Block of axoplasmic transport in vitro by vinca alkaloids

The three potent antimitotic vinca alkaloids: vincristine (VCR), vinblastine (VLB), and vindesine (VDS) were compared for their effect in blocking axoplasmic transport in vitro using a desheathed preparation of the peroneal branch of cat sciatic nerve. A range of vinca alkaloid concentrations from 1–100μM was examined. The relative order of potency in blocking axoplasmic transport was VCR > VLB > VDS at a concentration of 25μM. At the higher concentrations block occurred so rapidly that a statistically significant difference between these agents could not be obtained. The relation of vinca block ot the transport mechanism is discussed.     

mRNA-induced expression of the cardiac Na+-Ca2+ exchanger in Xenopus oocytes

Xenopus oocytes were injected with total mRNA isolated from hearts of 1-day-old chicks. After 5 days of incubation the follicular cell layers were removed and the oocytes were loaded with Na+ by incubation in hypertonic EGTA solution at 37 degrees C. The Na+-loaded oocytes accumulated 45Ca2+ from a Na+-free medium at a 3-18-fold higher rate than noninjected oocytes or oocytes injected with control solution containing no mRNA. Oocytes not subjected to the Na+-loading procedure showed no mRNA-dependent 45Ca2+ uptake. Size fractionation of the mRNA using sucrose density gradient centrifugation under denaturing conditions led to the identification of a 25 S fraction competent for induction of the Na+-Ca2+ exchange system.     

A rapid technique for measuring calcium uptake in mitogen-induced T and B lymphocytes.

The procedures for lymphocyte activation and for removing the cells from the radioactive loading solution in incubation medium were modified to routinely obtain significant and reproducible 45Ca2+ uptakes in mitogen-induced mouse T and B lymphocytes. Factors such as mouse strain, lymphocyte origin, and media pH were not critical to the 45Ca2+ uptake measurements. In contrast, factors such as lymphocyte cell concentration during mitogenic activation, filtering the 45Ca2+:3H2O mixtures, and the nature and purity of the B-cell mitogens were critical for obtaining maximal and reproducible 45Ca2+ uptakes. Centrifugation through silicone oil into sucrose was an efficient and rapid procedure for separating the cells from the radioactive loading solution in the incubation medium. Using optimal conditions, an approximate twofold increase in 45Ca2+ uptake (representing an influx of approximately 97 amol per lymphocyte and an increase in average cellular Ca2+ of approximately 0.72 mM) was routinely obtained with purified mouse lymphocytes activated with a variety of T- and B-cell mitogens (using concentrations resulting in maximal [3H]thymidine incorporation). A larger 45Ca2+ uptake was routinely obtained with mitogenic concentrations of A23187, a divalent cation ionophore stimulating T cells. Experiments employing [14C]sucrose and [14C]inulin with control and mitogen-induced lymphocytes showed that the trapped extracellular fluid measurements in the cell pellets should be used to correct the magnitude of the 45Ca2+ uptake measurements.     

Na+ Dependence of Tyrosine Transport Across the Synaptosomal Membrane Reflects Changes in the Morphology of Synaptosomes

The Na+ dependence of tyrosine uptake into rat brain synaptosomes and synaptosomal plasma membrane vesicles (SPMV) was examined in the present study. At low tyrosine concentrations, the isoosmotic substitution of Na+ by sucrose in the incubation medium led to an increase of tyrosine uptake in synaptosomes and to a decrease in SPMV. The removal of extracellular Ca2+ and Mg2+ and addition of isoosmotic sucrose completely prevented the augmented tyrosine uptake in Na+-free incubated synaptosomes. Morphological differences were found at the electron-microscopic level when synaptosomes were incubated in Na+-free and Na+-containing media. The internal volume measured for synaptosomes incubated in a Na+-free medium was almost half of that obtained in a Na+-containing medium, in good agreement with the observations made with the electron microscope. Also, the omission of Ca2+ and Mg2+ resulted in a specific swelling of only the synaptosomes incubated in Na+-free medium. When synaptosomes and SPMV were preloaded with several neutral amino acids, the tyrosine uptake rate was greatly increased, indicating fully operational exchange mechanisms for these amino acids. We propose that the enhancement of high-affinity synaptosomal tyrosine uptake observed in Na+-free medium is a consequence of a specific shrinkage of the synaptosomes and a parallel increase of the exchange rate with endogenous neutral amino acids.     

Dependence of batrachotoxin block of axoplasmic transport on sodium

Batrachotoxin (BTX) in the low concentration range of 19-190 nM blocks axoplasmic transport in the desheathed cat peroneal nerve in vitro. When the level of Na+ in the incubation medium was reduced to 10 mM, the blocking effect of BTX was much diminished, and in an Na+-free medium BTX had no effect on transport at all. The blocking action of BTX with Na+ present was inhibited by increasing the concentration of Ca2+ in the experimental medium. Relatively small increases were effective with a maximum protection seen when the Ca2+ concentrations were 7-10 mM. The results support the view that an increase in axonal Na+ is inhibitory to the transport mechanism. The results are discussed on the basis of the recently developed transport filament model of axoplasmic transport which takes into account an obligatory role for Ca2+ in transport and its axonal regulation. The possible relation of intraaxonal Na+ concentration to the Ca2+ level is also discussed.     

Vinflunine, a novel microtubule inhibitor, suppresses calmodulin interaction with the microtubule-associated protein STOP

Vinca alkaloids vinblastine and vincristine and some of their derivatives such as vinorelbine are widely used in therapy of leukemia and several solid tumors. Their action is associated with alterations of the mitotic spindle functions that prevent the cell cycle progression and lead to mitotic block. A number of studies show that some Vinca alkaloids inhibit CaM-target interaction. The newest microtubule inhibitor, vinflunine (Javlor), currently in clinical trials, is remarkably more active than vinblastine against a number of tumors. Moreover, vinflunine is significantly less toxic than other Vinca alkaloids. The high antitumor activity of this molecule is not well understood since it binds to tubulin with an overall affinity several-fold lower than that of vinblastine or vincristine. In this study, we examined the interaction of Ca2+-CaM with vinflunine, vinblastine, and stable tubule only polypeptide (STOP) by using a combination of thermodynamic and mass spectrometric approaches. We characterized the influence of Vinca alkaloids on Ca2+-CaM-STOP complex formation. Our results revealed different binding modes to Ca2+-CaM for vinflunine and vinblastine, highlighting that adding fluorine atoms on the cleavamine moiety of the Vinca alkaloid molecule is critical for the localization of the drug on calmodulin. We demonstrate that vinflunine is a better inhibitor for STOP binding to calmodulin than vinblastine. We suggest that vinflunine action on calmodulin can have an effect on microtubule dynamics. These data may contribute to a better understanding of the superior antitumor efficiency and lower toxicity of vinflunine.     

Radioimmunoassays for the Vinca alkaloids, vinblastine and vincristine.

Radioimmunoassays for the chemotherapeutic alkaloids, vinblastine (VLB) and vincristine (VCR) were developed that could determine less than 1 pmol of each compound in the homologous hapten-antibody reaction. The anti-VCR serum bound VCR 200 times more effectively than VLB, demonstrating a high specificity in spite of the structural similarity of the drugs. Several other chemotherapeutic drugs showed no significant cross-reactivity. The assays were used to measure plasma levels of the alkaloids in rabbits treated with the drugs and to determine immunoreactive drug equivalents in extracts of the periwinkle plant following fractionation by high-pressure liquid chromatography.     

Ca2+ transport through the brush border membrane of human placenta syncytiotrophoblasts.

The calcium (Ca2+) uptake by brush border membrane vesicles isolated from fresh human placentas has been characterized. This process was saturable and time- and concentration-dependent. It exhibited a double Michaelis-Menten kinetics, with apparent Km values of 0.17 +/- 0.03 and 2.98 +/- 0.17 mM Ca2+, and Vmax values of 0.9 +/- 0.13 and 2.51 +/- 0.45 pmol.micrograms-1.5 s-1. It was not influenced by the presence of Na+ or Mg2+ in the incubation medium. It was not increased by K+ or anion diffusion potentials, inside negative. At a steady state of 1 mM Ca2+ uptake, a large proportion (approximately 94%) of the Ca2+ was bound to the internal surface of the membranes. Preincubation of these membrane vesicles with voltage-dependent Ca2+ channel blockers (nifedipine and verapamil) had no influence on Ca2+ uptake. However, this uptake was very sensitive to pH. In the absence of a pH gradient, the Ca2+ uptake increased with alkalinity. When the intravesicular pH was kept constant while the pH of the incubation medium was increased, Ca2+ uptake was also stimulated by alkaline pH. In contrast, when the pH of the incubation medium was kept constant and the intravesicular pH was progressively increased, Ca2+ uptake was diminished with alkaline pH. Therefore, H+ gradient (H+ in trans-position greater than H+ in cis-position) favored Ca2+ transport, suggesting a H+/Ca2+ exchange mechanism. Finally, in contrast to the basal plasma membrane, the brush border membrane did not show any ATP-dependent Ca2+ transport activity.     

Characterization of Ca2+ uptake by guinea pig epididymal spermatozoa

Comparative studies of Ca2+-uptake by guinea pig spermatozoa were performed with fresh epididymal sperm and with cells preincubated in a chemically defined, Ca2+-free medium for capacitation. Calcium uptake was negligible in fresh spermatozoa, but increased dramatically after 20 min of incubation at 37 degrees C in the presence of pyruvate and lactate. Spermatozoa incubated in the absence of these substrates accumulated only 34% as much 45Ca2+ as was taken up by cells in complete medium. The monosaccharides glucose, fructose, and mannose and the nonmetabolizable sugars 2-deoxyglucose and sucrose inhibited the enhancement of Ca2+-permeability. In the presence of 6 mM sucrose 45Ca2+ uptake was not influenced by external sodium chloride concentration between 0 mM and 145 mM. The respiratory activity of the capacitated spermatozoa not only was higher than that of uncapacitated cells, but it was stimulated by Ca2+. No effect of Ca2+ on respiration of fresh spermatozoa was detected. An increase in calcium uptake was associated with increasing pH of the medium. It is possible that a regulatory mechanism through the calcium permeability of the plasma membrane of guinea pig spermatozoa exists and controls the development of physiological events related with the fertilization process. The sugar composition, the availability of the energy substrates lactate and pyruvate, and the pH of the reproductive tract fluids could play an important role in the accessibility of Ca2+ into the cells in vivo, as has been demonstrated in vitro. The enhancement of calcium permeability during the preincubation could be a useful indicator to verify if capacitation has occurred.     

Competitive interaction of cyclosporins with the Vinca alkaloid-binding site of P-glycoprotein in multidrug-resistant cells

The mechanism of reversal of resistance to Vinca alkaloids by cyclosporins is unclear. We investigated the molecular mechanism of reversal of Vinca alkaloid resistance by cyclosporin A (CsA) and its nonimmunosuppressive analog O-acetyl C9(1) CsA (SDZ 33-243) in multidrug resistant DC-3F/VCRd-5L Chinese hamster cells. CsA at 3 microM increased vincristine (VCR) sensitivity and almost totally reversed VCR resistance. SDZ 33-243 at 1 microM reduced the IC50 for VCR in resistant cells from 62.0 to 0.00062 microM. CsA and SDZ 33-243 at 10 microM increased [3H]vinblastine (VBL) accumulation in DC-3F/VCRd-5L cells by 27- and 22-fold, respectively. At 10 microM, these compounds also increased [3H]VCR accumulation by 3.5- and 4.0-fold, respectively. [3H]VCR uptake by membrane vesicles from DC-3F/VCRd-5L cells showed high and low affinity components with Michaelis-Menten kinetics, and apparent Km values were 0.140 +/- 0.0523 and 24.8 +/- 6.67 microM, respectively. Kinetic analysis of [3H]VCR uptake in membrane vesicles in the presence of 0.2 microM CsA revealed that CsA competitively inhibited the high affinity [3H]VCR uptake with an apparent inhibition constant (Ki) of 0.126 +/- 0.0173 microM. In addition, CsA and SDZ 33-243 inhibited VBL photoaffinity labeling of P-glycoprotein in a dose-dependent manner, with half-maximum inhibition at 0.5 and 0.4 microM, respectively, compared with that of VBL at 0.6 microM. These data confirm that cyclosporins modulate Vinca alkaloid resistance at least partially through interaction with P-glycoprotein.     

Calcium ion binding to lobster nerve membranes

The binding of 45Ca2+ to membrane material isolated from lobster walking leg nerves was studied using a rapid filtration technique. In solutions of high ionic strength (450 mM), the amount of 45Ca2+ bound to this membrane material was found to be highly dependent on the monovalent cation used in the incubating solution. The amount of 45Ca2+ bound was larger when the membranes were incubated in a KCl solution compared to when they were incubated in a NaCl solution. This difference was attributed to the ability of these closed membrane vesicles to accumulate Ca2+ into the vesicle when incubating in a KCl solution but not in a NaCl solution. This accumulation of Ca2+ was found to be independent of metabolic energy and depended primarily on the absence of Na+ from the incubation medium. At low ionic strength, the membranes formed open fragments and the amount of Ca2+ bound was no longer sensitive to the monovalent cation species in the incubation solution. The 45Ca2+ bound under these low ionic strength conditions was considered to be bound to anionic sites on the membranes.     

Inhibition of Ca2+-dependent K+ channels by lead in one-step inside-out vesicles from human red cell membranes

Pb2+ modified the apparent threshold sensitivity to Ca2+ of individual K+ channels with a biphasic time-course. At first, the sensitivity to Ca2+ was lowered with the result of a decrease of the fraction of activated vesicles at a given Ca2+ concentration. Later, Pb2+ increased the sensitivity to Ca2+ and the fraction of activated vesicles. The increase of Pb2+ concentration increased the extent of the initial inhibition but decreased its duration. The inhibitory effect was not observed when the addition of Ca2+ preceded the addition of Pb2+. The presence of Mg2+ in the incubation medium was also required. In the absence of Mg2+, Pb2+ decreased the rate of uptake of 86Rb, but no decrease in the fraction of activated vesicles could be demonstrated.     

Subcellular fractionation of pig coronary artery smooth muscle

A detailed procedure for subcellular fractionation of the smooth muscle from pig coronary arteries based on dissection of the proper tissue, homogenization, differential centrifugation and sucrose density gradient centrifugation is described. A number of marker enzymes and Ca2+ uptake in presence or absence of oxalate, ruthenium red and azide were studied. The ATP-dependent oxalate-independent azide- or ruthenium red-insensitive Ca2+ uptake, and the plasma membrane markers K+-activated ouabain-sensitive p-nitrophenylphosphatase, 5'-nucleotidase and Mg2+-ATPase showed maximum enrichment in the F2 fraction (15-28% sucrose) which was also contaminated with the endoplasmic reticulum marker NADPH: cytochrome c reductase, and to a small extent with the inner mitochondrial marker cytochrome c reductase, and also showed a small degree of oxalate stimulation of the Ca2+ uptake. F3 fraction (28-40% sucrose) was maximally enriched in the ATP- and oxalate-dependent azide-insensitive Ca2+ uptake and the endoplasmic reticulum marker NADPH: cytochrome c reductase but was heavily contaminated with the plasma membrane and the inner mitochondrial markers. The mitochondrial fraction was enriched in cytochrome c oxidase and azide- or ruthenium red-sensitive ATP-dependent Ca2+ uptake but was heavily contaminated with other membranes. Electron microscopy showed that F2 contained predominantly smooth surface vesicles and F3 contained smooth surface vesicles, rough endoplasmic reticulum and mitochondria. The ATP-dependent azide-insensitive oxalate-independent and oxalate-stimulated Ca2+ uptake comigrated with the plasma membrane and the endoplasmic reticulum markers, respectively, and were preferentially inhibited by digitonin and phosphatidylserine, respectively. This study establishes a basis for studies on receptor distribution and further Ca2+ uptake studies to understand the physiology of coronary artery vasodilation.     

Insulin action in isolated fat cells. I. Effects of divalent cations on the stimulation by insulin of glucose uptake.

The effects of divalent cations, in particular Ca2+ and Mg2+, on glucose uptake by rat isolated fat cells in the presence and absence of insulin have been studied. EDTA (disodium salt) was used to deplete the bovine serum albumin present in the incubation medium of endogenous divalent cations prior to incubation with the cells, but was not present in the incubation medium during the incubation of the cells. The removal of Ca2+ and Mg2+ from the incubation medium did not affect the basal glucose uptake, but abolished the ability of insulin to stimulate glucose uptake by the cells. Addition of 25 microM MgCl2 or CaCl2 to the incubation medium restored a significant insulin stimulation, and this stimulation was maximal when 0.1 mM MgCl2 or CaCl2 had been added. SrCl2 and BaCl2 were also effective in restoring the insulin stimulation, but did not substitute fully for Ca2+ and Mg2+ in the incubation medium. Possible explanation for these observations are discussed.     

Utilization of X-537A to distinguish between intravesicular and membrane-bound calcium ions in sarcoplasmic reticulum.

The Ca2+ ionophore X-537A is employed as a tool to distinguish between intravesicular Ca2+ and surface membrane-bound Ca2+ in sarcoplasmic reticulum isolated from rabbit skeletal muscle. When sarcoplasmic reticulum is incubated in 20 mM Ca2+ in the absence of ATP, 10-12 h are necessary for measurable amount of Ca2+ to penetrate into the vesicular space, as determined by the fact that X-537A releases Ca2+ from 'loaded' vesicles only after this period of incubation. A fraction of Ca2+ of 50-60 nmol/mg protein, rapidly taken up by sarcoplasmic reticulum, exchanges with Mg2+ and K+ in the medium and is readily released by ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid, but it is not released by X-537A. The slow-penetrating fraction of Ca2+ (30-40 nmol/mg protein) is rapidly released X-537A. The results indicate that most of the Ca2+ retained by sarcoplasmic reticulum under conditions of passive uptake is bound to the external side of the membrane. The fraction of Ca2+ that slowly penetrates the vesicles remains essentially free inside the vesicles and only a small part is bound to the internal side of the membrane.     

Localization of calcium in nerve fibers

Using the desheathed nerve preparation, a pyroantimonate precipitation method was used to examine the distribution of electron-dense particles seen in various organelles of the nerve fibers following exposure of nerve to various levels of Ca2+ in vitro. The presence of Ca2+ in the electron-dense particles was indicated by their extraction with EGTA and by the use of energy-dispersive X-ray microanalysis. In normal Ringer or in a Ca2+ -free medium, electron-dense particles were seen associated with the outer membrane of the mitochondria, with the smooth endoplasmic reticulum (SER), along the axolemma and yet others scattered throughout the axoplasm. When nerves were incubated in media containing higher than normal concentrations of 20-60 mM Ca2+, an increase in the number of such electron-dense particles was seen in the axoplasm and within the mitochondrial matrix. Nerves loaded with a high concentration of 60mM Ca2+ could be depleted of these particles after transfer to a Ca2+ -free or low Ca2+ Ringer medium. The sequestration of Ca2+ in axonal organelles is discussed with respect to Ca2+-regulatory mechanisms in the axon needed to maintain a low level of Ca2+ which is optimal for the support of axoplasmic transport.     

Studies on the efflux of metalloporphyrin from rat liver mitochondria. Effect of K+ and other cations.

The mechanism by which metalloporphyrins synthesized within the mitochondria escape to the incubation medium was studied in isolated rat liver mitochondria. In a low-ionic-strength sucrose medium, the efflux of metalloporphyrins is markedly decreased when K+ (greater than 10 mM) is added. The effect of K+ is not dependent on the energy state of the mitochondria and it can in part be abolished by adding globin, but not albumin. K+ also decreases the uptake of porphyrins by the mitochondria and thereby the rate of synthesis of metalloporphyrins. Qualitatively similar results are found with Na+, Li+, Mg2+ and Ca2+. Quantitatively, however, the efficiency of cations to inhibit the release of metalloporphyrins decreases in the order: Mg2+ greater than Ca2+ greater than K+ greater than Li+ greater than Na+. Co-protoporhyrin behaves essentially as Co-deuteroporphyrin. The results provide further evidence that the efflux of metalloporphyrins from the mitochondria depends on haem-binding ligands of the suspending medium and also on the ionic strength of the incubation medium.     

The study of Ca2+ influx in human erythrocytes in isotonic polyethylene (glycol) 1500 (PEG-1500) and sucrose media

Effects of isotonic solutions of polyethylene (glycol) 1500 (PEG-1500) and sucrose on Ca2+ influx into ATP-depleted red blood cells were studied using the Ca2+ -sensitive fluorescent dye fura-2AM. When incubated in isotonic low ionic strength media (containing 2 mM CaCl2 in addition to sucrose and PEG-1500), the initial rate of Ca2+ influx was higher than that for the cells in physiological (normal ionic strength) medium. After 20 minutes of incubation in the PEG-1500-containing solution, a 10-fold increase of Ca2+ influx was observed, whereas in the sucrose medium the rate of Ca2+ influx decreased compared to that in physiological medium. 1H-NMR data provided no evidence of direct interaction between PEG-1500 and the erythrocyte membrane. Moreover, PEG-1500 did not affect lipid peroxidation (LPO) induction in erythrocyte membranes. We propose that a change in the hydrogen environment of Ca2+ -ATPase of the erythrocytes suspended in the PEG-1500 solution is the primary cause of altered Ca2+ homeostasis in these cells. The activation of the Ca2+ -ATP-ase in sucrose medium may result in an incomplete suppression of the Ca2+-pump activity in ATP-depleted cells, which is accelerated when calmodulin binds with the Ca2+-ATP-ase under the conditions of rapid Ca2+ accumulation.     

Properties of calcium binding by Myxicola axoplasmic protein

The 45Ca2+ binding properties of axoplasmic protein from the Myxicola giant axon have been investigated using a centrifugal/concentration-dialysis technique. Scatchard plot analysis of these binding data suggest that Ca2+ is attached to a site with an equilibrium dissociation constant of 7.7 +/- 0.5 microM and a capacity of 4.4 +/- 0.2 mumol/g axoplasmic protein (n = 11). Addition of other cations--Cd2+, Mn2+, Al3+, Cu2+, Ba2+, and Zn2(+)--at concentrations up to 10 microM did not displace 0.2 microM 45Ca2+ from its binding site, probably because of buffering of these cations by amino acid residues within the protein solutions. The protein could be stored at 4 degrees C for up to 16 days with no appreciable change in the number of calcium sites. Ca2+ binding equilibrium took place in less than 30 min of incubation. Increasing the incubation temperature from 4 degrees C to 37 degree C reduced the number of Ca2+ sites. The binding capacity was reduced by one-half when the protein was dialyzed with 4 M urea or high ionic strength KCl (2 M). Calcium binding was examined as a function of pH. When the protein was dialyzed overnight at different pH values and all the binding was done at pH 7.0, the apparent number of Ca2+ sites decreased as the pH of the dialysis medium was increased. When the protein was dialyzed overnight at pH 7.0 and the binding was done at different pH values, the apparent binding capacity increased as pH increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of subcellular membranes from canine stomach smooth muscle

Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5'-nucleotidase and NADPH-cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18-32% (w/w) sucrose was maximally enriched (13-fold) in 5'-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH-cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32-40% (w/w) sucrose, was maximally enriched in NADPH-cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive. ATP-dependent Ca2+ uptake correlated very well with that of 5'-nucleotidase but less well with NADPH-cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role ih regulating intracellular Ca2+ concentration in canine corpus circular muscle.