomycin were present.4. It is concluded that the recovery of the extent of H⁺ uptake or the carotenoid change between flashes reflected the turnover of the electron transport chain, and that the increased recovery in the presence of uncoupling agents or valinomycin reflected the stimulation of electron flow under uncoupled conditions, or on dissipation of the membrane potential.     

The Ca2+-Na+ antiporter of heart mitochondria operates electroneutrally

The transmembrane potential of heart mitochondria during Na⁺-induced Ca²⁺ release has been monitored continuously with a Tetraphenylphosphonium-sensitive electrode. The data obtained indicate that the exchange process does not generate an electrical current, and provides direct support for the concept of a 2 Na⁺ per 1 Ca²⁺ exchange.     

Kinetic properties of Na+/Ca2+ exchange in basolateral plasma membranes of rat small intestine

The presence of an Na⁺/Ca²⁺ exchange system in basolateral plasma membranes from rat small intestinal epithelium has been demonstrated by studying Na⁺ gradient-dependent Ca²⁺ uptake and the inhibition of ATP-dependent Ca²⁺ accumulation by Na⁺. The presence of 75 mM Na⁺ in the uptake solution reduces ATP-dependent Ca²⁺ transport by 45%, despite the fact that Na⁺ does not affect Ca²⁺-ATPase activity. Preincubation of the membrane vesicles with ouabain or monensin reduces the Na⁺ inhibition of ATP-dependent Ca²⁺ uptake to 20%, apparently by preventing accumulation of Na⁺ in the vesicles realized by the Na⁺-pump. It was concluded that high intravesicular Na⁺ competes with Ca²⁺ for intravesicular Ca²⁺ binding sites. In the presence of ouabain, the inhibition of ATP-dependent Ca²⁺ transport shows a sigmoidal dependence on the Na⁺ concentration, suggesting cooperative interaction between counter transport of at least two sodium ions for one calcium ion. The apparent affinity for Na⁺ is between 15 and 20 mM. Uptake of Ca²⁺ in the absence of ATP can be enhanced by an Na⁺ gradient (Na⁺ inside > Na⁺ outside). This Na⁺ gradient-dependent Ca²⁺ uptake is further stimulated by an inside positive membrane potential but abolished by monensin. The apparent affinity for Ca²⁺ of this system is below 1 μM. In contrast to the ATP-dependent Ca²⁺ transport, there is no significant difference in Na⁺ gradient-dependent Ca²⁺ uptake between basolateral vesicles from duodenum, midjejunum and terminal ileum. In duodenum the activity of ATP-driven Ca²⁺ uptake is 5-times greater than the Na⁺/Ca²⁺ exchange capacity but in the ileum both systems are of equal potency. Furthermore, the Na⁺/Ca²⁺ exchange mechanism is not subject to regulation by 1α,25-dihydroxy vitamin D-3, since repletion of vitamin D-deficient rats with this seco-steroid hormone does not influence the Na⁺/Ca²⁺ exchange system while it doubles the ATP-driven Ca²⁺ pump activity.     

Synthetic peptide K+ carrier with Ca2+ inhibition

Based on a proposed solution conformation of the Ca²⁺ ion complex of the repeat hexapeptide of elastin, l-Val-l-Ala-l-Pro-Gly-l-Val-Gly, it is possible to modify the molecule making it more lipophilic for lipid bilayer permeation while retaining its complexation features. Therefore the two peptides, For-MeVal-Ala-Pro-Sar-Pro-Sar-OMe and For-MeVal-Ala-Pro-Sar-Pro-Sar-OH, were synthesized and evaluated for lipid bilayer activity and cation binding (For, N-formyl; Me, N-methyl; Sar, N-methyl glycine). Both peptides bound Ca²⁺ preferentially but did not exhibit the properties of a Ca²⁺ carrier. They were however active as K⁺ carriers although K⁺ ion titration curves showed a much lower affinity for K⁺ than for Ca²⁺. The addition of Ca²⁺ or Mg²⁺ to the bilayer system inhibited the peptide K⁺ carrier activity. Three possible explanations of this interesting Ca²⁺ inhibition of carrier activity are irreversible complexation of Ca²⁺, mixed ligand complex formation involving Ca²⁺, lipid and peptide, and impermeability of the lipid layer when peptide is complexed with a divalent cation.     

Effects of prostaglandins on the interaction of Ca2+ with mitochondria

Ca²⁺ stimulates the binding of a variety of prostaglandins (PG) to liver mitochondria. Optimal binding is observed at slightly acidic pH and at concentrations of Ca²⁺ between 200 and 500 μm. The stimulation of the binding requires the active transport of Ca²⁺ in mitochondria and is only observed in the absence of permeant anions. The maximal amount of PG bound is about 3 nmol/mg of mitochondrial protein. All PG tested induce efflux of the Ca²⁺ taken up by mitochondria without impairing the ability of mitochondria to actively accumulate it. Optimal stimulation of the efflux of Ca²⁺ requires concentrations of PG higher than those used in the PG-binding experiments and is associated with an evident uncoupling of the respiration that follows a Ca²⁺-induced O₂ uptake jump. The “uncoupling” by PG is explained by postulating the entrance of protonated PG into mitochondria, followed by their exit from the organelle as 2:1 complexes with Ca²⁺.     

Calmodulin stimulates polyamine-responsive protein kinase in the absence of Ca2+

A cyclic nucleotide-independent, polyamine-responsive protein kinase from the cytosol of Morris hepatoma 3924A, which phosphorylated heat-stable endogenous substrates and casein in the presence of polyamines (Criss, W.E., Yamamoto, M., Takai, Y., Nishizuka, Y. and Morris, H.P. (1978) Cancer Res. 38, 3540–3545) was observed to be stimulated by an endogenous protein activator. This protein activator was identified to be calmodulin. the polyamine-responsive protein kinase was also stimulated by purified calmodulin, but only in the presence of polyamines such as polylysine. This action of cadmodulin did not require Ca²⁺ for activation of the enzyme; and activation occured in the presence of EGTA. DNA and RNA inhibited the polyamine-responsive protein kinase, either in the presence or absence of Ca²⁺. Purified calmodulin, in the presence of cyclic AMP or cyclic GMP, did not activate the protein kinase. Therefore, polyamines such as polylysine are an absolute requirement for this expression of calmodulin action. The increased enzyme activity by calmodulin was accompanied with an increased V_max and with no changes in the F_m (ATP). High levels of cation, up to 100 mM Mg²⁺, did not effect the action of cadmodulin. These results indicate that tumor cytosolic polyamine-responsive protein kinase is regulated by calmodulin, the latter being increased in the tumor tissue.